The Gut Microbiome

The influence of the gut microbiome on human health and disease has been estab-
lished in recent years through advances in high-throughput DNA sequencing. The
Gut Microbiome: Bench to Table presents a scientific introduction to this topic
and analyzes the research on how the microbiome is affected by nutrients and how
dietary modifications can alter the microbiome.
The Gut Microbiome: Bench to Table is divided into three sections. The first sec-
tion details the current state of laboratory-scale analysis of gut microbiome samples
and how we can identify the communities and their functional repertoire. Section II
explains the next phase of translational research models such as preclinical animal
studies, proof of concept safety, and efficacy human trials. The third section demon-
strates the effectiveness of therapeutic treatments in larger populations. It addresses
how diet influences the gut microbiome and presents an array of approaches that
have been reported, including a discussion of issues of the safety of probiotics and
selected supplements and micronutrients.
This book is essential for clinicians, dietitians, and food and nutrition
professionals who wish to have the most up-to-date knowledge in understanding
gut microbiome and diet.
The Gut Microbiome
Bench to Table

Edited by
Vivian C.H. Wu
First edition published 2023
by CRC Press
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© 2023 Taylor & Francis Group, LLC

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Library of Congress Cataloging-in-Publication Data

Names: Wu, Vivian C. H., editor.
Title: The gut microbiome : bench to table / edited by Vivian C.H. Wu, Ph.D.
Description: First edition. | Boca Raton : CRC Press, 2023. |
Includes bibliographical references and index. |
Identifiers: LCCN 2022022597 (print) | LCCN 2022022598 (ebook) |
ISBN 9781032295442 (hardback) | ISBN 9781032295435 (paperback) |
ISBN 9781003302070 (ebook)
Subjects: LCSH: Gastrointestinal system–Microbiology. |
MESH: Gastrointestinal Microbiome | Gastrointestinal Tract–microbiology
Classification: LCC QR171.G29 G886 2023 (print) |
LCC QR171.G29 (ebook) | DDC 612.3/601579–dc23/eng/20220601
LC record available at https://lccn.loc.gov/2022022597
LC ebook record available at https://lccn.loc.gov/2022022598

ISBN: 9781032295442 (hbk)

ISBN: 9781032295435 (pbk)
ISBN: 9781003302070 (ebk)

DOI: 10.1201/b22970

Typeset in Times
by codeMantra
 Dedication

This book is wholeheartedly dedicated

to my parents and grandmother for their
dear unsurpassed love and support.
To my husband and the greatest gift
from God, my son for their love,
patience, and understanding.
To my brother, sister-in-law, and
nephews for their love and care.
To God for giving me strength and
power of mind and for enabling me
to live, learn, love, and inspire.
Contents
Preface.......................................................................................................................ix
Acknowledgments .....................................................................................................xi
Editor ..................................................................................................................... xiii
Contributors ............................................................................................................. xv
Introduction ............................................................................................................xvii

Section i t0 Benchwork, including

techniques for Microbiome Studies

Chapter 1 Tools and Resources Enabling Marker Gene–Based

Microbiome Studies. ............................................................................ 3
Jianan Liu and Robert W. Li

Chapter 2 Phageome in Gut Microbiome ........................................................... 45

Yujie Zhang, Yen-Te Liao, Logan Tom, Somanshu Sharma,
and Vivian C.H. Wu

Section ii t1 Preclinical Animal Studies, and

Safety and efficacy trial in Humans

Chapter 3 Microbiome and Nutrition – Why Do We Care?:

Discussing a Complex Relationship ................................................... 71
Angelos K. Sikalidis

Chapter 4 Models for Researching the Gut Microbiome ....................................97

Alison Lacombe, Vivian C.H. Wu, and Aleksandra S. Kristo

Chapter 5 Dietary Modulation of Gut Microbiota by Cultured Products ..............119

Salam A. Ibrahim, Rabin Gyawali, Raphael D. Ayivi,
Hafize Fidan, Saeed Paidari, and Reza V. Bakhshayesh

Chapter 6 Dietary Modulation of the Gut Microbiota by Prebiotics and

Other Dietary Nutrients and Relevant Health Effect(s) ................... 157
Alison Lacombe and Vivian C.H. Wu
vii
viii Contents

Section iii t2–3 effectiveness trials in General

Population and Regulatory Limitations

Chapter 7 Gut Microbiota and Polyphenols: Discussing a Powerful

Interplay and Its Effect on Health .................................................... 183
Aleksandra S. Kristo and Dorothy Klimis-Zacas

Chapter 8 Diet, Obesity, and Gut Microbiome ................................................. 211

Yu-Chieh Cheng, Alison Lacombe, and Vivian C.H. Wu

Chapter 9 Challenge, Future Research, and Influence in Food Science

and Product Development ................................................................ 239
Marta Vernero, Claudia Caglioti, and Gianluca Ianiro

Index ...................................................................................................................... 253

Preface
The human fascination with diet and its impact on health is not new and quite ancient.
The link between what goes into the body and what comes out is obvious and indis-
putable. The history of medicine in the West, the East, and the Americas focused on
the amelioration of disease through the impact of foods, herbs, and elixirs on diges-
tion. Stool testing was commonly used in medicine long before the understanding of
microbes and their role in the digestive process. A common historical example was
the madness of King George III. His intermittent bouts of mania were associated
with abdominal pain, constipation, and discoloration of the urine. Twentieth-century
scientists would later pore over meticulous documentation of stool samples and post-
humously diagnose the king with porphyria. While dissecting stool samples for signs
of melancholic dysphoria seems arcane, the medical profession at the time was ironi-
cally on the right track. Today medical science has reinforced the evidence suggest-
ing that the gut microbiome is associated with conditions like major depression.
What has evolved from this fascination with the elimination product from diges-
tion is the understanding of the individual components and key players in the diges-
tive process. Today there are numerous tools to investigate the gut microbiome
and the impact of diet. The influence of the gut microbiome on human health and
disease has been established through numerous advances in high-throughput DNA
sequencing. Genome-enabled biology provides an effective tool for investigating
the members of a microbial community, their adaption to the environment, and
downstream effects on their host. While we might seem quite advanced compared
with the standard medical practices of the 18th century, we are still excavating just
the tip of the iceberg. With every advance in genomics, metabolomics, proteomics,
and other -omics, scientists are coming up with more questions than answers. This
book will attempt to answer some of these questions and posit the question to be
addressed in the future.
The subtitle Bench to Table depicts the journey of knowledge from small labo-
ratory investigations to more extensive clinical trials. This book borrows from the
National Institutes of Health guidelines for translational medicine. Translational
research is the process of integrating laboratory, clinical, and population-based
research with the long-term aim of improving public health. The translational
approach uses a multidisciplinary team of experts to bring knowledge generated in
small-scale laboratory experiments to a clinical setting in an attempt to bridge the
gap between the bench and bedside. T0 research is conducted in laboratories with
the aim of achieving tools and resources enabling microbiome studies. T1 research
expedites the movement between laboratory research and clinical research by inves-
tigating the preclinical outcomes such as safety and efficacy. T2 research facilitates
the movement of knowledge gained toward the general population with clinical trials
investigating which factors contribute to better patient outcomes, implementation of
best practices, and improved health status in communities. T3 research promotes

ix
x Preface

the interaction between laboratory-based research and population-based research

through larger-scale clinical trials to stimulate a robust scientific understanding of
human health and disease. The book addresses the modulation of the gut microbiota
through dietary modulation. Such knowledge in understanding gut microbiome and
diet may offer innovative strategies for targeted product development.
Acknowledgments
This book would not have been possible without the trust and effort of each of the
chapter contributors. Special thanks go to Dr. Alison Lacombe for her countless
hours assisting with the chapters. We began our world of the microbiome in Maine
and accomplished the book of gut microbiome we promised in California.
I thank my publishers, CRC Press/Taylor & Francis Group, LLC, especially
Randy Brehm and Tom Connelly, for their assistance in this book project. Thanks
to Joseph Eckenrode for approaching me and encouraging me to develop this book.
Thanks also to Susan Farmer for connecting the right place for this book.
Acknowledgment is also given to Wu’s group from Maine to California and to all
over the world. If I have ever made an impact on the science of microbiology, that is
from the hard work of Wu’s group.
My deep gratitude and heartfelt appreciation go to Dr. Daniel Y. C. Fung,
my M.S. and Ph.D. advisor, for teaching and inspiring me with everything about
food microbiology.
I am grateful, as always, to my beloved family.

xi
Editor
Dr. Vivian C.H. Wu is the Research Leader of the Produce Safety and Microbiology
Research Unit, one of the six Research Units at the U.S. Dept. of Agriculture (USDA)-
Agriculture Research Service (ARS) Western Regional Research Center in Albany,
California. In 2021, she was the acting Associate Area Director of USDA-ARS
Pacific West Area (PWA), providing leadership and line management to the program
and staff in the PWA, which covers eight states with more than 2000 employees.
Before joining USDA ARS, Dr. Wu served as a Full Professor at the University of
Maine, where she led the Pathogenic Microbiology Laboratory. She currently serves
as an Adjunct Professor at Washington State University, Texas Tech University, and
National Chiao Tung University (Taiwan). Dr. Wu earned her Ph.D. in food science
with an emphasis on microbiology under Dr. Daniel Y. C. Fung from the Department
of Animal Sciences and Industry at Kansas State University. She was appointed to
the faculty at the University of Maine in 2003 and has developed an exceptionally
creative research program that led to attaining her international stature in micro-
bial food safety, pathogen detection, interventions, and functional foods. Since
August 2015, she has been serving as the Research Leader for the Produce Safety
and Microbiology Research Unit, USDA-ARS, where she oversees more than 60
scientists (PIs), technicians, support staff, and postdocs, in addition to student interns
and volunteers. Dr. Wu has published 145 peer-reviewed journal articles, four book
chapters, 18 proceedings, 161 peer-reviewed abstracts, one patent and six invention-
patent disclosures, and 51 technical reports (375 publications in total). Dr. Wu is a
fundholder of an $8M annual budget for three ARS CRIS research projects in addi-
tion to more than $18 million in external competitive grants she received for her
research program. Before ARS, she had garnered more than $12 million in external
competitive grants as the Principal or Co-Investigator to her research program dur-
ing her 11 years at the University of Maine. Additionally, Dr. Wu received the 2012
International Bimbo Pan-American Nutrition, Food Science and Technology Award
(out of 107 nominees) for her achievement in the development of biosensors, the
2011 Eastern Scholar Award from Shanghai Institutions of Higher Learning for her
research achievement, and the 2012 Distinguished Service Award from the Chinese
American Food Society for her service contribution to the society. She received
the Chinese American Food Society (CAFS) Lifetime achievement award in 2015
and CAFS Professional achievement award in 2020. She received National Chung-
Hsing University Distinguished Alumni Achievement Award in 2020. Her research
accomplishments have also been translated into successfully training undergraduate
and graduate students and postdoctoral research scientists, resulting in more than
28 prestigious national and international research awards. She has graduated seven
Ph.D. students, 24 M.S. thesis students, three undergraduate honors thesis students,
29 undergraduate research capstone students, and nine high school research capstone
students. She currently advises three postdocs and co-advises four Ph.D. students.
Dr. Wu’s internationally recognized expertise has resulted in invitations from the
USDA, FDA, universities, and research institutes in the U.S. and other countries

xiii
xiv Editor

to give talks about her research and insights on food safety. She gave 122 invited
presentations, including 54 international invitations, and made 135 presentations at
national and international meetings. She was the Spotlight Scientist selected by the
Center for Produce Safety at the University of California-Davis (2013), highlighting
her research achievement in produce safety. Dr. Wu was appointed to serve on the
Board of Agriculture to advise the Chancellor of the University of Maine System
and the President of the University of Maine on matters concerning agricultural
research and extension development. She received invitations to serve on prestigious
grant panels and ad hoc proposal reviews. Before ARS, She was invited to chair
an ARS review panel in the Quality and Utilization of Agricultural Crops National
Program. She is the Editor-in-Chief of the Journal of Food Safety. She serves on 16
international journal editorial boards. She served as the elected treasurer and several
standing committees of the Chinese American Food Society for ten years before
becoming the President of the Chinese American Food Society (CAFS), 2015-2016.
Contributors
Raphael D. Ayivi Hafize Fidan
Department of Food and Nutritional Department of Nutrition and Tourism
Sciences University of Food Technology
North Carolina A&T State University Plovdiv, Bulgaria
University of North Carolina
Greensboro, North Carolina Rabin Gyawali
Department of Nanoscience, Joint Department of Food and Nutritional
School of Nanoscience and Sciences
Nanoengineering North Carolina A&T State University
University of North Carolina Greensboro, North Carolina
Greensboro, North Carolina
Gianluca Ianiro
Reza V. Bakhshayesh Digestive Disease Center
Department of Food Biotechnology, Catholic University of Rome
Branch for Northwest & Rome, Italy
West Region, Agricultural
Research, Education and Salam A. Ibrahim
Extension Organization (AREEO) Department of Food and Nutritional
Agricultural Biotechnology Research Sciences
Institute of Iran North Carolina A&T State University
Tabriz, Iran Greensboro, North Carolina
Department of Food Science and
Technology Dorothy Klimis-Zacas
University of Tabriz School of Food and Agriculture
Tabriz, Iran University of Maine
Orono, Maine
Claudia Caglioti
Digestive Disease Center Aleksandra S. Kristo
Catholic University of Rome Department of Food Science and Nutrition
Rome, Italy California Polytechnic
State University
Yu-Chieh Cheng San Luis Obispo, California
Agricultural Biotechnology Research
Center Alison Lacombe
Academia Sinica Produce Safety and Microbiology
Taipei, Taiwan Research Unit, Agricultural
Biotechnology Center in Southern Research Service
Taiwan United States Department of
Academia Sinica Agriculture
Tainan, Taiwan Albany, California

xv
xvi Contributors

Robert W. Li Angelos K. Sikalidis

Agriculture Research Service, Animal Department of Food Science and
Genomics and Improvement Nutrition
Laboratory California Polytechnic State University
United States Department of Agriculture San Luis Obispo, California
Beltsville, Maryland
Logan Tom
Yen-te Liao Produce Safety and Microbiology
Produce Safety and Microbiology Research Unit, Agricultural
Research Unit, Agricultural Research Service, Western Regional
Research Service, Western Regional Research Center
Research Center United States Department of
United States Department of Agriculture
Agriculture Albany, California
Albany, California
Marta Vernero
Jianan Liu
Department of Medical Sciences
Agriculture Research Service, Animal
University of Pavia
Genomics and Improvement
Pavia, Italy
Laboratory
United States Department of Agriculture
Beltsville, Maryland Vivian C.H. Wu
Produce Safety and Microbiology
Saeed Paidari Research Unit, Agricultural
Department of Food Science and Research Service, Western Regional
Technology, Science and Research Research Center
Branch United States Department of
Islamic Azad University Agriculture
Tehran, Iran Albany, California

Somanshu Sharma Yujie Zhang

Produce Safety and Microbiology Produce Safety and Microbiology
Research Unit, Agricultural Research Unit, Agricultural
Research Service, Western Regional Research Service, Western Regional
Research Center Research Center
United States Department of United States Department of
Agriculture Agriculture
Albany, California Albany, California
Introduction
Our planet is inhabited by millions of species that have coevolved to either share
or compete for the same resources. The collective way multiple species interact is
generically known as symbiosis. There are many forms of symbiotic relationships
that are apparent to the naked eye, for example, the honeybee and flowering plants.
With nature, what is seen at the macrocosmic level is often reflected in the micro-
cosm. The result is an infinite “doll” of interconnected microorganisms forever
adapting and evolving in an ever-changing environment. The gut microbiome is a
perfect representation of the continuing dialogue that occurs with organisms in a
constantly changing environment. Fed by the host’s diet, the microorganisms that
inhabit the gastrointestinal tract (GIT) compete for resources, and the resulting rela-
tionship is categorized as follows: mutualism, commensalism, predation, parasitism,
and competition. The human large intestine is an intensively colonized area contain-
ing microorganisms that are health promoting as well as pathogenic. This has led
to functional food developments that fortify the former at the expense of the latter.
Translational research is the process of integrating laboratory, clinical, and
population-based research with the long-term aim of improving public health. The
translational approach uses a multidisciplinary team of experts to bring knowledge
generated in small-scale laboratory experiments to a clinical setting to bridge the gap
between the bench and bedside and table. Basic research (T0) is conducted in labo-
ratories with the aim of determining the efficacy of an intervention and its potential
mechanism. In the next stage, T1 research expedites the movement between labora-
tory research and clinical research by investigating the preclinical outcomes such
as safety and efficacy. T2 research facilitates the movement of knowledge gained
toward the general population with clinical trials investigating which factors con-
tribute to better patient outcomes, implementation of best practices, and improved
health status in communities. Finally, T3 research promotes the interaction between
laboratory-based research and population-based research through larger-scale clini-
cal trials to stimulate a robust scientific understanding of human health and disease.
To properly examine the interactions between human health and the gut microbiome,
each step of the translational approach is needed. For example, a specific phyto-
chemical may demonstrate promising anti-inflammatory properties in vitro, but have
poor bioavailability when studied in animals or humans.
This book will discuss some of the challenges, such as complex relationships
between human hosts, gut microbiome, and diet. The book will explore the popu-
lation complexity and diversity between individuals when studying gut microbiota
and how to improve the techniques available for microbiota research. To advance
the knowledge in this area and understand how gut microbiota adapts to humans’
changing lifestyle involves understanding the gut microbiomes’ involvement in
human development. In addition, we need detailed information about the specific
aspects of host–microbe relationships and the mechanisms underlying them. There
is a complex bidirectional symbiosis that exists between the gut microbiome and its
host. The holistic composition of the human microbiome is governed by genetics,

xvii
xviii Introduction

environment, and diet. In turn, the microbiome influences host metabolism, calorie
absorption, immune reactions, and behavior. The gut microbiome modulates its host
environment by releasing its own metabolites, modifying the metabolites of the host,
and synthesizing vitamins and enzymes.
Consumers are aware of the potential healing properties of dietary regimens and
foods and have fueled the demand for products that promote health. The desire for
food that meets nutritional needs and can improve health has led food scientists to
develop the concept of functional foods. Dietary supplements/nutrients offer new
tools for improving host health by either preventing or ameliorating the symptoms
of immune disorders, cancer, obesity, and other diseases. This book will discuss the
microbes used as probiotics and the best industrial practices for optimal use. One
future direction is optimizing the composition of infant formulas and other food
products in maintaining gut health.
As the concept of gut health continues to evolve, researchers investigate the poten-
tial benefits of enhancing beneficial species through diet or other interventions. There
are many possible sources of beneficial products for the gut, which can be found in
the natural environment or synthesized based on research indicating certain func-
tional groups with benefits. However, more work is needed to determine which prebi-
otic regimen is best for dietary recommendations, although recommendations cannot
probably be applied ubiquitously across the population. This opens a fascinating field
of inquiry into tailoring probiotic and prebiotic regimens for optimum health. Using
a translation approach with different models, research can begin to elucidate the
causal relationship between the diet, the host genetics, and the gut microbiome.
Section I
T0 Benchwork,
Including Techniques for
Microbiome Studies
 1 Tools and Resources
Enabling Marker
Gene–Based
Microbiome Studies
Jianan Liu and Robert W. Li
United States Department of Agriculture

CONTENTS
Introduction ................................................................................................................4
Historical Perspective of Microbiome Studies...........................................................6
High-Throughput Sequencing Technologies .............................................................8
454 Pyrosequencing and Supported Oligonucleotide Ligation
and Detection ........................................................................................................8
Illumina Platform ..................................................................................................9
Ion Torrent ........................................................................................................... 10
Long Read Sequencing Platforms ....................................................................... 11
SSU Reference Databases ........................................................................................ 12
Greengenes .......................................................................................................... 12
SILVA ..................................................................................................................13
Ribosomal Database Project ............................................................................... 13
EzBioCloud ......................................................................................................... 14
UNITE ................................................................................................................. 14
PR2....................................................................................................................... 15
Marker Gene Sequence Analysis Pipelines.............................................................. 15
QIIME 1 and QIIME 2 ........................................................................................ 15
mothur ................................................................................................................. 18
FROGS ................................................................................................................ 19
iMAP ...................................................................................................................20
Recent Advances in Computational Tool Development........................................... 21
Uniqueness of Marker Gene Survey Datasets ..................................................... 21
Statistical Testing and Differentially Abundant Taxon Identification ................. 21
Balances and Microbial Signature Identification ................................................24
Tools for Network Analysis and Their Applications ...........................................26

DOI: 10.1201/b22970-2 3
4 The Gut Microbiome: Bench to Table

Web-Based Tools for Marker Gene Survey Data ..................................................... 30

Conclusions .............................................................................................................. 35
Acknowledgments.................................................................................................... 35
References ................................................................................................................ 35

INTRODUCTION
The microbiome is referred to as “the entire habitat, including the microorgan-
isms (bacteria, archaea, lower and higher eukaryotes, and viruses), their genomes
(i.e., genes), and the surrounding environmental conditions” (Marchesi and Ravel,
2015). Trillions of microorganisms from archaea, bacteria, and eukarya and their
viruses colonize the gastrointestinal tract of humans and animals from birth and
play critical roles in their development and disease. It is still debatable whether the
placenta is sterile. Most studies show that the detection of the placental microbiome
is largely due to contamination (Kuperman et al., 2019; Theis et al., 2019). However,
a recent study of a human fetus microbiome revealed the possibility of the exposure
of the fetus to bacteria and their metabolites in utero (Stinson et al., 2019). Moreover,
questions have been raised about the widely quoted notion that the total number of
microbial cells outnumbers human cells by 10 to 1. The latest study suggests that the
estimated number of bacteria in the human body is 3.8 × 1013, approximately a 1:1
ratio with the number of human cells (Sender et al., 2016). Nevertheless, the micro-
biome plays vital functions in host physiology and nutrition, including modulating
the host immunity, protecting the host against invading pathogens, and affecting the
host organ development. It has long been known that microorganisms in the gastro-
intestinal tract act as a metabolic organ and are responsible for nutrient production
and utilization. For ruminant species, the microorganisms in the rumen and hindgut
convert carbohydrates (mainly plant fiber) into readily absorbable short-chain fatty
acids, which provide up to 75% of their total metabolizable energy (Li et al., 2012). In
monogastric species, such as humans and pigs, microbial fermentation in the hindgut
allows the host to harvest extra energy from otherwise indigestible carbohydrates.
Numerous gut bacteria, especially those from Prevotella, also participate in dietary
protein degradation, including oligopeptide degradation and deamination. In addi-
tion, gut microorganisms produce vitamins for the host (LeBlanc et al., 2013). As
a result, ruminants do not need a dietary supply of water-soluble vitamins, while
a significant portion of our daily vitamin requirement, particularly water-soluble
B vitamins and vitamin K, are synthesized by gut microbes. Gut microorganisms
also affect host organ development. Evidence accumulated from studies of germ-
free animals suggests the gut microbiome is essential for proper intestinal motility,
morphology, function, and epithelial renewal (Sommer and Bäckhed, 2013). Most
notably, the gut microbiome is instrumental in promoting the development of both
innate and adapted immune systems (Duerkop et al., 2009; Sansonetti and Di Santo,
2007). Indeed, the strong induction of innate immunity followed by the stimula-
tion of adaptive immune responses can be detected only four days after colonization
of germ-free mice with the total fecal microbial community (El Aidy et al., 2012).
Microbiome-derived metabolites, including short-chain fatty acids, directly regulate
innate and adaptive immune cells (Rooks and Garrett, 2016). However, disruption
Tools and Resources for Microbe Analysis 5

of the homeostasis of the host microbiome can result in severe pathological con-
sequences, such as chronic inflammation and metabolic dysfunction (Sommer and
Bäckhed, 2013). The gut microbiome has been linked to the development of obesity
and metabolic disorders, such as diabetes and insulin resistance (Ley, 2010). The
intestinal microbiome affects the pathogenesis of inflammatory bowel diseases, such
as Crohn’s disease and ulcerative colitis (Baker et al., 2009; Joossens et al., 2011;
Schwiertz et al., 2010). Inflammatory bowel disease patients tend to have an aberrant
microbiome, characterized by the depletion of commensal bacteria, notably members
of the phyla Firmicutes and Bacteroidetes. Furthermore, the altered microbiome has
been suggested to affect the carcinogenesis of several tumors, including colorectal
gastric tumors and cancerous liver tumors (Fox et al., 2010). Evidence also suggests
the gut microbiome affects viral infection (Wilks and Golovkina, 2012) and plays a
role in HIV progression (Ellis et al., 2011). As a result, the gut microbiome has been
touted as a therapeutic target or as a drug target (Cani and Delzenne, 2011).
While the structure and functions of the gut microbiome have been extensively
explored in the past few years, the complex interrelationships of the microbiome
within a host still require higher-resolution studies using comprehensive toolsets
to promote new discoveries (Heintz-Buschart and Wilmes, 2018). Because of the
complexity and functional importance of the microbiome, numerous methods and
bioinformatic tools have been developed in recent years to conduct comprehensive
microbiome studies using culture-independent approaches. For example, enzymes
that catalyze chemical reactions are encoded by genes present in microbial com-
munities from a broad spectrum of environmental conditions. Therefore, functional
screening has become an increasingly important tool to discover novel biomolecules
for applications in biotechnology and medicine. This approach, also termed func-
tional metagenomics, provides direct access to largely unexploited microbial genetic
diversity in the environment. In the meantime, the rapid advancement of ultra-low
cost and high-throughput or parallel sequencing technologies and the development
of bioinformatics tools and various databases have stimulated sequencing-based
microbiome studies, including whole genome shotgun–based computational metage-
nomics and marker gene–based microbiome studies. The former allows us to have a
holistic assessment of the functional capacity and metabolic potential of the micro-
bial community in its habitat.
Marker gene–based microbiome profiling is a rapid alternative to whole genome
shotgun–based approaches in quantifying the structure and composition of microbial
communities. Due to their functional constancy and highly conserved nature, small
subunits (SSUs) of the rRNA gene, especially the 16S rRNA gene, have been used
the most as phylogenetic markers for genus and species identification and taxonomic
classification of bacteria and archaea. The 16S rRNA gene is large enough (~1,500
nucleotides) compared to 5S rRNA genes to carry adequate information for com-
parisons and is small enough compared to 23S rRNA genes (~2,900 nucleotides)
to be conveniently analyzed. As a homologue of prokaryotic 16S rRNA genes, the
eukaryotic 18S rRNA gene is also widely used as a phylogenetic marker for fungi and
other eukaryotes. The presence of hypervariable (HV) regions in these genes reflects
the evolutionary divergence of microorganisms and can be explored to differentiate
even highly related species (Woese, 1987). The internal transcribed spacer (ITS),
6 The Gut Microbiome: Bench to Table

located between the 18S and 5.8S rRNA genes, is used as a marker specifically to
identify a broad range of fungi. The ITS is more variable than the 18S rRNA genes
and is therefore more appropriate as the genetic marker to study intraspecific genetic
diversity (Schoch et al., 2012). Furthermore, many protein-coding single-copy genes,
such as the RNA polymerase β subunit gene (rpoB), have also been explored as
molecular markers for microbial ecology studies (Case et al., 2007).
Many molecular methods have been developed in the past to characterize SSUs
of rRNA genes, such as polymerase chain reaction (PCR)–based cloning and tra-
ditional sequencing and culture-independent molecular approaches, including
denaturing gradient gel electrophoresis/temperature gradient gel electrophoresis,
PCR-restriction fragment length polymorphism (RFLP), and terminal restriction
fragment length polymorphism (TRFLP). Additionally, 16S probe-based fluorescent
in situ hybridization (FISH) is a powerful tool for the visualization and identification
of microorganisms in their native habitats (Crocetti et al., 2000; Ginige et al., 2004).
The recent renaissance of the microbial marker gene–based study to characterize the
gut microbiome is largely driven by rapid advances in next-generation DNA sequenc-
ing technologies (Gloor et al., 2010; Tringe and Hugenholtz, 2008) and the develop-
ment of related data analysis pipelines and databases (Balvočiūtė and Huson, 2017;
Caporaso et al., 2010). Moreover, several new concepts and computational tools are
in rapid development. In this chapter, we will focus on recent advances in marker
gene–based approaches and bioinformatics tools essential for microbiome studies.

HISTORICAL PERSPECTIVE OF MICROBIOME STUDIES

Traditional culturing methods, accompanied with morphological and biochemical
studies of cultured strains, were developed gradually for the characterization of sin-
gle microorganisms in the late 19th century (Schaechter, 2015). However, by the
1980s, it was recognized that the population of prokaryotes in the environment is
vast and incredibly diverse (Whitman et al., 1998). Because of the complexity of
microbial communities, only a limited portion of the bacteria in the environment
can be successfully isolated and cultured. The discovery of viable but nonculturable
microorganisms (Oliver, 2005) motivated scientists to develop alternative methods
to study them in their habitat. SSU rRNA genes gradually attracted attention. These
genes were later found to be an appropriate phylogenetic marker of microorganisms
because of their highly conservative sequences and functional specificity (Woese,
1987; Woese and Fox, 1977; Woese et al., 1990).
Since the invention of Sanger sequencing and PCR techniques, scientists have
developed culture-independent methods for sequencing cloned 16S rRNA genes
from microbial populations (Pace et al., 1986). The clone library of 16S and/or 18S
rRNA genes was sequenced, followed by phylogenetic analysis. This conventional
clone-and-sequencing method targeting SSU rDNA was developed to study complex
microorganism populations without prior microbial cultivation. The process involves
the cloning of targeted genes by creating a population of host bacteria, such as E. coli,
that carries plasmids containing the inserted DNA. With the development of avail-
able databases, taxonomy identification can be achieved for the obtained sequences,
with similarity thresholds from 80% at the phylum level to 97% at the species level
Tools and Resources for Microbe Analysis 7

(DeSantis et al., 2007). The advantage of this conventional cloning method is that
the clean sequence of the entire 16S rRNA gene can be obtained and then sequenced,
which provides a reliable and precise picture of prokaryotes in a microbial commu-
nity. However, the heavy workload makes the method extremely time-consuming,
laborious, and expensive (Green and Sambrook, 2012).
A variety of molecular methods were then developed to overcome the issues asso-
ciated with conventional cloning. Most of these methods utilize DNA hybridization
and DNA length and sequence polymorphisms to study microbial communities. In
1993, a method called denaturing gradient gel electrophoresis was applied to ana-
lyze the genetic diversity of complex prokaryotic communities (Muyzer et al., 1993).
Shortly after, a similar method called temperature gradient gel electrophoresis was
developed. Denaturing gradient gel electrophoresis examines microbial genetic pat-
terns via a chemical gradient to denature the PCR-amplified 16S molecules as they
move across an acrylamide gel, while temperature gradient gel electrophoresis is
based on melting point differences in DNA molecules with a linear temperature gra-
dient in the gel (Muyzer et al., 1993; Muyzer and Smalla, 1998). These two methods
are more rapid and reproducible than the conventional cloning method.
Two other efficient methods, PCR-RFLP and TRFLP, are based on RFLP. These
methods can be applied to 16S rRNA, ITS-5.8S rRNA-ITS2, or 23S rRNA genes
to characterize microbial diversity (Guillamön et al., 1998; Leaw et al., 2006).
For PCR-RFLP, PCR products amplified from SSU rRNA genes are digested with
restriction enzymes to generate DNA fragments that are then separated by gel
electrophoresis based on their size. In TRFLP, PCR products are generated with
5ʺ-fluorescent-labeled primers before endonuclease digestion to generate the termi-
nal restriction fragments (Marsh, 1999). The mixture of DNA fragments can then
be analyzed in an automated DNA sequencer, but only the terminal fragments with
labels can be detected and displayed. Several bioinformatics methods have been
developed for peak identification and taxonomic assignment for TRFLP results,
such as T-REX (T-R FLP analysis EX pedited) (Culman et al., 2009), web-based
TRFMA (Nakano et al., 2006), and Excel-based tools (Fredriksson et al., 2014).
TRFLP simplifies complex banding pattern analysis compared to PCR-RFLP and
allows for the more rapid identification of microbial composition in a community.
Although RFLP-based methods are able to provide fingerprint patterns to detect
community structure, problems exist, including the introduction of PCR bias and
variation caused by the choice of restriction enzymes (Osborn et al., 2000; v.
Wintzingerode et al., 1997). Phylogenetic oligonucleotide arrays, an amplification-
independent method, were developed to overcome these pitfalls. PhyloChip, devel-
oped at Lawrence Berkeley National Laboratory, is the most comprehensive and
high-density phylogenetic oligonucleotide array to detect the presence of bacteria and
archaea from direct microbial DNA or PCR products with a broad dynamic range.
The platform developed its own proprietary bioinformatics pipelines to identify vari-
ations in 16S rRNA gene sequences. The latest version of PhyloChip (PhyloChip G4),
currently available from Second Genome, Inc., can identify and measure the relative
abundance of more than 50,000 individual microbial taxa. PhyloChip has been used
to characterize microbial communities in a variety of samples and environments. In
one case, spatial patterns of microbial species in a rough environment, salt crusts
8 The Gut Microbiome: Bench to Table

of the hyper-arid Atacama Desert, were examined using PhyloChip G3. The results
from this study identified two dominant species, Halobacteriales and Bacteroidetes,
and the existence of a few algal and cyanobacterial species in low abundance levels
(Finstad et al., 2017) in this extreme habitat.
FISH is another powerful method that evolved from non-FISH in 1980 (Bauman
et al., 1980). FISH targeting microbial SSU rRNA genes can be used to determine the
spatial distribution and genome structure of microbes. Typically, probes targeting the
16S rRNA gene are generally 18–30 nucleotides in length with fluorescence labels.
These probes can penetrate through the cell membrane and eventually hybridize to
the target 16S rRNA genes in situ (Hugenholtz et al., 2002). Hybridized target genes
within the individual cells can then be visualized using a fluorescence or confocal
microcopy, which allow exploring the spatial location of the targeted genes in addi-
tion to taxonomic identifications. With recent technical innovations, FISH has been
combined with several other cutting-edge biological tools to visualize and elucidate
genome structures of microorganisms. A powerful example is the integration of
FISH with Hi-C to reveal the 3D structure of chromosomes (Abbas et al., 2019). This
method was also used to define the chromosome structure of Mycoplasma pneu-
moniae. By using Hi-C with a 10kb resolution to generate 3D models of the station-
ary M. pneumoniae chromosome and then applying FISH to visualize and measure
distances between genomic regions, Trussart et al. (2017) concluded that transcrip-
tional regulation can be affected by chromosome organization.

HIGH-THROUGHPUT SEQUENCING TECHNOLOGIES

In the past decade, rapid technological advances in next-generation sequencing (NGS)
and the concomitant development of bioinformatics tools and database resources
have revolutionized microbiome studies. Unlike traditional Sanger sequencing, NGS
platforms can generate (hundreds of) millions of sequences during a single run. The
development history of the traditional and NGS technologies, the basic features and
performance comparisons and the application potential between various sequencing
platforms have been extensively reviewed (Ambardar et al., 2016; Besser et al., 2018;
Gužvić, 2013. Lam et al., 2012). In this chapter, we limit our discussion to the tech-
nologies more relevant to SSU marker gene–based studies.

454 Pyrosequencing and suPPorted

oligonucleotide ligation and detection
The introduction of large-scale parallel pyrosequencing technology by 454 Life Sciences
in 2005 initiated the NGS revolution. Roche then acquired this technology and released
the 454 GS FLX Titanium system in 2008 with improved average read length (up to
700 bp) and accuracy (up to 99.997%). The GS FLX system can sequence ~400–600
Mb of DNA per 10-hour run (Voelkerding et al., 2009). The long read length facilitates
de novo sequencing and microbiome studies. However, the labor-intensive nature of
the sample preparation, the high reagent cost, and the high error rates in homopolymer
regions are some of the obstacles that are difficult to overcome. Because Roche later
decided to stop supplying the hardware and reagents, this platform is now defunct.
Tools and Resources for Microbe Analysis 9

Supported Oligonucleotide Ligation and Detection was originally invented by

Applied Biosystems Instruments and later acquired by Life Technologies (Thermo
Fisher Scientific). The platform relies on a ligation-based sequencing technology that
uses DNA ligase and fluorescently labeled oligonucleotide probes for sequencing in
either the 3ʹ to 5 ʹ direction or the 5 ʹ to 3ʹ direction. While the current 5,500 W Series
Genetic Analysis Systems offer a highly accurate and flexible sequencing solution
with adequate output, the short read length they generate (up to 75 bp) and the long
running time (days) make Supported Oligonucleotide Ligation and Detection subop-
timal for SSU amplicon sequencing.

Illumina Platform
The Illumina platform relies on the “sequencing by synthesis” technology invented
by Solexa (Bentley et al., 2008). In contrast to various costly enzymes used by
pyrosequencing in the 454 platform, sequencing by synthesis uses only DNA poly-
merase. Specifically, in the flow cell, DNA templates are amplified using isother-
mal bridging amplification, which relies on arching over and hybridizing to an
adjacent oligonucleotide anchor by DNA strands. Numerous amplification cycles
make the single-molecule DNA template into a clonally amplified arching cluster
of approximately 1,000 clonal molecules. As each Deoxynucleotide triphosphates
(dNTP) is inserted, a fluorescently marked reversible terminator is recorded and
then cleaved to allow the next base to be integrated. The ultimate result is a base-by-
base sequencing that allows a wide range of applications with accurate sequencing
data (Bentley et al., 2008).
Illumina has since acquired this technology from Solexa and marketed several
systems that differ in terms of sequencing capacities. HiSeq (2 × 150 bp) and MiSeq
(2 × 250 ~ 300 bp) series have long been the dominant high-throughput sequenc-
ers. In recent years, Illumina has launched several new systems, such as NovaSeq
6000. The HiSeq X system is the world’s first to break the $1,000 genome barrier for
human whole genome sequencing. The system contains a set of 10 HiSeq X ultra-
high-throughput instruments. Illumina will discontinue its support for HiSeq 2500
and X series after 2023 and 2024, respectively. NovaSeq 6000 is a relatively new
comprehensive Illumina system that enables cost-effective sequencing. Notably, it is
a scalable platform that allows the loading of individual flow cell lanes with differ-
ent library types to suit a wide range of project sizes or needs with the NovaSeq Xp
workflow. It also comes with single and dual flow cell modes with different flow cell
types to generate several combinations of read length and coverage depth, with read
lengths up to 2 × 250 bp (paired-end).
Illumina MiSeq is another powerful sequencer that enables the generation of
approximately 25 million sequencing reads with up to 2 × 300 bp read lengths. In
November 2013, the US Food and Drug Administration approved the marketing of
the NGS system, Illumina’s MiSeqDx, as a diagnostic device for human genome
sequencing. The marketing approval of the first high-throughput genome sequencer
represents a significant step forward in producing genomic data to aid diagnosis and
eventually improve patient care. MiSeq is suitable for amplicon-based microbiome
studies because of its longer read length. Merging its paired-end reads can result in
10 The Gut Microbiome: Bench to Table

up to 550 bp assembled reads while allowing for an overlap of 50 bp. As a result, it is

advisable to use paired-end MiSeq sequencing to cover more than one HV region on
SSU marker genes. The selection of target HV regions and primers is a critical factor
for consideration for SSU marker gene sequencing using MiSeq. Various HV regions
have discriminatory differences in terms of the groups of microbes (D’Amore et al.,
2016). It is known that V1 works best in differentiating across Staphylococcus aureus
and coagulase-negative Staphylococcus species, whereas V2 and V3 perform better
in distinguishing bacterial species up to the genus level, except for the closely related
species in Enterobacteriaceae. V6 differentiates across all select agents defined by
the Centers for Disease Control and Prevention (Chakravorty et al., 2007). More
recent studies demonstrate that V4 is generally more informative than other regions,
and combining HV regions is highly recommended (D’Amore et al., 2016; Soergel
et al., 2012). For example, the selection of 341F/785R (or 805R) across V3 to V4 regions
is recommended over using V4 only, as longer amplicon length is reported to improve
species-level assignment and provide higher phylogenetic diversity (Klindworth
et al., 2012; Thijs et al., 2017). Besides universal primers, archaea- and bacteria-
specific primers should also be selected depending on the objectives of a specific
project. A study of the human archaeome found that the archaea-specific primer pair
349af/519ar outperforms universal primers in identifying archaeal operational taxo-
nomic unit (OTU) and Amplicon Sequence Variant (ASV) (Koskinen et al., 2017).
One recent trend is full-length sequencing of the 16S rRNA gene using exist-
ing platforms with improved combinational chemistry and novel bioinformatic tools.
For example, the recently developed LoopSeq technique is able to sequence DNA
or mRNA molecules with synthetic long reads based on Illumina sequencers (Wu
et al., 2019). Its core technology lies in a set of unique barcodes that attach to each
individual DNA molecule. Every molecule is amplified with its unique barcode, and
the copies get sequenced next to each randomly distributed barcode. Short reads with
the same barcodes are assembled with linked-read de novo assembly. Full length in
combination with high sequence accuracy from proved sequencing platforms makes
species-level taxonomic specificity possible (Wu et al., 2019).

Ion Torrent
While the Ion Torrent platform uses sequencing by synthesis and emulsion PCR
somewhat similarly to Illumina chemistry and 454 pyrosequencing, respectively, its
method of detection is unique. If a nucleotide is incorporated into a strand of DNA,
an H+ ion is released. The charge from H+ ions alters the solution pH value, which
can be detected by a proprietary ion sensor. An Ion Torrent sequencer is essentially
a solid-state pH meter. Because the detection is direct and rapid without the need for
scanners, cameras, or light (and without the need for fluorescence and chemilumines-
cence reagents), the cost of sequencers is low, and running time is short (<7 hours).
While high error rates in sequencing homopolymer stretches and repeats (similar to
the 454 platform) are undesirable, Ion Torrent generates a mean read length up to 400
bp, which is quite appealing for SSU amplicon sequencing.
Salipante et al. (2014) compared the performance of two common benchtop NGS
sequencers, Illumina MiSeq and Ion Torrent Personal Genome Machine (PGM), for
Tools and Resources for Microbe Analysis 11

bacterial community profiling by 16S rRNA (V1–V2) amplicon sequencing using

a mock microbial community consisting of 20 known species and human samples.
Two issues with Ion Torrent PGM, comparatively higher error rates and premature
sequence truncation, are documented in this study. The latter results in organism-
specific biases in community profiles. While results are generally in good agreement
between the two platforms with protocol optimization in Ion Torrent, organism-
dependent differences in sequence error rates and premature sequence truncation
pose a challenge for certain microorganisms when using Ion Torrent technology.

Given limited read lengths, only one or two adjacent HV regions of the 16S rRNA
gene can be sequenced using the second-generation sequencing technologies. Short
reads may not be effective for species- or subspecies-level identification. New plat-
forms with long read sequencing, also termed the third-generation sequencing tech-
nologies—such as those developed by Pacific Biosciences (PacBio) and Oxford
Nanopore—have increasingly gained popularity.
PacBio Sequel Systems use a single-molecule, real-time sequencing technology
to provide highly accurate long reads. Single-molecule, real-time cells contain mil-
lions of zero-mode waveguides (ZMWs). A single DNA molecule is immobilized in
each ZMW and is repeat sequenced with real-time detection of base incorporation.
Each of the four nucleotides A, C, G, and T are labeled with different fluorescent
dyes on their terminal phosphates. Light is emitted during DNA synthesis through
the template in the ZMW and recorded as nucleotide sequences. The sequencing
reads can reach up to tens of Kb. However, one key issue with the technology in
the past is a high sequencing error rate, sometimes up to 11%–15%. PacBio tries to
reduce the error rate by utilizing a consensus accuracy function to reduce system-
atic errors. Specifically, both DNA strands in one ZMW can be sequenced multiple
times to generate multiple reads (subreads); a consensus sequence of these subreads
can yield higher accuracy (Rhoads and Au, 2015). The newest release is the Sequel
II System, which produces exceptional results with faster running time, affordable
expense, and highly accurate long reads. PacBio has recently been successfully used
in SSU sequencing projects to cover the full length of 16S rRNA genes (Callahan et
al., 2019; Earl et al., 2018).
Oxford Nanopore is another promising DNA sequencing technology designed
to have essentially no reagent costs, little sample preparation, and ultra-long read
length to reduce the need for complicated assembly algorithms and reveal the long-
range structure of the genome at the same time (Branton et al., 2010). The prin-
ciple of Nanopore is that when a strand of DNA passes through a nanopore, the
current is changed as the bases T, C, G, and A pass through in different combina-
tions. Several sequencers, such as MinION, GridION, and PromethION, that differ
in device size, nanopore channels, and sample prep procedures are currently avail-
able. Nanopore technology has also been successfully applied to sequence full-length
16S rRNA genes. One study analyzed the mouse gut microbiome by full-length 16S
rRNA amplicon sequencing using Nanopore and compared the results with short-
read sequencing data (Shin et al., 2016). The study found no significant differences
12 The Gut Microbiome: Bench to Table

in major taxonomic assignment between the two methods. However, the differ-
ence becomes apparent at species-level identification. Empowered with long reads,
Nanopore is able to identify more species than short-read platforms, which improves
the classification accuracy of community bacterial composition (Shin et al., 2016).
It is conceivable that this technology will play a critical role in field microbial ecol-
ogy and microbiome studies with anticipated significant reduction of the sequencing
error rate in the near future.

SSU REFERENCE DATABASES

The accuracy and robustness of taxonomic classification and phylogenetic affilia-
tion of sequence reads obtained in amplicon-based sequencing are dependent on the
coverage and quality of SSU reference databases. Resulting from many years of com-
munity effort, these databases are vital resources in microbiome studies.

Greengenes is a database for full-length 16S rRNA genes of bacteria and archaea
that aims to provide a curated taxonomy for users based on de novo tree inference
(DeSantis et al., 2006; McDonald et al., 2012). The latest Greengenes version is
gg_13_8, released in August 2013, and is a minor improvement over the previous
version (the gg_13_5 released in May 2013). The improvement addressed the missing
genus and species names. If a genus or species name is missing for a particular OTU,
the National Center for Biotechnology Information (NCBI) taxonomy of cluster
members was checked, and the most supported names were then selected to update
the taxonomy of representative sequences. The taxonomic name was then transferred
to all the lower similarity representative sequences. The number of sequences in
Greengenes and other databases is listed in Table 1.1.
Although Greengenes has not been updated since 2013, this curated, chimera-
checked database is still robust and is one of the most used databases in 16S gene
sequencing analysis. Several analysis tools, such as the Quantitative Insights into
Microbial Ecology (QIIME) analysis pipeline and the metagenome function prediction

TABLE 1.1
Commonly Used Databases on Marker Gene–Based Microbiome Studies and
Their Number of Sequences
Database Latest Version Release Date Number of Sequences
Greengenes gg_13_8 Aug 2013 1262,986 (16S rRNA sequence)
SILVA SILVA 132 Dec 2017 2,090,668 (SSU Ref), 695,171 (SSU Ref NR)
RDP Release 11.5 Sep 2016 3,356,809 (16S rRNA sequences), 125,525
(28S rRNA sequences)
EzBioCloud Version 20191112 Nov 2019 65,050 (16S rRNA sequences), bacterial genomes
UNITE Version 8.1 Sep 2019 98,183 (fungal SH with DOIs at 1.5% threshold)
PR2 Version 4.12.0 Aug 2019 183,949 (eukaryotic SSU)
Tools and Resources for Microbe Analysis 13

software package Phylogenetic Investigation of Communities by Reconstruction of

Unobserved States (PICRUSt) (Langille et al., 2013), have chosen Greengenes as
their default database or have been developed based on Greengenes.

SIL
SILVA (from Latin silva, forest) is a comprehensive rRNA gene web resource dedicated
to providing the taxonomy information for bacteria, archaea, and eukarya (Pruesse
et al., 2007; Quast et al., 2012). It contains the ribosomal RNA sequence datasets of
aligned small (16S/18S, SSU) and large subunit (23S/28S, LSU) rRNA sequences,
which are quality checked and regularly updated. The latest full release is the SILVA
SSU/LSU version 132, released in December 2017. The preliminary release informa-
tion of SILVA 138 is available on its website, and the version is expected to launch
around the end of 2019. SILVA is highly recommended by the widely used analy-
sis platform mothur as its default reference database. The SILVA database contains
different sections of rRNA genes. “SSU Parc” includes all aligned sequences with
an alignment identity value ≥ 50, alignment quality value ≥ 40, and base-pair score
or sequence quality ≥ 30. “SSU Ref” was created by excluding sequences shorter
than 1,200 bases (for bacteria and eukarya) and below 900 bases (for archaea) or an
alignment identity lower than 70 or an alignment quality value lower than 50 from
the SSU Parc dataset. Additionally, SILVA offers a nonredundant (NR) version of
the SSU Ref dataset, “SSU Ref NR,” where highly identical sequences are removed
by applying UCLUST with a 99% identity criterion, yet sequences from cultivated
species have been kept in all cases. By adding all sequences to the SSU Ref tree of
SILVA release 128, a guide tree was calculated. Similarly, for LSU rRNA databases,
SILVA also contains “LSU Parc” and “LSU Ref” datasets based on different criteria
of LSU gene sequences. “LSU Ref” also contains a calculated guide tree.
In addition, SILVA provides several online data processing and analysis functions,
including probe and primer evaluation with “TestProbe” and “TestPrime,” sequence
alignment and classification, and an online data analysis service, SILVAngs, for
rRNA NGS amplicon reads. SILVAngs uses the taxonomies and alignments of the
SILVA rRNA gene database, supports reads classification, and offers many user-
friendly output files in table, chart, and sequence formats.

The ribosome database project (RDP) offers quality-controlled bacterial and archaeal
SSU rRNA sequence alignments and annotation, as well as data analysis tools.
Besides the SSU rRNA genes of bacteria and archaea, the RDP also adds a collection
of fungal LSU 28S rRNA sequences (Cole et al., 2009; Cole et al., 2013). The latest
release is RDP 11.5, which was released in September 2016. The RDP implements
improved alignments and also provides collections of tools for rRNA analysis. Newly
obtained sequences are aligned and classified with the RDP Aligner and Classifier
with its own phylogenetic assessment. Specifically, the RDP utilizes the infernal sec-
ondary structure-aware aligner to improve its alignment quality, speed, and con-
sistency. The RDP Classifier is a powerful and widely used method in taxonomy
14 The Gut Microbiome: Bench to Table

classification for SSU rRNA data analysis. The RDP Classifier and RDP Hierarchy
Browser Bacteria and Archaea hierarchy model has been updated to training set
no. 16, to which over 300 new genera and 2000 new sequences have been added.
Notably, the RDP provides its resource files for the NCBI LinkOut service, which
enables users to switch from the sequence records in the Nucleotide and BioProject
databases of the NCBI directly to the corresponding RDP sequence records. The
RDP also provides an online RDPipeline for high-throughput amplicon analysis and
offers multiple tools, such as primer-probe examination, approximate phylogenetic
building of user-submitted sequences, chimera removal, and automated alignment
(Cole et al., 2013).

EzBioCloud is a curated and taxonomically united database of 16S rRNA gene

sequences and whole genome assemblies for bacteria and archaea (Yoon et al., 2017).
It filters the low-quality whole genome assemblies from the NCBI Assembly Database
and contains a bioinformatics composite identification pipeline using gene-based
searches followed by average nucleotide identity calculations to construct a database
with 61,700 species/phylotypes. These include 13,132 validly published names and
62 ,362 whole genome assemblies. Using a combination of a gene-based search and
Orthologous Average Nucleotide Identity (OrthoANI) (Lee et al., 2016) calculations,
all genomes are taxonomically defined at the genus, species, or subspecies level. The
hierarchy is based on the 16S maximum likelihood phylogenetic tree, taking into
account the currently accepted classification. The website also provides a complete
set of EzBioCloud 16S databases in formats compatible with QIIME or mothur pipe-
lines. One of the unique features of EzBioCloud is that it enables identifying a bacte-
rial isolate using either 16S rRNA or genome data. EzBioCloud also offers a cloud
service called Microbiome Taxonomic Profiling with several bioinformatics tools.
Users are able to compare their own data with the published microbiome data in this
Microbiome Taxonomic Profiling service and perform comprehensive species-level
profiling using closed-reference OTU picking.

UNITE
The ITS region is the most widely used fungal marker to identify fungi and explore
fungal diversity in the environment. The UNITE is a web-based database and
sequence management environment dedicated to the molecular identification of
fungi using ITS regions as markers (Nilsson et al., 2018). UNITE is arguably the
most commonly used database for classifying and annotating fungi with ITS high-
throughput sequencing. It includes ~1000,000 public fungal ITS sequences that
are clustered into ~459,000 species hypotheses for reference and assigned DOIs to
reduce ambiguity among studies. UNITE redesigned how it handles unclassifiable
species hypotheses, integrated the Global Biodiversity Information Facility’s taxo-
nomic backbone, and supports parallel taxonomic classification systems (Nilsson et
al., 2018). Users can also compare sequences to species hypotheses through Basic
Local Alignment Search Tool (BLAST), probabilistic method for taxonomical
Tools and Resources for Microbe Analysis 15

classification (PROTAX) (Abarenkov et al., 2018) and other query tools through
UNITE. In addition to taxonomic annotation, UNITE also adds relevant metadata,
such as the host of collection and voucher specimens. Importantly, UNITE supports
web-based third-party annotations to update taxonomy and nomenclature informa-
tion and to correct suboptimal taxonomic annotation and other metadata across pub-
lic DNA sequences (Nilsson et al., 2018). With these efforts, UNITE identifies fungi
from ITS sequence data with assembling and disseminating taxonomic, ecological,
and geographical metadata. UNITE acts as a data provider for a number of metaba-
rcoding software tools and exchanges data with all major fungal sequence databases
and other community resources on a regular basis.

Pr2
The Protist Ribosomal Reference (PR2) database offers exclusive access to the ribo-
somal RNA and DNA sequences of eukaryotic SSU 18S with curated taxonomy
(Guillou et al., 2013). The majority of sequences in this database are nuclear-encoded
protistan sequences. Yet PR2 also contains sequences from metazoans, land plants,
macrosporic fungi, and eukaryotic organelles (mitochondrion, plastid, and others),
which is useful for high-throughput sequencing data analysis. PR2 also carefully
checks introns and putative chimeric sequences. Additionally, there are web tools
one can use to search the database by sequence similarity (Guillou et al., 2013).

MARKER GENE SEQUENCE ANALYSIS PIPELINES

qiime 1 and qiime 2
To facilitate marker gene–based microbiome studies, several data analysis pipelines
have been developed (Figure 1.1). QIIME (Caporaso et al., 2010) is a widely used
pipeline for analyzing SSU rRNA amplicon sequencing data of microbial commu-
nity studies, including 16S, 18S, and ITS sequence data. QIIME incorporates many
third-party tools and algorithms that make it a robust pipeline to achieve a wide
range of analyses and visualizations. It can handle SSU data generated from various
sequencing platforms.
The QIIME pipeline starts with preprocessing raw sequences with a mapping file
(sample metadata) and performs primer removal, demultiplexing, and quality filter-
ing. For paired-end data, two methods, fastq-join (default) and SeqPrep, are available
to merge forward and reverse reads. Barcode and primer sequences from the map-
ping file are used to demultiplex and clean raw sequences. Low-quality sequence
reads can be removed based on Phred-score and user-defined parameters. After pre-
processing, three types of OTU-picking strategies, de novo, closed-reference, and
open-reference, are available. QIIME provides multiple OTU clustering tools, such
as cd-hit, mothur, BLAST, Trie, uclust, usearch, sumaclust, sortmerna, and swarm.
The default OTU-picking tool is uclust with a similarity threshold of 97% and sev-
eral flexible parameter options. The QIIME team recommends open-reference OTU
picking because it offers a trade-off between running time and the possibility of new
diversity discovery. During OTU clustering, a representative sequence is selected for
16 The Gut Microbiome: Bench to Table

Sample & metadata collecon

Total DNA extracon

MP Bio FastDNA™ SPIN Kit for Soil,
Qiagen Stool DNA kit with a modified bead-
beang step (Lysing Matrix E)

DNA sequencing
Targeted Hypervariable Primer selecon Sequencing pla–orm
regions F515/R806, Illumina MiSeq v3
Full length, V3-V4, V4 F341/R805 chemistry, 2x 250 cycles

Sequencing output Metadata

Fastq, fasta Mapping file

Pre-processing
Remove primers, demulplex, quality filter

OTU/ASV detecon
QIIME, Mothur, FROGS, iMAP

Alpha diversity Beta diversity

Diversity index: Chao1, Fisher's alpha, Distance matrix: Weighted Unifrac, Aitchison
PD Tree, Shannon, Simpson Ordinaon: PCoA, NMDS
Stascal tesng: ANOVA, Wilcoxon, Global stascal tesng: PERMANOVA,
or Kruskal-wallis PERMDISP, ANOSIM, MPPR, etc.

Differenal abundance test

ALDEx2, ANCOM

Balances or Molecular Signature

Gneiss, Selbal

Phylogenec tree visualizaon

SigTree

Global network inference

Stac (cross seconal) Dynamic (Time series)
MPLasso, SPIEC-EASI, SparCC SgLV-EKF, LIMITS

FIGURE 1.1 Recommended data analysis pipeline for marker gene-based microbiome studies.
Tools and Resources for Microbe Analysis 17

each OTU to represent the whole cluster. The default is to pick the most abundant
sequence from a cluster as its representative, as these sequences are less likely to
represent sequencing errors. When uclust or usearch is selected, the cluster seed is
used as the representative sequence. The QIIME team also recommends the addition
of chimera checking after OTU picking by UCHIME to exclude chimera in down-
stream analyses. The subsequent step in the pipeline includes assigning taxonomy
to representative sequences. The latest Greengenes database is used as the default.
The sequence alignment and phylogeny construction can then be performed with
Python Nearest Alignment Space Termination (PyNAST) and FastTree by default,
respectively. An OTU table is finally generated in the Biological Observation Matrix
(BIOM) format. For downstream analyses, OTU tables and phylogenetic trees gener-
ated from upstream analysis and corresponding mapping files are used. A second-
level quality filtering can be done to reduce the problem of spurious OTUs, followed
by a rarefaction step at a user-defined subsampling depth. After that, the OTU table
is collapsed into taxa to be summarized at each taxonomic level. A series of plots
including bar, pie, and area graphs can be generated and then visualized via a web
interface. QIIME pipeline integrates two diversity analyses, alpha and beta diver-
sity. Three alpha diversity metrics, phylogenetic diversity (PD)_whole_tree, Chao1,
and observed_OTU, are the default outputs. Dozens of other metrics are optional
in alpha_diversity.py script, for example, Simpson and Shannon indices. QIIME
recommends Unifrac (Lozupone and Knight, 2005) for beta diversity analysis and
generates weighted and unweighted Unifrac to analyze community differences.
Additionally, interactive diversity graphs, such as rarefaction curves based on alpha
diversity metrics and principal coordinate analysis (PCoA) based on community beta
diversity distance can be accessed through the output HTML documents. Further
statistical analysis of taxa and diversities can be performed within QIIME or with
external methods. In addition to common t-tests, QIIME also offers multivariate
analyses in the script compare_categories.py, including analysis of similarities
(ANOSIM) and Adonis, to examine beta diversity. OTU network analysis is also
integrated in QIIME.
Since 2017, the QIIME team has reengineered and developed a new system,
QIIME 2 (Bolyen et al., 2019). As a result, the conventional QIIME or QIIME 1 is no
longer officially supported. QIIME 2 also integrates third-party tools in the form of
plugins to allow others to contribute to its function. QIIME 2 provides better support
for paired-sample and time series analyses. Although several features in QIIME 1
are retained, QIIME 2 offers several key improvements over QIIME 1. One critical
aspect of QIIME 2 is using ASV instead of OTU as microbial features. The recom-
mended demultiplexing and denoising workflow in QIIME 2 for AVS identification
is the DADA2 algorithm. Deblur is also used as an optional denoising method to
combine q2-Vsearch to join reads before proceeding. Specifically, these denoising
methods exclude noisy sequences, correct marginal sequence errors (in DADA2),
perform chimera removal and singleton removal, join denounced paired-end reads
(in DADA2), and perform sequence dereplication. Both DADA2 and Deblur contain
internal chimera removal methods and abundance filtering during the denoising pro-
cess. However, QIIME 2 still keeps three OTU-picking options, similar to QIIME 1.
UCHIME and Vectorized search (Vsearch) are used to remove chimera sequences
18 The Gut Microbiome: Bench to Table

and perform OTU picking, respectively. In addition to the common downstream

analysis tools in QIIME 1, QIIME 2 implements several recently released tools, such
as q2-ALDEx2, q2-Gneiss, and q2-PICRUSt2, to enable multidimensional analy-
sis. Other new features include a retrospective data tracking system that records all
parameters and steps used during data generation. Furthermore, QIIME 2 adds a new
interactive visualization method (qiime2 view https://view.qiime2.org) that allows
data sharing and interaction without installing QIIME 2. Moreover, QIIME 2 devel-
ops a graphical user interface, QIIME 2 Studio (q2studio), and a Python 3 application
programmer interface, the artifact Application Programming Interface (API), to suit
the needs of both beginners and advanced users.

mothur

mothur is another widely used data pipeline for amplicon-based microbial commu-
nity analysis. As a comprehensive pipeline, mothur integrates several preexisting
tools and incorporates additional features to process raw community sequencing data
(Schloss et al., 2009).
Supporting documents and a discussion forum that contain procedures to pro-
cess sequence data generated from Sanger, PacBio, Ion Torrent, 454, and Illumina
platforms and provide corresponding standard operating procedures are available
for mothur users and developers. For example, the MiSeq standard operating pro-
cedure contains the procedures for raw sequence demultiplexing, quality control,
chimera removal, taxonomic classification, OTU clustering, diversity analysis, and
phylogeny-based analysis (Kozich et al., 2013). mothur merges forward and reverse
reads to make contigs and removes ambiguous sequences. Unique sequences are
then identified and aligned to the reference. mothur provides several alignment
databases containing 16S and 18S gene sequences compatible with the Greengenes
or SILVA alignments. mothur removes chimeras using the Vsearch algorithm and
identifies nonbacterial lineages, such as those related to chloroplasts and mito-
chondria. Multiple algorithms are available for sequence clustering, including
Opticlust (default), Vsearch distance-based greedy clustering, Vsearch abundance-
based greedy clustering, furthest neighbor, average neighbor, nearest neighbor, and
Unique. The pipeline also performs rarefaction and microbial diversity analyses. A
numbers of diversity metrics, such as ACE, Chao1, observed OTU, Simpson, and
Shannon, can be calculated. Distance metrics, such as weighted complete lineage,
unweighted Unifrac, and Jaccard, are optional with various commands. mothur
also contains tools for ordination analysis using PCoA and nonmetric dimensional
scaling (NMDS). The diversity results can be statistically tested within mothur
using correlation measurements of the relative abundance of each OTU or analy-
sis of molecular variance. Additionally, mothur implements nonparametric analysis
tools—Metastats and linear discriminant analysis (LDA) effect size (LEfSe)—to
examine if there are significantly different OTUs between groups of interest. mothur
workflows are also available in Galaxy.
Both QIIME and mothur share numerous functional features in sequence data
preprocessing, OTU clustering, and diversity index extraction, as well as the genera-
tion of OTU or feature tables. The major difference lies in interactive visualizations.
Tools and Resources for Microbe Analysis 19

QIIME 2 offers several newly developed interactive visualization tools, whereas

mothur focuses on generating results that can be easily imported and visualized
with other tools. Additionally, unlike QIIME that wraps some third-party tools in a
python environment, the mothur team translates many packages in C++ and makes
them open source for other contributors to modify and improve (Bolyen et al., 2019;
Schloss et al., 2009). A recent study compared the analysis results of QIIME and
mothur on a rumen microbiome dataset. The conclusion is that for the most abun-
dant genera, there are no statistical differences between the two pipelines, no matter
which reference databases are used (SILVA or Greengenes). However, for microbes
in low abundance, mothur is more sensitive than QIIME, but this difference is dimin-
ished, and the results become more consistent when SILVA is chosen as the reference
database in both pipelines (López-García et al., 2018).

FROGS
Find, Rapidly, OTUs with Galaxy Solution (FROGS) is a recently developed Galaxy-
supported pipeline that aims to analyze large sets of amplicon sequences (Escudié
et al., 2017). The pipeline includes a variety of functions, such as preprocessing, cluster-
ing, chimera removal, filtering, and taxonomic or phylogenetic affiliation assignment,
visualization, and tree construction. FROGS handles both single and paired-end
data from the Illumina platform and those generated using 454. The pipeline merges
paired-end reads using Flash with 10% mismatch tolerance and ambiguous reads
removal. Dereplication is also achieved in this step, and a graphical report is gener-
ated for posterior cleaned sequences. The pipeline then uses Swarm for sequence
clustering and Vsearch with de novo UCHIME to remove chimeras. Notably, an
innovative cross-sample validation process is applied to validate the chimeric status
on all samples. In each sample, chimeras are first identified separately, but in the
end, this sequence is considered chimeric only if it is marked as a chimera in all
samples where it exists. This is different from mothur, where only the redundant chi-
mera sequences are removed. FROGS also applies an abundance filter to screen clus-
ters and optionally delete clusters not present in all replications. Taxonomy can be
assigned up to the species level with the implemented RDP Classifier or BLASTn+.
Common SSU marker gene databases, such as Greengenes, SILVA, and EzBioCloud,
can be selected as the reference database. One unique characteristic of FROGS is
that it considers conflicting affiliations with BLASTn+, and if affiliation results find
multiple hits with different taxonomy, the first level of confliction and lower taxono-
mies are denoted as “multi-affiliation” in the final OTU table. Because of its modular
design, which allows users to choose their tools of preference or processing order,
FROGS is a fast, scalable, and parallelizable pipeline (Escudié et al., 2017).
FROGS allows in-depth analyses not only of 16S, 18S, and 23S rRNA genes but
also of amplicons from functional genes, such as dsrB. The latest version of FROGS
can also be used to analyze ITS data. The entire pipeline can be run in either com-
mand line or in Galaxy platform, which is user friendly for those without much bio-
informatic experience. FROGS has the advantage of using Swarm and its adaptive
sequence agglomeration instead of using a global similarity threshold. Additionally,
FROGS has a rigorous step of removing chimera and explicitly considers conflicting
20 The Gut Microbiome: Bench to Table

affiliations. Compared with QIIME, mothur, and UPARSE, FROGS performs better
than mothur and is more conservative than QIIME on the V4 region and better on
V3/V4 regions for in silico datasets. Furthermore, it performs relatively better for
staggered abundances and worse for uniform ones and produces less spurious OTUs
than other pipelines on real datasets (Escudié et al., 2017).

iMAP
The Integrated Microbiome Analysis Pipeline (iMAP) is a very recent development
that provides the research community with a user-friendly and portable tool that inte-
grates bioinformatics analysis and data visualization (Buza et al., 2019). The pipeline
includes bundles of commands wrapped individually in driver scripts for performing
various analyses and data visualization. It requires data files (sample metadata and
raw sequences), software, and reference databases to be in place before execution via
either a command line interface or from a Docker container command line interface.
Outputs from major steps can be conveniently transformed, visualized, and summa-
rized into a progress report. The default reference databases are up-to-date SILVA
seed or Greengenes for mothur and QIIME, respectively.
One of the interesting features of iMAP is that it allows exploratory analysis of
sample metadata or experimental/environmental variables. iMAP uses seqkit for
the general inspection of raw sequences, FastQC for base quality assessment, and
BBDuk for trimming and filtering low-quality or control reads. iMAP uses standard
approaches in mothur (default) and QIIME 2 for sequence processing and classifi-
cation. Moreover, it applies additional quality control and removes all undesirable
matches including the unknown and any sequences classified to nonbacterial lin-
eages, such as archaea, chloroplasts, eukaryotes, mitochondria, and viruses.
iMAP uses a fairly unique approach for OTU clustering and taxonomy assign-
ment. It relies on a combination of phylotype, OTU-based, and phylogeny methods
to assign conserved taxonomy to OTUs. Merged quality sequences are binned into
known phylotypes up to the genus level. All sequences are binned into clusters of
OTUs based on their similarity at ≥97% identity, and precision and the false dis-
covery rate (FDR) are then calculated using Opticlust, a default mothur function for
assigning OTUs. In the phylogeny method, a tree that displays consensus taxonomy
for each node is generated. The output from all three methods—phylotype, OTU-
based, and phylogeny—is manually reviewed, deduplicated, and integrated to form a
complete OTU taxonomy output.
The pipeline uses the R package for microbial diversity analysis. At least three
ordination methods—principal component analysis (PCA), PCoA, and NMDS—are
provided. Furthermore, phylogenetic annotation is done using Interactive Tree Of
Life (iTOL) tree viewer with the aid of selected annotation files, such as the species
richness, diversity, and relative abundances files.
While the iMAP pipeline uses an integrated approach for OTU clustering and
taxonomy assignment and demonstrates improvements over some widely used pipe-
lines, such as mothur and QIIME, it offers limited solutions for downstream analysis
beyond microbial diversity and a phylogenetic tree view, such as the identification
of differential abundant taxa and biomarker discovery using advanced statistical
Tools and Resources for Microbe Analysis 21

algorithms and inference of microbial interaction networks in both static (cross-sec-

tional) and time series data. Furthermore, while using Docker images is the first step,
the pipeline is still in a preliminary phase, and much effort is needed to address data
reproducibility concerns in relation to modern microbiome data workflows.

RECENT ADVANCES IN COMPUTATIONAL TOOL DEVELOPMENT

Uniqueness of Marker Gene Survey Datasets
Unlike species abundance data obtained in macroecology, OTU or ASV tables gener-
ated for the marker gene–based microbiome study have some unique challenges. The
absolute count data in the OTU table are largely dependent on sequencing depth and
are not a good proxy for the original abundance of microorganisms in a microbial com-
munity. As a result, the relative abundance or proportion is more biologically relevant
than absolute counts. Data, such as those in the OTU table, that are naturally described
as proportions or probabilities or as having a constant or irrelevant fixed sum (for exam-
ple, 1.0) are referred to as compositional (Gloor et al., 2017). The compositional data
pose special challenges for downstream statistical analyses and have been discussed in
detail (Aitchison, 1982; Fernandes et al., 2014). These challenges include total value
constraints, strong correlation with OTU or ASV, and subcompositional incoherence.
The second salient feature about the taxon (OTU) sample matrix is sparseness.
Only a small number of OTUs are detectable in all biological samples collected, and
a typical OTU table contains numerous zeros. These zeros include both structural
zeros (i.e., those representing the true or absolute absence of a taxon from a given
sample, regardless of sequencing depth) and technical or rounded zeros (Kaul et al.,
2017) that result from insufficient sampling or sequencing depth. The high frequency
of both types of zeros in the dataset requires special consideration. Consequently,
several methods have been developed for zero replacement (Brill et al., 2019; Quinn
et al., 2019; Rivera-Pinto et al., 2018).
The 16S dataset or OTU sample matrix is often multidimensional in nature, typi-
cally including a few thousand OTUs and up to a few hundred samples. Furthermore,
there may exist a correlation structure in 16S datasets, and a strong correlation
between OTUs negatively affects the performance of subsequent statistical testing
(Hawinkel et al., 2017). Several recent studies have clearly demonstrated that dealing
with compositionality in the 16S dataset is not optional (Gloor et al., 2017; Mandal
et al., 2015), and statistical tests ignoring these unique characteristics of 16S data
often fail to control the FDR (Hawinkel et al., 2017).

Statistical Testing and Differentially Abundant Taxon Identification

One of the key areas of interest in microbiome studies is to detect features (taxa or
ASV) displaying a statistically significant difference in relative abundance between two
groups of interest or pathophysiological conditions. These significant taxa or features
can be readily developed as biomarkers or signatures for therapeutic options (Hawinkel
et al., 2017). Over the years, many classical statistical tools have been used to iden-
tify significantly different taxa. For example, while t-tests and Analysis of variance
(ANOVA) are often used to compare alpha diversity indices between experimental
22 The Gut Microbiome: Bench to Table

groups, they are also used to detect significant taxa, for example, in a study involv-
ing HIV-positive patients with or without antiretroviral therapy and healthy con-
trols (McHardy et al., 2013). Similarly, nonparametric counterparts of these tests,
including the Wilcoxon rank sum test and the Kruskal–Wallis test by rank, are also
widely used in microbiome studies. LEfSe is an algorithm for high-dimensional bio-
marker discovery and identification of genomic features such as genes, pathways, or
taxa under experimental or pathophysiological conditions (Segata et al., 2011). The
algorithm includes three major steps—detecting features (taxa or ASV) significant
in relative abundance using the nonparametric Kruskal–Wallis test, investigating
biological consistency using the Wilcoxon rank sum test, and using LDA to estimate
the effect size of each differentially abundant feature. The method has been widely
used for microbiome data analysis, including the discovery of biomarkers or dif-
ferentially abundant taxa derived from OTU or ASV tables or biological pathway
data inferred using PICRUSt (Kageyama et al., 2019; Lo Presti et al., 2019; Puri et
al., 2018; Sims et al., 2019). The popularity of the method is partially driven by the
implementation of a user-friendly Galaxy version of the algorithm (http://hutten-
hower.sph.harvard.edu/galaxy/).
In several regards, OTU or ASV count data are similar to gene expression data
obtained using microarrays or RNAseq. As a result, many algorithms developed in
the early years of the millennium to detect differentially expressed genes, such as
DESeq2 and EdgeR, can be readily adapted to analyze marker gene survey data
(McMurdie and Holmes, 2014). In 2013, Paulson et al. proposed a novel normal-
ization procedure, cumulative sum scaling normalization, to correct the bias in the
assessment of differential abundance introduced by widely used total-sum normal-
ization (Paulson et al., 2013). A zero-inflated Gaussian (ZIG) distribution mixture
model that accounts for biases in differential abundance testing resulting from under-
sampling of the microbial community was also proposed in that paper. Unlike the
ad-hoc heuristic approach used in LEfSe, ZIG uses linear modeling and has been
shown to be more sensitive under simulated conditions. More differentially abundant
species can be identified using ZIG than LEfSe in real microbiome datasets (Paulson
et al., 2013). However, this method does not address the compositionality issue inher-
ent in marker gene datasets, which poses particular challenges, and traditional statis-
tical tools often become invalid.
In 2014, an algorithm dealing with compositional data typically seen in data-
sets involving multiple omics approaches was developed. This method, ALDEx2
(Fernandes et al., 2014), includes several straightforward steps. First, the counts are
converted to probabilities by Monte Carlo sampling from the Dirichlet distribution
with the addition of a uniform prior, usually 1/2. The resultant Monte Carlo Dirichlet
instance is then normalized using the centered log-ratio (CLR) approach. The value
is the base 2 logarithms of the abundance of the feature in each Dirichlet instance in
each sample divided by the geometric mean abundance of the Dirichlet instance of
the sample. Significance tests between groups of interest are then conducted using the
Welch’s t-test and the Wilcoxon rank sum test; P values obtained are corrected using
the Benjamini–Hochberg procedure; and the effect size is also estimated. Compared
to traditional statistical test tools, such as the widely used LEfSe, ALDEx2 tends
to reduce the number of false positives, especially when the sample size is small
Tools and Resources for Microbe Analysis 23

(Fernandes et al., 2014). Moreover, the method enables a unified analysis for high-
throughput sequencing datasets, including marker gene count tables, RNAseq, and
ChIPseq datasets.
Recently, a novel statistical framework called analysis of composition of micro-
biomes (ANCOM) has been developed to detect taxa differing significantly in two
or more populations or experimental groups (Mandal et al., 2015). The framework
is based on Aitchison log-ratios without distributional assumptions and is therefore
quite flexible. ANCOM uses the W statistic and selects an internal threshold (W val-
ues) for significance (no P values provided). One of the limitations of the framework
is the assumption that less than 25% of the taxa are changing significantly between
two groups, which limits its application when two populations or groups under com-
parison have drastic differences. Nevertheless, when compared to the conventional
t-test and the aforementioned ZIG method, ANCOM can reduce false positives by as
much as 68% or even more while improving detection power. Since its advent, the
method has become rapidly accepted (Iszatt et al., 2019; Peterson et al., 2019) and is
now implemented in the QIIME pipeline.
A detailed comparison of several methods designed for differential abundance
detection (originally intended for gene expression count data obtained using micro-
arrays or RNAseq) under various scenarios, including sample size, effect size, and
distributional assumptions, has been recently conducted (Hawinkel et al., 2017). For
example, the sensitivity of both ANCOM and ALDEx2 is fairly low (<25%) under
the negative binomial distribution with or without correlation. Of the methods tested,
DESeq2 and SAMseq have modest sensitivity when the sample size is small, while
metagenomeSeq is more powerful with a sensitivity > 50% when the sample size is
larger than 25. However, all methods fail to control the FDR. Compared to ALDEx2,
ANCOM has a much higher FDR under all the scenarios tested, which is in sharp
contrast to the original claim that the method has reasonable power to detect differ-
entially abundance log-ratios while controlling the FDR at a nominal level (<0.05).
Other studies have also suggested that ANCOM is unable to control the FDR, espe-
cially under the global null setting (Brill et al., 2019). Moreover, ANCOM is com-
putationally intensive when used at the OTU level without filtering out very rare
features. Weiss et al. (2017) compared several methods along with ANCOM and sug-
gested that ANCOM behaves better in terms of sensitivity and controlling the FDR
than those methods not specifically designed for handling compositionality.
During the preparation of this chapter, we conducted a direct comparison of sev-
eral popular methods using a published 16S rRNA gene dataset (Liu et al., 2019)
(Sequence Read Archive (SRA) accession: PRJNA534501). The part of the dataset
used for this comparison included two experimental groups, normal control mice
(N = 10) and colitis mice induced with dextran sulfate sodium (DSS) (N = 10). The
sample OTU matrix includes 20 × 1,666 data points, a typical dataset commonly seen
in controlled animal experiments.
The number of significant genera identified by all four non-ANCOM–related
methods is 22, whereas the number of significant genera identified by all six methods
is 15 (Table 1.2); 15 of the 16 genera identified by ANCOM (v2) are also detected by
all other methods. ALDEx2 performs well compared to other methods. For composi-
tional datasets, such as those obtained in marker gene–based microbiome studies, we
24 The Gut Microbiome: Bench to Table

TABLE 1.2
The Number of Differentially Abundant Genera Identified by
Six Algorithms Tested
ANCOM_ ANCOM_
Version Method LEfSe v1 v2 Wilcoxon DESeq2 ALDEx2
v1.0 LEfSe 35 23 16 31 24 24
v1.1-3 ANCOM_v1 23 16 22 21 20
v2.0 ANCOM_v2 16 15 15 15
v1.0 Wilcoxon 31 24 24
v1.12.4 DESeq2 25 22
v1.4.0 ALDEx2 24

All algorithm except LEfSe was at a cutoff value of the FDR < 0.05. The default cutoff for LEfSe was used
(absolute LDA > 2.0). The values in off-diagonal entries indicate the number of significant genera identi-
fied by both methods.

would recommend ALDEx2 as a default option for detecting taxa with significantly
different abundance between groups of interest.

Although changes in the relative abundance of individual taxa or ASV induced

by treatments or alterations in pathophysiological status can be compared using
the statistical methods discussed above, the interpretation of compositional data
is somewhat more difficult than real vectors in standard analysis (Egozcue and
Pawlowsky-Glahn, 2005). Moreover, in the real world, understanding the relation-
ship or balance (log ratio) of two or more features (parts) of the sample composition
within a group or between groups becomes more relevant. Recently, the balance con-
cept developed by Egozcue and Pawlowsky-Glahn (2005) has been applied to marker
gene–based microbiome analysis (Morton et al., 2017). Morton et al. developed a
method called Gneiss to construct balance trees using clustering tools or a phyloge-
netic tree and isometric log-ratio transformation. Once constructed, balances can be
analyzed using standard statistical methods, such as linear regression. Morton et al.
(2017) demonstrated that by focusing on subcommunities or groups of taxa instead
of individual taxa of a microbial community, the balance tree approach can bet-
ter yield novel insights into niche differentiation. The open-source software can be
accessed at https://github.com/biocore/gneiss. It is also implemented in the QIIME 2
pipeline. A nice video about this software is freely available at https://www.youtube.
com/watch?v=HAULM1WQkew.
Examining the treatment effect on individual OTUs or taxa is an important step
in microbiome studies. It is of particular interest to identify and visualize branches
in a phylogenetic tree whose members (OTUs or taxa) display a significant consensus
(overall) abundance response to treatments or changes in conditions. Motivated by
this, a new software package called SigTree (Stevens et al., 2017) has been developed.
Tools and Resources for Microbe Analysis 25

This software relies on meta-analytic principles and provides tools to use the results
of OTU-level significance tests (with meaningful one-sided P values) to identify and
visualize branches in a phylogenetic tree that are significantly responsive to experi-
mental interventions or changes in environmental conditions. SigTree uses P values
rather than raw data and allows users to select models appropriate for specific study
designs, including accounting for study-specific data distribution (Poisson, negative
binomial, or nonparametric). The package also provides two multiple testing cor-
rection options, the Hommel and Benjamini–Yekutieli corrections. Together, the
authors claim these flexible features allow SigTree to be applicable in any experi-
mental design and with any high-throughput technology. Compared to Gneiss, which
does not include convenient tree-level visualization tools, SigTree provides better
tree visualization. Moreover, SigTree does not have the constraints typically associ-
ated with the isometric log-ratio transformation used in Gneiss, including issues with
zero replacement.
A novel approach superficially similar to Gneiss has been proposed to identify
balances or microbial signatures, a group of taxa that can better predict treatment out-
comes or a phenotype of interest (Rivera-Pinto et al., 2018). This package, selbal, uses
a greedy stepwise algorithm for the selection of balances that preserves the principles
of compositional data analysis. The process of balance identification by selbal includes
two steps, modeling the response variable and identifying the smallest number of
taxa with the highest prediction or classification accuracy. The software also provides
several choices for zero replacement, including the default geometric Bayesian mul-
tiplicative replacement method. The variables can be categorical (dichotomous) or
numerical (continuous). To demonstrate its utilities, we analyzed the aforementioned
dataset (Liu et al., 2019) (SRA accession: PRJNA534501) using selbal with default
parameters. Three variables are considered, treatment (dichotomous, normal, or DSS),
spleen weight (g, numerical), and colon length (cm, numerical). The selbal algorithm
selects 20 variables (genera) with good classification accuracy that are compatible
with the 24 genera identified using ALDEx2. As Figure 1.2a shows, a microbial signa-
ture consisting of the balance (log ratio) of Streptococcus (numerator) to an unclassi-
fied genus in the family Peptostreptococcaceae (denominator) can readily distinguish
between DSS-induced colitis mice and normal control mice. The balance value for
DSS mice is negative, suggesting that the relative abundance of the unclassified genus
in Peptostreptococcaceae is much higher than that of Streptococcus. The cross-vali-
dation data indicate that this global balance is very robust and is selected 62% of the
time during the validation. Moreover, a balance, consisting of four genera, an unclas-
sified genus in the family Clostridiaceae and the genus Clostridium (numerator) and
Streptococcus and [Clostridium] in the family Peptostreptococcaceae, has a strong
predictive power for the spleen weight (R2 = 0.818) with a very low mean squared error
of 0.0041 (Figure 1.2b). Two of the four genera, Clostridium and Streptococcus, are
included in balance selection more than 60% of the time, further indicating that the
selected global balance is robust. In addition, the algorithm also identifies a microbial
signature for the colon length, but the association is modest with R2 = 0.47.
26 The Gut Microbiome: Bench to Table

FIGURE 1.2A A microbial signature consisting of the balance (log ratio) of Streptococcus
(numerator) to an unclassified genus in the family Peptostreptococcaceae (denominator).

FIGURE 1.2B Regression Analysis of the microbial signature in association with spleen weight.

Detecting robust microbial associations or interactions and identifying microbial co-

occurrence patterns or keystone species is one of the essential steps in understand-
ing the structure and function of the gut microbiome. Network algorithms are able
to encode the interactions between the components in complex systems. Microbial
Tools and Resources for Microbe Analysis 27

interaction network inference based on network theory and practice provides system-
level insight into potential ecological relationships of microorganisms in a microbial
community. For example, modularity, the extent to which microbial interactions are
organized into modules or subnetworks in a global network, may reflect habitat het-
erogeneity or phylogenetic clustering of closely related microbes, and modules with
closed-linked species may represent some key coevolution units (Olesen et al., 2007).
Furthermore, knowledge of interaction networks forms the foundation to predic-
tively model the interplay between environmental factors and microbial populations
(Kurtz et al., 2015) and may hold keys to the development of efficacious probiotics
and antibiotic therapeutics. Ultimately, the knowledge obtained by modeling micro-
bial interactions should facilitate microbial community engineering via the targeted
elimination and expansion of keystone species in a network. Motivated by these lofty
goals, dozens of algorithms have been developed to infer microbial co-occurrence
patterns and model microbial interaction network dynamics (Barberán et al., 2012;
Faust et al., 2012; Freilich et al., 2010; Ruan et al., 2006). For example, a meta-net-
work framework has recently been proposed to decipher microbial interactions and
construct more meaningful networks with biologically important clusters and hubs
(Yang et al., 2019). The framework starts with a loose definition strategy to recover
more correlations before correlation calculation. While direct relationships are dis-
covered, association rule mining is subsequently applied to detect complex forms
of correlations, including indirect correlations using Functional Similarity Weight
(FS-weight) and nonlinear correlations using the part mutual information adjusted by
path consistency algorithm, which enables the detection of interactions that are often
missed using they Pearson and Spearman approaches.
Habitat filtering, the co-occurrence of microbes due to habitats rather than biologi-
cal interactions being sampled, can induce apparent correlations, resulting in a network
dominated by habitat effects and the masking of true correlations of biological inter-
est (Berry and Widder, 2014). The phenomenon can confound microbial interaction
network inference when samples from different habitats are combined. To overcome
this issue, a novel algorithm to correct for habitat filtering effects has been proposed
(Brisson et al., 2019). This algorithm significantly improves correlation detection accu-
racy compared to Spearman and Pearson correlations, thus enabling the construction
of consensus correlation networks from datasets combining multiple habitats.
Weiss et al. (2016) conducted a comprehensive benchmarking comparison analy-
sis of the performance of eight microbial network inference methods. Commonly
used network inference tools are based on a broad range of underlying methodolo-
gies, such as correlation and regression-based tools (Compositionality Corrected by
REnormalizaion and PErmutation (CCREPE), Sparse Correlations for Compositional
data (SparCC), Random Matrix Theory (RMT)), graphical model inference tools
(Sparse Inverse Covariance Estimation for Ecological Association Inference
[SPIEC-EASI]), extended local similarity analysis (eLSA), and mutual information-
based tools (Mutual Information Clustering (MIC)). Weiss et al. (2016) concluded
that while different tools have various strengths and weaknesses in response to the
diverse challenges presented by microbiome data, all tools result in a high false-
positive rate. An ensemble approach, including CoNet, SparCC, and Spearman and
Pearson methods, tends to enhance the precision of detection. While paired with
28 The Gut Microbiome: Bench to Table

Pearson correlation, the RMT approach (Molecular ecological network analyses

(MENA)) also significantly improves precision. Furthermore, dataset characteristics
and desired ecological relationship discovery determine the selection of the better
tool. While datasets have high compositionality, SparCC is better at inferring linear
relationships with high precision. While sparsity is < 50% (after filtering to remove
very rare OTUs), local similarity analysis (LSA) is good for both time series (longi-
tudinal) and cross-sectional studies, while MIC is good for cross-sectional studies.
Otherwise, ensemble approaches tend to increase precision. Nevertheless, nonlinear
relationships are very difficult to detect using these tools.
To address compositionality and dimensionality challenges in the microbiome
data during network inference, Friedman and Alm (2012) pioneered the development
of SparCC, which is capable of estimating linear Pearson correlation values from
compositional data using CLR transformation. Since its advent, this popular algo-
rithm has been widely applied to microbiome studies and is subject to many bench-
marking comparisons (Deshpande et al., 2018; Friedman and Alm, 2012; Jackson
et al., 2018; Wang et al., 2018). However, SparCC does not consider the influence of
errors in compositional data, which may reduce the correlation estimation accuracy.
Moreover, the P value estimator used by SparCC may lead to bias and overestimating
significance. The limitations of SparCC have been discussed by Fang et al. (2015).
Recently, a fast and parallelizable implementation of the SparCC algorithm with an
unbiased P value estimator was published (Watts et al., 2018). This implementation
significantly reduces run time and memory consumption while maintaining perfor-
mance equivalent to the original SparCC. A novel strategy called SPIEC-EASI was
later developed to address some of the issues associated with SparCC (Kurtz et al.,
2015). SPIEC-EASI network inference consists of two steps. First, a CLR log trans-
formation is applied. Second, SPIEC-EASI estimates the interaction graph from the
transformed data using one of the methods, neighborhood selection (Meinshausen
and Bühlmann (MB) method) and sparse inverse covariance selection or graphical
Least Absolute Shrinkage and Selection Operator (LASSO). Unlike empirical cor-
relation or covariance estimation used in SparCC and CCREPE, SPIEC-EASI infers
an underlying graphical model using the concept of conditional independence. Under
various simulated scenarios, SPIEC-EASI, especially with neighborhood covariance
selection, outperforms SparCC and CCREPE in terms of network recovery. All
compositionality-based methods tested—SPIEC-EASI, SparCC, and CCREPE—
are better than the Pearson correlation method in detecting global network topology
features. Furthermore, the networks inferred by SPIEC-EASI are more reproduc-
ible than those obtained by SparCC and CCREPE. SPIEC-EASI infers considerably
sparser model networks than the other two methods.
A novel framework called Microbial Prior LASSO (MPLasso) has been recently
proposed (Lo and Marculescu, 2017). In their study, Lo and Marculescu use the
graphical LASSO algorithm to infer the microbial association network. The unique-
ness of the method is that the authors are among the first to integrate text mining
results from the scientific literature (i.e., experimentally verified biological informa-
tion) as prior knowledge to infer the microbial graph structure. To acquire experi-
mentally verified microbial associations, the PubMed database is searched via two
routes, microbial co-occurrence data in the literature are examined and compared to
Tools and Resources for Microbe Analysis 29

by chance alone and the machine learning–based is the method (Lim et al., 2016) to
extract the interaction information. MPLasso outperforms all methods tested, includ-
ing the aforementioned SPIEC-EASI, SparCC, and CCREPE, as well as Correlation
Inference for Compositional Data through Lasso (CCLasso) and regularized estima-
tion of the basis covariance based on compositional data (REBACCA) in terms of
area under the precision-recall curve and the accuracy (ACC) of network association
prediction in synthetic datasets. In the presence of prior knowledge, MPLasso can
achieve remarkably high accuracy in terms of the edge recovery rate, up to 95%.
Moreover, MPLasso can robustly and accurately estimate microbial interactions,
with reproducibility up to 90% in real microbiome datasets.
Microbial communities are dynamic in nature. The abundance of community
members fluctuates temporally or in response to environmental factors or perturba-
tions. Similarity-based network inferring tools (Bray–Curtis, Pearson, Spearman, or
LSA) treat time series data as a static snapshot and ignore their temporal dependen-
cies (Alshawaqfeh et al., 2017) and are generally unable to infer nonlinear relation-
ships, especially those with multispecies complex interactions. The Lotka–Volterra
equations, a pair of first-order nonlinear differential equations, have been frequently
used to describe the dynamics of biological systems in traditional macroecology.
Recently, the role of dynamic systems theory in understanding microbial commu-
nity dynamics, especially the utility of generalized Lotka–Volterra models, has been
extensively discussed (Gao et al., 2018; Gonze et al., 2018). For example, using gen-
eralized Lotka–Volterra dynamics modeling, Berry and Widder (2014) found that co-
occurrence networks can recapitulate interaction networks under certain conditions,
but they lose interpretability when the effects of habitat filtering become significant
(Berry and Widder, 2014). In particular, networks suffer from local hot spots of spu-
rious correlation in the neighborhood of keystone or hub species that engage in many
interactions. Recently, a new approach called Learning Interactions from Microbial
Time Series (LIMITS) has been developed (Fisher and Mehta, 2014). LIMITS uses
sparse linear regression with bootstrap aggregation to infer a discrete-time Lotka–
Volterra model for microbial dynamics and overcome some of the obstacles com-
monly encountered while inferring microbial interactions. The obstacles include
the observations that a correlation in the abundance between two microbial species
does not necessarily imply their actual interaction and that it is difficult to infer
parameters in time series data due to the sum constraint on the relative abundance
(compositionality) in typical microbiome datasets. When applied to real microbiome
datasets, LIMITS is able to successfully detect interaction networks dominated by
distinct keystone species.
Alshawaqfeh et al. (2017) proposed a stochastic generalized Lotka–Volterra
dynamic model that adopts the extended Kalman filter (SgLV-EKF) algorithm to
model the microbial interaction network dynamics (Alshawaqfeh et al., 2017). The
proposed stochastic model accounts for the uncertainty in the model by adding a
noise term in the dynamic equation, while extended Kalman filter, due to its ability
to estimate the parameters of nonlinear interactions from a limited number of obser-
vations, is used for the first time to estimate the abundance level of microbes and
their interactions. SgLV-EKF is also compared to five other algorithms, including
two similarity-based (Pearson correlation coefficient and LSA), one integral-based
30 The Gut Microbiome: Bench to Table

(Nelder and Mead, 1965), and two regression-based (Stein’s and LIMITS) algorithms
using both synthetic and real datasets. SgLV-EKF outperforms the other five algo-
rithms in identifying the structure of the interaction network. Moreover, SgLV-EKF
provides robust and reliable performance against the uncertainty in the dynamic
model at an accuracy level higher than 75% and that is also higher than that of
LIMITS, which provides reliable results with consistent accuracy of approximately
60%. Nelder’s algorithm generates results similar to those of SgLV-EKF under vary-
ing measurement noise levels but fails to compensate for randomness in the dynamic
system and is computationally intensive. Stein’s algorithm fails to detect the majority
of the interactions and exhibits very low sensitivity, while the two similarity-based
algorithms show a significant reduction in their accuracy due to the increase in the
process noise power. Moreover, the latter two algorithms lack mathematical model-
ing of the microbial community and are unable to predict network dynamics.

WEB-BASED TOOLS FOR MARKER GENE SURVEY DATA

In addition to the pipelines discussed above, such as iMAP, numerous resources and
tools have recently been developed and have become widely available to the research
community. For example, the mothur and FROGS pipelines are also implemented in the
Galaxy and are available online. Moreover, a complete workflow for 16S microbiome
data analysis consisting of various R packages, such as DADA2, phyloseq, DESeq2,
ggplot2, structSSI, and vegan, has provided good examples of how to use abundant
resources in R to conduct analyses from raw read filtering to community-level analyses,
including supervised analyses using Random Forest and nonparametric testing using
community networks (http://web.stanford.edu/class/bios221/MicrobiomeWorkflowII.
html). Several powerful platforms, such as MEtaGenome Analyzer (MEGAN) (Mitra
et al., 2011), the metagenomics Rapid Annotation using Subsystems Technology server
(MG-RAST) (https://www.mg-rast.org), and Visualization and Analysis of Microbial
Population Structures (VAMPS) (https://vamps2.mbl.edu), have been widely used to
visualize and analyze raw sequence data for microbial population structures and dis-
tributions. While these platforms provide good tools for data preprocessing, includ-
ing filtering, normalization, and taxonomic profiling, they generally do not contain
sophisticated algorithms for the identification of differentially abundant taxa and the
discovery of potential biomarkers and microbial co-occurrence patterns.
Next, we introduce several recent web-based tool suites for in-depth analyses of
marker gene–based microbiome data. MicrobiomeAnalyst (https://www.microbio-
meanalyst.ca) is a web-based tool suite for the comprehensive statistical, visual, and
meta-analysis of microbiome data (Dhariwal et al., 2017). The suite contains four
modules, the Marker Data Profiling (MDP) module, the Shotgun Data Profiling
module, the Taxon Set Enrichment Analysis module, and the Projection with Public
Data module. Both MDP and Projection with Public Data module are specially
designed for 16S rRNA maker gene survey data and will be discussed in detail.
The input file for MDP is count tables (OTUs or ASV) of various origin, including
both a tab delimited text (.txt) or in comma separated values (.csv) and BIOM files
from QIIME and mothur pipelines. MDP includes dozens of tools organized in five
categories—visual exploration, community profiling, clustering and correlation,
Tools and Resources for Microbe Analysis 31

comparison and classification, and functional prediction. The tool suite offers mul-
tiple choices for data scaling and normalization, including CLR transformation. For
visualization, the module provides standard methods for stacked bar or area plots,
interactive pie charts, rarefaction curves, and a phylogenetic tree view. Moreover,
the module provides a heat tree analysis based on the MetaCoder algorithm (Foster
et al., 2017). This unique feature uses the hierarchical structure of taxonomic clas-
sifications to quantitatively and statistically depict taxonomic differences between
microbial communities of interest. Under the community profiling, the suite enables
the generation of six alpha diversity indices—Abundance-based (ACE), Fisher’s
Alpha, Chao1, observed taxa, Shannon, and Simpson. For beta diversity evaluation,
the suite allows two popular ordination methods, PCoA and NMDS, based on five
distance matrixes, Bray–Curtis dissimilarity, Jensen–Shannon Divergence, Jaccard
Index, unweighted Unifrac measures, and weighted Unifrac measures. Moreover,
three algorithms, Permutational multivariate analysis of variance (PERMANOVA),
ANOSIM, and distance-based tests for homogeneity of multivariate dispersions
(Permutational analysis of multivariate dispersions (PERMDISP)), are provided for
global significance testing.
Under the comparative analysis category, multiple algorithms are provided for
users to identify taxa that are significantly different between groups of interest and
to discover potential biomarkers. In addition to classical univariate analysis (such
as t-tests and ANOVA) and nonparametric tests (such as the Mann–Whitney U test
or the Kruskal–Wallis rank sum test), MDP also offers metagenomeSeq, edegR,
DESeq2, LEfSe, and Random Forest.
The MDP module also provides two useful tools that enable functional prediction
from 16S rRNA count tables, PICRUSt for the Greengene database, and Tax4Fun for
the SILVA database. The result is a table containing relative abundance levels of Kyoto
Encyclopedia of Genes and Genomes Orthology. The Kyoto Encyclopedia of Genes
and Genomes Orthology profiles obtained from predictions can be used for functional
diversity profiling based on Kyoto Encyclopedia of Genes and Genomes pathway or
Clusters of Orthologous Groups annotation systems. Because one Kyoto Encyclopedia
of Genes and Genomes Orthology or Clusters of Orthologous Groups can be assigned
to multiple functional groups, MicrobiomeAnalyst offers different approaches to deal
with this issue, including simple sum, normalized sum, and weighted sum methods.
The results are presented as a stacked area plot organized by experimental factors to
help visualize patterns of variations across different conditions. The underlying abun-
dance table is available for downloading. A nice feature of the MicrobiomeAnalyst
suite is that R command line history is shown along with the data in the browser.
One of the unique features of the MicrobiomeAnalyst suite is that it contains a
module for meta-analysis. The Projection with Public Data module allows users to
visually explore their datasets within the context of a compatible public dataset for
context reference and pattern discovery using an interactive 3D PCoA plot. While
there is still room for improvement, the MicrobiomeAnalyst suite is a very user-
friendly online toolbox that enables comprehensive visualization and statistical test-
ing for both marker gene– and whole genome shotgun–based microbiome datasets.
Calypso is a recently developed web application with a powerful yet user-friendly
tool suite for the comprehensive analysis of bacterial, archaeal, viral, and eukaryotic
32 The Gut Microbiome: Bench to Table

communities (Zakrzewski et al., 2017). The package uses count data, metadata, and
an optional matrix of pair-wise community distances (such as Unifrac) as input files
to mine and visualize microbiome environment interactions. The suite emphasizes
multivariate analysis for hypothesis testing, which is one of its key strengths.
The package supports various formats of count data, either a .txt, .csv, or BIOM
file, with taxonomic assignments from phylum to OTU levels. Output files from sev-
eral popular pipelines, such as QIIME (Caporaso et al., 2010), mothur (Schloss et al.,
2009), MG-RAST (Meyer et al., 2008), and MetaPhlAn (Segata et al., 2012), can be
directly uploaded. The metadata file consists of a simple text file in either .txt or.csv
format and contains meta information for each sample, such as sample unique iden-
tifiers, sample groups, sample include/exclude options, and optional explanatory or
environmental variables, such as control vs. treated, disease status, and physiological
parameters. Both numeric and categorical variables are supported.
Calypso provides various tools for data filtering, rarefying, scaling, and transfor-
mation, such as asinh, cumulative sum scaling, log, quantile normalization, square
root, and total sum of squares (TSS). It also recommends the default choice of either
TSS combined with square root transformation or cumulative sum scaling + log2
transformation. Of note, Calypso also provides a CLR transformation, which is one
of the most widely used transformations designed for compositional data.
Calypso provides many graphical tools for data visualization, including bubble
plot, heatmap, interactive hierarchical tree, Krona plot, and standard bar charts, box
plots, and strip charts. Users can exclude samples, select colors, and change figure
resolution and dimensions. Output files include data tables in either .csv or .txt for-
mats and publication-quality images in PNG, PDF, or SVG formats.
Calypso allows users to calculate up to eight alpha diversity indices, such as ACE,
Chao1, Fisher’s Alpha, Evenness, Richness, Shannon, and Simpson. These alpha
diversity measures can be viewed using various bar charts, strip charts, box plots,
scatter plots, dot plots, and interactive boxplots. The significance can be tested using
ANOVA or nonparametric Wilcoxon and Kruskal–Wallis rank sum tests. Complex
associations between microbial diversity and multiple explanatory variables can be
identified by multiple linear regression. Calypso also supports diversity analysis using
mcpHill (Pallmann et al., 2012), which simultaneously investigates several diversity
measures by unifying them in the same mathematical family of indices. The advan-
tage of mcpHill is that it may provide biological insights that would remain hidden
when only examining one arbitrary or a priori committed measure of diversity.
Calypso emphasizes multivariate analysis and provides a broad range of ordi-
nation methods for beta diversity, including unsupervised, such as PCA, PCoA,
detrended correspondence analysis, and NMDS, and supervised methods, such as
redundancy analysis and canonical correspondence analysis, using several distance
matrixes. Moreover, the package offers several methods of global significance test-
ing. For example, associations between microbial community composition and a
single environmental variable can be identified by PERMDISP2 or by comparing
intragroup and intergroup community distances using ANOSIM. More complex
environment–microbiome associations can be identified and tested using redundancy
analysis, correspondence analysis, and PERMANOVA (or Adonis). These methods
Tools and Resources for Microbe Analysis 33

test if variance in community composition can be explained by variance in mul-

tiple explanatory or environmental variables. The package also has strong graphical
capability. For example, PCoA can be viewed in three ways, 2D, 3D, and interactive.
Furthermore, two different hierarchical clustering tools and a correlation heatmap
are provided.
Calypso offers a dozen tools for identifying taxa or features with significantly
different abundance between groups of interest. The relative abundance can be com-
pared using ANOVA (one-way, two-way, and nested) and t-tests (regular, paired, and
Bayesian) or nonparametric tests, such as the Wilcoxon rank test and the Kruskal–
Wallis rank sum test. Of particular interest is that the package offers two recent algo-
rithms specifically developed to handle compositional data, ALDEx2 (Fernandes
et al., 2014) and ANCOM (Mandal et al., 2015). No other web-based tool suites offer
these tools designed for compositionality. Calypso also provides mixed effect regres-
sion and machine learning tools, such as Random Forest. All P values are further
adjusted for multiple testing corrections, including, Bonferroni and FDR corrections.
Calypso offers four methods for feature or biomarker discovery, LEfSe, stepwise
regression, LASSO regularized regression, and Random Forest, more than other
similar web-based tools. Features or taxa selected by stepwise or LASSO regular-
ized regression are presented as bar charts that depict the importance of each taxa.
Random Forest results are also presented as bar charts, indicating the relative impor-
tance of taxa, as estimated by random permutation. Under the advanced section,
Calypso offers mixMC, a multivariate framework that takes into account the sparsity
and compositionality of microbiome count data (Lê Cao et al., 2016). The framework
enables a holistic understanding of microbial communities through insightful graphi-
cal output and highlighting features or taxa with discriminative differences between
environmental variables. When emphasizing repeated-measures design with a multi-
level variance decomposition, mixMC can be used in a more general case to compare
phenotypes or disease outcomes.
Calypso also includes several tools for the identification of co-occurrence patterns
using network analysis. Five algorithms are provided in Calypso for network infer-
ence, including those based on Pearson’s correlation or Spearman’s rho and weighted
gene correlation network analysis (Langfelder and Horvath, 2008). Moreover, the
LASSO regression-based approach involves the abundance of each taxon regress-
ing on all remaining taxa iteratively until the most relevant associations are identi-
fied. However, an ensemble-based approach is based on multiple similarity measures
combining Bray–Curtis dissimilarities with Pearson correlation and Spearman’s rho.
For each similarity/dissimilarity measure, the significance of the correlation is com-
puted. P values for Bray–Curtis dissimilarities are computed with permutation. P
values are further corrected using multiple testing by the FDR. Table 1.3 summarizes
some of the major features offered in three popular web-based tool suites, the two
discussed above and METAGENassist (Arndt et al., 2012).
Overall, Calypso is an easy-to-use web-based toolbox packed with many sophis-
ticated statistical algorithms for microbiome studies. It allows a scientist without
any programming skills to produce a rapid, robust, and comprehensive analysis of
compositional data.
34 The Gut Microbiome: Bench to Table

TABLE 1.3
Summarization of Feature Categories of Three Major Web-Based Tools for
Marker Gene Studies.
Category METAGEN-Assist MicrobiomeAnalyst Calypso
Web-based Yes Yes Yes
Implementation
Version v8.84
URL http://www. https://www. http://cgenome.net/calypso/
metagenassist.ca microbiomeanalyst.ca/
Data input OTU table, OTU/ASV table, OTU/ASV table, BIOM file
BIOM file BIOM file
Data processing Scaling, Rarefying, scaling, Rarefying, scaling,
transformation transformation transformation
Quantitative Bar chart, pie Bar chart, pie chart, Bar chart, bubble plot, heatmap,
visualization chart phylogenetic tree, heat scatterplot, strichart
tree
Clustering Dendrogram, Dendrogram, heatmaps Dendrogram, heatmaps, SOM
heatmaps, SOM
Hierarchical tree No Yes Yes
Alpha diversity No 6 indices 8 indices
Beta diversity
Distance matrix Yes Yes Yes
Ordination/ PCA, PSL-DA PCoA, NMDS PCA, PCoA, PLS, RDA, CCA,
multivariate DCA, NMDS
analysis
Global significance No ANOSIM, ANOSIM, PERMANOVA,
testing PERMANOVA, PERMDISP
PERMDISP
Univaraite analysis T-test, ANOVA, T-test, ANOVA, T-test, ANOVA, Wilcoxon,
Wilcoxon, Wilcoxon, Kruskal– Kruskal–Wallis, DESeq2,
Kruskal–Wallis Wallis, edgeR, DESeq2, ANCOM, ALDEx2
MicrobiomeSeq
Feature/biomarker Random Forest, LEfSe, Random Forest LEfSe, Random Forest, LASSO,
discovery SVM stepwise regression, SVM
Functional No PICRUSt and Tax4Fun PICRUSt
prediction
Correlation analysis Pearson, Pearson, Spearman, Pearson, Spearman, Kendall,
Spearman, Kendall, SparCC WGCNA, network analysis,
Kendall factor analysis
Multiple testing No Yes Yes
correction
Regression No Yes Yes

RDA: redundancy analysis; DCA: detrended correspondence analysis; CCA: canonical correspondence
analysis; WGCNA: Weighted correlation network analysis; PLS: Partial least squares; SVM: Support
Vector Machines.
Tools and Resources for Microbe Analysis 35

CONCLUSIONS
The advent of high-throughput sequencing technologies and the rapid development of
user-friendly bioinformatic algorithms have enabled a renaissance of marker gene–
based studies. Freely accessible reference databases have rapidly expanded. Several
user-friendly pipelines, such as QIIME, mothur, and FROGS, have been developed.
Numerous algorithms and sophisticated statistical testing, including machine learn-
ing, have been proposed to detect differentially abundant taxa, identify biomarkers
and taxa correlated with environmental variables, and infer microbial interaction
networks. Easy-to-use web-based tool suites for data analysis have also become
available. However, microbial network inference is still in its infancy, and biological
interpretation and validation of inferred microbial interactions are still extremely dif-
ficult. There are no public databases available for microbial interactions. While many
challenges remain, we strongly believe that knowledge of microbial association and
co-occurrence patterns will establish a foundation to predictively model the interplay
between environmental factors and microbial populations (Kurtz et al., 2015). This
knowledge will ultimately facilitate the development of microbial community engi-
neering by targeted elimination and/or expansion of keystone species in a microbial
community and hold keys to efficacious synbiotic and antibiotic therapeutics.

ACKNOWLEDGMENTS
Mention of trade names or commercial products in this publication is solely for
the purpose of providing specific information and does not imply recommenda-
tion or endorsement by the US Department of Agriculture. The US Department of
Agriculture is an equal opportunity provider and employer.

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 2 Phageome in
Gut Microbiome
Yujie Zhang, Yen-Te Liao, Logan Tom,
Somanshu Sharma, and Vivian C.H. Wu
United States Department of Agriculture

CONTENTS
The Origins and Roles of Phages, Phageome, and Its Composition in
Human Gut Microbiota ............................................................................................ 45
How Do Phages Shape the Human Gut Microbiota ................................................ 49
Phage Interaction with the Immune System ............................................................ 52
Phageome and Health and Disease .......................................................................... 53
Phage-Based Therapy for Human Gut-Related Disease Is a Promising
Approach to Control/Prevent Human Disease ......................................................... 56
The Challenges of Phageome in Human Gut Microbiome, Perspectives,
and Future Directions............................................................................................... 59
References ................................................................................................................ 62

THE ORIGINS AND ROLES OF PHAGES, PHAGEOME, AND

ITS COMPOSITION IN HUMAN GUT MICROBIOTA
Bacteriophages (phages) are viruses that infect bacteria and are the most abundant
biological entities in the biosphere, with estimated 1031 virions (Mushegian, 2020).
They are found wherever bacteria live, including soil, water, and the human body,
and for instance, approximately 140,000 phage sequences were found to reside in the
human intestines (Camarillo-Guerrero et al., 2021; Dalmasso et al., 2014; Roux et al.,
2016; Trubl et al., 2018). Within phage populations, there is an amazing diversity of
protein structure, genome, and host ranges, and researchers continue to reform the
understanding of phages (Hsu et al., 2019). Bacteriophages were first discovered by
British bacteriologist Frederick Twort in 1915 when he observed plaques of dead
bacteria on plates of bacterial lawns. In 1917, Felix d’Herelle reported an increase
of phages in the stools of recovering Shigella dysentery patients, paving the way
for phage therapy (Letarov, 2020). Although overshadowed by the discovery and
widespread use of penicillin antibiotics in 1942, phages have been used extensively
in Russia and Georgia to combat bacterial infections in humans. In addtion, bacterio-
phages have been useful instruments in numerous biological discoveries, such as the
mechanisms of translation and DNA replication (Keen, 2015).

DOI: 10.1201/b22970-3 45
46 The Gut Microbiome: Bench to Table

Phages infect bacteria through a lytic or lysogenic life cycle (Howard-Varona

et al., 2017; Hsu et al., 2019). Lytic phages (or virulent phages) inject their viral
genome into a host bacterium and use host’s cellular machinery to replicate their
genome and produce viral proteins that can form progeny. Eventually, the host cell
is lysed open to release the viral progeny, which goes on to infect more bacteria
(Manrique et al., 2017). Lysogenic phages (or temperate phages) insert their genome
into a host cell and integrate their DNA into the bacterial host’s genome as a prophage
or present as a free plasmid called phagemid; thereby, in the lysogenic life cycle, the
phage replicates whenever the cell does, without producing progeny (Howard-Varona
et al., 2017). After integration of the phage genome, the lytic cycle can be activated
via prophage induction from a number of triggers, such as antibiotic stress that is
commonly related to the gut environments for disease treatment (Allen et al., 2011;
Grif et al., 1998; Sutcliffe et al., 2021; Zhang et al., 2020).
In certain microbiota, phages do not simply live with and infect bacteria, but
they play dynamic roles to shape their surroundings. The community of phages
(also known as the phageome) in the gut has positive and negative impacts on
gastrointestinal fitness and impacts the bacterial community. Specifically, in the
human gut, phages in the gastrointestinal (GI) tract affect the microbiota com-
position by lysing bacteria depending on the density of bacterial species; this
drives microbial diversity and evolution and stabilizes the microbial population.
Phages can benefit from any viable host bacteria in the gut, where a slight increase
in bacteria can quickly lead to a large bloom in phages (Manrique et al., 2017).
Some lysogenic phages can transport important genes, such as those involved
in anaerobic respiration and macromolecule biosynthesis, between different host
cells through transduction, which contributes to the gut microbiota’s ecological
role (Kieft et al., 2021; Sutcliffe et al., 2021). Furthermore, phages can affect the
gut microbiota by removing gut bacteria. A study conducted by Sutcliffe et al.
reported that common oral medication, such as nonsteroidal anti-inflammatory
drugs (NSAID), inhibited the growth of bacteria in the human gut microbiota by
inducing prophages in a species-specific manner, increasing the phage population,
and changing the composition of the gut microbiota. However, the mechanisms
of functions of NSAIDS are yet to be studied with the gut microbiota (Sutcliffe
et al., 2021). The dynamics of phages in the gastrointestinal (GI) tract were further
observed by Zuo et al. when researchers collected fecal samples from COVID-
19 patients of varying disease severity and used shotgun metagenomics to char-
acterize the gut RNA and DNA viromes (Zuo et al., 2021). COVID-19 patients
were shown to have a significant decrease in multiple viruses, including phages,
compared to non-COVID-19 patients. Viruses found in COVID-19 patients con-
tained more inflammation- and virulence-associated genes, which affected the
metabolism and virulence of bacterial hosts and may contribute to the gastroin-
testinal symptoms seen in COVID-19 patients. These viruses, including phages,
were also inversely correlated with proinflammatory proteins and leukocytes in
the blood, which means that phages affect host body immunity. The imbalance of
the gut virome in COVID-19 patients again highlights that the proper healthy gut
phageome (HGP) is a balanced steady state in the body.
Phageome in Gut Microbiome 47

The diversity and abundance of the gut microbiota are not simply present at
birth, but in humans, it develops in a newborn’s first 2–3 years of life and stabi-
lizes over adult years. The microbiota colonizes within the first 1–4 days of life,
where the diversity of the phage community is initially high, but a further steady
decrease in overall viral diversity in the first 2.5 years of life leads to a rise in
microbial populations and diversity. This eventually revives the viral population
through prophage induction, and these induced prophages in the human microbiota
become a crucial source of phages in the gut (Manrique et al., 2017). Adult phage
communities are relatively stable, and most phages at this time contain dsDNA
or ssDNA. A larger Microviridae to Caudovirales ratio has been correlated with
healthier adults, and most are temperate phages (Townsend et al., 2021). At the
point of development, the phage virome is also highly varied and sensitive to the
individual host gut microbiota, and for each individual, multiple different phages
are intertwined and play vital roles in the gut, which cannot be replicated in other
individual adults because of diet, environment, and maternal lineages (Hallowell
et al., 2021). Although the gut phage composition is mostly unique to the indi-
vidual, there does exist a reservoir of common phages, notably one example being
crAssphage, which was isolated in 73% of 450 global fecal metagenomes. Out of
4,301 phages isolated from two study individuals, 23 “core phages” were found in
more than 50% of healthy individuals around the world, questioning the composi-
tion of these core phages in the HGP and the role they play in health; it even has
been proposed that some of the core phages might play more impactful roles in
human health (Norman et al., 2015). In addition, it is still debated whether common
phages occur among people in the same environment, such as household members,
or among unrelated individuals, and evidence exists to support each view. The
details of the HGP are still poorly understood, but partially through predator–prey
relationships and lytic–lysogenic balances, an active phageome is key to a healthy
and functional gut ecosystem (Manrique et al., 2016).
Ogilvie et al. studied a human gut-specific phage named φB124-14, and phage
φB124-14’s genome was compared with 611 other human gut phages and 48 chro-
mosomal sequences from its host Bacteroides fragilis, which is a normal gut
microbe that is essential to healthy GI function (Ogilvie et al., 2012). Although
its role in the gut was not precisely defined, researchers found both phages and
bacteria in the gut are closely related in their genomes, possibly due to horizon-
tal gene transfer. Therefore, phages and microbes in the gut likely share a very
similar ecological niche. In addition, phage φB124-14 still plays a specialized role
through its thymidylate synthase gene, which is rare in gut viral genomes and
possibly helps with the fitness and function of B. fragilis through lysogeny. Since
B. fragilis is important in human mucosal immunity and nutrition, the findings
indicated that phage φB124-1 facilitates this healthy role of gut microbes in the
body by transferring essential genes between host cells. The finding by Stern et al.
supported the concept that specific phages or taxa of phages in a common phage
reservoir in the human population are correlated with human health. Researchers
identified 991 phages from the gut microbiota of 124 individuals using sequences
found in bacterial CRISPR genes, and 78% of these phages were present in two or
48 The Gut Microbiome: Bench to Table

more individuals (Stern et al., 2012). In his study, Stern et al. also observed varying
ratios of phages and bacteria, some individuals showing dominance in phages or
host bacteria, and although temperate phages dominate the phage reservoirs, there
are even different ratios of lytic and lysogenic phages, suggesting that the HGP
changes over a lifetime and varies among individuals. These findings provide a
glimpse into the complex ecology of gut microbiota, and more research is required
to fully understand the effect of phages.
The GI tract is a heterogeneous ecosystem that not only contains diverse organ-
isms but also distinguished sub-environments. Different locations in the gut respond
differently to food macromolecules, microorganisms, and pathogens, and each
organ in the tract is structured differently to fit its role in the digestive system. As a
result, distinct microbial communities can grow in different locations in the GI tract
(Bushman and Liang, 2021). Because most of the phage genome is still unknown and
the gut microbiota is naturally individualized, it is difficult to sample and characterize
phages in detail across the GI tract. Therefore, not many studies on the locations of
phages in the human gut exist. However, through animal models, several studies have
provided an overall profile of phages in the gut to shed more light and provide trends
in mammalian gut microbiota. Looft et al. compared the intestinal metagenomes of
pigs and found that the ileum displays a significant abundance of phages and phage-
related genes, and these numbers remained stable over the month-long study, sug-
gesting that phages are concentrated and consistent in the ileum (Looft et al., 2014).
Ileal bacteria, mostly consisting of facultative anaerobes like Streptococcaceae, and
nutrients that are absorbed in the ileum, such as vitamin B12, can make the ileum a
more comfortable location for phages to settle, compared to the colon (Hsu et al.,
2021). Because of physical barriers in the GI tract, such as the ileocecal valve, the
phage profile among organs can be different; despite sharing common phages, the
virome of the small intestine and the virome of the large intestine and rectum are
ultimately unique, which lends caution to the bias of fecal samples to study the gut
virome (Guerin and Hill, 2020).
Phages are also present at high numbers in the mucosa, which lines the epi-
thelial layer of the GI tract, and this limits pathogen colonization in the body.
Bao et al. added 8 log PFU/mL of phage PA13076 (isolated from chicken feces)
or BP96115 (induced from Salmonella Pullorum Spu-109) into mouse models for
31 days to track phages throughout the GI tract; they observed that phages gradu-
ally increase toward the end of the GI tract in mice, with the highest phage titer
(close to 6.5 log PFU/g) present in feces (Bao et al., 2020). In their study, lytic
phages are dormant from the stomach to the jejunum and begin to increase in the
ileum and colon reaching their peak in feces, while lysogenic phages maintain
relatively higher titers from the stomach to the colon but are highest in feces and
the cecum. Generally, phages do not settle in the location of the GI tract that is the
active site of digestion, such as the stomach and duodenum, because of the hydro-
chloric acid and proteolytic enzymes present, respectively. Overall, the specific
profile of the phage gut community in various GI organs still requires research,
but studies already show that phages inhabit distinct areas in the gut based on their
structures and function in the body.
Phageome in Gut Microbiome 49

HOW DO PHAGES SHAPE THE HUMAN GUT MICROBIOTA

Phages play an active and important role in the human gut microbiota and the over-
all human body; and the longitudinal spread of phages in the GI tract points to
phages’ dominance in the body. Their composition and population across virtually
every organ and layer of the GI tract, especially epithelial tissue, suggest an impor-
tant role in humans (Townsend et al., 2021). Scanlan et al. observed lytic phages
playing an ecological role in the gut of phage cocktail-treated mice (Scanlan, 2020).
After analyzing the populations of host bacteria and phages, the gut bacteria exhib-
ited a source–sink ecological dynamic, where bacteria would move between differ-
ent areas of the gut, based on overpopulation, nutrient availability, and population
support. Here, because of phages’ density-dependent lysis, phages may contribute to
the habitat quality, thus fluctuating where gut microbes reside and how many there
are. Other observed dynamics with gut microbiota add to this role (Figure 2.1). For
example, phages and bacteria can illustrate the Red Queen dynamics, named from
Lewis Carroll’s Through the Looking-Glass when the Red Queen describes the
Looking-Glass Land to Alice and says, “Now, here, you see, it takes all the running
you can do, to keep in the same place.” In the Red Queen dynamics, there is a con-
tinual coevolution between bacterial host and phage to defend and counter-defend,
each species running to keep up with the other. Phages can also display the kill-
the-winner dynamics, which include phages lysing common, susceptible cells and
preventing bacterial dominance, or exhibit piggyback-the-winner dynamics, where

FIGURE 2.1 Phage–bacteria dynamics in the gut.

50 The Gut Microbiome: Bench to Table

lysogenic phages allow for coexistence by integrating with their host (Townsend
et al., 2021). The piggyback-the-winner dynamic is predominant in the absence of
pathogens and plays a crucial role in the genetic exchange. Prophages have shown up
to 5% of the functions in the human gut microbiota by sharing metabolism genes that
contribute to nutrient cycling and community stability, thus regulating cell growth
and competition (Oh et al., 2019b). Shkoporov and Hill reported the large titer of
lysogenic phages in human guts compared with lytic phages. Even in a high con-
centration of hosts, phages will choose to replicate with bacterial hosts (Shkoporov
and Hill, 2019). The gut microbiota abundantly adheres to the top mucin layer in
the GI tract where more temperate phages are found and piggyback-the-winner is
favored. Despite the risk of lysis, the gut microbiota benefits from phages in the GI
tract through lysogeny and regulation of colonization, and phages have formed an
important ecological niche with GI microbes.
From the discovery of core phages, such as crAssphage, different phages have
been correlated with healthy and ill individuals, posing the existence of the HGP,
a gut virome significantly correlated to human health. The disruption of this core
virome from equilibrium is either the cause or result of disease, so more investiga-
tion is required. Studying phages in relation to human health and gut microbiota
requires an overarching view of the GI ecosystem, which is still not well understood
(Garmaeva et al., 2019). One angle to this problem is studying the immune system.
To the human body, phages are foreign nonself entities that cross the gut epithelium
via transcytosis or phagocytosis and come into contact with innate immune cells,
such as macrophages and dendritic cells, eventually inducing an antiphage antibody
response in lymph nodes (Carroll-Portillo and Lin, 2019). The questions arise when
these interactions induce inflammation and immune signaling seen in responses
triggered by animal viruses; whether the interactions are beneficial or harmful
to the gut microbiota, it is yet clear that the ecological role of phages is tied to
human health. A phage-induced immune response can fight off a bacterial infection,
subsequently disrupting gut microbes. Phages in the mucosal layers modulate the
immune system likely through activating anti-inflammatory cytokines in the lam-
ina propria and suppressing inflammation in the gut, thus stabilizing the ­intestinal
environment and inhibiting nonspecific immune responses present in autoimmu-
nity, hypersensitivity, and infection in the GI tract (Carroll-Portillo and Lin, 2019;
Łusiak-Szelachowska et al., 2017).
Changes in the gut phageome have also been associated with diet, and it is well
established that diet has a large influence on gut microbiota. Food macromol-
ecules, such as proteins and digestible and nondigestible carbohydrates, induce
shifts in the gut microbiota, and subsequently affect overall diversity of gut micro-
biota (Singh et al., 2017). A study conducted by Townsend et al. had shown that
diet also impacts the structure and composition of the gut phageome (Townsend
et al., 2021). differences in diet linked with gut phageome differences have been
observed over the development of an individual human. Breast milk is high in fats,
and studies with mouse models with high-fat diets show increased Microviridae to
Siphoviridae ratios in the gut (Schulfer et al., 2020). As babies wean and eat solid
foods, phage composition continues to alter, and an increase in Pectobacterium
phages and a decrease in Lactobacillus phages and Streptococcus phages are seen
Phageome in Gut Microbiome 51

in adulthood. In addition, certain foods are richer sources of phages; fresh meats
and fermented foods, such as cheese, soybeans, and sauerkraut, are rich source
of phages, so diets concerning these foods may contribute to a wider and larger
gut phageome (Chukeatirote et al., 2018). Malnourishment, especially in infancy,
significantly disrupts a healthy phageome, which suggests that infancy is a crucial
period for phageome development. Malnourished youth contain a phageome that
is correlated with individuals with growth stunts and various gut illnesses (Reyes
et al., 2015).
Once the phageome stabilizes in adulthood, the individual diet has less of an effect
on gut phages; however, the diets from geographical communities and ethnic cultures
begin to play a larger role. Zuo et al. studied the gut phage DNA virome in 930
healthy individuals in Hong Kong and Yunnan, China, who spanned various ethnici-
ties and residencies, and geography played the most significant role in shaping both
individual’s gut virome and bacteriome (Zuo et al., 2020). It is not clear whether the
virome affects the bacteriome or vice versa, and geography is impacted by many fac-
tors, such as available food sources and region-specific food preparations. However,
certain diets in these regions that have been ethnically established, such as barley,
buttermilk tea, and Pu’er tea, contributed to virome differences. Urban and rural
residencies also affect gut virome; variations in personal hygiene and cleanliness in
the kitchen were associated with varying levels of viral diversity. Even though the
gut phage community and its regulation are still not fully understood, these studies
show the significant effect of environmental factors, diet in particular, on the human
gut phageome and bacteriome. Specifically, several foods have been shown to have a
direct effect on phages in the human gut, and by extension, gut bacteria as well. Oh
et al. reported that dietary fructose and short-chain fatty acids (SCFAs) promote the
induction of prophages in gut symbiont Lactobacillus reuteri in mouse models in a
RecA-dependent manner (Oh et al., 2019a). Researchers observed L. reuteri reducing
fructose from the diet to mannitol, and other gut microbes were able to convert man-
nitol to SCFA, which leads to prophage induction. This implies that diet affects gut
microbes, which alter the phageome; the phageome then alters gut bacteria by lysis
of lysogens. In the colon specifically, nutrient starvation was shown to drive phage
production. This likely has to be a consequence of changes in the gut microbiota
density and competition, possibly linked with the activation of the SOS response.
Garmaeva et al. studied phages in the guts of 11 healthy adults on a gluten-free diet
(GFD) and found a decrease of crAss-like phages, Microviridae, and Podoviridae
phages in GFD individuals (Garmaeva et al., 2021). Additionally, the comparison
of Bray–Curtis distances between the individuals with GFD and gluten-containing
diets revealed the change of the gut virome. Whether the addition of gluten can
reverse these changes was not studied, but researchers were able to see how changes
in the diet result in changes in the phageome. Cronin et al. researched the effects of
physical exercise and regular whey protein supplements on the human gut micro-
biota (Cronin et al., 2018). Out of 90 study individuals, most experiment participants
receiving whey protein experienced higher taxonomic richness and β-diversity in
their gut virome than control participants. Overall, diet significantly influences the
phages and bacteria in the human gut microbiota, although researchers are still try-
ing to understand mechanistic details.
52 The Gut Microbiome: Bench to Table

PHAGE INTERACTION WITH THE IMMUNE SYSTEM

Phages influence bacterial cells in the gut microbiota, subsequently affecting the
immune system. Gut microbes expose the body to a chronically low level of patho-
gen-associated molecular patterns (PAMPs), which stimulates the body with IgA
and CD4/CD8 T cells for future infections. Also, some gut bacteria, such as various
Clostridia species, induce regulatory T cells and IL-10 production, which suppress
immune responses and prevent inappropriate inflammation (Sinha and Maurice,
2019). However, phages can also negatively affect health by causing autoimmune
diseases or hypersensitivity when phages cause an inappropriate immune response.
Activated prophages from the gut microbiota can play a part in this when phages are
released from bacterial cells and access immune cells to elicit a response (Carroll-
Portillo and Lin, 2019). The lysing of bacterial host cells by phages would upset
immune signals from gut microbes in the gut and disrupt the balance of immunity.
In addition, the lysing of bacterial cells releases many PAMPs, including DNA and
endotoxins, that can activate the immune system, which can inflame and damage the
GI tract. Through transduction, phages could hypothetically carry virulence factors
from pathogens to any bacterial cell in the gut microbiota. However, because phages
usually have specific host ranges, this hypothesis still desires research to explain the
expansion of virulence (Carroll-Portillo and Lin, 2019; Sinha and Maurice, 2019).
Phages have been associated with various illnesses, which further describes
phages’ dynamic role in human health. Seth et al. studied the gut virome in Gulf
War illness (GWI) patients, who experienced bowel inflammation, headaches, neu-
roinflammation, and other unexplained symptoms (Seth et al., 2019). Increases in
gut viral populations and diversity, specifically in dsDNA phages, are observed in
GWI patients and animal models. The changes of virome have been positively cor-
related with gut bacterial dysbiosis and the loosening of GI epithelial tight junctions,
such as the activation of Toll-like receptor (TLR)- 6 and TLR-9 pathways, increasing
permeability inside the body. Increases in phages have also been associated with
increases in proinflammatory cytokines, such as IL-6. Whether or not changes in the
gut phageome or the bacteriome-caused symptoms in GWI could not be concluded,
and there may be independent or cumulative causes at play. However, in mouse GWI
models, the broad-spectrum antiviral ribavirin and the induction of gut sterility by
antibiotics both improved symptoms, and therapeutics for GWI can possibly target
the gut virome or bacteriome. On the other hand, phage therapy can change gut
microbiota to treat diseases. Febvre et al. studied the use of phages against general
GI inflammatory symptoms, and 106 PFU per dose of a commercial four-phage cock-
tail, PreforPro, that targets E. coli was orally administered to patients over 28 days
(Febvre et al., 2019). Most patients experienced symptom alleviation after the end of
the trial, and the phage cocktail additionally did not disturb the overall diversity and
richness of the gut microbiota. E. coil was generally reduced in the gut. Reductions
in Clostridium perfringens and increases in the genus Eubacterium were observed;
because Eubacterium spp. produced butyrate, this could lead to a decrease in inflam-
mation and clinical improvement in the patients. The detailed interaction between
gut phageome and human disease was further discussed in a later section.
Phageome in Gut Microbiome 53

PHAGEOME AND HEALTH AND DISEASE

The gut microbiota can be characterized as the community of microorganisms that
live in the gastrointestinal tracts. This community plays a vital role in the regulation
and maintenance of human health and is established prenatally with the transmission
of microorganisms from mother to fetus. Several properties of these gut-residing
microbes play an imperative role in the homeostasis of the body, and deviations or
disruptions in the ecology of the gut microbiota have been implicated in the emer-
gence of several diseases and metabolic disorders.
Gut microbiota diversity and interactions with hosts have been linked to energy
homeostasis and the emergence of conditions such as obesity (Bäckhed et al.,
2004; Ley et al., 2005; Ridaura et al., 2013). A study conducted by Bäckhed et al.
discovered a connection between the gut microbiome and fat storage with subse-
quent adipose tissue accumulation in humans (Bäckhed et al., 2004). The results
showed that microbiota in the gut could lead to the suppression of fasting-induced
adipocyte factor (Fiaf) protein, thus promoting fat storage, which, in excess, can
lead to obesity. While obesity is certainly a major health outcome following the
disruption of gut microbiota ecology, dysbiosis (harmful changes in gut flora) can
also lead to other significant health disorders (Cheng et al., 2020; Zhu et al., 2018).
One of these diseases is inflammatory bowel disease (IBD), which occurs when
there are regions of excessive inflammation along the gastrointestinal tract. Frank
et al. compared the GI tract tissue samples from patients with two IBDs: Crohn’s
disease (CD) and ulcerative colitis (UC), as well as non-IBD controls to deter-
mine changes in gut microbiomes; they found that IBD was correlated with lower
diversity of gut microbiota and lower levels of microbiota from the Firmicutes and
Bacteroidetes phyla (Frank et al., 2007). In addition, recent studies showcase that
the microbiome can actually affect the cardiovascular system and even influence
mental processes via the microbiota–gut–brain (MGB) axis. There are limited case
studies showing this link in humans, but several animal studies have corroborated
and emphasized the connection between gut microbiota and non-gastrointestinal
disease (Fröhlich et al., 2016; Gan et al., 2014; Gaykema et al., 2004; Kelly et al.,
2016). A study by Lam et al. investigated the effect of antibiotic-induced dysbio-
sis treatments on cardiovascular health in rats and showed that both treatments
led to a reduction in myocardial infarction rate by 27% and 29%, respectively
(Lam et al., 2016). The antibiotics help eliminate harmful bacteria in the gut that
could potentially increase the severity of myocardial infarction, thus showcasing
that reducing certain species of bacteria can help reduce the risk of myocardial
infarction. Work by Sudo et al. suggests a link between the gut microbiome and
neurodevelopmental pathways (Sudo et al., 2004). These researchers tested neural
development and stress response in germ-free (GF) mice, specific pathogen-free
(SPF) mice, and gnotobiotic mice. The results showcased that the GF mice had an
exaggerated hypothalamic–pituitary–adrenal stress response phenotype, which
could be rescued when these mice were transplanted with fecal microbiota from
SPF mice. Collectively, these studies demonstrated that the gut microbiome plays
a vital role in the proper functions and development of the human body.
54 The Gut Microbiome: Bench to Table

As previously described, phageome is a crucial part of shaping the gut micro-

biota and it plays a major role in the maintenance and defense of the healthy gut
microbiome. The effect of phages on the health of the gut microbiome was studied
by Duerkop et al., who performed experiments on mammalian intestinal bacterial
Enterococcus faecalis (Duerkop et al., 2012). The research found that an isolated
E. faecalis strain V583 revealed that this bacterial strain created a composite phage
ϕV1/7, which is derived from two prophages: ϕV1 and ϕV7. Moreover, a link between
bacteriophages and the health of the gut microbiome was observed since the infection
with ϕV1/7 likely rendered V583 bacterial strains a competitive advantage over other
strains in both in vitro and in vivo models. Thus, the dependence of the gut micro-
biota on production and infection by bacteriophages is evident. These viruses that
reside in the gut microbiota are also highly diverse and have many unique properties
and life cycles. Reyes et al. explored the diversity of viruses in the gut microbiome
by analyzing fecal samples during 1 year at three different time points from female
monozygotic twins and their mothers (Reyes et al., 2010). The results demonstrated
that the co-twins and mothers exhibited a significantly higher degree of virome simi-
larity compared to unrelated individuals. Additionally, Reyes et al. discovered that
even though there were large differences in intestinal viral content between different
people, the viral content within an individual often was stable over time; greater than
95% of the virotypes in individuals were retained over the one-year period of the
study. This is in stark contrast to the parasitic and competitive nature of bacteria–
phage dynamics observed in other ecological systems. Thus, the implication of these
results reveals that healthy gut microbiomes maintain a stable and diverse viral popu-
lation, which can benefit the bacterial species residing in the gastrointestinal tract.
Phages have been associated with various illnesses (Table 2.1). For example, a
study by Zuo et al. compared the virome data from rectal mucosa samples of both
patients with ulcerative colitis and healthy controls (Zuo et al., 2019). The results
showed that compared to healthy controls, in patients with ulcerative colitis, there
was an expansion of mucosa phages as well as a decrease in diversity and richness
of phages from the Caudovirales, whereas there was also an increase in Escherichia
phage and Enterobacteria phage populations in patients with ulcerative colitis family.
Notably, the ulcerative colitis patients also demonstrated a markedly increased num-
ber of diverse viral function defects and yet higher expression of phage genes related
to bacterial pathogenicity and bacterial host fitness. The phageome is not only related
to ulcerative colitis but can also play a role in other human diseases, such as diabetes.
A study by Tetz et al. revealed that there was an association between decreasing levels
of amyloid-producing Escherichia coli abundance in the gut and the emergence of
type I diabetes (Tetz et al., 2019). Furthermore, the researchers noted an increase in
the E. coli phage/E. coli ratio prior to the decreasing levels of E. coli; this phenom-
enon revealed that prophage induction was responsible for the depletion of the gut’s
amyloid-producing E. coli populations and thus the emergence of type 1 diabetes in
the patients. The enteric phageome also plays a role in diseases related to the immune
system. Particularly, an acquired immunodeficiency syndrome (AIDS), caused by the
human immunodeficiency virus (HIV), is linked to the gut virome in many ways.
Monaco et al. investigated both HIV-infected and HIV-uninfected Ugandan patients
to determine a link between certain viruses of the gut and AIDS and found that there
Phageome in Gut Microbiome 55

TABLE 2.1
Relationship between Gut Phageome and Human Diseases
Diseases Changes in the Gut Phageome Example References
Ulcerative colitis A decrease in diversity and richness of Caudovirales Zuo et al. (2019)
phages; an increase in abundance of Caudovirales
phages and Enterobacteria phages in particular
Each disease type and cohort have unique Norman et al. (2015)
bacteriophages; decreased bacterial diversity and
richness was inversely correlated to gut phageome
changes
Crohn’s disease A significant expansion of Caudovirales Norman et al. (2015)
bacteriophages
Temperate bacteriophages dominate the gut virome Clooney et al. (2019)
Decreased prevalence of core bacteriophages (phages Manrique et al. (2016)
preset in more than 20% of healthy individuals)
Largest abundance of phages in CD ileum tissue and Wagner et al. (2013)
CD gut wash sample
Type 1 diabetes Increase in the E. coli phage/E. coli ratio due to Tetz et al. 2019
prophage induction
Evenness of two major phage groups, Myoviridae and Townsend et al. (2021)
Podoviridae, was lower; diversity of intestinal E. coli
phages is significantly higher
Type 2 diabetes The relative numbers of the Myoviridae, Podoviridae, Ma et al. (2018)
Siphoviridae, and unclassified Caudovirales families
increased significantly
Abundance of phages specific to Enterobacteriaceae Chen et al. (2021)
hosts increased significantly
Acquired Increase in adenoviruses and viruses from Monaco et al. (2016)
immunodeficiency the Anelloviridae
syndrome (AIDS)
Stunting Lower phage diversity; a decrease in temperate phages Mirzaei et al. (2020)

was an increase in adenoviruses and viruses from the Anelloviridae that were associ-
ated with the HIV infection (Monaco et al., 2016). These studies thus solidify the link
between the gut phageome and the emergence of a variety of diseases.
Importantly, while there is limited information on the gut phageome, several stud-
ies indicated that most viral populations could not be assigned taxonomy and were
kept unknown as the viral “dark matter”. Finkbeiner et al. studied diarrhea samples
from 12 children and employed a novel “micro-mass sequencing” technique to detect
the presence of both known and unknown viruses in the samples (Finkbeiner et al.,
2008). The results showed that there were several known viruses in these samples,
such as rotaviruses, caliciviruses, astroviruses, and adenoviruses. Additionally, the
samples revealed the presence of several unknown viruses, which held very little
sequence similarity to viromes in the GenBank database. What this study shows
is that viruses are highly interconnected to gastrointestinal health, and understand-
ing and characterizing viral genomes can help lead to a broader understanding of
56 The Gut Microbiome: Bench to Table

both causes and treatments of prevalent diseases. Recently, several studies investi-
gated different techniques to explore this viral dark matter (Fitzgerald et al., 2021).
For instance, Benler et al. discussed their search for unknown human gut phages
using phage hallmark genes. Their studies led them to discover 3,738 complete phage
genomes from 451 different genera, thus illustrating how vast and unknown the
human gut phageome truly is (Benler et al., 2021). In the future, further studies and
techniques are needed to help explore this unknown gut phageome.

PHAGE-BASED THERAPY FOR HUMAN GUT-RELATED DISEASE IS A

PROMISING APPROACH TO CONTROL/PREVENT HUMAN DISEASE
It is clear that a diverse set of phages play an important role in human health and dis-
ease. In addition, infection with foreign viruses has been implicated in causing a vari-
ety of gastrointestinal diseases. The application of phage benchwork can help provide a
useful framework for better understanding and diagnosing pathogens of the human gut
microbiome. One such phage benchwork technique is virome metagenomics. The field
of virome metagenomics involves collecting and characterizing the genomic sequences
of viruses from a community of organisms. The application of virome metagenom-
ics in the context of the gut microbiome can help researchers determine the causative
factors of various gut-related diseases and understand the microbiome of the gut in
greater detail. Fernandes et al. studied the fecal viromes of children with ulcerative
colitis (UC), Crohn’s disease (CD), and healthy controls to understand the pathogenesis
of inflammatory bowel disease via virome metagenomics (Fernandes et al., 2019). The
results showed that a higher number of Caudovirales phages was found in patients
with CD but not UC. Additionally, compared to healthy controls, children with CD
had a lower richness of phages from Microviridae. These findings indicate that virome
metagenomics is a powerful tool in understanding the makeup and composition of
the gut microbiome and changes in this composition that lead to different diseases.
Virome metagenomics has also helped expand the study of the pathogenesis of autoim-
mune diseases. In particular, classifying gut viromes can help determine what kinds of
bacteria host may be responsible for triggering the immune system to target the host’s
own body. Kim et al. collected 182 fecal and plasma samples from case children with
islet autoimmunity and healthy controls (Kim et al., 2019). The researchers were able
to detect a total of 129 viruses that were statistically significantly different between the
case and control samples using metagenomics analysis. Additionally, five enterovirus
A species were detected at significantly higher levels in the islet autoimmune samples
than controls. These results highlight a potential causal link between enterovirus A
species and the emergence of islet autoimmunity in children. Furthermore, the results
underscore the effectiveness of gut virome metagenomics as a laboratory technique to
analyze the pathogenicity of diseases. The use of virome metagenomics and its rela-
tion to bacterial species of the gut can also help understand nutritional disorders. This
topic is the focus of research by Reyes et al., who investigated severe acute malnutrition
(SAM) in children and its connection to the gut microbiome (Reyes et al., 2015). The
researchers analyzed metagenomes of virus-like particles (VLPs) from fecal samples
of Malawian twin infants who were either concordant for healthy growth or discordant
for severe acute malnutrition. They discovered that phages from the Anelloviridae and
Phageome in Gut Microbiome 57

Circoviridae families were present at significantly different levels in the discordant

from concordant health pairs. Virome metagenomics has also been a useful technique
in identifying previously unknown etiological agents responsible for gastrointestinal
disorders or health problems. For example, while several pathogenic substances and
metabolic conditions could cause diarrhea, there was a rise in unexplained severe
diarrhea in children in Turkey in 2015. Altay et al. examined stool samples from
the Turkish children with diarrhea using virome metagenomic analyses to investi-
gate the possible etiological agents responsible for the disease (Altay et al., 2015). The
authors found that there was bufavirus (a type of parvovirus) DNA present in about
1.4% of the diarrhea samples but in none of the healthy samples (Altay et al., 2015).
Additionally, further analysis of this bufavirus showed that children with this virus
had more severe diarrhea compared to other children; the specific type of bufavirus
responsible for this diarrhea was bufavirus genotype 3. Another study also elucidated
the importance of virome metagenomics to identify etiological viruses responsible for
diarrhea (Yinda et al., 2019). These researchers analyzed the fecal samples from 221
Cameroonians with symptoms of gastroenteritis via virome metagenomic sequencing.
The results showcased that there was an abundance of viruses belonging to the families
of Adenoviridae, Astroviridae, Caliciviridae, Picornaviridae, and Reoviridae among
these samples. Additionally, orthoreovirus, picobirnavirus, and smacovirus were also
all found in the samples and showed a genetic similarity to viruses in fecal samples
from bats and other animals. Thus, these results highlight not only the importance of
virome metagenomics in determining the cause of diseases such as diarrhea but also
underscore viral metagenomics being potentially used to trace back the source of these
etiological viruses to other animal hosts.
The use of genetically engineered phages holds promising therapeutic appli-
cations. Phage therapy is an emerging field that uses bacteriophages to target and
eliminate specific pathogenic bacteria. As described earlier, dysbiosis (disruptions
in the microbiome) in the gut, through the imbalance of bacterial species, can lead
to various health problems. Thus, the selective elimination of these harmful bacteria
through phage therapy can be a major breakthrough in controlling several health
disorders. This novel approach also has its advantage over traditional antibiotics,
which are often not specific to harmful bacteria and can thus cause the side effect of
eliminating beneficial gut bacteria as well. The specificity and efficiency of phage-
based therapy can potentially make this the dominant medical procedure for curing
gastrointestinal diseases. While phage-based therapy certainly has its advantages,
there are definite disadvantages of this procedure in a medical setting. Excessive use
of phage therapy can lead to the potential evolution of pathogenic bacteria to become
phage-resistant. Thus, this would mean that in the future, stronger or more compli-
cated phage treatments, such as using more virulent phages or phage cocktails, may
be needed for therapeutic benefit. Additionally, errors in the engineering of phages
for phage therapy could also lead to disastrous health consequences, such as viru-
lence against the healthy gut microbiota or off-target effects. To overcome these bar-
riers, several studies have tested the efficacy of phage-based therapy in controlling
and preventing human gut-related diseases. Phage-based therapy is still a new and
emerging field, so most of these studies are in vitro and animal model studies with
potential future applications in human medicine.
58 The Gut Microbiome: Bench to Table

Contemporary phage therapy has relied on lytic phages and usually involves mul-
tiple types of phages to create “phage cocktails” for greater efficacy. As of today,
according to a review by Lin et al., phage therapy products have not been approved
for human use in the EU or the USA (Lin et al., 2017). However, phage therapy has
been approved and widely used in the biocontrol of foodborne pathogens by the food
industry (Kazi and Annapure, 2016; Niu et al., 2009). Research in the field of phage
therapy is still persistent, and the medical potential of phage therapy as an antibacte-
rial treatment is being studied globally. Work by Maura et al. tested the efficacy of
a three-phage cocktail (CLB_P1, CLB_P2, and CLB_P3) in eliminating enteroag-
gregative Escherichia coli (EAEC) O104:H4 55989Str strain using mouse models
(Maura et al., 2012). The results showcased that after 24 hours, the bacteriophage
treatment led to significantly lower concentrations of 55989Str in the ileum of the
mice and slightly lower concentrations in the fecal matter. Thus, the efficacy and
potential application of phages in eliminating pathogenic bacteria in the gut are evi-
dent. These decreases were only transient but with a return to baseline concentrations
of 55989Str 3 days after treatment. The results of bacterial regrowth after 3 days high-
light the limitations of contemporary phage therapy, indicating that more research is
needed to design long-lasting treatments against bacterial pathogens. On the other
hand, phage therapy has the potential as a supplemental treatment to aid other thera-
peutic procedures. One of the primary medical areas that phage-based therapy has
been tested on is cancer. Particularly, phage therapy has been explored to use in
combination with other conventional cancer therapies, like chemotherapy. Research
conducted by Zheng et al. studied mouse models with colorectal cancer to test the
ability of phages to aid chemotherapy in treating cancer (Zheng et al., 2019). The
bacteria Fusobacterium nucleatum tends to promote tumors along the gastrointesti-
nal tract, leading to colorectal cancer. Thus, the researchers used irinotecan-loaded
dextran nanoparticles covalently linked to azide-modified phages to selectively elim-
inate harmful Fusobacterium nucleatum in the gut. Their results revealed that this
phage-based treatment led to significantly more successful first-line chemotherapy
treatments in mice compared to controls. Additionally, the authors repeated the treat-
ment in piglets and found that there were no significant changes in hemocyte counts,
immunoglobulin and histamine levels, and liver and renal functions. The finding
suggests that phage-based therapy can potentially be used as a supplemental treat-
ment for colorectal cancer with little to no side effects.
In addition, there has been an interest in using phage therapy to deliver CRISPR-
Cas9 systems to the gut in order to make genomic edits for the microbiome. Lam
et al. used mouse models to study the effects of using engineered bacteriophages
as vehicles for CRISPR delivery into the gut (Lam et al., 2021). The researchers
engineered the filamentous bacteriophage M13 to deliver exogenous CRISPR-Cas9
DNA to Escherichia coli populations of the mouse GI tract. The results reported that
using phages as a delivery mechanism for CRISPR systems induced chromosomal
deletions in the selected bacteria in both in vitro and in vivo settings. However, some
conclusions showcased potential drawbacks of this technique, including bacterial
deletion of the CRISPR DNA to escape targeting. This study provides a proof of con-
cept for future studies and suggests that manipulating the gut microbiome is possible
by using a phage delivery system.
Phageome in Gut Microbiome 59

Yet another important benchwork tool for phage therapy applications to the human
gut is fecal viral transplants. Fecal viral transplants involve screening and obtaining
viral populations of stool samples from one healthy individual and transplanting the
viruses into the colon of another recipient individual (Bojanova and Bordenstein,
2016). Essentially, it allows the microbiota of the healthy individual to repopulate
the gut of the sick individual. This technique has been proposed as a therapeutic
treatment for gastrointestinal diseases, given the connection between gut microbiota
and human health and the role that phages play in this connection. One of the pri-
mary advantages of using fecal viral transplants is preventing and treating bacterial
infections of the gut. As described earlier, numerous pathogenic bacterial species
can disrupt the healthy gut microbiome and cause dysbiosis. Thus, it is important to
eliminate these pathogenic agents to restore a healthy gut. The use of bacteriophages
emerges as a possible solution. Zuo et al. compared the virome and bacterial microbi-
ome changes in Clostridium difficile infection (CDI) of the gut subjects treated with
fecal microbiota transplantation (FMT) or with vancomycin (Zuo et al., 2018). The
results showed that the FMT treatment significantly decreased Caudovirales DNA
in subjects with CDI. Additionally, FMT resulted in changes in both bacterial micro-
biota and virome in the gut, while the vancomycin treatment only changed the bacte-
rial composition of the gut. These findings suggest that FMT may be a beneficial way
to treat bacterial infection of the gut. Some applications of fecal viral transplantation
are still being tested in animal models. One such application is the use of fecal viral
transplantation in improving the restoration of the normal bacterial gut microbiota
after antibiotic treatment. Draper et al. disrupted the mice’s gut microbiome using
a combination of penicillin and streptomycin (Draper et al., 2020). After that, the
bacteriome of mice from either fecal viral transplants or fecal viral transplants with
heat and nuclease treatment (both of which killed the bacteriophages as controls)
was observed. The results indicated that the fecal viral transplanted mice showcased
a higher degree of resemblance to the pre-antibiotic treatment bacteriome. Moreover,
analysis of the gut viromes of both groups of mice demonstrated that the fecal viral
transplanted mice maintained the phages used in the transplantation over time, sug-
gesting long-term benefits. Thus, the results of this study suggest the role of fecal
viral transplantation is not only a primary treatment but also a way to mitigate side
effects and improve recovery after other medical treatments, such as antibiotics.

THE CHALLENGES OF PHAGEOME IN HUMAN GUT

MICROBIOME, PERSPECTIVES, AND FUTURE DIRECTIONS
As phage research continues to develop, phage therapy remains a safe and effective
therapeutic approach to bacterial infections and inflammatory illnesses associated
with the GI tract. Theoretically, phages are not dangerous to human tissue because
phages are viruses that specifically infect bacterial cells without affecting eukary-
otic cells. Phages’ host ranges target specific bacterial species or serotypes, so other
cells, including natural microbiota, are not infected (Lewis and Hill, 2020). Phages
have already been successfully used in medicine in several studies. Clarke et al. col-
lected clinical reports regarding 277 patients who received phage therapy for joint
and bone infections by various bacterial species from 1933 to 2020; over 95% of
60 The Gut Microbiome: Bench to Table

patients received significant clinical improvement, even total resolution of the infec-
tion (Clarke et al., 2020). Although localized pain and swelling were found in some
patients, researchers concluded that the phages were contaminated with endotox-
ins from raw phage lysates. Beatrix and Domingo-Calap reviewed similar results
concerning phage therapy clinical trials in GI diseases (Gutiérrez and Domingo-
Calap, 2020). Based on a randomized, double-blind, placebo-controlled trial treating
chronic diarrhea patients with a four-phage cocktail, patients experienced alleviated
symptoms after 28 days of oral treatment. This was correlated with reduced proin-
flammatory E. coli and reductions in inflammatory cytokines and enzymes, such
as aminotransferases. Alpha and beta diversity parameters were maintained, also
meaning the phages did not disrupt the gut microbiota. Phage therapy has been suc-
cessfully administered throughout clinical experiences, likely indicating the safety
and efficacy of phage therapy against GI diseases caused by pathogenic bacteria.
When studying phages in the human gut microbiota, there are still research gaps
and challenges. In general, some phage genomes are unknown and share little homol-
ogy with known phage sequences. Unknown and hypothetical viral sequences are
continually prevalent in phage research that often force current genomic and taxo-
nomic systems to think instead of seeing current data in a new light (Shkoporov
and Hill, 2019). Phages are constantly evolving, and the diversity of viral genomes
also hinders how well researchers can align phages with references. Metagenomic
approaches have successfully facilitated the characterization of different phages in
the environment and closely linked the gut virome and bacteriome; however, at this
time, pure genomic studies are not able to detail specific phage–host interactions.
As genomic and sequence-based tools become more popular, researchers will be
losing whole picture if the information from in vitro and in vivo studies is missing.
This would create a methodological bias that does not account for phage biology
and evolutionary history and low sensitivity toward small DNA yields from phages.
Moreover, unlike bacteria, phages lack universal phylogenetic markers, and thus, it is
very complicated to gather large quantities of viral genetic material within metage-
nomic data without contamination of bacterial sequences based on current protocols
(Garmaeva et al., 2019).
When utilizing phages in therapy or application, the stability of phages is also a
challenge because it is frequently variable across phage species and formulations,
which makes it difficult for researchers to maintain phage titers through experiments
and clinical trials (Garmaeva et al., 2019). If not frozen or cooled down, phages will
spontaneously mutate over long periods of storage, which can impair the fitness of
phages and research data (Garmaeva et al., 2019). In addition, there is generally a
lack of quality and safety guidelines for preparing phages, especially for therapy.
Although there are strict regulations for pharmaceutical products, few standards have
been addressed specifically for phage research and application. Also, phage research
currently lacks a simple, fast, and high-throughput method to screen phages. The
techniques, including double-layer agar plates, real-time PCR, and flow cytometry,
commonly used for phage research are not easily compatible with all phages while
producing quick results (Pires et al., 2020). To efficiently continue phage therapy, it is
required to improve experimental methods, including methods in genomics, molecular
biology, and microbiology; standards of pharmacokinetics and pharmacodynamics of
Phageome in Gut Microbiome 61

phages also need to be investigated (Luong et al., 2020). The optimal routes of admin-
istration, dosage, and the appropriate diseases for phage therapy are also research
gaps. Moreover, the phage resistance has not been currently addressed and remains
a barrier to more effective research and phage therapy (Yang et al., 2020). Therefore,
there can be uncertainty among researchers, patients, and consumers, regarding the
effectiveness of phages and the arms race between phages and their bacterial hosts.
As phages in the gut microbiota continue to be studied, there will still be gaps to be
investigated in the future, such as the exploration of the viral dark matter.
While understanding the multitude of gastrointestinal diseases and other health
disorders that arise from the dysbiosis of the gut microbiota remains a goal for
microbiology research, there are still many current and future studies focusing on
manipulating the gut microbiome for therapeutic benefit, despite the challenges and
research gaps. Since the microbiome is inherently linked to a variety of immune and
metabolic responses, ways to manipulate the microbiome by enriching or changing
its composition and diversity can hold the key to therapeutic treatments for a variety
of health conditions. One of the emerging techniques to use the gut microbiome for
therapeutic benefit is fecal microbiota transplantation which has shown successful
therapy in IBD clinical trials (Oka and Sartor, 2020). Still, it is not always effec-
tive. There have been many documented cases of fecal microbiota transfer failures,
where the technique failed to eliminate pathogenic bacterial species or resulted in the
recurrence of gastrointestinal problems, like diarrhea (Allegretti et al., 2018). Recent
research has been using fecal viral transplants instead of whole fecal microbiota
transplants. There have been a few studies recently that have shown proof of the
concept of this technique. Of note, Lin et al. researched the effectiveness of trans-
planting only VLPs, many of which are bacteriophages, in mice compared to per-
forming whole fecal microbiota transplants (Lin et al., 2019). The goal was to test the
efficacy of both techniques to treat small intestinal bacterial overgrowth (SIBO). The
results revealed that the transplantation of the VLPs was as effective as normal FMT.
Using only bacteriophages has several advantages over traditional FMT. First, fecal
microbiota transplants have the risk of transferring potentially dangerous pathogenic
bacterial species from the donor to the recipient. This risk is eliminated with fecal
viral transplants, as no bacteria are transferred during this procedure. Additionally,
fecal viral transplants could theoretically lead to higher efficacy than FMT since
researchers can carefully select and screen for specific phages or viruses from fecal
samples. While fecal viral transplants certainly have their advantages, there are still
numerous ways to improve the technique. Future research should focus on improving
the selection and isolation of phages from fecal samples to yield the most efficacious
results. Additionally, while this technique has been tested in animal models, future
studies will need to be conducted with human trials to see whether these results can
truly be extrapolated for human therapeutic benefit.
While fecal viral transplants can help add or eliminate bacterial species to the gut
microbiome, an emerging field of study focuses on the genetic manipulation of the
microbiome to combat diseases. One of the most interesting contemporary examples
of this has been using bacteriophages as vehicles to deliver CRISPR-Cas9 systems
to the gut, as previously mentioned. Additional research on CRISPR and phages
can potentially help revolutionize this field. There are several concerns regarding
62 The Gut Microbiome: Bench to Table

CRISPR and gene editing that must be taken into consideration and investigated by
future research. For example, one major topic of focus regarding current and future
studies is to reduce the off-target effects of the CRISPR-Cas9 system. Improving
specificity and screening of bacteriophages to be used as vehicles for the CRISPR
systems is also another potential topic for future research.
Overall, while several recent studies have already shown how effective under-
standing and manipulating the phageome of gut microbiota can be, there are many
mysteries in the field that remain to be discovered, as described above. Additionally,
there are many potential directions for the future of this expanding field, including
establishing core gut viral and genetic databases, studying the evolutionary adapta-
tions of the gut microbiota to changing viral environments, and observing specific
changes in host phenotypes in response to alterations in the gut phageome. Future
research will hopefully discover the underlying mechanism and interactions of gut
microbiota and find more efficient alternatives to these challenges related to human
health and diseases.

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GUTJNL-2017-313952
Section II
T1 Preclinical Animal
Studies, and Safety and
Efficacy Trial in Humans
 3 Microbiome and Nutrition –
Why Do We Care?
Discussing a Complex
Relationship
Angelos K. Sikalidis
California Polytechnic State University

CONTENTS
Introductory Remarks .............................................................................................. 71
The Human Gut Microbiome ................................................................................... 74
The Metabolome and Microbiome Connection ....................................................... 75
Host–Gut Microbiome Metabolic Interactions ................................................... 76
Microbiome – Diet Interactions ............................................................................... 78
The Gut Microbiome and Carbohydrate Metabolism .............................................. 79
General Considerations ....................................................................................... 79
Simple Carbohydrates .........................................................................................80
Complex Carbohydrates ...................................................................................... 82
Nondigestible Polysaccharides – Fiber ............................................................... 82
The Gut Microbiome and Fatty Acid Metabolism ...................................................84
General Considerations .......................................................................................84
Saturated Fatty Acids .......................................................................................... 85
Polyunsaturated Fatty Acids (PUFAs) – Omega-3 and -6 (ω-3 and ω-6)
Fatty Acids .......................................................................................................... 86
Short-Chain Fatty Acids ...................................................................................... 88
The Gut Microbiome and Amino Acid Metabolism ................................................ 89
The Microbiome and Weight Change ......................................................................90
Future Perspectives ..................................................................................................92
References ................................................................................................................92

INTRODUCTORY REMARKS
The term microbiome arises from the Greek words “μικρο” – micro meaning “small”
(in mathematics and sciences designated with the Greek letter μ (10 −6)) – “βίωμα” –
biome meaning “of life”, in Greek: “μικροβίωμα”. Prior to 2001, the term micro-
biome was in use, mostly to infer a rather small significantly complex ecological
niche incorporating plant and animal life. While the study of what is now known as

DOI: 10.1201/b22970-5 71
72 The Gut Microbiome: Bench to Table

the “human microbiome” can be traced as far back as Antonie van Leeuwenhoek
(1632–1723), “I then most always saw, with great wonder, that in the said matter there
were many very little living animalcules, very prettily a-moving”. Leeuwenhoek,
who is known to have made over 500 “microscopes”, of which fewer than ten have
survived to the present day, was a keen, laborious and systematic observer of the
microbial world.
In 2001, the term “microbiome” was re-discovered, re-surfaced and used by Nobel
laureate-microbiologist Joshua Lederberg to signify the microbial life in symbiosis
(under normal healthy conditions) with the human body (Prescott, 2017). In this con-
text, a fair definition of the microbiome could be: “the full complement of microbes
(bacteria, viruses, fungi, and protozoa), their genes, and genomes in or on the human
body”. Microbiota on the other hand, which is used similar to the term microbi-
ome, refers to an “ecological community of commensal, symbiotic and pathogenic
microorganisms found in all multicellular organisms studied to date from plants and
animals” (Prescott, 2017; Institute of Medicine, 2013). Hence, the difference between
microbiota and microbiome is that the latter could be more encompassing in the
sense that it refers not only to the organisms but also to the genome present and the
relevant implications.
The notion that something existing in the gut could produce health effects is indeed
ancient. Furthermore, the idea that fecal matter contains elements that could alter
favorable health outcomes to the recipients was also entertained since the antiquity.
The history of fecal microbial transplant (FMT) constitutes a characteristic example:
Although it is often considered a “novel” therapeutic strategy (Opritu, 2016), it was
being practiced while well-documented, certainly in terms of oral administration, in
various European medical crafts ever since ancient Greece. The practice is described
in numerous key works of medicine of 16th- and 17th-century Europe (Moore, 2019;
Faming et al., 2012), while further it was practiced in China since the Don-jin dynasty
(4th century AD) (Faming et al., 2012). FMT rectal delivery was used by American
doctor I.O. Wilson in 1910, particularly in the effort to instigate favorable changes in
fecal bacterial composition in patients with functional bowel disorders ameliorating
symptoms (Woodruff, 1910). Therefore, FMT cannot be considered novel, at least not
conceptually, whereas its historical and intercultural ubiquity may in all actuality pro-
vide solid support to an emerging scientific model, that of gut microbiome functioning
as a human body’s essential organ, itself comprised of several organisms which have
coevolved with human cells such that they are to some extent “us” and we are “them”.
On that basis, Alexander Khoruts, a pioneering researcher studying FMT, made
the argument that FTM-Clostridium difficile therapy must be viewed as a “trans-
plant” as opposed to a “drug” (Steve and Khoruts, 2014). Evidence regarding com-
mensal and symbiotic intestinal microbes continuously accumulates and supports
such an interpretation (Gray, 2017). Moreover, consistent with accepting the micro-
bial origin of human mitochondria (Gray, 2017), this organelle is another example
of coevolution through symbiosis where identifiable structures and unique DNA
(mtDNA) are evident (Moore, 2019).
Similarly, to several examples in biological sciences, the “microbiome” has
enjoyed significant biohype culminating to what some term a “microbiome zeitgeist”
Microbiome and Nutrition 73

(Prescott, 2017) leading to a “World microbiome day: June 27th”. Even though it is
well established and widely known both in the scientific community and in the public
that bacteria reside at various locales of the human body especially in the large intes-
tine (colon), the importance of the microbiome has gained significant attention due
to a significant body of scientific literature over the past 10–15 years indicating and
demonstrating that the size and type of microflora populations in the gut could well
influence health outcomes, spanning from immune responses to metabolic syndrome
(syndrome X), type 2 diabetes mellitus (T2DM), nonalcoholic steatohepatitis, cardio-
vascular disease (CVD) and some forms of cancer, particularly of colon (Bashiardes
et al., 2018). Thus, the microbiome is deemed integral to human physiology, health
and disease, while it arguably constitutes the most intimate connection that humans
extend to their external environment, mostly through diet. We typically consider the
large intestine (colon) as a site hosting a variety of populations of bacteria that reside
in a symbiotic mode at that habitat with the human body. While this is not the only
bodily location where bacteria reside, it appears to be a location with probably the
highest numbers and diversity of bacterial populations extending a rich variety of
metabolic and physiological influences.
The human microbiome, especially that of the gut, is being regarded an “essential
organ” (Bashiardes et al., 2018), with roughly 150 times more genes than the whole
human genome, while the human body contains at least 1,000 different species of
known bacteria. Microbiotic composition and function differ according to variations
in location, age, sex, race, and diet of the host (Steve and Khoruts, 2014). Although
determining precise numbers is rather challenging and of questionable accuracy, it
has been proposed that the number of human cells while at the order of 40 trillion
human cells (4 × 1013) in a typical human body is less than that of bacterial 1014 –1015
(Sender et al., 2016a, 2016b; Savage, 1977). There are recent estimates updating
the total number of bacteria in the 70 kg “reference man” to be 3.8 × 1013, while for
human cells, same estimates identify the dominant role of the hematopoietic lineage
to the total count (90%) and revise past estimates to 3.0 × 1013 human cells (Sender et
al., 2016a). The same analysis also updates the widely cited 10:1 ratio, showing that
the number of bacteria in the body is actually of the same order as the number of
human cells (Sender et al., 2016a).
The Human Microbiome Project (HMP) was initiated by the National Institutes of
Health (NIH) in 2007, with the majority of funding ($153 million of the $173 million
as of 2013 alone) coming from the NIH Common Fund (Institute of Medicine, 2013).
The project was initiated aiming to characterize the genomic makeup of all microbes
inhabiting the human body. Nonetheless, gradually even more so a growing number
of scientists place increasing emphasis on the importance of studying beyond the
identification on the microbes present but also the function of those microbes. HMP
used sequencing to examine the microbes associated with the human body. HMP’s
major purpose has been to generate resources for the research community, focusing
on building a “healthy cohort” reference database of human–microbiome genome
sequences (known as metagenomic sequences), computational tools for complex
metagenomic sequences analyses and clinical protocols for human–microbiome
sampling (Institute of Medicine, 2013).
74 The Gut Microbiome: Bench to Table

THE HUMAN GUT MICROBIOME

A variety of bacteria, archaea, yeasts, planctomycetes and filamentous fungi and
viruses, such as Senegal virus, are examples of human gastrointestinal (GI) residents
and evidence of the complexity characterizing the human GI microbiome. While
estimates of species numbers in the gut exhibit significant variation, the adult micro-
biota is generally accepted to consist of over 1,000 species, which in turn are grouped
into a few bacterial phyla, with over 7,000 strains (Thakur et al., 2014). Several dif-
ferentiating ecological habitats comprise the GI tract, while foods typically provide
microbes even after gastric treatment. Despite the reduction in the microbial load
after gastric emptying, significant populations remain in the chyme found in the
stomach, the small and later large intestine (Mondot and Lepage, 2016).
The most prominent phyla out of the several detected in the stomach are as fol-
lows: Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria and Fusobacteria.
Cultivable species mainly belong to the genera Streptococcus, Neisseria and
Lactobacillus, and very high interindividual variation is observed in the gastric
microbiome at the genus level. The bile and pancreatic secretions in the small intes-
tine together with peristalsis maintain low levels of the bacterial population com-
pared to that of colon. Respective to levels of bacterial population, intestinal contents
range from duodenum to the ileocecal segment as 104 to 106 –107 CFU/g, respectively
(Mondot and Lepage, 2016). Small intestine microbiota is mainly composed of facul-
tative aerobic–anaerobic species (Streptococcus, Lactobacillus, Enterobacteriaceae)
but also strict anaerobes (Clostridium and Veillonella). Fecal microflora samples,
while they may provide a view on the dominant intestinal microbiota composition,
only partially describe the distal colonic microbiota (Mondot and Lepage, 2016).
The colonic microbiota is characterized by the presence of four major phyla:
Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria, with Firmicutes and
Bacteroidetes accounting for over 90% of the total bacteria in the ecosystem. The
Firmicutes often dominate and are divided into two major classes of Gram-positive
bacteria in the gut: Clostridia (mainly Clostridium cluster IV and Clostridium XIVa)
and Bacilli (Bacillales and Lactobacillales). The phylum Bacteroidetes includes
most Gram-negative bacteria, is less diverse with respect to species and is subdivided
into three predominant genera: Bacteroides, Prevotella and Porphyromonas (Mondot
and Lepage, 2016).
To study microbial communities in a variety of environments, culture-indepen-
dent methods have been developed. These methods typically entail the use of small
subunit ribosomal RNA gene (16S rRNA) of the environmental microorganisms,
thus providing information on microbial diversity in the gut. In a mere decade of use,
these methods have described several species that significantly exceeds that of the
cultivated species. More specifically, out of over 1,200 microbes described in the gut,
only 12% were detected by the application of both a molecular approach and a cul-
ture, while the vast majority (roughly 75%) were observed only via 16S rRNA gene
sequencing (Institute of Medicine, 2013; Mondot and Lepage, 2016).
Genes involved in immunity, nutrient absorption, energy metabolism and intesti-
nal barrier function are modulated by the gut microbiota in, as demonstrated, both
humans and mouse models (Thakur et al., 2014; Sarkar et al., 2016). Moreover, there
Microbiome and Nutrition 75

are several factors implicated to extending higher or lower degrees of influence to gut
microbiota composition, including genetics, health status, mode of delivery at birth
and environmental exposures.

THE METABOLOME AND MICROBIOME CONNECTION

Personalized health approaches require integrative, systems-level tactics considering
human biocomplexity. In this context, microbiome research aligns powerfully with
this paradigm. When considering the gene–diet–microbial axis, genome is merely
one of the components that ultimately comprise the human “mega system”. Therefore,
even though genome-wide association studies (GWASs) are commonly performed,
they do not necessarily enlighten as revealingly since links are rarely deterministic,
while, moreover, statistical significance often times does not translate at all, or at
least equally powerfully, to biological significance of function. There are several
such examples of weak biological connections between the mere genetic association
and the actual biological phenotype as per metabolic profiles and functions.
For instance, researchers reported that the 32 statistically significant body mass
index (BMI)-associated genes identified accounted for a thin 1.47% of actual BMI
value variation (Speliotes et al., 2010). Other researchers have actually revealed an
association between obesity and microbiome (Ley et al., 2006; Turnbaugh et al.,
2006). This is just an example of the highly controversial and consensus-lacking obe-
sity–microbiome connection. While most of the scientific community recognizes the
possibility for a connection, it is rather difficult to determine what the relationship is,
what the underlying mechanism is and finally what is the order of events (i.e., obesity
informs the microbiome or is it the other way around? While if there is a cross talk,
how does such dynamic evolve?). Some investigators propose that the main underly-
ing cause for inconsistencies that lead to controversial and conflicting results regard-
ing the obesity–microbiome relationship stems from the fact that there are significant
differences regarding the level of phylogeny as well as the design of the experiments
conducted. These include lack of concise measures, models and translation to clinical
environment. Nonetheless, there seems to be no agreement as to standardization and/
or best practices regarding those types of approaches (Institute of Medicine, 2013).
Systems-level approach has shown that a tremendous number of human diseases are
genetically connected, which is biologically plausible, with oftentimes the same
gene or groups of genes implicated in different disorders. However, understanding
the human genome in itself is not enough toward personalized/precision health care.
The microbiome represents yet another new level of complexity and genetic link
(Institute of Medicine, 2013).
Beyond the varying degrees of disease risk that complex interactions between the
microbiome and its host generate, diverse metabolic phenotypes in turn associated
with pathologies are also observed (Holmes et al., 2008). Actually, while disease
risk levels and metabolic phenotypes are biologically and statistically associated
in such manner, measurement of metabolite levels (biomarkers) can assess disease
risk. The idea that changes in metabolic products are an indication of disease is cer-
tainly not new. However, as new high-throughput technologies are used to produce a
rather extensive bulk of information on metabolites and construct detailed metabolic
76 The Gut Microbiome: Bench to Table

profiles, the complexity of such information generated is significant primarily due to

the fact that not only are all human cells producing metabolites (with more than 500
functionally distinct cell types), but so do all microbial cells (Nicholson et al., 2005)
which may well add exponentially to the metabolite pools.
Microbes produce short-chain fatty acids (SCFAs), bile acids and related oxyster-
ols, vasoactive (aromatic) amines, cresols and aromatic acids, endocannabinoids, and
a host of other compounds (Hussein et al., 2022). Moreover, several microbial metab-
olites participate in human metabolism. For example, bile acids, which are critically
important host signaling molecules, are cometabolized by microbes, with significant
implications for liver and colonic disease risk (Nicholson et al., 2005). Bile acids are
biosynthesized hepatically daily, stored in the gallbladder, released as needed into
the mammalian gut, used as emulsifiers to drive lipid absorption and recycled via the
enterohepatic circulation thereby returning to the liver. However, as enterohepatic
circulation is not 100% efficient, there is always a smaller or larger portion of the
overall produced bile that will result in the colon where it will be deconjugated into
cholic acid by Lactobacillus and other gut-residing bacteria. The cholic acid, in turn,
can be dehydroxylated by yet other microbes into deoxycholic acid which is both
hepatoxic and carcinogenic. Furthermore, microbial cometabolism of bile acids may
also negatively impact lipid bioavailability (Nicholson et al., 2010).
Additional to its potential toward contributing to personalized medicine, metabolic
phenotyping carries potential for applications at the entire population level. Holmes
and coworkers (2008) introduced the concept of metabolome-wide association studies,
the metabolic equivalent of GWAS, and demonstrated significant geographic variation
in metabolic phenotypes. Similar concept has been entertained by Yap and colleagues,
while comparable metabolic variation has been associated with varying risks of car-
diovascular as well as other diseases (Yap et al., 2010). Hence, it seems that there is
mounting evidence to suggest that as humans coevolve with their natural/geographic
environment, part of such coevolution is the evolution of their microbiome as well as
their metabolic profile and ensuing risk for certain diseases of metabolic background.

While typically the consensus was that microbes were thought as from dangerous to
undesirable at best, the communities both scientific and lay do appreciate the vari-
ous aspects that the microbiome entails along with the beneficial ones that are health
promoting. Furthermore, it is increasingly more appreciated that it is in fact more
accurate to consider microbial communities (oftentimes diverse and in a dynamic
state) as opposed to microbes as units per se.
The development of a highly complex gut ecosystem in the human host begins
at or possibly even prior to birth, while the intestinal microbiota early colonization
depends on various environmental exposures. The taxa colonizing the otherways
relatively “sterile” newborn intestine are largely determined by the mode of deliv-
ery. Newborn intestinal colonization in the case of vaginal birth initiates while the
neonate passes through the birth canal. Neonatal’s ingestion of microorganisms
induces the gut microbiome and initiates its development. Moreover, infant feed-
ing depending on whether it is breastfeeding or formula-feeding further informs the
Microbiome and Nutrition 77

manner in which the intestinal microbiota will develop both in terms of quality and
quantity and overall bacterial demography. The early species are placed in the neo-
natal intestines through maternal feeding. Breastfeeding providing maternal milk
carbohydrates naturally selected to feed mutualist microbes in the infant’s micro-
biota provides significant support in the microbial growth. Beyond its nutritive value
both fort the infant and its intestinal microbiome, breastfeeding is further credited
with reducing infant mortality and limiting the risk of chronic diseases later in life.
Breastfed infants exhibit a Bifidobacteria-dominated microbiome inducing a tolero-
genic immune response in the gut, hence reducing the risk of pathogen colonization,
infection and overall gut inflammation (Wasielewski et al., 2016).
Human milk oligosaccharides (HMOs), the second most abundant milk carbohy-
drate after lactose, provide 5%–6% of milk’s energy content. Gut Bifidobacteria’s
growth is primarily supported by this type of carbohydrates. Interestingly, since
HMOs cannot be digested by humans, it requires microbial fermentation to produce
utilizable energy fuel for the host (i.e., human) in the form of SCFAs. Moreover, since
breast milk’s sialylated oligosaccharides prevent pathogen adhesion to epithelial cells,
they contribute significantly to infection prevention. Additionally, breast milk’s lipid
fraction further contributes to milk’s antipathogen properties. Breast milk’s triglycer-
ides are converted to antimicrobial fatty acids and monoglycerides by specific lipases
produced by the infant, which extend antiviral, -bacterial and -protozoan pathogen
properties, creating a hostile environment unsupportive of harmful microbe growth
(Wasielewski et al., 2016). Finally, breast milk is significantly advantageous in the
sense that is significantly enriched by a variety of antibodies reflective of mother’s
immunological profile as informed both by innate and by adaptive immunity.
Host-provided glycans are characterized by structural similarities with HMOs
and similar antimicrobial properties, offering an alternative to breast milk support
route for desirable microbes. The gel-like mucosal structure coating the intestinal
epithelium consists of proteins and O-glycosylated carbohydrate chains. This muco-
sal structure feeds the specific commensal bacteria, which in turn produce SCFAs,
while the host can capture the energy from SCFAs, thus contributing toward the
energy balance for manufacturing the mucus structure. This mutualistic relation-
ship results in benefit for the host because microbes occupy the glycan-binding sites,
thereby preventing pathogen crossing of the mucus layer which would cause inflam-
mation and ultimately infection (Wasielewski et al., 2016).
The symbiotic relationships between microbiota and host stem as a result of a
dynamically balanced habitat of symbionts and pathobionts. Perturbations affect-
ing the afore-described delicate balance leading to disturbance in the composition
of microbial communities, termed dysbiosis, typically result in disfavorable interac-
tions between the microbiome and the host, leading to increased disease suscepti-
bility. Various studies have demonstrated associations between intestinal dysbiosis
and chronic low-grade inflammation and metabolic dysfunctions. Thus, metabolic
syndrome, obesity and diabetes may be negatively impacted from changes in the gut
microbiome (Hemarajata and Versalovic, 2013; Thakur et al., 2014).
The gut microbiome may be affected by dietary alterations, infections and expo-
sure to antibiotics. Throughout infancy and early childhood, the aforementioned fac-
tors can alter the microbial balance in the individual’s gut. The infant’s intestinal
78 The Gut Microbiome: Bench to Table

microbiota is characterized by instability and relatively low degree of diversity.

Nevertheless, by the toddler years, the intestinal microbiota is similar in diversity
and stability to that of adults (Albenberg and Wu, 2014). In adulthood, the equiva-
lent of approximately 2 kg of microbes (yielding a number approximation of about
30 trillion) reside in the human body, with the majority residing in the gut, mostly
in the colon. The immune system can generally tolerate high density of microbes in
the distal gut, while comparable density in the small intestine causes severe illness.
However, in the event of microbial infection where microbes enter circulation, even
at low amounts, can cause cardiac collapse and death (Wasielewski et al., 2016).
Defending intestinal homeostasis and host health is achieved via cooperative
action of various functional microbial groups. By means of deploying an array of
glycoside hydrolases and polysaccharide lyases, the gut microbiome produces essen-
tial amino acids and some vitamins, thus facilitating the utilization of nondigestible
food compounds, while saccharide fermentation by gut microbiome is a major source
of energy for intestinal epithelial cells. Moreover, the bacterial metabolism of dietary
fiber to SCFAs can constitute a significant energy source for humans (Thakur et al.,
2014). Microbial de-polymerization of complex carbohydrates and proteins leads to
the production of a large variety of mono- and oligo-meric compounds, subsequently
fermented into SCFAs and ultimately to carbon dioxide and molecular hydrogen.
Interestingly, carbohydrate fermentation and SCFA production significantly improve
the absorption of calcium, magnesium and phosphorus (Thakur et al., 2014), hence
contributing to the nutritive efficiency of diet.

MICROBIOME – DIET INTERACTIONS

Intestinal microbiota functions as a metabolic organ that influences nutrient uptake,
bioavailability, metabolic signaling, energy balance, weight and disease risk in a
dynamic manner (Figure 3.1). Unfavorable nutritional environment for the microbi-
ome can lead to intestinal permeability, resulting in low-grade inflammation as well
as obesity, and associated chronic metabolic diseases such as nonalcoholic fatty liver
disease, dyslipidemia and insulin resistance. Thus, there is a special and rather sig-
nificant relationship between the type and the amount of nutrients becoming avail-
able to the microbiome in the gut, and how the microbiome changes in response to
such exposure to the nutrients in terms of its type, numbers and subsequent function
and metabolic signaling. This implies a series of events that will ultimately impact
metabolism of the host and finally risk for chronic diseases. More specifically, diet
exerts a major influence upon host immune function and the GI microbiota. Although
components of the human diet (including carbohydrates, fats and proteins) are essen-
tial sources of nutrition for the host, they also influence immune function directly
through interaction with innate and cell-mediated immune regulatory mechanisms
(Heras et al., 2022). Gut microbiome extends influences over many endocrine func-
tions such as adrenal steroidogenesis, thyroid function, sexual hormones, IGF-1 path-
way and peptides produced in the GI system. It is fundamental in glycemic control
and obesity, while also exerting an important function in modulating the immune
system and associated inflammatory disease. The result of this cross talk in gut
mucosa is the formation of the intestinal immunological niche (Rossella et al., 2022).
Microbiome and Nutrition 79

FIGURE 3.1 Conceptual scheme: microbiome – diet interactions can lead to metabolic sig-
naling, energetics and disease risk modulation. (Adapted from: Sikalidis and Maykish, 2020.)

THE GUT MICROBIOME AND CARBOHYDRATE METABOLISM

General Considerations
Carbohydrates constitute a key nutritional component for mammals and the mam-
malian microbiome, including that of the gut, typically conferring the majority of
energy fuel. In humans, carbohydrates are the most important food energy provider
among the macronutrients (between 40% and 80% of total energy intake). Mammals
can directly absorb simple sugars, fructose, galactose and glucose, in the proximal
jejunum via specialized transmembrane transporter proteins. Mammalian enzymes
hydrolyze disaccharides (sucrose, lactose, maltose) and starches to constituent
absorbable monomeric units (monosaccharides), but possess rather limited abilities
to hydrolyze other polysaccharides. Consequently, on a regular basis and depend-
ing on the diet consumed, a significant bulk of undigested plant polysaccharides
(cellulose, xylan and pectin) as well as partially digested starch reach the colonic
lumen where microbial communities reside. By hosting a vibrant and metaboli-
cally active microbiome well able to hydrolyze complex carbohydrates, mammals
are not required to invest significant energy, time and resources as well as genetic
capital toward the evolution/development and production of complex enzymes that
are required to catabolize the numerous polysaccharides in the diet. Microbes, by
contrast, possess a plethora of genes encoding various carbohydrate-active enzymes
(CAZymes) in the human microbiome (Devaraj et al., 2013; Cantarel et al., 2012).
Microbial CAZymes, which comprise a typical mammalian host repertoire, include
glycoside hydrolases, carbohydrate esterases, glycosyl transferases and polysaccha-
ride lyases (Cantarel et al., 2012). Approximately 40 g of dietary carbohydrates reach
the colon daily, escaping digestion by host enzymes. The main categories are resis-
tant starch, nonstarch polysaccharides and oligosaccharides, although some di- and
monosaccharides (e.g., sugar alcohols) may also reach the colon. The human gut
80 The Gut Microbiome: Bench to Table

microbiota is particularly well adapted to perform the degradation of polysaccha-

rides: 10,000 bacterial enzymes implicated in sugar digestion have been identified
(Arnone et al., 2021).
In the symbiotic relationship formed between the host and the gut microbiome,
the gut microbes gain effortless direct access to abundant readily fermentable car-
bon sources to be utilized in the absence of competition, which would otherwise be
wasted excreted by the host. Moreover, microbes by the same token may use those
complex carbohydrate substrates to support viable, functionally vigorous micro-
bial communities and produce bioactive compounds exerting signals that in turn
modulate mammalian metabolism. Intestinal bacterial taxa vary notably regarding
their aptitude to use dietary/host-derived carbohydrates (e.g., mucus components)
(Sonnenburg et al., 2010).
Bacteroidetes are shown to straightforwardly assimilate dietary carbohydrates as
well, since representatives of this bacterial phylum hold the ability to activate numer-
ous carbohydrate utilization pathways. Conversely, in cases of dietary carbohydrate
starvation, gut bacteria will typically aggressively catabolize mucins in the GI tract
yielding a carbohydrate source, thus possibly compromising the mucus layer adja-
cent to the epithelium, causing inflammation and likely permeation integrity prob-
lems depending on the extent of catabolism. Comparable to Bacteroides, strains of
the genus Bifidobacterium encompass genes encoding for glycan-foraging enzymes,
which in turn enable those gut bacteria to obtain nutrients from host-derived glycans
(Devaraj et al., 2013). Similar to their capacity to hydrolyze starch, gut microbes have
evolutionarily developed the capability to degrade several plant- and host-derived
glycoconjugates (glycans) and glycosaminoglycans such as cellulose, chondroitin
sulfate, hyaluronic acid, mucins and heparin. Catabolic enzymes of microbial origin
including endoglycosidases might act on dietary substrates, thereby releasing com-
plex N-glycans from human milk and other dairy sources (Garrido, 2012).
Dietary variations can extend functional consequences on both bacteria and the
host alike, leading to the “cannibalization” of indigenous mammalian carbohydrates,
which in turn could lead to expansion of beneficial features, prevention of diseases
or predisposition to different disease states depending on the specifics and the extent
of the phenomena. For instance, human milk oligosaccharide-grown Bifidobacteria
contribute to the stabilization of epithelial tight junctions’ formation while promot-
ing secretion of interleukin-10 (Chichlowski et al., 2012). The biogeography of the
microbiome may be relevant since specific genes/pathways such as simple carbohy-
drate transport phosphotransferase systems are more prominent in the small intestine
as opposed to the colon (Zoetendal et al., 2012), seemingly by design.

High levels of plasma lipopolysaccharides (LPS) with ectopic hepatic lipid accu-
mulation increase gut permeability, and low-grade endotoxemia have all been
demonstrated in animals whereby obesity was induced via exposure to high-fat or
high-fructose diets. High-fructose diets, in particular, have been strongly related to
both hepatic and extra-hepatic insulin resistance as well as obesity-related metabolic
disturbances via mechanisms involving gut microbiome and downstream intestinal
Microbiome and Nutrition 81

permeability (Ochoa et al., 2015). While sugars in general constitute a major car-
bon source modulating growth and virulence in a broad spectrum of gut pathogen
and pathobionts, refined/simple carbohydrates support overgrowth for opportunistic
bacteria including Clostridium difficile and Clostridium perfringens by induction of
biliary output (Wasielewski et al., 2016; Albenberg and Wu, 2014). Taken together,
these observations imply that high-fat/high-refined sugars diets can introduce unde-
sirable ecological alterations in the gut environment while intensifying host response
such as inflammation and differentiated glucose metabolism (Albenberg and Wu,
2014). Furthermore, simple carbohydrate overconsumption could produce unfavor-
able effects at both central and peripheral levels, such as fluctuations in secretion of
satiety-regulating peptides as well as neuropeptides. Further negative impact includes
increase of gut permeability leading to low-grade inflammation and liver disease as
well as induced blood–brain barrier (BBB) permeability, in turn affecting appetite and
mood/predisposition toward food, via the reward/dopamine axis related mechanism
(Ochoa et al., 2015). While fiber consumption seems to protect against inflammation,
the accumulation of simple sugars in the colon induces an increase of the osmotic
load, as well as the fermentation rate by the colonic microbiome, subsequently poten-
tially promoting abdominal pain, discomfort and various intestinal dysfunctions.
While enterocytes in the intestine will typically produce fatty acids, utilizing both
glucose and fructose as substrates, compared to glucose, fructose may be proven
a deprived substrate for de novo lipogenesis in enterocytes, while fatty acid accu-
mulation could worsen intestinal function. Consequently, excessive sucrose intake
has been recognized as a primary cause of metabolic syndrome, the toxicity result-
ing from excess fructose rather than sucrose, as fructose extends the induction of
glycation phenomena targeting proteins and, physiologically, its metabolic substrate
rapidly flows into de novo lipogenesis, which in turn strongly promotes liver inflam-
mation (MacDonald, 2016). Additionally, the pro-oxidative and pro-inflammatory
effects of fructose can lead to induced gut permeability and endotoxemia both of
which conditions do exacerbate chronic inflammation in the gut.
High sugar intake might disturb intestine homeostasis in a variety of mechanisms
and ways. The consumption of sugar-rich diets can alter the intestinal mucosa archi-
tecture. A high-sugar diet promotes intestinal stem cells differentiation into absorp-
tive enterocytes and secretive enteroendocrine cells in Drosophila, which causes the
gut to become thinner, and produces high levels of reactive oxygen species (Zhang et
al., 2017); specifically, high fructose intake can lead to reduced mucus thickness in
the colon and defensin secretion. Recent studies demonstrated that dietary fructose
worsens colitis in mice by altering the composition, localization and metabolism
of gut microbiota through mechanisms involving GLUT5 (glucose transporter 5)
expression (fructose transporter) (García-Montero et al., 2021).
Moreover, high glucose- and high fructose-fed mice have been shown to lose gut
microbial diversity (characterized by a reduced proportion of Bacteroidetes and a
markedly increased proportion of Proteobacteria) (Do et al., 2018). Dietary sugars
that are not absorbed at the intestinal level of the host and reach the colonic environ-
ment exposed to the microbiota also regulate gut colonization by beneficial microbes.
High-sucrose diets (when comprising 70% of the kilocalories from carbohydrates,
mainly in the form of sucrose) have previously been shown to elevate Clostridiales
82 The Gut Microbiome: Bench to Table

(a class of Firmicutes) while simultaneously reducing Bacteroidales (an order of the

phylum Bacteroidetes) abundance in adult rodents, thereby promoting gut dysbiosis
(Magnusson et al., 2015). Additionally, dietary fructose intake was negatively asso-
ciated with the abundance of the bacterial species Eubacterium eligens, while it is
known that E. eligens, along with other members of the phylum Firmicutes, produce
fewer polysaccharide-degrading enzymes compared to the members of the phylum
Bacteroidetes. Moreover, dietary fructose consumption is negatively associated with
microbes belonging to the genus Streptococcus, including the species S. thermophi-
lus, with this bacterium has been shown to effectively ferment lactose and sucrose
in addition to its ability to metabolize fructose (Jones et al., 2019). Recently, a study
underlined the importance of intestinal symbiosis to prevent chronic inflammation, as
well as the fact that the intestinal microbiota needs to also be studied from an ecosys-
tem perspective instead of merely focusing on select bacterial species whose relative
abundance correlates with the inflammatory level (Barnich and Chassaing, 2021).

Oligosaccharides and polysaccharides extend several functions toward the mainte-

nance of colonic health. Raffinose, stachyose, fructooligosaccharides and soluble
fiber are shown to be beneficial to gut microflora and overall health of the colon.
More specifically, making these carbohydrates available to the gut microbiome, bac-
teria synthesize specific vitamins (biotin and K), which can be utilized by the host.
Furthermore, such feeding conditions support the generation of beneficial fatty acids
that are shown to improve colonic health, lower inflammation and reduce risk of
colon cancer (Ochoa et al., 2015; Salonen and de Vos, 2014). Two most common oli-
gosaccharides considered prebiotics as they help desirable gut microflora to flourish
are raffinose, composed of three monosaccharide units, and stachyose, composed
of five monosaccharide units, primarily found in beans and other legumes. Another
oligosaccharide group is the fructo-oligosaccharides, comprised of varying numbers
of fructose molecules, found in artichokes, onions, garlic, bananas and other plant-
based foods (Salonen and de Vos, 2014). Furthermore, in work studying the in vitro
digestion of the whole blackberry fruit, whereby bioaccessibility, bioactive variation
of active ingredients and impacts on human gut microbiota were assessed, results
showed that the phenolics were mainly released in gastric phase while carbohydrates
in small intestinal phase. The bioaccessibility for phenolics and carbohydrates was
42.80% and 69.30%, respectively, indicating most of phenolics still remain in colon
and are available for intestinal flora (Dou et al., 2022).

Unrefined grains, starchy and nonstarchy vegetables, legumes, and fruit constitute
dietary sources of a broad variety of nondigestible polysaccharides. Fiber, a collective
term for nondigestible polysaccharides divided into soluble and insoluble fibers, is not
easily digested or absorbed in the small intestine, but rather typically metabolized in
the distal colon. Cellulose is degraded by Bacteroides species, primarily associated
with plant polysaccharide degradation, or Ruminococcus, and degradation results
Microbiome and Nutrition 83

in the production of SCFAs. Abundance in Bacteroides was observed in population-

wide studies with participants on rural (significantly rich plant-based and high fiber
foods) versus Western diet. Furthermore, Prevotella populations are typically high
in humans with a high intake of plant-based complex carbohydrates. These bacte-
ria utilize xylan, common hemicellulose in human diets, via a variety of metabolic
pathways conserved in the Bacteroidetes’ phylum. Shifts in diet composition when
introducing high plant polysaccharide intake lead to increased production of SCFAs,
while by contrast Western diet has been shown to reduce Bacteroides’ levels (Salonen
and de Vos, 2014; Sandhu et al., 2016). Inulins, plant storage polysaccharides, are
extensively studied and well-established prebiotics. Fermentation of inulin and several
of its subgroups enhances expansion of Bifidobacterium and Lactobacillus popula-
tions, in the gut. Inulins are predominantly found in wheat and a variety of fruits and
vegetables including onions, bananas, asparagus and artichokes (Sandhu et al., 2016).
The amount of Bifidobacteria in feces of humans on a low-fat Western diet supple-
mented with inulin is higher than no inulin supplementation. Additionally, inulin-
supplemented diets in dextran sulfate sodium-induced colitis murine model produced
a significant increase of colonic Lactobacilli and amelioration of colitis symptoms.
In studies with infants, where mothers received inulin and galacto-oligosaccharide
supplements during pregnancy and breastfeeding, newborns were more protected
against allergies, exhibited lower histamine levels and better bowel permeability
(Sandhu et al., 2016). A different polysaccharide β-glucan, commonly found in seeds
and grains like barley and oats, is shown to reduce hyperglycemia, hyperlipidemia and
hypertension (Dawson et al., 2016).
Moreover, in a recent human study conducted in the USA, tree-based analysis
of dietary diversity captured associations between fiber intake and gut microbiota
composition in a healthy U.S. adult cohort (Kable et al., 2021). More specifically,
two to three Automated Self-Administered 24-hour Dietary Recalls (ASA24) were
collected over 10–14 d together with a single stool sample from 343 healthy adults
in a cross sectional phenotyping study. The study examined a multi-ethnic cohort
balanced for age (18y-65y), sex and BMI (18.5–45). Further, the tree structure was
annotated with the average total grams of dry weight, fat, protein, carbohydrate or
fiber from each food item reported. The alpha and beta diversity measurements,
calculated using the tree structure, were analyzed relative to the microbial com-
munity diversity determined by QIIME 2 analysis of the bacterial 16S rRNA V4
region, sequenced from stool samples. K-means clustering was used to form groups
of individuals consuming similar diets, and gut microbial communities were com-
pared among groups using DESeq2. The authors reported that alpha diversity of
diet dry weight was significantly correlated with gut microbial community alpha
diversity (r = 0.171). The correlation observed was shown improved when diet was
characterized using grams of carbohydrates (r = 0.186) or fiber (r = 0.213). The
researchers concluded that consumption of fiber from fruit robustly associated
with abundance of pectinolytic bacterial genus, Lachnospira, in the gut of healthy
adults, revealing a strong association between the ingested fiber and the gut micro-
biota profile (Kable et al., 2021).
Interestingly, as mentioned earlier, polysaccharides for intestinal microbiome
metabolism are derived from mucus as well as diet. Underlying the ability of
84 The Gut Microbiome: Bench to Table

intestinal bacteria to switch substrate utilization depends on substrate availability/

source (Albenberg and Wu, 2014). Interestingly, low-fiber diets lead to loss of micro-
biome diversity which may well extend over three or four generations. Thus, consum-
ing a fiber-rich diet constitutes a critical factor contributing to an individual’s overall
health and sustained development of a diverse healthy gut microbiome (Dawson et
al., 2016; Sandhu et al., 2016).
In both animals and humans, by way of varying the suite and amounts of sug-
ars and sweeteners consumed, we generate a new gut environment that induces in
turn the alteration of the demography of our microbial community, changes in micro-
bial metabolism and excreted metabolites, and selects for different microbial strains.
Moreover, early-life sugar consumption significantly has been shown to alter the
gut microbiome independently of obesity and total caloric intake in a rodent model
(Arnone et al., 2021).

THE GUT MICROBIOME AND FATTY ACID METABOLISM

G

Intestinal bacteria produce a variety of fatty acids which in turn can extend health-
promoting effects. Bifidobacteria in the gut generate conjugated linoleic acid, which
is shown to influence fatty acid composition in the liver and adipose tissue in mouse
models (Devaraj et al., 2013; O’Shea et al., 2012). Additionally, as discussed earlier,
intestinal bacteria produce SCFAs such as acetate, butyrate, propionate via dietary
carbohydrate fermentation nondigestible by humans. Interestingly, de novo gluco-
neogenesis or lipid synthesis can be derived from certain SCFAs including propio-
nate, hence serving as a means for fuel production and energy yielding for the host
(human). Another interesting aspect of SCFAs is that they can extend signaling
properties and, in this sense, constitute the mode by which microbes can produce
metabolic signals affecting the metabolic regulation at the host level. In fact, SCFAs
can modulate carbohydrate metabolism as well as gut physiology via the stimulation
of mammalian peptide secretion while serving as fuel for gut epithelial cells. More
specifically, SCFAs can stimulate glucagon-like peptide 1 (GLP-1) secretion via the
G-protein-coupled receptor FFAR2 (free fatty acid receptor 2) in colon (Devaraj et
al., 2013). Through the stimulation of GLP-1 secretion, bacterially produced SCFAs
stipulate signals that downregulate glucagon secretion and stimulate glucose-depen-
dent insulin secretion, while promoting glucose homeostasis.
Peptide YY, a hormone released by ileal and colonic epithelial cells in response
to feeding seemingly able to suppress appetite, is also shown to be stimulated by
SCFAs (Zhou et al., 2006). Interestingly, butyrate-supplemented high-fat diets were
demonstrated to partially improve insulin resistance in diet-induced obese mice.
Simultaneously, diets low in specific types of carbohydrates are shown to yield
reduced butyrate-producing bacteria and fecal butyrate concentrations (Gao et al.,
2009), thus lending support to the notion that the type of fuel can determine bac-
terial metabolite output and by extension ensuing signaling. Propionate promotes
G-protein-coupled receptor 41 (GPR41)-mediated activation of sympathetic neurons,
in contrast to ketone bodies, thereby affecting energy homeostasis (Kimura et al.,
Microbiome and Nutrition 85

2011). This documented potential for sympathetic outflow modulation constitutes yet
another mechanism outlining the axis of gut microbiome–enteric nervous system–
energetics–metabolic homeostasis.
Interestingly, Roux-en-Y gastric bypass (RYGB) surgery, a major bariatric inter-
vention to treat morbid obesity, is shown to have an impact on the gut microbiome.
Enhanced populations of Proteobacteria at the expense of those of Bacteroidetes were
detected post-RYGB surgery. Such microbial population shifts plausibly result in
alterations of metabolite profiles as well as relative preponderance for various fatty
acids, including SCFAs. In animal experiments, nonobese rats subjected to RYGB
exhibited reduced amounts of both Firmicutes and Bacteroidetes while exhibited sig-
nificantly increased amounts (52-fold higher concentrations) of Proteobacteria com-
pared to sham-operated counterparts (Bouillot et al., 2010). While obesity can be
viewed as a chronic mild inflammatory or at least a pro-inflammatory state, the abun-
dance of butyrate-producing Faecalibacterium prausnitzii was demonstrated to be
negatively associated with biomarkers of inflammation prior and post-RYGB surgery,
indicative of the fact that such bacterial species could be contributors to a healthy gut
(Furet et al., 2010). Therefore, GI tract surgeries could extend a significant impact on
gut microbial profile ensuing SCFA production and immunological responses.
Antibiotic administration even at the subtherapeutic level constitutes a de facto
intervention in the GI tract and can alter the microbiome characteristics and by exten-
sion metabolic function. In an in vivo study, researchers administered subtherapeutic
doses of antibiotics to mice observing increase in adiposity and levels of incretin GIP-
1. Furthermore, significantly altered taxonomy in the microbiome was seen (increased
Lachnospiraceae and Firmicutes at the expense of Bacteroidetes). Moreover, changes
in metabolism-related genes (carbohydrates to SCFAs) were observed. More specifi-
cally, increased levels of acetate, propionate and butyrate were measured as well as
changes in the regulation of hepatic lipid and cholesterol metabolism. Therefore,
alteration of gut microbiota by antibiotic administration can result in the modula-
tion of murine metabolic homeostasis. In this context, significant concerns are posed
regarding antibiotics in the food chain and how these indirect exposures of consumers
to antibiotics, especially chronically, may influence the human gut microbiome and by
extension metabolic function and disease predisposition (Cho et al., 2012).
Additional microbial by-products with important relevance in host metabolism
and health include secondary bile acids, which affect glucose homeostasis by activat-
ing receptors similar to those activated by the parent compounds (e.g., farnesoid X
receptor-α), and trimethylamine (TMA), derived by choline degradation (Cella et al.,
2021). While TMA conversion to trimethylamine N-oxide is involved in CVD and
choline deficiency is related to nonalcoholic fatty liver disease and altered glucose
metabolism, secondary bile acids have been shown to be carcinogenic, thus signifi-
cantly increasing risk for colonic tumorigenesis (Ocvirk and O’Keefe, 2021).

Saturated fatty acids were reported to enhance dysbiosis as well as intestinal inflam-
mation in IL-10-deficient mice by inducing excessive growth of the bile-resis-
tant Gram-negative bacterium Bilophila wadsworthia. In separate experiments,
86 The Gut Microbiome: Bench to Table

feeding C57BL/6J mice high-saturated fat diet significantly promoted the growth
of 3 types of sulfidogenic bacteria in the colonic mucosa. These are hydrogen sul-
fide-producing bacteria as a metabolic by-product that damages intestinal barri-
ers while causing endotoxemia. Providing C57BL/6J mice with a high-saturated fat
dietary regime downregulated expression of tight junction proteins, contributing
to induced intestinal permeability, endotoxemia as well as elevated levels of LPS-
binding protein. Additionally, to higher fecal and plasma endotoxin levels, mice fed
the aforementioned diet exhibited lower Bifidobacteria populations but increased
Enterobacteriaceae populations in fecal culture. Laugerette et al. (2012) demon-
strated a significantly increased intestinal E. coli population along with elevated
both plasma and adipose inflammation in animals fed palm oil-based diets com-
pared to counterparts fed diets with unsaturated fat. Collectively, these data suggest
that diets high in saturated fat may negatively modulate the profile and subsequently
the function of gut microbiota, contributing to increased inflammation in animal
models (Alcock and Lin, 2015).
Researchers showed that high-fat diets resulted in a significant population
reduction of dominant bacteria and groups such as Bifidobacteria, Eubacterium,
Bacteroides and rectal Clostridium coccoides, constituting gut microflora. Such
microflora alterations in turn increase the ratio of Gram-negative to Gram-positive
bacteria including an increase in plasma LPS levels. Increased adiposity, body
weight, hepatic ectopic lipid accumulation, insulin resistance and type 2 diabetes
mellitus were all also associated with the aforementioned intestinal microflora
alterations (Estadella, 2013).
LPSs cause endothelial activation by way of receptor complex consisting of
Toll-like receptor 4 (TLR4), CD14 and myeloid differentiation factor 2 (MD2).
Endothelial activation is characterized as a pro-inflammatory state during which
the expression of leukocyte adhesion molecules increases, vascular integrity is lost,
cytokine production increases and human leukocyte antigen production is upreg-
ulated (Kelleher and Sikalidis, 2021). When the GI is in an inflammatory state,
metabolic endotoxemia occurs and bacterial LPS circulation increases by two- to
threefold (Bailey and Holscher, 2018). Therefore, a diet high in saturated fat may
further encourage a state of inflammation by upregulating LPS transport, while a
diet high in fiber may decrease the proportion of Gram-negative bacteria and subse-
quently prevent endotoxemia, thus contributing to an overall healthier gut environ-
ment (Kelleher and Sikalidis, 2021).

Polyunsaturated Fatty Acids (PUFAs) – Omega-3

and -6 (ω-3 and ω-6) Fatty Acids

The role of omega 3 (ω-3) and omega 6 (ω-6) polyunsaturated fatty acids (PUFAs),
particularly omega 3, in regulating microbial metabolism by supporting optimal gut
microbial demography more so during early life is of significant interest. The omega-
class PUFAs, biosynthetic derivatives of α-linoleic acid and linolenic acid (LA) are
found mostly in fatty fish and some plant oils (Sandhu et al., 2016).
The majority of beneficial anaerobic bacteria including Roseburia, Bifidobacteria
and Lactobacillus are widely found in the colon, where PUFA metabolism from
Microbiome and Nutrition 87

linoleic acid mainly occurs. Supplementation with omega 3 to an individual’s diet

during early-life stage supports prevention of gut microbiota changes, thereby reduc-
ing the risk for metabolic disorder, as well as chronic inflammation associated with
early-life exposure to antibiotics (Sandhu et al., 2016).
Moreover, in adult mice exposed to an omega-3-supplemented diet, in utero and
early life consistently increased both Bifidobacterium and Lactobacillus, but also
yielded a higher Bifidobacteria-to-Enterobacteriaceae ratio. Omega-3 in particular is
an extensively studied PUFA because of its effects on brain function including neu-
roprotection, restoration of energy metabolism, regulation of neurotransmitter levels
and maintenance of membrane structure and composition. Notably, the Western-diet
paradigm is typically characterized by high intake of saturated fat alongside with
low omega-3 fatty acid intake. High-saturated fat intake at the expense of omega-3,
among others, may be yet another reason as to why the “Western” dietary scheme is
not generally considered particularly gut microbiome friendly.
According to a number of studies, inclusion of omega-3 PUFAs in the diet could
restore inflammatory imbalances and mitigate subsequent harmful effects on cardio-
vascular diseases and metabolic syndrome (Rousseau G, 2021). A variety of mecha-
nisms may be involved in this regard and include the following: (1) a reduction of
bacteria-producing TMA; (2) an increase in bacteria-producing butyrate, which car-
ries anti-inflammatory properties; and (3) a decrease in the production of pro-inflam-
matory cytokines. Moreover, omega-3 PUFAs could promote maintaining optimal
integrity of the intestinal barrier, thereby preventing the translocation of intestinal
contents in circulation.
The anti-inflammatory properties of omega-3 PUFA could be related to their
incorporation in cell membrane phospholipids, largely at the expense of AA (omega-6
PUFA), with the latter being well established as pro-inflammatory (Simopoulos,
2008). Other possibilities were revealed through the identification of G-protein-
coupled receptors (GPCRs) that interact with fatty acids (GPR43, GPR120). More
specifically, DHA interacts with GPR120 and could inhibit iΚB kinase as well as
the production of pro-inflammatory cytokines, for example, tumor necrosis factor-
alpha (TNFα). Docosahexaenoic acid and eicosapentaenoic acid may also inhibit
NF-κB activity by the interaction with PPARγ or interference with early events prior
to NF-κB activation (Rousseau G, 2021).
Moreover, a diet high in omega-3 PUFA is cardioprotective via a mechanism
involving Akt activation (Rondeau et al., 2011), which is involved in a critical bio-
chemical pathway component, the reperfusion injury salvage kinase. When activated
at the onset of the reperfusion, these kinases confer cardioprotection by mPTP open-
ing inhibition, while DHA can also inhibit the opening of the mPTP and result in
a reduction of infarct size although the mechanism of action still remains elusive
(Rousseau G, 2021).
Further to the direct effect of omega-3 PUFA, the metabolites involved in the
resolution phase of inflammation known as resolvins (Rv) may constitute yet another
avenue through which omega-3 PUFAs can exert cardioprotective effects to humans.
It has been reported by Tran and colleagues (Tran et al., 2014), that RvD1 adminis-
tration prior to the onset of reperfusion effectively reduced myocardial infarct size
in a porcine model (Tran et al., 2014). Additional observations confirmed that when
88 The Gut Microbiome: Bench to Table

there is inhibition of the main enzymes involved in DHA transformation to RvD1

(COX-2 and 15-LOX), plasma RvD1 concentrations are significantly reduced and
the cardioprotection is abolished (Gilbert et al., 2015). Along similar lines, Keyes
et al. demonstrated that RvE1 administration in a rat model of myocardial infarction
significantly reduces infarct size while also increases Akt and Erk activity (Keyes
et al., 2010). These observations taken together illustrate a potential role for a number
of metabolites involved in omega-3 PUFAs-related cardioprotection observed with
omega-3 PUFAs-rich diets. Another cardioprotective effect that omega-3 PUFAs and
their metabolites could induce may be associated with the impact they may exert
on the composition of the microbiota, since omega-3 PUFAs can positively alter gut
microbiota demography.

Short-Chain Fatty Acids

Typically, main metabolic products of colonic fermentation are as follows: SCFAs
and varying gaseous volumes of H2 and CO2. SCFAs are generated by plant-derived
polysaccharides, whereas they contribute approximately 5%–10% of daily energy
requirement in humans. While butyrate is used as fuel, in situ by colonocytes, propi-
onate and acetate are largely absorbed and transported to the liver, muscles and other
peripheral tissues. Acetate supports resynthesizing of lipids in the liver, whereas pro-
pionate constitutes an important substrate for gluconeogenesis (Wasielewski et al.,
2016; Thakur et al., 2014; Salonen and de Vos, 2014).
Diets rich in fiber have been shown to increase the production of SCFAs by the
intestinal microbiota. SCFAs are very important metabolic products in the mainte-
nance of the intestinal barrier, with physiological concentrations having provable effects
on intestinal barrier function. Certain types of bacterial families such as Bacteroides,
Bifidobacterium, Propionibacterium, Eubacterium, Lactobacillus, Clostridium,
Roseburia and Prevotella produce SCFAs. SCFAs in turn can function as neurohor-
monal signaling molecules that can cross the BBB transported by monocarboxylate
transporters. Imaging studies demonstrated that microbiota-derived acetate can cross
the BBB where it subsequently affects hypothalamic gene expression. Butyrate has been
demonstrated to influence expression of tight junction proteins (claudin-2, occludin, cin-
gulin, and zonula occludens proteins, ZO-1, ZO-2), while it is shown to be able to reduce
bacterial translocation and extend antitumorigenic properties (Kelly et al., 2015).
Furthermore, SCFAs constitute a critical energy source for both colonic and ileal
cells while they further affect the intestinal epithelial barrier and defense functions
by regulating relevant gene expression and influencing inflammatory status of the
gut. Moreover, SCFAs regulate functions of innate immune cells contributing to the
immune systematic responses, such as macrophages, neutrophils and dendritic cells.
Additionally, SCFAs can extend regulation over the differentiation of T cells and B
cells as well as the antigen-specific adaptive immunity mediated by the former. Also,
SCFAs constitute potential precursors as in raw materials for glucose and lipid syn-
thesis, which stipulates a theoretical framework for examining the potential role of
SCFAs regarding the regulation of energy homeostasis and metabolism. Moreover,
there are also studies showing that SCFAs inhibit tumor cell proliferation and pro-
mote apoptosis (Yao et al., 2022).
Microbiome and Nutrition 89

More specifically, Yang and colleagues (2020) demonstrated that microbiota-

derived SCFAs promote interleukin 22 (IL-22), a member of the IL-10 family,
central to host protection against inflammatory insult in the intestine by induc-
ing antimicrobial peptides and promoting epithelial barrier function. Conducting
in vivo work, WT B6 mice were administrated with or without 200 mM butyrate
in drinking water for 3 weeks, and IL-22 was shown to be produced by CD4+ T
cells and ILCs through G-protein receptor 41 (GPR41) and inhibit histone deacety-
lase. The authors reported that SCFAs upregulate IL-22 production by promoting
aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1α (HIF1α) expres-
sion, which are differentially regulated by mTOR and Stat3. Mechanistically, they
showed that HIF1α binds directly to the Il22 promoter, and SCFAs increase HIF1α
binding to the Il22 promoter through histone modification. Hence, SCFA supple-
mentation enhances IL-22 production, which protects intestines from inflammation,
and SCFAs promote human CD4+ T-cell IL-22 production. Their reported findings
reveal significant roles for SCFAs in inducing IL-22 production in CD4+ T cells and
ILCs to maintain intestinal homeostasis and protect against inflammation while
optimizing immune responses.

THE GUT MICROBIOME AND AMINO ACID METABOLISM

Desirable gut bacteria like Bifidobacteria and Lactobacilli produce amino acid–derived
bioactive compounds, including several biogenic amines. Amino acids, di-, tri-peptides
and a few oligo-peptides from the diet as well as to a lesser degree partially hydrolized
proteins reach the colon and ultimately constitute sources yielding substrates for lumi-
nal bioconversion via the gut microbiome. Various microbiome-produced enzymes
contribute to amino acid metabolism by generating bioactive metabolites in the intes-
tine. A typical class of that type of enzymes is amino acid decarboxylases, widely
prevalent in gut microbes. These microbial enzymes, when combined with amino acid
transport systems, link dietary compounds with microbial metabolism and signal-
ing with the gut mucosa. Such an example of the gut microbiome is the bacterium
Lactobacillus reuteri, which can convert dietary L-histidine, to an immunoregulatory
signaling molecule, histamine, which in turn suppresses pro-inflammatory TNF pro-
duction via histamine type 2 receptors in the intestinal epithelium. Another example
of microbe-facilitated amino acid metabolism is the generation of γ-amino butyric
acid from glutamate, by glutamate decarboxylase (Devaraj et al., 2013), while another
is the production of putrescine from ornithine. Such anti-inflammatory amino acid
metabolites derived from microbial metabolism utilizing amino acids as substrates
may improve pathologies related to obesity, systemic inflammation and type 2 diabetes
mellitus status.
In an interesting in vivo study involving Sprague–Dawley rats, Li and colleagues
investigated the effect of protein feeding to the gut microbiome of rats using soybean
protein (Spro), soybean protein-derived peptides (SPre) and casein as feeding regimes
(Li et al., 2021). The cecum microflora composition of the rats was determined via
16S rDNA amplicon sequencing, showing that the SPro along with its derived pep-
tides significantly increased the abundance and uniformity of the gut microbiota after
35 days of feeding. The authors reported that Firmicutes/Bacteroidetes (F/B) ratios of
90 The Gut Microbiome: Bench to Table

the SPep, SPro and casein groups were 2.49 ± 0.60, 2.98 ± 1.12 and 2.59 ± 0.74, respec-
tively. Interestingly, while the rats fed SPro and SPep exhibited similar gut microbi-
ome profiles, SPep significantly promoted Lactobacillus and Phascolarctobacterium
growth. Their results indicated that SPep significantly increased the diversity of the
gut microbiota and elevated the probiotic portion of the bacterial gut population. The
authors concluded that both SPro and SPep were able to modify the demography of
gut microbiota in rats, with the effect of SPep being more desirable from a health
standpoint as judged by F/B ratio (Li et al., 2021).
In another study, Butteiger et al. demonstrated that soy protein compared to
milk protein in a Western-diet regime increases gut microbial diversity and reduces
serum lipids in golden Syrian hamsters (Butteiger et al. 2016). More specifically,
32, 6- to 8-week-old male golden Syrian hamsters were fed a Western diet contain-
ing 22% (%wt) milk protein isolate (MPI) as the single protein source for 3 week
followed by 6 week of one of four diets containing either of the following [22%
protein (%wt)]: MPI, soy protein concentrate, partially hydrolyzed soy protein
isolate (SPI1) or intact soy protein isolate. Serum lipids, hepatic gene expression
and gut microbial populations were evaluated. Sequencing of the 16S ribosomal
RNA gene revealed greater microbial diversity in each soy-fed group than in the
MPI-fed group (P < 0.05). The authors concluded that dietary protein sources in
male golden Syrian hamsters fed a Western diet differ in terms of how they affect
the gut microbiota with soy protein producing a favorable gut microbiome profile
while also potentially promoting the reduction of lipogenesis through alterations
of the gut microbial community (Butteiger et al. 2016). These in vivo studies lend
support to the notion that plant-based/-derived protein may constitute a dietary
approach that could extend health benefits, in addition to other shown aspects such
as N-balance with lower risk for allergies, also relative to the gut microbiome,
while also promoting sustainability and an eco-friendly approach to agriculture
and food production.

THE MICROBIOME AND WEIGHT CHANGE

Overnutrition leading to obesity is associated with a cluster of metabolic and sys-
temic disorders such as insulin resistance, type 2 diabetes mellitus, fatty liver dis-
ease, atherosclerosis and hypertension. Alarmingly, obesity rates have more than
doubled in the last 2 decades globally. A growing body of evidence suggests that
the gut microbiota represents an important factor contributing to energetics and by
extension to over-/undernutrition. A pivotal study by Turnbaugh et al. (2006) was
among the first studies to demonstrate that gut microbiota contributes to obesity. The
microbiome obtained from the colon of genetically obese leptin-deficient mice
(ob/ob) was compared against that of lean littermates. Researchers reported that
ob/ob mice microbiota included genes encoding enzymes that hydrolyze indigestible
dietary polysaccharides. Higher amounts of fermentation endproducts (i.e., acetate
and butyrate) and lower caloric content were identified in the obese mice fecal matter
compared to lean counterparts. The results imply that gut microbiota in the obese
mice induced extraction of additional calories from the diet by means of metabolic
Microbiome and Nutrition 91

rearrangement, concluding that gut microbiome profile appears important for body
weight regulation (Ley et al., 2006). Interestingly, when gut microbiota of either
ob/ob mice or lean mice was transplanted to lean gnotobiotic mice, in mere 2 weeks,
mice that received microbiota from the ob/ob mice extracted more calories from
diet, while demonstrating significantly higher adiposity compared to mice receiving
the microbiota of lean mice. The results obtained sustenance a critical part of gut
microbiome regarding the pathogenesis of obesity and by extension obesity-related
disorders. In human experiments, Ley and colleagues (2006) as well as Ravussin
and colleagues (2011) successively examined fecal gut microbiota of 12 obese par-
ticipants who followed an annual weight loss program by engaging in a fat-restricted
or a carbohydrate-restricted, but always low-calorie diet. Analogous to similar in
vivo experiments, abundance of gut bacteria of the Bacteroidetes and Firmicutes
phyla was detected, while the microbiome exhibited noteworthy intraindividual
consistency over time. Prior to the low-calorie diet initiation, increased populations
of Firmicutes at the expense of those of Bacteroidetes were observed in the obese
individuals compared to nonobese controls. After weight loss, higher populations of
Bacteroidetes (3%–15%) with decreased populations of Firmicutes were seen. More
interestingly, the changes observed correlated quantitatively with weight loss rather
than dietary caloric content changes in terms of percentages.
In terms of definite dietary schemes, a recent study demonstrated that the
Mediterranean diet can positively affect resting metabolic rate as well as salivary
microbiota in humans subjects (Daniele et al., 2021). More specifically, salivary
microbiota composition and metabolic profile were analyzed in participants with
vegan (VEG) or Mediterranean (MED) long-term dietary patterns. The MED par-
ticipants demonstrated significantly higher percentages of Subflava and Prevotella
bacterial species as compared to VEG participants. Moreover, the authors reported
that MED participants showed higher basal metabolic rate (BMR) and lower respi-
ratory quotient (RQ). Furthermore, Prevotella abundance was demonstrated to be
inversely correlated with RQ and carbohydrate consumption (which was seen to
be lower in the MED participants), whereas Subflava percentages were demon-
strated to be positively correlated to BMR. Lactobacillus abundance, which was
inversely related to Subflava presence in MED participants, was associated with
decreased BMR (Harris-Benedict) values. Taken together, these observations
demonstrate positive effects of the Mediterranean diet on BMR and on the abun-
dance of microbial species associated with a better macronutrient metabolism
(Daniele et al., 2021).
The Mediterranean diet is becoming increasingly more popular due to growing
evidence regarding the role it can play in chronic disease prevention and immune
response modulation. Characterized by a diet rich in fruits, vegetables, fish, olive
oil, red wine and refined cereal products, the foods that are part of Mediterranean
diet are typically rich in fiber, omega-3 fatty acids, polyphenols, SCFAs, PUFAs,
phytosterols and antioxidants. Together, these bioactive compounds collectively and
synergistically extend anti-inflammatory and antioxidative properties, which may
be needed in counteracting accumulation of TMA N-oxides and the endotoxin LPS
(Kelleher and Sikalidis, 2021).
92 The Gut Microbiome: Bench to Table

FUTURE PERSPECTIVES
Whereas the link between gut microbiome, food and health is becoming increas-
ingly clearer, researchers are struggling to successfully and effectively manipulate
the microbiome as a form of treatment. Even though we have gained more knowledge
and understanding than ever before as to how the microbiome influences chronic
disease, it remains elusive as to how to change a person’s microbiome in a particular
favorable direction. There appears to be a clear need to assess how delivery and/or
interactive systems in the gut’s interface can be employed to affect bacterial type
and population size in the gut (Kristo et al., 2015) promoting optimal microbiome
demography, including but not solely limited to dietary schemes (Sikalidis, 2019). It
is clearly important to consider nutrition/diet perspectives as related to implications
with clinical nutrition and medical nutrition therapy. Beyond the studied relationship
between the microbiome and the obesity, further, there is significant evidence to
suggest that there are strong associations between the demographics/quality of the
microbiome and risk toward chronic diseases such as T2DM and ensuing CVD, as
well as inflammation and related pathologies (Sikalidis and Maykish, 2020), extend-
ing through the gut–brain axis to potential roles in Alzheimer’s as well as other
neurodegenerative diseases (Shukla et al., 2021).
After the successful completion of the human genome project approximately
two decades ago, Relman and Falkow urged the scientific community to embark
on a “second human genome project”, a large-scale genomic survey of our endog-
enous microflora (Relman and Falkow, 2001). Since then, the field of microbiome
research has greatly expanded, leading to large-scale efforts such as the NIH, HMP
and the Earth Microbiome Project. Those efforts alongside a significant increase in
the interest and ensuing research of the microbiome generated a significant body of
literature aiming to shed light on this human–microbiome symbiotic relationship.
There is ample evidence that microbiomes are networked and interconnected primar-
ily by means of the flows of mobile genetic elements (MGEs) (Haraoui, 2022). The
diversity, demography and profile of those MGEs have significantly expanded over
the past decades responsive to continuously growing selective pressures primarily
due to several anthropogenic forces applied. A microbial ontology more attuned to
these phenomena considering the aforementioned process could potentially provide a
more effective theoretical framework toward studying the microbiome and revealing
potential ways to utilize its plasticity for the benefit of human health.

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 4 Models for Researching
the Gut Microbiome
Alison Lacombe and Vivian C.H. Wu
United States Department of Agriculture

Aleksandra S. Kristo
California Polytechnic State University

CONTENTS
Introduction...............................................................................................................97
Computational Models.............................................................................................. 98
In Vitro Models........................................................................................................ 100
Fecal Batch Cultures.......................................................................................... 100
Cell Culture........................................................................................................ 100
Ex Vivo Models................................................................................................... 101
3D Cell Culture.................................................................................................. 102
Microfluidics Systems and the “gut chip”.......................................................... 102
Simulator of the Human Intestinal Microbial Ecosystem (SHIME)....................... 103
Animal Models for Studying Gut Microbiota......................................................... 105
Insect Models..................................................................................................... 105
Avian Models..................................................................................................... 107
Murine Models................................................................................................... 107
Humanized Gnotobiotic Mice............................................................................ 109
Porcine Models.................................................................................................. 110
Zebrafish Model................................................................................................. 110
Fecal Microbiome Translocation Models............................................................... 111
Conclusion.............................................................................................................. 113
References............................................................................................................... 113

INTRODUCTION
Models are used to study gastrointestinal health to determine the impact of a
treatment, critical time points for administration, and potential mechanisms that drive
efficacy. For many studies pertaining to probiotics and prebiotics, complex microbio-
logical and metabolomics assays serve as proxy for microbiota function (Mabwi et al.,
2021). A “snapshot” of the gut microbiota composition and function can provide valu-
able insights into what factors alter compositional and metabolic changes subsequent
to dietary interventions (Mabwi et al., 2021). However, investigating discreet time
points only offers limited information regarding the dynamics of compositional and

DOI: 10.1201/b22970-6 97
98 The Gut Microbiome: Bench to Table

metabolic changes. Therefore, models to investigate alterations in gut function incurred

by dietary changes must be placed in the proper context of a defined research question.
Most research on the gut microbiota utilizes clinical studies or animal models.
Models attempt to address the intrinsic difficulty in accessing the human intestine
to study its microbial composition, metabolite and enzyme production, and inflam-
matory processes (Mabwi et al., 2021). However, studies with humans and animal
models can be limited by the lack of homogeneity with regard to the target popula-
tion. Due to their technical reproducibility, easy access, and monitoring, in vitro and
in silico models represent a valid alternative to study the microbial behavior of the
human gut microbiota. In previous chapters, we have discussed the recent advances
in high-throughput sequencing of the gut microbiome and the general impact of
nutrition on species profile composition. In this chapter, we will be providing a brief
overview of the models utilized to study the interactions between diet and gut micro-
biota. The intestinal microbiota has been studied in a variety of in vitro, in vivo, and
ex vivo models, each presenting distinct benefits and drawbacks, while all models can
provide useful insights into the mechanism of human digestion of prebiotics.

COMPUTATIONAL MODELS
Mathematical models can determine the statistical relationship between dietary
interventions and gastrointestinal health; however, these experiments are subject to
the assumptions used to build the models (Lamichhane, Sen, Dickens, Orešič, &
Bertram, 2018). “Snapshots” of the gut metagenome or metabolome often fail to
provide sufficient information on periodic patterns, interdependencies, or temporal
variations present within the microbiome (Lamichhane et al., 2018). Longitudinal
analysis can elucidate the changes over time and offer better context to the health
impacts of dietary interventions; however, this approach presents its own unique set
of challenges (Mabwi et al., 2021). Longitudinal studies in human populations are
often complicated by limited time points, sample dropout, and uneven sampling fre-
quency (Mabwi et al., 2021). Therefore, other models, such as animals or cell culture,
are often employed for complex computational analysis.
Interactions in a microbial community are driven by several mechanisms includ-
ing competition for nutrients, direct interactions between community members, and
interactions with the host and attachment sites in the host environment (Mabwi et al.,
2021). This network of bacterial interactions can be altered by the host’s age, diet,
disease initiation, or other exogenous perturbations (Mabwi et al., 2021). The combi-
nations of the correct models and computational tools are needed to infer ecological
relationships of microbiota and its associated metabolic functionality.
Temporal networks that depict how a connected system evolves with time have
emerged as useful tools for analyzing the gut microbiome. There are numerous uni-
variate and multivariate analytical methods that can test the significance of dietary
intervention at discrete time points or within a time series (Mabwi et al., 2021). These
methods have been applied to both metataxonomic and metabolomic temporal data.
For longitudinal studies where specific microbial features (e.g., taxa) are differen-
tially abundant, data smoothing, such as spline ANOVA, can discrete time points
into a polynomial curve (Mabwi et al., 2021). This analysis can estimate a difference
Models for Gut Microbiome 99

in function in the context of time with differential abundance. With the integration
of the difference function over a time interval, a statistical divergence in the data
set can be detected. Tools such as MetaDprof and MetaLonDA demonstrated the
specificity and sensitivity needed for detecting time periods of differentially abun-
dant features (Mabwi et al., 2021). Most notably, MetaLonDA can handle uneven
sample sizes and time intervals, which are common in human subject studies. For
example, using inputs from the DIABIMMUNE project, MetaLonDA found signifi-
cant differences between the Finnish and Russian cohorts in the relative abundances
of Bacteroides and Bifidobacterium species during the first year of life (Mabwi et
al., 2021). Additionally, MetaLonDA showed that Bacteroides is significantly more
abundant from day 96 to day 584 in Finnish infants, while Bifidobacterium is sig-
nificantly more abundant in Russian infants from day 96 to day 720 (Markowiak &
Ślizewska, 2017). This type of computational analysis can inform the treatment of
several gut-driven immune disorders often seen in children and provide valuable
regional population context.
Compared to taxonomic profiling and metagenomic data, there have been fewer
published works on metabolomic data. In dietary studies, the purpose of analyz-
ing metabolomic data is to determine whether two or more metabolite profiles are
significantly different. For a comparison of time series data from two experimental
groups, Hoteling-T2 statistic can be used as a measure of holistic differences (Chong
et al., 2019). When these studies employ multiple groups or levels of treatments,
multivariate empirical Bayes analysis of variance may be used to test for signifi-
cance (Chen et al., 2020). In cases where the specific metabolite might be unknown
or is overly complex to analyze, significant discriminatory biomarkers are used in
multivariate analysis (Park, Ufondu, Lee, & Jayaraman, 2020a). Linear mixed mod-
els offer an attractive option to investigate the longitudinal trend of metabolites and
identify metabolites that show significant concentration changes over time (Mabwi
et al., 2021). One promising technique is dynamic probabilistic principle coordi-
nate analysis, which models the correlations in multivariate data that occur due to
repeated measurements in time (Mabwi et al., 2021).
Integration of complex computation into microbiome studies is important. Even
with longitudinal studies, there is often a mismatch between microbial community
dynamics and sampling frequency. Most in vivo studies collect samples infrequently,
because of practical limitations, over days or weeks, whereas microbial community
dynamics in the gut microbiota can significantly vary within hours (Park, Ufondu,
Lee, & Jayaraman, 2020b). One possible solution is to use studies in parallel with
computational models to simulate the microbiome or metabolome dynamics to iden-
tify optimal sample collection (Park et al., 2020b). Computational and statistical
methods can rigorously address the challenges of microbiome temporal data analy-
sis. For mechanistic studies, a promising collection of software tools has become
available to support the analysis of large “omics” data sets from longitudinal studies
(Park et al., 2020b). There is no one size fits all regarding a standardized process for
reporting and comparing outcomes from different analyses. As a result, the choice
of a data analysis tool often relies on trial and error. In this regard, open-source plat-
forms that provide options to conduct multiple analyses on consistently formatted
and normalized data sets would be a powerful resource for elucidating diet.
100 The Gut Microbiome: Bench to Table

IN
In vitro models constitute an opportunity to study the gut microbiota in cases that an
in vivo study would be considered conceptually not feasible or unethical. In essence,
in vitro gut models function as chemostats, that is, bioreactors inoculated with fecal
microbiota and operated under physiological temperature, pH, and anaerobic condi-
tions (Ahmadi et al., 2019). Experimentation within these models is very flexible
with regards to studying multiple permutations/combinations of dietary ingredients
and their impact on gut microbial populations (Ahmadi et al., 2019). Ultimately,
cumulative in vitro studies can lead to robust in vivo approaches to investigating gut
microbiota functionality (Litten-Brown, Corson, & Clarke, 2010; Nguyen, Vieira-
Silva, Liston, & Raes, 2015; Ray & Dittel, 2015). In theory, each study yields com-
plementary results that strengthen the overall validity of each individual model and
distinguishes between the functionality of gut microbes and human processes.

Many of the microbes that reside within the gut are not cultivable using traditional
microbiological techniques such as agar Petri plates (Nagalingam, Kao, & Young,
2011). Preliminary studies of the human microbiome typically start with obtaining
donor specimens and culturing the microbes under anaerobic conditions in a biore-
actor (Ahmadi et al., 2019). The bioreactor setup can be as simple as an Erlenmeyer
flask inside a CO2 incubator on a stir plate or a complex system of pumps and valves
meant to closely simulate the physical system of the digestive tract (Ahmadi et al.,
2019). The complexity of the bioreactor setup is usually dictated by the question the
investigator is attempting to answer. For example, in determining if a potential food
ingredient is bifidogenic, investigators can quantify the presence of Bifidobacteria
using agar and simple biochemical tests. However, such setup cannot reveal the
ingredient’s effect on other important bacterial species or potential effects in human
physiology (Ahmadi et al., 2019; Vrieze et al., 2013a). For a comprehensive in vitro
or ex vivo analysis of microbiome shift with different dietary interventions, metage-
nomic analysis is still needed. For preliminary studies of the interactions of the
gut microbiome and specific nutrients, a video protocol can be found at www.jove.
com/v/59524/an-vitro-batch-culture-model-to-estimate-effects-interventional. This
protocol details a comprehensive and cost-effective way to use a bioreactor for study-
ing the compositional shift that occurs in a fecal sample following dietary amend-
ments (“An In Vitro Batch-culture Model to Estimate the Effects of Interventional
Regimens on Human Fecal Microbiota|Protocol,” n.d.).

In vitro human cell models are typically composed of polarized monolayers of single
or co-cultured carcinoma cells (Saygili, Dogan-Gurbuz, Yesil-Celiktas, & Draz, 2020).
These cell lines are distinguished based on their physiological properties and ability to
differentiate (Pearce et al., 2018). The intestinal epithelial consists of several cell types,
including enterocytes, goblet cells, stem cells, enteroendocrine cells, Tuft cells, M cells,
Models for Gut Microbiome 101

and Paneth cells, all of which can interact or sense the presence of the gut microbiota
and respond accordingly (Pearce et al., 2018). Caco-2 and mucus-secreting HT29-MTX
cell lines predominate the literature with regards to the study of probiotics and prebiot-
ics (Pearce et al., 2018). There are important considerations in selecting a cell culture
model, and decisions should be guided based on the research question asked.
Caco-2 cell line, derived from colorectal adenocarcinomas, is one of the oldest and
most utilized cell cultures in medical research (Pearce et al., 2018). This cell line can
function as an undifferentiated large intestinal cell or can spontaneously differenti-
ate into a cell closely resembling a small intestinal enterocyte (Ahmadi et al., 2012).
This ability to spontaneously differentiate is difficult to control and presents chal-
lenges in the reproduction of scientific studies, despite the fact that these Caco-2 cells
are widely used to study the protective effects of probiotics and prebiotics (Pearce
et al., 2018). Additionally, Caco-2 cells are commonly used in immunological studies
since they can express important pattern recognition receptors, Toll-like receptors,
which can provide information about the relative abundance of inflammatory mark-
ers known to contribute to the disease process (Payne et al., 2012).
HT-29 is another commonly utilized, polarized, and undifferentiated human
colorectal adenocarcinoma cell line (Paul et al., 2018). Recently, several subpopula-
tions have been selected to exhibit more “enterocyte-like” features, but in general,
spontaneous differentiation is less common compared to Caco-2 (Payne et al., 2012).
HT-29 cell lines have been extensively studied in the field of nutrition and host–
microbiome interactions because they produce important inflammatory cytokines,
such as interleukins and TNF-α (De Simone et al., 2015; Khan Mirzaei et al., 2016).
In addition, HT-29 provides an important benefit compared to Caco-2 in that HT-29
cells contain mucus-producing goblet cells. The ability to produce mucus is critical
for predicting immune function in the intestinal brush border and studying bacte-
rial–host interactions (Raja et al., 2012; Altamimi et al., 2016).
Prebiotic oligosaccharides have been shown to positively modulate both Caco-2
and HT-29 cells, and exhibit reduced inflammatory activity (Bode, 2009; Kelly,
2008). For example, the HT-29-MTX cell line, treated with methotrexate to produce
more mucin, has been a useful tool to show that oligosaccharides and a functional
mucin layer can reduce bacterial adhesion to the epithelium (Altamimi et al., 2016).
Studies with HT-29-MTX demonstrated that prebiotics can reduce the possibility of
an infection by reducing pathogens’ adhesion, migration, and internalization, includ-
ing Salmonella and E. coli. Co-cultures of Caco-2 and HT-29 render cell culture mod-
els more physiologically and functionally relevant, by combing the benefits of mucin
secretion and differentiation (Altamimi et al., 2016). In co-cultures of HT-29 Caco-2,
anti-adhesive effects of Streptococcus thermophilus and Lactobacillus acidophilus
against enteroinvasive E. coli were observed (Resta-Lenert and Barrett, 2003).

Ex ViVo models
Ex vivo models are models cultured outside of an organism, while closely mimic
functional live tissue systems and the complex cellular environments found in vivo
(Roeselers et al., 2013). These extended models are used to mimic the intestinal tract
in more detail by using advanced tissue engineering approaches. Ex vivo model
102 The Gut Microbiome: Bench to Table

systems can delve deeper into the complexity of the microbiome–host environment
and can probe the mechanism by which certain dietary components can improve
health outcomes (Roeselers et al., 2013).

3d cell culture
Modern 3D printing techniques allow for the creation of complex biological tissue
(Saygili et al., 2020). Bioprinting simultaneously combines living cells and biomateri-
als through a computer-aided design program to create 3D bioengineered living con-
structs that mimic natural tissue characteristics, including organs. This microscale
fabrication is commonly used for tissue engineering, regenerative medicine, micro-
biology, and the study of the gut microbiota (Saygili et al., 2020). In 3D bioprinting,
there are three major groups of techniques commonly used: layer-by-layer (stereo-
lithographic), line-by-line (extrusion-based), and droplet-based bioprinting (Saygili
et al., 2020). Extrusion-based bioprinting is the most common and affordable 3D
printing and creates complex, multilayered scaffolds and tissue constructs in bio-
medical applications. The stereolithography utilizes a photopolymerization process
in which UV light creates a pattern over a photopolymerizable liquid polymer, cross-
linking the light-sensitive polymers into a hardened layer (Patel, Lee, Park, Kim,
& Jeong, 2018). Droplet-based bioprinting utilizes multiple mechanisms to deposit
droplets of cell suspension in a high-throughput manner (Saygili et al., 2020). The
materials fed through the printer are typically hydrogels that have similar viscos-
elastic properties to that of biological material (Patel et al., 2018). Hydrogel-based
bio-ink materials typically have short-termed stability, the ability to retain nutrients,
and controlled cross-linking to facilitate bioprinter deposition (Patel et al., 2018).
The development of 3D tissue systems allowed for the modeling of host–bacte-
rial microbiome interactions in an organoid environment that closely resembles the
intestinal tract (Saygili et al., 2020). Organoid models for the gastrointestinal tract
were first developed in 2009 and have evolved into two distinct types, adult stem cell
(ASC) and pluripotent stem cell (PSC) derivatives (Saygili et al., 2020). ASC- derived
intestinal organoids are limited and consist only of basic epithelial cells such as pari-
etal cells, chief cells, and surface pit cells (Saygili et al., 2020). In comparison, PSC
has the capacity to differentiate into three germ layers (ectoderm, mesoderm, and
endoderm), mesenchymal cells, including smooth muscle cells, fibroblasts, and myo-
fibroblasts. However, to date, the cultivation of environmental cells, such as neural
cells, immune cells, and endothelial cells, is extremely difficult (Patel et al., 2018).
Without the environmental context of these other systems, organoids are prevented
from properly recapitulating the features of the actual organs designed to represent.

M
Recent advances in cell culture 3D printing and microfluidics have facilitated the
development of lab-on-a-chip systems that can be used to study the gut microbi-
ota. Gut chip and microfluidic systems typically house channels, chambers, sen-
sors, electrodes, and valves that allow for cellular growth media and air exchange
(Ashammakhi et al., 2020). Most microfluidic models of the gut involve an array of
Models for Gut Microbiome 103

microchannels that are separated by a porous and flexible membrane that allows for
nutrient exchange and elimination of waste, a design intended to simulate the bar-
rier between the intestinal lumen and the draining vasculature. The target cells are
seeded into channels and allowed to adhere, and then media is pumped through the
channels to simulate the microenvironment found in vivo (Ashammakhi et al., 2020).
Multiple cell types have been used within gut-on-a-chip devices depending on the
proposed research question. The most commonly used cell type is Caco-2 cells due
to their ease in culturing and capacity to differentiate in 3D cell culture, generating
an intestinal structure (von Martels et al., 2017). The presence of fluid flow to provide
biomimetic shear stress to cells is critical to this development, with the peristalsis-like
strain on cells inducing morphogenesis into 3D intestinal villi. However, the use of
Caco-2 if severely is limited by their lack of capacity to produce a significant mucosal
layer (von Martels et al., 2017). Therefore, co-culture with HT-29 cells and other types
of cells is an attractive option for many studies. In another gut model studying the
remodeling of extracellular matrix, Caco-2 cells were co-cultured with subepithelial
myofibroblasts to generate a full-thickness model of the intestine (Ashammakhi et al.,
2020). Co-cultures in 3D system can go beyond typical cell monolayers through the
formation of the villous structures, and the expression of tight junctions, presentation
of a brush layer, and production of mucous (von Martels et al., 2017).
The advent of gut chip technologies has allowed for the study of host–micro-
biota interaction, circumventing the use of costly animal models. A model micro-
fluidic environment consists of two compartments separated, one containing mixed
microbiota and the other enterocytes (Marzorati et al., 2014). To recreate physiologi-
cally relevant GIT conditions in order to study the gut microbiota, the following are
needed: (a) a mucosal area under shear stress for bacterial adherence; (b) transport
of low molecular weight metabolites; and (c) microaerophilic conditions. With this
model, it is possible to simulate bacterial adhesion and the indirect effect on cells.
Compared to traditional cell cultures, the constant nutrient cycling of microfluid-
ics systems allows for observations over longer periods with a lower risk of con-
tamination (Ashammakhi et al., 2020). Recent studies examined the intercellular
cross talk between Caco-2BBE cells and peripheral blood mononuclear cells in
response to challenges with dextran sulfate sodium (DSS), lipopolysaccharide (LPS)
endotoxin, probiotic VSL#3, and non-pathogenic E. coli (von Martels et al., 2017).
This system facilitated the isolation of each component of the signaling cascade to
identify the root cause of the inflammatory response. The gut epithelium disruption
with LPS activated the immune component of the model allowing for elucidation of
dysfunctional pathways, potentially not revealed by using complex animal models
(Ashammakhi et al., 2020).

SIMULATOR OF THE HUMAN INTESTINAL

MICROBIAL ECOSYSTEM (SHIME)
The SHIME was developed in 1993 as a multicompartment dynamic simulator of the
human gut (Molly et al., 1993). The development of SHIME® addresses the limita-
tions that in vivo studies have capturing colon microbiota in terms of community
composition and metabolic activity by adding some of the physical and mechanical
104 The Gut Microbiome: Bench to Table

aspects of digestion to the study design (Van De Wiele, Van Den Abbeele, Ossieur,
Possemiers, & Marzorati, 2015.). Simple fecal batch culture studies only utilize sin-
gle-stage chemostats to mimic colon conditions. These designs are often limited for
diet studies because environmental parameters such as pH, redox potential, available
nutrients, and microbial population dynamics constantly change throughout tran-
sit. To simulate in vivo conditions while still taking advantage of in vitro designs,
semicontinuous fermenters were developed where the intermittent supplementation
of nutritional medium (Van De Wiele et al., 2015). In contrast, fecal batch culture
studies that use one single fermenter ignore the heterogeneity in substrate availabil-
ity, fermentation activity, microbial composition, and other intrinsic characteristics.
The SHIME system is intended to mimic the microbiota, and the initial inoculum
originates from a donor’s fecal sample. Fecal samples capture a microbial com-
munity and metabolic shifts during transit from the proximal colon to the rectum.
It is important to consider that the fecal microbiome is significantly different from
the in vivo colon microbiome, both in terms of composition and metabolic activity
(Van De Wiele et al., n.d.). With that in mind, the colon is where the microbiome is
the most accessible sample for an individual intestinal community. The purpose of
the SHIME system is to adapt the fecal microbiome to the conditions present in the
different colon compartments (Van De Wiele et al., n.d.). The SHIME is typically
inoculated with fecal material sourced from individual donors, despite research due
to pool samples from several people. Pooling samples can partially account for the
interindividual variability that exists in microbiome composition and incorporates
properties from different enterotypes. Since there is enormous functional redun-
dancy of the gut microbiome, such pooled microbiome and the adaption process of
SHIME will shift the fermentation profile, not a strictly necessary step. However,
because SHIME models adapt the fecal microbiome to the simulated environment
of the colon, it often fails to capture function nuances. For example, the metabolism
of polyphenols, such as daidzein, isoxanthohumol, and catechins, is highly depen-
dent on an individual’s microbiome (van Duynhoven et al., 2011).
The mucosal microbiome is a crucial part of the gut microbial ecosystem because
of its proximity to host epithelial cells. It is thought to have an intrinsically higher
impact on human health because of the immunological activity that occurs (Van den
Abbeele et al. 2011). The mucosal microbiome is different from the luminal microbi-
ome in composition and, interestingly, the presence of important mucosal colonizers
such as Faecalibacterium prausnitzii (Willing et al. 2009). Access to the mucosal
environment in vivo is extremely difficult, and the development of gut simulators that
accurately mimic mucosal microbial colonization is needed. The SHIME can mimic
some of the mucosal microbial colonization by incorporation of mucin and other
compounds in which to establish a biofilm. One result of mucosal SHIME experi-
ments was the higher colonization rate of butyrate-producing Clostridium clusters IV
and XIVa (Van De Wiele et al., n.d.). This phylogenetic group is considered crucial
for delivering butyrate as a primary energy source to colonocytes and improves gut
barrier function by strengthening the tight junctions (Van De Wiele et al., n.d.).
This modular nature of SHIME is useful for diet studies and investigating the
microbiome’s ability to produce a bioactive metabolite (Possemiers et al. 2006). The
microbiome behavior in response to dietary inputs can be investigated by comparing
Models for Gut Microbiome 105

upper digestive tract to the colon. The residence times of the different gastric compart-
ments, the composition of the gastric juices, region-specific pH values, feed, feeding
regimes, and body temperatures are adapted in the SHIME setup leading to a simula-
tion of the targeted human or animal host. Finally, other features of the SHIME include
the gradual emptying of the gastric digest into the intestine compartment. The option of
running a dynamic pH profile in the gastric compartment enables the running experi-
ments with real food matrices or food constituents that need to undergo predigestion
and removal of sugar monomers or amino acids and peptides before the digest is trans-
ferred advancing to the colon compartment (Van De Wiele et al., n.d.).

ANIMAL MODELS FOR STUDYING GUT MICROBIOTA

As the microbiota of the human gut is gaining increasing research attention, the need
for the development of models for non-human microbiota studies is significant. There
are several possibilities for in vivo models utilized in microbiota research spanning
from mice and other rodents such as guinea pigs to primates, zebrafish, pigs, and
dogs (Nguyen et al., 2015). Most studies are using rodent models, with murine being
the most common, porcine, and zebrafish. In the following section of this chapter,
these commonly used models are discussed with particular emphasis on the murine
models being the most widely used currently.

Insects are the most diverse class of animals regarding species numbers and biomass
(Muñoz-Benavent, Pérez-Cobas, García-Ferris, Moya, & Latorre, 2021). The micro-
biota of insects, especially the gut microbiota, is as complex and rich as the phylogeny
and ecology of insects. Most insect species have endosymbiotic relationships with a spe-
cialized gut microbiota that contributes to health status and important nutritional roles
(Muñoz-Benavent et al., 2021). The insect intestine is inhabited by microorganisms
from all domains, including bacteria, fungi, archaea, protozoa, and viruses (Muñoz-
Benavent et al., 2021). As a result of their co-evolutionary histories with the host, these
microorganisms play essential roles in host physiology. Insect symbiotic systems are
either ectosymbiotic when the symbiont is on the surface of the host or endosymbiotic
when it lives inside specialized host cells (Muñoz-Benavent et al., 2021). For example,
termites gut endosymbiotic microbiota enables them to feed on a wood diet due to its
unique enzymatic capabilities. Cockroaches possess bacteriocytes, specialized cells
in the fat body of the insect, and an abundant and varied intestinal microbiota, whose
function is not fully understood (Muñoz-Benavent et al., 2021).
The combination of microorganisms populating the insect gut is driven by
several evolutionary and ecological factors, including phylogeny, diet, life stage,
and host environment (Muñoz-Benavent et al., 2021). Proteobacteria, Firmicutes,
Bacteroidetes, Actinobacteria, and Tenericutes are the most common phyla insect
guts, and the distribution of these phyla varies greatly among insect species. Diet is
a major driver of the gut microbiota composition in animals. Gene sequencing has
revealed the influence of diet on the gut microbiota of insects (Muñoz-Benavent
et al., 2021). With termites, there are major distinctions in the gut microbiota
106 The Gut Microbiome: Bench to Table

between dry-wood, damp-wood, and subterranean environments. A comparative

study including 18 species of higher termites identified the diet as a significant
factor of bacterial community structure in the termite gut using amplicon librar-
ies of 16S rRNA genes (Muñoz-Benavent et al., 2021). Lower termites have a
diet based on lignocellulose that is digested by symbiotic flagellates, which are
absent in higher termites and cockroaches (its closest relative) (Muñoz-Benavent
et al., 2021). However, in wood feeding, higher termites’ bacterial species from
Spirochaetes and Fibrobacteres phyla are responsible for digesting their diet [20].
The analysis of metagenomes from the hindgut microbiota of a wood-feeding
higher termite showed a large and diverse set of bacterial genes for cellulose and
xylan hydrolysis (Muñoz-Benavent et al., 2021). In addition, H2 metabolism, CO2-
reductive acetogenesis, and N2 fixation functions were identified as putative func-
tions of the microbiome (Muñoz-Benavent et al., 2021).
Drosophila melanogaster, the common house fly, is a universal model for genetic
and genomic studies and is used as a human disease model (Douglas, 2018). The fly
genome includes orthologs for many human disease genes. The Drosophila research
community has developed powerful resources to facilitate translation between the
fly and human disease including the Human Disease Model Report in FlyBase
(http://flybase.org/) (Douglas, 2018). Programs like FlyBase focus on the genetic
mechanisms of drug–microbiome interactions in Drosophila to the treatment of
human disease (Douglas, 2018). The Drosophila microbiome is dominated by two
groups of taxa: gut microorganisms localized to the gut lumen and endosymbionts,
which are transmitted with high fidelity from mother to offspring via the oocyte
(Douglas, 2018). Endosymbionts, which humans lack, can have substantial effects
on the metabolic and immune phenotype of flies (Douglas, 2018). Therefore, the use
of endosymbiont-free Drosophila strains is recommended for the study of drug–gut
microbiome interactions.
The intimate relationship between Drosophila and its food adds an important
dimension to microbiome interactions. The gut microbiome of laboratory Drosophila
is generally dominated by bacteria, usually Acetobacteraceae (a-proteo-bacteria)
and Lactobacillales (Firmicutes) (Douglas, 2018). Drosophila diets with high sugar
content favor Acetobacteraceae, while lactobacilli are favored by Drosophila diets
dominated by complex carbohydrates like cornmeal-based diets (Douglas, 2018).
The capacity of microorganisms to persist and proliferate in the Drosophila gut
varies among microbial isolates. Many of the bacteria that inhabit Drosophila
exploit the mobile fly as a vector for transmission among fruits at different stages of
decay (Douglas, 2018).
Genomic sequencing analysis demonstrated that diet contributed not only to dif-
ferential gut microbiota diversity but also to a distinct colonization resistance capac-
ity against pathogens. Another field of interest in medicine is the study of antibiotic
resistance genes and their transmission in bacteria through insects. The gut micro-
biota plays a crucial role in developing and maintaining the insect immune system,
so studying these communities is essential to better understand the pathogens they
carry and transmit or even find ways to prevent them. For example, the gut micro-
biota of the mosquitoes can prevent them from becoming infected with Plasmodium,
the malaria parasite (Muñoz-Benavent et al., 2021).
Models for Gut Microbiome 107

Birds represent a vertebrate class that plays a major role in natural ecosystems and
is with diverse gut microbiomes (Hird, Grond, Poulsen, & Jønsson, 2021). As with
most vertebrate species, the microbiome of birds is influenced to varying degrees by
host physiology and genetics, ecology and behavior, the environment, and diet (Hird
et al., 2021). Avian microbiomes are extremely diverse, and there is little similarity
between gut microbial community and host phylogeny. Low levels of host specificity
and high variability of the gut microbiome may result from the effects of a highly
diverse diet. Functional insights into avian gut bacteria stem mainly from poultry
studies, where gut microbes aid digestion, synthesize essential molecules for the host
(Hird et al., 2021), and interact with the immune system during microbiome estab-
lishment and development (Hird et al., 2021). However, poultry are unlikely to be
representative of all bird species. Nevertheless, the application of insights and meth-
ods developed for poultry may have implications for human diet and disease studies.
Birds represent important reservoirs and carriers of both animal and human
pathogens as well as antimicrobial resistance genes (Tellez et al., 2001). Bird micro-
biomes can reflect active infection status, and noninvasive methods exist to collect
fecal samples for migration and population-monitoring programs (Tellez et al., 2001).
Avian social behaviors impact the dynamics and assembly of gut bacterial communi-
ties (Tellez et al., 2001). Studies on social contact during breeding have yielded some
insights into the effects of family interactions on microbiomes. Group living may
also increase pathogen transfer among individuals who may select for diverse and
stable microbiomes that are more resilient to pathogen invasion, and such advantages
may be greater in social than in solitary species (Tellez et al., 2001).
Apart from chickens, few studies have investigated the cross talk between avian
immune systems and gut microbes, including gut symbiont responses to microbial
infections. In wild ducks, gut microbiome composition is significantly correlated with
influenza virus infections (Navarro-gonzalez et al., 2020), and in ostriches, coloniza-
tion of harmful bacteria can lead to dysbiosis and even death (Navarro-gonzalez et al.,
2020). Environmental microbial diversity can also impact both immune functions and
cloacal microbiomes of hosts (Zimmer-Faust, Steele, Griffith, & Schiff, 2020). Such
pattern-based and experimental manipulation of infections provides an initial foun-
dation to identify defensive microbial symbionts (Zimmer-Faust et al., 2020). These
insights can then be used to establish more precisely how microbes combat pathogens.

Rodents, mice, in particular, have become the most broadly and commonly used
model of choice for most studies in this emerging field. While both healthy human
and murine gut microbiota are dominated by the same two phyla (i.e., Bacteroidetes
and Firmicutes), studies by Ley et al. have demonstrated that approximately 85% of
the bacterial genera found in mice are not observed in humans under normal healthy
conditions (Ley et al., 2006). While similarities between the murine and human con-
dition have been observed, studies with mice have indicated changes in abundance
of bacterial phylotypes. In mice bacteria such as Tenericutes and Enterobacteriaceae
108 The Gut Microbiome: Bench to Table

(Nagalingam et al., 2011), explained at least to some extent by differential meth-

odological approaches (16S rRNA vs. stool sample analyses) as per the assessment
of the microbiota between humans and (Nguyen et al., 2015). Regardless of the
approach, a certain degree of differentiation is always seen.
Another question, as per the suitability of murine models for studying human
gut microbiome responses, is the extent to which murine gut microbiota is modi-
fied in response to various metabolic conditions and diseases in a similar manner
to that reported in the human gut. For example, studies investigating obesity and
the gut microbiota have shown a significant overlap in terms of responses between
humans and mice. Moreover, genome-wide association studies of obesity in mice
have revealed that genes associated with obesity overlap with some genes in human
obesity (Nguyen et al., 2015). A plethora of studies have been conducted on mice or
humanized mouse models (i.e., germ-free (GF) mice administered human gut micro-
biota), in which animals were fed diets high in fat or saturated/unsaturated fat to
investigate changes in the gut microbiota (Le Chatelier et al., 2013). Furthermore,
there is a similar change in the Bacteroidetes/Firmicutes (B/F) ratio in ob/ob obese
mice and obese humans (Murphy et al., 2010).
The development of inflammatory bowel disease (IBD) has been linked to the
composition of gut microbiota, among other factors (Ray & Dittel, 2015). A sig-
nificant challenge in assessing the alignment and translatability of findings from
murine models to human IBD patients is that IBD is essentially a cluster of dis-
eases, further distinguished by stages of activity. Various mouse models have been
developed in efforts to mimic the human pathophysiology of IBD by manipulating
the murine genome, chemical induction of IBD as well as pathogen-driven models.
While genetic models for IBD are a promising practice, targeted genes are often
involved in multiple pathways, thus potentially confounding the conclusions derived
based on the association between gut microbiota and function in the disease state.
For example, none of the approximately 60 colitis murine models mimic accurately
the human condition (Peloquin & Nguyen, 2013). Overall, obesity and IBD appear to
be quite differently translatable from murine models to humans, illustrating concerns
in the accuracy and validity of conclusions.
Interestingly, a noticeable parallel exists between dominant bacterial families of
the mouse and human enterotypes. Namely, one mouse enterotype is dominated by
Lachnospiraceae/Ruminococcaceae, similar to the human Ruminococcaceae entero-
type (also known as enterotype 3). Additionally, the second mouse enterotype, domi-
nated by Bacteroidaceae/Enterobacteriaceae, is similar to the human Bacteroides
enterotype (enterotype 1) (Arumugam et al., 2011). Interestingly, two enterotypes
were also identified in wild mice, dominated by Bacteroides and Robinsoniella,
respectively (Wang et al., 2016). Moreover, the laboratory mouse enterotypes were
found to correlate with species richness and inflammation, that is, mice belonging to
the low species-richness enterotype (Bacteroidaceae/Enterobacteriaceae) demon-
strated significantly higher levels of calprotectin, a biomarker of inflammation. This
result is consistent with studies of human obesity (Le Chatelier et al., 2013), in which
low species-richness individuals were found to have more pronounced inflammation,
with microbiota dominated by Bacteroidetes and Proteobacteria, the same bacterial
groups that dominated the high inflammation mouse enterotype.
Models for Gut Microbiome 109

Overall, these observations underline clear differences at the level of specific

genus/species abundances between the murine and human gut microbiota while
simultaneously indicating that overall community composition, as well as the driving
factors, might be similar (e.g., enterotypes). Although absolute comparisons might be
challenging and not straightforward, murine models are likely relevant for studying
the processes responsible for microbiota variation and shifts upon perturbations.
Murine models permit interventions for studying the causal role of gut microbiota
in health and disease, not otherwise attainable in humans. The use of murine models
generates comprehensive knowledge of mouse genetics and thus extends the avail-
ability of numerous genetically modified mouse models more than any other model.
Other advantages include the relatively low cost of maintenance, high reproductive
rate, and short life cycle, all of which increase the efficiency and efficacy of experi-
mental designs using mice. Mice are omnivorous mammals, with gut physiology
and anatomy comparable to that of humans. Moreover, murine microbiota models
allow for specificity in targeting genes/pathways in the complex gut microbiota–host
interactions by employing knockout models. Mouse models are inbred, providing a
homogenous genetic background, a cleaner system to dissect signals from gut–bacte-
ria–host interactions and improve the reproducibility of experiments with high power
in relatively low numbers of animals per group. Sources of variations such as diets
and housing conditions are generally controlled for in experiments, limiting unde-
sired background influence to gut microbiota.
Despite important similarities, with mice being different from humans in anat-
omy, genetics, and physiology, their use cannot fully recapitulate human systems. In
this regard, different mouse models can give rise to diverging shifts in gut microbiota
composition. Cross talk between gut microbiota and the host can be largely host-
specific; hence, observations in mice may be of limited translatability to humans.
Genetic homogeneity while positive in terms of reducing “genetic noise” also implies
that the inbred mouse strains cannot capture the inherent genetic variations naturally
occurring in the human population. Multiple factors, such as genetic background,
birth mode of delivery (caesarean versus vaginal), mode of feeding (breast ver-
sus bottle), diet, medical history, and social activities, all contribute to shaping the
“actual” gut microbiota in humans. The absence of these factors in mice implies that
gut microbiota in murine models cannot closely reflect a “real-life” gut microbiota.

Humanized gnotobiotic mice are produced by the inoculation of a human gut micro-
biota sample in GF mice. This murine model constitutes a powerful tool for gut
microbiota studies capturing a large part of the human gut microbiota phylogenetic
composition (100% of phyla, 11/12 classes, and ~88% of genus-level taxa) (Nguyen
et al., 2015). This approach has been widely employed in a plethora of studies since
it allows perturbations in a “human-like system” and is widely regarded as the gold
standard for confirming associations and trying to prove causality in gut microbiota
research (Faith, McNulty, Rey, & Gordon, 2011; Goodman et al., 2011).
Nevertheless, it must be emphasized that host–microbe relationships in human-
ized mouse models do not necessarily reflect the entire relationship spectrum seen
110 The Gut Microbiome: Bench to Table

in humans since the gut microbiota transplanted into a host (mouse) has not co-
evolved with the recipient. It has been reported that certain resident bacterial taxa
in the human gut microbiota are absent in the humanized mouse gut microbiota
(Turnbaugh et al., 2009).
Despite their limitations, humanized mice constitute one of the very few methods
to assess causality in microbiota research, and therefore, further development and
improvement of this approach is important.

Porcine Models
The porcine gut microbiota is increasingly and more rigorously studied due to the
scale of porcine husbandry industry, but also due to the similarities in anatomy,
physiology, and immunology to the human gastrointestinal tract (Litten-Brown
et al., 2010). An elegant and in-depth study of swine gut microbiota composition in
the Yorkshire pig breed demonstrated that human and pig microbiota shared similar
diversity patterns, with the two dominant phyla being Bacteroidetes and Firmicutes
(Lamendella, Santo Domingo, Ghosh, Martinson, & Oerther, 2011). However, at the
genus level, the swine gut microbiota harbors more Spirochaetes and Prevotella than
the human gut microbiota (Lamendella et al., 2011).
Another porcine model promising for microbiota research is the miniature pig.
Generally, miniature pigs develop obesity when fed ad libitum and are thus used as an
obesity and metabolic syndrome model. Specifically, the gut microbiota composition
of two miniature pig breeds, Gottingen and Ossabaw, was investigated for responses
to obesity induction (Meier & Bode, 2013; Pedersen et al., 2013), demonstrating that
the major phyla of miniature pig gut microbiota are Firmicutes and Bacteroidetes.
Interestingly, the two miniature pig breeds responded differently to an obesity-
inducing diet: Ossabaw gut microbiota displayed more of the characteristics of a
“healthy” obese microbiota, while Gottingen gut microbiota demonstrated alterations
analogous to metabolic syndromes, such as those found in gut microbiota profiles of
type 2 diabetic mice.

Zebrafish Model
Zebrafish is an attractive model used extensively for gut microbiota research due to
its small size, high fecundity, and full annotation of its genome. Given that several
gut functions and immune genes are conserved between zebrafish and mammals,
the zebrafish is an interesting model organism to investigate fundamental processes
underlying intestinal inflammation and injury (Brugman, 2016). As revealed by
genomic profiling, the zebrafish gut microbiota shares six bacterial divisions with
mice and five with humans, although marked differences exist in the relative abun-
dance of these phyla (Zhao et al., 2017). The microbiota of laboratory-reared zebraf-
ish intestine is dominated by Proteobacteria, while Firmicutes and Bacteroidetes
dominate in mice and humans (Rawls et al., 2004). Despite differences in the compo-
sition of their microbiota, the responses of zebrafish and mammals to microbial colo-
nization are similar. As an example, microarray analysis comparing the digestive
tracts of GF versus conventionally reared zebrafish revealed differential expression
Models for Gut Microbiome 111

of over 200 genes, of which nearly one-third were conserved in mice (Rawls et al.,
2004). The majority of these genes can be linked to epithelial proliferation, nutrient
metabolism, and immune responses. In terms of utilization of the zebrafish model,
Aryas-Jayo and colleagues investigated the effects of a high-fat diet (HFD) over a
period of 25 days on intestinal microbiota and inflammation in zebrafish. The con-
sumption of the HFD resulted in microbial dysbiosis, characterized by an increase in
the relative abundance of the phylum Bacteroidetes (Aryas-Jayo et al., 2018).

FECAL MICROBIOME TRANSLOCATION MODELS

The bidirectional relationships between fecal microbiota and human health have
been considered the next frontier for microbiome research and the amelioration of
disease (Wu et al., 2019). Recent studies demonstrated an association between intes-
tinal microbiota composition and human disease; however, direct causality remains
to be proven. The application of fecal microbiome translocation (FMT) is the closest
example of a causal relationship between gut microbiota composition and a resultant
cure of several disease states (Chong et al., 2019). Randomized controlled double-
blind trials have provided valuable insight into implementing FMT that might be
serving as a future diagnostic and therapy in human disease.
In clinical practice, FMT from healthy donors is applied in the treatment of spe-
cific dysbiosis-related diseases. The principle of FMT involves restoration of the
colonic microflora by introducing healthy bacterial flora through the infusion of
stool from a healthy donor. Clostridioides difficile is a disorder of the intestinal
flora often the result of antibiotics and microorganisms that do not respond to anti-
microbial therapy (Vrieze et al., 2013b). Fecal translocation demonstrated efficacy
against C. difficile infection and performed better than vancomycin. The overall
efficacy of FMT is an >90% reduction of C. difficile, and it is influenced by various
factors depending on the donor, transfer method, and colonic environment (Vrieze
et al., 2013a). The overall efficacy of FMT can be increased with multiple infusions,
higher initial dosages, and improved delivery, with a higher success rate if FMT is
performed by colonoscopy (Ianiro et al., 2018). New scientific studies have reported
that in patients suffering from C. difficile infection, FMT is followed immediately
by a repairing effect and disappearing symptoms of the disease, which relates to
normalization of the disturbed microbiota (CDC, 2020). As C. difficile infection
becomes common, FMT research has increased in prominence and has been used
experimentally to treat other gastrointestinal diseases including colitis, constipa-
tion, irritable bowel disease, and some neurological conditions such as Parkinson’s
disease (Vrieze et al., 2013c).
Many authors investigated the potential role of restoring eubiosis with FMT
among IBS patients’ community to obtain combined effects on intestinal and men-
tal disturbances. Microbiota analysis shows that, after FMT in C. difficile patients,
there was an increase in the number of the Bacteroidetes, Clostridium clusters IV
and XIVa, Faecalibacterium prausnitzii, Butyrivibrio crossotus, Enterococcus,
Lactobacillus, Veillonella, and a decrease in pathogenic bacteria (Antushevich,
2020a). A recent clinical trial demonstrated, in patients administrated FMT for
3 months, lower improvement of clinical symptoms, namely swelling of the liver,
112 The Gut Microbiome: Bench to Table

disappearance of abdominal pain syndrome, and normalization of the frequency

of defecation (Antushevich, 2020a). Fecal translocation may also be a promising
therapy for ulcerative colitis, and clinical trials demonstrated a decrease in diar-
rhea, hematochezia, and improved clinical scores (Antushevich, 2020a). Murine
models of dextran sulfate sodium (DSS)-induced colitis found that FMT reduces
the levels of TNF-α, IL-1β, and MPO activity and increases the level of IL-10 in the
colon (Antushevich, 2020a). Reduction of intestinal inflammation in DSS-induced
colitis mice was also observed (Antushevich, 2020a). In FMT group of rats, the
authors observed reducing the colonic expression of the pro-inflammatory cytokine
genes and bacterial antigens, decreasing weight loss, and increasing length of the
colon and antimicrobial peptides and mucins secretion (Vrieze et al., 2013c). Also,
an increase of Lactobacillaceae and Bifidobacteriaceae in the gut was observed
(Vrieze et al., 2013c).
It is important to consider the heterogeneity of fecal samples in the human popu-
lation and the collection of fecal specimen protocols. Fecal collection, transporta-
tion condition, storage status, and DNA extraction method all have some impacts
on sample quality (Vrieze et al., 2013a). Methodology studies are helpful for inves-
tigators to build more reasonable protocols for the fecal sampling and handling
process. Although knowledge and evidence were accumulating to contribute to the
quality control of fecal sampling of microbiome study, there remain some gaps
and challenges (Settanni, Ianiro, Bibbò, Cammarota, & Gasbarrini, 2021a). For
example, it might be debated whether the anaerobic condition should be maintained
for sample collection since the MetaHIT protocol contains an anaerobic bag in the
fecal collection bottle, but the HMP did not (Settanni, Ianiro, Bibbò, Cammarota, &
Gasbarrini, 2021b). Another controversy is whether to include the homogenization
process before aliquoting since the microbial population from different sampling
sites in poop can be quite divergent (Antushevich, 2020b). If homogenization is not
performed, how much biomass in a sample is enough to be representative for a com-
plete microbial population in a poop may become a concern. Besides, since most
of the comparative methodology studies were currently done at 16S DNA amplicon
analysis level, we just begin to learn the optimal condition for shotgun metagenome
study (Niederwerder, 2018).
Although it can be relatively simple to perform, cost challenges need to be
overcome before this procedure is widely accepted in mainstream clinical prac-
tice. Most of the solutions to these challenges already exist, but some need further
optimization and testing. Standardized fecal microbiota is being developed as a
therapeutic agent, although it clearly challenges some of the existing paradigms
of drug development, delivery, and regulation. The dietary record is an important
component for gut microbiome study for its potential to provide environmental
vectors to explain the compositional results of a relevant gut microbiome analy-
sis (Niederwerder, 2018). Food frequency questionnaire (FFQ) and 24 h dietary
record are useful tools for dietary surveillance and have been successfully applied
for microbiome studies (Vrieze et al., 2013c). However, it is worth noting that the
validity of FFQ and dietary record recalls relying heavily on the completeness
of the food composition database that may conventionally only include common
macro- and micronutrients.
Models for Gut Microbiome 113

CONCLUSION
To elucidate causal relationships between prebiotics, probiotics, and gastrointestinal
health, multiple investigative approaches are needed that combine appropriate mod-
els with sensitive methods for data analysis. Modern approaches include in vitro,
in vivo, and ex vivo models, along with metagenomic and metabolomic analysis of
the community composition, its functional repertoire, and the by-products produced.
This information is often used to determine the dominant set of taxa that govern the
dynamics of multiple metabolic functions at the community level. In addition, infor-
mation can be revealed about the dynamics of the microbiome and the correlation
between specific taxa’s abundance with a metabolic function. However, due to the
complexity of the human body, it is rare that causation can be unequivocally estab-
lished using only one model.

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 5 Dietary Modulation
of Gut Microbiota by
Cultured Products
Salam A. Ibrahim and Rabin Gyawali
North Carolina A&T State University

Raphael D. Ayivi
North Carolina A&T State University
University of North Carolina Greensboro

Hafize Fidan
University of Food Technologies

Saeed Paidari
Islamic Azad University

Reza V. Bakhshayesh
Agricultural Biotechnology Research Institute of Iran
University of Tabriz

CONTENTS
Introduction ............................................................................................................ 120
Biotherapeutic Properties of Cholesterol-Lowering Probiotics ............................. 121
Concept of Probiotics ........................................................................................ 121
Probiotic Microorganism................................................................................... 122
Important Criteria in Selecting Probiotics......................................................... 122
Synthesis and Metabolism of Plasma Lipoprotein ............................................ 124
Cholesterol-Lowering of Probiotics .................................................................. 125
Cholesterol-Lowering Effect Mechanisms........................................................ 126
Decongesting Bile Salts Using Bile Salt Hydrolyzing Enzyme........................ 126
Cholesterol Deposition with Conjugated Bile Salt ........................................... 128
Cholesterol Integration into Bacterial Cell Walls ......................................... 128
Lowering Serum Cholesterol by Probiotics ................................................. 129
Binding of Cholesterol ................................................................................. 130
Other Mechanisms........................................................................................ 131
Relationship between Nutrition and Gut Microbiota ................................... 132

DOI: 10.1201/b22970-7 119

120 The Gut Microbiome: Bench to Table

Role of the Gut Microbiota in Human Health and

Disease Prevention ....................................................................................... 134
Cardiovascular Disease ................................................................................ 135
Cultured Food Products..................................................................................... 140
Role of Cultured Products in Gut Microbiota Modulation .................................... 140
Yogurt ................................................................................................................ 140
Milk Kefir .......................................................................................................... 143
Probiotic Fermented Milk ................................................................................. 143
Role of Cultured Beverages in Gut Microbiota Modulation ............................. 144
Role of Fermented Vegetables in Gut Microbiota Modulation ......................... 145
Effects of Phenolic Compounds on Gut Microbiota .............................................. 145
Conclusion ............................................................................................................. 147
References .............................................................................................................. 147

INTRODUCTION
Proper diet and nutrition are fundamentally important for human health and meta-
bolic development and have been advocated by various stakeholders in the quest to
decrease the surging rate of health and metabolic diseases. More specifically, the
human gut microbiota has been associated with the proper regulation and functional-
ity of the human metabolic and immune system (Gentile & Weir, 2018). As a result of
the important role of the gut microbiota in combatting diseases and promoting health
and wellness, it is critical for the modulatory functionality of the gut microbiota to
be adequately supported in its functionality. Cultured food products, probiotics, and
fermented functional foods have all been proposed as conventional approaches to
naturally boosting the modulatory effect of the gut microbiota. Cultured food prod-
ucts allude to all fermented dairy or milk products or fermented food beverages with
lactic acid bacteria as a key culturing agent (Ayivi et al., 2020). Moreover, the grow-
ing demand by consumers and dietary health experts to adopt cleaner and greener
strategies in enhancing the human immune system warrants rapid attention and a
paradigm shift away from chemical supplements that could pose a human health
risk coupled with long-term side effects. The interplay between diet, nutrition, and
probiotic consumption results in a synergistic effect on the gut microbiota that boosts
the immune system in its fight against attacks from foreign bodies, pathogens, and
disease-causing organisms (Yang et al., 2020). Probiotics have been shown to pos-
sess a high therapeutic effect on human health. Moreover, studies have confirmed the
immunomodulatory properties of probiotics and diet on the gut microbiota through
the promotion of beneficial bacteria and the suppression of deleterious microor-
ganisms (Ibrahim et al., 2021). However, there is little available research that has
addressed the role of cultured products and diet together and their contingent effect
on the modulation of the gut microbiota.
This chapter thus embraces a nutraceutical concept and elucidates the promising
and potential role of diet, probiotics, and nutrition in the modulatory action of the gut
microbiota. We will also discuss the role of various factors such as cultured products,
probiotics, and fermented functional foods in gastrointestinal (GI) disease prevention,
Dietary Modulation of Gut Microbiota 121

cholesterol regulation, lactose intolerance (LI) mediation, and some cardiovascular

disease (CVD) prevention strategies. In addition, we wish to promote a comprehen-
sive background of pertinent information regarding the ways in which diet, nutrition,
and cultured probiotic food products could immensely enhance human health by
promoting the modulatory action of the gut microbiota.

BIOTHERAPEUTIC PROPERTIES OF
CHOLESTEROL-LOWERING PROBIOTICS
C

The commensal gut microbiome is fundamentally salient in immunological and meta-

bolic activities, inhibiting the development of unwanted microbes through antimicro-
bial agent production or direct competition for binding sites and resources. Probiotics,
as defined by the World Health Organization (WHO), are living and nonpathogenic
microbial supplements that, when administered in sufficient amounts to the host
organism, confer health benefits by increasing gut health and intestinal mucosal integ-
rity (FAO & WHO, 2002). ‘Probiotic’ is a Greek term meaning ‘for life’; however,
the definition has evolved over the years. The evolution of the probiotic term over the
years has significantly been associated with the increasing interest in the use of viable
bacterial supplements and the progress made in understanding their mechanisms of
action. Originally, the phrase referred to secretory compounds from specific bacteria
that were beneficial in the development of other species. Later, the probiotic term was
associated with extracts of tissue that increased microbial development, as well as
animal feed additives that improved intestinal microbiota (Fuller, 1999).
Probiotics improve gut health, improve intestinal mucosal integrity, and sup-
press potentially pathogenic microorganisms through competition for nutrients
and space for gut adherence, antihypertensive effects, reduction in allergic symp-
toms, cancer prevention, mineral absorption facilitation, cholesterol-lowering
effects, arthritis amelioration, and dermis symptoms. In 2013, the global market
for probiotics was USD 32.06 billion. In 2015, the value of the probiotics market
was estimated at USD 33.19 billion. By 2020, it was expected to reach USD 46.55
billion, USD 64.02 billion by 2022 (Byakika, Mukisa, Byaruhanga, & Muyanja,
2019). The probiotic market thus has promising growth potential. This market
is projected to further develop due to rising global consumer health awareness,
fueled by the usefulness of probiotics in preventing and treating a variety of health
issues, in addition to the soaring global demand for functional food products
(El-Saadony et al., 2021). These developments in recent years have influenced the
rising demand and consumption of functional foods, as many individuals are now
more interested in disease prevention than cure. The positive impact of a probi-
otic on its host is proportional to its concentration in the intestinal lumen, which
must be at least 107 CFU/g of fecal material. For the host organism to adequately
experience the efficiency of the probiotic, the product should have a minimum of
10 6 CFU/g or mL, and a total of 108 to 109 probiotic bacteria should be taken daily
(Kechagia et al., 2013).
122 The Gut Microbiome: Bench to Table

Probiotic microorganism
Lactobacillus and Bifidobacterium are considered as the most widely and commonly
used probiotic microorganisms nowadays (Nami et al., 2018). In addition, other
probiotic strains that have gained prominence and consumer acceptance include
Lactococcus (Shehata, El-Sahn, El Sohaimy, & Youssef, 2019), Streptococcus
(Shastri et al., 2020), Enterococcus (Nami et al., 2019), Bacillus (Lim, Oh, Yu, &
Kim, 2021), Saccharomyces (Rodríguez-Nogales et al., 2018), Propionibacterium
(Nair & Kollanoor Johny, 2018), Leuconostoc (Le & Yang, 2019), Weissella (Yadav,
Yadav, Singh, Singh, & Mani, 2019), or Escherichia coli originating from the GIT
(Behrouzi et al., 2020) or clostridia (Liu, Xie, Wan, & Deng, 2020) (Table 5.1).
Although pathogenic strains such as Bacillus anthracis and Bacillus cereus are nota-
ble in the Bacillus family, their toxicity is strain-dependent, and some strains do not
create enterotoxins, thus making them suitable for use as probiotics.

Probiotic applications in animal food and feed are also on the rise, but this must be
done with caution because the strain can spread from animal to human. Consequently,
stringent standards for probiotic safety and quality must be implemented and

TABLE 5.1
Some Known Lactic Acid and Probiotic Bacteria
Probiotic Microorganisms
Lactobacillus Bifidobacterium Other Lactic Acid
spp. spp. Bacteria Nonlactic Acid Bacteria
L. acidophilus B. adolescentis Enterococcus faecalis Bacillus cereus var. to yoi
B. coagulans GBI-30
L. casei B. animalis E. faecium E. coli strain nissle
L. crispatus B. bifidum Lactococcus lactis Propionibacterium freudenreichii
L. acidophilus B. breve Leuconostoc mesenteroides Saccharomyces cerevisiae
L. gallinarum B. infantis Pediococcus acidilactici S. boulardii
L. gasseri B. adolescentis Sporolactobacillus inulinus
L. johnsonii B. animalis Streptococcus thermophilus
L. paracasei B. bifidum Weissella paramesenteroides
L. plantarum B. breve
L. reuteri B. lactis
L. rhamnosus B. longum
L. brevis
L. fermentum
L. salivarius
L. helveticus
Dietary Modulation of Gut Microbiota 123

adhere to global requirements commencing with strain selection, manufacturing

and labeling, and ending with postmarket surveillance of adverse effects (Byakika
et al., 2019). According to several reports, some probiotics can change into oppor-
tunistic pathogens, causing illnesses including sepsis and pneumonia (Antoun,
Hattab, Akhrass, & Hamilton, 2020). Before a probiotic strain is approved for
entry into the consumer market, extra attention should be paid to many adverse
qualities such as gene transfer, translocation, the creation of toxic metabolites,
and immunomodulation. Even though probiotics (fermented foods) have long been
consumed by humans, their safety requires special care, as novel strains from
unusual sources and genetically engineered probiotic strains are currently in vari-
ous phases of commercialization.
The introduction of new probiotic strains generally consists of the isolation and
selection of appropriate strains from a diverse microbial population. This is done
by searching a set of cell banks, separating traditional dairy products, or isolating
healthy humans and animals. First, the isolates are isolated by culturing them in
vitro, after which the best probiotic strains are identified by simple in vitro methods
by creating conditions such as GI tract and intestinal epithelium (Vasiljevic & Shah,
2008). The features of probiotics are always strain-specific and cannot be general-
ized to other strains. The criteria of probiotic selection and validation should satisfy
key requirements and possess certain qualities that have been integral in probiotic
experiment investigations. The choice of probiotics requires well-established and
important criteria as follows (FAO & WHO, 2002):

Apart from adhesion to mucus and/or human epithelial cells and cell lines, the prin-
cipal tests described above are identical to the Indian Council of Medical Research
and Department of Biotechnology (ICMR-DBT) criteria for probiotic strain in vitro
characterization (Ganguly et al., 2011). The Joint FAO/WHO Working Group advises
at a minimum the following screening tests in terms of organism safety (FAO &
WHO, 2002):
124 The Gut Microbiome: Bench to Table

The following assays for microbiological safety evaluation are recommended by the
ICMR-DBT guidelines (Ganguly et al., 2011):

Nonetheless, the significance of these metrics is still debatable due to issues of rel-
evance, in vivo and in vitro inconsistencies, and a lack of uniformity of operating
protocols. Because no single criterion is required for all probiotic uses, the best
way to determine a strain’s qualities is to conduct studies on a specific popula-
tion and physiologic function. Evidence supporting a strain’s probiotic efficacy
should thus be disseminated in medical journals or as peer-reviewed scientific
work, according to the Joint FAO/WHO Working Group. Negative results also add
to the overall body of evidence supporting probiotic efficacy and should likewise
be published.

Two important organs in the body, the stomach and the liver, are responsible for
lipoprotein production. The liver synthesizes bile which is then transported to the
gallbladder and utilized there prior to being transported to the intestine via a cystic
duct. When a fatty meal reaches the small intestine, bile salts assist in the emulsifica-
tion of the fats. This allows the fatty meal to be digested and absorbed in the intes-
tines. Next, in the epithelial cells of the gut, fatty acids, triglycerides, and cholesterol
mix and are covered by a layer of protein, called chylomicrons (Kaplan, Pesce, &
Kazmierczak, 2003). A system of lymphatic cells then absorbs the chylomicrons
after which they are released into the bloodstream. Chylomicrons are thus converted
to cholesterol and triglycerides upon reaching the liver. It is noteworthy that lipids
are immiscible with the bile salts in the intestines. Lipids descend to the ileum, where
absorption of a greater proportion of the bile salts occurs and then reabsorbed into
the bloodstream. The liver, therefore, is responsible for the recirculation of returned
bile salts that stay in the gallbladder, together with bile, to be employed in the afore-
mentioned process once again. The small intestine, however, does not absorb all of
the bile salts, and some of them move into the colon, where they are discarded along
with feces. By manufacturing bile salts from its cholesterol store, the liver compen-
sates for the loss of bile salts. In addition to dietary sources of cholesterol, liver cells
produce cholesterol and are thus another important source of the body’s cholesterol
pool. A variety of factors, including heredity and food, influence the liver’s ability to
generate cholesterol.
Dietary Modulation of Gut Microbiota 125

Hypercholesterolemia is closely linked to the occurrence of ischemic heart disease

in both men and women. A slight reduction in serum cholesterol of 1% can lead
to a 2%–3% reduction in heart attacks. In previous years, the only treatment was
the use of lipid-lowering drugs such as statins, which prevent the synthesis of cho-
lesterol. However, not only is the cost of these drugs very high, but their use in
pregnant women and people with liver failure or kidney disease is very dangerous.
Moreover, even in otherwise healthy individuals, the use of lipid-lowering drugs can
cause abdominal pain, allergic reactions, unusual emotions, hair loss, changes in
vision, headaches, muscle cell breakdown, and decreased sexual potency. This has
naturally prompted patients to look for other safe options such as weight loss, dietary
supplements, lean diets, and exercise. Less than 300 mg of cholesterol per day is
recommended according to FDA guidelines published in 2010. However, in 2015,
in the absence of scientific evidence, the DGAC Advisory Committee retracted the
claim that there was a significant link between dietary cholesterol intake and serum
cholesterol. Because diet and lifestyle changes alone would not decrease the risks
associated with coronary heart disease (DGA, 2015), despite this claim, lowering
cholesterol intake seems to play a significant role in managing blood lipids. It is
more expensive to lower one’s blood fat than to modify their diet. The growing need
to lower blood cholesterol levels has thus provided an opportunity to explore new
alternative approaches to doing so including the use of plant sterols, soluble fiber,
and, most importantly, probiotics. For example, recent studies conducted on labora-
tory animals as well as humans suggest that foods containing probiotic lactic acid
bacteria may be effective in lowering blood lipids (Khare & Gaur, 2020; Thumu &
Halami, 2020). As a result, finding probiotics that have a hypocholesterolemic impact
is of considerable interest as probiotics are of Generally Recognized as Safe status
and are thus free of hazardous compounds. Moreover, probiotics are less expensive
than traditional cholesterol-lowering medications. A meta-analysis of 30 randomized
controlled studies found that probiotics reduced TC and LDLc by 7.8 mg/dL and
7.3 mg/dL, respectively, in 1624 patients compared to control subjects. High-density
lipoprotein (HDL) and TG values were unchanged (Galie et al., 2009). According to
meta-analysis research, the particular probiotic strains that lowered TC in patients
included L. plantarum (MD of 1.56 mg/dL), VSL #3 (MD of 11.04 mg/dL), and
B. lactis and L. acidophilus (MD of 8.30 mg/dL) (Wang et al., 2018). In another
study, the L. acidophilus strain decreased high levels of TC and LDLc (MD 0.35
mmol/L) in those who had normal-to-moderate hypercholesterolemia (Esmaeili,
Zamindar, Paidari, Ibrahim, & Mohammadi Nafchi, 2021; Shimizu, Hashiguchi,
Shiga, Tamura, & Mochizuki, 2015; Tarrah et al., 2021).
Although the ability of probiotic bacteria to lower cholesterol has been reported in
several different strains of these microorganisms, the primary mechanisms by which
they can lower cholesterol have remained largely unknown. Most of the hypotheses
proposed to date have been based on laboratory experiments, and little attempt has
been made to evaluate the possible mechanisms of hypercholesterolemia based on in
vivo examination.
126 The Gut Microbiome: Bench to Table

Biological molecules such as lipids have insoluble qualities in aquatic conditions

because they have a nonpolar, hydrophobic area that prevents their dissolving in
water (Croft et al., 1988). Phospholipids and glycolipids are present in eukaryotic and
bacterial cells. The arrangement of lipid and protein units in variable concentrations
forms HDLs, very-low-density lipoproteins, chylomicrons, and low-density lipopro-
teins (LDLs), and from chylomicrons to HDL, the number of proteins increases.
HDL eliminates excess cholesterol from the tissues and is re-transported in the liver,
whereas chylomicrons distribute ingested fat to the body tissues (Croft et al., 1988).
The rate and levels of synthesized cholesterol are regulated by the daily consump-
tion levels of cholesterol. De novo synthesis regulates cholesterol levels in the body,
keeping them between 150 and 200 mg/dL. In healthy adults, just 0.3 g of cholesterol
is excreted out of approximately 1 g produced each day. Cholesterol is an important
component of the production of bile acids (BAs), steroid hormones, and membrane
formation. Because bile salts with conjugated systems are extremely soluble, the
majority enter the enterohepatic circulation following absorption, resulting in blood
buildup (McAuliffe, Cano, & Klaenhammer, 2005). It has been reported that the
hypocholesterolemic function of proteins is related to cholesterol assimilation during
cell growth, which reduces the quantity of cholesterol accessible for absorption in the
intestine (Huey-Shi Lye, Rahmat-Ali, & Liong, 2010).
The hypothetical concept of cholesterol-lowering probiotic effects includes the
following: activity of BSH for the deconjugation of bile (Huey-Shi Lye et al., 2010;
H-S Lye, Rusul, & Liong, 2010), probiotic cell binding of cholesterol and choles-
terol molecules incorporation into the probiotic cellular membrane (De Preter et al.,
2007), production of short-chain fatty acids (SCFAs) from oligosaccharides (De
Preter et al., 2007), deconjugated bile coprecipitates with cholesterol (Kumar et al.,
2012), and coprostanol formation from converted cholesterol (Kumar et al., 2012)
are all part of the hypothesis of probiotics. Although numerous hypocholesterolemic
probiotic mechanisms exist, the activity of BSH has been attributed to the most sig-
nificant probiotic cholesterol-lowering mechanism. Although the hypocholesterol-
emic mechanism of probiotics is not entirely understood, cholesterol and bile salt
metabolism are known to be intertwined. For example, bile salts are produced by the
liver hepatocytes, which aid in the transport of fat and dietary cholesterol across the
intestinal epithelium.

The enzyme BSH (choloylglycine hydrolase; EC 3.5.1.24) produces free BAs and
amino acid residues upon hydrolyzing glycine- and/or taurine-conjugated bile salts
(Figure 5.1), with a preference for glycol-conjugated bile salts over tauro-conjugated
bile salts. Bile salt conjugation significantly depends on the characteristics of the
host, and in mammals, the site of conjugation depends on the type of host. For exam-
ple, the microbial flora of Lactobacillus in the small intestine of mice are present at
the site where bile salt degeneration occurs, whereas, in humans, substantial flora
begin only at the end of the ileum and develop fully in the large intestine. Several
Dietary Modulation of Gut Microbiota 127

FIGURE 5.1 (A) The enzyme BSH hydrolyzes conjugated bile salts. (b) The involvement
of BSH in hypocholesterolemia and cholesterol as a mediator in the synthesis of new BAs. R:
Amino acid glycine or taurine. RDCA: Glyco- or tauro-deoxycholic acid, DCA: Deoxycholic
acid. Adapted from: Jones, Chen, Ouyang, Metz, and Prakash (2004) and Anandharaj,
Sivasankari, and Parveen Rani (2014).

researchers have proposed that the activity of BSH activity should be considered in
the probiotic selection criteria as they possess the ability to lower cholesterol. This
is because some probiotic microorganisms lacking this enzyme are not able to sig-
nificantly reduce cholesterol. Therefore, if bile secretion is an essential mechanism
for lowering cholesterol levels, the cultures used in in vivo tests should be selected
from the appropriate source. Numerous studies have demonstrated the distribution of
BSH activity in Bifidobacterium and Lactobacillus is genus, species, or even strain-
dependent. Most probiotic lactic acid bacteria originating from the human intestines
and feces have shown BSH activity. However, it is important to note that strains
with BSH activity isolated from the intestine or feces can also survive without this
enzyme and grow under bile salt conditions. BAs also stimulate cholesterol assimi-
lation through anaerobic development. The microbial metabolism of intestinal BAs
and the enterohepatic cycle defines the makeup of the pool of human BAs. Bile,
which is held in the gallbladder, is comprised primarily of conjugated BAs. Only 5%
of the BAs released in bile are reabsorbed in the terminal ileum and are recirculated
in the liver via the enterohepatic circulation to the large intestines, whereby 95%
of BAs are expelled in feces. While an array of colonic bacteria perform decon-
jugation activities, 7a-dehydroxylation appears to be limited to a small number of
intestinal bacteria. Consequently, the BAs profile expelled in feces and consisting
primarily of secondary BAs is highly dependent on gut microbial metabolism. The
pool measure, metabolite anatomy, and compartment fixations are important aspects
of the BA results because they relate to the normal gut microbiota composition and
128 The Gut Microbiome: Bench to Table

how it metabolizes BAs and influences host metabolic processes and obesity (Joyce
et al., 2014). The organisms use BAs to change the expression of genes controlled
by the farnesoid X receptor, resulting in a unique activation by the acids and their
metabolites (Sayin et al., 2013). Consequently, the more bile salts are secreted, the
more cholesterol will be removed from the bloodstream. Recently, the use of LAB
degenerated bile salts to reduce serum cholesterol levels in patients with hyperten-
sion and prevent hypertension in normal people has been receiving more attention.
The induction of such physiological effects was strongly connected to probiotic con-
sumption, prompting a further investigation to determine whether the effects were
due to native microbiota regulation or specific metabolic action of the consumed
probiotic strains (Hassan et al., 2019). Until recently, only one probiotic product
associated with claims of cardiovascular health has been approved in the market by
Health Canada. Cardioviva, which is also available in the United States and Europe,
contains two billion encapsulated Lactobacillus reuteri NCIMB 30242 and has been
clinically proven to lower LDL cholesterol levels by 11.6% in people with high cho-
lesterol levels (Jones, Martoni, & Prakash, 2012).

Simultaneous removal of deconjugated bile salts associated with cholesterol, in vitro,

has been described by several researchers. Cholesterol may be deposited with bile
salts degraded at pH below 5.5 at the same time as bacterial fermentation and the
formation of SCFAs. At acidic pH, degraded bile salts protonate and precipitate, while
glycine-conjugated bile salts remain unreacted without heterolysis and precipitate to
some extent leaving taurine-conjugated bile salts ionized in solution (Dashkevicz &
Feighner, 1989). Scientific reports have confirmed that the removal of cholesterol from
an environment by disruption of unstable cholesterol micelles occurs as a result of the
loss of bile salts, followed by a decrease in pH and the deposition of cholesterol with
free bile salts (Jones et al., 2004; Molinero, Ruiz, Sánchez, Margolles, & Delgado,
2019). In vitro culture of L. casei significantly reduced cholesterol levels by desta-
bilizing cholesterol micelles and coprecipitating them with decongested bile salts at
pH less than six (Brashears, Gilliland, & Buck, 1998). Conjugated bile salts associ-
ated with the simultaneous deposition of cholesterol are ambient and pH-dependent.
Simultaneous deposition of cholesterol occurs at pH around 3.78–6.69 (Liong & Shah,
2005). The highest deposition of cholesterol with colic acid occurs at a pH of less than
five, and the lowest simultaneous deposition of cholesterol occurs in the presence of
sodium glycocholate regardless of the pH level. Thus, it can be inferred that choles-
terol deposition is not solely only pH-dependent (Bhat & Bajaj, 2020).

Cholesterol Integration into Bacterial Cell Walls

Cholesterol incorporation into bacterial cellular membranes reduces cholesterol
absorption from the intestine into the blood, resulting in a decrease in total serum
cholesterol. The amino acids that make up the peptidoglycan of probiotic bacteria’s
cell walls, and the exopolysaccharides released by these microbes, are principally
responsible for the close interaction between cholesterol in the medium and the pro-
biotic bacteria’s cell surface. The lipids of probiotic lactic acid bacteria are primarily
Dietary Modulation of Gut Microbiota 129

concentrated in the membrane, suggesting that cholesterol incorporated into mem-

brane cells altered the fatty acid composition of the cells (Esmaeili, Paidari, et al.,
2021). Moreover, membrane cells with incorporated cholesterol had increased con-
centrations of unsaturated and saturated fatty acids, thereby improving membrane
strength. The cholesterol is then incorporated into the cell membrane of the bac-
teria which would reduce cholesterol absorption from the intestine into the blood.
Interestingly, the content and peptidoglycan structure in bacterial cell walls could
also have an impact on each strain’s ability to accumulate cholesterol. In a study con-
sisting of 34 Bifidobacterium strains (Bifidobacterium animalis, Bifidobacterium bif-
idum, Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium
pseudocatenulatum), it was discovered that these bacteria were capable of assimilat-
ing 4–81 mg cholesterol/g of dry biomass. Among the different strains investigated,
B. bifidum MB 109 and B. bifidum MB 107 were able to digest the maximum quanti-
ties (Bordoni et al., 2013). Shehata et al. (2019) examined the bacterium Lactococcus
lactis subspecies in terms of cholesterol-lowering in vitro. The results showed that
43.70% of cholesterol was absorbed by growing cells, 12.93% by resting cells, and
6.35% by dead cells. Electron imaging also confirmed cholesterol attachment to bac-
terial cell walls (Shehata et al., 2019). This process is attributed to the relationship
between cholesterol incorporation and the pH of the growth medium. When com-
pared to cultures produced at a pH of 6.0, cell membranes from cultures grown with-
out pH control had considerably higher cholesterol. Moreover, cholesterol levels were
found to be greater in cell membranes from cultures that did not have pH control.
Cell membranes produced without pH control had greater cholesterol concentrations
than whole cells grown under the same circumstances, but there was no significant
difference in cholesterol content between cell membranes and entire cells grown at
pH 6.0. It has been confirmed that L. brevis strains in Iran can assimilate cholesterol
up to 80% after 9 hours of incubation compared to other isolated species of lactic acid
bacteria originating from traditional dairy products (ewe milk, traditional yogurt,
and sour buttermilk). However, the quantity of cholesterol eliminated in the L. aci-
dophilus membrane fraction did not account for the overall amount removed by the
culture. According to scientists, some cholesterol may loosely interact with the cells
and fail to be integrated into the membrane, resulting in loss during the isolation
process. Probiotics also reduced cholesterol levels by incorporating cholesterol into
cellular membranes during development. By using a fluorescent probe, the probable
locations of the cholesterol-binding process inside the phospholipid bilayer mem-
brane of probiotic cells were investigated. Although the absorption of cholesterol by
this pathway is strain- and growth-dependent, these findings are, however, yet to be
confirmed in vivo (Hassan et al., 2019).

Lowering Serum Cholesterol by Probiotics

Another mechanistic health characteristic of some lactic acid bacteria and bifido-
bacteria for decreasing cholesterol levels is cholesterol assimilation. Cholesterol is
largely assimilated during bacterial development, where it is attached to the cel-
lular surface without transformation and integrated into the membrane phospholipid
layer. In a high-cholesterol mouse model, Park et al. investigated the hypocholester-
olemic characteristic of L. acidophilus ATCC 43121. The consumption of probiotics
130 The Gut Microbiome: Bench to Table

by the mice reduced total serum cholesterol levels by up to 25% and confirmed a
significant (P < 0.05) reduction in LDL, intermediate-density lipoprotein, and LDL
cholesterol levels. Probiotics must exhibit viability and growth in order to be able
to absorb cholesterol. In one study, L. plantarum 49 and L. plantarum 201 were
shown to lower TC serum levels after 14 days of bacterium consumption (da Costa
et al., 2019). In another study, L. mesenteroides subsp. mesenteroides KDK411 and
L. curvatus KFP419 were found to have potential hypercholesterolemia action in rats
through the absorption and excreting of cholesterol per evidence in the feces (Park,
Kim, Shin, Kim, & Whang, 2007).
Despite these results, some in vivo investigations have yielded contradictory
results. Since the cholesterol content of the feces of rats fed with L. acidophilus
ATCC 43121 (3 × 107 CFU/d) did not change from that of the control group, Park
et al. did not ascribe this mechanism to the hypocholesterolemic effect of the
probiotics. Moreover, adult individuals’ serum TC, LDL cholesterol, HDL choles-
terol, and TAG concentrations were not affected by treatment with L. rhamnosus
LC705 (two capsules/d containing 2×1010 CFU, for 4 weeks), even with a small
rise in serum cholesterol. As a result, we can conclude that the ability of bacteria
to assimilate cholesterol in the medium appears to be strain-specific and reliant
on the strain’s growth. The occurrence of the hypocholesterolemic effect can be
hindered if the strain can remove cholesterol but does not survive during passage
through the GI tract.

Binding of Cholesterol
Growing bacterial cells maintained a considerable quantity of cholesterol, and soni-
cation from cells of Bifidobacterium breve ATCC 15700 exuded more than 40%
of cholesterol. Even after several washes, detachment of the absorbed cholesterol
was impossible, indicating a strong binding potential between cholesterol and the
growing cells. Additionally, nongrowing Lactococcus cells have been confirmed to
have the capability of absorbing cholesterol in vitro; thus, the mechanism of choles-
terol removal has been studied from different perspectives. Probiotics endowed with
cholesterol-lowering properties possess the attribute of cholesterol-binding potential
in the small intestine. Nami et al (2019) reported that probiotic L. plantarum YS5
exhibited an elevated cholesterol-removal capacity by growing cells (84%) and mod-
erate cholesterol-removal capacity by resting (41.14%) cells and dead (32.71%) cells.
Moreover, L. fermentum lowered cholesterol levels in vitro, according to Pereira
and Gibson (2002). It has been suggested that cholesterol assimilation by gut cells
may lower the quantity of cholesterol available for intestinal absorption (Miremadi,
Ayyash, Sherkat, & Stojanovska, 2014; Pigeon, Cuesta, & Gilliland, 2002). Viable
as well as heat-killed cells were capable of cholesterol removal from growing media
by L. lactis subsp. lactis N7. Viable cells were also able to substantially eliminate
more cholesterol than dead cells (Kimoto-Nira et al., 2007). L. gasseri strains have
been confirmed to eliminate cholesterol in vitro by adhering to the cellular surface,
although this capacity seemed to be growth- and strain-specific. The capacity of pro-
biotics to eliminate cholesterol throughout various growth conditions, notwithstand-
ing, bolstered the aforementioned result (Kimoto, Ohmomo, & Okamoto, 2002).
Dietary Modulation of Gut Microbiota 131

Other Mechanisms
Metabolic products such as SCFAs synthesized by probiotics have a pivotal role in
cholesterol removal. SCFAs serve as ligands to activate peroxisome proliferator-
activated receptors (PPARs). Angiopoietin-like protein 4 (ANGPTL4), a lipoprotein
lipase (LPL) inhibitor, is produced by brown and white fat in the liver and intestines
and is regulated by PPARγ (Hoda, 2011). According to Sheril et al. (2013), buty-
rates activate PPARs predominantly, followed by propionate, and finally acetate. As a
result of PPAR activation and the upregulation of ANGPTL4 levels, SCFAs decrease
LPL activities. This action suppresses fat storage by regulating fatty acid oxidation
in muscle and adipocytes. Therefore, to have a hypocholesterolemic impact, the pro-
biotic microorganisms must create enough propionate to counteract the effects of
acetate on cholesterogenesis. For example, L. plantarum MA2 (1011 cells/d) incor-
porated into a cholesterol-enriched diet and fed to Sprague–Dawley rats for 5 weeks
exhibited a higher fecal propionate concentration than that of the control group.
Cholesterol can be converted to coprostanol (5-cholestan-3-ol) and coprostanone
in small concentrations. These metabolites have a poor intestinal absorption rate
and are excreted in the feces with a decrease in cholesterol absorption. The cho-
lesterol reductase enzyme must be present in the cholesterol-lowering probiotics in
order to convert cholesterol. This enzyme is present in probiotic bacteria such as
Lactobacillus bulgaricus FTCC 0411, L. acidophilus FTCC 0291, L. acidophilus
ATCC 314, L. casei 1311 FTDC ATCC 393, B. bifidum PRL2010, and Eubacterium
coprostanoligenes ATCC 51222 (Gérard, 2014; H-S Lye et al., 2010; Zanotti et al.,
2015). There is limited information in the literature regarding the strains that are
positive for this enzyme; however, the activity of this enzyme is strain-specific.
The Niemann-Pick C1-Like 1 (NPC1L1) protein is found in the small intestines
on the brush surface membrane of enterocytes and plays an integral role in cho-
lesterol absorption (Jia, Betters, & Yu, 2011). In a study by Huang et al., hyper-
cholesterolemic rats were treated with L. acidophilus ATCC 4356 for 28 days, and
it was found that NPC1L1 levels in segments of the jejunum and the duodenum
were considerably decreased (P < 0.05) (Huang et al., 2013). This was corroborated
and confirmed by Duval et al. that activators of the liver X receptor (LXR) sup-
pressed the expression of the NPC1L1 gene in the gut. Interestingly, in mammals,
cholesterol metabolism is primarily regulated by LXRs. LXRα- and LXRβ- are the
two kinds of LXR that have been identified so far and regulate the metabolism of
lipids and carbohydrates. These LXRs are nuclear receptors and constitute mem-
bers of a superfamily that functions as ligand-activated transcription factors. Huang
and Zheng (2010) found that the expression of LXR-α and LXR-β was upregulated
by the L. acidophilus ATCC 4356 strain, whereas the expression of NPC1L1 was
downregulated in a dose- and time-dependent manner in Caco-2. In addition, L
plantarum LRCC 5273 effectively decreased hypercholesterolemia in mice by acti-
vating hepatic and intestinal LXR-, leading to BA excretion and increased fecal
cholesterol in the small intestine (Heo et al., 2018).
Choline, phosphatidylcholine, L-carnitine, and betaine were metabolized by the
intestinal microbiota to trimethylamine (TMA), which is then oxidized to trimethyl-
amine N-oxide (TMAO) by hepatic flavin monooxygenases (FMO3) (Zhou et al., 2020).
132 The Gut Microbiome: Bench to Table

TMAO levels in the blood have been linked to negative outcomes in CVD patients such
as chronic heart failure and coronary heart disease. Consequently, attention to TMAO
levels could be a potentially useful predictive indicator for unfavorable cardiac events
in patients with chronic heart failure following myocardial infarction. In addition, the
formation of gut microbiota-dependent TMAO is inhibited and has proven to be a via-
ble method for atherosclerosis therapy (Z. Wang et al., 2015). However, it is yet unclear
which gut microorganisms play the most important role in CVD, and the precise pro-
cesses involved need to be investigated further (Molinero et al., 2019).

Relationship between Nutrition and Gut Microbiota

The GI tract is one of the most significant and important systems in the human body.
For example, the small intestine itself is responsible for two important processes,
notably digestion and resorption. The lining of the small intestine is rich in glands
that secrete intestinal juice, which contains enzymes that catalyze polypeptides, fats,
and disaccharides. The small intestine is associated with the digestion of food due to
secreted secretions of the pancreas, liver, and mucous membranes. In addition, the
small intestine secretes hormones that participate in the body’s immune defenses.
The undigested, unabsorbed food that reaches the end of the small intestine passes
into the large intestine, which forms the last section of the digestive tract and has
no practical significance for digestion. The large intestine is thus simply a reservoir
of fecal masses periodically excreted from the body. However, the cavity that com-
prises the large intestine is inhabited by the normal intestinal microflora, the pres-
ence and standard composition of which are very important for overall human health.
The intestinal microflora is characterized by diverse species composition including
microorganisms such as fungi, bacteria, protozoa, and viruses that colonize the GI
tract (Valdes, Walter, Segal, & Spector, 2018). The large intestine microbiota breaks
down complex carbohydrates and sugars through the metabolism of SCFAs, acetate,
butyric acid, and propionate. Protein fermentation ends with a more diverse meta-
bolic profile. Some nitrogen products, especially N-nitroso compounds, are carcino-
genic and can cause mutations in DNA. These compounds occur endogenously due
to microbial fermentation of proteins in the colon or can be obtained as a result of
nutrition (Duncan et al., 2007).
Intestinal bacteria partially break down dietary fiber and other undigested food
residues. The bacterial breakdown of food residues results in end products that
include SCFAs and gases. Intestinal bacteria can also synthesize some vitamins
(from group B (biotin, pyridoxine, cobalamin, riboflavin, folate, nicotinic acid, thia-
mine) and vitamin K). Some representatives of the intestinal microflora are antago-
nists of food pathogens and inhibit their development, growth, and metabolic activity
(Holscher, 2017).
The digestion and resorption of nutrients occur in the intestinal tract, particularly
the small intestine. Chronic inflammation of the small intestine causes digestion
problems and impedes nutrient absorption. Dysfunction of the small intestine sig-
nificantly hampers the rate of digestion and resorption. In general, factors such as the
presence of vitamins, minerals, and other nutritional deficiencies, food intolerance,
and food allergies are all hindered when the functionality of the small intestine is
impaired (Gentile & Weir, 2018).
Dietary Modulation of Gut Microbiota 133

With regard to intestinal disorders, it is essential to exclude, from the diet, foods
that impair the motor function of the intestine. In other words, the food must be
mechanically, chemically, and thermally sparing. It is thus necessary to reduce fat
consumption as the quantity and quality of fats impact their degree of digestion by
the body. Therapeutic diets are also required to provide the necessary amount of
energy and protein. In addition, to address intestinal disorders, therapeutic diets
should include a substantial amount of vitamins and minerals (Cardona, Andrés-
Lacueva, Tulipani, Tinahones, & Queipo-Ortuño, 2013).
The primary bacterial groups that make up the intestinal microflora of the
human body are Firmicutes, Bacteroidetes, Proteobacteria, Verrucomicrobia, and
Actinobacteria. The amount and species composition of the intestinal microflora of
a healthy person varies with age and the composition of the food, and most of these
bacteria are commensal (they coexist with the human body without causing disease).
Children are born with a sterile digestive tract, which begins to colonize with micro-
organisms after a few hours. Enterobacteria and Bifidobacteria are the first settlers.
However, a healthy stomach and the proximal part of the small intestine contain very
few organisms, which is due to the bactericidal action of hydrochloric acid in gas-
tric juice. The colon, on the other hand, is dominated by anaerobic microorganisms,
while the small intestine is predominantly aerobic and anaerobic. Bacteria in the
intestinal lumen break down different types of sterols and steroids. The bacteria also
convert the cholic acid of bile salts into deoxycholate, break down toxins, produce
biotin and pantothenic acid, stimulate the immune system, and cause the fermenta-
tion of cellulose (Schmidt, Raes, & Bork, 2018).
Many factors affect the degree of microbial presence in the gut. Factors that
influence the ecosystem of the intestinal microbiota include the mode of birth, the
mother’s microbiota, breastfeeding, exposure to environmental bacteria, intake
of antibiotics/probiotics, and diet (Figure 5.2). It was found that in terms of com-
position, variety, and functional competencies, the microbiota of 2- to 3-year-old

FIGURE 5.2 Factors influencing the gut microflora.

134 The Gut Microbiome: Bench to Table

children is close to that of adults (Yang, Ye, Yan, He, & Xing, 2019). It is believed
that the composition of the microbiota after three years of age is complete and
functional. Moreover, eating habits and overall healthy lifestyles could generally
promote a healthy intestinal microbiota (Yang et al., 2019).
There is a strong link between nutrient intake and the composition of intestinal
microbiota. It is known that intestinal microorganisms can affect the synthesis of
specific vitamins or the breakdown of certain nutrients, that is, the effects of micro-
biota on nutrition. Moreover, the intestinal microbiota also differs among people
from different latitudes and is also buttressed by environmental and genetic factors,
as well as the use and administration of antibiotics (Goodrich et al., 2016). Nutrition,
however, is suggested as the main reason for the differences in the composition of the
gut microbiota between people.
Studies have shown that different food components are used by different bacterial
communities and ensure the colonization of this community; in this way, the domi-
nant bacterial species can be formed depending on the diet. Among the nutrients,
carbohydrates, fiber, protein, fat, phytochemicals, and vitamins play a significant
role in creating the intestinal microbiota (Duncan et al., 2007). Studies examining
the effects of a high-fat diet on the intestinal fat microbiota have shown that such a
diet reduces microbial diversity and increases the number of Bacteroides, Alistipe,
and Bilophila. High-fat diets increase the concentration of SCFAs in the feces com-
pared to low-fat diets and significantly reduce the levels of bifidobacteria (Heiman
& Greenway, 2016). Phytochemicals are biologically active substances derived from
plants that give plants their color, odor, and natural stability. It is known that a diet
rich in fruits, vegetables, grains, and legumes reduces the risk of various diseases.
The prophylactic effects, therefore, of these foods are partly due to antioxidants in
their composition. The beneficial health effects of foods with probiotics such as dairy
products have been well studied. For example, in recent years, we have seen increas-
ing evidence that probiotics may play an essential role in the proper functioning of
gut health and digestion, urogenital tract, immune system, respiratory features, and
allergies, and can significantly combat various infectious diseases (Yang et al., 2019).

Role of the Gut Microbiota in Human Health and Disease Prevention

The microflora in the digestive system plays a significant role in human health.
Prebiotics, probiotics, and symbiotics are used to protect this microflora from patho-
gens. For example, probiotic bacteria inhibit pathogenic microorganisms, increase
the digestibility of food, strengthen the immune system, lower blood cholesterol lev-
els, and increase the absorption of prebiotics. On the other hand, prebiotics are indi-
gestible carbohydrates that increase the number and activity of bacteria in the colon
and enhance the effect of probiotics. While prebiotics is used selectively by the ben-
eficial microflora in the colon, they prevent the proliferation of potential pathogens.
Probiotics and prebiotics can be used in combination with foods called symbiotics.
With this application, the life span of probiotic bacteria is extended, resulting in
enhanced colonization in the colon. Meanwhile, studies on the clinical use of probiotic
microorganisms continue to increase. The use of probiotics is particularly associated
with the treatment and enhanced outcomes of the following: diseases of the intestinal
system, liver, H. pylori, oral and dental health, diseases of the genitourinary system,
Dietary Modulation of Gut Microbiota 135

LI, symptoms of constipation, stimulating the immune system, allergies, cancer, pre-
vention of various types of diarrhea, and regulation of cholesterol levels (Liu, Tran,
& Rhoads, 2018). There are many studies on probiotic use in diarrhea due to the
use of antibiotics, and inflammatory bowel diseases (IBDs) such as ulcerative colitis
(UC), chronic illness, spastic irritable bowel syndrome, and necrotizing enterocolitis
(Ryma, Samer, Soufli, Rafa, & Touil-Boukoffa, 2021). Oligosaccharides cannot be
hydrolyzed or absorbed in the small intestine. However, they can be fermented by
Lactobacillus spp. and Bifidobacterium spp. and have prebiotic properties. In the
food industry, the most widely used oligosaccharides are the following: fructooligo-
saccharides, galactooligosaccharides (GOS), transgalactooligosaccharides, xylooli-
gosaccharides, gentio, lactulose, lactosaccharose, inulin, isomaltooligosaccharides,
preharisaccharides, and salt. Inulin has also been widely used in food in recent years
and can be fermented by Lactobacillus spp. and Bifidobacterium spp., and has prebi-
otic characteristics (Liu et al., 2018).

Cardiovascular Disease
Globally, CVD is extensive, and the death toll attributed to this disease is increas-
ing. Estimates by the WHO confirm that approximately 23 million people world-
wide will have CVD by 2023, affecting up to 22% of the global economy. As
human societies progress, lifestyle changes, and mobility declines, anomalies such
as impaired insulin production or insulin resistance have led to increased levels
of triglycerides, total cholesterol, low-density lipoprotein (LDL-C), and decreased
LPL-C lipoprotein. High (HDL-C) cholesterol rises in the blood, thus posing an
elevated CVD health risk in susceptible individuals. In addition, cholesterol oxi-
dized by the formation of arterial plaques is a major cause of coronary heart disease.
These factors, in combination with widespread smoking, contribute to the bulk of
critical modifiable risk factors, which are responsible for approximately half of all
deaths globally. Aortic and other peripheral vascular blood vessel inflammation is
also a less prevalent health concern linked to atherosclerosis. According to Lee et
al. (2012), CVD is attributed as one of the major precursors of death and sickness in
the world, accounting for 29% of overall worldwide mortality (Lee et al., 2013). In
addition to these variable basic factors, ACD mortality is influenced by extremely
widespread physiologic and metabolic changes. Dyslipidemias, obesity, high blood
sugar, hypertension, and insulin resistance are the most common diseases (Ramos,
Esteves, Prates, Moreno, & Santos, 2022). For example, people with hypercholes-
terolemia are three times more susceptible to having a heart attack than those with
a normal lipid profile.

Liver Disease
During the first years of use of lactulose in hepatic encephalopathy, the colon pH
decreases as a result of the bacterial metabolism of lactulose, which facilitates the
proliferation of Lactobacillus and other acidophilic bacteria that can tolerate acid
while preventing the formation of ammonia. Lactobacillus is thought to avoid the
proliferation of the responsible acidophobic, proteolytic bacteria. Some researchers
contend that bacterial proliferation in the colon increases with lactulose, that the
increased bacterial population uses ammonia as a source of nitrogen or that there
136 The Gut Microbiome: Bench to Table

is metabolic inhibition in bacteria due to lower pH in the colon ammonia-producing

bacteria not producing ammonia (Aller et al., 2011).
As is well known, probiotics can inhibit bacteria (especially gram-negative ones)
with fecal enzyme activity by various mechanisms. In addition, the administration
of lactulose alone or in combination with probiotics (especially Bifidobacteria) is
one of the most recommended treatment methods. At low pH, most ammonia is in
the form of ammonium ions (NH4 +), which the gut cannot absorb, and this reduces
the passage of ammonia into the blood. The use of lactulose in combination with
Bifidobacteria may support this protective effect (Xu, Wan, Fang, Lu, & Cai, 2011).
The second important mechanism associated with probiotics in treating encepha-
lopathy is that probiotics help to more effectively clear ammonia and other toxins
from the liver by reducing inflammation and oxidative stress in liver cells. Probiotics
can play a protective role against inflammation and cell damage in the liver.
Details on the inhibition of the absorption of toxins other than ammonia, the
last possible mechanism for the positive effects of probiotics, are not fully defined.
However, the potential mechanism is advanced kidney disease undergoing hemodi-
alysis (Aller et al., 2011).
Gut microbiota may cause nonalcoholic fatty liver disease by luminal ethanol
production. Probiotics are used in order to treat changes in microbiota that have been
reported in nonalcoholic fatty liver disease. A mixture of three genera of bacteria
(a multistrain formulation composed of Streptococcus, Thermophilus, and several
species of Bifidobacterium and Lactobacillus) administered to Lepob/ob mice over
4 weeks improved insulin sensitivity, total fatty acid content, serum alanine amino-
transferase levels, and the histological spectrum of liver damage (Meroni, Longo, &
Dongiovanni, 2019).

Dental Diseases
The activity of probiotics on the health of the oral cavity and teeth is related to
the ability of probiotics to produce an antimicrobial agent against oral pathogens.
Probiotic bacteria can release various antimicrobial substances such as organic acid,
hydrogen peroxide, carbon dioxide, diacetyl, bacteriocin, and adhesion inhibitors,
which have high antimicrobial potential. Probiotics can also adhere to the surface of
the teeth as well as the mouth surface. This is an essential point with regard to the
long-term probiotic effects of bacteria. Lactobacilli can adhere to hydroxyapatite
on teeth. Most adhesion experiments for probiotic purposes were based on the daily
consumption of cheese and yogurt. Another vital ability of probiotics is their effect
on the immune system. Probiotics are thought to have mechanisms that can alter
the host’s immune system. Dendritic cells scattered on the mucosa surface play a
crucial role in the pre-identification of bacteria and activation of T-cell responses.
Depending on the signal, either immune tolerance or an active immune response
against a particular antigen is established in the dendritic cells. Due to the ability of
probiotic bacteria to produce various antimicrobial substances and adhesion inhibi-
tors that affect the oral flora, probiotics can significantly affect the environmental
conditions necessary for the survival of bacteria by altering the pH of the environ-
ment and the redox potential (Haukioja, 2010).
Dietary Modulation of Gut Microbiota 137

Gastrointestinal Diseases
Inflammatory bowel Disease
Inflammatory bowel disease is an inflammatory, intermittent, chronic GI tract dis-
ease that recurs spontaneously. IBD is a term used to describe a group of complex
disorders of the GI tract. IBDs include two main forms: Crohn’s disease (CD) and UC,
which share similar clinical symptoms. Symptoms and signs of the disease include
stool changes in CD; however, recurrence is observed when stool change disappears.
Inflammation of the mucosa is reduced when broad-spectrum enteric-coated antibi-
otics are used for UC. Triggering environmental conditions, genetic predisposition,
and intestinal bacteria plays a role in the occurrence of IBD. Studies in humans and
animals have shown that the percentage of beneficial microorganisms such as probi-
otics decreases the incidence of IBD (Lakatos, 2006).
Manipulation of the intestinal flora has become an interesting approach to pre-
venting and treating IBD. Different results were obtained with other probiotic strains
and their combined use in experimental models of colitis (Ryma et al., 2021).
Ulcerative colitis: Another well-known chronic IBD is UC whereby inflamma-
tion occurs as a result of abnormal reactions of the immune system. Many studies
have shown that probiotics effectively maintain remission in UC. When fecal suspen-
sions of healthy individuals are given to patients with UC by enema, the symptoms
can be relieved and remission achieved.
In placebo-controlled studies, the incidence of relapse was found to be signifi-
cantly reduced in probiotic users. With the establishment of remission, NF-κB,
IL-1β, TNF-α decreased, and IL-10 increased (Baumgart & Carding, 2007; Ryma
et al., 2021).
Crohn’s disease: This is another IBD that leads to inflammation of the digestive
tract. The distribution of bacteria in the gut of individuals with CD is very different
from that of normal individuals. For this reason, although it was thought possible
to approximate the flora of normal individuals through the use of probiotics, it was
not possible to make a definite assessment of the results obtained (Nagao-Kitamoto,
Kitamoto, Kuffa, & Kamada, 2016). CD is characterized by a discontinuous pattern,
affecting the entire GI tract. In CD patients, the inflammation is transmural with
large ulcerations and granulomas.
Several clinical trials and experimental studies displayed the role of Saccharomyces
boulardii as a good biotherapeutic agent for preventing and treating several GI dis-
eases including CD. S. boulardii stimulates effects that resemble the protective
effects of the normal healthy gut flora (Kelesidis & Pothoulakis, 2012).
Pouchitis is inflammation occurring in the ileal region after ileal-anal anasto-
mosis, in which the irregularities in the intestinal flora act as a triggering factor.
In a study investigating the efficacy of a probiotic preparation (VSL#3) containing
5 × 10(11) per gram of viable lyophilized bacteria of four strains of Lactobacilli,
three strains of Bifidobacteria, and one strain of Streptococcus salivarius subsp.
thermophilus, it was confirmed that this probiotic preparation was as efficient as
compared to a placebo in the maintenance of remission of chronic pouchitis. The
results showed that fecal concentration of Lactobacilli, Bifidobacteria, and S. ther-
mophilus increased significantly from baseline levels only in the VSL#3-treated
138 The Gut Microbiome: Bench to Table

group. Thus, oral administration of the new probiotic preparation was effective in
preventing flare-ups of chronic pouchitis (Gionchetti et al., 2000). While 85% of
study subjects had a decrease in disease effects after 9 months, there was evidence of
disease recurrence in all individuals in the placebo group.

Complications after Surgery

The incidence of complications is high in patients who have undergone surgery and
are kept in the intensive care unit. Among the complications, infections occupy a vital
place. Early probiotic therapy and appropriate nutritional support have been valuable
and effective options compared to the prophylactic use of antibiotics to control these
complications. For example, perioperative care for patients with colorectal cancer
includes antibiotics that are used in postoperative care and provide some protection
against infections. Antibiotics carry risks of microbial resistance and disruption of
the microbiome. Probiotics, on the other hand, can help to maintain the balance of
the microbiome after surgery by preserving the integrity of the intestinal mucosa
and reducing bacterial translocation. The authors demonstrated that probiotics were
successful in postoperative infectious and noninfectious complications, analysis of
bacterial translocation rate, and assessment of intestinal permeability. There was a
tendency for lower levels of postoperative contagious and noninfectious complica-
tions with probiotics compared to placebo. Probiotics reduced bacterial translocation,
maintained the permeability of the intestinal mucosa, and provided a better balance
of beneficial pathogens (Pitsillides, Pellino, Tekkis, & Kontovounisios, 2021).

Cancer Treatment
Diet plays a role in the development of colorectal cancer. A diet rich in meat and
animal fats, and low in fiber, changes the distribution of colon flora. As Clostridium
and Bacteroides strains increase, Bifidobacterium strains decrease. Because flora
and the immune system play a role in tumor development, it is thought that, theoreti-
cally, prebiotics and probiotics that regulate flora can also be used to prevent tumor
development. For example, lactulose causes a decrease in 7-α dehydroxylase activity
through changes in the bacterial flora of the colon. 7-α dehydroxylase is the enzyme
that causes the formation of secondary BAs. Secondary BAs are considered cocar-
cinogens. In addition, secondary BAs enter the enterohepatic circulation and form
a continuous cycle, affecting BAs and cholesterol synthesis. The production of 7-α
dehydroxylase and other potentially toxic enzymes of bacterial origin is reduced after
administration of lactulose. Thus, lactulose administration helps in the reduction
of toxic substances such as ammonia, which is also a known potential carcinogen.
One hypothesis is that butyrate formed in the colon using lactulose has a DNA-
protective impact. In addition to the many positive effects on health, probiotics also
have anticancer, tumor-suppressant, and therapeutic effects (Pitsillides et al., 2021).
Studies investigating probiotic–cancer interactions have demonstrated the capabili-
ties of probiotics, such as the cancer-preventing effects. Probiotics enhance the host’s
immune response, deteriorate the structure of potentially carcinogenic compounds,
create qualitative and quantitative changes in the intestinal flora, and have antimu-
tagenic effects on the colon. Among the positive effects of probiotic action could be
the ability of probiotics to produce anticancer compounds, alteration of metabolic
Dietary Modulation of Gut Microbiota 139

activities, such as inhibition of the conversion of precancers into carcinogens in the

intestinal microflora, prevention or retardation of toxin absorption, and modification
of physicochemical conditions as enhanced intestinal barrier mechanisms.
Mohammed et al. (2010) reported that Alexandrium minutum possesses advan-
tageous, anticancerous activity with mild apoptotic effects (Altonsy, Andrews, &
Tuohy, 2010). Prebiotics are also efficient in the treatment of colon cancer due to their
ability to ferment dietary fibers and enhance the level of metabolites. For example,
wheat aleurone fermentation was reported to be enhanced by the addition of LGG/
Bb12, resulting in an increased concentration of butyrate (Altonsy et al., 2010). The
beneficial effects of the probiotic lactic acid bacteria Lactobacillus plantarum A7
and a commercial strain of Lactobacillus rhamnosus GG on human colon cancer
cell lines (Caco-2 and HT-29) and normal cells (L-929) have been reported. It was
determined that lactobacilli can exert antiproliferative effects on the various cancer
cell lines including colon cancer (Sadeghi-Aliabadi, Mohammadi, Fazeli, & Mirlohi,
2014). A wealth of data implicates that ErbB receptors have essential roles in tumor
development. Bacillus polyfermenticus has been clinically used in the growth of
tumors by suppressing ErbB2 and ErbB3 protein. Generally, probiotic bacteria are
known to exert anticancer activity in animal studies (Ma et al., 2010).

Lactose Intolerance
Lactose intolerance is one of the most common forms of food intolerance and occurs
when lactase activity is reduced in the small intestine lining. LI is characterized by
GI symptoms, including vomiting, diarrhea, flatulence, and abdominal pain, which
are caused by the fermentation of unabsorbed lactose in the colon. The severity of
LI and the symptoms mentioned above can vary considerably from individual to
individual. Lactase deficiency can be primary, secondary, or congenital. The most
common form is primary lactase deficiency, a consequence of lactase deficiency
characterized by a progressive decrease in lactase activity (Leis, de Castro, de
Lamas, Picáns, & Couce, 2020).
If there is no or low lactase activity in the gut, undegraded lactose disturbs the
osmotic balance and causes the accumulation of fluids and electrolytes in the gut. In
enlarged intestines, motility increases, and diarrhea occurs.
Lactose, which reaches the colon without being broken down, is fermented by
bacteria, resulting in hydrogen, methane, and carbon dioxide. Yogurt is the most
superior probiotic product due to its beneficial effects on LI (Gyawali et al., 2020;
Ibrahim et al., 2021). Because some microorganisms in yogurt contain the enzyme
lactase, they break down lactose before it reaches the colon and prevents LI symp-
toms and signs.
People who have digestive problems with lactose can tolerate yogurt because there
is beta-galactosidase activity in the bacteria found in yogurt. S. thermophilus and
Lactobacillus delbrueckii subsp. bulgaricus contain the enzyme beta-galactosidase
(lactase), which improves the digestion of lactose. Bacteria in yogurt metabolize lac-
tose by releasing bacterial lactase due to lysis through the effect of bile salts in the
small intestine after digestion. In addition, because fermented dairy products are
darker than milk, their passage time through the GI tract is longer which allows for
better digestion.
140 The Gut Microbiome: Bench to Table

Cultured foods (fermented foods) are defined as ‘foods or beverages produced

through controlled microbial growth, and the conversion of food components through
enzymatic action’ (Marco et al., 2017). Specifically, lactic acid bacteria such as
Streptococcus, Lactobacillus, and Leuconostoc are predominant in cultured foods. In
addition, other microorganisms such as fungi and yeast also have several applications
in food fermentation. Cultured foods are consumed worldwide, and the presence of
beneficial microorganisms and their metabolites is considered to be an alternative to
alleviate conditions associated with gut problems (Marco et al., 2017). These micro-
organisms known as probiotics are known to provide beneficial effects on gut micro-
flora. According to Fuller (1989), cultured food products that have beneficial effects
on host health by improving intestinal microbial balance are generally known to have
a probiotic effect (Fuller, 1999). Some health benefits of cultured foods include boost-
ing the immune system, improving intestinal health, alleviating the condition of LI,
decreasing the prevalence of allergic reactions, preventing CVD, and minimizing the
potential risk of certain cancers (Tamang, Shin, Jung, & Chae, 2016).
Daily consumption of cultured food products helps deliver commensal microbes
through the GI tract for modulation of the gut microbiota. Foods such as yogurt,
kefir, kimchi, cheese, and Kombucha contain microbial cells ranging between 6 and
9 log CFU/g or mL, and these cells are known to survive passage through the human
GI tract (Derrien & van Hylckama Vlieg, 2015). These microbes could help alter the
gut microbiota composition. Production of SCFAs, direct inhibition or stimulation of
competitors, and other indirect effects are responsible for such changes in the micro-
bial composition of the intestine (Derrien & van Hylckama Vlieg, 2015).

ROLE OF CULTURED PRODUCTS IN GUT

MICROBIOTA MODULATION
Recently, there has been significant interest in cultured foods due to their health
benefits and as carriers of beneficial microorganisms such as probiotics and health-
promoting metabolites. As a result of these foods, the diversity of the gut microbiota
is potentially influenced. Table 5.2 lists selected cultured food products shown to
influence gut microbiota.

Yogurt and other fermented products of dairy origin are generally considered by
consumers to be good sources of health-promoting organisms (Tremblay & Panahi,
2017). Yogurt by definition is a fermented cultured milk product containing two
strains of live bacteria, namely Lactobacillus delbrueckii subsp. bulgaricus and
Streptococcus thermophilus. These microbes should be at least 107cfu/g and live at
the time of consumption (Savaiano & Hutkins, 2021). Often other genera of probiotic
Lactobacillus and Bifidobacterium strains are also added into yogurt for additional
health benefits. For instance, yogurt-based diets have been linked to a decreased risk
of bladder cancer, reduced weight gain, and a reduced risk of metabolic syndrome
Dietary Modulation of Gut Microbiota 141

TABLE 5.2
Intervention Studies and Impact of Cultured Food Products on Gut Microbiota
Foods Study Population Intervention Findings References
Yogurt Thirty-eight Four-week ingestion of Reduced E. coli: Yang and
children with H. probiotics-containing Bifidobacterium spp. ratio in Sheu
pylori infection yogurt children infected in H. pylori (2012)
Yogurt Twenty-three Six months of daily Bifidobacterium and total C. Wang
drink healthy ingestion of a probiotic Lactobacillus increased, and et al.
preschool (Lactobacillus casei strain Enterobacteriaceae, (2015)
children Shirota) containing drink Staphylococcus, and
Clostridium perfringens
decreased
Yogurt Fifty-eight Four-week commercial Viable counts of fecal Savard
volunteers: yogurt consumption lactobacilli were et al.
healthy women supplemented with significantly higher, and (2011)
and men aged Bifidobacterium animalis those of enterococci were
between 18 and subsp. lactis and significantly lower
55 years Lactobacillus acidophilus
Kefir Forty-five (23 400 mL/day kefir was Lactobacillus bacterial load of Yılmaz,
male and 22 administered twice a day feces of all subjects in the Dolar, and
female) patients to the patients for 4 weeks, treatment group was Özpınar
completed the which contains between 104 and 109 CFU/g, (2019)
study Lactobacillus bacteria and the first and last
(treatment group, 25 measurements were
patients) statistically significant
Kefir Eighty-two Patients were given 250 mL Results showed a 14-day Bekar et al.
patients with of kefir twice daily (triple regimen of triple therapy (2011)
symptoms of therapy + kefir, n = 46) or with kefir is more effective
dyspepsia and H. 250 mL of milk in achieving H.
pylori infection containing placebo (triple pylori eradication than triple
therapy + placebo, n = 36) therapy alone
for 14 days
Probiotic Six healthy female The volunteers consumed Bacteroidetes increased Unno et al.
milk volunteers, two servings of drink during ingestion and (2015)
20–24 years of daily for 3 weeks, and decreased again after the end
age then stopped consuming of ingestion. Firmicutes
for an additional 3 weeks changed in the opposite way
of the study period
Probiotic Thirty-two Thirty-two subjects Probiotic milk product Veiga et al.
milk women (aged consumed (125 g/serving) containing dairy starters (2014)
20–69 years) twice a day either the and Bifidobacterium
probiotic milk (n = 17) or animalis potentiates colonic
an acidified milk product SCFA production and
(n = 15) for 4 weeks decreases abundance of a
pathobiont Bilophila
wadsworthia compared to a
milk product
(Continued)
142 The Gut Microbiome: Bench to Table

TABLE 5.2 (Continued)

Intervention Studies and Impact of Cultured Food Products on Gut Microbiota
Foods Study Population Intervention Findings References
Soy milk Twenty-eight The drink consisted of Bifidobacterium spp. Cheng
healthy adults 250 mL, twice a day and Lactobacillus spp. et al.
between meals, of either increased. The population of (2005)
fermented soy milk or coliform organisms
regular soy milk first for decreased when subjects
2 weeks, then switched to consumed fermented
the other drink after soymilk
2 weeks
Soy milk Ten healthy Subjects consumed During the administration of Inoguchi
volunteers (six fermented soy milk (100 fermented soy milk, the et al.
male, four g/day) and nonfermented number of bifidobacteria and (2012)
female, soy milk (100 g/day) for lactobacilli in the feces
21–25 years old) 2 weeks increased and clostridia
decreased
Kimchi Twenty-four obese Consuming 180 g of fresh Abundance of Bacteroides Han et al.
women aged or fermented kimchi per and Prevotella increased (2015)
30–60 years day (60 g/pkg × 3 meals) while that of Blautia was
for 8 weeks decreased after intake of
fermented kimchi
Kimchi Six participants Subjects consumed 300 g Did not show any therapeutic Kil et al.
(age range of kimchi with their effect on H. pylori in the (2004)
34–57) were normal diet. For control stomach. However, kimchi
assessed for H. subjects consumed 60 g of increased the population of
pylori infection kimchi Lactobacillus spp.

(Tremblay & Panahi, 2017). Over a century ago, Elie Metchnikoff proposed that
by modifying the intestinal microbiome with bacteria present in yogurt, one’s
health could be improved (Mackowiak, 2013). Odamaki et al. (2012) assessed 420
healthy people and found that frequent yogurt consumption containing probiotic
Bifidobacterium longum BB536 reduced the amount of enterotoxigenic Bacteroides
fragilis in the gut. These results also corroborated the conclusion that endotoxin-
producing bacteria are significantly suppressed by the habitual consumption of
yogurt (Odamaki et al., 2012). A four-week intervention study with yogurt contain-
ing probiotic bacteria reduced the intestinal Escherichia coli: Bifidobacterium spp.
ratio in children infected with Helicobacter pylori when compared with the control.
This study showed reduced H. pylori loads and elevated serum IgA concentrations
in infected children (Yang & Sheu, 2012). Donovan and Shamir (2014) confirmed
the enormous health benefits associated with yogurt consumption including the
prevention of GI diseases, as well as the maintenance of healthy gut microbiota
(Donovan & Shamir, 2014). For example, consumption of probiotic yogurt drink
fermented with the strain, L. casei Shirota, showed an increase in the total fecal
Lactobacillus count and decreased Enterobacteriaceae and Staphylococcus counts
Dietary Modulation of Gut Microbiota 143

(Tsuji et al., 2014; C. Wang et al., 2015). The consumption of fermented yogurt
containing Bifidobacterium lactis and Lactobacillus fermentum strains was also
shown to elevate fecal Bifidobacterium and Lactobacillus counts (Ahmed, Prasad,
Gill, Stevenson, & Gopal, 2007; Ibrahim et al., 2021; Savard et al., 2011; West et al.,
2011). It has been reported that consumption of yogurt is associated with an increase
in Lactobacillus gasseri and a decrease in Enterobacteriaceae and Staphylococcus
levels. Another important health benefit associated with yogurt is the alleviation
of LI symptoms due to the expression and release of β-galactosidase by the yogurt
cultures (Gyawali et al., 2020; Ibrahim et al., 2021).

Kefir is a cultured dairy product produced by the action of yeasts, lactic acid bac-
teria, and acetic acid bacteria fermentation of lactose in milk (Guzel-Seydim, Kok-
Tas, Greene, & Seydim, 2011). Recently, kefir drink has raised increased interest
among health-conscious consumers due to its superior health benefits. The impact
of kefir on gut microbiota was investigated by Santos et al. (2003), where isolated
kefir strains exhibited adhesion properties to the human enterocyte-like Cco-2 cells
indicating a potential ability to colonize the human gut (Santos, San Mauro, Sanchez,
Torres, & Marquina, 2003). Some studies on kefir strains have shown an increase
in Lactobacillus, Bifidobacterium, and Lactococcus populations, and a decrease in
Proteobacteria and Enterobacteriaceae counts in the gut microbiota (Kim et al.,
2015). In another human study (n = 45), Lactobacillus kefiri was identified in fecal
samples after 800 mL/day of kefir was consumed for 4 weeks. The total Lactobacillus
count in stool samples was significantly higher than as compared to the control sam-
ples in patients with CD (Bekar, Yilmaz, & Gulten, 2011). Likewise, a double-blind
randomized controlled trial investigated the impact of consumption of 500 mL/day
kefir on the eradication of Helicobacter pylori in patients with dyspepsia. The results
showed that the control group (250 mL/day milk) had a lower eradication rate of H.
pylori compared to the kefir group which had significantly higher levels of H. pylori
(Bekar et al., 2011). Kefir also helped to improve LI symptoms, improve the immune
system, lower cholesterol, and was shown to have anticarcinogenic and antimuta-
genic properties (Guzel-Seydim et al., 2011).

Probiotic fermented milk

Probiotic milk is an important functional food that is consumed globally. It is obtained
by milk fermentation which results in pH reduction. The common microbes involved
in this process are Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus ther-
mophilus, Lactobacillus acidophilus, and Lactobacillus kefiri (Ebringer, Ferenčík,
& Krajčovič, 2008). Probiotic fermented milk confers several health benefits. For
example, fermented milk consumption was associated with microbial changes in
the gut in a crossover intervention study of six healthy women. The study showed
an increase in the Firmicutes: Bacteroidetes ratio after 3 weeks of fermented milk
consumption (Unno et al., 2015). Similarly, Veiga et al. (2014) observed a decrease
144 The Gut Microbiome: Bench to Table

in Bilophila wadsworthia (a pathobiont) and higher counts in butyrate-producing

bacteria, SCFA (butyrate) in women with irritable bowel syndrome after fermented
milk consumption for 4 weeks (Veiga et al., 2014). Joseph et al. (2019) studied intes-
tinal microbial changes in school children (n = 42, normal and overweight) after con-
sumption of a probiotic drink containing Lactobacillus casei Shirota (Joseph et al.,
2019). The results suggested that frequent consumption of probiotic drinks helped to
significantly alter the composition of Bifidobacterium spp. and Lactobacillus spp. in
the gut microbiota. Elevated fecal SCFAs (butyric and propionic acid) were observed
in overweight children compared to normal-weight children which correspond to
the presence of a higher number of gut microbiota. Sidira et al. (2010) assessed the
effect of probiotic milk containing Lactobacillus casei ATCC 393 on modulation of
intestinal microbiota in a rat model (Sidira et al., 2010). Daily administration (9 days)
of milk led to significant reduction in staphylococci, enterobacteria, coliforms, and
streptococci counts indicating protection against pathogens.
In a double-blind, placebo-controlled trial, Miyazaki et al. (2015) confirmed
that frequent daily consumption of fermented milk products containing the pre-
biotic GOS and Bifidobacterium breve strain Yakult prevented dry skin in adult
women in good health (Miyazaki et al., 2015). The results showed that the pro-
biotic strain and GOS improved skin by limiting the production of phenols by
the gut microbiota. A similar study examined the effects of the consumption of
a fermented milk product containing the probiotic L. casei strain Shirota on the
stress response of healthy medical students. The study confirmed that the frequent
consumption of L. casei significantly lowered the percentage of Bacteroidaceae
compared to the placebo group (Kato-Kataoka et al., 2016).

Kombucha is the most popular nonalcoholic fermented drink obtained by sugared

tea fermentation with a cocktail of bacterial cultures (lactic acid and acetic acid bac-
teria) and symbiotic culture of bacteria and yeast. During fermentation, ethanol is
produced from sucrose conversion by yeast and carbon dioxide, plus organic acids,
while acetic acid and acetaldehyde are produced by acetic acid bacteria (Dufresne &
Farnworth, 2000).
An evaluation was performed on the effect of Kombucha tea and its associa-
tion with the gut microbiota on a progressive nonalcoholic fatty liver disease.
The study confirmed a reduction in Allobaculum, Turicibacter, and Clostridum
genera, whereas Lactobacillus was abundant in kombucha tea-fed mice (Jung et
al., 2019). Even though kombucha is rich in microbial culture, limited available
studies have explored the link between the composition of the gut microbiota and
the effect of kombucha consumption in mammals (Coton et al., 2017).
Fermented soymilk is a soy-based functional drink with known health benefits.
Increased counts of Lactobacillus and bifidobacteria spp. and decreased counts of
coliform and Clostridium perfringens were linked to the consumption of 250 mL soy-
bean milk twice daily for 2 weeks (Cheng et al., 2005). These results were in agreement
with the findings of Inoguchi et al. (2012) in which consumption of 100 g of fermented
soy milk (once/daily for 2 weeks) was associated with a decrease in Clostridia spp and
Dietary Modulation of Gut Microbiota 145

an increased number of Lactobacillus spp. (Inoguchi et al., 2012) in ten healthy adults.
Similarly, consumption of extracts of fermented plants for 8 weeks by 22 hypercho-
lesterolemic patients was found to increase bifidobacteria and Lactobacillus spp. and
decrease E. coli and C. perfringens counts (Chiu et al., 2017).

Kimchi is a traditionally fermented vegetable product of Korean origin and contains

various lactic acid bacteria such as Lactobacillus, Leuconostoc, Pediococcus, and
Weissella (Choi, Lee, & Paik, 2015). Kimchi also possesses several health-promoting
properties including antiaging, antihypertension, anticancer, anti-obesity, and anticon-
stipation effects (Das et al., 2020). An et al. (2019) investigated the effect of kimchi
on the intestinal microbiota of rats (An et al., 2019). The microbiota’s diversity was
higher in the diet supplemented with kimchi as compared to the control. It was reported
that obesity-associated Firmicutes decreased while leanness-associated Bacteroidetes
increased. These results thus indicate that fermented kimchi can help reduce obesity and
improve the host’s health by modulating gut microbiota. Han et al. (2015) were inter-
ested in understanding the anti-obesity impact of kimchi by evaluating the relationship
between gut microbiota and kimchi after its consumption (Han et al., 2015). The results
showed an increase in Prevotella and Bacteroides and a decrease in the genus Blautia
in the gut microbial population. This indicated that participants of the kimchi inter-
vention group aligned with a ‘lean enterotype’ pattern as Prevotella and Bacteroides
were associated with anti-obesity effects. In a randomized study with human subjects,
consumption of 300 g/day kimchi for 4 weeks increased counts of Lactobacillus and
Leuconostoc and decreased stool pH compared to 60 g/day of kimchi (Kil et al., 2004).
Based on these results, kimchi was found to be an excellent source for colon health as
it increased the population of beneficial bacteria and was also found to decrease toxic
enzyme activity (Anusha Siddiqui, Redha, Esmaeili, & Mehdizadeh, 2022).

EFFECTS OF PHENOLIC COMPOUNDS ON GUT MICROBIOTA

During the last few decades, scientists have conducted numerous research projects on
the effects of nutritional diet on gut microbiota. Their results have revealed the direct
impacts of nutritional habits on the microbial load, and survival, of essential bacteria
in the GI tract (Suganya, Son, Kim, & Koo, 2021). Therefore, the evaluation of dif-
ferent structural food materials seems to be essential for a precise determination of
their effects on gut microbiota and human health. In recent years, among diverse food-
stuffs, cultured probiotics have played a major role in transferring essential nutritional
compounds to the human body. This transfer has occurred via the development of the
intestinal microflora and continuous modulation of the load and species of the distal
gut as well as manipulation of the human gut microbiome (Shabbir et al., 2021). The
term cultured probiotics, as mentioned before, is designated to a wide range of food-
stuffs, and can be modified via the addition of various essential nutritional compounds,
for example, polyphenols and antioxidants. Polyphenols, for example, have been the
subject of a great deal of research due to their ability to prevent the formation of bioac-
tive oxygen as well as their potential for synergism with numerous body compounds.
146 The Gut Microbiome: Bench to Table

Polyphenols are considered to be a secondary plant metabolite containing a

minimum of two hydroxyl groups linked to a carious number of aromatic struc-
tures, mostly found in plant residues, stems, leaves, and flowers. Polyphenols are
mostly classified into flavonoids such as anthocyanins and nonflavonoids, for
example, phenolic acids (Pandey & Rizvi, 2009).
The human body gains its essential polyphenolic compounds by consumption of
various common agricultural products, namely tomatoes, onions, apple, berry, hazel-
nuts, plums, and black olive. Since polyphenol compounds consist of a large num-
ber of nutritional chemicals, there is not a prepared dietary reference intake value for
determining the daily or even monthly intake of their source foodstuff. According to
an investigation, Eastern countries consume an average of 2 g (per day) of polypheno-
lic compounds in comparison with 800 mg per day for Western countries due to the
Western countries’ typically lower consumption of natural food materials such as veg-
etables and fruits. Additionally, according to a report based on investigations in 2020,
an average of 0.9 g/person/day was consumed by the total global population, primarily
from coffee, tea, and fruits. Regarding various scientific reports, a deficiency of flavo-
noids and anthocyanins as the most important phenolics compounds can intensify the
risk of various illnesses and diseases such as cancer, pancreatitis, and GI problems. On
the contrary, excessive intake of polyphenols is reported to be hazardous due to their
high affinity to interact with drugs and body enzymes (Rong et al., 2017).
Phenolic compounds are poorly absorbed (lower than 10%) due to their struc-
ture which inhibits their application and in functionalized food material same as
in probiotics cultured foods. In this regard, modulated gut microbiota can help the
body to biotransform polyphenolic compounds to smaller flavonoids or anthocyanins
via different reactions in the GI tract same as is the case with oxidation, methyla-
tion, and isomerization. According to the results of Tao et al., gut microbiota can
effectively transform bundle side flavonoids to aglycone acacetin and then into other
various forms (Tao, Duan, Jiang, Qian, & Qian, 2016). Thus, it could be concluded
that the biotransformation of flavonoids can not only increase their absorbance but
also positively increase their stability in the GI tract. In this regard, there are vari-
ous reported microorganisms and probiotics that have the ability to biotransform
these phenolic compounds. Additionally, according to the results conducted on
Lactobacillus acidophilus, these bacteria can remarkably transform glycosides into
aglycones and aglycones that can then be beneficially used by host microorganisms.
There are numerous reports on the application of Gordonibacter, Urolithinfacients,
Lactobacillus plantarum, Streptococcus intermedious, etc., as probiotics that serve
to increase the bioavailability of polyphenols in the human intestine tract (Kasprzak-
Drozd, Oniszczuk, Stasiak, & Oniszczuk, 2021).
In conclusion, a healthy body is derived from the accurate and proper function-
ing of the GI tract which, in turn, is related to modulated dietary nutrition. The
consumption of various cultured probiotics-containing polyphenolic compounds can
thus beneficially increase the bioavailability and absorption of flavonoids and antho-
cyanins resulting in the preparation of proper chemicals for the consumption of gut
microbiota. Proper functioning of the gut microbiota via proper nutrition such as the
daily intake of cultured probiotics can be considered to be the easiest way not only to
increase body health but also to prevent various cancers, diabetes, and GI diseases.
Dietary Modulation of Gut Microbiota 147

CONCLUSION
In the last few decades, probiotic consumption has gained much attention due to its
association with health benefits. Additionally, there has been increasingly widespread
knowledge regarding the role of nutrition and diet in supporting a healthy gut micro-
biota. In vitro, experimental, and clinical researches have all demonstrated the syn-
ergistic effect of diet and probiotic use on enhancing the modulatory mechanism of
the gut microbiota in specifically addressing human health and disease challenges.
Consequently, it has also been established from the literature that cultured and fer-
mented functional food products exert a positive impact on the overall metabolic
functionality of the human system. The mechanism of probiotic action against dis-
ease-causing organisms helps to enhance the performance of the gut microbiota by the
production of metabolites that support proper coordination and hormonal imbalance.
Diet and nutrition are thus important key factors that influence the composition of the
gut microbiota and serve as a driving force in promoting human health and wellness.
As presented in this chapter, the importance of diet and cultured food products in the
modulation of the gut microbiome cannot be overemphasized. Thus, the blending of
diet, nutrition, and probiotic consumption as a part of an individual’s lifestyle will
result in a natural and therapeutic approach to disease prevention via the resultant
modulatory effect on the gut microbiota without compromising the immune system.

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 6 Dietary Modulation of
the Gut Microbiota by
Prebiotics and Other
Dietary Nutrients and
Relevant Health Effect(s)
Alison Lacombe and Vivian C.H. Wu
United States Department of Agriculture

CONTENTS
Introduction............................................................................................................. 157
Types of Prebiotics.................................................................................................. 158
Carbohydrate Prebiotics..................................................................................... 159
Oligosaccharides........................................................................................... 159
Human Milk Oligosaccharides...................................................................... 159
Polysaccharides............................................................................................. 161
Inulin............................................................................................................. 162
Non-Carbohydrate Prebiotics.................................................................................. 163
Polyphenols........................................................................................................ 163
Short-Chain Fatty Acids..................................................................................... 164
Metabolism of Nutrients by the Gut Microbiota..................................................... 164
Effect of Prebiotic on Immunity........................................................................ 164
Prebiotics as Functional Foods............................................................................... 166
Novel Prebiotic Formulations............................................................................ 168
Food-Processing Effects on Probiotics and Prebiotics....................................... 169
Factors That Influence Probiotic Viability......................................................... 170
Conclusion.............................................................................................................. 173
References............................................................................................................... 173

INTRODUCTION
A significant advancement in gastrointestinal microbiology realized the impact diet
extends on health status and how gut constituent affects the host’s health (David
et al. 2014). Within the last 30 years, science has transitioned from viewing the gas-
trointestinal tract (GIT) as an inert tube to a flourishing ecosystem akin to vibrant
and diverse dynamic communities in the Amazon rainforest. Similar to the deepest

DOI: 10.1201/b22970-8 157

158 The Gut Microbiome: Bench to Table

recesses of planet Earth, little is known about the gut community and, more impor-
tantly, what inputs keep it thriving. This chapter focuses on the relationship between
the gut microbiota, the host diet, and the resulting symbiotic relationship. Evidence
accumulated by in vitro work and recent evidence from in vivo studies and human
clinical trials collectively indicate that phytochemicals, fiber, and other nutrients
demonstrate prebiotic capabilities (David et al. 2014). Parsing through the panoply of
research requires a deep look into the chemical structure of prebiotics in the gut and
the metabolic orchestration of the host gut microbiome.
Prebiotic compounds cannot be digested in the human stomach and small intestine
and instead pass through to the large intestine. Prebiotics enrich a particular category
of bacteria in the large intestine that is proven beneficial to gut health. While numer-
ous bacteria provide health benefits to their host, the families of Bifidobacteriaceae
and Lactobacillaceae specifically dominate the published research even though they
merely comprise less than 0.01% of the intestinal microbiome. Before the 1960s,
Bifidobacteria was known as Lactobacillus bifidus because it was observed to ferment
carbohydrates into lactic acid. It was later discovered that Bifidobacteria employs
a unique fructose-6-phosphate phosphoketolase pathway to ferment carbohydrates.
This characteristic feature has allowed Bifidobacteria to dominate the scientific con-
versation around prebiotics. Advances in whole genome sequencing technology (see
the previous chapters) challenge the conventional consensus of probiotic definition.
However, this progress is impeded because many of these species cannot be cultured
and subsequently enumerated and characterized outside their host.

TYPES OF PREBIOTICS
As far back as 1921, several experiments described the enrichment of Lactobacilli
in humans following the consumption of carbohydrates (Gibson et al. 2017). The
first formal definition for prebiotics was established in 1995 as a “nondigestible food
ingredient that beneficially affects the host by selectively stimulating the growth and
activity of one or a limited number of bacteria already resident in the colon” (Cheplin
and Rettger 2011). The definition of prebiotics has expanded over the years as
advances in metagenomics and metabolomics have made it possible to take a clearer
snapshot of the gastrointestinal environment (Gibson et al. 2017). Genomic technolo-
gies have expanded the definitions of nondigestible foods, beneficial bacteria, and
the site of interaction (Gibson et al. 2017). This definition continues to evolve, and
emerging evidence demonstrates that synergistic activities between microorganisms
are not limited to the GIT and nondigestible carbohydrates (Gibson et al. 2017). Here,
we will define prebiotics as a food source established to sustain microorganisms. The
result is a symbiotic relationship with sustained benefits to the host. Here, we discuss
the specific nutrients and microorganisms that support such a beneficial relationship.
The popularity of gut health in the wellness industry has allowed the rapid expan-
sion of products that support beneficial microbes. Several products on the market of
undefined composition claim prebiotic capabilities (Hume, Nicolucci, and Reimer
2017). Here, we approach the nondigestible food ingredients/metabolites with an
evidence-based approach demonstrating stimulation of the growth and metabolism
of beneficial gut microbes. Prebiotics must confer specific positive changes in gut
Dietary Modulation of the Gut Microbiota by Prebiotics 159

microbial communities and preferentially stimulate the growth of beneficial gut

microbes. In addition, prebiotics should be resistant to gastric acidity and mamma-
lian digestive enzymes, allowing them to enter the colon intact.

The most common prebiotics with a strong basis of evidence are indigestible carbo-
hydrates (i.e., dietary fiber, such as inulin and oligosaccharides) (Collins and Reid
2016). Although prebiotics are derived from digestible fiber, not all fiber types are
prebiotics (Collins and Reid 2016). Bacteria differ in their carbohydrate processing
capabilities, and most gut microbiota members selectively metabolize mono- and
oligosaccharides. Complex polysaccharides often require multiple microorganisms
with specialized enzymatic repertoires for complete digestion to utilizable energy
units. Most commercially available prebiotic carbohydrates come from plants; how-
ever, emerging research on animals, algae, and bacteria provides promising alterna-
tives (Gurpilhares et al. 2019).
Numerous food compounds are resistant to digestion and are used for fermentation
by gut microflora. Saccharolytic fermentation occurs with nondigestible carbohy-
drates and favors the growth of some specific bacteria genera such as Bifidobacterium
and Lactobacillus. These saccharolytic bacteria produce short-chain fatty acids
(SCFA) in the colon, namely acetate, propionate, or butyrate, which contribute calo-
ries, absorbed by the gut, and extend the putative effect on systemic inflammation.

Oligosaccharides
Oligosaccharides are relatively small chains ranging from three to ten simple
saccharides, eliciting a variety of functions in cell biology and host immunity.
Oligosaccharides are classified by their monosaccharide constituents and linkages to
lipids (glycolipids) or amino acids (glycoproteins). Not all oligosaccharides occur as
components of glycoproteins or glycolipids. Raffinose is a storage or transport car-
bohydrate in plants, and maltodextrins result from the microbial breakdown of larger
polysaccharides such as starch or cellulose (Fernandes et al. 2017).
In this section, a particular focus is placed on specific examples of oligosaccha-
rides particularly interested in those with a prebiotic function, namely fructooligo-
saccharides (FOS) and galactooligosaccharides (GOS). FOS elicit between 30% and
50% of the sweetness that sucrose confers. They are different from fructans and
inulin, which can be precursors of oligosaccharides, and demonstrate a much higher
degree of polymerization than that of FOS and GOS (Figure 6.1).

Human Milk Oligosaccharides

Intestinal flora plays a crucial role in an infant’s physiological postnatal GIT develop-
ment (Fanaro et al. 2005). Several studies have demonstrated that the mode of deliv-
ery influences the infant’s gut colonization flora (Fanaro et al. 2005). Maternal milk
is arguably an essential source of oligosaccharides for humans since it is delivered
during critical development. Human milk contains remarkably more oligosaccha-
rides than bovines, 5.15 vs. 0.05 g/L, respectively. Oligosaccharides are an evolution-
ary boon to the infant that aids in establishing immunity. Many researchers attempt
160 The Gut Microbiome: Bench to Table

FIGURE 6.1 Building blocks of oligosaccharides representing the combinations of simple

sugars and glycosidic linkages found in natural products.

to formulate products enriched with human milk oligosaccharides (HMO) to reverse

a dysbiotic system. Nevertheless, whether the application of HMO in the adult diet
can shift the gastrointestinal microflora and if the shift can indeed produce beneficial
downstream effects remains unclear.
For HMO to function as prebiotics, GOS and FOS must enter the colon intact and
promote the growth of probiotics given that 10%–80% of the HMO is detected in the
infant feces depending on the stage of infant development. These data suggest that
the colonized microbiome can utilize more available substrates (Bode 2012) as the
infant grows (Kelly 2009; Bode 2012). In vitro experiments have proven that both
Bifidobacteria and Lactobacilli not merely survive but actually thrive on HMO. The
relative abundance of HMO translates to 90% GOS and 10% FOS. Based on the
existing data, it could be hypothesized that the 9/1 ratio is ideal for the growth of
these bacteria and possibly exerts a symbiotic effect (Kelly 2009). Small in vitro pilot
trials using the fecal flora obtained from breastfed infants produced the same SCFA
profile as isolated HMO with a formula of 9/1 ratio GOS/FOS (Mahovic, Tenney,
and Bartz 2007; Fanaro et al. 2005) (Figure 6.2). The results were significantly dif-
ferent from the data obtained from the control group comprised of infants fed a for-
mula supplemented with 0.8 g maltodextrins as a placebo (Fanaro et al. 2005). More
recently, it has been demonstrated that both ingredients of the prebiotic mixture are
detectable in stools. The experimental data and the results obtained in formula-fed
infants indicate that the FOS/GOS mixture can induce the development of an intes-
tinal flora quite similar to that of breastfed infants. While these initial findings are
promising, the therapeutic application of HMO still presents many challenges.
Dietary Modulation of the Gut Microbiota by Prebiotics 161

FIGURE 6.2 General structure of milk oligosaccharides and a comparison between human
and bovine oligosaccharides.

The advancement of metabolomics and proteomics has allowed for the in-depth
analysis of HMO constituents. The findings from these studies suggest that each
mother’s HMO composition constitutes a unique fingerprint rather than being some-
thing homogeneous across the population (Bode 2012; Azad et al. 2018). There
are several maternal determinants of the final composition of human breast milk.
Lifestyle, exercise, and health status are predictors of the relative abundance of
fats, proteins, and oligosaccharides overall (Moossavi et al. 2019; Azad et al. 2018;
Addison et al. 2019). However, the relative abundance of individual oligosaccharides
is driven primarily by genetics. Principle coordinate analysis of 10,000 human milk
samples taken from six continents shows two distinct clusters (Azad et al. 2018).
The differences have been linked to a single nucleotide polymorphism (SNP) that
encodes for an enzyme called fucosyltransferase II (fucII) that adds L-fucose to the
oligosaccharide chain forming a specific α-1,2 linkage (Bode 2012). These SNPs
map out geographically, as shown by genome-wide sequence analysis and transcrip-
tomics. For example, 90% of Peruvian women have an active fucII, but that number
goes down to 65% on the African continent (Kober, Zehe, and Bode 2013; Bode
2015). Several additional SNPs are associated with the formation of different HMO
patterns, and more information can be found at the University of California, San
Diego, MOMI Core Institute.

Polysaccharides
Polysaccharides are heterogeneous macromolecules composed of monosaccharides
linked by glycosidic linkages. Typical examples of biologically essential polysac-
charides are starches (glucose polymers), a mixture of amylose and amylopectin,
162 The Gut Microbiome: Bench to Table

and the animal counterpart glycogen, cellulose, chitin, and pectin (O’Connor et al.
2017). As with the previous section, most of the research on prebiotic polysaccha-
rides focuses on their bifidogenic effects. Research is still probing the “tip of the
iceberg” regarding polysaccharides and gut microbiota. Not all polysaccharides
enter the large intestine intact, but the ones that do tend to have bifidogenic effects
(O’Connor et al. 2017). Our current understanding suggests that this is because of the
“resistant” nature of the molecule itself and the enzymatic repertoire of bacteria like
Bifidobacteria. This assumption is not corroborated in human models when investi-
gating in depth the types of bacteria affected by dietary changes.

Inulin
Inulin is a generic term that covers all linear fructans with beta (2−1) fructosyl-fructose
glycosidic bonds (Kelly 2008). The beta configuration of the bonds between fructose
monomers and inulin-type fructans resists enzymatic hydrolysis by human salivary
constituents and small intestinal digestive enzymes allowing for them to pass to the
large intestine intact. As a result, inulin-type fructans are indigestible and fermented
in the colon (Kelly 2008). Inulin is present in over 3,000 vegetables and extensively
present in plants. Commercially available natural inulin mostly comes from chicory
root, dahlia, and Jerusalem artichoke, while synthetic inulin is prepared from sucrose.
Inulin is commonly used in processed foods and as a nondigestible carbohydrate to
replace sweeteners and is utilized in low-calorie foods (Shoaib et al. 2016).
Inulin stimulates the development and metabolic action of a limited number of
bacteria in the colon, particularly Bifidobacteria and Lactobacilli (Kelly 2008).
In vitro trials have demonstrated that colonic fermentation depends on inulin’s
chain length. Fermentation time is dependent on chain length, with longer chain
inulin allowing for the initiation of bacterial fermentation in the distal part of the
colon. The nutritional utilization of inulin and oligofructose provides a possible
way to encourage microbial balance and overcome the difficulties in colonizing
the gut mucosa. For this reason, inulin is used to reduce and replace sugar in food
products. Inulin has been demonstrated to enhance the growth sustainability of
L. acidophilus, L. rhamnosus, and B. lactis when added to milk, yogurt, and ice
cream (Kelly 2009).
The consumption of inulin has demonstrated numerous benefits to the host diges-
tive tract. Inulin has improved bowel movements, GI symptoms, quality of life, and
increased fecal output in various adult populations (Watson et al. 2019). These shifts
are generally attributed to the ability of inulin to reach the colon intact, where inulin
is metabolized by bacteria, leading to beneficial byproducts. These improvements
have generally been associated with modest changes in gut microbiota composition,
specifically Bifidobacteria, Anaerostipes, and Clostridium in constipated subjects
(Vandeputte et al. 2017). A recent randomized, double-blind, placebo-controlled
cross-over trial with inulin isolated chicory root attempted to elucidate the relation-
ship between diet, bowel movement, and colon microbiota. The study demonstrated
that 10 g/d of inulin improved stool frequency and consistency in older adults who
experience constipation (<1 bowel movement/day); however, they could not detect a
shift in gut microbiota (Watson et al. 2019).
Dietary Modulation of the Gut Microbiota by Prebiotics 163

NON-CARBOHYDRATE PREBIOTICS
Conventional dietary knowledge suggests that a diet high in plant material is ben-
eficial for the host’s gut microbiota. As discussed in previous chapters, numerous
studies in humans have examined the dietary impact on gut microbiota composition
and divided the population into two groups: those that eat fruits and vegetables over a
certain level and those that do not. In addition, the relative abundance of one phylum
of bacteria relative to another can predict the risk of obesity (Singh et al. 2017). The
same phyla are also associated with the consumption of fruits and vegetables. The
correlation between health status, diet, and gut composition is putatively linked to
nondigestible carbohydrates. However, some ingredients are excluded when examin-
ing the relationship between certain foods and gut fermentation.

PolyPhenols
Polyphenols represent a new category of potential prebiotics. Until recently, little was
known about the metabolic fate of polyphenols in the intestinal tract. Berries have
been used for centuries as elixirs to help with gastrointestinal symptoms. In humans,
the intestinal absorption of dietary polyphenols is often slow and largely incomplete;
up to 85% of anthocyanins enter the colon intact and can be used as substrates for
microbial metabolism (Kahle et al. 2006). The capability for anaerobic digestion
of berries in gastrointestinal environments is reflected by increased Bifidobacteria
populations and other microbes that catabolize the diverse berry compounds avail-
able, especially polyphenols (Lacombe et al. 2013).
The use of berries for their prebiotic effect has been tested in clinical trials to
improve the clinical picture and the well-being of patients. Promising beneficial
effects demonstrated with berries include positive shifts in gut microbiota com-
position. In vitro human fecal batch cultures have demonstrated the enrichment of
Lactobacillus and Bifidobacteria with 1 g/L of gallic acid and 200 mg/L of antho-
cyanins (Håkansson et al. 2009). These results demonstrate the benefits of dietary
enrichment of berries and its impact on bacterial communities with unique func-
tional repertoires by promoting their growth in competitive environments.
Bioprotective effects of polyphenols and other nutrients observed in vitro are not
entirely transferable to health effects observed in vivo; therefore, it is essential to use
in vivo models to understand mechanisms. Recent studies using the Sprague Dawley
(SD) rat model, fed polyphenols extracted from blueberry and blackberry via gavage,
demonstrated increased Lactobacillus and Bifidobacteria (Bò et al. 2009; Vendrame
et al. 2013). Actinobacteria, with known impacts on human health, namely Slackia,
Bifidobacteria, and Coriobacteriaceae spp, were detected in higher abundance
in SD rats fed a blueberry-enriched diet (Lacombe et al. 2013, 2012). In humans,
dietary treatment with blueberry increased the population of Bifidobacteria more
than two-fold, demonstrating the prebiotic activity of berry polyphenols (Vendrame
et al. 2013). For clinicians, these findings are significant due to the growing interest
in probiotic bacteria and the perceived benefit of increasing their numbers in the GIT
to attain their health benefits.
164 The Gut Microbiome: Bench to Table

Additional metabolic byproducts created by probiotic bacteria such as Bifidobacteria

and Lactobacillus are discussed below. We specifically look into the saccharolytic and
proteolytic byproducts of probiotics such as Bifidobacteria and lactic acid–producing
bacteria whereby small-chain fatty acids (SCFAs) such as butyrate, acetate, and pro-
pionate are produced (Younes et al. 2001). Downstream effects of SCFAs are utilized
as energy sources by host epithelial cells, modulate intestinal immunity, enhance bar-
rier function, and inhibit the adhesion of pathogenic bacteria (Younes et al. 2001).

METABOLISM OF NUTRIENTS BY THE GUT MICROBIOTA

The microbial role in the metabolism of ingested bioactive compounds is not well
understood. However, recent evidence suggests that bacterial metabolism has a
substantial potential for the bioactivation of essential nutrients and detoxification
(Lacombe et al. 2013, 2012). The microbial enzymatic biotransformations may also
be relevant for converting many classes of compounds, including flavonoids, iso-
flavonoids, lignans, phenolic acids, fiber, and tannins (Kahle et al. 2006; Heinonen
2007). Cleavage of the glucosidic bond in the anthocyanin structure is proposed as
the first step in bacterial anthocyanin bioconversion, involving β-glucosidase activ-
ity (Bò et al. 2009; Pap et al. 2021). New metabolites, including gallic, syringic, and
homogentisic acids appeared due to bacterial enzymatic action on malvidin, some of
which have been described as more bioavailable and bioactive than the former origi-
nal molecule (Wu et al. 2004). In addition, recent studies documented a 20% increase
in xenobiotic degradation and a two-fold increase in benzoate degradation in the
proximal colon of rats fed an 8% blueberry diet (Lacombe et al. 2013). The genome
sequence of Bifidobacterium longum encodes a large number of predicted proteins
(more than 8%) related to the catabolism of nondigestible plant polymers, including
enzymes involved in the degradation of complex polysaccharides and xenobiotics
(Gill et al. 2006; Claus et al. 2011). In termite hind guts, members of the phylum
Actinobacteria have demonstrated their involvement in xenobiotic metabolism (Claus
et al. 2011). These microorganisms could contribute to the degradation of benzoate
compounds derived from berries. Therefore, the protective anti-inflammatory effect
of blueberries may be credited with positive shifts in the composition of the micro-
biota and concomitant regulation of microbial metabolism (Del Bo’ et al. 2010). The
downstream effects of a berry-enriched diet are demonstrated by increased plasma
antioxidant capacity while protecting the lymphocytes against oxidative DNA dam-
age and lower vascular reactivity and sensitivity to an α-adrenergic agonist in the
aorta of SD rats (Vendrame et al. 2011).

Prebiotics can improve immune function by increasing the population of beneficial

bacteria. Irritable bowel syndrome (IBS) and Crohn’s disease (CD) are the most com-
mon causes of GIT stress and are directly associated with the nervous system, muco-
sal barrier, neurotransmitters, hormones, and immune system (Menezes and Da Silva
Dietary Modulation of the Gut Microbiota by Prebiotics 165

2017). Interaction of prebiotics with pattern recognition receptor attributes to altering

the expression of cytokines. The mechanism of immunomodulation by prebiotics
influences pro-inflammatory cytokines, interferons (IFN-γ), and interleukin-2 (IL-
2) in the gut lumen (Cavaglieri et al. 2003; Khangwal and Shukla 2019). Prebiotics
found in mushrooms such as proteoglycans and polysaccharides are renowned for
their antitumor activity (Danneskiold-Samsøe et al. 2019). The available glucans and
heteroglycans in mushrooms can activate macrophages, splenocytes, and thymocytes
(İspirli, Demirbaş, and Dertli 2018).
What we learn from the infant/mother relationship is essential for application
in the adult community. Inflammatory bowel diseases are increasing in the United
States’ adult population including a significant dietary component (Bode 2009).
Macrophages play a significant role in the inflammatory process of ulcerative coli-
tis and CD (Bode 2009). In cell models, HMO and macrophage combined, we see
a significant reduction in the inflammatory marker, interleukin-6 (IL-6) (Manthey
et al. 2014). When looking at the structure–function relationship between HMO and
reduction of IL-6, 3 sialyllactose (3-SL) demonstrates the most significant effect
(Bode 2018); however, determining the active binding site is very difficult. RNA-seq
studies demonstrated that the lipopolysaccharide-binding site, toll-like receptor 4
(TRL-4), and the sialic acid-binding site lectin are not the target for 3-SL (Manthey
et al. 2014). While the receptor for 3-SL is still unknown, the RNA-seq data dem-
onstrate that 90% of genes induced in the presence of 3-SL are involved in lipid
metabolism (Manthey et al. 2014). These are the same pathways involved in resolv-
ing the inflammatory process in the cell. Even though TRL-4 is not the receptor
binding site, HMO acts as an intermediary, switching off the macrophage recruit-
ment significantly faster (Manthey et al. 2014). This has significant implications for
other autoimmune diseases as well. Data from cell and rat models suggest that HMO
can diminish the progression and symptoms of rheumatoid arthritis using a similar
mechanism (Bode and Jantscher-Krenn 2012).
Prebiotics can stimulate the immune response by altering the inflammatory
cytokine cascade and decreasing the accumulation of disease-causing pathogens.
Various studies on animals and humans have demonstrated that prebiotics inhibit
pathogens by enhancing the Lactobacillus and Bifidobacteria population (Looijer-
van Langen and Dieleman 2009). Prebiotic monomers of mannose adhere to the
intestinal tract and diminish the infections by Salmonella (Revolledo, Ferreira, and
Ferreira 2009). In addition, mannose binds to the virulence factor type 1 fimbriae
and inhibits the infection (Revolledo, Ferreira, and Ferreira 2009). A study with
Wistar rats demonstrated that the inulin as a potential prebiotic, as it increased the
population of Bifidobacterium and Lactobacillus spp., activates toll-like receptors
(TLRs) such as TLR-2, TLR-5, and TLR-7, which ultimately improve the immunity
(Revolledo, Ferreira, and Ferreira 2009). A mixture of oligofructose and inulin and
FOS alone could improve antibody response against viral infections such as influ-
enza and measles (Langkamp-Henken et al. 2004). Apart from this, a supplement of
prebiotics reduced the usage of antibiotics and prevented the incidence of febrile sei-
zures in infants (Saavedra and Tschernia 2022). Fructans stimulate the serum IL-4,
CD282+/TLR-2+ myeloid dendritic cells, and TLR-2 (Saavedra and Tschernia 2022)
and decrease the IL-6 and phagocytosis in monocytes and granulocytes in healthy
166 The Gut Microbiome: Bench to Table

volunteers (Guigoz et al. 2002). Notably, infants supplemented with FOS have shown
a reduced risk of immune diseases (Moro et al. 2006). GOS improves the activity
of natural killer cells by increasing the IL-8, IL-10, and C-reactive protein (Vulevic
et al. 2013, 2015). It has also been reported to prevent the risks of atopic dermatitis
and eczema (Moro et al. 2006).
Necrotizing enterocolitis (NEC) is a severe gastrointestinal necrosis condition in
premature neonates. It is one of the most common and often fatal intestinal disor-
ders in preterm infants. Clinical trials have demonstrated a high correlation between
HMO in breast milk and positive outcome measures in NEC progression (Bode
2015; Autran et al. 2018). As previously discussed, HMOs have a bifidogenic effect
in the gut and protect neonates from NEC and pathogenic bacterial infection (Knol
et al. 2005). However, increased levels of bifidogenic oligosaccharides outcompet-
ing pathogens do not entirely describe HMOs’ beneficial effects on NEC (Bode
2015). A randomized study revealed that FOS and GOS enhance the population of
Bifidobacteria but had no significant consequence in controlling the progression
of NEC (Srinivasjois, Rao, & Patole, 2009). Research on HMOs has demonstrated
improved immunity benefits similar to breastfed infants (Bode 2015). Preclinical
research also shows that HMOs reduce intestinal discomfort, reduce food allergy
symptoms, and enhance cognition, leading to myriad health benefits for infants
(Autran et al. 2018). The link between HMO and necrotizing colitis is associated
with competitive binding within the intestinal glycocalyx (Bode 2015). For patho-
gens to survive in the gut, they must attach to epithelial cells; otherwise, they will be
eliminated through excretion. This process occurs through bacterial lectins attach-
ing to glycans on the surface of epithelial cells. In the case of necrotizing colitis,
there is a specific oligosaccharide called disialyllacto-N-tetraose (DSLNT), which
is associated with protective effects in mouse models (Bode 2006). A human cohort
study with preterm infants demonstrated that necrotizing colitis was lower in infants
who received higher amounts of DSLNT from their mothers (Bode 2006). DSLNT
content in breast milk is a potential noninvasive marker to identify infants at risk of
developing NEC and screen high-risk donor milk. In addition, DSLNT could serve
as a natural template to develop novel therapeutics against this devastating disorder.
This association is significant to note when considering therapeutic applications of
HMO in infant formula (Autran et al. 2018).

PREBIOTICS AS FUNCTIONAL FOODS

Consumers are aware of the potential healing properties of dietary regimens and
foods and have fueled the demand for products that promote health. The desire for
food that meets nutritional needs and can improve health has led food scientists to
develop the concept of functional foods. A formal definition of functional foods was
determined by the European Commission Concerted Action on Functional Food
Science, which states, “A food can be regarded as ‘functional’ if it is satisfacto-
rily demonstrated to affect beneficially one or more target functions in the body,
beyond adequate nutritional effects, in a way that is relevant to either an improved
state of health and well-being and reduction of risk of disease” (Wang et al. 2020b;
Bigliardi and Galati 2013). The definition of functional foods can be expanded to
Dietary Modulation of the Gut Microbiota by Prebiotics 167

include improving general body conditions, reducing the risk of specific diseases,
and potential use for the prevention and treatment of illnesses (Bigliardi and Galati
2013). Functional foods differentiate themselves from conventional foods consumed
in a standard diet by containing bioactive components whose physiological activity
is scientifically proven (Bigliardi and Galati 2013). Categories of functional foods
include those that naturally contain one of the bioactive components, fortified foods
in which the native bioactive level is enhanced, and processed foods in which the
bioactive compound is intentionally added (Bigliardi and Galati 2013).
A typical Western diet contains moderate levels of prebiotics through consum-
ing various fruits and vegetables. However, the number of prebiotics contained in
many fruits and vegetables is not sufficient or not consumed in an amount that would
significantly alter the composition of intestinal microflora (Jovanovic-Malinovska,
Kuzmanova, and Winkelhausen 2014). Therefore, consumers look toward supple-
mentation to increase the effective dose of many prebiotics. This affects the puta-
tive mechanism of action by altering the relative abundance of specific nondigestible
components in the diet. The mechanism by which prebiotics can attenuate the pro-
gression of diseases is of great interest to researchers and clinicians.
Fiber supplementation is one of the most common applications for prebiotics in
the Western diet. Most (90%) of the US population does not consume 15 g/d of rec-
ommended dietary fiber doses. Many consumers turn to fiber supplements, typically
isolated from a single food source (McRorie et al. 2015). However, of the fiber supple-
ments on the market today, only a minority possess the physical characteristics that
underlie the mechanisms driving clinically meaningful health benefits. Prebiotics
are clinically accepted and recommended as dietary supplements for the treatment of
constipation. These carbohydrates and prebiotics are fermented in the large intestine
and diminish the transport time of assimilated food by hydrating and increasing acid
production (Den Hond, Geypens, and Ghoos 2000). Intestinal-associated diseases
like IBS and CD are also thought to benefit from these fermentative processes.
Many prebiotic functional food formulations try to take advantage of the inher-
ent presence of probiotics in the food product. Dairy foods are a popular probiotic
food category worldwide and intrinsically contain nutrients that maintain the pro-
biotic organism. Examples of popular probiotic functional foods are yogurt, which
represents about 35% of sales overall, followed by cultured drinks, kefir, cheese,
ice cream, and infant formula (Tamime et al. 2006; Saavedra and Tschernia 2022).
Fermented products, such as yogurt, kefir, and cheese are commonly enhanced pro-
biotic cultures, and more recently, unfermented dairy matrices, such as ice cream
and butter, have also been enhanced with probiotic cultures (Koebnick et al. 2003).
Whey protein, which is rich in nutrients and potential prebiotics, has been forti-
fied with probiotics (Tenorio et al. 2010; Suomalainen et al. 2006). The functional
use of whey, a byproduct of cheesemaking that is often discarded, has been a focus of
functional food development (Tenorio et al. 2010). The commercially available oligo-
saccharides are usually derived from cow milk, plants, and fermented processes and
are very different structurally from what is found in HMO (Bode 2015). Commercial
GOS and FOS tend to be linear carbohydrate chains (Vulevic et al. 2013). However,
little is known about oligosaccharides’ structure–function relationship regarding
therapeutic applications (Vulevic et al. 2015).
168 The Gut Microbiome: Bench to Table

Current knowledge stems from associated genetic differences from cohort studies
and cell/animal models observed within populations. For example, we now know that
HMO is not just a preferential food source for Bifidobacteria but have antimicrobial
properties and can outcompete other bacteria for binding site in the intestinal tract
and directly modulate epithelial cell responses (Bode 2015). Applying HMO formu-
lation in the adult population is much more complicated than in infants. Although
there are many products on the market that claim they can provide increased gut
function, they do not take into account the diversity of HMO (Bode 2015). Adults
are not blank slates when it comes to gut microbiota composition, which means its
alteration is much more difficult. In addition, diet and lifestyle are very heterogone-
ous among humans, generating many variations in the human datasets (Bode 2015).
This variation makes causal relationships between the structure/function of HMO
and the gut microbiota challenging to establish.
Many consumers have allergies to milk proteins, lactose intolerance, and may
have adopted diet choices that partially or entirely exclude food of animal origin.
In response to the increased market for dairy products of nonanimal origin, plant-
based probiotic/prebiotic products are available to consumers. Many raw materials
of plant origin often contain prebiotic substances, which can stimulate the growth
of probiotic microorganisms both during the processing steps and during storage
(Veereman 2007; İspirli, Demirbaş, and Dertli 2018). The use of nuts and grains,
such as almonds, rice, and oats, has been widely adopted as animal milk as these
products are generally perceived as healthy by the consumer and have an appealing
sensory profile (Håkansson et al. 2009; Vulevic et al. 2015). These foods contain
additional functional benefits, such as fibers, vitamins, minerals, flavonoids, and
antioxidants. Enrichment with probiotic cultures is carried out through osmotic
dehydration or vacuum impregnation techniques or by directly adding the micro-
bial cells to juices. A fermentation step may occur in the latter case, creating addi-
tional bioactive nutrients.

Numerous novel prebiotic formulations are abundantly found in food-processing

and agricultural byproducts. The sources of potential prebiotics vary from the agri-
cultural silage from wheat, mushroom, and seaweed products to animal products
like dairy effluents (Shoaib et al. 2016; Scott et al. 2011; Danneskiold-Samsøe et al.
2019). Often, disposal of these byproducts confers negative impacts on the eco-
system by burdening the waste stream. Therefore, food-processing byproducts that
contain high nutritive values can be used as the primary source of functional or
nutraceutical ingredients in a process whereby nutritional valorization of these prod-
ucts is achieved. Manufacturers of prebiotics are shifting the utilization of their raw
materials to lower-cost alternative sources. Prominent raw materials or substrates
to produce prebiotics are roots of chicory and Jerusalem artichoke and lactose from
the agriculture and dairy industries, respectively. However, due to high demand, the
cost of these raw materials is exponentially increasing every year. Therefore, vari-
ous byproducts derived from food processing, including agricultural-based silage,
are being developed.
Dietary Modulation of the Gut Microbiota by Prebiotics 169

There is a burgeoning market for prebiotic formulations incorporated into various

foods such as dairy products, bread, cereals, dietary supplements, and others. For exam-
ple, soybean whey, a byproduct of tofu manufacturing, contains nondigestible polysac-
charides and oligosaccharides. Usually, this product is discarded, but soybean whey
may be used as a valuable ingredient in functional foods (Tenorio et al. 2010). The solid
wastes that are accumulated in malting industries—namely barley husks, spent grains,
and grain fragments—contain oligosaccharides and can be fermented to generate suc-
cinate, lactate, formate, acetate, propionate, and butyrate, exhibiting prebiotic poten-
tial (Gullón et al. 2021). Wang, Liang, and Liang (2010) studied that mung beans may
enhance the growth of Lactobacillus paracasei. Gullón, González-Muñoz, and Parajó
(2011b) investigated the prebiotic potential of FOS-rich refined product from apple pom-
ace, processed by simultaneous saccharification and solid-state fermentation.
Combining prebiotics and probiotics in the same functional food may present
a synergistic effect. The popularity of dairy products fortified with prebiotics and
probiotics is also motivated by consumer demand for appropriate texture and fla-
vor. The sensory profile of drinkable yogurts made with prebiotics, namely soluble
corn fiber, polydextrose, and chicory inulin, is an acceptable vehicle to deliver
the probiotics. Ice cream supplemented with Lactobacillus casei and 2.5% inulin
demonstrated improved nutritional and sensory properties (Di Criscio et al. 2010).
Different dehydrated prebiotic fibers, namely, oat bran, β-glucan, and green banana
flour, provided the substratum for adherence, and trehalose acted as a cell protec-
tant prolonging the viability of L. casei (Di Criscio et al. 2010). In a sensory evalu-
ation study, the prebiotic oat bran added to a dairy fruit beverage has been well
accepted by consumers (Guergoletto et al. 2010). Apple purees enriched with two
commercial FOS prebiotics [Beneo GR® (inulin) and HSI®] showed stability for
30-day storage at 4°C (Keenan et al. 2011). However, a large quantity of prebiotics
was required to deliver the desired effect, and high hydrostatic pressure posed the
risk of certain prebiotic hydrolysis.

Consumers are becoming increasingly health-conscious, leading to consumer

demand emphasizing functional foods. The shift of consumer preferences, needs,
and acceptances is a dynamic process; hence, the maintenance of food quality via
technological innovation is evidently important (Danneskiold-Samsøe et al. 2019).
The development of prebiotic food formulations is crucial for the future functional
food market. Therefore, there are constant concerns about recovering and maintain-
ing healthy intestines stocked with prebiotic nutrients that support the survival of
beneficial health agents. Understanding the molecular details of gut microorganism
metabolism of dietary ingredients is essential for creating prebiotic and probiotic for-
mulations that benefit consumers. There is a limited number of quantification meth-
ods that analyze complex oligosaccharides and other prebiotic molecules. This had
hindered the understanding of probiotic interactions with prebiotics.
In most cases, viable probiotic microorganisms are necessary to deliver the pur-
ported health benefits to the host gut. The industry for microbial viability must
be high to overcome the hurdles of cellular deterioration over time and adverse
170 The Gut Microbiome: Bench to Table

conditions of the host gut. Intrinsic features of the food matrix can strongly endanger
their viability in food, processing treatments applied, competitive background flora,
conditions, and duration of storage. Probiotic microorganisms are relatively sensitive
compared to some spoilage microorganisms, and the number of viable bacteria can
decrease by 1–2 log CFU (colony-forming unit)/g or even more during production
and storage. Minimum values of at least 106 –107 CFU/g are needed for residency
in the gut; therefore, the microbial dose in the original product needs to be 108–109
probiotic cells (Jayamanne and Adams 2006). Since the health effects depend on the
conditions of the product at the time of consumption, the maintenance of microbial
viability must be ensured over its entire shelf-life.

The carrier matrix has a significant effect on the quality of probiotic products, as its
composition and characteristics can alter the viability and effectiveness of probiot-
ics. One of the main obstacles is represented by the acid pH since Lactobacilli have
an optimal growth pH of 5.5–6.0 and Bifidobacteria of 6.0–7.0 (De Vuyst, 2000).
Acidic pH, which is relevant for fermented foods, fruit, and vegetables, leads to an
increase in the concentration of organic acids in the undissociated form, therefore
increasing the antimicrobial effect of these acids. Furthermore, at low pH values
(below 4.5), cells need significant energy to keep the intracellular pH constant, seiz-
ing adenosine triphosphate from other cellular needs, causing cell death. Low pH
(approx. 3.5) was shown to be the main culprit in the loss of viability of L. rhamno-
sus, L. plantarum, and L. casei in cherry juice, especially during refrigerated storage
(Nematollahi et al. 2016). In fermented milk, L. acidophilus tolerates acid conditions
better than Bifidobacteria, proving the careful selection of the strains used in each
probiotic food to be crucial (Tamime et al. 2006).
Water content in food formulations, whether expressed as moisture content or water
activity (aw), plays a determining role in the survival of probiotics in food. The aw, i.e.,
the availability of water for microbial growth, varies based on the product’s intended
shelf. If a food product has a water activity range above 0.85, it will have to be refrig-
erated or use another barrier to control pathogen growth (Gomand et al. 2019). At aw
above 0.85, there is a substantial loss of prebiotic viability as other spoilage microor-
ganisms compete for nutrients. In contrast, a food product with a water activity between
0.60 and 0.85 will not require refrigeration but will have a limited shelf-life due to
yeasts and molds. Lastly, food with a water activity below 0.60 will have an extended
shelf-life, even without refrigeration. With probiotic formulations, food manufacturers
can employ several tactics, including drying, freezing, or adding solutes like salt or
sugar, helping to reduce their products’ water activity levels, extending their shelf-life.
There is a potential benefit to adding probiotics to low moisture or intermedi-
ate moisture foods, such as chocolate, peanut butter, cereals, and dried fruit paste
because they are stable for prolonged periods (Finn et al. 2013). For example, a via-
bility loss of less than 1 log CFU/g of L. rhamnosus was observed in peanut butter
(aw approx. 0.35) stored at 25°C for 27 weeks (Klu et al. 2012). In addition, osmolarity
in foods containing high amounts of sugar or salt, such as fruit purees, jams, or aged
cheeses, plays a role in the survival of microorganisms in foods. Generally, sugar and
Dietary Modulation of the Gut Microbiota by Prebiotics 171

salt greatly influence the growth and survival of probiotics in food as their presence
increases osmotic pressure. Salt is widely used in foods to prolong its shelf-life, but
at high concentrations (~4%–6%), it can put the viability of probiotics at risk. The
tolerance to osmotic stress is strain-specific and depends on the phospholipidic com-
position of the membrane. Lactobacillus casei demonstrated slower growth in the
presence of high glucose concentrations, as well as Bifidobacteria, while L. planta-
rum demonstrated more resistance to osmotic stress (Homayouni et al. 2008).
Since most probiotic species are strictly anaerobic, dissolved oxygen in the matrix
is critical for their survival in foods, as hydrogen peroxide is formed through differ-
ent mechanisms both at intracellular and extracellular levels. Probiotic bacteria are
practically devoid of an oxygen scavenging system, which causes the reduction of
oxygen to hydrogen peroxide. The toxicity of hydrogen peroxide is due to its ability
to block fructose-6-phosphofructoketolase, a key enzyme for sugar metabolism. In
addition, superoxide and hydroxyl radicals are formed in dissolved oxygen due to the
oxidation of membrane lipids in the cell, which induces DNA damage and causes cell
death (Önneby et al. 2013). Different processing steps can contribute to the enrich-
ment of the matrix in dissolved oxygen, such as homogenization, cooling after heat
treatment, and mixing with ingredients. Although all probiotics are anaerobic, oxy-
gen tolerance varies enormously between genera and species. Indeed, Bifidobacteria
tend to be more sensitive than lactic acid bacteria. In the presence of oxygen, the most
sensitive bacteria change the profile of membrane fatty acids and show an elongation
of the lag phase and a substantial reduction in lactate produced (Looijer-van Langen
and Dieleman 2009; Patel and Denning 2013).
Probiotic microorganisms can be exposed to thermal stress when producing food-
containing probiotics. When heat treatments are used to inactivate pathogenic and
spoiler microorganisms in raw materials, thermal stress on probiotics can be easily
avoided by inoculating them downstream of the heat treatment or by using probiotic
species other than lactic acid bacteria and Bifidobacteria. The genus Bacillus coagulans
has been successfully used in the processing of various foods such as banana muffins
and waffles, brewed coffee, chocolate fudge frosting, and hot fudge topping, and the loss
of viability has been less than 1 log CFU/g in all cases (Majeed et al. 2016). However,
during fermentation, probiotics may undergo thermal stress. The production of yogurt
often requires that the milk is held at temperatures above 40°C–42°C, which can be
harmful to probiotics. Therefore, the industry has curated cultivars of probiotics more
resistant to thermal treatment. In yogurt making, the incubation temperature can be as
high as 44°C, which causes a loss in viability of L. acidophilus and Bifidobacterium
BB-12. At higher fermentation temperatures (e.g., 45°C), more heat-resistant L. brueckii
subsp. bulgaricus becomes the dominant species and produces a large amount of lactic
acid, hydrogen peroxide, and sometimes bacteriocins, which causes probiotic growth to
stop (Nematollahi et al. 2016). Therefore, to foster the biodiversity of probiotics, some
manufacturers inoculate species after the fermentation process.
Freezing is a critical process in the manufacture of ice cream. However, it presents
a technical challenge in the development of probiotic-enhancing products. Subzero
temperatures are detrimental to microbial viability because as the portion of fro-
zen water increases, the aw value of the liquid solution decreases. Furthermore,
osmotic pressure increases as water diffuses outside the cell, dehydrates, and freezes
172 The Gut Microbiome: Bench to Table

the extracellular water. The mechanical stress due to ice crystals damages the mem-
branes, the low temperatures shock the cells, the (harmful) solutes concentrate, and
the cells become dehydrated. Together with the progressive lowering of the tempera-
ture, all this determines the so-called cold shock, which causes cell death. In probi-
otic ice cream, L. rhamnosus loses viability during freezing of about 1.8 log CFU/g,
probably due to a combined effect of temperature lowering and oxygen incorporation
during mixing (Marino et al. 2017).
Developers of enhanced probiotic products attempt to harness the intrinsic pro-
tective substances that protect the viability of probiotics from the stresses accumu-
lated in the food matrix and during the production process and the GIT transit. The
compounds most frequently used for this purpose are carbohydrates, and many have
additional prebiotic effects. Sugars, especially those with low molecular weight,
interact with the polar heads in the cell membranes, replacing the water molecules
and stabilizing the membranes themselves during osmotic stresses. Low-molecular-
weight sugars, such as trehalose, sucrose, and lactulose, can improve the survival of
probiotics during freezing, dehydration, and storage both in products of animal and
plant origin (Betoret et al. 2017). Besides, they have also been shown to have direct
functional activities, such as a bifidogenic effect and an increase in the hydrophobic-
ity of the surface of probiotic cells, which is related to the ability to adhere to and
interact with the intestinal wall (Dlamini et al. 2019).
Even the most complex polysaccharides and fibers can improve the viability of
probiotics in food. These are dietary compounds that reach the gut in an undigested
form and are selectively fermented by the intestinal microbiota, thus stimulating
their growth and activity. However, their stimulation activity also aids the probiot-
ics added to foods. Consequently, the development of functional foods that contain
both probiotics and prebiotics (symbiotic foods) can provide a double physiologi-
cal advantage. Typically, prebiotic substances mainly come from plant food sources.
Inulin and FOS, widely used for this purpose, are commonly extracted from onions,
bananas, wheat, artichokes, garlic, and other whole foods. Other widely used oli-
gosaccharides, such as GOS and HMO, are of animal origin (Anadón, Martínez-
Larrañaga, Arés, & Martínez, 2016).
Even microorganisms are capable of producing extracellular substances that act
as prebiotics, such as exopolysaccharide (İspirli, Demirbaş, and Dertli 2018). The
effectiveness of prebiotics in the context of functional probiotic foods is linked to
the fact that these substances can be metabolized and used as a source of energy
by probiotics. Furthermore, owing to their physicochemical characteristics, they can
effectively protect cell envelopes, increase the glass transition temperatures in the
aqueous phase, retain water, and prevent the formation of ice crystals that may cause
mechanical damage to the cells themselves (Tymczyszyn et al. 2011). Due to these
activities, the effects essentially protect probiotics against acidity, high temperatures,
and dehydration. Their addition to food improves the stability of probiotics during
processing, storage, and in vitro digestion. In the blended carrot and orange juices,
the presence of 2% inulin improved the survival of L. plantarum during storage for
30 days at 4°C and during in vitro digestion, and a mixture of inulin, FOS, and GOS
had a similar effect on L. casei in a blended red fruit beverage (Bernal-Castro, Díaz-
Moreno, and Gutiérrez-Cortés 2019).
Dietary Modulation of the Gut Microbiota by Prebiotics 173

CONCLUSION
As the concept of prebiotics continues to evolve, researchers investigate the potential
benefits of their ingestion. There are many possible sources for prebiotics, which can
be found in the natural environment or be synthesized based on research indicating
certain functional groups with benefits. However, more work is needed to determine
which prebiotic regimen is best for dietary recommendations, while recommenda-
tions cannot probably be applied ubiquitously across the population. The future of
prebiotic supplementations may rely upon the intended hosts existing microbiome
and genetic profile. This opens a fascinating field of inquiry into tailoring probiotic
and prebiotic regimens for optimum health.

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Section III
T2–3 Effectiveness Trials
in General Population and
Regulatory Limitations
 7 Gut Microbiota and
Polyphenols: Discussing
a Powerful Interplay and
Its Effect on Health
Aleksandra S. Kristo
California Polytechnic State University

Dorothy Klimis-Zacas
University of Maine

CONTENTS
Introduction ............................................................................................................ 183
Overview of Polyphenols ....................................................................................... 185
Classification and General Description ............................................................. 185
Food Sources and Consumption........................................................................ 186
Overall Metabolism/Bioavailability and Excretion ............................................... 187
Microbiota Metabolism of Polyphenols and Health Effects .................................. 190
Gut-Derived Metabolites ................................................................................... 190
Health Effects of Polyphenol/Metabolite-Microbiota Interaction .................... 193
Considerations on Limitations of Human Studies, Novel Metabolic
Approaches, and Metabotypes .......................................................................... 201
References ..............................................................................................................205

INTRODUCTION
Gut microbiota and polyphenols, each in their own right, constitute research areas
of increasing interest due to accumulating scientific evidence indicating their sig-
nificant potential benefits and functions in human health and longevity. While
polyphenols have been investigated separately as bioactive compounds that can
confer notable health benefits in an array of metabolic conditions, the association
and interaction with the gut microbiome is less studied while is gaining increasing
interest lately.
Polyphenols, as plant-secondary metabolites minimally processed by human
metabolism, rely heavily on the metabolic capacities of the gut microbiota for
their absorption and potential biological actions. By producing bioactive poly-
phenol-derived compounds through enzymatic activities of a diverse community

DOI: 10.1201/b22970-10 183

184 The Gut Microbiome: Bench to Table

of microorganisms, gut microbiota can affect human health in a variety of ways.

Polyphenols can shape gut microbiota composition and function, thereby eliciting
beneficial changes in the gut ecosystem (Vendrame et al., 2011; Lacombe et al.,
2013). Elucidation of the specific bacterial populations and the metabolic processes
involved in producing the polyphenol-derived bioactive compounds, the specific
metabolites, and their mechanisms of action is deemed necessary to understand the
microbiota–polyphenol interplay and consequently implications for human health. In
this sense, one could argue that the current state of affairs in polyphenol and micro-
biota interplay research is found in its “infancy.”
Presently, human studies of microbial metabolism on polyphenols or polyphenol-
induced modification of microbiota are limited. Further, the evidence generated in the
available studies is inconsistent, owing to methodological challenges including the
variations in chemical nature, doses, single compounds or mixtures of polyphenols
under study, the dissimilar health status or the inherent interindividual microbiota
variations of the participants, the small study size, or the insufficient endpoints inves-
tigated. Therefore, drawing definite associations between the polyphenol–microbiota
interplay and health effects on the host as a result of such interplay can be daunting.
The multitude of polyphenols with different structures, signaling capabilities, syner-
gies, pathways involved, and physiological roles, makes it challenging to fully eluci-
date their short- and long-term health effects both positive and potentially negative,
let alone any additional effects due to interactions such as those with the microbiota.
The reported health benefits of plant-based diets, which to a significant degree
have been shown by the scientific literature, cannot be attributed to any specific
phytochemical(s). Rather benefits derive from the combined effects of various phy-
tochemicals including fiber, carotenoids, phytosterols, or polyphenols. The latter has
gained special research attention initiated by their early recognition as antioxidants
along with evidence indicating oxidative stress as the underlying cause of chronic
disease. Before realizing the function, metabolic capacity, and potential role of micro-
biota in human health, the bioavailability of polyphenols was often overlooked in ear-
lier in vitro studies of polyphenol compounds that employed native compounds and
pharmacological doses, highly unlikely to represent tissue exposure post-polyphenol
consumption, augmenting a limited view of polyphenols as mere antioxidants and in
general leading to inconsistencies in results and conclusions.
Polyphenols’ capacity to chelate metals and scavenge free radicals renders them
their antioxidant potential. Notably, however, the potency of polyphenol antioxidant
activity highly depends on their chemical structure and more specifically the num-
ber of hydroxyl groups augmenting, while glycosylation reducing their antioxidant
potential (Di Meo et al., 2018). In this regard, factors that would alter the chemical
structure of polyphenols could affect the latter’s ability to function as antioxidants.
There is a significant body of literature suggesting the beneficial health effects of
polyphenols due to their antioxidant capacity. Early evidence indicated reduced cap-
illary resistance and permeability in the case of human blood vessels as a result of the
activity of citrus flavonoids, hesperidin, and rutin (Bentsath et al., 1936).
As relevant research is being conducted for a number of decades now, a signifi-
cant body of literature currently exists to support cardiovascular and other potential
health effects of polyphenols. More specifically, consumption of plant-based food
Powerful Interplay of Gut Microbiota and Polyphenols 185

items such as apples, berries, citrus fruits, plums, beans, nuts, cocoa, tea, coffee, and
wine are considered good sources of polyphenols and have been to a greater or lesser
degree documented to confer health benefits related to chronic conditions including
cardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM) (Williamson,
2017), along with neurodegenerative diseases (Renaud and Martinoli, 2019). While
polyphenols gained early recognition for their antioxidant capacity, mechanistic
studies employing individual compounds are increasingly documenting their signal-
ing properties via multiple cell signaling mechanisms, commonly related to oxidative
or inflammatory status (Vendrame et al., 2015).
Recently, more emphasis has been placed on the potential interplay of polyphe-
nols and the microbiome. More specifically, work is being conducted to investigate
mechanisms of action involving microbiota-derived metabolites, as these relate to
polyphenol action. The gut microbiome can convert polyphenols into secondary
compounds that may extend health benefits.
Notably, the global polyphenol market, which includes a variety of their uses in
foods, beverages, pharmaceuticals, and cosmetics, was estimated to have exceeded
700 million USD in 2015 and is projected to reach 1.1 billion USD by 2022.
Interestingly, however, there is currently no uniform regulatory framework or rec-
ommendation consensus regarding polyphenol consumption and/or supplementation
or fortification in functional foods (Cory et al., 2018).

OVERVIEW OF POLYPHENOLS
C

Polyphenols constitute the largest group of phytochemicals, i.e., chemicals natu-

rally occurring in plants. Produced as secondary metabolites, polyphenols serve
to defend the plant from environmental stress related to oxidative damage, ultra-
violet radiation, extreme temperatures, pathogens, parasites, or plant predators.
Additionally, the bright colors of fruits and vegetables are attributed to polyphenols
(Zhang, 2015). While not considered essential nutrients, such substances may con-
siderably affect human health, especially with regard to chronic diseases includ-
ing CVD, diabetes, and cancer. A plethora of evidence indicates potential health
benefits of plant-based diets and polyphenol dietary sources, including fruits, veg-
etables, whole grains, legumes, nuts, and seeds, as well as tea, coffee, and wine
(Kristo et al., 2016). While originally classified as “vegetable tannins” because of
their tanning action on animal skin, polyphenols are currently moderately esti-
mated to comprise over 8,000 unique chemical compounds that extend pharmaco-
logical and/or therapeutic action (Renaud and Martinoli, 2019). From a chemistry
standpoint, it is primarily the presence of hydroxylated phenol (aromatic) rings that
characterizes this large heterogeneous group of naturally occurring compounds
(D’Archivio et al., 2007).
According to the number of phenolic rings and relative substitution groups, poly-
phenols are classified as flavonoid and non-flavonoid molecules. Flavonoids, which are
the largest subgroup in terms of the number of identified compounds, are further clas-
sified into flavanones, flavones, dihydroflavonols, flavonols, flavan-3-ols, or flavonols,
186 The Gut Microbiome: Bench to Table

isoflavones, anthocyanidins, and proanthocyanidins (PACs). Non-flavonoids on the

other hand include phenolic acids and other various phenolics (Cardona et al., 2013).
Along with flavonoids, phenolic acids (derived from benzoic and cinnamic
acid) and stilbenes constitute the main subclasses (Williamson and Clifford, 2017).
Interestingly, most polyphenols, particularly flavonoids, occur in glycosylated forms
(i.e., with a sugar moiety attached to the main polyphenolic structure), while esteri-
fied and/or polymerized versions are fairly common modifications also. Moreover,
flavonols, flavanones, and anthocyanins are typically found in planta as glycosides
with glucose and rhamnose units being the main saccharides attached. Flavanols
primarily occur in their free native form or alternatively in polymerized forms,
while finally can be found as galloylated versions such as in the case of green tea.
Polymerized flavonols, also referred to as PACs, constitute a widespread group of
compounds found in a variety of food items including cocoa. Phenolic acids are typi-
cally attached to quinic acid or other organic acids and are found in plant-based foods
specifically coffee and most fruits at very high and moderate levels, respectively
(Williamson and Clifford, 2017).

The type, variety, and number of polyphenols as well as concentrations in a particu-

lar food item can vary depending on where (soil/climate conditions), when the food
is grown (seasonality), how it is farmed and transported, how ripe it is, and how it is
cooked and/or prepared. Overall, polyphenols are the best food sources and hence
provide the most abundant antioxidants to our diet and are found as widespread con-
stituents of fruits (particularly apples, citrus fruits, plums, and berries), vegetables
(such as broccoli, celery, parsley, onion, and leek), cereal, olive, dry legumes, nuts,
chocolates, and beverages, such as tea, coffee, and wine (D’Archivio et al., 2007).
Not surprisingly, the differences in the dietary patterns among populations reflect
the differences in the type and amount of polyphenol intake. As a result, there is sig-
nificant variation in terms of both the type and amount of polyphenol intake among
humans as reasonably expected.
More specifically, Edmands and colleagues compared the polyphenol metabolome
from urine samples of 481 participants from four European countries as part of the
European Prospective Investigation on Cancer and Nutrition cohort. The researchers
concluded that the metabolome approach mapped the dietary intake, which, however,
is significantly diverse and produced variable results based on the type and amount
of polyphenol intake of the participants (Edmands et al., 2015). Similarly, in a sepa-
rate experiment conducted in the framework of the Adventist Health Study, different
dietary patterns were associated with notable differences in polyphenol intake, their
main classes, or sources of dietary polyphenols, corroborating the findings of others.
More specifically, high flavonoid intake was stated to signify vegetarian diets, while
phenolic acid intake, primarily due to significant coffee consumption, was found
higher in nonvegetarian diets (Burkholder-Cooley et al., 2016).
In an earlier comprehensive review of the evidence, Scalbert and Williamson con-
cluded that the main polyphenol dietary sources are fruits and beverages (fruit juice,
wine, tea, coffee, chocolate, and beer) and, to a lesser extent vegetables, dry legumes,
Powerful Interplay of Gut Microbiota and Polyphenols 187

TABLE 7.1
Dietary Polyphenols: Their Characteristic Bioactive Compounds and Typical
Food Sources
Dietary
Polyphenol Type Characteristic Bioactive Compound(s)a Dietary Source(s)
Phenolic acids Chlorogenic, caffeic, gallic, ferulic acids Coffee, berries, kiwi, apple, cherry
Phenolic alcohols Hydroxytyrosol Olive
Stilbenes Resveratrol Grapes, red wine (primarily)
Lignans Secolariciresinol Linseed
Flavonoids Genistein, daidzein (isoflavones), Soy, miso
Luteolin, apigenin (flavones) Celery, parsley
Hesperetin, naringenin (flavanones) Oranges, lemon
Quercetin, kaempferol, myricetin (flavonols) Onion, leek, broccoli
(Epi)catechins (flavanols) Grapes, wine, cocoa, apricots
(Epi)gallocatechins (flavanols) Beans, green tea
Epigallocatechin gallate (flavanols) Berries, aubergine
Delphinidin, cyanidin, malvidin
(anthocyanins)
Tannins Procyanidins (condensed tannins) Cocoa, chocolate, apples, grapes
Gallotannins, ellagitannins (hydrolyzable Mango, pomegranate
tannins)

a Polyphenol subclass designation in parentheses when applicable.

and cereals, while the total intake was estimated to be 1 g/d. Nonetheless, significant
uncertainties were due to the lack of accurate and comprehensive data on the content
of some main polyphenol classes in foods (Scalbert and Williamson, 2000). More
recently, the estimated intake of polyphenols through dietary sources and herbal
medicines and/or supplements combined was at 2 g/d, a higher amount than initially
anticipated (Espin et al., 2017). The types of dietary polyphenols, with characteristic
bioactive compounds and typical dietary sources, can be viewed in Table 7.1, while
the classification of compounds can be seen in Figure 7.1.

OVERALL METABOLISM/BIOAVAILABILITY AND EXCRETION

Certain plant bioactive compounds can be absorbed in their native form by the
stomach or gut, but the majority of the glycosylated, polymeric, or esterified native
plant compounds must be hydrolyzed prior to absorption, a step partly performed
by the gut microbiota. For the intestinal uptake of the bioactive compounds, both
passive and active mechanisms have been reported, as well as efflux transporters,
which lower the absolute influx of bioactive compounds through intestinal absorp-
tion. Nevertheless, our knowledge of the carriers involved in the various compounds
is still very scarce. When absorbed, hydrophilic plant food compounds typically
undergo first-pass metabolism in the intestine and liver with phase I (oxidation/
 188

Bioactive
compounds
D
i
e
t
a
r
y

S
o
u
r
c
e
s

FIGURE 7.1 Main bioactive compounds as they per tain to polyphenols and respective dieta r y sources displayed (Modified from: K r isto et al., 2016).
The Gut Microbiome: Bench to Table
Powerful Interplay of Gut Microbiota and Polyphenols 189

reduction reactions) and predominantly phase II (β-glucuronidation, sulfation, meth-

ylation, and glutathione conjugation) biotransformations.
In general, however, the metabolism of polyphenols is rather well understood due
to the xenobiotic nature of these compounds. Polyphenols specifically undergo metab-
olism in the small intestine, liver, and finally the colon by colonic microbes. While
significant metabolism and uptake of polyphenol derivatives occur in the colonic
environment, phenolic compounds are glucuronidated and sulfated in the liver and
intestinal tissues, with those metabolites detected in body fluids. More specifically,
absorbed metabolites produced by microbial transformation (enterolignans and dihy-
droxylated compounds) such as methylcatechol can be subjected to further glucuroni-
dation, methylation, sulfation, or glycination in the liver. While there is a significant
lag time from the interplay between the colon and the liver in terms of blood residence
time, ultimately, phenolic microbial metabolites are distributed to tissues and finally
excreted via urine, primarily as hepatic conjugates or in their free form (Bohn et al.,
2015). This is an interesting interplay that deems further research and understanding
of its kinetics. An interesting point is that small colon-derived phenolic acids may not
be accounted for in analyses of human or cell fluids due to their hydrophilic nature
and the challenges posed in terms of their identification and quantification.
Furthermore, gut microbiota-driven polyphenol metabolism also occurs via the
opening of carbon rings, breaking of C–C bonds, demethylation, reduction, and
hydrogenation/de-hydrogenation, all of which can eventually modulate their bio-
availability and/or biological activity. Therefore, whenever considering the effects
of polyphenols as they relate to the gut microbiome, metabotype characterization
emerges as the key toward interpreting those effects (Tressera-Rimbau et al., 2018),
while the individual epigenome needs to be also considered even though the role of
the individual epigenome in the variation of responses to plant food bioactive com-
pounds is still unexplored.
As previously mentioned, in human studies, there is often a significant interin-
dividual variation observed in terms of microbially derived metabolites and bio-
availability of polyphenols, echoed by different plasma concentrations and urinary
excretion levels of bioactive compounds or their metabolites in individuals with simi-
lar dietary intake (Manach et al., 2017).
Notably, in clinical trials of soy isoflavones and ellagitannins, the observed health
effect of those polyphenols was associated with certain gut metabotypes. Polyphenol
compounds, or by the same token polyphenol-rich foods, produce a positive impact on
desirable microbial species such as Akkermansia muciniphila, Faecalibacterium praus-
nitzii, and Roseburia spp. Oligomeric and polymeric polyphenols reaching the colonic
environment unmodified interestingly demonstrate probiotic-like effects (Williamson
and Clifford, 2017; Duenas et al., 2015). More specifically, polyphenols and polyphe-
nol-like compounds, as well as polyphenol-rich foods, have been demonstrated to sig-
nificantly induce the population of A. muciniphila, F. prausnitzii, and Roseburia spp.,
bacterial species associated with positive health effects (Tressera-Rimbau et al., 2018).
Observed inter-participant differences could arise due to variances in absorption,
metabolism, tissue distribution and turnover, excretion, or a combination of these
parameters. In nutrition, this intra-participant variation in the response of supple-
mentation of carotenoids for example has been demonstrated, thus leading research-
ers to introduce the concept of responsiveness degree, alluding to the classification of
190 The Gut Microbiome: Bench to Table

low, medium, and high responders to nutrient supplementation either through the diet
or other types of oral supplementation (Tressera-Rimbau et al., 2018). Interestingly,
a series of pharmacogenomics studies have demonstrated that for specific drugs,
including some phytochemicals of the alkaloid family like codeine, individuals can
be categorized into poor, intermediate, or extensive, and ultra-rapid metabolizers,
and dosing must be adapted to achieve equivalent plasma or tissue concentration of
the active metabolite (Filipski et al., 2016).
An interesting example of the approach toward a better understanding of the role
of genetic polymorphisms in the variability of the response to diet can be seen in
the case of the Mediterranean diet (MedDiet). More specifically, the benefit of the
Mediterranean diet on cardiovascular‐related outcomes has been shown to depend
significantly on genetic variants of the transcription factor 7‐like 2 (TCF7L2) gene
(Corella et al., 2013). This specific gene encodes for a protein functioning as a tran-
scription factor, and the TCF7L2‐rs7903146 polymorphism is known as a strong
genetic determinant of T2DM risk and fasting plasma glucose concentration regula-
tion (Palmer et al., 2011). More specifically, the prevalence of the 7903146T allele is
associated with higher T2DM risk. In an elegant study, Corella et al. demonstrated
that intervention with the MedDiet was more beneficial toward significantly attenuat-
ing cardiovascular risk factors and stroke incidence for individuals with the 7903146T
allele (Corella et al., 2013). Nonetheless, it must be underlined that the majority of
nutrigenetics studies thus far are observational and association studies, while obtained
results have not been fully confirmed in follow-up intervention experimental studies.

MICROBIOTA METABOLISM OF POLYPHENOLS

AND HEALTH EFFECTS
G

The biological properties of dietary polyphenols as they relate to health benefits and
relevant effects are determined to a large extent by the bioavailability of their gut-
derived metabolites. The human microbiota, composed of bacteria, fungi, archaea,
viruses, and protozoans, can be found throughout the body; as it colonizes the skin,
mouth, vagina, gastroenteric tube, and/or respiratory system. Over 70% of microbi-
ota colonizes the gastrointestinal tract and contains more than 100 trillion microbes.
The human microbiome, a synergistic community of microorganisms often con-
sidered an independent endocrine organ in a symbiotic relationship with the host, is
comprised of two primary phyla, namely Bacteroidetes and Firmicutes. These two
phyla represent 98% of the gut microbiota and are typically found in a ratio favor-
ing Bacteroidetes over Firmicutes (B/F >1) when we consider a healthy/desirable
gut phenotype. Notably, however, a universal definition of “healthy microbiota” as
it would pertain to a specific microbial profile is still lacking. Other phyla include
Proteobacteria, Verrucomicrobia, Fusobacteria, Cyanobacteria, and Actinobacteria.
Firmicutes can be grouped into three major classes: Clostridia, Negativicutes, and
Bacilli, while Firmicutes overall consist of over 200 genera, including Staphylococcus,
Lactobacillus, Ruminococcus, and Clostridium. The relevant phyla consist primar-
ily of gram-positive bacteria, except for those belonging to the Negativicutes class.
Powerful Interplay of Gut Microbiota and Polyphenols 191

Interestingly, Negativicutes possess an outer membrane with lipopolysaccharides

(LPS), making them stain gram-negative (Sikalidis and Maykish, 2020). Bacterial
species of the genera Lactobacillus and Bifidobacterium are considered beneficial;
thus, they are commonly used as probiotics, whereas the Clostridium, Eubacterium,
and Bacteroides genera are implicated in adverse health outcomes (Nash et al., 2018).
Eubiosis (from the Greek words ευ/eu: good and βίος/bios: life) refers to the normal/
healthy profile of gut microflora, as opposed to dysbiosis (from the Greek words δυς/
dys: nonfavorable/difficult and βίος/bios: life) which produces a profile that in turn
induces the risk for certain diseases; hence, in a healthy microbiome–host relationship
promoting the well-being of the host, the condition would be a eubiotic one. Notably,
the profile of gut microbiota is highly amenable to a variety of external factors includ-
ing diet, antibiotic use, hygiene, pharmaceuticals, exercise frequency, climate, geo-
graphical region, pollution level, toxins, and physiological and/or psychological stress
potentially resulting in dysbiosis, which is shown to be associated with increased risk
for a plethora of diseases particularly chronic ones such as inflammatory bowel dis-
ease, CVD, and T2DM. Endogenous factors, such as age, genetics, birth gestational
date, mode of delivery at birth, infant feeding method, weaning period, hormonal
changes, and health status, may also influence the microbiota composition (profile).
The microbiota composition may finally also vary depending on the colonization
location in the gut. Moreover, a significant body of literature discusses the differential
antimicrobial properties of polyphenols and their capacity to adversely affect patho-
genic bacteria (Ozdal et al., 2016; Maharjan et al., 2018; Luca et al., 2020).
Notably, a small fraction of polyphenol molecules can be absorbed in the small
intestine (namely the smaller molecules), while glycosylated and polymeric forms
(estimated at 90–95% of total ingested polyphenols) reach the colon in their native
form. Microbes of the gut then convert polyphenols into phenolic compounds of low
molecular weight, hence increasing absorption by intestinal epithelial cells (Pasinetti
et al., 2018; Aura, 2008).
Moreover, conjugation reactions, such as methylation, sulfation, and glucuronida-
tion, increase the hydrophilicity of polyphenolics, hence facilitating absorption by
the gut, in addition to biliary and urinary elimination (Filosa et al., 2018). A notewor-
thy variety of gut bacteria can perform enzymatic reactions such as deglycosylations,
while the production of certain metabolites such as urolithin by Gordonibacter uro-
lithinfaciens and Gordonibacter pamelaeae, and S-equol by Bifidobacterium breve
is a characteristic of gut bacteria with significant potential to support health (Marin
et al., 2015). In this context, it is important to develop a better and more accurate
understanding of this polyphenolic biotransformation, and it is important to grasp
the significant breadth of catalytic abilities for the various bacterial species. From an
absorption standpoint, interestingly, the best absorbed among flavonoids appear to
be isoflavones, followed by catechins, flavanones, and flavonol glycosides, while the
least absorbed are PACs, flavan-3-ol gallates, and anthocyanins (Crozier et al., 2010).
A conceptual schematic of the main biotransformations of dietary polyphenols can
be seen below (Figure 7.2).
In general, phenolic compounds are typically found conjugated to glycosides,
glucuronides, and organic acids, which can be hydrolyzed by gut microbiota, result-
ing in aglycones. Subsequently, post-colonic absorption, transformation into phase II
192 The Gut Microbiome: Bench to Table

Dietary Polyphenols Ingested

Monomers/Dimers Polymers

Small Intestine Colon

Esterification Hydrolysis
Acylation Esterification
Glycosylation Acylation

Epithelial Cells Uptake

Phase II
xenobiotic
metabolism

Distribution to tissues Hepatocyte uptake Excretion / re-uptake

FIGURE 7.2 Conceptual schematic depicting the major bio-transformations and relevant
locations of dietary polyphenols (the blue dashed line represents separation between the intes-
tinal lumen and the internal organismal space).

conjugates occurs (sulfated and glucuronidated conjugates) in the intestinal tissues

and the liver. Conjugated compounds are excreted into the gut as biliary constituents
via enterohepatic recirculation, and before being reabsorbed or transformed, microbial
enzymes deconjugate these compounds (Plamanda and Vondar 2021). Fecal micro-
bial enzymes—β-glucosidase, α-rhamnosidase, esterase, β-glucuronidase—catalyze
the deconjugation in the gut, while reactions such as ring and lactone fission, dehy-
droxylation, reduction, decarboxylation, demethylation, and isomerization are also
induced by the gut microbiota. The degree and type of microbial transformations are
significantly influenced by the phenolic structure, flavonoid and nonflavonoid factors,
polymerization degree, and spatial configuration (Makarewicz et al., 2021).
Flavonols, flavanones, flavan-3-ols, isoflavones, and anthocyanins, all belong-
ing to the flavonoid category, share the common basic structure: two benzene rings
(ring A and B), linked by a heterocyclic pyrone C-ring. In foods, they are found as
glycosides, O-glycosides, C-glycosides, and flavan-3-ols, which are not conjugated.
Flavan-3-ols can form either PACs or condensed tannins, and either procyanidins,
prodelphinidins, or propelargonidins, when they are solely comprised of one single
compound—epicatechin, epigallocatechin, epiafzelechin. Simple phenolics derived
from the A and B rings are released after the gut microbiota disrupts the C-ring
at different positions. The resulting type of phenolic compounds is affected by the
hydroxylation pattern and the position of the B-ring. Thus, in phenolic compounds,
such as flavonols, flavan-3-ols, PACs, rendering hydroxyphenyl-propionic acids and
hydroxyphenyl acid, the C-ring cleavages are produced at 1–2 and 4–10 bonds or
1–2 and 3–4 bonds. In flavanone and isoflavone groups, the resulting metabolites
indicate that the C-ring cleavage is produced at either position 1 and 2, or 4 and
10. Subsequent steps of flavonoid metabolism involving the gut microbiota include
demethylation and dehydroxylation reactions, while the majority of the resulting
metabolites are acid or aldehyde phenolics with one, two, or three hydroxyl and
Powerful Interplay of Gut Microbiota and Polyphenols 193

methyl ester radicals, for example, 3-(3,4-dihydroxyphenyl)-propionic acid from the

flavonol quercetin and equol from the isoflavone daidzein (Plamanda and Vondar
2021; Makarewicz et al., 2021).
Moreover, the gut microbiota is also able to transform gallic acid and ellagic acid.
More specifically, gallic acid is prone to decarboxylation and dihydroxylation reac-
tions, while ellagic acid is more susceptible to dehydroxylation. Following the dihy-
droxylation of ellagic acid, nasutin metabolites are produced, which are compounds
characterized by the removal of the two hydroxyl units. After ellagic acid is trans-
formed by way of lactone ring cleavage, decarboxylation, and dehydroxylation reac-
tions, the urolithin metabolites are formed (Catalkaya et al., 2020). After lactonases
open one of the lactone rings, luteic acid is produced, which is in turn converted
by decarboxylases in the gut, to produce pentahydroxyurolithin, a critical stage in
the production of different types of urolithins. Further, after dehydroxylations of
pentahydroxyurolithin, tetrahydroxyurolithins, and trihydroxyurolithins are cre-
ated, compounds that lead to the principal metabolites are typically detected in vivo,
namely dihydroxy-urolithins urolithin-A (Uro-A), isourolithin-A (IsoUro-A), and the
hydroxyurolothin (Uro-B) (Catalkaya et al., 2020; Plamanda and Vondar 2021).
In summary, the inherently markedly low bioavailability of polyphenols is essentially
greatly improved through a variety of gut microbial enzymatic reactions (dihydroxyl-
ation, deglycosylation, demethylation) that produce structurally simpler metabolites
such as aglycones and monomers that are well absorbed in the intestine and can be
subsequently detected in the systemic circulation for a longer time (Shortt et al., 2018).
Generally, extensive microbial metabolism ultimately truncates the structural diversity
of the native polyphenols to a limited number of mainly simple aromatic metabolites.
Other major metabolites that have been seen to be by-products of microbial metabo-
lism in the gut also associated with health effects are short-chain fatty acids (SCFAs)
with butyrate being the most prominent and well-studied, branched-chain amino
acids (BCAAs) with established signaling properties/functions and LPS (Sikalidis
and Maykish, 2020). The SCFAs and BCAAs are typically reported to extend positive
health effects by promoting eubiosis, especially in metabolic syndrome, CVD, and can-
cer, whereas LPS on the contrary appears to have a negative impact through dysbiosis.
While these are not derivatives of polyphenols, however, they add to the complexity of
the health effects extended to the host as a result of gut microbiome metabolism and the
interactions with dietary constituents of naturally consumed mixed diets.
While human studies indicate an increasing number of metabolites appearing at
high concentrations in the colon and systemic circulation, interestingly, the biologi-
cal relevance for most of these metabolites is largely unknown (van Duynhoven et al.,
2011). Synergistic deployment of in vitro and in vivo (humanized mouse) models, as
well as human intervention trials, is required in order to unlock the complex meta-
bolic fate of dietary polyphenols.

Health Effects of Polyphenol/Metabolite-Microbiota Interaction

A variety of common phenolic compounds have been investigated in the context
of studies conducted on berries. After their extraction and chemical characteriza-
tion typically through high-performance liquid chromatography, compounds such
as anthocyanins, flavonols, caffeic acid derivatives, ellagic acid derivatives, or
194 The Gut Microbiome: Bench to Table

ellagitannins were identified. An in-depth study conducted in 2020 by Baenas et al.

analyzed polyphenols from raspberry through an in vitro fermentation model and
metabolites, such as SCFAs. The results showed that the identified polyphenols were
mainly hydrolyzable polyphenols found in the insoluble fraction of fibers, and those
were identified as the primary compounds responsible for the raspberries’ prebi-
otic effect. The study by Baenas et al. concluded that, through their antimicrobial
and antioxidant effects, raspberry and/or raspberry extracts could be utilized as a
prebiotic substrate in foods, functional foods, as well as in dietary supplements. In
another work focusing on berries’ polyphenols and their impact on gut microbiota,
the authors reported that a high quantity of polyphenols can effectively reach the
colon, thus actively further producing a host of metabolites. Berries’ polyphenols
were shown to produce changes in the bacteria population, specifically enhanc-
ing the growth of Bifidobacterium, Lactobacillus, Akkermansia, Bacteroides, and
Eubacterium, while simultaneously decreasing the extent of the populations for
Pseudomonas, Salmonella, Staphylococcus, or Bacillus. The mechanism whereby in
vitro approaches are followed in studies to identify the effects of polyphenols on gut
microbiota still requires deeper understanding; however, the ability for high produc-
tion of SCFAs has been identified repeatedly and corroborated by several studies and
as such could lend strong support illustrating the prebiotic-like effect polyphenols
seem to extend (Plamanda and Vondar 2021).
A food particularly rich in polyphenols is grapes. Polyphenols in grapes can be
found mainly in the fruit in native form, but also wine or wine by-products, through
the grape pomace. The most common polyphenols identified in grapes are querce-
tin, anthocyanins, anthocyanosides, anthocyanidins, catechins, and PACs. Previous
studies conducted on the fruit, wine, wine industry by-products, and grape extracts
have demonstrated the ability of polyphenols to influence the intestinal bacteria, with
significant stimulation of the Lactobacillus and Bifidobacterium genera. Polyphenols
were able to be used as a carbon source by these beneficial bacteria. Another manu-
script published in 2018 showed modifications in the bacteria ratio from gut micro-
biota (Gil-Sánchez et al., 2018). There was an increase of Enterococcus, Prevotella,
Bacteroides, Bifidobacterium, Bacteroides uniformis, Eggerthella lenta, Blautia
coccoides–Eubacterium rectale groups, as well as a decrease of Actinobacteria,
Clostridium spp., C. histolyticum group. In the case of dealcoholized wine intake,
an increase in the Fusobacteria/Firmicutes population and a decrease in the
Actinobacteria population (Gil-Sánchez et al., 2018; Sáyago-Ayerdi et al., 2019) were
noted. In general, gram-positive (gram+) bacteria are prone to be enriched in the gut
rather than gram negatives (gram–) when polyphenols are ingested because of the
difference in their cell wall composition. Gram-negative bacteria are more impervi-
ous to flavan-3-ols than gram-positive bacteria.
Recent evidence from research on the gut microbiome has prompted a shift of
focus on health and disease, highlighting the idea that consumption of a healthier
diet reduces disease risk at the microbiota level which in turn reduces the disease risk
further, utilizing eubiotic mechanisms not yet fully understood. While the protective
role of polyphenols depends, to some extent, on interindividual variations of metabo-
lism by gut microbiota, polyphenol consumption is associated with protection against
CVD, metabolic syndrome, and cognitive decline (Derrien et al., 2017). Specific
polyphenols such as isoflavones, lignans, and ellagitannins are biotransformed by
Powerful Interplay of Gut Microbiota and Polyphenols 195

gut microbes to equol, enterolignans (enterolactone or enterodiol), and urolithins,

respectively. The binding of the produced phytoestrogens to estrogen receptors
can confer protection against breast and prostate cancer by suppressing oncogenes.
Moreover, glucosinolates, typically received via consumption of cruciferous plants
of the Brassicaceae family, are the precursors of isothiocyanates. Furthermore, bac-
terial or plant myrosinases convert glucosinolates into isothiocyanates, and gluco-
sinolates, post-ingestion of cooked crucifers, are converted into isothiocyanates by
gut microbial myrosinases. These transformations produce metabolites that extend
further protection against tumorigenesis (Derrien et al., 2017).
There is evidence to suggest that fruits, vegetables, cereals, and coffee contain
conjugated hydroxycinnamates, and antioxidant and anti-inflammatory compounds
that are activated post-microbial biotransformation. Interestingly, members of
Bifidobacterium, Lactobacillus, and Escherichia in the human gut have been shown
to hydrolyze esters of caffeic and ferulic acids, hence transforming the ester conju-
gates naturally found in plants to the respectively active compounds of caffeic acid,
ferulic acid, and p-coumaric acid that are shown to yield health benefits pertinent
to their antioxidant potential (Carmody and Turnbaugh, 2014). Other similar exam-
ples of microbial biotransformation of dietary-derived compounds producing bioac-
tives include the production of the nonsteroidal estrogen equol from the soy-derived
isoflavonoid daidzein and liberation of aglycones with anticancer properties from
anthocyanins (Tsuji et al., 2012; Selma et al., 2009). An important aspect to be con-
sidered is that the polyphenols are catabolized in the colon by the gut microbiota into
metabolites, which have greater bioavailability and more potent anti-inflammatory
properties compared to their precursors.
Studies have proposed potential mechanisms via which the polyphenol–microbi-
ota interaction may reduce the risk for carcinogenesis, i.e., via a reduction of inflam-
mation and/or abrogation of cell proliferation signaling combined with the induction
of apoptosis. The anti-inflammatory action of resveratrol for example involves inhi-
bition of pro-inflammatory moderators, modification of eicosanoid synthesis, and
inhibition of enzymes, such as COX-2, NF-κB, AP-1, TNF-α, IL-6, and VEGF. In
cells, several phenolic compounds impede COX-2 function, probably by complexing
with the enzyme (Namasivayam N, 2011). Notably, both urolithins A and B, being
the most representative microbial metabolites of dietary ellagitannins, have demon-
strated estrogenic function in a dose-dependent manner, even at high concentrations
(40 microM), without antiproliferative or toxic effects toward MCF-7 breast cancer
cells (Cardona et al., 2013). In an LT97 human adenoma cell line, it was demon-
strated that certain intestinal polyphenol metabolites 3,4-dihydroxyphenylacetic acid
and 3-(3,4-dihydroxyphenyl)-propionic acid, metabolites of quercetin and chloro-
genic acid/caffeic acid upregulate GSTT2 and downregulate COX-2, a combined
effect that could plausibly contribute to the chemopreventive potential of polyphe-
nols, post-gut biotransformation (Cardona et al., 2013).
Markedly, in a study by Monagas et al., the authors observed that dihydroxylated
phenolic acids (3,4-dihydroxyphenylpropionic acid, 3-hydroxyphenylpropionic acid,
and 3,4-dihydroxyphenylacetic acid) derived from microbial metabolism of PACs,
presented marked in vitro anti-inflammatory properties, reducing the secretion of
TNF-α, IL-1b, and IL-6 in LPS-stimulated peripheral blood mononuclear cells
from healthy participants (Monagas et al., 2009). It has been advocated that these
196 The Gut Microbiome: Bench to Table

microbial metabolites could be among the new generation of therapeutic agents for
the management of immunoinflammatory diseases such as atherosclerosis, but also
for stifling the inflammatory response to bacterial antigens, which plausibly extends
significant implications in terms of chronic inflammatory or autoimmune diseases
such as inflammatory bowel disease (Cardona et al., 2013).
Interestingly, Beloborodova et al. analyzed the role of phenolic acids of microbial
origin as biomarkers in the progress of sepsis. They reported that p-hydroxyphenylacetic
acid was able to prevent reactive oxygen species production in neutrophils. By acting on
neutrophils, there is retardation of immune responses, however, when acting on mito-
chondria, there is preclusion or attenuation of multiple organ failure. Thus, during the
development of bacteremia and purulent foci of infection associated with P. aerugi-
nosa and Acinetobacter baumanii, their metabolite, p-hydroxyphenylacetic acid, can
directly enter the systemic blood flow and inhibit the phagocytic activity of neutrophils
(Beloborodova et al., 2012).
While limited studies are investigating these relationships in humans, some rep-
resentative characteristic examples are discussed below. In healthy men, a single
intake of approximately 240 g of fresh blueberries, rich in polyphenols such as
anthocyanins, was shown to increase flow-mediated dilation (FMD), accompanied
by increases in plasma concentrations of phenolic metabolites such as vanillic acid,
homovanillic acid, benzoic acid, hippuric acid, and hydroxyhippuric acid. Hence,
sufficient residence time of blueberry metabolites in the circulation allows for their
vascular activity to be exerted. In addition, blueberries induced a dose-dependent
and biphasic increase in FMD, while the elevation in plasma polyphenol metabolites
occurred in tandem with FMD improvement (Rodriguez-Mateos et al., 2013).
Dietary intervention with 66 healthy men consuming polyphenols from Aronia
berry (extract or fruit) for 12 weeks demonstrated significant associations between
changes in endothelial function, plasma metabolites of Aronia berry polyphe-
nols, and specific gut microbial genera. FMD, used to assess endothelial function,
increased alongside changes in the gut microbiota. Aronia (poly)phenol-rich extract
(116 mg, 75 g berries) was shown to increase the growth of Anaerostipes and whole
fruit powder (12 mg, 10 g berries) caused a notable increase in Bacteroides, without
any changes in the diversity of gut microbiota in either treatment (Istas et al., 2019).
The authors concluded that in healthy men, Aronia consumption resulted in improved
endothelial function and positively modulated gut microbiota composition, indicat-
ing a potential benefit for maintaining cardiovascular health (Istas et al., 2019).
Moreover, A. muciniphila was reported to be induced post-consumption of
pomegranate extract (rich in hydrolyzable ellagitannins) in 16 out of the 20 healthy
participants that were producing urolithin-A (Li et al., 2015). Nevertheless, in a
separate study with 49 healthy overweight or obese participants consuming a higher
dose of a different pomegranate extract, no significant alterations were observed for
A. muciniphila (Gonzalez-Sarrias et al., 2017). A clinical trial with obese insulin-
resistant patients showed that resveratrol, a natural phenolic compound, increased
the abundance of A. muciniphila in Caucasians but not in other ethnic groups
(Verhoog et al., 2019). In conclusion, there is a variety of gut bacteria that can pro-
duce metabolic by-product compounds when exposed to polyphenols and may pro-
mote health in humans.
Powerful Interplay of Gut Microbiota and Polyphenols 197

There is a series of recent human studies that aimed to use different approaches to
delineate the effects of plant polyphenols on the gut microbiota. Basak et al. recently
studied the efficacy of curcumin on tumor suppression. More specifically, a double-
blind, randomized, placebo-controlled phase 1 clinical trial was conducted with
APG-157 in 13 normal subjects and 12 patients with oral cancer. Two doses of 100 or
200 mg were delivered transorally every hour for 3 hours. Blood and saliva were col-
lected before and 1, 2, 3, and 24 hours after treatment. Electrocardiograms and blood
tests did not demonstrate any toxicity. Curcumin was found in the blood and tumor
tissues. Inflammatory markers and Bacteroides species were found to be decreased
in the saliva, and immune T-cells were increased in the tumor tissue. APG-157 is
absorbed well, reduces inflammation, and attracts T-cells to the tumor, suggesting
its potential use in combination with immunotherapy drugs. Though endowed with
properties of tumor cell suppression due to its antioxidant and anti-inflammatory
actions, curcumin is poorly absorbed when administered orally and consequently
less effective. APG-157, a botanical drug containing curcumin among other poly-
phenols, on the other hand, is absorbed well and is reported to be potentially benefi-
cial when combined with immunotherapy by reducing inflammation and increasing
T-cell concentration in the tumor (Basak et al., 2020).
Vetrani and colleagues showed that a diet naturally rich in polyphenols and/or
long-chain n − 3 polyunsaturated fatty acids (LCn3) increased the diversity of the
predominant fecal bacteria; the polyphenols increased Clostridium leptum (clostrid-
ial cluster IV), which was directly associated with good glucose tolerance, by early
secretion of insulin. More specifically, 78 individuals with high waist circumference
and at least one additional component of the metabolic syndrome were randomized
to an isoenergetic 8-week diet: (a) low LCn3 and polyphenols; (b) high LCn3; (c) high
polyphenols; or (d) high LCn3 and polyphenols. Microbiota analysis was performed
on feces collected before and after the intervention. Denaturing gradient gel elec-
trophoresis (DGGE) analysis of the predominant bacteria, Eubacterium rectale and
Blautia coccoides group (Lachnospiraceae, EREC), C. leptum (Ruminococcaceae,
CLEPT), Bacteroides spp., Bifidobacteria, and Lactobacillus group was performed.
A quantitative real-time PCR was performed for the same group additionally includ-
ing the Atopobium cluster (Coriobacteriaceae). Before and after the intervention,
participants underwent a 75 g oral glucose tolerance test and a high-fat test meal
to evaluate glucose and lipid response. The authors reported that polyphenols sig-
nificantly increased microbial diversity and CLEPT but reduced EREC. LCn3 sig-
nificantly increased the numbers of Bifidobacteria. Τhus, it was concluded that diets
naturally rich in polyphenols or LCn3 influenced gut microbiota composition in
individuals at high cardiometabolic risk, with these modifications associated with
changes in glucose/lipid metabolism (Vetrani et al., 2020).
In an in vitro colon system study, polyphenol-rich fractions of blueberry-con-
taining anthocyanins/flavonol glycosides (ANTH/FLAV), PACs, sugar/acid frac-
tion (S/A), and total polyphenols (TPP) had a distinct effect on fecal microbiota
composition. Effective promotion of microbiome alpha diversity was observed with
ANTH/FLAV, and PAC fractions as opposed to the S/A and TPP fractions, which
has been attributed to the differentially responsive taxa. Older compared to the
younger group showed an abundance of gut microbiota diversity with blueberrry
198 The Gut Microbiome: Bench to Table

consumption, which correlated with increased plasma antioxidant activity (Ntemiri

et al., 2020).
Flavonoid-rich orange juice treatment, in contrast to flavonoid-low treatment,
resulted in an abundance of Lachnospiraceae family—namely Lachnospiraceae_uc,
Eubacterium_g4, Roseburia_uc, Coprococcus_g_uc, and Agathobacter_uc—and
showed a positive correlation with brain-derived neurotrophic factor. Treatment with
flavonoids also improved depression in young adults, probably due to an alteration of
the stool microbiome (Park et al., 2020).
A randomized, double-blinded, crossover clinical trial, comparing urinary excre-
tion of isoflavones and their metabolites, after a soybean meal and fermented soy-
bean meal (FSBM) consumption, suggested a benefit for FSBM, in terms of higher
excretion of all metabolites, a higher (67%) urinary recovery of isoflavones, and prev-
alence of O-demethylangolensin-producer metabotype (72%), while that of equol
producer was similar (11%) and nonproducer was low (17%). The findings suggest an
improvement in the bioavailability of isoflavones and a reduction in the impact of gut
microbiota on its metabolism following fermentation (de Oliveira Silva et al., 2020).
A crossover interventional trial studying the impact of increased intestinal per-
meability on the bioavailability of polyphenols and results showed a significant
difference in the urinary levels of phase II and microbiota-derived metabolites in
participants with healthier barrier integrity and those with disrupted integrity. The
disturbance in the gut microbial metabolism and phase II methylation process and
the microbiota-derived metabolites were found to be responsible for the biological
activity of dietary polyphenols against age-related intestinal permeability disruption
(Hidalgo-Liberona et al., 2020).
In a study comparing the efficacy of green tea and its extract epigallocatechin-
3-gallate (EGCG) as an antimicrobial in mouthwash in children, the results showed
a significant reduction in the two mutants of streptococci and lactobacilli in the oral
cavity after rinsing with EGCG solution. Even though ECGC demonstrated a better
antimicrobial activity, both EGCG and green tea were found to be alternatives to
chlorhexidine-based mouthwashes (Vilela et al., 2020).
An improved insulin sensitivity consequent to an increase in the phosphorylation
of adenosine monophosphate protein kinase in the skeletal muscles of obese partici-
pants was observed with consumption of genistein for 2 months. The improvement
in insulin sensitivity is also attributed to an increase in A. muciniphila, following
a decrease in gut microbiota dysbiosis and metabolic endotoxemia, after genistein
treatment (Guevara-Cruz et al., 2020).
A placebo-controlled interventional study in athletes elucidated a beneficiary
effect of a daily intake of 5 g of cocoa (425 mg of flavanols), with a significant reduc-
tion of body fat percentage, especially in the trunk, viscera, and lower limbs, associ-
ated with an elevation of plasma follistatin and decrease in leptin, while the myostatin
levels remained static. Despite the reduction in body fat, the performance status of
these athletes remained status quo (Ángel García-Merino et al., 2020).
Daily consumption of orange juice showed improvement in the blood biochemi-
cal parameters including low-density lipoprotein cholesterol, insulin sensitivity, and
glucose in young women. It also showed a positive and beneficial change in the
composition and metabolic activity of the microbiota and an increase in the popu-
lation of fecal Bifidobacterium spp. and Lactobacillus spp. A PCR-DGGE of the
Powerful Interplay of Gut Microbiota and Polyphenols 199

microbiota found that the composition of total bacteria was similar. A reduction
in ammonia and an increase in the production of SCFAs were also elucidated. The
study implies a positive effect on the daily consumption of orange juice in young
women (Lima et al., 2019).
Consumption of an olive pomace-enriched biscuit formulation (OEP), which
delivers 17.1 ± 4.01 mg/100 g of hydroxytyrosol and its derivative on analysis, showed
an upregulation of the microbial polyphenol biotransformation in the intestine, as
evidenced by a significant increase in the excretion of small phenolic acids in urine.
OEP also significantly elevated homovanillic acid and 3,4-dihydroxyphenylacetic
acid in fasted plasma samples, indicating clearance of these compounds from blood
and an extended release and uptake from the intestine. However, the study failed to
show any change in ox-LDL or urinary isoprostane (Conterno et al., 2019).
A study on the targets of curcumin natural polyphenols on nonalcoholic fatty liver
disease determined amino acids, TCA cycle, bile acids, and gut microbiota. Phospholipid
curcumin supplement led to a significant reduction in 3-methyl-2-oxovaleric acid,
3-hydroxyisobutyrate, kynurenine, succinate, citrate, α-ketoglutarate, methylamine, tri-
methylamine, hippurate, indoxyl sulfate, chenodeoxycholic acid, taurocholic acid, and
lithocholic acid (Chashmniam et al., 2019).
Dietary intervention with functional foods in patients with T2DM who exhibit
intestinal dysbiosis, characterized by an increase in Prevotella copri, showed a
reduction in P. copri and increased species with anti-inflammatory effects, namely
Faecalibacterium prausnitzii and A. muciniphilia. A significant reduction in area
under curve for glucose, total and LDL cholesterol, FFA, HbA1c, triglyceride, and
CRP, as well as an increase in antioxidant activity was also observed. The study sug-
gested a beneficial effect on fecal microbiota, pointing to novel avenues for improving
glycemic control, dyslipidemia, and inflammation with long-term adherence to high-
fiber, polyphenol-enriched, vegetable-protein-based diet (Medina-Vera et al., 2019).
In another study, Kim and colleagues showed that mango (Mangifera indica L.)
polyphenols reduce IL-8, growth-related oncogene (GRO), and GM-SCF plasma
levels and increase Lactobacillus species in a pilot study in patients with inflam-
matory bowel disease (Kim et al., 2020). In this study, ten participants received
a daily dose of 200–400 g of mango pulp for 8 weeks. Mango intake significantly
improved the primary outcome, Simple Clinical Colitis Activity Index score, and
decreased the plasma levels of pro-inflammatory cytokines including interleukin-8
(IL-8), GRO , and granulocyte macrophage colony-stimulating factor by 16.2%
(p = 0.0475), 25.0% (p = 0.0375), and 28.6% (p = 0.0485), all factors related to neu-
trophil-induced inflammation, respectively. Mango intake beneficially altered fecal
microbial composition by significantly increasing the abundance of Lactobacillus
spp., Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus lactis,
which was accompanied by increased fecal butyric acid production. Therefore, an
enriching diet with mango fruits or potentially other gallotannin-rich foods seems
to be a promising adjuvant therapy combined with conventional medications in the
management of IBD via reducing biomarkers of inflammation and modulating the
intestinal microbiota (Kim et al., 2020).
Yang and colleagues studied the effect of standardized grape powder consump-
tion, a rich source of polyphenols and fiber, on the gut microbiome of healthy par-
ticipants. More specifically, 46 g of whole grape powder, providing the equivalent
200 The Gut Microbiome: Bench to Table

of two servings of California table grapes, were assessed as per their effects on the
gut microbiome in healthy adults. The study included a 4-week standardization to a
low-polyphenol diet, followed by 4 weeks of 46 g of grape powder consumption while
continuing the low-polyphenol diet. Compared to the baseline, 4 weeks of grape pow-
der consumption significantly increased the alpha diversity index of the gut microbi-
ome. The authors concluded that grape powder consumption significantly modified
the gut microbiome (Yang et al., 2021).
In separate experiments through a small-scale pilot study, Ezzat-Zadeh and cowork-
ers reported that California strawberry consumption increased the abundance of gut
microorganisms related to lean body weight, health, and longevity in healthy partici-
pants (Ezzat-Zadeh et al., 2021). More specifically, 15 healthy adults consumed a beige
diet + 26 g of freeze-dried strawberry powder (SBP) for 4 weeks, followed by 2 weeks
of beige diet only. Stool samples were collected at 0, 4, and 6 weeks. Fecal microbiota
was analyzed by 16S rRNA sequencing. Fecal cholesterol, bile acid (BA), and micro-
bial metabolites were assessed via gas chromatography. Daily SBP altered the abun-
dance of 24 operational taxonomic units (OTUs). Comparing week 4 to baseline, the
most significant increases were observed for one OTU from Firmicutes\Clostridia\
Christensenellaceae\NA, one OTU from Firmicutes\Clostridia\Mogibacteriacea\NA,
one OTU from Verrucomicrobia\Verrucomicrobiaceae\Akkermansia muciniphila,
one OTU from Actinobacteria\Bifidobacteriaceae\Bifidobacterium\NA, and one OTU
from Bacteroidetes\Bacteroidia\Bacteroidaceae\Bacteroides and a decrease of one
OTU from Proteobacteria\Betaproteobacteria\Alcaligenaceae\Sutterella. Comparing
weeks 4–6, we observed a reversal of the same OTUs from C Christensenellaceae,
V muciniphilia, and C Mogibacteriaceae. Fecal SCFAs and most of the fecal mark-
ers including cholesterol, coprostanol, and primary and secondary BAs were not
changed significantly except for lithocholic acid, which was increased significantly
at week 6 compared to baseline (Ezzat-Zadeh et al., 2021).
Wilson and collaborators investigated SunGold kiwi fruit supplementation of
individuals with prediabetes and demonstrated that such supplementation alters gut
microbiota and improves vitamin C status as well as anthropometric and clinical
markers. More specifically, two SunGold kiwifruit per day over 12 weeks were con-
sumed by participants (n = 24). Venous blood samples were collected at each study
visit (baseline, 6, and 12 weeks) to determine glycemic indices, plasma vitamin C
concentrations, hormones, lipid profiles, and high-sensitivity CRP. Participants pro-
vided a fecal sample at each study visit. DNA was extracted from the fecal sam-
ples and a region of the 16S ribosomal RNA gene was amplified and sequenced to
determine fecal microbiota composition. Compared to a baseline measurement, the
results of 12 weeks showed a significant increase in plasma vitamin C (14 µmol/L,
p < 0.001). There was a significant reduction in both diastolic (4 mmHg, p = 0.029)
and systolic (6 mmHg, p = 0.003) blood pressure and a significant reduction in waist
circumference (3.1 cm, p = 0.001) and waist-to-hip ratio (0.01, p = 0.032). Results also
showed a decrease in HbA1c (1 mmol/mol, p = 0.005) and an increase in fasting
glucose (0.1 mmol/L, p = 0.046); however, these changes were small and were not
clinically significant (Wilson et al., 2018).
An interesting study investigating the effects of a dietary supplement on the
gut microbiota was a recent study by Shin and colleagues (Shin et al., 2021). The
Powerful Interplay of Gut Microbiota and Polyphenols 201

researchers evaluated the effects of Saengshik supplementation on the gut microbial

composition among healthy Korean adults in a pilot study. Saengshik is a type of
meal replacement product or dietary supplement comprising an uncooked and dried
plant-based food mixture with various health-promoting properties, such as anti-
diabetic, anti-dyslipidemic, antioxidant, and anticancer properties. The researchers
conducted a single-group design trial involving 102 healthy men and women who
received a 40 g/day dose of Saengshik powder as a dietary supplement for 8 weeks,
during which stool samples were collected at two fixed time points (baseline and
the endpoint) for gut microbiota profiling analysis. The authors reported a sig-
nificant decrease in the α-diversity of gut microbiota after Saengshik consumption
(p <0.05), with significant changes evident in the composition of major micro-
bial taxa, such as Bacteroidetes (p <0.0001), Proteobacteria, Actinobacteria, as
well as Verrucomicrobia (p <0.0001). Markedly, the gut microbial response was
strongly related to the interindividual variability of habitual dietary intake and
enterotype at baseline (Shin et al., 2021). The obtained results suggest that indi-
vidual habitual diet patterns, as well as gut microbial shapes, should be considered
as key aspects when supplementation strategies are aimed at the optimization of
gut microflora as designed.
Recent research and clinical trials have identified the beneficial effects of a plant-
based diet to increase bacterial diversity and ameliorate various disorders, includ-
ing intestinal disorders, obesity-related endotoxemia, and cardiovascular disorders
(Vazhappilly et al., 2019, 2021; Guglielmetti et al., 2020).

Even though there is a significantly large body of evidence in support of the gut
microbiome’s role and specific enzymes in the biotransformation of polyphenols,
there are still significant aspects of the interplay between the microbiome and poly-
phenol metabolism that need to be unlocked. It appears that gut bacteria possess
innate mechanisms, allowing them to generate de novo and potentially highly bioac-
tive compounds when exposed to dietary polyphenols.
In this regard, innovative approaches are essential to reveal the precise activities
of certain bacteria and the exact role they play in the biotransformation of poly-
phenols. One such approach entails gut bacterial population characterization, using
metagenomic binning of bacterial DNA methylation to create bacterial barcodes for
efficient identification of unique bacterial strains. Other efforts employ gnotobiotic
and germ-free mice to manipulate bacterial populations so as to better understand
host metabolism. Such novel approaches can enhance identification of the distinc-
tive metabolism of gut microbiota, different among individuals, as well as elucidate
how this may regulate bioavailability and modulate the bioactivity of polyphenolic
metabolites (Pasinetti et al., 2018). These approaches could provide more insights
as to the interpersonal variation usually observed in the microbiome and metabolite
types in human trials. Such complex polyphenol–microbiota interactions, which are
determined notably by interindividual variability leading to different polyphenol-
metabolizing phenotypes or “metabotypes”, may be the key in terms of a more
202 The Gut Microbiome: Bench to Table

personalized approach for the optimization of diet and health with the involvement
of the microbiome (Bolca et al., 2013). The term “metabotype” refers to a particu-
lar metabolic phenotype with specific gut microbiome-derived metabolites, which
characterize metabolism of the parent compound (i.e., nutrient, pharmaceutical, and
toxin) (Espin et al., 2017).
In general, limited human studies have studied metabotypes. More specifically, a
pilot study assessed the phenolic compound capsaicin and reported stronger effects
on metabolic and inflammatory markers in healthy participants of the Bacteroides
enterotype compared to those of the Prevotella enterotype (Kang et al., 2016). Other
researchers reported that an interaction was observed between gut bacterial profiles
and enterolignan production in response to lipid levels after 1 week of intervention
in healthy normal-weight participants (Lagkouvardos et al., 2015). Interestingly,
even though the ability to stratify the population based on their capacity to pro-
duce specific microbial-derived metabolites in response to certain polyphenols and
the impact of this differential on health has not been investigated significantly,
most available evidence pertains to the correlation of certain metabotypes to iso-
flavones and ellagitannins. More specifically, randomized clinical trials have dem-
onstrated that patients’ capacity to produce the microbial metabolites equol and
O-desmethylangolesin (ODMA) has been shown to reduce the risk for CVD (Espin
et al., 2017). Ellagitannins represent another interesting example of a nexus between
a specific metabotype and a polyphenolic compound(s). In this regard, there are
three distinct metabotypes: (a) type A which is characterized by excretion of Uro-A
and related conjugates as the final urolithin; (b) type B which produces Uro-B and/
or IsoUro-A additionally to Uro-A; and (c) type 0 which does not produce any of
these urolithins. Interestingly, metabotype B has been correlated with obesity, meta-
bolic syndrome, and colorectal cancer, hence suggesting a potential role in dysbiosis
for this metabotype (Espin et al., 2017). Furthermore, in certain human trials with
overweight participants, while metabotype A was positively associated with HDL
cholesterol, metabotype B was associated with VLDL (Selma et al., 2009). These
interesting results could at least partly contribute to a potential explanation for mixed
and nonconclusive results in the case of polyphenol-rich dietary supplementations
such as the earlier case of the pomegranate study, for example. Interindividual vari-
ability regarding the improvement of cardiovascular risk in 49 healthy overweight
or obese participants consuming pomegranate was explained by urolithin metabo-
types (Gonzalez-Sarrias et al., 2017). Although the exact mechanisms explaining the
gut microbiota/polyphenol interplay-related impact on human health remains under
investigation, it appears that microbiota-derived bioactive metabolites from dietary
polyphenols are primarily involved in such effects.
Finally, it is important to consider the overall diet in addition to the interventions
made in terms of polyphenols when microbiota is considered. There is strong evi-
dence supporting the plasticity of the microbiome and it is significantly influenced
by the dietary regimen of the individual regardless of other factors. For example, in a
cross-sectional study, Noh et al. investigated the association of habitual dietary intake
with the taxonomic composition and diversity of the human gut microbiota among
222 Koreans aged 18–58 years (Noh et al., 2021). Gut microbiota data were obtained
by 16S rRNA gene sequencing on DNA extracted from fecal samples. The habitual
Powerful Interplay of Gut Microbiota and Polyphenols 203

diet for the year previous to the study was evaluated through a food frequency ques-
tionnaire. After multivariable adjustment, intake of several food groups, including
vegetables, fermented legumes, legumes, dairy products, processed meat, and nonal-
coholic beverages, were associated with major phyla of the gut microbiota. A dietary
pattern related to higher α-diversity (HiαDP) derived by reduced rank regression
was characterized by higher intakes of fermented legumes, vegetables, seaweeds,
and nuts/seeds and lower intakes of nonalcoholic beverages. The HiαDP was found
to be positively associated with several genera of Firmicutes such as Lactobacillus,
Ruminococcus, and Eubacterium (p <0.05). Among enterotypes identified by prin-
cipal coordinate analysis based on the β-diversity, the Ruminococcus enterotype
exhibited higher HiαDP scores and was strongly positively associated with the intake
of vegetables, seaweeds, and nuts/seeds, compared to the two other enterotypes. The
authors concluded that a plant- and fermented food-based diet was positively associ-
ated with certain genera of Firmicutes indicative of improved gut microbial health.
Their conclusions indicate that diet is a very strong determinant, in its own right, of
the microbiota profile in humans, which should be appreciated and considered as
such in studies and therapeutic approaches.
Moreover, significant in size, human studies with long-term assessment of diet also
support further the point discussed above. More specifically, Yu et al. showed that
long-term diet quality is associated with gut microbiome diversity and composition in
urban Chinese adults (Yu et al., 2021). The studies comprised 1,920 men and women,
enrolled in two prospective cohorts (baseline 1996–2006), who remained free of
CVDs, diabetes, and cancer at stool collection (2015–2018) and had no diarrhea or
antibiotic use in the last 7 days before stool collection. The microbiome was profiled
by 16S rRNA sequencing. Long-term diet was assessed by repeated surveys at baseline
and follow-ups (1996–2011), with intervals of 5.2–20.5 years between dietary surveys
and stool collection. Associations of dietary variables with microbiome diversity and
composition were evaluated by linear or negative binomial hurdle models, adjusting
for potential confounders. False discovery rate (FDR) <0.1 was considered significant.
Diet quality was positively associated with microbiome α-diversity and abundance of
Firmicutes, Actinobacteria, Tenericutes, and genera/species within these phyla, includ-
ing Coprococcus, Faecalibacterium/Faecalibacterium prausnitzii, Bifidobacterium/
Bifidobacterium adolescentis, and order RF39 (all FDRs <0.1). Significant associations
were also observed for intakes of dairy, fish/seafood, nuts/legumes, refined grains, and
processed meat, including a positive association of dairy with Bifidobacterium and
inverse associations of processed meat with Roseburia/Roseburia faecis. Most asso-
ciations were similar, with or without adjustment for BMI and hypertension status or
excluding participants with antibiotic use in the past 6 months (Yu et al., 2021).
Overall, there are limited studies in humans, typically with a small number of
participants examining the effect of polyphenols on the configuration and activity of
the gut microbiome. This represents an important gap in our understanding regarding
the polyphenol–microbe interactions in humans. Nonetheless, there is some evidence
mostly in the form of in vitro, in vivo studies, while more rarely clinical studies,
suggesting that particular doses of specific polyphenols do extend modulation of the
gut microbial populations; hence, as certain bacterial groups can be inhibited, oth-
ers can thrive. While this concept carries biological plausibility, further research is
204 The Gut Microbiome: Bench to Table

warranted and more results need to be obtained for such a relationship to be estab-
lished and most critically a mechanistic explanation to be derived.
In conclusion, all the findings suggest that in addition to the dietary polyphenols,
their microbial metabolites must also be taken into consideration when assessing
the impact of polyphenols on the host’s health. Despite the recent surge of inter-
est in their relationship and our increased appreciation of this dynamic cross talk
between dietary polyphenols and gut microbiota, we are far from fully grasping their
therapeutic potential for employing informed strategies in medical nutrition therapy
and/or microbial therapeutic interventions. However, smartly manipulating the gut
microbiota through dietary interventions for the prevention or treatment of disorders
might lead to the enrichment of “personalized nutrition” to be more comprehensive
in its considerations and help better understand the effects of dietary bioactive com-
pounds on the host microbiome.
From a clinical perspective and through a lens of developing therapeutic pharma-
ceuticals and schemes, there is strong evidence from human studies that polyphenols
contribute to disease prevention and the overall properties (antioxidant, anti-inflam-
matory) contribute to the favorable health outcomes documented (Safe et al., 2021).
However, it is equally evident from mechanistic in vitro and in vivo studies that
individual compounds as well as their mixtures modulate activity and/or expression
of multiple genes and downstream responses at various levels and degrees. For exam-
ple, similarly to other flavonoids, genistein interacts directly with multiple tyrosine
kinases, G-protein coupled receptors, and intracellular receptors alike. Therefore,
a potential clinical application utilizing genistein that would target a single gene/
pathway is rather complicated due to its diverse activities and highly possible off-
target effects. Discerning the successful development of clinical applications of
polyphenols, a series of requirements pertinent to mechanistic precision medicine
approaches need to be considered and include the following aspects:

Another limitation in phenolic compound-related research and potential applica-

tions in human health is that typically, existing studies have generally ignored the
effect of food matrix and processing conditions. Food components interact, not
uncommonly highly selectively, with different forms (e.g., free, bound, and polym-
erized) of polyphenols. Such interactions significantly affect the bioaccessibility
Powerful Interplay of Gut Microbiota and Polyphenols 205

and subsequently the bioavailability that ultimately affect the phenolic compounds
and their metabolites. They can directly act on the epithelium to modulate local
immune system functions and absorb metabolites into the systemic circulation
to have systemic effects on the host. It is a very challenging task for the research
community to grasp the relationship fully and delineate the various interplays
among the complex gut microbiota, the diverse types, and forms of dietary poly-
phenols, the varied immunomodulatory ability of the hosts, and connections with
gut–brain functions.
Innovative approaches such as that of Foodomics, driven by the integration of
the use of advanced-omics technologies, such as transcriptomics, proteomics, and
metabolomics, together with biostatistics, chemometrics, and bioinformatics, to
allow the evaluation of complex biological systems, as well as the mechanisms
of bioactive food compounds that may affect them, are emerging (Valdés et al.,
2022). The utilization of such methods has brought a significant change in the field
of food science, as the performed research may be redirected to ascertain new-
fangled associations. For example, by way of Foodomics-related applications, our
knowledge regarding the binomial between food/diet and health has widened.
Foodomics expand the possibilities toward unraveling the enormous complexity
of the Foodome, which has been defined as the pool of all compounds present in
a food item and/or in a biological system interacting with the investigated food
at a given time (Valdés et al., 2022). The investigation in foods of bioactive com-
pounds which widely vary in chemical structure, including polyphenols, can be
meaningfully supported by Foodomics toward understanding function, but also
investigating the presence, bioavailability, and biological characteristics (such as
toxicity, antioxidant, antiproliferative, or anti-inflammatory properties) of these
interesting molecules, while considering different food matrices and processing
influences (Valdés et al., 2022). Such approaches may constitute an important and
most helpful tool toward advancing our current understanding of those interac-
tions and their effects.

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 8 Diet, Obesity, and
Gut Microbiome
Yu-Chieh Cheng
Academia Sinica

Alison Lacombe and Vivian C.H. Wu

United States Department of Agriculture

CONTENTS
Introduction ............................................................................................................ 211
Gut Microbiota Formation and Characteristic ....................................................... 214
Gut Microbial Composition, Dynamics, and Function ..................................... 214
Diversity and Composition of Gut Microorganisms Implicated in
Host Health Maintenance .................................................................................. 214
Energy Metabolism and Gut Microbiota ............................................................... 217
Gut Microbiota in Obesity ................................................................................ 217
Regulation on Host Metabolism........................................................................ 218
Modulating Feed Behavior and Satiety ............................................................. 220
Modulating Feed Behavior by Gut–Brain Axis ................................................ 222
Dietary Modulating Gut Microbiota Composition ................................................ 223
Dietary Styles in Relation to Gut Microbial Composition ................................ 223
Macronutrients ..................................................................................................224
Carbohydrates...............................................................................................224
Fat ...................................................................................................................... 225
Protein ............................................................................................................... 225
Micronutrients ................................................................................................... 226
Applications ........................................................................................................... 226
Clinical Therapeutics ........................................................................................ 227
Probiotics and Prebiotics Intervention .............................................................. 227
Conclusion ............................................................................................................. 230
References .............................................................................................................. 230

INTRODUCTION
Obesity and overweight have become serious public health issues worldwide. The
population of obese individuals has shown rapid growth over the past four decades.
From 1975 to 2016, obesity prevalence demonstrated nearly tripling growth. In 2016,
more than 1.9 billion adults (18 years and older) were defined as overweight, and

DOI: 10.1201/b22970-11 211

212 The Gut Microbiome: Bench to Table

650 million adults were obese (World Health Organization, 2020). Overweight and
obesity issues also reflect human physiological conditions that represent abnormal
or excessive fat accumulation. To define overweight and obesity in adults, body
mass index (BMI) is a typical approach that calculates the ratio of a person’s weight
expressed in kilograms divided by the square of his/her height expressed in meters.
For the BMI scale, according to the World Health Organization definition, over-
weight in adults is a BMI greater than or equal to 25, whereas obesity in adults is
a BMI greater than or equal to 30. BMI provides a useful and standardized mea-
sure that only needs simple and physiological data to readily classify overweight and
obese populations in adults (World Health Organization, 2020).
While there are complex and multiple reasons for obesity, the fundamental cause
of obesity is an energy imbalance when energy intake exceeds energy expenditure.
Macronutrients from dietary intake are used as a metabolic fuel and for maintaining
physiological function, while any excess calories promote the accumulation of adi-
pose tissue, leading to obesity. The most common obesogenic dietary pattern is that
of a high-fat and high-sugar diet. High–energy–density foods provide few portions
with extremely high calories, a condition that, when combined with lower levels of
physical activity due to an increase in sedentary behavior, leads to obesity. Excessive
energy intake is a cause of obesity but not the single obesogenic factor (Chooi et al.,
2019). Alterations in dietary and physical activity patterns are often linked to envi-
ronmental and societal changes as well as a lack of policies to support health and
agriculture, change the mode of transportation, urban planning, food overprocessing,
and lack of education.
Overweight and obesity have a detrimental relationship with human health, which
may lead to metabolic disorders such as type 2 diabetes, dyslipidemia, and hyper-
tension. Other systemic complications, cardiovascular diseases, and musculoskeletal
disorders are also related to obesity and overweight. There is evidence that obesity
may increase the risk of liver, gallbladder, and pancreatic cancers, as well as hema-
tological cancers and aggressive prostate cancer (Calle and Kaaks, 2004). Previous
reports revealed that obese patients exhibit a higher risk for hospitalization and
mechanical ventilation support due to insufficient expiratory reserve volume, func-
tional capacity, and respiratory system compliance. Thus, these functional disorders
can lead to significant increases in morbidity and mortality from COVID-19 (Caussy
et al., 2020; Dietz and Santos-Burgoa, 2020; Popkin et al., 2020).
Currently, the mortality of obese and overweight individuals is higher than
underweight individuals in the world. The high prevalence of obesity develops seri-
ous social problems and may squander unnecessary medical resources in society.
Fortunately, obesity and overweight rates could be attenuated through a healthy diet
and lifestyle, as well as increasing physical activity. According to the World Health
Organization suggestions, limiting energy intake from sugars and fats; increasing
fruit, vegetable, legume, whole grain, and nuts consumption; and enhancing regu-
lar physical activity frequency are advised to individuals to prevent overweight and
obesity. With obesity becoming a worldwide epidemic, insights into how obesity
contributes to metabolism and human homeostasis, as well as new interventions, are
urgently needed. Nowadays, numerous research teams are dedicated to inquiry on
the mechanism to provide more efficient resolutions for obesity.
Diet, Obesity, and Gut Microbiome 213

The human gastrointestinal (GI) tract is colonized by abundant microorganisms

from the external environment, including bacteria, archaea, eukarya, and viruses
that form an ecosystem so-called the “gut microbiota.” The gut microbiota has the
largest number of bacteria and the most diversity among other parts of the body,
which number approximately 100 trillion. (Rinninella et al., 2019; Thursby and Juge,
2017). Previous studies have estimated that the density of bacterial cells in the colon
is between 1011 and 1012/mL, making the colon one of the most intensive micro-
bial habitats on Earth (Ley et al., 2006). The formation of the gut microbiota is
exogenous. Infants are born to be uninhabited with gut microflora, but they receive
exogenous microbes and establish gut microflora in the GI tract from the environ-
ment and mother, developing into a complex microbial ecosystem gradually (Nejrup
et al., 2017). The establishment of the gut microbiome and its stability in the GI tract
requires approximately 2 years. Thus, the first colonizers of the human gut are crucial
to the host’s early life and are in turn linked to a range of diseases, immune system
development, and energy metabolism. The diversity of microorganisms in the gut is
increased from breastfeeding and kept stable when transferred to solid food due to
reduced food diversity (Zmora et al., 2019). Various studies have also demonstrated
that there is a correlation between the childhood gut microbiota composition and
immune and metabolic disorders later in life (Milani et al., 2017; Rao et al., 2021).
The composition of the gut microbiota is dynamic; microbial communities are a
complex dynamical system and change promptly following host lifestyle, gestational
age, host intestinal epithelium, immune ontogeny, environments, medication, and
diet (Gonze et al., 2018). Variations in the gut microbiota communities benefit host
health conditions by modulating host immunity, strengthening gut integrity, shaping
the intestinal epithelium, and protecting the pathogen invasion (Thursby and Juge,
2017). Moreover, the gut microbiota has been reported to have a clear influence on
human energy homeostasis, aging, exercise performance, circadian rhythm, and
brain function (Foster and Neufeld, 2013; O’Toole and Jeffery, 2015; Scheiman et al.,
2019; Voigt et al., 2016). If the commensal microflora is disrupted, known as micro-
bial dysbiosis, the host immune system would be damaged easily and develop intes-
tinal disorders such as irritable bowel syndrome and inflammatory bowel disease
(IBD). Microbial dysbiosis has been a serious consequence that leads to the patho-
genesis of colon, gastric, pancreatic, laryngeal, breast, hepatic, and gallbladder car-
cinomas as well (Kaakoush et al., 2012; Rinninella et al., 2019; Sobhani et al., 2011;
Xuan et al., 2014). Moreover, numerous studies have proven the correlation between
the gut microbiota and host energy metabolism, which could separate into multiple
parts, such as satiety modulation, fat accumulation, and energy production. When
dysbiosis occurs, the risks of metabolic disorders such as obesity, type 2 diabetes,
nonalcoholic liver disease, cardiometabolic diseases, and malnutrition are increased
significantly (Bäckhed et al., 2004; Fan and Pedersen, 2021). At present, scientists
are gaining knowledge concerning the interplay between the microbial community
and host physiology in order to develop promising therapeutic interventions. In this
chapter, the relationship between the gut microbiota and obesity is discussed. The
chapter focuses on critical factors that influence the bacteria–gut–host physiology
system in order to gain a clear understanding of the potential of the bacterium to
improve host metabolic health.
214 The Gut Microbiome: Bench to Table

GUT MICROBIOTA FORMATION AND CHARACTERISTIC

G

The gut microbiota composition is unique and has large diversity, which plays a vital
role in host metabolism, immunomodulation, and biosynthesis. The gut microbiota
involves not only nutrient absorption but also metabolite production, including lipids,
amino acids, bile acids, and short-chain fatty acids (SCFAs). Moreover, resident bac-
teria inhibit pathogen invasion by maintaining intestinal epithelium integrity, com-
peting for nutrients and space with pathogens by pH modification and antimicrobial
peptide secretions (Coyte and Rakoff-Nahoum, 2019). Microorganisms are classified
into phyla, classes, orders, families, genera, and species by using the taxonomy. The
dominant phyla in the gut microbes are Firmicutes, Bacteroidetes, Actinobacteria,
Proteobacteria, Fusobacteria, and Verrucomicrobia. Firmicutes and Bacteroidetes
account for the vast majority of microorganisms which are approximately 97% of the
gut microbiota. Over 200 genera of Firmicutes are found in this phylum, including
Lactobacillus, Bacillus, Clostridium, Enterococcus, and Ruminicoccus. In addition,
the Clostridium genus constituted 95% of the Firmicutes phylum. Bacteroides are
the most abundant and variable genera which contain Bacteroides and Prevotella.
The predominant genera in Bacteroidetes are Bacteroides and Prevotella. For
Actinobacteria, the most abundant genus is Bifidobacterium (Arumugam et al., 2011;
Rinninella et al., 2019; Rosenbaum et al., 2015).

The first understanding of this interaction relied on germ-free (GF) mice, which exist
in a sterile environment completely without microorganisms. It is illustrated that
GF animals had a lower body weight and had poorer intestinal function than con-
ventionally raised mice. An enlarged liver and lower concentrations of amino acids
in the gut were observed as well. Scientists used GF mice to colonize microbiota
from conventionally raised mice, and the results showed a significant increase in
body weight, fat accumulation, and relative insulin resistance (Bäckhed et al., 2004).
Interestingly, although body weight was increased, the energy expenditure grew by
approximately 30%, whereas energy intake declined by 30% approximately after
GF mice received a microbial community. Many GF animal studies proved resi-
dent microbes are implicated in the energy balance equation (Bäckhed et al., 2007;
Rosenbaum et al., 2015). An additional study mentioned that GF animals are pro-
tected against diet-induced obesity such as Western-style, high-sugar, and high-fat
diets (HFDs) (Bäckhed et al., 2007). Supporting this notion, increased expression of
glucagon-like peptide-1 (GLP-1), which is an obesity-related peptide in the brain and
can reduce food intake, was observed (Uzbay, 2019).
The species variation of gut microbiota is dynamic. The colonization of microbes
in the GI tract begins postpartum. Infants are born to be uncolonized with the gut
microbiota and then develop an unstable microflora receiving from the environment
and the maternal microbial reservoir. Infants temporarily acquire microbes and form
microflora from the maternal skin and vagina and then persistently accumulate from
Diet, Obesity, and Gut Microbiome 215

the maternal gut, and it has been proven that the strains are more adapted than other
sources (Ferretti et al., 2018). Many factors, such as types of birth delivery, gesta-
tional age at birth, methods of milk feeding, and antibiotic medications, impact the
composition of the neonatal gut microbiota. These pioneering microbes utilize lac-
tose, galactose, and sucrose as their energy sources; after infants switch diet into solid
food, the gut microbiome alternates to the pathway of carbohydrate fermentation and
vitamin biosynthesis (Zmora et al., 2019). However, dysbiosis has been revealed to
be related to several diseases during the early stage, such as allergies, asthma, type 1
diabetes, Crohn’s disease, IBD, necrotizing enterocolitis, and inflammation (Ferretti
et al., 2018; Renz and Skevaki, 2021). Developing a healthy gut microbial community
in the early stage is crucial for later life. Conversely, studies have indicated that the
human gut microbiome can be altered and develop into maturity over the different
life stages. Scientists have observed a decline in microbiota diversity during aging
in elderly people. There is a trend whereby saccharolytic bacteria decrease while
proteolytic bacteria increase (Bischoff, 2016). Elderly individuals have higher levels
of Escherichia coli, Bacteroidetes, and Proteobacteria than adults and infants but a
lower ratio of Firmicutes to Bacteroidetes than younger adults (Mariat et al., 2009).
The dominant population has been replaced by subdominant species, which may be
due to an age-related decrease in immune function and aging-associated pathologies
(Vaiserman et al., 2017).
Additionally, more factors alter the composition of the gut microbiome. Circadian
rhythms modulate daily events that affect feeding, sleeping, hormone secretion,
metabolism, and temperature, which rely on the oscillation of light during the 24 h
cycle (Liang and FitzGerald, 2017; Mu et al., 2016). The functions of the intestine, gut
motility, and nutrient absorption are also affected by the circadian rhythm (Hussain
and Pan, 2009). The gut microbiota shows diurnal variations modulated by circadian
rhythms. Bacteroidetes, Firmicutes, and Proteobacteria are reported to be related to
oscillation. Bacteroidetes were discovered to be the most sensitive phylum that exhib-
ited circadian rhythmicity. During the daytime, Bacteroidetes are more copious than
nighttime with abundance oscillations which impacts the gut microbiome assembly
(Liang et al., 2015; Zmora et al., 2019). In addition, disruption of the host circadian
clock drives loss of microbiota rhythms and dysbiosis which facilitates glucose intol-
erance and obesity in humans and mice (Thaiss et al., 2014).
Previous studies have recently shown the association between microbial profiles
and numerous human diseases, such as cardiovascular disease, IBD, irritable bowel
syndrome, obesity, and type 2 diabetes. Furthermore, accumulating evidence sug-
gests that the gut microbiota is involved in maintaining brain health. It has been
reported that the gut microbiota impacts the central nervous system homeostasis
indirectly by modulating neurotransmitter and host immune function and blood–
brain barrier integrity. Thus, the alterations react to stress responsivity, anxiety-like
behaviors, sociability, and cognition (Luczynski et al., 2016). Increasing validation
for the essential role of microbial metabolism in maintaining the host immune system
has been found in recent research analyzing the composition and function of individ-
ual microbial species and complex microbial communities. The crosstalk between
the gut microbiome and host immune system is complex and has systemic impacts
on the whole body. In general, resident bacteria produce metabolites by undigested
216 The Gut Microbiome: Bench to Table

nutrients, as well as endogenous compounds to modulate the host immune system

and lead to immune reactions or dysfunction. The GI tract has a single layer of epi-
thelial cells that boost microbial-produced compounds that can easily interact with
host cells (Rooks and Garrett, 2016). One of the factors that leads to gut immune
system development and functional maturation is the gut microbiota. Resident bac-
teria can interact with gut-associated lymphoid tissue, T helper 17 cells, inducible
regulatory T cells, IgA-producing B cells, and innate lymphoid cells. Furthermore,
the gut microbiota prevents pathogen invasion by direct competition for nutrients,
indirect innate immunity enhancement, and adaptive immunity promotion (Kamada
et al., 2013). One of the most direct and crucial elements that changes the gut micro-
biome is the dietary pattern. Many studies have demonstrated that different dietary
styles produce varying gut microbial profiles and could be modified by changing
the dietary pattern in just a few days. The change in accurate time depends on indi-
viduals, which is approximately 1–4 days (Zmora et al., 2019). Variations in the gut
microbiome with dietary conversion can have synergistic or opposing effects on the
host health. The details of food modulation will be further discussed later.
Resident bacteria in the gut produce a large number of metabolites from the fer-
mentation of undigested carbohydrate and host-derived products, and it can mediate
host physiology by signaling, stimulatory, and inhibitory. These bacterial metabo-
lomes can interact with host metabolomes and can be detected in host feces, urine,
and blood (Flint et al., 2015; Martin et al., 2007). The metabolites induce and medi-
ate host homeostasis in multiple ways, inducing both local and systemic effects. In
the gut lamina propria, these metabolites can affect barrier function and immune
cell populations. Additionally, metabolites may affect downstream organ systems as
well. Actively or passively transported metabolites can cross the intestinal epithelium
and directly affect distal organ functions or diseases. Moreover, the metabolites pro-
duced by groups of microbes can impact other members of the microbiota or patho-
gens, altering their composition or function. Changes both locally and systemically
can have indirect consequences for the host (McCarville et al., 2020).
SCFAs are a major product generated by microbial from the fermentation of
undigested fibers, including acetic acid, butyric acid, and propionic acid. Most of
SCFAs are produced at the distal colon and absorbed mostly (Topping and Clifton,
2001). Various species have been reported that have the ability to produce SCFAs.
Dietary patterns with rich fermentable fiber or microbe-accessible carbohydrates
provide a critical source for the bacteria that produce SCFAs (Sonnenburg et al.,
2016). In general, acetate and butyrate are the major energy providers in all body tis-
sues, while propionate participates in the gluconeogenic pathway which occurs in the
liver. Additionally, acetate can be used for lipogenesis and is associated with satiety-
regulating hormones. In colonocytes, approximately 60–70% of the energy is from
butyrate, and it is a preferential energy resource for colon cells by using diffusion or
plasma membrane transporters (Blaut, 2015; McCarville et al., 2020). SCFAs have
been reported to possess anti-inflammatory and antiapoptotic abilities and act as
histone deacetylase inhibitors that induce colon cancer cell cycle arrest, differentia-
tion, and apoptosis. Thus, SCFAs promote intestinal integrity and play an important
role in colorectal cancer and colitis prevention. G protein-coupled receptors are inte-
gral membrane proteins on the surface of epithelial cells and immune cells. SCFAs
Diet, Obesity, and Gut Microbiome 217

bind to G protein-coupled receptors to induce signal transduction into host cells,

resulting in the promotion of immune tolerance and gut homeostasis. The SCFAs–G
protein-coupled receptors interaction enhances forkhead box P3 expression, trans-
forming growth factor-β and interleukin-10, which are pivotal anti-inflammatory
activities in the host body (Rooks and Garrett, 2016). Moreover, inhibition of nuclear
factor-κB and modulation of interleukin-8 have also been reported to represent
mediating mechanisms of SCFA-induced effects on host immunity. In response to
SCFAs, enhanced mucin gene transcription by intestinal goblet cells and altered tight
junction permeability of intestinal epithelial cells are observed to enforce epithelial
barrier integrity, thereby preventing pathogen invasion (Rooks and Garrett, 2016;
Willemsen et al., 2003). Butyrate has been demonstrated in vitro to play an impor-
tant role in maintaining the gut barrier function, stabilizing transcription factors,
assembling tight junction proteins, and keeping mucin secreted even under anaerobic
conditions. Enhancing the integrity of the gut barrier can block the translocation of
lipopolysaccharide (LPS), which has been associated with insulin resistance, inflam-
mation, adiposity, hepatic fat, and metabolic endotoxemia (Cani et al., 2008). In addi-
tion, SCFAs also participate in appetite control (Byrne et al., 2015).
However, high levels of SCFAs are linked with obesity but inversely correlated
with gut microbiota diversity. Metagenomic studies have shown that the obese human
gut microbiome exhibits upregulation of the pathways related to the processing of
carbohydrates and SCFAs production (Turnbaugh et al., 2009). SCFAs overexcre-
tion demonstrates a positive correlation with gut dysbiosis, gut permeability, excess
adiposity, and cardiometabolic risk factors (la Cuesta-Zuluaga et al., 2019). This con-
tradictory situation may indicate the involvement of additional particular bacteria or
metabolites that may trigger multiple metabolic mechanisms into a systemic result.
LPSs are microbial metabolites produced by gram-negative bacteria particularly.
They are found in the outer membrane of bacteria and consist of lipids and polysac-
charides. For the host, LPSs are endotoxins that pass through the gut barrier into cir-
culation and induce inflammation (Manco et al., 2010). A high concentration of LPS
in circulation is closely related to metabolic disorders. Moreover, HFDs alter the gut
microbiota composition and induce LPS absorption, resulting in elevated LPS levels
in circulation (Heiss and Olofsson, 2018).

ENERGY METABOLISM AND GUT MICROBIOTA

G

Gut microbiota composition has been linked to BMI class and diet. The ratio of
Bacteroidetes and Firmicutes phyla shows variation in obese adults, in which
Bacteroidetes decrease dramatically and enrich the proportions of Firmicutes as
well as Proteobacteria. A clinical trial supports this notion. The individuals were
separated into two groups and received an underfeeding or overfeeding diet. The
results revealed that the energy intake has a positive correlation with the relative
amount of Firmicutes but a negative correlation with the relative abundance of
Bacteroidetes. These studies validated the gut microbiota is responsive to host energy
balance (Jumpertz et al., 2011). Moreover, scientists have discovered that differences
218 The Gut Microbiome: Bench to Table

at the genus and species level are more relevant to obesity. For example, the relative
amount of Bifidobacterium decreased while Lactobacillus increased conversely in
overweight to obese individuals (Million et al., 2012). Erysipelotrichaceae, the pre-
dominant family, has four groups of closely related species-level phylotypes that were
revealed to have different responses during dietary changes and metabolic pheno-
types (Zhao, 2013). Previous studies indicated lower proportions of Bifidobacterium
vulgatus in obese humans than in lean individuals (Bervoets et al., 2013; Rinninella
et al., 2019). These variations are very sensitive to energy balance and can be recov-
ered along with weight loss based on a healthy diet.
Similarly, the gene richness and diversity of the gut microbiome declines signifi-
cantly in obese adults compared to lean individuals and is enhanced after dietary
weight loss (Rosenbaum et al., 2015; Torres-Fuentes et al., 2017). Low microbial rich-
ness also has a relationship with host metabolic parameters, including serum insulin,
homeostasis model assessment insulin resistance, triglyceride levels, and free fatty
acids in plasma (Le Chatelier et al., 2013). This means that the variation in the gut
microbiota in obese populations results in dysbiosis and affects host metabolism and
health, which may lead to detrimental comorbidities and complications such as dia-
betes and metabolic syndrome. The low diversity of the gut microbiome also has a
high correlation with trunk fat mass and comorbidities such as type 2 diabetes and
hypertension (Aron-Wisnewsky et al., 2019). Furthermore, the obese microbiome is
transmittable. GF mice inoculated with bacteria from obese mice’s cecum exhibited
significant weight gain compared with those received from a lean donor. A decline
in fecal energy excretion is observed in colonization with microbiota from an obese
donor, and it has been illustrated that the gut microbiome from obese individuals can
enhance energy harvest capacity from the diet (Turnbaugh et al., 2006).

The crosstalk between the gut microbiome and host is implicated in metabolism and
energy homeostasis. Scientists have found this phenomenon by using the GF mouse
model. GF mice lacking gut microbiota are protected against diet-induced obesity
with an HFD. GF mice fed a lard-based HFD showed increased energy expenditure,
fecal fat content, and energy excretion, which led to diet-induced obesity. It has also
been observed that mice with microbiota depletion have more browning of the ingui-
nal subcutaneous and perigonadal visceral adipose tissue. This metabolic improve-
ment can enhance the volume of brown adipose tissue, improve insulin sensitivity, and
decrease white fat and adipocyte size (Suárez-Zamorano et al., 2015). In short, these
results indicate the reason why GF mice have the resistance to diet-induced obesity.
Additionally, accumulating evidence suggests that gut microbiome composition
and host metabolism are highly correlated. The alteration of the Bacteroidetes and
Firmicutes ratio in the gut depends on the difference in the host metabolism sta-
tus and it is illustrated that related to carbohydrate degradation and fermentation
(Ley et al., 2006). Moreover, scientists monitored the relationship between rest-
ing energy expenditure, body composition, and the gut microbiome. The popula-
tion of Firmicutes has demonstrated a positive correlation with fat mass, whereas
Diet, Obesity, and Gut Microbiome 219

the opposite relationship with resting energy expenditure and the maximal oxygen
consumption (VO2max) has been observed (Kocełak et al., 2013). The gut microbi-
ome composition can be altered by probiotics and prebiotic intervention. By using
inulin-type fructo-oligosaccharides as a supplement, an increase in health-promoting
species such as Bifidobacterium, Lactobacillus, Roseburia, and Faecalibacterium
has been observed. This alteration has been proven to decrease postprandial plasma
glucose responses after a standardized meal, also enhancing plasma GLP-1 and pep-
tide YY (PYY) concentrations which may contribute to host satiation and glucose
homeostasis (Cani et al., 2009).
Accumulating evidence proves that there are specific relevant functions in each
phylum. In Firmicutes, some species transfer indigested fiber into butyrate and join
symbiosis with pathogens. The other predominant phylum, Bacteroidetes, has been
reported to degrade polysaccharides and ferment fiber, and its major energy sources
are protein and grain-rich diets. Actinobacteria, which account for approximately 3%
of the total gut bacteria, have been illustrated to work on host vitamin biosynthesis.
In addition, although other minor phyla account for less than 1%, they are also impli-
cated in host metabolisms, such as iron degradation, vitamin synthesis, and carbohy-
drate fermentation (Consortium, 2012; Turnbaugh et al., 2009). Specific bacteria may
inhibit circulating lipoprotein lipase inhibitors in the intestinal epithelia, resulting in
host weight gain due to enhanced triglyceride synthesis, promoted fat accumulation
as well as reduced insulin sensitivity (Sun et al., 2018).
Furthermore, metabolites produced by resident microorganisms comprehensively
affect host energy homeostasis function. SCFAs, bacterial metabolites, provide an
important energy source for intestinal epithelial cells. The major consortiums of
bacteria that produce SCFAs were Faecalibacterium prausnitzii and Eubacterium,
Lactobacillus, and Bifidobacterium species. Bacteria produce SCFAs throughout
carbohydrate fermentation, and increased energy harvesting accounts for a sub-
stantial part of energy uptake. The gut microbiome also protects against Western
diet–induced obesity and metabolic disorders by SCFAs production. According
to previous studies, the intervention with butyrate and succinate in mouse models
improved glucose sensitivity and glucose tolerance and enhanced energy expenditure
(De Vadder et al., 2016; Gao et al., 2009). Type 2 diabetes patients have been discov-
ered with the α lower relative ratio of SCFA-producing bacteria in the gut microbiota.
Clinically, a high-fiber diet alters gut microbiota composition and improves patients’
glycated hemoglobin A1c (HbA1c) via induced GLP-1 production (Zhao et al., 2018).
Conversely, some studies have indicated that there is a negative correlation between
host metabolism and SCFAs, especially acetate and propionate. In the rodent model,
circulating acetate which is enriched by altering gut microbiota leads to activation of
the parasympathetic nervous system, which may drive insulin secretion and increase
the risk of obesity (Perry et al., 2016). Studies in humans have revealed that a diet
with propionate may induce insulin resistance and obesity. One of the reasons may
be that SCFAs are ligands of free fatty acid receptors (FFAR2 and FFAR3). Free
fatty acid receptors activation has a high correlation with satiety and insulin sensitiv-
ity (Tirosh et al., 2019). Hence, the beneficial effect of SCFAs on host metabolism is
controversial and inconsistent.
220 The Gut Microbiome: Bench to Table

Accumulating evidence suggests the implication between obesity and LPS

which are produced by gram-negative bacteria. A number of Enterobacteriaceae
and Desulfovibrionaceae families which belong to the phylum Proteobacteria were
enhanced in the obese population. These families are known to produce high con-
centrations of LPS (Zhao, 2013). LPS is recognized by toll-like receptor 4 which
induces cholecystokinin (CCK) secretion to impact the host gastrointestinal system.
Additionally, LPS has been reported as a triggering factor for insulin resistance in
adipose tissue. By consumption of HFDs, LPS levels increase intestinal permeability,
resulting in elevated systemic LPS levels and low-grade inflammation (Blaut, 2015).

Regulating feeding behavior is extremely complicated and could be separated into

homeostatic control and nonhomeostatic control. Both controls could respond to
host energy status and react to feeding behavior. Communication between the brain
and viscera relies on the vagus nerve, which is the longest cranial nerve in the
body. The vagus nerve participates as a coordinator in the regulation of the homeo-
static and nonhomeostatic feeding systems by connecting gastrointestinal hun-
ger and satiety signals and bidirectionally regulating higher-order brain regions.
According to previous studies, the microorganisms that inhabit the host gut have
the capacity to modulate appetite via the intestinal satiety pathway and conse-
quently impact obesity and eating disorders. In this mechanism, bacterial com-
ponents and microbiota-derived metabolites play major roles in modulating host
satiety (Yu and Hsiao, 2021). In animal models, accumulating evidence suggests
that gut microbiota has the ability to influence host dietary preference (Alcock
et al., 2014). The possible mechanisms may be that the gut microbiome affects oral
and intestinal taste receptors, while endocannabinoid signaling is also discussed as
a possible avenue (Méndez‐Díaz et al., 2012). By using the intervention of prebiot-
ics, the composition of the gut microbiome has been changed, which affects host
food intake by regulating hedonic and motivational drives for a food reward. Thus,
prebiotics alleviates dysbiosis of the gut microbiome and increases the sensitivity
of sweet taste, which suggests a high correlation between gut microbiota composi-
tion and taste (Bernard et al., 2019).
The hypothalamus has an important function to integrate external stimulation and
sensory, hormonal, and nutrient signals into regulating feeding behavior. In particu-
lar, the hypothalamus has been divided functionally into two parts to control appetite
which are anorexigenic arcuate pro-opiomelanocortin (POMC) neurons and orexi-
genic agouti-related protein/neuropeptide Y (AgRP/NPY)–coexpressing neurons.
POMC neurons, located in the arcuate nucleus of the hypothalamus, are activated
by energy surplus and inhibited by energy deficits. When POMC is activated, the
cells decrease appetite and promote weight loss (Rau and Hentges, 2019). Conversely,
AgRP/NPY neurons act as an anabolic promotor, producing peptides to promote
food intake and enhance body weight. In particular, AgRP/NPY neurons express
leptin and insulin receptors and are inhibited by these two hormones. When leptin
and insulin levels are decreased, AgRP/NPY neurons can be activated (Morrison
et al., 2005; Morton and Schwartz, 2001). Furthermore, the hypothalamus provides
Diet, Obesity, and Gut Microbiome 221

connective function between many brain regions, ensuring coordinated responses to

metabolic state and demand (Wright et al., 2016). Several animal studies have proven
that variations in the gut microbiota may implicate hypothalamic gene expression,
neuropeptide and neurotransmitter levels, and neuronal activity. One of the animal
trials revealed a decrease in Npy and Agrp expression but increased the Pomc expres-
sion compared with GF mice. Moreover, conventionally raised mice have less brain-
derived neurotrophic factor expression which is an anti-obesity neuropeptide (Schéle
et al., 2013). In contrast, another study indicated that conventionally raised mice
have lower hypothalamic Pomc and suppressor of cytokine signaling 3 expression
than GF mice. The discrepancy between these results and influences on the hypo-
thalamus may be caused by the variability of the diet and other rearing conditions.
The potential mechanisms are discussed, and microbial metabolism and microbiota
metabolites are involved in the potential modulation. SCFAs are regarded as modu-
lators that affect host feeding behavior via hypothalamic neurons. Acetate has been
demonstrated to be an anorectic signal by directly inducing hypothalamic neuronal
activation, while propionate and butyrate are reported to influence peripheral circuits
that innervate the hypothalamus (Blaak et al., 2020; Byrne et al., 2015). The gut
microbiota also has modulating ability on the host brainstem, which bidirection-
ally processes both descending neural signals from the midbrain and forebrain and
ascending signals from the vagus nerve, hormones, microbes, and host metabolites
(Liu and Kanoski, 2018). The brainstem is divided into two nuclei, the nucleus of the
solitary tract and the dorsal raphe nucleus. Previous reports have proven that both
the nucleus of the solitary tract and dorsal raphe nucleus can be modulated by the
gut microbiota and lead to feeding behavior alterations (Campos et al., 2016; Minaya
et al., 2020; Vaughn et al., 2017).
It has also been reported that variations in gut microbiota composition caused by
different nutritional status and physical activity could affect appetite-regulating hor-
mones such as leptin and ghrelin. Leptin, a hormone generated from adipose tissue,
is highly correlated with body fat mass. Leptin is transferred by blood circulation and
then passes through the blood–brain barrier to the hypothalamus, the place that has
leptin receptors and stimulates appetite suppression. This chain reaction results in
reduced food intake, inhibited fat accumulation, and ultimately reduced body weight
(Zhou and Rui, 2013). In the GF mouse model, lower levels of leptin were shown com-
pared with conventionally raised mice (Fetissov et al., 2008). In addition, previous
research found a significant positive correlation between circulating leptin concen-
tration and the quantity of Bifidobacterium and Lactobacillus and a negative correla-
tion with the number of Clostridium, Bacteroides, and Prevotella and serum leptin
levels (Queipo-Ortuño et al., 2013). However, leptin receptors become less sensitive
in obese individuals, in which the hypothalamus loses the responsiveness to leptin,
usually accompanied by hyperleptinemia (Andreoli et al., 2019). In a mouse model,
a gut microbiota modulation by a prebiotic treatment improved leptin sensitivity and
other metabolic parameters in diet-induced obesity (Everard et al., 2011). Another
report mentioned that probiotic intervention also improved leptin sensitivity in an
HFD mouse model (Cheng and Liu, 2020). The possible mechanism by which the gut
microbiota is involved in is the reduction of leptin resistance-associated suppressor
of cytokine signaling 3 expression in the hypothalamus and brainstem (Rosenbaum
222 The Gut Microbiome: Bench to Table

et al., 2015). In addition, Heiss et al. indicated that gut microbiota enhances leptin
sensitivity by regulating GLP-1–dependent mechanism (Heiss et al., 2021).
Furthermore, ghrelin is a gastrointestinal peptide hormone secreted from the
stomach and stimulates appetite by affecting the hypothalamic arcuate nucleus
(Kojima and Kangawa, 2005). Serum ghrelin levels were proven to have a negative
correlation with the ratio of Bacteroidetes and Firmicutes, as well as the number of
Faecalibacterium and Prevotellaceae, and a positive correlation with the number of
total bacteria, Clostridium, and Ruminococcus. For Bacteroides, Bifidobacterium,
and Lactobacillus, both positive and negative correlations with ghrelin were observed
(Leeuwendaal et al., 2021; Queipo-Ortuño et al., 2013). Numerous studies have indi-
cated that microbial metabolites play a key role in ghrelin secretion. SCFAs are able
to suppress ghrelin secretion and reduce ghrelin receptor (the growth hormone secre-
tagogue receptor type 1a) stimulation; LPS has been proven to decrease circulating
ghrelin levels. On the contrary, amino acids have the capacity to promote ghrelin
release (Leeuwendaal et al., 2021).

The gut microbiome modulates the gut–brain function and occupies an important
modulator of the microbiota–gut–brain axis, which affects host energy homeostasis.
By using the GF animal model, scientists showed the first evidence of the absence
of microbiome implicated in brain regulation and altered animal behavior. Scientists
have gained more understanding of the neuron alterations by specific strains on
myelination, neurotransmitters, microglia, neurogenesis, dendritic growth, and the
blood–brain barrier. Numerous studies have found that the administration of probi-
otics and prebiotics can reduce negative impacts and deterioration of brain function
(Cryan et al., 2019). Moreover, recent research has indicated that the gut microbiome
highly participates in with satiety, digestive function, and further cognitive and psy-
chological effects. The gut conveys nutritional status signals with the central nervous
system and engages energy modulation by using enteroendocrine cells (EECs), the
vagus nerve, and the enteric nervous system (van Son et al., 2021). In addition, the
metabolites produced by microbes can be involved in regulating signal transduction.
Interplay between the gut and brain is crucial for host energy homeostasis. In
recent years, it has become clear that signals from the gut play a major role in
appetite regulation, energy balance, glucose homeostasis, and satiety. One of the
regulations might be the activation of neuroendocrine mechanisms. EECs, which
are located in the GI tract from the stomach to rectum, secrete gut hormones and
neurotransmitters such as somatostatin, serotonin, ghrelin, CCK, PYY, and GLP-1
that are stimulated by luminal nutrients and provide a wide range of human physi-
ological modulations. After secretion, these gut hormones are transported to the
target neuron or enteric nervous system and exert their effectiveness throughout
the bloodstream. CCK, GLP-1, PYY, and oxyntomodulin are known as modula-
tors of on postprandial satiety. During the fasting stage, the group of gut hormones
that were highly increased was featured in promoting food intake and inhibiting
gastric acid secretion, which included ghrelin, motilin, insulin-like peptide 5, and
somatostatin. Conversely, hormones which secret postprandial such as gastrin,
Diet, Obesity, and Gut Microbiome 223

histamine, GLP-1, and CCK are characterized by enhancing digest efficiency, pro-
moting satiety, and preparing for storage of nutrients (Gribble and Reimann, 2016;
Symonds et al., 2015; van Son et al., 2021).
Beyond gut hormones, neuronal orchestration has been revealed as a key element
in the gut–brain function. The vagus nerve is a mixed nerve that interfaces with the
parasympathetic control of the heart, lungs, and digestive tract. Appetite, gastroin-
testinal motility, and anti-inflammatory modulation are all associated with the vagus
nerve. In addition, gut hormones (ghrelin, PYY, CCK) can trigger the vagus nerve
directly. According to previous studies, obesity and an HFD reduce the sensitivity of
vagus nerve afferent fibers that respond to gut hormones. The gut microbiome influ-
ences the vagus nerve indirectly by driving EECs to stimulate neurotransmitters. In
addition, dysbiosis has proven to be correlated with vagus nerve innervation and sig-
naling by increasing LPS and inflammatory cytokines. (Sen et al., 2017) (Browning
et al., 2017; van Son et al., 2021). Moreover, microbial metabolites converted by
the gut microbiome, such as SCFAs, also play a crucial role in the gut–brain axis.
SCFAs are derivatives from undigested fiber that modulate satiety and may increase
energy expenditure. Additionally, a study revealed that SCFAs affect insulin signal-
ing which leads to inhibition of fat accumulation in adipose tissue and promotes lipid
and glucose metabolism in other tissues (Kimura et al., 2013). A portion of SCFAs
can interact with EECs and stimulate PYY and GLP-1 secretion (Larraufie et al.,
2018; Tolhurst et al., 2012; van Son et al., 2021).

DIETARY MODULATING GUT MICROBIOTA COMPOSITION

D

Diet is a critical factor affecting the gut environment, gut transit time, pH, as well
as changes in the composition of gut microbiota and diversity that influence host
metabolism. An unhealthy style of diet may lead to microbiome dysbiosis and induce
several sequelae, including obesity and type 2 diabetes. (Kim et al., 2019). Thus, a
healthy diet could improve the composition of gut microbiota and reduce the risks of
host metabolic disorders. Previous studies have revealed the differences in diet pat-
terns such as Western diet, Mediterranean diet, vegetarian diet, and gluten-free diet
have their own gut microbiome profiles that have different impacts on host metabo-
lism (Lazar et al., 2019).
The Western diet, developed from the industrial evolution, is comprised of satu-
rated fats, sugar, salt, protein, additional additives, and lower fiber and micronutrients.
The Western diet has been proven to have a positive correlation with inflammation
that leads to metabolic disorders and obesity. The major gut microbiota profile of the
Western diet is the reduction in total bacterial volume and beneficial species includ-
ing Lactobacillus spp., Bifidobacterium spp., and Eubacterium spp. Due to suffi-
cient dietary fiber, some of the specific bacteria lack the sources to generate SCFAs.
The observation of SCFAs in the lumen is significantly lower when consuming a
Western diet that may enhance risks of inflammatory (Kim et al., 2019; Lazar et al.,
2019; Statovci et al., 2017). Conversely, vegan and vegetarian diets contain abundant
dietary fiber which is opposite to the Western diet. Dietary fiber provides a good
224 The Gut Microbiome: Bench to Table

energy source to beneficial bacteria that promote a stable gut microbiota profile and
increase the number of lactic acid bacteria. A low quantity of Bacteroides spp. and
Bifidobacterium spp. was observed in the group that consumed vegan or vegetarian
diets. In addition, these diets usually consume rich polyphenols which is from coffee,
artichokes, olive, asparagus, and berries, which can facilitate the growth of protec-
tive bacteria. Bifidobacterium and Lactobacillus which are known intestinal barrier
protectors, and butyrate-producing bacteria, including Faecalibacterium prausnitzii
and Roseburia, Bacteroides vulgatus, and Akkermansia muciniphila, are reported
to have an increase in vegan and vegetarian diets. Additionally, a decline in LPS
producers such as Escherichia coli and Enterobacter cloacae has also been observed
in vegan and vegetarian diets that lead to alleviation of inflammation (Glick-Bauer
and Yeh, 2014).
The gut microbiota profile of a gluten-free diet has a lower abundance of
Lactobacillus spp., Bifidobacterium spp., Ruminococcus bromii, and Roseburia faecis,
whereas the Victivallaceae and Clostridiaceae, E. coli, and total Enterobacteriaceae
demonstrate the opposite trend due to decreasing of polysaccharide and fiber intake
(De Palma et al., 2009). The gluten-free diet, which is characterized by a reduc-
tion or exclusion of alimentary fiber from specific foods (i.e., grain), now has been
considered as a therapeutic method for celiac disease (Reddel et al., 2019). The
Mediterranean diet consists of abundant vegetables, olive oil, legumes, fish, and a
low amount of red meat and dairy products that contain monounsaturated and poly-
unsaturated fatty acids as well as antioxidants and fiber. The gut microbiota profile
in the Mediterranean diet has a high amount of Lactobacillus spp., Bifidobacterium
spp., and Prevotella spp., but a low amount of Clostridium spp. This profile of the
gut microbiota can prevent obesity and improve lipid and cholesterol levels (Garcia-
Mantrana et al., 2018). Additionally, numerous studies have demonstrated that dif-
ference in the ratio intake of macronutrients and micronutrients can change the
composition of gut microbiota and diversity which can influence host metabolism.

Carbohydrates
Many research studies have highlighted the altered ability of carbohydrates. In gen-
eral, carbohydrates are separated into two types, which are digestible and nondigest-
ible carbohydrates, and the gut microbiota composition has different responses to
different types of carbohydrates. Starch and sugars such as glucose, lactose, fructose,
and sucrose constitute digestible carbohydrates. They release glucose into the circula-
tion and induce insulin to further respond after digestion in the intestine. The level of
glucose is positively correlated with Bifidobacteria and Prevotella but negatively cor-
related with Bacteroides (Holmes et al., 2012; Lazar et al., 2019). In addition, lactose
is related to the reduction of Clostridia (Francavilla et al., 2012). Furthermore, unlike
digestible carbohydrates, nondigestible carbohydrates are the main materials that
offer commensal bacteria to produce SCFAs. Quantity and types of fiber intake have
a huge effect on gut microbiota alternations. Negative association with Bacteroides
and Actinobacteria and positive association with Proteobacteria and Firmicutes were
discerned in fiber intervention, and Firmicutes and Actinobacteria were the main
Diet, Obesity, and Gut Microbiome 225

responders particularly (Holmes et al., 2012). According to previous reports, indi-

viduals who received a diet with resistant starch as a supplement had an enhanced
abundance of Bifidobacterium adolescentis, Ruminoccocus bromii, Eubacterium
rectale, and Parabacteroides distasonis (Makki et al., 2018). Conversely, galactooli-
gosaccharides intervention can increase the volume of Bifidobacterium spp. (Davis
et al., 2011). Faecalibacterium prausnitzii, a high SCFA producer, was not observed
in the gut microflora profile of diabetic patients. These results illustrated that carbo-
hydrates are attributed to the composition of gut microbiota (Machiels et al., 2014).
Artificial sweeteners such as sucralose, saccharin, and aspartame are designed as
healthier food additives, are becoming a new trend, and are widely used to reduce
energy intake for natural sugar. However, Suez et al. proved that artificial sweeteners
have a relationship with microbiota dysbiosis and metabolic abnormalities. This gut
microbiota compositional and functional alteration leads to metabolic disease and
glucose intolerance in healthy human subjects. An increase of Bacteroides spp. and
reduction of Lactobacillus reuteri are observed in the diet with artificial sweetener
usage (Suez et al., 2014).

Fats are generally divided into several types based on their structure. Consumption
of saturated and trans fats has been proven to enhance the risks of cardiovascular
disease. Conversely, monounsaturated and polyunsaturated fats are known as health-
promoting fats that alleviate the risk of chronic diseases. In general, a positive corre-
lation with Bacteroides and Actinobacteria and a negative correlation with Firmicutes
and Proteobacteria were observed in the diet with rich fats. Fats from dairy prod-
ucts have been reported to promote delta Proteobacteria and induce inflammation
(Holmes et al., 2012). Dietary with rich fat modifies the gut microbiota structure with
abundant LPS-expressing bacteria that lead to enhanced circulating LPS levels in
humans (Amar et al., 2008). LPS is related to weight gain, adiposity accumulation,
and the induction of inflammatory markers in white adipose tissue that facilitate
host physiology to a preinflammatory status. It is interesting that the adverse effect
of saturated fat seems to be specific to mice fed a lard-rich diet, and meanwhile,
enrichment of Bacteroides, Turicibacter, and Bilophila spp. were observed to be
associated with the white adipose tissue inflammation, adiposity, and insulin intoler-
ance. In contrast, mice fed a diet with unsaturated fat exhibited increased amounts
of Bifidobacterium, Akkermansia, and Lactobacillus spp. with no metabolic disor-
ders observed (Caesar et al., 2015). Nevertheless, there are inconsistent results in the
human clinical trials and individuals who switch diet patterns from saturated fat to
unsaturated fat as caloric sources did not influence the gut microbiota structure but
reduced the total bacterial volume (Fava et al., 2013).

Protein
Contrarily, consumption protein presents a comprehensively different gut micro-
biota profile. Sources of protein are important characteristics to impact gut micro-
biota. Both the diet with vegetarian protein (whey and pea) and animal protein are
226 The Gut Microbiome: Bench to Table

contributed to overall microbiota diversity (Singh et al., 2017). Generally, plant

protein intake has positively associated with commensal Bifidobacterium and
Lactobacillus, whereas negatively associated with pathogenic including Bacteroides
and Clostridium perfringens. Bifidobacterium and Lactobacillus are contributed to
SCFA production that optimizes the host gut mucosa barrier and reduces inflamma-
tion. By contrast, the diet rich in animal protein enhances Prevotella and Clostridia
levels as well as reduce the number of Bifidobacterium. Also, animal protein can
enhance the abundance of bile-tolerant anaerobes such as Alistipes spp., Bilophila
spp., and Bacteroides spp. that are related to trimethylamine N‐oxide formation and
promote the risk of atherosclerosis and IBD (David et al., 2014; De Filippis et al.,
2016; Singh et al., 2017).

Trace minerals and vitamins are known as micronutrients, and their intake is
crucial for physiological maintenance. Various studies have demonstrated the
interaction between micronutrients and gut microbiota and led to further meta-
bolic regulation. Iron supplementation suppresses the levels of Bifidobacterium
but enhances the counts of Bacteroides and E. coli. Another study revealed an
increase in the fecal Enterobacteriaceae family and a reduction in Lactobacillus.
These results are totally consistent with the argument that bacteria and some
pathogens are efficient iron scavengers. Thus, iron supplementation may give rise
to microbiota dysbiosis and pathogen invasion (Yilmaz and Li, 2018). Manganese
supplementation in mice increased the colonization of the heart and lethality of
Staphylococcus aureus infection, likely because the bacterium utilizes manganese
to protect itself from reactive oxygen species and neutrophil killing (Juttukonda
et al., 2017). Bacteroides vulgatus is a prominent responder to vitamin A, and
it increases in number in the absence of vitamin A. Bacteroides dorei decreases
when vitamin A is deficient (Hibberd et al., 2017). Vitamin D plays a vital role in
immune homeostasis maintenance due to its interaction with the gut microbiota.
It has been reported that vitamin D has a negative correlation with gram-negative
genera, such as Haemophilus and Veillonella. This variation may be promoted by
low intake or levels of vitamin D (Luthold et al., 2017).

APPLICATIONS
Cumulative evidence elucidates that gut microbiota composition and diversity influ-
ence host metabolism and has close relationships with obesity and metabolic disor-
ders. Currently, scientists gain more knowledge on the mechanism between resident
bacteria and host metabolism, which leads to the development of different ways to
manipulate gut microbiota, such as fecal microbiota transplantation, probiotics, and
prebiotic intervention. In addition, lifestyle and dietary patterns are well understood
to alter and optimize the gut microbiota profile, especially by enriching commensal
bacteria. In this section, we will review studies with different applications to gain
more comprehension on enhancing human wellness by fostering a healthy gut micro-
biota and environment.
Diet, Obesity, and Gut Microbiome 227

Fecal microbiota transplantation (FMT) is a novel method to alleviate dysbiosis

and complications. Patients receive healthy donor feces and transfer the donor’s
gut microbiota into the intestinal tract to restore the dysbiosis. In clinical, FMT has
become a robust method to treat recurrent or refractory Clostridium difficile infec-
tion (Smits et al., 2013). Additionally, various clinical experiments have proven
that FMT can relieve ulcerative colitis and reduce the relative pathogenic gastro-
intestinal symptoms (Gupta et al., 2016). Recently, persuasive evidence has shown
that FMT has become a treatment for obesity, metabolic syndrome, and diabetes
mellitus. The possible mechanisms of FMT are the alternation of gut microbiota,
enhancement of gut barrier integrity and immunomodulation, and inhibition of
pathogen growth. Furthermore, the level of SCFA-producing bacteria is increased
in the patients with metabolic syndrome after receiving microbiota from lean
donors and significantly improves insulin sensitivity and fasting glycemia (Vrieze
et al., 2012). Supporting this notion, Ng et al. revealed that FMT can increase
butyrate-producing bacteria in the group of obese patients with type 2 diabetes
mellitus. In particular, the abundances of Bifidobacterium and Lactobacillus were
increased after FMT with lifestyle intervention. The improvement of total and low-
density lipoprotein cholesterol and liver stiffness is observed in recipients with
lifestyle intervention (Ng et al., 2021). Thus, manipulating the human gut microbi-
ome may provide an effective treatment for obesity.

Probiotics and Prebiotics interVention

Probiotics are commensal bacteria that have a beneficial influence on host health.
Bifidobacterium and lactic acid bacteria such as Lactobacillus are the most well-
known and widely used probiotics that exist in functional food, fermented food, and
dietary supplements. Moreover, heat-killed probiotics have been proven to have the
advantage of physiological modulation (Plaza-Diaz et al., 2019). Probiotics have been
reported to interact with the host through four main mechanisms, including promot-
ing intestinal barrier function, competition with pathogens, immunomodulation, and
physiological influences (Sánchez et al., 2017). Previous studies prove the mitiga-
tion of severe digestive disorders, allergic disorders, Clostridium difficile–associ-
ated diarrhea, and inflammatory bowel disorders after administration of probiotics
(Plaza-Diaz et al., 2019).
Additionally, probiotics have also declared a resolution of metabolic disorders
and obesity due to their modulation capability. Supporting this notion, various
studies have enumerated evidence that probiotics have the antimetabolic disor-
ders and anti-obesity ability. In animal models, Lactobacillus (e.g., L. casei strain
Shirota (LAB13), L. gasseri, L. rhamnosus, L. plantarum) and Bifidobacterium
(e.g., B. infantis, B. longum, and B. breve B3) have been revealed to have anti-
obesity ability and reduce fat accumulation in well-established animal models of
obesity (Abenavoli et al., 2019). Clinically, Lactobacillus gasseri SBT2055 and
Lactobacillus plantarum have been proven to reduce the BMI in obese adults,
and L. gasseri SBT2055 decreases visceral fat area, while L. plantarum can
228 The Gut Microbiome: Bench to Table

especially alleviate hypertension (Kadooka et al., 2010; Sharafedtinov et al., 2013).

Bifidobacterium animalis subsp. lactis CECT 8145 led to a decrease in BMI, body
weight, as well as fat mass and waist circumference (Pedret et al., 2019). The com-
bination of probiotics with multiple strains is widely applied in clinical trials. A
commercial probiotic mixture, VSL#3®, combined with multiple bacterial strains,
including Lactobacillus spp., Bifidobacterium spp., and Streptococcus, provides
evidence of anti-obesity ability and inhibits adipose accumulation in young adults
(Cheng et al., 2020; Osterberg et al., 2015). Obese individuals who supplemented
Lactobacillus acidophilus La5 and Bifidobacterium animalis subsp lactis Bb12
can improve glycemic control (Ivey et al., 2014). Selected probiotics have been
demonstrated to improve fasting blood glucose levels, insulin sensitivity, and car-
bohydrate metabolism and reduce the metabolic stress in type 2 diabetes mellitus.
In addition, recent research shows that particular selected probiotics and symbiot-
ics improve the liver biochemical and metabolic parameters in nonalcoholic fatty
liver disease patients (Sáez-Lara et al., 2016).
Prebiotics are also critical for alleviating dysbiosis and improving obesity. In
2008, Food and Agriculture Organization defined prebiotics as a “nonviable food
component that confers a health benefit on the host associated with modulation of the
microbiota” (Pineiro et al., 2008). Prebiotics are common in fruits and vegetables, and
their presence has numerous health benefits. The major characteristics of prebiotics
is non-digested or partially digested and cannot be absorbed in the upper segments
of the alimentary tract, while prebiotics can be well fermented by probiotics which
are usually located in the colon. Carbohydrates and their derivates are the most com-
mon molecular structures of prebiotics and are normally found in animal and human
diets including disaccharides, oligosaccharides, and polysaccharides (Markowiak
and Śliżewska, 2017). Prebiotics may mediate microbiota composition and micro-
bial metabolic products such as SCFAs, suppression of pathogenic bacteria, trace
elements absorption, and the immune system. Lactobacilli and Bifidobacteria are
the populations that have the most significant blooms induced by prebiotics (Bindels
et al., 2015). In addition, accumulating evidence suggests the beneficial effects of
prebiotics on host metabolism. In a human study, overweight and obese adults con-
sumed oligofructose as supplementation and found a decrease in body weight, body
fat, and plasma glucose. In particular, oligofructose supplementation also resulted in
a lower energy intake due to the regulation of satiety hormones, including ghrelin and
PYY (Parnell and Reimer, 2009). Supplementation with prebiotics increased plasma
gut peptide production (GLP-1 and PYY), which contributes to altered appetite sen-
sations and glucose responses following meals (Cani et al., 2009). Another study
also found a similar result in overweight and obese children. A reduction in energy
intake and improvement in appetite control was observed with prebiotics interven-
tion (Hume et al., 2017). Moreover, prebiotics are helpful in the reduction of blood
lipids, including total cholesterol, low-density lipoprotein-cholesterol, and triacylg-
lycerol levels (Delzenne and Williams, 2002). Thus, scientists suppose that prebiotics
play a pivotal role in satiety control, glucose, and lipid catabolism (Markowiak and
Śliżewska, 2017).
In addition to administrating probiotics and prebiotics supplementation, consum-
ing fermented food is also a good way to intake sufficient probiotics. Fermented
Diet, Obesity, and Gut Microbiome 229

foods contain stable microorganisms, usually including diverse lactic acid bacteria
and their metabolites. The well-known fermented foods are yogurt, cheese, kefir,
cocoa, pickles, kimchi, sauerkraut, and red wine. The variety of bacteria that fer-
mented food contains may have health-promoting properties and modulated func-
tions on the gut microbiota. Probiotics in fermented food can interact with the host gut
microbiota and mediate the composition into a “healthy gut microbiota.” It has been
reported that dysbiosis can be prevented after consuming fermented food periodi-
cally (Stiemsma et al., 2020). Yogurt, fermented by cultured probiotics, ameliorates
human metabolic disorders, including insulin intolerance and hepatic fat fraction.
Additionally, a reduction in serum LPS levels and modulation of gut microbiota com-
position were observed in obese adults with metabolic syndrome and nonfatty liver
disease (Chen et al., 2019). The investigations of kefir were discrepancies about the
metabolism modulation. However, some studies have mentioned the improvement
of lipid metabolic parameters, fasting blood glucose, and HbA1c levels; in contrast,
body weight was not significantly different (Bourrie et al., 2020). Han et al. found
an increase in Bifidobacterium after kimchi consumption for 8 weeks, and the gene
correlated with metabolism, immunity, and digestion was upregulated in obese indi-
viduals (Han et al., 2015). For red wine, previous studies have revealed that the intake
of red wine increases Bifidobacterium, Prevotella, Bacteroides, Eggerthellalenta,
Enterococcus, Bacteroidesuniformis, and Blautiacoccoides/Eubacterium rectale
abundance in the gut microbiota. Meanwhile, a reduction in BMI and body weight
and amelioration of blood pressure, total cholesterol, high-density lipoprotein -cho-
lesterol levels are observed due to the rich polyphenols (Moreno-Indias et al., 2016;
Queipo-Ortuño et al., 2012).
Individuals with high-protein and low-carbohydrate diets have fewer lev-
els of Roseburia and Eubacterium rectale and lower levels of butyrate in their
feces, which may have detrimental effects (Russell et al., 2011). In addition, the
abundance of Prevotella and Bacteroides is increased after intake of red meat.
Furthermore, red meat contains L-carnitine, which induces the production of the
proatherogenic microbial metabolite trimethylamine N‐oxide production and may
induce the risk of metabolic syndrome (Lazar et al., 2019). HFDs have been posi-
tively correlated with metabolic syndrome and type 2 diabetes and have also been
known as obesogenic diets. In addition, saturated fats and trans fats are espe-
cially linked with cardiovascular disease. Consumption of monosaturated and
polyunsaturated fats can decrease the risk of chronic disease. For gut microbiota
modification, HFDs can enrich the numbers of Bacteroides and total anaerobic
microorganisms. In contrast, blooms of Bifidobacterium are observed after intake
of a low-fat diet and improve fasting glucose and total cholesterol (Fava et al.,
2013). As mentioned above, fruits and vegetables are full of dietary fiber, flavones,
and polyphenols. They are recommended for intake and bring multiple benefits
to optimize gut microbiota and GI tract health. A recent study used a multiomics
approach to demonstrate the intestinal microbiome response in vegetable-rich
diets. An increase in catalytic activities for carbohydrates and food proteins, an
increase of cell motility to access nutrients, and the release or synthesis of bio-
active metabolites and proteins that are beneficial to human health have been
observed (De Angelis et al., 2020).
230 The Gut Microbiome: Bench to Table

CONCLUSION
In this chapter, we elucidated the importance and characteristics of gut microbiota
and the interaction between bacteria and the host. In particular, we mentioned how
gut microbiota signals and contributes to host metabolism by microbial metabolites,
altering richness, and elevating or suppressing specific bacteria. The gut microbi-
ota plays a critical role in the host metabolism by modulating the gut environment,
immune system, metabolism, and appetite. The knowledge drives the development of
therapies that enhance human well-being by improving the gut microbiome composi-
tion and diversity. Thus, therapeutic applications of microbiota, including diet, probi-
otics, and prebiotics treatment, have become a novel approach to tackle the problem
of obesity and metabolic disorders.

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 9 Challenge, Future
Research, and Influence
in Food Science and
Product Development
Marta Vernero
University of Pavia

Claudia Caglioti and Gianluca Ianiro

Catholic University of Rome

CONTENTS
Food and Gut Microbiome ..................................................................................... 239
Microbiota Investigation Tools: Present and Future ..............................................240
Culturomics: A New Approach to Investigate Human Gut Microbiota ................. 242
Role of Functional Food in Microbiota Modulation .............................................. 243
The Journey of Microbiome through Human Evolution ........................................ 245
References ..............................................................................................................248

FOOD AND GUT MICROBIOME

Being more than 1014, bacteria are the most represented inhabitants of human micro-
biota, together with viruses and fungi. Human gut host approximately 1,000 species
of bacteria, most of which are anaerobes, mainly belonging to Bacteroidetes and
Firmicutes phyla, that all together represent the 90% of gut bacterial microbiome.
The remaining 10% is represented by Actinobacteria, Fusobacteria, Proteobacteria,
and Verrucomicrobia as well as by some other minor phyla. The individual composi-
tion of gut microbiota is widely different in each human depending on a variety of
genetic and environmental factors, including age, lifestyle, food intake, drug admin-
istration, and illnesses of the patient. As drugs are concerned, of course the most
relevant influence on gut microbiota composition is due to antibiotics administration
(Ianiro, Tilg, and Gasbarrini 2016; Jernberg et al. 2010; Slimings and Riley 2014;
Jakobsson et al. 2010; Zhang et al. 2013; Fouhy et al. 2012).
Another major factor influencing gut microbiome is diet; in fact, both short and
mostly long-term diet changes can influence permanently influence gut microorgan-
isms (Bibbò et al. 2016). For instance, an animal-based diet (high in meat and animal

DOI: 10.1201/b22970-12 239

240 The Gut Microbiome: Bench to Table

fat), especially if associated with high sugar and low fiber intake, increases bile-toler-
ant microorganisms including Bacteroides, Bilophila, Alistipes, and Ruminococcus,
decreasing those polysaccharides digesting species such as Firmicutes (Mukhopadhya
et al. 2012; David et al. 2014). Children living in Burkina Faso have a prevalence
of Bacteroidetes in their microbiome, while Italian children, with opposite dietary
habit, have a majority of Enterobacteriaceae.
This finding suggests that carbohydrate and fiber intake is the key to richness in gut
microbiota composition (Yatsunenko et al. 2012). Fibers divide into complex carbohy-
drates and oligosaccharides with different impacts on the gut. Complex carbohydrates
usually contain a huge part of indigestible material that is eliminated through stools,
called starch. These indigestible fibers can be divided into four subgroups, depending
on the resistance features: type 1 is characterized by plant cell wall polymers, type
2 is characterized by a granular structure, type 3 is derived from retro gradiation by
means of heating and coiling, and type 4 is represented by chemical cross-linking
(Flint et al. n.d.). Particularly, resistant starch type 2 increases Ruminococcus spp. and
Eubacterium rectale, whereas type 3 promotes Roseburia spp. and Ruminococcus
bromii (Leitch et al. 2007). Type 4 starch increases Parabacteroides distasonis but
reduces Euacterium rectale and Ruminococcus bromii. This last one also leads to a
reduction of Firmicutes while increasing Bacteroides and Actinobacteria (Martínez
et al. 2010). In addition, a diet high in soluble fibers is usually associated with an
increase in butyrate producing bacteria such as Clostridium leptum and Eubacterium
rectale and of other beneficial bacteria such as Bifidobacterium longum. Particularly,
in vegetarian individuals, the most represented bacteria is Prevotella spp. (Dewulf
et al. 2013). On the contrary, a low-fiber diet significantly decreases all of the afore-
mentioned bacteria (Bibbò et al. 2016). Oligosaccharides, including fructooligosac-
charides, galactooligosaccharides, and arabinoxylan-oligosaccharides, can influence
microbiota composition. For instance, inulin and fructooligosaccharides increase
Bifidobacterium spp. and Lactobacillus spp. and butyrate producing bacteria such as
Faecalibacterium prausnitzii (Ramirez-Farias et al. 2009). On the other hand, galac-
tooligosaccharides usually increases Bifidobacteria spp. (adolescentis and catenula-
tum) and Faecalibacterium prausnitzii, although its effect can be different in each
subject (Ramirez-Farias et al. 2009; Davis et al. 2011).
Additionally, the role of fatty acids in gut microbiota modulation has been exten-
sively studied: A high intake in monounsaturated fatty acids decreases Bifidobacteria
spp. and slightly improves Bacteroides spp. (Simões et al. 2013). In addition to that a
high-fat diet can lead to dysbiosis through reduction of Roseburia species (Neyrinck
et al. 2011).

MICROBIOTA INVESTIGATION TOOLS: PRESENT AND FUTURE

Analyzing gut microbiota ecosystem had become a major goal for scientists and
researchers due to its pivotal role in human health.
Culture-dependent methods, such as plating and Gram-staining techniques, rep-
resent the first attempt to study the complexity of human gut microbial community.
Unfortunately, this approach limited the range of detectable organisms to the aerobic
ones. This bacterial genus accounts for approximately only 0.1% of the microbes
Challenge, Future Research, and Influence 241

inhabiting the average human intestine, whereas the majority of microbial species
could never have been cultured, studied, or quantified in a laboratory. Great progress
has been made by the advent of molecular biology, a DNA-based culture-independent
method. By doing this, researchers received a key tool to investigate several aspects
of microbial communities (e.g., taxonomic composition and functional metagenom-
ics) and (theoretically) to deduce potential biological tasks carried out by a commu-
nity as a whole.
Starting from the earliest DNA-based methods by using fluorescent in-situ
hybridization (FISH), passing through polymerase chain reaction (PCR), we
arrived to the revolutionary technology of high-throughput sequencing, known
as next-generation sequencing (NGS). NGS exhibits substantial advances over
the Sanger method in terms of ease and cost of sequencing, as complete bacterial
genome sequences could be assayed and dissected in hours or days rather than
months or years (Hiergeist et al. 2015).
With the development of these methods, two different approaches became avail-
able: the 16S gene analysis and metagenomics. 16S DNA sequencing needs the DNA
extraction from the sample of choice and the use of a specific primer to allow the PCR
to begin. It usually looks for a fundamental part of the set of prokaryotic functional
genes; therefore, only bacteria can be detected. These hypervariable gene regions
could define the genetic fingerprint of a single microorganism. In fact, such regions
have been numbered from V1 to V9 and every one of them encodes specific primers.
To notice, some authors pointed out that there is no hypervariable region that alone
allows the diagnosis of all the bacterial genera examined, so it would be advisable to
investigate different regions (Martellacci et al. 2019).
On the other hand, metagenomics is the study of genetic material recovered
directly from environmental samples, such as fecal samples, in an untargeted (shot-
gun) way without the need to first cultivate these organisms or specific primers.
It relies on a labor-intensive cloning process and on NGS technologies such as
the 454/Roche, Illumina/Solexa, and Ion Torrent/Ion Proton platforms (Garza and
Dutilh 2015).
In particular, Shotgun metagenomics, which “fragments” and then “reads” the
entire DNA in the sample, is able to detect viruses, bacteria, and parasites.
Comparing these two methods is quite clear that metagenomics approach presents
several advantages: (i) it needs less amount of PCR amplification than 16S DNA
sequence analysis; (ii) it is able to quantify individual bacterial species in samples;
and (iii) only with metagenomic approach is possible to identify genetic segments
potentially associated with health or disease. However, it has to be considered that
16S DNA sequencing has lower costs and less computational requirements than
metagenomics, and for these reasons, it is often preferred by researchers (Martellacci
et al. 2019).
Overall, metagenomics revolutionized the understanding of the relations among
the human microbiome, health, and diseases and supplanted the long and labori-
ous culture-dependent methods of classic microbiology (Lagier et al. 2016). With
metagenomic approaches, we can discover the identity, evolution, gene composition,
distribution, and ecological patterns of uncultured microbes and viruses (Garza and
Dutilh 2015). However, it generates a countless number of sequences that have not
242 The Gut Microbiome: Bench to Table

been assigned to a known microorganism, raising the awareness that a complemen-

tarity use of both culture-dependent and culture-independent methods was needed.
Therefore, in 2012, a new strategy named “culturomics” was proposed for the study
of the human gut microbiota.

CULTUROMICS: A NEW APPROACH TO

INVESTIGATE HUMAN GUT MICROBIOTA
Culturomics is a high-throughput culture method that utilizes culture conditions,
matrix-assisted laser desorption ionization–time of flight mass spectrometry
(MALDI–TOF MS) and 16S rRNA sequencing for the identification of a wider
number of bacterial species. It allows the culture of organisms corresponding to
sequences previously not assigned (Lagier et al. 2012).
The first step of culturomics method is to divide the sample into different culture
conditions designed to suppress the culture of majority populations and to promote
the growth of fastidious microorganisms at lower concentrations. This technique is
the co-culture, which allows to culture bacteria from different environments at the
same time. In fact, according to environmental microbiology, some bacterial species
naturally grow in the same environment, as they provide each other growth promot-
ing factors. An important feature of culturomics is the rapid (less than 1 hour) iden-
tification by MALDI–TOF mass spectrometry which relies on the comparison of the
protein mass spectra of the isolate with an upgradable database. If identification fails,
the isolate is subjected to 16S rRNA sequencing. If there is less than 98.65% similarity
to the closest official strain, the isolate could be a new species. Then, the discovery of
new taxa is confirmed by genome sequencing and taxonogenomics is used to formally
describe the bacterium. Finally, all identification results are compared with a database
that contains bacterial species recovered from humans (Lagier et al. 2018).
Through culturomics, we can identify much more bacterial species, isolated at
least once in the human gut. Moreover, culturomics allows to cultivate even that spe-
cies “noncultivable” with standard approach. In the first reported culturomics study
by Lagier and his group, 212 different culture conditions generated more than 30,000
colonies. Among all the bacterial species isolated, 31 were new or belonged to rare
phyla (Lagier et al. 2012). To date, thanks to culturomics, 73 additional species from
the human gut, 13 new species from the urinary tract, 15 species from the vaginal
tract, nine species from the respiratory tract, two species from human skin, one from
human colostrum, and one from the foot of an individual with osteomyelitis have
been isolated (Lagier et al. 2018).
Moreover, the culturomic method, despite the long analytical times, has a greater
analytical depth allowing the detection of bacterial populations up to concentrations
of 102 cells per gram, while for metagenomics bacteria population, under 105 cells
per gram are undetectable (Dubourg et al. 2014). However, an important limitation
of culturomics is that the result can only be qualitative, as this approach is based on
the induction to the growth of the greatest number of bacterial species present on the
sample. No quantitative information can be drawn. Nonetheless, this negative aspect
can be avoided by combining culturomics with the real-time PCR that, unfortunately,
causes an increase in costs (Martellacci et al. 2019).
Challenge, Future Research, and Influence 243

In conclusion, to investigate microbial communities efficiently and completely (i.e.,

to detect all members including the least abundant), deep sampling and high-throughput
DNA sequencing are the approaches of choice at present, probably in combination with
other genome-wide analyses such as transcriptomics, proteomics, or metabolomics
(Hiergeist et al. 2015). In fact, metaproteomics enables functional activity information
to be gained from the microbiome samples, while metabolomics provides insight into
the overall metabolic states affecting/representing the host–microbiome interactions.
Combining these functional-omic platforms together with microbiome composition
profiling allows for a holistic overview on the functional and metabolic state of the
microbiome and its influence on human health (Peters et al. 2019).

ROLE OF FUNCTIONAL FOOD IN MICROBIOTA MODULATION

The gut microbiota is a complex ecosystem continuously shaped by many factors,
such as dietary habits, seasonality, lifestyle, stress, antibiotics use, or diseases.
Changes in habitual or available diet have a significant impact on the structure-
function activity of the gut microbiota, potentially impacting intestinal barrier func-
tions and the immune system. In fact, there is the growing perception that diet may
play a major role than host genetics in selective pressure on gut microbiota.
As diet has a major influence on microbiome dynamics, the study of how a specific
nutrient affects the gut microbiota composition represents a powerful tool, known as
nutritional ecology, even though it still needs to be fully understood (Murtaza, Ó
Cuív, and Morrison 2017; Rinninella et al. 2019). Both current dietary habits, result-
ing from a specific mixture of micro- and macronutrients, and food appearing to
exert positive effect on gut microbiome (functional foods) have been investigated.
In a review of Rinninella and colleagues, the effects of different types of mod-
ern diet on commensal bacterial species, mucus layer, and lamina propria hosting
immune cells have been discussed.
What emerges is that Western diet rich in total fat, animal proteins, refined sugars,
and additives may reduce gut microbial diversity in terms of phyla and genus leading
to dysbiosis, alteration of barrier function and permeability, abnormal activation of
immune cells, and a higher incidence of chronic diseases.
On the other hand, elimination diets such as low-FODMAP (fermentable oligo-,
di-, monosaccharides, and polyols) diet and gluten-free diet (GFD) seem to improve
the symptoms of some diseases like irritable bowel syndrome (IBS) and celiac dis-
ease (CD) in selected patients, but the long-term effects on gut microbiota require
elucidations. FODMAPs are a large class of small nondigestible carbohydrates
which are poorly absorbed in the small bowel. They can be found in a wide range
of foods such as fruits, vegetables, legumes, cereals, honey, sweeteners, milk, and
dairy products. Low-FODMAP diet strongly limits consumption of FODMAP
foods that are potential triggers of abdominal symptoms in the IBS patient (Bellini
et al. 2020). Unfortunately, such long-term limitation decreases the amount of
potential prebiotics (fructooligosaccharides and galactooligosaccharides) leading
to a reduction in beneficial bacteria, for example, Bifidobacteria, and fermentative
effects that may require an integration with probiotics to counteract gut microbiota
imbalances (Rinninella et al. 2019). Gluten-free diet is characterized by the complete
244 The Gut Microbiome: Bench to Table

elimination of gluten. It was recognized as a potential cure, restoring the normal

intestinal mucosa in coeliac patients. However, it has been demonstrated that long-
term GFD leads to a decrease of healthy bacteria such as Bifidobacterium and
Lactobacillus, to a diminution of SCFAs production and their beneficial meta-
bolic and host immunity effects, and to an increase of detrimental species such as
Enterococcus, Staphylococcus, Salmonella, Shigella, and Klebsiella.
To date, the Mediterranean diet (MD) seems to remain the evergreen solution to
optimally modulating microbiota diversity and stability as well as the regular perme-
ability and activity of immune functions of the human host (Rinninella et al. 2019).
The MD is based on the consumption of monounsaturated and polyunsaturated fatty
acids (MUFAs and PUFAs), polyphenols, and other antioxidants, a high intake of
prebiotic fiber and low-glycemic carbohydrates, and greater consumption of plant
proteins than animal proteins. It seems that this combination of nutrients linked to
many health benefits and to an increase of healthy bacteria and SCFAs, thus improv-
ing the diversity and richness of gut microbiota.
“Functional food” is a new term that indicates a food that is known to beneficially
affect one or more target functions in the body in addition to adequate nutritional
effects in a way to either the state of well-being and health or to a reduction in disease
incidence (Jones 2010).
To this extent, fermented foods (yogurt, kombucha, sauerkraut, tempeh, natto,
miso, kimchi, and sourdough bread) need a special mention due to the putative
impact they have.
They are defined as foods or beverages produced through controlled microbial
growth, contain potentially probiotic microorganisms, fermentation-derived metabo-
lites, prebiotics, and vitamins that may exert health benefit. For instance, lactic acid
bacteria (both dairy and nondairy fermented foods) generate bioactive peptides and
polyamines potentially influencing cardiovascular, immune, and metabolic health.
Moreover, fermentation can reduce toxins and antinutrients such as the reduction of
phytic acid concentrations and content of fermentable carbohydrates (e.g., ferment-
able oligosaccharides, disaccharides, monosaccharides and polyols, FODMAPs),
which may increase the tolerance in patients with functional disorders such as irri-
table bowel syndrome.
The most widely investigated fermented food is kefir, with evidence from at
least one randomized controlled trial, suggesting beneficial effects in both lac-
tose malabsorption and Helicobacter pylori eradication (Bekar, Yilmaz, and
Gulten 2011). Kefir is a fermented milk drink with a creamy texture, sour taste,
and subtle effervescence. It is produced by adding a starter culture named “kefir
grains” to milk. These grains contain symbiotic lactose-fermenting yeasts (e.g.,
Kluyveromyces marxianus), nonlactose fermenting yeasts (e.g., Saccharomyces
cerevisiae, Saccharomyces unisporus), and lactic and acetic acid producing bac-
teria, housed within a polysaccharide and protein matrix called kefiran. Kefir’s
antimicrobial activity has been investigated through several in vitro studies. Its
antimicrobial activity is attributed to competition with pathogens for available
nutrients, as well as the production of organic acids, bacteriocins, carbon dioxide,
hydrogen peroxide, ethanol, and diacetyl (Leite et al. 2013). These studies have
also shown that kefir exhibits antimicrobial activity against Candida albicans,
Challenge, Future Research, and Influence 245

Salmonella typhi, Salmonella enterica, Shigella sonnei, Escherichia coli, Bacillus

subtilis, Enterococcus faecalis, and Staphylococcus aureus (Chifiriuc, Cioaca, and
Lazar 2011). Fermentation-derived bioactive peptides produced from casein have
been shown to stimulate the immune system in animal models. It has also been
suggested a potential anti-oxidative, anti-hypertensive, anti-carcinogenic, cho-
lesterol, and glucose-lowering effects of kefir (Kwon et al. 2008; Liu, Chen, and
Lin 2005; Khoury et al. 2014; Liu et al. 2006). Kefir and its constituent strains
seem to have a considerable impact on gut microbiota composition, increasing
Lactobacillus, Lactococcus, and Bifidobacterium and reducing in Proteobacteria
and Enterobacteriaceae concentrations, being demonstrated in numerous animal
studies (Dimidi et al. 2019).
Overall, the use of combinations of pre- and probiotics has the potential to induce
more substantial effects on the gut microbiota and host health than isolated intake of
one of them, as the prebiotic component stimulates probiotic bacteria survival and
growth in the gastrointestinal tract. However, scientists are moving toward examining
the capacity of dietary patterns and personalized nutrition to modulate the intestinal
microbiota in pathological conditions. Their main struggle is to find a balance between
the effects of specific foods, nutrients, bioactive compounds, and macronutrients since
first ones are isolated nutrients rarely consumed, and Western diet, the most common
dietary pattern, is high in fat and simple carbohydrates while low in important fibers.
A recent study in patients with type 2 diabetes demonstrates that a dietary interven-
tion with functional foods significantly modifies gut microbiota by increasing alpha
diversity and modifying the abundance of specific bacteria, independently of antidia-
betic drugs. In this study, patients had an increase in two anti-inflammatory bacterial
species (Faecalibacterium prausnitzii and Akkermansia muciniphila) together with a
decrease in P. copri. Long-term adherence to a high-fiber polyphenol-enriched and
vegetable-protein-based diet provides benefits for the composition of gut microbiota
and may offer potential therapies for improvement of glycemic control, dyslipidemia,
and inflammation. After 2.5 months of dietary intervention, patients with type 2 dia-
betes also exhibit significant reductions in areas under the curve for glucose, total and
LDL cholesterol, FFAs, HbA1c, triglycerides, and C-reactive protein.
In conclusion, the observation that diet can modulate host–microbe interactions is
an indicator that future therapeutic approaches can be developed to modify the gut
microbiota and reduce the dysbiosis induced by diseases related with the nutrition
(Sánchez-Tapia, Tovar, and Torres 2019).

THE JOURNEY OF MICROBIOME THROUGH HUMAN EVOLUTION

Humans can be considered as ecosystems containing millions of microorganisms.
Our current gut microbiome derives from years of microorganisms selection due to
dietary, environmental, lifestyle cultural, and historical changes. How the composi-
tion of the microbiome has changed since humans diverged from other species, since
human populations diverged from one another, is a key point in the comprehension
of gut microbiome, but it is still difficult to investigate.
As previously discussed, gut microbiome plays a pivotal role in human body
homeostasis, influencing key functions such as digestion, mood and behavior,
246 The Gut Microbiome: Bench to Table

development, immunity, and the susceptibility of a vast range of acute and chronic
diseases (Marchesi and Ravel 2015). With the initiation of the Human Microbiome
Project in 2007, as a direct consequence of the improvement of DNA sequencing, it
has become increasingly clear that the study of human evolution could exclude the
study of human gut microbiome (Peterson et al. 2009).
Gut microbiome and its co-resident microbes contain a huge quantity of different
additional genes, functioning as a target for natural selection (Schnorr et al. 2016).
So, the study of gut microorganisms in human populations, non-human primates,
and past human populations is a key point in understanding human evolution, even
though this is a novel field still lacking strong evidences. In fact, up to date, the only
archaeological materials containing stable components allowing ancient microbiome
sequencing are coprolites (paleofeces) from mummified human rests and dental cal-
culus (Zeng et al. 2015). The majority of these materials come from samples origi-
nated in Europe and Americas.
Interestingly, environmental and lifestyle changes that have occurred through-
out human history and prehistory had a huge impact on microbiome composition
and physiology.
In fact, unlike the nuclear and mitochondrial genomes of the host, the micro-
biome continuously responds to external and internal changes and signaling
(Dewulf et al. 2013).
Particularly, gut microbiome can change through both vertical (parental trans-
mission) and horizontal transmission (more typical of microorganisms, information
derive from the environment) (Davenport et al. 2017).
Moeller et al. investigated the changes in the passage from apes microbiome
to human microbiome and found out that among apes, Prevotella was negatively
correlated with Bacterioides, but Bacterioides were positively correlated with
Ruminococcus and Parabacteroides. Most of these changes in the composition of
the human microbiome have functional implications for host nutrition. For instance,
the abundance of Bacteroides, which has been positively associated with diets rich
in animal fat and protein, has increased in humans. On the other hand, the archaeon
Methanobrevibacter, which promotes the degradation of complex plant polysac-
charides, has reduced within humans, as well as Fibrobacter (Moeller et al. 2014;
Kobayashi, Shinkai, and Koike 2008).
Interestingly, among primates, Old World monkeys and apes have the most similar
microbiome to actual human microbiome, rather than New World primates and lemurs.
In fact, captive primates consume less diverse, lower-fiber diets compared to their wild
counterparts mirroring the gradual transition to low-fiber diets of human evolution and
the contrast of modern Western and non-Western diets (Davenport et al. 2017).
Basing on biogeographic analyses, it is clear that there are some specific micro-
bial taxa depending host genotypes and environment. This is evident in a higher
presence of Prevotella, Catenibacterium, Succinivibrio, and Treponema among both
contemporary and ancient populations following the same lifestyle such as hunting
and gathering or agriculture (Gomez et al. 2016; Yatsunenko et al. 2012; Raul Y. Tito
et al. 2012; Raúl Y. Tito et al. 2008).
Recently, some samples recovered from Mexico provided DNA from numer-
ous human gut microbial symbionts (Tito et al. 2008, 2012), with taxonomic profile
Challenge, Future Research, and Influence 247

similar to those observed in contemporary rural and traditional human communities

(Yatsunenko et al. 2012; Obregon-Tito et al. 2015).
A further example of how environmental and dietary factors influence human
microbiome is the huge difference between nowadays US humans and apes. In
fact, this difference is more evident than what we would expect basing on the evo-
lutionary time from African apes to hominid and to actual human being (Moeller
et al. 2014). On average, there has been a reduction of ancestral diversity in the
passage from apes microbiome to human microbiome; this finding is more evident
among US population. A possible explanation could be due to the recent lifestyle
changes that possibly have depleted the human microbiome of microbial diversity
that was present in our wild-living ancestors (Blaser and Falkow 2009). Alongside
with this hypothesis, we know that diet has a strong impact on gut microbiome. In
fact, some authors sequenced oral microbiota from skeleton teeth deriving from
different eras human beings and found out that the most significant changes had
occurred during the passage from the hunter-gatherer Paleolithic era to the farm-
ing Neolithic era and the beginning of the industrialized period. In the first pas-
sage, the human diet shifted from meat based to fiber and carbohydrates based,
while in the second one, diet shifted to a more processed sugar and flour (Bibbò
et al. 2016; Chan, Estaki, and Gibson 2013; Adler et al. 2013). Last but not least,
the most recent change in human gut microbiome was at the beginning of antibi-
otic revolution due to the well-known influence of antibiotics on gut microbiome
(Ianiro, Tilg, and Gasbarrini 2016).
Not only host genes influence gut microbiome composition (together with diet
and energy substance available), but also gut microbiome can influence some gene
selection. For instance, human genetic variants that facilitate lactose tolerance and
dairying in adulthood (that differentiate human being from the other mammalian
and modern human being from ancient human being) were probably microbiome
influenced. As we know, lactase production is intensive during childhood and pro-
gressively decreases during life and a continued milk consumption in the lack of this
enzyme leads to an extensive microbial fermentation of lactose in colon. This mecha-
nism produces some metabolites that can induce lactose intolerance symptoms, but at
the same time facilitate the development of adaptive alleles allowing the persistence
of lactase expression (Schnorr et al. 2016; He et al. 2008).
Another example of this interaction is the presence of gluten degrading micro-
organisms in oral (Rothia mucilaginosa, Rothia aeria, Actinomyces odontolyticus,
Streptococcus mitis, Streptococcus sp., Neisseria mucosa, and Capnocytophaga
sputigena) and gut (Firmicutes and Actinobacteria, mainly from the genera
Lactobacillus, Streptococcus, Staphylococcus, Clostridium, and Bifidobacterium)
microbiome of human species after the advent of agriculture (Fernandez-Feo et al.
2013; Caminero et al. 2014).
Moreover, as gut microbes produce more than 90% of the body’s serotonin (Yano
et al. 2015) and modulate synthesis of metabolites affecting gene expression for
myelin production in the prefrontal cortex, host–microbe interactions have likely
influenced human brain development (Stilling et al. 2014). Particularly, human
behavior such as stress, anxiety, and novelty seeking is additionally reinforced by
microbial production of neuroactive compounds (Schnorr et al. 2016).
248 The Gut Microbiome: Bench to Table

So, the evolution of gut microbiome basically was dictated by the environmen-
tal changes (especially food related) that occurred during human evolution. The
most important difference between present microbiome composition and apes is the
decreased variability mainly due to fast lifestyle changes. In addition to that, nowa-
days, composition of microbiome is much more different between different individu-
als, depending on where they live and how they eat (Moeller et al. 2014; Bibbò et al.
2016; Ianiro, Tilg, and Gasbarrini 2016).

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Index
Note: Bold page numbers refer to tables; italic page numbers refer to figures.
aberrant microbiome 5 ASC see adult stem cell (ASC)
absorbable short-chain fatty acids 4 ASV see Amplicon Sequence Variant (ASV)
Acetobacteraceae (a-proteo-bacteria) 106 atherosclerosis 90
acidic pH 128, 170 Automated Self-Administered 24-hour Dietary
Acinetobacter baumanii 196 Recalls (ASA24) 83
acquired immunodeficiency syndrome (AIDS) avian microbiomes 107
54, 55 avian models 107
Actinobacteria 74, 105, 163, 190, 214, 229
adaptive immune responses 4 Bacillus
ad-hoc heuristic approach 22 B. anthracis 122
adult stem cell (ASC) 102 B. cereus 122
derived intestinal organoids 102 B. coagulans 171
Adventist Health Study 186 B. subtilis 245
AgRP/NPY neurons 220 bacteria
AhR see aryl hydrocarbon receptor (AhR) and metabolites 4
AIDS see acquired immunodeficiency syndrome phages infect 46
(AIDS) probiotic lactic acid, lipids of 128–129
Akkermansia muciniphila 189, 196, 198, 224 SCFA-producing 227
ALDEx2 22–23, 25 bacteria–gut–host physiology system 213
Alexandrium minutum 139 bacterial cell walls, cholesterol integration into
α-linoleic acid, biosynthetic derivatives of 86 128–129
Allobaculum 144 bacterial CRISPR genes 47
Amazon rainforest 157 bacterial host cells, lysing of 52
amino acid 128 bacterial metabolism of dietary fiber 78
metabolism, gut microbiome and 89–90 bacteriophages (phages) 45
Amplicon Sequence Variant (ASV) 10 Bacteroidaceae 108, 144
amplicon sequencing, full-length 16S rRNA 11 Bacteroides 82, 99
amplification cycles 9 abundance in 83
amyloid-producing Escherichia coli 54 Bacteroides 108, 138, 145, 224, 229
Anaerostipes 162 B. dorei 226
analysis of composition of microbiomes B. fragilis 47
(ANCOM) 23 B. vulgatus 224, 226
analysis of similarities (ANOSIM) 17 Bacteroidetes 5, 74, 80–83, 91, 105, 214, 215,
Analysis of variance (ANOVA) 21 217–219
ANCOM see analysis of composition of Bacteroidetes 53, 108, 111
microbiomes (ANCOM) basal metabolic rate (BMR) 91
Anelloviridae 55, 56 Basic Local Alignment Search Tool (BLAST) 14
animal protein, diet rich in 226 Bayesian multiplicative replacement method 25
anthocyanins 186, 192 BCAAs see branched-chain amino acids
antibiotics 135 (BCAAs)
anti-inflammatory action of resveratrol 195 Benjamini–Hochberg procedure 22
anti-inflammatory amino acid metabolites 89 berries’ polyphenols 194
antimicrobial activity against Candida betaine 131
albicans 244 β-glucan polysaccharide 83
arabinoxylan-oligosaccharides 240 Bifidobacteria 80, 86, 89
aromatic acids 76 dominated microbiome 77
aronia (poly)phenol-rich extract 196 Bifidobacteria 133, 136, 137, 144, 145, 158, 162,
artificial sweeteners 225 164, 170, 171, 243
aryl hydrocarbon receptor (AhR) 89 Bifidobacteriaceae 158

253
254 Index

Bifidobacterium 80, 83, 87, 99, 122, 136, 138, carbohydrate-active enzymes (CAZymes) 79
142, 159, 194, 219, 224, 226, 229 carbohydrate metabolism
B. adolescentis 225 gut microbiome and 79–84
B. animalis 228 complex carbohydrates 82
subsp. lactis CECT 8145 228 nondigestible polysaccharides–fiber
B. breve 144, 191 82–84
B. longum 142, 164 simple carbohydrates 80–82
breve ATCC 15700 130 carbohydrate prebiotics 159
BSH activity in 127 human milk oligosaccharides (HMO)
B. vulgatus 218 159–161, 160
bifidogenic effects 162 inulin 162
bile acids (BAs) 76, 126 oligosaccharides 159
microbial cometabolism of 76 polysaccharides 161–162
bile salt 124 carbohydrates 224–225
cholesterol deposition with conjugated 128 complex 82
conjugation 126 simple 80–82
hydrolyzing enzyme cardiovascular disease (CVD) 73, 135–139, 185
decongesting bile salts using 126–128, 127 carotenoids 189
removal of deconjugated 128 carrier matrix 170
bile salt hydrolase (BSH) activity, in casein 89
Bifidobacterium and Lactobacillus 127 catechins 191
Bilophila wadsworthia 85, 144 Caudovirales 54
bioactive compounds, plant 187 phages 56
bioactive polyphenol-derived compounds 183 CCK 223
biogeography of microbiome 80 CD see celiac disease (CD); Crohn’s disease (CD)
bioinformatics methods 7 CD4+ T cells 89
Biological Observation Matrix (BIOM) format 17 cecum microflora 89
bioprinting 102 celiac disease (CD) 243
droplet-based 102 cell culture 100–101
extrusion-based 102 3D 102
biotransformations, microbial enzymatic 164 cell membranes 129
bird microbiomes 107 cellulose 82
BLAST see Basic Local Alignment Search Tool centered log-ratio (CLR) approach 22
(BLAST) Centers for Disease Control and Prevention 10
blood–brain barrier (BBB) 888 C-glycosides 192
permeability 81 cholecystokinin (CCK) 220
BMI see body mass index (BMI) cholesterol
body mass index (BMI) 75, 212 binding of 130
brainstem 221 deposition of
branched-chain amino acids (BCAAs) 193 with colic acid 128
Bray–Curtis dissimilarities 33 deposition with conjugated bile salt 128
breastfeeding 77 integration into bacterial cell walls 128–129
breast milk 50 cholesterol-lowering effect mechanisms 126
triglycerides 77 cholesterol-lowering probiotic effects,
butyrate-producing Faecalibacterium hypothetical concept of 126
prausnitzii 85 cholesterol-lowering probiotics, biotherapeutic
butyrate-supplemented high-fat diets 84 properties of 121–140
Butyrivibrio crossotus 111 binding of cholesterol 130
cholesterol deposition with conjugated bile
Caco-2 103 salt 128
cell line 101 cholesterol integration into bacterial cell
cells 101 walls 128–129
co-cultures of 101 cholesterol-lowering effect mechanisms 126
Calypso 31–33 decongesting bile salts using bile salt
cancer treatment 138–139 hydrolyzing enzyme 126–128, 127
Candida albicans 244 gut microbiota in human health and disease
antimicrobial activity against 244 prevention 134–135
Index 255

probiotics gut virome in 46

cholesterol-lowering of 125 viruses in 46
concept 121–140 COX-2 195
lowering serum cholesterol by 129–130 cresols 76
microorganism 122 CRISPR-Cas9 systems 58, 61
selection criteria 122–144 Crohn’s disease (CD) 53, 55, 56, 137, 164
relationship between nutrition and gut cultured beverages, in gut microbiota modulation
microbiota 132–134, 133 144–145
synthesis and metabolism of plasma cultured foods (fermented foods) 140
lipoprotein 124 products 120, 140
cholesterol reductase enzyme 131 cultured products in gut microbiota modulation
cholic acid 76 140–145, 141–142
choline 131 milk kefir 143
choline degradation 85 probiotic fermented milk 143–144
chromosomes, 3D structure 8 yogurt 140
chronic inflammation 5 culturomics 242–243
of small intestine 132 curcumin 197
chylomicrons 124 CVD see cardiovascular disease (CVD)
Circoviridae 56 Cyanobacteria 190
clinical therapeutics 227 cytokines
Clostridiaceae 25 proinflammatory 52
Clostridiales 81 signaling 3 expression 221
Clostridia species 52
Clostridioides difficile 111 DADA2 algorithm 17
Clostridium 25, 138, 144, 162, 190 dairy foods 167
C. coccoides 86 decarboxylase, glutamate 89
C. difficile 81 de facto intervention in GI tract 85
associated diarrhea 227 degradation, choline 85
C. leptum 197 deleterious microorganisms 120
clusters IV 104 denaturing gradient gel electrophoresis
clusters IV and XIVa 111 (DGGE) 7, 197
C. perfringens 52, 81, 144, 145 dendritic cells 136
Clostridium difficile infection (CDI) 59, 227 de novo gluconeogenesis 84
colonic cells 88 de novo lipogenesis in enterocytes 81
colonic microbiota 74 de novo synthesis 126
colonization, newborn intestinal 76 density-dependent lysis phages 49
commensal bacteria 5 dental diseases 136
commensal microflora 213 deoxynucleotide triphosphates (dNTP) 9
complex carbohydrates 82, 240 de-polymerization, microbial 78
complex polysaccharides 159 dereplication 19
Compositional Data through Lasso (CCLasso) 29 DESeq2 23, 83
computational tool development, advances in Desulfovibrionaceae 220
21–30 dextran sulfate sodium (DSS) 23
balances and microbial signature induced colitis 112
identification 24–25, 26 DGGE see denaturing gradient gel
tools for network analysis and their electrophoresis (DGGE)
applications 26–30 DHA 87
uniqueness of marker gene survey datasets diabetes 5
21–24, 24 DIABIMMUNE project 99
CoNet 27 diet and nutrition 120
conjugated compounds 192 dietary fiber 223
conjugation reactions 191 bacterial metabolism of 78
contemporary phage therapy 58 dietary intervention, with functional foods 199
conventional clone-and-sequencing method 6 dietary modulation, of gut microbiota 119–147
conventional cloning method 7 dietary polyphenols 187, 192
correlation and regression-based tools 27 biological properties of 190
COVID-19 patients dietary sugars 81
256 Index

diets, low-fiber 84 E. eligens 82

digestive system, microflora in 134 E. rectale 225, 229
disease-causing pathogens 165 eukaryotic 18S rRNA gene 5
disialyllacto-N-tetraose (DSLNT) 166 European Commission Concerted Action on
DNA Functional Food Science 166
fragments 7 exopolysaccharides 128
hybridization 7 extrusion-based bioprinting 102
ligase 9 ex vivo models 101–102
sequencer 7 EzBioCloud 14
sequencing technology 11
next-generation 6 Faecalibacterium 219
strands in ZMW 11 F. prausnitzii 104, 111, 189, 219, 224, 225
synthesis 11 butyrate-producing 85
templates 9 false discovery rate (FDR) 20, 21
dNTP see deoxynucleotide triphosphates (dNTP) fasting-induced adipocyte factor (Fiaf)
droplet-based bioprinting 102 protein 53
Drosophila FastQC 20
D. melanogaster 106 fat 225
microbiome 106 fatty acid metabolism
secretive enteroendocrine cells in 81 gut microbiome and 84–89
dsDNA 47 polyunsaturated fatty acids (PUFAs)–
DSLNT see disialyllacto-N-tetraose (DSLNT) omega-3 and 6 (ω-3 and ω-6) fatty
dysbiosis 57 acids 86–88
microbial 213 saturated fatty acids 85–86
dyslipidemias 135 short-chain fatty acids 88–89
fatty acids
EECs see enteroendocrine cells (EECs) absorbable short-chain 4
eglycosylations 191 saturated 85–86
elimination diets 243 short-chain 88–89
ellagic acid 193 6 (ω-3 and ω-6) fatty acids 86–88
ellagitannins 189, 202 fatty liver disease 90
endocannabinoids 76 fecal batch cultures 100
endosymbionts 106 fecal microbial enzymes 192
endothelial activation 86 fecal microbial transplant (FMT) 72
energy metabolism and gut microbiota 217–223 rectal delivery 72
enteroaggregative Escherichia coli (EAEC) 58 fecal microbiome translocation (FMT) 111
Enterobacteria 54, 133 models 111–112
Enterobacteriaceae 10, 108, 143, 220, 226 fecal microbiota 199
Enterococcus 111 and human health 111
E. faecalis 54, 245 standardized 112
enterocytes, de novo lipogenesis in 81 fecal microbiota transplantation (FMT) 59, 61, 227
enteroendocrine cells (EECs) 222 fecal viral transplants 59, 61
environmental microbial diversity 107 feed behavior and satiety, modulating 220–222
enzymatic biotransformations, microbial 164 feeding behavior 220
enzymes 5 fermentation
BSH, hydrolyzes conjugated bile salts 126, 127 derived bioactive peptides 245
fecal microbial 192 endproducts 90
mammalian 79 of inulin 83
microbial 89 microbial 4
microbiome-produced 89 saccharolytic 159
polysaccharide-degrading 82 fermentative effects 243
epigallocatechin-3-gallate (EGCG) 198 fermented food 228–229
Erysipelotrichaceae 218 probiotics in 229
Escherichia coli 6, 52, 60, 101, 142, 145, 195, 245 fermented functional foods 120
amyloid-producing 54 fermented milk, probiotic 143–144
ethanol 144 fermented soybean meal (FSBM) consumption 198
Eubacterium 52 fermented soymilk 144
Index 257

fermented vegetables, in gut microbiota galactooligosaccharides (GOS) 144, 159, 240

modulation 145 Galaxy platform 19
fiber 172 gallic acid 193
diets rich in 88 gastrin 222
supplementation 167 gastrointestinal diseases 137–138
Find, Rapidly, OTUs with Galaxy Solution gastrointestinal microbiology 157
(FROGS) 19–20 gastrointestinal (GI) tract 48, 157, 213
pipelines 30 de facto intervention in 85
Firmicutes 5, 74, 91, 105, 214, 215, 217, 218 phages in 46
Firmicutes 53, 145 physical barriers in 48
Firmicutes/Bacteroidetes (F/B) ratios 89 gel electrophoresis
FISH see fluorescent in situ hybridization (FISH) denaturing gradient 7
flavan-3-ols 192 temperature gradient 7
flavanone 186, 191–192 GenBank database 55
flavonoid-rich orange juice treatment 198 gene–based microbiome profiling marker 5
flavonoids 185 gene–based microbiome studies marker 5
flavonol glycosides 191 gene sequencing 105
flavonols 186, 192 genetic homogeneity 109
polymerized 186 gene transfer 47
flow-mediated dilation (FMD) 196 Genomes Orthology profiles 31
fluorescent in situ hybridization (FISH) 241 genome-wide association studies (GWASs) 75, 76
16S probe-based 6 of obesity 108
targeting microbial SSU rRNA genes 8 genomic sequencing analysis 106
FMT see fecal microbiome translocation genomic technologies 158
(FMT); fecal microbiota germfree animals 44
transplantation (FMT) germ-free (GF) mice 53, 214
FODMAPs 243 model 221
food and gut microbiome 239–240 GFD see gluten-free diet (GFD)
food compounds, hydrophilic plant 187 ghrelin 222
food frequency questionnaire (FFQ) 112 GLP-1 223
Foodomics 205 secretion, stimulation 84
food-processing effects, on probiotics and glucosidic bond, cleavage of 164
prebiotics 169–170 glucuronidation 191
food products, cultured 120 glutamate decarboxylase 89
FOS see fructooligosaccharides (FOS) gluten-containing diets 51
freezing 171 gluten-free diet (GFD) 51, 224, 243–244
FROGS see Find, Rapidly, OTUs with Galaxy GLUT5 (glucose transporter 5) expression
Solution (FROGS) (fructose transporter) 81
fructans 165 glycans, host-provided 77
inulin-type 162 glycolipids 126
fructooligosaccharides (FOS) 159, 240 glycosides 192
fructose, pro-oxidative and pro-inflammatory Gordonibacter
effects of 81 G. pamelaeae 191
FTM-Clostridium difficile therapy 72 G. urolithinfaciens 191
fucosyltransferase II (fucII) 161 GOS see galactooligosaccharides (GOS)
full-length 16S rRNA amplicon sequencing 11 GPCRs see G-protein coupled receptors (GPCRs)
functional foods 244 G-protein coupled receptors (GPCRs) 87, 204
categories of 167 G-protein-coupled receptor 41 (GPR41)-mediated
defined 166–167 activation of sympathetic neurons 84
dietary intervention with 199 G-protein receptor 41 (GPR41) 89
in microbiota modulation 243–245 gram-staining techniques 240
prebiotics as 166–172 grapes, polyphenols in 194
novel prebiotic formulations 168–169 Greengenes 12, 12–13
functional metagenomics 5 GRO see growth-related oncogene (GRO)
Functional Similarity Weight (FS-weight) 27 growth-related oncogene (GRO) 199
Fusobacteria 74, 190, 214, 229 GS FLX system 8
Fusobacterium nucleatum 58 454 GS FLX Titanium system 8
258 Index

GSTT2 195 diversity and interactions 53

Gulf War illness (GWI) patients, gut virome in 52 ecological role 46
gut bacteria 4 effects of phenolic compounds on 145–146
gut–brain axis, modulating feed behavior by energy metabolism and 217–223
222–223 formation and characteristic 214–217
gut–brain function 223 formation of 213
gut chip, microfluidics systems and 102–103 modulatory effect of 120
gut-derived metabolites 190–193 nutrition and 132–134, 133
gut ecosystem in human host 76 in obesity 217–218
gut microbes 4, 52 origins and roles of phages, phageome, and its
gut microbial composition composition in human 45–48
dietary styles in relation to 223–224 phages shape 49–51
dynamics, and function 214 polyphenol metabolism 189
gut microbial response 201 and polyphenols 183
gut microbiome 5, 77, 78, 194 role in human health and disease prevention
and amino acid metabolism 89–90 134–135
and carbohydrate metabolism 79–84 species variation of 214
complex carbohydrates 82 gut microbiota modulation
nondigestible polysaccharides–fiber cultured beverages in 144–145
82–84 cultured products in 140–145, 141–142
simple carbohydrates 80–82 milk kefir 143
and fatty acid metabolism 84–89 probiotic fermented milk 143–144
polyunsaturated fatty acids (PUFAs)– yogurt 140
omega-3 and 6 (ω-3 and ω-6) fatty fermented vegetables in 145
acids 86–88 role of cultured products in 140–145
saturated fatty acids 85–86 gut microflora, factors influencing 133
short-chain fatty acids 88–89 gut microorganisms 4
food and 239–240 diversity and composition of 214–217
human 74–75 gut phageome
structure and functions of 5 changes in 50
gut microbiome, models for researching 97–113 vs. human diseases 55
animal models for studying gut microbiota gut-residing microbes 53
105–111 gut virome 56
avian models 107 in COVID-19 patients 46
humanized gnotobiotic mice 109–110 in Gulf War illness (GWI) patients 52
insect models 105–106 GWASs see genome-wide association studies
murine models 107–109 (GWASs)
porcine models 110
zebrafish model 110–111 habitat filtering 27
computational models 98–99 HDL see high-density lipoprotein (HDL)
fecal microbiome translocation models health and disease, phageome and 53–56
111–112 healthy gut phageome (HGP) 46
simulator of the human intestinal microbial healthy microbiota 190
ecosystem (SHIME) 103–105 heat-killed probiotics 227
in vitro models 100 Helicobacter pylori 142, 143
cell culture 100–101 eradication 244
ex vivo models 101–102 hemoglobin A1c (HbA1c) 219
fecal batch cultures 100 hepatic flavin monooxygenases (FMO3) 131
microfluidics systems and gut chip heterogeneous ecosystem 48
102–103 HGP see healthy gut phageome (HGP)
3D cell culture 102 HiαDP 203
gut microbiota 53 high (HDL-C) cholesterol 135
activated prophages from 52 high-cholesterol mouse model 129
communities, variations 213 high-density lipoprotein (HDL) 125
composition, dietary modulating 223–223 high–energy–density foods 212
dietary modulation of 119–147 high-fat diet (HFD) 110
diversity and abundance of 47 mouse model 221
Index 259

high-fructose diets 80 hybridized target genes 8

high-sucrose diets 81 hydrogen peroxide, toxicity of 171
high-throughput sequencing technologies 8–12 hydrolyzing enzyme, bile salt 126–128, 127
illumina platform 9–10 hydrophilicity of polyphenolics 191
ion torrent platform 10–11 hydrophilic plant food compounds 187
long read sequencing platforms 11–12 hypercholesterolemia 125
pyrosequencing and supported hypertension 90
oligonucleotide ligation and hypervariable (HV) regions 5
detection 8–9 hypothalamic neurons 221
HiSeq X system 9 hypothalamic–pituitary–adrenal stress response
10 HiSeq X ultrahigh-throughput instruments 9 phenotype 53
histamine 223 hypothalamus 220–221
HMOs see human milk oligosaccharides (HMOs) hypoxia-inducible factor 1 α (HIF1α) 89
HMP see Human Microbiome Project (HMP)
homeostasis of host microbiome 5 IBD see inflammatory bowel disease (IBD)
host and gut microbiome, symbiotic relationship 80 ILCs 89
host cell 46 ileal bacteria 48
host–gut microbiome metabolic interactions ileal cells 88
76–78 Illumina MiSeq 9, 10
host health illumina platform, high-throughput sequencing
intestinal homeostasis and 78 technologies 9–10
maintenance 214–217 illumina’s MiSeqDx 9
host metabolism, regulation on 218–220 IL-22 production 89
host microbiome, homeostasis of 5 iMAP see Integrated Microbiome Analysis
host-provided glycans 77 Pipeline (iMAP)
HT-29 immune response
cell lines 101 phage-induced 50
co-cultures of 101 stimulation of adaptive 4
MTX cell line 101 immune system, phage interaction with 52
human diseases vs. gut phageome 55 immunity, effect of prebiotic on 164–166
human evolution, microbiome through 245–248 immunomodulation by prebiotics 165
human fetus microbiome 4 indigestible fibers 240
human gut microbiome 74–75 infant’s intestinal microbiota 77–78
human gut microbiota 79–80 inflammation, chronic 5
approach to investigate 242–243 inflammatory bowel disease (IBD) 5, 53, 135,
human gut-related disease, phage-based therapy 137–138, 165, 213
for 56–59 development of 108
human health genetic models for 108
and disease prevention, gut microbiota role inhibitor, lipoprotein lipase (LPL) 131
134–135 insects, microbiota of 105
fecal microbiota and 111 insulin-like peptide 222
human immunodeficiency virus (HIV) 54 insulin resistance 5
humanized gnotobiotic mice 109–110 Integrated Microbiome Analysis Pipeline (iMAP)
human metabolism 183 20–21
human–microbiome 72, 73, 190 Interactive Tree Of Life (iTOL) 20
genome sequences 73 interleukin 22 (IL-22) 89
symbiotic relationship 92 internal transcribed spacer (ITS) 5–6
Human Microbiome Project (HMP) 73 intestinal bacteria 132
human microbiota 190 intestinal disorders 133, 213
human milk oligosaccharide-grown intestinal epithelium 77
Bifidobacteria 80 intestinal flora 159
human milk oligosaccharides (HMOs) 77, manipulation of 137
159–161, 160 intestinal homeostasis and host health 78
formulation 168 intestinal microbiome 5
human studies, considerations on limitations of intestinal microbiome metabolism,
201–205 polysaccharides for 83–84
hybridization, DNA 7 intestinal microbiota functions 78
260 Index

intestinal microflora 132 L. gasseri 130, 143

intestinal organoids, ASC-derived 102 L. gasseri SBT2055 227
inulin 162 L. kefiri 143
consumption of 162 L. lactis 199
fermentation of 83 L. plantarum 172, 199, 227
nutritional utilization of 162 MA2 (1011 cells/d) 131
inulin-type fructans 162 L. reuteri 89, 199
in vitro colon system study 197 in mouse models 51
In vitro experiments 160 L. reuteri NCIMB 30242 128
in vitro fermentation model 194 L. rhamnosus 139, 170
in vitro models L. rhamnosus 170
gut microbiome models for researching 100 Lactococcus
cell culture 100–101 cells 130
ex vivo models 101–102 L. brevis 129
fecal batch cultures 100 L. lactis 129
microfluidics systems and gut chip lactose intolerance 139, 168
102–103 lactulose 138
3D cell culture 102 LASSO see Least Absolute Shrinkage and
Ion Torrent Personal Genome Machine (PGM) Selection Operator (LASSO)
10, 11 L-carnitine 131
ion torrent platform, high-throughput sequencing LCn3 197
technologies 10–11 LDA see linear discriminant analysis (LDA)
Ion Torrent sequencer 10 ‘lean enterotype’ pattern 145
iron supplementation 226 Learning Interactions from Microbial Time
irritable bowel syndrome (IBS) 164, 213, 243 Series (LIMITS) 29, 30
isoflavones 192 Least Absolute Shrinkage and Selection Operator
isoflavonoid daidzein, soy-derived 195 (LASSO) 28
ITS see internal transcribed spacer (ITS) regression-based approach 33
LEfSe 22
Joint FAO/WHO Working Group 124 leptin 221
leptin receptors 221
Kalman filter 29 Leuconostoc 140, 145
kefir 142, 143, 244 L-histidine 89
kimchi 142, 145 454 Life Sciences 8
Kombucha tea 144 Life Technologies 9
Kruskal–Wallis test 22 linear discriminant analysis (LDA) 18
Kyoto Encyclopedia of Genes 31 linear mixed models 99
linolenic acid (LA), biosynthetic derivatives of 86
Lachnospiraceae 108 lipid-lowering drugs 125
lactic acid 122 lipid synthesis 84
bacteria 140, 244 lipopolysaccharide (LPS) 86, 191, 225
Lactobacillaceae 158 binding site 165
Lactobacillales (Firmicutes) 106 high levels of plasma 80
Lactobacilli 89, 136, 137, 158, 162, 170 translocation of 217
Lactobacillus 74, 76, 83, 86, 87 transport 86
Lactobacillus 111, 122, 126, 135, 136, 140, lipoprotein lipase (LPL) inhibitor 131
144–145, 159, 164, 190, 194, 199, lipoprotein plasma, synthesis and metabolism
219, 224 of 124
abundance 91 liver disease 135–136
BSH activity in 127 liver X receptor (LXR) 131
L. acidophilus 143 local similarity analysis (LSA) 28
ATCC 43121 129–130 long-chain n – 3 polyunsaturated fatty acids
ATCC 4356 strain 131 (LCn3) 197
La5 228 long read sequencing platforms, high-throughput
L. bifidus 158 sequencing technologies 11–12
L. casei 144, 171 Lotka–Volterra dynamic model 29
L. delbrueckii subsp. bulgaricus 139, 143 Lotka–Volterra equations 29
Index 261

Lotka–Volterra model 29 gut-derived 190–193

for microbial dynamics 29 microbial 223
lowering serum cholesterol, by probiotics microbiome-derived 4
129–130 microbiota-derived 220
low-fiber diets 84 plant-secondary 183
LPS see lipopolysaccharide (LPS) secondary 185
LSA see local similarity analysis (LSA) urolithin 193
LT97 human adenoma cell line 195 metabolome and microbiome connection
luminal nutrients 222 75–76
lymphatic cells 124 metabolomics 161
lysing of bacterial host cells 52 metabotypes 201–205
lysogenic life cycle 46 MetaDprof 99
lysogenic phages 46 MEtaGenome Analyzer (MEGAN) 30
lysogeny 47 metagenomics 5, 241
lytic life cycle 46 functional 5
lytic–lysogenic balances 47 Shotgun 241
lytic phages 46 virome 56
metagenomic sequences 73
macronutrients 224–226 metagenomics Rapid Annotation using
carbohydrates 224–225 Subsystems Technology server
fat 225 (MG-RAST) 30
protein 225–226 MetaHIT protocol 112
MALDI–TOF mass spectrometry 242 MetaLonDA 99
mammalian enzymes 79 meta-network framework 27
manganese supplementation in mice 226 methylation 191
mannose, prebiotic monomers of 165 MGEs see mobile genetic elements (MGEs)
Marker Data Profiling (MDP) module 30 MG-RAST see metagenomics Rapid Annotation
marker gene–based microbiome profiling 5 using Subsystems Technology server
marker gene–based microbiome studies 5, 12, 15 (MG-RAST)
data analysis pipeline for 16 microbes 76, 80
marker gene sequence analysis pipelines 15–21 microbial CAZymes 79
FROGS 19–20 microbial cometabolism of bile acids 76
Integrated Microbiome Analysis Pipeline microbial community 29, 74
(iMAP) 20–21 complexity of 6
QIIME 1 and QIIME 2 15–18 interactions in 98
marker gene survey datasets, uniqueness of microbial de-polymerization 78
21–24, 24 microbial dynamics, Lotka-Volterra model for 29
marker gene survey data, web-based tools for microbial dysbiosis 213
30–33, 34 microbial enzymes 89
maternal milk 159 biotransformations 164
MDP 30 fecal 192
Mediterranean diet (MedDiet) 91, 190, 224, 244 microbial fermentation 4
membrane formation 126 microbial interaction network 26–27
metabolic disorders 5 microbial marker gene–based study,
metabolic dysfunction 5 renaissance of 6
metabolic interactions, host–gut microbiome microbial metabolism 193
76–78 human studies of 184
metabolic products 131 microbial metabolites 223
metabolic syndrome 73, 77 microbial network inference 35
metabolism microbial ontology 92
microbial 193 microbial origin, catabolic enzymes of 80
of nutrients by gut microbiota 164–166 Microbial Prior LASSO (MPLasso) 28, 29
PUFA 86–87 microbial richness 218
metabolite-microbiota interaction 193–201 microbial signature identification, balances and
metabolites 131 24–25, 26
anti-inflammatory amino acid 89 microbiological safety evaluation, ICMR-DBT
bacteria and 4 guidelines 124
262 Index

microbiome 4, 71, 72; see also specific types milk proteins 168
aberrant 5 MiSeq 9, 10
altered 5 standard operating procedure 18
bifidobacteria-dominated 77 mobile genetic elements (MGEs) 92
biogeography of 80 molecular ecological network analyses
human fetus 4 (MENA) 28
nutritional environment for 78 monocarboxylate transporters 88
placental 4 monounsaturated fatty acids (MUFAs) 244
role in 4 Monte Carlo sampling 22
through human evolution 245–248 mothur 18
and weight change 90–91 motilin 222
Microbiome Analyst 30, 31 mouse models, Lactobacillus reuteri in 51
microbiome connection, metabolome and 75–76 MPI see milk protein isolate (MPI)
microbiome-derived metabolites 4 MPLasso see Microbial Prior LASSO (MPLasso)
microbiome–diet interactions 78, 79 mucosal layers, phages in 50
microbiome metabolic interactions mucosal microbiome 104
host–gut 76–78 murine models 107–109
microbiome-produced enzymes 89 Mycoplasma pneumoniae, chromosome
microbiome profiling, gene–based marker 5 structure of 8
microbiome studies myeloid differentiation factor 2 (MD2) 86
gene-based marker 5
historical perspective of 6–8 Nanopore, Oxford 11
Microbiome Taxonomic Profiling 14 National Center for Biotechnology Information
microbiome zeitgeist 72 (NCBI) taxonomy 12
microbiota 46, 72 NEC see necrotizing enterocolitis (NEC)
colonic 74 necrotizing enterocolitis (NEC) 166
colonizes 47 Neisseria 74
infant’s intestinal 77–78 Nelder’s algorithm 30
microbiota and host stem, symbiotic relationships neonatal’s ingestion of microorganisms 76
between 77 netagenomic approaches 60
microbiota composition 191 network algorithms 26
microbiota-derived metabolites 220 neuroendocrine mechanisms, activation of 222
microbiota–gut–brain (MGB) axis 53 neurohormonal signaling molecules 88
microbiota investigation tools 240–242 neurons
microbiota metabolism of polyphenols and health AgRP/NPY 220
effects 190–205 hypothalamic 221
microbiota modulation, role of functional food in pro-opiomelanocortin (POMC) 220
243–245 newborn intestinal colonization 76
microbiota of insects 105 next-generation DNA sequencing technologies 6
microbiota’s diversity 145 next-generation sequencing (NGS) 8, 241
microbiotic composition 73 NGS see next-generation sequencing (NGS)
microflora Niemann-Pick C1-Like 1 (NPC1L1) protein 131
commensal 213 nonalcoholic steatohepatitis 73
in digestive system 134 non-carbohydrate prebiotics 163–164
microfluidics systems and gut chip 102–103 polyphenols 163
micro-mass sequencing technique 55 short-chain fatty acids 164
micronutrients 226 nonculturable microorganisms 6
microorganisms 4, 214 nondigestible polysaccharides–fiber 82–84
evolutionary divergence of 5 non-flavonoids 186
neonatal’s ingestion of 76 nonparametric tests 31
nonculturable 6 nonsteroidal anti-inflammatory drugs
Microviridae 56 (NSAID) 46
to Caudovirales ratio 47 NovaSeq 6000 9
to Siphoviridae ratios NovaSeq Xp workflow 9
in gut 50 novel metabolic approaches, considerations on
milk kefir 143 limitations of 201–205
milk protein isolate (MPI) 90 novel prebiotic formulations 168–169
Index 263

NSAID see nonsteroidal anti-inflammatory PAMPs see pathogen-associated molecular

drugs (NSAID) patterns (PAMPs)
nutrition Parabacteroides distasonis 225
diet and 120 pathogen-associated molecular patterns
and gut microbiota 132–134, 133 (PAMPs) 52
pathogenic strains 122
obesity 5, 53, 90 PCA see principal component analysis (PCA)
genome-wide association studies of 108 PCR see polymerase chain reaction (PCR)
gut microbiota in 217–218 Pearson approach 27
and overweight 211–212 Pediococcus 145
obesogenic diets 229 peptides
O-desmethylangolesin (ODMA) 202 fermentation-derived bioactive 245
O-glycosides 192 YY 84
oligofructose 162 Peptostreptococcaceae 25
oligomeric polyphenols 189 PERMDISP2 32
oligonucleotide peroxisome proliferatoractivated receptors
arrays, phylogenetic 7 (PPARs) 131
ligation and detection 8–9 phage 48
oligosaccharides 135, 159, 240 cocktails 58
building blocks of 160 communities 47
human milk 159–161 density-dependent lysis 49
prebiotic 101 genome 46, 48
olive pomace-enriched biscuit formulation (OEP), host ranges 59
consumption of 199 in gastrointestinal (GI) tract 46
omega-3 fatty acid 87 infect bacteria 46
omega-3 PUFAs interaction with immune system 52
anti-inflammatory properties of 87 in mucosal layers 50
cardioprotective effect 88 shape, gut microbiota 49, 49–51
diet high in 87 phage-based therapy 57, 58, 60
direct effect of 87 for human gut-related disease 56–59
inclusion of 87 phage-induced immune response 50
related cardioprotection 88 phageome 46, 54
open-source software 24 in gut microbiome 45–62
operational taxonomic units (OTUs) 200 and health and disease 53–56
orange juice, daily consumption of 198 in human gut microbiome, perspectives, and
orexigenic agouti-related protein/neuropeptide future directions 59–62
Y (AgRP/NPY)–coexpressing stabilizes in adulthood 51
neurons 220 phenolic compounds 191
organoid models 102 effects of on gut microbiota 145–146
organoids, ASC-derived intestinal 102 phosphatidylcholine 131
Orthologous Average Nucleotide Identity phospholipids 126
(OrthoANI) 14 curcumin 199
orthoreovirus 57 PhyloChip 7
OTU PhyloChip G3 8
picking tool 15 PhyloChip G4 7
sample matrix 21 Phylogenetic Investigation of Communities by
table 17, 19, 21 Reconstruction of Unobserved States
overnutrition 90 (PICRUSt) 13
overweight phylogenetic oligonucleotide arrays 7
defined 212 physical barriers, in gastrointestinal (GI) tract 48
obesity and 211–212 phytochemicals 134
Oxford Nanopore 11 picobirnavirus 57
oxysterols 76 PICRUSt 22
piggyback-the-winner dynamic 50
Pacific Biosciences (PacBio) 11 placebo-controlled interventional study 198
Sequel Systems 11 placental microbiome 4
PACs see proanthocyanidins (PACs) plant-based diets, health benefits of 184
264 Index

plant bioactive compounds 187 effect of

metabolism/bioavailability and excretion on immunity 164–166
187–190, 188 food-processing effects on 169–170
plant protein 226 as functional foods 166–172
plant-secondary metabolites 183 immunomodulation by 165
plasma lipoprotein, synthesis and metabolism interaction of 165
of 124 intervention 227–229
Plasmodium 106 non-carbohydrate 163–164
pluripotent stem cell (PSC) 102 polyphenols 163
polymerase chain reaction (PCR) 6, 241 short-chain fatty acids 164
amplified 16S molecules 7 predator–prey relationships 47
PCR-DGGE of microbiota 198–199 Prevotella 4, 91, 145, 224, 229
PCR-RFLP 7 enterotype 201
products 7 P. copri 199
polymeric polyphenols 189 populations 83
polymerized flavonols 186 principal component analysis (PCA) 20
polyphenolics, hydrophilicity of 191 principal coordinate analysis (PCoA) 17
polyphenol metabolism proanthocyanidins (PACs) 186, 195
gut microbiota-driven 189 probabilistic method for taxonomical
polyphenol–microbiota interaction 195 classification (PROTAX) 14–15
polyphenols 163, 183, 184, 189 probiotic bacteria 122, 171
berries’ 194 probiotic-enhancing products 171
bioavailability of 184 probiotic fermented milk 143–144
bioprotective effects of 163 probiotic lactic acid bacteria, lipids of 128–129
classification and general description 185–186 probiotic microorganisms 170, 171
dietary 187, 192 viable 169
biological properties of 190 probiotic milk 142
food sources and consumption 186–187 probiotics 120, 136, 138, 227–229
gut microbiota and 183 cholesterol-lowering of 125
health effects of 193–201 concept of 121–140
molecules, fraction of 191 in fermented food 229
multitude of 184 food-processing effects on 169–170
oligomeric 189 heat-killed 227
polymeric 189 lowering serum cholesterol by 129–130
Polyphenols in grapes 194 microorganism 122
polysaccharides 83, 161–162, 172 positive impact of 121
β-glucan 83 selection criteria 122–144
complex 159 species 171
degrading enzymes 82 probiotic therapy 138
for intestinal microbiome metabolism 83–84 probiotic viability, factors influence 170–172
polyunsaturated fatty acids (PUFAs) 244 probiotic yogurt drink, consumption of 142
metabolism 86–87 proinflammatory cytokines 52
omega-3 86–88 Projection with Public Data module 30
porcine models 110 prokaryotic 16S rRNA gene 5
Pouchitis 137 pro-opiomelanocortin (POMC) neurons 220
PPARs see peroxisome proliferatoractivated PROTAX see probabilistic method for
receptors (PPARs) taxonomical classification
PR2 see Protist Ribosomal Reference (PR2) (PROTAX)
prebiotic compounds 158 protein 225–226
prebiotic food formulations development 169 fasting-induced adipocyte factor (Fiaf) 53
prebiotic formulations, novel 168–169 Proteobacteria 74, 85, 105, 190, 214, 217, 229
prebiotic functional food formulations 167 Proteobacteria 108, 110
prebiotic monomers of mannose 165 proteomics 161
prebiotic oligosaccharides 101 Protist Ribosomal Reference (PR2) 15
prebiotics 139, 158–159 PSC see pluripotent stem cell (PSC)
carbohydrate 159 Pseudomonas aeruginosa 196
defined 228 PUFAs see polyunsaturated fatty acids (PUFAs)
Index 265

PyNAST see Python Nearest Alignment Space Sequel II System 11

Termination (PyNAST) sequencing-based microbiome studies 5
Python Nearest Alignment Space Termination S-equol 191
(PyNAST) 117 serum lipids 90
severe acute malnutrition (SAM) 56
QIIME 15, 17, 18 SgLV-EKF 29–30
QIIME 1 17–19 Shigella
QIIME 2 17, 18, 20 dysentery patients 45
Quantitative Insights into Microbial Ecology S. sonnei 245
(QIIME) analysis 12 SHIME see simulator of the human intestinal
microbial ecosystem (SHIME)
raffinose 159 short-chain fatty acids (SCFAs) 51, 76, 77, 84,
Random Forest and nonparametric testing 30 88–89, 126, 131, 144, 159, 164, 193,
RDP see ribosomal database project (RDP) 216
Red Queen dynamics 49 absorbable 4
resolvins (Rv) 87 beneficial effect of 219
respiratory quotient (RQ) 91 downstream effects of 164
restriction fragment length polymorphism G protein-coupled receptors 217
(RFLP) 7 producing bacteria 227
methods 7 production of 140
resveratrol, anti-inflammatory action of 195 Shotgun Data Profiling module 30
ribosomal database project (RDP) 13–14 Shotgun metagenomics 241
Classifier 13–14 SIBO see small intestinal bacterial
ribosomal RNA gene (16S rRNA) of the overgrowth (SIBO)
environmental microorganisms 74 signaling molecules, neurohormonal 88
ribosomal RNA sequence 13 SigTree 24, 25
RNA polymerase β subunit gene (rpoB) 6 SILVA 13
RNAseq 22 SILVAngs 13
Robinsoniella 108 similarity-based network inferring tools 29
Roseburia 86, 189, 219, 224, 229 simple carbohydrates 80–82
Roux-en-Y gastric bypass (RYGB) surgery 85 simulator of the human intestinal microbial
rRNA gene ecosystem (SHIME) 103–105
small subunits (SSUs) of 5–7 single nucleotide polymorphism (SNP) 161
FISH targeting microbial 8 3-SL 165
ruminant species 4 smacovirus 57
Ruminoccocus bromii 225 small intestinal bacterial overgrowth (SIBO) 61
Ruminococcaceae 108 small intestine
enterotype 108 chronic inflammation of 132
Ruminococcus 190, 203 dysfunction of 132
microbiota 74
saccharolytic fermentation 159 small subunits (SSUs)
Saccharomyces boulardii 137 reference databases 12
Saengshik 201 EzBioCloud 14
Salmonella 101, 165 Greengenes 12, 12–13
S. enterica 245 Protist Ribosomal Reference (PR 2) 15
S. typhi 245 ribosomal database project (RDP) 13–14
SAM see severe acute malnutrition (SAM) SILVA 13
SAMseq 23 UNITE 14–15
Sanger sequencing 6 of rRNA gene 5–7
traditional 8 FISH targeting microbial 8
saturated fats 225 16S molecules, PCR-amplified 7
saturated fatty acids 85–86 SNP see single nucleotide polymorphism (SNP)
SCFAs see short-chain fatty acids (SCFAs) Solexa 9
16S dataset 21 somatostatin 222
secondary metabolites 185 soybean protein (Spro) 89
second human genome project 92 soybean protein-derived peptides (SPre) 89
Senegal virus 74 soy-derived isoflavonoid daidzein 195
266 Index

soy isoflavones 189 transporters, monocarboxylate 88

soy milk 142 T-REX (T-R FLP analysis EX pedited) 7
SparCC 27, 28 TRFLP 7
Spearman and Pearson method 27 TRFMA 7
Spearman approach 27 triglycerides, breast milk 77
species-richness enterotype 108 trimethylamine (TMA) 131
specific pathogen-free (SPF) mice 53 trimethylamine N-oxide (TMAO) 131
SPep 90 tumor necrosis factor alpha (TNFα) 87
SPIEC-EASI network inference 28 Turicibacter 144
Sprague–Dawley (SD) rats 89, 163 type 1 diabetes 55
16S probe-based fluorescent in situ type 2 diabetes 55, 219, 245
hybridization (FISH) 6 type 2 diabetes mellitus (T2DM) 73, 90, 185
16S ribosomal RNA gene sequencing 90 tyrosine kinases 204
16S rRNA (V1–V2) amplicon sequencing 11
16S rRNA amplicon sequencing, full-length 11
UCHIME 17
5S rRNA gene 5
ulcerative colitis (UC) 53, 54, 55, 56, 137
16S rRNA gene 5, 7, 10, 11
Unifrac 17
amplicon libraries of 106
UNITE 14–15
18S rRNA gene 5
urolithin metabolites 193
23S rRNA gene 5
16S rRNA V4 region 83
ssDNA 47 vagus nerve 220
Staphylococcus 10, 143, 190 VAMPS see Visualization and Analysis of
S. aureus 10, 245 Microbial Population Structures
Statistical Testing and Differentially Abundant (VAMPS)
Taxon Identification 21 vasoactive (aromatic) amines 76
stereolithography 102 vegetable tannins 185
steroid hormones 126 Veillonella 111
Streptococcaceae 48 Verrucomicrobia 190, 214, 229
Streptococcus 25, 74, 82, 136, 140 viable cells 130
S. salivarius subsp. thermophilus 137 viable probiotic microorganisms 169
S. thermophilus 82, 139, 143 viral genomes 55–56
stunting 55 viral transplants, fecal 61
Subflava 91 virome metagenomics 56, 57
sulfation 191 viruses in COVID-19 patients 46
virus-like particles (VLPs) 56
Taxon Set Enrichment Analysis module 30 Visualization and Analysis of Microbial
T2DM risk 190 Population Structures (VAMPS) 30
temperate phages 47 vitamins 226
temperature gradient gel electrophoresis 7 Vsearch algorithm 18
Tenericutes 105
Thermophilus 136 web-based tools, for marker gene survey data
3D cell culture 102 30–33, 34
3D tissue systems 102 Weissella 145
3D printing techniques 102 Welch’s t-test 22
TLR4 see toll-like receptor 4 (TLR4) Wilcoxon rank sum test 22
TMA see trimethylamine (TMA) wood feeding 106
TMAO 132 World Health Organization (WHO) 121
TNFα see tumor necrosis factor alpha (TNFα) 5,500 W Series Genetic Analysis Systems 9
toll-like receptor 4 (TLR4) 86, 165
toxicity of hydrogen peroxide 171 yogurt 142, 229
trace minerals 226
traditional Sanger sequencing 8 zebrafish model 110–111
transcription factor 7 like 2 (TCF7L2) gene 190 zero-inflated Gaussian (ZIG) distribution mixture
trans fats 225 model 22
translocation models, fecal microbiome 111–112 zero-mode waveguides (ZMWs) 11