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Text Book on Food Microbiology

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 FOOD MICROBIOLOGY
Authored by Mrs. R. Rajeshwari Anburaj

Edited by Dr. P. F. Steffi

************************************AUTHOR NAME
Author: Mrs. R. Rajeswari Anburaj

Editor: Dr. P. F. Steffi

ISBN: 978-81-947191-6-8

E-ISBN: 978-81-947191-7-5

Publisher: Ryan Publishers

B-3, Lakshmi Pride,

80 feet road, 10th Cross West,
Thillainagar, Trichy, 620018,
Tamil Nadu, India,
Ph- +91 6374561101

Copyright © 2020 by Ryan Publishers

No part of this book shall be reproduced or transmitted in any

form or by any means (electronic or mechanical including
photocopy, recording or any information storage or retrieval
system) without the permission in writing of the publisher.

2
Preface

The food industry continues to manufacture large number of

food microbiological products. The number of food industry is
increasing day by day. The quantity and variety of food
microbiological products are increasing and novel products are
regularly introduced in the market. This book contains five
chapter.

Chapter 1 deals about various scientist involved in identifying

food borne causing microorganism and the products
manufactured using microbiological practices. Various food
borne bacteria have been addressed, when food is mishandled it
leads to production of harmful pathogen, the role of microbes in
foods were discussed. In factors affecting microbial growth
contains information on physicochemical characteristics of
organisms. Lactic acid organism inhibitory effect was discussed.
Hurdle technology influencing multiplication of microorganism
in food were depicted.

Chapter 2 contains information about microorganism present in

specific foods, followed by microbiological assessment of food
which is essential to determine its quality. Characterization of
microbes were proceeded by means of physical and chemical
methods. Whole animal assays performed using various animals
have been experimented to determine the toxins in food.
Immunological assays deals with the antigen- antibody
interaction for detection of food borne pathogens.

Chapter 3 deals that the Nutritive value of food is not changed

when it is treated with chemicals, antibiotics, radiation, Low and
3
high temperature, followed by equipments used in aseptic
packaging of foods, utilization of high electric fields to destroy
microbes present in food. Administration of food safety and its
quality is determined by FDA, HACCP and [Link] determine
standards for the product these techniques have been established.

Chapter 4 contains information about food borne microbes

which leads to spoilage and cause hazardous effects in humans
and causes, diagnosis, treatment and prevention have been
discussed, followed by foods contaminated with various toxins
and particular species responsible for the release of toxins and
their harmful effects have been recognized.

Chapter 5 comprises utilization of microbes in food and the

products developed with the aid of microbes. Prebiotics found in
food stimulate the activity of microbes, which is essential for the
gastrointestinal tract. Probiotics consist of microbicidal
substances, competing with pathogens to avoid their adhesion to
the intestinal epithelium, Genetically engineered foods
developed by means of introducing new traits to the plants by
selective breeding. Moreover instrumentation technique
containing senses of element to determine biological origin and
to detect signals produced by them have been discussed.

This textbook has doubtless benefits from the extensive

academic teaching and intense research investigation of its
authors, all of whom are familiar authorities in their fields.

4
 Acknowledgment

I would like to thank Department of Microbiology, Nadar

Sarawathi Arts and Science College, Theni for being a
motivation for creation of this book. The author expresses
her gratitude to [Link], editor of this book for design
of the cover page and their co-operation, she managed the
books progress and ensuring the highest quality at every
stage. The authors are grateful to their families for their
patience during the preparation of the manuscript. Thanks
to [Link] for his constant and immense support.

-[Link]

5
Table of Contents

1 Chapter-1: History and development of food

Microbiology ...............................................................9
1.1 Common Food borne bacteria ....................................... 12
1.2 Significance of Microorganism in foods ....................... 19
1.3 Factors affecting microbial growth ............................... 20
1.4 Lactic antagonism.......................................................... 38
1.5 Hurdle Concept.............................................................. 41
2 Chapter-2: Microorganism in foods and
methods for detection ...............................................47
2.1 Fresh Meat ..................................................................... 48
2.2 Processed Meat .............................................................. 50
2.3 Poultry Meat .................................................................. 51
2.4 Culture, Microscopic and sampling method for detecting
microbes .................................................................................... 54
2.5 Physical methods ........................................................... 61
2.6 Chemical methods ......................................................... 67
2.7 Whole-Animal Assays ................................................... 74
2.8 Immunological methods ................................................ 77
3 Chapter-3: Food Preservation using Chemicals
83
3.1 Chemicals:-.................................................................... 83
3.2 Antibiotics ..................................................................... 87

6
3.3 Radiation........................................................................ 92
3.4 Food preservation by Low Temperature...................... 101
3.5 High Temperature Methods ......................................... 104
3.6 High pressure processing pulsed electric fields ........... 112
3.7 Aseptic packaging........................................................ 115
3.8 Mano-thermo-sonication ............................................. 117
3.9 FDA ............................................................................. 120
3.10 The Hazard Analysis and Critical Control Point
(HACCP) Concept: .................................................................. 123
3.11 Indian standard Institute .............................................. 125
4 Chapter 4: Microbial Food spoilage and Food
borne disease .......................................................... 128
4.1 Staphylococcus aureus food poisoning........................ 128
4.2 [Link] ............................................................................ 131
4.3 Salmonellosis ............................................................... 138
4.4 Shigellosis.................................................................... 142
4.5 Listerial infections ....................................................... 148
4.6 Mycotoxin.................................................................... 153
4.7 Alternaria toxin ............................................................ 157
4.8 Toxigenic phytoplanktons ........................................... 160
4.9 Viruses ......................................................................... 163
5 Chapter 5: Applications of Food Microbiology
171
5.1 Classification of Molds and Molds of Industrial
Importance. .............................................................................. 171
7
5.2 Prebiotics ..................................................................... 182
5.3 Probiotics ..................................................................... 188
5.4 Genetically modified Foods ........................................ 190
5.5 Biosensors in food ....................................................... 200
6 References .........................................................210

8
1 Chapter-1: History and development
of food Microbiology

Micro-organisms play a significant role in manufacture,

storage and utilization of foods. Most food chains initiate
wherever photosynthetic organisms are able to entrap light
energy and use it to produce large molecules from carbon
dioxide, water and mineral salts forming the carbohydrates,
fats and proteins which all other living beings used to
supply energy. The foods that we consume carry microbial
associations whose composition depends upon which
organisms gain contact and how they cultivate, stay alive
and interact in the food over time. The micro-organisms
present will originate from the normal flora of the raw
material and the route of microbes is through
harvesting/slaughter. Food preservation techniques were
urbanized empirically to arrest or retard the normal method
of decomposition. The staple foods for major parts of the
humankind were the seeds – rice, sorghum, maize and
barley – which would remain for one or two seasons if
efficiently dehydrated, and it seems feasible that most
timely methods of food maintenance depended basically on
water activity reduction in the form of sun drying, storing
in concentrated sugar solutions, salting, or smoking over a
fire. It is recognized that the harms of spoilage and food
poisoning were encountered early in prescientific era. Dead
sea from the salt was employed for the conservation of
various foods. The Chinese and Greeks used salted fish in
their diet, Followed by the people in greek and romans.
Another method of food preservation that it seems that
arose during this time was the utilization of oils such as
olive and sesame.
9
In 1659, Kircher demonstrated the occurrence of
bacteria in milk;
In 1765, Spallanzani boiled beef broth for an hour
and sealed which is used to maintain sterile
conditions.
In 1780, Scheele identified lactic acid as the
principal acid in sour milk
In 1782, Swedish chemist introduced the caning
canning of vinegar.
In 1809, Parisian confectioner preserved meats in
glass bottles and kept in boiling water at various
time intervals.
In 1813, Donkin, Hall, and Gamble used SO2 as
meat preservative.
In 1837 Pasteur introduced souring of milk caused
by microbes. He used heat to destroy undesirable
organism in beer and wine.
In 1853, [Link] Appert obtained a patent for
sterilization of food by autoclaving.
In 1867, Martin advanced the theory that cheese
ripening was similar to alcoholic, lactic, and butyric
fermentations.
In 1873, Lister was first to isolate Lactococcus
lactis in pure culture
In 1890, Mechanical refrigeration for fruit storage
was begun in Chicago.
In 1896, Van Ermengem first discovered
Clostridium botulinum
In 1906, the U.S. Federal Food and Drug Act was
passed by Congress.
10
In 1907, E. Metchnikoff and co-workers isolated
yogurt bacteria namely Lactobacillus bulgaricus
In 1912, the term osmophilic was coined by Richter
to describe yeasts that grow well in an environment
of high osmotic pressure.
In 1920, the general method for calculating thermal
process was discovered by Bigelow and Esty.
In 1928, the first commercial use of controlled
atmosphere storage of apples was made in Europe
In 1937, paralytic shell fish poisoning was
discovered.
In 1938, Outbreaks of Campylobacter enteritis were
traced to milk in Illinois.
In 1954, the antibiotic nisin was patented in
England for use in certain processed cheeses to
control clostridial defects.
In 1954, the Miller Pesticide Chemicals
Amendment to the Food, Drug, and Cosmetic Act
was passed by Congress.
In 1963, the FDA approved the use of irradiation for
the preservation of bacon in U.S.
In 1965, Foodborne disease giardiasis was reported.
In 1971, the First U.S. foodborne outbreak of Vibrio
parahaemolyticus gastroenteritis occurred in
Maryland.
In 1973, the state of Oregon adopted microbial
standards for fresh and processed retail meat.
In 1981 Foodborne listeriosis outbreak was
recognized in the United States.

11
 In 1986, Bovine spongiform encephalopathy (BSE)
was first diagnosed in cattle in the United Kingdom.

1.1 Common Food borne bacteria

Bacteria and viruses are the root cause of several food

poisoning cases, due to improper food handling. Several
bacteria, in minute amounts, are not harmful to most
healthy adults since the human body is prepared to fight
them off. The problem begins when certain bacteria and
other destructive pathogens multiply and spread, which can
happen when food is mishandled. Symptoms of food
poisoning differ and extend as fast as 30 minutes to as long
as a number of days after eating foodstuff that's been
infected. The bacterial vegetation can be occupied by
Gram-positive rods and cocci unless there has been a
extremely recent infectivity of the air by an aerosol
generated caused by water, animal and human. Bacteria
have no vigorous mechanisms for becoming airborne. They
are separated on dust particles disturbed by physical
agencies, in tiny droplets of water generated by the
progression which leads to the development of an aerosol,
and on minute rafts of skin constantly shed by many
animals including humans. The most apparent mechanisms
for generating aerosols are coughing and sneezing but
many other processes generate minute droplets of water.

Acinetobacter: These Gram-negative rods show some

resemblance to the family Neisseriaceae, and some that
were formerly Achromobacters and Moraxellae are placed
here. Also, some previous Acinetobacters are now in the
genus Psychrobacter. They are strict aerobes that do not
decrease nitrates. Although rod-shaped cells are produced
in juvenile cultures, mature cultures have many coccoid-
12
shaped cells. They are extensively dispersed in soil and
water and may be established on many foods, especially
refrigerated fresh products. It has been proposed, based on
DNA–rRNA hybridization data, that the genera
Acinetobacter, Moraxella, and Psychrobacter be positioned
in a new family (Moraxellaceae), but this proposal has not
been approved.

Bacillus: These are Gram-positive spore-forming rods that

are aerobes in distinction to the clostridia, which are
anaerobes. Although most are mesophiles, psychrotrophs
and thermophiles exist. The genus is comprised of only two
pathogens: B. anthracis (cause of anthrax) and B. cereus.
Although the majority strains of the second one are
nonpathogens, some is the root cause of foodborne
gastroenteritis. This genus has been enclosed by the
relocation of a number of its former species to new genera:
Alicyclobacillus, Brevibacillus, Paenibacillus and
Salibacillus.

Brochothrix: These Gram-positive nonspore forming rods

are closely related to the genera Lactobacillus and Listeria,
Even though they are not exact coryneforms, they abide
resemblance to this assemblage. Characteristically,
exponential-phase cells are rods, and adult cells are
coccoids, an attribute representative of coryneforms. Their
detached taxonomic status has been reaffirmed by rRNA
data, even though merely two species are recognized: B.
thermosphacta and B. campestris. They distribute a number
of features with the genus Microbacterium. They are
widespread on preserved meats that are preserved in gas-
impermeable parcels at frozen temperatures. In distinction
to B. thermosphacta, is rhamnose and hippurate positive.
They do not grow at 37°C.

13
Clostridium perfringens: Clostridium perfringens is
widespread in our surroundings. It can reproduce very
quickly under perfect conditions. Mostly, Infants, young
children and older adults are in threat. Illness habitually
occurs by eating foods infected with huge numbers of this
bacterium that fabricate enough toxin to cause illness in the
form of abdominal cramping and diarrhea. C. perfringens is
occasionally known as the "buffet germ" since it grows
most in bulk portions of food, such as gravies, casseroles,
stews and at room temperature in the danger zone. Live
bacterial contamination is due to uncooked food and cause
illness. Cook food carefully and maintain it out of the
danger zone, above a temperature of 140°F or below 40°F.
Practice leftover protection by dividing roasts and stews
into smaller quantities for faster cooling and refrigerate
right away. Before serving foods can be preserved at 165°F
or high.

Campylobacter: Campylobacter is a frequent reason for

diarrhea. In majority of Campylobacteriosis, the illness
caused by Campylobacter bacteria, are associated with
eating undercooked poultry and meat or from cross-
infection of other foods by these substance. Proper heating
of food is the key factor because freezing reduces the
quantity of Campylobacter bacteria on raw meat but will not
devastate them utterly. Campylobacteriosis occurs more
commonly in the summer and is most frequent in infants
and young children. Sources include consuming uncooked
poultry and other meats, unpasteurized dairy products and
untreated water or contaminated products. Cook all foods
thoroughly to their suitable internal temperatures, prevent
cross contamination by using separate cutting boards when
managing raw and cooked foods, don't drink unpasteurized
milk or untreated water and wash hands frequently. Proper
washing of food materials is an important requirement.
14
E. coli O157:H7: Escherichia coli are a large assemblage of
bacteria. Even though the major strains of E. coli are
nontoxic, a few can make you very sick. One strain, E. Coli
O157:H7 (STEC) is generally related with food poisoning
outbreaks as its effects can be tremendously severe. These
include eating raw or drinking unpasteurized beverages.
Proper washing of foods and maintenance of internal
temperatures; avoid unpasteurized dairy products, juices.
Also, don't consume water when playing or swimming in
lakes, ponds, streams or pools.

Enterococcus: This genus contains some of the Lancefield

serologic group D cocci. It has been extended to more than
16 species of Gram-positive ovoid cells that occur singly,
in pairs, or in short chains. They were once in the genus
Streptococcus. The genus is characterized more thoroughly,
and its phylogenetic relationship to other lactic acid
bacteria.

Flavobacterium: These Gram-negative rods are

characterized by their creation of yellow to red pigments on
agar and by their relationship with plants. A few are
mesotrophs, and others are psychrotrophs, contribute to the
spoilage of refrigerated meats and vegetables. A number of
the former flavobacterial species have been classified in the
subsequent five new genera: Empedobacter,
Chryseobacterium, Myroides, Sphingomonas, and
Sphingobacterium.

Micrococcus: These Gram-positive and catalase-positive

cocci are inhabitants of mammalian skin and can grow in
the existence of high levels of NaCl. This genus has the
following genera: Dermacoccus, Kytococcus and
Stomatococcus. At the current time, M. luteus and M. lylae
15
are the merely two micrococcal species.

Pseudomonas: These are distinctive soil and water bacteria

and they are extensively spread among fresh foods,
particularly in vegetables, meats, poultry, and seafood
products. 13 new genera are present, which includes
Acidovorax, Aminobacter, Brevundimonas, Burkholderia,
Comamonas, Delftia, Devosia, Herbaspirillium,
Hydrogenophaga, Marinobacter, Ralstonia,
Sphingomonas, Telluria, and Wautersia. P. fluorescens and
P. aeruginosa remain in the original Genus.
Salmonella: Salmonella is the name of an assemblage of
bacteria that causes the infection salmonellosis. It is one of
the most universal bacterial causes of diarrhea and the most
common source of foodborne-related hospitalizations and
deaths. Salmonella is more stern in those with a weakened
immune system. Because Salmonella bacteria can survive in
the intestinal tract of humans and other animals, it can
broaden easily unless you use suitable sanitation and
appropriate cooking methods.

Staphylococcus aureus: Staphylococcus aureus (Staph) is

normally found on the skin, throats and nostrils of healthy
people and animals. Therefore, it usually doesn't cause
sickness unless it is transmitted to food products where it
can reproduce and generate detrimental toxins.
Staphylococcal symptoms comprise nausea, stomach
cramps, diarrhea. Staphylococcal bacteria are heat resistant,
but in some cases they can be damaged by cooking. Staph
infection can affect anyone but some groups of people are
at greater risk, including people with constant conditions
such as diabetes, vascular disease, cancer, and lung disease.

Norovirus: Norovirus is one of the most important causes of

food poisoning and frequently results in symptoms
16
comparable to stomach flu for instance stomach cramping,
vomiting and diarrhea. Norovirus spreads by contact with
someone who is contaminated, particularly in swarming
areas. Food materials can become infected with the
norovirus. The infection can be particularly severe for
young children and older adults. You can get contaminated
with norovirus several times in your life.

Toxoplasma gondii: Toxoplasma leads to toxoplasmosis — a

sickness that leads to severe health problems in persons
who are affected by food poisoning, New born babies,
older adults pregnant women and people with weakened
immune systems. Symptoms can be comparable to flu and
comprise swollen lymph glands or trouble that remains for
months. Other symptoms affect the eyes, leading vision to
be reduced or blurred or cause sting, redness or tearing.

Fungi

Aspergillus: Chains of conidia are produced. Cleistothecia

with ascospores are formed; the ideal phase of those
originated in foods is Emericella, Eurotium, or
Neosartorya. Eurotium (the former A. glaucus) E.
herbariorum has been found to cause spoilage of grape
jams and jellies. Emericella forms white cleistothecia, and
E. nidulans is the teleomorph of Aspergillus nidulans.
Neosartorya turn out into white cleistothecia and colorless
ascospores. N. fischeri is heat resistance is alike to those of
Byssochlamys. The aspergilli appear yellow to green to
black on a bulk number of foods. They are originated in
hams and bacon. Several species are the source of spoilage
of oils, such as palm, peanut, and corn. A. oryzae and A.
soyae are implicated in the shogu fermentation which was
previously known as koji. A. glaucus forms katsuobushi, a
17
fermented fish product. The A. glaucus–A. restrictus group
comprises storage fungi that attack seeds and soybeans. A.
niger synthesise β-galactosidase, glucoamylase, invertase,
lipase, and pectinase and A. oryzae produces α-amylase.
Several species produce aflatoxins, and others produce
ochratoxin A and sterigmatocystin.

Botrytis: Long, slender, and regularly pigmented

conidiophores are formed. B. cinerea is the mostly
widespread in foods. They are prominent as the cause of
gray mold rot of apples, pears, raspberries, strawberries,
grapes, blueberries, citrus, and some stone fruits.

Cladosporium: Septate hyphae with dark, tree-like,

budding conidia variously branched, characterize this
genus. In culture, development is olive colored to black. C.
herbarum forms “black spot” on beef and freezing mutton.
Some spoil butter is classified as rot of stone fruits and
black rot of grapes. They develop on wheat grains and
barley. C. herbarum and C. cladosporiodes originates on
food materials.

Geotrichum (once known as Oidium lactis and Oospora

lactis): These yeast-like fungi are usually white. The
hyphae are septate, and reproduction occurs by
configuration of arthroconidia from vegetative hyphae.
They have flattened ends. G. candidum, the anamorph of
Dipodascus geotrichum, is the majority of vital species in
food products. It is referred to as “dairy mold” as it imparts
flavor and fragrance to various kind of cheese and as
“machinery mold” as it builds up on food contact utensils,
chiefly in tomato canning plants. It is the reason for sour rot
of citrus fruits and the putrefaction of dairy cream. It is
extensively spread and has been found on meats and many
vegetables. Some participate in the fermentation of gari.
18
Penicillium: When conidiophores and conidia are the only
mature structures present, this genus belongs to
Deuteromycota. Of the two teleomorphic genera,
Talaromyces is significant in foods. T. flavus is the
teleomorph of P. dangeardii, and it has been concerned in
the spoilage of fruit juice concentrates. It forms heat-
resistant spores. Conidia have the capacity to form
phialides. They are coloured from blue to blue-green. Rots
are found in several species of citrus fruits and blue mold
rot of apples, pears are also found. One species, P.
roqueforti, produces blue cheese. A number of species
produce citrinin, ochratoxin A, and other mycotoxins.

1.2 Significance of Microorganism in foods

Microorganisms have been used in the production of

foods for thousands of years. They are used for the purpose
of baking, brewing, pickiling and wine making. Buttermilk
is made as the result of souring of low fat milk, the unique
flavor comes from substances such as diacetyl and
acetaldehyde. They are formed by different species of
Streptococcus, leuconostoc and lactobacillus as they grow.
Food industry uses Lactobacillus and Bifidobacterium for
their production techniques.
Fungi play a major role in food production. The fungus
which is used for making are yeast. Molds need free
oxygen for development and hence cultivated on the
surface of contaminated food. Molds are used in food
industry and for making food. This yeast ferments the
carbohydrates in the dough to produce carbondioxide and
then small amount of ethanol and alcohol are produced.
The carbon dioxide produced causes the dough to rise when
the bubbles become trapped in dough and creates soft
texture. Various types of microorganismare are used in the
19
production of cheese.
Yeast is also used in manufacture of beer and alcoholic
beverages. A species of Bothrytiscinerea, is used in rotting
of grape for manufacture of wine. Saccharomyces
carlsbergensis is most frequently used in fermentation of
most beers. The yeast strains which are significant in
production include Brettanomyces, Candida, Debaryomyces,
Saccharomyces. Other foods that are made include olives,
coffee, soysauce, meat products such as salami and certain
food additives such as monosodium glutamate and citric
acid.

1.3 Factors affecting microbial growth

The plants and animals develop resistance against the
invasion and proliferation of microorganisms. The factors
that affecting microbial growth in foods, and therefore the
associations that develop, also decide the nature of spoilage
and any healthiness risks posed. Physico-chemical
characters of the food (intrinsic factors); situation of the
storage environment (extrinsic factors); properties and
connections of the microorganisms present (implicit
factors); and processing factors. The factors of plant and
animal tissues that are an innate part of the tissues are
known as intrinsic parameters. Various factors influencing
intrinsic parameters include:

Moisture content
Nutrient content
pH
Oxidation-reduction potential
Antimicrobial substances

20
Moisture Content

Life is totally reliant on the occurrence of water in its

liquid state. The reactions which are found in the cytoplasm
and in an aqueous atmosphere and the cytoplasm are
surrounded by a membrane which is usually permeable to
water molecules which travels from the cytoplasm to the
environment and vice versa. The conservation of foods by
drying is a direct result of elimination or binding of
moisture, without which microorganisms do not grow. It is
now generally acknowledged that the water equipment of
microorganisms is expressed in terms of the water activity
(aw) in the surroundings. This factor is distinct by the ratio
of the water vapor pressure of food substrate to the vapor
pressure of pure water at the same temperature: aw = p/po,
where p is the vapor pressure of the solution and po is the
vapor pressure of the solvent. This idea is related to relative
humidity (RH) in the following way: RH = 100 × aw.
In general, bacteria need maximum values of aw for
development than fungi, with Gram-negative bacteria
having superior requirements than Gram positives.
Spoilage molds can produce as little as 0.80 whereas
spoilage bacteria do not grow below aw = 0.91. The lowest
reported assessment for foodborne bacteria is 0.75 for
halophiles (literally, “salt-loving”), whereas xerophilic
molds and osmophilic yeast have been reported.
First, at any temperature, the capacity of
microorganisms to grow is reduced as the aw is lowered.
Second, the range of aw over which development occurs is
maximum at the optimum temperature for growth; and
third, the existence of nutrients increases the range of aw
over which the organisms can survive. The specific
standards should be considered only as reference points, as
a change in warmth or nutrient substance might permit
development at lower values of aw.
21
Effects of low aw

The common effect of lowering aw below optimum is to

enlarge the length of the lag phase of growth and to reduce
the growth rate and amount of final population. This result
may be expected to effect from adverse influences of
lowered water on all metabolic activities as all chemical
reactions of cells need an aqueous environment. In their
study of the effect of aw on the growth of Enterobacter
aerogenes in culture media, Wodzinski and Frazier
established that the lag phase and generation time were
increasingly lengthened until no development occurred
with a lowering of aw. When both the pH and temperature
of incubation were made critical, the minimum aw for
growth was higher. Although some Gram-positive bacteria
accumulate proline, its level is intense due to concentrated
and higher levels by Gram-negative bacteria. The three
transporter systems in E. coli and S. typhimurium are PutP,
ProP, and ProU, with ProP being the mainly efficient. The
sigma factor-B plays a major role in the regulation of
carnitine utilization in L. monocytogenes.
With regard to definite compounds used to lower water
activity, results akin to those seen with adsorption and
desorption systems have been analysed. In an analysis on
the least aw for the development and germination of
Clostridium perfringens, in another statement, glycerol was
found to be more inhibitory than NaCl to comparatively
salt-tolerant bacteria, but least inhibitory than NaCl to salt-
sensitive species when contrast to similar levels of aw in
complex media. The germination of clostridial spores was
entirely inhibited at aw = 0.95 with NaCl. The management
of aw with glycerol permitted catabolism to maintain aw
values below that for growth on glucose. NaCl was used by
this investigator to adjust the aw, substrate catabolism
22
ceased at an aw better than the minimum for growth,
whereas glycerol allowed catabolism at lower aw values
than the minimum for growth.

Osmophilic yeasts build up polyhydric alcohols to a

concentration commensurate with their extracellular aw.
According to Pitt, the xerophilic fungi gather well-matched
solutes or osmoregulators as an effect of the requirement
for high internal solutes if growth at a low aw is to be
possible. In a relative study of xerotolerant and
nonxerotolerant yeasts to water stress. A nontolerant S.
cerevisiae reacted to lessening of aw by synthesizing
additional glycerol however retaining less. It became
visible from this study that a low aw forces S. cerevisiae to
deflect an advanced quantity of its metabolic activity to
glycerol fabrication accompanied to amplify the quantity of
glucose consumed during development. It became visible
from this examination that a low aw forces S. cerevisiae to
deflect a larger amount of its metabolic action to glycerol
production accompanied to intensify the quantity of
glucose consumed during growth. It is documented that the
growth of at least some cells may happen in high numbers
at declined aw values. For instance, condensed aw results in
the termination of enterotoxin B production by S. aureus
although high numbers of cells are formed at the same time
In addition to the consequence on nutrients, a lowered
aw certainly has adverse effects on the functioning of the
cell membrane, which must be maintained in a fluid state.
The stability of water between cells and substrate occurs
and the drying of internal parts of cells would be expected
to occur upon placing cells in a medium of lowered aw to a
point. Every microbial cells may need the similar efficient
internal aw. Those that can grow under intense conditions
of a low aw apparently do so by virtue of their capability to
concentrate salts, polyols and amino acids (and possibly
23
other types of compounds) to internal levels sufficient not
only to avoid the cells from losing water, but that it may
permit the cell to extract water from the water-depressed
external environment.

Nutrient content

Micro-organisms can utilize foods as a resource of nutrients

and energy. As sources of energy, foodborne
microorganisms may use sugars, alcohols, and amino acids.
Some few microorganisms are able to employ mixture of
carbohydrates such as starches and cellulose as sources of
power by initially degrading these compounds to simple
sugars. From them, they obtain the chemical elements that
encompass microbial biomass, those molecules vital for
developments are used as an energy source. The
widespread use of food products such as meat or casein
digests (peptone and tryptone) is suitable for this purpose.
The major nitrogen sources employed by heterotrophic
microorganisms are amino acids. A huge number of other
nitrogenous compounds may supply this function for
various types of organisms. Some microbes, for example,
are able to make use of nucleotides and free amino acids,
whereas others are able to use peptides and proteins. The
inability of microbes to make use of a main component of a
food material will limit its development and put it at a
competitive disadvantage. Thus, the capacity to synthesize
amylolytic (starch degrading) enzymes will favour the
increase of an organism on cereals and other farinaceous
products. The accumulation of fruits comprising of sucrose
and other sugars to yoghurt amplify the variety of
carbohydrates. Microorganisms may need B vitamins in
low quantities, and almost all natural foods tend to have a
rich quantity for those organisms that are unable to produce
their essential requirements. Normally, gram-positive
24
bacteria are the less synthetic and must therefore be entire
with one or more of these compounds before they will
grow. The gram-negative bacteria and molds are capable to
produce majority of their requirements.
The absorption of key nutrients can, to some extent,
decide the speed of microbial growth. Monod equation, is
mathematically identical to the Michaelis– Menten
equation of enzyme kinetics, reflecting the reliance of
microbial growth on rate-limiting enzyme reactions:
µ ̳ µmS
S+Ks
Where µ is the precise multiplication rate; µm the
greatest exact development rate; S the concentration of
preventive nutrient; and in Ks the saturation is steady.
When S ˃˃Ks, a micro-organism will develop at a speed
approaching its maximum, but as S falls to values
approaching Ks, and the development rate. Values for Ks
have been calculated for a range of organisms and
nutrients; usually they are particularly low, of the order of
10-5M for carbon and energy sources, suggesting that in the
majority of cases, lack of nutrient is unlikely to be rate-
limiting. Local microenvironments may be deficient in vital
nutrients, or where nutrient constraint is used as a defence
against microbial disease, for instance the white part in the
hen’s egg.

pH

As calculated with the glass electrode, pH is equivalent to

the negative logarithm of the hydrogen ion action. The
action is proportional to concentration, the activity
coefficient.
pH ̳ -- log(aH) = log 1/(aH) ~log 1/[H+]
where (aH) is the hydrogen ion movement and [H+] the
hydrogen ion absorption.
25
 For water solution, pH 7 match to neutrality (since
[H+][OH_]=10-14 for water), pH values below 7 are acidic
and those above 7 specify an alkaline surroundings. As pH
is a logarithmic scale differences in pH of 1, 2 and 3 units
correspond to 10-, 100- and 1000-fold variation in the
hydrogen ion concentration
The acidity or alkalinity of surroundings has a profound
consequence on the activity and constancy of
macromolecules such as enzymes, progress and metabolism
of microorganism are affected by pH. In general, bacteria
nurture fastest in the pH range 6.0–8.0, yeasts 4.5–6.0 and
filamentous fungi 3.5–4.0. As with all generalizations there
are exceptions, mainly among those bacteria that produce
amount of acids as a result of their energy-yielding
metabolism. Pseudomonads produces soft rods have
function in spoilage of foods.
As a rule, fish spoil is faster than meat under chill
circumstances. Shewanella is involved in fish spoilage.
With the exemption of those soft drinks that contain
phosphoric acid, most foods owe their acidity to the
survival of weak organic acids. These do not divide
completely into protons and conjugate base in solution but
set up equilibrium:
HA→ H+ + A-
The equilibrium constant for this process, Ka, is given
by
Ka = H+[A]_A-
[HA]
where [ ] denotes concentration. This expression can be
rearranged:
1 = 1 [A-]
H+ Ka [HA]
If we take logarithms to the base 10 we get:
pH= pKa +log [A-]
[HA]
26
 Equation is known as the Henderson–Hasselbalch
equation and describes the relationship between the pH of a
solution, the strength of the acid and its degree of
dissociation. Table: 1 Depicts a list of some general food-
associated acids and their pKa values. This partial
dissociation of weak acids, such as acetic acid, is involved
as a major part in their competence to inhibit microbial
growth. It is well recognizable that, although accumulation
of strong acids has a new effect on pH pro rata.

Table: 1 pKa values of some common food acids

1. Acid pKa

2. Acetic 4.75

3. Lactic 3.86

4. Citric 3.14,4.77,6.39

5. Benzoic 4.19

6. Carbonic 6.37

7. Nitrous 3.37

Many necessary cell functions such as ATP synthesis in

bacteria, active transport of nutrients and cytoplasmic
regulation happen at the cell membrane and are reliant on
potential force stored in the membrane by means of a
proton motive force. This is an electrochemical potential
formed by the active translocation of protons from the cell
27
interior to the external environment. At this higher pH, the
equilibrium shifts in favor of the dissociated molecule, so
the acid ionizes producing protons which will acidify the
cytoplasm and break down the pH constituent of the proton
motive force. The cell will attempt to maintain its internal
pH by neutralizing or expelling the protons leaking in but
these will slower the development as it diverts energy from
growth-related functions.

Oxidation reduction Potential

Microorganisms exhibit unstable degrees of sensitivity to

the oxidation-reduction potential (O/R, Eh) of their growth
medium. The O/R potential of a substrate is known usually
as the ease with which the substrate loses or gains
electrons.
Cu Oxidation Cu + e
Reduction
Oxidation may also be attained by the addition of
oxygen, as represented in the following reaction:
2 Cu + O2 -» 2CuO
Therefore, a substance that willingly gives up electrons
is a superior reducing agent, and one that readily takes up
electrons is a high-quality oxidizing agent. This
dissimilarity may be measured by use of an appropriate
instrument, and expressed as millivolts (mV). The more
extremely oxidized a substance is the more positive will be
its electrical potential; the more highly condensed a
substance is, the more negative will be its electrical
potential.

The O/R potential is indicated by the symbol Eh.

Aerobic microorganisms necessitate positive Eh

28
 values (oxidized) for progress, whereas anaerobes
need negative Eh values (reduced)
The characteristic O/R possibility of the innovative
foodstuff
The poising ability; that is, the conflict to alteration
in the potential of the food
The oxygen tension of the environment about the
foodstuff
The contact that the atmosphere has to the food

With respect to Eh supplies of microorganisms, some

bacteria need reduced circumstances for growth initiation
(Eh of about -200 mV), whereas others require a positive
Eh intended for growth. In the former category are the
anaerobic bacteria such as the genus Clostridium; in the
latter belong aerobic bacteria such as some members of the
genus Bacillus. Examples of microaerophilic bacteria are
lactobacilli and campylobacters. Some bacteria have the
ability to grow under either aerobic or anaerobic
conditions. Such types are referred to as Facultative
anaerobes.
With regard to the Eh of foods, plant foods, especially
plant juices, tend to have Eh values of from 300 to 400.
With respect to the Eh of pre rigor as complementary to
post rigor muscles, Barnes and Ingram undertook a study of
the quantity of Eh in muscle over periods of up to 30 hours
postmortem and its effect on the growth of anaerobic
bacteria. These authors establish that the Eh of the
sternocephalicus muscle of the horse immediately after
death was 250 mV, at which time clostridia failed to
multiply. The authors confirmed for horse meat the finding
for whale meat: those anaerobic bacteria do not increase
until the onset of rigor mortis because of the high Eh in pre
29
rigor meat.

Eh effects

Microorganisms influence the Eh of their atmosphere

during development just as they do pH. This is accurate
especially for aerobes, which can inferior the Eh of their
environment while anaerobes cannot. As aerobes grow, O2
in the medium is exhausted, resulting in the lowering of Eh.
Growth is not slower, though, as much as might be
conventional due to the ability of cells to utilize O2-
donating or hydrogen-accepting substances in the medium.
The consequence is that the medium becomes poorer in
oxidizing and richer in reducing substances.
Eh is reliant on the pH of the substrate, and the direct
correlation between these two factors is the rH value
distinct in the subsequent way:
Eh = 2.303 RT (rH - 2 pH)
F
where R= 8.315 joules, F = 96,500 coulombs, and T is
the absolute temperature.53 Therefore, the pH of a
substrate should be declared when Eh is known. Normally
Eh is taken at pH 7.0 (expressed Eh'). When taken at pH
7.0, 25°C, and with all concentrations at 1.0M, Eh = Eh'
(simplified Nernst equation). In nature, Eh tends to be more
negative under progressively alkaline conditions.
While the development of anaerobes is usually believed
to happen at reduced values of Eh, the exclusion of O2 may
be essential for some anaerobes. When Clostridium
perfringens and Peptococcus magnus were cultivated in the
existence of O2, inhibition of development occurred even
when the medium was at a negative Eh of -50 mV.
Researchers found that development occurred in media
with an elevated Eh level as 325 mV when no O2 was
present. With regard to the consequence of Eh on lipid
30
production by Saccharomyces cerevisiae, it has been
revealed that anaerobically grown cells generate a lower
total level, an extremely changeable glyceride fraction, and
reduce phospholipid and sterol mechanism as compared to
aerobically grown cells. The lipid formed by anaerobically
grown cells was distinguished by a high content (up to 50%
of total acid) of 8:0 to 14:0 acids and a small level of
unsaturated fatty acid in the phospholipid fraction. In
aerobically grown cells, 80-90% of the fatty acid ingredient
was linked with glyceride, and the phospholipid was found
to be 16:1 and 18:1 acids. Unlike aerobically mature cells,
anaerobically developed S. cerevisiae cells were found to
have a lipid and sterol requirement.

Antimicrobial Substances

All foods were at some stage part of living organisms

and have been organized through the course of
development with ways in which possible damaging
microbial infections might be prevented or limited.
The first of these is the integument: a physical hurdle to
infection such as the skin, shell, husk or rind of a product.
Physical damage to the integument allows microbial attack
of the essential nutrient-rich tissues and it is a regularly
observed that damaged fruits and vegetables decline more
quickly than complete products, and that this progression is
initiated at the site of injury. As a result it is vital to the
farmer and food processor that harvesting and transport
continue these barriers as far as possible.
As a second line of defense, the product tissues may
have antimicrobial mechanism, the local concentration of
which frequently increases as a consequence of physical
damage. This occurs in plants such as mustard, horseradish,
watercress, cabbage and other brassicas to produce
antimicrobial isothiocyanates (mustard oils) and in Allium
31
species (garlic, onions and leeks) to produce thiosulfinates
such as allicin.
Many normal constituents of plant tissues such as
pigments, alkaloids and resins have antimicrobial
properties, but partial convenient is made use of these.
Benzoic and sorbic acids found in cranberries and mountain
ash berries in this order, they are notable exceptions that
are used in their pure forms as food preservatives.
Considerable attention has been intended for the
microbicidal properties of those plants used as herbs and
spices to flavour food identified compounds such as
eugenol from allspice (pimento), allicin in garlic, thymol
from thyme and oregano, cloves and cinnamon and
cinnamic aldehyde from cinnamon and cassia which have
considerable antimicrobial activity. For instance it has been
claimed that addition of cinnamon in raisin bread retards
mould spoilage.
Antimicrobial components varies in their spectrum of
activity and potency, they are present at varying
concentrations in the natural product, and are usually at
levels too little to have any consequence. Hops and their
extracts are ingredients to be always added in beer.
Humulones contained in the hop resin and isomers formed
during processing, impart the characteristic bitterness of the
product but have also been shown to own action against the
common beer spoilage organisms, lactic acid bacteria.
When first introduced into brewing, hops possibly
contributed to microbiological stability, but this is less
likely nowadays with the comparatively low hopping rates
used. In fact the capacity of lactic acid bacteria to attain
resistance to hop resins means that the brewery
environment possibly acts as a very resourceful normal
enhancement culture for humulone - tolerant bacteria, thus
negating any beneficial effects.
Animal products too, have a variety of non-specific
32
antimicrobial constituents. Possibly the absolute illustration
of this is the white or albumen of the hen’s egg which
possesses a whole battery of inhibitory components. Both
products contain the enzyme lysozyme which catalyses the
hydrolysis of glycosidic linkages in peptidoglycan, the
structural polymer responsible for the potency and firmness
of the bacterial cell wall. Destruction or weakening of this
layer causes the cell to rupture (lyse) under osmotic
pressure. Lysozyme is most dynamic against Gram-positive
bacteria, where the peptidoglycan is more readily available,
but it can also kill Gram-negatives if their protective outer
membrane is injured in some way.
Ovotransferrin in egg white and lactoferrin in milk are
proteins that scavenge iron from the medium. Iron is an
essential nutrient for all bacteria and many have evolved
means of overcoming iron limitation by forming their own
iron-binding compounds known as siderophores. In
addition, egg white has powerful cofactor-binding proteins
such as avidin and ovoflavoprotein which sequester biotin
and riboflavin control the development of those bacteria for
which they are vital nutrients.
Milk also has the ability to generate antimicrobials in the
occurrence of hydrogen peroxide. The enzyme
lactoperoxidase comprise about 0.5% of whey proteins and
catalyses the oxidation of thiocyanate by hydrogen
peroxide. Thiocyanate is evidently present in milk and its
level can be boosted by consumption of brassicas which are
rich in thiocyanate precursors. Hydrogen peroxide can be
produced by endogenous enzyme activity or by the aerobic
metabolism of lactic acid bacteria. The reaction forms short
lived oxidation products such as hypothiocyanate which
can kill Gram negative bacteria and inhibit Gram positives,
possibly by destructing the bacterial cytoplasmic
membrane.
Various factors in extrinsic parameters include
33
 • Relative humidity
• Gaseous atmosphere

Relative humidity

Relative humidity is used to quantify the water activity

of the gas phase. When food commodities having a little
water activity are accumulated in an atmosphere of high
relative humidity water will transport from the gas phase to
the food. Condensation may occur on surfaces giving rise
to localized regions of high water activity. Once micro-
organisms have started to produce and become
physiologically active they usually generate water as an end
product of respiration. Thus they increase the water
movement of their own immediate environment so that
ultimately micro-organisms requiring a high aw are able to
cultivate and destroy a food which was initially measured
to be microbiologically constant.
Such a circumstances can take place in grain silos or in
tanks in which concentrates and syrups are stored. Another
trouble in large-scale storage units such as grain silos occur
since the relative humidity of air is very susceptible to
temperature. If one side of a silo heats up during the day
due to contact to the sun then the relative humidity on that
side is reduced and there is a net migration of water
molecules from the colder side to equilibrate the relative
humidity. The temporary amplification in relative humidity
may be enough to cause local condensation onto the grain
with a localized raise in aw sufficient to allow germination
of fungal spores and consecutively spoilage of the grain.
The storage of fresh fruit and vegetables need
cautiousness due to relative humidity. If it is too low then
many vegetables will lose water and become flaccid. If it is
too elevated then condensation may take place and
34
microbial spoilage may be initiated.
Microbial growth can arise over a temperature varying
from about -8 °C up to 100°C at atmospheric pressure. The
most vital requirement is that water should be present in the
liquid state and thus accessible to support growth. No
single organism is able for growth over the whole of this
range; bacteria are usually restricted to a temperature span
of around 35°C and moulds relatively less, about 30°C.
Each organism exhibits at least, optimum and maximum
temperature at which growth can take place. They are
called as cardinal temperatures and are, to a huge extent,
characteristic of an organism, although they are prejudiced
by other environmental feature such as nutrient availability,
pH and aw. Micro-organisms can be categorized into
numerous physiological assembly based on their cardinal
temperatures. It is functional, if rather arbitrary,
convention, since the allocation of micro-organisms growth
temperature assortment is continuous throughout the
assortment. It is moreover appropriate to identify cardinal
temperatures as ranges more willingly than single values.
In food microbiology mesophilic and psychrotrophic
organisms are generally of greatest implication.
Mesophiles, with temperature optima around 37°C, are
normally of human or animal origin and include many of
the frequent foodborne pathogens such as Salmonella,
Staphylococcus aureus and Clostridium perfringens. As a
rule mesophiles grow more rapidly at their optima than
psychrotrophs and so destroy of perishable foodstuffs
stored in the mesophilic development range is more rapid
than spoilage under cool conditions. Because of the
different groups of organisms implicated, it can also be
diverse in character. Among the organisms capable in
growth at low temperatures, two grouping can be eminent:
the strict psychrophiles (‘cold loving’) have optimum
temperature of 12–15°C and will not grow above about
35
20°C. As a consequence of this sensitivity to quite
moderate temperatures, psychrophiles are largely confined
to polar regions and the marine environment. This patience
of a wider range of temperatures means that psychrotrophs
are found in a more varied assortment of habitats and as a
result are of better significance in the spoilage of chilled
foods.
As the temperature is reduced from the optimum the
development rate slows, partially as a result of the slow
enzymic reactions within the cell. The modification in
growth rate with temperature below the optimum might be
predicted to follow the Arrhenius Law which explains the
relationship between the rate of a chemical reaction and the
temperature. Microbial growth results from the activity of a
network of interacting and interregulating reactions and
describes a far higher order of complexity than simple
individual reactions.
A most essential contribution to the slow and eventual
cessation of microbial growth at low temperatures is now
careful to be changes in membrane structure that influence
the uptake and deliverance of nutrients to enzyme systems
within the cell. It has been revealed that many
microorganisms react to growth at lower temperatures by
rising the proportion of unsaturated and/or shorter chain
fatty acids in their membranes and that psychrotrophs
generally have superior levels of these acids than others.
Rising the degree of unsaturation or declining the carbon
chain length of a fatty acid reduces its melting point so that
membranes include these will remain fluid and hence
efficient at lower temperatures.
As the temperature amplify above the optimum, the
growth rate decrease much more sharply according to the
irreversible denaturation of proteins and the thermal
breakdown of the cell’s plasma membrane. At temperatures
above the maximum for growth, these changes are adequate
36
to kill the organism – the rate at which this take place with
rising temperature.

Gaseous atmosphere

Oxygen consist of 21% of the earth’s atmosphere and is

the the majority of vital gas in contact with foodstuff under
normal situation. Its existence and its control on redox
potential are significant determinants of the microbial
associations that expand and their rate of growth.
Carbon dioxide is not consistent in its effect on micro-
organisms. Moulds and oxidative Gram-negative bacteria
are main susceptible and the Gram-positive bacteria,
predominantly the lactobacilli, tend to be most. Some yeast
such as Brettanomyces spp. also show substantial tolerance
of high CO2 levels and govern the spoilage microflora of
carbonated beverages. Growth inhibition is usually superior
under aerobic circumstances than anaerobic and the
inhibitory effect amplify with decrease of temperature,
most probably due to the amplified solubility of CO2 at
lower temperatures. Some micro-organisms are killed by
extended contact to CO2 but frequently its result is
bacteriostatic.
The mechanism of CO2 inhibition is a mixture of several
processes whose precise individual contributions are so far
to be determined. One feature often recognized is the
consequence of CO2 on pH. Carbon dioxide dissolves in
water to generate carbonic acid which partly dissociates
into bicarbonate anions and protons. Probably of more
importance than its effect on the developing medium is the
ability of CO2 to act in the identical way as weak organic
acids, piercing the plasma membrane and acidifying the
cell’s interior.
Other causative factors are thought to comprise changes
in the physical properties of the plasma membrane
37
harmfully disturbing solute transport; inhibition of key
enzymes, mainly those involving carboxylation
/decarboxylation response in which CO2 is a reactant; and
reaction with protein amino groups which is the reason for
modification in their properties and action.

1.4 Lactic antagonism

The occurrence of a lactic acid bacterium inhibiting or
killing closely related and food-poisoning and food
spoilage organisms when in mixed culture has been
observed for more than 80 years known as lactic
antagonism. Among factors recognized are the production
of antibiotics, H2O2, diacetyl, and bacteriocins in addition
to pH depression and nutrient depletion. Lactic antagonism,
thus, is an example of microbial interference. In one study,
a pediocin-producing strain of Lactococcus lactis was
genetically enhanced to produce enough pediocin to control
growth of L. monocytogenes. In control cheese, the
pathogen increased to about 107/g after 2 weeks and then
reduce to about 103 after 6 months but in the investigational
cheese, the pathogen decline to 102/g within 1 week and
then to only 10/g within 3 months. Propionibacterium
freudenreichii sub sp. shermanii produces an ill-defined,
multicomponent inhibitory system when cultivated in
pasteurized skim milk that is efficient against Gram-
negative bacteria and molds in cottage cheese, for example
Microgard. Reuterin (3-hydroxypropionalaldehyde) is
formed by Lactobacillus reuteri from glycerol. At a
concentration of 100 AU/g, a 5-log reduction of E. coli
0157:H7 was attained in raw ground pork after 1 day at
7◦C. Reuterin at 4 AU/ml inhibited the multiplication of E.
coli and 8 AU/ml inhibited L. monocytogenes. This
inhibitor was even more effective in combination with
lactic acid.
38
 Lactobacillus reuteri and allyl isothiocyanate (AITC, at
ca. 1,300 ppm) alone and in combination were tested for
their effectiveness against a five-strain mixture of E. coli
0157:H7 in ground beef at 4°C for 25 days. The E. coli
inoculum levels were 3- and 6- log10 cfu/g, and 250 mM
glycerol/kg of meat was added. E. coli was killed in meat
with the two levels of glycerol before day 20 by L. reuteri.
AITC completely eliminated the 3-log10 inoculum of E.
coli 0157:H7 and at the higher concentration, it effected a
>4.5-log10 cfu/g reduction after 25 days.
To analyse the protecting result of a lactic organism,
Lactobacillus casei and its culture permeate were tested on
ready-to-use salad vegetables at 8°C. After 6 days of
storage, 3% culture permeate reduced APC from 6 to 1
log10 cfu/g, and suppressed coliforms, enterococci, and
Aeromonas hydrophila. The inhibitory principal produced
by a Staphylococcus equorum strain has been identified as
a macrocyclic peptide antibiotic, micrococcin P1, and the
bacterium was effective in inhibiting L. monocytogenes on
the surface of a soft cheese. Another antilisterial
compound, coagulin, was isolated from a strain of Bacillus
subtilis and placed in the pediocin family of bacteriocins.
The efficacy of five lactic acid bacteria to control L.
monocytogenes in pasteurized milkwas assessed by
inoculating the pathogen at ca. 104 cfu/ml with incubation
at 30°C. The lactics employed were Lactococcus lactis,
Leuconostoc cremoris, Lactobacillus plantarum, L.
delbrueckii subsp. bulgaricus, and Streptococcus
salivarious subsp. Thermophilus. An inhibition of 89–
100% was achieved with final pH of 4.17–4.21 at 30◦C or
37° C. Complete inhibition was achieved by the two
lactobacilli after 20 hours at 37°C and 64 hours at 30°C. In
another study, 49 isolates of lactic acid bacteria from ready-
to-eat products were screened on agar spot plates against L.
39
monocytogenes. The three most effective isolates were
identified as Lactobacillus casei, Lactobacillus paracasei,
and Pediococcus acidilactici. Using MRS broth incubated
at 5°C for 28 days, a 3.5-log reduction of L. monocytogenes
was found while in frankfurters, a 4.2–4.7 log reduction
occurred. In cooked ham stored at 5°C for 28 days under
vacuum packaging, a 2.6-log reduction was noted.

To inhibit foodborne pathogens in meats and meat

products. A meat starter culture of Pediococcus acidilactici
was shown to effect a 2.3-log reduction of E. coli 0157:H7,
L. monocytogenes, and Staphylococcus aureus during a
salami fermentation after 24 hours compared to a 1.3-log
reduction in controls. When added to the surfaces of beef
steaks inoculated with E. coli or S. typhimurium and stored
at 5°C, Lactobacillus delbrueckii subsp. lactis effected a
significant reduction of psychrotrophs and coliforms, and a
slight reduction in E. coli. Significant reductions of both
pathogens as well as psychrotrophs occurred when the
lactic culture was applied to the surfaces of freshly
slaughtered beef and pork carcass samples. The
effectiveness of Leuconostoc carnosum against L.
monocytogenes on cooked, sliced, and modified
atmosphere packaged cooked meat (saveloy) has been
demonstrated. The most effective method used was
spraying the lactic organism on meat surfaces, which kept
L. monocytogenes to 10 cfu/g for 4 weeks at 10°C
compared to controls where the pathogen increased to ca.
107 cfu/g. Regarding further reports on the protective
effects of lactics in meat products, a study in Norway using
cooked, sliced, modified-atmosphere-packaged ham and
sausage meats that were inoculated with 104–105/g of a
five-strain mixture of Lactobacillus sakei, it prevented the
growth of added L. monocytogenes and E. coli 0157:H7
when held at 8◦C for 21 days although a serotype 0:3 strain
40
of Yersinia enterocolitica was unaffected.20 All meats
were acceptable at the end of the storage period. When
1,180 psychrotrophic isolates from salad vegetables were
tested by agar plate assay against S. aureus, E. coli
0157:H7, L. monocytogenes, and S. Montevideo, (3.2%) of
the isolates displayed varying degrees of inhibition against
at least one of the four pathogens. Thirty-four of the 37
inhibitory cultures were Gram negative. A hydrogen-
peroxide-producing strain of Lactobacillus delbrueckii
subsp. lactis was added to several fresh-cut vegetables
along with E. coli 0157:H7 and L. monocytogenes and
incubated at 7°C for up to 6 days. There was no reduction
of pathogens, apparently because the catalase in the cut
vegetables destroyed the hydrogen peroxide by the lactic
culture. When a foodborne strain of Staphylococcus
equorum was tested against 95 Listeria strains (all species
included), all were inhibited. All but one of 131 other
species and strains of Gram-positive bacteria were inhibited
while 37 strains of 6 Gram-negative species were not.
Protective cultures refer to the microorganisms that can be
found in or added to a food product to effect
preservation/protection, and this concept was advanced.
The organisms noted above under lactic antagonism fit the
definition of protective cultures. Among the properties that
the latter should possess are: (1) they should present no
health risks, (2) provide beneficial effects on the product,
(3) have no negative impact on sensory properties, and (4)
serve as “indicators” under abuse conditions.

1.5 Hurdle Concept

The requirement for new and high-quality quality food

products has led to the appearance of hurdle technology.
Numerous methods are essential to hurdle technology.
There are four mechanisms by which hurdle technology
41
influence the multiplication of microbes in foods.

1. Homeostasis: In food preservation, homeostasis is the

key phenomenon. Homeostasis is the stable resemblance of
organisms to protect a steady and reasonable internal
temperature. The preservative aspect implementing as
hurdles can disturb the homeostasis mechanism to evade
the microbe from multiplication and making them to stay
inactive or even die. The low water activity, low pH, and
less redox potential perform on food synergistically. The
interference with homeostasis of microbes form a striking
and practical focus for development of food preservation
techniques. The result of preservation feature (hurdles) on
the homeostasis of microbes in a variety of foods may be
worthwhile.

2. Metabolic Exhaustion: The microbes in hurdle treated

stable goods utilize their energy for homeostasis, thereby,
happen to metabolically exhausted. This direct to auto-
sterilization of foodstuffs. Hence, microbiologically
constant foodstuff becomes safe for maintaining at ambient
room temperature.

3. Stress Reaction: Production of shielding stress shock

proteins is stimulated by numerous factors, such as water
activity, pH, heat, ethanol etc. Exposure to multiple stresses
is the cause of making the organism metabolically feeble.
As a result, multi-target preservation of foods can be the
elucidation to avoid synthesis of stress shock proteins.

4. Multitarget Preservation The theory of multi-target

preservation of food has been commenced by Leistner. The
multi-target conservation of foodstuff in which cleverly
useful gentle hurdles will have a synergistic property.
Therefore, purpose of frequent hurdles simultaneously
42
would lead to an optimal microbial stability and capable of
food preservation. It is applicable to mention that multi-
target attack of microbes may be a hopeful approach in
food microbiology.

Types of Hurdles

A plethora of hurdles are engaged in many countries of

the world to make the food secure to the consumers. Each
hurdle aims to abolish, inactivate or at least inhibit
unwanted organisms from the food for high-quality and
high security to the customer. Common salt or organic
acids can be used as hurdle to deal with the microbes in
food. Many normal antimicrobials such as nisin, natamycin,
and other bacteriosins, and essential oils resultant from
rosemary also work well. A complete evaluation on the
responsibility of bacteriosin as food preservative is
published by Pal and others in Beverage and Food World.
The category of hurdles, namely physical, physiochemical,
and microbial, which are used for the protection of variety
of foods, are briefly summarize in tabular form.

1. Physical: Ionizing radiation, low temperature (freezing),

aseptic wrapping, elevated temperature (blanching, baking,
evaporation, sterilization), adapted atmosphere, ultraviolet
radiation, electromagnetic force (radio frequency, pulse
magnetic fields, microwave), ultrasonification, elevated
force, photodynamic inactivation.

2. Physio-chemical: Sodium nitrate, sodium nitrite,

potassium sulphite, salt and herbs, carbon dioxide, oxygen,
surface treatment agents, phenols, lactic acid, low pH, low
water activity, lactoperoxidase, Maillard reaction products.

3. Microbial: Bacteriocins, protecting cultures, aggressive

43
flora, antibiotics. Hurdle is defined as a feature that avoids
the microbial growth and educe microbial load. At present,
further 50 hurdles are used in food processing trade all over
the world. The most significant hurdles employed are food
preservative such as nitrate, sulfite and sorbate; and also
competitive organisms like lactic acid bacteria. The hurdle
effect is the regularly used to analyse the essential
significance for the conservation of foods, since the barrier
in a stable product, manage the microbial spoilage, food
poisoning and desired fermentation processes. If the
strength of a fastidious hurdle in a food is too low, it should
be made stronger, and in case, if it is harmful to the food
quality, they should be lowered. Therefore, it is imperative
to mention that hurdles in the food should be kept in
optimum range for the security and quality of the food. The
microbes present in a food should not be capable to
overcome hurdles present during the storage of a product,
or else food will be blemished or even cause food
toxication. According to the kind and virulence of
pathogens, the strength of hurdles can be adjusted to meet
consumer preference in a profitable way without
compromising the protection of product. Some researchers
mentioned the chief barriers, which are used to conserve a
variety of foods.

Application of hurdle technology in foods

Hurdle technology has been used in a wide range of

foods, such as milk and dairy products, meat products,
poultry products, sea foods, canned products, bakery
products, juices, jams, spices etc. in order to intensify their
shelf life. This technology is used in food industry for
gentle but are expensive for conservation of food. The
sensory and dietary attributes of foods are sustained in
hurdle technology. It is significant to assert that
44
significance of a number of hurdles concurrently would
lead to an optimal microbial stability and efficient food
preservation.

Several hurdles such as water activity, heat treatment

and antimicrobials are functional to conserve high moisture
fruit products like papaya, pineapple, mango, and peach.
Lee (2004) depicts that hurdle technology can improve the
microbial safety of pickled fruits and vegetables. Gamma
radiation, osmotic dryness, and infrared drying can promote
the microbial load in pineapple slices and thereby rising
shelf life up to 40 days. Accomplishment of hurdle
technology is favorable, as it enlarged the shelf life of pork
sausages. Conservation of sugarcane juice by using
numerous hurdles like irradiation, heat treatment,
preservatives, and wrapping supplies was reported by
Sankhla and co-investigators. Lopez Malo and co-workers
(1994) equipped minimally processed shelf stable high
moisture grated papaya using diverse hurdles like mild heat
management, aw, pH decline and the addition of
preservatives. Hurdle technology was efficient in the
conservation of fresh scrapped coconut by means of heat
treatment and additives such as humectants, acidulants, and
preservatives (Gunathilake, 2005)

Function of hurdle technology was occupied to enhance

shelf life of chicken lollipop (Singh et al., 2014). The
efficiency on the use of hurdles such as antimicrobials,
partial dryness and wrapping in polymeric bags to develop
grated carrots fresh and microbiologically protected for
further 6 months at ambient temperature was carried out by
Vibhakara and others (2016).The research conducted by
these researchers evidently indicated that the hurdle treated
foods have better quality and complete shelf life. The
45
successful function of these hurdles shows that these can be
recommended for conservation of all type of food products
to increase the product safety and stability. Malik and
Sharma (2014) prepared a shelf stable ready to eat pickle
type spiced buffalo meat foodstuffs by controlling diverse
hurdles like pH, water activity, soluble hydroxyproline,
TBA values, nitrite content, and protein solubility. The
hurdle treated keema was shelf stable, and conventional up
to the fifth day unlike the usually prepared keema that is
highly perishable and was found satisfactory only for one
day.

46
2 Chapter-2: Microorganism in foods
and methods for detection

The microbiological report of meat products is due to

the animal health, situation under which it was reared,
processed, wrapped and stored conditions. Meat pathogens
are the reason for self-limiting human enteric diseases or
systemic and fatal illness of the immunocompromised, the
elderly and the young. Meat can act upon as a most
excellent substrate for microbial propagation. Major meat
associated pathogenic bacteria include Clostridium
perfringens, Salmonella spp, pathogenic strains of
Escherichia coli, Yersinia enterocolitica, Listeria
monocytogenes and Aeromonas hydrophila

Microorganism associated with meat while processing

Meat spoilages point toward (a) color changes (b)

textural changes and (c) progress of off-flavour or off-odor
or substance as a consequence of microbial
growth. Salmonella is the chief microbial challenge for
poultry. Listeria, which is an adulterant with zero tolerance,
is the main calamity for ready to eat meat products.
Treatment with organic acids, carcass pasteurization and
steam carcass vacuuming, trisodium phosphate, chlorine
dioxide, lactoferrins, sodium lactate, sodium acetate and
and radiation are used as microbial decontaminants.
47
Carcass cleansing with hot water of 80°C for 10 seconds
can decrease microbial loads by 2 logs. Current regulatory
policies and inspection in the meat industry include the
HACCP (Hazard Analysis Critical Control Point) food
safety system with an objective to supply protected food for
utilization and prevent chemical, physical and biological
hazards.

Gram-negative bacteria (Aerobes)

Neisseriaceae: Psychrobacter immobilis, P.

phenylpyruvica, Acinetobacter spp, A. twoffii, A.
Johnsonii, Pseudomonadaceae: Pseudomonas
fluorescens, P. lundensis, P. fragi, P. putida

Gram-positive bacteria:

Brochothrix thermosphacta, Staphylococcus spp.,

Clostridium casigenes, Clostridium algidixylanolyticum.

2.1 Fresh Meat

In contrast to fruits and vegetables, meats are comprised

mainly of protein and fats. Water content is 71–76% and
consequently moisture is not an issue except for spoilage
microbes on cured meats. High-quality hygienic practices
are vital to produce high quality meats. The quantity of
spoilage organisms on meat just after slaughter is a decisive
aspect in determining shelf life. The exterior surface of beef
carcasses may have colonies ranging from\101 to
107 cfu/cm2, the majority of which are cold loving
psychrotrophic microbes. Chopping and grinding of meats
can amplify the microbial load as more surface area is
exposed and more water and nutrients become available.
Microbes are found on fresh meat depending on parameters
48
such as pH, composition and texture of processed meats,
temperature and packaging atmosphere. Pseudomonas spp.
is the main spoilage bacteria in aerobically stored raw meat
and poultry. Once the preliminary low levels of glucose are
exhausted by various microbes, Pseudomonas has an
advantage because it can catabolize gluconates and amino
acids more voluntarily than other microbes. Dark, firm and
dry meat with a comparatively high pH of 6.0 spoils more
quickly because deamination of amino acids starts
formerly. At pH<6.0 microbes can produce sulfides and
ammonia even when glucose is still available. These
sulfides not only smell bad however they also cause color
changes in meat, and consequently Shewanella has an
elevated levels of spoilage possible on fresh, high pH meats
stored aerobically even when it is not a dominant
microbe. Brochothrix thermosphacta is often a major
spoilage organism on fresh meat stored aerobically at
refrigeration temperatures. Enterobacteriaceae, mainly
species of Serratia, Enterobacter, and Hafnia, are root
causes of spoilage in vacuum-packed, high pH fresh meats.
These organisms are facultative anaerobes that create
organic acids, hydrogen sulfide and greening of meats.
Lactic acid bacteria (LAB) grow on meat and poultry
package under vacuum and adapted atmospheres,
producing organic acids from glucose by fermentation. This
gives rise to acid uric off-odors which may be accompanied
by gas and slime development and greening of meat.
However, LAB are weakly proteolytic and so do not
produce enormous quantity of amines or sulfides, and
destroy of meat by LAB is not as offensive. Psychrophilic,
anaerobic Clostridium spp. is associated with spoilage of
vacuum-packaged meats. "Blown pack" meat spoilage is
described by excessive gas formation with off odors owing
to arrangement of butyric acid, butanol and sulfurous
compounds. Yeasts and molds cultivate gradually on fresh
49
meat and do not fight well with bacteria. Therefore, they
are the slightest component of spoilage flora.

2.2 Processed Meat

Addition of sodium chloride, nitrites and/or nitrates,

along with a variety of other seasonings, emulsifiers and
preservatives to ground or whole muscle meats alter the
environment considerably and also the spoilage flora of
processed meats. Dried and dry-fermented meats generally
do not bear microbial growth although process deviations
may permit development of some organisms. Spoilage
organisms can grow on new and cooked cured meats, so
they are best stored chilled, under a vacuum or modified
atmosphere. Pseudomonas spp. are not usually significant
reason of spoilage in processed meats because of their
sensitivity to curing salts and heat pasteurization and their
inability to grow well in meats filled with a vacuum or high
carbon dioxide atmosphere. However, when packages have
been released and there has been inadequate curing, these
bacteria may spoil refrigerated processed meats. Some
cold- and salt tolerant Enterobacteriaceae is the root cause
of decay in processed meats, such as ham or bacon.
Lactic acid bacteria (LAB) are the assemblage of
bacteria primarily associated with spoilage of processed
meats. They create sour off-flavors, gas, slime, and
greening, and this spoilage may be further harsh than in
fresh meat because of the existence of added carbohydrates.
Although, faulty cooking/cooling procedures, including
long cooling periods and temperature abuse, has permitted
growth of these organisms. Spores of these organisms may
be established with spices or other ingredients. Yeasts
cause some decay in processed meats but are normally only
important when sulfite is used as an additive or when meats
have been irradiated or are stored aerobically in the cold.
50
Spoilage under aerobic condition

1. Slime in bacteria is due to Pseudomonas acinetobacter,

Streptococcus, Bacillus.

2. Change in colour of meat pigment. The red colour of

meat may be altered to shades of green, brown or grey
by Lactobacillus and Leconostocs spp.

3. Sour odour is produced by volatile acids and

Actinomycetes produce earthy aroma. Yeast is the reason
for sliminess discoloration and off odor and taste
deficiency.

4. Aerobic molds are originated in meat. These are

stickiness, black-spot, white-spot, green patches rotten odor
and taste.

5. Spoilage under anaerobic situation.

i) Souring is due to production of formic, acetic, butyric,
lactic, succinic and propionic acid.
ii) Putrefaction is the root cause for decomposition of
proteins under anaerobic situation by Clostridium species.

2.3 Poultry Meat

Poultry meat like meat of other animals is also liable to

infection by a variety of sources. Contamination of skin and
lining of the body cavity occur through a mixture of
processing operations. The organisms of great consequence
in poultry are Salmonella spp. and Campylobacter jejuni. A
number of Gram negative psychrotropic bacteria are
isolated from poultry carcasses. Ground turkey also may
carry fecal Streptococci. It is vital to freeze the poultry fast
51
in order to keep it in good condition for several months.
Additionally freezing further reduces the quantity of
microorganisms in the poultry.

Meat Borne Diseases

Food borne microbial hazards have an overwhelming

impact on human suffering. Salmonella,
Campylobacter, Escherichia coli including serotype
O157:H7 and other enteric pathogens are the microbes to
be controlled in meat. Illness due to Listeria
monocytogenes following utilization of ready to eat meat
and poultry goods is a main problem in the recent years.
Also there are food borne disease caused by Yersinia
enterocolitica, Clostridium botulinum and Bacillus cereus.
Prevalence of some food borne pathogens recognized since
1970's include Vibrio cholerae, Vibrio vulnificus, Noro
virus, Enterobacter sakazakii, prions and resistant bacteria.
In current years the food borne disease correlate with
animal health pandemics include Avian Influenza (AI) and
Foot and Mouth Disease (FMD) viruses. Avian influenza
can be eliminated by suitable cooking with a temperature of
70°C and more. Development of antibiotic resistance in
food borne pathogens is very essential from public health
view point in present days and in the future.

Control of meat borne pathogens

Proficient management of pathogens in meat and

development of meat safety can be achieved by latent
infections among livestock, at the farm, during harvesting,
dressing and product processing, improvement in process
food hygiene and prospective application of new or novel
processing and preservation technologies. Animal stress
can damage meat quality and direct to more contamination
52
and increased pathogen shedding. Antimicrobial
intervention technologies can be used proficiently to
recover the microbiological quality of meats. These
technologies comprise decrease in contamination on the
raw manufactured goods, minimization of microbial
contact to the foodstuffs, decline in contamination that has
achieved the right to use the product, inactivation of the
microbes on the product devoid of cross contamination and
prevention and control of the growth of non-inactivated
microbes, which have increased access to the meat. An
effectual pathogen management at pre-harvest, postharvest,
processing, storage, allotment, merchandizing, preparation,
food-service and utilization of meat include activities
engaged during pre-harvest or in the field, through post-
harvest processing treatments for partial or complete
devastation of contaminants and antimicrobial procedures
for inhibition or retardation of microbial growth.
Management of sanitation operating system, operations
under the HACCP system to meet microbiological
standards, association of good manufacturing practices
(GMP) and high-quality hygiene practices (GHP). Low and
elevated temperature levels, non-thermal process like
irradiation and high pressure and packaging,
physiochemical interventions (low pH, modification of
oxidation reduction potential through packaging and
application of antimicrobial additives, and biological
interventions. So, there should be necessities to educate the
food handlers and customers predominantly on culinary
tips.

53
2.4 Culture, Microscopic and sampling method
for detecting microbes
It is habitually necessary to perform a microbiological
examination of food to decide its quality. This may be
essential to estimate its shelf life, its suitability for human
consumption or to confirm that it meets some established
microbiological criterion.

Conventional Standard Plate count

By the conventional SPC method, food samples are

blended or homogenized, serially diluted in an appropriate
diluent, plated onto a suitable agar medium, and incubated
at a suitable temperature for a specified time, after which
all evident colonies are calculated by use of a Quebec or
electronic counter.
Using SPC number of feasible count in bacteria can be
determined. Plating procedures are restricted by the degree
of inhibition and effectiveness of the selective and/or
differential agents employed. Although the SPC is
determined by pour plating, essentially equivalent results
can be acquired by surface plating. By the latter method,
prepared and hardened agar plates with dry surfaces are
engaged. The diluted specimens are planted onto the
surface of replicate plates, and, with the assistance of bent
glass rods ("hockey sticks"), the 0.1 -ml inoculum per plate
is cautiously and evenly dispersed over the entire surface. It
is the technique of choice when the colonial features of a
colony are imperative to its presumptive recognition and
for most selective media. Strict aerobes are evidently
favored by surface plating, but microaerophilic organisms
tend to grow slower. Among the disadvantages of surface
54
plating are the trouble of spreaders (when the agar surface
is not adequately dry prior to plating) and the crowding of
colonies.

Homogenization of Food Samples

Microorganisms were extracted from food specimens for

plating almost generally by use of mechanical blenders.
The Stomacher, a comparatively effortless device,
homogenizes specimens in a special plastic bag by the
vigorous pounding of two paddles. The pounding effects
the shearing of food specimens, and microorganisms are
released into the diluent. The Stomacher has been
compared to a high-speed blender for food investigation by
numerous researchers. Plate counts from stomacher treated
sample are comparable to those treated by blender.
Stomacher was shown to be less toxic than a blender to
Staphylococcus aureus, Enterococcus faecalis and
Escherichia coli. One researchers reported that counts by
using a Stomacher were considerably higher than when a
blender was used. Significantly elevated counts of gram-
negative bacteria were attained by Stomacher.

Spiral plating method

The enumeration of colonies on plates equipped with a

spiral plater is accomplished by use of a special counting
grid. Depending on the relative density of colonies,
colonies that appear in one or more specific areas of the
superimposed grid are counted. In this case, a whole
sample volume of 0.0018 ml was deposited, and the two
grid areas counted contained 44 and 63 colonies,
respectively, resulting in a total count of 6.1 x 104 bacteria
per milliliter. The spiral plating device here described was
devised by Gilchrist. It was contrast to the SPC method by
55
using 201 samples of raw and pasteurized milk; overall
excellent agreement was acquired. A collaborative study
from six analysts on milk samples demonstrated that the
spiral plater compared favorably with the SPC. In other
analysis, the spiral plater was compared with three other
methods (pour, surface plating, and drop count), and no
distinction was found among the methods at the 5% level of
significance. However, the spiral plate maker yielded
counts as those by the droplette method.
Advantages of the spiral plater over standard plating
are the following: less agar is used; fewer plates, dilution
blanks, and pipettes are required; and three to four times
more samples per hour can be examined. Also, 50-60 plates
per hour can be prepared, and little training is necessary for
its operation. It is more appropriate for use with liquid
foods such as milk. A laser-beam counter has been
industrialized.

Membrane filters

Membranes with a pore size that will retain bacteria.

Following the collection of bacteria upon filtering a given
volume, the membrane is located on an agar plate or an
absorbent pad saturated with the culture medium of choice
and incubated. Following growth, the colonies are
enumerated. Alternatively, a DMC can be made. The
microbes found in the membrane are observed and counted
microscopically. They are used to calculate low numbers of
bacteria.
On the whole efficiency of membrane filter methods for
determining microbial numbers by the DMC has been
enhanced by the use of fluorescent dyes. The utilization of
fluorescent dyes and epifluorescent microscopes to specify
bacteria in waters has been engaged rather widely since the
early 1970s. Cellulose filters were initially used; although,
56
polycarbonate Nucleopore filters recommend the assistance
of retaining all bacteria on top of the filter. When lake and
ocean waters were observed using the two kinds of
membranes, counts were twice as high with Nucleopore
membranes as with cellulose membranes.

Direct epifluorescence filter technique

The direct epifluorescent filter technique is used for

estimating bacterial counts in raw milk. The procedure was
developed in response to the need for a quick process for
judging the hygienic quality of farm milks. It accomplishes
a significantly increased sensitivity (103–104 bacteria ml-1)
over conventional microscopy techniques by concentrating
bacteria from a considerably larger volume of sample by
filtering it through a polycarbonate membrane filter. The
bacteria are counted directly under the epifluorescence
microscope. It may be required to pretreat the sample to
allow filtration thus, for instance, milk can be pretreated
with detergent and a protease enzyme. This is attained by
using a polycarbonate membrane where relatively
standardized pores are produced following neutron
bombardment of a plastic film, rather than cellulose acetate
filters which have tortuous pores where bacteria will be
held at various stages.

Agar Droplet method

This method was proposed by Sharpe and Kilsby, the

food homogenate is diluted in tubes of melted agar (at
45°C). Among the three tubes of agar the first tube being
inoculated with 1 mL of food homogenate. After mixing, a
sterile capillary pipette (ideally delivering 0.033 mL/drop)
is used to transfer a line of 5 x 0.1-mL droplets to the
bottom of an empty petridish. With the same capillary
57
pipette, three drops (0.1 mL) from the first 9-mL tube are
transmitted to the second tube, and, after mixing, another
line of 5 x 0.1-mL droplets is placed next to the first. This
step is repetitively used for the third tube of agar. Petri
plates containing the agar droplets are incubated for 24
hours, and colonies are enumerated with the aid of a 10x
viewer. Analysis using this method from pure cultures,
meats, and vegetables compared constructively to those
attained by conventional plate counts; droplet counts from
ground meat were slightly higher than plate counts. The
process was about three times faster, and 24-hour
incubations gave counts equal to those acquired after 48
hours by the conventional plate count. Dilution blanks are
not essential, and only one petridish per sample is required.

Most probable numbers

In this process, dilutions of food sample analysis are

arranged as for the SPC. Three successive aliquots or
dilutions are then planted into 9 or 15 tubes of appropriate
medium for the three- or five-tube method. Numbers of
organisms in the novel sample are acquired by use of
standard MPN tables. The technique is statistical in nature,
and MPN analyses are generally higher than SPC results.
This process was commenced by McCrady in 1915. 95%
confidence intervals for a three-tube test range from 21 to
395. When the three-tube test is used, 20 of the 62 possible
test combinations report for 99% of analysis, whereas with
the five-tube test, 49 of the possible 214 combinations
produce 99% of results. In a mutual study on coliform
densities in foods, a three-tube MPN value of 10 was found
to be as high as 34, whereas in another stage of the study,
the upper limit could be as high as 60. Although Woodward
accomplished that many MPN values are impossible.

58
Direct microscopic count

In its simplest form, the DMC consists of creating

smears of food specimens or cultures onto a microscope
slide, staining with a suitable dye, and screening and
counting cells with the help of a microscope (oil immersion
objective). DMCs are most widely used in the dairy
industry for assessing the microbial quality of raw milk and
other dairy products, and the precise method engaged is
that originally developed by R.S. Breed (Breed count).
Briefly, the method consists of adding 0.01 mL of a sample
to a 1-cm2 area on a microscope slide, and following fixing,
defatting of sample, and staining, the organisms or clumps
of organisms are analyzed. The latter involves the
utilization of a calibrated microscope.

The methods provide rapid microbiological examination

of other food products, for instance dried and frozen foods.
Among the advantages of DMC are that it is fast and
simple, cell morphology can be assessed, and it provides
fluorescent probes for better efficiency. Both viable and
nonviable cells are counted, food particles are not always
distinguishable from microorganisms, microbial cells are
not uniformly dispersed relative to single cells and clumps,
some cells do not take the stain well and may not be
counted, and DMC counts are invariably superior than
counts by SPC.
A slide method to become aware of and count viable
cells has been developed. The methods utilize the use of the
tetrazolium salt (p-iodophenyl-3-j9-nitrophenyl)-5-phenyl
tetrazolium chloride (INT). Cells are exposed to filter-
sterilized INT for 10 minutes at 37°C in a water bath
followed by filtration on 0.45-um membranes.
Subsequently followed by drying of membranes for 10
minutes at 500°C, the particular membranes are
59
accumulated in cottonseed oil and observed with coverslip
in place. The process was found to be realistic for pure
cultures of bacteria and yeasts, but it underestimated APC
by 1-1.5 log cycles while compared using milk. By
applying fluorescence microscopy and via blue (modified
aniline blue fluorochrome), viable yeast cells could be
distinguish from nonviable cells. Viable cells can be
determined by staining with acridine orange (0.01%)
subsequently followed by epifluorescence microscopy and
account of those that fluoresce orange.

Contact Plate

The replicate organism direct agar contact (RODAC)

technique use special Petri plates, which are poured with
15.5-16.5 mL of a suitable plating medium, resulting in a
raised agar surface. When the plate is inverted, the
hardened agar makes direct contact with the surface. When
surfaces are examined that have been cleaned with certain
detergents, it is necessary to comprise a neutralizer
(lecithin, Tween 80, and so on) in the medium. Plates are
enclosed and incubated, and the colonies were enumerated.
Plates with dried agar surfaces can be used by means of
selective media. The RODAC plate has been shown to be
the technique of choice when the surfaces to be observed
are smooth, firm, and nonporous. Although it is not
appropriate for seriously contaminated surfaces, it has been
predicted that a solution that contaminates a surface needs
to contain at least 10 cells per milliliter before results can
be attained either by contact or by swabs. The latter
investigators found that the contact plate detached only
about 0.1% of surface flora. This recommends that 10
cfu/cm2 identified by this process are preferable to a
surface that essentially contains about 104 cfu cm2. When
stainless-steel surfaces were contaminated by B. subtilis
60
endospores, 41% were recovered by the RODAC plate in
contrast to 47% by the swab method. In another analysis,
swabs were improved than contact plates when the
contamination level was 100 or more organisms per 21-25
cm2. On the other hand, contact plates give enhanced
results where low numbers exist.

Physical and chemical methods in detection of microbes

The methods are used for detecting and characterizing

microorganisms. To calculate approximately sum of cells
or cellular products. Most of them are based on metabolic
activity of microorganisms on specified substrates,
measurements of growth reaction, size of some part of cells
including nucleic acids.

2.5 Physical methods

Biosensors

In a wider sense, a biosensor is a device that can be used

to distinguish the incidence or activity of an organism
living or dead. A more brief definition is the transducer is
the component that converts the charge into a quantifiable
signal. Some biosensors are related to the principles of
physics (e.g., fiber optics) whereas others are based on
biological principles (e.g., lux gene luminescence).

Piezoelectric Crystals (Accoustical Biosensors)

It is owing to force functional in a crystalline material

like quartz. Quartz is a particularly receptive load indicator.
It can be employed to distinguish the calculation of its
homologous antigen. The efficient principle of a
piezoelectric biosensor is represented in Figure 1. With the
61
quartz coated with an antibody, the intention analyte is the
homologous antigen, when binds to the antibody, alters the
mass with a resulting decrease in frequency.

Figure: 1 Working Principle of Piezoelectric biosensor

In one study, gold-coated quartz crystal surfaces were

used to develop a FIA system to detect S. typhimurium. To
mobilize the antibody, crystals were initially coated with
DSP (dithiobissuccinimidly- propionate), then the S.
typhimurium antibody, and finally with S. typhimurium
cells. It can be depicted that ∆F (Hz) response for DSP
alone was 9.0; 123 for DSP+antibody; and 228 for
DSP+antibody+antigen. The hertz changes represent the
effect of attached material on the quartz surface. The
method was linear with S. Typhimurium from 105 to 109
cfu/ml with ∆F changing from 90 to 170 Hz.

Fiber Optics

An optical fiber is a “light wire” (optical waveguide)

made of glass or polymeric material, and the light waves
are propagated along the fiber by total internal reflection. A
62
fiber optic biosensor utilize electronic or optical
transduction to examine a biological reaction, and reports it
as an optical signal. The typical format of a fiber optic
system consists of a tapered fiber optic probe coated with
an antibody of interest. Light from a diode laser moves
from an all-fiber system to the fiber top and then penetrates
as an evanescent wave in the area outside the tips. When a
fluorescently-labeled homologous antigen binds to the
antibody on the fiber tip, it interacts with the evanescent
wave of the fiber optic waveguide and the fluorescent
signal radiates in all directions with some traveling back up
the fiber tip to the detection system.
With a surface plasmon resonance (SPR) fiber optic
system, antibodies bind to the surface of a thin film on a
precious metal that is on the reflecting surface of an
optically transparent glass wave guide. When visible or
near-infrared light is passed through the waveguide, a
reflection occurs from the waveguide. The reflected light
interacts with a plasma of electrons on the metal surface
and a resonance effect causes a strong absorbance which is
a consequence of the concentration of the antibody–antigen
complex on the reflecting surface of the waveguide. The
greater the antibody–antigen reaction, the longer the
wavelengths.
A convenient evanescent wave fiber optic biosensor
(Analyte 2000, developed at the U.S. Naval Research
Laboratories) was evaluated for its capacity to detect E. coli
0157:H7 in ground beef. Using two waveguides, the system
detected 9 × 103 and 5.2 × 102 cfu/g. No false-positive
reactions occurred, and results were obtained within 25
minutes of sample preparation. The above test system
employed a standard sandwich immunoassay and Cy5 to
illuminate the captured antigens. The Analyte 2000 was
used to detect L. monocytogenes, and with an inoculof<10
cfu/ml followed by a 20-hour enrichment, biosensor results
63
were obtained in 20–45 minutes.

Impedance

The idea of electrical impedance measurement of

microbial growth was formulated by G.N. Stewart in 1899.
Impedance is the visible resistance in an electric circuit to
the flow of alternating current, analogous to the actual
electrical resistance to a direct current. When
microorganisms grow in cultured media, they metabolize
substrates of low conductivity into foodstuffs of higher
conductivity and thereby decline the impedance of the
media. When the impedance of broth cultures is calculated,
the curves are reproducible for species and strains, and
mixed cultures can be recognized by use of specific growth
inhibitors. The method has been revealed capable of
distinguish as few as 10–100 cells is depicted in Table: 2

Table: 2

64
 Cell populations of 105–106/ml can be detected in 3–5
hours and 104–105/ml in 5–7 hours. The times noted are
required for the organisms in question to reach a threshold
of 106–107 cells per milliliter. The brewing industry test for
distinguishing spoilage organisms in beer was shortened
from 3 weeks or more to only 2–4 days by use of
impedance. Yeasts growing in wort caused to amplify the
impedance, while in bacteria the results have been declined.
In one study, 200 samples of pureed vegetables were
evaluated, and a 90–95% agreement was found between
impedance measurements and plate count consequences
comparative to unacceptable levels of bacteria. Impedance
analyses required 5 hours, and the method was found to be
relevant to cream pies, ground meat, and other foods.
Impedance measurement was used to evaluate growth
(negative test) or no growth (positive test) of E. coli
0157:H7 strains in a sorbitol-containing medium to which a
specific bacteriophage (AR1) was added and calibration
were obtained. At 30 minutes, the absence of conductance
changes in tubes that contained the AR1 phage was denoted
as positive. Only 1 of 155 E. coli 0157:H7 strains multiply
in the occurrence of the phage.

Microcalorimetry

This is the analysis of small heat changes: the extent of

65
the enthalpy change involved in the breakdown of growth
substrates. The heat formation that is calculated is casually
connected to the catabolic behavior of cells.
There are two types of calorimeters: batch and flow.
Most of the early work was done with batch-type
instruments. The thermal events calculated by
microcalorimetry are those from catabolic activities, are
noted. One of the most extensively used microcalorimeters
for microbiological work is the Calvet instrument, which is
sensitive to a heat flow of 0.01 cal/hour from a 10-ml
sample. With respect to its utilization as a rapid method,
most consideration has been devoted to the recognition and
depiction of foodborne organisms. Perry et al. successfully
describe commercial yeast strains. The latter method is one
in which a microcalorimeter is filled with a flow-through
calorimetric vessel. By the utilization of a chemically
defined medium containing seven sugars, thermograms
were formed by nine lactic acid bacteria (belonging to the
genera Enterococcus, Leuconostoc and Lactobacillus)
characteristic enough to recommend the process for their
detection.
This process has been used to analyse spoilage in canned
foods, to differentiate between the Enterobacteriaceae, to
recognize the presence of S. aureus, and to estimate
bacteria in ground meat. In identifying S. aureus, results
were achieved in 2 hours using a preliminary number of
107–108 cells per milliliter and in 12–13 hours when only 2
cells per milliliter were used.

Flow Cytometry

Flow cytometry is the discipline of quantify components

(cells) and the properties of individual cells in liquid
suspension. By flow channel the suspended cells are
brought to the detector. Fluidic devices under laminar flow
66
identify the trajectories and velocities that cells traverse the
detector, and among the cell properties that can be
identified are fluorescence, absorbance and light scatter. By
use of flow sorting, individual cells can be classified on the
basis of their calculated properties, and 1–3 or more global
properties of the cell can be measured. Flow cytometers
and cell sorters make use of one or more excitation sources
for instance argon, krypton, or helium–neon ion lasers and
one or two fluorescent dyes to assess and characterize
several thousand cells per second. When a dye is used, its
excitation spectrum must be equivalent the light
wavelengths of the excitation source. Two dyes may be
used in mixture to measure, for example, total protein and
DNA content. For example, both dyes must excite at the
same wavelength and emit at various wavelengths so that
the light emitted by each dye is calculated separately. The
early narration of flow cytometry has been reviewed by
Horan and Wheeless.

2.6 Chemical methods

Thermostable nuclease

The presence of S. aureus in considerable numbers in a

food can be determined by investigating the food for the
occurrence of thermostable nuclease (DNase). This is
possible because of the elevated correlation between the
production of coagulase and thermostable nuclease by S.
aureus strains, chiefly enterotoxin producers. For instance,
in one study, 232 of 250 (93%) enterotoxigenic strains
formed coagulase, and 242 or 95% produced thermostable
67
nuclease.
Spectrophotometric method was accepted out by
Chesbro and Auborn spectrophotometric method for
nuclease determination. As the number of cells increased in
ham sandwiches, there was amplification in the quantity of
extractable thermostable nuclease of staphylococcal origin.
They suggested that the presence of 0.34 unit of nuclease
indicated certain staphylococcal growth and that at this
level; it was unlikely that enough enterotoxin was present
to cause food poisoning. The 0.34 unit was shown to
correspond to 9.5 × 10−3μg of enterotoxin by S. aureus
strain. It has been found to be as good as coagulase in
testing for enterotoxigenic strains, and in another study, all
foods that consist of enterotoxin containing thermostable
nuclease, which was present in the majority of foods with 1
× 106 S. aureus cells per gram. On the other hand,
thermostable nuclease is produced by some enterococci
The cause for the decrease was found to be proteolytic
enzymes, and the decrease was reversed by the addition of
protease inhibitors. [Link] indication can be tested by
means of heat stable nuclease
1. Because of its heat-stable nature, the enzyme will
preserve even though if the bacterial cells are damaged by
temperature, chemicals, or bacteriophage or if they are
induced to l-forms.
2. The heat tolerant nuclease can be found earlier than
enterotoxin (about 3 hours versus several days).
3. The nuclease is produced by enterotoxigenic cells
before enterotoxins appear.

Limulus Lysate for Endotoxins

Gram-negative bacteria are characterized by their

manufacture of endotoxins, which consist of a
lipopolysaccharide (LPS) layer (outer membrane) of the
68
cell envelope and lipid A, which is buried in the outer
membrane. The LPS is pyrogenic and accountable for
symptoms that lead to disease caused by Gram-negative
bacteria. The Limulus amoebocyte lysate (LAL) test utilizes
a lysate protein attained from the blood (actually
hemolymph) cells (amoebocytes) of the horseshoe crab
(Limulus polyphemous). The lysate protein is the main
sensitive substance known for endotoxins.
The LAL test is achieved by adding aliquots of food
suspensions or other test materials to minor amount of a
lysate preparation, followed by incubation at 37°C for 1
hour. The existence of endotoxins causes gel formation of
the lysate material. LAL reagent availability can detect 1.0
pg of LPS. As E. coli cell contains about 3.0 fg of LPS, it is
possible to identify <300 Gram-negative cells. By
analyzing the reports the meatborne pseudomonads, was
found to contain 102 cfu/ml colonies.
The first food application was the utilization of LAL to
identify the microbial spoilage of ground beef. Endotoxin
titers amplify the proportion to viable counts of Gram-
negative bacteria. Since the normal spoilage of refrigerated
fresh meat is due to Gram-negative bacteria, the LAL test is
a high-quality, rapid indicator of the sum numbers of
Gram-negative bacteria. The method has been found to be
appropriate for the rapid assessment of the hygienic quality
of milk relative for the recognition of coliforms before and
after pasteurization. Because both viable and nonviable
Gram-negative bacteria are detected by LAL, simultaneous
plating is necessary to determine the numbers of cfu. This
method has been applied to monitor milk products,
microbial quality of raw fish, and cooked turkey rolls.

Adenosine Triphosphate measurement

Adenosine triphosphate (ATP) is the leading resource of

69
power in all existing live cells. It disappears within 2 hours
subsequently leading to cell fatality, and the quantity per
cell is normally constant, with values of 10−18 to 10−17 mole
per bacterial cell, which belongs to around 4 × 104 M
ATP/105 cfu of bacteria. Among procaryotes, ATP in
exponentially growing cells is regularly around 2–6 mole
ATP/mg dry weight regardless of mode of nutrition. The
absolute extraction and precise quantity of cellular ATP can
be equated to individual groups of microorganisms in the
similar method as endotoxins for Gram-negative bacteria.
One of the easy ways to calculate ATP is to make use of
the firefly luciferin–luciferase system; luminometer is used
to measure the light.
The application of ATP measurement is a quick method
for estimating microbial numbers has been used in clinical
microbiology. In the clinical laboratory, it has been
engaged to monitor urine samples. The victorious
utilization of the technique for bacteriuria and for assessing
the sludge content has been employed. It represents an
excellent potential method for the rapid estimation of
microorganisms in foods. In meats, the problem on
microbal ATP was addressed by Stannard and Wood by use
of a three-stage procedure consisting of centrifugation,
utilization of cation exchange resin, and filtration to
liberate food particles and accumulate the bacteria on 0.22-
μmfilters.
The ATP analysis has been used for detection of
microbial load. 190 Chicken carcasses were rinsed and
results were obtained within 10 minutes, but the method
could not reliably detect <1×104/ml due to carcass ATP. A
500-cm2 area for beef carcasses, and a 50-cm2 area for pork
were surface-wiped with an ATP-free sponge. The
complete test could be accomplished in about 5 minutes
with the least detectable amount being log 2.0 cfu/cm2 for
beef and log 3.2/cm2 for pork. The ATP assay is
70
extensively used as a quick and on-the-spot method for
monitoring food handling surfaces by swabbing selected
areas and interpreting the relative light units (RLU) from a
luminometer.

Radiometry

The radiometric recognition of microorganisms is based

on the integration of a 14C-labeled metabolite in a growth
medium so that when the organisms employ this
metabolite, 14CO2 is released and calculated by use of a
radioactivity counter. For organisms that make use of
glucose, 14C-glucose is usually engaged. For those that
cannot develop this compound, others such as 14C-formate
otherwise 14C-glutamate are used. Generally in this
process consists of using capped 15-ml serum vials to
which are further added somewhere from 12 to 36 ml of
medium consisting of the labeled metabolite. The vials are
completed either aerobic or anaerobic by sparging with
suitable gases and are then inoculated. Subsequently
followed by incubation, the headspace is experienced
occasionally for the existence of 14CO2. The moment
required to identify the labeled CO2 is inversely related to
the amount of organisms in a product. The Bactec is a
commercially available detection system.
The experimental finding of S. aureus, Salmonella
typhimurium and Clostridium botulinum in beef loaf was
studied by Previte. The inocula employed ranged from
about 104 to 106/ml of medium, and the revealing time
ranged from 2 hours for S. typhimurium to 5–6 hours for C.
botulinum spores. For this experiment, 0.0139 μCi of 14C-
glucose per milliliter of tryptic soy broth was employed.
Investigators have revealed that spores need 3–4 hours
longer for discovery than vegetable cells. From the findings
of Lampi et al., the radiometric recognition procedure
71
could be engaged as a selection process for foods
containing maximum numbers of organisms, for such foods
outcome of result was produced within 5–6 hours, whereas
those with lesser numbers essential for longer times. The
recognition of nonfermentors of glucose by this process is
probable when metabolites such as labeled formate and/or
glutamate are used. It has been revealed that a huge amount
of foodborne organisms can be quantified by this method in
1–6 hours. The radiometric discovery of 1–10 coliforms in
water within 6 hours was achieved by Bachrach and by
employing 14C-lactose with incubation at 37◦C in a liquid
medium. It is believable that a discrimination can be made
among fecal E. coli and total coliforms by employing
45.5°C incubation along with 37°C incubation.

Fluorogenic and chromogenic substrates

The substrates engaged in culture media in food

microbiology are as follows:
1. 4-methylumbelliferyl-β-d-glucuronide (MUG),
2. 4-methylumbelliferyl-β-d-galactoside (MUGal),
3. 4-methylumbelliferyl phosphate (MUP)
MUG is the most extensively used among the
fluorogenic substrates, and it is hydrolyzed by β-d-
glucuronidase (GUD) to discharge the fluorescent 4-
methylumbelliferyl moiety, which is analysed with long-
wave ultraviolet light. The value of MUG is that E. coli is
the main producer of GUD, and this substrate has found
extensively used as a disparity agent in media and methods
for this organism. A small number of Salmonellae and
Shigellae are also GUD positive, as are some
corynebacteria.
An essential phase of this process is the incidence of
fluorescence before gas production from lactose.
Employing the Feng–Hartman method, another group
72
analysed 1,020 specimens by a three-tube MPN and were
able to recognize more E. coli-positive samples than with a
conventional MPN. The greater efficiency of LTB-MUG
resulted because some E. coli strains are anaerogenic. In an
assessment of MUG added to lauryl sulfate broth (LSB), a
94.8% conformity was obtained on 270 product samples
with 4.8% false positives but no false negatives with LSB-
MUG. Oysters contain endogenous glucuronidase, but an
E. coli (EC) broth–MUG method was engaged effectively
in one study where 102 of 103 fluorescing tubes were
positive for E. coli. A 20-minute tube procedure employing
MUG was useful to 682 E. coli cultures, and 630 (92.4%)
were positive. Of 188 0157 strains of E. coli, 166 were
MUG negative and all were positive for the vero toxin. By
use of this 20-minute method, MUG-negative E. coli are
probable to be verotoxigenic.
MUG has established limited study as a fluorogenic
substrate, but in earlier report it was used to analyse fecal
coliforms in water by employing membrane filter method
where as few as 1 cfu/100 ml of water could be detected in
6 hours. It has also been used to distinguish species of
enterococci. The method engaged dyed starch along with
the substrate, together which were added to a medium
which is chosen for enterococci. By observing for starch
hydrolysis and fluorescence, 86% of enterococci from
environmental samples were correctly differentiated.
ONPG is a colorimetric substrate that is precise for
coliforms. The substrate is hydrolyzed by β-galactosidase
to fabricate a yellow color that can be quantitated at 420
nm. To find out E. coli in water, the organisms are
collected on a 0.45-μm membrane and incubated in EC
medium for 1 hour consequently followed by the addition
of filter-sterilized ONPG. Incubation is continued at 45.5°C
until color develops that can be read at 420 nm. The
sensitivity of ONPG is analogous to that of MUG, with
73
about 107 cells required to produce measurable hydrolysis.
ONPG is engaged in a modification of the classical
presence–absence method for coliforms in water. By this
customized technique, tubes that include coliforms become
yellow. To identify E. coli, each yellow tube is viewed with
a hand-held fluorescent lamp (366 nm); those that contain
E. coli fluoresce brightly. The Colilert and ColiQuik
systems employ both ONPG and MUG as sole nutrient
substrates where total coliforms are confirmed by a yellow
color; E. coli is indicated by MUG fluorescence.

2.7 Whole-Animal Assays

Mouse Lethality

This method was first used to detect foodborne

pathogens around 1920 and continues to be a significant
bioassay technique. To experiment for botulinal toxins in
foods, suitable extracts are made and portions are treated
with trypsin (for toxins of nonproteolytic Clostridium
botulinum strains). Pairs of mice are injected
intraperitoneally (IP) with 0.5 mL of trypsin-treated and
untreated preparations. Untreated preparations that have
been heated for 10 minutes at 1000°C are injected into a
pair of mice. All injected mice are observed for 72 hours
for symptoms of botulism or death. Mice injected with the
heat preparations should not die because the botulinal
toxins are heat labile. Therefore definite serologic type of
botulinal toxin can be determined.
Stark and Duncan used the method for Clostridium
perfringens enterotoxin. Mice were injected IP with
enterotoxin preparations and experimented for up to 72
hours for lethality. The mouse-lethal dose was expressed as
the reciprocal of the highest dilution that was lethal to the
mice within 72 hours. The mouse is the most widely used
74
animal for virulence evaluation of Listeria spp. The LD50
for L. monocytogenes in normal adult mice is 105-106, and
for 15-g infant mice, as few as 50 cells may be lethal.

Suncus Murinus

This small animal has been used in Japan as an

experimental model for emesis research using a variety of
drugs, respond to cereulide, and the emetic toxin of
Bacillus cereus. Suncus murinus is referred to as the
Japanese house shrew, and adults do not exceed 100 g in
weight. For investigational use, those weighing 50-80 g are
used. In their study of the emetic toxin of B. cereus,
ED50 (quantity of toxin required for emesis in one half of
the exposed animals) in Suncus by oral administration was
found to be 12.9 ug/kg. The ED50 by the intraperitoneal
route was 9.8 jig/kg.

Suckling (Infant) Mouse

This animal model was introduced mainly for

Escherichia coli enterotoxins and is now used for this and
some other foodborne pathogens. Typically, mice are
separated from their mothers and given oral doses of the
test substance consisting of 0.05-0.1 mL with the aid of a
blunt 23-gauge hypodermic needle. A drop of 5% Evans
blue dye per milliliter of test material may be used to find
out the presence of the test material in the small intestine.
The animals are regularly held at 25°C for 2 hours and then
killed. The entire small intestine is removed, and the
relative activity of test material is estimated by the ratio of
gut weight to bodyweight (GW/BW).
Test material may also be injected percutaneously
directly in the stomach through the mouse's translucent skin
or by administration orogastrically or intraperitoneally by
75
means of the suckling mouse model. For the selection of
large numbers of cultures, the intestines may be examined
visually for dilation and fluid gathering. Infant mice along
with 1 to 3 day old piglets are the animals of choice for E.
coli enterotoxin STa; STb is inactive in the suckling mouse
but active in piglets and weaned pigs.

Rabbit and Mouse Diarrhea

Rabbits and mice have been engaged to test for

diarrheagenic activity of several foodborne pathogens.
Employing young rabbits weighing 500-800 g, researchers
inoculated orogastrically just about 1010 cells of Y
enterocolitica suspended in 10% sodium bicarbonate.
Diarrhea developed in 87% of 47% rabbits after a mean
time of 5.4 days. Bacterial colonization occurred in all
animals regardless of dose of cells.
Infant rabbits have been used by Smith to assay
enterotoxins E. coli and Vibrio cholerae. Infant rabbits 6-9
days old are administered 1-5 mL of culture filtrate via
stomach tube. Following return to their mothers, they are
experimented for diarrhea. Diarrhea after 6-8 hours is a
positive response. If fatality of animals occurs, a large
volume of yellow fluid is estimated in the small and large
intestines. The quantitation of enterotoxin is achieved by
ascertaining the ratio of intestinal weight to total body
weight.

Monkey Feeding

The utilization of rhesus monkeys (Macaca mulatto) to

assay staphylococcal enterotoxins was developed in 1931
by Jordan and McBroom. Subsequently next to humans,
this is possibly the animal most sensitive to staphylococcal
enterotoxins. When enterotoxins are to be assayed by this
76
method, young rhesus monkeys weighing 2-3 kg are
selected. The food homogenate, frequently in solution in
50-mL quantities, is administered via stomach tube. The
animals are then observed constantly for 5 hours. Nausea
occurs in at least two of six animals indicates a positive
response. Rhesus monkeys have been revealed to respond
to levels of enterotoxins A and B as low as approximately 5
ug per 2-3 kg of body weight.

Kitten (Cat) Test

This method was industrialized by Dolman et al. as an

assay for Staphylococcal enterotoxins. The original test
employed the injection of filtrates into the abdominal cavity
of very young kittens (250-500 g). This outcome leads to
false positive results. The most normally used method
consists of administering the filtrates IV and observing the
animals continuously for emesis. When cats weighing 2-4
kg are used, positive responses occur in 2-6 hours. Emesis
has been reported to happen with 0.1 and 0.5 jug of
staphylococcal enterotoxin A (SEA) and SEB per kilogram
of body weight. The assessment tends to be scarce in the
specificity of the monkey-feeding test as staphylococcal
culture filtrates containing other byproducts may also
stimulate emesis. Kittens are much easier to acquire and
sustain than rhesus monkeys, and in this observation the
test has value.

2.8 Immunological methods

Fluorescent Antibody

This method had comprehensively used in both clinical

and food microbiology since it’s industrialized in 1942. An
antibody to a given antigen is made fluorescent by coupling
77
it to a fluorescent compound and when the antibody reacts
with its antigen, the antigen-antibody complex emits
fluorescence and can be analysed by the use of a
fluorescence microscope. The fluorescent markers
extensively used are rhodamine B, fluorescein isocyanate,
and fluorescein isothiocyanate. The direct method employs
antigen and specific antibody to which is coupled the
fluorescent compound (antigen coated by specific antibody
with fluorescent label). In the indirect method, the labeled
compound analyses the existence of the homologous
antibody; in the direct method, it detects the presence of the
antigen. The FA technique obviates the necessity of pure
culture isolations of salmonellae if H antisera are engaged.
A generally engaged conjugate is polyvalent salmonellae
OH globulin labeled with fluorescein isothiocyanate with
somatic groups A to Z represented. The first flourishing use
of the FA technique for the recognition of foodborne
organisms was made by Russian workers, who engaged the
technique to detect salmonellae in milk.

Enrichment serology

The use of enrichment serology (ES) is a fast method for

recovering salmonellae from foods than the conventional
culture method (CCM). Originally developed by Sperber
and Deibel. enrichment in a nonselective medium for 18
hours; selective enrichment in selenite-cystine and/or
tetrathionate broth for 24 hours; selective enrichment in M
broth for either 6-8 hours or 24 hours; and agglutination
with polyvalent H antisera at 500°C for 1 hour, results are
obtained in 50 hrs. Overall, the ES method provides results
in 32-50 hours compared to 92-120 hours for CCM, results
are comparable to both CCM and FA, and no specialized
equipment or training is needed.
Salmonella 1-2 Test
78
 This process is similar to ES. This relies on antibody
reaction with flagellated salmonellae strains. Unlike ES, the
1-2 test employs the use of a semisolid phase. The process
is conducted in a specially designed plastic device that has
two chambers, one for selective broth and the other for a
nonselective motility medium. In addition to selective
ingredients, the latter contains the amino acid L-serine,
which is selective for salmonellae. Following inoculation of
the discerning medium chamber, the apparatus is incubated,
during which time motile salmonellae move into the
nonselective medium chamber. The latter contains flagellar
antibodies, and when the motile organisms enter the
antibody area, an immunoband forms and confirms the
presence of antigen-antibody reaction, results are obtained
within 8-14 hrs. With the accumulation of a tetrathionate
brilliant green broth enhancement step for the 1-2 Test, 84
of 314 samples were salmonellae positive—3 more than the
culture method—and outcome could be obtained 1 day
before the culture method.

Radioimmunoassay

This method consists of adding a radioactive label to an

antigen, allowing the labeled antigen to react with its
specific antibody, and measuring the amount of antigen that
pooled with the antibody by the use of a counter to evaluate
radioactivity. Solid-phase radioimmunoassay (RIA) refers
to methods that employ solid materials or surfaces onto
which a monolayer of antibody molecules binds
electrostatically. The solid materials consist of
polypropylene, polystryene, and bromacetylcellulose.
When the free-labeled antigen is washed out, the
radioactivity dimensions are quantitative. Staphylococcal
enterotoxin A was extracted from a variety of foods,
79
including ham, milk products, and crabmeat, by Collins et
al. and considered by RIA, all within 3-4 hours. By
iodination of enterotoxins, solid-phase RIA can be engaged
to detect as little as 1 ng of toxin per gram. The RIA
method lends itself to the assessment of foods for other
biological hazards such as endotoxins, paralytic shellfish
toxins. The detection and recognition of bacterial cells
within 8-10 minutes have been achieved by use of 125I-
labeled homologous antibody filtered and washed on a
Millipore membrane.

ELISA

The enzyme-linked immunosorbent assay (ELISA,

enzyme immunoassay, or EIA) is an immunological
process analogous to RIA but the use of an enzyme coupled
to either an antigen or an antibody rather than a radioactive
isotope. A typical ELISA is done with a solid-phase
(polystyrene) coated with antigen and incubated with
antiserum. Following incubation and washing, an enzyme-
labeled preparation of anti-immunoglobulin is added. After
gentle washing, the enzyme remaining in the tube or
microtiter well is assayed to find out the amount of specific
antibodies in the initial serum. A commonly used enzyme is
horseradish peroxidase and its amount is calculated by the
addition of peroxidase substrate. The sum of enzyme
present is ascertained by the colorimetric analysis of
enzyme substrate. The ELISA technique is used widely to
detect and quantitate organisms and/or their products in
foods.

Botulinum Toxins

• For type A toxin a "double-sandwich" ELISA detected

50-100 mouse LD50 of type A and <100 mouse i.p. LD50
80
of type E; and a "double-sandwich" ELISA with alkaline
phosphatase and polystyrene plates has been shown capable
of detecting 1 mouse i.p. median lethal dose of type G
toxin.

E. coli Enterotoxins

• A monoclonal antibody definite for enterohemorrhagic

strains of E. coli (EHEC) was shown to be highly precise
when used in an ELISA to identify EHEC strains.
• Two "sandwich" ELISAs were developed based on
toxin-specific murine monoclonal capture antibodies and
rabbit polyclonal second antibodies specific for the Stxl and
Stx2 genes of E. coli. The Stxl ELISA could identify 200
pg of purified Stxl toxin, while the Stx2 could distinguish
75 pg of Stx2 toxin.

Gel Diffusion

Gel diffusion methods have been widely used for the

recognition and quantitation of bacterial toxins and
enterotoxins. The four most used are the single-diffusion
tube (Oudin), microslide double diffusion, micro-
Ouchterlony slide, and electroimmunodiffusion. They have
been engaged to measure enterotoxins of staphylococci and
C. perfringens and the toxins of C. botulinum. The micro-
Ouchterlony method can identify 0.1-0.01 jig of
staphylococcal enterotoxin, which is the same limit for the
Oudin test. The double-diffusion tube test can identify
levels as low as 0.1 ug/mL, but the incubation period
required for such low levels is 3-6 days.

Hemagglutination

Gel diffusion methods normally need at least 24 hours

81
for results, two comparable serologic methods yield results
in 2-4 hours: hemagglutination-inhibition (HI) and reverse
passive hemagglutination (RPH). In the HI test, precise
antibody is kept stable and enterotoxin (antigen) is diluted
out. Following incubation for about 20 minutes, treated
sheep red blood cells (SRBCs) are added.
Hemagglutination (HA) occurs only when antibody is not
bound by antigen contrast to HI, antitoxin globulin in RPH
is attached directly to SRBCs and used to detect toxin.
When diluted toxin preparations are added, the experiment
is read for HA after incubation for 2 hours. HA occurs only
where optimal antigen antibody levels arise.

82
3 Chapter-3: Food Preservation using
Chemicals

Introduction

Preservative for food may be distinct as any substance

and/or process, when applied to food, check alterations
caused by the intensification of microorganisms or enable
the physical properties, chemical composition and nutritive
value to stay unaltered by microbial growth. Some
chemicals have been used conventionally since a number of
decades as direct or indirect inhibitors of microbial growth
and are still generally used in spite of their boundaries.

3.1 Chemicals:-
The popular food preservation operations used
nowadays also make utilization of some kind
of chemical additive to reduce spoilage. Some well-known
examples of the previous class of food additives are sodium
benzoate and benzoic acid; and propionic acid; calcium,
potassium, sodium sorbate, and sorbic acid. Examples of
the latter class of additives include calcium, sodium
ascorbate, and ascorbic acid (vitamin C); and butylated
hydroxytoluene (BHT); and sodium and potassium sulfite
and sulphur dioxide.

83
Classification of Preservatives

According to FSSA rules → class I and class II

preservatives
Class I preservatives
[Link]
[Link]
[Link]
[Link]
[Link]
[Link]
[Link]
h. Edible vegetable oil

Addition of class I preservatives in any food is not

restricted, unless otherwise provide in the rule.

Class II preservatives

Use of class II preservatives is limited. They shall be

added further to only specified item for consumption and at
a concentration not higher than the quantity specified for
the product.
Use of more than one class II preservative is prohibited.
No human being shall use in or upon a foodstuff more than
one class II preservative

Benzoic acid and its salt

Commonly utilized as an antimicrobial agent. Benzoates

are more efficient but exhibit their action against yeasts and
bacteria than molds. Antimicrobial activity is achieved by
inhibition in enzymatic system of microbial cells,
distressing acetic acid metabolism, citric acid cycle and
84
oxidative phosphorylation.

Microbicidal action is influenced by pH of

medium. The highest inhibition occurs at pH value of 2.5 to
4.0 and it reduces when pH rises above 4.5.
The food products conserved with the benzoate include
fruit juices, salads, jams, pickles, dried fruits and sauce,
carbonated beverages, bakery items and other fat spreads,
spices.

Sulphur dioxide and sulfites

Sulphur dioxide (SO2) gas is one of the oldest antimicrobial

agents. It is a colourless, nonflammable gaseous compound
or liquid under pressure with a disgusting strong odour.
When dissolved in water of foods, it yields sulphurous acid
and its ions, owing to its solubility in water.
Sulphite salts such as potassium sulphite, sodium sulphite,
sodium metabisulphite, potassium metabisulphite used as
preservatives. When dissolved in water, form sulphurous
acid, bisulphite and ions. Sulphurous acid formed from
these compounds is a vigorous antimicrobial material. The
efficiency of sulphurous acid is higher at low pH values.
Microbicidal assessment of sulfites against yeasts, molds
and bacteria is discriminating, with certain species being
more susceptible to inhibition than others. Bacteria are
usually more susceptible to inhibition than yeasts and
molds. In addition to antimicrobial assessment, they are
also employed, to avoid enzymatic and non-enzymatic
alteration and discoloration in several foods. Sulphur
dioxide and sulphites are used in fruit products for instance
pickles fruit juice concentrate and chutneys.

85
Sorbic acid and its salts

Sorbic acid and its salts (calcium, potassium or sodium

salts) are proficient antimicrobial agents against yeast and
molds, as well as bacteria. They are less competent against
bacteria. Sorbate has an upper pH limit for activity
around 6.0-6.5. The food products conserved with
sorbates are tomato products, carbonated beverages, candy
jellies, chocolate syrup, smoked fish, fruit juices, cheese,
grains and [Link] acid and its salts
Propionic acid & its salts (Ca & Na) are used most
commonly in the deterrence of mold growth and rope
progression in baked supplies and for mold inhibition in
lots of cheese foods and spreads. They are more competent
against molds as compared to yeasts and bacteria.
Propionate has an upper pH limit for activity around 5 to 6.

Lactic acid and its salts

Lactic acid is produced during fermentation of lactose by

lactic acid bacteria. Lactic acid & its salts are not very
familiar & not easily accessible. It can be worn in pickles
(with acetic acid), crispy biscuits, some beverages, dairy &
meat foodstuffs. Calcium lactate is used as a firming agent
in fruits, vegetables and pickles. Na & K lactate are also
recommended with sodium diacetate for managing food
poisoning & other bacteria in meat product.

Acetic acid has antimicrobial properties. The execution

remains to be fixed rather than cidal. It is more proficient
against bacteria & yeast then molds. Vinegar is made of 5
to 10 % solution of acetic acid. mayonnaise, pickles, sauce,
pickled sausage etc are acetic acid used in the form of
vinegar

86
Sodiumchloride (common salt)

Microbicidal accomplishment of NaCl arises from its

lowering water activity (aw) of the food product. This
reduces accessible water in food to the certain level which
renders adverse for microbial growth. At higher
concentration it has a prominent bacteriostatic action. The
10% NaCl inhibits the multiplication of most bacteria.
Delaying action upon bacteria- creates lack of moisture in
microbial cell—by osmosis—varying results into plasmosis
of the cell. Oxygen level in food is decreased by
reduction in solubility of oxygen—decrease growth of
aerobic microorganisms. It is more efficient against
bacteria & mold in contrast to yeast.
One of the conventional method of food preservation.
Mainly used to preserve meat, pickles & fish. Fish is
usually salted by immersing in brine or by mixing with dry
salt. It is mostly significant as a preservative for cheese &
table butter. Depending upon type of cheese salt content
varied from 1 to 5 %. In table butter salt is added at a max
concentration as 3%.

Sucrose (sugar)

More effectual against bacteria & mold compared to yeast.

Microbicidal action of sucrose arises from, lowering water
activity (aw) of the food product which is unfavourable for
microbial growth. This creates dehydration of microbial
cell—by osmosis results into plasmosis of the cells. The
food products conserved with sugar are fruit products, dairy
products.

3.2 Antibiotics

Historically, secondary metabolites formed by

87
microorganisms that destroy a broad spectrum of other
microorganisms are known as antibiotics. The majority of
the constructive ones are produced by molds and bacteria
of the genus Streptomyces, and a the minority by Bacillus
and Paenibacillus spp. Consumers does not accept the
utilization of chemical preservatives in foods; the usage of
antibiotics is very low. Consumption of any food additive
cause harm, but the risks should not outweigh the benefits
overall
1. The antibiotic agent should destroy but not hinder the
flora and should preferably putrefy into harmless products
or be damaged on food preparation for products that need
cooking.
2. The antibiotic are not supposed to be inactivated by
food components or goods of microbial metabolism.
3. The antibiotic should not voluntarily arouse the
appearance of resistant strains.
4. The antibiotic are not to be utilized in foods if used
therapeutically or as an mammal nourishment additive.

Monensin

This antibiotic was standardized by the FDA as a cattle

feed additive in the 1970s, and it is used predominantly for
advancement of nourishment efficiency in ruminants. In
fistulated cows amino acid-sparing action has been
confirmed. It inhibits Gram-positive bacteria, and thus its
long-term use has the possibility of shifting the
gastrointestinal tract bacterial biota from one that is usually
Gram positive to one that is more Gram negative. Similar
to nisin, monensin is an ionophore (destroys selective
permeability of cell membranes), and the two agents to
assess constructively as feed additives.

Natamycin
88
 This antibiotic is a polyene that is quite effective against
yeasts and molds but not bacteria. Natamycin was isolated
from Streptomyces natalensis. In yielding the recognition
of natamycin as a food preservative, the joint Food and
Agriculture Organization/the World Health Organization
(FAO/WHO) Expert Committee took the following into
consideration: it does not affect bacteria, it stimulates an
unusually low level of resistance among fungi, it is rarely
engaged in cross-resistance among other antifungal
polyenes, and DNA transfer between fungi does not happen
to the extent that it does with some bacteria.

Table: 3 Properties of Some antibiotics

Also, from Table 3, it may be noted that its use is

restricted as a clinical agent, and it is not used as a feed
additive.
Natamycin has been shown by a number of investigators
to be effective against both yeasts and molds, and many of
these reports have been summarized. The relative
effectiveness of natamycin was compared to that of sorbic
acid and four other antifungal antibiotics by Klis et al. for
the inhibition of 16 different fungi (mostly molds), and
although 100–1,000 ppm of sorbic acid were required for
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inhibition, 1–25 ppm of natamycin were capable against the
same strains in the same media. To control fungi on
strawberries and raspberries, natamycin was compared with
rimocidin and nystatin, and it, along with rimocidin, was
effective at levels of 10–20 ppm, whereas 50 ppm of
nystatin were necessary for effectiveness. In controlling
fungi on salami, the spraying of fresh salami with a 0.25%
solution was found to be effective by one group of
investigators, but another investigator was unsuccessful in
his attempts to prevent surface-mold growth on Italian dry
sausages when they were dipped in a 2,000-ppm solution.
Natamycin spray (2 × 1,000 ppm) was as good as or
slightly better than 2.5% potassium sorbate. Natamycin
appears to act in the identical way as other polyene
antibiotics—by binding to membrane sterols and inducing
deformation of selective membrane permeability. Because
bacteria do not acquire membrane sterols, their lack of
sensitivity to this agent is thus explained.

Tetracyclines

Chlortetracycline (CTC) and oxytetracycline (OTC)

were approved by the FDA in 1955 and 1956, respectively,
at a level of 7 ppm to control bacterial spoilage in
uncooked refrigerated poultry, but these approvals were
subsequently rescinded. The efficiency of this group of
antibiotics in extending the shelf life of frozen foods was
first recognized by Tarr and associates working with fish in
Canada.
Succeeding research by a large number of workers in
many countries established the effectiveness of CTC and
OTC in delaying bacterial spoilage of not only fish and
seafoods but poultry, red meats, vegetables, raw milk, and
other foods. CTC is generally more efficient than OTC.
The surface treatment of chilled meats with 7–10 ppm
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typically results in shelf-life extensions of at least 3–5 days
and a shift in crucial spoilage biota from Gram-negative
bacteria to yeasts and molds. The tetracyclines are both
heat sensitive and storage labile in foods, and these factors
were significant in their initial recognition for food use.
They are used to treat diseases in humans and animals and
are used also in nutritive supplements in the United States.

Subtilin

This antibiotic was revealed and developed by scientists

at the Western Regional Laboratory of the USDA. It is
formed by some strains of Bacillus subtilis. Like nisin, it is
efficient against Gram-positive bacteria, is steady to acid,
and possesses enough heat resistance to withstand
destruction at 121°C for 30–60 minutes. Subtilin is
proficient in canned foods at levels of 5–20 ppm in
preventing the consequence of germinating endospores, and
its site of accomplishment is identical as for nisin (Figure
13–1). Like nisin, it is used neither in the management of
human or animal infections nor as a nutrition additive. This
antibiotic may be just as efficient as nisin, although it has
received slight consideration since the late 1950s.

Tylosin

This antibiotic is a nonpolyene macrolide, as are the

clinically functional antibiotics erythromycin,
oleandomycin and others. It is more inhibitory than nisin or
subtilin. Denny et al. were apparently the first to study its
possible use in canned foods. No spoilage occurred after 30
days with incubation at 54°C when 1 ppm was added to
cream-style corn containing flatsour spores and given a
“botulinal” cook. Similar results were made by others in the
1960s, and these have been summarized. Unlike nisin,
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subtilin, and natamycin, tylosin is used in animal feeds and
also to take concern of several diseases of poultry. Gram-
positive bacteria is sensitive to tylosin as a macrolide. It
inhibits protein synthesis by associating with the 50S
ribosomal subunit and shows at least imperfect cross-
resistance with erythromycin.

3.3 Radiation

Energy can be propagated through space by means of

electromagnetic radiation. It is characterized in terms of its
wavelength l, or its frequency n, and the product of these
two properties gives the speed, c, at which it travels (3×108
msec-1 in a vacuum).
E=h‫ע‬
where h is a constant (6.6×10-27 ergs sec_1) known as
Planck’s constant. Thus, elevated the frequency of the
radiation the superior its quantum energy. Three areas of
the e.m. spectrum is considered; microwaves, the UV
region and gamma rays.

Microwave Radiation

The microwave region of the e.m. spectrum occupies

frequencies between 109 Hz up to 1012 Hz and so has a
relatively low quantum energy. For the two frequencies
employed in food processing, 2450MHz and 915 MHz, this
is around 10-18 ergs or 10-6 eV. Domestic microwave
ovens use 2450MHz which is less penetrating than the
lower frequency. Microwaves act indirectly on micro-
organisms through the generation of heat. The dipolar
water molecules align themselves with the field, when a
food consisting of water is placed in a microwave field.
Each second the water molecules are persistently
92
oscillating as the field reverses its polarity about 2.5×109
times. Due to rapid increase in temperature throughout the
product this kinetic energy is transmitted to neighbouring
molecules.
A device initially developed in the UK during
investigation into radar during the Second World War are
microwaves are generated using a magnetron. Due to the
incidence of cold spots in the oven, and the non-uniform
dielectric properties of the food in household use of
microwaves is non-uniform heating of foods. These can
lead to cold spots in some microwaved foods and concern
over the risks related with employment of inadequately
heated meals has led to more explicit orders on
microwaveable foods. These often indicate a tempering
period after heating to permit the temperature to
equilibrate. Microwaves have been used to defrost frozen
blocks of meat prior to their processing into products such
as burgers and pies thus reducing wear and tear on
machinery. There has also been a incomplete application of
microwaves in the blanching of fruits and vegetables and in
the pasteurization of soft bakery goods and moist (30%
H2O) pasta to destroy yeasts and moulds.
In Japan, microwaves are included to pasteurize high-
acid foods, for example fruits in syrup, planned for
distribution at ambient temperature. These are packed
before processing and have an indefinite microbiological
shelf-life because of the heat process and their low pH.
However, the modest oxygen barrier properties of the pack
has intended that their biochemical shelf-life is limited to a
few months.

UV Radiation

UV radiation has wavelengths below 450nm (nC1015

Hz) and a quantum energy of 3–5 eV (10-12 ergs). The
93
quanta contain energy enough to excite electrons in
molecules from their ground state into higher energy
orbitals constructing the molecules more reactive.
Chemical reactions thus induced in micro-organisms can
cause the malfunction of critical metabolic processes
leading to injury or death.
Only quanta providing energy enough to excite these
photochemical reactions will inhibit micro-organisms, so
those wavelengths that are most proficient give us an
implication of the sensitive chemical targets within the cell.
The greatest lethality is shown by wavelengths around
260nm which correspond to a strong absorption by nucleic
acid bases. The pyrimidine bases appear particularly
sensitive, and UV light at this wavelength will, among
other things, encourage the formation of covalently linked
dimers between adjacent thymine bases in DNA. If left
intact these will avoid transcription and DNA replication in
affected cells. The resistance of micro-organisms to UV is
basically determined by their ability to repair such damage,
although some organisms such as micrococci also
synthesize protective pigments. Generally, the resistance to
UV irradiation follows the pattern:
Death of a population of UV-irradiated cells
demonstrates log-linear kinetics alike to thermal death and,
in a similar way, D values can be determined. These give
the amount necessary to construct a tenfold reduction in
existing numbers where the dose, expressed in ergs or
mWs, is the creation of the intensity of the radiation and the
time for which it is applied.

Determination of UV D values is not generally a

straightforward affair since the incident radiation can be
engaged by other medium components and has very low
penetration. Passage through 5 cm of clear water will
decrease the strength of UV radiation by two-thirds. This
94
consequence amplify with the concentration of solutes and
suspended material so that in milk 90% of the incident
energy will be absorbed by a layer only 0.1mm thick. Low-
pressure mercury vapour discharge lamps are used: 80% of
their UV emission is at a wavelength of 254nm which has
85% of the biological activity of 260 nm. Wavelengths
below 200nm are screened out by surrounding the lamp
with an absorbent glass since these wavelengths are
engaged by oxygen in the air producing ozone which is
harmful. The yield of these lamps falls off over time and
they need to be monitored frequently.

Air disinfection is only functional when the organisms

suspended in air can make a important involvement to the
product’s microflora and are possible to harm the product;
for instance, in the manage of mould spores in bakeries.
UV lamps have also been mounted in the head space of
tanks storing concentrates, the steadiness of which depends
on their low aw. Fluctuations in temperature can cause
condensation to form inside the tank. If this contacts the
product, then areas of locally elevated aw can form where
formerly latent organisms can grow, spoiling the product.

Process water can be disinfected by UV; this avoids the

risk of tainting sometimes associated with chlorination,
although the treated water will not have the residual
antimicrobial properties of chlorinated water. UV radiation
is normally used in the depuration of shellfish to sterilize
the water recirculated through the depuration tanks.
Chlorination would not be apt in this situation as residual
chlorine would cause the shellfish to prevent feeding thus
stopping the depuration process. Surfaces can be sanitized
by UV, although protection of microorganisms by organic
material such as fat can decrease its efficacy. Food
95
containers are frequently treated in this way and some meat
chill store rooms have UV lamps to hinder surface growth.
UV can however stimulate spoilage of products containing
unsaturated fatty acids where it accelerates the progress of
rancidity. Process workers must also be protected from UV
since the wavelengths are the root cause for burning of the
skin and eye disorders.

Ionizing Radiation

Ionizing radiation has frequencies better than 1018 Hz

and carries sufficient energy to eject electrons from
molecules it encounters. In practice three different types are
used.
(1) High-energy electrons in the form of b particles
created by radioactive decay or machine generated
electrons. Strictly speaking they are particles rather than
electromagnetic radiation, although in some of their
behaviour they do exhibit the properties of waves. Because
of their mass and charge, electrons have a tendency to be
less penetrating than ionizing e.m. radiation; for example,
5MeV b particles will normally enter food materials to a
depth of about 2.5 cm.
(2) X-rays generated by impinging high energy electrons
on a suitable target.
(3) Gamma g rays formed by the decay of radioactive
isotopes. The most commonly used isotope cobalt 60,
60Co, is formed by bombarding non-radioactive cobalt,
59Co, with neutrons in a nuclear reactor. It emits high-
energy g-rays (1.1 MeV) which can enter food up to a
depth of 20 cm (cf. b particles). An isotope of caesium,
137Cs, which is extracted from spent nuclear fuel rods, has
also been used but is less favoured for a number of reasons.
Ionizing radiation can influence micro-organisms directly
by interacting with key molecules within the microbial cell,
96
or indirectly during the inhibitory effects of free radicals
produced by the radiolysis of water. In the absence of water
these indirect effects play significant role, doses 2–3 times
superior are necessary to attain the same lethality. Removal
of oxygen also boost microbial resistance 2–4 fold and it is
thought that this may be due to the capacity of oxygen to
take part in free radical reactions and evade the repair of
radiation induced lesions. As with UV irradiation, the main
site of damage in cells is the chromosome. Hydroxyl
radicals cause single- and double-strand breaks in the DNA
molecule as a result of hydrogen abstraction from
deoxyribose followed by b-elimination of phosphate which
cleaves the molecule. They can also hydroxylate purine and
pyrimidine bases. Resistance to ionizing radiation depends
on the capability of the organism to repair the damage
caused. Inactivation kinetics is normally logarithmic,
although survival curves often appear sigmoidal exhibiting
a shoulder and a tail to the phase of log-linear death. The
shoulder is usually very small but is more evident with
bacteria which have more proficient repair mechanisms
where substantially more harm can be accumulated before
death ensues.

D values can be derived from the linear portion of these

curves 6D values (the dose to produce a million fold
reduction) reported for a quantity of foodborne organisms.
These are calculated in terms of the absorbed amount of
ionizing radiation which is measured in Grays (1 Gy¼1
joule kg_1).
Food-associated organisms do not display exceptional
resistance, although spores of several strains of Clostridium
botulinum type A have the the majority of radiation
resistant spores. As studies on food irradiation are in
progress, numerous bacteria which are highly resistant to
radiation have been isolated. Although one of these,
97
Deinococcus radiodurans, was initially isolated from meat,
their function in foods is not considerable in the common
measures. After the findings of radioactivity at the turn of
the 20th century, even though patents describing the
utilization of ionizing radiation in the management of food
appeared soon. This was largely due to technological
advances during the progress of nuclear weapons but also
to a strong wish to reveal that nuclear technology could
recommend the human race several other benefits. In
particular, food irradiation has the gain of being a much
more accurately controlled process than heating, since
penetration is deep, direct and identical.

This malfunction of low doses of radiation to generate

substantial chemical modification in the product has been a
barrier to the progress of easy experiment to decide
whether a food has been irradiated. Although availability of
such an analysis would assist international trade in
irradiated food, increase consumer confidence and help
enforce labeling regulations. Free radicals formed by
irradiation can be analysed using electron spin resonance
when they are trapped in solid matrices such as bone, seeds
and shells. The energy accumulated in grains of silicate
minerals as a result of irradiation can be calculated in foods
for instance herbs and spices using thermo luminescence
and long chain volatile hydrocarbons and 2-
alkylcyclobutanones formed by irradiation of fatty foods
can be detected using gas chromatography. One
microbiological test for irradiated food is based on the ratio
between an evaluation of total microbial numbers using the
DEFT method and a plate count to find out the number of
viable bacteria present. In 1981 an expert international
committee of the FAO/WHO and the International Atomic
Energy Authority recommended general approval of food
irradiation up to a level of 10 kGy. Further toxicological
98
examinations of such treated foods are therefore not
required and there is no toxicological risk.

It had been considered that irradiation could direct to

pathogens becoming further virulent but, apart from one or
two exemption, it has been analysed that where virulence is
affected it is diminished. In the exceptions illustrated, the
consequence was slight and not sufficient to compensate
for the overall reduction in feasible numbers. No example
has been found where a nonpathogenic organism has been
converted to a pathogen as a result of irradiation. Although
it has been depicted that spores of some mycotoxigenic
moulds which endure irradiation may yield cultures with
better mycotoxin production.

Morphological, biochemical and other changes which

may impede isolation and recognition and increased
radiation resistance have been well-known as a result of
repeated cyclic irradiation. However, these analyses were
performed under the most favourable circumstances and for
this to happen would involve extensive microbial regrowth
after each irradiation; a state that is readily avoidable by
good sanitary practices and is most unlikely to occur.

The levels of radiation proposed for foods are not

sufficient to stimulate radioactivity in the product and there
is no indication that consumption of irradiated foods is
detrimental. Food irradiation facilities do require severe
safety principles to guard workers but that is already in
place for the irradiation of other equipment such as the
sterilization of medical goods and disposables.

The greatest trouble to the more widespread utilize food

irradiation is not technical but sociological in the form of
extensive consumer resistance and distrust. Much of this is
99
based on inadequate information and false propaganda and
parallels very directly former arguments over the qualities
of milk pasteurization. Among the same objections raised
then were that pasteurization would be worn to mask
deprived quality milk and would encourage poor practices
in food preparation. While it has to be agreed that those
who take the most cynical view of human nature are often
proved correct, this did not prove to be the case with milk
pasteurization where the production standards and
microbiological quality of raw milk are at elevated levels
than they have ever been.

Depending on the lethality required, food irradiation can

be applied at two diverse levels. At high levels it can be
used to produce a safe shelf stable product in a treatment
known as radappertization. Though this has been
investigated in the context of military rations, it is unlikely
to be a commercial actuality in the forseeable future. C.
botulinum spores are the major radiation resistant known,
so very high doses are necessary to attain the least standard
of a 12D reduction (E45 kGy) for low acid foods. In the
event of a progression failure, the intensification of more
challenging, non-pathogenic clostridia would not act as a
caution as it can be done in thermal processing. High
radiation doses are also more likely to generate
unacceptable sensory changes and the product has to be
irradiated in the freezing state to decrease migration of the
radiolytic species that reason for such alterations. These
considerations would not be relevant when the food was
inhibitory to the multiplication of C. botulinum as a
consequence of low pH or the occurrence of agents such as
curing salts.
Two terms are used to differentiate various types of
radiation pasteurization. Radicidation is used to depict the
procedure where the objective is the eradication of a
100
pathogen, as, for instance, elimination of Salmonella from
meat and poultry. Radurization applies to development
intends to prolong shelf-life. This will invariably improve
both safety and shelf-life.

3.4 Food preservation by Low Temperature

In order to slow down the spoilage process and increase
the shelf life of foods for a longer period, a wide range of
preservation methods are applied to foods like
pasteurization, boiling, refrigeration, freezing, vacuum
treatment, addition of antimicrobial agents. In general, most
methods involve the removal or control of factors which
affect bacterial growth such as the use of low or high
temperatures, moisture control, dehydration and use of
certain chemicals as preservatives. In practice, there are
two important methods used to control or kill the
microorganism: 1. Physical methods: Mostly temperature
control methods like low or high temperatures. 2. Chemical
methods: It included certain antimicrobial chemicals such
as sorbate, benzoates, nitrates

Physical methods of food preservation

Physical preservation methods are mostly aimed at

extending the stability of foods by slowing down or
suppressing the spoilage mechanisms by the changing of
relevant parameters. Microbial growth and multiplication
can be influenced by temperature, water activity and high-
energy radiation. Generally, microorganisms are killed by
the application of high temperatures in combination with
the treatment times required in a given case. Comparatively
mild treatments affect only the vegetative forms
101
(pasteurization), while more destructive treatments are
required to kill spores as well (sterilization). The
pasteurized foods need additional cold storage to prevent
further germination of the spores. Use of low temperatures
processing, reduces and inhibits the growth of microbes (in
refrigeration) or prevent any microbial activity at deep-
freezing. High temperature methods such as canning,
sterilization and cooking are capable in complete
destruction of all forms of microbes and their toxins.
Hence, providing more safe food for consumption.
Application of ionizing radiation on certain food can be
used to reduce a potential risk to hygiene, to influence
physiological processes, to control insect’s populations and
to increase the shelf-life of fresh food within in the short
time. However, at the end the optimum product quality can
be retained by linking various preservation processes. Few
important and most widely accepted as well as used low
and high temperature based methods for food preservation
are discussed below.

Low Temperature Methods: Storage at low temperatures

extends the shelf life of many foods. Because, low
temperatures reduce the growth rates of microorganisms,
slows down the speed of enzyme reaction and slow many
of the physical and chemical reactions that happen in foods.
Hence, the use of low temperatures can prevent the growth
of most food borne pathogens and food spoilage causing
microorganisms. Preservation methods using low
temperatures include:

Refrigeration

It is a current food preservation method based on the

information that harmful foodborne bacteria do not grow at
refrigeration temperatures (0-5°C or below). In common,
102
refrigeration is considered as a provisional food
preservation process as it will slow down the development
of microbes (food spoilage microbes: bacteria and fungi)
but doesn't avoid it finally as freezing does. Hence
refrigeration conserves only food for days. Almost, the life
of lots of foods may be extended by storage at temperatures
below 4°C. Refrigeration cannot progress the quality of
decayed food as it can only retard microbial deterioration.
Most commonly refrigerated foods like fresh fruits and
vegetables, eggs, fish, dairy products, meats etc. Certain
foods like bananas cannot be refrigerated because it is
damaged if exposed to low temperatures. Limitation: There
is one issue of modern mechanical refrigeration that is
dehydration of stored foods because of moisture
condensation and though, it has been overcome through
humidity control mechanisms within the storage chamber
and by selecting suitable packaging techniques.

Freezing

It is one of the simple, less time-consuming and most

appropriate methods of food preservation. Freezing is very
high-quality at retain the dietary value and their normal
color, aroma and texture superior than when other methods
of food preservation are used for example fruits, meats,
breads, cakes etc. It is suggested that before freezing the
fresh vegetables, must be blanched first to stop the
enzymatic activity. For blanching, vegetables dropped into
a pot of boiling water. Allow them for 1-2 minutes and then
immediately stop the cooking process by removing them
from the boiling water and dropping them into ice water.
As freezing one of the most commonly used processes
commercially and domestically for preserving a very wide
range of food including prepared food stuffs that would not
have required freezing in their unprepared state, such as
103
potato waffles are stored in the freezer, but potatoes
themselves need only a cool dark place to ensure several
days storage. In general, freezing does not destroy spoilage
or contamination microorganisms but it stops their growth
as long as the food is kept at -180°C. Any microorganism
present will become active as the food thaws. Proper
packaging of food in freezer paper or containers can
prevents any deterioration in its quality and damage occurs
once food comes in contact with the dry air of a freezer.
Following important point should be taken into
consideration to avoid freezer burn: Avoid fluctuating
temperatures: Maintain the freezer closed as much as
possible.

3.5 High Temperature Methods

Heat is one of the oldest methods of destroying
microorganisms and their spores in food processing and
preservation. Growth of several elevated temperature based
process like boiling, roasting, baking and other heat
treatments are the greatest progress in food hygiene
because such technique kills all the forms of
microorganisms (vegetative and spore) and make the food
protected. Most commonly used methods of heat treatment
used for food preservation are discussed below.

Pasteurization

It is named after its inventor French chemist, Louis

Pasteur (1822-1895). He used the application of heat to
destroy human pathogens in foods. The principle of
pasteurization is to amplify milk safety for the customer by
devastating disease causing pathogenic microorganisms
that may be there in milk and to increase keeping the
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quality of milk products by destroying spoilage
microorganisms and inactivating enzymes that contribute to
the poor quality and shelf life of milk. In general,
pasteurization is a process of heat treatment of milk and
other beverages requires adequate holding time to assure
the thermal destruction of pathogens and microbes
accountable for food spoilage, without any changing in the
nutritional qualities. Milk is a food product that is
pasteurized worldwide, but some other foods are normally
pasteurized in certain parts of the world:

Canned foods: Such as meats, vegetables and fruits that

are generally heated in the can or container itself to kill the
microbes effectively.

Juices: Most of the Tetra Pak and bottled juices are

pasteurized first before.

Low-alcoholic beverages: Such as juices, these beverages

are also heated and cooled before filling.

Water: the bottled water is usually pasteurized and in

places where polluted water is available.

Practically, pasteurization involves heating the food to a

specific temperature for a definite time and then cooling
quickly. Usually, the temperature applied and the holding
time of pasteurization varies with the equipment available
and the type of milk or food product (Table 10.1). For
pasteurization of milk, the time temperature combination is
carefully selected on the basis of the thermal death time of
the most resistant pathogens (such as Mycobacterium
tuberculosis) that may be present in raw milk and keeping
the maximum temperature and time at which the nutritional
105
and other qualities of milk (like taste and palatability) are
retained. At dairy industries, regularly milk is pasteurized
at 62.8°C for minimum 30 minutes or at 71.7°C for 15
seconds or with ultra-high temperature (UHT) at 135°C for
1–2 seconds. UHT processed milk is sterilized (all forms of
life are killed) and this lengthens its storage time but does
affect the taste and other values.

Sterilization

It is a method of destruction of all microorganisms using

temperatures above 100°C. For complete sterilization of
foods, the time and temperature required are much
influenced by a number of factors like the type of
microorganisms found on the food, the size of the
container, the acidity or pH of the food and the method of
heating. In general, the thermal processes of canning are
generally designed to destroy the spores of the bacterium C.
botulinum (can easily grow under anaerobic conditions,
producing the deadly toxin that causes botulism). In
sterilization process, heating to temperatures greater than
100°C and However, C. botulinum is not viable in acidic
foods (pH less than 4.6), therefore, such foods can be
adequately processed by dipping in water at temperatures
just below 100°C. For low-acid foods (pH greater than 4.6),
the sterilization is generally carried out in steam vessels
called retorts at temperatures ranging from 116° to 129°C
and the retorts are controlled by programmed devices and
detailed records are kept of the time and temperature
treatments for each batch of processed cans. At the end of
the heating cycle, the cans are cooled under water sprays or
in water baths to about 38°C and then dried to prevent any
surface rusting. The cans are then labeled properly, placed
in fibre board cases either by hand or machine and stored in
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cool and dry storerooms.

Ultra-Heat Treatment (UHT)

It is also called, ultra pasteurization (UP), the most

widely used pasteurization process for milk in Europe and
throughout the world ( gaining popularity in the United
States and Canada too), is a more recently developed
process of sterilization of food by heating it for an
extremely short period, about 1–2 seconds, at a temperature
exceeding 135°C. Generally, such temperature is required
to kill spores in milk. Although, the most common UHT
product is milk, but the process is also used for fruit juices,
cream, soy milk, yogurt, wine, soups and honey. When
UHT process is coupled with sterile packaging (tetrapak),
creates an extended shelf life of milk UHT milk from six to
nine months, until opened. At industrial scale, the UHT
milk passes through heating and cooling stages in rapid
succession, and then it is immediately put into a sterile
Tetra Pak shelf-safe carton to avoid any re-infection. Such
product lasts up to six months without refrigeration or
addition of any preservatives. Specially, the Tetra Pak
made of Paperboard, obtained from wood from selectively
harvested (regrown trees), is used to make the package
more stable and lightweight. Thin layers of polyethylene (a
common plastic), are added to seal in the liquid and protect
it from external moisture content and a thin layer of
aluminum foil protects products from oxygen, flavors and
light. For the environment Tetra Pak cartons are
biodegradable and more efficient to transport than heavier
packages or refrigerated products.

Cooking

This method is usually used to improve palatability

107
rather than to improve storage quality of food. It is also
called boiling, is the process of using heat to water until the
temperature reaches about 100°C. Altough, boiling of foods
in water cannot completely destroy all microbes, but the
actively growing (vegetative) cells of bacteria and fungi
(yeasts and moulds) are generally quickly destroyed at this
temperature if maintained for a sufficiently long period of
time, helps the heat to completely penetrate the foods and
kill the microorganisms. Bacterial spores (extremely
resistant to heat) not killed at this temperature, although
their growth is prevented such as spores of Clostridium
perfringes and Clostridium botulinum (an organism present
in non-acid and semi-acid foods like peas, corn, green
beans, meat etc. and produces injurious toxins in food),
being highly resistant to heat. Botulin (toxin) is inactivated
by boiling foods for at least 10 minutes and the spores of
such bacteria are easily destroyed only if food is cooked
under pressure and destruction by heat is affected by time
and temperature variation. However, some enterotoxins
(produced by Staphylococci) are not easily inactivated. The
thermophilic microrganisms may survive the effects of
boiling and improperly handled cooked foods are rapidly
cause food spoilage whenever environmental conditions are
favourable for them.

Ohmic Heating

In most of the used heating techniques for liquid

depends on heat transfer from a hot surface and such heat
can be generated directly via an electrical heating element
or indirectly from a hot medium (like steam). In general,
such methods need a temperature gradient to transfer heat
to the process liquid and we know that these surfaces are at
a higher temperature than the product. Thus, this can cause
fouling of the surfaces for certain products which become
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burnt onto the hot surfaces reducing heat transfer rates and
negatively affecting the quality of product. An another
problem with heat transfer is found when heating very
viscous fluid and fluids with particulates where effective,
even heat transfer is very difficult to achieve without
compromising the quality of food product. To overcome,
these issues, an ohmic heater (also known as a joule heater)
can be used in which electrical heating device that uses a
liquid’s own electrical resistance to generate the heat. In
such process, the fluid is heated directly by passing an
electrical current (usually AC) through the product and its
own electrical resistance causes heating throughout giving
a uniform temperature increase without any loss in
performance for high viscosity or low flow rates. Hence,
the heat is produced directly within the fluid itself by Joule
heating as electrical current passes through it and is not
transmitted to it by means of temperature gradients or hot
surfaces and microbes are destroyed by the use of high-
voltage electric current through foods. Practically, Ohmic
heaters are very efficient with over 94% of the applied
electrical power converted to heat and have been revealed
to improve the colour and vitamin retention of foods and
provide an extended shelf life for pasteurised products
when compared to hot surface heating devices. Ohmic
heaters are extensively used in food industries for
successful heating of conductive and pumpable products
like dairy or milk based foods, tomato products, fruit juice,
liquid egg, jams, soups, casseroles etc.

Canning

It is one of the most widely used method of preserving

food, in which the food contents are processed (using heat
to appropriate temperature and for a prescribed time to
destroy micro-organisms, including Clostridium botulinum
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spores), and sealed in an airtight container. Canning
process was developed by Nicolas Appert. Generally, this
practice provides a typical shelf life extending from one
year to five years, though under specific circumstances a
freeze-dried canned product like canned dried lentils can
last as long as 30 years in an edible condition. In the
method of canning, there is a careful preparation of food
packed into a sealed tin, glass or plastic container which is
subjected to distinct high temperatures (above 100ºC) for a
suitable period of time and then cooled. During heating
there is a removal of oxygen and promote hermetic sealing
of containers to evade post-process infectivion and boiling
the food in the container to destroy all the microbes and
sealing the can to control and further avoid any new
microorganisms from getting in. After the thermal
processing, the sealed container must be cooled instantly to
a temperature of about 38ºC to avoid undesirable effects of
heat on the texture, flavour or colour of the foodstuff. Thus,
this sterilises the food so it will maintain for a extensive
period without any risk of destroy by unwanted
microorganisms. This process engages the following steps:

Sterilising the food products to be canned

• Aseptic stuffing (in sterile, sealed stainless metal, glass

or plastic bottles) and
• Hermetically sealing (with a complete, airtight seal)
Nowadays, a several number of canning methods are in
practiced but only two (as follows) are approved by the
United States Department of Agriculture (USDA):

a) Water-bath canning: It is known as the boiling-water

method of canning or as hot water canning, is the effortless
and simple process for conserve high-acid food. Filled jars
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are submerged in the water and heated to an internal
temperature (100°C) for an exact period of time. This
technique is sufficient to kill molds, yeasts, enzymes and
some bacteria, making it secure for utilization at a later
time. For example, acid foods such as fruit butters and
spreads, fruit pie fillings, sauerkraut, pickles and pickled
vegetables, jams and jellies can be strongly processed by
boiling water bath canning, because spoilage causing
microorganisms of foods are regularly damaged at boiling
temperatures. Tomatoes and its foodstuffs may be
processed by this way after little acidification.

b) Pressure canning: In pressure canning, a large kettle

used and steam is produced steam in a locked compartment.
The filled container in the kettle acquired an internal
temperature of 116°C under an exact pressure which is
calculated with gauge. It is valuable for processing
vegetables and other low-acid foods (i.e. meat, poultry,
seafoods etc.). In such process, Clostridium is destroyed in
low-acid foods when they are processed at the accurate
time and temperature in pressure canners. If C. botulinum
bacteria survive and grow inside a sealed jar of food, they
can produce a deadly toxin and even a tiny taste of food
containing this toxin can be deadly.

Before use any canned food we should inspect the

physical structure (damage or swollen) of can check
because a swollen, bulging can indicates that gas is being
produced on the inside and exhibits there is microbial
activity in the food, so it would not be safe for
consumption. Though, in canning we can keep our food
safe but once we open the can, there is a risk because
certain microorganism can enter and begin attacking the
food. Therefore, it is suggested that we should refrigerate
the contents after opening and use within the recommended
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time.

3.6 High pressure processing pulsed electric

fields
This physical technique consists of the function of short
pulses (microseconds) of high electric fields to foods
placed between two electrodes. It is a nonthermal process
and the toxic effect is effectively a function of pulse
intensity, pulse width, and pulse repetition rate. Pulsed
electric field (PEF) generation necessitate a pulsed power
supply and a treatment chamber.

The utilization of electric currents to destroy

microorganisms was studied in the 1920s, but those early
studies consisted of applying continuous current to liquid
foods, which resulted in heat buildup and free radical
formation. The use of PEF dates back to the mid-1960s.
The pulses used may be of the square-wave or the
exponentially decomposing types, and the earlier are more
toxic than the latter. In one study, a 99% decline in E. coli
numbers was formed by a square wave after 100
microseconds at 7°C compared to 93% by the
exponentially putrefying method.

Among the general properties and features of PEF as

applied to foods are the following:
1. Gram-negative bacterial cells are more susceptible
than Gram positives or yeasts.
2. Vegetative cells are more receptive than spores.
3. Microbial cells are more susceptible in the log phase
of development than in the stationary phase.
4. Cell death by PEF appears to be as a result of
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disruption of cell membrane function and by
electroporation (production of pores in membranes by the
electric current). It has been recommended that bacterial
inactivation by PEF may be an “all or nothing” event since
sublethal injury could not be identified.
5. Overall, the antimicrobial effects of PEF are function
of electric field strength, management time, and
temperature dealing, with cells being more susceptible
when treated at elevated temperatures.

A typical PEF application consists of the following

components listed in order:
• Pulse intensity (from 10 to 90 kV/cm is common)
• Pulse number (number of pulses varies widely from 10
to at least 70)
Pulse duration (in microseconds, 2μ is common)
• Flow rate (time in minutes/hour for given volume to
pass).
• Treatment parameters (the main ones are temperature
and pH; others could be aw, presence of
additives, and the like).

Some examples of PEF applications to food protection

are summarized below.
With L. monocytogenes, cells were more sensitive to
PEF when grown at 4°C than at 37°C; more resistant at low
aw; more sensitive under acidic conditions; and more
resistant in stationary phase than log phase of growth.
When orange juice was subjected to 30 kV/cm and 50
kVcm at 50°C, up to a 5-log cycle reduction of
Leuconostoc mesenteroides, E. coli, and Listeria innocua
was achieved.
At 50 kV/cm and 50°C, S. cerevisiae ascospores were
reduced by a maximum of 2.5 logs. In another study
employing orange juice, a reduction of the APC of >6 log
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cycles occurred in fresh orange juice with a treatment of 80
kV/cm, 20 pulses, pH 3.5, temperature of 44◦C, and 100
IU/ml nisin.20 The treated juice had a 40◦C-shelf life of at
least 28 days.
A log10 5.9 reduction of S. Typhimurium in orange juice
was demonstrated with 90 kV/cm and 50 pulses at 55°C.25
Nisin and lysozyme acted synergistically, and combined
with PEF, an additional 1.37 log-cycle reduction of the
pathogen was noted. The synergy between nisin and
lysozyme supports the view that the plasma membrane is
the PEF target. A 5.35-log10 cfu/ml reduction of E. coli
0157:H7 added to apple cider was achieved with 80 kV/cm
and 30 pulses at 42◦C.21 With 90 kV/cm and ten pulses at
42°C, a 5.91-log reduction occurred but when cinnamon
powder (2%) or nisin (2.5%) was added, a 6- to 8-log
reduction occurred.
Using raw skim milk, APC was reduced by 7 logs with
80 kV/cm, 50 pulses at 52°C, and both nisin (38 IU/ml) and
lysozyme (1,638 IU/ml) added.57 Against vegetative cells
of Bacillus cereus, a treatment with 16.7 kV/cm, 50 pulses
each at 2 μsec plus 0.06 ppm nisin, an increased reduction
of 1.8 log units was observed over what PEF and nisin
alone achieved.43 A 5-log reduction of E. coli 0157:H7
was obtained in a simulated milk medium by 5 kV/cm +
1,200 IU nisin/ml at aw of 0.95. It was found that NaCl and
nisin reduced the effectiveness of PEF.
In a study that compared PEF withHHPand heat for
controlling ascospores of Z. bailii in fruit juices, two pulses
of 32–36.5 kV/cm reduced vegetative cells 4.5–5 and
ascospores 3.5–4 log cycles, and this compared with a
nearly 5-log reduction of vegetative cells by HHP but only
a 0.5–1 log reduction of ascospores with a 5-minute
treatment at 300 MPa. Overall, two pulses of 32–36.5
kV/cm reduced vegetative cells or ascospores 3.5–5 log
cycles for each of the five juices tested. The ascospores
114
were 5–8 times more heat resistant than the vegetative
cells.
Regarding E. coli, when 106 cfu/ml were added to pea
soup and treated with two 16-pulse steps at 35 kV/cm for a
total of 2 sec, cells could not be identified by plate count. In
an earlier study, bacteriophages of Lactococcus cremoris
were found to be more susceptible to electric shock than
four species of bacteria, as well as spores of Bacillus
subtilis.

3.7 Aseptic packaging

In traditional canning methods, nonsterile food is
positioned in nonsterile metal or glass containers,
subsequently followed by container closure and
sterilization. In aseptic packaging, sterile food under
aseptic conditions is kept in sterile containers, and the
packages are sealed under aseptic conditions as well.
Although the methodology of aseptic covering was
patented in the early 1960s, the technology was little used
until 1981, when the Food and Drug Administration
accepted the use of hydrogen peroxide for the sterilization
of flexible multilayered wrapping materials used in aseptic
processing systems.
In general, any food that can be pumped through a heat
exchanger can be aseptically wrapped. The widest purpose
has been used to liquids such as fruit juices, and a broad
diversity of singleserve products of this type has resulted.
The technology for foods that contain particulates has been
more complicated to develop, with microbiological
considerations only one of the many troubles to conquer. In
determining the sterilization process for foods pumped
through heat exchangers, the fastest-moving components
are utilized, and where liquids and particulates are mixed,
115
the latter will be the slower moving. Heat diffusion rates
are not similar for liquids and solids, making it more
complex to establish minimum process necessities that will
efficiently destroy both organisms and food enzymes. Some
of the advantages of aseptic packaging are as follows:
• Products such as fruit juices are further flavorful and
do not have the metallic taste of those processed in metal
containers.
• Flexible multilayered cartons can be utilized as an
alternative of glass or metal containers.
• The time manufactured goods are subjected to high
temperatures is minimized when ultrahigh temperatures are
utilized.
• The equipment let the use of membrane filtration of
certain liquids.
• A Range of container headspace gases for instance
nitrogen may be employed.
Among the disadvantages are that parcels may not be
equal to glass or metal containers in avoiding the
permeation of oxygen, and the output is lesser than that for
solid containers. A wide assortment of aseptic packaging
method now survives are under development. Sterilization
of packages is accomplished in a variety of ways, one of
which occupy the continuous feeding of rolls of packaging
material into a machine where hot hydrogen peroxide is
used to achieve sterilization, followed by the forming,
filling with foodstuff, and close with the containers.
Sterility of the filling operation may be preserved by a
positive pressure of air or gas such as nitrogen. Aseptically
packaged fruit juices are shelf-stable at ambient
temperatures for 6-12 months or longer.
The spoilage of aseptically packaged foods fluctuates
from foods in metal containers. Whereas hydrogen swells
take place in high-acid foods in the latter containers, aseptic
packaging materials are nonmetallic. Seam leakage may be
116
estimated to be absent in aseptically packaged foods, but
the permeation of oxygen by the nonmetal and nonglass
containers may allow for other types of spoilage in low-
acid foods.

3.8 Mano-thermo-sonication
Manothermosonication (MTS) combines and synergises
the ultrasound with moderate temperature and pressure in
order to inactivate enzymes and/or micro-organisms. This
method has seen convincing developments in past three
decades for food preservation due to its ability to inactivate
microorganisms and endogenous enzymes while hold on to
nutrients and flavour. Harvey and Loomis (1929) first
documented the lethal effects of ultrasound on living
organisms and since then, its use has been continuously
suggested for disinfection and food preservation. Sound
waves having frequencies > 20 kHz are regarded as
ultrasounds and in perspective of food preservation, upper
limit is usually taken to be 5 MHz in gases and 500 MHz in
liquids and solids. The primary analysis is on high
hydrostatic pressure (HHP) conservation of foods were
conducted in early 1890s. It was confirmed that microbial
inactivation with ultrasound amplify when treatment is
useful under pressure.
The lethality of ultrasound under higher static pressure
was accounted to be remarkably greater within a given
pressure range (0 to 300 kPa). The application of MS
treatment simultaneously with heat surges the microbial
inactivation manifolds. A major benefit of utilizing MTS is
a advanced extent of specificity as acoustic energy is
absorbed specially at the interface of membranes causing
targeted heating. This heating effect has also supposed to
be in charge for raising the permeability of the living
117
membranes resulting in total loss of their selectivity. For
example, considerably increased rate of diffusion of sodium
ions through living frog skin under ultrasound was
established by Lehmann and Krusen. Since, MTS is
undertaken at relatively lower temperatures than
conventional thermal processes, a product with heat
sensitive components can be treated. The local molecular
temperature is still rising during the treatment. Therefore
careful temperature control is necessary. Also, the
treatment time is in fact longer during the destruction
and/or inactivation of microorganisms and/or enzymes
varying with product to product, which may cause high
energy requirement (Burgos, 1998). Thus, MTS is a
promising technology that professionally exploits the effect
of heat and ultrasonic waves synergised by pressure.

Mechanism

In MTS, major role of micro-organism and enzyme

inactivation is played by ultrasound and temperature while
pressure acts as a synergising energy that assists to
optimize the overall strength of the process. Here we can
think about the individual effect of pressure, ultrasound and
heat in order to understand their combined outcome.
Ultrasound is defined as sound waves with frequencies
beyond that of human hearing (typically higher than 18
kHz). These waves can be propagated in liquid media as
alternating compression. If ultrasound has sufficient
energy, cavitation takes place in the medium. This incident
involves the formation, growth, and sudden collapse of
microscopic bubbles. These collapsing bubbles deliver very
high temperatures (approximately 5000 K) and pressures
(estimated at 50000 kPa) momentarily to the liquid media.
High pressure results in physico-chemical changes, often
leading to longer shelf life. High pressure devastates the
118
cell membrane function, leading to cell leakage. Thus,
summing up the mechanism of MTS, the ultrasound
produces the cavitation or bubble implosion in the media.
These implosions can cause inactivation and/or devastation
of micro-organisms and enzymes. The instantaneous
pressure treatment maximizes the intensity of the
explosion, which results in greater levels of inactivation.
The method of microbial killing is mainly owing to
thinning of cell membranes, localized heating and
production of free radicals. Very strong shaking of
molecules takes place causing breakage of bonds. This
result in liberation of dipicolinic acid and some low
molecular weight polypeptides from the cortex of spores of
certain bacterial species. Rehydration of the protoplast
takes place resulting in loss of heat resistance. The loss in
heat resistance at acidic pH of the medium is also reason
for the rehydration of protoplast as a result of cortex
degradation (protonization). Another observed peculiarity
of MTS treatment in microbial and enzyme inactivation is
that it is a bi-phasic process i.e. a faster rate of inactivation
in initial stages of treatment subsequently followed by
decrease in inactivation for remaining course of treatment.
Although it is a recognized fact that efficiency of MTS
treatment increases with elevated temperature but after a
certain temperature increase, the lethality of MTS has been
observed to decrease. A possible reason for this weakened
consequence could be decreased strength of bubble
implosions because of rise of the water vapour pressure
inside the bubble with increase in temperature. It has
already been proved that the effect of heat and ultrasonic
waves in enzyme inactivation combines synergistically.
This is actually derived from the result that the inactivation
rate of combined treatment is larger than the sum of the
speed of inactivation by ultrasound at room temperature
and the rate of inactivation by simple heating. Microbial
119
and enzyme inactivation by MTS follows first order
kinetics and there are a number of models industrialized by
researchers to predict the process requirements that could
concern the industry.

3.9 FDA
The Food and Drug Administration is a Federal
agency of the United States Department of Health and
Human Services, one of the United States Federal
Executive departments.

The FDA was authorized by the United States

Congress to implement the Federal Food, Drug, and
Cosmetic Act, which provide the prime focus for the
Agency; the FDA also insist on other laws, notably Section
361 of the Public Health Service Act and associated policy,
many of which are not directly associated to food or drugs.
These comprise regulating lasers, cellular phones, condoms
and control of disease on products ranging from certain
household pets to sperm donation for assisted reproduction.

The FDA is led by the Commissioner of Food and

Drugs, selected by the President with the advice and
consent of the Senate. The Commissioner informs to
the Secretary of Health and Human Services. Stephen M.
Hahn, MD is the acting commissioner, as of December
2019.

The FDA has its headquarters in unincorporated White

Oak, Maryland. The agency as well has 223 field offices
and 13 laboratories situated throughout the 50 states,
the United States Virgin Islands, and Puerto Rico. In 2008,
the FDA begins to placement workers to foreign countries,
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as well as China, India, Costa Rica, Chile, Belgium, and the
United Kingdom.

The main dream is to attempt for pharmaceutical

distinction and health to guarantee the availability of safe,
efficient and quality food and drugs to the public and our
effort to continue with the time tested custom of sustaining
Goa Food and Drugs Administration as one of the best
FDA in the Country and pool into all sincere hard work to
place FDA – Goa on the global map as one of the greatest
regulatory institution.

The Directorate has its self-governing Food & Drugs

Laboratory for analysis of food and drugs sample at
Bambolim, which was build under the Capacity Building
Project of the Ministry of Health & Family Welfare,
Government of India by the support of World Bank; the
said independent laboratory was raised and commissioned
for operation since March 2007.

The building encompass of the ground floor catering to

the food analysis, whilst the initial floor caters for the drugs
investigation; Both the Food & drugs laboratory sector
have been adequately prepared with all the modern vital
and sophisticated equipments and instruments required for
testing of food and drugs samples.

The laboratory consists of separate independent staff of

Senior Scientific Officer, Junior Scientific Officer, Chemist
and Assistant Chemists, laboratory Attendant and
Laboratory Technicians discretely for the food and drugs
sections.

The laboratory conducts examination of food and drugs

samples referred by the Food Inspectors as well as Drugs
121
Inspectors below the respective food and drugs laws; Â In
addition imported food articles samples are referred by the
Port Health Authorities, liquor samples by the State Excise
Department and the narcotic samples by the Police and
Customs department. On an average, annually 600 food
samples and 800 drugs samples are examined by the
Laboratory

History of FDA

The Directorate of Food and Drugs Administration,

Government of Goa was well-known in November’ 1991
with its office performance from the building premises at
Campal, Panaji – Goa. Thereafter the office location was
shifted and functioned from the Old Goa Medical Complex
at Panaji, Goa; until October 2003 and thereafter the
Directorate function from the Old IPHB Complex at
Altinho, Panaji, Goa until February 2014.

Presently, the Directorate now role is due to the

Directorate of Food and Drug Administration,
'Dhanwantari', Opposite Shrine of the Holy Cross,
Bambolim - Goa 403202. Prior to the concern of an
independent Food and Drugs Administration, the
Directorate, was performing as an office of the Drugs
Controller as one of a variety of units under the Directorate
of Health Services at Campal and was entrusted to handle
all food and drugs related legislation.
The Directorate functions with its one wing concerned in
the administration and drugs/food activities through its
office located at Bambolim; whilst its Food and Drugs
Laboratory utilize from its independent building located at
Bambolim.

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Role of FDA

The Directorate of Food and Drugs Administration is

assign with the task of executing and enforcing all the Food
and Drugs related legislation, which comprise
controlling the quality of food articles and drugs,
manufactured and sold within the State as well as
manufactured outside the State but sold in the State.

The Directorate through its independent Food and

Drugs Testing Laboratory is entrusted with the
responsibilities in investigation of a variety of food and
drugs, samples drawn by the Food Inspectors and Drugs
Inspectors correspondingly, liquor samples, referred by the
Excise Department, Narcotic samples passed by the Police
and Customs Department as well as examination of
imported food articles referred by the Mormagoa Port
Health Authorities. The Directorate also standardize the
prevention on the sale, production and stocking of injurious
food article viz food article including tobacco under the
Goa Public Health (Amendment) Act 2005, which includes
Gutka, etc

3.10 The Hazard Analysis and Critical Control

Point (HACCP) Concept:
In the food industry today approach based on Good
Manufacturing Practice are being largely substituted by
function of the Hazard Analysis Critical Control Point
(HACCP) concept. This has enhanced due to traditional
practices by introducing a more efficient, rule-based
approach for concern our knowledge of food microbiology
to the control of microbiological quality.

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 The same system can also be approved with physical
and chemical factors affecting food safety or acceptability,
but here we will confine ourselves to microbiological
hazards. It should also be remember that HACCP is mainly
a preventative approach to quality assurance and used to
design quality to develop novel products during their
development.

HACCP was initially industrialized as part of the United

States space programme by the Pillsbury Company, the
National Aeronautics and Space Administration (NASA)
and the US Army Natick Laboratories who used it to apply
the same zero defects philosophy to food for astronauts as
to other items of their equipment.
It is based on an engineering system known as the
Failure Modes Analysis Scheme which observes a result
and all its components asking the question ‘What can go
wrong?’

In 1973 it was approved by the US Food and Drug

Administration for the examination of low-acid canned
food. It has since been more and more extensively applied
to all features of food manufacture, food processing and
food service, and to all scales of operation from large
industrial concerns, through to cottage industries and even
domestic food preparation.

The meaning of the terms hazard and threat in the

HACCP system change from their general everyday usage
as synonyms. In HACCP, a hazard is a source of risk;
defined as the unacceptable contamination, development or
survival of a micro-organism which can involve safety or
shelf-life, and/or the unacceptable creation or persistence in
food of microbial metabolites disturbing security or shelf-
life.
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 Individual hazards can be evaluated in terms of their
severity and threat. Clearly a hazard to food safety is more
strict than one to shelf-life, and botulism is a far more
severe hazard than say Staphylococcus aureus food
poisoning. Risk is an estimate of the likely incidence of a
hazard so, although C. botulinum is a more severe hazard,
epidemiological indication shows that the risk it poses is
normally very low.

A HACCP study is best conducted by a

multidisciplinary team including a microbiologist, a
process supervisor, an engineer and a quality guarantee
manager, all of whom will be able to convey their own
particular expertise and knowledge to bear on the problem.
Contribution of production personnel will also ensure
recognition with the plan by those who will have to execute
it.

Experience recommends that best results are acquired

when the study’s terms of indication specify particular
microbial hazards for consideration since this will allow the
team to characterize specific controls. The selection of
hazard considered will depend on whether there is
epidemiological verification linking a particular micro-
organism with the food in question.

In the absence of such confirmation, features such as the

product’s physical and chemical characteristics and the way
it is ultimately used by the customer must offer the basis
for selection.

3.11 Indian standard Institute

The ISI was recognized in the year 1947. It has been

125
renamed as the ‘Bureau of Indian Standards‘. Its main
purpose is to lay down quality principles for consumer and
industrial goods.

A producer who wants to implement a standard for his

product has to attain a license from the ISI under its
marking scheme. He has to approve certain measures for
quality control as approved by the license.

The inspectors of the ISI will regularly watch whether

the manufacturer maintains the particular quality or not.
They may collect samples for the purpose of examination at
any time even from the open market. These samples will
then be tested in the laboratories of the ISI.

If any customer has complaints against the value of ISI

marked products, he can inform the ISI officials regarding
the same. The ISI will take timely action on any such
precise complaint.

If any manufacturer is found to be using the ISI mark

fraudulently, he will be responsible for punishment. For the
purpose of analysing products, the ISI has set up
laboratories in diverse parts of India. In addition to these
actions, the ISI also furthers India’s interest in the field of
international standardization by working closely with
the International Standards Organization (ISO).

Functions of ISI

The functions of ISI may be stated as follows:

1. To organize principles for commodities, materials and
processes.
2. To assist in the production of quality goods.
3. To verify industrial goods.
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4. To pass information relating to standardization
5. To support universal standards mutually at the national
and international levels.
6. To guard the consumers by promising them high-quality
and product presentation.
7. To eradicate unnecessary selection.
8. To cut down the price of production.

A number of technical committees advise the ISI in the

entire process of standardization. Standardization will
ensure economy and the best exploitation of the material
resources.

ISI mark
The ISI mark is a standards-compliance mark for
industrial products in India since 1955. The mark certifies
that a product conform to an Indian standard (IS)
industrialized by the Bureau of Indian Standards (BIS), the
national standards body of India. The ISI mark is by far the
mainly recognized certification mark in the Indian
subcontinent. The ISI is an initialism of Indian Standards
Institution, the name of the national standards body until 1
January 1987, when it was renamed to the Bureau of Indian
Standards. The ISI mark is compulsory for certain products
to be sold in India, such as many of the electrical
equipment like switches, electric motors, wiring cables,
heaters, kitchen appliances, etc., and other foodstuffs
like Portland cement, LPG valves, LPG
cylinders, automotive tyres, etc.

Counterfeiting

It is very common in India to find products with fake ISI

marks. That is, industrial traders cheat consumers by
affixing ISI marks on the product without essentially
127
getting certified. Fake ISI marks usually do not carry the
following:
(i) The compulsory 7-digit licence number (of the format
CM/L-xxxxxxx, where x signifies a digit from the licence
number) necessary by BIS; and
(ii) The IS number on top of the ISI mark which indicate
the Indian standard a particular manufactured goods is in
compliance with.
For instance, if a kitchen grinder's box has a small ISI
mark on it with the ISI code of the appliance's wire, one
can conclude that the wire is BIS-certified but the piece of
equipment itself is not an BIS-certified product.
Counterfeiting ISI marks is a punishable offence by the
law, but enforcement is uncommon.

4 Chapter 4: Microbial Food spoilage

and Food borne disease

4.1 Staphylococcus aureus food poisoning

Staphylococcus aureus is a bacterium found in the nose
and on the skin of about 25 percent trusted Source of
healthy people and animals. S. aureus is accomplished of
making seven diverse toxins and is often the reason of food
poisoning. It is most normally transferred to foodstuffs like
milk and cheese during contact with food workers that
carry S. aureus. S. aureus food poisoning (SFP) is normally
not life-threatening. Most cases of SFP do not require
treatment as the condition will pass on its own. Mainly
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people get over food poisoning within two days.

Symptoms of Staphylococcus aureus food poisoning

SFP causes symptoms alike to a severe case of

gastroenteritis, or inflammation of the digestive tract.
Symptoms may show rapidly, sometimes in as little as 30
minutes after you’ve eaten contaminated food. But it
naturally takes up to six hours for symptoms to extend.

Symptoms of SFP include:

• diarrhea
• vomiting
• abdominal cramping
• Illness is generally mild, and most people
recover within one to three days

Causes for Staphylococcus aureus food poisoning

SFP is the reason for contamination of food products. S.

aureus has an elevated salt tolerance, and can cultivate in ham
and other meats, and in dairy products. The toxins that the
bacteria produce are heat resistant and cannot be damaged
through cooking. Once food has been infected, bacteria begin to
develop. Food products most normally associated with SFP are
milk and cheeses. And the most general reason for contamination
is through contact with food workers who carry the bacteria.
Foods that need a lot of handling and are stored at room
temperature are frequently involved with SPF.
These include:
• Sandwiches
• Cold salads, such as tuna, chicken or ham salad
129
 • Sliced meats
• Pastries

Figure: 2 Staphylococcus aureus

Diagnosis of Staphylococcus aureus food poisoning

In most cases, SFP does not require medical attention. It

often clears up with rest and fluids. But contact your doctor
if your illness lasts longer than three days, or if you are not
capable to drink enough fluids to prevent dehydration.
Your doctor can diagnose SFP with a physical assessment
and an analysis of your symptoms. They may also pose
inquiry about current activities and things you have eaten.
If symptoms are harsh, your doctor may organize blood
tests or a stool culture. This examination can assist to
decide if the S. aureus bacterium is existing, and may also
help your doctor rule out other possible causes.

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Treatment of Staphylococcus aureus poisoning

SFP generally lasts for a day or two. Medical

intervention is often unnecessary as this illness normally
disappears on its own. Treatment normally involves rest
and improved fluid intake. But some people may require
medical help. SFP may be hazardous in young children,
babies, older adults, and people who have HIV. Because
the most common complication of SFP is drying out,
treatment managing intravenous liquids may be necessary.
In severe cases, you may be hospitalized for observation in
order to avoid complications.

Prevention of Staphylococcus aureus food poisoning

To prevent food poisoning and the spread of bacteria,

take the following precautions:
• Avoid unpasteurized milk
• Wash hands and fingernails thoroughly before
cooking, eating, or serving food
• Sustain clean and sanitary surfaces for food
preparation
• Preserve hot foods at temperatures over 140˚F
(60˚C) and cold foods under 40˚F (4˚C)
• Do not organize food for others if you have injury
or sores on your hands or wrist.

4.2 [Link]

Serological Classification

For the genus E. coli, over 200 O serotypes have been

documented. Because the flagellar proteins are less
heterogeneous than the carbohydrate side chains that make
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up the O groups, significantly fewer H antigenic types
exist.

Enteroaggregative E. coli

This group (also designated enteroadherent) is

associated to EPEC but the aggregative adherence
displayed by these strains is unique. Strains display a
"stacked-brick-type" of adherence to HEp-2 cells, and carry
a 60-MDa plasmid that is desirable for the production of
fimbriae that are in charge for the aggregative expression,
and for a definite outer membrane protein (OMP).
Antibodies raised against the OMP of a prototype strain
prevented adherence to HEp-2 cells. An EAggEC DNA
probe has been created by using a 1.0-kilobase (kb)
fragment from the 60-MDa plasmid of the prototype strain
(03:H2), and it was found to be 99% specific for these
strains.

Figure: 3 Escherichia coli

Some EAggEC strains generate a heat stable enterotoxin

(ST), which has been designated EASTl. The plasmid-
borne gene for EASTl is astA, which encodes a 38-amino-
acid molecule in contrast to estA, which encodes the 72
amino acid enterotoxin STa. They generate an
enterotoxin/cytotoxin that is about 108 kDa, and it is
situated on the large virulence plasmid. The distinguishing
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clinical characteristic of EAggEC strains is a persistent
diarrhea that lasts >14 days, especially in children. These
strains are not the prime reason of traveler's diarrhea.

Enterohemorrhagic E. coli

These strains are both similar and dissimilar to EPEC

strains. They are similar to EPEC in their possession of the
chromosomal gene eaeA (or one that is similar) and in the
production of attachment-effacement lesions (see the
subsection on EPEC). In distinction to EPEC, EHEC strains
have an effect on only the large intestine (in piglet models)
and manufacture large quantities of Shigalike toxins (SLT,
Stx, see below). EHECs produce a 60-MDa plasmid that
encodes fimbriae that mediate attachment to culture cells,
and they do not occupy HEp-2 or INT407 cell lines,
although some strains have the capacity to invade some
human epithelial cell lines.

Toxins

Shigella dysenteriae produces a potent toxin that is

referred to as Shiga toxin (after K. shiga who first isolated
and studied the organism). The toxins of EHEC strains of
E. coli have been known as Shiga-like toxins (verotoxin,
verocytotoxin) and the two prototypes as SLT-I and SLT-
II. However, new terms has been functional, and what was
once SLT-I is now Stxl and the former SLT-II is Stx2.9
The genes for Stxl and Stx2 are determined by temperate
bacteriophages in some EHEC strains. Stx-sensitive cells
acquire the toxin receptor, globotriaosylceramide (Gb3),
and sodium butyrate appears to participate a function in
sensitizing cells to Stxs. Once toxins bind to Gb3,
internalization follows with transport to the trans-Golgi
network. Once inside host cells, the A subunit binds to and
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discharge an adenine residue from the 28S ribosomal RNA
(rRNA) of the 60S ribosomal subunit and this inhibits
protein synthesis. The B subunits form pentamers in
connection with a single a subunit and, thus, they are
accountable for the binding of the toxin to the neutral
glycolipid receptors.

Prevalence in Foods

Overall, the occurrence and prevalence of EHEC strains

in meat, milk, poultry, and seafood products are highly
changeable. Significantly more positives are found when
DNA probes are used to differentiate for EHEC strains than
when EC O157:H7 is tested for alone. The first published
study on the incidence in meats of EHEC strains was that
of Doyle and Schoeni, who tested for EC O157:H7 and
found this strain in 3.7% of 164 beef, 1.5% of 264 pork,
1.5% of 263 poultry, and 2.0% of 205 lamb samples. In a
more existing study of foods in the Seattle area following
the 1993 outbreak, 17.3% of 294 foods were positive for
colonies that contained Stxl and/or Stx2 strains. Additional
to radish seeds and incubated at 18-25°C for 7 days, the
organism was found in interior tissues and stomata of
cotyledons and also on the outer surfaces. It could not be
detached by immersion in 0.1% HgCl2.

Following the Pacific Northwest outbreak in 1993, the

Food Safety and Inspection Service (FSIS) of the USDA
undertake a multistate study of the occurrence and
incidence of EC O157:H7 in both beef and dairy herds. The
largest number per gram found was 15, and the standard
was around 4 cfu/g of fresh beef. It is generally believed
that dairy herds are the chief reservoirs of these organisms.
For instance, of 1,266 fecal samples from calves, heifers,
and cows, only 18 (1.42%) were positive for EC O157:H7.
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Only 1 of 662 cow samples was positive, whereas 5 of 210
calf and 12 of 394 heifer feces samples were positive.

Table: 4 Some of the O serotypes found among the five

virulence groups

Enteroinvasive E. coli

These strains normally do not generate enterotoxins as

do ETECs, but they enter and multiply in colonic epithelial
cells and then extend to adjacent cells in a manner similar
to the shigellae. Prior to the 1970s, some of these
organisms were referred to as "paracolons." Like the
shigellae, EIECs acquire 140-MDa enteroinvasive plasmids
(pINV) that are quite alike to those found in Shigella
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flexneri and are necessary for their invasiveness.
Plasmidless strains are not persistent. The typical EIEC
strains are also Sereny positive. Members of this group
have a predilection for the colon, and bloody or nonbloody
but voluminous diarrhea is an outcome. Dysentery is
unusual, and the very young and very old are the most risk
members of the population. The incubation period is
between 2 and 48 hours with an average of 18 hours. Some
of the serotypes that include EIEC strains are listed in
Table: 4. At least one, 0167, contains both EIEC and ETEC
strains.

Enteropathogenic [Link]

They acquire adherence factor plasmids that enable

adherence to the intestinal mucosa. After colonizing the
intestinal mucosa, attachment effacement (att-eff, A/E)
lesions are produced. The process starts upon as a
preliminary contact and is supposed to be aided by a
plasmid-encoded bundle-forming pilus. EPEC secreted
proteins (Esps) block phagocytosis and direct to
cytoskeletal rearrangement and tyrosine phosphorylation of
Tir. When Tir binds with the outer membrane protein
intimin, the attachment is intimate, resulting in devastation
of brush border microvilli and formation of pedestals.

Enterotoxigenic E. coli

These strains connect to and colonize the small intestine

by means of fimbrial colonization factor antigens (CFAs).
There are four types of CFA-1, II, III, and IV—and they
have been cloned and sequenced. CFAs are plasmid
encoded, normally on the same plasmid that encodes the
heat-stable enterotoxin, and they are not produced under
200°C. Once attached, they generate either one or two
136
enterotoxins. Some of the ETEC serotypes are listed in
Table: 4 . In a study of ETEC strains from 109 patients, the
strains that produced both ST and LT were more
constrained in O:K:H serotypes than those that produced
only one of these toxins. These toxins are further
characterized below. Unlike EPEC strains, which cause
diarrhea mainly in the very young, ETEC strains is the
reason for diarrhea in both children and adults? These
strains are among the primary causes of travelers' diarrhea.
The ETEC disease syndromes not often come with fever,
and the diarrhea is sudden. It has been projected that 108-
1010 cfu are necessary for diarrhea by an ETEC strain in
adult humans.

Prevention

In general, the prevention/avoidance of foodborne

illness by E. coli can be attained by examining several
factors. However, because of the consequences to young
children, special safety measures need to be experimented.
The heat sensitivity of these organisms is such that cases
should not occur when foods are appropriately cooked. In
the case of ground beef, the recommendation is that it be
cooked to 1600°F, or that the core temperature be brought
to a least of 155°C for at least 15 seconds and that the
juices are clear Because of unevenness of hamburger
patties, cooking at 155-1600°F offer a measure of safety.
Once cooked, hamburgers as well as other meats should not
be held between 400°F and 1400°F for more than 3-4
hours. Although the largest recorded foodborne outbreak
was related with ground beef, all raw meat, poultry, and
seafood should be regarded as potential vehicles for
hemorrhagic colitis

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4.3 Salmonellosis
This syndrome is caused by the ingestion of foods that
contain significant numbers of non-hostspecific species or
serotypes of the genus Salmonella. From the time of
ingestion of food, symptoms usually expand in 12–14
hours, although shorter and longer times have been
analysed. The symptoms comprise of nausea, abdominal
pain, headache and diarrhea. These symptoms are usually
come with by prostration, muscular weakness, faintness,
moderate fever and drowsiness. Symptoms usually
continue for 2–3 days.

Incidence and vehicle foods

The precise incidence of salmonellae food poisoning in

the United States is not known. However, the two chief
recorded outbreaks of salmonellosis take place under rather
unusual circumstances. The main occurred in 1994 and it
involved more than 224,000 persons. The vehicle food was
ice cream produced from milk that was transported in
tanker trucks that had formerly hauled liquid eggs. The
serovar was S. Enteritidis, and cases were seen in at least
41 U.S. states. The next largest outbreak takes place in
1985 and occupied close to 200,000 persons.

For the years 1981–1995, 3,504 (23.8% of all) bacterial

foodborne illnesses in Korea were due to salmonella while
in Japan for the same period, there were 101,395 (19.9%)
cases. Because in the United States S. enteritidis is
extremely connected with the consumption of raw or
undercooked eggs, the CDCP suggests the following:

(1) Raw or undercooked eggs should be avoided,

138
particularly by the young, elderly, and
immunocompromised;
(2) When eggs are not appropriately cooked, pasteurized
egg products should be used;
(3) Eggs should be cooked at ≥145◦F (63◦C) for 15
seconds, or until both yolk and white are firm, and then
should be eaten;
(4) Casseroles and other dishes containing raw eggs should
be cooked to 160◦F (71◦C);
(5) Raw eggs should be stored at ≤ 45◦F (7.2◦C) at all
times.

The possible routes of S. enteritidis to eggs are the following:

1. Transovarial
2. Translocation from peritoneum to yolk sac or oviduct
3. Penetration of shell by organisms as eggs passes through
the cloaca
4. Egg washing
5. Food handlers

The three outbreaks traced to fresh tomatoes in the

United States during the years 1990, 1993, and 1997–1998
were due to three dissimilar serotypes. S. Muenchen was
the etiologic agent in an orange juice outbreak in 1999 13;
and it was also the reason for alfalfa sprout outbreak during
the same year.

Figure: 4 Salmonella species

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Prevention and Control of Salmonellosis

The intestinal tract of humans and other animals is the

primary reservoir of the etiological agents. Animal fecal
matter is of greater significance than human, and animal
hides may become polluted from the fecal source.
Secondary contamination is another of the vital sources of
salmonellae in human infections. Their existence in meats,
eggs, and even air makes their presence in certain foods
inevitable through the agency of handlers and direct contact
of no contaminated foods with contaminated ones.
Improper preparation and management of foods in homes
and food service establishments continue to be the main
features in outbreaks.

With respect to the colonization of chickens by S.

enteritidis, one investigation used a phage type 8 strain
administered orally 108 to adult laying hens. Within two
days, the organism was found all over the body, including
the ovary and oviduct. It was identified in some forming
eggs, although its incidence was much lower in freshly laid
eggs. Researchers accomplished that forming eggs are
subject to descending infection from colonized ovarian
tissue, to ascending illness from colonized vaginal and
cloacal tissues, and to lateral disease from colonized upper
oviduct tissues.

Table: 5 Some of Salmonella strains ranking by year

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Reduction of Salmonella carriage in poultry

If young chicks become occupied with salmonellae, the

bacteria may be shed in feces, through which other birds
become polluted. Among the methods that may be engaged
to reduce or eradicate intestinal carriage is competitive
exclusion. Competitive exclusion is an occurrence whereby
feces from salmonellae-free birds, or a mixed fecal culture
of bacteria, are given to young chicks so that they will
colonize the similar intestinal sites that salmonellae utilize
and, thus, eliminate the subsequent attachment of
salmonellae or other enteropathogens.

The enteropathogen-free biota may be managed orally to

recently hatched chicks through drinking water or by spray
inoculation in the hatchery. Protection is established within
a few hours and normally carry on throughout the life of
the fowl or as long as the biota remains undisturbed. Older
birds can be taken care of by first governing antibacterial
agents to eliminate enteropathogens, and then competitive
exclusion biota is administered. Only viable cells are
effective, and both aerobic and anaerobic components of
the gut flora seem to be essential. The crop and ceca
emerge to be the major adherence sites, with the ceca being
superior in germ-free chickens. In another report, the
protective flora remains attached to cecal walls after four
successive washings.

The gist of competitive exclusion is that salmonellae and

the native gut biota struggle for the same adherence sites on
gut walls. Extracellular polysaccharides of a glycocalyx
nature may be implicated, and if so, management of young
chicks with this material may be as efficient as the
utilization of live cultures. The sugar mannose is a receptor
in the intestinal tract to which bacterial pathogens such as
141
salmonellae bind. Since the yeast strain Saccharomyces
cerevisiae var. boulardii contains mannose in its outer wall,
some have recommended that the feeding of this yeast to
susceptible poults should decrease the attachment of
salmonellae.

The possible use of probiotic cultures to exclude some

Gram-negative pathogens from the gut biota has been
investigated by several groups. When a 3-strain mixture of
probiotic bacteria (competitive exclusion of E. coli
strains)was used on weaned calves and challenged with E.
coli serotypes 0111:NM, 026:H11, and 0157:H7, the
probiotic-treated calves showed a significant reduction in
the shedding of two of the three pathogens but not E. coli
serotype 026:H11.55When ca. 109 cells/chicken of a
chicken isolate of Enterococcus faecium was orally
administered to 30-hour-old broiler chicks followed by a
challenge with 105 cells of S. Pullorum per chick, the
chicks survived. However, chicks that were infected on the
first day and then treated with the lactic culture died four
days later.

4.4 Shigellosis
People who are ill with shigellosis
have Shigella microbes in their stool (poop) while they
have diarrhea and for up to a week or two after the diarrhea
has gone away. Shigella is very infectious; just a little
amount of germs can make someone sick. People could get
sick by:

Getting Shigella germs on their hands and then touching

their food or mouth. This may happen after:

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 Touching surfaces polluted with germs from stool from a
sick person, such as toys, bathroom fixtures, shifting tables
or diaper pails.
Changing the diaper of a sick child or caring for a sick
person.
Eating food that was prepared by someone who is sick with
shigellosis.
Foods that are consumed raw are more likely to be
contaminated with Shigella germs.
Shigella germs can pollute fruits and vegetables if the fields
contain human waste.
Swallowing recreational water (for instance, lake or river
water) while swimming or drinking water that is
contaminated with stool (poop) containing the germ.
Having exposure to stool during sexual contact with
someone who is sick or recently (several weeks) recovered
from shigellosis.

Symptoms

People who are ill from Shigella infection usually

experience symptoms 1 to 2 days after contacting with the
germ. Symptoms of shigellosis consist of:

Diarrhea (sometimes bloody)

Fever
Stomach pain
Sensation of the need to pass stool [poop] yet when the
bowels are empty

Figure: 5 Shigella species

143
 Some people with shigellosis will not have any
symptoms. Symptoms usually last 5 to 7 days, but some
people may experience symptoms anywhere from a few
days to 4 or more weeks. In some cases, it may take several
months before bowel habits (for example, how repeatedly
someone passes stool and the evenness of their stool) are
entirely normal.
People with diarrhea should contact their healthcare
contributor if they have any of these symptoms:

Fever
Bloody diarrhea
Severe stomach cramping or tenderness
Dehydrated
Feel very sick.

People who are in deprived health or who have immune

systems weakened from illness such as HIV/AIDS, or
chemotherapy for cancer, are more likely to get sick for a
longer period of time if they have shigellosis. They should
contact their healthcare provider if they think they have
shigellosis to determine the best course of treatment.

Rare symptoms of Shigella infection

About 2% of people who are affected with the type

of Shigella called Shigella flexneri will experience post-
infectious arthritis, which causes joint pains, eye irritation,
and painful urination. The disease only occurs in people
who have precise genetic makeup that puts them at threat.
It can last for months or years, and can direct to chronic
arthritis. Post-infectious arthritis generally does not happen
in people who get sick from the other types
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of Shigella, called S. sonnei, S. boydii, or S. dystenteriae.

Diagnosis

Many type of germs can cause diarrhea. Knowing which

germ is causing an illness is significant to conduct
appropriate treatment. Healthcare contributor can organize
laboratory tests to recognize Shigella germs in the stool
(poop) of someone who is sick.

Treatment

Most people will convalesce from shigellosis without

treatment in 5 to 7 days. People who have shigellosis
should drink plenty of fluids to avoid dehydration. Contact
your healthcare provider if you, or one of your family
members, have a fever, bloody diarrhea, severe stomach
cramping or tenderness, are dehydrated, or feel very sick.
People who are in deprived health or who have damaged
immune systems, such as from HIV/AIDS or chemotherapy
treatment for cancer, also should get in touch with their
healthcare provider because they are more probable to get
ill for an extended period of time.
In some people, bismuth subsalicylate (for example,
Pepto-Bismol) can help to alleviate symptoms. People with
shigellosis should not use anti-diarrheal medication, such as
loperamide (for example, Imodium) or diphenoxylate with
atropine (for example, Lomotil). This medicine may make
indication poorer.
Healthcare providers may recommend antibiotics for
some people who have severe cases of
shigellosis. Antibiotics such as ciprofloxacin (common
treatment for adults), and azithromycin (common treatment
145
for children) are functional for severe cases of
shigellosis because they can help people get better earlier.
However, some antibiotics are not efficient against certain
types of Shigella bacteria. Healthcare contributors can
organize laboratory tests to determine which antibiotics are
likely to work.
People who have shigellosis should follow their healthcare
provider’s advice. If your healthcare provider prescribes
antibiotics, let them know if you do not get better within a
couple of days after starting the medication. They can do more
tests to learn whether your type of Shigella bacteria can be
treated effectively with the antibiotic you are taking. If not, your
doctor may prescribe another type of antibiotic.

Prevention

Shigella germs multiply easily from one person to another;

just a small amount of Shigella germs can make someone sick.
Understanding how to avoid the spread of Shigella germs can
help you shield yourself and your loved ones from getting sick.
People usually get sick from Shigella bacteria after putting
something in their mouth or swallowing something that has
come into contact with the stool (poop) of someone else who is
ill from Shigella bacteria. There is no vaccine to prevent
shigellosis. However, you can decrease your chance of getting
shigellosis by:

Cleansing your hands with soap and water during key

times
Before eating or organizing food for others
After shifting a diaper or serving to sanitize another person
who went to the bathroom
If you concern for a child in diapers who has shigellosis,
quickly throw away soiled diapers in a covered, lined
garbage can. Cleanse your hands and the child’s hands
carefully with soap and water instantly after changing the
146
 diapers. Clean up any leaks or spills of diaper stuffing
instantly.
Safe & healthy diapering in the home
Diaper-Changing Steps for Childcare Settings
Keep away from swallowing water from ponds, lakes, or
unprocessed swimming pools.
When wandering internationally, follow protected foodstuff
and water guidelines and wash hands repeatedly with soap
and water.
Avoid sexual activity with those who have diarrhea or who
newly (several weeks) improved from shigellosis.

If you are sick with shigellosis you can prevent others from
getting sick by:

Washing hands frequently, especially:

Before arranging food or eating
After utilizing the bathroom or changing diapers
Not preparing food if you are ill and not distributing food
with anyone if you or your family members are sick
Avoid swimming
NOT having sex (vaginal, anal, and oral) for one week after
you no longer have diarrhea. Aas Shigella germs may be in
stool for several weeks, follow secure sexual practices, or
ideally avoid having sex, for several weeks after you have
recovered.
Reside in home and be away from school or from
healthcare, food service, or childcare jobs while ill or until
your health section says it’s safe to return.

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4.5 Listerial infections

Characteristics of organism

L. monocytogenes is a gram-positive, nonsporeforming,

facultatively anaerobic rod which grows between -0.4 and
500°C. It is found to be oxidase negative and catalase
positive and expresses a P-hemolysin which forms zones of
clearing on blood agar. The hemolysin acts synergistically
with the 3-hemolysin of Staphylococcus aureus on sheep
erythrocytes. The organisms acquire peritrichous flagella,
which provide a characteristic tumbling, motility, occurring
only in a narrow temperature range. When the organism is
grown between 20 and 25°C, flagellin is both formed and
gathers at the cell surface, but at 370°C flagellin production
is obviously reduced.
Of several acids (acetic, lactic, citric, and hydrochloric
acids) used to lesser the pH of brain heart infusion broth
prior to using it as the growth medium for four L.
monocytogenes strains, acetic acid was the most efficient
growth inhibitor. The authors established that the least pH
is necessary for initiation of growth ranged from 5.0 to 5.7
at 4°C and from 4.3 to 5.2 at 30°C. Analysis on
carbohydrate fermentations by Listeria spp. were reported
by Pine et al. Under anaerobic environment only hexoses
and pentoses maintain growth; aerobically, maltose and
lactose, but not sucrose, also maintain growth. L.
monocytogenes and L. innocua make use of glucose,
lactose, and rhamnose under aerobic conditions.

Symptoms

Listeriosis can cause a range of symptoms, depending on

the person and the part of the body affected. Listeria leads
to fever and diarrhea analogous to other foodborne germs,
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but this type of disease is rarely identified. Symptoms in
people with invasive listeriosis, meaning the bacteria has
multiplied beyond the gut, depend on whether the person is
pregnant.

Pregnant women

Pregnant women normally experience only fever and

other flu-like sign, such as fatigue and muscle aches.
Though, infections during pregnancy can direct to
miscarriage, stillbirth, premature delivery, or life-
threatening infection of the newborn.
People other than pregnant women

Signs can include headache, stiff neck, confusion, loss

of balance, and convulsions in addition to fever and muscle
[Link] with invasive listeriosis usually report the
indication starting 1 to 4 weeks after eating food
contaminated with Listeria; some people have report
symptoms initial as late as 70 days after contact or as
initially as the same day of exposure.

Figure: 6 Listeria species

149
Diagnosis and Treatment

Listeriosis is usually detected when a bacterial culture (a

type of laboratory test) cultivate Listeria
monocytogenes from a body tissue or fluid, such as blood,
spinal fluid, or the placenta. Listeriosis is treated with
antibiotics.

Incidence of L. monocytogenes in foods

L. monocytogenes has the capacity to stay alive the

manufacture and ripening of many different cheeses,
existing best in cheeses such as Camembert and worst in
foodstuffs such as cottage cheese. L. monocytogenes is
typically concentrated in the curd, with only small numbers
of organisms being found in whey. L. monocytogenes
cultivate well in both naturally and artificially polluted
fluid dairy products at temperatures ranging from 4 to
35°C. The organism obviously cultivates in the presence of
common psychrotrophic bacteria in milk; it has been shown
that the existence of pseudomonads in milk may enhance
the growth of L. monocytogenes.
Chicken also seems to be profoundly polluted with L.
monocytogenes as surveys show contamination rates
ranging from 12 to 60%. Few studies have observed the
serotype predominance in chickens, but it emerge that, as
for beef, serotype 1 is the most prevalent. Bailey has
recently observed the reason influencing colonization of
broiler chickens with L. monocytogenes. It is
evident that poultry become contaminated either
environmentally during production or from healthy carrier
chickens in the processing plant. Glass and Doyle found
that development of L. monocytogenes on meat was
exceedingly dependent on product type and pH. The
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organism have a tendency to grow well on meat products
with a pH value near or above 6.0, while it grew poorly or
not at all on meats near or below pH 5.0.
L. monocytogenes newly found at low levels (1 and
8 CFU/ml) in 2 of 42 samples of commercially broken raw
liquid egg. It is found in positive samples. As with meats, it
appears that it can stay alive in frozen raw egg and grow
well in cooked eggs. Generation times for L.
monocytogenes growing in egg yolks, cooked whole eggs,
cooked yolks, and made without starter culture. This
diverse explanation of cooked albumen was 1.7, 1.9, 2.3
and 2.4 days at 5°C.
Although many diverse types of vegetables have been
analyzed for the presence of L. monocytogenes, only
potatoes and radishes appear to be regularly contaminated.
Although L. monocytogenes appears to grow quite well in
lettuce juice stored at 5sC, it could be cultivated in heat-
sterilized cabbage juice only when stored at 30°C and
containing 2.0% NaCl.
Weagant et al., upon investigating 57 samples of frozen
seafood products, established 15 samples along with
shrimp, crabmeat, lobster tail, fin fish, and surimi-based
seafood, to be positive for L. monocytogenes.

People at risk

CDC estimates that Listeria is the third principal cause of

fatality from food poisoning, in the United States. An
estimated 1,600 people get sick from Listeria each year, and
about 260 die. Listeria is most likely to sicken pregnant
women and their newborns, adults aged 65 or older, and
people with damaged immune systems. Other people can
be polluted with Listeria, but they rarely become seriously
ill.
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People with weakened immune system

CDC estimates that Listeria infection is the third leading

cause of death from food poisoning in the United States.
About 1,600 people get sick from Listeria each year, and
about 260 die. People with weakened immune systems due
to underlying medical conditions, such as cancer, diabetes,
liver or kidney disease, alcoholism, and HIV or AIDS, are
more likely to get a Listeria infection. Treatments that make
it more complex for the body to fight off illness, such as
steroids and chemotherapy, also can boost the chance
of Listeria infection

Prevention

Listeria is a harmful germ that can hide in many

foods. Outbreaks of Listeria infections in the 1990s were
mainly associated to deli meats and hot dogs.
Now, Listeria outbreaks are often linked to dairy products.
Investigators have traced recent outbreaks to soft cheeses,
celery, sprouts, cantaloupe, and ice cream.

Do not consume uncooked or lightly cooked sprouts

of any kind (including alfalfa, clover, and mung bean
sprouts).
Cook sprouts methodically to decline your threat for
getting ill. Thorough cooking destroys the dangerous
bacteria.
When you’re eating out, ask that raw sprouts not be
added to your food. If you buy a readymade sandwich,
salad, or Asian food, confirm to make sure it doesn’t
contain raw sprouts.
Cleaning sprouts with water will not eliminate
bacteria. Home-grown sprouts also can make you ill if
you consume them uncooked or lightly cooked.
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 Eat cut melon in an exact way or refrigerate it.
Keep cut melon refrigerated at 41° F or freezing and
not exceeding more than 7 days.
Throw away cut melons left at room temperature for
exceeding more than 4 hours
Do not eat cold smoked fish if not it is canned or
shelf-stable or it is in a cooked dish, such as a
casserole.
Only drink pasteurized milk and milk products,
including soft cheese, ice cream and yogurt. See
for the word “pasteurized” on the sticker. If
suspicious, don’t buy it!
Store milk and milk products frozen at 40°F or
colder.

4.6 Mycotoxin

Aflatoxins are a type of mycotoxin produced

by Aspergillus species of fungi, such as A. flavus and A.
parasiticus. The umbrella term aflatoxin refers to four
different types of mycotoxins produced, which are B1, B2,
G1, and G2. Aflatoxin B1, the most toxic, is a potent
carcinogen and has been directly correlated to adverse
health effects, such as liver cancer, in many animal species.
Aflatoxins are largely associated with commodities
produced in the tropics and subtropics, such as cotton,
peanuts, spices, pistachios, and maize.
Ochratoxin is a mycotoxin that comes in three secondary
metabolite forms, A B, and C. All are produced
by Penicillium and Aspergillus species. The three forms
vary in Ochratoxin B (OTB) is a nonchlorinated form of
Ochratoxin A (OTA) and that Ochratoxin C (OTC) is an
ethyl ester form Ochratoxin A. Aspergillus ochraceus is
established as a contaminant of a broad range of
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commodities together with beverages such as beer and
wine. Aspergillus carbonarius is the main species found on
vine fruit, which liberate its toxin during the juice making
process. OTA has been marked as a carcinogen and a
nephrotoxin, and has been associated to tumors in the
human urinary tract, even though research in humans is
incomplete by confounding features.
Citrinin is a toxin that was first isolated from P.
citrinum, but has been recognized in over a dozen species
of Penicillium and a number of species of Aspergillus.
Some of these species are used to produce human
foodstuffs such as cheese (Penicillium camemberti), sake,
miso and soy sauce (Aspergillus oryzae). Citrinin is related
with yellowed rice disease in Japan and acts as a
nephrotoxin in all animal species tested. Although it is
associated with many human foods (wheat, corn, oats, and
food colored with Monascus pigment). Citrinin can also
perform synergistically with Ochratoxin A to depress RNA
synthesis in murine kidneys.
Ergot is a compound formed as a toxic mixture of
alkaloids in the sclerotia of species of Claviceps, which are
common pathogens of various grass species. The ingestion
of ergot sclerotia from infected cereals, commonly in the
form of bread produced from contaminated flour, cause
ergotism the human disease historically known as St.
Anthony’s Fire. There are two forms of ergotism:
Gangrenous, disturbing blood supply to extremities, and
convulsive, affecting the central nervous system. Modern
technique of grain cleaning have considerably reduced
ergotism as a human disease, though it is still an significant
veterinary problem. Ergot alkaloids have been utilized
pharmaceutically.
Patulin is a toxin formed by
the [Link], Aspergillus, Penicillium,
and Paecilomyces fungal species. [Link] is mainly
154
related with a choice of moldy fruits and vegetables,
in particular decaying apples and Figs. It is damaged by the
fermentation process and so is not found in apple
beverages, such as cider. Although patulin has not been
revealed to be carcinogenic, it has been reported to damage
the immune system in animals. In 2004, the European
Community set limits to the concentrations of patulin in
food products. They currently stand at 50 μg/kg in all fruit
juice concentrations, at 25 μg/kg in solid apple products
used for direct consumption, and at 10 μg/kg for children’s
apple products, including apple juice.
Fusarium toxins are produced by over 50 species
of Fusarium and have a history of contaminating the grain
of developing cereals such as wheat and maize. They
include a range of mycotoxins, such as: the fumonisins,
which influence the nervous systems of horses and may
leads to cancer in rodents; the trichothecenes, which are
most strongly related with chronic and fatal contaminated
effects in animals and humans; and zearalenone, which is
not associated to any lethal toxic effects in animals or
humans. Some of the other major types of Fusarium toxins
include: beauvercin and enniatins, butenolide, equisetin,
and fusarins.

Health implications of eaten foods contaminated by

mycotoxins

The consumption of mycotoxin-contaminated

commodities is correlated to several acute and chronic
diseases in humans as well as in animals. While the
accurate cause and effect relationship has been recognized
for only a few of the diseases, speculation about the role of
mycotoxins in the aetiology of a variety of diseases has
been based on circumstantial proof in other cases. The
acute infection for which there is some verification of a
155
relationship with mycotoxins include: Aflatoxic hepatitis in
India and Kenya; enteric ergotism in India; vascular
ergotism in Ethiopia; and deoxynivalenol mycotoxicosis in
India and China. A common trait in all these outbreaks has
been the association of staple foods such as corn, wheat or
pearl millet, following unseasonable rains or drought
during either the growing season or harvest.
Among the mycotoxins, aflatoxins have been implicated
in human diseases including liver cancer, Reye’s syndrome,
Indian childhood cirrhosis, chronic gastritis, kwashiorkor
and certain occupational respiratory diseases in different
parts of the world, particularly in African and Asian
countries. In China, the Philippines, Thailand, Kenya,
Swaziland and Mozambique, higher levels of aflatoxins in
the food supply have been associated with aflatoxins and
their derivatives in human fluids which may be related with
liver cancer. Fusarium toxins have been supposed to have a
role in diseases such as Kashin Beck syndrome in the
USSR, China and Vietnam; Mseleni joint disease in
southern Africa; endemic familial arthritis in India;
alimentary toxic aleukia in the USSR; and oesophageal
cancer in southern Africa. Ochratoxins have been linked
with Balkan endemic nephropathy and urinary tract
tumours. Though, in most of these instances, conclusive
indication for the role of mycotoxins in disease causation
has been lacking.

Removal of mycotoxins in foods

In the feed and food industry it has become familiar

practice to add mycotoxin binding agents such as
montmorillonite or bentonite clay in order to affectively
adsorb the mycotoxins. To reverse the adverse effects of
mycotoxins, the following criteria are used to assess the
functionality of any binding additive:
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 Effectiveness of active Constituent established by
scientific data.
A low efficient inclusion rate.
Stability over a broad pH range.
High capacity to absorb high concentrations of
mycotoxins.
High affinity to absorb small concentrations of
mycotoxins.
Affirmation of chemical contact between mycotoxin
and adsorbent.
Verified in-vivo data with all major mycotoxins
Non-toxic, environmentally friendly constituent.

Since not all mycotoxins can be bound to such agents,

the newest approach to mycotoxin control is mycotoxin
deactivation. By means of enzymes (esterase, de-
epoxidase), yeast (Trichosporon mycotoxinvorans), or
bacterial strains (Eubacterium BBSH 797), mycotoxins can
be reduced during pre-harvesting contamination. Other
elimination methods include physical separation, washing,
milling, nixtamalization, heat-treatment, radiation,
extraction with solvents, and the use of chemical or
biological agents. Irradiation methods have confirmed to be
efficient treatment against mold development and toxin
production.

4.7 Alternaria toxin

Alternaria is a fungal genus that includes saprophytic

and pathogenic species and that is widespread in nature.
They can infect a wide variety of crops in the field and in
the postharvest stage causing considerable losses due to
fruit and vegetable decay. The most
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common Alternaria species include A. alternata, A.
tenuissima, A. arborescens, A. radicina, A. brassicae, A.
brassicicola, and A. infectoria. They colonize a range of
plants including cereals, tomatoes, apples, grapes, oil crops,
oilseeds, sunflower seeds, oranges, lemons, melons,
cucumbers, cauliflowers, peppers, and tangerines.

Toxicity of Alternaria mycotoxin

Toxicological data are restricted to the above-mentioned

major mycotoxins and even these data are incomplete, with
neither good bioavailability studies nor long-term clinical
studies available. Although little is known so far about their
properties and toxicological mechanisms, bioavailability,
and strength in the digestive tract, Alternaria toxins have
been shown to have damaging effects in animals, including
cytotoxicity, fetotoxicity, and teratogenicity. They have
been associated to a range of pathologies from
hematological disorders to esophageal cancer. They are
also mutagenic, clastogenic, and estrogenic in microbial
and mammalian cell systems and tumorigenic in rats.
In the lasts years diverse food matrices were
investigated for the existence of Alternaria mycotoxins,
including cereals (wheat, rice, maize, bakery products, and
bread); fruits and vegetables (tomato products, pepper,
onions, black radish apple, sweet cherry, strawberries,
blueberries, orange fruits, citrus, and pomegranate fruits);
beverages (beer; wine: red, white, and rosé; tea and herbal
infusions; cider; fruit; and vegetable juices).
Alternaria toxins are, together with aflatoxins,
ochratoxin A, and patulin, the most normally found
mycotoxins in fruits and their processed products. Due to
the limitations of current industrial development to
completely remove the rotten tissues and the reported
158
stability of some Alternaria mycotoxins (i.e., AOH, AME,
and ATXs) in fruit juices and during tomato processing, it
is clear that these mycotoxins are likely to be present in
commercial end products.
The maximum concentrations of Alternaria toxins
reported in commercial food products ranged from 1 up to
8700 μg/kg. The highest levels were found in samples
visibly infected with Alternaria rot, that is, in products
obviously not suitable for human consumption. According
to the European Food Safety Authority (EFSA), major
contributors to Alternaria mycotoxins dietary exposure are
grains and grain-based products, especially wheat. The
highest concentrations of TeA were reported for cereals,
followed by commercial beers, while AOH, AME, TeA,
and TEN were found in legumes, nuts, and oilseeds,
particularly in sunflower seeds.
Moreover, it was experimented that the cooccurrence
of Alternaria toxins with other mycotoxins is probable.
Owing to the different physical and chemical properties of
all the widespread mycotoxins in fruits, the purpose of trace
amounts of all them in food represents an particularly
challenging task.

Most Reported Mycotoxins

Among the 58 studies included in Table : 6, more than

80% analyzed AOH and AME, and around 50% included
TEN and TeA, followed by ALT (36%). These were
followed by Alternaria toxins ATXs included in 16% of the
analyzed studies, whereas only a few publications reported
other compounds such as macrosporin (9%), monocerin,
ALN, AALs, and infectopyrone (all <3%). Notably, limited
information was found about pyrenochaetic acid A (PyA),
alternarienonic acid (AIA), and alloTeA and isoALT,
recently reported in food samples for the first time and
159
metabolites such as AOH3S, AOH3G, AME3S, and
AME3G. In this way, AOH, AME, TeA, and TEN have
been shown to occur in food samples generally while the
incidence of ALT, isoALT, ATX-I, and AAL toxins is of
much lower prevalence, mainly due to shortcomings in
current analytical methodologies.
Slight differences were observed based on the analyzed
food matrices. Thus, AOH and AME (both 86%) were the
most examined ones in cereals and cereal by-products,
followed by TEN and TeA (59 and 41%, resp.). Other
mycotoxins calculated in these matrices were ALN (27%),
macrosporin (23%), ALT (14%), and lesser degree in
monocerin and infectopyrone (both 5%). The same trend
was observed in fruits, vegetables, and derived products,
with predominance of AOH and AME (both 79%),
followed by TEN (41%), TeA (38%), ALT (34%), and
ATXs (14%).

4.8 Toxigenic phytoplanktons

Paralytic Shellfish Poisoning

This situation is contracted by consumption of toxic

mussels, clams, oysters, scallops, or cockles. These
bivalves become toxic after supplying certain
dinoflagellates of which Gonyaulax catenella is found
along the North Atlantic Coast of the United States and
over to northern Europe, G. tamarensis is found. G.
acatenella is originated along the coast of British
Columbia. Masses or blooms of these toxic dinoflagellates
give rise to the red tide condition of seas. The paralytic
shellfish poison (PSP) is saxitoxin, Saxitoxin apply its
effect in humans during cardiovascular collapse and
respiratory failure. It blocks the propagation of nerve
impulses without depolarization, and there is no recognized
160
antidote. It is heat stable, water soluble, and usually
destroyed by boiling 3-4 hours at pH of 3.0.

Symptoms of PSP develop within 2 hours after ingestion

of toxic mollusks, known as paresthesia (tingling,
numbness, or burning), which initiate on the mouth, lips,
and tongue and later spreads over the face, scalp, and neck,
and to the fingertips and toes. The death rate is variously
reported to range from 1% to 22%. In 1990, there were 19
cases from two outbreaks in the states of Massachusetts and
Alaska alone. In the past, six fishermen became ill after
eating boiled mussels that enclosed 4,280 jig/100 g
saxitoxin. The raw mussels enclosed 24,400 jug/100g. The
greatest safe level of PSP toxins. Lig/100g. Mollusks may
become poisonous in the absence of red tides.
Detoxification of mollusks can be attained by their transfer
to clean water, and a month or more may be necessary.

Ciguatera Poisoning

In the years 1983-1992, 129 outbreaks were reported to

the CDC involving 508 persons with no deaths. This
disease is contracted from the ingestion of any one of over
300 fish species (barracuda, grouper, sea bass, etc.) that
nourish on herbivorous or reef fishes, which in turn feed on
phytoplankton, particularly the dinoflagellates. The
dependable dinoflagellate is Gambierdiscus toxicus, which
generate ciguatoxin. This toxin is intense more in fish
organs such as the liver than in muscle tissue. Upon
ingestion of toxic fish, symptoms take place within 3-6
hours (about the same as for staphylococcal food
poisoning), and consist of nausea and paresthesia about the
mouth, tongue, and throat and finally direct to respiratory
paralsysis when untreated.

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DomoicAcid

This is an uncommon amino acid that antagonizes

glutamic acid in the central nervous system. It is formed by
a diatom, Pseudonitzschia pungens, and its structure is as
indicated. (Diatoms. Domoic acid reason for amnesic
shellfish poisoning (ASP) subsequently followed by the
consumption of mussels or scallops harvested from marine
waters with a bloom of the diatom noted. The initial
recorded outbreak of human cases take place in eastern
Canada in 1988 subsequently followed by the consumption
of mussels from Prince Edward Island,and there were 107
victims and three deaths. ASP episode affected scallops in
northwest Spain in 1996. The main amount of domoic acid
was found in the hepatopancreas—from 52% to 88% of the
total. During frozen storage, some domoic acid transport to
other parts of the scallops. It was found that canning of
scallops did not devastate this toxic principal.

Pfiesteria piscicida

It is animal-like organisms that generate potent toxins.

One toxin stuns fish within a few seconds, and the animals
die within a few minutes. It is heat constant. Another toxin
causes the fish epidermis to slough off. The dinoflagellate
reproduces sexually after a fish kill, and it can encyst. The
exact character of the toxins is indistinct, as are their effect
on humans. Those who have been exposed have an account
of memory loss, confusion, severe skin burning, and
frequently general symptoms such as headache, skin rash,
muscle cramps.

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4.9 Viruses

Norovirus

Virological aspects

NoVs belong to the Family Caliciviridae, which is

divided into genera. Norovirus and Sapovirus are the two
out of five genera of the family Caliciviridae that contain
viruses that cause infections in humans. NoVs have also
been detected in pigs, cattle, mice, cats, dogs, and sheep,
and sapoviruses in pigs. In humans, NoVs cause
gastroenteritis, while the animal viruses can cause a range
of different clinical syndromes, including oral lesions,
systemic disease with hemorrhagic syndromes, upper
respiratory tract infections, and others. Furthermore, one
other potential genus comprising viruses detected in rhesus
macaques has been described. So far, the NoVs and
sapoviruses are the only caliciviruses known to cause
disease in humans, with the exception of anecdotal zoonotic
infection with vesiviruses. NoVs can be divided into distinct
genogroups, based on phylogenetic analyses of the capsid
protein. Viruses of GI, GII, and GIV are known to infect
humans. GII viruses have additionally been detected in
pigs, and GIV viruses have been detected in carnivores (a
lion cub and a dog). GIII viruses infect cattle and sheep,
and GV viruses infect mice. Recombination between
viruses from different genogroups is rare, suggesting that
this constitutes a species level in taxonomy. Within each
genogroup, viruses are further segregated into lineages,
termed genotypes. Where known, these seem to have a
global distribution, with little evidence for geographic
clustering. Direct comparison of data across countries is
challenging because of differences in study design and
163
laboratory diagnostics, resulting in poorly defined biases.
This is particularly the case when trying to establish causes
of food-borne illness. Here, the less common genotypes of
norovirus are likely to play a bigger role, and it is these
viruses that are less available for assay test validation
studies. The development of quality assurance schemes for
molecular diagnostics, therefore, is particularly important
for detection of such highly diverse viruses.

Epidemiology

The etiological importance of NoVs as causes of

diarrheal illness has been documented worldwide, but few
studies have been performed in a standardized way that
allows international comparison and true burden of disease
estimates. Community studies have provided evidence for
the abundance of NoVs and established that these viruses
are the number one cause of community-acquired
gastroenteritis, with one out of four or five persons infected
per year. The burden of illness is highest in young children
and the elderly. The best described feature is the dose
required for productive infection is very low (1-10
particles), and infected persons shed huge amounts of
viruses (up to 1010 million per gram of stool) In addition,
the interaction of NoVs with histo-bloodgroup antigens
determines the outcome of exposure, and strain-dependent
differences in host susceptibility have been observed.
Viruses are by far the most commonly identified outbreak
strain. In a study in hospitalized patients, the probability of
secondary transmission of NoVs differed by age and
genotype. In recent years, the incidence of norovirus
outbreaks has increased with the emergence of a particular
variant. More severe complications are seen in
immunocompromised patients, and mortality in the elderly.
164
Estimation of the burden of food-borne infection

In the EU, in 2008, crustaceans, shellfish, mollusks, and

products thereof were the most frequently implicated food
items in NoV and HAV outbreaks, but this may also reflect
an ascertainment bias, because testing for the presence of
viruses in shellfish is well established across Europe. The
use of epidemiological criteria in the United States
concluded that an estimated 28 percent of all reported
outbreaks with unknown etiology were likely caused by
NoVs. An important caveat in using these data is that
testing of patients with gastroenteritis for NoV is not yet an
established routine, although this is rapidly changing. In
addition to the recognized food-borne outbreaks, the high
rate of secondary infections in NoV outbreaks can rapidly
mask an initial food-borne introduction. only 5 percent of
all reported NoV outbreaks are fully confirmed, reflecting
the challenges of virus detection in or on food items.
Given the paucity of evidence, few studies have
attempted to quantify burden of food-borne illness
attributable to viruses. A recent analysis of available data
estimates that almost 60 percent of illness cases, 26 percent
of hospitalizations, and 11 percent of deaths from food-
borne illness are caused by NoV. Similarly, an estimate
based on data from Australia suggests that NoVs are
important causes of food-borne illness.
Also, the ultimate number of persons affected by a food-
borne outbreak is rarely known, and reported outbreaks are
likely to be biased. The average size of reported outbreaks
is limited, but there are examples of widespread
dissemination, for instance following consumption of
wedding cake, sandwiches from an ill baker, deli meat
165
during rafting trips down the Grand Canyon, frozen
shellfish, or a manually prepared salad. This variant was
found in association with four different capsids until
equilibrium was reached and the virus continued to
circulate in combination with GII3 capsid. These viruses
currently are the second most common cause of infection in
children hospitalized with NoV.

Hepatitis A (HAV)

Virology

The hepatitis A virus belongs to the family

Picornaviridae, genus Hepatovirus. Hepatoviruses have
only been found in humans and primates. Based on genetic
diversity, hepatitis A viruses are divided into six lineages or
genotypes, of which genotypes I-III infect humans.
Genotypes I and II contain subgenotypes (Ia, Ib, IIa, and
IIb). In regions with endemic HAV circulation, further
segregation into geographically defined clusters is
observed, a property that can be used to support source
tracing activities in food-borne outbreaks.

Epidemiology

HAV incidence is greatly reduced in regions with proper

sanitation and good hygienic conditions. These differences
also affect the level of population immunity and, thus, the
susceptibility to food-contamination events. In highly
endemic regions, HAV is one of the childhood infections
that, in the majority of cases, runs an asymptomatic course,
while triggering a protective immune response that lasts
long, possibly even lifelong. In such regions, sustained
circulation of HAV strains is found, resulting in
166
geographically distinct genetic fingerprints. Although this
geographical diversity is used to support investigations into
the possible source of an outbreak, or in defining where a
patient most likely contracted the disease.
In regions with high socioeconomic status, HAV
circulation is very limited and mostly restricted to risk
groups such as men who have sex with men, to immigrant
populations from regions with higher endemicity that may
reintroduce viruses when infected during family visits in
their country of origin, to travelers who contracted
infection while visiting an endemic country and may
transmit infection to nonimmune contacts, and to food- and
water-borne infection. In such regions, population
immunity builds up much slower, leading to an increase in
the size of the susceptible population, and a right shift of
first-time infections to higher age groups. With increasing
age, the probability of having symptomatic illness
increases, and complications such as fulminant hepatitis are
more common. This leads to the somewhat contrasting
situation that food-contamination events may have a greater
impact in regions with low endemicity of hepatitis A than
in highly endemic regions. This different epidemiological
pattern also has consequences for the use of molecular
typing in HAV source tracing; in low endemic regions,
most people with HAV will have contracted the infection in
a different region, and, as a consequence, a great diversity
of HAV strains may be seen, reflecting the geographic
fingerprints from the regions where they contracted the
illness. This basic pattern can be greatly influenced by
changing the population immune status through
vaccination. Vaccination confers clinical protection that is
thought to be long lasting. Whether vaccinated individuals
contribute to shedding also is not well known.

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Evidence for food-borne infection

HAV is quite stable outside a host and, therefore, can

persist on contaminated environments, food, and water.
Food- and water-borne outbreaks have been documented,
although again, as for NoVs, the most common mode of
transmission occurs between persons. Because of the risk
pattern described above, the biggest risk of food-borne
HAV currently is introduction through food into regions
where population immunity is relatively limited. Foods of
primary importance, therefore, are those susceptible to
contamination during the production phase, such as bivalve
filter-feeding mollusks (oysters, clams, mussels) or produce
that is irrigated with water that may be contaminated (e.g.,
lettuce, green onions, and soft fruits, such as raspberries
and strawberries). An extreme example of the potential
impact dates from 1988, when almost 300,000 cases were
caused by consumption of clams harvested from a sewage-
polluted area. A specific problem with shellfish is that the
current microbiological quality control criteria are based on
testing for bacterial contamination, which does not reliably
predict the presence or absence of viruses. Also, mildly
polluted products can be put on the market after “rinsing”
the shellfish by storing them for a period of time in clean
water in a process called depuration. Depurated shellfish
have been associated with outbreaks of norovirus, hepatitis
A, gastroenteritis, and other viral diseases. For NoV,
specific binding to histo-bloodgroup antigens in oyster
tissues has been demonstrated, possibly further explaining
the retention of viruses in these animals.

Estimation of the food-borne burden of illness

In the CDC assessment of food-borne pathogens,

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hepatitis A is the second virus listed and is considered a
significant cause of severe disease. This may be related to
the increased severity when HAV infection is first acquired
during adulthood, although there also are differences in
virulence between genotypes.

Rotaviruses

The first expressions of these viruses happen in 1973 in

Australia, and they were initially propagated in the
laboratory in 1981. Six groups have been recognized, and
three are known to be infectious for humans. Group A is
the most normally encountered among infants and young
children throughout the world. Group B leads to diarrhea in
adults, and they have been found only in China.
Rotaviruses belong to the family Reoviridae; they are about
70 nm in diameter, are nonenveloped, and contain double-
stranded RNA (dsRNA). The fecal–oral route is the
principal mode of transmission. Rotaviruses cause a
probable one-third of all hospitalizations for diarrhea in
children below age 5, and the peak season for illness take
place during the winter months. Most vulnerable are
children between the ages of 6 months and 2 years, and it
has been reported that virtually every child in the United
States is infected by age 4. Although the majority of
persons are immune by age 4, high inoculums or lowered
states of immunity can direct to milder illness among older
children and adults. These viruses are identified to be
transmitted among children in day care centers and by
water. A community waterborne outbreak took place in
Eagle-Vail, Colorado, in 1981, and 44% of 128 persons,
most of them adults, became sick. They are thought to be
only infrequent source of foodborne gastroenteritis. The
incubation period for rotavirus gastroenteritis is 2 days.
Vomiting occur for 3 days along with watery diarrhea for
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3–8 days, and often abdominal pain and fever also occur.
They are known to be associated with travelers’ diarrhea. It
appears that these viruses induce diarrhea by activating the
enteric nervous system (ENS) based on the inhibition of
ENS functions in mice and in vitro by four drugs that
inhibit ENS functions.
For the 23-month period between January 1989 and
November 1990, stool specimens were observe in the
United States, with 9,639 (20%) being positive for
rotavirus. The maximum percentage of positive stools
occurred in February (36%) and the lowest in October
(6%). Between 1979 and 1985, an annual average of 500
children died from diarrheal sickness in the United States,
and 20% were due to rotavirus infections
The host-cell-receptor protein for rotavirus also serve as
the β-adrenergic receptor. Once inside cells, they are
transported to lysosomes where uncoating take place.
Rotaviral infections can be analyzed by immunoelectron
microscopy, RT–PCR, enzyme-linked immunosorbent
assay (ELISA), and latex agglutination methods.

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5 Chapter 5: Applications of Food Microbiology

5.1 Classification of Molds and Molds of Industrial

Importance.

In the following only genera of industrial importance will

be shortly overviewed.

Genus Mucor (Mucor racemosus, Mucor rouxii): Mucors

are implicated in the spoilage of some foodstuffs and in the
production of others e.g. oriental fermented foods.
Genus Rhizopus: Rhizopus nigricans, sometimes called
bread mold”, is very common and is concerned in the
spoilage of many foods such as berries, fruits, vegetables,
bread, etc.
Genus Aspergillus: The members of this genus are very
widespread. Many are concerned in the spoilage of foods
and some are functional in preparation of fermented foods.
Many groups and hundreds of aspergillus species are
identified. Aspergillus niger is the foremost species
essential for food microbiologists. Preferred strains are
utilized for commercial production of citric and gluconic
acids.
Genus Penicillium: This is another prevalent genus
important in foods. Penicillium expansum, a green spored
species, is the reason soft rot of fruits. Penicillium
camemberti with grayish conidia, useful in the ripening of
Camembert cheese, and Penicillium roqueforti, used in
ripening of blue cheeses, are also well known members of
this genus.
Genus Bothrytis: The species Bothrytis cinerea leads to
the noble rot of grape in some wine producing areas such as
Tokay (Hungary).
Genus Alternaria: Molds of this genus are common
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reason for the spoilage of foods. Alternaria citri and
Alternaria brassicae are the common species.
Genus Neurospora (Monilia): The species of this genus
are developed on a variety of foods.

Citric Acid Production

Production by Fungi Since the early demonstration by

Wehmer in 1893 of the occurrence of citric acid in culture
media including sugar and inorganic salts with species of
Penicillium, a variety of fungi were screened for citric acid
production. At present Aspergillus niger is most frequently
used for industrial production of citric acid. Generally the
strains of Aspergillus niger require a fairly high initial
concentration (16-18%) of sugars in the medium. An
elevated concentration leads to greater quantity of residual
sugar, making the process uneconomic. On the other hand,
lower sugar concentration means lesser yield as well as
accumulation of oxalic acid. When molasses is used, it is
diluted to a sugar content of 16-20% and adjusted to pH of
5.5 to 6.5. Nitrogen sources are assured by inorganic salts
(e.g. ammonium nitrate). Accumulation of phosphate is
also common. The citric acid fermentation is an aerobic
fermentation, thus a suitable oxygen supply is vital. Citric
acid is precipitated from filtered fermentation liquor in the
form of calcium citrate. The filtered and washed calcium
citrate is treated with sulfuric acid. After elimination of
calcium sulfate the citric acid contain liquid is purified and
then concentrated in a vacuum and finally crystallized.

Butter

Butter is made from separated cream, which is usually

soured, since butter made from soured cream keeps better
than that made from sweet. The souring of the cream is
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brought about by organisms naturally present in the milk,
or the milk may first be pasteurized and specific starters
then added. Organisms concerned in the making of butter
include Streptococcus lactis and Streptococcus cremoris,
which ferment lactose and sour the cream, and two other
capsulated streptococcal-like organisms, Leuconostoc
citrovorum and L. dextranicum. The latter organisms attack
citric acid, a by-product of lactose fermentation, to produce
diacetyl which imparts a buttery aroma to the cream. Many
other identified strains are also present and add their
particular flavours to the butter. The temperature at which
the cream is soured is very important. Temperatures around
20°C are low enough to prevent growth of thermophilic
spoilage organisms, which have survived pasteurization,
and yet are sufficiently high to allow growth of the
desirable streptococci. In butter-making the soured cream is
churned. This causes the fat globules to aggregate to form
butter, leaving the buttermilk which can be drained off.
Bacteria are restricted to the moisture droplets throughout
the butter. Washing of butter to replace these droplets of
buttermilk by water deprives bacteria of nutrients and limits
their growth, thus improving the keeping quality of the
butter. 'Working' of butter to break the moisture into
smaller droplets also improves the keeping quality by
limiting the available nutrients within a droplet. This does
not apply to moulds, which can penetrate the surrounding
wall of fat and move to other droplets.

Cheese

Cheese is one of the most important milk products. The

widely used Cheddar cheese has a close texture, firm
mellow body, and a mild nutty flavour. The raw milk is
heated to 68°C for 15 seconds to destroy most vegetative
organisms, including coagulase-positive staphylococci
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(which have increased with the wider use of milking
machines) as well as the udder-derived coagulase-negative
strains. It is important that the milk be pasteurized without
delay to ensure that any strains of Staphylococcus aureus
which may be present would not have had time to produce
enterotoxins. The pasteurized milk is not sterile and will
contain thermoduric organisms. A starter is required to
produce lactic acid in situ in the curd; this can be either
Streptococcus lactis, Streptococcus cremoris, or a mixture
of these, with or without Leuconostoc cremoris, etc. Great
care must be taken not to build up specific bacteriophages
in cheese rooms, particularly where a single strain of
bacterium is always employed. Single strains are used
when waxy, bland-flavoured cheese is favoured, whereas
multi-strains commonly produce the more mellow, nutty
flavour. Antibiotics used to control mastitis cause problems
in cheese making and penicillinase-producing strains of
coagulase-negative staphylococci have been used both to
remove the antibiotic and because their lipolytic action can
contribute to the cheese flavour. The starter rapidly gives a
low redox potential, produces protein degradation product
and a pH of 5·0, all conditions essential to the development
of the lactobacilli and unfavourable to other bacteria;
clostridia are inhibited by the antibiotic nisin produced by
some strains of streptococci. If the streptococci fail, so also
do the lactobacilli and the cheese develops such faults as
taints and gas produced by coliforms and clostridia, and
faulty colour by a variety of organisms. Toxigenic
staphylococci would also find the conditions favourable.
Renneting with calf rennet follows immediately after the
starter which also produces the optimum pH for casein
decomposition and clotting. The coagulum is cut into small
pieces to release the whey, the degree of syneresis being
dependent on the temperature and the rate of acid
production by the bacteria. Finally the curd is consolidated
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into blocks under light pressure until they become plastic,
when they are broken into pieces, sprinkled with salt and
pressed into moulds. The shaped blocks are first dipped in
molten paraffin wax to exclude air and thus to prevent
external mould growth and weight loss, and then ripened at
13°C for quick process cheese or 5.5°C for the old slow-
ripening type. The optimum period of maturing varies from
2 to 14 months, according to the previous treatment and
type of cheese required. During cheese ripening the
numbers of lipolytic organisms are low; the lactic
streptococci used as starters are soon inhibited while the
numbers of lactobacilli increase progressively during the
later stages of ripening of all cheeses, especially the hard
varieties. The main flora consists of homofermentative
types (Lactobacillus casei and Lactobacillus plantarum);
Lactobacillus brevis and Lactobacillus. lactis occur much
less frequently. The total number of lactobacilli and
streptococci, both alive and dead, is extremely large and
probably accounts for as much as 0·1% of the weight of the
cheese. In Camembert cheese, Streptococcus lactis is
dominant during the first phase of growth, Penicillium and
Oidium in the second and L. casei in the third phase. Blue
cheeses are made from open textured cheese through the
action of varieties of the mould Penicillium roqueforti. The
drying cheese coat supports the growth of moulds, yeasts
(Torulopsis sp.), streptococci, and lactobacilli. The
microflora of the coat of Stilton cheese is characteristic of
the factory producing it. Clammy rind on Dutch cheese is
caused by the growth of corynebacteria that develop only at
pH 5·3 and is favoured by the prior development of yeasts
(such as Debaryomyces, Candida, Trichosporon sp.) that
decompose lactic acid and form ammonia from proteins,
thus altering the pH in favour of the corynebacteria. On the
other hand, surface ripened cheeses such as Liederkrantz,
Trappist, Tilsiter, etc., that have a brown surface smear of
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aerobic bacteria, have antibiotic activity due to
Brevibacterium linens.

Fermented milks

Many different strains of lactobacilli, either alone or in

combination with lactose fermenting yeasts and
streptococci, are used to produce characteristic fermented
beverages. In Europe and the Middle East yoghurt is made
from milk thickened by heating or the addition of dried
milk solids and fermented with Lactobacillus bulgaricus
and Streptococcus thermophilus. It has custard like
consistency and is often flavoured with ground fruit or nuts.
Kefir is a fizzy beverage made from fermented mares' milk
using 'grains' that contain yeasts, streptococci, lactobacilli,
and micrococci. The grains are strained off after their
action is complete and added to the next batch of milk.
Koumiss, also made from mares' milk, is a greyish-white
liquid with uncurdled casein, often bottled and allowed to
carbonate by the action of the micro-organisms. It normally
contains 1 - 2.5% v/v alcohol. Acidophilus milk has been a
fashionable drink for several decades since it was thought
that Lactobacillus acidophilus would survive in the gut and
replace so-called harmful bacteria. However, it is doubtful
whether this organism would survive without a high
carbohydrate diet rich in growth factors. The most frequent
usage for bacteria in food preparation is with dairy
fermentations. Yogurt and cheeses have been made for
centuries using bacteria. The ancients may not have known
precisely what kind of bacteria that was needed. All they
know was that the earlier batch was required to make a new
one. Many people lack the capability to break down and
absorb lactose, the sugar molecule in milk. As an effect, it
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enters the gut, producing acid and gas, causing pain and
diarrhea. Fermented milk products metabolize lactose into
lactic acid, which is more acceptable for many people. It is
found in two isomer form D and L. The mainly widespread
fermented milk product is yogurt. The lactobacilli used in
the production of many yogurts, though, may not be the
same type as found within the common flora of humans as
there are many diverse strains (Probiotics). Some of the
bacteria are utilized in the diary industry.
Lactic acid bacteria are a heterogenous family of mainly
low G+C Gram positive anaerobic, non sporulating and
acid tolerant bacteria. They can ferment various nutrients
through a homo or heterofermentative route into primarily
lactic acid, but also into by products such as acetic acid,
Formic acid, ethanol and carbon dioxide. They contribute
to rapid acidification of food products, but also to flavours,
texture and nutrition. The genome sequencing of food and
health related LAB has been booming. Around forty LAB
genomes have been entirely sequenced, including the
genera Lactobacilli, Lactococci, Streptococci, Pediococcus,
Oenococcus and Leuconotoc. Acidophilus milk is made
with Lactobacillus acidophilus. Butter is made from
pasteurized cream, to which a lactic acid starter has been
added. The starter contains, for example, Streptococcus
cremoris or S. lactis, but requires Lactobacillus
diacetylactis to give it its characteristic flavor and odor.
Cheese is regularly made with Streptococcus and
Lactobacillus bacteria. Fermentation lesser the pH, thus
serving in the initial coagulation of the milk protein, as well
as giving characteristic flavors. In such Swiss cheeses, the
typical flavor is the result of the utilization of
Propionibacterium. Cheese can be classified within two
groups -- ripened and unripened. Unripened cheeses consist
of cottage cheese, cream cheese, and Mozzarella. These are
soft cheeses and are finished by the lactic acid fermentation
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of milk. Many different bacteria are utilized to manufacture
the various cheeses, but Lactococcus lactis and
Leuconostoc cremoris are used most often. Soft cheeses
can take one to five months to ripen; hard cheeses, three
months to a year or more; and very hard cheeses, like
Parmesan, can take twelve to eighteen months. The blue
veins found in cheeses, are caused by growth of Penicillium
roqueforti, which is deliberately added to cheese. Initially,
it was found as a natural contaminant of the areas where it
was made. The holes in Swiss cheese are the result of
Propionibacterium shermanii. The surfaces of Camembert
and Brie are inoculated with Penicillium camembertii,
which then develops in a skin on the surface. Limburger is
soaked in brine to support the growth of Brevibacterium
linens. Kefir includes many various microbes, including
yeasts, lactobacilli, lactococci, and leuconostocs.
Depending on geographical locations, the precise types of
microbes will differ. Yogurt usually needs the addition of
Lactobacillus bulgaricus, Lactococcus thermophilus,
and/or Streptococcus thermophilus to the milk. Lactic acid
bacteria comprise of an ecologically diverse group of
microorganisms with the formation of lactic acid as the
primary metabolite of sugar metabolism. These bacteria
utilize sugars by either homo- or heterofermentative
pathways. (Figure: 7)

Figure: 7 Homo and heterofermentative pathways of

LAB

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Beer and lager

An alcoholic beverage is a drink that include ethanol,

commonly known as alcohol. Beer has been a part of
human culture for 8,000 years. In Germany, Ireland, the
United Kingdom, and many other European countries,
drinking beer (and other alcoholic beverages) in a local bar
or pub is a cultural practice. Non-alcoholic beverages are
drinks that usually contain alcohol, such as beer and wine,
but contain less than 0.5% alcohol by volume. This group
includes low-alcohol beer, non-alcoholic wine, and apple
cider. Wines are completed from a variety of fruits, such as
grapes, peaches, plums or apricots. The majority of wines
are produced from grapes. The soil in which the grapes are
grown and the weather conditions in the growing season
decide the quality and taste of the grapes which in turn
influence the flavor and value of wines. When ripe, the
grapes are crushed and fermented in large vats to produce
wine. Beer is also made by the process of fermentation. A
liquid mix, called wort, is prepared by mixing yeast and
malted cereal, such as corn, rye, wheat or barely.
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Fermentation of this liquid mix generate alcohol and carbon
dioxide. The method of fermentation is stopped before it is
completed to limit the alcohol content. The alcohol so
formed is called beer. It comprises 4 to 8 per cent of
alcohol.
Beer is made by fermenting a hop-flavoured extract of
barley malt with a top-fermentation strain of
Saccharomyces cerevisiae. The bottom-fermenting yeast,
S. carlsbergensis, is required for lager. Barley is
transformed into malt by allowing the soaked grain to
germinate under controlled conditions of temperature and
humidity, leading to the formation of α-and β-amylases,
and the breakdown of the protein, hordein, to amino acids.
The process is stopped by raising the temperature and
removing the rootlet and plumules. The exact treatment
depends on the type of malt required. In some countries, the
protein is fully converted into amino acids; lager malts still
contain appreciable quantities of protein, for beer
manufacture. Stout malts are dried at 105°C to give a dark-
coloured extract; malts from distilleries and vinegar
breweries receive no final kilning and still contain limit-
dextrinase.
Successful brewing is dependent not only on the
biochemical properties of a yeast strain but also on the
physical properties of the mass culture. In contrast, cider
and wine were made by controlling a natural flora of
yeasts, moulds, and bacteria, so that only organisms with
desirable properties were allowed to grow. The
microbiology of cider is being discussed here, that of wine
is similar in many respects. In the traditional cider-making
countries, the raw material consists of special varieties of
apples, high in tannin and low in acidity. Now a day these
are supplemented with dessert and culinary apples graded
as unsuitable for the fresh fruit market; other countries use
these latter sorts as their sole source of raw material. The
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fruit as it arrives at the factory carries both an internal and
an external microflora. Some of the common moulds are
Candida pulcherrima, Kloeckera apiculata, Torulopsis
jamata, Aspergillus niger, Botritis cinerea, Penicillium sp.
together with the 'black yeast' Aureobasidium pullulans and
occasionally rhodotorulae, Candida, and Torulopsis spp.
Saccharomyces spp. and bacteria are rare in sound fruit but
mould-damaged specimens carry large populations, not
only of mould spores but also of acetic acid bacteria and
fermenting yeasts. Both the moulds and acetic bacteria
produce sulphite binding compounds that are of great
significance at later stages of processing. Hence sound,
preferably washed fruit, is essential. The apples are milled
to a pulp and pressed hydraulically in cloth envelopes
between wooden drainage racks. Formerly the racks and
cloths were washed very infrequently, consequently they
soon supported a dense population of Saccharomyces
species; these were the yeasts mainly responsible for the
subsequent fermentation. At first Candida spp. and other
poorly fermenting yeasts are most numerous but they are
rapidly overgrown by Kloeckera sp. after a few degrees
drop in specific gravity. The latter then co-exist and in turn
are overgrown by Saccharomyces sp. while in the fully
fermented dry ciders film yeasts and acetic bacteria become
more important. The whole process is improved by
treatment of the freshly pressed juice with sulphur dioxide,
the actual amount being varied according to the pH. Below
pH 3.3, 100 ppm is sufficient, while 150 ppm is desirable
between 3.3 and 3.8; to be effective more S02 is required
above 3.8 than is allowed by law (200 ppm), but most
factories process a mixture of apples whose juice pH is
generally 3.5-3.6. It is vitally important that the
concentration of sulphite binding compounds be kept low
in the juice, otherwise no free sulphur dioxide remains in
the solution and this is the only effective moiety
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(sometimes referred to as undissociated sulphurous acid).
After treatment only Saccharomyces spp. remains, so that
in effect sulphiting selects a pure culture from the original
microflora. However, with higher standards of cleanliness
in the press rooms, the juices have a flora similar to that of
the fruit, i.e., virtually free of fermenting yeasts. Hence, the
addition of pure cultures of bottom-fermenting wine yeasts,
following sulphiting, is becoming almost universal in cider
factories. It also means that both the time needed for
fermentation and the flavour of the cider become more
predictable, since there is no longer any need to rely on
what were virtually factory contaminants. A second
fermentation of malic acid to lactic acid and carbon
dioxide, due to lactic acid bacteria, nearly always occurs
during either the yeast fermentation or storage of the cider.
The organisms that can bring about this change include
both homo-and heterofermentative lactic rods and cocci,
including the organisms responsible for producing
polysaccharide slime or ropiness. The bacterium found
most frequently is Lactobacillus pastorianus var. quinicus.
If citric acid is present, as in perry but not cider, diacetyl,
lactic, and acetic acids are also formed and the flavour is
thereby spoiled. The source of these bacteria is still in some
doubt since they appear to be absent from many sulphited
juices. Ciders stored in contact with air, especially those
held in small wooden containers, soon show surface
growths of Acetobacter xylinum and the film yeasts Pichia
membranaefaciens and Candida mycoderma. The problem
is now rare in large factories where juice sulphiting and
very large storage tanks are normal practice.

5.2 Prebiotics

Prebiotics are compounds in food that stimulate the

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growth or activity of useful microorganisms such as
bacteria and fungi. The most common case is in the
gastrointestinal tract, where prebiotics can alter the
composition of organisms in the gut microbiome.
Dietary prebiotics are typically
nondigestible fiber compounds that pass undigested
throughout the upper part of the gastrointestinal tract and
arouse the growth or activity of advantageous bacteria that
colonize the large bowel by using as substrate for them.
They were first recognized and named by Marcel
Roberfroid in 1995. As a functional food component,
prebiotics, like probiotics, are a theoretical intermediary
between foods and drugs. Depending on the jurisdiction,
they normally receive an intermediate level of regulatory
scrutiny, in particular of the health claims made regarding
them for marketing purposes.
The characterization of prebiotics and the food
ingredients that can fall under this classification, has
develop since its first definition in 1995. In its early
definition, the term prebiotics was used to refer to non-
digestible food ingredients that were expensive to the host
during their selective stimulation of specific bacteria within
the colon. As a result of research suggesting that prebiotics
could impact microorganisms outer surface of the colon, in
2016 the International Scientific Association for Probiotics
and Prebiotics (ISAPP) produced the following description
of prebiotics: a substrate that is selectively used by a host
microorganism to create a health benefit.
Compounds that can be classified as prebiotics must also
meet the subsequent following criteria:
Non-digestible and resistant to breakdown by stomach
acid and enzymes in the human gastrointestinal tract
Discriminating fermentation by intestinal
microorganisms
Selectively choosing the targeting and stimulating the
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 growth and activity of beneficial bacteria
Thus, consumption of prebiotics may assist in the health
of the host. Based on the preceding classifications, plant-
derived carbohydrate compounds
called oligosaccharides are the main source of prebiotics
that have been
recognized. Specifically, fructans and galactans are two
oligosaccharide sources which have been found to arouse
the activity and development of beneficial bacterial
colonies in the gut. Fructans are a group of carbohydrate
consisting of fructooligosaccharides (FOS) and inulins,
while galactans consist
of galactooligosaccharides (GOS). Other dietary fibers also
fit the meaning of prebiotics, such as resistant
starch, pectin, beta-glucans, and xylooligosaccharides.
The European Food Safety Authority (EFSA), the
regulatory agency for product labeling, differentiates
between "prebiotic" and "dietary fiber", condition that
"a cause and effect relationship has not been recognized
between the consumption of the food constituents which
are the subject of the health claims and a beneficial
physiological consequence related to rising numbers of
gastrointestinal microbiota".[12] As a result, under EFSA
rules individual ingredients cannot be labeled as prebiotics,
but only as dietary fiber and with no implication of health
benefits.

Function

Majority of the prebiotic research has focused on the

result that prebiotics present
on Bifidobacteria and Lactobacillus. These bacteria have
been emphasized as key probiotics and favorable gut
bacteria as they may have numerous useful effects on the
host in conditions of improving digestion and the
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effectiveness and intrinsic power of the immune
system. Both Bifidobacteria and Lactobacillus have been
exposed to have differing prebiotic precisely and to
selective ferment prebiotic fiber based on the enzymes
characteristic of the bacterial population. Thus, Lactobacilli
favor inulin and fructooligosaccharides,
whereas Bifidobacteria show specificity for inulin,
fructooligosaccharides, xylooligosaccahrides and
galactooligosaccharides. A product that arouse
bifidobacteria is depicted as a bifidogenic factor, a theory
that overlaps, but is not the same with, being prebiotic.
Analysis have also shown that prebiotics, besides inspiring
the growth of beneficial gut bacteria, can also hinder the
growth of damaging and potentially pathogenic microbes in
the gut, for instance clostridia.

Mechanism of action

Fermentation is the major mechanism of action by

which prebiotics are utilized by beneficial bacteria in the
colon. Both Bifidobacteria and Lactobacillus are bacterial
populations which make use of saccharolytic metabolism to
break down substrates. The bifidobacterial genome include
many genes that encode for carbohydrate-modifying
enzymes as well as genes that encode for carbohydrate
uptake proteins. The presence of these genes specify
that Bifidobacteria include specific metabolic pathways
specific for the fermentation and metabolism of plant-
derived oligosaccharides, or prebiotics. These pathways
in Bifidobacteria ultimately generate short chain fatty
acids, which have various physiological roles in body
functions.

Sources
Prebiotic sources must be verified to confer a benefit to
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the host in order to categorize as a prebiotic. Fermentable
carbohydrates resulting from fructans and xylans are the the
majority of the well-recognized example of prebiotics.

Endogenous

An endogenous reason for prebiotics in humans is

human breast milk, which consist of oligosaccharides
structurally similar to GOS, referred to as human milk
oligosaccharides (HMOs). These HMOs were identified to
increase the Bifidobacteria bacterial population in breastfed
infants, and to make the infant immune system
powerful. Furthermore, HMOs engage in recreation in the
concern of a healthy intestinal microbiota composition of
newborns.

Exogenous

Indigestible carbohydrate compounds categorized as

prebiotics are a type of fermentable fiber, and therefore can
be classified as dietary fiber. Though, not all dietary fiber
can be organized as a prebiotic source. Additionally the
food sources highlighted in the following table, raw oats,
unrefined barley, and whole grain breakfast cereals are also
classified as prebiotic fiber sources. The main type of
prebiotic fiber may differ according to the food. For
example, oats and barley have high quantity of beta-
glucans, fruits contain pectins, seeds contain gums,
onions and Jerusalem artichokes are prosperous
in inulin and oligofructose, and bananas and legumes
consist of resistant starch.
While there is no broad consensus on an ideal daily
serving of prebiotics, recommendations typically range
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from 4 to 8 grams (0.14–0.28 oz) for common digestive
health support, to 15 grams (0.53 oz) or more for those with
digestive disorders. Given an average 6 grams (0.21 oz)
serving, below are the quantity of prebiotic foods necessary
to accomplish a daily serving of prebiotic fiber (Table: 6)

Table: 6 Foods containing Prebiotics

Research

Preliminary study has demonstrated possible effects on

calcium and other mineral absorption, immune
system effectiveness, bowel acidity, reduction of colorectal
cancer threat, inflammatory bowel disease (Crohn's
disease or ulcerative
colitis), hypertension and defecation regularity. Prebiotics
may be efficient in decreasing the number of infectious
episodes requiring antibiotics and the entire amount of
infections in children aged 0–24 months. No good evidence
shows that prebiotics are efficient in avoiding or treat
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allergies.
While investigation reveal that prebiotics direct to
amplified production of short-chain fatty
acids (SCFA), more investigation is necessary to establish a
direct causal connection. Prebiotics may be valuable
to inflammatory bowel disease or Crohn's illness through
manufacture of SCFA as nourishment for colonic walls,
and mitigation of ulcerative colitis indication.
The rapid adding up of considerable amount of
prebiotics to the diet may boost the fermentation, foremost
to enlarged gas production, bloating or bowel
movement. Manufacture of SCFA and fermentation quality
are reduced during long-term diets of low fiber
intake. Until bacterial flora are regularly established to
rehabilitate or restore intestinal bacteria, nutrient absorption
may harm and colonic transit time provisionally increased
with a rapid adding up of elevated levels of prebiotic
intake.

5.3 Probiotics

Probiotics are live microbes that can be prepared into

various types of products, including food stuff, drugs, and
dietary supplements. Species of Lactobacillus and
Bifidobacterium are most normally utilized as probiotics,
however yeast Saccharomyces cerevisiae and some E. coli
and Bacillus species are also used as probiotics. Lactic acid
bacteria, together with Lactobacillus species, which have
been used for preservation of foodstuff by fermentation for
thousands of years, can provide a dual function by acting as
agents for food fermentation and, in addition, potentially
suggesting health benefits. Fermentation is globally
functional in the preservation of a variety of raw
agricultural materials (cereals, tubers, fruit and vegetables,
milk, fish etc.).
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Lactobacillus- a model probiotic

Lactic acid bacteria are Gram-positive, catalase-negative

bacterial species able to produce lactic acid as main end-
product of carbohydrate fermentation. Lactobacillus is a
type of bacterium having lots of different species. These are
"friendly" bacteria that usually live in our digestive,
urinary, and genital systems that don’t lead to disease. In
addition it is utilized to avoid and treat diarrhea related with
using antibiotics. Some people use lactobacillus for general
digestion problems; irritable bowel syndrome (IBS); colic
in babies; Crohn's disease; inflammation of the colon; and a
severe gut problem called necrotizing enterocolitis (NEC)
in babies born prematurely.
Probiotics have an antimicrobial consequence through
altering the microflora, secreting microbicidal substances,
competing with pathogens to avoid their adhesion to the
intestinal epithelium, competing for nutrients essential for
pathogen survival, generating an antitoxin effect and
reverse some of the consequences of disease on the
intestinal epithelium, such as secretory changes and
neutrophil migration. Probiotics stimulate T-cells, B-cells,
macrophages and NK-cells with smart messages that
promote specific immune responses. They also activate
cytokines and phagocytic cells directly to coordinate their
intelligent immune response.
Probiotics also produce acetic acids, formic acids
lipopolysaccharides, peptidoglycans, superantigens, heat
shock proteins and bacterial DNA-all in precise portions to
nourish each other, inhibit challengers and/or benefit the
host. Probiotics also produce antibacterial molecules called
bacteriocins. Lactobacillus plantarum produces lactolin.
Lactobacillus bulgaricus secretes bulgarican. Lactobacillus
acidophilus can produce acidophilin, acidolin, bacterlocin
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and lactocidin. These and other antibacterial substances
equip probiotic species with territorial mechanisms to
combat and reduce pathologies related to Shigella,
Coliform, Pseudomonas, Klebsiella, Staphylococcus,
Clostridium, Escherichia and other infective genera.

5.4 Genetically modified Foods

Genetically modified foods (GM foods), also

referred to as genetically engineered foods (GE foods),
or bioengineered foods are foods formed
from organisms that have had changes incorporated into
their DNA using the technique of genetic engineering.
Genetic engineering technique allow for the introduction of
new character as well as superior control over traits when
contrast to preceding methods, such as selective
breeding and mutation breeding.
Commercial sale of genetically modified foods began in
1994, when Calgene initially marketed its
unsuccessful Flavr Savr delayed-ripening tomato. Most
food alteration have mainly focused on cash crops in high
requirement by farmers such as soybean, corn, canola,
and cotton. Genetically modified crops have been
engineered for resistance to pathogens and herbicides and
for enhanced nutrient report.

History

Human-directed genetic manipulation of food began

with the domestication of plants and animals
during artificial selection at about 10,500 to 10,100 BC.
The development of selective breeding, in which organisms
with preferred traits are utilized to breed the next
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generation and organisms deficient in the trait are not bred,
is a precursor to the current idea of genetic modification
(GM). With the finding of DNA in the early 1900s and a
variety of advancements in genetic techniques through the
1970s it became probable to directly modify the DNA and
genes in food.
Genetically modified microbial enzymes were the initial
function of genetically modified organisms in food
production and were accepted in 1988 by the US Food and
Drug Administration. In the early 1990s,
recombinant chymosin was accepted for use in several
countries. Cheese had normally been made using the
enzyme complex rennet that had been extracted from cows'
stomach lining. Scientists modified bacteria to generate
chymosin, which was also able to clot milk, producing
cheese curds.
The first genetically modified food approved for release
was the Flavr Savr tomato in 1994. Developed by Calgene,
it was engineered to have a longer shelf life by inserting
an antisense gene that delayed ripening. China was the first
country to commercialize a transgenic crop in 1993 with
the introduction of virus-resistant tobacco. In 1995, Bacillus
thuringiensis (Bt) Potato was permitted for cultivation,
making it the first pesticide manufacturing crop to be
approved in the US. Other genetically modified crops
receiving marketing approval in 1995 were: canola with
modified oil composition, Bt maize, cotton resistant to the
herbicide bromoxynil, Bt cotton, glyphosate-
tolerant soybeans and delayed maturing tomato.
With the manufacture of golden rice in 2000, researchers
had genetically customized food to amplify its nutrient
value for the earliest time. By 2010, 29 countries had
planted commercialized biotech crops and a further 31
countries had approved regulatory support for transgenic
crops to be imported. The US was the foremost country in
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the manufacture of GM foods in 2011, with twenty-five
GM crops having received regulatory support. In 2015,
92% of corn, 94% of soybeans, and 94% of cotton
produced in the US were genetically customized strains.
The first genetically modified animal to be approved for
food use was AquAdvantage salmon in 2015. The salmon
were altered with a growth hormone-regulating gene from
a Pacific Chinook salmon and a promoter from an ocean
pout facilitate it to grow year-round as an alternative during
spring and summer.
In April 2016, a white button mushroom (Agaricus
bisporus) modified using the CRISPR technique
received de facto support in the United States, after the
USDA said it would not have to go through the agency's
regulatory process. The agency considers the mushroom
exempt because the editing process did not involve the
introduction of foreign DNA.
The most extensively planted GMOs are intended to
tolerate herbicides. By 2006 some weed populations had
developed to tolerate some of the identical
herbicides. Palmer amaranth is a weed that competes with
cotton. A native of the southwestern US, it passed through
east and was first established in resistant to glyphosate in
2006, fewer than 10 years after the introduction of GM
cotton.

Crops

Genetically modified crops (GM crops) are genetically

adapted plants that are utilized in agriculture. The first
crops industrialized were used for animal or human food
and offer resistance to certain pests, diseases,
environmental circumstances, spoilage or chemical
treatments (e.g. resistance to an herbicide). The second
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generation of crops intended to advance the quality, often
by changing the nutrient profile. Third generation
genetically modified crops could be employed for non-food
purposes, as well as the production of pharmaceutical
agents, biofuels, and other industrially functional goods,
additionally used for bioremediation. GM crops have been
formed to improve harvests through decreasing insect
pressure, increase nutrient value and endure diverse abiotic
stresses. As of 2018, the commercialised crops are
restricted mostly to cash crops like cotton, soybean, maize
and canola and the enormous majority of the introduced
traits offer either herbicide tolerance or insect resistance.
The majority of GM crops have been customized to be
resistant to preferred herbicides, usually
a glyphosate or glufosinate based one. Genetically modified
crops engineered to resist herbicides are now further
available than normally bred resistant varieties. The
majority of the currently accessible genes employed to
engineer insect resistance come from the Bacillus
thuringiensis (Bt) bacterium and code for delta endotoxins.
A few utilization of the genes that encode for vegetative
insecticidal proteins. The only gene commercially utilized
to provide insect protection that does not initiate from B.
thuringiensis is the Cowpea trypsin inhibitor (CpTI). CpTI
was initially permitted for use cotton in 1999 and is
presently undergoing test in rice. Less than one percent of
GM crops contained other traits, which comprise providing
virus resistance, delaying senescence and varying the plants
composition.

Fruits and vegetables

Papaya was genetically customized to resist the ringspot

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virus (PSRV). "SunUp" is a transgenic red-fleshed Sunset
papaya cultivar specifically homozygous for the coat
protein gene PRSV; "Rainbow" is a yellow-fleshed F1
hybrid urbanized by crossing 'SunUp' and nontransgenic
yellow-fleshed "Kapoho". The GM cultivar was permitted
in 1998 and by 2010 80% of Hawaiian papaya was
genetically engineered. The New York Times declared,
"without it, the state's papaya industry would have
collapsed". In China, a transgenic PRSV-resistant papaya
was industrialized by South China Agricultural
University and was first permitted for commercial planting
in 2006; as of 2012 95% of the papaya grown
in Guangdong province and 40% of the papaya grown
in Hainan province was genetically modified. In Hong
Kong, where there is an exemption on growing and
releasing any varieties of GM papaya, more than 80% of
grown and imported papayas were transgenic.
The Novel Leaf potato, a GM food developed
using Bacillus thuringiensis (Bt), was made to provide in-
plant safety from the yield-robbing Colorado potato beetle.
The New Leaf potato, brought to market by Monsanto in
the late 1990s, was industrialized for the fast food market.
In 2011, BASF requested the European Food Safety
Authority's approval for cultivation and marketing of its
Fortuna potato as feed and food. The potato was made
resistant to late blight by adding up resistant genes blb1 and
blb2 that initiate from the Mexican wild potato Solanum
bulbocastanum. In February 2013, BASF withdrew its
application. In 2014, the USDA accepted a genetically
modified potato industrialized by J. R. Simplot
Company that contained ten genetic modifications that
avoid bruising and produce fewer acrylamide when fried.
The modifications eradicate specific proteins from the
potatoes, via RNA interference, rather than pioneering
novel proteins.
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 In 2013, the USDA accepted the import of a GM
pineapple that is pink in color and that "overexpresses" a
gene expressed from tangerines and suppresses new genes,
rising production of lycopene. The plant's flowering cycle
was altered to supply for more uniform development and
quality. The fruit "does not have the capacity to propagate
and persevere in the environment just the once they have
been yielded", according to USDA APHIS. According to
Del Monte's compliance, the pineapples are commercially
developed in a "monoculture" that avoids seed production,
as the plant's flowers aren't uncovered to
compatible pollen sources. Importation into Hawaii is
banned for "plant sanitation".
In February 2015 Arctic Apples were permitted by the
USDA, becoming the initially genetically modified apple
agreed for sale in the US. Gene silencing is employed to
reduce the synthesis of polyphenol oxidase (PPO), thus
avoid the fruit from browning.

Corn

Corn utilized for food and ethanol has been genetically

altered to endure a variety of herbicides and to synthesis a
protein from Bacillus thuringiensis (Bt) that destroy certain
insects. About 90% of the corn developed in the US was
genetically modified in 2010. In the US in 2015, 81% of
corn acreage contained the Bt trait and 89% of corn acreage
contained the glyphosate-tolerant trait. Corn can be
developed into grits, meal and flour as an ingredient in
pancakes, muffins, doughnuts, breadings and batters, as
well as baby foods, meat products, cereals and some
fermented products. Corn-based flour and masa dough are
utilised in the manufacture of taco shells, corn chips and
tortillas.

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Soy

Soybeans accounted for half of all genetically modified

crops planted in 2014. Genetically modified soybean has
been altered to tolerate herbicides and manufacture
healthier oils. In 2015, 94% of soybean acreage in the U.S.
was genetically altered to be glyphosate-tolerant.

Rice

Golden rice is of the majority of the well known GM

crop that is intended at increasing nutrient value. It has
been engineered with three genes that biosynthesise beta-
carotene, a precursor of vitamin A, in the edible parts of
rice. It is projected to produce a fortified food to be
developed and consumed in areas with a shortage of dietary
vitamin A, a insufficiency which each year is predicted to
kill 670,000 children under the age of 5 and is the source
for an additional 500,000 cases of irreversible childhood
blindness. The original golden rice produced 1.6μg/g of
the carotenoids, with additional development rising this 23
times. In 2018 it increase its first approvals for utilize as
food.

Wheat

As of December 2017, genetically modified wheat has

been estimated in field trials, but has not been released
profitably.

Lecithin

Lecithin is an obviously occurring lipid. It can be found

196
in egg yolks and oil-producing plants. It is an emulsifier
and thus it is employed in many foods. Corn, soy and
safflower oil are root source of lecithin, though the majority
of lecithin profitably available and is a derivative of soy.
Adequately developed lecithin is frequently untraceable
with normal testing practices. According to the FDA, no
proof shows or imply hazard to the public when lecithin is
used at frequent level. Lecithin added to foods quantity to
only 2 to 10 percent of the 1 to 5 g
of phosphoglycerides consumed daily on
average. Nonetheless, customer concerns about GM food
extend to such products. This concern led to strategy and
regulatory changes in Europe in 2000, when instruction
(EC) 50/2000 was passed which is necessary for labelling
of food comprising additives derived from GMOs as well
as lecithin. Because of the complexity of identifying the
origin of derivatives like lecithin with existing testing
practices, European regulations necessitate those who wish
to sell lecithin in Europe to utilize a comprehensive system
of Identity preservation (IP).

Sugar

The US imports 10% of its sugar, whereas the left over 90%
is extracted from sugar beet and sugarcane. After deregulation in
2005, glyphosate-resistant sugar beet was widely implemented in
the United States. 95% of beet acres in the US were planted with
glyphosate-resistant seed in 2011. GM sugar beets are accepted
for cultivation in the US, Canada and Japan; the vast majority are
grown in the US. GM beets are accepted for import and
consumption in Australia, Canada, Colombia, EU, Japan, Korea,
Mexico, New Zealand, Philippines, the Russian Federation and
Singapore. Pulp from the refining process is employed as animal
feed. The sugar formed from GM sugar beets contains no DNA
or protein – it is just sucrose that is chemically indistinguishable
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from sugar formed from non-GM sugar beets. Independent
investigation carry out by internationally accepted laboratories
found that sugar from Roundup Ready sugar beets is equal to the
sugar from comparably grown conventional sugar beets.

Vegetable oil

There is a vanishingly small quantity of protein or DNA

from the original crop in vegetable oil. Vegetable oil is
prepared of triglycerides extracted from plants or seeds and
then refined and may be promoted and developed
via hydrogenation to turn liquid oils into solids. The
refining method removes all or nearly all non-triglyceride
ingredients. Medium-chain triglycerides (MCTs)
recommend an substitute to conventional fats and oils. The
length of a fatty acid manipulates its fat absorption during
the digestive process. Fatty acids in the middle position on
the glycerol molecules appear to be absorbed more
effortlessly and influence metabolism more than fatty acids
on the end positions. Unlike ordinary fats, MCTs are
metabolized like carbohydrates. They have exceptional
oxidative stability, and avoid foods from turning rancid
readily.

Animal feed

Livestock and poultry are raised on animal feed, much of

which is collected of the leftovers from processing crops,
including GM crops. For instance, around 43% of a canola
seed is oil. What remains after oil extraction is a meal that
turn out to be an ingredient in animal feed and contains
canola protein. Similarly, the bulk of the soybean crop is
grown for oil and meal. The high-protein defatted and
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toasted soy meal develop into livestock feed and dog food.
98% of the US soybean crop goes for livestock feed. In
2011, 49% of the US maize yield was utilized for livestock
feed. Even though technique that are appropriate more and
more sensitive, tests have not yet been capable to establish
a difference in the meat, milk, or eggs of animals
depending on the type of feed they are fed. It is not possible
to tell if an animal was fed GM soy just by looking at the
resulting meat, dairy, or egg products. The only way to
confirm the presence of GMOs in animal feed is to
investigate the origin of the feed itself."
Enzymes formed by genetically modified
microorganisms are also integrated into animal feed to
develop availability of nutrients and overall digestion.
These enzymes may also supply benefit to the
gut microbiome of an animal, as well
as hydrolyse antinutritional factors present in the feed.

Proteins

Rennet is a mixture of enzymes used to coagulate milk

into cheese. Originally it was accessible only from the
fourth stomach of calves, and was scarce and costly, or was
available from microbial sources, which often formed
unpleasant tastes. Genetic engineering made it possible to
extract rennet-producing genes from animal stomachs and
insert them into bacteria, fungi or yeasts to make them
generate chymosin, the key enzyme. The customized
microorganism is killed after fermentation. Chymosin is
isolated from the fermentation broth, so that
the Fermentation-Produced Chymosin (FPC) utilized by
cheese producers has an amino acid sequence that is the
same to bovine rennet. The majority of the applied
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chymosin is to hold in the whey. Trace quantities of
chymosin may stay in cheese.
FPC was the first artificially formed enzyme to be
accepted by the US Food and Drug Administration. FPC
products have been on the market since 1990 and as of
2015 had yet to be surpass in commercial markets. In 1999,
about 60% of US hard cheese was made with FPC. Its global
market contribute to 80% approach. By 2008, just about
80% to 90% of commercially made cheeses in the US and
Britain were finished using FPC.
In several countries, recombinant (GM) bovine
somatotropin (also called rBST, or bovine growth hormone
or BGH) is accepted for administration to increase milk
production. rBST may be present in milk from rBST treated
cows, but it is damaged in the digestive system and even if
directly injected into the human bloodstream, has no
observable effect on humans. The FDA, World Health
Organization, American Medical Association, American Dietetic
Association and the National Institutes of Health have
independently confirmed that dairy products and meat from
rBST-treated cows are secure for human
consumption. However, on 30 September 2010, the United
States Court of Appeals, Sixth Circuit, study submitted
verification, found a "compositional difference" between
milk from rBGH-treated cows and milk from untreated
[Link] court stated that milk from rBGH-treated cows
has: amplified levels of the hormone Insulin-like growth
factor 1 (IGF-1); higher fat content and lower protein
content when formed at certain points in the cow's lactation
cycle; and more somatic cell counts, which may "make the
milk turn sour more fast".

5.5 Biosensors in food

A biosensor can be defined as a quantitative or

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semiquantitative analytical instrumental technique
containing a sensing element of biological origin, which is
either integrated within or is in intimate contact with a
physicochemical transducer. A chemical sensor is a tool
that alter chemical information, ranging from the
concentration of a specific sample constituent to whole
composition analysis, into an analytically functional signal.
Chemical sensors typically contain two basic mechanism
connected in series: a chemical (molecular) recognition
system (receptor) and a physicochemical transducer uses
specific biochemical reactions mediated by isolated
enzymes, immunosystems, tissues, organelles or whole
cells to detect chemical compounds usually by electrical,
thermal or optical signals.

Prerequisites for a biosensor

While developing a biosensor system following

requirements are very essential and considered for
successful and commercially viable project.
a. Selectivity: The biosensor device should be highly
selective for the target analyte and show minimum or no
cross reactivity with moieties having similar chemical
structure.
b. Sensitivity: The biosensor device should be able to
measure in the range of interest for a given target analyte
with minimum additional steps such as pre cleaning and pre
concentration of the samples.
c. Linearity of response: The linear response range of
the system should cover the concentration range over which
the target analyte is to be measured.
d. Reproducibility of signal response: When samples
having same concentrations are analyzed several times,
they should give same response.
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 e. Quick response time and recovery time: The biosensor
device response should be quick enough so that real time
monitoring of the target analyte can be done efficiently.
The recovery time should be small for reusability of the
biosensor system.
f. Stability and operating life: As such most of the
biological compounds are unstable in different biochemical
and environmental conditions. The biological element used
should be interfaced such that the activity is retained for a
long time so as to make the device marketable and
practically useful in the field.

Calorimetric biosensors

Calorimetric biosensors determine the change in

temperature of the solution containing the analyte
following enzyme action and infer it in terms of the analyte
concentration in the solution. Since most of the enzyme
catalyzed reactions are exothermic, the heat generated by
the reaction is used to determine the analyte. Calorimetric
biosensors are extensively used for the detection of
pesticides and other enzymatic reactions

Optical biosensors

Optical biosensors are also known as “optodes” because

of their resemblance with electrodes (Figure- 8). These
include determining modification in light absorption among
the reactants and products of a reaction, or calculating the
light output by a luminescent process. Optical biosensors
integrate optical technique with a biological element to
identify chemical or biological species. Many optical
biosensors were developed based on surface Plasmon
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resonance, spectroscopy and evanescent waves etc.
Recently our group at our institute has published several
research papers to monitor pesticides, vitamins,
carcinogens and toxins based on chemiluminescence and
fluorescence.

Figure: 8 Optical Biosensors

Microbial biosensors

A microbial biosensor is an analytical device that

combines microorganisms with a transducer to facilitate
rapid, accurate and sensitive detection of target.
Conventional microbial biosensors employ the respiratory
and metabolic role of the microorganisms to identify a
substance that is either a substrate or an inhibitor of the
processes. Some of the main types of microbial biosensors
are amperometric, potentiometric, and conductometric
sensors. Amperometric microbial biosensor operates at
fixed potential with respect to a reference electrode and
involves the detection of the current generated by the
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oxidation or reduction of species at the surface of the
electrode. Amperometric microbial sensors are extensively
used for detection of biological oxygen demand in
industrial waste water and also used for detection of
ethanol, total sugars, organophosphates, cyanide, phenols
and phenolic compounds. Generally potentiometric
microbial biosensors consist of an ion-selective electrode or
a gas-sensing electrode coated with an immobilized
microbe layer. Potentiometric transducers measure the
potential difference between a working electrode and a
reference electrode, and the signal is correlated to the
concentration of analyte. The signals are expressed in
logarithmic relationship between the potential generated
and analyte concentration. Potentiometric microbial sensors
are used for the detection of organophosphates, penicillin,
tryptophan, urea, trichloroethylene, ethanol and sucrose.
Whole cell biosensor immobilization of Pseudomonas
alcaligenes MTCC 5264 having the capability to degrade
caffeine were used for the development of caffeine
biosensor. The biosensor system was able to identify
caffeine in solution over a concentration range of 0.1 to 1
mg/mL. Caffeine biosensor perform as a rapid study system
for caffeine in Fig. 5 Diagrammatic representation of
optical biosensor solutions.
Surface plasmon resonance (SPR) biosensors SPR is a
physical optics phenomenon which is used for development
of non-labelled/marker based biosensor system. SPR
biosensor is an analytical device, which works on optical
phenomenon in which plasmon waves are excited at the
metal dielectric interface. The surface plasmon waves are
enormously sensitive to the refractive index variation at the
sensor surface and are proportional to the sample mass.
SPR is a surface sensitive technique in which small change
at the surface due to binding or dissociation of
biomolecules brings variation in SPR signal. Antibodies are
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the commonly used biomolecules for the capture of the
analyte from the sample. For a consistent assay of
bimolecular interaction in a SPR biosensor, one species
should be immobilized. Covalent attachment of
biomolecules at the sensor surface is the the major
frequently utilized technique for reliable assays condition.
SPR biosensors recommend unique opportunity of rapid,
label free, real time and cost effective discovery and
identification of biomolecules. A simple method for the
immobilization of antibodies on gold substrates has been
developed for surface plasmon resonance (SPR)
applications (Schmid et al. 2006). Gold surface on a glass
slide was altered with Protein A with cross linker
dithiobissuccinimide propionate (DSP) to get uniform,
stable and sterically accessible antibody coating. The
deposition of the antibodies bound to Protein A was
analysed as a utility of the protein concentration, buffer pH,
buffer type and reaction time. The modified gold surface
was even for a number of weeks and the reproducibility
was agreeable. Thus this technique can be applied to
construct immunosensors or biochips.
Immunosensors
Immunosensors are affinity ligand-based biosensor
devices in which the immunochemical reaction is coupled
to a transducer (Figure: 9). They are solid state devices.
The essential basis of all immunosensors is the precise
method of the molecular identification of antigens by
antibodies to form an even complex which is similar to the
immunoassay methodology. Immunosensors can be
classified related to the recognition principle applied. The
major progress are electrochemical, optical and
microgravimetric immunosensors. While comparing to
immunoassay, modern transducer technology allow the
label-free detection and quantification of the immune
complex. Immunosensors based on Chemiluminescence for
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detection of vitamin B12.

Figure: 9 Schematic representation (a)

immunochemiluminescence based dipstick technique
for detection of vitamin B12, (b) Enzymatic
dephosphorylation of dioxetane by alkaline
phosphatase.

Benzene in soft drinks

Presence of benzene in soft drinks is of concern for

human health due to the carcinogenic nature of the
benzene. In 1993, benzene formation from benzoic acid in
the presence of vitamin C was reported by Gardner and his
coworker. The benzene results from decarboxylation of the
preservative benzoic acid in the presence of ascorbic acid
(vitamin C), E300 or erythorbic acid (E315), especially
under heat and light. Benzoic acid is commonly added as a
food preservative in the form of its salts sodium benzoate
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(E211), potassium benzoate (E212), or calcium benzoate
(E213). Citric acid is not considered to encourage
significant benzene production in grouping with benzoic
acid, but some proof recommend that in the presence of
ascorbic or erythorbic acid and benzoic acid, citric acid
may accelerate the production of benzene. Other factors
that affect the formation of benzene are heat and light.
Storing soft drinks in warm condition speeds up the
formation of benzene. Microbial biosensor based on flow
injection analysis was developed for the detection of
benzene. In the sensor cells Pseudomonas putida were
immobilized between two cellulose acetate membranes and
covered on a Clark dissolved oxygen electrode. The P.
putida aerobically degrades benzene and the working of the
biosensor is simple and at the same time it has good
reproduction of results.

Nitrosamine

In processed foods, nitrosamines are generally formed

from nitrites and secondary amines, which are found in the
form of proteins (Hinuma et al. 1990). Nitrosamines
formation occurs in case of strongly acidic conditions
during digestion in stomach (Scanlan 1983; Hinuma et al.
1990). High temperature processing such as frying, can
promote the formation of nitrosamines. The presence of
nitrosamines may be identified by the Liebermann’s
reaction. Nitrosamines is the root source of cancers in a
broad range of animal species, a character that recommend
that they may also be carcinogenic in humans. A biosensing
method for determination of nitrate in food and water
samples by using nitrate reductase (EC [Link]) was
developed based on the detection of oxidation peak current
of redox mediator, methyl viologen, related to nitrate
207
concentration. This method is selective and sensitive to
determine the nitrate levels of water samples and processed
meat samples. Detection limit of the method was 5.0–
90.0×10−9 M for nitrate concentration with a 10 s response
time.

Freshness of fish

Generally enzyme based biosensors are used to evaluate

the freshness of fish. Different types of metabolites are
determined to estimate the freshness of fish like
hypoxanthine, inosine, octopine and amines, histidine,
cadaverine, putrescine, polyamines and sulphates.

Free radicals and antioxidants

Antioxidants are one of the major ingredients that shield

food attributes by avoiding oxidation that take place during
processing, supply and end preparation of food. Different
types of antioxidants are added to food products to increase
the attributes of food product. Amperometric biosensors are
generally preferred for the determination of antioxidants in
various food products.

Biosensor for ascorbic acid analysis

Work has been carried out on the development of a

tissue based biosensor for L-ascorbic acid analysis in food
and pharmaceutical samples. An immobilized Ascorbic
acid oxidase enzyme was used for the detection of ascorbic
acid oxidase obtained from cucumber peels. We found that
ascorbic acid oxidase was suitable enzyme for the
development of several biosensor systems for detection of
pesticides, vitamin C and copper ions.
208
Detection of copper ions by biosensor

An ascorbic acid oxidase based system was used for the

detection of Cu++ ions. This enzyme contains Cu++ in its
active site. Based on its folding and unfolding activity, a
biosensor was constructed. It was able to detect Cu++ ions
in water sample.

209
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