Submitted By

Aman
Roll No. 251903002
5TH SEM
SESSION 2019 – 2023
In partial fulfillment of the degree of

[Link] IN BIOTECHNOLOGY
Under the supervision of
Dr. Vinod Kumar Gupta
Research Scientist
Rapture Biotech

Submitted To

DEPARTMENT OF BIOTECHNOLOGY
UNIVERSITY INSTITUTE OF ENGINEERING AND TECHNOLOGY
KURUKSHETRA UNIVERSITY
KURUKSHETRA , HARYANA
 ACKNOWLEDGEMENT

Throughout the writing of this report, I have received a great deal of support
and assistance.
I wish to express my sincere gratitude to Dr. Vinod Kumar Gupta, for providing
me an opportunity to do my training in his lab. I thank him for his
encouragement and guidance in carrying out this training. His dedication and
keen interest above all his overwhelming attitude to help his students had
been mainly responsible for completing my work. His timely suggestions have
enabled me to complete this report.
I would also like to thank Mr. Mayank Raj Bhardwaj, the CEO of Rapture
Biotech, for providing his support and guidance in fulfilling my training.
In addition, I would like to thank my parents for their blessings. Their support
always encouraged me to move forward and achieve my aim. Support of my
friends helped me a lot throughout my training period. Their well wishes
proved to be my strength in order to complete my work.
 CONTENTS

1. MEDICAL AND
CLINICAL
MICROBIOLOG
Y
Introduction about medical and clinical microbiology, Overview and
types of Sterilization techniques, Basic principle, standard operating
procedure (SOP) and application of instruments (Autoclave, pH
meter, Laminar air flow, incubator, microscope and colony counter),
Classification of bacteria on the basis of Gram staining, types of
media, concept of basic calculation, solution preparation, General
and safety rules for working in Lab, Types of media, Preparation
and sterilization of selective media (EMB/MSA/Blood Agar),
Isolation of pathogenic bacteria (Salmonella sp. Staphylococcus sp.
Enterococcus sp. Proteus sp., E. coli etc.), Inoculation of
pathogenic bacteria into nutrient broth medium, Different method for
the detection of antimicrobial activity, Basic concept of positive and
negative controls, Antimicrobial activity test against isolated
pathogenic bacteria by disc diffusion method, Minimum inhibitory
concentration (MIC) via Agar diffusion method, Minimum
bactericidal concentration (MBC) through Broth dilution method.

1.1 INTRODUCTION
Medical microbiology, the large subset of microbiology that is applied to medicine,
is a branch of medical science concerned with the prevention, diagnosis and
treatment of infectious diseases. In addition, this field of science studies various
clinical applications of microbes for the improvement of health. There are four kinds
of microorganisms that cause infectious
disease: bacteria, fungi, parasites and viruses, and one type of infectious protein
called prion.
A medical microbiologist studies the characteristics of pathogens, their modes of
transmission, mechanisms of infection and growth. The academic qualification as a
clinical/Medical Microbiologist in a hospital or medical research centre generally
requires a Masters in Microbiology along with Ph.D. in any of the life-sciences
(Biochem, Micro, Biotech, Genetics, etc).[1] Medical microbiologists often serve as
consultants for physicians, providing identification of pathogens and suggesting
treatment options. Using this information, a treatment can be devised. Other tasks
may include the identification of potential health risks to the community or monitoring
the evolution of potentially virulent or resistant strains of microbes, educating the
community and assisting in the design of health practices. They may also assist in
preventing or controlling epidemics and outbreaks of disease. Not all medical
microbiologists study microbial pathology; some study common, non-pathogenic
species to determine whether their properties can be used to develop antibiotics or
other treatment methods.
Epidemiology, the study of the patterns, causes, and effects
of health and disease conditions in populations, is an important part of medical
microbiology, although the clinical aspect of the field primarily focuses on the
presence and growth of microbial infections in individuals, their effects on the human
body, and the methods of treating those infections. In this respect the entire field, as
an applied science, can be conceptually subdivided into academic and clinical sub-
specialties, although in reality there is a fluid continuum between public health
microbiology and clinical microbiology, just as the state of the art in clinical
laboratories depends on continual improvements in academic medicine and research
laboratories.

Clinical microbiology is a discipline that encompasses a broad range of testing

methodologies, and it is complex in terms of organisms and methods used to isolate
and identify them. Although significant improvements in testing methodologies have
been made, clinical microbiology remains heavily reliant on culture-based methods
and phenotypic methods for identification of culture organisms. The wide variety of
pathogens and testing methods that are available makes microbiological testing
challenging, and thus error detection and correction are important components of
quality microbiology laboratory testing. Errors may occur at all stages of testing (pre-
analytical, analytical, and post-analytical), and an error in one stage of testing is
likely to overlap with or lead to errors in other stages (e.g., incorrect specimen
collection can lead to culture, identification, and reporting of organisms that are not
involved in the disease process and to incorrect or unnecessary antimicrobial
therapy as a result). In the clinical microbiology laboratory, as in every other
discipline, the frequency of analytical errors has been reduced considerably with the
implementation of quality control and quality assurance programs. Despite the
improvements in microbiological testing, microorganisms remain a constant
challenge, and errors do occasionally occur. This chapter discusses some of the
common interferences in the clinical microbiology laboratory.

1.2 BASIC INSTRUMENT HANDLING

In medical and clinical microbiology we perform various kind of
experiments and for these experiments basic handling of different
instruments like Autoclave, pH meter, Laminar air flow, incubator,
microscope and colony counter is required.

1.2.1 AUTOCLAVE
An autoclave is a machine used to carry out industrial and scientific processes requiring
elevated temperature and pressure in relation to ambient pressure/temperature. Autoclaves are
used in medical applications to perform sterilization and in the chemical industry to cure coatings
and vulcanize rubber and for hydrothermal synthesis. Industrial autoclaves are used in industrial
applications, especially in the manufacturing of composites.
Many autoclaves are used to sterilize equipment and supplies by subjecting them to
pressurized saturated steam at 121 °C (250 °F) for around 15–20 minutes depending on the size
of the load and the contents.[1] The autoclave was invented by Charles Chamberland in 1879,
[2]
although a precursor known as the steam digester was created by Denis Papin in 1679.[3] The
name comes from Greek auto-, ultimately meaning self, and Latin clavis meaning key, thus a
self-locking device.[4]
Sterilization autoclaves are widely used
in microbiology and mycology, medicine and prosthetics fabrication, tattooing and body piercing,
and funerary practice. They vary in size and function depending on the media to be sterilized and
are sometimes called retort in the chemical and food industries.
Typical loads include laboratory glassware, other equipment and waste, surgical instruments,
and medical waste.[5][6]
A notable recent and increasingly popular application of autoclaves is the pre-disposal treatment
and sterilization of waste material, such as pathogenic hospital waste. Machines in this category
largely operate under the same principles as conventional autoclaves in that they are able to
neutralize potentially infectious agents by using pressurized steam and superheated water. A
new generation of waste converters is capable of achieving the same effect without a pressure
vessel to sterilize culture media, rubber material, gowns, dressings, gloves, etc. It is particularly
useful for materials which cannot withstand the higher temperature of a hot air oven. [7]
Autoclaves are also widely used to cure composites, especially for melding multiple layers
without any voids that would decrease material strength, and in the vulcanization of rubber. [8] The
high heat and pressure that autoclaves generate help to ensure that the best possible physical
properties are repeatable. Manufacturers of spars for sailboats have autoclaves well over 50 feet
(15 m) long and 10 feet (3 m) wide, and some autoclaves in the aerospace industry are large
enough to hold whole airplane fuselages made of layered composites.[9]
Other types of autoclaves are used to grow crystals under high temperatures and pressures.
Synthetic quartz crystals used in the electronics industry are grown in autoclaves. Packing of
parachutes for specialist applications may be performed under vacuum in an autoclave, which
allows the chutes to be warmed and inserted into their packs at the smallest volume.
A thermal Effluent Decontamination System functions as a single-purpose autoclaves designed
for the sterilization of liquid waste and effluent.

1.2.2 pH METER
A pH meter is a scientific instrument that measures the hydrogen-ion activity in water-based
solutions, indicating its acidity or alkalinity expressed as pH.[2] The pH meter measures the
difference in electrical potential between a pH electrode and a reference electrode, and so the
pH meter is sometimes referred to as a "potentiometric pH meter". The difference in electrical
potential relates to the acidity or pH of the solution. [3] The pH meter is used in many applications
ranging from laboratory experimentation to quality control.[4]

The rate and outcome of chemical reactions taking place in water often depends on the acidity of
the water, and it is therefore useful to know the acidity of the water, typically measured by means
of a pH meter.[5] Knowledge of pH is useful or critical in many situations, including chemical
laboratory analyses. pH meters are used for soil measurements in agriculture, water
quality for municipal water supplies, swimming pools, environmental remediation; brewing of
wine or beer; manufacturing, healthcare and clinical applications such as blood chemistry; and
many other applications.[4]
Advances in the instrumentation and in detection have expanded the number of applications in
which pH measurements can be conducted. The devices have been miniaturized, enabling direct
measurement of pH inside of living cells.[6] In addition to measuring the pH of liquids, specially
designed electrodes are available to measure the pH of semi-solid substances, such as foods.
These have tips suitable for piercing semi-solids, have electrode materials compatible with
ingredients in food, and are resistant to clogging.[7]

1.2.3 LAMINAR AIR FLOW

Laminar airflow is defined as air moving at the same speed and in the same direction,
with no or minimal cross-over of air streams (or “lamina”). By contrast, turbulent flow creates
swirls and eddies that deposit particles on surfaces randomly and unpredictably.
The principle of laminar flow cabinet is based on the laminar flow of air through the
cabinet. The device works by the use of inwards flow of air through one or more HEPA
filters to create a particulate-free environment.
Laminar air flow systems are used in various applications such as life science research,
mushroom cultivation, microbiology, IVF, IUI and histopathology / pathology lab, plant tissue
and cell culture and pharmaceutical and electronics industry and many more.
A laminar flow cabinet or tissue culture hood is a carefully enclosed bench designed to
prevent contamination of semiconductor wafers, biological samples, or any particle
sensitive materials. Air is drawn through a HEPA filter and blown in a very smooth, laminar
flow towards the user.

1.2.4 INCUBATOR
An incubator is a device used to grow and maintain microbiological cultures or cell cultures. The
incubator maintains optimal temperature, humidity and other conditions such as the
CO2 and oxygen content of the atmosphere inside. Incubators are essential for much
experimental work in cell biology, microbiology and molecular biology and are used to culture
both bacterial and eukaryotic cells.
The simplest incubators are insulated boxes with an adjustable heater, typically going up to 60 to
65 °C (140 to 150 °F), though some can go slightly higher (generally to no more than 100 °C).
The most commonly used temperature both for bacteria such as the frequently used E. coli as
well as for mammalian cells is approximately 37 °C (99 °F), as these organisms grow well under
such conditions. For other organisms used in biological experiments, such as the budding
yeast Saccharomyces cerevisiae, a growth temperature of 30 °C (86 °F) is optimal.
More elaborate incubators can also include the ability to lower the temperature (via refrigeration),
or the ability to control humidity or CO2 levels. This is important in the cultivation of mammalian
cells, where the relative humidity is typically >80% to prevent evaporation and a slightly
acidic pH is achieved by maintaining a CO2 level of 5%.

1.2.5 MICROSCOPE
A microscope (from Ancient Greek: μικρός mikrós 'small' and σκοπεῖν skopeîn 'to look (at);
examine, inspect') is a laboratory instrument used to examine objects that are too small to be
seen by the naked eye. Microscopy is the science of investigating small objects and structures
using a microscope. Microscopic means being invisible to the eye unless aided by a microscope.
There are many types of microscopes, and they may be grouped in different ways. One way is to
describe the method an instrument uses to interact with a sample and produce images, either by
sending a beam of light or electrons through a sample in its optical path, by detecting photon
emissions from a sample, or by scanning across and a short distance from the surface of a
sample using a probe. The most common microscope (and the first to be invented) is the optical
microscope, which uses lenses to refract visible light that passed through a thinly
sectioned sample to produce an observable image. Other major types of microscopes are
the fluorescence microscope, electron microscope (both the transmission electron
microscope and the scanning electron microscope) and various types of scanning probe
microscopes.[1]
 1.2.6 COLONY COUNTER
The colony counter is an instrument that is commonly used to count colonies of bacteria or
other microorganisms on a plate containing a gelled growth medium. A fully automated
system would essentially consist of collecting images using any one of the digital image
capturing devices such as document scanner/charge coupled device digital camera/webcam
or video equipment. Colonies can be counted from pictures of plates using software tools. In
automated systems, the objects and their backgrounds can be varied widely. Usually, one of
the three methods of illumination can be selected to enhance visibility and increase
accuracy. These are the following: transmission method, reflection method, and dark field
method. The colony counter is used for microbiology applications for fast and accurate
counting of bacterial and mould colonies.
 1.3 STERILIZATION TECHNIQUES
STERILIZATION : Sterilization refers to any process that removes, kills, or deactivates all
forms of life (in particular referring to microorganisms such
as fungi, bacteria, spores, unicellular eukaryotic organisms such as Plasmodium, etc.) and
other biological agents like prions present in a specific surface, object or fluid, for example food
or biological culture media.[1][2] Sterilization can be achieved through various means,
including heat, chemicals, irradiation, high pressure, and filtration. Sterilization is distinct
from disinfection, sanitization, and pasteurization, in that those methods reduce rather than
eliminate all forms of life and biological agents present. After sterilization, an object is referred to
as being sterile or aseptic.

 TYPES OF STERILIZATION TECHNIQUES :

 Ionizing Radiation.
 Dry-Heat Sterilizers.
 Liquid Chemicals.
 Performic Acid.
 Filtration.
 Microwave.
 Glass Bead “Sterilizer”
 Vaporized Hydrogen Peroxide
(VHP®)
  Ozone
 Formaldehyde Steam
 Gaseous Chlorine Dioxide
 Vaporized Peracetic Acid
 Infrared Radiation

BASIC PRINCIPLE : The basic principle of steam sterilization, as accomplished in an

autoclave, is to expose each item to direct steam contact at the required temperature
and pressure for the specified time. ... These temperatures (and other high
temperatures)830 must be maintained for a minimal time to kill microorganisms.

1.3 CLASSIFICATION OF BACTERIA ON THE

BASIS OF GRAM STAINING
The Gram stain characterizes bacteria based on the structural characteristics of their cell
walls. By combining morphology and Gram-staining, most bacteria can be classified as
belonging to one of 4 groups (Gram-positive cocci, Gram-positive bacilli, Gram-negative
cocci, and Gram-negative bacilli).

1.4 PREPARATION OF SELCETIVE MEDIA

EMB :
In 100 ml
Peptpone = 0.5g
Agar = 1.5g
NaCl = 0.5g
pH = 7.0
We need to prepare solution for 25 ml
1.5 STERILIZATION OF SELCETIVE MEDIA
Media sterilization is the destruction or removal of all forms of microbial life from the
aqueous feedstock. In industrial fermentations, components such as vessels, pipework,
media, inlet air, and exhaust gases are frequently sterilized by a combination of wet-heat
and filtration methods.

1.6 ISOLATION OF PATHOGENIC

BACTERIA
1. Introduction
 In recent years, outbreaks of human infections associated with the consumption of
fresh or minimally processed fruits and vegetables have increased, despite their
nutritional and health benefits [1,2]. Human disease outbreaks have been recognized
as being caused by contaminated vegetable and fruits consumption, several studies
have been published describing the bacterial contamination of intact vegetables and
fruits in open markets [3]. Raw vegetables host a variety of pathogenic
microorganisms that may be spread over the plants or occur in the plant tissues as
micro colonies [2]. Some diverse factors may be affected the variation of microbial
profile of vegetables including normal microflora of soil, animal manure derived flora,
irrigation or sewage water, transportation and unconscious handling by retailers
[Ref?]. Microbial contamination is commonly exposed to fruits and vegetables by
contact with dirt, dust and water and by handling at harvest or during post-harvest
processing [4,5]. Therefore, a wide range of microorganisms are present including
plant and human pathogens. Due to lack of surveillance and inadequate screening of
these raw vegetables in Chittagong region, most outbreaks have become undetected
and there is very little information available in the literature so far [6].
In another study, it was found that bacterial contamination of fruits and salad
vegetables (tomato, cucumber etc.) was correlated with the fact that it is typically
eaten without thermal treatment. A major contributing factor to contamination is use
of untreated wastewater and manure as fertilizers for the production of fruits and
vegetables [7]. In Bangladesh various fruits and vegetables are sold in open markets
and most of them are eaten raw, which is suitable medium for bacterial contamination
[8]. In developing countries like Bangladesh, both poverty and poor sanitation is
common, faecal contamination of domestic and commercial food is likely to occur, and
illness has been traced to the ingestion of faecally infected food in multiple outbreaks
[9]. The ingestion of infected fresh vegetables and fruits has been related to many
outbreaks of human gastro-enteritis [10].
The use of untreated waste water and manure as fertilizers for the cultivation of fruit
and vegetables is a significant contributor to contamination [11,12].
Fresh fruits and vegetables can harbor large and diverse populations of bacteria.
However, most of the research on fresh fruits and vegetables has focused on their
isolation and, as a result, we know far less about the overall diversity and molecular
characteristics of those bacterial communities [13]. We addressed these knowledge
gaps by isolating bacteria from different fruits and vegetables and assessing their
molecular characteristics through 16 S rRNA sequencing approach.
The present study was undertaken to isolate and identify pathogenic bacteria from fresh
fruits and vegetables that are very popular in Chittagong region, Bangladesh.
2. Materials and Methods
Sample collection
A total of 7 samples of fresh fruits and vegetables were collected from two open
markets (Baluchara, Masjid Market and Oxygen market) of Chittagong city. The
samples were Cucumber (Cucumis sativus), Carrot (Daucus carota), Tomato (Solanum
lycopersicum), Hog plum (Spondias mombin), Guava (Psidium guajava), Apple (Malus
domestica) and Jujube (Ziziphus). All samples were collected in a sterile polythene
bag in an insulated ice box to maintain temperatures ranging from 4° to 6°C and
analyzed within one hour of receipt.
Preparation of samples
Within an hour of collection, the samples were taken into the laboratory and rinsed for
each with 100 ml of distilled water, then diluted 10 fold serial. After washing the
vegetables surface 10 ml of washed aqueous suspension of each sample was mixed
with 90 ml Luria Bertani (LB) broth and incubated for 24 h at 37°C. This overnight
culture in LB broth was used for the isolation and identification of strains on selective
media in streak plate technique [14,15].
Isolation and identification of bacteria
The bacteria were isolated and enumerated by growing them on selective and non-
selective mediums such as nutrient agar was used for total viable bacterial count
 (TVBC) and for total coliform (TC) and fecal coliform (FC) count MacConkey broth was
used. One loop full culture from LB broth was streaked over selective media and kept
it for incubation overnight at 37°C.
Eosin methylene blue (EMB) for E. coli and thiosulfate citrate bile sucrose agar (TCBS
Agar) for Vibrios Spp., Vibrio cholera like organism (VCLO), xylose lysine deoxycholate
agar (XLD agar) and salmonella-shigella sgar (S-S Agar) for Salmonella, tomato juice
agar for Lactobacillus spp. Cetrimide agar for Pseudomonas spp. and mannitol salt
phenol-red agar was used for Staphylococcus spp.
For identification confirmation IMViC tests were done for every species [16].
Bacterial enumeration
Spread plate method was used for bacterial enumeration to determine the number of
colony forming units (CFUs) [17].
Statistical Analysis
For statistical analysis, the SPSS program (V12) was used and the T test was performed
to determine the significance of the difference between groups. At 95 percent conviction
(p<0.05), the significance between the values was assessed [18].
3. Results
Total Viable Bacterial Count
In this study, all sampled fruits and vegetables were contaminated. The microbial load
of fruit and vegetables varied with the types. Range of microbial count of guava was
2.1×104 CFU/ml, apple was 4.3×104 CFU/ml, tomato was 8.2×104 CFU/ml, cucumber
was 5.0×104 CFU/ml, carrot was 1.3×104 CFU/ml, hog Ppum (Amra) was
7.5×104 CFU/ml and jujube (Boroi) was 1.2×104.
Isolation and identification
A total of 4 bacteria were isolated from each of the samples and they were identified
on the basis of the cultural, morphological and biochemical characteristics (Table: 1).

Table 1. Biochemical results

Yellow colonies from TCBS plates were Vibrio cholera, black centers on XLD
were Salmonella spp.., opaque colonies from tomato juice agar plates
were Lactobacillus spp., blue-green colonies from cetrimide agar plate
was Pseudomonas spp. (Fig: 1).

Figure 1. A) Vibrio spp. B) Salmonella Spp. C) Lactobacillus

spp. D) Pseudomonas spp

Among the samples-

Pseudomonas spp. (50%), Vibrio spp. (20%), Lactobacillus spp. (15%), (0.37%)
and Salmonella spp. (0.37%).
Highly presence of these bacteria is serious concern for the people of this region.
Necessary step should be taken as early as possible to eliminate the presence of these
bacteria for avoiding any future chance of outbreak related to food borne pathogen.
4. Discussion
 During cultivation in fields or orchards or during harvesting, post-harvest handling,
refining and distribution, vegetables and fruits become infected with pathogenic
microorganisms. [19].
The presence of all four types of bacteria in the fruits and vegetable samples is
probably a reflection of the nature of the two retail outlets in the study [14]. This
result highlights the fact that fresh vegetables could be contaminated with pathogenic
bacteria and thus could possibly act as a transmission vehicle of many diseases. The
prevalence of coliform in guava was 2.1×104 CFU/ml, apple was 4.3×104 CFU/ml,
tomato was 8.2×104 CFU/ml, cucumber was 5.0×104 CFU/ml, carrot was
1.3×104 CFU/ml, hog plum (Amra) was 7.5×104 CFU/ml and jujube (Boroi) was
1.2×104 which clearly indicated that the unhygienic condition of open markets.
The Pseudomonas spp. was present in almost all the samples where presence
of Salmonella spp. was less in among the samples. The detection of Vibrio cholera in
particularly 20% of the samples is a matter of concern.
Identification of Vibrio spp., Lactobacillus spp.,
Pseudomonas spp. and Salmonella spp. is a clear sign of serious health crisis alert for
of the people living in this area.
This was clear that samples from wet markets yielded a higher proportion of bacteria
because it could be seen that the way vegetables were treated on the wet markets was
less hygienic. The surroundings and places for vegetable displays on markets were not
clean and tidy, and the handlers did not wear gloves while the vegetables were being
handled. Contamination might be occurred through a contaminated container for
transporting and improper handling [20]. Apart from that, the vegetables at
supermarkets sometimes have a long holding time, which could contribute to the
accumulation of pathogenic bacteria [21].
5. Conclusions
This study demonstrated the alarming presence of pathogenic bacteria among the most
common and popular fresh fruits and vegetable in this area. The result also indicated
that the current hygiene condition of selling and buying zone of fresh fruits and
vegetable in Chittagong. This study provided a general overview of the microbiological
quality of fresh fruits and vegetable in Chittagong which will help to take necessary step
to make sure the hygiene condition during fruits and vegetables selling and buying.

1.7 MINIMUM INHIBITORY

CONCENTRATION [MIC]
In microbiology, the minimum inhibitory concentration (MIC) is the lowest concentration of a
chemical, usually a drug, which prevents visible growth of a bacterium or bacteria. MIC depends
on the microorganism, the affected human being (in vivo only), and the antibiotic itself. [1] It is often
expressed in micrograms per milliliter (μg/mL) or milligrams per liter (mg/L).
The MIC is determined by preparing solutions of the chemical in vitro at increasing
concentrations, incubating the solutions with separate batches of cultured bacteria, and
measuring the results using agar dilution or broth microdilution. Results have been graded into
susceptible (often called sensitive), increased exposure, or resistant to a particular antimicrobial
by using a breakpoint. Breakpoints are agreed upon values, published in guidelines of a
reference body, such as the U.S. Clinical and Laboratory Standards Institute (CLSI), the British
Society for Antimicrobial Chemotherapy (BSAC) or the European Committee on Antimicrobial
Susceptibility Testing (EUCAST).[2] There have been major discrepancies between the
breakpoints from various European countries over the years, and between those from the
European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the US Clinical and
Laboratory Standards Institute (CLSI).
While MIC is the lowest concentration of an antibacterial agent necessary to inhibit visible
growth, minimum bactericidal concentration (MBC) is the minimum concentration of an
antibacterial agent that results in bacterial death. The closer the MIC is to the MBC, the more
bactericidal the compound.[4]
The first step in drug discovery is often the screening of a library drug candidate for MICs against
bacteria of interest.[5] As such, MICs are usually the starting point for larger pre-clinical
evaluations of novel antimicrobial agents.[6] The purpose of measuring the minimum inhibitory
concentration is to ensure that antibiotics are chosen efficiently to increase the success of
treatment.

History
After the discovery and commercialization of antibiotics, microbiologist, pharmacologist, and
physician Alexander Fleming developed the broth dilution technique using the turbidity of the
broth for assessment.[7] This is commonly believed to be the conception point of minimum
inhibitory concentrations.[8] Later in the 1980s, Clinical and Laboratory Standards Institute has
consolidated the methods and standards for MIC determination and clinical usage. Following the
discovery of new antibacterials, pathogens and their evolution, the protocols by CLSI are also
continually updated to reflect that change.[9] The protocols and parameters set by CLSI are
considered to be the "gold standard" in the United States and are used by regulatory authorities,
such as the FDA, to make evaluations.[10]

Clinical usage
Nowadays, the MIC is used in antimicrobial susceptibility testing. The MIC is reported by
providing the susceptibility interpretation next to each antibiotic. The different susceptibility
interpretations are: S (Sensitive), I (Intermediate), and R (Resistant). These interpretations were
created and implemented by the Clinical and Laboratory Standards Institute (CLSI). In clinics,
more often than not, exact pathogens cannot be easily determined by symptoms of the patient.
Then, even if the pathogen is determined, different serotypes of pathogens, such
as Staphylococcus aureus, have varying levels of resistance to antimicrobials. As such, it is
difficult to prescribe correct antimicrobials.[11] The MIC is determined in such cases by growing the
pathogen isolate from the patient on plate or broth, which is later used in the assay. [12] Thus,
knowledge of the MIC will provide a physician valuable information for making a prescription.
Accurate and precise usage of antimicrobials is also important in the context of multidrug-
resistant bacteria. Microbes such as bacteria have been gaining resistance to antimicrobials they
were previously susceptible to.[13] Usage of incompatible levels of antimicrobials provides the
selective pressure that has driven the direction and evolution of resistance of bacterial
pathogens.[14] This has been seen at sub-MIC levels of antibiotics.[15] As such, it is increasingly
important to determine the MIC in order to make the best choice in prescribing antimicrobials.
MIC is used clinically over MBC because MIC is more easily determined.[9] Minimum bactericidal
concentration (MBC), which is the minimum antibacterial concentration resulting in microbial
death, is defined by the inability to re-culture bacteria. In addition, drug effectiveness is generally
similar when taken at both MIC and MBC concentrations because the host immune system can
expel the pathogen when bacterial proliferation is at a standstill.[16] When the MBC is much higher
than the MIC, drug toxicity makes taking the MBC of the drug detrimental to patient. Antimicrobial
toxicity can come in many forms, such as immune hypersensitivity and off-target toxicity. [17]

Methods
Broth dilution assay
Broth Dilution Assay. The MIC is determined by evaluation of turbidity of tubes with constantly increasing
concentration of antimicrobial agent.

There are three main reagents necessary to run this assay: the media, an antimicrobial agent,
and the microbe being tested. The most commonly used media is cation-adjusted Mueller Hinton
Broth, due to its ability to support the growth of most pathogens and its lack of inhibitors towards
common antibiotics.[18] Depending on the pathogen and antibiotics being tested, the media can be
changed and/or adjusted. The antimicrobial concentration is adjusted into the correct
concentration by mixing stock antimicrobial with media. The adjusted antimicrobial is serially
diluted into multiple tubes (or wells) to obtain a gradient. The dilution rate can be adjusted
depending on the breakpoint and the practitioner's needs. The microbe, or the inoculating agent,
must come from the same colony-forming unit, and must be at the correct concentration. This
may be adjusted by incubation time and dilution. For verification, the positive control is plated in a
hundred fold dilution to count colony forming units. The microbes inoculate the tubes (or plate)
and are incubated for 16–20 hours. The MIC is generally determined by turbidity. [18]

1.8 MINIMUM BACTERIAL

CONCENTRATION [MBC]
The minimum bactericidal concentration (MBC) is the lowest concentration of an antibacterial
agent required to kill a particular bacterium.[1] It can be determined from broth dilution minimum
inhibitory concentration (MIC) tests by subculturing to agar plates that do not contain the test
agent. The MBC is identified by determining the lowest concentration of antibacterial agent that
reduces the viability of the initial bacterial inoculum by ≥99.9%. The MBC is complementary to
the MIC; whereas the MIC test demonstrates the lowest level of antimicrobial agent that inhibits
growth, the MBC demonstrates the lowest level of antimicrobial agent that results in microbial
death. This means that even if a particular MIC shows inhibition, plating the bacteria onto agar
might still result in organism proliferation because the antimicrobial did not cause death.
Antibacterial agents are usually regarded as bactericidal if the MBC is no more than four times
the MIC.[2] Because the MBC test uses colony-forming units as a proxy measure of bacterial
viability, it can be confounded by antibacterial agents which cause aggregation of bacterial cells.
Examples of antibacterial agents which do this include flavonoids[3] and peptides.[4]
 2. COMPUTATIO
NAL &
STRUCTURAL
BIOLOGY
[BIOINFORMA
TICS]
Introduction to the field of bioinformatics, historical overview and
conceptual understanding about molecular biology, Information
Search and Data retrieval: electronic libraries, tools for web search
and data mining, Bioinformatics applications, bioinformatics tools,
server, pipelines and resources, Introduction of databases: major
databases in bioinformatics, data management and analysis, Tools
for similarity search and alignments: FASTA, BLAST and other
program, DNA/RNA/Protein Sequences: alignments and analysis,
Introduction to Phylogenic Studies and Clustal W. Conceptual
understanding of molecular biology and Structural Biology, Sources
of biological data: major databases in bioinformatics, data files and
analysis. Sequence data, searching and alignment, genome
sequencing, genome analysis, genetic variation, gene (Genebank,
EMBL, Unigene, Ensemble etc). Computational methods for
pathways and system biology (KEGG, BRITE, Genepath and
GeNET), Structural Studies (DNA, RNA and Protein): MFold,
RNAfold, IFold, PDB. Concept of homology modeling and molecular
docking (Zdock, Hexdoc and Autodock).

BIOINFORMATICS

The term “Bioinformatics” was first coined by Paulien Hogeweg in 1970.

Bioinformatics is defined as the field of science that involves the application of
various computational tools for the analysis and interpretation of biological
data. It is an interdisciplinary field, which make use of computer science,
mathematics, physics, and biology.
Bioinformatics focuses on three main areas i.e., creation of databases,
updating the databases, and development of new tools and software to
analyze the data.
As a large amount of data has been generated due to various sequencing
projects in human and other organisms, the demand for the analysis and
interpretation of these data also increased. So, bioinformatics is essential for
the management of data in biology and medicine.
Bioinformatics play a major role in biotechnology as it speeds up the research
involving sequence data and drug design. Bioinformatics helps in sequence
analysis and alignment, molecular modeling, docking, annotation and dynamic
simulation, thereby, accelerating the biotechnological research.
Bioinformatics includes computational bioinformatics, post – genome
applications, bioinformatics and medicines, biodiversity, gene expression, gene
network, sequence analysis, structure network, and molecular evolution.

Molecular and Structure biology

The study of biological micro/macromolecules' architecture, shape, and

dynamics is important in understanding the fundamental mechanisms that
drive the vital functions of all life. The driving forces and interactions
which characterize the three dimensional forms and dynamics of
biomollecules are dealt with in structural biology. Use of different
structure databases for modeling of 3-D structures of target and ligand
molecules and visualization.

Computer Aided Drug Designing

Identification of potential drug targets for lead molecules/peptides to know

their mode of action through ligand-protein and/or protein-protein
interaction (molecular modeling, docking and saimulation studies).

Next Generation Sequencing

Introduction of Next Generation Sequencing, Read/contig assembly

methods, popular Next Generation Technologies, Tools for NGS data
analysis, Application of NGS in agriculture and life sciences
 3. NANO-
BIOTEC
HNOLOG
Y
Introduction about Nano-Biotechnology and its application, General
and safety rules for working in Lab, Demonstration of instruments,
Good laboratory practices (GLP), Basic concept of calculation
(Percentage, Molarity and Normality), Micropipette handling
(forward and reverse pipetting techniques), SOP of magnetic stirrer
with hot plate, Different methods for the preparation of
nanoparticles, Synthesis of Ag-nanoparticles/Cu-nanoparticles/Mg-
nanoparticles through chemical reduction methods, Screening of
Medicinal plants for green synthesis of nanoparticles, Green
synthesis of Ag- nanoparticles/Cu- nanoparticles/ Mg- nanoparticles
through different medicinal plants, Principle, SOP and application of
spectrophotometer, characterization of different nanoparticles
through UV-Vis spectroscopy scanning method, Different method
for the detection of antimicrobial activity, Biological screening of
different Nanoparticles (Antimicrobial Susceptibility Testing
(AST)/Antioxidant activity).

3.1 INTRODUCTION
Nanobiotechnology, bionanotechnology, and nanobiology are terms that refer to the
intersection of nanotechnology and biology.[1] Given that the subject is one that has only emerged
very recently, bionanotechnology and nanobiotechnology serve as blanket terms for various
related technologies.
This discipline helps to indicate the merger of biological research with various fields of
nanotechnology. Concepts that are enhanced through nanobiology include: nanodevices (such
as biological machines), nanoparticles, and nanoscale phenomena that occurs within the
discipline of nanotechnology. This technical approach to biology allows scientists to imagine and
create systems that can be used for biological research. Biologically inspired nanotechnology
uses biological systems as the inspirations for technologies not yet created.[2] However, as with
nanotechnology and biotechnology, bionanotechnology does have many potential ethical
issues associated with it.
The most important objectives that are frequently found in nanobiology involve applying
nanotools to relevant medical/biological problems and refining these applications. Developing
new tools, such as peptoid nanosheets, for medical and biological purposes is another primary
objective in nanotechnology. New nanotools are often made by refining the applications of the
nanotools that are already being used. The imaging of native biomolecules, biological
membranes, and tissues is also a major topic for nanobiology researchers. Other topics
concerning nanobiology include the use of cantilever array sensors and the application
of nanophotonics for manipulating molecular processes in living cells.[3]
Recently, the use of microorganisms to synthesize functional nanoparticles has been of great
interest. Microorganisms can change the oxidation state of metals.[citation needed] These microbial
processes have opened up new opportunities for us to explore novel applications, for example,
the biosynthesis of metal nanomaterials. In contrast to chemical and physical methods, microbial
processes for synthesizing nanomaterials can be achieved in aqueous phase under gentle and
environmentally benign conditions. This approach has become an attractive focus in current
green bionanotechnology research towards sustainable development.[4]
Nanotechnology is a novel scientific approach that involves materials and equipments capable of
manipulating physical as well as chemical properties of a substance at molecular levels. On the other
hand, biotechnology uses the knowledge and techniques of biology to manipulate molecular, genetic
and cellular processes to develop products and services and is used in diverse fields from medicine to
agriculture. Nanobiotechnology is considered to be the unique fusion of biotechnology and
nanotechnology by which classical micro-technology can be merged to a molecular biological
approach in real. Through this methodology, atomic or molecular grade machines can be made by
mimicking or incorporating biological systems, or by building tiny tools to study or modulate diverse
properties of a biological system on molecular basis. Nanobiotechnology may, therefore, ease many
avenues of life sciences by integrating cutting-edge applications of information technology &
nanotechnology into contemporary biological issues. This technology has potential to remove obvious
boundaries between biology, physics and chemistry to some extent, and shape up our current ideas
and understanding. For this reason, many new challenges and directions may also arise in education,
research & diagnostics in parallel by the extensive use of nanobiotechnology with the passage of time.

Nanobiotechnology at a glance

Biotechnology and nanotechnology are two of the 21st century’s most promising technologies.
Nanotechnology (sometimes referred to as nanotech) is defined as the design, development and
application of materials & devices whose least functional make up is on a nanometer scale .
Generally, nanotechnology deals with developing materials, devices, or other structures possessing at
least one dimension sized from 1 to 100 nanometers. Meanwhile, Biotechnology deals with metabolic
and other physiological processes of biological subjects including microorganisms. Association of
these two technologies, i.e. nanobiotechnology can play a vital role in developing and implementing
many useful tools in the study of life.

Nanotechnology is very diverse, ranging from extensions of conventional device physics to

completely new approaches based upon molecular self-assembly, from developing new materials with
dimensions on the nanoscale to investigating whether we can directly control matters on/in the atomic
scale/level. This idea entails the application of fields of science as diverse as surface science, organic
chemistry, molecular biology, semiconductor physics, microfabrication, etc.

Advantages of nanobiotechnology

The pathophysiological conditions and anatomical changes of diseased or inflamed tissues can
potentially trigger a great deal of scopes for the development of various targeted nanotechnological
products. This development is like to be advantageous in the following ways:

1. Drug targeting can be achieved by taking advantage of the distinct pathophysiological features of
diseased tissues

2. Various nanoproducts can be accumulated at higher concentrations than normal drugs

3. increased vascular permeability coupled with an impaired lymphatic drainage in tumors improve
the effect of the nanosystems in the tumors or inflamed tissues through better transmission and
retention .

4. Nanosystems have capacity of selective localization in inflammed tissues .

5. Nanoparticles can be effectively used to deliver/transport relevant drugs to the brain overcoming
the presence of blood–brain barrier (meninges) .

6. Drug loading onto nanoparticles modifies cell and tissue distribution and leads to a more selective
delivery of biologically active compounds to enhance drug efficacy and reduces drug toxicity .

Applications of nanobiotechnology in medical and clinical fields

A number of clinical applications of nanobiotechnology, such as disease diagnosis, target-specific

drug delivery, and molecular imaging are being laboriously investigated at present. Some new
promising products are also undergoing clinical trials . Such advanced applications of this approach to
biological systems will undoubtedly transform the foundations of diagnosis, treatment, and prevention
of disease in future. Some of these applications are discussed below.

(a)
Diagnostic applications: Current diagnostic methods for most diseases depend on the
manifestation of visible symptoms before medical professionals can recognize that the patient
suffers from a specific illness. But by the time those symptoms have appeared, treatment may
have a decreased chance of being effective. Therefore the earlier a disease can be detected,
the better the chance for a cure is. Optimally, diseases should be diagnosed and cured before
symptoms even manifest themselves. Nucleic acid diagnostics will play a crucial role in that
process, as they allow the detection of pathogens and diseases/diseased cells at such an early
symptomless stage of disease progression that effective treatment is more feasible. Current
technology, such as- polymerase chain reaction (PCR) leads toward such tests and devices,
but nanotechnology is expanding the options currently available, which will result in greater
sensitivity and far better efficiency and economy.

1. Detection:
Many currently used/conventional clinical tests reveal the presence of a molecule or a disease
causing organism by detecting the binding of a specific antibody to the disease-related target.
 Traditionally, such tests are performed by conjugating the antibodies with inorganic/organic
dyes and visualizing the signals within the samples through fluorescence microscopy or
electronic microscopy. However, dyes often limit the specificity and practicality of the
detection methods. Nanobiotechnology offers a solution by using semiconductor nanocrystals
(also referred to as "quantum dots"). These minuscule probes can withstand significantly
more cycles of excitations and light emissions than typical organic molecules, which more
readily decompose [14].

2. Individual target probes

Despite the advantages of magnetic detections, optical and colorimetric detections will
continue to be chosen by the medical community. Nanosphere (Northbrook, Illinois) is one of
the companies that developed techniques that allow/allowing doctors to optically detect the
genetic compositions of biological specimens. Nano gold particles studded with short
segments of DNA form the basis of the easy-to-read test for the presence of any given genetic
sequence. If the sequence of interest in the samples, it binds to complementary DNA tentacles
on multiple nanospheres and forms a dense web of visible gold balls. This technology
allows/facilitates the detection of pathogenic organisms and has shown promising results in
the detection of anthrax, giving much higher sensitivity than tests that are currently being
used [15].

3. Protein chips
Proteins play the central role in establishing the biological phenotype of organisms in healthy
and diseased states and are more indicative of functionality. Hence, proteomics is important in
disease diagnostics and pharmaceutics, where drugs can be developed to alter signaling
pathways. Protein chips can be treated with chemical groups, or small modular protein
components, that can specifically bind to proteins containing a certain structural or
biochemical motif [16]. Two companies currently operating in this application space are
Agilent, Inc. and NanoInk, Inc. Agilent uses a non-contact ink-jet technology to produce
microarrays by printing oligos and whole cDNAs onto glass slides at the nanoscale. NanoInk
uses dip-pen nanolithography (DPN) technology to assemble structure on a nanoscale of
measurement [17].

4. Sparse cell detection

Sparse cells are both rare and physiologically distinct from their surrounding cells in normal
physiological conditions (e.g. cancer cells, lymphocytes, fetal cells and HIV-infected T cells).
They are significant in the detection and diagnosis of various genetic defects. However, it is a
challenge to identify and subsequently isolate these sparse cells. Nanobiotechnology presents
new opportunities for advancement in this area. Scientists developed nanosystems capable of
effectively sorting sparse cells from blood and other tissues. This technology takes advantage
of/exploits the unique properties of sparse cells manifested in differences in deformation,
surface charges and affinity for specific receptors and/or ligands. For example, by inserting
electrodes into microchannels, cells can be precisely sorted based on surface charge. They can
also be sorted by using biocompatible surfaces with precise nanopores. The nano-
biotechnology center at Cornell University (NBTC) is currently using these technologies to
develop powerful diagnostic tools for the isolation and diagnosis of various diseases [18].

5. Nanotechnology as a tool in imaging

Intracellular imaging can be made possible through labelling of target molecules with
quantum dots (QDs) or synthetic chomophores, such as fluorescent proteins that will facilitate
direct investigation of intracellular signalling complex by optical techniques, i. e. confocal
fluorescence microscopy or correlation imaging [19, 20].
(b)
Therapeutic applications:Nanotechnology can provide new formulations of drugs with less
side effects and routes for drug delivery.

1. Drug Delivery:
Nanoparticles as therapeutics can be delivered to targeted sites, including locations that
cannot be easily reached by standard drugs. For instance, if a therapeutic can be chemically
attached to a nanoparticle, it can then be guided to the site of the disease or infection by radio
or magnetic signals. These nanodrugs can also be designed to "release" only at times when
specific molecules are present or when external triggers (such as infrared heat) are provided.
At the same time, harmful side effects from potent medications can be avoided by reducing
the effective dosage needed to treat the patient. By encapsulating drugs in nanosized materials
(such as organic dendrimers, hollow polymer capsules, and nanoshells), release can be
controlled much more precisely than ever before. Drugs are designed to carry a therapeutic
payload (radiation, chemotherapy or gene therapy) as well as for imaging applications [21].
Many agents, which cannot be administered orally due to their poor bioavailability, will now
have scope of use in therapy with the help of nanotechnology [22, 23]. Nano-formulations
offer protection for agents vulnerable to degradation or denaturation when exposed to extreme
pH, and also prolong half-life of a drug by expanding retention of the formulation through
bioadhesion [24, 25]. Another broad application of nanotechnology is the delivery of antigens
for vaccination [26, 27]. Recent advances in encapsulation and development of suitable
animal models have demonstrated that microparticles and nanoparticles are capable of
enhancing immunization [28].

2. Gene delivery
Current gene therapy systems suffer from the inherent difficulties of effective pharmaceutical
processing and development, and the chance of reversion of an engineered mutant to the wild
type. Potential immunogenicity of viral vectors involved in gene delivery is also problematic
[29, 30]. To address this issue, nanotechnological tools in human gene therapy have been
tested and nanoparticle-based nonviral vectors (usully 50-500 nm in size) in transportation of
plasmid DNA described. Therefore, successful introduction of less immunogenic nanosize
gene carriers as a substitution of the disputed viral vectors seems beneficial in repairing or
replacing impaired genes in human [31].

3. Liposomes
A liposome being composed of a lipid bilayer can be used in gene therapy due to its ability to
pass through lipid bilayers and cell membranes of the target. Recent use of several groups of
liposomes in a local delivery has been found to be convincingly effective [32, 33]. Liposomes
can also help achieve targeted therapy. Zhang et al demonstrated widespread reporter
expression in the brains of rhesus monkeys by linking nanoparticle (such as polyethylene
glycol) treated liposomes to a monoclonal antibody for human insulin reporter [34]. These
successful trials reflect the future of targeted therapy and the importance of nanometer-sized
constructs for the advancement of molecular medicine.

4. Surfaces
In nature, there are a multitude of examples of the complicated interactions between
molecules and surfaces. For example, the interactions between blood cells and the brain or
between fungal pathogens and infection sites rely on complex interplays between cells and
surface characteristics. Nanofabrication unravels the complexity of these interactions by
modifying surface characteristics with nanoscale resolutions, which can lead to hybrid
biological systems. This hybrid material can be used to screen drugs, as sensors, or as medical
 devices and implants. Nanosystems, owned by the Irish drug company Elan, developed a
polymer coating capable of changing the surface of drugs that have poor water solubility [35].

1. Biomolecular Engineering
The expense and time involved in traditional biomolecule designing limit the availability of
bioactive molecules. Nanoscale assembly and synthesis techniques provide an alternative to
traditional methods. Improvements can be achieved due to the ability to carry out chemical
and biological reactions on solid substrates, rather than through the traditional solution based
processes. The use of solid substrate usually means less waste and the ability to manipulate
the biomolecule far more precisely. EngeneOS (Waltham, Massachusetts) pioneered the field
of biomolecular engineering. The company developed the engineered genomic operating
systems that create programmable biomolecular machines employing natural and artificial
building blocks. These biomolecule machines have broad range of commercial applications-as
biosensors, in chemical synthesis and processing, as bioelectronic devices and materials, in
nanotechnology, in functional genomics and in drug discovery.

2. Biopharmaceuticals
Nanobiotechnology can develop drugs for diseases that conventional pharmaceuticals cannot
target. The pharmaceutical industry traditionally focuses on developing drugs to treat a
defined universe of about five hundred confirmed disease targets. But approximately 70 to 80
percent of the new candidates for drug development fail, and these failures are often
discovered late in the development process, with the loss of millions of dollars in R&D
investments. Nanoscale techniques for drug development will be a boon to small companies,
which cannot employ hundreds of organic chemists to synthesize and test thousands of
compounds. Nanobiotechnology brings the ability to physically manipulate targets, molecules
and atoms on solid substrates by tethering them to biomembranes and controlling where and
when chemical reactions take place, in a fast process that requires few materials (reagents and
solutions). This advance will reduce drug discovery costs, will provide a large diversity of
compounds, and will facilitate the development of highly specific drugs. Potentia
Pharmaceuticals (Louisville, Kentucky) is an early-stage company that is attempting to
streamline the drug development process with the use of nanotechnologies (Harvard Business
School 2001).

3. Nanotechnology in cardiac therapy

Nanotechnology is currently offering promising tools for applications in modern
cardiovascular science to explore existing frontiers at the cellular level and treat challenging
cardiovascular diseases more effectively. These tools can be applied in diagnosis, imaging
and tissue engineering [36]. Miniaturized nanoscale sensors like quantum dots (QDs),
nanocrystals, and nanobarcodes are capable of sensing and monitoring complex immune
signals in response to cardiac or inflammatory events [20]. Nanotechnology can also help
detect and describe clinically-significant specific mechanisms implicated in cardiac disorders.
In addition, it is useful in designing atomic-scale machines that can be incorporated into
biological systems at the molecular level. Introduction of these newly designed nanomachines
may positively change many ideas and hypotheses in the treatment of critical cardiovascular
diseases. Nanotechnology could also have great impact in tackling issues like unstable
plaques and clarification of valves. Thus, this approach could be a real milestone of success in
achieving localized and sustained arterial and cardiac drug therapy for the management of
cardiovascular diseases [37].

4. Nanotechnology in dental care

Nanotechnology will have future medical applications in the field of dentistry. The role of
nanodentistry by means of the use of nanomaterials [38, 39], biotechnology [40, 41], and
 nanorobotics will ensure better oral health. Millions of people currently receiving poor dental
care will benefit from such remarkable breakthrough in the science of dental health [42, 43].
Moreover, nanodental techniques in major tooth repair may also evolve. Reconstructive
dental nanorobots could be used in selective and precise occlusion of specific tubules within
minutes, and this will facilitate quick and permanent recovery. The advantage of
nanodentistry in natural tooth maintenance could also be significant [44]. Covalently-bonded
artificial materials like sapphire may replace upper enamel layer to boost the appearance and
durability of teeth [43].

9. Nanotechnology in orthopedic applications

Nanomaterials sized between 1 and 100 nm have role to play as new and functional
constituents of bones being also made up of nanosized organic and mineral phases [45, 46].
Nanomaterials, nanopolymers, carbon nanofibers, nanotubes, and ceramic nanocomposites
may help with more efficient deposition of calcium-containing minerals on implants. Based
on these evidences and observations, nanostructure materials represent a unique realm of
research and development that may improve the attachment of an implant to the surrounding
bone matters by enhancing bone cell interactions, and this will indeed aid in improving
orthopedic implant efficacy while drastically minimizing patient-compliance problems.

3.2 CONCEPT OF BASIC CALCULATIONS

While performing various tests in laboratories one must know about the basic
calculations as they are used for the preparation of various reagents, samples
and other solutions.
1. Molarity: - It is defined as the number of moles of solutes present in one
liter of a solution. It is known as the molar concentration of a solution.
Molarity (M) = number of moles of solutex1000 / volume in ml
Or
Molarity (M) = weight of solutex1000 / molecular weight x volume in ml
2. Normality: - It is defined as the number of mole equivalents per liter of
solution.
Normality (N)=number of mole equivalent/1L of solution.
Mole equivalent =moles of the reagent / moles of the limiting reagent.
Normality = Molarity x Molar mass / equivalent mass
For acidic solution,
Normality =Molarity x Basicity
For basic solution,
Normality=Molarity x Acidity
N1 V1 = N2 V2
Where, N1 = Normality of the acidic solution
V1 = Volume of the acidic solution
N2 = Normality of the basic solution
 V2 = Volume of the basic solution
3. For volume and concentration
C1 V1 = C2 V2
C1 = Initial concentration of solution
V2 = Initial volume of solution
C2 = Final concentration of solution
V2 = Final volume of solution
4. Percentage

3.3 BASIC INSTRUMENT HANDLING

3.3.1 MICROPIPETTE (Gilson Micropipette)

Micropipette is a laboratory instrument used to measure and to transfer small

volume of solutions in the range of µl (5-50µl, 50-100µl and 100-1000µl). They
are most commonly used in biological and biochemistry labs. They release the
solution by volumetric displacement of air by the vertical movement of an
internal piston.
For the usage we need a pipette, tip box, vessel (receiving and transferring
vessel) and a discarder. Now set the pipette volume by turning the plunger
clockwise and anticlockwise to reach a desired volume. Set the tip and ensure
that the tip is firmly attached on the pipette. Press the plunger down until the
first stop, then depress the plunger before you put your pipette into the
solution otherwise bubbles will form. Now carefully place the pipette tip into
the desired solution, slowly release the plunger and the set amount of solution
will be pulled up into the tip. After this remove the tip of the pipette. Place the
tip into the receiving vessel and slowly push the plunger down all the way to
the first stop, then the second stop to make sure all solution has been pushed
out of the tip. Remove the pipette from the vessel and discard the tip by
pressing down the tip ejector button.
There are two pipette techniques, one is forward pipetting and the other is
reverse pipetting. Forward pipetting is used for non-viscous or low-viscous
liquids. For forward pipetting press the plunger down until the first stop only to
aspirate the liquid and also to dispense the liquid in the receiving vessel.
Reverse pipetting is used for highly viscous liquids and also for dispensing very
small volumes. For reverse pipetting press the plunger down until the second
stop to aspirate the liquid and to dispense the liquid in the receiving vessel
press the plunger until the first stop.
Handle pipette with care to avoid dysfunction like when tip attached to pipette
do not lay down the pipette horizontally and do not allow any liquid to enter
the pipette. Avoid allowing a temperature difference between pipette, tip, and
liquid as this may lead to incorrect dispensing volumes and do not clean the
pipette with aggressive solution.

3.3.2 SPECTROPHOTOMETER

Spectrophotometer is an instrument that measures the concentration of a

known substance in a solution. The amount of the substance can be
determined measuring the absorbance. Spectrophotometers are used in
chemistry, physics, biochemistry, clinical, and materials testing labs. A
spectrophotometer consists of a light sample, a method to collect the light
from the sample, a monochromator to separate the light into its component
wavelengths and a detector to measure the intensity of light at each intensity.
Spectrophotometers use the basic principle that, “Each compound absorbs or
transmits light over a certain range of wavelength”. There are two different
types of spectrophotometers: Single beam spectrometer and double beam
spectrometer. In single beam spectrometer the light intensity of the sample
and the reference are measured separately one by one while in double beam
spectrometer the light intensity of the reference and the sample can be
measured at the same time because here the light source is split into two
separate beams, one beam passes through the reference and the other
through the sample. The double beam spectrometer can be classified into:
Visible, UV and IR spectrometer.
To use the spectrophotometer, the cuvette must be completely clean. Wipe
both sides of the cuvette before and after putting the solution in the cuvette.
While exchanging the contents in the sample cuvette rinse the cuvette several
times with the standard solution. Place the cuvette in the spectrophotometer
and check the absorbance of the sample. Then compare the values of different
samples and plot a graph to know about the concentration of the sample in a
given amount.
Spectrophotometers are used in various laboratories for different purposes
such as for concentration measurement, detection of impurities, chemical
kinetics, detection of functional groups and for molecular weight
determination.

3.4 METHODS FOR THE

PREPARATION OF NANOPARTICLES
3.5 Screening Of Medicinal Plants For
Green Synthesis Of Nanoparticles
A green synthetic route for the production of silver nanoparticles (AgNPs) using five different
aqueous plant extracts, namely, Berberis vulgaris, Brassica nigra, Capsella bursa-
pastoris, Lavandula angustifolia and Origanum vulgare, was investigated in this study. The
present work demonstrates the influence of plant extract composition (antioxidant and total
phenolic content) on the size and morphology of the produced AgNPs. The biosynthetic
procedure was rapid and simple and was easily monitored via colour changes and ultraviolet
and visible (UV-Vis) spectroscopy. Subsequently, measurement of zeta potential (ZP),
photon cross-correlation spectroscopy (PCCS), X-ray diffraction (XRD), Fourier-transform
infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and selected area
electron diffraction (SAED) analysis were employed to characterise the as-synthesised
nanoparticles. The XRD investigation confirmed the presence of Ag0 in the nanoparticles,
and interactions between the bioactive compounds of the plants and the produced AgNPs
were evident in the FTIR spectra. TEM indicated that the nanoparticles exhibited a bimodal
size distribution, with the smaller particles being spherical and the larger having a truncated
octahedron shape. In addition, the antimicrobial activity of the AgNPs was tested against five
bacterial strains. All synthesised nanoparticles exhibited enhanced antimicrobial activity at a
precursor concentration of 5 mM compared to the control substance, gentamicin sulphate,
with the best results observed for AgNPs prepared with B. nigra and L. angustifolia extracts.

3.6 Method For The Detection Of

Antimicrobial Activity
A variety of laboratory methods can be used to evaluate or screen the in vitro antimicrobial
activity of an extract or a pure compound. The most known and basic methods are the disk-
diffusion and broth or agar dilution methods. Other methods are used especially for
antifungal testing, such as poisoned food technique.

3.6.1 Diffusion method

Agar disk-diffusion method

Agar disk-diffusion testing developed in 1940 [8], is the official method used in many
clinical microbiology laboratories for routine antimicrobial susceptibility testing. Nowadays,
many accepted and approved standards are published by the Clinical and Laboratory
Standards Institute (CLSI) for bacteria and yeasts testing [9], [10]. Although not all fastidious
bacteria can be tested accurately by this method, the standardization has been made to test
certain fastidious bacterial pathogens like streptococci, Haemophilus
influenzae, Haemophilus parainfluenzae, Neisseria gonorrhoeae and Neisseria meningitidis,
using specific culture media, various incubation conditions and interpretive criteria for
inhibition zones [9].
In this well-known procedure, agar plates are inoculated with a standardized inoculum of the
test microorganism. Then, filter paper discs (about 6 mm in diameter), containing the test
compound at a desired concentration, are placed on the agar surface. The Petri dishes are
incubated under suitable conditions. Generally, antimicrobial agent diffuses into the agar and
inhibits germination and growth of the test microorganism and then the diameters of
inhibition growth zones are measured (Fig. 1A). Table 1 shows the growth media,
temperature, period of incubation and inoculum size required by CLSI standards.
Agar diffusion methods: (A) disk-diffusion method of microbial extract using C. albicans as test
microorganism, (B) agar well diffusion method of essential oil using Aspergillus niger as test
microorganism, and (C) agar plug diffusion method of Bacillus sp. against C. albicans.

3.6.2 Dilution methods

Dilution methods are the most appropriate ones for the determination of MIC values, since
they offer the possibility to estimate the concentration of the tested antimicrobial agent in the
agar (agar dilution) or broth medium (macrodilution or microdilution). Either broth or agar
dilution method may be used to quantitatively measure the in vitro antimicrobial activity
against bacteria and fungi. MIC value recorded is defined as the lowest concentration of the
assayed antimicrobial agent that inhibits the visible growth of the microorganism tested, and
it is usually expressed in µg/mL or mg/L. There are many approved guidelines for dilution
antimicrobial susceptibility testing of fastidious or non-fastidious bacteria, yeast and
filamentous fungi. The most recognized standards are provided by the CLSI and the
European Committee on Antimicrobial Susceptibility Testing (EUCAST). As advised, these
guidelines provide a uniform procedure for testing that is practical to perform in most clinical
microbiology laboratories. The development of such methodologic standards does not
guarantee the clinical relevance of such testing. Nevertheless, it does allow the bioassay to be
performed in a standardized approach in order to evaluate the clinical relevance of
results [55].

[Link] Broth dilution method

Broth micro- or macro-dilution is one of the most basic antimicrobial susceptibility testing
methods. The procedure involves preparing two-fold dilutions of the antimicrobial agent (e.g.
1, 2, 4, 8, 16 and 32 µg/mL) in a liquid growth medium dispensed in tubes containing a
minimum volume of 2 mL (macrodilution) or with smaller volumes using 96-well
microtitration plate (microdilution) (Fig. 2). Then, each tube or well is inoculated with a
microbial inoculum prepared in the same medium after dilution of standardized microbial
suspension adjusted to 0.5 McFarland scale (Fig. 3). After well-mixing, the inoculated tubes
or the 96-well microtitration plate are incubated (mostly without agitation) under suitable
conditions depending upon the test microorganism (Table 1). The experimental methodology
to perform accurately the microdilution is schematized in Fig. 4.

Open in a separate window

Broth microdilution method of plant extract against B. subtilis using resazurin as growth indicator.
 Open in a separate window

0.5 McFarland microbial inoculum preparation by the direct colony suspension as recommended by
CLSI guidelines.
 Open in a separate window

Broth microdilution for antibacterial testing as recommended by CLSI protocol.

The MIC is the lowest concentration of antimicrobial agent that completely inhibits growth of
the organism in tubes or microdilution wells as detected by the unaided eye [56]. Unlike
microdilution method, the main disadvantages of the macrodilution method are the tedious,
manual undertaking, risk of errors in the preparation of antimicrobial solutions for each test,
and the comparatively large amount of reagents and space required [11]. Thus, the
reproducibility and the economy of reagents and space that occurs due to the miniaturization
of the test are the major advantages of the microdilution method. Nevertheless, the final result
is significantly influenced by approach, which must be carefully controlled if reproducible
results (intralaboratory and interlaboratory) are to be attained [56]. For the determination of
MIC endpoint, viewing devices can facilitate reading microdilution tests and recording results
with high ability to discern growth in the wells. Moreover, several colorimetric methods
based on the use of dye reagents have been developed. Tetrazolium salts, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2,3-bis {2-methoxy-4-
nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide} (XTT), are often used in the
MIC endpoint determination for both antifungal and antibacterial microdilution
assays [57], [58], [59], [60]. The Alamar blue dye (resazurin), an effective growth indicator,
can also be used for this purpose [61], [62], [63], [64].
It is well known that the inoculum size [65], the type of growth medium [66], the incubation
time and the inoculum preparation method can influence MIC values [67], [68]. Therefore,
broth dilution has been standardized by CLSI for testing bacteria that grow aerobically [56],
yeast [69] and filamentous fungi [70]. The EUCAST broth dilution method is principally
similar to that of CLSI with modifications usually concerning some of the test parameters
such as inoculum preparation, inoculum size, and the MIC reading method which is visual in
CLSI assay and spectrophotometric in EUCAST guidelines [71].
As regards to the conidium and spores forming fungi, the microdilution standardized by CLSI
involves an inoculum of spores adjusted spectrophotometrically to 0.4×104–5×104 CFU/mL.
However, in the EUCAST assay, the inoculum can be adjusted to (2–5)×105 CFU/mL by
haemocytometer counting [72]. Numerous studies showed the importance of inoculum
preparation by haemocytometer counting for reproducible and suitable preparation
independent of the color and size of conidia [68], [73], [74].
The determination of minimum bactericidal concentration (MBC) or minimum fungicidal
concentration (MFC), also known as the minimum lethal concentration (MLC), is the most
common estimation of bactericidal or fungicidal activity. The MBC is defined as the lowest
concentration of antimicrobial agent needed to kill 99.9% of the final inoculum after
incubation for 24 h under a standardized set of conditions described in document M26-
A [75], in which the MBC can be determined after broth macrodilution or microdilution by
sub-culturing a sample from wells or tubes, yielding a negative microbial growth after
incubation on the surface of non-selective agar plates to determine the number of surviving
cells (CFU/mL) after 24 h of incubation. The bactericidal endpoint (MBC) has been
subjectively defined as the lowest concentration, at which 99.9% of the final inoculum is
killed [75]. MFC is also defined as the lowest concentration of the drug that yields 98%–
99.9% killing effect as compared to the initial inoculum [71]. Several studies have been
carried out for evaluation of different test parameters for determination of MFC of various
drugs against Candida isolates [76], Aspergillus [77] and other molds [78].

[Link] Agar dilution method

The agar dilution method involves the incorporation of varying desired concentrations of the
antimicrobial agent into an agar medium (molten agar medium), habitually using serial two-
fold dilutions, followed by the inoculation of a defined microbial inoculum onto the agar
plate surface. The MIC endpoint is recorded as the lowest concentration of antimicrobial
agent that completely inhibits growth under suitable incubation conditions (Table 1).
This technique is suitable for both antibacterial and antifungal susceptibility testing. If
multiple isolates are being tested against a single compound, or if the compound (or extract)
tested masks the detection of microbial growth in the liquid medium with its coloring, agar
dilution method is often preferred to broth dilution for the MIC determination. Nowadays,
commercially produced inoculum replicators are available and can transfer between 32 and
60 different bacterial inocula to each agar plate. Agar dilution is often recommended as a
standardized method for fastidious organisms [79] such as anaerobes
and Helicobacter species. It has been also used for antifungal agent-drugs combinations
against Candida sp., Aspergillus, Fusarium and dermatophytes [80], [81], [82], [83].
This method presents a good correlation with Etest mostly for antibacterial testing against
both Gram-positive and Gram-negative bacteria. Moreover, category comparisons of agar
dilution, disk-diffusion and broth microdilution methods give excellent results [25].
3.7 ANTIMICROBIAL SUSCEPTIBILITY
TESTING
Antibiotic sensitivity testing or antibiotic susceptibility testing is the measurement of
the susceptibility of bacteria to antibiotics. It is used because bacteria may have resistance to
some antibiotics. Sensitivity testing results can allow a clinician to change the choice of
antibiotics from empiric therapy, which is when an antibiotic is selected based on clinical
suspicion about the site of an infection and common causative bacteria, to directed therapy, in
which the choice of antibiotic is based on knowledge of the organism and its sensitivities. [1]
Sensitivity testing usually occurs in a medical laboratory, and uses culture methods that expose
bacteria to antibiotics, or genetic methods that test to see if bacteria have genes that confer
resistance. Culture methods often involve measuring the diameter of areas without bacterial
growth, called zones of inhibition, around paper discs containing antibiotics on agar culture
dishes that have been evenly inoculated with bacteria. The minimum inhibitory concentration,
which is the lowest concentration of the antibiotic that stops the growth of bacteria, can be
estimated from the size of the zone of inhibition.
Antibiotic susceptibility testing has been needed since the discovery of the beta-
lactam antibiotic penicillin. Initial methods were phenotypic, and involved culture or dilution.
The Etest, an antibiotic impregnated strip, has been available since the 1980s, and genetic
methods such as polymerase chain reaction (PCR) testing have been available since the early
2000s. Research is ongoing into improving current methods by making them faster or more
accurate, as well as developing new methods for testing, such as microfluidics.

Uses
In clinical medicine, antibiotics are most frequently prescribed on the basis of a
person's symptoms and medical guidelines. This method of antibiotic selection is called empiric
therapy,[1] and it is based on knowledge about what bacteria cause an infection, and to what
antibiotics bacteria may be sensitive or resistant.[1] For example, a simple urinary tract
infection might be treated with trimethoprim/sulfamethoxazole.[2] This is because Escherichia
coli is the most likely causative bacterium, and may be sensitive to that combination antibiotic.
[2]
However, bacteria can be resistant to several classes of antibiotics.[2] This resistance might be
because a type of bacteria has intrinsic resistance to some antibiotics,[2] because of resistance
following past exposure to antibiotics,[2] or because resistance may be transmitted from other
sources such as plasmids.[3] Antibiotic sensitivity testing provides information about which
antibiotics are more likely to be successful and should therefore be used to treat the infection. [1]
Antibiotic sensitivity testing is also conducted at a population level in some countries as a form
of screening.[4] This is to assess the background rates of resistance to antibiotics (for example
with methicillin-resistant Staphylococcus aureus), and may influence guidelines and public
health measures.[4]

Methods
Once a bacterium has been identified following microbiological culture, antibiotics are selected
for susceptibility testing.[5] Susceptibility testing methods are based on exposing bacteria to
antibiotics and observing the effect on the growth of the bacteria (phenotypic testing), or
identifying specific genetic markers (genetic testing).[6] Methods used may be qualitative,
meaning that a result indicates resistance is or is not present; or quantitative, using a minimum
inhibitory concentration (MIC) to describe the concentration of antibiotic to which a bacterium is
sensitive.[6]
There are many factors that can affect the results of antibiotic sensitivity testing, including failure
of the instrument, temperature, moisture, and potency of the antimicrobial agent. Quality
control (QC) testing helps to ensure the accuracy of test results.[7] Organizations such as
the American Type Culture Collection and National Collection of Type Cultures provide strains of
bacteria with known resistance phenotypes that can be used for quality control. [8]

Clinical practice

Antibiotic resistance tests: Bacteria are streaked on dishes with white disks, each impregnated with a
different antibiotic. Clear rings, such as those on the left, show that bacteria have not grown—indicating
that these bacteria are not resistant. The bacteria on the right are fully resistant to all but two of the seven
antibiotics tested.[31]

Ideal antibiotic therapy is based on determining the causal agent and its antibiotic sensitivity.
Empiric treatment is often started before laboratory microbiological reports are available. This
might be for common or relatively minor infections based on clinical guidelines (such
as community-acquired pneumonia), or for serious infections, such as sepsis or bacterial
meningitis, in which delayed treatment carries substantial risks.[1] The effectiveness of individual
antibiotics varies with the anatomical site of the infection, the ability of the antibiotic to reach the
site of infection, and the ability of the bacteria to resist or inactivate the antibiotic. [32]
Specimens for antibiotic sensitivity testing are ideally collected before treatment is started. [1] A
sample may be taken from the site of a suspected infection; such as a blood culture sample
when bacteria are suspected to be present in the bloodstream (bacteraemia), a sputum sample
in the case of a pneumonia, or a urine sample in the case of a urinary tract infection. Sometimes
multiple samples may be taken if the source of an infection is not clear. [1] These samples are
transferred to the microbiology laboratory where they are added to culture media, in or on which
the bacteria grow until they are present in sufficient quantities for identification and sensitivity
testing to be carried out.[33][28]
When antibiotic sensitivity testing is completed, it will report the organisms present in the sample,
and which antibiotics they are susceptible to.[28] Although antibiotic sensitivity testing is done in a
laboratory (in vitro), the information provided about this is often clinically relevant to the
antibiotics in a person (in vivo).[34] Sometimes, a decision must be made for some bacteria as to
whether they are the cause of an infection, or simply commensal bacteria or contaminants,
[28]
such as Staphylococcus epidermidis[35] and other opportunistic infections. Other considerations
may influence the choice of antibiotics, including the need to penetrate through to an infected site
(such as an abscess), or the suspicion that one or more causes of an infection were not detected
in a sample.[1]
4. NATURA
L
PRODUC
T
RESEAR
CH
Introduction of natural product research, Basic concept of
calculation, Micropipette handling (Reverse and Forward pepetting),
Principle - Standard operating procedure and application of different
instruments (Autoclave, Laminar air flow and pH meter), Basic
knowledge about Types of extraction, Standard procedure of extract
preparation by Maceration/Decoction method, Introduction about
phytochemicals, Quantitative estimation of
Phenolic/Carbohydrates/Protein through UV-Visible
spectrophotometer, Standard Curve preparation and Data
Interpretation, Knowledge about types of media, different methods
for the detection of antimicrobial activity, Antimicrobial susceptibility
testing (AST) of plant extract against pathogenic bacteria (E. coli/
S. aureus/Proteus mirabilis etc.) through Disc diffusion, Basic
concept of Synergistic and antagonistic effect of herbal drug,
Overview of chromatography, Separation of Antioxidant compound
by TLC (Thin layer chromatography), Basic concept of Purification
of herbal extract by Column chromatography.
Basic Concepts
Preparation of different percentages;
Formulae;
Volume required to be added in the distilled water or the solvent/Weight.
(g=ml, mg=micro liter) 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑡𝑜𝑐𝑘 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 =𝐷𝑒𝑠𝑖𝑟𝑒𝑑 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛𝑆𝑡𝑜𝑐𝑘
𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 ×𝐹𝑖𝑛𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒
For liquid stock; The volume of solvent required = The final volume – volume of stock required
Molarity(M);
It is defined as number of moles of the solute in one liter of the solvent.
1 mole = 6.022x1023\ Units (Avogadro number)
1 mole = The mass of chemical required/Molar mass
Molarity = 𝑇ℎ𝑒 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑚𝑜𝑙𝑒𝑠𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 𝐹𝑖𝑛𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒 ×1000 𝑚𝑙
Molality(m);
The number of moles in one kilo grams of the solvent.
Molality = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 1 𝑘𝑔 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
Useful; It does not change with temperature.
Normality(N);
Number of equivalent grams in one liter of the solvent.
Normality = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑔𝑟𝑎𝑚 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡𝑅𝑒𝑞𝑢𝑖𝑟𝑒𝑑 𝑉𝑜𝑙𝑢𝑚𝑒 ×1000𝑚𝑙
EQ = MW/n
MW = Molecular weight
n = basicity/acidity
Note; The molarity of the solutes with basicity or acidity of one is equal to the molarity of the
solute.
Density to molarity;
Density = Normality x 6.916667 x 10-2 for HCL
Molarity = 𝑑𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑚𝑎𝑠𝑠 ×1000𝑚𝑙
Things to be taken care;
• Whenever using any liquid chemical read the following carefully:

- Name
- Molecular weight
- Formula
- Concentration
- Read the precautions
- Storage method
- Biohazard
• When solutions are prepared from salts or solid solvents the solvent should be added in the
volume of solvent which is less than the final volume (May be 2-3ml less than the final volume)
and after the dissolution make up the volume to its required final value.

• If the solution is required to be at a certain pH, in initial stage the addition of the stock and
solvent should be less than the final volume so that the pH can be set by addition of base or
acid. After the pH maintenance the volume should be made up to the final volume

Molarity and normality;

We are solving it with an example;
Let’s get accustomed with the molarity of H2SO4;
M = Moles of the solute/Volume
Moles = Mass required / molar mass
Mass required to make 1 M = Mol. mass x moles
Where number of moles = 1, molecular mass = 98.02
So, Mass required = 98.02 x 1 = 98.02g in 1000ml of solvent
Now, Normality(N);
N = Eq/Volume where, Eq = Molecular mass/n (n = basicity or acidity)
The mass of the solute required to prepare one N solution is called as gram equivalent(x)
X = (98.02/2) = 49.01
So, 49.01g of solute require to prepare one normal solution which is half the mass of the solute
required to prepare 1M solution.
Which also means that we need to multiply molarity of the H2SO4 by 2 to get the normality.
Which also means that 1 Normal H2SO4 is 0.5 molar solution.
As the acidity of the H2SO4 is 2, which leads us to the conclusion that;
Normality = Molarity x the acidity or basicity of the acid/base.
There are others way to solve the above-mentioned problem. This approach is very frequently
used by me. I used others ways to derive the relationship between normality and molarity but
faced so many problems. As the result of that I used this particular approach to solve this.
Information;
• HCl is commercially available at 32-37 percentage where as H2SO4 at 98 percentage.
• Dried HCl does not change the color of the litmus paper as there are no proton acceptors. Dry
HCl does not behave as an acid.

WHY SO?
Problem; Why is HCl commercially available at 32-37 percentage?
Things to be considered;
• Solubility
• Concentration
• Energy
• Economical

To answer this question, we need understand the process by which HCL is prepared.
Preparation of HCL;
420K
NaCl + H2SO4 NaHSO4+ HCl(g)
823K
NaHSO4 + NaCl Na2SO4 + HCl(g)
Post this process the Hydrogen chloride is dried by passing it through concentrated H2SO4.
Note; HCl gas is liquid at 189k and solid at 159k.
Now, on the basis of frequent observations, it is observed that HCl when saturated in the
solvent it reaches its saturation point at 37 percentage at the room temperature and 1
atmospheric pressure. Even we saturate the HCl above 38 percent the extra HCL gas will
vaporize.
We can saturate it even above 38 percent at very low temperature and high pressure* but
following issues are involved;
• It will make storage of highly concentrated HCl very difficult; has to be stored in low
temperature and high pressure; it will make HCl expensive.
• The process of preparing HIGHLY concentrated HCl is energetically expensive and economically
exhaustive; it will make HCl exorbitant and will make it economically unavailable.
• The highly concentrated HCl is difficult and high-costed to transport and any malfunctioning in
the temperature regulators and pressurized containers
may lead to lethal accidents and cause severe damage to surrounding life. Because of increase
in temperature and decrease in pressure which will lead to increase in relative vapor pressure of
the acid and bottle will bast off. (I haven’t confirmed it experimentally).
• Working with HCl will become much more dangerous.

For H2SO4; (reference; brainly);

• The H2SO4 is prepared by contact process which is energetically more efficient and economic
than the other available methods. 98 percent Sulfuric acid is very frequently available for
laboratory uses but it is possible to prepare 100 percent sulfuric acid. Problem is that Sulfuric
acid is highly hygroscopic and preparing 100 percent of it will be costly and more energetically
exhaustive. (Ref; Bruno Maes)

Osmolarity;
It is defined as total number of osmols in solution. It means number of osmols per liter of
solution.
Relationship between molarity and osmolarity
Osmolarity = Molarity x number of the osmotically active molecules.
Osmotic Pressure;
The minimum pressure required to be applied to prevent flow of pure solvent across
semipermeable membrane.
Van’t Hoff derived a formula explaining the calculations of osmotic pressure;
Π = iCRT
Where, i = Van’t Hoff factor
C = molar concentration
R = Universal gas constant
T = 300K
It also means that;
OP is directly proportional to molarity, normality.
It is only applicable for solvents who almost behave like ideal solutions.
Ideal solution is one which obeys Roult’s law;
The freezing point and boiling point of an ideal solution are respectively depresses and elevates
by an amount of mole fraction of the solvent (tea science);
Micro pipetting;
1. Forward;
- For pipetting in = Single Press
- For pipetting out = Double press

Importance = For liquids with density similar to dihydromonoxide in liquid form.

2. Reverse Pipetting;
- For pipetting in = Double Press
- For pipetting out = Single press

Importance = For Viscous fluids (similar honey so forth and so on).

Principles, Results and Inferences
The Plant Sample Used; Syzygium cumini leaf extract
List of experiments performed
• Qualitative Analysis of carbohydrates by using DNSA method
• Antimicrobial susceptibility test
• TLC
Here, only principles, inferences and precautions will be discussed
Qualitative Analysis of Carbohydrates
Aim; To estimate quantity of carbohydrates in a given sample
Principle;
Carbohydrates are polymers of sugars. Carbohydrates are degraded into reducing sugars like
glucose and Mannose. In the presence of reducing sugars DNSA is converted into 3-amino-5-
nitrosalicylic acid which gives a reddish color on heating. The known concentrations of dextrose
are used to determine the carbohydrate concentrations by using spectrophotometer (VIS) at
540nm.
Precautions;
• Every apparatus should be clean.
• While using cuvette fingers should only touch the opaque side.

Inferences:
• Relativity is used here to estimate concentration.
• Standard graph is helpful in estimating concentrations of other molecules than carbohydrates

Antimicrobial susceptibility test

Aim; To analyze antimicrobial properties of the given sample by disc diffusion method.
Principle; An absorbent disc is used to expose bacteria to a particular antibiotic to compare
antimicrobial properties of the sample. It is done in nutritional agar media by inoculating
bacteria and then different concentrations of antibiotic are introduced by disc. Before it, plate is
divided in different compartments by using water insoluble ink marker.
Bacteria plated; E. coli
Antibiotic used; Erythromycin
Mode of action; It binds to ribosomal subunit and blocks the other subunit to bind to it. This
leads to inhibition of protein synthesis.
Inference; Only very high concentrations of Erythromycin produced a significant zone of
inhibition. The most plausible explanation is that Erythromycin is bactericidal to gram positive
bacteria and bacteriostatic to gram negative bacteria. As, E. coli is gram negative bacteria and
some strains of it are resistant to Erythromycin which could be the reason of very minimal zone
of inhibition by high concentrations of it (10mg/ml).
Precautions;
• Switch off UV and switch on blower before opening the Laminar air flow hood.
• Always disinfect your hands inside the hood (With 70 percent Ethanol).
• Don’t talk while working in LAF or wear double mask
• Be cautious to disinfect your hands after completion of the work in LAF.
• Avoid touching surfaces.
• While steaking, sterilize the loop and let it cool down to room temperature, don’t touch
bacterial colonies with hot loop.
• Don’t put the lid on unless the agar media solidifies.

Thin Layer Chromatography

Aim; To confirm presence of antioxidants in the plant sample
Principle;
Thin layer chromatography uses glass, plastic or aluminum along with silica gel as stationary
phase. Non-polar solvents like hexanes are used as mobile phase. This technique works on the
principle that the retention factor depends upon the affinity of the analyte towards the mobile
and stationary phase. Analytes having more affinity to mobile phase will have high retention
factor than the compounds with more affinity to the stationary phase. The stationary phase has
UV binders. The retention factors can be used to identify the separated compound of the
analyte.
What are antioxidants? For understanding antioxidants first, we have to understand what are
reactive oxygen species or ROS?
ROS are highly reactive free radicle species which are formed during cellular oxidation or
Electron transport chain in mitochondria. Usually, these ROS are taken care of by cytochrome C.
These ROS damage protein, fats, carbohydrates and DNA. The damage to DNA can lead cancer
and other autoimmune diseases. They are also very important in cell signaling of apoptosis.
Sequential reduction of O2 forms number of ROS.
The antioxidants are scavengers of ROS. The antioxidants block the free radicles and makes
them harmless.
The DPPH stands for 2,2-diphenyl-1-picrylhydrazyl. It is stable free radicle provider which will
be inhibited by the antioxidants. After running TLC, the DPPH is sprayed on the TLC sheet. The
entire sheet will appear purple under UV C and the antioxidant bands will appear yellow in color
as antioxidants will inhibit the DPPH.
• Nine different kinds of antioxidants were observed in the Jamun leaf extract.

Precautions;
• Only led pencil should be used to mark sample spot on TLC sheet.
• After the drying of sheet, the distance travelled by the solvent should be
immediately marked with lead pencil.
• The DPPH is biohazardous and carcinogenic and should not be touched with bare
hands. It should be sprayed on TLC sheet in a separate room other than main
laboratory with proper ventilation.
• Mask should be on while using DPPH.
• Cover the set up with plastic paper as methanol and chloroform used as mobile
phase are volatile in nature.
• Do not leave Chloroform bottle open. Wear mask while working with it and in
case of excessive inhalation or ingestion seek medical help as soon as possible.

5. BIOPROC
ESS
 ENGINEE
RING
AND
FERMEN
TATION
TECHNO
LOGY
Introduction about Fermentation, Future prospect in fermentation
technology, Basic concept of upstream and downstream process,
General and safety rules for working in Lab, Demonstration of
instruments, Concept of basic calculation (percentage, molarity and
normality), basic concept of reagent preparation, Micropipette
handling (forward and reverse pipetting techniques), Overview of
media preparation, Isolation and identification of citric acid
producing microorganism from soil sample, Preparation of
fermentation media, Production of citric acid by submerged
fermentation methods, Qualitative analysis of citric acid by titrimetric
method, Basic concept of wine production parameter analysis by
different physico-chemical methods, wine production from different
fruit juices, Principle-standard operating procedure and application
of spectrophotometer, estimation of ethanol by potassium
dichromate method through UV-VIS spectrophotometer, Standard
Curve preparation and Data Interpretation.

5.1 FERMENTATION
Fermentation is a metabolic process that produces chemical changes in
organic substrates through the action of enzymes. In biochemistry, it is narrowly defined as the
extraction of energy from carbohydrates in the absence of oxygen. In food production, it may
more broadly refer to any process in which the activity of microorganisms brings about a
desirable change to a foodstuff or beverage.[1] The science of fermentation is known
as zymology.
In microorganisms, fermentation is the primary means of producing adenosine
triphosphate (ATP) by the degradation of organic nutrients anaerobically. The word equation for
fermentation is: glucose → ethanol + carbon dioxide, or C6H12O6 (aq) → 2C2H5OH (l)
+ 2CO2 (g).[2] Humans have used fermentation to produce foodstuffs and beverages since
the Neolithic age. For example, fermentation is used for preservation in a process that
produces lactic acid found in such sour foods as pickled cucumbers, kombucha, kimchi,
and yogurt, as well as for producing alcoholic beverages such as wine and beer. Fermentation
also occurs within the gastrointestinal tracts of all animals, including humans. [3]

Definitions
Below are some definitions of fermentation. They range from informal, general usages to more
scientific definitions.[4]
1. Preservation methods for food via microorganisms (general use).
2. Any large-scale microbial process occurring with or without air (common definition used
in industry).
3. Any process that produces alcoholic beverages or acidic dairy products (general use).
4. Any energy-releasing metabolic process that takes place only under anaerobic conditions
(somewhat scientific).
5. Any metabolic process that releases energy from a sugar or other organic molecule,
does not require oxygen or an electron transport system, and uses an organic molecule
as the final electron acceptor (most scientific).

Biological role
Along with aerobic respiration, fermentation is a method to extract energy from molecules. This
method is the only one common to all bacteria and eukaryotes. It is therefore considered the
oldest metabolic pathway, suitable for primeval environments – before plant life on Earth, that is,
before oxygen in the atmosphere.[5]: 389
Yeast, a form of fungus, occurs in almost any environment capable of supporting microbes, from
the skins of fruits to the guts of insects and mammals to the deep ocean. Yeasts convert (break
down) sugar-rich molecules to produce ethanol and carbon dioxide.[6][7]
Basic mechanisms for fermentation remain present in all cells of higher
organisms. Mammalian muscle carries out fermentation during periods of intense exercise where
oxygen supply becomes limited, resulting in the creation of lactic acid.[8]: 63 In invertebrates,
fermentation also produces succinate and alanine.[9]: 141
Fermentative bacteria play an essential role in the production of methane in habitats ranging
from the rumens of cattle to sewage digesters and freshwater sediments. They produce
hydrogen, carbon dioxide, formate and acetate and carboxylic acids. Then consortia of microbes
convert the carbon dioxide and acetate to methane. Acetogenic bacteria oxidize the acids,
obtaining more acetate and either hydrogen or formate. Finally, methanogens (in the
domain Archea) convert acetate to methane.[10]
Biochemical overview

Comparison of a aerobic respiration and most known fermentation types in eukaryotic cell.[11] Numbers in
circles indicate counts of carbon atoms in molecules, C6 is glucose C6H12O6, C1 carbon
dioxide CO2. Mitochondrial outer membrane is omitted.

Fermentation reacts NADH with an endogenous, organic electron acceptor.[2] Usually this
is pyruvate formed from sugar through glycolysis. The reaction produces NAD+ and an organic
product, typical examples being ethanol, lactic acid, and hydrogen gas (H2), and often
also carbon dioxide. However, more exotic compounds can be produced by fermentation, such
as butyric acid and acetone. Fermentation products are considered waste products, since they
cannot be metabolized further without the use of oxygen.[12]
Fermentation normally occurs in an anaerobic environment. In the presence of O2, NADH, and
pyruvate are used to generate ATP in respiration. This is called oxidative phosphorylation. This
generates much more ATP than glycolysis alone. It releases the chemical energy of O 2.[12] For this
reason, fermentation is rarely used when oxygen is available. However, even in the presence of
abundant oxygen, some strains of yeast such as Saccharomyces cerevisiae prefer fermentation
to aerobic respiration as long as there is an adequate supply of sugars (a phenomenon known as
the Crabtree effect).[13] Some fermentation processes involve obligate anaerobes, which cannot
tolerate oxygen.[citation needed]
Although yeast carries out the fermentation in the production of ethanol in beers, wines, and
other alcoholic drinks, this is not the only possible agent: bacteria carry out the fermentation in
the production of xanthan gum.[citation needed]

Products of fermentation
Ethanol
In ethanol fermentation, one glucose molecule is converted into two ethanol molecules and
two carbon dioxide molecules.[14][15] It is used to make bread dough rise: the carbon dioxide forms
bubbles, expanding the dough into a foam.[16][17] The ethanol is the intoxicating agent in alcoholic
beverages such as wine, beer and liquor.[18] Fermentation of feedstocks,
including sugarcane, corn, and sugar beets, produces ethanol that is added to gasoline.[19] In
some species of fish, including goldfish and carp, it provides energy when oxygen is scarce
(along with lactic acid fermentation).[20]
The figure illustrates the process. Before fermentation, a glucose molecule breaks down into two
pyruvate molecules (Glycolysis). The energy from this exothermic reaction is used to bind
inorganic phosphates to ADP, which converts it to ATP, and convert NAD+ to NADH. The
pyruvates break down into two acetaldehyde molecules and give off two carbon dioxide
molecules as waste products. The acetaldehyde is reduced into ethanol using the energy and
hydrogen from NADH, and the NADH is oxidized into NAD+ so that the cycle may repeat. The
reaction is catalyzed by the enzymes pyruvate decarboxylase and alcohol dehydrogenase.[14]

Lactic acid
Homolactic fermentation (producing only lactic acid) is the simplest type of fermentation.
Pyruvate from glycolysis[21] undergoes a simple redox reaction, forming lactic acid.[22][23] Overall,
one molecule of glucose (or any six-carbon sugar) is converted to two molecules of lactic acid:
C6H12O6 → 2 CH3CHOHCOOH
It occurs in the muscles of animals when they need energy faster than the blood can supply
oxygen. It also occurs in some kinds of bacteria (such as lactobacilli) and some fungi. It is
the type of bacteria that convert lactose into lactic acid in yogurt, giving it its sour taste.
These lactic acid bacteria can carry out either homolactic fermentation, where the end-
product is mostly lactic acid, or heterolactic fermentation, where some lactate is further
metabolized to ethanol and carbon dioxide[22] (via the phosphoketolase pathway), acetate, or
other metabolic products, e.g.:
C6H12O6 → CH3CHOHCOOH + C2H5OH + CO2
If lactose is fermented (as in yogurts and cheeses), it is first converted into glucose and
galactose (both six-carbon sugars with the same atomic formula):
C12H22O11 + H2O → 2 C6H12O6
Heterolactic fermentation is in a sense intermediate between lactic acid
fermentation and other types, e.g. alcoholic fermentation. Reasons to go further and
convert lactic acid into something else include:
 The acidity of lactic acid impedes biological processes. This can be beneficial to
the fermenting organism as it drives out competitors that are unadapted to the
acidity. As a result, the food will have a longer shelf life (one reason foods are
purposely fermented in the first place); however, beyond a certain point, the
acidity starts affecting the organism that produces it.
 The high concentration of lactic acid (the final product of fermentation) drives the
equilibrium backwards (Le Chatelier's principle), decreasing the rate at which
fermentation can occur and slowing down growth.
 Ethanol, into which lactic acid can be easily converted, is volatile and will readily
escape, allowing the reaction to proceed easily. CO2 is also produced, but it is
only weakly acidic and even more volatile than ethanol.
 Acetic acid (another conversion product) is acidic and not as volatile as ethanol;
however, in the presence of limited oxygen, its creation from lactic acid releases
additional energy. It is a lighter molecule than lactic acid, forming fewer
hydrogen bonds with its surroundings (due to having fewer groups that can form
such bonds), thus is more volatile and will also allow the reaction to proceed
more quickly.
  If propionic acid, butyric acid, and longer monocarboxylic acids are produced
(see mixed acid fermentation), the amount of acidity produced per glucose
consumed will decrease, as with ethanol, allowing faster growth.
Hydrogen gas
Hydrogen gas is produced in many types of fermentation as a way to regenerate
NAD+ from NADH. Electrons are transferred to ferredoxin, which in turn is oxidized
by hydrogenase, producing H2.[14] Hydrogen gas is
a substrate for methanogens and sulfate reducers, which keep the concentration of
hydrogen low and favor the production of such an energy-rich compound,[24] but
hydrogen gas at a fairly high concentration can nevertheless be formed, as in flatus.
[citation needed]

For example, Clostridium pasteurianum ferments glucose to butyrate, acetate,

carbon dioxide, and hydrogen gas:[25] The reaction leading to acetate is:
C6H12O6 + 4 H2O → 2 CH3COO− + 2 HCO3− + 4 H+ + 4 H2
Alternative protein

Fermentation is used to generate the heme protein found in the Impossible Burger.

Fermentation can be applied to generate alternative protein sources. For

instance, plant based protein foods such as tempeh are produced using
fermentation. However, fermentation can also be used to culture animal
products made from non-living material in vitro. Eggs, honey, cheese and milk
are all examples which are made of various proteins. These proteins can be
produced using this particular application of fermentation. Substances that are
made using fermentation and which resemble milk are called milk substitutes.
Substances that resemble cheese are called cheese analogue and substances
that resemble eggs are called egg substitutes.[citation needed]
Some companies have started providing fermentation services to farmers
(Farming as a Service).[26][27]
Heme is a protein which gives meat its characteristic texture, flavour and aroma.
[28]
Impossible Foods used fermentation to generate a particular strand of heme
derived from soybean roots, called soy leghemoglobin, which was integrated
into the Impossible Burger to mimic meat flavor and appearance.[28]

5.2 FUTURE PROSPECT IN

FERMENTATION TECHNOLOGY
Fermentation is the use of living organisms (mainly microorganisms), typically on a large scale,
to produce commercial products or to carry out important chemical transformations.
Industrial fermentation is an interdisciplinary science that applies principles associated with
biology and engineering. The biological aspect focuses on microbiology and biochemistry. The
engineering aspect applies fluid dynamics and materials engineering. Industrial fermentation is
associated primarily with the commercial exploitation of microorganisms on a large scale. The
microbes used may be natural species, mutants, or microorganisms that have been genetically
engineered. Many products of considerable economic value are derived from industrial
fermentation processes. Common products such as antibiotics, cheese, pickles, wine, beer,
biofuels, vitamins, amino acids, solvents, and biological insecticides and pesticides are produced
via industrial fermentation.
The goal of industrial fermentation is to improve biochemical or physiological processes that
microbes are capable of performing while yielding the highest quality and quantity of a particular
product. The development of fermentation processes requires knowledge from dis-
disciplines such as microbiology, biochemistry, genetics, chemistry, chemical and bioprocess
engineering, mathematics, and computer science. The major microorganisms used in industrial
fermentation are fungi (such as yeast) and bacteria.
Careers and Course Work
The knowledge and skills developed in the Fermentation program have relevance to numerous
professional fields and careers. Students will be exposed to a wide range of applications to
broaden their career scope and assure long-term success. There are several career options for
people who interested in being trained in fermentation technology. Food-biotechnology,
microbiology, pharmaceutical, chemical, and biofuel companies are the biggest employers in the
area. Students who are interested in conducting research in industrial fermentation can find jobs
in university, government, and industry laboratories.
Following is the list of prospective job opportunities in this field:
• Quality Control/QC (the most common one)
• Analytical testing (a step ahead of QC)
• Production (the operation handler, working on big reactors, packaging units, and sterilizers)
• Research & Development ( many options: Therapeutic research, GMOs, Biopesticides,
Biofertilizers, Flavor research etc)
• Marketing
• process development
• technical services

Some industrial fermentation specialists may be considered as

• genetic engineers (using DNA techniques to modify living organisms)

• Bioprocess or chemical engineers (optimizing bioreactors and biochemical pathways for the
desired product).

When choosing a career in industrial fermentation, one should be prepared for an

interdisciplinary science. Students should obtain skills in microbiology, molecular biology;
bioengineering, plant biology, organic chemistry, biochemistry, agriculture, bioprocess
engineering, and chemical engineering.
Most professionals in industrial fermentation have a bachelor’s degree in biology, microbiology,
or biotechnology. Individuals who have managerial responsibilities often have a master’s or
doctorate in biology, microbiology, fermentation, molecular biology, biochemistry, biotechnology,
bioprocess or chemical engineering, or genetics. A career in industrial fermentation presents a
variety of work options.
Applications of Fermentation
• Fermentation has a number of commercial applications beyond those described thus far. Many
occur in the food preparation and processing industry. A variety of bacteria are used in the
production of olives, cucumber pickles, and sauerkraut from the raw olives, cucumbers, and
cabbage, respectively. The selection of exactly the right bacteria and the right conditions (for
example, acidity and salt concentration) is an art in producing food products with exactly the
desired flavors. An interesting line of research in the food sciences is aimed at the production of
edible food products by the fermentation of petroleum.
• In some cases, antibiotics and other drugs can be prepared by fermentation if no other
commercially efficient method is available. For example, the important drug cortisone can be
prepared by the fermentation of a plant steroid known as diosgenin. The enzymes used in the
reaction are provided by the mold Rhizopus nigricans.
• One of the most successful commercial applications of fermentation has been the production of
ethyl alcohol for use in gasohol. Gasohol is a mixture of about 90% gasoline and 10% alcohol.
The alcohol needed for this product can be obtained from the fermentation of agricultural and
municipal wastes. The use of gasohol provides a promising method for using renewable
resources (plant material) to extend the availability of a nonrenewable resource (gasoline).
• Another application of the fermentation process is in the treatment of wastewater. In the
activated sludge process, aerobic bacteria are used to ferment organic material in wastewater.
Solid wastes are converted to carbon dioxide, water, and mineral salts.
• Industrial fermentation plays a major role in providing food, chemicals, and fuels. End users are
consumers, farmers, medical doctors, and industrialists. Industrial fermentation is changing the
course of history

5.3 BASIC CONCEPT OF UPSTREAM

AND DOWNSTREAM PROCESSES
5.3.1 UPSTREAM PROCESS
Upstream process is the first step of bioprocess from early cell isolation
and cultivation, to cell banking and culture development of the cells until
final harvest where the desired quantity is reached. Since this is the early
stage of bioprocessing, the quality of the product is of critical importance.
Therapeutic cell manufacturing processes can be separated into upstream
processes and downstream processes. The upstream process is defined as the
entire process from early cell isolation and cultivation, to cell banking and culture
expansion of the cells until final harvest (termination of the culture and collection
of the live cell batch).
Aside from technology challenges, concerning the scalability of culture apparatus,
a number of raw material supply risks have emerged in recent years[when?],
including the availability of GMP grade fetal bovine serum[citation needed].
The upstream part of a bioprocess refers to the first step in which microbes/cells
are grown, e.g. bacterial or mammalian cell lines (see cell culture), in bioreactors.
Upstream processing involves all the steps related to inoculum development,
media development, improvement of inoculum by genetic engineering process,
optimization of growth kinetics so that product development can improve
tremendously. Fermentation has two parts: upstream and downstream. After
product development, the next step is the purification of product for desired
quality. When they reach the desired density (for batch and fed-batch cultures)
they are harvested and moved to the downstream section of the bioprocess.

5.3.2 DOWNSTREAM PROCESS

The downstream part of a bioprocess refers to the part where the cell mass from
the upstream are processed to meet purity and quality requirements.
Downstream processing is usually divided into three main sections: cell
disruption, a purification section and a polishing section. The volatile products
can be separated by distillation of the harvested culture without pre-treatment.
 Distillation is done at reduced pressure at continuous stills. At reduced pressure,
distillation of product directly from fermentor may be possible. The steps of
downstream processing are:
1. Separation of biomass: separating the biomass (microbial cells) generally carried out by
centrifugation or ultra-centrifugation. If the product is biomass, then it is recovered for
processing and spent medium is discarded. If the product is extra cellular the biomass
will be discarded. Ultra filtration is an alternative to the centrifugation.
2. Cell disruption: If the desired product is intra cellular the cell biomass can be disrupted
so that the product should be released. The solid-liquid is separated by centrifugation or
filtration and cell debris is discarded.
3. Concentration of broth: The spent medium is concentrated if the product is extracellular.
4. Initial purification of metabolites: According to the physico-chemical nature of the product
molecule several methods for recovery of product from the clarified fermented broth
were used (precipitation, etc.)
5. De-watering: If low amount of product is found in very large volume of spent medium, the
volume is reduced by removing water to concentrate the product. It is done by vacuum
drying or reverse osmosis.
6. Polishing of metabolites: this is the final step of making the product 98 to 100% pure.
The purified product is mixed with several inert ingredients called excipients. The
formulated product is packed and sent to the market for the consumers.

5.4 Production Of Citric Acid By

Submerged Fermentation
Citric acid is the most important organic acid produced in tonnage and is extensively used in food
and pharmaceutical industries. It is produced mainly by submerged fermentation using
Aspergillus niger or Candida sp. from different sources of carbohydrates, such as molasses and
starch based media. However, other fermentation techniques, e.g. solid state fermentation and
surface fermentation, and alternative sources of carbon such as agro-industrial residues have
been intensively studied showing great perspective to its production. This paper reviews recent
developments on citric acid production by presenting a brief summary of the subject, describing
micro-organisms, production techniques, and substrates, etc.

Citric acid (C6H8O7, 2 - hydroxy - 1,2,3 - propane tricarboxylic acid), a natural constituent and
common metabolite of plants and animals, is the most versatile and widely used organic acid in
the field of food (60%) and pharmaceuticals (10%). It has got several other applications in
various other fields. Currently, the global production of citric acid is estimated to be around
736000 tones/year (Química e Derivados, 1997), and the entire production is carried out by
fermentation. In Brazil, almost the entire demand of citric acid is met through imports. There is
constant increase (3.5-4%) each year in its consumption, showing the need of finding new
alternatives for its manufacture.

Applications of citric acid

Citric acid is mainly used in food industry because of its pleasant acid taste an its high solubility
in water. It is worldwide accepted as "GRAS" (generally recognized as safe), approved by the
Joint FAO/WHO Expert Committee on Food Additives. The pharmaceutical and cosmetic
industries retain 10% of its utilization and the remainder is used for various other purposes. Table
1 presents main applications of citric acid.

MICRO-ORGANISMS USED FOR CITRIC ACIC PRODUCTION

A large number of micro-organisms including bacteria, fungi and yeasts have been employed
to produce citric acid. Most of them, however, are not able to produce commercially
acceptable yields. This fact could be explained by the fact that citric acid is a metabolite of
energy metabolism and its accumulation rises in appreciable amounts only under conditions
of drastic imbalances. Kubicek and Rohr (1986) reviewed the strains reported to produce
citric acid. Table 2 shows the micro-organisms used to produce citric acid. Among these,
only A. niger and certain yeasts such as Saccharomycopsis sp. are employed for commercial
production. However, the fungus A. niger has remained the organism of choice for
commercial production. The main advantages of using this micro-organism are: (a) its ease of
handling, (b) its ability to ferment a variety of cheap raw materials, and (c) high yields.

5.5 Basic Concept Of Wine

Production Parameter Analysis

Blending different fruits as well as adding medicinal herbs improves important physicochemical and
sensorial properties of fruit wine. The present study aimed at investigating prominent
physicochemical and sensory properties of wine produced from cactus pear and Lantana camara fruit
juice blend. Both fruit juices were characterized based on pH, sugar, titratable acidity, total phenol,
and organic acid contents. The fermentation process was made at previously optimized fermentation
temperature of 24.8°C, pH of 3.4, inoculum concentration (Saccharomyces cerevisiae) of 10.16%
(v/v), and Lantana camara fruit juice concentration of 10.66% (v/v). The final wine was characterized
as having pH of 3.47 ± 0.04, 4.6 ± 0.02 g/L sugar equivalent to dextrose, 0.33 ± 0.006% titratable
acidity (w/v citric acid), total phenol of 696.1 ± 22.1 mg/L equivalent to gallic acid, and 4.35 ± 0.4
mg/mL organic acid equivalent to citric acid composition. Predominant color intensity, ethanol,
methanol, total sulfite, and sensory value of the final wine were measured as 48.07 ± 2.66% of
yellowish color, 8.6 ± 0.68% (v/v), 124.4 ± 9.5 mg/L, 129.94 ± 4.04 mg/L, and 8.65 ± 0.92,
respectively. The blended Lantana camara fruit enhanced total phenol, color, and sensory value of the
final wine. Titratable acidity and methanol and sulfite contents of the final wine are in an acceptable
limit compared to standards for commercial wines. Utilizing cactus pear fruit by
incorporating Lantana camara fruit for health-enhancing functional food development such as fruit
wines could solve the current postharvest loss of both fruits and be a means of alternative beverage.

5.6 Wine Production From Different

Fruit Juices
Introduction
Wine is an alcoholic beverage, made of fermented fruit juice, usually from grapes. The natural
chemical balance of grapes lets them ferment without the addition of sugars, acids, enzymes, or
other nutrients. Grape wine is produced by fermenting crushed grapes using various types of
yeast. Yeast consumes the sugars in the grapes and converts them into alcohol. Different
varieties of grapes and strains of yeasts produce different types of wine. Wines made from other
fruits, such as apples and berries, are normally named after the fruit from which they are
produced (for example, apple wine orelderberry wine) and are generically called fruit wine or
country wine. During fermentation, yeast interacts with sugars in the juice to create ethanol,
commonly known as ethyl alcohol, and carbon dioxide (as a by-product). In winemaking, the
temperature and speed of fermentation are important considerations as well as the levels of
oxygen present in the must at the start of the fermentation. In winemaking, there are different
processes that fall under the title of “Fermentation” but might not follow the same procedure
commonly associated with wine fermentation.

Grape wine is perhaps the most common fruit juice alcohol. Because of the commercialisation of
the product for industry, the process is well known and documented.

The production of grape wine is quite straight forward and can be carried out at the small-scale,
without the need for very expensive or specialised equipment. It does, however, require a basic
understanding of the processes involved, tightly controlled fermentation conditions to ensure a
high quality product and a strict adherence to cleanliness and hygiene to prevent contamination
of the wine by spoilage bacteria.

Essentially, wine production involves the following basic steps;

 crushing the grapes to extract the juice

 alcoholic fermentation
 bulk storage and maturation of the wine in a cellar
 clarification and packaging.
There are really two distinctive types of wine made from grapes – red wine and white wine. The
main difference in the two types is the variety of grape used as raw material and the removal of
grape skins in the production of white wine. Grapes contain a number of chemical compounds
that all contribute to the flavour and colour of wine. Tannins are one group of compounds that
give the wine a bitterness and astringency. The tannins are found in the grape skins, therefore
red wines tend to be more astringent than white wines.

Principles of winemaking
Wine making uses the following basic principles:
  The sugars present in the fruit (and any sugar that is added to the fruit) are fermented by
yeast that is added to the mixture. There are natural yeasts present on the skins of fruits,
but these are usually not sufficient to carry out the fermentation on their own.
 When sugar is fermented by yeast, it is converted into alcohol (ethanol) and carbon
dioxide gas is released.
 The fermentation has to take place without oxygen (it is an anaerobic fermentation). If
oxygen gets into the system during the fermentation, the alcohol will be converted into
acid (this is what happens when you make vinegar, which is acetic acid). Wine that has
spoiled because it has been exposed to the air may taste very acidic.
 There are lots of bacteria and yeasts around in the air and on the surface of the fruits.
They all have the potential to spoil the wine. It is extremely important that these bacteria
do not start to grow in the fermenting grape juice. Particular care must be taken with the
cleanliness of the equipment and personal hygiene.
 All equipment must be sterilized with a solution of sodium or potassium metabisulphite
before it is used
Requirements
1. Reagents/Supplies
2. Fruits (apple or grapes)
3. Potassium metabisulphite
4. S. cerevesiae culture
Procedure
Production of red grape wine

Red grape wine is an alcoholic fruit drink of between 10 and 14% alcoholic strength that is made
from grapes. The colour ranges from a light red to a deep dark red depending on the grape
variety and the length of fermentation and maturation. The skins of the grape are included in the
production of red wine, to allow for the extraction of colour and tannins, which contribute to the
flavour.

Production of white grape wine

White grape wine is an alcoholic fruit drink of between 10 and 14% alcoholic strength. White
wine has a pale yellow colour. The skins are removed from the grapes before fermentation
begins.

The fermentation process is very similar for both types of wine:

Raw material preparation

Select healthy, ripe, undamaged grapes. The fruit should taste sweet, ripe and slightly tart. Make
sure they are ripe by squashing two handfuls, straining the juice and measuring the sugar level
with a refractometer if you have one available. The total soluble sugars should be about 22° Brix,
which is equivalent to a specific gravity of 1.0982 or 11% potential alcohol. Remove the grapes
from the stems (stems make the wine taste bitter). Discard any that are rotten or unripe. Wash
them well in clean water to remove dust. Crush the grapes to yield the juice plus skins, which is
known as must. Traditionally grapes are crushed in large open vessels by people walking on
them with bare feet. This really is not very hygienic and is not recommended. It is preferable to
use a sterilised potato masher or very clean hands.

Sterilise the equipment

It is essential to sterilise all the equipment before use. Wash the equipment in boiling water. Use
a solution of sodium or potassium metabisulphite to clean the fermentation vessel and the bottles
for storage. Add 3 tablespoons of potassium metabisulphite to 4.5 litres of water and mix well.
Rinse the bottles well with boiled water afterward to get rid of any residual sulphite.

Processing

Red wine

Transfer the crushed grapes plus skins to a large fermentation vessel, such as a plastic bucket
with a lid. Seal the lid, place in a warm room (21-24°C) and leave to ferment for between 24
hours and three weeks. The ethanol produced during this initial fermentation helps with the
extraction of pigment from the skins. The longer the fermentation, the darker the wine.

Remove the skins and transfer the partially fermented wine to a separate tank to complete the
fermentation. Add yeast to the fermenting grape must, close the top of the fermentation vessel
with an airlock that contains water, place in a warm place (21-24°C) and leave to ferment until all
the sugar has been converted to alcohol or the alcohol content of the wine has reached a high
enough level. You know this has happened when the bubbles stop appearing in the water in the
airlock. You can measure the specific gravity of the wine with a hydrometer. This gives an
indication of the amount of alcohol that is present.

White wine

Strain the extracted grape juice into a fermentation bucket. Add the wine yeast, seal the
fermentation vessel and leave in a warm place (12-18°C) for 7 to 14 days to ferment. The low
temperature and slow fermentation encourages the retention of volatile compounds which give
the wine flavour.

Adjusting the Juice

Controlling the acidity, sugar content and temperature of the juice (must) are all critical to
producing good quality wine. The acid content can be measured using a titration kit. The ideal
acid content is 6 to 7 grams per liter for dry reds and 6.5 to 7.5 grams per litre for dry whites. If
the acidity is to low, add tartaric acid (in very small amounts) until the acidity reaches the desired
level.

The sugar level should be about 22° Brix for both red and white wines. If it is lower than this,
increase it by adding a sugar syrup to the juice. Make the sugar syrup by dissolving one cup
sugar into one-third cup of water. Bring it to a boil in a saucepan and immediately remove from
the heat. Cool before adding in small amounts, one tablespoon at a time, until the desired
degrees Brix is reached. To lower the sugar level, simply dilute the must or juice with water.

The temperature of the must should be adjusted to provide optimum conditions for the yeast to
grow. The optimum temperature of the juice is about 22-24°C for red wines and 12-18°C for
white wines. If the juice is colder than this, warm it by gentle heating, but do not boil as this
affects the flavour of the wine.

Racking the Wine

“Racking” means transferring the fermenting wine away from the sediment at the bottom of the
bucket. Use a clear plastic tube to siphon off the wine into a sterilized fermentation jug. Do not
disturb the sediment at the base of the bucket – it is important to have a clear wine without
cloudiness or debris. Seal the top of the fermentation jug. Leave to ferment until no more carbon
dioxide gas can be seen escaping via the air lock (this means that all the available sugar has
been converted into alcohol, or the yeast has died and the fermentation is complete).
Bottling the Batch

After fermentation, the wine is bottled by siphoning it off into clean, sterilised bottles. Do not fill
the bottles to the top (leave about 5cm of head space) to allow room for fermentation in the bottle
if it happens. Insert a cork into the bottle using a hand corking machine.

Some wines can be drunk immediately, however most develop distinctive flavours and aromas
by leaving them to age for a while. The bottles should be laid on their sides during ageing to
keep the cork wet. If the cork dries out, it may allow air into the wine, which causes it to oxidize
and spoil

5.7 Preparation of Standard Curves

Principle

Many laboratory tests require the measurement of concentration be evaluated or read in a

photometer (colorimeter or spectrophotometer). Since these instruments are capable of only
measuring the amount of light being allowed to pass through the cuvette, their readout devices
display % of light transmitted or mathematically derived absorbance. One method of obtaining
concentration from % transmittance or absorbance is through the use of a standard curve. For our
purposes, standard curves are defined as a graphs with absorption or %T plotted on the Y axis, and
increasing concentrations of standard along the X axis. If Beer’s Law is followed, the resulting line
representing absorbance vs concentration will be straight. A standard curve is constructed after
obtaining the %T/Abs readings from a number of solutions of known concentration (standards) used
in a reaction or procedure. After the readings are obtained each is plotted on semi-log (%
transmittance) or linear (absorbance) paper against the corresponding concentration. If the
procedure follows Beer's Law, the points plotted will generally lie such that a straight line can be
drawn through them. The concentration of controls and other unknowns (patient samples) can be
determined by locating their %T/Abs reading on the line, then dropping an imaginary line down from
that point to intersect the concentration axis. Once the curve is drawn, a number of things must be
considered to determine its acceptability. The majority of the curve’s points should be on or close to
the line. There could be many reasons for a point not being on the line. If the standards are formed
from a series of dilutions, the accuracy of the dilutions must be suspect. Calculations of the dilutions
and spectrophotometer errores are other possibilities. Whether or not the curve passes through the
point of origin (the “0"), varies with the procedure. If Beer’s law is followed and the procedure is
linear at the lower concentrations, the curve’s line generally goes through the zero.

Uv/ Vis. Spectrophotometry

When a beam of radiation passes through matter, apportion is frequently absorbed; this process
involves a transfer of radiant energy to the system, thus electrons of atoms or molecules are excited
to higher energy levels
The quantity of the absorbed radiation by a certain species is a function of its
concentration. The relationship between energy absorption (Absorbance: A) and
the concentration (C) of the absorbing species is given by:
A= ε c l (ε = molar absorbtivity l = path length )
Components of a Spectrophotometer
SPECTROPHOTOMETRIC ANALYSIS OF ASPIRIN
The chemical name for aspirin is acetylsalicylic acid. It is an ester derivative of
salicylic acid
A colored complex is formed between aspirin and the iron (III) ion. The
intensity of the color is directly related to the concentration of aspirin present;
therefore, spectrophotometric analysis can be used. A series of solutions with
different aspirin concentrations will be prepared and complexed. The
absorbance of each solution will be measured and a calibration curve will be
constructed. Using the standard curve, the amount of aspirin in a commercial
aspirin product can be determined
First the acetylsalicylic acid is reacted with sodium hydroxide to form the
salicylate dianion. Then the addition of acidified iron(III) ion produces the
violet tetraaquosalicylatroiron (III) complex.
Procedure
Part I / STANDARD CURVE
1- Mass 100 mg of acetylsalicylic acid in a 125 mL Erlenmeyer flask. Add 10
mL of a 1 M NaOH solution to the flask and heat to boiling.
2- Quantitatively transfer the solution to a 250 mL volumetric flask and dilute
with distilled water to the mark.
3- transfer 1,2,3,4 and 5 ml of standard aspirin solution to a (10for group A and
15ml for group B) mL volumetric flask or graduated cylinder. Dilute to the 10
mL mark with buffered 0.02 M iron(III) chloride solution.
4- Measure the absorbance of each solution starting with lower concentration
first with a spectrophotometer set at 530 nm. Use the iron (III) solution as a
blank. Record the results on the data sheet.

5- Draw the standard curve using excel program on your computer or ordinary
sheet
Part II: Making a Solution of Unknown Concentration from a Tablet.
1. Place one aspirin tablet (record the brand and mg of ASA as indicated on the
bottle) in a 125 mL flask. Add 10 mL of 1 M NaOH solution to the flask, and
heat until the contents begin to boil and the entire tablet has dissolved.
2. Quantitatively transfer the solution to a 250 mL volumetric flask, and dilute
with distilled water to the mark.
3. Pipet a 1.00 mL sample of this aspirin tablet solution to a 10 mL volumetric
flask. Dilute to the mark with a 0.02 M Fe3+ solution. Label this solution
"unknown,"
Note : 7g FeCl3 in 2 L H2O

5.8 DATA INTERPRETATION

Data interpretation is the process of reviewing data through some predefined
processes which will help assign some meaning to the data and arrive at a
relevant conclusion. It involves taking the result of data analysis, making
inferences on the relations studied, and using them to conclude.
Therefore, before one can talk about interpreting data, they need to be
analyzed first. What then, is data analysis?
Data analysis is the process of ordering, categorizing, manipulating, and
summarizing data to obtain answers to research questions. It is usually the
first step taken towards data interpretation.
It is evident that the interpretation of data is very important, and as such
needs to be done properly. Therefore, researchers have identified some data
interpretation methods to aid this process.
Data Interpretation Methods?
Data interpretation methods are how analysts help people make sense
of numerical data that has been collected, analyzed and presented. Data,
when collected in raw form, may be difficult for the layman to understand,
which is why analysts need to break down the information gathered so that
others can make sense of it.
For example, when founders are pitching to potential investors, they must
interpret data (e.g. market size, growth rate, etc.) for better understanding.
There are 2 main methods in which this can be done, namely; quantitative
methods and qualitative methods.
Qualitative Data Interpretation Method
The qualitative data interpretation method is used to analyze qualitative data,
which is also known as categorical data. This method uses texts, rather than
numbers or patterns to describe data.
Qualitative data is usually gathered using a wide variety of person-to-person
techniques, which may be difficult to analyze compared to the quantitative
research method.
Unlike the quantitative data which can be analyzed directly after it has been
collected and sorted, qualitative data needs to first be coded into numbers
before it can be analyzed. This is because texts are usually cumbersome, and
will take more time and result in a lot of errors if analyzed in its original
state. Coding done by the analyst should also be documented so that it can be
reused by others and also analyzed.
There are 2 main types of qualitative data, namely; nominal and ordinal data.
These 2 data types are both interpreted using the same method, but ordinal
data interpretation is quite easier than that of nominal data.
In most cases, ordinal data is usually labelled with numbers during the
process of data collection, and coding may not be required. This is different
from nominal data that still needs to be coded for proper interpretation.
Quantitative Data Interpretation Method
The quantitative data interpretation method is used to analyze quantitative
data, which is also known as numerical data. This data type contains numbers
and is therefore analyzed with the use of numbers and not texts.
Quantitative data are of 2 main types, namely; discrete and continuous data.
Continuous data is further divided into interval data and ratio data, with all
the data types being numeric.
Due to its natural existence as a number, analysts do not need to employ the
coding technique on quantitative data before it is analyzed. The process of
analyzing quantitative data involves statistical modelling techniques such as
standard deviation, mean and median.
Some of the statistical methods used in analyzing quantitative data are
highlighted below:
 Mean
The mean is a numerical average for a set of data and is calculated by
dividing the sum of the values by the number of values in a dataset. It is used
to get an estimate of a large population from the dataset obtained from a
sample of the population.
For example, online job boards in the US use the data collected from a group
of registered users to estimate the salary paid to people of a particular
profession. The estimate is usually made using the average salary submitted
on their platform for each profession.
 Standard deviation
This technique is used to measure how well the responses align with or
deviates from the mean. It describes the degree of consistency within the
responses; together with the mean, it provides insight into data sets.
In the job board example highlighted above, if the average salary of writers
in the US is $20,000 per annum, and the standard deviation is 5.0, we can
easily deduce that the salaries for the professionals are far away from each
other. This will birth other questions like why the salaries deviate from each
other that much.
With this question, we may conclude that the sample contains people with
few years of experience, which translates to a lower salary and people with
many years of experience, translating to a higher salary. However, it does not
contain people with mid-level experience.
 Frequency distribution
This technique is used to assess the demography of the respondents or the
number of times a particular response appears in research. It is extremely
keen on determining the degree of intersection between data points.
Some other interpretation processes of quantitative data include:
  Regression analysis
 Cohort analysis
 Predictive and prescriptive analysis

6. FOOD
INDUSTR
IES –
QUALITY
ANALYSI
S AND
CONTRO
L SKILLS
Introduction of food industries, General and safety rules for working
in Lab, Overview of adulteration and its disadvantages according to
FSSAI guidelines, Demonstration of instruments, Micropipette
handling (forward and reverse pipetting techniques), Principle-SOP
and application of hot air oven, Determination of moisture and ash
value, Quality and adulteration analysis of different brands of milk,
Overview of gluten, Presence of gluten in different brands of flours,
Difference between artificial and natural coloring agents, Detection
of Artificial coloring agent in vegetables, Presence of synthetic and
natural caramel in chocolates and candies via Seliwanoff’s test,
Determination of calcium, iron & Reducing sugar in different food
products, Extraction and analysis of caffeine from tea/coffee,
Introduction of chicory, Determination of chicory in coffee, Different
adulterants in red chilli powder, Adulteration analysis in red chilli
powder, Quality analysis of turmeric powder.

6.1 INTRODUCTION TO FOOD INDUSTRIES

The food industry is a complex, global network of diverse businesses that supplies most of
the food consumed by the world's population. The term food industries covers a series of
industrial activities directed at the production, distribution, processing, conversion, preparation,
preservation, transport, certification and packaging of foodstuffs. The food industry today has
become highly diversified, with manufacturing ranging from small, traditional, family-run activities
that are highly labor-intensive, to large, capital-intensive and highly mechanized industrial
processes. Many food industries depend almost entirely on local agriculture, produce, or fishing.[1]
It is challenging to find an inclusive way to cover all aspects of food production and sale. The
UK Food Standards Agency describes it as "the whole food industry – from farming and food
production, packaging and distribution, to retail and catering."[2] The Economic Research
Service of the USDA uses the term food system to describe the same thing, stating: "The U.S.
food system is a complex network of farmers and the industries that link to them. Those links
include makers of farm equipment and chemicals as well as firms that provide services to
agribusinesses, such as providers of transportation and financial services. The system also
includes the food marketing industries that link farms to consumers, and which include food and
fiber processors, wholesalers, retailers, and foodservice establishments."[3] The food industry
includes:
 Agriculture: raising crops, livestock, and seafood. Agricultural economics.
 Manufacturing: agrichemicals, agricultural construction, farm machinery and supplies, seed,
etc.
 Food processing: preparation of fresh products for market, and manufacture of prepared
food products
 Marketing: promotion of generic products (e.g., milk board), new products, advertising,
marketing campaigns, packaging, public relations, etc.
 Wholesale and food distribution: logistics, transportation, warehousing
 Foodservice (which includes catering)
 Grocery, farmers' markets, public markets and other retailing
 Regulation: local, regional, national, and international rules and regulations for food
production and sale, including food quality, food security, food safety, marketing/advertising,
and industry lobbying activities
 Education: academic, consultancy, vocational
 Research and development: food science, food microbiology, food technology, food
chemistry, and food engineering
 Financial services: credit, insurance
Areas of research such as food grading, food preservation, food rheology, food storage directly
deal with the quality and maintenance of quality overlapping many of the above processes.
Only subsistence farmers, those who survive on what they grow, and hunter-gatherers can be
considered outside the scope of the modern food industry.
The dominant companies in the food industry have sometimes been referred to as Big Food, a
term coined by the writer Neil Hamilton.

Food production

A soybean field in Argentina

Most food produced for the food industry comes from commodity crops using conventional
agricultural practices. Agriculture is the process of producing food, feeding products, fiber and
other desired products by the cultivation of certain plants and the raising of domesticated animals
(livestock). On average, 83% of the food consumed by humans is produced using terrestrial
agriculture.[8] Other food sources include aquaculture and fishing.[8]
Scientists, inventors, and others devoted to improving farming methods and implements are also
said to be engaged in agriculture. One in three people worldwide are employed in agriculture,
[9]
yet it only contributes 3% to global GDP.[10] In 2017, on average, agriculture contributes 4% of
national GDPs.[8] Global agricultural production is responsible for between 14 and 28% of global
greenhouse gas emissions, making it one of the largest contributors to global warming, in large
part due to conventional agricultural practices, including nitrogen fertilizers and poor land
management.[8]
Agronomy is the science and technology of producing and using plants for food, fuel, fibre,
and land reclamation. Agronomy encompasses work in the areas of plant genetics, plant
physiology, meteorology, and soil science. Agronomy is the application of a combination of
sciences. Agronomists today are involved with many issues including producing food, creating
healthier food, managing the environmental impact of agriculture, and extracting energy from
plants.[11]
Food processing

Packaged meat in a supermarket.

Food processing includes the methods and techniques used to transform raw ingredients into
food for human consumption. Food processing takes clean, harvested or slaughtered and
butchered components and uses them to produce marketable food products. There are several
different ways in which food can be produced.
One-off production: This method is used when customers make an order for something to be
made to their own specifications, for example, a wedding cake. The making of one-off products
could take days depending on how intricate the design is.
Batch production: This method is used when the size of the market for a product is not clear, and
where there is a range within a product line. A certain number of the same goods will be
produced to make up a batch or run, for example a bakery may bake a limited number
of cupcakes. This method involves estimating consumer demand.
Mass production: This method is used when there is a mass market for a large number of
identical products, for example chocolate bars, ready meals and canned food. The product
passes from one stage of production to another along a production line.
Just-in-time (JIT) (production): This method of production is mainly used in restaurants. All
components of the product are available in-house and the customer chooses what they want in
the product. It is then prepared in a kitchen, or in front of the buyer as in sandwich
delicatessens, pizzerias, and sushi bars.

Industry influence
The food industry has a large influence on consumerism. Organizations, such as The American
Academy of Family Physicians (AAFP), have been criticized for accepting monetary donations
from companies within the food industry, such as Coca-Cola.[12] These donations have been
criticized for creating a conflict of interest and favoring an interest such as financial gains

6.2 DETERMINATION OF MOISTURE

AND ASH VALUE
Abstract:

Wheat flour is widely used on an industrial scale in baked goods, pasta, food concentrates, and
confectionaries. Ash content and moisture can serve as important indicators of the wheat flour’s
quality and use, but the routinely applied assessment methods are laborious. Partial least squares
regression models, obtained using Raman spectra of flour samples and the results of reference
gravimetric analysis, allow for fast and reliable determination of ash and moisture in wheat flour,
with relative standard errors of prediction of the order of 2%. Analogous calibration models that
enable quantification of carbon, oxygen, sulfur, and nitrogen, and hence protein, in the analyzed
flours, with relative standard errors of prediction equal to 0.1, 0.3, 3.3, and 1.4%, respectively, were
built combining the results of elemental analysis and Raman spectra. Keywords: wheat flour; ash;
moisture; protein; elemental analysis; multivariate analysis

1. Introduction

Wheat flour is the most important product of wheat milling. It is used on an industrial scale in
baking and in producing confectionaries, pasta and food concentrate. Ash is one of the major
indicators of wheat flour’s quality and use [1,2]. The ash obtained from flours consists of mineral
compounds of phosphorous, potassium, calcium, magnesium, iron, zinc, and copper. Phosphorus
(approximately 45%), potassium (approximately 38%), magnesium, and calcium (approximately 13%
and 3%, respectively) are the main elements present in ash, while the other elements amount to
only 1% [3,4]. The whole wheat grain contains 1.17–2.96% of the mineral constituents [5]. This
variation is caused by the genotype, wheat class and cultivar as well as the growing location and year
[4]. Minerals in the kernel are distributed unevenly. The aleurone layer and pericarp contain
approximately 68%, the starch endosperm 20%, and the embryo 12% of the total minerals [6]. Flour
characterized by a higher ash level is usually less purified and contains more particles of fine bran
and endosperm adjacent to the bran. Therefore, ash is a widely used index of flour purity and its
extraction rate during milling [4]. However, it should be noted that some wheat types, e.g., durum
wheat, naturally have a higher level of endosperm ash due to genetic factors and soil conditions.
From a nutritional point of view, an increase in the ash content in flour combined with an increase in
the content of dietary fiber, vitamins, and non-gluten proteins is desirable [7]. However, the
technical quality of high-ash flour is lower because it is characterized by a darker color and greater
activity of proteolytic and amylolytic enzymes. Dietary fiber and non-gluten proteins disintegrate and
weaken the protein matrix during dough formation [2,8]. Therefore, the ash content in flour is an
important parameter in the assessment of flour quality. Measurement of the ash content is routinely
performed using a standard ash analysis method in which the sample is burned at 550 ◦C for soft
wheat flours and 575–590 ◦C for hard wheat flours. Incinerating is carried out until light gray ash is
obtained or until a constant weight is reached. The time of this determination is long and varies from
5 to 7 h. In an industrial practice, this method is not frequently used because the time needed is too
long and does not allow the wheat flour’s quality to be verified effectively [9,10]. Numerous
instrumental techniques have been proposed for ash and moisture analysis in different types of flour
samples. Undoubtedly the most important and often applied in an industrial practice, is near-
infrared spectroscopy (NIR) [11]. Other techniques include ATR (attenuated total reflection), infrared
transmission and laser-induced breakdown spectroscopy [9,12–14] In this report, we present a new,
fast, and reliable Raman spectroscopic method for ash, moisture, and protein quantification in
wheat milling fractions. Portable Raman spectrometers, widely available now, can be used to adapt
this type of analysis in production facilities. Raman spectroscopy is a versatile analytical method that
delivers unique information about molecules on the basis of their oscillations. It enables qualitative
and quantitative analysis of different compounds present in the studied samples, and it is much
more specific than NIR spectroscopy [15].

2. Materials and Methods

2.1. Milling Process

 Flours obtained from milling 15 different cultivars of common wheat (Triticum aestivum) were used.
Wheat grain (ten spring wheat genotypes cv. Kamelia, Katoda, Monsun, Narwa, Ostka Smolicka,
Raweta, Goplana, Tybalt, Fala, Kandela and five winter genotyps cv. Bogatka, Smuga, Pokusa,
Tonacja, Skagen) was obtained from the Research Centre for Cultivar Testing COBORU (Cicibór Du
˙zy, Poland). All wheat samples were cleaned using mechanical dockage testers with air cyclones to
remove impurities before milling. After cleaning, all samples were tempered to 15% moisture and
conditioned for 24 h. After conditioning, the wheat samples’ moisture content was determined again
to confirm the desired level. A second tempering event, up to 16.5% moisture, was carried out 20
min before the milling process. It was applied to toughen the skin so it would resist powdering
during milling. Milling was performed using three-roller laboratory Sadkiewicz Instruments mill
(Figure S1 in Supplementary Materials). Four samples of stream flour (A, B, C, D), two bran fractions
and one shorts fraction were collected for each wheat cultivar (Figure S1 and Table S1 in
Supplementary Materials). To diversify the ash content, some of the samples were prepared by
mixing, in different proportions, flours and bran obtained from the same wheat cultivar (Table S2 in
Supplementary Materials). The granulation of samples was maintained to be smaller than 160 µm.

2.2. Moisture and Ash Analysis Moisture analysis was performed according to American Association
of Cereal Chemists (AACC) method 44-15A [16]. The flour samples (3 g) were measured into glass
weighing bottles and placed in a laboratory dryer for 3 h. The samples were dried at 105 ◦C to
constant weight. After cooling, the samples were weighed, and the moisture contents were
calculated (Table S2 in Supplementary Materials). The ash content was determined using AACC
method 08–01 [17]. The flour samples were measured into ash dishes in amounts of 3–5 g. Then
samples were placed in a muffle furnace at 550 ◦C. They were incinerated until light gray ash or
constant weight was obtained (7 h). After cooling, the samples were weighed, and the ash contents
were calculated (Table S2 in Supplementary Materials). Moisture and ash analysis was performed in
triplicate. The obtained data were used to calculate mean values and standard deviations.

2.3. Elemental Analysis Elemental analysis was performed using an Elementar Vario EL Cube CHNS
combustion analyzer with a thermal conductivity detector. Samples weighing 10 mg were collected
from the analyzed flours in duplicate. Nitrogen, carbon, hydrogen, and sulfur content was
determined, while oxygen content was calculated as the difference between the total weight and
the other elements’ content.

2.4. Raman Spectra Raman spectra were recorded using a Thermo Scientific iS50 Raman Module
equipped with an InGaAs detector and CaF2 beamsplitter. Samples in the form of pellets were
placed on an XYZ motorized stage. The spectra were excited using an Nd:YAG laser operating at 1064
nm, with power at the sample equal to 150 mW. Backscattered radiation was collected. Due to the
samples’ inhomogeneity, the spectra were recorded from 16 different points. For each of them,
interferograms were averaged over eight scans, and Happ-Genzel apodized and Fourier transformed
using a zero-filling factor of 2 to yield spectra in the 200–3700 cm−1 range at a resolution of 8 cm−1 .
Finally, the spectra were averaged.

2.5. Data Analysis Partial least squares (PLS) models were built using Turbo Quant Analyst version 9
chemometrics software, which utilizes the nonlinear iterative partial least squares (NIPALS)
algorithm [18]. A cross-validation procedure using the leave-two-out technique was performed to
estimate the performance of the models. To determine the predictive abilities of the obtained
models, relative standard errors of prediction were calculated for calibration and validation samples,
according to the procedure described elsewhere [19]. The root means square error of cross-
validation (RMSECV) was calculated to select the optimal number of PLS factors. Regression models
were constructed combining MSC (multiplicative scatter correction) corrected spectra and the
results of reference gravimetric and elemental analysis.

3. Results and Discussion

In Figure 1, the Raman spectra are presented for the five types of wheat flours with different ash
content that were analyzed; they are labeled according to PN-A-74022:2003. The broad band with a
maximum of around 3320 cm−1 corresponds to the O-H and N-H stretching bands. The massive band
in the 2800–3000 cm−1 range consists of the C-H stretching vibration bands of the various flour
ingredients. The bands with maxima located at 1630, 1530, and 1235 cm−1 are related to the I, II
and III amide vibration modes. Their intensity increases with the ash content. The bands with
maxima at 1460, 1385, 1339, 1128, and 1082 cm−1 can be assigned to different deformation modes
of CH, COC, and CCC moieties in starch and dietary fiber. An intense, characteristic band of COC
deformation vibration of starch molecules is observed at 479 cm−1 [20]. The fluorescent background
is more pronounced for the samples with higher ash content.

Forty-nine samples, characterized by an ash content in the 0.5–2.5% range and moisture content in
the 7.6–14.3% range, were used for training purposes, while 12 were randomly selected for
validation of the obtained models and six were omitted (Table S2 in Supplementary Materials). For
ash modeling, 975–1790 and 2770–3580 cm−1 spectral ranges were applied. In the case of moisture
analysis, slightly different regions were utilized (814–1770 and 2850–3696 cm−1 ). Regression
coefficients plots for ash and moisture are presented in Figure S2 in Supplementary Materials. In
these plots, characteristic spectral features mentioned above, can be easily identified. In the case of
ash analysis, they include ν(CH), ν(C-C), and ν(C-O) vibrations together with amide I, II and III modes,
and deformation motions of CH, COC, and CCC moieties. For moisture, except for ν(OH) and δ(OH)
bands, contributions from different functional groups forming hydrogen bonds with water molecules
can be recognized. The number of latent variables, determined from the RMSECV plots, was set to
four for both analytes. The prediction plots and regression residuals for ash and moisture
determination based on the Raman spectra are shown in Figure 2. The detailed model parameters
are collected in Table 1. It follows from the presented results that the quantification errors of ash
and moisture determination for wheat flour samples are of the order of 2%. What is important is
that both analytes were determined for each sample from its Raman spectrum collected in 2 min.

The model elaborated for ash determination can be easily modified for ash content up to 5% by
incorporation of the data for the whole wheat flour samples (Tables S2 and S3 and Figure S3 in
Supplementary Materials). The established amounts of carbon, hydrogen, nitrogen, oxygen and
sulfur in the studied flours varied in the 40.9–42.6, 6.0–7.2, 1.9–3.0, 47.8–50.2, and 0.07–0.14%
(w/w) ranges, respectively. The quantification error, expressed as the relative range, was 2–3 times
higher for hydrogen quantification than for the other elements, except for sulfur. Detailed
information on the results of this analysis is presented in Table S4 in Supplementary Materials. By
combining spectra and the elemental analysis results, PLS calibration models that enabled the
determination of N, C, S, and O content in the analyzed flours based on their Raman spectra were
obtained. The parameters of these models are gathered in Table 2, while the prediction plots and
residual errors are presented in Figures S4–S7 in Supplementary Materials. We were unable to
construct a reliable model for hydrogen quantification. The protein amount in the analyzed flours
can be determined by multiplying the nitrogen content by a factor of 5.52 [21] (Figure 3).

4. Conclusions
FT-Raman spectroscopy, combined with chemometric methods, appears to be an attractive tool for
accurate and effective analysis of wheat flour samples. It enables quantitative determination of ash,
moisture, and protein content in the analyzed samples and is much faster than the methods
commonly used now. Supplementary Materials: The following are available online at
[Link] Table S1: Ash and moisture content in the products
of milling; in % (w/w), Table S2: Ash and moisture content in the analyzed samples; in % (w/w), Table
S3: Calibration parameters of the PLS model for ash quantification in the 0.5–5% range, Table S4:
Elemental composition of the analyzed flours; in % (w/w), Figure S1 Mill flow for common wheat
(Triticum aestivum) using the Sadkiewicz Laboratory mills, Figure S2: Regression coefficients plots for
PLS modeling of ash, moisture and protein, Figure S3: Prediction plot and regression residuals for ash
quantification in the 0.5–5% range based on Raman spectra, Figure S4: Prediction plot and
regression residuals for nitrogen quantification based on Raman spectra, Figure S5: Prediction plot
and regression residuals for carbon quantification based on Raman spectra, Figure S6: Prediction
plot and regression residuals for sulfur quantification based on Raman spectra, Figure S7: Prediction
plot and regression residuals for oxygen quantification based on Raman spectra.

6.3 OVERVIEW OF GLUTEN

Gluten is a protein that appears in foods processed from wheat and other cereal grains,
including barley and rye. It gives elasticity to dough, helping it to rise and keep its shape, and
often giving the final product a chewy texture.
Gluten, when dried and milled to powder and added to ordinary flour dough, improves rising
and increases the bread's structural stability and chewiness. On digestion, the 33-amino
acid gluten protein breaks down into smaller peptide units. There are two main groups of
proteins in gluten, called the gliadins and glutenins.
The demand for cereal grain products labeled as ‘gluten free’ has grown over the past few
years. One cause for the demand is the increase of people diagnosed with celiac disease.
Celiac disease, also known as gluten-sensitive enteropathy, nontropical sprue, and celiac
sprue, is a genetic disorder in which people are predisposed to an autoimmune response
that causes damage to the small intestine when gluten proteins are contained in certain
foods. Approximately three million people, 1% of the population, have been diagnosed with
celiac disease.
Australia, Europe, and the USA have diagnosed celiac disease. The Food and Drug
Administration (FDA) has proposed a gluten-free labeling rule for voluntary use in foods. The
current standard proposed by FDA is that food mostly contain no more than 20 ppm to claim
a gluten-free status. A gluten-free label means that the food being produced does not
contain any species of prohibited grains such as, wheat, rye, barley, or a crossbred hybrid.
In addition, it does not contain any ingredients that is derived from prohibited grains and has
not been processed to remove gluten.
Manufactures that want to make ‘gluten free’ claims need to view gluten in the same manner
as an allergen. Companies must design policies and systems within their facilities based on
the presence or absence of gluten-containing products. Facilities that are not completely free
of gluten-containing products increase the chance of not meeting the proposed labeling rule.
However, systems can be designed to have the needed segregations and sanitation
procedures. Plants must have segregated areas for receiving raw ingredients and develop
systems to avoid cross-contamination. Processing equipment must be properly sanitized to
ensure the absence of the gluten protein.
Gluten-free products may be made of naturally gluten-free cereals such as corn, rice,
sorghum, and millet as well as pseudocereals such as buckwheat, amaranth, and quinoa.
Additionally, gluten-containing raw materials from wheat, rye, or barley may be rendered
gluten-free through specialized processing such as extensive washing for
starch, peptidase treatment for beverages, or use of gluten-deficient strains. Improving
textural and flavor attributes, especially of breads and beer, is still a challenge, although
considerable progress has been achieved also in improving nutritional value. According to
Codex Alimentarius Standard 118-1979, the gluten level in gluten-free products must not
exceed 20 mg/kg. With the exception of Australia and New Zealand, food labeling legislation
in the European Union, Canada, and the United States largely follows the Codex. The
Crossed Grain symbol is internationally recognized to identify products for celiac disease
(CD) patients. To guarantee the safety of gluten-free products, several analytical methods
for gluten quantitation have been developed. After an appropriate extraction of gluten
proteins from the food matrix, enzyme-linked immunosorbent assays (ELISA) based on
specific antibodies are most widely used for quantitation of gluten. Nonimmunochemical
methods such as polymerase chain reaction (PCR), column chromatography, or mass
spectrometry of gluten peptides as specific markers are promising alternatives. However, an
accurate quantitation of gluten in a variety of food matrices still poses a challenge owing to
the complexity of gluten as the target analyte, and appropriate reference materials to
calibrate the analytical methods are urgently needed.

6.4 Difference Between Artificial And

Natural Coloring Agents
A color is deemed natural if its origin is vegetal, microbiological,
animal or mineral. Whereas, artificial colors were created in labs
(and sometimes accidentally) by chemists. From a chemical
point of view it is the chromophore which is responsible for the
color and occurs when a molecule has lots of conjugated
double bonds and the energy difference between two different
molecular orbitals falls within the range of light which we can
visualize.

6.5 Extraction And Analysis Of

Caffeine From Tea/Coffee
Abstract:

Caffeine is a chemical found in coffee, tea, cola, guarana, mate, and other [Link] is one of
the most commonly used stimulants among athletes. Taking caffeine, within limits, is allowed by the
National Collegiate Athletic Association (NCAA). Urine concentrations over 15 mg/mL are prohibited.
It takes most people about 8 cups of coffee providing 100 mg/cup to reach this urine concentration.
The aim of this study is to determine the concentration of caffeine in reputed tea types and coffee.
The Technique used here is Liquid-Liquid Extraction to extract caffeine. The Study also focused
whether we can extract a significant amount of caffeine using different extracting solvents and
different bases.

1. INTRODUCTION

Caffeine is a naturally occurring chemical stimulant found in the leaves, seeds and fruits of a
numerous plant species of a group of compounds called trimethylxanthine. Its chemical formula is
C8 H10 N4 O2. Caffeine is most commonly used to improve mental alertness, but it has many other
uses. Caffeine is used by mouth or rectally in combination with painkillers (such as aspirin and
acetaminophen) and a chemical called ergotamine for treating migraine headaches. It is also used
with painkillers for simple headaches and preventing and treating headaches after epidural
anesthesia.

[Link] of Caffeine

 Systematic name:1,3,7-trimethyl-1H-purine- 2,6(3H,7H)-Dione

 Other name: 1,3,7-trimethylxanthine & 1,3,7-trimethyl-2,6-dioxopurine

 Molecular formula: C8H10N4O2

 Molecular mass: 194.19 g/mole

 Melting point: 238°C

 Solubility in water: slightly soluble

[Link] Benefits of Caffeine

 Research indicates that caffeine may help protect human brain cells, which lowers the risk of
developing some diseases, such as Parkinson’s.

 Regular cups of coffee may stimulate the gallbladder and reduce the risk of gallstones.

 Caffeine causes the blood vessels to constrict, which may help relieve some headache pain.

 Coffee reduces inflammation and may help prevent certain heart related illnesses.

 Treats Migraine.

 Relieves Asthma Attack

 Increases the potency of analgesics.

 Caffeine is also used for weight loss and type 2 diabetes

Very high doses are used, often in combination with ephedrine, as an alternative to illegal
stimulants.
Caffeine creams are applied to the skin to reduce redness and itching in dermatitis.

1.3. Adverse Effects of Caffeine

 There is a significant association between drinking caffeinated coffee and the decrease of bone
mineral density, which leads to osteoporosis.
 The daily consumption of caffeinated drinks can increase blood sugar levels and cause problems for
people with diabetes.
 Caffeine is a diuretic and can cause dehydration.
 Caffeine can prevent some from falling asleep and interferes with deep sleep, which can lead to
fatigue during the day.
The level of caffeine can vary depending on what is consumed. A piece of chocolate may have as
little as five milligrams while energy drinks contain as much as 160 milligrams. Make sure to read the
labels of pain medications and diet pills as products can have levels of caffeine as high as 200
milligrams. Michigan State University Extension recommends moderate doses of caffeine, 200 to 300
milligrams per day, which is equivalent to two to four cups of brewed coffee and is considered safe
for most adults. If you are consuming more than 500 to 600 milligrams of caffeine per day, which
equals four to seven cups of coffee, you may be prone to health problems including insomnia,
nervousness, nausea or gastrointestinal problems, elevated heartbeat, headaches, etc. If you are
experiencing unusual side-effects associated with the consumption of foods with caffeine, you should
consult your physician.
1.4. How Does Caffeine Works?

Caffeine works by stimulating the central nervous system (CNS), heart, muscles, and the centers that
control blood pressure. Caffeine can raise blood pressure, but might not have this effect in people who
use it all the time. Caffeine can also act like a “water pill” that increases urine flow. But again, it may
not have this effect in people who use caffeine regularly. Also, drinking caffeine during moderate
exercise is not likely to cause dehydration.

Caffeine Content of Common Food and Drugs

Espresso 120 mg per 2 Oz

Coffee, Regular, Brewed 103 mg per cup
Instant Coffee 57mg per cup
Coffee, Decaffeinated 2 to 4 mg per cup
Tea 30-75 mg per cup
Cocoa 5-40mg per cup
Milk Chocolate 6mg per Oz
Baking Chocolate 35mg per Oz
Coca-Cola Classis 46mg per 12 Oz
Jolt Cola 72mg per 12 Oz
Anacin Bromo Seltzer Midol 32mg per pill
Excedrin Extra Strength 65mg per pill
DexatrimDietacVivarin 200mg per pill
Dristan 16mg per pill
No-Doz 100mg per pill
In table 1 the mentioned beverages and drugs are frequently used and it reveals that among them,
Espresso contains the maximum amount of caffeine as compared to other beverages and drugs. In
its pure form, caffeine is a white crystalline powder that tastes very bitter. It is medically useful to
stimulate the heart and also serves as increasing the rate of urine excretion. It is one of the most
studied ingredients in the food supply. The most commonly known sources of caffeine are coffee
and cocoa beans, guarana, and tea leaves. The amount of caffeine in food and beverage products
varies depending on the serving size, the type of product and preparation method. Tea which we
generally drink is made from the leaves of an Asian evergreen known as Camellia sinensis. The
presence of caffeine in plants helps to prevent them from insects and other herbivores with the
compound’s bitter taste and stimulating qualities. The caffeine content of tea leaves depends on the
variety and where they were grown; most tea has 3-5% by weight. The optical transition properties
of caffeine were measured in different solvents (dichloromethane, water, chloroform and ethyl
acetate). Caffeine has highest optical transitions in dichloromethane than the other solvents.
Caffeine can be extracted more at the boiling temperature than at 30°C. Caffeine had been widely
used in the food and pharma industry. The cost of extraction of caffeine from natural source is more.
Research has been taken to extract it from natural source more economically.
Table2. Caffeine Content in Tea/Coffee Sample (Extraction with water)

TEA/COFFEE SAMPLES AMOUNT OF CAFFEINE (gm)

Brook Bond Red Label 0.01
AVT 0.03
Eastern Eastea 0.02
Palat 0.04
3 Roses 0.02
Kannan Devan 0.01
Bru gold Coffee 0.68
AVT Coffee 0.62

2. MATERIALS AND METHODS

Liquid–liquid extraction (LLE) is a method to separate compounds or metal complexes, based on their
relative solubilities in two different immiscible liquids, usually water (polar) and an organic solvent
(non-polar). There is a net transfer of one or more species from one liquid into another liquid phase,
generally from aqueous to organic. The transfer is driven by chemical potential, i.e. once the transfer
is complete, the overall system of protons and electrons that make up the solutes and the solvents are
in a more stable configuration (lower free energy). The solvent that is enriched in solute(s) is called
extract. The feed solution that is depleted in solute(s) is called the raffinate. LLE is a basic technique
in chemical laboratories, where it is performed using a variety of apparatus, from separatory funnels
to countercurrent distribution equipment called as mixer settlers. This type of process is commonly
performed after a chemical reaction as part of the work-up, often including an acidic work-up.
Extraction is a method used for the separation of organic compound from a mixture of compound.
This technique selectively dissolves one or more compounds into an appropriate solvent. The solution
of these dissolved compounds is referred to as the extract. In the case of Caffeine extraction from tea
powder, the solubility of caffeine in water is 22mg/ml at 25°C, 180mg/ml at 80°C, and 670mg/ml at
100°C. Here the organic solvent Dichloromethane is used to extract caffeine from aqueous extract of
tea powder because caffeine is more soluble in dichloromethane (140mg/ml) than it is in water
(22mg/ml).The dichloromethane - caffeine mixture can then be separated on the basis of the different
densities of dichloromethane and water because dichloromethane is much denser than water and
insoluble in it. Residual water is separated from dichloromethane by drain out the dichloromethane
through separating funnel, thus dichloromethane passed through the funnel while polar solvents such
as water is still remaining in the funnel.
In the first phase of experimentation screening was carried out in order to determine the maximum
content of caffeine among black tea, green tea and coffee. The procedure was as follows: 10gm of tea,
green tea and coffee sample was taken and boiled for 15 minutes along with the addition of 6gm of
sodium carbonate which acts like a base which reacts with tannins to form sodium salts of tannins.

Next step is to filter the solution using vacuum filtration technique. The filtrate obtained is then used
for liquid-liquid extraction to extract the caffeine into a organic solvent. Dichloromethane is used as
solvent in liquid-liquid extraction because caffeine has higher solubility in Dichloromethane as
compared to other solvents. After separation of organic layer from the separating funnel it is then
kept for evaporationso as to evaporate the dichloromethane present in it. Now raw crude yellowish
caffeine is further sent to recrystallisation in order to obtain pure white caffeine. Ethanol is used for
recrystallisation as solvent.

Now after carrying out the above experiment and comparing the quantities of the caffeine obtained,
we came to the results that caffeine content is higher in coffee as compared to green tea and black
tea.

The next attempt of experimentation is to extract caffeine using different solvents and different
bases. Using coffee for further extraction procedure because caffeine content is more in coffee as
compared to green tea, black tea and coffee. First of all, keeping the base as constant i.e. sodium
carbonate and varying solvents we are going to extract caffeine from coffee. 10 gm of coffee was
boiled for 15-20 mins with sodium carbonate as base. This step is called as Solid-Liquid Extraction.
Now the next step is filtration which is carried using vacuum filtration instead of gravity filtration so
as to minimize the time required for filtration. Filtrate obtained is used for liquid-liquid extraction
using different solvents such as dichloromethane, acetone and ethanol. These solvents are not used
simultaneously. For each solvent, different liquid liquid extraction is carried out and then the
product obtained which is present in the organic layer is kept for evaporation. Then the quantity of
caffeine from each of the solvent used is compared in the results.

The final step of the experimentation is to determine what happens to the quantity and quality of
caffeine when we change the base used during solid-liquid Extraction. For this again 10 gm of coffee
is used and boiled along with the addition of sodium hydroxide as base instead of sodium carbonate.
Then again vacuum filtration is carried out in order to separate the particles of coffee beans present.
Then liquid-liquid extraction is used to separate caffeine in organic layer. Then organic layer
obtained is kept for evaporation and the product obtained is compared.

The procedure along with some snapshots are depicted as follows:

In order to extract caffeine from tea, several techniques are accompanied. First, a solid-liquid
extraction must take place in order to get the solid natural product into the liquid solvent. This can
be done by boiling tea leaves with the addition of sodium carbonate as a base. Further to separate
the tannins vacuum filtration is used.
Fig2. Solid-Liquid Extraction

Fig3. Vacuum Filtration

After Vacuum Filtration, Liquid-Liquid extraction is used to separate caffeine in organic layer.
Solvent used for solid liquid extraction is sodium carbonate whereas solvent used for liquid-liquid
extraction is Dichloromethane (CH2Cl2) (Note- Dichloromethane can irritate your skin so do not
handle Dichloromethane bare handedly).
Fig4. Liquid-Liquid Extraction

Now the next step is to keep the organic layer for evaporation of solvent which is dichloromethane.
After evaporation of solvent the left product is raw crude caffeine which is further sent to analysis.

Fig5. Crude Caffeine

Note: The sodium carbonate acts as a base - you could use sodium hydroxide instead. When you boil
tea leaves tannins dissolve in the water as well as the caffeine. If you do not use a base the tannins
will also be extracted into the solvent (i.e. methylene chloride) used in the subsequent extraction.
The base converts the tannins into their sodium salts - being ionic these salts are not soluble in
solvents like methylene chloride so remain in the aqueous layer during extraction. This allows purer
caffeine to be extracted.
After carrying out repeated extractions and using vacuum filtration we get crude white crystalline
caffeine as a product. Getting pure form of crystalline caffeine from crude caffeine, we need to carry
out recrystallisation. Recrystallisation is a fast and easy way to purify the caffeine.

The Success Of extraction involving a natural product is often expressed as percentage recovery

%Recovery= 𝐺𝑟𝑎𝑚𝑠 𝑜𝑓 𝑐𝑎𝑓𝑓𝑒𝑖𝑛𝑒 𝑅𝑒𝑐𝑜𝑣𝑒𝑟𝑒𝑑/𝐺𝑟𝑎𝑚𝑠 𝑜𝑓 𝑡𝑒𝑎 𝑙𝑒𝑎𝑣𝑒𝑠

The percentage recovery is called the purified percent recovery or crude percent recovery. The
extraction with the highest percent recovery is considered the most successful extraction.

2.1. Analysis Techniques for Caffeine

Thin Layer Chromatography (TLC)

There are different types of chromatographic methods such as paper chromatography, thin-layer
chromatography, column chromatography, gas chromatography, etc. They have the same principle:

1. Different solutes have different solubility in a solvent /different solutes have different degrees of
tendency to be dissolved in the same solvent.
2. As the solution (contains the solvent with the dissolved solutes) moves along a stationary solid
surface (a solid surface), different solutes adsorbed onto the solid surface in different extent as they
have different degree of adsorption characteristics (due to the different degrees of dissolve tendency)
3. The “less soluble” solute will be retained first, and the “more soluble” solutes will be retained
afterwards. (Note: No two substances have the same solubility and adsorption characteristics.
4. Different solutes will then be separated on the different positions of the solid surface.
5. Retention Factor (RF) of each component is calculated as follow

Rf= 𝐺𝑟𝑎𝑚𝑠 𝑜𝑓 𝑐𝑎𝑓𝑓𝑒𝑖𝑛𝑒 𝑅𝑒𝑐𝑜𝑣𝑒𝑟𝑒𝑑𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒 𝑓𝑟𝑜𝑚 𝑡ℎ𝑒 𝑏𝑎𝑠𝑒𝑙𝑖𝑛𝑒/𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦
𝑡ℎ𝑒 𝑠𝑜𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑚 𝑡ℎ𝑒 𝑏𝑎𝑠𝑒𝑙𝑖𝑛𝑒

Pure caffeine and the extract are analyzed in the same TLC plate and compare any differences of their
Rf.
 Spike Test

By adding known amount of standard caffeine in distilled water and raw coffee solution, then carry
out solvent extraction. By comparing the extraction results, we can analyze the recovery percentage of
the spiked caffeine and efficiency of solvent extraction.
 Iodometric Back Titration

Iodometric Back Titration Caffeine reacts with excess accurately known amount of iodine in acidic
environment, forming insoluble precipitate. Then the insoluble precipitate is removed by filtration.
Using titration by a standard sodium thio-sulphate solution with starch solution as indicator, we can
determine the amount of remaining iodine, and thus the amount of caffeine can be found. Here are the
chemical equations:

C8H10N4O2 + 2 I2 + KI + H2SO4 → [Link] .I4 + KHSO4 I2 + 2 Na2S2O3 → 2NaI + Na2S4O6

The Analysis Technique used here is iodometric back titration.

The procedure is as follows: Caffeine solution was prepared using sulphuric acid as an acidic
medium. Iodine was added to it and then the solution was titrated against sodium thiosulphate till
the solution becomes pale brown in color. Then starch was added as indicator after the solution
becomes pale brown. Again, the solution is titrated against sodium thiosulphate and the endpoint of
the reaction is dark blue to colorless solution.

Fig6. Before addition of starch

Before the addition of starch the color of the solution is pale brown but after the addition of starch
indicator the color of the solution changes to dark blue which then after titration with sodium
thiosulphate the color of the solution turns colorless
Fig7. After addition of starch

The solution is colorless that means the end point is reached and now calculating the amount of
unreacted iodine with caffeine we can easily calculate the amount of reacted iodine with caffeine
and hence by stoichiometry the amount of caffeine or the concentration of caffeine is determined.

Fig8. Endpoint blue to colorless

3. RESULTS

Table3. Screening Results: To determine the maximum content of caffeine in different types of beverages.
Type of Beverage Amount of SampleTaken (gm) Extracted Raw Crude
Caffeine(mg)
Green tea 10 1.6
Black Tea 10 2
Coffee 10 40

Here Solvent used was Dichloromethane and base used was sodium Carbonate for extracting
caffeinefrom above types of tea and coffee.
S. Parvathy ,Adlet Luiz and Jaya T. Vakrey in 2014 carried out the same analysis and they extracted
more amount of caffeine from black tea as compared to our experiment because they used acidified
water during solid-liquid extraction.
Amber Nawab, Quratulam Waseem, Javeria Asif, Fatima Ahmed in 2016 extracted caffeine from
black tea and they got 3% caffeine in the sample.
Muthanna [Link],Firas A. Al-Bayati in 2008 also extracted caffeine from tea and they also got
3% caffeine as the result. The variations in the caffeine content of the coffee and tea is because of
using different techniques during extraction of caffeine.
Now for further experimentation coffee was used with different solvents and bases, as the caffeine
content of coffee is much higher as compared to other types of tea and coffee.
R.R Shinde, N.H Shinde in 2017 extracted caffeine using acetone as solvent and the results were
quite satisfying i.e. they obtained 11.27% of caffeine using acetone as solvent. We got more amount
of caffeine as compared to them because we have used sodium carbonate as base during Solid-
Liquid Extraction.

4. CONCLUSION
Tea is very rich in antioxidants. It is the most widely used beverage all over the world. It also has
medicinal properties. In this study teas will be decaffeinated using dichloromethane as a solvent. This
study will be carried out to check the amount of caffeine in used tea leaves. It is acceptable that the
amount of caffeine decreased with every use. Caffeine from tea is extracted by liquid-liquid extraction
followed by recrystallization. Caffeine is the most commonly used psychoactive drug in the world. It
is a pharmacological active substance and depending on the dose, can be a mild central nervous
system stimulant. Approximately 80% of the world’s Population Consumes Caffeine on daily basis.
The purified caffeine is then analyzed by using high performance liquid chromatography or
Iodometric back titration method. The serious concern about potential use of caffeine for pathogenic
effects has made it one of the most broadly studied drugs. In the present study Caffeine content of
different tea and coffee samples were studied and it is found that the caffeine content varies from 1-
5%. The values generally agree well with literature quoted values of 2-5%. The Series of experiments
that have been conducted, we can conclude that the caffeine content of coffee is relatively high as
compared to other beverages and therefore we can also state that the caffeine is highly soluble in
Dichloromethane as compared to other solvents and also the Tannins are more soluble in sodium
carbonate as compared to other bases.
ACKNOWLEDGEMENT Authors acknowledge the immense help received from the scholars whose
articles are cited and included in the references of the manuscript. Authors are also grateful to the
project guide and the college for the immense help they have provided during experimentation.

6.6 Introduction Of Chicory

Common chicory (Cichorium intybus)[4] is a somewhat woody, perennial herbaceous plant of
the daisy family Asteraceae, usually with bright blue flowers, rarely white or pink. Many varieties
are cultivated for salad leaves, chicons (blanched buds), or roots (var. sativum), which are
baked, ground, and used as a coffee substitute and food additive. In the 21st century, inulin,
an extract from chicory root, has been used in food manufacturing as a sweetener and source
of dietary fiber.[5]
Chicory is grown as a forage crop for livestock.[6] It lives as a wild plant on roadsides in its native
Europe, and is now common in North America, China, and Australia, where it has become
widely naturalized.[7][8][9] "Chicory" is also the common name in the United States for
curly endive (Cichorium endivia); these two closely related species are often confused.[10]

Names
Common chicory is also known as blue daisy, blue dandelion, blue sailors, blue
weed, bunk, coffeeweed, cornflower, hendibeh, horseweed, ragged sailors, succory, wild
bachelor's buttons, and wild endive[11] (note: "cornflower" is commonly applied to Centaurea
cyanus). Common names for varieties of var. foliosum include endive, radicchio, radichetta,
Belgian endive, French endive, red endive, sugarloaf, and witloof (or witlof).

Description
When flowering, chicory has a tough, grooved, and more or less hairy stem. It can grow to 1.5
metres (4 ft 11 in) tall.[12] The leaves are stalked, lanceolate and unlobed; they range from 10–
32 cm (4–12+1⁄2 in) in length and 2–8 cm (3⁄4–3+1⁄4 in) wide.[12] The flower heads are 3–4 cm (1+1⁄4–
1+1⁄2 in) wide,[12] and usually light purple or lavender; it has also been described as light blue, and
rarely white or pink.[12] Of the two rows of involucral bracts, the inner is longer and erect, the outer
is shorter and spreading. It flowers from July until October.

Culinary uses
Root chicory
Root chicory (Cichorium intybus var. sativum) has long been cultivated in Europe as a coffee
substitute.[13] The roots are baked, roasted, ground, and used as an additive, especially in
the Mediterranean region (where the plant is native). As a coffee additive, it is also mixed
in Indian filter coffee, and in parts of Southeast Asia, South Africa, and the southern United
States, particularly in New Orleans. In France a mixture of 60% chicory and 40% coffee is sold
under the trade name Ricoré. It has been more widely used during economic crises such as
the Great Depression in the 1930s and during World War II in Continental Europe. Chicory,
with sugar beet and rye, was used as an ingredient of the East German Mischkaffee (mixed
coffee), introduced during the "East German coffee crisis" of 1976–79. It is also added to coffee
in Spanish, Greek, Turkish, Syrian, Lebanese and Palestinian cuisines.[14]
Some beer brewers use roasted chicory to add flavor to stouts (commonly expected to have a
coffee-like flavor). Others have added it to strong blond Belgian-style ales, to augment the hops,
making a witlofbier, from the Dutch name for the plant.
The roots can also be cooked like parsnips.[15]

Leaf chicory
Wild
While edible raw, wild chicory leaves usually have a bitter taste, especially the older leaves.
[16]
The flavor is appreciated in certain cuisines, such as in the Ligurian and Apulian regions
of Italy and also in the southern part of India. In Ligurian cuisine, wild chicory leaves are an
ingredient of preboggion and in Greek cuisine of horta; in the Apulian region, wild chicory leaves
are combined with fava bean puree in the traditional local dish fave e cicorie selvatiche.
[17]
In Albania, the leaves are used as a spinach substitute, mainly served simmered and
marinated in olive oil, or as ingredient for fillings of byrek.[citation needed]
By cooking and discarding the water, the bitterness is reduced, after which the chicory leaves
may be sautéed with garlic, anchovies, and other ingredients. In this form, the resulting greens
might be combined with pasta[18] or accompany meat dishes.[19]
Cultivated
Chicory may be cultivated for its leaves, usually eaten raw as salad leaves. Cultivated chicory is
generally divided into three types, of which there are many varieties:[20]
 Radicchio usually has variegated red or red and green leaves. Some only refer to the white-
veined red-leaved type as radicchio, also known as red endive and red chicory. It has a bitter
and spicy taste, which mellows when it is grilled or roasted. It can also be used to add color
and zest to salads. It is largely used in Italy in different varieties, the most famous being the
ones from Treviso (known as radicchio rosso di Treviso),[21][22] from Verona (radicchio di
Verona), and Chioggia (radicchio di Chioggia), which are classified as an IGP.[23] It is also
common in Greece, where it is known as radiki and mainly boiled in salads, and can be used
in pies.[citation needed]

Witloof, Belgian endive

 Belgian endive is known in Dutch as witloof or witlof ("white leaf"), and also as witloof in the
United States,[24] indivia in Italy, endivias in Spain, chicory in the UK, as witlof in
Australia, endive in France, and chicon in parts of northern France, in Wallonia and (in
French) in Luxembourg. It has a small head of cream-colored, bitter leaves. The harvested
root is allowed to sprout indoors in the absence of sunlight, which prevents the leaves from
turning green and opening up (etiolation). It is often sold wrapped in blue paper to protect it
from light, so to preserve its pale color and delicate flavor. The smooth, creamy white leaves
may be served stuffed, baked, boiled, cut and cooked in a milk sauce, or simply cut raw. The
tender leaves are slightly bitter; the whiter the leaf, the less bitter the taste. The harder inner
part of the stem at the bottom of the head can be cut out before cooking to prevent
bitterness. Belgium exports chicon/witloof to over 40 countries. The technique for
growing blanched endives was accidentally discovered in the 1850s at the Botanical Garden
of Brussels in Saint-Josse-ten-Noode, Belgium.[25] Today France is the largest producer of
endive.[26]
 Catalogna chicory (Cichorium intybus var. foliosum), also known as puntarelle, includes a
whole subfamily (some varieties from Belgian endive and some from radicchio)[27] of chicory
and is used throughout Italy.

Leaves unlobed and pointed

Inflorescences of a blue-flowered form, showing the two rows of bracts

Although leaf chicory is often called "endive", true endive (Cichorium endivia) is a different
species in the genus, distinct from Belgian endive.

Chicory root and inulin

Around 1970, it was found that the root contains up to 20% inulin, a polysaccharide similar
to starch. Inulin is mainly found in the plant family Asteraceae as a storage carbohydrate (for
example Jerusalem artichoke, dahlia, yacon, etc.). It is used as a sweetener in the food industry
with a sweetening power 10% that of sucrose[28] and is sometimes added to yogurts as a
'prebiotic'.[29]
Fresh chicory root may contain between 13 and 23% inulin, by total weight.[30]

Nutrition
Raw chicory leaves are 92% water, 5% carbohydrates, 2% protein, and contain
negligible fat (table). In a 100 gram reference amount, raw chicory leaves provide 23 calories and
significant amounts (more than 20% of the Daily Value) of vitamin K, vitamin A, vitamin C,
some B vitamins, and manganese. Vitamin E and calcium are present in moderate amounts.
Raw endive is 94% water and has low nutrient content.

Agents responsible for bitterness

The bitter substances are primarily the two sesquiterpene lactones, lactucin and lactucopicrin.
Other ingredients are aesculetin, aesculin, cichoriin, umbelliferone, scopoletin, 6,7-
dihydrocoumarin, and further sesquiterpene lactones and their glycosides.[31]

Traditional medicine
Chicory root contains essential oils similar to those found in plants in the related
genus Tanacetum.[32] In traditional medicine, chicory has been listed as one of the 38 plants used
to prepare Bach flower remedies

Forage
Chicory is highly digestible for ruminants and has a low fiber concentration.[34] Chicory roots are
an "excellent substitute for oats" for horses due to their protein and fat content.[35] Chicory
contains a low quantity of reduced tannins[34] that may increase protein utilization efficiency in
ruminants.[citation needed]
Some tannins reduce intestinal parasites.[36][37] Dietary chicory may be toxic to internal parasites,
with studies of ingesting chicory by farm animals having lower worm burdens, leading to its use
as a forage supplement.[38][39][40] Although chicory might have originated in France, Italy, and India,
[41]
much development of chicory for use with livestock has taken place in New Zealand. [42]

6.7 ADULTERATION IN RED CHILLI POWDER

Chili powder is usually adulterated with brick powder, salt powder, or talc powder.
Crushed husk or wood can be used to increase the weight of packaged spices or you may
also find the use of artificial colors that improve the look & feel of the spices.

ADULTERATION ANALYSIS
This study on “detection of food adulterants in chilli powder, turmeric powder and coriander powder using
physical and chemical methods.” Was conceived and carried out with the objective of identifying the presence
of adulteration in chilli powder, turmeric powder and coriander powder (the major spices used for cooking in
India). Various samples of the above mentioned spices were collected from Vellore .Both branded and
unbranded samples were selected for the study to determine the adulteration levels and the qualitative difference
between them. The tests were carried out by chemical analysis in a majority of products and through visual
inspection in few of the products. After the tests, the products containing adulterants were identified in branded
and unbranded food products. This study is attempted to bring in awareness to the public on the important
subject of food adulteration and various simple methods available to detect food adulteration

INTRODUCTION:
Food is one of the basic necessities of life. Any individual spends a lot of money on food. [4] But at the end of the
day he finds that he is eating dyes, stones, brick, dung and other contaminate matter. The act of intentionally
debasing the quality of food offered for sale either by the admixture or substitution by inferior substances or by
the removal of some valuable ingredient is known as adulteration. [1][3] . Adulteration in foods decrease our moral
and social value. According to Beckman a leading food researcher, in our daily life there are so many
unhygienic and contaminated things which are harmful to our health. When these things enter our body (in the
form of adulterants) through the food we eat, many harmful diseases like Cancer are caused. Some diseases
caused by adulterants in the spices we dealt with in this project are listed in [Link] study shows that almost
all the samples we collected from Vellore local market contain some adulteration. [2][3]

Table-1 Disorders caused due to adultered chilli, turmeric and coriander

Spices Adulterant Disease/disorder
A. Chilli powder Brick Stomach disorder
Lead soluble salts Metal toxicity, Cancer, Lead
poisoning
Rodamine B Cancer
Oil soluble tar Heart disease, damage to
liver, tumor
B. Turmeric Aniline dye Cancer
Powder
Yellow lead salts Cancer
Metanil Yellow Cancer, toxicity
Chalk Indigestion
Tapioca starch Stomach disorder
C. Coriander Common salt High blood pressure
powder
Dung Stomach problem

METHODOLOGY:
The methods adopted for detection of adulterants are given below-
8 random samples are collected and the following methods are referred to check adulteration

CHILLI POWDER:
a. To detect the presence of red lead salts:
Dilute nitric acid is added to the sample of chilli powder. The solution is filtered. Next 2 drops of Potassium
Iodide is added to the filtrate. Formation of yellow coloured precipitate indicates the presence of red lead salts.

b. To detect the presence of oil soluble coal tar:

2 g of chilli powder is taken in a test tube. Few ml of ether solvent is added and the test tube is shaken well.
Ether layer is decanted into a test tube containing 2 ml of dilute Hydrochloric acid. It is shaken properly.
[1]
Distinct pink to red colour of the lower acid layer will indicate the presence of oil soluble coal tar.

c. To detect the presence of brick powder:

Chilli powder is added in a beaker containing water. Brick powder settles down while pure chilli powder
floats. [1]

d. To detect the presence of Rodamine B:

2 g of chilli powder is taken in a test tube.5 ml of acetone is added. Immediate red colouration indicates the
presence of Rodamine B.

TURMERIC POWDER:
a. To detect the presence of yellow lead salts
2 g of turmeric powder is taken in a test tube. Conc. Hydrochloric acid is added to it. Magenta colouration
indicates presence of yellow oxides of lead.
b. To detect the presence of chalk:
2 g of turmeric powder is taken in a test tube. Few drops of water and then few drops of Hydrochloric acid is
added to it. Effervescence will indicate the presence of chalk.
c. To detect the presence of Metanil yellow:
A sample of turmeric powder is taken. To it 13N sulphuric acid is added. Disappearance of red colour on adding
distilled water indicates the presence of metanil yellow.[1]
d. To detect the presence of aniline dyes:
To a sample of turmeric powder few drops of water is added. To it 5 ml of spirit is added. Immediate
disappearance of yellow colour indicates the presence of aniline dye.
e. To detect the presence of starch of maize, wheat and rice
Microscopic view reveals that pure turmeric is yellow in colour and bigger in size.

CORIANDER POWDER:
a. To detect the presence of dung powder:
Soak a sample of coriander powder in water. Dung/sawdust will float and can easily be detected by its foul
smell.

b. To detect the presence of common salt:

A sample of coriander powder is taken. To it 5 ml of water is added. Next few drops of silver nitrate is added to
it. White precipitate confirms presence of salt.

STREAKING OF CULTURE MEDIA:

Chilli powder, coriander powder and turmeric powder samples are taken. All the three spices belong to the same
brand. After media preparation and its solidification streaking is done.

RESULTS AND DISCUSSION:

CHILLI POWDER:
1 Samples- 1,5 and 7 do not contain lead .
2. All the samples contain brick powder. Among them sample -6 has maximum amount of brick powder.
3. The 3rd sample contains oil soluble tar.
4. Samples-8,2,5 and 1 contain no rodamine b.

TURMERIC POWDER:
1 Sampe-1 contains yellow lead salts
2 Sample -1 contains chalk as adulterant
3 5th sample contains metanil yellow as adulterant
4 None of the samples contain aniline dyes

CORIANDER POWDER:
1. Dung is present as an adulterant only in the first sample.

After 24 hrs. pf incubation, growth of bacterial colonies is observed in cultures containing chilli and coriander
powder.

ACKNOWLEDGEMENT:
All the authors express their heartiest gratitude to Dr. G Viswanathan, honorable Chancellor, VIT University for
his encouragement, support and for providing good lab facilities to carry out this research work.

CONCLUSION:
Adulterated food not only consists of the physical adulterated particles other than food, but it also hosts
pathogens which can cause harmful diseases. Adulterated food causes both physical and mental disorders along
with malnutrition. Hence we must avoid eating such food. Also the government needs to take necessary actions
against the companies and individuals who for the sake of their own profit are manufacturing and selling
adulterated products to consumers.