A PROJECT REPORT

PCR METHODS FOR IDENTIFICATION AND SPECIFIC DETECTION OF PROBIOTIC

LACTIC ACID BACTERIA

In partial fulfilment for the award of degree

Of
BACHELOR OF SCIENCES [ LIFE SCIENCES]

Submitted by:

GOLI HARINI PATEL -112322487011

YELLA ROSHINI -112322487023
PATHURI SRUTHI -112322487020

UNDER THE GUIDELINES OF

Dr J.SWATHI Dr SABITHA

[HOD OF BIOTECHNOLOGY] [ HEAD OF CIENCIA

LABS ]
 MNR DEGREE COLLEGE
(Affiliated to Osmania University)
HYDERABAD-72

CERTIFICATE

This is to certify that Goli Harini Patel-112322487011,Yella Roshini 112322487023 and

Pathuri Sruthi- 112322487020 studying in B.sc -Mb.Bt.c have finished their project work
on probiotics successfully I appreciate their hard work and sincerity. I wish them good
success.

External Examiner Principal Internal Examiner

Dr.J.SWATHI
 ACKNOWLEDGEMENT
I would like to express my special thanks to my teacher Mrs.J.SWATHI ,
who gave us the golden opportunity to do this productive project work
PCR METHODS FOR IDENTIFICATION AND SPECIFIC DETECTION OF PROBIOTIC
LACTIC ACID BACTERIA, who also helped me in completing my project.

I would also like to extend my gratitude to the CIENCIA LABS [DR.SABITHA] for
providing with all the facility that was required.

I would like to thank our principal of MNR Degree College SMT P.VANI SRI GARU for
giving us encouragement and permissions required to complete our project.
ABSTRACT
 CONTENTS

S.NO TITLE PAGE NO

1. ABSTRACT

2. INTRODUCTION 1-15

3. REVIEW OF LITERATURE

4. AIM AND OBJECTIVE

5. METHADOLOGY

6. RESULTS & DISCUSSION

Introduction

1.1 Overview of Probiotics and Their Importance

Probiotics are live microorganisms that confer health benefits when administered in

appropriate amounts. These beneficial microbes primarily colonize the gastrointestinal tract,

but they are also found in various other parts of the human body, including the oral cavity,

skin, and urogenital tract. They exert a positive impact on host health by modulating the gut

microbiota, strengthening the intestinal barrier, producing bioactive compounds, and

interacting with the host immune system.

Probiotics play a crucial role in gut health, immune system modulation, and food

fermentation processes. They help maintain a symbiotic relationship between the host and

resident microbial populations by competing with pathogenic bacteria for nutrients and

adhesion sites, producing antimicrobial peptides such as bacteriocins, and regulating the

production of short-chain fatty acids (SCFAs), which contribute to gut homeostasis. The most

commonly studied probiotic genera include Lactobacillus, Bifidobacterium, Streptococcus,

and Lactococcus [1]. These bacteria are frequently used in fermented foods such as yogurt,

kefir, sauerkraut, kimchi, and miso, where they enhance flavor, texture, and nutritional value.

Probiotic bacteria are widely used in food and pharmaceutical industries for their ability to

enhance digestion, prevent gastrointestinal infections, and maintain microbial balance in the

gut [2]. Their application in functional foods and dietary supplements has increased due to

growing consumer awareness of gut health and the role of the microbiome in overall well-

being. Probiotics have been extensively researched for their roles in improving

gastrointestinal disorders such as irritable bowel syndrome (IBS), inflammatory bowel

disease (IBD), and antibiotic-associated diarrhea by modulating inflammatory responses,

reinforcing mucosal integrity, and restoring microbial balance. Recent studies have also

highlighted their potential applications in metabolic disorders, including obesity, type 2

diabetes, and cardiovascular diseases [3]. Emerging evidence suggests that probiotics may

influence lipid metabolism, reduce insulin resistance, and lower blood pressure by interacting

with gut microbiota and host metabolic pathways. Furthermore, probiotics are being

investigated for their effects on mental health, as the gut-brain axis plays a crucial role in

mood regulation and cognitive function, with some probiotic strains showing promise in

alleviating symptoms of anxiety and depression.

1.1.1 Mechanisms of Probiotic Action

Probiotics exert their health benefits through several mechanisms, including:

Microbial balance regulation: Probiotics help maintain a balanced gut microbiota,

preventing dysbiosis that could lead to disease conditions [4].

Production of antimicrobial substances: Some probiotic strains produce bacteriocins and

organic acids, which inhibit the growth of pathogenic bacteria [5].

Modulation of the immune system: Probiotics interact with gut-associated lymphoid tissues

(GALT), stimulating immune responses [3].

Enhancing gut barrier function: Certain probiotics increase the expression of tight junction

proteins, reducing intestinal permeability [2].

Metabolism and vitamin synthesis: Probiotic bacteria contribute to short-chain fatty acid

(SCFA) production and the biosynthesis of essential vitamins such as B12 and K [4].

1.2 Common Probiotic Strains and Their Functions

Lactic acid bacteria (LAB) are a group of Gram-positive, acid-tolerant, generally non-

sporulating, non-respiring, rod-shaped (bacilli) or spherical (cocci) bacteria that produce

lactic acid as the major end product of carbohydrate fermentation. They are commonly found
in decomposing plants and dairy products and play a significant role in food fermentation

processes.

Here is an overview of the genera Lactobacillus, Streptococcus, Enterococcus,

Bifidobacterium, and Lactococcus, including their classification, family, and notable species.

1. LACTOBACILLUS

● Family: Lactobacillaceae
● Classification:
○ Phylum: Firmicutes
○ Class: Bacilli
○ Order: Lactobacillales
● Characteristics: Gram-positive, facultative anaerobic or microaerophilic rod-
shaped bacteria. They are a major part of the lactic acid bacteria group and can
convert hexose sugars to lactic acid, producing an acidic environment that
inhibits the growth of harmful bacteria.[2]
● Notable Species:
○ Lactobacillus acidophilus
○ Lactobacillus casei
○ Lactobacillus plantarum
○ Lactobacillus rhamnosus

2. STREPTOCOCCUS

● Family: Streptococcaceae
● Classification:
○ Phylum: Firmicutes
○ Class: Bacilli
○ Order: Lactobacillales
● Characteristics: Gram-positive cocci that often form chains or pairs. They are known
for their role in various fermentation processes and can be pathogenic or non-
pathogenic.
● Notable Species:
○ Streptococcus thermophilus: Used in yogurt production.
○ Streptococcus pneumoniae: Pathogenic, causing pneumonia.
○ Streptococcus pyogenes: Pathogenic, causing strep throat.

3. ENTEROCOCCUS
 ● Family: Enterococcaceae
● Classification:
○ Phylum: Firmicutes
○ Class: Bacilli
○ Order: Lactobacillales
● Characteristics: Gram-positive cocci that are facultative anaerobes. They are part of
the normal intestinal flora but can be opportunistic pathogens.[3]
● Notable Species:
○ Enterococcus faecalis
○ Enterococcus faecium

4. BIFIDOBACTERIUM

● Family: Bifidobacteriaceae
● Classification:
○ Phylum: Actinobacteria
○ Class: Actinobacteria
○ Order: Bifidobacteriales
● Characteristics: Gram-positive, non-motile, often branched anaerobic bacteria. They
are one of the major genera that make up the gastrointestinal tract microbiota in
mammals.
● Notable Species:
○ Bifidobacterium bifidum
○ Bifidobacterium longum
○ Bifidobacterium breve

5. LACTOCOCCUS

● Family: Streptococcaceae
● Classification:
○ Phylum: Firmicutes
○ Class: Bacilli
○ Order: Lactobacillales
● Characteristics: Gram-positive cocci used extensively as starter cultures in dairy
fermentation, such as cheese and buttermilk production[5].
● Notable Species:
○ Lactococcus lactis: Widely used in the dairy industry for fermentation
processes.
○ These genera play crucial roles in food fermentation, human health, and
disease. Their classification has evolved with advances in molecular biology
and phylogenetic analysis, leading to more accurate taxonomy and
understanding of their functions.
1.2.1 Vitamins Produced Probiotics

Probiotics, the beneficial bacteria residing in our gut, play a crucial role in
synthesizing various essential vitamins, thereby contributing significantly to our overall
health. Here's a detailed overview of the vitamins produced by these microorganisms:

VITAMIN B :

Several strains of probiotic bacteria synthesize B-group vitamins, which are vital for
numerous bodily functions:

Thiamine (Vitamin B1): Certain strains, such as Bifidobacterium bifidum and

Bifidobacterium longum, have been identified to produce thiamine [5].

Riboflavin (Vitamin B2): Lactobacillus fermentum is known to synthesize riboflavin [5].

Niacin (Vitamin B3): Probiotic bacteria contribute to the production of niacin, essential for
energy metabolism [7].

Pantothenic Acid (Vitamin B5): Some probiotics are involved in synthesizing this vitamin,
crucial for synthesizing coenzyme A [7].

Pyridoxine (Vitamin B6): Certain probiotic strains contribute to the production of vitamin
B6, important for amino acid metabolism [7].
Biotin (Vitamin B7): Probiotics synthesize biotin, essential for fatty acid synthesis and
energy production [7].

Folate (Vitamin B9): Strains like Lactobacillus plantarum, Streptococcus thermophilus,

Bifidobacterium infantis, Bifidobacterium breve, and Bifidobacterium longum are capable of
producing folate, which is crucial for DNA synthesis and repair [5].

Cobalamin (Vitamin B12): Some probiotics, such as Lactobacillus plantarum, have been
noted to produce vitamin B12, essential for nerve function and red blood cell formation [5].

Probiotic bacteria, particularly those in the colon, synthesize vitamin K2 (menaquinone),

which is essential for blood clotting and bone health. Genera such as Lactobacillus,
Lactococcus, Enterococcus, Leuconostoc, and Streptococcus are known producers of vitamin
K2 [7].
AMINO ACIDS :

Many probiotic strains can produce amino acids, including:

Aromatic Amino Acids: Tryptophan, tyrosine, and phenylalanine are synthesized by certain
bacteria. These amino acids are precursors to neurotransmitters like serotonin, dopamine, and
norepinephrine, which regulate various biological processes [7].

Branched-Chain Amino Acids (BCAAs): Leucine, isoleucine, and valine are produced by
some probiotic strains. BCAAs support muscle metabolism, protein synthesis, growth,
healing, digestion, and energy production [7].

Understanding the role of probiotics in vitamin synthesis underscores the importance of

maintaining a healthy gut microbiota for optimal nutrient status and overall well-being.

The Importance of Probiotics in Gut Health:

Probiotics are live microorganisms, primarily beneficial bacteria and yeasts, that provide
numerous health benefits when consumed in adequate amounts. They are essential for
maintaining a balanced gut microbiome, which is crucial for digestion, nutrient absorption,
immune function, and overall well-being. Research has increasingly demonstrated their
significance in preventing and managing various gastrointestinal disorders and even
influencing mental health through the gut-brain axis.

1. The Gut Microbiome and Its Role in Health

The human gut is home to trillions of microorganisms, including bacteria, viruses, and fungi,
collectively known as the gut microbiome. This ecosystem plays a vital role in digestion,
metabolic functions, and immune defense. A well-balanced microbiome contains a higher
proportion of beneficial bacteria, such as Lactobacillus and Bifidobacterium, which compete
with harmful pathogens, aid in food breakdown, and produce essential compounds like short-
chain fatty acids (SCFAs).

2. How Probiotics Help Maintain Gut Microbial Balance

A disrupted gut microbiome, known as dysbiosis, can lead to digestive issues, infections, and
inflammatory conditions. Factors such as poor diet, stress, antibiotic use, and environmental
toxins can disturb this balance. Probiotics help restore microbial equilibrium by:

Inhibiting the growth of pathogenic bacteria through the production of antimicrobial

substances.

Enhancing the colonization of beneficial bacteria that improve gut health.

Reducing inflammation and strengthening the gut lining to prevent leaky gut syndrome.
3. Digestive Health Benefits of Probiotics

a) Improved Digestion and Nutrient Absorption

Probiotics aid in the breakdown of complex carbohydrates, proteins, and fats, making it easier
for the body to absorb essential nutrients such as vitamins and minerals. They also help in
synthesizing certain B vitamins and vitamin K.

b) Relief from Constipation and Diarrhea

Studies have shown that strains like Bifidobacterium lactis and Lactobacillus rhamnosus
improve bowel regularity and stool consistency. Probiotics are particularly beneficial for:

Antibiotic-Associated Diarrhea (AAD): Antibiotics kill both harmful and beneficial bacteria,
leading to diarrhea. Probiotics restore the gut flora, preventing AAD.

Traveler’s Diarrhea: Certain probiotics help prevent diarrhea caused by contaminated food
and water.

Irritable Bowel Syndrome (IBS) and Inflammatory Bowel Disease (IBD): Probiotics alleviate
symptoms such as bloating, pain, and irregular bowel movements by reducing gut
inflammation.

4. Role of Probiotics in Strengthening the Immune System

Approximately 70% of the immune system resides in the gut. Probiotics enhance immune
function by:

Stimulating the production of protective mucus in the intestines, which acts as a barrier
against pathogens.

Regulating immune responses by modulating the activity of immune cells such as

macrophages and T-cells.

Competing with harmful bacteria for nutrients and attachment sites, reducing the risk of
infections.
5. Gut-Brain Connection: Impact of Probiotics on Mental Health
Emerging research has revealed a strong link between gut health and mental well-being,
known as the gut-brain axis. Probiotics can influence brain function by:

Producing neurotransmitters like serotonin and GABA, which regulate mood and anxiety.

Reducing inflammation and oxidative stress, both of which are linked to mental disorders.

Lowering cortisol levels, thereby alleviating stress and depression.

Strains like Lactobacillus helveticus and Bifidobacterium longum have shown potential in
reducing symptoms of anxiety and depression.

6. Specific Probiotic Strains and Their Health Benefits

Different probiotic strains offer unique benefits:

Lactobacillus rhamnosus GG – Helps prevent diarrhea and boosts immune function.

Bifidobacterium bifidum – Aids in digestion and prevents infections.

Saccharomyces boulardii – A beneficial yeast that treats antibiotic-associated and traveler’s

diarrhea.

Lactobacillus reuteri – Supports oral health and reduces inflammation.

7. Sources of Probiotics
Probiotics can be obtained from:

Fermented Foods: Yogurt, kefir, sauerkraut, kimchi, miso, and kombucha.

Dietary Supplements: Capsules, powders, and probiotic-enriched drinks.

8. Future Research and Clinical Applications

Scientists are exploring the role of probiotics in conditions such as obesity, diabetes, and
neurodegenerative diseases. Personalized probiotic treatments based on an individual’s gut
microbiome profile may become a key approach in precision medicine.
1.2.2 Role of Polymerase Chain Reaction (PCR) in Probiotic

Identification

Traditional microbiological methods for bacterial identification, such as culture-based

techniques, are time-consuming and lack precision. The advent of molecular techniques,

particularly PCR, has revolutionized the identification and characterization of probiotic

strains. PCR-based methods offer high specificity, sensitivity, and rapid detection of

probiotics in complex microbiological environments [3].

PCR has become an indispensable tool in the field of microbial genomics. It allows the

amplification of specific DNA sequences, enabling researchers to study microbial

communities, detect pathogens, and perform strain-level differentiation. The sensitivity of

PCR makes it ideal for detecting probiotics in low-abundance samples, ensuring quality

control in food products and clinical applications [4].

1.3 Types of PCR Techniques for Probiotic Identification

1.3.1 Conventional PCR

Conventional PCR involves the amplification of specific genetic markers such as the 16S

rRNA gene, which is widely used for bacterial classification. This technique provides a

qualitative assessment of bacterial presence but lacks quantitative precision [4].

1.3.2 Real-Time Quantitative PCR (qPCR)

Quantitative PCR (qPCR) enables both absolute and relative quantification of probiotic

bacteria. qPCR is extensively used for gene expression analysis, bacterial quantification in

food products, and microbial community profiling [5]. It relies on fluorescence-based

detection, which allows real-time monitoring of DNA amplification.

1.3.3 Multiplex PCR

Multiplex PCR allows the simultaneous amplification of multiple target genes in a single

reaction, making it an efficient tool for differentiating multiple probiotic strains within a

sample. This technique is particularly useful in dairy and fermented food industries for

detecting various probiotic species [3].

1.3.4 Digital PCR (dPCR)

Digital PCR is an advanced technique that enhances the accuracy and sensitivity of bacterial

quantification by partitioning the sample into thousands of micro-reactions. This method is

particularly useful for detecting low-abundance probiotic strains and assessing their viability

[1].

1.4 Molecular Markers for Probiotic Detection

PCR-based identification of probiotic bacteria relies on the amplification of conserved

genetic markers, including:

● 16S rRNA gene – Universal marker for bacterial identification [2].

● recA and hsp60 genes – Used for species differentiation within Lactobacillus and

Bifidobacterium genera [4].

● gyrB gene – Provides high-resolution identification of lactic acid bacteria (LAB) [5].

● Specific functional genes – Encode probiotic traits such as adhesion, acid resistance,

and antimicrobial production [3]

1.5 Applications of PCR in Probiotic Research

PCR-based methodologies have numerous applications in probiotic research, including:

● Strain authentication and regulatory compliance – Ensuring the correct strain is

used in probiotic formulations [4].

● Quantification of probiotic populations – Monitoring bacterial load in food and

pharmaceutical products [2].

● Assessment of probiotic viability – Differentiating between live and dead cells using

viability PCR [3].

● Antibiotic resistance screening – Detecting resistance genes to ensure probiotic

safety [5].

● Metagenomic studies – Analyzing the composition of gut microbiota and the impact

of probiotics on microbial ecology [1].

1.6 Challenges in PCR-Based Probiotic Identification

Despite its advantages, PCR-based probiotic identification faces several challenges:

● Primer specificity issues – Designing primers that accurately differentiate closely

related strains [2].

● PCR inhibitors in complex samples – Food matrices and biological samples contain

inhibitors that affect amplification efficiency [3].

● Standardization of protocols – Lack of universally accepted PCR conditions leads to

variability in results across laboratories [4].

● Cost and technical expertise – Advanced techniques such as dPCR require

specialized equipment and expertise [5].

1.7 Advances in Lactic Acid Bacteria Research

Lactic acid bacteria (LAB) are extensively studied due to their probiotic properties and

industrial applications. Research in LAB focuses on:

● Genome sequencing of LAB strains – Understanding genetic variations that

contribute to probiotic efficacy [3].

● Metabolic engineering – Enhancing the ability of LAB to produce bioactive

compounds such as bacteriocins and exopolysaccharides [5].

● Synbiotic formulations – Combining probiotics with prebiotics to improve

colonization and health benefits [1].

● LAB as delivery systems – Using genetically modified LAB for vaccine delivery and

therapeutic applications [2].

1.8 Future Perspectives in Probiotic Identification

The future of probiotic identification lies in integrating PCR with next-generation sequencing

(NGS) and artificial intelligence-based bioinformatics tools. The combination of these

technologies will enhance the precision, efficiency, and automation of probiotic detection.

Emerging trends include:

● Machine learning for data analysis – AI-based models can optimize primer design

and improve strain differentiation [1].

● Portable PCR devices – On-site probiotic detection for food safety and clinical

applications [3].

● CRISPR-based diagnostics – Targeted identification of probiotic strains with high

specificity [4].
Conclusion

PCR-based methods have revolutionized the identification and characterization of

probiotic lactic acid bacteria by providing rapid, precise, and highly sensitive
detection techniques. Probiotics, particularly lactic acid bacteria, play a critical role in
gut health, immune function, and various industrial applications. Traditional culture-
based identification methods are being replaced by advanced molecular approaches,
such as qPCR, multiplex PCR, and digital PCR, which enable strain-level
differentiation, quantification, and viability assessment.

Despite challenges such as primer specificity, PCR inhibitors, and standardization

issues, ongoing advancements in sequencing technologies, bioinformatics, and AI-
driven analysis are enhancing the efficiency and accuracy of probiotic identification.
The integration of PCR with next-generation sequencing (NGS) and CRISPR-based
diagnostics holds great promise for the future, allowing for precise characterization
and quality control of probiotic strains in food, pharmaceuticals, and clinical research.
 REVIEW OF LITERATURE

Probiotics
Probiotics are live microorganisms that confer health benefits when administered in
adequate amounts. Research has focused on their role in gut health, immune
modulation, and disease prevention. Studies by Gill and Guarner (2008) highlight the
mechanisms by which probiotics influence gut microbiota, including competitive
exclusion of pathogens, production of antimicrobial compounds, and modulation of
immune responses. Furthermore, Lactobacillus and Bifidobacterium strains have been
extensively studied for their probiotic properties in gastrointestinal health (Ouwehand
et al., 2002).

Recent advancements explore the potential of probiotics in mental health,

known as the gut-brain axis. Studies suggest that probiotics may help alleviate
symptoms of anxiety and depression (Mayer et al., 2015). Additionally, research is
expanding into their role in metabolic disorders, allergy prevention, and anti-
inflammatory properties (Plaza-Diaz et al., 2019).

Historical Perspective of Probiotics The concept of probiotics was first introduced by

Elie Metchnikoff in the early 20th century, who proposed that consuming fermented
dairy products containing beneficial bacteria could promote longevity. Over time,
extensive research has led to the identification and characterization of various
probiotic strains with specific health benefits (Fuller, 1989).

Role of Lactic Acid Bacteria in Probiotics LAB are considered key probiotic
organisms due to their ability to survive gastrointestinal conditions, adhere to
intestinal mucosa, and modulate gut microbiota. They produce metabolites such as
bacteriocins, hydrogen peroxide, and organic acids, which inhibit pathogenic
microorganisms and support gut homeostasis (Gibson & Rastall, 2003).

2. B1 and B2 Estimations

Vitamin B1 (thiamine) and B2 (riboflavin) are essential water-soluble vitamins

involved in energy metabolism and cellular function. Various analytical methods have
been employed to estimate their levels in biological and food samples.

High-performance liquid chromatography (HPLC) has been widely used for accurate
estimation. A study by Ndaw et al. (2000) discusses the use of reversed-phase HPLC
with fluorescence detection to quantify thiamine and riboflavin. Additionally,
spectrophotometric and fluorometric methods have been employed for routine
estimations, though they may have lower sensitivity compared to HPLC (Kasper et al.,
2003).

Recent studies focus on advanced chromatographic techniques, including liquid

chromatography-mass spectrometry (LC-MS), which offer higher specificity and
sensitivity for detecting low concentrations of these vitamins in complex matrices
(Zhang et al., 2018).

Probiotics as a Source of Vitamin B1 Several studies have demonstrated the ability of

probiotics, particularly Lactobacillus, Bifidobacterium, and Enterococcus species, to
produce B1. The biosynthesis of thiamine in probiotics depends on the metabolic
pathways and growth conditions of the bacterial strains (LeBlanc et al., 2011).
Probiotic fortification with B1 has been explored to enhance its dietary availability,
especially in fermented foods (Saubade et al., 2017)

Biosynthesis of Vitamin B2 by Probiotic LAB Several LAB strains exhibit the ability
to produce riboflavin during fermentation. Studies have shown that specific strains,
such as Lactobacillus fermentum, Lactobacillus reuteri, and Bifidobacterium breve,
can enhance B2 levels in fermented dairy and plant-based products (Capozzi et al.,
2012). The genetic regulation of riboflavin biosynthesis in these bacteria has been
extensively studied, highlighting the role of rib operons and metabolic engineering
approaches to increase production (Russo et al., 2014)

3. Polymerase Chain Reaction (PCR)

PCR is a widely used molecular biology technique for amplifying specific DNA
sequences. It has applications in medical diagnostics, genetic research, and forensic
science. Mullis and Faloona (1987) first described PCR, revolutionizing genetic
studies by allowing the amplification of minute DNA quantities.

Several variations of PCR have been developed to enhance specificity and sensitivity.
Quantitative PCR (qPCR) enables real-time monitoring of DNA amplification,
making it valuable in pathogen detection and gene expression studies (Heid et al.,
1996). Another innovation, digital PCR (dPCR), provides absolute quantification of
DNA without the need for standard curves, improving precision in applications like
viral load estimation and mutation detection (Hindson et al., 2011).

PCR techniques are crucial in microbiome research, aiding in the identification and
quantification of probiotic strains. Metagenomic studies often use 16S rRNA gene
sequencing combined with PCR to analyze microbial diversity (Caporaso et al., 2012).
Vitamin B1 (Thiamine) and Vitamin B2 (Riboflavin) are essential water-soluble
vitamins that play crucial roles in energy metabolism, cellular function, and overall
health. Accurate estimation of these vitamins in biological samples, food products,
and pharmaceutical formulations is important for nutritional and clinical assessments.
Various analytical techniques have been developed over the years to improve the
sensitivity, accuracy, and efficiency of B1 and B2 quantification.

Spectrophotometric Methods Early methods for B1 and B2 estimation were based on

UV-visible spectrophotometry. These techniques relied on the characteristic
absorption spectra of thiamine and riboflavin. However, spectrophotometric methods
often lacked specificity due to interference from other biomolecules and required
derivatization steps to enhance sensitivity (Zhang et al., 2000)
 AIM & OBJECTIVE

AIM - PCR METHODS FOR IDENTIFICATION AND SPECIFIC DETECTION OF

PROBIOTIC LACTIC ACID BACTERIA.

OBJECTIVE- 1. Serial dilution

2. Colony counting / morphology

3. Pure culture

4. Glycerol stocks

5. Bile salt tolerance

6. Acid Tolerance

7. Nacl tolerance

8. Gram staining

9. H2S

10. Catalyse

11. MRVP

12. Arginine test

13. Vitamin B1

14. Vitamin B2

15. PCR - Polymerase Chain Reaction

16. Gel electrophoresis

METHADOLOGY
Identification of B complex vitamin in producing probiotic bacteria

Materials and Method

Source of Materials

The test samples for sourcing lactic acid bacteria (LAB) includes idly batter (fermented
batter), were sourced randomly from the open market by random selection.

Sample Preparation

Samples were collected into sterile bottles and were taken into the laboratory for
microbiological examination. They were homogenized by adding 1 milliliter (ml) of idly
batter into 9 ml of sterile peptone physiological saline solution.

Isolation of Lactic Acid Bacteria

Preparation of Lactobacillus De Mann Rogassa Sharpe (MRS) Agar Media forisolation

of Lactic acid bacteria

Table 4.1. MRS Agar Media Preparation

Composition g/100ml

Peptone 1g

Beef extract 1g

Yeast extract 0.4 g

Glucose 2g

Sodium acetate trihydrate 0.5 g

Polysorbate 80/Tween 80 0.1 g

Dipotassium hydrogen phosphate 0.2 g

Triammonium citrate 0.2 g

Magnesium sulphate heptahydrate 0.02 g

Magnesium sulphate tetrahydrate 0.005 g

Agar 1.5 g

pH adjusted 6.5 + 0.2 at 25oC

The final volume of solution is made up to 100ml with distilled water.

Isolation of Lactic Acid Bacteria using MRS Agar

Based on the method described by N gene, et al., in 2019, the fermented idly batter is
subjected to serial dilutions. From the serially diluted preparation, the 10-5, 10-6, 10-7 and
10-8 samples are selected for spread-plate method and a volume of 100 µL is poured and
spread on the MRS agar plates and incubated at 37 for 48 hours in anaerobic conditions. The
colony morphology of different strains are observed after 48 hours. After 48 hours of
anaerobic incubation on the MRS agar plates, the colonies are counted using a colony counter
and the colony forming units per ml is determined using the following formula:

CFU/mL = No. of colonies counted

_________________________________________
Volume of sample plated (mL) * Dilution factor

The random colonies are selected and streaked on the MRS Agar for getting pure culture. The
selected pure cultures are grown in liquid nutrient broth and in sterile 3ml vials, a volume of
0.5 ml culture is added with 80% of glycerol and stored in - 20°C for future experiments [50].

Microbiological and Biochemical screening of the Lactic acid bacteria

Gram staining for identification of Lactic acid bacteria

As per the method described in Cappuccino manual, the Gram staining is a technique for
differentiating and categorizing bacterial species into the two major categories of gram-
positive and gram-negative bacteria. The isolated colony is kept on a clean glass slide using a
sterile inoculating loop and heat fixed. The smear is gently soaked with crystal violet and
allow to remain for one minute. The slides are washed under running tap water and are
fixated with Gram's iodine for 1 minute. The slides are washed again under running water
and a decolorizer is added containing 95% ethyl alcohol drop by drop for 5–10 seconds until
the alcohol clears the violet colour. Then the slide is counter-stained with safranin, and
allowed to remain for 45 seconds. The slides are washed and examined using a compound
microscope with oil immersion at a 100x magnification.

4.3.2 Primary identification of Lactic acid bacteria using Catalase test

 The surface of a clean glass slide is placed with a little amount of an isolated
bacterial colony, and a drop of 3% hydrogen peroxide (H2O2) is added. Based on the
presence of catalase positive organism, air bubbles are observed within 0 to 10 secs, the false
positive result is observed from 11th to 20th second and negative samples won’t show any air
bubbles.

Secondary screening of Lactic Acid Bacteria by Biochemical analysis tests

Carbohydrate fermentation and Gas production test

The broth media is prepared by mixing all ingredients in distilled water and heated
gently to dissolve it. The two types of sugars one hexose-glucose and one pentose-ribose are
mixed depending on the requirement. The test tubes of 13 x 100 mm are filled with 4 – 5 ml
of phenol red carbohydrate broth with inverted Durham tube without air bubbles. The
medium is sterilized by autoclaving at 121°C for 15 mins. The final pH of the media is made
to 7.4 + 0.2. Each test tube is aseptically inoculated with the test strain using 0.1 % (100 μL)
of overnight culture and incubated at 35 – 37°C for 18-24 hours [55].

Table 4.2. Composition of Fermentation broth

Fermentation broth composition g/100ml

Peptone 1g

Sugars(glucose / ribose) 0.5 g

Sodium chloride 0.15 g

Phenol red 1.8 g

Distilled water 100 ml

The final volume of solution is made up to 100ml with distilled water.

4.3.3.2 Hydrogen sulphide production test

Hydrogen sulfide (H2S) production test is used for the detection of hydrogen sulfide (H2S)
gas produced by an organism. H2S is produced by certain bacteria through the reduction of
sulfur-containing amino acids through the reduction of inorganic sulfur compounds such as
thiosulfates, sulfates, or sulfites. The hydrogen sulfide production could be detected by
incorporating a heavy metal salt containing iron or lead as an H2S indicator to a nutrient
culture medium containing cysteine and sodium thiosulfates as the sulfur substrates.
Hydrogen sulfide, a colorless gas, if produced reacts with the metal salt-forming visible
insoluble black precipitate of ferrous sulfide indicating a positive result. The labelled tubes
with SIM agar are inoculated by stabbing needle for inoculation and incubated at 37°C for
24-48 hours and observed for H2S production by the inoculated bacteria.

Table 4.3. Composition of SIM Agar Media

Composition of SIM agar media g/100ml

Beef Extract 3g

Peptone 3g

Sodium thiosulphate 0.0025 g

Ferrous ammonium sulfate 0.02 g

Agar 0.05 g

Final pH At 25°C, 7.3 ± 0.2

The final volume of solution is made up to 100ml with distilled water.

4.3.3.3 Methyl Red – Voges Proskauer (MR – VP) Test Table 4.5. Composition of MR –
VP Broth
Composition of MR – VP Broth g/100ml

Buffered peptone 0.7 g

Glucose / Dextrose 0.5 g

Dipotassium phosphate 0.5 g

The final volume of solution is made up to 100ml with distilled water.

a) Methyl Red Test

The bacteria cultures are inoculated into 5 ml of MR – VP broth and incubated
anaerobically at 37˚C for 24 hours. After 24 hours, a drop of methyl red solution is added in
the overnight grown broth and observed for red or dark pink colour formation to confirm the
positive result [50].

b)Voges Proskauer Test

 The bacterial cultures are inoculated into 5 ml of MR – VP Broth and incubated
anaerobically at 37˚C for 48 hours. A drop of 5% alpha-naphthol and a drop of 40% KOH is
added to the culture tubes and mixed well. The culture is observed to have colour change
after the addition of the reagents [50].

Citrate utilization Test

Simons Citrate Agar is used for this test. 3.15 g of Simons Citrate Agar is added to 130ml of
distilled water and sterilized by autoclaving at 121˚C for 15 mins at 15 lbs. The media is then
poured into sterilized test tubes and kept in slant position at room temperature. After
solidification of the media, the media is stab using inoculation loop with the inoculum of
overnight grown bacterial cultures. The tubes are incubated for 24 hours at 35 – 37˚C. After
24 hours the bacterial cultures changing the green colour into blue are considered as positive
result and the tubes which are remaining with same colour are considered negative result [56]

4.4 In vitro Probiotic evaluation of selected Lactic acid bacterial strains

Bile Salt Tolerance Test – Qualitative method

Bile salt tolerance test is done by preparing 300ml nutrient agar (4.5 gm agar) supplemented
along with 1%, 2.5%, and 5% concentration of bile salt and then autoclaved. Bile salt
tolerance of the bacterial culture are determined by streaking on the different nutrient agar
plates and grown overnight for isolating active cultures by incubating at 35 – 37oC for 24
hours and 48 hours under anaerobic conditions. The blank medium is used as a control [57].

NaCl Tolerance Test – Qualitative method

NaCl tolerance test is done by preparing 300ml of nutrient agar (with 4.5 gm agar)
supplemented along with 1%, 2.5%, and 5% concentration of NaCl salt and then autoclaved.
NaCl tolerance of the bacterial culture are determined by streaking on the different nutrient
agar plates and grown overnight for isolating active cultures by incubating at 35 – 37oC for
24 hours and 48 hours under anaerobic conditions. The blank medium is used as a control
[58].

Acid Tolerance Test – Qualitative method

For the acid tolerance test, 100ml Nutrient broth is taken and added with 2ml (2%) conc. HCl
and another set of 100ml broth with 4ml (4%) conc. HCl is added. Then 5ml of broth is filled
in red cap tubes and marked which is later kept for autoclaving. After sterilization, the tubes
are inoculated with the overnight grown selected bacterial cultures and kept for incubation for
24 hrs and the growth is observed [57].
 Estimation of Thiamine (vitamin B1)

Materials:

1. Sample

2. Ethanolic sodium hydroxide (For 50ml; 0.21g of NaOH dissolved in 0.25ml dis.
Water and 50ml of ethanol is added in it; Allowing the solution to stand in tightly
stoppered bottle for 24 hrs and then quickly decant the clear liquid in another
suitable bottle.)

3. 0.1% Potassium Dichromate

Methodology:

1. Centrifuge the culture broth for 5min at 10000rpm and take 1ml of supernatant.
.
2. Add 5ml of Ethanolic sodium hydroxide mix well and centrifuge and take
Supernanatant.

3. Add 1ml of 0.1% Potassium Dichromate to supernatant.

4. Absorbance was measured at 360 nm.

Standard graph:

Standard Graph for B1 Vitamin

3.768
4
3.5 3.14
f(x) = 0.621942857142857 x − 0.0141333333333336
Absorbance at 360 nm

3 R² = 0.994591379037816
2.3
2.5
1.884
2
1.5 1.256
1 0.628
0.5
0
0 1 2 3 4 5 6 7
Concentration of Vitamin B1 (mg)

Estimation of Riboflavin (Vitamin B2)

Materials:

1. Sample

2. Bromothymol blue (0.2%)

3. Chloroform

4. Distilled water

Methodology:

1. Centrifuge the culture broth for 5min at 10000rpm and take 1ml of supernatant.

2. To that 2ml of bromothymol blue (0.2%) was added and the total volume of the
aqueous phase was made upto 10ml with distilled water.

3. About 5ml of chloroform was added to each funnel and the contents were vortex for
2mins, the two phases were allowed to separate and the absorbance of the upper layer
was measured at 440nm against the corresponding reagent blank.

4. The amount of riboflavin present in the sample solution was computed from its
calibration curve.

Standard graph:

Standard Graph for B2 Vitamin

1.6
1.4
1.4
f(x) = 1.455 x − 0.0769999999999999
1.2 1.05
R² = 0.997514488997785
1
OD 440 nm

0.81
0.8
0.6 0.5

0.4 0.22
0.2
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1

concentration mg/ml

RESULT & DISCUSSION :

1) Pour Plate Method

2) Streak Plate Method

3) Colony Counting

4) Bile Salt Tolerance Test

5) Ncal Tolerance Test
6) Acid Tolerance

1 H L B

O M K
O
CATALASE TEST

Catalase Test
3% Hydrogen Peroxide
which is 300ul for 10ml.

Isolated strain placed on a glass slide

Glass slide dried with a loop

3% H2O2 dropped on the slide

Mixed well
RESULT :
 We observed no air bubble formation, indicating all the bacterial strains negative.
 All the 8 strains are negative

Strain +/-
J7 -
H6 -
L6 -
B4 -
K5 -
O5 -
M2 -
O4 -

Catalase test for all the selected probiotic strains

Mr Test

Result
Vp Test

Result :

Arginine Test
Result:

Citrate
Result:
References

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2.

3.

4.

5. Evaluation of three PCR primers based on the 16S rRNA gene

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