Introduction to PCR Methods for Identification and Specific Detection of Probiotic Lactic Acid Bacteria
Introduction to PCR Methods for Identification and Specific Detection of Probiotic Lactic Acid Bacteria
Submitted by:
Dr J.SWATHI Dr SABITHA
CERTIFICATE
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
1. ABSTRACT
2. INTRODUCTION 1-15
3. REVIEW OF LITERATURE
5. METHADOLOGY
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
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
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
reinforcing mucosal integrity, and restoring microbial balance. Recent studies have also
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
Modulation of the immune system: Probiotics interact with gut-associated lymphoid tissues
Enhancing gut barrier function: Certain probiotics increase the expression of tight junction
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].
Lactic acid bacteria (LAB) are a group of Gram-positive, acid-tolerant, generally non-
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.
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:
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].
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].
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].
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.
Reducing inflammation and strengthening the gut lining to prevent leaky gut syndrome.
3. Digestive Health Benefits of Probiotics
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.
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.
Stimulating the production of protective mucus in the intestines, which acts as a barrier
against pathogens.
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.
Strains like Lactobacillus helveticus and Bifidobacterium longum have shown potential in
reducing symptoms of anxiety and depression.
7. Sources of Probiotics
Probiotics can be obtained from:
Identification
techniques, are time-consuming and lack precision. The advent of molecular techniques,
strains. PCR-based methods offer high specificity, sensitivity, and rapid detection of
PCR has become an indispensable tool in the field of microbial genomics. It allows the
PCR makes it ideal for detecting probiotics in low-abundance samples, ensuring quality
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
Quantitative PCR (qPCR) enables both absolute and relative quantification of probiotic
bacteria. qPCR is extensively used for gene expression analysis, bacterial quantification in
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
Digital PCR is an advanced technique that enhances the accuracy and sensitivity of bacterial
particularly useful for detecting low-abundance probiotic strains and assessing their viability
[1].
● recA and hsp60 genes – Used for species differentiation within Lactobacillus and
● gyrB gene – Provides high-resolution identification of lactic acid bacteria (LAB) [5].
● Specific functional genes – Encode probiotic traits such as adhesion, acid resistance,
● Assessment of probiotic viability – Differentiating between live and dead cells using
safety [5].
● Metagenomic studies – Analyzing the composition of gut microbiota and the impact
● PCR inhibitors in complex samples – Food matrices and biological samples contain
Lactic acid bacteria (LAB) are extensively studied due to their probiotic properties and
● LAB as delivery systems – Using genetically modified LAB for vaccine delivery and
The future of probiotic identification lies in integrating PCR with next-generation sequencing
technologies will enhance the precision, efficiency, and automation of probiotic detection.
● Machine learning for data analysis – AI-based models can optimize primer design
● Portable PCR devices – On-site probiotic detection for food safety and clinical
applications [3].
specificity [4].
Conclusion
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).
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
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).
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)
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.
3. Pure culture
4. Glycerol stocks
6. Acid Tolerance
7. Nacl tolerance
8. Gram staining
9. H2S
10. Catalyse
11. MRVP
13. Vitamin B1
14. Vitamin B2
METHADOLOGY
Identification of B complex vitamin in producing probiotic bacteria
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.
Composition g/100ml
Peptone 1g
Beef extract 1g
Glucose 2g
Agar 1.5 g
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:
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].
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.
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].
Peptone 1g
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.
Beef Extract 3g
Peptone 3g
Agar 0.05 g
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
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]
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 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].
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.)
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.
Standard graph:
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)
1. Sample
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:
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
3) Colony Counting
1 H L B
O M K
O
CATALASE TEST
Catalase Test
3% Hydrogen Peroxide
which is 300ul for 10ml.
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 -
Mr Test
Result
Vp Test
Result :
Arginine Test
Result:
Citrate
Result:
References
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