Isolation and Characterization of Probiotic Cultures from Poultry Sample

International Journal of Bio-Technology and Research (IJBTR) ISSN (P): 2249-6858; ISSN (E): 2249-796X Vol. 10 Issue 1, Jun 2020, 1–14 Š TJPRC Pvt. Ltd.

ISOLATION AND CHARACTERIZATION OF PROBIOTIC CULTURES FROM POULTRY SAMPLE SHAIK HASEENABI1 & KONDAPALLI KASTURI2 1 2

Research Scholar, Department of Biotechnology, Acharya Nagarjuna University, Guntur, India

Assistant Professor, Department of Biotechnology, Acharya Nagarjuna University, Guntur, India

ABSTRACT Probiotic cultures were isolated from the poultry samples and these cultures were characterized for probiotic characters.

These cultures were analyzed for probiotic nature and allowed for PCR gene confirmation and agar gel electrophoresis for gene sequencing. Lactic acid bacillus, Bacillus subtilis, Saccharomyces cerevisiae and Lactobacillus rhamnoses cultures were isolated from the poultry samples. Lactic acid bacillus was isolated from the intestinal samples, Bacillus subtilis was isolated from chicken gizzard, E. Coli and salmonella were isolated from the chicken feces samples.

Probiotic characters like bile salt resistance, tolerance to acid, antimicrobial resistance, antibiotic resistance, heat resistance, gastric juice resistance tests were conducted for all probiotic isolates. All probiotic culture isolates were tested for probiotic characters and allowed for the gene confirmation. The probiotic character tests were analyzed by paired T-

Original Article

test.

KEYWORDS: Probiotic Cultures, Chicken Meat & Poultry Samples

Received: Oct 28 2019; Accepted: Nov 18, 2019; Published: Nov 29, 2019; Paper Id.: IJBTRJUN20201

INTRODUCTION The major infection causing agents to the poultry industry were the pathogens (e.g. Escherichia coli, Clostridium perfringens and Salmonella sp.). Eradication of the pathogen infections are done by antibiotics.

Without antibiotics, the industry becomes susceptible to pathogen infections. Due to the risk of antibiotic usages, researchers found an alternative solution, which was probiotics. Probiotics was used instead of antibiotics. The broilers were fed with probiotics and this gave better output in terms of growth and disease resistance than the control group. Lactic acid-producing bacteria such as lactobacilli, streptococci, bifidobacteria, Bacillus spp. and fungi-like Saccharomyces cerevisiae, Saccharomyces boulardii and Aspergillus oryzae (Fuller, 1992; Asmahan Azhari Ali, 2011). These were the most commonly used probiotic cultures. However, lactic acid bacteria (LAB) was treated as a good probiotic culture isolated from the ileum flora of broiler chicks (Saavedra, 2001). So many culture strains were isolated from the ileum sample but the main thing was to isolate the probiotic characters containing cultures- a live microbial feed supplement, which beneficially affects the host animal by improving its intestinal balance, defined as the probiotics (Fuller 1989).

Antimicrobial substances such as organic acids, diacetyl hydrogen peroxide and bacteriocins were produced by probiotics to inhibit the growth of the pathogens (Cleveland et al. 2001). Bacteriocins were synthesized from peptides or proteins ribosomally with antimicrobial properties that often targeted bacterial species by livestock (Diez-Gonzalez 2007). The probiotic species like Bifidobacterium sp., Streptococcus sp., (Patterson and Burkholder, 2003) Lactobacillus sp., Enterococcus sp. were non-spore-former cultures. These cultures were sensitive to harsh environments, so there was the need to use spore formers for the poultry industry to resist the www.tjprc.org

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heat and chemicals (Nicholson et al., 2002; Setlow, 2006; Cartman et al., 2007). Spore formers like B. subtilis, B. pumilus, B. licheniform is isolated from fecal materials of broilers and tested for resistance to gastrointestinal tract conditions. Chaiyawan, N. et al./Thai. J. Vet. Med. 2010 40(2): 207–214. 209. The major concept of this study was the isolation and characterization of probiotic properties were performed.

MATERIALS AND METHODS Samples Collection and Isolation of Cultures A total of 35 samples were collected from the 30 days old chicks from a poultry farm. The collected samples like gizzard, ileum and cecum have been stored in phosphate buffer for storage. The total samples have been transferred to the 100 ml of phosphate buffer. These samples have been transferred into the pre-sterilized conical flasks separately. From the sample, collected the 1 ml of sample and serially diluted the sample up to 10–10 dilutions. The final dilution samples have been plated by using the spread plate method. For this method, kept the 3 replicas for every 3 dilutions. A loopful of a sample has been used for streaking, streaked on pre-sterilized MRS agar and nutrient agar plate. Then, incubated the agar plates for 24–72 hours for incubation. After 24–72 hours incubation, colonies were observed on agar plates.

Culture’s Confirmation and Isolation All colonies have been identified by microscopic observation. For isolation, each colony were separately streaked on agar plates. Each isolate was tested for spore formation. The spore formation was identified by 48 hours’ incubation.

Sporulation was observed by the microscopic observation. Each isolate has been used by picking up the colonies separately and streaked on separate plates.

Identification of Lactic Cultures Colonies from the isolates were streaked on the MRS agar plates, tested for sporulation by 48 hours incubation and confirmed by microscopic observation. They have gram-positive and able to produce spores. PH was decreased for the production of lactic acid in the broth cultures. The cultures were sent for 16s. RNA test for genetical confirmation. These isolates were stored by using the 20% glycerol stocks for the future purpose for preservation.

Identification of Bacillus Cultures Colonies of isolates were streaked on the nutrient agar plates. Colonies were inoculated on broth media and kept for 48 hours’ incubation, then observed on the microscope for morphological studies and spore formers were separately isolated on agar plates, tested the colony morphology by direct colony observation and genetical confirmation was done by using the 16s RNA. The cultures were stored in the form of glycerol stocks by using the 20% glycerinin – 20 degrees freezer.

Screening of Probiotic Properties Bile Salt Resistance Grown isolates were inoculated into MRS broth with oxgall treated as bile salt with concentrations of 0.0, 0.3 and 0.5 after 24 hours’ incubation. After incubation, enumerated the isolates for viability test. The grown culture plates have the colonyforming units at 5 log10 CFU (105 CFU) (Awan and Rahman, 2005). Isolated colonies were preserved in the form of glycerol stocks by using 20% glycerin.

Impact Factor (JCC): 5.9084

NAAS Rating: 3.80

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Tolerance to Acidic pH MRS medium was used for this test. The medium PH was adjusted to PH 7.0, 4.0 and 2.0 by using the 1 M HCL and 0.5 M NaOH. Then inoculated the 1 ml of grown culture to the broth and incubated the broth for 24 hours, then poured 1 ml of culture into the petri plates and added with agar plates at 10–5 dilutions. Incubated the petri plates for 24 hours at 37 degrees temperature and isolated the stable cultures for preservation for future purpose.

Survival to Simulate Gastric Juice and Simulated Intestinal Juice As described by Dunne et al. (2001). For this purpose, each isolated bacterial culture was mixed with suspension of spores or vegetative cells were inoculated into simulated gastric juice pH 2 containing isotonic buffer (1.24% K2HPO4, 0.76% KH2PO4, 0.1% Trisodium citrate, 0.1% [NH4]2SO4) and 0.4% pepsin (Sigma) or simulated intestinal juice (isotonic buffer pH 7 with 0.3% bile salt [Sigma]). Incubation was carried out at 370C with vigorous shaking at 250 pm. The viable count was done at 0, 30, 60, 90 and 120 min after exposure by spread plate technique on the appropriate isolation media.

The resistance of each isolate was reported as % survival.

Antimicrobial Activity Agar diffusion method by Fleming et al. (1985) using this method 24 hours of incubated culture broth was used for this purpose. Agar plate was seeded with E. coli and diffused the agar plate with 8 mm filled with isolates culture broth, incubated the plates for 24 to 72 hours for results.

Resistance to Heat The ability of the isolates to resist wet heat was carried out at 80oC and 100oC, a range of temperatures used as water bath.

The isolates of grown broths were exposed to the different temperature time intervals; 0, 15, 30, 60 and 180 min. Culture viability was exposed by enumeration method.

Viability of Bacteria during Storage The storage viability of isolated species was recorded weekly at -200C, 40C and room temperature. The test tubes were inoculated with 105 CFU of each culture suspension. These inoculated test tubes were stored at -200C (with 10% V/V glycerol), 40C and room temperature for 6 weeks. The growth was monitored weekly by the plate count method.

Analysis of 16S Ribosomal DNA (rDNA) Sequence •

DNA was isolated from bacterial sample provided by the client using Biopure™ kits (Bioaxis DNA Research Centre) for Bacterial genomic DNA isolation).

•

16S rRNA gene was amplified by PCR from the above-isolated DNA. •

•

The primers used for the amplification of the 16S rRNA gene: •

F

AGAGTTTGATCCTGGCTCAG

•

R

TACGGTTACCTTGTTACGACTT

PCR Conditions

1 cycle: 94°C for 5 min (Initial denaturing)

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35 cycles: 94°C for 60 sec (denaturing) 53°C for 45 sec (annealing) 68°C for 90 sec (extension) 1 Cycle: 68°C for 10 min (final extension) •

Amplified PCR product was subjected to electrophoresis using Agarose gel 1% along with 1 kb Marker in TAE buffer and visualized by staining with ethidium bromide.

•

PCR product was purified by washing with sodium acetate and 70% of ethanol eluted from the gel.

•

Forward and reverse sequencing reactions of PCR amplicon were carried out on ABI 3730XL sequencer to obtain the sequence.

•

The assembled DNA sequence was used to carry out BLAST with the database of NCBI.

RESULTS AND DISCUSSIONS Table -1 showed the probiotic cultures’ isolations and isolation sources. L. rhamnosus, lysinibacillus, L. acidophilus, B. subtilis, gizzard source isolates, B. subtilis, B. coagulens. Table-2 Oxygal concentration was increased as plate count increased at log 10 also increased like 0.0 is low concentration, high concentration is 1.0. Table-3 showed that probiotic cultures’ growth at different PH levels. High PH showed high in plate counts than at pH low showed low plate counts.

Table-4 showed that probiotic cultures growth at different gastric juices at a different time like 30 60 90 & 120 minutes.

Table-5 antimicrobial activity at different zones by E. coli and Salmonella. Highest zone at Lactobacillus acidophilus and low at lysinibacillus. Table-6 probiotic cultures stores at 200 C temperature and table-7 showed 400 C temperature.

Figure -1 displayeded probiotic cultures growth at 200 C and Figure -2 displayed probiotic cultures growth at 400 C. Figure -3 displayed antibiotic sensitivity studies. Figure-4 displayed antimicrobial activities of probiotic cultures on E. coli and salmonella. Graph-5 displayed viable counts of probiotic cultures at different times in the gastric juice.

CONCLUSIONS Table 1: Identification of isolates by DNA was isolated from bacterial sample provided by the client using Biopure™ kits (Bioaxis DNA Research Centre) for Bacterial genomic DNA isolation. L. rhamosus st. Pro 7, Lysinibacillus sp. B-1-43, Lactobacillus acidophilus st. IDCC 3301 isolated from the intestinal contents and bacillus subtilis st. and Bacillus coagulans st., ATCC 7050 isolated from the gizzard contents.

Sample Type Intestinal contents

Gizzard

Table 1 Species Identified Identification (%) L. rhamosus st. Pro 7 100% Lysinibacillus sp. B-1-43 99% Lactobacillus acidophilus st. IDCC3301 100% Bacillus subtilis st. SRI 93% Bacillus coagulans st. ATCC7050 100%

Table 2: Mean values of plate count (Log 10) of isolated lactobacilli at different oxgall concentrations. The concentration of oxgall 0.0 to 1.0 increases as the CFU growth at log10 shows decreases, as the concentration was increased.

Impact Factor (JCC): 5.9084

NAAS Rating: 3.80

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Table 2 Oxgall PHconc.(%) 0.0 0.3 0.5 1.0

L. Acidophilus,

L. Rhamnosus

B. Subtilis,

8.16 8.66 7.78 6.3

7.37 7.42 6.85 5.76

8.47 5.28 4.26 3.76

Lactic Acid Bacillus, 7.86 7.75 7.87 6.35

Lysini Bacillus 7.5 7.6 6.58 5.76

Table 3: Mean values of plate count (log10) of isolated lactobacilli at different pH values. PH values 7, 4, 2 when the values decreases the growth also decreases.

Table 3 L. Acidophilus 5.31 7.46 1.34

pH 7.0 4.0 2.0

L. Rhamnosus

B. Subtilis

4.81 5.73 2.81

5.46 7.27 1.93

lactic Acid Bacillus 6.14 0.81 1.85

Lysini Bacillus 4.56 6.89 2.04

Table 4: Mean values of viable count (log10) of isolated lactobacilli after gastric transit incubation time (min).

The incubation time was increased as the growth rate was decreased.

Table 4 Time 30 60 90 120

L. Acidophilus 5.83 5.75 4.94 3.93

L. Rhamnosus

B. Subtilis

5.69 5.47 4.92 2.97

3.56 1.72 0.00 0.00

Lactic Acid Bacillus 5.90 5.77 5.76 3.52

Lysini Bacillus 5.81 5.92 5.67 4.22

Table 5: Mean values of the zone of inhibition (mm) for antimicrobial activity of isolated lactobacilli against E. coli and Salmonella Isolated spp. Highest zone was observed at salmonella with L. acidophilus culture.

L. acidophilus Lynsini bacillus L. acid bacillus L. rhamnosus Bacillus subtilis

Table 5 E. coli (29922) 7.00 0.00 6.50 6.40 5.80

Salmonella (29923) 8.60 1.00 10.00 4.10 7.60

Table 6: Mean values of viable count (log10) of isolated lactobacilli during storage at -200C weeks of storage.

When the time duration was increased, the growth of the culture was also decreased.

Table 6

1st 2nd 3rd 4th 5th 6th

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L. Acidophilus 4.91 4.95 4.82 4.29 4.61 4.45

L. Rhamnosus

B. Subtilis

4.90 4.90 4.84 4.81 4.73 4.55

4.99 4.97 4.96 4.87 4.63 4.44

Lactic Acid Bacillus 4.91 4.85 4.9 4.74 4.60 4.31

Lysini Bacillus 4.95 4.98 4.92 4.91 4.82 4.71

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Table 7: Viable count (log10) of isolated lactobacilli during storage at 40C weeks of storage. The incubation time was increased as the counts of the cultures also decreased.

1st 2nd 3rd 4th 5th 6th

L. acidophilus 4.91 4.90 4.82 4.29 4.62 4.24

L. rhamnosus 4.90 4.88 4.84 4.81 4.74 4.54

Table 7 B. subtilis 4.99 4.97 4.95 4.88 4.68 4.43

Lactic acid bacillus 4.91 4.90 4.90 4.75 4.75 4.75

Lysini bacillus 4.95 4.95 4.93 4.92 4.84 4.70

Figure-1 Growth at 200C for different cultures. While the duration of the cultures increases as the growth of the cultures decreases. The final 6th week shows the low culture growth.

Paired t test data 6

Control Treated Legend

4

Legend Legend

2

6t h

5t h

4t h

3r d

2n d

1s t

0

Figure 1 Figure 2 Growth at 200 C for different cultures, Figure 1 Growth at 200 C for different cultures. While the

6t h

5t h

4t h

3r d

2n d

1s t

duration of the culture increases, the growth of the cultures decreases. The final 6th week shows low culture growth.

Figure 2 Figure 3 Antibiotic sensitivity zone diameters of cultures. The x-axis different concentrations of the antibiotic and the y-axis the growth of the cultures. The growth of the cultures decreases as the antibiotic concentration decreases.

Impact Factor (JCC): 5.9084

NAAS Rating: 3.80

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Figure 3

Figure 4: Cultures Growth was Low when Treated with E. Coli and Salmonella.

Table 8 Table analyzed Paired t-test data Repeated measures ANOVA summary Assume sphericity?

No F 1.389 P value 0.3040 P-value summary ns Statisticallysignificant (P < 0.05)?

No Geisser-Greenhouse's epsilon 0.3333 R square 0.2577 Was the matching effective?

F 26.01 P value <0.0001 P-value summary **** Is there significant matching (P < 0.05)?

Yes R square 0.8655 Table 9 ANOVA Table Treatment (between columns) Individual (between rows) Residual (random) Total Data summary Number of treatments (columns) Number of subjects (rows) www.tjprc.org

SS

DF

MS

6.272 156.6 18.07 181

3 4 12 19

2.091 39.16 1.506

F (DFn, DF P-value D) F (1, 4) = 1.389 P = 0.3040 F (4, 12) = 26.01 P<0.0001

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Figure 5 Growth at gastric juice. The cultures growth was decreased as the time of the incubation was increased.

30

60

90

0 12

Figure 5 Sample S1 Sequence TGCAGTCGAACGAGTTCTGATTATTGAAAGGTGCTTGCATCTTGATTTAATTTTGAACGAGTGGCGGACGGG TGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAAT CCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAG CTAGTTGGTGAGGTAACGGCTCACCAAGGCAATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTG GGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGA TGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGC AGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGT AATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGAT GTGAAAGCCCTCGGCTTAACCGAGGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGG AACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCT GTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAA CGATGAATGCT BLAST Reference

Impact Factor (JCC): 5.9084

NAAS Rating: 3.80

Isolation and Characterization of Probiotic Cultures from Poultry Sample

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Inference The sequence obtained was 100% identical to the partial gene sequence of 16S rRNA of lactobacillus rhamnosus st. Pro7.

Sample S2 Sequence ACCTACATCTCCAGGCGGAGTGCTTATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGC ACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTC AGTTACAGACCAGAAAGTCGCCTTCGCCACTGGTGTTCCTCCAAATCTCTACGCATTTCACCGCTACACTTGG AATTCCACTTTCCTCTTCTGCACTCAAGTCCCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTC ACATCAGACTTAAAGGACCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTA TTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTAATAAGGTACCGTCAAGGTACAGCCAGTTACTA CTGTACTTGTTCTTCCCTTACAACAGAGTTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATC AGGCTTTCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGT GTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATG CGCCGCGGGCCCATCCTATAGCGACAGCCGAAACCGTCTTTCAGTCTTTTGCCATGAAGCAAAAGAGATTAT TCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAAACTATAGGGTAGGTTGCCCACGTGTTACTCACCCGTCC GCCGCTAACGTCAAAGGAGCAAGCTCCTTTTCTGTTCGCTCGACTTGCATGTATTAGGCACGCCGCCGGCGTT CGTCCTGA BLAST Reference

Inference The sequence obtained was 99% identical to the partial gene sequence of 16S rRNA of Lysini bacillus SPB-1-43.

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Sample S3 Sequence CGGCCCGCTTCTCCCCAAAATTGGGATTCAAGCGTTATCAGCAGGACTCTTCGCTCTTTTAACCCCCCCTACT TACCAATCATTCTTTTTTGGGTGAACTACCGGGAATCTAATCTCGCTCCCTCCAACGCTTGTCTCCTCAGCGTT ATTCTAGACAACACAGTCTCCTTCGCCACTGGTGTTCCTCCCCATCTCTACGCATTTTACCGCTACACGTGGA ATTCTACTCTCCTCTTCTGCTCTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCAC ATCAGACTTAACAAACCGCCTGCGAGCCCTTTACGCCCATTAATTCCGGACAACGCTTGCCACCTACCTATTA CCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCCAG GTACCGCCCTATTCCAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTC ACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTACGAGTCTGGG CCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTAC CTCACCTACTAGCTAATGCGCCGCGGGGTCCATCTTGTAAGTGGTAGCCAAATCCACCTTTTAATGTTTGAAC CATGCGGCTCAAACTATCATCCGGGATTAGCCCCGGTTTCCCGGATTATCCCAGTCTTACTGGCAGGTTACCA CGTGTTACTCACCCGTCGGCGCTAACATCTGGAGCAGCTCCCATCTGTCCGCTCG ACTTGCTTGTACTAGGCAGCCGTCGCGTTCGTCCTG BLAST Reference

Inference The sequence obtained was 93% identical to the partial gene sequence of 16S rRNA of bacillus subtilis SR1.

Sample Bacillus coagulans Sequence TGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGTGCGGACCTTTT AAAAGCTTGCTTTTAAAAGGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGG Impact Factor (JCC): 5.9084

NAAS Rating: 3.80

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ATAACGCCGGGAAACCGGGGCTAATACCRGATAGTTTTTTCCTCCGCATGGAGGAAAAAGGAAAGGCGGCT TCGGCTGCCACTTACAGATGGGCCCGCGGCGCATTAGCTAGTTGGCGGGGTAACRGCCCACCAAGGCAACG ATGCGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGC AGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAAGAAGGCCTTCG GGTCGTAAAACTCTGTTGCCGGGGAAGAACAAGTGCCGTTCGAACAGGGCGGCGCCTTGACGGTACCCGGC CAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATT GGGCGTAAAGCGCGCGCAGGCGGCTTCTTAAGTCTGATGTGAAATCTTGCGGCTCAACCGCAAGCGGTCATT GGAAACTGGGAGGCTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATG TGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCGCG AAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTA BLAST Reference

Inference The sequence obtained was 100% identical to the partial gene sequence of 16S rRNA of Bacillus coagulans st. ATCC 7050.

The identity of the sample is bound by the limitations of sample integrity and sequencing-based molecular methodology. Correlation with biochemical characterization is suggested.

Sample Lactobacillus Acidophilus Sequence GTACGCAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATC GTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAA GTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGT AAAGCGAGCGCAGGCGGAAGAATAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCGGAAA www.tjprc.org

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CTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAG AACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAG GATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGC TGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGG GGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCT AGTGCAATCCGTAGAGATACGGAGTTCCCTTCGGGGACACTAAGACAGGTGGTGCATGGCTGTCGTCAGCTC GTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCCAGCATTAAGTTGGG CACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTA BLAST Reference

Inference The sequence obtained was 100% identical to the gene sequence of 16S rRNA of Lactobacillus acidophilus st. IDCC 3301.

The identity of the sample is bound by the limitations of sample integrity and sequencing-based molecular methodology. Correlation with biochemical characterization is suggested.

Gel picture of the Amplicons

S1 Impact Factor (JCC): 5.9084

S2

S3

M NAAS Rating: 3.80

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S1 = Sample 1 S2 = Sample 2 S3 = Sample 3 M = DNA Marker The identity of the sample is bound by the limitations of sample integrity and sequencing-based molecular methodology. Correlation with biochemical characterization is suggested.

ACKNOWLEDGMENTS The authors gratefully acknowledge Intron Life Sciences for allowing experiments and cooperations and Department Of Biotechnology, Acharya Nagarjuna University for providing the facilities to carry out this work.

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AUTHORS PROFILE

Mrs. Shaik. Haseenabi has completed my Bsc of Biotechnology Acharya Nagarjuna university of sriji college in 2007. I got my Msc biotechnology degree from Bharathidasan university from J.J College from Trichi of 2009. Pursuing the Phd from 2016 in Acharya Nagarjuna university, Guntur, India Biotechnology. I published several research papers in field of Biotechnology.

Dr. Kondapalli Kasturi, Assistant Professor, Department of Biotechnology, Acharya Nagarjuna University, Guntur, India.

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