The document evaluates the antimicrobial activity of bacteriocin produced by Lactobacillus isolated from curd samples against human pathogens. Lactic acid bacteria were isolated from curd using MRS media and identified through morphological and biochemical tests as Lactobacillus species. The isolated Lactobacillus strains were screened for their ability to produce bacteriocin exhibiting antagonistic activity against indicator strains like E. coli and Staphylococcus aureus using agar well diffusion assays. Clear inhibition zones were observed around the wells indicating the isolated Lactobacillus strains could produce bacteriocin with antimicrobial activity against pathogens.

1. AN EVALUATION OF ANTIMICROBIAL ACTIVITY
OF BACTERIOCIN PRODUCING LACTOBACILLUS
AGAINST HUMAN PATHOGENS
Presented by the fulfillment of
Bsc. Biotechnology degree under
M.G.University
Conducted at UniBiosys
research lab.
Submitted by,
Teena joy
Roll no:12155194
M.E.S.College

2. INTRODUCTION
• The intent of the study is to determine the antimicrobial activity of
Lactobacillus producing bacteriocin isolated from curd sample and to
characterize the bacteriocin.
• Lactic acid bacteria (LAB) are generally recognized as safe and play an
important role in food and feed fermentation and preservation.
• Lactic acid bacteria is a group of industrial microorganism involved in
the processing of various fermented food which include vegetables and
sausages, dietary adjuncts, probiotics and even cosmetic ingredients.

3. • Bacteriocins, are defined as bioactive peptides or protein with an
antimicrobial activity towards gram positive bacteria including closely
related species and/or food spoilage and pathogenic bacteria.
• The bacterial strains includes are E coli, Bacillus cereus, Clostridium
botulinum, Staphylococcus aureus and Listeria monocytogenes.
• Lactic acid bacteria (LAB) which are predominantly present in the
fermented foods are well known for producing bacteriocins.
• The inhibitory activity towards spoilage microorganisms is the
result of the metabolic product secreted by these LAB which acts
as antimicrobial compounds.

4. •Bacteriocins are extracellularly produced primary compounds
of bacterial ribosomal synthesis which have a relatively narrow
spectrum of bactericidal activity .
•Nisin, is the bacteriocin produced by Lactococcus lactis, is
most widely used as food preservative.
•Isolated Bacteriocin producing species of lactobacilli may be
used as protective cultures to improve the microbial safety of
food.

5. Aim and objectives
•The purpose of this study is to screen Lactobacillus isolated from
different curd samples.
• The Lactobacillus is screen for their ability to produce bacteriocin
which exhibit antagonistic activity against indicator strains.
•Lactic acid bacteria (LAB) were isolated using MRS media from
different curd samples ,were screened for bacteriocin production.
• The agar diffusion assay was used to screen for bacteriocin
producing LAB .
•Also, Microtitre plate technique is used for accurate evaluation of
inhibitory activity

6. MATERIALS AND METHODS
Isolation of lactic acid bacteria
•Biochemical tests
•Microscopical observation
Effect of antimicrobial activity
Identification of LAB

7. Isolation of lactic acid bacteria
• Lactic acid bacteria strains were isolated from curd sample.
• sample was serial diluted and appropriate dilution were
streaked on de Man, Rogosa and Sharpe (MRS) agar plates.
• The plates were incubated for 48 hours under anaerobic
condition at 37°C .
• Isolated colonies with typical characteristics of LAB were
picked from each plate and sub cultured into MRS broth and
nutrient agar slant.

8. Identification of Lactic acid bacteria (LAB)
•The cultures were identified according to their morphological,
cultural, physiological and biochemical Characteristics based on
bergeys manual of systematic bacteriology.
•The morphology was identified using Gram’s staining method and
spore forming test.
.
Biochemical tests
•Cultures of Lactobacillus sp. were further characterised for Spore
forming test, Catalase test,Methyl Red test, VogesPrauskeur test,Citrate
test, Starch hydrolysis and Glucose fermentation test
•The results of the biochemical tests were used to confirm
Lactobacillus sp. and differentiate the biovariants based on their
response to glucose fermentation analyses.

9. Screening for bacteriocin production
•The cultures of the indicator strains (Staphylococcus aureus and E.
coli) were prepared by spreading 0.1ml of the inoculum onto MHA
plates.
•4 wells of diameter 8 mm were made in each of the plates using
sterilized well cutter.
•The sample was subjected to 100% ammonium sulphate
precipitation.
1. Agar well diffusion assay
•The two wells were filled with different concentrations of sample,
one well with 30µl antibiotic (chloramphenicol) and other kept as
control.
•The inhibition zone was measured in millimeter.

10. RESULTS
Isolation of lactic acid bacteria
• The colonies were isolated from curd sample using MRS agar
medium.
• The colonies were identified as creamy white, transparent, mucoid
and smooth round in shape.

11. Fig:1 Growth of Lactobacillus in MRS media

12. Identification of morphology
Gram’s staining
Violet coloured Gram positive rod bacteria was observed
under microscope.
Fig:2 Microscopical observation of
Lactobacillus

13. Biochemical reactions
Table:1 Biochemical tests of of Lactobacillus
TEST RESULT
Spore forming test Negative
Catalase test Negative
Methyl Red Positive
Voges Prauskeur test Negative
Citrate Negative
Starch hydrolysis Positive
Glucose fermentation test Positive (acid and gas)
Tentative identification Lactobacillus sp

14. Bergeys manual of systematic bacteriology

15. Screening for bacteriocin production
Agar well diffusion assay
• The Lactobacillus strains were able to produce bacteriocin
whose antibacterial activity was analyzed by agar well diffusion
assay.
• The antibacterial substance produced by Lactobacillus
inhibited pathogen such as Staphylococcus aureus and E. coli.
• Clear zone was observed around the wells.
• The inhibition zone obtained in E.coli plate :- 5-8mm
• The inhibition zone obtained in Staphylococcus plate :-4-9mm.
• The effect of antimicrobial activity is also detected by ELISA
plate technique.

16. Fig:3 Plate showing inhibition towards
E.coli
Fig:4 Plate showing inhibition towards
Staphylococcus aereus

17. Thank you…