ASSESSING THE ANTIBACTERIAL PROPERTIES OF

PROBIOTICS AGAINST FOOD BORNE PATHOGENS BY: SANEEA IMRAN | SUPERVISOR: DR. RUMEZA HANIF

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INTRODUCTION

Probiotics are live microorganisms that when administered in adequate amount confer a health benefit on the host.

Probiotics in food products have been consumed for thousands of years to provide health benefits to the body.

Earlier studies conducted by Metchnikoff (1911), Rettger (1935) , Bohnoff (1954) and Freter (1955) established the therapeutic properties of probiotics

Common probiotic strains belong to Lactic Acid Bacteria (LAB). Some examples include L casei, L reuteri, L plantarum and B bifidum, B breve, B. animalis etc

Probiotics are isolated from Human (e.g. breast milk, fecal sample) and animal sources (e.g. gut of duck) and fermented food products (e.g. milk, kefir, yogurt)

References:

Rettger, Leo F. "Some of the newer conceptions of milk in its relation to health." The Scientific Monthly (1917): 64-79.

Rettger, Leo Frederick, et al. "Lactobacillus acidophilus and its Therapeutic Application." Lactobacillus acidophilus and its Therapeutic Application.(1935).

Bohnhoff, Marjorie, Barbara L. Drake, and C. Phillip Miller. "Effect of streptomycin on susceptibility of intestinal tract to experimental Salmonella infection." Proceedings of the Society for Experimental Biology and Medicine86.1 (1954): 132-7.

Freter, Rolf. "The fatal enteric cholera infection in the guinea pig, achieved by inhibition of normal enteric flora." The Journal of infectious diseases (1955): 57-65.

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BENEFITS OF PROBIOTICS

Probiotics are helpful in treating gut related disorders such as Inflammatory Bowel Disease, Irritable Bowel

Syndrome, Lactose Intolerance, and Diarrhea

Probiotics also have antagonistic effects on food borne pathogens like Campylobacter spp., C. botulinum, Listeria

monocytogenes, E. coli and Shigella.

Several mechanism of action of probiotics are:

1. Enhancement of epithelial barrier of the intestine

2. Increased Adhesion to Intestinal Mucosa

3. Competitive exclusion of pathogenic organisms

4. Release of Antimicrobial substances

5. Immuno-modulation

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AIMS AND OBJECTIVES

Probiotic bacteria can be used to prevent or cure commonly occurring bacterial infections.

1. We aim to assess the antibacterial activity of probiotic bacteria, Lactobacilli and Bifidobacterium, isolated from local

sources within Pakistan against food borne pathogenic bacteria, Shigella and E. coli.

2. and to enhance the current understanding of the possible mechanism of action of probiotics.

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METHODOLOGY

Collection of sources

Isolation of probiotics from

sources

Characterization of the isolated

strains

Agar Well Diffusion Assay

Collection of clinical isolates of pathogens

Preparation of probiotic cell free extracts

Observation of the zone of inhibition

Calculation of percentage

activity

Statistical analysis of

results

1. Infant fecal

sample,

2. gut of a duck

1. Gram

Staining,

2. F6PPK test

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RESULTS

The results of gram staining revealed that the isolated bacterial strains were gram-positive which is a characteristic of

LAB (Lactic Acid Bacteria)

Results of gram staining- L: strain 1, R: strain 7

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ISOLATED BACTERIAL SPECIES

Strain Number Source from which isolated Specie name

1 Infant Fecal Sample Bifidobacterium animalis

2 Infant Fecal Sample Bifidobacterium animalis

3 Infant Fecal Sample Bifidobacterium animalis

4 Duck Sample Bifidobacterium animalis

5 Duck Sample Lactobacillus plantarum

6 Duck Sample Bifidobacterium animalis

7 Duck Sample Lactobacillus plantarum

8 Duck Sample Lactobacillus plantarum 8

ISOLATED BACTERIAL SPECIES

Strain number Result

1 F6PPK (+)

2 F6PPK (+)

3 F6PPK (+)

4 F6PPK (+)

5 F6PPK (-)

6 F6PPK (+)

7 F6PPK (-)

8 F6PPK (-)9

Zone of

Inhibition

given against

Shigella and E.

coli

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Fig 1: Antibacterial activity of probiotics against

Shigella

n=4±SEM

• The highest antibacterial activity is given by strain 7 (7N) and

strain 4 (4H) [Fig 1]

• The lowest antibacterial activity is given by strain 3 (3N) and

strains 2 and 6 (2H and 6H)

• Heating produced, significant differences in the antibacterial

activity of strains 3, 5, 6, 7 and 8

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Where, N= Probiotic Extracts

H= heat treated probiotic extracts

Fig 2: Antibacterial activity of probiotics against E. coli

• The highest antibacterial activity is given by strain 1

(1N) and strain 4 (4H)

• The lowest antibacterial activity is given by strain 3

(3N) and strain 8 (8H)

• Heating produced, significant differences in the

antibacterial activity of strains 1,2 and 3

n=4±SEM

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UNDERSTANDING THE MECHANISM OF ACTION

1. Bacterial Proteins

Bacterial proteins (BIF, Mucin Binding Protein) contribute to the antibacterial activity.

Heat stability of the proteins?

Heat Shock Proteins

Alteration of mechanism after heat treatment?

References:

Buck BL, Altermann E, Svingerud T, Klaenhammer TR: Functional analysis of putative adhesion factors in Lactobacillus acidophilus NCNCFM. Appl Environ Microbiol 2005; 71: 8344–8351.

Dr S. Fujiwara, Technology and Research Institute, Snow Brand Milk Products Co., Ltd, 1–1-2 Minamidai, Kawagoe, Saitama, 350–1165 Japan

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CONT…

2. Lowering of pH through the release

of Organic Acids

The antibacterial activity is independent

of the pH [Fig 3]

E.g. strain 1 has the lowest pH but does

not show good antibacterial activity

Strain 2 has a near to neutral pH (6.35)

but shows strong antibacterial activity.

Fig 3: Antibacterial activity of heat treated probiotics against Shigella and E. coli w.r.t pH

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Fig 3: Antibacterial activity of heat treated probiotics against Shigella and E.

coli w.r.t pH

• Strain 4 has an acidic pH but shows

highest antibacterial activity against

both pathogens.

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HEAT STABILITY OF THE PROBIOTICS

The antibacterial agents are heat stable

This behavior is supported by the findings of Asahara et al. (2001).

Strain 3 shows increased antibacterial activity on heating in case of both Shigella and E. coli.

Heating may lead to breakdown of the responsible antimicrobial agent into more active products.

References:

Asahara, Takashi, et al. "Antimicrobial activity of intraurethrally administered probiotic Lactobacillus casei in a murine model of Escherichia coli urinary tract infection." Antimicrobial agents and chemotherapy 45.6 (2001): 1751-1760.

Antibacterial activity of probiotics against Shigella Antibacterial activity of probiotics against E. coli 16

CONCLUSION

Probiotic extracts have antibacterial activity and thereby, insert an inhibitory effect on the growth of pathogenic

bacteria, Salmonella and E. coli.

different probiotic strains give varying antibacterial activity against a particular pathogen.

Multiple factors seem to contribute towards the antibacterial activity.

The antibacterial activity shown by the extracts is resistant to heat.

Low pH of probiotics doesn’t necessarily mean high antibacterial activity

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FUTURE PROSPECTS

The exact nature, composition and concentration of the substances which produce the antibacterial activity needs to

be determined

The role of proteins and organic acids should be studied more precisely

It should also be studied if a single probiotic strain can show more than mechanisms of action under varying

conditions.

Probiotics can be added into food products and sought as an alternative or adjunctive

preventive or therapeutic option against commonly occurring bacterial infections of the

GI tract.

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THANK-YOU!I am thankful to Prinicipal ASAB, Dr. Peter John and to the whole staff of Atta-ur-Rehman School of Applied Biosciences

including the Head of Department, Dr. Attya Bhatti for providing me the resources to carry out this project.

I am also thankful to my supervisor, Dr. Rumeza Hanif for her guidance at every step and her confidence in my abilities

which kept me strong.

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