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Antimicrobial Activity of Lactic Acid Bacteria Against Pathogens Causing Food Borne Diseases

Dipanjana Datta
Department of Mathematics and Natural Sciences Biotechnology Program BRAC University Dhaka, Bangladesh

Abstract/ Executive Summary:

Food-borne diseases are quite prevalent in the current situation of the world. Adulteration and contamination of food lead to occurrence of several food borne diseases, which can be fatal, especially for infants. Moreover, the rise of new and antibiotic-resistant pathogenic strains has lead to the search for other alternative solutions. Probiotics can be an excellent solution to treat many common food borne diseases. Probiotics are described as live microorganisms which help in the maintenance of the health and well being of the hosts by improving the intestinal microbial balance. Lactic Acid Bacteria (LAB) are known to have many health benefits and are mostly used as probiotics. As they can cause inhibition of food pathogens by the reduction of pH due to lactic acid production, hydrogen peroxide production and production of antimicrobial compounds such as bacteriocin. In this study lactic acid bacteria were isolated from several food items and screened for antimicrobial activity against the pathogens such as Bacillus subtilis, Bacillus cereus, Streptococcus pneumonia, Staphylococcus aureus, E. coli and Salmonella typhi. Fourteen isolates were obtained, among which only five were identified as Lactic Acid Bacteria by biochemical tests. The LAB isolates showed inhibitory activity against all the pathogens in the agar spot test but no zone of inhibition was observed in the case of agar diffusion test even after the addition of ammonium sulphate to precipitate proteins present in the supernatant. This suggests that the antimicrobial activity might not be due to bacteriocin production. Instead it might have been due to the production of lactic acid and hydrogen peroxide. In conclusion, LAB is still effective as probiotics against food borne diseases as they have antimicrobial properties but more sophisticated methods and equipment should be used to isolate and purify bacteriocin.

Keywords: Food borne diseases, Antibiotic resistant pathogens, Antimicrobial compounds, Lactic Acid Bacteria, pathogenic bacteria

Location: The Microbiology and Biotechnology Laboratory of the Department of Mathematics and Natural Sciences, BRAC University, Bangladesh

Start Date:

End Date:

Program Area: Food Safety and Security

Commodity: Diseases

Objectives:

* As there is an increase in the emergence of new pathogens and antibiotic resistant pathogens, the purpose of this study is to find new and more antimicrobial compounds among the local species of Lactic Acid Bacteria.

* To find new strains of Lactic Acid Bacteria that produce anti-microbial compounds.

* To compare the well diffusion assay and spot-on-lawn method and to determine the most reliable method for detection of anti-microbial activity against other pathogenic bacteria.

Methodology:

This research work was carried out at the Microbiology and Biotechnology Laboratory of the Department of Mathematics and Natural Sciences, BRAC University. Different types of media were used for selective growth, enrichment culture and indication of specific properties. Media preparation and sterilization were done according to the protocol and standard recipe. For biochemical tests, specific media were prepared. Nutrient Agar is a common microbiological growth medium. Nutrient agar typically contains 0.5% peptone, 0.3% beef extract/yeast extract, 1.5% agar, 0.5% NaCl, 97.2% distilled water. De Man, Rogosa and Sharpe Agar and broth were designed to encourage the growth of the lactic acid bacteria‘ which includes species of the following genera: Lactobacillus, Streptococcus, Pediococcus and Leuconostoc. It typically contains 1.0 % peptone, 1.0 % beef extract, 0.4 % yeast extract, 2.0 % glucose, 0.5 % sodium acetate trihydrate, 0.1 % polysorbate 80 (also known as Tween 80), 0.2 % dipotassium hydrogen phosphate, 0.2 % triammonium citrate, 0.02 % magnesium sulfate heptahydrate, 0.005 % manganese sulfate tetrahydrate and the desired amount of distilled water. It can be used as a solid medium by adding 1.0% agar. Mueller and Hinton developed Mueller Hinton Agar (MHA) which is more commonly used for the routine susceptibility testing of non-fastidious microorganism by the Kirby-Bauer disk diffusion technique. It contains 0.2% beef extract, 1.75% casein hydrolysate, 0.15% starch and 1.7% agar. Various types of fruits, vegetables and dairy products were chosen as samples for the isolation of Lactic Acid Bacteria (LAB) which were sourced from banana, tomato, carrot, pointed gourd, cabbage, cauliflower, radish, yogurt made from raw cow milk, local cheese and honey. Each of the samples was purchased from the local market named Karwan Bazar in Dhaka and carried in an aseptic bag or container to the laboratory. The fruits and vegetables were chopped up, mixed with sterile water and allowed to ferment in closed conical flasks filled to the top with water for two weeks. Two ml of each of the sample solutions was individually added to 10 ml MRS broth and incubated for 24 hours at 37°C. From each of the MRS broth solutions 200 µl was spread plated onto MRS agar plates and incubated further. The different bacterial colonies based on their morphology were selected in a way to ensure no two colonies displayed the same characteristics. These were four-way streaked on MRS agar plates to obtain single colonies of pure isolates. The plates were sealed with parafilm, refrigerated at 4°C and were sub cultured every two weeks. The agar spot test was a modification of that described by Schillinger and Lucke (1989). Colonies of each isolates were picked and stabbed on MRS agar medium and incubated anaerobically for 24 hours at 37°C. The plates were later overlaid with 10 ml of Nutrient soft agar (0.5%). The overlay agar was seeded with 104 cfu/ml of the pathogenic bacteria to be tested for sensitivity (Escherichia coli, Bacillus subtilis, Bacillus cereus, Streptococcus pneumonia, Staphylococcus aureus and Salmonella typhi). After incubation for 18- 24 h at 37 °C the plates were checked for inhibition zone of inihibition. Inhibition was scored as positive if the diameter of the clear zone around the colonies of the producer strain was 10 mm or larger. For lipid hydrolysis test Tributyrin agar base was prepared by adding 23 grams to 990 ml distilled water followed by 10 ml Tributyrin addition to the flask. The media was sterilized by autoclaving at 15 lbs pressure (121°C) for 15 minutes followed by transfer of media to glass petri dishes. After solidification, the plates were streaked with the samples and incubated at 37°C for 24 hours.Three milliliter of T1N1 agar was inoculated through stabbing each bacterium from nutrient agar plate. The vial was incubated for 5 hours to allow the bacteria to acquire log phase. Two hundred microlitre of sterile glycerol was next added and the vial sealed with parafilm and stored at room temperature.

Source of Information: Thesis (Bachelor of Science in Biotechnology) [118], Department of Mathematics and Natural Sciences, BRAC University

Article Link (Online Journal): http://hdl.handle.net/10361/7882

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In conclusion, it can be stated that Lactic Acid Bacteria can be used as probiotics against food borne pathogens as this research have shown that LAB produce inhibitory properties. Lactic Acid Bacteria can be used as live cells as probiotics since they have a 'Generally Recognized as Safe' status although inhibition by bacteriocin might be more effective as well. Further research needs to be done to find the antimicrobial property by bacteriocin produced by LAB. A bigger and more diverse sample collection should be chosen for the study and the number of isolates should be increased to alleviate the possibility of obtaining bacteriocin producing strains of LAB. Since bacteriocins are proteins produced against closely related species (Klaenhammer, 1988), to determine optimal parameters for the bacteriocin production, it is necessary to determine the ideal conditions of growth of the lactic strains and the composition of the culture medium. Moreover, bacteriocin should be precipitated and further purified by more sophisticated methods such as protein dialysis membrane and equipment to get better results.

Knowledge Management: Journal