International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009, Vol-5, Iss-2, Spl. Issue-2 Jun.-2017 http://iraj.in SCREENING OF LACTOBACILLUS FROM CURD AND IDLI BATTER FOR PRODUCTION OF BACTERIOCINS
1MEGHANA TELI, 2SOJAL MAHAJAN, 3ISHA MAHAJAN, 4JITENDRA RAJPUT, 5SARITA MAHAJANI
1,2,3,4,5Sinhgad College of Engineering, Pune Email: [email protected],[email protected], [email protected]
Abstract - Bacteriocins are ribosomal proteins of bacteria that are synthesized during the logarithmic phase of cell growth cycle. These are antimicrobial products that kill or inhibit the growth of other closely related bacteria. As these are naturally synthesized, they are believed to be less toxic than artificially synthesized antibiotics and hence are ‘Generally Recognized as Safe’ (GRAS). In addition to this anticipatory role of alternatives to conventional antibiotics, bacteriocins are used as preservatives in food and beverage industries. They are also sought to be highly specific therapeutic agents in cancer therapy. Studies show that bacteriocins are widely produced by lactic acid bacteria (LAB). The following study aims at isolating novel bacteriocin producing strain from sources rich in LAB namely curd and Idli batter. The strains were isolated based on their antagonistic activity against indicator strains using cross-streak technique and agar well diffusion. Extraction and purification of the strains is currently carried out using Chloroform extraction. Characterization of the isolated strains will be done using gel electrophoresis and High Performance Liquid Chromatography (HPLC) to further decode the properties of the bacteriocin.
Index terms - Bacteriocins, ribosomal proteins, antimicrobial products, Cross-streak method, Agar well diffusion.
I. INTRODUCTION In addition to their contribution in pharmaceutical industries, bacteriocins also play a major role in food Bacteria produce a unique kind of ribosomal protein and beverage industries. One special characteristic of called Bacteriocin that shows antimicrobial activity bacteriocin is its ability to prevent formation of against other related or non related bacteria species. biofilm by food spoiling bacteria like Listeria These toxic components are lethal to the survival of monocytogenes and Staphylococcus aureus. bacteria other than the producing strain. Furthermore, Therefore, they are used as preservatives to inhibit these bioactive peptides exhibit antagonistic action the growth of microbes resulting in increasing shelf against both narrow and broad spectrum bacteria [1]. life and maintaining nutritive value of the product. Bacteriocins are primary metabolites, in other words they are produced during the logarithmic stage of the II. CLASSIFICATION cell growth cycle and hence help in sustainable cell growth. They are found to be chemically and Both Gram positive and Gram negative bacteria structurally stable against varying temperature produce bacteriocins. These are however divided into conditions. different classes.
Bacteriocins are the innate immune weapon system of A. Gram Positive – From LAB bacteria; due to their status of ‘naturally synthesized’ Lactic acid bacteria are Gram positive bacteria that product they are observed to be less harmful and produce bacteriocins. These have been classified in highly target specific. They produce minimum or no three groups on the basis of their biochemical side effects on their consumption and hence have characteristics: acquired a position in ‘Generally Recognized as Safe’ 1. Class I: Also known as lantibiotics, they are (GRAS) category proposed by American Food and small, membrane-active, heat-stable peptides Drug Administration. The association validates food containing unusual thioether amino acids, like and drug items based on their quality, ability of lanthionine and B-methyllanthione. The model generating side effects and classify them as safe when bacteriocin of this group is isolated from they are fit for human consumption [2]. Thus, Lactococcus lactic and is called nisin[4]. bacteriocins prove to be excellent alternatives to 2. Class II: Bacteria belonging to this class show hazardous conventional antibiotics. Moreover, due to better biological activity and physiochemical their target specificity, bacteriocins can also be used property than other classes of bacteria. They are as therapeutic agents in cancer therapy. thus most promising bacteriocin candidates for food preservation.
Screening of Lactobacillus from Curd and Idli Batter for Production of Bacteriocins
30 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009, Vol-5, Iss-2, Spl. Issue-2 Jun.-2017 http://iraj.in 3. Class III: This class of bacteriocins are large colonies was determined. Strains were inoculated in (>30kDa) and secreted by the bacterial 100 mL Nutrient broth and incubated for 48 hrs at preprotein translocase. This group is not well 37°C. These samples were than centrifuged at 4500 documented [4]. rpm for 40 minutes to obtain the cell-free supernatant, 50 µl of which was then was aliquoted in the wells of B. Gram Negative – From E. coli Nutrient Agar Plates. These were seeded with A bacteriocin named Colicin now known as microcin indicator strains viz. Gram Positive-Listeria. is produced by Gram negative bacteria like E. coli. monocytogenes, Staphylococcus aureus and Gram They are the longest studied bacteriocins [4]. Negative Pseudomonas aeruginosa. The zone of inhibition was noted for further analysis. III. ISOLATION VII. TEMPERATURE STABILITY Lactic acid bacteria were isolated from curd made from cow milk and idli batter (mixture of rice and Characterization of the strains was done beginning split black gram in water and fermented overnight.) with the test of heat stability. Bacteriocin producing on growth medium. For this purpose, de-Mann, strains were fermented for 40 hours in Nutrient broth Rogosa and Sharpe (MRS) agar plates (peptone 1% , and the spent media was centrifuged at 4500 rpm for beef extract 1%, Yeast extract 0.4%, glucose 2%, 40 minutes at room temperature. The supernatant agar 1%, sodium polysorbate 0.1%, acetate trihydrate obtained was divided into three parts and stored at 0.5%, tri-ammonium citrate 0.2%, magnesium different temperature conditions viz. 4ºC, 37ºC, 60ºC sulphatehepta-hydrate 0.02%, manganese sulphate for 24 hours. The stability of peptide was determined tetra-hydrate 0.005%, di-potassium hydrogen using agar well diffusion against Staphylococcus phosphate 0.2%, pH 6.2) were prepared that enable aureus. specific growth of LABs. Samples were diluted with saline water to dilution factors of the order of 1010- VIII. RESULTS 1013 and plated using spread plate technique. These plates were incubated for 24 hours at room 1. ISOLATION temperature. Single colonies were picked based on A total of 6 strains were obtained from the above their morphology and were sub-cultured to obtain mentioned sources. These were named sequentially pure colonies using 4 streak method. Furthermore, for convenience. MRS agar slants of obtained isolated colonies were prepared and stored at 4°C. Table 1: Colonies isolated from samples Source Strain number
IV. PRELIMINARY SCREENING Curd Cd1, Cd2
Antimicrobial activity of isolated colonies was Idli batter Id1, Id2, Id3, Id4 examined through cross-streak method using indicator strain Listeria monocytogenes (MTCC 2. PRELIMINARY SCREENING 657/ATCC 19111) on Nutrient Agar plates. Strains Strains showing antimicrobial action were selected that did not show inhibitory activity against indicator for further analysis. were discarded. Table 2: Cross streak technique V. ACID PRODUCTION TEST Source Strain number Curd Cd2 Isolated cultures were fermented for 2 days and spent Idli batter Id2, Id3, Id4 medium was collected, centrifuged at 10000 rpm for 20 minutes, room temperature. The supernatant obtained were analyzed for acid production using bromothymol blue solution. Acid producing colonies changed the color of the solution to yellow while bacteriocin producing colonies did not show any color change.
VI. AGAR WELL DIFFUSION
The strains obtained after acid production test were inferred to be bacteriocin producing. Quantitative analysis of the colonies was done using agar well diffusion. Three indicator strains were used and the activity of the bacteriocin produced by selected Figure 2: Cross streak of curd and Idli samples
Screening of Lactobacillus from Curd and Idli Batter for Production of Bacteriocins
31 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009, Vol-5, Iss-2, Spl. Issue-2 Jun.-2017 http://iraj.in 3. ACID PRODUCTION TEST Following strains were selected as they did not show CONCLUSION any color change in bromothymol blue solution and were thought to produce bacteriocin. Isolation of bacteria responsible for bacteriocin production from curd and idli batter was successfully Table 3: Acid production done. Their antagonistic activity against major food Source Strain number spoiling bacteria shows their potential as Curd Cd2 preservatives in food industries. As these food borne Idli batter Id2 pathogens affect the quality of dairy products, the inhibitory effect shown by the microbes prove to be a turning point in the process of food preservation using living organisms. Also, as these are naturally synthesized they pose minimum threat to the health of the consumer. Therefore, it is safe to say that in coming years, treatment of diseases using conventional antibiotics will be replaced by bacteriocins as therapeutic agents and that they are the “next wave of antibiotics.” In addition, these
Figure 3: Acid production test peptides are active against both broad and narrow spectrum bacteria and thus can act against multiple 4. AGAR WELL DIFFUSION drug resistant bacteria. The characterization and Quantitative analysis of the above mentioned strains extraction of the bacteriocins produced by the strains was done by observing their activity against indicator is currently being investigated. strains. REFERENCES Table 4: Agar well diffusion Indicator strain Cd2 Id2 [1] Juan L. Arques, Eva Rodriguez, Susana Langa, Jose Maria L. monocytogenes + ++ Landete, and Margarita Medina, “Antimicrobial Activity of P. aeruginosa + +++ Lactic Acid Bacteria in Dairy Products and Gut: Effect on S. aureus ++ ++ Pathogens”, Hindawi Publishing Corporation, BioMed + sign indicates inhibition. +,++,+++ denote the number of Research International 2013. plates that show inhibition. [2] Shih-Chun Yang, Chih-Hung Lin, Calvin T. Sung and Jia- You Fang, “Antibacterial activities of bacteriocins: application in foods and pharmaceuticals”, Frontiers in 5. TEMPERATURE STABILITY Microbiology 2014. Bacteriocins produced by both the strains were found [3] R Lagos, “Bacteriocins”, Elsevier 2013. to be temperature stable. The activity of the protein [4] “Characterization of bacteriocin produced by Lactic Acid Bacteria isolated from dairy products”, Review of Literature- was retained after storing it at 3 different temperature Chpt. 2. conditions. [5] Lipsy Chopra, Gurdeep Singh, Kautilya Kumar Jena &Debendrra K. Sahoo, “Sonorensin: A new bacteriocin with potential of an anti-biofilm agent and a food preservative”, Nature- scientific reports 2015. [6] R. D. Joerger, “Alternatives to Antibiotics: Bacteriocins, Antimicrobial Peptides and Bacteriophages”, Department of Animal and Food Sciences, University of Delaware, Newark, Delaware. [7] Cavera VL, Arthur TD, Kashtanov D, Chikindas ML, “Bacteriocins and their position in the next wave of conventional antibiotics”, International Journal of Antimicrobial Agents 2015. [8] Paul D. Cotter, R. Paul Ross and Colin Hill, “Bacteriocins — a viable alternative to antibiotics” , Nature Reviews Microbiology 2012. [9] Aran H-Kittikun et al,” Bacteriocin-producing Enterococcus faecalis KT2W2G isolated from mangrove forests in southern Thailand: Purification, characterization and safety evaluation”, Food Control 2014 Figure 4: Heat stability
Screening of Lactobacillus from Curd and Idli Batter for Production of Bacteriocins
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