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Isolation and Characterization of Potential Bacteriocin Producing Lactic Acid Bacteria from Fermented Food Products

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Isolation and Characterization of Potential Bacteriocin Producing Lactic Acid Bacteria from Fermented Food Products Isolation and Characterization of Potential Bacteriocin Producing Lactic Acid Bacteria From Fermented Food Products S. Keerthini ( [email protected] ) University of Jaffna R. Kapilan University of Jaffna S. Vasantharuba University of Jaffna Research Article Keywords: Antibacterial activity, bacteriocin, fermented food products, food spoilage organisms, LAB Posted Date: August 24th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-815679/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/9 Abstract Background Probiotic, Lactic Acid Bacteria (LAB) are consumed by human through the traditional food products for several decades due to their benecial health effects. LAB is Gram positive, non motile bacteria that produce antimicrobial peptides for their defense mechanism. Among the antimicrobial peptides, Bacteriocins are widely investigated because of their possible utilization in food sector especially food security actions, where bacteriocin kills or inhibits the growth of other bacteria. Usage of metabolic products of LAB and LAB are generally recognized as safe. Therefore, this study was aimed to isolate LAB species that produce bacteriocin showing wide spectrum antibacterial activity. Results Lactic acid bacteria were isolated from yoghurt, curd, dosa batter, idli batter and soaked and ground rice batter using (De Man, Rogosa and Sharpe agar) MRS agar and incubated at room temperature (30±2ºC) for 24-72 h aerobically and anaerobically. When agar well diffusion method was employed to detect the antibacterial activity of the twenty ve isolates against food spoilage organisms (Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, E.coli, Klebsiella pneumoniae, Serratia marcescens, Salmonella sp., Proteus sp., Micrococcus sp., and Bacillus sp.,). Three bacterial species showing broad spectrum of antibacterial activity, were identied and characterized by biochemical and molecular methods. The wide spectrum antibacterial bacterial species were identied as Lactobacillus reuteri AF182723 isolated from curd, Lactobacillus rhamnosus AY299488 isolated from rice batter and Lactobacillus acidophilus AF182726 isolated from yoghurt. Among all the isolates, Lactobacillus reuteri AF182723 showed signicant wide spectrum antibacterial activity against diverse Gram positive and Gram negative bacterial species. Conclusion The production of bacteriocin from Lactobacillus reuteri AF182723 and incorporating it in the food would inhibit the food spoilage organisms. Usage of this bacteriocin at appropriate concentration in food needs to be studied further. Background Lactic acid bacteria (LAB) are long been consumed by people in several fermented foods. Lactic acid bacteria display numerous antimicrobial activities due to the production of organic acids, bacteriocins and antifungal peptides. Bacteriocins are antibacterial proteins and posttranslational modied peptides produced by bacteria that have a bacteriocidal or bacteriostatic effect on other bacterial species (Cleveland et al., 2001). Bacteriocins are generally recognized as "natural" compounds that can inuence the safety and quality of foods. Since food safety has become an increasingly important international concern, the application of bacteriocin from lactic acid bacteria, which target food pathogens without toxic or other adverse effects, has received great attention (Cleveland et al., 2001). Though Bacteriocins could be categorized as antibiotics; they are not produced during the primary phase of growth. Bacteriocins from LAB are peptides with antimicrobial activity synthesized by ribosomes as part of their defense mechanism and potential to cover a very broad eld of application, including both the food industry and the medical sector (Vuyst and Leroy, 2007). Most of LAB bacteriocins are small (< 10 kDa) cationic, heat-stable, amphiphilic and membrane permeabilizing peptides. Nowadays usage of bacteriocins in food industry has been extensively investigated (Zacharof and Lovittb, 2012). Bacteriocins produced by LAB have the potential to cover a very broad eld of application in food industry. Usage of LAB and their metabolic products generally considered as safe (Zacharof and Lovittb, 2012). Therefore, this study was aimed to isolate of LAB from fermented food products and to screen and identify the bacterial species showing wide spectrum antibacterial activity. Results Isolation of lactic acid bacteria from fermented food products Lactic acid bacteria (LAB) were isolated by using MRS agar containing bromocresol green as an indicator for acid formation. Bromocresol green will change to yellow colour in acidic condition (Fhoula et al., 2013). Therefore, colonies with yellow colour zone were randomly picked 24–72 h intervals and streaked repeatedly on MRS agar. Resulted pure colonies were streaked on MRS agar substituted with 1% cycloheximide to conrm the presence of yeast. Twenty-ve bacterial isolates were obtained from the fermented food products such as curd, yoghurt, rice batter, dosa batter and idli batter by using of MRS agar plate substituted with 1% cycloheximide were gram positive and catalase negative. Therefore, these 25 isolates were considered as LAB bacteria. From the experiment, four LAB isolates were isolated from curd, ve from yoghurt, ve from rice batter, six from dosa batter and ve from idli batter. Detection of antibacterial activity of isolated lactic acid bacteria Results of the antibacterial activity study of the isolated LAB are explained in Table 1. The potential three bacterial genera which inhibited the highest number of food spoilage organisms were selected for further studies. Page 2/9 Table 1 Average inhibitory zone of isolated lactic acid bacteria from fermented food products Isolate Staphylococcus Enterococcus E.coli Pseudomonas Klebsiella Serratia Salmonella Proteus Micrococcus Bacillus aureus faecalis aeruginosa pneumoniae marcescens sp sp sp sp C1 11.8 ± 0.2 11.57 ± 0.2 - - - - - - - 11.98 ± 0.1 C2 - - - 11.67 ± 0.1 - 10.41 ± 0.3 - 10.67 11.20 ± 0.4 - ± 0.1 C3 11.50 ± 0.2 11.34 ± 0.1 11.50 10.92 ± 0.3 10.75 ± 0.3 10.92 ± 0.2 11.25 ± 0.2 - 10.34 ± 0.1 11.86 ± ± 0.1 0.2 C4 10.54 ± 0.1 - - 10.79 ± 0.2 - - - - - - Y1 - - 11.73 - 11.84 ± 0.4 - - - 11.19 ± 0.3 10.67 ± ± 0.2 0.2 Y2 - - - - - - - 10.87 - - ± 0.1 Y3 - - - - - 10.97 ± 0.1 - 11.30 10.61 ± 0.2 11.35 ± ± 0.2 0.3 Y4 - - 12.80 12.05 ± 0.2 - - - 13.38 - - ± 0.1 ± 0.3 Y5 12.27 ± 0.2 - - 10.75 ± 0.4 11.62 ± 0.2 - 10.59 ± 0.3 11.65 - 10.45 ± ± 0.1 0.2 R1 - - 12.16 - 11.52 ± 0.1 - - 11.88 12.07 ± 0.1 11.50 ± ± 0.1 ± 0.2 0.2 R2 10.93 ± 0.1 - 11.16 - - 10.81 ± 0.1 - 11.27 10.81 ± 0.1 10.61 ± ± 0.2 ± 0.1 0.2 R3 12.97 ± 0.3 11.69 ± 0.2 12.14 12.00 ± 0.1 - - - - 14.01 ± 0.2 - ± 0.2 R4 - - 11.32 - - - - - - - ± 0.2 R5 - - 10.90 - 13.43 ± 0.2 - - 14.40 11.26 ± 0.1 - ± 0.1 ± 0.1 D1 11.11 ± 0.2 13.87 ± 0.2 - - 10.57 ± 0.2 15.52 ± 0.3 - - 15.13 ± 0.4 - D2 - - 11.40 - - - - 10.64 - - ± 0.2 ± 0.1 D3 11.41 ± 0.2 - - - - - 13.09 ± 0.1 11.49 - - ± 0.2 D4 - - - 10.92 ± 0.3 - - 12.73 ± 0.4 10.79 11.62 ± 0.1 - ± 0.1 D5 - - - - - - 10.66 ± 0.2 - 11.78 ± 0.1 - D6 - - - - - - 10.50 ± 0.1 10.92 11.22 ± 0.4 - ± 0.1 I1 - 13.85 ± 0.2 - - - 12.04 ± 0.1 - - - - I2 - 11.41 ± 0.1 - 10.73 ± 0.4 - - - 10.99 - - ± 0.1 I3 11.29 ± 0.2 - - - - - 11.35 ± 0.2 11.06 - - ± 0.3 I4 - - - - - - 10.66 ± 0.1 - 12.14 ± 0.1 - I5 11.21 ± 0.1 - - - - - 10.35 ± 0.2 - 11.56 ± 0.4 - Clear zones were measured in mm against control. MRS broth was used as control. Results represent the mean ± standard deviation of three replicates. Here C – strains from curd, Y- strains from yoghurt, I- strains from idli batter, D- strains from dosa batter, R- strains from rice batter. Physiological characterization of selected bacterial strains Physiological characterizations of the selected strains were shown in Table 2. The isolates C3 & Y5 showed growth at 25ºC and 45ºC respectively. But they didn’t show any growth at 15ºC and 55ºC. The isolate R2 showed growth at 15ºC, 25ºC and 45ºC. The isolates C3, R2 & Y5 did not grow at pH 1.0 and 2.0. The Page 3/9 isolate C3 showed growth between pH 3.0 and pH 8.0, while the isolate R2 grew well between pH 3.0 and pH 1.0. The isolate Y5 showed growth between pH ranges 4.0 to 9.0. All the selected isolates showed growth up to the 12% NaCl concentration. Table 2 Physiological characterization of the selected lactic acid bacterial isolates C3 R2 Y5 15ºC - + - 25ºC + + + 45ºC + + + 55ºC - - - pH 1.0 - - - pH 2.0 - - - pH 3.0 + + - pH 4.0 + + + pH 5.0 + + + pH 6.0 + + + pH 7.0 + + + pH 8.0 + + + pH 9.0 - + + pH 10.0 - + - pH 11.0 - - - 1% − 4% NaCl + + + 4% − 8% NaCl + + + 8% − 12% NaCl + + + 12%- 16% NaCl - - - + = Positive reaction - = Negative reaction v = variable Biochemical characterization of selected bacterial strains The biochemical characterization of selected bacterial isolates was shown in Table 3. All the selected bacterial isolates didn’t show motility and gave negative results for endospore staining. Similarly all the bacterial isolates were citrate positive and showed negative reaction to gelatin hydrolysis. R2 showed negative reaction to Arginine hydrolysis while C3 and Y5 showed variable reactions (positive and negative response) to Arginine hydrolysis. Different carbohydrate fermentation patterns observed for selected bacterial isolates were shown in Table 3. The isolate C3 utilized glucose, sucrose, lactose, galactose, fructose, xylose, maltose, mannose, arabinose and ribose, showed positive and negative response to sorbitol and mannitol and didn’t utilize starch and ranose. The isolate R2 utilized glucose, sucrose, lactose, galactose, sorbitol, fructose, xylose, maltose, ranose, mannitol, mannose and ribose, didn’t hydrolyze starch and arabinose.
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