Optimizing the Production of Bacteriocins by Lactic Acid Bacteria Isolated from Foods Using Improved Deferred Antagonism Assay.

Abstract

Bacteriocins are antimicrobial peptides or proteins produced by certain bacteria. Lactic acid bacteria (LAB) are common bacteriocin-producers and often used in the production of fermented food. These bacteria and bacteriocins they produce can inhibit certain bacteria causing food spoilage and foodborne disease. It is time consuming and labor intensive to isolate bacteriocin producing LAB from food. This study aimed to (1) improve the bacteriocin-producer isolation method, deferred antagonism assay, by optimizing media composition; (2) isolate and identify bacteriocin producing LAB from fermented foods; and (3) determine the influence of different growth conditions on the production of bacteriocins by those LAB isolates. To identify more appropriate media to isolate bacteriocin-producers, three types of media (de Man, Rogosa and Sharpe [MRS] agar, M17 agar, and Elliker agar) with two types of buffering salts (disodium-β-glycerophosphate and the combination of Na2HPO4 and NaH2PO4) at different initial media pH (5.5-6.9) were tested with known six LAB strains via deferred antagonism assay. Tween 80 and ethanol were added at 1% to the isolation media to assess their effect on bacteriocin production. Both bacteriocin producing and bacteriocin non-producing LAB formed inhibition zones on MRS agar with two types of buffering salts. There was no inhibition zone formed by bacteriocin non-producers in the other two types of media (M17 agar and Elliker agar). The bacteriocin-producers generated significantly larger inhibition zones in Elliker agar than in M17 agar. The buffering salts did not significantly affect the size of inhibition zones. But disodium-β-glycerophosphate was known to inhibit the growth of Lactobacillus bulgaricus in previous study. The size of inhibition zones, formed by the tested bacteriocin producing LAB, enlarged with the application of higher initial media pH and the supplement 1% Tween 80. Therefore, the Elliker agar with buffering salts (Na2HPO4 and NaH2PO4) and 1% Tween 80 at pH 6.9 was the most appropriate bottom media in deferred antagonism assay. The improved deferred antagonism assay was employed to isolate bacteriocin producing LAB from kimchee, sauerkraut, yogurt and kefir. Bacteriocin-encoding genes in the isolates were amplified and sequenced. In addition, the inhibition spectrum of the isolates’ bacteriocins was also determined by inoculated with four common pathogenic bacteria. A total of 10 bacteriocin producing LAB were isolated from those fermented food samples, which included 8 Lactococcus lactis and 2 Lactobacillus plantarum. Based on the result of random amplification of polymorphic DNA-polymerase chain reaction, the Lactococcus lactis isolates and Lactobacillus plantarum isolates could be divided into 4 groups and 1 group, respectively. Isolated Lactococcus lactis carried nisin Z and lactococcin 972 genes, and isolated Lactobacillus plantarum carried plantaricin S gene. Three types of the Lactococcus lactis isolates not only inhibited the growth of Listeria monocytogenes but also showed antimicrobial activity against Staphylococcus aureus. To optimize the production of bacteriocins by those LAB isolates, the influence of different liquid culture media (MRS, M17 and Elliker broth) at different initial pH (5.5-6.9) with 1% Tween 80 and/or 1% ethanol on their antimicrobial activity was determined. The Lactococcus lactis isolates produced more bacteriocins in MRS broth than in M17 broth and Elliker broth. In comparison, the type of culture medium did not significantly affect bacteriocin production by the Lactobacillus plantarum isolates. Higher initial medium pH increased the bacteriocin production. The bacteriocin units of all isolates at pH 6.9 were more than three-fold higher than at pH 5.5. As for the supplements, 1% Tween 80 effectively increased bacteriocin production by the Lactococcus lactis isolates, and 1% ethanol showed remarkable enhancement in bacteriocin production by the Lactobacillus plantarum isolates. In summary, the optimized bottom media improves the deferred antagonism assay and provides a more effective approach to isolating bacteriocin-producers. The bacteriocin producing LAB isolates identified in this study can potentially be used as starter cultures in fermented foods to improve their quality and safety. Further work is needed to purify their bacteriocins and test them as natural antimicrobials in food preservation.