Bacteriocins and bacteriocin producers present in kefir and kefir grains
Powell, Jillian Elizabeth
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Kefir is a traditional fermented milk that is carbonated, has a sharp acidic taste, yeasty flavour and contains a low percentage alcohol (less than 2% (v/v)). The beverage is manufactured by fermenting milk with Kefir grains, comprised of microorganisms, polysaccharides and milk proteins. The microbial population of Kefir grains primarily include lactic acid bacteria (LAB), namely lactococci and lactobacilli, yeasts, Acetobacter and filamentous fungi. Kefir exhibits antimicrobial activity in vitro against some fungi, and Grampositive and Gram-negative bacteria. Although the exact cause of this inhibition in Kefir is not known, the ability of LAB to inhibit the growth of closely related bacteria is well known. This inhibition of pathogenic and spoilage microbes may be due to the production of organic acids, hydrogen peroxide, acetaldehyde, diacetyl, carbon dioxide or bacteriocins. Acid is not the only contributor to the antimicrobial activity of Kefir and Kefir grains, and bacteriocins may play a role in the inhibitory activity. The bacteriocin producer Lactobacillus plantarum ST8KF, isolated from Kefir and Kefir grains, produces a bacteriocin 3.5 kDa in size. The mode of activity of bacteriocin ST8KF (bacST8KF) is thought to be bacteriostatic in exponential cultures of Enterococcus faecalis E88, Lactobacillus casei LHS, Lactobacillus curvatus DF38, Lactobacillus sakei DSM 20017, Lactobacillus salivarius 241 and Listeria innocua F and LMG 13568. The peptide is sensitive to proteolytic enzymes and does not adsorb to the surface of the producer cell. The bacteriocin is stable between pH 2.0 and 10.0, and for 20 min at 121°C. Maximum bacteriocin activity was observed in modified MRS medium supplemented with glucose or saccharose, meat extract, KH2PO4, glycerol, thiamine or cyanocobalamin, or in modified MRS medium without tri-ammonium citrate. Maximum levels of adsorption of bacST8KF (80%) to Lb. casei LHS and Lb. sakei DSM 20017 were recorded. Adsorption (80%) of the bacteriocin to Lactobacillus paraplantarum ATCC 700211T and Streptococcus caprinus ATCC 700066, which are not sensitive to the bacteriocin was also recorded. Optimal adsorption to E. faecalis E88 was recorded at 25°C at pH 2.0, and to L. innocua LMG 13568 at 4°C, 10°C and 25°C at pH 6.0. Potassium ions, MgCl2, Tris, NH4- citrate, Na-acetate, Na2CO3, EDTA and SDS led to decreased adsorption to both sensitive strains, while NaCl and mercaptoethanol resulted decreased adsorption to E. faecalis E88, but not to L. innocua LMG 13568. Methanol resulted in lower levels of adsorption to L. innocua LMG 13568 but not to E. faecalis E88. Triton X-100 and Triton X-114 increased the adsorption of bacST8KF by 40%, and ethanol and chloroform had no effect on bacteriocin adsorption. The growth of Lb. plantarum ST8KF and L. innocua LMG 13568 in a mixed culture resulted in an increase of bacST8KF production. Cells treated with bacST8KF secreted DNA and galactosidase. As bacST8KF remains stable under a variety of conditions, the bacteriocin may have application, if awarded GRAS (generally regarded as safe) status, in various food products as a natural additive or preservative. The genes encoding bacteriocin production are located on a 3.9 kilo base (kb) plasmid. Curing of the plasmid resulted in a mutant strain of Lb. plantarum ST8KF, and the Lb. plantarum strains ST8KF(+) and ST8KF(-) differed with regards to antibiotic resistance and carbohydrate fermentation reactions. The wild type and the cured strain were incorporated into Kefir grains during mass cultivation. The survival of the bacST8KF sensitive Enterococcus mundtii ST4SA added to the milk during Kefir production using the enriched mass cultured grains was monitored using fluorescent in situ hybridization. Enterococcus mundtii ST4SA was present in higher numbers in the ST8KF(-) Kefir system when compared to the ST8KF(+) system. It can, therefore, be concluded that Lb. plantarum ST8KF(+) contributes to the antimicrobial activity of Kefir through the production of bacteriocin ST8KF.
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