Bacteriocins and bacteriocin producers present in kefir and kefir grains
Date
2006-03
Authors
Journal Title
Journal ISSN
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Publisher
Stellenbosch : University of Stellenbosch
Abstract
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.
Description
Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2006.
Keywords
Dissertations -- Food science, Theses -- Food science, Kefir, Bacteriocins, Grain, Lactobacillus plantarum, Antimicrobial activity of kefir