Nine mixed-strain starters were examined for their abilities to produce gamma-aminobutyric acid. Six commercial starters were found to produce gamma-aminobutyric acid in a skim milk culture. The bacterium that produced gamma-aminobutyric acid was isolated from the mixed-strain starters, identified as citrate-utilizing Lactococcus lactis ssp. lactis (formerly L. lactis ssp. lactis biovar diacetylactis) and designated as strain 01-7. A cell extract showed glutamate decarboxylase activity, for which the optimum pH was 4.7. In pH-controlled cultivation, gamma-aminobutyric acid was generated at pH 5.0 but not above pH 5.5. Cheeses were prepared experimentally using strain 01-7 to determine the relationship between the pH values and the production of gamma-aminobutyric acid during cheese ripening. gamma-Aminobutyric acid increased linearly in the experimental cheeses as the pH of the cheese decreased. Based on these results, gamma-aminobutyric acid was concluded to be produced by the cheese starters during ripening.
Glutamate decarboxy~ase, which is associated with a glutamate-dependent acid-resistance mechanism, was purified from Lactococcus lactis subsp. lactis by a three-step procedure. The specific activity was increased about 114-fold with a yield of 16%. The N-terminal amino acid sequence of the enzyme was determined. The gene encoding this enzyme was cloned in kcherichia coli, and its nucleotide sequence was determined. The deduced amino acid sequence suggests that the enzyme is produced as a mature form (466 amino acid residues), not as a precursor protein. The subunit molecular mass of L. lactis glutamate decarboxylase was calculated to be 53926 Da. The enzyme was maximally active a t pH 4 7 and reacted only with L-glutamate among 20 a-amino acids. The apparent Km value was calculated to be 0.51 mM. The activity was stable at acidic pH values; there was no activity in the neutral pH range. A t pH 4 1 the enzyme activity was retained at temperatures up to 70 "C in 10 min incubations. L. lactis glutamate decarboxylase behaved as a single protein when the enzyme was purified. A single band corresponding to the glutamate decarboxylase gene was detected on Southern blot analysis. These data suggest that there is one glutamate decarboxylase gene in L. lactis.
Lactococcus lactis strains were examined for their ability to produce y-aminobutyric acid (GABA). Results showed that strains of L. lactis subsp. lactis were able to produce this acid, whereas L. lactis subsp. cremoris were not. GABA production thus represents another effective characteristic for distinguishing L. lactis subsp. lactis from L. lactis subsp. cremoris.
The most widely used probiotic bacteria are lactobacilli and bifidobacteria, which have been isolated from the human gastrointestinal tract. The development of new probiotic strains, which are more feasible and beneficial organisms, is awaited in the dairy industry. Lactococci would be promising species because of their extensive usage in manufacturing dairy products such as cheese and fermented milk. However, there have been few studies on the probiotic activity of lactococci since they are traditionally not considered to be natural inhabitants of the human gastrointestinal tract. Recently, several works showed the possibility of the presence of lactococci in the flora of the human or animal gastrointestinal tract. In this review, we would like to propose Lactococcus sp. as new probiotic bacteria.
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