Elevated serum cholesterol in humans is generally a risk factor correlated with the development of coronary heart disease. It has been reported that a culture of Lactobacillus acidophilus actively taking up cholesterol from a laboratory medium would function in vivo to exert a hypocholesterolemic effect. In the present study, seven strains of the genus Lactococcus were examined for their ability to remove cholesterol from laboratory media during growth. All strains of lactococci tested could remove cholesterol from media without degrading cholesterol. The amount of cholesterol removed was strain specific. Among them, Lc. Lactis subsp. lactis biovar diacetylactis N7 could remove as much cholesterol as L. acidophilus ATCC 43121, which had a beneficial influence on serum cholesterol levels in pigs. The manner of cholesterol removal by strain N7 corresponded to the manner of its growth. The growth of strain N7 (growth yield and growth efficiency) was enhanced. The fatty acid composition of the cells of strain N7 was altered by removing cholesterol from the media. The ability to remove cholesterol was also observed in the heat-killed cells of strain N7. However, the amount of cholesterol removed by the cells during growth was significantly higher than that removed by the heat-killed cells. Thus, strain N7 has the ability to remove cholesterol from media independently of whether cells are viable. These results indicate that administration of strain N7 in vivo may well be promising on the hypocholesterolemic effect.

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.

There have been few studies on the probiotic activity of Lactococcus strains although they are commonly used as starter bacteria in manufacturing many kinds of fermented dairy products. Nine strains of the genus Lactococcus were examined for their probiotic properties, such as adherence to human enterocyte‐like Caco‐2 cells and tolerance to acid and bile. Six strains were adhesive and the highest adhesion was observed with Lactcoccus lactis ssp. lactis NIAI527. This strain adhered to the microvilli of cells as observed by scanning electron microscopy and also tolerated low pH and bile. These properties should make strain 527 a potential new probiotic strain.

Few studies exist dealing with the probiotic activity of lactococci, which are commonly used as starter bacteria in the manufacture of many kinds of fermented dairy products. Fifteen strains of the genus Lactococcus were examined for their probiotic activities, such as immunomodulatory effects. Six strains induced the production of cytokines (IL-12, IL-6, and TNF-␣) in macrophage-like cell line J774.1, and the highest induction was observed with Lactococcus lactis subsp. lactis G50. The cytokine induction in the J774.1 cell line was almost entirely sustained after heat-killing of the strain. Spleen cells from BALB/c mice fed G50 culture produced more IL-12 and IFN-␥ and slightly less IL-4 and IL-6 than the control (i.e., without strain G50), indicating that strain G50 can enhance Th1-type immune response in vivo. The effect of the oral administration of strain G50 on antibody response in mice was also investigated. Mice were immunized with ovomucoid (OVM), a potent egg allergen, and the antibody level in the serum was then determined. The total IgE antibody level in the group treated with strain G50 was significantly lower than that of the control. The response of OVM-specific IgG1 and IgE antibodies tended to be low in the group that was administered strain G50, compared with the response of the control group. These results suggest that strain G50 has an ability to suppress the Th2 response. Thus, Lactococcus lactis subsp. lactis G50 is a potential probiotic strain for the suppression of hypersensitive reactions caused by the Th2 response.

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.