Aims:  The aim of this study was to obtain new Lactococcus lactis strains from nondairy materials for use as milk fermentation starters. The genetic and phenotypic traits of the obtained strains were characterized and compared with those of L. lactis strains derived from milk. It was confirmed that the plant‐derived bacteria could be used as milk fermentation starters. Methods and Results:  About 2600 lactic acid bacteria were subjected to screening for L. lactis with species‐specific PCR. Specific DNA amplification was observed in 106 isolates. Forty‐one strains were selected, including 30 strains of milk‐derived and 11 of plant‐derived, and their phenotypic traits and genetic profiles were determined. The plant‐derived strains showed tolerance for high salt concentration and high pH value, and fermented many more kinds of carbohydrates than the milk‐derived strains. There were no remarkable differences in the profiles of enzymes, such as lipases, peptidases and phosphatases. Isolates were investigated by cluster analysis based on randomly amplified polymorphic DNA profiles. There were no significant differences between isolates from milk and those from plant. The L. lactis subsp. cremoris strains were clustered into two distinct groups, one composed of the strains having the typical cremoris phenotype and the other composed of strains having a phenotype similar to subsp. lactis. Fermented milk manufactured using the plant‐derived strains were not inferior in flavour to that manufactured using the milk‐derived strains. Conclusions:  Plant‐derived L. lactis strains are genetically close to milk‐derived strains but have various additional capabilities, such as the ability to ferment many additional kinds of carbohydrates and greater stress‐tolerance compared with the milk‐derived strains. Significance and Impact of the Study:  The lactic acid bacteria obtained from plants in this study may be applicable for use in the dairy product industry.

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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.

The effects of oral administration of a lactococcal strain on physiological changes associated with ageing were investigated using senescenceaccelerated mice (SAM). SAM develop normally, but then show an early onset and irreversible advancement of senescence. SAMP6 is a SAM strain that develops osteoporosis with ageing. Oral administration of heat-killed Lactococcus lactis subsp. cremoris H61 (strain H61) to aged SAMP6 mice was associated with reduced bone density loss, a suppression of incidence of skin ulcers and reduced hair loss, compared with controls. Spleen cells from mice fed strain H61 produced more interferon-g and IL-12 than those from control mice, suggesting that administration of strain H61 altered immune responses. The numbers of viable cells of Bifidobacterium sp., Bacteroides sp. and Enterococcus sp. in faeces were similar for mice fed the strain H61 and control diets, but counts for Staphylococcus sp. were significantly lower (P,0·05) in mice fed strain H61. Mice fed strain H61 had similar serum concentrations of thiobarbituric acid-reactive substances as in controls, indicating a lack of effect on lipid peroxidation status. Administration of living cells of strain H61 or fermented milk containing strain H61 was also associated with a suppression of incidence of skin ulcers and reduced hair loss. These results indicate that oral administration of strain H61 has the potential to suppress some of the manifestations associated with ageing.

One of the important properties of probiotics is the ability to survive in the intestine. There have been few studies on the probiotic property of lactococci, since they are formally not considered to be natural inhabitants of the intestine. To evaluate lactococci as probiotic bacteria, we investigated their ability to survive during gastric transit by in vitro and in vivo tests. When exposed to an in vitro simulated gastrointestinal environment, such as low pH and bile, only Lactococcus lactis subsp. lactis bv. diacetylactis N7 showed a moderate survival rate among the four strains tested. The tested strains were orally administered to mice, and intestinal passage of the ingested strains was monitored by two methods: antibiotics and PCR. Viable cells of strain N7 were recovered from feces within 24-48 h after administration but not at 72 h. Lactococcus lactis subsp. cremoris ATCC 19257, which had a poor survival rate in vitro test, was also detected at 12 h but not at 24 h. These results indicate that lactococci can reach the mouse intestine alive, but not colonize it. If administered daily, viable strain N7 may exist continuously in the intestine. The effect of strain N7 on intestinal microbial balance and on animal health will be the subject of a further study.