Effect of Growth Media and Extended Incubation on the Appearance of Lactose - Nonfermenting Variants in Lactococci

Growth and storage were investigated for the development of Lac^- (lactose-nonfermenting) variants of Lactococcus lactis subsp. lactis C2, ML3 and L. lactis subsp. cremoris ML1, SC607 under different conditions, in an unbuffered medium (M17^-), and in media buffered with inorganic or organic phosphates. Strains were grown overnight (16-18 h) at 32°C and subsequently held at 32 and 22°C. The cell survival was much higher at 22°C than at 32°C after storage for 96 h. Most of the survivors in M17^- broth were of the Lac^- phenotype. Lac^- variants were also observed when the cultures were grown in skim milk at 32°C and then held at that temperature for 96 h. These results showed that the lactose-fermenting ability of lactococcus in general is lost when overnight cultures in M17^- broth are kept either at room temperature (22°C) or at 32°C for an extended period. However, the cultures in buffered media under similar conditions showed little or no loss of lactose-fermenting ability, suggesting that phosphate in the media had a stabilizing effect on plasmid-encoded lactose-fermenting gene(s). These observations indicate the possibility of utilizing this method as a simple technique for isolating mutants deficient in plasmid-linked genetic traits in lactococci.