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.