Detection and vitality of Lactococcus lactis during ripening of model cheeses.

Recently, the presence, during late cheese ripening, of metabolically active populations of Lactococcus lactis, commonly used as starter culture, was investigated. Thus, the main goal of the present study was to assess the persistence and vitality of this microorganism throughout manufacturing and ripening of model cheeses. Eleven commercial starters, constituted of L. lactis subsp. lactis, L. lactis subsp. cremoris and L. lactis subsp. lactis biovar diacetylactis, were inoculated in pasteurized milk, and miniature cheeses were manufactured and ripened for six months. Samples were analyzed at different steps (milk after inoculum, curd after cutting, curd after pressing and draining, cheese immediately after salting and cheese at 7, 15, 30, 60, 90, 120, 150 and 180 days of ripening) and submitted to both culture-dependent and -independent analysis. Quantitative-PCR (qPCR) and RT-qPCR protocols, to detect selectively L. lactis in cheese, were optimized in a previous study. On the basis of RNA analysis, L. lactis was found, in miniature cheeses, with loads up to 104 colony forming units (CFU)/g until the sixth month of ripening. Moreover, in order to investigate the possible presence of viable but not cultivable (VBNC) cells of L. lactis in ripened cheeses, traditional plating was also performed on the same samples. Lactococci were isolated on selective medium M17 and the isolates were submitted to species-specific PCR for L. lactis, which was not found in a few cheese samples, even when its presence was detected by RT-qPCR. Thus, it could be hypothesized that L. lactis starter populations were mainly present in VBNC state during ripening and, for this reason, culture-dependent methods have to be complemented with direct analysis of microbial RNA. As future prospective, it will be important to support these data with studies on large-scale and, eventually, investigate the possible role of L. lactis during the ripening process.