Study of Lactococcus lactis during advanced ripening stages of model cheeses characterized by GC-MS.

Lactococcus lactis, is extensively used as starter culture in dairy products. Nevertheless, it has recently been detected in cheese, as metabolically active cells, in advanced ripening stages. In this study, we assessed the viability of L. lactis subsp. lactis in model cheeses up to 180 days of ripening by both culture-dependent and -independent methods. In addition, we studied the expression of metC and als genes involved in the production of aroma compounds detected by Gas Chromatography-Mass Spectrometry (GC-MS). Three L. lactis subsp. lactis commercial starters were inoculated in pasteurized milk and model cheeses were manufactured and ripened for six months. Samples were analysed at manufacturing and ripening steps, in terms of viability of L. lactis by both traditional plating and direct analysis of RNA by reverse transcription quantitative PCR (RT-qPCR), and in terms of aroma profile by GC-MS. Relatively to RT-qPCR analysis, L. lactis was found viable throughout the whole process of cheesemaking and aging, with final average loads of 3-4 Log CFU/g at 180 days. On the contrary, the microorganism was not detected, in ripened samples, by traditional plating on M17 medium, suggesting its entering in a viable but not cultivable (VBNC) state. The aroma profiles of the cheeses highlighted the presence of volatile compounds related to cheese flavor as acetoin, diacetyl, 2,3-butanediol and dimethyl disulfide, whose presence was partially correlated to metC and als genes expression. These results add new insights on the capability of L. lactis to persist during late cheese ripening and suggest a potential contribution of the microorganism to cheese flavor formation.