Some recent evidences support the probable presence, during cheese late ripening, of microbial species belonging to starter populations as Lactococcus lactis. The detection and vitality of L. lactis starter cultures were investigated, by culture-dependent and -independent techniques, in commercial and artisanal ripened cheeses. Quantitative PCR (qPCR) and Reverse Transcriptase (RT)-qPCR protocols were optimized and standard curves were constructed from serially diluted cells of L. lactis in physiological solution and inoculated in grated cheese used as biological model. Ten grams of each cheese sample were submitted to direct extraction of nucleic acids and qPCR and RT-qPCR protocols. In this way, a quantification of L. lactis populations was reached in terms of both L. lactis total cells (at DNA level) and metabolically active populations (at RNA level). The cheese samples resulted positive for the presence of active L. lactis populations by RT-qPCR, were submitted to traditional culture-dependent analysis on lactococci selective medium M17 agar, in order to check for L. lactis colonies. DNA extraction and L. lactis species-specific PCR were carried out on the colonies isolated in order to assign their belonging to this species. Culture-independent analysis highlighted the presence of metabolically active populations of L. lactis in most of the cheeses examined at advanced stage of ripening. On the contrary, traditional plating on selective medium underlined a low number of L. lactis colonies probably due to the presence of microbial cells viable and metabolically active but not cultivable (VNC), and to the microbial competition on selective medium. These results highlight, once again, the limitations due to culture-dependent approach and the possibility to overcome it by culture-independent method. Further studies will be carried on the technological role of L. lactis in ripened cheeses.
Vitality of Lactococcus lactis throughout cheese ripening
RUGGIRELLO, MARIANNA;DOLCI, Paola;COCOLIN, Luca Simone
2013-01-01
Abstract
Some recent evidences support the probable presence, during cheese late ripening, of microbial species belonging to starter populations as Lactococcus lactis. The detection and vitality of L. lactis starter cultures were investigated, by culture-dependent and -independent techniques, in commercial and artisanal ripened cheeses. Quantitative PCR (qPCR) and Reverse Transcriptase (RT)-qPCR protocols were optimized and standard curves were constructed from serially diluted cells of L. lactis in physiological solution and inoculated in grated cheese used as biological model. Ten grams of each cheese sample were submitted to direct extraction of nucleic acids and qPCR and RT-qPCR protocols. In this way, a quantification of L. lactis populations was reached in terms of both L. lactis total cells (at DNA level) and metabolically active populations (at RNA level). The cheese samples resulted positive for the presence of active L. lactis populations by RT-qPCR, were submitted to traditional culture-dependent analysis on lactococci selective medium M17 agar, in order to check for L. lactis colonies. DNA extraction and L. lactis species-specific PCR were carried out on the colonies isolated in order to assign their belonging to this species. Culture-independent analysis highlighted the presence of metabolically active populations of L. lactis in most of the cheeses examined at advanced stage of ripening. On the contrary, traditional plating on selective medium underlined a low number of L. lactis colonies probably due to the presence of microbial cells viable and metabolically active but not cultivable (VNC), and to the microbial competition on selective medium. These results highlight, once again, the limitations due to culture-dependent approach and the possibility to overcome it by culture-independent method. Further studies will be carried on the technological role of L. lactis in ripened cheeses.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.