Parmigiano-Reggiano (PR) is a long-ripened cheese made from raw cow’s milk supplemented with natural whey starter rich in thermophilic lactic acid bacteria (LAB). The use of culture-dependent methods to study microbial succession could underestimate the less-abundant components of microflora, which are equally important for cheese ripening and flavor development. A detailed knowledge of LAB dynamics during the manufacturing and ripening stages is necessary for a deeper insight into the complex processes which contribute to the development of this product. In the present study, length heterogeneity PCR (LH-PCR) was used to monitor the microbial dynamics during 24 months of PR ripening for both the whole and lysed cells.
Samples were collected from milk to cheese ripened for 24 months. In order to recover a heterogeneous bacterial population, different types of nutritional media were used. A total of 187 strains were isolated and the genomic DNA was extracted. All species identified by 16S rRNA gene sequencing were fingerprinted. Strains belonging to eight different species or belonging to the same species but with a different randomly amplified polymorphic DNA (RAPD-PCR) profile and coming from different samples, were chosen to generate the LH-PCR database. To investigate evolution of the microbial community in PR cheese, DNA was extracted from samples both from lysed cells and whole cells and the LH-PCR was performed.
The dendrogram obtained reveal a high biodiversity among strains and species present in a complex system like PR cheese. Starter LAB and non-starter LAB dynamically evolve during ripening. When starter thermophilic species underwent autolysis, species non starter coming from milk were able to grow. These results are also important to understand the moments of release of cytoplasmatic enzymes important for the casein peptides proteolysis in ripening.
Monitoring both entire and lysed cells through LH-PCR allows to coming aware of the importance of this two fractions in PR cheese production and ripening. For the first time, the LH-PCR technique was applied to study a fermented food. This approach opens perspectives for insight microbial evolution in fermented food environment. The new findings in this study contribute to a better understanding of microbial dynamics in a complex fermented ecosystem.