Identificazione di batteri lattici mediante RSA e pentaplex PCR nel sieroinnesto per Grana Padano.

Lactic acid bacteria present in wheystarter are a key factor for obtaining typical raw milk cheeses such as Grana Padano. Determination of microbial composition of natural wheystarter is therefore an important point to control the production process and consequently to achieve the typical and appreciated sensory characteristics of cheese. In this work we have compared two analytical methods for LAB identification in wheystarter culture: a Pentaplex PCR assay and the analysis of 16S-23S rDNA polymorphic region (RSA). The Pentaplex PCR was optimized to identify simultaneously Lactobacillus helveticus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, L. fermentum and S. thermophilus, that constitute the main component of wheystarter in dairy products such as Grana Padano and Parmigiano-Reggiano. The analysis of RSA is commonly used for rapid identification of bacterial isolates; the amplification of this hypervariable region allows specific amplifications for particular bacterial groups. In both cases, the protocol provides for the amplification of total DNA extracted directly from wheystarter. In this work, 35 wheystarter for Grana Padano, collected during the period between January and April 2010 from two dairy farms located in Lombardy, were analyzed. The results obtained with the Pentaplex PCR showed the presence of L. helveticus and L. delbrueckii subsp. lactis in all the wheystarter analyzed. S. thermophilus and L. fermentum were found respectively in 13 (37.1%) and 15 (42.8%) samples analyzed. No wheystarter culture was positive for L. delbrueckii subsp. bulgaricus. The RSA analysis did not allow the identification of the different rod-shaped species present in association the wheystarters. However, it was possible to detect in 13 samples the presence of S. thermophilus, confirming the results obtained with the Pentaplex-PCR.