Microbial succession during Parmigiano-Reggiano cheesemaking was monitored by length heterogeneity PCR (LH-PCR), considering the intact and lysed cells at different stages of cheese production and ripening. When starter species underwent autolysis, species coming from milk were able to grow. For the first time, the LH-PCR technique was applied to study a fermented food.Parmigiano-Reggiano (PR) is a protected-designation-of-origin cheese, produced in specific areas of Northern Italy. It is a hard-textured, cooked, and long-ripened cheese made from raw cow's milk supplemented with natural whey starter rich in thermophilic lactic acid bacteria (LAB). Microbiological features of PR have been studied, isolating on traditional growth media a large number of strains from a large amount of samples representative of production and of the earlier and advanced stages of ripening (2, 3). Other studies have focused on the biodiversity of different strains of Lactobacillus helveticus isolated from natural whey cultures (6, 7). These previous studies showed the microbial biodiversity of PR but not its real microbial succession during production and ripening, and the use of culture-dependent methods could have underestimated the less-abundant components of microflora, which are equally important for cheese ripening and flavor development (15). Thus, the microbial ecology of PR still appears not to be completely understood. From this perspective, 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 appreciated cheese.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. The availability of PR twin wheels allowed us to have samples representative of the subsequent stages of the same cheesemaking process.In our opinion, the new findings in this article contribute to a better understanding of microbial dynamics in a complex fermented ecosystem.Cheesemaking. A unique cheese factory was selected for its equipment and technological conditions in order to obtain a suitable number of PR twin wheels. The same milk was distributed in equal volumes in eight vats and was worked according to PR production standards.Thirty-four liters of natural whey starter was added in each vat, containing 1,070 liters of milk (525 liters of partially skimmed evening milk and 545 liters of whole morning milk), bringing the pH of the mixture to 6.20. After cheesemaking and molding, the wheels, two from each vat, were held for 3 days and frequently turned to enable complete whey drainage. They were then salted by immersion in brine at 300 g NaCl ⅐ liter Ϫ1 for 23 days. Ripening was carried out in aging rooms with 85% relative humidity and a temperature of about 18°C for 24 months.Cheese sampling and bacterial recovery. Samples were collected from milk to cheese ripened for 24 months. Aliquots of the total mass of raw...

Lactobacillus helveticus CNRZ 32 is recognized for its ability to decrease bitterness and accelerate flavor development in cheese, and has also been shown to release bioactive peptides in milk. Similar capabilities have been documented in other strains of Lb. helveticus, but the ability of different strains to affect these characteristics can vary widely. Because these attributes are associated with enzymes involved in proteolysis or AA catabolism, we performed comparative genome hybridizations to a CNRZ 32 microarray to explore the distribution of genes encoding such enzymes across a bank of 38 Lb. helveticus strains, including 2 archival samples of CNRZ 32. Genes for peptidases and AA metabolism were highly conserved across the species, whereas those for cell envelope-associated proteinases varied widely. Some of the genetic differences that were detected may help explain the variability that has been noted among Lb. helveticus strains in regard to their functionality in cheese and fermented milk.

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-Parmigiano Reggiano is a Protected Designation of Origin, long-ripened cheese, made from cow's milk supplemented with natural whey starter, which thus contains a large microbial biodiversity. The aim of this study was to understand the population dynamics of the total lactic microflora throughout the manufacture and ripening of this cheese. Several approaches were combined to determine the quantitative changes in the different bacterial populations during 20 months of ripening of Parmigiano Reggiano cheeses from the same cheesemaking. Total and viable cells were enumerated after fluorescent labeling. Culturable bacteria were enumerated on different plate count agar media, including original media prepared from curd and ripened cheese. Six peptidase activities were quantified in curd and cheese samples free from cells. While the total bacterial cultivable population remained high and similar for the first six months, a decrease in viable starter lactic acid bacteria was observed during the first 48 h. The non-starter lactic acid bacteria populations, initially present in low numbers, began to grow after the brining and remained at high levels (about 10 7 CFU·g −1 ) for at least 10 months. During ripening, a strong decrease in the total bacterial population and a marked increase in 4 out of 6 peptidase activities were observed. In the external and internal zones of Parmigiano Reggiano cheese different trends in microbial growth, cell autolysis and peptidase activity were observed. This study gives for the first time a global view of the possible contribution of total, viable, cultivable and lysed bacterial cells throughout the ripening of Parmigiano Reggiano cheese.Parmigiano Reggiano cheese / ripening / lactic acid microflora / bacterial cell lysis / cell viability / peptidase activity