Survey on the community and dynamics of lactic acid bacteria in Grana Padano cheese

Grana Padano (GP) is a Protected Designation of Origin cheese made with raw milk and natural whey culture (NWC) that is characterised by a long ripening period. In this study, six GP productions were considered in order to evaluate the trend of microbial dynamics and compare lactic acid bacteria (LAB) population levels in cheeses during the entire cheese-making process. To reach this goal, for each GP production, samples of vat raw milk, NWC and cheeses at 48 h, 2, 6, 9 and 13 months were subjected to plate counts and direct counts by fluorescence microscopy, as well as amplicon length heterogeneity-PCR (LH-PCR). Statistical analysis was applied to the results and ecological indices were estimated. It was demonstrated that the LAB able to grow in the cheese-environment conditions could arise from both raw milk and NWC. Starter lactobacilli (SLAB) from NWC were the main species present during acidification, and non-starter LAB (NSLAB), mainly from milk but also from NWC, were able to grow after brining and they dominated during ripening. The peak areas of LH-PCR profiles were used to determine ecological indices during manufacture and ripening. Among cheese ecosystems with different ageing times, diversity, Evenness and Richness were different, with highest bacterial growth and diversity occurring in cheese ripening at 2 months. At this time point, which seemed to be a crucial moment for GP microbial evolution, cell lysis of both SLAB and NSLAB was also observed.     

Inventory of non starter lactic acid bacteria from ripened Parmigiano Reggiano cheese as assessed by a culture dependent multiphasic approach

The objective of this study was to investigate microbial species diversity and strain complexity of the cultivable non starter lactic acid bacteria (NSLAB) occurring in 31 ripened Parmigiano Reggiano (PR) cheeses. Dereplication of 127 lactobacilli isolates by (GTG)(5)-PCR fingerprinting yielded a total of 51 genotypes. Phylogenetic relatedness of all the genotypes with known Lactobacillus species was determined by a novel combined amplified 16S rDNA restriction analysis (16S-ARDRA), species-specific PCR assays and 16S rRNA gene sequencing. The species Lactobacillus rhamnosus and Lactobacillus paracasei comprise the largest portions of the cultivable NSLAB community in PR cheese, with an inter-individual diversity ranging from one to four dominant genotypes per sample. Lactobacillus casei, Lactobacillus harbinensis and Lactobacillus fermentum species were also detected at low frequency. The data showed differences in cultivable NSLAB population, with an overall decrease in diversity and complexity from early to advanced stages of ripening. Finally the de-replicated collection of genotypes resulting from this work is the bases for further functional screening.