Shift in raw bovine milk microbiota after spontaneous fermentation as revealed by 16S rDNA metagenomic analysis

Several traditional dairy products are still fermented using unselected starter cultures largely represented by natural whey cultures (NWCs). Microbial biodiversity present in NWCs employed for the production of high moisture Mozzarella cheese areresponsible for differences in primary and secondary proteolysis as well as for the production of aroma compounds. In this work, Illumina (MiSeq)16S rDNA metagenomics analysis of raw milk samples and derived NWCs,was employed to investigate microbial changes occurring during natural fermentation.The microbiota composition, reported as average Operational Taxonomic Units (OUTs)per sample, showed significant differences immediately after the end of the first fermentation step (2h at 45°C) and after the firstandsecond overnight fermentations at 37°C (Pairwise-Wilconoxtest, p value 0.008, 0.008 and 0.014 respectively). More than 100 different OTUs were identified in raw milk samples, which were reduced to 32 at the end of the second overnight fermentation step. This switch in natural microbiota were mainly represented by Pseudomonadaceae (75%) and Lactococcusgarviae (7%) in raw milk to more than 80% of Streptococcusspp. and homofermentative Lactobacillus spp. after the second or third overnight fermentation steps. The application of metagenomic approach allowed us to identify 54 bacterial genus and 29 species and to monitor in detail changes occurring in microbiota when a NWC is produced from raw milk. Many of these species, accounting for less than 1% of total average OTUs per sample, were never reported isolated from and/or identified in milk or in NWC samples. In addition, the technological fermentation approach applied in this study made it feasible to enrich NWCs of useful lactic acid bacteria occurring in low amount in raw milk samples.