Composition and dynamics of microbiota in different dairy ecosystems

Cheese is a biologically and biochemically dynamic food containing microorganisms both deliberately added as starters and non-starter adventitious contaminants. The composition of microbial population changes under the influence of continuous shifts in environmental conditions and interactions occurring among microorganisms during manufacturing and ripening. In cheese manufacturing, the selection of technological parameters can influence and even induce several biochemical processes needed for this product. The microbiota present in cheese is complex and its growth and activity represent the most important, but the least controllable steps. The aim of the present thesis is the study of microbial diversity and dynamics of starter, non-starter and adventitious microorganisms involved in the cheese manufacturing and ripening processes. Thus, different ecosystems from raw milk to cheese and rind were investigated in order to comprehend the specific role played by microorganisms in each cheese making phase and to correlate the occurrence of certain microbial species with desired flavors and textures. In particular, the microbial ecosystems in natural whey starters, hard Italian cheeses (i.e. Grana Padano and Parmigiano Reggiano), and natural rind of mold-ripened cheese (i.e. blue cheese) were investigated throughout cheese manufacturing and ripening by culture-dependent and culture-independent techniques. By studying the microbiota in natural whey starters and cheese starting from the first days of manufacture, it was possible to describe the fermentative activity of starter lactic acid bacteria (SLAB) determining the ripening progress of the cheese. Whey starter titratable acidity did not seem to be related to the cell amount (total and cultivable cells) nor to the different species contribution. High concentrations of lactic acid and free aminoacids were found in cheeses with higher levels of Lactobacillus helveticus species and cultivable thermophilic bacterial densities. The presence of residual galactose was associated with higher contents of Streptococcus thermophilus species. In addition, the biotype composition of whey starters seemed to be far more important than the species composition in ensuring their good performances. A direct correlation between the composition of SLAB species and the acidifying efficacy in whey starters was not found. Contrarily, in the cheese matrix, the SLAB fermentative activity seemed to be species-dependent. In hard cheese ecosystems, both SLAB and non-starter LAB (NSLAB) seemed to contribute to acidification and ripening. However, SLAB, and in particular L. helveticus, resulted to be the species mainly subjected to the lysis occurring at 2 months of ripening. NSLAB were able to grow after brining, and became more relevant during ripening. NSLAB could arise both from the raw milk and the natural whey starter but their contribution to the development of cheese characteristics is still unknown. Furthermore, the presence of L. helveticus and Lactobacillus delbrueckii subsp. lactis in a non-cultivable state, up to 13 months of ripening, suggests that these species could play a different but still unknown role in cheese ripening. The microbiota on the surface of a blue cheese during the natural rind development showed microbial diversity comprising fourteen genera of bacteria (Enterococcus; Lactococcus; Leuconostoc; Macrococcus; Staphylococcus; Klebsiella; Brevibacterium; Corynebacterium; Brachybacterium;, Nocardiopsis;, Cobetia; Psychrobacter; Halomonas; Haererehalobacter), two yeasts genera (Candida; Debaryomyces) and one filamentous fungal genus (Penicillium). High and comparable densities of viable bacteria and yeasts were observed. Bacterial succession was observed during rind formation and no genus remained constant throughout ripening. The Staphylococcus genus dominated the early stages and then Brevibacterium the later stages. By using interaction experiments, inhibition and stimulation were observed among several species; these interactions could explain how some microorganisms contribute to community formation. Candida catenulata and Debaryomyces hansenii enhanced the growth of Staphylococcus equorum while C. catenulata inhibited D. hansenii growth. However, thorough studies need to be performed in order to evaluate whether the above mentioned bacteria, yeasts and molds can be beneficial and play a role in flavor and texture development of other cheese varieties with similar natural rinds. Overall, thanks to culture-dependent and culture-independent complementary approaches it was possible to identify which microorganisms were mainly involved in each dairy matrix and to address the importance of their presence that, if balanced, can help obtaining the distinctive features of each product.