Abstract
Raw cow’s milk microbiota and diversity has been a subject of many studies for more than 30 years. As it is a highly nutritious substrate, raw cow´s milk supports a consistent (103 -105 cfu/ml), diverse and complex microbiota with multifaceted roles: starting/facilitating dairy fermentations, contributing to human health, and, conversely, causing spoilage and disease. Culture-dependent approaches are widely used to isolate and characterize the raw cow milk microbial diversity but is limiting on providing information from the live microbiota. Culture-independent approaches based on high-throughput sequencing allows an indepth study of the microbial diversity, potentially also revealing the evolution of the microbial consortia. In addition, the advances of next generation sequencing methods fortify further our understanding on the potential functionality of the total microbiome or even a part of it. Further, is now evident that the complexity and diversity of the raw cow milk microbiome, is associated with many farming management systems, conditions, and contamination sources. To date, significant effort has been made to associate the farming system, other abiotic and biotic factors to the raw cow milk microbiome assessing combination of those factors, but not as a whole system.
Therefore, this thesis aims to elucidate the composition and diversity of raw cow’s milk microbiome from a statistically meaningful and representative number of farms from northern Italy (model system) and point the main farming conditions that correlate with the total and lactic microbiome composition utilizing both culture-dependent and culture-independent approaches. Assessing altogether numerous farming conditions for a relevant number of dairy farms, this study had the chance to assess their interactive and complementary associations. Once understood who was populating milk, shotgun metagenome analyses could explain what the microbiome can do.
Another milestone was to deepen the investigation of the microbiome and its functionality towards the lactic acid bacteria (LAB) and especially the non-starter lactic acid bacteria (NSLAB). Among traditional processes, cheese is known to be essentially fermented by LAB. This group of microbes can be either homofermentative or heterofermentative, with lactic acid as the primary product or lactic acid, CO2, and ethanol/acetic acid as products, respectively. Starter lactic acid bacteria (SLAB), which are mostly homofermentative are driving the acidification of the curd and non-starter lactic acid bacteria (NSLAB), which are heterofermentative are continuing the ripening process. Since NSLAB are considered major contributors for the sensory and organoleptic characteristics of ripened cheeses and especially the ones produced with raw milk, the functionality investigation was focused on the identified species of this group with more attention to the pathways related to proteolysis.
Concerning the total raw cow milk microbiome apart from the compositional and diversity analyses, a further investigation of the annotated genes related to virulence factors and antibiotic resistance genes (ARG) provided further literature in respect to the potential transfer of ARG between bacteria via horizontal gene transfer (HGT). Finally, autochthonous mesophilic LAB isolated from the raw cow milk (2095) were assessed with a high throughput in situ model for their potentiality to be used in combinations as starters for cheesemaking.