Evolutionary study over space and time of lactic acid bacteria used as starter in cheeses manufacturing

Microorganisms have been mutating and evolving on Earth for billions of years. Undoubtedly, microorganisms in general and bacteria in particular are extremely successful in inhabiting and adapting to diverse ecological niches available in the biosphere. That’s because bacteria are characterized by a short generation times and large population sizes, but also thanks to transposons, plasmids and bacteriophages, that play a key role, moving and rearranging the presence and the order of genes in the chromosome. Food matrices can be viewed as different ecosystems, characterized by diverse stressful and selective conditions, leading to bacteria to adapt rapidly, acquiring or maintaining genes useful for their survival or losing genes no more helpful for the cell in those conditions. Lactic acid bacteria (LAB) are the most important bacteria traditionally associated with food fermentation due to their long and safe history of application. LAB have a spread ecological distribution; they are to be found in different environments, from milk and dairy products, vegetable and plants until cereals and meat. Besides, their presence in different ecological niches, LAB are commonly associated to dairy environment, in particular to starter cultures, which ‘start’ the fermentative process, producing lactic acid; their primary purpose in cheese manufacture. Many studies have demonstrated that these starter cultures have a large variability at strain level, rather than at species level. Even if, LAB biodiversity was extensively assessed in different works, a depth exploration of technological traits, lost or acquired, over the years, and in different ecological niches, have been very poorly investigated. Based on these assumptions, the aim of this PhD thesis is to investigate how selective pressures, due to environmental or technological drivers, shape the bacterial genome of LAB and microbial communities over the time and/or through different ecological niches (space). The main object of this doctoral project was to lay the foundations for understanding the factors that contribute to the selection, in order to direct the technology to favour a biotype rather than another in a specific environment. This PhD thesis was organized in four main sections. The first section was devoted to study a new protocol to perform in a quickly, cheaply and simply way, Amplified Fragment Length Polymorphism (AFLP) analysis. The second section was focused on Streptococcus thermophilus strains isolated from Pecorino Toscano ecosystem over time. It has been investigated if the strains isolated in different years from this niche, were able to produce histamine and tyramine, in order to understand if there has been an evolution over time of genetic traits involved in biogenic amines production. The third section has concerned the genome study of Lactobacillus helveticus strains isolated from fully different ecosystems, to gain an insight into the role of selective pressures in the genome evolution and to identify highly desirable traits with technological potential. Throughout the last section the microbial community of natural starter culture, used in Parmigiano Reggiano manufacturing, was investigated to understand how selective and environmental forces (like the seasonality and different temperatures in the technological process) shape the structure of this bacterial consortium. The results obtained highlighted the importance to study how technological and environmental pressures, over time and space, change the ecosystem scenario, leading bacteria to acquire, maintain or lose genetic traits. Overall, the biodiversity, found in different ecological niches, should be perceived as a great richness; the leak of these biotypes could mean losing technological traits that may reoccur useful for various applications.