Aspetti di sicurezza e proprietà antagonistiche di ceppi di Streptococcus thermophilus provenienti da prodotti caseari

Food fermentation is the oldest biotechnological application and one of the cheaper and more efficient ways for the biopreservation of food products. It is also involved in many of the qualitative, organoleptic and nutritional properties of fermented products (taste, aroma, texture, consistency, availability of nutrients). The microorganisms that are mostly involved in food fermentation processes are lactic acid bacteria (LAB) and yeasts. In the dairy production, starters are cultures containing high amounts of LAB that are added to the milk in order to target the fermentation processes. The selected starter cultures most used in Italy consist mainly of thermophilic LAB, associations of different Streptococcus thermophilus biotypes or mixtures of S. thermophilus and other bacterial species. Even if the species S. thermophilus is genetically correlated to pathogenic species of the Streptococcus genus, it is safely used in food from many centuries. The non-pathogenicity of S. thermophilus is supported by its long-term safety use in foods (Iyer et al., 2010), by its “Generally Recognised As Safe” (GRAS) status declared by the Food and Drug Administration (FDA) in the USA and by its inclusion in the “Qualified Presumption of Safety” (QPS) list of the European Food Safety Authority (EFSA). In the last decades the European Community underlined the importance of a correct identification of the microorganisms used in food production and the need to verify the absence of hazard traits such as antibiotic resistances and the production of toxic compounds like biogenic amines. For this reason further studies are needed to better understand the positive and negative traits of S. thermophilus and its ability to adapt to different environmental and microbiological growth conditions. The present PhD project fits in this context and it has the purpose to evaluate the biodiversity, the antagonistic properties and the safety aspects of the species S. thermophilus. The first part of the research regards the identification and genetic typing of a collection of S. thermophilus strains, derived from the collections of different Italian research groups and isolated from dairy products of different type and geographical origin. The study of the intraspecific variability of the collected strains, obtained through a combination of different molecular techniques (specie-specific PCR, RAPD-PCR, RAPD-PCR combined with enzymatic restriction and plasmidic profiles) has allowed to establish the biodiversity among the strains and to detect some similarity groups. The results of this experiment represented the starting point for a wider study regarding this bacterial species. The second part of the doctoral thesis concerned the evaluation of the antagonistic activity of S. thermophilus strains towards other species used as starters or towards pathogens and contaminants. Some strains of the collection showed a higher activity than others, especially towards other starter species such as Streptococcus gallolyticus subsp. macedonicus, Lactobacillus delbruekii subsp. lactis, Lactobacillus helveticus and other strains of S. thermophilus. Some strains also displayed an antagonistic activity towards Staphylococcus aureus, Escherichia coli and Enterococcus faecium, even if in a lower extent respect those towards the other starters. Some chemico-physical features of the inhibitory substances and their production kinetic were also studied. The chemico-physical characterization of the molecules produced by the most performing strains was due to protein compounds with a size higher than 3 kDa, probably thermophilins. The produced thermophilins are thermoresistant, stable in a pH range from 2 to 7 and are produced at the beginning of the growth phase of the strains. The presence and diffusion of the genes coding for the thermophilins 9, 13 and 1277, the unique with known sequences till now, were also assessed. Three primer pairs were designed ad hoc to detect these determinants and two out of three genes, i.e. those for thermophilins 9 and 13, were found in the strains at different frequencies. The third part of the doctoral thesis regards the study of the antibiotic resistance traits in the S. thermophilus collection strains, and in particular the tetracycline resistance. This research includes the presence and expression of the genetic traits (tet genes) that confer resistance to this antibiotic, the presence of mobile genetic elements able to transfer resistance genes and the real ability of some strains to transfer antibiotic resistance to other food-borne bacterial species, especially pathogens and contaminants. The results showed the presence of three [tet(L), tet(M) and tet(S)] out of the many genes that give tetracycline resistance described in literature. All except one strains carried the tet(S) gene, half of the strains carried tet(L) and some strains tet(M). More than 70% of the resistant strains hold two tet genes at the same time. Expression studies of tet(L), tet(M) and tet(S) displayed that only tet(S) is always expressed while the other two genes are expressed only in some strains. The study of the transferability of the tet genes has revealed the presence of the Tn916-Tn1545 transposon family together with tet(M) gene, and the presence of plasmids of different sizes in some of the analysed strains. Such plasmids are found to carry the tet(S) or tet(M) gene. Conjugation trials conducted using as recipient strains of S. thermophilus, Enterococcus faecalis and Listeria innocua did not show gene transfer events. In the last part of the doctoral thesis the presence and expression of genes involved in the production of the most important biogenic amines, histamine and tyramine, were performed in S. thermophilus, and studies were conducted to determine the nucleotide sequence of these determinants. Some strains of the collection were positive for the presence of genes coding for hystidine (hdec) and tyrosine (tdec) decarboxylases, involved in the production of histamine and tyramine. The expression of hdec and tdec genes was evaluated by RT-PCR and Real-Time RT-PCR in different cultural conditions. The presence or not of the precursor in an available form in the growth media was considered, together with the occurrence of small amounts of fermentable sugars and the addition of salt. The data obtained from RT-PCR experiments displayed that the presence of the precursor can be a fundamental factor leading to an higher production of the two amines. Gene transcription, analysed by Real-Time RT-PCR, and amine production were determined in a cultural medium (Skim Milk) and in growth conditions that simulate the starting of a dairy production process. In the tested conditions, hdec gene was always expressed, with an increased expression just after two hours from the addition of the precursor to the substrate. On the contrary, the expression levels of the tdec gene were quite low and a high level tyramine production was observed only after seven days of incubation. To obtain the nucleotide sequence of the hdec and tdec genes of S. thermophilus different experimental strategies were followed: the design and use of new degenerated specific primers, also in combination with others already available in literature, and the setting up of Uneven PCR reactions (Chen & Wu, 1997). This multipurpose approach allowed to obtain the complete sequence of the hdec gene of the species S. thermophilus and the almost complete sequence of tdec gene. In conclusion, this doctoral thesis have deepen and understand some different aspects and characteristics of the species S. thermophilus and important innovative contributions were added to the knowledge of the species, in the view of its safe use in food.