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Background: In recent years consumers, due to the growing interest for functional foods, have put particularly a ention on prod- ucts enriched with probiotic lactic acid bacteria for their ability to give human bene t by their assumption. Microorganisms to be considered as probiotic should be able to survive to gastrointestinal juices and colonize the intestinal tract to provide bene ts to human health. The aim of the study consisted on the in vitro investigation of novel LAB strains, isolated during cheesemaking and ripening of Caciocavallo cheese, and on their probiotic potential for food application. Methods: In order to select potential probiotic bacteria, LAB isolates were preliminarily screened for their survival to simulated gastro-intestinal transitand assayed for others activities of probiotic and functional interest. Thanks to promising a itude as poten- tial probiotics, four strains were tested to evaluate their adhesion ability on Caco-2 cell lines used as intestinal cell model. Microbiological data were analysed by one-way analysis of variance (ANOVA). Comparisons of means were carried out using post-hoc Tukey’s test. For vitro adhesion experiments, statistical signi cance was evaluated by Kruskal-Wallis test for equal medi- ans. Results: Twenty- ve LAB, presumptively identi ed as Lactobacillus spp., were isolated from cheese during 60 days of ripening and screened for their survival to simulated gastrointestinal transit. Sixteen isolates that showed a survival rate ≥85% were identi edas 10 di erent pro les of Lb. paracasei subsp. paracasei. The 10 strains tolerated high concentration of bile salts by Minimal Inhibitory Concentration and growth rate assays and showed susceptibility or moderate susceptibility against antibiotics of human and vet- erinary importance, except for the resistanceto the class of aminoglycoside antibiotics. Eight out 10 strains showed in vitro choles- terol-lowering ability, whereas all strains showed antioxidant activity of their cell-free supernatants. Moreover, the four strains with highest survival to simulated gastrointestinal transit showed the ability to adhere to Caco-2 cells. Conclusion: The results suggest that some strains may be e ective probiotics to be use as tool to design probiotic dairy products after con rmation probiotic activities in further in vivo studies. Findings of the present study suggest that four strains showed good or strong adherent ability on Caco-2 cell monolayer, that is one of prerequisite that probiotic bacteria must have to perform their functional properties.

Fermentation is a widespread technological process in culinary traditions all over the world, not only because assures the preservation and the safety of products but also because provides foods with excellent sensory and organoleptic characteristics. Moreover, the growing interest in functional foods has led to involve fermented foods as excellent food matrices by which to design functional products. Today is known that fermented foods are characterized by complex microbial communities that play needful and specific role of pro-technological interest improving the food safety, the shelf-life and the sensory profile of the end product. Moreover, in the last decades the demand by consumers for foods able to improve the human health is growing. In that context, fermented foods containing probiotic microorganisms cover a large slice in the sector of functional foods. This research thesis was finalized on the study of microbial ecosystems of fermented foods and the selection of functional strains and their potential use to design safer and healthier products. The goals were pursued through the study of microbial ecosystem of fermented foods and the monitoring of starter and pathogenic bacteria by next generation sequencing (NGS) techniques and dependent-culture methods, and the isolation and characterization of starter and probiotic cultures and their application in food matrices. In this research project the use of both NGS techniques and culture-dependent methods to investigate the microbial diversity of fermented foods provided a complete overview of microbial populations that occur in fermented foods. Culture-independent methods, as NGS techniques, are able to describe the bacterial ecosystem at species level, which are hard to highlight using culture-based methods. Nevertheless, isolation and characterization are still needed to date because represent the unique way to select strains to use as starter, protective, functional and probiotic cultures in fermented foods. On this purpose, one of the goals of the research is focused on the screening of LAB strains and their selection for abilities of pro-technological, protective and probiotic interest for the industry of fermented foods.

Lactobacillus curvatus 54M16 produced bacteriocins sak X, sak Tα, sak Tβ and sak P. The aim of this study was to investigate the anti-listerial activity of the bacteriocins-producing strain against Listeria monocytogenes in vitro co-culture experiments and during the manufacture of fermented sausages. In MRS broth, Lb. curvatus 54M16 was able to inhibit L. monocytogenes to undetectable levels after 48 h at 20 °C or 5 days at 15 °C. Anti-listerial activity was lower during the production of fermented sausages with pathogen inoculation at levels of approximately 4 Log CFU g-1. However, total inhibition of L. monocytogenes native to the raw ingredients was achieved over the course of the fermentation. Moreover, 16S rRNA-based analysis revealed the ability of Lb. curvatus 54M16 to dominate and affect the bacterial ecosystem, whereas spoilage-associated bacterial genera, such as Brochothrix, Psychrobacter, Pseudomonas and some Enterobacteriaceae, were found until the end of ripening in sausages without Lb. curvatus 54M16. The use of the bacteriocins-producing Lb. curvatus 54M16 in fermented sausages could be an important contribution to product safety, provided that eco-physiological factors and other preservation methods are maintained at levels required for the inhibition of pathogens in controlled conditions.

Traditional Caciocavallo of Castelfranco is a semi-hard "pasta-filata" cheese produced from raw cows' milk in Campania region. The aim of the present research is mainly focused on the study, by 16S rRNA gene pyrosequencing and viable counts, of the dynamics of bacterial communities during manufacture and ripening of traditional Caciocavallo cheese. Moreover, the possible correlation between cheese microbiota and cows' feeding based on silage or hay was also evaluated. In general, except for enterococci, the technological process significantly affected all the microbial groups. According to 16S rRNA, raw cows' milk was dominated by Streptococcus thermophilus, L. lactis and Pseudomonas sp. in hay cheese production, whereas Lactococcus lactis and Acinetobacter sp. dominated silage production. Differences in the taxonomic structure of the milk's microbiota within diet groups were not related to silage and hay cows' feeding. Moreover, S. thermophilus was the unique species that dominate from raw milks to fermented intermediates and cheese in both hay and silage cheese productions. Feeding and ripening time influenced significantly sensory characteristics of the cheeses.