Criteri funzionali e di sicurezza per la valutazione di batteri lattici e lieviti da impiegare nella produzione di alimenti fermentati e come probiotici

Over the last years consumers from industrialized countries have become more and more demanding about foods which not only can satisfy nutritional and safety needs, but also assure sensorial peculiarity and quality, and/or that contain probiotics or microbial metabolites with positive effects on health. These foods, which are recognized as “high quality foods”, have a high added-value and focus the attention of food industry areas interested in the evolution of traditional products and in the development of novel ones, with a huge growing potential in new market segments. Nowadays, through knowledge breakthroughs and the latest advanced methods deriving from biotechnology and molecular biology, it is possible to carry out research for studying in depth microorganisms responsible for transformations during production of these foods, and to use them specifically for answering consumers and modern food industry demands. In this context, the aim of this PhD project titled “Functional and safety criteria for the evaluation of lactic acid bacteria and yeasts to use in the production of fermented foods and probiotics” was the individuation of new LAB (Lactic acid bacteria) and yeasts cultures having peculiar technological, healthy and safety characteristics suitable to obtain high quality fermented foods. The study deals with the selection and the analysis of autochthonous microorganisms from fermented foods obtained without starters. The attention was in particular focused on two important groups of fermented foods: dairy products and wine. These products are, in fact, strongly influenced by microbial populations growing in, which widely affect production processes and quality. Then, these spontaneously fermented matrices could be the source of microbial strains in which starter, protective, support and probiotic cultures, technologically and functionally very useful, can be identified. To study biodiversity and isolates’ genotypic and phenotypic properties, different innovative biotechnological criteria were considered. These criteria, indeed, with the traditional parameters examined for microbial strains selection, can highlight the presence of technological and/or functional properties able to improve quality and thus to provide an added value to final products. The first part of this thesis (chapters 2 and 3) was dedicated to the study of dairy LAB and of a probiotic strain. In detail, chapter 2 regards activities carried out in a project aimed at preservation, characterization and exploitation of lactic microflora in cheeses produced in Val Venosta “malga”. The research began with the screening of 359 dairy isolates for some technological properties, like proteolytic and milk coagulation and acidification activities, useful for the individuation of starters. Among functional properties, antagonistic activity toward some undesired pathogenic or food-spoiling microorganisms was investigated. These preliminary analyses allowed selecting about a hundred isolates on which further physiological characterization was conducted to verify their capability to grow in cheese, where they could perform their biochemical activities, and their resistance to human gastrointestinal tract, fundamental condition for defining a microorganism “probiotic”. These assays allowed to individuate about thirty isolates which were identified at species and strain levels by using some molecular techniques and in which the presence of genes codifying for undesired characteristics, like antibiotic resistances and biogenic amines production ability was verified. Antagonistic properties were further investigated, for enterococci and lactococci, assaying the presence of genes codifying the most spread bacteriocins in these microorganisms. The peculiar technological and functional traits detected in laboratory for a Lactococcus lactis subsp. lactis strain were validated in cheese-making trials. Moreover, most of the isolates belonging to the more interesting dairy species were assayed for their ability to produce compounds inhibiting the Angiotensin I-converting enzyme (EC 3.4.15.1) (ACE), a key enzyme involved in the regulation of blood pressure in animals, including humans. The presence of ACE inhibitors was tested after growth in milk by a spectrophotometric assay. Some Lactobacillus casei strains gave positive results. In this study the presence of the prt gene, codifying a proteinase, identified in Lactobacillus helveticus CP790 and probably involved in bioactive peptides production, was also examined. Chapter 3 reported the results of feeding a probiotic strain (Lactobacillus acidophilus D2/CSL) to broiler chickens. This microorganism is, nowadays, registered at European Community level for laying hens but needs authorization for broilers feeding. The strain was automatically dosed and administered to animals through water. In two trials, broilers received coccidiostats through the feed, while in the third trial chickens were previously vaccinated. Treated and control groups were allocated in two identical and adjacent barns. Treatment effects were verified by chicken growth detection like average weight gain, mortality and feed efficiency calculation (conversion and performance index). Statistically processed data showed significant differences between treated and control groups. Microbiological analysis of crop and intestine contents, instead, did not exhibit significant differences because of the high heterogeneity present between samples of the same group and the limited number of slaughtered animals. The second part of the thesis (chapter 4 and 5) deals with the study of microorganisms (yeasts and bacteria) of oenological origin, aimed at selecting some strains suitable to be proposed like starters or support cultures, for being able to exalt typicality of autochthonous vines present in the Veneto region. It is actually well-known that the strains present on the market limit the expression of the area peculiarities, because obtained from selection program carried out on different terroir. The object of chapter 4 is a study conducted on 37 Oenococcus oeni strains previously isolated from wines in spontaneous malolactic fermentation (MLF). The research began with the examination of the strains safety with respect to their ability to produce biogenic amines and ethylcarbamate; then their b- glucosidase activity was evaluated. This property is very important because it is involved in flavours release from their odourless glucosilated precursors, and in the production of resveratrol, a compound naturally present in the grape skin like glucosyl derivative and widely studied for its antioxidant and anticoagulation properties, effective against cardiovascular diseases and carcinogenesis. The enzymatic assay showed that activity was associated to whole cells and was higher in the late exponential growth phase. The high intra-species variability allowed to select the more active strains. Concentrations of ethanol over 10% strongly inhibited the enzyme, while lower values showed a stimulation of the straindependent activity. PCR-based molecular protocols, developed to detect the presence and the expression of genes codifying for this enzyme, found a low genetic heterogeneity, even if RT-PCR assays detected a moderate expression variability, suggesting a strain-specific regulation of factors involved in gene transcription process. The last analysis concerned the high pressure liquid chromatography (HPLC) quantification of resveratrol produced by the selected O. oeni strains in synthetic wine, in two different kinds of red wine, Merlot and Recioto passito, and in a white wine. Micro wine-making trials were carried out and, at the end of MLF, the resveratrol presence was detected only in synthetic wine. Probably, the complex composition and the chemical-physical conditions of wine negatively influence O. oeni strains’ b-glucosidase activity. The last part of this PhD project (chapter 5) concerned the selection of yeasts to be use like starter or added cultures in the production of Veneto region wines. From spontaneous alcoholic fermentations of musts, obtained from grapes directly collected in fields and from the dripping of farm trailers carrying grapes to the winery, 529 yeasts were isolated. Several characterization tests were done: some for the detection of undesired activities (sulphur hydroxide and phenols production) and some for the evaluation of desired properties, as those involved in the release of flavours and in the wine stability. Particular attention was dedicated to examine some innovative characteristics, like, for example, glycosidase activities. Another important assayed trait was the yeasts proteolytic activity that could contribute to the stabilization and the clarification of wines, avoiding or decreasing turbidity. Molecular characterization was performed by pulsed field gel electrophoresis, which was able to identify isolates as Saccharomyces cerevisiae and to detect the presence of clones, excluded from the next analyses. Afterwards, micro-fermentations trials with 80 selected strains were carried out. Collected data highlighted a good fermentative behaviour of the strains, and wines analysis allowed to select seven strains to test in winery wine-making trials. One strain from these trials was found very interesting, and produced a Valpolicella wine with good sensorial quality traits. In conclusion, this PhD thesis collected different research projects in food microbiology field, which were carried out using a common innovative approach based on the application of new investigation methodologies and on the examination of unconventional parameters. The work allowed the development of new protocols for the survey of functional properties and the search of negative traits to be excluded in microorganisms for fermented foods production. The performed selections highlighted the biodiversity richness in spontaneously fermented foods, which were identified as an interesting source of microorganisms for their potential effects on fermented products quality and on consumer health. This last consideration was also verified in the zootechnical field, underlining the importance of using LAB in this sector, in which issues associated to antibiotic use are currently of great interest and directly involve consumer health. The individuation of some LAB and yeasts, potentially interesting for industrial application as starters for the production of traditional and novel fermented food with high quality and value, was also very relevant.