Rossana Romaniello

Diversity of Saccharomyces cerevisiae Strains Isolated from Two Italian Wine-Producing Regions

Numerous studies, based on different molecular techniques analyzing DNA polymorphism, have provided evidence that indigenous Saccharomyces cerevisiae populations display biogeographic patterns. Since the differentiated populations of S. cerevisiae seem to be responsible for the regional identity of wine, the aim of this work was to assess a possible relationship between the diversity and the geographical origin of indigenous S. cerevisiae isolates from two different Italian wine-producing regions (Tuscany and Basilicata). For this purpose, sixty-three isolates from Aglianico del Vulture grape must (main cultivar in the Basilicata region) and from Sangiovese grape must (main cultivar in the Tuscany region) were characterized genotypically, by mitochondrial DNA restriction analysis and MSP-PCR by using (GTG)5 primers, and phenotypically, by determining technological properties and metabolic compounds of oenological interest after alcoholic fermentation. All the S. cerevisiae isolates from each region were inoculated both in must obtained from Aglianico grape and in must obtained from Sangiovese grape to carry out fermentations at laboratory-scale. Numerical analysis of DNA patterns resulting from both molecular methods and principal component analysis of phenotypic data demonstrated a high diversity among the S. cerevisiae strains. Moreover, a correlation between genotypic and phenotypic groups and geographical origin of the strains was found, supporting the concept that there can be a microbial aspect to terroir. Therefore, exploring the diversity of indigenous S. cerevisiae strains can allow developing tailored strategies to select wine yeast strains better adapted to each viticultural area.

Control of inoculated fermentations in wine cellars by mitochondrial DNA analysis of starter yeast

The main purpose of this work was to test the effective dominance of the inoculated strain during the fermentation process. During this research activity, two Saccharomyces cerevisiae selected strains, isolated from Aglianico del Vulture grapes, were tested during inoculated fermentations at pilot scale in three wine cellars producing Aglianico del Vulture wine and characterized by different typologies. Yeast colonies sampled during the processes of fermentation were identified by restriction analysis of the internal transcribed spacer (ITS) region, and S. cerevisiae strains were differentiated by restriction fragment length polymorphism of mitochondrial DNA (RFLP-mtDNA). Analysis of the yeast population during the inoculated fermentations evidenced a significant presence of non-Saccharomyces, which varied with the cellar. The presence of non-Saccharomyces yeasts at different levels affected the aromatic composition of the experimental wines, as determined by gas-chromatographic analysis. The RFLP-mtDNA of S. cerevisiae isolates revealed the differing dominance of inoculated strains as a function of the wine cellar. In two cellars, all the isolates showed the same restriction profile, which was identical to that of the starter pattern. In contrast, in the third cellar, a significant percentage of S. cerevisiae isolates exhibited mtDNA-RFLP patterns different from the yeast starter profile, showing that, in this case, the starter exhibited low dominance in the fermentation. Our results demonstrate that, although the inoculated strains were found with high frequency, other yeasts (S. cerevisiae and non-Saccharomyces) developed, contributing to the fermentative process and to organoleptic quality of the final wine.

Impact of yeast starter formulations on the production of volatile compounds during wine fermentation

The most diffused starter formulation in winemaking is actually represented by active dry yeast (ADY). Spray‐drying has been reported as an appropriate preservation method for yeast and other micro‐organisms. Despite the numerous advantages of this method, the high air temperatures used can negatively affect cell viability and the fermentative performance of dried cells. In the present study, 11 wine S. cerevisiae strains (both indigenous and commercial) were submitted to spray‐drying; different process conditions were tested in order to select the conditions allowing the highest strain survival. The strains exhibited high variability for tolerance to spray‐drying treatment. Selected strains were tested in fermentation at laboratory scale in different formulations (free fresh cells, free dried cells, immobilized fresh cells and immobilized dried cells), in order to assess the influence of starter formulation on fermentative fitness of strains and aromatic quality of wine. The analysis of volatile fraction in the experimental wines produced by selected strains in different formulations allowed identification of > 50 aromatic compounds (alcohols, esters, ketones, aldehydes and terpenes). The results obtained showed that the starter formulation significantly influenced the content of volatile compounds. In particular, the wines obtained by strains in dried forms (as both free and immobilized cells) contained higher numbers of volatile compounds than wines obtained from fresh cells. Copyright

Comparative study of Saccharomyces cerevisiae wine strains to identify potential marker genes correlated to desiccation stress tolerance.

The most diffused formulation of starter for winemaking is active dry yeast (ADY). ADYs production process is essentially characterized by air-drying stress, a combination of several stresses, including thermal, hyperosmotic and oxidative and cell capacity to counteract such multiple stresses will determine its survival. The molecular mechanisms underlying cell stress response to desiccation have been mostly studied in laboratory and commercial yeast strains, but a growing interest is currently developing for indigenous yeast strains which represent a valuable and alternative source of genetic and molecular biodiversity to be exploited. In this work, a comparative study of different Saccharomyces cerevisiae indigenous wine strains, previously selected for their technological traits, has been carried out to identify potentially relevant genes involved in desiccation stress tolerance. Cell viability was evaluated along desiccation treatment and gene expression was analyzed by real-time PCR before and during the stress. Our data show that the observed differences in individual strain sensitivity to desiccation stress could be associated to specific gene expression over time. In particular, either the basal or the stress-induced mRNA levels of certain genes, such as HSP12, SSA3, TPS1, TPS2, CTT1 and SOD1, result tightly correlated to the strain survival advantage. This study provides a reliable and sensitive method to predict desiccation stress tolerance of indigenous wine yeast strains which could be preliminary to biotechnological applications.

Identification by phenotypic and genetic approaches of an indigenous Saccharomyces cerevisiae wine strain with high desiccation tolerance

During active dry yeast (ADY) production process, cells are exposed to multiple stresses, such as thermal, oxidative and hyperosmotic shock. Previously, by analysing cells in exponential growth phase, we selected an indigenous Saccharomyces cerevisiae wine strain, namely CD‐6Sc, for its higher tolerance to desiccation and higher expression of specific desiccation stress‐related genes in comparison to other yeast strains. In this study, we performed a desiccation treatment on stationary phase cells by comparing the efficacy of two different methods: a ‘laboratory dry test’ on a small scale (mild stress) and a treatment by spray‐drying (severe stress), one of the most appropriate preservation method for yeasts and other micro‐organisms. The expression of selected desiccation‐related genes has been also assessed in order to validate predictive markers for desiccation tolerance. Our data demonstrate that the ‘mild’ and the ‘severe’ desiccation treatments give similar results in terms of cell recovery, but the choice of marker genes strictly depends on the growth phase in which cells undergo desiccation. The indigenous CD‐6Sc was ultimately identified as a high dehydration stress‐tolerant indigenous strain suitable for ADY production. This study highlights the exploitation of natural yeast biodiversity as a source of hidden technological features and as an alternative approach to strain improvement by genetic modifications. Copyright

Pre-cultivation with Selected Prebiotics Enhances the Survival and the Stress Response of Lactobacillus rhamnosus Strains in Simulated Gastrointestinal Transit

In our study, we dwelled upon combinations of lactobacilli/prebiotics, considering four different strains belonging to the Lactobacillus rhamnosus species, including Lactobacillus rhamnosus GG (LGG), and different prebiotics often found in commercial synbiotic products, such as inulin, lactulose and polyols mannitol and sorbitol. In the first step of the research, the survival, the growth kinetic parameters and the protein expression of Lb. rhamnosus strains cultivated in presence of the different prebiotics as a unique carbon source were evaluated. In the second step, the influence of pre-cultivation in medium added of metabolizable prebiotics on the strains survival to simulated gastrointestinal (GI) transit, assayed without prebiotics addition, was estimated. Our results showed that the presence in the medium of certain low fermented prebiotics, specific for each strain, represents a stress factor that significantly affects the growth of Lb. rhamnosus strains, inducing the up-regulation of several proteins. In detail, all added prebiotics used as unique carbon source caused a growth retard compared with glucose, as testified by increased values of the lag phase and decreased values of the μmax. Mannitol evidenced intermediate μmax values between those registered with glucose and those detected with the other assayed prebiotics. Moreover, the cultivation with prebiotics induced the over expression of 7 protein bands. Interestingly, we found a correlation between the up-regulation of two specific stress proteins, called P4 (ATP-binding subunit Clpx) and P7 (GrpE), and the death kinetic parameters (resistance and cells viability) registered during the simulated GI transit of strains pre-cultivated with specific, low fermented prebiotics. Specifically, the highest resistance and gastric-vitality scores were highlighted for the strain AT195 when pre-cultivated in presence of sorbitol. Conversely, the lowest values were found in the case of DSM20021 pre-cultivated with mannitol. Among the up-regulated stress proteins, P7 resulted involved in the response to the starvation. Finally, it is possible to conclude that the pre-cultivation with certain prebiotics as a unique carbon source represents a strain-specific, sub-lethal stress able to enhance the resistance of Lb. rhamnosus strains and consequently their viability under simulated GI transit.

Yeast Starter as a Biotechnological Tool for Reducing Copper Content in Wine.

Copper is widely used in agriculture as a traditional fungicide in organic farming to control downy mildew on grapes, consequently it is possible to find this metal during all stages of the vinification process. Low amounts of copper play a key role on the function of key cell enzymes, whereas excess quantities can exert amount-dependent cytotoxicity, resulting in general cellular damage. Nowadays the excessive copper ions in wines is removed by addition of adsorbents, but these additives can influence the sensory characteristics of wine, as well as detrimental to the health of consumers. It is well known that high concentrations of Cu2+ can be toxic to yeasts, inhibiting growth and activity, causing sluggish fermentation and reducing alcohol production. In this study, 47 S. cerevisiae strains were tested for copper tolerance by two different tests, growth on copper added medium and fermentative activity in copper added grape must. The results obtained by the two different tests were comparable and the high strain variability found was used to select four wild strains, possessing this characteristic at the highest (PP1-13 and A20) and the lowest level (MPR2-24 and A13). The selected strains were tested in synthetic and natural grape must fermentation for ability to reduce copper content in wine. The determination of copper content in wines and yeast cells revealed that at the lowest copper residual in wine corresponded the highest content in yeast cells, indicating a strong strain ability to reduce the copper content in wine. This effect was inversely correlated with strain copper resistance and the most powerful strain in copper reduction was the most sensitive strain, MPR2-24. This wild strain was finally tested as starter culture in cellar pilot scale fermentation in comparison to a commercial starter, confirming the behavior exhibited at lab scale. The use of this wild strain to complete the alcoholic fermentation and remove the copper from wine represents a biotechnological sustainable approach, as alternative to the chemical-physical methods, ensuring at the same time a completed alcoholic fermentation and organoleptic quality of wine.

Use of Saccharomyces cerevisiae var. boulardii in co-fermentations with S. cerevisiae for the production of craft beers with potential healthy value-added

In recent years, the awareness of consumers about the impact of food on health is constantly increasing. A high amount of dietary antioxidant intake can be supplied by beverages widely consumed, such as wine, coffee, beer. Recently, an increase in the consumer interest was observed for beer, in consequence of the high phenolic antioxidants and low ethanol content present in this beverage. Among all beer types, in recent years, consumption of craft beers has gained popularity. Being an unpasteurized and unfiltered, craft beer is potentially a new vehicle for delivering health effects. While health benefits of lactic acid bacteria as probiotics are well known, few data are available on probiotic yeasts in fermented food. Therefore, this study was aimed to analyse the effect of integrating the well-known probiotic yeast strain of S. cerevisiae var. boulardii (S.b) in mixed cultures with S. cerevisiae strains for production of beers with increased healthy benefits. The probiotic strain of S.b was tested in mixed cultures with selected S. cerevisiae strains, during wort fermentation. As the viability during processing operations is one of the criteria for selecting suitable strains of probiotic microorganisms, the survival of probiotic yeast during the fermentation and the presence of highly viable cells at the end of fermentations were evaluated. In almost all the mixed fermentations, at the end of the process the probiotic yeast was predominant on S. cerevisiae strain, and the experimental beers contained a high number of viable cells of S.b strain (ranging between 8 × 106 and 7.0 × 107/mL). The analysis of experimental beers for the content of main volatile compounds showed that the inclusion of S.b strain in mixed starter did not affect negatively beer aroma. Moreover, the inclusion of S.b strain in mixed starters determined an increase in the antioxidant activity and polyphenols content, in comparison to beers from single starter fermentations, indicating the influence of S.b strain on these parameters. Some mixed starter cultures tested in this study resulted a very promising tool to increase the healthy quality of the product, such as the improve the antioxidant activity and polyphenols content of beer.