Simona Guerrini

Indigenous Saccharomyces cerevisiae strains persist within individual cellars and contribute to the character of spontaneously fermented wines, yet the molecular basis of their oenological differences remains largely unknown. In this work, eight predominant strains isolated from spontaneous fermentations in four Tuscan wineries were characterized by technological traits of oenological relevance and laboratory-scale fermentations of both a synthetic must and Sangiovese grape must. In each must, the strains reached similar final ethanol concentrations, although lag phase and maximum fermentation rate differed significantly, whereas the resulting wines differed significantly in glycerol, acetic acid and phenolic alcohol content and in the scores assigned by a trained sensory panel. Four strains spanning the observed range of behaviors were hence analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-ToF MS) at an early and a mid-fermentation stage, yielding 30–46 differentially abundant proteins identified per strain. Strain-specific abundance of proteins involved in glycerol formation, acetaldehyde oxidation and sulfur amino acid metabolism paralleled the corresponding differences in glycerol, acetic acid and hydrogen sulfide production, whereas glycolytic enzyme abundance varied without any corresponding variation in ethanol. Proteomic analysis thus identified candidate molecular correlates of the traits that distinguish strains, suggesting that it could complement traditional screening in the selection of indigenous starter cultures.

The principal critical issue in the production of Vin Santo passito wine is the high likelihood of fermentation arrests or sluggish fermentation, resulting in low sensory quality. A potential solution is to select indigenous yeasts from well-conducted spontaneous fermentations. In this study, a novel mixed starter culture consisting of two indigenous strains, Saccharomyces cerevisiae Ris III and Zygosaccharomyces rouxii Zr186, was evaluated. After selecting the strains and testing their compatibility, the mixed culture was used as a starter in the winemaking process of dried grapes, which was conducted in barrels of various sizes. The fermentations were monitored microbiologically for a period of 100 days to verify the ability of the inoculated strains to dominate and thus facilitate the fermentation process. Both strains dominated the fermentations: S. cerevisiae showed a 100% isolation rate immediately, while Z. rouxii achieved dominance within a month. The mixed starter demonstrated a 25% improvement in fermentation purity for the same barrel size. Sensory analyses revealed that the wines produced with the mixed starter were very consistent with one another, contrasting with those obtained through spontaneous fermentations, and received the highest scores for gustatory descriptors. In conclusion, this mixed culture represents an effective strategy for standardizing fermentation and improving the sensory quality of Vin Santo.

Second cheese whey (SCW), a major by-product of ricotta cheese production, poses significant environmental challenges due to its high organic load. Biohydrogen (bio-H₂) and poly-β-hydroxybutyrate (PHB) production offer a sustainable reuse of SCW, that provides ideal nutrients for microbial growth. This study aimed to convert SCW into Bio-H₂ and PHB using a 5-liter tubular bioreactor in a sequential lactic fermentation and photofermentation system. Two lighting conditions were tested: white LED (WL) and selected LED (SL). Optimal results were achieved with a co-inoculum of Lactococcus lactis MK L84 and Lacticaseibacillus paracasei MK L49 at pH 4.5–5.5, followed by photofermentation with Rhodopseudomonas palustris 42OL under SL condition. The process yielded an average of 0.47 L of H₂ per liter of substrate and 1.66% wPHB/wCDW. This approach successfully transformed dairy waste into high-value products, promoting circular economy principles.

Recent studies on yeast-insect associations demonstrated that social wasps of the genera Polistes and Vespula act as a reservoir for the conservation of yeasts and as vectors capable of transferring such yeasts on the grapes. This work aimed to assess yeast species associated with social wasps and to obtain new strains to be used as bio-protection agents in winemaking. The wine yeast communities present on the exoskeleton, intestine of social wasps, and on the surface of grapes sampled in the vineyards of three Tuscan wineries were determined. Regardless of the wasp species, yeasts were mostly associated with female workers and found mainly in their intestine (up to 7 × 10 ⁵ CFU/mL). The identification revealed 20 species belonging to 10 genera; Aureobasidium pullulans and Metschnikowia pulcherrima were isolated from wasps of all the wineries, the latter occurring at the highest frequency. Strain-level characterization highlighted that three strains present on grapes were also present in the gut of wasps from the same vineyard. All the isolated M. pulcherrima strains underwent in vitro tests to select the most suitable for use as bio-protective cultures. Three strains showed good inhibitory activity against Kloeckera apiculata and Brettanomyces bruxellensis ; hence, they were selected for bio-protection trials on artificially contaminated grapes. Results highlighted the reduction of non- Saccharomyces populations, suggesting the effectiveness of M. pulcherrima as a biocontrol agent. The study confirmed the role of social wasps as yeast vectors in the vineyard and as a reservoir of yeast strains to be exploited for biotechnological applications in oenology. Key points: • Social wasps can carry yeast strains that are also present on the surface of grapes • Metschnikowia pulcherrima was found within the wasp gut as the prevalent yeast species • M. pulcherrima proved to be a suitable biocontrol agent on grapes

Cereal grains have been dietary staples for millennia, providing essential nutrients alongside their primary carbohydrate content. Recently, the search for sustainable, nutrient-rich alternatives has drawn attention to spelt (Triticum aestivum ssp. spelta L.), a low-input crop with promising nutritional properties. Spelt supplies a higher content of unsaturated fatty acids and minerals, such as iron, zinc, and magnesium and exhibits lower levels of phytic acid compared to common wheat. This study explores the nutraceutical potential of fermented bakery products made from spelt and wheat flours using sourdough fermentation, a process driven by lactic acid bacteria (LAB) and yeasts. Breads produced with baker’s yeast were included for comparison. Specifically, this manuscript focuses on the generation of bioactive peptides (BPs), which have demonstrated anti-oxidant, anti-inflammatory, and gut-protective effects by modulating oxidative stress and inflammatory signaling pathways. By comparing aqueous extracts from breads prepared with varying flours and fermentation methods, optimal conditions for producing functional baked goods could be defined. The findings may offer new avenues for developing bakery products that potentially increase intestinal health while promoting sustainable agriculture through the use of spelt.

Vin Santo is a passito wine produced mainly in Tuscany. In the traditional production of Vin Santo, fermentation occurs naturally. Only a few reports have explored the microbial ecology of Vin Santo. Therefore, the present study aimed to investigate the microbial ecology and its impact on the fermentative kinetics in traditional processes of Vin Santo carried out in two different Tuscan wineries. Despite the different systems used for drying the grapes, both wineries showed similar microbial ecology. Non-Saccharomyces yeasts were the dominant microbial population during grape drying in different succession, even though in the end, the dominant species (at different percentages) in both were Metschnikowia pulcherrima, Kloeckera apiculata, and Starmerella bacillaris. The spontaneous fermentations were instead both dominated by Saccharomyces cerevisiae, however in different concentration throughout the process, leading to a different ethanol content—12% (v/v) and 10.8% (v/v) in winery A and B, respectively. In both wineries, acetic bacteria and moulds did not grow. Considering the intraspecific biodiversity of S. cerevisiae populations, the vinifications of both wineries displayed very similar biodiversity indices. No single strain of S. cerevisiae dominated the entire fermentation process. The analysis identified 30 distinct genetic patterns in the fermentations of winery A and 23 in the fermentations of winery B. The work provided an insight into the microbial communities and their metabolomic interactions during Vin Santo production which could improve the management and control of the process.

Low-temperature treatments can be applied to grapes or must before alcoholic fermentation to enhance the wine’s sensory characteristics. Several studies have shown that such practices have a positive effect on the polyphenol profile of the wine, but only a few surveys have examined the effect of these treatments on the yeast microbiota of grapes and wine. Therefore, this study aimed to evaluate how cryoextraction (freezing the grape with liquid nitrogen) and cold pre-fermentative maceration (at 5 °C for 48 h) affect the Saccharomyces and non-Saccharomyces populations during the winemaking process of red grapes, cv Sangiovese, conducted at two temperatures (20 and 30 °C). This research analyzed the concentration of various yeast species, their fermentation abilities, and the resulting wine’s aromatic profile. The Principal Component Analysis performed on yeast concentrations during the fermentations of various wines did not group the experimental wines based on treatment. However, the same groupings were highlighted when the concentrations of the volatile compounds, quantified in the experimental wines, were processed using the same statistical approach. Therefore, cryoextraction and cold pre-fermentative maceration seem to contribute less to the aromatic profile than the yeasts involved in the fermentation process.

Recent interest in the special beer category has encouraged the search for novel brewing materials, including new ingredients and novel yeast strains, in order to differentiate the finished products. The aim of this work was to select non-brewing S. cerevisiae strains for the production of a fruit beer with raspberry. The in vitro tests and the wort fermentations allowed the selection of two sourdough S. cerevisiae strains, showing high maltose and maltotriose consumption, high ethanol production, and high viability. Fruit beers (FB) and control beers (CB) without raspberries were prepared. Fruit addition accelerated sugar consumption (7 days compared to 13 days) and increased ethanol and glycerol production by yeasts. Raspberry addition and the inoculated yeast strongly affected the aroma profile of beers. FB samples showed a higher amount of volatile organic compounds (VOCs); the most represented classes were alcohols, followed by esters and acids. FB inoculated by the selected S. cerevisiae SD12 showed the highest VOCs concentration (507.33 mg/L). Results highlighted the possible application of sourdough yeast strains for the brewing process, which, combined with raspberry addition, can be exploited for the production of beers with enhanced aromatic features and suitable chemical properties.