Yeasts have been widely used as a model to better understand cell cycle mechanisms and how nutritional and genetic factors can impact cell cycle progression. While nitrogen scarcity is well known to modulate cell cycle progression, the relevance of nitrogen excess for microorganisms has been overlooked. In our previous work, we observed an absence of proper entry into the quiescent state in Hanseniaspora vineae and identified a potential link between this behavior and nitrogen availability. Furthermore, the Hanseniaspora genus has gained attention due to a significant loss of genes associated with DNA repair and cell cycle. Thus, the aim of our study was to investigate the effects of varying nitrogen concentrations on H. vineae\'s cell cycle progression. Our findings demonstrated that nitrogen excess, regardless of the source, disrupts cell cycle progression and induces G2/M arrest in H. vineae after reaching the stationary phase. Additionally, we observed a viability decline in H. vineae cells in an ammonium-dependent manner, accompanied by increased production of reactive oxygen species, mitochondrial hyperpolarization, intracellular acidification, and DNA fragmentation. Overall, our study highlights the events of the cell cycle arrest in H. vineae induced by nitrogen excess and attempts to elucidate the possible mechanism triggering this absence of proper entry into the quiescent state.

New nonthermal technologies, including pulsed electric fields (PEF), open a new way to generate more natural foods while respecting their organoleptic qualities. PEF can reduce wild yeasts to improve the implantation of other yeasts and generate more desired metabolites. Two PEF treatments were applied; one with an intensity of 5 kV/cm was applied continuously to the must for further colour extraction, and a second treatment only to the must (without skins) after a 24-hour maceration of 17.5 kV/cm intensity, reducing its wild yeast load by up to 2 log CFU/mL, thus comparing the implantation and fermentation of inoculated non-Saccharomyces yeasts. In general, those treated with PEF preserved more total esters and formed more anthocyanins, including vitisin A, due to better implantation of the inoculated yeasts. It should be noted that the yeast Lachancea thermotolerans that had received PEF treatment produced four-fold more lactic acid (3.62 ± 0.84 g/L) than the control of the same yeast, and Hanseniaspora vineae with PEF produced almost three-fold more 2-phenylethyl acetate than the rest. On the other hand, 3-ethoxy-1-propanol was not observed at the end of the fermentation with a Torulaspora delbrueckii (Td) control but in the Td PEF, it was observed (3.17 ± 0.58 mg/L).

Global warming is causing serious problems, especially, in warm regions, where musts with excess sugars and high pH produce wines with decreased freshness and unstable evolution. This study aimed to determine biocompatibility between yeast species, the capacity for microbiological acidification, and the aromatic profile produced in ternary fermentations in which Lachancea thermotolerans has been co-inoculated with Hanseniaspora vineae, Torulaspora delbrueckii, or Metschnikowia pulcherrima, and the fermentation process is subsequently completed with sequential inoculation of Saccharomyces cerevisiae. For this purpose, different cell culture media and instruments were used such as infrared spectroscopy, enzymatic autoanalyzer, chromatograph coupled with a flame ionization detector, spectrophotometric analysis, among others. The behavior of these yeasts was evaluated alone and in co-inoculation, always finishing the fermentation with sequential inoculation of S. cerevisiae, at a stable temperature of 16°C and with a low level of sulfites (25 mg/L) in white must. Significant results were obtained in terms of biocompatibility using population counts (CFU/ml) in differential plating media that permitted monitoring. Quantification of the five species was studied. Concerning acidification by L. thermotolerans in co-inoculations, we showed some metabolic interactions, such as the inhibition of acidification when H. vineae/L. thermotolerans were used, generating just over 0.13 g/L of lactic acid and, conversely, a synergistic effect when M. pulcherrima/L. thermotolerans were used, achieving 3.2 g/L of lactic acid and a reduction in pH of up to 0.33. A diminution in alcohol content higher than 0.6% v/v was observed in co-inoculation with the L. thermotolerans/M. pulcherrima yeasts, with total sugar consumption and very slow completion of fermentation in the inoculations with H. vineae and T. delbrueckii. The aromatic composition of the wines obtained was analyzed and a sensory evaluation conducted, and it was found that both L. thermotolerans and co-inoculations retained more aromatic esters over time and had a lower evolution toward the yellow tones typical of oxidation and that the best sensory evaluation was that of the Lt + Mp co-inoculation. Lachancea thermotolerans and co-inoculations produced wines with low levels of volatile acidity (<0.4 g/L). This work shows that good consortia strategies with binary and ternary fermentations of yeast strains can be a powerful bio-tool for producing more complex wines.

Hanseniaspora vineae is a non-Saccharomyces yeast that has a powerful impact on the sensory profile of wines. Its effect on the aromatic profile of non-aromatic grape varieties, such as Albillo Mayor (Vitis vinifera, L), during vinification is a useful biotechnology to improve sensory complexity. Fermentation in steel barrels using Hanseniaspora vineae and sequential inoculation with Saccharomyces cerevisiae have been used to study the formation of terpenes and cell lysis in the production of Albillo white wines. The GC-MS analysis profile shows a significant effect of H. vineae fermentation on the contents of terpenes (≈×3), mainly in linalool (>×3), β-citronellol (>×4), geraniol (>×2) and α-terpineol (≈×2). The contents of several polyoxygenated terpenes and some volatile phenols with a spicy aroma were increased during fermentation. In summary, Hanseniaspora vineae releases a large number of cell wall polysaccharides during fermentation that affect wine palatability and structure. Hanseniaspora vineae is a powerful bio-tool to enhance the fruitiness, floral notes and freshness in non-aromatic white varieties.

Vitis vinifera flowers and grape fruits are one of the most interesting ecosystem niches for native yeasts development. There are more than a 100 yeast species and millions of strains that participate and contribute to design the microbial terroir. The wine terroir concept is understood when grape and wine micro-regions were delimited by different quality characteristics after humans had been growing vines for more than 10,000 years. Environmental conditions, such as climate, soil composition, water management, winds and air quality, altitude, fauna and flora and microbes, are considered part of the \"terroir\" and contribute to a unique wine style. If \"low input winemaking\" strategies are applied, the terroir effect will be expected to be more authentic in terms of quality differentiation. Interestingly, the role of the microbial flora associated with vines was very little study until recently when new genetic technologies for massive species identification were developed. These biotechnologies allowed following their environmental changes and their effect in shaping the microbial profiles of different wine regions. In this chapter we explain the interesting positive effects on flavor diversity and wine quality obtained by using \"friendly\" native yeasts that allowed the microbial terroir flora to participate and contribute during fermentation.

The use of Saccharomyces and non-Saccharomyces yeast species as mixed starters has potential advantages over pure culture fermentation due to increased wine complexity based on modification of metabolites of oenological interest. In this work, the effects of initial oxygenation on fermentation performance, chemical and volatile composition of French Colombard wine fermented with Hanseniaspora vineae and Saccharomyces cerevisiae in sequential inoculations were investigated in 1 L flasks. Although dominated by S. cerevisiae at the middle-end of fermentation, initial aeration for 1 day boosted H. vineae populations, and allowed H. vineae to coexist longer with S. cerevisiae in mixed cultures compared to no aeration, and suppressed S. cerevisiae later in the fermentation, which resulted in extended fermentation time. More important, the major fermentation products and volatile compounds were significantly modified by aeration and different from no aeration fermentation. The wines produced by aeration of mixed fermentations were characterized with higher amounts of glycerol, lactic acid and acetate esters, and lower levels of ethanol, higher alcohol and ethyl fatty acid esters. The aeration had more potential to shape the quality of wines and diversify the aromatic characteristics relative to simple mixed inoculation, as indicated by PCA analysis. Our results suggested that the impact of early aeration on yeast physiology extends beyond the aeration phase and influences fermentation activity, chemical and aromatic compounds in the following anaerobic stage. The aeration for a short time during the cell growth stage in mixed fermentation is therefore a potential means to increase the aromatic diversity and quality of wine, possibly providing an alternative approach to meet the expectations of wine consumers for diverse aromatic qualities.

This work presents the attempt to enhance the flavor complexity of cider fermented by different non-Saccharomyces species. Pichia kluyveri and Hanseniaspora vineae pure cultures were used as reference ciders. Mixed cultures between all 4 species gave 5 fermentations, where Hanseniaspora uvarum or Torulaspora quercuum were included for apple juice fermentation. Chemical composition and sensorial properties of all ciders were studied. The results indicated that the growth of P. kluyveri and H. vineae were interreacted and also affected by H. uvarum and T. quercuum. H. vineae was more capable of consuming sugar than P. kluyveri. Ciders from the single culture fermentation with P. kluyveri (Pk), as well as from mixed fermentation with P. kluyveri and H. uvarum (Pk-Hu), had high residual sugar, sugar/acid ratio, and glucose-fructose consumption ratio. Large shifts in the consumption and production of organic acids and polyphenols among all ciders were observed. The calculation of the relative odor activity value (rOAV) showed that 17 volatile compounds had an rOAV >1 in at least one sample, and acetate esters and ethyl esters were the groups with the highest number of volatile compounds of importance to the cider aroma. Among these 17 compounds, 3-methylbutyl acetate, 2-methylbutyl acetate, ethyl hexanoate, ethyl octanoate, and β-damascenone exhibited high rOAVs in some ciders and might contribute fruity, floral, and sweet features to the cider aroma. Besides, the tropical fruity aroma from 3-methylbutyl acetate was only perceived in Pk and Pk-Hu. The partial least squares regression (PLSR) analysis revealed that acetate esters contributed positively to the roasted and cooked odor of all ciders. This is the first study evaluating simultaneous fermentation of two non-Saccharomyces yeasts to produce cider, which provides new insights into cider production.

This study examines the effect of juices and non-Saccharomyces yeasts on physicochemical properties and sensory quality for fruit wines. Here, fruit wines produced from six kinds of fruit juices and eight non-Saccharomyces yeasts were evaluated, and the results show significant phenotypic diversity within these yeasts for wine fermentation on a range of substrates. Substantial variations in indicators such as total acid, sugar content and organic acid content were observed, which were caused by selection of both yeasts and juice types. These differences in characteristics had an impact on consumer preference. Considering overall acceptance by the panelists, correspondence analysis showed that Pichia kluyveri X31-10 (Pk31) was the most suitable strain for apple juice fermentation, and Fuji apple juice was the best substrate for this fermentation. A partial least-squares regression model of aroma characteristics against aroma components revealed that acetate esters were the primary contributors to tropical fruit aroma notes of ciders from Pk31. Additionally, 1-butanol, 3-methyl-, acetate; acetic acid, hexyl ester; and acetic acid, pentyl ester have been implicated as characteristic volatiles associated with Pk31.

Using novel non-Saccharomyces strains is regarded as an effective way to improve the aroma diversity of wines to meet the expectations of consumers. The non-Saccharomyces Hanseniaspora vineae and Metschnikowia pulcherrima have good aromatic properties useful for the production of table wine. However, no detailed information is available on their performances in icewine fermentation. In this study, simultaneous and sequential fermentation trials of indigenous M. pulcherrima CVE-MP20 or H. vineae CVE-HV11 with S. cerevisiae (SC45) were performed in 50-L fermenters of Vidal icewine, respectively. The results showed that SC45 cofermented with different non-Saccharomyces strains could generate a distinct aroma quality of icewine compared with four S. cerevisiae strain monocultures as evidenced by principal component analysis (PCA). Mixed fermentation of MP20/SC45 produced higher contents of acetate esters and β-damascenone with lower C6 alcohols relative to SC45 monoculture. Interestingly, HV11/SC45 generated the highest amounts of C6 alcohols [(Z)-3-hexen-1-ol and (E)-3-hexen-1-ol], higher alcohols (isobutanol, isopentanol, and 2-phenylethanol), acetate esters (2-phenethyl acetate and isoamyl acetate), cis-rose oxide, β-damascenone, and phenylacetaldehyde. Compared with simultaneous inoculation, sequential inoculation could achieve higher aroma diversity and produce higher intensity of fruity, flowery, and sweet attributes of icewine as assessed by calculating the odor activity values (OAVs). Our results verified the desired enological characteristics of H. vineae strain in icewine fermentation and also demonstrated that using indigenous non-Saccharomyces and Saccharomyces strains is a feasible way to improve aroma diversity of icewine products, which could provide an alternative way to meet the requirement of wine consumers for diversified aromatic quality.

Benzyl alcohol and other benzenoid-derived metabolites of particular importance in plants confer floral and fruity flavors to wines. Among the volatile aroma components in Vitis vinifera grape varieties, benzyl alcohol is present in its free and glycosylated forms. These compounds are considered to originate from grapes only and not from fermentative processes. We have found increased levels of benzyl alcohol in red Tannat wine compared to that in grape juice, suggesting de novo formation of this metabolite during vinification. In this work, we show that benzyl alcohol, benzaldehyde, p-hydroxybenzaldehyde, and p-hydroxybenzyl alcohol are synthesized de novo in the absence of grape-derived precursors by Hanseniaspora vineae. Levels of benzyl alcohol produced by 11 different H. vineae strains were 20-200 times higher than those measured in fermentations with Saccharomyces cerevisiae strains. These results show that H. vineae contributes to flavor diversity by increasing grape variety aroma concentration in a chemically defined medium. Feeding experiments with phenylalanine, tryptophan, tyrosine, p-aminobenzoic acid, and ammonium in an artificial medium were tested to evaluate the effect of these compounds either as precursors or as potential pathway regulators for the formation of benzenoid-derived aromas. Genomic analysis shows that the phenylalanine ammonia-lyase (PAL) and tyrosine ammonia lyase (TAL) pathways, used by plants to generate benzyl alcohols from aromatic amino acids, are absent in the H. vineae genome. Consequently, alternative pathways derived from chorismate with mandelate as an intermediate are discussed.