Insight into the growth and metabolic characteristics of indigenous commercial S. cerevisiae NX11424 at high and low levels of yeast assimilable nitrogen based on metabolomic approach.

Yeast assimilable nitrogen (YAN) is one of the important factors affecting yeast growth and metabolism. However, the nitrogen requirement of indigenous commercial S. cerevisiae NX11424 is unclear. In this study, metabolomics was used to analyze the metabolite profiles of the yeast strain NX11424 under high (433 mg/L) and low (55 mg/L) YAN concentrations. It was found that yeast biomass exhibited different trends under different YAN conditions and was generally positively correlated with the initial YAN concentration, while changes of key biomarkers of yeast strain NX11424 at different stages of fermentation showed a similar trend under high and low YAN concentrations. The YAN concentration affected the metabolite levels of the yeast strain NX11424, which resulted in the significant difference in the levels of pyruvic acid, α-oxoglutarate, palmitoleic acid, proline, butane-2,3-diol, citrulline, ornithine, galactinol, citramalic acid, tryptophan, alanine, phosphate and phenylethanol, mainly involving pathways such as central carbon metabolism, amino acid metabolism, fatty acid metabolism, purine metabolism, and energy metabolism. Yeast strain NX11424 could utilize proline to produce protein under a low YAN level. The intracellular level of citrulline and ornithine under high YAN concentration was higher than that under low YAN level. Yeast strain NX11424 is more suitable for fermentation at lower YAN level. The results obtained here will help to rational utilize of YAN by S. cerevisiae NX11424, and is conducive to precise control of the alcohol fermentation and improve wine quality.

Influence of flor yeast starters on volatile and nitrogen compounds during a controlled biological aging.

Fino Sherry wine undergoes biological aging carried out by a velum of flor yeast within a traditional dynamic system known as "criaderas and solera". The complex microbiota of biofilm-forming Saccharomyces cerevisiae strains play a crucial role in shaping the distinctive organoleptic profile of these types of wines. For this reason, the aim of this study is to analyze the changes produced by different flor yeast strains in the volatilome and the aminogram of different wines from the criaderas and solera system during biological aging in the laboratory, simulating a flor yeast velum condition at different stages of the system. Results suggest that each strain metabolizes wine differently, finding that depending on the wine, some strains are better suited for the process than others. In addition, it is found that the content of biogenic amines in Fino Sherry wines, previously attributed to malolactic bacteria, varies according to the yeast strain metabolizing the wine, suggesting that flor yeast could be used to modify biogenic amines content during biological aging. Results indicate that the use of selected flor yeast starters in biological aging may be of interest to modulate some parameters during Fino Sherry wine aging.

Oxygen alters redox cofactor dynamics and induces metabolic shifts in Saccharomyces cerevisiae during alcoholic fermentation.

Environmental conditions significantly impact the metabolism of Saccharomyces cerevisiae, a Crabtree-positive yeast that maintains a fermentative metabolism in high-sugar environments even in the presence of oxygen. Although the introduction of oxygen has been reported to induce alterations in yeast metabolism, knowledge of the mechanisms behind these metabolic adaptations in relation to redox cofactor metabolism and their implications in the context of wine fermentation remains limited. This study aimed to compare the intracellular redox cofactor levels, the cofactor ratios, and primary metabolite production in S. cerevisiae under aerobic and anaerobic conditions in synthetic grape juice. The molecular mechanisms underlying these metabolic differences were explored using a transcriptomic approach. Aerobic conditions resulted in an enhanced fermentation rate and biomass yield. Total NADP(H) levels were threefold higher during aerobiosis, while a decline in the total levels of NAD(H) was observed. However, there were stark differences in the ratio of NAD+/NADH between the treatments. Despite few changes in the differential expression of genes involved in redox cofactor metabolism, anaerobiosis resulted in an increased expression of genes involved in lipid biosynthesis pathways, while the presence of oxygen increased the expression of genes associated with thiamine, methionine, and sulfur metabolism. The production of fermentation by-products was linked with differences in the redox metabolism in each treatment. This study provides valuable insights that may help steer the production of metabolites of industrial interest during alcoholic fermentation (including winemaking) by using oxygen as a lever of redox metabolism.

Curse or blessing: Growth- and laccase-modulating properties of polyphenols and their oxidized derivatives on Botrytis cinerea.

Infection of grapevines with the grey mold pathogen Botrytis cinerea results in severe problems for winemakers worldwide. Browning of wine is caused by the laccase-mediated oxidation of polyphenols. In the last decades, Botrytis management has become increasingly difficult due to the rising number of resistances and the genetic variety of Botrytis strains. During the search for sustainable fungicides, polyphenols showed great potential to inhibit fungal growth. The present study revealed two important aspects regarding the effects of grape-specific polyphenols and their polymerized oxidation products on Botrytis wild strains. On the one hand, laccase-mediated oxidized polyphenols, which resemble the products found in infected grapes, showed the same potential for inhibition of growth and laccase activity, but differed from their native forms. On the other hand, the impact of phenolic compounds on mycelial growth is not correlated to the effect on laccase activity. Instead, mycelial growth and relative specific laccase activity appear to be modulated independently. All phenolic compounds showed not only inhibitory but also inductive effects on fungal growth and/or laccase activity, an observation which is reported for the first time. The simultaneous inhibition of growth and laccase activity demonstrated may serve as a basis for the development of a natural botryticide. Yet, the results showed considerable differences between genetically distinguishable strains, impeding the use of a specific phenolic compound against the genetic variety of wild strains. The present findings might have important implications for future understanding of Botrytis cinerea infections and sustainable Botrytis management including the role of polyphenols.

Effect of sequential fermentation with indigenous non-Saccharomyces cerevisiae combinations and Saccharomyces cerevisiae on the chemical composition and aroma compounds evolution of kiwifruit wine.

To unlock the potential of indigenous non-Saccharomyces cerevisiae and develop novel starters to enhance the aromatic complexity of kiwifruit wine, Zygosaccharomyces rouxii, Pichia kudriavzevii and Meyerozyma guilliermondii were pairwise combined and then used in sequential fermentation with Saccharomyces cerevisiae. The impact of different starter cultures on the chemical composition and flavor profile of the kiwifruit wines was comprehensively analyzed, and the aroma evolution during alcoholic fermentation was investigated by examining the changes in key volatiles and their loss rates. Compared with Saccharomyces cerevisiae, mixed starter cultures not only improve antioxidant capacity but also increase esters and alcohols yields, presenting intense floral and fruity aromas with high sensory acceptability. The results indicated that sequential inoculation of non-Saccharomyces cerevisiae combination and Saccharomyces cerevisiae promoted the development of volatiles while maintaining the stability of key aroma compounds in the winemaking environment and reducing the aroma loss rates during alcoholic fermentation.

Technological affinity index for interaction between lactic acid bacteria and Saccharomyces cerevisiae strains to modulate the fruity and floreal aroma of Catarratto wines.

Microbial interactions during the fermentation process influence the sensory characteristics of wines. Alongside alcoholic fermentation, malolactic fermentation also plays a crucial role in determining the aromatic traits of wines. The time (t), rate (m) and volatile organic compounds (VOCs) of malolactic fermentation are linked to the interaction between yeasts and lactic acid bacteria. The study investigated the interactions between Lactiplantibacillus plantarum or Oenococcus oeni with Saccharomyces cerevisiae by using the Technological Affinity Index (TAIndex). The co-inoculation of L. plantarum/S. cerevisiae resulted in a higher TAIndex than the co-inoculation of O. oeni/S. cerevisiae conditions. A low TAIndex led to increased aromaticity of the wines. The time and rate of malolactic fermentation have a strong impact on the synthesis of VOCs with a high olfactory impact. Therefore, knowledge of the TAIndex could play a decisive role in improving winemaking planning to produce wines with higher fruit and floral perceptions.

Impact of Bacillus subtilis on Chinese yellow rice wine (Huangjiu) fermentation: Method variations and flavor analysis.

This investigation explores the impact of various fermentation techniques and the inoculation of Bacillus subtilis spores on the physicochemical properties and principal flavor profiles of Huangjiu. Employing sensory analysis, headspace solid-phase microextraction, gas chromatography-tandem mass spectrometry (HS-SPME-GC-MS), and orthogonal partial least squares discriminant analysis (OPLS-DA), we observed that these variables significantly alter the physicochemical attributes of Huangjiu. Our analysis, integrating volatile organic compounds (VOCs) with odor activity values (OAV), revealed that while B. subtilis inoculation modifies the concentrations of key flavor compounds, it does not affect their types. Notably, the inoculation enhances the concentrations of 13 primary flavor compounds, thereby enriching floral and fruity notes while reducing higher alcohol levels. These findings contribute valuable insights into the flavor formation mechanisms of Huangjiu and guide the optimization of fermentation processes.

Culturable yeast community associated with grape must and honey bees sampled from apiaries located in the vineyards.

AIM: In this study, we investigated culturable yeast community, present in grape must sampled from vineyards with apiaries on the borders, and in honey bees collected in these apiaries. METHODS AND RESULTS: To this aim, yeasts isolated from spontaneously fermented grapes randomly collected in two vineyards (P1 and P2) with apiaries on the borders (A1 and A2) were compared to those isolated from spontaneously fermented grapes collected from a vineyard without apiary (P4). At the same time, yeast community was analyzed on bees collected in each apiary placed in the vineyards, in comparison to yeasts isolated from an apiary (A3) located far from the vineyards. The analysis was performed for two consecutive years (2021 and 2022). The isolated yeasts were identified by restriction analysis of amplified ITS region, followed by sequencing of ITS fragment.Our research showed that the presence of apiaries seems to increase yeast counts of grape must, in particular of Saccharomyces cerevisiae; furthermore, the permanence of apiaries in the vineyards allowed the recovering of these yeasts also from bees. CONCLUSIONS: Our findings seem to corroborate the role of bees as vectors and reservoirs of oenologically relevant yeasts, such as a source of non-conventional yeasts with potential biotechnological applications.

Screening, identification, and characterization of molds for brewing rice wine: Scale-up production in a bioreactor.

Rice wine, well known for its unique flavor, rich nutritional value, and health benefits, has potential for extensive market development. Rhizopus and Aspergillus are among several microorganisms used in rice wine brewing and are crucial for determining rice wine quality. The strains were isolated via Rose Bengal and starch as a combined separation medium, followed by oenological property and sensory evaluation screening. The strain exhibiting the best performance can be screened using the traditional rice wine Qu. The strains YM-8, YM-10, and YM-16, which exhibited strong saccharification and fermentation performance along with good flavor and taste, were obtained from traditional rice wine Qu. Based on ITS genetic sequence analysis, the YM-8, YM-10, and YM-16 strains were identified as Rhizopus microsporus, Rhizopus arrhizus, and Aspergillus oryzae. The optimum growth temperature of each of the three strains was 30°C, 32°C, and 30°C, and the optimum initial pH was 6.0, 6.5, and 6.5, respectively. The activities of α-amylase, glucoamylase, and protease of YM-16 were highest at 220.23±1.88, 1,269.04±30.32, and 175.16±1.81 U/g, respectively. The amino acid content of rice wine fermented in a 20-L bioreactor with the three mold strains was higher than that of the control group, except for arginine, which was significantly lower than that of the control group. The total amino acid content and the total content of each type of amino acid were ranked as YM-16 > YM-8 > YM-10 > control group, and the amino acid content varied greatly among the strains. The control group had a higher content, whereas YM-8 and YM-16 had lower contents of volatile aroma components than the control group and had the basic flavor substances needed for rice wine, which is conducive to the formation of rice wine aroma. This selected strain, YM-16, has strong saccharification and fermentation ability, is a rich enzyme system, and improves the flavor of rice wine, thereby demonstrating its suitability as a production strain for brewing.

Physical cell-cell contact elicits specific transcriptomic responses in wine yeast species.

Fermenting grape juice provides a habitat for a well-mapped and evolutionarily relevant microbial ecosystem consisting of many natural or inoculated strains of yeasts and bacteria. The molecular nature of many of the ecological interactions within this ecosystem remains poorly understood, with the partial exception of interactions of a metabolic nature such as competition for nutrients and production of toxic metabolites/peptides. Data suggest that physical contact between species plays a significant role in the phenotypic outcome of interspecies interactions. However, the molecular nature of the mechanisms regulating these phenotypes remains unknown. Here, we present a transcriptomic analysis of physical versus metabolic contact between two wine relevant yeast species, Saccharomyces cerevisiae and Lachancea thermotolerans. The data show that these species respond to the physical presence of the other species. In S. cerevisiae, physical contact results in the upregulation of genes involved in maintaining cell wall integrity, cell wall structural components, and genes involved in the production of H2S. In L. thermotolerans, HSP stress response genes were the most significantly upregulated gene family. Both yeasts downregulated genes belonging to the FLO family, some of which play prominent roles in cellular adhesion. qPCR analysis indicates that the expression of some of these genes is regulated in a species-specific manner, suggesting that yeasts adjust gene expression to specific biotic challenges or interspecies interactions. These findings provide fundamental insights into yeast interactions and evolutionary adaptations of these species to the wine ecosystem.IMPORTANCEWithin the wine ecosystem, yeasts are the most relevant contributors to alcoholic fermentation and wine organoleptic characteristics. While some studies have described yeast-yeast interactions during alcoholic fermentation, such interactions remain ill-defined, and little is understood regarding the molecular mechanisms behind many of the phenotypes observed when two or more species are co-cultured. In particular, no study has investigated transcriptional regulation in response to physical interspecies cell-cell contact, as opposed to the generally better understood/characterized metabolic interactions. These data are of direct relevance to our understanding of microbial ecological interactions in general while also creating opportunities to improve ecosystem-based biotechnological applications such as wine fermentation. Furthermore, the presence of competitor species has rarely been considered an evolutionary biotic selection pressure. In this context, the data reveal novel gene functions. This, and further such analysis, is likely to significantly enlarge the genome annotation space.

Metatranscriptomics reveals microbial community function succession and characteristic flavor formation mechanisms during black rice wine fermentation.

Black rice wine (BRW) is a traditional Chinese rice wine with unique flavors; however, the formation pathways of flavor compounds driven by microbiota remain unclear. This study employed HPLC and GC-MS to reveal that during BRW fermentation, free amino acids increased sevenfold, volatile compounds doubled, and 28 key characteristic flavor compounds were identified. Metatranscriptomic analysis indicated that during fermentation, driven by physicochemical factors and microbial interactions, Saccharomyces gradually became the dominant active microorganism (relative abundance 87.01%-97.70%). Other dominant microorganisms (relative abundance >0.1%), including Saccharomycopsis, Pediococcus, Wickerhamomyces, and Weissella, significantly decreased. Meanwhile, the microflora's signature functions underwent succession: transcription early, carbohydrate metabolism mid-stage, and autophagy late. These microbial and functional successions facilitated the accumulation of flavor compounds. Metabolic network reconstruction revealed that Saccharomyces was pivotal in substrate degradation and flavor formation, while other dominant microorganisms actively promoted these processes. This study provides insights into regulating BRW's flavor through microorganisms.

Influence of different stress factors during the elaboration of grape must's pieddecuve on the dynamics of yeast populations during alcoholic fermentation.

The pieddecuve (PdC) technique involves using a portion of grape must to undergo spontaneous fermentation, which is then used to inoculate a larger volume of must. This allows for promoting autochthonous yeasts present in the must, which can respect the typicality of the resulting wine. However, the real impact of this practice on the yeast population has not been properly evaluated. In this study, we examined the effects of sulphur dioxide (SO2), temperature, ethanol supplementation, and time on the dynamics and selection of yeasts during spontaneous fermentation to be used as PdC. The experimentation was conducted in a synthetic medium and sterile must using a multi-species yeast consortium and in un-inoculated natural grape must. Saccharomyces cerevisiae dominated both the PdC and fermentations inoculated with commercial wine yeast, displaying similar population growth regardless of the tested conditions. However, using 40 mg/L of SO2 and 1% (v/v) ethanol during spontaneous fermentation of Muscat of Alexandria must allowed the non-Saccharomyces to be dominant during the first stages, regardless of the temperature tested. These findings suggest that it is possible to apply the studied parameters to modulate the yeast population during spontaneous fermentation while confirming the effectiveness of the PdC methodology in controlling alcoholic fermentation.

Development of a qPCR method for classification of botrytized grape berries originated from Tokaj wine region.

One of the best-known Hungarian products on world wine market is Aszú, which belongs to the family of Tokaj wine specialties and is made from aszú berries. An important condition for the formation of aszú berries is the noble rot of technologically mature grapes, which is caused by Botrytis cinerea. At the same time botrytized sweet wines are produced not only in Hungary, but in many locations of wine-producing areas of Europe as well as in certain wine growing regions of other continents. The determination of botrytization is mostly based on sensory evaluations, which is a highly subjective procedure and largely depends on the training and experience of the evaluator. Currently, the classification of aszú berries (class I and class II) is based only on visual inspection and determination of sugar content. Based on these facts the primary goal of our work was to develop a qPCR assay capable for objective rating and classification of aszú berries. The developed qPCR is highly specific and sensitive as can clearly distinguish between B. cinerea and other filamentous fungi and yeast species occur on grapes. Moreover, it is suitable for categorizing berries colonized by B. cinerea to varying degrees. Thus, the developed qPCR method can be a useful technique for classification of the grape berries into four quality groups: healthy, semi-shrivelled, Aszú Class II and Aszú Class I.

Simultaneous inoculation of non-Saccharomyces yeast and lactic acid bacteria for aromatic kiwifruit wine production.

To further explore strain potential and develop an aromatic kiwifruit wine fermentation technique, the feasibility of simultaneous inoculation by non-Saccharomyces yeast and lactic acid bacteria was investigated. Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, and Limosilactobacillus fermentum, which have robust ß-glucosidase activity as well as good acid and ethanol tolerance, were inoculated for simultaneous fermentation with Zygosaccharomyces rouxii and Meyerozyma guilliermondii, respectively. Subsequently, the chemical compositions and sensory characteristics of the wines were comprehensively evaluated. The results showed that the majority of the simultaneous protocols effectively improved the quality of kiwifruit wines, increasing the content of polyphenols and volatile compounds, thereby enhancing sensory acceptability compared to the fermentation protocols inoculated with non-Saccharomyces yeast individually. Particularly, the collaboration between Lacp. plantarum and Z. rouxii significantly increased the diversity and content of esters, alcohols, and ketones, intensifying floral and seeded fruit odors, and achieving the highest overall acceptability. This study highlights the potential significance of simultaneous inoculation in kiwifruit wine production.

The formation of volatiles in fruit wine process and its impact on wine quality.

Fruit wine is one of the oldest fermented beverages made from non-grape fruits. Owing to the differences in fruit varieties, growing regions, climates, and harvesting seasons, the nutritional compositions of fruits (sugars, organic acids, etc.) are different. Therefore, the fermentation process and microorganisms involved are varied for a particular fruit selected for wine production, resulting in differences in volatile compound formation, which ultimately determine the quality of fruit wine. This article reviews the effects of various factors involved in fruit wine making, especially the particular modifications differing from the grape winemaking process and the selected strains suitable for the specific fruit wine fermentation, on the formation of volatile compounds, flavor and aroma profiles, and quality characteristics of the wine thus produced. KEY POINTS: • The volatile profile and fruit wine quality are affected by enological parameters. • The composition and content of nutrients in fruit must impact volatile profiles. • Yeast and LAB are the key determining factors of the volatile profiles of fruit wines.

Unraveling the Metabolic Behavior and Interspecific Interaction Pattern of Lactic Acid Bacteria within Chinese Rice Wine.

The synthetic community of lactic acid bacteria (LAB) is commonly utilized in the food industry for manipulating product properties. However, the intermediate interactions and ecological stability resulting from metabolic differences among various LAB types remain poorly understood. We aimed to analyze the metabolic behavior of single and combined lactic acid bacteria in China rice wine based on microbial succession. Three-stage succession patterns with obligate heterofermentative LAB dominating prefermentation and homofermentative LAB prevailing in main fermentation were observed. Facultative heterofermentative LAB exhibited significant growth. Pairwise coculture interactions revealed 63.5% positive, 34.4% negative, and 2.1% neutral interactions, forming nontransitive and transitive competition modes. Nontransitive competitive combinations demonstrated stability over ∼200 generations through amino acid (mainly aspartic acid, glutamine, and serine) cross-feeding and lactic acid detoxification, which also showed potential for controlling biogenic amines and developing LAB starter cultures. Our findings offer insights into the mechanistic underpinnings of LAB interaction networks.

Commercial wine yeast nitrogen requirement influences the production of secondary metabolites (aroma, hydroxytyrosol, melatonin and other bioactives) during alcoholic fermentation.

During alcoholic fermentation, Saccharomyces cerevisiae synthesizes different compounds, which are crucial for product quality: volatile compounds with sensory impact, and bioactive compounds such as melatonin (MEL) and hydroxytyrosol (HT), linked to health benefits. As many of these compounds are related with yeast's nitrogen metabolism, their production have been studied in four different commercial strains with different nitrogen requirement (Red Fruit, Uvaferm VRB, Lalvin Rhone 2323 and Lalvin QA23) being, Uvaferm UVR the higher nitrogen demander strain. All strains produced the secondary metabolites, notably Uvaferm UVR produced the highest HT concentration, despite its low growth. Uvaferm UVR emerged also as a significant producer of MEL, indicating a potential role in fermentation related stress. Moreover, Uvaferm UVR shows the highest total concentrations of volatile compounds. Multivariate analysis revealed distinct clustering based on nitrogen requirements of the strains, highlighting the strain-dependent metabolic responses.

Potential to take root in viticulture? An evaluation of mycorrhizal inoculants on the growth and nutrient uptake of young wine grapes planted in live field soil.

AIMS: Incorporating biofertilizers, such as arbuscular mycorrhizal fungal (AM) fungal inoculants, into vineyard management practices may enhance vine growth and reduce environmental impact. Here, we evaluate the effects of commercially available and local AM fungal inoculants on the growth, root colonization, and nutrient uptake of wine grapes (Vitis vinifera) when planted in a field soil substrate. METHODS AND RESULTS: In a greenhouse experiment, young wine grapes were planted in a field soil substrate and inoculated with one of three commercially available mycorrhizal inoculant products, or one of two locally collected whole soil inoculants. After 4 months of growth, inoculated vines showed no differences in plant biomass, colonization of roots by AM fungi, or foliar macronutrient concentrations compared to uninoculated field soil substrate. However, vines grown with local inoculants had greater shoot biomass than vines grown with mycorrhizal inoculant products. CONCLUSIONS: Although effects from inoculations with AM fungi varied by inoculant type and source, inoculations may not improve young vine performance in field soils with a resident microbial community.

Inoculation of Zaopei with biofortification: Inducing variation in community structure and improving flavor compounds in Nongxiangxing baijiu.

The deterioration of the quality of raw liquor caused by the low content of ethyl hexanoate in Nongxiangxing baijiu has become a pervasive problem in the baijiu industry. Therefore, this study attempted to increase the synthesis of ethyl hexanoate by microorganisms with high esterase activity to increase Zaopei fermentation. The results showed that biofortification was a feasible and important way to improve the quality of the raw liquor and increase the ethyl hexanoate content. Adding Bacillus subtilis, Staphylococcus epidermidis, and Millerozyma farinosa for biofortified fermentation disturbed the microbial community structure of Zaopei and increased the abundance of Wickerhamomyces, Saccharomyces, and Thermoascus. The contents of ethyl hexanoate, ethyl valerate, ethyl caprylate, and ethyl heptanoate also increased noticeably in baijiu. The results of E-nose and sensory analysis tested and verified that the baijiu in the fortified group had better flavor characteristics.

Advancements and challenges for brewing aroma-enhancement fruit wines: Microbial metabolizing and brewing techniques.

Aroma, a principal determinant of consumer preference for fruit wines, has recently garnered much attention. Fruit wines brewing was concomitant with complex biochemical reactions, in which a variety of compounds jointly contribute to the aroma quality. To date, the mechanisms underlying the synthesis of aroma compounds and biological regulation methods in fruit wines have remained ambiguous, hindering the further improvement of fruit wines sensory profiles. This review provides a detailed account of the synthesis and regulatory mechanisms of typical aroma compounds and their contributions to the characteristics of wines. Additionally, Comprehensive involves between microflora and the formation of aroma compounds have been emphasized. The microflora-mediated aroma compounds evolution can be controlled by key fermentation techniques to protect and enhance. Meanwhile, the genes impacting key aroma compounds can be identified, which provide references for the rapid screening of aroma-enhanced strains as well as target formation of aroma by modifying relative genes.