Metabolomics and HS-SPME-GC-MS-based analysis of quality succession patterns and flavor characteristics changes during the fermentation of Lycium barbarum and Polygonatum cyrtonema compound wine.

This work set out to investigate how the physicochemical markers, volatiles, and metabolomic characteristics of mixed fermented the fermentation of Lycium barbarum and Polygonatum cyrtonema compound wine (LPCW) from S. cerevisine RW and D. hansenii AS2.45 changed over the course of fermentation. HS-SPME-GC-MS combined with non-targeted metabolomics was used to follow up and monitor the fermentation process of LPCW. In total, 43 volatile chemical substances, mostly alcohols, esters, acids, carbonyl compounds, etc., were discovered in LPCW. After 30 days of fermentation, phenylethyl alcohol had increased to 3045.83 g/mL, giving off a rose-like fresh scent. The biosynthesis of valine, leucine, and isoleucine as well as the metabolism of alanine, aspartic acid, and glutamic acid were the major routes that led to the identification of 1385 non-volatile components in total. This study offers a theoretical foundation for industrial development and advances our knowledge of the fundamental mechanism underlying flavor generation during LPCW fermentation.

Fungal-Bacterial Mutualism: Species and Strain-Dependent Simultaneous Modulation of Branched-Chain Esters and Indole Derivatives in Fermented Sausages through Metabolite Cross-Feeding.

The precise impact of species and strain diversity on fungal-bacterial interactions and the overall community functioning has remained unclear. First, our study revealed how Debaryomyces hansenii influences diverse bacteria to accumulate key metabolites in a simulated fermented food system. For flavor, D. hansenii promoted the accumulation of branched-chain esters in Staphylococcus xylosus by promoting growth and facilitating the precursor branched-chain acids transformations but hindered the accumulation of Staphylococcus equorum. Furthermore, fungal-bacterial interactions displayed diversity among S. equorum strains. For bioactive compounds, species and strain diversity of lactic acid bacteria (LAB) also influences the production of indole derivatives. Then, we investigated specific metabolic exchanges under reciprocal interaction. Amino acids, rather than vitamins, were identified as the primary drivers of the bacterial growth promotion. Moreover, precursor transformations by D. hansenii played a significant role in branched-chain esters production. Finally, a synthetic community capable of producing high concentrations of branched-chain esters and indole derivatives was successfully constructed. These results provide valuable insights into understanding and designing synthetic communities for fermented sausages.

Impact of phenylalanine on Hanseniaspora vineae aroma metabolism during wine fermentation.

Hanseniaspora vineae exhibits extraordinary positive oenological characteristics contributing to the aroma and texture of wines, especially by its ability to produce great concentrations of benzenoid and phenylpropanoid compounds compared with conventional Saccharomyces yeasts. Consequently, in practice, sequential inoculation of H. vineae and Saccharomyces cerevisiae allows to improve the aromatic quality of wines. In this work, we evaluated the impact on wine aroma produced by increasing the concentration of phenylalanine, the main amino acid precursor of phenylpropanoids and benzenoids. Fermentations were carried out using a Chardonnay grape juice containing 150 mg N/L yeast assimilable nitrogen. Fermentations were performed adding 60 mg/L of phenylalanine without any supplementary addition to the juice. Musts were inoculated sequentially using three different H. vineae strains isolated from Uruguayan vineyards and, after 96 h, S. cerevisiae was inoculated to complete the process. At the end of the fermentation, wine aromas were analysed by both gas chromatography-mass spectrometry and sensory evaluation through a panel of experts. Aromas derived from aromatic amino acids were differentially produced depending on the treatments. Sensory analysis revealed more floral character and greater aromatic complexity when compared with control fermentations without phenylalanine added. Moreover, fermentations performed in synthetic must with pure H. vineae revealed that even tyrosine can be used in absence of phenylalanine, and phenylalanine is not used by this yeast for the synthesis of tyrosine derivatives.

The Role of Glucose-6-phosphate Dehydrogenase in the Wine Yeast Hanseniaspora uvarum.

Hanseniaspora uvarum is the predominant yeast species in the majority of wine fermentations, which has only recently become amenable to directed genetic manipulation. The genetics and metabolism of H. uvarum have been poorly studied as compared to other yeasts of biotechnological importance. This work describes the construction and characterization of homozygous deletion mutants in the HuZWF1 gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), which provides the entrance into the oxidative part of the pentose phosphate pathway (PPP) and serves as a major source of NADPH for anabolic reactions and oxidative stress response. Huzwf1 deletion mutants grow more slowly on glucose medium than wild-type and are hypersensitive both to hydrogen peroxide and potassium bisulfite, indicating that G6PDH activity is required to cope with these stresses. The mutant also requires methionine for growth. Enzyme activity can be restored by the expression of heterologous G6PDH genes from other yeasts and humans under the control of a strong endogenous promoter. These findings provide the basis for a better adaptation of H. uvarum to conditions used in wine fermentations, as well as its use for other biotechnological purposes and as an expression organism for studying G6PDH functions in patients with hemolytic anemia.

Insights into the mechanisms driving microbial community succession during pepper fermentation: Roles of microbial interactions and endogenous environmental changes.

Elucidating the driving mechanism of microbial community succession during pepper fermentation contributes to establishing efficient fermentation regulation strategies. This study utilized three-generation high-throughput sequencing technology, microbial co-occurrence network analysis, and random forest analysis to reveal microbial community succession processes and driving mechanisms during pepper fermentation. The results showed that more positive correlations than negative correlations were observed among microorganisms, with positive correlation proportions of 60 %, 51.03 %, and 71.43 % between bacteria and bacteria, fungi and fungi, and bacteria and fungi in sipingtou peppers, and 69.23 %, 54.93 %, and 79.44 % in zhudachang peppers, respectively. Microbial interactions, mainly among Weissella hellenica, Lactobacillus plantarum, Hanseniaspora opuntiae, and Kazachstania humillis, could drive bacterial and fungal community succession. Notably, the bacterial community successions during the fermentation of two peppers were similar, showing the transition from Leuconostoc pseudomesenteroides, Lactococcus lactis, Weissella ghanensis to Weissella hellenica and Lactobacillus plantarum. However, the fungal community successions in the two fermented peppers differed significantly, and the differential biomarkers were Dipodascus geotrichum and Kazachstania humillis. Differences in autochthonous microbial composition and inherent constituents brought by pepper varieties resulted in different endogenous environmental changes, mainly in fructose, malic acid, and citric acid. Furthermore, endogenous environmental factors could also drive microbial community succession, with succinic acid, lactic acid, and malic acid being the main potential drivers of bacterial community succession, whereas fructose, glucose, and succinic acid were the main drivers of fungal community succession. These results will provide insights into controlling fermentation processes by raw material combinations, optimization of environmental parameters, and microbial interactions.

Spontaneous fermentation of Marastina wines: The correlation between autochthonous mycobiota and phenolic compounds.

Understanding fungal community dynamics during fermentation is important for assessing their influence on wine's phenolic content. The present study represents the first effort to explore the correlation between the autochthonous mycobiota of Marastina grapes collected from Dalmatian winegrowing sub-regions in Croatia and the phenolic composition, as well as the physicochemical parameters of wines produced through spontaneous fermentation. The metataxonomic approach revealed Metschnikowia pulcherrima, Metschnikowia fructicola and Hanseniaspora uvarum as the core mycobiota detected at the initial phase of fermentation. By contrast, Saccharomyces cerevisiae took over the dominance starting from the middle stage of fermentation. The wine's phenolic compounds were revealed by high-performance liquid chromatography, with tyrosol being the most abundant. Rhodotorula babjevae and Botrytis cinerea showed a positive correlation with p-hydroxybenzoic acid, gentisic acid, caffeic acid and cinnamic acid, while demonstrating a negative correlation with protocatechuic acid and chlorogenic acid. Heterophoma novae-verbascicola exhibited the opposite behaviour regarding the same phenolic compounds. The concentration of lactic acid was positively correlated with B. cinerea and negatively correlated with Het. novae-verbascicola. These findings serve as a foundation for in-depth investigations into the role of autochthonous grape mycobiota in phenolic transformation during spontaneous fermentation, potentially leading to the production of high-quality wines with unique terroir characteristics. Future studies should aim to explore the specific role played by individual yeast isolates in the formation of phenolic compounds.

Integrating shotgun metagenomics and metabolomics to elucidate the dynamics of microbial communities and metabolites in fine flavor cocoa fermentation in Hainan.

The aim of this study was to investigate the dynamic profile of microorganisms and metabolites in Hainan Trinitario cocoa during a six-day spontaneous box fermentation process. Shotgun metagenomic and metabolomic approaches were employed for this investigation. The potential metabolic functions of microorganisms in cocoa fermentation were revealed through a joint analysis of microbes, functional genes, and metabolites. During the anaerobic fermentation phase, Hanseniaspora emerged as the most prevalent yeast genus, implicated in pectin decomposition and potentially involved in glycolysis and starch and sucrose metabolism. Tatumella, possessing potential for pyruvate kinase, and Fructobacillus with a preference for fructose, constituted the primary bacteria during the pre-turning fermentation stage. Upon the introduction of oxygen into the fermentation mass, acetic acid bacteria ascended to dominant within the microflora. The exponential proliferation of Acetobacter resulted in a decline in taxonomic richness and abundance. Moreover, the identification of novel species within the Komagataeibacter genus suggests that Hainan cocoa may serve as a valuable reservoir for the discovery of unique cocoa fermentation bacteria. The KEGG annotation of metabolites and enzymes also highlighted the significant involvement of phenylalanine metabolism in cocoa fermentation. This research will offer a new perspective for the selection of starter strains and the formulation of mixed starter cultures.

Lentils protein isolate as a fermenting substrate for the production of bioactive peptides by lactic acid bacteria and neglected yeast species.

In the current trend where plant-based foods are preferred over animal-based foods, pulses represent an alternative source of protein but also of bioactive peptides (BPs). We investigated the pattern of protein hydrolysis during fermentation of red lentils protein isolate (RLPI) with various lactic acid bacteria and yeast strains. Hanseniaspora uvarum SY1 and Fructilactobacillus sanfranciscensis E10 were the most proteolytic microorganisms. H. uvarum SY1 led to the highest antiradical, angiotensin-converting enzyme-inhibitory and antifungal activities, as found in low molecular weight water soluble extracts (LMW-WSE). The 2039 peptide sequences identified by LMW-WSE were screened using BIOPEP UWM database, and 36 sequences matched with known BPs. Fermentation of RLPI by lactic acid bacteria and yeasts generated 12 peptides undetected in raw RLPI. Besides, H. uvarum SY1 led to the highest abundance (peak areas) of BPs, in particular with antioxidant and ACE-inhibitory activities. The amino acid sequences LVR and LVL, identified in the fermented RLPI, represent novel findings, as they were detected for the first time in substrates subjected to microbial fermentation. KVI, another BP highly characteristic of RLPI-SY1, was previously observed only in dried bonito. 44 novel potential BPs, worthy of further characterization, were correlated with antifungal activity.

Exploring future applications of the apiculate yeast Hanseniaspora.

As a metaphor, lemons get a bad rap; however the proverb 'if life gives you lemons, make lemonade' is often used in a motivational context. The same could be said of Hanseniaspora in winemaking. Despite its predominance in vineyards and grape must, this lemon-shaped yeast is underappreciated in terms of its contribution to the overall sensory profile of fine wine. Species belonging to this apiculate yeast are known for being common isolates not just on grape berries, but on many other fruits. They play a critical role in the early stages of a fermentation and can influence the quality of the final product. Their deliberate addition within mixed-culture fermentations shows promise in adding to the complexity of a wine and thus provide sensorial benefits. Hanseniaspora species are also key participants in the fermentations of a variety of other foodstuffs ranging from chocolate to apple cider. Outside of their role in fermentation, Hanseniaspora species have attractive biotechnological possibilities as revealed through studies on biocontrol potential, use as a whole-cell biocatalyst and important interactions with Drosophila flies. The growing amount of 'omics data on Hanseniaspora is revealing interesting features of the genus that sets it apart from the other Ascomycetes. This review collates the fields of research conducted on this apiculate yeast genus.

Selection of yeast strains in naturally fermented cured meat as promising starter cultures for fermented cured beef, a traditional fermented meat product of northern China.

BACKGROUND: Fermented meat products are meat products with a unique flavor, color, and texture as well as an extended shelf life under natural or artificially controlled conditions. Microorganisms or enzymes are used to ferment the raw meat so that it undergoes a series of biochemical and physical changes. Common fermentation strains are lactic acid bacteria, yeasts, staphylococci, molds, and so forth. Studies on the inhibitory effect of yeast fermentation strain on N-nitrosamines in fermented meat products have not been reported. Two excellent yeast starters were identified to solve the problem of nitrosamines in fermented meat products. RESULTS: Meyerozyma guilliermondii and Debaryomyces hansenii led to weak acid production, strong resistance to NaCl and NaNO2 , and high tolerance to low acidic conditions. The inoculated fermented beef exhibited decreased lightness, moisture content, water activity, pH, protein content, nitrite content, and N-nitrosamine content in comparison with the control group fermented bacon. M. guilliermondii had a better effect, reducing pH from 5.69 to 5.41, protein content from 254.24 to 221.92 g·kg-1 , nitrite content from 28.61 to 25.33 mg·kg-1 and N-nitrosamine by 18.97%, and giving the fermented beef the desired meat color, mouthfeel, odor, taste, and tissue quality. CONCLUSION: In this study, two strains of yeast fermenters that can degrade N-nitrosamine precursors were identified, which to some extent solves the problem of the high risk of generating nitrosamines such as N-nitrosodiethylamine (NDEA) by processing fermented meat products with nitrites as precursors. These two strains are likely to be used as starter cultures for fermented meat products. © 2023 Society of Chemical Industry.

Identification of key yeast species and microbe-microbe interactions impacting larval growth of Drosophila in the wild.

Microbiota consisting of various fungi and bacteria have a significant impact on the physiological functions of the host. However, it is unclear which species are essential to this impact and how they affect the host. This study analyzed and isolated microbes from natural food sources of Drosophila larvae, and investigated their functions. Hanseniaspora uvarum is the predominant yeast responsible for larval growth in the earlier stage of fermentation. As fermentation progresses, Acetobacter orientalis emerges as the key bacterium responsible for larval growth, although yeasts and lactic acid bacteria must coexist along with the bacterium to stabilize this host-bacterial association. By providing nutrients to the larvae in an accessible form, the microbiota contributes to the upregulation of various genes that function in larval cell growth and metabolism. Thus, this study elucidates the key microbial species that support animal growth under microbial transition.

Genome-scale metabolic models reveal determinants of phenotypic differences in non-Saccharomyces yeasts.

BACKGROUND: Use of alternative non-Saccharomyces yeasts in wine and beer brewing has gained more attention the recent years. This is both due to the desire to obtain a wider variety of flavours in the product and to reduce the final alcohol content. Given the metabolic differences between the yeast species, we wanted to account for some of the differences by using in silico models. RESULTS: We created and studied genome-scale metabolic models of five different non-Saccharomyces species using an automated processes. These were: Metschnikowia pulcherrima, Lachancea thermotolerans, Hanseniaspora osmophila, Torulaspora delbrueckii and Kluyveromyces lactis. Using the models, we predicted that M. pulcherrima, when compared to the other species, conducts more respiration and thus produces less fermentation products, a finding which agrees with experimental data. Complex I of the electron transport chain was to be present in M. pulcherrima, but absent in the others. The predicted importance of Complex I was diminished when we incorporated constraints on the amount of enzymatic protein, as this shifts the metabolism towards fermentation. CONCLUSIONS: Our results suggest that Complex I in the electron transport chain is a key differentiator between Metschnikowia pulcherrima and the other yeasts considered. Yet, more annotations and experimental data have the potential to improve model quality in order to increase fidelity and confidence in these results. Further experiments should be conducted to confirm the in vivo effect of Complex I in M. pulcherrima and its respiratory metabolism.

Comparing the hierarchy of inter- and intra-species interactions with population dynamics of wine yeast cocultures.

In winemaking, the development of new fermentation strategies, such as the use of mixed starter cultures with Saccharomyces cerevisiae (Sc) yeast and non-Saccharomyces (NS) species, requires a better understanding of how yeasts interact, especially at the beginning of fermentation. Despite the growing knowledge on interactions between Sc and NS, few data are available on the interactions between different species of NS. It is furthermore still unclear whether interactions are primarily driven by generic differences between yeast species or whether individual strains are the evolutionarily relevant unit for biotic interactions. This study aimed at acquiring knowledge of the relevance of species and strain in the population dynamics of cocultures between five yeast species: Hanseniaspora uvarum, Lachancea thermotolerans, Starmerella bacillaris, Torulaspora delbrueckii and Sc. We performed cocultures between 15 strains in synthetic grape must and monitored growth in microplates. Both positive and negative interactions were identified. Based on an interaction index, our results showed that the population dynamics seemed mainly driven by the two species involved. Strain level was more relevant in modulating the strength of the interactions. This study provides fundamental insights into the microbial dynamics in early fermentation and contribute to the understanding of more complex consortia encompassing multiple yeasts trains.

Metabolic cross-feeding enhances branched-chain aldehydes production in a synthetic community of fermented sausages.

Microbial interactions play an important role in regulating the metabolic function of fermented food communities, especially the production of key flavor compounds. However, little is known about specific molecular mechanisms that regulate the production of key flavor compounds through microbial interactions. Here, we designed a synthetic consortium containing Debaryomyces hansenii D1, Staphylococcus xylosus S1, and Pediococcus pentosaceus PP1 to explore the mechanism of the microbial interactions underlying the branched-chain aldehydes production. In this consortium, firstly, D. hansenii secreted amino acids that promoted the growth of P. pentosaceus and S. xylosus. Specifically, D. hansenii D1 secreted alanine, aspartate, glutamate, glutamine, glycine, phenylalanine, serine, and threonine, which were the primary nutrients for bacterial growth. P. pentosaceus PP1 utilized all these eight amino acids through cross-feeding, whereas S. xylosus S1 did not utilize aspartate and serine. Furthermore, D. hansenii D1 promoted the production of branched-chain aldehydes from S. xylosus and P. pentosaceus through cross-feeding of α-keto acids (intermediate metabolites). Thus, the accumulation of 2-methyl-butanal was promoted in all co-culture. Overall, this work revealed the mechanism by which D. hansenii and bacteria cross-feed to produce branched-chain aldehydes in fermented sausages.

Interaction and Metabolic Function of Microbiota during the Washed Processing of Coffea arabica.

Coffee fermentation is crucial for flavor and aroma, as microorganisms degrade mucilage and produce metabolites. This study aimed to provide a basis for understanding the impact of microorganisms on Coffea arabica from Yunnan, China, during washed processing. The microbial community structure and differentially changed metabolites (DCMs) of C. arabica beans during washed processing were analyzed. The results indicated that the top five predominant microorganisms at the genera level were Achromobacter, Tatumella, Weissella, Streptococcus, and Trichocoleus for bacteria and Cystofilobasidium, Hanseniaspora, Lachancea, Wickerhamomyces, and Aspergillus for fungi. Meanwhile, the relative content of 115 DCMs in 36 h samples decreased significantly, compared to non-fermentation coffee samples (VIP > 1, p < 0.05, FC < 0.65), and the relative content of 28 DCMs increased significantly (VIP > 1, p < 0.05, FC > 1.5). Furthermore, 17 DCMs showed a strong positive correlation with microorganisms, and 5 DCMs had a strong negative correlation (p < 0.05, |r| > 0.6). Therefore, the interaction and metabolic function of microbiota play a key role in the formation of coffee flavor, and these results help in clarifying the fermentation mechanisms of C. arabica and in controlling and improving the quality of coffee flavor.

Co-Fermentation of Agri-Food Residues Using a Co-Culture of Yeasts as a New Bioprocess to Produce 2-Phenylethanol.

Whey is a dairy residue generated during the production of cheese and yogurt. Whey contains mainly lactose and proteins, contributing to its high chemical oxygen demand (COD). Current environmental regulations request proper whey disposal to avoid environmental pollution. Whey components can be transformed by yeast into ethanol and biomolecules with aroma and flavor properties, for example, 2-phenyethanol (2PE), highly appreciated in the industry due to its organoleptic and biocidal properties. The present study aimed to valorize agri-food residues in 2PE by developing suitable bioprocess. Cheese whey was used as substrate source, whereas crab headshells, residual soy cake, and brewer's spent yeast (BSY) were used as renewable nitrogen sources for the yeasts Kluyveromyces marxianus and Debaryomyces hansenii. The BSYs promoted the growth of both yeasts and the production of 2PE in flask fermentation. The bioprocess scale-up to 2 L bioreactor allowed for obtaining a 2PE productivity of 0.04 g2PE/L·h, twofold better productivity results compared to the literature. The bioprocess can save a treatment unit because the whey COD decreased under the detection limit of the analytical method, which is lower than environmental requirements. In this way, the bioprocess prevents environmental contamination and contributes to the circular economy of the dairy industry.

Exploring the effects of the fermentation method on the quality of Lycium barbarum and Polygonatum cyrtonema compound wine based on LC-MS metabolomics.

This study aimed to examine the effect of fermentation methods on the quality of Lycium barbarum and Polygonatum cyrtonema compound wine (LPW) by combining non-targeted metabolomic approaches with chemometrics and path profiling to determine the chemical and metabolic properties of LPW. The results demonstrated that SRA had higher leaching rates of total phenols and flavonoids, reaching 4.20 ± 0.10 v/v ethanol concentration. According to LC-MS non-targeting genomics, the metabolic profiles of LPW prepared by different mixtures of fermentation methods (Saccharomyces cerevisiae RW; Debaryomyces hansenii AS2.45) of yeast differed significantly. Amino acids, phenylpropanoids, flavonols, etc., were identified as the differential metabolites between different comparison groups. The pathways of tyrosine metabolism, biosynthesis of phenylpropanoids, and metabolism of 2-oxocarboxylic acids enriched 17 distinct metabolites. SRA stimulated the production of tyrosine and imparted a distinctive saucy aroma to the wine samples, providing a novel research concept for the microbial fermentation-based production of tyrosine.

Effect of selected agents for ochratoxin A biocontrol on the colour, texture and volatile profile of dry-cured fermented sausages.

BACKGROUND: Traditional dry-cured fermented sausages favour the growth of an autochthonous microbial population, which plays an important role in their sensory aspects. However, some moulds can produce mycotoxins such as ochratoxin A (OTA). The biocontrol agents (BCAs) Debaryomyces hansenii FHSCC 253H and Staphylococcus xylosus FHSCC Sx8 have been demonstrated to reduce OTA production in dry-cured meat products, but their influence in the sensory characteristics of sausages has to be tested. The aim of this study was to evaluate the effect of these BCAs on the colour, texture and volatile profile of dry-cured fermented sausages. RESULTS: D. hansenii caused few differences in the tested parameters with respect to the control batch. S. xylosus modified the texture and colour, although the values found were within the range expected for dry-cured fermented sausages 'salchichón'. Additionally, the volatile profile revealed the potential antioxidant effect of both BCAs and their ability to produce compounds associated with the ripened aroma that could increase product acceptability. CONCLUSION: The results indicate that there were no inconveniences in implementing both BCAs during the processing of dry-cured fermented sausages 'salchichón'. Moreover, D. hansenii FHSCC 253H could improve the volatile profile of this product. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Use of brewer's residual yeast for production of bacterial nanocellulose with Gluconacetobacter hansenii.

Bacterial nanocellulose (BNC) has attained elevated interest due to its versatile structure and high resistance characteristics. Accordingly, efforts have been made in order to reduce its production costs, such as the employment of its by-products as a nutrient broth to yield the microorganism. Residual brewer's yeast is an excellent recourse, due to its high nutritional value and availability. Therefore, research which aimed to contribute to the development of a low cost, efficient and biosustainable technology for BNC production with Gluconacetobacter hansenii was carried out. BNC was obtained from residual brewer's yeast hydrolysate at pH 7.0 and five days of incubation at 30 °C in static culture. The hydrolysate was characterized by the amount of sugars, fatty acids, total proteins and ash content. Subsequently, BNC obtained was characterized in terms of yield, carbon conversion ratio, hydrodynamic size, crystallinity, morphology, Fourier-transform infrared spectroscopy, and surface analysis. Residual brewer's yeast hydrolysate proved to be efficient in BNC production via gluconeogenesis with consumption of alanine, threonine and glycerol, obtaining 1.9 times the yield of the chemically defined broth adopted as standard. Additionally, properties observed in the obtained BNC were equal to those obtained from conventional chemical medium. The research contributed to bacterial nanocellulose production using by-products from the brewing industry.

Yeast diversity during the spontaneous fermentation of wine with only the microbiota on grapes cultivated in Japan.

Making wine via spontaneous fermentation without sulfur dioxide and commercial yeast (spontaneous winemaking) is increasing in recent year, but there is scant research regarding microbial communities present in Japan during spontaneous winemaking using culture-independent molecular methods. We analyzed fungal communities and populations during laboratory-scale spontaneous winemaking using sterilized labware to avoid winery-resident microbes. In the spontaneous fermentation of four grape varieties (Pinot Noir, Riesling, Koshu, and Koshusanjaku) grown in the same Japanese vineyard, our analysis of yeast and other fungal species by next-generation sequencing based on the ITS1 region demonstrated that Saccharomyces cerevisiae was eventually dominant in seven of 12 fermentation batches (three replications for each grape variety), whereas non-Saccharomyces species (e.g., Schizosaccharomyces japonicus, Lachancea dasiensis, and Hanseniaspora valbyensis) became dominant in four batches at the end of fermentation. In another batch, lactic acid bacteria (LAB) became dominant and the fermentation remained incomplete. Diverse microbes were involved in the spontaneous fermentation (particularly in Koshusanjaku), indicating that residual sugar remained and lactic and acetic acid largely increased. Compared to the control wine made with SO2 and commercial yeast, the concentration of lactic acid was 47-fold higher in the must dominated by L. dasiensis, and the concentrations of acetic acid and lactic acid were 10-fold and 20-fold higher in the must dominated by LAB, respectively. Even when indigenous S. cerevisiae became dominant, the finished wines obtained high sensory-analysis scores for complexity but low scores for varietal typicality, indicating the risk of fermentation with unselected wild yeast on the grapes grown in Japan.