Identification of Key Parameters Inducing Microbial Modulation during Backslopped Kombucha Fermentation.

The aim of this study was to assess the impact of production parameters on the reproducibility of kombucha fermentation over several production cycles based on backslopping. Six conditions with varying oxygen accessibility (specific interface surface) and initial acidity (through the inoculation rate) of the cultures were carried out and compared to an original kombucha consortium and a synthetic consortium assembled from yeasts and bacteria isolated from the original culture. Output parameters monitored were microbial populations, biofilm weight, key physico-chemical parameters and metabolites. Results highlighted the existence of phases in microbial dynamics as backslopping cycles progressed. The transitions between phases occurred faster for the synthetic consortium compared to the original kombucha. This led to microbial dynamics and fermentative kinetics that were reproducible over several cycles but that could also deviate and shift abruptly to different behaviors. These changes were mainly induced by an increase in the Saccharomyces cerevisiae population, associated with an intensification of sucrose hydrolysis, sugar consumption and an increase in ethanol content, without any significant acceleration in the rate of acidification. The study suggests that the reproducibility of kombucha fermentations relies on high biodiversity to slow down the modulations of microbial dynamics induced by the sustained rhythm of backslopping cycles.

Competition for Nitrogen Resources: An Explanation of the Effects of a Bioprotective Strain Metschnikowia pulcherrima on the Growth of Hanseniaspora Genus in Oenology.

As a biological alternative to the antimicrobial action of SO2, bioprotection has been proposed to winemakers as a means to limit or prevent grape musts microbial alteration. Competition for nitrogenous nutrients and for oxygen are often cited as potential explanations for the effectiveness of bioprotection. This study analyses the effect of a bioprotective M. pulcherrima strain on the growth of one H. valbyensis strain and one H. uvarum strain. Bioprotection efficiency was observed only against H. valbyensis inoculated at the two lowest concentrations. These results indicate a potential species-dependent efficiency of the bioprotective strain and a strong impact of the initial ratio between bioprotective and apiculate yeasts. The analysis of the consumption of nitrogen compounds revealed that leucine, isoleucine, lysine and tryptophan were consumed preferentially by all three strains. The weaker assimilation percentages of these amino acids observed in H. valbyensis at 24 h growth suggest competition with M. pulcherrima that could negatively affects the growth of the apiculate yeast in co-cultures. The slowest rate of O2 consumption of H. valbyensis strain, in comparison with M. pulcherrima, was probably not involved in the bioprotective effect. Non-targeted metabolomic analyses of M. pulcherrima and H. valbyensis co-culture indicate that the interaction between both strains particularly impact lysin and tryptophan metabolisms.

Influence of pH on Oenococcus oeni metabolism: Can the slowdown of citrate consumption improve its acid tolerance?

Oenococcus oeni is the lactic acid bacteria most suited to carry out malolactic fermentation in wine, converting L-malic acid into L-lactic acid and carbon dioxide, thereby deacidifying wines. Indeed, wine is a harsh environment for microbial growth, partly because of its low pH. By metabolizing citrate, O. oeni maintains its homeostasis under acid conditions. Indeed, citrate consumption activates the proton motive force, helps to maintain intracellular pH, and enhances bacterial growth when it is co-metabolized with sugars. In addition, citrate metabolism is responsible for diacetyl production, an aromatic compound which bestows a buttery character to wine. However, an inhibitory effect of citrate on O. oeni growth at low pH has been highlighted in recent years. In order to understand how citrate metabolism can be linked to the acid tolerance of this bacterium, consumption of citrate was investigated in eleven O. oeni strains. In addition, malate and sugar consumptions were also monitored, as they can be impacted by citrate metabolism. This experiment highlighted the huge diversity of metabolisms between strains depending on their origin. It also showed the capacity of O. oeni to de novo metabolize certain end-products such as L-lactate and mannitol, a phenomenon never before demonstrated. It also enabled drawing hypotheses concerning the two positive effects that the slowing down of citrate metabolism could have on biomass production and malolactic fermentation occurring under low pH conditions.

Impact of Saccharomyces cerevisiae yeast inoculation mode on wine composition.

Inoculation modes are known to affect yeast behavior. Here, we characterized the impact of ADY and pre-culturing on the composition of the resulting wine, fermented by four commercial strains of Saccharomyces cerevisiae. Classical oenological parameters were not affected by the yeast inoculation mode. Using an untargeted metabolomic approach, a significant distinction in wine composition was noted regardless of the strain between the two inoculation modes, each associated with a specific metabolomic signature. 218 and 895 biomarkers were annotated, respectively, for ADYs associated with the preservation of wine polyphenols, and for pre-cultures related to the modulation of yeast nitrogen metabolism. Volatilome analysis revealed that the ester family was that most impacted by the inoculation mode whatever the strain. Ester production was enhanced in ADY condition. For the first time, the complete reprogramming of the yeast metabolism was revealed as a function of yeast preparation, which significantly impacts its volatilome and exometabolome.

Bioprotection Efficiency of Metschnikowia Strains in Synthetic Must: Comparative Study and Metabolomic Investigation of the Mechanisms Involved.

Three Metschnikowia strains marketed as bioprotection yeasts were studied to compare their antimicrobial effect on a mixture of two Hanseniaspora yeast strains in synthetic must at 12 °C, mimicking pre-fermentative maceration by combining different approaches. The growth of the different strains was monitored, their nitrogen and oxygen requirements were characterised, and their metabolomic footprint in single and co-cultures studied. Only the M. fructicola strain and one M. pulcherrima strains colonised the must and induced the rapid decline of Hanseniaspora. The efficiency of these two strains followed different inhibition kinetics. Furthermore, the initial ratio between Metschnikowia and Hanseniaspora was an important factor to ensure optimal bioprotection. Nutrient consumption kinetics showed that apiculate yeasts competed with Metschnikowia strains for nutrient accessibility. However, this competition did not explain the observed bioprotective effect, because of the considerable nitrogen content remaining on the single and co-cultures. The antagonistic effect of Metschnikowia on Hanseniaspora probably implied another form of amensalism. For the first time, metabolomic analyses of the interaction in a bioprotection context were performed after the pre-fermentative maceration step. A specific footprint of the interaction was observed, showing the strong impact of the interaction on the metabolic modulation of the yeasts, especially on the nitrogen and vitamin pathways.

Influence of spontaneous, "pied de cuve" and commercial dry yeast fermentation strategies on wine molecular composition and sensory properties.

While most producers in recent decades have relied on commercial yeasts (ADY) as their primary choice given their reliability and reproducibility, the fear of standardising the taste and properties of wine has led to the employment of alternative strategies that involve autochthonous yeasts such as pied de cuve (PdC) and spontaneous fermentation (SF). However, the impact of different fermentation strategies on wine has been a subject of debate and speculation. Consequently, this study describes, for the first time, the differences between the three kinds of fermentation at the metabolomic, chemical, and sensory levels in two wines: Chardonnay and Pinot Noir. The results showed how the yeast chosen significantly impacted the molecular composition of the wines, as revealed by metabolomic analysis that identified biomarkers with varying chemical compositions according to the fermentation modality. Notably, higher numbers of lipid markers were found for SF and PdC than ADY, which contained more peptides. Key molecules from the metabolic amino acid pathway, which are addressed in this article, showed evidence of such variations. In addition, the analysis of volatile aromatic compounds revealed an increase in groups of compounds specific to each fermentation. The sensorial analysis of Chardonnay wine showed a more qualitative sensory outcome (Higher fruit intensity) for ADY and SF compared to PdC. Our finding challenges the common speculation among wine producers that autochthonous yeast fermentations may offer greater complexity and uniqueness in comparison to commercial yeast fermentations.

Must protection, sulfites versus bioprotection: A metabolomic study.

The reduction of chemical inputs in wine has become one of the main challenges of the wine industry. One of the alternatives to sulfites developed is bioprotection, which consists in using non-Saccharomyces strains to prevent microbial deviation. However, the impact of substituting sulfites by bioprotection on the final wine remains poorly studied. For the first time, we characterized this impact on Chardonnay wine through an integrative approach. Interestingly, physico-chemical analysis did not reveal any difference between both treatments regarding classical oenological parameters. Nevertheless, bioprotection did not seem to provide as much protection against oxidation as sulfites, as observed through phenolic compound analysis. At a deeper level, untargeted metabolomic analyses revealed substantial changes in wine composition according to must treatment. In particular, the specific footprint of each treatment revealed an impact on nitrogen-containing compounds. This observation could be related to modifications in S. cerevisiae metabolism, in particular amino acid biosynthesis and tryptophan metabolism pathways. Thus, the type of must treatment seemed to impact metabolic fluxes of yeast differently, leading to the production of different compounds. For example, we observed glutathione and melatonin, compounds with antioxidant properties, which were enhanced with sulfites, but not with bioprotection. However, despite substantial modifications in wines regarding their chemical composition, the change in must treatment did not seem to impact the sensory profile of wine. This integrative approach has provided relevant new insights on the impact of sulfite substitution by bioprotection on Chardonnay wines.

Bio-protection in oenology by Metschnikowia pulcherrima: from field results to scientific inquiry.

Finding alternatives to the use of chemical inputs to preserve the sanitary and organoleptic quality of food and beverages is essential to meet public health requirements and consumer preferences. In oenology, numerous manufacturers already offer a diverse range of bio-protection yeasts to protect must against microbiological alterations and therefore limit or eliminate sulphites during winemaking. Bio-protection involves selecting non-Saccharomyces yeasts belonging to different genera and species to induce negative interactions with indigenous microorganisms, thereby limiting their development and their impact on the matrix. Although the effectiveness of bio-protection in the winemaking industry has been reported in numerous journals, the underlying mechanisms are not yet well understood. The aim of this review is to examine the current state of the art of field trials and laboratory studies that demonstrate the effects of using yeasts for bio-protection, as well as the interaction mechanisms that may be responsible for these effects. It focuses on the yeast Metschnikowia pulcherrima, particularly recommended for the bio-protection of grape musts.

To be or not to be required: Yeast vitaminic requirements in winemaking.

Although vitamins are prime actors in yeast metabolism, the nature and the extent of their requirement in Saccharomyces cerevisiae in winemaking remains little understood. To fill this gap, the evolution of 8 water-soluble vitamins and their diverse vitamers during its alcoholic fermentation in a synthetic must medium was monitored, providing the first evidence of the consumption of vitamers by five commercial S. cerevisiae strains, and highlighting the existence of preferential vitameric sources for its nutrition. The vitamins required by the yeast, B1, B5, and B8, were then identified, and the nature of their requirement characterized, strongly asserting the required trait of B1 for fermentation, B8 for growth, and B5 for both processes. The extent of the requirement for B5, that with the most impact of the three vitamins, was then quantified in three S. cerevisiae strains, resulting in the conclusion that 750 µg.L-1 should prove sufficient to cover the yeast's requirements. This investigation offers the first insight into S. cerevisiae vitaminic requirements for winemaking.

To each their own: Delving into the vitaminic preferences of non-Saccharomyces wine yeasts.

Considering the growing interest in non-Saccharomyces wine yeasts, and notably in the context of mixed fermentations with S. cerevisiae, understanding their nutritional behaviors is essential to ensure better management of these fermentations. The vitaminic consumption of three non-Saccharomyces yeasts (Starmerella bacillaris, Metschnikowia pulcherrima and Torulaspora delbrueckii) was investigated during their growth in wine-like conditions, providing initial evidence that they consume different vitamers. The vitamin consumption profiles during their growth highlighted releases of certain vitamers by the yeasts before re-assimilation, strongly suggesting the existence of synthesis pathways. Not only did the essential character of vitamin B1, in particular, appear to be a trait common to these yeasts, since all its vitamers are consumed, this investigation also provided evidence of the existence of species-dependent preferences for their vitaminic sources. These different behaviors were quite striking in certain vitamers, as was observed in nicotinamide: while it was consumed by T. delbrueckii, it was left untouched by S. bacillaris and produced by M. pulcherrima during growth. Furthermore, this offers grounds for further investigation into these yeasts' requirements, and provides the first tool for managing vitamin resources during mixed fermentations with S. cerevisiae, and for preventing nutritive deficiencies from occurring.

Thiamine and Biotin: Relevance in the Production of Volatile and Non-Volatile Compounds during Saccharomyces cerevisiae Alcoholic Fermentation in Synthetic Grape Must.

Vitamins are major cofactors to numerous key metabolic pathways in enological yeasts, and both thiamine and biotin, notably, are believed to be essential to yeast fermentation and growth, respectively. In order to further assess and clarify their role in winemaking, and in the resulting wine, alcoholic fermentations of a commercial Saccharomyces cerevisiae active dried yeast were conducted in synthetic media containing various concentrations of both vitamins. Growth and fermentation kinetics were monitored and proved the essential character of biotin in yeast growth, and of thiamine in fermentation. The synthetic wine volatile compounds were quantified, and notable influences of both vitamins appeared, through a striking positive effect of thiamine on the production of higher alcohols, and of biotin on fatty acids. Beyond the evidence of this influence on fermentations and on the production of volatiles, this work proves, for the first time, the impact held by vitamins on wine yeasts' exometabolome, investigated through an untargeted metabolomic analysis. This highlighted chemical differences in the composition of synthetic wines through a notably marked influence of thiamine on 46 named S. cerevisiae metabolic pathways, and especially in amino acid-associated metabolic pathways. This provides, overall, the first evidence of the impact held by both vitamins on the wine.

Experimental evolution forcing Oenococcus oeni acid tolerance highlights critical role of the citrate locus.

Oenococcus oeni is the main lactic acid bacterium associated with malolactic fermentation (MLF) of wines. MLF plays an important role in determining the final quality of wines. Nevertheless, due to the stressful conditions inherent to wine and especially acidity, MLF may be delayed. This study aimed to explore by adaptive evolution improvements in the acid tolerance of starters but also to gain a better understanding of the mechanisms involved in adaptation toward acidity. Four independent populations of the O. oeni ATCC BAA-1163 strain were propagated (approximately 560 generations) in a temporally varying environment, consisting in a gradual pH decrease from pH 5.3 to pH 2.9. Whole genome sequence comparison of these populations revealed that more than 45% of the substituted mutations occurred in only five loci for the evolved populations. One of these five fixed mutations affects mae, the first gene of the citrate operon. When grown in an acidic medium supplemented with citrate, a significantly higher bacterial biomass was produced with the evolved populations compared to the parental strain. Furthermore, the evolved populations slowed down their citrate consumption at low pH without impacting malolactic performance.

Use of Oenological Tannins to Protect the Colour of Rosé Wine in a Bioprotection Strategy with Metschnikowia pulcherrima.

Although bioprotection is now recognised as an alternative to SO2 for limiting microbial spoilage, it does not guarantee protection against oxidation. This limits its application, more specifically for rosé winemaking. Oenological tannins present antioxidant properties, which could represent an interesting alternative to SO2 to protect must and wines against oxidation. A combination of the inoculation of a bioprotectant yeast strain and the addition of oenological tannins was tested to eliminate sulfites during the pre-fermentative step of rosé winemaking. In this experiment carried out in a winery, two oenological tannins were compared: quebracho and gall nut tannins. The antioxidant efficiency of tannins was compared to that of SO2. Colorimetric assays associated with chemical analyses of anthocyanins and phenolic compounds confirmed that the use of bioprotection alone did not protect the wine from oxidation. An addition of oenological tannins on musts stabilized the colour of bioprotected rosé wine in a similar way that SO2 addition did. Quebracho tannins appeared more efficient than gall nut tannins. The colour differences observed cannot be explained either by the concentration or forms of anthocyanins. However, the addition of tannins led to better protection of oxidation-sensitive phenolic compounds comparable to that obtained with the addition of sulfites.

Exploring the unexplored: A characterization of vitamins and vitamers in white grape musts by high-performance liquid chromatography.

Although prime compounds in yeast metabolism, vitamins in oenology have remained mostly unexplored for decades. Here, a premier characterization of the vitamers in white grape musts has been drawn. A RP-HPLC method has therefore been developed for their direct analysis in musts, allowing for the determination of 19 different vitamers from 8 water-soluble vitaminic groups, including thiamine forms T, TMP and TPP, with LODs between 0.1 and 45.9 µg.L-1 and LOQs between 0.4 and 137.8 µg.L-1. A resulting characterization of 85 grape musts has been drawn from their vitaminic composition. Plus, the use of neither sulfites nor filtration affects the must vitamin content. The method stands as a useful tool for the later determination of yeast requirements, or impact of winemaking products on vitamins. The method has, overall, proven as practical and sensitive, for rapid identification of vitamins and vitamers in musts.

Citrate metabolism in lactic acid bacteria: is there a beneficial effect for Oenococcus oeni in wine?

Lactic acid bacteria (LAB) are Gram positive bacteria frequently used in the food industry for fermentation, mainly transformation of carbohydrates into lactic acid. In addition, these bacteria also have the capacity to metabolize citrate, an organic acid commonly found in food products. Its fermentation leads to the production of 4-carbon compounds such as diacetyl, resulting in a buttery flavor desired in dairy products. Citrate metabolism is known to have several beneficial effects on LAB physiology. Nevertheless, a controversial effect of citrate has been described on the acid tolerance of the wine bacterium Oenococcus oeni. This observation raises questions about the effect of citrate on the capacity of O. oeni to conduct malolactic fermentation in highly acidic wines. This review aims to summarize the current understanding of citrate metabolism in LAB, with a focus on the wine bacterium O. oeni. Metabolism with the related enzymes is detailed, as are the involved genes organized in cit loci. The known systems of cit locus expression regulation are also described. Finally, the beneficial effects of citrate catabolism on LAB physiology are reported and the negative impact observed in O. oeni is discussed.

Oxygen management during kombucha production: Roles of the matrix, microbial activity, and process parameters.

Oxygen plays a key role in kombucha production, since the production of main organic acids, acetic and gluconic acids, is performed through acetic acid bacteria's oxidative metabolism. Oxygen consumption during traditional kombucha production was investigated by comparing kombucha to mono and cocultures in sugared tea of microorganisms isolated from kombucha. Two yeasts, Brettanomyces bruxellensis and Hanseniaspora valbyensis and one acetic acid bacterium Acetobacter indonesiensis were used. Results showed that tea compounds alone were mainly responsible for oxygen depletion during the first 24 h following inoculation. During the first 7 days phase of production in open vessel, the liquid surface was therefore the only access to oxygen for microorganisms, as anaerobic conditions were sustained below this area. During the 5 days second phase of production after bottling, comparison of cultures with different microbial compositions showed that oxygen was efficiently depleted in the head space of the bottles in 3-6 h if the acetic acid bacterium was present. Lower access to oxygen after bottling stimulated ethanol production in B. bruxellensis and H. valbyensis cocultures with or without A. indonesiensis. This study provides insights into the management of oxygen and the roles of the tea and the biofilm during kombucha production.

eGFP Gene Integration in HO: A Metabolomic Impact?

Integrating fluorescent genes including eGFP in the yeast genome is common practice for various applications, including cell visualization and population monitoring. The transformation of a commercial S. cerevisiae strain by integrating a cassette including a gene encoding an EGFP protein in the HO gene was carried out using CRISPR-Cas9 technology. Although this type of integration is often used and described as neutral at the phenotypic level of the cell, we have highlighted that under alcoholic fermentation (in a Chardonnay must), it has an impact on the exometabolome. We observed 41 and 82 unique biomarkers for the S3 and S3GFP strains, respectively, as well as 28 biomarkers whose concentrations varied significantly between the wild-type and the modified strains. These biomarkers were mainly found to correspond to peptides. Despite similar phenotypic growth and fermentation parameters, high-resolution mass spectrometry allowed us to demonstrate, for the first time, that the peptidome is modified when integrating this cassette in the HO gene.

Use of a Minimal Microbial Consortium to Determine the Origin of Kombucha Flavor.

Microbiological, chemical, and sensory analyses were coupled to understand the origins of kombucha organoleptic compounds and their implication in the flavor of the kombucha beverage. By isolating microorganisms from an original kombucha and comparing it to monocultures and cocultures of two yeasts (Brettanomyces bruxellensis and Hanseniaspora valbyensis) and an acetic acid bacterium (Acetobacter indonesiensis), interaction effects were investigated during the two phases of production. 32 volatile compounds identified and quantified by Headspace-Solid Phase-MicroExtraction-Gas Chromatography/Mass Spectrometry (HS-SPME-GC/MS) were classified according to their origin from tea or microorganisms. Many esters were associated to H. valbyensis, while alcohols were associated to both yeasts, acetic acid to A. indonesiensis, and saturated fatty acids to all microorganisms. Concentration of metabolites were dependent on microbial activity, yeast composition, and phase of production. Sensory analysis showed that tea type influenced the olfactive perception, although microbial composition remained the strongest factor. Association of B. bruxellensis and A. indonesiensis induced characteristic apple juice aroma.

Microbial Interactions in Kombucha through the Lens of Metabolomics.

Kombucha is a fermented beverage obtained through the activity of a complex microbial community of yeasts and bacteria. Exo-metabolomes of kombucha microorganisms were analyzed using FT-ICR-MS to investigate their interactions. A simplified set of microorganisms including two yeasts (Brettanomyces bruxellensis and Hanseniaspora valbyensis) and one acetic acid bacterium (Acetobacter indonesiensis) was used to investigate yeast-yeast and yeast-acetic acid bacterium interactions. A yeast-yeast interaction was characterized by the release and consumption of fatty acids and peptides, possibly in relationship to commensalism. A yeast-acetic acid bacterium interaction was different depending on yeast species. With B. bruxellensis, fatty acids and peptides were mainly produced along with consumption of sucrose, fatty acids and polysaccharides. In opposition, the presence of H. valbyensis induced mainly the decrease of polyphenols, peptides, fatty acids, phenolic acids and putative isopropyl malate and phenylpyruvate and few formulae have been produced. With all three microorganisms, the formulae involved with the yeast-yeast interactions were consumed or not produced in the presence of A. indonesiensis. The impact of the yeasts' presence on A. indonesiensis was consistent regardless of the yeast species with a commensal consumption of compounds associated to the acetic acid bacterium by yeasts. In detail, hydroxystearate from yeasts and dehydroquinate from A. indonesiensis were potentially consumed in all cases of yeast(s)-acetic acid bacterium pairing, highlighting mutualistic behavior.

Evolution in Composition of Kombucha Consortia over Three Consecutive Years in Production Context.

Kombucha is a traditional drink obtained from sugared tea that is transformed by a community of yeasts and bacteria. Its production has become industrialized, and the study of the microbial community's evolution is needed to improve control over the process. This study followed the microbial composition of black and green kombucha tea over three consecutive years in a production facility using a culture-dependent method. Microorganisms were isolated and cultivated using selective agar media. The DNA of isolates was extracted, amplified using 26S and 16S PCR, and sequenced. Identities were obtained after a comparison to the NCBI database. Dekkera/Brettanomyces bruxellensis, Hanseniaspora valbyensis and Saccharomyces cerevisiae were the major yeast species, and the major bacterial genera were Acetobacter and Liquorilactobacillus. Results highlight the persistence of yeast species such as B. bruxellensis detected in 2019. Some yeasts species appeared to be sensitive towards stressful events, such as a hot period in 2019. However, they were resilient and isolated again in 2021, as was the case for H. valbyensis. Dominance of B. bruxellensis was clear in green and black tea kombucha, but proportions in yeasts varied depending on tea type and phase (liquid or biofilm). Composition in acetic acid and lactic acid bacteria showed a higher variability than yeasts with many changes in species over time.