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.

Microbiological and technological parameters impacting the chemical composition and sensory quality of kombucha.

Kombucha is a beverage made from sugared tea transformed by yeasts and acetic acid bacteria. Being originally homemade, it has become an industrially produced soft drink whose quality standards are poorly defined and whose production process is still not fully controlled. Based on current knowledge in beverages, links between kombucha's chemical composition and sensorial compounds are drawn. Macromolecules create turbidity, whereas uncharacterized tea pigments derivatives participate in the color. Residual sugars bring sweetness and organic acids produced by acetic acid bacteria form its characteristic sour taste. Acetic acid is also part of its aroma profile, although little data are available on the smell of kombucha. Carbon dioxide, potentially polyphenols, and residual ethanol are involved in the mouthfeel. In this review, after defining the key compounds that shape the characteristic sensory properties of kombucha, the impact of different production parameters is discussed. Water composition is determinant in the extraction of tea compounds along with the tea type and infusion duration and temperature. The type and amount of sweeteners play a role in the sweetness and influences the production kinetics. Similarly, the amount of inoculum and its microbial composition have an effect on the production, but the role of the vessels' geometry and temperature are also essential parameters that can be used to adjust the acidification phase's duration. Despite the amount of research carried out, further investigations of kombucha's sensory characteristics are needed. Such research could lead to a better definition of kombucha's quality and to an improved control over its production process.

Influence of nitrogen status in wine alcoholic fermentation.

Nitrogen is an essential nutrient for yeast during alcoholic fermentation. Nitrogen is involved in the biosynthesis of protein, amino acids, nucleotides, and other metabolites, including volatile compounds. However, recent studies have called several mechanisms that regulate its role in biosynthesis into question. An initial focus on S. cerevisiae has highlighted that the concept of "preferred" versus "non-preferred" nitrogen sources is extremely variable and strain-dependent. Then, the direct involvement of amino acids consumed in the formation of proteins and volatile compounds has recently been reevaluated. Indeed, studies have highlighted the key role of lipids in nitrogen regulation in S. cerevisiae and their involvement in the mechanism of cell death. New winemaking strategies using non-Saccharomyces yeast strains in co- or sequential fermentation improve nitrogen management. Indeed, recent studies show that non-Saccharomyces yeasts have significant and specific needs for nitrogen. Moreover, sluggish fermentation can occur when they are associated with S. cerevisiae, necessitating nitrogen addition. In this context, we will present the consequences of nitrogen addition, discussing the sources, time of addition, transcriptome changes, and effect on volatile compound composition.

Dehydration stress responses of yeasts Torulaspora delbrueckii, Metschnikowia pulcherrima and Lachancea thermotolerans: Effects of glutathione and trehalose biosynthesis.

In food industry and winemaking, the use of active dehydrated yeast (ADY) Saccharomyces cerevisiae is a frequent practice because of the long-term stability and high efficiency of ADY. Nowadays, there is an increasing interest for new yeasts strains, such as Torulaspora delbrueckii (Td), Metschnikowia pulcherrima (Mp) and Lachancea thermotolerans (Lt). However, the yeasts transformation processes into the solidified form generate several stresses that reduce the cell viability. In this case, understanding the phenomena of yeast cell resistance before, during and after dehydration is of great importance. In this study we analyzed two compounds associated with resistance to stress and produced by cells, glutathione (total, oxidized and reduced) and trehalose, at different stages of the process. The impact of growing and dehydration conditions on cell viability was analyzed by flow cytometry and two-photon laser scanning microscopy. The results showed that cells naturally enriched in glutathione or trehalose acquired resistance to dehydration, preventing the oxidation of glutathione in a growth/dehydration condition dependent manner. This is the first time that simultaneous metabolic and dehydration responses were observed in three non-Saccharomyces strains. These findings represent an opportunity to better understand the yeast's dehydration resistance phenomena and thus to promote the efficient industrial production of new dried yeasts.

Wine microbiome: A dynamic world of microbial interactions.

Most fermented products are generated by a mixture of microbes. These microbial consortia perform various biological activities responsible for the nutritional, hygienic, and aromatic qualities of the product. Wine is no exception. Substantial yeast and bacterial biodiversity is observed on grapes, and in both must and wine. The diverse microorganisms present interact throughout the winemaking process. The interactions modulate the hygienic and sensorial properties of the wine. Many studies have been conducted to elucidate the nature of these interactions, with the aim of establishing better control of the two fermentations occurring during wine processing. However, wine is a very complex medium making such studies difficult. In this review, we present the current state of research on microbial interactions in wines. We consider the different kinds of interactions between different microorganisms together with the consequences of these interactions. We underline the major challenges to obtaining a better understanding of how microbes interact. Finally, strategies and methodologies that may help unravel microbe interactions in wine are suggested.

Cyclopropane fatty acid synthase from Oenococcus oeni: expression in Lactococcus lactis subsp. cremoris and biochemical characterization.

Bacterial cyclopropane fatty acid synthases (CFA synthases) catalyze the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the double bond of a lipid chain, thereby forming a cyclopropane ring. CFAs contribute to resistance to acidity, dryness, and osmotic imbalance in many bacteria. This work describes the first biochemical characterization of a lactic acid bacterium CFA synthase. We have overexpressed Oenococcus oeni CFA synthase in E. coli in order to purify the enzyme. The optimum cyclopropanation activity was obtained at pH 5.6 and 35.8 °C. The high K(m) (AdoMet) value obtained (2.26 mM) demonstrates the low affinity of O. oeni enzyme toward the L. lactis subsp. cremoris unsaturated phospholipids. These results explain the partial complementation of the L. lactis subsp. cremoris cfa mutant by the O. oeni cfa gene and suggest a probable substrate specificity of the O. oeni enzyme. The current study reveals an essential hypothesis about the specificity of O. oeni CFA synthase which could play a key function in the acid tolerance mechanisms of this enological bacterium.

High­throughput sequencing of amplicons for monitoring yeast biodiversity in must and during alcoholic fermentation.

We compared pyrosequencing technology with the PCR-ITS-RFLP analysis of yeast isolates and denaturing gradient gel electrophoresis (DGGE). These methods gave divergent findings for the yeast population. DGGE was unsuitable for the quantification of biodiversity and its use for species detection was limited by the initial abundance of each species. The isolates identified by PCR-ITSRFLP were not fully representative of the true population. For population dynamics, high-throughput sequencing technology yielded results differing in some respects from those obtained with other approaches. This study demonstrates that 454 pyrosequencing of amplicons is more relevant than other methods for studying the yeast community on grapes and during alcoholic fermentation. Indeed, this high-throughput sequencing method detected larger numbers of species on grapes and identified species present during alcoholic fermentation that were undetectable with the other techniques.

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.

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.

Unsaturated fatty acids from food and in the growth medium improve growth of Bacillus cereus under cold and anaerobic conditions.

In a chemically defined medium and in Luria broth, cold strongly reduced maximal population density of Bacillus cereus ATCC 14579 in anaerobiosis and caused formation of filaments. In cooked spinach, maximal population density of B. cereus in anaerobiosis was the same at cold and optimal temperatures, with normal cell divisions. The lipid containing fraction of spinach, but not the hydrophilic fraction, restored growth of B. cereus under cold and anaerobiosis when added to the chemically defined medium. This fraction was rich in unsaturated, low melting point fatty acids. Addition of phosphatidylcholine containing unsaturated, low melting point, fatty acids similarly improved B. cereus anaerobic growth at cold temperature. Addition of hydrogenated phosphatidylcholine containing saturated, high melting point, fatty acids did not modify growth. Fatty acids from phospholipids, from spinach and from hydrogenated phosphatidylcholine, although normally very rare in B. cereus, were inserted in the bacterium membrane. Addition of phospholipids rich in unsaturated fatty acids to cold and anaerobic cultures, increased fluidity of B. cereus membrane lipids, to the same level as those from B. cereus normally cold adapted, i.e. grown aerobically at 15 °C. B. cereus is therefore able to use external fatty acids from foods or from the growth medium to adapt its membrane to cold temperature under anaerobiosis, and to recover the maximal population density achieved at optimal temperature.

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.

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.

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.

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.

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.

Yeast-yeast interactions revealed by aromatic profile analysis of Sauvignon Blanc wine fermented by single or co-culture of non-Saccharomyces and Saccharomyces yeasts.

There has been increasing interest in the use of selected non-Saccharomyces yeasts in co-culture with Saccharomyces cerevisiae. The main reason is that the multistarter fermentation process is thought to simulate indigenous fermentation, thus increasing wine aroma complexity while avoiding the risks linked to natural fermentation. However, multistarter fermentation is characterised by complex and largely unknown interactions between yeasts. Consequently the resulting wine quality is rather unpredictable. In order to better understand the interactions that take place between non-Saccharomyces and Saccharomyces yeasts during alcoholic fermentation, we analysed the volatile profiles of several mono-culture and co-cultures. Candida zemplinina, Torulaspora delbrueckii and Metschnikowia pulcherrima were used to conduct fermentations either in mono-culture or in co-culture with S. cerevisiae. Up to 48 volatile compounds belonging to different chemical families were quantified. For the first time, we show that C. zemplinina is a strong producer of terpenes and lactones. We demonstrate by means of multivariate analysis that different interactions exist between the co-cultures studied. We observed a synergistic effect on aromatic compound production when M. pulcherrima was in co-culture with S. cerevisiae. However a negative interaction was observed between C. zemplinina and S. cerevisiae, which resulted in a decrease in terpene and lactone content. These interactions are independent of biomass production. The aromatic profiles of T. delbrueckii and S. cerevisiae in mono-culture and in co-culture are very close, and are biomass-dependent, reflecting a neutral interaction. This study reveals that a whole family of compounds could be altered by such interactions. These results suggest that the entire metabolic pathway is affected by these interactions.

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.

Non-Targeted Metabolomic Analysis of the Kombucha Production Process.

Kombucha is a traditional fermented beverage obtained from the transformation of sugared black tea by a community of yeasts and bacteria. Kombucha production recently became industrialized, but its quality standards remain poorly defined. Metabolomic analyses were applied using FT-ICR-MS to characterize the impacts of production phases and the type of tea on the non-volatile chemical composition of kombucha. Independently from tea type, the first phase of acidification in open vessel was characterized by the release of gluconate and gallate from acetic acid bacteria metabolism and probably from polymeric polyphenols, respectively. The second phase of carbonation in closed vessel induced a consumption or transformation of oleic acid that could be consecutive of oxygen limitation. The first phase had the most impact on molecular diversity, but tea type mainly influenced the global composition in polyphenol profile. Black tea polyphenols were more impacted by microbial activity compared to green tea polyphenols.

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.

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.