Implementation of a Novel Method for Processing Proteins from Acetic Acid Bacteria via Liquid Chromatography Coupled with Tandem Mass Spectrometry.

Acetic acid bacteria (AAB) and other members of the complex microbiotas, whose activity is essential for vinegar production, display biodiversity and richness that is difficult to study in depth due to their highly selective culture conditions. In recent years, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has emerged as a powerful tool for rapidly identifying thousands of proteins present in microbial communities, offering broader precision and coverage. In this work, a novel method based on LC-MS/MS was established and developed from previous studies. This methodology was tested in three studies, enabling the characterization of three submerged acetification profiles using innovative raw materials (synthetic alcohol medium, fine wine, and craft beer) while working in a semicontinuous mode. The biodiversity of existing microorganisms was clarified, and both the predominant taxa (Komagataeibacter, Acetobacter, Gluconacetobacter, and Gluconobacter) and others never detected in these media (Asaia and Bombella, among others) were identified. The key functions and adaptive metabolic strategies were determined using comparative studies, mainly those related to cellular material biosynthesis, energy-associated pathways, and cellular detoxification processes. This study provides the groundwork for a highly reliable and reproducible method for the characterization of microbial profiles in the vinegar industry.

Functional metaproteomic analysis of alcohol vinegar microbiota during an acetification process: A quantitative proteomic approach.

Vinegar is elaborated using a semi-continuous submerged culture of a complex microbiota of acetic acid bacteria. The genus Komagataeibacter provides much of the proteins of the metaproteome, being K. europaeus the main species working in this environment. In this work, the protein profile of the vinegar microbiota, obtained by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in samples from different cycle times of an acetification process using an alcohol medium, has been used to describe the functional metaproteome throughout the process. The analysis was focused on Komagataeibacter species which supplied about 90% of the metaproteome and particularly K. europaeus which accounts for more than 70%. According to these results, the natural behaviour of a microbial community in vinegar has been predicted at a quantitative proteomic level. The results revealed that most of the identified proteins involved in the metabolism of amino acids, biosynthesis of proteins, and energy production related-metabolic pathways increased their expression throughout the cycle loading phase and afterwards experimented a decrease coming into play other proteins acting against acetic acid stress. These findings may facilitate a better understanding of the microbiota's role and contributing to obtain a quality product.

Effect of endogenous CO2 overpressure on the yeast "stressome" during the "prise de mousse" of sparkling wine.

Sparkling wines elaboration by the "Champenoise" method involves a second fermentation of a base wine in hermetically sealed bottles and a subsequent aging period. The whole process is known as "prise de mousse". The endogenous CO2 pressure produced during the second fermentation by the yeast Saccharomyces cerevisiae could modify the sub-proteome involved in the response to different stresses, or "stressome", and cell viability thus affecting the wine organoleptic properties. This study focuses on the stressome evolution along the prise de mousse under CO2 overpressure conditions in an industrial S. cerevisiae strain. The results reveal an important effect of endogenous CO2 overpressure on the stress sub-proteome, cell viability and metabolites such as glycerol, reducing sugars and ethanol. Whereas the content of glycerol biosynthesis-related proteins increased in sealed bottle, those involved in the response to toxic metabolites like ROS, ethanol, acetaldehyde and acetic acid, decreased in content. Proteomic profile obtained in this study may be used to select suitable wine yeast strains for sparkling wine elaboration and improve their stress tolerance.

New insights on yeast and filamentous fungus adhesion in a natural co-immobilization system: proposed advances and applications in wine industry.

Fungi possess extraordinary strength in attachment to biotic and abiotic surfaces. This review focuses on adhesion mechanisms of yeast and filamentous fungi and the proposed combination of the adhesive forces of both organisms in an immobilization system called yeast biocapsules, whereby Saccharomyces cerevisiae cells are attached to the hyphae of Penicillium chrysogenum. The natural adherent properties of each organism, one multicellular and another unicellular, allow yeast to be fixated securely on the filamentous fungi and complete alcoholic fermentation. Following alcoholic fermentation, the hyphae become an inert support for yeast cells while maintaining shape and integrity. Biocapsules have been used successfully in both wine and bioethanol production. Investigation of the potential genes involved in fungal-yeast fusion suggests that natural hydrophobic interactions of both organisms play a major role. Analysis of the possible mechanisms involved in fungus and yeast adhesion, future perspectives on improving yeast immobilization, and proposed applications of the biocapsules are explored.

Comparative analysis of intracellular metabolites, proteins and their molecular functions in a flor yeast strain under two enological conditions.

Flor yeasts confer a wide range of organoleptic properties to Sherry-type wines during a process called "biological aging" that takes place after alcoholic fermentation. These kinds of yeasts adapt to a biological aging condition by forming a biofilm known as "flor velum" and by changing from fermentative to oxidative metabolism. It has been reported that some functions such as increase of cell surface hydrophobicity or changes to lipid metabolism are enhanced when yeasts switch to biofilm lifestyle. Here, we attempt to reveal intracellular metabolites and protein molecular functions not documented before that are relevant in biofilm formation and in fermentation by an endometabolome and proteome screening. We report that at early stages of biofilm formation, flor yeasts accumulate mannose, trehalose, glycerol, oleic and stearic acids and synthesize high amounts of GTPases, glycosylases and lipoproteins. On the other hand, in early fermentation, flor yeasts rapidly consume glucose and phosphoric acid; and produce abundant proteins related to chromatin binding, transcription factors and methyl transferases.

Recent advances in applying omic technologies for studying acetic acid bacteria in industrial vinegar production: A comprehensive review.

Vinegar and related bioproducts containing acetic acid as the main component are among the most appreciated fermented foodstuffs in numerous European and Asian countries because of their exceptional organoleptic and bio-healthy properties. Regarding the acetification process and obtaining of final products, there is still a lack of knowledge on fundamental aspects, especially those related to the study of biodiversity and metabolism of the present microbiota. In this context, omic technologies currently allow for the massive analysis of macromolecules and metabolites for the identification and characterization of these microorganisms working in their natural media without the need for isolation. This review approaches comprehensive research on the application of omic tools for the identification of vinegar microbiota, mainly acetic acid bacteria, with subsequent emphasis on the study of the microbial diversity, behavior, and key molecular strategies used by the predominant groups throughout acetification. The current omics tools are enabling both the finding of new vinegar microbiota members and exploring underlying strategies during the elaboration process. The species Komagataeibacter europaeus may be a model organism for present and future research in this industry; moreover, the development of integrated meta-omic analysis may facilitate the achievement of numerous of the proposed milestones. This work might provide useful guidance for the vinegar industry establishing the first steps towards the improvement of the acetification conditions and the development of new products with sensory and bio-healthy profiles adapted to the agri-food market.

Latest Trends in Industrial Vinegar Production and the Role of Acetic Acid Bacteria: Classification, Metabolism, and Applications-A Comprehensive Review.

Vinegar is one of the most appreciated fermented foods in European and Asian countries. In industry, its elaboration depends on numerous factors, including the nature of starter culture and raw material, as well as the production system and operational conditions. Furthermore, vinegar is obtained by the action of acetic acid bacteria (AAB) on an alcoholic medium in which ethanol is transformed into acetic acid. Besides the highlighted oxidative metabolism of AAB, their versatility and metabolic adaptability make them a taxonomic group with several biotechnological uses. Due to new and rapid advances in this field, this review attempts to approach the current state of knowledge by firstly discussing fundamental aspects related to industrial vinegar production and then exploring aspects related to AAB: classification, metabolism, and applications. Emphasis has been placed on an exhaustive taxonomic review considering the progressive increase in the number of new AAB species and genera, especially those with recognized biotechnological potential.

Unraveling the Role of Acetic Acid Bacteria Comparing Two Acetification Profiles From Natural Raw Materials: A Quantitative Approach in Komagataeibacter europaeus.

The industrial production of vinegar is carried out by the activity of a complex microbiota of acetic acid bacteria (AAB) working, mainly, within bioreactors providing a quite specific and hard environment. The "omics" sciences can facilitate the identification and characterization analyses of these microbial communities, most of which are difficult to cultivate by traditional methods, outside their natural medium. In this work, two acetification profiles coming from the same AAB starter culture but using two natural raw materials of different alcoholic origins (fine wine and craft beer), were characterized and compared and the emphasis of this study is the effect of these raw materials. For this purpose, the composition and natural behavior of the microbiota present throughout these profiles were analyzed by metaproteomics focusing, mainly, on the quantitative protein profile of Komagataeibacter europaeus. This species provided a protein fraction significantly higher (73.5%) than the others. A submerged culture system and semi-continuous operating mode were employed for the acetification profiles and liquid chromatography with tandem mass spectrometry (LC-MS/MS) for the protein analyses. The results showed that neither of two raw materials barely modified the microbiota composition of the profiles, however, they had an effect on the protein expression changes in different biological process. A molecular strategy in which K. europaeus would prevail over other species by taking advantage of the different features offered by each raw material has been suggested. First, by assimilating the excess of inner acetic acid through the TCA cycle and supplying biosynthetic precursors to replenish the cellular material losses; second, by a previous assimilation of the excess of available glucose, mainly in the beer medium, through the glycolysis and the pentose phosphate pathway (PPP); and third, by triggering membrane mechanisms dependent on proton motive force to detoxify the cell at the final moments of acetification. This study could complement the current knowledge of these bacteria as well as to expand the use of diverse raw materials and optimize operating conditions to obtain quality vinegars. Clinical Trial Registration: [www.ClinicalTrials.gov], identifier [PXD031147].

Combining omics tools for the characterization of the microbiota of diverse vinegars obtained by submerged culture: 16S rRNA amplicon sequencing and MALDI-TOF MS.

Vinegars elaborated in southern Spain are highly valued all over the world because of their exceptional organoleptic properties and high quality. Among the factors which influence the characteristics of the final industrial products, the composition of the microbiota responsible for the process and the raw material used as acetification substrate have a crucial role. The current state of knowledge shows that few microbial groups are usually present throughout acetification, mainly acetic acid bacteria (AAB), although other microorganisms, present in smaller proportions, may also affect the overall activity and behavior of the microbial community. In the present work, the composition of a starter microbiota propagated on and subsequently developing three acetification profiles on different raw materials, an alcohol wine medium and two other natural substrates (a craft beer and fine wine), was characterized and compared. For this purpose, two different "omics" tools were combined for the first time to study submerged vinegar production: 16S rRNA amplicon sequencing, a culture-independent technique, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), a culture-dependent method. Analysis of the metagenome revealed numerous taxa from 30 different phyla and highlighted the importance of the AAB genus Komagataeibacter, which was much more frequent than the other taxa, and Acetobacter; interestingly, also archaea from the Nitrososphaeraceae family were detected by 16S rRNA amplicon sequencing. MALDI-TOF MS confirmed the presence of Komagataeibacter by the identification of K. intermedius. These tools allowed for identifying some taxonomic groups such as the bacteria genera Cetobacterium and Rhodobacter, the bacteria species Lysinibacillus fusiformis, and even archaea, never to date found in this medium. Definitely, the effect of the combination of these techniques has allowed first, to confirm the composition of the predominant microbiota obtained in our previous metaproteomics approaches; second, to identify the microbial community and discriminate specific species that can be cultivated under laboratory conditions; and third, to obtain new insights on the characterization of the acetification raw materials used. These first findings may contribute to improving the understanding of the microbial communities' role in the vinegar-making industry.

Metabolic Changes by Wine Flor-Yeasts with Gluconic Acid as the Sole Carbon Source.

Gluconic acid consumption under controlled conditions by a Saccharomyces cerevisiae flor yeast was studied in artificial media. Gluconic acid was the sole carbon source and the compounds derived from this metabolism were tracked by endo-metabolomic analysis using a Gas Chromatography-Mass Spectrometry (GC-MSD) coupled methodology. After 6 days, about 30% of gluconic acid (1.5 g/L) had been consumed and 34 endo-metabolites were identified. Metabolomic pathway analysis showed the TCA cycle, glyoxylate-dicarboxylate, glycine-serine-threonine, and glycerolipid metabolic pathway were significantly affected. These results contribute to the knowledge of intracellular metabolomic fluctuations in flor yeasts during gluconic acid uptake, opening possibilities for future experiments to improve their applications to control gluconic acid contents during the production of fermented beverages.

Relationship between changes in the total concentration of acetic acid bacteria and major volatile compounds during the acetic acid fermentation of white wine.

BACKGROUND: In the scope of the wine vinegar production, this paper provides comprehensive information about the evolution of some volatile compounds during the biological acetification cycle. These data were compared with the acidity, cell concentration and ethanol concentration. Such information may allow a better understanding of the complex biological processes involved. RESULTS: The volatile compounds 2-phenylethanol, diethyl succinate (diethyl butanedioate), meso-2,3-butanediol (meso-butane-2,3-diol), levo-2,3-butanediol (levo-butane-2,3-diol), methanol and ethyl acetate exhibited no significant changes between the starting wine and produced vinegar, whereas the rest [acetoin (3-hydroxybutan-2-one) excepted] ethyl lactate (ethyl 2-hydroxypropanoate), isoamyl alcohols (3-methylbutan-1-ol and 2-methylbutan-1-ol), isobutanol (2-methylpropan-1-ol), 1-propanol (propan-1-ol), and acetaldehyde were consumed in substantial amounts during the process. Additionally, their specific evolution patterns alongside bacterial cell concentrations, acidity and ethanol concentration are shown. CONCLUSION: Concentrations of acetic acid bacteria at the end of the acetification cycle were found to vary because of cell lysis, a result of the high acidity and low ethanol concentration of the medium. Variations were similar to those in some volatile compounds, which suggests their involvement in the metabolism of acetic bacteria. The results testify to the usefulness of this pioneering study and suggest that there should be interest in similar, more detailed studies for a better knowledge of the presence of certain volatile compounds and metabolic activity in cells effecting the acetification of wine.

Metaproteomics of microbiota involved in submerged culture production of alcohol wine vinegar: A first approach.

Acetic acid bacteria form a complex microbiota that plays a fundamental role in the industrial production of vinegar through the incomplete oxidation reaction from ethanol to acetic acid. The organoleptic properties and the quality of vinegar are influenced by many factors, especially by the raw material used as acetification substrate, the microbial diversity and the technical methods employed in its production. The metaproteomics has been considered, among the new methods employed for the investigation of microbial communities, since it may provide information about the microbial biodiversity and behaviour by means of a protein content analysis. In this work, alcohol wine vinegar was produced through a submerged culture of acetic acid bacteria using a pilot acetator, operated in a semi-continuous mode, where the main system variables were monitored and the cycle profile throughout the acetification was obtained. Through a first approach, at qualitative level, of a metaproteomic analysis performed at relevant moments of the acetification cycle (end of fast and discontinuous loading phases and just prior to unloading phase), it is aimed to investigate the microbiota existent in alcohol wine vinegar as well as its changes during the cycle; to our knowledge, this is the first metaproteomics report carried out in this way on this system. A total of 1723 proteins from 30 different genera were identified; 1615 out of 1723 proteins (93.73%) belonged to the four most frequent (%) genera: Acetobacter, Gluconacetobacter, Gluconobacter and Komagataeibacter. Around 80% of identified proteins belonged to the species Komagataeibacter europaeus. In addition, GO Term enrichment analysis highlighted the important role of catalytic activity, organic cyclic compound binding, metabolic and biosynthesis processes throughout acetic acid fermentation. These findings provide the first step to obtain an AAB profile at omics level related to the environmental changes produced during the typical semi-continuous cycles used in this process and it would contribute to the optimization of operating conditions and improving the industrial production of vinegar.

Mapping the intracellular metabolome of yeast biocapsules - Spherical structures of yeast attached to fungal pellets.

Co-culture conditions are beneficial for study due to the advances which arise from symbiotic interactions and which cannot be replicated under pure culture conditions. Here, the focus is on the connection between two fungi - a yeast, Saccharomyces cerevisiae, and a filamentous fungus, Penicillium chrysogenum - in a yeast immobilization system termed' yeast biocapsules', where the yeast and filamentous fungus are strongly attached to one another, forming spherical structures. This co-culture condition hinders filamentous fungal biomass growth, while immobilization of yeast cells continues to increase. The effect of the co-culture condition on endometabolites or intracellular metabolites were tracked during the beginning and end of the yeast biocapsule formation period, and metabolites analyzed by Gas Chromatography-Mass Spectrometry Detector (GC-MSD). Distinct metabolite profiles were found between single culture conditions, involving each organism separately, and with the co-culture condition, where there were differences in 54 endometabolites. Specifically, co-culture condition compounds such as fructose, glycolic acid and glyceric acid were present in higher concentrations at the end of biocapsule formation. These results shed light on the mechanisms and biochemical impact of the interaction between the yeast and filamentous fungus and serve as a basis to apply and further develop yeast biocapsules as a new biotechnological tool with benefits for industry.

Effects of ADH2 overexpression in Saccharomyces bayanus during alcoholic fermentation.

The effect of overexpression of the gene ADH2 on metabolic and biological activity in Saccharomyces bayanus V5 during alcoholic fermentation has been evaluated. This gene is known to encode alcohol dehydrogenase II (ADH II). During the biological aging of sherry wines, where yeasts have to grow on ethanol owing to the absence of glucose, this isoenzyme plays a prominent role by converting the ethanol into acetaldehyde and producing NADH in the process. Overexpression of the gene ADH2 during alcoholic fermentation has no effect on the proteomic profile or the net production of some metabolites associated with glycolysis and alcoholic fermentation such as ethanol, acetaldehyde, and glycerol. However, it affects indirectly glucose and ammonium uptakes, cell growth, and intracellular redox potential, which lead to an altered metabolome. The increased contents in acetoin, acetic acid, and L-proline present in the fermentation medium under these conditions can be ascribed to detoxification by removal of excess acetaldehyde and the need to restore and maintain the intracellular redox potential balance.

Yeast Immobilization Systems for Alcoholic Wine Fermentations: Actual Trends and Future Perspectives.

Yeast immobilization is defined as the physical confinement of intact cells to a region of space with conservation of biological activity. The use of these methodologies for alcoholic fermentation (AF) offers many advantages over the use of the conventional free yeast cell method and different immobilization systems have been proposed so far for different applications, like winemaking. The most studied methods for yeast immobilization include the use of natural supports (e.g., fruit pieces), organic supports (e.g., alginate), inorganic (e.g., porous ceramics), membrane systems, and multi-functional agents. Some advantages of the yeast-immobilization systems include: high cell densities, product yield improvement, lowered risk of microbial contamination, better control and reproducibility of the processes, as well as reuse of the immobilization system for batch fermentations and continuous fermentation technologies. However, these methods have some consequences on the behavior of the yeasts, affecting the final products of the fermentative metabolism. This review compiles current information about cell immobilizer requirements for winemaking purposes, the immobilization methods applied to the production of fermented beverages to date, and yeast physiological consequences of immobilization strategies. Finally, a recent inter-species immobilization methodology has been revised, where yeast cells are attached to the hyphae of a Generally Recognized As Safe fungus and remain adhered following loss of viability of the fungus. The bio-capsules formed with this method open new and promising strategies for alcoholic beverage production (wine and low ethanol content beverages).

Influence of two yeast strains in free, bioimmobilized or immobilized with alginate forms on the aromatic profile of long aged sparkling wines.

Production of sparkling wines involve a second alcoholic fermentation and contact with yeast less over an extended period of time, which influences the aroma composition and sensory quality of the resulting wines. Sparkling wines obtained with two yeast strains inoculated as free cells, immobilized in alginate bed and bioimmobilized as biocapsules, were aged during 32 months. Among the volatile compounds, high Odor Activity Values were obtained with isoamyl acetate, ethyl propanoate, ethyl butanoate, ethyl 3-methylbutanoate, ethyl hexanoate, ethyl octanoate, hexanol, 2-methoxy-4-vinylphenol, decanal, octanoic acid, decanoic acid and TDN. Taken together these contribute more than 70% of the overall aromatic series value. Although some results rely more on the yeast strain than the inoculation format, specific aroma compounds were associated with the immobilization format, allowing the classification of sparkling wines by PCA. As a result the aroma quality of sparkling wines could be improved using immobilized yeasts.

FLO1, FLO5 and FLO11 Flocculation Gene Expression Impacts Saccharomyces cerevisiae Attachment to Penicillium chrysogenum in a Co-immobilization Technique.

A reoccurring flaw of most yeast immobilization systems that limits the potential of the technique is leakage of the cells from the matrix. Leakage may be due to weakly adherent cells, deterioration of the matrix, or to new growth and loss of non-adherent daughter cells. Yeast biocapsules are a spontaneous, cost effective system of immobilization whereby Saccharomyces cerevisiae cells are attached to the hyphae of Penicillium chrysogenum, creating hollow spheres that allow recovery and reutilization. This attachment is based on naturally occurring adherent properties of the yeast cell surface. We hypothesized that proteins associated with flocculation might play a role in adherence to fungal hyphae. To test this hypothesis, yeast strains with overexpressed and deleted flocculation genes (FLO1, FLO5, and FLO11) were evaluated for biocapsule formation to observe the impact of gene expression on biocapsule diameter, number, volume, dry mass, and percent immobilized versus non-immobilized cells. Overexpression of all three genes enhanced immobilization and resulted in larger diameter biocapsules. In particular, overexpression of FLO11 resulted in a five fold increase of absorbed cells versus the wild type isogenic strain. In addition, deletion of FLO1 and FLO11 significantly decreased the number of immobilized yeast cells compared to the wild type BY4742. These results confirm the role of natural adherent properties of yeast cells in attachment to fungal hyphae and offer the potential to create strongly adherent cells that will produce adherent progeny thereby reducing the potential for cell leakage from the matrix.

Flor Yeast: New Perspectives Beyond Wine Aging.

The most important dogma in white-wine production is the preservation of the wine aroma and the limitation of the oxidative action of oxygen. In contrast, the aging of Sherry and Sherry-like wines is an aerobic process that depends on the oxidative activity of flor strains of Saccharomyces cerevisiae. Under depletion of nitrogen and fermentable carbon sources, these yeast produce aggregates of floating cells and form an air-liquid biofilm on the wine surface, which is also known as velum or flor. This behavior is due to genetic and metabolic peculiarities that differentiate flor yeast from other wine yeast. This review will focus first on the most updated data obtained through the analysis of flor yeast with -omic tools. Comparative genomics, proteomics, and metabolomics of flor and wine yeast strains are shedding new light on several features of these special yeast, and in particular, they have revealed the extent of proteome remodeling imposed by the biofilm life-style. Finally, new insights in terms of promotion and inhibition of biofilm formation through small molecules, amino acids, and di/tri-peptides, and novel possibilities for the exploitation of biofilm immobilization within a fungal hyphae framework, will be discussed.

Potential application of a glucose-transport-deficient mutant of Schizosaccharomyces pombe for removing gluconic acid from grape must.

Musts from rotten grapes typically contain high levels of gluconic acid, which can raise severe problems in winemaking processes. In this work, the ability of the glucose-transport-deficient mutant YGS-5 of Schizosaccharomyces pombe to completely or partly remove gluconic acid from a synthetic glucose-containing medium and the potential use of this yeast strain for the same purpose in musts and wines were examined. Surprisingly, the S. pombe YGS-5 strain successfully removed 93% of the initial gluconic acid (2.5 gL(-1)) and 80% of the initial malic acid (1.0 gL(-1)) within 30 h after inoculation. Also, the yeast strain produced no volatile compounds other than those obtained in fermentations conducted with the wine yeast Saccharomyces cerevisiae. S. pombe YGS-5 could thus be used to remove gluconic acid present in musts from rotten grapes. On the basis of these results, various ways of using S. pombe YGS-5 to treat musts containing gluconic acid in order to solve the problems due to the high gluconic acid concentrations in botrytized grape must are proposed.

Effect of Schizosaccharomyces pombe on aromatic compounds in dry sherry wines containing high levels of gluconic acid.

Volatile compounds have been determined in control dry sherry wines and those supplemented with gluconic acid, which were inoculated with the Schizosaccharomyces pombe 1379 (ATCC 26760) yeast strain. These compounds were grouped, according to volatiles exhibiting the identical odor quality, into nine groups of the same odor character (aromatic series) as a way of establishing the aroma profile for the studied wines. Control and supplemented wines showed changes in the balsamic, spicy, roasty, and fruity aromatic series, and tasters judged the aroma as typical of wines subjected to biological aging. This fission yeast may be used as a treatment to reduce gluconic acid contents in wines obtained from rotten grapes, making feasible the incorporation of these wines into the biological aging process. In addition, this procedure may also help to accelerate the traditional biological aging in sherry winemaking due to the contribution of some specific compounds by S. pombe to the wine.