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.

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.

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.

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].

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.

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.

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.