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.

Enhance Wine Production Potential by Using Fresh and Dried Red Grape and Blueberry Mixtures with Different Yeast Strains for Fermentation.

Red grapes and blueberries are known for their high content of bioactive compounds and antioxidant properties. In Mediterranean winemaking, traditional sun-drying can be replaced by controlled-airflow-chamber-drying, which provides better quality, higher phenolic content, and increased antioxidants. This study aimed to increase the sugar content and phenolic compounds of the must by drying the fruits to fifty per cent of their original moisture content. Two musts were prepared: the first one was prepared by combining fresh red grapes and dried blueberries (M1), while the other was created using dried red grapes and fresh blueberries (M2), followed by fermentation at 25 °C with M05 Mead and X5 yeast strains. The M2 must showed the highest levels of phenolic compounds, red color (A520), total anthocyanins, and antioxidant activity. During fermentation, the anthocyanin content increased mainly in the dried blueberry macerates, where it increased between 4- to 5.5-fold. More bioactive compounds were extracted from the wines produced using yeast inoculation despite the shorter maceration times. A sensory analysis demonstrated consumers' acceptance of the wines in terms of color, flavor, and aroma. In conclusion, the use of red grapes in the production of blueberry red wine proved to be effective, providing higher sugar and must yields, while the dried fruits improved the fermentable sugar content obtaining wines with an alcoholic content between 10 and 11% (v/v). The higher levels of bioactive compounds increased the antioxidant capacity of the resulting red fruit wines.

Heteroresistance to colistin in wild-type Klebsiella pneumoniae isolates from clinical origin.

IMPORTANCE: Colistin is one of the last remaining therapeutic options for dealing with Enterobacteriaceae. Unfortunately, heteroresistance to colistin is also rapidly increasing. We described the prevalence of colistin heteroresistance in a variety of wild-type strains of Klebsiella pneumoniae and the evolution of these strains with colistin heteroresistance to a resistant phenotype after colistin exposure and withdrawal. Resistant mutants were characterized at the molecular level, and numerous mutations in genes related to lipopolysaccharide formation were observed. In colistin-treated patients, the evolution of K. pneumoniae heteroresistance to resistance phenotype could lead to higher rates of therapeutic failure.

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.

Microbial diversity in sherry wine biofilms and surrounding mites.

Sherry wines are film wines produced in the Jerez-Xérès-Sherry and Montilla-Moriles regions in southern Spain which require an aging process under flor biofilms, known as "biological aging". The presence of mites in Sherry wine wineries has been reported and associated with improved wine volatile properties. This work analyzes the microbial diversity in flor biofilms and mites in Sherry wine wineries using Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) and ITS/gene amplification. Two mite species, Carpoglyphus lactis and Tyrophagus putrescentiae, were spotted in the sampled winery and 32 microorganism species were identified in their exoskeleton or surrounding biofilms. To our knowledge, 26 of these species were never described before in sherry wine environments. We hypothesized that mites feed on the flor biofilms as well as another type of biofilm located in barrel cracks, known by winemakers as "natas" (cream in English). These non-studied biofilms showed the highest microbiome diversity among all samples (followed by C. lactis spotted nearby) thus, representing a niche of microorganisms with potential biotechnological interest. Besides mites, Drosophila flies were spotted in the sampling areas. The role of flies and mites as vectors that transport microorganisms among different niches (i.e., flor biofilms and natas) is discussed.

Flor yeast immobilization in microbial biocapsules for Sherry wine production: microvinification approach.

Sherry wine is a pale-yellowish dry wine produced in Southern-Spain which features are mainly due to biological aging when the metabolism of biofilm-forming yeasts (flor yeasts) consumes ethanol (and other non-fermentable carbon sources) from a previous alcoholic fermentation, and produces volatile compounds such as acetaldehyde. To start aging and maintain the wine stability, a high alcohol content is required, which is achieved by the previous fermentation or by adding ethanol (fortification). Here, an alternative method is proposed which aims to produce a more economic, distinctive Sherry wine without fortification. For this, a flor yeast has been pre-acclimatized to glycerol consumption against ethanol, and later confined in a fungal-based immobilization system known as "microbial biocapsules", to facilitate its inoculum. Once aged, the wines produced using biocapsules and free yeasts (the conventional method) exhibited chemical differences in terms of acidity and volatile concentrations. These differences were evaluated positively by a sensory panel. Pre-acclimatization of flor yeasts to glycerol consumption was not successful but when cells were immobilized in fungal pellets, ethanol consumption was lower. We believe that immobilization of flor yeasts in microbial biocapsules is an economic technique that can be used to produce high quality differentiated Sherry wines.

Endogenous CO2 Overpressure Effect on Higher Alcohols Metabolism during Sparkling Wine Production.

Higher alcohols produced by yeast during the fermentation of sparkling wine must have the greatest impact on the smell and taste of wine. At present, the metabolic response to methanol and higher alcohols formation of Saccharomyces cerevisiae under endogenous CO2 overpressure has not been fully elucidated. In this work, a proteomics and metabolomics approach using a OFFGEL fractionator and the LTQ Orbitrap for the protein identification, followed by a metabolomic study for the detection and quantification of both higher alcohols (GC-FID and SBSE-TD-GC-MS) and amino acids (HPLC), was carried out to investigate the proteomic and metabolomic changes of S. cerevisiae in relation to higher alcohols formation under a CO2 overpressure condition in a closed bottle. The control condition was without CO2 overpressure in an open bottle. Methanol and six higher alcohols were detected in both conditions, and we have been able to relate to a total of 22 proteins: 15 proteins in the CO2 overpressure condition and 22 proteins in the control condition. As for the precursors of higher alcohols, 18 amino acids were identified in both conditions. The metabolic and proteomic profiles obtained in both conditions were different, so CO2 overpressure could be affecting the metabolism of higher alcohols. Furthermore, it was not possible to establish direct correlations in the condition under CO2 overpressure; however, in the condition without pressure it was possible to establish relationships. The data presented here can be considered as a platform that serves as a basis for the S. cerevisiae metabolome-proteome with the aim of understanding the behavior of yeast under conditions of second fermentation in the production of sparkling wines.

Heteroresistance to Colistin in Clinical Isolates of Klebsiella pneumoniae Producing OXA-48.

Heteroresistance to colistin can be defined as the presence of resistant subpopulations in an isolate that is susceptible to this antibiotic. Colistin resistance in Gram-negative bacteria is more frequently related to chromosomal mutations and insertions. This work aimed to study heteroresistance in nine clinical isolates of Klebsiella pneumoniae producing OXA-48 and to describe genomic changes in mutants with acquired resistance in vitro. Antimicrobial susceptibility was determined by broth microdilution (BMD) and heteroresistance by population analysis profiling (PAP). The proteins related to colistin resistance were analyzed for the presence of mutations. Additionally, PCR of the mgrB gene was performed to identify the presence of insertions. In the nine parental isolates, the PAP method showed colistin heteroresistance of colonies growing on plates with concentrations of up to 64 mg/L, corresponding to stable mutant subpopulations. The MICs of some mutants from the PAP plate containing 4×MIC of colistin had absolute values of ≤2 mg/L that were higher than the parental MICs and were defined as persistent variants. PCR of the mgrB gene identified an insertion sequence that inactivated the gene in 21 mutants. Other substitutions in the investigated mutants were found in PhoP, PhoQ, PmrB, PmrC, CrrA and CrrB proteins. Colistin heteroresistance in K. pneumoniae isolates was attributed mainly to insertions in the mgrB gene and point mutations in colistin resistance proteins. The results of this study will improve understanding regarding the mechanisms of colistin resistance in mutants of K. pneumoniae producing OXA-48.

Yeast cell vacuum infusion into fungal pellets as a novel cell encapsulation methodology.

Immobilized yeast cells are used industrially in winemaking processes such as sparkling wine and Sherry wine production. Here, a novel approach has been explored for the infusion and immobilization of yeast cells into filamentous fungal pellets, which serve as a porous natural material. This was accomplished through vacuum application to force the yeast cells towards the core of the fungal pellets followed by culture in YPD medium to promote their growth from the interior. This method represents an improved variation of a previous approach for the assembly of "yeast biocapsules," which entailed the co-culture of both fungal and yeast cells in the same medium. A comparison was made between both techniques in terms of biocapsule productivity, cell retention capacity, and cell biological activity through an alcoholic fermentation of a grape must. The results indicated a substantial increase in biocapsule productivity (37.40-fold), higher cell retention within the biocapsules (threefold), and reduction in cell leakage during fermentation (twofold). Although the majority of the chemical and sensory variables measured in the produced wine did not exhibit notable differences from those produced utilizing suspended yeast cells (conventional method), some differences (such as herbaceous and toasted smells, acidity, bitterness, and persistence) were perceived and wines positively evaluated by the sensory panel. As the immobilized cells remain functional and the encapsulation technique can be expanded to other microorganisms, it creates potential for additional industrial uses like biofuel, health applications, microbe encapsulation and delivery, bioremediation, and pharmacy. KEY POINTS: • New approach improves biocapsule productivity and cell retention. • Immobilized yeast remains functional in fermentation. • Wine made with immobilized yeast had positive sensory differences.

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.

Effect of Bentonite Addition to Pedro Ximénez White Grape Musts before Their Fermentation with Selected Yeasts on the Major Volatile Compounds and Polyols of Wines and Tentative Relationships with the Sensorial Evaluation.

In this work, we study the effect of bentonite addition to the grape must before alcoholic fermentation on the chemical composition and sensorial profile of the obtained wines. Fermentations were carried out with two Saccharomyces cerevisiae commercial active dry yeasts treated or not with bentonite and were compared with a control wine obtained by spontaneous fermentation (using the grape must microbiota). Several significant effects on the chemical and sensorial attributes were established by statistical treatments. The selection by multiple variable analysis of seven volatile molecules (ethyl acetate; methanol; 1-propanol; isobutanol; 2-methyl-1-butanol; 3-metyl-1-butanol and 2-phenylethanol) provided several footprints that provide an easy visualization of bentonite effects on wine volatile compounds. A Principal Component Analysis carried out with all the compounds quantified by Gas-Chromatography revealed that the first two Principal Components explain 60.15 and 25.91%, respectively, of the total variance and established five groups that match with the five wines analyzed. Lastly, predictive models at p ≤ 0.05 level for the attributes sight, smell and taste were obtained by Partial Least Squared regression analysis of selected chemical variables.

Population Pharmacokinetic/Pharmacodynamic Modelling of Daptomycin for Schedule Optimization in Patients with Renal Impairment.

The aims of this study are (i) to develop a population pharmacokinetic/pharmacodynamic model of daptomycin in patients with normal and impaired renal function, and (ii) to establish the optimal dose recommendation of daptomycin in clinical practice. Several structural PK models including linear and non-linear binding kinetics were evaluated. Monte Carlo simulations were conducted with a fixed combination of creatinine clearance (30-90 mL/min/1.73 m2) and body weight (50-100 kg). The final dataset included 46 patients and 157 daptomycin observations. A two-compartment model with first-order peripheral distribution and elimination kinetics assuming non-linear protein-binding kinetics was selected. The bactericidal effect for Gram+ strains with MIC ≤ 0.5 mg/L could be achieved with 5-12 mg/kg daily daptomycin based on body weight and renal function. The administration of 10-17 mg/kg q48 h daptomycin allows to achieve bactericidal effect for Gram+ strains with MIC ≤ 1 mg/L. Four PK samples were selected as the optimal sampling strategy for an accurate AUC estimation. A quantitative framework has served to characterize the non-linear binding kinetics of daptomycin in patients with normal and impaired renal function. The impact of different dosing regimens on the efficacy and safety outcomes of daptomycin treatment based on the unbound exposure of daptomycin and individual patient characteristics has been evaluated.

Use of yeast biocapsules as a fungal-based immobilized cell technology for Indian Pale Ale-type beer brewing.

Immobilized cell technologies (ICT) have been used in wort fermentation, beer maturation, or production of alcohol-free or low-alcohol beer. The purpose of ICT is to restrict intact cells to a specific location while allowing biological function. It improves cell stability, operational flexibility, and control in brewing, as well as ease in executing continuous operations. We investigated the use of yeast biocapsules for Indian Pale Ale (IPA) type beer wort fermentation, a novel ICT in brewing. Yeast biocapsules are a spherical yeast immobilization system in which yeast cells are encapsulated and connected to the hyphae of an inactivated hollow filamentous fungus pellet. Fermentations with yeast encapsulated in alginate beads, as the standard immobilization practice, and in free (non-immobilized) forms were carried out in parallel. We found that yeast biocapsules are a better option for cell reutilization than alginate beads, but worse for beer must clarity. Beer brewed with yeast biocapsules differed in concentration for five volatile compounds (acetaldehyde, diacetyl, ethyl acetate, 1,1-diethoxyethane, and isoamyl alcohol) and three sensory characters (persistency of the foam, malt, and yeast character). KEY POINTS: • Yeast biocapsules were investigated for beer wort fermentation • Biocapsules improve cell reutilization but are limited for beer clarification • Beer brewed with biocapsules is chemically different than conventional beer • Most sensory features did not differ between biocapsule and control beer.

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].

Impact of CO2 overpressure on yeast mitochondrial associated proteome during the "prise de mousse" of sparkling wine production.

The "prise de mousse" stage during sparkling wine elaboration by the traditional method (Champenoise) involves a second fermentation in a sealed bottle followed by a prolonged aging period, known to contribute significantly to the unique organoleptic properties of these wines. During this stage, CO2 overpressure, nutrient starvation and high ethanol concentrations are stress factors that affect yeast cells viability and metabolism. Since mitochondria are responsible for energy generation and are required for cell aging and response to numerous stresses, we hypothesized that these organelles may play an essential role during the prise de mousse. The objective of this study is to characterize the mitochondrial response of a Saccharomyces cerevisiae strain traditionally used in sparkling wine production along the "prise de mousse" and study the effect of CO2 overpressure through a proteomic analysis. We observed that pressure negatively affects the content of mitochondrion-related proteome, especially to those proteins involved in tricarboxylic acid cycle. However, proteins required for the branched-amino acid synthesis, implied in wine aromas, and respiratory chain, also previously reported by transcriptomic analyses, were found over-represented in the sealed bottles. Multivariate analysis of proteins required for tricarboxylic cycle, respiratory chain and amino acid metabolism revealed differences in concentrations, allowing the wine samples to group depending on the time and CO2 overpressure parameters. Ethanol content along the second fermentation could be the main reason for this changing behavior observed at proteomic level. Further research including genetic studies, determination of ROS, characterization of mitochondrial activity and targeted metabolomics analyses is required. The list of mitochondrial proteins provided in this work will lead to a better understanding of the yeast behavior under these conditions of special interest in the wine 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.

Towards a better understanding of the evolution of odour-active compounds and the aroma perception of sparkling wines during ageing.

A native veil-forming yeast and a commercial yeast strain were used to elaborate sparkling wines by the Champenoise method with a grape variety traditionally used for the production of still wines. Wines aged on lees for fifteen months were sampled at five points and their physicochemical and sensory indices were analysed. Unsupervised and supervised statistical techniques were used to establish a comparison between 81 volatile compounds and eight odour descriptors (chemical, fruity, floral, fatty, balsamic, vegetal, empyreumatic and spicy). Principal component analysis of both datasets showed good separation among the samples in relation to ageing time and yeast strain. By using a partial least squares regression-based criterion, 38 odour active compounds were selected as the most influential for the ageing factor and out of them, only 27 were unique to certain aroma descriptors. These results contribute to a better understanding of the aroma perception of sparkling wines.

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.

Revealing the Yeast Diversity of the Flor Biofilm Microbiota in Sherry Wines Through Internal Transcribed Spacer-Metabarcoding and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry.

Flor yeast velum is a biofilm formed by certain yeast strains that distinguishes biologically aged wines such as Sherry wine from southern Spain from others. Although Saccharomyces cerevisiae is the most common species, 5.8 S-internal transcribed spacer (ITS) restriction fragment length polymorphism analyses have revealed the existence of non-Saccharomyces species. In order to uncover the flor microbiota diversity at a species level, we used ITS (internal transcribed spacer 1)-metabarcoding and matrix-assisted laser desorption/Ionization time of flight mass spectrometry techniques. Further, to enhance identification effectiveness, we performed an additional incubation stage in 1:1 wine:yeast extract peptone dextrose (YPD) before identification. Six species were identified: S. cerevisiae, Pichia manshurica, Pichia membranifaciens, Wickerhamomyces anomalus, Candida guillermondii, and Trichosporon asahii, two of which were discovered for the first time (C. guillermondii and Trichosporon ashaii) in Sherry wines. We analyzed wines where non-Saccharomyces yeasts were present or absent to see any potential link between the microbiota and the chemical profile. Only 2 significant volatile chemicals (out of 13 quantified), ethanol and ethyl lactate, and 2 enological parameters (out of 6 quantified), such as pH and titratable acidity, were found to differ in long-aged wines. Although results show a low impact where the non-Saccharomyces yeasts are present, these yeasts isolated from harsh environments (high ethanol and low nutrient availability) could have a potential industrial interest in fields such as food microbiology and biofuel production.