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Chitosan exerts a significant influence on various chemical parameters affecting the quality of wine produced using multiple strains of Lachancea thermotolerans. The impact of chitosan on these parameters varies depending on the specific strain studied. We observed that, under the influence of chitosan, the fermentation kinetics accelerated for all examined strains. The formation of lactic acid increased by 41% to 97% across the studied L. thermotolerans strains, depending on the specific strain. This effect also influenced acidity-related parameters such as total acidity, which increased by 28% to 60%, and pH, which experienced a decrease of over 0.5 units. The consumption of malic acid increased by 9% to 20% depending on the specific strain of L. thermotolerans. Nitrogen consumption also rose, as evidenced by all L. thermotolerans strains exhibiting a residual value of Primary Amino Nitrogen (PAN) of below the detection limit, and ammonia consumption increased by 90% to 100%, depending on the strain studied. However, certain parameters such as acetic acid, succinic acid, and glycerol showed contradictory results depending on the strain under investigation. In terms of volatile composition, chitosan supplementation led to increased production of i-butanol by 32% to 65%, 3-methylbutanol by 33% to 63%, and lactic acid ethyl ester by 58% to 91% across all studied strains of L. thermotolerans. Other analyzed aroma compounds exhibited varying changes depending on the specific strain of L. thermotolerans.
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Assyrtiko is a rare ancient grape variety of Greece, which is known to produce Protected Designation of Origin (PDO) Santorini white wines. Besides the famous character of the volcanic terroir, Assyrtiko of Santorini is also marked by a low pH value and sharp acidity. The aim of the present study was to apply a new inoculation procedure that modulates the fermentation process by maintaining the unique sensorial characteristics of Assyrtiko wines based on acidity. For this purpose, the Lachancea thermotolerans species, known for the formation of lactic acid, was tested in sequential fermentation with three different Saccharomyces cerevisiae strains. At the end of the fermentation process, implantation control for S. cerevisiae strains (interdelta sequence profile analysis) was performed, oenological parameters were determined according to the OIV protocols, and the volatile compounds produced were measured by gas chromatography-mass spectrometry (GC/MS). Finally, all produced wines were evaluated by quantitative descriptive analysis by two groups of experts; the Greek team of oenologists from Santorini Island specialized in Assyrtiko wines, and the French team of oenologists specialized in wine from Bordeaux. As expected, the inoculated strain was the one that dominated the fermentation process, but nine S. cerevisiae indigenous strains were also identified in the produced wines. Lachancea thermotolerans produced 1 g/L of lactic and also modulated the volatile profile of the wines independently of the S. cerevisiae strain used. The origin of the panelists played an important role in bringing up sensorial traits, such as acidity. Our results led to a new interesting application of L. thermotolerans for white wine production adapted to climate change claims.
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Botrytis cinerea poses a recurring threat to viticulture, causing significant yield losses each year. The study explored the biocontrol capabilities of commercially used winemaking yeasts as a strategy to manage B. cinerea in grape berries. The winemaking yeast strains-Saccharomyces cerevisiae ES181, Saccharomyces pastorianus KBG6, S. cerevisiae BCS103, Lachancea thermotolerans Omega, and Torulaspora delbrueckii TD291-reduced B. cinerea growth and conidiation in vitro. Furthermore, they demonstrated a decreased disease severity and number of conidia in grape berries. Among these strains, S. cerevisiae BCS103 was the most effective, inducing the expression of the defense-related gene PR4 in berries. Its diffusible compounds and volatile organic compounds also reduced the expression of BcLTF2, a positive regulator of B. cinerea conidiogenesis. The examined winemaking yeast strains, especially S. cerevisiae BCS103, demonstrated effective inhibition of B. cinerea in vitro and in grape berries, influencing key defense genes and reducing BcLTF2 expression, offering potential solutions for disease management in viticulture. The study underscores the promise of commercially available winemaking yeast strains as eco-friendly tools against B. cinerea in viticulture. Leveraging their safety and existing use in winemaking offers a potential avenue for sustainable disease management.
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Vitis , Vinho , Saccharomyces cerevisiae/metabolismo , Botrytis/genética , Vinho/análiseRESUMO
The study explores diverse strains of Lachancea thermotolerans in single-inoculum wine fermentation conditions using synthetic grape must. It aims to analyze the role of the species without external influences like other microorganisms or natural grape must variability. Commercial strains and selected vineyard isolates, untested together previously, are assessed. The research evaluates volatile and non-volatile chemical compounds in final wine, revealing significant strain-based variations. L. thermotolerans notably produces lactic acid and consumes malic acid, exhibiting moderate ethanol levels. The volatile profile displays strain-specific impacts, affecting higher alcohol and ester concentrations compared to S. cerevisiae. These effects vary based on the specific compounds. Using a uniform synthetic must enables direct strain comparisons, eliminating grape-related, environmental, or timing variables in the experiment, facilitating clearer insights into the behavior of L. thermotolerans in wine fermentation. The study compares for the first time all available commercial strains of L. thermotolerans.
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Evaluating domestication signatures beyond model organisms is essential for a thorough understanding of the genotype-phenotype relationship in wild and human-related environments. Structural variations (SVs) can significantly impact phenotypes playing an important role in the physiological adaptation of species to different niches, including during domestication. A detailed characterization of the fitness consequences of these genomic rearrangements, however, is still limited in non-model systems, largely due to the paucity of direct comparisons between domesticated and wild isolates. Here, we used a combination of sequencing strategies to explore major genomic rearrangements in a Lachancea cidri yeast strain isolated from cider (CBS2950) and compared them to those in eight wild isolates from primary forests. Genomic analysis revealed dozens of SVs, including a large reciprocal translocation (~16 kb and 500 kb) present in the cider strain, but absent from all wild strains. Interestingly, the number of SVs was higher relative to single-nucleotide polymorphisms in the cider strain, suggesting a significant role in the strain's phenotypic variation. The set of SVs identified directly impacts dozens of genes and likely underpins the greater fermentation performance in the L. cidri CBS2950. In addition, the large reciprocal translocation affects a proline permease (PUT4) regulatory region, resulting in higher PUT4 transcript levels, which agrees with higher ethanol tolerance, improved cell growth when using proline, and higher amino acid consumption during fermentation. These results suggest that SVs are responsible for the rapid physiological adaptation of yeast to a human-related environment and demonstrate the key contribution of SVs in adaptive fermentative traits in non-model species.IMPORTANCEThe exploration of domestication signatures associated with human-related environments has predominantly focused on studies conducted on model organisms, such as Saccharomyces cerevisiae, overlooking the potential for comparisons across other non-Saccharomyces species. In our research, employing a combination of long- and short-read data, we found domestication signatures in Lachancea cidri, a non-model species recently isolated from fermentative environments in cider in France. The significance of our study lies in the identification of large array of major genomic rearrangements in a cider strain compared to wild isolates, which underly several fermentative traits. These domestication signatures result from structural variants, which are likely responsible for the phenotypic differences between strains, providing a rapid path of adaptation to human-related environments.
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Saccharomyces cerevisiae , Saccharomycetales , Humanos , Saccharomyces cerevisiae/genética , Domesticação , Saccharomycetales/genética , Bebidas Alcoólicas , Translocação GenéticaRESUMO
In the current situation, wine areas are affected by several problems in a context of global warming: asymmetric maturities, pH increasing, high alcohol degree and flat wines with low freshness and poor aroma profile. The use of emerging biotechnologies allows to control or manage such problems. Emerging non-Saccharomyces as Lachancea thermotolerans are very useful for controlling pH by the formation of stable lactic acid from sugars with a slight concomitant alcohol reduction. Lower pH improves freshness increasing simultaneously microbiological stability. The use of Hanseniaspora spp. (specially H. vineae and H. opuntiae) or Metschnikowia pulcherrima promotes a better aroma complexity and improves wine sensory profile by the expression of a more complex metabolic pattern and the release of extracellular enzymes. Some of them are also compatible or synergic with the acidification by L. thermotolerans, and M. pulcherrima is an interesting biotool for reductive winemaking and bioprotection. The use of bioprotection is a powerful tool in this context, allowing oxidation control by oxygen depletion, the inhibition of some wild microorganisms, improving the implantation of some starters and limiting SO2. This can be complemented with the use of reductive yeast derivatives with high contents of reducing peptides and relevant compounds such as glutathione that also are interesting to reduce SO2. Finally, the use of emerging non-thermal technologies as Ultra High-Pressure Homogenization (UHPH) and Pulsed Light (PL) increases wine stability by microbial control and inactivation of oxidative enzymes, improving the implantation of emerging non-Saccharomyces and lowering SO2 additions. GRAPHICAL ABSTRACT.
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Yeasts are ubiquitously present in different natural sources. Some of these yeasts have interesting characteristics for the production of fermented food products. This study characterized Lachancea thermotolerans and L. quebecensis isolated from insects to determine their brewing potential. The yeasts were evaluated according to their fermentative potential in glucose and maltose-defined media and their resistance to ethanol and hop. Finally, craft beer was elaborated at a laboratory scale (10 L). The yeasts utilized glucose as the only carbon source and produced 3.25 ± 1.77, and 4.25 ± 1.06% (v/v), of ethanol for L. thermotolerans and quebecensis, respectively. While in the maltose-defined medium, ethanol content reached 3.25 ± 0.45, and 3.92 ± 0.36, respectively. The presence of alpha acids and ethanol affected the growth of L. quebecensis, which showed lower growth at 90 IBU and 8 ethanol% (v/v) mixtures. The craft beer brewed with L. quebecensis in monoculture experiments showed fruity flavors associated with ethyl acetate and isoamyl acetate. The ethanol content reached 3.50 ± 0.46% (v/v). The beer pH was 4.06 ± 0.20, with a lactic acid concentration of 1.21 ± 0.05 g/L. The sensory panel identified the beer as "fruity", "floral", "hoppy", "sweet", and "sour". To our knowledge, this is the first time L. quebecensis was reported as a potential candidate for sour beer production with reduced ethanol content.
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Spent coffee grounds (SCG) is a solid waste generated from coffee brewing. We recently developed a novel SCG hydrolysate-derived alcoholic beverage fermented with Lachancea thermotolerans Concerto and Lactiplantibacillus plantarum ML Prime. To further understand the potential health benefits of the fermented SCG hydrolysate alcoholic beverage, an untargeted metabolomics-based approach (UPLC-QTOF-MS/MS) was applied to detect and identify bioactive metabolites especially low molecular weight compounds. Our results showed that, compared to yeast monoculture-fermented SCG alcoholic beverages, yeast-lactic acid bacterial coculture enriched the beverage with a range of bioactive compounds especially aromatic and branched-chain amino acid derivatives (e.g., 4-hydroxyphenyl lactic acid, phenyl lactic acid, indole lactic acid, (S)-(-)-2-hydroxyisocaproic acid, and 4-hydroxyphenyl ethanol). Although some endogenous phenolic compounds were metabolized during fermentation, many phenolic metabolites (e.g., vinyl phenols, dihydrocaffeic acid, 3,4-dihydroxybenzoic acid, 4-hydroxycoumarin) were produced. Our study provided a theoretical basis for further valorization of SCG hydrolysates from the health benefits point of view and the findings may be extended to other fermented products.
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Café , Saccharomyces cerevisiae , Bebidas Alcoólicas , Café/química , Ácido Láctico/metabolismo , Fenóis/metabolismo , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em Tandem , Lactobacillus plantarumRESUMO
The discrete steps of transcriptional rewiring have been proposed to occur neutrally to ensure steady gene expression under stabilizing selection. A conflict-free switch of a regulon between regulators may require an immediate compensatory evolution to minimize deleterious effects. Here, we perform an evolutionary repair experiment on the Lachancea kluyveri yeast sef1Δ mutant using a suppressor development strategy. Complete loss of SEF1 forces cells to initiate a compensatory process for the pleiotropic defects arising from misexpression of TCA cycle genes. Using different selective conditions, we identify two adaptive loss-of-function mutations of IRA1 and AZF1. Subsequent analyses show that Azf1 is a weak transcriptional activator regulated by the Ras1-PKA pathway. Azf1 loss-of-function triggers extensive gene expression changes responsible for compensatory, beneficial, and trade-off phenotypes. The trade-offs can be alleviated by higher cell density. Our results not only indicate that secondary transcriptional perturbation provides rapid and adaptive mechanisms potentially stabilizing the initial stage of transcriptional rewiring but also suggest how genetic polymorphisms of pleiotropic mutations could be maintained in the population.
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Redes Reguladoras de Genes , Fatores de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Mutação , FenótipoRESUMO
Climate change is causing a lack of acidity during winemaking and oenologists use several solutions to cope with such a problem. Lachancea thermotolerans, which has the potential to tolerate the harsh physicochemical conditions of wine, has emerged as a promising alternative for pH management during winemaking and, currently, it is the most valuable yeast used for acidity control in wine. In this work a manageable method for L. thermotolerans genotyping based on a multiplexed microsatellite amplification in 6 different loci was developed. The proposed method was used to distinguish between 103 collection strains obtained from different geographical and isolation sources, and then challenged against a 429 L. thermotolerans isolates from several wineries and harvests. The procedure was also tested for fermentation monitoring and strain implantation. This approach was conceived to simplify the methodology available for L. thermotolerans genotyping, making it easy for applying in wine-related laboratories. This method can be applied to distinguish between L. thermotolerans strains in selection programs and to follow implantation of inoculated strains during winemaking with optimal results.
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Saccharomycetales , Vinho , Vinho/análise , Fermentação , Saccharomyces cerevisiae , Repetições de Microssatélites/genéticaRESUMO
BACKGROUND: The vineyard is a great reservoir of autochthonous yeast strains whose composition is defined by different regional (edaphology, orography or climatology) and anthropological factors (cultivation systems or cultural practices). Most of this yeast diversity corresponds to non-Saccharomyces strains, some of which have potential use in winemaking. METHODS: The oenological potential of 29 different native non-Saccharomyces strains belonging to 4 species (Lachancea thermotolerans, Torulaspora delbrueckii, Starmerella bacillaris and Metschnikowia spp.) was evaluated, using the autochthonous Saccharomyces cerevisiae XG3 strain as a control. Microfermentations with pure culture of each strain were performed in duplicate and the basic parameters and major volatiles of wines were analysed following official methodology. The best strain within each species was selected using a quantification matrix including the relevant oenological characteristics. RESULTS: The fermentative ability of non-Saccharomyces was lower than S. cerevisiae in all cases, but with differences among species. L. thermotolerans and T. delbrueckii showed higher fermentation rates than Starm. bacillaris, whereas Metschnikowia spp. presented a low fermentative power. At chemical level all non-Saccharomyces strains reduced the alcoholic content, the higher alcohols and the volatile acidity of wines and increased the content of glycerol, with differences among strains within a given species. T. delbrueckii and L. thermotolerans increased the total acidity of wines. The latter and Metschnikowia spp. strains produced lactic acid, which decreased the wine pH in the case of L. thermotolerans. According to their oenological traits the best rated strains of each species were Lt93, Td315, Mf278 and Sb474. In addition, the data obtained in pure fermentations were correlated to those chemical and aromatic compounds obtained with these non-Saccharomyces strains in sequential fermentations. CONCLUSIONS: Autochthonous strains of non-Saccharomyces yeast species contribute distinctive chemical characteristics to the wines. The correlations observed between wines fermented with the different non-Saccharomyces indigenous strains in pure and sequential fermentations suggest that their contribution to wine properties remains stable regardless of must composition or winemaking techniques.
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Vitis , Vinho , Saccharomyces cerevisiae , Vitis/química , Vinho/análise , Glicerol/análiseRESUMO
Non-Saccharomyces (NS) yeasts are gaining popularity in modern winemaking for improving wine quality. Climate change is one of the biggest challenges winegrowing now faces in warm regions. Here, Lachancea thermotolerans LtS1 and Torulaspora delbrueckii TdS6 combined with Saccharomyces cerevisiae ScS13 isolated from Assyrtiko grapes from Santorini island were evaluated in grape must fermentation with the aim to mitigate major consequences of temperature rise. Different inoculation protocols were evaluated, including simultaneous and sequential mixed-strain inoculations, displaying significant variation in the chemical and kinetic characteristics. Both LtS1 and TdS6 could raise the titratable acidity (TA). TdS6 also reduced the volatile acidity (VA) and was thus chosen for further evaluation in microvinifications and pilot-scale fermentations. Consistent with lab-scale trials, sequential inoculation exhibited the longest persistence of TdS6 resulting in minimum VA levels. Diethyl succinate, ethyl propanoate, and ethyl isobutyrate were significantly increased in sequential inoculations, although a decline in the net total ester content was observed. On the other hand, significantly higher levels of TA, succinic acid, and 2-methylpropanoic were associated with sequential inoculation. The overall performance of TdS6 coupled with a high compatibility with S. cerevisiae suggests its use in the fermentation of Santorini-Assyrtiko or other high sugar musts for the production of structured dry or sweet wines.
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The study performed sequential fermentations of red grape juice using several strains of Lachancea thermotolerans and one strain of Saccharomyces cerevisiae. Due to the new conditions imposed by climate change, wine acidity must be affected as well as the volatile profile. Non-Saccharomyces yeasts such as L. thermotolerans are real alternatives to soften the impact of climate change in winemaking. The L. thermotolerans strains included three commercially available strains and two wine-related natural isolates. L. thermotolerans showed significant statistical differences in basic chemical parameters such as lactic acid, malic acid, or ethanol concentrations as well as in the volatile profile. S. cerevisiae clearly produced some volatile compounds in higher amounts than the studied L. thermotolerans strains while others showed the opposite effect. Sequential fermentations involving any of the studied strains of L. thermotolerans with S. cerevisiae showed an increased volatile profile compared to the S. ceresisiae single fermentation, highlighting the synergic effect between the studied species.
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Saccharomycetales , Vitis , Vinho , Vinho/análise , Saccharomyces cerevisiae , FermentaçãoRESUMO
Climate change increases sugar content in grapes, resulting in unwanted increase in ethanol content of wine. Lachancea thermotolerans ferments glucose and fructose into both ethanol and lactate, decreasing final ethanol content and positively affecting wine acidity. Reported Lachancea thermotolerans strains show big variation in lactate production during fermentation. However, a mechanistic understanding of this lactate producing phenotype is currently lacking. Through a combination of metabolomics, transcriptomics, genomics and computational methods we show that the lactate production is induced by amino acid limitation in a high lactate producing strain. We found in fermentations in synthetic grape juice media that lactate production starts in the last stages of growth, marked by decreased growth rate and increased expression levels of stress related genes. This onset of lactate production is specific for the high lactate producing strain and independent of oxygen availability. The onset of lactate production was changed by increased amino acid content of the media, and it is shown by both computational methods and amino acid measurements that at the onset of lactate production amino acids become limiting for growth. This study shows that lactate production of Lachancea thermotolerans is directly linked to nitrogen availability in the media, an insight that can further aid in the improvement of wine quality.
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Ácido Láctico , Saccharomycetales , Etanol , Aminoácidos , Meios de CulturaRESUMO
Yeast selection for the wine industry in Spain started in 1950 for the understanding of the microbial ecology, and for the selection of optimal strains to improve the performance of alcoholic fermentation and the overall wine quality. This process has been strongly developed over the last 30 years, firstly on Saccharomyces cerevisiae, and, lately, with intense activity on non-Saccharomyces. Several thousand yeast strains have been isolated, identified and tested to select those with better performance and/or specific technological properties. The present review proposes a global survey of this massive ex-situ preservation of eukaryotic microorganisms, a reservoir of biotechnological solutions for the wine sector, overviewing relevant screenings that led to the selection of strains from 12 genera and 22 species of oenological significance. In the first part, the attention goes to the selection programmes related to relevant wine-producing areas (i.e. Douro, Extremadura, Galicia, La Mancha and Uclés, Ribera del Duero, Rioja, Sherry area, and Valencia). In the second part, the focus shifted on specific non-Saccharomyces genera/species selected from different Spanish and Portuguese regions, exploited to enhance particular attributes of the wines. A fil rouge of the dissertation is the design of tailored biotechnological solutions for wines typical of given geographic areas.
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Vitis , Vinho , Saccharomyces cerevisiae , Vinho/análise , Portugal , Fermentação , BiotecnologiaRESUMO
Global warming poses a threat to winemaking worldwide, especially in dry-warm regions such as Southern Italy. Must fermentation with non-Saccharomyces yeast starter is a possible approach to limit the negative effects of climate change, leading to desirable effects such as an increase in total acidity and/or aroma improvement. The aim of this study was to evaluate the effects of the use of a non-Saccharomyces starter (Lachancea thermotolerans) on the chemical and sensory properties of wines obtained by the the fermentation of two autochthonous Apulian grape cultivars, namely Bombino nero and Minutolo, as compared to the traditional Saccharomyces cerevisiae-driven fermentation. Bombino and Minutolo wines fermented with either Lachancea thermotolerans or Saccharomyces cerevisiae were characterized for their oenological parameters, volatile profiles, and sensory properties. Both chemical and sensory properties were affected by the yeast starter. Inoculation of L. thermotolerans increased sensory complexity, with different floral and sweet-like attributes for both cultivars. Bombino nero, a neutral cultivar, showed a clear effect on wine composition, with both an increase in lactic acid and a change in the volatile profile. On the contrary, the impact of L. thermotolerans was partially masked in Minutolo due to the strong primary aroma background of this highly terpenic cultivar. In this work, we evidenced a notable cultivar × yeast interaction, showing how generalizations of the effects of non-Saccharomyces yeasts on vinification are difficult to achieve, as they show a cultivar-specific outcome.
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Spent coffee grounds (SCG) disposal is an environmental problem. These residues from coffee brewing and instant coffee production have potential to produce novel alcoholic beverages. SCG valorization through sequential alcoholic and malolactic fermentation was investigated using a yeast, Lachancea thermotolerans Concerto and a lactic acid bacterium (LAB), Oenococcus oeni Lalvin 31 in this study. Our results showed that sequential inoculation prevented early death of yeast confronted when simultaneous inoculation was adopted, allowing for growth and persistence of both yeast and LAB till the end of fermentation. Adequate ethanol production (4.91 ± 0.13 %, v/v) with low residual sugar content was also attained. In addition, relatively lower levels of acetic, lactic, and succinic acids were produced by sequential inoculation than that of simultaneous inoculation. Furthermore, SCG hydrolysates fermented via sequential inoculation had the widest variety of volatiles (e.g. esters and ketones). Overall, our results indicated that sequential inoculation of L. thermotolerans and O. oeni in SCG hydrolysates might be a way to develop novel beverages with pleasant flavor profiles.
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Climate change is generating several problems in wine technology. One of the main ones is lack of acidity and difficulties performing malolactic fermentation to stabilize wines before bottling. Among the different available acidity management technologies, such as direct acid addition, ion exchange resins, electro-membrane treatments, or vineyard management, the microbiological option is reliable and deeply studied. The main approach is the increase in malic acid content because of the metabolism of specific Saccharomyces strains and to increase lactic acid because of the metabolism of Lachancea genus. Other non-Saccharomyces yeasts, such as Starmerella bacillaris or Candida stellata can also acidify significantly because of the production of pyruvic or succinic acid. Wine industry needs the removal of malic acid in most red wines before bottling to achieve wine stability. Oenococus oeni performs the malolactic fermentation of red wines on most conditions because of the metabolization of malic acid into lactic acid. However, modern oenology challenges such as high ethanol concentrations, high pH or low levels of malic acid have made researchers to look for other options to reduce potential risks of deviation. Other wine-related microorganisms able to de-acidify malic acid have appeared as interesting alternatives for specific difficult scenarios. Lactiplantibacillus plantarum and Schizosaccharomyces genus make up nowadays the main studied alternatives.
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Oenococcus , Schizosaccharomyces , Vinho , Etanol/metabolismo , Fermentação , Ácido Láctico/metabolismo , Malatos/metabolismo , Oenococcus/metabolismo , Schizosaccharomyces/metabolismo , Vinho/microbiologiaRESUMO
Viognier is a warm climate grape variety prone to loss of acidity and accumulation of excessive sugars. The yeast Lachancea thermotolerans can improve the stability and balance of such wines due to the partial conversion of sugars to lactic acid during alcoholic fermentation. This study compared the performance of five L. thermotolerans strains in co-inoculations and sequential inoculations with Saccharomyces cerevisiae in high sugar/pH Viognier fermentations. The results highlighted the dichotomy between the non-acidified and the bio-acidified L. thermotolerans treatments, with either comparable or up to 0.5 units lower pH relative to the S. cerevisiae control. Significant differences were detected in a range of flavour-active yeast volatile metabolites. The perceived acidity mirrored the modulations in wine pH/TA, as confirmed via "Rate-All-That-Apply" sensory analysis. Despite major variations in the volatile composition and acidity alike, the varietal aromatic expression (i.e., stone fruit aroma/flavour) remained conserved between the treatments.
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The first committed step in the leucine biosynthetic pathway is catalyzed by α-isopropylmalate synthase (α-IPMS, EC 2.3.3.13), which in the Saccaromycotina subphylum of Ascomycete yeasts is frequently encoded by duplicated genes. Following a gene duplication event, the two copies may be preserved presumably because the encoded proteins diverge in either functional properties and/or cellular localization. The genome of the petite-negative budding yeast Lachancea kluyveri includes two SAKL0E10472 (LkLEU4) and SAKL0F05170 g (LkLEU4BIS) paralogous genes, which are homologous to other yeast α-IPMS sequences. Here, we investigate whether these paralogous genes encode functional α-IPMS isozymes and whether their functions have diverged. Molecular phylogeny suggested that the LkLeu4 isozyme is located in the mitochondria and LkLeu4BIS in the cytosol. Comparison of growth rates, leucine intracellular pools and mRNA levels, indicate that the LkLeu4 isozyme is the predominant α-IPMS enzyme during growth on glucose as carbon source. Determination of the kinetic parameters indicates that the isozymes have similar affinities for the substrates and for the feedback inhibitor leucine. Thus, the diversification of the physiological roles of the genes LkLEU4 and LkLEU4BIS involves preferential transcription of the LkLEU4 gene during growth on glucose and different subcellular localization, although ligand interactions have not diverged.