Succinic acid production by wine yeasts and the influence of GABA and glutamic acid / Producción de ácido succínico por levaduras enológicas y la influencia del GABA y el ácido glutámico

As a consequence of alcoholic fermentation (AF) in wine, several compounds are released by yeasts, and some of them are linked to the general quality and mouthfeel perceptions in wine. However, others, such as succinic acid, act as inhibitors, mainly of malolactic fermentation. Succinic acid is produced by non-Saccharomyces and Saccharomyces yeasts during the initial stages of AF, and the presence of some amino acids such as γ-aminobutyric acid (GABA) and glutamic acid can increase the concentration of succinic acid. However, the influence of these amino acids on succinic acid production has been studied very little to date. In this work, we studied the production of succinic acid by different strains of non-Saccharomyces and Saccharomyces yeasts during AF in synthetic must, and the influence of the addition of GABA or glutamic acid or a combination of both. The results showed that succinic acid can be produced by non-Saccharomyces yeasts with values in the range of 0.2–0.4 g/L. Moreover, the addition of GABA or glutamic acid can increase the concentration of succinic acid produced by some strains to almost 100 mg/L more than the control, while other strains produce less. Consequently, higher succinic acid production by non-Saccharomyces yeast in coinoculated fermentations with S. cerevisiae strains could represent a risk of inhibiting Oenococcus oeni and therefore the MLF.(AU)

Effect of abiotic and biotic factors on Brettanomyces bruxellensis bioadhesion properties.

Biofilms are central to microbial life because of the advantage that this mode of life provides, whereas the planktonic form is considered to be transient in the environment. During the winemaking process, grape must and wines host a wide diversity of microorganisms able to grow in biofilm. This is the case of Brettanomyces bruxellensis considered the most harmful spoilage yeast, due to its negative sensory effect on wine and its ability to colonise stressful environments. In this study, the effect of different biotic and abiotic factors on the bioadhesion and biofilm formation capacities of B. bruxellensis was analyzed. Ethanol concentration and pH had negligible effect on yeast surface properties, pseudohyphal cell formation or bioadhesion, while the strain and genetic group factors strongly modulated the phenotypes studied. From a biotic point of view, the presence of two different strains of B. bruxellensis did not lead to a synergistic effect. A competition between the strains was rather observed during biofilm formation which seemed to be driven by the strain with the highest bioadhesion capacity. Finally, the presence of wine bacteria reduced the bioadhesion of B. bruxellensis. Due to biofilm formation, O. oeni cells were observed attached to B. bruxellensis as well as extracellular matrix on the surface of the cells.

Preliminary Study on Yeasts Associated with the Production of "Tostado"-a Traditional Sweet Wine from Galicia (NW Spain).

BACKGROUND: Tostado is a traditional sweet wine from the Designations of Origins (DOs) of Ribeiro and Valdeorras in Galicia (NW Spain). The harvested grapes are air-dried and pressed to increase the concentrations of sugars, acids, and flavour compounds. Therefore, knowledge of the yeasts involved in fermentation under these conditions is essential to guarantee the quality and uniqueness of the valuable, distinctive, and expensive Tostado wines. METHODS: Saccharomyces and non-Saccharomyces yeasts were identified using Wallerstein Laboratory (WL) Nutrient Agar and lysine plating, followed by polymerase chain reaction (PCR) amplification, enzymatic digestion, and sequencing. Saccharomyces cerevisiae isolates were further characterised at the strain level using mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP). Statistical analyses were also performed, including different diversity indices, Similarity Percentage (SIMPER) analysis, principal component analysis (PCA), neighbor-joining clustering, parsimony-phylogram, and network plot. In addition, the total acidity, volatile acidity, reducing sugars, and alcoholic strength by volume of the Tostado wines were analysed. RESULTS: A wide diversity of autochthonous yeasts was found, which were predominantly species of oenological relevance, such as Lachancea thermotolerans, Starmerella bacillaris, Hanseniaspora uvarum, Debaryomyces hansenii, Torulaspora delbrueckii, Pichia spp., and Saccharomyces cerevisiae from the must and paste stages of Tostado wine. In addition, 19 different S. cerevisiae strains were identified. This high yeast diversity, which changed from the early stages of fermentation, could contribute to the distinctive characteristics observed in Tostado wine. CONCLUSIONS: Characteristic and differentiating chemical and microbiological profiles were found as early as the pre-fermentation stages, which adds value to these special wines that have rarely been studied.

Application of white wine lees for promoting lactic acid bacteria growth and malolactic fermentation in wine.

In the context of ecological transition, the use of wine by-products for industrial applications is a major challenge. Wine lees, the second wine by-product in terms of quantity, represent a source of nutrients that can be used for stimulating the growth of microorganisms. Here, white wine lees were used as a stimulating agent for the growth of wine lactic acid bacteria (LAB) and to promote wine malolactic fermentation (MLF) driven out by Oenococcus oeni. By adding freeze-dried wine lees to wines under different conditions - including different wine lees at different concentrations and different O. oeni strains at various initial populations - it was observed that wine lees can enhance the growth of LAB and reduce the duration of MLF. The chemical composition of wines was also evaluated, proving that wine lees do not compromise the quality of the wines. In addition, wine lees did not seem to promote the growth of spoilage microorganisms like as Brettanomyces bruxellensis. Altogether, this work reports the possibility of recovering the lees of white wine to obtain a product favoring the MLF of red wines. More general, we propose a recycling strategy of wine by-products to obtain new products for winemaking.

Effect of overexpression of partial TDH1 and TDH2/3 gene sequences in a starter strain of industrial bioethanol fermentation on the Brettanomyces bruxellensis contaminant growth.

Selected Saccharomyces cerevisiae strains, such as the commercial Ethanol-Red (ER) strain, are used as starters in the bioethanol industry. Yet, bioethanol fermentations are prone to microbial contaminations, mainly by Brettanomyces bruxellensis and lactic acid bacteria. Chemicals, such as sulphuric acid and antibiotics, are commonly used to combat those contaminations, but they have negative environmental impacts. Recently, ER strain was found to secrete antimicrobial peptides (AMPs) active against B. bruxellensis. Therefore, the partial TDH1 and TDH2/3 genes sequences that codify those AMPs were inserted into the pSR41k plasmid and cloned in ER strains. The relative expression levels (plasmidic/genomic) of those sequences in the respective modified ER strains were quantified by real-time quantitative polimerase chain reaction (RT-qPCR), confirming their overexpression. The effect of the modified strains on B. bruxellensis (Bb) growth was then evaluated during synthetic must (SM) and carob syrup (CS) fermentations, co-inoculated with 105 cells ml-1 of ER and Bb in SM and with 106 of ER and 5 × 103 cells ml-1 of Bb in CS. Results showed that modified ER strains exerted a much higher inhibitory effect against B. bruxellensis (72-fold in SM and 10-fold in CS) than the non-modified ER strain. In those fermentations, 90-100 g l-1 of ethanol was produced in 3-6 days.

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.

Bioprotection by non-Saccharomyces yeasts in oenology: Evaluation of O2 consumption and impact on acetic acid bacteria.

Bioprotection by yeast addition is increasingly used in oenology as an alternative to sulfur dioxide (SO2). Recent studies have also shown that it is likely to consume dissolved O2. This ability could limit O2 for other microorganisms and the early oxidation of the grape must. However, the ability of yeasts to consume O2 in a context of bioprotection was poorly studied so far considering the high genetic diversity of non-Saccharomyces. The first aim of the present study was to perform an O2 consumption rate (OCR) screening of strains from a large multi species collection found in oenology. The results demonstrate significant inter and intra species diversity with regard to O2 consumption. In the must M. pulcherrima consumes O2 faster than Saccharomyces cerevisiae and then other studied non-Saccharomyces species. The O2 consumption was also evaluate in the context of a yeast mix used as industrial bioprotection (Metschnikowia pulcherrima and Torulaspora delbrueckii) in red must. These non-Saccharomyces yeasts were then showed to limit the growth of acetic acid bacteria, with a bioprotective effect comparable to that of the addition of sulfur dioxide. Laboratory experiment confirmed the negative impact of the non-Saccharomyces yeasts on Gluconobacter oxydans that may be related to O2 consumption. This study sheds new lights on the use of bioprotection as an alternative to SO2 and suggest the possibility to use O2 consumption measurements as a new criteria for non-Saccharomyces strain selection in a context of bioprotection application for the wine industry.

Single nucleotide polymorphisms associated with wine fermentation and adaptation to nitrogen limitation in wild and domesticated yeast strains.

For more than 20 years, Saccharomyces cerevisiae has served as a model organism for genetic studies and molecular biology, as well as a platform for biotechnology (e.g., wine production). One of the important ecological niches of this yeast that has been extensively studied is wine fermentation, a complex microbiological process in which S. cerevisiae faces various stresses such as limited availability of nitrogen. Nitrogen deficiencies in grape juice impair fermentation rate and yeast biomass production, leading to sluggish or stuck fermentations, resulting in considerable economic losses for the wine industry. In the present work, we took advantage of the "1002 Yeast Genomes Project" population, the most complete catalogue of the genetic variation in the species and a powerful resource for genotype-phenotype correlations, to study the adaptation to nitrogen limitation in wild and domesticated yeast strains in the context of wine fermentation. We found that wild and domesticated yeast strains have different adaptations to nitrogen limitation, corroborating their different evolutionary trajectories. Using a combination of state-of-the-art bioinformatic (GWAS) and molecular biology (CRISPR-Cas9) methodologies, we validated that PNP1, RRT5 and PDR12 are implicated in wine fermentation, where RRT5 and PDR12 are also involved in yeast adaptation to nitrogen limitation. In addition, we validated SNPs in these genes leading to differences in fermentative capacities and adaptation to nitrogen limitation. Altogether, the mapped genetic variants have potential applications for the genetic improvement of industrial yeast strains.

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.

Microbial diversity on grape epidermis and wine volatile aroma in spontaneous fermentation comprehensively driven by geography, subregion, and variety.

On their journey from the wine grape to the resulting wine, microbiota from grape surfaces controlled by multiple factors is transferred to wine spontaneous fermentation process with indisputable consequences for wine quality parameters. The associated microbiota was regionally distinct (defined to microbial terroir) but how these microbial patterns with significantly regional distinctiveness quantitatively drive the wine regional characteristics are not definite within a complete grape ecosystem at different geographical (> 300 km), subregional (< 10 km), and varietal scales. Here, we collected 24 samples (containing two grape varieties) from four subregions of two regions in Xinjiang wine production area to investigate fungal distribution patterns and the association with wine chemical composition at different evaluation scales. Meanwhile, the relationships were established between geographical, subregional, varietal community of fungi, and wine volatile aroma using partial least squares regression (PLSR) and structural equation modeling (SEM). Results show that microbial and volatile samples present the significantly regional difference inside the complete ecosystem. Microbiota showed a stronger heterogeneity at geography scales, which drove the distributions of subregional and varietal microbiota thereby influencing the volatile composition of finished wines. Moreover, geographical microbiota seems to weaken the effects of varietal community on wine aroma compounds. Microbial communities respond to environmental changes within a completely set grape-related ecosystem at different scales, and these responses resulted in the wine regional distinctiveness based on the volatile profiles. Our findings further confirmed the important role of microbial terroir in shaping wine styles and provided the new cerebration for the terroir drivers of microbiota.

Comparison of microbial communities and volatile profiles of wines made from mulberry and grape.

In this study, three kinds of wines separately made from mulberry (MW), grape (GW), or mulberry/grape (MGW) were developed and their enological parameters, sensory scores, volatile components, and microbiota were investigated and compared. Contrary to the order of residual sugar and acidity of the three kinds of wines, the order of alcohol content from high to low is GW, MW, and MGW. A total of 60 volatile components (VCs), including esters (17), alcohols (12), acids (6), aldehydes (7), ketones (3), alkenes (3), amines (3), alkanes (4), pyrazines (2), benzene (1), sulfide (1), and thiazole (1), were identified by gas chromatography-ion mobility spectrometer (GC-IMS). The fingerprint of VCs and principal component analysis revealed that the volatile profiles of MGW and GW were more similar in comparison to that of MW and were significantly correlated with the mass ratio of mulberry to grape. Lactobacillus, Weissella, Pantoea, Leuconostoc, Lactococcus, Paenibacillus, Pediococcus, and Saccharomyces were identified as the main microflora at the genus level shared by the MW, MGW, and GW, suggesting that the heterolactic bacteria may contribute more to the high content of volatile acids in MW and MGW. The heatmap of core microbiota and main VCs of MW, MGW, and GW suggested the complicated and significant correlation between them. The above data implied that the volatile profiles were more closely related to the raw materials of winemaking and markedly affected by the fermentation microorganisms. This study provides references for evaluation and characterization of MGW and MW and improvement of MGW and MW winemaking process. KEY POINTS: • Fruit wine enological parameters, volatile profile, and microbiota were compared. • Sixty volatile compounds were identified by GC-IMS in three types of fruit wines. • Winemaking materials and microbiota affect volatile profiles of the fruit wines.

Alcoholic fermentation drives the selection of Oenococcus oeni strains in wine but not in cider.

Oenococcus oeni is the predominant lactic acid bacteria species in wine and cider, where it performs the malolactic fermentation (MLF). The O. oeni strains analyzed to date form four major genetic lineages named phylogroups A, B, C and D. Most of the strains isolated from wine, cider, or kombucha belong to phylogroups A, B + C, and D, respectively, although B and C strains were also detected in wine. This study was performed to better understand the distribution of the phylogroups in wine and cider. Their population dynamics were determined by qPCR all through wine and cider productions, and the behavior of the strains was analyzed in synthetic wines and ciders. Phylogroups A, B and C were all represented in grape must and throughout the alcoholic fermentation, but on the transition to MLF, only phylogroup A remained at high levels in all wine productions. In the case of cider, phylogroups A, B and C were detected in stable levels during the process. When they were tested in synthetic wine and cider, all phylogroups performed MLF, but with different survival rates depending on the ethanol content. In this sense, ethanol and fermentation kinetics are the main agent that drives the selection of phylogroup A strains in wine, while B and C strains dominates in cider containing less ethanol.

Directed evolution of Saccharomyces cerevisiae for low volatile acidity during winemaking under aerobic conditions.

The use of yeast respiratory metabolism has been proposed as a promising approach to solve the problem of increasing ethanol content in wine, which is largely due to climate change. The use of S. cerevisiae for this purpose is mostly hampered by acetic acid overproduction generated under the necessary aerobic conditions. However, it was previously shown that a reg1 mutant, alleviated for carbon catabolite repression (CCR), showed low acetic acid production under aerobic conditions. In this work directed evolution of three wine yeast strains was performed to recover CCR-alleviated strains, expecting they will also be improved concerning volatile acidity. This was done by subculturing strains on galactose, in the presence of 2-deoxyglucose for around 140 generations. As expected, all evolved yeast populations released less acetic acid than their parental strains in grape juice, under aerobic conditions. Single clones were isolated from the evolved populations, either directly or after one cycle of aerobic fermentation. Only some clones from one of three original strains showed lower acetic acid production than their parental strain. Most clones isolated from EC1118 showed slower growth. However, even the most promising clones failed to reduce acetic acid production under aerobic conditions in bioreactors. Therefore, despite the concept of selecting low acetic acid producers by using 2-deoxyglucose as selective agent was found to be correct, especially at the population level, the recovery of strains with potential industrial utility by this experimental approach remains a challenge.

Malolactic fermentation of lactic acid bacteria isolated from southern Brazilian red wine.

The objective was to isolate lactic acid bacteria (LAB) from southern Brazil's wines and investigate their potential as starter cultures for malolactic fermentation (MLF) in Merlot (ME) and Cabernet Sauvignon (CS) wines through the fermentative capacity. The LAB were isolated from CS, ME, and Pinot Noir (PN) wines in the 2016 and 2017 harvests and evaluated for morphological (color and shape of the colonies), genetic, fermentative (increase in pH, acidity reduction, preservation of anthocyanins, decarboxylation of L-malic acid, yield of L-lactic acid, and content of reduced sugars), and sensory characteristics. Four strains were identified as Oenococcus oeni [CS(16)3B1, ME(16)1A1, ME(17)26, and PN(17)65], one as Lactiplantibacillus plantarum [PN(17)75], and one as Paucilactobacillus suebicus [CS(17)5]. Isolates were evaluated in the MLF and compared to a commercial strain (O. oeni), as well as a control (without inoculation and spontaneous MLF), and standard (without MLF). CS(16)3B1 and ME(17)26 isolates finished the MLF for CS and ME wines, respectively, after 35 days, similar to the commercial strain, and CS(17)5 and ME(16)1A1 isolates ended the MLF in 45 days. In the sensory analysis, ME wines with isolated strains received better scores for flavor and overall quality than the control. Compared to the commercial strain, CS(16)3B1 isolate obtained the highest scores for buttery flavor and taste persistence. CS(17)5 isolate received the higher scores for a fruity flavor and overall quality and the lowest for a buttery flavor. The native LAB displayed MLF potential, regardless of the year and grape species from which they were isolated.

Biotechnological applications of biofilms formed by osmotolerant and halotolerant yeasts.

Many microorganisms are capable of developing biofilms under adverse conditions usually related to nutrient limitation. They are complex structures in which cells (in many cases of different species) are embedded in the material that they secrete, the extracellular matrix (ECM), which is composed of proteins, carbohydrates, lipids, and nucleic acids. The ECM has several functions including adhesion, cellular communication, nutrient distribution, and increased community resistance, this being the main drawback when these microorganisms are pathogenic. However, these structures have also proven useful in many biotechnological applications. Until now, the most interest shown in these regards has focused on bacterial biofilms, and the literature describing yeast biofilms is scarce, except for pathological strains. Oceans and other saline reservoirs are full of microorganisms adapted to extreme conditions, and the discovery and knowledge of their properties can be very interesting to explore new uses. Halotolerant and osmotolerant biofilm-forming yeasts have been employed for many years in the food and wine industry, with very few applications in other areas. The experience gained in bioremediation, food production and biocatalysis with bacterial biofilms can be inspiring to find new uses for halotolerant yeast biofilms. In this review, we focus on the biofilms formed by halotolerant and osmotolerant yeasts such as those belonging to Candida, Saccharomyces flor yeasts, Schwannyomyces or Debaryomyces, and their actual or potential biotechnological applications. KEY POINTS: • Biofilm formation by halotolerant and osmotolerant yeasts is reviewed. • Yeasts biofilms have been widely used in food and wine production. • The use of bacterial biofilms in bioremediation can be expanded to halotolerant yeast counterparts.

Comparative genomic analysis of the genus Weissella and taxonomic study of Weissella fangxianensis sp. nov., isolated from Chinese rice wine starter.

In this study, three lactic acid bacteria, namely, HBUAS51963T, HBUAS51964 and HBUAS51965, were isolated from Chinese rice wine starter sampled in Fangxian County, PR China. All were non-motile, non-spore-forming and Gram-positive spherical cells. Their taxonomic status was characterized using a polyphasic approach. Genome-based analysis revealed that all three strains were phylogenomically related to Weissella thailandensis KCTC 3751T and Weissella paramesenteroides ATCC 33313T. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the three strains and the phylogenetically related type strains were less than 54.8 and 93.8 %, respectively, and thus, they were below the thresholds of dDDH and ANI for species definition. The genomic DNA G+C content was 38.6 mol %. The predominant fatty acid methyl esters (>10 %) were C16 : 0, C19 : 0 cyc11 and summed feature 10 (C18 : 1 cyc11 and/or ECL 17.834). The polar lipids in the cells of strain HBUAS51963T were mainly phosphatidylglycerol, diphosphatidylglycerol, unidentified glycolipids, phospholipids and lipids. Finally, the three strains were capable of producing d-lactic acid (4.29 g l-1) and various organic acids such as tartaric, acetic, lactic and succinic acids. Overall, the results of genotypic, phenotypic and genomic analyses suggest that the three strains represent a new species of the genus Weissella, for which the name Weissella fangxianis sp. nov. is proposed. The type strain is HBUAS51963T (=GDMCC 1.3506T= JCM 35803T).

Oenological characteristics of two indigenous Starmerella bacillaris strains isolated from Chinese wine regions.

To broaden knowledge about the oenological characteristics of Starmerella bacillaris, the influence of two Chinese indigenous S. bacillaris strains on the conventional enological parameters and volatile compounds of Cabernet Sauvignon wines were investigated under different inoculation protocols (single inoculation and simultaneous/sequential inoculation with the commercial Saccharomyces cerevisiae EC1118). The results showed that the two S. bacillaris strains could complete alcohol fermentation alone under high sugar concentrations while increasing the content of glycerol and decreasing the content of acetic acid. Compared with wines fermented by EC1118 single inoculation, S. bacillaris single inoculation and S. bacillaris/EC1118 sequential inoculation increased the contents of isobutanol, ethyl isobutanoate, terpenes, and ketones and decreased the contents of isopentanol, phenylethyl alcohol, fatty acids, acetate esters, and total ethyl esters. Furthermore, for S. bacillaris/EC1118 simultaneous inoculation, the concentrations of ethyl esters were increased, contributing to a higher score of "floral" and "fruity" notes in agreement with sensory analysis. KEY POINTS: • S. bacillaris single and simultaneous/sequential inoculation. • Conventional enological parameters and volatile compounds were investigated. • S. bacillaris/EC1118 simultaneous fermentation increased ethyl esters.

Experimental evolution forcing Oenococcus oeni acid tolerance highlights critical role of the citrate locus.

Oenococcus oeni is the main lactic acid bacterium associated with malolactic fermentation (MLF) of wines. MLF plays an important role in determining the final quality of wines. Nevertheless, due to the stressful conditions inherent to wine and especially acidity, MLF may be delayed. This study aimed to explore by adaptive evolution improvements in the acid tolerance of starters but also to gain a better understanding of the mechanisms involved in adaptation toward acidity. Four independent populations of the O. oeni ATCC BAA-1163 strain were propagated (approximately 560 generations) in a temporally varying environment, consisting in a gradual pH decrease from pH 5.3 to pH 2.9. Whole genome sequence comparison of these populations revealed that more than 45% of the substituted mutations occurred in only five loci for the evolved populations. One of these five fixed mutations affects mae, the first gene of the citrate operon. When grown in an acidic medium supplemented with citrate, a significantly higher bacterial biomass was produced with the evolved populations compared to the parental strain. Furthermore, the evolved populations slowed down their citrate consumption at low pH without impacting malolactic performance.

New malic acid producer strains of Saccharomyces cerevisiae for preserving wine acidity during alcoholic fermentation.

In the context of climate change, the chemical composition of wines is characterized by a massive drop of malic acid concentration in grape berries. Then wine professionals have to find out physical and/or microbiological solutions to manage wine acidity. The aim of this study is to develop wine Saccharomyces cerevisiae strains able to produce significant amount of malic acid during the alcoholic fermentation. By applying a large phenotypic survey in small scale fermentations, the production level of malic acid in seven grape juices confirmed the importance of the grape juice in the production of malic acid during the alcoholic fermentation. Beside the grape juice effect, our results demonstrated that extreme individuals able to produce up to 3 g/L of malic acid can be selected by crossing together appropriate parental strains. A multivariate analysis of the dataset generated illustrate that the initial the amount of malic acid produced by yeast is a determining exogenous factor for controlling the final pH of wine. Interestingly most of the acidifying strains selected are particularly enriched in alleles that have been previously reported for increasing the level of malic acid at the end of the alcoholic fermentation. A small set of acidifying strains were compared with strains able to consume a large amount of malic acid previously selected. The total acidity of resulting wines was statistically different and a panelist of 28 judges was able to discriminate the two groups of strains during a free sorting task analysis.

Screening of Saccharomyces cerevisiae and Torulaspora delbrueckii strains in relation to their effect on malolactic fermentation.

The use of Torulaspora delbrueckii in the alcoholic fermentation (AF) of grape must is increasingly studied and used in the wine industry. In addition to the organoleptic improvement of wines, the synergy of this yeast species with the lactic acid bacterium Oenococcus oeni is an interesting field of study. In this work, 60 strain combinations were compared: 3 strains of Saccharomyces cerevisiae (Sc) and 4 strains of Torulaspora delbrueckii (Td) in sequential AF, and four strains of O. oeni (Oo) in malolactic fermentation (MLF). The objective was to describe the positive or negative relationships of these strains with the aim of finding the combination that ensures better MLF performance. In addition, a new synthetic grape must has been developed that allows the success of AF and subsequent MLF. Under these conditions, the Sc-K1 strain would be unsuitable for carrying out MLF unless there is prior inoculation with Td-Prelude, Td-Viniferm or Td-Zymaflore always with the Oo-VP41 combination. However, from all the trials performed, it appears that the combinations of sequential AF with Td-Prelude and Sc-QA23 or Sc-CLOS, followed by MLF with Oo-VP41, reflected a positive effect of T. delbrueckii compared to inoculation of Sc alone, such as a reduction in L-malic consumption time. In conclusion, the obtained results highlight the relevance of strain selection and yeast-LAB strain compatibility in wine fermentations. The study also reveals the positive effect on MLF of some T. delbrueckii strains.