Factors affecting yeast ethanol tolerance and fermentation efficiency.

Alcohol fermentation is a key process in wine, beer, alcoholic beverage production, bioethanol production by means of carbohydrate sources, and food industry byproducts. There are three key points in these kinds of processes determining their efficiency; enzymatic cellulose lysis into simple sugar molecules, alcohol fermentation rate, and ethanol tolerance of yeast cells. The first process is usually carried out by either the use of pure cellulolytic enzymes, which is a high cost procedure, or by the production of these enzymes from cellulolytic bacteria and filamentous fungi. Lately, Saccharomyces cerevisiae and several other yeasts were genetically modified to express recombinant cellulases in media or display them on the cell surface. Many studies have indicated that the genetic engineering of yeast cells can be a useful approach in increasing the alcoholic fermentation rate as well as their ethanol tolerance. These modifications could be the overexpression of a key protein using a strong promoter or the modification of a specific domain or amino acid which can also lead to the desired outcome. This review focuses on the modifications of a single protein and/or pathways that can lead to the augmentation of ethanol tolerance and alcoholic fermentation efficiency of Saccharomyces cerevisiae.

Apple bagasse as a substrate for the propagation of Patagonian wine yeast biomass.

AIMS: A culture medium based on apple bagasse was designed and tested as a substrate for biomass production of conventional and unconventional native wine yeasts. METHODS AND RESULTS: The physicochemical characterization of the apple bagasse was carried out and its potential utility as a constituent of a complete culture medium for the production of yeast biomass was analysed using the experimental statistical designs. Growth parameters of conventional and nonconventional Patagonian wine yeasts were analysed with Placket-Burman designs and response surface methodology, comparing in each assay the apple bagasse substrate with the commonly used substrate for biomass development, cane molasses. Culture media composition was optimized and models were validated. CONCLUSIONS: This study demonstrates that, both from a nutritional and from an economic point of view, apple bagasse constitutes a more advantageous substrate than cane molasses for the propagation of native yeasts from Patagonia. SIGNIFICANCE AND IMPACT OF THE STUDY: We used an alternate carbon-rich material, generously available in our region, originally generated as fruit industrial waste, to transform it into a source of sustainable, economically profitable and environmentally friendly energy resource.

Mucor indicus and Rhizopus oryzae co-culture to improve the flavor of Chinese turbid rice wine.

BACKGROUND: One of the most important species used to ferment Chinese turbid rice wine (CTRW) at an industrial-scale level is Rhizopus oryzae, although the flavor of CTRW fermented by pure R. oryzae is inferior to that of traditional CTRW. RESULTS: Mucor indicus was used as a cooperative species to improve the flavor of CTRW presented by R. oryzae. The flavor compounds in different fermentation stages were determined by headspace solid-phase microextraction-gas chromatography-mass spectrometry and high-performance liquid chromatography. It was noted that the M. indicus and R. oryzae co-culture changed the profiles of flavor compounds in CTRW, including esters, higher alcohols, amino acids and organic acids, and also significantly enhanced the concentration of sweet amino acids, fruity and floral esters, and higher alcohols. Sensory evaluation demonstrated that the CTRW fermented by M. indicus and R. oryzae had a more intense aroma, harmonious taste, continuation and full body mouth-feel because of more abundant flavor compounds. CONCLUSION: Mucor indicus is a promising species for co-culture with R. oryzae to improve the flavor of CTRW. © 2019 Society of Chemical Industry.

Torulaspora delbrueckii produces high levels of C5 and C6 polyols during wine fermentations.

Non-Saccharomyces yeasts impact wine fermentations and can diversify the flavor profiles of wines. However, little information is available on the metabolic networks of most of these species. Here we show that unlike the main wine yeast Saccharomyces cerevisiae, Torulaspora delbrueckii and to a lesser extent Lachancea thermotolerans produce significant concentrations of C5 and C6 polyols under wine fermentation conditions. In particular, D-arabitol, D-sorbitol and D-mannitol were produced at significant levels. Their release into the extracellular matrix started when that of glycerol ceased. The data also show that polyol production is influenced by initial sugar concentration, repressed by acetic acid and induced in ethanol supplemented media. Moreover, unlike glycerol and sorbitol, mannitol was partially re-assimilated when populations started to decline. The findings suggest that polyol synthesis is a physiological adaptation to stressful conditions characteristic of alcoholic fermentation and that these polyols may serve a similar purpose as glycerol production in S. cerevisiae, including osmoadaptation and redox balancing.

Cell wall polysaccharides released during the alcoholic fermentation by Schizosaccharomyces pombe and S. japonicus: quantification and characterization.

The present work demonstrates that yeasts belonging to the Schizosaccharomyces genus release a high quantity of polysaccharides of cell wall origin starting from the onset of the alcoholic fermentation. By the end of the alcoholic fermentation, all of the Schizosaccharomyces yeast strains released a quantity of polysaccharides approximately 3-7 times higher than that released by a commercial Saccharomyces cerevisiae yeast strain under the same fermentative conditions of synthetic juice. A higher content of polysaccharide was found in media fermented by Schizosaccharomyces japonicus with respect to that of Schizosaccharomyces pombe. Some of the strains evaluated were also able to produce high levels of pyruvic acid, which has been shown to be an important compound for color stability of wine. The presence of strains with different malic acid consumption patterns along with high polysaccharide release would enable production of naturally modified wines with enhanced mouth feel and reduced acidity. The chemical analysis of the released polysaccharides demonstrated divergence between the two yeast species S. pombe and S. japonicus. A different mannose/galactose ratio and a different percentage of proteins was observed on the polysaccharides released by S. pombe as compared to S. japonicus. Analysis of the proteins released in the media revealed the presence of a glycoprotein with a molecular size around 32-33 kDa only for the species S. japonicus. Mass spectrometry analysis of carbohydrate moieties showed similar proportions among the N-glycan chains released in the media by both yeast species but differences between the two species were also observed. These observations suggest a possible role of rapid MALDI-TOF screening of N-glycans compositional fingerprint as a taxonomic tool for this genus. Polysaccharides release in the media, in particular galactomannoproteins in significant amounts, could make these yeasts particularly interesting also for the industrial production of exogenous polysaccharide preparations.

Yeast strain affects phenolic concentration in Pinot noir wines made by microwave maceration with early pressing.

AIMS: This study examined the effects of yeast strains in a novel winemaking process that had been designed to optimize phenolic extraction and improve production efficiency for Pinot noir winemaking. METHODS AND RESULTS: Microwave maceration with early pressing and co-inoculation of yeast and malolactic bacteria for simultaneous alcoholic and malolactic fermentation was investigated. Yeast treatments (Saccharomyces cerevisiae RC212 and EC1118, and Saccharomyces bayanus AWRI1176) were co-inoculated with Oenococcus oeni PN4 immediately after must microwave maceration. Alcoholic and malolactic fermentation were complete 17 days postinoculation for all three yeast treatments. At 16-month bottle age, the AWRI1176-treated wines had approximately twice the nonbleachable pigment and colour density of wines fermented by EC1118 and RC212. CONCLUSIONS: The novel winemaking process produced Pinot noir wine that was stable 37 days after fruit had been harvested and yeast strain choice significantly impacted the stability and phenolic character of wine. SIGNIFICANCE AND IMPACT OF STUDY: Successful simultaneous alcoholic and malolactic fermentation in 17 days, and a demonstrated lack of inhibition between the yeast strains and malolactic strain applied in this study, provide proof of concept for very rapid red winemaking using the novel winemaking approach described herein. Further investigation would be required to assess strain effects on wine aroma, mouth feel and taste, however, this novel winemaking approach may offer significant industry efficiencies.

Silicification of wood adopted for barrel production using pure silicon alkoxides in gas phase to avoid microbial colonisation.

The paper presents a new approach, covering wood with silica-based material in order to protect it from spoilage due to microbial colonisation and avoiding the loss of the natural features of the wood. Wood specimens derived from wine barrels were treated with methyltriethoxysilane in gas phase, leading to the deposition of a silica nanofilm on the surface. (29)Si and (13)C solid state Nuclear Magnetic Resonance and Scanning Electron Microscope-Energy Dispersive X-ray analysis observations showed the formation of a silica polymeric film on the wood samples, directly bonding with the wood constituents. Inductively Coupled Plasma-Mass Spectroscopy quantification of Si showed a direct correlation between the treatment time and silica deposition on the surface of the wood. The silica-coated wood counteracted colonisation by the main wine spoilage microorganisms, without altering the migration from wood to wine of 21 simple phenols measured using a HPLC-Electrochemical Coulometric Detection.

Adaptation of the wine bacterium Oenococcus oeni to ethanol stress: role of the small heat shock protein Lo18 in membrane integrity.

Malolactic fermentation in wine is often carried out by Oenococcus oeni. Wine is a stressful environment for bacteria because ethanol is a toxic compound that impairs the integrity of bacterial membranes. The small heat shock protein (sHsp) Lo18 is an essential actor of the stress response in O. oeni. Lo18 prevents the thermal aggregation of proteins and plays a crucial role in membrane quality control. Here, we investigated the interaction between Lo18 and four types of liposomes: one was prepared from O. oeni grown under optimal growth conditions (here, control liposomes), one was prepared from O. oeni grown in the presence of 8% ethanol (here, ethanol liposomes), one was prepared from synthetic phospholipids, and one was prepared from phospholipids from Bacillus subtilis or Lactococcus lactis. We observed the strongest interaction between Lo18 and control liposomes. The lipid binding activity of Lo18 required the dissociation of oligomeric structures into dimers. Protein protection experiments carried out in the presence of the liposomes from O. oeni suggested that Lo18 had a higher affinity for control liposomes than for a model protein. In anisotropy experiments, we mimicked ethanol action by temperature-dependent fluidization of the liposomes. Results suggest that the principal determinant of Lo18-membrane interaction is lipid bilayer phase behavior rather than phospholipid composition. We suggest a model to describe the ethanol adaptation of O. oeni. This model highlights the dual role of Lo18 in the protection of proteins from aggregation and membrane stabilization and suggests how modifications of phospholipid content may be a key factor determining the balance between these two functions.

Orthonasal vs. retronasal: Studying how volatiles' hydrophobicity and matrix composition modulate the release of wine odorants in simulated conditions.

In this study a Retronasal Aroma Simulator was employed to compare the release of volatiles from two different white wine matrices (TW: table, SW: sweet) with and without the addition of human or artificial saliva to simulate retronasal and orthonasal conditions, respectively. The headspace volatiles were isolated by Solid Phase Microextraction under dynamic conditions and identified and quantified by Gas-Chromatographic analyses. Compared to the orthonasal, the retronasal conditions modified the release of odorants from both wines and the observed trends cannot be ascribed only to dilution consequent to saliva addition. The relative amounts of volatiles belonging to different chemical classes were modified in the presence of saliva with possible sensory implications concerning some fruity (esters), oxidative (furans) and varietal (linalool, vitispirane) odorants. Regression analyses show that the impact of saliva depends on the volatile (concentration and hydrophobicity) and the non-volatile (residual sugars) composition of the wine. The highly significant linear models (TW: R2 = 0.988; SW: R2 = 0.993) indicate that the release of volatiles is logP octanol/water dependent in both the wines but the slopes change with matrix composition. This suggest that in the presence of human saliva the release of odorants with similar hydrophobicity vary as a linear function of their initial headspace concentration above the wine and is modulated by the composition of the wine matrix. Differences between artificial and human saliva confirmed that the retronasal release of wine odorants is affected by the whole salivary composition and suggest that salivary components different from mucin and α-amylase are involved in the retention of the most hydrophobic volatiles as well as in the metabolization of some aromas.

Influence of wine composition on consumer perception and acceptance of Brettanomyces metabolites using temporal check-all-that-apply methodology.

Brettanomyces spoilage in wine is due to the production of metabolites, which together create the distinctive 'Bretty' aroma and flavor profile associated with wine. The objective of this study was to assess the influence of three wine flavor matrices on consumer acceptance and the temporal sensory properties of wines containing high and low concentrations of Brettanomyces-metabolites. A commercial Shiraz red wine was altered through additions of whiskey lactone (oaky) and 2-isobutyl-3-methoxypyrazine (green). The Shiraz wines (unaltered, oak and green) were spiked with either low or high concentrations of 4-ethylphenol (4-EP), 4-ethylguaiacol (4-EG), and isovaleric acid (IA). All wines were evaluated by consumers (n = 105) using check-all-that-apply (CATA) for wine aroma. In-mouth flavor and mouthfeel perceptions were evaluated with temporal check-all-that-apply (TCATA) and a ranking evaluation where the top three most prominent attributes were reported. Lastly, consumers evaluated each sample on overall liking. Consumers were classified as having low, medium, or high wine knowledge level, in addition to wine industry experience. Differences in flavor and aroma attribute citation across all wine samples were described by consumers. In comparing oak and green treatments, the presence of whiskey lactone in the oak wine more strongly masked Brettanomyces associated aromas than did a 2-isobutyl-3-methoxypyrazine in the green wine. Brettanomyces metabolite-associated flavor terms commonly increased in citations by consumers when concentrations of 4-EP, 4-EG, and IA were increased from the low to high Brett levels (p < .05). At the high Brett treatments, citations of Band-Aid®, smoky, and leather flavor attributes were all significantly lower when oak was present. Consumers identified as having wine industry experience had lower liking ratings for the wine samples as compared to those without experience (p < .05). Results demonstrated the influence of wine composition on the perception of Brettanomyces metabolites, and provided valuable information to the wine industry as to how composition, or further wine style may influence the perception of wine spoilage aroma and flavors.

Evaluation of the ability of commercial wine yeasts to form biofilms (mats) and adhere to plastic: implications for the microbiota of the winery environment.

Commercially available active dried wine yeasts are regularly used by winemakers worldwide to achieve reliable fermentations and obtain quality wine. This practice has led to increased evidence of traces of commercial wine yeast in the vineyard, winery and uninoculated musts. The mechanism(s) that enables commercial wine yeast to persist in the winery environment and the influence to native microbial communities on this persistence is poorly understood. This study has investigated the ability of commercial wine yeasts to form biofilms and adhere to plastic. The results indicate that the biofilms formed by commercial yeasts consist of cells with a combination of different lifestyles (replicative and non-replicative) and growth modes including invasive growth, bud elongation, sporulation and a mat sectoring-like phenotype. Invasive growth was greatly enhanced on grape pulp regardless of strain, while adhesion on plastic varied between strains. The findings suggest a possible mechanism that allows commercial yeast to colonise and survive in the winery environment, which may have implications for the indigenous microbiota profile as well as the population profile in uninoculated fermentations if their dissemination is not controlled.

Aroma Precursors in Grapes and Wine: Flavor Release during Wine Production and Consumption.

Pioneering investigations into precursors of fruity and floral flavors established the importance of terpenoid and C13-norisoprenoid glycosides to the flavor of aromatic wines. Nowadays flavor precursors in grapes and wine are known to be structurally diverse, encompassing glycosides, amino acid conjugates, odorless volatiles, hydroxycinnamic acids, and many others. Flavor precursors mainly originate in the grape berry but also from oak or other materials involved in winemaking. Flavors are released from precursors during crushing and subsequent production steps by enzymatic and nonenzymatic transformations, via microbial glycosidases, esterases, C-S lyases, and decarboxylases, and through acid-catalyzed hydrolysis and chemical rearrangements. Flavors can also be liberated from glycosides and amino acid conjugates by oral microbiota. Hence, it is increasingly likely that flavor precursors contribute to retronasal aroma formation through in-mouth release during consumption, prompting a shift in focus from identifying aroma precursors in grapes to understanding aroma precursors present in bottled wine.

Genomics Perspectives on Metabolism, Survival Strategies, and Biotechnological Applications of Brettanomyces bruxellensis LAMAP2480.

Wine production is an important commercial issue for the liquor industry. The global production was estimated at 275.7 million hectoliters in 2015. The loss of wine production due to Brettanomyces bruxellensis contamination is currently a problem. This yeast causes a "horse sweat" flavor in wine, which is an undesired organoleptic attribute. To date, 6 B. bruxellensis annotated genome sequences are available (LAMAP2480, AWRI1499, AWRI1608, AWRI1613, ST05.12/22, and CBS2499), and whole genome comparisons between strains are limited. In this article, we reassembled and reannotated the genome of B. bruxellensis LAMAP2480, obtaining a 27-Mb assembly with 5.5 kb of N50. In addition, the genome of B. bruxellensis LAMAP2480 was analyzed in the context of spoilage yeast and potential as a biotechnological tool. In addition, we carried out an exploratory transcriptomic analysis of this strain grown in synthetic wine. Several genes related to stress tolerance, micronutrient acquisition, ethanol production, and lignocellulose assimilation were found. In conclusion, the analysis of the genome of B. bruxellensis LAMAP2480 reaffirms the biotechnological potential of this strain. This research represents an interesting platform for the study of the spoilage yeast B. bruxellensis.

Influence of the dominance of must fermentation by Torulaspora delbrueckii on the malolactic fermentation and organoleptic quality of red table wine.

Torulaspora delbrueckii can improve wine aroma complexity, but its impact on wine quality is still far from being satisfactory at the winery level, mainly because it is easily replaced by S. cerevisiae yeasts during must fermentation. New T. delbrueckii killer strains were selected to overcome this problem. These strains killed S. cerevisiae yeasts and dominated fermentation better than T. delbrueckii non-killer strains when they were single-inoculated into crushed red grape must. All the T. delbrueckii wines, but none of the S. cerevisiae wines, underwent malolactic fermentation. Putative lactic acid bacteria were always found in the T. delbrueckii wines, but none or very few in the S. cerevisiae wines. Malic acid degradation was the greatest in the wines inoculated with the killer strains, and these strains reached the greatest dominance ratios and had the slowest fermentation kinetics. The T. delbrueckii wines had dried-fruit/pastry aromas, but low intensities of fresh-fruit aromas. The aroma differences between the T. delbrueckii and the S. cerevisiae wines can be explained by the differences that were found in the amounts of some fruity aroma compounds such as isoamyl acetate, ethyl hexanoate, ethyl octanoate, and some lactones. This T. delbrueckii effect significantly raised the organoleptic quality scores of full-bodied Cabernet-Sauvignon red wines inoculated with the killer strains. In particular, these wines were judged as having excellent aroma complexity, mouth-feel, and sweetness.

Anaerobic treatment of rice winery wastewater in an upflow filter packed with steel slag under different hydraulic loading conditions.

Rice-washing drainage (RWD), a strong organic wastewater, was anaerobically treated using an upflow filter filled with blast-furnace slag. The continuous performance of the reactor was examined at varying hydraulic retention times (HRTs). The reactor achieved 91.7% chemical oxygen demand removal (CODr) for a 10-day HRT (0.6 g COD/Ld organic loading rate) and maintained fairly stable performance until the HRT was shortened to 2.2 days (CODr > 84%). Further decreases in HRT caused process deterioration (CODr < 50% and pH < 5.5 for a 0.7-day HRT). The methane production rate increased with decreasing HRT to reach the peak level for a 1.3-day HRT, whereas the yield was significantly greater for 3.4-day or longer HRTs. The substrate removal and methane production kinetics were successfully evaluated, and the generated kinetic models produced good performance predictions. The methanogenic activity of the reactor likely relies on the filter biofilm, with Methanosaeta being the main driver.

Gluconic acid consumption in wines by Schizosaccharomyces pombe and its effect on the concentrations of major volatile compounds and polyols.

Schizosaccharomyces pombe 1379 (ATCC 26760) yeast strain in wine substantially increases acetaldehyde and 1,1-diethoxyethane concentrations and to decreases levo-2,3-butanediol, glycerol, acetoin, and gluconic acid concentrations. In this study, S. pombe has been used for the first time to reduce gluconic acid in wine under aerobic conditions. Only acetaldehyde and acetoin exhibited significantly higher levels in the wines containing gluconic acid. The high in vitro specific activity of alcohol dehydrogenase observed may be directly related to the high production of acetaldehyde by the studied fission yeast.

Foam aptitude of trepat and monastrell red varieties in cava elaboration. 1. Base wine characteristics.

The foam properties of base wines made from red autochthonous varieties (Trepat and Monastrell) were studied. Four wines of each variety were elaborated (fermented off skins at industrial scale in two consecutive harvests) and blended at different proportions with the white traditional variety (Macabeo, Xarel.lo, and Parellada) wines to elaborate Cava (closed-bottle-fermented sparkling wine). When crescent amounts of Trepat were added to the traditional white blend, the foamability and the color intensity (CI) of the wine increased polinomically. The increase of the CI depended on the year of harvest. Thus, oenologists could decide the blend proportion most suitable to elaborate either a "blanc de noirs" sparkling wine or a new type of Cava.

Effect of freeze-dried immobilized cells on delignified cellulosic material in low-temperature and ambient-temperature wine making.

In this article, we report on wine making by freeze-dried immobilized cells on delignified cellulosic material for ambient and low temperatures. Biocatalyst supported by freeze-dried delignified cellulosic (FDC) material recovered after the first repeated-batch fermentations the fermentation efficiency and startup, which become about equal to those of biocatalyst supported by wet delignified cellulosic material. The FDC biocatalyst was suitable for wine making at low temperatures (5-15 degrees C), and produced wine of 12% alcoholic degree, with the main volatiles contained in the wine and reduced by a decrease in temperature. The fermentation efficiency was not affected by total acidity of must, while an increase in initial Be density improved percentages of higher alcohols and ethyl acetate. The quality of the wine was validated by a preliminary taste test to be in the range of acceptable to excellent.

Winemaking from Gaglioppo grapes with hybrid strains of Saccharomyces.

The fermentative behavior of two hybrid wine yeast strains (first-generation hybrid-strain 12,233 x 6167--obtained by hybridization of the cryotolerant strain S. bayanus 12,233 with the mesophilic strains S. cerevisiae 6167, and TT254 x 6392 arising by hybridization of the thermotolerant strain S. cerevisiae TT254 with the mesophilic strain S. cerevisiae 6392) was compared with that of a commercial wine yeast strain S. cerevisiae K1 in must from black grapes of the Calabrian variety Gaglioppo. The goal was to obtain wines with a high content of 'polyphenols' from a grape must with a limited phenolic content such as the Gaglioppo must. The progress of the winemaking was estimated according to residual sugars; at the end of fermentation, the wines were decanted, bottled and principal physico-chemical characteristics determined. Our results point to the possibility to select wine yeasts (significantly differing in the above parameters) by their ability to interact with phenolic compounds.

[Immobilization of champagne yeasts by inclusion into cryogels of polyvinyl alcohol for preventing cell escape from carrier matrix]. / Immobilizatsiia shampanskikh drozhzhei vkliucheniem v kriogel' PVS, puti predotvrashcheniia vykhoda kletok iz matritsy nositelia.

Wine champagnizing, a process involving the use of champagne yeasts immobilized by inclusion into cryogels of polyvinyl alcohol, has been studied. Treatment of yeast cells with the autoregulatory factor d1 was proposed as a means of preventing the cell escape from the carrier matrix. Such a treatment inhibited growth and proliferation processes in yeasts cells, without affecting the activity of fermentation; the resulting champagne had the same organoleptic and chemical characteristics as its counterparts obtained using traditional techniques.