Fate of carbon in synthetic media fermentations containing Metschnikowia pulcherrima or Meyerozyma guilliermondii in the presence and absence of Saccharomyces cerevisiae.

While sequentially inoculating non-Saccharomyces yeasts with Saccharomyces cerevisiae can lower the alcohol contents of wine, the abilities of these yeasts to utilize/produce ethanol or generate other byproducts remained unclear. Metschnikowia pulcherrima or Meyerozyma guilliermondii were inoculated into media with or without S. cerevisiae to assess byproduct formation. Both species metabolized ethanol in a yeast-nitrogen-base medium but produced the alcohol in a synthetic grape juice medium. In fact, Mt. pulcherrima and My. guilliermondii generated less ethanol per gram of metabolized sugar (0.372 and 0.301 g/g, respectively) compared to S. cerevisiae (0.422 g/g). Sequentially inoculating each non-Saccharomyces species with S. cerevisiae into grape juice media achieved up to 3.0% v/v alcohol reduction compared to S. cerevisiae alone while producing variable glycerol, succinic acid, and acetic acid concentrations. However, neither non-Saccharomyces yeasts released appreciable CO2 under fermentative conditions regardless of incubation temperature. Despite equivalent peak populations, S. cerevisiae produced more biomass (2.98 g/L) than the non-Saccharomyces yeasts while sequential inoculations yielded higher biomass with Mt. pulcherrima (3.97 g/L) but not My. guilliermondii (3.03 g/L). To reduce ethanol concentrations, these non-Saccharomyces species may metabolize ethanol and/or produce less from metabolized sugars compared to S. cerevisiae but also divert carbon towards glycerol, succinic acid, and/or biomass.

Sensory properties of 6- and 18-month-stored wines made with pectinase-producing non-Saccharomyces yeasts.

Recently, the use and commercial availability of non-Saccharomyces yeasts (NSY) in winemaking to reduce alcohol content have increased. However, research exploring the influence on sensory quality of the wine, particularly during storage, is limited. Therefore, the objective of this study was to characterize the sensory profiles of Merlot and Chardonnay wines made with pectinase-producing NSY, with added substrate, that is, pectin. Apple pectin (0 or 0.5 g/L) was added to Merlot and Chardonnay grape musts after inoculation with (a) only Saccharomyces cerevisiae or (b) a three species mixture of NSY; after 3 days, S. cerevisiae was added. Addition of NSY with added pectin resulted in higher concentrations of d-galacturonic acid and glycerol concentration in the wines after 6 months of aging. However, mouthfeel (viscosity or weight) of wines with or without added pectin as determined by a sensory evaluation panel was not altered by the presence of these yeasts. Significant interactions among the yeast utilized, pectin addition, and 6-month aging affected some flavors (solvent) of Merlot, while addition of NSY increased other attributes (cherry) during aging. No sensory differences were perceived among Chardonnay samples due to NSY; however, aging from 6 to 18 months increased the intensity of 40 sensory attributes. Though mouthfeel was not specifically affected, the utilization of NSY may be a useful tool to alter wine quality in Merlot by increasing specific aromas during storage. PRACTICAL APPLICATION: We found that must fermented with pectinase-producing non-Saccharomyces yeasts (NSY) modified the chemical composition of the final young wine. After one additional year of aging, an increase in cherry flavor was observed in Merlot wines made with NSY, which may increase perceived quality. Thus, the use of these pectinase-producing NSY may be a useful tool for winemakers.

Improving the chemical and sensory characteristics of red and white wines with pectinase-producing non-Saccharomyces yeasts.

This study examined the influence of pectinase-producing non-Saccharomyces yeasts on the chemical and sensory attributes of red and white wines with added pectin. Merlot and Chardonnay wines were produced with or without a mixture of pectinase-producing non-Saccharomyces yeasts (Cryptococcus adeliensis, Issatchenkia orientalis, and Pichia kluyveri) added to the must prior to alcoholic fermentation conducted by a commercial strain of Saccharomyces cerevisiae. To ensure sufficient substrate was present, varying concentrations of apple pectin (up to 1.25 g/L for red wines and 1.00 g/L for white wine) were added at the start of fermentation. After bottling, trained panelists (n = 10) analyzed these wines for aroma, flavor, taste, and mouthfeel attributes. For both wines, significant interactions were noted between the presence of non-Saccharomyces yeasts and pectin addition which affected pH, titratable acidity, and concentrations of D-galacturonic acid. While no significant sensory differences were observed among the red wines, limited changes were noted for white wines. However, a strong positive correlation was found between the D-galacturonic acid and buttery aroma for Chardonnay and with flavor for Merlot. Increasing D-galacturonic acid concentrations, through utilization of non-Saccharomyces yeasts, may improve the wine quality as a buttery aroma is often associated with high-quality Chardonnay. For both red and white wines, the utilization of these particular non-Saccharomyces yeasts significantly influenced chemical properties but yielded minor sensory changes without any faults. PRACTICAL APPLICATION: With the recent trend to reduce alcohol content in commercial wines, the interest in non-Saccharomyces yeasts has grown. This study showed that the addition of non-Saccharomyces yeasts, perhaps due to their pectinase activity, influenced the chemical characteristics of red and white wines with limited sensory differences, making these yeasts a useful tool for winemakers to modify wine properties.

Native yeast and non-yeast fungal communities of Cabernet Sauvignon berries from two Washington State vineyards, and persistence in spontaneous fermentation.

To address a knowledge gap about the grape berry mycobiome from Washington State vineyards, next-generation sequencing of the internal transcribed spacer region (ITS1) was used to identify native yeast and fungal species on berries of cultivar 'Cabernet Sauvignon' from two vineyards at veraison and harvest in 2015 and 2016. Four hundred fifty-six different yeast amplicon sequence variants (ASV), representing 184 distinct taxa, and 2467 non-yeast fungal ASV (791 distinct taxa) were identified in this study. A set of 50 recurrent yeast taxa, including Phaeococcomyces, Vishniacozyma and Metschnikowia, were found at both locations and sampling years. These yeast species were monitored from the vineyard into laboratory-scale spontaneous fermentations. Taxa assignable to Metschnikowia and Saccharomyces persisted during fermentation, whereas Curvibasidium, which also has possible impact on biocontrol and wine quality, did not. Sulfite generally reduced yeast diversity and richness, but its effect on the abundance of specific yeasts during fermentation was negligible. Among the 106 recurring non-yeast fungal taxa, Alternaria, Cladosporium and Ulocladium were especially abundant in the vineyard. Vineyard location was the primary factor that accounted for the variation among both communities, followed by year and berry developmental stage. The Washington mycobiomes were compared to those from other parts of the world. Sixteen recurrent yeast species appeared to be unique to Washington State vineyards. This subset also contained a higher proportion of species associated with cold and extreme environments, relative to other localities. Certain yeast and non-yeast fungal species known to suppress diseases or modify wine sensory properties were present in Washington vineyards, and likely have consequences to vineyard health and wine quality.

Impacts of non-Saccharomyces species and aeration on sequential inoculation with Saccharomyces cerevisiae to produce lower alcohol Merlot wines from Washington state.

BACKGROUND: Species of non-Saccharomyces yeasts isolated from Washington vineyards were evaluated for their abilities to reduce alcohol contents of wines. As many of these yeasts benefit from some oxygen, the effect of limited aeration was also studied. RESULTS: Although fermentations of a high sugar Merlot grape must (310 g L-1 ) did not reach dryness, inoculation of Metschnikowia chrysoperlae, Mt. pulcherrima, Meyerozyma guillermondii, Pichia kluyveri, or P. membranifaciens yielded in wines with lower amounts of ethanol without excessive levels of acetic acid. Aeration frequently resulted in wines with less ethanol but with more acetic acid compared to non-aerated fermentations. Inoculation of Mt. pulcherrima or My. guilliermondii into another Merlot grape must that contained a lower initial amount of fermentable sugar (266 g L-1 ) resulted in dry wines that contained less alcohol. CONCLUSIONS: Inoculation of My. guilliermondii or Mt. pulcherrima before primary alcoholic fermentation resulted in wines with reduced alcohol contents without excessive acetic acid production. © 2020 Society of Chemical Industry.

Carbohydrate Accumulation and Differential Transcript Expression in Winter Wheat Lines with Different Levels of Snow Mold and Freezing Tolerance after Cold Treatment.

Winter wheat (Triticum aestivum L.) undergoes a period of cold acclimation in order to survive the ensuing winter, which can bring freezing temperatures and snow mold infection. Tolerance of these stresses is conferred in part by accumulation of carbohydrates in the crown region. This study investigates the contributions of carbohydrate accumulation during a cold treatment among wheat lines that differ in their snow mold tolerance (SMT) or susceptibility (SMS) and freezing tolerance (FrT) or susceptibility (FrS). Two parent varieties and eight recombinant inbred lines (RILs) were analyzed. The selected RILs represent four combinations of tolerance: SMT/FrT, SMT/FrS, SMS/FrT, and SMS/FrS. It is hypothesized that carbohydrate accumulation and transcript expression will differ between sets of RILs. Liquid chromatography with a refractive index detector was used to quantify carbohydrate content at eight time points over the cold treatment period. Polysaccharide and sucrose content differed between SMT and SMS RILs at various time points, although there were no significant differences in glucose or fructose content. Glucose and fructose content differed between FrT and FrS RILs in this study, but no significant differences in polysaccharide or sucrose content. RNAseq was used to investigate differential transcript expression, followed by modular enrichment analysis, to reveal potential candidates for other mechanisms of tolerance, which included expected pathways such as oxidative stress, chitinase activity, and unexpected transcriptional pathways. These differences in carbohydrate accumulation and differential transcript expression begin to give insight into the differences of wheat lines when exposed to cold temperatures.

Impacts of depectinization of pear juice on alcoholic fermentation and indole formation.

BACKGROUND: Recently, a producer of fermented ciders observed 'vinyl' off-odors formed during fermentation of pear juice previously depectinized at ≥ 49 °C but not if depectinized at lower temperatures. The objective of this study was to investigate the source of this spoilage and evaluate factors that affect formation. RESULTS: Analysis of untainted and tainted samples obtained from the producer determined the causative agent to be indole, a compound sometimes produced by yeast (Saccharomyces cerevisiae) during fermentation. To mimic commercial depectinization conditions, pectinases were added to pear juices held at 35 °C for 45 min (Treatment A), 49 °C for 45 min (Treatment B), or 49 °C for 90 min (Treatment C). Juice processing conditions did not affect yeast growth nor progress of alcoholic fermentation. Although neither yeast strain (DV10 or MERIT) synthesized indole during fermentation of Treatment A juices, the compound was produced by MERIT in Treatments B (27.05 µg L-1 ) and C (469.9 µg L-1 ). Supplementation of Treatment C juice with pyridoxine (vitamin B6 ) prior to fermentation resulted in no detectable indole formed. However, juices from Treatments A, B, or C contained similar concentrations of pyridoxine and non-detectable amounts of tryptophan, a potential precursor to indole. Furthermore, indole was not detected during fermentations of a synthetic pear juice medium without pyridoxine. CONCLUSION: Supplementation of cider musts with pyridoxine prior to fermentation and choice of yeast strain can lower the risk of formation of off-odors caused by indole. However, other unidentified factors are present which affect its formation in perry. © 2019 Society of Chemical Industry.

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.

Growth and metabolism of non-Saccharomyces yeasts isolated from Washington state vineyards in media and high sugar grape musts.

Utilization of carbohydrates and amino acids/ammonium by selected non-Saccharomyces yeasts and impacts on alcoholic fermentation was evaluated using media and high sugar grape musts (>270 g/L). Consumption patterns of single cultures were ascertained in synthetic media and a Chardonnay grape must. While the non-Saccharomyces species maintained >106 cfu/mL after >40 days, concentrations of residual sugars ranged from 103 g/L (Wickerhamomyces anomalus) to 155 g/L (Candida californica), amino acids and ammonium were generally depleted (>85%), and excessive amounts of volatile acidity (>0.8 g/L) were sometimes produced (e.g., C. oleophila). To minimize problems associated with nutrient depletion before alcoholic fermentation, non-Saccharomyces yeasts were inoculated six days ahead of S. cerevisiae into Syrah grape musts. Syrah ferments inoculated with C. californica or Metschnikowia pulcherrima contained lower concentrations of residual sugar and ethanol compared to those with only S. cerevisiae. Furthermore, the presence of non-Saccharomyces yeasts influenced concentrations of glycerol and volatile aroma compounds. These results suggested potential use of some non-Saccharomyces yeasts towards reducing alcohol concentrations without risking slower alcoholic fermentations.

Characterization of Bacillus megaterium, Bacillus pumilus, and Paenibacillus polymyxa isolated from a Pinot noir wine from Western Washington State.

This report provides the first confirmed evidence of Bacillus-like bacteria present in a wine from Washington State. These bacteria were isolated from a 2013 Pinot noir wine whose aroma was sensorially described as being 'dirty' or 'pond scum.' Based on physiological traits and genetic sequencing, three bacterial isolates were identified as Bacillus megaterium (strain NHO-1), Bacillus pumilus (strain NHO-2), and Paenibacillus polymyxa (strain NHO-3). These bacteria grew in synthetic media of low pH (pH 3.5) while some survived ethanol concentrations up to 15% v/v. However, none tolerated molecular SO2 concentrations ≥0.4 mg/l. Growth of strains NHO-1 and NHO-3 in a Merlot grape juice resulted in increases of titratable and volatile acidities while decreases in titratable acidity were noted for NHO-2.

Impact of available nitrogen and sugar concentration in musts on alcoholic fermentation and subsequent wine spoilage by Brettanomyces bruxellensis.

The level of yeast assimilable nitrogen (YAN) supplementation required for Saccharomyces cerevisiae to complete fermentation of high sugar musts in addition to the impact of non-metabolized nitrogen on post-alcoholic spoilage by Brettanomyces bruxellensis was studied. A 2 × 3 factorial design was employed using a synthetic grape juice medium with YAN (150 or 250 mg N/L) and equal proportions of glucose/fructose (230, 250, or 270 g/L) as variables. S. cerevisiae ECA5 (low nitrogen requirement) or Uvaferm 228 (high nitrogen requirement) were inoculated at 10(5) cfu/mL while B. bruxellensis E1 or B2 were added once alcoholic fermentation ceased. Regardless of YAN concentration, musts that contained 230 or 250 g/L glucose/fructose at either nitrogen level attained dryness (mean = 0.32 g/L fructose) while those containing 270 g/L generally did not (mean = 2.5 g/L fructose). Higher concentrations of YAN present in musts yielded wines with higher amounts of α-amino acids and ammonium but very little (≤ 6 mg N/L) was needed by B. bruxellensis to attain populations ≥ 10(7) cfu/mL. While adding nitrogen to high sugar musts does not necessarily ensure completion of alcoholic fermentation, residual YAN did not affect B. bruxellensis growth as much as ethanol concentration.

Impact of sulfur dioxide and temperature on culturability and viability of Brettanomyces bruxellensis in Wine.

Brettanomyces is a major threat to red wine quality, causing off-odors such as "medicinal," "barnyard," or even "sewage" during aging. Although sulfites (SO2) are used to limit spoilage by these yeast cells, reduced storage temperatures may lessen SO2 requirements. To test this hypothesis, a 4 | 4 factorial experimental design with molecular SO2(mSO2) concentration (0.0, 0.2, 0.5, or 1.1 mg/liter) and storage temperature (22, 18, 15, or 10°C) was devised. Of three strains evaluated, B5 was the lone strain to regain culturability following exposure to 0.5 mg/liter mSO2 (18°C), whereas only F3 remained culturable in the absence of mSO2 at 10°C. Application of fluorescence microscopy using two different probes and quantitative PCR assays revealed only a 2-log reduction in metabolically active cells from wines with SO2 that were not culturable on nonselective media. Culturability in these wines eventually returned regardless of the concentration of mSO2 present. In addition, 4-ethylphenol production ceased upon addition of SO2. These findings provide additional support that Brettanomyces can enter a "viable-but-not-culturable" state upon exposure to sulfites. Given the diversity among strains, maintaining conditions of ≤15°C and ≥0.4 mg/liter mSO2 will help limit spoilage by Brettanomyces but will not lead to its complete eradication.

Metabolism of nonesterified and esterified hydroxycinnamic acids in red wines by Brettanomyces bruxellensis.

While Brettanomyces can metabolize nonesterified hydroxycinnamic acids found in grape musts/wines (caffeic, p-coumaric, and ferulic acids), it was not known whether this yeast could utilize the corresponding tartaric acid esters (caftaric, p-coutaric, and fertaric acids, respectively). Red wines from Washington and Oregon were inoculated with B. bruxellensis, while hydroxycinnamic acids were monitored by HPLC. Besides consuming p-coumaric and ferulic acids, strains I1a, B1b, and E1 isolated from Washington wines metabolized 40-50% of caffeic acid, a finding in contrast to strains obtained from California wines. Higher molar recoveries of 4-ethylphenol and 4-ethylguaiacol synthesized from p-coumaric and ferulic acids, respectively, were observed in Washington Cabernet Sauvignon and Syrah but not Merlot. This finding suggested that Brettanomyces either (a) utilized vinylphenols formed during processing of some wines or (b) metabolized other unidentified phenolic precursors. None of the strains of Brettanomyces studied metabolized caftaric or p-coutaric acids present in wines from Washington or Oregon.

Identification and characterization of Dekkera bruxellensis, Candida pararugosa, and Pichia guilliermondii isolated from commercial red wines.

Yeast isolates from commercial red wines were characterized with regards to tolerances to molecular SO(2), ethanol, and temperature as well as synthesis of 4-ethyl-phenol/4-ethyl-guaiacol in grape juice or wine. Based on rDNA sequencing, nine of the 11 isolates belonged to Dekkera bruxellensis (B1a, B1b, B2a, E1, F1a, F3, I1a, N2, and P2) while the other two were Candida pararugosa (Q2) and Pichia guilliermondii (Q3). Strains B1b, Q2, and Q3 were much more resistant to molecular SO(2) in comparison to the other strains of Dekkera. These strains were inoculated (10(3)-10(4)cfu/ml) along with lower populations of Saccharomyces (<500 cfu/ml) into red grape juice and red wine incubated at two temperatures, 15 degrees C and 21 degrees C. Although Saccharomyces quickly dominated fermentations in grape juice, B1b and Q2 grew and eventually reached populations >10(5)cfu/ml. In wine, Q3 never entered logarithmic growth and quickly died in contrast to Q2 which survived >40 days after inoculation. B1b grew well in wine incubated at 21 degrees C while slower growth was observed at 15 degrees C. Neither Q2 nor Q3 produced 4-ethyl-phenol or 4-ethyl-guaiacol, unlike B1b. However, lower concentrations of volatile phenols were present in wine incubated at 15 degrees C compared to 21 degrees C.

Inhibition of malolactic fermentation by a peptide produced by Saccharomyces cerevisiae during alcoholic fermentation.

The ability of Saccharomyces to inhibit Oenococcus oeni during the alcoholic fermentation by mechanisms other than SO(2) production was investigated. During fermentation in synthetic grape juice, S. cerevisiae strain RUBY.ferm inhibited the malolactic fermentation by O. oeni while strain EC1118 did not despite both strains producing similar amounts of SO(2). The bacterial inhibition exerted by RUBY.ferm was diminished when the wine was treated with proteases but not through the addition of nutrients. Wine fermented by RUBY.ferm was fractionated based on molecular weight and each fraction tested for the ability to inhibit the growth of O. oeni. The fraction containing compounds larger than 3 kDa was the sole inhibitory fraction. The inhibitory fraction was analyzed by SDS PAGE and showed a 5.9 kDa protein band present in wine fermented by RUBY.ferm that was not present in wine fermented by a non-antagonistic yeast, S. cerevisiae strain Saint Georges S101. The ability of the peptide to inhibit O. oeni seemed to be dependent on the presence of SO(2).

Quantitative determination of geosmin in red beets (Beta vulgaris L.) using headspace solid-phase microextraction.

An improved analytical method for the determination of geosmin in red beets was developed using headspace solid-phase microextraction (HSPME). Volatiles of beet juice were extracted in headspace for 2 h using a polydimethylsiloxane/divinylbenzene fiber, thermally desorbed from the fiber, and analyzed by gas chromatography. The HSPME method was determined to be suitable for geosmin analysis as evidenced by high relative recovery (99.2%), low relative standard deviation (7.48%), and reasonable detection limit (1 microg/kg of beet root tissue). The concentrations of geosmin in four beet cultivars ranged from 9.69 +/- 0.22 to 26.7 +/- 0.27 microg/kg, depending on cultivar.

Biosynthetic origin of geosmin in red beets (Beta vulgaris L.).

Geosmin provides the characteristic but sometimes undesirable "earthy" flavor to red table beets. To date, it is not known whether geosmin is a byproduct of beet metabolism or synthesized by soil-borne microorganisms and taken up by the beets during maturation. Analysis of mature beet roots revealed that peels contained 6 times the amount of geosmin compared to the bodies and cores. Sterilized beet seeds were aseptically grown in a basal medium prior to analysis for the presence of geosmin. Using a headspace solid-phase microextraction (HSPME) method, the relative recovery of geosmin from beet seedling extracts was 72.0 +/- 4.2% with (-)-menthone as the internal standard. The presence of geosmin in aseptically grown beet seedlings was confirmed by gas chromatography-mass spectrometry using authentic geosmin as the standard. During aseptic growth, the concentration of geosmin in seedlings remained constant for up to 5 months but increased at 6 months. Geosmin added to the growth medium was not absorbed by the seedlings. These studies support the conclusion that red beets are capable of endogenous synthesis of geosmin.