MET2 affects production of hydrogen sulfide during wine fermentation

The production of hydrogen sulfide (H₂S) during yeast fermentation contributes negatively to wine aroma. We have mapped naturally occurring mutations in commercial wine strains that affect production of H₂S. A dominant R₃₁₀G mutant allele of MET2, which encodes homoserine O-acetyltransferase, is present in several wine yeast strains as well as in the main lab strain S288c. Reciprocal hemizygosity and allele swap experiments demonstrated that the MET2 _R310G allele confers reduced H₂S production. Mutations were also identified in genes encoding the two subunits of sulfite reductase, MET5 and MET10, which were associated with reduced H₂S production. The most severe of these, an allele of MET10, showed five additional phenotypes: reduced growth rate on sulfate, elevated secretion of sulfite, and reduced production in wine of three volatile sulfur compounds: methionol, carbon disulfide and methylthioacetate. Alleles of MET5 and MET10, but not MET2, affected H₂S production measured by colour assays on BiGGY indicator agar, but MET2 effects were seen when bismuth was added to agar plates made with Sauvignon blanc grape juice. Collectively, the data are consistent with the hypothesis that H₂S production during wine fermentation results predominantly from enzyme activity in the sulfur assimilation pathway. Lower H₂S production results from mutations that reduce the activity of sulfite reductase, the enzyme that produces H₂S, or that increase the activity of l-homoserine-O-acetyltransferase, which produces substrate for the next step in the sulfur assimilation pathway.     

Development of a method to measure hydrogen sulfide in wine fermentation

A hydrogen sulfide (H2S) detecting tube was developed for the quantitative determination of H2S produced by yeast during laboratory scale wine fermentations. The detecting tube consisted of a small transparent plastic tube packed with an H2S-sensitive color-indicating medium. The packed medium changed color, with the color change progressing upward from the bottom of the tube, upon exposure to H2S produced by yeast during fermentation. A calibration study using a standard H2S gas showed that the length of the portion that darkened was directly related to the quantity of H2S (microg) with a high correlation coefficient (r2=0.9997). The reproducibility of the H2S detecting tubes was determined with five repetitive measurements using a standard H2S solution [5.6 microg/200 ml (28 ppb)], which resulted in a coefficient of variation of 3.6% at this level of H2S. With the sulfide detecting tubes, the production of H2S was continuously monitored and quantified from laboratory scale wine fermentations with different yeast strains and with the addition of different levels of elemental sulfur to the grape juice. This sulfide detecting tube technology may allow winemakers to quantitatively measure H2S produced under different fermentation conditions, which will eventually lead winemakers to better understand the specific factors and conditions for the excessive production of H2S during wine fermentation in a large production scale.     

Occurrence of hydrogen sulfide in wine and in fermentation: influence of yeast strain and supplementation of yeast available nitrogen

Hydrogen sulfide (H₂S) is a powerful aroma compound largely produced by yeast during fermentation. Its occurrence in wines and other fermented beverages has been associated with off-odors described as rotten egg and/or sewage. While the formation of hydrogen sulfide (H₂S) during fermentation has been extensively studied, it is the final H₂S content of wine that is actually linked to potential off-odors. Nevertheless, factors determining final H₂S content of wine have received little attention, and it is commonly assumed that high H₂S-forming fermentations will result in high final concentrations of H₂S. However, a clear relationship has never been established. In this report, we investigated the contribution of yeast strain and nitrogen addition to H₂S formation during fermentation and its consequent occurrence the resulting wines. Five commercial Saccharomyces cerevisiae wine yeast strains were used to ferment a Chardonnay juice containing 110 mg/l of YAN (yeast assimilable nitrogen), supplemented with di-ammonium phosphate (DAP) to increase YAN concentration to moderate (260 mg/l) and high (410 mg/l) levels. In contrast to the widely reported decrease in H₂S production in response to DAP addition, a non-linear relationship was found such that moderate DAP supplementation resulted in a remarkable increase in H₂S formation by each of the five wine yeasts. H₂S content of the finished wine was affected by yeast strain, YAN, and fermentation vigor. However, we did not observe a correlation between concentration of H₂S in the finished wines and H₂S produced during fermentation, with low-forming fermentations often having relatively high final H₂S and vice versa. Management of H₂S in wine through nitrogen supplementation requires knowledge of initial YAN and yeast H₂S characteristics.     

Biomass content governs fermentation rate in nitrogen-deficient wine musts

Problematic fermentations are common in the wine industry. Assimilable nitrogen deficiency is the most prevalent cause of sluggish fermentations and can reduce fermentation rates significantly. A lack of nitrogen diminishes a yeast's metabolic activity, as well as the biomass yield, although it has not been clear which of these two interdependent factors is more significant in sluggish fermentations. Under winemaking conditions with different initial nitrogen concentrations, metabolic flux analysis was used to isolate the effects. We quantified yeast physiology and identified key metabolic fluxes. We also performed cell concentration experiments to establish how biomass yield affects the fermentation rate. Intracellular analysis showed that trehalose accumulation, which is highly correlated with ethanol production, could be responsible for sustaining cell viability in nitrogen-poor musts independent of the initial assimilable nitrogen content. Other than the higher initial maintenance costs in sluggish fermentations, the main difference between normal and sluggish fermentations was that the metabolic flux distributions in nitrogen-deficient cultures revealed that the specific sugar uptake rate was substantially lower. The results of cell concentration experiments, however, showed that in spite of lower sugar uptake, adding biomass from sluggish cultures not only reduced the time to finish a problematic fermentation but also was less likely to affect the quality of the resulting wine as it did not alter the chemistry of the must.     

Impact of different malolactic fermentation inoculation scenarios on Riesling wine aroma

During malolactic fermentation (MLF), lactic acid bacteria influence wine aroma and flavour by the production of volatile metabolites and the modification of aroma compounds derived from grapes and yeasts. The present study investigated the impact of different MLF inoculation strategies with two different Oenococcus oeni strains on cool climate Riesling wines and the volatile wine aroma profile. Four different timings were chosen for inoculation with bacteria to conduct MLF in a Riesling must/wine with a high acidity (pH 2.9–3.1). Treatments with simultaneous inoculation showed a reduced total fermentation time (alcoholic and malolactic) compared to the sequential inoculations. No negative impact of simultaneous alcoholic and malolactic fermentation on fermentation success and on the final wine volatile aroma composition was observed. Compared to sequential inoculation, wines with co-inoculation tended to have higher concentrations of ethyl and acetate esters, including acetic acid phenylethylester, acetic acid 3-methylbutylester, butyric acid ethylester, lactic acid ethylester and succinic acid diethylester. Results of this study provide some alternatives to diversify the number of wine styles by safely conducting MLF in low-pH, cool-climate white musts with potential high alcohol content.     

Mitotic recombination and genetic changes in Saccharomyces cerevisiae during wine fermentation

Natural strains of Saccharomyces cerevisiae are prototrophic homothallic yeasts that sporulate poorly, are often heterozygous, and may be aneuploid. This genomic constitution may confer selective advantages in some environments. Different mechanisms of recombination, such as meiosis or mitotic rearrangement of chromosomes, have been proposed for wine strains. We studied the stability of the URA3 locus of a URA3/ura3 wine yeast in consecutive grape must fermentations. ura3/ura3 homozygotes were detected at a rate of 1 x 10(-5) to 3 x 10(-5) per generation, and mitotic rearrangements for chromosomes VIII and XII appeared after 30 mitotic divisions. We used the karyotype as a meiotic marker and determined that sporulation was not involved in this process. Thus, we propose a hypothesis for the genome changes in wine yeasts during vinification. This putative mechanism involves mitotic recombination between homologous sequences and does not necessarily imply meiosis.     

Impact of substrates acclimation strategy on simultaneous biodegradation of hydrogen sulfide and ammonia

Three columns were differentiated with feeding mixture of H₂S and NH₃ (MFC), feeding NH₃ followed by H₂S (NFC), and feeding H₂S followed by NH₃ (SFC). Removal performance, biodegradation capacity and microbial community structures in the three columns were compared. The results show that NFC has a shorter acclimation period for the removal of NH₃ gas and nitrification than MFC. Under the high loading of H₂S and NH₃ at 164 and 82 g m⁻³ h⁻¹, respectively, NFC exhibited high removal efficiency of NH₃ (>95%) while the removal efficiencies were obtained at 63 and 75% in MFC and SFC, respectively. The removal of NH₃ gas in NFC was significantly attributed to nitrification (over 50%), while adsorption and chemical reaction contributed to the removal of NH₃ in MFC and SFC. The different biodegradation capacities of NH₃ could be due to the dissimilarity in the microbial population presented in each column.     

Evaluating fermentation characteristics of Kazachstania spp. and their potential influence on wine quality

World Journal of Microbiology and Biotechnology - The current study is the first one to demonstrate the wine fermentation potential of members of several species of the genus Kazachstania including...     

Impact of pitching rate on yeast fermentation performance and beer flavour

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e. higher inoculum size). However, the impact of the pitching rate on crucial fermentation and beer quality parameters has never been assessed systematically. In this study, five pitching rates were applied to lab-scale fermentations to investigate its impact on the yeast physiology and beer quality. The fermentation rate increased significantly and the net yeast growth was lowered with increasing pitching rate, without affecting significantly the viability and the vitality of the yeast population. The build-up of unsaturated fatty acids in the initial phase of the fermentation was repressed when higher yeast concentrations were pitched. The expression levels of the genes HSP104 and HSP12 and the concentration of trehalose were higher with increased pitching rates, suggesting a moderate exposure to stress in case of higher cell concentrations. The influence of pitching rate on aroma compound production was rather limited, with the exception of total diacetyl levels, which strongly increased with the pitching rate. These results demonstrate that most aspects of the yeast physiology and flavour balance are not significantly or negatively affected when the pitching rate is changed. However, further research is needed to fully optimise the conditions for brewing beer with high cell density populations.     

The therapeutic potential of hydrogen sulfide: separating hype from hope

Hydrogen sulfide (H(2)S) has become the hot new signaling molecule that seemingly affects all organ systems and biological processes in which it has been investigated. It has also been shown to have both proinflammatory and anti-inflammatory actions and proapoptotic and anti-apoptotic effects and has even been reported to induce a hypometabolic state (suspended animation) in a few vertebrates. The exuberance over potential clinical applications of natural and synthetic H(2)S-"donating" compounds is understandable and a number of these function-targeted drugs have been developed and show clinical promise. However, the concentration of H(2)S in tissues and blood, as well as the intrinsic factors that affect these levels, has not been resolved, and it is imperative to address these points to distinguish between the physiological, pharmacological, and toxicological effects of this molecule. This review will provide an overview of H(2)S metabolism, a summary of many of its reported "physiological" actions, and it will discuss the recent development of a number of H(2)S-donating drugs that show clinical potential. It will also examine some of the misconceptions of H(2)S chemistry that have appeared in the literature and attempt to realign the definition of "physiological" H(2)S concentrations upon which much of this exuberance has been established.     

Diurnal changes in pore water sulfide concentrations in the seagrass Thalassia testudinum beds: the effects of seagrasses on sulfide dynamics

The dynamics of the seagrass-sulfide interaction were examined in relation to diel changes in sediment pore water sulfide concentrations in Thalassia testudinum beds and adjacent bare areas in Corpus Christi Bay and lower Laguna Madre, Texas, USA, during July 1996. Pore water sulfide concentrations in seagrass beds were significantly higher than in adjacent bare areas and showed strong diurnal variations; levels significantly decreased during mid-day at shallow sediment depths (0-10 cm) containing high below-ground tissue biomass and surface area. In contrast, diurnal variations in sediment sulfide concentrations were absent in adjacent bare patches, and at deeper (>10 cm) sediment depths characterized by low below-ground plant biomass or when the grasses were experimentally shaded. These observations suggest that the mid-day depressions in sulfide levels are linked to the transport of photosynthetically produced oxygen to seagrass below-ground tissues that fuels sediment sulfide oxidation. Lower sulfide concentrations in bare areas are likely a result of low sulfate reduction rates due to low organic matter available for remineralization. Further, high reoxidation rates due to rapid exchange between anoxic pore water and oxic overlying water are probably stimulated in bare areas by higher current velocity on the sediment surface than in seagrass beds. The dynamics of pore water sulfides in seagrass beds suggest no toxic sulfide intrusion into below-ground tissues during photosynthetic periods and demonstrate that the sediment chemical environment is considerably modified by seagrasses. The reduced sediment sulfide levels in seagrass beds during photosynthetic periods will enhance seagrass production through reduced sulfide toxicity to seagrasses and sediment microorganisms related to the nutrient cycling.     

Carvedilol induces endogenous hydrogen sulfide tissue concentration changes in various mouse organs

Carvedilol, a third generation non-selective adrenoreceptor blocker, is widely used in cardiology. Its action has been proven to reach beyond adrenergic antagonism and involves multiple biological mechanisms. The interaction between carvedilol and endogenous 'gasotransmitter' hydrogen sulfide (H2S) is unknown. The aim of the study is to assess the influence of carvedilol on the H2S tissue level in mouse brain, liver, heart and kidney. Twenty eight SJL strain female mice were administered intraperitoneal injections of 2.5 mg/kg b.w./d (group D1, n=7), 5 mg/kg b.w./d (group D2, n=7) or 10 mg/kg b.w./d of carvedilol (group D3, n=7). The control group (n=7) received physiological saline in portions of the same volume (0.2 ml). Measurements of the free tissue H2S concentrations were performed according to the modified method of Siegel. A progressive decline in H2S tissue concentration along with an increase in carvedilol dose was observed in the brain (12.5%, 13.7% and 19.6%, respectively). Only the highest carvedilol dose induced a change in H2S tissue level in the heart - an increase by 75.5%. In the liver medium and high doses of carvedilol increased the H2S level by 48.1% and 11.8%, respectively. In the kidney, group D2 showed a significant decrease of H2S tissue level (22.5%), while in the D3 group the H2S concentration increased by 12.9%. Our study has proven that carvedilol affects H2S tissue concentration in different mouse organs.     

Isolation of sulfite reductase variants of a commercial wine yeast with significantly reduced hydrogen sulfide production

The production of hydrogen sulfide (H₂S) during fermentation is a common and significant problem in the global wine industry as it imparts undesirable off-flavors at low concentrations. The yeast Saccharomyces cerevisiae plays a crucial role in the production of volatile sulfur compounds in wine. In this respect, H₂S is a necessary intermediate in the assimilation of sulfur by yeast through the sulfate reduction sequence with the key enzyme being sulfite reductase. In this study, we used a classical mutagenesis method to develop and isolate a series of strains, derived from a commercial diploid wine yeast (PDM), which showed a drastic reduction in H₂S production in both synthetic and grape juice fermentations. Specific mutations in the MET10 and MET5 genes, which encode the catalytic α- and β-subunits of the sulfite reductase enzyme, respectively, were identified in six of the isolated strains. Fermentations with these strains indicated that, in comparison with the parent strain, H₂S production was reduced by 50–99%, depending on the strain. Further analysis of the wines made with the selected strains indicated that basic chemical parameters were similar to the parent strain except for total sulfite production, which was much higher in some of the mutant strains.     

硫化氢抗大鼠动脉粥样硬化作用研究

目的:探讨硫化氢(H2S)对大鼠动脉粥样硬化(AS)的作用及其机制。方法:体重(210±10)g的健康雄性SD大鼠125只,随机分为:对照组、AS模型组、AS+低剂量NaHS(2.8μmol/(kg.d))组、AS+中剂量NaHS(14μmol/(kg.d))组及AS+高剂量NaHS(28μmol/(kg.d))组。采用高脂饲料加大剂量VitD3注射复制大鼠AS模型。NaHS腹腔注射,连续用药12周。分别在喂养前及喂养后3、6、9、12周各处死动物。用生化分析仪检测血脂,去蛋白法检测血浆硫化氢,HE染色观察血管病理损伤程度及病变评分,免疫组化法检测血管组织中血管内皮生长因子(VEGF)的表达。结果:与相同时期的对照组相比,AS模型组在喂养后3、6、9、12周,血清甘油三脂(TG)和胆固醇(TC)均明显升高;主动脉病变评分从第6周到12周明显增加(P0.01),并出现明显的动脉粥样硬化病变,表现为阳性区域的脂质斑块;血清H2S浓度明显降低,从喂养前的(44.98±2.06)μmol/L到第3、6、9、12周分别为(38.56±2.26),(32.96±2.38),(28.63±0.92),(23.55±0.92)μmol/L,并分别低于同时期各对照组的(44.72±0.85),(43.71±0.59),(41.96±0.97),(39.87±1.25)μmol/L(P0.01);血管组织中VEGF的表达明显增强(P0.01)。与模型组比较,低剂量NaHS组,各指标均无明显变化;中剂量NaHS组大鼠血清H2S含量于第6周开始明显高于模型组(36.13±0.73)vs(32.96±2.38)μmol/L,P0.05;于9、12周时,分别为(33.07±1.14)vs(28.63±0.92)μmol/L,(30.16±0.62)vs(23.55±0.92)μmol/L,P0.01;高剂量NaHS组大鼠血中H2S浓度于第3周开始到12周,分别为:(41.25±0.80),(38.71±0.46),(35.31±0.62),(33.38±0.78)μmol/L,均明显高于模型组(P0.01);中、高剂量NaHS组血清TC均从第3周开始到12周明显降低(P0.01),TG分别从第3、第6周开始到12周明显降低(P0.05,P0.01),血管组织病变评分与VEGF的表达均于第6周开始到12周明显降低(P0.05)。相关分析显示血清中硫化氢的浓度与动脉粥样硬化的病变评分及血管VEGF的表达呈明显的负相关(r=-0.917,P0.01,r=-0.885,P0.01),而与血清甘油三脂和胆固醇之间无显著相关性。结论:动脉粥样硬化病变的形成与发展与内源性硫化氢的降低密切相关,补充外源性H2S可提高动脉粥样硬化大鼠血清中硫化氢浓度,减轻血管损伤程度,抑制VEGF的表达。     

Whole tissue hydrogen sulfide concentrations are orders of magnitude lower than presently accepted values

Hydrogen sulfide is gaining acceptance as an endogenously produced modulator of tissue function. The present paradigm of H(2)S (diprotonated, gaseous form of hydrogen sulfide) as a tissue messenger consists of H(2)S being released from the desulfhydration of l-cysteine at a rate sufficient to maintain whole tissue hydrogen sulfide concentrations of 30 microM to >100 microM, and these tissue concentrations serve a messenger function. Utilizing physiological concentrations of l-cysteine and aerobic conditions, we found that catabolism of hydrogen sulfide by mouse liver and brain homogenates exceeded the rate of enzymatic release of this compound such that measureable hydrogen sulfide release was less with tissue-containing vs. tissue-free buffers. Analyses of the gas space over rapidly homogenized mouse brain and liver indicated that in situ tissue hydrogen sulfide concentrations were only about 15 nM. Human alveolar air measurements indicated negligible free H(2)S concentrations in blood. We conclude rapid tissue catabolism of hydrogen sulfide maintains whole tissue brain and liver concentrations of free hydrogen sulfide that are three orders of magnitude less than conventionally accepted values and only 1/5,000 of the hydrogen sulfide concentration (100 microM) required to alter cellular function in vitro. For hydrogen sulfide to serve as an endogenously produced messenger, tissue production and catabolism must result in intracellular microenvironments with a sufficiently high hydrogen sulfide concentration to activate a local signaling mechanism, while whole tissue concentrations remain very low.