Genotype-dependent sulphite tolerance of Australian Dekkera (Brettanomyces) bruxellensis wine isolates

Aims: The aim of this study was to determine sulphite tolerance for a large number of Dekkera bruxellensis isolates and evaluate the relationship between this phenotype and previously assigned genotype markers. Methods and Results: A published microplate‐based method for evaluation of yeast growth in the presence of sulphite was benchmarked against culturability following sulphite treatment, for the D. bruxellensis type strain (CBS 74) and a reference wine isolate (AWRI 1499). This method was used to estimate maximal sulphite tolerance for 41 D. bruxellensis isolates, which was found to vary over a fivefold range. Significant differences in sulphite tolerance were observed when isolates were grouped according to previously assigned genotypes and ribotypes. Conclusions: Variable sulphite tolerance for the wine spoilage yeast D. bruxellensis can be linked to genotype markers. Significance and Impact of the Study: Strategies to minimize risk of wine spoilage by D. bruxellensis must take into account at least a threefold range in effective sulphite concentration that is dependent upon the genotype group(s) present. The isolates characterized in this study will be a useful resource for establishing the mechanisms conferring sulphite tolerance for this industrially important yeast species.     

添加芦荟汁对红葡萄酒发酵的抑菌效果

二氧化硫（SO₂）危害人体健康。为了寻找用于葡萄酒酿造的SO₂替代品,在赤霞珠葡萄酒自然发酵和接种发酵中比较了添加芦荟汁（80 g/L）与SO₂（60 mg/L）的抑菌和发酵效果。结果表明,添加芦荟汁促进接种酵母和内源酵母生长,SO₂抑制内源酵母生长,接种酵母、添加芦荟汁和添加SO₂显著抑制细菌和霉菌生长,SO₂比芦荟汁对细菌和霉菌生长的抑制作用大。添加芦荟汁和添加SO₂同样提高乙醇产量和减小酸度下降,降低葡萄酒的残糖量和褐变度。添加芦荟汁提高了挥发性酸度和高级醇产量,降低了接种发酵的葡萄酒色度和提高乙酸产量,降低了自然发酵的乙醛产量,而SO₂作用相反。添加芦荟汁具有减少SO₂用量的可能性。     

Evaluation of the acetaldehyde production and degradation potential of 26 enological <Emphasis Type="Italic">Saccharomyces</Emphasis> and non-<Emphasis Type="Italic">Saccharomyces</Emphasis> yeast strains in a resting cell model system

Acetaldehyde is relevant for wine aroma, wine color, and microbiological stability. Yeast are known to play a crucial role in production and utilization of acetaldehyde during fermentations but comparative quantitative data are scarce. This research evaluated the acetaldehyde metabolism of 26 yeast strains, including commercial Saccharomyces and non-Saccharomyces, in a reproducible resting cell model system. Acetaldehyde kinetics and peak values were highly genus, species, and strain dependent. Peak acetaldehyde values varied from 2.2 to 189.4 mg l⁻¹ and correlated well (r ² = 0.92) with the acetaldehyde production yield coefficients that ranged from 0.4 to 42 mg acetaldehyde per g of glucose in absence of SO₂. S. pombe showed the highest acetaldehyde production yield coefficients and peak values. All other non-Saccharomyces species produced significantly less acetaldehyde than the S. cerevisiae strains and were less affected by SO₂ additions. All yeast strains could degrade acetaldehyde as sole substrate, but the acetaldehyde degradation rates did not correlate with acetaldehyde peak values or acetaldehyde production yield coefficients in incubations with glucose as sole substrate.     

Early diagnosis of SO2-stress by volatile emissions in some crop plants

Summary Fumigation experiments with SO₂ performed on the seedlings of three plant species viz, tomato (Lycopersicon esculentum), mung bean (Vigna radiata) and maize (Zea mays) resulted in the emission of volatiles. Acetaldehyde and ethanol were produced in the fumigated plants. In addition, there was also an increased production of ethylene and ethane. The production of these volatiles was positively correlated to the SO₂ concentrations of 4.2 and 8.3 mol m^–3 (0.1 and 0.2 ppm). Ethylene was emitted primarily from SO₂-stressed yet healthy leaves, whereas high ethane levels were detected in leaves with visible injury symptoms. However, with the appearance of visible injury symptoms, there was a decline in ethylene, acetaldehyde and ethanol emissions. Synthesis of ethylene and ethane seems to be a result of different metabolic pathways. Ethane evolution and its inhibition by antioxidants indicate SO₂-mediated lipid peroxidation by free radical species formed during sulphite oxidation. Perturbation in the cellular respiratory machinery results in the formation of acetaldehyde and ethanol. Since the rates of emissions of ethane, acetaldehyde and ethanol fromplant species were positively correlated to their relative resistance to SO₂, the production of these gases could be used as a reliable diagnostic tool for biomonitoring air pollution (SO₂) stress.Abbreviations ADH alcohol dehydrogenase - NaHSO₃ sodium metabisulphite - O ₂ ^– superoxide radical - OH hydroxyl radical - pO₂ oxygen partial pressure - SO₂ sulphur dioxide - SO ₃ ^– sulphite radical - SOD superoxide dismutase     

Control of Flavor Development in Wine during and after Malolactic Fermentation by Oenococcus oeni

During malolactic fermentation in wine by Oenococcus oeni, the degradation of citric acid was delayed compared to the degradation of malic acid. The maximum concentration of diacetyl, an intermediary compound in the citric acid metabolism with a buttery or nutty flavor, coincided with the exhaustion of malic acid in the wine. The maximum concentration of diacetyl obtained during malolactic fermentation was strongly dependent on the oxygen concentration and the redox potential of the wine and, to a lesser extent, on the initial citric acid concentration. The final diacetyl concentration in the wine was also dependent on the concentration of SO₂. Diacetyl combines rather strongly with SO₂ (K_f = 7.2 × 10³ M⁻¹ in 0.1 M malate buffer [pH 3.5] at 30°C). The reaction is exothermic and reversible. If the concentration of SO₂ decreases during storage of the wine, the diacetyl concentration increases again.     

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.     

Kinetics and mathematical model of killer/sensitive interaction under different physicochemical conditions of must/wine: a study from a biological point of view

Fermentation of grape must to wine is carried out by a complex microbial mixture, which also involves spoilage yeasts of wine. The latter yeasts produce organoleptic changes that cause significant economic losses to the wine industry. SO₂ is traditionally used to control this spoilage populations, but because of its harmful effects on human health, biocontrol has emerged as an alternative treatment. Although studies have been carried out to select biocontroller yeasts and examine their underlying mechanisms of action, reports on their application have not been published yet. To better understand the interaction and the successful application of biocontrol, the use of mathematical models, among other methods, is important, as they facilitate the prediction of success or failure of the antagonist. The objective of the present study was to use an existing mathematical model to obtain information about the yeast’s interaction assayed and to validate its predictive use under different physicochemical conditions during the wine fermentation, and eventually predict biocontrol kinetics. The mathematical model was applied to the fermentation conditions and provided information on the kinetic parameters of the biocontrol interaction and allowed interpretations about other parameters. The model was applied in the different physicochemical conditions for the biocontrol and did not fit correctly to experimental data, and therefore an improvement was proposed which was successful and presented new hypotheses.     

Occurrence of Lactic Acid Bacteria During the Different Stages of Vinification and Conservation of Wines

We showed that the growth of lactic acid bacteria during alcoholic fermentation depends on the composition of the must. We illustrated how the addition of sulfur dioxide to the must before fermentation and the temperature of storage both affect the growth of these bacteria in the wine. Whereas species of Lactobacillus and Leuconostoc mesenteroides were isolated from grapes and must, Leuconostoc oenos was the only species isolated after alcoholic fermentation. This organism was responsible for the malolactic fermentation. Isolates of this species varied in their ability to ferment pentoses and hexoses. The survival of Leuconostoc oenos in wines after malolactic fermentation depended on wine pH, alcohol concentration, SO₂ concentration, and temperature of storage.     

Transcriptive complex: isolation by cesium sulfate-centrifugation

Formation of the “CsCl complex” (an RNA polymerase-DNA complex isolatable by equilibrium centrifugation in CsCl) was further shown to require the presence of more than two species of nucleoside triphosphate substrates and double-stranded DNA suggesting that the formation at least of a single phosphodiester bond renders the RNA polymerase-promoter DNA complex resistant to dissociation by such high concentration of CsCl. Growing RNA chains were still attached to RNA polymerase-DNA complex even after centrifugation whereas completed chains were recovered as released from the complex and banded at the position of free RNA in Cs₂SO₄ equilibrium centrifugation.     

Impact of acetaldehyde- and pyruvic acid-bound sulphur dioxide on wine lactic acid bacteria

Aims: To investigate the impact of acetaldehyde‐ and pyruvic acid‐bound sulphur dioxide on wine lactic acid bacteria (LAB). Methods and Results: Growth studies were performed where Oenococcus oeni, Pediococcus parvulus, Ped. damnosus and Lactobacillus hilgardii were inoculated into media containing various concentrations of acetaldehyde or pyruvic acid and an equimolar concentration of SO₂ at pH 3·50 and 3·70. Low concentrations of acetaldehyde‐ and pyruvic acid‐bound SO₂ were inhibitory to the growth of all bacteria although acetaldehyde‐bound SO₂ was generally more inhibitory than pyruvic acid‐bound SO₂. Inhibition was greater at pH 3·50 than 3·70, and Lact. hilgardii was the most sensitive to acetaldehyde‐bound SO₂, while O. oeni was the most sensitive to pyruvic acid‐bound SO₂. Degradation of SO₂‐bound acetaldehyde was observed for all LAB, and aside from O. oeni, there was also complete degradation of SO₂‐bound pyruvic acid at both pH values. O. oeni only degraded pyruvic acid at pH 3·70. Degradation of SO₂‐bound acetaldehyde or pyruvic acid did not correlate with bacterial growth as inhibition was always observed in media containing bound SO₂. Conclusions: Acetaldehyde‐ and pyruvic acid‐bound SO₂ were inhibitory to wine LAB growth at concentrations as low as 5 mg l^?1. Despite this inhibition, all wine LAB degraded SO₂‐bound acetaldehyde and pyruvic acid suggesting that bound SO₂ may have a bacteriostatic rather than bacteriocidal action. Significance and Impact of the Study: Sulphur dioxide bound to acetaldehyde or pyruvic acid is inhibitory to growth of wine LAB and must be considered when conducting the malolactic fermentation or controlling the growth of spoilage bacteria such as Pediococcus and Lactobacillus.     

A survey ofSaccharomyces populations associated with wine fermentations from the Apulia region (South Italy)

The aim of this paper was to investigate the genetic and phenotypic characteristics of yeasts isolated from samples of grape musts collected from four different areas of Apulia region. The 68 yeast isolates were identified asSaccharomyces cerevisiae by PCR-RFLP of 5.8S-ITS region of the rRNA gene. Individual isolates were differentiated by RAPD-PCR and AFLP. The following oenological traits were studied: fermentation power, resistance to cycloheximide, alcohol and SO₂, formation of SO₂ and H₂S, β-glucosidase activity, and production of biogenic amines and secondary compounds. Many phenotypes were common to several yeasts isolated from the four different areas, such as high SO₂ resistance and fermentation power. In addition, someS. cerevisiae isolates showed a β-glucosidase activity and others had a high resistance to cycloheximide. All the strains formed biogenic amines. Solid Phase Microextraction was used to determine secondary compounds produced in wine by the single yeast cultures.     

Transgenic tobacco plants overexpressing the Met25 gene of Saccharomyces cerevisiae exhibit enhanced levels of cysteine and glutathione and increased tolerance to oxidative stress

Summary. The cysteine biosynthesis pathway differs between plants and the yeast Saccharomyces cerevisiae. The yeast MET25 gene encoded to O-acetylhomoserine sulfhydrylase (AHS) catalyzed the reaction that form homocysteine, which later can be converted into cystiene. In vitro studies show that this enzyme possesses also the activity of O-acetyl(thiol)lyase (OASTL) that catalyzes synthesis of cysteine in plants. In this study, we generated transgenic tobacco plants expressing the yeast MET25 gene under the control of a constitutive promoter and targeted the yeast protein to the cytosol or to the chloroplasts. Both sets of transgenic plants were taller and greener than wild-type plants. Addition of SO₂, the substrate of the yeast enzyme caused a significant elevation of the glutathione content in representative plants from each of the two sets of transgenic plants expressing the yeast gene. Determination of non-protein thiol content indicated up to four-folds higher cysteine and 2.5-fold glutathione levels in these plants. In addition, the leaf discs of the transgenic plants were more tolerant to toxic levels of sulphite, and to paraquat, an herbicide generating active oxygen species.     

Effect of wine inhibitors on the proteolytic activity of papain from Carica papaya L. latex

The influence of potential inhibitors naturally present in wine on the proteolytic activity of papain from Carica papaya latex was investigated to evaluate its applicability in white wine protein haze stabilization. Enzymatic activity was tested against a synthetic tripeptide chromogenic substrate in wine‐like acidic medium that consisted of tartaric buffer (pH 3.2) supplemented with ethanol, free sulfur dioxide (SO₂), grape skin and seed tannins within the average ranges of concentrations that are typical in wine. The diagnosis of inhibition type, performed with the graphical method, demonstrated that all of tested wine constituents were reversible inhibitors of papain. The strongest inhibition was exerted by free SO₂, which acted as a mixed‐type inhibitor, similar to grape skin and seed tannins. Finally, when tested in table white wines, the catalytic activity of papain, even when if it was ascribable to the hyperbolic behavior of Michaelis‐Menten equation, was determined to be strongly affected by free SO₂ and total phenol level. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:48–54, 2015     

The significance of stemflow chemistry for epiphytic lichen diversity in a dieback-affected spruce forest on Mt Brocken,northern Germany

Epiphytic lichen diversity in a boggy stand of Norway spruce (Picea abies) was studied in the eastern Harz Mountains, northern Germany. Spruce trees at wet sites were affected by forest dieback, whereas trees on drier sites remained unaffected. Lichen diversity was higher on dieback-affected trees than on healthy ones. The foliose lichen Hypogymnia physodes was significantly more frequent on dead trees, whereas the crustose, extremely toxitolerant Lecanora conizaeoides occurred more frequently on healthy trees. Stemflow concentrations of NH₄⁺, NO₃⁻, PO₃⁻, and SO₄²⁻ were lower on affected trees. This is attributed to reduced interception from the atmosphere due to needle loss. Cover of H. physodes decreased with increasing mean SO₄²⁻ concentration in stemflow. The total of lichen species per sample tree also decreased with increasing SO₄²⁻ concentration in stemflow, indicating that most species reacted in a similar way as H. physodes. Cover of L. conizaeoides increased with increasing SO₄²⁻ concentration, but decreased at higher SO₄²⁻ concentrations. Bark chemistry had a minor influence on lichen diversity.     

Physicochemical properties of salt-soluble,unsheared chromatin

Salt-dependent structural changes of rat liver chromatin isolated by an extraction procedure not involving shear and exogeneous nucleases were investigated by sedimentation and light scattering methods. The effects observed are complex involving changes of the molecular weight and expansion. Between 0.1 M and 0.2 M (NH₄)₂SO₄ where histone H1 is released, a fragmentation into molecules of half molecular weight is found which is accompanied by an expansion into a more extended conformation gradually increasing to 0.4 M (NH₄)₂SO₄. The H1-free chromatin does not exhibit the reduction in molecular weight but undergoes this expansion. The original conformation is not reversible on re-decreasing the salt concentration to 0.1 M (NH₄)₂SO₄.     

贺兰山东麓优良本土酿酒酵母筛选及发酵特性分析

【背景】商业酵母的使用造成葡萄酒同质化问题严重,发掘优良本土酿酒酵母具有十分重要的意义。【目的】从168株宁夏本土酿酒酵母菌株中筛选出性能优良、具有出色葡萄酒发酵能力的菌株。【方法】基于杜氏管发酵试验和乙醇、高糖等耐受性试验分析产H2S能力及生长曲线测定的方法,筛选出发酵力好、耐受性强、低产H2S的本土酿酒酵母进行赤霞珠葡萄酒发酵试验,测定葡萄酒样基础理化指标、酚类物质和挥发性成分,探究筛选出的酿酒酵母发酵特性。【结果】初步筛选出发酵快速,能适应13%乙醇、350 g/L葡萄糖、250 mg/L SO2、pH 1.0的生存环境且低产H2S的4株本土酿酒酵母YC-E8、QTX-D17、QTX-D7、YQY-E18。菌株YC-E8产甘油能力强,所发酵酒样香气与商业酵母XR、F33最为接近,适用于赤霞珠葡萄酒的发酵。菌株QTX-D17发酵酒样中酒精、单宁、总酚和花色苷含量最高,表现出本土酿酒酵母优良的发酵特性。菌株QTX-D7所发酵酒样香气中乙酸乙酯、辛酸乙酯、1-壬醇等物质含量较高,赋予了葡萄酒香蕉味、苹果味、菠萝味、椰子味等愉悦花果香。【结论】最终筛选出3株优良本土酿酒酵母QTX-D17...     

Continuous glycerol production by the sulphite process with immobilized cells of Saccharomyces cerevisiae

Summary Glycerol fermentations by the sulphite process with immobilized yeast cells were carried out successfully in continuous culture in a fixed-bed column reactor. In comparison to continuous glycerol fermentations with free yeast cells, fourfold higher dilution rates were obtained. The stability of the immobilized cell system was dependent on dilution rate (D) and temperature. Glycerol yields were influenced by the ratio of sugar to Na₂SO₃ in the feed. At a flow rate of D = 0.06 h ^–1 glycerol concentrations up to 25 g1 ^–1 were measured in the effluent with an average volumetric productivity of 35 g 1 ^–1 per day. A constant production rate was maintained for nearly 9 months.Offprint requests to: H.-J. Rehm     

Biosynthesis of sulphur amino acids in Saccharomyces cerevisiae

A number of strains of Saccharomyces which produce sulphite by sulphate reduction were examined from an enzymatic and genetic point of view.There are a number of mechanisms that regulate this activity. All of these mechanisms involve the sulphite-reducing activity. In the strains examined, reduced function as a result of mutation in the Sr-locus (affecting H₂S-NADP oxidoreductase EC 1.8.1.2), repression of biosynthesis of the enzyme because of a mutation below the specific locus, and inhibition of the enzyme by endogenous factors were found to be responsible. The production of sulphite can also be connected with a complex state of heterozygosity.It is probably this multiplicity of biochemical and genetic mechanisms that accounts for the frequency with which the production of sulphite is observed in wild strains in nature.This investigation was supported by a research grant of C.N.R. (Consiglio Nazionale delle Ricerche, Roma).     

Isolation and partial characterization of a 51Cr complex from Brewers' yeast

A butanol: H₂O extract of Brewers' yeast grown in a medium which contained ⁵¹Cr was analyzed by gel-filtration chromatography. A single radioactive peak was eluted at an elution volume which suggested a molecular weight of approximately 400–600 daltons. Subsequent examination of pooled radioactive fractions obtained from gel-filtration chromatography demonstrated that the ⁵¹Cr complex was eluted in a single peak from both cation- and anion-exchange resins. The elution characteristics of the ⁵¹Cr complex indicated that the compound is a single anionic species. The ⁵¹Cr complex was purified by a combination of gel-filtration chromatography and ion-exchange chromatography and subsequently analyzed by thin-layer chromatography. The results indicated that the ⁵¹Cr complex from Brewers' yeast is a peptide that contains at least six amino acids. When a partially purified preparation of the ⁵¹Cr complex from yeast was administered orally to rats, the absorption and retention of ⁵¹Cr was significantly greater than that in rats given ⁵¹Cr in the form of CrCl₃·6H₂O. These experiments demonstrate that chromium is associated with a single metal-binding peptide in Brewers' yeast and indicate that the chromium in this complex is absorbed and retained more efficiently than chromium salts.     

Chemiluminescence determination of sulphite using a cyclometalated iridium complex as chemiluminescence reagent

A simple, fast and accurate chemiluminescence (CL) method for the determination of sulphite has been developed, based on its sensitizing effect on the CL reaction between a novel water‐soluble iridium complex, [(dpci)₂Ir(bvbbi)](PF₆) (dpci = 3,4‐diphenylcinnoline; bvbbi = N,N′‐bivinylester‐¹H,^1′H‐[2,2′] bibenzimidazole) and cerium(IV). Under the optimal experimental conditions, the increased CL response was linear, with the concentration of sulphite over the range 5.0 × 10^–7–5.0 × 10^–4 mol/L. The detection limit of the method was 1.6 × 10^–7 mol/L, with a relative standard deviation (RSD) of 2.7% for nine repetitive determination of 1.0 × 10^–4 mol/L sulphite. The method was successfully applied to the quantitative analysis of sulphite in sugar samples. The possible reaction mechanism of sulphite on the [(dpci)₂Ir(bvbbi)](PF₆)–cerium(IV) system is also briefly discussed. Copyright © 2012 John Wiley & Sons, Ltd.