Determination of 2,3-butanediol in high and low acetoin producers of Saccharomyces cerevisiae wine yeasts by automated multiple development (AMD)

P. ROMANO, G. SUZZI, V. BRANDOLINI, E. MENZIANI AND P. DOMIZIO. 1996. High performance thin layer chromatography with automated multiple development was used to determine 2,3-butanediol levels in wine produced by high and low acetoin-forming strains of Saccharomyces cerevisiae . An inverse correlation between acetoin and 2,3-butanediol content was found suggesting a leaky mutation in acetoin reductase of the low 2,3-butanediol producing strains.     

Properties of 2,3-Butanediol Dehydrogenases from Lactococcus lactis subsp. lactis in Relation to Citrate Fermentation

Two 2,3-butanediol dehydrogenases (enzymes 1 and 2; molecular weight of each, 170,000) have been partially purified from Lactococcus lactis subsp. lactis (Streptococcus diacetylactis) D10 and shown to have reductase activity with either diacetyl or acetoin as the substrate. However, the reductase activity with 10 mM diacetyl was far greater for both enzymes (7.0- and 4.7-fold for enzymes 1 and 2, respectively) than with 10 mM acetoin as the substrate. In contrast, when acetoin and diacetyl were present together, acetoin was the preferred substrate for both enzymes, with enzyme 1 showing the more marked preference for acetoin. meso-2,3-Butanediol was the only isomeric product, with enzyme 1 independent of the substrate combinations. For enzyme 2, both the meso and optical isomers of 2,3-butanediol were formed with acetoin as the substrate, but only the optical isomers were produced with diacetyl as the substrate. With batch cultures of strain D10 at or near the point of citrate exhaustion, the main isomers of 2,3-butanediol present were the optical forms. If the pH was sufficiently high (>pH 5), acetoin reduction occurred over time and was followed by diacetyl reduction, and meso-2,3-butanediol became the predominant isomer. Interconversion of the optical isomers into the meso isomer did occur. The properties of 2,3-butanediol dehydrogenases are consistent with diacetyl and acetoin removal and the appearance of the isomers of 2,3-butanediol.     

Cloning, expression and characterization of meso-2,3-butanediol dehydrogenase from Klebsiella pneumoniae

The budC gene encoding the meso-2,3-BDH from Klebsiella pneumoniae XJ-Li was expressed in E. coli BL21 (DE3) pLys. Hypothetical amino acid sequence alignments revealed that the enzyme belongs to the short chain dehydrogenase/reductase family. After purification and refolding, the recombinant enzyme had activities of 218 U/mg for reduction of acetoin and 66 U/mg for oxidation of meso-2,3-butanediol. Highest activities were at pH 8.0 and 9.0 respectively. These are higher than other meso-2,3-butanediol dehydrogenases from K. pneumoniae. The low K (m) value (0.65 mM) for acetoin indicated that the enzyme can easily reduce acetoin to meso-2,3-butanediol. There were no significant activities towards 2R,3R-2,3-butanediol, 1,4-butanediol and 2S,3S-2,3-butanediol, suggesting that the enzyme has a high stereospecificity for the meso-dihydric alcohol.     

Characterization and functional role of Saccharomyces cerevisiae 2,3-butanediol dehydrogenase

Using a conserved sequence motif, a new gene (YAL060W) of the MDR family has been identified in Saccharomyces cerevisiae. The expressed protein was a stereoespecific (2R,3R)-2,3-butanediol dehydrogenase (BDH). The best substrates were (2R,3R)-2,3-butanediol for the oxidation and (3R/3S)-acetoin and 1-hydroxy-2-propanone for the reduction reactions. The enzyme is extremely specific for NAD(H) as cofactor, probably because the presence of Glu223 in the cofactor binding site, instead of the highly conserved Asp223. BDH is inhibited competitively by 4-methylpyrazole with a K(i) of 34 microM. Yeast could grow on 2,3-butanediol or acetoin as a sole energy and carbon sources, and a 3.6-fold increase in BDH activity was observed when cells were grown in 2,3-butanediol, suggesting a role of the enzyme in 2,3-butanediol metabolism. However, the disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions, and the disrupted strain could also grow in 2,3-butanediol and acetoin. This suggests that other enzymes, in addition to BDH, can also metabolize 2,3-butanediol in yeast.     

Isolation of mutants of Alcaligenes eutrophus unable to derepress the fermentative alcohol dehydrogenase

Eight representative strains of Alcaligenes eutrophus, two strains of Alcaligenes hydrogenophilus and three strains of Paracoccus denitrificans were examined for their ability to use different alcohols and acetoin as a carbon source for growth. A. eutrophus strains N9A, H16 and derivative strains were unable to grow on ethanol or on 2,3-butanediol. Alcohol-utilizing mutants derived from these strains, isolated in this study, can be categorized into two major groups: Type I-mutants represented by strain AS1 occurred even spontaneously and were able to grow on 2,3-butanediol (t _d=2.7–6.4 h) and on ethanol (t _d=15–50 h). The fermentative alcohol dehydrogenase was present on all substrates tested, indicating that this enzyme in vivo is able to oxidize 2,3-butanediol to acetoin which is a good substrate for wild type strains. Type II-mutants represented by strain AS4 utilize ethanol as a carbon source for growth (t _d=3–9 h) but do not grow on butanediol. In these mutants the fermentative alcohol dehydrogenase is only present in cells cultivated under conditions of restricted oxygen supply, but a different NAD-dependent alcohol dehydrogenase is present in ethanol grown cells. Cells grown on ethanol, acetoin or 2,3-butanediol synthesized in addition two proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity and acetate thiokinase. An acylating acetaldehyde dehydrogenase (EC 1.2.1.10) was not detectable. Applying the colistin- and pin point-technique for mutant selection to strain AS1, mutants, which lack the fermentative alcohol dehydrogenase even if cultivated under conditions of restricted oxygen supply, were isolated; the growth pattern served as a readily identifiable phenotypic marker for the presence or absence of this enzyme.     

Role of Saccharomyces cerevisiae Oxidoreductases Bdh1p and Ara1p in the Metabolism of Acetoin and 2,3-Butanediol

NAD-dependent butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae reversibly transforms acetoin to 2,3-butanediol in a stereospecific manner. Deletion of BDH1 resulted in an accumulation of acetoin and a diminution of 2,3-butanediol in two S. cerevisiae strains under two different growth conditions. The concentrations of (2R,3R)-2,3-butanediol are mostly dependent on Bdh1p activity, while those of (meso)-2,3-butanediol are also influenced by the activity of NADP(H)-dependent oxidoreductases. One of them has been purified and shown to be d-arabinose dehydrogenase (Ara1p), which converts (R/S)-acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol. Deletion of BDH2, a gene adjacent to BDH1, whose encoded protein is 51% identical to Bdh1p, does not significantly alter the levels of acetoin or 2,3-butanediol in comparison to the wild-type strain. Furthermore, we have expressed Bdh2p with a histidine tag and have shown it to be inactive toward 2,3-butanediol. A whole-genome expression analysis with microarrays demonstrates that BDH1 and BDH2 are reciprocally regulated.Acetoin and 2,3-butanediol are minor products generated by Saccharomyces cerevisiae during alcohol fermentation. Their sensory impacts on wine are poorly documented. Acetoin may affect the wine bouquet, although its perception threshold in wine is relatively high, around 150 mg/liter (21, 31). On the other hand, 2,3-butanediol is odorless (33) and cannot be expected to appreciably affect the sensory quality of wine. However, the compound may contribute to the wine body (28).Acetaldehyde, pyruvate, and α-acetolactate are the main precursors of acetoin in S. cerevisiae. Acetoin can be formed from acetaldehyde and/or pyruvate through an anomalous reaction of pyruvate decarboxylase. Thus, although its main activity is to irreversibly decarboxylate pyruvate to acetaldehyde, it can also catalyze carbon-carbon bond formation, yielding acetoin from pyruvate and/or acetaldehyde (2, 4). In addition, α-acetolactate would produce acetoin through its nonenzymatic decarboxylation to diacetyl and subsequent reduction to acetoin through the action of several NADH- and NADPH-dependent oxidoreductases (12). However, the situation is more complex in wine fermentation, where other yeasts and bacteria display supplementary enzymatic activities capable of producing both acetoin and 2,3-butanediol (1, 27).We have previously characterized a butanediol dehydrogenase (Bdh1p) as a medium-chain dehydrogenase/reductase (MDR) that can reversibly transform R-acetoin and S-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol, respectively, in a NAD(H)-dependent reaction (10). BDH2 is a gene adjacent to BDH1 whose uncharacterized protein product (Bdh2p) shares 51% sequence identity with Bdh1p. To evaluate the in vivo roles of Bdh1p and Bdh2p, we compared the levels of several extracellular metabolites in cultures of wild-type and deficient strains. The results show that, although Bdh1p is the main enzyme in 2,3-butanediol production [essentially the (2R,3R)-2,3-butanediol stereoisomer], some meso-2,3-butanediol is still produced by the bdh1Δ strains. We have characterized Ara1p as an oxidoreductase that can reduce racemic acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol in the presence of NADPH.Furthermore, we have overexpressed Bdh2p with a histidine tag at its carboxyl terminus and have shown it to be inactive toward acetoin and 2,3-butanediol. A microarray study indicated that BDH1 and BDH2 are reciprocally regulated under the conditions studied.     

Enhanced production of 2,3-butanediol by engineered <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Production of 2,3-butanediol by Bacillus subtilis takes place in late-log or stationary phase, depending on the expression of bdhA gene encoding acetoin reductase, which converts acetoin to 2,3-butanediol. The present work focuses on the development of a strain of B. subtilis for enhanced production of 2,3-butanediol in early log phase of growth cycle. For this, the bdhA gene was expressed under the control of P _alsSD promoter of AlsSD operon for acetoin fermentation which served the substrate for 2,3-butanediol production. Addition of acetic acid in the medium induced the production of 2,3-butanediol by 2-fold. Two-step aerobic–anaerobic fermentation further enhanced 2,3-butanediol production by 4-fold in comparison to the control parental strain. Thus, addition of acetic acid and low dissolved oxygen in the medium are involved in activation of bdhA gene expression from P _alsSD promoter in early log phase. Under the conditions tested in this work, the maximum production of 2,3-butanediol, 2.1 g/l from 10 g/l glucose, was obtained at 24 h. Furthermore, under the optimized microaerophilic condition, the production of 2,3-butanediol improved up to 6.1 g/l and overall productivity increased by 6.7-fold to 0.4 g/l h in the engineered strain compared to that in the parental control.     

Biotechnological suitability of Saccharomycodes ludwigii for fermented beverages

Nineteen Saccharomycodes ludwigii strains were tested for the production of secondary products in grape must fermentation. A predominant metabolic pattern characterized by high production of isobutyl alcohol, acetoin and ethyl acetate was obtained. The occurrence of some strains producing enhanced amounts of these compounds suggests a potential utilization of this species for industrial applications. Feijoa juice was inoculated with a selected S'codes ludwigii strain in comparison to a control strain of S. cerevisiae and evaluation of the fermented products was carried out by 30 consumers with respect to the odour, flavour and taste. The sample fermented by S'codes ludwigii was characterized by a fresh odour with a fruity flavour, identified as flavour of apple and kiwi-fruit. This product was compared to apple juice, with a more acid taste. Despite the high concentrations of acetic acid, this beverage might be considered a potential summer refreshing drink, addressed to a target of consumers who prefer fruit drinks that leave a slightly acid and little sugary taste in the mouth.     

Exocellular products from Bifidobacterium adolescentis as immunomodifiers in the lymphoproliferative responses of mouse splenocytes

Abstract Pelobacter carbinolicus strain GraBd1 fermented methylacetoin, which is a good carbon source for growth ( μ = 0.16 h⁻¹) of this strict anaerobic bacterium, to acetone, acetate and ethanol (main products), acetoin, 2,3-butanediol and methylbutanediol (minor products). During growth on 2,3-butanediol, acetoin and methyl-acetoin the formation of a protein exhibiting acetoin: DCPIP oxidoreductase activity is induced. This enzyme amounts to a substantial portion of the soluble proteins. In vitro, it cleaves acetoin into acetate and acetaldehyde but reacts also with diacetyl or methylacetoin. We discussed four different models for the degradation of acetoin in the cells and came to the conclusion that in vivo the oxidative-thiolytic acetoin cleavage model is most probably realized in P. carbinolicus .     

Evaluation of selective media for enumeration of yeasts during wine fermentation

The growth of individual species of yeasts during wine fermentations was measured by plating wine samples on malt extract, ethanol sulphite and lysine agars. Colonies of Saccharomyces cerevisiae dominated on plates of malt extract agar and sometimes masked the presence of other non- Saccharomyces species. Lysine agar suppressed the growth of S. cerevisiae and enabled the enumeration of non- Saccharomyces species such as Kloeckera apiculata, Candida stellata and Saccharomycodes ludwigii. The growth of non- Saccharomyces yeasts on ethanol sulphite agar was variable.     

Effect of deletion of 2,3-butanediol dehydrogenase gene (bdhA) on acetoin production of Bacillus subtilis

The present work aims to block 2,3-butanediol synthesis in acetoin fermentation of Bacillus subtilis. First, we constructed a recombinant strain BS168D by deleting the 2,3-butanediol dehydrogenase gene bdhA of the B. subtilis168, and there was almost no 2,3-butanediol production in 20?g/L of glucose media. The acetoin yield of BS168D reached 6.61?g/L, which was about 1.5 times higher than that of the control B. subtilis168 (4.47?g/L). Then, when the glucose concentration was increased to 100?g/L, the acetoin yield reached 24.6?g/L, but 2.4?g/L of 2,3-butanediol was detected at the end of fermentation. The analysis of 2,3-butanediol chiral structure indicated that the main 2,3-butanediol production of BS168D was meso-2,3-butanediol, and the bdhA gene was only responsible for (2R,3R)-2,3-butanediol synthesis. Therefore, we speculated that there may exit another pathway relating to the meso-2,3-butanediol synthesis in the B. subtilis. In addition, the results of low oxygen condition fermentation showed that deletion of bdhA gene successfully blocked the reversible transformation between acetoin and 2,3-butanediol and eliminated the effect of dissolved oxygen on the transformation.     

Isolation and identification of an acetoin high production bacterium that can reverse transform 2,3-butanediol to acetoin at the decline phase of fermentation

Acetoin (3-hydroxy-2-butanone), a very popular food spice is now used in many industries (pharmaceuticals, chemicals, paint, etc.). In this study, an acetoin high producing strain, numbered as JNA-310, was newly isolated and identified as Bacillus subtilis which is safe on food industry, based on its physiological, biological tests and 16S rDNA sequence analysis. When glucose was used as carbon source in fermentation, the fermentation characterizations of this strain were analyzed, and a new phenomenon of reverse transforming 2,3-butanediol which was synthesized from glucose in the fermentation broth to acetoin was detected. Before 96 h, glucose which was mainly transformed to 2,3-butanediol and acetoin was totally consumed, and the yield of the two products were 41.7 and 21.0 g/l respectively. Acetoin was only a by product in the fermentation broth at prophase of fermentation. At the end of fermentation, the yield of acetoin was greatly improved and the yield of 2,3-butanediol was declined and the yield of them were about 42.2 and 15.8 g/l, respectively. The results indicated that 2,3-butanediol was reversely transformed to acetoin.     

D-2,3-butanediol production due to heterologous expression of an acetoin reductase in Clostridium acetobutylicum

Acetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions, Clostridium acetobutylicum ATCC 824 (and strains derived from it) generates both d- and l-stereoisomers of acetoin, but because of the absence of an ACR enzyme, it does not produce 2,3-butanediol. A gene encoding ACR from Clostridium beijerinckii NCIMB 8052 was functionally expressed in C. acetobutylicum under the control of two strong promoters, the constitutive thl promoter and the late exponential adc promoter. Both ACR-overproducing strains were grown in batch cultures, during which 89 to 90% of the natively produced acetoin was converted to 20 to 22 mM d-2,3-butanediol. The addition of a racemic mixture of acetoin led to the production of both d-2,3-butanediol and meso-2,3-butanediol. A metabolic network that is in agreement with the experimental data is proposed. Native 2,3-butanediol production is a first step toward a potential homofermentative 2-butanol-producing strain of C. acetobutylicum.     

Cloning of the Alcaligenes eutrophus alcohol dehydrogenase gene

Mutants of Alcaligenes eutrophus which are altered with respect to the utilization of 2,3-butanediol and acetoin were isolated after transposon mutagenesis. The suicide vehicle pSUP5011 was used to introduce the drug resistance transposable element Tn5 into A. eutrophus. Kanamycin-resistant transconjugants of the 2,3-butanediol-utilizing parent strains CF10141 and AS141 were screened for mutants impaired in the utilization of 2,3-butanediol or acetoin. Eleven mutants were negative for 2,3-butanediol but positive for acetoin; they were unable to synthesize active fermentative alcohol dehydrogenase protein (class 1). Forty mutants were negative for 2,3-butanediol and for acetoin (class 2). Tn5-mob was also introduced into a Smr derivative of the 2,3-butanediol-nonutilizing parent strain H16. Of about 35,000 transconjugants, 2 were able to grow on 2,3-butanediol. Both mutants synthesized the fermentative alcohol dehydrogenase constitutively (class 3). The Tn5-labeled EcoRI fragments of genomic DNA of four class 1 and two class 3 mutants were cloned from a cosmid library. They were biotinylated and used as probes for the detection of the corresponding wild-type fragments in a lambda L47 and a cosmid gene bank. The gene which encodes the fermentative alcohol dehydrogenase in A. eutrophus was cloned and localized to a 2.5-kilobase (kb) SalI fragment which is located within a 11.5-kb EcoRI-fragment. The gene was heterologously expressed in A. eutrophus JMP222 and in Pseudomonas oxalaticus. The insertion of Tn5-mob in class 3 mutants mapped near the structural gene for alcohol dehydrogenase on the same 2.5-kb SalI fragment.     

Improved Production of 2,3-Butanediol in Bacillus amyloliquefaciens by Over-Expression of Glyceraldehyde-3-Phosphate Dehydrogenase and 2,3-butanediol Dehydrogenase

Background

Previously, a safe strain, Bacillus amyloliquefaciens B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, B. amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production.

Methodology/Principal Findings

In the 2,3-butanediol metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of 3-phosphate glyceraldehyde to 1,3-bisphosphoglycerate, with concomitant reduction of NAD⁺ to NADH. In the same pathway, 2,3-BD dehydrogenase (BDH) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD⁺. In this study, to improve 2,3-BD production, we first over-produced NAD⁺-dependent GAPDH and NADH-dependent BDH in B. amyloliquefaciens. Excess GAPDH reduced the fermentation time, increased the 2,3-BD yield by 12.7%, and decreased the acetoin titer by 44.3%. However, the process also enhanced lactate and succinate production. Excess BDH increased the 2,3-BD yield by 16.6% while decreasing acetoin, lactate and succinate production, but prolonged the fermentation time. When BDH and GAPDH were co-overproduced in B. amyloliquefaciens, the fermentation time was reduced. Furthermore, in the NADH-dependent pathways, the molar yield of 2,3-BD was increased by 22.7%, while those of acetoin, lactate and succinate were reduced by 80.8%, 33.3% and 39.5%, relative to the parent strain. In fed-batch fermentations, the 2,3-BD concentration was maximized at 132.9 g/l after 45 h, with a productivity of 2.95 g/l·h.

Conclusions/Significance

Co-overexpression of bdh and gapA genes proved an effective method for enhancing 2,3-BD production and inhibiting the accumulation of unwanted by-products (acetoin, lactate and succinate). To our knowledge, we have attained the highest 2,3-BD fermentation yield thus far reported for safe microorganisms.     

Mechanism for the formation of 2,3-butanediol stereoisomers in Bacillus polymyxa

The mechanism of the formation of 2,3-butanediol isomers in Bacillus polymyxa was studied. We proposed a new model with NADPH-linked diacetyl reductase (S-acetoin forming) and R(−)-2,3-butanediol dehydrogenase. The two enzymes were separated by Blue Sepharose CL-6B and their stereospecificities were identified using all of the pure isomers of 2,3-butanediol (R(−), S(+)m, and meso), acetoin (R(−) and S(+)) and the separation and measurement of these isomers. The presence of acetoin or butanediol racemase was not confirmed in our experiments.     

Molecular characterization of the Pseudomonas putida 2,3-butanediol catabolic pathway

Abstract The 2,3-butanediol dehydrogenase and the acetoin-cleaving system were simultaneously induced in Pseudomonas putida PpG2 during growth on 2,3-butanediol and on acetoin. Hybridization with a DNA probe covering the genes for the E1 subunits of the Alcaligenes eutrophus acetoin cleaving system and nucleotide sequence analysis identified acoA (975 bp), acoB (1020 bp), acoC (1110 bp), acoX (1053 bp) and adh (1086 bp) in a 6.3-kb genomic region. The amino acid sequences deduced from acoA , acoB , and acoC for E1α ( M _r 34639), E1β ( M _r 37268), and E2 ( M _r 39613) of the P. putida acetoin cleaving system exhibited striking similarities to those of the corresponding components of the A. eutrophus acetoin cleaving system and of the acetoin dehydrogenase enzyme system of Pelobacter carbinolicus and other bacteria. Strong sequence similarities of the adh translational product (2,3-butanediol dehydrogenase, M _r 38361) were obtained to various alcohol dehydrogenases belonging to the zinc- and NAD(P)-dependent long-chain (group I) alcohol dehydrogenases. Expression of the P. putida ADH in Escherichia coli was demonstrated. The aco genes and adh constitute presumably one single operon which encodes all enzymes required for the conversion of 2,3-butanediol to central metabolites.     

Bacterial 2,3-butanediol dehydrogenases

Enterobacter aerogenes, Aeromonas hydrophila, Serratia marcescens and Staphylococcus aureus possessing L(+)-butanediol dehydrogenase produced mainly meso-butanediol and small amounts of optically active butanediol; Acetobacter suboxydans, Bacillus polymyxa and Erwinia carotovora containing D(-)-butanediol dehydrogenase produced more optically active butanediol than meso-butanediol. Resting and growing cells of these organisms oxidized only one enantiomer of racemic butanediol. The D(-)-butanediol dehydrogenase from Bacillus polymyxa was partially purified (30-fold) with a specific activity of 24.5. Except NAD and NADH no other cofactors were required. Optimum pH-values for oxidation and reduction were pH 9 and pH 7, respectively. The optimum temperature was about 60°C. The molecular weight was 100000 to 107000. The K _m-values were 3.3 mM for D(-)-butanediol, 6.25 mM for meso-butanediol, 0.53 mM for acetoin, 0.2 mM for NAD, 0.1 mM for NADH, 87 mM for diacetyl, 38 mM for 1,2-propanediol; 2,3-pentanedion was not a substrate for this enzyme. The L(+)-butanediol dehydrogenase from Serratia marcescens was purified 57-fold (specific activity 22.3). Besides NAD or NADH no cofactors were required. The optimum value for oxidation was about pH 9 and for reduction pH 4.5. The optimum temperature was 32–36°C. The molecular weight was 100000 to 107000. The K _m-values were 5 mM for meso-butanediol, 10 mM for racemic butanediol, 6.45 for acetoin, 1 mM for NAD, 0.25 mM for NADH, 2.08 mM for diacetyl, 16.7 mM for 2,3-pentanedion and 11.8 mM for 1,2-propanediol.Abbreviations Bud 2,3-butanediol - DH dehydrogenase     

关于四个酵母菌种对乙醇耐受性的研究

本文报道了从存活,生长,发酵能力和氢离子渗漏等角度研究和比较四种酵母菌(Zygosaccharomyces bailii NCYC 1427,Saccharomyces cerevisiae NCYC 431,Harisenula anomala NCYC 711和 Kloeckera apiculata NCYC 468)对乙醇的耐受性。乙醇对酵母在上述特性的抑制程度,取决于乙醇的浓度。Z．Bailii NCYC 1427是四个酵母中乙醇耐受性最强的。在2M的乙醇溶液中,其存活率与无乙醇的对照组一样高。在低于1.5M的乙醇中,其生长率高于 S．Cerevisiae NCYC 431,氢离子渗入细胞的速率增长则低于 S．Cerevisiae 和 H．Anomala。S．Cerevisiae NCYC 431对于乙醇抑制生长的效应耐受性很强,在低于IM的乙醇溶液中,其发酵能力下降得较为缓慢,存活率几乎没有变化。H．Anomala NCYC 711在生长、发酵能力和氢离子渗漏方面对乙醇的抑制作用均表现敏感,但其存活率变化却与S．Cerevisiae NCYC 431很相似。K．Apiculata NCYC 468在存活、生长和发酵能力方面对乙醇的效应是四个菌种中最敏感的。研究结果还表明,乙醇对生长、发酵能力和氢离子渗漏的作用比它对存活的影响大得多。环境条件也影响酵母对乙醇的耐受性。     

Changes in gluconic acid, polyols and major volatile compounds in sherry wine during aging with submerged flor yeast cultures

The traditional biological process by which sherry wines are aged can be accelerated by using submerged Saccharomyces cerevisiae var. capensis (G1) strain cultures previously grown in glycerol. The used controlled shaking conditions raise the acetaldehyde, acetoin, and meso 2,3-butanediol contents in the wine, and increases the consumption of gluconic acid by flor yeast relative to traditional biological aging under flor yeast velum.