Metabolic responses of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 upon transition from glucose limitation to glucose excess

When chemostat cultures of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621, grown under glucose limitation, were pulsed with excess glucose, both organisms initially exhibited similar rates of glucose and oxygen consumption. However, striking differences were apparent between the two yeasts with respect to the production of cell mass in the culture and metabolite excretion. Upon transition from glucose limitation to glucose excess, S. cerevisiae produced much ethanol but the growth rate remained close to that under glucose limitation. C. utilis, on the other hand, produced little ethanol and immediately started to accumulate cell mass at a high rate. This high production rate of cell mass was probably due to synthesis of reserve material and not caused by a high rate of protein synthesis. Upon a glucose pulse both yeasts excreted pyruvate. In contrast to C. utilis, S. cerevisiae also excreted various tricarboxylic acid cycle intermediates, both under steady-state conditions and after exposure to glucose excess. These results and those of theoretical calculations on ATP flows support the hypothesis that the ethanol production as a consequence of pyruvate accumulation in S. cerevisiae, occurring upon transition from glucose limitation to glucose excess, is caused by a limited capacity of assimilatory pathways.     

Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation.

Addition of benzoate to the medium reservoir of glucose-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 growing at a dilution rate (D) of 0.10 h-1 resulted in a decrease in the biomass yield, and an increase in the specific oxygen uptake rate (qO2) from 2.5 to as high as 19.5 mmol g-1 h-1. Above a critical concentration, the presence of benzoate led to alcoholic fermentation and a reduction in qO2 to 13 mmol g-1 h-1. The stimulatory effect of benzoate on respiration was dependent on the dilution rate: at high dilution rates respiration was not enhanced by benzoate. Cells could only gradually adapt to growth in the presence of benzoate: a pulse of benzoate given directly to the culture resulted in wash-out. As the presence of benzoate in cultures growing at low dilution rates resulted in large changes in the catabolic glucose flux, it was of interest to study the effect of benzoate on the residual glucose concentration in the fermenter as well as on the level of some selected enzymes. At D = 0.10 h-1, the residual glucose concentration increased proportionally with increasing benzoate concentration. This suggests that modulation of the glucose flux mainly occurs via a change in the extracellular glucose concentration rather than by synthesis of an additional amount of carriers. Also various intracellular enzyme levels were not positively correlated with the rate of respiration. A notable exception was citrate synthase: its level increased with increasing respiration rate. Growth of S. cerevisiae in ethanol-limited cultures in the presence of benzoate also led to very high qO2 levels of 19-21 mmol g-1 h-1. During growth on glucose as well as on ethanol, the presence of benzoate coincided with an increase in the mitochondrial volume up to one quarter of the total cellular volume. Also with the Crabtree-negative yeasts Candida utilis, Kluyveromyces marxianus and Hansenula polymorpha, growth in the presence of benzoate resulted in an increase in qO2 and, at high concentrations of benzoate, in aerobic fermentation. In contrast to S. cerevisiae, the highest qO2 of these yeasts when growing at D = 0.10 h-1 in the presence of benzoate was equal to, or lower than the qO2 attainable at mu(max) without benzoate. Enzyme activities that were repressed by glucose in S. cerevisiae also declined in K. marxianus when the glucose flux was increased by the presence of benzoate.(ABSTRACT TRUNCATED AT 400 WORDS)     

不同食品酵母对葡萄糖的流加强度和热激压力的生理响应

微生物在发酵过程中往往会面临一系列诸如高温、高渗透压、乙醇等压力。该研究探究了4种食品酵母对葡萄糖的流加强度和热激压力的生理响应差异性。结果表明,Pichia kudriavzevii和Saccharomyces cerevisiae的生长几乎没有受到升温压力的影响。高速流加的葡萄糖和梯度升温抑制了Cyberlindnera fabianii的还原糖消耗,而对其他酵母没有影响。P.kudriavzevii和S.cerevisiae的胞内海藻糖表现为对葡萄糖的流加和低压力热激(35、37℃)高度敏感,C.fabianii的胞内海藻糖均表现出低敏感性,而Candida tropicalis的胞内海藻糖仅在严重压力下(>39℃)才开始积累。在所有酵母中,P.kudriavzevii的海藻糖含量在发酵调控后变化最显著,增长了3.7倍。此外,除C.fabianii外,高速流加的葡萄糖有利于P.kudriavzevii、S.cerevisiae和C.tropicalis乙醇的积累,特别是P.kudriavzevii的乙醇含量最高,达到48 g/L。该研究结果可以用于高耐受性菌株的筛选,为生物乙醇和酿造食品的应用提供指导。     

3株酿酒酵母发酵过程中有机酸含量变化分析

通过高效液相色谱法对3株酿酒酵母(Saccharomyces cerevisiae)发酵过程中葡萄糖、甘油、乙醇及有机酸含量进行测定分析。结果表明:S.cerevisiae1946发酵体系中甘油和乙醇的含量显著高于S cerevisiae P1和S.cerevisiae 32788发酵体系(P0.05);3株酿酒酵母发酵液中7种有机酸存在明显的差异性和规律性;S.cerevisiae 1946发酵体系中有机酸的含量低于S.cerevisiae P1和S.cerevisiae 32788发酵体系,S cerevisiae的产酸水平与产乙醇能力呈负相关关系。     

外源乙醇对果糖与葡萄糖酒精发酵的影响

研究了不同体积分数外源乙醇对酵母GJ2008利用等量果糖与葡萄糖混合发酵过程的影响,旨在为高浓度甘蔗汁酒精发酵提供参考和依据。用YPDF培养基模拟甘蔗汁,并调节乙醇体积分数为0%、6%、9%和12%,以初始酵母数为1.12×108个/mL进行酒精发酵,高效液相色谱法测定发酵过程中果糖与葡萄糖含量,并采用曲线下面积法对果糖与葡萄糖代谢曲线进行分析。在外源乙醇体积分数为0%~9%时,酵母GJ2008可以快速利用果糖和葡萄糖进行酒精发酵,但对葡萄糖有着明显的偏好;在外源乙醇体积分数为12%时,果糖和葡萄糖消耗缓慢,生物量得不到有效增加。等质量浓度果糖与葡萄糖混合发酵表明:外源乙醇对果糖代谢的影响明显大于对葡萄糖代谢的影响,外源乙醇体积分数达6%以上时,糖转化乙醇的产量减少了19%~27%。     

酿酒酵母QY-1发酵过程中有机酸及游离氨基酸变化分析

该研究对酿酒酵母（Saccharomyces cerevisiae）QY-1发酵过程中的OD600 nm值、pH值、葡萄糖消耗量、乙醇生成量、有机酸及游离氨基酸种类及生成量进行监控和分析。结果表明,随着发酵时间的延长,发酵液的pH值与OD600 nm值呈负向耦合;乙醇的生成量与葡萄糖消耗量呈负向耦合,发酵36 h时,乙醇含量最高,为（26.87±2.76） g/L;有机酸含量呈现先升高后稳定的趋势,最高达（3.242±0.213） g/L,其中乙酸含量最高;游离氨基酸含量呈现先升高后降低的趋势,最高达（5.57±0.08） mg/L,其中谷氨酸含量最高。S. cerevisiae QY-1具有较强的产酸和氨基酸能力。     

Fermentative stress adaptation of hybrids within the Saccharomyces sensu stricto complex

Along the fermentation process yeasts are affected by a succession of stress conditions that affect their viability and fermentation efficiency. Among the stress conditions the most relevant are high sugar concentration and low pH in musts, temperature and, as fermentation progresses, ethanol accumulation. Nowadays, due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing search for particular wine yeast strains possessing a wide range of optimized, improved or novel enological characteristics. Traditionally, the species S. cerevisiae and S. bayanus within the Saccharomyces sensu stricto species are considered some of the most important yeast species involved in fermentation processes. However, in the last years, hybrid strains between the species S. cerevisiae, S. bayanus and S. kudriavzevii have been described as yeasts conducting the alcoholic fermentations and some of them are commercially available. Our results indicate that yeasts in the Saccharomyces sensu stricto complex were not affected by low pH or high glucose content in the media; however temperature and ethanol concentration variables appreciably affected their growth. The strains pertaining to S. cerevisiae were able to tolerate high temperature stress, whereas strains within S. bayanus and S. kudriavzevii were better adapted to growth at lower temperatures. Regarding to alcohol tolerance, S. cerevisiae is tolerating alcohol better than S. bayanus or S. kudriavzevii. Surprisingly, the natural hybrids between these species have adapted to growth under ethanol and temperature stress by inheriting competitive traits from one or another parental species. These results open new perspectives in the construction of new hybrid strains with biotechnological interest, as the characteristics of the parents may result in interesting combinations in the hybrids.     

发酵条件对一步法发酵菊芋生产乙醇的影响

使用产菊粉酶的酿酒酵母可实现一步法发酵菊芋生产乙醇.该试验以菊芋汁为发酵培养基,研究了起始糖浓度、无机盐、温度、种子培养基、接种量和氮源对高菊粉酶活酿酒酵母Y05-步发酵菊粉生成乙醇的影响.试验结果表明:(1)菊芋汁中最适起始总糖浓度为250g/L.菊芋汁含有的较多无机盐对乙醇发酵不但没有抑制作用,反而有促进作用;(2)最适乙醇发酵温度为37℃;(3)种子培养基中使用菊粉做为碳源有利于酿酒酵母Y05产生较多菊粉酶,进而促进其乙醇发酵;(4)最适乙醇发酵接种量为15％;(5)尽管菊芋汁是一个良好乙醇发酵培养基,但补加氮源仍是必要的.补加5.0g/L玉米浆可显著酿酒酵母Y05的提高总糖利用率、最终乙醇浓度和乙醇得率.     

Sugar consumption and ethanol fermentation by transporter-overexpressed xylose-metabolizing Saccharomyces cerevisiae harboring a xyloseisomerase pathway

Four kinds of transporters, HXT1 and HXY7 from Saccharomyces cerevisiae, and GXF1 and GXS1 from Candida intermedia, were overexpressed in xylose-metabolizing S. cerevisiae harboring a xyloseisomerase-based pathway. Overexpression of transporter enhanced sugar consumption and ethanol production, and GXF1 was efficient for ethanol fermentation from both glucose and xylose.     

3株哈密瓜酒酵母的分子生物学鉴定

通过对从哈密瓜酒生产中分离获得的3株哈密瓜酒酵母进行发酵性能分析,发现其中1株发酵异常.采用26S rDNA D1/D2区序列分析法对酵母进行分子鉴定,经序列比对及构建系统发育树分析,得出1号和3号酵母为酿酒酵母,与酿酒酵母模式菌株序列相似性在99％以上;2号酵母鉴定为高里毕赤酵母,与高里毕赤酵母(Pichia guilliermondii)相似性为99％.进一步对3株酵母的同源序列与其他种之间的发育关系进行了分析,发现有很好的相似度,表明26S rDNA D1/D2区域序列分析方法能够应用于酿酒酵母的分子生物学鉴定,并可以作为生产中酵母污染检测的工具.     

Effects of salts on Debaryomyces hansenii and Saccharomyces cerevisiae under stress conditions

The effect of Na+ and K+ on growth and thermal death of Debaryomyces hansenii and Saccharomyces cerevisiae were compared under stress conditions as those commonly found in food environments. At the supraoptimal temperature of 34 degrees C both cations at concentrations of 0.5 M stimulated growth of D. hansenii, while K+ had no effect and Na+ inhibited growth of S. cerevisiae. At 8 degrees C, close to the minimum temperature for growth in both species, both cations inhibited both yeasts, this effect being more pronounced with Na+ in S. cerevisiae. At extreme pH values (7.8 and 3.5) both cations at concentrations of 0.25 M stimulated D. hansenii while Na+ inhibited S. cerevisiae. K+ inhibited this yeast at pH 3.5. Thermal inactivation rates, measured at 38 degrees C in D. hansenii and at 48 degrees C in S. cerevisiae, decreased in the presence of both cations. This protective effect could be observed in a wider range of concentrations in D. hansenii. These results call the attention to the fact that not all yeasts have the same behaviour on what concerns synergy or antagonism of salt together with other stress factors and should be taken into consideration in the establishment of food preservation procedures.     

The co-action of osmotic and high temperature stresses results in a growth improvement of Debaryomyces hansenii cells

Debaryomyces hansenii is a salt tolerant yeast species, often isolated from sea water or found among other spoilage yeasts in several types of food. In this work, we examined the influence of temperature and increased osmotic pressure (two parameters also important in food industry) on D. hansenii growth. Several other authors showed that its growth at the normal yeast cultivation temperature (28 to 30 degrees C) is stimulated by the presence of sodium, in contrast to the growth of Saccharomyces cerevisiae, which is inhibited by the presence of sodium under the same experimental conditions. Here we show that the previously reported growth stimulation by sodium is temperature dependent in D. hansenii and can be observed under conditions that already amount to high temperature stress for D. hansenii. At a lower temperature (more convenient for D. hansenii cultivation), we found no significant improvement or even an inhibition of cell growth in the presence of Na(+). The growth of D. hansenii at high temperatures is also improved by the presence of potassium or sorbitol. Moreover, the temperature dependence of stimulatory effects of increased osmotic pressure in media does not seem to be unique for D. hansenii; similar relationships between the growth, cultivation temperature and presence of osmolytes we also observed for S. cerevisiae and Schizosaccharomyces pombe.     

Significance of yeasts in the fermentation of maize for ogi production

Saccharomyces cerevisiae, Candida krusei, C. tropicalis, Geotrichum candidum, G. fermentans and Rhodotorula graminis were isolated during the fermentation of maize for ogi production. All the isolates except Geotrichum fermentans and Rhodotorula graminis were able to degrade phytate. All the yeasts strains exhibited lipase and esterase activities. Only S. cerevisiae (2.60%) and C. krusei (7.41%) exhibited amylase activities. Candida sp. produced wider zone of inhibition than the other yeasts strains tested during lipase activity while S. cerevisiae strains produced significantly wider zone of clearing as compared to the other yeasts for esterase activities. The study of inter-relationships between Lactobacillus plantarum and yeasts (C. krusei and S. cerevisiae) showed that the growth of the yeast strains were enhanced during fermentation by the presence of the lactic acid bacteria, but the growth of the L. plantarum strain was significantly enhanced especially by the C. krusei.     

Ethanol yield and volatile compound content in fermentation of agave must by Kluyveromyces marxianus UMPe-1 comparing with Saccharomyces cerevisiae baker's yeast used in tequila production

In tequila production, fermentation is an important step. Fermentation determines the ethanol productivity and organoleptic properties of the beverage. In this study, a yeast isolated from native residual agave must was identified as Kluyveromyces marxianus UMPe-1 by 26S rRNA sequencing. This yeast was compared with the baker's yeast Saccharomyces cerevisiae Pan1. Our findings demonstrate that the UMPe-1 yeast was able to support the sugar content of agave must and glucose up to 22% (w/v) and tolerated 10% (v/v) ethanol concentration in the medium with 50% cells survival. Pilot and industrial fermentation of agave must tests showed that the K. marxianus UMPe-1 yeast produced ethanol with yields of 94% and 96% with respect to fermentable sugar content (glucose and fructose, constituting 98%). The S. cerevisiae Pan1 baker's yeast, however, which is commonly used in some tequila factories, showed 76% and 70% yield. At the industrial level, UMPe-1 yeast shows a maximum velocity of fermentable sugar consumption of 2.27g·L(-1)·h(-1) and ethanol production of 1.38g·L(-1)·h(-1), providing 58.78g ethanol·L(-1) at 72h fermentation, which corresponds to 96% yield. In addition, the major and minor volatile compounds in the tequila beverage obtained from UMPe-1 yeast were increased. Importantly, 29 volatile compounds were identified, while the beverage obtained from Pan1-yeast contained fewer compounds and in lower concentrations. The results suggest that the K. marxianus UMPe-1 is a suitable yeast for agave must fermentation, showing high ethanol productivity and increased volatile compound content comparing with a S. cerevisiae baker's yeast used in tequila production.     

Development of culture media containing spent yeast cells of Debaryomyces hansenii and corn steep liquor for lactic acid production with Lactobacillus rhamnosus

To develop a cost-effective fermentation medium, biomass of Debaryomyces hansenii coming from xylitol production and corn steep liquor was evaluated for lactic acid production by Lactobacillus rhamnosus in glucose-containing media. Glucose consumption and lactic acid generation were followed using a variety of media made with different nutrient supplementation. Under selected conditions, media containing glucose, biomass of D. hansenii and corn steep liquor as unique components led to product yields similar to those obtained in a fully supplemented medium.     

Headspace flavour compounds produced by yeasts and lactobacilli during fermentation of preferments and bread doughs.

Production of volatile flavour compounds during fermentation with pure cultures of Saccharomyces cerevisiae and Candida guilliermondii, Lactobacillus brevis and Lactobacillus plantarum have been investigated, using wheat doughs and several preferements as substrates. For yeast, preferments consisted of 10% (w/v) glucose, maltose and sucrose solutions, whereas for lactobacilli they consisted of supplemented and unsupplemented (3% and 10% (w/v)) glucose solutions, and a 10% (w/v) wheat flour slurry. Seven volatile compounds (acetaldehyde, acetone, ethyl acetate, ethanol, hexanal+isobutyl alcohol, and propanol) were detected when using yeasts. All these compounds, except propanol, appeared for all the substrates assayed, with ethanol as the predominant component. Generally, S. cerevisiae produced higher amounts of the different components than C. guilliermondii. Both yeasts produced larger amounts of volatile flavour compounds during fermentation in glucose and sucrose solutions than in maltose or wheat dough. In general the yeasts examined produced more flavour components than the lactobacilli. For the lactobacilli the highest number of volatile flavour compounds were observed for substrates containing flour.     

Functionality of selected strains of moulds and yeasts from Vietnamese rice wine starters

The role of starch-degrading mycelial fungi, and the alcohol production and ethanol tolerance of the yeasts isolated from selected Vietnamese traditional rice wine starters were examined, and optimum conditions for these essential steps in rice wine fermentation were determined. Of pure isolates from Vietnamese rice wine starters, mould strains identified as Amylomyces rouxii, Amylomyces aff. rouxii, Rhizopus oligosporus and Rhizopus oryzae, were superior in starch degradation, glucose production and amyloglucosidase activity during the saccharification of purple glutinous rice. A. rouxii was able to produce up to 25%w/w glucose with an amyloglucosidase activity up to 0.6 Ug(-1) of fermented moulded mass. Five yeast isolates identified as Saccharomyces cerevisiae were selected for their superior alcohol productivity. They were able to deplete a relatively high initial percentage of glucose (20% w/v), forming 8.8% w/v ethanol. The ethanol tolerance of S. cerevisiae in challenge tests was 9-10% w/v, and 13.4% w/v as measured in fed-batch fermentations. Optimum conditions for the saccharification were: incubation for 2 d at 34 degrees C, of steamed rice inoculated with 5 log cfu g(-1); for the alcoholic fermentation 4 d at 28.3 degrees C, of saccharified rice liquid inoculated with 5.5 log cfu mL(-1).     

Identification and characterization of Saccharomyces cerevisiae strains isolated from West African sorghum beer

The occurrence and characterization of yeasts isolated from sorghum beer produced in Ghana and Burkina Faso, West Africa, were investigated. The yeasts involved in the fermentations were found to consist of Saccharomyces spp. almost exclusively. Of the isolates investigated, 45% were identified as Saccharomyces cerevisiae, whereas more than half of the isolates (53%) had physiological properties atypical of S. cerevisiae or any other member of the complex sensu strictu, as they were able to assimilate only glucose, maltose and ethanol as carbon sources. Both ITS-PCR RFLP and PFGE strongly indicated that these isolates were related to S. cerevisiae, regardless of their phenotypic characteristics. Sequencing of the D1/D2 domain of the 26S rDNA confirmed the close relatedness to S. cerevisiae with 0.5% nucleotide differences. The MAL1 and MAL3 loci were found for all isolates as the only recognized MAL loci. Besides, for 40% of the isolates the MAL61 probe hybridized to a position of about 950 kbp, which has not formerly been described as a MAL locus. The results showed that the spontaneous fermentation of West African sorghum beer is dominated by a variety of strains of S.cerevisiae not previously described, among which starter cultures should be selected.     

糖浓度对酒精酵母GJ2008果糖与葡萄糖利用差异性的影响

在不同糖浓度条件下,以YPDF培养基模拟甘蔗汁进行酒精发酵,测定过程中各参数的变化,并对果糖与葡萄糖消耗过程进行曲线拟合,以拟合方程计算出果糖与葡萄糖代谢一半和代谢完全所需时间.结果表明,糖浓度为90gL～270g/L,酵母GJ2008始终会偏用葡萄糖,果糖利用一直受到葡萄糖的竞争性抑制.糖浓度为90g/L时,细胞生长受糖浓度抑制程度最小,但乙醇产率较低;糖浓度为230g/L,发酵液中葡萄糖含量较低时,果糖受葡萄糖的竞争性抑制得到了解除,果糖的利用急剧加快;糖浓度为250g/L和270g/L,发酵液中葡萄糖含量较低时,果糖受葡萄糖的竞争性抑制得到了解除,但果糖的利用并没有加快,表现为后期酵母数维持值较低.糖浓度对果糖代谢的影响要大于对葡萄糖代谢的影响,较低糖浓度有利于后期果糖与葡萄糖利用差异性的缩小.     

Characterization of the β-Glucosidase Activity Produced by Enological Strains of Non-Saccharomyces Yeasts

ABSTRACT: The β-glucosidase activities of 20 wine-related non- Saccharomyces yeasts were quantified, characterized, and assessed for their efficiency in releasing aroma-enhancing compounds during the winemaking process. Of these enzymatic activities, the β-glucosidase activity of Debaryomyces pseudopolymorphus revealed the most suitable combination of properties in terms of functionality at wine pH, resistance to wine-associated inhibitory compounds (glucose, ethanol, and sulfur dioxide), high substrate affinity, and large aglycone-substrate recognition. Its potential as a wine aroma-enhancing enzyme was confirmed by the significantly increasing concentrations of free volatiles (citronellol, nerol, and geraniol) during the fermentation of Chardonnay juice inoculated with both D. pseudopolymorphus and a widely used commercial starter culture strain of Saccharomyces cerevisiae , VIN13.