纤维乙醇用酵母菌的驯化

酵母菌菌种性能的优劣直接关系纤维乙醇发酵质量的好坏及设备的利用率等,进而影响企业的经济效益.该文通过对纤维乙醇生产用酵母菌株的驯化,获得一株驯化酵母菌株:并将该菌株与1308菌株、1300菌株和安琪酵母发酵、耐乙酸、耐糠醛、耐羟甲基糠醛能力进行了对比试验,结果表明该驯化酵母菌株发酵能力、耐受性均优于其他供试菌株;更适宜于在纤维乙醇生产中应用.     

ISOLATION AND SELECTION OF ETHANOL-RESISTANT AND OSMOTOLERANT YEASTS FROM REGIONAL AGRICULTURAL SOURCES IN MEXICO

In Mexican alcohol distilleries using sugarcane molasses, one reason for low alcoholic fermentation efficiency is the use of inferior yeast cultures. The objective of the present study was to isolate and select yeast strains from alcoholic fermentations of natural sources (sugarcane molasses, grape juice, cane juice and pineapple) from Veracruz city market and Mexican distilleries, and to evaluate their performance under laboratory conditions in an effort to select superior strains for industrial fermentations. Ethanol production, glucose composition, growth rate, "Killer" activity, ethanol and glucose tolerance of the most promising strains were monitored on synthetic and molasses media. A total of 112 yeast strains were isolated by their capacity to produce ethanol, and from these, only 58 were selected on the basis of best ethanol theoretical yields (88–96%). These strains were exposed several times to high concentrations of glucose and ethanol in order to select ethanol- and glucose-tolerant yeast; 10 were obtained that adapted best to these conditions and that showed "Killer" activity. Of these strains, U3-11, M11, JC10 and U2-10 (obtained from grape juice, sugarcane molasses and cane juice) demonstrated the highest adaptation to both ethanol (5–7% w/v) and glucose (20% w/v). The maximum yield obtained was 0.46 g/g (90% theoretical yield) in a 20-L bioreactor with cane molasses under nonsterile conditions.

PRACTICAL APPLICATIONS

The selected yeasts could be introduced into industrial processes in Mexican distilleries using sugarcane molasses in order to improve productivity and diminish contamination problems.     

High-cell-density fermentation of Saccharomyces cerevisiae for the optimisation of mead production

Mead is a traditional drink that contains 8%–18% (v/v) of ethanol, resulting from the alcoholic fermentation of diluted honey by yeasts. Mead fermentation is a time-consuming process and the quality of the final product is highly variable. Therefore, the present investigation had two main objectives: first, to determine the adequate inoculum size of two commercial wine-making strains of Saccharomyces cerevisiae for the optimisation of mead fermentation; and second, to determine if an increase in yeast pitching rates in batch fermentations altered the resulting aroma profiles. Minor differences were detected in the growth kinetics between the two strains at the lowest pitching rate. With increasing pitching rates net growth of the strain ICV D47 progressively decreased, whereas for the QA23 the increasing inoculum size had no influence on its net growth. The time required to reach the same stage of fermentation ranged from 24 to 96 h depending on the inoculum size. The final aroma composition was dependent on the yeast strain and inoculum size. Fourteen of the twenty-seven volatile compounds quantified could contribute to mead aroma and flavour because their concentrations rose above their respective thresholds. The formation of these compounds was particularly pronounced at low pitching rates, except in mead fermented by strain ICV D47, at 10⁶ CFUs/mL. The esters isoamyl acetate, ethyl octanoate and ethyl hexanoate were the major powerful odourants found in the meads. The results obtained in this study demonstrate that yeast strain and inoculum size can favourably impact mead's flavour and aroma profiles.     

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).     

酿酒活性干酵母生理特性的研究

建立一种酿酒活性干酵母生理活性的简易评价方法,比较并筛选一株适合以木薯粉水解液发酵发酵生产燃料酒精的活性干酵母。优化2株酿酒活性干酵母的活化条件、生长温度和生长pH值,以木薯粉水解液为发酵液,在最适生长条件下比较两株酵母的生长曲线、发酵强度、耐糖能力、耐温能力和耐酒精能力。选择酿酒活性干酵母Ⅰ作为木薯粉水解液发酵酵母,该酵母在前36h发酵强度高于1g(/L.h),36h后发酵强度迅速下降;耐糖能力为20%,耐受温度是55℃以及耐酒精浓度是7%。     

Modulation of Glycerol and Ethanol Yields During Alcoholic Fermentation in Saccharomyces cerevisiae Strains Overexpressed or Disrupted for GPD1 Encoding Glycerol 3-Phosphate Dehydrogenase

The possibility of the diversion of carbon flux from ethanol towards glycerol in Saccharomyces cerevisiae during alcoholic fermentation was investigated. Variations in the glycerol 3-phosphate dehydrogenase (GPDH) level and similar trends for alcohol dehydrogenase (ADH), pyruvate decarboxylase and glycerol-3-phosphatase were found when low and high glycerol-forming wine yeast strains were compared. GPDH is thus a limiting enzyme for glycerol production. Wine yeast strains with modulated GPD1 (encoding one of the two GPDH isoenzymes) expression were constructed and characterized during fermentation on glucose-rich medium. Engineered strains fermented glucose with a strongly modified [glycerol] : [ethanol] ratio. gpd1Δ mutants exhibited a 50% decrease in glycerol production and increased ethanol yield. Overexpression of GPD1 on synthetic must (200 g/l glucose) resulted in a substantial increase in glycerol production (×4) at the expense of ethanol. Acetaldehyde accumulated through the competitive regeneration of NADH via GPDH. Accumulation of by-products such as pyruvate, acetate, acetoin, 2,3 butane-diol and succinate was observed, with a marked increase in acetoin production. © 1997 John Wiley & Sons, Ltd.     

Identification of <Emphasis Type="Italic">Zygosaccharomyces mellis</Emphasis> strains in stored honey and their stress tolerance

To screen yeast with high sugar tolerance and evaluate their stress tolerance, six yeast strains were selected from 17 stored honey samples. The species were identified through 26S rRNA sequencing. Their stress tolerance was determined via the Durham fermentation method and ethanol production ability was determined via flask fermentation. The results demonstrated that all the six strains were Zygosaccharomyces mellis. Their sugar, ethanol, and acid tolerance ranges were 500–700 g/L, 10–12% (v/v), and pH 2.5–4.5, respectively. The SO₂ tolerance was 250 mg/L. Among the six strains, 6-7431 had the best stress tolerance with sugar tolerance of 700 g/L, ethanol tolerance of 12% (v/v), and acid tolerance of pH 2.5. Furthermore, the strain of 6-7431 had the highest percentage of ethanol production at the same initial sugar content as the other strains. Therefore, the selected six yeast strains would be promising fermentation yeasts for wine-making, ethanol production, or other fermentation purposes.     

CAMBRIDGE PRIZE LECTURE IMPROVING YEAST FERMENTATION PERFORMANCE

A number of factors affecting yeast fermentation performance have been investigated. These include the mode of substrate feeding, nutrient supplementation, temperature, osmotic pressure, oxygen, intracellular ethanol accumulation, and yeast ethanol tolerance. Nutrient supplementation was observed to play a key role in yeast fermentations employing high gravity media and at high temperatures. Furthermore, complete attenuation of high gravity wort (25°P) could be achieved by optimizing the yeast pitching rate, fermentation temperature, and level of wort oxygenation. Genetic manipulation techniques, such as spheroplast fusion, were successfully employed to obtain ethanol and osmotolerant yeast strains. In addition, a number of stable 2-deoxy-D-glucose resistant mutants, isolated from brewing and non-brewing yeast strains, were observed to rapidly utilize maltose and maltotriose in the presence of high concentrations of glucose. Fermentation and ethanol production rates were increased in these strains. Therefore, employing strategies of optimized fermentation conditions and strain development have resulted in improvements in yeast fermentation performance.     

Altered Patterns of Maltose and Glucose Fermentation by Brewing and Wine Yeasts Influenced by the Complexity of Nitrogen Source

Maltose and glucose fermentations by industrial brewing and wine yeasts strains were strongly affected by the structural complexity of the nitrogen source. In this study, four Saccharomyces cerevisiae strains, two brewing and two wine yeasts, were grown in a medium containing maltose or glucose supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low sugar concentration for brewing and wine strains, independent of nitrogen supplementation, and the type of sugar. At high sugar concentrations altered patterns of sugar fermentation were observed, and biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for brewing and wine strains. In maltose, high biomass production was observed under peptone and casamino acids for the brewing and wine strains, however efficient maltose utilization and high ethanol production was only observed in the presence of casamino acids for one brewing and one wine strain studied. Conversely, peptone and casamino acids induced higher biomass and ethanol production for the two other brewing and wine strains studied. With glucose, in general, peptone induced higher fermentation performance for all strains, and one brewing and wine strain produced the same amount of ethanol with peptone and casamino acids supplementation. Ammonium salts always induced poor yeast performance. The results described in this paper suggest that the complex nitrogen composition of the cultivation medium may create conditions resembling those responsible for inducing sluggish/stuck fermentation, and indicate that the kind and concentration of sugar, the complexity of nitrogen source and the yeast genetic background influence optimal industrial yeast fermentation performance.     

Structural Complexity of the Nitrogen Source and Influence on Yeast Growth and Fermentation

The structural complexity of the nitrogen source strongly affects both biomass and ethanol production by industrial strains of Saccharomyces cerevisiae, during fermentation in media containing glucose or maltose, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low glucose and maltose concentrations independent of nitrogen supplementation. At high sugar concentrations diauxie was not easily observed, and growth and ethanol production depended on the nature of the nitrogen source. This was different for baking and brewing ale and lager yeast strains. Sugar concentration had a strong effect on the shift from oxido‐fermentative to oxidative metabolism. At low sugar concentrations, biomass production was similar under both peptone and casamino acid supplementation. Under casamino acid supplementation, the time for metabolic shift increased with the glucose concentration, together with a decrease in the biomass production. This drastic effect on glucose fermentation resulted in the extinction of the second growth phase, probably due to the loss of cell viability. Ammonium salts always induced poor yeast performance. In general, supplementation with a nitrogen source in the peptide form (peptone) was more positive for yeast metabolism, inducing higher biomass and ethanol production, and preserving yeast viability, in both glucose and maltose media, for baking and brewing ale and lager yeast strains. Determination of amino acid utilization showed that most free and peptide amino acids present, in peptone and casamino acids, were utilized by the yeast, suggesting that the results described in this work were not due to a nutritional status induced by nitrogen limitation.     

96孔板高通量选育产乙醇酵母

以巴斯德毕赤酵母(Pichia pastoris),安琪酵母(Angel yeast),酿酒酵母(Sacchro-myces cerevisiae)等4株菌为出发菌株,经过紫外诱变,96孔板高通量筛选出60株,复筛选出4株.最大乙醇产量提高到76.6g/L,糖醇转化率达到了0.488g/g.对上述4株菌进行耐乙醇性能驯化,发酵耐乙醇能力达到15%.     

Overexpression of vacuolar H+‐ATPase‐related genes in bottom‐fermenting yeast enhances ethanol tolerance and fermentation rates during high‐gravity fermentation

Vacuolar H⁺‐ATPase (V‐ATPase) is thought to play a role in stress tolerance. In this study it was found that bottom‐fermenting yeast strains, in which the V‐ATPase‐related genes DBF2, VMA41/CYS4/NHS5 and RAV2 were overexpressed, exhibited stronger ethanol tolerance than the parent strain and showed increased fermentation rates in a high‐sugar medium simulating high‐gravity fermentation. Among the strains examined, the DBF2‐overexpressing bottom‐fermenting yeast strain exhibited the highest ethanol tolerance and fermentation rate in YPM20 medium. Using this strain, high‐gravity fermentation was performed by adding sugar to the wort, which led to increased fermentation rates and yeast viability compared with the parent strain. These findings indicate that V‐ATPase is a stress target in high‐gravity fermentation and suggests that enhancing the V‐ATPase activity increases the ethanol tolerance of bottom‐fermenting yeast, thereby improving the fermentation rate and cell viability under high‐gravity conditions. Copyright © 2012 The Institute of Brewing & Distilling     

耐高糖酵母筛选及其高糖胁迫机制的研究进展

耐高糖酵母菌在浓醪酒精发酵过程中起着重要的作用。目前,国内外对耐高酒精度酵母菌有较为广泛深入的研究,而对耐高糖酵母菌的研究还尚有不足。该文介绍了耐高糖酵母的筛选及其高糖胁迫应急机制的研究进展,并对耐高糖酵母的发展趋势进行展望,指出酒精发酵所需酵母菌应生理耐受性较好,能耐高糖度、高酒度、高渗压等,能够抵抗极端不良环境,在工业生产中能够有效提高乙醇的产量。通过筛选耐高糖酵母菌,旨在为酵母菌高糖胁迫机理的研究提供参考。     

Cellular mechanism for the improvement of multiple stress tolerance in brewer's yeast by potassium ion supplementation

The ethanol fermentation efficiency was affected by multiple stress tolerance of yeast during brewing and bioethanol industry. The effect of KCl on the multiple stress tolerance of yeast cells was examined. Results showed that KCl addition significantly enhanced the tolerance of yeast cells to osmotic and ethanol stress, which correlated with the decreased membrane permeability, the increased intracellular ergosterol and ATP content, and the improved activity of complex II and complex III in yeast cells. Biomass and viability of yeast cells under osmotic and ethanol stress were increased significantly by KCl addition. Supplementation of 4 and 10 g L⁻¹ KCl exhibited the best promotion activity for yeast cells present in medium with 500 g L⁻¹ sucrose and 10% (v v⁻¹) ethanol, respectively. These results suggested that exogenous potassium addition might be an effective strategy to improve yeast tolerance and fermentation efficiency during industrial very-high-gravity (VHG) fermentation.     

SPME–GC method as a tool to differentiate VOC profiles in Saccharomyces cerevisiae wine yeasts

The aim of this work was to study the variability of 36 Saccharomyces cerevisiae wild strains isolated from different grape varieties and from two very distant zones, located in Northern and Southern Italy. The strains were differentiated on the basis of parameters of technological interest, such as resistance to antimicrobial compounds frequently present in wine, and the production of volatile aromatic compounds (VOC), determined by SPME procedure in the experimental wines obtained by inoculated fermentations. The VOC profile allowed to differentiate the yeasts in function of isolation area: S. cerevisiae isolated from Southern Italy grapes were able to produce more volatile compounds than those from Northern Italy. The compounds synthesized by all the yeasts, besides the ethanol, were 3-methyl-1-butanol and ethyl acetate. The production of acids during the alcoholic fermentation was a characteristic of Southern yeast strains. The screening of S. cerevisiae strains for technological parameters, such as sulphur dioxide, copper and ethanol resistance or hydrogen sulphide production, revealed similar behaviour for sulphur dioxide resistance among Northern and Southern S. cerevisiae strains. Copper resistance and sulphur dioxide production were correlated to isolation area: S. cerevisiae “Northern” strains showed higher copper resistance and lowest hydrogen sulphide production than that exhibited from “Southern” strains.     

野木瓜酵母菌株抗性及发酵特性评价

以从野木瓜自然发酵过程中筛选出的8株发酵力强、产香好的酵母菌,对其进行抗性（温度耐受性、葡萄糖耐受性、酒精耐受性等）评价。供试菌株中,在NaCl浓度为140 g/L时,有4株菌仍可生长发酵并具耐高渗性,可用于高盐环境发酵中;菌株B7-2、C-4、BL-5的耐高温性较好,在55℃时仍可生长,可用于工业生产的高温环境;所有供试菌株都有很好的SO2耐受性,筛选的5株优良酵母模拟酒精发酵实验中,菌株C-4、B1-3产酒精度高于商品酵母RC212,可以作为优良酵母进一步开发利用。菌株B7-2产酒精度为0,但产香很好,可作为产香酵母在发酵过程中适量添加以增加香味,或者用于面包和无醇饮料的开发。     

Bioethanol production from ball milled bagasse using an on-site produced fungal enzyme cocktail and xylose-fermenting Pichia stipitis

Sugarcane bagasse is one of the most promising agricultural by-products for conversion to biofuels. Here, ethanol fermentation from bagasse has been achieved using an integrated process combining mechanical pretreatment by ball milling, with enzymatic hydrolysis and fermentation. Ball milling for 2 h was sufficient for nearly complete cellulose structural transformation to an accessible amorphous form. The pretreated cellulosic residues were hydrolyzed by a crude enzyme preparation from Penicillium chrysogenum BCC4504 containing cellulase activity combined with Aspergillus flavus BCC7179 preparation containing complementary β-glucosidase activity. Saccharification yields of 84.0% and 70.4% for glucose and xylose, respectively, were obtained after hydrolysis at 45 °C, pH 5 for 72 h, which were slightly higher than those obtained with a commercial enzyme mixture containing Acremonium cellulase and Optimash BG. A high conversion yield of undetoxified pretreated bagasse (5%, w/v) hydrolysate to ethanol was attained by separate hydrolysis and fermentation processes using Pichia stipitis BCC15191, at pH 5.5, 30 °C for 24 h resulting in an ethanol concentration of 8.4 g/l, corresponding to a conversion yield of 0.29 g ethanol/g available fermentable sugars. Comparable ethanol conversion efficiency was obtained by a simultaneous saccharification and fermentation process which led to production of 8.0 g/l ethanol after 72 h fermentation under the same conditions. This study thus demonstrated the potential use of a simple integrated process with minimal environmental impact with the use of promising alternative on-site enzymes and yeast for the production of ethanol from this potent lignocellulosic biomass.     

桓仁产区优良冰酒酵母菌株的筛选及酿造特性评价（英文）

为获得可用于东北桓仁地区威代尔冰酒生产的酿酒酵母菌株,采用一种快速的酵母菌株筛选方法,通过测定菌株乙醇和二氧化硫耐受性,从威代尔冰酒自然发酵过程中筛选到9株酿酒酵母菌株。进一步酿造实验结果显示,所筛选的酿酒酵母可以顺利完成冰酒发酵,产生的香气轮廓与商业酵母DV10相比也不同(主成分分析结果),最终获得了2株具有高发酵活力且香气特征与商业酵母差异显著的酵母菌株SC42和SC45,其中SC42能够高产高级醇和酯类物质,并且低产乙酸,而SC45能够产生高含量的甘油、酯类物质以及反式玫瑰醚和β-大马士酮。结果表明,采用本研究的筛选方法能够快速有效地筛选到具有应用潜力的冰酒生产菌株,同时也证明了使用本土野生酵母菌株能够有效地改善冰酒香气品质,生产出与接种商业酵母不同风格的冰酒产品。     

Fermentative capacity of Kluyveromyces marxianus and Saccharomyces cerevisiae after oxidative stress

Volatile compound production during alcoholic fermentation has been studied in the production of many beverages. Temperature, yeast strain, nutrients and pH have been identified as important factors in the production of volatile compounds. In addition, other factors could influence this production during the fermentation process as well. Oxidative stress could occur during yeast biomass production because oxygen is an essential nutrient that is added to the growth medium. The fermentation parameters and the volatile compound production of one Saccharomyces cerevisiae strain (MC4) and two Kluyveromyces marxianus strains (OFF1 and SLP1) were evaluated in relation to fermentation parameters after oxidative stress induced by hydrogen peroxide or menadione. These yeasts were compared with S. cerevisiae W303–1A and showed significant differences in ethanol production, ethanol yield and maximum ethanol production rate. K. marxianus (OFF1) showed better fermentative capacity after oxidative stress. The higher alcohol production decreased after oxidative stress by >35% after 72 h fermentation time, and the amyl alcohol decreased at a higher level (>60%); however, the isobutanol production increased after oxidative stress between 1.5 and 4 times. The yeasts produced significant concentrations of esters however ethyl lactate, ethyl caprylate and the ethyl caproate were not detected in the control fermentation, while in the stress fermentation they accounted for up to 3 mg/L. These results demonstrate that oxidative stress can play an important role in the final aroma profile; but it is necessary to guarantee adequate yeast growth to obtain the volatile compounds desired. Copyright © 2017 The Institute of Brewing & Distilling