Yeast responses to stresses associated with industrial brewery handling

During brewery handling, production strains of yeast must respond to fluctuations in dissolved oxygen concentration, pH, osmolarity, ethanol concentration, nutrient supply and temperature. Fermentation performance of brewing yeast strains is dependent on their ability to adapt to these changes, particularly during batch brewery fermentation which involves the recycling (repitching) of a single yeast culture (slurry) over a number of fermentations (generations). Modern practices, such as the use of high-gravity worts and preparation of dried yeast for use as an inoculum, have increased the magnitude of the stresses to which the cell is subjected. The ability of yeast to respond effectively to these conditions is essential not only for beer production but also for maintaining the fermentation fitness of yeast for use in subsequent fermentations. During brewery handling, cells inhabit a complex environment and our understanding of stress responses under such conditions is limited. The advent of techniques capable of determining genomic and proteomic changes within the cell is likely vastly to improve our knowledge of yeast stress responses during industrial brewery handling.     

固态发酵下酵母自溶的工艺优化

固态下酵母自溶可以有效促进菌体内多种活性物质的释放,进而提高酵母类产品的品质。通过优化自溶温度、自溶时间及自溶促进剂锌离子浓度以获得固态发酵下酵母自溶的最佳工艺,对固态发酵物料中游离氨基酸、可溶性蛋白、α-氨基氮含量和A260/A280等指标的分析来确定固态酵母自溶工艺条件,在此基础上以自溶温度40 ℃、50 ℃、55 ℃;作用时间12、18、24 h;锌离子添加浓度2、4、8 mg/kg设置L9(33)正交试验,进一步优化固态酵母自溶的工艺参数。结果表明酵母自溶的最佳工艺条件为：自溶温度55 ℃、作用时间18 h、锌离子浓度2 mg/kg,此时其可溶性蛋白含量可达9.31 mg/g、游离氨基酸14.36 mg/g、α-氨基氮10.16 μg/g、A260/A280为1.73。经工艺优化后,可显著提高酵母自溶产物可溶性蛋白、游离氨基酸和α-氨基氮的含量,从而明显提高了复合菌培养物的品质。     

土霉素菌渣源酵母菌的筛选及其培养条件的优化

【背景】随着制药行业的蓬勃发展,中国每年产生大量的抗生素菌渣,已造成了不可忽视的环境污染和资源浪费等问题。由于抗生素菌渣中含有丰富的蛋白质等有机物质,利用其蛋白质等进行二次生产或将成为解决抗生素菌渣处理问题的一种有效方法。【目的】从土霉素菌渣中筛选出天然酵母菌菌株,并利用土霉素菌渣作为酵母菌生长的主要培养基成分,通过其培养条件的优化,实现土霉素菌渣的资源化利用。【方法】以土霉素菌渣为样品,运用稀释涂布法进行酵母菌的筛选,通过其形态学观察和18S rRNA基因序列分析等方法对菌株进行鉴定。通过单因素试验与Box-Behnken Design试验结合,对培养条件进行优化,确定筛选菌株在以土霉素菌渣为主要成分培养基中的最佳生长条件。【结果】筛选的土霉素菌渣源酵母菌为一株希腊接合囊酵母菌(Zygoascus hellenicus)Y1,其最佳培养条件为:土霉素菌渣添加量为5%,葡萄糖添加量为0.5%,接菌量为2%,在pH 5.0、32℃、160r/min条件下培养24h。【结论】筛选到一株希腊接合囊酵母菌Y1,该菌能够很好地利用土霉素菌渣进行生长,实现了土霉素菌渣的资源化利用,大大减少了菌渣的...     

废糟液全循环条件下絮凝酵母乙醇连续发酵

丙酸是以玉米为原料自絮凝酵母乙醇连续发酵系统废糟液全循环过程中积累的主要抑制物。基于丙酸对酵母细胞抑制机理,开发了3种废糟液全循环条件下乙醇连续发酵工艺策略。首先根据高温导致丙酸生成的现象,去除了物料灭菌环节,使发酵液丙酸浓度显著降低,生物量和乙醇浓度分别提高了59.3%和7.4%。其次,以丙酸浓度达到半数抑制浓度(IC50)40 mmol/L为目标,通过拟合丙酸积累数据预测废糟液全循环的最长运行时间,发酵装置运行应控制在此时间范围内。再次,较低的环境pH值提高了丙酸毒性,而实验证明发酵液pH为5.5时,丙酸对细胞生长的抑制影响最小,因此控制发酵过程中的pH有利于弱化丙酸毒性。     

Effect of the size of yeast flocs and zinc supplementation on continuous ethanol fermentation performance and metabolic flux distribution under very high concentration conditions

Taking continuous ethanol fermentation with the self‐flocculating yeast SPSC01 under very high concentration conditions as an example, the fermentation performance of the yeast flocs and their metabolic flux distribution were investigated by controlling their average sizes at 100, 200, and 300 µm using the focused beam reflectance online measurement system. In addition, the impact of zinc supplementation was evaluated for the yeast flocs at the size of 300 µm grown in presence or absence of 0.05 g L^?1 zinc sulfate. Among the yeast flocs with different sizes, the group with the average size of 300 µm exhibited highest ethanol production (110.0 g L^?1) and glucose uptake rate (286.69 C mmol L^?1 h^?1), which are in accordance with the increased flux from pyruvate to ethanol and decreased flux to glycerol. And in the meantime, zinc supplementation further increased ethanol production and cell viability comparing with the control. Zinc addition enhanced the carbon fluxes to the biosynthesis of ergosterol (28.6%) and trehalose (43.3%), whereas the fluxes towards glycerol, protein biosynthesis, and tricarboxylic acid cycle significantly decreased by 37.7%, 19.5%, and 27.8%, respectively. This work presents the first report on the regulation of metabolic flux by the size of yeast flocs and zinc supplementation, which provides the potential for developing engineering strategy to optimize the fermentation system. Biotechnol. Bioeng. 2010;105: 935–944. © 2009 Wiley Periodicals, Inc.     

高生物量富铁酵母菌的选育及其发酵条件的研究

对402株不同种属的酵母菌株进行初筛、复筛,筛选到一株生物量较高的二倍体菌株ZY-46(Saccharomyces cerevisiae)和一株铁富集量较高的二倍体菌株ZY-173(Saccharomyces kluyveri)。然后以它们为出发菌,分别进行单倍体分离、硫酸二乙酯(DES)诱变,并通过原生质体融合,得到一株高生物量富铁酵母融合菌株ZYF-15。在优化的发酵条件下,该融合菌株生物量可达11．2g／L,细胞铁含量达24．5mg／g干细胞,细胞总铁含量分别比原始亲株ZY-46和ZY-173提高了2．6倍和1．9倍。     

发酵抑制物对絮凝酵母戊糖发酵的影响

将絮凝剂加入酵母溶液中,使酵母絮凝成颗粒以此作为固定化酵母进行戊糖发酵。研究了常见发酵抑制物(甲酸、乙酸、糠醛和乳酸等)对絮凝酵母发酵木糖的影响。结果表明:在60.0g/L木糖发酵液中,经过24h发酵,木糖利用率达94.6%,当分别添加抑制物甲酸、乙酸、糠醛、乙醇和乳酸时,聚氧乙烯絮凝酵母分别对其的耐受浓度为0.5、0.5、1.0、30.0和8.0g/L。当抑制物添加量超过各自的耐受浓度后,对絮凝酵母发酵会产生明显的抑制作用。     

Dynamics of the yeast transcriptome during wine fermentation reveals a novel fermentation stress response

In this study, genome-wide expression analyses were used to study the response of Saccharomyces cerevisiae to stress throughout a 15-day wine fermentation. Forty per cent of the yeast genome significantly changed expression levels to mediate long-term adaptation to fermenting grape must. Among the genes that changed expression levels, a group of 223 genes was identified, which was designated as fermentation stress response (FSR) genes that were dramatically induced at various points during fermentation. FSR genes sustain high levels of induction up to the final time point and exhibited changes in expression levels ranging from four- to 80-fold. The FSR is novel; 62% of the genes involved have not been implicated in global stress responses and 28% of the FSR genes have no functional annotation. Genes involved in respiratory metabolism and gluconeogenesis were expressed during fermentation despite the presence of high concentrations of glucose. Ethanol, rather than nutrient depletion, seems to be responsible for entry of yeast cells into the stationary phase.     

Biotechnological impact of stress response on wine yeast

Wine yeast deals with many stress conditions during its biotechnological use. Biomass production and its dehydration produce major oxidative stress, while hyperosmotic shock, ethanol toxicity and starvation are relevant during grape juice fermentation. Most stress response mechanisms described in laboratory strains of Saccharomyces cerevisiae are useful for understanding the molecular machinery devoted to deal with harsh conditions during industrial wine yeast uses. However, the particularities of these strains themselves, and the media and conditions employed, need to be specifically looked at when studying protection mechanisms.     

RIM21基因缺失对拉格啤酒酵母絮凝性能的影响

拉格啤酒酵母是我国啤酒酿造的主要菌种.细胞絮凝是啤酒酵母重要的生产性状,在不影响发酵性能的情况下适度提高酵母的絮凝能力,有助于发酵结束时细胞和产物的分离,有利于工业化啤酒生产,具有较高的经济价值.前期在对一株工业用拉格啤酒酵母G03及其絮凝突变株的研究中,挖掘到一个可能影响啤酒酵母絮凝性的候选基因RIM21.为了验证该...     

Functional yeast starter cultures for cocoa fermentation

The quest to develop a performant starter culture mixture to be applied in cocoa fermentation processes started in the 20th century, aiming at achieving high-quality, reproducible chocolates with improved organoleptic properties. Since then, different yeasts have been proposed as candidate starter cultures, as this microbial group plays a key role during fermentation of the cocoa pulp-bean mass. Yeast starter culture-initiated fermentation trials have been performed worldwide through the equatorial zone and the effects of yeast inoculation have been analysed as a function of the cocoa variety (Forastero, Trinitario and hybrids) and fermentation method (farm-, small- and micro-scale) through the application of physicochemical, microbiological and chemical techniques. A thorough screening of candidate yeast starter culture strains is sometimes done to obtain the best performing strains to steer the cocoa fermentation process and/or to enhance specific features, such as pectinolysis, ethanol production, citrate assimilation and flavour production. Besides their effects during cocoa fermentation, a significant influence of the starter culture mixture applied is often found on the cocoa liquors and/or chocolates produced thereof. Thus, starter culture-initiated cocoa fermentation processes constitute a suitable strategy to elaborate improved flavourful chocolate products.     

Identification of genes and proteins induced during the lag and early exponential phase of lager brewing yeasts

AIMS: The aim of the present study is to identify genes and proteins whose expression is induced in lager brewing yeast during the lag phase and early exponential growth. METHODS AND RESULTS: Two-dimensional gel electrophoresis was used to identify proteins induced during the lag and early exponential phase of lager brewing yeast in minimal medium. The identified, early-induced proteins were Ade17p, Eno2p, Ilv5gp, Sam1p, Rps21p and Ssa2p. For most of these proteins, the patterns of induction differed from those of the corresponding genes. However, the genes had similar early expression patterns in minimal medium as observed during lager brewing conditions. The expression of previously identified early-induced genes in Saccharomyces cerevisiae grown in minimal medium, ADO1, ALD6, ASC1, ERG4, GPP1, RPL25, SSB1 and YKL056C, was also early induced in lager yeast under brewing conditions. CONCLUSIONS: The results indicate that the above-mentioned genes in general are induced during the lag phase and early exponential growth in Saccharomyces yeasts. The processes in which these genes take part are likely to play an important role during growth initiation. SIGNIFICANCE AND IMPACT OF THE STUDY: Increased knowledge regarding the early growth phase of lager brewing yeast was obtained. Further, the universality of the identified expression patterns suggests new methodologies for optimization and control of growth initiation during brewing fermentations.     

Modeling of yeast metabolism and process dynamics in batch fermentation

Much is known about yeast metabolism and the kinetics of industrial batch fermentation processes. In this study, however, we provide the first tool to evaluate the dynamic interaction that exists between them. A stoichiometric model, using wine fermentation as a case study, was constructed to simulate batch cultures of Saccharomyces cerevisiae. Five differential equations describe the evolution of the main metabolites and biomass in the fermentation tank, while a set of underdetermined linear algebraic equations models the pseudo-steady-state microbial metabolism. Specific links between process variables and the reaction rates of metabolic pathways represent microorganism adaptation to environmental changes in the culture. Adaptation requirements to changes in the environment, optimal growth, and homeostasis were set as the physiological objectives. A linear programming routine was used to define optimal metabolic mass flux distribution at each instant throughout the process. The kinetics of the process arise from the dynamic interaction between the environment and metabolic flux distribution. The model assessed the effect of nitrogen starvation and ethanol toxicity in wine fermentation and it was able to simulate fermentation profiles qualitatively, while experimental fermentation yields were reproduced successfully as well.     

Direct ethanol fermentation from lignocellulosic biomass by Antarctic basidiomycetous yeast Mrakia blollopis under a low temperature condition

Antarctic basidiomycetous yeast Mrakia blollopis SK-4 has unique fermentability for various sugars under a low temperature condition. Hence, this yeast was used for ethanol fermentation from glucose and also for direct ethanol fermentation (DEF) from cellulosic biomass without/with Tween 80 at 10 °C. Maximally, 48.2 g/l ethanol was formed from 12% (w/v) glucose. DEF converted filter paper, Japanese cedar and Eucalyptus to 12.2 g/l, 12.5 g/l and 7.2 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80, ethanol concentration increased by about 1.1–1.6-fold compared to that without Tween 80. This is the first report on DEF using cryophilic fungi under a low temperature condition. We consider that M. blollopis SK-4 has a good potential for ethanol fermentation in cold environments.     

Continuous secondary fermentation using immobilised yeast

An immobilised yeast, two-stage reactor system was applied to laboratory fermentations of wort. The first stage, the primary fermentation process was carried out in an up-flow gas-lift bioreactor, and in the second stage, fermentation was processed in column reactors consisting of eight packed-beds filled with both yeasts entrapped in three different polysaccharide hydrogels (calcium alginate, calcium pectate, sodium carrageenan) and with free yeast in parallel. The residence time for the two-stage immobilised system varied from 74 to 108 h. © Rapid Science Ltd. 1998     

Effect of carbon dioxide on yeast growth and fermentation

Inhibition of yeast function by ethanol and by high substrate concentrations is well recognized and, to a limited extent, quantified. The role of carbon dioxide in affecting yeast metabolism (particularly growth processes) is not clear although inhibition is generally found at moderate to high concentrations of the dissolved gas. A similar situation exists with other microorganisms and with other fermentation systems. An understanding of the role of carbon dioxide, and particularly of its inhibitory effects on enzyme action and membrane function, is required if the observed global inhibition of yeasts and other fermentation systems is to be partitioned to its appropriate causes.