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.     

Production of Food and Fodder Yeasts

Abstract

A decade or so ago, there was considerable interest in developing single cell protein production from raw materials. Many factors have influenced the development of fodder yeast technology, notably the biochemistry and physiology of the yeast.

It is shown that those considerations have led to the choice of a continuous fermentation technology.     

乙醇脱氢酶Ⅱ活性低谷胱甘肽含量高的啤酒酵母工程菌

微生物代谢工程是当前国内外研究的热点.在食品、能源、环境等领域,通过遗传修饰改变微生物的物质和能量代谢流向以获得期望产物的研究已广泛地开展.乙醛是啤酒中重要的风味物质之一,过高的乙醛含量已成为国内啤酒风味改良的瓶颈,一直难有突破.     

Production of ethanol from guava pulp by yeast strains

Guava pulp used for ethanol production by three yeast strains contained 10% (w/v) total sugars and was pH 4.1. Ethanol production at the optimum sugar concentration of 10%, at pH 4.1 and 30°C was 1.5%, 3.6% and 3.9% (w/v) by Saccharomyces cerevisiae MTCC 1972, Isolate-1 and Isolate-2, respectively, at 60 h fermentation. Higher sugar concentrations at 15 and 20% were inhibitory for ethanol production by all test cultures. The maximum production of ethanol at optimum natural sugar concentration (10%) of guava pulp, was 5.8% (w/v) at pH 5.0 by Isolate-2 over 36 h fermentation, which was only slightly more than the quantity of ethanol produced by Saccharomyces cerevisiae (5.0%) and Isolate-1 (5.3%) over 36 and 60h fermentation, respectively.     

Enhancement of maltose utilisation by Saccharomyces cerevisiae in medium containing fermentable hexoses

Saccharomyces cerevisiae are unable to maintain high rates of fermentation during transition from catabolism of hexoses to maltose. This phenomenon, termed ‘maltose lag’, presents problems for the baking, brewing and distilling industries, which rely on yeast catabolism of mixtures of hexoses and maltose. Maltose utilisation requires the presence of maltose permease and α-glucosidase (maltase), encoded by MAL genes. Synthesis of these is induced by maltose and repressed by glucose. One strain of baker’s yeast used in this work exhibited a marked maltose lag, whereas a second strain exhibited a shorter lag during conversion from hexose to maltose metabolism. The extent of the lag was linked to the levels of maltose permease and maltase in cells at the time of inoculation into mixed sugar medium. This view is supported by results showing that pulsing yeast with maltose to induce expression of MAL genes prior to inoculation into mixed sugar medium, enhanced sugar fermentation. Maltose pulsing of yeasts could therefore be useful for enhancing some fermentations relevant to baking and other yeast industries. Received 24 December 1988/ Accepted in revised form 18 March 1999     

低乙醇脱氢酶Ⅱ活性的抗老化啤酒酵母工程菌的构建

采用自克隆技术,破坏啤酒酵母工业菌株YSF31的ADH2基因,在ADH2基因位点插入来源于YSF31的编码γ-谷氨酰半胱氨酸合成酶的GSH1基因和铜抗性筛选标记CUP1基因.通过铜抗性筛选转化子,经PCR和乙醇脱氢酶Ⅱ(ADH Ⅱ)活性测定验证,获得了1株啤酒酵母工程菌.10°P麦芽汁发酵实验显示,自克隆菌株的乙醇脱氢酶Ⅱ活性是受体菌的65%,谷胱甘肽含量比受体菌YSF31的高34%.其他发酵指标并没有发生明显改变.由于DNA操作过程中没有外源基因介入,因此啤酒酵母工程菌为生物安全的自克隆菌株,具有重要的应用价值.     

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.     

l-Isoleucine Production by Analog-resistant Mutants Derived from Threonine-producing Strain of Corynebacterium glutamicum

A thiaisoleucine-resistant mutant, ASAT–372, derived from a threonine producer of Corynebacterium glutamicum, KY 10501, produced 5 mg/ml each of l-isoleucine and l-threonine. l-Isoleucine productivity of ASAT–372 was improved stepwise, with concurrent decrease in threonine production, by successively endowing it with resistivity to such substances as ethionine, 4-azaleucine and α-aminobutyric acid. The mutant strain finally selected, RAM–83, produced 9.7 mg/ml of l-isoleucine with a medium containing 10% (as sugar) molasses.

l-Isoleucine production was significantly affected by the concentration of ammonium sulfate in the fermentation medium. At 4% ammonium sulfate l-isoleucine production was enhanced whereas l-threonine production was suppressed. At 2% ammonium sulfate l-threonine production was stimulated while l-isoleucine production decreased.     

Production of 1,3-Propanediol from Glycerol by Clostridium acetobutylicum and Other Clostridium Species

Glycerol was fermented with the production of 1,3-propanediol as the major fermentation product by four strains of Clostridium acetobutylicum, six of C. butylicum, two of C. beijerinckii, one of C. kainantoi, and three of C. butyricum. 1,3-Propanediol was identified by its retention times in gas chromatography and high-pressure liquid chromatography and by its mass spectrum. During growth of C. butylicum B593 in a chemostat culture at pH 6.5, 61% of the glycerol fermented was converted to 1,3-propanediol. When the pH was decreased to 4.9, growth and 1,3-propanediol production were substantially reduced.     

Production of an Antifungal Chitinase from Enterobacter sp. NRG4 and its Application in Protoplast Production

Summary In this study flake chitin, crab shell chitin, mushroom stalk, fungal cell wall, wheat bran and rice bran were used as substrate for chitinase production by Enterobacter sp. NRG4 under submerged and solid state fermentation (SSF) conditions. Enterobacter sp. NRG4 produced 72 and 49.7 U/ml of chitinase in presence of cell walls of Candida albicans and Fusarium moniliforme in submerged fermentation. Under SSF, maximum chitinase production was 965 U/g solid substrate with flake chitin and wheat bran (1:3 ratio) at 75% moisture level after 144 h. The purified chitinase inhibited hyphal extension of Fusarium moniliforme, Aspergillus niger, Mucor rouxi and Rhizopus nigricans. The chitinase was effective in release of protoplasts from Trichoderma ressei, Pleurotus florida, Agaricus bisporus and Aspergillus niger. Protoplasts yield was maximum with 60 mg of 24 h old fungal mycelium incubated with 60 U of chitinase and 60 U of cellulase.     

山稻蝗及相关物种Cyt b基因序列及其遗传关系

中国7个不同地理区域的山稻蝗Oxya agavisa共16个个体的线粒体DNA细胞色素b基因中间部分序列被测定,分析和比较,同时与小稻蝗O.inricata,日本稻蝗O.japonica和中华稻蝗O.chinensis相应序列进行比较,用瘤锥蝗科的曲尾似橄蝗Pseudomorphacris hollisi和蚱科的日本蚱Tetrix japonica作外群,构建了山稻蝗不同单倍型及其相关物种的NJ分子系统树。在获得的山稻蝗432bp的序列中,A＋T约占71．0％,其中9个核苷酸位点存在变异（约占所测核苷酸的2．08％）,但权有1个位点引起了氨基酸的变异,就每个氨基酸密码子来看,第三位点的A＋T含量较,达88．3％。分子系统树显示,山稻蝗6个单倍型聚为一个簇,彼此之间有一定的分歧,其分枝与地理分布没有直接的关系;该种与日本稻蝗关系较近,与中华稻蝗和小稻蝗相对较远。并对此进行了讨论。     

绿僵菌Ma83几丁质酶的发酵研究

本实验从虫生真菌中筛选出金龟子绿僵菌M a83菌株,它的几丁质酶合成能力最强。其产酶的适宜条件是,碳源为胶体几丁质加葡萄糖,氮源为NaNO3,培养温度为28℃,培养基起始pH 6.0;接种量为5 mL液态种,最适装液量为5 mL,添加维生素C可以提高酶活;正交实验表明培养因子的最佳组合是:NaNO31 g/L,胶体几丁质0.6 g/L,酵母膏0.05 g/L,葡萄糖0.10 g/L。根据液态培养产酶过程结果可知,当M a83菌培养6天时,几丁质酶活力达到8.1 U/mL。     

一种耐热几丁质酶的产生及其稳定性研究

疏绵状嗜热丝孢菌(Thermomyces lanuginosus)SY2 在以胶状几丁质为唯一碳源的诱导培养基中产生了胞外几丁质酶.该酶在50℃保温1 h,酶活稳定;65℃时半衰期为25 min;酶液在室温下保存到12周,残余酶活性为45%左右.该酶有较宽的pH范围,3.0～9.0之间保持稳定,pH值为2.5时,仍具有70%的剩余酶活性.Ca2 对几丁质酶的活性有显著的激活作用;高浓度变性剂对酶有抑制作用.结果表明该酶是一种热稳定性高且耐酸碱的新型几丁质酶,能在酸性和高温环境中发挥作用,这些特性赋予了T.lanuginosus几丁质酶在几丁质的生物转化及其它生物技术中极大的应用优势.     

Production of high-purity isomalto-oligosaccharides syrup by the enzymatic conversion of transglucosidase and fermentation of yeast cells

A method for the production of high-purity isomalto-oligosaccharides (IMO) involving the transglucosylation by transglucosidase and yeast fermentation was proposed. The starch of rice crumbs was enzymatically liquefied and saccharified, and then converted to low-purity IMO syrup by transglucosylation. The low-purity IMO produced either from rice crumbs or tapioca flour as the starch source could be effectively converted to high-purity IMO by yeast fermentation to remove the digestible sugars including glucose, maltose, and maltotriose. Both Saccharomyces carlsbergensis and Saccharomyces cerevisiae were able to ferment glucose in the IMO syrup. Cells of S. carlsbergensis harvested from the medium of malt juice were also able to ferment maltose and maltotriose. A combination of these two yeasts or S. carlsbergensis alone could be used to totally remove the digestible sugars in the IMO, coupled with the production of ethanol. The resultant high-purity IMO, including mainly isomaltose, panose, and isomaltotriose made up more than 98% w/w of the total sugars after a 3-day fermentation. When the low-purity IMO was produced from the starch of tapioca flour, 3-day fermentation under the same conditions resulted in IMO with purity lower than that from rice crumbs. For low-purity IMO from rice crumbs, fermentation with washed S. carlsbergensis cells harvested at log phase was the most effective. However, for the low-purity IMO from tapioca flour, incubation with S. cerevisiae for the first 24 h and then supplementing with an equal amount of S. carlsbergensis cells for further fermentation was the most effective approach for producing high-purity IMO.     

一种耐热几丁质酶的产生及其稳定性研究

疏绵状嗜热丝孢菌(Thermomyces lanuginosus)SY2在以胶状几丁质为唯一碳源的诱导培养基中产生了胞外几丁质酶。该酶在50℃保温1 h, 酶活稳定; 65℃时半衰期为25 min; 酶液在室温下保存到12周, 残余酶活性为45％左右。该酶有较宽的pH范围, 3.0~9.0之间保持稳定, pH值为2.5时, 仍具有70％的剩余酶活性。Ca2+ 对几丁质酶的活性有显著的激活作用; 高浓度变性剂对酶有抑制作用。结果表明该酶是一种热稳定性高且耐酸碱的新型几丁质酶, 能在酸性和高温环境中发挥作用, 这些特性赋予了T. lanuginosus几丁质酶在几丁质的生物转化及其它生物技术中极大的应用优势。     

粘质沙雷氏菌XJ-01几丁质酶合成条件的优化

目的:对粘质沙雷氏菌(Serratia marcescens)XJ-01几丁质酶合成条件优化.方法:研究了碳源、氮源、温度、pH等单个因素对该菌几丁质酶合成的影响,并通过正交实验确定了该菌的最适酶合成条件.结果:该茵几丁质酶最优合成条件为:胶体几丁质5g/L、硫酸铵5 g/L、培养温度32℃、最适pH 8.结论:优化了S.marcescens XJ-01几丁质酶的合成条件.     

Maltotriose fermentation by Saccharomyces cerevisiae

Maltotriose, the second most abundant sugar of brewer's wort, is not fermented but is respired by several industrial yeast strains. We have isolated a strain capable of growing on a medium containing maltotriose and the respiratory inhibitor, antimycin A. This strain produced equivalent amounts of ethanol from 20 g l⁻¹ glucose, maltose, or maltotriose. We performed a detailed analysis of the rates of active transport and intracellular hydrolysis of maltotriose by this strain, and by a strain that does not ferment this sugar. The kinetics of sugar hydrolysis by both strains was similar, and our results also indicated that yeast cells do not synthesize a maltotriose-specific α-glucosidase. However, when considering active sugar transport, a different pattern was observed. The maltotriose-fermenting strain showed the same rate of active maltose or maltotriose transport, while the strain that could not ferment maltotriose showed a lower rate of maltotriose transport when compared with the rates of active maltose transport. Thus, our results revealed that transport across the plasma membrane, and not intracellular hydrolysis, is the rate-limiting step for the fermentation of maltotriose by these Saccharomyces cerevisiae cells. Journal of Industrial Microbiology & Biotechnology (2001) 27, 34–38. Received 13 January 2001/ Accepted in revised form 29 May 2001