Proline accumulation in baker's yeast enhances high-sucrose stress tolerance and fermentation ability in sweet dough

During bread-making processes, yeast cells are exposed to various baking-associated stresses. High-sucrose concentrations exert severe osmotic stress that seriously damages cellular components by generation of reactive oxygen species (ROS). Previously, we found that the accumulation of proline conferred freeze-thaw stress tolerance and the baker's yeast strain that accumulated proline retained higher-level fermentation abilities in frozen doughs than the wild-type strain. In this study, we constructed self-cloning diploid baker's yeast strains that accumulate proline. These resultant strains showed higher cell viability and lower intracellular oxidation levels than that observed in the wild-type strain under high-sucrose stress condition. Proline accumulation also enhanced the fermentation ability in high-sucrose-containing dough. These results demonstrate the usefulness of proline-accumulating baker's yeast for sweet dough baking.     

游离高表达Mal62基因对面包酵母耐冷冻性的影响

通过测定胞内海藻糖积累量、冷冻前后相对发酵力以及存活率的变化,对比游离高表达麦芽糖酶基因（Md62）的突变株BYCPM与亲本BY14的海藻糖合成能力,研究Ma／62基因游离高表达与酵母耐冷冻性之间的关系。结果表明．Ma／62基因游离高表达与酵母耐冷冻性有一定的相关性,突变株耐冷冻性改善,其在烘焙产业中具有潜在商业价值。     

面包酵母耐冷冻性影响因子的研究进展

论述了影响面包酵母耐冷冻性的各种因子。耐冷冻面包酵母是冷冻面团技术的关键,许多因素与酵母冷冻耐受性有关,如酵母胞内海藻糖含量、甘油含量、酒精含量、热休克蛋白含量、不同脂肪成分的比例、某些带电荷的氨基酸含量、呼吸能力、控制质膜的水运送的基因的表达等等,而海藻糖在酵母胞内起到的耐冷冻作用被广泛证明。     

Differential importance of trehalose accumulation in Saccharomyces cerevisiae in response to various environmental stresses

Trehalose is believed to play an important role in stress tolerance in the yeast Saccharomyces cerevisiae. In this research, the responses to various environmental stresses, such as high ethanol concentration, heat, oxidative, and freezing stresses, were investigated in a strain with deletion of the NTH1, NTH2, and ATH1 genes encoding trehalases that are involved in trehalose degradation and the triple deletion strains overexpressing TPS1 or TPS2, both of which encode trehalose biosynthesis enzymes in S. cerevisiae. The contents of trehalose constitutively accumulated in the TPS1- and TPS2-overexpressing triple deletion strains were higher than that in the original triple deletion strain. High trehalose accumulation and growth activity were observed in the TPS2-overexpressing triple deletion strain after ethanol stress induction. The same was also observed in the triple deletion and the TPS1- and TPS2-overexpressing triple deletion strains after heat stress induction. In case of freezing stress, all the recombinant strains with high constitutive trehalose content showed high tolerance. However, in case of oxidative stress, trehalose accumulation could not make the yeast cells tolerant. Our results indicated that high trehalose accumulation can make yeast cells resistant to multiple stresses, but the importance of this accumulation before or after stress induction is varied depending on the type of stress.     

Characterization of a Torulaspora delbrueckii diploid strain with optimized performance in sweet and frozen sweet dough

Torulaspora delbrueckii is a baker's yeast that is highly tolerant to freeze-thaw stress, making it suitable for frozen dough technology. The T. delbrueckii strain PYCC5321, isolated from traditional bread dough, combines this tolerance with a high degree of ionic and osmotic stress resistance. However, the industrial use of this strain for frozen and sweet frozen baking is hampered by its small cell size, which causes clogging problems at the filtering stage. Here, we report the construction of a stable diploid strain of T. delbrueckii PYCC5321, which we named Td21-2n. The new strain was more than 2.7-fold bigger than their haploid counterpart, whereas biomass yield, stress resistance and sweet dough leavening ability were found to be similar in both strains. Moreover, the gassing power of the diploid after dough freezing also remained unaltered. Thus, Td21-2n meets the requirements necessary for industrial production and is suitable for application in frozen sweet baking products.     

高表达MAL62基因对面包酵母耐高糖的影响

面包酵母(Saccharomyces cerevisiae)的抗逆性对于烘焙工业至关重要。以前期获得的耐冷冻酵母突变株B+MAL62为研究对象,测定其在高糖环境下的生长特性、形态特征、胞内海藻糖与甘油的积累以及产气的变化,并与市售高糖酵母进行对比。研究发现在质量分数为40%~60%的糖胁迫环境下,B+MAL62菌株的胞内海藻糖与甘油水平分别比对照菌株提升55. 03%~64. 27%与1. 2~1. 3倍,且高糖环境下B+MAL62具有更好的细胞形态稳定性,其产气速度以及最终产气量可优于市售高糖酵母。结果表明,麦芽糖酶编码基因MAL62高表达可增强面包酵母耐高糖能力。     

海藻糖对面包酵母高糖耐性的影响

通过高温刺激使面包酵母积累较高的胞内海藻糖,考察胞内海藻糖含量对面包酵母高糖发酵力和高渗存活率的影响。研究结果表明,发酵结束前2h培养温度提高至40℃后,菌株BY-6胞内海藻糖含量由4.21%提高到9.76%,高糖发酵力提高了17.2%,高渗存活率也有一定提高。另外对3株面包酵母的海藻糖积累能力和面团发酵力的比较表明,海藻糖积累能力强的酵母菌株高糖耐性较好。     

冷冻面团中酵母抗冻保护剂的优选

为改善冷冻面团品质,减少低温对酵母的损害,对常见的几种酵母抗冻保护剂进行配方优化。单因素试验测定冷冻7 d后酵母存活率和面团发酵力,筛选出保护剂及其添加量水平,再运用二次回归正交旋转组合设计进行优选试验,在对存活率影响试验中,因素主次顺序为：海藻糖＞甘油＞脯氨酸;在对发酵力影响试验中,因素主次顺序为：海藻糖＞脯氨酸＞甘油。验证得到最优试验条件为：海藻糖、甘油和脯氨酸添加量分别为2.23%、3.43%和1.55%。在此条件下,酵母存活率和面团发酵力分别为85.86%和322 mL。     

Effect of proline and arginine metabolism on freezing stress of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the PUT1-encoded proline oxidase and the PUT2-encoded delta1-pyrroline-5-carboxylate dehydrogenase are required to convert proline to glutamate. We recently showed that a put1 disruptant accumulated higher levels of proline intracellularly and conferred higher resistance to freezing stress. Here, we determined the effect of put2 disruption on yeast cell viability under freezing stress. When grown on arginine as the sole nitrogen source, the put2 disruptant showed a significant decrease in cell viability after freezing despite the high proline and arginine contents. This result suggests that delta1-pyrroline-5-carboxylate or glutamate-gamma-semialdehyde, a proline catabolism intermediate, is toxic to yeast cells under freezing stress. In contrast, the survival rate of the wild-type and the put1-disruptant strains was found to increase after freezing in proportion to their arginine contents. This indicates that arginine has a cryoprotective function in yeast. Furthermore, the yeast cells accumulated proline as well as arginine in the vacuole, suggesting that there is a system for the transport of excess proline to the vacuole and that this vacuolar accumulation may be important in the freezing resistance of yeast cells.     

Effect of trehalose accumulation on response to saline stress in Saccharomyces cerevisiae

To examine the effect of trehalose accumulation on response to saline stress in Saccharomyces cerevisiae, we constructed deletion strains of all combinations of the trehalase genes ATH1, NTH1 and NTH2 and examined their growth behaviour and intracellular trehalose accumulation under non‐stress and saline‐stress conditions. Saline stress was induced in yeast cells by NaCl addition at the exponential growth phase. All deletion strains showed similar specific growth rates and trehalose accumulation to their parent strain under non‐stress conditions. However, under the saline stress condition, one single deletion strain, nth1Δ, two double deletion strains, nth1Δ ath1Δ and nth1Δ nth2Δ, and the triple deletion strain nth1Δnth2Δ ath1Δ, all of which carry the nth1Δ deletion, showed increased trehalose accumulation as compared to the parent and other deletion strains. In particular, our statistical analysis revealed that the triple deletion strain showed a higher growth rate under the saline stress condition than the parent strain. Moreover, some deletion strains showed further trehalose accumulation under non‐stress conditions by overexpression of the TPS1 or TPS2 genes encoding the enzymes related to trehalose biosynthesis at the mid‐exponential phase. Such increased trehalose accumulation prior to NaCl addition could improve the growth of these strains under saline stress. Our results indicate that high trehalose accumulation prior to NaCl addition, rather than after NaCl addition, is necessary to achieve high growth activity under stress conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

酵母内海藻糖积累条件的优化

对面包酵母 (BY 11)海藻糖积累条件进行了较系统的研究 ,采用了PB和响应曲面设计法 ,得到了 1个优化的培养基。采用此培养基 ,以 3 g/L(以酵母干固物计 )的接种量 ,3 7℃震荡培养 3h可积累海藻糖 0 96g/L ,从而大大提高了海藻糖的产量 ,为工业生产提供了可行性。     

Quality parameters and RAPD-PCR differentiation of commercial baker's yeast and hybrid strains

Baker's yeast, Saccharomyces cerevisiae, is a key component in bread baking. Total of 12 commercial baker's yeast and 2 hybrid strains were compared using traditional quality parameters. Total of 5 strains with high leavening power and the 2 hybrid strains were selected and evaluated for their alpha-amylase, maltase, glucoamylase enzymes, and compared using random amplified polymorphic DNA (RAPD). The results revealed that all selected yeast strains have a low level of alpha-amylase and a high level of maltase and glucoamylase enzymes. Meanwhile, the Egyptian yeast strain (EY) had the highest content of alpha-amylase and maltase enzymes followed by the hybrid YH strain. The EY and YH strains have the highest content of glucoamylase enzyme almost with the same level. The RAPD banding patterns showed a wide variation among commercial yeast and hybrid strains. The closely related Egyptian yeast strains (EY and AL) demonstrated close similarity of their genotypes. The 2 hybrid strains were clustered to Turkish and European strains in 1 group. The authors conclude that the identification of strains and hybrids using RAPD technique was useful in determining their genetic relationship. These results can be useful not only for the basic research, but also for the quality control in baking factories.     

面包酵母自交提升耐高糖发酵力的研究

用工业耐高糖面包酵母BH3制备了102株单倍体,通过四分体分析发现,控制生物量和耐高糖发酵力的主效基因在此菌株中是杂合的,并且控制生物量的主效基因和控制接合型的基因MAT极大可能是连锁的。结果表明,通过自交和人工选择相结合的方式,筛选出耐高糖且发酵活力优良的18株单倍体,构建27个交配组合,其中有11个组合的自交菌株高于亲本的耐高糖发酵力水平,最高能达到亲本水平的116.73%,体现出菌株BH3在自交系选育上有进步空间。     

Trehalose reserve in Saccharomyces cerevisiae: phenomenon of transport, accumulation and role in cell viability

Strains of Saccharomyces cerevisiae deleted for TPS1 encoding trehalose-6-phosphate synthase still accumulate trehalose when harbouring a functional MAL locus. We demonstrate that this accumulation results from an active uptake of trehalose present in the 'yeast extract' used to make the enriched culture media and that no accumulation is observed in mineral media. The uptake of trehalose was shown to be mediated by the alpha-glucoside transporter encoded by AGT1, the expression of which is linked to the presence of a functional MAL locus. Deletion of this gene in a MAL+ tps1 mutant abolished trehalose accumulation on a maltose or galactose mineral medium. However, small amounts of disaccharide were still detected in a agt1 tps1 double mutant when the medium was supplemented with 10 g trehalose l(-1), indicating the existence of a non-concentrative low-affinity sugar transporter. The presence of the high-affinity trehalose permease allowed us to investigate the effect of increasing exogenous trehalose from 0 to 10 g(-1) on intracellular accumulation. A maximum of ca. 10% (wt/wt dry cells) trehalose was attained in the presence of only 1 g l(-1) of disaccharide in the medium. The capability to monitor the intracellular content of trehalose by varying its extracellular concentration, independent of genetic alterations of the trehalose metabolic machinery, allowed the remarkable contribution of this molecule in stress tolerance to be demonstrated, as the higher the trehalose content, the longer the cell survival to a severe heat shock and to glucose starvation.     

Inhibitory effect of arginine on proline utilization in Saccharomyces cerevisiae

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. Although the presence of other nitrogen sources under fermentation conditions is likely to interfere with proline utilization, the inhibitory mechanisms of proline utilization remain unclear. In this study, we examined the effect of arginine on proline utilization in S. cerevisiae. We first constructed a proline auxotrophic yeast strain and identified an inhibitory factor by observing the growth of cells when proline was present as a sole nitrogen source. Intriguingly, we found that arginine, and not ammonium ion, clearly inhibited the growth of proline auxotrophic cells. In addition, arginine prevented the proline consumption of wild-type and proline auxotrophic cells, indicating that arginine is an inhibitory factor of proline utilization in yeast. Next, quantitative polymerase chain reaction (PCR) analysis showed that arginine partially repressed the expression of genes involved in proline degradation and uptake. We then observed that arginine induced the endocytosis of the proline transporters Put4 and Gap1, whereas ammonium induced the endocytosis of only Gap1. Hence, our results may involve an important mechanism for arginine-mediated inhibition of proline utilization in yeast. The breeding of yeast that utilizes proline efficiently could be promising for the improvement of wine quality.     

低糖适应性耐冻酵母菌与普通酵母菌细胞构成成分的比较

采用气相、液相色谱法和分光光度比色法,对筛选出的低糖适应性耐冻面包酵母BY-03和FY-03与普通高糖面包酵母（PT）细胞构成成分,即胞内海藻糖含量,脂肪酸组成,麦角固醇,磷脂和氨基酸组成进行了分析与比较,进而了解其对酵母耐冻性的影响。结果表明,FY-03海藻糖含量是盯含量的1．40倍,BY-03的海藻糖含量与PT含量基本持平。从3种酵母中皆检出18种氨基酸,其中有助于提高酵母耐冻性的3种带电荷氨基酸即精氨酸,脯氨酸和谷氨酸的含量,BY-03和FY-03均高于PT。从3种酵母中检出了8种饱和与不饱和的脂肪酸,其中BY-03、FY-03的不饱和脂肪酸比例分别为79．82％和78．62％,明显高于胛64．96％,其中棕榈油酸C16：1和油酸C18：1的含量相差较大。麦角固醇的含量BY-03和FY-03分别为4．99mg／g和4．77mg／g显著高于胛的2．56mg／g,而三者的磷脂含量没有明显差别。     

面包酵母冻干粉保护剂筛选及其生物活性分析

目的:优化真空冷冻干燥过程中保护剂配方,得到发酵性能较好的面包酵母3G-28冻干粉。方法:在对脱脂奶粉、蔗糖、吐温?80、阿拉伯胶、β-环状糊精、甘油单因素实验基础上,采用响应面实验对复合冻干保护剂的配方进行了优化,并对酵母冻干粉的发酵力、海藻糖含量、蔗糖酶活力以及发酵液中的风味物质进行了研究。结果:各因素对面包酵母冻干粉活菌数量影响顺序为:甘油浓度 > 蔗糖浓度 > 脱脂奶粉浓度 > β-环状糊精浓度;最佳复合冻干保护剂的配方为:甘油4.7%,脱脂奶粉20%,β-环状糊精15%,蔗糖5%;面包酵母冻干粉活菌数量为36.89×109 个/mL。面包酵母3G-28冻干粉的发酵能力为214 mg/h/g干酵母,海藻糖含量为44.22 mg/g干酵母,蔗糖酶活力20.27 U/g干酵母,与市售酵母菌冻干粉和初始菌株酵母冻干粉相比表现出较高的生物活性。在风味物质方面,面包酵母3G-28冻干粉与其它两者相比醇类的含量最高,表现出了良好的实用性。结论:本文探讨了面包酵母3G-28冻干粉的最佳冻干保护剂配方,得到了生物活性较优的面包酵母冻干粉,在改善面包产品品质及微生物工业化应用中具有很好的前景。     

Tolerance mechanism of the ethanol-tolerant mutant of sake yeast

Several ethanol-tolerant mutants have been bred from industrial sake yeasts, but the mechanism of ethanol tolerance in these mutants has not been elucidated. After the determination of the entire genome sequence of Saccharomyces cerevisiae, various methods to monitor the whole-gene expression of the yeast have been developed. In this study, we used a commercially available nylon membrane on which virtually every gene of S. cerevisiae was spotted to compare expression profiles between the ethanol-tolerant mutant and its parent sake yeast to investigate the mechanism of ethanol tolerance in this mutant. As a result, we found that several genes were highly expressed only in the ethanol-tolerant mutant but not in the parent strain. These genes were known to be induced in cells that were exposed to various stresses, such as ethanol, heat, and high osmolarity, or at the stationary-phase but not at the log-phase. In the ethanol-tolerant mutant, the expression level of these stress-responsive genes was further increased after exposure to ethanol. We also found that substances such as catalase, glycerol and trehalose that may have protective roles under stressful conditions were accumulated in high amounts in the ethanol-tolerant mutant. The ethanol-tolerant mutant also exhibited resistance to other stresses including heat, high osmolarity and oxidative stress in addition to ethanol tolerance. These results indicate that the mutant exhibits multiple stress tolerance because of elevated expression of stress-responsive genes, resulting in accumulation of stress protective substances.     

Identification of genes whose expressions are enhanced or reduced in baker's yeast during fed-batch culture process using molasses medium by DNA microarray analysis

Genes whose expression levels are enhanced or reduced during the cultivation process that uses cane molasses in baker's yeast production were identified in this study. The results showed that baker's yeast grown in molasses medium had higher fermentation ability and stress tolerance compared with baker's yeast grown in synthetic medium. Molasses apparently provided not only sugar as a carbon source but also provided functional components that enhanced or reduced expression of genes involved in fermentation ability and stress tolerance. To identify the genes whose expression is enhanced or reduced during cultivation in molasses medium, DNA microarray analysis was then used to compare the gene expression profile of cells grown in molasses with that of cells grown in synthetic medium. To simulate the commercial baker's yeast production process, cells were cultivated using a fed-batch culture system. In molasses medium, genes involved in the synthesis or uptake of vitamins (e.g., biotin, pyridoxine and thiamine) showed enhanced expression, suggesting that vitamin concentrations in molasses medium were lower than those in synthetic medium. Genes involved in formate dehydrogenase and maltose assimilation showed enhanced expression in molasses medium. In contrast, genes involved in iron utilization (e.g., siderophore, iron transporter and ferroxidase) showed enhanced expression in synthetic medium, suggesting that iron starvation occurred. The genes involved in the metabolism of amino acids also showed enhanced expression in synthetic medium. This identification of genes provides information that will help improve the baker's yeast production process.     

Properties of Wheat Dough at Sub-Zero Temperatures and Freeze Tolerance of a Baker's Yeast (Saccharomyces cerevisiae)

ABSTRACT: This work studied the relationship between the freeze tolerance of a baker's yeast ( Saccharomyces cerevisiae ) and the physical properties of the frozen wheat dough. The behavior of wheat dough, of the gluten phase, and of the liquid phase at sub-zero temperatures was examined by differential scanning calorimetry (DSC) and cryomicroscopy. The effect on yeast viability of dough water content, freezing and storage temperatures, and prefermentation before freezing was studied. Yeast viability was mostly affected by the freezing and storage temperatures, with temperatures below the glass transition temperature (T_g) giving the highest survival ratios. At temperatures above T_g, viability after freezing treatment and during storage seemed to be governed by different mechanisms, encompassing osmotic and mobility factors. Those factors were also found to influence the freeze tolerance of growing yeast in the presence of ethanol.