Simultaneous accumulation of proline and trehalose in industrial baker's yeast enhances fermentation ability in frozen dough

Freeze tolerance is a necessary characteristic for industrial baker's yeast because frozen-dough baking is one of the key technologies for supplying oven-fresh bakery products to consumers. Both proline and trehalose are known to function as cryoprotectants in yeast cells. In order to enhance the freeze tolerance of yeast cells, we constructed a self-cloning diploid baker's yeast strain with simultaneous accumulation of proline, by expressing the PRO1-I150T allele, encoding the proline-feedback inhibition-less sensitive γ-glutamyl kinase, and trehalose, by disrupting the NTH1 gene, encoding neutral trehalase. The resultant strain retained higher tolerance to oxidative and freezing stresses than did the single proline- or trehalose-accumulating strain. Interestingly, our results suggest that proline and trehalose protect yeast cells from short-term and long-term freezing, respectively. Simultaneous accumulation of proline and trehalose in industrial baker's yeast also enhanced the fermentation ability in the frozen dough compared with the single accumulation of proline or trehalose. These results indicate that baker's yeast that accumulates both proline and trehalose is applicable for frozen-dough baking.     

高适应性面包酵母菌株的杂交选育

为了改善耐高糖酵母在不同含糖量面团中的发酵力,首先制备了2 4 0株耐高糖酵母单倍体,通过高麦芽糖发酵筛选培养基筛选出了2 8株麦芽糖发酵性能良好的单倍体菌株,通过测定这些单倍体在无糖面团中的发酵力,发现9株单倍体菌株的发酵力优于或等于其亲本,其中4株为a型,5株为α型。通过它们之间的杂交得到2 0 0株杂交株,在无糖、中糖、高糖面团中测定这些杂交株的发酵力,获得4株在不同含糖量(0～30 % )面团中都具有高发酵力的高适应性面包酵母杂交株。研究表明,来自同一亲本单倍体之间的杂交有可能改善面包酵母的某些特征。     

Characterization of a new set of mutants deficient in fermentation-induced loss of stress resistance for use in frozen dough applications

In frozen dough applications a prefermentation period during the preparation of the dough is unavoidable and might also be important to obtain bread with a good texture. A major disadvantage of the prefermentation period is that it is associated with a rapid loss of the freeze resistance of the yeast cells. A major goal for the development of new baker's yeast strains for use in frozen dough applications is the availability of strains that maintain a better freeze resistance during the prefermentation period. We have isolated mutants that retain a better stress resistance during the initiation of fermentation. Some of these showed the same growth rate and fermentation capacity as the wild type cells. These mutants are called 'fil', for deficient infermentation induced loss of stress resistance. First we used laboratory strains and heat stress treatment, given shortly after the initiation of fermentation, as the selection protocol. The first two mutants isolated in this way were affected in the glucose-activation mechanism of the Ras-cAMP pathway. The fil1 mutant had a partially inactivating point mutation in CYR1, the gene encoding adenylate cyclase, while fil2 contained a nonsense mutation in GPR1. GPR1 encodes a member of the G-protein coupled receptor family which acts as a putative glucose receptor for activation of the Ras-cAMP pathway. In a next step we isolated fil mutants directly in industrial strains using repetitive freeze treatment of doughs as selection protocol. Surviving yeast strains were tested individually for maintenance of fermentation capacity after freeze treatment in laboratory conditions and also for the best performing strains in frozen doughs prepared with yeast cultivated on a pilot scale. The most promising mutant, AT25, displayed under all conditions a better maintenance of gassing power during freeze-storage. It was not affected in other commercially important properties and will now be characterised extensively at the biochemical and molecular level.     

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.     

Construction and analysis of self-cloning sake yeasts that accumulate proline

We constructed self-cloning diploid sake yeast strains that accumulate proline. The appropriate proline level is important for its protective effect against ethanol stress in yeast cells. Sake brewed with the proline-accumulating strains contained two- to threefold more proline than the sake brewed with the parent strain. It was also suggested that intracellular proline does not affect overall fermentation profiles, but reduces fermentation time in terms of ethanol production rate.     

面包酵母发酵力测定方法的探讨

探讨了面包酵母发酵力的测定方法。试验结果表明 ,采用发酵 0 .5h内面团的产气量 (mL)作为面包酵母发酵力的指标 ,能较好地反映面包酵母产品的质量 ;面团浮水法设备简单、重复性好 ,且与排水法之间有良好的相关关系 ,亦可作为面包酵母发酵力检测的方法。     

面包酵母高糖发酵力与蔗糖酶活力关系的研究

本文研究了面包酵母高糖耐性与蔗糖酶活性的关系。通过对八株酵母菌株的蔗糖酶活性和高糖面团发酵力比较分析,其中ADY2蔗糖酶酶活最大,BY-6最小,分别为128.70 U/g干酵母和30.55 U/g干酵母,而在高糖面团中发酵力却是BY-6最大,ADY2最小,CO2的产生量分别为850 ccm和225 ccm,证实了较低蔗糖酶活性的面包酵母菌株具有在高糖面团中发酵力较高的特性。通过测定蔗糖酶酶活相差较大的株菌BY-6和ADY2在蔗糖模拟面团中的蔗糖消耗和葡萄糖积累曲线,结果表明ADY2不仅蔗糖消耗速度比BY-6快,且其积累葡萄糖的速度比BY-6快,同时所积累的最高葡萄糖量也比BY-6高,分别为5.89?10-2和4.50?10-2 g/mL。此外,即便是蔗糖酶酶活低且高糖面团发酵力大菌株BY-6在蔗糖模拟面团培养基中仍有较多葡萄糖积累,因此选育蔗糖酶水解生成葡萄糖速度与其利用葡萄糖速度一致或相差不大的菌株是我们选育耐高糖面包酵母菌株的一个控制靶点。     

面包酵母耐冷冻性能的评价方法

讨论了面包酵母耐冷冻能力的评价方法,通过对酵母在普通面团中冷冻后的相对发酵力、在液体模拟面团中冷冻后的相对发酵力和酵母细胞冷冻存活率之间的比较,发现3者之间具有显著的线性正相关性。而应用液体模拟面团测定酵母耐冷冻能力的方法简便、易控制,可用于耐冷冻酵母菌种的选育和耐冷冻酵母产品的检测中。     

Disruption of ubiquitin-related genes in laboratory yeast strains enhances ethanol production during sake brewing

Sake yeast can produce high levels of ethanol in concentrated rice mash. While both sake and laboratory yeast strains belong to the species Saccharomyces cerevisiae, the laboratory strains produce much less ethanol. This disparity in fermentation activity may be due to the strains' different responses to environmental stresses, including ethanol accumulation. To obtain more insight into the stress response of yeast cells under sake brewing conditions, we carried out small-scale sake brewing tests using laboratory yeast strains disrupted in specific stress-related genes. Surprisingly, yeast strains with disrupted ubiquitin-related genes produced more ethanol than the parental strain during sake brewing. The elevated fermentation ability conferred by disruption of the ubiquitin-coding gene UBI4 was confined to laboratory strains, and the ubi4 disruptant of a sake yeast strain did not demonstrate a comparable increase in ethanol production. These findings suggest different roles for ubiquitin in sake and laboratory yeast strains.     

Bread Dough and Baker's Yeast: An Uplifting Synergy

Yeast‐mediated dough fermentation is an important phase in the bread making process. The fermentative performance of yeast cells during fermentation is of critical importance for final bread quality, since yeast cells produce CO₂ and other metabolites that have an influence on dough rheology and bread texture, volume, and taste. Different factors affect the fermentative performance of yeast cells during dough fermentation, including dough ingredients, fermentation conditions, the type of yeast strain used and yeast pregrowth conditions. Bread dough is a complex matrix that contains several ingredients that can affect the fermentation rate of yeast cells. Although the individual effects of sugar availability and salt level on the leavening ability of yeast have been studied extensively, a comprehensive overview of the relationship between bread dough constituents, fermentation conditions and yeast functionality is still lacking. Moreover, the dough environment is highly variable as several types of dough like lean, sweet or frozen doughs are currently produced by commercial bread producers. For optimal fermentation rates in different types of dough, the use of appropriate yeast strains with specific phenotypic traits is required. Therefore, many researchers have focused on the improvement of yeast strains for optimal fermentation in different types of dough like lean, sweet or frozen dough. Against this background, this review summarizes the current knowledge on the interaction between bread dough and baker's yeast and how to improve this interaction, thereby providing a useful background for further research concerning the functionality of yeast in bread dough.     

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.     

The influence of a baker's yeast pre-inoculum on bread-dough leavening

In order to investigate the influence of a pre-inoculum on the fermentation performance of compressed baker's yeast used for bread-dough leavening, pre-inocula were made by adding compressed baker's yeast to a premix-sugar broth and cultivating it for 30, 60, 90 and 120 min. The pre-inocula were added separately to the dough and the gas production measured with the aid of a fermento-manometer. The pre-inoculum cultivated for 120 min presented the highest increase (43·6%) in maximum gas produced (P_max) after 90 min of dough fermentation. The possibility of reducing lag time during dough fermentation, by using a cost-effective pre-inoculum, was assessed and results showed a significant (P ≤ 0·05) increase in gas production which could be highly beneficial to the bread industry.     

基于代谢组学和转录组学分析工业面包酵母（Saccharomyces cerevisiae）ABY3冷冻胁迫应答机制

为研究面包酵母（Saccharomyces cerevisiae）响应冷冻胁迫的机理，对面包酵母－20 ℃处理7 d前后的发酵菌液进行胞内的代谢组学和转录组学分析。在－20 ℃、7 d的环境胁迫下，面包酵母不加糖模拟面团发酵后的存活率为43%，发酵力下降42%。冷冻胁迫下，面包酵母胞内24 种代谢物的变化与494 种基因的表达差异与应答机制相关。通过差异代谢通路分析得出：冷冻胁迫下，胞内氨基酸的匮乏与质膜僵硬化可能是影响细胞生长和发酵性能的主要原因，而胞内不饱和脂肪酸相对含量的增加和海藻糖的积累并不能消除低温对细胞的损伤。研究结果可完善酵母冷冻胁迫应答机理，为耐性调节机制的研究提供思路，对冷冻面团的优化和技术发展具有重要意义。     

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.     

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。     

不同酵母对面团发酵特性及馒头品质的影响

就不同酵母对面团发酵特性和馒头品质的影响进行了试验.通过比较不同酵母发酵面团的产气量和面团膨发状态,进而确定相同条件38℃,88%RH,酵母添加量0.8%,不同酵母的最适发酵时间,然后对其发酵的馒头进行感官评价和质构测定,结果显示:不同酵母发酵面团时的产气量和面团膨发状态不同,Y-1、Y-2、Y-3和Y-4的最适发酵时间依次为40、30、25和30 min.由感官评价和质构测定结果还得出结论:不同酵母显著影响馒头产品的感官评价总分,且感官评价和质构测定参数与其面团发酵性能相一致.     

Use of selected sourdough lactic acid bacteria to hydrolyze wheat and rye proteins responsible for cereal allergy

This work was aimed at showing the capacity of selected sourdough lactic acid bacteria to hydrolyze wheat and rye allergens. Hydrolysis was investigated after wheat sourdough fermentation and after treatment of wheat and rye sourdough breads with pepsin, trypsin and pancreatin, which mimicked the digestive process. As shown by immunoblotting with sera from allergic patients, wheat sourdough fermentation caused the disappearance of some IgE-binding proteins (albumins/globulins and gliadins mainly) with respect to the chemically acidified dough used as the control. The IgE-binding protein profile of wheat and rye sourdough breads differed from those of baker's yeast breads. The signals of the IgE-binding proteins contained in the sourdough breads disappeared after in vitro digestion with pepsin, trypsin and pancreatin. The same effect by digestive enzymes was not found for baker's yeast breads which showed persistent IgE-binding proteins. As shown by ELISA inhibition assays, the presence of IgE-binding low molecular weight proteins/peptides in sourdough breads significantly decreased with respect to baker's yeast breads. Proteolytic activity by selected sourdough lactic acid bacteria may have an importance during food processing to produce pre-digested wheat and rye dough which contains IgE-binding proteins degradable by digestive enzymes.     

几种面包活性干酵母发酵不加糖面团性能的比较

对市场上的5种面包活性干酵母(ADY)进行了发酵性能测定。结果表明,不同面包酵母对低糖面团与不加糖面团发酵力存在差异,其中ADY1基本无差别,ADY2的差别较小(3.2%),而ADY4(11.9%)、ADY5(9.4%)的差别较大。通过对5种酵母麦芽糖发酵速度和葡萄糖阻遏程度的分析表明,干酵母ADY1、ADY2发酵麦芽糖速度较快,葡萄糖阻遏程度较小;而ADY4、ADY5发酵麦芽糖速度较慢,葡萄糖阻遏程度较高。可见,麦芽糖发酵速度快、葡萄糖阻遏程度小的酵母品种比较适合于不加糖面团的发酵。     

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。