Isolation of a lager yeast with an increased copy number of the YCK1 gene and high fermentation performance

The selection of yeast with good fermentation characteristics is critical for producing beer with desirable qualities. A yeast population was selected with an enhanced fermentation rate, referred to as high‐fermentation yeast (HFY), which was derived from the wild‐type Sacchromyces pastorianus yeast population (WTY). To identify genes that contribute to the fermentation performance, we compared the genetic profiles of the WTY and HFY populations by next‐generation sequencing. Several chromosomal regions were found to exhibit markedly different sequence coverage, suggesting chromosomal duplications and deletions, which might have occurred during selection of the HFY population. Among the genes with altered coverage, the copy number of the Saccharomyces eubayanus‐type YCK1 (SeYCK1) gene was almost two times higher in the HFY population than in the WTY population. The gene which is involved in glucose sensing in Saccharomyces cerevisiae was at a higher level in the HFY population throughout fermentation. These findings suggest that the chromosomal duplication of a region including the SeYCK1 gene locus of the HFY population is at least partially responsible for the differences in the fermentation properties between the WTY and HFY populations. © 2018 The Institute of Brewing & Distilling     

Use of spent brewer's yeast in L‐(+) lactic acid fermentation

The application of by‐products from the brewing industry in lactic acid (LA) production was investigated in order to replace expensive nitrogen sources (such as yeast extract) with cheaper and renewable nitrogenous materials such as brewer's yeast (BY). In this study, brewer's spent grain (BSG) hydrolysate was used for L‐(+)‐LA fermentation by Lactobacillus rhamnosus ATCC 7469. The effect of pH control during the fermentation and the addition of various BY contents (5–50 g/L) in BSG hydrolysate on fermentation parameters was evaluated. BY addition significantly increased free amino nitrogen (FAN) concentration (by 25.2% at 5 g/L to 616% at 50 g/L). A strong positive correlation between FAN concentration in the hydrolysate and concentration of L‐(+)‐LA produced was observed (correlation coefficient of 0.913). A high cell viability of L. rhamnosus ATCC 7469 (1.95–3.32 × 10⁹ CFU/mL at the end of fermentation) was achieved in all fermentations with the addition of brewer's yeast. The addition of BY increased L‐(+)‐lactic acid yield and volumetric productivity up to 8.4% (5 g/L) and 48.3% (50 g/L). The highest L‐(+)‐LA yield (89%) and volumetric productivity (0.89 g/L h^?1) were achieved in fermentation of BSG hydrolysate with 50 g/L of BY. © 2019 The Institute of Brewing & Distilling     

Characterization of the Saccharomyces bayanus‐type AGT1 transporter of lager yeast

Transport of maltose and maltotriose into the yeast cell is thought to be rate‐limiting in the utilization of these sugars. The maltose and maltotriose transporters Malx1, Agt1, Mtt1 and Mphx are present in different combinations in brewer's yeast strains, conferring different maltose and maltotriose transport characteristics on the strains. A new putative maltose/maltotriose transporter ORF was identified during whole genome sequencing of the lager strain WS34/70 (Y. Nakao et al., DNA Res., 2009, 16, 115–129). Sequence comparisons suggested that this putative α‐glucoside transporter might be a Saccharomyces bayanus counterpart of the Agt1 (Saccharomyces cerevisiae type) transporter. In the present work, the transporter coded by a SbAGT1 gene from a lager strain, A15 (and with the same sequence as the corresponding gene in WS34/70) was characterized. It is shown that this SbAGT1 encodes a functional α‐glucoside transporter with a wide‐substrate range, including maltose and maltotriose. Trehalose, α‐methylglucoside and sucrose were inhibitors, suggesting they are also substrates. The SbAgt1 transporter had similar affinities for maltose and maltotriose (17 ± 7 and 22 ± 2 m m , respectively) and a higher V_max for maltose than maltotriose (21 ± 7 and 12 ± 2 µmol min^?1 g dry yeast^?1, respectively). Copyright © 2012 The Institute of Brewing & Distilling     

酿酒酵母工业菌株胁迫条件耐受性分析

对酿酒酵母（Saccharomyces cerevisiae）工业菌株胁迫条件，包括高浓度酒精、高渗透压、高温、营养饥饿、氧化胁迫、糠醛毒性的耐受性进行了分析，同时测定了抗生素G418对这些菌株的最低抑菌浓度。结果表明，所测定的酵母菌株对这些逆境条件的耐受性有明显差别，表现出良好耐受性的是6508和安琪酵母菌株，同时多倍性的酿酒酵母工业菌株的耐受性均比单倍性实验室菌株高。     

Determinants of flocculence of brewer's yeast during fermentation in wort

Ability of Saccharomyces cerevisiae MPY3 cells to flocculate during fermentation in wort was found to be triggered after growth limitation by oxygen shortage and to coincide with a sharp increase in cell surface hydrophobicity of the cells. Presence of oxygen in the pitching wort influenced final cell number, flocculence of the cells and cell surface hydrophobicity. Flocculation ability of cells grown in air-depleted pitching wort was hampered, concomitant with a decrease in final cell number and in final cell surface hydrophobicity. Addition of ergosterol and Tween 80 to air-depleted wort restored normal growth of the cells as well as flocculation ability and the increase in cell surface hydrophobicity. The same parameters increased in value after addition of ergosterol and Tween 80 to a fermentation with air-saturated pitching wort. Hydrophobicity of a non-flocculent mutant of S. cerevisiae strain MPY3, fermenting in air-saturated pitching wort, did not increase at cell division arrest. These results support the hypothesis that cell surface hydrophobicity is a major determinant for yeast cells to become flocculent, and suggest that shortage of sterols and unsaturated fatty acids precedes flocculence under brewing conditions.     

Lactic acid fermentation of brewer's spent grain hydrolysate by Lactobacillus rhamnosus with yeast extract addition and pH control

Lactic acid (LA) is a versatile chemical with a wide range of applications in food, pharmaceutical, cosmetic, textile and polymer industries. Brewer's spent grain (BSG) is the most abundant brewing by‐product. In this study BSG hydrolysates were used for LA fermentation by Lactobacillus rhamnosus ATCC 7469. The aim of this study was to evaluate the effects of pH control during fermentation, reducing sugar content and yeast extract content in BSG hydrolysate on LA fermentation parameters. The pH control greatly increased reducing sugar utilization, l ‐(+)‐LA content, yield and volumetric productivity. The highest l ‐(+)‐LA yield and volumetric productivity were achieved with the reducing sugar content of 54 g/L. Yeast extract addition significantly increased reducing sugar utilization, l ‐(+)‐LA content, L. rhamnosus cell viability, l ‐(+)‐LA yield and volumetric productivity. The highest l ‐(+)‐LA content (39.38 g/L), L. rhamnosus cell viability (9.67 log CFU/mL), l ‐(+)‐LA yield (91.29%) and volumetric productivity (1.69 g/L/h) were achieved with the reducing sugar content of 54 g/L and yeast extract content of 50 g/L. Copyright © 2017 The Institute of Brewing & Distilling     

酿酒酵母与产香酵母混合发酵鸭梨酒工艺条件优化

产香酵母通过不同的代谢途径产生多种挥发性香气成分,增加了酒体的香气。该文利用产香酵母与酿酒酵母协同发酵酿造鸭梨果酒,以期改善单一酿酒酵母发酵酒体的风味与口感,更大程度地开发水果的酿酒潜力。从6株产香酵母中优选1株产香酵母B(异常毕赤酵母(Pichia anomala))与安琪SY果酒酵母进行共酵,在单因素试验的基础上,选择发酵时间、接种量和接种时间进行响应面优化试验,以感官评分为响应值,确定最佳工艺为,发酵初始接种2%的产香酵母B,24 h后接种3%的果酒酵母SY,24℃发酵8 d,所得鸭梨酒酒精度达11.6%vol,可溶性固形物为5 Brix。通过理化及顶空固相微萃取-气相色谱-质谱分析发现,酯类物质是构成酒体香气的主要成分,混菌发酵酒体的总酯含量为3.21 g/L,是单菌发酵酒体的2.6倍,混菌发酵酒体中辛酸乙酯、己酸乙酯、乙酸乙酯、癸酸乙酯相对含量都比单菌发酵高。所得酒体呈亮黄色,澄清透亮,果香清雅,香味协调。     

Identification of regulatory elements in the AGT1 promoter of ale and lager strains of brewer's yeast

Agt1 is an interesting α-glucoside transporter for the brewing industry, as it efficiently transports maltotriose, a sugar often remaining partly unused during beer fermentation. It has been shown that on maltose the expression level of AGT1 is much higher in ale strains than in lager strains, and that glucose represses the expression, particularly in the ale strains. In the present study the regulatory elements of the AGT1 promoter of one ale and two lager strains were identified by computational methods. Promoter regions up to 1.9 kbp upstream of the AGT1 gene were sequenced from the three brewer's yeast strains and the laboratory yeast strain CEN.PK-1D. The promoter sequence of the laboratory strain was identical to the AGT1 promoter of strain S288c of the Saccharomyces Genome Database, whereas the promoter sequences of the industrial strains diverged markedly from the S288c strain. The AGT1 promoter regions of the ale and lager strains were for the most part identical to each other, except for one 22 bp deletion and two 94 and 95 bp insertions in the ale strain. Computational analyses of promoter elements revealed that the promoter sequences contained several Mig1- and MAL-activator binding sites, as was expected. However, some of the Mig1 and MAL-activator binding sites were located on the two insertions of the ale strain, and thus offered a plausible explanation for the different expression pattern of the AGT1 gene in the ale strains. Accordingly, functional analysis of A60 ale and A15 lager strain AGT1 promoters fused to GFP (encoding the green fluorescent protein) showed a significant difference in the ability of these two promoters to drive GFP expression. Under the control of the AGT1 promoter of the ale strain the emergence of GFP was strongly induced by maltose, whereas only a low level of GFP was detected with the construct carrying the AGT1 promoter of the lager strain. Thus, the extra MAL-activator binding element, present in the AGT1 promoter of the ale strain, appears to be necessary to reach a high level of induction by maltose. Both AGT1 promoters were repressed by glucose but their derepression was different, possibly due to a distinct distribution of Mig1 elements in these two promoters.     

Isolation and partial purification of mannose-specific agglutinin from brewer's yeast involved in flocculation

Yeast cell-agglutinating activity, designated agglutinin (possible lectin), was isolated from cell walls of both non-flocculent and flocculent brewer's yeast cells. Agglutinin-mediated aggregation of yeast cells in a manner similar to flocculation with respect to specific mannose-sensitivity, pH-dependence and calcium-dependence. Agglutinating activity was found to be heat-stable and protease-insensitive. Furthermore, addition of agglutinin to flocculent cells strongly stimulated the flocculation ability of the cells, whereas addition to non-flocculent cells rendered these cells weakly flocculent. Agglutinin was found to be released from flocculent cells during the course of a flocculation assay, but not from non-flocculent cells. Presence of mannose during the assay inhibited release of agglutinin. Our results suggest that (i) mannose-specific agglutinin is continuously synthesized during growth of brewer's yeast cells, (ii) agglutinin is present in cell walls of non-flocculent cells but is unable to bind its ligand on other cells, and (iii) the ability of yeast cells to flocculate in a flocculation assay depends, among other factors, on release of agglutinin from the cells. A 10-kDa polypeptide might represent one form of agglutinin.     

Identification of bottom-fermenting yeast genes expressed during lager beer fermentation

It has been proposed that bottom-fermenting yeast strains of Saccharomyces pastorianus possess at least two types of genomes. Sequences of genes of one genome [S. cerevisiae (Sc)-type] have been found to be highly homologous (more than 90% identity) to S. cerevisiae S288C sequences, while those of the other [Lager (Lg)-type] are less so. To identify and discriminate Lg-type from Sc-type genes expressed during lager beer fermentation, normalized cDNA libraries were constructed and analysed. From approximately 22 000 ESTs, 3892 Sc-type and 2695 Lg-type ORFs were identified. Expression patterns of Sc- and Lg-type genes did not correlate with particular cell functions in KEGG classification system. Moreover, 405 independent clones were isolated that have no significant homology with sequences in the S288C database, suggesting that they include the bottom-fermenting yeast-specific (BFY) genes. Most of BFY genes have significant homology with the S. bayanus genome.     

优良耐酸酿酒酵母的筛选及发酵特性研究

该研究采用酸性选择培养基、氯化三苯基四氮唑（TTC）显色法、杜氏小管发酵法从实验室保藏的87株酿酒酵母（Saccharomyces cerevisiae）中筛选耐酸性强的优良菌株,并测定其最适生长条件、耐受性及发酵性能。结果表明,最终筛选出一株耐受性强及发酵性能优良的菌株SC-62,其最适生长温度为28 ℃、最适pH为4.0,可耐受乙醇体积分数14%、NaCl 100 g/L、葡萄糖500 g/L,具有发酵力[5.93 g CO2/（100 mL·24 h）]强,酒精产量（7.55%vol）高,延滞期（12 h）短、适应性强等优点。该菌株的成功筛选为后续高效生产酒精提供了良好的菌种来源。     

酵母可同化氮含量对低产硫化氢酿酒酵母发酵特性的影响

该研究以3株本土低产硫化氢酿酒酵母（Saccharomyces cerevisiae）41y5、182y12和174y1为研究对象,研究酵母可同化氮（YAN）的质量浓度对其发酵特性的影响,并对不同菌株各指标间的相关性进行分析。结果表明,随着初始YAN质量浓度的升高,酵母的生物量越大,发酵周期越短;发酵后模拟酒的挥发酸含量和pH值升高;初始YAN质量浓度对菌株产H2S的影响不同。初始YAN质量浓度与CO2平均释放速率、挥发酸含量、pH值呈极显著正相关（P＜0.01）,与最大生物量呈显著正相关（P＜0.05）,与H2S释放量无显著相关性（P＞0.05）,且在发酵过程中H2S的释放量与发酵后模拟酒的pH值存在显著的正相关（P＜0.05）。与酿酒酵母41y5和182y12相比,酿酒酵母174y1在4个初始YAN质量浓度下,生物量均最高,发酵周期均最短,发酵性能优良。     

富硒酵母的选育及其发酵条件的优化试验

通过对一株耐硒酵母进行UV和60Co逐级诱变,获得一株产量高的突变株Y7,发酵液的硒总含量达19mg/L,是出发菌株的1.58倍,经多次传代试验,证明其稳定性良好。通过单因素、L16(37)正交试验对其发酵培养基和培养条件进行优化,试验表明富硒酵母发酵培养基的最佳配方是:蔗糖6%,牛肉膏1%,蛋白胨1%,K2HPO4 0.15%,亚硒酸钠35μg/L;最佳培养条件:初始pH为4.0,温度32℃,装液量60mL/250mL摇瓶,转速200r/min,10%(v/v)接种量,培养48h。富硒酵母的硒总含量达23mg/L以上,是出发菌株的1.92倍。     

Effects of growth with ethanol on fermentation and membrane fluidity of Saccharomyces cerevisiae

Saccharomyces cerevisiae HSc was grown with ethanol at concentrations up to 10% (v/v). The immediate effects of additions of externally added ethanol on CO₂ production and O₂ consumption of washed organisms were studied by stopped-flow membrane inlet quadrupole mass spectrometry. Fermentative activities of organisms grown with ethanol (0–5% v/v) showed similar sensitivities to inhibition by ethanol, whereas those grown with 10% (v/v) ethanol had become protected and were markedly less sensitive. The fluidity of subcellular membrane fractions was measured by determination of the temperature dependence of the rotational order parameter of the spin label 5-doxyl stearic acid (free radical) by electron spin resonance. Mitochondria prepared from yeasts grown with 0, 7 and 9% (v/v) ethanol showed similar overall fluidity, although differences in temperature-dependent behaviour indicate altered lipid composition or lateral phase separations. On the other hand the microsomal fraction from organisms grown with 9% ethanol showed a remarkable increase in fluidity. These data suggest that the protective effects of growth with ethanol near the limit of tolerance on fermentative activities may arise from altered plasma membrane fluidity properties.     

常压室温等离子体诱变选育耐酸酿酒酵母菌株

利用常压室温等离子体(ARTP)诱变方法对实验室保藏的酿酒酵母(Saccharomyces cerevisiae)SC-62进行诱变,通过试验确定最佳诱变条件为处理时长80 s,此条件下菌株SC-62致死率84%。将诱变获得的菌株进行初筛、复筛和发酵性能测定。结果显示,筛选出一株耐酸性强、发酵性能优良的正突变菌株A-107,其在pH为2.5的发酵培养基上培养6 d后测得的发酵力[6.21 g CO2/(100 mL·24 h)]和酒精产量(11.52%vol)较出发菌株SC-62分别提高了37%和30%,突变菌株A-107可耐受16%乙醇、100 g/L NaCl、500 g/L葡萄糖,耐受性和遗传稳定性良好。     

常压室温等离子体诱变选育耐酸酿酒酵母菌株

利用常压室温等离子体(ARTP)诱变方法对实验室保藏的酿酒酵母(Saccharomyces cerevisiae)SC-62进行诱变,通过试验确定最佳诱变条件为处理时长80 s,此条件下菌株SC-62致死率84%。将诱变获得的菌株进行初筛、复筛和发酵性能测定。结果显示,筛选出一株耐酸性强、发酵性能优良的正突变菌株A-107,其在pH为2.5的发酵培养基上培养6 d后测得的发酵力[6.21 g CO2/(100 mL·24 h)]和酒精产量(11.52%vol)较出发菌株SC-62分别提高了37%和30%,突变菌株A-107可耐受16%乙醇、100 g/L NaCl、500 g/L葡萄糖,耐受性和遗传稳定性良好。     

Disruption of URA7 and GAL6 improves the ethanol tolerance and fermentation capacity of Saccharomyces cerevisiae

Screening of the homozygous diploid yeast deletion pool of 4741 non-essential genes identified two null mutants (Deltaura7 and Deltagal6) that grew faster than the wild-type strain in medium containing 8% v/v ethanol. The survival rate of the gal6 disruptant in 10% ethanol was higher than that of the wild-type strain. On the other hand, the glucose consumption rate of the ura7 disruptant was better than that of the wild-type strain in buffer containing ethanol. Both disruptants were more resistant to zymolyase, a yeast lytic enzyme containing mainly beta-1,3-glucanase, indicating that the integrity of the cell wall became more resistance to ethanol stress. The gal6 disruptant was also more resistant to Calcofluor white, but the ura7 disruptant was more sensitive to Calcofluor white than the wild-type strain. Furthermore, the mutant strains had a higher content of oleic acid (C18 : 1) in the presence of ethanol compared to the wild-type strain, suggesting that the disruptants cope with ethanol stress not only by modifying the cell wall integrity but also the membrane fluidity. When the cells were grown in medium containing 5% ethanol at 15 degrees C, the gal6 and ura7 disruptants showed 40% and 14% increases in the glucose consumption rate, respectively.