Screening of non-Saccharomyces wine yeasts for the presence of extracellular hydrolytic enzymes

Twenty-six strains of yeasts, belonging to the genera Candida, Debaryomyces, Hanseniaspora, Hansenula, Kloeckera, Metschnikowia, Pichia, Saccharomyces and Torulaspora previously isolated from wines, were screened for the production of extracellular pectinases, amylases, lipases, proteases and β-glucosidases. Some strains of Candida species and Hanseniaspora uvarum/Kloeckera apiculata produced extracellular proteolytic or lipolytic activities. Most yeasts exhibited β-glucosidase activity, but particularly high activity was observed in strains of Pichia anomala/Candida pelliculosa (formerly Hansenula anomala ) and Hanseniaspora uvarum/Kloeckera apiculata . The potential impact of these enzymes on wine quality is discussed.     

Amino acid uptake by wild and commercial yeasts in single fermentations and co-fermentations

Musts require nitrogen-containing compounds in order to ensure yeast development. This study examined the nitrogen-nutrient requirements of two commercial yeasts and three wild strains isolated from inoculated fermentations. The results showed that wild strains generally consumed lower amounts of amino acids than commercial yeasts. Most amino acids were assimilated during the exponential growth phase; only a few – including asparagine and histidine – were metabolized until the end of fermentation. The study also sought to determine whether industrial drying affected yeast nitrogen requirements.     

Cork-borne bacteria and yeasts as potential producers of off-flavours in wine

Bacteria and yeasts were found to be present within cork lenticels, covered by mucous or fibrous substances. They survived heating, peroxide treatment and contact with the alcohol and sulfur dioxide of wine. 187 bacteria and 36 yeast strains were isolated from cork stoppers of wine bottles and, during various stages of production, from corkwood and new cork stoppers. After culturing, a number of isolates showed the ability to modify the aroma of model systems consisting of dilute or full strength wine and pulverised cork. The aromas produced by isolates of varying cork origin are tabled. A small number of isolates methylated 2,4,6-trichlorophenol, yielding 2,4,6-trichloroanisole, responsible for the typical cork taint. During the boiling of cork slabs, the internal temperature on the inside of a box made from cork slices did not exceed 87°C.     

昌黎产区产酶酵母多样性及其应用潜力分析

利用优选酵母混菌发酵,是改善葡萄酒品质及增香的有效途径。以赖氨酸培养基、WL培养基和产酶筛选培养基为基础,分析了昌黎4个葡萄园土样中产酶酵母的多样性,结果表明,该地区产酶酵母丰富,50%的产酶酵母能同时产2种或2种以上的酶。对产酶酵母酿造因子耐性分析表明,菌株GY1、M9和J24具有高糖耐性,适合于高糖低醇果酒酿造;菌株GY13和M9具有高酒精耐性,酒精含量为15.000%时活性较强;菌株J24和GY1具有高酸耐性,pH 2.5时具有较高活性;7株菌15℃时具有较高活性。综合分析发现,菌株M9(Torulaspora delbrueckii)同时产β-葡萄糖苷酶、果胶酶和纤维素酶,除酸耐性较差外,具有高糖、高酒精度和低温耐性,在高糖低醇果酒酿造中具有很大应用潜力。     

Volatile composition and aromatic attributes of wine made with Vitisvinifera L.cv Cabernet Sauvignon grapes in the Xinjiang region of China: effect of different commercial yeasts

The Xinjiang region is a major grape- and wine-production area in China, but the region’s notably high temperatures in the summer and year-round intense sun exposure play negative roles in the aroma, complexity, and elegance of Cabernet Sauvignon wine. In this study, Cabernet Sauvignon grapes harvested in this region were fermented on an industrial scale using four commercial yeast strains (L2323, D254, RVA, and CECA) and spontaneous yeast (NF). The results showed that a total of 123 volatile compounds were detected and 15 volatile compounds significantly contributed their flavor notes to the wine’s overall aroma. The use of RVA and CECA strains resulted in wine with higher concentrations of higher alcohols, terpenes and norisoprenoids. However, the D254-fermented wine showed high level of esters and carbonyl compounds. Wine fermented with the L2323 and D254 strain possessed a stronger fruity aroma, whereas the RVA strain enhanced the herbaceous, chemical, and fatty aromas in wine. Principal component analysis revealed that a significant aromatic feature difference was observed in these wines after alcoholic and malolactic fermentation. The use of different commercial yeast strains altered the aromatic profile of Cabernet Sauvignon wine.     

Modulation of Glycerol and Ethanol Yields During Alcoholic Fermentation in Saccharomyces cerevisiae Strains Overexpressed or Disrupted for GPD1 Encoding Glycerol 3-Phosphate Dehydrogenase

The possibility of the diversion of carbon flux from ethanol towards glycerol in Saccharomyces cerevisiae during alcoholic fermentation was investigated. Variations in the glycerol 3-phosphate dehydrogenase (GPDH) level and similar trends for alcohol dehydrogenase (ADH), pyruvate decarboxylase and glycerol-3-phosphatase were found when low and high glycerol-forming wine yeast strains were compared. GPDH is thus a limiting enzyme for glycerol production. Wine yeast strains with modulated GPD1 (encoding one of the two GPDH isoenzymes) expression were constructed and characterized during fermentation on glucose-rich medium. Engineered strains fermented glucose with a strongly modified [glycerol] : [ethanol] ratio. gpd1Δ mutants exhibited a 50% decrease in glycerol production and increased ethanol yield. Overexpression of GPD1 on synthetic must (200 g/l glucose) resulted in a substantial increase in glycerol production (×4) at the expense of ethanol. Acetaldehyde accumulated through the competitive regeneration of NADH via GPDH. Accumulation of by-products such as pyruvate, acetate, acetoin, 2,3 butane-diol and succinate was observed, with a marked increase in acetoin production. © 1997 John Wiley & Sons, Ltd.     

Effect of aromatic precursor addition to wine fermentations carried out with different Saccharomyces species and their hybrids

This work explores the ability of different yeast strains from different species of the genus Saccharomyces (S. cerevisiae, S. uvarum and S. kudriavzevii) and hybrids between these species to release or form varietal aroma compounds from fractions of grape odourless precursors. The de novo synthesis by the yeasts of some of the varietal aroma compounds was also evaluated. The study has shown that de novo synthesis affects some lipid derivatives, shikimic derivatives and terpenes in all species and hybrids, with some remarkable differences amongst them. The release or formation of aroma compounds from precursors was found to be strongly linked to the yeast or hybrid used, and the triple hybrid S. cerevisiae × S. bayanus × S. kudriavzevii in particular and secondarily the hybrid S. cerevisiae × S. bayanus were highly efficient in the production of most varietal aroma compounds, including γ-lactones, benzenoids, volatile phenols, vanillin derivatives and terpenols. The presence of precursors in the fermenting media caused a surprising levelling effect on the fermentative aroma composition. Altogether, these results suggest that it is possible to modulate wine aroma by employing different yeast species in order to create new wines with different aromatic notes.     

酒精发酵过程中酿酒酵母海藻糖代谢的研究

研究了酿酒酵母在酒精发酵过程中酵母细胞内海藻糖的代谢。结果表明海藻糖的代谢受几种因素如底物浓度、发酵温度以及其他条件的调节与控制。在试验中对酿酒酵母细胞内海藻糖在整个酒精发酵过程中的生物功能作了简要的评价。     

Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae

Non-Saccharomyces yeasts are metabolically active during spontaneous and inoculated must fermentations, and by producing a plethora of by-products, they can contribute to the definition of the wine aroma. Thus, use of Saccharomyces and non-Saccharomyces yeasts as mixed starter cultures for inoculation of wine fermentations is of increasing interest for quality enhancement and improved complexity of wines. We initially characterized 34 non-Saccharomyces yeasts of the genera Candida, Lachancea (Kluyveromyces), Metschnikowia and Torulaspora, and evaluated their enological potential. This confirmed that non-Saccharomyces yeasts from wine-related environments represent a rich sink of unexplored biodiversity for the winemaking industry. From these, we selected four non-Saccharomyces yeasts to combine with starter cultures of Saccharomyces cerevisiae in mixed fermentation trials. The kinetics of growth and fermentation, and the analytical profiles of the wines produced indicate that these non-Saccharomyces strains can be used with S. cerevisiae starter cultures to increase polysaccharide, glycerol and volatile compound production, to reduce volatile acidity, and to increase or reduce the total acidity of the final wines, depending on yeast species and inoculum ratio used. The overall effects of the non-Saccharomyces yeasts on fermentation and wine quality were strictly dependent on the Saccharomyces/non-Saccharomyces inoculum ratio that mimicked the differences of fermentation conditions (natural or simultaneous inoculated fermentation).     

桓仁产区优良冰酒酵母菌株的筛选及酿造特性评价（英文）

为获得可用于东北桓仁地区威代尔冰酒生产的酿酒酵母菌株,采用一种快速的酵母菌株筛选方法,通过测定菌株乙醇和二氧化硫耐受性,从威代尔冰酒自然发酵过程中筛选到9株酿酒酵母菌株。进一步酿造实验结果显示,所筛选的酿酒酵母可以顺利完成冰酒发酵,产生的香气轮廓与商业酵母DV10相比也不同(主成分分析结果),最终获得了2株具有高发酵活力且香气特征与商业酵母差异显著的酵母菌株SC42和SC45,其中SC42能够高产高级醇和酯类物质,并且低产乙酸,而SC45能够产生高含量的甘油、酯类物质以及反式玫瑰醚和β-大马士酮。结果表明,采用本研究的筛选方法能够快速有效地筛选到具有应用潜力的冰酒生产菌株,同时也证明了使用本土野生酵母菌株能够有效地改善冰酒香气品质,生产出与接种商业酵母不同风格的冰酒产品。     

产4-羟基-2,5-二甲基-3(2H)-呋喃酮酵母菌的分离、筛选及分子生物学鉴定

以菠萝、草莓等水果为分离源,分离、筛选利用D-果糖产香料HDMF酵母菌株,并对其高产菌株进行分子生物学鉴定。采用稀释涂布法分离酵母菌株,高效液相色谱法检测HDMF产量,26SrDNAD1/D2区序列分析及系统发育分析鉴定菌株。共分离得到46株酵母,5株可利用D-果糖产HDMF;产量较高的两株菌为:C5(6.84mg/L)、P3(10.96mg/L),分别约为已报道毕赤氏酵母属(Pichia capsulata)利用L-(+)-鼠李糖产HDMF的3倍和5倍;26S rDNA D1/D2区序列分析及系统发育分析结果显示,P3与Pichia caribbica(毕赤酵母属),C5与Hanseniaspora sp.(有孢汉逊酵母属)相似性均在99%以上,分子生物学法鉴定P3为Pichia caribbica,C5为Hanseniaspora sp.     

Cloning and sequencing of the Saccharomyces cerevisiae gene LYP1 coding for a lysine-specific permease

The LYP1 gene of Saccharomyces cerevisiae was cloned by complementation in lysine-permease-deficint recipient yeast cells, and its nucleotide sequence was determined. An open reading frame of 1833 nucleotides was found encoding a polypeptide of 611 amino acids, with a calculated molecular weight of 68 118. Analysis of the deduced primary structure of the protein revealed ten membrane-spanning regions and three potential N-glycosylation sites. Analysis of the deduced sequence of protein LYP1 indicates homology with other yeast amino-acid permeases, in particular with CAN1, and also the lysine-specific permease of Escherichia coli. The strain transformed by a multi-copy plasmid harbouring the LYP1 gene, showed a 20-fold increase in the maximum velocity of lysine uptake over that in the wild type, with no changes in the affinity of the permease for its substrate.     

Comparison of expression systems in the yeasts Saccharomyces cerevisiae,Hansenula polymorpha,Klyveromyces lactis,Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalbøge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4·5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1α and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters, Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning. GenBank Accession Numbers: TEF gene promoter sequence: AF054508; RPS7 gene promoter sequence: AF054509. © 1998 John Wiley & Sons, Ltd.     

酿酒酵母的发酵优化与动力学研究

研究了酿酒酵母在游离生长条件下细胞生长、乙醇生成和葡萄糖消耗动力学.通过耐高温酿酒活性干酵母在35℃的复合培养基中不通风生长的三水平正交实验,用Gauss-Newton非线性最小二乘法拟合了发酵动力学参数.当发酵液中的葡萄糖浓度远大于饱和生长系数Ks时,酵母细胞的生长代谢规律受葡萄糖的影响很小.乙醇总是减缓酿酒酵母的生长代谢速率,当乙醇浓度达到完全抑制值KP时细胞就停止生长.酵母细胞的自由生长存在极限浓度Kx,而在细胞浓度为Kx/2处获得最大细胞增长率.当发酵液中乙醇浓度低于KP[1-β/(αμmax)]时,在细胞浓度为Kx[1-β/(αμmax)]/2处获得最大乙醇发产率.而高于此乙醇浓度时,乙醇产率随细胞浓度单调增加.在发酵动力学方程的基础上,可根据发酵目标对发酵条件进行优化.     

红酵母的鉴定及其固态发酵产类胡萝卜素的研究

利用26SrDNAD1/D2区域序列分析法对黄酒酒糟中筛选出的红酵母b-5进行序列比对鉴定,结果表明,该酵母菌的序列与黏性红圆酵母(Rhodotorulamucilaginosa)模式菌株的序列同源性100%。通过固态发酵该菌生产类胡萝卜素条件的优化研究和探讨变温培养对固态发酵的影响,初步确定黏性红圆酵母RM-1固态发酵的最适条件:籼米:水=1:2.5、1%葡萄糖,pH6.5、15%接种量、培养时间96h、前48h发酵温度33℃、后48h发酵温度28℃,此时类胡萝卜素产量可达11.73μg/g干基,与恒温培养相比较,类胡萝卜素产量提高了44%,可为工业生产类胡萝卜素提供参考。     

Application of pure and mixed probiotic lactic acid bacteria and yeast cultures for oat fermentation

Fermentation of a prebiotic containing oat substrate with probiotic lactic acid bacteria and yeast strains is an intriguing approach for the development of new synbiotic functional products. This approach was applied in the present work by using pure and mixed microbial cultures to ferment a heat‐treated oat mash. Results show that the strains studied were appropriate for oat fermentation and the process could be completed for 6–10 h depending on the strain. The viable cell counts achieved within this time were above the required levels of 10⁶–10⁷ cfu ml⁻¹ for probiotic products. Both single lactic acid bacteria strains and mixed cultures of the same strains with yeast were found suitable for oat fermentation. However, the pure LAB cultures attributed better flavour and shelf life of the oat drinks. The content of the prebiotic oat component beta‐glucan remained within 0.30–0.36% during fermentation and storage of the drinks obtained with each of the strains used. Thus, these products would contribute diet with the valuable functional properties of beta‐glucan. Also, the viability of pure and mixed cultures in the oat products was good: levels of cell counts remained above the required numbers for probiotic products throughout the estimated shelf‐life period. Copyright © 2005 Society of Chemical Industry     

Review: The Cct eukaryotic chaperonin subunits of Saccharomyces cerevisiae and other yeasts

All eight of the CCT1-CCT8 genes encoding the subunits of the Cct chaperonin complex in Saccharomyces cerevisiae have been identified, including three that were uncovered by the systematic sequencing of the yeast genome. Although most of the properties of the eukaryotic Cct chaperonin have been elucidated with mammalian systems in vitro, studies with S. cerevisiae conditional mutants revealed that Cct is required for assembly of microtubules and actin in vivo. Cct subunits from the other yeasts, Candida albicans and Schizosaccharomyces pombe, also have been identified from partial and complete DNA sequencing of genes. Cct8p from C. albicans, the only other completely sequenced Cct protein from a fungal species other than S. cerevisiae, is 72% and 61% similar to the S. cerevisiae and mouse Cct8 proteins, respectively. The C. albicans CCT8 sequence has been assigned the Accession Number U37371 in the GenBank/EMBL database.