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).     

非酿酒酵母与酿酒酵母混合发酵对葡萄酒香气的影响

为研究自选非酿酒酵母对葡萄酒香气物质的影响,选取毕赤克鲁维酵母（Pichia kluyveri）HSX-5、长孢洛德酵母（Lodderomyces elongisporus）MNS-6和戴尔有孢圆酵母（Torulaspora debrueckii）YQX-8与酿酒酵母（Saccharomyces cerevisiae）AWRI 796混合发酵赤霞珠葡萄酒,采用顶空固相萃取-气相色谱质谱联用（HS-SPME-GC-MS）技术检测其挥发性香气物质,并对结果进行主成分分析（PCA）。结果表明,相较于酿酒酵母单菌发酵酒样,菌株HSX-5和YQX-8混合发酵酒样中己酸乙酯提升了2倍,菌株HSX-5、YQX-8、MNS-6混合发酵酒样中的辛酸乙酯分别提升76.6%、35.5%、5.5%,乙酸乙酯含量增加均为10%,混合发酵增强了葡萄酒花果类香气及醇香;同时混合发酵各组均产生了新物质,增加了葡萄酒风味体系的丰度。PCA结果表明,菌株HSX-5和YQX-8混合发酵的发酵酒样主要贡献香气种类较多且气味活度值（OAV）较高,对葡萄酒香气的影响显著。     

Multi-enzyme production by pure and mixed cultures of Saccharomyces and non-Saccharomyces yeasts during wine fermentation

Saccharomyces and non-Saccharomyces yeasts release enzymes that are able to transform neutral compounds of grape berries into active aromatic compounds, a process that enhances the sensory attributes of wines. So far, there exists only little information about enzymatic activity in mixed cultures of Saccharomyces and non-Saccharomyces during grape must fermentations. The aim of the present work was to determine the ability of yeasts to produce extracellular enzymes of enological relevance (β-glucosidases, pectinases, proteases, amylases or xylanases) in pure and mixed Saccharomyces/non-Saccharomyces cultures during fermentation. Pure and mixed cultures of Saccharomyces cerevisiae BSc562, Hanseniaspora vinae BHv438 and Torulaspora delbrueckii BTd259 were assayed: 1% S. cerevisiae/99% H. vinae, 10% S. cerevisiae/90% H. vinae, 1% S. cerevisiae/99% T. delbrueckii and 10% S. cerevisiae/90% T. delbrueckii. Microvinifications were carried out with fresh must without pressing from Vitis vinifera L. c.v. Pedro Jiménez, an autochthonous variety from Argentina. Non-Saccharomyces species survived during 15-18days (BTd259) or until the end of the fermentation (BHv438) and influenced enzymatic profiles of mixed cultures. The results suggest that high concentrations of sugars did not affect enzymatic activity. β-Glucosidase and pectinase activities seemed to be adversely affected by an increase in ethanol: activity diminished with increasing fermentation time. Throughout the fermentation, Saccharomyces and non-Saccharomyces isolates assayed produced a broad range of enzymes of enological interest that catalyze hydrolysis of polymers present in grape juice. Vinifications carried out by a pure or mixed culture of BTd259 (99% of T. delbrueckii) showed the highest production of all enzymes assayed except for β-glucosidase. In mixed cultures, S. cerevisiae outgrew H. vinae, and T. delbrueckii was only detected until halfway the fermentation process. Nevertheless, their secreted enzymes could be detected throughout the fermentation process. Our results may contribute to a better understanding of the microbial interactions and the influence of some enzymes on vinification environments.     

非酿酒酵母与酿酒酵母混合发酵柿子酒特性的研究

基于非酿酒酵母代谢产物可提升酒的香气和风味物质,将两种非酿酒酵母柠檬形克勒克酵母（Klockera apiculata）和东方伊萨酵母（Issatchenkia orientalis）用于柿子酒发酵,研究两种非酿酒酵母对酒精、二氧化硫、单宁的耐受性和产β-葡萄糖苷酶的能力;对它们分别与酿酒酵母（Saccharomyces cerevisiae）混合发酵柿子酒的特性进行研究。结果表明,东方伊萨酵母在各方面耐受性均优于柠檬形克勒克酵母,但产β-葡萄糖苷酶的能力却较弱;两种非酿酒酵母单菌发酵性能都较弱,东方伊萨酵母单菌发酵结束酒精度为4.51%vol,而柠檬形克勒克酵母单菌发酵酒精度只有3.05%vol;两种非酿酒酵母与酿酒酵母混菌发酵7 d后,酒精度均达到10%vol以上。     

Cellular death of two non-Saccharomyces wine-related yeasts during mixed fermentations with Saccharomyces cerevisiae

The early death of two non-Saccharomyces wine strains (H. guilliermondii and H. uvarum) during mixed fermentations with S. cerevisiae was studied under enological growth conditions. Several microvinifications were performed in synthetic grape juice, either with single non-Saccharomyces or with mixed S. cerevisiae/non-Saccharomyces inocula. In all mixed cultures, non-Saccharomyces yeasts grew together with S. cerevisiae during the first 1-3 days (depending on the initial inoculum concentration) and then, suddenly, non-Saccharomyces cells began to die off, regardless of the ethanol concentrations present. Conversely, in both non-Saccharomyces single cultures the number of viable cells remained high (ranging 10(7)-10(8) CFU ml(-1)) even when cultures reached significant ethanol concentrations (up to 60-70 g l(-1)). Thus, at least for these yeast strains, it seems that ethanol is not the main death-inducing factor. Furthermore, mixed cultures performed with different S. cerevisiae/ H. guilliermondii inoculum ratios (3:1; 1:2; 1:10; 1:100) revealed that H. guilliermondii death increases for higher inoculum ratios. In order to investigate if the nature of the yeast-yeast interaction was related or not with a cell-cell contact-mediated mechanism, cell-free supernatants obtained from 3 and 6 day-old mixed cultures were inoculated with H. guilliermondii pure cultures. Under these conditions, cells still died and much higher death rates were found for the 6 days than for the 3 day-old supernatants. This strongly indicates that one or more toxic compounds produced by S. cerevisiae triggers the early death of the H. guilliermondii cells in mixed cultures with S. cerevisiae. Finally, although it has not been yet possible to identify the nature of the toxic compounds involved in this phenomenon we must emphasise that the S. cerevisiae strain used in the present work is killer sensitive with respect to the classical killer toxins, K1, K2 and K28, whereas the H. guilliermondii and H. uvarum strains are killer neutral.     

Outlining a future for non-Saccharomyces yeasts: selection of putative spoilage wine strains to be used in association with Saccharomyces cerevisiae for grape juice fermentation

The use of non-Saccharomyces yeasts that are generally considered as spoilage yeasts, in association with Saccharomyces cerevisiae for grape must fermentation was here evaluated. Analysis of the main oenological characteristics of pure cultures of 55 yeasts belonging to the genera Hanseniaspora, Pichia, Saccharomycodes and Zygosaccharomyces revealed wide biodiversity within each genus. Moreover, many of these non-Saccharomyces strains had interesting oenological properties in terms of fermentation purity, and ethanol and secondary metabolite production. The use of four non-Saccharomyces yeasts (one per genus) in mixed cultures with a commercial S. cerevisiae strain at different S. cerevisiae/non-Saccharomyces inoculum ratios was investigated. This revealed that most of the compounds normally produced at high concentrations by pure cultures of non-Saccharomyces, and which are considered detrimental to wine quality, do not reach threshold taste levels in these mixed fermentations. On the other hand, the analytical profiles of the wines produced by these mixed cultures indicated that depending on the yeast species and the S. cerevisiae/non-Saccharomyces inoculum ratio, these non-Saccharomyces yeasts can be used to increase production of polysaccharides and to modulate the final concentrations of acetic acid and volatile compounds, such as ethyl acetate, phenyl-ethyl acetate, 2-phenyl ethanol, and 2-methyl 1-butanol.     

Rapid asymmetrical evolution of Saccharomyces cerevisiae wine yeasts

Genetic instability causes very rapid asymmetrical loss of heterozygosity (LOH) at the cyh2 locus and loss of killer K2 phenotype in some wine yeasts under the usual laboratory propagation conditions or after long freeze-storage. The direction of this asymmetrical evolution in heterozygous cyh2(R)/CYH2(S) hybrids is determined by the mechanism of asymmetrical LOH. However, the speed of the process is affected by the differences in cell viability between the new homozygous yeasts and the original heterozygous hybrid cells. The concomitant loss of ScV-M2 virus in the LOH process may increase cell viability of cyh2(R)/cyh2(R) yeasts and so favour asymmetrical evolution. The presence of active killer K2 toxin, however, abolishes the asymmetrical evolution of the hybrid populations. This phenomenon may cause important sudden phenotype changes in industrial and pathogenic yeasts.     

Screening of non-Saccharomyces wine yeasts for the production of beta-D-xylosidase activity

Fifty-four yeast strains belonging to the genera Candida, Dekkera, Hanseniaspora, Metschnikowia, Pichia, Rhodotorula, Schizosaccharomyces and Zygosaccharomyces, mainly isolated from grapes and wines, were screened for the production of beta-D-xylosidase activity. Beta-D-xylosidase activity was only detected in eight yeast strains belonging to the genera Hanseniaspora (H. osmophila and H. uvarum) and Pichia (P. anomala). Beta-D-xylosidase preparations active against p-nitrophenyl-beta-D-xyloside were characterised with respect to their optimal pH and temperature conditions. H. uvarum 11105 and 11107 and P. anomala 10320 beta-D-xylosidase preparations were active at pH and temperature ranges and at concentrations of glucose and ethanol usually found during winemaking processes.     

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.     

梅鹿辄葡萄自然发酵酵母菌群研究

利用WL营养培养基对梅鹿辄葡萄自然发酵过程中分离到的98株酵母菌进行初步鉴定,进而对代表性的菌株进行5.8S rDNA-ITS区PCR产物的限制性内切酶酶切分析.结果表明,供试的98株酵母菌属于3属3个种,分别为酿酒酵母(Saccharomyces cerevisiae)、东方伊萨酵母(Issatchenkia orientalis)、克鲁维毕赤酵母(Pichia kluyver),3种酵母菌分别占分离自该葡萄品种总酵母菌株的80.61％、13.26％、6.13％.在自然发酵的初期和中期,酵母菌的比例因是否添加SO2而有所差异,但发酵末期均以S.cerevisiae为优势菌.     

非酿酒酵母在发酵过程中的代谢产物对葡萄酒质量的影响

非酿酒酵母广泛存在于葡萄园及酿酒环境中,虽然其在发酵过程中能产生各种异味物质影响酒的品质,但某些非酿酒酵母的代谢产物却能增强葡萄酒的香气、风味和复杂性。该文综述了非酿酒酵母在发酵过程中的代谢产物及其对葡萄酒质量的影响,以期对我国筛选非酿酒酵母用于生产具地区特色的葡萄酒提供参考。     

丝氨酸对葡萄酒酿造过程中酿酒酵母产H2S的形成研究

为探究丝氨酸对葡萄酒酿造过程中酿酒酵母产H2S的影响,以自主筛选的本土酿酒酵母32y12为研究对象,分别在正常氮源（300 mg N/L）和低浓度氮源（150 mg N/L）条件下,比较了外源丝氨酸的添加对酿酒酵母32y12发酵过程中产H2S的影响。结果显示,相对于正常氮源水平,低氮条件下外源添加140.8 mg/L（5倍）和281.5 mg/L（10倍）丝氨酸时,H2S释放量显著增加,分别达到573.1 μL/L和798.6 μL/L。为了进一步研究酵母自身丝氨酸合成对H2S产生的影响,借助Cre-Loxp系统敲除了丝氨酸合成关键基因SER33,结果发现在不同氮源水平下,基因SER33的敲除均显著降低H2S产量（P＜0.05）,并且外源添加不同浓度丝氨酸也并不会显著增加基因SER33敲除菌株的H2S释放量（P＞0.05）。该结果为工业生产中针对低氮葡萄原料选择发酵助剂提供了理论支持。     

野生猕猴桃果酒酵母的筛选鉴定及耐受性的研究

文中主要筛选适合野生猕猴桃果酒发酵的酵母.通过显微镜观察和WL培养基的颜色变化,初筛出酵母菌136株.采用杜氏管发酵法,CO2失重比较法,猕猴桃果汁发酵法等从中复筛出两株发酵能力好、絮凝能力强、产香较好的菌株:YM-7和YM-210,初步鉴定均为酵母属的酿酒酵母.对YM-7菌种进行了耐受性研究,结果表明YM-7对葡萄糖、酒精度、酸度、NaC1和SO2具有较高耐受性,可作为野生猕猴桃发酵专用菌种或选育野生猕猴桃专用菌种的出发菌株.     

不同有孢汉逊酵母与酿酒酵母混合发酵对威代尔冰葡萄酒香气的影响

为了研究菌株和接种方式对冰葡萄酒非挥发性产物和挥发性香气成分的影响,选用2株自筛非酿酒酵母——仙人掌有孢汉逊酵母(Hanseniaspora opuntiae)和葡萄汁有孢汉逊酵母(Hanseniaspora uvarum),分别与商业酿酒酵母DV10混合发酵。结果表明:H.uvarum与DV10同时接种混合发酵可以显著降低乙酸产量(降低26.90%);H.opuntiae与DV10同时接种混合发酵促进了酯类和萜烯类物质的合成,总量分别增产88.61%和21.40%,其中乙酸苯乙酯含量提高14.60倍,β-大马士酮增产8.85%。除此之外,感官品评结果显示H.opuntiae与DV10混合发酵可显著增强花果香和黄油味。综合分析,认为H.opuntiae更适合应用于冰葡萄酒发酵,为冰酒混合发酵的进一步研究提供了数据基础和理论依据。     

酵母菌FW-sc-08的生长及发酵特性研究

通过对影响菌体生长因素、发酵指标和香气成分的测定,研究了从自然发酵黑莓酒中筛选出的酵母菌FW-sc-08的生长和发酵特性。结果显示,酵母菌FM-sc-08对数生长期为3～9 h,对SO2最大耐受质量浓度为800 mg/L;酵母菌FM-sc-08适宜发酵的可发酵糖最大含量为30%,对糖最大耐受量为40%,可发酵酒精度最高16.3%vol。通过测定不同温度对发酵指标的影响,最终确定酵母菌FM-sc-08的最适发酵温度为25 ℃;对比20 ℃和25 ℃发酵的黑莓果酒酒样中的香气成分,可知酵母菌FM-sc-08在20 ℃发酵黑莓酒产香气成分较多（35种）。     

酿酒酵母ZGJ-1在猕猴桃果酒发酵中的研究及应用

为了将专利菌ZGJ-1在工业上进行广泛的应用并为猕猴桃产业开辟一条深加工新途径,分别以游离和固定化的酵母菌ZGJ-1为研究对象,采用单因素试验和正交试验,研究了猕猴桃果酒的最优发酵工艺条件。结果表明,固定化酿酒酵母ZGJ-1发酵果酒的最佳工艺参数为接种量5%,SO2添加量100 mg/L,起始糖度200 g/L。在此优化条件下,猕猴桃果酒酒精度为9.6%vol,残糖为32.28 g/L,发酵速度快,所产果酒香味浓郁,各项品质指标均达到相关国标标准。抗氧化能力试验结果表明,固定化酿酒酵母ZGJ-1发酵果酒对 ·OH、DPPH·、O2-·清除率分别为42%、96%、80%,显示其较好的抗氧化能力。     

河北产区优选酿酒酵母的中试研究

将分离自河北产区的3株本土优良葡萄酒酿酒酵母菌株NJ5、N19、NJ17用于葡萄酒中试发酵,考查不同酵母菌株的发酵性能、所发酵葡萄酒的理化特征、多酚类物质含量和挥发性香气成分组成.结果表明,3株本土酿酒酵母具有良好的发酵性能,可生产具有典型地域特征的葡萄酒,有望于用于河北地区特色葡萄酒的生产.