葡萄酒的理化性质对酒酵母的影响

葡萄酒的理化性质对酒酵母的影响 Influence of wine-related physicochemical factors on the growth and metabolism of non-Saccharomyces and Saccharomyces yeasts in mixed culture 葡萄酒的理化学因素对葡萄酒中非酵母属的酵母在混合体系中的增 长和新陈代谢的影响 Abstract(摘要) The influence of two physicochemical factors involved in winemaking, temperature and SO2, on the kinetics and metabolic behavior of Kloeckera apiculata and Saccharomyces cerevisiae was examined. (检验参与酿酒的两个理化因素:温度和SO2，对柠檬形克勒克酵母和酿酒酵母 在动力学和代谢行为方面的影响。) Highest biomass was reached at 15 and 25? for K. Apiculata and S.cerevisiae, respectively. Pure cultures of K. Apiculatadied off early with increasing temperature, but in co-culture withS. cerevisiaeit showed higher viability and a change in the death curve from exponential to linear. (柠檬形克勒克酵母和酿酒酵母分别在温度达到15?、25?时菌落数达到最高。柠檬形克 勒克酵母纯培养，菌落数会随着温度的升高而降低，但是与酿酒酵母混合培养时，则可显示 出较高的生存能力。死亡曲线也会呈指数性变化。)Statistical analysis revealed that metabolite production was significantly different for the three cultures and also at the different fermentation temperatures. Besides, the interaction between culture type and temperature was significant. (统 计分析表明，三个组分的代谢产物具有显著的差异，且发酵温度不同。此外，各组分之间的 交互类型和温度是显著的。)At temperatures from 15 to 30? the mixed culture showed similar ethanol and lower acetic acid production compared with a pure culture ofK. apiculata.SO2 addition slightly increased survival of the non-Saccharomyces species in pure and mixed cultures. (温度为15-30?时，混合培养与柠檬形克勒克酵母纯培养相比，乙醇的产量相似，乙酸的 产量较低。在纯培养与混合培养过程中，SO2的少量添加，可以增加非酿酒酵母的存活率。) Statistical evaluation indicated that culture type and SO2 addition significantly affected metabolite production, but the interaction between culture and SO2 was not significant. (统计分析表明，组 分类型与SO2添加量对代谢产物的影响显著，但是组分与SO2的交互作用影响不显著。) These results contribute to current knowledge of enological factors and their effect on prevalence and fermentative activities of the composite yeast flora and the statistical significance emphasizes the importance of the combined influence of the culture type and physicochemical factors on the production of fermentation metabolites.(这些结果，有助于了解葡萄酒工程的影响因素及他们 对植物复合酵母的生长率及促发酵能力的影响，且统计学分析强调了各复合组分类型和理化 因素对发酵代谢产物产量的综合影响的重要性。) Keywords(关键词) K. apiculata (柠檬形克勒克酵母) Mixed culture(混合组分) Temperature (温度)Sulfur dioxide (二氧化硫) Winemaking(酿酒) Introduction() In the production of wine, natural grape juice fermentation is carried out by a succession of different yeast populations. (在葡萄酒生产过程中,一系列不同的酵母菌种群对天然葡萄汁进 行发酵。)Recently, several groups have examined various non-Saccharomyces yeasts as potential adjuncts (or alternatives) to Saccharomyces cerevisiaein an effort to modify wine flavor and improve product quality [18, 21, 31]. (最近,一些组织已经检验了各种非酿酒酵母作为潜在的 辅料(或替代)帮助酿酒酵母，努力修改酒风味,提高产品质量。)Apiculate wine yeasts (K. apiculata/Hanseniaspora uvarumand Hanseniaspora guilliermondii) have become an object of interest as they are frequently found in grapes and are also dominators of the early stages of must fermentation [15, 17]. (最好的葡萄酒酵母(柠檬形克勒克酵母、葡萄汁有孢汉逊酵母 、季 也蒙有孢汉逊酵母)已经成为一个有趣的对象，它们常常在葡萄酒发酵的早期阶段被发现， 并且是引导发酵的主宰者。)Their intolerance to high concentrations of ethanol, the high sugar concentration and the low available oxygen conditions during fermentation are the main reasons why S. Cerevisiae becomes dominant and keeps its activity until the end of fermentation [8, 10, 14, 18, 23, 32]. (在发酵过程中，它们不能耐受高浓度的乙醇,高浓度糖和低氧的条件，这就是 酿酒酵母可以成为主导者并且可以保持活性知道发酵的最后的原因。)Mendoza et al. [24] reported that non-Saccharomyces apiculate yeast and Saccharomyces strain growing together reached lower maximal biomass than its respective pure cultures, but the apiculate cell showed longer survival. (Mendoza et al.报道指出，,非酵母属的细尖酵母和酿酒酵母菌株共同培养可 达到的最大菌落数比它们各自的纯培养低。但是细尖细胞具有较长的细胞周期。)Thus, the metabolites formed by this non-Saccharomyces species may contribute to wine quality. The persistence of non-Saccharomyces fermentation species during fermentation may depend, however, upon many factors. (因此，由这个非酵母属菌种产生的代谢物可能有助于葡萄酒 的质量。非酿酒发酵菌种的持久性在发酵过程中也许是可信赖的，当然，是在考虑很多因素 的基础上)The fermentation temperature is one of the important vinification factors that affect the rate of yeast growth and the alcoholic fermentation. Also the number of different species, as well as their endurance during alcoholic fermentation, is conditioned by both the temperature of the must and the temperature during fermentation. (发酵温度是在葡萄酒酿造中非常重要的影响 因素，它可以影响酵母的生长速率和酒精发酵过程。不同菌种的数量以及它们在酒精发酵过 程中的耐受能力也都受制于必需生长温度和发酵温度。)These changes determine the chemical and organoleptic qualities of the wine [14].(这些变化都决定了葡萄酒的化学品质和感官品 质。) Addition of SO2to grapes or must to control oxidation reactions and restrict the growth of the indigenous yeast population is a well established practice in winemaking [1] and so is inoculation of the grape juice with selected yeasts. (在酿酒过程中添加SO2，对葡萄或者未发酵葡萄汁控制 氧化反应，限制本地酵母菌数量增长是一个很好的常用。所以要在葡萄之中接种选定的 酵母。)Sulfur dioxide is highly toxic to most non-Saccharomyces yeasts, while production strains of Saccharomyces in general are quite resistant to it [2, 13, 30, 36]. The total concentration of SO2 in grape juice during fermentation consists of bound and free forms. (对大多数非酿酒酵母而言， 二氧化硫是有剧毒的，普通的酵母属生产菌株对它是有抗性的。二氧化硫在葡萄汁发酵过程 中有结合态和游离态两种存在形式。)The free SO2will consist mostly in bisulfite anion and a small proportion of molecular SO2. The undissociated molecular form of free sulfur dioxide is the most important antimicrobial agent. (游离的二氧化硫主要是以酸性亚硫酸盐阴离子和一个少 量的小分子二氧化硫形式存在。以未离解的分子形式存在的二氧化硫是最重要的抗菌剂。) At pH 3.0–4.0, it exists mainly as the bisulfite ion (94–98%), and only a very small proportion (2–6%) occurs in the molecular form [30]. Generally, it accepts that 0.5 to 2.0 mg/l of molecular SO2 is necessary to obtain a good biological stability.(在pH值为3.0——-4.0时,它的以亚硫酸 氢离子(94 - 98%)的形式存在,只有很小的比例(2 - 6%)是以分子形式存在。通常认为0.5到2.0 mg / l的二氧化硫分子浓度，对保持一个良好的生物稳定性是有必要的。) Several studies have reported on the influence of vinification conditions on the dynamics of yeast populations and their effects on the characteristics of finished wines [10, 11, 23]. (几项研 究已经报道了葡萄酒酿造条件对发酵酵母菌落生长动态及成品葡萄酒特征的影响。) However, few studies have carried out a statistical evaluation of the effect of enological factors on growth and physiology of the microorganisms involved [27].(然而,，很少有研究对葡萄酒工程 的影响因素对参与发酵的微生物的生理特性及其生长繁殖的影响进行了统计。) The aim of the present work was to study the influence of different fermentation temperatures and sulfur dioxide concentrations on the kinetics and metabolic behavior of a mixed culture of Saccharomycesand non-Saccharomyces wine yeasts. (现阶段工作的目的是研究不同发酵温度 和二氧化硫浓度对酿酒和非酿酒葡萄酒酵母混合培养的动力学及其代谢行为的影响。) Statistical analysis was applied to evaluate the correlation between the different factors and culture type on the fermentative activities of the microorganisms.(统计分析是用于评价不同因素和各 组分微生物发酵行为之间的相关性。) Materials and methods(材料与方法) Microorganisms(微生物) Kloeckera apiculata mc1 and S. Cerevisiae mc2 were isolated from Argentinean wines. (从 阿根廷葡萄酒分离得到克勒克酵母mc1和酿酒酵母酵母mc2 。 Culture medium and growth conditions.培养基和生长条件。) The wine yeasts were inoculated in basal medium (10 g/l yeast extract, 1 ml/l tween 80 and 170 ml/l grape juice),pH was adjusted to 3.5. The medium was heated at 90? for 10 min and then 6inoculated with 10cells/ml of 14 h pre-cultures grown in the same medium as follows(酿酒酵 母接种在基础培养基(10 g / l酵母提取物,1毫升/ l吐温80和170毫升/ l葡萄汁),pH值调整 6到3.5。将培养基在90?加热10分钟,然后在相同培养基中接种10细胞/毫升纯培养14 h 后 的接种液，如下: (1) pure cultures of K. Apiculata and S. Cerevisiae and (2) a mixed culture of both yeast strains cultured at a 1:1 ratio.( (1)克勒克酵母和酿酒酵母酵母的纯培养液，(2)两个 酵母菌株比例为1:1的混合培养液。) All cultures were incubated statically at 25? during 10 days. When the temperature effect was studied, incubation was carried out at 15, 25, 30 and 35?(所有的菌株都是在.25?静态培养10 天。对温度效应进行了研究,潜伏期温度设定为15、25和35?、30。) In order to study the influence of SO2the basal medium was supplemented with 50, 100 and 250 mg/l of sodium metabisulfite. (为了研究SO2的影响，基础培养基增补50、100和250毫克 /升的焦亚硫酸钠。) Samples were taken every day during fermentation and yeast growth was monitored by successive dilutions. Differential enumeration of wine yeasts was carried out by plating them onto selective malt agar medium (20 g/l glucose, 1 g/l peptone, 20 g/l yeast extract, 3 g/l malt extract and 20 g/l agar); pH 6.8. (在发酵过程中，每天都要采样，并且通过连续稀释对酵母的生长 进行监测。酿酒酵母的分离纯化，是通过平板接种到麦芽汁琼脂培养基(20 g / l葡萄糖、 1 g / l蛋白胨、20 g / l酵母提取物、3 g / l麦芽提取物和20 g / l琼脂)进行选择性培养;pH值为 6.8。)This medium allows non-Saccharomyces species to be distinguished morphologically from Saccharomyces colonies. Plates were incubated at 25? for 3–4 days. (这种培养基可以从形态 上区别出非酵母属的微生物菌落与酵母菌菌落。在25?下，在培养板培养3 - 4天。)S. cerevisiae developed brilliant, white-cream colonies with spherical and protuberant forms and regular borders.(酵母菌菌落呈乳白色、有光泽的凸起球形，菌落边缘整齐。)K. Apiculata colonies were of an opaque white–gray color, smoothed or flattened with slightly irregular borders, which differed in tonality of the rest of the colony [16].(克勒克酵母菌菌落与其他菌落 不太一样，是不透明的灰白色，平滑或者扁平状，菌落边缘不整齐。) Analytical determinations (分析检测) Determination of the concentrations of sugars and fermentation products (测定糖的浓度与 发酵产物) Cell-free samples were obtained by centrifugation of the growth medium.(将培养液离心处 理，得到脱细胞样品。) Samples were stored at -20 ?until analysis. (进行分析前，将样品 于-20?储藏。)The ethanol, glucose, fructose, acetic acid and glycerol concentrations were determined by HPLC (ISCO 2350, Software Peak Simple II) using a column HPX.87H(Bio-Rad, 30097.8 mm). (乙醇、葡萄糖、果糖、乙酸和丙三醇浓度由高效液相色谱(ISCO 2350,软件 峰简单II)测定，色谱柱规格为HPX。87H(bio rad 30097.8毫米))。The column was eluted at 45?with a degassed mobile phase containing 5 mM H2SO4at a flow rate of 0.6 ml/min.(色谱柱 用脱气后的5 mM H2SO4以0.6 ml/min的流速，在45?时，进行洗脱。) All the compounds were determined with a RI detector (Knauer). (所有化合物均采用折射率侦测器 (Knauer)测 定。)Samples were analyzed in duplicate. The identification and quantification of compounds were carried out by comparing retention time and concentration with standard solution (Sigma)(样品一式两份，进行分析。通过与溶液比较保留时间和浓度，对化合物进行识别与量 化)