酒类酒球菌对葡萄酒中相关物质代谢的影响

酒类酒球菌是触发葡萄酒苹果酸-乳酸发酵(malolactic fermentation,MLF)的主要乳酸菌,其代谢产物如乳酸、乙酸、双乙酰、乙偶姻、生物胺等对葡萄酒的感官品质有重要作用。本文对酒类酒球菌在葡萄酒MLF过程的代谢情况进行总结,阐述糖、有机酸、含氮物质和酚类化合物的代谢情况,以及在不同影响因素下酒类酒球菌生长和代谢途径的改变状况,已经证明pH值、SO2、乙醇体积分数、葡萄糖/果糖比等都是影响代谢的因素。随着研究的不断深入,酚类化合物在代谢中的作用也受到越来越多的关注。酚类化合物一方面通过刺激或者抑制酒类酒球菌的生长而间接影响MLF的进行;另一方面,不同的酚类物质也会影响细菌对其他酚类的代谢,从而影响葡萄酒的口感和质量。通过全面综合了解酒类酒球菌在葡萄酒中的代谢情况有助于更好地调控MLF过程。     

The influence of malolactic fermentation and Oenococcus oeni strain on glycosidic aroma precursors and related volatile compounds of red wine

Free and glycosidically bound volatile compounds of red wine were measured after malolactic fermentation (MLF) with four different commercial starter cultures of Oenococcus oeni. MLF resulted in a significant decrease in the concentration of total glycosides, expressed as phenol‐free glycosyl glucose. Gas chromatographic analyses of wine enzyme hydrolysates showed that the extent of hydrolysis of glycosides during MLF was dependent on both bacterial strain and chemical structure of the substrate. The highest decrease was observed for glycosidic precursors of primary terpene alcohols. Glycoside‐related aroma compounds such as linalool, farnesol, and β‐damascenone were increased after MLF with all the bacterial strains tested. Two of the strains were also able to release significant amounts of vinylphenols during MLF. Copyright © 2006 Society of Chemical Industry     

Use of a polymerase chain reaction for specific detection of the malolactic fermentation bacterium Oenococcus oeni (formerly Leuconostoc oenos) in grape juice and wine samples

A PCR method has been developed that enables rapid and direct identification of the malolactic bacterium Oenococcus oeni from grape must or wine samples. Two primers, based on unique, highly conserved regions within the 16S rRNA gene of O. oeni , were used to amplify a 995 bp fragment which is specific for O. oeni . Other species of bacteria from Lactobacillus, Pediococcus and Acetobacter which may be found in grape must or wine were not detected using this technique. This diagnostic test is able to specifically detect in the order of 10³colony forming units per mL of O. oeni in a wine sample, and can be used for monitoring bacterial growth during malolactic fermentation.     

不同发酵条件对模拟葡萄酒中酒酒球菌柠檬酸代谢的影响

以酒酒球菌31-DH进行模拟酒苹果酸-乳酸发酵,使用离子排斥色谱法测定相关代谢物质的含量,研究苹果酸-乳酸发酵期间及发酵后p H、乙醇和葡萄糖对柠檬酸代谢的影响。结果表明:该方法耗时短、前处理简单、分离度良好、回收率为87%¹08%、精密度为0.77%⁴.33%,满足分析要求。当发酵液处于低p H,高酒精度,低葡萄糖浓度时,柠檬酸代谢缓慢,乳酸、乙酸及2,3-丁二醇的产量较低,双乙酰含量峰值较高。当发酵液中不含有乙醇时,苹果酸、柠檬酸、葡萄糖代谢迅速,伴随菌体的大量生长,乳酸、乙酸、2,3-丁二醇含量较高,但无双乙酰的产生。当发酵液中不含有葡萄糖时,柠檬酸代谢速率加快,乳酸产量较低,2,3-丁二醇及双乙酰产量较高。p H、乙醇、葡萄糖均显著影响酒酒球菌31-DH对柠檬酸的代谢。      

Potential formation of ethyl carbamate in simulated wine inoculated with Oenococcus oeni and Lactobacillus plantarum

The production of ethyl carbamate (EC) and its precursor citrulline by two strains of Oenococcus oeni and one of Lactobacillus plantarum during malolactic fermentation (MLF) was studied in different conditions of pH, temperature, and ethanol and l -malic acid concentrations. The potential EC, defined as the concentration of EC after wine is heated at 80 °C for 48 h, was also investigated. The O. oeni strains were able to degrade arginine in the conditions studied and to excrete some citrulline. In these strains, the conditions that led to a slight increase in EC were a high ethanol concentration, low pH and a high l -malic acid concentration. However, the potential EC increased with higher pH. In the case of L. plantarum , arginine was not degraded and citrulline was not produced, although the potential EC was comparable with that of the O. oeni strains studied.     

Inducing malolactic fermentation in Chardonnay musts and wines using different strains of Oenococcus oeni

Malolactic fermentations (MLF) were induced in a commercially prepared Washington State Chardonnay must to evaluate the influence of timing of inoculation and pre-culture conditions of Oenococcus oeni strains MCW, EQ-54, and WS-8. The must (pH 3.62, 21.5°Brix) was divided into lots and inoculated with Saccharomyces cerevisiae strain CY3079. Strains of O. oeni were pre-cultured by growing in diluted juice or by re-hydration of freeze-dried strains. Bacteria were inoculated into the musts before (Day 0) or after completion of the alcoholic fermentation (Day 22). Yeast populations exceeded 10⁷cfu/mL in all fermentations that proceeded to dryness. However, the viability of most strains of O. oeni quickly declined after inoculation regardless of the timing of inoculation or the strain used. MLF was induced in the wines inoculated with strains EQ-54 and WS-8 but not with MCW, and the rate depended on the time of inoculation. The method used to prepare bacterial starter cultures had no apparent influence on the completion of MLF. Values for volatile acidity were slightly higher (P< 0.05) in wines inoculated with O. oeni before alcoholic fermentation compared with those inoculated after alcoholic fermentation.     

中国主要葡萄酒产区酒酒球菌糖苷酶活性

在苹果酸乳酸发酵（malolactic fermentation,MLF）过程中,一些酒酒球菌产生的糖苷酶活性受葡萄酒环境的影响,筛选并利用在酿酒环境中具有高活力糖苷酶的菌株进行MLF,有助于提升葡萄酒的香气复杂性。本实验以中国5 个酿酒产区19 株酿酒特性优良的酒酒球菌和一株商业菌为实验菌株,通过测定5 种糖苷酶活性,对其中5 株糖苷酶活性高的菌株研究其在葡萄酒环境中糖苷酶的酶学性质。结果表明：20 株菌在相应底物作用下均含有可检测到的糖苷酶活力,不同菌株酶活性差异显著,地区间差异不显著。5 株糖苷酶活性高的菌株最适pH值为4.0,最适温度为45 ℃,在低水平（4%乙醇＋0.1 g/100 mL果糖）时有促进作用,高水平（14%乙醇＋2 g/100 mL果糖）时抑制作用显著,葡萄糖则表现为抑制作用,但各种酶活性表现为菌株依赖性。在模拟酒条件下,菌株相对酶活力仅是菌株酶活力的9.805%～32.331%。总之,在所选菌株中,SD-1f在葡萄酒环境中的糖苷酶活性最高。     

Differentiation of Australian wine isolates of Oenococcus oeni using random amplified polymorphic DNA (RAPD)

Intraspecific variation of Oenococcus oeni , the preferred lactic acid bacteria species for inducing malolactic fermentation in wine, was studied using the randomly amplified polymorphic DNA (RAPD) strain fingerprinting technique. Ten of fifteen isolates of O. oeni from Australian wineries situated in different wine regions could be distinguished by the RAPD technique. Strains of O. oeni which originated from the same winery were either indistinguishable or closely related to each other. Six different commercially available O. oeni strains could be differentiated with the four RAPD primers used and their genetic similarity determined. Analysis of O. oeni present in wines from a single source of fruit (Cabernet Sauvignon, vintage 2002) that underwent spontaneous malolactic fermentation revealed wide genetic variation amongst the isolates. Each fermentor contained several different O. oeni strains, which were present throughout alcoholic and malolactic fermentation. These data highlight the sensitivity of RAPDs when suitable primers are applied to O. oeni of unknown genetic origin, thus enabling O. oeni strains with desirable sensory and fermentation properties to be genetically analysed.     

Malolactic Fermentation and Secondary Metabolite Production by Oenoccocus oeni Strains in Low pH Wines

Abstract: Different wine varieties, including some with low pH, were studied to determine the ability to grow and produce secondary metabolites of a previously selected autochthonous Oenococcus oeni strain (C22L9), compared with a commercial strain. Monitoring of malolactic fermentation (MLF) was carried out by microbiological and chemical analysis of wines. The concentration of some major volatile compounds and biogenic amines in wines before and after malolactic fermentation was also determined. The results showed major differences in MLF duration both between wines and strains, although the differences between strains were slight for most of the analyzed compounds. Statistically significant differences in citric acid degradation were found in all wine varieties and it was confirmed that O. oeni C22L9 is a poor degrader of citric acid; this means that MLF can be prolonged without the risk of producing high concentrations of diacetyl and acetoin. Sensory analysis of wines after MLF showed similar characteristics in wines from both strains. This study thus shows that O. oeni C22L9 possesses even better sensory and fermentation properties than the commercial strain and can be used in wines with different characteristics, which makes it highly valuable for industrial use. Practical Application: The increasingly use of grape varieties of low pH in winemaking and the higher alcohol content of wines, as a consequence of the climatic change, make interesting the study of the behavior during MLF of O. oeni strains in order to determine their ability to grow, when growth conditions are not optimal, and to produce secondary metabolites. A comparative study was conducted using an autochthonous O. oeni strain (C22L9) and a commercial O. oeni strain and 4 wine varieties.     

酒类酒球菌新菌株苹果酸-乳酸发酵条件的研究

酒类酒球菌(Oenococcus oeni)普遍应用于葡萄酒生产中,它可以触发苹果酸-乳酸发酵,促进葡萄酒中的苹果酸转化为乳酸,降低总滴定酸,提高酒体生物稳定性,改善酒体口感。本文针对3株自主筛选的酒类酒球菌,研究了酒精度、pH、SO2浓度、发酵温度、接种量等因素对葡萄酒苹-乳发酵过程的影响。     

Influence of different pH values and inoculation time on the growth and malolactic activity of a strain of Oenococcus oeni

The present study was undertaken to evaluate the influence of medium pH and inoculation time on the growth and malolactic activity of an Oenococcus oeni culture. Samples of a commercial white grape juice adjusted to pH 3.2, 3.4 or 3.6, and inoculated with Saccharomyces cerevisiae strain CY3079, were inoculated with a malolactic culture ( Oenococcus oeni strain 31) at the beginning, middle, and end of alcoholic fermentation. The results obtained from this single case study show that it is possible to inoculate the bacterial culture at the three different times during alcoholic fermentation without slowing down or stopping alcoholic fermentation or causing failure of MLF. However, pH and timing of bacterial inoculation were critical to how rapidly MLF starts. At pH 3.2 a lowering of bacterial viability was observed, but a more important reduction was recorded at all tested pH levels when the bacteria were inoculated halfway through alcoholic fermentation. When inoculation was carried out at the end of alcoholic fermentation, the presence of yeast seemed to favour bacterial viability and activity and bacteria performed MLF even in difficult conditions such as pH values around 3. In all wines malolactic fermentation was accompanied by total degradation of malic and citric acids and production of L-lactic acid, D-lactic and acetic acids.     

基于RNA-seq分析酒酒球菌SD-2a酸胁迫应答机制

通过RNA-seq检测酒酒球菌SD-2a对短期酸胁迫（3 h）的反应,利用基因本体论和京都基因与基因组百科全书数据库分析不同处理间的转录组数据。此外,还测定了一些与应激反应相关的生理指标。在此基础上,绘制胁迫响应机理示意图。在酸性胁迫条件下,菌株合成更多的ATP,将H＋泵出细胞,SD-2a的ATPase活性显著升高,适宜pH值可以维持适宜的酶反应环境进行生命活动。通过调节总不饱和脂肪酸和总环脂肪酸的含量,降低细胞膜的流动性。综上所述,胞内能量生产显著增加,一些有利于抗酸胁迫的耗能反应显著增加,而一些不相关的耗能反应受到抑制。此外,为了应对酸胁迫造成的损伤,DNA修复基因和伴侣蛋白基因的表达水平大大提高。本研究结果为酒酒球菌中与酸应激反应相关的调控网络提供了一定参考。     

苹果乳酸发酵生产葡萄酒

二次发酵-苹果乳酸发酵（MLF）在葡萄酒生产过程中起非常重要的作用.酿酒需要选择合适的酿酒酵母和细菌,乳酸菌接种方案以及工艺条件控制系统.本文介绍了MLF发酵过程的优缺点、效益和技术缺陷.讨论了启动和运行MLF的方法及葡萄酒生产过程中各种微生物之间的相互作用.微生物主要是指酵母菌属中的酿酒酵母和酒类酒球菌属中的酿酒细菌.     

紫外诱变法选育酒酒球菌乙醇胁迫耐受菌株及其发酵性能研究

该研究以酒酒球菌（Oenococcus oeni）SD-2a为研究对象,采用紫外诱变法选育乙醇胁迫耐受突变菌株,评价其在乙醇胁迫条件下的生长能力,并测定其产β-葡萄糖苷酶活性及其在模拟酒中苹果酸、乳酸含量及活菌数的变化。结果表明,分离筛选到3株乙醇胁迫耐受性好的突变菌株,分别编号为UVe1、UVe2、UVe3,在高体积分数乙醇（12%和14%）胁迫环境下,3株突变菌株的生长速度均显著高于出发菌株SD-2a（P＜0.05）;突变菌株UVe2的β-葡萄糖苷酶活性显著高于出发菌株SD-2a和对照菌株L-450（P＜0.05）;在模拟酒中,3株突变菌株的苹果酸降解与乳酸生成速度均显著高于出发菌株SD-2a（P＜0.05）,且突变菌株UVe1和UVe2的存活率显著高于出发菌株SD-2a（P＜0.05）;综上,突变菌株UVe2具有优良商业酒酒球菌的潜力。     

Yeast viability during fermentation and sur lie ageing of a defined medium and subsequent growth of Oenococcus oeni

Interactions between the yeast strain used for primary oenological fermentation and the bacterium used to conduct subsequent malolactic fermentation were studied under model winemaking conditions. A commercial Saccharomyces cerevisiae wine yeast (strains, EC 1118, AWRI 835 and CY-3079) was grown in a defined medium whose composition approximated grape juice. Fermentations by all strains reached dryness, and retained a cell viability of greater than 90% upon completion of fermentation. Highest total viable cell number and percentage of viable cells were recorded for EC 1118. A sur lie ageing of the fermented medium over a 12 week period revealed a bi-phasic decay of culture viability for all strains. Thus 99% of cells had died within 2 weeks post-fermentation. Viabilities were then stable for the subsequent 4–6 week period before a second decline phase ensued and ended in either a minimal ( ca 100 CFU/mL, EC 1118) or no viable cells being detected at 12 weeks of ageing. The growth response of an Oenococcus oeni inoculum to yeast culture supernatants, previously aged for up to 12 weeks in the presence or absence of yeast lees, was evaluated in a bio-assay. In this way, yeast strains could be designated as being either inhibitory, neutral or stimulatory to the growth of O. oeni (strain Lc5p). Inhibition by supernatants of strain EC 1118 was evident, but found to be reduced by ageing the supernatant (with or without lees). Conversely, longer ageing on yeast lees increased the magnitude of the stimulatory response in O. oeni (strain Lc5p) to the supernatant from the wine yeast (strain CY-3079).     

酒酒球菌在青梅汁中的生长及苹果酸乳酸发酵特性的研究

以青梅汁为原料,利用酒酒球菌的苹果酸-乳酸发酵（MLF）对青梅汁进行生物降酸的研究。结果表明:接种量为2.0×108CFU/mL,青梅汁的pH≥3.4时,MLF能正常进行,并使样品的酸度降低61.35%以上;接种量为2.6×107CFU/mL时,青梅汁的pH为3.6才能触发MLF,样品的酸度降低了77.60%;接种量为2.3×108CFU/mL,在pH3.6和4.0的青梅汁中,添加1.0g/100g的葡萄糖的样品,与不添加葡萄糖的样品相比,其酸度分别降低了27.04%和34.63%;在柠檬酸-柠檬酸钠缓冲体系中,酒酒球菌使体系酸度降低了26.40%,证实酒酒球菌可利用柠檬酸作为碳源,进行MLF。