酒酒球菌SD-2a原生质体制备的研究

通过单因素和正交试验,研究了菌龄、酶浓度、酶解温度及渗透压稳定剂等因素对酒酒球菌原生质体制备及再生的影响.确定其制备与再生的最佳条件为:菌龄34h,酶浓度1mg/mL,酶解温度37℃,0.8mol/L KCI为渗透压稳定剂,再生培养基也添加0.8mol/L KCI,在此条件下,其原生质体形成率与再生率之积为10.65%.     

酒类酒球菌SD-2a高密度培养的研究

对酒类酒球菌SD-2a液体培养条件和半连续高密度培养进行了研究,研究结果表明,酒类酒球菌SD-2a的适宜培养条件为:培养温度25℃,接种量5%,pH值4.8.在培养过程中,每4 h添加一次CaCO3溶液,调整pH值至最适值.并结合优化培养条件,对酒类酒球菌SD-2a进行半连续法高密度培养,使其菌体密度达到了6.5×1010 cfu/mL,比二次培养前提高了近10倍.     

酒类酒球菌SD-2a营养需求的研究

以西北农林科技大学葡萄酒学院优选酒类酒球菌SD-2a为菌种，对其苹果酸-乳酸发酵过程中所需的营养物质及其最佳比例进行了研究。结果表明，酒类酒球菌SD-2a所需的营养物质及其最佳配比为番茄汁30％，异亮氨酸、甲硫氨酸、缬氨酸、亮氨酸、天门冬氨酸各0．03g/L，精氨酸、脯氨酸、色氨酸、盐酸半胱氨酸各0．01g／L，硫酸镁0．2g/L、硫酸锰0．05g/L。     

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。     

酒类酒球菌SD-2a活性干粉与国外干粉生产性能比较

利用酒类酒球菌SD-2a活性干粉、450PreAc和OENOS干粉在生产条件下进行对比苹果酸-乳酸发酵(MLF),通过发酵过程的监测和对葡萄酒品质的影响测定其生产性能.结果表明,SD-2a活性干粉、450PreAc和OENOS干粉及对照酒样经MLF后,总酸、挥发酸、总酚等成分含量均发生显著变化,其中总酸含量分别降低18.6%,16.4%,11.7%和2.9%;品尝鉴定表明,经过MLF后葡萄酒口感变得柔和、润口、协调,酒质得到提高,SD-2a和450PreAc无明显差异,但优于OENOS和对照,说明SD-2a活性干粉有优良的生产性能.     

Oenococcus oeni and malolactic fermentation – moving into the molecular arena

Malolactic fermentation (MLF) is the bacterial‐driven decarboxylation of L‐malic acid to L‐lactic acid and carbon dioxide, and brings about deacidification, flavour modifications and microbial stability of wine. Pasteur first described the presence of ‘bacteria’ in wine nearly one‐hundred‐and‐fifty years ago and the subsequent elucidation of the bacterial‐driven malolactic reaction was described about fifty years later. Over the following years the occurrence of MLF became apparent in wines worldwide, and eventually Oenococcus oeni was identified as the principal organism involved in the process. O. oeni is remarkable in its ability to tolerate the nutritionally poor and challenging, harsh wine environment; however, it can be a difficult and sometimes unreliable organism to work with in the winery. A greater knowledge of its biology would undoubtedly facilitate the development of strains and practices, with improved performance outcomes. We already know a considerable amount about the biochemistry and physiology of O. oeni, and ironically, although we know little about its genetics, its genome has been sequenced. With this groundwork in place and molecular biology techniques at our disposal we are poised to increase our knowledge and understanding of this organism enormously.     

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。      

Molecular cloning, heterologous expression, and characterization of Ornithine decarboxylase from Oenococcus oeni

Ornithine decarboxylase (ODC) is responsible for the production of putrescine, the major biogenic amine found in wine. Oenococcus oeni is the most important lactic acid bacterium in the winemaking process and is involved in malolactic fermentation. We report here the characterization of ODC from an O. oeni strain isolated from wine. Screening of 263 strains isolated from wine and cider from all over the world revealed that the presence of the odc gene appears to be strain specific in O. oeni. After cloning, heterologous expression in Escherichia coli, and characterization, the enzyme was found to have a molecular mass of 85 kDa and a pI of 6.2 and revealed maximal activity at pH 5.5 and an optimum temperature of 35°C. Kinetic studies showed that O. oeni ODC is specific for L-ornithine with a K(m) value of 1 mM and a V(max) of 0.57 U·mg(-1). The hypothesis that cadaverine, which results from lysine decarboxylation, may be linked to putrescine production is not valid since O. oeni ODC cannot decarboxylate L-lysine. As no lysine decarboxylase was detected in any of the O. oeni genomes sequenced, cadaverine synthesis may result from another metabolic pathway. This work is the first characterization of an ODC from a lactic acid bacterium isolated from a fermented product.     

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.     

Characterisation of malolactic conversion by Oenococcus oeni to reduce the acidity of apple juice

The high concentration of malic acid is responsible for the acidity and sourness in apple juice. Bio‐conversion of malic acid to lactic acid through malolactic conversion (MC) in apple juice using Oenococcus oeni was investigated. When apple juice was inoculated with O. oeni (1 × 10⁶ CFU mL^?1), over 90% of malic acid was converted into lactic acid within 96 h at room temperature. When pH of apple juice was adjusted to 4.1 prior to inoculation, MC was completed within 60 h. MC was enhanced at a higher temperature (30°C) when compared with room temperature. The rate of MC was directly proportional to the number of bacteria added and MC was completed within 24 h at 1 × 10⁹ CFU mL^?1 initial cell density. MC occurred equally under aerobic and anaerobic conditions. The sensory analysis of partial MC‐applied juice when compared against control revealed potential for use of MC for manufacture of low‐acid apple juice.     

Release of glycosidically bound flavour compounds of Chardonnay by Oenococcus oeni during malolactic fermentation

Glycosidases, produced by Oenococcus oeni strain Lalvin EQ54 during malolactic fermentation (MLF) performed in a chemically defined wine (CDW) medium, contributed to the release of volatile aglycons from their glycosylated precursors, present in a Chardonnay wine glycosidic extract. The liberation of wine volatiles during MLF was limited by the low activity of these enzymes in this strain. Six different aglycons examined were 3-hydroxydamascone, alpha-terpineol, vanillin, methyl vanillate, 4-hydroxybenzoic acid and tyrosol. Using p-nitrophenyl synthetic substrates, it was shown that O. oeni Lalvin EQ54 has β-glucosidase, and limited α-l-rhamnopyranosidase and α-l-arabinofuranosidase activities. Release of aglycons from the Chardonnay wine glycosidic extract was increased when purified α-l-rhamnopyranosidase and α-l-arabinofuranosidase were added to the CDW medium together with the malolactic bacteria culture. The results obtained confirmed that O. oeni has the necessary glycosidases for the sequential liberation of the disaccharide sugars from wine glycosides. The rate of MLF proceeded much faster in the CDW supplemented with the Chardonnay wine glycoside extract (8 days compared to 20 days), even though the bacterial growth was unaffected.     

Evidence of mixed wild populations of Oenococcus oeni strains during wine spontaneous malolactic fermentations

Two hundred and four bacterial isolates from Rioja red wines undergoing spontaneous malolactic fermentation (MLF) were studied. Bacterial species was determined both by microbiological identification methods and by specific PCR analysis. Oenococcus oeni was shown to be the predominant species (98.5% of total isolates). Pulsed field gel electrophoresis (PFGE) of chromosomal DNA digested with SfiI was used to differentiate individual strains of O. oeni. A wide variety of restriction digest patterns were detected, which indicated a rich biodiversity of indigenous strains. Most fermentations (37 out of 41) showed from 2 to 6 clones growing in the same tank. Five O. oeni strains were the most frequently found, appearing in more than three of the 13 studied wineries, and most times in combination with other less frequently found strains. PFGE was shown to be a suitable method for strain differentiation, for monitoring individual strains and determining which strains actually survive and carry out MLF. A high genotypic heterogeneity of wild O. oeni strains was demonstrated and 90% of the studied wines showed mixed populations of O. oeni strains during MLF.     

Phenotypic and genotypic characterization of Oenococcus oeni strains isolated from Italian wines

A phenotypic and genotypic characterization of 84 Oenococcus oeni isolates from Italian wines of different oenological areas was carried out. Numerical analysis of fatty acid profiles grouped the isolates into two clusters at low level of similarity (63%), the minor cluster containing seven isolates besides the type and the reference strains. Forthy-eight O. oeni isolates, representative of the two clusters, showed no differences in their metabolic properties (heterolactic fermentation pattern, citrate degradation capability and formation of some secondary metabolites). Moreover, the analysis of species-specific randomly amplified polymorphic DNA and 16S-23S rDNA intergenic spacer region polymorphism as well as the sequence-specific separation of V3 region from 16S rDNA by denaturing gradient gel electrophoresis demonstrated a substantial homogeneity among the isolates. On the basis of ApaI Pulsed Field Gel Electrophoresis (PFGE) restriction patterns, the 84 isolates were grouped into five different clusters at 70% similarity, but no correlation with the phenotypic groups could be demonstrated. However, by combining phenotypic and genotypic data, the 84 O. oeni isolates grouped into eight phenotypic-genotypic combined profiles and a relationship between the origin of the isolates and their combined profile became evident, so that a sort of strain specificity can be envisaged for each wine-producing area.     

A new approach for selection of Oenococcus oeni strains in order to produce malolactic starters

The lactic acid bacterium Oenococcus oeni, mainly responsible for malolactic fermentation (MLF), is used in new winery process as starter culture for direct inoculation. The difficulty to master MLF according to the wine led us to search a new approach to select effective O. oeni strains. Biochemical and molecular tests were performed in order to characterize three strains of O. oeni selected for malolactic starter elaboration. Malolactic and ATPase activities that appeared as a great interest in MLF were measured and the expression of a small heat shock protein Lo18 was evaluated by immunoblotting and real-time PCR. These results were correlated with the performances of strains in two red wines. Physiological and molecular characteristics of the three strains showed significant differences for the global malolactic activity on intact cell at pH 3.0 and at the level of induction of the small heat shock protein Lo18. These two parameters appeared of interest to evaluate in the ability of O. oeni strains to survive into wine after direct inoculation and to perform MLF. Indeed, a tested strain that presented the highest malolactic activity on intact cells at pH 3.0 and a high level of Lo18 induction showed a high growth rate and a high specific kinetic of malate consumption. The techniques used in this work carry out more quickly and more reliable than usual for the selection of effective strains intended for direct inoculation in wines.     

酒酒球菌蛋白质提取方法研究

以裂解液为提取介质,分别采用超声法、反复冻融法和玻璃珠破碎3种方法提取细菌蛋白质,通过分析细菌破碎程度、蛋白质SDS-PAGE电泳及蛋白质质量浓度,比较各提取方法的效果。结果表明,超声法细胞破碎明显,提取蛋白质条带丰度最高,蛋白质质量浓度最大,有较好的稳定性和重复性,且操作简单。     

The effects of freezing and freeze-drying of Oenococcus oeni upon induction of malolactic fermentation in red wine

Summary The use of Oenococcus oeni starter cultures for the induction of malolactic fermentation (MLF) in wine permits control over the timing of the process and the quality of the wine. Successful inoculation of bacterial starter cultures into wine depends on the selection of suitable strains and on the preparation and conservation of those cultures. Medium for Leuconostoc oenos (MLO) is the best medium for easy and rapid growth of O. oeni cultures under laboratory controlled conditions for isolation and identification. However, this study showed that O. oeni cells inoculated in MLO failed to induce MLF in wine while cells grown in Medium of Preculture (MP) or wine, stored at −20 °C or freeze-dried retained the ability to induce MLF when inoculated in wine. Our results suggest that the use of freeze-dried cultures of O. oeni previously grown in MP is the best choice for industrial application.     

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.     

葡萄酒中酒类酒球菌的分离——抑制剂对酵母菌生长的抑制效应

从葡萄酒中分离酒类酒球菌时 ,酵母菌是最主要的干扰菌。作者通过在ATB和改良MRS琼脂培养基中添加放线菌酮、山梨酸和制霉菌素 ,研究了不同抑制剂对酵母生长的抑制效应。结果表明 ,采用ATB + 5 0mg/L放线菌酮、改良MRS + 5 0mg/L放线菌酮、改良MRS + 10 0mg/L山梨酸、改良MRS + 5 0mg/L制霉菌素选择性培养基 ,能够完全抑制酿酒酵母的生长。