葡萄酒生境对乳酸菌代谢的影响

在葡萄酒酿造中,为了提高其稳定性及质量,经常利用乳酸菌进行苹果酸.乳酸发酵.苹果酸一乳酸发酵一般自发进行,也可以接种乳酸菌.本文从酿酒酵母与乳酸菌的交互作用及酚类物质和酿酒工艺对乳酸菌的作用等方面进行了综述,讨论了葡萄酒生态环境对乳酸菌代谢的影响,为苹果酸一乳酸发酵的有效控制提供一些参考.     

苹果酸-乳酸发酵的相关酶和基因的研究进展

对苹果酸-乳酸发酵的相关酶及其基因的研究进展作了简要综述。     

苹果酸降解相关基因在酿酒酵母中的表达

微生物降酸是现代葡萄酒酿造重要工艺。将裂殖酵母苹果酸通透酶基因(mae1)和苹果酸酶基因(mae2)克隆到酿酒酵母中,构建了苹果酸酒精酵母;将mae1基因和乳酸乳球菌的苹果酸乳酸酶基因(mleS)克隆到酿酒酵母中,构建了苹果酸乳酸酵母。构建的酵母重组子能够有效地分解发酵基质中的苹果酸。     

苹果酸-乳酸发酵能量产生机理

苹果酸乳酸发酵(MLF)是现代葡萄酒酿造工艺中重要的生物降酸手段。MLF是L苹果酸在乳酸菌的苹果酸乳酸酶催化下转变成L乳酸的酶促反应过程,该过程没有底物水平的磷酸化,但在MLF过程中苹果酸确实能够刺激细菌的比生长速率,增加细菌生物量。进一步的研究表明,代谢能的产生并非由于苹果酸的脱羧反应,而主要源于跨膜的L苹果酸摄入和(或)L乳酸的流出,从而产生跨膜质子电动势(pmf)(一小部分能量可能由L苹果酸的代谢中间产物丙酮酸产生)。按照化学渗透理论,pmf驱动F0F1ATPase合成ATP用于细菌生长。本文还对苹果酸的运输机制进行了综述。     

Oenococcusoeni苹果酸-乳酸酶基因克隆及其在酿酒酵母中的表达

苹果酸-乳酸酶是苹果酸-乳酸发酵过程中负责苹果酸转化为乳酸的功能酶。在进行酒酒球菌SD2a的苹果酸-乳酸酶基因(mleA)克隆测序基础上,以PGK1强启动子和ADH1终止子为调控元件,以大肠杆菌-酵母菌穿梭质粒YEp352为载体,构建了重组表达质粒并转化酿酒酵母YS58。酵母转化子用SD/Ura平板筛选鉴定。斑点杂交检测表明目的基因mleA转化到受体菌中,SDSPAGE检测表明获得的转化子表达了约60kDa的目标蛋白。获得的转化子在添加了L苹果酸的培养基中培养4d;取培养液上清用HPLC检测L苹果酸及L乳酸含量,采用t检验进行差异显著性分析,结果表明mleA基因进行了功能性的表达,将L苹果酸转化成L乳酸,L苹果酸和L乳酸含量分别与对照差异极显著和显著,苹果酸的相对降低率平均为20.95%。在有选择压力条件下,重组质粒相对稳定,而在无选择压力条件下,传代培养10d后大约有65%的重组质粒丢失。     

乳酸菌在苹果酒酿造中的应用

乳酸菌用于苹果酒酿造中 ,可以触发苹果酸 乳酸发酵 ,通过分解苹果酸 ,产生乳酸 ,并引起其他有机酸的变化而使苹果酒的口感质量得以改善。供试的 3个乳酸菌种中 ,L3由于具有较高的苹果酸分解速率 ,发酵的苹果酒感官质量优良而成为苹果酒苹果酸 乳酸发酵的优良菌种。pH、温度、二氧化硫、酒度通过影响乳酸菌的活动而对苹果酸 乳酸发酵产生一定的影响     

酒酒球菌苹果酸-乳酸酶基因的测序及分析

苹果酸乳酸酶是乳酸菌进行苹果酸乳酸发酵(MLF)的关键酶。以携带酒酒球菌(Oenococcusoeni)优良菌系OenococcusoeniSD2a的苹果酸乳酸酶基因mleA的重组质粒pLmleA作为测序质粒,进行测序分析。测序结果表明,克隆到的mleA基因序列与已报道的序列同源性为99%。mleA基因序列中有2个碱基与报道不同,其中1614碱基的改变导致错意突变,编码的氨基酸由报道的Asp变为Glu,这一改变使得原有的BamHI位点不再存在。     

酒明串珠菌31DH酿酒特性的研究

研究了四个品种干红葡萄酒的酒明串珠菌（Leuconostocoenos）31DH的酿酒特性。结果表明,18～20℃为该菌进行苹果酸-乳酸发酵（MLF）的最适温度;PH＜3.1时,MLF触发困难;31DH对总SO2的抗性达60mg／L且能耐12.8％的酒精度。葡萄酒经过MLF后,总酸下降2．0～3．5g／L,挥发酸和挥发酯分别上升0．20～0．30g／L和0．15～0．22g／L,风味平衡指数达5以上,口感变得柔和、润口、协调,酒质得到提高。     

葡萄酒苹果酸-乳酸菌精氨酸代谢研究概况

葡萄酒苹果酸-乳酸菌的精氨酸代谢会导致葡萄酒中氨基甲酸乙酯含量的增加,从而严重影响葡萄酒的饮用安全性。近年来研究表明,葡萄酒苹果酸-乳酸菌的精氨酸代谢途径是精氨酸脱亚氨基酶途径(Arginine deiminasepathway,简称ADI途径)。系统分析苹果酸-乳酸菌的ADI途径、精氨酸转运机制、ADI途径酶的调节等方面的研究进展,阐明葡萄酒苹果酸-乳酸菌的精氨酸代谢对酿造优质葡萄酒具有重要的理论和实际意义。     

苹果酸—乳酸发酵能量产生机理

苹果酸－乳酸发酵（MLF）是现代葡萄酒酿造工艺中重要的生物降酸手段。MLF是L－苹果酸在乳酸菌的苹果酸－乳酸酶催化下转变成L－乳酸的酶促反应过程,该过程没有底物水平的磷酸化,但在MLF过程中苹果酸确实能够刺激细菌的比生长速率,增加细菌生物量。进一步的研究表明,代谢能的产生并非由于苹果酸的脱羧反应,而主要源于跨膜的L－苹果酸摄入和（或）L－乳酸的流出,从而产生跨膜质子电动势（pmf)(一小部分能量可能由L－苹果酸的代谢中间产物丙酮酸产生）。按照化学渗透理论,pmf驱动F0F1-ATPase合成ATP用于细菌生长。本文还对苹果酸的运输机制进行了综述。     

Saccharomyces cerevisiae and Oenococcus oeni immobilized in different layers of a cellulose/starch gel composite for simultaneous alcoholic and malolactic wine fermentations

The production of a two-layer composite biocatalyst for immobilization of two different microorganisms for simultaneous alcoholic and malolactic fermentation (MLF) of wine in the same bioreactor is reported. The biocatalyst consisted of a tubular delignified cellulosic material (DCM) with entrapped Oenococcus oeni cells, covered with starch gel containing the alcohol resistant and cryotolerant strain Saccharomyces cerevisiae AXAZ-1. The biocatalyst was found effective for simultaneous low temperature alcoholic fermentation resulting to conversion of malic acid to lactic acid in 5 days at 10 °C. Improvement of wine quality compared with wine fermented with S. cerevisiae AXAZ-1 immobilized on DCM was attributed to MLF as well as to increased ester formation and lower higher alcohols produced at low fermentation temperatures (10 °C) as shown by GC and headspace SPME GC/MS analysis. Scanning electron microscopy showed that the preparation of a three-layer composite biocatalyst is also possible. The significance of such composite biocatalysts is the feasibility of two or three bioprocesses in the same bioreactor, thus reducing production cost in the food industry     

Inhibitory effect of sulfur dioxide and other stress compounds in wine on the ATPase activity of Oenococcus oeni

Malolactic fermentation (MLF) is carried out by Oenococcus oeni under very harsh conditions. This paper shows that stress compounds in wine such as SO(2), fatty acids and copper have an inhibitory effect on cell growth and MLF duration, and relates this effect to an inhibition of ATPase activity. Of the stress compounds, SO(2) and dodecanoic acid had the strongest effect, decreasing the ATPase specific activity to 37% and 58%, respectively. It can be concluded that ATPase is a good indicator of the physiological state of the cells and their ability to lead MLF.     

A multiplex PCR method for simultaneous species identification and strain typification of Oenococcus oeni

Selected starter cultures of Oenococcus oeni are widely used to initiate malolactic fermentation (MLF) in wine. Nevertheless, the inoculated culture does not always develop as expected and undesired strains can grow causing wine spoilage. Therefore, methods that can reliably differentiate O. oeni strains are essential to monitor the population dynamics of MLF. This work presents a new multiplex PCR method that allows the simultaneous species identification and strain typification of O. oeni, based on the combined use of species-specific PCR primers and a Random Polymorphic DNA (RAPD)-PCR primer. This method represents an useful tool for the control of wine MLF.     

Timing of malolactic fermentation inoculation in Shiraz grape must and wine: influence on chemical composition

Malolactic fermentation (MLF) is an integral step in red winemaking, which in addition to deacidifying wine can also influence the composition of volatile fermentation-derived compounds with concomitant affects on wine sensory properties. Long-established winemaking protocols for MLF induction generally involve inoculation of bacteria starter cultures post alcoholic fermentation, however, more recently there has been a trend to introduce bacteria earlier in the fermentation process. For the first time, this study shows the impact of bacterial inoculation on wine quality parameters that define red wine, including wine colour and phenolics, and volatile fermentation-derived compounds. This study investigates the effects of inoculating Shiraz grape must with malolactic bacteria at various stages of alcoholic fermentation [beginning of alcoholic fermentation (co-inoculation, with yeast), mid-alcoholic fermentation, at pressing and post alcoholic fermentation] on the kinetics of MLF and wine chemical composition. Co-inoculation greatly reduced the overall fermentation time by up to 6 weeks, the rate of alcoholic fermentation was not affected by the presence of bacteria and the fermentation-derived wine volatiles profile was distinct from wines produced where bacteria were inoculated late or post alcoholic fermentation. An overall slight decrease in wine colour density observed following MLF was not influenced by the MLF inoculation regime. However, there were differences in anthocyanin and pigmented polymer composition, with co-inoculation exhibiting the most distinct profile. Differences in yeast and bacteria metabolism at various stages in fermentation are proposed as the drivers for differences in volatile chemical composition. This study demonstrates, with an in-depth analysis, that co-inoculation of yeast and bacteria in wine fermentation results in shorter total vinification time and produces sound wines, thus providing the opportunity to stabilise wines more rapidly than traditional inoculation regimes permit and thereby reducing potential for microbial spoilage.     

The occurrence of malolactic fermentation in brandy base wine and its influence on brandy quality

AIMS: In this study we determined the extent to which lactic acid bacteria (LAB) occurred in brandy base wines, their ability to catalyse the malolactic fermentation (MLF) and the effect of MLF on the quality of the base wine and the brandy distillate. METHODS AND RESULTS: Lactic acid bacteria were isolated and enumerated from grape juice, experimental and commercially produced brandy base wines. Spontaneous MLF occurred in approximately 50% of the commercial base wines. The occurrence of MLF had an influence on the quality of the base wines and the resulting distillates. In samples where MLF occurred there was a loss of fruitiness and in the intensity of aroma. Volatile compounds like iso-amyl acetate, ethyl acetate, ethyl caproate, 2-phenethyl acetate and hexyl acetate decreased in samples having undergone MLF, while ethyl lactate, acetic acid and diethyl succinate increased in the same samples. CONCLUSIONS: Spontaneous malolactic fermentation does occur in commercial brandy base wines and it has an influence on base wine and brandy quality. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that MLF influences the quality of the base wine and the resulting distillate and with this in mind commercial base wine producers should be able to produce brandy of higher quality.     

Molecular and biochemical diversity of Oenococcus oeni strains isolated during spontaneous malolactic fermentation of Malvasia Nera wine

The diversity of indigenous Oenococcus oeni strains was investigated by molecular and biochemical characterization of isolates from Malvasia Nera wine, an economically important red wine of the Salento Region (Apulia, Italy), during spontaneous malolactic fermentation (MLF). A total of 82 isolates of this species, identified by species-specific PCR and 16S rDNA sequence analysis, was molecularly characterized by the amplified fragment length polymorphism (AFLP) technique. Three main groups resulted from cluster analysis and showed intraspecific homology higher than 50%, and a total of seven subgroups, with similarity values ranged from 80% to 98%, were obtained within these groups. Enzymatic activities, such as esterase, β-glucosidase, protease, and the consumption rate of l-malic acid, citric acid, acetaldehyde and arginine were assessed in the representative strains, according to AFLP analysis. The results showed different enzymatic activities and consumption rates of the tested metabolites among the strains. No correlation between molecular and biochemical data was observed. The evidence of biochemical variability observed among Malvasia Nera strains demonstrated that the wine aroma can be modulated depending on the strains involved in MLF. Hence, the heterogeneity existing within natural O. oeni populations represents an interesting ecological source that can be useful for technological purposes.     

Impact of different malolactic fermentation inoculation scenarios on Riesling wine aroma

During malolactic fermentation (MLF), lactic acid bacteria influence wine aroma and flavour by the production of volatile metabolites and the modification of aroma compounds derived from grapes and yeasts. The present study investigated the impact of different MLF inoculation strategies with two different Oenococcus oeni strains on cool climate Riesling wines and the volatile wine aroma profile. Four different timings were chosen for inoculation with bacteria to conduct MLF in a Riesling must/wine with a high acidity (pH 2.9–3.1). Treatments with simultaneous inoculation showed a reduced total fermentation time (alcoholic and malolactic) compared to the sequential inoculations. No negative impact of simultaneous alcoholic and malolactic fermentation on fermentation success and on the final wine volatile aroma composition was observed. Compared to sequential inoculation, wines with co-inoculation tended to have higher concentrations of ethyl and acetate esters, including acetic acid phenylethylester, acetic acid 3-methylbutylester, butyric acid ethylester, lactic acid ethylester and succinic acid diethylester. Results of this study provide some alternatives to diversify the number of wine styles by safely conducting MLF in low-pH, cool-climate white musts with potential high alcohol content.     

Implications of new research and technologies for malolactic fermentation in wine

The initial conversion of grape must to wine is an alcoholic fermentation (AF) largely carried out by one or more strains of yeast, typically Saccharomyces cerevisiae. After the AF, a secondary or malolactic fermentation (MLF) which is carried out by lactic acid bacteria (LAB) is often undertaken. The MLF involves the bioconversion of malic acid to lactic acid and carbon dioxide. The ability to metabolise l-malic acid is strain specific, and both individual Oenococcus oeni strains and other LAB strains vary in their ability to efficiently carry out MLF. Aside from impacts on acidity, LAB can also metabolise other precursors present in wine during fermentation and, therefore, alter the chemical composition of the wine resulting in an increased complexity of wine aroma and flavour. Recent research has focused on three main areas: enzymatic changes during MLF, safety of the final product and mechanisms of stress resistance. This review summarises the latest research and technological advances in the rapidly evolving study of MLF and investigates the directions that future research may take.     

Methionine catabolism and production of volatile sulphur compounds by OEnococcus oeni

AIMS: During malolactic fermentation (MLF), the secondary metabolisms of lactic acid bacteria (LAB) contribute to the organoleptic modification of wine. To understand the contribution of MLF, we evaluated the capacity of various wine LAB to metabolize methionine. METHODS AND RESULTS: Using gas chromatography (GC) coupled either with mass spectrometry (MS) or a flame photometry detector in sulphur mode (FPD), we studied this metabolism in laboratory media and wine. In laboratory media, several LAB isolated from wine were able to metabolize methionine. They formed methanethiol, dimethyl disulphide, 3-(methylsulphanyl)propan-1-ol and 3-(methylsulphanyl)propionic acid. These are known to have powerful characteristic odours and play a role in the aromatic complexity of wine. In various red wines, after MLF only the 3-(methylsulphanyl)propionic acid concentration increased significantly, as verified with several commercial starter cultures. This compound, which is characterized by chocolate and roasted odours, could contribute to the aromatic complexity produced by MLF. CONCLUSIONS: This study shows that LAB isolated from wine, especially OEnococcus oeni strains, the major species in MLF, are able to metabolize methionine to form volatile sulphur compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate the capacity of wine LAB to metabolize methionine.