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-乳酸发酵相关基因克隆及其在酵母中的表达

苹果酸乳酸发酵是不同葡萄酒酿造过程中至关重要的降酸步骤。近20年来,围绕苹果酸乳酸发酵相关基因的克隆以及在酿酒酵母中表达的研究取得了一些进展,就该研究进展和存在的问题进行了综述。此外,也论及了苹果酸-乙醇发酵相关基因的研究,这对于一些不适合苹果酸-乳酸发酵的葡萄酒具有重要意义 。     

22株中国优良酒酒球菌代谢生物胺的检测

葡萄酒苹果酸-乳酸菌在葡萄酒苹果酸-乳酸发酵中对氨基酸进行脱羧反应产生具有毒性作用的生物胺.利用PCR技术及HPLC检测方法对22株中国优良酒酒球菌的生物胺代谢安全性进行研究.结果表明,PCR检测结果与HPLC测定结果一致,从我国葡萄产区筛选的优良酒酒球菌均不产生组胺、酪胺和腐胺,从生物胺角度.我国筛选的22株优良酒酒球菌具有较高的生物胺代谢安全性.     

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。     

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。     

豆豉中高产乳酸乳酸菌的筛选及其产酸条件的优化研究

目的 从云南传统发酵豆豉中分离并筛选得到一株高产乳酸的乳酸菌菌株,并对其产酸培养基组成及条件进行优化探讨.方法 运用紫外分光光度法,对豆豉由来高产有机酸菌株YM-4-3的碳源进行优化.结果 分子生物学鉴定结果表明该乳酸菌菌株属于植物乳杆菌,并将之命名为Lactobacillus plantarum YM-4-3,碳源优化结果表明,乳糖是L.plantarum YM-4-3 菌株生物合成乳酸的最佳碳源,而果糖则是最利于各种主要有机酸合成的碳源,由于此时各常见有机酸的含量均高于其他碳源时的含量,且其总酸含量高达9696.32 mg/L.结论 云南传统发酵豆豉可以成为功能性乳酸菌筛选的菌种资源库,所分离得到的L plantarum YM-4-3产酸能力较强,它可被应用于有机酸发酵及豆豉发酵的酸化处理.     

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     

两株乳酸菌的产酸性能及其对抗生素敏感性研究

从市售酸奶中分离出2株乳酸菌,经鉴定为嗜热链球菌(St)与保加利亚乳杆菌(Lb),并对其产酸性能及对抗生素敏感性进行了研究。结果表明:St与Lb 1∶1混合发酵效果优于单菌发酵;乳酸菌对4种抗生素类药物敏感性较弱,服用该类药物对人体肠道内乳酸菌的有益作用产生的影响较小。     

苹果酸酶基因转化酿酒酵母的研究进展

微生物降酸是现代葡萄酒酿造工艺中重要环节之一。利用现代生物技术将粟酒裂殖酵母中的苹果酸酶基因和苹果酸通透酶基因共同转化到酿酒酵母中,构建苹果酸-酒精酵母,使之既能进行酒精发酵,又能分解苹果酸。主要对近些年粟酒裂殖酵母苹果酸酶性质、基因结构及其转化酿酒酵母的研究做了回顾与总结,并指出了有待于解决的问题。     

酒酒球菌(Oenococcus oeni)胁迫适应性反应机制

苹果酸-乳酸发酵有利于提高葡萄酒品质,为了获得高活性的直投式酒酒球菌发酵制剂,从生理和分子生物学的角度理解该菌种胁迫耐受性增强的机制是必要的.本文就酒酒球菌利用苹果酸-乳酸发酵和膜结合的H -F0F1-ATP酶以维持细胞内环境的稳定和能量供给;胁迫适应过程中细胞膜组分的调整;小热休克蛋白Lo18等胁迫蛋白及其相应的基因的表达和调控等方面进行了综述.胁迫适应性反应机制的研究对发酵剂菌株的筛选、发酵剂的制备及其他工程菌株的构建具有重要意义.     

Oenococcus oeni cells immobilized on delignified cellulosic material for malolactic fermentation of wine

Oenococcus oeni ATCC 23279 cells immobilized on delignified cellulosic material (DCM) were used for malolactic fermentation (MLF). In first, eleven repeated alcoholic fermentation batches of white must of 11-12 degrees Be initial density were performed by Saccharomyces cerevisiae cells immobilized on delignified cellulosic material at 20 degrees C. Subsequently, the induction of MLF in the eleven taken wine batches by O. oeni cells immobilized on DCM took place at 27 degrees C. From the 3rd MLF batch up to 10th, the malic acid degradation was 53.1 up to 67.4% and the cfu of the immobilized cells/g of biocatalyst remained stable. The produced lactic acid was less than the stoichiometric yield and acetic acid content was significantly reduced after MLF not contributing in an important increase of the volatile acidity of wine. Ethanol, higher alcohols acetaldehyde and diacetyl contents in wines after MLF were in acceptable levels.     

Application of directed evolution to develop ethanol tolerant Oenococcus oeni for more efficient malolactic fermentation

Applied Microbiology and Biotechnology - Malolactic fermentation (MLF) is an important step in winemaking, which can be notoriously unreliable due to the fastidious nature of Oenococcus oeni. This...     

Development of a sequence-characterized amplified region marker-targeted quantitative PCR assay for strain-specific detection of Oenococcus oeni during wine malolactic fermentation

Control over malolactic fermentation (MLF) is a difficult goal in winemaking and needs rapid methods to monitor Oenococcus oeni malolactic starters (MLS) in a stressful environment such as wine. In this study, we describe a novel quantitative PCR (QPCR) assay enabling the detection of an O. oeni strain during MLF without culturing. O. oeni strain LB221 was used as a model to develop a strain-specific sequence-characterized amplified region (SCAR) marker derived from a discriminatory OPA20-based randomly amplified polymorphic DNA (RAPD) band. The 5' and 3' flanking regions and the copy number of the SCAR marker were characterized using inverse PCR and Southern blotting, respectively. Primer pairs targeting the SCAR sequence enabled strain-specific detection without cross amplification of other O. oeni strains or wine species of lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeasts. The SCAR-QPCR assay was linear over a range of cell concentrations (7 log units) and detected as few as 2.2 × 10(2) CFU per ml of red wine with good quantification effectiveness, as shown by the correlation of QPCR and plate counting results. Therefore, the cultivation-independent monitoring of a single O. oeni strain in wine based on a SCAR marker represents a rapid and effective strain-specific approach. This strategy can be adopted to develop easy and rapid detection techniques for monitoring the implantation of inoculated O. oeni MLS on the indigenous LAB population, reducing the risk of unsuccessful MLF.     

Genetic and physiological studies on Oenococcus oeni PsuI in response to ethanol stress

Malolactate fermentation (MLF), which is known to decreases total acidity and improves the stability and quality of cider is conducted by Oenococcus oeni; the principal microorganism responsible for MLF under stress conditions. Understanding O. oeni physiology in stress conditions can be used to generate tools based on molecular and physiological approaches allowing more precise characterization of strains. Regarding intracellular protein, the results showed an increase in the levels of amino acids under ethanol stress. To study the expressed genes under ethanol stress, one gene were sequenced. An outer-membrane lipoprotein carrier protein precursor, Lo1A was expressed under ethanol stress conditions. Scanning electron microscopy was used to study the effect of ethanol stress on cell morphology. SEM revealed aggregation of bacterial cells as the level of ethanol increases in culture medium in comparison to controls.     

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.     

Activity of ethanol-stressed Oenococcus oeni cells: a flow cytometric approach

Aims:  To study the effect of ethanol on Oenococcus oeni activity at the single cell level.
Methods and Results:  The active extrusion of the fluorescent probe carboxy fluorescein (cF) was used to assess the metabolic activity of ethanol-stressed O. oeni cells. Subsequent flow cytometric analysis revealed that O. oeni cells extrude the accumulated cF upon energizing with l -malic acid. However, O. oeni cells exposed to 12% (v/v) ethanol for 1 h showed a decreased capacity for active extrusion of cF. Moreover, two subpopulations could be distinguished, one of which being able to extrude cF and the other one remaining cF fluorescent. Growing cells in the presence of 8% (v/v) ethanol resulted in robust cells that maintained the capacity to actively extrude cF after being exposed to 12% (v/v) ethanol, which in turn correlated with the high levels of ATP observed in these ethanol stressed, malolactic fermentation (MLF) performing cells.
Conclusion:  From our results, it becomes evident that active extrusion of cF can be used to assess malolactic activity in O. oeni .
Significance and Impact of the Study:  The present study provides information for the development of a rapid method to assess the malolactic activity of individual O. oeni cells performing MLF during wine production.     

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。     

Characterization and technological properties of Oenococcus oeni strains from wine spontaneous malolactic fermentations: a framework for selection of new starter cultures

Aims:  To characterize the genetic and phenotypic diversity of 135 lactic acid bacteria (LAB) strains isolated from Italian wines that undergone spontaneous malolactic fermentation (MLF) and propose a multiphasic selection of new Oenococcus oeni malolactic starters.
Methods and Results:  One hundred and thirty-five LAB strains were isolated from 12 different wines. On the basis of 16S amplified ribosomal DNA restriction analysis (ARDRA) with three restriction enzymes and 16S rRNA gene sequencing, 120 O. oeni strains were identified. M13-based RAPD analysis was employed to investigate the molecular diversity of O. oeni population. Technological properties of different O. oeni genotypes were evaluated in synthetic medium at increasing selective pressure, such as low pH (3·5, 3·2 and 3·0) and high ethanol values (10, 11 and 13% v/v). Finally, the malolactic activity of one selected strain was assessed in wine by malolactic trial in winery.
Conclusions:  The research explores the genomic diversity of wine bacteria in Italian wines and characterizes their malolactic metabolism, providing an efficient strategy to select O. oeni strains with desirable malolactic performances and able to survive in conditions simulating the harsh wine environment.
Significance and Impact of the Study:  This article contributes to a better understanding of microbial diversity of O. oeni population in Italian wines and reports a framework to select new potentially O. oeni starters from Italian wines during MLF.     

Impact of winemaking practices on arginine and citrulline metabolism during and after malolactic fermentation

AIMS: To study arginine degradation and carcinogenic ethyl carbamate precursor citrulline formation during and after malolactic fermentation (MLF). METHODS AND RESULTS: MLF was induced in white wine with two commercial Oenococcus oeni strains under different winemaking conditions regarding the type of alcoholic fermentation (spontaneous, induced) and the lees management (racked, on lees). Arginine degradation and citrulline formation did not occur during malic acid degradation in any treatment. In five of the six treatments in which arginine degradation took place, it occurred 3 weeks after malic acid depletion and significant amounts of citrulline were formed. Presence of yeast lees in wines led to increased citrulline formation. Conclusions: This study suggests that arginine metabolism is inhibited in oenococci at low pH values (< 3.5) and that in the postalcoholic fermentation phase, citrulline formation from arginine degradation can be avoided if MLF is induced by pure cultures of O. oeni with inhibition of the bacterial biomass after malic acid depletion. Residual yeast lees in the wine have been identified as a significant risk factor for increased citrulline formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Conclusions drawn from this study allow reducing the risk of carcinogenic ethyl carbamate formation from citrulline excretion by wine lactic acid bacteria.