苹果酒酿造中的苹果酸-乳酸发酵

在苹果酒酿造中进行苹果酸-乳酸发酵,乳酸菌通过分解苹果酸,产生乳酸,引起其他有机酸的变化,使苹果酒的口感、质量得以改善。pH、温度、SO_2、酒度通过影响乳酸菌的活动而影响苹果酸-乳酸发酵的进行。保证苹果酒苹果酸-乳酸发酵进行的条件为:温度16～18℃,总SO_2含量<70mg/L,pH<3.70,酒精体积分数低于13％。     

苹果酒苹果酸乳酸菌顺序发酵工艺的研究

本文对乳酸菌Oenococcus oeni L4在起泡苹果酒(cider)中的具体应用工艺和影响因素进行了研究。结果发现,采用顺序发酵是三种发酵方式中产乙酸的量最少,发酵温度在20℃、接种量为6×105cfu/ml最有利于苹果酸乳酸发酵的进行。研究还发现,在相同的发酵时间内,发酵初始苹果酸含量在6676.95mg/L仍能够顺利完成MLF发酵。在此工艺条件下酿制的苹果酒不仅实现了苹果酸向乳酸的转化,并且苹果酒的口感能够得到较大的改善。     

苹果酸乳酸发酵的研究进展

苹果酸乳酸发酵是现代葡萄酒、苹果酒酿造工艺中非常重要的二次发酵过程。葡萄酒、苹果酒经过苹果酸乳酸发酵以后 ,原有的酸涩和粗糙感降低 ,而变得柔和、圆润且具有果香味。文中根据国内外研究成果 ,分析总结了苹果酸乳酸发酵的机理并且对苹果酒酿造过程中乳酸菌的一些生长特性进行了阐述。在此基础上 ,论述了现代生物技术在苹果酒和葡萄酒生产中的新应用。     

自然发酵肉制品中乳酸菌的分离及特性研究

以MRS和改良TJA(改良番茄汁琼脂培养基)培养基为基础培养基,从自然发酵肉品中分离得到8个菌株,其中4株为乳酸菌,3株为杆菌,1株为球状菌。进一步测试表明:4株乳酸菌在30℃培养时,生长最好;在pH3.5～4.0时生长良好;4株乳酸菌在10%NaCl的环境中均能生长,其中以2%～6%的NaCl浓度内生长最适宜;属兼性厌氧菌;对大肠杆菌产生一定的抑制作用。分离出的4株乳酸菌均适宜作为发酵肉制品的发酵剂。     

分离自藏灵菇的乳酸菌的益生特性

从藏灵菇中分离纯化5株乳酸菌,初步鉴定2株为嗜酸乳杆菌,3株为乳酸乳球菌。选取其中2株菌研究其益生特性。结果表明,从藏灵菇中分离出的乳酸菌具有良好的益生特性,2株乳酸菌在pH值为4～6可生长良好;耐热范围为30～60℃;胆盐耐受性为0.1%～0.5%;发酵液对金黄色葡萄球菌、沙门氏菌、大肠杆菌等肠道病原菌有抑制作用;对抗生素有不同程度的耐药性。     

肠膜明串珠菌在苹果酒苹果酸乳酸发酵中的应用探讨

通过在苹果酒中添加肠膜明串珠菌启动苹果酸乳酸发酵,研究了温度、pH和酒精度等因子对苹果酸-乳酸发酵的影响表明,肠膜明串珠菌能够启动苹果酸-乳酸发酵,其中温度、pH和酒精度均对苹果酒的柔和指数和感官评价有显著影响,不同发酵条件下酿造的苹果酒柔和指数在1.33～7.94之间,感官评分在73.7～91.3之间.添加肠膜明串珠菌后,苹果酸的浓度变化在322.45mg/L～692.44mg/L之间,pH值提高了约0.06～0.11,苹果酒的口感变得柔和,风味得到明显改善.     

酸和盐胁迫对乳酸菌活性的影响

该研究以13株食品常见乳酸菌为研究对象,考察酸和盐胁迫对其活性的影响,探究这13株乳酸菌的最适酸、盐生长条件及耐酸、耐盐能力。结果表明,适当的低盐浓度（1%、2%）对部分乳酸菌的生长有促进作用,植物乳杆菌和副干酪乳杆菌的最适盐浓度为1%,保加利亚乳杆菌和嗜酸乳杆菌的最适盐浓度为2%;盐浓度进一步增大,13株乳酸菌的生长受到抑制;盐浓度为10%时,除戊糖片球菌、乳酸乳球菌乳酸亚种、嗜热链球菌和乳酸片球菌外,其余9株菌的生长抑制率都＞95%。当pH值为5～7时,13株乳酸菌都生长良好,pH=6时,7株乳酸菌活性均最高;随着酸胁迫的增强,13株乳酸菌生长受抑制程度增大;pH为1～3时,13株乳酸菌的生长抑制率都＞90%,高盐及高酸对9株杆菌的抑制作用强于4株球菌。     

耐受重金属铅乳酸菌的分离筛选及鉴定

通过对包头尾矿库区、白云鄂博采矿区重金属污染严重区域土壤样品中的乳酸菌进行分离、筛选,共得到62株乳酸菌,并对分离得到的乳酸菌进行耐受铅离子的测定,62株乳酸菌对铅离子均有一定的耐受能力,不同菌株对铅离子的耐受能力有所不同;铅离子浓度为1200~9600 mg/L时,随着培养时间的增长,菌株对耐受铅离子的修复能力逐渐增强;30℃下分离得到的菌株对铅离子的修复能力明显优于37℃下分离得到的菌株;菌株BY-Fa4、BG-5b3、BG-8b2等13株菌可在铅离子浓度为7200 mg/L的环境下生长,菌株BY-Fa1、BY-Fc2、BG-6b1可在铅离子浓度为8400 mg/L的环境下生长。实验结果得出筛选得到的13株乳酸菌对铅离子不仅具有较强的耐受能力,而且还具有较强的修复能力。对筛选出13株高耐受铅离子的菌株进行16S rRNA序列分析,鉴定结果为戊糖片球菌2株、清酒乳杆菌1株、食窦魏斯氏菌5株、戊糖乳杆菌2株、植物乳杆菌3株。     

甘南州牧区优良乳酸菌耐受特性的研究

对分离自甘南州牧区酸牦牛奶中的4株优良乳酸菌的生长性能、耐酸能力、耐胆盐能力、耐渗透压能力和抑菌能力进行了研究。结果表明:4株乳酸菌均有较好的生长特性;球菌A-2-1和杆菌F-2-1在极限pH1.5仍能生长;当胆酸盐浓度达到0.5%时,杆菌B-4-2-1生长受到抑制,其余3株菌均能生长;杆菌F-2-1和G-3-2对浓度高达8%的NaCl均表现出一定的耐受力;同时4株乳酸菌也表现出良好的抑菌效果,杆菌B-4-2-1对金黄色葡萄球菌抑制作用较强,抑菌圈直径19mm,在对大肠杆菌和枯草芽孢杆菌的抑制作用中菌株F-2-1效果明显,抑菌圈直径分别为18mm和22mm,研究结果可以为其在发酵乳制品中的应用提供参考依据。     

分离自传统自然发酵食品中降胆固醇乳酸菌的筛选与评价

为了筛选优良的降胆固醇乳酸菌,本分析了从传统自然发酵食品中分离的30株乳酸菌,从中筛选出WS1、LP2、LP3、LP4、LP5和LP6 6株具有较强降胆固醇能力的乳酸菌,并通过对其胆盐耐受性及在含胆盐MRS培养基中的降胆固醇效率的分析,复筛出其中的WS1、LP2、LP3和LP6 4株乳酸菌。对4株乳酸菌的生长特征、产酸特征、不同生长阶段的种子液对胆固醇的降低效率的影响、耐酸特征及人工胃酸的耐受性进行分析和评价。结果显示,4株优选的乳酸菌降胆固醇能力较好,在胆盐中具有一定的存活力。胆盐的存在对降胆固醇能力具有一定的促进作用,4株菌在常规MRS培养基中生长和产酸特征稳定。不同生长时期的种子液对乳酸菌的降胆固醇能力有一定影响,对数期优于稳定期。耐酸性分析表明, pH越高,乳酸菌耐受性越好。pH 2.5,处理2h,乳酸菌依然具有一定的存活力;pH 3.5,乳酸菌存活力和存活时间都有所提高,pH 4.5时的存活力基本和自然pH的保持一致。在人工胃酸条件下,4株复筛乳酸菌在pH 2的条件下,存活时间超过4 h,随着pH的升高,存活率和存活时间都有所提高。本试验结果表明,4株乳酸菌均具有降胆固醇的特征,为后续优良菌株的降胆固醇机理研究打下了基础。     

Effects of Inhibitory Environmental Factors on Growth of Oenococcus oeni CCSYU2068 for Malolactic Fermentation of Cider Production

The effects of several inhibitory factors (sulfur dioxide, pH and ethanol) on the growth of lactic acid bacteria and the subsequent malolactic fermentation (MLF) were studied by inoculation of different culture strains of Oenococcus oeni, the major lactic acid bacteria (LAB) in cider production. After comparing their organoleptic properties, three strains of Oenococcus oeni were selected from indigenous and commercial sources and their inhibitory effects on cell growth and MLF examined. The malolactic bacteria expressed variations in tolerance to the environmental conditions of pH, sulfur and ethanol concentration. Isolated from an indigenous cider production facility, O. oeni L4 had a better capacity with constant growth even when the concentration of SO₂ was 50 ppm, ethanol 10% (v/v) and pH 3.0. O. oeni L4 showed better properties for metabolizing the major acids: malic, lactic and acetic acid. The decomposition mean rate of malic acid was as high as 228.52 mg/L per day with a low acetic acid concentration of 101.78 mg/L under the stress conditions of cider production.     

酒酒球菌（Oenococcus oeni）耐酸突变株苹果酸-乳酸发酵能力分析

目的：研究酒酒球菌（Oenococcus oeni）耐酸突变菌株的抗胁迫能力和苹果酸-乳酸发酵能力,为耐酸突变菌株开发为商业发酵剂提供参考。方法：以采用离子注入诱变,分离纯化后筛选出耐酸突变菌株b1为研究对象,酒酒球菌SX-1b和商业菌株31-DH为对照,探究单因素胁迫环境、复合因素胁迫条件及模拟酒环境对菌株b1生长能力、L-苹果酸降解速率和β-葡萄糖苷酶活性的影响,评价菌株b1的苹果酸-乳酸发酵能力。结果：单因素试验结果显示,当pH?3.0、乙醇体积分数14%、L-苹果酸质量浓度3?g/L时,菌株b1的L-苹果酸降解速率和β-葡萄糖苷酶活性均高于其余菌株;正交试验进一步确定各因素对菌株生长能力、L-苹果酸降解速率和β-葡萄糖苷酶活性的影响程度为：pH值＞乙醇体积分数＞L-苹果酸质量浓度;当模拟酒的乙醇体积分数为14%时,菌株b1的累积L-苹果酸降解量为1.493?2?g/L,分别为SX-1b与31-DH的1.41?倍和1.26?倍,且菌株b1的β-葡萄糖苷酶活性最高。结论：耐酸突变菌株b1表现出良好抗胁迫能力和苹果酸-乳酸发酵能力。因此,菌株?b1具有成为商业发酵剂的潜能。     

Genotyping by Amplified Fragment Length Polymorphism and malate metabolism performances of indigenous Oenococcus oeni strains isolated from Primitivo wine

The Amplified Fragment Length Polymorphism (AFLP) technique was applied for the first time to investigate the genotyping of Oenococcus oeni, the most important species involved in malolactic fermentation (MLF) in wine. A total of 87 out of 220 lactic acid bacteria, isolates from "Primitivo" wine (Apulia, Italy) undergoing MLF, identified as O. oeni by species-specific PCR and 16S rRNA sequence analysis, were studied by AFLP analysis. Four main clusters were distinguished and three of them showed intraspecific homology higher than 60%. A total of 28 strains, representative of AFLP clusters, were tested for malate metabolism in order to gain information on their malolactic performances. Significant differences were observed among strains for malic acid consumed, biomass produced and specific malic acid consumption rate. These findings indicated that AFLP technique is reliable for typing O. oeni strains and that, together with metabolism studies it may be used to individuate possible candidates as industrial malolactic starters.     

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.     

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.     

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.     

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.     

Characterization and amino acid metabolism performances of indigenous Oenococcus oeni isolated from Chinese wines

Oenococcus oeni is a multiple physical stress-tolerant lactic acid bacterium that plays an important role in wine making. It is often added as a starter culture to carry out malolactic fermentation (MLF). In this study, a total of 22 out of 127 lactic acid bacteria, isolated from Chinese wines undergoing MLF, were identified as O. oeni by species-specific PCR and 16S rRNA sequencing. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis showed that all strains could be typed under these conditions, and three main groups were determined by cluster analysis, which showed intraspecific homology higher than 69 %. Eight strains, representative of SE-AFLP clusters, were tested for malolactic activity. Significant differences were observed among strains with regard to the amount of malic acid consumed. Seventeen amino acids in different wines that were inoculated by 4 O. oeni strains, respectively, were analyzed before and after MLF. The results indicated that the amino acid metabolism of the 4 strains was significantly different between each strain.     

Metabolism of SO₂ binding compounds by Oenococcus oeni during and after malolactic fermentation in white wine

Sulfur dioxide SO? is the key additive for the preservation of wines. Carbonyl and keto compounds in wine can bind to SO? and decrease its efficacy, resulting in higher total SO? requirements. Increased consumer demand for low sulfite and organic wines pose production challenges if SO? binders have not been properly managed during vinification. Malolactic fermentation (MLF) has been known to reduce bound SO? levels but detailed time course studies are not available. In this work, the kinetics of major SO? binding compounds and malic acid were followed during MLF in wine with 12 commercially available strains of Oenococcus oeni. Pyruvic acid, acetaldehyde and α-ketoglutaric acid were degraded to various degrees by O. oeni, but galacturonic acid was not. At the time of malic acid depletion, percent degradation of pyruvate, α-ketoglutaric acid and acetaldehyde was 49%, 14% and 30%, respectively. During MLF, the decrease in average bound SO? levels, as calculated from carbonyl metabolism, was 22%. The largest reduction in wine carbonyl content occurred in the week after completion of MLF and was 53% (107 mg/L to 34 mg/L) calculated as bound SO?. Prolonged activity of bacteria in the wines (up to 3 weeks post malic acid depletion) resulted only in reduced additional reductions in bound SO? levels. The results suggest that microbiological wine stabilization one week after malic acid depletion is an effective strategy for maximum removal of SO? binders while reducing the risk of possible post-ML spoilage by O. oeni leading to the production acetic acid and biogenic amines.