枇杷酒中苹果酸乳酸发酵优良乳酸菌的分离与鉴定

目的:分离获得高耐受二氧化硫的枇杷酒苹果酸乳酸发酵(MLF)菌,为果酒生物降酸提供优良乳酸菌资源.方法:采用平板划线法,从自然发酵的枇杷酒中分离乳酸菌,并通过耐受性试验筛选优良的MLF乳酸菌;通过形态学特征、生态学特征、生理生化特征和16S rDNA序列同源性分析法对目标菌进行鉴定.结果及结论:R23和R35分别被鉴定为植物乳杆菌(Lactobacillus plantarum R23)和干酪乳杆菌(Lactobacillus casei R35);R23与R35均具有MLF能力,且可耐受120mg/L的总SO2浓度,是2株优良的枇杷酒MLF菌.     

苹果酸乳酸转化高活力菌株的选育

选育苹果酸乳酸转化活力高的乳酸菌,为果酒的苹果酸乳酸发酵生物降酸提供新菌源。采用适宜的紫外亚硝基胍复合诱变条件处理植物乳杆菌R23,以降酸量及苹果酸乳酸转化活力为指标,对诱变获得的菌株进行筛选。紫外亚硝基胍复合诱变的适宜条件是:菌液浓度107cfu/mL,亚硝基胍质量浓度1.8 mg/mL,诱变时间60min。紫外线照射用功率15 W的紫外灯,距离20 cm,照射时间20 s。最终选育出1株降酸能力和苹果酸乳酸转化活力分别比出发菌提高77.5%与15.4%,遗传性能稳定的优良菌株RF17。     

肠膜明串珠菌肠膜亚种Z_25在苹果酒中发酵特性的研究

从发酵温度、接种量、酒精度、起始苹果酸浓度等方面研究了肠膜明串珠菌肠膜亚种Z25的苹果酸乳酸发酵（MLF）能力,确定了肠膜明串珠菌MLF适宜条件为温度20℃、接种量6%、酒精度10%（v/v）以及起始苹果酸浓度4.0g/L。按此工艺酿制,发酵时间12d后苹果酒中的乳酸含量由0.99g/L提高到了3.5g/L,苹果酸含量从4g/L下降到0.25g/L,且苹果酸降解发生在菌株Z25的对数生长阶段。显然,辅助肠膜明串珠菌肠膜亚种Z25到苹果酸乳酸发酵中,可以改善苹果酒的品质。     

响应面法优化植物乳杆菌R23产苹果酸乳酸酶条件

为获得高产量的苹果酸乳酸酶(MLE),以植物乳杆菌R23为发酵菌株,在前期单因素试验的基础上,采用响应面设计(RSD),建立二次多项数学模型,优化植物乳杆菌R23的产MLE条件。试验结果表明,对植物乳杆菌R23产MLE的总活力的影响次序是:起始L-苹果酸浓度>发酵温度>起始pH值;植物乳杆菌R23产MLE的最佳培养条件是:发酵温度33.5℃、pH值6.2、L-苹果酸质量浓度3.8g/L,在此条件下植物乳杆菌R23厌氧发酵28h的产酶量为1.34mg/mL,酶活力462.33u,总活力619.52U。     

酒类酒球菌的分离及发酵适应性研究

对我国葡萄酒主产区的酒类酒球菌进行了分离和筛选。通过考查分离菌株苹果酸—乳酸发酵性能后,对鉴定为酒类酒球菌的127株菌株进行了pH、SO2和酒精单因素耐受性试验,然后再选择抗性较好的初选菌株(8株)进行复合因子(pH×酒精×SO2)梯度筛选实验。结果表明,有4株酒类酒球菌分离株具有较强的苹果酸—乳酸发酵适应性,其中分离菌株O.oeniSD-2a对胁迫条件的适应性显著高于对照商业菌株O.oeni31DH。     

野生猕猴桃酒苹果酸-乳酸发酵优良乳酸菌的筛选与耐受性研究

该研究通过形态观察及生理生化试验,采用酸性番茄（ATB）培养基从自然启动苹果酸-乳酸发酵（MLF）的野生猕猴桃酒中筛选MLF优良乳酸菌,并对其SO2、酒精、pH耐受性进行测定。结果表明,经初筛得到6株乳酸菌,分别编号为R6、R7、R11、R14、R15、R18。其中,菌株R6、R15可耐受SO2 80 mg/L,菌株R7可耐受SO2 100 mg/L;菌株R6、R7可耐受酒精度14%vol,菌株R15可耐受酒精度12%vol;菌株R6、R7可耐受pH 3.2,菌株R15可耐受pH 3.0。综合分析,菌株R6、R7和R15具有较强的SO2、酒精及pH耐受能力,为MLF优良乳酸菌。     

酿酒酵母JP2发酵枇杷汁的有机酸代谢及其产香特征分析

为了探讨酿酒酵母JP2在发酵枇杷酒时的降酸途径及其产香特征,以活性干酵母为对照菌,分别采用高效液相色谱法和气-质联用法测定2种酵母菌对枇杷汁发酵前、后有机酸组分、相对含量以及枇杷酒中主要香气成分的影响。结果表明:酿酒酵母JP2具有较强的降酸能力,在发酵枇杷果汁时能消耗代谢苹果酸,产生口感柔和的乳酸,生成少量的乙酸和琥珀酸,使枇杷酒的总酸下降16.60%,而对照菌发酵后总酸反而升高9.10%;酿酒酵母JP2发酵的枇杷酒中总酯种类多达21种,总相对含量高达88.20%,而干酵母发酵的酯类为17种,相对含量仅61.11%。     

苹果酒苹果酸乳酸发酵乳酸菌的筛选

首先根据乳酸菌对苹果酒风味的贡献情况筛选出 3株乳酸菌 ,再从微生物角度对乳酸菌的生理特征以及环境因素对乳酸菌生长的影响进行了研究和比较 ,发现OenococcusoeniL4能够在SO2 和乙醇体积分数分别为 5 0mg/L和 6% ,pH 3 .2时良好的生长 ;该菌具有良好的苹果酸降解能力 ,达到 2 2 8 5 2mg/(L·d) ,表明O oeniL4能够适应于我国起泡苹果酒的酿造 ,是 1株优良的苹果酒苹果酸乳酸发酵菌株。     

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。     

新疆酿酒葡萄表皮乳酸菌的分离鉴定及耐受性分析

以新疆酿酒葡萄表皮为原料,经分离、鉴定和耐受性分析,筛选适合新疆葡萄发酵的乳酸菌。结果表明,使用MRS培养基从新疆酿酒葡萄表皮中共分离纯化出37株乳酸菌,主要分为4类。通过形态观察及16S rDNA基因序列分析,其中菌株axb81、axb80、axb79均被鉴定为柠檬明串珠菌（Leuconostoc citreum）,菌株axb74被鉴定为乳酸乳球菌乳酸亚种（Lactococcus lactis subsp. lactis）。菌株axb74和axb79的pH、乙醇、SO2、盐的耐受性分析结果表明,菌株axb74最高耐受12%乙醇、pH 3.5、80 mg/L SO2、8%氯化钠,菌株axb79最高耐受11%乙醇、pH 3.0、80 mg/L SO2、9%氯化钠。     

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.     

干红葡萄酒中苹果酸—乳酸发酵的研究 Ⅰ.苹果酸—乳酸发酵工艺条件的选择

利用两菌株:酒明串珠菌(Leuconostoc oenos)ML34和31DH,接入到四个葡萄品种(宝石解百纳、梅鹿辄、赤霞珠和佳醩酿)的葡萄酒中,在同样的传统工艺条件下,选出了适合苹果酸—乳酸发酵(简称MLF)酿制干红葡萄酒的优良葡萄品种和较好的菌种。根据实际生产情况,选择不同的pH(3.0、3.2、3.4),总SO_2浓度(5.55、100mg/1)及温度(20℃、25℃、30℃),进行三因子三水平的正交试验,考察了三个因素对MLF的影响,确定在25℃及较低SO_2浓度下,MLF速率较快,同时观察到当葡萄酒的pH高于3.2时,MLF较易诱发。经试验还发现不同菌种在不同时间接种对MLF速率的影响不同,而且经MLF后,葡萄酒中的有些成分略有差异。自然诱发MLF获得成功,但其效果不如人工接种好,甚至不如控制未产生MLF的酒。     

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。     

干红葡萄酒中苹果酸-乳酸发酵的研究 Ⅱ.苹果酸-乳酸发酵所导致干红葡萄酒有关成分的变化及其对酒质的影响

利用两菌株:酒明串珠菌(Leuconostoc oenos)ML34和31DH,接种到四个葡萄品种(宝石解百纳、梅鹿辄、赤霞珠和佳醴酿)的干红葡萄酒中,在同样的工艺条件下,分析MLF过程中酒的主要成分的变化。其结果表明:经MLF后,苹果酸几乎全部消失,转化为乳酸;酒石酸的含量略有下降。总酸下降在1.4～2.5g/1之间,pH升高在0.10～0.25个单位,挥发酸含量增高,平均增加0.31g/1。经MLF的葡萄酒滋味柔和、润口、协调。双乙酰,乙偶姻含量有大幅度增加,其含量比对照酒高一倍。挥发酯也有提高,有益于干红葡萄酒质的改善,增加了酒香,使得经MLF的酒具有典型的老酒风格。 进行了体积为5000L的中试,成功地诱发了MLF,酒的质量有一定提高,预期可在实际应用中获得较好的经济效益。     

植物乳杆菌R23在枇杷酒中生长及苹果酸乳酸发酵特性研究

目的:为植物乳杆菌R23菌剂的研发及其在果酒生物降酸上的应用提供理论依据.方法:以LH16为培养基,在不同温度、pH值条件下培养,测定生物量,确定最适生长条件;以枇杷酒为基酒,研究R23在酒中消长规律和苹果酸乳酸发酵(MLF)特性.结果与结论:植物乳杆菌R23的最适温度30℃、pH 6.0.植物乳杆菌在酒中的生长总体呈...     

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.     

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.     

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。