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。     

酒精、SO2和pH值对酒类酒球菌生长特性影响

选用酒类酒球菌31DH和SD-1进行了试验,在ATB培养基中考察了环境因子低pH值、酒精和SO2对于菌株的影响以及这些限制因子之间的协同作用.结果显示低pH值是酒类酒球菌生长的主要抑制因子;低pH值与酒精之间存在协同作用,10%的酒精度是工艺控制的关键点.SO2的抑菌效果受pH值影响,pH值越低SO2的抑制作用越强烈;结合态SO2与酒精存在协同作用,但当结合态SO2浓度＞40(mg·L-1)时,其浓度的增加对酒精抑制作用的增强效果并不显著.     

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。     

菠萝果酒酵母筛选及发酵性能测试

从不同成熟度的菠萝皮中分离酵母菌,通过一级筛选,得到45株产香和产酒较好的菌株,再通过杜氏管法筛选得到发酵速度较快的12株产香和产酒较优良的优势菌,三级筛选是将这12株菌接种到菠萝汁发酵液中,筛选出一株适合菠萝汁发酵的产酒和产香性能较优的菌株。并对该菌株的温度、pH值、初始糖度、SO2及酒精度耐受性进行了测试。结果表明,35℃以上的温度条件下酵母菌酒精产率下降,30℃产酒率最高;pH5.0酵母菌酒精达到最大;初糖浓度超过25°Be就对酵母菌产生抑制;SO2添加量为60mg/L时,酒精产率提高,产酒率随着添加量继续增加而减小;发酵液中酒精体积分数达到5%时,酵母菌的生长受到抑制,当达到8%,抑制效果加剧,超过11%以后,基本达到酵母菌的耐受极限。     

甘肃河西走廊葡萄酒产区本土酒酒球菌发酵耐受性分析

为筛选出具有良好发酵耐受性的本土酒酒球菌(Oenococcus oeni),以甘肃河西走廊葡萄酒产区分离鉴定的7株O. oeni为供试菌株,分别对其进行pH值、乙醇、SO_2质量浓度和发酵温度等酿造因素的耐受性考察,选择耐受性较好的菌株进行复合因素(pH值×乙醇×SO_2质量浓度)适应性分析。结果表明,7株本土O. oeni发酵耐受性存在差异,且同一菌株对不同因素的抗胁迫性也不相同;菌株MG-1、MG-7、QL-11、ZX-1在pH 3.2、乙醇体积分数14%及SO_2质量浓度40 mg/L条件下均可生长增殖;影响菌株MG-1和MG-7生长的因素主次为乙醇体积分数SO_2质量浓度pH值,影响菌株QL-11和ZX-1生长的因素主次为乙醇体积分数pH值SO_2质量浓度。综合分析,供试菌株中ZX-1的发酵耐受性最强,其次为菌株MG-1、MG-7和QL-11,均具有一定的产业化应用潜力。     

昌黎赤霞珠葡萄相关酿酒酵母的分离与筛选

以昌黎地区赤霞珠葡萄为样本,从中分离出5种酵母。通过菌落特征、菌体形态及赖氨酸培养基鉴定,初步确定其中4株为葡萄酒酿酒酵母。通过高浓度酒精(10%~18%)、高浓度SO2(100~400mg/L),高糖浓度(10%~60%)和高温(37、42℃)的耐受性实验,初步筛选出两株可耐受14%浓度酒精、300mg/L SO2、50%的葡萄糖浓度及42℃高温的菌株。与市售的两种酿酒酵母进行葡萄酒发酵实验,将所得酒样进行理化分析及感官品评,结果表明菌株a发酵力强,较市售酵母菌发酵后挥发酸含量低,残还原糖含量为0.40g/L,且葡萄酒感官评价最高。最终选出菌株a为赤霞珠葡萄酿酒酵母最佳菌株。     

葡萄酒MLF高耐受性优良乳酸菌的筛选与鉴定

以活菌数为指标,以L450为对照菌株,考察了从自然发酵葡萄酒中分离的14株乳酸菌对酒精度、SO2浓度、p H值的耐受能力,筛选出高耐受性优良菌株。通过菌落形态、菌体形态、生理生化特征和16S r DNA分子测序进行优良菌株鉴定。结果表明:菌株G23与L42综合耐受能力最强且优于对照菌株L450,二者被鉴定为植物乳杆菌,可作为葡萄酒MLF高耐受性优良乳酸菌。     

不同酒类酒球菌苹果酸—乳酸发酵对葡萄酒中氨基酸的影响

不同酒类酒球菌茵株完成MLF后,葡萄酒中氨基酸均发生显著变化:种类增多,诸多氨基酸含量提高。SD-2a发酵酒样中的丙氨酸和谷氨酸、SD-1b发酵酒样中甘氨酸、丝氨酸和脯氨酸、SD-2h酒样中谷氨酸、甘氨酸、丝氨酸、脯氨酸含量明显增加;含量呈极显著增加的氨基酸为:在SD-2h发酵酒样中的丙氨酸、在SD-1b发酵酒样中的丙氨酸、缬氨酸和谷氨酸;对照菌株31DH发酵酒样中各种氨基酸的增减没有达到显著水平。葡萄酒学院分离筛选的3个菌株发酵酒样中的精氨酸含量增加,而对照菌株31DH则有所降低。因此,3个菌株代谢特性良好,在葡萄酒中不会导致致癌物质——氨基甲酸乙酯的前体物脲、瓜氨酸等的过多合成。鉴于各种氨基酸独特的生化和生理特性,3株酒类酒球菌菌株完成MLF后,氨基酸种类、含量的增加可以提高葡萄酒的营养价值和保健功能。     

无花果果酒酵母的筛选及发酵性能研究

本文主要筛选适合无花果果酒发酵的酵母。从无花果果皮和无花果果酒酒渣中初步分离得到41株酵母菌,经过一级筛选得到12株产酒产香较好的菌株,经过杜氏管法筛选得到6株起酵快的优势菌株,再经过无花果浆汁发酵法筛选得到1株起酵快、产酒产香较好的酵母。再对这株菌进行耐受性测试,结果表明,酵母菌Z20对糖度、酸度、SO2以及乙醇的耐受性较好,可作为无花果酒发酵的专用酵母。     

酒类酒球菌对葡萄酒中相关物质代谢的影响

酒类酒球菌是触发葡萄酒苹果酸-乳酸发酵(malolactic fermentation,MLF)的主要乳酸菌,其代谢产物如乳酸、乙酸、双乙酰、乙偶姻、生物胺等对葡萄酒的感官品质有重要作用。本文对酒类酒球菌在葡萄酒MLF过程的代谢情况进行总结,阐述糖、有机酸、含氮物质和酚类化合物的代谢情况,以及在不同影响因素下酒类酒球菌生长和代谢途径的改变状况,已经证明pH值、SO2、乙醇体积分数、葡萄糖/果糖比等都是影响代谢的因素。随着研究的不断深入,酚类化合物在代谢中的作用也受到越来越多的关注。酚类化合物一方面通过刺激或者抑制酒类酒球菌的生长而间接影响MLF的进行;另一方面,不同的酚类物质也会影响细菌对其他酚类的代谢,从而影响葡萄酒的口感和质量。通过全面综合了解酒类酒球菌在葡萄酒中的代谢情况有助于更好地调控MLF过程。     

中国葡萄酒产区酒酒球菌种质资源遗传多样性分析

为了解我国葡萄酒产区酒酒球菌种质资源遗传多样性,对22 株筛选自我国不同葡萄酒产区的乳酸细菌进行了种特异性聚合酶链式反应（species-specific polymerase chain reaction,PCR）分析、16S rRNA序列分析和扩增片段长度多态性分析（amplified fragment length polymorphism,AFLP）基因分型。种特异性PCR和16S rRNA序列分析表明22 株分离株为酒酒球菌（Oenococcus oeni）。建立了O. oeni基于HindⅢ和MseⅠ为内切酶的AFLP分析体系,对16 对引物组合进行了筛选,结果表明HT-MA、HT-MT、HT-MC、HG-MA、HG-MT、HC-MT为O. oeni AFLP分析的最佳引物组合,且实验重复性在98%以上。对O. oeni的AFLP分析结果显示：22 株O. oeni分为3 个簇群,且簇群间遗传相似性系数较小。所以,以HindⅢ和MseⅠ为内切酶的AFLP技术是研究O. oeni基因分型的有效方法,我国葡萄酒产区的O. oeni具有丰富的遗传多样性,O. oeni菌株间的遗传相似性系数不仅仅与其生态地理分布有关,可能还与其所处的微生态和其他因素有关。     

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。     

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.     

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.     

Restriction fragment length polymorphism analysis of 16S rRNA genes in lactic acid bacteria isolated from red wine

Various lactic acid bacteria isolated from wine and alcoholic drinks attempted to identified by restriction fragment length polymorphism (RFLP). Oenococcus oeni strains exhibited unique RFLP patterns by HaeIII-digestion of 12 reference strains. We performed RFLP analysis using the AccII or HaeIII enzyme for 44 strains isolated from red wine and the results indicated profiles identical to O. oeni type strain. O. oeni does not exhibit interspecific diversity. Thus, the use of RFLP analysis of 16S rDNA is useful in the identification of O. oeni strains isolated from wines.     

Inhibition of malolactic fermentation by a peptide produced by Saccharomyces cerevisiae during alcoholic fermentation

The ability of Saccharomyces to inhibit Oenococcus oeni during the alcoholic fermentation by mechanisms other than SO(2) production was investigated. During fermentation in synthetic grape juice, S. cerevisiae strain RUBY.ferm inhibited the malolactic fermentation by O. oeni while strain EC1118 did not despite both strains producing similar amounts of SO(2). The bacterial inhibition exerted by RUBY.ferm was diminished when the wine was treated with proteases but not through the addition of nutrients. Wine fermented by RUBY.ferm was fractionated based on molecular weight and each fraction tested for the ability to inhibit the growth of O. oeni. The fraction containing compounds larger than 3 kDa was the sole inhibitory fraction. The inhibitory fraction was analyzed by SDS PAGE and showed a 5.9 kDa protein band present in wine fermented by RUBY.ferm that was not present in wine fermented by a non-antagonistic yeast, S. cerevisiae strain Saint Georges S101. The ability of the peptide to inhibit O. oeni seemed to be dependent on the presence of SO(2).     

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β-葡萄糖苷酶活性与耐酸胁迫能力的相关性分析

目的:β-葡萄糖苷酶是葡萄酒中结合态香气物质释放的关键酶,但其活性受诸多因素的影响。本研究旨在从酒酒球菌自身耐酸能力的视角去分析评估菌株糖苷酶活性,探索酸胁迫下不同耐酸表型酒酒球菌与其β-葡萄糖苷酶活性之间存在的相关性关系。方法:结合利用离子注入诱变与胁迫环境筛选的方式,获得耐酸(p H 3.0)、酸敏(p H 9.0)突变酒酒球菌菌株,对其β-葡萄糖苷酶的活力进行测定,并筛选3组酸胁迫下表型差异大的β-葡萄糖苷酶基因送样测序。结果:耐酸突变酒酒球菌的β-葡萄糖苷酶酶活力是出发菌株的2~4倍,是相应酸敏突变株的2~7倍。测序结果显示,除菌株a3的β-葡萄糖苷酶基因在108位(G置换成C)和1 232(A置换成T)位处发生了突变外,其余菌株β-葡萄糖苷酶的基因均未发生改变。结论:酒酒球菌β-葡萄糖苷酶活性与菌株酸胁迫能力显著相关(P0.05)。耐酸胁迫能力越强的菌株,β-葡萄糖苷酶活性越高。     

紫外诱变法选育酒酒球菌乙醇胁迫耐受菌株及其发酵性能研究

该研究以酒酒球菌（Oenococcus oeni）SD-2a为研究对象,采用紫外诱变法选育乙醇胁迫耐受突变菌株,评价其在乙醇胁迫条件下的生长能力,并测定其产β-葡萄糖苷酶活性及其在模拟酒中苹果酸、乳酸含量及活菌数的变化。结果表明,分离筛选到3株乙醇胁迫耐受性好的突变菌株,分别编号为UVe1、UVe2、UVe3,在高体积分数乙醇（12%和14%）胁迫环境下,3株突变菌株的生长速度均显著高于出发菌株SD-2a（P＜0.05）;突变菌株UVe2的β-葡萄糖苷酶活性显著高于出发菌株SD-2a和对照菌株L-450（P＜0.05）;在模拟酒中,3株突变菌株的苹果酸降解与乳酸生成速度均显著高于出发菌株SD-2a（P＜0.05）,且突变菌株UVe1和UVe2的存活率显著高于出发菌株SD-2a（P＜0.05）;综上,突变菌株UVe2具有优良商业酒酒球菌的潜力。     

中高温大曲和曲房细菌群落的相关性

为了解多粮浓香型白酒厂中高温大曲和曲房细菌群落的相关性,采用改良NA培养基和高氏I号培养基对细菌进行分离。经过表观去除冗余后,从多家规模多粮浓香型白酒厂的中高温大曲生产环境(曲房)和大曲分离得到217株细菌。对这217株菌进行16S rRNA基因的系统发育分析及纤维素维素分解能力、淀粉水解能力、7%乙醇耐受能力和pH 4.5耐受能力进行了检测。发现中高温大曲和曲房细菌群落均呈现出一定的多样性,二者的优势菌均为Bacillus,且分别以Staphylococcus和Streptomyces为次优势属;中高温大曲和曲房环境中均存在一定数量具有应用前景的微生物资源;2个细菌群落组成及具有纤维素分解能力、淀粉水解能力和7%乙醇耐受能力的菌株分布均存在高度的一致性,环境对中高温大曲细菌群落影响巨大。