葡萄汁有孢汉逊酵母和酿酒酵母的混合酒精发酵动力学

利用课题组前期优选的具有高β-葡萄糖苷酶活性的葡萄汁有孢汉逊酵母（Hanseniaspora uvarum）研究其与酿酒酵母（Saccharomyces cerevisiae）菌株混合发酵的动力学,为其葡萄酒增香酿造的应用提供理论和技术支持。实验选用陕西杨凌的爱格丽葡萄为原料,设计优选菌株提前酿酒酵母48 h接种和同时接种两个发酵处理,接种量1.0×106 CFU/mL,同时用YPD液体培养基进行两个处理的模拟发酵实验,实验以纯酿酒酵母发酵为对照。发酵过程中监测不同酵母菌数量、酒精体积分数、还原糖等指标,建立动力学模型。结果表明,提前接种处理中优选菌株生存数量最多,生存时间最长,在其对数生长期酒精生成和还原糖消耗速率最慢,但整体酒精发酵正常,酒精生成量不受影响,说明发酵过程中不良副产物生成量有限。因此,优选菌株提前酿酒酵母接种的混合发酵具有葡萄酒增香酿造的应用可能。     

酿酒酵母和异常汉逊酵母混合发酵黄酒工艺研究

以酿酒酵母和异常汉逊酵母为黄酒发酵菌种,分别考察2种酵母对18 °Bx糖化液的发酵特性,结果表明酿酒酵母产酒精(9.9% vol)能力强于异常汉逊酵母(6.8% vol),异常汉逊酵母产挥发酯(2.41 g/L)显著高于酿酒酵母(0.53 g/L)。比较混菌发酵途径,顺序混合发酵酒精度(8.1% vol)略低于同时混合发酵酒精度(8.7% vol),挥发酯含量(1.59 g/L)显著高于同时混合发酵(0.56 g/L)(P<0.05),结合发酵过程理化指标和感官评价结果,选择顺序混合发酵途径较为合适。通过单因素和Box-Benhnken中心组合试验设计,选择主发酵温度、pH值、酵母接种量3个因素对顺序混合发酵黄酒产品的挥发酯含量进行响应面优化,结果显示:顺序混合发酵黄酒的最佳参数为:发酵温度31.5 ℃,接种量1.0×107个/mL,pH 4.2。验证试验表明,在此条件下顺序混合发酵黄酒的挥发酯含量为(1.66±0.028)g/L,与预测值1.70 g/L相差不大,比酿酒酵母单独发酵提高213%。本研究为非酿酒酵母在黄酒发酵上的应用奠定了基础。     

菌种和底物因素对荔枝发酵液主要功效酶活性的影响

目的探究酵母和乳酸菌菌种及底物因素对荔枝汁发酵特性的影响。方法采用不同的酵母、乳酸菌及组合对荔枝汁进行发酵,测定发酵液中超氧化物岐化酶(SOD酶)、淀粉酶活性及总酚含量;结合单因素和Box-Benhnken设计试验进行响应面统计分析,以优化发酵底物参数工艺条件。结果荔枝汁发酵基质接入葡萄酒酵母与果酒酵母混菌(3:7,V:V),接种量为4%,25℃下发酵24 h;以保加利亚乳杆菌和嗜热链球菌菌种(8:2,V:V)混合菌种进行乳酸菌发酵,接种量为2%,37℃下发酵16 h,发酵液中SOD酶与淀粉酶活性较高。荔枝汁发酵底物优化的参数为:初始可溶性固形物含量为20°Brix,N源添加量为1.0%,pH为3.0,终发酵液的SOD酶活性可达52.17 U/mL、淀粉酶活性为12.15 U/mL、总酚含量为12.76 mg/100 mL,与模型预测值相近。结论荔枝汁通过混菌发酵,可制备具有一定功效成分的发酵饮料。     

桑葚酒用非酿酒酵母的筛选及特性研究

为筛选出适合桑葚酒发酵的非酿酒酵母,改善桑葚酒的口感及香味,以桑葚及桑园土壤样品中筛选得到的72株酵母菌为出发菌株,经杜氏管发酵、酒精发酵与感官品评进行初筛。将初筛得到的酵母分别与酿酒酵母（Saccharomyces cerevisiae）BO213按1∶1的比例接种到桑葚汁中发酵,通过感官品评与理化分析优选出一株非酿酒酵母JM-7,经鉴定该酵母为异常威克汉逊酵母（Wickerhamomyces anmalus）。其与菌株BO213混酿的桑葚酒感官评分为90.2分,总酯含量为3.21 g/L,还原糖含量为3.9 g/L,酒精度、总酸、挥发酸含量分别为11.7%vol、7.97 g/L、0.51 g/L。研究菌株JM-7生长曲线及耐受性发现,该酵母生长速率快,5 h即进入对数期,36 h达到稳定期,最高菌体浓度为4.5×108 CFU/mL,对温度、pH、SO2都有较好的耐受性,在16～36 ℃、pH 2～10、SO2质量浓度0～100 mg/L均能正常生长,但对酒精的耐受性较差,在酒精度为8%vol时生长受到抑制,在12%vol时不能生长。     

三种非酿酒酵母对贵人香白葡萄酒发酵进程及挥发性组分的影响

本文以‘贵人香’葡萄为原料,利用三种商业化非酿酒酵母（戴尔有孢圆酵母、美极梅氏酵母和耐热克鲁维酵母）与酿酒酵母混合发酵酿制白葡萄酒,并对酵母的发酵进程、葡萄酒理化指标、挥发性物质含量以及香气感官品质进行比较,旨在阐述非酿酒酵母/酿酒酵母混合发酵对白葡萄酒香气质量的影响。结果表明,三种非酿酒酵母与酿酒酵母的混合发酵均能顺利完成,但美极梅氏酵母易受到酿酒酵母的抑制。理化指标检测结果显示混合发酵白葡萄酒的酒精含量低于酿酒酵母单独发酵。挥发性香气物质采用气相色谱-离子迁移谱技术进行检测,共检测到34种化合物,接种美极梅氏酵母的葡萄酒中己酸乙酯、异戊酸乙酯、乙酸己酯、γ-壬内酯、δ-辛内酯、己醇、芳樟醇等物质的信号强度最高,整体香气风格最突出。接种戴尔有孢圆酵母和耐热克鲁维酵母的酒样中辛酸乙酯、乙酸丙酯、丙酸乙酯、α-松油烯等组分的含量较高,香气感官特征存在相似性,且均优于酿酒酵母单独发酵的葡萄酒。主成分分析结果能较好地区分不同混合发酵工艺生产的葡萄酒。综上,非酿酒酵母/酿酒酵母混合发酵对提升白葡萄酒香气质量有积极作用。     

酿酒酵母与球拟酵母混合发酵对白酒风味物质的影响

为研究球拟酵母在白酒发酵过程中对风味物质生成量的影响,将工业酿酒酵母AY15、高产酯酿酒酵母MY15分别以不同接种体积比、不同接种顺序与球拟酵母WY2进行混合发酵,以3种酵母单独接种发酵作为对照,检测其发酵过程中发酵性能、乙酸酯及高级醇生成量的变化。在不同接种体积比试验中,高产酯酿酒酵母和球拟酵母接种体积比为1∶2时,乙酸乙酯生成量达到351.65 mg/L,比MY15菌株纯种发酵提高27.78%,且发酵周期缩短至4 d。在不同顺序接种试验中,先接种酿酒酵母AY15和MY15,再接种球拟酵母,对应的乙酸乙酯生成量分别提高了24.55%和12.78%,而异戊醇生成量分别降低了10.58%和24.24%。结果表明,酿酒酵母与球拟酵母混合发酵时,球拟酵母对酿酒酵母的产酯能力具有强化作用,且不影响酿酒酵母的产酒精能力。     

德尔布有孢圆酵母与酿酒酵母纯种及混合发酵苹果酒过程中的酶活变化

本研究以富士苹果为原料,将德尔布有孢圆酵母(Torulaspora delbrueckii)与酿酒酵母(Saccharomyces cerevisiae)按纯培养和混合培养(MST1:10~5 cfu/mL S.cerevisiae/10~6 cfu/mL T. delbrueckii;MST2:10~4 cfu/mL S.cerevisiae/10~6 cfu/mL T. delbrueckii)接种到苹果汁中,研究不同发酵方式酿造苹果酒中β-葡萄糖苷酶、果胶酶、蛋白酶及淀粉酶活性的变化。研究结果表明:在纯培养中,除了α-淀粉酶和果胶酶以外,酿酒酵母32168产生的β-葡萄糖苷酶、酸性蛋白酶的最大酶活性值及酶曲线下面积都高于德尔布有孢圆酵母1004,最大酶活性值为2.03 nmol/(min·mL)和43.93 nmol/(min·mL),酶曲线下面积为10.65和223.73。而果胶酶曲线下面积在德尔布有孢圆酵母1004中较高,达到了14.17,而α-淀粉酶无明显差异。混合培养中,除了酸性蛋白酶和α-淀粉酶以外,β-葡萄糖苷酶和果胶酶最大的酶活性值及酶曲线下面积都高于纯培养,最大酶活性值最高为2.37nmol/(min·mL)和4.56mg/(h·mL),酶曲线下面积最大为15.11和19.71。德尔布有孢圆酵母与酿酒酵母纯培养与混合培养在整个发酵过程中都产生了大量的酶活性物质,这些酶有助于催化苹果汁中天然前体聚合物的水解和提高苹果酒的品质,而混合接种可以作为不同于酿酒酵母纯种发酵的另一种发酵方式。     

酿酒酵母与2株非酿酒酵母混酿培养中的增香特性

非酿酒酵母与酿酒酵母混合发酵可提升风味物质种类及含量,以高粱浸出液培养基（sorghum hydrolyzate medium,SHM）为基质,以酿酒酵母（Saccharomyces cerevisiae）S15单菌发酵为对照组,东方伊萨酵母（Issatchenkio orientalis）F10和发酵毕赤酵母（Pichia fermentans）F8单菌发酵,以及F10、F8分别与S15以体积比为1∶1顺序混合发酵为试验组。与对照组相比,东方伊萨酵母与酿酒酵母混合发酵显著提升总酯含量（P<0.05）,提高了105.33% ,其中具有花果香的乙酸乙酯含量增加了208.1% 。发酵毕赤酵母与酿酒酵母混合发酵显著提升异戊醇、苯乙醇含量,分别增加52.99% 和2.95% 。结果表明,东方伊萨酵母F10和发酵毕赤酵母F8在混菌体系中提升挥发性物质含量方面具有潜在的应用价值。     

苹果白兰地酿造中酿酒酵母与异常汉逊酵母的相互作用

通过构建酿酒酵母与异常汉逊酵母在富士苹果汁中的共培养发酵体系,采用两种酵母混合接种和顺序接种的发酵方式,比较不同培养体系中的生物量和发酵力的变化,分析两种酵母之间的相互作用。结果表明,共培养发酵体系中,异常汉逊酵母的生长受到了酿酒酵母生长的抑制,酿酒酵母的接种方式对共培养体系发酵力的影响大于异常汉逊酵母。     

高产糖苷酶非酿酒酵母菌株筛选、鉴定及其发酵过程中酶活性变化

为探究发酵过程中本土非酿酒酵母菌株的发酵能力和糖苷酶活性,利用WL培养基、七叶苷培养基从宁夏贺兰山东麓产区自然发酵的葡萄汁中初步分选非酿酒酵母菌株;通过对硝基苯酚法测定β-D-葡萄糖苷酶、β-D-木糖苷酶、α-L-鼠李糖苷酶和α-L-阿拉伯糖苷酶活性,比较筛选高产糖苷酶菌株;并在模拟葡萄汁发酵过程中动态监测菌株的生长动力学和糖苷酶活性。结果表明：经26S rDNA的D1/D2区鉴定为Hanseniaspora opuntiae、Metschnikowia pulcherrima、3 株Hanseniaspora uvarum、Torulaspora delbrueckii共6 株非酿酒酵母菌株具有较高的糖苷酶活性,且不同菌株间表现出一定差异性。在发酵过程中,T. delbrueckii表现出较好的发酵能力和最大的糖苷酶累积活性（94.10～127.70 mU/mL）,是对照菌株的1.96～2.30 倍;供试菌株M. pulcherrima具有较高的α-L-鼠李糖苷酶和α-L-阿拉伯糖苷酶活性;H. opuntiae和H. uvarum-3表现出高水平β-D-木糖苷酶和α-L-阿拉伯糖苷酶活性。本研究优选的本土非酿酒酵母具有较高的糖苷酶活性,具有葡萄酒增香酿造的应用潜力。     

混菌发酵葡萄汁对葡萄糖苷类芳香前体物水解作用的影响

探究非酿酒酵母对葡萄汁中葡萄糖苷类芳香前体物的水解作用,以内蒙古西部地区分离到的具有β-葡萄糖苷酶活性的分属5个属5株酵母菌株为材料,对部分酵母细胞进行胞壁透化处理。随后,采用酿酒酵母（Saccharomyces cerevisiae）单独接种或与非酿酒酵母non-Saccharomyces混合接种的方式进行发酵,探究酿酒酵母与非酿酒酵母混合（1∶1）发酵对发酵进程及发酵液中葡萄糖苷类芳香前体物水解作用的影响。结果表明,除星形假丝酵母（Candida stellata）外,其他3株非酿酒酵母与酿酒酵母混合接种对发酵结果均无明显影响（P＞0.05）,且均可显著降低发酵液中葡萄糖苷类物质的浓度（P＜0.05）,其中尤以萄萄汁有孢汉逊酵母（Hanseniaspora uvarum）及异常毕赤酵母（Pichia anomala）的能力最强,葡萄糖苷类物质的浓度分别为3.04 mmol/L和2.66 mmol/L。     

Application of fluorescence in situ hybridisation (FISH) to the analysis of yeast population dynamics in winery and laboratory grape must fermentations

To analyse the yeast population diversity during wine fermentations, specific fluorescein-labelled oligonucleotide probes targeted to the D1/D2 region of the 26S rRNA of different yeast species known to occur frequently in this environment were designed and tested with reference strains. The probes were then used to identify wine must isolates and to follow, in combination with plate counts, the evolution of yeast populations in two winery fermentations of white and red grape musts. In both cases, a high diversity of non-Saccharomyces yeast species was detected, including Candida stellata, Hanseniaspora uvarum, H. guilliermondii, Kluyveromyces marxianus, K. thermotolerans and Torulaspora delbrueckii. Some of these species (e.g., K. marxianus, K. thermotolerans and T. delbrueckii) were present in significant amounts during the tumultuous fermentation stage, despite the predominance of Saccharomyces cerevisiae cells following the inoculation of the wine musts with a starter strain. To further clarify the yeast population dynamics at the late phase of the fermentations, and because winery conditions do not allow a reliable control of experimental variables, strains isolated from the industrial musts were used to conduct two laboratory microvinifications in synthetic grape juice, using different ratios of S. cerevisiae/non-Saccharomyces in the inocula. Under these conditions, the results were similar to those obtained in the winery, showing a yeast profile with mixed species throughout the first fermentation stage, i.e. until about 40-50% of the total sugar was consumed. Non-Saccharomyces yeasts were outgrown by S. cerevisiae only after ethanol reached concentrations around 4-5% (v/v), which argues in favour of a potential important role of non-Saccharomyces in the final organoleptic characteristics of the wine.     

Multi-enzyme production by pure and mixed cultures of Saccharomyces and non-Saccharomyces yeasts during wine fermentation

Saccharomyces and non-Saccharomyces yeasts release enzymes that are able to transform neutral compounds of grape berries into active aromatic compounds, a process that enhances the sensory attributes of wines. So far, there exists only little information about enzymatic activity in mixed cultures of Saccharomyces and non-Saccharomyces during grape must fermentations. The aim of the present work was to determine the ability of yeasts to produce extracellular enzymes of enological relevance (β-glucosidases, pectinases, proteases, amylases or xylanases) in pure and mixed Saccharomyces/non-Saccharomyces cultures during fermentation. Pure and mixed cultures of Saccharomyces cerevisiae BSc562, Hanseniaspora vinae BHv438 and Torulaspora delbrueckii BTd259 were assayed: 1% S. cerevisiae/99% H. vinae, 10% S. cerevisiae/90% H. vinae, 1% S. cerevisiae/99% T. delbrueckii and 10% S. cerevisiae/90% T. delbrueckii. Microvinifications were carried out with fresh must without pressing from Vitis vinifera L. c.v. Pedro Jiménez, an autochthonous variety from Argentina. Non-Saccharomyces species survived during 15-18days (BTd259) or until the end of the fermentation (BHv438) and influenced enzymatic profiles of mixed cultures. The results suggest that high concentrations of sugars did not affect enzymatic activity. β-Glucosidase and pectinase activities seemed to be adversely affected by an increase in ethanol: activity diminished with increasing fermentation time. Throughout the fermentation, Saccharomyces and non-Saccharomyces isolates assayed produced a broad range of enzymes of enological interest that catalyze hydrolysis of polymers present in grape juice. Vinifications carried out by a pure or mixed culture of BTd259 (99% of T. delbrueckii) showed the highest production of all enzymes assayed except for β-glucosidase. In mixed cultures, S. cerevisiae outgrew H. vinae, and T. delbrueckii was only detected until halfway the fermentation process. Nevertheless, their secreted enzymes could be detected throughout the fermentation process. Our results may contribute to a better understanding of the microbial interactions and the influence of some enzymes on vinification environments.     

混菌发酵对贺兰山东麓‘赤霞珠’干红葡萄酒香气的影响

为研究非酿酒酵母与酿酒酵母混合发酵对贺兰山东麓‘赤霞珠’葡萄酒香气品质的影响,确定最适的混菌发酵接种工艺,以单一酿酒酵母发酵作为对照,选用葡萄汁有孢汉逊酵母（Hanseniaspora uvarum）Yun268、发酵毕赤酵母（Pichia fermentans）Z9Y-3和毕赤克鲁维酵母（Pichia kluyveri）C735三种优选非酿酒酵母,分别与酿酒酵母进行不同比例接种（10∶1、1∶1）及不同时间接种（提前48 h顺序接种、同时接种）的发酵方案酿造‘赤霞珠’干红葡萄酒,评价酒样理化及香气指标。结果表明：与对照相比,所有混菌发酵处理均提高了0.38～1.11 g/L葡萄酒甘油产量,同时降低了0.33%～1.9%乙醇体积分数。发酵毕赤酵母Z9Y-3与酿酒酵母按10∶1比例同时接种,在确保乙醇发酵顺利完成的同时,还能够显著增加干浸出物含量23.6%,增加甘油含量4.6%,此外在香气方面能够最大程度发挥其产香特性,显著增加芳樟醇、β-大马士酮、辛酸乙酯、乙酸苯乙酯、癸酸甲酯和辛酸异戊酯等30 种香气物质的含量,使挥发性成分总量提升45%,并在感官上增强了葡萄酒的浆果香、热带水果香、花香和坚果香,因此可作为贺兰山东麓‘赤霞珠’葡萄酒的理想混菌发酵方案。     

混菌发酵中酵母菌生长状况及代谢产物分析

研究3种非酿酒酵母与酿酒酵母在混菌发酵过程中生长的变化及对酒风味的影响.试验结果表明:非酿酒酵母的存在会降低酿酒酵母的发酵速率,与混合培养相比,连续培养能够提高非酿酒酵母的存活抗性,克鲁维酵母和圆酵母参与的混合培养表现出较低的醋酸产量,圆酵母和汉逊酵母相似纯培养时表现出发酵不完全的特性,非酿酒酵母的存在能够提高酒乙酸乙酯的含量.     

Growth of non-Saccharomyces yeasts affects nutrient availability for Saccharomyces cerevisiae during wine fermentation

Yeast produces numerous secondary metabolites during fermentation that impact final wine quality. Although it is widely recognized that growth of diverse non-Saccharomyces (NS) yeast can positively affect flavor complexity during Saccharomyces cerevisiae wine fermentation, the inability to control spontaneous or co-fermentation processes by NS yeast has restricted their use in winemaking. We selected two NS yeasts from our Uruguayan native collection to study NS-S. cerevisiae interactions during wine fermentation. The selected strains of Hanseniaspora vineae and Metschnikowia pulcherrima had different yeast assimilable nitrogen consumption profiles and had different effects on S. cerevisiae fermentation and growth kinetics. Studies in which we varied inoculum size and using either simultaneous or sequential inoculation of NS yeast and S. cerevisiae suggested that competition for nutrients had a significant effect on fermentation kinetics. Sluggish fermentations were more pronounced when S. cerevisiae was inoculated 24h after the initial stage of fermentation with a NS strain compared to co-inoculation. Monitoring strain populations using differential WL nutrient agar medium and fermentation kinetics of mixed cultures allowed for a better understanding of strain interactions and nutrient addition effects. Limitation of nutrient availability for S. cerevisiae was shown to result in stuck fermentations as well as to reduce sensory desirability of the resulting wine. Addition of diammonium phosphate (DAP) and a vitamin mix to a defined medium allowed for a comparison of nutrient competition between strains. Addition of DAP and the vitamin mix was most effective in preventing stuck fermentations.     

高酵母接种量的抑菌特性及其在蔗汁生产乙醇中的应用

研究了高酵母菌接种量的抑菌特性及对蔗汁乙醇发酵过程的影响。混合杂菌与酵母以不同浓度配比接入蔗汁发酵培养基中发酵,当增加酵母接种量达到5×10~7个/mL时,醪液中乙醇浓度没有明显降低。利用鲜甘蔗汁直接发酵时,接种量为5×10~7个/mL,乙醇产量达10.54%(20℃,v/v),与使用经高温灭菌的甘蔗汁作培养基,酵母接种量为2×10~7个/mL时相当,但发酵速度明显快于后者。其发酵上清液中未检出抑菌成分,说明提高酵母的接种量可以有效抑菌,其原因可能是酵母的生长处于竞争优势,抑制了杂菌的生长代谢。该研究为蔗汁发酵生产燃料乙醇,降低染菌风险提供了有效的尝试。