冬果梨果酒酵母的筛选及鉴定

以冬果梨作为富集培养基,对野生酵母菌株进行收集、分离,得到980株;采用TTC平板法、耐乙醇性能测定以及产乙醇性能测定共三级筛选,最终筛得一株可耐受18%vol乙醇、乙醇产量达7.79%vol、发酵过程中糖利用率92.75%的菌株L-0301。通过对筛选所得菌株L-0301的形态特征观察、26S rDNA D1/D2区序列分析方法对菌株进行分类鉴定,结果显示,菌株L-0301为酵母亚科酵母属酿酒酵母菌（Saccharomyces cerevisiae）。     

佛手果醋乙醇发酵优良酵母菌的选育及鉴定

佛手汁酿制果醋的过程需要优先利用酵母菌将糖发酵为酒精,但佛手汁中的柠檬苦素会抑制酵母生长。为避免其对酵母酒精发酵能力的影响,作者利用佛手为原料,结合醋酸发酵的选育方式,筛选适合佛手果醋乙醇发酵的酵母菌株。从自然发酵的佛手汁中,分离得到85株酵母菌株。经过杜氏小管初筛,以产乙醇能力、起酵速度、有机酸生产能力为指标进行复筛和醋酸发酵试验,最终得到优良菌株FS-ZJQ。经18S r DNA序列鉴定该酵母菌株为酿酒酵母。其在糖度18°Bx的佛手汁中,25℃发酵7 d可产乙醇8.49%,发酵液中萜烯类化合物、酯类化合物质量分数高,分别占挥发性风味成分的58.83%、16.44%;经醋酸发酵后,非挥发酸质量浓度可达5.06mg/mL。菌株FS-ZJQ对糖度、pH、乙醇的耐受性较好。     

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。     

湖南民间酒酿中酵母菌多样性的研究

从湖南民间收集的3种酒曲中共分离得到9株酵母菌株。利用26S rDNA的D1/D2区序列分析并结合形态学生理学特征对这些菌株进行鉴定,确定它们分别属于克鲁维毕赤酵母属和酿酒酵母属。其中4株属于克鲁维毕赤酵母属,5株属于酿酒酵母属。     

传统诱变与基因组重排技术相结合选育耐高温酿酒酵母菌株

以酿酒酵母AY12为出发菌株,采用传统人工诱变育种与基因组重排技术相结合的方法选育出耐高温且高产乙醇的酿酒酵母菌株.通过将出发菌株进行紫外诱变,获得酿酒酵母突变群体,并从中筛选出耐高温且高产乙醇的23株突变株作为正向突变文库;将该文库通过酵母高效产孢与杂交反应实现突变文库的全基因组重排,筛选得到的基因组重排最优菌LYQ-F1的高温耐受性较出发菌株AY12明显提高,且在高温模拟发酵工艺中其乙醇产量较出发菌株提高了22.1％.     

空心李酵母菌多样性及酿酒特性分析

为研究空心李酵母菌群多样性及其酿酒特性,采用WL营养琼脂鉴定培养基和26S rDNA D1/D2区序列分析法对空心李果皮富集培养液中酵母的种类进行鉴定,光密度法分析酵母生长特性、生理耐受性,对硝基苯基-β-D吡喃葡萄糖苷(p-NPG)显色法检测酵母菌产β-糖苷酶特性,亚硫酸铋培养法分析酵母菌硫化氢产生能力。结果表明,从空心李果皮上共分离到5大类酵母菌,分别为酿酒酵母(Saccharomyces cerevisiae)、热带假丝酵母(Candida tropicalis)、库德里阿兹威(氏)毕赤酵母(Pichia kudriavzevii)、葡萄汁有孢汉逊酵母(Hanseniaspora uvarum)和茶叶籽酵母(Meyerozyma caribbica)。H36菌株稳定期后菌体浓度低于商业化酿酒酵母X16,其他种类酵母生长性能高于X16;菌株H18、H3具有较好的生理耐受性,菌株H8乙醇耐受性较差,菌株H5乙醇和酸耐受性较差;菌株H3高产β-糖苷酶,其他菌株产β-糖苷酶量与X16较为接近或低于X16。菌株H36、H18产硫化氢能力较弱,而菌株H3产硫化氢能力较强;菌株H18和H5产硫化氢能力与对照组X16较为接近。空心李酵母种类相对偏少,且存在一些优质酵母资源,具有较好的应用潜能。研究空心李酵母菌的多样性,为其酵母资源多样性的开发和利用奠定基础。     

浓香型白酒醅中高产酒精酵母菌的定向筛选及分子鉴定

为了获得适用于酿酒生产的高产酒精酵母菌株,从浓香型白酒醅样品中,经不同浓度乙醇和pH条件下的定向富集和耐受性筛选以及发酵实验,获得3株能在酒精度13%vol、pH3.50的条件下稳定生长且产酒能力较强的酵母菌)XP-3、XP-7和XP-8,其中XP-3的产酒力达87.04%,粮食出酒率可达44.71%,高于一般酿酒酵母.18S rDNA序列分析结果表明,3株酵母菌均与酿酒酵母(Saccharomyces cerevisiae)具有极高的相似性.     

YE专用高蛋白高RNA酵母菌株的筛选及鉴定

为筛选适合生产酵母抽提物(Yeast extract,YE)的高蛋白、高RNA酵母菌株,对来自安琪酵母公司野外采集保藏的酵母菌进行摇瓶活化、稀释涂布和划线纯化,获得酵母菌150株。通过摇瓶培养进行初筛,得到5株高蛋白、高RNA酵母。经过30 L小试发酵复筛,获得1株酵母菌株J-5。该酵母菌发酵后蛋白质含量平均达59.4%,RNA含量达9.86%,干重41.58 g/L。并对菌株J-5发酵活力、海藻糖含量测定,结果表明该菌株发酵活力、海藻糖积累量均较低,为酵母抽提物生产优良菌株。通过形态学、生理生化及26S r DNA序列同源性分析,初步鉴定菌株J-5为酵母属酿酒酵母(Saccharomyces cerevisiae)。     

无花果内源酵母的筛选及鉴定

为了获得无花果酿酒酵母的优选菌株,从自然发酵的无花果汁中分离纯化,初步筛选出两株酵母菌SUYX-02和SUYX-03。对比研究其在不同温度、pH值、接种量条件下的生长特性并探究其二氧化硫和酒精的耐受性,最终选择了酵母菌SUYX-03作为发酵菌株并测试该菌株的发酵性能。结果表明,该酵母菌的适宜生长温度为30 ℃,适宜pH值为5.5,具有14%vol的酒精耐受性和300 mg/L的二氧化硫耐受性。对该酵母菌进行形态学和生理学特征鉴定以及内转录间隔区（ITS）序列分析,最终鉴定出菌株SUYX-03属于酵母属（Saccharomyces）中的酿酒酵母（Saccharomyces cerevisiae）。     

酿酒酵母的筛选及其发酵性能应用研究

以葡萄、草莓、苹果、桃子为研究材料筛选、分离和纯化出4株酵母菌,并和实验室前期筛选出的未知酵母菌进行共同培养研究。通过形态学观察和生理生化反应鉴定,识别出酿酒酵母,并对其进行生长曲线测定和耐受性分析。结果表明：试验成功分离筛选出5株酵母菌,其中J-1和J-3菌株经鉴定为酿酒酵母,J-2和J-5为克鲁维酵母,J-4菌株为季也蒙毕赤酵母。在相同生长时间下,分离筛选出的酿酒酵母J-1相较J-3生长速率更快;J-1菌株耐受性方面综合性能优于J-3菌株。相较于未添加酵母菌的米酒,当酵母菌添加量达10⁸ CFU/mL时,J-1菌株在感官评分、乙醇体积分数方面整体稍优于J-3菌株;但在出汁量和活菌落数方面J-1菌株和J-3菌落没有明显差异。     

改良Genomeshuffling技术选育埃博霉素B高产菌株

目的：用改genomeshuffling技术选育埃博霉素高产菌株。方法：本研究对genomeshu御ing技术进行了改良,具体是在递归原生质体融合过程中对原生质体进行了紫外诱变;通过UV和DES复合诱变方法获得T4株出发菌株,并用改良genomeshuffling技术选育埃博霉素B高产菌株。结果：通过三轮基因组重组成功选育到了2株遗传稳定的高产埃博霉素B重组菌株,其中一株重组菌株S0073-45的埃博霉素B产量达到T42．5mg／L。结论：该研究证明改良后的基因组重组技术不仅能有效提高埃博霉素B的产量,而且比传统的基因组重组技术更为高效。     

原生质体融合提高产谷氨酰胺转氨酶菌株产量

目的：研究并建立产谷氨酰胺转氨酶茂原链霉菌原生质体制备技术,通过原生质体融合技术筛选高产菌株。方法：以溶菌酶处理茂原链霉菌获得原生质体,采用原生质体融合技术,通过96 孔板高通量初筛、试管复筛、摇瓶验证选育高产菌株。结果：茂原链霉菌形成的原生质体再生出的菌落直径比较小,而菌丝生成的菌落直径比较大,两种形态的菌落96 孔板发酵后酶活力有显著性差异。不同的茂原链霉菌株制备原生质体,酶解程度、原生质体纯度、再生率有很大的差异,再生率最高可达1 804.25%,最低仅为12.76%。原生质融合后的高产菌株,选取96 孔板初筛酶活力前3%的菌株进行试管发酵,得到高产菌株比例为32.2%,酶活力比亲本高22.4%,经摇瓶验证产酶比对照提高16.28%。结论：利用基因组重排技术,可以初步对茂原链霉菌进行高产菌株选育。     

植物乳杆菌细菌素高产菌株的诱变选育及其对肉丸的防腐保鲜作用

为了提高细菌素产量并研究细菌素对肉制品的保鲜效果,本研究以植物乳杆菌JL-A65为出发菌株,对其进行常温等离子(ARTP)诱变、甲基硝基亚硝基胍(MNNG)诱变与基因组改组,并将细菌素与双乙酸钠复配后添加到肉丸中。结果表明,ARTP诱变最佳处理时间为40 s,经筛选得到两株突变株A7-10和A8-110,细菌素产量提高率分别为45.1%和48.9%。MNNG诱变的最佳处理浓度为1.5 mg/mL,经筛选得到两株突变株M2-58和M7-111,细菌素产量提高率分别为46.6%和31.3%。对植物乳杆菌进行基因组改组最终得到一株融合菌株F4-23,细菌素产量为413 mg/L,较原始菌株提高了103.48%。细菌素与双乙酸钠之间存在协同作用,可以使肉制品保质期较对照延长5 d。结论:利用理化诱变结合基因组改组可以获得细菌素高产菌株,细菌素与双乙酸钠复配后可以使肉丸保质期较对照延长5 d。细菌素对肉丸具有较好的防腐保鲜效果。     

Identification of <Emphasis Type="Italic">Zygosaccharomyces mellis</Emphasis> strains in stored honey and their stress tolerance

To screen yeast with high sugar tolerance and evaluate their stress tolerance, six yeast strains were selected from 17 stored honey samples. The species were identified through 26S rRNA sequencing. Their stress tolerance was determined via the Durham fermentation method and ethanol production ability was determined via flask fermentation. The results demonstrated that all the six strains were Zygosaccharomyces mellis. Their sugar, ethanol, and acid tolerance ranges were 500–700 g/L, 10–12% (v/v), and pH 2.5–4.5, respectively. The SO₂ tolerance was 250 mg/L. Among the six strains, 6-7431 had the best stress tolerance with sugar tolerance of 700 g/L, ethanol tolerance of 12% (v/v), and acid tolerance of pH 2.5. Furthermore, the strain of 6-7431 had the highest percentage of ethanol production at the same initial sugar content as the other strains. Therefore, the selected six yeast strains would be promising fermentation yeasts for wine-making, ethanol production, or other fermentation purposes.     

基因组改组:几株同源酱油曲霉的多亲株电融合育种

酱油酿造中原料的全氮利用率是一个主要的生产指标，通过育种提高生产菌株的中性蛋白酶活是一个有效的方法。利用一株用曲精中分离、纯化的酱油曲霉，制备其分生孢子的原生质体，并优化了原生质体制备的条件。将分生孢子的原生质体进行紫外诱变，得到7株酶活提高幅度较大的紫外诱变株U，以此作为候选株文库，采用基因组改组（Genome shuffling），进行多亲株灭活电融合，获得9株酶活进一步提高的菌株。     

Reduced acetaldehyde production by genome shuffling of an industrial brewing yeast strain

High levels of acetaldehyde produced by yeast during fermentation can be of concern to product quality. A novel approach, based on genome shuffling, was applied to reduce the production of acetaldehyde by industrial brewing strain YS86. Four isolates with different impacts of acetaldehyde concentration were obtained from populations generated by ultraviolet irradiation and nitrosoguanidine mutagenesis. These yeast strains were then subjected to recursive pool‐wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a recombinant YSF2–9 strain produced less acetaldehyde than wild‐type strain YS86, by 64.5 and 66.2% in laboratory and pilot plant fermentations, respectively. The shuffled yeast strain YSF2–9 was genetically stable and may have a potential application in brewing industry for managing acetaldehyde in beer. Copyright © 2017 The Institute of Brewing & Distilling     

Molecular identification and osmotolerant profile of wine yeasts that ferment a high sugar grape must

The objective of this study was to examine the Saccharomyces and non-Saccharomyces yeast populations involved in a spontaneous fermentation of a traditional high sugar must (Vino cotto) produced in central Italy. Molecular identification of a total of 78 isolates was achieved by a combination of PCR-RFLP of the 5.8S ITS rRNA region and sequencing of the D1/D2 domain of the 26S rRNA gene. In addition, the isolates were differentiated by RAPD-PCR. Only a restricted number of osmotolerant yeast species, i.e. Candida apicola, Candida zemplinina and Zygosaccharomyces bailii, were found throughout all the fermentation process, while Saccharomyces cerevisiae prevailed after 15 days of fermentation. A physiological characterization of isolates was performed in relation to the resistance to osmotic stress and ethanol concentration. The osmotolerant features of C. apicola, C. zemplinina and Z. bailii were confirmed, while S. cerevisiae strains showed three patterns of growth in response to different glucose concentrations (2%, 20%, 40% and 60% w/v). The ability of some C. apicola and C. zemplinina strains to grow at 14% v/v ethanol is noteworthy. The finding that some yeast biotypes with higher multiple stress tolerance can persist in the entire winemaking process suggests possible future candidates as starter for Vino cotto production.     

Isolation and characterization of tyramine-producing Enterococcus faecium strains from red wine

Enterococcus faecium strains were isolated from red wines undergoing malolactic fermentation and identified by comparison of their 16S rDNA gene sequences with those included in the GenEMBL Databases. The tyrosine decarboxylase gene was identified in all the strains analysed by PCR using gene-specific primers and the ability to produce tyramine in a synthetic media was analysed by RP-HPLC. Survival of an E. faecium strain was also evaluated in microvinification assays using two different musts with different ethanol concentrations (10% and 12% (v/v)). Tyramine production was monitored during the vinification trials. Our results suggest that E. faecium strains isolated from wine are able to produce tyramine and tolerate wine conditions following a pre-acidic stress.     

基于基因组改组的米曲霉沪酿3.042多亲株PEG介导融合育种

利用经过分生孢子的原生质体紫外线-氯化锂、NTG复合诱变得到的米曲霉沪酿3.042的8株突变株作为候选株文库,采用基因组改组(genome shuffling),利用原生质体,进行多亲株双灭活PEG介导融合,优化融合条件。以酪蛋白平板初筛,以及固体发酵测定中性蛋白酶酶活复筛,通过2轮融合操作,筛选出6株高产中性蛋白酶的融合株。其中融合菌株FII-41酶活达到7412U/g(干基),比原始出发菌株4212U/g(干基)提高1.76倍,且遗传稳定。