刺梨自然发酵过程中非酿酒酵母多样性分析

【目的】分析刺梨果实自然发酵过程中非酿酒酵母菌群特征,为筛选优质刺梨非酿酒酵母提供参考。【方法】基于Illumina MiSeq高通量测序技术和WL营养琼脂鉴定培养基纯种分离技术,分析刺梨果实自然发酵1 d(F1)、3 d(F3)、5 d(F5)和15 d(F15)4个阶段及YPD培养基富集培养样本中非酿酒酵母种群组成和多样性。【结果】高通量测序分析结果共获得182个OTUs(operational taxonomic units,OTUs),归属于81个属107个种;物种多样性分析结果表明,刺梨果实自然发酵前期,优势非酿酒酵母为汉逊酵母(Hanseniasporasp.)和伯顿丝孢毕赤酵母(Hyphopichiaburtonii),二者在样本F1中分别占42.59%和26.85%;随着自然发酵的不断进行,二者的比例逐渐降低,在第15天(F15),Hanseniaspora sp.和H.burtonii比例降低至7.73%和0.52%。相反,Pichia sporocuriosa和未培养的酵母,随着自然发酵不断进行所占比例逐渐增大,分别由F1中的0.23%和0.33%增至F15中的37.26%和32.62%。此外,采用WL营养琼脂鉴定培养基纯种分离和鉴定技术,从刺梨上分离到Hanseniasporasp.、H.burtonii、克鲁维毕赤酵母(Pichia kluyveri)、P.sporocuriosa和异常威克汉姆酵母(Wickerhamomyces anomalus)5种类型的可培养非酿酒酵母。【结论】刺梨果实上存在着丰富的非酿酒酵母菌资源,研究刺梨自然发酵过程中非酿酒酵母多样性,为酵母资源开发和利用奠定基础。     

赤霞珠葡萄酒自然发酵中酿酒酵母的菌株区分

采用Interdelta指纹图谱分析, 对分离自宁夏地区赤霞珠葡萄自然发酵过程中的45个酿酒酵母单菌落进行菌株区分, 研究发酵过程中酿酒酵母菌株的变化, 为发酵的有效控制及选育优良酿酒酵母菌株提供依据。结果发现, 本研究分离到的45个酿酒酵母单菌落中, 产生5种指纹图谱, 代表5种不同的基因型, 基因型I-V分别占所分离单菌落的71%、13%、9%、5.0%、2.0%, 基因型I是发酵过程中的优势菌株。本研究中, 二氧化硫处理影响自然发酵过程中酿酒酵母菌株的类型、数目及比例, 但其影响不是很大。     

利用微卫星标记分析新疆老面酵头中酿酒酵母遗传多样性

本研究对分离自新疆不同地区老面酵头中433株酿酒酵母进行微卫星标记技术分析,研究新疆老面酵头中酿酒酵母遗传多样性。结果表明,新疆老面酵头中酿酒酵母观测等位基因数为1.396 6–1.8599,平均有效等位基因数为1.0463–1.1082,基因多样性指数为0.0356–0.0850,Shannon指数为0.1583–0.0682,其中,南疆酿酒酵母遗传多样性指数高于北疆。新疆各地域居群具有明显简单重复序列(simple sequence repeat, SSR)基因特征,主要表现为7个类群(XJⅠ–XJⅦ),在6个微卫星位点上具有显著的基因型差异,并在120–160 bp和250–300 bp扩增位点多样性均明显少于参考菌株葡萄酒酵母EC1118。居群XJⅢ、XJⅣ、XJⅥ、XJⅦ在250–400bp扩增位点少于XJⅠ、 XJⅡ、 XJⅤ;XJⅣ、 XJⅤ、XJⅦ丰度明显大于XJⅠ、 XJⅡ、XJⅢ、 XJⅥ和EC1118。XJⅠ、XJⅤ、XJⅥ扩增位点在300–400 bp少于XJⅡ、XJⅢ、XJⅣ、XJⅦ;XJⅠ、XJⅡ丰度比XJⅢ、XJⅣ、XJⅤ、XJⅥ、XJⅦ和EC1118低,...     

酿酒酵母Saccharomyces cerevisiae重组菌株木糖醇发酵的初步研究

木糖还原酶催化木糖为木糖醇的反应,是木糖代谢的第一步。将木糖还原酶的原因ＸＹＬ１引入酿酒酵母中,构建得到儿表达ＸＹＬ１基因的重组酿酒酵母菌株ＨＹＥＸ２,该重组菌株的木糖还原酶比活力为７．４７Ｕ／ｍｇ。研究表明,该菌株获得转化木糖产生木糖醇的能力,当辅助碳源葡萄糖的浓度为２％,并在发酵３０ｈ左右添加木糖,木糖醇的转化率可达到０．９７ｇ／ｇ。     

混合糖发酵重组酿酒酵母的菌株构建和菊芋秸秆同步糖化发酵研究

【目的】构建可用于纤维素乙醇高效生产的混合糖发酵重组酿酒酵母菌株,并利用菊芋秸秆为原料进行乙醇发酵。【方法】筛选在木糖中生长较好的酿酒酵母YB-2625作为宿主菌,构建木糖共代谢菌株YB-2625 CCX。进一步通过r DNA位点多拷贝整合的方式,以YB-2625 CCX为出发菌株构建木糖脱氢酶过表达菌株,并筛选得到优势菌株YB-73。采用同步糖化发酵策略研究YB-73的菊芋秸秆发酵性能。【结果】YB-73菌株以90 g/L葡萄糖和30 g/L木糖为碳源进行混合糖发酵,乙醇产量比出发菌株YB-2625 CCX提高了13.9%,副产物木糖醇产率由0.89 g/g降低至0.31 g/g,下降了64.6%。利用重组菌YB-73对菊芋秸秆进行同步糖化发酵,48 h最高乙醇浓度达到6.10%(体积比)。【结论】通过转入木糖代谢途径以及r DNA位点多拷贝整合过表达木糖脱氢酶基因可有效提高菌株木糖发酵性能,并用于菊芋秸秆的纤维素乙醇生产。这是首次报道利用重组酿酒酵母进行菊芋秸秆原料的纤维素乙醇发酵。     

楸树种质生长和叶部性状的遗传多样性

为提高楸树遗传资源的利用效率,本研究对从楸树基因库中选出的53个无性系进行地径、苗高、节间距等12个性状指标进行了遗传多样性分析。研究结果表明,53个楸树无性系间地径、苗高的差异极显著,遗传变异系数分别为18.53%、16.29%,遗传多样性指数分别为1.84、1.79。地径、苗高与叶长、叶柄长、叶片总数、叶面积、皮孔密度呈现极显著的正相关,且叶长、叶柄长、叶片总数、叶面积、皮孔密度的遗传变异系数分别为8.57%、17.74%、18.56%、24.21%和26.91%,遗传多样性指数(Shannon-Weaver)分别为1.91、1.87、1.80、1.80和1.94,具较大变异且受强遗传控制,多样性丰富且分布均匀。来源不同的种质资源差别较大,其中来源于河南地区的种质生长指标最优,来源于山东地区的种质变异系数最高。聚类分析将53个无性系分为5大类,第I类的地径、苗高、叶片总数、皮孔密度均值最高,第Ⅱ类生长最差,第Ⅲ类的节间距、叶长、叶宽、叶柄长、叶面积、叶周长均值最高,第IV类和第V类的叶绿素含量和比叶重均值分别最高。     

产虫草素酿酒酵母工程菌株的构建与发酵优化

虫草素作为药用真菌蛹虫草的主要活性成分,具有抗肿瘤、抗病毒等多种生理功能。现阶段虫草素主要通过蛹虫草液体发酵生产,但发酵周期长、生产强度低,制约了其大规模开发利用。文中在酿酒酵母Saccharomyces cerevisiae S288C中异源表达虫草素合成关键基因ScCNS1和ScCNS2,成功构建了产虫草素的酵母工程菌SHC16,发酵240 h虫草素产量可达67.32 mg/L;基因表达分析显示,发酵后期磷酸戊糖途径、嘌呤代谢及虫草素合成途径关键酶编码基因ZWF1、PRS4、ADE4、ScCNS1及ScCNS2表达水平显著上调。进一步地,通过优化发酵培养基组成,确定以50 g/L葡萄糖为初始底物结合一次补料、添加5 mmol/L Cu2+和1.0 g/L腺嘌呤为最适培养基组成。基于此在5 L搅拌发酵罐中开展补料分批发酵,144 h虫草素产量达到137.27 mg/L,生产强度达0.95 mg/(L·h),较未优化发酵体系提高240%。     

不同等位HXT3基因对酿酒酵母EC1118发酵特性的影响

葡萄酒酿酒酵母果糖利用能力与酒精发酵中止和发酵不彻底密切相关,其果糖利用分子机制的研究备受关注。以高果糖利用葡萄酒酿酒酵母菌EC1118为试验材料,利用重组技术获得了HXT3基因敲除菌,通过酶切和基因测序鉴定,首次发现EC1118具有2个不同的HXT3等位基因,并研究了不同HXT3等位基因缺失对菌体发酵过程中葡萄糖、果糖利用的影响。结果表明,突变的HXT3-champ基因对保证菌体发酵后期高果糖利用能力起到了重要的作用,对筛选和构建高果糖利用优良葡萄酒酿酒酵母研究具有重要指导意义。     

工业用糖蜜酿酒酵母菌株耐受性分析研究

采用休止细胞梯度生长法, 对工业糖蜜酿酒酵母(Saccharomyces cerevisiae)菌株进行高浓度酒精、高温和高渗透压, 以及糠醛毒性、苯酚毒性、乙酸毒性和抗生素G418毒性的耐受性分析。结果表明, 所测定的工业酵母菌株对这些逆境条件的耐受性有明显的差别; 其中AS2.1189和AS2.1190对测定的胁迫条件均表现出相对较好的耐受性; 396对乙酸毒性和G418毒性具有很好的耐受性; 2610对高温表现出较强的耐受性。     

商业酵母在不同品种葡萄酒工业化生产中的定殖差异

[背景]酵母菌在葡萄酒酿造中起到重要的作用,接种商业活性干酵母(active dry yeast,ADY)进行葡萄酒酿造在国内较为普遍,然而商业酿酒酵母(Saccharomyces cerevisiae)对我国本土酵母菌资源的影响及二者竞争关系的相关报道不多.[目的]比较商业酿酒酵母在不同品种葡萄酒工业化生产中的定殖差...     

Saccharomyces cerevisiae biodiversity in spontaneous commercial fermentations of grape musts with 'adequate' and 'inadequate' assimilable-nitrogen content

AIM: To evaluate whether intraspecific diversity of Saccharomyces cerevisiae in wine fermentations is affected by initial assimilable-nitrogen content. METHODS AND RESULTS: Saccharomyces cerevisiae isolates from two spontaneous commercial wine fermentations started with adequate and inadequate nitrogen amounts were characterized by mitochondrial DNA restriction analysis. Several strains occurred in each fermentation, two strains, but not the same ones, being predominant at frequencies of about 30%. No significant differences were detected by comparing the biodiversity indices of the two fermentations. Cluster analysis demonstrated that the strain distribution was independent of nitrogen content, the two pairs of closely related dominant strains grouping into clusters at low similarity. CONCLUSIONS: The genetic variability of S. cerevisiae in wine fermentations seemed not to depend on the nitrogen availability in must. SIGNIFICANCE AND IMPACT OF THE STUDY: Nitrogen content did not affect the genetic diversity but may have induced a 'selection effect' on S. cerevisiae strains dominating wine fermentations, with possible consequences on wine properties.     

Selective antimicrobial action of chitosan against spoilage yeasts in mixed culture fermentations

The effect of chitosan on Saccharomyces cerevisiae (the yeast that carries out alcohol fermentation), Brettanomyces bruxellensis and Brettanomyces intermedius (contaminants of alcohol fermentations), was investigated. The effect of chitosan was tested on each yeast, as well as on mixed cultivations of S. cerevisiae + B. bruxellensis and S. cerevisiae + B. intermedius. Chitosan enhanced the lag period of both strains of Brettanomyces (80 h for B. bruxellensis and 170 h for B. intermedius with 6 and 2 g/l chitosan, respectively). The growth rate of S. cerevisiae was inversely proportional to the chitosan concentration; the former was 50% when 6 g/l polysaccharide was used. Moreover, in mixed cultivations of S. cerevisiae and Brettanomyces strains, it was found that both B. bruxellensis and B. intermedius failed to grow while growth of S. cerevisiae was not affected (using 3 and 6 g/l chitosan, respectively). An interesting collateral result was that the presence of chitosan accelerated the consumption of glucose in the mixed cultivations (60 h instead of 120 h).     

Analytical differentiation of wine fermentations using pure and mixed yeast cultures

Summary Thirty-three fermentations of Pedro Ximénez grapes, collected in three degrees of ripeness, were carried out by inoculation with three types of inoculum: pure cultures ofSaccharomyces cerevisiae races and ofTorulaspora delbrueckii, indigenous yeasts, and mixed cultures of indigenous yeasts enriched with the pure cultures. By means of variance analysis 21 compounds were determined whose final concentrations in the wines significantly depended on the musts, the inocula or both. Eleven products that depended significantly on the inocula were subjected to a discriminant analysis in which most of the pure cultures gathered in a discriminant space area different from that occupied by the indigenous yeasts. The centroids corresponding to most of the mixed cultures were shifted to the central area of the discriminant space, moved away from their corresponding pure cultures and approached the indigenous yeasts. The results show a high similarity between the fermentations carried out with mixed cultures with the addedS. cerevisiae races and those fermentations carried out with the indigenous yeasts, with regard to those compounds which were significantly dependent on the inocula.     

微生物遗传多样性的挖掘和代谢工程应用

随着近年来系统生物学研究的深入,微生物的基因组、转录组、蛋白组及代谢组等不同层次的组学信息不断增加。我国具有丰富的微生物多样性,但目前对多样性的研究大多集中在物种多样性及生态多样性方面,对微生物菌株水平遗传多样性的研究还刚刚起步。以酿酒酵母和链霉菌为例,结合本课题组的成果,总结了近年来利用其基因组序列及转录组蛋白质等功能基因组信息,开发利用其遗传多样性的研究进展。在工业酿酒酵母中发现了多个独特的功能基因,包括絮凝基因及与环境胁迫耐性相关的调节蛋白基因,还发现了独特的启动子序列。此外,在海洋放线菌基因组中也发现了独特的调节基因。对微生物遗传多样性的挖掘利用,不仅有助于深入理解微生物不同菌株中独特的调节方式,也为微生物的代谢工程改造提供了大量新的可利用的遗传组件。     

Metabolic diversity of Saccharomyces cerevisiae strains from spontaneously fermented grape musts

One hundred and fifteen Saccharomyces cerevisiae strains from Aglianico del Vulture, a red wine produced in Southern Italy, were characterized for the production of some secondary compounds involved in the aroma and taste of alcoholic beverages. The strains exhibited a uniform behaviour in the production levels of n-propanol, active amyl alcohol and ethyl acetate, whereas isobutanol, isoamyl alcohol and acetaldehyde were formed with a wide variability. Only five strains produced wines close to the reference Aglianico del Vulture wine for the traits considered. Of these, two strains were selected, underwent to tetrad analysis and the single spore cultures were tested in grape must fermentation. The progeny of one strain showed a significant metabolic variability, confirming the necessity to test starter cultures for the segregation of traits of technological interest. Our findings suggest the selection of specific strains for specific fermentations as a function of the vine variety characteristics in order to take the major advantage from the combination grape must/S. cerevisiae strain.     

Arginase activity is a useful marker of nitrogen limitation during alcoholic fermentations

Nitrogen deficiency in musts is one of the causes of sluggish or stuck fermentations. In this work we propose that arginase activity determination can be useful for detecting nitrogen starvation early in vinification. CAR1 and YGP1 genes are not specifically induced under conditions of nitrogen starvation. However, a significant increase in the enzymatic activity of arginase, the product of the CAR1 gene, is detected in vinifications carried out with musts containing limiting amounts of nitrogen. Moreover, on adding ammonia to a nitrogen-deficient vinification, even at late stages, this enzymatic activity is repressed, and growth rate is restored simultaneously. We also investigate the role of ethanol toxicity in nitrogen starvation. The results suggest that ethanol produced during vinification or exogenously added up to 8% (v/v) concentration does not cause nitrogen starvation under the conditions tested because arginase activity is not increased.     

Influence of yeast population on characteristics of the wine obtained in spontaneous and inoculated fermentations of must from <Emphasis Type="Italic">Vitis vinifera</Emphasis> Lado

This work describes the influence of yeast population on the chemical characteristics of wine obtained by spontaneous and inoculated fermentation of must from Vitis vinifera Lado, a minor white grapevine autochthonous to Galicia (NW Spain). The study was carried out for two consecutive years. The results showed that musts derived from Lado presented a high acidity though the potential alcohol level was acceptable. The genetic diversity of S. cerevisiae strains isolated from spontaneous fermentations was low, probably due to must characteristics, although these did not interfere with the implantation of the commercial strains used. Analyses showed that the wines subsequently produced had high alcoholic levels and very high acidities (pH 3.0) as was expected from must composition. Wines obtained from spontaneous fermentations had a lower alcohol content but higher total acidity than those from inoculated fermentations. Monovarietal wines produced from Lado were poorly evaluated in sensorial tests because of their unbalanced structure and sourness; however, when they were mixed with other autochthonous white varieties with less acidity, the resulting wines were well accepted.     

基于DNA高通量测序分析生料酿醋过程中的真菌多样性

【目的】了解生料酿醋不同阶段的真菌群落结构及其变化规律,为生料酿醋工艺优化提供理论指导。【方法】从山西一家生料酿醋企业采集原料、麸曲、发酵缸醋醅、熏醋样、淋醋样等涉及生料酿醋各阶段的样品共51份,扩增真菌ITS1区序列并利用高通量测序技术分析真菌多样性。【结果】除5份样品未扩增成功外,在剩余46份样品中共检测到489个真菌OTU,以子囊菌为主(占88.3%)。原料、麸曲、发酵缸醋醅、熏醋样、淋醋样等不同组别间在真菌群落结构方面存在显著差异。原料和麸曲中的真菌物种丰富度最低,发酵缸醋醅的真菌物种丰富度最高,熏醋样和淋醋样中的真菌物种丰富度又有所降低。原料和麸曲中的优势真菌分别为酿酒酵母和黑曲霉,是发酵阶段真菌的重要来源,但发酵缸醋醅中也检测到大量可能来源于发酵室环境的真菌。发酵缸醋醅在不同发酵时期间也存在明显的真菌群落结构差异,并可据此划分成发酵前期(包括发酵第2–13天的样品)和发酵后期(包括发酵第17–46天的样品)。酿酒酵母和亮白曲霉的丰度在发酵前期显著高于发酵后期,而黑曲霉、一种小戴卫霉科真菌等的丰度在发酵后期显著高于发酵前期。【结论】生料酿醋的不同阶段和发酵缸醋醅发酵的不同时期,其真菌群落结构都存在明显差异。酿酒酵母和黑曲霉是发酵阶段的优势真菌。本研究为生料酿醋工艺优化提供了理论依据。     

Acceleration of high gravity yeast fermentations by acetaldehyde addition

In high gravity Saccharomyces cerevisiae fermentations containing 300 g glucose l^–1, daily addition of acetaldehyde to a total of 93 mM shortened the time required to ferment the first 250 g glucose l^–1 from 790 h to 585 h. Acetaldehyde feeding had no effect on the ethanol yield but increased by 135%, 78% and 77% the final concentrations of 2,3-butanediol, 2-methylpropanol and acetate, while decreasing that of glycerol by 14%. Controlled acetaldehyde feeding has potential as a technique for accelerating high gravity fuel or industrial ethanol fermentations and may be useful in preventing incomplete fermentations.