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。     

酵母菌的高密度发酵

高密度发酵是酵母工业的发展趋势。本文分析了限制酵母菌高密度发酵的可能的因素,并讨论了高密度发酵的研究进展和发展方向。最后介绍了高密度发酵的清洁生产工艺。     

高效发酵木糖生产乙醇酵母菌株的构建

获得高效发酵木糖生产乙醇的酵母菌株是木质纤维素生物转化生产燃料乙醇的重要前提。在4%乙醇驯化的基础上,选择了乙醇耐性提高的休哈塔假丝酵母（Candida shehatae）CICC1766菌株进一步进行紫外诱变,得到了木糖发酵性能较强的呼吸缺陷型突变体,并与乙醇发酵性能良好的酿酒酵母（Saccharomyces cerevisiae）ATCC4126进行原生质体融合。采用单亲灭活法对休哈塔假丝酵母原生质体进行紫外灭活,在聚乙二醇（PEG）诱导下融合,对得到的融合子进行木糖发酵能力测定,选择到了一株能够更好地利用木糖产乙醇,并且木糖发酵性能比亲本得到明显提高的融合子F6,此融合子发酵50 g/L木糖,最高乙醇浓度达到18.75g/L,乙醇得率为0.375,达到理论转化值0.511的73.4%。与原始出发菌株CICC1766相比,乙醇产量提高了28%。     

葡萄酒相关酵母的香气形成及香气特征

一系列相关微生物的代谢及相互作用贯穿在葡萄酒的酿造过程中,其中各种酵母菌的代谢产物是影响葡萄酒感官特征的关键,不同菌种代谢产生不同的挥发性物质,造成对葡萄酒香气的最直接影响。介绍了酿酒酵母以及非酿酒酵母的代谢特征与差别,由此所引起的葡萄酒气味的不同表现,以及发酵过程中不同菌种的相互作用,总结了近些年来对非酿酒酵母酿酒特性的研究与利用。目前已证实非酿酒酵母的一些代谢特征对葡萄酒香气和香气结构具有一些积极的作用,并且酿酒酵母和有些非酿酒酵母的结合使用对改善葡萄酒感官也具有良好的作用与发展前景。     

甘蔗糖蜜酒精废液混菌发酵菌体蛋白的研究

本文报导利用甘蔗糖蜜酒精废液混菌发酵高质量菌体蛋白的研究,通过热带假丝酵母Ｃａｎｄｉｄａ ｔｒｏｐｉｃａｌｉｓ种合融合株Ｃｔ－３配伍其他菌株,混菌发酵时间缩短２￣４ｈ,生物量可达２０ｇ／Ｌ,粗蛋白质含量５０￣５３％,灰份≤１０％,水分为５￣８％,与Ｃｔ－３单菌发酵相比,蛋白质提高４￣６％。     

葡萄酒相关酵母的香气形成及香气特征

一系列相关微生物的代谢及相互作用贯穿在葡萄酒的酿造过程中,其中各种酵母菌的代谢产物是影响葡萄酒感官特征的关键,不同菌种代谢产生不同的挥发性物质,造成对葡萄酒香气的最直接影响。介绍了酿酒酵母以及非酿酒酵母的代谢特征与差别,由此所引起的葡萄酒气味的不同表现,以及发酵过程中不同菌种的相互作用,总结了近些年来对非酿酒酵母酿酒特性的研究与利用。目前已证实非酿酒酵母的一些代谢特征对葡萄酒香气和香气结构具有一些积极的作用,并且酿酒酵母和有些非酿酒酵母的结合使用对改善葡萄酒感官也具有良好的作用与发展前景。     

混菌发酵中不同分子量代谢产物对非酿酒酵母胞内蛋白及酒体有机酸的影响

本研究采用透析袋发酵法研究了酿酒酵母产生的不同分子量代谢产物对非酿酒酵母胞内蛋白和酒体有机酸的影响。当截留分子量为3.5 kD和10.0 kD时非酿酒酵母的存活时间分别延长至18 d和22 d,表明通过改变菌种之间物质的交换可以调节非酿酒酵母的存活时间。蛋白质解析发现共有65个蛋白质斑点表达出现差异,占蛋白质总数的13%,经质谱鉴定表明与这些蛋白同类固醇、赖氨酸、有机酸和ATP的生物合成有关。与截留分子量为10 kD的透析袋发酵相比,在截留分子量为3.5 kD的混菌发酵中,酒石酸含量升高5.1%、醋酸含量下降44.3%,说明通过限定菌体间代谢产物的沟通可以调整酒的滴定酸度和挥发酸度。     

发酵过程中的防染菌防治研究

发酵是生物体对有机物的一种分解过程,是人类接触较早的化学生物反应之一。随着化学生物技术的不断发展与进步,发酵应用的领域越来越广泛,涉及生物工业、化学工业和食品工业。染菌是发酵过程中出现的严重问题,染菌率不断升高会导致生产成本不断增加,降低企业的生产水平,还会给环境造成极大的污染,威胁社会与环境的和谐发展。本文主要从加强染菌的管理和监督、染菌的有效处理和严格控制发酵操作环节三个方面,研究发酵过程中防染菌的防治措施。     

葡萄酒苹果酸—乳酸发酵接种时间选择的研究

分别在佳利酿葡萄汁酒精发酵前期（第Ｉ阶段）、中后期（第Ⅱ阶段）和结束时（第Ⅲ阶段）接种酒明串珠菌（Ｌｅｕｃｏｎｏｓｔｏｃ ｏｅｎｏｓ）３１ＤＨ,进行苹果酸－乳酸发酵（ＭＬＦ）接种时间选择的研究,结果表明：不同阶段接种经ＭＬＦ后总酸降幅均为２．６ｇ／Ｌ;挥发酸含量上升且第Ｉ阶段＞第Ⅱ阶段＞第Ⅲ阶段;挥发酯以第Ⅱ阶段含量最高,为０．３７ｇ／Ｌ,同其他两阶段含量相比较,差异显（α＝０．０５）;接种时间     

Influence of Williopsis saturnus yeasts in combination with Saccharomyces cerevisiae on wine fermentation

Aim: To examine the growth and survival of Williopsis saturnus strains along with wine yeast Saccharomyces cerevisiae in grape must. Methods and Results: For this study, fermentations were performed in sterilized grape must at 18°C. Inoculum level was 5 × 10⁶ cells per ml for each yeast. The results showed that W. saturnus yeasts exhibited slight growth and survival depending on the strain, but they died off by day 5. Saccharomyces cerevisiae, however, dominated the fermentation, reaching the population of about 8 log CFU ml^?1. It was observed that ethanol formation was not affected. The concentrations of acetic acid, ethyl acetate and isoamyl acetate were found higher in mixed culture experiments compared to control fermentation. The results also revealed that higher alcohols production was unaffected in general. Conclusion: Fermentations did not form undesirable concentrations of flavour compounds, but production of higher levels of acetic acid in mixed culture fermentations may unfavour the usage of W. saturnus in wine making. Significance and Impact of the Study: This study provides information on the behaviour of W. saturnus together with S. cerevisiae during the alcoholic fermentation.     

Acetaldehyde production in Saccharomyces cerevisiae wine yeasts

Abstract Eighty-six strains of Saccharomyces cerevisiae were investigated for their ability to produce acetaldehyde in synthetic medium and in grape must. Acetaldehyde production did not differ significantly between the two media, ranging from a few mg/l to about 60 mg/l, and was found to be a strain characteristic. The fermentation temperature of 30°C considerably increased the acetaldehyde produced. This study allowed us to assign the strains to different phenotypes: low, medium and high acetaldehyde producers. The low and high phenotypes differed considerably also in the production of acetic acid, acetoin and higher alcohols and can be useful for studying acetaldehyde production in S. cerevisiae , both from the technological and genetic point of view.     

崇明老白酒酿造过程中酵母菌的鉴定及其特性初探

从八二酒曲及酿造崇明老白酒过程中分离纯化得到1株白色酵母菌和1株红色酵母菌,采用分子生物学方法进行鉴定,并对其酿造老白酒的特性进行了分析。结果显示,八二酒曲及崇明老白酒酿造过程中的优势酵母菌为酿酒酵母(Saccaromyces cerevisiae),从酿酒过程中分离的红色酵母菌为粘红酵母(Rhodotorula mucilaginosa)。采用粘红酵母和米根霉曲酿造的酒液的酒精度为11.9%vol,残余还原糖含量为11.2 g/100 m L,总酸含量为4.59 g/L,总酯含量为4.42 g/L。纯化的酿酒酵母和米根霉曲酿成的酒液口味醇和爽口,酒曲的纯化有助于开发出口感更爽口的老白酒。混合酵母和米根霉曲酿造的酒液呈典型的崇明老白酒风味,粘红酵母的参与对崇明老白酒口味风格的形成有一定的作用。     

Genetic diversity in commercial wineries: effects of the farming system and vinification management on wine yeasts

Aims: Analysis of the diversity and distribution of wine yeasts isolated from organically and conventionally grown grapes, and during the subsequent fermentation with or without starter cultures in six different commercial wineries. Methods and Results: PCR‐RFLP screening of isolates revealed the involvement of ten different species. Saccharomyces cerevisiae, scarcely isolated from grapes, was the dominant species during the latter phases of fermentation, identifying 108 different genotypes by means of SSR analysis. Species and strains’ diversity and presence were strongly influenced by the farming system used to grow the grapes and the system of vinification. Conclusions: Organic farming management was more beneficial in terms of diversity and abundance than the conventional one. Induced fermentation generated a great replacement of native yeasts. Although winery‐resident yeasts resulted to be predominant in the process, some noncommercial strains originally in the vineyard were found in final stages of the fermentation, confirming that autochthonous strains of S. cerevisiae are capable to conduct the fermentation process up to its end. Significance and Impact of the Study: The study of natural yeast communities from commercial vineyards and wineries is an important step towards the preservation of native genetic resources. Our results have special relevance because it is the first time that the real situation of the yeast ecology of alcoholic fermentation in commercial wineries belonging to the relevant wine‐producing Appellation of Origin ‘Vinos de Madrid’ is shown.     

Acetoin production in Saccharomyces cerevisiae wine yeasts

Abstract One hundred strains of Saccharomyces cerevisiae were examined for the capacity to produce acetoin in synthetic medium and in grape must. The low production of acetoin was found to be the more common pattern in this species. Most strains exhibited a similar distribution in both media, production ranging from non-detectable amounts to 12 mg 1⁻¹. Only four strains produced high quantities of acetoin, up to 29.5 mg l⁻¹ in synthetic medium and up to 194.6 mg l⁻¹ in grape must. This biometric study showed the existence of two phenotypes, "low and high acetoin production", that could be selected for conferring a desirable flavour of the final product.     

The effect of pyrimethanil on the growth of wine yeasts

Aims:  The toxicity of the fungicide pyrimethanil on the growth of wine yeasts was evaluated using in vivo and in vitro experimentation.
Methods and Results:  The effect of pyrimethanil in the must was studied during the spontaneous wine fermentation of three consecutive vintages and by the cultivation of Hanseniaspora uvarum and Saccharomyces cerevisiae yeasts in a liquid medium. The residues of the fungicide were measured using gas chromatography-mass spectrometry system and the sugar concentration in the must using HPLC-RI. Molecular and standard methods were used for identifying the yeast species. Although the pyrimethanil residues in grapes were below the maximum residue limits, they significantly affected the reduced utilization of sugars in the first days of fermentation. Its residues controlled the growth of H. uvarum during the fermentation and during in vitro cultivation as well.
Conclusions:  The fungicide pyrimethanil had an effect on the course and successful conclusion of spontaneous wine fermentation that was correlated with the initial concentration of yeasts in the must.
Significance and Impact of the Study:  The impact of pyrimethanil on the indigenous mixed yeast flora in fermenting must was investigated for the first time. The results showed that its residues might play an important role in the growth and succession of yeast during spontaneous wine fermentation.     

Genetically different wine yeasts isolated from Austrian vine-growing regions influence wine aroma differently and contain putative hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii

To evaluate the influence of the genomic properties of yeasts on the formation of wine flavour, genotypic diversity among natural Saccharomyces cerevisiae strains originating from grapes collected in four localities of three Austrian vine-growing areas (Thermenregion: locations Perchtoldsdorf and Pfaffst?tten, Neusiedlersee-Hügelland: location Eisenstadt, Neusiedlersee: location Halbturn) was investigated and the aroma compounds produced during fermentation of the grape must of 'Grüner Veltliner' were identified. Amplified fragment length polymorphism analysis (AFLP) showed that the yeast strains cluster in four groups corresponding to their geographical origin. The genotypic analysis and sequencing of the D1/D2 domain of 26S rRNA encoding gene and ITS1/ITS2 regions indicated that the Perchtoldsdorf strains were putative interspecies hybrids between S. cerevisiae and Saccharomyces kudriavzevii. Analysis of the aroma compounds by GS/MS indicated a region-specific influence of the yeasts on the chemical composition of the wines. The aroma compound profiles generated by the Perchtoldsdorf strains were more related to those produced by the Pfaffst?tten strains than by the Eisenstadt and Halbturn strains. Similar to the Pfaffst?tten yeasts, the putative hybrid strains were good ester producers, suggesting that they may influence the wine quality favourably.     

Analysis of Saccharomyces cerevisiae hexose carrier expression during wine fermentation: both low- and high-affinity Hxt transporters are expressed

The transport of glucose and fructose into yeast cells is a critical step in the utilization of sugars during wine fermentation. Hexose uptake can be carried out by various Hxt carriers, each possessing distinct regulatory and transport-kinetic properties capable of influencing yeast fermentation capacity. We investigated the expression pattern of the hexose transporters Hxt1 to 7 at the promoter and protein levels in Saccharomyces cerevisiae during wine fermentation. The Hxt1p carrier was expressed only at the beginning of fermentation, and had no role during stationary phase. The Hxt3p carrier was the only one to be expressed throughout fermentation, displaying maximal expression at growth arrest and slowly decreasing in abundance over the course of the stationary phase. The high-affinity carriers Hxt6p and Hxt7p displayed similar expression profiles, with expression induced at entry into stationary phase and persisting throughout the phase. The expression of these two carriers occurred despite the presence of high amounts of hexoses, and the proteins were stably expressed when the cells were starved for nitrogen. The Hxt2p transporter was only transiently expressed during lag phase, which suggests a role for the protein in growth initiation. Characterization of glucose transport kinetics indicated the presence of a shift in the low-affinity component that is consistent with a predominant expression of Hxt1p during growth phase and of Hxt3p during stationary phase. In addition, a high-affinity uptake component consistent with functional expression of Hxt6p/Hxt7p was identified during stationary phase.