清香型白酒发酵过程中酵母群落结构及其与尿素代谢的关系

【目的】探索清香型白酒发酵过程中酵母群落结构及演变,分析潜在的关键尿素代谢酵母及其环境调控因素,为降低发酵过程中氨基甲酸乙酯的含量提供理论依据。【方法】通过相关性分析明确发酵过程中氨基甲酸乙酯主要前体物质及其代谢微生物类群,利用高通量测序技术解析酵母群落结构组成,并结合偏最小二乘回归分析寻找潜在的关键尿素代谢酵母。采用冗余分析评价发酵过程中环境因素对酵母群落结构的影响。【结果】尿素是清香型白酒发酵过程中氨基甲酸乙酯的主要前体物质,酵母是尿素代谢的主要微生物。高通量测序结果显示,在97%的相似度下进行操作分类单元聚类后,共鉴定出22个酵母种。其中,嗜高压有孢汉生酵母(Hanseniaspora osmophila)、发酵毕赤酵母(Pichia fermentans)和酿酒酵母(Saccharomyces cerevisiae)与尿素合成存在正相关性,库德毕赤酵母(Pichia kudriavzevii)与尿素降解存在正相关性。酒醅含水量、p H、乙醇和精氨酸是影响发酵过程中酵母群落演替的重要环境因素。【结论】环境因素影响潜在的关键尿素代谢酵母,为降低发酵过程中尿素与氨基甲酸乙酯含量提供理论依据。     

酱香型白酒发酵中酵母群落结构及其对风味组分的影响

【目的】研究酱香型白酒发酵过程中酵母群落结构,及其对酱香型白酒生产的影响。【方法】通过变性梯度凝胶电泳(DGGE)和实时荧光定量PCR(RT-q PCR)技术分别对产量、酒质较高的第五轮次和较低的第七轮次发酵过程中的酵母群落结构进行解析,同时利用顶空固相微萃取气质联用(HS-SPME-GC-MS)以及高效液相色谱(HPLC)技术分析酒醅中的代谢组分,初步分析了酵母群落对酱香型白酒产量及品质的影响。【结果】用DGGE方法从酒醅中的检测到Issatchenkia orientalis、Torulaspora delbrueckii、Pichia galeiformis、Schizosaccharomyces pombe、Galactomyces geotrichum、Trichosporon asahii、Zygosaccharomyces bailii、Saccharomyces cerevisiae和Pichia fabianii等9种含量丰富的酵母,其中G.geotrichum和S.cerevisiae是第五轮的优势酵母,而第七轮的优势酵母增加了I.orientalis和Z.bailii两种酵母,且Sc.pombe未检测到。第七轮发酵过程中5种酵母在发酵后期衰亡明显,第五轮酵母群落结构相对更稳定;发酵前期第五轮酵母总量是第七轮的2–5倍,而发酵结束时第五轮乙醇含量达到第七轮次的2.45倍。酒醅中挥发性风味组分种类丰富,第五轮中酯类等白酒中重要的风味物质含量明显高于第七轮,有机酸和杂醇油含量低于第七轮。【结论】酱香型白酒酿造过程中酵母菌群多样性较丰富,酵母菌群结构变化对白酒产量及品质影响明显,这为酱香型白酒酿造机制研究奠定了基础。     

清香型白酒中乳酸菌和酵母菌的相互作用

【目的】探究清香型白酒中不同乳酸菌和酵母菌的相互作用,了解不同菌株的发酵性能,为更深入地认识白酒发酵机理、实现发酵过程优化提供理论基础。【方法】利用程序控温和固态发酵模拟清香型白酒酿造环境,测定纯培养和共培养中菌株的理化指标、活菌数以及主要代谢产物的变化。【结果】Saccharomyces cerevisiae YJ1糖消耗快产乙醇和酯类物质多,Lactobacillus plantarum JMRS4糖消耗快产酸较多。共培养中乳酸菌对Saccharomyces cerevisiae YJ1的生长和产乙醇抑制较大,对Candida aaseri MJ7产乙醇几乎无影响。乳酸菌对Pichia kudriavzevii MJ14的生物量和乙醇代谢抑制作用较小,还对其产己酸乙酯、乙酸乙酯和异戊醇等代谢产物有促进作用;而反过来Pichia kudriavzevii MJ14对3株乳酸菌产乳酸均有抑制作用,对产乙酸则有促进作用。【结论】建立了一种固态培养方法,结合清香型白酒发酵温度变化规律,有效模拟了实际发酵环境。Pichia kudriavzevii MJ14在与乳酸菌共培养中受到的抑制较小并能有效抑制乳酸菌产乳酸,Saccharomyces cerevisiae YJ1能代谢产生多种风味物质,对清香型白酒酿造有重要意义。     

酱香型与清香型白酒发酵过程中乳酸菌菌群的差异性分析

【目的】为认识乳酸菌在中国白酒酿造过程中的作用与影响, 分析、比较了酱香型与清香型白酒发酵过程中乳酸菌的菌群结构及其差异。【方法】运用PCR-DGGE技术分析酱香型与清香型白酒发酵过程中乳酸菌群的演变规律。并利用传统微生物分离筛选方法进一步确定酱香型白酒发酵中的主要乳酸菌种。【结果】DGGE图谱表明, 白酒发酵过程中的主要乳酸菌种是乳杆菌。但两种香型白酒发酵过程中乳酸菌群组成及动态变化均呈现出明显的差异。清香型白酒酒醅中Lactobacillus fuchuensis是优势菌种, 而酱香型白酒发酵中检测到多种含量较高的乳酸菌种。利用MRS培养基从酱香型白酒酒醅中共筛选获得5种乳酸菌种。通过两种方法, 确定Lactobacillus homohiochii是酱香型白酒发酵过程中含量最高的乳酸菌。【结论】深入研究白酒发酵过程中乳酸菌的组成及分布规律, 对于更好地认识中国白酒酿造中主要的细菌类群——乳酸菌的作用, 具有重要的理论意义和实践价值。     

基于26S rDNA D1/D2序列分析酱香型白酒酒醅中酵母菌的群落结构

【目的】比较分析不同发酵阶段的酱香型白酒北大仓酒醅中酵母菌的群落结构。【方法】酒醅样本采集自黑龙江省北大仓白酒不同阶段的发酵窖池,采用平板分离技术获得大量酵母菌纯培养物;基于26S rDNA D1/D2区域的碱基序列,准确鉴定酒醅中的酵母菌;根据酵母菌的数量变化、种类组成、相对频率、多样性指数及其相似性系数,研究酱香型北大仓白酒酒醅中酵母菌的群落结构特征。【结果】北大仓白酒酒醅中酵母菌数量在酿造过程中的变化,遵循着"升高-降低-升高-降低"的规律;第1轮"升高-降低"的过程表现出急剧的"速升速降"特点,而第2轮"升高-降低"的过程则具有相对来说比较温和的"缓升缓降"特征。酒醅内的酵母菌鉴定为8属13种,总体多样性指数处于较高的水平,发酵初期酵母菌多样性指数逐渐增加,最高的多样性指数(1.89)出现在发酵第5 d的酒醅内;然后又逐渐下降,发酵至第11 d时降至最低(0.66);酒醅发酵至13 d时开始直至发酵末期,多样性指数呈现出波动性变化。北大仓白酒酒醅中酵母菌的种类组成总体来说比较接近,大多数情况下相似性系数都高于0.5,说明在白酒酿造过程中酵母菌的组成和分布处于一个相对稳定的状态。对于北大仓白酒酒醅内存在的常见酵母菌来说,Issatchenkia orientalis、Pichia kudriavzevii和Saccharomyces cerevisiae可能在酿造过程中发挥更重要的作用。【结论】北大仓等酱香型白酒的酒醅内存在着丰富的酵母菌资源,阐明酒醅内酵母菌的群落结构可以为解析酱香型白酒的发酵机理提供基础数据和理论基础。     

荧光定量PCR法定量白酒固态发酵过程中产土味素的链霉菌

【目的】荧光定量PCR法具有特异性高、灵敏度高等优点,在定量复杂环境中微生物数量方面得到广泛应用。本文采用荧光定量PCR方法对固态白酒发酵过程产土味素链霉菌进行定量分析并验证其准确性。【方法】通过优化大曲和酒醅中微生物基因组提取方法,并建立相应的大曲和酒醅两种基质条件下的荧光定量PCR标准曲线,并对方法的精度和准确度进行验证分析。采用荧光定量PCR方法对大曲和酒醅中产土味素链霉菌进行定量分析。【结果】结果表明大曲中产土味素链霉菌数量在105数量级,并且清茬曲中此类链霉菌数量最高。酒醅发酵起始阶段产土味素链霉菌数量在104数量级,而后随着发酵的不断进行,酒醅中此类链霉菌数量有所减少。【结论】荧光定量PCR方法能够对白酒固态发酵过程中产土味素链霉菌准确进行定量分析,对采用此方法定量其他微生物具有借鉴意义。     

配糟对清香型白酒发酵中环境因子及微生物群落的影响

【背景】环境因子是影响微生物生长代谢的重要因素,解析半开放条件下酿造过程中环境因子对微生物群落演替的作用对于清香型白酒生产调控具有重要意义。配糟在白酒发酵过程中起着调节发酵速度的作用,其对微生物群落组成变化的影响尚不明确。【目的】揭示使用不同发酵周期配糟对清香型白酒发酵过程中环境因子及微生物群落演替的影响。【方法】采用PacBio测序平台和多元统计分析比较使用2种配糟酒醅中微生物群落结构组成,结合蒙特卡洛置换检验明确环境因子对微生物群落的影响。【结果】与使用正常发酵周期配糟酒醅相比,使用延长发酵周期配糟酒醅水分较低,而酸度、氨基酸态氮、总游离氨基酸、还原糖和残余淀粉较高;微生物多样性和丰富度分析发现,使用延长发酵周期配糟酒醅中细菌α多样性极显著高于使用正常发酵周期配糟酒醅(P<0.001),而真菌α多样性显著/极显著低于使用正常发酵周期配糟酒醅(P<0.05, P<0.001);通过组间差异性分析发现,细菌群落共产生28个差异指示种,而真菌群落共产生15个差异指示种;水分、酸度、氨基酸态氮、还原糖、残余淀粉和总游离氨基酸对微生物群落结构的影响显著(P<0.05)...     

酱香型白酒发酵中两株主要乳酸菌对酿造微生物群体的影响

【目的】研究从酱香型白酒发酵酒醅中分离得到的2株主要乳酸菌Lactobacillus homohiochii XJ-L1和Lactobacillus buchneri XJ-L2对酱香型白酒发酵中酿造微生物群体的作用,并探索该种相互作用对酱香型白酒品质的影响。【方法】结合抑菌实验和组合发酵实验研究L. homohiochii XJ-L1和L. buchneri XJ-L2对酿造微生物群体生长的影响,通过对纯培养和共培养体系中代谢物的比较,研究2株优势乳酸菌对主要酿造酵母风味相关代谢产物的影响。【结果】L. buchneri XJ-L2能够抑制3株芽孢杆菌(Bacillus amyloliquefaciens XJ-B1,Bacillus subtilis XJ-B2,Bacillus licheniformis XJ-B3)、5株霉菌(Aspergillus oryzae XJ-M1,Aspergillus niger XJ-M2,Aspergillus flavus XJ-M3,Aspergillus albicans XJ-M4,Rhizopus oryzae XJ-M5)、2株酵母(Schizosaccharomyces pombe XJ-Y4,Geotrichum candidum XJ-Y5)的生长;L. homohiochii XJ-L1和L. buchneri XJ-L2能够促进3株主要酵母(Saccharomyces cerevisiae XJ-Y1,Zygosaccharomyces bailii XJ-Y2,Pichia galeiformis XJ-Y3)的生长,同时促进其酸类、醇类、酯类等风味物质的代谢。【结论】L. homohiochii XJ-L1和L. buchneri XJ-L2可促进3株主要酵母的生长代谢,同时L. buchneri XJ-L2明显抑制细菌、霉菌和少数酵母的生长,以此促进和维持主要酵母在酱香型白酒发酵过程中的生态地位,从而影响酒中酸类、醇类、酯类等风味物质的形成,保证酱香型白酒的品质。因此,适当比例的乳酸菌对维持酿造微生物区系平衡,生产典型酱香品质白酒具有重要意义。     

芝麻香型酒醅堆积过程中微生物结构变化分析

【背景】高温堆积发酵过程是芝麻香型白酒生产过程中的一个重要环节,是芝麻香型白酒典型风格形成的关键工序。【目的】解析堆积发酵过程中原核和真核微生物群落的演替规律。【方法】分别对高温堆积酒醅样本的细菌16S rDNA V3-V4区和真菌ITS2区进行Illumina高通量测序,解析高温堆积过程中酒醅的真菌和细菌菌群结构变化规律,同时解析发酵过程中温度、水分、酸度、淀粉、还原糖的变化规律。【结果】酒醅堆积初期0-6 h,占优势的原核微生物菌属是Bacillus、Weissella和Acetobacter。堆积20 h后微生物群落结构变化显著,Weissella的比例降低,Acetobacter的比例显著增加。堆积初期,占优势的真核微生物菌属是Aspergillus、Saccharomyces和Pichia。堆积6 h后,Aspergillus比例下降,而Pichia和Saccharomyces则逐步增多,其绝对优势地位一直保持至堆积结束。酒醅微生物群落结构受翻堆操作的短暂影响(堆积20 h时),最终恢复至平衡状态。高温堆积36 h以后,酒醅中的原核和真核微生物群落均趋于稳定。还原糖数据显示堆积前期微生物糖化作用迅速,36 h后糖化趋于稳定,温度、酸度、淀粉含量参数基本平稳。【结论】酒醅高温堆积发酵过程中,原核微生物和真核微生物有其不同的变化规律。原核微生物的变化是以20 h时的翻堆为结点,翻堆前的优势菌属是Bacillus、Weissella和Acetobacter;堆积后的优势菌属是Weissella和Acetobacter,其中后者占绝对优势。与原核微生物相比,真菌微生物的结构逐步集中,到发酵结束时毕赤酵母和酵母两者占真菌总量的93%以上。芝麻香型白酒发酵过程的高温堆积环节,原核和真核微生物的菌种繁殖以及相应的菌群结构变化为稍后的高温发酵奠定了微生物基础。     

酱香型白酒酿造酒醅中酵母菌多样性研究

为解析酱香型白酒酿造酒醅中酵母菌的菌群结构,获取酒醅中的主要酵母菌,采用高通量测序法分析酱香型白酒酒醅中酵母菌多样性及主要功能菌群,同时采用可培养分离方法获取酒醅中酵母菌活性菌株。从酱香型白酒下沙至五轮次酒醅中共检出59个属、129个种的酵母菌,分离得到酵母菌活性菌株41种,检测到的酵母菌种类与获得的酵母菌活菌在各香型白酒中最多。不同时期酒醅中的酵母菌种类和数量差异明显,其中下沙、造沙轮次以Pichia kudriavzevii为绝对优势酵母菌;一至五轮次随着轮次的递增,酒醅中优势酵母菌的种类增多,其中主要的优势酵母菌有Pichia kudriavzevii、Pichia manshurica、Zygosaccharomyces bailii、Saccharomyces cerevisiae、Candida apicola。酱香型白酒酒醅中蕴藏着极其丰富的酵母菌资源,对酵母菌菌群结构的解析有助于科学地认识酱香型白酒酿造过程中产酒与风味代谢机理,为发酵过程的调控提供一定依据。     

In Situ Analysis of Metabolic Characteristics Reveals the Key Yeast in the Spontaneous and Solid-State Fermentation Process of Chinese Light-Style Liquor

The in situ metabolic characteristics of the yeasts involved in spontaneous fermentation process of Chinese light-style liquor are poorly understood. The covariation between metabolic profiles and yeast communities in Chinese light-style liquor was modeled using the partial least square (PLS) regression method. The diversity of yeast species was evaluated by sequence analysis of the 26S ribosomal DNA (rDNA) D1/D2 domains of cultivable yeasts, and the volatile compounds in fermented grains were analyzed by gas chromatography (GC)-mass spectrometry (MS). Eight yeast species and 58 volatile compounds were identified, respectively. The modulation of 16 of these volatile compounds was associated with variations in the yeast population (goodness of prediction [Q²] > 20%). The results showed that Pichia anomala was responsible for the characteristic aroma of Chinese liquor, through the regulation of several important volatile compounds, such as ethyl lactate, octanoic acid, and ethyl tetradecanoate. Correspondingly, almost all of the compounds associated with P. anomala were detected in a pure culture of this yeast. In contrast to the PLS regression results, however, ethyl lactate and ethyl isobutyrate were not detected in the same pure culture, which indicated that some metabolites could be generated by P. anomala only when it existed in a community with other yeast species. Furthermore, different yeast communities provided different volatile patterns in the fermented grains, which resulted in distinct flavor profiles in the resulting liquors. This study could help identify the key yeast species involved in spontaneous fermentation and provide a deeper understanding of the role of individual yeast species in the community.     

新疆地区酸马奶中酵母菌的鉴定及其生物多样性分析

从新疆少数民族牧民家庭采集的28份传统工艺酿造酸马奶样品中分离出87株酵母菌,并对其进行了生理生化鉴定、分子生物学鉴定和生物多样性分析。生化试验结果表明,新疆地区酸马奶中的酵母菌为Saccharomyces unisporus(占总分离株的48.3%),Kluyveromyces marxianus(27.6%),Pichia membranaefaciens(15.0%)和Saccharomyces cerevisiae(9.2%)。选取其中的6株酵母菌和1株参考菌株,进行大亚基(26S)rDNA D1/D2区域(600bp左右)碱基序列分析,并通过GenBank进行同源序列搜索以确定各菌株的归属,进一步验证生理生化方法的正确性。从得到的结果中可以看出,S.unisporus和K.marxianus为新疆地区酸马奶中的优势菌。     

Occurrence and dominance of yeast species in naturally fermented milk from the Tibetan Plateau of China

To determine which yeasts are present in the naturally fermented milks of China, 69 samples made by the nomads of Tibet were collected from the Tibetan Plateau in China. From these samples, 225 strains of yeast were isolated and identified using conventional microbiological analysis and gene sequencing analysis of the D1/D2 domain of the large subunit (26S) ribosomal DNA. The results showed that the total concentration of yeasts in these samples ranged from 5.01 to 8.97 log10 colony-forming units (CFU)/mL (6.91?± 1.02 log10 CFU/mL; mean?± SD). The number of cultivable yeasts was higher in the samples from Qinghai (7.55?± 0.75 log10 CFU/mL) than those from Tibet (6.21?± 0.79 log10 CFU/mL, P?< 0.05). Moreover, there were 15 phylotypes in these 69 samples. Among these phylotypes, Kluyveromyces marxianus (49.3%, frequency percentage), Saccharomyces cerevisiae (62.3%), and Pichia fermentans (46.4%) appeared frequently and can be considered the most common culturable species in naturally fermented milk products. Traditional fermented Mongolian cow milk featured a wide diversity of yeast species, including Issatchenkia orientalis, Kazachstania unisporus, Rhodotorula mucilaginosa, Candida pararugosa, Torulaspora delbrueckii, Geotrichum sp., Kazachstania unisporus, Geotrichum fragrans, Debaryomyces hansenii, Yarrowia lipolytica, Trichosporon gracile, and Pichia membranifaciens. This study provides new data on yeast composition in naturally fermented milk and shows the yeast biodiversity of fermented milk products from the Tibetan Plateau of China.     

Characterization and comparison of microbial community of different typical Chinese liquor Daqus by PCR-DGGE

Aims: To identify and compare microbiota in Chinese liquor Daqu, which were produced in the different regions using different production process. Methods and Results: The DNA exacted from Daqu samples was used as a template for PCR with universal primers of 16S rRNA, 26S rRNA and 18S rRNA, respectively. The amplicons were analysed using denaturing gradient gel electrophoresis (DGGE). It was observed that the bacterial DGGE profile indicated high diversity and predominance of lactic acid bacteria. The results showed that Saccharomycopsis fibuligera and Pichia anomal were dominant yeast species and that several non‐Saccharomyces yeasts including Hanseniaspora guilliermondii, Debaryomyces hansenii, Issatchenkia orientalis and Trichosporon asahii were also detected. As for fungal DGGE, Aspergillus oryzae and Absidia blakesleeana were the most common species amongst different samples. Based on the DGGE analysis, a few differences in community structure were found between Daqu samples. Conclusions: A variety of bacteria, yeast and moulds were identified in Daqu samples, in addition to the present knowledge obtained mainly through the traditional culture‐dependent methods. Moreover, production temperature played a more decisive role on the formation of micro‐organism composition in Daqu than geographical region. Significance and Impact of the Study: PCR–DGGE technique was used in this study to fully observe and asses all microbial community (including bacteria, yeast and mould) in Chinese liquor Daqu for the first time and proved to be effective in profiling Daqu microbial diversity.     

高温大曲中地衣芽胞杆菌(Bacillus licheniformis CGMCC 3963)的耐高温特征

【目的】高温大曲的使用是中国酱香型白酒酿造的特征之一,耐高温的细菌是高温大曲中重要的优势功能菌,它们对酱香型白酒的酿造具有重要的价值。研究高温大曲中细菌的耐高温特征对于认识酱香型白酒的酿造特征具有一定的作用。【方法】本文以中国白酒高温大曲中筛选获得的一株具有自主知识产权的耐高温功能细菌地衣芽胞杆菌(Bacillus licheniformis)CGMCC 3963为对象,测定其耐高温特征,并首次通过细胞形态学的分析,以及转录组学等技术的应用系统研究了B.licheniformis CGMCC 3963的耐高温机制。【结果】地衣芽胞杆菌B.licheniformis CGMCC 3963能够耐受55℃高温生长。在高温条件下,该菌仍具有较旺盛的代谢生长能力,且产生大量荚膜,同时大量I类热休克蛋白基因以及聚谷氨酸合成基因的表达水平均得到明显提高。【结论】B.licheniformis CGMCC 3963在高温下,I类热休克蛋白以及以聚谷氨酸为主要成分的荚膜大量产生,对功能细菌耐高温具有重要的作用。该结果首次从分子水平认识了中国白酒酿造微生物的耐高温特征,丰富了白酒酿造微生物学的科学理论。     

Identification of yeast species from orange fruit and juice by RFLP and sequence analysis of the 5.8S rRNA gene and the two internal transcribed spacers

Yeast isolates from orange fruit and juice in a spontaneous fermentation were identified and classified by two molecular techniques. The first was analysis of the restriction pattern generated from the polymerase chain reaction (PCR)-amplified 5.8S rRNA gene and the two internal transcribed spacers (ITS) using specific primers. The second technique was sequence analysis of the ITS regions using the same two primers. Nine different restriction profiles were obtained from the size of the PCR products and the restriction analyses with three endonucleases (CfoI, HaeIII and HinfI). These groups were identified as Candida tropicalis, Clavispora lusitaniae, Hanseniaspora uvarum, Pichia anomala, Pichia fermentans, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Saccharomyces unisporus, and Trichosporon asahii. Checking against identification according to morphological, physiological and biochemical traits corroborated this molecular identification. A total concordance was found in the identification with PCR-restriction fragment length polymorphism of the ITS region after analysing certified yeast strains from two different culture collections. Consequently, a rapid and reliable identification of the yeast populations was achieved by using molecular techniques.