芝麻香型白酒酿造微生物多样性分析
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摘要
芝麻香型白酒是近几十年来才发展起来的新香型，该香型白酒风格突出、自成一格。尽管目前对工艺技术特点和香味成分的基本特征有了初步认识，但其微生物的消长规律及香味特征成分的形成机理，以及工艺技术的进一步改进，仍需要进一步的探索。因此，研究芝麻香型白酒高温大曲、堆积过程及窖泥中微生物，有利于揭示芝麻香型白酒香味的形成机制。本论文综合运用PLFA、DGGE等免培养方法研究了芝麻香型白酒高温大曲、堆积过程和窖泥微生物的生物量及其群落结构特征。通过鉴定和评价芝麻香型白酒麸曲中的酵母菌，为提高芝麻香型白酒品质和产量的稳定性提供参考。
PLFAs谱图分析表明偶数碳不饱和脂肪酸和直链饱和脂肪酸是高温大曲和整个堆积过程的优势脂肪酸,而不同窖泥中的优势PLFAs存在差异。同时，细菌是高温大曲、整个堆积过程及窖泥中的优势微生物种群，G-是细菌中的优势菌群。高温大曲的细菌生物量和真菌生物量较接近，制曲温度较低时细菌/真菌生物量的比值变小。但是代表细菌特征性脂肪酸的种类较多，真菌的种类较少。在整个堆积过程，PLFAs的种类和数量呈现一定的变化规律，可以反映微生物群落结构的变化。堆积的时间和温度对生物量的含量影响显著。厌氧菌和好氧菌在不同的样品中差异较大。
通过PCR-DGGE图谱分析高温大曲、堆积过程及窖泥细菌群落结构，并对目的条带进行测序确定其优势细菌的种属信息。结果表明，芝麻香型白酒高温大曲主要由Thermoactinomyces, Staphylococcu, Lactobacillus, Solitalea, Pediococcus,Acinetobacter构成。其中，Staphylococcus xylosus和Thermoactinomyces是细菌微生物中的优势菌。同时，制曲温度对大曲微生物的多样性影响显著，温度越高，多样性越低。整个堆积过程的优势细菌为Thermoactinomyces和Bacillus，Lactobacillus和Acinetobacter是堆积后期的次优势菌。同时，堆积后期微生物的多样性明显高于前期。而窖泥微生物结构简单，新窖泥和老窖泥微生物的组成存在差异。
麸曲含有的5种酵母菌，经过分子鉴定分别属于Wickerhamomyces酵母属和酿酒酵母属。利用PCR-SSCP谱图分析酵母菌的类别表明，PCR扩增区域的保守性及片段大小对结果影响较大。通过生长曲线和发酵力的比较发现，5种酵母中的酿酒酵母生长迅速，发酵力较强。
The Sesame-flavor Liquor is a new flavor which has been only developed inrecently years. The flavor style prominent, unique flavor, and the basic of technologycharacteristics and flavor components have got an initial understanding, but themicrobial variation, the formation mechanism of characteristic flavor and furtherimprovement in technology need further exploration. So it is helpful to reveal theformation mechanism of the Sesame-flavor Liquor through analysis of microorganismin high temperature Daqu, stacking fermentation and Pit Mud. In this paper, wecomprehensively used two types of culture-independent methods as PLFA and DGGEtechnology to research the biomass and the microorganism’s community structuralfeatures of high temperature Daqu, stacking fermentation and Pit Mud. Throughidentification and evaluation5yeasts of bran koji of the Sesame-flavor Liquor,References would be provided to improve the quality and yield.
PLFA profiles showed Straight chain saturated fatty acids and even-carbonunsaturated fatty acids were the predominant PLFAs in high temperature Daqu andstacking fermentation, but they have differences in different Pit Mud. Besides, thepredominant microorganism was bacteria and G-was the predominant bacteria in hightemperature Daqu, stacking fermentation and Pit Mud. Bacterial and fungal biomassof different high temperature Daqu were almost the same, the ratio of bacterial/fungalbiomass was smaller when the producing temperature of high temperature Daqu islower. There were more types of bacteria PLFAs and fewer types of fungal PLFAs.The types and numbers of microorganisms PLFAs show certain variation rules duringstacking fermentation can reflect the changes in microbial community structure. Theimpacts of stack temperature and time on microbial biomass were obvious. Theanaerobic and aerobic bacteria are quite different in different samples.
Bacteria community structure of high temperature Daqu, stacking fermentation andPit Mud were investigated using PCR-DGGE. The sequencing ofDGGE-distinguished bands was proceeded to obtain the dominant bacterialpopulation information. The profile of DGGE showed that bacteria community structure of high temperature Daqu was composed of Thermoactinomyces,Staphylococcus, Lactobacillus, Solitalea, Pediococcus, Acinetobacter. And thedominant bacteria were Staphylococcus xylosus and Thermoactinomyces. The hightemperature Daqu microbial diversity decreased as the craftwork temperature of Daquincreasing. The dominant bacteria were Thermoactinomyces and Bacillus duringstacking fermentation, the second dominant bacteria were Lactobacillus andAcinetobacter in the late stage of stacking fermentation. The microbial diversity in thelate stage of stacking fermentation was higher than the early stage. The bacterialcommunity structure of Pit Mud was simple, and it has differences between new PitMud and old Pit Mud.
The5yeasts of bran koji belong to Wickerhamomyces and Saccharomycescerevisiae after molecular identification. PCR-SSCP fingerprinting analysis of thetype of yeasts showed the conserved and size of PCR amplified region has greaterimpact on results. Comparison of fermentation capacity and the growth curve of5yeasts indicated Saccharomyces cerevisiae growth rapidly and fermentation capacitywas higher.
PLFAs谱图分析表明偶数碳不饱和脂肪酸和直链饱和脂肪酸是高温大曲和整个堆积过程的优势脂肪酸,而不同窖泥中的优势PLFAs存在差异。同时，细菌是高温大曲、整个堆积过程及窖泥中的优势微生物种群，G-是细菌中的优势菌群。高温大曲的细菌生物量和真菌生物量较接近，制曲温度较低时细菌/真菌生物量的比值变小。但是代表细菌特征性脂肪酸的种类较多，真菌的种类较少。在整个堆积过程，PLFAs的种类和数量呈现一定的变化规律，可以反映微生物群落结构的变化。堆积的时间和温度对生物量的含量影响显著。厌氧菌和好氧菌在不同的样品中差异较大。
通过PCR-DGGE图谱分析高温大曲、堆积过程及窖泥细菌群落结构，并对目的条带进行测序确定其优势细菌的种属信息。结果表明，芝麻香型白酒高温大曲主要由Thermoactinomyces, Staphylococcu, Lactobacillus, Solitalea, Pediococcus,Acinetobacter构成。其中，Staphylococcus xylosus和Thermoactinomyces是细菌微生物中的优势菌。同时，制曲温度对大曲微生物的多样性影响显著，温度越高，多样性越低。整个堆积过程的优势细菌为Thermoactinomyces和Bacillus，Lactobacillus和Acinetobacter是堆积后期的次优势菌。同时，堆积后期微生物的多样性明显高于前期。而窖泥微生物结构简单，新窖泥和老窖泥微生物的组成存在差异。
麸曲含有的5种酵母菌，经过分子鉴定分别属于Wickerhamomyces酵母属和酿酒酵母属。利用PCR-SSCP谱图分析酵母菌的类别表明，PCR扩增区域的保守性及片段大小对结果影响较大。通过生长曲线和发酵力的比较发现，5种酵母中的酿酒酵母生长迅速，发酵力较强。
The Sesame-flavor Liquor is a new flavor which has been only developed inrecently years. The flavor style prominent, unique flavor, and the basic of technologycharacteristics and flavor components have got an initial understanding, but themicrobial variation, the formation mechanism of characteristic flavor and furtherimprovement in technology need further exploration. So it is helpful to reveal theformation mechanism of the Sesame-flavor Liquor through analysis of microorganismin high temperature Daqu, stacking fermentation and Pit Mud. In this paper, wecomprehensively used two types of culture-independent methods as PLFA and DGGEtechnology to research the biomass and the microorganism’s community structuralfeatures of high temperature Daqu, stacking fermentation and Pit Mud. Throughidentification and evaluation5yeasts of bran koji of the Sesame-flavor Liquor,References would be provided to improve the quality and yield.
PLFA profiles showed Straight chain saturated fatty acids and even-carbonunsaturated fatty acids were the predominant PLFAs in high temperature Daqu andstacking fermentation, but they have differences in different Pit Mud. Besides, thepredominant microorganism was bacteria and G-was the predominant bacteria in hightemperature Daqu, stacking fermentation and Pit Mud. Bacterial and fungal biomassof different high temperature Daqu were almost the same, the ratio of bacterial/fungalbiomass was smaller when the producing temperature of high temperature Daqu islower. There were more types of bacteria PLFAs and fewer types of fungal PLFAs.The types and numbers of microorganisms PLFAs show certain variation rules duringstacking fermentation can reflect the changes in microbial community structure. Theimpacts of stack temperature and time on microbial biomass were obvious. Theanaerobic and aerobic bacteria are quite different in different samples.
Bacteria community structure of high temperature Daqu, stacking fermentation andPit Mud were investigated using PCR-DGGE. The sequencing ofDGGE-distinguished bands was proceeded to obtain the dominant bacterialpopulation information. The profile of DGGE showed that bacteria community structure of high temperature Daqu was composed of Thermoactinomyces,Staphylococcus, Lactobacillus, Solitalea, Pediococcus, Acinetobacter. And thedominant bacteria were Staphylococcus xylosus and Thermoactinomyces. The hightemperature Daqu microbial diversity decreased as the craftwork temperature of Daquincreasing. The dominant bacteria were Thermoactinomyces and Bacillus duringstacking fermentation, the second dominant bacteria were Lactobacillus andAcinetobacter in the late stage of stacking fermentation. The microbial diversity in thelate stage of stacking fermentation was higher than the early stage. The bacterialcommunity structure of Pit Mud was simple, and it has differences between new PitMud and old Pit Mud.
The5yeasts of bran koji belong to Wickerhamomyces and Saccharomycescerevisiae after molecular identification. PCR-SSCP fingerprinting analysis of thetype of yeasts showed the conserved and size of PCR amplified region has greaterimpact on results. Comparison of fermentation capacity and the growth curve of5yeasts indicated Saccharomyces cerevisiae growth rapidly and fermentation capacitywas higher.
引文
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