【作者】 高亦豹；

【导师】 徐岩；

【作者基本信息】 江南大学 ， 发酵工程， 2010， 硕士

【摘要】 酒曲酿造是我国优势传统发酵食品白酒酿造的技术特征和关键技术。大曲以小麦等谷物为原料固态生料发酵而成,大曲作为白酒生产中微生物的主要来源,其质量直接关系到酒的出酒率及酒质。长期以来,由于研究技术手段的限制,对白酒大曲微生物区系的研究和认识还非常有限。为克服培养方法的局限性,采用建立在分子生物学技术基础上的未培养技术,从未培养角度探究大曲微生物群落结构组成对于深入白酒大曲微生物群落特征的认识,丰富我国传统酿造食品微生物学的机理的研究,具有实践和理论意义和价值。本文运用PCR-DGGE方法对国内具有代表性的不同工艺白酒大曲的微生物群落结构进行了研究。分别基于细菌的16S rRNA V3基因、酵母的26S rRNA D1基因和真菌的18S rRNA基因对大曲中的细菌、酵母和真菌的群落结构进行了分析。通过DGGE优势条带的切胶回收及测序鉴定获取了大曲微生物种群结构信息。研究结果表明:1)与传统分离培养方法研究相比,本研究首次基于细菌的16S rRNA V3基因DGGE揭示了白酒大曲中乳酸菌的种群多样性,多种乳酸菌存在于不同工艺大曲中。所鉴定出的乳酸菌包括Weissella cibaria、Lactobacillus panis、L. helveticus、L. fermentum、L. pontis。细菌是中国大曲最具特点的微生物。同时DGGE检测到了传统培养方法未检测到的嗜麦芽窄食单胞菌、假单胞菌、葡萄球菌及高温放线菌;2)在真菌的分析研究中,针对传统培养方法和已报道未培养方法研究多集中在细菌群落结构的状况,分布基于酵母的26S rRNA D1基因和真菌的18S rRNA基因对酵母和真菌群落结构进行了研究。酵母26S rRNA D1区基因的DGGE研究显示Saccharomycopsis fibuligera、Pichia anomala普遍存在于所有大曲中,且在整个酵母群落中处于优势地位,弥补了传统培养方法在反应优势菌群分布方面的缺陷。传统培养方法研究认为大曲酵母群落认为大曲中酵母主要为酿酒酵母、汉逊酵母、毕氏酵母、拟内孢霉四大类酵母,本研究使用DGGE方法均检测到了此四大类酵母,且还检测到了Trichosporon asahii、Debaryomyces hansenii、Hanseniaspora guilliermondii等酿酒工业中认为重要的非酿酒酵母。犁头霉及米根霉在真菌18S rRNA DGGE图谱中显示出明显的优势地位。除犁头霉、米曲霉及烟曲霉以外,利用18S rRNA引物还检测到了Candida tropicalis、Candida allociferrii、Saccharomyces bulderi等酵母。真菌18S rRNA DGGE图谱Shannon-Wiener多样性分析结果显示单粮曲真菌多样性均低于杂粮曲;3)地衣芽孢杆菌和枯草芽孢杆菌均为大曲中优势芽孢杆菌。PCR扩增的偏好性会引起细菌16S rRNA V3区引物很难将芽孢杆菌进行DGGE分离。通过利用巢式PCR获取芽孢杆菌专一性基因,将芽孢杆菌的16S rRNA V9区基因作为DGGE靶基因成功构建了大曲芽孢杆菌群落结构分析手段,该方法可较好的反应不同工艺大曲中芽孢杆菌群落信息。4)为进一步研究工艺因素对大曲细菌群落组成影响,分析了同一产地5种高温和中温白酒大曲细菌群落结构差异,通过优势条带切胶鉴定,确定了大曲中优势细菌种属信息。结果表明Weissella cibaria、L. helveticus、L. fermentum、L. panis等乳酸菌普遍存在于5种大曲中,Thermoactinomyces sanguinis仅存在于高温曲酱曲中。不同工艺大曲细菌群落结构存在明显差异,随着制曲温度的升高,大曲细菌多样性指数有下降趋势。综上,本研究从未培养角度较全面和系统地分析了不同工艺大曲微生物群落结构组成,获取了不同工艺大曲共有的优势种群信息,并研究了工艺因素对微生物种群结构的影响。运用PCR-DGGE基因指纹图谱技术分析白酒大曲的微生物群落结构,将其与传统的菌种分离鉴定、白酒风味组成分析等手段相结合,对判断和鉴定白酒生产中与特征风味物质相关的关键微生物、指导生产工艺改进,从而将传统的经验酿造提升到科学的水平具有重要的理论和实践意义。更多还原

【Abstract】 Solid state fermentation based on starters (Daqu in Chinese) is a major characteristic of Chinese liquor which is well known Chinese traditional fermented food. Daqu is prepared by solid state fermentation with uncooked grain such as wheat and barley. As the main provider of microorganism, the quality of Daqu directly affects the yield and quality of liquor. For a long period, due to the disperse research condition and backward technologies, researchers know very limited about the microbial communities in Daqu.To overcome the limitation of culture-dependent method and get the Daqu microbial community from a culture-independent method, the microbial diversity of Chinese liquor Daqu was investigated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The investigation of Daqu microbial community based on culture independent method would have theory and practice sense in getting better understanding of Daqu microbial community structure and the mechanism of traditional brewing microbe.10 typical Daqu samples collected from different regions were analyzed for bacteria, yeast and fungi by PCR-DGGE as a culture independent method. Extracted DNA was used as a template for PCR to amplify 16S rRNA gene V3 region, 26S rRNA gene D1 region and 18S rRNA gene partial region using universal primers. The Daqu microbial community information was confirmed by sequencing of dominant bands cutting from DGGE gel.The result indicated that1)For bacteria, compared with culture-dependent method, the bacterial DGGE profile displayed high diversity. Lactic acid bacterias were one of the dominant bacterial groups in different Daqu samples. The LAB included Weissella cibaria, L. helveticus, L. fermentum, L. panis and L. pontis. Thermoactinomyces sanguinis, Pseudomonas spp., Staphylococcus xylosus, and Stenotrophomonas maltophilia were also detected in bacterial DGGE while they were not detected in.2)As for fungi, researches based on traditional culture dependent method and culture independent method published in recent years mainly focused on bacteria, so 26S rRNA gene D1 region and 18S rRNA gene partial region DGGE was proceeded in Daqu samples. The main yeast detected by traditional culture-dependent method were Saccharomyces cerevisiae, Hansenula spp., Pichia spp. and Endomycopsis spp.. Besides these yeast, the DGGE method also detected yeast such as Hanseniaspora guilliermondii, Debaryomyces hansenii, Trichosporon asahii and Issatchenkia orientalis. And the non-Saccharomyces yeasts revealed its high diversity in this study. The predominance of Saccharomycopsis fibuligera and Pichia anomala were proved in all the samples. The fungal 18S rRNA DGGE proved the dominance of Absidia spp. and Aspergillus spp. in Daqu. Saccharomycopsis fibuligera, Saccharomyces bulderi, Candida allociferrii and Candida tropicalis were identified as yeast in the fungal analysis. DGGE profiles of fungal 18S rRNA gene showed fewer bands than bacterial and yeast DGGE profiles. Daqu manufactured with only wheat showed lower Shannon-Wiener diversity than those manufactured with more than one variety of grains. 3)Bacillus licheniformis and Bacillus subtilis were proved to be dominant in Daqu Bacillus species. Due to the limitation of PCR amplification by bacterial universal primer, 16S rRNA V3 DGGE failed to discriminate Bacillus species in the Daqu samples. A nested-PCR based DGGE method was established. The gene which was special for Bacillus species was obtained by first round PCR and the Bacillus 16S rRNA V9 gene was obtained by the second round PCR. The 16S rRNA V9 gene DGGE was proved to be effective in profiling Bacillus community in Daqu.4)To get a better understanding of correlation between craftwork and bacterial community structure in Daqu. The bacterial community structure of 5 high and medium temperature Chinese liquor Daqu from the same region were investigated using PCR-DGGE. DNA sequencing was proceeded to obtain the dominant bacterial population information. The result of DGGE profiles showed that Weissella cibaria, L. helveticus, L. fermentum and L. panis were commonly detected in all the five Daqu. Thermoactinomyces sanguinis was detected only in the Jiangqu sample. The correlation between the craftwork of Daqu and the bacterial community structure was obvious. The Daqu bacterial Shannon-Wiener index decreased as the craftwork temperature of Daqu increasing. PCR-DGGE was proved to be a powerful tool for gaining detailed insight into the bacterial diversity of the Chinese liquor Daqu.Microbial community of different Chinese liquor Daqu was investigated using culture-dependent method in this study and information of dominant species in Daqu was obtained and the affection of craftwork on microbial community structure was studied. The combined analysis of microorganism community structure in Daqu obtained by DNA fingerprint methods with interrelated results using other experiment means, such as traditional microbial identification based on culture and separation, detection of liquor solid-form fermentation and analysis of aroma components in liquor, will help to isolate and identify important microbes corresponding to the formation of characteristic key flavor components, and direct improvement of production techniques.更多还原