【作者】 杜海；

【导师】 徐岩；

【作者基本信息】 江南大学 ， 发酵工程， 2013， 博士

【摘要】 我们前期的研究已发现，萜类化合物土味素（geosmin）产生的土霉异味是影响中国清香型白酒风味品质的主要原因。异味物质的存在，很大程度上表征着发酵不正常。其中包括正常酿造微生态的平衡的破坏，酿造功能的下降。最终体现为酒体品质的下降，酿造原料的浪费。因而，本研究中针对导致白酒中土霉异味的异味物质的产生机制，以及在异常发酵状态下对酿造微生态及功能的影响机制进行深入、系统的研究，不仅对于提高白酒的品质和食品安全具有重要应用价值，而且对于认识中国传统酿造食品的发酵机制和丰富微生物学理论也具有重要意义。本研究运用风味导向学术思想，首次明确了白酒中的土霉味物质的来源为微生物代谢合成。并采用现代分子生物学手段建立基于土味素关键合成酶编码基因实时荧光定量PCR方法对产土味素的链霉菌进行快速检测和跟踪。发现并证实产土味素链霉菌的重要代谢物对固态发酵微生物群落结构的调节规律和机制，建立了揭示中国白酒复杂体系中异味物质微生物产生机理的研究方法。主要创新性结论如下：1.运用风味导向技术，从复杂的白酒酿造菌群结构中成功筛选得到五株产生明显土霉气味的土味素代谢菌株。根据其菌落、细胞形态和生理生化特征，以及放线菌/链霉菌特异分类鉴定引物鉴定，确定这五株菌株均为链霉菌（Streptomyces）。选择基于放线菌16S核糖RNA特异引物——Com2xf/Ac1186r，采用变性梯度凝胶电泳（DGGE）技术分析白酒生产过程中可能产生土味素的放线菌的群落结构。发现白酒大曲中的放线菌结构较为简单。清茬大曲中放线菌菌群最为丰富，并随着大曲培养时间的延长菌群的的多态性更为明显。土味素产生菌的菌群结构会受到工艺，地理环境和气候等因素的影响。2.系统研究白酒异常酿造微生态中各菌种间生长规律，发现产土味素链霉菌与白酒酿造中的功能微生物有明显的竞争关系。同一酿造环境中，链霉菌的生长代谢对酿造功能微生物有不同程度的抑制作用。其中，菌株QC-2对贡献主要白酒风味的真菌类微生物，如酿酒酵母（Saccharomyces cerevisiae）、异常毕赤酵母（Pichia anomala）、东方伊萨酵母（Issatchenkia orientalis）和异常汉逊酵母（Hansenula anomala），具有明显抑制作用。同时，影响由功能酵母代谢生成的醇类、酯类等白酒重要风味物质的形成。进一步研究发现酵母，霉菌等功能微生物的繁殖周期较短，白酒酿造系统中大量的功能微生物生长对产土味素链霉菌也有一定的抑制作用，揭示了群体微生物固态发酵体中土味素产生菌与功能微生物之间的相互影响的机制。3.围绕产土味素链霉菌抑制群体微生物的分子机制，通过超高效液相色谱（UPLC）-二极管阵列检测器（PDA）检测，证实白酒酿造中具有明显抑制酿造真菌特性的链霉菌代谢产物中存在9个可能具有抗真菌活性的组分，且均在360nm、380nm、400nm三波段有明显吸收的特征紫外吸收谱。经过进一步超高压液相色谱-四级杆飞行时间串联质谱（UPLC-Q-TOF-MS/MS）检测，分子模拟，添加标准品等方式，确定抗真菌活性物质为一系列七烯大环内酯类同系物的抗生素。从而明确了产土霉异味链霉菌对白酒酿造群落结构的影响机制。4.针对群体微生物固态酿造的复杂性和菌体鉴别定量的难题，采用分子生物学手段钓取土味素合成酶的编码基因（geoA），证实所筛选到的链霉菌中存在与合成土霉味萜烯类化合物等萜类物质相关的土味素合成酶。通过考察不同微生物中的土味素合成酶编码基因的同源性，建立了基于土味素合成酶编码基因保守序列的实时荧光定量PCR方法。该方法对白酒酿造过程中常见不产土味素微生物无非特异性扩增。与应用放线菌特异引物荧光定量PCR结果相比较，发现白酒大曲中约1~10%的放线菌含有geoA基因。通过对合成土味素编码基因的核酸量进行定量分析，可实现在白酒酿造复杂基质中与土霉异味直接相关的灵敏、快速的生物定量分析，为可疑风味污染源预测、传播环境调查、产生菌溯源等工作奠定基础。5.针对白酒群体微生物固态酿造体系的特征，系统研究了酿造环境因素（温度、水分、pH、乙醇含量等）对产土味素链霉菌生长代谢的影响规律。研究发现：产土味素链霉菌最适生长温度为30℃，最适生长的基质含水量为50%。菌体在中性偏碱性（pH=7~8）的环境中可以大量繁殖。并会产生弱碱性的吡嗪类物质调节周围生长环境。在酸性（pH=3~5）环境中，酒精度高于10%vol的酿造环境中，菌体生长受到完全抑制。上述研究发现为白酒酿造过程中通过控制关键环境因素达到控制或抑制该化合物的合成提供可靠的理论依据，从而实现从根源上降低或消除白酒中土霉气味的目的。更多还原

【Abstract】 Our preliminary studies have illustrated that geosmin, a degraded sesquiterpeneterpenoids, was the main cause of the frequent and serious flavor deterioration of Chineselight-aroma type liquor. The presence of off-odor substance indicates that the fermentation isnot normal, including the imbalance of the normal brewing microflora and the functiondecline of brewing system. Finally, it is embodied in the decline of liquor quality and the costwaste. Thus, in-depth studies of the generation mechanism of off-odor substances in Chineseliquor, and the brewing microecological and functional changes corresponding to theabnormal fermentation are very necessary.In this study, with the flavor-oriented academic idea, the source of geosmin in Chineseliquor was characterized as the synthesis of microbial metabolism for the first time. And areal-time quantitative PCR method, based on the gene encoding a dual-function geosminsynthase, was developed to rapidly and sensitively detect geosmin-producing Streptomyces.The regulation mechanisms of important metabolites of geosmin-producing Streptomyces forthe microbial community during solid-state fermentation were discovered. The researchmethods were established to reveal microbial mechanism of the off-odor substances formingin the complex system of Chinese liquor. The major innovative conclusions are as follows:1. Through the flavor-oriented screening technology, five strains of Streptomyces wereisolated from Daqu and identified as geosmin producer. The ecology of these Streptomycesspecies were analyzed using denaturing gradient gel electrophoresis (DGGE) of amplifiedActinobacteria-specified ribosomal DNA with Com2xf/Ac1186r. The results showed thatStreptomyces were widely distributed during Daqu incubation, and multiple processing,geographic and climate factors could affect their distribution and diversity.2. There were significant interactions of diverse Streptomyces spp. acting as geosminproducer with the functional microbials in Chinese liquor brewing system. Somegeosmin-producing Streptomyces could secrete anti-fungal compounds against the functionalyeasts and moulds. Therefore, the presence of Streptomyces caused the imbalance of microbialcommunity of brewing system. Accordingly, the contribution of yeasts to liquor flavor weredecreased, such as alcohols, esters. The study has also shown that some dominant yeast andfilamentous fungi adapted well to Daqu associated niches producing flavor and enzyme hadthe capacity to competitively exclude geosmin producers.3. Nine components were detected in strain QC-2broth by UPLC-PDA detector, withcharacteristic ultraviolet absorption spectrum at360nm,380nm,400nm. The maincompontents produced by strain QC-2were identified and confirmed byUPLC-Q-TOF-MS/MS, standard spiking, molecular simulation and mass spectrometry. Theantifungal active compontents were a series of heptaene macrolide antibiotics.4. The genes associated with geosmin production were characterized in fourgeosmin-producing Streptomyces strains, all of which were isolated fromgeosmin-contaminated Daqu. Based on the information, a real-time PCR method wasdeveloped, enabling the detection of traces of Streptomyces in complex solid-state matrices. The primer targeted the gene coding for geosmin synthase (geoA). The real-time PCR methodwas found to be specific for geosmin-producing Streptomyces and did not show anycross-reactivity with geosmin-negative isolates, which were frequently present in Chineseliquor brewing process. Quantification of geoA in Chinese liquor making process couldpermit the monitoring of the level of geosmin producers prior to the occurrence of geosminproduction. Comparison of the qPCR results based on gene encoding geosmin synthase andActinobacteria-specified ribosomal DNA showed that about1-10%of the Actinobacteriacarry the geosmin synthesis gene.5. Through the investigation of water content, temperature, pH, ethanol content, and otherfactors, it was clear about the relation of geosmin production and brewing factors in thespecial liquor brewing system. Our studies have revealed that geosmin-producingStreptomyces could be widely distributed during Daqu incubation, and that multipleprocessing and climate factors could affect their distribution and diversity. More importantly,the growths of geosmin-producing Streptomyces were inhibited at pH values below5andalcohol contents over10%vol, both in liquid state and in solid state. The above discoveriesprovide reliable theory and path to reduce or eliminate the earthy off-flaovr in Chinese liquorby controlling the key environmental factors.更多还原