【作者】 吴建峰；

【导师】 徐岩；

【作者基本信息】 江南大学 ， 发酵工程， 2013， 博士

【摘要】 四甲基吡嗪(tetramethylpyrazine，TTMP)又名川芎嗪(1igustiazine)，是我国首先从常用中药川芎中提取出的生物碱，它是川芎治疗心脑血管疾病的有效成分之一，临床上已广泛应用于治疗缺血性心脑血管疾病及慢性心衰、肾衰等并且取得了良好的疗效。白酒作为我国特有的酒种，早在上世纪80年代，在进行白酒中风味物质分析时就已经在白酒中发现了四甲基吡嗪，但是由于含量较低，没有引起重视。作者在2006年率先提出四甲基吡嗪是白酒中的主要功能性成分之一，在不同香型白酒中普遍存在，引起行业的关注。由于白酒中四甲基吡嗪的合成机理尚不清晰，在本论文开展研究工作之前尚没有有效的方法用于提高白酒中四甲基吡嗪的含量。而高含量四甲基吡嗪是成功塑造中国白酒是“健康白酒”的关键。芝麻香型白酒是建国后白酒两大创新香型之一，以其优雅舒适的香气，醇和细腻的风味深受消费者喜爱，现在已经成为中国高档白酒的代表之一，虽然芝麻香型白酒总吡嗪含量较高，但是四甲基吡嗪含量相对较低。针对白酒中四甲基吡嗪含量相对较低这一共性问题，本论文以江苏今世缘芝麻香型白酒酿造过程为研究模型，以提升酒体中四甲基吡嗪含量为主要导向，紧紧围绕四甲基吡嗪的合成机理来进行，为高四甲基吡嗪含量芝麻香型白酒的酿造提供实验基础、理论依据与新思路。主要研究内容如下：（1）构建了酒曲、酒醅与白酒酒体中包括四甲基吡嗪在内的含氮化合物的定性（GC×GC-TOF-MS）与定量（GC-NPD）测定技术与方法体系。针对本论文在开展研究时尚没有成熟的技术与方法体系用于酒曲、酒醅与白酒酒体中四甲基吡嗪分析这一问题，本论文首先基于GC×GC-TOF-MS对顶空固相微萃取（HS-SPME）和液-液萃取的效果进行了比对于分析，结果表明:前者萃取效果优于后者，然后通过运用顶空固相微萃取和GC×GC-TOF-MS联用对白酒中功能性成份实现全扫描。同时从萃取头类别、萃取时间、萃取温度和盐离子浓度角度对HS-SPME萃取条件进行优化，得到了从酒曲、酒醅与白酒酒体中提取吡嗪类物质提取的最佳条件：萃取头CAR/DVB/PDMS，萃取温度50°C，时间40min，NaCl浓度0.3g/ml；最后利用优化后的顶空固相微萃取和GC-NPD联用技术实现了白酒中9种吡嗪类物质的快速、准确检测方法。在所测范围内，吡嗪类化合物均有较好的线性关系(R2>0.999)，检测限均小于150ng/L，相对标准偏差在2.51%-10.28%之间，加标回收率在85.13%-108.05%之间，同时发现芝麻香型白酒中四甲基吡嗪含量相对较低。（2）确认了芝麻香型白酒酿造过程中，细菌曲制曲阶段所生成的四甲基吡嗪是酒中四甲基吡嗪的主要来源之一。系统的对酒曲、酒醅与白酒酒体中的吡嗪类物质进行分析，酒曲中吡嗪类物质变化规律为：多粮曲高于纯小麦曲；高温曲高于中温曲；细菌曲中吡嗪类物质含量最高，酵母曲和白曲中吡嗪化合物含量很低。芝麻香酒醅堆积前后吡嗪类物质含量进行比较分析，大部分吡嗪物质在堆积后含量增加，四甲基吡嗪含量是堆积外层含量高于堆积内层。比较细菌曲及堆积过程中四甲基吡嗪增量发现，细菌曲与堆积阶段所产生的四甲基吡嗪贡献率分别为：60%和40%。这表明：细菌曲是芝麻香型白酒中四甲基吡嗪的主要来源之一。（3）从细菌曲中获得一株具有高产四甲基吡嗪能力的菌株Bacillus subtilis S12。对细菌曲中4株菌分别进行纯培养，并测定四甲基吡嗪含量，发现菌株S12产四甲基吡嗪含量最高。根据菌落形态特征、细胞形态特征以及生理生化特性，参考《常见细菌系统鉴定手册》和《伯杰氏系统细菌学手册》，同时对S12的16S rDNA序列进行测序利用Clustal X、Bioedit和MEGA4.0选择Kimura2-parameter模型构建系统发育树，由系统发育树结果结合蛋白组MALDI-TOF分析，鉴定S12为枯草芽孢杆菌。（4）发现并证明:在固态发酵条件下是Bacillus subtilis S12所产生氨基酸脱氢酶实现从氨基酸到氨的转化，氨和3-羟基丁酮通过缩合作用合成四甲基吡嗪,因此氨基酸脱氢酶是合成四甲基吡嗪的关键酶。进而提出并验证了Bacillus subtilis S12固态发酵中四甲基吡嗪合成的新理论：基于酶-化学联合反应的四甲基吡嗪合成机制。通过试验证明：3-羟基丁酮和氨基酸不能合成四甲基吡嗪，只有3-羟基丁酮和氨通过缩合作用才能合成四甲基吡嗪。Bacillus subtilis S12在固态发酵的条件下，代谢产生3-羟基丁酮，分泌蛋白酶将蛋白质降解为氨基酸，然后由细菌曲中Bacillus subtilis S12产生的氨基酸脱氢酶（如谷氨酸脱氢酶和苯丙氨酸脱氢酶）将氨基酸脱氢得到氨。在白酒细菌曲制曲过程中，四甲基吡嗪的合成分为两个阶段：第一阶段是0到24小时，最适温度下（37oC），细菌代谢产生3-羟基丁酮和氨；第二阶段是在高温条件下3-羟基丁酮和氨进行缩合反应，合成四甲基吡嗪。其中温度对第二阶段四甲基吡嗪合成影响很大，高温利于反应的进行。（5）构建了Bacillus subtilis S12固态条件下两阶段酶-化学联合反应的四甲基吡嗪合成动力学模型。在0-24小时的第一阶段，四甲基吡嗪的形成为0级动力学反应，动力反应常数为0.002；在24-48小时，四甲基吡嗪的合成为一级动力学反应，分数转化技术可以成功用于描述四甲基吡嗪的合成与积累，频率因子常数A0的取值为1.4×105min-1，活化能Ea的数值为18kcal/mol，从37oC到62oC，预测值与实验值均具有极高的吻合性。利用模型预测的结果指导机械化制曲和酿酒生产，与传统方法对比：曲中四甲基吡嗪含量提高4倍，酒体中四甲基吡嗪提高7.5倍。更多还原

【Abstract】 Tetramethylpyrazine (TTMP) also Known as1igustiazine, which is an alkaloid extractedfrom Chinese herbal medicine Ligusticum chuanxiong Hort. It also is the key component partin Ligusticum chuanxiong Hort to cure cardiovascular and cerebrovascular diseases.Pharmacological studies proved that TTMP have the ability for dilating blood vessels,increasing coronary blood flow and inhabitation of platelet aggregation and so on. In addition,TTMP also is an important flavoring agent, the author previously found that TTMP existed inChinese liquors and presented a seminar related to TTMP as the functional component inChinese liquors at “The First Symposium on Chinese Liquor and Health” held in2006. Thefinding of the presence of TTMP in Chinese liquors made it similar to red wine as thefunctional nutraceutical wine for health improvement in addition to being an alcoholicbeverage. Sesame flavor liquor is one of two major innovative flavors discovered after1950.It has become top grade Chinese liquors in China as its excellent and comforting aroma.Although the content of pyrazines in sesame flavor liquor is higher compared to otherflavored Chinese liquors in which the content of TTMP is low. To improve the level of TTMPin sesame flavored liquor, this research thesis takes sesame flavored liquor brewing process asthe research model from the Jiangsu Jinshiyuan company, and mainly focused on (1)Development of the method and technology based on GC-NPD for determination the level ofpyrazines in Chinese liquors, Qu and fermentation grains;(2) Determined the main stage forTTMP formation of the enetire fermentation process from Qu preparation to distilled liquorwhich were analyzed by GC-MS technology;(3) Improvement of the yield of TTMPprecursors-acetoin for strain S12through screening and cultivation of the bacteria strainswhich exist in Qu and classified S12as Bacillus subtilis S12by comparing the16S rDNAsequence of S12, physiological and biochemical analysis as well as MALDI-MS analysis;(4)A two-stage synthetic mechanism " enzyme/thermal catalytic synthesis of TTMP " wasdeveloped through analysis of the levels of TTMP in the reaction mixture which containscarbon source (glucose or acetoin) and nitrogen (amino acid or ammonium ions) at varioustemperatures, as well as construction of an mathematical dynamics model to describe thesynthesis of TTMP, and application of the model in the industrial preparation of bacteria Qu.The main research accomplishments are shown as follows:(1) Established the technology and methodology for the determination the compoundscontaining nitrogen, including TTMP using GC-NPD and GC-MSMS in Qu，fermentationgrain and liquor. Since there are still no available technology and methodology for thedetermination of the nitrogen compounds in Qu，fermentation grain and liquor before. In thisthesis, the author developed the method of headspace solid-phase microextraction combined with GC-NPD technology. And determined the optimal condition for pyrazines extractionfrom Qu, fermentation grain and liquor by optimization of the condition of headspacesolid-phase microextraction (HS-SPME): Extraction was done with CAR/DVB/PDMS.extraction temperature was50°C for40min. The NaCl concentration was0.3g/ml; by usingheadspace solid-phase microextraction combined with GC-NPD technology,9pyrazines wereaccurately detected in Qu, fermentation grain and liquor. Pyrazine compounds had good linearrelationship (R2>0.999) in the measuring range, the detection limits were less than150ng/L.The relative standard deviation was between2.51%-10.28%. The recovery is between85.13%-108.05%. The author found that TTMP level is low in liquor.(2) Confirming tetramethylpyrazine generated in the preparation of bacteria koji stage isthe main source of TTMP in sesame flavor liquor. Systematic studies was conducted tocompare the pyrazines contents in various Qu used for sesame flavored liquor brewing，fermentation grain and liquor. The results shown that the levels of pyrazines in the grain quwas higher than pure wheat starter, and high temperature Daqu was higher than that ofmedium temperature. However, the pyrazines level in the bacteria starter culture was thehighest, and the level of pyrazines in yeast Qu and Bai Qu was low. The accumulation ofpyrazines level in fermented grains before and after “duiji” was analyzed. When the level ofmost pyrazines in fermented grains were increased, the accumulation of pyrazines in outerpacking were higher than that of inner layer. In addition, Comparing with the increasing ofpyrazines level in bacteria culture stages during which the increase of pyrazines in thefermentation grain was low.(3) Screening for high yield of TTMP strain S12and identification of it as Bacillussubtilis S12. Four strains were screened from the bacteria Qu, The highest yield of TTMP wasproduced by S12and its TTMP yield was67.4mg/kg. According to the colonial morphology,cell morphological characteristics and physiological and biochemical characteristics, also withreference to "common bacterial system identification manual" and "Bergy’s Manual ofSystematic Bacteriology", as well as the genetic tree was constructed based on the16S rDNAsequence of S12using Clustal X, Bioedit and MEGA4.0, all the results combined withproteomic analysis of S12using MALDI-TOF, S12identified the culture as Bacillus subtilisS12.(4) A new theory is proposed and demonstrated to be related to TTMP synthesis inChinese liquor by solid-stage fermentation with enzyme/thermal dynamic coupling catalysismechanism for tetramethylpyrazine synthesis. Bacillus subtilis S12producing amino aciddehydrogenase which changed the amino acid to ammonium; acetoin and amino acid which could not synthesize TTMP. TTMP production can proceed with3-hydroxy-2-butanone andammonium ion. Under the synthetic process with the bacterial Qu culture, synthesis of TTMPcan be divided into two stages: the first stage is0-24hours,3-hydroxy-2-butanone is mainlysynthesized in this stage; the second stage is the thermal power stage, under high temperature,acetoin and ammonium ion undertake chemical reaction and produce TTMP. Hightemperature enhanced TTMP production.(5) Proposed kinetics model for TTMP synthesis in the preparation process of bacteriaQu with Bacillus subtilis S12was formulated. In the first stage of0-24hours, the chemicalreaction of TTMP formation is0grade, the dynamic reaction constant is0.002; In the secondstage of24-48hours, the chemical reaction of TTMP formation belong to first-order kinetics,fraction conversion technology was successfully used to describe TTMP synthesis andaccumulation, A0was0.14, and activation energy Ea is18kcal/mol, the data used to developthe model in this work were proven to be accurately fitting the experimental data. Thebacteria Qu preparation of mechanized production was guided by the model. Comparing withtraditional methods, the TTMP levels in Qu increased4-fold and7.5-fold in the originalsesame flavored liquor.更多还原