Study On The Biosynthesis Mechanism Of Tetra-methylpyrazine During The Chinese Liquor Brewing

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.