Fermentation Control And Mechanism Of Two-step Tetramethylpyrazine Production By Bacillus Subtilis

2,3,5,6-tetramethylpyrazine (TTMP) associated with other alkylpyrazines are a group of heterocyclic nitrogen-containing compounds widely existed in raw and processed food and alcoholic beverages; they are generally considered as important aroma compounds and give tonalities of nutty, roasty, and toast. Besides its flavoring additives properties, TTMP as the main bioactive ingredient of alkaloid isolated from the rhizome of Ligusticum wallichii, was also proved to have pharmacological activity on cardial-cerebrovascular disease, and protective effects on cisplatin-induced oxidative stress, apoptosis and nephrotoxicity.Since TTMP was firstly isolated from the culture of Bacillus natto in 1962, the research on microbial TTMP production started, however, only a few applications of TTMP production were reported, and diverse viewpoints focused on the mechanism of TTMP formation in microbial environments. Although several microbial processes have been described, the numbers of industrial applications are limited till now. Reasons for this are in most cases low final product yield, low biotransformation rates, precursor acetoin inhibition, toxicity of acetoin and TTMP towards the microorganisms and etc. Therefore, screening of potential microbes with enhanced metabolic flux to precursor acetoin excretion endogenously from glucose or other carbon sources, exploring the mechanism of TTMP formation in microbial system, and constructing optimal processes were essential for high production of TTMP.In this study, an endogeneous precursor screening strategy was established with the purpose of screening TTMP-producing strains, and a B. subtilis XZ1124 was isolated from a high temperature Daqu. Based on the kinetic analysis of fermentation under different cultivation conditions, several control strategies were according proposed and verified. Based on the verification of enzyme-catalyzed reactions in TTMP formation in microbial system, combined process of microbial precursor acetoin fermentation and chemical synthesis of TTMP was propsed and demonstrated an effective method for high TTMP production. Meanwhile, waste cells of B. subtilis were reutilized as biocatalysts to catalyze glucose into precursor acetoin, and a combined process of multi-conversion of glucose to acetoin and chemical synthesis of TTMP was established. The main contents of this dissertation are as follows:(1) Based on the relationship of molecular structures of acetoin and TTMP, an endogenous precursor strategy was established for screening of flavor compound producers, and a strain XZ1124 was obtained from the Chinese Maotai-flavor Daqu by using the screening strategy above. The strain was characterized as Bacillus subtilis according to its morphological, physiological and biochemical properties as well as partial 16S rRNA gene sequences. Preliminary optimization was carried out and 4.08 g/l TTMP was obtained under the optimized medium composition (100 g/l sucrose, 40 g/l soy meal, 5 g/l yeast extract, 30 g/l diammonia phosphate, initial pH 7.5) and cultivation conditions (50/250 ml, 200 rpm 37°C). Precursor acetoin reached peak value (22.3 g/l) at 66 h of cultivation, and the conversion rate was 23.7%, which is higher than the value (1%) reported in the literature.(2) Based on the kinetic analysis of TTMP fermentation under different pH-controlled process, a two-stage pH-shifted strategy was developed as follows: pH was controlled at 5.5 during the first 48 h of cultivation to allow rapid cell proliferation and acetoin accululation, and then switched to 7.0 to enhance TTMP synthesis from acetoin. By applying the strategy, a final concentration of 7.43 g/l TTMP was obtained, increased by 22.2% compared with that of fermentation with constant pH controlled at 7.0. Based on the kinetic analysis of acetoin and TTMP fermentation under different rotation speed, combined with the chemical nature of TTMP synthesis from acetoin and ammonia, a multiple-stage rotation speed and cultivation temperature strategy was developed as follows: temperature was controlled at 37 oC with rotation speed at 700 rpm during the first 12 h of cultivation, and then rotation speed switched to 500 rpm before 48 h of cultivation, and then temperature was switched to 55 oC till cultivation ended. By applying the strategy, a high accumulation of precursor acetoin was obtained, and TTMP production reached 7.12 g/l, increased by 38.5% and 25.1% compared with that of fermentation under constant cultivation temperature (37 oC) and constant rotation speed (500 r/min), respectively. Based on the kinetic analysis of TTMP fermentation under different initial glucose concentrations, a glucose-feeding strategy was developed as follows: initial glucose concentration was 80 g/l, and then 50 g/l glucose was added into the broth at 36 h of cultivation. A large amount of precursor HB (40.9 g/l) was accumulated in the first stage of fermentation, and TTMP synthesis (6.54 g/l) was thus effectively stimulated in the conditions of higher temperature and precursor concentration in later stage of fermentation, increased by 96.6% and 28.2%, respectively, compared with that of control fermentation.(3) The effects of ammonium salts on tetramethylpyrazine (TTMP) production were tested, and diammonium phosphate (DAP) was found to have a predominant effect on stimulating TTMP synthesis. Higher concentrations of DAP favored TTMP production, while both the ammonium and phosphate ions exhibited inhibitory effects on the cell growth and precursor 3-hydroxy-2-butanone (HB) accumulation. Based on the results above, a DAP feeding strategy was developed and verified in further experiments. By applying the strategy, the maximum TTMP concentrations reached 9.10 and 7.34 g/l in flask and fermentor, increased by 55.1% and 29.0% compared to that of the batch TTMP fermentation, respectively.(4) The mechanism of TTMP formation in microbial fermentative system was examined by verification of enzyme-catalyzed reactions in acetoin/diammonia phosphate model systems and in situ fermentative reaction systems. The results demonstrated that no intracellular or extracellular enzyme(s) participated in the process of TTMP synthesis. However, low molecular compounds, which could stimulate TTMP formation from acetoin and ammonia, were existed in microbial environment.(5) Based on the requirement of proton donor and acceptor to facilitate proton transfer during the Schiff base formation between ammonia and acetoin, the effects of microbial metabolites on TTMP synthesis were investigated, and a series of organic acid such as acetate and citrate, were shown to be beneficial for TTMP formation. The effects of acetoin and ammonia concentrations in microbial environment, as well as different reaction conditions on TTMP synthesis were further investigated, and a high yield of TTMP (45.1%) was obtained after 8 h of reaction under optimized microbial fermentative system. Based on the biological availability of acetoin accumulation by microbial metabolic activity, and the chemical nature of TTMP synthesis from acetoin and ammonia, a combined process of microbial acetoin fermentation under weak acid conditions (pH 5.5) and chemical reaction in optimized conditions was proposed. A final concentration of 16.8 g/l TTMP was obtained, which demonstrated the effectiveness of the process in surporting high TTMP production.(6) Waste cells were reutilized as biocatalyst to utilize glucose by multi-conversion into precursor acetoin. Preliminary optimization was carried out and 41.4 g/l acetoin was obtained under the optimized reaction mixture (140 g/l glucose, 100 g/l wet cell, 0.5 M PBS buffer initial pH 7.0) and reaction conditions (20/50 ml, 150 rpm, 35°C, gauze sealed, 96 h), which is equivalent to that of microbial acetoin fermention under optimized conditions, however, reaction time was reduced by 20% compared with that of microbial acetoin fermentation. A combined process of multi-conversion of glucose to acetoin and chemical synthesis of TTMP from acetoin was proposed and demonstrated, and a final concentration of 16.5 g/l TTMP was obtained, which demonstrated the effectiveness of the process in surporting high TTMP production.