枯草杆菌两步法生产四甲基吡嗪的调控及机制的研究
|
摘要
四甲基吡嗪(tetramethylpyrazine,TTMP),又称川芎嗪,是吡嗪环碳原子上均连接甲基的含氮杂环化合物。TTMP作为天然香料,具有烘烤、花生、榛子和可可香气,可用于烘烤食品、冷饮、肉类、乳制品、饮料酒和卷烟等香精的调配;同时,TTMP是中药材川芎(Ligusticum wallichii)根茎的主要活性生物碱成分,具有扩张血管、改善微循环及抑制血小板集聚等作用,广泛应用于临床。
微生物发酵法生产TTMP的研究起步较晚,相关文献报道较少,且发酵体系中前体乙偶姻与氨反应生成TTMP的机制仍存在争议。目前,鲜有大规模TTMP发酵生产的工业化应用实例,其原因主要为TTMP产量较低、前体乙偶姻的有效转化率不高(<1%)、外源添加的乙偶姻存在产物抑制现象且乙偶姻和TTMP均对细胞具有毒性等。因此,开发具有优良特性的TTMP生产菌株、研究微生物利用葡萄糖代谢产生乙偶姻以及乙偶姻和氨反应生成TTMP的机制、并基于不同产生机制建立合适的TTMP生产工艺是提高微生物发酵生产TTMP的有效手段,为工业化应用奠定基础。
本文以获得高产TTMP菌株为目标,首先建立了一种内源前体筛选策略,从酱香型高温大曲中获得一株高产TTMP的菌株Bacillus subtilis XZ1124;根据其在不同培养条件下的发酵动力学分析,建立相应的发酵策略;对发酵体系中TTMP的生成机制进行探索,并基于实验结果建立有效的微生物发酵偶联TTMP合成工艺;同时,利用发酵结束废弃的菌体作用生物催化剂,建立枯草杆菌多步转化葡萄糖生成乙偶姻的工艺及其偶联化学合成生产TTMP的工艺,具体内容和结果如下:
(1)基于前体乙偶姻和TTMP分子结构的相关性,建立了一种适合于风味化合物TTMP筛选的内源前体筛选策略;并应用该策略,从酱香型高温大曲中获得一株TTMP产生菌XZ1124,该菌株能利用葡萄糖代谢产生大量前体乙偶姻,从而有效促进了TTMP的合成;根据菌落、细胞形态特征和生理生化特性以及16S rDNA序列分析,将该菌株鉴定为枯草杆菌(Bacillus subtilis);通过单因素优化,确定了最优的培养基组成和培养条件:蔗糖100 g/l、豆饼粉40 g/l、酵母膏5 g/l、磷酸氢二铵30 g/l、初始pH 7.5、装液量50/250 ml、摇床转速200 rpm、培养温度37°C;在此条件下,B. subtilis XZ1124摇瓶培养120 h,TTMP产量达4.08 g/l,前体乙偶姻积累量在培养66 h时达到峰值(22.3 g/l),前体的有效利用率达23.7%,远高于文献报道值(1%)。
(2)根据不同pH控制下发酵罐培养的动力学分析,建立了适合于枯草杆菌TTMP发酵的两阶段pH控制策略,通过控制培养前期的发酵液为弱酸性,保证细胞的快速增殖和前体乙偶姻的大量积累,培养后期调节发酵液为中性,促进枯草杆菌转化乙偶姻生成TTMP(7.43 g/l);根据枯草杆菌在7.5 L发酵罐培养时不同搅拌转速下的动力学分析,结合发酵体系中前体乙偶姻和氨的化学反应特性,建立了多阶段搅拌转速偶联温度控制的发酵策略:0~12 h,控制培养温度37oC、搅拌转速为700 rpm,12~48 h,控制培养温度37oC、搅拌转速为500 rpm,48~120 h,控制培养温度55oC、搅拌转速为500 rpm,该发酵策略可有效促进前体乙偶姻的大量积累,发酵结束时TTMP的产量达7.12 g/l,比恒温(37oC)和恒定搅拌转速(500 rpm)培养时分别提高了38.5%和25.1%;根据不同初始葡萄糖浓度下的发酵动力学分析,建立了葡萄糖补加策略:初始葡萄糖浓度为80 g/l、培养36 h补加50 g/l葡萄糖,该补加策略有效提高了前体乙偶姻的积累量,发酵结束时前体乙偶姻的积累量达40.9 g/l,与对照发酵相比提高了96.6%;TTMP浓度达6.54 g/l,比对照发酵提高了28.2%。
(3)考察了不同铵盐对枯草杆菌发酵产TTMP的影响,结果表明磷酸氢二铵(DAP)对TTMP合成的促进作用最为显著,且NH4+和PO43-同时存在时该促进作用尤为突出;提高培养基中初始DAP的浓度(或单独提高NH4+和PO43-浓度)可有效促进发酵体系中TTMP的生成,然而,高浓度的NH4+和PO43-离子不利于细胞的增殖、糖的消耗以及前体乙偶姻的积累;基于细胞增殖、前体乙偶姻积累和产物TTMP合成过程对DAP需求的差异,提出DAP补加策略,并应用于枯草杆菌摇瓶发酵和发酵罐培养过程,TTMP的最大生成速率分别提高了46.8%和76.8%,发酵结束时TTMP产量分别为9.10和7.34 g/l。
(4)建立了酶促反应模型体系,该模型体系为含有一定浓度乙偶姻和30 g/l磷酸氢二铵的溶液(pH 7.0),通过向该模型体系中加入胞内粗酶液和胞外粗酶液检测TTMP的变化;并进一步应用原位反应体系验证TTMP生成过程中的酶促催化反应,将枯草杆菌全细胞作为催化剂,考察了胞内酶和胞外酶对体系中乙偶姻和氨反应生成TTMP的影响;结果表明,发酵体系中TTMP的合成过程不存在酶促催化反应;但枯草杆菌培养液中可能存在一种或多种小分子代谢物,该代谢物能够促进乙偶姻和氨化学反应合成TTMP。
(5)考察了不同质子供体对乙偶姻和氨反应生成TTMP的影响,磷酸盐在pH 7.0时主要以磷酸二氢根和磷酸一氢根共轭对形式存在,能够提供足量的质子参与乙偶姻和氨反应生成Schiff碱的过程,促进了TTMP的合成;通过考察糖代谢途径中有机酸对枯草杆菌发酵体系中TTMP生成的影响,发现乳酸、丙酮酸、草酸、琥珀酸和柠檬酸均可促进发酵体系中TTMP的生成;进一步考察了乙偶姻和DAP浓度、pH、温度和搅拌条件以及供氧条件对枯草杆菌原位发酵体系中TTMP和YTTMP/HB的影响,确定了最优的TTMP合成条件,将100 g/l乙偶姻和200 g/l DAP的原位发酵体系于90oC、200 rpm的水浴摇床中反应8 h后,TTMP的产量达45.1 g/l;在此基础上,建立了枯草杆菌发酵生产乙偶姻偶联TTMP化学合成的工艺:在前体乙偶姻的积累阶段,采用pH 5.5的胁迫环境促进枯草杆菌的快速增殖和前体乙偶姻的大量积累;培养120 h后,控制反应条件为TTMP合成最优条件,反应结束时TTMP的浓度达16.8 g/l,该结果验证了枯草杆菌发酵生产前体乙偶姻偶联TTMP化学合成工艺提高TTMP产量的有效性。
(6)建立了枯草杆菌全细胞多步转化葡萄糖生成乙偶姻的工艺,确定了最适的转化条件:葡萄糖浓度140 g/l,细胞浓度100 g/l,以0.5 M的PBS缓冲(pH7.0)为反应介质,装液量为20/50 ml,纱布密封,于35°C、150 rpm摇床中反应96 h;在上述条件下,枯草杆菌转化体系中乙偶姻的产量达41.4 g/l,与枯草杆菌在最优发酵条件下的乙偶姻产量相当,但转化周期比发酵周期缩短了20%;同时,建立了枯草杆菌多步转化乙偶姻偶联TTMP化学合成工艺:在前体乙偶姻的积累阶段,枯草杆菌全细胞可以转化140 g/l葡萄糖生成41.4 g/l前体乙偶姻;然后,控制反应条件为TTMP合成最优条件,反应结束时TTMP的浓度达16.5 g/l,该结果验证了枯草杆菌多步转化乙偶姻偶联TTMP化学合成工艺提高TTMP产量的有效性。
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.
微生物发酵法生产TTMP的研究起步较晚,相关文献报道较少,且发酵体系中前体乙偶姻与氨反应生成TTMP的机制仍存在争议。目前,鲜有大规模TTMP发酵生产的工业化应用实例,其原因主要为TTMP产量较低、前体乙偶姻的有效转化率不高(<1%)、外源添加的乙偶姻存在产物抑制现象且乙偶姻和TTMP均对细胞具有毒性等。因此,开发具有优良特性的TTMP生产菌株、研究微生物利用葡萄糖代谢产生乙偶姻以及乙偶姻和氨反应生成TTMP的机制、并基于不同产生机制建立合适的TTMP生产工艺是提高微生物发酵生产TTMP的有效手段,为工业化应用奠定基础。
本文以获得高产TTMP菌株为目标,首先建立了一种内源前体筛选策略,从酱香型高温大曲中获得一株高产TTMP的菌株Bacillus subtilis XZ1124;根据其在不同培养条件下的发酵动力学分析,建立相应的发酵策略;对发酵体系中TTMP的生成机制进行探索,并基于实验结果建立有效的微生物发酵偶联TTMP合成工艺;同时,利用发酵结束废弃的菌体作用生物催化剂,建立枯草杆菌多步转化葡萄糖生成乙偶姻的工艺及其偶联化学合成生产TTMP的工艺,具体内容和结果如下:
(1)基于前体乙偶姻和TTMP分子结构的相关性,建立了一种适合于风味化合物TTMP筛选的内源前体筛选策略;并应用该策略,从酱香型高温大曲中获得一株TTMP产生菌XZ1124,该菌株能利用葡萄糖代谢产生大量前体乙偶姻,从而有效促进了TTMP的合成;根据菌落、细胞形态特征和生理生化特性以及16S rDNA序列分析,将该菌株鉴定为枯草杆菌(Bacillus subtilis);通过单因素优化,确定了最优的培养基组成和培养条件:蔗糖100 g/l、豆饼粉40 g/l、酵母膏5 g/l、磷酸氢二铵30 g/l、初始pH 7.5、装液量50/250 ml、摇床转速200 rpm、培养温度37°C;在此条件下,B. subtilis XZ1124摇瓶培养120 h,TTMP产量达4.08 g/l,前体乙偶姻积累量在培养66 h时达到峰值(22.3 g/l),前体的有效利用率达23.7%,远高于文献报道值(1%)。
(2)根据不同pH控制下发酵罐培养的动力学分析,建立了适合于枯草杆菌TTMP发酵的两阶段pH控制策略,通过控制培养前期的发酵液为弱酸性,保证细胞的快速增殖和前体乙偶姻的大量积累,培养后期调节发酵液为中性,促进枯草杆菌转化乙偶姻生成TTMP(7.43 g/l);根据枯草杆菌在7.5 L发酵罐培养时不同搅拌转速下的动力学分析,结合发酵体系中前体乙偶姻和氨的化学反应特性,建立了多阶段搅拌转速偶联温度控制的发酵策略:0~12 h,控制培养温度37oC、搅拌转速为700 rpm,12~48 h,控制培养温度37oC、搅拌转速为500 rpm,48~120 h,控制培养温度55oC、搅拌转速为500 rpm,该发酵策略可有效促进前体乙偶姻的大量积累,发酵结束时TTMP的产量达7.12 g/l,比恒温(37oC)和恒定搅拌转速(500 rpm)培养时分别提高了38.5%和25.1%;根据不同初始葡萄糖浓度下的发酵动力学分析,建立了葡萄糖补加策略:初始葡萄糖浓度为80 g/l、培养36 h补加50 g/l葡萄糖,该补加策略有效提高了前体乙偶姻的积累量,发酵结束时前体乙偶姻的积累量达40.9 g/l,与对照发酵相比提高了96.6%;TTMP浓度达6.54 g/l,比对照发酵提高了28.2%。
(3)考察了不同铵盐对枯草杆菌发酵产TTMP的影响,结果表明磷酸氢二铵(DAP)对TTMP合成的促进作用最为显著,且NH4+和PO43-同时存在时该促进作用尤为突出;提高培养基中初始DAP的浓度(或单独提高NH4+和PO43-浓度)可有效促进发酵体系中TTMP的生成,然而,高浓度的NH4+和PO43-离子不利于细胞的增殖、糖的消耗以及前体乙偶姻的积累;基于细胞增殖、前体乙偶姻积累和产物TTMP合成过程对DAP需求的差异,提出DAP补加策略,并应用于枯草杆菌摇瓶发酵和发酵罐培养过程,TTMP的最大生成速率分别提高了46.8%和76.8%,发酵结束时TTMP产量分别为9.10和7.34 g/l。
(4)建立了酶促反应模型体系,该模型体系为含有一定浓度乙偶姻和30 g/l磷酸氢二铵的溶液(pH 7.0),通过向该模型体系中加入胞内粗酶液和胞外粗酶液检测TTMP的变化;并进一步应用原位反应体系验证TTMP生成过程中的酶促催化反应,将枯草杆菌全细胞作为催化剂,考察了胞内酶和胞外酶对体系中乙偶姻和氨反应生成TTMP的影响;结果表明,发酵体系中TTMP的合成过程不存在酶促催化反应;但枯草杆菌培养液中可能存在一种或多种小分子代谢物,该代谢物能够促进乙偶姻和氨化学反应合成TTMP。
(5)考察了不同质子供体对乙偶姻和氨反应生成TTMP的影响,磷酸盐在pH 7.0时主要以磷酸二氢根和磷酸一氢根共轭对形式存在,能够提供足量的质子参与乙偶姻和氨反应生成Schiff碱的过程,促进了TTMP的合成;通过考察糖代谢途径中有机酸对枯草杆菌发酵体系中TTMP生成的影响,发现乳酸、丙酮酸、草酸、琥珀酸和柠檬酸均可促进发酵体系中TTMP的生成;进一步考察了乙偶姻和DAP浓度、pH、温度和搅拌条件以及供氧条件对枯草杆菌原位发酵体系中TTMP和YTTMP/HB的影响,确定了最优的TTMP合成条件,将100 g/l乙偶姻和200 g/l DAP的原位发酵体系于90oC、200 rpm的水浴摇床中反应8 h后,TTMP的产量达45.1 g/l;在此基础上,建立了枯草杆菌发酵生产乙偶姻偶联TTMP化学合成的工艺:在前体乙偶姻的积累阶段,采用pH 5.5的胁迫环境促进枯草杆菌的快速增殖和前体乙偶姻的大量积累;培养120 h后,控制反应条件为TTMP合成最优条件,反应结束时TTMP的浓度达16.8 g/l,该结果验证了枯草杆菌发酵生产前体乙偶姻偶联TTMP化学合成工艺提高TTMP产量的有效性。
(6)建立了枯草杆菌全细胞多步转化葡萄糖生成乙偶姻的工艺,确定了最适的转化条件:葡萄糖浓度140 g/l,细胞浓度100 g/l,以0.5 M的PBS缓冲(pH7.0)为反应介质,装液量为20/50 ml,纱布密封,于35°C、150 rpm摇床中反应96 h;在上述条件下,枯草杆菌转化体系中乙偶姻的产量达41.4 g/l,与枯草杆菌在最优发酵条件下的乙偶姻产量相当,但转化周期比发酵周期缩短了20%;同时,建立了枯草杆菌多步转化乙偶姻偶联TTMP化学合成工艺:在前体乙偶姻的积累阶段,枯草杆菌全细胞可以转化140 g/l葡萄糖生成41.4 g/l前体乙偶姻;然后,控制反应条件为TTMP合成最优条件,反应结束时TTMP的浓度达16.5 g/l,该结果验证了枯草杆菌多步转化乙偶姻偶联TTMP化学合成工艺提高TTMP产量的有效性。
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.
引文
1. Burdock G A. Fenaroli's Handbook of Flavor Ingredients, 5 ed. Boca Raton, Fla: CRC Press, 2004
2. Masuda H, Mihara S. Olfactive properties of alkylpyrazines and 3-substituted
2-alkylpyrazines [J]. Journal of Agricultural and Food Chemistry, 1988, 36(3):584-587
3.胡国芬,王建平.川芎嗪的药理作用及临床应用进展[J].中国药物与临床, 2006, 6(10):773-774
4.胡明一,王中.食用香料乙偶姻[J].精细与专用化学品, 2002, (1):20-21
5.吴建峰.中国白酒中健康功能性成分四甲基吡嗪的研究[J].酿酒科技, 2007, 151:117-120
6. Seitz E W. Bioprocess production of flavor, fragrance, and color ingredients [M]. New York: Wiley, 1994:95-134
7. Fan W L, Xu Y, Zhang Y H. Characterization of pyrazines in some Chinese liquors and their approximate concentrations [J]. Journal of Agricultural and Food Chemistry, 2007, 55(24):9956-9962
8.邓泽庭,陶德胜,罗荣,等.四甲基吡嗪生产方法[P]. 2006
9.韩丽,赵祥颖,刘建军. 3-羟基丁酮的研究现状[J].食品与发酵工业, 2006, (10)
10.何坚,孙里国.香料化学与工艺学[M].北京:化学工业出版社, 1999
11.肖旭辉.抗菌药伦氨苄西林盐酸盐合成[J].精细化工中间体, 1998, 34(10):6-10
12. Saito S, Inoue H, Ohno K.α-hydroxyketone derivatives, liquid crystal compositions containing said derivatives, and liquid crystal devices using said compositions [P]. United States, 1992
13.杨虹,刘璐,张海菊.川芎嗪提取工艺及质量标准研究[J].中国民族民间医药, 2009, 18(7):24-25
14. Li H B, Chen F. Preparative isolation and purification of chuanxiongzine from the medicinal plant Ligusticum chuanxiong by high-speed counter-current chromatography [J]. Journal of Chromatography A, 2004, 1047(2):249-253
15.刘本, Dean J R, Price R.法多索溶剂和超临界流体提取川芎中的川芎嗪[J].中国医药工业杂志, 1999, 30(5):196-198
16. Amranihemaimi M, Cerny C, Fay L B. Mechanisms of formation of alkylpyrazines in the Maillard reaction [J]. Journal of Agricultural and Food Chemistry, 1995,43(11):2818-2822
17. Jalbout A F, Shipar M A H. Formation of pyrazines in hydroxyacetaldehyde and glycine nonenzymatic browning Maillard reaction: a computational study [J]. Food Chemistry, 2007, 103(4):1208-1216
18.王向宇,侯昭胤,陆维敏,等.二乙酰一肟催化加氢制四甲基吡嗪的研究[J].化学反应工程与工艺, 2000, 16(2):153-158
19. Fischer H, Walach B. Synthese der 2,4-dimethyl-5-?thylpyrrol-3-propions?ure [J]. Justus Liebig's Annalen der Chemie, 1926, 447(1):38-48
20. Cenker M, Jackson D R, Langdon W K, et al. Synthesis of pyrazines: vapor-phase dehydrogenation of piperazines [J]. Industrial and Engineering Chemistry Product Research and Development, 1964, 3(1):11-14
21.章思规.实用精细化学品手册(下) [M].北京:化学工业出版社, 1996
22. Shoji T, Nakaishi T, Mikata M. Preparation of pyrazines as synthetic intermediates [P]. Germany, DE 19629258 1997
23. Yasuda S. Pyrazines [P]. Japan, JP 60258168 1985
24. Winans C F, Adkins H. The synthesis and reactions of certain nitrogen ring compounds over nickel [J]. Journal of the American Chemical Society, 1933, 55(10):4167-4176
25. Kosuge T, Kamiya H. Discovery of a pyrazine in a natural product - tetramethylpyrazine from cultures of a strain of Bacillus subtilis [J]. Nature, 1962, 193(4817):776
26. Reineccius G A, Keeney P G, Weissberger W. Factors affecting the concentration of pyrazines in cocoa beans [J]. Journal of Agricultural and Food Chemistry, 1972, 20(2):202-206
27. Yamaguchi N, Toda T, Teramoto T, et al. Effect of sugars on microbiological pyrazine formation by Bacillus natto in synthetic liquid-medium [J]. Journal of the Japanese Society for Food Science and Technology-Nippon Shokuhin Kagaku Kogaku Kaishi, 1993, 40(12):841-848
28. Besson I, Creuly C, Gros J B, et al. Pyrazine production by Bacillus subtilis in solid-state fermentation on soybeans [J]. Applied Microbiology and Biotechnology, 1997, 47(5):489-495
29. Larroche C, Besson I, Gros J B. High pyrazine production by Bacillus subtilis in solid substrate fermentation on ground soybeans [J]. Process Biochemistry, 1999, 34(6-7):667-674
30. Demain A L, Jackson M, Trenner N R. Thiamine-dependent accumulation oftetramethylpyrazine accompanying a mutation in isoleucine-valine pathway [J]. Journal of Bacteriology, 1967, 94(2):323-326
31. Kim K H, Kwang-soo, lee H-j, et al. Optimum conditions for the production of retramethylpyrazine dlavor compound by aerobic fed-batch culture of Lactococcus lactis subsp. lactis biovar. diacetylactis FC1 [J]. Journal of Microbiology and Biotechnology, 1994, 4(4):327-332
32. Xiao Z J, Xie N Z, Liu P H, et al. Tetramethylpyrazine production from glucose by a newly isolated Bacillus mutant [J]. Applied Microbiology and Biotechnology, 2006, 73(3):512-518
33. Kosuge T, Kamiya H, Adachi T. Isolation of tetramethylpyrazine from culture of bacillus natto, and biosynthetic pathways of tetramethylpyrazine [J]. Nature, 1962, 195(4846):1103
34. Adachi T, Kamiya H, Kosuge T. Studies on the metabolic products of Bacillus subtilis. ii. the production of tetramethylpyrazine by natto [J]. Yakugaku Zasshi, 1964, 84:451-452
35. Adachi T, Kamiya H, Kosuge T. Studies on the metabolic products of Bacillus subtilis. III. relation between amino acids and tetramethylpyrazine production [J]. Yakugaku Zasshi, 1964, 84:543-545
36. Rizzi G P. Formation of pyrazines from acyloin precursors under mild conditions [J]. Journal of Agricultural and Food Chemistry, 1988, 36(2):349-352
37. Huang T C, Fu H Y, Ho C T. Mechanistic studies of tetramethylpyrazine formation under weak acidic conditions and high hydrostatic pressure [J]. Journal of Agricultural and Food Chemistry, 1996, 44(1):240-246
38. Huang T C. Combined effects of a buffer and solvent on tetramethylpyrazine formation in a 3-hydroxy-2-butanone/ammonium hydroxide system [J]. Biosystems Biotechnology and Biochemistry, 1997, 61(6):1013-1015
39. Zhu B F, Xu Y, Fan W L. High-yield fermentative preparation of tetramethylpyrazine using an endogenous precursor approach by Bacillus sp. [J]. Journal of Industrial Microbiology & Biotechnology, 2010, 37:179-186
40. Larroche C, Gros J B. Special transformation processes using fungal spores and immobilized cells [J]. Advances in Biochemical Engineering/Biotechnology, 1997, 55:179-220
41. Schrader J. Flavours and Fragrances [M]. 2007:507-574
42. Zhu B F, Xu Y. A feeding strategy for tetramethylpyrazine production by Bacillus subtilis based on the stimulating effect of ammonium phosphate [J]. Bioprocess andBiosystems Engineering, 2010, doi: 10.1007/s00449-010-0419-5
43.东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社, 2001
44.张克旭.氨基酸发酵工艺学[M]:中国轻工业出版社, 1995
45. Chen X, Chen S, Sun M, et al. Medium optimization by response surface methodology for poly-γ-glutamic acid production using dairy manure as the basis of a solid substrate [J]. Applied Microbiology and Biotechnology, 2005, 69(4):390-396
46. Miller G L. Use of dinitrosalicylic acid reagent for determination of reducing sugar [J]. Analytical Chemistry, 1959, 31(3):426-428
47. Wang C L, Shi D J, Gong G L. Microorganisms in Daqu: a starter culture of Chinese Maotai-flavor liquor [J]. World Journal of Microbiology & Biotechnology, 2008, 24(10):2183-2190
48. Buchanan R E, Gibbons N E. Bergey's Manual of Determinative Bacteriology, 8th ed. Maryland: Williams & Wilkins, 1974
49. Nakashimada Y, Kanai K, Nishio N. Optimization of dilution rate, pH and oxygen supply on optical purity of 2, 3-butanediol produced by Paenibacillus polymyxa in chemostat culture [J]. Biotechnology Letters, 1998, 20(12):1133-1138
50. Xiao Z J, Xu P. Acetoin metabolism in bacteria [J]. Critical Reviews in Microbiology, 2007, 33(2):127-140
51. Grundy F J, Turinsky A J, Henkin T M. Catabolite regulation of Bacillus subtilis acetate and acetoin utilization genes by CcpA [J]. Journal of Bacteriology, 1994, 176(15):4527-4533
52. Johansen L, Bryn K, Stormer F C. Physiological and biochemical role of the butanediol pathway in Aerobacter (Enterobacter) aerogenes [J]. Journal of Bacteriology, 1975, 123(3):1124-1130
53. Shu C K, Lawrence B M. Formation of 2-(1-hydroxyalkyl)-3-oxazolines from the reaction of acyloins and ammonia precursors under mild conditions [J]. Journal of Agricultural and Food Chemistry, 1995, 43(11):2922-2924
54. Maestri O, Joset F. Regulation by external pH and stationary growth phase of the acetolactate synthase from Synechocystis PCC6803 [J]. Molecular Microbiology, 2000, 37(4):828-838
55. Adachi T, Kamiya H, Kosuge T. Studies on the metabolic products of Bacillus subtilis. IV. determination and mechanism of formation of tetramethylpyrazine [J]. Yakugaku Zasshi, 1964, 84:545-548
56. Shu C K. Pyrazine formation from amino acids and reducing sugars, a pathway other than Strecker degradation [J]. Journal of Agricultural and Food Chemistry, 1998,46(4):1515-1517
57. Horton R, Moran L A, Scrimgeour G, et al. Principles of Biochemistry [M]. New York: Prentice Hall, 1968
58. Shibamoto T, Bernhard R A. Investigation of pyrazine formation pathways in glucose-ammonia model systems [J]. Agricultural and Biological Chemistry, 1977, 41(1):143-153
59. Xiao Z J, Liu P H, Qin J Y, et al. Statistical optimization of medium components for enhanced acetoin production from molasses and soybean meal hydrolysate [J]. Applied Microbiology and Biotechnology, 2007, 74(1):61-68
60. Yu E K C, Saddler J N. Fed-batch approach to production of 2,3-butanediol by klebsiella pneumoniae grown on high substrate concentrations [J]. Applied and Environmental Microbiology, 1983, 46(3):630-635
61. Gupta K G, Yadav N K, Dhawan S. Laboratory-scale production of acetoin plus diacetyl by Enterobacter cloacae ATCC 27613 [J]. Biotechnology and Bioengineering, 1978, 20(12):1895-1901
62. Zeng A P, Biebl H, Deckwer W D. Production of 2,3-butanediol in a membrane bioreactor with cell recycle [J]. Applied Microbiology and Biotechnology, 1991, 34(4):463-468
63. Ui S, Mimura A, Okuma M, et al. The production of d-acetoin by a transgenic Escherichia coli [J]. Letters in Applied Microbiology, 1998, 26(4):275-278
64. Kaneko T, Takahashi M, Suzuki H. Acetoin fermentation by citrate-positive Lactococcus-lactis subsp lactis 3022 grown aerobically in the presence of hemin or Cu2+ [J]. Applied and Environmental Microbiology, 1990, 56(9):2644-2649
65. Faveri D D, Torre P, Molinari F, et al. Carbon material balances and bioenergetics of 2,3-butanediol bio-oxidation by Acetobacter hansenii [J]. Enzyme and Microbial Technology, 2003, 33(5):708-719
66. Ui S, Masuda H, Muraki H. Laboratory-scale production of acetoin isomers (d(-) and l(+)) by bacterial fermentation [J]. Journal of Fermentation Technology, 1983, 61(3):253-259
67. Afschar A S, Bellgardt K H, Rossell C E, et al. The production of 2,3-butanediol by fermentation of high test molasses [J]. Applied Microbiology and Biotechnology, 1991, 34(5):582-585
68. Ui S, Masuda H, Muraki H. Laboratory-scale production of acetoin isomers (d(-) and l(+)) by bacterial fermentation [J]. Journal of Fermentation Technology, 1984, 62(2):151-156
69. Moes J, Griot M, Keller J, et al. A microbial culture with oxygen-sensitive product distribution as a potential tool for characterizing bioreactor oxygen-transport [J]. Biotechnology and Bioengineering, 1985, 27(4):482-489
2. Masuda H, Mihara S. Olfactive properties of alkylpyrazines and 3-substituted
2-alkylpyrazines [J]. Journal of Agricultural and Food Chemistry, 1988, 36(3):584-587
3.胡国芬,王建平.川芎嗪的药理作用及临床应用进展[J].中国药物与临床, 2006, 6(10):773-774
4.胡明一,王中.食用香料乙偶姻[J].精细与专用化学品, 2002, (1):20-21
5.吴建峰.中国白酒中健康功能性成分四甲基吡嗪的研究[J].酿酒科技, 2007, 151:117-120
6. Seitz E W. Bioprocess production of flavor, fragrance, and color ingredients [M]. New York: Wiley, 1994:95-134
7. Fan W L, Xu Y, Zhang Y H. Characterization of pyrazines in some Chinese liquors and their approximate concentrations [J]. Journal of Agricultural and Food Chemistry, 2007, 55(24):9956-9962
8.邓泽庭,陶德胜,罗荣,等.四甲基吡嗪生产方法[P]. 2006
9.韩丽,赵祥颖,刘建军. 3-羟基丁酮的研究现状[J].食品与发酵工业, 2006, (10)
10.何坚,孙里国.香料化学与工艺学[M].北京:化学工业出版社, 1999
11.肖旭辉.抗菌药伦氨苄西林盐酸盐合成[J].精细化工中间体, 1998, 34(10):6-10
12. Saito S, Inoue H, Ohno K.α-hydroxyketone derivatives, liquid crystal compositions containing said derivatives, and liquid crystal devices using said compositions [P]. United States, 1992
13.杨虹,刘璐,张海菊.川芎嗪提取工艺及质量标准研究[J].中国民族民间医药, 2009, 18(7):24-25
14. Li H B, Chen F. Preparative isolation and purification of chuanxiongzine from the medicinal plant Ligusticum chuanxiong by high-speed counter-current chromatography [J]. Journal of Chromatography A, 2004, 1047(2):249-253
15.刘本, Dean J R, Price R.法多索溶剂和超临界流体提取川芎中的川芎嗪[J].中国医药工业杂志, 1999, 30(5):196-198
16. Amranihemaimi M, Cerny C, Fay L B. Mechanisms of formation of alkylpyrazines in the Maillard reaction [J]. Journal of Agricultural and Food Chemistry, 1995,43(11):2818-2822
17. Jalbout A F, Shipar M A H. Formation of pyrazines in hydroxyacetaldehyde and glycine nonenzymatic browning Maillard reaction: a computational study [J]. Food Chemistry, 2007, 103(4):1208-1216
18.王向宇,侯昭胤,陆维敏,等.二乙酰一肟催化加氢制四甲基吡嗪的研究[J].化学反应工程与工艺, 2000, 16(2):153-158
19. Fischer H, Walach B. Synthese der 2,4-dimethyl-5-?thylpyrrol-3-propions?ure [J]. Justus Liebig's Annalen der Chemie, 1926, 447(1):38-48
20. Cenker M, Jackson D R, Langdon W K, et al. Synthesis of pyrazines: vapor-phase dehydrogenation of piperazines [J]. Industrial and Engineering Chemistry Product Research and Development, 1964, 3(1):11-14
21.章思规.实用精细化学品手册(下) [M].北京:化学工业出版社, 1996
22. Shoji T, Nakaishi T, Mikata M. Preparation of pyrazines as synthetic intermediates [P]. Germany, DE 19629258 1997
23. Yasuda S. Pyrazines [P]. Japan, JP 60258168 1985
24. Winans C F, Adkins H. The synthesis and reactions of certain nitrogen ring compounds over nickel [J]. Journal of the American Chemical Society, 1933, 55(10):4167-4176
25. Kosuge T, Kamiya H. Discovery of a pyrazine in a natural product - tetramethylpyrazine from cultures of a strain of Bacillus subtilis [J]. Nature, 1962, 193(4817):776
26. Reineccius G A, Keeney P G, Weissberger W. Factors affecting the concentration of pyrazines in cocoa beans [J]. Journal of Agricultural and Food Chemistry, 1972, 20(2):202-206
27. Yamaguchi N, Toda T, Teramoto T, et al. Effect of sugars on microbiological pyrazine formation by Bacillus natto in synthetic liquid-medium [J]. Journal of the Japanese Society for Food Science and Technology-Nippon Shokuhin Kagaku Kogaku Kaishi, 1993, 40(12):841-848
28. Besson I, Creuly C, Gros J B, et al. Pyrazine production by Bacillus subtilis in solid-state fermentation on soybeans [J]. Applied Microbiology and Biotechnology, 1997, 47(5):489-495
29. Larroche C, Besson I, Gros J B. High pyrazine production by Bacillus subtilis in solid substrate fermentation on ground soybeans [J]. Process Biochemistry, 1999, 34(6-7):667-674
30. Demain A L, Jackson M, Trenner N R. Thiamine-dependent accumulation oftetramethylpyrazine accompanying a mutation in isoleucine-valine pathway [J]. Journal of Bacteriology, 1967, 94(2):323-326
31. Kim K H, Kwang-soo, lee H-j, et al. Optimum conditions for the production of retramethylpyrazine dlavor compound by aerobic fed-batch culture of Lactococcus lactis subsp. lactis biovar. diacetylactis FC1 [J]. Journal of Microbiology and Biotechnology, 1994, 4(4):327-332
32. Xiao Z J, Xie N Z, Liu P H, et al. Tetramethylpyrazine production from glucose by a newly isolated Bacillus mutant [J]. Applied Microbiology and Biotechnology, 2006, 73(3):512-518
33. Kosuge T, Kamiya H, Adachi T. Isolation of tetramethylpyrazine from culture of bacillus natto, and biosynthetic pathways of tetramethylpyrazine [J]. Nature, 1962, 195(4846):1103
34. Adachi T, Kamiya H, Kosuge T. Studies on the metabolic products of Bacillus subtilis. ii. the production of tetramethylpyrazine by natto [J]. Yakugaku Zasshi, 1964, 84:451-452
35. Adachi T, Kamiya H, Kosuge T. Studies on the metabolic products of Bacillus subtilis. III. relation between amino acids and tetramethylpyrazine production [J]. Yakugaku Zasshi, 1964, 84:543-545
36. Rizzi G P. Formation of pyrazines from acyloin precursors under mild conditions [J]. Journal of Agricultural and Food Chemistry, 1988, 36(2):349-352
37. Huang T C, Fu H Y, Ho C T. Mechanistic studies of tetramethylpyrazine formation under weak acidic conditions and high hydrostatic pressure [J]. Journal of Agricultural and Food Chemistry, 1996, 44(1):240-246
38. Huang T C. Combined effects of a buffer and solvent on tetramethylpyrazine formation in a 3-hydroxy-2-butanone/ammonium hydroxide system [J]. Biosystems Biotechnology and Biochemistry, 1997, 61(6):1013-1015
39. Zhu B F, Xu Y, Fan W L. High-yield fermentative preparation of tetramethylpyrazine using an endogenous precursor approach by Bacillus sp. [J]. Journal of Industrial Microbiology & Biotechnology, 2010, 37:179-186
40. Larroche C, Gros J B. Special transformation processes using fungal spores and immobilized cells [J]. Advances in Biochemical Engineering/Biotechnology, 1997, 55:179-220
41. Schrader J. Flavours and Fragrances [M]. 2007:507-574
42. Zhu B F, Xu Y. A feeding strategy for tetramethylpyrazine production by Bacillus subtilis based on the stimulating effect of ammonium phosphate [J]. Bioprocess andBiosystems Engineering, 2010, doi: 10.1007/s00449-010-0419-5
43.东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社, 2001
44.张克旭.氨基酸发酵工艺学[M]:中国轻工业出版社, 1995
45. Chen X, Chen S, Sun M, et al. Medium optimization by response surface methodology for poly-γ-glutamic acid production using dairy manure as the basis of a solid substrate [J]. Applied Microbiology and Biotechnology, 2005, 69(4):390-396
46. Miller G L. Use of dinitrosalicylic acid reagent for determination of reducing sugar [J]. Analytical Chemistry, 1959, 31(3):426-428
47. Wang C L, Shi D J, Gong G L. Microorganisms in Daqu: a starter culture of Chinese Maotai-flavor liquor [J]. World Journal of Microbiology & Biotechnology, 2008, 24(10):2183-2190
48. Buchanan R E, Gibbons N E. Bergey's Manual of Determinative Bacteriology, 8th ed. Maryland: Williams & Wilkins, 1974
49. Nakashimada Y, Kanai K, Nishio N. Optimization of dilution rate, pH and oxygen supply on optical purity of 2, 3-butanediol produced by Paenibacillus polymyxa in chemostat culture [J]. Biotechnology Letters, 1998, 20(12):1133-1138
50. Xiao Z J, Xu P. Acetoin metabolism in bacteria [J]. Critical Reviews in Microbiology, 2007, 33(2):127-140
51. Grundy F J, Turinsky A J, Henkin T M. Catabolite regulation of Bacillus subtilis acetate and acetoin utilization genes by CcpA [J]. Journal of Bacteriology, 1994, 176(15):4527-4533
52. Johansen L, Bryn K, Stormer F C. Physiological and biochemical role of the butanediol pathway in Aerobacter (Enterobacter) aerogenes [J]. Journal of Bacteriology, 1975, 123(3):1124-1130
53. Shu C K, Lawrence B M. Formation of 2-(1-hydroxyalkyl)-3-oxazolines from the reaction of acyloins and ammonia precursors under mild conditions [J]. Journal of Agricultural and Food Chemistry, 1995, 43(11):2922-2924
54. Maestri O, Joset F. Regulation by external pH and stationary growth phase of the acetolactate synthase from Synechocystis PCC6803 [J]. Molecular Microbiology, 2000, 37(4):828-838
55. Adachi T, Kamiya H, Kosuge T. Studies on the metabolic products of Bacillus subtilis. IV. determination and mechanism of formation of tetramethylpyrazine [J]. Yakugaku Zasshi, 1964, 84:545-548
56. Shu C K. Pyrazine formation from amino acids and reducing sugars, a pathway other than Strecker degradation [J]. Journal of Agricultural and Food Chemistry, 1998,46(4):1515-1517
57. Horton R, Moran L A, Scrimgeour G, et al. Principles of Biochemistry [M]. New York: Prentice Hall, 1968
58. Shibamoto T, Bernhard R A. Investigation of pyrazine formation pathways in glucose-ammonia model systems [J]. Agricultural and Biological Chemistry, 1977, 41(1):143-153
59. Xiao Z J, Liu P H, Qin J Y, et al. Statistical optimization of medium components for enhanced acetoin production from molasses and soybean meal hydrolysate [J]. Applied Microbiology and Biotechnology, 2007, 74(1):61-68
60. Yu E K C, Saddler J N. Fed-batch approach to production of 2,3-butanediol by klebsiella pneumoniae grown on high substrate concentrations [J]. Applied and Environmental Microbiology, 1983, 46(3):630-635
61. Gupta K G, Yadav N K, Dhawan S. Laboratory-scale production of acetoin plus diacetyl by Enterobacter cloacae ATCC 27613 [J]. Biotechnology and Bioengineering, 1978, 20(12):1895-1901
62. Zeng A P, Biebl H, Deckwer W D. Production of 2,3-butanediol in a membrane bioreactor with cell recycle [J]. Applied Microbiology and Biotechnology, 1991, 34(4):463-468
63. Ui S, Mimura A, Okuma M, et al. The production of d-acetoin by a transgenic Escherichia coli [J]. Letters in Applied Microbiology, 1998, 26(4):275-278
64. Kaneko T, Takahashi M, Suzuki H. Acetoin fermentation by citrate-positive Lactococcus-lactis subsp lactis 3022 grown aerobically in the presence of hemin or Cu2+ [J]. Applied and Environmental Microbiology, 1990, 56(9):2644-2649
65. Faveri D D, Torre P, Molinari F, et al. Carbon material balances and bioenergetics of 2,3-butanediol bio-oxidation by Acetobacter hansenii [J]. Enzyme and Microbial Technology, 2003, 33(5):708-719
66. Ui S, Masuda H, Muraki H. Laboratory-scale production of acetoin isomers (d(-) and l(+)) by bacterial fermentation [J]. Journal of Fermentation Technology, 1983, 61(3):253-259
67. Afschar A S, Bellgardt K H, Rossell C E, et al. The production of 2,3-butanediol by fermentation of high test molasses [J]. Applied Microbiology and Biotechnology, 1991, 34(5):582-585
68. Ui S, Masuda H, Muraki H. Laboratory-scale production of acetoin isomers (d(-) and l(+)) by bacterial fermentation [J]. Journal of Fermentation Technology, 1984, 62(2):151-156
69. Moes J, Griot M, Keller J, et al. A microbial culture with oxygen-sensitive product distribution as a potential tool for characterizing bioreactor oxygen-transport [J]. Biotechnology and Bioengineering, 1985, 27(4):482-489