Xiao Z J, Xie N Z, Liu P H, Hua D L, Xu P
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China.
Appl Microbiol Biotechnol. 2006 Dec;73(3):512-8. doi: 10.1007/s00253-006-0491-6. Epub 2006 Jun 27.
2,3,5,6-Tetramethylpyrazine (TTMP) was produced using a newly isolated Bacillus mutant. Culture medium optimization studies showed that soytone, an enzyme-hydrolysate of soybean meal, with the supplementation of vitamins, can fully replace yeast extract plus peptone in supporting TTMP production from glucose. In a 5-l fermenter, using the optimized medium which contained 20% glucose, 5% soytone, 3% (NH(4))(2)HPO(4), and vitamin supplements, fermentations were carried out with stirring at 700 rpm, air flow at 1.0 vvm, controlled pH at 7.0, and temperature at 37 degrees C. TTMP reached 4.33 g l(-1) after 64.6 h cultivation. A product recovery method was described, which involved evaporation, crystallization, and lyophilization. The product purity was 99.88%, determined by GC with the normalization method. The main impurities were 2,3,5-trimethylpyrazine (0.09%) and 2-ethyl-3,5,6-trimethylpyrazine (0.02%), which were identified by GC/MS. (13)C NMR determination also gave a consistent result. Natural and high purity of the product and the utilization of cheap green renewable materials make this process promising to compete with TTMP chemical synthetic methods.
使用新分离的芽孢杆菌突变体生产2,3,5,6 - 四甲基吡嗪（TTMP）。培养基优化研究表明，大豆蛋白胨（豆粕的酶水解产物）在补充维生素后，能够完全替代酵母提取物加蛋白胨，以支持从葡萄糖生产TTMP。在5升发酵罐中，使用含有20%葡萄糖、5%大豆蛋白胨、3%磷酸氢二铵（NH₄）₂HPO₄和维生素补充剂的优化培养基，在700转/分钟搅拌、1.0体积比每分钟通气量、pH值控制在7.0以及温度37℃的条件下进行发酵。培养64.6小时后，TTMP达到4.33克/升。描述了一种产品回收方法，该方法包括蒸发、结晶和冻干。采用气相色谱归一化法测定产品纯度为99.88%。主要杂质为2,3,5 - 三甲基吡嗪（0.09%）和2 - 乙基 - 3,5,6 - 三甲基吡嗪（0.02%），通过气相色谱/质谱联用仪（GC/MS）鉴定。碳 - 13核磁共振（¹³C NMR）测定也给出了一致的结果。产品的天然性和高纯度以及对廉价绿色可再生材料的利用使得该工艺有望与TTMP化学合成方法竞争。
