淡豆豉炮制中产γ-氨基丁酸微生物在不同pH值和不同温度下产酶能力比较研究  

A comparative study on enzyme production ability of microorganism producing γ-aminobutyric acid in processing of Sojae Semen Praeparatum at different pH and temperatures

作　　者：周姝含 黄越燕[2] 熊京京 刘宇星 苏明声[1] 王立元[1] 龙凯[1] 谢卫华[1] 谢小梅[1] ZHOU Shu-han;HUANG Yue-yan;XIONG Jing-jing;LIU Yu-xing;SU Ming-sheng;WANG Li-yuan;LONG Kai;XIE Wei-hua;XIE Xiao-mei(Jiangxi University of Traditional Chinese Medicine,Nanchang 330004,China;Medical College of Jiaxing University,Jiaxing 314001,China)

机构地区：[1]江西中医药大学,江西南昌330004 [2]嘉兴学院医学院,浙江嘉兴314001

出　　处：《中草药》2020年第16期4176-4182,共7页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金项目(81660664);江西省重点研发计划一般项目(20192BBGL70051);江西省教育厅科学技术研究项目(GJJ170719);江西省教育厅科学技术研究项目(GJJ180655);江西省教育厅科学技术研究项目(GJJ190634)。

摘　　要：目的阐明淡豆豉炮制中微生物调控γ-氨基丁酸(γ-amino butyric acid,GABA)的形成机制。方法采用Berthelot比色法、福林酚法测定枯草芽孢杆菌、鸟肠球菌、屎肠球菌、解淀粉芽孢杆菌、黑曲霉、黄曲霉、溜曲霉、桔青霉、极细支孢霉、白腐菌、米根霉、鲑色锁掷酵母菌12种微生物在不同pH值和不同温度条件下产谷氨酸脱羧酶(glutamicacid decarboxylase,GAD)、蛋白酶(中性蛋白酶、碱性蛋白酶、酸性蛋白酶)能力。结果 12种微生物在pH 5~7、28~37℃时产GAD和蛋白酶酶活较高。其中黄曲霉产GAD酶活最高,为41.97 U/h,最适pH 7、温度28℃;其次是鲑色锁掷酵母菌、黑曲霉、米根霉、枯草芽孢杆菌,酶活分别为29.04、25.78、22.42、19.43 U/h。枯草芽孢杆菌产中性蛋白酶活最高,为24.80 U/mL,最适pH 7、温度37℃;其次是解淀粉芽孢杆菌、米根霉、鸟肠球菌,酶活分别为16.86、12.51、9.18 U/mL。解淀粉芽孢杆菌产碱性蛋白酶活最高,为13.29U/m L,最适pH7、温度34℃;其次是枯草芽孢杆菌、米根霉,酶活分别为8.86、6.20 U/mL。12种微生物产酸性蛋白酶能力普遍较差。结论所选的12种微生物产酶最适pH值、温度与淡豆豉自然炮制基本一致,其中真菌有较强的产GAD能力,细菌有较强的产中性蛋白酶能力,淡豆豉高富集GABA是多菌种共同作用所致。Objective To lay a foundation for elucidating the mechanism of microbial regulation of γ-amino butyric acid(GABA) formation in the processing of Sojae Semen Praeparatum. Methods The ability of glutamic acid decarboxylase and protease(neutral protease, alkaline protease and acid protease) production of Bacillus subtilis, Enterococcus avium, Enterococcus faecium, Bacillus amyloliquefaciens, Aspergillus niger, Aspergillus flavus, Aspergillus tamarii, Penicillium citrinum, Cladosporium tenuissimum, Phanerochaete sordida, Rhizopus oryzae, Sporidiobolus salmonicolor in different pH and temperature conditions were determined by Berthelot colorimetry and Folin phenol method. Results The GAD and protease activities of the twelve strains were stronger at pH 5-7 and temperature 28-37 ℃. The highest GAD enzyme activity from A. flavus was 41.97 U/h, the optimum pH was 7 and the temperature was 28 ℃, followed by S. salmonicolor, A. niger, R. oryzae, and B. subtilis, respectively. The enzyme activities were 29.04, 25.78, 22.42 and 19.43 U/h. The highest neutral protease activity of B. subtilis was 24.80 U/mL, the optimum pH was 7 and the temperature was 37 ℃, followed by B. amyloliquefaciens, R. oryzae and E. avium. The enzyme activities were 16.86, 12.51 and 9.18 U/mL. The highest alkaline protease activity of B. amyloliquefaciens was 13.29 U/m L, the optimum pH was 7 and the temperature was 34 ℃, followed by B. subtilis and R. oryzae. The enzyme activities were 8.86 and 6.20 U/mL, respectively. The ability of 12 kinds of microorganisms to produce acid protease was generally poor. Conclusion The optimal pH and temperature of the 12 kinds of microorganisms selected for this experiment are basically consistent with the natural processing of Sojae Semen Praeparatum. Among them, fungi have stronger GAD production capacity and bacteria have stronger neutral protease production capacity. The high GABA concentration of Sojae Semen Praeparatum is caused by the joint action of multiple strains.

关 键 词：淡豆豉 Γ-氨基丁酸 微生物 谷氨酸脱羧酶 蛋白酶 pH值 温度 Berthelot比色法 福林酚法 枯草芽孢杆菌 鸟肠球菌 屎肠球菌 解淀粉芽孢杆菌 黑曲霉 黄曲霉 溜曲霉 桔青霉 极细支孢霉 白腐菌 米根霉 鲑色锁掷酵母菌 

分 类 号：R283.1[医药卫生—中药学]