摘要
目的:对胆南星发酵过程中的微生物进行分离与鉴定,筛选优势菌种并对胆南星复合菌种发酵进行探索,以解决胆南星杂菌发酵工艺产品质量及稳定性不易控制的问题。方法:取胆南星发酵过程中的深层培养物进行菌种分离与纯化,运用细菌、真菌多相鉴定检测方法及全自动微生物分析系统,比对DNA序列,鉴定微生物,将分离鉴定出的菌种分别进行纯种发酵并筛选优势菌种,将筛选出的优势菌种进行复合菌种发酵探索,比较复合菌种发酵胆南星与传统发酵胆南星的指标成分含量差异。采用超高效液相色谱-三重四极杆串联质谱法(UPLC-QqQ-MS/MS)对传统发酵胆南星和复合菌种发酵胆南星中的指标成分进行含量测定,流动相0.1%甲酸乙腈溶液(A)-0.1%甲酸水溶液(B)梯度洗脱(0~2 min,35%~45%A;2~10 min,45%~48%A;10~12 min,48%~100%A;12~12.01 min,100%~35%A;12.01~15 min,35%~65%A),流速0.35 mL·min-1,采用电喷雾电离源(ESI),负离子采集模式,扫描方式为多反应监测(MRM)模式,采集范围m/z 50~1 000。结果:从胆南星深层培养物中共分离鉴定了8种微生物,筛选出肠球菌属Enterococcus sp.(厌氧)和铅黄肠球菌E.casseliflavus是胆南星发酵过程中的优势菌种,以二者作为复合菌种发酵制得的胆南星与传统发酵法样品比较,游离型胆酸中的鹅去氧胆酸、猪去氧胆酸和猪胆酸质量分数分别增加1.76、0.06、0.19 mg·g-1,结合型胆酸中的甘氨鹅去氧胆酸、牛磺鹅去氧胆酸、甘氨猪去氧胆酸、牛磺猪去氧胆酸、甘氨猪胆酸、牛磺猪胆酸含量较传统发酵胆南星分别降低0.63、0.23、0.26、0.16、0.03、0.04 mg·g-1。结论:复合菌种(肠球菌属和铅黄肠球菌)发酵能够使胆南星发酵更加完全,缩短发酵周期,并能够提高产品的质量及其稳定性。
Objective:In order to solve the problem that the quality and stability of Arisaema Cum Bile in the fermentation process with hybrid bacteria were not easy to control,the microorganism in the fermentation process of Arisaema Cum Bile was isolated and identified,the dominant strains were screened and the fermentation process of Arisaema Cum Bile with compound bacteria was investigated.Method:The submerged culture during the fermentation process of Arisaema Cum Bile was taken out for strain separation and purification.Bacteria and fungi multiphase identification and detection methods and automatic microbial analysis system were used to analyze and compare DNA sequences and identify microorganisms.The isolated and identified strains were respectively inoculated and fermented.After screening the dominant strains,a preliminary exploration of compound strain fermentation were carried out.The contents of index components in Arisaema Cum Bile fermented by compound strain and traditional Arisaema Cum Bile were compared by ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLC-QqQ-MS/MS).Mmobile phase was0.1% formic acid acetonitrile solution(A)-0.1% formic acid aqueous solution(B)for gradient elution(0-2 min,35%-45%A;2-10 min,45%-48%A;10-12 min,48%-100%A;12-12.01 min,100%-35%A;12.01-15 min,35%-65%A),the flow rate was set at 0.35 mL·min-1.The mass spectrographic analysis employed electrospray ionization(ESI),negative ion acquisition mode and multiple reaction monitoring(MRM)scanning mode were adopted to collect information,the collection range was m/z 50-1 000.Result:Eight microorganisms were isolated and identified from the submerged culture of Arisaema Cum Bile.Among them,Enterococcus sp.(anaerobic)and E.casseliflavus were selected as the dominant strains in the fermentation process.Compared with the traditional fermentation method,the contents of chenodeoxycholic acid,hyodeoxycholic acid and hyocholic acid in free cholic acid increased by 1.76,0.06,0.19 mg·g-1,respectively.In bound chol
作者
施宇
刘晓峰
单丽倩
高慧
SHI Yu;LIU Xiaofeng;SHAN Liqian;GAO Hui(School of Pharmacy,Liaoning University of Traditional Chinese Medicine,Dalian 116600,China;Liaoning Vocational College of Medicine,Shenyang 110000,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第17期150-156,共7页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家发改委中医药行业专项(201507004-03)
国家中药标准化项目(ZYBZH-Y-ZY-45)。
关键词
胆南星
发酵工艺
菌种鉴定
中药炮制
优势菌种
胆酸
超高效液相色谱-三重四极杆串联质谱法(UPLC-QqQ-MS/MS)
Arisaema Cum Bile
fermentation process
strain identification
traditional Chinese medicine processing
dominant strain
bile acid
ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLC-QqQ-MS/MS)
