淡豆豉炮制中黄曲霉毒素产毒株的筛选鉴定和产毒能力测定  

Screening,identification and toxin-producing ability analysis of aflatoxinproducing strains in processing of Sojae Semen Preaparatum

作　　者：李春玲 贺婧 王立元[1] 李翠英 龙凯[1] 翁美芝[1] 冯驰[3] 谢小梅[1] LI Chun-ling;HE Jing;WANG Li-yuan;LI Cui-ying;LONG Kai;WENG Mei-zhi;FENG Chi;XIE Xiao-mei(Jiangxi University of Chinese Medicine,Nanchang 330004,China;Institute of Translational Medicine,Nanchang University,Nanchang 330031,China;The Affiliated Hospital of Jiangxi University of Chinese Medicine,Nanchang 330006,China;Department of Urology,The Sixth Affiliated Hospital of Guangzhou Medical University,Qingyuan 511500,China)

机构地区：[1]江西中医药大学,江西南昌330004 [2]南昌大学转化医学研究院,江西南昌330031 [3]江西中医药大学附属医院,江西南昌330006 [4]广州医科大学附属第六医院泌尿外科,广东清远511500

出　　处：《中草药》2022年第5期1411-1417,共7页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金项目(82060709);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);江西省教育厅科技研究项目(GJJ190634);江西省科技计划项目(20203BAAW208025)。

摘　　要：目的对淡豆豉炮制过程中产黄曲霉毒素(aflatoxins,AFTs)的微生物(简称产毒菌)进行筛选鉴定、定量分析和产毒能力测定。方法运用紫外荧光法初筛淡豆豉炮制中各样本的产毒菌并菌落计数;对初筛菌株的18S rDNA序列进行PCR扩增、测序,测序结果经NCBI同源性比对、MEGA 7.0软件构建系统发育树进行分子生物学鉴定;应用超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)检测各产毒菌培养液中AFTs含量,确定各产毒菌的产毒能力。结果紫外荧光法结合分子生物学共筛选鉴定出15株产毒菌株,为黄曲霉和溜曲霉;“黄衣上遍”阶段产毒菌菌落数逐渐增多,至发酵第6天时最多,为1×10^(6.30) CFU/g,进入“再闷”阶段产毒菌数量逐渐减少,从再闷第9天开始至第15天均未检测到产毒菌;经UPLC-MS/MS法确定15株菌中有5株不产毒,10株产毒,且产毒能力各不相同并均低于5 ng/mL,远低于黄曲霉标准株(654.90 ng/mL)。结论淡豆豉炮制过程中存在产AFTs能力不同的黄曲霉、溜曲霉且产毒菌数量呈现先升后降,将为探讨淡豆豉炮制中AFTs消长机制奠定基础,并首次从安全性角度证实了淡豆豉“再闷”的重要性和“再闷”时间的合理性。Objective Aflatoxins(AFTs)producing strains(referred to as toxin-producing microorganism)during the processing of Sojae Semen Preaparatum(SSP)were screened,identified,quantitatively analyzed,and tested for their toxin-producing ability.Methods Preliminary screening of various toxin-producing microorganism in SSP processing by ultraviolet fluorescence method,then count the colonies;Analyse the 18S rDNA of those strains by PCR amplification and sequencing.The sequences are compared by NCBI homology,and the phylogenetic tree be constructed by MEGA 7.0 for molecular biology identification;Ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)were used to detect the AFTs content in the culture solution of each strain to determine their toxin-producing ability.Results Fifteen toxin-producing strains were screened and identified by ultraviolet fluorescence&molecular biology analyze,which were Aspergillus flavus or A.tamarii;During the processing of SSP,the toxinproducing microorganism gradually increased during the‘yellow cladding’stage,and it became the maximum at the 6th day,which was 1×10^(6.30) CFU/g,then it gradually decreased in the‘secondary fermentation’stage,even no toxin-producing strains be found during day 9 to day 15;With UPLC-MS/MS,we found that five strains did not produce toxin,10 strains produced toxin.Though they can make toxin,all the toxin the microorganism made below 5 ng/mL.It’s much lower than A.flavus standard strain(654.90 ng/mL).Conclusion In the processing of SSP,some A.flavus and A.tamarii with different toxin-producing ability were found,and these strains increased at first and decreased later.This study will lay the foundation for studying the law and mechanism of AFTs content changing in SSP processing.It also proved the importance of'secondary fermentation'and the rationality of'secondary fermentation'time,from the perspective of safety for the first time.

关 键 词：淡豆豉 炮制过程 黄曲霉毒素 产毒能力 超高效液相色谱-串联质谱技术 黄曲霉 溜曲霉 

分 类 号：R283.6[医药卫生—中药学]