摘要
目的明确淡豆豉Sojae Semen Praeparatum炮制中不产毒黄曲霉菌Aspergillus flavus的分布特征及其拮抗能力。方法按实验室前期已建立的规范炮制工艺制备淡豆豉,获取淡豆豉炮制中9个不同时间点的样本,各样本用氯硝胺18%甘油培养基(DG-18)进行培养、分离纯化,经形态学初筛、分子生物学鉴定为黄曲霉菌。通过紫外荧光法初筛和超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)测定黄曲霉菌产毒能力,确定为不产毒黄曲霉菌(简称:不产毒菌)。用平板对峙法检测不产毒菌及其代谢产物对产毒黄曲霉菌标准株(产毒菌)生长的影响。结果从淡豆豉炮制过程中共筛选出21株不产毒菌,其中“黄衣上遍”过程中的第3、6天分别筛选出3、6株,“再闷”过程中的第3、6、9天分别筛选出2、7、3株,再闷第6天筛选到的不产毒菌最多。不产毒菌对产毒菌的生长抑制率在26.75%~36.69%,抑制效果最好的是F6-L8(指第1批在“黄衣上遍”阶段的发酵第6天样品中筛选到的第8株疑似黄曲霉菌),为36.69%,发酵上清液对产毒菌的抑制率在14.29%~43.23%,抑制效果最好的是Z3-X1(指第2批在“再闷”阶段的第3天样品中筛选到的第1株疑似黄曲霉菌),达43.23%。结论淡豆豉炮制过程中存在不产毒黄曲霉菌,且在炮制的不同时间点其数量变化呈现“上升-下降-再上升-再下降”的独特趋势,不产毒菌及其发酵液具有抑制产毒菌生长的作用。为进一步研究不产毒黄曲霉菌在淡豆豉炮制过程中的作用、黄曲霉毒素污染的生物防控提供实验依据。
Objective To clarify the distribution characteristics and antagonistic ability of Aspergillus flavus during the processing of Dandouchi(Sojae Semen Preaparatum,SSP).Methods The fermenting process of SSP was based on the established a standardized fermentation processing technology of own laboratory,and samples were taken at nine different time points during the fermenting process of SSP.A.flavus from each samples at different time points in the fermenting process of SSP were cultured,isolated and purified by clonitramine 18%glycerol medium(DG-18),and were identified by morphological primary screening and molecular biology.The toxin-producing ability of A.flavus was determined by ultraviolet fluorescence method primary screening and ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS),and it was determined that it was a non-toxigenic A.flavus(atoxigenic A.flavus).The effects of atoxigenic A.flavus and their metabolites on the growth of toxigenic A.flavus standard strain(toxigenic A.flavus)was detected by plate confrontation method.Results A total of 21 strains of atoxigenic A.flavus were screened out from the process of SSP,among which three and six strains were screened out on the 3rd and 6th d of the“yellow cladding”stage respectively.During the“secondary fermentation”stage,two,seven,and three strains were screened out on the 3rd,6th and 9th d respectively.The number of atoxigenic A.flavus screened on the 6th d of the“secondary fermentation”stage was the most.The growth inhibition rate of atoxigenic A.flavus to toxigenic A.flavus was between 26.75%and 36.69%,and the best inhibitory effect was F6-L8(referred to the eighth suspected A.flavus screened from the first batch of samples on the 6th d of fermentation in the“yellow cladding”stage),which was 36.69%.The inhibition rate of fermentation supernatant to toxigenic A.flavus was 14.29%—43.23%,and Z3-X1(referred to the first suspected A.flavus screened from the second batch of samples on the 3rd d of the“secondary fermentation”stage)had the best inhibitory effect,reaching 43.23%.Conclusion Atoxigenic A.flavus was found in the processing of SSP and its quantity change showed a unique trend of“rising-decreasing-rising-re-decreasing”at different time points during processing.The atoxigenic A.flavus and its fermentation supernatant have an inhibitory effect on the growth of toxigenic A.flavus.This result provides an experimental basis for further research on the role of atoxigenic A.flavus in the processing of SSP and the biological control of aflatoxin pollution.
作者
李翠英
贺婧
戴家齐
龙凯
王立元
周立分
杨安金
徐佳
翁美芝
谢小梅
LI Cui-ying;HE Jing;DAI Jia-qi;LONG Kai;WANG Li-yuan;ZHOU Li-fen;YANG An-jin;XU Jia;WENG Mei-zhi;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)
出处
《中草药》
CAS
CSCD
北大核心
2023年第6期1775-1782,共8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)
国家自然科学基金项目(82060699)
江西省教育厅科技研究项目(GJJ190634)。
关键词
淡豆豉
黄曲霉毒素
不产毒黄曲霉菌
超高效液相色谱-串联质谱技术
拮抗能力
黄衣上遍
再闷
Sojae Semen Preaparatum(SSP)
aflatoxins
atoxigenic Aspergillus flavus
ultra performance liquid chromatographytandem mass spectrometry
antagonistic ability
yellow cladding
secondary fermentation
