基于主成分分析结合正交设计的百药煎纯菌种发酵炮制工艺研究  

Pure strain fermentation process of Baiyaojian based on principal component analysis combined with orthogonal design

作　　者：曹文文 王瑞生[1,2,3,4] 李柯柯 张振凌 CAO Wenwen;WANG Ruisheng;LI Keke;ZHANG Zhenling(Henan University of Chinese Medicine,Zhengzhou 450008,China;Collaborative Innovation Center of Research and Development on the Whole Industry Chain of Yu Yao,Henan Province,Zhengzhou 450008,China;Henan Engineering Research Center of Chinese Traditional Medicine Special Processing Technology,Zhengzhou 450008,China;Henan Provincial Key Laboratory of Traditional Chinese Medicine Decoction Pieces Processing,Zhengzhou 450008,China;Central Hospital of Yongcheng,Shangqiu 476610,China;Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases co-constructed by Henan Province&Education Ministry of P.R.China,Zhengzhou 450008,China)

机构地区：[1]河南中医药大学,河南郑州450008 [2]豫药全产业链研发河南省协同创新中心,河南郑州450008 [3]河南省中药特色炮制技术工程研究中心,河南郑州450008 [4]河南省中药饮片炮制中医药重点实验室,河南郑州450008 [5]永城市中心医院,河南商丘476610 [6]呼吸疾病中医药防治省部共建协同创新中心,河南郑州450008

出　　处：《中草药》2024年第21期7257-7265,共9页Chinese Traditional and Herbal Drugs

基　　金：河南省级科技研发计划联合基金项目(222301420076);国家重点研发计划(2018YFC1707200);国家公益性行业专项(00104296)。

摘　　要：目的 优化百药煎的纯菌种发酵工艺,以实现百药煎质量稳定、可控,保证临床应用安全、有效,为国家及地方炮制规范提供技术支持。方法 收集根霉曲、宝山酒曲、中药特曲、红薯酒曲和安琪酿酒曲;采用酒曲发酵百药煎,以百药煎中没食子酸含量和外观性状为指标,筛选最佳发酵酒曲;从筛选出的酒曲分离发酵优势菌种,采用显微形态学及分子生物学方法对菌种进行鉴定;采用L_9(3~4)正交设计,以发酵温度、发酵时间、菌种接种量作为考察因素,以百药煎中没食子酸、(-)-表没食子儿茶素、没食子酸甲酯、2,4,6-三-O-没食子酰-α-D-葡萄糖、2,4,6-三-O-没食子酰-β-D-葡萄糖的含量作为考察指标,采用主成分分析(principal component analysis,PCA)法对5种成分进行综合评价,以计算出的总因子得分(F)为综合指标,对百药煎的纯菌种发酵工艺进行优化。结果 5种酒曲发酵百药煎中没食子酸质量分数分别为14.41%、13.73%、9.41%、9.78%、7.16%,根霉曲发酵百药煎表面长有白色菌丝,与传统发酵百药煎性状最接近。从根霉曲中分离出1个菌种,菌丝无隔膜,具分枝和假根,顶端有孢子囊,孢子囊近似球状,孢子呈球形或近似球状;测序结果通过BLAST与NCBI(www.ncbi.nlm.nih.gov)Gen Bank数据库中的菌株序列进行比对,结合传统的形态学鉴定和分子生物学鉴定,最终鉴定结果为米根霉Rhizopus oryzae。正交法优化米根霉发酵百药煎最佳工艺为每五倍子粉末100 g,加绿茶叶10 g,茶叶加15倍量蒸馏水煎煮20 min,煎煮3次,过滤,合并滤液,浓缩约100 mL;将茶渣、适量茶汁与五倍子粉混合均匀制软材,使软材含水量约45%,并于115℃高压蒸汽灭菌锅中灭菌30 min,放凉;接种9%浓度为1×10^(8)个/mL的米根霉孢子混悬液,在温度34℃、湿度85%的条件下发酵时间52 h,取出,烘干。5种成分没食子酸、(-)-表没食子儿茶素、没食子酸甲酯、2,4,6-三-O-没食子�Objective Optimizing the pure strain fermentation process of Baiyaojian to achieve stable and controllable quality of Baiyaojian and ensure safe and effective clinical application,and provide technical support for national and local processing standards.Methods Collecting Rhizopus koji,Baoshan koji,Traditional Chinese Medicine special koji,Sweet Potato koji,and Angel koji.Using the above five types of koji to ferment Baiyaojian,selecting the best fermented koji based on the appearance characteristics and gallic acid content of Baiyaojian.Isolating the dominant fermentation strains from the screened koji,and identifying the strains by using microscopic morphology and molecular biology methods.Using L9(34)orthogonal design,fermentation temperature,fermentation time,and strain inoculation amount were used as factors,the contents of gallic acid,(-)-epigallocatechin,methyl gallate,2,4,6-tria-O-galloyl-α-D-glucose,2,4,6-tria-O-galloyl-β-D-glucose in Baiyaojian were used as the evaluation indicator,and principal component analysis(PCA)was used to comprehensively evaluate these five components,the calculated total factor score(F)was used as the comprehensive indicator to optimize the pure strain fermentation process of Baiyaojian.Results The content of gallic acid in Baiyaojian fermented with five types of koji was 14.41%,13.73%,9.41%,9.78%,and 7.16%,respectively.The Baiyaojian fermented by Rhizopus koji had white hyphae on its surface,which was closest to the traditional fermentation Baiyaojian in terms of properties.One strain was isolated from Rhizopus koji,with no septa in the hyphae but branching and pseudoroots.The top of the hyphae had a sporangium,which was approximately spherical in shape,and the spores were spherical or nearly spherical in shape.The sequencing results were compared with the effectively published strain sequences in the GenBank database through BLAST,combined with traditional morphological and molecular biological identification,and the final identification result was Rhizopus oryzae.The optim

关 键 词：百药煎 米根霉 纯菌种发酵 工艺研究 没食子酸 (-)-表没食子儿茶素 没食子酸甲酯 2 4 6-三-O-没食子酰-α-D-葡萄糖 2 4 6-三-O-没食子酰-β-D-葡萄糖 

分 类 号：R283.6[医药卫生—中药学]