14种易霉变饮片微生物污染状况及其表面真菌多样性研究

Research on Microbial Contamination of 14 Kinds of Mold-prone TCM Decoction Pieces and the Diversity of Fungi on the Surface

目的考察14种易霉变饮片的微生物污染水平,并对其污染真菌进行多样性研究。方法参考2020年版《中国药典》四部通则1108项中药饮片微生物限度检查法,对易霉变饮片的需氧菌总数(TAMC)、霉菌和酵母菌总数(TYMC)、耐热菌总数(HRMC)及3类控制菌进行检查并采用平板法培养样品表面真菌,顶端纯化法获得单一菌株,通过菌落形态及菌体显微特征观察、分子鉴定等方法进行菌种鉴定。结果31批次易霉变饮片TAMC分布范围为250~2.45×10^(6)cfu·g^(-1),TYMC分布范围为5~6.6×10^(5)cfu·g^(-1),HRMC分布范围为0~30 cfu·g^(-1),除1批远志和1批甘草样品外,其余样品耐胆盐革兰阴性菌均未超过1×10^(4)cfu·g^(-1),1批陈皮样品中检出大肠埃希菌,所有样品均未检出沙门菌,但控制菌检查项中检出重要条件致病菌,典型菌包括阴沟肠杆菌、肺炎克雷伯菌和阪崎克罗诺杆菌等。通过内转录间隔区(ITS)序列测序比对共鉴定106株真菌,归为16个属,其中曲霉属(Aspergillus)、青霉属(Penicillium)和枝孢属(Cladosporium)为优势菌属,分别占比31%、18%、17%。曲霉属中,鉴定出11株黑曲霉、7株黄曲霉、2株烟曲霉、1株杂色曲霉等致病性真菌。结论通过对易霉变饮片微生物污染及表面真菌多样性研究,可为其潜在的真菌毒素污染提供早期风险预警。

OBJECTIVE To evaluate the microbial contamination levels of 14 kinds of mold-prone TCM decoction pieces and the diversity of fungi on the surface.METHODS The total aerobic microbial count(TAMC),total yeasts and molds count(TYMC),heat-resistant microbial count(HRMC),and three types of control bacteria of mold-prone TCM decoction pieces were analyzed by referring the Chinese Pharmacopoeia 2020 Edition VolumeⅣ1108 microbiological limit test method.Furthermore,the fungi on the surface were cultivated using the plate method,and the single strain was isolated by the top purification method,which was further identified by colony morphology and molecular identification.RESULTS The results showed that TAMC of sample ranged 250-2.45×10^(6) cfu·g^(-1),TYMC ranged 5-6.6×10^(5) cfu·g^(-1),and HRMC ranged 0-30 cfu·g^(-1).The remaining samples of cholesterol-resistant progestin-negative bacteria did not reach 1×10^(4) cfu·g^(-1),with the exception of one batch of Polygalae Radix and one batch of Glycyrrhizae Radix et Rhizoma.Although only one batch of Citri Reticulatae Pericarpium tested Escherichia coli and no Salmonella was detected in all samples,significant conditional pathogenic bacteria such as Enterobacter cloacae,Klebsiella pneumoniae,and Cronobacter sakazakii were found in the control bacterial examination items.ITS sequence sequencing comparison identified 106 strains in total,grouped into 16 genera,with Aspergillus,Penicillium,and Cladosporium accounting for 31%,18%,and 17%,respectively,of the major bacteria.Among the genus Aspergillus,11 strains of A.niger,7 strains of A.flavus,2 strains of A.fumigatus,and 1 strain of A.miscellaneous were isolated as pathogenic fungi.CONCLUSION It can provide early risk warning for its potential fungi toxins contamination through research on microbial pollution and surface fungi diversity.