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Article(id=1248601550881448468, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1248601546695528820, articleNumber=1001-2494(2024)07-0571-08, orderNo=null, doi=10.11669/cpj.2024.07.002, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1689609600000, receivedDateStr=2023-07-18, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1775619407624, onlineDateStr=2026-04-08, pubDate=1712505600000, pubDateStr=2024-04-08, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1775619407624, onlineIssueDateStr=2026-04-08, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1775619407624, creator=13701087609, updateTime=1775619407624, updator=13701087609, issue=Issue{id=1248601546695528820, tenantId=1146029695717560320, journalId=1190317699101192196, year='2024', volume='59', issue='7', pageStart='561', pageEnd='656', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1775619406627, creator=13701087609, updateTime=1775619979013, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1248603947515142525, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1248601546695528820, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1248603947515142526, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1248601546695528820, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=571, endPage=578, ext={EN=ArticleExt(id=1248601551355404825, articleId=1248601550881448468, tenantId=1146029695717560320, journalId=1190317699101192196, language=EN, title=Research on Microbial Contamination of 14 Kinds of Mold-prone TCM Decoction Pieces and the Diversity of Fungi on the Surface, columnId=null, journalTitle=Chinese Pharmaceutical Journal, columnName=null, runingTitle=null, highlight=null, articleAbstract=

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 Volume Ⅳ 1108 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 250-2.45×106 cfu·g-1, TYMC ranged 5-6.6×105 cfu·g-1, and HRMC ranged 0-30 cfu·g-1. The remaining samples of cholesterol-resistant progestin-negative bacteria did not reach 1×104 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.

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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250~2.45×106 cfu·g-1,TYMC分布范围为5~6.6×105 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1×104 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。

, correspAuthors=刘绪平, 刘卫德, authorNote=null, correspAuthorsNote=
*刘绪平,男,主任药师,硕士生导师 研究方向:药品微生物与质量控制研究 Tel:(0791)88158128；
刘卫德,男,主管药师 研究方向:药品质量和标准研究 Tel:(0791)88158796
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王燕,女,硕士研究生 研究方向:中药质量控制

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王燕,女,硕士研究生 研究方向:中药质量控制

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王燕,女,硕士研究生 研究方向:中药质量控制

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Microbiol China(微生物学通报), 2017, 44(5):1089-1098., articleTitle=Community structure analysis of fungi isolated from citrus surfaces, refAbstract=null), Reference(id=1249073378011849705, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, doi=null, pmid=null, pmcid=null, year=2021, volume=72, issue=4, pageStart=112, pageEnd=121, url=null, language=null, rfNumber=[31], rfOrder=30, authorNames=GUADALUPE VILLA-MARTINEZ B, PEREZ-CRUZ C, DOLORES PEREZ-SANTIAGO A, journalName=J Food Saf Food Qual, refType=null, unstructuredReference=GUADALUPE VILLA-MARTINEZ B, PEREZ-CRUZ C, DOLORES PEREZ-SANTIAGO A, et al. Aflatoxins: Latent danger in the daily diet[J]. J Food Saf Food Qual, 2021, 72(4):112-121., articleTitle=Aflatoxins: Latent danger in the daily diet, refAbstract=null), Reference(id=1249073378104124396, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, doi=null, pmid=null, pmcid=null, year=2017, volume=15, issue=11, pageStart=661, pageEnd=674, url=null, language=null, rfNumber=[32], rfOrder=31, authorNames=VAN DE VEERDONK F L, GRESNIGT M S, ROMANI L, journalName=Nat Rev Microbiol, refType=null, unstructuredReference=VAN DE VEERDONK F L, GRESNIGT M S, ROMANI L, et al. Aspergillus fumigatus morphology and dynamic host interactions[J]. Nat Rev Microbiol, 2017, 15(11):661-674., articleTitle=et al. 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Chin J Pharm Anal(药物分析杂志), 2019, 39(11):1945-1953., articleTitle=Microbial community composition analysis of six Chinese herbal pieces through 16S rRNA high-throughput sequencing, refAbstract=null), Reference(id=1249073378527749113, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, doi=null, pmid=null, pmcid=null, year=2022, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[35], rfOrder=34, authorNames=YU J S H, journalName=Fungal identification on the surface of Chinese herbal materials through high throughput sequencing-Case studies of six herbal materials, refType=null, unstructuredReference=YU J S H. Fungal identification on the surface of Chinese herbal materials through high throughput sequencing-Case studies of six herbal materials[D]. Beijing: Peking Union Medical College, 2022., articleTitle=null, refAbstract=null), Reference(id=1249073378632606715, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, doi=null, pmid=null, pmcid=null, year=2023, volume=58, issue=24, pageStart=2282, pageEnd=2293, url=null, language=null, rfNumber=[36], rfOrder=35, authorNames=SUN Y, GUO X P, XU Q K, journalName=Chin Pharm J, refType=null, unstructuredReference=SUN Y, GUO X P, XU Q K, et al. Community characteristics of contaminated microorganisms in six kinds of Gansu authentic Chinese herbal pieces based on high-throughput sequencing[J]. Chin Pharm J (中国药学杂志), 2023, 58(24):2282-2293., articleTitle=Community characteristics of contaminated microorganisms in six kinds of Gansu authentic Chinese herbal pieces based on high-throughput sequencing, refAbstract=null)], funds=[Fund(id=1249073373121291129, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, awardId=81960715, language=CN, fundingSource=国家自然科学基金项目资助(81960715), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1249073364514579139, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, xref=1, ext=[AuthorCompanyExt(id=1249073364518773443, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, companyId=1249073364514579139, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 Jiangxi University of Chinese Medicine, Nanchang 330004, China), AuthorCompanyExt(id=1249073364527162052, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, companyId=1249073364514579139, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 江西中医药大学, 南昌 330004)]), AuthorCompany(id=1249073364678157002, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, xref=2, ext=[AuthorCompanyExt(id=1249073364682351307, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, companyId=1249073364678157002, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 Jiangxi Institute for Drug Control, NMPA Key Laboratory of Quality Evaluation of Traditional Chinese Patent Medicine, Jiangxi Province Engineering Research Center of Drug and Medical Device Quality, Nanchang 330029, China), AuthorCompanyExt(id=1249073364690739916, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, companyId=1249073364678157002, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 江西省药品检验检测研究院, 国家药品监督管理局中成药质量评价重点实验室, 江西省药品与医疗器械质量工程技术研究中心, 南昌 330029)])], figs=[ArticleFig(id=1249073372064326471, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, language=EN, label=Fig.1, caption=Microbial quantity of 14 kinds of mold-prone TCM decoction pieces

TAMC-Total aerobic microbial count;TYMC-Total yeasts and molds count;HRMC-Heat-resistant microbial count;YZT-Polygalae Radix; BJT-Morindae Officinalis Radix; YZ-Polygonati Odorati Rhizoma; DH-Angelicae Pubescentis Radix; HZ-Polygoni Cuspidati Rhizoma et Radix; GC-Glycyrrhizae Radix et Rhizoma; PDH-Sterculiae Lychnophorae Semen; BL-Arecae Semen; YR-Coicis Semen; CP-Citri Reticulatae Pericarpium; JXT-Spatholobi Caulis; DQY-Isatidis Folium; QX-Scorpio; MH-Mume Flos.

, figureFileSmall=QzIGcGKR8JdM8f1/IGx+wg==, figureFileBig=icH5dYWlKPLMrSuxaf3ISQ==, tableContent=null), ArticleFig(id=1249073372156601162, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, language=CN, label=图1, caption=14种易霉变中药饮片微生物污染载量计数结果

TAMC-需氧菌总数;TYMC-霉菌和酵母菌总数;HRMC-耐热菌总数;YZT-远志;BJT-巴戟天;YZ-玉竹;DH-独活;HZ-虎杖;GC-甘草;PDH-胖大海;BL-槟榔;YR-薏苡仁;CP-陈皮;JXT-鸡血藤;DQY-大青叶;QX-全蝎;MH-梅花。

, figureFileSmall=QzIGcGKR8JdM8f1/IGx+wg==, figureFileBig=icH5dYWlKPLMrSuxaf3ISQ==, tableContent=null), ArticleFig(id=1249073372290818896, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, language=EN, label=Fig.2, caption=Microbial quantity of 6 types of mold-prone TCM decoction pieces

TAMC-Total aerobic microbial count; TYMC-Total yeasts and molds count; HRMC-Heat-resistant microbial count.

, figureFileSmall=tLy1Y3y3t7g/SXgi4oZo1Q==, figureFileBig=bIjv8fkgMCzQzPsHIL3Rvw==, tableContent=null), ArticleFig(id=1249073372357927765, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, language=CN, label=图2, caption=6类中药饮片微生物污染载量计数结果

TAMC-需氧菌总数;TYMC-霉菌和酵母菌总数;HRMC-耐热菌总数。

, figureFileSmall=tLy1Y3y3t7g/SXgi4oZo1Q==, figureFileBig=bIjv8fkgMCzQzPsHIL3Rvw==, tableContent=null), ArticleFig(id=1249073372429230936, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, language=EN, label=Fig.3, caption=Morphological and microscopic characteristics of surface fungi colonies of 14 kinds of mold-prone TCM decoction pieces (Part)

A1,A2-Mucor circinelloides; B1,B2-Aspergillus flavus; C1,C2-Aspergillus fumigatus; D1,D2-Aspergillus pseudoglaucus.

, figureFileSmall=16WSc2iQiRTwz9d0hwGesw==, figureFileBig=77c2uVwDrNCRLYAOeFqmUg==, tableContent=null), ArticleFig(id=1249073372504728412, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, language=CN, label=图3, caption=14种易霉变饮片表面真菌的菌落形态与显微特征图(部分)

A1,A2-卷枝毛霉;B1,B2-黄曲霉;C1,C2-烟曲霉;D1,D2-假灰绿曲霉。

, figureFileSmall=16WSc2iQiRTwz9d0hwGesw==, figureFileBig=77c2uVwDrNCRLYAOeFqmUg==, tableContent=null), ArticleFig(id=1249073372601197406, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, language=EN, label=Fig.4, caption=Construction of phylogenetic tree of surface fungi based on ITS sequences by NJ method

Note:The branch colors indicate the genus, and the circle colors indicate its corresponding family.

, figureFileSmall=K9I3O+7VGFIN3P5WDjEfkA==, figureFileBig=2olFTgnWRJpAl+FKco7GeA==, tableContent=null), ArticleFig(id=1249073372689277795, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, language=CN, label=图4, caption=基于内转录间隔区(ITS)序列运用邻接法(NJ法)构建的系统发育树

注:分支颜色表示属,圈层颜色表示其对应的科。

, figureFileSmall=K9I3O+7VGFIN3P5WDjEfkA==, figureFileBig=2olFTgnWRJpAl+FKco7GeA==, tableContent=null), ArticleFig(id=1249073372756386664, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, language=EN, label=Tab.1, caption=

Sample information of 14 kinds of mold-prone TCM decoction pieces

, figureFileSmall=null, figureFileBig=null, tableContent=
Samples Number Medicinal parts Sources(in Chinese)
Polygalae Radix YZT1 Roots Shanxi(山西)
Morindae Officinalis Radix BJT1 Roots Guangdong(广东)
Polygonati Odorati Rhizoma YZ1-3 Rhizomes Hunan(湖南)
Angelicae Pubescentis Radix DH1-3 Roots Hubei(湖北)
Polygoni Cuspidati Rhizoma et Radix HZ1 Roots and rhizomes Jiangxi(江西)
Glycyrrhizae Radix et Rhizoma GC1-3 Roots and rhizomes Inner Mongolia(内蒙古)
Sterculiae Lychnophorae Semen PDH1 Seeds Guangxi(广西)
Arecae Semen BL1-6 Seeds Hainan(海南)
Coicis Semen YR1-5 Seeds Guizhou、Yunnan(贵州、云南)
Citri Reticulatae Pericarpium CP1 Peels Jiangxi(江西)
Spatholobi Caulis JXT1 Stems Guangxi(广西)
Isatidis Folium DQY1-3 Leaves Jiangsu(江苏)
Scorpio QX1 Whole worm Shandong(山东)
Mume Flos MH1 Buds Anhui(安徽)
), ArticleFig(id=1249073372852855664, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1248601550881448468, language=CN, label=表1, caption=

14种易霉变中药饮片样品信息

, figureFileSmall=null, figureFileBig=null, tableContent=
Samples Number Medicinal parts Sources(in Chinese)
Polygalae Radix YZT1 Roots Shanxi(山西)
Morindae Officinalis Radix BJT1 Roots Guangdong(广东)
Polygonati Odorati Rhizoma YZ1-3 Rhizomes Hunan(湖南)
Angelicae Pubescentis Radix DH1-3 Roots Hubei(湖北)
Polygoni Cuspidati Rhizoma et Radix HZ1 Roots and rhizomes Jiangxi(江西)
Glycyrrhizae Radix et Rhizoma GC1-3 Roots and rhizomes Inner Mongolia(内蒙古)
Sterculiae Lychnophorae Semen PDH1 Seeds Guangxi(广西)
Arecae Semen BL1-6 Seeds Hainan(海南)
Coicis Semen YR1-5 Seeds Guizhou、Yunnan(贵州、云南)
Citri Reticulatae Pericarpium CP1 Peels Jiangxi(江西)
Spatholobi Caulis JXT1 Stems Guangxi(广西)
Isatidis Folium DQY1-3 Leaves Jiangsu(江苏)
Scorpio QX1 Whole worm Shandong(山东)
Mume Flos MH1 Buds Anhui(安徽)
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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
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王燕 1 , 章红 2 , 段和祥 2 , 刘绪平 1, 2, * , 刘卫德 2, *
中国药学杂志 | 论著 2024,59(7): 571-578
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中国药学杂志 | 论著 2024, 59(7): 571-578
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
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王燕1, 章红2, 段和祥2, 刘绪平1, 2, *, 刘卫德2, *
作者信息
  • 1 江西中医药大学, 南昌 330004
  • 2 江西省药品检验检测研究院, 国家药品监督管理局中成药质量评价重点实验室, 江西省药品与医疗器械质量工程技术研究中心, 南昌 330029

    • 王燕,女,硕士研究生 研究方向:中药质量控制

通讯作者:

*刘绪平,男,主任药师,硕士生导师 研究方向:药品微生物与质量控制研究 Tel:(0791)88158128；
刘卫德,男,主管药师 研究方向:药品质量和标准研究 Tel:(0791)88158796
Research on Microbial Contamination of 14 Kinds of Mold-prone TCM Decoction Pieces and the Diversity of Fungi on the Surface
Yan WANG1, Hong ZHANG2, Hexiang DUAN2, Xuping LIU1, 2, *, Weide LIU2, *
Affiliations
  • 1 Jiangxi University of Chinese Medicine, Nanchang 330004, China
  • 2 Jiangxi Institute for Drug Control, NMPA Key Laboratory of Quality Evaluation of Traditional Chinese Patent Medicine, Jiangxi Province Engineering Research Center of Drug and Medical Device Quality, Nanchang 330029, China
出版时间: 2024-04-08 doi: 10.11669/cpj.2024.07.002
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摘要
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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250~2.45×106 cfu·g-1,TYMC分布范围为5~6.6×105 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1×104 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。

易霉变饮片  /  微生物污染  /  分子鉴定  /  真菌多样性

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 Volume Ⅳ 1108 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 250-2.45×106 cfu·g-1, TYMC ranged 5-6.6×105 cfu·g-1, and HRMC ranged 0-30 cfu·g-1. The remaining samples of cholesterol-resistant progestin-negative bacteria did not reach 1×104 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.

mold-prone TCM decoction piece  /  microbe contamination  /  molecular identification  /  fungal diversity
王燕, 章红, 段和祥, 刘绪平, 刘卫德. 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. 中国药学杂志, 2024 , 59 (7) : 571 -578 . DOI: 10.11669/cpj.2024.07.002
Yan WANG, Hong ZHANG, Hexiang DUAN, Xuping LIU, Weide LIU. Research on Microbial Contamination of 14 Kinds of Mold-prone TCM Decoction Pieces and the Diversity of Fungi on the Surface[J]. Chinese Pharmaceutical Journal, 2024 , 59 (7) : 571 -578 . DOI: 10.11669/cpj.2024.07.002
正文
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中药饮片来源于天然植物、动物或矿物,除去自身携带的微生物以外,在采收、炮制、运输及贮藏过程中也极易受到外源性微生物侵染[1]。其中饮片霉变问题尤为突出[2-5],是饮片储藏、养护过程最易出现的难题之一。特别是我国南方地区多高温、高湿天气,更容易受到霉菌侵染从而引发中药饮片霉变。饮片一旦发生霉变,不仅会降低其有效成分、降低疗效或失效[6-7],更为严重的是产生霉菌毒素,给患者带来潜在的严重风险。Meng等[8]对地龙饮片表面真菌的多样性研究,分离到黄曲霉等9种真菌,Zhang等[9]从龟甲饮片表面分离到烟曲霉等10种真菌,真菌特别是产毒真菌的普遍存在严重威胁中药饮片质量[3-4]。此外,中药饮片受微生物污染的报道亦屡见不鲜[10],检出率较高的是耐热菌[11]和耐胆盐革兰阴性菌[12],二者均有致病报道。目前笔者尚未见有学者专门针对易霉变饮片进行表面真菌研究,本研究以14种临床常用、具有代表性(包括动植物药、不同用药部位、质地等)、2020年版《中国药典》[13]要求检测黄曲霉毒素或在“贮存”项下标注“防霉”的饮片作为研究对象,初步掌握了其微生物污染负载水平。在此基础上,对其表面污染真菌进行鉴定和研究,为中药饮片微生物污染风险评估和微生物标准的制定提供数据支撑,对易霉变饮片贮藏安全、使用安全有着重要意义。
1 材料
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1.1 样品
从14种共计31批中药饮片,见表1,市售。经江西中医药大学谌瑞林主任中药师鉴定为正品。所有样品置于阴凉干燥处储存,直至检测前,饮片表面均未观察到肉眼可见的霉变现象。
1.2 主要仪器与试剂
生物安全柜(AB2-5S1型,山东博科生物产业有限公司)、光学显微镜(德国徕卡仪器有限公司)、全自动微生物快速检测分析系统(VITEK 2 Compact,法国梅里埃公司)、定性PCR仪(C1000型)、基础型电泳仪(PowerPac Basic)、凝胶成像仪(ChemiDoc MP)(美国BIO-RAD公司)。
硫酸庆大霉素注射液(华中药业股份有限公司);乳酸酚棉蓝染色液(青岛高科技工业园海博生物技术有限公司);真菌基因组DNA提取试剂盒(北京索莱宝科技有限公司);Taq PCR Master Mix(2×,blue dye)(BBI生命科学有限公司);100 bp DNA Marker、50×TAE缓冲液、琼脂糖(上海生工生物工程股份有限公司);GoldView核酸染色剂(上海玉博生物科技有限公司)。
1.3 培养基
沙氏葡萄糖琼脂(SDA)培养基、胰酪大豆胨琼脂(TSA)培养基、胰酪大豆胨液体(TSB)培养基、pH 7.0氯化钠蛋白胨缓冲液、肠道增菌肉汤培养基、紫红胆盐葡萄糖琼脂培养基、麦康凯液体培养基、麦康凯琼脂培养基、沙门菌增菌肉汤(RV)培养基、木糖赖氨酸脱氧胆酸盐琼脂培养基(北京陆桥技术有限责任公司)。
1.4 标准菌株
金黄色葡萄球菌(Staphylococcus aureus)[CMCC(B)26003]、铜绿假单胞菌 (Pseudomonas aeruginosa)[CMCC(B)10104]、枯草芽孢杆菌(Bacilllus subtilis)[CMCC(B)63501]、白色念珠菌(Candida aibicans)[CMCC(F)98001]、大肠埃希菌(Escherichia coli)[CMCC(B)44102]、乙型副伤寒沙门菌(Salmonella paratyphi B)[CMCC(B)50094]、黑曲霉(Aspergillus niger)[CMCC(F)98003]均来自中国食品药品检定研究院。
1.5 PCR引物
真菌鉴定通用引物:ITS1(TCCGTAGGTGAACCTGCG)、ITS4 (TCCTCCGCTTATTGATATGC)(上海生工生物工程股份有限公司)。
2 方法
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2.1 菌液制备
按2020年版《中国药典》四部通则1105、1106项,制备金黄色葡萄球菌、铜绿假单胞菌、枯草芽孢杆菌、白色念珠菌、大肠埃希菌和沙门菌的菌悬液以及黑曲霉的孢子悬液。
2.2 供试液的制备
取供试品25 g(由于大青叶、全蝎和梅花质量较轻,为保证分散均匀,称取样品10 g置90 mL pH 7.0氯化钠蛋白胨缓冲液),置225 mL pH 7.0氯化钠蛋白胨缓冲液中制成1∶10供试液,充分振摇荡洗15 min,将供试液用pH 7.0氯化钠蛋白胨缓冲液做10倍系列稀释。
取上述1∶10供试液适量,置沸水浴30 min处理后迅速冷却,作为耐热菌点数(HRMC)测定用供试液。
2.3 方法适用性试验
由于样品中的污染微生物过多,为保证验证试验结果的准确性,取“2.2”项下供试液(1∶10)置沸水浴30 min后,照2020年版《中国药典》四部通则1108中药饮片微生物限度检查法,以常规法进行需氧菌总数(TAMC)、霉菌和酵母菌总数(TYMC)、HRMC、耐胆盐革兰阴性菌、大肠埃希菌、沙门菌的微生物方法适用性试验。
2.4 供试品微生物限度检查
2.4.1 需氧菌、霉菌和酵母菌、耐热菌检查
经“2.3”项下确认的方法进行试验。
2.4.2 控制菌检查
经“2.3”项下确认的方法进行试验。
2.4.3 可疑菌株鉴定
对“2.4.2”项下控制菌检查得到的污染菌株进行分离纯化,采用VITEK全自动微生物快速检测分析系统进行鉴定。
2.5 表面污染真菌的分离与纯化
将“2.4.1”项下TYMC测定中SDA培养基(含庆大霉素0.1 g·L-1)中生长的真菌,根据菌落形态的不同,采用菌丝顶端纯化法获得纯化的真菌,接种到新的SDA培养基(含庆大霉素0.1 g·L-1)中,每个培养基接种3个点,23 ℃避光培养5 d。获得单一菌株,用于观察菌体在培养基中菌落形态与生长情况。待纯化后转移到SDA斜面试管中培养,用于菌种鉴定。
2.6 表观鉴定
取单一菌落,观察菌落的形态、颜色、质地、直径等。在载玻片上滴加2滴乳酸酚棉蓝染色液,使用镊子夹取少许菌丝于载玻片,使分散均匀,制片,放置于光学显微镜下放大40倍,观察真菌菌丝特点、颜色、孢子结构特征、足细胞等,结合真菌的菌落形态与显微特征,依据《真菌鉴定手册》[14]对其进行鉴定。
2.7 分子鉴定
2.7.1 真菌DNA的提取
根据真菌基因组DNA提取试剂盒说明书提取各菌株DNA。
2.7.2 ITS扩增与测序
使用真菌鉴定通用引物ITS1和ITS4以“2.7.1”项下的DNA溶液为模板进行PCR扩增,PCR扩增反应体系:总体积30 μL,Taq PCR Master Mix 15 μL,ITS1和ITS4引物各1 μL,DNA Template 2 μL,纯化水11 μL。ITS-PCR扩增程序:94 ℃预变性5 min;94 ℃变性45 s,54 ℃退火45 s,72 ℃延伸1 min,35个循环;最后延伸10 min。将PCR扩增产物以质量分数1%琼脂糖凝胶电泳分离后,选择条带单一清晰,大小正确(约500 bp)的样品送到上海生工生物工程股份有限公司测序。测序结果进行拼接后在美国国家生物技术信息中心(NCBI)数据库中进行BLAST比对,找出与获得片段同源性最高的真菌菌株的参考序列,以确定其种属。
2.8 数据处理
本研究数据采用Origin 2023软件绘图。利用NCBI将测试结果进行BLAST比对分析,查找相似度最高的菌种,用MEGA 11.0软件按邻接法(NJ法)构建系统发育树。
3 结果
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3.1 微生物限度检查法的建立
在常规法进行微生物计数法适用性试验中,金黄色葡萄球菌、铜绿假单胞菌、枯草芽孢杆菌、白色念珠菌和黑曲霉的回收率均在0.5~2.0内,耐胆盐革兰阴性菌、大肠埃希菌、沙门菌的阳性对照组均能检出,阴性对照组均无菌生长,符合药典相关规定,说明常规法能够用于本研究中14种易霉变饮片的微生物限度及控制菌检查。
3.2 微生物限度检查
本研究收集的14种易霉变饮片微生物载量见图1(微生物负载水平以测定结果的对数值表示,需氧菌、霉菌和酵母菌、耐热菌检测1∶10无菌生长则赋值0.5)。结果显示,各样品TAMC分布范围为250~2.45×106 cfu·g-1,其中,远志TAMC是所有饮片中最高的,污染量大于1×106 cfu·g-1。全蝎TAMC低于其他饮片品种,污染量为1×102 cfu·g-1。各样品TYMC分布范围为5~6.6×105 cfu·g-1。其中,远志TYMC亦高于其他样品,污染量大于1×105 cfu·g-1。各样品HRMC分布范围为0~30 cfu·g-1,其中,陈皮HRMC高于其他饮片。
参照2020年版《中国药典》四部直接口服及泡腾饮片微生物限度标准,1批远志和1批槟榔TAMC高于限量标准1×105 cfu·g-1,占6.5%。远志、玉竹、甘草、槟榔和陈皮各有1批样品TYMC高于限量标准1×103 cfu·g-1,占16.1%。
共有21批检出耐胆盐革兰阴性菌,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1×104 cfu·g-1。除1批陈皮样品外,其余样品均未检出大肠埃希菌。所有样品均未检出沙门菌。有6批饮片在控制菌检查中发现可疑菌落,经鉴定主要为以下6类菌:肺炎克雷伯菌Klebsiella pneumoniae(GC-1、BL-1、PDH-1、YZT-1)、阪崎克罗诺肠杆菌Cronobacter sakazakii(GC-2、BL-1)、阴沟肠杆菌Enterobacter cloacae(GC-1)、泛菌属Pantoea spp(YR-4)、蜡样芽孢杆菌Bacillus cereus(BL-1)和考氏科萨克氏菌Kosakonia cowanii(YR-4)。其中,前4类菌为寄生于呼吸道或肠道的革兰阴性条件致病菌,蜡样芽孢杆菌为革兰阳性条件致病菌[15]。当此类条件致病菌集聚部位改变、菌群失调或机体抵抗力降低等特定条件下具有不同程度的致病风险,虽然现行药典中未对上述条件致病菌进行限制性规定,但其危害性不容忽视。
根据不同药用部位将14种饮片分为6类,其微生物载量见图2。根茎类饮片TYMC明显高于其他5类饮片,其次是果实种子类,推断可能是由于根茎类药材长期接触土壤,导致原药材中霉菌载量相对较高,而果实种子类饮片营养通常较丰富,易出现霉菌滋生。除动物类饮片微生物载量较小外,其余种类饮片TAMC均值差异不明显。对于HRMC,6类饮片污染量均相对较低。
3.3 表面污染真菌的分离与纯化
根据不同的菌落形态特征,“2.6”项下分离纯化得到106株真菌,部分菌株菌落形态与显微特征图见图3。从独活、甘草、槟榔、薏苡仁及大青叶饮片中分离得到10株以上真菌,其余饮片分离得到的菌株2~7株不等,污染率达93.3%,表明选取的14种易霉变饮片表面真菌污染情况较为普遍。
3.4 表面污染真菌鉴定
将“2.8”项下得到的106株真菌测序结果进行BLAST比对分析,可归为2门5纲9目12科16属,所有菌株ITS序列与数据库中收载同源序列最大相似性均达98%以上。在纲分类水平上,优势菌群分别为散囊菌纲(Eurotiomycetes)、座囊菌纲(Dothideomycetes)、粪壳菌纲(Sordariomycetes)和毛霉纲(Mucoromycetes),分别占比52%、19%、13%、13%;在科分类水平上,优势菌群分别为曲霉科(Aspergillaceae)、枝孢菌科(Cladosporiaceae)、根霉科(Rhizopodaceae)和毛霉科(Mucoraceae),分别占比52%、17%、7%、5%;在属分类水平上,曲霉属(Aspergillus)、青霉属(Penicillium)和枝孢属(Cladosporium),分别占比31%、18%、17%。采用NJ法构建系统发育树见图4,图中可见,所有菌株可聚类至12科16属,并且均与NCBI数据库中BLAST比对获得的同源性最高已知序列处于同一簇,这进一步验证了前述BLAST比对分类鉴定结果。
在远志、陈皮、甘草、薏苡仁、梅花饮片中均分离到黄曲霉,而薏苡仁饮片中还同时分离到杂色曲霉、烟曲霉和镰刀菌产毒菌株,提示上述饮片可能有真菌毒素污染的风险,后期将重点针对分离出产毒菌株的饮片开展真菌毒素研究。另外值得注意的是,本研究中分离到多株植物病原真菌[16],包括:胖大海、甘草、独活、玉竹中均分离到少根根霉(根腐病、软腐病);薏苡仁中分离到镰刀菌(镰孢茎腐病、枯萎病);大青叶中分离到稻黑孢(稻褐纹病、黑孢菌穗腐病、叶尖枯病)。而上述植物病原真菌一般在植物生长和成熟过程中增殖,被认为是田间真菌,说明中药材在生长过程中易受到植物病原真菌侵染,而后续炮制加工处理未能去除该类真菌导致残留,提示我们不仅要注重人工种植过程中的植物病害防治,同时也要采取有效手段降低此类真菌及其他有害微生物的负载水平,如控制炮制工艺参数、采用适宜的灭菌技术[17]等。
4 结论与讨论
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本研究结果表明,中药饮片微生物污染普遍存在,与饮片品种、药用部位、种植采收、炮制工艺及产地气候等诸多因素存在一定程度的关系。本研究收集的31批次中药饮片均检测到大量微生物,这间接说明易霉变饮片的微生物控制措施还需进一步提高。其中微生物污染量最高的远志饮片为“远志筒”,其TAMC、TYMC和耐胆盐革兰阴性菌均超过2020年版《中国药典》对中药口服及泡服饮片的微生物限度标准,并分离到肺炎克雷伯菌和黄曲霉等条件致病菌,提示该饮片微生物污染严重,风险较大。而根据本研究结果,甘草、玉竹等饮片的TYMC载量亦较高,究其原因可能是此类根及根茎类饮片药用部位直接与土壤接触[18],且表面大多具有较密并深陷的横纵皱纹和裂纹,易导致各类微生物残留而发生霉变。因此,对于根及根茎类易霉变饮片,应加强药材采收净制过程中表面泥土及微生物的去除,必要时进行适当的灭菌处理。此外,本研究中微生物污染较为严重的还有1批陈皮饮片,其TYMC超出药典限量标准,且检出大肠埃希菌、黄曲霉和黑曲霉等致病菌或条件致病菌,本身含水分、糖分高于其他饮片,为霉菌等微生物滋生提供必要养分而导致其负载水平偏高。胖大海、槟榔均产于温暖潮湿的南方地区,适宜的温湿度为霉菌等微生物生长繁殖提供了有利条件。虽然饮片表面均未观察到肉眼可见的霉变现象,但此类以果实种子入药的饮片,内部种子团或种仁部位由于水分“分布表里不一”而易发生局部霉变。因此,控制饮片的水分含量是避免其霉变的关键因素之一。大青叶入药部位为干燥叶,因其表面积较大,多皱缩卷曲,易附着粉尘而滋生微生物。梅花需氧菌负载水平较高,原因可能是其仅采用净制的方法进行简单炮制,难以去除表面附着的微生物,可导致其残留、滋生[19]。动物类饮片含有丰富蛋白质,可为真菌生长提供营养,容易导致饮片霉变。但本研究收集的全蝎采用清水煮法炮制而来,经沸水后不仅能使毒性蛋白变性而降低毒性,同时也能杀灭绝大多数饮片携带的微生物,因此与其他饮片相比,全蝎微生物负载量较低,各项微生物指标均在规定限度内。由此可见,炮制工艺对微生物污染量存在一定影响。综上,影响饮片微生物污染的因素复杂多样,应对易霉变品种积极采取预防措施,通过科学种植管理、优化采收及炮制工艺参数、控制水分含量、改善储藏条件、建立科学的监控体系等,从源头降低中药饮片微生物污染风险,从而有效减少下游产品微生物负载量。
大量研究从中药饮片中多次检出重要条件致病菌,主要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[20-22]。本研究中也检出上述菌株。阴沟肠杆菌是医院感染的主要病原菌,通过胃肠道和皮肤接触入侵机体导致感染,可导致新生儿脑膜炎、呼吸道和尿路感染等[23]。阪崎克罗诺杆菌属于A类致病菌,是婴幼儿奶粉中的检验控制菌,易引发坏死性小肠结肠炎、败血症和脑膜炎等[24]。研究表明,该菌可能是饮片加工过程中由生产设备或环境或水体污染所致[21]。肺炎克雷伯菌是少数能够引起原发性肺炎的革兰阴性杆菌之一[25],易入侵免疫功能低下的人群,严重可导致肺炎、血流感染、尿路感染、软组织疾病和自发性细菌性腹膜炎等[26]。该菌是医院感染的主要致病菌之一[27],在世界范围内具有极强的患病率和高死亡率[28]。此外,本研究中16批样品检出耐热菌,而中药饮片主要通过简单煎煮制成汤剂服用,尤其是近年来流行的药食两用中药还可作养生茶直接泡服饮用(如本研究中的甘草、胖大海、陈皮等饮片),并不能彻底杀灭具有耐热性的致病菌,这对患者用药安全存在一定的风险隐患[29]。但现行药典中尚未对上述重要条件致病菌和耐热菌做限制要求,因此,建议进一步开展中药饮片微生物危害的风险评估工作,完善其相关标准的制定。
本研究通过菌落形态及菌体显微特征观察、分子鉴定等方法共鉴定真菌106株,可归为16个属,其中曲霉属、青霉属是易霉变饮片中分离真菌的共有优势菌属,这与Zhang等[9]人的研究结果一致。有文献报道,陈皮表面污染真菌黑曲霉代谢转化能够促进陈皮中黄酮类药效成分的增加,从而阐明陈皮“陈久者良”的科学内涵[30]。由此可见,中药表面真菌与其药效之间有一定关系。但部分污染真菌在特定环境下会代谢产生危害人体健康的真菌毒素,例如黄曲霉、杂色曲霉、烟曲霉和镰刀菌等。黄曲霉次级代谢产生的黄曲霉毒素具有严重肝毒性,甚至产生致畸、致癌和致突变等危害,被列为Ⅰ级致癌物[31]。杂色曲霉次级代谢产生的杂色曲霉毒素与黄曲霉毒素B1的化学结构极其相似,其毒性仅次于黄曲霉毒素,被国际癌症研究机构归类为2B级[24]。烟曲霉是病死率极高的侵袭性曲霉菌病的主要病原菌,属于人类临床常见的三大机会性致病真菌之一[32]。镰刀菌能产生多种高致癌性的真菌毒素,包括单端孢霉烯族毒素、伏马菌素和玉米赤霉烯酮等,对人类健康构成严重威胁[33]。上述致病性真菌的检出对易霉变饮片的贮藏条件提出预警,也为其用药安全提供参考。
通过平板法分离纯化单一菌株进行菌种鉴定,方便学者进行微生物的生理生化特性和生物学功能研究。但该法具有较强的筛选功能,部分难以培养或无生命现象的微生物会被漏检,因此分离获得的菌株可能远远小于饮片表面微生物的真实数量,无法全面地反映中药饮片表面微生物群落的真实结构。近年来,随着高通量测序技术的迅猛发展,该技术也逐步应用于中药饮片微生物污染的研究领域[4,8-9,34-36]。与平板法相比,该法无需培养即可快速高效地获得微生物群落完整的结构和功能信息,扩大了表面微生物的研究范围,较为真实地呈现了优势微生物物种。在今后的研究中,可综合两者共同分析中药饮片污染微生物的群落构成,能够更科学、精确、全面地研究饮片污染微生物的种类,利于开展后续风险评估工作。
本研究仅选取14种31批次易霉变饮片,代表性稍显不足,因此相关结果仅针对以上31批次。后期将在此基础上增加饮片品种、样品批数及产地,从而提供更多数据支撑,以便更全面地评估中药饮片微生物污染状况。
基金
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  • 国家自然科学基金项目资助(81960715)
文献
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参考文献 引证文献
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2024年第59卷第7期
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doi: 10.11669/cpj.2024.07.002
  • 接收时间：2023-07-18
  • 首发时间：2026-04-08
  • 出版时间：2024-04-08
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  • 收稿日期：2023-07-18
基金
国家自然科学基金项目资助(81960715)
作者信息
    1 江西中医药大学, 南昌 330004
    2 江西省药品检验检测研究院, 国家药品监督管理局中成药质量评价重点实验室, 江西省药品与医疗器械质量工程技术研究中心, 南昌 330029

通讯作者:

*刘绪平,男,主任药师,硕士生导师 研究方向:药品微生物与质量控制研究 Tel:(0791)88158128；
刘卫德,男,主管药师 研究方向:药品质量和标准研究 Tel:(0791)88158796
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https://castjournals.cast.org.cn/joweb/zgyxzz/CN/10.11669/cpj.2024.07.002
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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