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Article(id=1218291755917693339, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1218291750003724554, articleNumber=1001-2494(2024)13-1246-06, orderNo=null, doi=10.11669/cpj.2024.13.010, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1695571200000, receivedDateStr=2023-09-25, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1768392989376, onlineDateStr=2026-01-14, pubDate=1720368000000, pubDateStr=2024-07-08, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1768392989376, onlineIssueDateStr=2026-01-14, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1768392989376, creator=13701087609, updateTime=1768392989376, updator=13701087609, issue=Issue{id=1218291750003724554, tenantId=1146029695717560320, journalId=1190317699101192196, year='2024', volume='59', issue='13', pageStart='1173', pageEnd='1272', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1768392987967, creator=13701087609, updateTime=1768394537396, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1218298248834503031, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1218291750003724554, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1218298248838697336, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1218291750003724554, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1246, endPage=1251, ext={EN=ArticleExt(id=1218291756160962988, articleId=1218291755917693339, tenantId=1146029695717560320, journalId=1190317699101192196, language=EN, title=Viable Bacteria Content and Gene Safety Analysis of Bifidobacterium Microbial Products, columnId=null, journalTitle=Chinese Pharmaceutical Journal, columnName=null, runingTitle=null, highlight=null, articleAbstract=

OBJECTIVE To investigate the influencing factors and test methods of viable bacterial count in probiotic products of nature Bifidobacterium, and provide reference and suggestions for improving the quality control of such products. METHODS The effects of storage temperature and viable count determination method on viable count were studied, and the pretreatment methods and diluent composition were optimized, and the drug resistance genes and virulence genes of 11 strains contained in four products were analyzed by whole genome sequencing and functional genomics analysis. RESULTS The results showed that temperature had a great influence on the results of viable bacteria count of Bifidobacteria products, and the optimized method of live bacteria count could effectively improve the count results. Several strains of live bacteria products contained drug resistance genes and virulence genes. CONCLUSION It is suggested that we should pay attention to the influence of temperature on products in production storage and circulation. The count method of live bacteria can consider the characteristics of strains and production technology, and optimize the influencing factors such as pre-treatment and diluent. And there is a need for systematic safety assessment of all strains involved in commercially live bacteria product.

, correspAuthors=Jing QIAN, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Xiaoling ZHENG, Yinhuan WANG, Junhao CHEN, Ling QIAN, Wei CAO, Xin WU, Jue LI, Wanzi GONG, Jing QIAN), CN=ArticleExt(id=1218291756572004806, articleId=1218291755917693339, tenantId=1146029695717560320, journalId=1190317699101192196, language=CN, title=双歧杆菌类微生态活菌制品的活菌含量研究及基因安全性分析, columnId=1190352405612040510, journalTitle=中国药学杂志, columnName=论著, runingTitle=null, highlight=null, articleAbstract=

目的 聚焦双歧杆菌类微生态活菌制品活菌含量影响因素及测定方法、基因安全性等核心问题,开展相关研究和分析,为该类产品质量控制的提升提供参考和建议。方法 研究贮藏温度和活菌数测定方法对该类产品活菌计数的影响,并进行前处理方式和稀释液成分的优化;采用全基因组测序和功能基因组分析方法开展4种产品包含的11株菌的耐药基因和毒力基因分析。结果 研究结果表明,温度对双歧杆菌类微生态活菌制品的活菌计数结果影响较大,优化后的活菌计数方法可有效提高计数结果,该类活菌制品中有多株菌含有耐药基因和毒力基因。结论 建议在生产、储藏和流通等环节重点关注温度对产品的影响;活菌计数方法需考虑菌株特性和生产工艺等,对前处理方式和稀释液等影响因素进行优化;有必要对已上市的微生态活菌制品涉及的所有菌株进行系统性的安全性评估。

, correspAuthors=钱璟, authorNote=null, correspAuthorsNote=
* 钱璟,女,硕士,副主任药师 研究方向:药品技术审评 Tel:(0571)81060409
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郑小玲,女,硕士,副主任药师 研究方向:微生物检验与质量控制

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郑小玲,女,硕士,副主任药师 研究方向:微生物检验与质量控制

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Count results of 8 strains at different stroage temperatures

, figureFileSmall=null, figureFileBig=null, tableContent=
Dosage form Bacteria Results of storage at
15-30 ℃/CFU·g-1		 Results of storage at
2-8 ℃/CFU·g-1
Live Bifidobacterium Preparation, Oral Bifidobacterium adolesc-entis 3.0×107 1.6×109
Combined Bifidobacterium, Lactobacillus, Enterococcus and Bacillus cereus Tablets, Live Bifidobacterium infantis 2.0×107 5.3×108
Enterococcus faecalis 4.0×106 2.7×107
Lactobacillus acidophilus 1.0×107 2.1×108
Bacillus cereus 6.0×106 7.9×106
Live Combined Bifidobacterium, Lactobacillus and Enterococcus Capsules, Oral Bifidobacterium longum 1.0×105 2.1×106
Enterococcus faecalis 1.5×109 1.6×109
Lactobacillus acidophilus 1.6×108 2.1×108
), ArticleFig(id=1218291763098342051, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291755917693339, language=CN, label=表1, caption=

不同贮藏温度下8株菌的活菌计数结果

, figureFileSmall=null, figureFileBig=null, tableContent=
Dosage form Bacteria Results of storage at
15-30 ℃/CFU·g-1		 Results of storage at
2-8 ℃/CFU·g-1
Live Bifidobacterium Preparation, Oral Bifidobacterium adolesc-entis 3.0×107 1.6×109
Combined Bifidobacterium, Lactobacillus, Enterococcus and Bacillus cereus Tablets, Live Bifidobacterium infantis 2.0×107 5.3×108
Enterococcus faecalis 4.0×106 2.7×107
Lactobacillus acidophilus 1.0×107 2.1×108
Bacillus cereus 6.0×106 7.9×106
Live Combined Bifidobacterium, Lactobacillus and Enterococcus Capsules, Oral Bifidobacterium longum 1.0×105 2.1×106
Enterococcus faecalis 1.5×109 1.6×109
Lactobacillus acidophilus 1.6×108 2.1×108
), ArticleFig(id=1218291763190616742, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291755917693339, language=EN, label=Tab.2, caption=

Count results of 8 strains under different methods

, figureFileSmall=null, figureFileBig=null, tableContent=
Dosage form Bacteria Results of standard
method/CFU·g-1		 Results of optimization
method/CFU·g-1
Live Bifidobacterium Preparation, Oral Bifidobacterium adoles-centis 1.6×109 1.4×109
Combined Bifidobacterium, Lactobacillus, Enterococcus and Bacillus cereus Tablets, Live Bifidobacterium infantis 5.3×108 1.0×109
Enterococcus faecalis 2.7×107 4.3×107
Lactobacillus acidophilus 2.1×108 4.5×108
Bacillus cereus 7.9×106 2.5×107
Live Combined Bifidobacterium, Lactobacillus and Enterococcus Capsules, Oral Bifidobacterium longum 2.1×106 3.1×106
Enterococcus faecalis 1.6×109 2.5×109
Lactobacillus acidophilus 2.1×108 3.3×108
), ArticleFig(id=1218291763270308521, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291755917693339, language=CN, label=表2, caption=

8株菌在不同计数方法下的活菌计数结果

, figureFileSmall=null, figureFileBig=null, tableContent=
Dosage form Bacteria Results of standard
method/CFU·g-1		 Results of optimization
method/CFU·g-1
Live Bifidobacterium Preparation, Oral Bifidobacterium adoles-centis 1.6×109 1.4×109
Combined Bifidobacterium, Lactobacillus, Enterococcus and Bacillus cereus Tablets, Live Bifidobacterium infantis 5.3×108 1.0×109
Enterococcus faecalis 2.7×107 4.3×107
Lactobacillus acidophilus 2.1×108 4.5×108
Bacillus cereus 7.9×106 2.5×107
Live Combined Bifidobacterium, Lactobacillus and Enterococcus Capsules, Oral Bifidobacterium longum 2.1×106 3.1×106
Enterococcus faecalis 1.6×109 2.5×109
Lactobacillus acidophilus 2.1×108 3.3×108
), ArticleFig(id=1218291763383554735, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291755917693339, language=EN, label=Tab.3, caption=

Drug resistance gene results of 8 strains

, figureFileSmall=null, figureFileBig=null, tableContent=
Types of drug resistance genes Drug resistance gene Source
Tetracyclines resistance tetW Bifidobacterium adoles-centis、Bifidobacterium infantis、2 strains of Bifidobacterium longum
Mupirocin resistance ileS Bifidobacterium adoles-centis、Bifidobacterium infantis、2 strains of Bifidobacterium longum
Rifampin resistance rpoB Bifidobacterium adoles-centis、Bifidobacterium infantis、2 strains of Bifidobacterium longum
Fluoroquinolone resistance EmeA、efrA、efrB 3 strains of Enterococcus faecalis
Lincosamides resistance IsaA 3 strains of Enterococcus faecalis
Diaminopyrimidine resistance dfrE 3 strains of Enterococcus faecalis
Glycopeptide resistance vanZF、vanYA、vanSA、vanRA Bacillus cereus
Phosphonomycin resistance FosB Bacillus cereus
Cephalosporins resistance BcI Bacillus cereus
), ArticleFig(id=1218291763488412340, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291755917693339, language=CN, label=表3, caption=

8株菌的耐药基因分析

, figureFileSmall=null, figureFileBig=null, tableContent=
Types of drug resistance genes Drug resistance gene Source
Tetracyclines resistance tetW Bifidobacterium adoles-centis、Bifidobacterium infantis、2 strains of Bifidobacterium longum
Mupirocin resistance ileS Bifidobacterium adoles-centis、Bifidobacterium infantis、2 strains of Bifidobacterium longum
Rifampin resistance rpoB Bifidobacterium adoles-centis、Bifidobacterium infantis、2 strains of Bifidobacterium longum
Fluoroquinolone resistance EmeA、efrA、efrB 3 strains of Enterococcus faecalis
Lincosamides resistance IsaA 3 strains of Enterococcus faecalis
Diaminopyrimidine resistance dfrE 3 strains of Enterococcus faecalis
Glycopeptide resistance vanZF、vanYA、vanSA、vanRA Bacillus cereus
Phosphonomycin resistance FosB Bacillus cereus
Cephalosporins resistance BcI Bacillus cereus
), ArticleFig(id=1218291763584881336, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291755917693339, language=EN, label=Tab.4, caption=

Virulence gene results of 8 strains

, figureFileSmall=null, figureFileBig=null, tableContent=
Coding gene function Virulence gene Source
Immune inhibitor inhA Bacillus cereus
Hemolysin alo、hblA、hblC、hblD Bacillus cereus
Enterotoxin nheA、nheB、nheC Bacillus cereus
Cytotoxin cytK Bacillus cereus
Exoenzyme EF3023 3 strains of Enterococcus faecalis
EF0818 1 strain of Enterococcus faecalis
Fibrinogen binding protein fss1 3 strains of Enterococcus faecalis
fss2 1 strain of Enterococcus faecalis
fss3 2 strains of Enterococcus faecalis
bopD 3 strains of Enterococcus faecalis
Capsule polysacharides cpsA、cpsB 3 strains of Enterococcus faecalis
cpsC、cpsD、cpsE、cpsF 1 strain of Enterococcus faecalis
cpsG、cpsH、cpsI、cpsJ
cpsK
Endocarditis antigen efaA 3 strains of Enterococcus faecalis
Quorum sensing fsrA、fsrB 1 strain of Enterococcus faecalis
fsrC 3 strains of Enterococcus faecalis
Gelatinase gelE 3 strains of Enterococcus faecalis
Serine protease sprE 3 strains of Enterococcus faecalis
Adhesion srtC、ebpA、ebpB、ebpC 3 strains of Enterococcus faecalis
), ArticleFig(id=1218291763664573115, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1218291755917693339, language=CN, label=表4, caption=

8株菌的毒力基因结果分析

, figureFileSmall=null, figureFileBig=null, tableContent=
Coding gene function Virulence gene Source
Immune inhibitor inhA Bacillus cereus
Hemolysin alo、hblA、hblC、hblD Bacillus cereus
Enterotoxin nheA、nheB、nheC Bacillus cereus
Cytotoxin cytK Bacillus cereus
Exoenzyme EF3023 3 strains of Enterococcus faecalis
EF0818 1 strain of Enterococcus faecalis
Fibrinogen binding protein fss1 3 strains of Enterococcus faecalis
fss2 1 strain of Enterococcus faecalis
fss3 2 strains of Enterococcus faecalis
bopD 3 strains of Enterococcus faecalis
Capsule polysacharides cpsA、cpsB 3 strains of Enterococcus faecalis
cpsC、cpsD、cpsE、cpsF 1 strain of Enterococcus faecalis
cpsG、cpsH、cpsI、cpsJ
cpsK
Endocarditis antigen efaA 3 strains of Enterococcus faecalis
Quorum sensing fsrA、fsrB 1 strain of Enterococcus faecalis
fsrC 3 strains of Enterococcus faecalis
Gelatinase gelE 3 strains of Enterococcus faecalis
Serine protease sprE 3 strains of Enterococcus faecalis
Adhesion srtC、ebpA、ebpB、ebpC 3 strains of Enterococcus faecalis
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双歧杆菌类微生态活菌制品的活菌含量研究及基因安全性分析
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郑小玲 1 , 王银环 1 , 陈君豪 1 , 钱凌 1 , 曹炜 1 , 吴鑫 1 , 李珏 1 , 龚万紫 2 , 钱璟 3, *
中国药学杂志 | 论著 2024,59(13): 1246-1251
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中国药学杂志 | 论著 2024, 59(13): 1246-1251
双歧杆菌类微生态活菌制品的活菌含量研究及基因安全性分析
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郑小玲1, 王银环1, 陈君豪1, 钱凌1, 曹炜1, 吴鑫1, 李珏1, 龚万紫2, 钱璟3, *
作者信息
  • 1 浙江省食品药品检验研究院,国家药品监督管理局药品微生物检测与预警重点实验室, 浙江省药品接触材料质量控制研究重点实验室, 杭州 310004
  • 2 中国药科大学, 南京 211198
  • 3 浙江省药品化妆品审评中心, 杭州 310012

    • 郑小玲,女,硕士,副主任药师 研究方向:微生物检验与质量控制

通讯作者:

* 钱璟,女,硕士,副主任药师 研究方向:药品技术审评 Tel:(0571)81060409
Viable Bacteria Content and Gene Safety Analysis of Bifidobacterium Microbial Products
Xiaoling ZHENG1, Yinhuan WANG1, Junhao CHEN1, Ling QIAN1, Wei CAO1, Xin WU1, Jue LI1, Wanzi GONG2, Jing QIAN3, *
Affiliations
  • 1 NMPA Key Laboratory for Testing and Risk Warning of Pharmaceutical Microbiology, Key Laboratory of Drug Contacting Materials Quality Control of Zhejiang Provincial, Zhejiang Institute for Food and Drug Control, Hangzhou 310004, China
  • 2 China Pharmaceutical University, Nanjing 211198, China
  • 3 Zhejiang Center for Drug and Cosmetic Evaluation, Hangzhou 310012, China
出版时间: 2024-07-08 doi: 10.11669/cpj.2024.13.010
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摘要
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目的 聚焦双歧杆菌类微生态活菌制品活菌含量影响因素及测定方法、基因安全性等核心问题,开展相关研究和分析,为该类产品质量控制的提升提供参考和建议。方法 研究贮藏温度和活菌数测定方法对该类产品活菌计数的影响,并进行前处理方式和稀释液成分的优化;采用全基因组测序和功能基因组分析方法开展4种产品包含的11株菌的耐药基因和毒力基因分析。结果 研究结果表明,温度对双歧杆菌类微生态活菌制品的活菌计数结果影响较大,优化后的活菌计数方法可有效提高计数结果,该类活菌制品中有多株菌含有耐药基因和毒力基因。结论 建议在生产、储藏和流通等环节重点关注温度对产品的影响;活菌计数方法需考虑菌株特性和生产工艺等,对前处理方式和稀释液等影响因素进行优化;有必要对已上市的微生态活菌制品涉及的所有菌株进行系统性的安全性评估。

双歧杆菌  /  活菌计数  /  耐药基因  /  毒力基因  /  微生态活菌制品

OBJECTIVE To investigate the influencing factors and test methods of viable bacterial count in probiotic products of nature Bifidobacterium, and provide reference and suggestions for improving the quality control of such products. METHODS The effects of storage temperature and viable count determination method on viable count were studied, and the pretreatment methods and diluent composition were optimized, and the drug resistance genes and virulence genes of 11 strains contained in four products were analyzed by whole genome sequencing and functional genomics analysis. RESULTS The results showed that temperature had a great influence on the results of viable bacteria count of Bifidobacteria products, and the optimized method of live bacteria count could effectively improve the count results. Several strains of live bacteria products contained drug resistance genes and virulence genes. CONCLUSION It is suggested that we should pay attention to the influence of temperature on products in production storage and circulation. The count method of live bacteria can consider the characteristics of strains and production technology, and optimize the influencing factors such as pre-treatment and diluent. And there is a need for systematic safety assessment of all strains involved in commercially live bacteria product.

Bifidobacterium  /  viable bacterial count  /  drug resistance gene  /  virulence gene  /  live bacteria product
郑小玲, 王银环, 陈君豪, 钱凌, 曹炜, 吴鑫, 李珏, 龚万紫, 钱璟. 双歧杆菌类微生态活菌制品的活菌含量研究及基因安全性分析. 中国药学杂志, 2024 , 59 (13) : 1246 -1251 . DOI: 10.11669/cpj.2024.13.010
Xiaoling ZHENG, Yinhuan WANG, Junhao CHEN, Ling QIAN, Wei CAO, Xin WU, Jue LI, Wanzi GONG, Jing QIAN. Viable Bacteria Content and Gene Safety Analysis of Bifidobacterium Microbial Products[J]. Chinese Pharmaceutical Journal, 2024 , 59 (13) : 1246 -1251 . DOI: 10.11669/cpj.2024.13.010
正文
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双歧杆菌为一类革兰阳性、不运动、严格厌氧细菌的属名,是人和动物肠道内一种重要的有益微生物,对人体健康具有生物屏障、营养作用、抗肿瘤、免疫增强、改善胃肠道功能及抗衰老等多种重要的生理功能[1]。特别是随着近年肠道微生物组学研究的发展,人们越来越认识到双歧杆菌的重要性。双歧杆菌类活菌药物是一类能治疗菌群失调或二重感染的重要的微生态活菌制品,该类活菌制品起步较早,是国内外研究最热门的益生菌制品之一。目前国外已经有70多种相关产品问世[2],国内也有7个品种在售,是市售最多的活菌药物,主要包括片剂、胶囊剂、肠溶胶囊和散剂,常见的如双歧杆菌活菌胶囊、三联活菌片、双歧杆菌四联活菌片,以及双歧杆菌三联活菌胶囊等。在超级细菌和众多耐药细菌出现的今天,该类药物也越来越得到国内外广大临床医生的认可和应用,其质量的好坏直接关系到临床用药安全和有效。
调研分析国内活菌药物产业现状,主要面临两大难题:一是活菌制品质量控制保障不完善,二是活菌应用的安全问题尚未完全解决。目前双歧杆菌类微生态活菌制品的质控项目主要包括鉴别、理化检查、活菌数测定、杂菌检查。该类产品的质控标准和检测方法难以全面反映产品质量,存在以下问题亟须解决:①贮藏条件对活菌含量的影响。贮藏条件如储存温度和时间等对活菌制品的稳定性影响较大。双歧杆菌类活菌制品在贮藏和流通过程中需要低温环境,温度变化会导致活菌的死亡。因此,该类产品在货架期内,活菌数一般会逐渐下降。②计数方法对活菌含量的影响。影响活菌计数的因素包括:前处理方式、稀释液、培养基、包埋技术、菌株特性和生产工艺等。目前大部分活菌制品活菌计数结果差异较大,结果重复性差、偏差大,直接影响产品质量水平,对用药安全及有效性存在不可控性。③菌种安全性问题。微生态活菌制品相关品种已被批准多年,之前可能由于技术等原因,相关菌种安全性评价程序缺失。随着检测技术的发展和相关研究的深入,益生菌菌株携带耐药基因、毒力基因以及产生有毒代谢产物等安全问题开始慢慢暴露出来[3]。
本研究对上述存在的问题先进行了贮藏温度和活菌计数影响因素的探索性研究,再采用全基因组测序及分析开展该类药物的毒力基因和耐药基因的评价研究,为相关产品的质量提升和安全性评价奠定基础。
1 材料
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1.1 仪器
Grid ION MK1测序仪(英国Oxford Nanopore Technologies 公司);BioDrop超微量蛋白核酸分析仪(中国宝予德有限公司);Qsep100(中国台湾光鼎生物科技股份有限公司);Oubit4.0 [赛默飞世尔科技(中国)有限公司];全自动柔性核酸纯化仪BJNAP-240(杭州柏炬科技有限公司);电泳仪(美国Bio-rad公司);AN2CTS三气培养箱(荷兰 Anoxomat公司);ME2002E电子天平(瑞士梅特勒-托利多公司);HFsafe-1200生物安全柜(上海力康生物有限公司);MLS-3781-PC高压蒸汽灭菌器(日本三洋公司);5424离心机(德国艾本德公司);TW20精密恒温水浴槽(德国优莱博公司);HTY-761匀浆仪(浙江泰林生物技术股份有限公司)。
1.2 试剂和耗材
细菌基因组DNA提取试剂盒(批号AI82587A,宝生物工程有限公司);溶菌酶(索莱宝生物科技有限公司); Oubit dsDNA HS AssayKit [赛默飞世尔科技(中国)有限公司]; 连接法测序多样本DNA建库辅助试剂盒(杭州柏熠科技有限公司);SQK-LSK110测序试剂盒、无扩增条形码Native barcode(EXP-NBD104+EXP-NBD114)、R9.4.1 Flow-cell测序芯片(英国 Oxford Nanopore Technologies公司);5k DNA marker(全式金生物科技有限公司);灭菌生理盐水(批号LP19121709,青岛蓝雁绿检生物技术有限公司);L-半胱氨酸(批号20210713,上海佰奥生物科技有限公司);纳米孔测序由杭州宝诚生物技术有限公司完成。
MRS培养基(批号20210923)、BA培养基(批号20210728)、EC培养基(批号20210728)、TPY培养基(批号20210608)(青岛海博生物技术有限公司)。
双歧杆菌四联活菌片、双歧杆菌三联活菌胶囊、双歧杆菌活菌胶囊和双歧杆菌三联活菌肠溶胶囊购自浙江省内不同经营单位和使用单位。
2 方法与结果
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2.1 贮藏温度对活菌含量影响的研究
因目前不同微生态活菌制剂的储藏温度主要涉及常温(15~30 ℃)储藏和冷藏(2~8 ℃)两种情况,故将双歧杆菌活菌胶囊、双歧杆菌四联活菌片和双歧杆菌三联活菌胶囊各取6批,分别置常温(15~30 ℃)和冰箱冷藏(2~8 ℃)贮藏1个月后,按照2020年版《中国药典》三部微生态活菌制品总论附录2要求进行各活菌数的测定。上述品种的活菌数测定方法按标准均为无菌称取样品3.0 g,加入27.0 mL稀释液,振摇稀释后依法培养。结果平均值见表1。
常温贮藏对于双歧杆菌活菌胶囊中的青春双歧杆菌活菌计数结果影响较大,与冷藏条件相比,活菌计数结果下降至冷藏条件的1/53;双歧杆菌四联活菌片中除蜡样芽孢杆菌活菌数降低至冷藏条件的1/2,其余菌的活菌计数均降至1/6甚至更少,其中双歧杆菌数量降低至1/27;双歧杆菌三联活菌胶囊中长双歧杆菌数量降低至冷藏条件的1/21,其余菌活菌数下降不大。
由此可见,含双歧杆菌的微生态活菌制品中虽然包含多种不同活菌,但是贮藏温度对双歧杆菌活菌数量的影响远大于对其他益生菌(粪肠球菌、嗜酸乳杆菌和蜡样芽孢杆菌)的影响。由试验结果可知温度是影响双歧杆菌类微生态活菌制品质量的重要因素,该类产品虽然一般都要求冷藏,但在生产、储藏和流通等环节均需重点关注温度的影响。
2.2 活菌数测定方法的优化研究
活菌数测定方法中稀释液成分、前处理方法、操作过程以及培养条件等因素都会影响活菌计数的结果。双歧杆菌活菌胶囊、双歧杆菌四联活菌片和双歧杆菌三联活菌胶囊3个品种的活菌数测定方法相似,前处理方法皆为振摇数分钟后逐级稀释,之后取样涂布培养,且对整个前处理过程温度未进行控制。优化活菌数测定方法时先将前处理方法采用15 000 r·min-1匀浆2 min进行供试液的均匀分散,因“2.1”中试验结果表明在储藏环节温度高低对双歧杆菌影响较大,温度上升可能会引起该菌活性下降,因此,优化方法时将所涉及的稀释液均预先降温至4 ℃,并在稀释液中增加0.1%聚山梨酯80作为表面活性剂帮助样品的扩散与溶解。将双歧杆菌活菌胶囊、双歧杆菌四联活菌片和双歧杆菌三联活菌胶囊各取3批,分别按标准方法和优化后的活菌计数方法进行活菌数测定,结果平均值见表2。
由表2结果可见,优化后的活菌计数方法对双歧杆菌活菌胶囊中的青春双歧杆菌活菌计数结果影响不大。优化活菌计数方法后,与之前相比,双歧杆菌四联活菌片中,双歧杆菌、粪肠球菌和嗜酸乳杆菌的活菌数计数结果均提升2倍左右,蜡样芽孢杆菌活菌计数结果提升3倍左右。双歧杆菌三联活菌胶囊中长双歧杆菌、嗜酸乳杆菌和粪肠球菌活菌数计数结果均提升1.6倍左右。上述结果显示,如果生产中添加碳酸钙类辅料(如蜡样芽孢杆菌菌粉),在稀释液中添加一定浓度的聚山梨酯80作为表面活性剂有助于样品分散均匀从而提高活菌数测定结果。供试液处理采用匀浆方法比振摇更有利于活菌的分散均匀,但是匀浆过程中容易产热,因此可以降低稀释液温度,减少产热升温对菌活性的影响。
2.3 耐药基因及毒力基因研究
采用纳米孔测序技术对4种产品中的总计11株菌(包括1株青春双歧杆菌、1株婴儿双歧杆菌、2株长双歧杆菌、3株嗜酸乳杆菌、3株粪肠球菌和1株蜡样芽孢杆菌)分别进行全基因组测序,研究每株菌的耐药基因和毒力基因。
2.3.1 核酸提取和纳米孔测序
取上述不同菌的新鲜培养物适量,分别加入200 μL(10 mg·mL-1)溶菌酶消化,37 ℃孵育30 min。使用全自动柔性核酸纯化仪进行核酸提取,使用Qubit4.0及Qubit dsDNA HS Assay Kit对纯化后DNA进行核酸浓度定量。 取纯化后核酸样本采用连接法测序多样本DNA建库辅助试剂盒建库,并使用GridION MK1测序仪及R9.4.1 Flowcell芯片进行测序。
2.3.2 数据分析
对质控过滤完成的数据进行无参组装,使用abricate软件进行耐药基因分析和毒力基因分析,耐药基因分析使用CARD数据库,毒力基因分析使用VFDB数据库。
2.3.3 耐药基因和毒力基因分析结果
上述11株菌的耐药和毒力基因结果详见表3~4,其中青春双歧杆菌、婴儿双歧杆菌和2株长双歧杆菌中均不含有毒力基因,但同时含有相同的3个耐药基因,主要对莫匹罗星、利福平和四环素类抗生素的耐药基因。3株嗜酸乳酸杆菌中均不存在耐药和毒力基因,因此无相关耐药基因转移和毒力因子风险。
蜡样芽孢杆菌含有7个耐药基因,主要对糖肽类、磷霉素和头孢菌素类抗生素的耐药基因。另含有9个毒力基因,分别为侵袭免疫系统的inhA基因、溶血素ALO基因(alo)、溶血素BL基因(hblA、hblC、hblD)、非溶血性肠毒素基因(nheA、nheB、nheC)和细胞毒素K基因(cytK)等。
3株粪肠球菌所含的耐药基因一样,均包含5个耐药基因,主要对喹诺酮类、林可酰胺类和二氨基嘧啶类等的耐药基因。但是3株粪肠球菌所含的毒力基因不一样,其中1株粪肠球菌有26种编码不同功能蛋白的毒力基因,另外2株粪肠球菌有14种毒力基因。根据基因功能分类, 携带毒力基因的功能主要是参与编码胞外酶、表面蛋白、荚膜多糖、心内膜炎抗原、群体感应系统和黏附素等[4-6]。
3 讨论
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虽然储藏温度对活菌制剂中双歧杆菌活性影响较大,但在前期研究中发现同品种活菌制品不同批次间双歧杆菌活菌数差异也较大,主要原因可能是目前标准中一般只要求活菌数的最低限度却缺乏最高限值规定,而温度和氧气等因素对双歧杆菌的活性影响较大,因此在生产投料中一般为确保活菌数量,每批次菌粉的投料量会有差异。微生态活菌制品中不同批次间菌粉投料量不一致的情况一直存在。如果同种产品不同批次间活菌数量相差较大,对临床使用效果可能会存在较大影响。
活菌数量是评价微生态活菌制品安全有效的重要质控指标。目前双歧杆菌类微生态活菌制品相关标准和检测方法难以全面反映该类产品质量。不同种微生物特性不一样,对营养条件要求也不一致,因此多联制剂中每种活菌的计数方法也不尽相同[7]。2020年版《中国药典》对微生态活菌制品的活菌数测定规定为无菌称取样品3.0 g,加入27.0 mL稀释液,稀释后涂布在选择性琼脂培养皿上测定。目前该类检测方法存在活菌数测定结果偏低、不同时间和人员测定结果差异大和多联制剂活菌互相干扰测定结果等问题。活菌计数结果应尽量真实反映产品的活菌数量。影响计数方法准确性的因素主要包括供试液成分、样品处理和培养条件等。如活菌制剂的辅料是碳酸钙类的易沉颗粒,可通过研究在供试液制备时添加合适浓度(如吐温-80类)的表面活性剂[8-9],防止辅料快速沉降影响计数结果,但也要注意表面活性剂是否对菌活性有影响[10]。样品处理时充分混匀样品对准确测定活菌数至关重要,目前常见混匀方式包括振荡混匀和匀浆混匀等。如采取人工振荡混匀,振荡的频率和时间受人员影响较大。匀浆混匀时应考虑匀浆速率和匀浆时间等影响因素,尤其是匀浆时可能引起供试液温度的上升,会对菌活性产生影响。传统微生物培养计数方法存在着培养时间长、操作繁琐等问题,随着快速微生物检测方法的发展,新技术如聚合酶链式反应(PCR)技术、流式细胞技术等开始应用于微生物活菌计数[11-12],Zheng等[13-14]也考察了基于标记微生物和流式细胞计数原理的流式细胞仪进行微生态活菌制剂的活菌数测定,结果显示与平板培养方法的计数结果无显著性差异。因此评估并应用快速准确的新技术替代传统的微生物培养计数方法也是微生物检测技术发展的趋势。
以往研究中涉及本文中的微生物菌株,主要集中来源于环境、肠道和临床,对药品来源菌株的耐药性和毒力基因等比对分析研究相对较少。目前,国内外很多法规要求对益生菌进行安全性评价,提出开展菌株的耐药性和毒力性方面的安全评估。欧盟于2007年正式提出了安全资格认证理念,对添加到食品及饲料中的微生物实施上市前的风险评估,排除含有耐药基因和毒力基因的不良菌株。国内近两年发布的《食品用菌种安全性评价程序》和《食品用益生菌通则》也提出了耐药基因及毒力基因等的分析和风险评估。因此对于批准多年的微生态活菌制品中相关菌株毒力基因和耐药基因的分析和评估也迫在眉睫。
研究中涉及的双歧杆菌菌株虽然携带3种耐药基因,但这3种耐药基因在双歧杆菌中均被认为是内在耐药性,在双歧杆菌中无特殊影响;但是同时也存在着产品中耐药基因转移问题,需要警惕转移至宿主生物或者微生物间耐药性传递的风险,尤其是和致病微生物间的转移[15-16]。本研究中的蜡样芽孢杆菌含有的耐药基因与乳制品等食品来源的菌株耐药基因不一样[17-18],其中含有的溶血素hbl、非溶血性肠毒素nhe和细胞毒素cytK被认为是腹泻疾病相关的毒力因子[19]。在临床及动物源肠球菌中检出率较高的耐药基因telL、telM、erm[20],包括临床上常见的耐万古霉素基因型vanA和vanB[21],在本研究中的3株粪肠球菌中均未发现。3株粪肠球菌含有的毒力因子种类都类似,且与其他环境分离株的毒力基因也无显著差异。总之微生态活菌制品类药物,一方面可以调节肠道菌群,另一方面也可能具备传播耐药基因和毒力基因的风险,因此有必要对已上市的微生态活菌制品涉及的所有菌株进行系统性的安全性评估,加强对与致病相关的毒力基因或耐药基因等的存在情况开展进一步研究和分析。
基金
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  • 浙江省药品监督管理局2023年度科技计划项目(2023011)
  • 浙江省市场监督管理局科研计划项目(20210128)
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参考文献 引证文献
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2024年第59卷第13期
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doi: 10.11669/cpj.2024.13.010
  • 接收时间：2023-09-25
  • 首发时间：2026-01-14
  • 出版时间：2024-07-08
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  • 收稿日期：2023-09-25
基金
浙江省药品监督管理局2023年度科技计划项目(2023011)
浙江省市场监督管理局科研计划项目(20210128)
作者信息
    1 浙江省食品药品检验研究院,国家药品监督管理局药品微生物检测与预警重点实验室, 浙江省药品接触材料质量控制研究重点实验室, 杭州 310004
    2 中国药科大学, 南京 211198
    3 浙江省药品化妆品审评中心, 杭州 310012

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* 钱璟,女,硕士,副主任药师 研究方向:药品技术审评 Tel:(0571)81060409
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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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