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Article(id=1153433694841852349, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433686872679135, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20240830004, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1724947200000, receivedDateStr=2024-08-30, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1752929622612, onlineDateStr=2025-07-19, pubDate=1744646400000, pubDateStr=2025-04-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1752929622612, onlineIssueDateStr=2025-07-19, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1752929622612, creator=13701087609, updateTime=1752929622612, updator=13701087609, issue=Issue{id=1153433686872679135, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='7', pageStart='1', pageEnd='322', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1752929620712, creator=13701087609, updateTime=1757656380159, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1173259152974561742, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433686872679135, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1173259152978756047, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433686872679135, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=255, endPage=261, ext={EN=ArticleExt(id=1153433696070783451, articleId=1153433694841852349, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Quantitative determination of Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus plantarum ST-III in fermented milk by propidium monoazide-qPCR, columnId=1151895321388347923, journalTitle=Journal of Food Safety & Quality, columnName=Food Analysis and Detection, runingTitle=null, highlight=null, articleAbstract=

Objective To establish a quantitative detection method for the quantitative determination of the viable bacteria number of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in fermented milk, by combining propidium monoazide (PMA) with real-time fluorescence quantitative polymerase chain reaction (qPCR). Methods Firstly, the specific primer probe of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III was designed and verified, the standard curve was established using standard strains, and the reaction conditions of the PMA were optimized. Secondly, the quantities of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in the fermented milk samples were quantitatively detected. Results The optimal mass concentration of damaged bacteria of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III was 10 μg/mL, and the optimal exposure time was 10 min. The PMA-qPCR detection method could accurately detect Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in fermented milk. Conclusion In this study, a rapid and accurate PMA-qPCR detection method is established for the detection of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in fermented milk system, which provides certain reference significance for the quantitative detection of probiotics in fermented milk products.

, correspAuthors=Yu-Lv ZHANG, 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=Yu-Lv ZHANG, Jing BAO, Lin-Ling WANG), CN=ArticleExt(id=1153433720993337539, articleId=1153433694841852349, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=叠氮溴化丙锭-qPCR定量检测发酵乳中动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III, columnId=1151895321958773274, journalTitle=食品安全质量检测学报, columnName=食品分析与检测, runingTitle=null, highlight=null, articleAbstract=

目的 将叠氮溴化丙锭(propidium monoazide, PMA)与实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction, qPCR)联合, 建立一种发酵乳体系中动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-Ⅲ的活菌定量检测方法。方法 首先设计动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-Ⅲ的特异性基因序列的引物探针, 验证引物探针特异性, 优化PMA反应条件, 建立标准曲线, 验证灵敏度, 最后定量检测发酵乳样品中动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-Ⅲ菌株的数量。结果 动物双歧杆菌乳亚种BB-12与植物乳植杆菌ST-Ⅲ经PMA处理受损菌的最佳质量浓度为10 μg/mL, 处理菌液样品的最佳曝光时间为10 min, PMA-qPCR方法能够准确检测发酵乳体系中的动物双歧杆菌乳亚种BB-12与植物乳植杆菌ST-Ⅲ。结论 本研究建立了一种快速、准确PMA-qPCR检测方法, 用于检测发酵乳体系中的动物双歧杆菌乳亚种BB-12与植物乳植杆菌ST-Ⅲ, 为发酵乳产品中益生菌的定量检测提供一定的借鉴意义。

, correspAuthors=张玉律, authorNote=null, correspAuthorsNote=
* 张玉律(1990—), 女, 硕士, 中级工程师, 主要研究方向为乳品科学与加工、乳酸菌基础研究。E-mail:
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Factors influencing the entry of Vibrio cholerae into the viable but non-culturable state and regulatory mechanisms[D]. 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Primer sequence of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株 引物序列
动物双歧杆菌乳亚种BB-12 正向: 5'-GATGGACGAGGCCGGAGTGC-3'
反向: 5'-CCTGCGTGAAGCGGAAGATG-3'
探针: 5'-VIC-GATGTGCAGAATCAGGACGTG-TAMRA-3'
植物乳植杆菌ST-III 正向: 5'-AGCTTGAAAGATGGCTTCGG-3'
反向: 5'-GGTCGGCTACGTATCATTGC-3'
探针: 5'-FAM-ACGCCGCGGGACCATCCAAA-TAMRA-3'
), ArticleFig(id=1173278579761754377, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694841852349, language=CN, label=表1, caption=

动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III的引物序列

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株 引物序列
动物双歧杆菌乳亚种BB-12 正向: 5'-GATGGACGAGGCCGGAGTGC-3'
反向: 5'-CCTGCGTGAAGCGGAAGATG-3'
探针: 5'-VIC-GATGTGCAGAATCAGGACGTG-TAMRA-3'
植物乳植杆菌ST-III 正向: 5'-AGCTTGAAAGATGGCTTCGG-3'
反向: 5'-GGTCGGCTACGTATCATTGC-3'
探针: 5'-FAM-ACGCCGCGGGACCATCCAAA-TAMRA-3'
), ArticleFig(id=1173278579812086026, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694841852349, language=EN, label=Table 2, caption=

Reaction system of qPCR (20 μL)

, figureFileSmall=null, figureFileBig=null, tableContent=
20 µL探针法PCR反应体系 使用量/μL
2×探针法PCR预混液 10
50×ROX II 0.4
上游引物(10 μmol/L) 0.4
下游引物(10 μmol/L) 0.4
探针(10 μmol/L) 0.2
DNA模板(1~20 ng/μL) 3.0
RNase Free dH2O 5.6
总计 20.0
), ArticleFig(id=1173278579887583499, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694841852349, language=CN, label=表2, caption=

qPCR反应体系(20 μL)

, figureFileSmall=null, figureFileBig=null, tableContent=
20 µL探针法PCR反应体系 使用量/μL
2×探针法PCR预混液 10
50×ROX II 0.4
上游引物(10 μmol/L) 0.4
下游引物(10 μmol/L) 0.4
探针(10 μmol/L) 0.2
DNA模板(1~20 ng/μL) 3.0
RNase Free dH2O 5.6
总计 20.0
), ArticleFig(id=1173278579942109452, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694841852349, language=EN, label=Table 3, caption=

Screening of thermal lethal time of lactic acid bacteria

, figureFileSmall=null, figureFileBig=null, tableContent=
热致死时间/min 平板计数结果/(CFU/mL)
0 1.2×108
5 0
10 0
20 0
), ArticleFig(id=1173278580021801229, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694841852349, language=CN, label=表3, caption=

乳酸菌热致死时间的筛选

, figureFileSmall=null, figureFileBig=null, tableContent=
热致死时间/min 平板计数结果/(CFU/mL)
0 1.2×108
5 0
10 0
20 0
), ArticleFig(id=1173278580105687310, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694841852349, language=EN, label=Table 4, caption=

Quantitative test results of Bifidobacterium animalis subsp. lactis BB-12 in fermented milk samples

, figureFileSmall=null, figureFileBig=null, tableContent=
样品编号 贮藏时间/d 未经PMA处理 经PMA处理 平板计数
/(CFU/g)
Ct值 稀释倍数 qPCR检测活菌数 Ct值 稀释倍数 qPCR检测活菌数
样品1 15 33.28 100 1.6×107 33.63 100 1.6×107 1.0×107
样品2 15 30.61 50 1.4×108 31.08 50 4.9×107 1.0×107
样品3 15 24.36 100 1.3×108 25.18 100 1.1×107 6.3×107
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发酵乳样品中动物双歧杆菌乳亚种BB-12定量检测结果

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样品编号 贮藏时间/d 未经PMA处理 经PMA处理 平板计数
/(CFU/g)
Ct值 稀释倍数 qPCR检测活菌数 Ct值 稀释倍数 qPCR检测活菌数
样品1 15 33.28 100 1.6×107 33.63 100 1.6×107 1.0×107
样品2 15 30.61 50 1.4×108 31.08 50 4.9×107 1.0×107
样品3 15 24.36 100 1.3×108 25.18 100 1.1×107 6.3×107
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Quantitative test results of Lactobacillus plantarum ST-III in fermented milk samples

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样品编号 贮藏时间/d PMA未处理 PMA处理 平板计数
/(CFU/g)
Ct值 稀释倍数 qPCR检测活菌数 Ct值 稀释倍数 qPCR检测活菌数
样品1 15 17.40 50 1.3×108 18.63 50 6.4×107 5.1×107
样品2 15 16.98 50 1.7×108 17.57 50 1.2×108 6.0×107
样品3 15 20.94 100 2.3×108 21.10 100 6.0×107 3.5×107
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发酵乳样品中植物乳植杆菌ST-III定量检测结果

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样品2 15 16.98 50 1.7×108 17.57 50 1.2×108 6.0×107
样品3 15 20.94 100 2.3×108 21.10 100 6.0×107 3.5×107
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叠氮溴化丙锭-qPCR定量检测发酵乳中动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III
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张玉律 1, * , 包静 2 , 王琳玲 2
食品安全质量检测学报 | 食品分析与检测 2025,16(7): 255-261
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食品安全质量检测学报 | 食品分析与检测 2025, 16(7): 255-261
叠氮溴化丙锭-qPCR定量检测发酵乳中动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III
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张玉律1, * , 包静2, 王琳玲2
作者信息
  • 1.乳业生物技术国家重点实验室, 上海乳业生物工程技术研究中心, 光明乳业股份有限公司乳业研究院, 上海 200436
  • 2.华测检测认证集团股份有限公司, 深圳 518101

通讯作者:

* 张玉律(1990—), 女, 硕士, 中级工程师, 主要研究方向为乳品科学与加工、乳酸菌基础研究。E-mail:
Quantitative determination of Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus plantarum ST-III in fermented milk by propidium monoazide-qPCR
Yu-Lv ZHANG1, * , Jing BAO2, Lin-Ling WANG2
Affiliations
  • 1. State Key Laboratory of Dairy Biotechnology, Shanghai Engineering Research Center of Dairy Biotechnology, Dairy Research Institute of Bright Dairy Co., Ltd., Shanghai 200436, China
  • 2. Centre Testing International Group Co., Ltd., Shenzhen 518101, China
出版时间: 2025-04-15 doi: 10.19812/j.cnki.jfsq11-5956/ts.20240830004
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摘要
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目的 将叠氮溴化丙锭(propidium monoazide, PMA)与实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction, qPCR)联合, 建立一种发酵乳体系中动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-Ⅲ的活菌定量检测方法。方法 首先设计动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-Ⅲ的特异性基因序列的引物探针, 验证引物探针特异性, 优化PMA反应条件, 建立标准曲线, 验证灵敏度, 最后定量检测发酵乳样品中动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-Ⅲ菌株的数量。结果 动物双歧杆菌乳亚种BB-12与植物乳植杆菌ST-Ⅲ经PMA处理受损菌的最佳质量浓度为10 μg/mL, 处理菌液样品的最佳曝光时间为10 min, PMA-qPCR方法能够准确检测发酵乳体系中的动物双歧杆菌乳亚种BB-12与植物乳植杆菌ST-Ⅲ。结论 本研究建立了一种快速、准确PMA-qPCR检测方法, 用于检测发酵乳体系中的动物双歧杆菌乳亚种BB-12与植物乳植杆菌ST-Ⅲ, 为发酵乳产品中益生菌的定量检测提供一定的借鉴意义。

叠氮溴化丙锭  /  实时荧光定量聚合酶链式反应  /  动物双歧杆菌乳亚种BB-12  /  植物乳植杆菌ST-III

Objective To establish a quantitative detection method for the quantitative determination of the viable bacteria number of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in fermented milk, by combining propidium monoazide (PMA) with real-time fluorescence quantitative polymerase chain reaction (qPCR). Methods Firstly, the specific primer probe of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III was designed and verified, the standard curve was established using standard strains, and the reaction conditions of the PMA were optimized. Secondly, the quantities of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in the fermented milk samples were quantitatively detected. Results The optimal mass concentration of damaged bacteria of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III was 10 μg/mL, and the optimal exposure time was 10 min. The PMA-qPCR detection method could accurately detect Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in fermented milk. Conclusion In this study, a rapid and accurate PMA-qPCR detection method is established for the detection of Bifidobacterium animalis subsp. lactis BB-12 and Lactiplantibacillus plantarum ST-III in fermented milk system, which provides certain reference significance for the quantitative detection of probiotics in fermented milk products.

propidium monoazide  /  real-time fluorescence quantitative polymerase chain reaction  /  Bifidobacterium animalis subsp. lactis BB-12  /  Lactiplantibacillus plantarum ST-III
张玉律, 包静, 王琳玲. 叠氮溴化丙锭-qPCR定量检测发酵乳中动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III. 食品安全质量检测学报, 2025 , 16 (7) : 255 -261 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20240830004
Yu-Lv ZHANG, Jing BAO, Lin-Ling WANG. Quantitative determination of Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus plantarum ST-III in fermented milk by propidium monoazide-qPCR[J]. Journal of Food Safety & Quality, 2025 , 16 (7) : 255 -261 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20240830004
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0 引言
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乳酸菌主要是革兰氏阳性、不产孢子的兼性厌氧细菌, 可从植物、动物和人类等多种来源中分离得到[1]。乳酸菌在发酵食品中发挥着重要作用, 鼠李糖乳酪杆菌GG和动物双歧杆菌乳亚种(Bifidobacterium animalis subsp. lactis) BB-12等很多乳酸菌被证明具有益生功能[2-3]。目前公认的益生菌的定义是2001年提出的: 摄入足够数量时, 对宿主产生健康益处的活性微生物[4]。近年来REID等[5]重新界定并进一步阐明了益生菌的定义, 指出益生菌必须是活的微生物, 必须经过安全评估并证明是安全的, 其有益作用必须有足够的证据, 并且益生菌的健康作用具有菌株特异性并且与其剂量有关。近年来, 菌株分型与菌株特异性的研究也取得了一定进展[6-9]。根据Web of Science数据库的数据, 2022年全球益生菌相关研究发表量继续增加, 达到了6916篇, 其中包含研究型文章5051篇[10]。益生菌具有改善肠道健康、增强免疫力、辅助治疗肠易激综合征等多种功效[11-13], 被广泛应用于食品、保健品、药品等诸多领域。目前市场上的益生菌产品种类繁多, 质量也良莠不齐。常见的益生菌菌株主要有植物乳植杆菌、嗜酸乳杆菌、干酪乳酪杆菌和动物双歧杆菌乳亚种等。
目前QB/T 5165《食品用乳酸菌鉴定技术指南》仅适用于可用于食品的双歧杆菌属(Bifidobacterium)、乳杆菌属(Lactobacillus)和嗜热链球菌(Streptococcus thermophilus)的种水平鉴定, 食品安全国家标准GB 4789.35—2023《食品安全国家标准 食品微生物学检验 乳酸菌检验》无法特异性定量检测发酵乳制品中的植物乳植杆菌。平板计数法是基于传统倾倒平板培养的定量方法, 需要大量的培养基和后续生化鉴定等步骤, 不仅操作烦琐, 而且检测周期长(3~7 d), 特别是容易受到样品中其他潜在杂菌的干扰[14-15], 并且受到培养基、取样、稀释、人员、设备和环境等因素的影响[16]。
利用叠氮溴化丙锭(propidium monoazide, PMA)检测活菌数已有报道。利用PMA-PCR技术鉴别水样中传染性噬菌体T4与受热损伤颗粒, 结果显示PMA-实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction, qPCR)方法的高效性[17], 使用PMA-qPCR评估幽门螺杆菌对酸性环境和尿素存在条件下的敏感性, 为细菌耐酸能力研究提供了重要参考[18], 应用PMA-qPCR方法区分活的和死的肺炎杆菌, 为细菌活力评估提供了新的可能性和技术方法[19]。
PMA是一种能和DNA共价交联的光敏材料, 可以选择性穿过死菌破损的细胞膜, 在钨灯强光照射下, PMA的叠氮基团转化为氮宾自由基, 然后与DNA中的碳氢氧键结合生成稳共价氮碳键, 从而抑制了死菌DNA扩增导致的qPCR检测中假阳性信号的产生, PMA特性温和、难以通过活菌的细胞膜, 不影响活菌DNA的扩增[20-24]。qPCR方法广泛应用于乳制品中的大肠杆菌、双歧杆菌活菌检测[25-28]。近年来研究者更倾向用PMA结合qPCR技术研究活菌检测, 且使用PMA-qPCR方法可以比平板计数更快地获得可靠的结果[29], 大大提高了检测的效率和准确性。因此PMA和qPCR技术相结合在定量检测活菌方面有巨大的发展潜力。
动物双歧杆菌乳亚种(Bifidobacterium animalis subsp. lactis) BB-12是一种安全、耐受性良好的益生菌菌株, 在婴儿配方奶粉、膳食补充剂和发酵乳制品中有着悠久的使用历史[30-33]。植物乳植杆菌属于同型发酵乳酸菌, 具有减轻肠易激综合征的症状、降低胆固醇和保护肝脏的作用[34-36], 被广泛应用于酸奶、干酪和发酵泡菜等食品中[37-39]。植物乳植杆菌(Lactiplantibacillus plantarum) ST-III是从泡菜中分离得到的具有多种益生功能的乳酸菌[40]。近年来, 动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-Ⅲ的定量检测技术备受关注。传统的平板计数法虽然是一种常用的方法, 但其操作烦琐、耗时长且容易受到其他杂菌的干扰。因此, 需要发展一种更为快速准确的定量检测方法, 以满足对动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III数量的精准测量需求。在本发酵乳体系中, 共有德氏乳杆菌保加利亚亚种、唾液链球菌嗜热亚种、动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III 4株乳酸菌, 本研究基于核酸共价交联技术, 从分子水平寻找细菌活的状态的标志物, 建立了基于探针法的PMA-qPCR方法, 可以有效解决传统方法存在的烦琐、耗时长、干扰多等问题, 将其应用于快速定量检测发酵乳体系中动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III, 以期为发酵乳产品中益生菌的定量检测提供一定的借鉴意义。
1 材料与方法
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1.1 材料与试剂
动物双歧杆菌乳亚种BB-12、发酵剂(科汉森中国有限公司); 植物乳植杆菌ST-III(光明乳业股份有限公司); PMA染料(美国Biotium公司); 二甲基亚砜(上海阿拉丁生化科技股份有限公司); 细菌基因组DNA提取试剂盒、淀粉酶K、溶菌酶[天根生化科技(北京)有限公司]; 目标基因引物、TB Green™ Premix Ex Taq™等PCR扩增试剂[生工生物工程(上海)股份有限公司]。
1.2 仪器与设备
LUYOR-3419 PMA光解仪(美国路阳公司); ABI 7500 fast实时荧光定量PCR仪(美国ABI公司); Q3000核酸蛋白分析仪(美国Quawell公司); PL303电子天平(精度0.001 g, 梅特勒-托利多仪器上海有限公司)。
1.3 实验方法
1.3.1 受损菌液的制备
标准菌株BB-12和ST-III的菌悬液分别在MRS (De Man, Rogosa and Sharpe)培养基厌氧培养24 h, 用MRS培养基稀释10倍, 取10‒2菌液于离心管中, 沸水浴5 min, 吸取1 mL涂布于MRS琼脂培养基, 经平板法培养24 h均未有菌落长出。
1.3.2 PMA试剂的配制
准确称量1 mg PMA染料, 溶于20%二甲基亚砜中制成1 mg/mL的储存液, 置于‒20 ℃避光保存。将PMA储存液稀释5倍后作为工作液。
1.3.3 PMA处理条件的优化
通过沸水浴5~10 min获得无活菌生长的BB-12和ST-III的受损菌液, 作为PMA条件优化的菌液。对PMA工作浓度(0~50 μg/mL)、曝光时间(0~30 min)处理条件进行优化: 取9份500 mL受损菌样品, 加入PMA工作液, 使得PMA染料的最终质量浓度分别为0、2、5、7、10、20、30、40、50 mg/mL, 并充分混匀, 避光孵育10 min, 然后将离心管置于光解仪中分别曝光0、2、10、20、30 min进行光反应。随后用试剂盒提取基因组DNA, 进行荧光PCR检测, 最终确定区分PMA处理受损菌的最佳浓度和最佳曝光时间。
1.3.4 引物探针的设计与筛选
为实现对动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III的特异性检测和绝对定量分析, 本研究选取双歧杆菌具有种属特异性且高度保守的单拷贝植物乳杆菌素基因(plnF, GenBank no. AP018405.1)作为靶序列, 通过美国国家生物技术信息中心(National Center of Biotechnology Information, NCBI)在线工具进行序列分析和比对, 利用Prime Express软件V3.0 (ABI, Foster City, CA, USA)设计出探针5’端标记VIC, 3’端标记TAMRA。引物/探针由上海生工生物有限公司合成。本研究植物乳杆菌选取GB 4789.35中探针5’端标记FAM, 3’端标记TAMRA。引物序列见表1。
1.3.5 实时荧光PCR反应
按照表2的qPCR反应体系, 将制备得到20 μL的qPCR反应体系, 置于荧光PCR仪上进行扩增, 50 ℃持续2 min, 95 ℃预变性3 min, 进而94 ℃持续15 s、60 ℃退火延伸40 s, 总共40个循环。反应完成后利用Excel软件(Microsoft Office专业增强版2019)进行分析。以10倍梯度稀释纯菌液中提取的DNA作为标准品, 根据公式(1)计算出的标准品浓度的对数值为横坐标, 以对应循环数阈值(cycle threshold, Ct)值为纵坐标, 绘制qPCR标准曲线, 根据回归公式和qPCR检测Ct值计算未知样品的靶基因拷贝数。
基因组DNA的拷贝浓度按公式(1)计算:
细菌浓度(C)/(CFU/g)=基因拷贝数/L= [m×10^(-9)]/(n×660)×(6.02×10^23)
式中: m代表核酸蛋白仪测得的核酸浓度, ng/L; n代表细菌基因组的长度, bp, 根据NCBI上已经发布的测序数据, 植物乳杆菌基因组平均长度为3.23×10^6 bp, 动物双歧杆菌基因组平均长度为1.94×10^6 bp。
1.3.6 平板计数
将经过10倍系列稀释后的菌液各取1 mL加入无菌平皿中, 每个平皿加入15~20 mL MRS琼脂培养基, 在厌氧条件下于36 ℃培养48 h±2 h。选取菌落数在30~300 CFU之间、无蔓延菌落生长的平板计数来确定菌落总数, 每个稀释度的菌落数应取2个平板计数结果的平均值, 最终得到植物乳杆菌的计数结果(CFU/mL)。在选择2~3个连续适宜的稀释度后, 吸取1 mL样品匀液于灭菌平皿中, 每个稀释度需做两个平皿。将稀释液移入平皿后, 将冷却至48~50 ℃的莫匹罗星锂盐和半胱氨酸盐酸盐MRS琼脂培养基倾倒入平皿15~20 mL, 转动平皿均匀混合。培养基凝固后倒置于36 ℃厌氧条件下培养48 h, 可得双歧杆菌的计数结果(CFU/mL)。
1.3.7 人工添加样品及发酵乳样品的检测
选择无植物乳杆菌、双歧杆菌添加的发酵乳样品作为添加基质, 基质中添加了德氏乳杆菌保加利亚亚种、唾液链球菌嗜热亚种。采用传统的平板计数和PMA-qPCR方法分别检测发酵乳制品中活菌数。为保证发酵乳制品中存在的PCR抑制剂不会影响qPCR的结果, 称取待测发酵乳样品1 g, 经PBS缓冲液稀释50~100倍后, 取1 mL加入到2 mL离心管中, 按照1.3.1制备受损菌液后, 细菌基因组提取试剂盒说明书进行DNA的提取, 最终将核酸溶解在60 μL TE [Tris-乙二胺四乙酸(Tris-ethylenediaminetetraacetic acid, Tris-EDTA)]缓冲液中。用核酸蛋白仪测定DNA浓度和纯度, 确保提取A260/A280在1.7~2.0之间。
1.4 数据处理
实际样本验证时, 采用SPSS 26.0软件对实验数据进行统计学分析, 两组数据间的比较采用独立样本T检验进行分析, P<0.05时存在显著性差异。
2 结果与分析
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2.1 PMA实验条件的确认
通过热致死法分别制备动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III的受损菌, 加入PMA处理, 并对PMA工作浓度、曝光时间等处理条件进行优化。由表3、图1和图2可看出, 最终确定区分PMA处理受损菌的最佳质量浓度为10 μg/mL, 处理菌液样品的最佳曝光时间为10 min, 处理发酵乳样品的最佳曝光时间需要延长至20 min。导致PMA处理时间差异的原因可能是发酵乳中蛋白质会影响蓝光的穿透率, 进而干扰PMA处理效果。
2.2 植物乳杆菌和双歧杆菌标准曲线的建立
将含有德氏乳杆菌保加利亚亚种、唾液链球菌嗜热亚种基础发酵乳培养6 h后, 分别添加高、中、低不同浓度的动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III标准菌株培养物, 混合物经PMA处理后, 提取核酸, 进行荧光PCR反应。以不同拷贝数的阳性模板的对数为横坐标、荧光定量PCR反应过程中达到的Ct值为纵坐标绘制标准曲线, 结果如图3和图4所示, 表明Ct值与Log (DNA拷贝数)存在良好的线性关系, 在培养物水平扩增的Ct值均未受到影响, 抗干扰能力较好。纯菌液稀释的最低浓度可达103 CFU/mL, 而实际发酵乳中样品稀释的最低浓度是103 CFU/mL, 低于103 CFU/mL时荧光信号杂乱, 因而该方法中发酵乳里动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III的最低检出限均为103 CFU/mL。
2.3 实际样品的检测及重复性验证
动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III的标准菌株培养至108 CFU/mL, 梯度稀释并提取DNA做阳性模板, PMA处理后, 提取总DNA, 进行荧光PCR扩增。以标准菌株浓度的对数值作为横坐标, 以对应的Ct值作为纵坐标, 绘制标准曲线。得出模板质量与Ct值之间的关系公式, 并求出R2值。不同批次发酵乳样品经PMA处理后, 提取总DNA为模板, 进行荧光PCR反应。将扩增所得Ct值带入标准曲线方程, 并根据公式推算出实际样品中所添加的菌种数量。同时采用传统的平板计数法、PMA-qPCR的方法同时检测不同批次发酵乳储存期间的活菌数变化, 结果如表4和表5所示。由于传统的平板计数法无法检测活的非可培养(viable but non-culturable, VBNC)状态下的细菌, 因此利用qPCR检测活菌数通常会略微高于平板计数。VBNC状态是细菌的一种存活方式, 处于该状态下的细菌, 往往无法在常规培养基上形成菌落, 但仍具有代谢活性。在特定条件下, 处于VBNC状态的细菌可以重新激活、恢复在培养基上形成菌落的能力, 并恢复其原有的致病性[41]。
根据已建立的方法进行实际样品的检验, 不同批次的产品均可以同时检出动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III, 检测值与平板计数之间的差异较小, 证明在符合方法质控的要求下, 实验室实际达到的荧光PCR法重复性符合实验室规定的要求, 可准确地检测出样品中添加的活菌数量。
实际样品检测结果表明, PMA处理后的荧光pCR活菌数检出的样品结果与平板培养法结果无明显差异, 证明建立的方法是可行的。本研究建立了PMA-PCR计数方法, 并用此方法对常见的发酵乳体系进行检测, 将平板计数法的检测结果与两者对产品中活菌的检测结果进行对比, 最终实验结果证明该方法可以准确、快速地检测样品中的活菌数, 并准确地检测出植物乳杆菌和双歧杆菌的含量。检测准确性高、重复性好, 与国标法对比, 两种检测结果与平板计数法一致, 该方法可以用于发酵乳中植物乳杆菌和双歧杆菌的定量检测, 成为企业监控发酵乳中乳酸菌含量的快速检测方法。
3 结论与讨论
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本研究采用PMA和qPCR相结合的方法, 成功建立了同时快速检测发酵乳中动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III的定量方法。动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III经过PMA处理后, 动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III受损菌的最佳质量浓度为10 μg/mL, 处理菌液样品的最佳曝光时间为10 min。检测结果与传统的平板计数法无明显差异。在方法优化过程中, 研究人员设计了特异性基因序列的引物探针, 并验证了其特异性。通过优化PMA反应条件、建立标准曲线以及验证灵敏度, 研究人员成功实现了对发酵乳样品中动物双歧杆菌乳亚种BB-12和植物乳植杆菌ST-III菌株数量的定量检测。
本研究为发酵乳产品中益生菌的定量检测提供了一种快速、准确的方法, 检测效率高、重复性好、准确度高, 并为相关领域的研究和实践带来了新的启示, 为进一步全面开展发酵乳、奶酪等乳制品甚至整个食品领域中乳酸菌的定量分析提供了数据支撑。
基金
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  • 国家重点研发计划项目(2022YFD2100705)
  • 市国资委能级提升项目(2022013)
文献
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2025年第16卷第7期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20240830004
  • 接收时间：2024-08-30
  • 首发时间：2025-07-19
  • 出版时间：2025-04-15
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  • 收稿日期：2024-08-30
基金
国家重点研发计划项目(2022YFD2100705)
市国资委能级提升项目(2022013)
作者信息
    1.乳业生物技术国家重点实验室, 上海乳业生物工程技术研究中心, 光明乳业股份有限公司乳业研究院, 上海 200436
    2.华测检测认证集团股份有限公司, 深圳 518101

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* 张玉律(1990—), 女, 硕士, 中级工程师, 主要研究方向为乳品科学与加工、乳酸菌基础研究。E-mail:
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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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