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Article(id=1216517519406449656, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1216517514570417012, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20250422002, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1745251200000, receivedDateStr=2025-04-22, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1767969978429, onlineDateStr=2026-01-09, pubDate=1755187200000, pubDateStr=2025-08-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1767969978429, onlineIssueDateStr=2026-01-09, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1767969978429, creator=13701087609, updateTime=1767969978429, updator=13701087609, issue=Issue{id=1216517514570417012, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='15', 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=null, createTime=1767969977276, creator=13701087609, updateTime=1768211590858, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1217530915467743720, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1216517514570417012, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1217530915467743721, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1216517514570417012, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=308, endPage=314, ext={EN=ArticleExt(id=1216517519750381588, articleId=1216517519406449656, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Optimization of incubation time and plate load capacity for molds enumeration, columnId=1151895321388347923, journalTitle=Journal of Food Safety & Quality, columnName=Food Analysis and Detection, runingTitle=null, highlight=null, articleAbstract=

Objective To study on the improvement of incubation time and plate load capacity for molds enumeration. Methods Mold quality control samples were tested according to GB 4789.15—2016 National food safety standard-Food microbiological examination-Enumeration of moulds and yeasts, with additional colony counting performed on the second day of incubation. The plate size was increased from 90 mm to 150 mm, and statistical analysis was employed to assess the differences between colony counts and the reference value of the quality control samples. Results Significant discrepancies were observed between the fifth-day counting result at the critical dilution (1:10) and the reference value. A sharp decline in colony counts was noted when the colony-forming units exceeded 30 CFU per 90 mm plate, which was defined in this study as the maximum load capacity of mold plates under acceptable accuracy. By adopting 150 mm plates with a 2-day incubation period, the original load capacity was overcome, yielding counting results that aligned well with the reference value. Under these optimized conditions, the linear coefficient of determination (R²) between sample means and dilution factors improved from R290=0.614 to R2150=0.998. Conclusion The optimized testing conditions effectively addressed the issues of colony overgrowth and poor linearity in mold plate count experiments, thereby enhancing the accuracy of test results.

, correspAuthors=Shuo 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=Shuo ZHANG, Sha-Sha HAN), CN=ArticleExt(id=1216517522690588850, articleId=1216517519406449656, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=霉菌平板计数培养时间和平板承载能力优化研究, 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 霉菌平板计数培养时间和平板承载能力的改进研究。方法 使用GB 4789.15—2016《食品安全国家标准 食品微生物学检验 霉菌和酵母计数》检验霉菌质控样品, 增加培养至第2 d的菌落计数, 将平板规格由90 mm变为150 mm, 并使用统计分析检验菌落计数结果与质控样品参考值之间的差异。结果 关键稀释度(1:10)第5 d计数结果与参考值之间存在显著差异; 单个90 mm平板在菌落形成单位超过30 CFU后会出现菌落计数的大幅衰减, 本次研究将该阈值定义为可接受准确度下的霉菌平板承载能力。使用150 mm规格平板, 2 d培养时间的实验条件可突破原有承载能力, 使计数结果与参考值之间呈现良好的一致性。在该条件下各样本均值与稀释因子之间线性决定系数由R290=0.614提升至R2150=0.998。结论 优化后的检验条件解决了霉菌平板计数实验中菌落过度生长和线性不佳的问题, 提升了检验结果的准确度。

, correspAuthors=张烁, authorNote=null, correspAuthorsNote=
*张烁(1983—), 男, 高级工程师, 主要研究方向为食品安全检测和检验检测机构质量管理。E-mail:
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Springer US, 2009., articleTitle=null, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1217127900663436044, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, xref=null, ext=[AuthorCompanyExt(id=1217127900676018959, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, companyId=1217127900663436044, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Jinan Center for Food and Drug Control, Jinan 250102, China), AuthorCompanyExt(id=1217127900688601872, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, companyId=1217127900663436044, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=济南市食品药品检验检测中心, 济南 250102)])], figs=[ArticleFig(id=1217127904106959826, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=EN, label=Fig.1, caption=Decay in colony-forming units, figureFileSmall=IAM/quNXjrHSAEzHBW6CkQ==, figureFileBig=bfTP1KnFQMcYD7HctAnnUw==, tableContent=null), ArticleFig(id=1217127904228594648, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=CN, label=图1, caption=菌落计数的衰减, figureFileSmall=IAM/quNXjrHSAEzHBW6CkQ==, figureFileBig=bfTP1KnFQMcYD7HctAnnUw==, tableContent=null), ArticleFig(id=1217127904371200994, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=EN, label=Fig.2, caption=Comparative analysis of 2 day testing data (90 mm mold plates), figureFileSmall=7ri7m1Gc+GR6OH/c8YUk0g==, figureFileBig=KWM/VK4aegzyrsMSVDHr+A==, tableContent=null), ArticleFig(id=1217127904509613036, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=CN, label=图2, caption=2 d检验数据比对分析(90 mm霉菌平板), figureFileSmall=7ri7m1Gc+GR6OH/c8YUk0g==, figureFileBig=KWM/VK4aegzyrsMSVDHr+A==, tableContent=null), ArticleFig(id=1217127904618664947, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=EN, label=Fig.3, caption=Comparative analysis of 2 day testing data (150 mm mold plates), figureFileSmall=YZ28qBdme4LVgmXPY4hl6g==, figureFileBig=RPuNVgccfxgM+06JA5XEYw==, tableContent=null), ArticleFig(id=1217127904710939641, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=CN, label=图3, caption=2 d检验数据比对分析(150 mm霉菌平板), figureFileSmall=YZ28qBdme4LVgmXPY4hl6g==, figureFileBig=RPuNVgccfxgM+06JA5XEYw==, tableContent=null), ArticleFig(id=1217127904819991551, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=EN, label=Fig.4, caption=Linear graph of 90 mm mold plates, figureFileSmall=PdEwyA0DI75j3wv1PIX8fA==, figureFileBig=D7XloQ/LSF/XmDSfLxBfbA==, tableContent=null), ArticleFig(id=1217127904966791179, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=CN, label=图4, caption=90 mm霉菌平板线性图, figureFileSmall=PdEwyA0DI75j3wv1PIX8fA==, figureFileBig=D7XloQ/LSF/XmDSfLxBfbA==, tableContent=null), ArticleFig(id=1217127905105203221, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=EN, label=Fig.5, caption=Linear graph of 150 mm mold plates, figureFileSmall=8ZxX2AqGFGklnrgU5n3QFA==, figureFileBig=9JpfZrVIIqmZa1U/fCpMjg==, tableContent=null), ArticleFig(id=1217127905256198172, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=CN, label=图5, caption=150 mm霉菌平板线性图, figureFileSmall=8ZxX2AqGFGklnrgU5n3QFA==, figureFileBig=9JpfZrVIIqmZa1U/fCpMjg==, tableContent=null), ArticleFig(id=1217127905356861478, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=EN, label=Table 1, caption=

Statistical measures and parameters involved in the study

, figureFileSmall=null, figureFileBig=null, tableContent=
名称 表述 作用
均值 某个稀释度各平板上菌落数的算术平均值 表示某个稀释度直接的计数结果集中趋势
实际均值 均值与相应稀释倍数的乘积 表示稀释度统一之后的计数结果集中趋势
实际参考值 质控样品证书给出的参考值, 固定为270 CFU/mL 用于和实际均值作比较
参考值 实际参考值与稀释因子(1/稀释倍数)的乘积 用于和均值作比较
实际均值误差 实际均值与实际参考值之差的绝对值 误差越小则实验结果正确度更高
), ArticleFig(id=1217127905457524785, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=CN, label=表1, caption=

研究涉及的统计量和参数

, figureFileSmall=null, figureFileBig=null, tableContent=
名称 表述 作用
均值 某个稀释度各平板上菌落数的算术平均值 表示某个稀释度直接的计数结果集中趋势
实际均值 均值与相应稀释倍数的乘积 表示稀释度统一之后的计数结果集中趋势
实际参考值 质控样品证书给出的参考值, 固定为270 CFU/mL 用于和实际均值作比较
参考值 实际参考值与稀释因子(1/稀释倍数)的乘积 用于和均值作比较
实际均值误差 实际均值与实际参考值之差的绝对值 误差越小则实验结果正确度更高
), ArticleFig(id=1217127906774536249, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=EN, label=Table 2, caption=

Enumeration results of incubation time validation experiment

, figureFileSmall=null, figureFileBig=null, tableContent=
实验编号(实验类别-计数时间-稀释度) 均值/CFU 参考值/CFU 实际均值/CFU 实际均值误差/CFU 菌落直径/mm
1-2 d-1:2 47.75±3.85 135.00 95.50 174.50 11.4±1.8
1-2 d-1:3 36.38±4.53 90.00 109.13 160.88 11.5±2.3
1-2 d-1:4 34.75±4.65 68.00 139.00 131.00 12.6±1.7
1-2 d-1:10 24.63±3.38 27.00 246.25 23.75 12.0±1.9
1-2 d-1:25 10.75±2.19 11.00 268.75 1.25 12.5±2.3
1-5 d-1:3 28.75±4.83 90.00 86.25 183.75 致密边缘相交
1-5 d-1:4 28.38±4.98 68.00 113.50 156.50 致密边缘相交
1-5 d-1:10 20.13±1.55 27.00 201.25 68.75 25.5±3.0
1-5 d-1:25 10.38±2.07 11.00 259.38 10.63 28.0±2.8
), ArticleFig(id=1217127906912948290, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=CN, label=表2, caption=

培养时间验证实验计数结果

, figureFileSmall=null, figureFileBig=null, tableContent=
实验编号(实验类别-计数时间-稀释度) 均值/CFU 参考值/CFU 实际均值/CFU 实际均值误差/CFU 菌落直径/mm
1-2 d-1:2 47.75±3.85 135.00 95.50 174.50 11.4±1.8
1-2 d-1:3 36.38±4.53 90.00 109.13 160.88 11.5±2.3
1-2 d-1:4 34.75±4.65 68.00 139.00 131.00 12.6±1.7
1-2 d-1:10 24.63±3.38 27.00 246.25 23.75 12.0±1.9
1-2 d-1:25 10.75±2.19 11.00 268.75 1.25 12.5±2.3
1-5 d-1:3 28.75±4.83 90.00 86.25 183.75 致密边缘相交
1-5 d-1:4 28.38±4.98 68.00 113.50 156.50 致密边缘相交
1-5 d-1:10 20.13±1.55 27.00 201.25 68.75 25.5±3.0
1-5 d-1:25 10.38±2.07 11.00 259.38 10.63 28.0±2.8
), ArticleFig(id=1217127907063943246, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=EN, label=Table 3, caption=

Single-sample t-test analysis of incubation time validation study

, figureFileSmall=null, figureFileBig=null, tableContent=
单样本t检验 差值95%置信区间 正态性检验(夏皮洛-威尔克)
实验编号 t 自由度 双尾 平均值差值 下限 上限 统计 自由度 显著性
1-2 d-1:2 -64.178 7 0.00 -174.50 -180.92 -168.07 0.84 8 0.08
1-2 d-1:3 -33.456 7 0.00 -160.88 -172.25 -149.50 0.91 8 0.36
1-2 d-1:4 -19.911 7 0.00 -131.00 -146.56 -115.44 0.95 8 0.74
1-2 d-1:10 -1.989 7 0.09 -23.75 -51.99 4.49 0.86 8 0.13
1-2 d-1:25 -.065 7 0.95 -1.25 -46.97 44.47 0.96 8 0.83
1-5 d-1:3 -35.846 7 0.00 -183.75 -195.87 -171.62 0.87 8 0.14
1-5 d-1:4 -22.204 7 0.00 -156.50 -173.17 -139.83 0.90 8 0.26
1-5 d-1:10 -12.524 7 0.00 -68.75 -81.73 -55.77 0.95 8 0.73
1-5 d-1:25 -.582 7 0.58 -10.63 -53.80 32.55 0.94 8 0.62
), ArticleFig(id=1217127907189772370, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=CN, label=表3, caption=

培养时间验证实验的单样本t检验结果

, figureFileSmall=null, figureFileBig=null, tableContent=
单样本t检验 差值95%置信区间 正态性检验(夏皮洛-威尔克)
实验编号 t 自由度 双尾 平均值差值 下限 上限 统计 自由度 显著性
1-2 d-1:2 -64.178 7 0.00 -174.50 -180.92 -168.07 0.84 8 0.08
1-2 d-1:3 -33.456 7 0.00 -160.88 -172.25 -149.50 0.91 8 0.36
1-2 d-1:4 -19.911 7 0.00 -131.00 -146.56 -115.44 0.95 8 0.74
1-2 d-1:10 -1.989 7 0.09 -23.75 -51.99 4.49 0.86 8 0.13
1-2 d-1:25 -.065 7 0.95 -1.25 -46.97 44.47 0.96 8 0.83
1-5 d-1:3 -35.846 7 0.00 -183.75 -195.87 -171.62 0.87 8 0.14
1-5 d-1:4 -22.204 7 0.00 -156.50 -173.17 -139.83 0.90 8 0.26
1-5 d-1:10 -12.524 7 0.00 -68.75 -81.73 -55.77 0.95 8 0.73
1-5 d-1:25 -.582 7 0.58 -10.63 -53.80 32.55 0.94 8 0.62
), ArticleFig(id=1217127907311407194, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=EN, label=Table 4, caption=

Enumeration results of mold plate carrying capacity experiment (CFU)

, figureFileSmall=null, figureFileBig=null, tableContent=
实验编号(实验类别-平板规格-稀释度) 均值 参考值 实际均值 实际均值误差
2-90-1:4 34.00±3.27 68.00 136.00 134.00
2-90-1:8 30.10±6.06 34.00 240.80 29.20
2-90-1:9 30.90±5.30 30.00 278.10 8.10
2-90-1:10 26.30±3.23 27.00 259.00 7.00
2-90-1:25 10.30±1.16 11.00 257.50 12.50
2-150-1:4 65.80±6.34 68.00 263.20 6.80
2-150-1:8 35.00±2.40 34.00 271.20 10.00
2-150-1:9 29.60±7.38 30.00 266.40 3.60
2-150-1:10 28.00±3.23 27.00 280.00 10.00
2-150-1:25 10.90±1.45 11.00 272.50 2.50
), ArticleFig(id=1217127907470790757, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=CN, label=表4, caption=

霉菌平板承载能力实验计数结果(CFU)

, figureFileSmall=null, figureFileBig=null, tableContent=
实验编号(实验类别-平板规格-稀释度) 均值 参考值 实际均值 实际均值误差
2-90-1:4 34.00±3.27 68.00 136.00 134.00
2-90-1:8 30.10±6.06 34.00 240.80 29.20
2-90-1:9 30.90±5.30 30.00 278.10 8.10
2-90-1:10 26.30±3.23 27.00 259.00 7.00
2-90-1:25 10.30±1.16 11.00 257.50 12.50
2-150-1:4 65.80±6.34 68.00 263.20 6.80
2-150-1:8 35.00±2.40 34.00 271.20 10.00
2-150-1:9 29.60±7.38 30.00 266.40 3.60
2-150-1:10 28.00±3.23 27.00 280.00 10.00
2-150-1:25 10.90±1.45 11.00 272.50 2.50
), ArticleFig(id=1217127907617591402, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=EN, label=Table 5, caption=

Single sample t-test of mold plate carrying capacity experiment

, figureFileSmall=null, figureFileBig=null, tableContent=
单样本t检验 差值95%置信区间 正态性检验(Shapiro-Wilk)
实验编号 t 自由度 双尾 平均值差值 下限 上限 统计 自由度 显著性
2-90-1:4 -32.436 9 0.00 -134.00 -143.35 -124.65 0.93 10 0.45
2-90-1:8 -1.904 9 0.09 -29.20 -63.90 5.50 0.90 10 0.19
2-90-1:9 0.537 9 0.60 8.10 -26.03 42.23 0.95 10 0.61
2-90-1:10 -0.685 9 0.51 -7.00 -30.13 16.13 0.97 10 0.88
2-90-1:25 -1.364 9 0.21 -12.50 -33.24 8.24 0.88 10 0.12
2-150-1:4 -0.848 9 0.42 -6.80 -24.94 11.34 0.95 10 0.72
2-150-1:8 1.644 9 0.13 10.00 -3.76 23.76 0.98 10 0.94
2-150-1:9 -0.171 9 0.87 -3.60 -51.12 43.92 0.85 10 0.06
2-150-1:10 0.978 9 0.35 10.00 -13.12 33.12 0.96 10 0.83
2-150-1:25 0.218 9 0.83 2.50 -23.42 28.42 0.86 10 0.08
), ArticleFig(id=1217127907747614835, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=CN, label=表5, caption=

霉菌平板承载能力实验的单样本t检验

, figureFileSmall=null, figureFileBig=null, tableContent=
单样本t检验 差值95%置信区间 正态性检验(Shapiro-Wilk)
实验编号 t 自由度 双尾 平均值差值 下限 上限 统计 自由度 显著性
2-90-1:4 -32.436 9 0.00 -134.00 -143.35 -124.65 0.93 10 0.45
2-90-1:8 -1.904 9 0.09 -29.20 -63.90 5.50 0.90 10 0.19
2-90-1:9 0.537 9 0.60 8.10 -26.03 42.23 0.95 10 0.61
2-90-1:10 -0.685 9 0.51 -7.00 -30.13 16.13 0.97 10 0.88
2-90-1:25 -1.364 9 0.21 -12.50 -33.24 8.24 0.88 10 0.12
2-150-1:4 -0.848 9 0.42 -6.80 -24.94 11.34 0.95 10 0.72
2-150-1:8 1.644 9 0.13 10.00 -3.76 23.76 0.98 10 0.94
2-150-1:9 -0.171 9 0.87 -3.60 -51.12 43.92 0.85 10 0.06
2-150-1:10 0.978 9 0.35 10.00 -13.12 33.12 0.96 10 0.83
2-150-1:25 0.218 9 0.83 2.50 -23.42 28.42 0.86 10 0.08
), ArticleFig(id=1217127907881832570, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=EN, label=Table 6, caption=

Linear comparison of results between 90 mm and 150 mm mold plates

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模型摘要 参数估算值
平板规格参数 R2 F 显著性 常量 系数
90 mm 0.614 4.771 0.117 14.089 96.735
150 mm线性 0.998 1984.424 0.000 1.413 260.278
), ArticleFig(id=1217127907986690181, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517519406449656, language=CN, label=表6, caption=

90 mm与150 mm霉菌平板结果的线性比较

, figureFileSmall=null, figureFileBig=null, tableContent=
模型摘要 参数估算值
平板规格参数 R2 F 显著性 常量 系数
90 mm 0.614 4.771 0.117 14.089 96.735
150 mm线性 0.998 1984.424 0.000 1.413 260.278
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霉菌平板计数培养时间和平板承载能力优化研究
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张烁 * , 韩莎莎
食品安全质量检测学报 | 食品分析与检测 2025,16(15): 308-314
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食品安全质量检测学报 | 食品分析与检测 2025, 16(15): 308-314
霉菌平板计数培养时间和平板承载能力优化研究
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张烁* , 韩莎莎
作者信息
  • 济南市食品药品检验检测中心, 济南 250102

通讯作者:

*张烁(1983—), 男, 高级工程师, 主要研究方向为食品安全检测和检验检测机构质量管理。E-mail:
Optimization of incubation time and plate load capacity for molds enumeration
Shuo ZHANG* , Sha-Sha HAN
Affiliations
  • Jinan Center for Food and Drug Control, Jinan 250102, China
出版时间: 2025-08-15 doi: 10.19812/j.cnki.jfsq11-5956/ts.20250422002
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摘要
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目的 霉菌平板计数培养时间和平板承载能力的改进研究。方法 使用GB 4789.15—2016《食品安全国家标准 食品微生物学检验 霉菌和酵母计数》检验霉菌质控样品, 增加培养至第2 d的菌落计数, 将平板规格由90 mm变为150 mm, 并使用统计分析检验菌落计数结果与质控样品参考值之间的差异。结果 关键稀释度(1:10)第5 d计数结果与参考值之间存在显著差异; 单个90 mm平板在菌落形成单位超过30 CFU后会出现菌落计数的大幅衰减, 本次研究将该阈值定义为可接受准确度下的霉菌平板承载能力。使用150 mm规格平板, 2 d培养时间的实验条件可突破原有承载能力, 使计数结果与参考值之间呈现良好的一致性。在该条件下各样本均值与稀释因子之间线性决定系数由R290=0.614提升至R2150=0.998。结论 优化后的检验条件解决了霉菌平板计数实验中菌落过度生长和线性不佳的问题, 提升了检验结果的准确度。

霉菌平板计数  /  准确度  /  培养时间  /  平板承载能力

Objective To study on the improvement of incubation time and plate load capacity for molds enumeration. Methods Mold quality control samples were tested according to GB 4789.15—2016 National food safety standard-Food microbiological examination-Enumeration of moulds and yeasts, with additional colony counting performed on the second day of incubation. The plate size was increased from 90 mm to 150 mm, and statistical analysis was employed to assess the differences between colony counts and the reference value of the quality control samples. Results Significant discrepancies were observed between the fifth-day counting result at the critical dilution (1:10) and the reference value. A sharp decline in colony counts was noted when the colony-forming units exceeded 30 CFU per 90 mm plate, which was defined in this study as the maximum load capacity of mold plates under acceptable accuracy. By adopting 150 mm plates with a 2-day incubation period, the original load capacity was overcome, yielding counting results that aligned well with the reference value. Under these optimized conditions, the linear coefficient of determination (R²) between sample means and dilution factors improved from R290=0.614 to R2150=0.998. Conclusion The optimized testing conditions effectively addressed the issues of colony overgrowth and poor linearity in mold plate count experiments, thereby enhancing the accuracy of test results.

mold enumeration  /  accuracy  /  incubation time  /  plate load capacity
张烁, 韩莎莎. 霉菌平板计数培养时间和平板承载能力优化研究. 食品安全质量检测学报, 2025 , 16 (15) : 308 -314 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250422002
Shuo ZHANG, Sha-Sha HAN. Optimization of incubation time and plate load capacity for molds enumeration[J]. Journal of Food Safety & Quality, 2025 , 16 (15) : 308 -314 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250422002
正文
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0 引言
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霉菌是造成食品污染的主要生物类因素之一[1-3], 该类微生物不但可以造成食品霉变腐败, 某些种类的霉菌生成的生物毒素也会对食用者的身体健康造成很大的危害[4-6], 所以霉菌指标一直是食品安全工作中一个重要的组成部分。随着科技的发展, 霉菌检测领域出现了一些新兴的快速测试方法[7-8], 如PCR技术[9-10]、无损检测技术[11]、高光谱检测技术[12], 但现阶段这些方法实验室通用性往往不高且大幅增加了实验室在设备、耗材、环境方面的成本[13-14]。为保证实验室通用性, 我国食品安全监督抽检工作规定其霉菌项目使用基于平板计数法的GB 4789.15—2016《食品安全国家标准 食品微生物学检验 霉菌和酵母计数》进行检验。由于霉菌是抽检工作中重要的微生物指标菌[15-16], 相关检验检测机构应当积极提升该项目的检验质量, 防止霉菌超标的食品对消费者的身体健康造成危害。
检验结果的准确度是评定霉菌检验质量的重要参数[17]。准确度是测试结果与接受参照值间的一致程度, 包含正确度和精密度两方面含义。为避免由于霉菌的生物特性造成计数结果准确度降低, GB 4789.15—2016《食品安全国家标准 食品微生物学检验 霉菌和酵母计数》在之前版本的基础上对检验条件做出了优化[18], 但在检验实践中该标准所依据的平板计数法依然存在一定的问题。本实验室在进行霉菌能力验证时发现培养48 h后霉菌菌落已基本形成, 菌落直径随培养时间延长逐渐增大, 菌落之间出现界限不清或相互叠加, 进而造成计数结果的衰减。在培养时间方面, 国标中“观察并记录培养至第5 d的结果”的表述较为模糊, 未明确是否需多次观察、观察的起始时间以及如果多次观察的结果之间出现差异应最终报出哪个结果。
通过横向比较可以发现现行2016版国标与相关国际、国外标准存在检验条件和过程细节上的不同, 主要表现在试剂种类、水活度、培养温度和方式、观察时间和次数、霉菌和酵母形态描述等方面[19]。ISO 21527-1:2008, 规定在霉菌生长过快时可在培养的第2 d、第5 d分别计数菌落。BAM Chapter 18: 2001规定如果平板上存在大量霉菌则150 CFU的计数上限可能会降低[20], 而无论是该标准还是GB 4789.15—2016均未对霉菌单独的计数上限作出确定的要求。ISO 6611│IDF 94: 2004认为霉菌的计数方法并不精确, 各稀释度检验结果常出现非线性, 这与霉菌孢子在稀释时的均匀程度、大量菌落产生竞争抑制作用等因素有关。该标准建议通过分散进样的方式降低单个平板的霉菌数量, 在产生色素的菌落形态下计数培养第2 d和第5 d的结果以应对霉菌快速生长。通过以上标准可以看到不确定性因素较少的低稀释度由于霉菌蔓延或可能存在的计数上限等问题无法被准确计数, 稀释度的提高会引入更多的不确定性因素, 最终也会影响结果的准确度[21-22]。
综上所述, 目前基于平板计数法的霉菌检验仍存在影响检验结果准确度的各种问题, 而现有的相关研究主要关注培养基的改良以及温湿度、样品匀质等条件[23-24], 较少有针对培养时间和平板计数上限的优化和验证, 且文献中对计数上限的描述也较为模糊。本次研究的创新性在于给出计数上限明确的定义并优化了被先前研究忽视的培养时间和平板规格等检验条件。由于食品安全抽检任务中微生物学项目具有不可复检性, 当检测结果处于临界值时, 其准确度对产品能否被正确判定具有重要影响。为最终达到提高霉菌检验结果准确度的研究目的, 本次研究确定了两阶段实验的研究目标: 一是验证对于食品中的霉菌检验, 尤其是高污染样品, 第2 d的计数结果比第5 d的准确度更高; 二是探索在可接受准确度下霉菌平板计数上限的阈值以及提高该上限的方法。以期通过优化检验条件提升霉菌平板计数结果的准确度, 并维护相关消费者和食品生产经营者合法权益。
1 材料与方法
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1.1 材料与试剂
孟加拉红琼脂[25-26](北京陆桥技术股份有限公司); 氯化钠(分析纯, 国药集团化学试剂有限公司); 培养皿(90 mm、150 mm, 青岛金典生化器材有限公司); 霉菌、酵母计数质控样品[27](FTQC-15-01, 中国工业微生物菌种保藏管理中心)。
1.2 仪器与设备
KB 240型低温培养箱(±0.1 ℃, 德国Binder公司); BSC-1300IIA2型生物安全柜(苏州安泰空气技术有限公司); CJY1500-Y型洁净工作台(山东新华医疗器械股份有限公司); BAGMIXER® 400型均质器(法国Interscience公司); MLS-3781L-PC型高压蒸汽灭菌器(日本松下健康医疗器械株式会社); CzoneG6T型菌落计数器(杭州迅数科技有限公司)。
1.3 实验方法
本研究使用GB 4789.15—2016作为基础检验方法。第一阶段实验作为预实验, 控制霉菌培养时间条件, 2 d为实验组, 5 d为对照组, 利用单样本t检验分析两组实验各稀释度结果与参考值之间的差异。在第一阶段实验结果的基础上, 第二阶段实验固定培养时间2 d, 并控制平板规格条件, 150 mm规格为实验组, 90 mm规格为对照组, 利用单样本t检验分析两组实验各稀释度结果与参考值之间的差异, 进而使用回归分析证明优化后的实验条件有助于检验方法准确度的提升。本次研究所使用的样品为适用于食品中霉菌检验的CICC FTQC-15-01霉菌和酵母计数质控样品。该样品的微生物组成为黑曲霉和酿酒酵母, 其最大优点是具有精确的参考值。相较于难以获得准确定量的标准菌株, 该样品更加适合本次研究所使用的单样本t检验方法。
实验严格按质控样品说明书要求将原始样品加入无菌生理盐水复溶后制成待测样品。在第一阶段中, 为达到分散1:1、1:10菌液浓度的目的, 分别将待测液加入适量无菌生理盐水制成1:2、1:3、1:4、1:10、1:25稀释液。每一步稀释过程样品均反复吹吸以保证其匀质状态, 防止孢子沉积于容器底部。由于已知样品参考值为270 CFU/mL, 除原液外其他稀释度上的预估霉菌菌落数应在11 CFU至135 CFU之间, 符合国标中10~150 CFU的合理计数范围。在进行稀释的同时, 每个稀释度分别吸取1 mL样品匀液于90 mm无菌平皿内, 并做空白对照。将20 mL冷却至46 ℃的孟加拉红琼脂倾注平皿, 并转动平皿使其混合均匀。为防止交叉污染, 平板凝固后被封闭于无菌袋中, 于28 ℃±1 ℃培养箱中正置培养, 袋中保留适量空气以保证霉菌的正常生长[28]。分别观察并记录培养至第2 d和第5 d的结果, 实验仅计数霉菌菌落, 酵母则作为背景菌。在第二阶段研究中, 除稀释梯度变为1:4、1:8、1:9、1:10、1:25, 平板规格变为150 mm, 培养时间固定为2 d外其他操作与第一阶段相同。
为了更为科学的对数据进行分析, 本次研究对实验数据统计量及相关参数进行如表1所示定义。针对文献中平板上菌落计数上限较为模糊的描述, 本次研究在实验所涉及领域和条件范围之内给出“可接受准确度下的霉菌平板承载能力”的定义: 当使用平板计数法进行霉菌检测时, 在可接受准确度下单个平板上霉菌菌落形成单位的最大值, 如果进样时的霉菌数量大于该数值, 则最终的计数结果将会与相应参考值之间出现统计学显著差异。
1.4 数据处理
本次研究使用SPSS 26等软件进行统计分析、制图, 实验所涉及的置信度均为95%。
2 结果与分析
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2.1 霉菌培养时间优化的验证实验
本阶段研究目的在于验证霉菌培养2 d后计数的可行性, 为进一步的研究打下基础。为提升检验结果的准确度, 实验采用双人8平行测试[29]。各组样本采用“实验类别-计数时间-稀释度”的方式进行编号, 除样本1-2 d-1:1、1-5 d-1:1、1-5 d-1:2出现菌落蔓延导致无法计数外, 其他实验结果如表2所示。由表2内数据可知各样本实际均值误差随稀释度的增大而逐渐减少, 而同稀释度2 d时的实际均值误差均小于5 d时相应值。在计数的同时, 使用菌落计数器测量各平板上霉菌菌落直径。每个平板随机选取8个菌落测量, 被测菌落应尽可能为独立菌落, 其致密菌丝边缘不宜与其他菌落相交。实验中发现培养时间2 d时黑曲霉菌落呈白色绒毛状, 边界较为清晰, 较为适合测量。培养时间为5 d时1:3、1:4外虽然可以较为清晰地计数, 但由于菌落致密菌丝边缘开始出现融合而无法测量菌落直径。经统计所有菌落的直径均值为2 d: 12.0 mm±2.1 mm、5 d: 26.8 mm±3.1 mm。
本阶段数据的正态性检验和检验值为270(实际参考值)单样本t检验数据如表3所示。使用SPSS 26对实验数据进行分析发现各组数据中均不存在异常值, 正态性检验结果均未达到显著性水平(Shapiro-Wilk检验, P>0.05), 各组数据均服从正态分布。由各组数据的单样本t检验结果可知样本1-2 d-1:2、1-2 d-1:3、1-2 d-1:4各自的实际均值与270之间存在显著差异(P<0.05), 而样本1-2 d-1:10、1-2 d-1:25的实际均值与270均无显著差异(P>0.05)。特别值得注意的是, 1-2 d-1:25组的检验结果(P=0.95)表明, 1:25稀释度培养2 d, 平板上的平均菌落数为11 CFU时的实际均值(268.75)与270具有良好的一致性。
由样本1-5 d-1:3、1-5 d-1:4的单样本t检验结果(P<0.05)可见延长培养时间并不能提升其实际均值与270的一致程度。对比样本1-2 d-1:10检验结果(P=0.09), 样本1-5 d-1:10的检验结果(P<0.05)表明1:10稀释度实际均值随着培养时间的延长与270出现显著差异, 计数结果未出现偏离的稀释度范围进一步缩小。对比样本1-2 d-1:25检验结果(P=0.95), 样本1-5 d-1:25的检验结果(P=0.58)表明当稀释度提升至1:25之后, 单个平板上平均菌落数依然仅为10左右, 时间的延长对实际均值的影响被削弱。由此可见, 在此次实验中时间参数影响最大的稀释度是1:10, 由于菌落直径的增大和菌落之间的叠加, 平均菌落数由24.63降低到20.13。1:10稀释度是方法规定的稀释梯度之一, 可见霉菌平板计数法存在计数衰减的风险。如图1所示, 计数衰减可以用某个稀释度2、5 d两次计数的实际均值之差表示。
由于计数衰减现象的出现, 第5 d的计数结果实际上只有1:25可用, 而该稀释度也是本次实验计数衰减量最少的稀释度, 可见无论在可计数稀释度范围还是在各稀释度准确度方面霉菌在第2 d计数都要优于第5 d。在有限的稀释度下, 计数衰减峰值出现在1:10, 可以预估在可接受准确度下的霉菌平板承载能力可能出现在1:4与1:10平均菌落数之间, 即约25~35 CFU/皿。
2.2 可接受准确度下的霉菌平板承载能力的测定
为了更准确地测定该承载能力, 本阶段采用双人10平行测试。为了减少单位面积菌落数, 降低竞争抑制作用,实验使用150 mm平板作为实验组, 90 mm平板作为对照组, 两组的其他检验条件保持一致。平板直径的增大使实验组平板内培养基含量扩大至对照组的约2.8倍, 这增加了霉菌获得的营养量, 同时也降低了单位面积内的霉菌数量, 减少了菌落蔓延和相互叠加对计数结果影响的可能性。依据前一阶段实验结果, 本阶段稀释系列调整为1:4、1:8、1:9、1:10、1:25, 其中1:8和1:9为出现计数衰减峰值稀释度(1:10)的微调稀释度。各组样本采用“实验类别-平板规格-稀释度”方式进行编号。由如表4所示实验结果可知当使用150 mm规格平板后低稀释度样本的实际均值误差出现明显减小, 如1:4稀释度由134.00锐减到6.80。
本阶段数据的正态性检验和检验值为270的单样本t检验数据如表5所示。使用SPSS 26对实验数据进行分析后发现各样本数据中均不存在异常值, 正态性检验结果均未达到显著性水平(Shapiro-Wilk检验, P>0.05), 各组数据均服从正态分布。由样本2-90-1:4的单样本t检验结果可知在“90 mm平板、稀释度1:4”的实验条件下每个霉菌平板平均生成34个菌落, 与理论上的参考值68相差一倍, 而其实际均值也与270之间存在显著差异(P<0.05)。与之靠近的1:8稀释度的实际均值与270已无显著差异(P=0.09), 且每个平板平均生成30个菌落, 这与该稀释度参考值34的差距已经较小, 且均值的略微降低都有可能使P降低到0.05以下。
如图2所示, 将90 mm平板各稀释度均值与对应的参考值进行比较后发现, 当菌落计数均值为34时, 其仍与参考值差异很大, 而当其小于30以后, 数据开始呈现一致性。基于以上实验证据, 结合霉菌检验精确度较低的特点, 本次研究认为在可接受准确度下90 mm规格平板的霉菌承载能力为30 CFU/皿左右。
2.3 可接受准确度下的霉菌平板承载能力的改进
表4数据表明使用150 mm规格平板的实验组所有稀释度样本的实际均值与270均无显著差异(P>0.05), 如图3所示, 将150 mm平板各稀释度均值与对应的参考值进行比较, 可以发现各稀释度均值与参考值基本一致。尤其是原本具有30 CFU/皿承载能力的1:4稀释度, 其平均菌落数提升为66 CFU/皿, 原有的承载能力被突破。
针对之前的文献中提及霉菌检验结果线性不佳的情况对表4中各样本均值与相应稀释因子进行线性回归分析, 数据如表6所示。150 mm平板均值与稀释因子呈现强线性关系(R2=0.998, P<0.001), 相较于90 mm的线性不显著(R2=0.614, P=0.117)有了很大的提升, 图4、图5也直观的展现了这一对比结果。依据表6数据可以得出使用优化后的实验条件150 mm规格平板, 2 d培养时间进行霉菌质控样品检验的线性方程为: Y=1.413+260.278X, X为对应的稀释因子(1/稀释倍数); Y为计数结果的均值。
3 结论与讨论
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本次研究经过两阶段实验和数据分析得出以下结论: 在利用平板计数法检测霉菌质控样品时, 第2 d的计数结果比第5 d的准确度更高, 这主要表现在关键稀释度(1:10)的实际均值与实际参考值之间差异显著性的变化上(P2d=0.09, P5d<0.05)。90 mm规格平板存在可接受准确度下的霉菌平板承载能力(30 CFU/皿), 计数时如果发现某个平板上的霉菌数量大幅超过该数值, 则其计数结果并不能良好地反映进样时平板中的霉菌数量。使用150 mm规格平板, 2 d培养时间的实验条件可以有效突破原有承载能力, 使各稀释度样本实际均值与实际参考值间表现出更强的一致性, 并使各稀释度计数结果呈现良好的线性。
本次研究利用实验数据验证了文献中提及的培养48 h后菌丝会影响观察计数[30]的描述。JI等[31]利用点种法测量黑曲霉生长曲线, 其中2~4 d为霉菌指数生长期, 菌丝快速辐射状延伸, 菌落直径随之快速增大。本次研究验证了霉菌平板计数在进入指数生长期后即可计数的可行性。为了进一步验证承载能力的存在, 可利用几何学圆填充(Circle Packing)理论进行平板内霉菌菌落数最大理论值的估计。假设霉菌菌落为大小均一且具有12.0 mm(2 d时所有被测菌落直径均值)直径的正圆, 为防止菌落间距离较近造成菌落融合, 霉菌菌落应以不相交圆的形式在平板内排列。90 mm平板在此假设下容纳极值为42个菌落, 这与国标中150 CFU的计数上限相差明显。以上由几何估计的菌落数是在非常理想的圆排列下得出的, 而实际情况由于菌落的分散性, 实际值应比理论值还要小, 这也验证了本次研究得出在30 CFU左右承载能力的存在。由“150 mm规格平板, 2 d培养时间”条件下的线性方程可知, 在该条件下各稀释度的计数结果受稀释度的影响已经很小, 本次研究解决了ISO 6611—2004等标准提及的霉菌多稀释度结果线性不佳的问题, 该结论也可以推广到含有相似微生物组成的样品霉菌检验工作中, 如能力验证、测量审核等。
通过以上实验结论, 本次研究还发现了一些可供探讨的问题。例如当使用90 mm平板进行霉菌和酵母共同计数时宜单独限制霉菌的有效计数范围, 直接套用GB 4789.15—2016中10~150 CFU的区间是具有风险的; 霉菌检验在计数时应选取菌落数量适当的平板, 实验室可以利用调节稀释度、分散进样等技术手段尽可能将每个平板上的菌落数控制在承载能力之内。由于霉菌包含多种种属, 如曲霉属、青霉属等, 而此次使用的质控样品仅含有黑曲霉一类霉菌。尽管黑曲霉在食品霉菌检测相关工作中具有较强的代表性, 但本次研究所得出的结论是否适合其他含复杂微生物组成的样品, 以及是否还有除平板规格和培养时间之外的可提升霉菌平板承载能力的因素还需要进一步的研究。
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2025年第16卷第15期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20250422002
  • 接收时间：2025-04-22
  • 首发时间：2026-01-09
  • 出版时间：2025-08-15
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  • 收稿日期：2025-04-22
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    济南市食品药品检验检测中心, 济南 250102

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*张烁(1983—), 男, 高级工程师, 主要研究方向为食品安全检测和检验检测机构质量管理。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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