ATP生物发光法快速检测电子烟雾化液菌落总数
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摘要
利用三磷酸腺苷(ATP)生物发光法建立快速检测电子烟雾化液微生物菌落总数的方法并进行优化,与国家标准微生物检测方法进行对比分析。结果表明:(1)ATP荧光检测仪的最优检测温度为25℃,反应时间为30s,ATP检出限为2.36×10~(-12) mol/L,RSD为3.5%~8.2%,加标回收率为97%~108%;(2)滤膜过滤富集检测可实现低活菌数样品的检测,混合纤维素酯滤膜的富集检测效果优于聚碳酸酯滤膜和醋酸纤维滤膜,富集后检测ATP生物荧光的相对光度值与富集前活菌总数具有良好的对数线性相关关系(r~2>0.96);(3)采用ATP检测方法估算的活菌总数结果与平板计数检测结果没有显著性差异(P>0.05),对电子烟雾化液的卫生情况评价结果一致。ATP生物发光检测方法可快速计算电子烟雾化液菌落总数,对于电子烟安全卫生风险评估具有建设意义。
In order to fast evaluate the hygiene situation of electronic cigarette liquids,the fast examination of total colony numbers in electronic cigarette liquids has been achieved which was based on the adenosine triphosphate(ATP)bioluminescence method.The method has been optimized,and the examination results of total colony by ATP method was compared with the results by National standard microbial detection method.Results showed:(1)the optimal temperature for ATP examination was 25 ℃,optimal time for bioluminescence reaction was 30 s,the detection limit of ATP was 2.36 ×10~(-12) mol/L,RSD was 3.5%~8.2%,and the sample recovery rate was 97% ~108%.(2)Samples with low Colony-Forming Units(CFU)were examined after enriching with filtration membrane.Mixed cellulose esterfiltration membrane had better enrichment effect than poly-carbonate membrane and cellulose acetate membrane.It showed good logarithmically linear correlation between the fluorescent light after membrane filtration and thetotal colony before filtration(r~2>0.96).There was no significant deference on the total colony numbers calculated between the ATP method and standard plate count method(P>0.05).And the evaluation results of electronic cigarette liquids are identical.Therefore,ATP bioluminescence method could instantly calculate the total colony numbers in eliquids,which was constructive in safety and hygiene risk evaluation in electronic cigarette industry.
In order to fast evaluate the hygiene situation of electronic cigarette liquids,the fast examination of total colony numbers in electronic cigarette liquids has been achieved which was based on the adenosine triphosphate(ATP)bioluminescence method.The method has been optimized,and the examination results of total colony by ATP method was compared with the results by National standard microbial detection method.Results showed:(1)the optimal temperature for ATP examination was 25 ℃,optimal time for bioluminescence reaction was 30 s,the detection limit of ATP was 2.36 ×10~(-12) mol/L,RSD was 3.5%~8.2%,and the sample recovery rate was 97% ~108%.(2)Samples with low Colony-Forming Units(CFU)were examined after enriching with filtration membrane.Mixed cellulose esterfiltration membrane had better enrichment effect than poly-carbonate membrane and cellulose acetate membrane.It showed good logarithmically linear correlation between the fluorescent light after membrane filtration and thetotal colony before filtration(r~2>0.96).There was no significant deference on the total colony numbers calculated between the ATP method and standard plate count method(P>0.05).And the evaluation results of electronic cigarette liquids are identical.Therefore,ATP bioluminescence method could instantly calculate the total colony numbers in eliquids,which was constructive in safety and hygiene risk evaluation in electronic cigarette industry.
引文
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[7]PISTELOK F,POHL A,STUCZYNSKI T,et al.Using ATPtests for assessment of hygiene risks[J].Ecological Chemistry&Engineering S,2016,23(2):259-270.
[8]WHITELEY G S,DERRY C,GLASBEY T,et al.The perennial problem of variability in adenosine triphosphate(ATP)tests for hygiene monitoring within healthcare settings[J].Infection Control&Hospital Epidemiology,2015,36(6):658-663.
[9]MORRIS H C.The benefits of rapid microbiological testing of finished products using ATP bioluminescence[J].International Journal of Cosmetic Science,2010,20(1):63-67.
[10]CARRASCOSAA C,MILLA N R,JABER J R,et al.Monitoring of cleanliness and disinfection in dairies:Comparison of traditional microbiological and ATP bioluminescence methods[J].Food Control,2012,28(2):368-373.
[11]COSTA P D,ANDRADE N J D,PASSOS F J V.ATP-bioluminescence as a technique to evaluate the microbiological quality of water in food industry[J].Brazilian Archives of Biology&Technology,2004,47(3):399-405.
[12]HECKER W,MEIER R,THEVENIN J P,et al.Comparative studies of gelatin and cellulose ester membrane filters for their suitability in determining the microorganism count in the air[J].Zentralbl Bakteriol Mikrobiol Hyg B,1983,177(5):375-393.
[2]HAJEK P,ETTER J F,BENOWITZ N,et al.Electronic cigarettes:review of use,content,safety,effects on smokers and potential for harm and benefit[J].Addiction,2014,109(11):1 801-1 810.
[3]中华人民共和国卫生部.GB/T 5750.12-2006生活饮用水标准检验方法微生物指标[S].北京:[出版者不详],2006:1-36.
[4]中华人民共和国深圳出入境检验检疫局.SZDB/Z157-2015电子烟雾化液产品通用技术要求[S].深圳:[出版者不详],2015:1-7.
[5]VARLET V,FARSALINOS K,AUGSBURGER M,et al.Toxicity assessment of refill liquids for electronic cigarettes[J].International Journal of Environmental Research&Public Health,2015,12(5):4 796-4 815.
[6]中华人民共和国卫生部.GB 4789.2-2010食品安全国家标准食品微生物学检验菌落总数测定[S].北京:[出版者不详],2010:1-12.
[7]PISTELOK F,POHL A,STUCZYNSKI T,et al.Using ATPtests for assessment of hygiene risks[J].Ecological Chemistry&Engineering S,2016,23(2):259-270.
[8]WHITELEY G S,DERRY C,GLASBEY T,et al.The perennial problem of variability in adenosine triphosphate(ATP)tests for hygiene monitoring within healthcare settings[J].Infection Control&Hospital Epidemiology,2015,36(6):658-663.
[9]MORRIS H C.The benefits of rapid microbiological testing of finished products using ATP bioluminescence[J].International Journal of Cosmetic Science,2010,20(1):63-67.
[10]CARRASCOSAA C,MILLA N R,JABER J R,et al.Monitoring of cleanliness and disinfection in dairies:Comparison of traditional microbiological and ATP bioluminescence methods[J].Food Control,2012,28(2):368-373.
[11]COSTA P D,ANDRADE N J D,PASSOS F J V.ATP-bioluminescence as a technique to evaluate the microbiological quality of water in food industry[J].Brazilian Archives of Biology&Technology,2004,47(3):399-405.
[12]HECKER W,MEIER R,THEVENIN J P,et al.Comparative studies of gelatin and cellulose ester membrane filters for their suitability in determining the microorganism count in the air[J].Zentralbl Bakteriol Mikrobiol Hyg B,1983,177(5):375-393.