Microsoft Word _张凤兰-二校915

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1 7 3 Vol. 7 No Journal of Food Safety and Quality Mar., 2016 ATP * 张凤兰, 徐潇, 王海燕, 高飞, 单纯, 崔生辉 (, ) 摘要 : 目的 ATP, 2 方法 3 ATP, ATP ;, ATP, ATP, 2 结果 3 ATP ATP ;, 3 ATP RLU ATP 3 ATP, ATP 结论 3 ATP ATP,,, ATP 关键词 : ATP ; ; ; Evaluation of microorganism on tableware by ATP bioluminescence ZHANG Feng-Lan, XU Xiao, WANG Hai-Yan, GAO Fei, SHAN Chun, CUI Sheng-Hui * (National Institutions for Food and Drug Control, Beijing , China) ABSTRACT: Objective To evaluate the detection limit and detection rate of the ATP bioluminescent method and the plate count method for tableware microbial contamination, and investigate the correlation between the 2 methods. Methods The standard solution with different concentrations were detected by the different ATP bioluminescent detectors produced by domestic companies (A, B, and C, respectively) and the standard curve was drawn after detection. Using different concentrations of Escherichia coli, Staphylococcus aureus bacteria liquid and artificial inoculation of E. coli tableware as goods, the ATP bioluminescence detector and the plate count method were used to detect them respectively, and then the standard curve of detection value between the 2 methods for quantity of Escherichia coli, Staphylococcus aureus and tableware in artificial pollution and the correlation curve were drawn between the 2 methods. Results The detection limit of ATP standard solution, Escherichia coli and Staphylococcus aureus were different among the 3 kinds of ATP bioluminescence detectors from different companies; but while in a detection range, the 3 kinds of different ATP bioluminescence detectors had a good linear correlation. And the 2 different methods have a good linear correlation. Conclusion There is an obvious instrument error between the different detectors from 3 companies on the detection limit for Escherichia coli and Staphylococcus aureus. And the difference is obvious, * 通讯作者 :,,, cuishenghui@aliyun.com *Corresponding author: CUI Sheng-Hui, Professor, National Institutions for Food and Drug Control, No.2 Tiantan Xili, Beijing , China. cuishenghui@aliyun.com

2 912 7 therefore, the ATP bioluminescent method cannot replace the traditional plate count method for evaluating the microbial contamination condition of tableware at present. KEY WORDS: ATP bioluminescence; plate counting; tableware; microorganism 1 引言, [1],,,, ATP, [2-6] ATP(adenosine triphosphate), ATP ATP, Mg 2, ATP D-, ATP, (relative light unit, RLU) ( ) D- +ATP+O 2 Oxy- +AMP+PPi+H 2 O Omidbakhsh [7] 4 ATP, 3 ATP, ATP, [8], ATP 2 材料与方法 2.1 菌株与试剂 : (ATCC29213) (ATCC25922)( ATCC) (ATP, Sigma ) 2.2 仪器与设备 ATP ( ): A B C, A B ATP, C ATP ; ( Thermo ); ( Thermo ) 2.3 实验方法 ATP 生物发光检测仪的 ATP 标准曲线建立 10-2 mol/l ATP, 1:10 1: mol/l ATP 10 μl(a B ) 50 μl(c ), RLU (n=3) ( ) ATP RLU, ATP 生物发光检测仪的细菌量标准曲线建立 (ATCC25922) (ATCC29213) (TSB), h, 3000 r/min 10 min, ; 0.85%, 3000 r/min 10 min,,, 0.85%,, MCF, 0.85% 1:10 1:10 2 1:10 3 1:10 4 1:10 5 GB [8], (0.85% ) 10 μl(a B ) 50 μl(c ) ATP, RLU (n=3) (0.85% ) (CFU), ATP RLU, 餐 ( 饮 ) 具人工污染样品的检测 120, 4 5 cm 5 cm, 160

3 3, : ATP h 120 5, 24 : (5, 4 ) 4 (0.85% ) : :10 1:10 2 1:10 3 1:10 4 1:10 5 (ATCC25922) 20 μl, 25 cm 2, 4 24, 0.85%, 25 cm 2, GB ,,, 1 CFU 3 ATP 25 cm 2, (CFU), ATP RLU, 3 结果与分析 3.1 ATP 生物发光法的标准曲线 A B C ATP ATP : fmol(1 fmol=10-15 mol)( 1), mol 10-9 mol, ATP RLU, ATP 生物发光方法与大肠杆菌量的标准曲线 A B C ATP : CFU( 1) CFU CFU, 3 ATP RLU, ATP Fig. 2 Standard curve of ATP bioluminescence and the amount of E. coli 3.3 ATP 生物发光方法与金黄色葡萄球菌量的标准曲线 A B C ATP : CFU( 1) CFU CFU, 3 ATP RLU, ATP Fig. 1 Standard curve of ATP bioluminescence 3 ATP Fig. 3 Standard curve of ATP bioluminescence and the amount of S. aureus

4 914 7 表 1 ATP 生物发光检测仪对 ATP 标准溶液 大肠杆菌和金黄色葡萄球菌的检出限 Table 1 The detection limit of ATP standard solution, E. coli and S. aureus by ATP meter ATP(fmol) (CFU) (CFU) (CFU) A B C ATP 生物发光法与平板计数法的相关性, GB CFU, CFU B C ATP CFU ; A ATP CFU, CFU CFU CFU, 3 ATP RLU, ATP Fig. 4 Linear correlation of ATP bioluminescence method and plate counting method 4 结论与讨论, 3 ATP A B ATP,,,, 9999; C ATP,,,, 10 6, 50 μl, 10 μl A B C ATP 10 fmol, 10 fmol 10 6 fmol, ATP RLU 0.95, [9,10] A B C, C, B C 2~3 Log, ATP Omidbakhsh [7] 4 ATP, 3 Log ATP Mg 2+, ATP, ATP [11-15] Turner [10] Charm Sciences novalum 10 2 CFU, Omidbakhsh [7] Charm Sciences 10 5 CFU, Turner, ATP, Omidbakhsh, ATP, ATP,, 3, 2 ATP

5 3, : ATP CFU, CFU, , ATP, [2,16],, VBNC(viable but not culturable),, 1/100, ATP,, ATP [9,17] ;, ATP [18], ATP [7],,, ( ) ATP,, A B ATP,,,,, ATP GB 参考文献 [1] [S]. Food Safety Law of the People's Republic of China [S]. [2],,,. ATP [J]., 2004, 31(2): Cao LR, Zhang W, Zhao R, et al. ATP bioluminescence method in the application of the railway station tableware hygiene inspection [J]. Railway Labor Saf Health Environ Prot, 2004, 31(2): [3],,,. ATP [J]., 2014, 26(11): Xu ZR, Guo ZC, Yao HB, et al. Effect of disinfect in cleaning tableware by ATP bioluminescence [J]. Zhejiang Prev Med, 2014, 26(11): [4] Champiat D, Matas N, Monfort B, et al. Applications of biochemiluminescence to HACCP [J]. Luminescence, 2001, 16(2): [5] Aycicek H, Oguz U, Karci K. Comparison of results of ATP bioluminescence and traditional hygiene swabbing methods for the determination of surface cleanliness at a hospital kitchen [J]. Int J Hyg Environ Health, 2006, 209(2): [6] Chen FC, Godwin SL. Comparison of a rapid ATP bioluminescence assay and standard plate count methods for assessing microbial contamination of consumers' refrigerators [J]. J Food Prot, 2006, 69(10): [7] Omidbakhsh N, Ahmadpour F, Kenny N. How reliable are ATP bioluminescence meters in assessing decontamination of environmental surfaces in healthcare settings [J]. PLoS One, 2014, 9(6). [8] GB [S]. GB National food safety standard-food microbiological examination: Aerobic plate count [S]. [9] Vogel SJ, Tank M, Goodyear N. Variation in detection limits between bacterial growth phases and precision of an ATP bioluminescence system [J]. Lett Appl Microbiol, 2014, 58(4): [10] Turner DE, Daugherity EK, Altier C, et al. Efficacy and limitations of an ATP-based monitoring system [J]. J Am Assoc Lab Anim Sci, 2010, 49(2): [11] Davidson CA, Davidson CA, Griffith CJ, et al. Evaluation of two methods for monitoring surface cleanliness-atp bioluminescence and traditional hygiene swabbing [J]. Luminescence, 1999, 14(1): [12] Moore G, Griffith C. Problems associated with traditional hygiene swabbing: the need for in-house standardization [J]. J Appl Microbiol, 2007, 103(4): [13] Landers TF, Hoet A, Wittum TE. Swab type, moistening and preenrichment for Staphylococcus aureus on environmental surfaces [J]. J Clin Microbiol, 2010, 48(6): [14],,,. ATP [J]., 2008, 20(9): Wu J, Zhu HH, Zhang JJ, et al. Research on effect ofquaternary ammonium surfactants in extract of ATP [J]. Jiangxi Acta Agric, 2008, 20(9): [15] Ishida A, Yoshikawa T, Nakazawa T, et al. Enhanced firefly bioluminescence assay of ATP in the presence of ATP extractants by using diethylaminoethyl-dextran [J]. Anal Biochem, 2002, 305(2): [16],,,. [J]., (7):

6 916 7 Zhang ZJ, Gu J, Wu ZH, et al. Invetigation of health status of tableware concentrate disinfection trade of Nantong [J]. Chin J Disinf, 2012, 29(7): [17] Vartoukian SR, Palmer RM, Wade WG. Strategies for culture of 'unculturable' bacteria [J]. FEMS Microbiol Lett, 2010, 309(1): 1 7. [18] Lappalainen J, Loikkanen S, Lappalainen J, et al. Microbial testing methods for detection of residual cleaning agents and disinfectants-prevention of ATP bioluminescence measurement errors in the food industry [J]. J Food Prot, 2000, 63(2): ( 责任编辑 : 杨翠娜 ) 作者简介 张凤兰, 博士, 副研究员, 主要研究方向为食品化妆品安全性评价 flzhang2007@163.com 崔生辉, 博士, 研究员, 主要研究方向为食品微生物 cuishenghui@aliyun.com