ATP生物发光检测技术的建立及应用可行性分析

建立了测定食品中细菌总数的ATP生物发光检测技术,考察了各种理化因素对生物发光反应的影响。反应体系最优化条件:Ln浓度为70mg/L,FL浓度为50mg/L,Mg2+浓度为0.25mmoL/L,pH为7.2,最适温度为23℃。在10-10～10-15mol/mL范围内,ATP浓度与生物发光强度之间有较好的线性关系,相关系数R2=0.978。方法检出限为10-15mol/mL,批内变异和批间变异分别小于7%和8%。将建立好的ATP生物发光反应体系应用于食品样品中细菌总数的检测,加标回收率范围为82.2%～112.4%,检测结果与平板计数结果相关性良好。因此,建立的ATP生物发光检测技术用于检测食品中细菌总数是可行的。     

ATP生物发光法快速测定生乳中微生物总数的研究

对ATP生物发光法应用于生乳中微生物检测进行了初步摸索,确定了加体细胞裂解液次数为三次;乳样最佳稀释倍数为20倍;用ATP生物发光法对大批乳样进行检测,同时用国标法进行了平皿菌落培养,结果显示,乳中微生物总数对数与光值对数呈正的直线相关（r=0.9485）,相关程度为显著相关（P<0.001）,二者回归方程为y=1.0496x+2.4。      

ATP生物发光法快速测定生乳中微生物总数的研究

对ATP生物发光法应用于生乳中微生物检测进行了初步摸索,确定了加体细胞裂解液次数为三次;乳样最佳稀释倍数为20倍;用ATP生物发光法对大批乳样进行检测,同时用国标法进行了平皿菌落培养,结果显示,乳中微生物总数对数与光值对数呈正的直线相关(r=0.9485),相关程度为显著相关(P<0.001),二者回归方程为Y=1.0496x 2.4.     

ATP生物发光法在饮料微生物检验中的应用分析

ATP生物发光法是一种快速的微生物检测方法,具有准确、简便、可实时检测等优点。ATP生物发光法与涂抹法结合能够实现饮料行业在原辅料、机械设备表面、人员及其装备、包装材料、生产环境等关键控制点的卫生状况现场监测与跟踪测定,测定结果与国标法相吻合,该方法能够帮助饮料企业提高日常的卫生管理工作水平,指导对工厂设施、设备、环境等实施清扫、洗涤和杀菌作业,及时发现卫生不良事故并提出解决方案。与滤膜法结合,可增加取样量,快速测定含菌量低的液体样品,又能消除样品中抑菌物质的干扰,能够对CIP系统清洗效果进行评估和指导。     

ATP生物发光法微生物速测技术研究获突破

ATP生物发光法微生物数量快速测定技术由广东省微生物研究所攻关完成。这一被列为广东省重点科技攻关的项目，近期通过了由广东省科技厅组织的成果鉴定。据介绍，微生物数量检测是微生物分析检测中的重要项目，传统的方法需进行长时间微生物培养，检测周期长，检测条件要求较高，不能满足食品、     

ATP生物发光法在提高复杂样品细菌检测方面的应用进展

微生物污染对食品、医药、卫生等领域造成严重危害,因此,开发微生物快速灵敏检测技术至关重要。ATP生物发光法相比于其他微生物检测技术,具有检测速度快、可实时检测等优点。近年来,随着检测技术与设备的不断改进,该技术在复杂样品中的检测应用能力得到了进一步提升。本文系统介绍了ATP生物发光法的原理和局限,从技术和设备层面综述了近年来创新性ATP生物发光技术和新兴检测设备,总结了该技术及设备在复杂样品细菌检测方面的应用现状,以期为提高复杂样品细菌检测能力提供参考。     

ATP生物发光法检测布拉氏酵母菌总数的研究

利用ATP生物发光法建立了布拉氏酵母菌与相对发光值的定量线性模型,对比研究了十六烷基三甲基溴化铵(CTAB)和苯扎溴铵(BAB)两种提取剂在不同的浓度、提取时间下对酵母菌相对发光值的影响。结果表明,浓度为0.015%CTAB作用时间4min的提取效果最好。同时,对检出的RLU值的对数值与菌落总数的对数值作统计学线性相关分析发现,R~2=0.9919,具有统计学意义。ATP生物发光法对布拉氏酵母菌的最低检测限是10~3cfu/mL。      

ATP生物发光法检测布拉氏酵母菌总数的研究

利用ATP生物发光法建立了布拉氏酵母菌与相对发光值的定量线性模型,对比研究了十六烷基三甲基溴化铵(CTAB)和苯扎溴铵(BAB)两种提取剂在不同的浓度、提取时间下对酵母菌相对发光值的影响.结果表明,浓度为0.015％ CTAB作用时间4min的提取效果最好.同时,对检出的RLU值的对数值与菌落总数的对数值作统计学线性相关分析发现,R2 =0.9919,具有统计学意义.ATP生物发光法对布拉氏酵母菌的最低检测限是103cfu/mL.     

采用ATP生物发光法分析6株常见细菌ATP含量差异

以ATP生物发光法为基础,辅以国标平板计数法,分析和测定了大肠埃希氏菌、枯草芽孢杆菌、蜡状芽孢杆菌、乳酸链球菌、乳酸片球菌等6株常见细菌中ATP含量的差异。结果显示:当单位体积菌悬液的ATP荧光值相同时,6株细菌菌落总数无明显差异;当菌悬液的菌落总数相同时,6株细菌的单位体积菌悬液ATP含量无明显差异。研究结果同时说明ATP生物发光法能够定量测定常见区域中的细菌总数。     

ATP生物发光法快速测定物体表面的菌落总数

以大肠埃希氏菌作为指示菌考量了ATP生物发光法测定物体表面卫生状况的准确性,并在实验室内模拟食品企业卫生管控,采用ATP生物发光法对具有代表性的台面进行细菌总数测定,与国家标准微生物检测方法进行了比对分析。结果显示:大肠埃希氏菌的ATP荧光值与菌落总数的线性相关性良好,相关系数R2为0.982 6;以实验室内指定台面作为盲样进行对比测定,其ATP荧光值与菌落总数的线性相关性良好,相关系数R2达到0.915 9,说明ATP生物发光法能够准确评估和测定物体表面的卫生状况。     

ATP荧光检测法快速筛查冷荤间加工现场凉拌菜的细菌污染状况

探讨ATP(adenosine triphosphate,三磷酸腺苷)荧光检测法检测冷荤间加工食品细菌总数的可行性,为食品细菌污染现场快速检测提供科学依据。方法 从本市餐饮单位中随机抽取20家各类餐饮单位,采集4大类80份菜品作为冷荤间加工食品检测样品,分别应用ATP荧光检测法与实验室平板计数法检测细菌总数。结果 ATP荧光检测法所得菌落数与实验室平板计数法检测菌落总数对比,假阳性率18.8%,假阴性率20.4%。结论 ATP荧光检测法适用于冷荤间加工食品微生物的快速检测。     

Evaluation of cleaning procedures for allergen control in a food industry environment

The hygiene of chicken processing surfaces and retention of the wheat protein gliadin and of protein in general on those surfaces were compared in 15 trials after 3 increasingly rigorous cleaning steps. Eleven different chicken products with wheat derivatives as a batter were prepared on 3 processing lines in 15 production runs selected at random over 6 mo (5 runs were thus replicates). Using surface swabs, surface hygiene was monitored by adenosine triphosphate (ATP) bioluminescence, gliadin by immunoassay, and protein by the Coomassie dye method. Gliadin was monitored in 14 trials, protein in 5, and all trials were monitored by ATP bioluminescence. In a typical trial, gliadin values normalized to uncleaned values fell from 100000 arbitrary units, to 6000 after rinsing, to 30 (foam, rinse), to not detected (sanitize, rinse). Parallel ATP bioluminescence values also decreased, but crucially, the relative gliadin value was less than the relative ATP value after foam and rinse in all 14 trials, a result unchanged after sanitize and rinse. In trials comparing ATP and protein, the relative ATP values exceeded the relative protein values in 4 of 5 trials after foaming and after sanitizing. Thus, for these 11 products, ATP bioluminescence was a surrogate indicator of residual gliadin and probably of residual protein. Absolute gliadin concentration on an uncleaned processing line was also the basis of modeling the risk of cross-contamination of gliadin in follow-up product, where the line was hypothetically left uncleaned between production runs. The results show that all follow-up product could be declared "gluten-free" under proposed legislation, and suggest that some industrial cross-contamination risks are currently overestimated.     

生物发光快速测定生乳菌落总数的方法

为消除利用ATP生物发光法测定生乳菌落总数时非细菌ATP对测定结果的干扰,建立了一种样品前处理方法。利用ATP生物发光法对经过前处理的生乳样品进行检测,结果表明,生乳菌落总数对数值与生乳细菌ATP发光对数值呈现较好的线性关系(R2=0.982),相关程度为显著相关(P<0.01),说明该前处理方法能够有效排除非细菌ATP的干扰,有利于提高ATP生物发光法定量测定生乳菌落总数准确性。     

Application of ATP bioluminescence for evaluation of surface cleanliness of milking equipment

The ATP bioluminescence method was used to evaluate the cleanliness of milking equipment surfaces (teat cup rubbers, teat dip containers, milk receivers, and pipeline joints) in dairy farms in Galicia (northwest Spain) with parlour, pipeline tie-stall or bucket tie-stall milking systems. The cleanest surfaces were teat cup rubbers. The use of non-chlorinated water for cleaning, and of pipeline or bucket tie-stall milking systems, was associated with high ATP bioluminescence values. However, ATP bioluminescence values only explained 12% of the variability in bulk-tank bacterial count; this is attributable to the importance of other factors (notably the correct functioning of the tank cooling system) for maintenance of low bacterial count.     

The benefits of rapid microbiological testing of finished products using ATP bioluminescence

Microbiological quality control of personal care products using traditional methods can take between 5 and 7 days to complete. This is frequently the rate limiting step in product release. Companies are looking to improve manufacturing efficiencies and to maximize resources by releasing products faster. ATP bioluminescence has been shown to be applicable to the detection of low levels of contaminating organisms in a diverse range of personal care products including detergent-based and soap-based products, cosmetics and toothpastes. The system described here has demonstrated the detection of less than 10 colony forming units per sample of a range of typical contaminants in these product types after enrichment for 24 h.     

Bioluminescence ATP monitoring as a surrogate marker for microbial load on hands and surfaces in the home

To evaluate the validity and reliability of adenosine triphosphate (ATP) monitoring as a surrogate marker for cleanliness, the kitchen table surface in 225 inner-city homes was sampled by microbiologic culture and by two types of biomass monitoring systems (HY-LiTEtrade mark 2 ATP System and HY-RiSE Colour Hygiene Test Strip, EM Science, Gibbstown, New Jersey, USA). A randomly selected hand of the homemaker (n = 225) was also cultured and sampled with the ATP monitoring system immediately after handwashing. Log microbial counts on hands ranged from 3.2 to 7.0 and from the table, 1.0 to 5.5. While the traditional ATP readings (HY-LiTE) and the color strips were significantly correlated (R = 0.18, P = 0.01), there was no significant correlation between the ATP monitor readings and the colony-forming units counts on either the hands (R = 0.03, P = 0.62) or the table (R = 0.04, P = 0.58). Such biomass measurements are not a substitute for quantitation of microbial load.     

食品中单核细胞增生李斯特氏菌两种定量检测方法的比较

研究比较不同杂菌污染条件下平板计数(Plate counting)法和稀释培养计数(Most probable number counting)法检测食品中单核细胞增生李斯特氏菌(Listeria monocytogenes,LM)含量的准确性,并探讨冷藏和冷冻食品,MPN保存过程中LM的数量增长情况。采用国标法中的平板计数法和MPN计数法对不同程度人工污染杂菌的牛奶、凉拌菜和盐水鸭分别进行LM含量的检测,并用MPN计数法对冷藏(2~8℃)和冷冻(-20℃)条件下保存的牛奶、凉拌菜和盐水鸭进行LM含量的检测。结果表明,杂菌染菌浓度较低时(LM含量与杂菌含量比为10:1), LM检出限较高(≥ 100 CFU/g (mL)),平板计数法检测LM含量的准确性较高,而杂菌染菌的初始浓度较高时(LM含量与杂菌含量比为1:10),LM检出限较低(< 100 CFU/g (mL)),MPN计数法检测LM含量的准确性较高;牛奶、凉拌菜和盐水鸭在经过不同冷藏和冷冻保存时间后LM数量对比差异有统计学意义(p<0.05),且食品中LM数量随着冷藏和冷冻保存时间的增加而增多。     

荧光素酶表达载体构建及其在巴斯德毕赤酵母的表达

萤火虫荧光素酶(Firefly Luciferase,FL)是ATP快速检测技术的核心组件,通过荧光强度与ATP浓度的对应关系,在食品行业检测微生物数量发挥着重大作用。本文为实现荧光素酶在重组毕赤酵母GS115中的异源表达,通过从载体pGL2-control中扩增荧光素酶基因luc,克隆到真核表达载体p PIC9K中,线性化后电击转化毕赤酵母GS115菌株,筛选阳性重组菌株。在甲醇的诱导下进行酶的表达,对粗酶液进行生物活性发光分析,然后对粗酶进行超滤、阴离子层析和分子筛凝胶层析三步纯化。甲醇诱导表达96 h发现胞外和胞内粗酶液均有相对较高的酶发光活性,酶活分别为1.45×10~6 RLU/mL和1.58×10~9 RLU/mL。SDS-PAGE与Western blot分析重组荧光蛋白大小约为70 ku,最终纯化得到的荧光素酶,其比活为7.0×10~8 RLU/mg,纯化倍数达到19.3倍,产量为48 mg/L。以上结果表明,荧光素酶能够在毕赤酵母表达系统获得较好的表达和纯化效果。     

基于ATP再生体系快速检测乳品中微生物

基于焦磷酸（pyrophosphoric acid,PPi）再生三磷酸腺苷（adenosine triphosphate,ATP）建立乳品中微生物快速检测方法。通过单因素试验优化PPi再生ATP反应条件,并考察方法的灵敏度、准确度、精密度和稳定性。结果表明,PPi再生ATP最佳反应条件为腺苷酰硫酸（adenosine phosphosulfate,APS）浓度10 μmol/L,ATP硫酸化酶（ATP sulfurylase,ATPS）活力0.15 U/mL、反应pH7.8。在最佳的ATP再生条件下偶联生物发光法,对ATP标准品、大肠杆菌、铜绿假单胞菌的检测限分别为10－17mol/mL、102CFU/mL和102CFU/mL。工作曲线在102～107CFU/mL范围内线性关系良好,对乳品基质的回收率为81.33%～97.78%,变异系数为14.24%～22.17%,与国标平板计数法对比显示两种方法检测结果相关性良好,相关系数为0.96。本方法快速、简单、灵敏、稳定,适用于乳品中微生物快速监测。