乳中ALP、LPO、γ-GGT检测方法的建立
摘要
牛乳在饮用之前,要经过合理的巴氏消毒,适当的巴氏消毒不仅可以杀死引起人类疾病的致病菌,还能保留牛乳的营养成份。而碱性磷酸酶(ALP)、乳过氧化物酶(LPO)、γ—谷氨酰转肽酶(γ—GGT)作为乳中的内源酶,对热比较敏感。当牛乳经过不同程度的热处理时,ALP、LPO、γ—GGT可作为热处理的指标。因此,本文对ALP、LPO、γ—GGT分别建立一种简便、快速的检测方法,通过检测酶的活力来反映乳品的热处理,以保证消费者安全。
系列Ⅰ碱性磷酸酶检测方法的建立
以4—甲基伞形酮磷酸酯(4—MUP)为底物,在碱性磷酸酶的催化作用下生成具有荧光性的4—甲基伞形酮,利用960CRT荧光光度计,采用连续性检测法对其检测荧光值,与标准曲线比较计算待测样品中碱性磷酸酶活力。分别从以下几个影响因素来优化反应条件:扫描波长、作用时间、灵敏度、pH、底物浓度、底物缓冲液浓度、酶浓度、温度等,探讨最佳条件为:最佳波长为激发波长365nm、发射波长449nm,最佳作用时间为前6min,灵敏度为2,最适pH为9,底物浓度为纯品4—MUP 100 uL,底物缓冲液浓度为0.1mol/L,酶浓度为0.165U/mL～0.0103U/mL,温度为常温。对建立的检测方法加以评价:回收率100.17%,最低检测限0.0103U/mL,相关性0.9979,批内变异系数2.81%,批间变异系数4.34%;与国标法比较后得到:建立方法简便、快速、回收率高、精密度好,但是本法要使用荧光分光光度计。
系列Ⅱ乳过氧化物酶检测方法的建立
以2、2｀-连氮基-双(3-乙基苯并噻吡咯啉-6磺酸)(ABTS)为底物,在H_2O_2的氧化作用下,经过氧化物酶的催化作用生成绿色的氧化型ABTS,利用可见分光光度计,采用动力学模式对其检测吸光度值,与标准曲线比较计算待测样中过氧化物酶活力。分别从以下几个影响因素来优化反应条件:扫描波长、作用时间、pH、底物浓度、H_2O_2浓度、酶浓度、温度等,探讨最佳条件为:最佳吸收波长416nm,作用时间前100s,最适pH为5.5,最佳底物浓度2mM,H_2O_2浓度5mg,酶浓度0.23125u/mL～1.85u/mL,温度为常温。对建立的检测方法加以评价:回收率98.84%,批内变异系数为2.39%,批间变异系数为3.42%,相关性0.9953,与愈创木酚法比较后得到:建立方法简便、快速、回收率高、精密度好、成本也比较低,对仪器要求不高。
系列Ⅲγ—谷氨酰转肽酶检测方法的建立
本方法原理是γ—GGT能将γ—谷氨酰基团从供体L—γ—谷氨酰—P—硝基苯胺(γ—GPNA)转移到受体双甘氨肽上,同时释放的P—硝基苯胺在390nm有强烈的吸收作用,其吸光率的增加与γ—GGT的活性成正比。分别从以下几个影响因素探讨最佳反应条件:扫描波长、作用时间、pH、底物最佳比、底物浓度、温度、澄清剂等,得出:最佳吸收波长390nm,作用时间前15min,最适pH为7,底物最佳比(双甘氨肽:γ—GPNA)=30,底物浓度为双甘氨肽为0.24moL·L~(-1)、γ—GPNA为0.008moL·L~(-1),水浴温度选用40℃。最后对检测方法评价:批内变异系数2.19%,批间变异系数3.73%,相关性0.9979,与试剂盒法(重氮试剂法)比较得到:建立方法简便、精密度高、操作过程简单易行、试剂无需避光保存、检测样品量多。但是时间稍长,最低检测限稍高。
Milk should be rationally pasteurized before drinking,because it can not only kill the pathogenic bacteria arousing diseases of human,but also preserve the nutrients of the milk.However,the alkaline phosphatase(ALP),the lactoperoxidase(LPO)and the Gammma-glutamyl transferase(γ-GGT)are sensitive to heat as the indigenous enzymes in milk,So ALP,LPO andγ-GGT may be indicators of different heat treatment in milk.Therefore,the main purpose is to establish a simple,convenient and quick method in order to assay activity of ALP, LPO,γ-GGT in milk.It can reflect the quality of milk through the activity of enzyme,and it can ensure the safety of the consumers.
ⅠEstablishing the testing method of ALP activity in milk
Taking 4- methylumbelliferyl phosphate liquid(4-MUP)as the substrate,the production 4- methylumbelliferone(4-MU)has the fluorescence in the catalytic of alkaline phosphatase with the 960CRT fluorophotometer,at last we can determine the fluorescence value with the kinetics determination method,and calculate the enzyme activity in comparison with the standard curve.The response condition can be optimized from the following several influence factors separately:the scanning wave,time,sensitivity,pH,concentration of substrate,concentration of substrate buffer solution,concentration of enzyme,temperature and so on.The results illustrate that the excitation wave is 365nm,emission wave is 449nm,time is the first 6min,sensitivity is 2,pH is 9,concentration of substrate is 100μL that equal to the standard solution,concentration of substrate buffer solution is 0.1mol/L, concentration of enzyme is 0.165U/mL～0.0103U/mL,temperature is selected as the common temperature.Appraises to the establishment examination method is that the recovery test experiments is 100.17%,the lowest detectable limit is 0.0103U/mL,the coefficient of correlation is 0.9979,the coefficient of variation in the same group is 2.81%,the coefficient of variation in the different group is 4.34%;compared with GB code law,the establishment method is simple,quick,the recovery ratio is high and the accuracy is good,but this method applies to using the fluorescence spectrophotometer.
ⅡEstablishing the testing method of LPO activity in milk
Taking ABTS as the substrate,the production has the chlorinated in the catalytic of Lactoperoxidation with the spectrophotometer,at last we can determine the OD value with the kinetics determination method,and calculate the enzyme activity in comparison with the standard curve.The response condition can be optimized from the following several influence factors separately:the scanning wave,time,pH,concentration of substrate,concentration of H_2O_2 solution, concentration of enzyme,temperature and so on.the results illustrate that scanning wave is 416nm,time is the first 100s,pH is 5.5,concentration of substrate is 2mM, concentration of H_2O_2 solution is 5mM,concentration of enzyme is 0.23125u/mL～1.85u/mL,temperature is selected as the common temperature.Appraises to the establishment examination method is that the recovery test experiments is 98.84%,the coefficient of correlation is 0.9953,the coefficient of variation in the same group is 2.39%,the coefficient of variation in the different group is 3.42%;comparison to guaiacol method:The establishment method is simple and quick,the recovery ratio is high,the accuracy is good and it costs very low.
ⅢEstablishing the testing method ofγ—GGT activity in milk
The principle of experiment is thatγ—glutamyl transfers fromγ—GPNA to glycylglycine,meantime,P-nitroaniline has the highest absorption at 390nm,it is directly in proportion toγ—GGT.The response condition can be optimized from the following several influence factors separately:the scanning wave,time,pH, concentration of substrate,the optimization ratio of substrate,concentration of enzyme,temperature,cleaning reagent and so on.the results illustrate that scanning wave is 390nm,time is the first 15min,pH is 7,the optimization ratio of substrate is 30,concentration of glycylglycine is 0.24moL·L~(-1),concentration ofγ—GPNA solution is 0.008moL·L~(-1),temperature is selected as 40℃.Appraises to the establishment examination method is that the coefficient of variation in the same group is 2.19%,the coefficient of variation in the different group is 3.73%,the coefficient of correlation is 0.9979,;compared to kit method,The establishment method is simple and quick,the recovery ratio is high,the accuracy is good,and it costs very low,the regent is in no need of being away from light,and it can detect many samples at the same time,but it costs a long time.
系列Ⅰ碱性磷酸酶检测方法的建立
以4—甲基伞形酮磷酸酯(4—MUP)为底物,在碱性磷酸酶的催化作用下生成具有荧光性的4—甲基伞形酮,利用960CRT荧光光度计,采用连续性检测法对其检测荧光值,与标准曲线比较计算待测样品中碱性磷酸酶活力。分别从以下几个影响因素来优化反应条件:扫描波长、作用时间、灵敏度、pH、底物浓度、底物缓冲液浓度、酶浓度、温度等,探讨最佳条件为:最佳波长为激发波长365nm、发射波长449nm,最佳作用时间为前6min,灵敏度为2,最适pH为9,底物浓度为纯品4—MUP 100 uL,底物缓冲液浓度为0.1mol/L,酶浓度为0.165U/mL～0.0103U/mL,温度为常温。对建立的检测方法加以评价:回收率100.17%,最低检测限0.0103U/mL,相关性0.9979,批内变异系数2.81%,批间变异系数4.34%;与国标法比较后得到:建立方法简便、快速、回收率高、精密度好,但是本法要使用荧光分光光度计。
系列Ⅱ乳过氧化物酶检测方法的建立
以2、2｀-连氮基-双(3-乙基苯并噻吡咯啉-6磺酸)(ABTS)为底物,在H_2O_2的氧化作用下,经过氧化物酶的催化作用生成绿色的氧化型ABTS,利用可见分光光度计,采用动力学模式对其检测吸光度值,与标准曲线比较计算待测样中过氧化物酶活力。分别从以下几个影响因素来优化反应条件:扫描波长、作用时间、pH、底物浓度、H_2O_2浓度、酶浓度、温度等,探讨最佳条件为:最佳吸收波长416nm,作用时间前100s,最适pH为5.5,最佳底物浓度2mM,H_2O_2浓度5mg,酶浓度0.23125u/mL～1.85u/mL,温度为常温。对建立的检测方法加以评价:回收率98.84%,批内变异系数为2.39%,批间变异系数为3.42%,相关性0.9953,与愈创木酚法比较后得到:建立方法简便、快速、回收率高、精密度好、成本也比较低,对仪器要求不高。
系列Ⅲγ—谷氨酰转肽酶检测方法的建立
本方法原理是γ—GGT能将γ—谷氨酰基团从供体L—γ—谷氨酰—P—硝基苯胺(γ—GPNA)转移到受体双甘氨肽上,同时释放的P—硝基苯胺在390nm有强烈的吸收作用,其吸光率的增加与γ—GGT的活性成正比。分别从以下几个影响因素探讨最佳反应条件:扫描波长、作用时间、pH、底物最佳比、底物浓度、温度、澄清剂等,得出:最佳吸收波长390nm,作用时间前15min,最适pH为7,底物最佳比(双甘氨肽:γ—GPNA)=30,底物浓度为双甘氨肽为0.24moL·L~(-1)、γ—GPNA为0.008moL·L~(-1),水浴温度选用40℃。最后对检测方法评价:批内变异系数2.19%,批间变异系数3.73%,相关性0.9979,与试剂盒法(重氮试剂法)比较得到:建立方法简便、精密度高、操作过程简单易行、试剂无需避光保存、检测样品量多。但是时间稍长,最低检测限稍高。
Milk should be rationally pasteurized before drinking,because it can not only kill the pathogenic bacteria arousing diseases of human,but also preserve the nutrients of the milk.However,the alkaline phosphatase(ALP),the lactoperoxidase(LPO)and the Gammma-glutamyl transferase(γ-GGT)are sensitive to heat as the indigenous enzymes in milk,So ALP,LPO andγ-GGT may be indicators of different heat treatment in milk.Therefore,the main purpose is to establish a simple,convenient and quick method in order to assay activity of ALP, LPO,γ-GGT in milk.It can reflect the quality of milk through the activity of enzyme,and it can ensure the safety of the consumers.
ⅠEstablishing the testing method of ALP activity in milk
Taking 4- methylumbelliferyl phosphate liquid(4-MUP)as the substrate,the production 4- methylumbelliferone(4-MU)has the fluorescence in the catalytic of alkaline phosphatase with the 960CRT fluorophotometer,at last we can determine the fluorescence value with the kinetics determination method,and calculate the enzyme activity in comparison with the standard curve.The response condition can be optimized from the following several influence factors separately:the scanning wave,time,sensitivity,pH,concentration of substrate,concentration of substrate buffer solution,concentration of enzyme,temperature and so on.The results illustrate that the excitation wave is 365nm,emission wave is 449nm,time is the first 6min,sensitivity is 2,pH is 9,concentration of substrate is 100μL that equal to the standard solution,concentration of substrate buffer solution is 0.1mol/L, concentration of enzyme is 0.165U/mL～0.0103U/mL,temperature is selected as the common temperature.Appraises to the establishment examination method is that the recovery test experiments is 100.17%,the lowest detectable limit is 0.0103U/mL,the coefficient of correlation is 0.9979,the coefficient of variation in the same group is 2.81%,the coefficient of variation in the different group is 4.34%;compared with GB code law,the establishment method is simple,quick,the recovery ratio is high and the accuracy is good,but this method applies to using the fluorescence spectrophotometer.
ⅡEstablishing the testing method of LPO activity in milk
Taking ABTS as the substrate,the production has the chlorinated in the catalytic of Lactoperoxidation with the spectrophotometer,at last we can determine the OD value with the kinetics determination method,and calculate the enzyme activity in comparison with the standard curve.The response condition can be optimized from the following several influence factors separately:the scanning wave,time,pH,concentration of substrate,concentration of H_2O_2 solution, concentration of enzyme,temperature and so on.the results illustrate that scanning wave is 416nm,time is the first 100s,pH is 5.5,concentration of substrate is 2mM, concentration of H_2O_2 solution is 5mM,concentration of enzyme is 0.23125u/mL～1.85u/mL,temperature is selected as the common temperature.Appraises to the establishment examination method is that the recovery test experiments is 98.84%,the coefficient of correlation is 0.9953,the coefficient of variation in the same group is 2.39%,the coefficient of variation in the different group is 3.42%;comparison to guaiacol method:The establishment method is simple and quick,the recovery ratio is high,the accuracy is good and it costs very low.
ⅢEstablishing the testing method ofγ—GGT activity in milk
The principle of experiment is thatγ—glutamyl transfers fromγ—GPNA to glycylglycine,meantime,P-nitroaniline has the highest absorption at 390nm,it is directly in proportion toγ—GGT.The response condition can be optimized from the following several influence factors separately:the scanning wave,time,pH, concentration of substrate,the optimization ratio of substrate,concentration of enzyme,temperature,cleaning reagent and so on.the results illustrate that scanning wave is 390nm,time is the first 15min,pH is 7,the optimization ratio of substrate is 30,concentration of glycylglycine is 0.24moL·L~(-1),concentration ofγ—GPNA solution is 0.008moL·L~(-1),temperature is selected as 40℃.Appraises to the establishment examination method is that the coefficient of variation in the same group is 2.19%,the coefficient of variation in the different group is 3.73%,the coefficient of correlation is 0.9979,;compared to kit method,The establishment method is simple and quick,the recovery ratio is high,the accuracy is good,and it costs very low,the regent is in no need of being away from light,and it can detect many samples at the same time,but it costs a long time.
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