Study On Establishment Of The Testing Method Of ALP,LPO,γ-GGT Activity In Milk

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.â… Establishing the testing method of ALP activity in milkTaking 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.â…¡Establishing 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 H₂O₂ 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 H₂O₂ 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.â…¢Establishing the testing method ofγ—GGT activity in milkThe 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.