Evaluation And Application Of Methods In Detecting The Activity Of Alkaline Phosphatase And Lactoperoxidase In Dairy Products

Bovine milk can provide a lot of nutrients for human body,but there are lots of pathogenic microorganisms in it.Appropriate pasteurization process can inactive pathogenic microorganisms in milk,also can maintain the nutrients undestroyed.Activity of alkaline phosphatase and lactoperoxidase can be used for evaluation of heat treatment process in pasteurized milk.But in China,alkaline phosphatase detection standard NY/T 801-2004 has some disadvantages,such as:complicated operation,long time-consuming,low sensitivity and also it can't meet the needs of dairy production process control for health and safety.Meanwhile,there is no assay standard for lactoperoxidase activity.Therefore,this study was focus on evaluating the method of detecting activity of alkaline phosphatase according to ISO 22160-2007,and another method of detecting activity of lactoperoxidase according to the commercialization kits(Evergreen TERA^TM),which can provide data basis for the establishment of the new industry standard;This study researched the change of activities of alkaline phosphatase and lactoperoxidase in the pasteurized milk under different heat treatment conditions,which can provide reference data for evaluation of pasteurization process.This study also detected activity of two enzymes in commercial pasteurized milk products to provide data base for evaluation of milk production and milk quality.The main test content and the results are as follows:Evaluation of the detection method for activity of alkaline phosphatase:This study optimized the method of ISO 22160-2007 and tested the selectivity of instrument and matrix?interference influence and stability of samples.The results showed that the method can be used for detection activity of alkaline phosphatase in pasteurized milk.The detection limit,linearity,recovery rate,the difference in and between days,the repeatability and reproducibility of the method are validated and evaluated.The results showed that:detection limit results is 18.4 mU/L,agreed with 20 mU/L in according to ISO 22160-2007;Milk content and activity of alkaline phosphatase had good linear relationship within 0.00¹.00%,the regression equation is y=10⁶x-85.068,the correlation coefficient R²=0.9971.Recovery ranges from 85.3%to 105.6%.Relative standard deviation ranges from 2.5%to 22.1%;Relative standard deviation of is in-days difference ranges from 9.4%to 10.7%,relative standard deviation of between-days difference is 10.7%;Comparative verification experiment was carried out in three department.The results showed good results of repeatability R and reproducibility limit r.This study set up a new method of sample confirmation to ensure the exclusion of false positive test results,This experiment compared this method with agricultural standard NY/T 801-2004.The results showed that this method is more sensitive,fast and accurate.The evaluation of the detection method for lactoperoxidase according to commercial kits(Evergreen TERA^TM):the results show that the method detection limit as low as 2.83 U/L,within the range of 0-400 U/L has a good regression relation,R² is 0.998,Recovery ranges from 99.2%to99.2%,relative standard deviation ranges from 4.5%to 17.2%,which all infers that the method of kit is accuracy.In addition,different matrix also has not affected the result of the assay,but long retention time of samples will make the results on the low side.The application of two detecting methods for activity of alkaline phosphatase and lactoperoxidase:This study researched the change of activities of alkaline phosphatase and lactoperoxidase in the pasteurized milk under different heat treatment conditions,and detected activity of two enzyme in commercial pasteurized milk product,then made evaluation to Chinese pasteurization process.The results show that with the higher temperature,activities of alkaline phosphatase and lactoperoxidase decreased;Alkaline phosphatase inactivates at 72?,15 s,lactoperoxidase inactivates at 75-80?,15 s;Alkaline phosphatase of commercial pasteurized milk products is generally negative,lactoperoxidase is inactivated in most samples,only a small number can be detected actively,which infers that ultra-pasteurization existed in dairy industry.