Development And Application Of Rapid Detection Methods Of Lactoperoxidase In Pasteurized Milk

Milk is rich in various nutrients and active proteins which are very beneficial to human health.In order to eliminate the pathogenic microorganisms in milk and prolong the shelf life,raw milk needs heat treatment.Lactoperoxidase is one of the most heat resistant enzymes with antibacterial properties in milk,which act as an indicator of heat load of dairy products,especially for over-pasteurized milk.This article aimed to develop rapid and qualitative detection methods of lactoperoxidase in milk,which were verified by experimental and commercially available milk samples.We tested lactoperoxidase activity in different heat treatment milk.Subsequently,we evaluated the activity in several pasteurized milk in local markets,and providing basis for process and quality evaluation of milk We developed a rapid,efficient,low-cost purification process of lactoperoxidase.It provided basic materials for the verification of the detection method and preparation of standard of product.The main results are as follow:(1)We developed methods for rapidly determining lactoperoxidase in milk using potassium iodide and p-phenylenediamine assay based on the principle of enzymatic reaction.The requirement for the volume of reagent solutions were optimized.LOQs of potassium iodide assay and p-phenylenediamine assay were 1400 U/L and 370 U/L,respectively,and both of them had an accurate within-run precision and between-run precision with a coefficient of variation less than 10%.The periods of validity for potassium iodide assay and p-phenylenediamine assay were 20 days and 14 days,respectively.The inactivation of lactoperoxidase in pasteurized milk can be quickly determined by using Colour Chart.In summary,these rapid detection methods of lactoperoxidase in milk could be used to detect the different heat treatment of pasteurized milk samples.(2)We studied lactoperoxidase activity in milk under different heat treatment conditions.The activity of lactoperoxidase in 72℃,75℃,80℃,85℃,90℃,95℃,100℃,105℃,110℃,115℃,120℃ for 15 s heat treated milk were studied by rapid detection method.The results showed that the potassium iodide assay could roughly determine the activity of lactoperoxidase in raw milk and heat-treated milk below80℃.The activity decreased progressively when temperature increased,and it inactivated lactoperoxidase when temperature above 80 ℃.The p-phenylenediamine assay could draw the similar conclusions.(3)We studied lactoperoxidase activity in pasteurized milk in market.15 batchesof pasteurized milk were collected in local supermarkets,and the activity of lactoperoxidase were tested and analyzed.The results indicated that only 2 batches of pasteurized milk were up to standard,and most of milk had the risk of overheating processing.We recommended that the detection of lactoperoxidase could be included in the pasteurized milk relevant national standard.(4)We developed a technology of protein purification process of lactoperoxidase in milk.First of all,the concentration of the most preferred ammonium sulfate precipitation was selected to be 50% by the ammonium sulfate concentration gradient test.Secondly,Octyl Sepharose 4 FF was chosed as the optimal hydrophobic chromatography packing from another 5 prepacked column.Finally,lactoperoxidase in milk was purified using optimal conditions,resulting in more than 90% purity.It provided the basic materials for the verification of the detection method and applications of antibacterial properties.The rapid detection method for LPO in pasteurized milk established in this paper did not require the pre-processing steps,large-scale equipments and complex detection procedures.It was a quick,efficient method,which could satisfy the needs of on-site acquisition of dairy products,and control of pre-processing processes,and rapid detection of dairy acceptance and testing.At the same time,it is proposed to add the detection of LPO into the pasteurized milk relevant national standard.