Study On The Determination Of The Whey Protein Components In Milk And Dairy Products

Whey protein refers to the protein dissolved and dispersed in the whey.The whey protein in milk accounts for about 18%to 20%of the total protein content.The functional proteins contained in whey protein mainly include ?-lactoglobulin,?-lactalbumin,lactoferrin,bovine serum albumin,immunoglobulin and other components.They accounted for 46.6%,17.5%,2.9%,5.8%and 11.7%of total whey protein,respectively.In addition,depending on the concentration and separation process,there may be varying amounts of glycomacropeptide in whey protein.Whey protein contains all 20 amino acids that make up protein,in sufficient quantity and reasonable composition,which is a full-price protein.Therefore,as a high-quality protein with high nutritional value,whey protein is widely used in dairy products and formula foods.However,the product is subjected to different degrees of heat treatment during processing,and the heat treatment process will inevitably lead to the main functional components of whey protein(?-lactoglobulin,?-lactalbumin,lactoferrin,immunoglobulin,etc.)Denaturation occurs,and the stronger the heat treatment of milk,the greater the loss of functional protein.At the same time,the heat treatment process causes the loss or even loss of the activity of the biologically active components in whey protein,which will also affect the body's bioavailability of the main functional whey protein components in dairy products.However,due to the lack of accurate and quantitative detection methods for undenatured whey protein components in milk and dairy products,manufacturers cannot control product quality,and regulatory agencies cannot effectively supervise and manage terminal products.Therefore,it is an urgent need of the industry to carry out research on the detection methods of undenatured whey protein components,as well as the needs of national regulatory authorities,and it is a guarantee of consumer health and safety.The study conducts research on the detection of undenatured whey protein components from the following aspects:1.Determination of undenatured ?-lactoglobulin in milk and dairy products by high performance liquid chromatographyThe pH of the sample solution was adjusted to 4.6 with glacial acetic acid,and the supernatant was centrifuged.The undenatured ?-lactoglobulin in the supernatant was separated by high performance liquid chromatography,measured by an ultraviolet detector,and quantified by an external standard method.The limit of quantification of undenatured?-lactoglobulin in raw milk and liquid milk is 25.0 mg/kg,and the limit of quantification in milk powder is 50.0 mg/kg.In the daily and intra-day recovery rate and precision experiments of this method,ultra-high temperature sterilized milk and milk powder were used as the sample matrix,and ?-lactoglobulin was added at three concentrations of 500 mg/kg,1000 mg/kg and 2000 mg/kg.Each concentration level is subjected to 3 repeated tests for 3 consecutive days.The results show that the recovery rate of ?-lactoglobulin in UHT milk is 103.1%?113.6%,and the RSD is 0.4%?1.8%;milk powder The recovery rate of ?-lactoglobulin in the medium is 101.5%?111.0%,and the RSD is 0.1%?3.2%.The test results show that the undenatured ?-lactoglobulin detection method established in this experiment can be used for the detection of different milk and dairy products2.Determination of undenatured lactoferrin in milk and dairy products by high performance liquid chromatographyThe sample was extracted with phosphate buffer solution,centrifuged and the supernatant was taken,and the undenatured lactoferrin in the supernatant was enriched and purified by a heparin affinity column.The undenatured lactoferrin was separated by high performance liquid chromatography and detected by UV Quantification by external standard method.The limit of quantification of undenatured lactoferrin in raw milk and liquid milk is 5.0 mg/kg,and the limit of quantification in milk powder is 10.0 mg/kg.In the daytime and intraday recovery and precision experiments of this method,ultra-high temperature sterilized milk was used as the sample matrix,and lactoferrin 5 mg/kg,20 mg/kg and 50 mg/kg were added;milk powder was used as the sample matrix.Sample matrix,adding lactoferrin at three concentration levels of 10 mg/kg,50 mg/kg and 100 mg/kg.Each concentration level was subjected to 3 repeated tests for 3 consecutive days.The results showed that:in UHT milk The recovery rate of lactoferrin is 92.9%?109.3%,and the RSD is 1.5%?3.2%;the recovery rate of lactoferrin in milk powder is 97.6%?107.7%,and the RSD is 1.4%?3.1%.The test results show that the undenatured lactoferrin detection method established in this experiment can be used for the detection of different milk and dairy products.3.Determination of undenatured immunoglobulin G in milk and dairy productsThe sample was extracted with phosphate buffer solution,centrifuged,and the supernatant was taken.The protein G immunoaffinity column was used to enrich and purify the undenatured IgG in the supernatant.The undenatured IgG was separated by high performance liquid chromatography and measured by UV detector.,Quantification by external standard method.The limit of quantification of undenatured IgG in raw milk,liquid milk and milk powder is 100.0 mg/kg.In the daily and intra-day recovery and precision experiments of this method,ultra-high temperature sterilized milk and milk powder were used as the sample matrix,and the IgG concentration was 100 mg/kg,200 mg/kg and 400 mg/kg.Three repeated tests were performed for each concentration level for 3 consecutive days.The results showed that the recovery rate of IgG in UHT milk was 92.0%?103.9%,and the RSD was 1.2%?4.6%;the recovery rate of IgG in milk powder 105.0%?122.5%,RSD is 1.2%?4.2%.The test results show that the undenatured lactoferrin IgG detection method established in this experiment can be used for the detection of different milk and dairy products.4.Determination of whey protein content in infant formula poeders by capillary gel electrophoresis(SDS-PAGE)Weigh 135±5 mg skimmed milk powder or 500±20 mg infant formula powders into a 10 mL graduated centrifuge tube,dilute to 5 mL with deionized water,and vortex to mix evenly.The 1%SDS sample solution and 10 kDa IS protein were mixed in a ratio of 84:1 to prepare an analysis preparation solution.Pipette 10 ?L of sample solution into a 2.0 mL microcentrifuge tube,add 85 ?L of analysis preparation solution and 5 ?L of?-mercaptoethanol to the microcentrifuge tube,mix well,and then heat in a water bath at 95±5? for 10 minutes.After cooling to room temperature,it was centrifuged at 7000 r/min for 1 min at room temperature.The supernatant was separated by capillary gel electrophoresis and detected at 220 nm ultraviolet wavelength.The casein and whey protein in skimmed milk powder or infant formula are separated into two groups of protein peaks that do not interfere with each other.According to the peak areas of these two groups of protein groups,and the mass-to-area correction coefficient of whey protein to casein Calculate the percentage of whey protein in the total protein of infant formula milk powder by using the peak area normalization method.The recovery rate of this method is based on skimmed milk powder as the sample matrix.By adding different concentrations of concentrated whey protein,three repeated tests are performed at each concentration level for 3 consecutive days.The recovery rate is 91.3%?99.8%;the method is precise With skimmed milk powder and infant formula powders as the sample matrix,each sample is subjected to 3 repeated tests for 3 consecutive days,and the RSD is 0.1%?2.9%.The test results show that the test method for total whey protein in infant formula powders established in this experiment meets the methodological requirements and is suitable for the test of infant formula powders.