Identification Of Whey Protein And Quantitative Detection Of Lactoferrin Based On Differential Scanning Fluorescence Method

The type and content of whey protein are important indicators of the nutritional and commercial value of milk and dairy products.Therefore,whey protein detection methods are essential for the identification of the authenticity of milk sources,the optimization of dairy processing and the assessment of the effective nutritional value of dairy products.The available methods for whey protein detection,such as electrophoresis,chromatography and chromatographymass spectrometry,can not widely apply due to their high cost,tedious manual operation and time consuming.Thus,it is necessary to establish the fast and efficient methods to detect the main whey protein which can be used in practical production.Differential scanning fluorescence detection technology(DSF)is a relatively new method with the significant advantages of simple operation,fast detection and less sample consumption.In the study,the method for the identification of whey proteins was developed,based on the DSF and the intrinsic fluorescence signal of whey proteins to obtain thermal stability difference curves.The method is applied to the differentiation of milk from different heating treatments and the rapid identification of milk from different animal species.In addition to that,the accurate quantitation of native bovine lactoferrin in dairy products was achieved by DSF.The contents and results were detailed as follows:(1)DSF-based identification of bovine whey proteins.Five whey protein specimens,including β-lactoglobulin,α-lactalbumin,bovine immunoglobulin,bovine serum albumin and lactoferrin,were analyzed by DSF.The characteristics of the thermal stability difference curves of each whey protein were extracted and statistically analyzed to establish a method for the identification of the five bovine whey proteins.The method was applied for the differentiation of dairy products with different heating treatments.The results showed that stable characteristic melting curves could be obtained at>0.2 mg/mL protein and pH 4.0-7.0.Melting information with significant differences and characteristics could be extracted by using melting curves of 350 nm,350 nm/330 nm or first derivative curves of the above two melting curves.The five types of bovine whey proteins were accurately identified by date processing,and the accuracy of blind sample testing reached 100%.Finally,the differentiation of different heating treatments bovine milk products was successfully achieved by extracting whey proteins to overcome the interference with endogenous fluorescent casein.(2)DSF-based identification of milk from different animal species.DSF melting curves of whey proteins from different animal species were found to be independent and significantly different from each other,so that a rapid identification method for the authenticity of milk sources was established.The results showed that the melting curves of human-,bovine-and sheep-derived IgG,human-,bovine-and sheep-derived LF,human-and bovine-derived serum albumin specimens were significantly different at protein concentrations>0.1 mg/mL and pH 4.0-7.0,indicating that the differences in thermal stability of whey proteins from different sources could be identified by DSF.Whey proteins were extracted by acetate buffer solution at pH 4.10 in order to reduce the interference of casein.The DSF melting curves of β-lactoglobulin from different animal sources were significantly different when the concentration of whey protein was higher than 1.0 mg/mL,which could be used for the accurate identification of adulterated bovine milk in sheep and camel milk.The detection limit of the adulteration ratio bovine whey protein to goat whey protein was as low as 5%,indicating that the method has a strong potential for practical application in the detection of authenticity of milk sources.(3)DSF-based detection of lactoferrin content in milk and dairy products.According to the properties that only lactoferrin increases its thermal stability significantly after binding Fe3+,and its melting temperature increases from 69℃to 90℃,a specific qualitative and quantitative method for the determination of lactoferrin content was established.The results show that the melting peak curve of 350 nm/330 nm is suitable for the qualitative determination of LF,while the melting peak curve of 350nm is more effective for the quantitative detection of LF.There was a good linear relationship between the melting peak area of at 350nm and the concentration of LF,y=0.01794x-0.28011(R2=0.99825).The detection limit of LF in PBS buffer was 0.03 mg/mL,but it increased to 0.2 mg/mL in simulated whey protein,which could not meet the requirements of actual sample detection.The preheating method could not remove the interference of other heatunstable whey proteins in the dairy matrix,but the interference was greatly reduced after heparin afinity column pretreatment.The quantitative detection of LF in raw milk and milk powder was successfully realized,the spiked recovery rate was 70.05%-126.67%,and the results were consistent with HPLC.