Study On The Differential Detection Technology Of Bovine And Sheep Milk Based On ?-Lactoglobulin

Goat and bovine milk are two kind of important dairy sources.Compared with bovine milk,the goat milk has the properties of high dry matter content,small fat globule,hypoallergenic,and similar casein to albumin ratio to the human milk.So goat milk is more apted to be digested and absorpted than that of bovine milk.Due to the small amount of milk lactation,the breeding scale of goat is small and goat milk production is influenced by the resources and seasonal factors,making the high production costs.Therefore,the prices of goat milk are higher than milk resulting in part of the adulteration behaviors of goat mixing with bovine milk,which damage the interests of consumers and the normal goat milk products market.In this study,we analyzed the differences of proteins in bovine and goat milk using the gel electrophoresis and found that the goat milk?-lactoglobulins(?-LG)differed from bovine milk,which was taken as the antigen to prepared the polyclonal and monoclonal antibodies.Three colloidal gold immuno-chromatography and an ELISA method were established based on the specific reaction of antigen and antibody to identify goat and bovine dairy products.The main results are as follows:(1)Two kinds of?-LG were isolated from cow and sheep milk.Determined by the recovery of whey protein,1.0 mol/L HCl precipitated casein resulted in higher protein content than 0.5 mol/L HCl precipitated casein,with higher purity showed by electrophoretic analysis.The optimum pH value for separating?-LG from whey by acid method was 3.9 for cow milk and 3.3 for goat milk.The protein yield of?-LG in milk by citric acid method was more than 20%higher than that by hydrochloric acid method.Under SDS-PAGE conditions,there were only one?-LG band both in cow and goat milk,and the molecular weight was about 17 KDa.However,under native electrophoresis conditions,the most obvious difference of protein bands in cow and goat milk is the low molecular weight of whey protein.The migration rate of?-LGA and?-LGB in cow milk is the fastest of the proteins,appeared at the bottom of gel,while there is only one?-LG band of goat milk,which is in the middle of the gel.Therefore,it is possible to use?-LG as a characteristic target protein to identify goat milk and bovine milk.(2)Preparation and detection of bovine?-LG based on polyclonal antibody.New Zealand white rabbits were immunized with the bovine?-LG isolated.After four-time immunization,the antiserum titer reached 1:20000.Protein A affinity column was used to purified?-LG-IgG and yielded the antibody protein content of 1.28 mg/mL,with its purity over 90%and a titer of 1:102400.There were no obvious cross-reaction.It had good specificity and could be used in subsequent experiments.An indirect enzyme-linked immunosorbent assay(ELISA)was established for the analysis of bovine milk adulterated in goat milk.With the secondary antibody concentration of 1:4000,the best antigen coated amount of?-LG-IgG was 1:3200 mg/L,and the optimum dilution of milk sample was 2~5.When the volume fraction of adulteration is between 2%and 50%,the curve shows a linear relationship.The regression equation is Y=0.2044+0.0112X,R~2=0.994.The lowest detectable amount of skim milk in goat milk was 3.5%,and the coefficient of variation(CV)of milk ingredients in goat milk was less than 5%,which could effectively distinguish between cow and goat.Three colloidal gold immuno chromatographic(GICA)strip assays based on polyclonal antibodies were established.Antibody conjugated with colloidal gold at the optimum pH 8.0 with the amount of labeled protein of 10?L.The sample pad of the gold standard pad selection of glass fiber membrane models were VL68,blocked with PEG20000;Nitrocellulose membrane(NC membrane)selected SartoriusCN 95,test line(T-line)and quality control line(C-line)were coated with?-LG antibody and goat anti-rabbit antibody,crossed antibody concentration of 1.0 mg/mL use of 6?L,respectively.Detection of goat milk adulteration,bovine milk incorporation of 5%in goat milk can be detected.The encapsulation amount of competitive?-LG was the same as that of sandwich method,T-line coating with?-LG,and the lowest detectable incorporation amount of bovine milk was 10%.For double test strip,adding the T-line of?-CN-IgG,the minimum amount of bovine milk can be detected was still 5%.In the detection of commercial samples,the double test strip can avoid the false positive caused by?-LG denaturation,and the results are more stable than sandwich method.(3)Monoclonal antibodies against bovine?-LG were prepared to and improved the specificity of the detection.Mice were immunized with bovine?-LG and fused with mouse myeloma cells in semi-solid medium.Six hybridoma cell-positive cells were screened out,which were 1B9,2D3,3A8,4G7,5E4 and 5F12.Monoclonal antibodies were prepared from ascites of mice.The titers of the six antibodies were all over 1:10 000,and the cross reaction with goat?-LG was low and the specificity was high.The titer of monoclonal antibody labeled with colloidal gold was 1:1.28×10~5,which was used to establish sandwich immuno chromatographic strips.After optimizing the test conditions,the minimum amount of bovine milk adulteration was 2%by using 6?L,0.5 mg/mL?-LG-IgG and secondary antibody coated T-line and C-line.The test strip had lower detection limit,high specificity and reliable and stable detection results.The structure and immunogenicity of?-LG in cow milk and goat milk are different.Based on this aspect,an specific immunological detection method can be established for adulteration of bovine milk in goat milk.The sandwich immuno colloidal gold strip method based on monoclonal antibody has the advantages of stability,fast detection speed,low cross-reaction and low detection limit.It is suitable for rapid detection of large-scale samples in the field.