Study On Differences Of Caseins From South China Buffalo And Other Breeds Milk

Several technologies were used to study morphological characteristics, quantitative analysis and peptide mass fingerprinting differences of caseins from water buffalo milk in South China and other breeds milk protein, respectively. Three caseins particles with the differences in particle size distribution, morphological structure, zeta potential and surface characteristics in natural state were analyzed by nanoparticles size analyzer and AFM. Caseins fractions were separated and quantitatively detected by CZE and RP-HPLC, and the peptides differences were also studied by use of 2-DE coupling MALDI-TOF-MS. LTQ-Orbitrap coupled to liquid chromatography techniques were applied to study the differences in fine structure of three caseins. Results were as follows:The particle size range of casein was 43.8～955 nm in water buffalo milk,37.8～625.1 nm in bovine milk and 88.2～1484 nm in goat milk, and distribution proportions of particle size from 40 nm to 300 nm were 54.5%,58% and 74% in three caseins, respectively. The average zeta potential were-28.7 mV,-25.1 mV and-31.5 mV, respectively. Results indicated that water buffalo casein with middle the size distribution and average zeta potential shown certain stability in solution. The morphological structure of three caseins were observed in spherical or ellipsoidal shape and the particle distribution of water buffalo casein was more uniform than bovine's and goat's casein by means of AFM. The results suggested that bovine's and goat's caseins aggregation were obviously more heavier than water buffalo's casein.Used CZE to separate and analyze the caseins fraction. The effect of several parameters, including buffer system and concentration, pH value, urea concentration and injection time on the separation efficiency of water buffalo caseins was investigated using separation voltage of 25kV and detection wavelength of 214nm at 25℃. Under the optimal conditions, the calibration curves were linear in the range of 0.5-5.0g/L forαS-CN,β-CN andκ-CN with correlation coefficients more than 0.9980. The spiked recoveries of three main caseins were in the range of 85%～106%. The relative standard deviations (RSDs) of migration time, peak area and peak high were less than 4.9%,5.0% and 5.1%, respectively.RP-HPLC was used to separate and quantitatively detect the casein fractions from water buffalo in South China. Gradient elution was carried out at a flow-rate of 1.0 ml/min and a UV detector at 214 nm by using a mixture of solvents A with 0.1% trifluoroacetic acid (TFA) and solvent B with 100% acetonitrile. The results showed that linear relationship (r>0.9991) between the concentrations of casein fractions and peak areas was observed and recoveries rate for casein fractions was found within 86% and 97%. RSDs of Retention time and peak area were less than 0.41% and 4.60%, respectively. Different chromatographic profiles and contents among water buffalo, bovine and goat caseins were also compared by use of RP-HPLC procedure.The major fractions of caseins from three different milk were separated by 2-DE to study the differences in caseins subunit structure. The results showed thatαS1-CN,αS2-CN,β-CN andκ-CN were located in different region according to their molecular weight and isoelectric point.21 differential proteins existed only in water buffalo milk were identified according to comparing the profiles of caseins matched with software of ImageMaster 2D Platinum. Four casein fractions and two new fractions which were high homology components with water buffalo casein (need to verify) were determined with the further analysis by MALDI-TOF/TOF-MS.To investigate amino acid sequence differences derived from the different resource of caseins, LTQ-Orbitrap coupled to liquid chromatography techniques were also applied to analyze substitution of amino acids in caseins fractions. Amino acids sequences of caseins fractions from experiment were acquired and matched with the amino acids sequences from the database, results shown that the substitution of amino acids from water buffalo and bovine of caseins were smaller than that from goat. That means stability of amino acids from water buffalo and bovine of caseins higher than the goat protein.All the results provide the theoretical basis for the development of antibacterial peptide, biological activity products of casein and the accurate identification of milk protein.