Study On Separation And Purification Technology Of South China Buffalo Caseins And The Differences Of Peptide Mass Fingerpringting From Other Non-milk Proteins

The southern buffalo milk was used as raw material in this study. Calcium-ureaprecipitation and ion-exchange chromatography were used to separate the main ingredients ofthe southern buffalo milk casein. And polyacrylamide gel electrophoresis (SDS-PAGE), reversedphase high performance liquid chromatography (RP-HPLC) and LTQ Orbitrap massspectrometer were used to analysis the main ingredients of caseins. These methods provided atheoretical basis for the bulk separation of casein and the technical means for fast qualitydetection and identification of the southern buffalo milk in food field. And laid the foundationfor the research of the bioactive peptides from the different components of the casein anddevelopments of functional foods respectively. Follows as the results:The results of chemical precipitation separation of casein showed that high purity of κ-CNwas got from milk when the concentration of CaCl2was0.09mol/L using the Calciumprecipitation, its purity could reach95.84%and the yield was approximately384mg/100mL.High-purity β-CN was obtained using calcium-low temperature system.its purity could be up to99%and the yield was approximately113mg/100mL, but low purity αs-CN was obtained usingthis system. the highest purity was63.72%and the yield was1696mg/100mL. αs-CN and β-CNcomponent could be better separation by using urea calcium precipitation system, and the puritycould reach83.43%and69.80%,And achieved943mg/100mL and468mg/100mL respectively.An excessively high concentration of urea was not suitable for the separation of the caseincomponent in the system.A method of separating the main component of casein, using ion-exchange chromatographywas established.The separation was carried out using Z series column (φ1.0×40cm) filled withDEAE-Sepharose CL-6B ion-exchange media.50mmol/L of tris-HCl buffer solution (pH8.0)including3mol/L urea and0.1%β-mercaptoethanol (β-ME) was used as the equilibriumsolution and different concentrations of NaCl solution was used as elution solution. Thedetection wavelength is280nm at the rate of2mL/min. The results showed that purified αs-CN(purity up to99.90%) could be obtained when the gradient elution concentrations of NaCl were0.1mol/L,0.2mol/L and0.3mol/L. And κ-CN，β-CN and αs-CN were separated when thegradient elution concentrations of NaCl were0.16mol/L,0.20mol/L and0.24mol/L,and the puritywere92.0%，99.5%，99.8%respectively detected by RP-HPLC. RP-HPLC was used to analysis the caseins. Gradient elution was carried out at a flow-rateof1.0ml/min and a UV detector at214nm by using0.1%trifluoroacetic acid (TFA) as theelution solvents A and100%acetonitrile as the elution solvent B. The results showed that thefour components of the casein, κ-CN, αs2-CN, αs1-CN, β-CN were eluted respectively and thecontent of them and peak areas had good linear relationship with the correlation coefficientgreater than0.9990in a certain protein concentration range, the recoveries was ranged between86%and97%. And good repeatability and reproducibility were observed. The comparativeanalysis of the differences of casein, soy protein and casein added soy protein by highperformance liquid chromatogram showed that characteristic peaks were found at the retentiontime of approximately8.5min,14.8min,20~27min in the casein added soy protein. This methodprovided a basis to detect cheap protein in milk protein.The study of this work was the establishment of the peptide mass fingerpringting of southbuffalo milk caseins and soybean protein components and comparation their amino acidsequences using the LTQ Orbitrap XL mass spectrometer. According to the results, the peptidesgot from enzyme treatment by trypsin of south buffalo milk casein would not found in soybeanprotein. And the number of amino acid, composition and proportion and the arrangement ofamino acid between south buffalo milk casein, which contains αs-CN, β-CN and κ-CN,andsoybean protein, contains glycinin(glycinin G1~G5subunit) and beta-conglycinin(α,α’,β subunit)were significant different. The established method provided a high precision and accuracy toolfor the identification and analysis the cheap proteins in milk protein. And it would be a powerfuland reliable analytical tool for identifying the low level components from complex mixture.