Separation And Identification Of β-casein From Chinese Human Milk

Human milk is the safest and the most economical food for infants. It contains all nutrientswhich are necessary to infants’ growth and development. Human milk play an important biologicalfunction, such as, enhance baby resistance and promote tissue growth. At present, the breastfeedingrate of the infants which are under6months is less than40%, and more than sixty percent of infantsneed infant formula which can provide nutrients for them. Along with young children’s need thatthe market demand of infant formula is enormous. Therefore, carrying out a research to understandthe human milk protein components will be of great practical importance in developmenting infantformula milk. Related products will effectively solve the problem of the lack of breastfeeding.Selective precipitation of whole casein was achieved through centrifugal degreasing andregulating protein isoelectric point precipitation. Human β-casein was purified with anion exchangechromatography. The eluent peak was identified with Western-Blot and matrix-assisted laserdesorption/ionization time of flight/time of flight mass spectrometry （MALDI-TOF/TOF MS） afterthe casein processed with anion exchange chromatography. The result proved that β-casein can besuccessfully separated. The main research work and conclusions were showed as follows:⑴Selective precipitation of whole casein from skimmed milk was achieved by the additionof calcium salt at acidic pH. Effects of pH, centrifugal force and final concentration of CaCl₂onseparation of were analyzed by measuring purity of final products using sodium dodecylsulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, along with effects of the former threefactors on separation of whole casein. The results showed that whole casein with the highest puritycould obtained when the pH was4.3, the centrifugal force was40000g and the final concentrationof CaCl₂was60mmol/L.⑵Human milk casein was purified by anion-exchange liquid chromatography. Then allfractions were subjected to analysis by SDS-PAGE. It can be preliminary predicted that the eluentpeak III is human milk β-casein, and the purity of β-casein was more than90%.⑶The eluent peak III was identified with Western-Blot. The results of Western-Blot showedthat peak III can combine specificity with human β-casein antibody, and it is human milk β-casein.⑷The eluent peak III was identified with matrix-assisted laser desorption/ionization time offlight/time of flight mass spectrometry （MALDI-TOF/TOF MS）. The eluent peak III’s peptide massfingerprints were analyzed by Mascot searching to identify the protein, sequence coverage is50%,and further determine it is human milk β-casein.