Effects of enzymatic dephosphorylation on properties of bovine casein.

摘要

Milk proteins represent an important source of protein ingredients due to their distinctive physico-chemical, nutritional, technological and functional properties. Casein content of milk represents about 80% of milk proteins. The distinguishing property of phosphorylation provides important properties to caseins. The objectives of this research were to investigate enzymatic dephosphorylation of caseins, to characterize products of dephosphorylation and to examine the effects of dephosphorylation on biological properties of caseins.;Bovine whole casein, alpha-casein and beta-casein were dephosphorylated with potato acid phosphatase; optimum dephosphorylation conditions were 37°C, pH 5.8 for 6 h. The extents of dephosphorylation accounted for 71.6, 89.2 and 73.7% for whole casein, alpha-casein and beta-casein, respectively. The apparent Vmax and apparent K m for dephosphorylation of whole casein were 0.283 mumol P/mg casein min and 9.951 mg casein/l, respectively. SDS-alphaPAGE, urea-PAGE, RP-HPLC and ESI-MS demonstrated effects of dephosphorylation on the caseins. Urea-PAGE and ESI-MS confirmed the identities of the individual fractions. ESI-MS established (a) the molecular weight for alpha-casein and beta-casein as 23, 612 and 24, 017 Da, respectively; (b) random removal of 1, 2, 4, 6, 7 and 8 phosphate groups from alpha-casein and 1, 2, 3, 4 and 5 phosphate groups from beta-casein and (c) effects of incubation conditions. The effects of dephosphorylation of alpha-casein and beta-casein on the action of pepsin and trypsin were evaluated. Peptide mapping by RP-HPLC indicated that both proteases generated a complex mixture of peptides, with dephosphorylated peptides showing an increase in retention time. LC-ESI-MS and MS/MS in conjunction with the use of advanced bioinformatics software resulted in the identification of the peptides generated. Dephosphorylated alpha-casein and beta-casein showed the presence of peptides in which phosphate groups were removed, and were not observed in peptides from the corresponding native protein. Several of the peptides identified contained sequences that have been reported to be biologically active. Residual allergenicity of dephosphorylated whole casein, alpha-casein and beta-casein as well as peptic and tryptic products of these caseins was determined by an ELISA technique. The results demonstrated that removal of phosphate groups from whole casein, alpha-casein and beta-casein reduced allergenicity by 33, 31.2 and 24.4%, respectively. Proteolysis and dephosphorylation resulted in a significantly (p 0.05) higher reduction in the antigen-antibody binding capacity compared to non-hydrolyzed and non-dephosphorylated caseins, particularly in the highly allergenic alpha-casein.