Occurrence of β-casein fragments in cold-stored and curdled river buffalo (Bubalus bubalis L.) milk

a) Sodium dodecyl sulfate-PAGE electrophoretograms of fresh (1) and frozen (2) river buffalo milk; b) cation exchange chromatography (cationic ion-exchange chromatography) separation of casein from frozen buffalo milk. Collected fractions were analyzed by SDS-PAGE (see Figure 2).

Sodium dodecyl sulfate-PAGE electrophoretograms of fractions collected from cationic ion-exchange chromatography. Protein bands correspond to whole casein of frozen buffalo milk; lane 1 = β-CN; lane 2 = β-CN with the additional 15.3 kDa component; lanes 3, 4, and 5 = κ-CN; lanes 6 and 7 = α_S-CN.

Reversed phase (RP)-HPLC chromatograms of the cationic ion-exchange chromatography fraction 2 showing 2 components. The matrix-assisted laser desorption ionization time of flight spectrum of the RP-HPLC fraction at 39.8min (inset) confirmed the presence of the 15,748.8Da protein component.

Matrix-assisted laser desorption ionization time of flight mass spectra of peptides arising from tryptic digestion of a) dephosphorylated river buffalo β-CN and b) protein component A (m/z 15,748.8Da), isolated by reversed-phase HPLC.

Sodium dodecyl sulfate-PAGE analysis of in vitro action of plasmin on bovine and river buffalo β-CN. Lane 1 = pure buffalo β-CN; lanes 2, 3, 4, and 5 = buffalo β-CN incubated with plasmin after 2, 4, 16, and 32min, respectively; lane 6 = river buffalo β-CN containing β-CN fragment (69–209); lane 7 = molecular weight standards; lane 8 = pure bovine β-CN; lanes 9, 10, 11, and 12 = bovine β-CN incubated with plasmin for 2, 4, 16, and 32min, respectively.

Sodium dodecyl sulfate-PAGE analysis of in vivo action of plasmin in cooled and frozen river buffalo milk. Fresh milk cooled at 4°C was sampled after 1, 2, and 4 d (lanes 1, 3, and 4, respectively); frozen milk at −20°C was sampled after 2, 4, 10, 15, and 30 d (lanes 2, 5, 6, 7, and 8, respectively).

Sodium dodecyl sulfate-PAGE electrophoretograms of milk and curd stored at −20°C; lane 1 = molecular weight standards; lanes 2, 3, and 4 = bovine milk after 3, 6, and 5 mo, respectively; lanes 5, 6, and 7 = river buffalo milk after 3 mo, 1 yr, and 6 mo, respectively; lanes 8 and 9 = bovine curd and lanes 10, 11, and 12 = river buffalo curd after 1 yr of storage. River buffalo milk was characterized by the presence of β-CN fragment (69–209); curds were distinguished by the presence of para-κ-CN.

Matrix-assisted laser desorption ionization time of flight spectra of protein extract from a) bovine and b) river buffalo Mozzarella cheese. To simplify, the first decimal figure in the molecular mass values has been omitted.