Effect of washing conditions on the recovery of milk fat globule membrane proteins during the isolation of milk fat globule membrane from milk

Experimental isolation procedure of milk fat globule membrane (MFGM) material from milk.

Sodium dodecyl sulfate-PAGE pattern of milk fat globule membrane (MFGM) samples obtained from different washing solutions: MU = milk ultrafiltrate; P5=50mM phosphate, pH 6.8, and 0.15 M NaCl; K=1.5g of KCl/L of water; D = deionized water. The sample load on each lane was 100ng of total protein. Subscripts 1, 2, and 3 indicate replicates 1, 2, and 3, respectively. Other lanes: SM = 100ng of skimmed milk proteins; c1, c5, and c10 = pure proteins (BSA, α_S-CN, β-CN, κ-CN, β-LG, and α-LA) loaded at 1, 5, and 10ng of each protein, respectively; and M12 = molecular weight indicator. Identification of MFGM proteins according to Mather (2000) and other proteins is given on the left; MUC1 = mucin 1; XDH/XO = xanthine dehydrogenase/oxidase; PAS III = periodic acid Schiff III; CD36 = cluster of differentiation 36, BTN = butyrophilin; PAS 6/7 = periodic acid Schiff 6/7; ADPH = adipophilin; PP3 = proteose peptone 3. The asterisk (*) indicates variants of the fragment of BTN released by proteolysis and lacking the C-terminus (Mather, 2000). One of the bands at the arrows could be apolipoprotein E (Fong et al., 2007).

Sodium dodecyl sulfate-PAGE pattern of milk fat globule membrane (MFGM) samples obtained from different washing temperatures: 38, 42, and 46°C. The sample load of total protein on each lane was 100ng. Subscripts 3 and 4 indicate replicates. Other lanes: SM = 100ng of skim milk proteins; c1, c5, and c10 = pure proteins (BSA, α_S-CN, β-CN, κ-CN, β-LG, and α-LA) loaded at 1, 5, and 10ng of each protein, respectively; and M12 = molecular weight indicator. Lanes 38w, 42w, and 46w were loaded with 0.5μL of the third discharged washing solutions of isolation at 38, 42, and 46°C. The lanes contained 30, 36, and 41ng of total proteins, respectively. Identification of MFGM proteins according to Mather (2000) and other proteins is given on the left; MUC1 = mucin 1; XDH/XO = xanthine dehydrogenase/oxidase; PAS III = periodic acid Schiff III; CD36 = cluster of differentiation 36, BTN = butyrophilin; PAS 6/7 = periodic acid Schiff 6/7; ADPH = adipophilin; PP3 = proteose peptone 3. The asterisk (*) indicates variants of the fragment of BTN released by proteolysis and lacking the C-terminus (Mather, 2000). One of the bands at the arrows could be apolipoprotein E (Fong et al., 2007).

Sauter mean diameter [d_(3,2)] of fat globules of milk, separated cream (cream), and that after the first (1w), second (2w), and third (3w) wash at 38 (—♦—), 42 (—■—) and 46°C (—▴—). The samples were dispersed in a) water and b) SDS/EDTA solution before measuring.

Fat globule size distributions in volume percentage versus droplet diameter of milk (—) and separated cream (----) and cream after the first (—△—), the second (—×—), and the third wash (—○—), which were determined after dispersing the samples in water. Cream separation and washing was done at a) 38, b) 42, and c) 46°C. The arrow points to a small and broad peak between 10 and 100μm.

Sodium dodecyl sulfate-PAGE pattern of milk (M) and milk fat globule membrane (MFGM) sample (E) isolated using procedure E (2 washes with 4.5L of deionized water) under the staining with Simply Blue (Invitrogen, Merelbeke, Belgium). The sample load on each lane was 20μg of total protein. M12 = molecular weight indicator. Identification of MFGM proteins according to Mather (2000) and other proteins is given on the right; MUC1 = mucin 1; XO = xanthine oxidase; CD36 = cluster of differentiation 36, BTN = butyrophilin; PAS 6/7 = periodic acid Schiff 6/7; ADPH = adipophilin; PP3 = proteose peptone 3.