Fish oil was incorporated into milk under different homogenization temperatures (50 and 72 degrees C) and pressures (5, 15, and 22.5 MPa). Subsequently, the oxidative stability of the milk and changes in the protein composition of the milk fat globule membrane (MFGM) were examined. Results showed that high pressure and high temperature (72 degrees C and 22.5 MPa) resulted in less lipid oxidation, whereas low pressure and low temperature (50 degrees C and 5 MPa) resulted in faster lipid oxidation. Analysis of protein oxidation indicated that especially casein was prone to oxidation. The level of free thiol groups was increased by high temperature (72 degrees C) and with increasing pressure. Furthermore, SDS-PAGE and confocal laser scanning microscopy (CLSM) indicated that high temperature resulted in an increase in beta-lactoglobulin adsorbed at the oil-water interface. This was even more pronounced with higher pressure. Less casein seemed to be present at the oil-water interface with increasing pressure. Overall, the results indicated that a combination of more beta-lactoglobulin and less casein at the oil-water interface gave the most stable emulsions with respect to lipid oxidation.

The present work reports the characterization of carbohydrate structures and the distribution of the newly identified mucin MUC15, a highly glycosylated protein associated with the bovine milk fat globule membrane (MFGM). Distribution of MUC15 was investigated in various fractions of bovine milk by densitometric scanning of Western blots. In raw milk, MUC15 was shown to constitute 0.08% (wt) of the protein and approximately 1.5% (wt) of the MFGM-associated proteins. Surprisingly, this study showed that in addition to the fat-containing fractions, such as MFGM and buttermilk, MUC15 was present in nonfat-containing fractions as well, such as skim milk and whey. Compositional and structural studies of the carbohydrates of bovine milk MUC15 showed that the glycans are composed of fucose, galactose, mannose, N-acetylgalactosamine, N-acetylglycosamine, and sialic acid. The carbohydrate was shown to constitute 65% of the total molecular weight, and the molar ratios of the individual sugars to protein of the O-linked glycans were determined. The glycan structures of MUC15 were further studied by enzymatic deglycosylation experiments using different endo- and exoglycosidases as well as a panel of lectins. The N-linked glycans were shown to contain mainly hybrid-type N-glycans. In addition, the N-glycans were shown to be sialylated and contain terminal poly-lactosamine structures. The O-linked glycans were found to constitute some unsubstituted Core-1 structures and a substantial number of sialylated Core-1 O-linked glycans. By comparing the results of peanut agglutinin lectin binding, enzymatic deglycosylation, and monosaccharide composition analysis, we concluded that bovine MUC15 also contains more complex O-glycans containing high amounts N-acetylglucosamine residues. Furthermore, a small subset of the O-linked glycans is decorated with lactosamine on their terminal ends.

The glycoprotein MUC15 (mucin 15) was initially isolated from the bovine milk fat globule membrane. The present work demonstrates the existence of immunologically similar proteins ( approximately 130 kDa) in ovine, caprine, porcine, and buffalo milk samples. Purification and N-terminal amino acid sequencing confirmed the presence of ovine and caprine MUC15 orthologs in milk fat globule membranes. Expression of MUC15 in human milk was demonstrated by immunostaining ( approximately 150 kDa) as well as by mass spectrometry. Screening of a human multiple tissue expression array showed abundant MUC15 gene expression in placenta, salivary gland, thyroid gland, trachea, esophagus, kidney, testis, and the leukemia K-562 cell line. Furthermore, moderate expression was seen in the pancreas, adult and fetal lung, fetal kidney, lymph node, adult and fetal thymus, and parietal lobe. Structural motifs for interactions (epidermal growth factor receptor and Src homology 2 domains) are identified in the intracellular region. Implication of the mucin in signal transduction and the potential physiological function of MUC15 are discussed.

The milk protein proteose peptone component 3 (PP3), also called lactophorin, is a small phosphoglycoprotein that is expressed exclusively in lactating mammary tissue. The C-terminal part of the protein contains an amphipathic helix, which, upon proteolytic liberation, shows antibacterial activity. Previous studies indicate that PP3 forms multimeric structures and inhibits lipolysis in milk. PP3 is the principal component of the proteose peptone fraction of milk. This fraction is obtained by heating and acidifying skimmed milk, and in the dairy industry milk products are also typically exposed to treatments such as pasteurization, which potentially could result in irreversible denaturation and inactivation of bioactive components. We show here, by the use of CD, that PP3 undergoes reversible thermal denaturation and that the a-helical structure of PP3 remains stable even at gastric pH levels. This suggests that the secondary structure survives treatment during the purification and possibly some of the industrial processing of milk. Finally, asymmetric flow field-flow fractionation and multi-angle light scattering reveal that PP3 forms a rather stable tetrameric complex, which dissociates and unfolds in guanidinium chloride. The cooperative unfolding of PP3 was completely removed by the surfactant n-dodecyl-b-D-maltoside and by oleic acid. We interpret this to mean that the PP3 monomers associate through hydrophobic interactions via the hydrophobic surface of the amphipathic helix. These observations suggest that PP3 tetramers act as reservoirs of PP3 molecules, which in the monomeric state may stabilize the milk fat globule. Structured digital abstractl PP3 and PP3 bind by circular dichroism (View interaction) l PP3 and PP3 bind by molecular sieving (View interaction) l PP3 and PP3 bind by fluorescence technology (View interaction) l PP3 and PP3 bind by molecular sieving (View interaction) Abbreviations AF4, asymmetric flow field-flow fractionation; DDM, n-dodecyl-b-D-maltoside; DOPC, 1,2-dioleoylphosphatidylcholine; DOPG, 1,2-dioleoylphosphatidylglycerol; GdmCl, guanidinium chloride; MALS, multi-angle light scattering; MRE, mean residue ellipticity; OA, oleic acid; PP3, proteose peptone component 3; RI, refractive index; SEC, size exclusion chromatography; TFE, trifluoroethanol.

The milk protein proteose peptone component 3 (PP3, also known as lactophorin) is a small phosphoglycoprotein, which is exclusively expressed in the lactating mammary gland. A 23-residue synthetic peptide (lactophoricin, Lpcin S), corresponding to the C-terminal amphipathic α-helix of PP3, has previously been shown to permeabilize membranes and display antibacterial activity. Lactophorin readily undergoes proteolytic cleavage in milk and during dairy processing, and it has been suggested that PP3-derived peptides are part of milk's endogenous defense system against bacteria. Here, we report that a 26-residue C-terminal peptide (Lpcin P) can be generated by trypsin proteolysis of PP3 and that structural and functional studies of Lpcin P indicate that the peptide has antibacterial properties. The Lpcin P showed α-helical structure in both anionic and organic solvents, and the amount of α-helical structure was increased in the presence of lipid vesicles. Oriented circular dichroism showed that Lpcin P oriented parallel to the membrane surface. However, the peptide permeabilized calcein-containing vesicles efficiently. Lpcin P displayed antibacterial activity against Streptococcus thermophilus, but not against Staphylococcus aureus and Escherichia coli. The PP3 full-length protein did not display the same properties, which could indicate that PP3 functions as a precursor protein that upon proteolysis, releases a bioactive antibacterial peptide.