title: In vitro digestion of purified β-casein variants A(1), A(2), B, and I: effects on antioxidant and angiotensin-converting enzyme inhibitory capacity.
abstract: Genetic polymorphisms of bovine milk proteins affect the protein profile of the milk and, hence, certain technological properties, such as casein (CN) number and cheese yield. However, reports show that such polymorphisms may also affect the health-related properties of milk. Therefore, to gain insight into their digestion pattern and bioactive potential, β-CN was purified from bovine milk originating from cows homozygous for the variants A(1), A(2), B, and I by a combination of cold storage, ultracentrifugation, and acid precipitation. The purity of the isolated β-CN was determined by HPLC, variants were verified by mass spectrometry, and molar extinction coefficients at λ=280nm were determined. β-Casein from each of the variants was subjected to in vitro digestion using pepsin and pancreatic enzymes. Antioxidant and angiotensin-converting enzyme (ACE) inhibitory capacities of the hydrolysates were assessed at 3 stages of digestion and related to that of the undigested samples. Neither molar extinction coefficients nor overall digestibility varied significantly between these 4 variants; however, clear differences in digestion pattern were indicated by gel electrophoresis. In particular, after 60min of pepsin followed by 5min of pancreatic enzyme digestion, one ≈4kDa peptide with the N-terminal sequence (106)H-K-E-M-P-F-P-K- was absent from β-CN variant B. This is likely a result of the (122)Ser to (122)Arg substitution in variant B introducing a novel trypsin cleavage site, leading to the changed digestion pattern. All investigated β-CN variants exhibited a significant increase in antioxidant capacity upon digestion, as measured by the Trolox-equivalent antioxidant capacity assay. After 60min of pepsin + 120min of pancreatic enzyme digestion, the accumulated increase in antioxidant capacity was ≈1.7-fold for the 4 β-CN variants. The ACE inhibitory capacity was also significantly increased by digestion, with the B variant reaching the highest inhibitory capacity at the end of digestion (60min of pepsin + 120min of pancreatic enzymes), possibly because of the observed alternative digestion pattern. These results demonstrate that genetic polymorphisms affect the digestion pattern and bioactivity of milk proteins. Moreover, their capacity for radical scavenging and ACE inhibition is affected by digestion.
authors.affiliation: Department of Food Science, Aarhus University, 8830 Tjele, Denmark.
authors.forename: B
authors.initials: B
authors.lastname: Petrat-Melin
authors.affiliation: Department of Food Science, Aarhus University, 8830 Tjele, Denmark.
authors.forename: P
authors.initials: P
authors.lastname: Andersen
authors.affiliation: Department of Molecular Biology and Genetics-Molecular Nutrition, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: J T
authors.initials: JT
authors.lastname: Rasmussen
authors.affiliation: Department of Food Science, Aarhus University, 8830 Tjele, Denmark.
authors.forename: N A
authors.initials: NA
authors.lastname: Poulsen
authors.affiliation: Department of Food Science, Aarhus University, 8830 Tjele, Denmark.
authors.forename: L B
authors.initials: LB
authors.lastname: Larsen
authors.affiliation: Department of Food Science, Aarhus University, 8830 Tjele, Denmark. Electronic address: JetteF.Young@agrsci.dk.
authors.forename: J F
authors.initials: JF
authors.lastname: Young
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002364:Caseins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D002417:Cattle; D002611:Cheese / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004063:Digestion; D005260:Female; D066298:In Vitro Techniques; D008892:Milk / Q000737:chemistry; D008894:Milk Proteins / Q000032:analysis; D010434:Pepsin A / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A; D011110:Polymorphism, Genetic; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
chemical_list: D000596:Amino Acids; D000806:Angiotensin-Converting Enzyme Inhibitors; D000975:Antioxidants; D002364:Caseins; D008894:Milk Proteins; D010455:Peptides; D007703:Peptidyl-Dipeptidase A; D014357:Trypsin; D010434:Pepsin A
keywords: bioactive peptide; genetic polymorphism; milk protein; β-casein