Characterization of major radical scavenger species in bovine milk through size exclusion chromatography and functional assays.
Radical scavenging activities of bovine milk components were quantified following size exclusion chromatography (SEC) with postcolumn characterization of fractions using the scavenging of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radicals (ABTS*(+)) in the Trolox equivalent antioxidant capacity (TEAC) assay and peroxyl radicals formed from cleavage of 2,2'-azobis(2-amidinopropane) (AAPH) in the oxygen radical absorbance capacity (ORAC) fluorometric assay. Caseins were quantitatively the major radical scavenger species in both assays, whereas beta-lactoglobulin (beta-lg) and alpha-lactalbumin (alpha-la) were much less active and only in the peroxyl radical assay. The radical scavenging activity of the caseins could be quantitatively accounted for by their constituent amino acids, as there were no effects of denaturing agents or complete digestion with proteases. In contrast, the activities of the whey proteins were dependent on denaturation or partial hydrolysis and dominated by the free thiol in beta-lg. A component in milk serum with a molecular mass of approximately 100 kDa contributed significantly to both ABTS*(+) and peroxyl radical scavenging but was absent in whey. This radical scavenger was identified as beta-casein. The only significant low molecular weight radical scavenger species were identified as ascorbate and urate in both assays.
Clausen
Morten R
MR
Department of Food Science, Faculty of Agricultural Sciences, Aarhus University, DK-8830 Tjele, Denmark.
Skibsted
Leif H
LH
Stagsted
Jan
J
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000975:Antioxidants / Q000032:analysis / Q000737:chemistry; D052160:Benzothiazoles; D002364:Caseins / Q000737:chemistry; D002417:Cattle; D002839:Chromans; D002850:Chromatography, Gel; D016166:Free Radical Scavengers / Q000032:analysis* / Q000737:chemistry*; D007768:Lactalbumin / Q000737:chemistry; D007782:Lactoglobulins / Q000737:chemistry; D008892:Milk / Q000737:chemistry*; D008894:Milk Proteins / Q000737:chemistry; D010545:Peroxides / Q000737:chemistry; D017510:Protein Folding; D013451:Sulfonic Acids; D000067816:Whey Proteins
D000596:Amino Acids; D000975:Antioxidants; D052160:Benzothiazoles; D002364:Caseins; D002839:Chromans; D016166:Free Radical Scavengers; D007782:Lactoglobulins; D008894:Milk Proteins; D010545:Peroxides; D013451:Sulfonic Acids; D000067816:Whey Proteins; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; C049375:perhydroxyl radical; D007768:Lactalbumin; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid