Lactoperoxidase-induced protein oxidation in milk.
The reaction between lactoperoxidase (LPO) and H(2)O(2) in the presence of bovine serum albumin (BSA), beta-lactoglobulin, or casein was investigated for the formation of protein radicals by freeze-quench electron spin resonance (ESR) and by the formation of the protein oxidation product, dityrosine. The presence of BSA resulted in a dramatic change after 1 min of reaction in the obtained ESR spectrum compared with the spectrum obtained for LPO and H(2)O(2) alone. Furthermore, experiments employing BSA or beta-lactoglobulin resulted in the formation of long-lived protein radicals detectable 10 min after initiation of the reaction. The presence of casein resulted in a minor change in the fine structure of the ESR spectrum after 1 min of reaction compared with LPO and H(2)O(2) alone, but no difference between the two reaction mixtures could be observed after 10 min of reaction. The formation of dityrosine could be detected in reaction mixtures containing LPO and H(2)O(2) after 1 and 10 min of incubation at 25 degrees C both in the absence and in the presence of BSA, beta-lactoglobulin, or casein. The presence of casein resulted in an increased dityrosine concentration compared with the reaction with LPO and H(2)O(2) alone. Endogenous LPO in unpasteurized milk was activated at 25 degrees C by adding 1 mM H(2)O(2). Radical species could be detected directly in the milk by freeze-quench ESR during the initial phase of the reaction, and dityrosine could be measured after 4 h of incubation. The role of LPO activity in the formation of ESR detectable radical species and dityrosine in milk was further verified in ultrahigh temperature (UHT) milk with no endogenous enzyme activity, as the formation of ESR detectable radical species and dityrosine took place in UHT milk only upon the addition of both H(2)O(2) and exogenous LPO.
Ostdal
H
H
Department of Animal Product Quality, Danish Institute of Agricultural Sciences, Research Centre Foulum, P.O. Box 50, DK-8830 Tjele, Denmark. Henrik.Ostdal@agrsci.dk
Bjerrum
M J
MJ
Pedersen
J A
JA
Andersen
H J
HJ
D000818:Animals; D002364:Caseins / Q000737:chemistry; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D007782:Lactoglobulins / Q000737:chemistry; D007784:Lactoperoxidase / Q000737:chemistry*; D008894:Milk Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D012710:Serum Albumin, Bovine / Q000737:chemistry
D002364:Caseins; D007782:Lactoglobulins; D008894:Milk Proteins; D012710:Serum Albumin, Bovine; D007784:Lactoperoxidase