No abstract

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.

Oxidation in 3 types of bovine milk with different fatty acid profiles obtained through manipulation of feed was evaluated by analytical methods quantifying the content of potential antioxidants, the tendency of formation of free radicals, and the accumulation of primary and secondary oxidation products. The milk samples were evaluated in parallel by descriptive sensory analysis by a trained panel, and the correlation between the chemical analysis and the descriptive sensory analysis was evaluated. The fatty acid composition of the 3 types of milk was found to influence the oxidative and lipolytic changes occurring in the milk during chill storage for 4 d. Sensory analysis and chemical analysis showed high correlation between the typical descriptors for oxidation such as cardboard, metallic taste, and boiled milk and specific chemical markers for oxidation such as hexanal. Notably, primary oxidation products (i.e., lipid hydroperoxides) and even the tendency of formation of radicals as measured by electron spin resonance spectroscopy were also highly correlated to the sensory descriptors for oxidation. Electron spin resonance spectroscopy should accordingly be further explored as a routine method for detection of early events in lipid oxidation in milk to predict shelf-life.

Water-binding properties of myofibrils extracted from porcine muscle, and added hemoglobin with and without exposure to H2O2, were characterized using low-field proton NMR T2 relaxometry. The effects of pH and ionic strength in the samples were investigated as pH was adjusted to 5.4, 6.2, and 7.0 and ionic strength was adjusted to 0.29, 0.46, and 0.71 M, respectively. The formation of dityrosine as a measure of oxidative protein cross-linking revealed a significant increase in dityrosine concentrations upon H2O2 activation. The formation of dityrosine was strongly pH-dependent and increased with decreasing pH. In addition, increased levels of thiobarbituric acid reactive substances were observed upon addition of H2O2, implying that lipid oxidation was enhanced, however, with a different oxidation pattern as compared to the myofibrillar proteins. Low-field NMR relaxation measurements revealed reduced T2 relaxation times upon H2O2 activation, which corresponds to reduced water-holding capacity upon oxidation. However, a direct relationship between degree of oxidation and T2 relaxation time was not observed with various pH values and ionic strengths, and further studies are needed for a complete understanding of the effect of oxidation on myofibrillar functionality.