Free Radical Oxidation and Antioxidant Status of Milk from Different Cow Breeds

The dependence of lipid peroxidation (LP) processes in the milk on cow breed was revealed...


Introduction
Milk is a unique mix of nutrients containing more than 200 species that are not present in this combination in other human food products. Due to significant number of somatic cells with prevalence of neutrophils, milk is traditionally considered as a highly reduction-oxidation (redox) resistant polycomponent system, consisting of substances possessing antioxidant and health-beneficial features. At the same time, apoptotic death and "respiratory burst" of neutrophils in milk activates a free-radical lipid peroxidation (LP), because of the presence of unsaturated fatty acids. The features of the dynamics of indices, characterizing pro-oxidant-antioxidant homeostasis of milk as a biological fluid on the whole were shown in works of several researchers [1][2][3][4][5].
Nowadays, an approach allowing integration of a great amount of well-known earlier and described recently facts, being fragmented previously, was gained a further development in the concept of biological regulation mechanisms [5]. In particular, the existence of a direct dependence between biochemical reactions and accompanying physiological processes from one side and the redox status of biological system and a surrounding medium was shown by many authors [6][7][8][9][10]. However, the possibility of milk antioxidant effects prolongation by manipulation with cattle genealogy, their feeding, and introduction of antioxidants into the ration, milk and dairy products needs further investigation. Therefore, to shed more light on this question we studied models of the antioxidant status estimation, lipid peroxidation and other oxidative processes in milk of three most widely used breeds of cows.

Materials and Methods
The plan of research was reviewed and approved by Lipid peroxidation was estimated according to [11] and expressed in nM/ml, taking the molar extinction' coefficient of pure malondialdehyde (MDA, Sigma Aldrich) equal to 1.56·105. The determination of active products, interacting with thiobarbituric acid (TBA) was carried according to [12].
In short, the method is based on the interaction between MDA and TBA in acid medium at high temperature with the formation of colored trimethine complex, having absorption maximum at 532 nm. The concentration of diene conjugates (DC) extracted from milk was measured at 233 nm [11]. The mean molecular weight fraction of oligopeptides was determined a follow. At first, the 0. saline. The free-radical oxidation indexes were measured using chemiluminometer HLM-003. The intensity of Н 2 О 2 -induced chemoluminescence was expressed in imp/s. The content of glutathione was estimated according to the [13]. The content of protein sulfhydryl groups in milk was determined according to [14], using the initial protein precipitation by means of perchloric acid followed by solubilization and interaction of sulfhydryl groups with Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid). Total milk antioxidant activity was estimated according to its ability to inhibit the accumulation of TBA-active products of LP under incubation of egg yolk lipoproteins as described in [15]. Lipid antioxidant activity was expressed as percentage of lipoprotein oxidation inhibition by egg yolk lipoproteins. Free-radical oxidation products (diene conjugates, ketodienes, trienes, Schiff's bases) were determined in isopropanol phase of milk at a wavelength of 232, 278 and 400 nm respectively. The result was expressed in oxidation index units, representing the absorption' ratio of oxidation products relative to independent double bonds number (measured at 220 nm), namely Е232/220, E278/220 and Е400/220.
Statistic parameters were calculated in the accordance with [16].
The data were presented in the form of median and interquartile range from 25 (low quartile) to 75 (high quartile). Comparison between groups was performed using the Mann-Whitney U test, while comparing more than 2 groups, the Kruskal-Wallis test was used [17]. Correlation analysis was performed using Spearman (rS) and Kendel (rK) tests [18]. Differences were considered statistically significant at p >0.999.

Results and Discussion
After studying the content of free-radical oxidation products, Fenton's medium. This reaction was modeled as follows: with a data on total amount of accumulated TBA-active products (Table 1). To evaluate the free radicals' amount and antioxidant properties of milk the luminol-dependent chemoluminescence was used. The generation of free radicals stimulated by presence of milk' somatic cells can be promoted by an addition of luminol.

The latter increases quantum efficiency of the reaction and it leads
to a boost in functional activity of leukocytes. This "respiratory burst" results in generation of -О 2 , O 2 and OH* [20]. The data on chemoluminescence of combined (daily) milk of cows of different breeds are presented in Table 3. The biggest changes were observed in the group (II) both for rapid and slow flashes. It was found that maximum intensity (Imax) parameter, most likely characterizing the free radicals' amount and thus LP speed, decreases in the row group (II) > group (III) > group (I) ( Table 3      Similarly, to luminol case, the highest S and Imax were observed in group (II) both for hydroperoxide-induced and own luminescene of milk, compared to other test groups ( Table 4)