Distribution of xanthine oxidase and xanthine dehydrogenase activity in bovine milk: Physiological and technological implications

The aim of this study was to evaluate the distribution of xanthine oxidoreductase (XOR) and its two forms, xanthine oxidase (XO) and xanthine dehydrogenase (XD), in milk fractions. XOR associated with milk phospholipid membranes was found to be distributed among an intra-membranous pool in which it takes the form of a mixture of XO and XD, with a clear predominance of XD, and a free pool of XO, of which 33% is found in the outer surface of milk fat globule membrane, 20.5% in the outer surface of whey membrane particles, and the remaining 46.7% in apparent solution. The inner-membrane XOR may play a nonenzymatic role in fat secretion, whereas extra-membranous XO is freely available for a role in the innate gland immune system and may affect milk quality.     

Relationship between activity of some fat globule membrane enzymes and the lipidic fraction in ewes' milk: Preliminary studies

The aim of this preliminary study was to improve the knowledge about milk fat globules by studying the relationships between the morphometric characteristics of fat globules, the fatty acid composition and the activity of membrane enzymes evaluated in 14 milk samples from Massese ewes: xanthine oxidase (XO), xanthine dehydrogenase (XDH), γ-glutamyl transpeptidase (γ-GT), alkaline phosphatase (AP) and 5′-nucleotidase (5′-N). A negative correlation was found between the activity of XO and XDH and fat globule size, and a positive correlation between AP activity, the number of globule per mL and medium fat globules (2–5 μm). A significant relationship was found between the enzymatic activity of γ-GT, XO, XDH, 5′-N and various fatty acids in milk.     

Physicochemical and kinetic properties of purified sheep's milk xanthine oxidoreductase

Xanthine oxidoreductase (XOR) was purified for the first time from sheep's milk. The ultraviolet-visible absorption spectrum was essentially identical to those of the corresponding bovine, human, and goats' milk enzymes and showed an A280/A450 ratio of 5.35 +/- 0.24, indicating a high degree of purity. Like milk XOR from other species, sheep's milk enzyme showed a single band on SDS-PAGE corresponding to a subunit with approximate Mr 150,000. Xanthine oxidase activity of purified sheep's milk XOR (0.69 +/- 0.04 micromole urate min(-1) mg(-1)) was low relative to that of the bovine milk enzyme (1.83 +/- 0.02 micromole urate min(-1) mg(-1)), but higher than those of human or goats' milk XOR. As in the latter 2 cases, the low activity of sheep's milk XOR can be attributed to its relatively low molybdenum content (0.18 atoms per subunit), compared with that of the bovine milk enzyme (0.56 atoms Mo per subunit). Consistent with this, NADH oxidase activity of sheep's milk XOR was similar to that of enzymes purified from bovine, human, or goats' milk. The presence of desulpho-enzyme in sheep's milk XOR was demonstrated by resulfuration experiments, whereby xanthine oxidase activity was increased by approximately 75%.     

The variation of superoxide dismutase (SOD) and xanthine oxidase (XO) activities in milk using an improved method to quantitate SOD activity

An improved method for determination of superoxide dismutase (SOD) in milk has been developed. The interference of endogenously occurring xanthine oxidase (XO) has been subs'antially reduced by addition of an ultrafiltration step, which removed 61 ± 10 (mean ± SD) % of the XO activity. This method was used to determine the SOD activity in milk serum from 15 Swedish Red and White (SRW) cows and 18 Swedish Friesian (SF) cows at various stages of lactation. The XO activity in raw milk was also determined. The milk from these cows had a mean SOD activity of 0.83 ± 1.14 U ml^?1 milk serum and a mean XO activity of 49 ± 22 mU ml^?1 raw milk (n = 127). A difference between breeds could be noted in XO activity, ie milk from SF cows had significantly (P < 0.05) higher mean XO levels compared with milk from SRW cows, 54 ± 23 mU ml^?1 than 44 ± 21 mU ml^?1. A significantly increased level of XO activity in milk from SRW cows during the lactation was found. No significant effect of breed or stage of lactation on SOD activity could be detected. Individual cows with high and low milk SOD levels during the entire lactation have been identified.     

Goats' milk xanthine oxidoreductase is grossly deficient in molybdenum

Xanthine oxidoreductase (XOR) was purified from goats' milk. The u.v.-visible absorption spectrum was essentially identical to those of the corresponding bovine and human milk enzymes and showed an A280/A450 ratio of 5.20+/-0.12, indicating a high degree of purity. Like bovine and human milk XORs, enzyme purified from goats' milk showed a single band on SDS-PAGE corresponding to a subunit with approximate Mr 150,000. On Western blotting, mouse monoclonal anti-human XOR antibody cross-reacted with purified caprine and bovine XORs. The specific xanthine oxidase activity of goats' milk XOR, however, was very much lower than that of bovine XOR, although NADH oxidase activities of XOR from the two sources were similar. In these respects, the caprine milk XOR mirrors the human milk enzyme, in which case the kinetic effects have previously been attributed to relatively low molybdenum content. The molybdenum content of goats' milk XOR also was shown to be relatively low, with 0.09 atoms Mo per subunit, compared with 055 atoms Mo per subunit for the bovine enzyme. A parallel purification of human milk XOR showed 0.03 atoms Mo per subunit. The possible physiological significance of the low molybdenum content of the caprine milk enzyme and of its correspondingly low enzymic activity is discussed.     

Effect of Lenient Steam Injection (LSI) heat treatment of bovine milk on the activities of some enzymes,the milk fat globule and pH

This study investigated the effects of Lenient Steam Injection (LSI) treatment at temperatures 70–150 °C on the enzymatic activities of the indigenous milk enzymes alkaline phosphatase, lactoperoxidase (LPO), xanthine oxidase (XO), lipoprotein lipase (LPL) and plasmin in comparison with two reference heat treatments of 63 °C for 30 s and of 72 °C for 15 s by indirect heating. Milk fat globule (MFG) size distributions and pH were also monitored. Alkaline phosphatase, LPO, XO and LPL activities decreased with increasing LSI temperature. Plasmin activity was increased at temperatures <80 °C and decreased at temperatures above 90 °C. Milk fat globule homogenisation was observed at temperatures above 110–130 °C.     

Oxidation of aldehydes by xanthine oxidase located on the surface of emulsions and washed milk fat globules

The enzymatic activity of xanthine oxidase (XO) located within the native and recombined milk fat globule membrane (MFGM) on emulsion surfaces was investigated after successive washing with deionised water (DW) or simulated milk ultrafiltrate (SMUF). DW was more effective than SMUF at removing non-MFMG proteins from the globule surface. Surface XO activity on a protein basis increased after three washes due to loss of MFGM proteins to the serum phase. Recombined emulsions had higher surface enzymatic activity than washed native fat globule samples. The oxidation of acetaldehyde, 3-methylbutanal, 2-methylpropanol and 2-methylbutanal to the corresponding acids by XO on the globule surface was analysed. A higher proportion of aldehydes were depleted at higher XO activity when the initial aldehyde concentration was constant, and for higher initial concentrations of aldehydes. The recovery of aldehydes and acids was above 79%, indicating that the depleted aldehydes were mostly converted to the corresponding acids.     

Free and membrane-bound xanthine oxidase in bovine milk during cooling and heating

The effect of cold storage (5 C, 24 h) and heat treatment (60 C, 5 min) of milk on activities of free and membrane-bound xanthine oxidase has been studied. Both treatments enhanced total xanthine oxidase activity in milk. Activity of membrane-bound xanthine oxidase increased and free xanthine oxidase decreased in buttermilk while it increased in skim milk on cold storage. Heat of milk increased free and membrane-bound xanthine oxidase activities in both buttermilk and skim milk. The state of xanthine oxidase activity in skim milk from reconstituted milk, which was prepared by mixing xanthine oxidase inactivated skim milk and fresh cream, showed that only the free enzyme migrated from the cream phase to skim milk on cold storage. Very little xanthine oxidase activity was detectable in skim milk on heat treatment of the reconstituted milk sample. The overall increased activity of xanthine oxidase in milk during cold storage or heat treatment may not be due to the release of fat globule membrane enzyme to skim milk.     

Milk xanthine oxidase: Properties and physiological roles

Xanthine oxidoreductase (XOR) is a complex molybdoflavoenzyme that occurs as a major protein component of the milk fat globule membrane surrounding lipid droplets in milk. The enzyme has broad substrate specificity and is capable of reducing oxygen to generate the reactive oxygen species (ROS), superoxide and hydrogen peroxide. It can also reduce nitrite, yielding reactive nitrogen species (RNS), such as nitric oxide and peroxynitrite. By virtue of its capacity to generate ROS and RNS, milk XOR may play an antimicrobial defensive role in the neonatal gut, complementing endogenous enzyme of the intestinal epithelium. XOR is also involved in secretion of milk fat globules in a process dependent on the enzyme protein rather than on its enzymic activity, which is known to vary greatly with time after parturition and also between species. Some potential hazards and benefits of ingestion of cows milk XOR are briefly discussed.     

Instant infusion pasteurisation of bovine milk. II. Effects on indigenous milk enzymes activity and whey protein denaturation

Direct heat treatment of two milk types, skimmed and nonstandardised full‐fat, was performed by instant steam infusion and compared with indirect heating. Infusion conditions were temperatures of 72–120°C combined with holding times of 100–700 ms, and indirect heat conditions were 72°C/15 s and 85°C/30 s. The activity of indigenous enzymes such as alkaline phosphatase, lactoperoxidase, xanthine oxidase and γ‐glutamyl transpeptidase was evaluated. Infusion temperature was the main determinant of inactivation. Whey protein denaturation represented by β‐lactoglobulin increased significantly with infusion temperature. The nonstandardised milk had a higher denaturation rate than skimmed milk. The effect of instant infusion on pH and milk fat globule size in relation to whey protein denaturation and association is discussed.     

The impact of cream churning conditions on xanthine oxidase activity and oxidation–reduction potential in model emulsion systems

Six types of milk fat globule membrane (MFGM) fractions were isolated from fresh raw cream using various churning conditions at different temperature (10, 15 or 20 °C) and pH values (5.5 or 6.6) and characterised for protein composition, xanthine oxidase (XO) content and activity, and oxidation–reduction potential (E_h). A greater proportion of non-membrane proteins was found at higher temperatures and higher cream pH, and the XO content decreased significantly under these conditions. XO activity was greater at lower temperatures and pH, yielding a more positive E_h value for the emulsions. Storage at 4 °C for 14 d significantly reduced the Eh of the emulsions to between −320 mV and −580 mV for low pH/low temperature, and high pH/high temperature emulsions, respectively. This study suggests that it is possible to obtain MFGM fractions with specific Eh reducing ability by altering churning conditions.     

Influence of storage, heat, and homogenization upon xanthine oxidase activity of milk

Xanthine oxidase activity in milk was determined by measuring the rate of formation of vanillic acid from vanillin. Raw milk received at a dairy plant had .208 units xanthine oxidase activity per ml and after 24-h storage at 4 C, .228 units per ml. Upon further storage activity decreased. Heating the fresh raw milk in a water bath to 55 C increased xanthine oxidase activity to .236 units per ml. Partial inactivation of the enzyme occurred when milk was heated at 60, 65, or 70 C for 5 min, and destruction was almost complete with heat at 75 C for 5 min. Raw milk heated at 48 C for 5 min and homogenized at pressures between 70.3 and 281.2 kg/cm2 had xanthine oxidase activities which were a linear function of pressure and showed that each additional kg/cm2 pressure resulted in additional xanthine oxidase activity of .16 milliunits per ml of milk.     

Transport of bovine milk xanthine oxidase into mammary glands of the rat

This research investigated transport of bovine milk xanthine oxidase into mammary glands of the lactating rat. Transport capability suggested an exogenous, nonmammary, source for the enzyme. Five lactating rats were injected intracardially with 100 microgram of purified iodine-125 labeled xanthine oxidase and five were injected with 100 microgram of the enzyme unpurified. Four hours later the rodents were hand-milked, and radiation was confirmed in all samples by liquid scintillation counting. Counts were recorded per volume of milk and the percentage radiation was computed. Autoradiographs of the rats indicated radiation almost exclusively associated with the mammary glands. Greatest concentration of radioactivity was in the micellar casein fraction of milk, and a compound of high molecular weight, presumably [iodine-125]xanthine oxidase, was confirmed by gel filtration of the casein. Results suggest that the compound was transported into the mammary glands. The degree of transport was dependent upon the stage of lactation.     

Impact of different milk fat globule membrane preparations on protein composition,xanthine oxidase activity,and redox potential

The protein composition, redox potential (E_h), and xanthine oxidase (XO) content and activity were determined for anhydrous milk fat emulsions containing milk fat globule membrane (MFGM) fractions derived from either buttermilk or commercial sources of bovine α-serum, β-serum, and buttermilk powder (BMP). Caseins were the dominant proteins in the MFGM fractions isolated from BMP and buttermilk, whereas fractions from α- and β-serum contained higher amounts of membrane proteins. The XO content and activity was >70-fold and >700-fold higher in α- and β-serum samples, respectively, compared with the BMP fraction. The E_h values of the recombined emulsions were highest for α-serum (196 mV), and β-serum (169 mV), followed by BMP (131 mV). These positive values contrasted with the highly negative E_h of the buttermilk emulsion (−580 mV). This study demonstrates how milk-processing methods can alter the composition and functionality of the MFGM.     

In vitro antioxidant and in vivo xanthine oxidase inhibitory activities of Pandanus amaryllifolius in potassium oxonate‐induced hyperuricemic rats

Xanthine oxidase (XO) plays an important role in the regulation of uric acid and prevents it from being overproduced as in hyperuricemia disease. The combined effects of antioxidant and xanthine oxidase inhibitor would become a promising approach for hyperuricemia treatment. In this research, the antioxidant and xanthine oxidase inhibitory activities of Pandanus amaryllifolius leaf were evaluated. The leaf water extract (PA‐W) showed highest total phenols, and petroleum ether extract (PA‐PE) showed highest total flavonoids contents. The antioxidant activity of DPPH, metal chelating and hydrogen peroxide was highest in PA‐W extract. The treatment of PA‐W extract at 1000 mg kg^?1 body weight in potassium oxonate‐induced hyperuricemic rats showed significant (P < 0.001) decrease in serum uric acid level by 85% and XO activity by 64%, respectively, as compared to the hyperuricemic rats. In conclusion, the P. amaryllifolius possess the dual effect of antioxidant and XO inhibition as potential therapeutic agents in the hyperuricemia treatment.     

Homogenized milk: is it really the culprit in dietary-induced atherosclerosis?

Xanthine oxidase activity was assayed in commercial samples of homogenized milk subjected to pH ranging from 6.7 to 2.0 and held at room temperature for 5 min. Activity decreased sharply between pH 5.5 and 3.2. Below pH 3.2 no activity was detected. Also, rabbit anti-bovine xanthine oxidase failed to crossreact immunologically with xanthine oxidase of mouse milk. These results cast doubt on the hypothesis that dietary xanthine oxidase participates in the formation of atherosclerotic plaques.     

桑色素对黄嘌呤氧化酶活性的抑制作用

在磷酸盐缓冲体系（pH 7.5）中,利用紫外光谱法、荧光光谱法和圆二色谱法,结合分子模拟技术研究了桑色素对黄嘌呤氧化酶（xanthine oxidase,XO）活性的抑制机理。结果表明：桑色素是一种有效的可逆性混合型抑制剂,其半数抑制浓度（IC50）和抑制常数（Ki）分别为1.35×10–5 mol/L和1.21×10–5 mol/L;桑色素通过疏水作用力与XO形成基态复合物导致XO内源荧光的猝灭,并诱导XO的二级结构发生改变;分子模拟结果进一步证实桑色素主要通过疏水作用力结合到XO的活性中心,与Phe914、Phe649、Phe1009、Leu648、Leu1014和Leu873等主要氨基酸发生作用。推测桑色素进入XO的疏水腔,阻碍了底物黄嘌呤进入XO活性中心并影响了XO活性中心的形成,从而抑制了XO对黄嘌呤的催化活性。     

Antioxidant Activity and Protective Effect on DNA Cleavage of Resveratrol

ABSTRACT: The major natural polyphenols of wine are flavonoids. Red wine also contains a natural phytoalexin called resveratrol which recently has been the subject of conflicting reports regarding its protective role against cardiovascular diseases. We have investigated the free radical scavenging capacity of resveratrol, its effects on xanthine oxidase (XO) activity, spontaneous membrane lipid oxidation, and DNA cleavage. Resveratrol showed a dose-dependent free radical scavenging activity, significant inhibition of XO activity, an anti-lipoperoxidative capacity, and a protective effect on DNA cleavage. The antioxidant capacity of resveratrol is ascribed to the concomitant activities of scavenging free radicals, metal chelating, and inhibition of some enzymes involved in free radical generation.     

牛乳和双峰驼乳中乳脂球膜蛋白的差异分析

为阐明双峰驼乳中乳脂球膜蛋白（milk fat globule membrane proteins,MFGMP）的组成以及与牛乳MFGMP之间的差异,以牛乳和双峰驼乳中的MFGMP为研究对象,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、双向电泳和液相色谱-串联质谱（liquid chromatograph-tandem mass spectrometry,LC-MS/MS）技术对其中的高丰度蛋白质进行表征,并对双峰驼乳MFGMP提取方法进行选择。结果显示,双峰驼乳和牛乳MFGMP中含量最高的蛋白组分依次为嗜乳脂蛋白、黄嘌呤氧化还原酶/脱氢酶、乳凝集素（MFG-E8或PAS6/7）、脂肪分化相关蛋白（ADRP或PLIN2）,3 种方法分析双峰驼乳和牛乳中MFGMP中高丰度蛋白组分的结果具有一致性。基于LC-MS/MS技术对双峰驼乳的MFGMP进行鉴定,并将对鉴定出的蛋白质进行基因本体功能注释和京都基因与基因组百科全书路径分析,发现这些蛋白主要是细胞膜、线粒体、内质网膜等组分,参与的生物途径有翻译和翻译的起始、氧化还原过程、细胞内蛋白质转运、蛋白质折叠、细胞氧化还原稳态等,具有结合活性和分子催化等分子功能。     

Possible implications of milk pasteurization on the manufacture and sensory quality of ripened cheese

Cheese made from raw milk represents an important proportion of the traditional cheeses, particularly in South European countries. Besides destruction of pathogenic bacteria, the most significant changes in milk relevant to cheesemaking, which are induced by pasteurization are:

• a partial elimination of the milk microorganisms which may grow in cheese during ripening,

• a partial or total activation or inhibition of the plasmin/plasminogen complex, cathepsin D, lipoprotein lipase and alkaline phosphatase. Enzymes from psychrotrophic bacteria, acid phosphatase and xanthine oxidase, which may be active during ripening, withstand pasteurization.,

• a slight (7%) denaturation of serum proteins and little or no modification of the cheesemaking properties (coagulation, acidification by lactic acid bacteria).

From experimental work carried out on several cheese varieties, comparing pasteurized or microfiltered milk and raw milk cheeses, it was found that facultatively heterofermentative lactobacilli, Micrococcaceae, enterococci, and propionibacteria in Swiss-type cheese, are found at higher levels in raw milk cheese. The main biochemical modification of cheese during ripening concerns the nature and extent of proteolysis. Although there is no clear trend in the breakdown of _s1- and β-caseins, milk pasteurization leads to a significant decrease of the amount of small peptides and free amino acids and to different HPLC profiles. Experiments carried out with sensory analysis show that, in all cases, pasteurized or microfiltered milk cheeses have received lower flavour intensity scores than raw milk cheeses. From this review, it is concluded that the indigenous milk microflora, with its diversity of species and strains, appears to be mainly responsible of the specific sensory properties of raw milk cheeses.