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.     

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%.     

Effects of caffeine and 3-isobutyl 1-methyl xanthine on caprine milk secretion

The objective of these studies was to determine if methyl xanthines can be used to alter milk production or composition in ruminants by enhancing adipose tissue mobilization. Three trials were conducted, one with intravenous caffeine infusions, one with intramuscular caffeine injections, and one with intramuscular injections of 3-isobutyl 1-methyl xanthine. Results indicate that: 1) continuous intravenous infusions of caffeine (720 mg/d) may reverse the milk fat depression of intravenously infused glucose in dairy goats; 2) intramuscular injections of caffeine (200 mg twice daily) do not reverse the milk fat-depressing effects of pelleted dairy goat diets during the 4th mo of lactation; and 3) intramuscular injections of 3-isobutyl 1-methyl xanthine (50 mg twice daily) do not consistently affect milk production of early lactation dairy goats.     

Some properties of set yoghurt made from caprine milk and bovine–caprine milk mixtures fortified by ultrafiltration or the addition of skim milk powder

Set yoghurt was produced from caprine milk (A), 70% caprine-30% bovine (B) and 50% caprine–50% bovine milk (C) mixtures, and stored for 14 days at ±4°C. Two methods of fortification, namely ultrafiltration (UF) and the addition of bovine skim milk powder (SMP), were applied to the milk mixtures. Some chemical, physical, microbiological and sensory properties of the six samples were analysed on the 1st, 7th and 14th days of storage. The effects of milk type, concentration method and storage period on the physicochemical and microbiological properties of the samples were investigated statistically.     

Nutritional and therapeutic value of fermented caprine milk

Caprine milk is a nutritional and therapeutic food. The unique and beneficial characteristics of caprine milk that are superior to bovine milk include: better digestibility; greater buffering capacity; fat globules that are smaller in diameter and better distributed in the milk emulsion; higher content of short‐chain fatty acids in the milk fat; higher content of zinc, iron and magnesium; stronger lactoperoxidase (antimicrobial) system as well as better immunological and antibacterial characteristics. The larger amounts of some minerals, such as calcium, zinc and magnesium, in caprine milk may influence the growth of lactic acid bacteria since they are a normal part of some enzymatic complexes involved in lactose fermentation. The higher whey protein content could also be significant because Lactobacillus acidophilus and bifidobacteria grow better in the presence of higher levels of some amino acids (valine, glycine, hystidine). The use of caprine and ovine milk in cheesemaking is well known, but the production of fermented caprine milk via probiotics has not yet been developed, although many studies have highlighted the requirements for production of that kind of healthy food. During fermentation caprine milk loses its characteristic ‘goaty’ taste, which is unacceptable to many consumers. Moreover, the nutritive value of caprine milk increases during fermentation. The rise in the number of goat farms in Croatia has created the need to find other products that can be produced using caprine milk. According to the present situation in Croatia, there is no real possibility of producing fermented caprine milk for the global market, but many studies of fermented caprine milk have been performed.     

Variation in caprine milk composition and coagulation as affected by udder health indicators

Milk lactose, pH, somatic cell count (SCC), bacterial count and NaCl are indirect markers of the mammary gland status; they can be used to screen milk quality and its technological properties. The variability of milk composition and coagulation properties from 1272 individual goat milk samples as a function of the udder health indicators was investigated. High lactose concentrations were associated with reduced coagulation time, but weakened curd firmness traits. High pH values impaired coagulation, while high SCC had delayed coagulation time but reduced curd-firming rate. Samples with high bacterial count were characterised by softer curds, but had faster curd-firming and larger syneresis rates. High concentrations of NaCl were associated with reduced fat, protein, and casein content, and impaired coagulation traits. These results show that the concurrent analysis of these markers can be highly informative and suitable to monitor the quality of milk destined for cheese-making.     

In vitro digestion of bovine and caprine milk by human gastric and duodenal enzymes

In vitro digestion was performed by human proteolytic enzymes on bovine and caprine individual milks. Two types of caprine milk were investigated: with high and low contents of α_S1-casein (CN). In addition the influence of heating of the milk on digestion was examined. The digestion was performed in two steps using human gastric and duodenal juice. Protein and peptide profiles were studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Caprine milk proteins were digested faster than bovine milk proteins. This was confirmed by the degradation profile obtained for both cows’ and goats’ milk, and was most evident for β-lactoglobulin. Comparing the digestion of milk protein from two groups of goats, high and low in α_S1-CN content, respectively, did not show significant differences. Heat treatment of milk had a strong and significant effect on the level of digestion. Raw milk was degraded faster than the heat-treated milk, and the effect of heating was different for bovine and caprine milk.     

Nucleotides and nucleosides in ovine and caprine milk during lactation

The aim of this study was to determine the nucleoside and nucleotide content in ovine and caprine milks at the colostral, transitional, and mature stages of lactation. Samples from 18 dairy sheep and 18 dairy goats were collected at 1, 2, 3, 4, 5, and 15 d postpartum. Separation and quantitation of the 5′-nucleotides (NT) and the nucleosides (NS) was performed by reverse phase HPLC. For each compound measured, considerable interindividual variation was recorded in both species of milk. The total NS content ranged from 57 to 132 μmol/L and from 54 to 119 μmol/L in ovine and caprine milk, respectively. The major NS identified in both species of milk was uridine, representing more than 60% of the total NS pool. The mean levels of inosine and guanosine were comparable between ewe and goat milk. Instead, the mean level of cytidine across the sampling period was much higher in ewe milk (11.9 μmol/L compared with 4.5 μmol/L in goat milk) and exhibited a peak value on the fourth day of lactation. The adenosine content was at least 3-fold higher in caprine milk compared with its ovine counterpart. The total NS and orotic acid contents did not differ significantly between the 2 species. However, in the case of total NT content, interspecies differences were significant, with NT levels ranging from 294 to 441 μmol/L in ovine milk and from 166 to 366 μmol/L in caprine milk. The NT content in colostrum (1-3 d) of both species was higher than in mature milk (15 d), and uridine monophosphate was the dominant NT in all samples.     

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.     

Analysis of major caprine milk proteins by reverse-phase high-performance liquid chromatography and electrospray ionization-mass spectrometry

Major proteins from caprine milk were separated by preparative gel permeation and cation-exchange fast protein liquid chromatography and were characterized by flow injection analysis by electrospray ionization mass spectrometry. In addition, proteins from whole skim milk and whole casein were analyzed by coupling reverse-phase HPLC and electrospray ionization mass spectrometry by two different chromatographic methods. These methods successfully resolved the major caprine milk proteins and main casein variants. The experimental molecular masses of major milk proteins and variants were: 19,302 for kappa-CN 2P; 25,599 for alphas2-CN A-11P; 25,514 for alphas2-CN B-10P; 23,370 for alphas1-CN A-8P; 23,345 for alphas1-CN B-8P; 23,264 for alphas1-CN E-8P; 18,817 for alphas1-CN F-3P; 23,835 for beta-CN 6P; 18,181 for beta-LG; 14,180 for alpha-LA and 66,318 for serum albumin.     

Cysteines involved in the interconversion between dehydrogenase and oxidase forms of bovine xanthine oxidoreductase

Mammalian xanthine oxidoreductase exists intracellularly in its dehydrogenase form. However, outside of this reducing milieu the enzyme quickly transforms into an oxidase form. Interconversion can be controlled by sulfhydryl reactive reagents, suggesting that disulfide bridging is linked to this phenomenon. The present work identified cysteines involved in the interconversion process. Purified enzyme was subjected to mild reduction with 1,4-dithioerythriol to regain dehydrogenase activity, and the accessible cysteines were labeled with specific radioactive alkylation reagents, iodoacetic acid. This partial alkylation stabilizes the dehydrogenase form, presumable by hindering formation of disulfide bond(s). Six of 38 cysteines were found to be labeled (residues 169, 170, 535, 992, 1317, and 1325). The significance of this labeling of bovine xanthine oxidoreductase is discussed in relation to structural knowledge about the enzyme, and especially by comparison with the AA sequences of avian and invertebrate enzymes, which do not undergo conversion. Cysteines 535 and 992 are the most likely marked residues to be involved in the interconversion, whereas the other cysteines are located too far from the cofactorbinding areas in xanthine oxidoreductase.     

Mathematical modelling of NADH oxidation catalyzed by new NADH oxidase from Lactobacillus brevis in continuously operated enzyme membrane reactor

NADH oxidase from Lactobacillus brevis was kinetically characterized in two different buffers: Tris-HCl and glycine-sodium pyrophosphate (pH 9.0). Reaction kinetics was described using the Michaelis-Menten model with product (NAD(+)) inhibition. It was found that this type of inhibition is uncompetitive. Experiments in the continuously operated enzyme membrane reactor revealed a strong enzyme deactivation at two different residence times: 12 and 60 min. A stronger deactivation was observed at the lower residence time in the glycine-sodium pyrophosphate buffer. Enzyme deactivation was assumed to be of the first order. The developed mathematical model for the continuously operated enzyme membrane reactor described these experiments very well. The mathematical model simulations revealed that a high enzyme concentration (up to 30 g cm(-3)) is necessary to obtain and maintain the stationary NADH conversion near 100% for a longer period of time.     

黄嘌呤和谷氨酰胺对枯草芽孢杆菌XGL产腺苷的影响

该研究以枯草芽孢杆菌（Bacillus subtilis）XGL为出发菌株,探究黄嘌呤和谷氨酰胺添加量及添加方式对其产腺苷的影响。结果表明,底物中添加100 mg/L黄嘌呤并在发酵16 h后持续向发酵液中流加3 g/L的黄嘌呤溶液200 mL可使腺苷产量达到34.4 g/L,相比于不流加黄嘌呤时腺苷产量（11.2 g/L）提高207%。在此基础上,再向底物中添加6 g/L谷氨酰胺,发酵32 h之后持续向发酵液中流加6 g/L的谷氨酰胺,腺苷产量达到45.8 g/L,相比于不添加谷氨酰胺腺苷产量（34.4 g/L）提高33%。因此,在B. subtilis XGL发酵过程中,可以通过底物添加和流加补料的发酵方式加入一定量的黄嘌呤和谷氨酰胺以提高腺苷产量。     

Milk fat globules by confocal Raman microscopy: Differences in human,bovine and caprine milk

Infant formulas are usually based on bovine milk and caprine milk. Pasteurization, homogenization and spray-drying are basic processes for the production of infant formulas. In this study, confocal Raman spectroscopy had been used to rapidly determine the differences between human, bovine and caprine milk fat globules (MFGs). The unsaturated fatty acids in human colostrum and mature MFGs were over 2 times more than in bovine and caprine MFGs. The phospholipid bands at 860 cm^− 1 of human colostrum MFGs were 5 times more than human mature, bovine and caprine MFGs. The carotenoid content varied according to the species of milk, with human MFGs containing more carotenoids than bovine and caprine MFGs. And the cholesterol content was not correlated with the size of the MFGs. Human MFGs were more mobile than bovine and caprine MFGs. The lipid composition and mobility of MFGs were underwent significant changes under different processing conditions. These results laid the foundations for the improvement and development of powdered milk formulas.     

Detection of bovine milk adulteration in caprine milk with N-acetyl carbohydrate biomarkers by using 1H nuclear magnetic resonance spectroscopy

In a return to tradition, the popularity of caprine milk is on the rise. However, particularly in countries with developed dairy industries based on bovine milk, there is the risk of adulteration with bovine milk, which is a cheaper alternative. Thus, a rapid, robust, and simple method for the detection of bovine milk added to caprine milk is necessary, and ¹H nuclear magnetic resonance spectroscopy appears to provide a solution. A matrix of 115 pure and artificially adulterated pasteurized milk samples was prepared and used to discover biomarkers of bovine milk that are independent of chemical and biological variation caused by factors such as genetics, diet, or seasonality. Principal component analysis and orthogonal projections to latent structures discriminant analysis of pure bovine milk and pure caprine milk revealed spectral features that were assigned to the resonances of 4 molecules. Of these, the peaks corresponding to protons in the N-acetylglucosamine and N-acetylgalactosamine acetyl moieties showed significant applicability for our method. Receiver operating characteristic curve analysis was used to evaluate the performance of the peak integrals as biomarkers of adulteration. This approach was able to distinguish caprine milk adulterated with 5% of bovine milk with 84.78% accuracy and with 10% of bovine milk an excellent 95.65% accuracy. This study demonstrates that N-acetyl carbohydrates could be used as biomarkers for the detection of bovine milk in caprine milk and could help in protecting caprine milk authenticity.     

Feta cheese texture: the effect of caprine and ovine milk concentration

Feta cheese was produced commercially with different caprine to ovine milk ratios. Milk fat concentrations, moisture and salt contents were similar for all the batches. However, the hardness and adhesive characteristics of the cheeses differed in relation to the milk ratio. The hardness of the cheese appeared to be correlated to increased goat milk content. Cryo-scanning electron microscopy (cryo-SEM) of the cheese samples showed that feta cheese with a higher proportion of caprine milk had a more compact and less porous appearance than feta produced from purely ovine milk. This difference in cheese structure helps to explain the difference in hardness between the samples.     

Characteristics of purified cows' milk xanthine oxidase and its submolecular characteristics

Xanthine oxidase (EC 1.2.3.2) was purified from fresh cows' milk by differential centrifugation and hydroxylapatite chromatography in the absence of reducing agents and proteases. The purified isolate possessed an absorbance at 280 nm:absorbance at 450 nm ratio of 4.84; an absorbance (1 cm at 280 nm 1%) of 11.9; an activity:absorbance at 450 nm of 141, a specific activity of 3.59 units/mg; and detectable dehydrogenase activity. The enzyme preparation was obtained in a reversible oxidase form that could be partially converted to xanthine dehydrogenase in the presence of 10mM dithiothreitol or 1% mercaptoethanol. Amino acid analyses revealed that the enzyme was hydrophobic in nature and that lysine constituted its N-terminal residue. The protein contained 22 disulfide and 38 sulfhydryl groups, four of which were detectable in the undenatured protein complex. Discontinuous PAGE in the presence of selected dissociation agents did not result in further resolution. Sodium dodecyl sulfate-PAGE of the purified enzyme revealed a sharp zone with a molecular weight of 151,000 +/- 4000 (i.e., monomer). The purified enzyme exhibited oxidase activity in the presence of 6 M urea and following limited proteolysis by trypsin, chymotrypsin, plasmin, pancreatin, pepsin, and papain. Proteolyzed xanthine oxidase migrated as a single zone in polyacrylamide gels in the presence and absence of dissociating agents such as 1% mercaptoethanol and 6 M urea. Restricted digestion of xanthine oxidase by proteases was indicated by the presence of three major zones with molecular weights ranging from 85,000 to 100,000, 30,000 to 35,000, and 18,000 to 20,000 commonly observed in SDS gels. Amino acid profiles of the principal peptidyl fragments of trypsin-cleaved xanthine oxidase indicated their hydrophobic nature and lysine as the N-terminal residue for all fragments.     

Sulfide oxidation by gene expressions of sulfide-quinone oxidoreductase and ubiquinone-8 biosynthase in Escherichia coli

Sulfides (S2),SH-) such as hydrogen sulfide belong to a class of sulfur compounds with unpleasant odors. In order to confer sulfide-oxidizing ability on the intestine-inhabiting bacteria, the sulfide-quinone oxidoreductase gene (sqr) in Rhodobacter capsulatus DSM-155 and genes for quinone biosynthesis (ubiC, ubiA and ispB) in Escherichia coli XL1 Blue-MRF' were transduced into E. coli BL21(DE3). Plasmids pT7-7 and pSTV were used as vectors of sqr, and ubiCA and ispB, respectively. The recombinants sqr-BL21(DE3) and ubiCA,ispB-sqr-BL21(DE3) were successfully constructed. The maximal sulfide-removing activities of the whole cells and membrane fractions of sqr-BL21(DE3) attained at pH 8.0 and 7.8, were 267 nmol/mg cells (dry weight)/min and 1250 nmol/mg membrane fraction (protein)/min, respectively. The molecular ratio of sulfide (S2-) oxidized and oxygen (O2) consumed was 2:1. SQR activity in the recombinant cells was positively restricted under anaerobic conditions and also by the addition of electron transfer inhibitors. Ubiquinone-8 (UQ-8) biosynthesis in the cells of ubiCA,ispB-sqr-BL21(DE3) increased as much as 2.2-fold compared with that of (pSTV)-sqr-BL21(DE3) during the 12-16 h incubation period. The maximal sulfide removal in the quinone-raised E. coli was attained slightly earlier, however, SQR activities thereafter were lower than those in (pSTV)-sqr-BL21(DE3).     

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.