Effect of commercial or depurinized milk on rat liver growth-regulatory kinases,nuclear factor-kappa B,and endonuclease in experimental hyperuricemia: Comparison with allopurinol therapy

Hyperuricemia is a biochemical hallmark of gout, renal urate lithiasis, and inherited purine disorders, and may be a result of enormous ATP breakdown or purine release as a result of cardiovascular disease, hypertension, kidney disease, eclampsia, obesity, metabolic syndrome, psoriasis, tumor lysis syndrome, or intense physical training. The beneficial role of dairy products on hyperuricemia management and prevention is well documented in the literature. The primary aim of our experimental study was to examine the effect of milk dietary regimen (commercial 1.5% fat UHT milk or patented depurinized milk) compared with allopurinol therapy on experimental hyperuricemia induced by oxonic acid in rats. Principal component analysis was applied on a data set consisting of 11 variables for 8 different experimental groups. Among the 11 parameters measured (plasma uric acid and the liver parameters NFκB-p65, Akt kinase/phospho-Akt kinase, ERK kinase/phospho-ERK kinase, IRAK kinase/phospho IRAK kinase, p38/phospho-p38, and DNase), Akt/phospho Akt and ERK/phospho-ERK signaling were extracted as the most discriminating. We also compared the content of various potentially toxic compounds (sulfur compounds, ketones, aldehydes, alcohols, esters, carboxylic acids, and phthalates) in untreated commercial milk and depurinized milk. Of all the compounds investigated in this study that were observed in commercial milk (24 volatile organic compounds and 4 phthalates), 6 volatile organic compounds were not detected in depurinized milk. For almost all of the other compounds, significant decreases in concentration were observed in depurinized milk compared with commercial milk. In conclusion, a depurinized milk diet may be recommended in nutritional treatment of primary and secondary hyperuricemia to avoid uric acid and other volatile, potentially toxic compounds that may slow down liver regeneration and may induce chronic liver diseases.     

Lipid and Cholesterol Oxidation in Whole Milk Powder during Processing and Storage

The influence of animal feed quality on lipid and cholesterol oxidation in whole milk powder was investigated. Powders from a herd receiving a ‘supplemented’ diet showed reduced PV (p < 0.01) and TBARS (p < 0.09) compared to a ‘restricted’ herd, after storage in both vacuum and sachet-packs and less (p < 0.003) cholesterol oxidation products (COPs). High pre-heating temperatures resulted in higher levels of PV, TBARS and COPs in fresh whole milk powders than low pre-heat temperatures, but after storage the reverse occurred. Superior animal feed quality and proper control of processing and storage conditions enhanced oxidative stability of whole milk powder. Lipid and cholesterol oxidation were positively correlated (p < 0.001).     

Effects of dry period length on milk production and health of dairy cattle

Holstein cows (n = 781) in a commercial dairy herd were used in a randomized design to evaluate 2 dry period (DP) management strategies on milk production, milk components, milk quality, colostrum quality, and incidence of metabolic disorders. Cows were randomly assigned to a traditional 55 d (T) or shortened 34 d (S) DP. Cows assigned to T were fed a low-energy diet until 34 d before expected calving at which time all cows were fed a moderate-energy transition diet until calving. Postpartum, cows assigned to T produced more milk and tended to produce more solids-corrected milk than cows on S. Treatment differences in milk and solids-corrected milk yield were accounted for by cows in their second lactation. Milk fat percentage did not differ between treatments, but milk protein percentage was greater for cows assigned to S. Colostrum quality measured as IgG concentration did not differ between management strategies. Somatic cell score and cases of mastitis were not affected by management strategy. There was a tendency for prepartum nonesterified fatty acid (NEFA) to be lower for cows assigned to T compared with S. However, postpartum, cows assigned to S had significantly lower NEFA concentrations than those assigned to T. The incidences of ketosis, retained placenta, displaced abomasum, and metritis did not differ between treatments. Postpartum energy balance, as indicated by plasma NEFA, may have been improved for cows assigned to S; there was no detectable effect on animal health.     

Effect of feed supplementation based on extruded linseed meal and fish oil on composition and sensory properties of raw milk and ultra-high temperature treated milk

The objectives of this study were to test the influence of a recently developed rumen protected feed supplement containing extruded linseed meal and fish oil (LFO), which was fed to lactating Holstein-Friesian cows for 10 weeks at the rate of 800 g day⁻¹ per animal, on the chemical and fatty acid (FA) composition of raw and ultra-high temperature (UHT) treated milks and to evaluate changes in sensory properties of UHT milk by both instrumental analysis and a panel of human assessors. Inclusion of LFO in the diet did not affect milk yield or the protein and fat contents of raw and UHT milks; however, it improved the FA composition of the milk fat by increasing the concentrations of health-enhancing polyunsaturated FA and beneficially decreasing the n-6/n-3 FA ratio without adversely affecting the sensory properties of the final product (UHT milk).     

Development of an improved extraction and HPLC method for the measurement of ascorbic acid in cows' milk from processing plants and retail outlets

An improved extraction (2.5% HPO₃, 5 mm dithiothreitol) and HPLC quantification methodology using a C–18 column at 35 °C and 0.1 m acetic acid (98%) and acetonitrile (2%) mobile phase was developed to quantify total ascorbic acid (AA) in commercial whole/semi‐skim/skim raw/pasteurised/UHT milk packaged in opaque bags, transparent plastic, cardboard and Tetra Brik^?. AA content ranged from 0.21 to 10 and from 3.4 to 16 mg L^?1 in milk from retail outlets and processing plants, respectively, and was higher in organic milk. For same processor/lot samples, pasteurised milk showed higher AA content than UHT milk. This was not true for retail outlets samples. AA content was similar for whole/semi‐skim and semi‐skim/skim milk, but not for whole/skim comparisons. Among UHT samples, the AA content trend was whole<semi‐skim<skim and lower for UHT milk in opaque plastic and Tetra Brik^? container. After 14 days at 4 °C in the dark, AA losses ranged 35–83% depending on milk type and preservation method with a higher AA retention in unopened containers.     

Short communication: Detection of stx2 and elt genes in bovine milk by using a multiplex PCR system

The aim of this study was to detect 2 important toxin genes from diarrheagenic Escherichia coli (DEC) in bovine milk using a new multiplex PCR. To standardize the multiplex PCR, the stx2 and elt genes were investigated for the detection of Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC), respectively. The DNA template was prepared with a thermal procedure (boiling) and a commercial kit. Samples consisted of UHT and pasteurized milk, both skimmed, and STEC and ETEC were tested in concentrations between 10¹ and 10⁹ cfu/mL. With the thermal procedure, the multiplex PCR system detected both pathotypes of E. coli at 10⁹ cfu/mL in UHT and pasteurized milk. When the commercial kit was used for template preparation, STEC and ETEC could be detected at concentrations as low as 10⁴ cfu/mL in UHT and pasteurized milk. Negative controls (Listeria monocytogenes, Salmonella Typhimurium, Salmonella Enteritidis, and Escherichia coli strain APEC 13) were not amplified with the multiplex PCR. These results indicate that the multiplex PCR was a rapid (less than 6 h) and efficient method to detect STEC and ETEC in milk using different methods for DNA preparation; however, the commercial kit was more sensitive than the thermal procedure.     

P NMR spectroscopic investigations of caseins treated with microbial transglutaminase

Caseins - the main constituents of bovine milk proteins - self-assemble into large supramolecular aggregates, so-called casein micelles. The enhancement of the stability of casein micelles is advantageous with respect to technological milk processing. A promising approach to accomplish this goal is the cross-linking of caseins using microbial transglutaminase (mTG). The present paper describes the combined use of liquid- and solid-state ³¹P NMR spectroscopy as well as dynamic light scattering in order to characterize the influence of an mTG treatment upon the structure of micelles in ultrahigh temperature (UHT)-treated skim milk at a molecular level. Liquid-state ³¹P NMR spectroscopy was applied to characterize milk, milk serum and casein dispersions. A narrow SerP signal in the liquid-state ³¹P NMR spectra of UHT-treated milk is shown to be due to casein molecules in the milk serum whereas the casein molecules bound in the micelles give rise to broad signals. Most of the caseins contribute to a 3 kHz broad background signal which can be visualized in the spectrum of suspensions of re-dispersed micellar material derived from UHT-treated milk. Treatment with mTG results in a cross-linking of caseins, which could be followed by liquid-state ³¹P NMR spectroscopy. Especially, the cross-linking of β-casein was demonstrated by quantitative liquid-state ³¹P NMR experiments. Furthermore, the stability of cross-linked micellar aggregates against EDTA could be investigated by liquid-state ³¹P HR NMR in combination with dynamic light scattering (DLS). Solid-state ³¹P NMR was used to show that the motional state of the κ-caseins located at the outer surface of the micelles derived from UHT-treated milk is not significantly changed by the applied mTG treatment.     

Changes in phosphoglyceride composition during storage of ultrahigh-temperature milk, as assessed by 31P-nuclear magnetic resonance: possible involvement of thermoresistant microbial enzymes

Soluble phosphoglycerides were studied in ultrahigh-temperature (UHT) milk by 31P-nuclear magnetic resonance. It was shown that, during storage of UHT milk, manufactured from raw milk with poor microbial quality, glycerophosphocholine and glycerophosphoethanolamine disappeared in parallel with an increase in alpha-glycerophosphate (GP). Storage at 10, 20, and 30 degrees C showed a faster transformation as the temperature increased. UHT milk samples manufactured from raw milks with better microbial quality and submitted to severe heat processes did not display changes in phosphoglycerides during storage. Screening of commercial UHT milks showed variations regarding the presence of GP, while in pasteurized milk samples, the appearance of GP occurred when the commercial life had been exceeded. Inoculation of sterile milk with Pseudomonas fluorescens NCIB9046 and incubation at 10 degrees C supported that changes in phosphoglycerides could be the consequence of a phosphodiesterase activity of bacterial origin, able to survive UHT processing. A similar behavior was observed between this activity and proteolytic activity. The potential application of the detection of these compounds as spoilage predictor indices is discussed.     

Assessing the effects of severe heat treatment of milk on calcium bioavailability: in vitro and in vivo studies

Thermal processing of milk is a common practice. As milk is the main source of dietary calcium, this study aimed to assess the effects of overheating milk on calcium availability. Thus, thermally damaged milk (overheated, OH, milk; 3 cycles of sterilization at 116°C, 16 min) was compared with UHT milk (150°C, 6 s) in 2 types of assays: in vitro and in vivo (rats). In addition, the greater Maillard reaction rate associated with thermal treatment in OH milk was confirmed by determining specific (furosine) and unspecific markers (CieLab color). A negative effect on calcium solubility was observed after in vitro digestion of OH milk compared with UHT milk. Feeding rats the diet containing OH milk as the protein source led to significantly lower values of apparent calcium absorption and retention than those found among animals fed the UHT milk diet. Whereas reducing the absorption appears to result mainly from the decreased food intake, the negative effect on retention seems to be due to factors derived from milk thermal damage, such as the formation of Maillard reaction products. It was concluded that milk-processing conditions warrant special attention to prevent impaired dietary calcium utilization. This may be especially important in situations where milk and dairy products are the main dietary components, such as in early infancy.     

The detection of viable vegetative cells of Bacillus sporothermodurans using propidium monoazide with semi-nested PCR

Bacillus sporothermodurans produces highly heat-resistant spores that can survive ultra-high temperature (UHT) treatment in milk. Therefore, we developed a rapid, specific and sensitive semi-nested touchdown PCR assay combined with propidium monoazide (PMA) treatment for the detection of viable B. sporothermodurans vegetative cells. The semi-nested touchdown PCR alone proved to be specific for B. sporothermodurans, and the achieved detection limit was 4 CFU/mL from bacterial culture and artificially contaminated UHT milk. This method combined with PMA treatment was shown to amplify DNA specifically from viable cells and presented a detection limit of 10² CFU/mL in UHT milk. The developed PMA-PCR assay shows applicability for the specific detection of viable cells of B. sporothermodurans from UHT milk. This method is of special significance for applications in the food industry by reducing the time required for the analysis of milk and dairy products for the presence of this microorganism.     

A metabolomics approach to characterize raw,pasteurized, and ultra-high temperature milk using ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry and multivariate data analysis

We developed a metabolomics workflow using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry to determine the effect of thermal treatment on milk composition and metabolites based on multivariate data analysis. We analyzed raw, pasteurized, and UHT milk samples. The samples were first centrifuged to remove the fat layer and mixed with methanol to precipitate proteins. Subsequently, the supernatant was analyzed by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry in electrospray negative mode. Mass spectral data were acquired in MS^E mode, a technique whereby both precursor and fragment mass spectral are simultaneously acquired by alternating between low and high collision energy (CE) during a single analytical run, to enable metabolite identification. Based on multivariate data analysis, these markers were significantly affected by thermal treatment. Among the 8 potential markers, we identified 7 oxylipids (9-hydroxydecanoic acid, 12-hydroxydodecanoic acid, 2-hydroxymyristic acid, 3-hydroxytetradecanoic acid, 5-hydroxyeicosatetraenoic acid, 3-hydroxyhexadecanoic acid, and 10-hydroxyoctadecanoic acid) and 1 phospholipid (LysoPE, hexadecanoyl-lysophosphatidylethanolamine). The oxylipids seemed to be adequate for distinguishing UHT milk from raw and pasteurized milk. The structures of the 8 potential markers were identified and characterized using informatics software. Our metabolomics workflow provides a fast approach for the identification of various types of milk.     

Principles and limitations of stable isotopes in differentiating organic and conventional foodstuffs: 2. Animal products

In this review, we examine the variation in stable isotope signatures of the lighter elements (δ²H, δ¹³C, δ¹⁵N, δ¹⁸O, and δ³⁴S) of tissues and excreta of domesticated animals, the factors affecting the isotopic composition of animal tissues, and whether stable isotopes may be used to differentiate organic and conventional modes of animal husbandry. The main factors affecting the δ¹³C signatures of livestock are the C3/C4 composition of the diet, the relative digestibility of the diet components, metabolic turnover, tissue and compound specificity, growth rate, and animal age. δ¹⁵N signatures of sheep and cattle products have been related mainly to diet signatures, which are quite variable among farms and between years. Although few data exist, a minor influence in δ¹⁵N signatures of animal products was attributed to N losses at the farm level, whereas stocking rate showed divergent findings. Correlations between mode of production and δ²H and δ¹⁸O have not been established, and only in one case of an animal product was δ³⁴S a satisfactory marker for mode of production. While many data exist on diet–tissue isotopic discrimination values among domesticated animals, there is a paucity of data that allow a direct and statistically verifiable comparison of the differences in the isotopic signatures of organically and conventionally grown animal products. The few comparisons are confined to beef, milk, and egg yolk, with no data for swine or lamb products. δ¹³C appears to be the most promising isotopic marker to differentiate organic and conventional production systems when maize (C4) is present in the conventional animal diet. However, δ¹³C may be unsuitable under tropical conditions, where C4 grasses are abundant, and where grass-based husbandry is predominant in both conventional and organic systems. Presently, there is no universal analytical method that can be applied to differentiate organic and conventional animal products.     

Determination of whey protein to total protein ratio in UHT milk using fourth derivative spectroscopy

A fourth derivative spectroscopy method was applied for the quantification of whey protein to total protein ratio in UHT milks. Some analytical features such as model compounds, selection of wavelength, linearity, repeatability and interference of milk fat were studied. The effect of refrigerated storage of raw milk, UHT treatment, and storage of UHT milk at room temperature on whey protein to total protein ratio was evaluated. No significant (p<0.05) differences among samples were found in any case. The ratio of whey protein to total protein was also determined in batches of whole (n=28) and skimmed (n=27) commercial UHT milks from different Spanish geographic areas processed by direct or indirect UHT systems in different periods of the year. The mean value was 18.1% for both whole and UHT skimmed milks. The analysis of laboratory-made mixtures of UHT milk with acid and rennet whey (2.5–15% of whey in milk expressed in protein) indicated that adulterations of UHT milk with whey up from 5% could be detected by the proposed method.     

Growth of Pseudomonas weihenstephanensis,Pseudomonas proteolytica and Pseudomonas sp. in raw milk: Impact of residual heat-stable enzyme activity on stability of UHT milk during shelf-life

One of the reasons for spoilage of UHT milk during shelf-life is the presence of residual proteolytic activity produced from Pseudomonas spp. during storage of raw milk. The aim of this study was to describe the product defects occurring in indirectly heated UHT milk during shelf-life, and to establish a correlation between proteolytic activity and onset of product spoilage. UHT milk was produced from raw milk incubated with different Pseudomonas strains, and examined over four months during storage at 20 °C. Inactivation kinetics of the peptidases were determined. In UHT milk, product defects occurred in the order: bitterness – particles – creaming – sediment – gelation in all the samples containing peptidases (apparent enzyme activity ≥ 0.03 pkat mL⁻¹). A linear correlation was found between proteolytic activity and onset of product defects, apart from onset of gelation.     

高效液相色谱-四级杆/静电场轨道阱质谱法鉴别 复原乳的掺假

目的以高效液相色谱-四级杆/静电场轨道阱质谱为检测平台,研究巴氏灭菌乳、UHT乳和复原乳中全部内源性物质的变化。方法首先建立乳制品的总离子流色谱图,运用SIMCA软件结合模型方法对数据进行分析,建立复原乳和非复原乳的主成分分析模型和正交偏最小二乘-判别分析模型。结果主成分分析得分图的结果显示,巴氏灭菌乳、UHT乳和复原乳的指纹图谱间存在一定差异。正交偏最小二乘-判别分析模型的S-plot和变量重要性因子筛选出对分类影响显著的物质。其中正、负离子模式分别筛选出18种和8种可能与复原乳相关的标志物。结论本研究所选标志物能够为复原乳的掺假鉴别研究提供参考依据,为执法部门的监督工作提供技术支持。这些标记物的结构鉴定仍有待研究。     

Lipolysis and Oxidation in Ultra‐High Temperature Milk Depend on Sampling Month,Storage Duration,and Temperature

During storage, some factors (for example, storage duration and temperature) can affect milk stability and consumer acceptability. Thiobarbituric acid reactive substances (TBARSs), lipid classes, and fatty acid profiles in stored ultra‐high temperature (UHT) milk were analyzed to assess the effects of storage time and temperature on lipid oxidation and lipolysis. With storage duration up to 12 months, the milk fat phase was separated and showed high levels of oxidation and lipolysis, manifested as increased levels of TBARS and free fatty acids. High oxidation levels decreased the percentage of unsaturated fatty acids (UFAs) in triacylglycerol and phospholipids. Higher storage temperatures (20, 30, and 37 °C) resulted in a higher degree of fat aggregation, oxidation, and lipolysis compared with refrigerated storage (4 °C). Additionally, sampling month of raw milk (May, July, and November) affected the lipid profiles of UHT milk during storage, with more UFA oxidized in July than in the other 2 months.     

Short communication: effect of production variables on the cis-9, trans-11 conjugated linoleic acid content of cows' milk

Although there have been numerous studies investigating effects of nutrition and individual variation on the concentration of cis-9, trans-11 conjugated linoleic acid (rumenic acid; RA) in milk, there is limited information on relationships among RA content of milk and production variables. The objective of the current analysis was to examine the effects of production variables on RA content and desaturase index of milk fat. A total of 430 samples were collected from cows fed a commercial total mixed ration in winter and grazing in summer. Across a >6-fold range in production variables, RA content of milk ranged from 1 to 32 mg/g of fatty acids and desaturase index ranged from 0.03 to 0.15. Days in milk, milk yield, milk fat content, and milk fat yield had minimal or no effect on RA content of milk fat or desaturase index (R² values all <0.08). Thus, whereas nutrition and individual variation are major factors affecting RA content and desaturase index of milk fat, these values are minimally affected by days in milk, milk yield, milk fat content, and milk fat yield. Differences in these parameters do not need to be considered, therefore, when designing management strategies to increase RA content of milk fat.     

Available lysine,protein digestibility and lactulose in commercial infant formulas

Available lysine, in vitro protein digestibility and lactulose values were determined in 23 commercial infant formulas. The mean available lysine content of the formulas based on dairy proteins was 66.7±9.5 mg g⁻¹ protein, similar to that of human milk, while that of soy based formulas was considerably lower (45.0±8.3 mg g⁻¹ protein). In vitro protein digestibility values ranged 85.5–88.9% for soy-based formulas and 90.5–98.3% for formulas based on dairy proteins. Formulas based on milk enriched with whey had higher lactulose content than those based on cow's milk. However, all values were below the limit of 600 mg L⁻¹ recommended for UHT milk.     

Total Cholesterol and Desmosterol Contents in Raw, UHT, Infant Formula Powder and Human Milks Determined by a New Fast Micro-HPLC Method

A simple, fast, sensitive, and environmental friendly micro-method is described for the determination of desmosterol and cholesterol in milk samples using isocratic normal phase high performance liquid chromatography and diode array detection. After sample micro-saponification, the chromatographic separation is achieved in 10 min, further reduced to 6 min when only cholesterol is expected to be present. The method is accurate, presenting cholesterol and desmosterol recoveries >96% and inter-day RSD lower than 3%. Quantification limits were clearly below the requirements for any milk sample, with 40 and 20 μg/100 mL for cholesterol and desmosterol, respectively. Several raw and UHT commercial milk samples, infant powder formulas, and human breast milks were analyzed, with cholesterol and desmosterol (present only in the latter) contents within reported amounts. The proposed method proved to be simple and feasible for determination of a large number of samples, requiring less solvent consumption (9–13 mL of hexane for all saponification/extraction/chromatographic steps) and simpler equipment than most ones reported, and a further possibility of partial solvent recycling due to the isocratic chromatographic mode.     

Effect of dietary supplementation of gallic acid and linoleic acid mixture or their synthetic salt on egg quality

The effect of a dietary supplementation of gallic acid and linoleic acid mixture (MGL) and their synthetic salt, sodium 2,3-dihydroxy-5-(((9E,12E)-octadeca-9,12-dienyloxy)carbonyl)phenolate (NGL), on egg quality was investigated. A total of 120 laying hens were allotted into five groups over 4 weeks of the experimental period. Birds were fed the following diets: (1) control [commercial diet (CD)], (2) 0.05% MGL (w/w, GA:LA = 1:1, equal molar ratio), (3) 0.1% MGL, (4) 0.05% NGL, (5) 0.1% NGL. The performance of the hen, the anti-oxidative potential of egg albumen and yolk, and the fatty acid composition and cholesterol content of egg yolk were measured. The TBARS value of egg yolk from hens fed 0.1% MGL and 0.05% NGL was lower than that fed control diet after storage for 14 days. The ABTS⁺ reducing activity of egg albumen was significantly improved by MGL and NGL, but only NGL had an effect on yolk (p < 0.05). The dietary supplementation of 0.05% or 0.1% MGL, and 0.05% NGL raised the PUFAs composition in egg yolk. The cholesterol content of egg yolk from hens fed control diet was higher than those fed 0.1% MGL, 0.05% or 0.1% NGL (p < 0.05). In conclusion, a diet consisting of MGL and NGL can improve the antioxidative potential of egg and the fatty acid quality of egg yolk while lowering the cholesterol level.