Characterization of the purified actinidin as a plant coagulant of bovine milk

In this work, actinidin was characterized in view of its possible suitability as a coagulant enzyme in the manufacturing process of cheese. The results show that actinidin does exhibit milk-clotting activity, which is correlated with the enzyme concentrations. The combined use of urea and SDS–PAGE led to the conclusion that the milk clot is clearly separated from the whey proteins and corresponds to casein coagulum. Moreover, both the enzyme dependence on pH and temperature and the stability profiles are fully suitable with the chemical–physical conditions adopted during the cheese-making procedure. The analysis of the kinetic constants as well as the electrophoretic pattern of the hydrolysis products suggests that β-casein is the preferred substrate of actinidin, whereas κ-casein seems to be hydrolyzed only in a few large fragments.     

蛋白质组学在人乳及牛乳蛋白研究中的应用现状

乳蛋白是生命最初阶段中最重要的营养物质,利用蛋白质组学技术能够直观、整体地研究乳蛋白质的组成与调控生命的活动规律,可以更全面、深入地阐明乳蛋白质的表达信息。通过阐述蛋白质组学在人乳与牛乳蛋白研究中的应用进展,以及乳酪蛋白质组学、乳清蛋白质组学、乳脂肪球膜蛋白质组学、乳铁蛋白质组学的研究,为寻找疾病的临床诊断和治疗、乳品加工保存条件、开发新型婴幼儿食品提供重要的理论依据。     

Distribution and fatty acid content of phospholipids from bovine milk and bovine milk fat globule membranes

The phospholipid (PL) content was determined comparatively in the milk fat globule membrane (MFGM) and whole milk including their fatty acid profiles. The possible role of milk PLs in defence against pathogens was also addressed. The MFGM and whole milk showed a similar distribution of PL species; however, the fatty acid contents of the PL species were different. Total PL from MFGM showed a decrease in C18:0 content in parallel with an increase in C18:1 and C18:2 and very long-chain fatty acid (more than C20) content. No significant differences in the fatty acid content of phosphatidylcholine and sphingomyelin from either source were found. However, the phosphatidylethanolamine from MFGM had more C18:1 and C18:2 and less C14:0 and C16:0 than that from whole milk. A similar but less pronounced result was found for phosphatidylserine/phosphatidylinositol. Enterotoxigenic Escherichia coli strains failed to bind to PL, which had been previously separated by high-performance thin-layer chromatography.     

Binding of zinc to bovine and human milk proteins

Zn binding by whole bovine and human casein and by purified bovine caseins and whey proteins was investigated by equilibrium dialysis. Bovine alpha s1-casein had the greatest Zn-binding capacity (approximately 11 atoms Zn/mol). Protein aggregation was observed as Zn concentration was increased and the protein precipitated at a free Zn concentration of 1.7 mM. Zn binding increased with increasing pH in the range 5.4-7.0 and decreased with increasing ionic strength. Competition between Zn and Ca was observed for binding to alpha s1-casein indicating common binding sites for these two metals. Bovine beta-casein bound up to 8 atoms Zn/mol and precipitated at a free Zn concentration of approximately 2.5 mM, while kappa-casein bound 1-2 atoms Zn/mol. Whole bovine and human casein bound 5-8 atoms Zn/mol and precipitated at a free Zn concentration of approximately 2.0 mM. Scatchard plots for Zn binding to caseins showed upward convexity, possibly due to Zn-induced association of caseins. Apparent average association constants (Kapp) for all caseins were similar (log Kapp 3.0-3.2). Enzymic dephosphorylation of alpha s1- or whole bovine casein markedly reduced, but did not eliminate, Zn binding. Thus, phosphoserine residues appeared to be the primary Zn-binding sites in caseins. With the exception of bovine serum albumin, which bound over 8 atoms Zn/mol, the bovine whey proteins, beta-lactoglobulin, alpha-lactalbumin and lactotransferrin, had little capacity for Zn binding.     

Autoxidation of purified myoglobin from two bovine muscles

To elucidate the behavioural differences between beef muscles from the viewpoint of colour stability, oxymyoglobin was extracted at 2 h post mortem, purified from two different muscles (longissimus lumborum (LL), stable and psoas major (PM), unstable) and the autoxidation rate was measured. Oxymyoglobin was isolated after separation from metmyoglobin by chromatography on DEAE-Sepharose and TSK SW 2000 columns, and its purity was controlled by electrophoresis and IEF. Over a wide range of pH values (5-9), temperatures (20-50°C) and ionic strength (0-500 mM), no difference was noted between autoxidation rates of LL and PM oxymyoglobin extracted at 2 h post mortem. Conversely, when myoglobin was extracted at 192 h post mortem, the autoxidation rate of PM oxymyoglobin was higher than LL myoglobin, particularly at elevated temperatures. These differences in autoxidation rates after extraction of myoglobin at 2h and 192 h post mortem were not associated with differences in Ea (approximately 23 kcal/mol).     

脂肪酶中具有壳聚糖酶活力组分的作用模式及鉴定

以甲壳四糖为底物,研究了脂肪酶中具有壳聚糖降解活力酶组分的作用方式.高效液相(HPLC)和薄层层析(TLC)结果均表明,该酶的作用模式是从底物的末端依次切下单糖即氨基葡萄糖残基,并且最终完全生成氨基葡萄糖,证明该酶为目前报道较少的外切β-D-氨基葡萄糖苷酶.另外,该酶不能生成聚合度高于底物的产物,表明不具有转苷酶的活性.     

Determination of molecular weight of a purified fraction of colloidal calcium phosphate derived from the casein micelles of bovine milk

Colloidal calcium phosphate (CCP) plays a key role in the formation and integrity of casein (CN) micelles. However, limited information is available on the molecular weight (M_w) of CCP. Recently, we theoretically derived the M_w of CCP and the objectives of this study were to experimentally determine the M_w of CCP. We used 2 methods to prepare CCP fractions: skim milk was enzymatically digested with either trypsin or a combination of papain and proteinase enzymes to remove most CN. The CN phosphopeptides are resistant to trypsin hydrolysis. Digestion was carried out in a membrane tube that was dialyzed against the same bulk milk used in sample preparation to remove small peptides and to minimize perturbation of CCP. After digestion, the protein contents of the enzyme-treated milks were 0.92 and 0.36% for the trypsin and papain-proteinase treatments, respectively. Size-exclusion chromatography, coupled with multi-angle laser light scattering, was used to separate the CCP-phosphopeptide fraction from the digested mixture. Simulated milk ultrafiltrate was used as a mobile phase during size-exclusion chromatography separation to try to preserve the integrity of CCP. Size-exclusion chromatography peaks, which had higher Ca and P contents than the baseline, were identified as the likely fractions containing the phosphopeptide-stabilized CCP; this peak eluted with retention times of 100 to approximately 110 min for trypsinated samples. The papain-proteinase treatment caused excessive loss of CN that were needed to stabilize CCP, which resulted in no obvious peak that had elevated Ca and P contents. Debye plots at these retention times indicated that the weight-average M_w for the fraction prepared by trypsin was 17,450 g/mol. Attempts to estimate the M_w of the phosphopeptides associated with CCP using sodium dodecyl sulfate-PAGE were not successful, as we did not observe any peptide bands in these gels, presumably because of their low concentration in the isolated, unconcentrated fraction. Assuming that 4 CN phosphopeptides stabilized each CCP and if the M_w of each of these phosphopeptides was about 2,500 g/mol, then the M_w of CCP would be around 7,450 g/mol. This experimental value was close to the theoretically-derived M_w of 4,897 and 9,757 g/mol for tetrahedron and bi-pyramid shaped objects, respectively, when using the brushite form of calcium phosphate.     

Technical note: Direct enzyme immunoassay of progesterone in bovine milk whey

A simple extraction-free or direct quantitative ELISA for progesterone in bovine milk whey was developed. Whey samples are easy to collect, transport, and store. This method also allows for monitoring progesterone levels in cattle, which is important in reproductive management. The assay was designed to cover the concentration range 0.05 to 2 ng/mL, and the sensitivity of the method was 1.5 pg/mL. The intra- and interassay coefficients of variation were 8 and 12%, respectively. A high correlation (r = 0.90) between ELISA and radioimmunoassay measurements of progesterone in the same milk whey samples was obtained. The method can be easily applied in practice because samples can be stored at room temperature (22 to 26°C) for 4 d. Moreover, because analysis requires milk coagulation, that process can be initiated during transport by standard mail services to the laboratory. Upon arrival at the laboratory, whey can be kept refrigerated for 1 wk before analysis. This tool is useful for monitoring luteal activity of dairy cows.     

Considerable variation in the concentration of osteopontin in human milk, bovine milk, and infant formulas

Osteopontin (OPN) is a multifunctional bioactive protein that is implicated in numerous biological processes such as bone remodeling, inhibition of ectopic calcification, and cellular adhesion and migration, as well as several immune functions. Osteopontin has cytokine-like properties and is a key factor in the initiation of T helper 1 immune responses. Osteopontin is present in most tissues and body fluids, with the highest concentrations being found in milk. In the present study, ELISA for human and bovine milk OPN were developed and OPN concentration in human breast milk, bovine milk, and infant formulas was measured and compared. The OPN concentration in human milk was measured to approximately 138 mg/L, which corresponds to 2.1% (wt/wt) of the total protein in human breast milk. This is considerably higher than the corresponding OPN concentrations in bovine milk (∼18 mg/L) and infant formulas (∼9 mg/L). Moreover, bovine milk OPN is shown to induce the expression of the T helper 1 cytokine IL-12 in cultured human lamina propria mononuclear cells isolated from intestinal biopsies. Finally, the OPN concentration in plasma samples from umbilical cords, 3-mo-old infants, and pregnant and nonpregnant adults was measured. The OPN level in plasma from 3-mo-old infants and umbilical cords was found to be 7 to 10 times higher than in adults. Thus, the high levels of OPN in milk and infant plasma suggest that OPN is important to infants and that ingested milk OPN is likely to induce cytokine production in neonate intestinal immune cells.     

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.     

Antioxidative and antibacterial peptides derived from bovine milk proteins

The search for alternative preservatives is on the rise due to safety issues linked with the application of synthetic antioxidants and the extensive increase in bacterial resistance to several conventional antibiotics. Therefore, the quest for finding suitable alternatives including bioactive peptides has received attention. This article reports a comprehensive insight concerning antioxidative and antibacterial peptides derived from milk proteins, a prolific source of peptides having various bioactivities. Caseins and whey proteins have also been evaluated for antioxidative and antibacterial potential using the BIOPEP database. A notable number of potentially active peptides are present in milk proteins. Technological approaches are here reported for the production of these peptides. The findings of this review show potentiality of utilizing dairy derived antioxidative and antibacterial peptides in the development of a superior alternative to the current generation of preservatives and therapeutic agents, as well as a functional ingredient in dietetic or pharmaceutical applications.     

牛乳中乳铁蛋白的纯化和抗菌活性研究

采用凝胶过滤层析和离子交换法层析分离纯化乳铁蛋白,通过SDS-PAGE和IEF-PAGE鉴定所得样品;同时利用抗菌实验(滤纸片法)进行样品的抗菌活性研究,并研究了乳铁蛋白分离纯化过程中抗菌活性的变化。结果表明,离子交换层析纯化乳铁蛋白的效果优于凝胶过滤层析,抗菌活性证实乳铁蛋白样品具有广谱抗菌作用,以蜡状芽孢杆菌为指示菌时,比活为3077.3AU/mg(试管法),纯化倍数提高了20.2倍。     

水解牛乳酪蛋白的抗原性研究

采用间接竞争抑制Elisa法测定水解酪蛋白中的残留抗原性,从而间接测定其致敏性。选择7种蛋白酶在各自适宜条件下酶解酪蛋白,观察酪蛋白抗原性随酶解过程的变化情况,并讨论了水解酪蛋白的抗原性随分子质量的变化以及深度水解酪蛋白与适度水解酪蛋白的抗原性。结果表明,不同蛋白酶对酪蛋白的抗原性影响不同,这可能是由于不同的蛋白酶具有不同特异性,其中,中性蛋白酶降低酪蛋白抗原性的效果最佳,抗原抑制率为20.91%;水解酪蛋白的分子质量越小,其残留抗原性越小,当其分子质量小于3 000Da时,抗原抑制率仅为6.61%;深度水解酪蛋白的抗原性较适度水解酪蛋白低,这主要是由于水解度及分子质量的不同。     

Inhibitory effects of human and bovine milk constituents on rotavirus infections

Among etiologic agents, rotavirus is the major cause of severe dehydration diarrhea in infant mammals. In vitro and in vivo studies have indicated that the human milk-fat globule protein lactadherin inhibits rotavirus binding and protects breast-fed children against symptomatic rotavirus infection. The present work was conducted to evaluate the effect of lactadherin, along with some other milk proteins and fractions, on rotavirus infections in MA104 and Caco-2 cell lines. It is shown that human, and not bovine, lactadherin inhibits Wa rotavirus infection in vitro. Human lactadherin seems to act through a mechanism involving protein-virus interactions. The reason for the activity of human lactadherin is not clear, but it might lie within differences in the protein structure or the attached oligosaccharides. Likewise, in our hands, bovine lactoferrin did not show any suppressive activity against rotavirus. In contrast, MUC1 from bovine milk inhibits the neuraminidase-sensitive rotavirus RRV strain efficiently, whereas it has no effect on the neuraminidase-resistant Wa strain. Finally, a bovine macromolecular whey protein fraction turned out to have an efficient and versatile inhibitory activity against rotavirus.     

蛋白质分析技术在牛乳和人乳鉴别方面的应用

概要归纳了人乳、牛乳和大豆中的主要蛋白质在质量分数和特性上的差别,综述了将电泳、色谱、质谱、光谱和免疫组化等蛋白质定量、定性分析技术应用于各类蛋白的分离、鉴定和纯度检测的文献报道,比较了各种检测方法的优缺点,认为变性凝胶电泳技术因其操作简便和成本低廉更适用于人乳产业化初期的原料乳纯度检测.     

Streptomycin and dihydrostreptomycin residues in bovine milk from the Brazilian retail market

Pasteurised bovine milk from retail markets in the State of São Paulo, Brazil, was analysed for the presence of streptomycin (STP) and dihydrostreptomycin (DHSTP) residues. An ELISA kit was used for screening and a LC–APCI–MS/MS QToF method for confirmatory analysis. Both methods were intra-laboratory validated and found suitable for screening and confirmatory testing, respectively, for STP and DHSTP residues in pasteurised bovine milk at concentration levels below the maximum residue limit (MRL) established for these substances (200 µg kg^?1 expressed as the sum of the concentrations of STP and DHSTP). No residues of STP and DHSTP at detectable levels were found in the analysed samples (n = 299).     

The analysis of milk components and pathogenic bacteria isolated from bovine raw milk in Korea

Bovine mastitis can be diagnosed by abnormalities in milk components and somatic cell count (SCC), as well as by clinical signs. We examined raw milk in Korea by analyzing SCC, milk urea nitrogen (MUN), and the percentages of milk components (milk fat, protein, and lactose). The associations between SCC or MUN and other milk components were investigated, as well as the relationships between the bacterial species isolated from milk. Somatic cell counts, MUN, and the percentages of milk fat, protein, and lactose were analyzed in 30,019 raw milk samples collected from 2003 to 2006. The regression coefficients of natural logarithmic-transformed SCC (SCCt) on milk fat (−0.0149), lactose (−0.8910), and MUN (−0.0096), and those of MUN on milk fat (−0.3125), protein (−0.8012), and SCCt (−0.0671) were negative, whereas the regression coefficient of SCCt on protein was positive (0.3023). When the data were categorized by the presence or absence of bacterial infection in raw milk, SCCt was negatively associated with milk fat (−0.0172), protein (−0.2693), and lactose (−0.4108). The SCCt values were significantly affected by bacterial species. In particular, 104 milk samples infected with Staphylococcus aureus had the highest SCCt (1.67) compared with milk containing other mastitis-causing bacteria: coagulase-negative staphylococci (n = 755, 1.50), coagulase-positive staphylococci (except Staphylococcus aureus; n = 77, 1.59), Streptococcus spp. (Streptococcus dysgalactiae, n = 37; Streptococcus uberis, n = 12, 0.83), Enterococcus spp. (n = 46, 1.04), Escherichia coli (n = 705, 1.56), Pseudomonas spp. (n = 456, 1.59), and yeast (n = 189, 1.52). These results show that high SCC and MUN negatively affect milk components and that a statistical approach associating SCC, MUN, and milk components by bacterial infection can explain the patterns among them. Bacterial species present in raw milk are an important influence on SCC in Korea.     

Occurrence of flunixin residues in bovine milk samples from the USA

5-Hydroxy-flunixin concentrations in milk samples were quantified by two commercially available screening assays – CHARM^® and enzyme-linked immunoabsorbant assay (ELISA) – to determine whether any concentrations could be detected above the tolerance limit of 2 ng g^?1 from different regions in the United States. Milk samples came from large tanker trucks hauling milk to processing plants, and had already been screened for antibiotics. Positive results for flunixin residues based on a screening assay were confirmed by ultra-HPLC with mass spectrometric detection. Of the 500 milk samples analysed in this study, one sample was found to have a 5-hydroxy-flunixin concentration greater than the tolerance limit. The results of this study indicate that flunixin residues in milk are possible. Regulatory agencies should be aware that such residues can occur, and should consider incorporating or expanding flunixin screening tests as part of routine drug monitoring in milk. Larger studies are needed to determine the true prevalence of flunixin residues in milk from other regions in the United States as well as different countries.