Characterisation of whey proteins of camel (Camelus dromedarius) milk and colostrum

Variation in the composition of whey proteins from camel (Camelus dromedarius) colostrum and milk was recorded over a 192 h period following parturition. Whey proteins were separated by cation-exchange fast protein liquid chromatography and identified by polyacrylamide gel electrophoresis. The main components of whey proteins in camel milk and colostrum were similar to that in bovine, except for the lack in β-lactoglobulin. Serum albumin was the major whey protein present in camel milk, with an average concentration of 10.8 g/l. Camel colostrum was rich in immunoglobulins G, which consist of IgG1, and the enzyme inhibitory antibodies IgG2 and IgG3. The concentration of these proteins decreased rapidly 48 h post partum. Lactophorin (proteose peptone-component 3) and basic whey protein were detected only within 48 h after parturition, reaching a level of 4.9 and 3.1 g/l at 192 h post partum, respectively. The maximum level of lactoferrin (2.3 g/l) was observed at 48 h after parturition. Camel milk and colostrum were shown to be rich in protective proteins, especially IgG2 and IgG3, which revealed to be a potential source of inhibitory antibodies.     

Purification of camel milk lysozyme and its lytic effect on Escherichia coli and Micrococcus lysodeikticus

1. Camel milk lysozyme was purified using heparin-Sepharose 4B, Sephadex G-75 and hydroxyapatite chromatography. By this procedure lysozyme was separated from lactoferrin and a low molecular weight protein. 2. The lytic effect of camel milk lysozyme was assayed using Escherichia coli and Micrococcus lysodeikticus and its activity was compared with that of lysozyme from human milk and egg white. 3. The specific activity of camel milk lysozyme was found to be lower than that of lysozyme from human milk or from egg white. 4. Camel milk lactoferrin did not show a lytic effect on bacteria, while the low molecular weight protein showed lytic activity.     

Characterization of oligosaccharides in milk of bearded seal (Erignathus barbatus)

Carbohydrates were extracted from milk of a bearded seal, Erignathus barbatus (Family Phocidae). Free neutral oligosaccharides were separated by gel filtration, anion-exchange chromatography and preparative thin layer chromatography, while free acidic oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and high performance liquid chromatography. Oligosaccharide structures were determined by 1H-NMR spectroscopy. The structures of the neutral oligosaccharides were as follows; lactose, 2'-fucosyllactose, lacto-N-fucopentaose IV, difucosyl lacto-N-neohexaose and difucosyl decasaccharide which contained a lacto-N-neohexaose unit as well as an additional Gal(beta1-4)GlcNAc(beta1-3) unit and two residues of non-reducing Fuc(alpha1-2). The acidic oligosaccharides were thought to contain an Neu5Ac(alpha2-6) residue linked to GlcNAc or a sulfate linked to Gal at OH-3. The sialyl oligosaccharides and sulfated oligosaccharides had a lacto-N-neohexaose unit and two non-reducing Fuc(alpha1-2) residues and some of them had in addition one or two Gal(beta1-4)GlcNAc(beta1-3) units. The milk oligosaccharides of the bearded seal were compared to those of the harbour seal, which had been studied previously.     

Characterization of a camel milk protein rich in proline identifies a new beta-casein fragment

A camel milk whey protein has been isolated by reverse-phase high performance liquid chromatography. The protein is, like caseins, rich in proline (25% of the whole protein). The N-terminal amino acid sequence shows that the protein is homologous with a C-terminal region of beta-caseins analyzed from other species. The protein is concluded to be a fragment of beta-casein, derived from a non-tryptic type of cleavage of the parent molecule, and increasing the multiplicity of known casein products.     

Purification and partial characterisation of camel milk xanthine oxidoreductase

Xanthine oxidoreductase (XOR) was purified in the presence of dithiothrietol from camel milk with yields of up to 22.2mg/l that were comparable to those obtained from bovine and human milk sources. On SDS-PAGE, the freshly purified camel milk XOR had a protein flavin (A280/A450) ratio of 5.3 +/- 0.4 and appeared homogenous with a single major band of approximately Mr 145.3 KDa. Surprisingly, in all the batches (n = 8) purified camel milk XOR showed no detectable activity towards xanthine or NADH. The molybdenum content of camel XOR was comparable to human and goat milk enzymes. After resulphuration, camel milk XOR gave a specific activity of 1.1 nmol/min/mg and 13.0 nmol/min/mg enzyme towards pterin (fluorimetric assay) and xanthine (spectrophotometric assay) respectively. This activity was markedly lower than that of human, bovine and goat enzymes obtained under the same conditions. These findings suggest that the molybdo-form of camel enzyme is totally under desulpho inactive form. It is possible that camel neonates are equipped with an enzymic system that reactivates XOR in their gut and consequently generates antibacterial reactive oxygen species.     

Characterization of a heterogeneous camel milk whey non-casein protein

A milk protein, occurring in the whey fraction, has been characterized from camel milk. Determination of the primary structure reveals the existence of two related types of chain with residue differences in at least the N-terminal region. A fragment representing an N-terminal part of the protein was also recovered (heterogeneous at the same positions). The absence of cysteine residues in the protein shows that no disulphide bridges are present. The pattern of fragments and a parent protein resembles that for casein and its fragments, showing that fragments and a multiplicity of forms may be typical for different milk proteins.     

A camel milk whey protein rich in half-cystine. Primary structure, assessment of variations, internal repeat patterns, and relationships with neurophysin and other active polypeptides

The amino acid sequence of a recently isolated camel milk protein rich in half-cystine has been determined by peptide analyses. The 117-residue protein has 16 half-cystine residues, concluded to correspond to disulfide bridges and suggesting a tight conformation of the molecule. Comparisons of the structure with those of other proteins reveal several interesting relationships. The camel protein is clearly homologous with a previously reported rat whey phosphoprotein of possible importance for mammary gland growth regulation, and with a mouse protein of probable relationship to neurophysins. The camel, rat and mouse proteins may represent species variants from a rapidly evolving gene. Residue identities in pairwise comparisons are 40% for the camel/rat proteins and 33% for the camel/mouse proteins, with 38 positions conserved in all three forms. The camel protein also reveals an internal repeat pattern similar to that for the other two proteins. The homology between the three milk whey proteins has wide implications for further relationships. Thus, previously noticed similarities, involving either of the milk proteins, include limited similarities to casein phosphorylation sites for the camel protein, to neurophysins in repeat and half-cystine patterns for the mouse and rat proteins, and to an antiprotease for the rat protein. These similarities are reinforced by the camel protein structure and the recognition of the three whey proteins as related. Finally a few superficial similarities with the insulin family of peptides and with some other peptides of biological importance are noticed. Combined, the results relate the camel protein in a family of whey proteins, and extend suggestions of relationships with some binding proteins.     

The primary structure of alpha-lactalbumin from camel milk

The primary structure of camel alpha-lactalbumin was determined by analysis of the intact protein, and of CNBr fragments and enzymatic peptides from the carboxymethylated protein chain. Results show that camel alpha-lactalbumin has 123 residues and a molecular mass of 14.6 kDa. The amino acid sequence is strictly homologous to alpha-lactalbumins characterized, but also exhibits extensive differences: 39 residues differ in relation to the bovine protein and only 35 residues are conserved among hitherto known alpha-lactalbumins with characterized structures. All residues ascribed critical structural or functional roles are strictly invariant in the camel protein.     

Proteomics of the milk fat globule membrane from Camelus dromedarius

Camel milk has been widely characterized with regards to casein and whey proteins. However, in camelids, almost nothing is known about the milk fat globule membrane (MFGM), the membrane surrounding fat globules in milk. The purpose of this study was thus to identify MFGM proteins from Camelus dromedarius milk. Major MFGM proteins (namely, fatty acid synthase, xanthine oxidase, butyrophilin, lactadherin, and adipophilin) already evidenced in cow milk were identified in camel milk using MS. In addition, a 1D‐LC‐MS/MS approach led us to identify 322 functional groups of proteins associated with the camel MFGM. Dromedary MFGM proteins were then classified into functional categories using DAVID (the Database for Annotation, Visualization, and Integrated Discovery) bioinformatics resources. More than 50% of MFGM proteins from camel milk were found to be integral membrane proteins (mostly belonging to the plasma membrane), or proteins associated to the membrane. Enriched GO terms associated with MFGM proteins from camel milk were protein transport (p‐value = 1.73 × 10^?14), translation (p‐value = 1.08 × 10^?11), lipid biosynthetic process (p‐value = 6.72 × 10^?10), hexose metabolic process (p‐value = 1.89 × 10^?04), and actin cytoskeleton organization (p‐value = 2.72 × 10^?04). These findings will help to contribute to a better characterization of camel milk. Identified MFGM proteins from camel milk may also provide new insight into lipid droplet formation in the mammary epithelial cell.     

Chemical characterization of the oligosaccharides in milk of high Arctic harbour seal (Phoca vitulina vitulina)

Carbohydrates were extracted from high Arctic harbour seal milk, Phoca vitulina vitulina (family Phocidae). Free neutral oligosaccharides were separated by gel filtration and preparative thin layer chromatography, while free sialyl oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and high performance liquid chromatography. Oligosaccharide structures were determined by 1H-NMR spectroscopy. The structures of the neutral oligosaccharides were as follows: lactose, 2'-fucosyllactose, lacto-N-neotetraose, lacto-N-neohexaose, monofucosyl lacto-N-neohexaose and difucosyl lacto-N-neohexaose. Thus, all of the neutral saccharides contained lactose or lacto-N-neotetraose or lacto-N-neohexaose as core units and/or non-reducing alpha(1-2) linked fucose. These oligosaccharides have also been found in hooded seal milk. The structures of the silalyl oligosaccharides were: monosialyl lacto-N-neohexaose, monosialyl monofucosyl lacto-N-neohexaose, monosialyl difucosyl lacto-N-neohexaose and disialyl lacto-N-neohexaose. These oligosaccharides contained lacto-N-neohexaose as core units, and one or two alpha(2-6) linked Neu5Ac, and/or non-reducing alpha(1-2) linked Fuc. The Neu5Ac residues were found to be linked to GlcNAc or penultimate Gal residues. The acidic oligosaccharides are the first to have been characterized in the milk of any species of seal.     

用聚丙烯酰胺凝胶电泳和微流体芯片电泳鉴别6种液态奶

目的:研究6种液态奶制品蛋白电泳图谱的区别,建立奶制品的蛋白质学鉴别方法。方法:以5种纯牛奶、羊奶、水牛奶、骆驼奶、牦牛奶、黄豆浆为研究对象,通过SDS-PAGE和Agilent 2100微流体芯片电泳法进行分析比较。结果:骆驼奶、黄豆浆与其他研究对象的图谱有明显区别,而牛奶、羊奶、水牛奶、牦牛奶的差异却不是很大;采用微流体芯片电泳可有效地对豆奶、骆驼奶进行区分,还可在一定程度上鉴别牛奶、羊奶、水牛奶和牦牛奶。结论:Agilent2100系统作为一种新型半自动微流体芯片技术,可以快速、高效、准确地应用于液态奶制品的蛋白成分分析及鉴别。     

Proteomic tools to characterize the protein fraction of Equidae milk

The principal components of the protein fraction in pony mare's milk have been successfully identified and partially characterized using proteomic tools. Skimmed pony mare's milk was fractionated by either reversed phase-high-performance liquid chromatography (RP-HPLC) on a C4 column or a bi-dimensional separation technique coupling RP-HPLC in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) in the second dimension (two-dimensional RP-HPLC/SDS-PAGE). The fractions thus obtained were analyzed by Edman N-terminal microsequencing and mass determination, with or without tryptic digestion, on a matrix-assisted laser desorption/ionization-time of flight spectrometer. Based on the sequence and molecular mass information obtained, identifications were achieved through a protein database search using homology or pattern research algorithms. This methodological approach was shown to be rapid, efficient and reliable in identifying the principal proteins in pony mare's milk. kappa-, alpha(s1)-, alpha(s2)-, and beta-casein, lysozyme C, alpha-lactalbumin and beta-lactoglobulin I and II were thus identified. alpha(s1) and beta-caseins displayed polymorphic patterns, probably due to alternative splicing processes leading to casual exon skipping events involving exons 7 and 14 in alpha(s1)-casein and exon 5 in beta-casein. Edman N-terminal microsequencing over 35 amino acid residues, for pony alpha(s1)-casein, clearly demonstrated the occurrence, in Equidae, of a splicing pattern similar to that reported in rodents, characterized by the constitutive outsplicing of exon 5. Pony mare's milk SDS-PAGE and RP-HPLC patterns were compared with those obtained for other milks (cow, goat and human), as were the relative levels of caseins and major whey proteins in these milks. Our results provide further evidence to support the notion that Equidae milk is closer to human breast milk than milk from bovine and caprine with respect to the casein and lysozyme C contents and casein/whey proteins ratio.     

Amino acids content and electrophoretic profile of camel milk casein from different camel breeds in Saudi Arabia

This study aimed to evaluate amino acids content and the electrophoretic profile of camel milk casein from different camel breeds. Milk from three different camel breeds (Majaheim, Wadah and Safrah) as well as cow milk were used in this study.Results showed that ash and moisture contents were significantly higher in camel milk casein of all breeds compared to that of cow milk. On the other hand, casein protein of cow milk was significantly higher compared to that of all camel milk breeds. Molecular weights of casein patterns of camel milk breeds were higher compared to that of cow milk.Essential (Phe, Lys and His) and non-essential amino acids content was significantly higher in cow milk casein compared to the casein of all camel milk breeds. However, there was no significant difference for the other essential amino acids between cow casein and the casein of Safrah breed and their quantities in cow and Safrah casein were significantly higher compared to the other two breeds. Non-essential amino acids except Arg and the essential amino acids (Met, Ile, Lue and Phe) were also significantly higher in cow milk α-casein compared to α-casein from all camel breeds. Moreover, essential amino acids (Val, Phe and His) and the non-essential amino acids (Gly and Ser) content was significantly higher in cow milk β-casein compared to the β-casein of all camel milk breeds and the opposite was true for Lys, Thr, Met and Ile. However, Met, Ile, Phe and His were significantly higher for β-casein of Majaheim compared to the other two milk breeds. The non-essential amino acids (Gly, Tyr, Ala and Asp) and the essential amino acids (Thr, Val and Ile) were significantly higher in cow milk κ-casein compared to that for all camel milk breeds. There was no significant difference among all camel milk breeds in their κ-casein content of most essential amino acids.Relative migration of casein bands of camel milk casein was not identical. The relative migration of α_s-, β- and κ-casein of camel casein was slower than those of cow casein. The molecular weights of α_s-, β- and κ-casein of camel caseins were 27.6, 23.8 and 22.4 KDa, respectively. More studies are needed to elucidate the structure of camel milk.     

The antiplatelet activity of camel milk in healthy and aluminum chloride-intoxicated rats

This study examined the effect of camel milk on some marker of blood coagulation markers in aluminum chloride (ALCl₃)-treated rats. Rats (n = 6) were assigned as control, control + fresh camel milk (1 ml), ALCl₃ (0.5 mg/kg), and ALCl₃ + fresh camel milk (1 ml and 0.5 mg/kg, respectively). Treatments were conducted orally for 30 days and daily. Administration of camel milk to control and ALCl₃-intoxicated rats significantly increased platelet count, bleeding time, and collagen epinephrine (CEPI)-induced platelet aggregation. It also lowered plasma levels of thromboxane B2 and hepatic levels of glutathione (GSH) and the activities of antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD). While the treatment with camel milk has no effect on the liver structure, values of activated partial prothrombin time (aPPT), and levels of prothrombin time (PT) in control rats, it improved liver architectures and decreased serum levels alanine and aspartate aminotransferases (ALT and AST, respectively), and reduced values of both aPTT and PT in ALCl₃-intoxicated rats. In conclusion, camel milk inhibits platelets activity and aggregation in both control and ALCl₃-intoxicated rats.     

Evidence for membrane affinity of the C-terminal domain of bovine milk PP3 component

Component PP3 is a phosphoglycoprotein isolated from bovine milk with unknown biological function, which displays in its C-terminal region a basic amphipathic alpha-helix, a feature often involved in membrane association. According to that, the behaviour of PP3 and of a synthetic peptide from the C-terminal domain (residues 113-135) was investigated in lipid environment. Conductance measurements indicated that the peptide was able to associate and form channels in planar lipid bilayers composed of neutral or charged phospholipids. Electrostatic interactions seemed to promote voltage-dependent channel formation but this was not absolutely required since the pore-forming ability of the 113-135 C-terminal peptide was also detected with the zwitterionic lipid bilayer. Additionally, a spectroscopic study using circular dichroism argues that the peptide adopts an alpha-helical conformation in interaction with neutral or charged micelles. Thus, the conducting aggregates in bilayers might be composed of a bundle of peptides in helical conformation. Besides, similar conductance measurements performed with the whole PP3 protein did not induce any channel fluctuations. However, with the latter, an early breakdown of the bilayers occurred, a finding that can be tentatively explained by a massive incorporation of PP3. In the light of the present results, it could be inferred that PP3 membrane attachment may be achieved by oligomerization of the C-terminal amphipathic helical region.     

Carbohydrates of the milk of the platypus

Twelve samples of milk of the platypus, Ornithorhynchus anatinus, had a mean content of 3 X 3% hexose. Of this, almost half was L-fucose. Of the total monosaccharides present in acid hydrolysates of the water-soluble carbohydrates, L-fucose constituted 33%, D-galactose 29%, glucosamine 20%, D-glucose 11% and sialic acid 7%. Free lactose was found in only trace amounts. In all samples, the major oligosaccharide was difucosyllactose, which represented 39-52% of the total hexose. Five higher neutral oligosaccharides (from penta- to nonasaccharides) were isolated and their monosaccharide compositions determined. Each contained one or more residues of fucose, glucosamine and galactose and one residue of glucose. The presence in the milk of 4-O-acetyl-N-acetylneuraminlactose was detected by thin-layer chromatography. All milk samples examined contained protein material (probably glycoprotein), which was not precipitated by chloroform-methanol extraction. No evidence was obtained for quantitative or qualitative changes in carbohydrates during the course of the lactation season except for a small decline in total hexose towards the end of the season.     

A small camel-milk protein rich in cysteine/half-cystine

A small protein (M_r about 14 000) rich in cysteine/half-cystine has been isolated from camel milk by exclusion chromatography and reverse-phase high-performance liquid chromatography. The N-terminal amino acid sequence shows a region with several positional identities with and -caseins, which however lack cysteine residues; postions 16–20 are identical and involve the serine residues that have been found to be phosphorylated in -caseins.     

Proteome mapping of human skim milk proteins in term and preterm milk

The abundant proteins in human milk have been well characterized and are known to provide nutritional, protective, and developmental advantages to both term and preterm infants. However, relatively little is known about the expression of the low abundance proteins that are present in human milk because of the technical difficulties associated with their detection. We used a combination of electrophoretic techniques, ProteoMiner treatment, and two-dimensional liquid chromatography to examine the proteome of human skim milk expressed between 7 and 28 days postpartum by healthy term mothers and identified 415 in a pooled milk sample. Of these, 261 were found in human skim milk for the first time, greatly expanding our understanding of the human skim milk proteome. The majority of the proteins identified were involved in either the immune response (24%) or in cellular (28%) or protein (16%) metabolism. We also used iTRAQ analysis to examine the effects of premature delivery on milk protein composition. Differences in protein expression between pooled milk from mothers delivering at term (38-41 weeks gestation) and preterm (28-32 weeks gestation) were investigated, with 55 proteins found to be differentially expressed with at least 90% confidence. Twenty-eight proteins were present at higher levels in preterm milk, and 27 were present at higher levels in term milk.     

Structures of acidic O-linked polylactosaminoglycans on human skim milk mucins

O-Linked glycans were isolated from human skim milk mucins or mucin-derived high-molecular weight glycopeptides and fractionated by anion exchange chromatography into neutral and acidic alditols. Major oligosaccharides contained in the acidic fraction were purified by high performance liquid chromatography and structurally characterized by a combination of fast atom bombardment mass spectrometry, methylation analysis and 500 MHz 1H-nuclear magnetic resonance spectroscopy. The structural aspects exhibited by these major species in the acidic fraction resemble those established previously for the neutral oligosaccharides from human skim milk mucins: 1) the size of the alditols varies from tri- to decasaccharides, 2) the core structure is of the ubiquitous type 2, 3) the backbone sequences are of the poly-N-acetyllactosamine type with a particular preponderance of linearly extended GlcNAc beta(1-3)Gal (major) or GlcNAc beta(1-6)Gal units (minor).     

Presence of immunoreactive endothelin in human milk

Endothelin-like immunoreactivity was detected in human milk at a concentration of 6.8 +/- 1.6 pmol/l (mean +/- SEM; n = 16) using a highly sensitive radioimmunoassay. Gel filtration and fast protein liquid chromatography (FPLC) verified the identity of the endothelin. FPLC revealed 4 peaks, one eluting just after the void volume, and the other three in the positions of endothelin-1, -2, and -3, respectively.