Topic:Casein
Casein is a family of phosphoproteins predominantly found in mammalian milk, and it constitutes a significant portion of the protein content in equine milk. In horses, casein plays a vital role in the nutritional profile of mare's milk, providing essential amino acids and serving as a primary source of protein for foals. The composition and structure of casein in equine milk can influence the digestibility and absorption of nutrients. Variations in casein content can occur due to factors such as lactation stage, diet, and individual mare differences. This page compiles peer-reviewed research studies and scholarly articles that explore the composition, function, and nutritional implications of casein in equine milk, as well as its impact on foal development and health.
Identification of phosphorylation sites of equine beta-casein isoforms. Equine beta-casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P-7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion-exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium d...
Equine alpha S1-casein: characterization of alternative splicing isoforms and determination of phosphorylation levels. alpha(S1)-Casein was isolated from Haflinger mare's milk by hydrophobic interaction chromatography and displayed great micro-heterogeneity by 2-dimensional electrophoresis, probably because of a variable degree of phosphorylation and alternative splicing events. The aim of the present work was to investigate the complexity of the mare's alpha(S1)-casein. The different isoforms present in milk were submitted to a double treatment of dephosphorylation, first by using alkaline phosphatase and then acid phosphatase to achieve complete dephosphorylation. The apoforms were then analyzed by electrosp...
Comparison of different extenders with defined protein composition for storage of stallion spermatozoa at 5 degrees C. To maintain the fertility of stallion spermatozoa during cooled storage, extender media are added to semen. In this study, three semen extenders were compared: EquiPro which contains defined caseinates and whey proteins instead of dried skim milk. The extender is provided in dry form and dissolved in distilled water prior to use. EquiPro TM has the same composition as EquiPro but is provided in a sterilized ready-to-use liquid form. AndroMed-E contains soybean lecithin as protein source. Semen was collected from seven stallions. Ejaculates were divided into three aliquots, diluted with the dif...
Behavior, milk yield, and milk composition of machine-and hand-milked Murgese mares. A 25-d trial was performed to assess the effects of machine and hand milking on behavior, and milk yield and composition of mares; the trial involved 8 Murgese mares. Milk yield was higher (7.69 vs. 4.91 kg) and milking time was shorter (1.80 vs. 5.40 min) in machine-milked than in hand-milked mares. Machine milking of mares also resulted in a greater fat content of milk (1.63%) than did hand milking (1.06%). Milking system did not affect casein content, nitrogen fractions, or somatic cell counts. No differences in behavioral activities and in response to humans emerged. Results indicate that ...
Composition, physiochemical properties, nitrogen fraction distribution, and amino acid profile of donkey milk. This study investigated the changes in chemical composition, nitrogen fraction distribution, and AA profile of milk samples obtained during lactation from the Jiangyue breed of donkey in Northwest China. Results showed that donkey milk contained 9.53% total solids, 1.57% protein, 1.16% fat, 6.33% lactose, and 0.4% ash on average, which is more similar to mare and human milk than to the milk of other mammals. Throughout the lactation investigated, pH and density were constant, protein and ash content showed an apparent negative trend (an increase in lactose content during 120 d postpartum, foll...
The primary structure of a low-Mr multiphosphorylated variant of beta-casein in equine milk. Highly phosphorylated casein with a low molecular mass was isolated from Haflinger mare's milk by RP-HPLC. It accounts for 4.0% of the casein content. Its mass was determined by LC-ESI-MS before and after treatment by alkaline phosphatase. The molecular mass found for the apo-form (10,591 +/- 2 Da) is in agreement with its primary structure, which was established by ESI-MS/MS from tryptic peptides. It appeared that this short protein (94 amino acid residues) is an internally truncated form of the full-length equine beta-casein (226 residues). This low-Mr variant of equine beta-casein displays ...
Determination of the phosphorylation level and deamidation susceptibility of equine beta-casein. beta-Casein was isolated from Haflinger mare's milk by RP-HPLC, and displayed microheterogeneity by urea-electrophoresis and 2-DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine beta-casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo-form of Haflinger mare's beta-casein (25 514 +/- 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region...
Comparison of an extender containing defined milk protein fractions with a skim milk-based extender for storage of equine semen at 5 degrees C. A problem of semen extenders based on milk or egg yolk is the fact that these biological products consist of a variety of substances. Extenders containing only components with clearly protective effects on spermatozoa would thus be an advantage. In this study, we have compared the effects of an extender containing defined caseinates and whey proteins only (EquiPro, defined milk protein extender) with skim milk extender on equine spermatozoa during cooled storage. The defined milk protein extender was used with and without the antioxidant N-acetyl cysteine (NAC). In a second experiment, semen w...
Proteomic evaluation of milk from different mammalian species as a substitute for breast milk. As milk represents the main source of nutrition for infants, the question of an effective human milk substitute becomes mandatory when a formula-fed baby is allergic to cows' milk proteins. In this case, formulas containing extensively hydrolysed milk proteins should be preferred, but even such a formula may cause allergic reactions in highly sensitive patients. If there is evidence of cows' milk allergy with IgE-associated symptoms, after 6 mo of age, a soy bean formula may be recommended only when tolerance to soy protein has been established by clinical challenge. In infants with allergic r...
Co-infection between influenza virus and flagellated bacteria. Trypsin is required in the hemagglutinin (HA) cleavage to in vitro influenza viruses activation. This HA cleavage is necessary for virus cell entry by receptor-mediated endocytosis. Bacteria in the respiratory tract are potential sources of proteases that could contribute to the cleavage of influenza virus in vivo. From 47 samples collected from horses, pigs, and from humans, influenza presence was confirmed in 13 and these samples demonstrated co-infection of influenza with flagellated bacteria, Stenotrophomonas maltophilia from the beginning of the experiments. Despite treatment with antibio...
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, wit...
Characterization of equine cDNA sequences for alphaS1-, beta- and kappa-casein. Here we report the entire cDNA sequences for equine alphaS1-, beta- and kappa-casein. Based on interspecies comparison, nine exons were found in equine beta-casein and five in kappa-casein. In equine alphaS1-casein cDNA the exon 5 was missing, which resulted in the total of 18 exons instead of 19 theoretically possible exons in alphaS1-casein cDNA. Comparison of DNA sequences representing exon 5 in other species with corresponding equine genomic region confirmed the presence of cryptic exon in horse genomic DNA. Equine alphaS1-casein mRNA was present in three forms in the lactating mammary gla...
Separation and characterization of mares’ milk alpha(s1)-, beta-, kappa-caseins, gamma-casein-like, and proteose peptone component 5-like peptides. The equine alpha(s1)- and beta-caseins (CN) were purified by chromatography on DEAE-cellulose and by reversed-phase HPLC. The alpha(s1)-, beta-, and kappa-CN were characterized either by monodimensional urea-PAGE or sodium dodecylsulfate (SDS)-PAGE or by bidimensional electrophoresis. Kappa-casein was characterized after electrophoresis by glycoprotein-specific staining. To identify alpha(s1)-CN without ambiguity, internal sequences were determined after trypsin or chymosin digestion of purified alpha(s1)-CN. These sequences, that could be estimated to correspond to 62% of the full protein, pr...
Primary structure of kappa-casein isolated from mares’ milk. In this work the purification and the complete primary structure of kappa-casein from equine milk are reported for the first time. Mares' milk casein was separated by RP-HPLC into four fractions. Complete primary sequence was obtained by sequence analysis of the protein in the fastest eluting peak isolated by chromatography. This sequence was 95% identical to that reported for the C-terminal portion of the zebras' kappa-casein and showed high similarity with kappa-caseins from sources other than Equidae, confirming that this protein was indeed kappa-casein in equine milk. The presence of post-...
Preservation of stallion sperm quality by native phosphocaseinate: a direct or indirect effect? Milk-based diluents are generally considered efficient for survival of stallion spermatozoa in vitro. However, milk is a complex and variable medium and native phosphocaseinate is a milk component that is more efficient for preservation of sperm motility and fertility, although the mechanisms involved in this protection have not yet been elucidated. The aim of the present study was to characterize the interactions between native phosphocaseinate and equine spermatozoa. No binding between sperm membranes and native phosphocaseinate was observed using indirect immunofluorescent staining or elect...
Effect of milk fractions on survival of equine spermatozoa. Milk-based semen diluents are known to be practical and effective in protecting equine spermatozoa during storage. Due to complex composition of milk, the components which are beneficial or harmful to spermatozoa are unknown. To address these unknowns the effect of various milk fractions on motility of stallion spermatozoa was evaluated. The fractions tested were native phosphocaseinate (NPPC), beta-casein, whey protein concentrate (WPC), alpha-lactalbumin, beta-lactoglobulin, microfiltrate, and ultrafiltrate. The standard reference diluents were INRA 82, commercial skim milk, and Hank's salts...
The comparison of pepsin and trypsin action on goat, cow, mare and human caseins. The degree of proteolysis of micellar caseins of human, goat's, mare's and two breeds (Black&White and Red Polish) of cow's milk was compared for pepsin and trypsin action in vitro. Human and goat's caseins were hydrolysed in 100% and 96%, respectively, mare's casein--92%, Black&White cow's casein--90%, Red Polish cow's casein--76%. The differences can be related to the micelle structure, especially to the prevalence of beta casein in the human and goat's casein. The significant dissimilarity between the two breeds of investigated cows is surprising and indicates a different geometry o...
Protein and nitrogen composition of equine (Equus caballus) milk during early lactation. Separation of whey protein from casein in equine milk was achieved by adjustment of pH to 4.3 without addition of calcium, and by ultracentrifugation at 189,000 g for 1 hr. True protein, whey protein, and casein decreased significantly during the first 28 days of lactation with the magnitude of decrease being greatest for whey protein. The proportion of nitrogen in whey protein:casein decreased from 85:15 to 54:46 during the 28 day time period. The concentration of non-protein nitrogen remained relatively constant at 500 mg nitrogen/l but increased in proportion from 2 to 13% of the total nitr...
Cellular sources of proteolytic enzymes in equine joints. Isolated equine blood and articular cells were investigated for proteolytic enzyme production by means of gel filtration and analysis on 14C-acetylated collagen and casein substrates. Significant amounts of collagenase and caseinase activity were produced by cultured synoviocytes stimulated with equine interleukin 1, although large amounts of collagenase also originated from neutrophils.
Proteolytic enzymes in equine joints with infectious arthritis. Significant amounts of collagenase and caseinase activity were detected in infected synovial fluid samples. Partial characterisation of the enzymes by gel filtration suggested that synovial fluid from cases of infectious arthritis may contain enzymes from both the synovial cells and neutrophils. This finding was also supported by analysis of sequential synovial fluid samples from 4 infected joints. In 3 joints the concentration of caseinase and in 1 joint collagenase paralleled the decline in total nucleated cell count. However, in 3 joints the concentration of collagenase remained high after ...
Effect of protein source in liquid formula diets on food intake, physiologic values, and growth of equine neonates. The effects of 2 liquid formula diets differing in protein source were evaluated in orphan foals. The response of 7 foals fed a diet containing casein as the protein source, and 6 foals fed a diet containing a combination of whey and casein, was compared with the response in a reference group of 8 mare-raised foals. Orphaned foals were fed 150 kcal/kg of body weight/d, divided into 6 equal feedings of 25 kcal/kg. Formula intake was comparable among the experimental groups, and foals fed the liquid formula diet grew as well as mare-raised foals. There was no difference among groups in mean dail...
Pharmacologic evaluation of factor XIIIa*-like enzyme activity in equine plasma as a potential therapeutic avenue for the inhibition of fibrinous tissue. Several pharmaceutical compounds were evaluated for their ability to selectively inhibit activated coagulation factor-XIII-like enzyme activity (eg, XIIIa*) in pooled equine plasma. Presence of coagulation factor-XIIIa*-like enzyme activity in plasma was established by assay procedures involving incorporation of the fluorescent amine compound, monodansylcadaverine, into purified casein, which served as a protein substrate. Pharmaceuticals inhibitory to coagulation factor-XIIIa*-like enzyme activity were recognized by plasma gel formation of high spectrophotometric transmittance (transparency),...
Late-stage mediators of the inflammatory response: identification of interleukin-1 and a casein-degrading enzyme in equine acute inflammatory exudates. Interleukin-1 and a casein-degrading enzyme have been identified in an experimental system for studying acute inflammation in the horse. The levels of both the cytokine and the proteinase increased over the first 24 hours following initiation of the inflammatory response, and remained at high levels through to the last sample collected at 48 hours. This is in marked contrast to prostaglandin E2 concentrations which were low initially, peaked at four to eight hours and had returned to low levels by 12 to 24 hours. It is likely that interleukin-1 and various proteinases are involved in the later...
Yield and composition of milk from lactating mares: effect of lactation stage and individual differences. The yields and composition of milk from nursing mares were studied during the first two months of lactation in 11 mares of heavy breeds (784 kg). Daily yield increased from 21.7 to 24.6 kg between weeks 1 and 8 of lactation. Fat, protein, gross energy and Ca concentrations significantly decreased when lactose content increased during this period. Individual variations were higher for yield than for composition. Casein, whey protein and non-protein N (56, 34 and 10% of crude protein, respectively) and amino acid composition did not vary between weeks 1 and 8 of lactation.
Comparison of the binding of Ca2+ and Mn2+ to bovine alpha-lactalbumin and equine lysozyme. The enthalpy change of the binding of Ca2+ and Mn2+ to equine lysozyme was measured at 25 degrees C and pH 7.5 by batch microcalorimetry: delta H degrees Ca2+ = -76 +/- 5 kJ mol-1, delta H degrees Mn2+ = -21 +/- 10 kJ mol-1. Binding constants, log KCa2+ = 6.5 +/- 0.2 and log KMn2+ = 4.1 +/- 0.5, were calculated from the calorimetric data. Therefore, delta S degrees Ca2+ = -131 +/- 20 JK-1 mol-1 and delta S degrees Mn2+ = 8 +/- 44 JK-1 mol-1. Removal of Ca2+ induces small but significant changes in the circular dichroism spectrum, indicating the existence of a partially unfolded apo-conformatio...
The effect of drugs used in the treatment of osteoarthrosis on stromelysin (proteoglycanase) of equine synovial cell origin. There is increasing evidence that the proteoglycan-degrading neutral metalloproteinase, stromelysin, is a key enzyme in the pathogenesis of osteoarthrosis. Equine synovial lining cells were stimulated in vitro to produce stromelysin, and phenylbutazone, flunixin, betamethasone, sodium hyaluronate and polysulphated glycosaminoglycan (PSGAG) were tested for their ability to inhibit the action of this enzyme on 14C-labelled casein substrate. Only PSGAG possessed inhibitory activity at concentrations likely to be achieved therapeutically in the equine fetlock joint.
Structural, histochemical and biochemical observations on horse milk-fat-globule membranes and casein micelles. Horse milk fat globules (MFGs) and casein micelles were studied using freeze fracturing, freeze etching and thin-section electron microscopy, as well as lectin histochemistry, gel electrophoresis, and Western blotting. Horse MFGs were found to be relatively small, their average volume-surface diameter being about 2.75 microns. The MFG membrane is composed of three layers: an inner proteinaceous coat occasionally having a paracrystalline substructure, a unit membrane, and a prominent filamentous glycocalyx. The last is rich in glycoconjugates, as revealed by its binding of various lectins. In a...
Serum triiodothyronine and thyroxine concentrations in weanling horses fed carbohydrate by direct gastric infusion. Plasma glucose and serum insulin, thyroxine, and triiodothyronine concentrations were monitored in 6 weanling Thoroughbreds after direct gastric infusion of solutions containing sucrose or casein. Neither plasma glucose nor serum hormone concentrations were affected by infusions of water or by infusions of 326 or 424 g of casein/250 kg of body weight. However, glucose and hormone concentrations increased significantly (P less than 0.001) after infusions of 649 or 844 g of sucrose/250 kg. Initial rates of increase were more rapid and increases were subsequently reversed more rapidly when 844 g ...
Interrelationships of constituents and partition of salts in milk samples from eight species. Skim-milk samples from rat, rabbit, pig, sheep, goat, horse and man were analysed for lactose, casein and the total and ultrafilterable concentrations of the main salts. Results are compared with data for the cow. The ultrafiltrate concentrations of Ca and Mg were positively correlated with that of citrate and the colloidal concentrations of Ca, Mg and citrate were positively correlated with that of Pi, suggesting that common, general, principles determine the partition of salts in milks. Casein concentration in the skim-milks was inversely related to that of lactose in accordance with a recen...