Characterisation of proteins in the milk of fur seals

Milk protein composition was investigated throughout the lactation periods of the Australian fur seal (Arctocephalus pusillus doriferus) and Antarctic fur seal (Arctocephalus gazella). The mean protein content of the milk was found to be 10.9% and 10.6% respectively. The concentration of total protein did not change during lactation, although a decline in casein content of the milk in late lactation was apparent. Milk protein concentration during a foraging/suckling cycle of the Antarctic fur seal analysed at the time of arrival on shore, and 24 h and 72 h after arrival was 12.8%, 11.4% and 12.5% respectively. Re-feeding animals at 72 h resulted in a significant increase in milk protein content to 14.9%. Characterisation of milk protein by SDS-PAGE analysis revealed 5 casein and 10 major whey protein bands. Amino-terminal sequencing indicated that the majority of the whey fraction of the milk is beta-lactoglobulin (beta-LG). The limited amino acid sequence indicated 3 different beta-LGs were secreted in the milk. Subsequently, RT-PCR was used to extend the sequence of one of the beta-LGs and translation of the 464 bp fragment indicated that it shared 79% sequence identity with feline beta-LG II.     

Milk composition of three free-ranging African elephant (Loxodonta africana africana) cows during mid lactation

Data are presented that indicate the dynamic changes of nutrients in milk from three free ranging African elephant (Loxodonta africana africana) cows during lactation. At the respective collection times of 12, 14 and 18 months of lactation the nutrient content was 47.3, 52.0 and 68.6 g protein; 60.7, 87.4 and 170.8 g fat; 1.6, 2.1 0.5 g lactose and 20.9, 21.5 and 8.6 g oligosaccharides per kg milk. The protein fraction respectively consisted of 18.0, 31.7 and 45.9 g caseins/kg milk and of 29.3, 20.3 and 22.7 g whey proteins/kg milk. Electrophoresis and identification of protein bands showed that polymorphs of one whey protein may be present in elephant's milk similar to polymorphs of alpha-lactalbumin found in cow's milk. From the middle of the lactation time lactose was replaced by oligosaccharides as major carbohydrate, and the major compound of these was identified as isoglobotriose by 1H NMR spectroscopy. The lipid fraction contains a high content, of capric and lauric acids, approximately 70% of the total fatty acids, and low content of myristic, palmitic and oleic acids. During these lactation times the content of short chain fatty acids, capric and caprylic acids increased, while fatty acids lauric acid and longer decreased.     

Southern elephant seal (Mirounga leonina) II. Studies of milk protein fractions by gel electrophoresis

 Milk protein fractions during various stages of lactation in the southern elephant seal Mirounga leonina were analysed. Twelve milk samples were taken from ten females throughout the lactation period during 1990 and 1991 at Stranger Point, King George Island, South Shetland Islands. Milk samples were subjected to polyacrylamide gel electrophoresis (PAGE). Samples from different days of lactation gave similar qualitative electrophoretic patterns. True protein content was significantly higher (P<0.05) at the beginning of lactation, and then remained constant until weaning. Caseins and whey proteins each consisted of several protein entities (four and five distinct bands respectively). Casein constituted only about 30% of the protein nitrogen, the remaining 70% being derived from whey proteins. There was some variation in concentration of casein and whey proteins as a function of time (P<0.0.5). Received: 28 July 1993/Accepted: 25 July 1995     

Prenatal and postnatal transfer of fatty acids from mother to pup in the hooded seal

Unlike most mammals, hooded seal (Cystophora cristata) pups are born with a substantial layer of adipose tissue. Subsequently, during the brief lactation period of only 4 days, fasting mothers mobilize enormous amounts of lipid from blubber and secrete milk (60% fat) at rates of 10 kg·day^-1. Pups gain 7 kg·day^-1 due primarily to the deposition of fat in blubber. We measured blubber content and fatty acid composition of blubber and milk in hooded seal mother-pup pairs at birth and over the 4-day lactation period to examine the nature and source of fetal lipids, the incorporation of maternal blubber fatty acids into milk lipid, and patterns of fatty acid deposition in suckling young. The fatty acid composition of the blubber of the newborn was notably different from that of its mother. Fetal deposition was likely due to a combination of both fetal synthesis and direct placental transfer of maternal circulating fatty acids. The blubber of the newborn was characterized by high levels (>90% of total fatty acids) of saturated and monounsaturated fatty acids of primarily endogenous origin. In particular, the fetus appeared to have high Δ-9 desaturase activity as evidenced by the large amounts of 14:1n-5 (4.2%) and 16:1n-7 (37.0%) in newborn blubber compared to maternal blubber (0.2% and 14.1%, respectively). Nevertheless, essential and long-chain polyunsaturated fatty acids of the n-3 and n-6 families, which could only have originated by direct transfer from the mother, comprised>7% of pup blubber fatty acids and indicated greater rates of placental transfer than found in humans. In hooded seal mothers, rapid lipid transfer during the brief lactation period appeared to be facilitated by direct incorporation of mobilized fatty acids into milk. Although some differences in proportions of specific fatty acids were found between milk and maternal blubber, most of these differences declined over the course of lactation. However, selective mobilization of 20:5n-3 from maternal blubber into milk was apparent throughout lactation and resulted in elevated levels in pup blubber at weaning compared to maternal blubber. Ingested fatty acids were deposited directly and without modification into the blubber of pups, and by 4 days the fatty acid composition of pup blubber was virtually identical to that of the milk consumed.     

Milk composition of a free-ranging African elephant (Loxodonta africana) cow during early lactation

Only one study previously reported comprehensively on the composition of African elephant's (Loxodonta africana) milk that was collected from 30 dead animals. In the current study milk was obtained from a tame but free-ranging African elephant cow without immobilization during the period when she was 4-47 days postpartum. At the respective collection times the nutrient content was 21.8 and 25.0 g protein; 56.0 and 76.0 g fat; 71.1 and 26.0 g sugars per kilogram of milk. The protein fraction, respectively, consisted of 10.0 and 14.0 g caseins/kg milk and of 11.8 and 11 g whey proteins/kg milk. During lactation the lactose content dropped from 52.5 to 11.8 g/kg milk, while the oligosaccharide content increased from 11.8 to 15.2 g/kg milk. The oligosaccharide was characterized as a galactosyllactose, which is digestible by cellulase. Electrophoresis and identification of protein bands showed a similar migrating sequence of proteins as seen in cow's milk, but some of the corresponding proteins were less negatively charged. The lipid fraction contains a high content of capric and lauric acids, approximately 60% of the total fatty acids, and low content of myristic, palmitic and oleic acids.     

Milk composition of free-ranging springbok (Antidorcas marsupialis)

Milk was obtained from three free-ranging springbok ewes of the Karoo, South Africa. The nutrient content was 74.4+/-13.8 g protein; 145.2+/-4.5 g fat; and 42.3+/-16.4 g lactose/kg milk. Small amounts of glucose, galactose and fucose were noted, and 0.3+/-0.4 g oligosaccharides. The protein fraction respectively consisted of 60.0+/-13.7 g caseins/kg milk and of 14.1+/-4.5 g whey proteins/kg milk. The lactation stage of the springbok ewes was not known, but variation in milk composition among individuals indicates that they were at different stages. Electrophoresis and identification of protein bands showed a similar migrating sequence of proteins as seen in caprine milk. The lipid fraction contains 604.0+/-26.5 g saturated fatty acids/kg milk fat, and 278.2+/-20.5 and 45.2+/-3.6 g/kg mono and poly-unsaturated fatty acids respectively. Compared to domesticated dairy species, a low content of short chain length fatty acids was observed, while stearic acid was at higher, and arachidonic acid at lower levels. Substantial levels of uneven carbon chain fatty acids were also observed. Springbok milk is much more concentrated than the milks of most ruminants, with higher fat and oligosaccharide contents.     

Disulphide bonds in casein micelle from milk

Mammary epithelial cells synthesised and secreted caseins, the major milk proteins in most mammals, as large aggregates called micelles into the alveolar lumen they surround. We investigated the implication of the highly conserved cysteine(s) of kappa-casein in disulphide bond formation in casein micelles from several species. Dimers were found in all milks studied, confirming previous observation in ruminants. More importantly, the study of interchain disulphide bridges in mouse and rat casein micelles revealed that any casein possessing a cysteine is engaged in disulphide bond interchange; these species express four or five cysteine-containing caseins, respectively. We found that the main rodent caseins form both homo- and heterodimers. Additionally, disulphide bond formation among milk proteins was specific since the interaction of the caseins with cysteine-containing whey proteins was not observed in native casein micelles.     

Chemical and immunochemical characterization of caseins and the major whey proteins of rabbit milk.

Caseins were separated from whey proteins by acid precipitation of skimmed rabbit milk. Whole casein was resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis into three major bands with apparent relative molecular masses (Mr of 31 000, 29 000 and 25 000. On agarose/urea-gel electrophoresis whole casein gave three bands with electrophoretic mobilities alpha, beta and gamma. The three components were purified by DEAE-cellulose chromatography under denaturing and reducing conditions. Each was shown to have a different amino acid, hexose and phosphorus content, as well as non-identical peptide fragments after proteinase digestion. The 31 000 Da (dalton) protein, of alpha-electrophoretic mobility, had a high phosphorus content (4.38%, w/w); the 29 000 Da peptide, of gamma-mobility, had the highest hexose content (2.2%, w/w), contained 0.8 cysteine residue per 100 amino acid residues and was susceptible to chymosin digestion corresponding thus to kappa-casein; the 25 000 Da protein migrated to the beta-position. The rabbit casein complex is composed of at least three caseins, two of which (alpha- and kappa-caseins) are analogous to the caseins from ruminants. Although caseins are poor immunogens, specific antibodies were raised against total and purified polypeptides. The antiserum directed against whole casein recognized each polypeptide, each casein corresponding to a distinct precipitation line. The antisera directed against each casein polypeptide reacted exclusively with the corresponding casein and no antiserum cross-reaction occurred between the three polypeptides. From whey, several proteins were isolated, characterized and used as antigens to raise specific antibodies. An iron-binding protein with an apparent Mr of 80 000 was shown to be immunologically and structurally identical with serum transferrin.     

Casein and alpha-lactalbumin messenger RNAs during the development of mouse mammary gland. Isolation, partial purification, and translation in a cell-free system

Messenger RNAs for the milk proteins, casein and α-lactalbumin, were isolated and partially purified from lactating mouse mammary glands by oligo(dT)cellulose chromatography followed by sucrose density gradient centrifugation. The translation of poly(A)⁺ mRNA in a wheat germ cell-free system yielded three casein polypeptides and a putative precursor form of α-lactalbumin which were precipitated by specific antibodies and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The casein polypeptides synthesized in vitro had a molecular weight that was no greater than that of the caseins in mouse milk. The presence of individual casein mRNAs coding for these polypeptides was demonstrated by the translation of various fractions of mRNA obtained by sucrose density gradient centrifugation of poly(A)⁺ mRNA. Casein mRNA activity increased about 250-fold between midpregnancy and the 10th–12th days of lactation, amounting to 50–60% of the total mRNA activity in that tissue. A similar study of α-lactalbumin mRNA showed an increase during lactation amounting to 0.2–0.4% of the total mRNA activity, which corresponds to the percentage of α-lactalbumin in total mouse milk protein.     

Genomic analysis of the major bovine milk protein genes.

The genomic arrangement of the major bovine milk protein genes has been determined using a combination of physical mapping techniques. The major milk proteins consist of the four caseins, alpha s1 (CASAS1), alpha s2 (CASAS2), beta (CASB), and kappa (CASK), as well as the two major whey proteins, alpha-lactalbumin (LALBA) and beta-lactoglobulin (LGB). A panel of bovine X hamster hybrid somatic cells analyzed for the presence or absence of bovine specific restriction fragments revealed the genes coding for the major milk proteins to reside on three chromosomes. The four caseins were assigned to syntenic group U15 and localized to bovine chromosome 6 at q31-33 by in situ hybridization. LALBA segregated with syntenic group U3, while LGB segregated with U16. Pulsed-field gel electrophoresis confirmed genetic mapping results indicating tight linkage of the casein genes. The four genes reside on less than 200 kb of DNA in the order CASAS1-CASB-CASAS2-CASK. Multiple restriction fragment length polymorphisms were also found at the six loci in three breeds of cattle.     

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.     

Bioavailability of zinc and its binding to casein in milks and formulas

Differences in zinc bioavailability among milk and formulas may be attributed to binding of zinc to various ligands. We determined the distribution of zinc and protein at different pHs and zinc and calcium concentrations. We used radiolabelled cow's milk, human milk, whey-predominant (WPF) and casein-predominant (CPF) infant formula. Lowering the pH changed zinc and protein distribution: zinc shifted from pellet (casein) to whey in cow's milk, from fat to whey in human milk and from fat and pellet to whey in formulas. Protein shifted from whey to pellet in human milk and from whey and pellet to fat in formulas. Increasing zinc and calcium concentrations shifted protein and zinc from pellet to whey for cow's milk and from whey and pellet to fat for the formulas. Protein distribution was not affected by calcium or zinc addition in human milk or CPF, while zinc shifted from whey to fat in human milk and from fat and pellet to whey in CPF. Zinc and calcium binding to isolated bovine or human casein increased with pH. At 500 mg/L of zinc, bovine casein bound 32.0 +/- 1.8 and human casein 10.0 +/- 0.9 mg zinc/g protein. At 500 mg/L of calcium, calcium was preferentially bound over zinc. Adding calcium and zinc resulted in 32.0 +/- 1.8 mg zinc/g bound to bovine casein and 17.0 +/- 0.8 mg zinc/g to human casein, while calcium binding was low. Suckling rat pups dosed with 65Zn labelled infant diets were killed and individual tissues were gamma counted. Lower zinc bioavailability was found for bovine milk at pH = 4.0 (%65Zn in liver = 18.7+1.4) when compared to WPF (22.8 +/- 1.6) or human milk (26.9 +/- 0.8). Lowering the pH further decreased zinc bioavailability from human milk, but not from cow's milk or WPF. Knowledge of the compounds binding minerals and trace elements in infant formulas is essential for optimizing zinc bioavailability.     

The composition of serval (Felis serval) milk during mid-lactation

Milk was obtained from three captive servals. The average nutrient content was 158.3+/-44.4 g protein; 152.6+/-62.3 g fat; and 68.7+/-31.4 g lactose per kg milk. The protein fraction respectively consisted of 117.7+/-44.8 g caseins per kg milk and of 40.6+/-6.7 g whey proteins per kg milk. Electrophoresis and identification of protein bands showed a similar migrating sequence of proteins as seen in cheetah and cat milk, with small differences in the beta-caseins. The lipid fraction contains 313.3+/-18.8 g saturated and 338.6+/-11.9 g mono unsaturated fatty acids per kg milk fat respectively. The high content of 292.4+/-24.9 g kg(-1) milk fat of polyunsaturated fatty acids is due to a high content in linolenic acid. No short chain fatty acids, but substantial levels of uneven carbon chain fatty acids were observed. In general, serval milk has a higher protein and fat content than that of the domestic cat and cheetah, and a lower content of unsaturated fatty acids than that of the domestic cat.     

INHIBITION OF LACTOGENIC ACTIVITIES OF BOVINE MAMMARY GLAND EXPLANTS BY THE WHEY FRACTION OF BOVINE MILK

The regulation of milk constituents, synthesis and secretion in tissue cultures of the bovine mammary gland was altered by a whey fraction of bovine milk. α-Casein gene expression, casein secretion and fatty acid synthesis were inhibited by the whey fraction in a dose-dependent manner. The whey fraction inhibited the enhancement activity of prolactin on α-casein gene expression and fatty acid synthesis, and also inhibited casein secretion to the medium, in explants cultured in a medium with or without prolactin. No effect on the expression of the β-lactoglobulin gene was found.     

Model process for removal of caseins from milk of transgenic animals

We describe a method for selective removal of caseins from milk. The method was developed as a model for transgenic milk processing. Raw cow milk spiked with nonmilk proteins was chosen as the model to resemble transgenic animal milk containing recombinant proteins. The most important elements of the process are (1) "deconstruction" of casein micelles in milk by destroying their Ca(2+) core using a chelating agent (EDTA), thus freeing any protein that might be entrapped in casein aggregates, and (2) "reconstruction" of micelles by providing them with a new Ca(2+) core, thus precipitating them away from the whey proteins, and the protein of interest. Calcium phosphate particles (CAP) were used to reform the disrupted casein micelles. The crystal clear supernatant fraction generated by this method provided >90% recovery and 6- to 13-fold concentration of the desired protein. Product-rich supernatant contained no detectable casein residues, as silver-stained SDS-PAGE and Western blot analyses demonstrated.     

A lactational study of the composition and integrity of casein micelles from the milk of the tammar wallaby (Macropus eugenii)

The amount of casein found in the milk of the tammar wallaby increases as lactation progresses. The increase is due to increasing amounts of β-casein; the α-casein remains largely constant. The α-casein is the more highly phosphorylated; the most abundant form is the 10-P, throughout lactation. The level of phosphorylation of β-casein shifts to lower average values in late lactation, possibly indicating the enzymatic reaction is overloaded by the increasing amounts of β-casein. Unlike bovine casein micelles, the wallaby micelles are not completely disrupted at pH 7.0 by sequestration of their calcium content with ethylene diamine tetraacetic acid (EDTA). Complete disruption only follows the addition of sodium dodecyl sulphate, indicating considerably greater importance for hydrophobic bonds in maintaining their integrity. This micellar behaviour indicates that, despite the evolutionary divergence of marsupials millennia ago, the caseins of wallaby milk assemble into micelles in much the same fashion as in bovine milk.     

A Novel Method for Separation of Caseins from Milk by Phosphates Precipitation

Milk protein of farm animals is difficult to isolate because of the presence of casein micelles, which are hard to separate from whey by using centrifugation or filtration. Insoluble casein micelles also create an obstacle for purification instruments to operate efficiently. The conventional method, to precipitate caseins by lowering pH to 4.6 and then recover the whey fraction for further purification using chromatography techniques, is not applicable to proteins having an isoelectric point similar to caseins. In addition, the acid condition used for casein removal usually leads to significantly poor yields and reduced biological activities. In this study, a novel method of precipitating caseins under neutral or weak acidic conditions is presented. The method employs a phosphate salt and a freeze–thaw procedure to obtain a casein-free whey protein fraction. This fraction contains more than 90% yield with little loss of bioactivity of the target protein, and is readily available for further chromatographic purification. This method was successfully applied to purify recombinant human factor IX and recombinant hirudin from the milk of transgenic pigs in the presented study. It is an efficient pretreatment approach prior to chromatographic purification of milk protein from farm animals and particularly of great value to collect those recombinants secreted from transgenic livestock.     

The composition of cheetah (Acinonyx jubatus) milk

Milk was obtained from two captive bred cheetahs. The nutrient content was 99.6 g protein; 64.8 g fat; and 40.21 g lactose per kg milk. Small amounts of oligosaccharides, glucose, galactose and fucose were noted. The protein fraction respectively consisted of 34.2 g caseins per kg milk and of 65.3 g whey proteins per kg milk. Very little variation in milk composition among the individual cheetahs was noted. Electrophoresis and identification of protein bands showed a similar migrating sequence of proteins as seen in lion's and cat's milk, with small differences in the β-caseins. The lipid fraction contains 290.4 g saturated and 337.3 g mono-unsaturated fatty acids per kg milk fat respectively. The high content of 279.5 g kg^− 1 milk fat of polyunsaturated fatty acids is due to a high content in α-linolenic acid. No short chain fatty acids, but substantial levels of uneven carbon chain fatty acids were observed.     

Casein: a milk protein with diverse biologic consequences

The consequences of bovine milk consumption are diverse, some of which are potentially deleterious. Although certain cultures shun cow's milk or milk-based products, Western societies consume large quantities of cow's milk. Although there are stronger similarities between bovine whey proteins and human whey proteins, the quantity and nature of casein in cow's milk differ markedly from human milk. We propose that the consequences of diets based on bovine casein should be more closely evaluated and certainly expanded beyond the simplistic approach of growth. What is good for the goose may be good for the gander, but what is good for the cow could be harmful to the human.     

Milk protein composition in purebred Holsteins and in first/second-generation crossbred cows from Swedish Red,Montbeliarde and Brown Swiss bulls

The aim of this study was to analyze milk protein composition in purebred and crossbred dairy cattle and estimate the effects of individual sources of variation on the investigated traits. Milk samples were collected from 505 cows from three commercial farms located in Northern Italy, some of which had originated from crossbreeding programs, although most were purebred Holsteins (HO). The basic crossbreeding scheme was a three-breed rotational system using Swedish Red (SR) semen on HO cows (SR×HO), Montbeliarde (MO) semen on SR×HO cows (MO×(SR×HO)) and HO semen again on MO×(SR×HO) cows. A smaller number of purebred HO from each of the herds were mated inverting the breed order (MO×HO and SR×(MO×HO)) or using Brown Swiss (BS) bulls (BS×HO) then MO bulls (MO×(BS×HO)). Milk samples were analyzed by reverse-phase HPLC to obtain protein fraction amounts (g/l) and proportions (% of total true protein). Traits were analyzed using a linear model, which included the fixed effects of herd-test-day (HTD), parity, days in milk and breed combination. Results showed that milk protein fractions were influenced by HTD, stage of lactation, parity and breed combination. The increase in protein concentration during lactation was due in particular to β-casein (β-CN), α_S1-CN and β-lactoglobulin (β-LG). The higher protein content of primiparous milk was mainly due to higher concentrations of all casein fractions. The milk from crossbred cows had higher contents and proportions of κ-CN and α-lactalbumin (α-LA), lower proportions of β-LG and greater proportion of caseins/smaller in whey proteins on milk true protein than purebred HO. The three-way crossbreds differed from two-way crossbreds only in having greater proportions of α-LA in their milk. Of the three-way crossbreds, the SR sired cows yielded milk with a smaller content and proportion of β-LG than the MO sired cows, and, consequently, a higher proportion of caseins than whey proteins. Results from this study support the feasibility of using crossbreeding programs to alter milk protein profiles with the aim of improving milk quality and cheese-making properties.