Physiological and pathological factors influencing bovine serum albumin content of milk

Concentrations of albumin from bovine blood serum were measured by radial immunodiffusion of 480 milk samples. Content in milk was not affected by content in serum, lactation number, or location of quarters. Bovine serum albumin concentrations in milk increased at the end of lactation (270 days) compared to the beginning (30 days) and at midlactation (150 days). Uninfected quarters had a mean concentration of serum albumin of .193 mg/ml. This was less than means for quarters infected by minor pathogens (.242 mg/ml) and by major pathogens (.284 mg/ml). Distributions of concentrations related to infection status, however, overlapped substantially. Somatic cell count was correlated .53 with concentrations of blood serum albumin in milk. About 32% of quarters infected by major pathogens had fewer than 500 x 10(3) cells/ml, whereas 47.5% of them had serum albumin content less than .2 mg/ml. For subclinical infections, concentration of serum albumin markedly increased when somatic cells were more than 1,000 x 10(3) cells/ml.     

Physiological and pathological factors influencing bovine immunoglobulin G1 concentration in milk

Bovine immunoglobulin G1 concentration was determined by radial immunodiffusion in 349 milk samples of uninfected quarters, 95 of infected quarters, and 118 blood serum samples from 42 Holstein-Friesian cows taken at days 30, 150, and 270. In lactation, immunoglobulin G1 concentration in milk was not affected by immunoglobulin G1 concentration in blood serum or location of quarters. The immunoglobulin G1 concentrations increased at the end of lactation and in samples collected from cows beyond the third lactation. Uninfected quarters had a mean immunoglobulin G1 concentration of .46 mg/ml. This was less than means from quarters infected by minor or major pathogens. Quarter infection by Staphylococcus aureus resulted in an increase of immunoglobulin G1 concentration in blood serum (9.22 to 11.3 mg/ml). When Corynebacterium bovis was persistent throughout the lactation, immunoglobulin G1 concentration in blood serum was increased (11.26 mg/ml). There was no correlation between somatic cell count and immunoglobulin G1 concentration in uninfected quarters. There was a slight correlation between bovine serum albumin and immunoglobulin G1 concentration in identical quarters (.23). Infection of quarters increased in varying degrees the correlation between immunoglobulin G1 concentration and bovine serum albumin concentration or somatic cell count in milk.     

High pressure-induced denaturation of alpha-lactalbumin and beta-lactoglobulin in bovine milk and whey: a possible mechanism

In this study, high pressure (HP)-induced denaturation of alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) in dairy systems was examined. In both milk and whey, beta-lg was less baroresistant than alpha-la; both proteins were considerably more resistant to HP-induced denaturation in whey than in milk. HP-induced denaturation of alpha-la and beta-lg increased with increasing proportion of milk in mixtures of milk and whey. Addition of a sulphydryl-oxidising agent, KlO3, to milk or whey increased HP-induced denaturation of beta-lg, but reduced the denaturation of alpha-la. Denaturation of both alpha-la and beta-lg was prevented by adding a sulphydryl-blocking agent, N-ethylmaleimide, to milk or whey prior to HP treatment, highlighting the crucial role of sulphydryl-disulphide interchange reactions in HP-induced denaturation of alpha-la and beta-lg. Removal of colloidal calcium phosphate from milk also reduced HP-induced denaturation of alpha-la and beta-lg significantly. The higher level of HP-induced denaturation of alpha-la and beta-lg in milk than in whey may be the result of the abscence of the casein micelles and colloidal calcium phosphate from whey, which facilitate HP-induced denaturation of alpha-la and beta-lg in milk.     

Antioxidant nature of bovine milk beta-lactoglobulin

Heating is necessary for processing milk in the dairy industry, which evidently produces a conformational change in β-lactoglobulin (β-LG). β-Lactoglobulin, a major protein that accounts for approximately 10 to 15% of total milk proteins, is a globular protein consisting of 162 AA with a relative molecular mass of 18.4 kDa. The purpose of the present study was to determine the antioxidant role of β-LG in milk and the possible mechanism involved. We showed that β-LG is a mild antioxidant whose potency is less than that of vitamin E and probucol (the latter being an antioxidant used for clinical therapy). The conversion of the β-LG monomer to dimer was responsible, in part, for the mode of action in protecting low-density lipoproteins against copper-induced oxidation. Cross-linking the free thiol groups of β-LG by heating (100°C for 2 min), or chemically modifying the β-LG by carboxymethylation to block the thiol groups resulted in a substantial loss of antioxidant activity. The data suggest that Cys-121 plays an essential role in the antioxidant nature of β-LG. By using an anti-LG antibody affinity column to deplete the β-LG from milk, we observed from the lost antioxidant activity that β-LG contributes approximately 50% of the total activity. Because β-LG is extremely sensitive to thermal denaturation, to maintain its antioxidant nature, dairy products consumed daily should not be overheated in order to maintain its antioxidant nature.     

Serum concentrations of the milk proteins alpha-lactalbumin and beta-lactoglobulin in pregnancy and lactation: correlations with milk and fat yields in dairy cattle.

Serum concentrations of alpha-lactalbumin and beta-lactoglobulin in first pregnancy, parturition, lactation, involution, and second parturition in 37 Holstein cattle were determined and used as an index of mammary status and in predicting milk yield. During first pregnancy, serum alpha-lactalbumin increased in the last 3 mo and reached a peak at parturition (approximately 1100 ng/ml). Changes in alpha-lactalbumin could not be described by a simple exponential equation, whereas changes in serum beta-lactoglobulin were described by a single exponential from second trimester until 4 wk prepartum and reached a peak at parturition (approximately 460 ng/ml). By 2 wk after parturition, alpha-lactalbumin had dropped to approximately 140 ng/ml, and beta-lactoglobulin dropped to approximately 25 ng/ml. In late lactation, alpha-lactalbumin was approximately 70 ng/ml and beta-lactoglobulin approximately 20 ng/ml. Short-term elevations were found after cessation of milking in both alpha-lactalbumin and beta-lactoglobulin in serum. The concentrations of alpha-lactalbumin and beta-lactoglobulin at second parturition were similar to those at first parturition with no differences found between parity. Both alpha-lactalbumin and beta-lactoglobulin in serum were functionally associated with mammary growth and development. In heifers late in pregnancy, both serum concentrations of alpha-lactalbumin and beta-lactoglobulin were positively correlated with mature equivalent milk and fat yields in the subsequent lactation. Serum beta-lactoglobulin concentrations at 16 wk prepartum in heifers were highly correlated with the sum of first and second lactation milk (r = .60) and fat (r = .60) yields. The potential value of using serum beta-lactoglobulin as an index for prescreening of heifers for lactation potential is discussed.     

Enzyme-linked immunosorbent assays for bovine alpha-lactalbumin and beta-lactoglobulin in serum and tissue culture media.

Enzyme-linked immunosorbent assays for bovine alpha-lactalbumin and beta-lactoglobulin have been developed for measurements of serum and tissue culture samples. Either alpha-lactalbumin or beta-lactoglobulin antiserum was coated on ELISA plates. Biotinylated proteins were used in competition with unknown amount of proteins in samples. After unbound proteins were washed off, ExtrAvidin-peroxidase and tetramethylbenzidine were then used as a detection system. Crossreactivity of caseins or bovine serum albumin was less than .0001% in either alpha-lactalbumin or beta-lactoglobulin ELISA. Parallel curves from serial dilutions were obtained in serum and media samples. The additivity of alpha-lactalbumin and beta-lactoglobulin ELISA was validated in either serum or medium samples. The intraassay and interassay coefficients of variation for alpha-lactalbumin and beta-lactoglobulin ELISA were below 10% over 51 and 47 assays. The ELISA are useful in mammary gland biology studies for measuring milk whey protein in serum or culture media.     

Effect of beta-lactoglobulin polymorphism and seasonality on bovine milk composition

The objective was to evaluate the effect of beta-lactoglobulin (beta-lg) polymorphism and seasonality on milk composition (fat, lactose, total solids, milk urea nitrogen, total protein, true protein, casein and somatic cell counts) of Holstein and Girolando cows. Milk and blood samples from 278 Holsteins cows and 156 Girolando cows were taken during two dry seasons and two rainy seasons, for milk composition analysis and to determine beta-lg genotypes, respectively. BB genotype was the most frequent for both breeds, followed by AA genotype for Holstein (BB>AA>AB) and by AB for Girolando cows (BB>AB>AA). No differences were found in milk compositional characteristics among genetic variants of beta-lg (AA, AB and BB) either between Holstein or Girolando cows. No association between milk composition and beta-lg genetic polymorphism was observed. During the dry season, independently of the breed considered, higher contents of lactose, true protein, casein and casein:true protein ratio were found.     

Purification and physicochemical characterization of ovine beta-lactoglobulin and alpha-lactalbumin

Ovine whey proteins were fractionated and studied by using different analytical techniques. Anion-exchange chromatography and reversed-phase high-performance liquid chromatography (HPLC) showed the presence of two fractions of beta-lactoglobulin but only one of alpha-lactalbumin. Gel permeation and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis allowed the calculation of the apparent molecular mass of each component, while HPLC coupled to electrospray ionisation-mass spectrometry (ESI-MS) technique, giving the exact molecular masses, demonstrated the presence of two variants A and B of ovine beta-lactoglobulin. Amino acid compositions of the two variants of beta-lactoglobulin differed only in their His and Tyr contents. Circular dichroism spectroscopy profiles showed pH conformation changes of each component. The thermograms of the different whey protein components showed a higher heat resistance of beta-lactoglobulin A compared to beta-lactoglobulin B at pH 2, and indicated high instability of ovine alpha-lactalbumin at this pH.     

Radioimmunoassay for measurement of bovine alpha-lactalbumin in serum, milk and tissue culture media

A sensitive, specific radioimmunoassay for bovine alpha-lactalbumin (alpha-la) suitable for measurement of serum, tissue culture media and milk was developed. alpha-La was not detected in serum from prepubertal male or female cattle, but was detected as early as 60 d of gestation in nulliparous Holstein heifers, the level being greatly increased during the last one third of gestation. Serum from cross bred beef heifers contained less alpha-la and it was not detected until late gestation. Concentrations of alpha-la in serum samples from pregnant multiparous Holstein cows decreased at drying-off and subsequently increased just before parturition. Secretion of alpha-la by mammary tissue explants from steroid-primed prepubertal Holstein heifers was induced by the addition of bovine prolactin, ovine prolactin or human growth hormone to tissue culture media.     

Caprine immunoglobulin G,beta-lactoglobulin,alpha-lactalbumin and serum albumin in colostrum and milk during the early post partum period

Colostrum and milk samples from 20 goats were analysed for concentrations of immunoglobulin G (IgG), beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and serum albumin (CSA) throughout the first 14 milkings post partum (7 d of lactation) using single radial immunodiffusion assay. Concentrations (mg/ml, means +/- SD) at first milking were IgG 47.9 +/- 25.5, beta-lg 30.7 +/- 10.4, alpha-la 2.77 +/- 0.82 and CSA 2.97 +/- 2.46 mg/ml. Large variations were recorded for IgG concentrations (19.9-94.5 mg/ml) and beta-lg (9.3-49.8 mg/ml). Concentrations of IgG, beta-lg and CSA dropped abruptly in the subsequent milkings and alpha-la concentration decreased slowly. Mean IgG concentration was < 2 mg/ml after 7 milkings and < 1 mg/ml after 11 milkings. However, IgG concentration does not differ significantly, at the 1% level, from milkings 7-14. The contribution of beta-lg to the increase in whey proteins in early milks was greater than that of IgG from milkings 5 to 14. The results were tabulated to make it possible to calculate the excess of whey proteins that would be obtained if early milks were illegally added to milk supply.     

Bovine immunoglobulin G, beta-lactoglobulin, alpha-lactalbumin and serum albumin in colostrum and milk during the early post partum period.

Colostrum and milk samples from 60 Holstein-Friesian cows were analysed for concentrations and yields of immunoglobulin G (IgG), beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and serum albumin (BSA) throughout the first 16 milkings post partum (8 d of lactation) using a single radial immunodiffusion assay. Concentrations (mg/ml, means +/- SD) at first milking were IgG 59.8 +/- 28.5, beta-lg 14.3 +/- 4.6, alpha-la 2.04 +/- 0.6, BSA 1.21 +/- 0.44. Large variations were recorded for IgG concentrations (15.3-176.2 mg/ml) and yields (0.2-925 g). Cows in their first lactation produced significantly lower concentrations and yields of colostral IgG than cows in later lactations. A colostral yield of IgG below the 100 g required to prevent calf hypo-gamma-globulinaemia was found in 18.3% of the cows. The concentrations of IgG, beta-lg and BSA dropped abruptly in subsequent milkings and alpha-la concentration decreased slowly. The mean IgG concentration was < 2 mg/ml after eight milkings and < 1 mg/ml after fifteen milkings. However, IgG concentration did not differ significantly, at the 1% level, during milkings 11-15. The results were tabulated to make it possible to calculate the excess of whey proteins that would be obtained if early milks were illegally added to the milk supply.     

The binding ability of alpha-lactalbumin and beta-lactoglobulin to mutagenic heterocyclic amines.

The binding ability of bovine milk proteins with mutagenic heterocyclic amines was investigated. Binding was determined with 2 mg of beta-lactoglobulin and 20 micrograms of heterocyclic amine in .4 ml of pH 7.4, 50 mM phosphate buffer, at 37 degrees C, in a shaker for 10 min. The unbound heterocyclic amine in protein-free ultrafiltrate was analyzed by HPLC method. The binding of alpha-lactalbumin, beta-lactoglobulin A and beta-lactoglobulin B were 90.44, 81.38, and 89.18%, respectively, with 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole; 37.85, 34.04, and 43.90%, respectively, with 3-amino-1-methyl-5H-pyrido[4,3-b]indole; and 49.11, 43.25, and 57.44%, respectively, with 2-amino-6-methyldipyrido[1,2-a:3',2'-d] imidazole. Binding of beta-lactoglobulin and alpha-lactalbumin to 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole and 3-amino-1-methyl-5H-pyrido [4,3-b]-indole was higher at pH conditions above 7.4, and binding was lost at pH less than 5.5. Maximum binding of both proteins to 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole was at pH 7.4, and binding was inhibited at pH conditions above 8.5 and less than 6.5.     

Determination of alpha-lactalbumin in human milk.

Alpha-lactalbumin is a major protein in human milk and of considerable nutritional importance to the infant. However, there are discrepancies in the literature on content of this protein in human milk, which indicate a need for a method that would permit estimation from a large number of samples. The technique of immunodiffusion was adopted for human alpha-lactalbumin. Purification of alpha-lactalbumin for production of antigen is described as is the statistical evaluation of the method. The alpha-lactalbumin contents of pooled human milk samples as well as of the milk from one mother during 2 mo of lactation were analyzed. The alpha-lactal-bumin content of human milk showed considerable variation. This variation and its relation to factors such as length of lactation, and nutritional status of the mother should be studied further.     

17Beta-estradiol and estrone concentrations in plasma and milk during bovine pregnancy

Estrone (E₁) and 17β-estradiol (E₂) are present in milk, but the mechanism(s) that regulate their appearance in milk are not known. The objectives of this study were to determine the impact of stage of pregnancy on the concentrations of E₁ and E₂ in plasma and milk and to determine the correlations between plasma and milk E₁ and E₂ and with milk components throughout pregnancy. Blood and milk samples were collected from 13 cows every 28 d throughout pregnancy. The E₁ and E₂ were quantified in plasma and milk using RIA after organic solvent extractions and Sephadex LH-20 column chromatography. Plasma E₁ concentrations averaged 0.8, 16.9, and 41.8 pg/mL in trimesters 1, 2, and 3, respectively. The respective E₁ concentrations in milk averaged 0.6, 7.9, and 27.1 pg/mL. The E₂ concentrations in plasma averaged 0.5, 0.9, and 2.0 pg/mL; milk E₂ averaged 0.3, 0.9, and 5.0 pg/mL. Plasma and milk E₂ concentrations were greater in trimester 3 compared with trimesters 1 and 2. The E₁ concentrations in milk were significantly correlated with plasma E₁ concentrations (r = 0.77), percentage of milk fat (r = 0.50), and milk yield (r = −0.43). The E₂ concentrations in milk were significantly correlated with plasma E₂ concentrations (r = 0.93), percentage of milk protein (r = 0.63), and milk yield (r = −0.57). The milk-to-plasma ratio of E₂ increased from 0.4 during trimester 1 to 2.2 in trimester 3, which suggested that the mechanism(s) regulating the appearance of E₂ in milk may change over the course of pregnancy.     

Does beta-lactoglobulin occur in human milk?

Although beta-lactoglobulin (beta-lg) has been considered to be absent from human milk, recent results of other workers, based on immunological reactions between human milk and rabbit antiserum to bovine beta-lg, suggest that this protein may be present. Although our results show similar immunological reactions, we consider that lactoferrin is responsible for these, as it was the only reactive protein species which could be prepared to homogeneity. Indeed two types of antibodies were found by ELISA test in the antisera to bovine beta-lg. One of them would be able to bind loosely to human lactoferrin, but its binding sites would not be antigenic in the rabbit.     

牛乳的凝固特性及其影响因素研究进展

本文综述了近年来有关牛乳凝固特性的研究进展,重点针对影响牛乳凝固能力的遗传和非遗传因素的研究进展进行了分析论述,包括不同来源牛乳的凝固特性、乳蛋白组分和工艺条件等对牛乳凝固能力的影响。旨在厘清影响牛乳凝固能力的主要因素。     

Molecular characterization of peptides released from beta-lactoglobulin and alpha-lactalbumin via cardosins A and B

Crude mixtures of aspartic proteases from flowers of the plant Cynara cardunculus have been studied frequently, as have activities of such enzymes (in pure form) on caseins from bovine, ovine, and caprine sources. This research study addressed pure bovine whey protein as substrates; that is, α-lactalbumin (αLA) and β-lactoglobulin (α-LG), submitted to hydrolysis by 1 of 2 aspartic proteases (cardosins A and B), previously extracted and purified from C. cardunculus. Samples collected, following incubation at 55°C and pH 5.2, were assayed by fast protein liquid chromatography, reversed phase-high performance liquid chromatography, and tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis; the major peptides released were then collected and sequenced by Edman degradation. Cardosin B and, to a lesser degree, cardosin A showed proteolytic activity toward α-LA, but the hydrolyzates produced were characterized by distinct peptide profiles. Cardosin B possesses a broad specificity, and produces several hydrophobic peptides (at least 5, with molecular mass in the range 2 to 8 kDa) in the early stages, which eventually become more hydrophilic (with molecular mass below 2 kDa) at later stages of hydrolysis. Cardosin A was found to cleave α-LA at the peptide bonds Phe28-Arg29, Gly54-Tyr55, Ala59-Ile60, Leu71-Phe72, and Leu105-Thr106, whereas cardosin B cleaved Ala19-Glu20, Phe28-Arg29, Glu30-Leu31, Tyr37-Gly38, Trp45-Val46, Phe50-His51, Ala59-Ile60, Ser66-Thr67, Leu71-Phe72, Phe72-Gln73, Gln73-Ile74, Ile78-Trp79, Leu115-Asp116, and Leu124-Ala125. Conversely, cardosins A and B are apparently not active on β-LG.