Improved purification of beta-lactoglobulin from acid whey by means of ceramic hydroxyapatite chromatography with sodium fluoride as a displacer

The successful separation of beta-lactoglobulin from other bovine whey proteins was performed by ceramic hydroxyapatite chromatography with a fluoride ion gradient in phosphate buffer as displacement agent. The method was applied to acid whey originating from milk of healthy as well as of mastitic cows. beta-Lactoglobulin was completely eluted in one peak at a fluoride concentration of about 0.6 mol/l. The purity of beta-lactoglobulin in this fraction was at least 96% if whey from healthy milk was processed. Co-eluted contaminants are traces of immunoglobulin G, serum albumin and lactoferrin. In case of mastitic whey the proportion of beta-lactoglobulin is diminished as the amounts of immunglobulin G, serum albumin and lactoferrin are increased within this fraction. Size exclusion chromatography on Superdex 75 pg effectively removed contaminants resulting in a purity for beta-lactoglobulin from normal whey of approximately 99%. The yield of beta-lactoglobulin from physiological whey was 50-55% referring to the fraction highly enriched with beta-lactoglobulin by hydroxyapatite chromatography. In case of mastitic milk the higher amounts of contaminants were also removed successfully by size exclusion chromatography.     

Proteomic characterization by 2-DE in bovine serum and whey from healthy and mastitis affected farm animals

Acute phase proteins (APP) have been identified in whey and sera from healthy and mastitis cows through the proteomic analysis using two-dimensional electrophoresis (2-DE) coupled with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Although normal and mastitis serum samples show relatively similar protein composition, marked differences in expression levels and patterns can be observed. Conversely, normal and mastitis whey showed a very different composition, likely due to extravasation of blood proteins to the mammary gland. Different isoforms from the most abundant protein in milk, casein, were detected in both normal and mastitis whey. Other proteins, such as lactotransferrin, were only detected in the inflamed animal samples. Immunoglobulins showed different patterns but not increased levels in the inflamed whey. Also, many cellular proteins in mastitis cow's whey, that were absent from healthy cow's milk. They are responsible for the great change in composition between normal and mastitis whey, especially those which exert a biological function related to immune defense. Data collected in this work are of interest for gaining information about physiological changes in protein patterns in different fluids and, the correspondent modifications as result of an acute phase process in farm. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Isolation of alpha-lactalbumin,beta-lactoglobulin,and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity

The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Globulins were then precipitated from whey by half-saturated ammonium sulfate and beta-lactoglobulin was purified further using Sephadex G-50 gel filtration. The proteins in the supernatant were also fractionated using diethylaminoethyl cellulose chromatography in which beta-lactoglobulin was separated from alpha-lactalbumin and bovine serum albumin. The latter two proteins that co-eluted in anion-exchange chromatography were then gently isolated from each other by Sephadex G-50 gel filtration. Pure beta-lactoglobulin was also obtained by anion-exchange chromatography of the ammonium sulfate-precipitated globulins. Using enzyme-linked immunosorbent assay (ELISA), Western blotting, and ELISA inhibition assay, antigenicity of the purified proteins was evaluated. Our results showed high purity and well-preserved antigenicity of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin thus purified.     

A proteomic characterization of water buffalo milk fractions describing PTM of major species and the identification of minor components involved in nutrient delivery and defense against pathogens

Water buffalo has been studied in relation to the exclusive use of its milk for the manufacture of high-quality dairy products. Buffalo milk presents physicochemical features different from that of other ruminant species, such as a higher content of fatty acids and proteins. We report here a detailed proteomic analysis of buffalo skim milk, whey and milk fat globule membrane fractions. Notwithstanding the poor information available on buffalo genome, identification of protein isoforms corresponding to 72 genes was achieved by a combined approach based on 2-DE/MALDI-TOF PMF and 1-DE/muLC-ESI-IT-MS-MS. Major protein components, i.e. alpha(Sl)-, alpha(S2)-, beta-, kappa-caseins, alpha-lactalbumin and beta-lactoglobulin, were characterized for PTM, providing a scientific basis to coagulation/cheese making processes used in dairy productions. Minor proteins detected emphasized the multiple functions of milk, which besides affording nutrition to the newborn through its major components, also promotes development and digestive tract protection in the neonate, and ensures optimal mammary gland function in the mother. Defense against pathogens is guaranteed by an arsenal of antimicrobial/immunomodulatory proteins, which are directly released in milk or occur on the surface of secreted milk-lipid droplets. Proteins associated with cell signaling or membrane/protein trafficking functions were also identified, providing putative insights into major secretory pathways in mammary epithelial cells.     

Feline whey proteins: identification, isolation and initial characterization of alpha-lactalbumin, beta-lactoglobulin and lysozyme

1. Both alpha-lactalbumin and beta-lactoglobulin-like proteins were detected in the whey fraction of feline milk by immunoblotting with rabbit antisera to alpha-lactalbumin and beta-lactoglobulin, respectively. 2. alpha-Lactalbumin was found to occur in both glycosylated and unglycosylated forms in approximately equal concentrations. No polymorphism of feline alpha-lactalbumin was found. 3. Feline beta-lactoglobulin-like proteins produced complex electrophoretic patterns that appear to be determined by three distinct loci. Between two and five genetic variants are expressed by each locus. 4. Lysozyme was detected at levels of approximately 1 mg/ml in skim milk. 5. The identifications of the proteins as alpha-lactalbumin, beta-lactoglobulin and lysozyme were confirmed by determination of N-terminal amino acid sequences.     

Selective separation of beta-lactoglobulin from sweet whey using CGAs generated from the cationic surfactant CTAB

The selective separation of whey proteins was studied using colloidal gas aphrons generated from the cationic surfactant cetyl trimethyl ammonium bromide (CTAB). From the titration curves obtained by zeta potential measurements of individual whey proteins, it was expected to selectively adsorb the major whey proteins, i.e., bovine serum albumin, alpha-lactalbumin, and beta-lactoglobulin to the aphrons and elute the remaining proteins (lactoferrin and lactoperoxidase) in the liquid phase. A number of process parameters including pH, ionic strength, and mass ratio of surfactant to protein (M(CTAB)/M(TP)) were varied in order to evaluate their effect on protein separation. Under optimum conditions (2 mmol/l CTAB, M(CTAB)/M(TP) = 0.26-0.35, pH 8, and ionic strength = 0.018 mol/l), 80-90% beta-lactoglobulin was removed from the liquid phase as a precipitate, while about 75% lactoferrin and lactoperoxidase, 80% bovine serum albumin, 95% immunoglobulin, and 65% alpha-lactalbumin were recovered in the liquid fraction. Mechanistic studies using zeta potential measurements and fluorescence spectroscopy proved that electrostatic interactions modulate only partially the selectivity of protein separation, as proteins with similar surface charges do not separate to the same extent between the two phases. The selectivity of recovery of beta-lactoglobulin probably occurs in two steps: the first being the selective interaction of the protein with opposite-charged surfactant molecules by means of electrostatic interactions, which leads to denaturation of the protein and subsequent formation and precipitation of the CTAB-beta-lactoglobulin complex. This is followed by the separation of CTAB-beta-lactoglobulin aggregates from the bulk liquid by flotation in the aphron phase. In this way, CGAs act as carriers which facilitate the removal of protein precipitate.     

Large scale extraction of alpha-lactalbumin and beta-lactoglobulin from bovine whey by precipitation with polyethylene glycol and partitioning in aqueous two-phase systems.

The milk proteins alpha-lactalbumin and beta-lactoglobulin have been isolated from bovine whey by fractional precipitation with polyethylene glycol (PEG) and hydrophobic partitioning in aqueous PEG-hydroxypropylstarch two-phase systems using PEG-bound palmitate as hydrophobic ligand. The possible use of this combination for large scale purification of these whey proteins is discussed.     

Proliferation of the MAC-T bovine mammary epithelial cell line in the presence of mammary secretion whey proteins.

Development of the MAC-T bovine mammary epithelial cell line by stable transfection with simian virus-40 large T-antigen should greatly assist study of possible intrinsic (local) and extrinsic (systemic) factors regulating bovine mammary epithelial cell development, differentiation, and function. This study evaluated the influence of mammary secretion whey proteins alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), lactoferrin (LF), transferrin (TF) and serum albumin (SA) on MAC-T cell proliferation in the absence and presence of 10% fetal bovine serum (FBS). Concentration of whey proteins in culture ranged from 0 to 625 micrograms/ml. MAC-T cell proliferation in the absence of FBS was significantly lower than in the presence of 10% FBS. Alpha-lactalbumin and LF significantly decreased MAC-T proliferation in both the absence and presence of 10% FBS. Transferrin significantly increased MAC-T cell proliferation only in the absence of FBS. There were no significant differences in MAC-T cell proliferation cultured in the presence of BLG or SA. These experiments illustrate the potential usefulness of MAC-T cells for the study of factors involved in mammary cell proliferation. Results identified ALA, LF and TF as possible intrinsic factors associated with regulation of mammary epithelial cell proliferation.     

Whey proteins as a model system for chromatographic separation of proteins

Although chromatographic separation of whey proteins has been considered too expensive, whey may serve as an excellent model mixture to investigate and validate the use of simulation tools in the development and optimization of chromatographic separations and the outcome could easily be utilized since the model system has an intrinsic value. Besides, milk from transgenic animals could be an attractive source of pharmaceuticals which must be separated from the other proteins in the milk. Several whey proteins are of interest especially, alpha-lactalbumin, beta-lactoglobulins, immunoglobulins, lactoperoxidase, and lactoferrin. The scope of the project is to develop a consistent set of chromatographic data for whey proteins including isotherms, transport properties and scale-up studies and to develop the appropriate models for the anion exchangers Q-Sepharose XL, Source 30Q, Ceramic Q-HyperD F, and Merck Fractogel EMD TMAE 650 (S). In this work we have determined and correlated gradient and isocratic retention volumes in the linear range of the isotherm for alpha-lactalbumin, beta-lactoglobulin A and B, and bovine serum albumin at a pH from 6 to 9 at various NaCl concentrations.     

Secretion of whey acidic protein and cystatin is down regulated at mid-lactation in the red kangaroo (Macropus rufus)

Milk collected from the red kangaroo (Macropus rufus) between day 100 and 260 of lactation showed major changes in milk composition at around day 200 of lactation, the time at which the pouch young begins to temporarily exit the pouch and eat herbage. The carbohydrate content of milk declined abruptly at this time and although there was only a small increase in total protein content, SDS PAGE analysis of milk revealed asynchrony in the secretory pattern of individual proteins. The levels of alpha-lactalbumin, beta-lactoglobulin, serum albumin and transferrin remain unchanged during lactation. In contrast, the protease inhibitor cystatin, and the putative protease inhibitor whey acidic protein (WAP) first appeared in milk at elevated concentrations after approximately 150 days of lactation and then ceased to be secreted at approximately 200 days. In addition, a major whey protein, late lactation protein, was first detected in milk around the time whey acidic protein and cystatin cease to be secreted and was present at least until day 260 of lactation. The co-ordinated, but asynchronous secretion of putative protease inhibitors in milk may have several roles during lactation including tissue remodelling in the mammary gland and protecting specific proteins in milk required for physiological development of the dependent young.     

Thermal denaturation of whey proteins and its effect in dairy technology

The irreversible denaturation of the most important whey protein fractions, namely beta-lactoglobulin A and B and alpha-lactalbumin were studied. The orders of the reactions, the rate constants and the activation energies were determined. The experiments were extended to include whey protein solutions of different concentrations and mixtures of whey proteins and caseins in different proportions. The kinetic data found by experiment make it possible to calculate in advance the precise degree of irreversible denaturation. It was found that the denaturation of beta-lactoglobulin was a good test parameter in technological studies and that there was a close correlation between the degree of denaturation and the results of important dairy processes.     

Synthesis and evolution of concentration of beta-lactoglobulin and alpha-lactalbumin from cow and sheep colostrum and milk throughout early lactation

Synthesis of beta-lactoglobulin and alpha-lactalbumin by explants of ovine mammary gland and evolution of concentration of these proteins in cow and sheep colostrum and milk throughout early lactation have been studied. The evolution of both proteins was similar in cow and sheep species. The highest concentration was found in the first milking (19 and 2 mg/ml for beta-lactoglobulin and alpha-lactalbumin, respectively). Then, levels of beta-lactoglobulin decreased sharply and those of alpha-lactalbumin slowly during the first days of lactation, reaching stable values during the second week postpartum (4 and 1.5 mg/ml). The concentration ratio beta-lactoglobulin/alpha-lactalbumin was four times greater in colostrum than in mature milk. On the other hand, synthesis of these proteins represented about 25-30% of the synthesized total soluble proteins. The synthesis ratio beta-lactoglobulin/alpha-lactalbumin in explants obtained at 12 and 30 hours postpartum was found to be 3.5 and 1.7. These results indicate that the synthesis and secretion of beta-lactoglobulin are comparatively greater than those of alpha-lactalbumin during colostral period, suggesting that beta-lactoglobulin could have some specific function during this period.     

Isolation of lactoperoxidase, lactoferrin, alpha-lactalbumin, beta-lactoglobulin B and beta-lactoglobulin A from bovine rennet whey using ion exchange chromatography

A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. beta-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, alpha-lactalbumin could not be adsorbed onto the resin. alpha-Lactalbumin and beta-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. alpha-Lactalbumin and beta-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. alpha-Lactalbumin was eluted using a linear (0-0.15 M) concentration gradient of NaCl in 0.05 M Tris-HCl buffer (pH 8.5). Subsequently, beta-lactoglobulin B and beta-lactoglobulin A were eluted from the column with 0.05 M Tris-HCl (pH 6.8), using a linear (0.1-0.25 M) concentration gradient of NaCl. The yields were 1260 mg alpha-lactalbumin, 1290 mg beta-lactoglobulin B and 2280 mg beta-lactoglobulin A from 1 l rennet whey.     

Presence of adropin,nesfatin-1, apelin-12, ghrelins and salusins peptides in the milk,cheese whey and plasma of dairy cows

Biological fluids (milk and serum/plasma) and cheese whey milk-derived fluid contain numerous molecules, especially amino acids and proteins. Therefore, the purpose of this study was to find out whether cheese whey (n:6), cow milk (n:6) and its blood (n = 6) have adropin, nesfatin-1, apelin-12, ghrelins and salusin peptides. Adropin, nesfatin-1, apelin-12 concentrations were measured by ELISA, whereas ghrelin and salusin concentrations were measured by EIA methods. It was found that adropin, nesfatin-1, apelin-12, des-acylated ghrelin and salusins in cheese whey were higher than in the corresponding milk peptides and plasma of dairy cows, with the exception of salusin alpha and acylated ghrelin in milk being the same than that of the corresponding cheese whey concentration and plasma of dairy cows. A correlation was also found between milk peptides and cheese whey, as also with plasma of dairy cows. The data suggest that peptides in cow milk might be an important and nutritious food for (neonatal) calves and human diet due to their biological and physiological properties.     

Multi-origins of milk serum albumin in the lactating goat.

L-[U-14C]Leucine was infused into the right-hand mammary glands of lactating goats. Milk from both glands of the animals was sampled at intervals for 36 h. After 3 h the specific activity of milk serum albumin from the infused glands was more than six times that from the non-infused glands. The specific activity of milk serum albumin was considerably lower than that of alpha-lactalbumin or beta-lactoglobulin which are exclusively synthesized by mammary secretory cells. Following the intravenous injection of 125I-labeled serum albumin, maximum specific activity of this protein appeared in milk in 12 h. The specific activity of serum albumin in milk attained no more than 45% of the specific activity of the serum albumin in blood. It is concluded that milk serum albumin has multiple origins and that a portion of it, at least (10-20%), is made in the mammary gland.     

Identification of alpha-lactalbumin and beta-lactoglobulin in cynomolgus monkey (Macaca fascicularis) milk.

1. An electrophoretic analysis of whey protein from cynomolgus monkey milk revealed that its constituents are more similar to bovine milk than human milk, i.e. cynomolgus monkey milk whey contains, besides alpha-lactalbumin-like protein (LaP), another predominant component similar to bovine beta-lactoglobulin (LgP), in its electrophoretic behavior on both disc- and SDS-polyacrylamide gel electrophoreses. 2. The amino acid composition of LaP shows close similarity to that of human alpha-lactalbumin, and LaP forms an immunoprecipitin line with anti-human alpha-lactalbumin rabbit antiserum. The homology between LaP and alpha-lactalbumin was further confirmed by an analysis of the N-terminal amino acid sequence. 3. LgP is not immunologically identical to bovine beta-lactoglobulin, but its amino acid composition is similar. The result of the N-terminal amino acid sequence analysis of LgP (up to the 26th residue) strongly suggests homology between this protein and beta-lactoglobulin.     

Features of the milk whey protein partitioning in polyethyleneglycol-sodium citrate aqueous two-phase systems with the goal of isolating human alpha-1 antitrypsin expressed in bovine milk

Partitioning behaviour of the bovine whey proteins (bovine serum albumin, alpha-lactoalbumin and beta-lactoglobulin) and human alpha-1 antitrypsin in aqueous two-phase systems prepared with polyethyleneglycol (molecular masses: 1000, 1450 and 3350)-sodium citrate was analysed at pH 5.2, 6.2 and 8.2. Alpha lactoalbumin concentrated in the polyethyleneglycol rich-phase, while beta-lactoglobulin, bovine serum albumin and alpha-1 antitrypsin showed affinity for the citrate rich-phase. In aqueous two-phase systems of high medium pH and high polyethyleneglycol molecular mass the protein partitioning equilibrium is displaced to the citrate rich-phase. The polyethyleneglycol 1450-pH 5.2 system with a top/bottom phase-volume ratio of 3 showed to have the best capability of recovering the alpha-1 antitrypsin from a mixture prepared with natural milk whey and human alpha-1 antitrypsin. The recovery of this protein in the bottom phase was of 90% and the purity of the obtained product was of 98%. The method appears to be suitable as a starting point to isolate other human proteins expressed in transgenic bovine milk.     

Ghrelin and orotic acid increased in subclinical mastitis

Hormone ghrelin and orotic acid accelerate wound healing as well as controlling inflammation and immunity. We have, therefore, investigated the serum and milk levels of ghrelin and orotic acid in dairy cows with (n = 21) or without (n = 21) subclinical mastitis. Acylated and des-acylated ghrelin as well as orotic acid concentration were detected by using high performance liquid chromatography (HPLC). The results revealed that ghrelin level in milk and serum was significantly higher in dairy cows with subclinical mastitis than that of dairy cows without subclinical mastitis. This was also the case when the orotic acid concentrations in dairy cows with subclinical mastitis were compared with those dairy cows without subclinical mastitis. In conclusion, ghrelin and orotic acid occur in particularly high concentrations in subclinical mastitis, and might, therefore, be required in greater amounts for tissue repair and may be also used as a indicator for subclinical mastitis.     

Effects of soybean saponin on protease hydrolyses of beta-lactoglobulin and alpha-lactalbumin

Effects of Soybean Saponin on Protease Hydrolyses of beta-Lactoglobulin and alpha-Lactalbumin The effects of soybean saponin on tryptic and chymotryptic hydrolyses of whey proteins were evaluated. beta-lactoglobulin and alpha-lactalbumin became more sensitive to both trypsin and chymotrypsin by interacting with saponin in contrast to serum albumin. Soybean saponin was shown to have different effects on various proteins according to their nature.     

The growth of compact and diffuse variants of Staphylococcus aureus in bovine mastitic and normal whey

Strains of Staphylococcus aureus producing either diffuse or compact colonies in serum-soft agar were grown in bovine normal and mastitic whey. Bacterial growth was followed by automated turbidometry. Compact strains multiplied faster and to higher final numbers in mastitic whey than diffuse strains, whereas diffuse strains grew to higher numbers in normal whey. Nutrients (hemolysed bovine blood, bovine serum, proteose-peptone) were added to normal whey to enhance bacterial growth as in mastitic whey. The growth-promoting effect of these nutrients was dose-dependent for compact strains but not for diffuse strains. The difference in the growth characteristics of diffuse and compact strains in bovine whey explains some aspects of the pathogenesis of bovine mastitis.