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PubMed2004

Effect of genetic variation on the tryptic hydrolysis of bovine beta-lactoglobulin A, B, and C.

The structure, stability, and hydrolysis characteristics of beta-lactoglobulin (LG) A are different from those of either beta-LG B or beta-LG C. Purified samples of these proteins were hydrolyzed with trypsin and the rates of loss of native monomeric beta-LG structure were measured using sodium dodecyl sulfate PAGE. At the same time, the appearance of many individual peptides were identified and followed in time by HPLC, measuring their concentration as a function of solution pH, temperature, protein concentration, and added urea or palmitate. The identity of the peptides was confirmed by liquid chromatography-mass spectrometry. This semiquantitative exploration showed that the rate of hydrolysis was in the order beta-LG A > beta-LG B > beta-LG C under most circumstances, and that 12 of the 18 trypsin-susceptible bonds were cleaved at very similar rates that were governed by the variant type. Consequently, the rate of hydrolysis of the intact protein was related to the overall structural stability of the individual proteins and the accessibility of certain peptide bonds to the enzyme. The hydrolysis of mixtures of 2 or more variants or of denatured beta-LG gave more heterogeneous peptide mixtures.

Animals↗
PubMed2005

Effect of kappa-casein and beta-lactoglobulin loci on milk production traits and reproductive performance of Holstein cows.

The effects of kappa-casein (kappa-CN) and beta-lactoglobulin (beta-LG) loci on milk production traits (milk, fat, protein, and lactose yield, fat, protein, and lactose content) and reproductive performance (gestation length, calving interval, age at first and second calving, number of services per conception) was estimated for 278 Holstein cows in the first 2 lactations. Genotypes of kappa-CN and beta-LG were determined by alkaline and acidic polyacrylamide gel electrophoresis. Milk production was recorded daily. Single-trait, mixed, linear models were used for the statistical analysis of the data. Results indicated that kappa-CN genotypes affected significantly protein yield and content (genotype AB > genotype AA). A tendency for increased milk and fat yield of animals having AB kappa-CN genotype was also found. Fat content and lactose yield and content were not affected. In the beta-LG system, significant differences were detected for milk yield (AB > AA), fat yield (BB and AB > AA), fat content (BB > AA and AB), and lactose yield (AB > AA). A tendency for higher protein yield was also observed (AB > AA). The beta-LG locus had no significant effect on protein and lactose content. No associations between polymorphisms at the kappa-CN locus and reproductive performance were found. There was a tendency, however, for cows with AB genotype to have older age at first and second calving. In the beta-LG system, cows with AA genotype had significantly shorter gestation length than did those with AB or BB genotype. No differences were detected between beta-LG polymorphisms for the other reproductive traits.

Animals↗
PubMed2005

Beta-lactoglobulin is a thermal marker in processed milk as studied by electrophoresis and circular dichroic spectra.

As much of the sterilization process involves heat treatment during the preparation of milk on an industrial scale, the unpredictable measures of the process are an essential issue in determining the quality of the milk. The purpose of the present study was to investigate the major protein change(s) of whey proteins in processed milk and extend the knowledge for future reference in the dairy industry. Using a native polyacrylamide gel electrophoresis, we showed almost a 90% loss and denaturation of beta-lactoglobulin (LG), but not alpha-lactalbumin (LA), in some brands of the processed and dry milks. Immunochemical analysis using Western blotting revealed that part of the loss was attributed to the formation of large multiple forms of LG in the processed product. Such denaturation was presumably associated with the heating procedure used in the process. Essentially, LG was the only major fraction converted to aggregates in milk heated at 95 degrees C for 30 min on 2-dimensional PAGE. The detailed thermal denaturation of purified LG and LA at various temperatures (50 to 95 degrees C) and time (5 to 960 s) were investigated using a circular dichroic analysis. The maximal changes of ellipticity at 205 nm (converting beta-structure to disordered structure) were correlated to heating temperature and time. There were no significant conformational changes of LG at temperatures below 70 degrees C for as long as 480 s. Pronounced and rapid changes occurred between 80 to 95 degrees C in a time-dependent manner. Fifty percent of the maximal changes could be reached within 15 s. In conclusion, the unique chemical and immunochemical loss and conformational changes made LG a superior marker for evaluating the thermal processing of milk. The detailed thermal denaturation curves of LG constructed with its time and temperature in this study provide a valuable reference for the dairy industry. We postulate that heat treatment over 80 degrees C in 15 s may induce a significant denaturation of milk LG.

Amino Acid Sequence↗
PubMed2006

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, alpha-lactalbumin (alphaLA) and beta-lactoglobulin (alpha-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 degrees 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 alpha-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 alpha-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 beta-LG.

Amino Acid Sequence↗
PubMed2006

Aberrant low expression level of bovine beta-lactoglobulin is associated with a C to A transversion in the BLG promoter region.

Beta-lactoglobulin (beta-LG) is the major whey protein in cow's milk. It is well established that the predominant 2 genetic variants, beta-LG A and B, are differentially expressed. Extensive investigation of the genetic variation in the promoter region of the BLG gene revealed the existence of specific haplotypes associated with the A and B variants, respectively. However, the genetic basis for the differential expression of BLG A and B alleles is still elusive. We have previously reported a quantitative beta-LG B variant, characterized by a very low beta-LG protein expression level. Here, we report that the corresponding BLG allele (BLG B*) shows a correspondingly low mRNA expression level. Comparative DNA sequencing of 7,670 bp of the BLG B* allele and the established BLG B allele revealed a unique difference of a C to A transversion at position 215 bp upstream of the translation initiation site (g.-215C>A). This mutation segregated perfectly with the differential phenotypic expression in a paternal half-sib family and could be confirmed in 2 independent cases. The sequence of the BLG B allele in the region of the mutation is highly conserved among 4 related ruminant species. The site of the mutation corresponds to a putative consensus-binding sequence for the transcription factors c-Rel and Elk-1 as predicted by searching the TRANSFAC database. The beta-LG B* site might be relevant in the natural production of milk of low beta-LG content.

Alleles↗
PubMed1996

Purification of beta-lactoglobulin from whey protein concentrate by pepsin treatment.

beta-Lactoglobulin was purified from whey protein concentrate by a combination of pepsin treatment and membrane filtration. Porcine pepsin was added to whey protein (1:200, wt/wt), and the mixture was then incubated at pH 2.0 and 37 degrees C for 60 min. The protein fraction was collected by ammonium sulfate precipitation, and the precipitate was either dialyzed against water using a dialysis membrane (20-kDa pore size) or filtered using an UF membrane (30-kDa pore size). The beta-LG did not differ from standard beta-LG as measured by chromatography, SDS-PAGE, native PAGE, differential scanning calorimetry, or UV spectrum. Based on the results, a simplified procedure was developed, consisting of pepsin treatment and UF, to purify beta-LG directly from whey.

Ammonium Sulfate↗
PubMed1997

Binding of retinoids to beta-lactoglobulin isolated by bioselective adsorption.

Binding of the retinoids, all-trans-retinol, all-trans-retinal, all-trans-retinyl acetate, and all-trans-retinoic acid, to beta-lactoglobulin (LG) (96% purity) that had been prepared by bioselective adsorption on N-retinyl-Celite was determined from changes in the fluorescence quenching (332 nm) of the protein tryptophanyl residues. High affinity binding of all of these compounds occurred at pH 7.0, and the apparent dissociation constant ranged from 1.7 to 3.6 x 10(-8) M. Furthermore, a stoichiometry of 1.0 mol.mol-1 of protein was obtained for each case, indicating that all of the sites in the protein preparation were available. When beta-LG in whey protein isolate (57.4% beta-LG) was studied, a stoichiometry of 0.65 to 0.82 mol.mol-1 of protein was obtained, indicating that a large number of the sites already had bound lipid or that the protein had been denatured. As the pH was lowered toward 5.15, the affinity decreased about fourfold, but the stoichiometry of binding was unchanged. Far UV circular dichroism spectra indicated that the secondary structure of the protein was not significantly affected by ligand binding; however, the near UV spectra were changed, indicating that the flexibility of tryptophanyl residues decreased. The latter effect is consistent with the change in fluorescence quenching and suggests that a tryptophan is in the binding site.

Adsorption↗
PubMed1997

Binding of vitamin D and cholesterol to beta-lactoglobulin.

beta-Lactoglobulin was isolated directly from acidic whey by bioselective adsorption on N-retinyl-Celite, yielding preparations of > or = 96% purity. Interactions of these preparations with vitamin D2, vitamin D3, ergosterol, cholesterol, and 7-dehydrocholesterol were examined by following changes in the fluorescence spectra. Both the excitation and emission spectra indicated that energy was transferred between the tryptophanyl residues of the protein and the chromophore of the ligand. Analyses of the fluorescence changes that occurred upon titration of beta-LG with the various ligands allowed determination of the dissociation constant for the complex and the number of moles bound per mole of protein. The affinity for vitamin D2 (dissociation constant of 4.91 nM) was 10-fold higher than that of the other compounds, except for ergosterol, which was 5-fold larger than the others. Also, the affinity was 10-fold higher than that typically reported for the retinoids. Furthermore, the value obtained for the number of moles bound per mole of protein was 2 mol.mol-1 for each of the ligands examined in this study; it has been well established that all of the retinoids are bound with a stoichiometry of 1.0. These results suggest that beta-LG may be a better carrier of vitamin D than of vitamin A.

Adsorption↗
PubMed1997

Effect of atropine on milk protein yield by dairy cows with different beta-lactoglobulin phenotypes.

The objective of this study was to determine the response of individual milk proteins to a reduction in amino acid (AA) availability induced by atropine and to determine whether the response was different in cows with different beta-lactoglobulin (LG) phenotypes. Six cows that were homozygous for the A variant of beta-LG and six cows that were homozygous for the B variant of beta-LG were each given a single subcutaneous injection of saline or 20 mg of atropine. In both groups of cows, atropine decreased milk yield by 30% and reduced the concentration of alpha-lactalbumin (LA) by 25 to 30% at 8 h following injection. Eight hours after atropine injection, yield of beta-LG was 41% lower than it was following saline injection, and yield of beta-casein (CN) after atropine injection declined 16% relative to saline. Concentrations of BSA and the ratio of gamma-CN to beta-CN, which reflects plasmin activity in milk, were significantly increased after administration of atropine. Although the response to atropine tended to be more pronounced in cows that were homozygous for beta-LG B, they were not significantly different from the response of cows that were homozygous for beta-LG B, they were not significantly different from the response of cows that were homozygous for beta-LG A. The differential response of individual proteins to a reduction in AA concentrations in whole blood suggested that susceptibility to restriction in substrate availability differed for individual proteins. The concentration of lactose in plasma did not change, which implied that the integrity of the mammary epithelial barrier was not compromised when AA derived from blood were diminished. The consistent concentration of lactose combined with the minimal increase in total yield of BSA in milk following atropine treatment indicated that the increased concentration in milk of proteins derived from serum was due to the concentrating effect of lower milk volume.

Animals↗
PubMed1997

Polymorphisms of bovine beta-lactoglobulin promoter and differences in the binding affinity of activator protein-2 transcription factor.

Differential production of the two most common allelic variants of beta-lactoglobulin (LG), beta-LG A and beta-LG B, has been observed using PAGE. This study evaluated 733 bp of the beta-LG promoter region and 92 bp of the first exon for possible polymorphisms using denaturing gradient gel electrophoresis and nucleotide sequence analysis. Within this region, 13 single nucleotide substitution polymorphisms were detected. Twelve polymorphisms were allele specific, and one appeared to be polymorphic only for the B allele. Several potential binding sites for transcription factors were found within the promoter sequence. This study investigated the role of the G to C transversion within a consensus binding site for activator protein-2 at position-430 bp upstream from the transcription initiation site. Using the DNase-I footprint assay, we confirmed the functional importance of this point mutation and showed different binding affinities of activator protein-2 for both alleles. We discuss the possible regulatory role of activator protein-2 in the transcriptional regulation of the beta-LG gene and propose the activator protein-2 transcription factor as a modulator of gene expression of beta-LG.

Alleles↗
PubMed1997

Production and epitopic characterization of monoclonal antibodies against bovine beta-lactoglobulin.

Sixty-nine murine monoclonal antibodies were produced against the bovine beta-lactoglobulin (beta-LG) variant B. Thirty-eight specificity groups of monoclonal antibodies were defined according to the reactivity in indirect or capture ELISA with bovine beta-LG B, for bovine beta-LG B coated at pH ranging from 2.3 to 9.4, and for beta-LG with naturally occurring residue substitutions such as bovine beta-LG variant A and ovine, caprine, porcine, and equine beta-LG. Ten monoclonal antibodies appeared to be monospecific for bovine beta-LG, 58 monoclonal antibodies recognized only ruminants beta-LG, and 1 recognized all species. The specificity of the monoclonal antibodies was also studied with sequenced tryptic peptides of beta-LG variant B. Critical residues involved in the epitopes of nine classes of monoclonal antibodies were characterized. Moreover, 5 monoclonal antibodies were able to discriminate between a fresh solution of beta-LG and a solution that had been stored for > or = 2 d at 4 degrees C, and 3 monoclonal antibodies distinguished bovine beta-LG that has been purified by gel filtration from that purified by salt fractionation. This array of monoclonal antibodies could be efficient probes for characterizing conformational structures involved in biological properties of beta-LG (e.g., interaction with ligands and hypersensitivity reactions) and for studying conformational changes occurring upon physical treatments such as heat denaturation.

Amino Acid Sequence↗
PubMed1997

Characterization by ionization mass spectrometry of lactosyl beta-lactoglobulin conjugates formed during heat treatment of milk and whey and identification of one lactose-binding site.

The extent of the early stage of the Maillard-type reaction that impaired functional properties of whey proteins was evaluated by electrospray ionization mass spectrometry. Under conditions of mild heat treatment (63 degrees C for 20 s) applied to milk before whey separation at room temperature 23 degrees C), a modification of the relative molecular mass of beta-lactoglobulin (beta-LG) was observed that differed from that of the native form by 324. This specific modification of beta-LG occurred in acidified whey as well as in sweet whey and increased with the extent of the heat treatment. Incubation of purified beta-LG dissolved in milk ultrafiltration permeate or in lactose solution at 50 to 80 degrees C demonstrated the presence of a lactosyl residue that was covalently bound to beta-LG; beta-casein, used as a control, showed no mass modification. Studies of kinetics showed that a maximum of 35% of the beta-LG was lactosyl-beta-LG conjugate after heat treatment at 70 degrees C for 1 h. This study provides the first direct evidence of specific lactosylation of beta-LG during the initial stage of the Maillard reaction. One of the first lactose-binding sites was identified as a Lys47 by protease mapping and analysis by means of on-line liquid chromatography combined with mass spectrometry. In addition, collision-activated dissociation performed on the lactosylated peptide beta-LG (f 46-51) showed the rearrangement reactions occurring during the fragmentation process by electrospray. A mechanism is proposed.

Animals↗
PubMed2003

Further evidence regarding the effect of dietary protein on oral tolerance against beta-lactoglobulin through Th1-mediated immune response in mice.

Oral tolerance is a potential strategy for preventing or minimizing aberrant immune responses. Although, oral tolerance has been extensively studied, to date the effects of dietary protein on the induction of oral tolerance are poorly understood. We have previously shown that restricted dietary protein induces oral tolerance to ovalbumin. This study was designed to investigate whether or not such tolerance occurs with beta-lactoglobulin (BLG) instead of ovalbumin (OVA) and if the tolerance resulting from this feeding regimen involves Th1-mediated immune response. Female BALB/c mice fed either 20% or 5% dietary protein were given 5 mg BLG or water orally for four consecutive days and then immunized intraperitoneally (ip) twice with BLG at 3-wk intervals. Oral tolerance induction was compared in BLG-fed and water-fed mice by measuring total IgE, BLG-specific antibodies, footpad reactions, splenocyte proliferation, and cytokine production. When mice were given BLG orally before ip immunization, the Th1-mediated immune responses (production of IL-2, IFN-gamma, and IgG2a) were significantly reduced, whereas the Th2-mediated immune responses (production of IL-4 and IgG1) were unchanged. The Th1-mediated immune responses were markedly down-regulated in mice fed 5% protein as compared to those in mice fed 20% protein. Moreover, the production of total IgE, BLG-specific IgE, splenocyte proliferation, and footpad reactions were more reduced in mice fed 5% protein than those in mice fed 20% protein. The present study provides evidence that dietary protein plays an important role in the induction of oral tolerance against BLG as the result of, clear down-regulation of Th1 helper activity accompanied by a reduction in IgE.

Administration, Oral↗
PubMed1975

RAST studies : IgE antibodies to Dermatogoides pteronyssinus (house dust mite), Aspergillus fumigatus and beta-lactoglobulin in sudden death in infancy syndrome (SDIS).

The incidence of 2.5 SDIS cases per 1,000 live births found in Western Australia is in agreement with figures reported for other centres. While the age range of SDIS victims extended from two weeks to 15 months, 57 per cent of deaths occurred in children of two to four months of age. Boys outnumbered girls 1.6:1. Environmental factors are implicated in that the majority of deaths occurred in a biphasic distribution - autumn and late winter months. No significant differences were observed in total IgE levels in serum from SDIS victims, post mortem children who died in trauma of known aetiology and live control children of the same age range. Serum IgE antibodies to D.pteronyssinus were found in 37% of SDIS victims compared with 7% of matched controls (post mortem plus live groups). IgE antibodies to beta-lactoglobulin, the major allergen of cow's milk, appeared with twice the frequency in SDIS vs. control group but both groups showed a similar incidence of antibodies to the allergens of Aspergillus fumigatus. The prevalence of IgE antibodies to D.pteronyssinus in SDIS victims who died in the late winter -- early spring period was double that found in the group who died in the autumn period. Sixtyfour percent of the SDIS victims had antibodies to two or more of the three allergens tested while the control sera were positive to only one allergen. These results support the hypothesis that anaphylaxis induced by immediate hypersensitivity to D.pteronyssinus in particular may be one of the causative factors in SDIS in Western Australia.

Allergens↗
PubMedDate not supplied

[Thermodynamic functions of lysozyme, myoglobin, beta-lactoglobulin, albumin, and poly-L-tryptophane in the temperature range 0-330 K].

The temperature dependence of heat capacity for lysozyme, myoglobin, beta-lactoglobulin, albumin, and poly-L-tryptophan was studied in the temperature range 5-330 K using an adiabatic vacuum calorimeter. The accuracy of measurements was approximately 0.2%. From the data obtained, the thermodynamic functions of these proteins and the polyamino acid Cp degree (T), H degree (T)-H degree (0), S degree (T)-S degree (0), G degree (T)-H degree (0) in the temperature range 0-330 K, and the values of fractal dimension D and intrinsic temperature theta max were calculated.

Animals↗
PubMed2002

[Oral tolerance induced by poly (lactide-co-glycolide) containing B lactoglobulin].

Allergies to milk proteins are frequently encountered in the new born population. In order to prevent this allergy by inducing oral tolerance, one of the major allergenic milk protein, B lactoglobulin (BLG) was entrapped into biodegradable Poly (lactide-co-glycolide) microspheres and was then orally given to mice. Microspheres are able to protect proteins against degradation by intestinal proteolytic enzymes and to target the Peyers patches which are one important priming site of the mucosal immune system. Microspheres were prepared by the multiple emulsion solvent evaporation method. The goal of the formulation study was to associate large amounts of proteins to the smallest amount of polymer so that a minimal quantity of microspheres would be administered. It was shown that introducing tween 20 in the formulation was able to increase the encapsulation efficiency and to better control protein release reducing the burst release effect. Moreover, Oral administration of microspheres containing BLG reduced significantly (by 10.000) the amount of protein necessary to decrease both specific anti BLG IgE and DTH response. In conclusion, microspheres appear to be optimal systems to induce oral tolerance.

Drug Carriers↗
PubMed2002

[Prevention of an IgE response to bovine beta-lactoglobulin by gene immunization in mice].

The therapeutic routes for the treatment of allergy have as their objective to prevent or diminish the specific IgE responsible for the appearance of the allergic reaction. This allergic reaction survives in the genetically predisposed subject and results in a dis-equilibrium of the "Th1-Th2 balance" by increasing the Th2 response. This Th2 response induces the production of IgE against environmental antigens, which from that time on become allergens. In this context, use of gene immunisation seems to be very interesting. The immunisation consists of the injection of an expression vector of bacterial origin, containing the cDNA of a protein of interest. Different studies have shown that the injection of such a plasmid into mice initiates a specific immune response of Th1 (IgG2a) type, stopping all further response of the Th2 (IgG1 and IgE) type. This "non-allergic" response is due to the intrinsic properties of the bacterial ADN, notably the presence of sequences of immunostimulants, the adjuvant of the Th1 response. We have sought to show such a preventative effect in the case of a food protein, bovine beta-lactoglobulin (BLG), a major allergen of cow's milk. Firstly, we made a expression plasmid that contained the cDNA of BLG. Intramuscular administration of this plasmid to mice allowed expression of the BLG in the native form at the site of the injection. The primary response induced by this type of immunisation is characterised by a mixed IgG1/IgG2a response and an absence of anti-BLG IgE. In addition, pre-immunisation of the mice with a plasmid that contained the cDNA of BLG prevented all further sensitisation with the protein administered by the intra-peritoneal route in the presence of alum, an adjuvant of the Th2 response. It appeared further that the preventative effect is dependent on the latent time between gene and protein immunisation. Prevention of the anti-BLG IgE response seems to result in an active inhibition by the cytokines such as interferon-gamma and interleukin 10, rather than a reduction in the production of type Th2 cytokines. Use of efficacious non-pathogenic vectors, that may be administered by the digestive route, could envisage a specific protection in the case of severe food allergies.

Allergens↗
PubMed1991

An aqueous hydroxypropylstarch-poly(ethylene glycol) two-phase system for extraction of alpha-lactalbumin and beta-lactoglobulin from bovine whey.

The partitioning of alpha-lactalbumin and beta-lactoglobulin from bovine whey has been studied in an aqueous poly(ethylene glycol) (PEG)-hydroxypropylstarch two-phase system. The influence of several parameters including concentrations of polymers, sodium phosphate buffer, KSCN, and of PEG palmitate, with and without the presence of Ca2+, on the partitioning of the proteins has been investigated. The separation of the two proteins was demonstrated by counter-current distribution. A purification procedure for both proteins has been developed by using PEG-hydroxypropylstarch two-phase system. This system is compared with the more costly standard system based on PEG and dextran. The possible use of the aqueous two-phase systems for batch extraction for large scale purification of these whey proteins is discussed.

Animals↗