Major Whey Proteins Research Articles

Whey Protein Soluble Aggregates from Heating with NaCl: Physicochemical, Interfacial, and Foaming Properties

Whey protein isolate was heat-treated at 85 degrees C for 15 min at pH ranging from 6.0 to 7.0 in the presence of NaCl in order to generate the highest possible amount of soluble aggregates before insolubility occurred. These whey protein soluble aggregates were characterized for composition, hydrodynamic diameter, apparent molecular weight, zeta-potential, surface hydrophobicity index, activated thiol group content, and microstructure. The adsorption kinetics and rheological properties (E', etad) of these soluble aggregates were probed at the air/water interface. In addition, the gas permeability of a single bubble stabilized by the whey protein soluble aggregates was determined. Finally, the foaming and foam-stabilizing properties of these aggregates were measured. The amount of whey protein soluble aggregates after heat treatment was increased from 75% to 95% from pH 6.0 to pH 7.0 by addition of 5 mM to 120 mM NaCl, respectively. These soluble aggregates involved major whey protein fractions and exhibited a maximum of activated thiol group content at pH > 6.6. The hydrodynamic radius and the surface hydrophobicity index of the soluble aggregates increased from pH 6.0 to 7.0, but the molecular weight and zeta-potential decreased. This loss of apparent density was clearly confirmed by microscopy as the soluble aggregates shifted from a spherical/compact structure at pH 6.0 to a more fibrillar/elongated structure at pH 7.0. Surface adsorption was faster for soluble aggregates formed at pH 6.8 and 7.0 in the presence of 100 and 120 mM NaCl, respectively. However, interfacial elasticity and viscosity measured at 0.01 Hz were similar from pH 6.0 to 7.0. Single bubble gas permeability significantly decreased for aggregates generated at pH > 6.6. Furthermore, these aggregates exhibited the highest foamability and foam liquid stability. Air bubble size within the foam was the lowest at pH 7.0. The coarsening exponent, alpha, fell within predicted values of 1/3 and 1/2, except for very dry foams where it was 1/5.

Beta-lactoglobulin polymorphism in Girgentana goat breed

Beta-lactoglobulin (β-lg) is a globular protein belonging to the lipocalin family. It is the major whey protein in the milk of ruminants. It is also present in the milk of most mammals but is lacking in rodents, lagomorphs and humans. A large number of variants have been reported for cow and sheep milk. Several studies have shown association between β-lg variants and milk production and composition, even if the results are not always concordant. In goat, no β-lg variants related with amino acid change have been characterized at DNA level, but some authors described the presence of polymorphisms in the 3’UTR and in the proximal promoter region. Mutations in the promoter region could be those most likely responsible for different level of gene expression. The aim of this work was to study the genetic polymorphism at DNA level of β-lg gene in Girgentana goat breed. A total of 238 genomic DNA samples of Girgentana breed were genotyped. A fragment of 709 bp, including 588 bp of proximal promoter region and 121 bp of exon 1, was amplified using primers GOAPF3 and GoatE1R2. PCR-RFLP procedure was used for fast detection of two single nucleotide substitutions as described by Graziano et al. (2003). The base substitutions originating the polymorphic sites consist of: 1. a transition T›C at position -341 and 2. a transition C›T at position -60. A FspBI PCR-RFLP protocol was used to detect the mutation -341 (T/C) and a SmaI PCR-RFLP protocol for the mutation -60 (C/T) of the proximal promoter region. The allelic frequencies and the Hardy-Weinberg equilibrium were estimated using the GENEPOP software. Girgentana goat breed shows no significant deviation from Hardy-Weinberg equilibrium for the allele frequencies found in both polymorphic sites considered. The genotypic frequencies for both mutations resulted in 0.65 (T/T), 0.33 (T/C) and 0.02 (C/C) for the position -341, and 0.82 (C/C), 0.17 (C/T) and 0.01 (T/T) for the position -60. These results are in agreement with the previous obtained by Graziano et al. (2003) in the same breed. Further analysis are in progress to investigate the possible effect of these variants on the expression of β-lg gene, on the milk protein composition and on milk production traits.The third author was supported by a Marie Curie Transfer ok Knowledge Grant of the Union European Community program ‘Quality of Life’, contract number MTKD/I-CT-2004-14412.

The influence of β-lactoglobulin genetic polymorphism on morphometric characteristics of milk fat globules and milk fatty acids composition in Italian Friesian cow

β-lactoglobulin (β-Lg) is the major whey protein in the milk of ruminants and some non-ruminants. It was first isolated 60 years ago and a wealth of physico-chemical and biological information has been accumulated. Results of studies on the effect of β-Lg genotypes on milk production traits (milk yield, protein and fat yield) and on milk coagulation parameters have been rather consistent. In this study we evaluated the relationship between genetic polymorphism of β-Lg morphometric characteristics of fat globules and fatty acids composition of cow milk. The trial was carried out on 89 unrelated pluriparous Friesian dairy cows reared in the same herd in a medium-temperate climatic zone in the province of Pisa. All animals were fed the same diet. Individual milk samples were collected and analyzed in a triplicate. One milk sample of milk was collected for each cow and no preservative was used. Genotypes were determined by isoelectric focusing analysis of single milk sample. Milk fatty acids composition and morphometric characteristics of fat globules were analysed on each milk sample in triplicate. The frequency distribution of total measured milk fat globules was evaluated according to their size. Fat globule diameters were divided into nine classes of 1.5 µm width: from class 1 (1.5-3 µm) to class 9 (>13.5 µm). Finally, fat globules were further classified into two groups: group S (1.5-6 µm), and group L (>6 µm). The analysis of variance was carried out to evaluate the effect of β-Lg genotype on all evaluated parameters with simultaneous adjustment for sampling season, parity, and days from calving as a covariate. As reported in literature, a higher gene frequency of the B variant of β-Lg was observed (0.59 vs 0.41). Significant effects of β-Lg genotype were observed for the percentage of the number of fat globules in the 8th (12-13.5 µm) and 9th class (>13.5 µm) which were higher in the AB genotype than in the other genotypes. The C20:4ω6 content was significantly higher in the AB genotype (P<0.05) and C15:1 content was significantly higher in the AA genotype (P<0.05) than in the other genotypes. Although β-Lg genotypes would seem to have limited relationships with the dimensions of fat globules of cows’ milk, the higher content of C20:4ω6 in AB genotype is of particular interest from the nutritional point of view.

Aberrant Low Expression Level of Bovine β-Lactoglobulin Is Associated with a C to A Transversion in the BLG Promoter Region

β-Lactoglobulin (β-LG) is the major whey protein in cow's milk. It is well established that the predominant 2 genetic variants, β-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 β-LG B variant, characterized by a very low β-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 β-LG B* site might be relevant in the natural production of milk of low β-LG content.

Investigation of the Lactosylation of Whey Proteins by Liquid Chromatography−Mass Spectrometry

Heat treatment of milk induces a reaction between the milk proteins and lactose, resulting in lactosylated protein species. The lactosylation of the two major whey proteins alpha-lactalbumin and beta-lactoglobulin was investigated by reversed phase liquid chromatography-mass spectrometry (LC-MS). Three sample series, consisting of aqueous model solutions of each whey protein separately and in mixture and whole milk, were heated for different time periods, and the progression of the lactosylation reaction was monitored. The observed degrees of lactosylation and the reaction kinetics showed that the lactosylation of beta-lactoglobulin was not influenced by the presence of other components, whereas the lactosylation of alpha-lactalbumin was enhanced in whole milk compared to the aqueous model systems. An in-depth evaluation of the LC-MS data yielded information regarding changes of physicochemical properties of the whey proteins upon lactosylation. Whereas retention time shifts indicated changes in hydrophobicity for both alpha-lactalbumin and beta-lactoglobulin, changes in the charge state distribution denoting conformational alterations were observed only for beta-lactoglobulin. The analysis of different liquid and solid milk products showed that the lactosylation patterns of the whey proteins can be used as indicators for the extent of heat treatment.

The challenge of cow milk protein allergy

Hypersensitivity to cow milk proteins is one of the main food allergies and affects mostly but not exclusively infants, while it may also persist through adulthood and can be very severe. Different clinical symptoms of milk allergy have been established. The diagnosis of milk allergy differs widely due to the multiplicity and degrees of symptoms, and can be achieved by skin or blood tests. Cow milk contains more than 20 proteins (allergens), that can cause allergic reactions. Casein fractions and β-lactoglobulin are the most common cow milk allergens. Human milk is free of β-lg, similar to camel milk. On the contrary, β-lg is a major whey protein in cow, buffalo, sheep, goat, mare and donkey milk. Caseins in milk of the different species differ in fraction number, amino acid composition, and their peptide mappings. β-Casein is the major fraction in goat casein, which is similar to human casein and different from cow casein. The peptide mappings of goat α-la and β-lg are completely different from those of cow milk. Different procedures can reduce the allergenicity of cow milk proteins by heat or enzymatic treatment to some degree. Allergies to milk proteins of non-bovine mammals have also been documented due to cross-reactivity between cow milk proteins and their counterpart in other species, and even between goat and sheep caseins. Genetic polymorphisms of milk proteins play an important role in eliciting different degrees of allergic reactions. Goat milk lacking α-s1-casein, which is the main casein in cow milk, is less allergenic than goat milk with α-s2-casein, which is more typical for many goat breeds. Several studies have reported real and dramatic benefits from using goat, camel, mare or even soy milk as alternatives in cases of cow milk allergy and they can be considered hypoallergenic. However, therapeutic benefits vary with the degree of severity of the allergy and may be only around 60% of all cases, since other studies revealed allergenicity to occur also for any of those other milks.

Ca2+-Induced Cold-Set Gelation of Whey Protein Isolate Fibrils

Abstract In this paper we describe the rheological behaviour of Ca2+-induced cold-set gels of whey protein mixtures. Cold- set gels are important applications for products with a low thermal stability. In previous work [1], we determined the state diagram for whey protein mixtures that were heated for 10 h at pH 2 at 80°C. Under these conditions, the major whey protein, β-lactoglobulin (β-lg), forms fibrils. When whey protein mixtures are heated at protein concentrations in the liquid solution regime of the state diagram, cold-set gels can be formed by adding Ca2+ ions at pH 7. We studied the rheological behaviour of cold-set gels for various sample compositions for whey protein mixtures. When keeping the total whey protein concentration constant, the elastic modulus, G’, for the cold-set gels decreased for increasing a-lactalbumin and bovine serum albumin ratios, because less material (β- lg fibrils) was available to form a gel network. In the cold-set gels the interactions between the β-lg fibrils induced by the calcium ions are dominant. The β-lg fibrils are forming the cold-set gel network and therefore determine the gel strength. a-Lactalbumin and bovine serum albumin are not incorporated in the stress-bearing structure of the gels.

Influence of pH in the recovery of lactoferrin from whey with ceramic membranes

A wide collection of proteins in relatively low concentrations but with an important biological activity is present in bovine whey. Among them, lactoferrin shows strong bacterial and antiviral properties, preventing the growth of pathogenic organisms in the gut, stimulating the immune system and enhancing iron absorption [1]. Lactoferrin recovery from whey is therefore of great interest since it can be used in the development of improved infant formula, functional foods and nutraceuticals. To this end, methods have been devised employing, for instance, surfactant-stabilized microbubbles [2] or ion exchage chromatography [3]. In the field of membrane technology, high performance tangential flow [4] filtration has been suggested as a feasible technique to allow the separation of lactoferrin at industrial scale [5]. In this work, the effect of pH in the selectivity of lactoferrin ultrafiltration through a ceramic membrane with respect to the major whey proteins has been studied. The results obtained are useful to design an optimal strategy to exploit the electrostatic protein–membrane interactions that takes place in this process. 2. Experimental

Changes in peptic digestibility of bovine β-lactoglobulin as a result of food processing studied by capillary electrophoresis and immunochemical methods

Digestion studies constitute a functional tool for allergen characterisation. This strategy for investigating allergenic proteins relates to the observation of increased proteolytic resistance of some proteins recognised to exhibit allergenic potential. beta-Lactoglobulin (betaLG) is one of the major whey proteins, a potent milk allergen and shows a high stability against peptic hydrolysis in its native form. In order to study the impact of milk fermentation process on its digestibility, two complementary analytical methods were applied: capillary zone electrophoresis (CZE) to quantitatively study proteolytic degradation of betaLG isolated from different fermented bovine milk products, and enzyme linked immunosorbent assay (ELISA) to assess differences in immunoreactivity. betaLG, isolated from either raw or pasteurised cow's milk (CM), as expected, showed only minimal digestibility (less than 10% in 2 h). However, when raw milk or pasteurised milk was fermented, the rate of peptic digestion of the protein significantly increased (up to 45% in 2 h). In accordance with changes in digestibility, the immunochemical response for all fermented samples was lower than that of non-fermented references. Raw and pasteurised milk "naturally" fermented in our laboratory only resulted in a slight reduction (betaLG detected is still in the range of milligrams per gram sample), whereas the industrially manufactured sour milk as well as the "Acidophilus milk" reflected a remarkably lower level of immunoreactivity (55-56 microg/g sample).

Fluorescence spectroscopy as a tool for monitoring solubility and aggregation behavior of β‐lactoglobulin after heat treatment

Denaturation and aggregation of whey proteins are of interest to the food and pharmaceutical industry due to the importance of final structure in functionality, impact on food texture, and the chemical stability of the final product. In this study, we demonstrate the potential of fluorescence spectrometry combined with multivariate chemometric methods for quantifying solubility and aggregation behavior of beta-lactoglobulin (beta-LG); a major whey protein and a frequent food ingredient. Heat-induced aggregation of beta-LG was studied under different conditions including pH, temperature and heating durations. Results showed very good agreement between the fluorescence-based predictions and measurements obtained by HPLC and gravimetric analysis regardless of the conditions. Standard normal variate (SNV), a signal preprocessing and filtering tool, was found to enhance the predictive accuracy and robustness of the fluorescence-based model.

Characterisation of whey proteins of camel ( Camelus dromedarius) milk and colostrum

Variation in the composition of whey proteins from camel ( Camelus dromedarius) colostrum and milk was recorded over a 192 h period following parturition. Whey proteins were separated by cation-exchange fast protein liquid chromatography and identified by polyacrylamide gel electrophoresis. The main components of whey proteins in camel milk and colostrum were similar to that in bovine, except for the lack in β-lactoglobulin. Serum albumin was the major whey protein present in camel milk, with an average concentration of 10.8 g/l. Camel colostrum was rich in immunoglobulins G, which consist of IgG1, and the enzyme inhibitory antibodies IgG2 and IgG3. The concentration of these proteins decreased rapidly 48 h post partum. Lactophorin (proteose peptone-component 3) and basic whey protein were detected only within 48 h after parturition, reaching a level of 4.9 and 3.1 g/l at 192 h post partum, respectively. The maximum level of lactoferrin (2.3 g/l) was observed at 48 h after parturition. Camel milk and colostrum were shown to be rich in protective proteins, especially IgG2 and IgG3, which revealed to be a potential source of inhibitory antibodies.

Screening for lactic acid bacteria with potential to reduce antigenic response of β-lactoglobulin in bovine skim milk and sweet whey

The whey protein β-lactoglobulin (β-lg) is in about 80% of all cases the main elicitor of milk allergies for children and infants. β-lg is the major whey protein in milk and milk products and it is of particular interest since it is the only whey protein of cow's milk not present in human milk. Lactic acid bacteria were screened for their ability to reduce the β-lg antigenity in skim milk and sweet whey. The antigenic response, measured as β-lg concentration equivalent was determined by means of an indirect competitive ELISA, using polyclonal antibodies from chicken egg yolk. Lactic acid fermentation was found to be suitable for decreasing milk immunoreactivity. In addition, synergism regarding the reduction of the antigenity were observed when using 1:1 mixtures of lactic acid bacteria with Streptococcus thermophilus subsp. salivarius strains. A 90% reduction of the initial β-lg antigenity of skim milk was observed for some of the lactic acid bacteria. In sweet whey more than 70% reduction was achieved. In general, lactic acid fermentation can attenuate β-lg antigenity in skim milk and sweet whey, but did not eliminate antigenity. Industrial relevance The research demonstrated that some lactic acid bacteria feature potential to reduce the antigenic response of β-lg in sweet whey and skim milk. The antigenity was quantified by an ELISA specific to some of the epitopes responsible for allergenic reaction according to the literature. Reduction of more than 70% in sweet whey and more than 90% in skim milk compared to the initial value was detected. Some of the strains studied reduced the β-lg antigenity significantly better in sweet whey, some in skim milk. The enzymes are more or less specific and some enzymes released from microorganisms are even better for reducing antigenity in milk and/or whey, as well as the different composition of milk and whey is of note. These results are promising for new fermented milk products with reduced antigenic properties. In addition, synergism regarding the reduction of antigenity was observed when using 1:1 mixtures of lactic acid bacteria with S. thermophilus subsp. salivarius strains. However, only the antigenity of β-lg was tested by means of an ELISA and not the allergenicity. The laboratory experiments demonstrate that fermented milk products or milk based formula may be prepared with reduced antigenity. Further research will be addressed to produce such products in pilot scale for clinical tests in order to test allergenicity or the allergy-reduced characteristic of the product.

A Novel Conformation-Dependent Monoclonal Antibody Specific to the Native Structure of β-Lactoglobulin and Its Application

Molten globules are thought to be general intermediates in protein folding and unfolding. β-lactoglobulin (β-LG) is one of the major bovine whey proteins, constituting ∼10 to 15% of total milk proteins. We have recently identified β-LG as a superior marker for evaluating thermally processed milk. Strand D of β-LG participates in irreversible thermal unfolding as probed by a monoclonal antibody (mAb) specific to thermally denatured β-LG. In the present study, we used native β-LG as an immunogen to test the hypothesis that a specific mAb against the native β-LG could be established. As result, a mAb (4H11E8) directed against the native structure of β-LG was made. The antibody did not recognize the heat-denatured form of β-LG, such as its dimer and aggregates. Immunoassay using this “native” mAb showed that the stability of β-LG was at temperatures ≤70°C. β-Lactoglobulin began to deteriorate between 70 and 80°C over time. The denaturation was correlated with the transition temperature of β-LG. Further chemical modification of Cys (carboxymethylation) or positively charged residues (acetylation) of β-LG totally abolished its immunoreactivity, confirming the conformation-dependent nature of this mAb. Using competitive ELISA, the 4H11E8 mAb could determine the native β-LG content in commercially processed milks. Concentrations of native β-LG varied significantly among the local brands tested. From a technological standpoint, the mAb prepared in this study is relevant to the design and operation of appropriate processes for thermal sanitation of milk and of other dairy products.

Binding of the Pepper Alkaloid Piperine to Bovine β-Lactoglobulin: Circular Dichroism Spectroscopy and Molecular Modeling Study

The pepper alkaloid piperine is a nontoxic, natural dietary compound with a broad range of physiological activity. The present work is the first demonstration of its interaction with a mammalian protein. Circular dichroism (CD) spectroscopy was used to reveal and analyze the binding of piperine to a lipocalin protein. Induced CD spectra measured in pH 7.7 phosphate buffer at 37 degrees C demonstrated reversible, non-covalent association of piperine with bovine beta-lactoglobulin (BLG), the major whey protein in milk. The binding parameters (K(a) approximately 8 x 10(4) M(-1), n = 0.8) determined from the CD titration data showed no significant differences between the piperine binding properties of the two main genetic variants of BLG (A and B). The vanishing extrinsic CD signal obtained upon acidification of the piperine-BLG sample solution (Tanford transition) suggested that the ligand binds in the central hydrophobic cavity of the beta-barrel. The cavity binding concept was further supported by a CD displacement experiment using palmitic acid, the well-known hydrophobic ligand of BLG. Molecular docking calculations showed that piperine can be efficiently accommodated within the calyx of BLG. Additional molecular modeling calculations indicated that the beta-barrel of human tear lipocalin, human serum retinol binding protein, and human neutrophil gelatinase associated lipocalin might also accommodate a piperine molecule.

Pressure-Induced Unfolding and Aggregation of the Proteins in Whey Protein Concentrate Solutions

Whey protein concentrate solutions (12% w/v, pH 6.65 +/- 0.05) were pressure treated at 800 MPa for 20-120 min and then examined using size exclusion chromatography (SEC), small deformation rheology, transmission electron microscopy, and various types of one-dimensional (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE). The pressure-treated samples showed a time-dependent loss of native whey proteins by SEC and 1D PAGE and a corresponding increase in non-native proteins and protein aggregates of different sizes. These aggregates altered the viscosity and opacity of the samples and were shown to be cross-linked by intermolecular disulfide bonds and by noncovalent interactions using 1D PAGE [alkaline (or native), sodium dodecyl sulfate (SDS), and SDS of reduced samples (SDS(R))] and 2D PAGE (native:SDS and SDS:SDS(R)). The sensitivity of the major whey proteins to pressure was in the order beta-lactoglobulin B (beta-LG B) > beta-LG A > bovine serum albumin (BSA) > alpha-lactalbumin (alpha-LA), and the large internal hydrophobic cavity of beta-LG may have been partially responsible for its sensitivity to high-pressure treatments. It seemed likely that, at 800 MPa, the formation of a beta-LG disulfide-bonded network preceded the formation of disulfide bonds between alpha-LA or BSA and beta-LG to form multiprotein aggregates, possibly because the disulfide bonds of alpha-LA and BSA are less exposed than those of beta-LG either during or after pressure treatment. It may be possible that intermolecular disulfide bond formation occurred at high pressure and that hydrophobic association became important after the high-pressure treatment.

Immunochemical quantification of heat denaturation of camel (Camelus dromedarius) whey proteins

The major whey proteins IgG, serum albumin and alpha-lactalbumin were purified from camel milk using gel permeation and ion-exchange chromatography. Specific antisera against each of them were raised and used to quantify their heat denaturation in early or mature milk over a range of 60-90 degrees C for 10-60 min using the single radial immunodiffusion technique. The heat denaturation midpoints for the mature milk heated 30 min were 67.2, 73.0 and 77.5 degrees C for IgG, albumin and alpha-lactalbumin respectively. The early milk was more sensitive to heat treatment with coagulation at low temperature and heat denaturation midpoints of 64.8, 71.6 and 72.6 degrees C respectively. This difference was related to the high IgG content of the early milk (12.6 mg/ml v. 0.5 mg/ml for the mature milk) and stresses the importance of monitoring the IgG level of milk to assess the absence of colostrum.

Precipitation behaviour of caseinomacropeptides and their simultaneous determination with whey proteins by RP-HPLC

Abstract A reversed-phase high-performance liquid chromatographic (RP-HPLC) method on polyvinylbenzene/divinylbenzene copolymer resin was applied for the quantitative determination of glycosylated and non-glycosylated caseinomacropeptides (CMP), in presence of the major whey proteins ( α -lactalbumin and β -lactoglobulin). The peak profile of the CMP-fractions was calibrated using the response factor determined by amino acid analysis. By omitting any critical precipitation step (e.g. trichloroacetic acid, TCA) usually applied to remove whey proteins, the method allows the simultaneous determination of the major whey proteins and CMP—the complete native spectrum—in a single run. As a prerequisite for forthcoming kinetic studies of CMP release, the precipitation sensitivity of individual whey proteins or CMP fractions against TCA or perchloric acid (PCA) was studied systematically. All CMP fractions were partially precipitated even at low TCA concentrations, whereas PCA treatment resulted in negligible decrease of CMP solubility.

Genetic polymorphism of alpha-lactalbumin gene in riverine buffalo

Alpha-lactalbumin (alpha-LA) is a major whey protein found in milk. Polymorphs of alpha-LA gene are reported to be significantly associated with milk production and constituent traits. Therefore, the present study was undertaken to detect polymorphism in alpha-LA at the genic level and to explore allelic variability at this locus. A total of 196 animals, belonging to four breeds of riverine buffalo viz. Bhadwari, Mehsana, Surti and Murrah were included under the present investigation. Two fragments i.e. 133 bp (Exon 1) and 159 bp (Exon 2) of alpha-LA gene were amplified by polymerase chain reaction and subsequently, single strand confirmation polymorphism (SSCP) study was carried out to identify different allelic pattern and genotypes of the animal included in the study. Both fragment of alpha-LA gene was found to be polymorphic in all the four breeds of riverine buffalo. Number of genotypes and allele varied breed to breed for both the fragments. In case of 133 bp fragment, four alleles A, B, C and D were found among different breeds of buffalo whereas in 159 bp fragment, five alleles namely A, B, C, D and E was found in different breeds. Nucleotide sequence data of different alleles showed the presence of both silent as well as functional mutation leading to variability in polypeptide chain.

Development of a method for quantitative analysis of the major whey proteins by capillary electrophoresis with on‐capillary derivatization and laser‐induced fluorescence detection

The main whey proteins have been derivatized on-capillary with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and analyzed using a laboratory-made capillary electrophoresis apparatus provided with a laser-induced fluorescence detector. Several parameters controlling on-capillary derivatization of proteins, including pH, mixing time, reaction time, concentration of the reagents (potassium cyanide and FQ), and reaction temperature, were optimized. Coefficient variations were lower than 1% for migration time and 7% for peak height. Assay detection limits for the different proteins were in the range 5 nM to 10 nM. The method developed was applied to the separation of the major whey proteins in a laboratory-made cheese whey and in an infant food formulated with milk. In addition, the beta-LG content of these samples was quantitated. The results showed good agreement with those given by an RP-HPLC method and with those reported in the literature.

Selective separation of β‐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.