Major Whey Proteins Research Articles

Low-allergenic hydrolysates of whey proteins with natural bioactive peptides

Hydrolysate of whey protein concentrate (WPC) has been obtained under conditions that ensure retention of natural bioactive peptides. Prior to this, the WPC was characterised by electrophoresis, which revealed the presence of major whey proteins that can cause allergies and be precursors of bioactive peptides. The electrophoretic studies have allowed establishing that by the 120th minute, the proteolysis of the main protein allergens was almost complete. That is why this sample of WPC hydrolysate was used for further studies. Sephadex G-50 gel filtration has shown that 23.4% to 27.5% of proteolytic products soluble in trichloroacetic acid are low-molecular-weight peptides with a molecular weight up to 1500 Da, while the control WPC sample contains less than 3 % of them. The hydrolysate obtained under physiological conditions was tested for allergenicity. The study was conducted in 18 rats divided into three groups. Animals of the first group (control) were given water, the second group whey protein concentrate, the third group pancreatin hydrolysate of whey proteins. According to the results of the experiment, the concentration of IgE in the 2nd group is significantly higher compared with the control (49%), and in the 3rd group, does not differ from the control values. To detect possible sensitisation in the experimental animals, we used the specific leucocyte agglomeration reaction, the leucocyte specific lysis reaction, the values of the change in the concentration of circulating immune complexes, and the neutrophil damage index. The studies have shown that in the animals receiving WPC hydrolysate, no signs of an allergic reaction were detected, while the animals sensitised with WPC developed type I hypersensitivity (by the value of the IgE content).

Low‐Temperature and Water‐Based Biotemplating of Nanostructured Foam‐Like Titania Films Using ß‐Lactoglobulin

AbstractGiven the broad use of nanostructured crystalline titania films, an environmentally friendly and more sustainable synthesis route is highly desirable. Here, a water‐based, low‐temperature route is presented to synthesize nanostructured foam‐like crystalline titania films. A pearl necklace‐like nanostructure is introduced as tailored titania morphology via biotemplating with the use of the major bovine whey protein ß‐lactoglobulin (ß‐lg). It is shown that titania crystallization in a brookite‐anatase mixed phase is promoted via spray deposition at a comparatively low temperature of 120 °C. The obtained crystallites have an average grain size of (4.2 ± 0.3) nm. In situ grazing incidence small‐angle and wide‐angle X‐ray scattering (GISAXS/GIWAXS) are simultaneously performed to understand the kinetics of film formation and the templating role of ß‐lg during spray coating. In the ß‐lg:titania biohybrid composites, the crystal growth in semicrystalline titania clusters is sterically directed by the condensing ß‐lg biomatrix. Due to using spray coating, the green chemistry approach to titania‐based functional films can be scaled up on a large scale, which can potentially be used in photocatalytic processes or systems related to energy application.

Dietary Linolenic Acid Increases Sensitizing and Eliciting Capacities of Cow's Milk Whey Proteins in BALB/c Mice.

α-Lactalbumin (BLA) and β-lactoglobulin (BLG) are the major whey proteins causing allergic reactions. Polyunsaturated fatty acids (PUFAs) stand among the extrinsic factors of the food matrix that can bind BLA and BLG and change their bioactivities, but their contribution to change the allergenic properties of these proteins has not been investigated. Here, we aimed to determine how PUFAs influence BLA and BLG to sensitize and trigger allergic responses in BALB/c mice. First, tricine–SDS–PAGE and spectroscopic assays identified that α-linolenic acid (ALA, as a proof-of-concept model) can induce BLA and BLG to form cross-linked complexes and substantially modify their conformation. Then, BALB/c mice (n = 10/group) were orally sensitized and challenged with BLA and BLG or ALA-interacted BLA and BLG, respectively. Allergic reactions upon oral challenge were determined by measuring clinical allergic signs, specific antibodies, levels of type-1/2 cytokines, the status of mast cell activation, and percentage of cell populations (B and T cells) in different tissues (PP, MLN, and spleen). Overall, systemic allergic reaction was promoted in mice gavage with ALA-interacted BLA and BLG by disrupting the Th1/Th2 balance toward a Th2 immune response with the decreased number of Tregs. Enhanced induction of Th2-related cytokines, as well as serum-specific antibodies and mast cell activation, was also observed. In this study, we validated that ALA in the food matrix promoted both the sensitization and elicitation of allergic reactions in BALB/c mice.

Chromatographic profile of major whey proteins in some dairy beverages based on milk serum.

The aim of this study was the determination of chromatographic profiles of major whey proteins (WP): α-lactalbumin (α-La), β-lactoglobulin A and B, (β-Lg A and B), bovine serum albumin (BSA) in dairy beverages based on Zonar milk serum. The studied WP were separated by high-performance liquid chromatography (HPLC) on Aeris XB-C18 column using gradient elution with 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 80% acetonitrile, at 214 nm detection. The HPLC method was validated for system suitability, selectivity/specificity, linearity (R2 ≥ 0.996), precision (RSD% ≤ 2.01), trueness (recovery 96.29%-102.08%), sensitivity (limits of quantification (LOQ) 1.35-10.08 μg/mL), and robustness. The total of studied whey proteins (WPT) in dairy beverages based on Zonar milk serum varied between 1.42 g/L and 3.04 g/L. The results obtained were evaluated by principal component analysis (PCA) for correlation between the types of studied dairy beverages (natural and with additives added), the HPLC dataset of four whey proteins and WPT (active variables) to differentiate type of dairy beverages samples. The obtained results confirm that the studied dairy beverages constitute a valuable source of bioactive components with benefits in human healthy nutrition and medical purposes.

Characterization and functional properties of Maillard reaction products of β-lactoglobulin and polydextrose

The Maillard reaction products (MRPs) between polydextrose (PDX), a popular polysaccharide in formula powder, and β-lactoglobulin (β-LG), a major whey protein, were studied in aggregation degree, structure, hydrophobic, antigenic and antioxidant activity changes of β-LG. Incubation of PDX and β-LG (60 ℃, 79% relative humidity) for up to 72 h yielded MRPs with increases in furosine, UV absorbance, fluorescence intensity and loss of free amino groups. High molecular weight β-LG-PDX MRPs were observed by SDS-PAGE. Circular dichroism spectroscopy revealed negligible change in β-LG secondary structure. Changes in the tertiary structure of β-LG were detected by tryptophan fluorescence spectroscopy consistent with an increase in surface hydrophobicity of heated β-LG-PDX. Antigenicity reduction of β-LG in β-LG-PDX reached its peak when heated for 24 h. After heating for 72 h, DPPH radical-scavenging activity of β-LG-PDX increased by 7.4-fold, and the ferric reducing antioxidant power reached 61.1 µmol ascorbic acid/g protein.

Dual-gating pH-responsive membranes with the heterogeneous structure for whey protein fractionation

Whey protein is a mixture of globular proteins with high nutritional value and excellent functional properties availability. Purified individual whey proteins exhibit better functionality than in their original mixture, thus there is an increasing interest in developing more efficient methods to recover individual whey protein fractions, especially membrane technology. However, whey protein fractionation from the protein mixture is still a huge challenge for membrane separation. Herein, a dual-gating pHresponsive membrane with the heterogeneous structure was developed for whey protein fractionation. The heterogeneous structure of the membranes was obtained by grafting the dimethylaminoethyl methacrylate (DMAEMA) and 4-vinylpyridine (4VP) near the topside and inside the pores of the ethylene vinyl alcohol copolymer (EVAL) membranes based on selective modification of grafting sites. The PDMAEMA and P4VP gates responded to pH individually and provided a multi-stage regulation of the surface charge and sieving property of the membranes. The major whey protein fractionation from the mixture could be achieved by single dual-gating pH-responsive membrane via the sequential stepwise pH gradient from 3.0 to 8.0. The α-Lactalbumin (α-La), β-Lactoglobulin (β-Lg), lactoferrin (LF) and bovine serum albumin (BSA) were separated and enriched in the permeate at pH 3.0, 4.2, 6.4 and 8.0 by the diafiltration process, respectively. This work not only proposed a novel concept of pHresponsive membrane, but also offered a sustainable solution to fractionate the whey protein from the complicated mixture.

Phycocyanobilin-modified β-lactoglobulin exhibits increased antioxidant properties and stability to digestion and heating

β-lactoglobulin (BLG) is a major whey protein with numerous techno-functional properties desirable for the food industry. Phycocyanobilin (PCB), a bioactive pigment of Arthrospira platensis with health-promoting effects, covalently binds to BLG at physiological pH. This study investigated the effects of this covalent modification on BLG functional properties. The BLG–PCB adduct possesses enhanced antioxidant properties, and bound PCB protects BLG against free radical-induced oxidation. Despite the similar thermal stabilities of BLG and BLG–PCB, BLG–PCB is less susceptible to covalent and noncovalent aggregation under moderate heat treatment (63 °C, 30 min). Blocked thiol group and reduced hydrophobicity due to hindering of hydrophobic residues by bound PCB, as well as the heat-induced transition of β-sheet to α-helix, contributed to the low susceptibility of BLG–PCB to aggregation. BLG–PCB has a higher resistance to pepsin and pancreatin digestion than BLG and unaltered IgE-binding properties. The improved functional properties of BLG–PCB make it a useful ingredient in the food industry.

Comparative Transcriptome Analysis of Milk Somatic Cells During Lactation Between Two Intensively Reared Dairy Sheep Breeds.

In dairy sheep industry, milk production dictates the value of a ewe. Milk production is directly related to the morphology and physiology of the mammary gland; both being designated targets of breeding strategies. Although within a flock breeding parameters are mutual, large differences in milk production among individual ewes are usually observed. In this work, we tested two of the most productive dairy sheep breeds reared intensively in Greece, one local the Chios breed and one foreign the Lacaune breed. We used transcriptome sequencing to reveal molecular mechanisms that render the mammary gland highly productive or not. While highly expressed genes (caseins and major whey protein genes) were common among breeds, differences were observed in differentially expressed genes. ENSOARG00000008077, as a member of ribosomal protein 14 family, together with LPCAT2, CCR3, GPSM2, ZNF131, and ASIP were among the genes significantly differentiating mammary gland’s productivity in high yielding ewes. Gene ontology terms were mainly linked to the inherent transcriptional activity of the mammary gland (GO:0005524, GO:0030552, GO:0016740, GO:0004842), lipid transfer activity (GO:0005319) and innate immunity (GO:0002376, GO:0075528, GO:0002520). In addition, clusters of genes affecting zinc and iron trafficking into mitochondria were highlighted for high yielding ewes (GO:0071294, GO:0010043). Our analyses provide insights into the molecular pathways involved in lactation between ewes of different performances. Results revealed management issues that should be addressed by breeders in order to move toward increased milk yields through selection of the desired phenotypes. Our results will also contribute toward the selection of the most resilient and productive ewes, thus, will strengthen the existing breeding systems against a spectrum of environmental threats.

Energetics and mechanism of β-lactoglobulin binding to dextran sulfate

Interactions of a major whey protein, β-lactoglobulin (BLG), with an anionic polysaccharide, dextran sulfate (DS), were investigated by nephelometric titration and high-sensitivity differential scanning calorimetry at pH 2.5–7.0 and NaCl concentrations of 0–500 mM. The pH-dependences of the light scattering of the BLG−DS system revealed a maximum. Its position was dependent on the polysaccharide/protein ratio and salt concentration. Thermodynamic parameters of the denaturation of BLG in mixtures with DS were determined at different pH and salt concentrations. Conformational stability of BLG in the BLG–DS complexes was shown to either increase or decrease depending on the solution conditions and complex composition. The binding curves of BLG to DS were calculated from the light scattering and calorimetric data and analyzed using various binding models including the Langmuir and McGhee–von Hippel models as well as a stoichiometric interpolyelectrolyte reaction. Mechanisms responsible for changes in the BLG oligomer structure and conformational stability induced by the complex formation with the polysaccharide were proposed.

The effects of denatured major bovine whey proteins on the digestive tract, assessed by Caco-2 cell differentiation and on viability of suckling mice.

Alpha-lactalbumin (α-LA) and β-lactoglobulin (β-LG) are contained in bovine milk whey. Chemical and physical treatments are known to alter the conformation of these proteins and we have previously reported that α-LA denatured with trifluoroethanol (TFE) and isolated from sterilized market milk inhibited the growth of rat crypt IEC-6 cells. In the present study, we aimed to evaluate the effects of TFE-treated α-LA and β-LG on cell growth using cultured intestinal cells and on their safety using a suckling mouse model. First, we investigated the effect of the TFE-treated whey proteins on human colonic Caco-2 cells at various differentiation stages. In the undifferentiated stage, we assessed cell growth by a water-soluble tetrazolium-1 method. The native whey proteins enhanced cell proliferation, whereas the TFE-treated whey proteins strongly inhibited cell growth. We investigated cell barrier function in the post-differentiated stage by measuring transepithelial electrical resistance (TER). Not only native but also the TFE-treated whey proteins increased TER. Next, we evaluated whether the TFE-treated α-LA and β-LG have adverse effects on healthy suckling mice. No mice given by the TFE-treated samples showed any adverse symptoms. We also performed a safety test using a human rotavirus infected gastrointestinal disease suckling mice model. Even the TFE-treated whey proteins appeared to prevent the development of diarrheal symptoms without any adverse effects. Although we cannot know the effect of long-term ingestion of denatured whey proteins, these results suggest that they have no adverse effects on differentiated intestinal cells and digestive tract, at least in short-term ingestion.

One Health in allergology: A concept that connects humans, animals, plants, and the environment.

One Health is "a collaborative effort of multiple disciplines - working locally, nationally, and globally - to attain optimal health for people, animals, and the environment", the so-called One Health triad1 (Fig. 1).Historically, aspects of One Healthwere already discussed in the fourth century by Hippocrates, who realized that a clean environment is very important for human health1 . More recently, Rudolf Virchow (1821-1902) coined the term "zoonosis" in 1880and fathered modern One Healthin the sense of OneMedicine by strongly supporting the view of human and animal health being linked to each other.1.

Quinoline yellow dye stimulates whey protein fibrillation via electrostatic and hydrophobic interaction: A biophysical study

Amyloid fibrilformation ofproteins is associated with a number of neurodegenerative diseases. Several small molecules can accelerate the amyloid fibril formationin vitro and in vivo. However, the molecular mechanism of amyloid fibrillation is still unclear. In this study, we investigated how the food dye quinoline yellow (QY) induces amyloid fibrillation in α-lactalbumin (α-LA), a major whey protein, at pH 2.0. We used several spectroscopy techniques and a microscopy technique to explore how QY provokes amyloid fibrillation in α-LA. From turbidity and Rayleigh light scattering experiments, we found that QY promotes α-LA aggregation in a concentration-dependent manner; the optimal concentration for α-LA aggregation was 0.15 to 10.00mM. Below 0.1 mM, no aggregation occurred. Quinoline yellow-induced aggregation was a rapid process that escaped the lag phase, but it depended on the concentrations of both α-LA and QY. We also demonstrated that aggregation switched the secondary structure of α-LA from α-helices to cross-β-sheets. We then confirmed the amyloid-like structure of aggregated α-LA by transmission electron microscopy measurements. Molecular docking and simulation confirmed the stability of the α-LA-QY complex due to the formation of 1 hydrogen bond with Lys99 and 2 electrostatic interactions with Arg70 and Lys99, along with hydrophobic interactions with Leu59 and Tyr103. This study will aid in our understanding of how small molecules induce aggregation of proteins inside the stomach (low pH) and affect the digestive process.

Variations in N-linked glycosylation of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) whey protein: Intercow differences and dietary effects

In bovine milk serum, the whey proteins with the highest N-glycan contribution are lactoferrin, IgG, and glycosylation-dependent cellular adhesion molecule 1 (GlyCAM-1); GlyCAM-1 is the dominant N-linked glycoprotein in bovine whey protein products. Whey proteins are base ingredients in a range of food products, including infant formulas. Glycan monosaccharide composition and variation thereof may affect functionality, such as the interaction of glycans with the immune system via recognition receptors. It is therefore highly relevant to understand whether and how the glycosylation of whey proteins (and their functionality) can be modulated. We recently showed that the glycoprofile of GlyCAM-1 varies between cows and during early lactation, whereas the glycoprofile of lactoferrin was highly constant. In the current study, we evaluated intercow differences and the effects of macronutrient supply on the N-linked glycosylation profiles of the major whey proteins in milk samples of Holstein-Friesian cows. Overall, approximately 60% of the N-glycan pool in milk protein was sialylated, or fucosylated, or both; GlyCAM-1 contributed approximately 78% of the total number of glycans in the overall whey protein N-linked glycan pool. The degree of fucosylation ranged from 44.8 to 73.3% between cows, and this variation was mainly attributed to the glycans of GlyCAM-1. Dietary supplementation with fat or protein did not influence the overall milk serum glycoprofile. Postruminal infusion of palm olein, glucose, and essential AA resulted in shifts in the degree of GlyCAM-1 fucosylation within individual cows, ranging in some cases from 50 to 71% difference in degree of fucosylation, regardless of treatment. Overall, these data demonstrate that the glycosylation, and particularly fucosylation, of GlyCAM-1 was variable, although these shifts appear to be related more to individual cow variation than to nutrient supply. To our knowledge, this is the first report of variation in glycosylation of a milk glycoprotein in mature, noncolostral milk. The functional implications of variable GlyCAM-1 fucosylation remain to be investigated.

Polymorphisms, Molecular Characteristics of LGB Gene in River and Swamp Buffalo (Bubalus bubalis)

Beta-lactoglobulin (β-LG) is the major whey protein associated with lactation traits in ruminants and has a potential impact on human health. So far, the polymorphisms and molecular characteristics of buffalo LGB gene have not been well understood. In this study, the complete coding sequence (CDS) of buffalo LGB were isolated and its polymorphisms were detected using direct sequencing of PCR product. The CDS of LGB gene for river and swamp buffalo was 543 nucleotides in length, encoding a peptide with 180 amino acid residues. A total of eight single nucleotide polymorphisms (SNPs) was identified in the CDS of LGB gene in two types of buffalo. Among them, c.46G>A and c.131C>T were non-synonymous, which led to an amino acid substitution from Ala to Thr at the 16th position of the signal peptide and a p.Ala26Val substitution in the mature peptide of buffalo β-LG, respectively. The prediction showed that the p.Ala26Val may affect the function of buffalo β-LG. Seven LGB haplotypes were defined based on the SNPs observed in buffalo, and accordingly, two variants and two synonymous variants of buffalo β-LG were inferred, named variant A, A1, A2 and B, respectively. All the variants determined in buffalo did not exist in the animals of Bos genus. The sequence alignment of β-LG mature peptide showed that there were two different amino acids (p.Ile1Leu and p.Val162Ile) between buffalo and the animals of Bos genus. The signal peptide of buffalo β-LG was two amino acids longer than that of Bos genus and there was an differential amino acid at the 13th position of signal peptide, which may lead to different functions of signal peptide between buffalo and Bos genus. The hydrophilicity of buffalo β-LG variants A and B were slightly different, and their physicochemical characteristics were similar to those of cattle β-LG B. The mature peptides of β-LG in buffalo and Bos genus had a lipocalin domain, which revealed that they had the same function. This study will give us new insights into the variation, physicochemical characteristics and biological function of buffalo LGB gene.

Investigating the Binding Interaction between β-Lactoglobulin and Vitamin B12: A Spectroscopic and Computational Approach

Binding interaction of the major whey protein β-Lactoglobulin (βLG) with vitamin B12 is crucial for understanding the potency of βLG as a transporter for vitamin B12. Hence, the binding interaction of the βLG with vitamin B12 was studied using spectroscopic (UV-Visible absorption spectroscopy, steady-state & time-resolved fluorescence spectroscopy, synchronous fluorescence spectroscopy (SFS), circular dichroism (CD), and fluorescence correlation spectroscopy (FCS)) and computational (molecular docking and molecular dynamics (MD) simulation) tools. The quenching of the intrinsic fluorescence of βLG by vitamin B12 confirms the binding of vitamin B12 with βLG. The nature of the quenching is found to be static due to ground state complex formation. The calculated thermodynamic parameters suggest the binding process is hydrophobic. Synchronous fluorescence spectroscopy revealed that vitamin B12 affects the tryptophan residue microenvironment of the protein. Vitamin B12 does not alter the secondary and tertiary structure of βLG, as concluded from the CD and FCS experiments. From the molecular docking results, vitamin B12 is found to bind at the β- barrel site of βLG. The MD simulation results suggest that vitamin B12 bound βLG complex is stable. The secondary structural element analysis of MD simulation results show no alteration in protein secondary structure after binding with vitamin B12, which is matched with the experimental findings. This biophysical binding interaction study of βLG with vitamin B12 might have promising applications in the pharmaceutical and food industries.

Capillary electrophoresis with dual C4D/UV detection for simultaneously determining major metal cations and whey proteins in milk.

A reliable and simple CE method with dual C4D and UV detection modes for simultaneous determination of major metal cations and whey proteins in milk samples was developed. Sample pretreatment comprised dilution, acidification to pH 4.55 with 10 mM AcOH and centrifugation. The complete separation of metal cations K+, Ca2+, Na+, and Mg2+ and whey proteins α-Lac, and β-Lg could be achieved respectively within 10 min and 20 min in a simple BGE composed of 1.0 M AcOH, 12 mM l-His and 2 mM 18-crown-6 with pH 2.74 at a voltage/current of +15 kV/12.5 μA. The samples were injected hydrodynamically by a pressure of 50 mbar for 5 s, the excitation voltage and excitation frequency of the C4D detector were 80 V and 1000 kHz, respectively and the detection wavelength of UV detection was set at 200 nm. In cation analysis, the range of the detection limit was 0.05-0.10 mg L-1 for C4D detection and 0.10-0.50 mg L-1 for UV detection, respectively, and the relative standard deviations (RSD%, n = 5) of intraday and interday analysis were 0.37-0.55% and 0.46-0.79% for the relative migration time, and 2.51-4.12% and 3.65-4.91% for the peak area, respectively. In whey protein analysis, the detection limits of β-Lg and α-Lac analysis were 5 mg L-1 and 3 mg L-1, respectively and the relative standard deviations (RSD%, n = 5) of intraday and interday analysis were 0.29-0.31% and 0.43-0.48% for the migration time and 2.89-3.25% and 3.29-4.18% for the peak area, respectively. The content of four major metal cations and two whey proteins in various types of milk samples was obtained. The results indicated that the content of metal cations varied little in milk samples of different brands and prices, while the content of whey proteins, as thermosensitive active proteins, varied greatly among different heat-treated milk samples.

Interaction of vitamin B12 with β-lactoglobulin: a computational study

The β-Lactoglobulin (βLG) is a major whey protein that has the potential to bind various ligands; hence it is used as a model protein in protein-ligand interaction studies. Vitamin B12 is an essential nutrient for the human body, which helps in the synthesis of DNA, proteins, and the production of red blood cells. Binding interaction of vitamin B12 with βLG will help to understand the potency of βLG as a transporter for vitamin B12. Our experimental findings already showed that βLG binds with vitamin B12 successfully (Swain et al., 2020). Nevertheless, to further support our experimental results firmly, here, we have employed computational tools such as molecular docking and molecular dynamics (MD) simulation. The molecular docking technique was used to elucidate the probable binding sites and binding affinity of vitamin B12 on βLG. The docked complex of vitamin B12 with βLG was subjected to MD simulation to investigate its stability and other interaction properties over a time frame. The study revealed that the compound is stable, and vitamin B12 imposes no change to the secondary structure of the βLG. The computational results agree reasonably well with our experimental study.

Development and validation of an RP-HPLC DAD method for the simultaneous quantification of minor and major whey proteins

Whey represents a valuable protein source for human nutrition. Whey composition varies with respect to process characteristics during milk processing. For efficient exploitation of this dairy side stream, reliable analytical methods are essential. The aim of this study was to develop and validate an RP-HPLC-DAD method for the simultaneous quantification of the minor (lactoferrin, lactoperoxidase, bovine serum albumin) and major (α-lactalbumin, β-lactoglobulin) whey proteins. Seven RP-columns were compared and the composition of the mobile phase was optimized to achieve baseline separation. In validation experiments the limits of detection (LOD < 8 mg/L) and quantification (LOQ < 24 mg/L) were determined. Validity was proofed by precision (>96%), accuracy (95% − 103%) and recovery (96% − 102%) measurements. Peak homogeneity was confirmed by SDS-PAGE. The individual working ranges were adjusted to the estimated protein concentrations in whey, allowing direct analysis without sample preparation at a method runtime of 23 min.

Distinction between glycomacropeptide and β-lactoglobulin with ‘stains all’ dye on tricine SDS-PAGE gels

Identification of glycomacropeptide (GMP) and β-lactoglobulin (β-lg) present in cheese whey is difficult on SDS-PAGE due to their close proximity during electrophoresis and poor sensitivity of commonly used staining dye ‘coomassie brilliant blue’ (CBB) towards GMP. A simple method has been developed for the detection of GMP and β-lg by staining acrylamide gel after tricine SDS-PAGE using cationic ‘stains all’ dye. After staining and destaining major whey proteins, viz. ɑ-lactalbumin (ɑ-la) and β-lg appear red while GMP stains blue. The method can be used for the identification of these macromolecules in cheese whey and the detection of adulteration of milk with rennet whey.

Negative regulation of αS1-casein (CSN1S1) improves β-casein content and reduces allergy potential in goat milk

Compared with other milk proteins, αS1-casein (encoded by CSN1S1) is associated with higher rates of allergies in humans. Although some studies have revealed that CSN1S1 affects a variety of cellular physiological processes such as immune response and proliferation in various cells, whether CSN1S1 regulates other milk proteins in ruminants is not known. In this study, we observed a negative Pearson correlation between the contents of αS1-casein and β-casein in goat milk. Thus, we used isolated primary goat mammary epithelial cells along with adenoviral infection or small interference RNA to alter abundance of CSN1S1 and examine its role in milk protein synthesis regulation. Overexpressing CSN1S1 through adenoviral transfection decreased β-casein mRNA (CSN2) and protein abundance, whereas interference of CSN1S1 resulted in a significant increase in β-casein abundance. CSN1S1 reduced phosphorylation level of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5a (STAT5a). The transcription factor STAT5a activates CSN2 transcription by binding with its promoter region and then promotes β-casein synthesis. Furthermore, CSN1S1 inhibited CSN2 promoter activity and β-casein synthesis by modulating the JAK2/STAT5a signaling pathway. No changes in abundance were detected for αS2-casein (CSN1S2), κ-casein (CSN3) and major whey proteins (LALBA, BLG). Overall, our results underscored a mechanism whereby CSN1S1 inhibits β-casein synthesis via inhibition of STAT5a. The data suggested that knocking down CSN1S1 not only reduced the content of αS1-casein, but also increased the abundance of major milk proteins including β-casein. Thus, the present study provides a theoretical basis for manipulating αS1-casein mRNA to improve quality of ruminant milk for human consumption.