Major Bovine Milk Proteins Research Articles

Genetic variation in bovine milk proteins: Implications for functional and nutritional properties

The six major bovine milk proteins are encoded by highly polymorphic chromosomes. Genetic variation has become a topic of interest and controversy in recent years due to supposed adverse health effects, beginning with the A2 variant of β‐casein. The functional implications of genetic variants have been well characterised, specifically heat stability and coagulation (i.e. rennet and acid) properties of milk, which have found that the B variant of β‐casein, κ‐casein and β‐lactoglobulin are the most beneficial. Genetic variants of milk proteins provide considerable potential to create new dairy products to add value to milk.

Orally administered milk-derived tripeptide improved cognitive decline in mice fed a high-fat diet.

The functions of the brain, which is thought of as an organ highly independent from the periphery, are often affected by the peripheral environment. Indeed, epidemiologic studies demonstrated that diabetes was a risk factor for dementia. It was also reported that the intake of dairy products, such as milk, reduces the risk of developing dementia. We found that mice on a short-term high-fat diet (HFD) for 1 wk had reduced cognitive function. Thus, using this acute model, we investigated the effects of milk-derived peptide on cognitive decline induced by HFD. Tyr-Leu-Gly (YLG), a tripeptide derived from αS1-casein, a major bovine milk protein, is released by gastrointestinal proteases. We found that orally administered YLG improved cognitive decline induced by 1-wk HFD intake in the object recognition test. YLG also improved cognitive decline in the object location test. Thus, we found that YLG improved cognitive decline induced by HFD. Next, we examined the effects of YLG on the hippocampus, a brain area essential for cognitive function. HFD intake decreased the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells, and this decrease was improved by YLG administration. HFD intake decreased nerve growth factor (NGF) and glial cell line-derived neurotrophic factor, whereas YLG increased NGF and ciliary neurotrophic factor, suggesting that these neurotropic factors play a role in hippocampal neurogenesis after YLG administration. In conclusion, we demonstrated that 1-wk HFD reduced cognitive function. Furthermore, we found that YLG, a milk-derived tripeptide, improved cognitive decline in mice on HFD. The HFD reduced neural stem cell proliferation, and YLG improved this reduction. YLG is the first reported milk peptide to improve cognitive decline induced by HFD intake.-Nagai, A., Mizushige, T., Matsumura, S., Inoue, K., Ohinata, K. Orally administered milk-derived tripeptide improved cognitive decline in mice fed a high-fat diet.

Allergenicity reduction of bovine milk β-lactoglobulin by proteolytic activity of lactococcus lactis BMC12C and BMC19H isolated from Iranian dairy products

Nowadays health benefits of bioactive food constituents, known as probiotic microorganisms, are a growing awareness. Cow's milk is a nutritious food containing probiotic bacteria. However, milk allergenicity is one of the most common food allergies. The milk protein, β-lactoglobulin (BLG), is in about 80% of all main cases of milk allergies for children and infants. With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated new proteolytic strains of cocci lactic acid bacteria from traditional Iranian dairy products. The proteases produced by these strains had strong proteolytic activity against BLG. Proteolysis of BLG, observed after sodium dodecyl sulfate-PAGE, was confirmed by the analysis of the peptide profiles by reversed-phase HPLC. The two isolates were submitted to 16S rDNA sequencing and identified as Lactcoccus lactis subsp. cremoris and Lactcoccus lactis subsp. hordniea. The competitive ELISA experiments confirmed that these isolates, with high proteolytic activity, reduce significantly the allergenicity of BLG. Accordingly, these isolates can reduce the immunoreactivity of bovine milk proteins, which can be helpful for the production of low-allergic dairy products.

Interaction between 2-(p-toluidino)-6-naphthalenesulfonic acid sodium salt (TNS) and β-lactoglobulin

The major bovine milk protein β-lactoglobulin (β-LG), a member of the lipocalin superfamily, can bind a wide range of ligands and act as a transporter. In the present study, the combination of the hydrophobic molecule 2-(p-toluidino)-6-naphthalenesulfonic acid sodium salt (TNS) with β-LG was analyzed using fluorescence spectroscopy and AutoDock modeling to discern the major binding sites of the protein and to determine the capacity of other small ligands to bind with β-LG by utilizing TNS as a reference. The experimental data indicate that in a neutral pH environment, TNS is located in the hydrophobic domain of the protein, 2.5 nm away from the Trp19 residues of β-LG. The binding constant of the small molecule to β-LG is (3.30 ± 0.32) × 106 (mol L–1)−1. An interaction model between the ligand and β-LG was developed, and AutoDock modeling also demonstrates that the ligand is located in the central hydrophobic calyx of β-LG within the regions covered by the Förster radius of the Trp19–ligand pair. Although the interaction between the ligand and β-LG is affected by increasing ion strength, pH change, and heat treatment, the complex is maintained until the secondary structure of β-LG is destroyed. Additionally, the ligand binding stabilizes the folding of β-LG. The binding constants of sodium dodecyl sulfate (SDS) and sodium dodecylbenzene sulfonate (SDBS) to β-LG were obtained using competitive ligand binding measurements. With a sensitive fluorescence signal and stable complex, the ligand could be utilized as a reference to detect the binding of other small ligands to β-LG.

Proteolytic activity of Enterococcus faecalis VB63F for reduction of allergenicity of bovine milk proteins

With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated a new proteolytic strain of Enterococcus faecalis (Ent. faecalis VB63F) from raw bovine milk. The proteases produced by this strain had strong activity against caseins (αS1-, αS2-, and β-casein), in both skim milk and sodium caseinate. However, only partial hydrolysis of whey proteins was observed. Proteolysis of Na-caseinate and whey proteins, observed after sodium dodecyl sulfate-PAGE, was confirmed by analysis of peptide profiles by reversed-phase HPLC. Inhibition of proteolysis with EDTA indicated that the proteases produced by Ent. faecalis VB63F belonged to the group of metalloproteases. The optimal conditions for their activity were 42°C and pH 6.5. The majority of assessed virulence genes were absent in Ent. faecalis VB63F. The obtained results suggest that Ent. faecalis VB63F could be efficient in reducing the immunoreactivity of bovine milk proteins.

Interaction between N-phenyl-1-naphthylamine and β-lactoglobulin / N-fenil-1-naftilamin and β-laktoglobulin arasındaki etkileşim

Abstract Objective: We aimed to explore the interaction between hydrophobic molecule N-phenyl-1-naphthylamine (AN) and major bovine milk protein β-lactoglobulin (β-LG) in aqueous solution. Methods: We use a combination of experiment and theoretical calculation to study how the β-lactoglobulin and AN interact. Fluorescence spectra were recorded on an F-4500 fluorescence spectrometer. The absorption spectra of AN were recorded on a 2250 UV-vis spectrophotometer. The crystal structure of β-LG (1B0O) is obtained from Protein Data Bank. Molecular docking was carried out using the AutoDock4.2 software package. The PyMOL molecular viewer and the MGLTools are used to render the output and to calculate the distance between nearest atoms. Results: A 1: 1 complex were found to be formed and the N-phenyl-1-naphthylamine binding site and Trp-19 residues of β-lactoglobulin was calculated to be 2.28 nm. The interaction force between the molecule and β-lactoglobulin was suggested to be mainly hydrophobic force through the calculated thermodynamic parameters of the reaction. Autodock research also further proved the interaction mode. The binding affects no conformation of the β-lactoglobulin. Moreover, it stabilizes the fold of the protein. The binding constants of oleate to β-lactoglobulin is also calculated by competitive binding experiments. Conclusion: β-lactoglobulin, by virtue of their ability to enhance solubility and stability of AN, can provide more information about its function as a versatile carrier for small bioactive molecules. AN could also be utilized as a reference to detect the binding of other small ligands to the protein by calculating the binding constants of oleate to β-lactoglobulin as a example.

Nanocomplex formation between riboflavin and β-lactoglobulin: Spectroscopic investigation and biological characterization

Light activated drug delivery systems with the use of photosensitizers are attracting increasing interest in both the medical and non-medical fields. Riboflavin (RF) is an endogenous photosensitizer that interacts with proteins located in the cell membrane and induces damages to biological systems including tumor tissues. Spectroscopic methods were used to demonstrate interaction and energy transfer between β-lactoglobulin (βlg), the major bovine milk protein, and RF. The findings reveal the formation of a ground-state nanocomplex between βlg and RF with minor impact on the structure of the protein. βlg can bind and transport RF, which has great implications for the food industry in terms of food fortification. The impact of these interactions on RF was determined by assessing the anti-proliferative activity of the βlg/RF nanocomplex by irradiating skin melanoma cancer cell lines with UV-A light according to the NCI/NIH Developmental Therapeutics Program. The βlg/RF nanocomplex exhibited important anti-proliferative activity in the micromolar range. The cytotoxicity is likely due to the generation of reactive radical and oxygen species as the result of the interaction between RF and βlg. This study provides important information on the potential use of the βlg/RF nanocomplex in photodynamic therapy, which is applied in the medical field against tumor cells as well as in the food industry against food-borne pathogens.

Effects of gastrointestinal pH conditions on the stability of the β-lactoglobulin/vitamin D3 complex and on the solubility of vitamin D3

β-Lactoglobulin (βlg) is the major bovine milk protein with important biological and functional properties including a transport role for small hydrophobic ligands. However, βlg is prone to structural changes triggered by modification of its environment such as pH variation. An unfavorable environment during formulation, manufacture or storage of food products or transit in the gastrointestinal track can have a dramatic impact on the stability of the βlg/ligand complex, resulting in the deterioration of its binding ability and premature release of the ligand. This can impair the biological properties of the ligands and reduce their beneficial effects on health. In the present study, vitamin D3 (D3) was used as a nutraceutical ligand model to study the pH-stability of the βlg/D3 complex as well as the consequences of the complex formation on the solubility of the vitamin. Fluorescence spectroscopy was used to monitor the stability of the βlg/D3 complex at pH1.2, 2.0, 3.0, 5.0, 6.8, 7.0 and 8.0. U-HPLC was used to evaluate the solubility of D3 by preparing the βlg/D3 complex in different ratios using a static concentration of D3 and increasing concentrations of βlg. The binding of D3 to βlg was not significantly affected by pH. Furthermore, the data allowed determination of the fractional residual fluorescence representing the fraction of βlg not bound to the ligand, which indicated that the βlg/D3 complex remained stable at all pH values. Therefore, D3 might be retained during formulation and storage of food products at different pH values and during passage in the stomach, which has important implications for the food industry. The solubility of D3 was also significantly increased as a result of binding to βlg, which confirms its role as a carrier to improve the uptake of D3 and consequently the beneficial effects of D3 on health.

Alteration of the N‐glycome of bovine milk glycoproteins during early lactation

Milk provides nutritional, immunological and developmental components for newborns. Whereas identification of such components has been performed by targeting proteins and free oligosaccharides, structural and functional analyses of the N-glycome of milk glycoproteins are scarce. In this study, we investigated, for the first time, the alterations of the bovine milk N-glycome during early lactation (1 day, 1, 2, 3 and 4 weeks postpartum), characterizing more than 80 N-glycans. The glycomic profile of colostrum on day 1 after calving differed substantially from that in other periods during early lactation. The proteins in colostrum obtained 1 day postpartum were more highly sialylated than milk samples obtained at other time points, and the N-glycolylneuraminic acid (Neu5Gc)/N-acetylneuraminic acid (Neu5Ac) ratio was significantly higher on day 1, showing a gradual decline with time. In order to dissect the N-glycome of colostrum, alterations of the N-glycosylation profile of major bovine milk proteins during the early lactation stage were elucidated, revealing that the alteration is largely attributable to qualitative and quantitative N-glycosylation changes of IgG, the major glycoprotein in colostrum. Furthermore, by preparing and analyzing IgGs in which the N-glycan structure and subtypes were well characterized, we found that the interaction between IgG and FcRn was not affected by the structure of the N-glycans attached to IgG. We also found that bovine FcRn binds IgG(2) better than IgG(1) , strongly suggesting that the role of FcRn in the bovine mammary gland is to recycle IgG(2) from the udder to blood, rather than to secrete IgG(1) into colostrum.

Determination of Major Bovine Milk Proteins by Reversed Phase High Performance Liquid Chromatography

Abstract A reversed-phase high-performance liquid chromatography (RP-HPLC) for the quantification of six major bovine milk proteins (κ-casein (κ-CN), αs2-casein (αs2-CN), αs1-casein (αs1-CN), β-casein (β-CN), immunoglobulins (IgG) and whey) was developed. The separation and quantification of these major proteomic components were achieved with an Agilent Zorbax 300SB-C8 analytical column (250 mm × 4.6 mm I.D., 5 μm) at 45 °C with detection wavelength at 214 nm. The gradient elution was carried out with solvents of 0.1% TFA in water and 0.1% TFA in acetonitrile at a flow rate of 0.8 ml min−1. The method validation consisted of testing the linearity, repeatability, reproducibility and accuracy. The recoveries of six target proteins spiked in milk were in the range from 74.8% to 132.5%. Nine different commercial milk brands were used, the difference of the concentration and relative ration of κ-CN, αs2-CN, αs1-CN, β-CN and whey were found.

Evidence for β‐lactoglobulin involvement in vitamin D transport in vivo– role of the γ‐turn (Leu‐Pro‐Met) of β‐lactoglobulin in vitamin D binding

Beta-lactoglobulin (LG) is a major bovine milk protein, containing a central calyx and a second exosite beyond the calyx to bind vitamin D; however, the biological function of LG in transporting vitamin D remains elusive. Crystallographic findings from our previous study showed the exosite to be located at the pocket between the alpha-helix and beta-strand I. In the present study, using site-directed mutagenesis, we demonstrate that residues Leu143, Pro144 and Met145 in the gamma-turn loop play a crucial role in the binding. Further evidence is provided by the ability of vitamin D(3) to block the binding of a specific mAb in the gamma-turn loop. Using the mouse (n = 95) as an animal model, we initially demonstrated that LG is a major fraction of milk proteins responsible for uptake of vitamin D. Most interestingly, dosing mice with LG supplemented with vitamin D(3) revealed that native LG containing two binding sites gave a saturated concentration of plasma 25-hydroxyvitamin D at a dose ratio of 2 : 1 (vitamin D(3)/LG), whereas heated LG containing one exosite (lacking a central calyx) gave a ratio of 1 : 1. We have demonstrated for the first time that LG has a functional advantage in the transport of vitamin D, indicating that supplementing milk with vitamin D effectively enhances its uptake.

Crystal structure of a secondary vitamin D3 binding site of milk β‐lactoglobulin

Beta-lactoglobulin (beta-LG), one of the most investigated proteins, is a major bovine milk protein with a predominantly beta structure. The structural function of the only alpha-helix with three turns at the C-terminus is unknown. Vitamin D(3) binds to the central calyx formed by the beta-strands. Whether there are two vitamin D binding-sites in each beta-LG molecule has been a subject of controversy. Here, we report a second vitamin D(3) binding site identified by synchrotron X-ray diffraction (at 2.4 A resolution). In the central calyx binding mode, the aliphatic tail of vitamin D(3) clearly inserts into the binding cavity, where the 3-OH group of vitamin D(3) binds externally. The electron density map suggests that the 3-OH group interacts with the carbonyl of Lys-60 forming a hydrogen bond (2.97 A). The second binding site, however, is near the surface at the C-terminus (residues 136-149) containing part of an alpha-helix and a beta-strand I with 17.91 A in length, while the span of vitamin D(3) is about 12.51 A. A remarkable feature of the second exosite is that it combines an amphipathic alpha-helix providing nonpolar residues (Phe-136, Ala-139, and Leu-140) and a beta-strand providing a nonpolar (Ile-147) and a buried polar residue (Arg-148). They are linked by a hydrophobic loop (Ala-142, Leu-143, Pro-144, and Met-145). Thus, the binding pocket furnishes strong hydrophobic force to stabilize vitamin D(3) binding. This finding provides a new insight into the interaction between vitamin D(3) and beta-LG, in which the exosite may provide another route for the transport of vitamin D(3) in vitamin D(3) fortified dairy products. Atomic coordinates for the crystal structure of beta-LG-vitamin D(3) complex described in this work have been deposited in the PDB (access code 2GJ5).

Identification and quantification of major bovine milk proteins by liquid chromatography

In the field of food quality, bovine milk products are of particular interest due to the social and economic importance of the dairy products market. However, the risk of fraudulent manipulation is high in this area, for instance, replacing milk powder by whey is very interesting from an economic point of view. Therefore, there is a need to have suitable analytical methods available for the determination of all milk components, which is currently not the case, especially for the main proteins. The detection of potential manipulations requires then a clear analytical characterisation of each type of bovine milk, what constitutes the goal of this work. The separation of the major milk proteinic components has been carried out by ion-pair reversed-phase HPLC with photodiode array detection, using a C 4 column. The overall optimisation has been achieved using a statistical experimental design procedure. The identification of each protein was ascertained using retention times, peak area ratios and second derivative UV spectra. Quantification was based on calibration curves drawn using purified proteins. Major sources of uncertainty were identified and the full uncertainty budget was established. The procedure was initially developed using the skimmed milk powder certified reference material CRM 063R and then applied to various types of commercial milks as well as to raw milk. The method is able to separate and quantify the seven major proteins (κ-casein, α s2-casein, α s1-casein, β-casein, α-lactalbumin, β-lactoglobulin B and β-lactoglobulin A) in one run and also to provide precise determinations of the total protein concentration. These are important results towards the further development of a reference method for major proteins in milk. In addition, the use of a certified material reference is suggested in order to make comparisons of method performances possible.

Separation and Quantification of Bovine Milk Proteins by Reversed-Phase High-Performance Liquid Chromatography.

Current analytical methods for milk proteins lack the capacity to simultaneously separate and quantify the six major bovine milk proteins and their genetic variants. A method is described that simultaneously separates and quantifies the six major bovine milk proteins. The separation is based on reversed-phase partitioning of the six major milk proteins and several genetic variants of kappa-casein, beta-casein, and beta-lactoglobulin. The described method has for each of the six milk proteins a linear quantitative response, precision (coefficient of variation below 5.1% within days of analysis, and below 7.1% between days of analysis), resolution (over 2.5 between proteins), peak efficiency (theoretical plate numbers between 8 000 and 50 000), and a sample treatment without filtration steps that together with the analysis takes 2 h. The composition of protein from milk of each of 234 cows was determined using the current method, and the results were similar to reference values for milk proteins.

Analysis of major bovine milk proteins by on-line high-performance liquid chromatography and electrospray ionization-mass spectrometry

Summary - Proteins from skim milk, paracaseinate and whey have been successfully analyzed by reverse-phase high-pertormance liquid chromatography (RP-HPLC) cou pied with electrospray ionization mass spectrometry (ESI-MS). The major milk proteins were identified by comparison of molecular masses determined by ESI-MS to molecular masses calculated from amino acid composition deduced from primary structures and cDNA sequences for some proteins. This method has permilted the simultaneous identification of caseins and whey protein variants. Observed molecular masses of major milk proteins were found to be 19038.0 ± 2.2 Da forK-CN A-l P (number of experiments n =6); 19007.0 ± 1.1 Da for K-CN B-1P (n = 4); 25230.0 ± 2.1 Da for as2-CN A (n = 9); 23617.2 ± 1.3 Da for as1-CN B-8P (n = 14); 24 092.0 ± 1.7 Da for ~-CN B-5P (n = 6); 24 025.3 ± 1.0 Da for ~-CN A 1-5P (n =14); 23 984.8 ± 0.7 Da for ~-CN A2_5P (n =14); 18278.3 ± 2.2 Da for the monomeric form of ~-LG B (n = 5); 18 364.8 ± 1.6 Da for the monomeric form of ~-LG A (n = 5); 14 179.21 ± 3.14 Da for a-LA (n =5). Hence an accuracy of 0.01 % was obtained by ESI-MS analysis. It was also shown that the on-line coupling of HPLC with ESI-MS offers a very promising alternative for studying the proteolysis and determining the specificity of used enzymes in some technological treatments as shown for milk-clotting enzymes.

Comparison of Milk Proteins Using Preparative Isoelectric Focusing Followed by Polyacrylamide Gel Electrophoresis

The major proteins in milks from bovine, caprine, porcine, and murine animals and from humans were compared using a two-dimensional analysis method. In the first dimension, proteins were separated by their isoelectric points using preparative isoelectric focusing in pH gradient of 3 to 10. Twenty fractions from each sample were then analyzed by urea-PAGE and SDS-PAGE. Two-dimensional gels showed characteristic patterns for each milk. Major bovine milk proteins were identified and used as reference for proteins of other mammals. Additionally, some peptides resulting from plasmin hydrolysis were characterized. Caprine milk proteins showed a pattern similar to that of bovine milk except for the absence of αs1-caseins. α-Lactalbumin of bovine and caprine milks resolved as two bands in an immunoblot using bovine α-lactalbumin antibody. Each band corresponded to normal and glycosylated α-lactalbumin. Human, porcine, and murine milk proteins were totally different from those of ruminant milks on the two-dimensional gels. Two-dimensional analysis using preparative isoelectric focusing, followed by PAGE, was a useful method to compare major milk proteins in several mammals because of the rapid simultaneous separation into 20 fractions. This fractionation allows additional analytical procedures for more efficient comparison of chemical and physical properties of the proteins.

Genomic analysis of the major bovine milk protein genes

The genomic arrangement of the major bovine milk protein genes has been determined using a combination of physical mapping techniques. The major milk proteins consist of the four caseins, alpha s1 (CASAS1), alpha s2 (CASAS2), beta (CASB), and kappa (CASK), as well as the two major whey proteins, alpha-lactalbumin (LALBA) and beta-lactoglobulin (LGB). A panel of bovine X hamster hybrid somatic cells analyzed for the presence or absence of bovine specific restriction fragments revealed the genes coding for the major milk proteins to reside on three chromosomes. The four caseins were assigned to syntenic group U15 and localized to bovine chromosome 6 at q31-33 by in situ hybridization. LALBA segregated with syntenic group U3, while LGB segregated with U16. Pulsed-field gel electrophoresis confirmed genetic mapping results indicating tight linkage of the casein genes. The four genes reside on less than 200 kb of DNA in the order CASAS1-CASB-CASAS2-CASK. Multiple restriction fragment length polymorphisms were also found at the six loci in three breeds of cattle.

Iron absorption in humans as influenced by bovine milk proteins

The effect of the two major bovine milk protein fractions on the dialyzability of iron in vitro under simulated gastrointestinal conditions and on the absorption of Fe by humans was studied. Liquid-formula meals were prepared from hydrolyzed maize starch, corn oil, and either spray-dried egg white or a milk-protein product. In meals containing egg white, 3.32% of the Fe was dialyzable. The substitution of casein and whey protein products reduced the dialyzable fraction to 0.19-0.56% and 0.86-1.60%, respectively. Percentage Fe absorption was also reduced by the substitution of casein or whey protein for egg white. Mean absorption values fell from 6.67 to 3.65% and 2.53 to 0.98%, respectively. When the intact milk-protein products were replaced by enzyme- or acid-hydrolyzed preparations, the dialyzable fraction increased markedly and in proportion to the extent of hydrolysis. A similar but much smaller effect on absorption was observed. These studies suggest that bovine casein and whey proteins are responsible at least in part for the poor bioavailability of the Fe in some infant formulas.