Bovine Milk Β-lactoglobulin Research Articles

The bile salt content of human bile impacts on simulated intestinal proteolysis of β-lactoglobulin

The gastrointestinal hydrolysis of food proteins has been portrayed in scientific literature to predominantly depend on the activity and specificity of proteolytic enzymes. Human bile has not been considered to facilitate proteolysis in the small intestine, but rather to assist in intestinal lipolysis. However, human bile can potentially influence proteins that are largely resistant to gastric digestion, and which are mainly hydrolysed after they have been transferred to the small intestine. We used purified and food-grade bovine milk β-lactoglobulin (βLg) to assess the impact of bile salts (BS) on the in vitro gastrointestinal digestion of this protein. Quantitative analysis showed that the proteolysis rate increased significantly with increasing BS concentration. The effect was consistent regardless of whether individual BS or real human bile samples, varying in BS concentrations, were used. The total BS content of bile was more important than its BS composition in facilitating the proteolysis of βlg. We also show that the impact of human bile observed during the digestion of purified βLg and βLg-rich whey protein isolate can be closely replicated by the use of individual BS mixed with phosphatidylcholine. This could validate simple BS/phosphatidylcholine mixtures as human-relevant substitutes of difficult-to-obtain human bile for in vitro proteolysis studies.

Cross-seeding of alpha-synuclein aggregation by amyloid fibrils of food proteins

The aggregation of the protein α-synuclein (aSyn) into amyloid fibrils in the human brain is associated with the development of several neurodegenerative diseases, including Parkinson's disease. The previously observed prion-like spreading of aSyn aggregation throughout the brain and the finding that heterologous cross-seeding of amyloid aggregation occurs in vitro for some proteins suggest that exposure to amyloids in general may pose a risk for disease development. To elucidate which protein fibril characteristics determine if and how heterologous amyloid seeding can occur, we investigated the potential of amyloid fibrils formed from proteins found in food, hen egg white lysozyme, and bovine milk β-lactoglobulin to cross-seed aSyn aggregation in the test tube. We observed that amyloid fibrils from lysozyme, but not β-lactoglobulin, potently cross-seeded the aggregation of aSyn as indicated by a significantly shorter lag phase of aSyn aggregation in the presence of lysozyme fibrils. The cross-seeding effect of lysozyme was found to be primarily driven by a surface-mediated nucleation mechanism. The differential seeding effect of lysozyme and β-lactoglobulin on aSyn aggregation could be explained on the basis of binding affinity, binding site, and electrostatic interactions. Our results indicate that heterologous seeding of proteins may occur depending on the physicochemical characteristics of the seed protein fibril. Our findings suggest that heterologous seeding has the potential to determine the pathogenesis of neurodegenerative amyloid diseases.

IIAEK Targets Intestinal Alkaline Phosphatase (IAP) to Improve Cholesterol Metabolism with a Specific Activation of IAP and Downregulation of ABCA1.

IIAEK (Ile-Ile-Ala-Glu-Lys, lactostatin) is a novel cholesterol-lowering pentapeptide derived from bovine milk β-lactoglobulin. However, the molecular mechanisms underlying the IIAEK-mediated suppression of intestinal cholesterol absorption are unknown. Therefore, we evaluated the effects of IIAEK on intestinal cholesterol metabolism in a human intestinal model using Caco-2 cells. We found that IIAEK significantly reduced the expression of intestinal cholesterol metabolism-associated genes, particularly that of the ATP-binding cassette transporter A1 (ABCA1). Subsequently, we chemically synthesized a novel molecular probe, IIXEK, which can visualize a complex of target proteins interacting with photoaffinity-labeled IIAEK by fluorescent substances. Through photoaffinity labeling and MS analysis with IIXEK for the rat small intestinal mucosa and intestinal lipid raft fractions of Caco-2 cells, we identified intestinal alkaline phosphatase (IAP) as a specific molecule interacting with IIAEK and discovered the common IIAEK-binding amino acid sequence, GFYLFVEGGR. IIAEK significantly increased IAP mRNA and protein levels while decreasing ABCA1 mRNA and protein levels in Caco-2 cells. In conclusion, we found that IIAEK targets IAP to improve cholesterol metabolism via a novel signaling pathway involving the specific activation of IAP and downregulation of intestinal ABCA1.

Characteristics of the interaction mechanisms of xylitol with β-lactoglobulin and β-casein: Amulti-spectral method and docking study.

Proteins and functional polyols are essential food ingredients coexisting in the food matrix, and therefore, interactions between them inevitably occur. In this study, the interaction mechanisms of xylitol (XY) with bovine milk β-lactoglobulin (β-LG) and β-casein (β-CN) were studied using multispectral techniques and molecular docking. It was found that XY strongly quenched the intrinsic fluorescence of β-LG and β-CN by static quenching. The values of the binding constants were KA(β-LG-XY)=3.369×104L/mol and KA(β-CN- XY)=7.821×104L/mol, indicating that the binding affinity of XY to β-CN was higher than that for β-LG. Hydrogen bonding and van der Waals forces played a major role in the interactions of XY with β-LG and β-CN, and both interactions were exothermic. Simultaneous fluorescence, three-dimensional fluorescence, and circular dichroism spectroscopy showed that binding of XY did not change the secondary structure of β-LG. However, XY interaction with β-CN led to the conversion of α-helices to random coils and structural loosening. In addition, molecular docking predicted the most likely binding sites of XY in both proteins and the interaction forces involved in binding, confirming the spectroscopic results. This study improves the understanding of the interactions of XY with β-LG and β-CN in functional dairy products and provides a theoretical basis for the addition of XY in a functional milk base.

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.

An experimental platform using human intestinal epithelial cell lines to differentiate between hazardous and non-hazardous proteins

Human intestinal epithelial cell lines (T84, Caco-2, and HCT-8) grown on permeable Transwell™ filters serve as models of the gastrointestinal barrier. In this study, this in vitro model system was evaluated for effectiveness at distinguishing between hazardous and non-hazardous proteins. Indicators of cytotoxicity (LDH release, MTT conversion), monolayer barrier integrity ([3H]-inulin flux, horseradish peroxidase flux, trans-epithelial electrical resistance [TEER]), and inflammation (IL-8, IL-6 release) were monitored following exposure to hazardous or non-hazardous proteins. The hazardous proteins examined include streptolysin O (from Streptococcus pyogenes), Clostridium difficile Toxins A and B, heat-labile toxin from enterotoxigenic Escherichia coli, listeriolysin O (from Listeria monocytogenes), melittin (from bee venom), and mastoparan (from wasp venom). Non-hazardous proteins included bovine and porcine serum albumin, bovine fibronectin, and ribulose bisphosphate carboxylase/oxygenase (RuBisco) from spinach. Food allergenic proteins bovine milk β-lactoglobulin and peanut Ara h 2 were also tested as was the anti-nutritive food protein wheat germ agglutinin. Results demonstrated that this model system effectively distinguished between hazardous and non-hazardous proteins through combined analysis of multiple cells lines and assays. This experimental strategy may represent a useful adjunct to multi-component analysis of proteins with unknown hazard profiles.

Binding study of novel anti-diabetic pyrimidine fused heterocycles to β-lactoglobulin as a carrier protein

Bovine milk β-lactoglobulin (β-LG) demonstrates significant resistance against both gastric- and simulated duodenal digestions. Therefore, it seems a realistic protein candidate for safe delivery and protection of particularly pH sensitive drugs in stomach. Recently, pyrimidine fused heterocycles (PFHs) revealed inhibitory properties against α-glucosidase (α-Gls) which is an important target enzyme for those drugs playing significant role in treatment of type-II diabetes and HIV/AIDS infection. The delivery of these compounds to small intestine where the enzyme plays its biological function is of great importance. Therefore, in this work the interaction of PFH compounds with β-LG, as a carrier protein has been investigated. Fluorescence, circular dichroism (CD) and UV–vis spectroscopic studies were used to examine the binding parameters and binding modes of the interaction. Moreover, the effects of PFH complexation on the secondary structures of β-LG were studied. All of these compounds significantly quenched the fluorescence intensity of β-LG due to a ground state complex formation. The binding and thermodynamic parameters were calculated. While hydrophobic interactions were proved to play significant role in the interaction of L1, L2 and L3, hydrogen bonding was shown to be important in the complexation of L4. The secondary structures of β-LG were preserved upon interaction of these synthetic compounds. Based on the achieved results, these potentially therapeutic agents can significantly bind to β-LG. Consequently, this protein might be useful for delivery of PFH compounds to small intestine where representing their potential ability to inhibit α-Gls and to reduce the postprandial hyperglycemia in diabetic patients.

Ionic Liquid-Induced Formation of the α-Helical Structure of β-Lactoglobulin

Structural modification of bovine milk β-lactoglobulin (β-LG) in aqueous 1-butyl-3-methylimidazolium nitrate ([bmim][NO3]) and ethylammonium nitrate ([EAN][NO3]) solutions has been investigated by Fourier transform infrared and circular dichroism spectroscopy. Remarkably, high ionic liquid (IL) concentrations (>15 mol %IL) caused formation of a non-native α-helical structure of β-LG and disruption of its tertiary structure. Furthermore, while [bmim][NO3] promoted protein aggregation, [EAN][NO3] inhibited it probably owing to differences in the unique solution structure (nanoheterogeneity) of the ILs by the different cationic species. The IL-induced α-helical formation of β-LG shows a behavior similar to the alcohol denaturation, but a disordered structure-rich state was observed in the β-α transition process by adding IL, in contrast to the case of an aqueous alcohol solution of protein. We propose that the molten salt-like property of aqueous IL solutions strongly support α-helical formation of proteins.

β‐Lactotensin derived from bovine β‐lactoglobulin exhibits anxiolytic‐like activity as an agonist for neurotensin NTS2 receptor via activation of dopamine D1 receptor in mice

β-Lactotensin (His-Ile-Arg-Leu) is a bioactive peptide derived from bovine milk β-lactoglobulin, acting as a natural agonist for neurotensin receptors. We found that β-lactotensin exhibited anxiolytic-like activity in an elevated plus-maze test after its intraperitoneal (i.p.) administration in mice. β-Lactotensin was also orally active. The anxiolytic-like activity of β-lactotensin after i.p. administration was blocked by levocabastine, an antagonist for the neurotensin NTS(2) receptor. β-Lactotensin had anxiolytic-like activity in wild-type but not Ntsr2-knockout mice. β-Lactotensin increased intracellular Ca(2+) flux in glial cells derived from wild-type mice but not Ntsr2 knockout mice. These results suggest that β-lactotensin acts as an NTS(2) receptor agonist having anxiolytic-like activity. The anxiolytic-like activity of β-lactotensin was also blocked by SCH23390 and SKF83566, antagonists for dopamine D(1) receptor, but not by raclopride, an antagonist for D(2) receptor. Taken together, β-lactotensin may exhibit anxiolytic-like activity via NTS(2) receptor followed by D(1) receptor.

The Hypocholesterolemic Activity of Transgenic Rice Seed Accumulating Lactostatin, a Bioactive Peptide Derived from Bovine Milk β-Lactoglobulin

Lactostatin is a novel pentapeptide (IIAEK) derived from bovine milk β-lactoglobulin with greater hypocholesterolemic activity than β-sitosterol, the drug commonly used to treat hypercholesterolemia. We developed transgenic rice expressing lactostatin as a fusion protein with seed storage protein (SSP) glutelins under the control of three different endosperm-specific promoters. Lactostatin accumulated in transgenic rice seed at approximately 1.6 mg/g seeds (dry seeds) without any apparent influence on seed traits such as endogenous SSP expression levels or alterations in the intracellular structures of endosperm cells. Short-term (three day) oral administration of the glutelin fraction containing lactostatin (namely three times of 300 mg/kg body weight/day) extracted from transgenic rice seeds resulted in hypocholesterolemic activity in rats; namely, the serum low-density-lipoprotein (LDL) cholesterol level was significantly reduced accompanied by a significant increase in beneficial serum high-density-lipoprotein (HDL) cholesterol.

Silver‐ and Coomassie‐staining protocols: Detection limits and compatibility with ESI MS

Staining protocols for PAGE have to be sensitive and should not impair further MS analysis of selected samples. In this study, the MS compatibility of different silver- and Coomassie-staining protocols with a nano-LC-MS/MS system was systematically elucidated. Altogether, 13 different silver-staining, 1 imidazole-staining and 2 Coomassie-staining protocols were used and compared to each other for their achieved sequence coverage and their detection sensitivity. Three proteins were used as model proteins (bovine serum albumin, rabbit L-lactate dehydrogenase, bovine milk beta-lactoglobulin) in decreasing concentration (12 pmol down to 30 fmol) and different staining protocols were applied. The conclusion of this study is that two silver-staining protocols (Blum, H. et al.,. Electrophoresis 1987, 8, 93-99 and Shevchenko, A. et al.,. Anal. Chem. 1996, 68, 850-858) combine good sequence coverage and good sensitivity and are recommended for nano-LC-MS/MS analysis.

A novel regulatory pathway for cholesterol degradation via lactostatin

Our group previously discovered a novel hypocholesterolemic pentapeptide (IIAEK: Ile-Ile-Ala-Glu-Lys, or what we describe as “lactostatin”) derived from bovine milk β-lactoglobulin. To clarify the mechanism of the hypocholesterolemic action of lactostatin, we screened the target gene and signal transducing pathway induced by lactostatin in HepG2, a human liver cell line. Unexpectedly, we found that water-soluble lactostatin can activate cholesterol 7α-hydroxylase (CYP7A1) gene expression. Treatment with mitogen-activated protein kinase (MAPK) inhibitor or calcium (Ca 2+) channel blocker blocked this activation. We also found that lactostatin regulates the phosphorylation of extracellular signal-regulated kinase (ERK) and intracellular Ca 2+ concentration. Here, we show the involvement of a new regulatory pathway in the calcium-channel-related MAPK signaling pathway of lactostatin-mediated cholesterol degradation. Oligopeptide shows promise as a new molecule for the development of medicines and functional foods to prevent and improve hypercholesterolemia and atherosclerosis.

Effect of β-lactotensin on acute stress and fear memory

β-Lactotensin (β-LT) is a bioactive peptide derived from bovine milk β-lactoglobulin and is a natural ligand for neurotensin receptors. We examined the effect of β-LT on restraint stress and fear memory in mice. Mice subjected to acute restraint stress exhibited a decreased number of head-dips and increased head-dip latency compared to non-stressed controls in the hole-board test, reflecting increased stress-induced behaviors. However, prior administration of β-LT improved the behaviors caused by stress. The anti-stress effect of β-LT was blocked by levocabastine, a neurotensin receptor subtype 2 (NTR2) antagonist. In the fear-conditioning test, the duration of freezing responses by cued fear conditioning was significantly reduced in mice administered β-LT compared with control mice. These results suggest that β-LT has an anti-stress effect and promotes the extinction of fear memory, which may be mediated by NTR2.

Amino acid residue substitution at T-cell determinant-flanking sites in beta-lactoglobulin modulates antigen presentation to T cells through subtle conformational change.

We compared T-cell responses to regions in residues 21-40 of A and B variants of bovine milk beta-lactoglobulin (beta-LG) that vary by two different amino acid residues at 64 and 118. Results showed that T cells from C57/BL6 and C3H/HeN mice immunized with peptide 21-40 or BALB/c mice immunized with peptide 21-32 or 25-40 responded more vigorously to beta-LG B than to beta-LG A. This difference in response to 25-40 in BALB/c mice was not observed when beta-LGs B and A were denatured, suggesting that the conformation difference affects display of the determinant 25-40. Reactivity of anti-beta-LG monoclonal antibodies and molecular modeling using molecular dynamics calculations revealed subtle differences in the three-dimensional structure of these two variants. Furthermore, substitution of two amino acid residues at sites distant from the T-cell determinant induced differential determinant display on antigen-presenting cells, possibly due to subtle conformational changes in beta-LG.

Identification of Novel Hypocholesterolemic Peptides Derived from Bovine Milk β-Lactoglobulin

This study was designed to clarify the mechanisms of hypocholesterolemic action of β-lactoglobuline tryptic hydrolysate (LTH) and to identify the novel hypocholesterolemic peptide derived from LTH by screening using Caco-2 cells and animal studies. Serum and liver cholesterol levels were significantly lower in rats fed LTH than in those fed casein tryptic hydrolysate (CTH). The present study suggests that the inhibition of micellar solubility of cholesterol which causes the suppression of cholesterol absorption by a direct interaction between cholesterol mixed micelles, and LTH in the jejunal epithelia is part of the mechanism underlying the hypocholesterolemic action of LTH. Though no one could trace the hypocholesterolemic peptide to any protein origin, we identified, for the first time, a novel hypocholesterolemic peptide, Ile-Ile-Ala-Glu-Lys (IIAEK). Surprisingly, the present study provides the first direct evidence that a new hypocholesterolemic peptide derived from β-lactoglobuline can powerfully influence serum cholesterol levels and exhibit a greater hypocholesterolemic activity in comparison with that of medicine, β-sitosterol, in animal studies.