Bovine Β-lactoglobulin Research Articles

Change in conformational, digestive and immunological characteristics of bovine allergen β-lactoglobulin induced by metal ions in combination with heating

Aggregation of bovine β-lactoglobulin is affected easily by external factors. In this study, effects of metal ions combining with temperature on aggregation of β-lactoglobulin were explored. The conformational characteristics of aggregates were detected by environment scanning electron microscope, CD spectrum and free sulfhydryl group, respectively. Digestive and immunological characteristics were assessed by simulated digestion in vitro and ELISA respectively. The results showed that the morphology of β-lactoglobulin aggregates became more amorphous in Cu2+ and Mg2+ treated samples and more constricted in Zu2+-induced protein. Among them, Cu2+ altered the secondary structure of β-lactoglobulin aggregates and free sulfhydryl content most as well as that in gastric digestion. However, all ion-treated groups had similar digestive stability in intestinal digestion. Specially, Ca2+ and Mg2+ made the antigenicity and potential allergenicity of β-lactoglobulin aggregates decrease, which helps us understand the role of metal ions in immunological characteristics.

Bulk phase behaviour vs interface adsorption: Effects of anions and isotopes on β-lactoglobulin (BLG) interactions

HypothesisProtein adsorption is highly relevant in numerous applications ranging from food processing to medical implants. In this context, it is important to gain a deeper understanding of protein-protein and protein-surface interactions. Thus, the focus of this investigation is on the interplay of bulk properties and surface properties on protein adsorption. It was hypothesised that the type of solvent and ions in solution should significantly influence the protein’s bulk and interface behaviour, which has been observed in literature and previous work for other net negatively charged, globular proteins such as bovine serum albumin (BSA). ExperimentsThe phase behaviour of β-lactoglobulin (BLG) with lanthanum chloride (LaCl3) and iodide (LaI3) in normal water H2O(l) and heavy water (D2O(l)) was established via optical microscopy and ultraviolet-visible spectroscopy. The formation of an adsorption layer and its properties such as thickness, density, structure, and hydration was investigated via neutron reflectivity, quartz-crystal microbalance with dissipation, and infra-red measurements. Findingsβ-lactoglobulin does not show significant anion-induced or isotope-induced effects - neither in bulk nor at the solid-liquid interface, which deviates strongly from the behaviour of bovine serum albumin. We also provide a comprehensive discussion and comparison of protein-specific bulk and interface behaviour between bovine serum albumin and β-lactoglobulin dependent on anion, cation, solvent, and substrate properties. These findings pave the way for understanding the transition from adsorption to crystallisation.

Binding Sites for Oligosaccharide Repeats from Lactic Acid Bacteria Exopolysaccharides on Bovine β-Lactoglobulin Identified by NMR Spectroscopy.

Lactic acid bacterial exopolysaccharides (EPS) are used in the food industry to improve the stability and rheological properties of fermented dairy products. β-Lactoglobulin (BLG), the dominant whey protein in bovine milk, is well known to bind small molecules such as fatty acids, vitamins, and flavors, and to interact with neutral and anionic polysaccharides used in food and pharmaceuticals. While sparse data are available on the affinity of EPS–milk protein interactions, structural information on BLG–EPS complexes, including the EPS binding sites, is completely lacking. Here, binding sites on BLG variant A (BLGA), for oligosaccharides prepared by mild acid hydrolysis of two EPS produced by Streptococcus thermophilus LY03 and Lactobacillus delbrueckii ssp. bulgaricus CNRZ 1187, respectively, are identified by NMR spectroscopy and supplemented by isothermal titration calorimetry (ITC) and molecular docking of complexes. Evidence of two binding sites (site 1 and site 2) on the surface of BLGA is achieved for both oligosaccharides (LY03-OS and 1187-OS) through NMR chemical shift perturbations, revealing multivalency of BLGA for EPS. The affinities of LY03-OS and 1187-OS for BLGA gave KD values in the mM range obtained by both NMR (pH 2.65) and ITC (pH 4.0). Molecular docking suggested that the BLGA and EPS complexes depend on hydrogen bonds and hydrophobic interactions. The findings provide insights into how BLGA engages structurally different EPS-derived oligosaccharides, which may facilitate the design of BLG–EPS complexation, of relevance for formulation of dairy products and improve understanding of BLGA coacervation.

Impact of denaturing agents on surface properties of myoglobin solutions

The addition of denaturants strongly influences the surface properties of aqueous myoglobin solutions. The effect differs from the results for mixed solutions of the denaturants and other globular proteins, for example, bovine serum albumin (BSA), lysozyme and β-lactoglobulin (BLG), although the surface properties of the solutions of the pure proteins are similar. The kinetic dependencies of the dynamic surface elasticity of myoglobin solutions with guanidine hydrochloride (GuHCl) reveal at least two adsorption steps at denaturant concentrations higher than 1 M: a very fast increase of the dynamic surface elasticity to approximately 30 mN/m at the beginning of adsorption, and a slower growth to abnormally high values of 250–300 mN/m. At the same time, the surface elasticity of BSA/GuHCl, BLG/GuHCl and lysozyme/GuHCl solutions is a non-monotonic function of the surface age, and does not exceed 50 mN/m close to equilibrium. The high surface elasticity of myoglobin/GuHCl solutions may be associated with protein aggregation in the surface layer. The formation of aggregates is confirmed by ellipsometry and Brewster angle microscopy. The addition of ionic surfactants to protein solutions leads to the formation of myoglobin/surfactant complexes, and the kinetic dependencies of the dynamic surface elasticity display local maxima indicating multistep adsorption kinetics, unlike the corresponding results for solutions of other globular proteins mixed with ionic surfactants. Ellipsometry and infrared reflection-absorption spectroscopy allow tracing the adsorption of the complexes and their displacement from the interface at high surfactant concentrations.

Energetic and structural effects of the Tanford transition on ligand recognition of bovine β-lactoglobulin

Bovine β-lactoglobulin, an abundant protein in whey, is a promising nanocarrier for peroral administration of drug-like hydrophobic molecules, a process that involves transit through the different acidic conditions of the human digestive tract. Among the several pH-induced conformational rearrangements that this lipocalin undergoes, the Tanford transition is particularly relevant. This transition, which occurs with a midpoint around neutral pH, involves a conformational change of the E-F loop that regulates accessibility to the primary binding site. The effect of this transition on the ligand binding properties of this protein has scarcely been explored. In this study, we carried out an energetic and structural characterization of β-lactoglobulin molecular recognition at pH values above and below the zone in which the Tanford transition occurs. The combined analysis of crystallographic, calorimetric, and molecular dynamics data sheds new light on the interplay between self-association, ligand binding, and the Tanford pre- and post-transition conformational states, revealing novel aspects underlying the molecular recognition mechanism of this enigmatic lipocalin.

Crocin inhibits urea-induced amyloid formation by bovine β-lactoglobulin

Crocin stabilizes the native structure of β-lactoglobulin and attenuates urea-induced unfolding and loss of β-sheet structure during amyloidogenesis.

IgE reactivity to milk components in dogs with cutaneous adverse food reactions.

We investigated the IgE reactivity to crude and purified milk antigens in the sera of 112 dogs with cutaneous adverse food reactions (CAFRs). Of the 112 dogs, 33 (29%) had specific IgE for crude milk antigens. In the dogs with milk-specific IgE, IgE reactivity to casein, bovine serum albumin (BSA), α-lactalbumin, β-lactoglobulin, and bovine IgG were 81%, 85%, 39%, 27%, and 35%, respectively. Casein and BSA may be important allergens in dogs with CAFRs. Some canine vaccines contain casein hydrolysate as a stabilizer and the pooled serum with anti-casein IgE showed IgE reactivity to the vaccines containing it. Information about IgE reactivity to casein in dogs with CAFRs could be useful for predicting adverse reactions to the vaccines including casein hydrolysate.

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.

Modulation of amyloid fibrillation of bovine β-lactoglobulin by selective methionine oxidation.

Deposition of oxidation-modified proteins during normal aging and oxidative stress are directly associated with systemic amyloidoses. Methionine (Met) is believed to be one of the most readily oxidisable amino acid residues of protein. Bovine beta-lactoglobulin (β-lg), a model globular whey protein, has been presented as a subsequent paradigm for studies on protein aggregation and amyloid formation. Herein, we investigated the effect of t-butyl hydroperoxide (tBHP)-induced oxidation on structure, compactness and fibrillation propensity of β-lg at physiological pH. Notably, whey protein modification, specifically Met residues, plays an important role in the dairy industry during milk processing and lowering nutritional value and ultimately affecting their technological properties. Several bio-physical studies revealed enhanced structural flexibility and aggregation propensity of oxidised β-lg in a temperature dependent manner. A molecular docking study is used to predict possible interactions with tBHP and infers selective oxidation of methionine residues at 7, 24 and 107 positions. From our studies, it can be corroborated that specific orientations of Met residues directs the formation of a partially unfolded state susceptible to fibrillation with possible different cytotoxic effects. Our studies have greater implications in deciphering the underlying mechanism of different whey proteins encountering oxidative stress. Our findings are also important to elucidate the understanding of oxidation induced amyloid fibrillation of protein which may constitute a new route to pave the way for a modulatory role of oxidatively stressed proteins in neurological disorders.

PH – Responsive colloidal carriers assembled from β-lactoglobulin and Epsilon poly-L-lysine for oral drug delivery

Site specific oral delivery of many biopharmaceutical classification system (BCS) class II and IV drugs is challenging due to their poor solubility, low permeability and degradation in the gastrointestinal tract. Whilst colloidal carriers have been used to improve the bioavailability of such drugs, most nanocarriers based drug delivery systems suffer from multiple disadvantages, including low encapsulation efficiency (liposomes, polymeric nanoparticles), complex synthesis methods (silica, silicon-based materials) and poorly understood biodegradability (inorganic nanoparticles). Herein, a novel pH responsive nanocolloids were self-assembled using natural compounds such as bovine β-lactoglobulin (BLG) and succinylated β-lactoglobulin (succ. BLG) cross-linked with epsilon poly l-lysine (BCEP and BCP), and found to possess high loading capacity, high aqueous solubility and site-specific oral delivery of a poorly soluble nutraceutical (curcumin), improving its physicochemical properties and biological activity in-vitro and ex-vivo. Our optimized synthesis formed colloids of around 200 nm which were capable of encapsulating curcumin with ~100% encapsulation efficiency and ~10% w/w drug loading. By forming nanocomplexes of curcumin with BLG and succ. BLG, the aqueous solubility of curcumin was markedly increased by ~160-fold and ~86-fold, respectively. Encapsulation with BLG increased the solubility, whereas succ. BLG prevent release of encapsulated curcumin when subjected to gastric fluids as it is resistant to breakdown on exposure to pepsin at acidic pH. In conditions mimicking the small intestine, Succ. BLG was more soluble resulting in sustained release of the encapsulated drug at pH 7.4. Additionally, crosslinking succ. BLG with E-PLL significantly enhanced curcumin’s permeability in an in-vitro Caco-2 cell monolayer model compared to curcumin solution (dissolved in 1% DMSO), or non-crosslinked BLG/succ. and BLG. In a mouse-derived intestinal epithelial 3D organoid culture stimulated with IL-1β, BLG-CUR and crosslinked BCEP nanoparticles reduced the production of inflammatory cytokines and chemokines such as Tnfα and Cxcl10 more than curcumin solution or suspension while these nanoparticles were non-toxic to organoids. Overall this work demonstrates the promise of nutraceutical-based hybrid self-assembled colloidal system to protect hydrophobic drugs from harsh gastrointestinal conditions and improve their solubility, dissolution, permeability and biological activity.

Charge variant analysis of protein-based biopharmaceuticals using two-dimensional liquid chromatography hyphenated to mass spectrometry

The profile of charge variants represents an important critical quality attribute of protein-based biopharmaceuticals, in particular of monoclonal antibodies, and must therefore becontrolled. In this work, 2D-LC methods for charge variant analysis were developed using a strong cation-exchange chromatography (SCX) as first dimension (1D) separation. Non-porous SCX (3 µm) particle columns and different mobile phases were evaluated using a test mixture of some standard proteins of different size and pI (comprising myoglobin, bovine serum albumin, cytochrome c, lysozyme and β-lactoglobulin) and two monoclonal IgG1 antibodies (NIST mAb and Secukinumab). The most promising 1D eluent for SCX was a salt-mediated pH-gradient system using a ternary mobile phase system with 2-(N-morpholino)ethanesulfonic acid, 1,3-diamino-2-propanol and sodium chloride. For the second dimension (2D), a desalting reversed-phase liquid chromatography (RP-LC) was chosen to enable the hyphenation of the charge variant separation with mass spectrometric (MS) detection. While for intact mAbs the 2D just served for desalting without additional selectivity, the 2D contributed some orthogonal selectivity for the mAb fragment separation. Various core-shell and monolithic columns were tested and variables such as gradient time and flow rate systematically optimized. Unexpectedly, a C4 400 Å column (3.4 µm diameter with 0.2 µm porous shell) provided higher peak capacities compared to the same 1000 Å column (2.7 µm diameter with 0.5 µm porous shell). A thinner shell appeared to be more advantageous than wider pores under high flow regime. An ultra-fast RP-LC method with a run time of one minute was developed using trifluoroacetic acid which was later replaced by formic acid as additive for better MS compatibility. The successful hyphenation of the two orthogonal separation modes, SCX and RP-LC, could be demonstrated in the multiple heart-cutting and the full comprehensive mode. MS analysis using a high-resolution quadrupole time-of-flight instrument enabled to identify different glycoforms and some major charge variants of the antibody at the intact protein level as well as on the subunit level (Fc/2, Lc, Fd’) in a middle-up approach by 2D-LC-ESI-MS analysis.

Structural mechanism of the Tanford transition of bovine β-lactoglobulin through microsecond molecular dynamics simulations

X-ray analysis has provided structural data about a pH-driven conformational change in β-lactoglobulin (BLG) known as the Tanford transition, which occurs at around pH 7 and involves the EF loop, which acts as a lid closing the internal cavity of the protein below pH 7 and opening it above pH 7. NMR studies using wild-type BLG have encountered problems trying to explain the Tanford transition, however, they have provided important insight using a dimeric BLG mutant, revealing that the opening and closure of the EF loop consists of two types of motions in the microsecond and milliseconds timescales. This provides valuable information indicating that the dimeric state is a good model to study the Tanford transition, although the understanding of this structural change is still lacking at the atomic level. We performed microsecond molecular dynamics (MD) simulations starting from different conformations of BLG in the monomeric and dimeric state, with protonated and deprotonated E89, in order to explore the Tanford transition. Our results provide structural information for the transition from the closed to the open conformation in BLG and show it occurs in the dimeric state in the microsecond timescale, in line with the fast motion observed through NMR experiments. In addition, MD simulations coupled to MMGBSA approach indicated that the most populated conformer of BLG in the open state is able to bind ligands with similar affinity to that of BLG at neutral pH obtained through crystallographic experiments. Communicated by Ramaswamy H. Sarma

Effect of Ligands on HP-Induced Unfolding and Oligomerization of β-Lactoglobulin

To probe intermediate states during unfolding and oligomerization of proteins remains a major challenge. High pressure (HP) is a powerful tool for studying these problems, revealing subtle structural changes in proteins not accessible by other means of denaturation. Bovine β-lactoglobulin (BLG), the main whey protein, has a strong propensity to bind various bioactive molecules such as retinol and resveratrol, two ligands with different affinity and binding sites. By combining in situ HP-small-angle neutron scattering (SANS) and HP-ultraviolet/visible absorption spectroscopy, we report the specific effects of these ligands on three-dimensional conformational and local changes in BLG induced by HP. Depending on BLG concentration, two different unfolding mechanisms are observed in situ under pressures up to ∼300 MPa: either a complete protein unfolding, from native dimers to Gaussian chains, or a partial unfolding with oligomerization in tetramers mediated by disulfide bridges. Retinol, which has a high affinity for the BLG hydrophobic cavity, significantly stabilizes BLG both in three-dimensional and local environments by shifting the onset of protein unfolding by ∼100 MPa. Increasing temperature from 30 to 37°C enhances the hydrophobic stabilization effects of retinol. In contrast, resveratrol, which has a low binding affinity for site(s) on the surface of the BLG, does not induce any significant effect on the structural changes of BLG due to pressure. HP treatment back and forth up to ∼300 MPa causes irreversible covalent oligomerization of BLG. Ab initio modeling of SANS shows that the oligomers formed from the BLG-retinol complex are smaller and more elongated compared to BLG without ligand or in the presence of resveratrol. By combining HP-SANS and HP-ultraviolet/visible absorption spectroscopy, our strategy highlights the crucial role of BLG hydrophobic cavity and opens up new possibilities for the structural determination of HP-induced protein folding intermediates and irreversible oligomerization.

Impact of Protein Corona on Noncovalent Molecule-Gold Nanoparticle-Based Sensing.

Gold nanoparticle (AuNP)-based sensors have been extensively applied for sensing or imaging. It is known that a protein shell named protein corona (PC) formed around the nanomaterials could not only block the desired function of nanomaterials but also affect their behavior, which is a hot and important issue needing consideration. Therefore, we hypothesize that the formation of PC around AuNPs could inevitably affect the AuNP-based target assay. In this work, the effects of PC on the detection results in sensors based on AuNPs were studied. Three types of noncovalent molecule-AuNP sensors including AuNP-dichlorofluorescein, AuNP-aptamer, and AuNP-antibody-DNA were constructed, and several typical proteins (bovine serum albumin, fibrinogen, hemoglobin, and β-lactoglobulin), milk, and fetal bovine serum were selected as models for the formation of PCs. This study shows that the PC could cause the loss of detection signals (up to 80%) and result in positive deviation of the measuring value compared with the true value. Moreover, the loss of detection signals could also increase the limits of detection (almost 10 times), decreasing the sensitivity of the three types of sensors, as proposed in this work compared to that without PC. Moreover, the polyethylene glycol backfilling strategy could not resolve the negative effects of PC on noncovalent molecule-AuNP sensors. The impacts of PC on detection results from noncovalent molecule-AuNP sensors would cause misdiagnosis or wasted production, which needs careful reconsideration of the AuNP-based detection in application fields like clinic diagnosis, food safety control, and so forth.

Spectroscopic, molecular docking and molecular dynamic simulation studies on the complexes of β-lactoglobulin, safranal and oleuropein

Herbal bioactive compounds have captured pronounced attention considering their health-promoting effects as well as their functional properties. In this study, the binding mechanism between milk protein bovine β-lactoglobulin (β-LG), oleuropein (OLE) and safranal (SAF) found in olive leaf extract and saffron, respectively via spectroscopic and in silico studies. Fluorescence quenching information exhibited that interactions with both ligands were spontaneous and hydrophobic interactions were dominant. Also, the CD spectroscopy results demonstrated the increase in β-sheet structure and decrease in the α-helix content for both ligands. Size of β-LG-OLE complex was higher than β-LG-SAF due to the conformation and larger molecular size. Molecular docking and simulation studies revealed that SAF and OLE bind in the central calyx of β-LG and the surface of β-LG next to hydrophobic residues. Lastly, OLE formed a more stabilized complex compared to SAF based on the molecular dynamic simulation results.

Studies on Molecular Interactions between Bovine β-Lactoglobulin and Silver Nanoparticles.

Silver Nanoparticles (AgNPs) were found to modulate the fibrillation of Bovine Β-Lactoglobulin (BLG). To gain an insight regarding the mechanism of BLG aggregation modulation by AgNPs at molecular level, studies on the interactions between BLG and AgNPs were carried out. Protein-ligand interactions were studied based on Trp fluorescence quenching (at four different temperatures), synchronous and three-dimensional fluorescence and circular dichroism spectroscopy (far-UV and near-UV). Protein-nanoparticles association constant was in the range of 106 -1010 M-1 and the quenching constant was determined as ~107 M-1. Ground state complexation between the protein and nanoparticles was predicted. Change in polarity surrounding the Trp residue was not detected by synchronous and three-dimensional fluorescence spectroscopy. AgNPs caused a global change in the secondary and tertiary structure of the protein as revealed from far-UV and near-UV CD spectroscopy. Enthalpy driven complexation between the protein and nanoparticles indicates the involvement of hydrogen bonding and/or van der Waals interactions. Modulation of BLG aggregation by AgNPs is due to strong binding of the nanoparticles with BLG, which also causes structural perturbations of the protein.

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.

Selective Protein Separation Based on Charge Anisotropy by Spherical Polyelectrolyte Brushes.

Protein purification is of vital importance in the food industry, drug discovery, and other related fields. Among many separation methods, polyelectrolyte (PE)-based phase separation was developed and recognized as a low-cost purification technique. In this work, spherical polyelectrolyte brushes (SPBs) with a high specific surface area were utilized to study the protein accessibility and selective protein binding on highly charged nanoparticles (NPs) as well as the selective phase separation of proteins. The correlation between charge anisotropy, protein binding, and phase separation was investigated on various protein systems including those proteins with similar isoelectric points (pI) such as bovine serum albumin (BSA) and β-lactoglobulin (BLG), proteins with similar molecular weights such as BSA and hemoglobin (HB), and even protein variants (BLG-A and -B) with a tiny difference of amino acids. The nonspecific electrostatic interaction studied by turbidimetric titrations and isothermal calorimetry titration (ITC) indicates a specific binding between proteins and SPBs arising from the charge anisotropy of proteins. An optimized output based on selective protein binding on SPBs could be correlated for efficient protein separation through tuning external conditions including pH and ionic strength. These findings, therefore, proved that phase separation based on selective protein adsorption by SPBs was an efficient alternative for protein separation compared with the traditional practice.

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.

Antioxidant ferulic acid prevents the aggregation of bovine β-lactoglobulin in vitro

Amyloids, a well-ordered β-sheet-enriched structural network, can be broadly defined as insoluble protein aggregates that are linked to a wide variety of diseases including systemic amyloidosis and some neurodegenerative disorders. Ferulic acid (FA), a phenolic acid, abundant in antioxidant and efficient pharmaceutical has beneficial effects against several ailments. Based on this, we have investigated the protective role of FA on amyloid formation of bovine β-lactoglobulin (β-lg), a model globular protein. Using a set of in vitro biophysical methods, such as UV-Vis spectroscopy, fluorescence, circular dichroism, transmission electron microscopy, etc., our research group has concluded that FA significantly inhibits the heat-induced amyloid formation of β-lg and this inhibitory effect is dose-dependent. Exposed surface hydrophobicity of β-lg amyloid fibrils decreased significantly in the presence of FA. Docking study revealed that ionic and hydrogen bonding interactions between FA and β-lg prevented protein conformational changes leading to fibrillation. We anticipate that our finding would give an insight into the protein aggregation inhibited by the antioxidant compound, FA and pave the way for finding and developing other new small molecules (protein misfolding inhibitors) that give similar result against amyloid fibril formation and its allied neurodegenerative disorders. Ferulic acid inhibits the aggregation of β-lg in a dose-dependent manner.