Aggregation and conformational changes of bovine β-lactoglobulin subjected to dynamic high-pressure microfluidization in relation to antigenicity

Our previous research indicated that dynamic high-pressure microfluidization (DHPM) had a significant effect on the antigenicity of β-lactoglobulin (β-LG). In this study, aggregation and conformational changes subjected to DHPM (0.1-160 MPa) were investigated in relation to antigenicity. When DHPM pressure increased from 0.1 to 80 MPa, disaggregation of β-LG samples and partial unfolding of the molecule were accompanied by an increase in β-LG antigenicity, which was reflected in the decrease of particle size, increase of free sulfhydryl (SH) contents and β-strands contents, and slight exposure of aromatic amino acid residues. At pressures above 80 MPa, the reaggregation of β-LG may contribute to the decrease in antigenicity, which was reflected by an increase in particle size, the formation of aggregates, a decrease of in SH and β-strands contents, and slight changes in aromatic amino acid residues. Aggregation and conformational changes of β-LG under DHPM was related to its antigenicity.     

Effect of high-pressure treatment on denaturation of bovine β-lactoglobulin and α-lactalbumin

The effect of high-pressure treatment on denaturation of β-lactoglobulin and α-lactalbumin in skimmed milk, whey, and phosphate buffer was studied over a pressure range of 450–700 MPa at 20 °C. The degree of protein denaturation was measured by the loss of reactivity with their specific antibodies using radial immunodiffusion. The denaturation of β-lactoglobulin increased with the increase of pressure and holding time. Denaturation rate constants of β-lactoglobulin were higher when the protein was treated in skimmed milk than in whey, and in both media higher than in buffer, indicating that the stability of the protein depends on the treatment media. α-Lactalbumin is much more baroresistant than β-lactoglobulin as a low level of denaturation was obtained at all treatments assayed. Denaturation of β-lactoglobulin in the three media was found to follow a reaction order of n = 1.5. A linear relationship was obtained between the logarithm of the rate constants and pressure over the pressure range studied. Activation volumes obtained for the protein treated in milk, whey, and buffer were −17.7 ± 0.5, −24.8 ± 0.4, and −18.9 ± 0.8 mL/mol, respectively, which indicate that under pressure, reactions of volume decrease of β-lactoglobulin are favoured. Kinetic parameters obtained in this work allow calculating the pressure-induced denaturation of β-lactoglobulin on the basis of pressure and holding times applied.     

Effect of high-pressure treatment on in-vitro digestibility of β-lactoglobulin

The effect of high-pressure (HP)-treatment on β-lactoglobulin (β-Lg) was investigated using in-vitro pepsin digestion under simulated gastric conditions. HP-treatment of β-Lg at 400 MPa for 10 min only slightly increased its subsequent hydrolysis by pepsin. However, higher pressure treatments (600 and 800 MPa) resulted in rapid digestion of β-Lg. After these higher pressure treatments, β-Lg disappeared in less than 1 min of pepsin incubation as determined by SDS-PAGE analysis. Mass spectrometry analysis of the digestion products at corresponding incubation times revealed rapid and progressive degradation of β-Lg. Most (> 90%) of the peptide products following pepsin digestion of HP-treated β-Lg were less than 1500 Da in size. Peptide products from pepsin digestion were identified and mapped to β-strand regions (Leu₃₂–Leu₅₄ and Phe₈₂–Leu₁₀₄) and to the N- and C-terminals regions (Leu₁–Leu₁₀ and Ser₁₅₀–Leu₁₅₆) of β-Lg. While these regions corresponded to known IgE epitopes of β-Lg, the predominant peptides resulting from 60 s of incubation were short (7–10 residues) in length. These results demonstrate that HP-treatment increased the digestibility of β-Lg and represents a promising processing technology for reducing the allergenicity of known allergens in a wide variety of food materials.Industrial relevanceHigh-pressure treatment is widely used to enhance the functional attributes of food proteins. The potential for enhanced nutritional value of β-Lg was also demonstrated here by its increased digestibility. High-pressure treatment followed by incubation with proteases may represent a method for the commercial production of bioactive peptides such as inhibitors of angiotensin converting enzyme. More importantly, high-pressure-induced unfolding of milk proteins may reduce their allergenicity. Unfolded proteins are less likely to become agents of immunological sensitization because they are more readily hydrolyzed. Thus high-pressure treatment applied to food ingredients such as whey protein isolate may contribute to the development of hypoallergenic foods.     

Effect of glycation on the gastrointestinal digestibility and immunoreactivity of bovine β-lactoglobulin

Immunoreactivity of bovine β-lactoglobulin (β-Lg) hydrolysates obtained after a simulated gastrointestinal digestion and previously glycated via Maillard reaction with galactose, tagatose, and dextran of 10 or 20 kDa has been determined, with a view to study the effect of glycation and aggregation degree of β-Lg on its residual immunoreactivity. High levels of glycation impaired β-Lg proteolysis and, consequently, increased the IgG- and IgE-reactivities of hydrolysates, regardless of the carbohydrate used. Protein aggregation during the advanced stages of Maillard reaction had a masking effect on β-Lg epitopes, counteracting the negative effect of the lower digestibility of glycated protein on its allergenicity. Finally, the use of polysaccharides as glycation agents did not contribute to enhancement of the masking effect of the attached carbohydrate on β-Lg epitopes. These findings stress the importance of evaluating the impact of glycation on protein gastrointestinal digestibility prior to investigation of the immunoreactivity of protein Maillard complexes.     

Steady-state kinetics of tryptic hydrolysis of β-lactoglobulin after dynamic high-pressure microfluidization treatment in relation to antigenicity

Our previous research revealed that dynamic high-pressure microfluidization (DHPM) increased the antigenicity of β-lactoglobulin (β-Lg) below 80 MPa, which was related to the unfolding of protein. To test the hypothesis that the unfolding of protein may change proteolytic susceptibility of β-Lg and modulate its antigenicity during the digestion, we developed that the steady-state kinetics of tryptic hydrolysis of β-Lg subjected to DHPM (0.1–80 MPa) have been investigated in relation to the antigenicity in this study. According to the steady-state kinetics analysis, the improved digestion of β-Lg was accompanied with the obvious decrease of antigenicity during the hydrolysis with pressure increasing, reflected by the increase of k _c , the decrease of K _m, the increase of overall catalytic efficiency (k _c/K _m), and the increase of the binding volume. It was indicated that although DHPM can increase the antigenicity of β-Lg, the enhanced digestibility of β-Lg at elevated pressure contributed to a decrease of antigenicity during the hydrolysis.     

Effect of glycation of bovine β-lactoglobulin with galactooligosaccharides on the growth of human faecal bacteria

The in vitro fermentation selectivity of purified galactooligosaccharides (GOS) after their conjugation with bovine β-lactoglobulin (β-LG) via the Maillard reaction and a subsequent simulated gastrointestinal digestion was evaluated. Changes in human faecal bacterial populations, lactic acid and short-chain fatty acids after 10 h and 24 h of fermentation of the digested β-LG:GOS conjugates revealed that this mixture of glycated peptides had a similar bifidogenic activity to the unconjugated GOS. These findings could open up new applications for Maillard reaction products in the functional foods field.     

Antigenicity of β-lactoglobulin reduced by combining with oleic acid during dynamic high-pressure microfluidization: Multi-spectroscopy and molecule dynamics simulation analysis

Some food components can modulate the antigenicity of β-lactoglobulin (β-LG). This study investigated the role of oleic acid (OA) in reducing the antigenicity of β-LG. The results indicate the antigenicity of β-LG gradually decreased from 15 (sample with no OA) to 9.86, 7.51, and 6.01 µg/mL when interacting with OA during dynamic high-pressure microfluidization treatment at 0.1, 80, and 160 MPa. Although binding sites (n) of β-LG combined with OA at 0.1, 80, and 160 MPa decreased from 0.79 to 0.5 and 0.66, β-LG had a higher binding affinity (K_a) to OA than that of untreated β-LG. The values of K_a for β-LG/OA at 0.1, 80, and 160 MPa were 5.51 × 10⁶, 17.43 × 10⁶, and 49.75 × 10⁶M^?1, respectively. The molecule dynamic simulation showed that the OA molecules located at both β-barrel (site 1) interacted with Lys60, Glu62, and Lys69 and outer surface site 2 consisting of Tyr20, Tyr42, Ser21, Glu157, and His161. Additionally, when binding with OA during the dynamic high-pressure microfluidization treatment, the conformation of β-LG changed, reflected by the decrease of fluorescence intensity and total sulfhydryl group content, the increase of surface sulfhydryl group content, and secondary structure changes of β-LG. These results deduce that some epitopes may be masked by OA or modified by the conformational changes, resulting in the decline of antigenicity of β-LG molecules.     

Effect of enzymatic hydrolysis and polysaccharide addition on the β-lactoglobulin adsorption at the air-water interface

The effect of enzymatic hydrolysis and polysaccharide addition on the interfacial adsorption of β-lactoglobulin (β-LG) was investigated in this work. The enzymatic treatment was performed in the hydrolysis degree (HD) range of 0.0-5.0% using bovine α-chymotrypsin II immobilized on agarose beads. Anionic non-surface active polysaccharides (PS), sodium alginate (SA) and λ-carrageenan (λ-C) were studied in the concentration range of 0.0-0.5 wt.%. The adsorption process at the air-water interface was evaluated by means of tensiometry and surface dilatational rheology. Biopolymer interactions in solution were analyzed by extrinsic fluorescence spectroscopy. The enzymatic hydrolysis improved β-LG interfacial properties. On the other hand, at low HD (1.0%), PS addition enhanced surface and elastic properties of β-LG hydrolysate films probably due to a higher repulsion between biopolymers in solution. However, at high HD (3.0-5.0%), SA addition caused a deterioration of surface and elastic properties of β-LG hydrolysate films probably due to the segregation and hydrolysate aggregation in solution, whereas λ-C addition could promote the formation of soluble complexes leading to a better control of elastic properties of β-LG hydrolysate films.     

The enhancement of gastrointestinal digestibility of β-LG by dynamic high-pressure microfluidization to reduce its antigenicity

Dynamic high-pressure microfluidization (DHPM) can increase the antigenicity of β-lactoglobulin (β-LG) in previous researches. We observed in this study that the antigenicity of DHPM-modified β-LG at pressures of 0.1, 80 and 160 MPa after in vitro digestion declined from 13.41 to 12.27 and 7.19 μg mL⁻¹, gradually. The enhancement of gastrointestinal digestibility of β-LG was related to the aggregation state and conformational changes induced by DHPM and was reflected by the electrophoretic bands of low molecular weight (5 and 10 kDa) shown in electrophoresis, the reduction of particle size, the generation of smaller peptide fragments (m/z 837.444, 955.492 and 1215.616) detected by mass spectrum and the increase in surface hydrophobicity. These changes contributed to the decrease in antigenicity. Simultaneously, a schematic diagram was proposed to demonstrate the conceivable mechanism of antigenicity changes of modified β-LG after in vitro digestion.     

Effect of pulsed-light processing on the surface and foaming properties of β-lactoglobulin

Pulsed-light processing was used to treat β-lactoglobulin (BLG) solutions. The impact of pulsed light (PL) on the structural properties of this protein was explored through far-UV, CD spectral analysis, size exclusion chromatography, surface hydrophobicity and NMR spectroscopy. Changes on these physicochemical properties were related to surface rheology, surface tension, foam stability and foam capacity of the non-treated and treated BLG to elucidate adsorption mechanism and consequences on foaming capacity. Conformational modification of BLG was related with PL total fluence as important conformational changes increased when total fluence was higher. Consequently, adsorption rate of treated BLG at the air/water interface was faster than native BLG. Additionally, treated BLG formed highly elastic interfaces. This was found to have an impact on the foam stability. Pulsed-light treatment seemed to enhance the overall strength of the interface, resulting in more stable foams.     

Antigenicity and conformational changes of β-lactoglobulin by dynamic high pressure microfluidization combining with glycation treatment

The combined effect of previous dynamic high-pressure microfluidization treatment (40, 80, 120, and 160 MPa) and subsequent glycation with galacto-oligosaccharides (GOS) on the antigenicity of β-lactoglobulin (β-LG) was investigated. The antigenicity of β-LG-GOS decreased at relatively low pressure (≤120 MPa). Surface sulfhydryl group content of β-LG-GOS increased and surface hydrophobicity of β-LG-GOS decreased. Additionally, protein unfolding in β-LG-GOS samples was reflected by quenching of fluorescence intensity, the red-shift of fluorescence spectra, decreased UV absorption, and circular dichroism analysis, indicating tertiary and secondary structural changes of β-LG. The conformational changes may contribute to the alteration of antigenicity.     

Effect of underwater high-current discharge on the properties of low-concentration β-lactoglobulin solutions

Solutions of 0.5–2 mg/ml β-lactoglobulin (β-LG) at pH 7.0 were treated with underwater high-current discharges (UHCD) using a bench top system constructed in our laboratory. The short time duration of the UHCD (less than 3 μs) and the high energy delivered to the discharge (∼800 J) produced strong shock waves, in a microsecond time scale, as well as a powerful flash of light with a broad spectrum, ranging from soft X-rays to IR. Structural changes in β-LG were evaluated using hydrophobicity fluorescence of the β-LG molecule, differential scanning calorimetry, reactivity of the free thiol group to Ellman's reagent, small angle X-ray scattering (SAXS) and circular dichroism (CD). The results indicated that the application of the UHCD to β-LG leads to the increase of its surface hydrophobicity by approximately 40%. The enthalpy (ΔH_UHCD) of UHCD treated samples decreased as much as 40% compared to the enthalpy (ΔH_native) of native untreated proteins, indicating extensive but incomplete unfolding of the protein structure with no aggregation. It was found that no refolding occurred after the UHCD treatment. Also, it was shown that the reactivity of the free thiol group of UHCD treated samples increased 10-fold. Tryptophan residues in β-LG were markedly modified, as observed by UV absorption, indicating a change in its position. CD spectra indicated slight modifications in both secondary and tertiary structure.     

The effect of gel structure on the kinetics of simulated gastrointestinal digestion of bovine β-lactoglobulin

The structure and properties of protein gels depend on the conditions under which they are formed. Here, we assessed the susceptibility of protein to simulated gastro-duodenal digestion of weak gels with contrasting structures, produced from either purified bovine β-lactoglobulin (β-Lg) or whey protein isolate (WPI) at pH ranging from 2.5 to 6.5 and using different heating regimes. Gels formed close to the isoelectric point proved to be very resistant to simulated gastric digestion, with more than 85% of β-Lg remaining and in the simulated duodenal phase of digestion. The sample heated to 85 °C was most resistant with over 40% remaining. In the WPI sample heated to 85 °C, more than 20% of the original β-Lg content remained undigested after simulated gastro-duodenal proteolysis. These results suggest that firm particulate gels can persist longer in the GI tract and may be useful in inducing satiety and thus provide another weapon in the fight against obesity.     

Proteolytic action of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 reduces antigenic response to bovine β-lactoglobulin

The whey protein β-lactoglobulin (BLG) is highly allergenic. Lactic acid bacteria can degrade milk proteins. The capacity of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 to hydrolyse the major BLG epitopes (V41–K60; Y102–R124; L149–I162) and decrease their recognition by IgE of allergic patients was evaluated. The intensity of BLG degradation was analysed by Tricine SDS–PAGE and RP-HPLC. Peptides released were identified by LC–MS/MS and the hydrolysates were tested for their capacity to inhibit IgE binding by ELISA test. L. delbrueckii subsp. bulgaricus CRL 656 degraded BLG (35%, 8 h). The sequence analysis of the released peptides indicated that this strain degraded three main BLG epitopes. BLG-positive sera (3–5 year old children) were used for testing IgE binding inhibition of BLG hydrolysates from the Lactobacillus strain. The hydrolysates were less immuno-reactive (32%) than the heated BLG. L. delbrueckii subsp. bulgaricus CRL 656 could be used for developing hypoallergenic dairy products.     

Thermal denaturation of bovine β-lactoglobulin in different protein mixtures in relation to antigenicity

Denaturation of β-lactoglobulin (BLG) was studied in relation to its antigenicity at two heat treatments in several native protein mixtures; allergenicity was determined by enzyme-linked immunosorbent assay based on BLG capacity to bind with immunoglobulin G (IgG) antibodies. The influence of other proteins on BLG denaturation correlated with altered antigenicity. Treatment at 72 °C/15 s enhanced antigenicity in a BLG+α-lactalbumin (ALA) mixture, possibly due to exposed epitopes in the unfolded structure. Treatment at 100 °C/30 s mostly resulted in BLG-led protein aggregation through thiol/disulphide interactions and decreased antigenicity by fragmentation and masking of epitopes, the extent of which was mixture-dependent. The presence of IgG resulted in diminished antigenicity in BLG + ALA + IgG at 100 °C/30 s in comparison with BLG + ALA. ALA governed whey protein denaturation over BLG in BLG + ALA + IgG + bovine serum albumin (BSA), possibly catalysed by BSA at 100 °C/30 s, resulting in a higher retention of antigenicity than in other mixtures.     

Effect of ascorbic acid and protein concentration on the molecular weight profile of bovine serum albumin and β-lactoglobulin by γ-irradiation

The effects of ascorbic acid and protein concentration on the molecular weight size distribution of BSA and β-lactoglobulin were examined after irradiation of proteins at various doses. Gamma-irradiation of protein solutions caused disruption of the ordered structure of protein molecules resulting in degradation, cross-linking, and aggregation of the polypeptide chains. SDS–PAGE and gel permeation chromatography study showed that ascorbic acid protected the aggregation and degradation of proteins by scavenging oxygen radicals produced by irradiation and the effect of irradiation on protein conformation was more significant at lower concentrations of proteins.     

Effect of different β-glucans on the gelatinisation and retrogradation of rice starch

Four β-glucan preparations, i.e., curdlan (CL), oat (OG), barley (BG) and yeast (YG) β-glucans, were compared for their effects on the gelatinisation and retrogradation of rice starch (RS). Rapid visco-analysis (RVA) showed that addition of any of these β-glucans significantly increased the peak, breakdown, final, and setback viscosities of RS, whereas the pasting temperatures were significantly decreased by OG or CL addition, but were unaffected by BG or YG addition. Differential scanning calorimetry (DSC) demonstrated that all the β-glucans had a negligible effect on the onset (T_o), peak (T_p), and conclusion (T_c) temperatures but slightly decreased the gelatinisation enthalpy (△H₁) of RS. Storage of all the gels at 4 °C resulted in a marked decrease in the T_o, T_p, T_c, and melting enthalpy (△H₂) values. The retrogradation ratio (△H₂/△H₁) and the phase transition temperature range (T_c–T_o) of all the gels increased with storage time. Dynamic viscoelastic measurements revealed weak gel-like behaviour of all the gels, in which their storage modulus (G′) increased and their loss tangent (tan δ) decreased during storage. Steady flow tests illustrated time-dependent shear-thinning (thixotropic) behaviour of all the gels. The hysteresis loop area and the gel hardness increased with storage time. However, the rate and extent of retrogradation and the rheological and textural changes of the RS gels were reduced by addition of any of these β-glucans. The extent of the aforementioned effects differed among the different β-glucan preparations, generally in the order OG ≈ BG > CL ≈ YG.     

Effect of plant polyphenols on the formation of advanced glycation end products from β-lactoglobulin

Dietary exposure to advanced glycation end products (AGEs) formed from proteins and reducing sugars is of increasing concern to human health. AGEs may form in protein-based powders containing sugars for instant beverages during drying and storage of the product. Chlorogenic acid, a plant phenol characteristic of coffee, was found to protect against the formation of AGEs at a concentration of 50mM during heating of β-lactoglobulin in the presence of glucose as a reducing sugar in 30% aqueous ethanol at 70°C. Epicatechin, a plant phenol characteristic of green tea, had no similar effect for the equivalent concentration of phenol on the formation of AGEs. Immunochemical detection (ELISA) using polyclonal antibodies raised against AGEs showed a dose-dependent effect of protection by chlorogenic acid on AGE formation and is recommended for routine quality control of sugar containing milk-based powders for instant beverages.     

Antimicrobial activity of bovine β-lactoglobulin against mastitis-causing bacteria

Bovine mastitis is one of the most economically deleterious diseases affecting dairy herds and results from an infection of the udder by pathogenic microorganisms such as Staphylococcus aureus, Streptococcus uberis, and Escherichia coli. The mammary gland is capable of preventing and combating bacterial infection by means of a complex network of innate and adaptive immune mechanisms. Lactoferrin is an 86-kDa protein with antibacterial activity that plays a role in the mammary gland's defense against infection. β-Lactoglobulin (β-LG) is an 18-kDa protein that is present in most mammals but is notably absent in humans, rodents, and lagomorphs. Different genetic variants of this protein exist, with β-LG A and β-LG B being the most common. In spite of being well studied, the biological function of β-LG is not thoroughly understood, and most noticeably, no reports exist on the effects of the native protein on bacterial growth. Hence, the objective of this study was to assess the potential antibacterial activity of β-LG against mastitis agents. To do this, we purified β-LG from normal bovine milk using a mild, nondenaturing method and performed in vitro growth inhibition assays with Staph. aureus, E. coli, and Strep. uberis. β-Lactoglobulin inhibited the growth of Staph. aureus and Strep. uberis but had no effect on E. coli. The antimicrobial activity against Staph. aureus and Strep. uberis was concentration dependent and was elicited by the intact protein because Tricine-sodium dodecyl sulfate-PAGE and analytical gel filtration chromatography did not reveal the presence of short degradation peptides. Analysis of the genetic variants of β-LG showed that β-LG A has higher inhibitory activity against Staph. aureus and Strep. uberis than β-LG B. Coincubation of β-LG and lactoferrin resulted in an augmented antibacterial activity against Staph. aureus, suggesting an additive effect of the proteins. This result, along with the proteins’ complementary spectrum of action, suggests that β-LG and lactoferrin may complement each other in the mammary gland's defenses against bacterial infection.