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.     

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.     

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 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.     

Investigation on the influence of high protein concentrations on the thermal reaction behaviour of β-lactoglobulin by experimental and numerical analyses

In this study, treatments at various temperature–time profiles were performed for β-lactoglobulin samples at different concentrations (50–70%) using a special rheometer as processing device. Rheological measurements, offline protein chemical analyses, and molecular dynamics analyses were performed to investigate the influence of high protein concentrations and treatment temperature on the denaturation and aggregation behaviour of β-lactoglobulin. Under these conditions, the degree of denaturation and aggregation decreased with increasing protein concentration. This corresponded to a strongly decreased diffusion and increased stability of exposed surface protein regions at high concentrations. Irreversible denaturation was observed for temperatures above 60 °C. Increasing thermal treatment intensity resulted in an increase of aggregation. Depending on the thermal treatment conditions, different protein–protein interactions were measured. By increasing the treatment temperature, the resulting aggregates were increasingly stabilised by covalent bonds. In addition to disulphide bonds, non-disulphide covalent cross-links were formed at temperatures above 100 °C.     

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 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.     

Influence of the overall charge and local charge density of pectin on the complex formation between pectin and β-lactoglobulin

The complex formation between β-lactoglobulin (β-lg) and pectin is studied using pectins with different physicochemical characteristics. Pectin allows for the control of both the overall charge by degree of methyl-esterification as well as local charge density by the degree of blockiness. Varying local charge density, at equal overall charge is a parameter that is not available for synthetic polymers and is of key importance in the complex formation between oppositely charged (bio)polymers. LMP is a pectin with a high overall charge and high local charge density; HMP_B and HMP_R are pectins with a low overall charge, but a high and low local charge density, respectively. Dynamic light scattering (DLS) titrations identified pH_c, the pH where soluble complexes of β-lg and pectin are formed and pH_?, the pH of phase separation, both as a function of ionic strength. pH_c decreased with increasing ionic strength for all pectins and was used in a theoretical model that showed local charge density of the pectin to control the onset of complex formation. pH_? passed through a maximum with increasing ionic strength for LMP because of shielding of repulsive interactions between β-lg molecules bound to LMP, while attractive interactions were repressed at higher ionic strength. Potentiometric titrations of homo-molecular solutions and mixtures of β-lg and pectin showed charge regulation in β-lg–pectin complexes. Around pH 5.5–5.0 the pK_as of β-lg ionic groups are increased to induce positive charge on the β-lg molecule; around pH 4.5–3.5 the pK_a values of the pectin ionic groups are lowered to retain negative charge on the pectin. Since pectins with high local charge density form complexes with β-lg at higher ionic strength than pectins with low local charge density, pectin with a high local charge density is preferred in food systems where complex formation between protein and pectin is desired.     

Effect of combined treatment of hydrolysis and polymerization with transglutaminase on β-lactoglobulin antigenicity

The effect of combined treatments of hydrolysis with different proteases, and subsequent polymerization with transglutaminase on the antigenic activity of β-Lg was studied. For the hydrolysis of β-Lg using Alcalase, Neutrase or bromelain, the reaction conditions were 3?% β-Lg and enzyme:substrate 25?U?g^?1 of protein, as was defined using factorial study. Under these conditions, the degree of hydrolysis (DH) of the hydrolysates was 12.6?% when obtained with Alcalase and approximately 4?% with Neutrase or bromelain. Post-hydrolysis polymerization did not result in an increase in molecular mass of the protein, but these samples presented a lower DH, determined by trinitrobenzenosulfonic acid (TNBS) method, suggesting that polymerization had occurred. Hydrolysis with the three enzymes reduced the β-Lg antigenicity, as evaluated by ELISA and immunoblotting analyses. The IgE-binding responses were practically null (<9?μg?mL^?1), 22.82 and 55.73?μg?mL^?1 towards the hydrolysates obtained with Alcalase, bromelain, and Neutrase, respectively. The post-hydrolysis polymerization increased or had no significant effect (P?≥?0.05) on the antigenic response of the hydrolysates.     

Effect of ultraviolet irradiation on molecular properties and immunoglobulin production-regulating activity of β-lactoglobulin

To utilize ultraviolet (UV) irradiation as a means to prepare hypo-allergenic food, we investigated the molecular weight profile, secondary structure content, fluorescence spectrum, and immunoglobulin (Ig) production-regulating activity of β-lactoglobulin after UV-irradiation. UV-irradiation caused a change on the molecular size distribution, disruption of the ordered structure, and decrease of emission intensity of β-lactoglobulin. The alteration on Igs production regulating activity of β-lactoglobulin by UV-irradiation was observed in the mouse spleen lymphocytes system. These results suggest that UV-irradiation is effective for alteration of molecular properties and antigenicities of β-lactoglobulin and such treatment should be useful for the preparation of low allergenic foods.     

Effect of pectin, starch, and locust bean gum on the interfacial activity of monostearin and β-lactoglobulin

The behavior of some hydrocolloids widely used as stabilizers of low-oil-content water emulsions (starch, pectins, and a locust bean gum-pectin blend) at the air-water and model oil-water interface is analyzed. Their influence on the surface and interface activity of typical food emulsifiers, such as β-lactoglobulin and monostearin, is also considered. It is demonstrated that the greatest interfacial activity is provided by one of the commercial pectins studied. It is capable of modifying the characteristics of monostearin and β-lactoglobulin interfacial films in a different way depending on both the nature of the oil phase and the type of surfactant used. PRACTICAL APPLICATION: This research may contribute not only to enhance the final-consumer life quality by optimizing low-oil-content food emulsion formulations which contain "natural" stabilizers, but also to increase the added value of by-products of some fruit juices as well as of sugar factories since pectin can be manufactured not only from citrus and apple peels but also from sugar beet pulps.     

Formation of conjugates between β-lactoglobulin and allyl isothiocyanate: Effect on protein heat aggregation,foaming and emulsifying properties

Whey proteins are widely used food ingredients due to their nutritional and functional properties (gelling, emulsifying, foaming). Owning to their structure (free thiol group, lysine residues, hydrophobic pocket), they can also be used as carriers for bioactives. In this study, conjugates between β-lactoglobulin (β-lg), and a bioactive metabolite from Brassicaceae vegetables, allyl isothiocyanate (AITC) were formed. Heat aggregation behavior (85 °C, 15 min), foaming and emulsifying properties of conjugates, at pH 4.0 and 7.1, were evaluated.     

Effect of α-lactalbumin and β-lactoglobulin on the oxidative stability of 10% fish oil-in-water emulsions depends on pH

The objective of this study was to investigate the influence of pH on lipid oxidation and protein partitioning in 10% fish oil-in-water emulsions prepared with different whey protein isolates with varying ratios of α-lactalbumin and β-lactoglobulin. Results showed that an increase in pH increased lipid oxidation irrespective of the emulsifier used. At pH 4, lipid oxidation was not affected by the type of whey protein emulsifier used or the partitioning of proteins between the interface and the water phase. However, at pH 7 the emulsifier with the highest concentration of β-lactoglobulin protected more effectively against oxidation during emulsion production, whereas the emulsions with the highest concentration of α-lactalbumin were most stable to oxidation during storage. These differences were explained by differences in the pressure and adsorption induced unfolding of the individual protein components.     

Effect of salts on the alkaline degradation of β-lactoglobulin gels and aggregates: Existence of a dissolution threshold

The effect of NaCl and CaCl₂ on the alkaline degradation of β-lactoglobulin gels and aggregates, and particularly on the onset of dissolution, is studied. For gels, measurements of solubility in 0.063–0.5 M NaOH at 20 °C show the existence of a practical dissolution threshold in NaCl concentration, lying between 0.24 and 0.47 M. For aggregates, destruction of soluble β-lactoglobulin in alkali, followed by size exclusion chromatography, yields similar results. Furthermore, during dissolution of a gel in alkali at high NaCl concentrations, the protein aggregates released are very large (e.g. ∼40% are larger than 200 kDa). CaCl₂ is found to cause similar inhibition of dissolution to NaCl, but at concentrations about 30× lower (∼10 mM). The threshold is hypothesised to arise from a combination of physical entanglements caused by the high protein concentration under conditions where little swelling occurs, and hydrophobic/electrostatic interactions between aggregates favoured by the high concentration of salts.     

Influence of calcium on β-lactoglobulin denaturation kinetics: Implications in unfolding and aggregation mechanisms

Much research dealing with the processing of milk by-products in heat exchangers has noted the key role of calcium in β-lactoglobulin (β-LG) fouling behavior. Nevertheless, the manner by which Ca affects β-LG denaturation has rarely been quantified using reliable kinetic and thermodynamic data. To this end, the influence of Ca on β-LG denaturation mechanisms in simulated lactoserum concentrates was studied on the laboratory-scale under 100°C by HPLC analysis. The heat-treated solutions were composed of 53.3 g/L β-LG and were enriched in Ca at various concentrations (0, 66, 132, and 264 mg/kg). The kinetic parameters (reaction order, activation energy, and frequency factor) associated with β-LG denaturation, along with the unfolding and aggregation thermodynamic parameters were deduced from these experiments and discussed with respect to Ca content. We found that the multistage process characterizing β-LG thermal denaturation is not greatly affected by Ca addition. In fact, the general model subdividing β-LG denaturation mechanisms in 2 steps, namely, unfolding and aggregation, remained valid for all tested Ca concentrations. The change in the predominant mechanism from unfolding to aggregation was observed at 80°C across the entire Ca concentration range. Moreover, the classical 1.5 reaction order value was unaffected by the presence of Ca. Interpretation of the acquired kinetic data showed that Ca addition led to a significant increase in kinetic rate, and more so in the aggregation temperature range. This indicates that Ca principally catalyzes β-LG aggregation, by lowering the Coulombian repulsion between the negatively charged β-LG reactive species, bridging β-LG proteins, or via an ion-specific conformational change. To a lesser extent, Ca favors β-LG unfolding, probably by disturbing the noncovalent binding network of native β-LG. Simultaneously, Ca has a slight protective role on the native and unfolded β-LG species, as shown by the increase in activation energy with Ca concentration. The calculation of thermodynamic parameters related to β-LG denaturation confirmed this observation. A threshold effect in Ca influence was noted in this study: no further significant kinetic rate change was observed above 132 mg/kg of Ca; at this concentration, the studied solution was an almost equimolar mixture of β-LG and Ca. Finally, we simulated the temporal evolution of β-LG species concentrations at diverse Ca contents at 3 holding temperatures. The simulations were based on the acquired kinetic parameters. This permitted us to highlight the greater effect of Ca on β-LG denaturation at high Ca content or for short-time heat treatments at temperatures near 100°C, as in heat exchangers.     

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.     

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.     

Effect of dynamic high-pressure microfluidization at different temperatures on the antigenic response of bovine β-lactoglobulin

The antigenic response of β-lactoglobulin (β-Lg), treated by dynamic high-pressure microfluidization (DHPM) at different temperatures, was determined by an indirect competitive enzyme-linked immunosorbent assay using polyclonal antibodies from rabbit serum. DHPM treatment causes changes in the protein structure and may influence the antigenicity of β-Lg. DHPM treatment of β-Lg at 90 °C showed significant effects with the antigenic response of 5.2 μg mL⁻¹ (untreated), 45 μg mL⁻¹ (40 MPa), 79 μg mL⁻¹ (80 MPa), 132 μg mL⁻¹ (120 MPa), and 158 μg mL⁻¹ (160 MPa). In combination with temperature treatment (70–90 °C), the antigenic response enhanced as the temperature increased at 160 MPa. The β-Lg antigenicities were about 14, 108, and 158 μg mL⁻¹ at 70, 80, and 90 °C, respectively. However, the influence of DHPM pressures on the antigenic response of β-Lg standards was different. DHPM modified β-Lg standards showed a remarkable increase in antigenicity when treated to 80 MPa. Above 80 MPa, the antigenic response decreased.     

Effect of ultrasound treatment on the wet heating Maillard reaction between β-conglycinin and maltodextrin and on the emulsifying properties of conjugates

Soy β-conglycinin (7S) was grafted with maltodextrin (MD) by combined ultrasound treatment and wet heating Maillard reaction in this work. The physicochemical and emulsifying properties of 7S–MDH (classical wet heating) and 7S–MDUH (ultrasound-assisted wet heating grafting) have been investigated. Ultrasound treatment could speed up the conjugation process and obtain a protein–polysaccharide conjugate that exhibits superior functionality compared with the one obtained simply by wet heat treatment. The degree of grafting of 32.73 % was obtained in 30 min by ultrasound-assisted wet heating , whereas classical wet heating required 60 min. SDS-PAGE analysis indicated that 7S globulin had become complex with MD to form conjugates of higher molecular weight. Reduction in the contents of lysine and arginine during the graft reaction indicated that these two amino acid residues had the covalent linkage between 7S and MD. The results of secondary structure showed that grafted 7S had decreased α-helix level and increased β-sheet and unordered coil levels. In addition, ultrasound-assisted treatment significantly changes in surface hydrophobicity (H ₀), emulsifying activity index and emulsifying stability index of 7S (p < 0.05). Moreover, the emulsions of 7S–MDUH were stable under unfavorable conditions, such as extreme pH, ionic strength and heat treatment temperature, which is mainly due to changes in surface hydrophobicity (H ₀) and secondary structure, resulting in a reduction in protein molecular steric hindrance in an increase in molecular flexibility. This study demonstrated that combined ultrasound treatment and wet heating Maillard reaction could potentially be applied as a method to prepare 7S–MD conjugates.