Tuning the properties of β-lactoglobulin nanofibrils with pH,NaCl and CaCl2

We investigated the effects of pH (1.6–2.4), NaCl and CaCl₂ (0–100 mm) on the kinetics of β-lactoglobulin fibril formation during heating at 80 °C. The morphology of fibrils was also examined. At pH 1.8–2.4 fibril formation occurred slightly faster with decreasing pH. At pH 1.6 fibril formation during the growth phase occurred much faster than at any other pH. Fibril morphology was unchanged between pH 1.6 and pH 2.0. Addition of NaCl or CaCl₂ accelerated fibril formation during the growth phase, and CaCl₂ shortened the lag phase as well. Worm-like fibrils were seen at ≥60 mm NaCl or ≥33 mm CaCl₂, and these had a persistence length which was much shorter than the long semi-flexible fibrils formed without salts. The efficiency of fibril formation can be substantially enhanced by varying pH and salt concentration.     

Effects of heat-treated β-lactoglobulin and its aggregates on foaming properties

The effects on foaming properties of the aggregates formed by heating concentrate beta-lactoglobulin solutions (55 mg mL⁻¹, pH 6.8) at 85 °C from 1 to 15 min were investigated. Structural characteristics (size and molecular conformation), hydrophobicity and protein aggregates proportion were also studied. All tested methods pointed at 3 min of heating as a critical time, in terms of conformational changes and aggregation processes. At this time, the most significant conformational changes took place: non-native monomers were present and the greatest amount of dimers and trimers was produced, which was proved with the results of gel densitometry of SDS-PAGE, fluorescence quenching and circular dichroism tests. Foamability and foam stability were both improved by pre-heating the protein. A constant proportion among beta-lactoglobulin species (monomer 51%, dimer 33% and trimer 16%), regardless the protein concentration, led to the same results on foaming properties, confirming the link with structural changes. Aggregates formed by heating beta-lactoglobulin up to 10 min produced more stabilized foams, slowing down disproportionation, because of the formation of stiffer films. The increase in surface hydrophobicity was considered a decisive factor in the improved foamability and hydrophobic interactions improved the foam stability trough the rapid formation of a viscoelastic film.     

Microstructure and rheology of β-lactoglobulin–galactomannan aqueous mixtures

The effect of the addition of a galactomannan (locust bean gum, LBG, or tara gum, TG) on the microstructure and rheological properties of a globular protein (β-lactoglobulin, β-Lg) solution was studied at pH 7.0, when the protein bears a net negative charge. Confocal laser scanning microscopy was used to explore the microstructure. Steady shear and dynamic oscillatory measurements were performed with a controlled stress rheometer AR2000 (TA Instruments) fitted with a cone-and-plate geometry. Mixtures were prepared with 6.5 wt% β-lactoglobulin concentration and 0.31–0.82 wt% LBG or 0.23–0.71 wt% TG concentration. All mixed systems were two-phase. The microstructure was clearly dependent on the concentration of the galactomannan in the mixture: the systems evolved from a continuous matrix of β-lactoglobulin enriched phase containing some small inclusions of the galactomannan, to a matrix of galactomannan-enriched continuous phase containing aggregates of β-lactoglobulin. Modifications of the flow and viscoelastic properties with respect to the individual components were clearly evidenced for the mixed systems. Phase inversion detected by microscopy could also be detected by rheology as a modification in the flow/viscoelastic behaviour.     

Interfacial and foaming properties of bovine β-lactoglobulin: Galactose Maillard conjugates

In this paper, the effect of the initial and advanced steps of glycosylation by Maillard reaction (MR) (glycation) of β-lactoglobulin (β-Lg) with galactose on the interfacial and foaming (foamability and foam stability) properties of this protein has been studied at both pH 7 and pH 5. Hardly any effect of glycation was observed at pH 7. However, a pH 5, due to its increased solubility, β-Lg glycated at 50 °C during 48 h (advanced steps of MR) presented the best dynamic of adsorption which lead to an increase of the surface dilatational modulus of adsorbed film. This resulted in a better foaming capacity, as well as higher stability of foams of β-Lg glycoconjugates with respect to native and control heated protein. These results could extend the applicability of β-Lg as a foaming agent, particularly in acid foods.     

Rheological properties of patatin gels compared with β-lactoglobulin, ovalbumin, and glycinin

BACKGROUND: The thermal unfolding and rheological properties of patatin gels were compared with those of commonly used proteins (β‐lactoglobulin, ovalbumin, glycinin). RESULTS: A significant difference between these proteins was observed in both the denaturation temperature (59 °C for patatin; about 20 °C lower than the other proteins) and the onset temperature of gel formation (50–60 °C, compared to 70–85 °C for the other proteins). At low ionic strength the minimal concentration was only 6% (w/v) for patatin, compared to 8–11% for the other proteins. This effect was attributed to the relatively high exposed hydrophobicity of patatin as determined by hydrophobic interaction chromatography. For gels compared at ‘iso‐strength’, the frequency dependence was found to be close to identical, while small differences were observed in the strain at fracture. CONCLUSIONS: Patatin was found to form gels with comparable small‐deformational rheological properties as typical food proteins. In addition, at concentrations where the elastic modulus was similar for all proteins, the frequency and strain dependence were also comparable. From this it is concluded that patatin is a promising protein to be used in food applications as a gelling agent. Copyright © 2010 Society of Chemical Industry     

Thermal denaturation of mixtures of α-lactalbumin and β-lactoglobulin: a differential scanning calorimetric study

The thermal denaturation of α-lactalbumin (α-lac), β-lactoglobulin (β-lg) and a mixture of the two proteins in the presence of several sugars, sodium salts and at various pH values was studied by differential scanning calorimetry. The effects of N-ethylmaleimide (NEM), cysteine, urea and sodium dodecyl sulfate (SDS) were also investigated. The temperature of denaturation (T_d) of β-lg decreased from 71.9°C in the absence of α-lac to 69.1°C in its presence. In contrast, an increase of 2.5°C was observed in the T_d of apo-α-lac when heated in the presence of β-lg suggesting that α-lac was made more thermally stable in the presence of β-lg. Glucose and galactose had the greatest effect in stabilizing the proteins against thermal denaturation with the effect being greater for β-lg than for α-lac. A decrease in thermal stability of both proteins was observed in the presence of sodium bicarbonate; sodium ascorbate, however, had a stabilizing effect. Renaturation of α-lac was prevented in the presence of cysteine and NEM, but not in urea or SDS. Translucent gels were formed when the α-lac/β-lg mixtures were heated in the presence of all five sugars and in the presence of cysteine, urea and SDS but not in NEM. This suggests that disulfide–sulfhydryl interchange reactions may be primarily responsible for the gelation of α-lac/β-lg mixtures.     

Heat-induced aggregation of β-lactoglobulin AB at pH 2.5 as influenced by ionic strength and protein concentration

Heat-induced (80°C) aggregation of β-lactoglobulin AB at pH 2.5 was studied using size-exclusion chromatography in combination with multi-angle laser light scattering, dynamic light scattering and electrophoretic techniques. Upon heating, large aggregates with molar masses of 10⁶–10⁷ Da were formed, whereas the concentration of intermediate-sized aggregates was very low. The rate of disappearance of native-like β-lactoglobulin increased with increasing protein concentration (reaction order 2) and ionic strength. Aggregate size increased slightly with heating time and ionic strength, but was independent of protein concentration. Aggregates were held together entirely with non-covalent bonding.     

The effect of glycation on foam and structural properties of β-lactoglobulin

The goal of the present work was to evaluate whether the Maillard reaction, with glucose and lactose as substrates, improves the foaming properties of β-lactoglobulin. Lactose led to the lowest degree of modification without significant differences by reaction time and by protein:sugar molar ratio. However, in the case of glucose, the degree of glycation increases with reaction time and molar ratio. The results obtained by UV fluorescence, surface hydrophobicity and differential scanning calorimetry clearly showed differences in the degree of folding of β-lactoglobulin upon modification with different sugars or thermal treatment, with changes in the foaming capacity of β-lactoglobulin. All the modified samples exhibited a significant increase (α ? 0.05) in draining stability (Kg) as compared to the non-thermally treated sample. In addition, foams formed by lactose-glycated samples were more stable than those formed by glucose-glycated ones. A significant increase (α ? 0.05) of foam stability with reaction time was also detected, particularly in glucose-glycated samples.     

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.     

Influence of buffer type and concentration on the peptide composition of trypsin hydrolysates of β-lactoglobulin

Proteins acquire conformation in aqueous media due to the influence of the buffer salt composition and concentration. Such influence may have impact on the enzyme–substrate interaction and somehow steer the enzyme attack properties, leading to release of dissimilar products. Our group has sought to investigate the influence of the hydrolysis environment on the trypsinolysis of a model protein, β-lactoglobulin (β-Lg). This work was aimed at investigating the effect of different buffers and their concentrations on the trypsinolysis patterns of β-Lg. The traditional NaOH-buffered water, in comparison to Tris–HCl and potassium-phosphate buffer at 62.5 mM–1.0 M were used at pH 8.5 for the pH drop and pH 7.8 for the hydrolysis. Bovine trypsin (EC 3.4.21.4) was used at an enzyme-to-substrate ratio of 1%. The samples were analysed for mass composition, using LC-ESI-TOF/MS and MALDI-TOF/MS for monitoring time-dependence of peptide evolution. In all buffer types and concentrations, peptides f(1–8), f(15–40, f(125–138) and f(142–148) were detected, implicating ease of hydrolysis of the terminal regions of β-Lg. A peptide from f(9–14), with sequence Gly-Leu-Asp-Ile-Gln-Lys, was detected at >0.5 M Tris–HCl only, while peptide f(71–75) was unique to <125 mM Tris–HCl and >250 mM potassium-phosphate buffer. Hydrolysis under buffer produced trypsin-specific peptides, numerous chymotrypsin-like non-specific peptides but no disulphide-linked peptides. Trypsinolysis shifted to the N-terminal region of lysine under some conditions. Hydrolysis under buffer holds potential for the avoidance of some peptides with undesirable characteristics while preserving a diversity of different peptides with possible bioactive properties.     

Membrane fractionation of a β-lactoglobulin tryptic digest: Effect of the pH

The tryptic digestion of β-lactoglobulin leads to the release of a large range of biologically active peptides. Ultrafiltration/nanofiltration technology can be used to fractionate protein hydrolysates in order to obtain permeate products with increased functionality and free from intact proteins and enzymes. The influence of the pH in the fractionation of the hydrolysate through a polyethersulfone membrane (MWCO 5 kDa) was investigated in this work. In this case peptide transmission was mainly governed by charge mechanisms and reached its maximum value when the pH value is close to the peptide isoelectric point. Almost complete rejection of acidic peptides was achieved at basic pH values due to electrostatic repulsive forces with the negatively charged membrane and, in agreement with Donnan theory, positively charged peptides shown lower transmission than the neutral species. The highest peptide recovery and the best separation factor between bioactive and non-bioactive peptides were obtained following nanofiltration at pH 8.0.     

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.     

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.     

Rheological properties of acid gels prepared from heated milk fortified with whey protein mixtures containing the A,B and C variants of β-lactoglobulin

The effect of fortification of reconstituted skim milk with different levels of a whey protein mixture containing a 1:2 ratio of α-lactalbumin (α-la) and different genetic variants of β-lactoglobulin (β-LG) on the rheological properties of acid milk gels, formed by acidification with glucono-δ-lactone, was investigated. Milk samples were either unheated or heated at 80°C for 30 min before acidification. Acid gels prepared from unheated skim milk had very low G′ values, long gelation times and low gelation pH. Samples prepared from heated milk had markedly higher G′ values, a reduced gelation time and an increased gelation pH. The addition of increasing levels of whey protein mixtures containing β-LG B or β-LG C to the milk prior to heating and acidification caused an almost linear increase in the G′. In contrast, whey protein mixtures containing β-LG A caused a progressive increase in the G′ with added protein levels up to about 0.7% (w/w) but little further change at higher addition levels. A mixture of the A and B variants of β-LG gave an intermediate behaviour between those of the A and B variants. In all samples, the G′ value at 5°C was approximately twice that at 30°C so that the relative differences as a result of the β-LG genetic variants were similar for the two temperatures.     

A large-scale isolation of native β-lactoglobulin: characterization of physicochemical properties and comparison with other methods

A novel method for β-lactoglobulin (β-lg) isolation from whey was investigated. The method comprised a peptic treatment of whey and membrane filtration under gentle conditions for isolation of native β-lg. The method was applied to 10,000 L batches of processed whey and the product quality obtained was compared with pilot-scale results of three other procedures: 3% trichloroacetic acid (TCA) precipitation, a salting-out procedure and a selective thermal precipitation. The calculated yields of native β-lg were 67.3, 44.9, 46.7 and 49.6% of the β-lg present in whey using the four methods, respectively. The composition of the different β-lg preparations was found to be comparable, however, analysis of purity revealed slight differences as indicated by electrophoresis and fast protein liquid chromatography (FPLC). The isolated β-lg retained a high degree of purity and native properties. The new method for isolation of native β-lg was found to be reproducible, selective and robust as processing of three 10,000 L batches did not indicate any technical problems or variations in the purity of the isolated β-lg.     

Calcium Addition,pH and High Hydrostatic Pressure Effects on Soybean Protein Isolates—Part 2: Emulsifying Properties

Soybean protein isolates (SPI) represent an important source of proteins that are used to prepare oil-in-water (o/w) emulsions. The influence of an innovative treatment (high hydrostatic pressure, HHP) combined with calcium addition at different pH levels and protein concentrations on the formation and stability of o/w SPI emulsions was evaluated in this work. When applied separately, calcium addition or HHP treatment produced different effect at pHs 5.9 and 7.0. Calcium addition led to stable emulsions with decreased flocculation index (FI) at pH 5.9 and low protein concentration (5 g L^?1), whereas at pH 7.0, this effect was observed at high protein concentration (10 g L^?1). In these conditions, calcium would favor the arrival of big aggregates to interface, which would be modified and adsorbed during homogenization. Treatment with HHP decreased FI and stabilized emulsions during storage at pH 7.0 (but not at pH 5.9) when prepared from 10 g L^?1 protein dispersions. In these conditions, protein unfolding due to HHP-induced denaturation, and high ζ-potential would be responsible for emulsion improvement. Combination of calcium addition and HHP treatment impaired both formation and stabilization abilities of SPI at both pHs. Bridging flocculation was enhanced in these samples while interfacial protein concentration and percentage of adsorbed protein were increased. Thus, soybean proteins that were subjected to combined calcium addition and HHP treatment exhibited a great ability to associate each other, what can be useful to improve other functional properties such as gelation.     

Effects of oxidative treatment on the physicochemical,rheological and functional properties of oat β-glucan

The objective of this work was promote oxidation of β-glucan from oat bran with hydrogen peroxide at different concentration levels (0.3%, 0.6% and 0.9% H₂O₂) and reaction times (30 and 60 min), and evaluate the physicochemical and functional properties of isedoxidised β-glucan with in-vitro tests. An increase in carbonyl and carboxyl groups and alterations in swelling power were verified in the oxidised β-glucan. The cholic acid binding capacity increased in the oxidised β-glucan; however, the fat binding capacity was not affected. After chemical digestion, the available glucose of the oxidised β-glucan was increased. Oxidation with hydrogen peroxide decreased the viscosity, hardness, adhesiveness and gumminess of the β-glucan gels. More studies are necessary to determine the effect of the oxidative treatment of β-glucan on its technological properties in food products, and biological properties should be examined with in-vivo studies.     

Modulation of physicochemical properties of lipid droplets using β-lactoglobulin and/or lactoferrin interfacial coatings

There is considerable interest in the development of food-grade delivery systems to encapsulate, protect and release bioactive lipids. In this study, emulsion-based delivery systems were prepared consisting of lipid droplets covered by β-lactoglobulin (BLG) and/or lactoferrin (LF) coatings. BLG and LF are globular proteins with relatively low (pI ∼ 5) and high (pI ∼ 8) isoelectric points, respectively. Mixed systems were prepared by adding LF to BLG-stabilized corn oil-in-water emulsions (BLG/LF-coated droplets) or by adding BLG to LF-stabilized corn oil-in-water emulsions (LF/BLG-coated droplets) at pH 7, where there is an electrostatic attraction between the proteins. The influence of pH (3–7), ionic strength (0–60 mM CaCl₂ or 0–200 mM NaCl, pH 7), and thermal treatment (21–90 °C, pH 7, 20 min) on the physical stability of the resulting emulsions was examined. Emulsions with good stability to pH, salt, and thermal processing could be created using mixed interfacial coatings. In addition, we found that the lipids in these emulsions could still be digested using an in vitro digestion model to simulate small intestine conditions. This work may lead to the formation of emulsion-based delivery systems with improved physicochemical and functional performance.