Glycation of α-lactalbumin with different size saccharides: Effect on protein structure and antigenicity

The effect of the size of saccharides on protein structure and antigenicity after glycation was studied. α-Lactalbumin (α-LA) was incubated in a dry state with glucose, maltose or maltooligosaccharides (up to maltopentaose), at 55 °C and 65% relative humidity. The extent of glycation, aggregation processes, antigenicity, glycation sites and structural changes were investigated. Structural changes were observed when the protein was incubated with larger saccharides. Furthermore, the degree of glycation decreased with an increase in the size of the saccharide. The antigenicity of all samples significantly decreased when glycation increased, but the greatest reduction of antigenicity was observed with maltotriose-α-LA. By comparing the glycation sites of all the glycated samples, glycation of Lys58 was dominant in glucose-α-LA and maltotriose-α-LA, whose antigenicity reductions were the highest. These results suggest that when the main epitope was glycated, the degree of antigenicity reduction increased with the increase in the size of the saccharide.     

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.     

Effects of high hydrostatic pressure on the structure of bovine α-lactalbumin

The effects of high hydrostatic pressure (HHP) processing (at 200 to 600 MPa, 25 to 55°C, and from 5 to 15 min) on some structural properties of α-lactalbumin was studied in a pH range of 3.0 to 9.0. The range of HHP processes produced a variety of molten globules with differences in their surface hydrophobicity and secondary and tertiary structures. At pH values of 3 and 5, there was a decrease in the α-helix content concomitant with an increase in β-strand content as the pressure increased. No changes in molecular size due to HHP-induced aggregation were detected by sodium dodecyl sulfate-PAGE. All samples showed higher thermostability as the severity of the treatment increased, indicating the formation of a less labile structure related to the HHP treatment.     

Impact of milieu conditions on the α-lactalbumin glycosylation in the dry state

Maillard reaction is influenced by protein and sugar properties, water activity (a_w) as well as the glycosylation time and temperature. The aim of this work was to investigate the influence of environmental parameters on the glycosylation reaction kinetics and to develop a technology platform for protein glycosylation as a possible substrate pre-treatment. The glycosylation reaction of bovine α-lactalbumin (α-La) was performed with lactose and maltodextrin in the dry-state at 40, 50 or 60 °C performed at a_w of 0.33, 0.44 or 0.58 for reaction times of 8, 24 or 48 h. The degree of glycosylation (DG) was determined as the loss of lysine using the ortho-phthalaldehyde (OPA) method. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) with Coomassie and glycoprotein staining was also performed. The reaction with lactose reached higher DG values in all cases as compared to reactions with maltodextrin (maximum DG of 85% and 31%, respectively, at a_w = 0.58 after 48 h). Lactosylation kinetics showed that the second order rate constants increased with increasing temperature and were highest at a_w = 0.58 in all cases. The activation energies were determined as 97.1 ± 37.7, 193.9 ± 9.1 and 136.6 ± 15.6 kJ/mol for a_w = 0.33, 0.44 and 0.58, respectively and showed an increasing trend with increasing temperature. Glycosylation of α-La offers a new process for improvement of functional properties as well as being a substrate pre-treatment process to control enzymatic digestion in order to generate tailor-made peptides as food additives with important health benefits like probiotics due to glycoprotein resistance to further enzyme hydrolysis.     

Ultrasonic effect on physicochemical and functional properties of α-lactalbumin

Ultrasound is the sound whose frequency is too high for humans to hear which is within the frequency range of 20 Hz-20 kHz, and the frequency of ultrasound is above 20 kHz. The aim of this study was to observe the effect of ultrasound and sonication on α-lactalbumin (α-LA) with a view to improving its physicochemical and functional properties. In this work both low-intensity ultrasound (500 kHz bath) and the high-intensity ultrasound (20 kHz probe and 40 kHz bath) were used. Ten per cent wt (g g⁻¹ dry matter) protein model suspensions of α-lactalbumin (α-LA) were treated with ultrasound probe (20 kHz for 15 and 30 min) and ultrasound baths (40 kHz and 500 kHz for 15 and 30 min).Changes in pH values, electrical conductivity, solubility measurements, foaming properties, as well as rheological and freezing-thawing properties have been examined. The protein fractions of α-lactalbumin were analyzed before and after ultrasound treatment by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).The result showed that pH did not change significantly upon ultrasound however conductivities increased significantly after 20 kHz sonication. Electrical conductivity decreased significantly for ultrasound treatments in baths at 40 kHz and 500 kHz for all samples. Solubility increased significantly for all samples at 20 kHz. Foam capacities and foam stabilities were improved after ultrasound treatments for both 20 kHz and 40 kHz treatments. Foaming properties were not improved for protein model suspensions for 500 kHz treatments. The molecular weight of the protein decreased significantly after ultrasound treatments both using a 20 kHz probe and 40 kHz bath. The flow behaviour of α-lactalbumin was observed to be shear-thickening after all treatments. Apparent viscosity data calculated with power law equation (R² = 0.983-0.999) have not been changed significantly after all treatments. A remarkable decrease of initial freezing point was obtained after 20 kHz treatments.     

Effect of heating on solid β-carotene

The effect of heating on isomerisation and stability of solid β-carotene was investigated, and the products generated by heating were analysed by a number of analytical techniques, including high-performance liquid chromatography (HPLC), UV/VIS-spectroscopy (UV) and gel permeation chromatography (GPC). For the first time, isomerisation of cis- to all-trans-isomer was demonstrated in partly melted solid β-carotene when β-carotene was heated at 90 and 140 °C. Only a few high molecular weight components were detected by GPC when β-carotene was heated in a nitrogen environment. In contrast, more high molecular weight polymers, as well as low molecular fragments, were produced when β-carotene was heated and exposed to air, suggesting that polymerisation was one of the dominant side-reactions of β-carotene change, in addition to degradation.     

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.     

Effects of Maillard reaction conditions on the antigenicity of α-lactalbumin and β-lactoglobulin in whey protein conjugated with maltose

The effects of Maillard reaction conditions (weight ratio of protein to sugar, temperature and time) on the antigenicity of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) in conjugates of whey protein isolate (WPI) with maltose were investigated. Response surface methodology was used to establish models to predict the antigenicity of α-LA and β-LG and find an optimal reaction condition under which the antigenicity of α-LA and β-LG reduces to minimum value. Conjugating WPI with maltose was an effective way to reduce the antigenicity of α-LA and β-LG. The antigenicity of α-LA decreased from 32.25 μg mL⁻¹ to 10.91 μg mL⁻¹. And the antigenicity of β-LG decreased from 272.4 μg mL⁻¹ to 38.17 μg mL⁻¹. Temperature had the greatest effect on the antigenicity of α-LA, while weight ratio of WPI to maltose was the most significant factor on the antigenicity of β-LG.     

Effect of temperature on molecular mobility,oxygen permeability,and dynamic site heterogeneity in amorphous α-lactalbumin films

We investigated molecular mobility and oxygen diffusion in amorphous solid films of α-lactalbumin (α-La) using phosphorescence from the xanthene probe erythrosin B in order to elucidate the molecular mechanism(s) that control oxygen permeability in amorphous solid proteins. Emission peak energy and bandwidth were determined by fitting spectra to a lognormal bandwidth function; intensity decays were fit to a stretched exponential function to determine the lifetime and the stretching exponent. Peak emission energy decreased gradually over the range from −20 to over 120 °C, indicating a gradual increase in the matrix dipolar relaxation rate. Bandwidths were constant at low temperature but increased dramatically above ∼50 °C, indicating that the dynamic heterogeneity of the protein matrix increased at high temperature. Emission lifetimes decrease gradually at low and more steeply at high temperature, indicating that the rate of matrix collisional quenching increased with temperature. Arrhenius analysis of the rate constant for non-radiative decay showed a gradual increase in quenching indicative of matrix softening. Comparison of lifetimes in air and N₂ (±oxygen) monitored oxygen permeability. Oxygen permeability became detectable at about 0 °C and appeared to correlate with matrix mobility. The emission spectrum shifted to higher energy as a function of time following excitation, whereas the phosphorescence lifetime decreased with increasing emission wavelength; both behaviors provided strong evidence for distinct sites within the protein matrix varying in molecular mobility. Phosphorescence spectroscopy thus provides a simple tractable tool for monitoring conditions that activate oxygen diffusion in amorphous solid foods.     

Identification and characterization of yak α-lactalbumin and β-lactoglobulin

α-Lactalbumin (α-LA) and β-lactoglobulin (β-LG) were isolated from yak milk and identified by mass spectrometry. The variant of α-LA (L8IIC8) in yak milk had 123 amino acids, and the sequence differed from α-LA from bovine milk. The amino acid at site 71 was Asn (N) in domestic yak milk, but Asp (D) in bovine and wild yak milk sequences. Yak β-LG had 2 variants, β-LG A (P02754) and β-LG E (L8J1Z0). Both domestic yak and wild yak milk contained β-LG E, but it was absent in bovine milk. The amino acid at site 158 of β-Lg E was Gly (G) in yak but Glu (E) in bovine. The yak α-LA and β-LG secondary structures were slightly different from those in bovine milk. The denaturation temperatures of yak α-LA and β-LG were 52.1°C and 80.9°C, respectively. This study provides insights relevant to food functionality, food safety control, and the biological properties of yak milk products.     

Taste modification of amino acids and protein hydrolysate by α-cyclodextrin

The objective of this work was to characterize the formation of amino acid inclusion complexes with α-cyclodextrin (α-CD) and evaluate the influence of added α-CD on the taste perception of amino acids and hydrolyzed soy protein at pH 4.5. The formation of the inclusion complexes of phenylalanine, tryptophane, tyrosine, isoleucine, proline and histidine with α-CD were detected by nuclear magnetic resonance techniques (ROESY and DOSY) and the sensory characteristics of soy hydrolysates were judged by a panel of trained tasters. It was concluded that these amino acids form inclusion complexes with α-CD and the order of affinity for the α-CD cavity is phenylalanine ≈ tryptophane > proline > isoleucine ≈ tyrosine ≈ histidine. α-CD alters the bitter taste perception of the amino acids and reduces the bitter taste from hydrolyzed soy protein. These results indicate a potential use of α-CD for debittering protein hydrolysates in acidic beverages.     

Partitioning of α-lactalbumin and β-lactoglobulin in whey protein concentrate/hydroxypropylmethylcellulose aqueous two-phase systems

Whey protein concentrate (WPC) was fractionated by using hydroxypropylmethylcellulose (HPMC) at pH 6.5. Incompatible mixtures with different proportions of HPMC and WPC were prepared. After phase separation, the protein concentration in both phases was determined by the Kjeldahl method and the proportion of each protein by SDS-PAGE combined with image analysis. The results show that the low molecular weight proteins α-lactalbumin (α-lac) and β-lactoglobulin (β-lg) were retained in high proportion in the upper phase (about 90% compared to 64% of WPC). The most efficient condition to fractionate β-lg and α-lac was the phase separation of an incompatible mixed system with a high initial concentration of WPC and a low initial concentration of HPMC i.e., WPC 20%, wt/wt/HPMC 0.5%, w/w. It can be concluded that the thermodynamic incompatibility which arises from mixing WPC with HPMC could be used as a method for fractionation of whey proteins.     

Proliferative effects of synthetic peptides from β-lactoglobulin and α-lactalbumin on murine splenocytes

Eleven peptides, selected on the basis of physicochemical characteristics and their theoretical release from β-lactoglobulin (β-Lg) and α-lactalbumin (α-La) by trypsin or chymotrypsin, were chemically synthesised to evaluate their immunomodulating properties. Murine splenocyte proliferation in the absence and presence of mitogen and different peptide concentrations were measured after 72–96 h incubations. β-Lg f78–83 had no effect on proliferation; β-Lg f15–20, f55–60, f84–91, f92–105, f139–148, f142–148 and α-La f10–16 stimulated proliferation to different extents; β-Lg f1–8, f102–105 and α-La f104–108 showed a cytotoxic effect. Regression analysis revealed the relationship of positive charge, hydrophobicity and length to the stimulatory proliferative effect. β-Lg f15–20, f55–60 and f139–148 also induced various inhibiting and/or stimulating effects on cytokine secretion. The results confirm that peptides releasable by digestive enzymes from α-La and β-Lg have the potential to influence the specific immune response through the modulation of splenocyte proliferation and cytokine secretion.