Evolution of β-lactoglobulin and α-lactalbumin content during yoghurt fermentation

The evolution of the concentrations of β-lactoglobulin (BLG) and α-lactalbumin (ALAC) was studied during the early stages of yoghurt fermentation by YC 191, a mixed strain culture from Chr Hansen containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Radial immunodiffusion of samples taken at different times indicated that the concentration of both proteins remained constant during fermentation. Electrophoresis performed on 12% polyacrylamide slab gels confirmed the results obtained with radial immunodiffusion. In model experiments, the strains were incubated either separately or in combination with both whey proteins, one by one or together. BLG proteolysis required a longer time than that used during yoghurt fermentation. ALAC was susceptible to proteolysis, especially by Streptococcus thermophilus. Despite evident possession of adequate proteolytic system, the strains used for yoghurt production did not cleave detectable amounts of the whey proteins during yoghurt fermentation.     

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.     

In vitro iron absorption of α-lactalbumin hydrolysate-iron and β-lactoglobulin hydrolysate-iron complexes

To study the feasibility of promoting iron absorption by peptides derived from α-lactalbumin and β-lactoglobulin, the present work examined the transport of iron across Caco-2 monolayer cell as in vitro model. Caco-2 cells were seeded in bicameral chambers with α-lactalbumin hydrolysate-Fe (α-LAH-Fe) complex and β-lactoglobulin hydrolysate-Fe (β-LGH-Fe) complex, α-LAH and iron mixture, β-LGH and iron mixture, FeSO₄ and ascorbic acid mixture, and FeSO₄. In addition, the cytotoxicity of α-LAH-Fe and β-LGH-Fe complexes were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The iron absorption and ferritin content were assessed using the coupled in vitro digestion/Caco-2 cell model. Results support that peptide-iron complexes can promote ferritin formation and it is possible to apply β-LGH-Fe complexes as iron-fortified supplements with high iron absorbability.     

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.     

Preparation and characterization of β-lactoglobulin hydrolysate-iron complexes

The purpose of this study was to determine the best preparation condition of β-lactoglobulin hydrolysate-iron complexes and characterize its structural transformation both before and after binding using the UV-visible absorption spectrum, Fluorescence spectrum, and Fourier transform infrared spectroscopy. Results showed that β-lactoglobulin hydrolysates obtained with alcalase after hydrolysis for 6h possessed the highest iron-binding capacity. The highest yield of complexes was obtained when the mass ratio between β-lactoglobulin hydrolysate and Fe(3+) reached 40:1, with the optimal pH value of 7.0. All of the spectra indicated that some sites such as amido bonds transformed during chelation, and nitrogen atoms could chelate with Fe(3+) to form coordinate bonds by offering electron pairs. Therefore, β-lactoglobulin hydrolysate-iron complexes may be good carriers for iron and possess great potential to be used as iron supplements.     

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.     

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.     

Heat-induced interactions of β-lactoglobulin and α-lactalbumin with the casein micelle in pH-adjusted skim milk

Skim milks, adjusted to pH 6.48, 6.60 or 6.83, were heated for various temperature–time combinations in a pilot-scale ultra-high temperature (UHT) plant. Heat-treated samples were ultracentrifuged and their supernatants analysed by quantitative polyacrylamide gel electrophoresis in order to measure the extent of β-lactoglobulin (β-lg) and α-lactalbumin (α-la) denaturation and their subsequent association with the casein micelle. The activation energy of β-lg denaturation decreased as the pH increased. In contrast, there was no apparent trend for α-la. The extent of β-lg and α-la association with the micelle increased with heating time and temperature. The association of both proteins with the micelle was markedly affected by the milk pH. The rate and extent of association were greatest at pH 6.48, and least at pH 6.83. α-La continued to associate with the micelle although most of the β-lg had already associated. It was possible that α-la interacted at the micelle surface with β-lg that had previously associated with the micelle. A pseudo-first-order mathematical model was used to calculate the apparent rate constant for β-lg association with the micelle.     

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 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.     

Structure and antioxidant activity of Maillard reaction products from α-lactalbumin and β-lactoglobulin with ribose in an aqueous model system

Maillard reaction products (MRPs) were prepared from aqueous model mixtures containing 3% (w/w) ribose and 3% (w/w) of the dairy proteins α-lactalbumin (α-LA) or β-lactoglobulin (β-LG), heated at 95 °C, for up to 5 h. The pH of MRPs decreased significantly during heat treatment of α-LA-Ribose and β-LG-Ribose mixtures from 8.4 to 5.3. The amino group content in MRPs, derived from the α-LA-Ribose and β-LG-Ribose model system, was decreased noticeably during the first hour and did not change thereafter. The loss of free ribose in MRPs was higher for β-LG-Ribose than for α-LA-Ribose. During the Maillard reaction, the concentration of native and non-native α-LA, or β-LG, decreased and the formation of aggregates was observed. Fluorescence intensity of the β-LG-Ribose MRPs reached maximum within 1 h, compared to 2 h for α-LA-Ribose MRPs. Meanwhile, modification of the UV/vis absorption spectra for α-LA and β-LG was mainly due to a condensation reaction with ribose. Dynamic light scattering showed a significant increase in the particle size of the MRPs. Size exclusion chromatography of MRPs revealed the production of both high and low molecular weight material. Electrophoresis of MRPs indicated polymerization of α-LA and β-LG monomers via inter-molecular disulfide bridge, but also via other covelant bonds. MRPs from α-LA-Ribose and β-LG-Ribose exhibited increased antioxidant activities, therefore theses MRPs may be used as natural antioxidants in food products.     

Comparison of the gel-forming ability and gel properties of α-lactalbumin,lysozyme and myoglobin in the presence of β-lactoglobulin under high pressure

α-Lactalbumin (α-La) and lysozyme (LZM) each contain four disulfide bonds but no free SH group, whereas myoglobin (Mb) possesses no disulfide bond or free SH group. In this work, the pressure-induced gelation of α-La, LZM and Mb in the absence and in the presence of β-lactoglobulin (β-Lg) was studied. Solutions of α-La, LZM and Mb (1–24%, w/v) did not form a gel when subjected to a pressure of 800 MPa and circular dichroism analysis revealed that both α-La and LZM are pressure-resistant proteins. In the presence of β-Lg (5%, w/v), however, a pressure-induced gel formed for α-La and LZM (each 15%, w/v) but not for Mb (15%, w/v). One- and two-dimensional SDS-PAGE demonstrated the disulfide cross-linking of proteins was responsible for the gelation. Although α-La and LZM are homologous and have the same disulfide bond arrangement, the texture and appearance of the gels formed from α-La/β-Lg and LZM/β-Lg were markedly different even when induced under the same experimental conditions. Microscopic analysis indicated that phase separation occurs during the gelation of LZM/β-Lg but not during the gelation of α-La/β-Lg. NMR relaxation measurement revealed that the association of water molecules with the protein matrix in the α-La/β-Lg gel is tighter compared to that in the LZM/β-Lg gel. These results indicate that the gel-forming ability of a globular protein under high pressure is related to the primary structure of the protein, and that the gel properties depend on the cross-linking reaction and on the phase behavior of protein dispersion under high pressure.     

One-step method for the isolation of α-lactalbumin and β-lactoglobulin from cow’s milk while preserving their antigenicity

Milk is a highly nutritional food, and separation of major allergens from milk has become important to people who are allergic. The aim of this study was to establish a simple and repeatable method for the isolation of α-lactalbumin and β-lactoglobulin from cow’s milk while preserving their antigenicity. Fractions of α-lactalbumin and β-lactoglobulin were salted-out using 50% ammonium sulfate from whey that was collected from cow’s milk after pH adjustment and then purified by anion-exchange chromatography with diethylaminoethyl-Sepharose Fast Flow. The antigenicity of the purified proteins was evaluated by indirect competitive enzyme-linked immunosorbent assay. The results showed that the purities of the α-lactalbumin and β-lactoglobulin collected were 84.85 and 94.91% and the cross-reactivities of the purified proteins were 93.2 and 95.4%, respectively. Therefore, this simple and efficient strategy consisting of a one-step process for α-lactalbumin and β-lactoglobulin is suitable for purifying the major allergens in cow’s milk. In addition, a scientific experimental basis for the preparation of non-allergenic milk was also offered in this study.     

Identification and characterization of novel α-amylase and α-glucosidase inhibitory peptides from camel whey proteins

This study explores the inhibitory properties of camel whey protein hydrolysates (CWPH) toward α-amylase (AAM) and α-glucosidase (AG). A general full factorial design (3 × 3) was applied to study the effect of temperature (30, 37, and 45°C), time (120, 240, and 360 min), and enzyme (pepsin) concentration (E%; 0.5, 1, and 2%). The results showed that maximum degree of hydrolysis was obtained when hydrolysis was carried out at higher temperature (45°C; P < 0.05), compared with lower temperatures of 30 and 37°C. Electrophoretic pattern displays degradation of all protein bands upon hydrolysis by pepsin at various hydrolysis conditions applied. All the 27 CWPH generated showed significant AAM and AG inhibitory potential as indicated by their lower IC₅₀ values (mg/mL) compared with intact whey proteins. In total 196 peptides were identified from selected hydrolysates and 15 potential peptides (PepSite score > 0.8; http://pepsite2.russelllab.org/) were explored via in silico approach. Novel peptides PAGNFLMNGLMHR, PAVACCLPPLPCHM, MLPLMLPFTMGY, and PAGNFLPPVAAAPVM were identified as potential inhibitors for both AAM and AG due to their high number of binding sites and highest binding probability toward the target enzymes. CCGM and MFE, as well as FCCLGPVPP were identified as AG and AAM inhibitory peptides, respectively. This is the first study that reports novel AG and AAM inhibitory peptides from camel whey proteins. The future direction for this research involves synthesis of these potential AG and AAM inhibitory peptides in a pure form and investigate their antidiabetic properties in the in vitro, as well as in vivo models. Thus, CWPH can be considered for potential applications in glycaemic regulation.     

Interactions of α-ionone, β-ionone and vanillin with the primary genetic variants of β-lactoglobulin

Interactions between bovine β-lactoglobulin (β-Lg) genetic variants (A, B and a mixture thereof) and α-ionone, β-ionone and vanillin were studied by tryptophan fluorescence spectroscopy under various conditions of pH and ionic strength. When β-ionone was added to β-Lg, we found progressive increase in quenching from pH 3.0 to pH 8.0 and relative decrease at pH 11.0. Fluorescence quenching progressively increased from pH 3.0 through to pH 11.0 when vanillin was added. Small differences in quenching of variants β-Lg variants A and B were observed at pH 8.0 for β-ionone, but not for vanillin at any pH. NaCl affected the fluorescence quenching of β-Lg by β-ionone at pH 7.0 and 8.0 and by vanillin at pH 8.0 and 11.0. No apparent interaction between α-ionone and β-Lg was observed under the conditions studied. The results suggest that β-ionone and vanillin bind at different domains of β-Lg, possibly with different binding mechanisms.     

In vitro release of α-tocopherol from emulsion-loaded β-lactoglobulin gels

Cold-set oil-loaded protein gels based on an emulsifying step followed by Ca²⁺-induced gelation of pre-denatured β-lactoglobulin (β-LG) have been recently developed. In vitro release and stability of a fat-soluble compound (α-tocopherol) therein were investigated in this work. Release of α-tocopherol was found to be controlled mainly by matrix erosion due to protein degradation. Compound release and matrix erosion were almost complete after incubation under gastric or intestinal conditions for 6.5 h. However, both processes were basically inhibited upon changing the dissolution medium from the gastric to the intestinal type, possibly due to β-LG partial hydrolysis products with greater emulsifying capacity anchoring to the surface of gel oil droplets. The stability of released α-tocopherol was apparently improved by binding to protein and/or hydrolysis products thereof.     

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.     

Foaming behaviour of EDTA-treated α-lactalbumin

The foaming properties of partially denatured α-lactalbumin was investigated. The partially denatured state was produced by removing bound Ca²⁺ by treatment with ethylenediaminetetraacetic acid (EDTA) at pH 8.0 and 25°C. Surface tension measurements showed that partially denatured α-lactalbumin unfolds easily at liquid interfaces compared with the native protein. The results of foam volume and stability measurements were consistent with the results of surface tension measurements. In the presence of EDTA a considerable amount of foam was obtained at low concentrations, such as 0.1 mg/ml, and the foam stability was improved. This indicates the importance of the protein structure on the adsorption of molecules at liquid interfaces. The presence of Ca²⁺ also resulted in an increase in the foamability and foam stability of α-lactalbumin compared with native protein, due to the saturation of the surface charges. This shows the binding affinity of protein to Ca²⁺. The investigation of the effect of Ca²⁺ on the surface behaviour of β-lactoglobulin, another whey protein, also showed an improvement in the foaming properties of protein.     

Isolation of β-lactoglobulin from buffalo milk and characterization of its structure changes as a function of pH and ionic strength

β-lactoglobulin (β-lg) is one of the most widely used proteins in food industry. Purification of β-lg from buffalo milk while maintaining its native structure is of practical influence to the industry. This research demonstrated that β-lg can be separated efficiently from whey using 70 g kg^??1 NaCl at pH 2.0, which showed higher yield and activity of β-lg. It was found that the presence of NaCl or the changing of pH had no influence on the secondary structure of β-lg while they significant influenced the tertiary structure of β-lg. More hydrophobic groups were exposed with increasing NaCl concentration. Presence of low concentration of NaCl inhibited the exposure of Trp while the exposure of Trp was increased in the presence of high concentration of NaCl. In contrast, pH changes did not influence the exposure of Trp while the decrease of pH in the range of 7.5–5.5 decreased the exposure of hydrophobic groups to the hydrophilic environment.