Comparative study on the effects of d-psicose and d-fructose in the Maillard reaction with β-lactoglobulin

d-Psicose, recognized as a noncaloric sweetener, has shown a great potential in food industry. In the present study, d-psicose and d-fructose were used to modify bovine β-lactoglobulin (β-Lg) through Maillard reaction. The Maillard reaction process and the physicochemical and structural properties of the modified proteins were also investigated. The result showed that compared to d-fructose, d-psicose played a more effective role in the Maillard reaction, especially after the initial stage of the reaction. Moreover, the modified β-Lg with d-psicose had more polymeric compounds, higher antioxidant activity, but lower thermal stability than that with d-fructose. These findings, especially the structural changes of the modified proteins, supplied detail information on the Maillard reaction of d-psicose, and could provide some guidance to the practical applications of this rare sugar on food industry.     

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.     

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.     

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.     

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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effect of β-lactoglobulin A and B whey protein variants on cheese yield potential of a model milk system

Cheese yield mainly depends on the amount and proportion of milk constituents; however, genetic variants of the proteins present in milk may also have an important effect. The objective of this research was to study the effect of the variants A and B of β-lactoglobulin (LG) on cheese yield using a model system consisting of skim milk powder fortified with different levels of a mixture containing α-lactalbumin and β-LG genetic variants (A, B, or A-B) in a 1:2 ratio. Fortified milk samples were subjected to pasteurization at 65°C for 30 min. Miniature cheeses were made by acidifying (pH = 5.9) fortified milk and incubating with rennet for 1 h at 32°C. The clot formed was cut, centrifuged at 2,600 × g for 30 min at 20°C and drained for determining cheese yield. Cheese-yielding capacity was expressed as actual yield (grams of cheese curd per 100 g of milk) and dry weight yield (grams of dried cheese curd per 100 g of milk). Free-zone capillary electrophoresis was used for determining β-LG A or B recovery in the curd during rennet-induced coagulation. The presence of β-LG variant B resulted in a significantly higher actual and dried weight cheese yield than when A or A-B were present at levels ≤0.675% of whey protein (WP) addition. Results of free-zone capillary electrophoresis allowed us to infer that β-LG B associates with the casein micelles during renneting, as shown by an increase in the recovery of this variant in the curd when β-LG B was added up to a maximum at 0.45% (equivalent to 0.675% WP). In general, actual or dried weight cheese yield increased as WP addition was increased from 0.225 to 0.675%. However, when WP addition ranged from 0.675 to 0.90%, a drastic drop in cheese yield was observed. This behavior may be because an increase in the aggregation of casein micelles with a concomitant inclusion of whey protein in the gel occurs at low levels of WP addition, whereas once the association of WP with the casein micelles reach a saturation point at addition levels higher than 0.675%, rearrangements of the gel network result in larger whey expulsion and syneresis. This knowledge is expected to be useful to maximize cheese yield and optimize processing conditions during cheese and cheese analogs manufacturing.     

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.     

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.     

Effects of lipid supplementation on the yield and composition of milk from cows with different β-lactoglobulin phenotypes

Responses to lipid supplementation differ between dairy breeds and genetic lines suggesting nutrition by genotype interactions. β-Lactoglobulin phenotype is associated with changes in yield and composition of milk. The response of cows with different β-lactoglobulin phenotypes to lipid supplementation has not been examined. Furthermore, we examined whether lipid supplementation alters milk protein composition. By using a randomized block design, we fed Holstein cows for 3 wk either a control diet containing 2.8% crude fat (n = 19) or an experimental diet that was supplemented with 4.2% tallow (n = 20). Before randomization, all cows were fed the supplemental tallow diet for at least 2 wk. Dry matter intake, body weight, milk yield, and milk composition were measured in the last week before and during the experimental period. Feeding supplemental tallow increased dry matter intake and yields of milk and milk components, including casein content, without decreasing milk component content or altering milk protein composition. On the low-fat control diet, cows with the β-lactoglobulin allele B had a greater milk and milk component yield than cows with the A allele, whereas no differences by β-lactoglobulin phenotype were observed in cows on the tallow supplement diet. Our results suggest that cows that differ in β-lactoglobulin phenotype respond differently to a low-fat diet and that feeding cows 4.2% of additional tallow increases milk yield without affecting milk component content and milk protein composition.     

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.