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 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.     

Steady-state kinetics of tryptic hydrolysis of β-lactoglobulin after dynamic high-pressure microfluidization treatment in relation to antigenicity

Our previous research revealed that dynamic high-pressure microfluidization (DHPM) increased the antigenicity of β-lactoglobulin (β-Lg) below 80 MPa, which was related to the unfolding of protein. To test the hypothesis that the unfolding of protein may change proteolytic susceptibility of β-Lg and modulate its antigenicity during the digestion, we developed that the steady-state kinetics of tryptic hydrolysis of β-Lg subjected to DHPM (0.1–80 MPa) have been investigated in relation to the antigenicity in this study. According to the steady-state kinetics analysis, the improved digestion of β-Lg was accompanied with the obvious decrease of antigenicity during the hydrolysis with pressure increasing, reflected by the increase of k _c , the decrease of K _m, the increase of overall catalytic efficiency (k _c/K _m), and the increase of the binding volume. It was indicated that although DHPM can increase the antigenicity of β-Lg, the enhanced digestibility of β-Lg at elevated pressure contributed to a decrease of antigenicity during the hydrolysis.     

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.     

Influence of salts on hydrolysis of β-lactoglobulin by free and immobilised trypsin

Immobilised trypsin is an alternative to free trypsin for producing protein hydrolysates with increased functionalities. However, the influence of hydrolytic conditions on this process remains unclear. The influence of salts on β-lactoglobulin (β-Lg) hydrolysis by free and immobilised trypsin was compared. For both forms of trypsin, 0.1 m Tris accelerated the release of most final peptides except f (71–75), and had no significant effects on the hydrolysis of intact β-Lg. Increasing NaCl concentrations from 0 to 0.02 m increased the degree of hydrolysis (DH) by 22.4% for free trypsin versus 62.1% for immobilised trypsin. The presence of 0.1 or 0.5 m NaCl hindered the release of peptides associated with the breakdown of intact protein. This led to 2–4 fold decreases in depleting intact β-Lg and DH, except immobilised trypsin at 0.1 m NaCl (DH increased by 44.3% versus without NaCl). Potential mechanisms underlying the effects of salts are discussed.     

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 polysaccharide charge on formation and properties of biopolymer nanoparticles created by heat treatment of β-lactoglobulin–pectin complexes

Biopolymer nanoparticles can be formed by heating globular protein/polysaccharide mixtures above the thermal denaturation temperature of the protein under pH conditions where the two biopolymers are weakly electrically attracted to each other. In this study, the influence of polysaccharide linear charge density on the formation and properties of these biopolymer nanoparticles was examined. Mixed solutions of globular proteins (β-lactoglobulin) and anionic polysaccharides (high and low methoxyl pectin) were prepared. Micro-electrophoresis, dynamic light scattering, turbidity and atomic force microscopy (AFM) measurements were used to determine the influence of protein-to-polysaccharide mass ratio (r), solution pH, and heat treatment on biopolymer particle formation. Biopolymer nanoparticles (d < 500 nm) could be formed by heating protein–polysaccharide complexes at 83 °C for 15 min at pH 4.75 and r = 2:1 in the absence of added salt. The biopolymer particles formed were then subjected to pH and salt adjustment to determine their stability. The pH stability was greater for β-lactoglobulin-HMP complexes than for β-lactoglobulin-LMP complexes. The addition of 200 mM sodium chloride to heated complexes greatly improved the pH stability of HMP complexes, but decreased the pH stability of LMP complexes. The biopolymer particles formed consisted primarily of β-lactoglobulin, which was probably surrounded by a pectin coating at low pH values. AFM measurements indicated that the biopolymer nanoparticles formed were spheroid in shape. These biopolymer particles may be useful as delivery systems or fat mimetics.     

Effect of various calcium concentrations on the interactions between β-lactoglobulin and epigallocatechin-3-gallate

β-Lactoglobulin (β-lg) is a globular protein accounting for 11% of all milk proteins. Recently, the use of β-lg as vehicles for the delivery of bioactive compounds such as green tea catechins has been explored. The objective of this study was to determine the impact of various calcium concentrations on the interactions between β-lg and epigallocatechin-3-gallate (EGCG), the most abundant green tea catechins. Five different calcium concentrations were tested for their effect on complex formation and characteristics. Our results showed that when EGCG was present, the increase in calcium concentration influenced ζ-potential and more importantly, drastically increased particle size. The presence of EGCG was essential to the formation of larger complexes, stabilized mainly by electrostatic interactions via calcium bridging when calcium is present.     

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.     

Evaluation of air-dried soy protein isolate-rice noodles prepared via combined treatment with microbial transglutaminase and glucono-δ-lactone

In this study, the effects of steaming treatments during the preparation of structurally enhanced air-dried rice noodles were investigated. Soy protein isolate-rice noodles (RNS) were combined (COM) with microbial transglutaminase (MTG) and glucono-δ-lactone (GDL), and steamed for 5 (S5) or 10 (S 10) min followed by air-drying to yield air-dried RNS-COM-S5 and RNS-COM-S10, respectively. Control samples were air-dried rice flour noodles (ADRN), steamed for 5 or 10 min before air-drying (ADRN-S5 and ADRN-S10). Compared to other air-dried RNS prepared without steaming, RNS-COM-S5 and RNS-COM-S10 showed a reduction in cooking time (5.35 min) and cooking loss (7.11%) and increased cooking yield (125%), supposedly due to low gelatinisation enthalpy, while retaining textural and mechanical properties, significantly higher (P < 0.05) than the controls. The scanning electron micrographs showed that all steamed air-dried noodles also had larger hollows, which could be responsible for the improved cooking qualities. In general, the relative order of starch crystallinity resulting from steaming treatment decreased in all steamed noodles, and RNS-COM1-S10 showed the highest order. The treatment of RNS with MTG and GDL yielded air-dried noodles with a more compact structure. Steaming is necessary to improve the rehydration characteristics of the air-dried noodles without sacrificing textural and mechanical qualities.     

Identification of glutaminyl sites on β-lactoglobulin for threadfin bream liver and microbial transglutaminase activity by MALDI-TOF mass spectrometry

The cross-linking of β-lactoglobulin (BLG) was efficiently catalysed by microbial transglutaminase (MTG) but not by fish (threadfin bream) liver transglutaminase (FTG). BLG cross-linking was inhibited by 2 mM 5-(biotinamido) pentylamine (BPNH₂) and MTG incorporated BPNH₂ into BLG ∼5 times more than was FTG. The glutaminyl sites for the incorporation of BPNH₂ into BLG by FTG and MTG were identified using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS analyses showed that MTG and FTG incorporated 4 and 1 residues of BPNH₂ per molecule of BLG, respectively. The BPNH₂-tagged BLG was digested by trypsin and BPNH₂-tagged peptides were selectively purified by avidin-affinity chromatography. Amino acid sequences of BPNH₂-tagged peptides were identified by comparing their MALDI-TOF mass spectra with the theoretical mass profiles from the MASCOT database. The BPNH₂-modification sites catalysed by MTG were glutamine (Q)13, Q68, Q15 or Q20, Q155 or Q159, whilst FTG only incorporated BPNH₂ into BLG at Q68. The different reactivities between FTG and MTG might be due to the different accessibilities of these TGases to the Q residues as well as to differences in substrate specificities.     

Effect of glucono-δ-lactone and κ-carrageenan combined with high pressure treatment on the physico-chemical properties of restructured pork

In this study, response surface methodology (RSM) was used to evaluate the combined effects of NaCl, glucono-δ-lactone (GdL), and κ-carrageenan concentration (0.25%, 0.5%, and 0.75%) on the binding properties of restructured pork under hydrostatic pressure. All the generated RSM models showed no lack-of-fit and significance at the 0.001 level. The addition of both NaCl and GdL had a significant effect on color. A significant decrease in pH was shown when the GdL level increased, and subsequently led to a decrease in WHC. However, increasing the GdL level increased the binding strength. Therefore, the results indicate that a reduction in the NaCl level during meat restructuring, under pressure treatment, can be achieved by using GdL; and even a low GdL concentration allows for palatable binding properties in meat restructuring when κ-carrageenan is added.     

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.     

Effect of plasma treatment and grafting of β-cyclodextrin on color properties of wool fabric dyed with Shrimp shell extract

The aim of this study was to evaluate the effect of low temperature plasma (LTP) treatment and grafting of beta-cyclodextrin (β-CD) on dyeing and fastness properties of wool fabrics using natural dye extracted from shrimp shell. Aluminum sulfate, iron sulfate, tin chloride, copper sulfate, and sodium dichromate were used as mordant and the dyeing of samples was conducted after the pre-mordanting process. Scanning electron microscopy and Fourier transform infrared spectroscopy (FTIR) were used to study the morphological and chemical changes of the fibers. Also, the antimicrobial properties of fabrics were quantitatively evaluated against Staphylococcus aureus according to AATCC 100-1993 test method. The results showed using of sodium dichromate as mordant could be attained to green shade after dyeing fabrics. The color difference and color strength of samples were improved by increasing the sodium dichromate concentration and β-CD in the after-treatment process. FTIR analysis indicated a significant increase in the absorption peak of hydroxyl group after grafting of β-CD on LTP-treated samples. Antimicrobial results of samples indicated that LTP-treated fabric demonstrated no antimicrobial behavior, while the dyed and β-CD-grafted sample possessed significant reduction in the number of bacteria. Moreover, the β-CD-treated samples showed very good to excellent light and washing fastness properties.     

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.     

Antigenicity and conformational changes of β-lactoglobulin by dynamic high pressure microfluidization combining with glycation treatment

The combined effect of previous dynamic high-pressure microfluidization treatment (40, 80, 120, and 160 MPa) and subsequent glycation with galacto-oligosaccharides (GOS) on the antigenicity of β-lactoglobulin (β-LG) was investigated. The antigenicity of β-LG-GOS decreased at relatively low pressure (≤120 MPa). Surface sulfhydryl group content of β-LG-GOS increased and surface hydrophobicity of β-LG-GOS decreased. Additionally, protein unfolding in β-LG-GOS samples was reflected by quenching of fluorescence intensity, the red-shift of fluorescence spectra, decreased UV absorption, and circular dichroism analysis, indicating tertiary and secondary structural changes of β-LG. The conformational changes may contribute to the alteration of antigenicity.     

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.     

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.     

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.     

Aggregation and conformational changes of bovine β-lactoglobulin subjected to dynamic high-pressure microfluidization in relation to antigenicity

Our previous research indicated that dynamic high-pressure microfluidization (DHPM) had a significant effect on the antigenicity of β-lactoglobulin (β-LG). In this study, aggregation and conformational changes subjected to DHPM (0.1-160 MPa) were investigated in relation to antigenicity. When DHPM pressure increased from 0.1 to 80 MPa, disaggregation of β-LG samples and partial unfolding of the molecule were accompanied by an increase in β-LG antigenicity, which was reflected in the decrease of particle size, increase of free sulfhydryl (SH) contents and β-strands contents, and slight exposure of aromatic amino acid residues. At pressures above 80 MPa, the reaggregation of β-LG may contribute to the decrease in antigenicity, which was reflected by an increase in particle size, the formation of aggregates, a decrease of in SH and β-strands contents, and slight changes in aromatic amino acid residues. Aggregation and conformational changes of β-LG under DHPM was related to its antigenicity.