Formation Of Soluble Protein Aggregates Research Articles

Improvement in the functional properties of quinoa (Chenopodium quinoa) protein isolates after the application of controlled heat-treatment: Effect on structural properties

Quinoa protein isolates (QPIs) were subjected to controlled heat-treatment (80−100 °C) at variable time (15−30 min). Results showed that heat-treatment induced structural changes in QPIs. Circular dichroism showed loss of secondary structure of QPIs, particularly α-helix and β-sheet. Total and exposed SH groups were significantly (p ≤ 0.05) lower in heat-treated QPIs. SDS-PAGE profile showed formation of soluble protein aggregates with smaller particle size. Particle size decreased when QPIs were heated at 80 °C for 30 min. Solubility and turbidity of heat-treated QPIs improved significantly. Decline in crystallinity was due to denaturation of protein molecules by heat-treatment. Water binding capacity, oil binding capacity, emulsifying activities and stabilities were found lower in QPIs heat-treated at higher thermal temperatures for longer time. This might be due to decline in total and exposed SH contents, accelerated conversion of SH groups into disulfide bonds and formation of large molecular weight polymers or protein aggregates due to polymerization reactions. Higher foaming capacity (89.99 %) and foaming stability values of 167.80 % (10 min) and 124.15 % (20 min) were found in QPIs heated at 80 °C for 30 min. The results thus suggests that heat-treatment carried out at lower heating temperatures had improved the functional properties of QPIs.

Effects of malondialdehyde‐induced protein oxidation on the structural characteristics of rice protein

SummaryMalondialdehyde (MDA) was selected to represent a secondary by‐product of lipid peroxidation during rice ageing. This study aimed to investigate the effects of MDA modification on the structural characteristics of rice protein. The results showed that as MDA concentration increased, rice protein carbonyl and disulphide groups increased, but sulphydryl content decreased. The blue shift of maximum fluorescence peak, the decrease of rice protein intrinsic fluorescence intensity and the reduction of surface hydrophobicity indicated the formation of protein aggregates caused by MDA oxidative modification. The results of molecular weight distribution and particle size distribution showed that MDA modification resulted in the formation of soluble protein aggregates, and the decrease of rice protein solubility indicated that insoluble protein aggregates were formed. Results of protein electrophoresis showed that MDA modification contributed to rice protein aggregation via non‐disulphide covalent bonds. The results showed that rice protein gradually aggregated with increasing MDA concentration.

Influence of pulsed light treatment on the aggregation of whey protein isolate

The effect of pulsed light (PL) on the aggregation of whey protein isolate (WPI) solutions was investigated. PL fluence values from 4 to 16J/cm2 were used to treat WPI (1% w/v) solutions in sodium phosphate buffer (pH=7.5). Whey protein structural modification and aggregation were assessed through the determination of free SH-groups and UV-absorption spectra. Additionally, covalent and non-covalently linked protein-protein interactions were identified through the measurement of turbidity, aggregation index, particle size distribution, and SDS-PAGE.WPI upon PL treatment showed structural changes as demonstrated by the immediate increase of free SH-group content (unfolding) and the subsequent formation of a small fraction of aggregation of unfolded proteins, due to both hydrophobic interactions and the formation of disulphide bonds. Turbidity, mean particle size, and aggregation index increased in samples treated at PL fluence from 4 to 16J/cm2. Furthermore, particle size distribution analysis of samples treated at higher fluence indicated that WPI dimer dissociation and formation of larger particles were likely to occur. The association of intermediate and larger protein molecules as well as the formation of soluble aggregates between β-lactoglobulin and α-lactalbumin were also observed in gel electrophoresis analysis. In conclusion, the results of this investigation demonstrated the potential of PL treatments to induce protein denaturation, with a minimal formation of soluble protein aggregates.

Physicochemical and functional properties of cowpea protein isolates treated with temperature or high hydrostatic pressure

Abstract The effect of thermal (TT, 70 and 90 °C) and high hydrostatic pressure (HHPTs, 200, 400 and 600 MPa) treatments on physicochemical and functional properties of cowpea protein isolates (CPIs) extracted at pH 8.0 (A8) and pH 10.0 (A10) was analyzed. The pH of protein extraction affected some physicochemical properties (surface hydrophobicity (Ho) and denaturation temperature), without affecting the functional properties. Treatments led to the formation of soluble protein aggregates stabilized by disulfide bonds, especially with TT at 90 °C. TT and HHPTs shifted the wavelengths of maximum emission to red and to blue, respectively. All treatments induced unfolding and denaturation. HHPTs was more efficient than TT to enhance gelation and water holding capacities. Interestingly, treated and untreated CPIs exhibited high values of solubility (72–97%). TT and HHPT induced greater changes in physicochemical and functional properties of A8 than in those of A10. Remarkably, functional properties were improved from the less energetic treatments (70 °C, 200 MPa). Industrial relevance The comparison between treatments (one traditional and one corresponding to an emerging technology) gives information about the possibility of obtaining modified proteins for different functional purposes. The modified cowpea protein isolates may be used in beverages because of high solubility, in desserts because of gel formation capacity and/or as additives in other foodstuff because of improved water holding capacity. This knowledge would increase the added value of a local production currently marketed without processing.

Design and characterization of soluble biopolymer complexes produced by electrostatic self-assembly of a whey protein isolate and sodium alginate

The aim of this study was to design and characterize soluble biopolymer complexes formed by self-assembly of an anionic polysaccharide (sodium alginate, SA) and a whey protein isolate (WPI). First, conditions for producing stable protein aggregates were studied by analysing the effect of concentration (2, 4, 6% w/w) and heating temperature (55, 70, 85 °C) on WPI structural characteristics, using spectroscopic techniques UV–Vis and fluorescence. As heat treatment of WPI at the highest temperature induced the formation of soluble protein aggregates with a greater exposure of hydrophobic patches on their surface, this condition was selected to form protein particles. Secondly, from aqueous solutions of 6% w/w heat-treated WPI at 85 °C, WPI/SA systems were obtained at different ratios (2:1, 4:1 and 6:1) and transmittance measurements as a function of pH (6.0–3.0) were made. Dynamic behaviour of biopolymers in WPI/SA systems was discussed in terms of transition pHs, pHc and pHϕ, corresponding to the formation of WPI-SA soluble complexes and the beginning of associative phase separation (coacervation), respectively. Finally, with these pH values, phase diagrams were constructed for each system. From these diagrams, pH 4.0 was selected to perform deposition of SA onto WPI aggregates surface, as at this pH value formation of WPI-SA soluble complexes was observed for all WPI:SA ratios. Resultant self-assembled biopolymer complexes were then characterized by the same set of spectroscopic techniques. Higher WPI:SA ratios produced soluble self-assembled biopolymer particles where hydrophobic patches of protein aggregates would be more occluded ensuring the protection of potential lipophilic bioactive agents attached inside.

Effects of pH, calcium-complexing agents and milk solids concentration on formation of soluble protein aggregates in heated reconstituted skim milk

The proportion of protein present in the supernatant after centrifugation and the formation of soluble protein aggregates in heated (90 °C for 10 min) reconstituted skim milk were investigated as a function of (a) pH, (b) milk concentration 9–21%, w/w (milk solids non-fat, MSNF) and (c) addition of calcium-complexing agents (orthophosphate or EDTA). Compositional changes in milk, resulting from pH adjustment or salt addition, altered the distribution of caseins between the serum and micellar phases of milk prior to heating, and influenced the amount and composition of soluble aggregates formed during heat treatment. Although milk pH was a good predictor of the aggregation behaviour of the proteins during heating of milk of different solids concentrations, neither the proportion of protein in the supernatant prior to heating nor the pH alone could predict the aggregation behaviour when the composition was adjusted with mineral salts.

Tetrameric N5-(l-1-Carboxyethyl)-l-Ornithine Synthase: Guanidine · HCl-Induced Unfolding and a Low Temperature Requirement for Refolding

Guanidine · HCl (GdnHCl)-induced unfolding of tetrameric N5-(l-1-carboxyethyl)-l-ornithine synthase (CEOS; 141,300 Mr) from Lactococcus lactis at pH 7.2 and 25°C occurred in several phases. The enzyme was inactivated at ∼1 M GdnHCl. A time-, temperature-, and concentration-dependent formation of soluble protein aggregates occurred at 0.5–1.5 M GdnHCl due to an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomer was observed between 2 and 3.5 M GdnHCl (without observable dimer or trimer intermediates), as evidenced by tyrosyl and tryptophanyl fluorescence changes, sulfhydryl group exposure, loss of secondary structure, size-exclusion chromatography, and sedimentation equilibrium data. GdnHCl-induced dissociation and unfolding of tetrameric CEOS was concerted, and yields of reactivated CEOS by dilution from 5 M GdnHCl were improved when unfolding took place on ice rather than at 25°C. Refolding and reconstitution of the enzyme were optimal at ≤15°C and yields of active tetramer increased as the concentration of unfolded subunits decreased. Refolding of unfolded subunits and active tetramer assembly upon 100-fold dilution from 5 M GdnHCl at 0°C also was increased two- or fourfold (to 44 or 28% reactivation for 0.08 or 0.28 μM subunit, respectively) when incubated at 15°C, pH 7.2, for 4 h with the Escherichia coli molecular chaperonin GroEL, ATP, MgCl2, and KCl.

Spray-Drying of Air–Liquid Interface Sensitive Recombinant Human Growth Hormone

□ Spray-drying is an attractive method for preparing fine recombinant human growth hormone (rhGH) powders if the detrimental effect of protein degradation at the air–liquid interface on the protein can be minimized. In this study, we demonstrated that rhGH degradation (insoluble and soluble aggregate formation), as the consequence of air–liquid interfacial degradation, could be prevented using the appropriate formulation. Adding polysorbate-20 surfactant into the liquid feed (with no presence of sugar protectant) significantly reduced the formation of insoluble protein aggregates, while adding the divalent metal zinc ion effectively suppressed the formation of soluble protein aggregates. The combination of the two yielded a spray-dried rhGH powder having insignificant protein degradation. Our data suggest that the two components might protect the protein through different mechanisms. Polysorbate molecules occupy the air–liquid interface of spray droplets, thereby reducing the chance for rhGH to form insoluble aggregates by surface denaturation. Two zinc ions associate with two rhGH molecules to form a dimer complex that can resist the formation of soluble protein aggregates. Characterization of spray-dried powders by scanning electron microscopy suggests that both formulation and drying conditions have a strong influence on particle morphology and shape. Overall, spherical rhGH powders of smooth surface and good biochemical quality can be prepared by spray-drying using this formulation with no addition of sugar protectant.

Mechanism of physical modification of insoluble soy protein concentrate

AbstractThe mechanism for solubilization of alcohol‐leached soy protein concentrate (ALSPC) by physical modification was studied from the standpoint of molecular interactions, which are related to the differences in protein solubility under different conditions. The low solubility of ALSPC is caused by both noncovalent and covalent forces, but the noncovalent forces do not affect the solubility of modified soy protein concentrate (MSPC). Gel filtration shows that the major constituents of soluble protein from ALSPC and MSPC are protein molecules and protein aggregates, respectively. Physical modification promotes the formation of aggregates that are readily soluble in buffer. Fluorescence spectroscopy further proved that the hydrophobic groups are located in the interior of the aggregates. The reason for the formation of soluble protein aggregates during physical modification of ALSPC is discussed.