Cold-set Gels Research Articles

Effect of thermomechanical treatment on the aggregation behaviour and colloidal functionality of β-Lactoglobulin at high concentrations

Thermomechanical treatment of β-Lactoglobulin (βLG) protein systems at concentrations above 50% (w/w) leads to the formation of aggregates stabilised by disulphide bonds and non-disulphide covalent bonds. Although thermomechanically treated samples had lower solubility, depending on the treatment conditions, the viscosity of dispersions containing 10% untreated and treated βLG increased from 0.01 Pa·s to 300 Pa·s, respectively. Additionally, the interfacial tension at the oil-water interface decreased from 13.5 mN m−1 to 11 mN m−1 depending on treatment conditions, probably due to the presence of native and small water-soluble aggregates in the continuous phase. Consequently, the emulsions prepared with treated samples were more stable and showed smaller Sauter mean diameters. Furthermore, samples with high degree of aggregation showed an increased thermal stability and formed cold-set gels when dispersed in water. In contrast, heating dispersions of partially aggregated samples resulted in the formation of heat-set gels due to a second gelation process.

Effects of moderate electric fields on cold-set gelation of whey proteins – From molecular interactions to functional properties

Whey protein gelation and final gel properties are dependent of the gel forming solution characteristics (e.g. protein concentration, pH, ionic strength), and physical variables involved in the method used for gel preparation. Ohmic heating (OH) is an emerging technology in food processing and its application in heat-induced gelation of whey proteins has demonstrated its capacity to influence the physicochemical properties of protein gels. In this work, we studied the OH process and its inherent moderate electric field (MEF) variables - i.e. electric field (EF) strength and frequency - in order to establish their influence in protein aggregation and gelation during WPI cold-set gels formation. The presence of the EF during OH, particularly at higher EF strengths conjugated with lower frequencies, contributed to the formation of smaller aggregates with lower content of reactive thiol groups and lower viscosity. The cold-set gels produced from the aggregates’ suspension presented distinctive properties, influenced by the EF variables. – i.e. higher EF strength and lower frequency. EF treatments give rise to more fine-stranded gels with lower disulphide crosslinking but higher number of hydrophobic interactions and hydrogen bonds. The EF effects during the treatments resulted in weaker, more elastic gels with higher water retention and swelling capacity. These results open novel perspectives for the use of OH as a tool for fine-tuning protein gel networks aiming at enhanced functionality for various applications (e.g. use as texturizer or encapsulating agents).

Effect of cysteine addition and heat treatment on the properties and microstructure of a calcium-induced whey protein cold-set gel

A model gel of whey protein isolate (WPI) was prepared by cold gelation with calcium. This system was modified by the addition of free cysteine residues (Cys) at different steps of the process. The WPI cold-set gels obtained were then subjected to heat treatment at 90°C. First, the effect of Cys addition on the heat-induced aggregation of WPI was studied through Atomic Force Microscopy (AFM) and infrared spectroscopy (ATR-FTIR), while Cys' effect on cold gelation was observed by AFM, Confocal Laser Scanning Microscopy (CLSM) and oscillatory rheology (amplitude sweeps). The impact of heating on the microstructure and the viscoelastic properties of the WPI cold-set gels were finally investigated through several techniques, including DSC, ATR-FTIR, CLSM, cryo-SEM, and rheological measurements (temperature sweeps). When added during the first step of cold gelation, Cys modified heat-induced aggregation of WPI, resulting in the formation of a denser gel network with a fractal dimension (Df) of 2.8. However, the addition of Cys during the second step of cold gelation led to the formation of highly branched clusters of WPI and a looser gel network was observed (Df = 2.4). In this regard, the use and limitations of oscillatory rheology and the “Kraus model” to determine the Df of WPI cold-set gels was discussed. The viscoelastic properties and the microstructure of the WPI cold-set gels were irreversibly modified by heating. Gels were stiffer, more brittle, and coarser after heat treatment. New disulfide bonds and calcium bridges formed, as well as H-bonded β-sheets, all contributing to the formation of the final gel network structure.

Heat treatments of defatted soy flour: Impact on protein structure, aggregation, and cold-set gelation properties

Abstract This study reveals that mild heat treatments of defatted soy flour promote Maillard reaction and modify its protein techno-functional properties such as solubility, aggregation, and cold-set gelation. Glycation was promoted by treatments of defatted soy flour (DSF) at 60 °C for 12, 24, and 48 h, with and without relative humidity control (RHC and WRHC, respectively) at 79%. All samples presented a significant increase of glycation extent (GE), reaching the highest value after 48 h at RHC. Despite all samples presented a similar protein denaturation degree, the increase in GE was accompanied by a decrease of antitryptic activity. Protein solubility (PS) of DSF remained constant for treated samples WRHC. However, PS decreased progressively with the treatment time at RHC. SDS-PAGE of soluble proteins revealed a positive relation between band intensities and PS. Despite sample dispersions showed a protein particle size increment with treatment time, further aggregation after heat-treatments at 100 °C produced a similar protein size distribution among samples. Rheological and microstructural studies of cold-set gels of samples obtained WRHC revealed no changes in the maximum elastic modulus (G’max) and a slight increase of its pore sizes. However, samples obtained with RHC showed cold-set gels with a progressive G’max decrease with the treatment time, which could be related to a coarser gel microstructure. In the more extreme condition, the sample obtained after 48 h at RHC showed a total loss of gelation capability. These results can be used to address the development of new tofu-like food products with different rheological properties.

Fabrication and characterization of acid-induced gels from thermally-aggregated egg white protein formed at alkaline condition

Cold gelation of egg white proteins (EWP) has still remained a challenge because they suffer from heat-induced gelation at low protein concentrations during the preheating step. This study suggested that adjusting pH of EWP solution at relatively high alkaline pH (i.e. 11.3) could inhibit the heat-induced gelation of EWP, allowing to produce thermally aggregated EWP (TA-EWP) at relatively high protein concentrations during the preheating step. Regarding SDS-PAGE, free –SH groups, circular dichroism, and surface hydrophobicity data, the preheating step at alkaline pH probably caused the TA-EWP are assembled from the partially unfolded protein molecules which stabilized by numerous disulfide bonds as well as hydrophobic interactions that develop a network of the β-sheet structures. The TA-EWPs are able to form acid-induced cold-set gels. SEM images showed the TA-EWP acid-induced gels had a honeycomb-like microstructure contained large serum pools. With increasing preheating temperature and protein content, the microstructure of the gels became more compact and all texture parameters including hardness, springiness, chewiness, cohesiveness as well as Young's modulus, true fracture stress (σ) and Hencky strain tend to increase. Furthermore, the water holding capacity (WHC) improved with increasing protein content and preheating temperature. All TA-EWP acid-induced gels exhibited a weak dependence of storage modulus (G′) with frequency and the G′ value of the acid-induced gels increased with increasing preheating temperature and protein content.

Phase separation in ternary composite cold-set gel of egg yolk/κ-carrageenan/xanthan: Study on rheological and proton mobility properties

The effects of sodium tripolyphosphate and sugar on cold-set gel properties of egg yolk/κ-carrageenan/xanthan gum mixed dispersions were investigated by zeta potential, rheological measurements, gel textural analysis and low-field nuclear magnetic resonance (LF-NMR). As the yolk content increased to the 5 g/100 mL in aqueous systems, phase separation of mixed gel could be observed, due to the relatively rough gel appearance. Furthermore, the addition of phosphate could significantly improve the phase separation of mixed gels by elevating the pH and reducing zeta potential. The increase in the content of yolk and sugar brought an enhancement in gel storage modulus, while the addition of phosphate induced a dramatic decrease in the gelation rate of mixed gels. Textural analysis confirmed that hydrogen bonding interaction was the important force, which determined gel strength of the ternary composite cold-set gelation system. The result of LF-NMR revealed that there was a negative correlation between the decrease of proton mobility of immobilization water and the increase of gel strength in different sample series.

Properties of β-Lactoglobulin Aggregates and Gels as Affected by Ternary Emulsifier Mixtures of Tween 20, Lecithin, and Sucrose Palmitate.

The influence of sucrose palmitate, Tween 20, and lecithin on the properties of heat-induced aggregates and cold-set gels of β-lactoglobulin was studied based on an experimental mixture design with a fixed total emulsifier concentration. Emulsifiers were added to the protein solution before heating. Aggregate size and absolute values of ζ potential increased with the addition of emulsifiers, among which lecithin had the most pronounced effect. The water retention of the aggregates correlated positively with the aggregate size. Gels had reduced fracture stress and strains with increasing sucrose palmitate and decreasing Tween 20 contents. The fracture properties correlated with the ζ potentials of the aggregates, and larger aggregates led to gels with higher water-holding capacities. The emulsifiers hence influenced the gel properties indirectly via the aggregate properties. The impact of emulsifiers on food structures should therefore be considered when a food product is designed.

Microstructural and textural characteristics of soy protein isolate and tara gum cold-set gels

Soy protein isolates (SPI) are capable of forming cold-set gels. This techno-functional property can be affected by the presence of tara gum (TG). Under certain conditions, these SPI/TG systems may also form water-in-water (W/W) emulsions. The aim of this study was to evaluate acid gels formed from soy protein isolates (SPI) and tara gum (TG) aqueous mixtures, and to find the conditions in which the W/W emulsions of SPI droplets dispersed in a TG continuous phase can be stabilized by SPI gelation as a strategy to prevent emulsion destabilization. Cold-set gels of SPI 0.3 g/L at different TG concentrations (0–0.05 g/L) showed different microstructures, a consequence of a different balance between gelation and segregative phase separation processes. SPI gels showed a homogenous and compact microstructure. When TG was present at 0.01 g/L and 0.02 g/L, the protein network was less interconnected, showing coarse-stranded and bicontinuous gels, respectively. At TG > 0.03 g/L, stable W/W emulsions were formed, revealing an abrupt decrease in gel firmness, a significant loss of fracture capacity, and a decrease in the water holding capacity. These findings may be used as a starting point for the application of these gelled systems as thickeners, texture modifiers, and coating materials for delivery of bioactive compounds.

Effect of the Solid Fat Content on Properties of Emulsion Gels and Stability of β-Carotene.

Whey-protein-isolate-based emulsion gels were prepared through a cold-set gelation process, and the effect of the solid fat (coconut oil) content in the oil phase on gel properties and β-carotene stability was investigated. An increase in solid fat content (0, 20, 50, 80, and 100% of the oil phase) resulted in a smaller droplet size, higher viscosity, and improved creaming stability of the emulsions. When glucono-δ-lactone was added to initiate gelation, a higher solid fat content contributed to an earlier onset of gelation and a higher storage modulus of the gels. Textural analysis indicated that the increase in the solid fat content allowed for an increase in fracture stress and Young's modulus of the emulsion gels. Microscopic observation revealed that emulsions containing a higher solid fat content formed gels with a denser and more uniform particulate network structure. The stability of β-carotene against thermal treatment (55 °C for 12 days) and ultraviolet light exposure (8 h) was determined. The results suggested that the solidification of the oil phase can improve the stability of β-carotene, and gels with higher hardness were capable of retaining more β-carotene after the treatments. These findings indicated that emulsion gels with a solidified oil phase could be potential delivery systems for lipophilic bioactive compounds.

Characterization and embedding potential of bovine serum albumin cold-set gel induced by glucono-δ-lactone and sodium chloride

Bovine serum albumin (BSA) was induced to form cold-set gel by using glucono-δ-lactone (GDL) and sodium chloride (NaCl), and characteristics of the gel and its embedding potential to bioactive molecules were investigated. The results from texture analyzer indicated that hardness and springiness of BSA cold-set gel were improved by increasing GDL or NaCl, but the gel with lower concentration of GDL (1.0%) and NaCl (0.2 M) had better water holding capacity (WHC). GDL had a significant influence on storage modulus (G’) and loss modulus (G″) but it did not appear to be applicable to NaCl. Light transmittance at 500 nm and average particle size were used to characterize formation of BSA cold-set gel as pH values decreased, and the results suggested that BSA molecules had aggregated at the pH value of the gel point. Microstructure and fractal analysis from confocal laser scanning microscope (CLSM) further suggested that electrostatic and hydrophobic interaction played important roles in the formation of the cold-set gel and it could be deduced that structure and properties of the gel were closely related to the forces. BSA cold-set gel showed an excellent embedding performance to encapsulate bioactive molecules (used quercetin as representative), and achieved effective slow-release action, especially in simulated intestinal juice of pH 7.5. BSA cold-set gel may have important potential application in encapsulation, protection, delivery and controlled-release of some fragile bioactive components, so the research provides a theoretical basis for its application.

Influence of the Maillard reaction on the properties of cold-set whey protein and maltodextrin binary gels

The Maillard conjugation of proteins and reducing saccharides is used to modify the technological functionality of whey proteins. In this study, whey protein isolate (WPI) was conjugated with maltodextrin (at 1:1 ratio and two total solid contents of 100 and 200 mg mL−1) through the Maillard reaction and used to form cold-set gels. The glycation reaction increased the strength of hydrogen bonding of whey proteins and preferentially modified α-lactalbumin, in comparison with β-lactoglobulin. It also increased the reducing power of binary protein-saccharide solution and allowed formation of self-standing cold-set WPI gel at a low protein content (i.e., ≈50 mg mL−1). Microscopic imaging showed micro-phase separated maltodextrin domains, interrupting the protein network, in gels made of protein-maltodextrin physical mixtures, whereas Maillard conjugation resulted in more homogenous microstructures at both total solid contents. The Maillard reaction increased gel firmness and water-holding capacity and caused a reduction in the extent of gel swelling.

Effect of Persian gum on whey protein concentrate cold-set emulsion gel: Structure and rheology study

In this study, the influence of Persian gum (PG) on the properties of whey protein concentrate (WPC) emulsion gel prepared through cold set gelation method (incorporation of CaCl2) was investigated. The mean droplet size of emulsions was analyzed prior to gelation and the emulsion gel samples were characterized by rheological studies, scanning electron microscopy (SEM) and water holding capacity (WHC) measurement. Results showed PG affected the droplet size of the initial emulsions and the matrix of gel systems. Emulsions containing 0.3% w/w PG revealed higher heat stability compared to the control. A viscoelastic behavior was observed for all emulsion gels with G′ being greater than G″ over the whole frequency range tested. Increasing the concentration of PG led to a change in the gel structure at pH 7. SEM images exhibited that PG at 0.3% w/w gave rise to an ordered honeycomb-like microstructure with an interconnected porous matrix of thin walls. In contrast, adding 0.6% w/w PG resulted in a non-uniform network with large pores of thick and rough walls and reduced the WHC. These findings suggest the viscoelastic properties and microstructure of WPC emulsion gel can be tailored by adjusting PG concentration for food formulations.

Rheological and structural characterisation of whey protein acid gels co-structured with chia (Salvia hispanica L.) or flax seed (Linum usitatissimum L.) mucilage

The effects of different plant seed mucilage (PSM) extracts, namely chia seed (CSM) and flaxseed (FSM), on the kinetics of δ-glucono-lactone induced acidification and gelation phenomena of whey proteins (5% w/w WPI) were investigated. The rheological and microstructural properties of mixed whey protein-PSM (0.05–0.75% w/w) cold-set gels produced at 30 or 37 °C were studied by means of oscillatory rheology and confocal microscopy. On exceeding 0.125% of PSM, a significant reduction of the gelation time due to the formation of loosely entangled whey protein soluble aggregates was observed. The impact of PSM on the gelation rates was closely related to the PSM type and concentration. CSM addition induced a gradual reduction of maximal gelation rate over the entire concentration range tested. On the other hand, FSM conferred a steep impedance of the gelation when exceeded 0.375%, which was associated with the occurrence of segregative phase separation. Fitting the elastic modulus – gelation time data to a model adapted to the Flory-Stockmayer theory, it was demonstrated that the presence of PSM inhibits the whey protein crosslinking capacity under both tested acidification regimes, leading to the formation of shorter protein crosslinks and therefore, to lower gel stiffness. However, the formation rate of elastically active chain networks was found to be increasing for CSM and FSM contents up to 0.5 and 0.25% respectively, suggesting a synergistic acid gel structuring effect of PSM under these conditions.

Thermal Versus High Hydrostatic Pressure Treatments on Calcium-added Soybean Proteins. Protein Solubility, Colloidal Stability and Cold-set Gelation

The effects of thermal treatment (TT) and high hydrostatic pressure treatment (HHPT) on calcium-added soybean protein 1% (w/w) aqueous dispersions at pH 7.0 were compared. High hydrostatic pressure, but not thermal treatment, improved protein solubility and colloidal stability. Despite the fact that the glycinin solubility is more affected by calcium than that of β-conglycinin, glycinin could remain in dispersion in the presence of calcium when denatured by HHPT (calcium added before or after treatment), but not when denatured by TT or without denaturing treatment. Thus, polypeptide composition of soluble aggregates depended on type of treatment. Colloidal stability and molecular weight of soluble aggregates depended on the order of application of calcium and denaturing treatment: when calcium was present during either HHPT or TT, the dispersions had higher stability and higher proportion of soluble aggregates with high molecular weight than when calcium was added after treatments. After freeze drying and re-dispersing at higher protein content (10% w/w) calcium-added dispersions subjected to HHPT formed cold-set gels that were transparent and exhibited excellent water holding capacity. Our results provide the basis for the development of ready-to-use functional ingredients.

Stability, rheology, and β-carotene bioaccessibility of high internal phase emulsion gels

High internal phase emulsions (HIPEs) were prepared using preheated whey protein isolate (WPI). The influence of cold-set gelation induced by addition of glucono-δ-lactone (GDL) on the microstructure, rheology, water holding capacity (WHC), and stability of HIPEs was determined. In the presence and absence of GDL, the mean particle diameters of HIPEs decreased with increasing WPI concentration. The addition of GDL to the HIPEs did not alter their mean particle diameter or microstructure. The gel strength, hardness, WHC, and stability of HIPEs increased with increasing WPI concentration and with GDL addition. An in vitro digestion experiment showed that β-carotene retention and bioaccessibility was higher after GDL was added to the HIPEs. These findings may be useful for the development of HIPE-based delivery systems for food and other applications.

Tailoring egg white proteins by a GRAS redox pair for production of cold-set gel

The present study aimed to modify egg white protein (EWP) to produce gelable aggregates by a redox pair (ascorbic acid + hydrogen peroxide). This technique eliminates the primary heating step and leads to gelation of non-heat-treated EWP. The redox pair modification mainly modified the egg white proteins by the formation of the disulfide bonds, as evidenced by a dramatic decrease in the free SH groups. Circular dichroism, Fourier-transform infrared spectroscopy (FTIR), and fluorescence analysis indicated that exposure of EWP to the redox pair caused an alteration in the secondary and tertiary structure of the egg white proteins. Furthermore, the particle size data revealed that protein aggregation occurred in the modified egg white proteins (MEWP). The pH at which the modification was performed had a significant effect on the production of gelable aggregates, where only EWP samples modified at pH 10 and 11 had the ability for development of cold-set gels. It sounds the redox pair-induced increase in particle size and surface hydrophobicity of egg white proteins had a dominant contribution to the ability of the MEWP to form a cold-set gel.

Effect of Partial Substitutes of NaCl on the Cold-Set Gelation of Grass Carp Myofibrillar Protein Mediated by Microbial Transglutaminase

The objectives of this study were to investigate the changes and the relationship between structure and physiochemical properties of low sodium salt substitutes (NaCl partially replaced by KCl, CaCl2, and MgCl2) on grass carp myofibrillar protein gels mediated by microbial transglutaminase during cold-set gels and to provide more information about the gel characteristics. The gel strength, water holding capacity, whiteness, rheological characteristics, differential scanning calorimeter (DSC), and Raman spectra of cold-set gels were determined. The Raman spectra data were fitted to four secondary structures (α-helix, β-sheet, β-turn, and random coil). The gel properties of cold-set gels varied both with the low sodium salt types and incubation time. Myofibrillar protein (MP) gels added with NaCl and KCl had significantly higher water holding capacity than the MgCl2, CaCl2, and control groups. Additionally, the results showed that the gel strength and G’ value increased with the incubating time. No significant difference was detected in whiteness between the NaCl group and partial substituted groups. Cold-set gels added with the same molar amount of NaCl and KCl had fairly similar gel properties. There is a strong correlation between structural properties and gel properties of MP gels determined by DSC during the cold-set gelation process.

Interactions between whey protein and inulin in a model system.

Inulin is a commonly used prebiotic ingredients for functional food formulation. The effect of inulin on the gelation properties of whey protein was investigated using whey protein and inulin (WP/inulin) and polymerized whey protein and inulin (PWP/inulin) mixtures at different levels of protein (4-8%, w/v) and inulin (1-5%, w/v). WP/inulin mixture was prepared by heating protein and inulin together while the latter by heating protein alone and then mixed with inulin. Both mixtures were analyzed for turbidity, zeta potential, particle size, and rheological properties. Dispersions became more opaque with increasing protein but there was no significant difference between the two mixtures. A small shift towards larger size and significantly decreased negative zeta potential with increasing inulin addition (1-5%) were observed for both mixtures. WP/PWP and inulin mixtures exhibited a shear thinning behavior. Transition temperature of whey protein increased with inulin addition. WP/PWP and inulin mixtures were induced into cold-set gels by calcium and the gels were analyzed for hardness. Hardness of WP/PWP and inulin gels increased with the increasing inulin. Results indicated that interactions between whey protein and inulin had impact on the gelation properties of whey protein regardless the way inulin added.

On the reversibility of ethanol-induced whey protein denaturation

The functional properties of whey proteins after exposure to ethanol were studied. The reversibility of the ethanol-induced whey protein denaturation was investigated using different methodologies to remove ethanol, such as 10-fold dilution with water, oven-drying and freeze-drying of whey protein mixed water/ethanol solutions. The effect of ethanol on whey protein denaturation, before and after ethanol removal, was measured by analysis of difference-UV spectra, rheological measurements, confocal microscopy and differential scanning calorimetry. The results showed a retention of the denatured character of whey proteins (27–34%) after removal of ethanol. The study of the combined effect of ethanol and heat on whey protein denaturation at concentrations ranging from 10 to 70%, revealed that at an ethanol concentration of 50%, the initial extent of denaturation was maximum, while heat did not contribute any additional denaturation effect. A whey protein mixture, previously incubated in 50% ethanol and subsequently freeze-dried to completely remove ethanol, produced a gel in a cold-set gelation procedure, where gelation was promoted by addition of glucono-δ-lactone, without an initial heating step. The confocal microscope revealed significant microstructural characteristics suggesting that whey protein solutions retain a significant degree of denaturation even after ethanol removal. Differential scanning calorimetry results further showed that the presence of 50% ethanol decreased the denaturation temperature of whey proteins by more than 15 °C, while removal of ethanol by freeze-drying, similarly decreased the denaturation temperature by about 2 °C. Overall, whey proteins, exposed in ethanol, retain a significant degree of denaturation after removal of the alcohol, with possible applications as functional ingredients capable of modifying the mechanical properties of food systems.

Cold-Set Gelation of Commercial Soy Protein Isolate: Effects of the Incorporation of Locust Bean Gum and Solid Lipid Microparticles on the Properties of Gels

This study aimed to evaluate the ability of commercial soy protein isolate (SPI) to form cold-set gels under different pHs (5–11), pre-heating temperatures (60 °C, 80 °C), CaCl2 (0–15 mM) and SPI (5–15%, w/v) concentrations, and also select a formulation for the investigation of the effects of incorporating locust bean gum (LBG) (0–0.3%, w/v) and solid lipid microparticles (SLM) on gels rheological and microstructural properties. Gels were evaluated in terms of visual aspect, water-holding capacity, microstructure (using confocal laser scanning microscopy and cryo-scanning electronic microscopy) and rheological properties. SPI showed higher solubilities at pHs 7 (32.0%), 9 (51.6%) and 11 (100%). Self-supported gels were obtained under several conditions at alkaline pHs. At pH 7, only systems pre-heated to 80 °C with 15% (w/v) SPI and 10 or 15 mM CaCl2 gave self-supported gels. At neutral pH, samples showed relative structural instability, which was minimized with LBG incorporation. Formulations GSPI (pH 7, preheated to 80 °C, 15% (w/v) SPI, 10 mM CaCl2) and GMIX (pH 7, preheated to 80 °C, 15% (w/v) SPI, 0.2% (w/v) LBG, 15 mM CaCl2) were selected for emulsion-filled gels (EFG) production. Power law parameters (K′, K″), calculated from frequency sweep results, revealed that non-filled GMIX (K′: 472.1; K″: 77.6) was stronger than GSPI (K′: 170.4; K″: 33.6). Besides, GMIX showed microphase separation. SLM stabilized with Tween 80-Span 80 were active fillers in EFG, altering microstructures and increasing G’, G” and the Young’s modulus (1.8 to 2.1 kPa for GSPI and 1.4 to 2.2 kPa for GMIX).