Modulation of thermal stability and heat-induced gelation of β-lactoglobulin by high glycerol and sorbitol levels

The influence of glycerol and sorbitol on the thermal stability and heat-induced gelation of β-lactoglobulin (β-lg) in aqueous solutions was investigated. The thermal stability of β-lg was characterized by measuring the thermal denaturation temperature (T_m) using differential scanning calorimetry, while its gelation properties were characterized by measuring the gelation temperature (T_gel) and final gel rigidity (G^∗) using dynamic shear rheology. All experiments were carried out using aqueous solutions containing 10% (w/w) β-lg, glycerol (0–70% w/w) or sorbitol (0–55% w/w), and 5 mM phosphate buffer (pH 7.0). No salt was added to these solutions so that there was a relatively strong electrostatic repulsion between the protein molecules, which usually prevents gelation. When the cosolvent concentration was increased from 0% to 50%, T_m increased from 74 to 86 °C for sorbitol, but only from 74 to 76 °C for glycerol, which indicated that sorbitol was much more effective at stabilizing the native state of the globular protein than glycerol. Protein solutions containing sorbitol (0–55%) did not form a gel after heating, but those containing glycerol formed gels when the cosolvent concentration exceeded about 10%, with G^∗ increasing with increasing glycerol concentration. We attribute these effects to differences in the preferential interactions of polyols and water with the surfaces of native and heat-denatured proteins, and their influence on the protein–protein collision frequency.     

Impact of sorbitol on the thermostability and heat-induced gelation of bovine serum albumin

The influence of sorbitol (0–40 wt.%) on the thermal denaturation and gelation of bovine serum albumin (BSA, pH 7.0) in aqueous solution has been studied. The effect of sorbitol on heat denaturation of 0.5 wt.% BSA solutions was measured using ultrasensitive differential scanning calorimetry. The unfolding process was irreversible and was characterized by the thermal denaturation temperature (T_m). As the sorbitol concentration increased from 0 to 40 wt.%, T_m increased from 73.0 to 80.9 °C. The rise in T_m was attributed to the increased thermal stability of the globular state of BSA relative to its native state. The dynamic shear rheology of 4 wt.% BSA solutions containing 200 mM NaCl was monitored as they were heated from 30 to 90 °C at 1.5 °C min⁻¹, held at 90 °C for 120 min, and then cooled back to 30 °C at −1.5 °C min⁻¹. Sorbitol increased the protein gelation temperature (ΔT_gel +10 °C for 40 wt.% sorbitol), decreased the isothermal gelation rate at 90 °C, but increased the final shear modulus of the gels cooled to 30 °C. The impact of sorbitol on gel characteristics was attributed to its ability to increase protein thermal stability, increase the attractive force between proteins and decrease the protein–protein collision frequency.     

Physicochemical basis for cosolvent modulation of β-lactoglobulin functionality: Interfacial tension study

The impact of neutral cosolvents on the thermal stability of globular proteins in aqueous solutions depends on the nature of the cosolvent, e.g., sorbitol causes a pronounced increase in the thermal denaturation temperature (T_m) of β-lactoglobulin (β-lg), whereas glycerol does not. When a protein unfolds there is a change in the exposed surface area and in the type of molecular interactions that occur at the protein–solvent–cosolvent interface. These changes contribute to the free energy change associated with protein denaturation and depend on cosolvent type. In this study we measured the equilibrium interfacial tensions of aqueous glycerol (0–70% w/w) and sorbitol (0–55% w/w) solutions as a function of temperature to provide insights into the role of the interfacial energy on the thermal stability of β-lg. There was a slight increase in interfacial tension with increasing sorbitol concentration, indicating its preferential exclusion from the oil–water interface. On the other hand, there was an appreciable decrease in interfacial tension with increasing glycerol concentration, indicating its preferential accumulation at the oil–water interface. These changes were largely independent of the measurement temperature (30–80 °C). Our results suggest that sorbitol increases the T_m of β-lg mainly through a steric exclusion effect, whereas glycerol has little effect on T_m because the steric exclusion effect is counter-balanced by a differential interaction effect.     

Impact of cosolvents (polyols) on globular protein functionality: Ultrasonic velocity, density, surface tension and solubility study

The objective of this study was to understand how cosolvents influence the molecular and functional properties of globular proteins in aqueous solutions. The ultrasonic velocity, density and adiabatic compressibility of cosolvent solutions (0–50 wt% sorbitol or glycerol) were measured in the absence and presence of a globular protein (1 wt% β-lactoglobulin) at 30 °C. These measurements were used to calculate the partial specific apparent volume and adiabatic compressibility of the protein. The protein's volume decreased and its compressibility increased in the presence of high cosolvent concentrations, which were attributed to changes in the properties of the protein interior and solvation layer. Sorbitol was more effective than glycerol at decreasing the protein volume at high cosolvent concentrations, which may be because glycerol has some surface activity and may therefore accumulate around hydrophobic regions on the protein surface. Our data were used to account for the observation that sorbitol is more effective than glycerol at increasing the thermal stability and self-association of the β-lactoglobulin. A better understanding of the influence of protein–cosolvent–solvent interactions on the functionality of globular proteins may facilitate the design of protein-based products.     

Sodium Caseinate/Sunflower Oil Emulsion-Based Gels for Structuring Food

Protein gels have attired attention since they allow structuring foods with no trans or saturated fats. The effects of protein concentration and sucrose addition on gelation kinetics and on physical properties of sodium caseinate (NaCas)/sunflower oil emulsion-based gels were studied by two methods: a new application of backscattering of light (BS) using a Turbiscan equipment and by dynamic oscillatory rheology. Structure of gels was also described by confocal laser scanning microscopy (CLSM) and small angle X-ray scattering (SAXS). T _gel values decreased with increasing sucrose or NaCas concentration. BS method sensed early changes in structure, while rheological measurements were less sensitive to those changes. However, tendencies found by rheological measurements were the same as the ones found by BS experiments. CLSM images of gels formed from emulsions containing high sucrose and protein concentrations had big oil droplets that were not present in initial emulsions. Gels with sucrose concentrations between 15 and 30 wt/wt% released oil. SAXS patterns showed that NaCas nanoaggregate sizes in the aqueous phase were smaller with increasing sucrose concentration. Polar groups of protein interacted with sucrose, and therefore, interactions among protein molecules diminished. As a result of weaker protein molecule interactions, nanoaggregates were smaller. However, this effect was beneficial. In the macroscale, rheological properties and visual appearance of gels were improved. The gel formulated with 5 wt/wt% NaCas and 10 wt/wt% sucrose had a smooth surface and was stable to syneresis and oil release. This formulation was a good alternative to trans fat.     

Thermal denaturation kinetics of whey proteins at high protein concentrations

A detailed kinetic study of the thermal reaction kinetics of whey protein concentrate was conducted at high protein concentrations. Whey protein solutions with protein concentrations of up to 40% (w/w) were heated at different temperatures for varying periods of times. The denaturation of β-lactoglobulin followed a reaction order of 1.5 and depended strongly on temperature and protein concentration. The rate of denaturation was shown to increase with increasing temperature. This could be explained by the strong influence of the temperature on the unfolding reaction. Furthermore, the protein concentration induced a faster thermal denaturation, most likely due to the increased probability of collision between whey protein molecules with increasing protein concentration which promotes protein aggregation. The results of this study are of industrial relevance for extrusion processes and the production of protein concentrates in evaporators where high protein concentrations are frequently used.     

Thermal denaturation and gelation of vicilin-rich protein isolates from three Phaseolus legumes: A comparative study

The thermal denaturation and gelation properties of vicilin-rich protein isolates from red bean, red kidney bean and mung beans (further denoted as RPI, KPI and MPI) were investigated by differential scanning calorimetry (DSC) and dynamic oscillatory measurements. The relation between the properties of these proteins to their free sulphydryl (SH)/disulfide bond (SS) contents was also evaluated. DSC analyses showed that many DSC characteristics of major endothermic peak (corresponding to vicilin component), including its denaturation temperature (T_d), enthalpy changes (ΔH) and width at half-peak height (ΔT_1/2), significantly varied with the type of protein isolates. Furthermore, the heat-induced gelation, including onset of gelation and the development of mechanical moduli, was also dependent on the type of protein isolates. The thermal denaturation of these proteins was nearly unaffected by the presence of reducing agent dithiothreitol (DTT), while the presence of DTT weakened the gel formation. The T_d of vicilin components and the mechanical moduli of corresponding formed gels were positively related to their SS contents. Additionally, the formed gels were thermo-irreversible, and heat pretreatment (carried out at temperatures higher than or close to the T_d of these vicilins) could improve the gel network formation. These results confirm that vicilin-rich protein isolates from various legumes show different patterns of thermal denaturation and gelation, and these properties are to a great extent related to their SH and/or SS contents.     

Effect of Sucrose Addition and Heat Treatment on Egg Albumen Protein Gelation

ABSTRACT: Studies were conducted to evaluate the effect of sucrose (0% to 60% w/w) on the heat denaturation and gelation of egg albumen solutions (pH 7.0) by thermal analysis and rheological measurements. Temperature and frequency sweeps were carried out under small deformations to determine the temperature at which structure development began (T_s) and the storage modulus plateau (G₀), respectively. The latter was used to estimate the molecular weight of the internal chains between cross-links (M_c) using the rubber elasticity theory. Gels were prepared under several heat intensities (70, 80, and 90°C/30 min), and the mechanical properties were determined under small (stress relaxation) and large (stress and strain at fracture) deformations. It was observed that increments in sucrose concentration could act in the stabilization of the proteins, promoting a significant increase in the onset values and temperature of denaturation. The gelling process showed aT_s increase with higher sucrose concentration. Gelation and the gel mechanical properties and network density showed a strong dependence on sucrose concentration and heat treatment temperature. In a general way, the sucrose promoted an improvement in the gel network up to a determined value of sugar concentration. At higher sugar concentrations, the values for the rheological properties decreased, which could be related to an incomplete unfolding of the oval-bumin under these conditions.     

Optical techniques to determine thermal effects on proteins

Optical rotation (OR) and transmitted light (TL) measurements were conducted on 1%, 2.5% and 5% (w/v) bovine serum albumin (BSA) in 0.01 m phosphate buffer at pH 7 and ionic strength 0.08. Denaturation temperatures (T_d) obtained from OR measurements were consistent with reported differential scanning calorimetry values. Protein concentration did not affect T_d in agreement with most reports. Changes in TL reflecting gel formation and protein aggregation were influenced by BSA concentration. Sugar concentration in the range used in this study (0–5%) did not affect the thermal stability of BSA. The lack of difference in sucrose, trehalose and sorbitol effects on the thermal stability of BSA was consistent with some but not all reports. The optical system used to study protein denaturation had acceptable accuracy (consistency with published T_d values) and precision (coefficient of variation under 3.5%) levels.     

Influence of sucrose on NaCl-induced gelation of heat denatured whey protein solutions

A solution of heat-denatured whey proteins was prepared by heating 10 wt.% whey protein isolate at pH 7.0 to 80°C for 10 min in the absence of salt. This treatment caused the globular protein molecules to partially unfold and aggregate. When the heat-denatured whey protein solution was cooled to room temperature and mixed with 200 mM NaCl it formed a gel. The influence of sucrose (0 to 10 wt.%) in the protein solutions prior to NaCl addition on the gelation rate was investigated. At relatively low concentrations (0–8 wt.%) sucrose decreased the gelation rate, presumably because sucrose increased the aqueous phase viscosity. At higher concentrations (> 8 wt.%) sucrose increased the gelation rate, probably because it decreased the thermodynamic affinity of the globular proteins for the aqueous solution, which increased the attraction between proteins. This data has important implications for the application of cold-setting whey protein ingredients in sweetened food products such as deserts and beverages.     

Ionic strength and buffering capacity of emulsifying salts determine denaturation and gelation temperatures of whey proteins

Ionic conditions affect the denaturation and gelling of whey proteins, affecting the physical properties of foods in which proteins are used as ingredients. We comprehensively investigated the effect of the presence of commonly used emulsifying salts on the denaturation and gelling properties of concentrated solutions of β-lactoglobulin (β-LG) and whey protein isolate (WPI). The denaturation temperature in water was 73.5°C [coefficient of variation (CV) 0.49%], 71.8°C (CV 0.38%), and 69.9°C (CV 0.41%) for β-LG (14% wt/wt), β-LG (30% wt/wt), and WPI (30% wt/wt), respectively. Increasing the concentration of salts, except for sodium hexametaphosphate, resulted in a linear increase in the denaturation temperature of WPI (kosmotropic behavior) and an acceleration in its gelling rate. Sodium chloride and tartrate salts exhibited the strongest effect in protecting WPI against thermal denaturation. Despite the constant initial pH of all solutions, salts having buffering capacity (e.g., phosphate and citrate salts) prevented a decrease in pH as the temperature increased above 70°C, resulting in a decline in denaturation temperature at low salt concentrations (≤0.2 mol/g). When pH was kept constant at denaturation temperature, all salts except sodium hexametaphosphate, which exhibited chaotropic behavior, exhibited similar effects on denaturation temperature. At low salt concentration, gelation was the controlling step, occurring up to 10°C above denaturation temperature. At high salt concentration (>3% wt/wt), thermal denaturation was the controlling step, with gelation occurring immediately after. These results indicate that the ionic and buffering properties of salts added to milk will determine the native versus denatured state and gelation of whey proteins in systems subjected to high temperature, short time processing (72°C for 15 s).     

Glass Transition Explanation for the Effect of Polyhydroxy Compounds on Protein Denaturation in Dehydrated Solids

Dehydrated proteins are frequently subjected to thermal stress in the presence of other components. The effect such substances may have upon protein structure, and therefore function, has not been fully investigated. Thus, the effect of added polyhydroxy components on the denaturation of lyophilized β-lactoglobulin, ovalbumin, and ribonuclease, as determined by differential scanning calorimetry, was evaluated. The denaturation temperature, T_m, of the globular proteins decreased in the dry state after the addition of sucrose, sorbitol, or glycerol. The thermal stability (based on T_m) of the dehydrated proteins appeared to correlate with the glass transition temperature (T_g) of the polyhydroxy component, which was assumed to be related to the T_g of the mixture. The lower the T_g of the component, the greater was the degree of protein destabilization. Thus, glass transition data may be used to predict the effect that a component would have on denaturation of dehydrated proteins at elevated temperatures.     

Stress Relaxation of Acid-induced Milk Gels

The effects of the addition of sugar (sucrose) or salt (KCl/NaCl mixture) on the rheological properties of acid milk gels formed under different process and composition conditions were studied using an experimental design and surface response analysis. The variables evaluated were: sodium caseinate, whey protein concentrate, carrageenan, sucrose or KCl/NaCl concentration, and the mixing temperature of the components. The stress relaxation measurements were made within the linear viscoelasticity domain, and the data obtained were fitted to the generalized Maxwell model. Five parameters were obtained from the fitting: three elastic elements and two viscous elements. In addition, the denaturation process of whey protein with different solute concentrations was evaluated using differential scanning calorimetry. Denaturation temperature increased with increases in the solute concentrations, but enthalpy values did not change independent of solute type and concentration. The results showed that the elastic and viscous moduli increased with sucrose concentration, i.e., the gel network was reinforced, while the opposite occurred with the addition of the saline mixture. Increasing ionic strength of saline mixture and the process temperature were the more important effects with respect to the rheological properties of gels with added saline mixture. On the other hand, the biopolymer concentrations showed the most significant effects on the properties of gels with added sugar.     

Trout skin gelatin-based edible film development

Edible biopolymer films were developed from gelatin extracted from trout skin (TSG) using thermal protein denaturation conditions and plasticizer (glycerol) concentration as variables. The amino acid composition of the TSG, elastic modulus, viscous modulus, and the viscosity of film-forming solutions, and tensile properties, water vapor permeability, solubility in water, and color of TSG-based films were determined. A 6.8% (w/w, wet basis) trout skin-extracted gelatin solution containing 9, 17, or 23% (w/w, dry basis) glycerol was heated at 80, 90, or 100 °C for 30, 45, or 60 min to prepare a film-forming solution. TSG can be characterized as a gelatin containing high contents of methionine and aspartic acid. The gelation temperature of the film-forming solution was 7 °C and the solution was subjected to heating to form a stable matrix for a film. Increased heating time of the film-forming solution reduced the film solubility (P < 0.05). Heating at 90 °C for 30 min was suggested as the requirement for film formation. As the concentration of glycerol in the film increased, film strength and moisture barrier properties decreased, while film stretchability increased (P < 0.05). Trout skin by-products can be used as a natural protein source for fabricating biopolymer films stable at ambient conditions with certain physical and moisture barrier properties by controlling thermal treatment conditions and glycerol concentrations. Practical Application: The fishing industry produces a significant amount of waste, including fish skin, due to fish processing. Trout skin waste has potential value as a protein source that can be used to form biopolymer edible films for packaging low and intermediate water activity food products, and thus may have practical applications in the food industry, which could be one way to cut waste disposal in the trout processing industry.     

Influence of pH on rheological properties of porcine myofibrillar protein during heat induced gelation

Texture of meat products is dependent on the gelation characteristics of myofibrillar protein. Gaining an understanding of the gelation mechanism of meat gel systems is beneficial for the development of processed meat products as well as maintaining quality in meat products. The aim of this study was to investigate the impact of pH (5.6, 6.0, 6.5, and 7.0) on heat-induced gelation properties of myofibrillar proteins from porcine semimembranosus muscle. Dynamic rheological measurements were taken as the temperature increased by 1°C/min from 20 to 85°C, followed by a holding phase at 85°C for 3min to ensure complete gelation and during a subsequent cooling where the temperature dropped from 85 to 5°C at a rate of 5°C/min. Storage modulus (G') increased as gel formation occurred, but decreased after reaching the temperature of myosin denaturation (52°C) until approximately 60°C when the gel strength increased again. This resulted in a peak and subsequent depression in the data. This depression in the curve was more pronounced with increasing pH. Results indicate protein denaturation and gel formation are pH dependent. Furthermore, rate of gelation appears to influence water-holding capacity.     

Modulating the gel properties of soy glycinin by crosslinking with tyrosinase

The gelation progression and gel properties of enzymatically crosslinked soy glycinin were evaluated in comparison to non-crosslinked glycinin. Glycinin was initially crosslinked using tyrosinase from Bacillus megaterium (TyrBm) and was later used to form gel upon heating. Gelation was evaluated by small deformation rheological measurements and revealed a significant increase in storage modulus (G′) obtained in the crosslinked gel. This was confirmed by temperature sweep and frequency sweep measurements that supported the results and proved that the difference in modulus was not frequency dependent. Texture profile analysis showed an increase in hardness and decrease in elasticity of the crosslinked gels. Scanning electron microscopy (SEM) images displayed a more structural network with larger pore size in the crosslinked gel. The less dense structure of the crosslinked glycinin gel network led to a slight decrease in the water holding capacity. Finally, thermal analysis using differential scanning calorimetry (DSC) confirmed no change in the gelation point induced by denaturation, however thermal gravimetric analysis (TGA) did show a difference in the decomposition profile of the crosslinked protein compared with non-crosslinked glycinin. The results suggest that by applying TyrBm mediated crosslinking we may modulate the protein gel properties for tailoring the texture of food products.     

菊粉对小麦面筋蛋白理化性质的影响

利用差示扫描量热仪、傅里叶变换红外光谱仪和扫描电子显微镜研究菊粉添加量对小麦面筋蛋白乳化特性和热特性的影响,并对小麦面筋蛋白的结构进行表征。结果表明,较低添加量的菊粉对小麦蛋白乳化性能的影响较显著,10%的菊粉可以使其乳化活性提高14.4%,7.5%的菊粉可以使蛋白质乳化稳定性提高18.7%。热力学实验表明菊粉可以提高蛋白质变性温度,降低焓变值,添加12.5%的菊粉可以最大程度提高蛋白质变性温度。面筋蛋白网络结构随着菊粉的添加更加致密。通过对小麦面筋蛋白的结构分析表明：添加20%的菊粉会使蛋白质中二硫键相对含量增加2.22 倍,小麦蛋白的β-转角结构在添加菊粉后向β-折叠结构和无规卷曲结构转化。     

Influence of Cosolvent Systems on the Gelation Mechanism of Globular Protein: Thermodynamic,Kinetic, and Structural Aspects of Globular Protein Gelation

How thermostability and gelation of globular protein are affected by cosolvent systems present in food systems is critical to understanding their functionality. The expression of these functional attributes depends on the molecular structure and thermal‐mechanical history of the protein, as well as its chemical environment. To improve the design of processing protein‐containing food systems, one must fully understand the thermodynamic, kinetic, and structural impact of cosolvent on globular protein gelation. This review focuses on the impact of weakly interacting neutral cosolvent systems (for example, sugars and polyols) on the gelation of globular proteins. The physicochemical mechanisms by which these cosolvent systems can modulate protein gelation are highlighted from a thermodynamic, kinetic, and structural point of view.     

Thermal denaturation and coagulation of whey proteins: effect of sugars

The thermal coagulation of unfractionated whey proteins was inhibited by various sugars. The disaccharides, sucrose and lactose, were most effective, and the amino sugar, glucosamine, least effective in this respect. Ultraviolet absorption and light-scattering measurements on the thermal denaturation and coagulation of both unfractionated and individual whey proteins (alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin) showed that sucrose promotes the denaturation of these proteins but inhibits their subsequent coagulation. These results are interpreted in terms of the effect of sucrose on the hydrophobic interactions between solvent and protein.     

Effects of inulin/oligofructose on the thermal stability and acid-induced gelation of soy proteins

ABSTRACT:  Differential scanning calorimetry (DSC) and dynamic oscillatory shear testing were performed to study the influence of inulin (Raftiline^® HP-gel and Raftiline^® ST-gel) and oligofructose (Raftilose^® P95) on the thermal stability and gelation (using glucono-δ-lactone [GDL] as a coagulant) of soy protein isolate (SPI) dispersions. Addition of 10% (w/v) inulin/oligofructose or sucrose increased ( P < 0.05) the peak denaturation temperatures ( T_m ) of 7S and 11S soy proteins in SPI dispersion (5%[w/v], pH 7.0) by an average of 1.9 and 2.3 °C, respectively. GDL induced SPI thermal gelation, and the gel rheology was affected by both the pH decline and the specific temperature of heating. Addition of inulin/oligofructose (8%, w/v) improved the gelling properties of preheated SPI dispersion (8%, w/v) coagulated with GDL, showing 14.4 to 45.6% increase ( P < 0.05) in gel rigidity ( G ' value) at the end of heating (81 °C). Microstructural examination revealed a denser protein cross-linking structure and reduced pore sizes in SPI gels containing inulin/oligofructose. In general, inulin was more capable of improving SPI gelation than oligofructose, suggesting that the degree of fructose polymerization in the fructans was of thermal and rheological importance.