Heat-induced Whey Protein Gels Research Articles

Heat-induced gel formation by whey protein isolate-Lycium barbarum polysaccharides at varying pHs

Effects of different levels (0, 0.1, 0.5, and 1% w/v) of Lycium barbarum polysaccharides (LBP) were investigated on the gel properties of heat-induced whey protein gels forming at varying pHs (3.0, 5.2 and 7.0). The textural, rheological, hydration properties and microstructure of gels were evaluated. Results exhibited that the composite gel containing 0.1% (w/v) LBP showed the greatest functional valorization, whereas higher concentrations of LBP (1% w/v) yielded hydrogels with higher porosity, which was confirmed by scanning electron microscopy. Moreover, whey protein formed white particulate gels and transparent fibrillar gels at pH values near to and far from the isoelectric point, respectively. As for hydration properties, no difference in water holding capacity and swelling property was observed at all pHs. This study simulated the conditions used in practical production, aiming to provide a theoretical basis for the application of LBP on composite gels and develop tunable gel-type food to satisfy different consumer preferences.

Formulation of Heat-Induced Whey Protein Gels for Extrusion-Based 3D Printing.

This study investigated the extrusion-based 3D printability of heat-induced whey protein gels as protein rich food inks. In particular, the effects of ionic strength by the addition of NaCl (0–250 mM), protein content (10%, 15%, 20%), fat content (0%, 10%), and partial substitution of whey protein isolate (WPI) with microparticulated whey protein (MWP) or micellar casein isolate (MCI) on printability were assessed. Texture analysis, specifically Young’s modulus, rheological measurements including yield stress, and creep–recovery behavior were used to characterize the gels. Modifications of the formulation in terms of ionic strength, increased protein content, and the formation of emulsion gels were insufficient to maintain a continuous extrusion process or shape stability after printing. However, the substitution of WPI with MWP created more viscoeleastic gels with improved printability and shape retention of the 3D cube structure after deposition. The partial replacement of WPI with MCI led to phase separation and 3D-printed cubes that collapsed after deposition. A narrow range of rheological material properties make WPI and MWP emulsion gels promising food inks for extrusion-based 3D printing.

Influence of chaperone-like activity of caseinomacropeptide on the gelation behaviour of whey proteins at pH 6.4 and 7.2.

The effect of caseinomacropeptide (CMP) on the heat-induced denaturation and gelation of whey proteins (2.5–10%, w/v) at pH 6.4 and 7.2, at a whey protein:CMP ratio of 1:0.9 (w/w), was investigated using differential scanning calorimetry (DSC), oscillatory rheology (90 °C for 20 min) and confocal microscopy. Greater frequency-dependence in the presence of CMP suggested that the repulsive interactions between CMP and the whey proteins affected the network generated by the non-heated whey protein samples. At pH 6.4 or 7.2, CMP increased the temperature of denaturation of β-lactoglobulin by up to 3 °C and increased the gelation temperature by up to 7 °C. The inclusion of CMP strongly affected the structure of the heat-induced whey protein gels, resulting in a finer stranded structure at pH 6.4 and 7.2. The presence of CMP combined with a lower heating rate (2 °C/min) prevented the formation of a solid gel of whey proteins after heating for 20 min at 90 °C and at pH 7.2. These results show the potential of CMP for control of whey protein denaturation and gelation.

Modification of heat-induced whey protein gels by basic amino acids

Various amino acids have been studied as gelation enhancers. This study investigated the effect of histidine, lysine, and arginine on gelling properties of heat-induced whey protein isolate (WPI) gels at different pHs. Basic amino acids modified WPI gels in a pH- and amino acid-dependent manner. Hardness and gumminess of the WPI gel was improved by arginine at pH 7.59 while springiness was enhanced by histidine at pHs 7.59 and 9.74 and by lysine at pH 7.59 (P < 0.05). At pH 2.0, WPI formed a weak gel. Lysine and arginine facilitated β-lactoglobulin cross-linking at pH 2.0 and reduced protein leach out from the gel (P < 0.05). At pH 5.2, WPI formed a particulate gel with poor water holding capacity (WHC). Lysine improved WHC of the WPI gel at pH 5.2 by changing the structure of the gel network. At pHs away from 5.2, basic amino acid treatments resulted in a more uniform and porous gel matrix and a greater WHC (P < 0.05). In conclusion, different basic amino acids may be applied as WPI gel enhancers depending on the pH and desired attributes of the product.

Influence of γ-aminobutyric acid on gelling properties of heat-induced whey protein gels

γ-Aminobutyric acid (GABA) is a non-protein amino acid that has attracted considerable attention due to its various health promoting benefits. Great efforts have been made to enrich GABA in food while few studies have considered that GABA may influence the protein functional properties, which are imperative to food quality. The present study investigated the effects of GABA on gelling properties of heat-induced whey protein gels at different pHs. GABA greatly strengthened the whey protein gels at pH 2.0 while weakened the gels at pH 6.5 and 8.0 (P < 0.05). At pH 2.0, GABA decreased protein leachability and enhanced water holding capacity (P < 0.05) by encouraging the disulfide cross-linking of α-lactalbumin and β-lactoglobulin, which resulted in a well-structured gel network that can resist deformation and trap water. At pH 6.5, GABA increased the leach-out of β-lactoglobulin which explained the reduced gel hardness and improved gel swelling ratio (P < 0.05). At pH 8.0, GABA treatment resulted in a less cohesive gel despite a decreased protein leachability (P < 0.05) and an increased incorporation of α-lactalbumin, β-lactoglobulin, and BSA into the gel network. In summary, the presence of GABA exhibited a pH-dependent impact on the gelling properties of heat-induced whey protein gels.

Citric acid cross-linking of heat-set whey protein hydrogel influences its textural attributes and caffeine uptake and release behaviour

Heat-induced whey protein gels were either pre-loaded with caffeine, then cross-linked with citric acid, or vice versa. Based on the results of Fourier transform infrared spectroscopy, the pre-loading then cross-linking procedure caused less cross-linkage formation than the cross-linking then caffeine loading (post-loaded) procedure. The latter also resulted in a firmer gel. The caffeine-to-protein ratio was approximately 8.5 μg mg−1 in the non-cross-linked hydrogel; those of the caffeine-loaded then cross-linked and the cross-linked then caffeine-loaded hydrogels were 4.5 and 2.9 μg mg−1, respectively. Cross-linking decreased swelling of the freeze-dried gels upon subsequent rehydration. It also influenced the extent of caffeine release from the protein hydrogel. Pre-loaded and post-loaded samples retained ∼78% and ∼88%, respectively, of the entrapped caffeine after immersion in water for 4 h; the non-cross-linked gel retained <55% of the initially loaded caffeine. Caffeine loading decreased gel water-holding capacity; subsequent citric acid cross-linking increased water-holding capacity.

Combined confocal microscopy and large deformation analysis of emulsion filled gels and stirred acid milk gels

Abstract The microstructural breakdown properties of heat-induced whey protein (WP) gels prepared at pH 7.0 or 5.4 and containing emulsified sunflower oil (7%, w/w) were studied using notch propagation tensile testing in combination with dynamic confocal laser scanning microscopy (CLSM). In addition, the microstructural breakdown properties of stirred acid milk gels containing added emulsified oil (0–15%, w/w) or Konjac glucomannan (0.05%, w/w) were studied using compressive rheological deformation in combination with dynamic CLSM imaging. The structural breakdown properties (Young's modulus and stress/strain at fracture), the microstuctural behaviour of the protein phase, the emulsified oil phase and the pattern of notch propagation during large deformation tensile testing of the WP emulsion filled gel prepared at pH 7.0 (fine stranded gel) differed from that of the WP emulsion filled gel prepared at pH 5.4 (particulate gel). The protein aggregates, emulsified oil droplets and the Konjac phase in the stirred acid milk gels flowed on compression; however, the flow pattern changed from “frictional” flow with no emulsified oil or Konjac to “slip” flow in the presence of emulsified oil or Konjac. Also, compression of stirred acid milk gels below a certain critical height led to disintegration of the aggregated protein structure, serum release and reduced stability of the oil droplets in the stirred gels. The critical compression height was influenced by emulsified oil level and the presence of added Konjac.

Influence of Run Time and Aging on Fouling and Cleaning of Whey Protein Deposits on Heat Exchanger Surface

In the cleaning operations of heat exchange surfaces in dairy processing plants, the effect of heating/run time (HT) and aging on the fouling/cleaning of heat exchangers is not well understood. Longer heating time of the same deposit is expected to result in the formation of stronger and perhaps more cleaning resistant deposit. In this study, the phenomenon of heating and aging on the formation and cleaning of dairy fouling is investigated using the heat induced whey protein gels (HIWPG) produced in laboratory. The processes were also investigated with the whey protein deposits formed in pilot-scale plant trails. The HIWPGs were produced in tubular capsules for various heating (or run) times (60, 120, 240, 1440 and 2880 min respectively) and then dissolved in aqueous sodium hydroxide (0.5 wt %). The heating process here would have been of ‘pure’ aging. The dissolution rate was calculated based on the previously established UV Spectrophotometer analysis. The structure and texture of the gels were analysed using Scanning Electron Microscope (SEM) and texture analyser. In the pilot-scale plant study, whey protein fouling layers were generated by recirculating whey protein solution (6 wt %) for various heating periods (30, 60, and 90 min respectively). The deposit layers were then removed by recirculating aqueous sodium hydroxide (0.5 wt %) and the cleaning efficiency was monitored in the form of the recovery of heat transfer coefficient while both fluid electric conductivity and turbidity were recorded as indications of cleaning completion. It was found that increasing the HIWPG heating time significantly increased the gel hardness and dissolution time, implicating the difficulty in cleaning. Similarly to these results on gels, increasing the pilot-scale plant heating/run time increased the extent of fouling. The fouling layer formed next to the metal surface experienced the longest period of aging and the slope of the heat transfer coefficient increase seen at the final cleaning stage is related to this aging effect. The rate of cleaning for deposits formed initially on the metal surface is lower indicating a ‘pure’ aging effect of the deposit near surface.

Effect of whey protein concentration on the fouling and cleaning of a heat transfer surface

In the studies of fouling and cleaning of heat exchange surfaces in dairy plants, whey protein deposits and heat induced whey protein gels (HIWPG) are considered as suitable model material to simulate the proteinaceous based type “A” milk fouling. Protein concentration of the fouling solution may significantly influence the formation of milk deposits on heat exchange surfaces, hence affecting the cleaning efficiency. In this study, a laboratory produced heat induced whey protein gels (HIWPG) and a pilot plant heat exchanger fouling/cleaning were used to investigate the effect of protein concentration on formation and cleaning of dairy fouling. Here, HIWPGs made from different protein concentrations were formed in capsules and then dissolved in aqueous sodium hydroxide (0.5 wt%). The dissolution rate calculation based on the UV spectrophotometer analysis. In the pilot-scale plant study, whey protein fouling deposits were formed by recirculating whey protein solutions with different concentrations through the heat exchange section in different runs, respectively. The deposit layers were then removed by recirculating aqueous sodium hydroxide (0.5 wt%) and the cleaning efficiency was monitored in the form of the recovery of heat transfer coefficient while both fluid electric conductivity and turbidity were monitored as indications of cleaning completion. It was found that increasing the protein concentration of the HIWPG significantly increased the gel hardness and the dissolution time. In addition, increasing the protein concentration significantly increased both, the amount of the fouling on the pilot-scale plant and the time required to clean the fouling deposit.

Heat-Induced Whey Protein Gels: Effects of pH and the Addition of Sodium Caseinate

The effects of pH (6.7 or 5.8), protein concentration and the heat treatment conditions (70 or 90 °C) on the physical properties of heat-induced milk protein gels were studied using uniaxial compression, scanning electron microscopy, differential scanning calorimetry, and water-holding capacity measurements. The systems were formed from whey protein isolate (10–15% w/v) with (5% w/v) or without the addition of caseinate. The reduction in pH from 6.7 to 5.8 increased the denaturation temperature of the whey proteins, which directly affected the gel structure and mechanical properties. Due to this increase in the denaturation temperature of the β-lactoglobulin and α-lactalbumin, a heat treatment of 70 °C/30 min did not provide sufficient protein unfolding to form self-supporting gels. However, the presence of 5% (w/v) sodium caseinate decreased the whey protein thermo stability and was essential for the formation of self-supporting gels at pH 6.7 with heat treatment at 70 °C/30 min. The gels formed at pH 6.7 showed a fine-stranded structure, with great rigidity and deformability as compared to those formed at pH 5.8. The latter had a particulate structure and exuded water, which did not occur with the gels formed at pH 6.7. The addition of sodium caseinate led to less porous networks with increased gel deformability and strength but decreased water exudation. The same tendencies were observed with increasing whey protein concentration.

Heat-Induced Whey Protein Gels: Protein−Protein Interactions and Functional Properties

Heat-induced gelation (80 degrees C for 30 min or 85 degrees C for 60 min) of whey protein concentrate (WPC) solutions was studied using small deformation dynamic rheology, small and large deformation compression, and polyacrylamide gel electrophoresis (PAGE). The WPC solutions (15% w/w, pH 6.9) were prepared by dispersing WPC powder in water (control), 1% (w/w) sodium dodecyl sulfate (SDS) solution, and N-ethylmaleimide (NEM) solution at a protein/NEM molar ratio of 1:1 or in 10 mM dithiothreitol (DTT) solution. PAGE analyses showed that the heat treatment of control solutions contained both disulfide and non-covalent linkages between denatured protein molecules. Only disulfide linkages were formed in heated SDS-WPC solutions, whereas only non-covalent linkages were formed in DTT-WPC and NEM-WPC solutions during heating. In heated NEM-WPC solutions, the pre-existing disulfide linkages remained unaltered. Small deformation rheology measurements showed that the storage modulus (G') values, compared with those of the control WPC gels (approximately 14000 Pa), were 3 times less for the SDS-WPC gels (approximately 4000 Pa), double for the NEM-WPC gels (approximately 24000 Pa), and even higher for the DTT-WPC gels (approximately 30000 Pa). Compression tests suggested that the rubberiness (fracture strain) of the WPC gels increased as the degree of disulfide linkages within the gels increased, whereas the stiffness (modulus) of the gels increased as the degree of non-covalent associations among the denatured protein molecules increased.

Comparison between the dissolution of whey protein gels and of synthetic polymers

AbstractThe dissolution behavior of heat‐induced whey protein gels in alkali is compared with the literature on the dissolution of (1) uncrosslinked synthetic polymers with chemical reaction, (2) uncrosslinked polymers in a good solvent, and (3) crosslinked hydrogels. Diffusion of hydroxide and acid–base reactions, which are key processes in the dissolution of phenolic resins in alkali (Case 1), are found not to be very important with protein gels. The strong dependency of the dissolution rate on the free volume observed in protein gels is described by a large scaling factor, as predicted for Case 2 theory, indicating that the disentanglement of long chains before they are released is important. The cleavage of intermolecular crosslinks, which is important in Case 3, is also identified as a key dissolution step, where the complexity of protein chemistry comes into play. Synthetic polymer mechanisms are therefore able to elucidate several aspects of protein gel dissolution, but are not yet sufficient to explain all aspects of the process. © 2008 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 1007–1021, 2008

Dissolving heat-induced protein gel cubes in alkaline solutions under natural and forced convection conditions

Heat-induced whey protein gels (HIWPG) were formed as cubes and dissolved in aqueous sodium hydroxide (0.25–0.50 wt%) at 25–60 °C, a range of agitation speeds, and with or without additional chemicals. The solution temperature was found to be the most important parameter affecting dissolution behaviour. Increasing the stirring speed reduced the time for complete dissolution, albeit marginally. Significant dissolution was observed under natural convection conditions and particulate release was evident under all conditions, indicating that the ‘dissolution’ process features both molecular solubilization and gel breakdown.

Influence of Galactomannans with Different Molecular Weights on the Gelation of Whey Proteins at Neutral pH

The effect of locust bean gum, a galactomannan, with different molecular weights on the microstructure and viscoelastic properties of heat-induced whey protein gels has been studied using confocal laser scanning microscopy and small-deformation rheology. The results obtained clearly showed that differences in the molecular weight of the polysaccharide have a significant influence on the gel microstructure. Homogeneous mixtures and phase-separated systems, with dispersed droplet and bicontinuous morphologies, were observed by changing the polysaccharide/protein ratio and/or the molecular weight. At 11% whey protein, below the gelation threshold of the protein alone, the presence of the nongelling polysaccharide induces gelation to occur. At higher protein concentration, the main effect of the polysaccharide was a re-enforcement of the gel. However, at the higher molecular weight and concentration of the nongelling polymer, the protein network starts to lose elastic perfection, probably due to the formation of bicontinuous structures with lower connectivity.

The roles of disulphide and non-covalent bonding in the functional properties of heat-induced whey protein gels.

Heat-induced gelation (80 degrees C, 30 min or 85 degrees C, 60 min) of whey protein concentrate (WPC) solutions was studied using transmission electron microscopy (TEM), dynamic rheology and polyacrylamide gel electrophoresis (PAGE). The WPC solutions (150 g/kg, pH 6.9) were prepared by dispersing WPC powder in water (control), 10 g/kg sodium dodecyl sulphate (SDS) solution or 10 mM-dithiothreitol (DTT) solution. The WPC gels containing SDS were more translucent than the control gels, which were slightly more translucent than the gels containing DTT. TEM analyses showed that the SDS-gels had finer aggregate structure (approximately equal to 10 nm) than the control gels (approximately equal to 100 nm), whereas the DTT-gels had a more particulate structure (approximately equal to 200 to 300 nm). Dynamic rheology measurements showed that the control WPC gels had storage modulus (G) values (approximately equal to 13,500 Pa) that were approximately equal to 25 times higher than those of the SDS-gels (approximately equal to 550 Pa) and less than half those of the DTT-gels after cooling. Compression tests showed that the DTT-gels were more rigid and more brittle than the control gels, whereas the SDS-gels were softer and more rubbery than either the control gels or the DTT-gels. PAGE analyses of WPC gel samples revealed that the control WPC solutions heated at 85 degrees C for 10 min contained both disulphide bonds and non-covalent linkages. In both the SDS-solutions and the DTT-solutions, the denatured whey protein molecules were in the form of monomers or small aggregates. It is likely that, on more extended heating, more disulphide linkages were formed in the SDS-gels whereas more hydrophobic aggregates were formed in the DTT-gels. These results demonstrate that the properties of heat-induced WPC gels are strongly influenced by non-covalent bonding. Intermolecular disulphide bonds appeared to give the rubbery nature of heat-induced WPC gels whereas non-covalent bonds their rigidity and brittle texture.

Fine-stranded and particulate aggregates of heat-denatured whey proteins visualized by atomic force microscopy.

beta-Lactoglobulin and whey protein isolate (WPI) were heated in aqueous solutions at pH 2 and 7 at 80 degrees C, spread onto freshly cleaved mica surfaces, and visualized under butanol using atomic force microscopy. Fine-stranded aggregates were formed at pH 2, the diameter of strands being ca. 4 nm for beta-lactoglobulin and 10 nm for WPI. At pH 7, aggregates were composed of ellipsoidal particles, regardless of the concentration of added NaCl. This observation supports the previously proposed two-step aggregation model at neutral pH (Aymard, P.; Gimel, J. C.; Nicolai, T.; Durand, D. J. Chim. Phys. 1996, 93, 987-997), consisting of the formation of primary globular particles and the subsequent aggregation of those primary particles. The AFM provides the first direct evidence for the anisotropic shape of these primary particles. The heights of primary particles increased from ca. 11 to 27 nm with increasing concentrations of added NaCl from 0 to 0.3 M in the case of WPI. The rate of aggregation was also accelerated with increasing NaCl concentrations, which appeared to induce transitions in gel networks from fine-stranded toward particulate networks. The present study provides structural information essential for understanding the diverse physical properties of heat-induced whey protein gels.

Factors Determining Fracture Stress and Strain of Fine-Stranded Whey Protein Gels

Whey proteins form translucent heat-induced gels at low and high pH that differ in fracture properties. Gels formed at pH 7.0 and 6.5 were strong (fracture stress of 59−92 kPa) and rubbery (fracture strain of 1.7−1.2). Gels formed at pH 3.0 and 2.5 were weak (fracture stress of 17−19 kPa) and brittle (fracture strain of 0.33−0.39). Altering disulfide bond formation changed gel texture. Addition of a disulfide reducing agent (dithiothreitol) at concentrations ≥ 25 mM, at pH 7.0, caused the gel to weaken (>46% reduction in fracture stress) and become brittle (fracture strain of 0.30−0.32). Increasing the number of disulfide bonds in gels formed at pH values ≤3.5 increased fracture strain to 2.0 while causing a minor increase in fracture stress. Formation of disulfide bonds appears to be essential to the deformability of heat-induced whey protein gels. Keywords: Gelation; fracture properties; disulfide bonding; whey proteins

Effects of Limited Proteolysis on the Microstructure of Heat-Induced Whey Protein Gels at Varying pH

Whey protein solutions, reconstituted 12% commercial whey protein isolate, were hydrolyzed at pH 7.0 by one of three proteases: trypsin, Neutrase® (from Bacillus subtilis), and a Bacillus licheniformis protease. Heat-induced gels were made from the hydrolysates after pH adjustment, and their microstructure was examined by transmission electron microscopy and compared with the structure of control gels made from unhydrolyzed whey protein solutions.The gels formed from the unhydrolyzed whey protein solutions, as expected, were fine-stranded when set at pH 3.0 and 7.0 and particulate when set at pH 5.2. The enzymatic treatments caused alterations in the microstructure of the heat-induced gels. The changes were small at pH 3.0, marked at pH 5.2, and strongest at pH 7.0. Of special interest was the dramatic change in the microstructure of the gels set after treatment with Bacillus licheniformis protease. The Bacillus licheniformis protease gels consisted of small aggregates (∼0.1 μm) arranged in open clusters (pH 5.2) or tightly packed in a regular pattern (pH 7.0), corresponding to a high gel strength.Limited hydrolysis is thus a way to change the microstructure and properties of gels at weakly acidic and neutral pH, a range that is suitable for the production of many foods.