Influence of pH on the dry heat-induced denaturation/aggregation of whey proteins

The effect of pH on the heat-induced denaturation/aggregation of whey protein isolate (WPI) in the dry state was investigated. WPI powders at different pH values (6.5, 4.5, and 2.5) and controlled water activity (0.23) were dry heated at 100 °C for up to 24 h. Dry heating was accompanied by a loss of soluble proteins (native-like β-lactoglobulin and α-lactalbumin) and the concomitant formation of aggregated structures that increased in size as the pH increased. The loss of soluble proteins was less when the pH of the WPI was 2.5; in this case only soluble aggregates were observed. At higher pH values (4.5 and 6.5), both soluble and insoluble aggregates were formed. The fraction of insoluble aggregates increased with increasing pH. Intermolecular disulphide bonds between aggregated proteins predominated at a lower pH (2.5), while covalent cross-links other than disulphide bonds were also formed at pH 4.5 and 6.5. Hence, pH constitutes an attractive tool for controlling the dry heat-induced denaturation/aggregation of whey proteins and the types of interactions between them. This may be of great importance for whey ingredients having various pH values after processing.     

Whey protein aggregate formation during heating in the presence of κ-carrageenan

The effect of a negatively charged polymer, κ-carrageenan, on the aggregation behaviour of whey proteins during heating was studied. Aqueous solutions of whey protein isolate (WPI) at 0.5% were heated in the presence of κ-carrageenan (0.1%) at pH 7.0. This concentration was chosen as optimal in the detection of the intermediate aggregates during chromatographic analysis. The residual unaggregated protein, the intermediate aggregates and the soluble aggregates were all examined as a function of heating time and temperature, using size-exclusion chromatography coupled with light scattering detection. The presence of κ-carrageenan did not affect the aggregation of whey proteins heated at 75 °C; however, a change in the mechanism of aggregation seemed to occur at higher temperatures, and intermediates with higher molecular mass formed at 85 °C. At 90 °C, in the presence of κ-carrageenan, the extent of WPI aggregation was much larger, as soluble aggregates were no longer present and less residual protein was recovered in the unaggregated peak.     

Microemulsions as nanoreactors to produce whey protein nanoparticles with enhanced heat stability by thermal pretreatment

Native whey proteins (NWPs) may form gels or aggregates after thermal processing. The goal of this work was to improve heat stability of NWPs by incorporating protein solutions in nanoscalar micelles of water/oil microemulsions to form whey protein nanoparticles (WPNs) by thermal pretreatment at 90 °C for 20 min. The produced WPNs smaller than 100 nm corresponded to a transparent dispersion. The WPNs produced at NWP solution pH of 6.8 had a better heat stability than those produced at pH 3.5. The salt concentration (0–400 mM NaCl) in NWP solutions did not significantly change the size of corresponding WPNs. Compared to NWPs, the 5% (w/v) dispersion of WPNs at pH 6.8, 100 mM NaCl did not form a gel after heating at 80 °C for 20 min. The improved heat stability and reduced turbidity of WPNs may enable novel applications of whey proteins in beverages.     

Effect of pH at heat treatment on the hydrolysis of κ-casein and the gelation of skim milk by chymosin

Skim milk was adjusted to pH values between 6.5 and 7.1 and heated at 90 °C for times from 0 to 30 min. After heat treatment, the samples were re-adjusted to the natural pH (pH 6.67) and allowed to re-equilibrate. High levels of denatured whey proteins associated with the casein micelles during heating at pH 6.5 (about 70-80% of the total after 30 min of heating). This level decreased as the pH at heating was increased, so that about 30%, 20% and 10% of the denatured whey protein was associated with the casein micelles after 30 min of heating at pH 6.7, 6.9 and 7.1, respectively. Increasing levels of κ-casein were transferred to the serum as the pH at heating was increased. The loss of κ-casein and the formation of para-κ-casein with time as a consequence of the chymosin treatment of the milk samples were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The loss of κ-casein and the formation of para-κ-casein were similar for the unheated and heated samples, regardless of the pH at heating or the heat treatment applied. Monitoring the gelation properties with time for the chymosin-treated milk samples indicated that the heat treatment of the milk markedly increased the gelation time and decreased the firmness (G^′) of the gels formed, regardless of whether the denatured whey proteins were associated with the casein micelles or in the milk serum. There was no effect of pH at heat treatment. These results suggest that the heat treatment of milk has only a small effect on the primary stage of the chymosin reaction (enzymatic phase). However, heat treatment has a marked effect on the secondary stage of this reaction (aggregation phase), and the effect is similar regardless of whether the denatured whey proteins are associated with the casein micelles or in the milk serum as nonsedimentable aggregates.     

Impact of Residual Lactose on Dry Heat-Induced Pre-texturization of Whey Proteins

Controlling denaturation/aggregation of whey proteins during their pre-texturization is highly critical to avoid variability in their functional properties. We investigated how the dry heat-induced (16 h at 100 °C and a_w?=?0.23) pre-texturization of whey protein isolate (WPI) is affected by traces of remaining lactose (0.3–2.0%) and how it influences its subsequent gelling properties. Lactose even in trace quantities developed intense browning of WPI. Dry-heating conditions used in this study mainly developed soluble aggregates stabilized by covalent crosslinks other than disulfide bonds. The extent of aggregation and size of aggregates were drastically increased with increasing lactose. Intermediate quantity (39–46%) of soluble aggregates improved the gel strength, while excessive aggregation (>?50%) resulted in loss of gel strength. Elasticity of gels was also increased by increasing protein aggregates. This study suggests that the traces of lactose that remain in WPI are critical for controlling its pre-texturization by dry heating and its subsequent gelling properties.     

Distinction of different heat-treated bovine milks by native-PAGE fingerprinting of their whey proteins

Native-PAGE (polyacrylamide gel electrophoresis) was used for the simultaneous qualitative and quantitative analysis of whey proteins of raw, commercial and laboratory heat-treated bovine milks. Four whey protein bands, including β-lactoglobulin variants (β-LG A and B), could be distinctively separated in the gel. The results showed that levels of the major whey proteins were reduced by less than 23% in the pasteurized milks and by more than 85% in the UHT milks as compared with raw milk. The α-lactalbumin (α-LA) exhibited the strongest heat-tolerance: about 32% of it remained in its native state after the milk was heated at 100 °C for 10 min. About 42% of β-LG A and 53% of β-LG B were lost after the milk was heated at 75 °C for 30 min. Blood serum albumin (BSA) was lost almost completely when the milk at pH 5.0 was heated at a temperature of 75 °C or higher. The β-LGA and β-LGB were much more stable at low pH than in neutral conditions.     

Effect of microwave heating on the low-salt gel from silver carp (Hypophthalmichthys molitrix) surimi

Gels from silver carp (Hypophthalmichthys molitrix) surimi were obtained using microwave (MW) heating (15 W/g power intensity for 20–80 s) at different levels of salt (0 g/100 g, 1 g/100 g, or 2 g/100 g). And the gel heated by MW was compared with the gel obtained by conventional water-bath heating (85 °C for 30 min). The gel strength increased when the salt level was increased. The mechanical and functional properties of non-salted, low-salt and regular-salt products were improved by MW heating for 60 s and 80 s, significantly (p < 0.05), except for the cook loss. The content of TCA-soluble peptides indicated that the MW heating inhibited the autolysis of proteins significantly (p < 0.05) during gelling. The SDS-PAGE and total content of –SH group proved that MW enhanced the cross-linking of proteins effectively through disulphide bonds and non-disulphide covalent bonds. The microstructure of the samples revealed that a fine compact network, with particles of protein aggregates, was formed in the low-salt gels (1 g/100 g) heated by MW for 60 s. All of these properties might be responsible for the formation of a superior textural low-salt gel induced by MW.     

Thermodynamic incompatibility between denatured whey protein and konjac glucomannan

The current study investigated the effect of a neutral polysaccharide, konjac glucomannan, on the heat-induced gelation of whey protein isolate (WPI) at pH 7. Oscillatory rheology (1 rad/s; 0.5% strain), differential scanning calorimetry and confocal laser scanning microscopy were used to investigate the effect of addition of konjac in the range 0-0.5% w/w, on the thermal gelation properties of WPI. The minimum gelling concentration for WPI samples was 11% w/w; the concentration was therefore held constant at this value. Gelation of WPI was induced by heating the samples from 20 to 80 °C, holding at 80 °C for 30 min, cooling to 20 °C, and holding at 20 °C for a further 30 min. On incorporation of increasing concentrations of konjac the gelation time decreased, while the storage modulus (G′) of the mixed gel systems increased to ∼1450 Pa for 11% w/w WPI containing 0.5% w/w konjac gels, compared to 15 Pa for 11% w/w WPI gels (no konjac). This increase in gel strength was attributed to segregative interactions between denatured whey proteins and konjac glucomannan.     

Comparative effect of thermal treatment on the physicochemical properties of whey and egg white protein foams

The optimization of the functionalities of commercial protein ingredients still constitutes a key objective of the food industry. Our aim was therefore to compare the effect of thermal treatments applied in typical industrial conditions on the foaming properties of whey protein isolate (WPI) and egg white proteins (EWP): EWP was pasteurized in dry state from 1 to 5 days and from 60 °C to 80 °C, while WPI was heat-treated between 80 °C and 100 °C under dynamic conditions using a tubular heat exchanger. Typical protein concentrations of the food industry were also used, 2% (w/v) WPI and 10% (w/v) EWP at pH 7, which provided solutions of similar viscosity. Consequently, WPI exhibited a higher foamability than EWP. For WPI, heat treatment induced a slight decrease of overrun when temperature was above 90 °C, i.e. when aggregation reduced too considerably the amount of monomers that played the key role on foam formation; conversely, it increased foamability for EWP due to the lower aggregation degree resulting from dry heating compared to heat-treated WPI solutions. As expected, thermal treatments improved significantly the stability of WPI and EWP foams, but stability always passed through a maximum as a function of the intensity of heat treatment. In both cases, optimum conditions for foam stability that did not impair foamability corresponded to about 20% soluble protein aggregates. A key discrepancy was finally that the dry heat treatment of EWP provided softer foams, despite more rigid than the WPI-based foams, whereas dynamically heat-treated WPI gave firmer foams than native proteins.     

Stability and mechanism of whey protein soluble aggregates thermally treated with salts

The formation of whey protein aggregates, often termed soluble aggregates, with specific physicochemical properties has been shown to result in improved functionality in gels, foams, emulsions, encapsulation, films and coatings. This work evaluated the potential of whey protein soluble aggregates to improve thermal stability in the presence of salts and determine the mechanism of improved thermal stability. Solutions of whey protein isolate (WPI) or β-lactoglobulin (β-lg) (7% w/w, pH 6.8) were heated for 10 min at 90 °C to form soluble aggregates. Native proteins and soluble aggregates were diluted to 3% w/w in solutions containing 0–108 mM NaCl and thermally treated (90 °C, 5 min). Turbidity, solubility, and viscosity were evaluated, in addition to ζ-potential and S_o (surface hydrophobicity). Size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) and dynamic light scattering were used to determine aggregate size and transmission electron microscopy (TEM) was used to evaluate aggregate shape. Use of soluble aggregates improved thermal stability due to their altered aggregate shape and higher charge, and resulted in final aggregates that were smaller and less dense, leading to reduced viscosity and turbidity, and increased solubility compared to native proteins. It is concluded that soluble aggregates formed under the appropriate conditions to produce the desirable physicochemical properties can be used to improve whey protein thermal stability with a possible application in beverages.     

Heat-induced aggregation of whey proteins in the presence of κ-casein or sodium caseinate

Aggregates were formed by heating mixtures of whey protein isolate (WPI) and pure κ-casein or sodium caseinate at pH 7 and 0.1 M NaCl. The aggregates were characterized by static and dynamic light scattering and size exclusion chromatography. After extensive heat-treatment at 80 °C for 24 h, almost all whey proteins and κ-casein formed mixed aggregates, but a large proportion of the sodium caseinate did not aggregate. At a given WPI concentration the size of the aggregates decreased with increasing κ-casein or sodium caseinate concentration, but the overall self-similar structure of the aggregates was the same. The presence of κ-casein or caseinate therefore inhibited growth of the heat-induced whey protein aggregates. The results were discussed relative to the reported chaperone-like activity of casein molecules towards heat aggregation of globular proteins.     

Effect of pH at heating on the acid-induced aggregation of casein micelles in reconstituted skim milk

Reconstituted skim milk samples at pH between 6.5 and 7.1 (heating pH) were heated at 80°C, 90°C or 100°C for 30 min (heating temperature). The particle size of the casein micelles was measured at pH 4.75-7.1 (measurement pH) and at temperatures of 10°C, 20°C and 30°C (measurement temperature) using photon correlation spectroscopy. The particle size of the casein micelles, at a measurement pH of 6.7 and a measurement temperature of 20°C, was dependent on the heating pH and heating temperature to which the milk was subjected. The casein micelle size in unheated milk was about 215 nm. At a heating pH of 6.5, the casein micelle size increased by about 15, 30 and 40 nm when the milk was heated at 80°C, 90°C or 100°C, respectively. As the heating pH of the milk was increased, the size of the casein micelles decreased so that, at pH 7.1, the casein micelles were ∼20 nm smaller than those from unheated milk. Larger effects were observed as the heating temperature was increased from 80°C to 100°C. The size differences as a consequence of the heating pH were maintained at all measurement temperatures and at all measurement pH down to the pH at which aggregation of the micelles was observed. For all samples, size measurements at 10°C showed no aggregation at all measurement pH. Aggregation occurred at progressively higher pH as the measurement temperature was increased. Aggregation also occurred at a progressively higher measurement pH as the heating pH was increased. The particle size changes on heating and the aggregation on subsequent acidification may be related to the pH dependence of the association of whey proteins with, and the dissociation of κ-casein from the casein micelles as milk is heated.     

Effect of milk solids concentration on the gels formed by the acidification of heated pH-adjusted skim milk

Reconstituted skim milk of 10–25% total solids was adjusted to pH values between about 6.2 and 7.1 and heated at 80 °C for 30 min. Gels were formed from the heated milks by slow acidification to pH 4.2 and the gelation process and final gels were analyzed for their rheological properties. At each milk concentration, the final acid gel firmness (final G′) and breaking stress could be changed markedly by manipulation of the pH during heating. The final gel firmness and breaking stress could also be modified by changing the concentration of the milk solids prior to heating and acidification. The results indicated that similar gel firmness and breaking stress could be achieved over a range of milk concentrations by control of the pH of the milk during heating. When expressed as a percentage change in final G′ or breaking stress relative to that obtained at the natural pH, plots of the change in final G′ or breaking stress versus pH fell close to a single curve, indicating that the same mechanism may influence the gelation properties at all milk concentrations. The final G′ and breaking stress were related to the denaturation and interaction of the whey proteins with the casein micelles, and the formation of non-sedimentable casein when the milk was heated.     

Influence of muscle type on rheological properties of porcine myofibrillar protein during heat-induced gelation

The gelation characteristics of myofibrillar proteins are indicative of meat product texture. Defining the performance of myofibrillar proteins during gelation is beneficial in maintaining quality and developing processed meat products and processes. This study investigates the impact of pH on viscoelastic properties of porcine myofibrillar proteins prepared from different muscles (semimembranosus (SM), longissimus dorsi (LD) and psoas major (PM)) during heat-induced gelation. Dynamic rheological properties were measured while heating at 1 °C/min from 20 to 85 °C, followed by a holding phase at 85 °C for 3 min and a cooling phase from 85 to 5 °C at a rate of 5 °C/min. Storage modulus (G′, the elastic response of the gelling material) 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 depression in the thermogram. Following 60 °C, the treatments maintained observed trends in gel strength, showing SM myofibrils produced the strongest gels. Myofibrillar protein from SM and PM formed stronger gels at pH 6.0 than at pH 6.5. Differences may be attributed to subtle variations in their protein profile related to muscle type or postmortem metabolism. Significant correlations were determined between G′ at 57, 72, 85 and 5 °C, indicating that changes affecting gel strength took effect prior to 57 °C. Muscle type was found to influence water-holding capacity to a greater degree than pH.     

Heating of milk alters the binding of curcumin to casein micelles. A fluorescence spectroscopy study

Curcumin, a polyphenolic compound present in turmeric, is a hydrophobic molecule that has been shown to bind to casein micelles. The present work tested the hypothesis that surface changes in the casein micelles caused by heat-induced interactions with the whey proteins would affect the binding of curcumin. Binding was quantified by direct and tryptophan quenching fluorescence spectroscopy. Curcumin binds to the hydrophobic moieties of the casein proteins, with a 10 nm blue shift in its fluorescence emission peak, and causes quenching of the intrinsic fluorescence spectra of the proteins. The fluorescence intensity of curcumin increased after heating of milk at 80 °C for 10 min; a similar trend in the binding constants was also observed with casein micelles separated from the soluble proteins by centrifugation. There was an increase in the non-specific interactions with heating milk at 80 °C for 10 min, both in milk as well as in casein micelles separated from the serum proteins. The increased capacity of milk proteins to bind curcumin after heat treatment can be attributed to whey protein denaturation, as whey proteins bind to the surface of casein micelles with heating.     

Effect of chymotrypsin digestion followed by polysaccharide conjugation or transglutaminase treatment on functional properties of millet proteins

Millet protein was solubilized by chymotrypsin; the soluble protein was conjugated to galactomannan under controlled conditions (60 °C, 76% RH) or polymerised by transglutaminase (TGase). SDS–PAGE patterns showed that the conjugated and polymerised proteins had higher molecular mass bands above the stacking gel. SDS–PAGE patterns also indicated that the digest was conjugated to galactomannan and polymerised by TGase. The free amino groups (OD₃₄₀) of the conjugated and polymerised digest were greatly reduced. Although the chymotrypsin digest was considerably insoluble between pH 2.0 and 5.0, galactomannan conjugate was completely soluble at all levels of pH. TGase polymer was slightly insoluble at pH 4.0. Galactomannan conjugate resisted heat-induced aggregation, even after heating at 90 °C for 20 min, while TGase polymer resisted heat-induced aggregation up to 70 °C, after which its solubility started to decline. The emulsifying properties of the conjugate and the polymerized proteins were greatly improved, compared to the native and chymotrypsin digests.     

Effect of thermal pretreatment of raw soymilk on the gel strength and microstructure of tofu induced by microbial transglutaminase

The influence of thermal pretreatment of raw soymilk on the gel hardness and microstructure of tofu, induced by microbial transglutaminase (MTGase), was investigated in this paper. Modulated differential scanning calorimetry analysis showed that individual proteins in soymilk were to a various extent denatured by different thermal pretreatments. The viscosity of the soymilk and the gel hardness of MTGase-induced tofu were more highly related with the heating rate (up to 90 °C) than the mode of heating. At any enzyme concentration of MTGase, the tofus prepared from soymilk heated at 75 °C for 10 or 30 min showed highest gel hardness among all tested ones (P?0.05). Scanning electron microscopy analysis indicated that the microstructure of the tofu from soymilk heated at 75 °C for 30 min had a unique coral-like structure, much more continuous and homogenous than that from soymilk at 95 °C for 5 min. These results confirmed that the appropriate heat pretreatment (e.g. in the present, at 75 °C for 10-30 min) remarkably improved the gel strength of tofu by means of MTGase, and strengthened the tofu gel structure.     

Dynamic oscillatory rheological measurement and thermal properties of pea protein extracted by salt method: Effect of pH and NaCl

The effects of two important factors, pH (3.0-10.0) and NaCl (0-2.0 M), on pea protein gelation properties were studied using dynamic oscillatory rheometer and differential scanning calorimeter (DSC). The strongest gel stiffness was achieved at 0.3 M NaCl; higher or lower salt concentrations lead to weakening of the gel. The gelation temperature was also influenced by ionic strength; salt had a stabilization effect which inhibited pea protein denaturation at higher salt concentrations resulting in higher gelling points (p < 0.05). At a NaCl concentration 2.0 M, pea protein gelation was completely suppressed at temperatures ?100 °C. The pH also played an important role in gel formation by pea protein isolates since acid and base cause partial or even total protein denaturation. In this paper the maximum gel stiffness occurred at pH 4.0 in 0.3 M NaCl; higher or lower pH values resulted in reduced gel stiffness (p < 0.05). pH also altered the denaturation temperature of the pea protein; higher pH values resulted in higher denaturation temperatures and higher enthalpies of denaturation (p < 0.05). At pH 3 pea proteins seem like completely denatured by acid as the DSC curve showed a straight line. The gelation temperature (gelling point) peaked at pH ∼6.0 (89.1 °C). Careful adjustment of pH and NaCl concentration would enable the food industry to effectively utilize the salt-extracted pea protein isolate as a gelling agent.     

Physical properties of acid milk gels prepared at 37°C up to gelation but at different incubation temperatures for the remainder of fermentation

We investigated the effect of altering temperature immediately after gels were formed at 37°C. We defined instrumentally measurable gelation (IMG) as the point at which gels had a storage modulus (G′) ≥5 Pa. Gels were made at constant incubation temperature (IT) of 37°C up to IMG, and then cooled to 30 or 33.5, or heated to 40.5 or 44°C, at a rate of 1°C/min and maintained at those temperatures until pH 4.6. Control gel was made at 37°C (i.e., no temperature change during gelation/gel development). Gel formation was monitored using small strain dynamic oscillatory rheology, and the resulting structure and physical properties at pH 4.6 were studied by fluorescence microscopy, large deformation rheology, whey separation (WS), and permeability (B). A single strain of Streptococcus thermophilus was used to avoid variations in the ratios of strains that could have resulted from changes in temperature during fermentation. Total time required to reach pH 4.6 was similar for samples made at constant IT of 37°C or by cooling after IMG from 37 to either 30 or 33.5°C, but gels heated to 40 or 44°C needed less time to reach pH 4.6. Cooling gels after IMG resulted in an increase in G′ values at pH 4.6, a decrease in LT_max, WS, and B, and an increase in the area of protein aggregates of micrographs compared with the control gel made at constant IT of 37°C. Heating gels after IMG resulted in a decrease in G′ values at pH 4.6 and an increase in LT_max values and WS. The physical properties of acid milk gels were dominated by the temperature profile during the gel-strengthening phase that occurs after IMG. This study indicates that the final properties of yogurt greatly depend on the environmental conditions (e.g., temperature, time/rate of pH change) experienced by the casein particles/clusters during the critical early gel development phase when bonding between and within particles is still labile. Cooling of gels may encourage inter-cluster strand formation, whereas heating of gels may promote intra-cluster fusion and the breakage of strands between clusters.     

Mixtures of whey protein microgels and soluble aggregates as building blocks to control rheology and structure of acid induced cold-set gels

Whey proteins (WP) today offer an extremely high potential for innovative development of functional and nutritious food products. Acid cold-set gels present an interesting approach of gelation at low temperature upon acidification of preformed whey protein (WP) aggregates. In the present work, we aimed to demonstrate how structure and rheological properties of acid gels can be controlled by combining two types of WP aggregates with different structural and chemical properties. Whey protein microgels (WPM) and soluble aggregates (WPSA) were generated upon heating WP isolate in specific pH conditions and temperature, leading to Z-average hydrodynamic diameters close to 270 nm for WPM and 100 nm for WPSA. Mixtures of WPM and WPSA were prepared at different weight ratios ranging from 100% WPM to 100% WPSA. The total protein concentration was set to 4 or 8%wt. Acidification was performed at 40 °C by addition of 1%wt glucono-δ-lactone (GDL). Gelation was followed using turbidimetry and small deformation rheology as function of pH. Microstructures of the gel were investigated at different length scales using various microscopy techniques (CLSM, SEM, AFM). When the WPM/WPSA ratio decreased, the pH of gelation and the gel strength increased because of the different structure and chemical reactivity of the two types of WP aggregates. The final pH had a strong impact on the structure of the gels. When final pH decreased below pH 4.3, a structure change was suggested by turbidimetry measurements. This resulted in a non self-supporting gel or in a decrease of gel strength. For pH above 4.3, self supporting gel were obtained. The rheological properties of the gel could therefore be modulated depending on the properties of the building blocks used (WPM versus WPSA). Interestingly, the gel microstructures observed for WPM/WPSA mixtures or WPM were comparable to those of acidified skimmed milk gels ranging from coarse structures with clumps of aggregates or to homogeneous fine networks (WPSA only) that have been described for WP gels obtained upon direct heating at various pH.