Effects of heating rate and pH on fracture and water-holding properties of globular protein gels as explained by micro-phase separation

The effect of heating rate and pH on fracture properties and held water (HW) of globular protein gels was investigated. The study was divided into 2 experiments. In the 1st experiment, whey protein isolate (WPI) and egg white protein (EWP) gels were formed at pH 4.5 and 7.0 using heating rates ranging from 0.1 to 35 °C/min and holding times at 80 °C up to 240 min. The 2nd experiment used one heating condition (80 °C for 60 min) and probed in detail the pH range of 4.5 to 7.0 for EWP gels. Fracture properties of gels were measured by torsional deformation and HW was measured as the amount of fluid retained after a mild centrifugation. Single or micro-phase separated conditions were determined by confocal laser scanning microscopy. The effect of heating rate on fracture properties and HW of globular protein gels can be explained by phase stability of the protein dispersion and total thermal input. Minimal difference in fracture properties and HW of EWP gels at pH 4.5 compared with pH 7.0 were observed while WPI gels were stronger and had higher HW at pH 7.0 as compared to 4.5. This was due to a mild degree of micro-phase separation of EWP gels across the pH range whereas WPI gels only showed an extreme micro-phase separation in a narrow pH range. In summary, gel formation and physical properties of globular protein gels can be explained by micro-phase separation. PRACTICAL APPLICATION: The effect of heating conditions on hardness and water-holding properties of protein gels is explained by the relative percentage of micro-phase separated proteins. Heating rates that are too rapid require additional holding time at the end-point temperature to allow for full network development. Increase in degree of micro-phase separation decreases the ability for protein gels to hold water.     

Combining protein micro-phase separation and protein–polysaccharide segregative phase separation to produce gel structures

The ability of protein micro-phase separation and protein–polysaccharide segregative phase separation to generate a range of gel structures and textures was evaluated. Whey protein isolate/κ-carrageenan mixed gels were prepared with 13% (w/v) whey protein isolate, 0–0.6% (w/w) κ-carrageenan and 50, 100 or 250 mM NaCl. The microstructure of gels, determined by confocal laser scanning microscopy, varied from homogenous to protein continuous, bicontinuous, coarse stranded or κ-carrageenan continuous, depending on the κ-carrageenan concentration. Microstructure also varied from stranded to particulate (micro-phase separated) depending on the salt concentration. The rheological behavior of mixed gels corresponded to the shift in the continuous phase from protein to κ-carrageenan. At small concentrations of κ-carrageenan, where carrageenan-rich droplets were dispersed in a continuous protein-rich matrix, gel strength (fracture stress) and firmness (G′) increased due to increased local concentration of proteins caused by phase separation. At higher κ-carrageenan concentrations, gels were substantially less firm, weaker and less deformable (fracture strain). The change in the continuous phase from protein continuous to carrageenan continuous explained the major change in mechanical properties and water-holding properties. The shift in microstructure occurred at lower concentrations of κ-carrageenan when whey proteins were under micro-phase separation conditions. The results demonstrated how the combined mechanisms of ion-induced micro-phase separation of proteins and protein–polysaccharide phase separation and inversion can be used to alter gel structure and texture.     

Effects of pork collagen on thermal and viscoelastic properties of purified porcine myofibrillar protein gels

The objectives of this study were to examine the thermal, water-binding and viscoelastic properties of mixed protein systems containing purified myofibrils from porcine semimembranosus (MP) and pork collagen (PC) during gelation and subsequent cooling. MP:PC mixtures (100:0, 90:10, 80:20, 70:30, 60:40, 50:50) normalized to 4% protein were evaluated. No significant differences in thermal characteristics of these mixtures could be detected using differential scanning calorimetry. A primary peak was observed near 66?°C. Using small-strain oscillatory testing, the rheological properties during gelling and cooling were quantified. Storage modulus (G') increased upon heating for all treatments, but the rate of gel firming and the G' value at 85?°C were significantly lower (P<0.05) as PC was added to the mixed protein system. Upon cooling, gels revealed a significantly lower (P<0.05) rate of gel firming and significantly lower (P<0.05) G' value at 5?°C in samples with 20% inclusion of PC and higher. Addition of PC yielded a significant linear (R(2)=0.65; P<0.01) increase in the water-holding capacity (WHC) of the gels, indicating that the matrix formed in MP:PC gels had a greater ability to entrap water than that of the control MP gels. The inclusion of 10% PC resulted in gels with significantly higher (P<0.05) WHC and similar firmness when compared with gels comprised of MP as the only protein source.     

Gelation characteristics of muscle proteins from pale, soft, exudative (PSE) pork

Thermally induced protein gels were made by using extracted salt-soluble proteins from normal pigs and stress-susceptible pigs determined to have pale, soft, exudative (PSE) muscle. Effects of heating rates (17,39 and 93° C/h) at various protein concentrations (23, 34, 48 and 54 mg/ml) were evaluated. Gel strength of PSE extracts was 45% of the controls at equivalent protein concentration. Gel strength of normal and PSE-muscle protein gels from the first compression curve increased with increasing protein concentrations at all heating rates; however, gel strength was greater for slow heating rates than for fast heating rates in both PSE and normal samples. Percent water loss was greater for PSE extracts than for controls at the same protein concentration. Losses of 47% and 36% for PSE and controls, respectively, were observed at a a protein level of 54 mg/ml protein. There was no heating rate effect on water losses in either case. Protein loss was less, for both PSE and control, at low protein concentrations than at high protein content in the range studied. Slow heating rates resulted in less protein loss, for both PSE and control, whereas faster heating rates gave greater protein losses in the exuded water.     

Enzymatic modification of selected functional properties of myofibril preparation obtained from mechanically recovered poultry meat

The effect of addition of 3 g/L of commercially available transglutaminase preparation to protein extracts obtained from mechanically recovered poultry meat was studied. The content of free thiol groups (–SH), thermal drip and gel texture were determined. After pre-incubation at 7–8 °C for 1, 3, 5 and 24 h, the samples were subjected to one-step heating at 50, 60, 70 and 80 °C and two-step heating at 50/80, 55/80 and 60/80 °C. The addition of preparation and the extension of pre-incubation time led to decrease of free -SH groups content. After heating, the number of thiol groups decreased, the texture was improved, but thermal drip from gels increased. The amount of –SH groups in gel extracts subjected to one-step heating decreased with simultaneous increase of mechanical strength of gels. Protein gels subjected to two-step heating exhibited higher firmness than gels subjected to one-step heating. Thus, the 3 g/L addition of transglutaminase preparation in combination with one-step thermal processing at 70 °C and pre-incubation for 3 h contributed to improvement of texture properties of model gels and low thermal drip.     

Characterisation of heat-set milk protein gels

Milk protein solutions [10% protein, 40/60 whey protein/casein ratio containing whey protein concentrate (WPC) and low-heat or high-heat milk protein concentrate (MPC)] containing fat (4% or 14%) and 70–80% water, form gels with interesting textural and functional properties if heated at high temperatures (90 °C, 15 min; 110 °C, 20 min) without stirring. Adjustment of pH before heating (HCl or glucono-δ-lactone) produces soft, spoonable gels at pH 6.25–6.6, but very firm, cuttable gels at pH 5.25–6.0. Gels made with low-heat MPC, WPC and low fat gave some syneresis; high-fat gels were slightly firmer than low-fat gels. Citrate markedly reduced gel firmness; adding calcium had little effect on firmness, but increased syneresis of low-heat MPC/WPC gels. The gels showed resistance to melting, and could be boiled or fried without flowing. Microstructural analysis indicated a network structure of casein micelles and fat globules interlinked by denatured whey proteins.     

Short communication: Effects of nanofiltration and evaporation on the gel properties of milk protein concentrates with different preheat treatments

This study aimed to evaluate the effects of different concentration methods (nanofiltration and evaporation) and heat treatments on the gel properties of milk protein concentrate (MPC). The MPC gels were produced using glucono-δ-lactone (GDL) as an acidifier with different preheat treatments (30 min at 80°C and 5 min at 92°C). We then evaluated the effect of preheat treatments on MPC gel properties, including storage modulus (G′), loss tangent (tan δ), firmness, whey separation, and microstructure. The results indicated that without preheating, evaporation (EP)-MPC had higher G′ and firmness, and lower tan δ and whey separation than nanofiltration (NF)-MPC. These results suggest that EP-MPC produced a better acid-induced gel than NF-MPC when no preheat treatments were performed. After preheating, however, except for a very small difference in the final G′ (EP-MPC was higher), the 2 MPC did not differ significantly in firmness, final tan δ, or whey separation. Additionally, compared with the gel of unheated MPC, both preheat-treated gels (NF-MPC and EP-MPC) achieved increased G′ and firmness and decreased tan δ and whey separation. The preheat-treated MPC also displayed a more flexible-stranded network. These findings demonstrate that, given a suitable heating treatment, NF-MPC compares favorably with EP-MPC in achieving desired gel properties.     

Heat-induced gelation of actomyosin

Rheological properties of actomyosin gels were markedly affected by protein concentration, pH and heating temperature. Gel strength increased with increasing protein concentration (30-60 mg ml(-1)) and heating temperature (55-75°C), but decreased with increasing pH (5·5-9·0). Low heating temperatures (50-55°C) favoured the formation of more cohesive actomyosin gels than the higher heating temperatures (60-75°C). Gels formed at low pH (5·5 and 6·0) were less cohesive than those formed at high pH (7·5-9·0). Addition of ATP and pyrophosphate (10 mm) prior to heating decreased gel strength and cohesiveness, whereas EDTA (1-5 mM) reduced gel strength but did not affect gel cohesiveness.     

Novel foods with microalgal ingredients – Effect of gel setting conditions on the linear viscoelasticity of Spirulina and Haematococcus gels

Microalgae represent an alternative and innovative source of natural ingredients that can be used in the development of novel food products. Biologically active compounds (e.g. carotenoids) are naturally encapsulated within microalgal cells, being able to resist harsh technological conditions involved in food technology processes. The aim of this work was to study the effect of adding Haematococcus pluvialis and Spirulina maxima microalgal biomass on the linear viscoelastic behaviour of vegetarian food gels prepared from pea protein, κ-carrageenan and starch. The gelation process was monitored in situ through dynamic oscillatory measurements, under different thermal profile conditions. Increasing temperature (70–90 °C, 5 min) resulted in more structured gels, while the effect of time (5–30 min, at 90 °C) was less pronounced. The effect of heating and cooling rates on gel setting was also studied. Haematococcus gels were highly structured and less dependent on gel setting conditions. Spirulina gels presented lower values of viscoelastic functions than the control (gel matrix without microalgae), but this was overcome when using lower heating/cooling rates.     

The effect of pH on gel structures produced using protein–polysaccharide phase separation and network inversion

Forming heat-induced gels through combined effects of micro-phase separation of whey protein isolate (WPI; 5%, w/v, 100 mm NaCl) by pH change (5.5, 6.0, and 6.5), and addition of κ-carrageenan (0–0.3%, w/w), were evaluated. The microstructure of WPI gels was homogeneous at pH 6.0 and 6.5 and micro-phase separated at pH 5.5. Addition of 0.075% κ-carrageenan to WPI solutions caused the microstructure of the gel to switch from homogeneous (pH 6.0 and 6.5) to micro-phase separated; and higher concentrations led to inversion of the continuous network from protein to κ-carrageenan. Protein solutions containing 0.075% (w/w) κ-carrageenan produced gels with increased storage modulus (G′) at pH 6.5 and decreased G′ at pH 5.5. All gels containing 0.3% (w/w) κ-carrageenan had κ-carrageenan-continuous networks. It was shown that microstructural and rheological changes were different in WPI and κ-carrageenan mixed gels when micro-phase separation was caused by pH rather than ionic strength.     

Interaction of Myosin-Albumin and Myosin-Fibrinogen to Form Protein Gels

Myosin, fibrinogen, albumin, myosin-fibrinogen and myosin-albumin gels were formed by heating in pH 6.0 phosphate buffer at two heating rates. Gel strength was measured with an annular pump and soluble protein was determined. Myosin and fibrinogen interacted to form a gel which was stronger than the sum of the gel strengths for the two individual proteins. The strength of myosin-fibrinogen gels formed at 50°C was not affected by heating method; however, the strength of gels developed between 55°C and 70°C was related to heating method. Myosin and albumin did not interact to form a gel matrix until 80°C where sufficient thermal alteration of albumin had occurred.     

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.     

The effect of shear on the rheology and structure of heat‐induced whey protein gels

The influence of mechanical shearing on the small deformation properties and microstructure of heat‐induced whey protein gel has been studied. The viscoelastic properties of these gels at different concentrations of 10% and 20% (w/w) exposed to different shear rates of 0, 50, 100, 200 and 500 s^?1 during gelation were measured using dynamic oscillatory rheometry. The structure of both the shear treated and unsheared gels was then investigated using light microscopy. The results showed that the storage modulus of the gels at both concentrations was increased by increasing the shear rate exposure during gelation while the shear‐treated gels were more elastic and showed frequency‐independent behaviour. As the total protein concentration of the gel increased, the viscoelastic properties of the gels also increased significantly and the gels showed greater elasticity. The gels obtained from the higher shear rate exposure were stronger with higher elastic moduli at both protein concentrations. Images of the gels obtained using light microscopy showed that shearing resulted in phase separation and some aggregation in the structure of the gels at both concentrations. However, the shearing rates applied in this study were not enough to cause aggregation breakdown in the gel network.     

Improvement of rheological properties of firm acid gels by skim milk heating is conserved after stirring

The enhancement of the strength of set acid gels by heating milk was related to rheological parameters (water retention capacity, storage modulus) of corresponding stirred gels. To obtain accurate rheological data from stirred gel it was necessary to maintain a constant granulometry of gel particles and to recognize time after stirring as a contributing factor. Two hours after stirring, the gel exhibited a higher storage modulus when milk was heated above 80 degrees C. A measurement of viscosity of just-stirred yoghurt was sufficient to predict correctly the quality of a stirred gel analysed by viscoelastic measurements. Increased resistance to syneresis of just-stirred gels was related to higher viscosity. The quantity of beta-lactoglobulin (beta-Ig) bound to casein micelles explains the improvement of these gel qualities. We have considered that the structure of the initial firm gel (mesostructure level) was conserved in fragments within the stirred gel. Consequently, the explanation given by various authors for the effect of heating milk on the properties of set gels can also be applied to stirred gels. The same mechanism, described in literature for structure formation of set gels from acidified milk is purposed to explain the role of heating milk on the recovery of gel structure after stirring. The beta-Ig association with casein micelles during heating favoured micelle connections during the acidification. It also favoured the association of gel fragments after stirring during the recovery in gel structure.     

Utilizing whey protein isolate and polysaccharide complexes to stabilize aerated dairy gels

Heated soluble complexes of whey protein isolate (WPI) with polysaccharides may be used to modify the properties of aerated dairy gels, which could be formulated into novel-textured high-protein desserts. The objective of this study was to determine the effect of polysaccharide charge density and concentration within a WPI-polysaccharide complex on the physical properties of aerated gels. Three polysaccharides having different degrees of charge density were chosen: low-methoxyl pectin, high-methoxyl type D pectin, and guar gum. Heated complexes were prepared by heating the mixed dispersions (8% protein, 0 to 1% polysaccharide) at pH 7. To form aerated gels, 2% glucono-δ-lactone was added to the dispersions of skim milk powder and heated complex and foam was generated by whipping with a handheld frother. The foam set into a gel as the glucono-δ-lactone acidified to a final pH of 4.5. The aerated gels were evaluated for overrun, drainage, gel strength, and viscoelastic properties. Without heated complexes, stable aerated gels could not be formed. Overrun of aerated gel decreased (up to 73%) as polysaccharide concentration increased from 0.105 to 0.315% due to increased viscosity, which limited air incorporation. A negative relationship was found between percent drainage and dispersion viscosity. However, plotting of drainage against dispersion viscosity separated by polysaccharide type revealed that drainage decreased most in samples with high-charge-density, low-methoxyl pectin followed by those with low-charge-density, high-methoxyl type D pectin. Aerated gels with guar gum (no charge) did not show improvement to stability. Rheological results showed no significant difference in gelation time among samples; therefore, stronger interactions between WPI and high-charge-density polysaccharide were likely responsible for increased stability. Stable dairy aerated gels can be created from WPI-polysaccharide complexes. High-charge-density polysaccharides, at concentrations that provide adequate viscosity, are needed to achieve stability while also maintaining dispersion overrun capabilities.     

β-Lactoglobulin Gelation and Modification: Effect of Selected Acidulants and Heating Conditions

ABSTRACT: The effect of acidulant selection, heating temperature, and heating rate on the properties of low-pH β-lactoglobulin (β-Lg) gels and powders derived from these gels was investigated by rheological and microscopic techniques. As isothermal gelation temperature was increased from 75 to 85 °C, gels made with hydrochloric and lactic acid showed more rapid gel formation and increased stress at gel fracture. Thickening and water-holding properties of powders derived from these gels also increased with temperature. Increases in gel strength and derivatized powder functionality appeared to plateau above 85 °C. Gels and derivatized powders prepared with phosphoric acid exhibited attributes similar to samples prepared with HCl and lactic acid at lower temperatures. The ion-specific ability of phosphate to increase denaturation temperature was responsible for the shift in properties of gels made with phosphoric acid. Microscopy revealed temperature effects on network building block size, but variations in rheological properties could not be linked to changes in gel micrographs. Alteration of heating rates from 2.0 to 0.2 °C/min during gelation affected the observed gelation temperature, but had little effect on final gel mechanical properties. Acid selection and gelation temperature offer alternatives to control β-Lg gel strength and the functional properties of instant thickening protein ingredients.     

Interactions between sodium caseinate and LBG in acidified systems: Rheology and phase behavior

The behavior of model systems formed by different concentration of sodium caseinate and locust bean gum (LBG) was investigated using confocal microscopy (CSLM) and rheological measurements in order to determine the interaction between these two ingredients in acidified dairy products. A visual phase diagram was constructed at different biopolymer concentrations and four different types of systems were observed for the mixtures in the isoelectric point of protein: one-phase gels (G1P), two-phase gels (G2P), one-phase liquid mixtures (L1P) and two-phase liquid mixtures (L2P). These different systems resulted from the different kinetics of phase separation and gel formation. The one-phase systems (gels and liquid mixtures) showed a micro-phase separation when visualized by CSLM, although they were homogeneous at macroscopic level. In a general way, the micro or macro phase separation led to a biopolymer concentration on separated phases, resulting in an increase of the stress at fracture for the gels or the viscosity for the liquid mixtures. However, the behavior of the whole system did not correspond to the sum of the mechanical properties of separated phases.     

Effect of Heating Rate and Protein Concentration on Gel Strength and Water Loss of Muscle Protein Gels

The effects of heating rates (17, 38 and 85°C/hr) and protein concentration (10, 20, 30, 40, and 50 mg/mL) on gels made of extracted salt-soluble proteins were evaluated. Gel strength decreased when heating rate was increased. A greater water loss from compressed gels occurred at protein concentrations of 10 and 20 mg/mL than at 30 to 50 mg/mL Protein loss in the expelled water, after compression, was less for the slower heating rates while the total amount of protein in the expelled water increased with increasing protein concentration in the system. SDS gel electrophoresis demonstrated a change in some of the expelled proteins at different heating rates.     

Effects of added whey protein aggregates on textural and microstructural properties of acidified milk model systems

Non-fat milk model systems containing 5% total protein were investigated with addition of micro- or nanoparticulated whey protein at two levels of casein (2.5% and 3.5%, w/w). The systems were subjected to homogenisation (20 MPa), heat treatment (90 °C for 5 min) and chemical (glucono-delta-lactone) acidification to pH 4.6 and characterised in terms of denaturation degree of whey protein, particle size, textural properties, rheology and microstructure. The model systems with nanoparticulated whey protein exhibited significant larger particle size after heating and provided acid gels with higher firmness and viscosity, faster gelation and lower syneresis and a denser microstructure. In contrast, microparticulated whey protein appeared to only weakly interact with other proteins present and resulted in a protein network with low connectivity in the resulting gels. Increasing the casein/whey protein ratio did not decrease the gel strength in the acidified milk model systems with added whey protein aggregates.