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The microstructural and large deformation rheological properties of model food gels were studied by performing notch propagation tensile testing on the gels using a tensile stage and observing changes in the microstructure of the gels during tensile testing using confocal laser scanning microscopy (CLSM). Heat-set whey protein (WP) gels containing either added sodium caseinate (NaCN) or sunflower oil droplets emulsified with WP or NaCN as the emulsifier protein were prepared in 0 or 50 mM NaCl. The WP gel structure strengthened in the presence of added NaCl and NaCN. The rheological properties of WP gels containing sunflower oil droplets emulsified with WP or NaCN were influenced by the NaCl concentration, oil concentration and extent of oil droplet aggregation in the gel or by the type of emulsifier protein used. During tensile testing, the notch length in all gels increased above a certain critical stress, leading to fracture of the gels through the notch. Also, the microstructural changes in the oil phase of emulsion filled gels subjected to tensile testing were influenced by the structural properties of the WP gel matrix and the proximity of the oil droplet to the fracture path.

In the initial part of this study, the high temperature (85 °C) microscopic phase behaviour of β-lactoglobulin (0.4–6%, w/w)–konjac (0.05–0.75%, w/w) mixtures containing 50 mM NaCl was established using confocal laser scanning microscopy (CLSM). Also, the effects of heating time (heating temperature: 78 °C) and NaCl concentration (0–75 mM) on protein denaturation kinetics and the phase behaviour in 2%, w/w, β-lactoglobulin–0.4%, w/w, konjac mixtures were investigated using turbidimetry, protein denaturation measurement, CLSM and image analysis techniques. Segregative phase separation occurred in heat-treated β-lactoglobulin–konjac mixtures containing biopolymer and NaCl concentrations exceeding certain critical levels, due to heat and NaCl induced β-lactoglobulin denaturation/aggregation. The microstructural properties of selected heated (to 85 °C for 30 min) and cooled (to 25 °C) β-lactoglobulin–konjac mixtures containing different NaCl levels were studied using CLSM and rheological measurements and the results showed that the microstructure can be distinguished as miscible, phase separated or phase separated containing stable protein inclusions dependent on NaCl concentration. Response surface methodology was used to determine the minimum NaCl concentrations required for phase separation and for formation of phase separated systems containing stable inclusions in a wide concentration range of heated and cooled β-lactoglobulin (0.8–2%, w/w)–konjac (0.2–0.75%, w/w) mixtures. The results show that the microstructural and rheological properties of β-lactoglobulin–konjac mixtures can be controlled by selecting appropriate mixture biopolymer and NaCl concentrations and heating conditions.

The aim of this study was to evaluate the suitability of Methylene Blue (MB) and Vitamin B 12 (Vit-B 12 ) as water soluble inner aqueous phase (W 1 ) markers for measuring the encapsulation efficiency and stability of water-in-oil-in-water (W 1 /O/W 2 ) double emulsions stabilized by sodium caseinate (NaCN). The encapsulation efficiency and stability were determined by centrifugation of the double emulsion to separate the cream phase (W 1 /O) and the outer aqueous phase (W 2 ) and measuring the concentration of marker in W 2 by absorbance spectrophotometry. To validate this method the marker concentration measurable and the stability of the marker in W 2 were measured. Both markers could be accurately measured in W 2 and there was no change in the concentration of marker on storage of a W 2 solution for 7 days at 45 °C. The recovery yields of MB and Vit-B 12 in the recovered W 2 of an oil-in-water (O/W 2 ) emulsion, determined using the procedure normally used for measuring encapsulation efficiency and stability, were ∼78% and ∼99%, respectively, and ∼52 and ∼100%, respectively. Double emulsions had encapsulation efficiency of 61.9 ± 21.4% and 16.6 ± 1.1% and encapsulation stability of 62.0 ± 22.6% and 10.7 ± 0.7% for MB and Vit-B 12 , respectively. Recovery yield and encapsulation efficiency/stability data for MB indicate that it is not a suitable marker for measuring the encapsulation properties of NaCN stabilized double emulsions while similar data for Vit-B 12 indicate that it is a suitable marker for studying the encapsulation properties of double emulsions stabilized with NaCN. Methods used in other studies to measure encapsulation properties of double emulsions are discussed in light of the results obtained in this study.

Gelling mixtures of Na + κ -carrageenan with whey protein isolate (WPI) at pH 7.0 have been studied rheologically and by differential scanning calorimetry (DSC), with comparative measurements for the individual constituents of the mixtures. The concentration of WPI was held fixed at 10.0 wt% and carrageenan concentration was varied in the range 0.05–3.0 wt%. Ca 2+ cations, which have been shown previously to be particularly effective in inducing gelation of κ -carrageenan, were introduced as CaCl 2 . The concentration of CaCl 2 used in most of the experiments was 8 mM, but other concentrations were also studied. Mixtures were prepared in the solution state at 45 °C, and showed no evidence of either phase separation or complex formation. Rheological changes were monitored by low-amplitude oscillatory measurements of storage modulus, G ′, during (i) cooling (1 °C/min) and holding at 5 °C, to induce gelation of the carrageenan in the presence of non-gelled WPI; (ii) heating and holding at 80 °C to dissociate the carrageenan network and induce gelation of WPI; (iii) cooling and holding again at 5 °C, to give composite networks with both components gelled; and (iv) re-heating to 80 °C to dissociate the carrageenan network. Gel structure was characterised further by creep–recovery measurements at the end of each holding period, and by torsion measurements at 5 °C, before and after thermal gelation of WPI. In 8 mM CaCl 2 , the presence of 10.0 wt% WPI promoted earlier gelation of κ -carrageenan during cooling, and displaced the disorder–order transition in DSC to higher temperature. These changes are attributed to segregative interaction with non-gelled WPI. The resulting networks, however, were weaker that those formed by κ -carrageenan alone, which was traced to binding of Ca 2+ cations to the WPI. At higher concentrations of CaCl 2 , where availability of Ca 2+ cations was no longer a limiting factor in gelation of the κ -carrageenan, the effect of segregative interactions could be seen as an increase in gel strength. On heating to gel the WPI, the presence of κ -carrageenan led to the earlier appearance of an aggregation exotherm in DSC and to network formation at shorter times, which can similarly be explained by enhanced self-association of denatured WPI in response to segregative interactions with disordered carrageenan. An initial large (∼10 fold) increase in gel strength with increasing concentration of carrageenan to ∼0.25 wt% was followed by a reduction at higher concentrations, attributed to excessive aggregation (incipient precipitation) of the WPI. The magnitude of the changes in G ′ observed on subsequent cooling suggest that gelation of κ -carrageenan within the pores of an existing network of gelled WPI gives rise to a bicontinuous co-gel structure. Comparison of moduli at 80 °C before and after cooling to 5 °C demonstrated that formation and dissociation of the carrageenan network caused no disruption of the network of gelled WPI.

The effect of two common emulsifiers, lecithin and monoglycerides, on the heat stability of a model infant formula emulsion was investigated. During homogenization the droplet diameter of the emulsion was more effectively reduced by monoglycerides than lecithin. The maximum in the heat coagulation time (HCT)-pH profile at 140 °C of the emulsion with no added emulsifier coincided with the unadjusted pH (∼6.8). Addition of lecithin (0–5 g l −1 ) progressively increased the maximum heat stability of the emulsion from 18 to 25 min and at 3–5 g l −1 slightly shifted the maximum in the HCT-pH profile to more acidic pH values. The addition of monoglycerides on the other hand decreased the heat stability; the emulsion containing 5 g l −1 had a maximum heat stability of 12 min. Particle size distribution and average particle diameter ( D [4,3]) measurements following heating confirmed that lecithin conferred stability to heat-induced aggregation while monoglyceride destabilized the emulsions. The zeta potential of oil droplets/particles in the emulsions became increasingly less negative with increasing monoglycerides concentration which is indicative of protein displacement; this may explain the low heat stability of the monoglyceride containing emulsions. The addition of lecithin on the other hand had no effect on zeta potential possibly because negatively charged lecithin replaced interfacial protein or interacted with interfacial protein. Progressively, less hydrophobic sites were available for binding of a hydrophobic probe as the concentration of either surfactant increased; interaction of surfactant with exposed hydrophobic regions of aqueous phase and/or interfacial protein is a possible explanation and this type of interaction may explain the high heat stability of lecithin containing emulsions.

The effect of the non-gelling polysaccharide guar gum on thermal gelation of whey protein isolate (WPI) was investigated by compression testing, oscillatory rheology and differential scanning calorimetry (DSC). All samples were prepared in 100 mM NaCl, with the concentration of WPI held constant at 3.0 wt%. Under these conditions, DSC heating scans (Setaram microcalorimeter; sample mass ≈ 850 mg; 1 °C/min) for WPI in the absence of guar gum showed an endothermic denaturation process with maximum heat flow at ∼76 °C and an exothermic aggregation process at ∼87 °C. On incorporation of increasing amounts of guar gum (0.0–0.5 wt%) the exotherm moved to progressively lower temperature, until eventually, at 0.5 wt% guar gum, it was obscured by the more intense denaturation endotherm. This is attributed to segregative interactions (“thermodynamic incompatibility”) with guar gum promoting aggregation of thermally denatured whey protein. After heating to 80 °C and cooling to induce gelation of the whey protein, Young's modulus ( E ) at 20 °C rose to a maximum at ∼0.05 wt% added guar gum and decreased at higher concentrations, until at 0.5 wt% guar gum the mixture was liquid-like. A similar initial increase and subsequent decrease with increasing concentration of guar gum was observed by oscillatory measurements (1 rad s −1 ; 0.5% strain) of storage modulus ( G ′), but the maximum occurred at ∼0.1 wt% guar gum. The difference in optimum concentration is ascribed to use of a different time–temperature regime in preparing the samples for compression testing. The reduction in gel strength observed by both techniques at high concentrations of guar gum is attributed to excessive aggregation of WPI in response to segregative interactions, with consequent collapse of the crosslinked network. However, at the very low optimum concentration of ∼0.1 wt% guar gum a large (∼12-fold) enhancement in gel strength ( G ′) was evident in comparison to WPI alone.

Polymerisation of whey proteins increases their potential use as food ingredients. In this investigation, mixtures of 6 wt% whey protein isolate (WPI) with 1–4 wt% type B (alkaline extract) gelatin, before and after polymerisation of WPI (heating to 85 °C and quenching to 5 °C), were characterised by measurements of solution viscosity at 45 °C (above the gelation temperature of gelatin) and by changes in optical rotation and turbidity (measured as optical density at 600 nm) over the temperature range 45–5 °C. Viscosities for mixtures of gelatin with un-polymerised WPI were only slightly higher than for gelatin alone, but after polymerisation they rose steeply above those of the individual components as the gelatin concentration was increased, giving values of 1.84 mPa s for 6 wt% polymerised WPI, 4.07 mPa s for 4 wt% gelatin, and 36.5 mPa s for the mixture of these. The large enhancements in viscosity are attributed to self-association of polymerised WPI in response to segregative interactions with gelatin. On cooling to 5 °C to gel the gelatin component, the unheated mixtures containing un-polymerised WPI showed only slight increases in turbidity, similar to those observed for WPI alone (polymerised or un-polymerised). Cooled mixtures of gelatin and polymerised WPI, however, gave very large increases in turbidity, which were fully reversible on re-heating to 45 °C. Optical rotation measurements showed no indication of direct association between gelatin and polymerised WPI at any temperature in the range studied (45–5 °C). It is suggested that association of gelatin molecules in the early stages of gelation triggers phase separation, giving a continuous matrix of gelatin gel with the polymerised whey protein dispersed through it in small droplets (which increase turbidity by scattering light).

Co-gels of whey protein isolate (WPI) and phosphate-crosslinked waxy maize starch (PCS) were formed by heating from 50 to 80 °C at 1 °C/min, holding for 30 min at 80 °C, cooling to 20 °C at 1 °C/min and holding for 30 min at 20 °C, with measurement of storage modulus (G′) and loss modulus (G″) at 1 rad s−1 and 0.5% strain throughout. G′ and G″ remained essentially constant on holding at 20 °C, and average values of log G′ during the holding period were used in analysis of co-gel moduli by the Takayanagi isostrain and isostress blending laws. WPI in the absence of starch and PCS in the absence of WPI were subjected to the same time–temperature regime as the mixtures, to provide calibration curves for use in the analyses. As anticipated, WPI and PCS showed independent thermal transitions in DSC heating scans, with no indication of any associative interactions between the two materials. During cooling, the mixtures showed appreciable increases in G′, similar to those observed for WPI alone, whereas slight decreases were observed for PCS, indicating a continuous WPI phase and dispersed starch phase. The magnitude of the increase in G′ observed on gelation of WPI also indicated development of a continuous protein network. Blending law analyses in which the starch phase was assumed to consist of fully swollen PCS granules dispersed individually through a WPI gel matrix were unsuccessful in matching the observed values of log G′ for the composites, irrespective of whether the swelling volume of PCS was held fixed at the value of Q≈11.9 mL/g determined experimentally by the polymer-exclusion method using blue dextran, or treated as a variable parameter, along with the modulus of individual swollen granules, which was varied in both analyses. However, an analysis in which PCS was regarded as a second gelling polymer gave a high standard of agreement (r2=0.994) between observed and fitted values of log G′ for all mixtures studied (18 in total, with concentrations of PCS ranging from 1 to 5 wt% and concentrations of WPI ranging from 1 to 10 wt%). The only variable in this analysis was the “solvent avidity parameter”, p, defined as the ratio of water to polymer in the (continuous) WPI phase divided by the corresponding ratio for the (dispersed) PCS phase. The best fit was obtained with p≈3.3. This high relative affinity of WPI for water implies restricted swelling of PCS except at low concentrations of either or both components of the mixtures. The changes in G′ observed on heating indicated that at high concentrations of WPI separation into a dispersed starch phase in a continuous protein matrix occurred prior to gelatinisation of PCS. At lower concentrations of WPI, however, gelatinisation of PCS was accompanied by large increases in G′, indicating formation of a starch network which was then fragmented by gelation of WPI. Fragments of starch network (on the length scale of ∼10–100 μm) embedded in a WPI gel matrix were visualised directly by light microscopy.

Gelation of globular proteins on heating involves two separate stages. The first is partial unfolding (denaturation) of the native globular structure; the second is intermolecular aggregation. Denaturation involves dissociation of intramolecular bonds (non-covalent and, in some cases, disulfide) and is therefore an endothermic process. The aggregation step involves formation of new bonds between protein molecules, and would therefore be expected to give a differential scanning calorimetry (DSC) exotherm on heating, but numerous previous studies of the thermal gelation of whey proteins, carried out on conventional (fast scanning) DSC calorimeters (typical sample mass ∼15–50 mg), have shown only endothermic transitions. In the present work, however, we have observed the endothermic (denaturation) and exothermic (aggregation) processes in thermogelation of whey protein isolate (WPI) as separate transitions in DSC heating traces recorded on a Setaram microcalorimeter (sample mass ∼850 mg). Under conditions where aggregation occurs much more slowly than denaturation (low protein concentration; low ionic strength) the two transitions are well resolved, with the exotherm from aggregation following the endotherm from denaturation. The position of the exotherm, however, appears to be time-dependent rather than temperature-dependent. On reduction in heating rate, the apparent peak-maximum temperature of the aggregation exotherm decreases towards the (essentially constant) position of the denaturation endotherm, and, at sufficiently low scan rates, the exotherm becomes obscured by the more intense endotherm. Progressive displacement of the exotherm into the temperature range of the denaturation endotherm also occurs in response to changes that accelerate intermolecular aggregation and accompanying gelation (addition of salt; increasing protein concentration). The absence of a detectable exotherm in previous studies using conventional calorimeters is attributed to the much smaller sample mass than in the Setaram instrument, giving much faster heat transfer, which may cause the exothermic heat flow from the slow aggregation process to be swamped by the endothermic heat flow from the more rapid denaturation process.

The microstructural and rheological properties of glucono-delta-lactone induced acid casein gels made from micellar casein dispersed in water and in lactose-free simulated milk ultrafiltrate (SMUF) containing 40, 70, 100 and 200% of the salts normally present in milk were studied. Confocal scanning laser microscopy (CSLM) and low amplitude oscillatory rheology (LAOR) were used to monitor structural development during acidification. Image analysis of CSLM micrographs was used for measurement of mean apparent equivalent circular diameter of pores, mean volume-weighted size (star volume) of pores and for measurement of homogeneity by grey-level pixel variance. Transmission electron microscopy (TEM) was used to determine mean particle diameter and star volume of protein aggregates within the gel networks. At low salt concentrations, gel networks formed more rapidly with consequently larger pores compared to more slowly forming, homogeneous gels being formed on acidification at higher salt concentrations. TEM analysis showed increased fusion of casein particles as salt concentration decreased, indicating significant destabilisation of micellar caseins. Dynamic LAOR indicated differences in storage modulus and phase angle depending on ionic strength. Results indicated an inverse relationship between pore or particle size and SMUF concentration. Some length-scale dependence was noted for pixel variance and apparent equivalent circular diameter.

The influence of pH on the heat stability characteristics, at 140 °C, of model milk protein ingredient systems and model infant formulae emulsions prepared using reconstituted skim milk powder (RSMP) and reconstituted electrodialysed whey (REDW) was studied. The heat coagulation time (HCT) of RSMP solutions (0.75–3 g protein 100 ml−1) was influenced by pH and protein concentration while the HCT of REDW solutions (0.75–3 g protein 100 ml−1) was largely unaffected by protein concentration and displayed a broad range of high heat stability at pH values of 6.5–7.4. HCT–pH profiles of blends (1.5 g protein 100 ml−1) of RSMP and REDW with whey protein:casein (W:C) ratios in the range 100:0–20:80 were determined; each blend displayed a distinct pH range of maximum stability which was dependent on the W:C ratio. The pH region of high stability coincided with the formation of colloidal casein–β-lg complexes upon heating. Two model infant formulae emulsions (3.5 or 4.2 g fat 100 ml−1) with W:C ratios of 60:40 were prepared by homogenizing oil in two RSMP–REDW blends with different levels of added lactose and CaCl2. Added lactose reduced the heat stability of the blends without altering the HCT–pH profiles while added CaCl2 shifted the pH of maximum heat stability to more alkaline values. The shape of the HCT–pH profile of the emulsions and corresponding fat-free phases were similar, however, the emulsions were slightly less heat stable. These emulsions had unadjusted pH values of 6.72 and 6.59, respectively, and it was necessary to adjust the pH of these emulsions to >6.9 and >7.2, respectively, values coinciding with high heat stability, otherwise aggregation and/or coagulation occurred on heating (140 °C×80 s). A casein dominant (20:80 W:C ratio) model infant formula emulsion (3.5 g fat 100 ml−1) was stable upon heating at pH values in the range 6.5–7.4.

Rennet casein is widely used in the manufacture of Mozzarella cheese analogues. Wide variation in the functional performance of different rennet caseins during the manufacture of such cheese analogues is encountered, resulting in considerable variability in the functional properties of the cheese analogue. Samples of 44 batches of edible rennet caseins were studied for performance in Mozzarella cheese analogue manufacture in two pilot-scale cheese analogue manufacturing processes and scoring systems for performance in the two processes were devised. Rennet casein performance during cheese analogue manufacture was found to vary widely and also differed depending on the pilot-scale process used. Of the protein, moisture and ash contents of the caseins only the ash content showed significant correlation to the performance scores achieved in cheese analogue manufacture, but this was not a suitable parameter for predicting performance. The hydration characteristics of the rennet casein when dispersed in solutions of disodium orthophosphate (DSP) were determined using a rheological method and compared to the performance of the rennet caseins in the two pilot-scale cheese analogue production processes. Wide variations in the maximum viscosity index, in the time taken to reach the maximum viscosity index and in the viscosity stability of rennet caseins dispersed in 0.4% (w/w) DSP were observed. In a non-linear graphical model, the time taken to reach maximum viscosity and the maximum viscosity index reached in 0.4% (w/w) DSP were found to be good indicators of the performance of rennet caseins in both the pilot-scale cheese analogue manufacturing processes. The model, or a suitable development of the model, should prove useful to manufacturers and users of rennet casein alike in assessing the potential performance of different batches of rennet casein in the manufacture of cheese analogues.

Rennet casein is used in the manufacture of Mozzarella cheese analogues. Hot solutions of calcium chelating salts are used to facilitate hydration of the protein which subsequently acts as an emulsifier for the oil phase dispersed in the protein/aqueous phase of the cheese analogue. On cooling, a matrix resembling cheese in texture and other rheological properties is produced. Considerable variations in the functional performance of different rennet caseins during cheese analogue manufacture have been observed. Commercial rennet caseins have been obtained and are being studied to establish the nature and causes of these variations in performance. A simplified model system to study rennet casein hydration was developed. The method involves dispersing rennet caseins in solutions of chelating salt at 55°C with stirring. The changes in viscosity index that occur as the rennet casein hydrated were followed using a controlled shear rate rheometer equipped with a custom designed paddle stirrer. Hydration of the rennet casein particles was found to occur in a number of stages including particle swelling, clumping, network formation and subsequent breakdown of the network. Considerable variations in the times taken for different rennet caseins to swell, clump and reach maximum viscosity index and in the stability of the hydrated protein dispersions were found. The rheological properties of the rennet caseins during hydration were highly dependent on the concentration of the chelating salt in the hydrating solution.

Small strain oscillatory techniques were used to study the effects of added sodium caseinate or casein fractions on the rheology of 1% ι-carrageenan gels containing 0.01 moll dm3 CaCl2 or 0.02 moll dm3 KCl at 5°C. The mechanical spectra (frequency dependence of G′ (storage modulus), G″ (loss modulus) and tan δ (G″/G′)) of all mixed casein/ι-carrageenan systems differed from those of corresponding ι-carrageenan gels. Added κ-casein transformed a relatively weak Ca2+-free ι-carrageenan gel to a strong, elastic gel, indicating increased crosslinking which is attributed to electrostatic interactions between κ-casein and ι-carrageenan. Changes to the rheology of Ca2+-free ι-carrageenan gels on addition of αs- or β-casein are attributed to excluded volume effects, with monomeric β-casein exerting a greater excluded volume effect than aggregated αs-casein. Added αs-or β-casein also altered the rheology of Ca2+-containing ι-carrageenan gels, attributable to Ca2+-mediated interactions of αs- and β-caseins with ι-carrageenan. The effects of these caseins were reduced on dephosphorylation of the caseins indicating the importance of phosphate groups for the interaction of these caseins with ι-carrageenan in systems containing Ca2+. Low levels of sodium caseinate increased both the rigidity and elasticity of Ca2+-free ι-carrageenan gels and Ca2+-containing ι-carrageenan gels. However, higher levels of sodium caseinate reduced elasticity in both cases. Sodium caseinate had a greater effect on the rheology of ι-carrageenan gels in the presence of Ca2+, presumably due to Ca2+-mediated interactions of αs- and β-caseins with ι-carrageenan.

Controlled stress oscillatory rheology was used to monitor the thermoreversible gelation of 1% ι-carrageenan and mixtures of 1% ι-carrageenan and 2% sodium caseinate, αs-, β- or κ-casein. All samples were prepared in a 10 mmol/dm3 imidazole-HCl buffer, pH 7.0, containing 0.01 mol/dm3 CaCl2. The addition of each casein altered the gelation and melting characteristics of ι-carrageenan, with the effects being most pronounced on the addition of κ-casein and least when αs-casein was added. The viscoelastic properties of formed gels were determined at 5°C using both oscillatory and creep techniques. The rigidity of the ι-carrageenan gel, as measured by storage modulus and instantaneous compliance, increased on the addition of the caseins. The rigidity of gels was in the order κ-casein/ι-carrageenan > β-casein/ι-carrageenan > sodium caseinate/ι-carrageenan > αs-casein/ι-carrageenan > ι-carrageenan. The elasticity of the ι-carrageenan gel was more frequency dependent than the elasticity of casein/carrageenan mixed gels.

The effects of increasing levels (0–1.0 mol/dm3) of protein-structuring (chloride, NaCl; sulfate, Na2SO4) and -destructuring anionic ions (thiocyanate, NaSCN) on rheological properties and electron microstructures of protein gels were examined. The effects of the ions varied with concentration and species. At concentrations above 100 mM they enhanced gel strength in the order SCN− > Cl− > SO42−. The hardness of gels increased with increasing concentrations up to 0.1, 0.2 and 1.0 mol/dm3 with SO42−, Cl− and SCN− respectively. Electron microstructures of the gels showed that distribution of the protein in the gel matrix became less ordered with increasing ion concentration.