Effect of pH on the gel properties and secondary structure of fish myosin

The relationships between gel properties and the secondary structures of silver carp myosin were investigated at pH 5.5–9.0 using dynamic rheological measurement, circular dichroism and scanning electron microscopy. The gel properties of fish myosin were strongly pH and temperature dependent. During heating at 1 °C/min, myosin formed gels in the pH range 5.5–7.5, but not at pH 8.0–9.0. α-Helix was the predominant structure at pH 7.0. The α-helix fraction declined with increasing temperature and the pH away from 7.0, whilst the other secondary structure fractions increased. The α-helix structure of myosin was more susceptive to acid-treatment than alkali-treatment. As pH increased, the gelation rate and gel strength decreased, and the water-holding capacity (WHC) showed an increasing trend followed by a plateau. High β-sheet and β-turn fractions prior to heating could improve G′ at 90 °C, but they depressed the WHC. A compact and uniform gel of fish myosin was obtained at pH 7.0.     

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

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

Highly acetylated pectin from cacao pod husks (Theobroma cacao L.) forms gel

A pectin (OP) obtained from cacao pod husk with a high acetyl content, which is a structural feature that could disturb the pectins' gel formation, was able to form gels at low pH and a high sucrose content. Pectin gels (1.32% GalA equivalent, w/w) were prepared at pH 2.5–3.3 in the presence of 60% sucrose (w/w). Rheological analyses were performed to determine the optimal pH for further studies. Next, the OP samples were prepared at pH 2.7 in concentrations ranging from 0.33 to 1.98% GalA (w/w) with 60% sucrose (w/w) and subjected to rheological analysis. Dynamic oscillatory experiments at 25 °C indicated the presence of gels for all of the analysed concentrations. Measurements of the elastic (G′) and viscous (G″) moduli at 25 °C also indicated that increasing the pectin concentration resulted in stronger gels. Rotational experiments revealed a shear-thinning behaviour in which the apparent viscosities of the samples increased as the concentration increased. Although the OP had a high degree of acetylation, this pectin was able to form gels, which suggests its potential for use as a gelling and thickening additive.     

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.     

Rheology and microstructure of myofibrillar protein-plant lipid composite gels: Effect of emulsion droplet size and membrane type

Composite gels were prepared from 2% myofibrillar protein (MP) with 10% imbedded pre-emulsified plant oils (olive and peanut) of various particle sizes at 0.6 M NaCl, pH 6.2. Dynamic rheological testing upon temperature sweeping (20-70 °C at 2 °C/min) showed substantial increases in G′ (elastic modulus) of MP sols/gels with the addition of emulsions, and the G′ increases were inversely related to the emulsion droplet size. Furthermore, gels containing emulsified olive oil had a greater (P < 0.05) hardness than those containing emulsified peanut oil. Regardless of oil types, MP-coated oil droplets exhibited stronger reinforcement of MP gels than Tween 80-stablized oil droplets; the latter composite gels had considerable syneresis. Light microscopy with paraffin sectioning revealed a stable gel structure when filled with protein-coated oil droplets, compared to gels with Tween 80-treated emulsions that showed coalesced oil droplets. These results suggest that rheological characteristics, hardness, texture, and water-holding capacity of MP gels were influenced by type of oils, the nature of the interfacial membrane, and the size of emulsion droplets.     

Dynamic rheological and thermal study of the heat-induced gelation of tomato-seed proteins

The structural aspects of proteins prepared from tomato seeds were studied by dynamic rheology, modulated differential scanning calorimetry (MDSC) and Fourier transform infrared (FTIR) spectroscopy. The critical gel point was determined from storage (G’) and loss (G”) moduli as functions of time and frequency. The findings indicate that tomato-seed proteins can create gels during heat treatment at 90–95 °C. Mechanical spectra of the tomato-seed protein gels were classified as weak gels based on the frequency sweep, complex viscosity (η∗) and tan δ results. Moreover, G’ and G” changes were found to be dependent on both concentration and frequency. MDSC showed two peaks, with gelation temperatures (T_d) of 70 and 87 °C, which we attribute to starch and globular protein fractions, respectively. The FTIR spectra show an increase in the absorbance of amides I and II after increasing the solid content from 1 to 10% w/w, indicating mostly β-sheet and α-helical conformations.     

Starch–cassia gum interactions: A microstructure – Rheology study

We present for the first time the interactions of starch and cassia gum – a novel galactomannan recently approved for use in food processing. Viscoelastic, pasting and microstructural characterization of various starches (waxy; high amylose; normal; cross-linked waxy corn starch; potato starch) containing different levels of the cassia gum was carried out. Significant changes were observed in the morphology of granule remnants formed during gelatinization in the starch pastes prepared with and without the addition of cassia gum. The freeze-dried starch–cassia gum pastes presented a shrunken and tight arrangement of the starch granule remnants, when studied by scanning electron microscopy. A significant reduction in the granule remnant size was also calculated using laser diffraction particle size analysis. The extent of interaction with cassia gum differed significantly among the various starch types. All the unmodified corn starches recorded an increase in peak viscosity at all levels of the cassia gum addition. An increase in the final viscosity of these starches was also observed by the addition of cassia gum, with high amylose and normal corn starch showing the maximum. Similarly, the extent of breakdown and setback viscosity also differed among the different starch types. Ranges of dynamic rheological measurements (temperature, time and frequency sweeps) were performed within the viscoelastic zones. Rheological parameters, such as storage modulus (G′), loss modulus (G″) and the gelatinization temperature (T_gel), of the corn starches during the heating cycle were observed to increase, when cassia gum was present at lower levels. The starch–gum systems also exhibited higher tan δ values during both the heating and the cooling cycles, indicating the dominance of the viscous modulus. The G′ and G″ of all the corn starch gels containing cassia gum showed higher values throughout the frequency sweep range. However, the increase in G′ and G″ of different starches was not always consistent with the increase in cassia gum levels. The changes in rheological behaviour during storage of the starch gels, aged on the plate of the rheometer and then studied through time sweeps at 5 °C and frequency sweeps at 25 °C, suggested that the starch gels containing cassia gum had less pronounced changes in the rheological parameters than had their control counterparts.     

Viscoelastic properties of high pressure and heat induced tofu gels

Tofu gels were rheologically examined to determine their storage or elastic (G′) and loss or viscous (G″) moduli as a function of frequency within their linear viscoelastic limits. The tofu gels were made using either glucono-δ-lactone (GDL) or calcium sulphate (CaSO₄·2H₂O), followed by either heat treatment (heated soymilk at ?97 °C prior to coagulation and subsequently held at 70 °C for 60 min, HT) or high pressure treatment (400 MPa at 20 °C for 10 min, HP). The overall moduli values of the GDL gels and CaSO₄·2H₂O gels of both physical treatments were similar, each gave frequency profiles expected for weak viscoelastic materials. However, although both temperature and high pressure treatments could be used to produce tofu gels, the final products were not the same. Pressure formed gels, despite having a higher overall “consistency” (increasing values of their moduli), had a proportionately higher contribution from the loss modulus (increased tan δ). Differences could also be observed using confocal scanning laser microscopy. While such treatment may give rise to differing systems/structures, with new or modified organoleptic properties, the more “open” structures obtained by pressure treatment may well cause processing difficulties if subsequent reworking or moulding is required.     

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.     

Effect of storage time and temperature of milk protein concentrate (MPC85) on the renneting properties of skim milk fortified with MPC85

This study investigated the effect of storage temperature (20–50 °C) and time (0–60 days) on the renneting properties of milk protein concentrate with 85% protein (MPC85). Reconstituted skim milk was fortified with the MPC85 (2.5% w/w) and the renneting properties of the skim milk/MPC85 systems were investigated using rheology. It was found that the final complex modulus (final G∗) and the yield stress of the rennet-induced skim milk/MPC85 gels decreased exponentially with storage time of the MPC85 for storage temperatures greater than 20 °C, with a greater effect at the higher storage temperatures. Changes in the solubility of MPC85 with storage time were correlated with the rheological properties. The primary phase of renneting (cleavage of κ-casein) was not affected by the storage of the MPC85; hence the effect was related to the secondary stage of renneting (aggregation/coagulation of rennet-treated casein micelles). Using a temperature–time superposition method, a master curve was formed from the final G∗, yield stress and solubility results. This suggested that the same physical processes affected the solubility and rennet gelation properties of the milks. It is proposed that the MPC85 protein in rennet-treated skim milk/MPC85 solutions may transform from an interacting material, when solubility is high, to an inert or weakly interacting material, when solubility is low, and that this results in the reduced final G∗ and yield stress of the rennet gels when MPC85 is stored at elevated temperatures for long periods.     

Rheological,thermal and structural behavior of poly(ε-caprolactone) and nanoclay blended films

Poly(ε-caprolactone)/nanoclay composite (PCLNC) films were prepared by solvent casting method using a wide range of layered silicate (2.5–10%) and were characterized by different techniques. Nanofiller dispersions in PCL matrix were studied by wide-angle X-ray diffraction (XRD) and transmission electron microscopy (TEM), and results indicated the formation of some intercalated nanostructure of PCLNC. Rheological and thermal properties of PCLNC were measured by parallel-plate oscillatory rheometry and differential scanning calorimetry (DSC), respectively. Rheological study indicated that the predominating liquid-like properties (viscous modulus, G″ > elastic modulus, G′) of neat PCL gradually transformed to solid-like (G′ > G″) behavior after incorporation of clay in the temperature range of 90–120 °C. A plot of G′ vs. G″ provide information on intercalation and microstructure of nanocomposite. Applicability of time–temperature superposition (TTS) principle and van Gurp–Palmen plot (phase angle vs. absolute complex modulus) on rheological data of clay incorporated PCL were employed and found that the results failed to follow the rules. Incorporation of the nanoclay into PCL matrix increased the crystallization temperature (T_c) and melting temperature (T_m) of neat PCL from 28.7 to 32.3 °C and 56.3 to 59.2 °C, respectively due to the nucleating effect, but the glass transition temperature (T_g) (≈−65 °C) was remained unaffected. The decrease in crystallinity with increase in clay concentration was confirmed by both XRD and DSC data.     

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.     

Conformational changes and dynamic rheological properties of fish sarcoplasmic proteins treated at various pHs

The conformational changes and rheological properties of soluble sarcoplasmic proteins isolated from striped catfish (Pangasius hypophthalmus), treated at various pHs (2–12), were investigated. Isoelectric point of striped catfish sarcoplasmic proteins was determined to be pH 5. SDS–PAGE of sarcoplasmic proteins treated at various pHs, showed molecular masses ranging from 11 to 97 kDa. Most sarcoplasmic proteins, regardless of treated pHs, showed a molecular mass of 43 kDa. A decrease in total sulfhydryl content was observed when the pH was shifted away from 6, indicating disulfide formation at pH lower and higher than 6. Gradual increases of S₀-ANS and S₀-PRODAN were observed as pH increased from 6 to 12, indicating the unfolding of sarcoplasmic proteins during alkaline extraction. DSC thermograms of sarcoplasmic proteins treated at pH 5–9 exhibited an exothermic transition peak, probably due to disulfide bond formation, and/or hydrophobic interactions, which was highly related to the onset temperature of G′ rising. Gel network formation of sarcoplasmic proteins did not take place at extreme pHs (<4 or >9) where proteins were highly charged while the viscoelastic properties of sarcoplasmic proteins were observed at pH 5.5–9. The highest G′ value at 90 °C was observed at pH 5.5 and 8 (P ? 0.05). The gel point, a temperature at which G′ = G″, increased to higher temperature as pH was shifted away from 7.     

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.     

Small and large deformation viscoelastic behaviour of selected fibre blends with gelling properties

Dietary fibres can be used as valuable functional ingredients in baked goods, as thickeners and gelling agents as a result of their ability to modify the structural properties of the matrix in which they are embedded. Viscoelastic behaviour of 12 selected gel–fibre blends (carboxymethylcellulose, hydroxypropylmethylcellulose, locust bean gum, high ester pectin, fructo-oligosaccharide and gluco-oligosaccharide) prepared at 10% concentration (w/v) was investigated at 25 °C and 95 °C by applying both fundamental and empirical rheological techniques to explore their usefulness/suitability as structural ingredients in diluted and weakened baking systems such as gluten free matrices. Mechanical and thermo-mechanical properties were recorded by using a controlled stress rheometer, measuring the storage modulus (G′), the loss modulus (G″) and the complex viscosity (η*). Textural characteristics were assessed by using a TAXTplus Texture Analyser with different attachments. Penetration and back extrusion tests were used for solid and liquid-like samples, respectively. The overall results indicated that (i) carboxymethylcellulose and pectin formed the strongest and the weakest gels, respectively, and that (ii) temperature had a significant effect on gel strength improvement especially for locust bean gum. A 30% substitution of hydrated fibres (cellulose derivates, galactomanans and high ester pectin) by prebiotics (fructo-oligosaccharides and gluco-oligosaccharides) led to a significant decrease of gel structure rigidity when compared to an identical system without prebiotic addition. Only locust bean gum exhibited an opposite behaviour inducing an increase in values of both dynamic moduli (G′ and G″) and static hardness. Significant relationships between dynamic (rheometry) and static (texture analysis) methods were found. Strengthening and structuring ability of some fibre blend gels endorsed them to be used as promising functional ingredients to make gluten-free bread by using low cost thickeners' agents.     

Characterization of fish gelatin from surimi processing wastes: Thermal analysis and effect of transglutaminase on gel properties

Fish gelatin extraction from wastes of fish Herring species (Tenualosa ilisha) was carried out by a series of pretreatment with 0.2 M Ca(OH)₂ followed by 0.1 M citric acid and final water extraction at 50 °C for 3 h. The resulting fish gelatin preparation was evaluated for its dynamic viscoelastic properties, gelling and melting temperatures and gel strength. The gelling and melting temperatures of gelatin samples (at 6.67%, w/v) were obtained from differential scanning calorimetry and rheological studies. The melting temperature of extracted fish gelatin (EFG) obtained ranged from 16.2 to 16.7 °C compared to that of commercial fish gelatin gel (CFG), from 23.7 to 25.6 °C and halal bovine gelatin (HBG), from 26.5 to 28.7 °C. On the other hand, gelling temperatures of EFG, CFG and HBG ranged from 5.1 to 5.2 °C, 11.9 to 17.46 °C, and 12.6 to 19.33 °C, respectively. EFG gave gels with a considerably lower G′ values than CFG and HBG. The bloom strength of EFG gels at 6.67% (w/v) was 69.03 g which was much lower than HBG (336.2 g) and CFG (435.9 g). Enzyme transglutaminase was added in the amounts of 0.5, 1.0, 3.0 and 5.0 mg/g gelatin to modify the gel properties of the extracted fish gelatin. The modified EFG gels obtained had higher gel strengths of 101.1 g and 90.56 g with added transglutaminase of 1.0 and 3.0 mg/g, respectively. However with addition of 5.0 mg/g enzyme the gel strength increased only up to 75.06 g. SDS-PAGE of extracted gelatin gel showed protein band intensities for α1-chains and 53 kDa but in gels added with higher concentration of transglutaminase, these protein band intensities seemed to disappear.     

Improvement of gelling properties of porcine blood plasma using microbial transglutaminase

The effect of microbial transglutaminase (MTGase) on the texture and water-holding capacity (WHC) of heat-induced gels prepared from porcine blood plasma at pH 5.5 was investigated. Different concentrations of commercial MTGase were added to plasma and incubated for several times under specific conditions of temperature and pH. From the results obtained, it can be postulated that enzymatic treatment enhances textural properties and WHC of plasma gels at pH 5.5, especially when incubated with 3% of the commercial product for 3 h at 30 °C and pH 7. This treatment increased by 0.4 N the hardness of gels and reduced by 3% the water released after gel centrifugation with respect to the control samples. SDS–PAGE confirmed that cross-linking took place when MTGase was added to plasma solutions. However, the results suggest that the sole addition of MTGase was not effective enough to improve the gelling properties of plasma proteins under acidic conditions.     

Mechanical and gelling properties of comminuted sausages containing chicken MDM

The present investigation focuses on the physicochemical properties, rheological behaviors and texture of raw and cooked emulsions containing different mechanically deboned meat (MDM) from chicken. MDMs were produced from the neck, backs and thighs. The texture and mechanical profile analyses were performed using a small deformation dynamic oscillation in a shear and instrumental texture analyzer. The mechanical spectra of the raw and cooked MDM emulsion gels were classified as weak gels based on their frequency sweep and tan δ results. Both the G′ and G″ values increased with increasing frequency in the temperature of 4 and 10 °C. The MDM from thighs had the lowest water holding capacity (WHC) and emulsifying (EC) values, while the MDM from backs had significant values of 2.41 and 128.87, respectively. The cooked emulsion containing backs showed the highest hardness and cohesiveness values, while the emulsion containing necks and thighs had the lowest texture parameters.     

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.     

The effects of sodium alginate and calcium levels on pea proteins cold-set gelation

A multi-scale investigation of pea proteins – alginate cold-set gels was proposed in this study. The gel preparation followed a two-steps procedure. Globular pea proteins were first denatured and aggregated by a pre-heating step. Sodium alginate was then added at different concentrations. Thereafter the in situ gelation process was induced at 20 °C using glucono-δ-lactone (GDL) and two calcium carbonate (CC) levels; calcium cations were released as the pH decreased. Small-amplitude rheology measurements (storage modulus G′) showed that stronger mixed gels were obtained than single-biopolymer solutions. Confocal laser scanning microscopy (CLSM) revealed phase-separating microstructures of mixed gels, foremost owing to biopolymers incompatibility. Phase separation was kinetically entrapped by gelation at different evolution stages. According to the co-occurrence method and microstructure classification, image texture analysis disclosed that a continuous protein network dispersing small gelled alginate microdomains corresponded to the strongest mixed gels. Transmission electron microscopy (TEM) evidenced that during gelation, the pre-aggregated proteins were mainly associated into large agglomerates with no peculiar pattern. Higher cohesiveness between both networks was hypothesized, since protein agglomerates could expose “anchoring points” for alginate chains. Depending on both protein – alginate initial composition and calcium availability, non-specific inter-biopolymer cross-links via calcium were assumed to be the key factor of synergism within mixed gels.