Gelling Properties of Gellan Solutions Containing Monovalent and Divalent Cations

Gelling temperatures of gellan solutions with the addition of Na⁺ and K⁺ ranging from 15 to 450 mM or Ca⁺⁺ and Mg⁺⁺ from 2 to 40 mM were determined by dynamic rheological testing at four polymer concentrations between 0.4 and 2.0% (w/w). Gelling temperatures were much higher for gellan solutions containing divalent cations than for those containing the same amount of monovalent cations. Solutions containing K⁺ gelled at higher temperatures than those containing Na⁺. Effects of Ca⁺⁺ and Mg⁺⁺ on gelling temperatures were not significantly different. A general model was developed to predict the gelling temperature of gellan solutions as functions of cation and polymer concentrations.     

EFECT OF pH BUFFERS ON MECHANICAL PROPERTIES OF GELLAN GELS

The strength and deformability of calcium cross-linked gellan gels as affected by pH 3.5 and 5.0 citrate and acetate buffers were measured by large compressive deformation test until failure. The trend of dependence of gel strength on polymer and calcium concentrations was similar to gels formed in distilled water without pH adjustment. A critical calcium concentration was observed for each gellan concentration. Gels formed at the critical calcium concentration exhibited the maximum strength. The chelating effect of pH 5.0 citrate buffer greatly increased the critical calcium concentration. The failure strain, representing the deformability, of gellan gels formed in buffers behaved differently from gels formed in distilled water. In the pH 3.5 buffer systems, gellan gels were brittle regardless of gellan and calcium concentrations. In the pH 5.0 buffer systems, gellan gels were brittle at high calcium concentrations and ductile at calcium concentrations less than 24 mM in citric buffer and less than 6 mM in acetate buffer.     

Gelling Temperature of Gellan Solutions Containing Calcium Ions

Gelling temperatures were measured using a dynamic rheological testing method, a spectrophotometric method and direct visual examination combined with use of a thermocouple. Results from the dynamic testing method were most consistent. Gelling temperatures of gellan solutions increased from 30 to 72°C, as polymer concentrations increased from 0.4 to 2.0% w/v and Ca⁺⁺ concentrations increased from 2 to 40 mM. A mathematical model was developed based on the van't Hoff equation to relate gelling temperatures to composition and polymer molecular weight. The heat of cross-linking in Ca-gellan gels was estimated to be 428.6 kJ/mole, about 1.4 to 2.1 times greater than for gelatin gels.     

Polymer and Ion Concentration Effects on Gellan Gel Strength and Strain

Failure stresses and strains were measured in compressive, tensile and torsional modes on gellan gels at four polymer (0.6–1.8% w/v) and seven Ca⁺⁺ (1.5–60 mM) concentrations. Shear stresses at failure were equal in all three testing modes and proportional to gellan content. Low calcium gels increased linearly in strength with Ca⁺⁺ concentration until it reached a level of about 0.5 calcium ions per repeat tetrasaccharide unit of gellan gum polymer. Gel strength decreased linearly with Ca⁺⁺ at higher concentrations. Low calcium gels were extensible with failure strains decreasing as the logarithm of Ca⁺⁺; whereas high calcium gels were brittle and failed at a constant strain, the value of which was twice as high in compression and torsion as in tension.     

The effect of pH and calcium ion on rheological behaviour of β‐lactoglobulin‐basil seed gum mixed gels

Effect of pH (4.5–7.5) and Ca²⁺ (0.01–0.5 m ) on gelation of single and mixed systems of 10% β‐lactoglobulin (BLG) and 1% basil seed gum (BSG) was investigated. The gelling point of BLG and BSG gels was strongly pH‐dependent, and stiffer gels formed at higher pH. The BLG gels were formed upon heating to 90 °C and reinforced on cooling to 20 °C; however, the gelation of BSG occurred at temperatures below 70 °C. By increasing Ca²⁺ concentration, storage modulus of BLG and BSG gels were increased, although pH had a greater effect than Ca²⁺. In contrast, mixed systems showed two distinct types of behaviour: BLG gel formation and BSG network, suggesting that phase‐separated gels were formed. In addition, higher strength was obtained for BLG‐BSG mixture at higher Ca²⁺ concentration.     

PURIFICATION AND CHARACTERIZATION OF ACIDIC PROTEASES FROM THE STOMACH OF THE DEEPWATER FINFISH ORANGE ROUGHY (HOPLOSTETHUS ATLANTICUS)

Acidic proteases were extracted and purified from the stomach of orange roughy (Hoplostethus atlanticus). Protease I and II were glycoproteins with molecular weights of 33.5 and 34.5 KDa, respectively. Protease I had an isoelectric point of 5.30. The two forms of protease II (a and b) had isoelectric points of 4.35 and 4.40, respectively, and N-terminal sequence identity for 12 amino acids. The proteases exhibited optimal temperature activity at 37C. They had high activity at low temperatures and low thermal stability compared to mammalian pepsins. They were stable in the pH range of 2–4.5 and unstable above pH 6.5. Protease I and II had pH optima of 2.5 and 3.5, respectively, and K _m’values for the hydrolysis of hemoglobin (pH 3.0, 37C) of 124 μM and 517 μM, respectively. Enzyme activities were inhibited by pepstatin A and high NaCl concentrations, and were slightly stimulated by Ca²⁺ and Cu²⁺.     

Formation of Calcium-Mediated Junction Zones at the Onset of the Sol-Gel Transition of Commercial κ-Carrageenan Solutions

ABSTRACT: Gelling temperatures of commercial κ-carrageenan solutions were measured using dynamic oscillatory shear rheometry, in response to varying polymer (0.4% to 1.6%, w/w) and added Ca²⁺ (5 to 20 mM) salts. Gelling temperatures were found to shift to higher temperatures with increased polymer and Ca²⁺ concentration. The Tang model was used to estimate the number of Ca²⁺-mediated cross-links formed with κ-carrageenan at the onset of junction zone formation. Based on this model, 6 κ-carrageenan double helices were estimated to be cross-linked by 5 Ca²⁺ ions through ionic bridging and electrostatic attraction, within a cross-linking region equivalent to one pitch of the double helix.     

Characterization of gelation time and texture of gelatin and gelatin–polysaccharide mixed gels

The effects of cooling rate, holding temperature, pH and polysaccharide concentration on gelation characteristics of gelatin and gelatin–polysaccharide mixtures were investigated using a mechanical rheometer which monitored the evolution of G′ and G″. At low holding temperatures of 0 and 4 °C, elastic gelatin gels were formed whereas a higher holding temperature of 10 °C produced less elastic gels. At slow cooling rates of 1 and 2 °C/min, gelling was observed during the cooling phase in which the temperature was decreased from room temperature to the holding temperature. On the other hand, at higher cooling rates of 4 and 8 °C/min, no gelation was observed during the cooling phase. Good gelling behavior similar to that of commercial Strawberry Jell-O^® Gelatin Dessert was observed for mixtures of 1.5 and 15 g sucrose in 100 ml 0.01 M citrate buffer containing 0.0029–0.0066 g low-acyl gellan. Also, these mixed gels were stronger than Strawberry Jell-O^® Gelatin Desserts as evidenced by higher G′ and gel strength values. At a very low gellan content of 0.0029 g, increasing pH from 4.2 to 4.4 led to a decrease in the temperature at the onset of gelation, G′ at the end of cooling, holding and melting as well as an increase in gel strength. The gelation time was found to decrease to about 40 min for gelatin/sucrose dispersions in the presence of 0.0029 g gellan at pH 4.2 whereas the corresponding time at pH 4.4 was higher (79 min). In general, the gelation time of gelatin/sucrose dispersions decreased by a factor of 2 to 3 in the presence of low-acyl gellan. The addition of low-acyl gellan resulted in an increase in the gelation rate constant from 157.4 to 291 Pa. There was an optimum low-acyl gellan content for minimum gelation time, this optimum being pH dependent. Addition of guar gum also led to a decrease in gelation time to 73 min with a corresponding increase in the gelation rate constant to 211 Pa/min though these values were not sensitive to guar gum content in the range of 0.008–0.05 g. The melting temperature of gelatin/sucrose/gellan as well as gelatin/sucrose/guar mixtures did not differ significantly from that of pure gelatin or Strawberry Jell-O^® Gelatin Desserts. At pH 4.2, the melting rate constant was highest at a low-acyl gellan content of 0.0029 g whereas the rate constant was insensitive to low-acyl gellan content at pH 4.4. Addition of guar did not seem to affect the melting temperature or the melting rate constant.     

Microstructure and rheological properties of psyllium polysaccharide gel

The strong gelling property of psyllium polysaccharides is closely related to its health benefits and applications, such as use as a binding agent in the landscape industry. However, information about gelling properties of psyllium polysaccharides is very limited. To explore the gel properties of psyllium, the alkaline extracted gel fraction (AEG) of psyllium polysaccharides was studied in this investigation. The rheological and low-temperature electron microscopy methods including cryo-scanning electron microscopy (Cryo-SEM) and freeze-substitution for transmission electron microscopy (TEM) were used to investigate Ca²⁺ influence on the gel properties of AEG. AEG formed a weak gel with a fibrous appearance. AEG did not have a sharp melting point and exhibited no thermal hysteresis during heating and cooling procedure. The origin of this gelling behaviour was due to the fibrillar gel structure of psyllium polysaccharide. It was found that Ca²⁺ had a significant influence on the gel properties and microstructure. Elastic modulus G′ of gel increased as Ca²⁺ concentration increased. Critical strain S at first increased and then decreased as Ca²⁺ concentration increased. AEG gel became more resistant to temperature change on addition of Ca²⁺. Psyllium gel changed to aggregated gel with added Ca²⁺. The strands of gel appeared thicker and the density of the junction zone increased with increasing Ca²⁺ concentration as revealed by SEM and TEM. The changing bulk gelling properties of psyllium polysaccharide by adding Ca²⁺ was attributed to the change in gel structure.     

Mechanical Properties of Gellan Gels in Relation to Divalent Cations

Mechanical behavior of gels formed with gellan polymer crosslinked by calcium and magnesium ions was studied to determine the influence of divalent ion type and polymer concentration. Failure strength and deformation were measured in compression and related to concentrations of gellan and bound cations in gel matrices. Insufficient cations formed weak, extensible gels. Maximum gel strength was achieved at 0.5 divalent cations/repeat tetrasaccharide unit, assumed to be the condition for maximal numbers of complete junction zones. At optimum cation levels gels with Ca⁺⁺ were about 1.2 times stronger than gels with Mg⁺⁺ at the same polymer concentration. Excessive cations weakened the gels. Twice as much reduction in gel strength resulted from additional Ca⁺⁺ as compared to the same additional amount of Mg⁺⁺. Differences between strengths of the gels may be attributable to polymer configurations at junction zones in relation to cation size.     

Structural and foaming properties of egg albumen subjected to different pH-treatments in the presence of calcium ions

The influence of low (pH 1.5–3.5) and high (10.5–12.5) pH-induced unfolding followed by refolding to pH 4.5–8.5 in the presence of Ca²⁺ on the structural, functional and viscoelastic properties of egg albumen was investigated. pH-induced unfolding significantly improved overrun, stability and reduced liquid drainage of egg albumen for most pH-treatments compared to untreated control. Unfolding at pH 12.5 led to the largest improvements, overall. Improvements in foam overrun and stability were found to be mostly due to pH-treatments, with Ca²⁺ having a small effect when compared to prior studies. Inclusion of Ca²⁺ did however reduce liquid drainage, and was found to significantly modify the rheological properties of the foams, making them stiffer and stronger. Inclusion of Ca²⁺ during pH-treatment led to more exposed –SH groups compared to albumen pH-treated in its absence. The pH-treatments also produced albumens with higher surface hydrophobicity, which was determined mostly to come from the pH unfolding, with Ca²⁺ having little impact.     

Firming of Cooked Sweet Potatoes as Affected by Alum Treatment

Sweet potato slices were cooked for 5 min in distilled water, alum (aluminum potassium sulfate), AICI₃ and several pH level buffer solutions. Slices preheated (60°C) in these solutions were boiled in water. The slices cooked at about pH 4 were firmest among those in the range pH 2-pH 12. Firmness was (greatest to least); preheated in alum > preheated in water > cooked in buffer (pH 3.5) >AICI₃ (pH 3.5) > alum (pH 3.5) > water. Preheating, Al³⁺ and acid prevented softening of slices. Acid-treated slices were firmer than Al³⁺ treated slices. Alum contains K⁺ and S0₄^-2, which accelerate solubilization of pectin, thus slices cooked in alum were softer than those in AICI₃. Scanning electron microscopy showed the middle lamella separated when cooked in water but not when cooked in other solutions.     

Mechanical and physical properties of calcium-treated gellan films

The physicochemical properties of Ca²⁺-treated gellan films plasticized with glycerol were investigated as a function of CaCl₂ concentration (0–20%, w/w) in an aqueous soaking solution. Films were examined based on their mechanical properties, water vapor permeability (WVP), swelling index (SI), thickness and opacity. The SI was lower for Ca²⁺-treated films relative to a control, however, above 13% (w/w) CaCl₂ no differences in SI were found. At 13% (w/w) CaCl₂, tensile and puncture strengths reached a maximum. Tensile elongation, puncture deformation, film thickness and WVP were lower for CaCl₂-treated films than untreated, however, all increased with CaCl₂ concentration. Opacity of gellan films increased with the Ca²⁺-treatment relative to the untreated film, however declined as CaCl₂ concentration increased. In general, Ca²⁺-treated gellan films were stronger, acted as better water vapor barriers, swelled less when in contact with water, and became less pliable and transparent.     

Effects of Cation Properties on Sol-gel Transition and Gel Properties of κ-carrageenan

The phase transition temperatures, rheological properties and gel‐network characteristics for gelation of κ‐carrageenan‐salt (NaCl, KCl and CaCl₂) solutions and their aged gels were investigated. The effectiveness of increasing gelling and gel‐melting temperatures at the salt concentrations examined followed the sequence of K⁺ > Ca2⁺ > Na⁺. This sequence was also true for the gel strength and the melting enthalpy (DH) of the most crosslinked junction zones of aged gels at low salt concentrations. Nonetheless, a different order (Ca⁺⁺ > K⁺ and Na⁺) was found for increasing storage modulus and gelation rate during early‐stage gelation, thermal hysteresis and the DH of aged gels in some salt‐carrageenan systems.     

Release and partial characterization of cell-envelope proteinases fromLactococcus lactis subsp.lactis IFPL 359 andLactobacillus casei subsp. casei IFPL 731 isolated from raw goat,s-milk cheese

Different methods of releasing the cell-envelope proteinase (CEP) fromLactococcus lactis IFPL 359 (Lc-CEP) andLactobacillus casei IFPL 731 (Lb-CEP) have been tested. Release of Lc-CEP was higher in Ca^2+-free buffer than in the presence of lysozyme and Ca^2+-. Lb-CEP was not soluble in Ca^2+--free buffer, making necessary the use of chelating agents such as ethylenediaminetetraacetate (EDTA) to attain release yields of 15–20%. Solubilizing the cell wall oflb.casei using lysozyme and mutanolysin improved CEP release yields, even in the presence of Ca^2+-. Two differently charged chromophoric peptides were degraded by whole cells and the soluble fractions studied at different hydrolysis rates in both the strains considered. Based on the specificity of these CEPs for the different substrates, the two proteinases can be placed in the same class as the CEP_I/III mixed-type variants that have been identified in lactococcal proteinases. In both strains ß-casein was hydrolysed more rapidly thanα _s-cascin.     

Diffusion of probe polymer in gellan gum solutions during gelation process studied by gradient NMR

The diffusion coefficients of pullulan added in gellan gum solutions as a probe polymer were measured using pulsed-field-gradient stimulated-spin-echo (PFG-Ste) ¹H NMR in order to investigate the gelation mechanism and gel structure. The echo intensity of gellan was steeply decreased at around the gelling temperature T_gel indicating stiffening of the gellan chains upon aggregation and formation of the network. The diffusion coefficient of pullulan D_pull increased with decreasing temperature below T_gel. This result suggests that the decrease in concentration of solute gellan in the interspaces of the network below T_gel, as evidenced by the decrease in its echo intensity, leads to an overall decrease of hydrodynamic interactions between gellan and pullulan. The characteristic hydrodynamic shielding length ξ was calculated from the relation D_pull/D_pull,0 = exp(−R_h/ξ), where D_pull,0 is D_pull in dilute solution and R_h is the hydrodynamic radius of pullulan. The temperature dependence of ξ, which was investigated for varying concentrations of several cations, was found to follow closely the gelling temperature, and in particular, showing a very similar thermal hysteresis.     

Carp Sarcoplasmic Reticulum Changes Due to Heat Treatment

Electron microscopy revealed that heat treatment in the range 35–55°C caused no observable change in membrane structures of the sarcoplasmic reticulum (SR). Proteins with molecular weights about 200 and 400 kDa, (probably dimers and tetramers of Ca²⁺-ATPase, respectively) were observed in SDS-PAGE when the light SR was treated at 49 and 55°C respectively. Ca²⁺ leakage from the SR was markedly enhanced with increasing temperatures, especially for the heavy SR. Results suggest that the decrease of Ca²⁺ uptake ability following heat treatment is at least partly caused by the acceleration of Ca²⁺ leakage together with irreversible denaturation of Ca²⁺-AT-Pase protein.     

Gelation of gellan – A review

Gellan is an anionic extracellular bacterial polysaccharide discovered in 1978. Acyl groups present in the native polymer are removed by alkaline hydrolysis in normal commercial production, giving the charged tetrasaccharide repeating sequence: → 3)-β-d-Glcp-(1 → 4)-β-d-GlcpA-(1 → 4)-β-d-Glcp-(1 → 4)-α-l-Rhap-(1 →. Deacylated gellan converts on cooling from disordered coils to 3-fold double helices. The coil–helix transition temperature (T_m) is raised by salt in the way expected from polyelectrolyte theory: equivalent molar concentrations of different monovalent cations (Group I and Me₄N⁺) cause the same increase in T_m; there is also no selectivity between different divalent (Group II) cations, but divalent cations cause greater elevation of T_m than monovalent. Cations present as counterions to the charged groups of the polymer have the same effect as those introduced by addition of salt. Increasing polymer concentration raises T_m because of the consequent increase in concentration of the counterions, but the concentration of polymer chains themselves does not affect T_m. Gelation occurs by aggregation of double helices. Aggregation stabilises the helices to temperatures higher than those at which they form on cooling, giving thermal hysteresis between gelation and melting. Melting of aggregated and non-aggregated helices can be seen as separate thermal and rheological processes. Reduction in pH promotes aggregation and gelation by decreasing the negative charge on the polymer and thus decreasing electrostatic repulsion between the helices. Group I cations decrease repulsion by binding to the helices in specific coordination sites around the carboxylate groups of the polymer. Strength of binding increases with increasing ionic size (Li⁺ < Na⁺ < K⁺ < Rb⁺ < Cs⁺); the extent of aggregation and effectiveness in promoting gel formation increase in the same order. Me₄N⁺ cations, which cannot form coordination complexes, act solely by non-specific screening of electrostatic repulsion, and give gels only at very high concentration (above ∼0.6 M). At low concentrations of monovalent cations, ordered gellan behaves like a normal polymer solution; as salt concentration is increased there is then a region where fluid “weak gels” are formed, before the cation concentration becomes sufficient to give true, self-supporting gels. Aggregation and consequent gelation with Group II cations occurs by direct site-binding of the divalent ions between gellan double helices. High concentrations of salt or acid cause excessive aggregation, with consequent reduction in gel strength. Maximum strength with divalent cations comes at about stoichiometric equivalence to the gellan carboxylate groups. Much higher concentrations of monovalent cations are required to attain maximum gel strength. The content of divalent cations in commercial gellan is normally sufficient to give cohesive gels at polymer concentrations down to ∼0.15 wt %. Gellan gels are very brittle, and have excellent flavour release. The networks are dynamic: gellan gels release polymer chains when immersed in water and show substantial recovery from mechanical disruption or expulsion of water by slow compression. High concentrations of sugar (∼70 wt % and above) inhibit aggregation and give sparingly-crosslinked networks which vitrify on cooling. Gellan forms coupled networks with konjac glucomannan and tamarind xyloglucan, phase-separated networks with kappa carrageenan and calcium alginate, interpenetrating networks with agarose and gelling maltodextrin, and complex coacervates with gelatin under acidic conditions. Native gellan carries acetyl and l-glyceryl groups at, respectively, O(6) and O(2) of the 3-linked glucose residue in the tetrasaccharide repeat unit. The presence of these substituents does not change the overall double helix structure, but has profound effects on gelation. l-Glyceryl groups stabilise the double helix by forming additional hydrogen bonds within and between the two strands, giving higher gelation temperatures, but abolish the binding site for metal ions by changing the orientation of the adjacent glucuronate residue and its carboxyl group. The consequent loss of cation-mediated aggregation reduces gel strength and brittleness, and eliminates thermal hysteresis. Aggregation is further inhibited by acetyl groups located on the periphery of the double helix. Gellan with a high content of residual acyl groups is available commercially as “high acyl gellan”. Mixtures of high acyl and deacylated gellan form interpenetrating networks, with no double helices incorporating strands of both types. Gellan has numerous existing and potential practical applications in food, cosmetics, toiletries, pharmaceuticals and microbiology.     

Influence of pH and temperature upon calcium accumulation and release by bovine sarcoplasmic reticulum

Sarcoplasmic reticulum (SR) was prepared from fresh beef sternomandibularis muscle and shown to be relatively free from contamination by lysosomes, sarcolemma and mitochondrial membranes. Ca^{2 +} accumulation by SR from fresh and cold-shortened muscle was 51 and 39 nmoles/mg protein, respectively. The Ca^{2 +} accumulating ability of fresh SR vesicles decreased with lowering of pH (7·3, 6·8, 6·2, 5·5 and 5·0) at all temperatures (0, 15 and 38°C). Lowering the temperature from 38 to 0°C at pH 6·6 resulted in the release of 48% of the total accumulated Ca^{2 +}, whereas the corresponding value on lowering the temperature from 38 to 15°C at the same pH was only 12%. Thus, low temperatures accelerate the release of Ca^{2 +} by SR. Although simultaneously lowering pH and temperature also increased Ca^{2 +} release by SR, the amount of Ca^{2 +} released was less than if pH and temperature were altered independently. The findings are discussed in the light of explaining cold shortening.     

Pectin–sucrose–Ca2+ interactions: effects on rheological properties

The effects of varying concentrations of pectin (4.5–6.5%, w/w), sucrose (40–60%, w/w) and calcium (20–60 mg/g pectin) on the viscoelastic properties of pectin dispersions at pH 3.0 were investigated. Pectin samples used were extracted from pomelo fruit peels (Citrus grandis) grown in Malaysia. The dynamic rheological parameters (G′, G″, δ and η*) of pectin–sucrose–calcium dispersion were determined at 1.5% strain from 90–20°C at a cooling rate of 3°C min⁻¹. Plots of G′ and G″ against frequency (rad s⁻¹) showed G″>G′ throughout the frequency range with no occurrence of crossover for most of the pectin dispersions. In addition both storage (G′) and loss (G″) moduli of the dispersions increase on cooling. Increasing pectin, sucrose and calcium concentrations increased G′ and G″ with pectin having the greatest effect. Interactions amongst the three factors were also studied. At lower pectin concentrations, addition of Ca²⁺ increased G′ at all temperatures. This effect was also observed at higher pectin concentrations at 20°C but not at 90°C. The opposite effect was observed with the addition of sucrose, i.e. addition of sucrose at a higher pectin concentration increased G′ whereas at a lower pectin concentration no effect was observed. Interaction between calcium and sucrose gave rise to an increase in G′ when Ca²⁺ was added at high sucrose concentrations, but a decrease in G′ was evident at low sucrose concentrations. Dispersions of pectin alone or in combination with sucrose exhibited a more liquid-like behaviour with G″>G′. However, in the presence of Ca²⁺, mechanical spectra of G′>G″ were obtained.