Gellan gum fluid gels: influence of the nature and concentration of gel-promoting ions on rheological properties

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: http://dx.doi.org/10.1007/s00396-018-4396-6” 1 Gellan gum fluid gels. Influence of the nature and concentration of gel-promoting 1 ions on rheological properties. 2 M Carmen García, Luis A. Trujillo, José Muñoz, M Carmen Alfaro* 3 Departamento de Ingeniería Química. Facultad de Química. Universidad de Sevilla. C/ 4 Profesor García González, s/n. 41012 Sevilla. Spain 5 * Corresponding author 6 e-mail: [email protected] 7 ORCID: 0000-0002-0110-2290 8 Tel: +34 954 55 64 47 9 Fax: +34 954 55 64 41 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 2 Abstract 30 Low-acyl gellan gum fluid gels were obtained using two different gel-promoting cations 31 under the same processing conditions and their rheological properties were investigated. 32 For that reason, stress and frequency sweeps measurements and flow curves tests were 33 carried out. Small amplitude oscillatory shear assays demonstrated that these materials 34 exhibit weak gel behaviour despite the shape of their mechanical spectra being similar to 35 those obtained from strong gels. However, the existence of a weak gel structure was 36 confirmed by the fact that the difference between the elastic and viscous components was 37 of only one order of magnitude. In addition, a reduction in the storage and lost modules 38 and the viscosity as a function of the applied shear was observed with increasing cation 39 concentration. Finally, in order to produce fluid gels, a higher concentration of the 40 monovalent cation was required than when divalent cations were employed in the low- 41 acyl gellan gum fluid gels formulation. 42 43 Keywords 44 Low-acyl gellan gum, fluid gel, Rheology, Viscoelasticity, Very shear thinning, Gel- 45 promoting cation. 46 47 Introduction 48 It is well known that fluid gels can be produced by polysaccharides capable of forming 49 strong gels. This is possible when a strong gel manufactured under quiescent conditions 50 is disturbed by submitting the sample to mechanical energy [1]. Consequently, the 51 resulting sample shows a heterogeneous microstructure consisting of an irregularly- 52 shaped cluster with gel-like behaviour suspended in an aqueous phase whose 53 polysaccharide concentration is smaller than that present in the microgels formed. Among 54 the biopolymers which can produce fluid gels are gellan gum [2, 3], agar, K-carrageenan, 55 whey protein [4] or alginate [5]. Norton et al. [6] proposed a model to explain the form 56 and stability of fluid gels. The latter model establishes a as a consequence of a gelation 57 3 stage takes place the particles or clusters form when the biopolymers aqueous solution is 58 cooled under mechanical treatment. The cluster sizes are dependent of the shear 59 conditions. Following, the molecular ordering is attained. The particle size and volume 60 fraction achieved are as a function of the applied shear during process and the polymer 61 concentration in the fluid gel formulation. In addition, the fluid gels stability is maintained 62 while it is not submitted at the melting temperature. 63 Although the sample presents rheological behaviour somewhere between that of a strong 64 gel and weak gel, it is closer to the latter. These rheological properties make possible the 65 current and future applications of fluid gels as suspension [3] and satiety [7] agents, 66 emulsion stabilizers [8]. Additionally, other applications can be mentioned in 67 pharmaceutical and biomedical fields such topical administration of diclofenac [9] or in 68 the paint formulation [10]. In the food industry, low-acyl gellan gum, as a fluid gel, is 69 widely used, since this biopolymer has been utilized as a food additive in the USA from 70 1992 and, in addition, its use is currently authorized in other countries. The code 71 established by The International Numbering System for food additives for gellan gum is 72 INS 418 [11]. 73 Gellan gum is a biopolysaccharide obtained by microbial fermentation from 74 Sphingomonas elodea (ATCC 31461) [3] which can produce strong gels when gel- 75 promoting ions are added to the gellan gum solution under cooling conditions. The 76 disturbance of the formation of this strong gel gives rise to the development of a fluid gel, 77 as mentioned above. The nature and ionic strength of gel-promoting ions chosen to 78 prepare the fluid gels determine their final rheological properties. The main applications 79 of gellan gum as a fluid gel are as a suspending agent in some drinks, such as citrus drinks 80 or chocolate milk. On another hand, it is also utilized to suspend coloured particles in 81 translucent drinks [12]. 82 4 The main objectives of this work are the study of the effect of the nature of the salt 83 employed to provide gel-promoting ions and the influence of the cation concentration on 84 the rheological properties of fluid gels containing 0.2 wt% gellan gum. For these 85 purposes, small amplitude oscillatory shear and steady-state measurements were carried 86 out. 87 88 Materials and methods 89 90 Materials 91 Low-acyl gellan gum (Kelcogel® FTM) cordially provided by CP-Kelco (San Diego, 92 USA) was employed as obtained and it has molecular weight of 2-3·105 Da. This 93 substance will be designated as LA-gellan in the rest of this paper. Its concentration in 94 the fluid gels formulation was 0.2 wt %. To acquire Na+ or Ca2+ gel-promoting ions, 95 99.5% purity NaCl [3] supplied from Panreac (Barcelona, Spain) and 98% purity CaCl2 96 provided by Merck were used. CaCl2 concentrations assessed were 9.0·10-3 M, 1.1·10-2 97 M, 1.3·10-2 M, 1.6·10-2 M and 1.8·10-2 M, and NaCl concentrations studied were 0.22 M, 98 0.24 M and 0.27M. Deionised water was always utilized and 0.1 wt % sodium azide was 99 used to preserve the fluid gels. 100 101 Fluid gel preparation 102 LA-gellan fluid gels were prepared in batches of 600 g following the steps suggested by 103 Sworn (2009), as was done in a previous work [13]. In order to disperse the LA-gellan 104 gum powder, it was added slowly to a vessel placed within a water-bath at 80º C. The 105 hydration step was carried out at 80º C while stirring at 700rpm for 25 min was maintained 106 using an Ika-Visc MR-D1 homogenizer (Ika, Germany) equipped with a sawtooth-type 107 impeller. The impeller diameter/container diameter ratio was 0.85. Once the 25 minutes 108 had elapsed, the required amount of CaCl2 or NaCl was added and the solution was 109 maintained under mechanical treatment at 80º C for 5min. Water loss by evaporation was 110 corrected by adding the appropriate quantity of deionised water. Afterwards, the sample 111 vessel was placed in a thermostatic bath at 20ºC and the gelation step was carried out by 112 submitting the solution to mechanical treatment under 700 rpm with the aforementioned 113 5 Ika-Visc homogenizer for 1500s [13, 14, 15]. The cooling rate was 2.4ºC/min. Finally, 114 the fluid gels were stored at 4.5 ºC for at least 48 h before performing the rheological 115 study. 116 117 Rheological Characterization 118 In order to perform stress sweep and frequency sweep measurements, a controlled-stress 119 rheometer AR2000 (TA Instruments, Crawley, United Kingdom) was employed. These 120 tests were carried out at 20 ºC. A parallel plate with serrated surfaces, 40mm diameter 121 and a measuring gap of 1mm was used as a sensor system. It should be noted that in order 122 to avoid wall depletion phenomena, sensor systems with rough surfaces were required, 123 since in previous works it was demonstrated that the gellan gum fluid gel structure 124 consists of microgels in aqueous phase, this being a heterogeneous microstructure [1]. To 125 prevent losses as a consequence of evaporation during measurements, a thin film of Dow- 126 Corning 200® (Dow Chemical Co.) silicon oil fluid (kinematic viscosity, 20 cSt) was 127 applied to enclose the plate rim. The estimation of the dynamic linear viscoelastic range 128 (DLVR) was performed by stress sweeps under oscillatory shear measurements at three 129 different frequencies (0.1, 1, 3 Hz), from 0.008 to 3 Pa. The frequency sweep tests were 130 conducted in the 3-0.01 Hz frequency range as this is within the linear range of response. 131 Flow behaviour was studied using a Haake RS100 controlled stress rheometer (Thermo 132 Scientific, Karlsruhe, Germany), with a parallel plate sensor system with serrated surfaces 133 (60mm diameter and a measuring gap of 1mm). The shear rate variation with the plate 134 radius in the flow curves data was corrected by using the following equations [16]: 135 136  corr ( .  R) =  N [1+ (m/4)] 137 (1) 138  corr ( .  R) =  N [1+ (m/4)]· .  R 139 (2) 140 where,  newt = (2·H/(  ·R4))*(M/Ω); m= d(log  newt)/d(log .  R) = nPL-1; .  R is the rim 141 shear rate;  corr is the corrected shear stress; H is the gap between plates; Ω is the angular 142 velocity; R is the plate radius, M is the torque applied,  newt is the viscosity calculated at 143 the rim of the parallel plate geometry and nPL is the power law flow index. 144 6 The measurements were carried out in the 0.5-33Pa shear stress range by a step-wise 145 procedure. 146 The samples were maintained in a rest state in the measuring gap for 600 s of equilibration 147 time, before conducting the rheological test, in order to avoid any effects of recent 148 mechanical history after the loading procedure. 149 Each type of measurement was carried out at least 4 times and in all of them, fresh sample 150 was used. 151 In order to determine the existence of the significant differences between the results in 152 this work, the standard deviations of all assessed parameters are shown in each table. For 153 this purpose, the ANOVA ONE WAY method using Statgraphic Plus software was used. 154 155 Results and discussion 156 157 Dynamic linear viscoelastic range (DLVR) 158 Fig. 1 shows the influence of the Ca2+ concentration on the stress sweep results obtained 159 to determine the DLVR at the three studied frequencies chosen in order to avoid 160 destruction of the sample structure [12]. This Figure exhibits constant values of G´ and 161 G´´ up to a certain shear stress, the critical stress, which is related to the inception of the 162 non-linear zone characterized by the dependence of the shear stress applied on the 163 viscoelastic moduli. The microstructural reorganization before breakdown entails energy 164 dissipation which brings about an increase in G´´ values up to a peak value at the 165 beginning of the non-linear response [16, 17]. It is interesting to mention that, from that 166 moment, the G´ value began to decrease. As can be observed in Fig.1, the Ca2+ 167 concentration had no influence on the extension of the linear viscoelastic range. The 168 critical stress values shown in Table 1 demonstrate that there is a slight decrease in their 169 values as the frequency increases. However, this tendency is not clear in the critical strain 170 values. Additionally, data show a decrease in the viscoelastic modules with an increase 171 in the Ca2+ concentration. 172 Figure 1 173 Table 1 174 Similar behaviour is observed when the stress sweep measurements were carried out over 175 the gellan gum fluid gels containing Na+ as a gel-promoting cation (Fig.2), that is, there 176 was a decrease in the G´ and G´´ values with an increase in the cation concentration. 177 7 However, there was a slight reduction in the DLVR with increasing Na+ concentration. 178 This is also supported by Table 2. 179 Figure 2 180 Table 2 181 182 Mechanical spectra 183 The frequency sweep results are displayed in Fig.3. The data show the storage module 184 (G´) is larger than the loss module (G´´) over the whole frequency range studied. In 185 addition, the mechanical spectra of gellan gum fluid gels containing both gel-promoting 186 cations exhibited the typical behaviour shown by strong gels which present a) a slow 187 frequency dependence on G´ characteristic slopes and b) a minimum of G´´in the mid- 188 section of the frequency range. However, they cannot be considered strong gels since 189 there is only a difference of one order of magnitude between the storage module values 190 and the loss module values. Additionally, as can be observed, both an increase in Ca2+ 191 concentration (Fig.3a) and Na+ concentration (Fig.3b) provoked a fall in the viscoelastic 192 moduli. However, this trend started to be significant when the value of Ca2+ reached 193 1.8·10-2 M, while significant differences in the case of Na+ were found for the sample 194 containing 0.27 M NaCl. 195 Figure 3a 196 Figure 3b 197 This effect of the concentration of the gel-promoting ions on the mechanical spectra is 198 due to the fact that the gellan gum is an anionic biopolymer and an increase in the cation 199 concentration causes a rise in the charge screen effect resulting in a decrease in the 200 repulsive interactions between the gellan gum macromolecules. Previous works have 201 reported that the existence of cations in solutions of polyelectrolyte provokes the 202 screening of the charge along the chains of polysaccharide involving a little and closely 203 packed structure resulting in a decrease in their rheological parameters [18, 19]. In 204 addition, the presence of inorganic salts with a degree of hydration causes the thickness 205 formation around the polysaccharide molecules provoking a reduction in these parameters 206 [20]. 207 Fig.3a shows the occurrence of a minimum peak in the G´´ curves of the order of 208 magnitude of 0.6 rad/s which makes it possible to estimate the plateau module value, GN0, 209 [21, 22]. The plateau module gives an idea of the level of the interactions among the 210 gelled particles forming the fluid gel. Fig.3c illustrates the plateau module, GN0, as a 211 8 function of the CaCl2 concentration. The decrease in the GN0 in 1.8·10-2 M CaCl2 indicates 212 that the concentration needed to form the gels is exceeded. This fact can be associated 213 with the charge screen effect between the gellan macromolecules since it is an anionic 214 polyelectrolyte. 215 Figure 3c 216 Additionally, it can be observed that the mechanical spectra shape did not change in the 217 CaCl2 concentration range studied, indicating the existence of the same microstructure. 218 This fact is supported by the representation of exponent (n´) of the power equation which 219 relates G´ to the angular frequency (G´   n´) (Fig.3d). The value of n´ was practically 220 constant at about 0.02 which is very close to zero. This result is typical of strong gels. 221 However, according to the criteria proposed by Ross-Murphy [23], the gellan gum fluid 222 gels cannot be classified as strong gels since the strong gels undergo shear-induced 223 fracture under flow, their linear viscoelastic region is broad and the differences between 224 G’ and G” are great enough to support the occurrence of a self-supporting gel. Here, the 225 difference between G´ and G´´ was of only one order of magnitude, which is more 226 characteristic of weak gels and justifies the fluid gel consistency. 227 228 Figure 3d 229 Moreover, it must be noted that smaller Ca2+ concentrations lead to similar, or even 230 higher, G´ and G´´ values in the mechanical spectra than those shown by the samples 231 involving higher Na+ concentrations in their formulation (Fig.3). This fact has been 232 attributed to the ionic strength and the nature of the salt employed [24]. The single 233 divalent ion is capable of substituting each monovalent ion-water-monovalent ion 234 interaction between two carboxylates [25]. 235 236 Steady state test 237 The influence of the concentration of gel-promoting ions can be observed in Fig.4 (Ca2+ 238 cations in Fig.4a and Na+ cations in Fig.4b) where the viscosity is plotted against the shear 239 stress. 240 It must be mentioned that the most noticeable aspect of the flow curves, for both Ca2+ and 241 Na+, is their “very shear thinning” [26] behaviour for all concentrations studied. This 242 behaviour is characterized by showing high plateau viscosity values at low shear stresses 243 and zero shear viscosity. However, as reported in several works [14,15] the high time 244 necessary to achieve the real steady state at low shear stresses or rates makes it necessary 245 15 1.6·10-2 2.7 1.8·10-2 4.1 425 Table 3b. Fitting parameters of the Herschel-Bulkley equation for fluid gels containing 426 0.2 wt% LA-gellan gum and CaCl2 or NaCl as gel-promoting ions, prepared by different 427 mechanical protocols. Temperature: 20ºC. E stands for standard error of fitting 428 parameters 429 430 CaCl2 M 0 (Pa) E0 1 -0 (Pa) E1 -0 n En R2 9.0·10-3 3.5 0.7 0.83 0.34 0.45 0.06 0,978 1.1·10-2 2.5 0.6 0.20 0.08 0.63 0.05 0,986 1.3·10-2 2.6 0.5 0.11 0.05 0.69 0.06 0,983 1.6·10-2 2.1 0.4 0.05 - 0.81 0.06 0,988 1.8·10-2 2.4 0.6 0.12 0.07 0.71 0.08 0,983 NaCl M 0 (Pa) E0 1 -0 (Pa) E1 -0 n En R2 0.22 2.36 0.51 0.06 - 0.75 0.010 0.979 0.24 3.68 0.08 0.04 - 0.82 0.001 0.999 0.27 1.90 0.31 0.10 - 0.71 0.004 0.990 431 432 Figure captions 433 434 Fig.1a. Stress sweep tests performed at 0.1Hz for fluid gels containing 0.2 wt% LA-gellan 435 gum and CaCl2 as gel-promoting ions. Temperature: 20ºC 436 437 Fig.1b. Stress sweep tests performed at 1Hz for fluid gels containing 0.2 wt% LA-gellan 438 gum and CaCl2 as gel-promoting ions. Temperature: 20ºC 439 440 Fig.1c. Stress sweep tests performed at 3 Hz for fluid gels containing 0.2 wt% LA-gellan 441 gum and CaCl2 as gel-promoting ions. Temperature: 20ºC 442 443 16 Fig.2a. Stress sweep tests performed at 0.1Hz for fluid gels containing 0.2 wt% LA-gellan 444 gum and NaCl as gel-promoting ions. Temperature: 20ºC 445 446 Fig.2b. Stress sweep tests performed at 1Hz for fluid gels containing 0.2 wt% LA-gellan 447 gum and NaCl as gel-promoting ions. Temperature: 20ºC 448 449 Fig.2c. Stress sweep tests performed at 3Hz for fluid gels containing 0.2 wt% LA-gellan 450 gum and NaCl as gel-promoting ions. Temperature: 20ºC 451 452 Fig.3a. Mechanical spectra obtained at fixed shear stress of 0.02 Pa for fluid gels 453 containing 0.2 wt% LA-gellan gum and CaCl2 as gel-promoting ions. The error bars at 454 different frequencies have been included to indicate the reproducibility of the test. 455 Temperature: 20ºC 456 457 Fig.3b. Mechanical spectra obtained at fixed shear stress of 0.1Pa for fluid gels containing 458 0.2 wt% LA-gellan gum and NaCl as gel-promoting ions. The error bars at different 459 frequencies have been included to indicate the reproducibility of the test. Temperature: 460 20ºC 461 462 Fig.3c. CaCl2 concentration dependence on the plateau module (G0N) for fluid gels 463 containing 0.2 wt% LA-gellan gum and CaCl2 as gel-promoting ions. Temperature: 20ºC 464 465 Fig.3d. CaCl2 concentration dependence on exponent (n´) of the power equation which 466 relates G´ to the angular frequency (G´  n´) for fluid gels containing 0.2 wt% LA-gellan 467 gum and CaCl2 as gel-promoting ions. Temperature: 20ºC 468 469 Fig.4a. Corrected shear stress dependence of viscosity for fluid gels containing 0.2 wt% 470 LA-gellan gum and CaCl2 as gel-promoting ions. Temperature: 20ºC 471 472 Fig.4b. Corrected shear stress dependence of viscosity for fluid gels containing 0.2 wt% 473 LA-gellan gum and NaCl as gel-promoting ions. Temperature: 20ºC 474 475 17 Fig.5a. Shear rate dependence of corrected shear stress for fluid gels containing 0.2 wt% 476 LA-gellan gum and CaCl2 as gel-promoting ions and fitting curve of the Herschel- 477 Buckley model. Temperature: 20ºC 478 479 Fig.5b. Shear rate dependence of corrected shear stress for fluid gels containing 0.2 wt% 480 LA-gellan gum and NaCl as gel-promoting ions and fitting curve of the Herschel-Buckley 481 model. Temperature: 20ºC 482 483 484 485 486 487 1E-3 0.01 0.1 1 10 100 0.01 0.1 1 10 100 G´ G´´ /Pa / Pa 0.0·10-3 M CaCl2 1.1·10-2 M CaCl2 1.3·10-2 M CaCl2 1.6·10-2 M CaCl2 1.8·10-2 M CaCl2 Figure 1a 1E-3 0.01 0.1 1 10 100 0.01 0.1 1 10 100 G´ G´´/ Pa / Pa 9.0·10-3 M CaCl2 1.1·10-2 M CaCl2 1.3·10-2 M CaCl2 1.6·10-2 M CaCl2 1.8·10-2 M CaCl2 Figure 1b 1E-3 0.01 0.1 1 10 100 1 10 100 G´ G´´/ Pa / Pa 9.0·10-3 M CaCl2 1.1·10-2 M CaCl2 1.3·10-2 M CaCl2 1.6·10-2 M CaCl2 1.8·10-2 M CaCl2 Figure 1c 1E-3 0.01 0.1 1 10 100 0.01 0.1 1 10 100 G´ G´´/ Pa / Pa 0.22 M NaCl 0.24 M NaCl 0.27 M NaCl Figure 2a 1E-3 0.01 0.1 1 10 100 0.1 1 10 100 G´ G´´/ Pa / Pa 0.22 M NaCl 0.24 M NaCl 0.27 M NaCl Figure 2b 1E-3 0.01 0.1 1 10 100 1 10 100 G´ G´´/ Pa / Pa G´ G´´ 0.22 M NaCl G´ G´´ 0.24 M NaCl G´ G´´ 0.27 M NaCl Figure 2c Figure 3a Figure 3b 0.010 0.015 0.020 10 20 40 60 80 100 G0 N M CaCl2 G0 N (Pa) Figure 3c 0.008 0.010 0.012 0.014 0.016 0.018 0.020 0.00 0.02 0.04 n` n` M CaCl2 Figure 3d 0.1 1 10 100 1E-3 0.01 0.1 1 10 100 1000 10000 100000 1000000 1E7 9.0·10-3 M CaCl2 1.1·10-2 M CaCl2 1.3·10-2 M CaCl2 1.6·10-2 M CaCl 1.8·10-2 MCaCl / Pa·s / Pa Figure 4a 0.1 1 10 100 1E-3 0.01 0.1 1 10 100 1000 10000 100000 1000000 / Pa·s 0.22 M NaCl 0.24 M NaCl 0.27 M NaCl / Pa Figure 4b -200 0 200 400 600 800 1000 1200 1400 1600 1800 0 5 10 15 20 25 30 9.0·10-3 M CaCl2 1.1·10-2 M CaCl2 1.3·10-2 M CaCl2 1.6·10-2 M CaCl2 1.8·10-2 MCaCl2 corrected (Pa) / (s-1) . Figure 5a -200 0 200 400 600 800 1000 1200 1400 1600 1800 0 5 10 15 20 0.22 M NaCl 0.24 M NaCl 0.27 M NaCl corrected (Pa)  (s-1) . Figure 5b