2023 年 29 巻 3 号 p. 171-195
This article reviews different studies on the gelation mechanisms and network structural characteristics in polysaccharide gels at the different length scales of observation: macroscopic, microscopic, and molecular. Rheological and micro-DSC (Differential Scanning Calorimetry) measurements represent the macroscopic viewpoints on the gelation of pure kappa-carrageenan (KC), iota-carrageenan (IC) and KC/IC mixtures. Microscopic rheological properties observed using particle tracking experiments have been used to elucidate the formation of heterogeneous networks of KC-rich and IC-rich domains in mixed KC/IC gels, and facilitate observations of their aging process. Nuclear magnetic resonance (NMR) relaxation time measurements provide information on the tumbling motion and local mobility; these molecular-level experiments are presented for agar, agarose, native gellan and deacylated gellan chains. NMR diffusion measurements of gelling and probe polymers give insights into the conformational changes, aggregation and network formation, and aging mechanisms, and are presented for all studied polysaccharides.
In recent decades, polysaccharides have been produced commercially from seaweeds, bacteria, plants, and animals, and have drawn much attention in food applications due to their promising viscoelastic and gelling properties, water affinity, biocompatibility and biodegradability (Venugopal, 2011). Some of these polysaccharides are renewable and have interesting physical properties, such as film-forming (Mostafavi and Zaeim, 2020; Velásquez et al., 2022), gelling and thickening properties (Scholten and Pierik, 1998; Venkatesan, Anil, and Kim, 2017), which are highly sought after in different industries (Haug and Draget, 2009; Rinaudo, 2008; Stephen, Phillips, and Williams, 2006). In the food industry, polysaccharides have played an important role in making food gels and regulating their textural and sensory characteristics based on consumer preferences for a long time (Barrangou, Daubert, and Foegeding, 2006; Barrangou, Drake, Daubert, and Foegeding, 2006; Yang, Li, Li, Sun, and Guo, 2020). Depending on the intrinsic physicochemical properties and the extrinsic gelation factors, polysaccharide chains in aqueous solution can self-assemble into a variety of network structures by different mechanisms, which ultimately leads to gelation (Rinaudo, 2008; Stanley, 2006; Graham Sworn and Stouby, 2020). The network structures formed in this way do not only affect the local microenvironments of polysaccharide gels, but they also influence their macroscopic mechanical responses to applied stress. As newer polysaccharide gels are continuously introduced in the food industry, it is important to elucidate their physical properties at macroscopic, and more importantly, microscopic and molecular, scales. The physical characteristics of these polysaccharides are closely related to their applicability in food formulations, as they determine flavor release and regulate food texture (Barrangou et al., 2006; Barrangou et al., 2006).
Macroscopic rheology has been applied widely to understand and predict the physical and mechanical properties of food gels (Lapasin and Pricl, 1995). However, the focus of bulk rheology approaches in gel texture studies is the relationship between applied force and deformation (Chen and Stokes, 2012). Understanding the complex structures and dynamics of polysaccharides at various length scales is of utmost importance for food researchers who elucidate the underlying mechanisms that affect the interactions between food components and their physical properties (Moschakis, 2013).
Recently, nuclear magnetic resonance (NMR) spectroscopy and passive particle tracking have been developed as powerful tools for observing the network structure of polysaccharide gels at the molecular and microscopic levels (Brenner, Shimizu, Nantarajit, and Matsukawa, 2014; Matsukawa, Tang, and Watanabe, 1999; Matsukawa and Watanabe, 2007; Moschakis, Lazaridou, and Biliaderis, 2012; Moschakis, Lazaridou, and Biliaderis, 2014). NMR spectroscopy is a non-invasive tool that can be used to elucidate the molecular mobility and dynamics of food gels, and the method has been used to investigate the structural changes that occur in polysaccharides during gelation and aging (Dai and Matsukawa, 2013; Descallar and Matsukawa, 2020a). Measurements of the spin-lattice relaxation (T1) and spin-spin relaxation times (T2) provide information on the local mobility and flexibility of polysaccharide molecules (Dai and Matsukawa, 2012; Matsukawa et al., 1999; Zhang, Matsukawa, and Watanabe, 2004). Pulse-field-gradient (PFG) NMR measurements can provide information on the mobility of polymer chains, as the decay of echo signal intensity of the polymer chains reflects chain displacement due to self-diffusion, and can be used to calculate the diffusion coefficient, D (Shimizu, Brenner, Liao, and Matsukawa, 2012; Zhao, Brenner, and Matsukawa, 2013). The value of D gives information about the local interspatial network environment; for example, D can be used to estimate the hydrodynamic mesh size and the local viscosity of the medium (Dai and Matsukawa, 2013; Descallar and Matsukawa, 2020b; Price, 2009). This information is not accessible by using other noninvasive analytical techniques, such as small-angle X-ray scattering (SAXS) (Djabourov, Nishinari and Ross-Murphy, 2013) and dynamic light scattering (DLS) (Nicolai and Durand, 2013). NMR also has the advantage of being able to provide information about the molecular mobilities for each species in mixtures, even for concentrated solutions, owing to the differences in chemical shifts in spectra.
On the other hand, passive particle tracking is an emerging experimental technique in food science that has the potential to clarify the physical properties of food gels that are not accessible using bulk rheological measurement methods (Moschakis, Murray, and Dickinson, 2010; Moschakis, 2013; Shabaniverki and Juarez, 2017). Passive particle tracking is commonly performed by observing the intrinsic diffusion—driven by Brownian motion—of particles embedded in the sample, and is analyzed quantitatively in terms of the local viscoelastic properties of the surrounding medium. It is also non-invasive, i.e., does not affect the network structure at any length scale (Geonzon, Descallar, Du, Bacabac, and Matsukawa, 2020). The corresponding mean squared displacement (msd) of the probe particles is converted to microrheological properties, such as elastic and viscous moduli, using the generalized Stokes-Einstein equation (Crocker and Grier, 1996; Geonzon, Bacabac, and Matsukawa, 2019; Mason and Weitz, 1995).
Although many investigations have been conducted to clarify the physical and macroscopic properties of food gels, few reports have investigated their associated gelation mechanisms at microscopic and molecular scales. Herein, we review recent studies on the gelation mechanisms and network structures of polysaccharide gels (i.e. agar, agarose, carrageenan, and gellan gum). We highlight the different length scales of observation, i.e., macroscopic, microscopic and molecular length scales probed using macroscopic rheology, particle tracking and NMR spectroscopy, respectively. The information derived from different length scales is expected to provide a more comprehensive physical understanding of the molecular mechanisms and network structures of polysaccharide gels.
Carrageenan is a high-molecular weight, linear polysaccharide that is extracted from certain species of red seaweed (Rhodophyceae), including Eucheuma cottonii and E. spinosum (Falshaw and Furneaux, 1998; Imeson, 2009; Nussinovitch, 1997b). Carrageenan is comprised of repeating galactose units and 3, 6-anhydro galactose (3, 6 AG), both sulfated and non-sulfated, joined by alternating α-(1,3) and β-(1,4) glycosidic links. The main carrageenan types (lambda, kappa and iota) are differentiated by the number and position of sulfate side groups (S) in their molecular backbone, with different carrageenan types showing varying gelation properties (Imeson, 2009). For kappa-carrageenan (KC), strong, brittle, and opaque gels are produced on cooling a heated KC solution, especially in the presence of potassium ions. The gelation mechanism involves a coil-to-helix transition followed by extensive aggregation of the double helices (Liu, Chan, and Li, 2015). However, iota-carrageenan (IC) is only slightly affected by potassium ions. IC can interact with calcium ions to produce soft and elastic gels, while salts have nearly no effect on the properties of lambda-carrageenan (LC) (Imeson, 2009). However, it has recently been reported that LC can specifically bind trivalent cations, such as Fe3+ and Al3+, to form gels (Cao et al., 2018).
Differential scanning calorimetry (DSC) and rheology measurements are generally used to study the macroscopic changes that occur in polysaccharide solutions during gelation (Du, Brenner, Xie, and Matsukawa, 2016; Miyoshi, Takaya, and Nishinari, 1994). Fig. 1 shows the DSC traces of KC, IC and their mixture on cooling and reheating. As shown in the figure, only one exothermic and one endothermic peak appeared for KC solutions during the cooling and reheating processes. The onset temperature of KC on cooling (corresponding to the initial stage of helix formation) was almost independent of polymer concentration, but the offset temperature on heating (corresponding to the end of the helix-to-coil transition) increased with increasing polymer concentration. A possible explanation for this is that higher polymer concentrations led to increased helical aggregation, generating discernible thermal hysteresis (Du et al., 2016; Du, Lu, Geonzon, Xie, and Matsukawa, 2016). For IC solutions, both the onset temperature on cooling and the offset temperature on heating increased with an increase in IC concentration, and concentration dependence was stronger than that in KC. This result indicates that no helical aggregation was involved in the gelation process of IC. The DSC cooling traces of KC/IC mixtures exhibited two exothermic peaks at higher polymer concentrations (1.5–3.0 wt%), with the onset temperatures closely corresponding to the onset temperatures in solutions of the individual polysaccharides. The same observation was also made for the DSC traces upon reheating, where the offset temperatures of the mixed KC/IC solutions agreed well with the offset temperatures of individual KC and IC solutions. The DSC results indicate that the mixed KC/IC solutions formed two independent networks, with IC forming a network at a higher temperature and KC forming a network at a lower temperature. Fig. 2 shows the storage modulus G′ of KC, IC and their mixtures as a function of temperature during cooling and reheating. For KC/IC mixtures, two distinct increases in G′ were observed at all concentrations, which coincided with the increase in G′ in the individual KC and IC solutions. On heating, however, no distinct steps could be detected as G′ gradually decreased, probably because KC and IC networks melted over a similar temperature range.
DSC traces for solutions of KC (a, b), KC/IC mixture (c, d) and IC (e, f) with 10 mM KCl on cooling (a, c, e) and heating (b, d, f). The scanning rate was 0.5 °C/min. Reproduced from (Du et al., 2016) with permission from Elsevier.
Temperature dependence of the storage shear modulus (G′) for solutions of KC (a, b), KC/IC mixture (c, d) and IC (e, f) with 10 mM KCl on cooling (a, c, e) and heating (b, d, f). Reproduced from (Du et al., 2016) with permission from Elsevier.
Passive particle tracking (PPT) is a non-invasive technique that is commonly used to monitor the Brownian motion of particles embedded in media. The motion trajectories of the particles are recorded and used to calculate the msd, which is further analyzed to deduce the local physical properties of the host material (Geonzon, Bacabac, and Matsukawa, 2019b). Using particle tracking, spatial differences in the local physical properties and structural heterogeneity of the host material can be probed without affecting the developing network structure.
Fig. 3 shows the msd of neutral fluorescent-labeled probe particles (0.1 µm, Green, Thermo Fisher Scientific) embedded in a 1.5 wt% carrageenan solution as a function of lag time τ at various temperatures. The exponent α, which describes the particle diffusion in the relationship msd ∼ τα, can be obtained as the slope of the curve from double logarithmic plots of msd against τ. Particle diffusion with α = 1 implies unrestricted diffusion, while a value of α between 0 and 1 indicates restriction of the diffusion space within the network structure. When α = 0, i.e., when msd is independent of lag time, the probe particles are considered to be trapped in the gel network. As seen in Fig. 3, at temperatures above the gelation temperatures (∼35 °C for IC and ∼23 °C for KC, as obtained from rheological measurements), the msd of all particles in both KC and IC solutions was approximately proportional to τ. For KC, the magnitude and distribution of the msd curves of the particles changed drastically below the gelation temperature, while for IC, the msd curves did not change markedly even below the gelation temperature, indicating that these particles still exhibited free diffusional behavior.
Individual mean squared displacement (msd) plot against lag time of 0.1 µm probe particles embedded in 1.5 % carrageenan solutions with 10 mM KCl at different temperatures on cooling. A, B, C are for KC, and D, E, F are for IC, respectively. Reproduced from (Geonzon et al., 2019) with permission from Elsevier.
Because the msd at short τ is considered to only reflect local motion of the particles, the temperature dependence of msd at τ = 10 s in the KC and IC solutions was further calculated and plotted in Fig. 4. As shown in the figure, for KC, the msd remained almost constant at temperatures above the gelation temperature (∼23 °C) and decreased markedly below 23 °C, indicating a restriction of particle motion by the KC network. In IC solutions, the msd showed high values at temperatures above the gelation temperature (∼ 35 °C) indicating that the particles were present in a microenvironment with a low local viscosity. With decreasing temperature, the msd also showed a decrease below the gelation temperature, reflecting the gradual network formation of IC aggregates. A schematic representation describing the change in microscopic environments of KC and IC during gelation is shown in Fig. 5. As shown in the figure, KC chains rapidly formed thick aggregates that percolated to generate a permanent gel network upon cooling to the gelation temperature. For IC gels, there are two possible explanations for this behavior: 1) at the gelation temperature, IC chains formed heterogeneous clusters of aggregates, but did not percolate; and 2) IC chains formed a loose network structure with pores larger than 100 nm (the size of the probe particles). On further storage, however, an eventual permanent gel network of IC was formed.
Temperature dependence of ⟨msd⟩ at τ = 10 of 0.1 µm probe particles embedded in KC (circles) and IC (squares). ⟨msd⟩ for KC was multiplied with 3 for clarity. Error bars indicate one standard deviation. Reproduced from (Geonzon et al., 2019b) with permission from Elsevier.
Graphical representation of the proposed gelation mechanism of KC and IC solutions on cooling. Reproduced from (Geonzon et al., 2019b) with permission from Elsevier.
The microrheology of mixed KC/IC gels was also studied by particle tracking. Fig. 6 shows the distribution of msd at τ = 10 s as a function of α at different KC/IC mixing ratios for different solutions of 1.5 wt% carrageenan. As shown in the figure, the msd of the particles in the IC gel was higher and had an α value of ∼ 1, while the msd in the KC gel was lower and had an α value of α ∼ 0, implying that there was a distinct difference in the microrheology of pure KC and IC gels. In the mixed KC/IC gels, two groups of particles with fast and slow mobilities were detected. The faster particles were considered to be present in IC-rich domains, while the slower particles were considered to exist in a microenvironment rich in KC. As the KC/IC ratio increased, the values of msd and α approached those observed in the pure KC gels. In particular, in the KC50IC50 gel broad distributions of msd and α were observed. The particles in the IC-rich domain showed a lower mobility than that observed in the pure IC gel, while the particles in the KC-rich domain showed a higher mobility than in the pure KC gel. A possible interpretation is that non-negligible amounts of KC and IC were present in IC-rich and KC-rich domains, respectively. The presence of KC chains in IC-rich domains increased the local viscosity of the IC-rich domains, and the IC chains in KC-rich domains decreased the local obstruction of the KC domains. Thus, the structural inhomogeneity observed in the KC/IC mixture suggested a possible structure that phase-separated into KC-rich and IC-rich domains, both of which were expected to have domain sizes larger than the size of probe particles (100 nm).
Distribution of msd and α (τ = 10 s). The solid lines indicate Gaussian probability distributions of KC and IC. In Fig.s b–g, the red dashed lines indicate bimodal Gaussian distributions. Reproduced from (Geonzon, Bacabac, Matsukawa, 2019a) with permission from ECS.
NMR is a powerful tool for studying the structural changes of polysaccharide chains during gelation and for providing information about molecular mobility in the network structure. Measurements of the T1 and T2 relaxation times provide information on the local mobility and flexibility of the polymer chains. On the other hand, the mobility of an entire molecule can be determined by pulsed-field-gradient (PFG) NMR, where the decay of the echo signal intensity with increasing gradient strength reflects the displacement of the molecule, which can be further used to calculate the molecule's diffusion coefficient D.
Agar Agar is the generic name for a family of structurally related polysaccharides extracted from the Rhodophyceae phylum, first exclusively from Gelidium amansii, and is composed of alternating D- and L-galactopyranose units (Araki, 1956; Armisén and Gaiatas, 2009; Nussinovitch, 1997a). The wide range of agar applications is based on its ability to form strong gels in aqueous solutions. On cooling, the gelation of agar solutions is initiated by a coil-to-helix transition, followed by the aggregation of the helices to form a network structure (Arnott et al., 1974; Nussinovitch, 1997a). The physicochemical, mechanical and rheological properties of different agar gels depend on the source and seaweed variety. Despite the fact that agaropectin does not form a gel by itself, the presence of agaropectin is shown to enhance the gelation of agar (Nishinari and Fang, 2017).
Molecular mobility of agar chains Stacked Pulsed-Field Gradient Stimulated Echo (PFG-STE) 1H NMR spectra for 2.3 wt% agar solution at various temperatures are shown in Fig. 7. The deuterated water proton HDO peaks were eliminated completely due to the fast diffusion of water, leaving only the signals from agar. A marked decrease in the intensity of the agar peaks can be observed during cooling, and a slight increase in agar peak intensity during heating was also observed. The intensity of the peaks at chemical shifts of 3.0–5.8 ppm was analyzed. The equation for the attenuation of peak intensity in the spin-echo spectra is given by equation 1, where I(g) and I(0) represent the echo intensities at t = 2t2 + t1 with and without the field gradient, respectively, and γ is the gyromagnetic ratio of 1H. Therefore, the D of agar (Dagar) was determined by fitting the equation 1 to the data points, which also yielded I(0) for the agar (Iagar(0)).
Stacked 1H NMR spectra of the 2.3 wt% agar solution during cooling (a–d), 15 h of storage at 24 °C (e), and reheating process (f–h). Reproduced from (Dai and Matsukawa, 2012) with permission from Elsevier.
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The temperature dependences of Iagar(0) and Dagar are shown in Fig. 8, where Iagar(0) values were normalized based on Iagar (0) at 60 °C. During cooling, Iagar(0) remained constant over the temperature range 60 to 50 °C (stage 1), and decreased sharply from 50 to 35 °C (stage 2) (Fig. 8a). Polysaccharide chains in agar are thought to adopt a random and semi-stiff coil conformation with some degree of segmental mobility when dissolved in water at high temperatures. This structure results in relatively long 1H T 1 and T2 values, both of which are much longer than 2τ2. In contrast, the polysaccharide chains in agar involved in the aggregation of helices have strongly restricted segmental mobility because of their rigid structure (Arnott, Fulmer and Scott, 1974), which led to much shorter 1H T2 values and the disappearance of corresponding agar peaks from the NMR spectrum. Further, only agar chains that were present as random semi-stiff coils contributed to the intensity of Iagar(0). Thus, a further decrease in Iagar(0) during stage 2 was thought to indicate a decline in the number of random and semi-stiff coils that facilitated an increase in the number of helix aggregates, which in turn formed thick bundles and produced the gel network (Aymard et al., 2001).
Temperature dependence of (a) 1H peak intensities of agar at g = 0, Iagar(0), and (b) the diffusion coefficient of agar, Dagar, in a 2.3 % agar solution during cooling (closed triangles) and heating (open triangles). Reproduced from (Dai and Matsukawa, 2012) with permission from Elsevier.
On the other hand, Dagar decreased slightly in stage 1 due to a decrease in molecular mobility with decreasing temperature (Fig. 8b). The high value of Dagar observed at 60 °C could have been caused by overestimation of the displacement due to convection effects (Matsukawa, Sagae and Mogi, 2009; Price, 2009; Déléris et al., 2010; Walderhaug, Söderman and Topgaard, 2010). The value of Dagar increased significantly with decreasing temperature in stage 2. A large proportion of polysaccharide chains in agar solution formed aggregates of helices in this stage, as evidenced by the change in Iagar (0). The polysaccharide chains in agar solutions are believed to comprise both gelling (agarose) and non-gelling chains, the latter of which (agaropectin) carry ionic charges (Nussinovitch, 1997; Araki, 1956; Arndt and Stevens, 1994; Arnott et al., 1974; Tako and Nakamura, 1988). If the larger or higher Mw polysaccharide chains in agar were preferentially involved in the aggregation, then the molecular weight distribution of the non-aggregated solute agar left to diffuse in the interspaces of the forming network should shift to lower values, causing an increase in the measured value of D. Such an increase in D values was indeed in the monomodal diffusion data (Dai and Matsukawa, 2012). Based on these results, a schematic representation of the agar chains at different stages during cooling and heating is shown in Fig. 9. In stage 1, polysaccharide chains exist in a semi-stiff and disordered coil conformation in the hot solution. In stage 2, these coils form ordered helices that subsequently aggregate into thick bundles, generating a network structure on cooling below the sol-to-gel transition temperature (Ts-g). In stage 3, some of the coil (solute) agar chains gradually form loose aggregates upon further cooling. On reheating, these loose aggregates dissociate with relative ease, as was observed in stage 4. Ultimately, in stage 5, a portion of the thick bundles melts gradually when the temperature is increased further.
Schematic representation of the conformation of agar molecules at different stages of cooling and heating. Reproduced from (Dai and Matsukawa, 2012) with permission from Elsevier.
Dendrimer self-diffusion in agar solutions Intermolecular interactions between probe molecules and network chains greatly affect molecular diffusion (Cukier, 1984b). The diffusion coefficient of host molecules can decrease due to hydrodynamic interactions, hydrogen bonding and hydrophobic interactions. In polymer gels, the network size, which affects the hydrodynamic interactions, depends on the polymer concentration. In many polymer solutions, the network is formed through polymer aggregation. If a probe molecule is involved in the aggregation, or associated with the gel network structure, its molecular mobility is strongly restricted, resulting in a decrease in D (Matsukawa and Ando, 1999). However, if the probe is not involved in the aggregation and is free to diffuse in network interspaces, then D is influenced by hydrodynamic interactions with the aggregated network and the number of entanglements changes due to aggregation. Consequently, D varies even if the total polymer concentration is constant throughout the aggregation and gelation process. Regarding the molecular mobility of polymers, the tumbling and translational motions of chains change markedly during aggregation and gelation, which affects the value of D and NMR relaxation times. The D measured by field gradient NMR reflects the displacement of a molecule by self-diffusion, which in turn describes the interspaces in the gel (Matsukawa et al., 1999a).
The structural changes and gelation mechanisms of agar have been proposed based on the results of the field-gradient NMR measurements of the molecular mobility of the agar chains. To elucidate the effects of the local polymer concentrations on agar gels, a probe polymer (dendrimer) was added to the agar solution and its D was measured at various temperatures. In the PFG-STE 1H NMR spectra of a 2.3 wt% agar solution containing 0.1 wt% dendrimer, the peaks of the ethylene protons in the dendrimer appeared in a separate region that was free of agar peaks, from 2.01 to 3.03 ppm (data not shown), and were therefore used for the analysis. Fig. 10a and 10b show the temperature dependence of I(0) and D for the dendrimer (Idend(0) and Ddend, respectively) in this solution. Idend(0) exhibited a gradual and continuous increase with decreasing temperature, and remained at a slightly higher value during reheating. This increase in Idend(0) was probably caused by longer T1 and T2 relaxation times for the dendrimer upon dilution of the coil (solute) agar, as the presence of solute agar restricted dendrimer mobility and thus attenuated its signal. The continuous change in Idend(0) indicated that the dendrimer was not involved in the aggregation of the agar chains, which meant that hydrodynamic interactions of the dendrimer with the agar chains was likely the dominant factor affecting dendrimer diffusion. Ddend showed a decrease in stage 1, a slight increase in stage 2 and a decrease with decreasing temperature in stage 3 (these stages correspond well to the stages shown in Fig. 9). The dendrimer concentration used in this study (0.1 %) was considerably lower than its critical overlapping concentration (c*, 27 %), which was calculated from its molecular weight (Mw) and hydrodynamic radius (RH) using the formula c* = 3Mw/4NA RH (Bieze, van der Maarel, Eisenbach, and Leyte, 1994). At this low concentration, Ddend,0 is considered to be identical to that of an infinitely dilute solution. Therefore, the RH value of the dendrimer was estimated from Ddend,0 at 24.3 °C using the Stokes-Einstein equation:
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where, kB, T and ηs are the Boltzmann constant, absolute temperature and viscosity of the solvent, respectively. Because RH of the dendrimer is considered to be constant due to its highly branched structure, the Ddend,0 values at various temperatures were calculated using the following equation:
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Temperature dependence of (a) 1H-NMR peak intensities of the dendrimer at g = 0, Idend(0), (b) the dendrimer diffusion coefficient, Ddend, and (c) Ddend/Ddend,0, measured in a 2.3 % agar solution with 0.1 % dendrimer. Closed and open triangles indicate cooling and heating. Values of Ddend,0 in dilute D2O solution calculated using Eq. (3) are indicated with a dashed line in (b). Theoretical ξ values, calculated using Din Host/Din Pure = exp(-RH/ξ), are shown on the right-hand axis of (c). Reproduced from (Dai and Matsukawa, 2012) with permission from Elsevier.
When Ddend is compared with Ddend,0, which was calculated using equation (3) and is shown as a dashed line in Fig. 10b, Ddend is substantially lower than Ddend,0. This result indicated that the diffusion of the dendrimer was restricted in the agar solution and that the decrease in Ddend reflected the degree of restriction. As a measure of this restriction, the ratio Ddend/Ddend,0 was calculated at each temperature and plotted in Fig. 10c. As shown in the figure, Ddend/Ddend,0 was constant during stage 1, increased (i.e., the restriction decreased) in stage 2, and reached a plateau in stage 3. Ddend/Ddend,0 then remained almost constant during the reheating process (stage 4) and decreased slightly in stage 5.
The diffusion of probe molecules in the solution of a host polymer, which has no intermolecular interactions with the probe except hydrodynamic interactions mediated by the motion of the solvent fluid and is generally larger than the probe, is expressed as follows (Cukier, 1984a; De Gennes, 1976; Matsukawa et al., 1999b),
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where, D in Host and Din Pure are diffusion constants of the probe molecules in the host polymer solution and in the pure dilute solution, respectively, and ξ is the hydrodynamic shielding length, considered to represent the hydrodynamic mesh size formed by the host polymer. The pure diluted state is considered to be the concentration at which it is much lower than the overlapping concentration (c*), and the restriction of the diffusion of probe is within the hydrodynamic region where RH is less than ξ, and no other intermolecular interactions exist between the host polymer and the probe (Bieze et al., 1994; Descallar and Matsukawa, 2020a). The ξ values calculated from Ddend/Ddend,0 using equation (4) are shown on the right-hand axis in Fig. 10c. In the agar solution containing the dendrimer, the solute agar, rather than the aggregated bundles, should predominantly assume the role of host polymer. Since the dendrimer is considered to be the probe polymer, the ξ value should to represent the hydrodynamic mesh size of the solute agar chains. As shown in Fig. 10c, the change in ξ captured well the change in the degree of restriction discussed earlier and provided insight into the microscopic gelation mechanisms of agar.
Agarose Agarose is the sulfate-free and neutral (non-ionic) gelling fraction of agar. It consists of repeating units of alternating (1➔3)-linked β-D-galactopyranose and (1➔4)-linked 3, 6 anhydro-α-L-galactopyranose units, designated as G and A residues, respectively (Araki, 1956; Gamini, Toffanin, Murano, and Rizzo, 1997). The gelation of agarose is considered to be initiated by the coil-to-helix transition, and proceeds with further aggregation of the helices (Dai and Matsukawa, 2013; Khalil et al., 2017; Tako and Nakamura, 1988). Agarose chains adopt a random and stiff coil conformation at high temperatures (Guenet, Brulet, and Rochas, 1993). Upon cooling below the gelation temperature, the coils order to form helices and subsequently aggregate into a three-dimensional network of thick bundles that form a gel (Arnott et al., 1974). Due to this highly ordered proposed structure, agarose typically forms rigid and opaque gels and shows a significant degree of hysteresis between cooling (gelling) and on heating (melting) (Indovina, Tettamanti, Micciancio-Giammarinaro, and Palma, 1979).
Molecular mobility of agarose chains In the pulse-field-gradient stimulated echo (PFG-STE) 1H NMR spectra of 2.1 wt% agarose solution containing 0.1 wt% probe polymer dendrimer, the peak at 5.16 ppm, assigned to the 1H on the 4-linked 3,6-anhydro-α-L-galactopyranose unit of agarose (Gamini et al., 1997) and the peaks of the dendrimer-associated ethylene protons appearing between 2.33 and 3.11 ppm, were used for the analysis (data not shown) (Dai and Matsukawa, 2013). Fig. 11 shows the temperature dependencies of Iaga(0) (determined from the PFG-STE and CPMG (Carr-Purcell-Meiboom-Gill) measurements) and Daga. During cooling, the Iaga(0) values, determined from both PFG-STE and CPMG measurements, remained relatively constant when the temperature decreased from 55 to 50 °C (stage 1), markedly decreased as the temperature decreased from 50 to 35 °C (stage 2) and gradually decreased below 35 °C (stage 3) (Fig. 11a). This decrease in the peak intensity during stage 2 was due to the formation of aggregated bundles with a highly restricted segmental mobility, as only the coil (solute) agarose chains still contributed to the CPMG signal and the echo intensity. The higher-molecular weight agarose chains in the aggregates had strongly restricted mobility that resulted in a short 1H spin-spin relaxation time (T2) and caused a fast decay during the echo times. It should be noted that Iaga(0) began to decrease at around 50 °C and continued to decrease down to approximately 30 °C, passing through Ts-g of 41 °C. This result suggested that the aggregated bundles were formed above Ts-g and a three-dimensional network structure was formed when approximately half of the agarose chains had aggregated. From the observed gradual decrease in Iaga(0) during stage 3, it was inferred that the solute agarose was progressively involved in forming bundles as the temperature was lowered. The value of Iaga(0) at 30 °C was only about 2 % of its value at 55 °C, which reflects the high gelling ability of agarose; in other words, almost all of the agarose chains were in the aggregated form as the temperature decreased to 30 °C.
Temperature dependence of (a) Iaga(0) on cooling (PFG-STE: filled squares, CPMG: open circles) and reheating (PFG-STE: crosses), and (b) Daga on cooling (filled squares) and reheating (crosses), as measured for a 2.1 % agarose solution. Reproduced from (Dai and Matsukawa, 2013) with permission from Elsevier.
As shown in Fig. 11b, the D of agarose chains (Daga) remained constant during cooling in stage 1, but increased markedly in stage 2. This increase reflected the formation of the aggregated bundles which led to a decrease in the concentration of solute agarose chains. The friction between the diffusing (solute) agarose chains and network bundles was reduced, which decreased Daga. In addition, Daga showed a tendency that was different from that observed for the agar solution, where the coil (solute) agar chains eventually formed loose aggregates and led to a decrease in the D value of agar chains (Dai and Matsukawa, 2012). On the other hand, the 1H T2 values showed only a small change on cooling, suggesting the presence of opposing effects of the decrease in temperature and a shift in the molecular weight of agarose to lower values as larger chains aggregated; the latter has a weaker effect on the segmental mobility than on molecular diffusion. In stage 3, the observed increase in Daga was attributed to the presence of only agarose chains with small Mw in the coil conformation.
Aging mechanism of agarose gels Diffusion of pullulan as a probe molecule in agarose gels was investigated to clarify the structural changes in gels during aging. Fig. 12 shows the NMR spectra of agarose gels, showing protons H-1 and H-4 of the 3, 6 anhydro-α-L-galactopyranose unit (Gamini et al., 1997)) at different storage times. As shown in the figure, the agarose peaks appeared to vanish after three days of storage. It is assumed that the solute agarose chains formed loose aggregates during early storage before eventually being involved in the aggregation of the network structure, which resulted in the disappearance of the agarose peaks. On the other hand, the signal intensity of the pullulan peaks remained unchanged. This result confirmed that pullulan was not involved in the aggregation of agarose chains and retained high molecular mobility.
Stacked PFG-STE 1H NMR (g = 150 G/cm) spectra of a 2.0 % agarose gel with 0.1 % pullulan at different storage time: (a) day 0, (b) 15 h, (c) day 1, (d) day 2, (e) day 3, (f) day 7, (g) day 14, (h) day 21 and (i) day 28. Reproduced from (Descallar and Matsukawa, 2020a) with permission from Elsevier.
To estimate the degree of restriction of pullulan diffusion by the agarose aggregates, the ratio of Dpull to Dpull,0 (Dpull/Dpull,0) was calculated. The value of Dpull/Dpull,0 is shown as a function of storage time, as indicated by the left-hand axis of Fig. 13. This ratio showed a marked increase after three days of storage and remained constant during further storage. The increase in Dpull/Dpull,0 could be related to the formation of thicker bundles of agarose aggregates, which would leave larger spaces for the pullulan to diffuse faster through the gel. The corresponding ξ value was considered to represent the hydrodynamic mesh size of the agarose aggregates, which started to increase from approximately 7 nm to approximately 30 nm after three days of storage, indicating a pronounced change in the network structure. According to a previous NMR study on the formation agarose networks (Dai and Matsukawa, 2013), the diffusion of the probe polymer was retarded by hydrodynamic interactions with both the solute agarose and aggregated bundles. In this study, the small NMR peaks from agarose chains that were observed on day 0 disappeared after three days of storage. Therefore, it was considered that the solute agarose chains formed loose aggregates, and the bundles of loosely aggregated agarose underwent densification, which acted to enlarge the space available for pullulan diffusion (Descallar and Matsukawa, 2020a; Descallar, Wang, and Matsukawa, 2022). The microscopic changes associated with agarose network aging are illustrated in Fig. 14.
Storage time dependence of Dpull/Dpull,0 in a 2.0 % agarose (diffusion time ◿ = 20 ms. Corresponding ξ values are displayed on the right-hand axis. Reproduced from (Descallar and Matsukawa, 2020) with permission from Elsevier.
Schematic depiction of the conformation of agarose network during storage. Reproduced from (Descallar and Matsukawa, 2020) with permission from Elsevier.
Gellan gum Gellan gum is biosynthesized by the bacterium Pseudomonas elodea under sterile fermentation conditions with strict control of aeration, agitation, temperature and pH (Sworn, 2009). At the end of the fermentation, the polysaccharide can be recovered in different ways. Direct recovery by alcohol precipitation from the fermentation broth gives the native state of gellan gum, which shows a relatively high degree of acylation. Alternatively, treatment of the broth with alkali before alcohol precipitation yields the deacylated form of gellan gum, which is referred to simply as “gellan” (Morris, Nishinari, and Rinaudo, 2012; Sworn, 2009; Sworn and Stouby, 2020). In the presence of gel-promoting cations, deacylated gellan (DG) can form gels on cooling of hot polysaccharide solutions, and the gelation mechanism is thought to be initiated by the coil-to-helix transition and proceeds by helical aggregation (Ogawa, 1996; Takahashi et al., 2004). On heating, DG gels show a significantly higher melting temperature than the gelformation temperature, or thermal hysteresis. However, for native gellan (NG), the presence of glyceryl and acetyl groups affects significantly the gelation process, with the glyceryl substituents contributing to the stability of the helical structures and the acetyl substituents hindering further helical aggregation (Morris, Gothard, Hember, Manning, and Robinson, 1996). On reheating, NG gels exhibit no thermal hysteresis (Morris, Nishinari, and Rinaudo, 2012). In addition, mixing NG and DG to produce gels with intermediate textures has been studied (De Silva, Poole-Warren, Martens, and in het Panhuis, 2013; Huang, Tang, Swanson, and Rasco, 2003; Mao, Tang, and Swanson, 2000; Noda et al., 2008). These studies stimulated the study of the molecular mechanisms involved in the gelation of NG/DG mixtures.
Water 1H T 2 measurements in gellan gum In NMR, measurements of water 1H T2 provide important information on the mobility of polysaccharide chains, as values are affected by chemical exchange between water protons and exchangeable polysaccharide protons (Hu, Lu, Zhao, and Matsukawa, 2017). On cooling, the chain mobility of polysaccharides tends to be strongly restricted at the gelation temperature, which leads to a steep decrease in the T2 of water. Water T2 measurements for DG solutions have been conducted previously. It was demonstrated that water T2 significantly changed as the result of conformational changes of DG, such as the random coil-double helix transition and the formation of helical aggregates. These conformational changes affect the rate of proton exchange between water and the hydroxyl groups of gellan (Matsukawa et al., 1999). In order to further elucidate the molecular mechanisms of gelation in gellan gum, the temperature dependence of water T2 for DG, NG and their mixtures at different Ca2+ concentrations during cooling and reheating was analyzed (Fig. 15). In the DG solutions, the T2 relaxation time of water decreased gradually during initial cooling, followed by a steep decrease around the gelation temperature of each solution, indicating that DG chains aggregated rapidly and showed restricted chain mobility at this temperature. For NG, the water T2 relaxation time also decreased sharply, but less so than for DG, suggesting that NG gelation was less extensive. In each of the NG and DG solutions, the temperature corresponding to the steep decrease in water T2 increased with increasing Ca2+ concentration, indicating that the addition of Ca2+ promoted the gelation of both NG and DG. In addition, water T2 relaxation showed higher sensitivity to Ca2+ for DG solutions than for NG solutions, confirming that DG gelation is more strongly affected by Ca2+ addition (Morris et al., 2012). The higher sensitivity of DG to Ca2+ was likely attributed to the absence of acyl groups. Previously, it was reported that L-glyceryl groups could stabilize the double helix structure, but also remove the binding site for metal ions by changing the orientation of the adjacent glucuronate residues and the carboxyl groups (Morris et al., 1996). Moreover, helical aggregation was further inhibited by acetyl groups located on the periphery of the double helix (Morris et al., 2012). In mixed NG and DG solutions, the water T2 relaxation time exhibited a two-stepped decrease at the temperatures corresponding to the temperatures observed in separate NG and DG solutions. This result indicates that NG/DG mixtures undergo a two-stepped gelation process, and that double helices always involved two chains of the same type. This result is consistent with the rheological, DSC and circular dichroism data presented in an earlier study (Matsukawa and Watanabe, 2007).
Temperature dependence of water T2 for DG (circles), NG (triangles) and DG/NG mixtures (squares) with different concentrations of Ca2+ during cooling (a, c, e, g) and reheating (b, d, f, h). The total polysaccharide content was fixed to 1 % in all samples. Reproduced from (Hu et al., 2017) with permission from Elsevier.
On reheating, the water T2 showed pronounced hysteresis of all DG gels. In contrast, water T2 values showed no hysteresis of any NG gels. Notably, for mixed NG/DG solutions, the increase in water T2 was steeper at higher temperatures than that in NG solutions. This trend was maintained at all Ca2+ concentrations. These findings imply that the formation of DG aggregates increased the stability of NG aggregates.
Molecular mobility of DG chains Fig. 16 shows the pulsed-field-gradient 1H NMR spectra of a 1.0 % DG solution with 5 mM CaCl2 and 0.1 % pullulan added as the probe polymer at 30 °C. As shown in the figure, with an increase in gradient strength (g), the peak of the water proton (at 4.7 ppm) rapidly decreased due to the high diffusivity of water. The peak at 1.3 ppm (assigned to the methyl protons on the rhamnose unit of DG) exhibited a small decrease, indicating the slow diffusion of coiled (solute) DG. The peak at approximately 5.3 ppm, assigned to the anomeric proton on pullulan, also slowly decreased with an increase in g, reflecting the slow diffusion of pullulan. The peaks at chemical shifts of 1.3 and 5.3 ppm were used for further analysis.
Pulsed-ffield-gradient 1H NMR spectra for a 1.0 % DG solution with 5 mM CaCl2 containing 0.1 % pullulan at 30 °C. Reproduced from (Shimizu et al., 2012) with permission from Elsevier.
Fig. 17 shows the echo signal intensity at 1.3 ppm and the corresponding diffusion coefficient D of gellan as a function of temperature for 1.0 % DG solutions with various cationic contents. The echo signal intensity I0 of each solution was normalized with its value at 60 °C. As shown in the figure, in the cooling process, I0 was almost constant and started to decrease markedly at around 36, 43, 38 and 45–50 °C for solutions with 2 mM CaCl2, 5 mM CaCl2, 40 mM KCl and 80 mM KCl, respectively. The decrease in NMR signal intensity indicates the marked decrease in chain mobility. For the 2 mM CaCl2 solution, the change in I0 during cooling was highly consistent with that in the subsequent heating process, as only a slight decrease in I0 on heating was observed. For the 5 mM CaCl2 solution, a greater decrease in I0 on heating was observed, indicating that higher Ca2+ concentrations promoted the formation of helical aggregates and thus produced a higher level of thermal hysteresis; a similar phenomenon was also observed in the presence of KCl. On reheating, I0 showed hysteresis, the extent of which depended on the cation present and its concentration. This thermal hysteresis is generally considered to reflect the thermal stability of the gellan helical aggregate (Morris et al., 2012).
Temperature dependence of the echo signal intensity at 1.3 ppm, I(0), and the diffusion coefficient (Dgel) for 1.0 % DG solutions with 0.1 % pullulan and different salt contents. Closed and open circles represent cooling and heating. Reproduced from (Shimizu et al., 2012) with permission from Elsevier.
The diffusion coefficient of gellan chains (Dgel) remained relatively constant above 36, 43 and 38 °C in the 2 mM CaCl2, 5 mM CaCl2 and 40 mM KCl solutions, respectively. Further cooling resulted in a slight increase in Dgel. A possible explanation for this observation was that, on cooling, the concentration of coiled (solute) gellan chains decreased markedly at around these temperatures (corresponding to the coil-to-helix transition), which reduced the local viscosity of the solutions and made it easier for the remaining solute gellan chains with lower molecular weight to diffuse. However, Dgel increased as the temperature was decreased to the coil-to-helix transition temperature for the 80 mM KCl solution, and then remained almost constant on further cooling. This observation likely occurred because, at higher cation concentrations, the electrostatic repulsion between negative charges on the carboxylate groups can be suppressed to a larger extent and the solute gellan concentration decreased more rapidly.
Diffusion of pullulan in DG gels Pullulan was used as a probe molecule to elucidate the dynamics of DG solutions during gelation (Shimizu, Brenner, Liao, and Matsukawa, 2012). The self-diffusion coefficient D of pullulan (Dpul) was measured, as shown in Fig. 18. For all DG solutions studied, Dpul decreased in the temperature range above the coil-to-helix transition temperature, indicating a decrease in molecular mobility. The solution viscosity increased on cooling, which is a simple explanation for the reduction in Dpul. Dpul in the solutions containing 5 mM CaCl2 and 80 mM KCl increased slightly at 43 °C and 50 °C, respectively, possibly due to less restriction by DG chains as they began to form helices and aggregate. At the gelation temperatures, DG chains with higher molecular weight preferentially aggregated into thick bundles and formed a network, leaving less coiled (solute) gellan in the interspaces of the network and more space for pullulan to diffuse in. On further cooling, the Dpul values in all solutions decreased concomitantly with a decrease in the amount of solute gellan chains (Shimizu et al., 2012). On reheating, the Dpul values in all solutions increased monotonically, and the values were consistently higher than those during cooling, suggesting that the concentration of solute gellan chains was lower than it had been during the original cooling step.
Temperature dependence of the diffusion coefficient of pullulan (Dpul) and Dpul/Dpul,0 in 1.0 % DG solutions containing 0.1 % pullulan and different salt contents. Closed and open circles represent cooling and heating. Reproduced from (Shimizu et al., 2012) with permission from Elsevier.
Dpull/Dpull,0 was calculated as a measure of the degree of restriction on pullulan diffusion. As shown in Fig. 18, Dpull/Dpull,0 values showed pronounced hysteresis loops that depended on the cation present and its concentration. For the solutions containing 2 mM CaCl2 and 40 mM KCl, Dpull/Dpull,0 showed slightly thermal hysteresis above 36 and 38 °C on heating, whereas a thermal hysteresis was higher in the solutions containing 5 mM CaCl2 and 80 mM KCl. This result is consistent with the Dgel measurements.
Molecular mobility in mixed NG and DG gels Due to different gelation mechanisms, NG and DG give gels very different textural characteristics. NG forms soft, elastic and non-brittle gels, whereas DG produces hard, brittle and non-elastic gels (Sworn, 2009). Thus, NG and DG mixtures have been suggested to produce gels with intermediate textures (Huang et al., 2003; Mao et al., 2000). The gelation mechanism in mixed NG/DG solutions, however, is not well understood. Fig. 19 shows the NMR spectra of a mixed NG/DG solution as measured by PFG-STE NMR. The signal of water (at 4.7 ppm) was eliminated completely due to rapid diffusion, leaving only the signals from the gellan protons. As the temperature decreased, signal intensities decreased, but the linewidths of all peaks were almost unchanged. This result indicates that there were two polysaccharide fractions with different chain mobilities, i.e., one fraction with a highly restricted mobility and a short T2 relaxation time that does not appear in the NMR spectrum, and another fraction with a higher mobility and a long enough T2 relaxation time that is not affected by temperature, as reflected in the unchanged linewidths of all proton signals. The signals at 1.3 and 2.1 ppm are assigned to the methyl protons of rhamnose and the protons of the acetate group on (1,3) β-D-glucose, respectively. The methyl protons from rhamnose correspond to both NG and DG chains, while the acetate group is present only in the high-acyl gellan (Matsukawa and Watanabe, 2007). Thus, NG chain mobility is reflected by the intensity of the signals at both 1.3 and 2.1 ppm, while DG chain mobility affects only the signal at 1.3 ppm.
PFG-STE NMR spectra of a mixed gellan solution (0.5 % NG + 0.5 % DG) at various temperatures during cooling. Reproduced from (Matsukawa and Watanabe, 2007) with permission from Elsevier.
The signal intensities at 1.3 and 2.1 ppm (normalized by their respective values at 80 °C) are shown as a function of temperature in Fig. 20. For the 1.0 % NG solution, the signal intensities of both 1.3 and 2.1 ppm peaks, expressed as I1 .3 and I 2.1, decreased markedly on cooling from 72 to 60 °C, followed by a gradual decrease with further cooling. The steep decrease in I1.3 and I 2.1 was attributed to the formation of double helices, which resulted in highly restricted chain mobility. The increase in I1.3 and I 2.1 in the temperature range of 85 to 72 °C was likely due to the increase in signal recovery during the repetition time due to the short T1 (Matsukawa and Watanabe, 2007). For the mixed solution, both I1.3 and I 2.1 started to decrease at 72 °C, but the extent of the decrease in I1.3 is half that of I 2.1. Given that I1.3 contains signals from both DG and NG while I 2.1 contains only signals from NG, this result indicates that only DG chains were involved in the formation of the double helix structure in this temperature range. On further cooling to 25 °C, I1.3 showed a steep decrease, which was also attributed to the formation of a double helix structure by DG chains.
Temperature dependence of the signal intensities at 1.3 (solid symbols) and 2.1 ppm (open symbols) of (a) 1.0 % NG and (b) 0.5 % NG + 0.5 % DG solutions. The signal intensities were normalized by their respective values at 80 °C. Reproduced from (Matsukawa and Watanabe, 2007) with permission from Elsevier.
T2 measurements on carrageenan T2 relaxometry was also applied to clarify the molecular mobility and dynamics of KC, IC and their mixtures during gelation (Hu, Du, and Matsukawa, 2016). Fig. 21 shows the temperature dependences of I0, T2 relaxation times and the diffusion coefficients D for KC, IC and a KC/IC mixture. The I0 of KC remained generally constant during cooling at temperatures above 35 °C and a steep decrease at temperatures less than 30 °C, indicating that the coil-to-helix transition of KC chains occurred at approximately 30 °C. In contrast, the I0 of IC (I0,IC) showed a gradual decrease throughout the cooling process, implying that IC aggregates were less rigid than KC aggregates. During cooling, T2 relaxation times in KC solutions (T2,KC) decreased slightly at temperatures above 35 °C, probably due to the decreased segmental mobility of the KC chains. With further cooling from 35 to 25 °C, T2,KC increased markedly, probably due to the formation of aggregates by most of the KC chains. These findings imply that most KC had a highly restricted segmental mobility with very short T2 relaxation times. Thus, only coiled (solute) KC chains, which probably have lower molecular weight, could still contribute to the echo signal intensity. For IC, the T2 values (T2,IC) were essentially constant in the high temperature range from 60 to 45 °C and increased slightly at temperatures below 41 °C, suggesting that longer IC chains were preferentially involved in the formation of aggregates and leaving only shorter IC chains as solute. During cooling, the diffusion coefficient D of KC chains was relatively constant and showed a significant increase with a decrease in temperature from 35 to 25 °C. This increase in D was much larger than expected from the decrease in the local restriction, suggesting that longer KC chains were preferentially involved in the formation of aggregates, shifting the average molecular weight of coiled (solute) KC chains to lower values. For IC, the D value was constant in the high temperature range from 60 to 45 °C, followed by an increase with further cooling, suggesting that longer IC chains were initially involved in the formation of helical aggregates, in a manner similar to the aggregation of KC. On reheating, the I0, 1H T2 and D values showed clear hysteresis in KC solutions, but not in IC solutions, which is consistent with the rheological and DSC measurements mentioned above.
Temperature dependence of the signal intensity I0 at echo time τ = 0 (a and d), T2 (b and e) and the diffusion coefficient D (c and f) of KC (triangles) and IC (circles) in a 2.0 % KC/IC mixture containing 30 mM KCl during cooling (open) and reheating (closed). I0 for IC was multiplied with 1.5 for clarity. Reproduced from (Hu et al., 2016) with permission from Elsevier.
Diffusion of polyethylene oxide in mixed KC/IC gels NMR diffusion measurements of polyethylene oxide (PEO), which was added as a probe molecule, were also used to clarify the microscopic environment of mixed KC/IC solutions (1.0 % each) during gelation (Hu et al., 2016). On cooling this mixed KC/IC solution, the diffusion coefficient of PEO (DPEO) decreased gradually above 35 °C, showed a plateau between 35 and 30 °C, and decreased further at temperatures below 30 °C. DPEO/DPEO,0 values were calculated, with DPEO,0 representing the diffusion coefficient of PEO in dilute solution. DPEO/DPEO,0 in pure KC solution decreased slightly on cooling in the temperature range from 60 to 35 °C, and increased markedly between 35 and 30 °C. The initial decrease was due to the increased local viscosity of the KC solution during cooling, and the significant increase was ascribed to the formation of KC aggregates at the critical transition temperature, which left larger pores in theforming KC gel network for PEO to diffuse in. On the other hand, DPEO/DPEO,0 in pure IC solution did not show any clear changes during cooling, indicating that, in contrast to the behavior of KC chains, IC chains did not form thick aggregates. Instead, IC chains might form loose and fine aggregates. In the mixed KC/IC solution, DPEO/DPEO,0 exhibited a similar tendency to that observed for the pure KC and IC solutions at temperatures above 40 °C, followed by a slight increase at around 35 °C. On further cooling, DPEO/DPEO,0 remained constant, which is an intermediate behavior between those observed for the pure KC and IC solutions. The diffusion of PEO in the mixed KC/IC solution could be described using a monomodal decay. Thus, for the diffusion time employed in these experiments (10 ms), PEO diffusion reflected a single environment. These results suggested two possible gel structures: 1) individual aggregates of KC and IC formed a homogeneous interpenetrating network (IPN) in which all and, 2) KC and IC microphase separated on a characteristic length scale smaller than the mean squared displacement (450 nm) observed in the diffusion experiments of PEO. These two possible structures are shown in Fig. 22.
Schematic representation of the two possible structures of KC/IC gels during cooling. Black spheres indicate the PEO probe polymers. Reproduced from (Hu et al., 2016) with permission from Elsevier.
Rheology and DSC analysis were used to clarify the viscoelasticity and gelation temperature characteristics of polysaccharides, i.e., to characterize gelation at a macroscopic scale, as was done successfully for KC and IC. Particle tracking, which is a microrheology technique, was effective for elucidating the different microscopic environments of pure KC and IC gels, as well as for inferring the formation of a heterogeneous network in mixed KC/IC gels. NMR relaxation time measurements provided useful information on the tumbling motion and local mobility of polysaccharide chains and were used to study structural changes of agar, agarose, carrageenan and gellan gum at a molecular level during gelation. In addition, NMR diffusion measurements of polysaccharides and probe polymers provided further insights into the aggregation, network formation and network mesh size of the different polysaccharide gels. By combining different experimental techniques, phenomena can be studied that encompass the macroscopic, microscopic, and molecular aspects of polysaccharide gelation and gel network structures.
Acknowledgements Lester Geonzon is grateful for the financial support of JSPS International Research Fellow, Grant No. 20F20388. Xi Yang acknowledges the financial assistance from JSPS Grant No. P22093.
Conflict of interest There are no conflicts of interest to declare.
