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CsI solutions from rheology and cryo-TEM
International Journal of Biological Macromolecules 25 (1999) 317 – 328
Conformation and association of k-carrageenan in the presence of locust bean gum in mixed NaI/CsI solutions from rheology and cryo-TEM Ioannis S. Chronakis *, Johan Borgstro¨m, Lennart Piculell Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund Uni6ersity, Box 124, S-221 00 Lund, Sweden Received 30 August 1998; accepted 25 January 1999
Abstract Mixtures of locust bean gum (LBG) with k-carrageenan (KC) in 0.1 M aqueous solutions of the mixed salts NaI/CsI were investigated by cryo-transmission electron microscopy (cryo-TEM) and dynamic viscoelastic measurements. Previous studies have shown that as the cesium content is increased in such mixed salt solutions, a transition occurs from molecularly dispersed helices to ‘superhelical rods’ of KC. We now found that LBG stabilises the superhelical rods, shifting the transition to a lower content of Cs for the mixtures than for KC alone. The formation of superhelical rods was evidenced both by cryo-TEM images and by an onset of thermal hysteresis in the coil–helix transition of KC. In the mixtures, the transition temperatures on cooling and heating were insensitive to the proportions of LBG and KC present at all cesium contents. Under conditions where no helix aggregation occurred (no hysteresis) the mixtures showed high tan d values and low storage moduli. Under aggregated conditions, gels formed, and gels with added LBG had enhanced moduli compared to gels with KC alone. On the basis of these results we propose that LBG associates to the super-helical rods of KC. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Coil – helix transitions; k-Carrageenan; LBG; Cryo-TEM; Rheology
1. Introduction Galactomannans consist of (14)-b-D mannopyranosyl main chains to which are attached to a single a-D-galactopyranosyl residues at O(6) on a proportion of the mannose residues. In the case of locust bean gum (LBG), a plant polysaccharide from the seed endosperm of Ceratonia siliqua, the galactose-substituted ‘hairy’ regions occur in blocks of about 25 residues with non-substituted ‘smooth’ mannose regions 60 residues in length [1]. These numbers represent average values since the distribution of galactose along the mannan backbone is non-regular, while such fluctuations in the substitution pattern are important [1,2]. In solution, LBG adopts a conformationaly-disordered random coil structure. LBG is known to self-associate under certain conditions (freeze-thaw treatment, long ageing) [1,3]. * Corresponding author. E-mail address: [email protected] (I.S. Chronakis)
The carrageenans are a family of polymeric sulfated galactans extracted from various species of red seaweed (i.e. Eucheuma cottoni, Eucheuma spinosum, Gigartina acicularis) [4]. The gel-forming members have linear backbones based on a repeating disaccharide sequence of 1,3-linked b-D-galactopyranose and 1,4-linked 3,6anhydro-D-galactopyranose residues. k-Carrageenan (KC) forms thermoreversible gels under certain conditions, strongly influenced by the type and quantity of ions present in the medium. The essential step in the gelation is a coil–helix conformational transition induced by lowering the temperature [5]. It has been shown that ‘specific’ cations such as K + , Cs + , NH4+ and Rb + bind to size selective sites on the double helix, promoting a subsequent aggregation of helices and increasing the gelling ability [6–9]. Other monovalent cations such as Li + , Na + , and (CH3)4N + are less effective since they bind primarily by long-range Coulomb interactions [10]. Not only cations but also anions specifically affect the conformational transition of KC [11–14]. Thus, SCN − and I − anions bind to the helix
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and promote the helix formation but prevent further aggregation of helices. The nature of the ion-binding sites on the carrageenan helix is not clear. When added to KC, LBG can considerably increase the gel strength, increase the elasticity, counteract syneresis and reduce the minimum concentration for gelation [1,15–20]. There have been numerous studies of the interactions between LBG and carrageenans and the gel structures of the mixtures [1,15 – 27]. A number of recent studies have given strong support to the model for the mixed gels originally proposed by Dea and co-workers with some extensions [28 – 33]. It has been demonstrated that mixed aggregates are formed, where the galactomannan chains take part in some direct association process involving bundles of self-aggregated KC. Mixed heterotypic aggregation has been demonstrated by means of rheology, differential scanning calorimetry, optical rotation, electron spin resonance spectroscopy [30 – 33], NMR [24,29], small angle X-ray scattering [27], static light scattering [34], and swelling experiments [35]. However, much remains to be clarified regarding the nature of the mixed aggregates, the structure of the heterotypic junctions and the conditions for their stability. The mixed aggregation depends not only on the type of galactomannan (galactose substitution) but also on the degree or state of self-aggregation of the carrageenan. A prerequisite for heterotypic aggregation seems to be a self-aggregation of carrageenan helices, as evidenced by a thermal hysteresis in the coil –helix transition. The heterotypic association then shows up most markedly as an increase in the melting temperature of the aggregate, i.e. an additional stability is conferred to these aggregates. There is no evidence of association with i-carrageenan nor for KC in NaI [34,36] where no hysteresis is found. Therefore, the case of KC is interesting since for this carrageenan, the extent of aggregation (as manifested by the degree of thermal hysteresis) can be varied by choice of salt conditions as pointed out above; from none at all (in certain iodide salts) to massive aggregation in salts like KCl and CsCl. Recent experiments in our laboratory for KC alone showed that when the cation content in mixed salts of NaI and CsI is varied (the molar fraction of the cesium salt, XCs = [CsI]/ ([CsI]+ [NaI], was varied between 0 and 1), an onset of aggregate formation occurs at a well-defined Cs content [37,38]. Thus, in 0.1 M NaI/CsI mixtures the aggregate formation in KC appears at XCs =0.4. This onset is evident from a number of observable properties such as a thermal hysteresis in the coil – helix transition (monitored by optical rotation, dynamic oscillation measurements), a substantial increase of the storage modulus and viscosity and an appearance of visible structures in cryo-transmission electron microscopy (cryo-TEM). For the KC samples with XCs =0 – 0.3 no microstruc-
ture was observed by cryo-TEM, while long (300–400 nm) thin (5 nm diameter) and very rigid fibers, ‘superhelical rods’, appeared at XCs = 0.4. On further increase of the cesium content, the superhelical rods did not seem to grow in size, but they aggregated into higherlevel aggregates [37]. The rheological studies showed, furthermore, that at low cesium contents, but at sufficiently high concentrations of KC, ‘weak gels’ (with no thermal hysteresis) were formed. Taken together, this recent evidence suggests that KC alone may form gels or gel-like networks by, at least, two different routes depending on the salt content: 1. coil helixgel (reversible association); and 2. coil helixsuperhelical rod gel (permanent aggregation). The intermediate structures of superhelical rods and aggregates of them, are still not understood. The internal structure of these rods cannot be resolved from the micrographs but no variation in thickness was seen regardless of the degree of aggregation [37]. This suggest that the fibers correspond to an aggregate with a well-defined structure. These rods have been referred to earlier also as ‘superstrands’ consisting of densely packed helices [39]. In the present study we use the same strategy of mixed NaI/CsI salts for the composite KC/LBG, to study to what extent the self-aggregation of KC is affected by added galactomannan. Since the successive steps in the aggregation of KC can be resolved by gradually changing XCs, it should also be possible by this strategy to achieve a more detailed information about the type of KC aggregate that is involved in the mixed aggregates with LBG. Both rheology and cryoTEM measurements are used in the study. We also compare the results on the mixtures with those from the earlier studies on KC alone. To facilitate this comparison we have looked at a few strategic proportions of the mixed systems. A preliminary account of part of this work has been published previously [36].
2. Materials and methods
2.1. Preparation of polysaccharide sample The KC (Envoi No 12869) and LBG (Lygomme 6, B 7756) samples were kindly donated by Sanofi Bio Industries (France), with molecular weights of 3 ×105 (in the coil form) and 106 g/mol, respectively. The sodium salt form of KC was prepared by cation exchange on a Dowex-50W strong acid cation exchange resin from Sigma. The resin was first converted to the H + form by elution with HCl, and then to the Na + form by using appropriate chloride salts. Ion exchange of the polysaccharide (1.5% w/w) was carried out at 95°C to maintain the polymer in the disordered coil form. The resultant
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samples were then freeze-dried and stored. To check for possible degradation of the polysaccharide the elastic modulus was measured before and after the ion exchange and no substantial difference was observed. Aqueous NaI and CsI (AnalaR grade from Sigma) solutions of a total concentration of 0.1 M were prepared and mixed in the desired proportions. KC and LBG were added to the appropriate salt solution at room temperature and then heated at 95°C at least for 20 min while stirring. Millipore® water was used throughout. All concentrations are given in weight percent (w/w).
2.2. Low amplitude oscillatory measurements Rheological measurements under low amplitude oscillatory shear were performed on a controlled stress Carri-Med CSL100 rheometer (TA Instruments, UK) using a parallel plate geometry (40 mm radius; 1 mm separation) at a frequency of 1.6 Hz. All measurements were performed with 0.5% strain since strain sweeps on selected gels demonstrated that the working deformation was well within the linear viscoelastic region. To avoid slippage due to syneresis [38,40], as repeatedly observed when CsI was predominating, a cross-hatched acrylic upper plate (40 mm diameter, 0.5 mm separation) was used at XCs ]0.4. Each sample was loaded as a hot solution on the platen of the rheometer pre-set at 80°C. The sample periphery was coated with silicone oil to minimize loss of solvent or adsorption of atmospheric moisture. The rheological parameters were monitored on cooling at 1°C/min to 20°C. Cooling scans were followed by a 30-min isothermal run and a frequency sweep between 0.01 and 10 Hz (about another 30 min). The rheological routine was completed with a heating run from 20 to 95°C (1°C/min). The above sequence of experimental procedures allowed recording of the storage modulus (G%), the loss modulus (G%%), tan d (=G%%/G%) and the complex viscosity (h*= (G%2 +G%%2)1/2/v) as functions of time, temperature, and frequency of oscillation. The coil–helix and helix – coil transition temperatures reported are ‘helix onset temperatures’, taken as the points where the storage modulus just exceeds the noise (for example see Fig. 1a, b). The increase in G% is frequently so steep that the noise level does not significantly affect the value of the transition temperature.
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imens were stored under liquid nitrogen until they were transferred to the cryo-holder. The cryo-holder was cooled by liquid nitrogen to a temperature lower than − 170°C. The electron microscope used was a Philips CM 120 Bio-twin run at an accelerating voltage of 120 kV equipped by a Gatan multi scan CCD and an Oxford cryo-holder. At least 20 micrographs were recorded of each solution and some solutions were observed several times. To facilitate the formation of thin liquid films, a small amount of C12E5 was added (1.62×10 − 6% w/w), giving a concentration in the solution of approximately 70% of critical micelle concentration (cmc). Three different salt mixtures were investigated by cryo-TEM: XCs = 0, 0.2 and 0.4. Due to the difficulty of preparing viscous solutions with this technique, the polymer concentration was kept low and an ultrasonically degraded KC was used [37]. Our studies
2.3. Cryo-TEM A small drop of the liquid was placed on a perforated carbon film, supported by a 200-mesh copper grid, and most of the liquid was removed by blotting with a filter paper. The thin liquid films formed across the holes were subsequently vitrified by plunging the grid into liquid ethane at its freezing point (− 183°C). The spec-
Fig. 1. Rheology of 0.75% KC and 0.75% LBG in 0.1 M mixture of NaI and CsI. a: Temperature dependence on cooling and heating of storage moduli G% ( — ) and loss moduli G%% (---) in XCs =0.2; and b: variation of G% ( —), G%% (---) on cooling and G% (– – – ), G%% (– – –) on heating for XCs =0.8. (1°C/min, 1.6 Hz, 0.5% strain).
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established that the microstructure of both intact and degraded KC solutions, as observed with cryo-TEM, does not differ substantially [37]. The polymer concentration was 0.1% for the pure polymer systems but for the mixed system (LBG+KC) the concentration of each component was 0.1%, giving a total polymer concentration of 0.2%. All solutions were stored at least over night before any preparations for the cryo-TEM were made.
3. Results The compositions of the mixtures for the rheological studies were chosen using the 1.5% pure KC systems of our previous study as a point of reference. Thus, in the first set of mixtures studied here (0.75% KC/0.75% LBG), half of the KC content of the pure KC systems was replaced by LBG, while, in the second set of mixtures (1.5% KC/0.75% LBG), the same amount of LBG (0.75%) was added. Finally, the third set of mixtures (0.75% KC/1.5% LBG) had the same amount of KC as the first set of mixtures, but twice the amount of LBG. As will be shown below, these compositions demonstrated consistent trends in the rheology of the mixtures. Sometimes, however, small deviations were seen for the 0.75% KC/1.5% LBG mixtures, which possibly may be due to the fact that problems with slippage in the measurements at high cesium were particularly severe for these mixtures.
3.1. Effects on hysteresis and aggregation Fig. 1 shows the temperature dependencies of the storage and loss moduli during conformational ordering and melting for the mixture of 0.75 KC and 0.75 LBG. As for 1.5% KC alone [38], different types of behavior was observed at low and high cesium contents, here represented by at XCs =0.2 and 0.8, respectively. In the presence of low amounts of cesium (Fig. 1a), the helix formation was accompanied by a small increase in rigidity (G%) with no detectable thermal hysteresis on melting. A higher cesium content displaced the conformational transition of the composite to higher temperatures and caused a more co-operative onset of gelation (more steep traces) with a substantial increase in G% (Fig. 1b). When cesium dominated a strong thermal hysteresis appeared in the setting-melting transitions, demonstrating the formation of aggregated helices of KC. Similar traces were obtained for the other proportions of the mixtures. Fig. 2 illustrates the coil –helix formation and melting transition temperatures, as defined in the experimental section, for the different ratios of the mixed polysaccharides, and at different
Fig. 2. Temperature dependence of coil – helix transitions (filled symbols) on cooling and helix – coil on melting (open symbols) vs. cesium content for KC/LBG mixtures; 0.75/075 (squares); 0.75/1.5 (triangles); 1.5/0.75 (circles); 1.5/0 (line, dotted lines on melting); (1°C/min, 1.6 Hz, 0.5% strain).
ratios of NaI and CsI. At any salt content, the observed transition temperature for a mixture on cooling was close to that observed for the algal polysaccharide alone [38,41]. For all systems, the cooling transition temperatures increased smoothly as a function of XCs, and at high cesium content, the transitions for the mixtures occurred a few degrees above the corresponding process for KC alone. The transitions were fully reversible for XCs 5 0.2. However, thermal hysteresis and aggregation was initiated at XCs : 0.25 and further developed with increasing cesium content. Obviously, added LBG caused the KC helices to aggregate at a lower cesium content than for carrageenan alone, i.e. at XCs : 0.25 instead of 0.4 [37,38,41]. In accordance with previous findings [30,32,33], there was also an increase in the extent of the thermal hysteresis when LBG was present. Note that the thermal hysteresis increased with increasing XCs. Thus, the differences in the transition temperatures varied from about 8 to 17°C for the 0.75/0.75 ratio. On the other hand, the melting transition temperatures—and thus, in all probability, the nature of the final aggregated states—in the mixtures were rather independent of the proportions and total concentrations of the two polysaccharides. Our investigation was further continued with cryoTEM studies to detect when the superhelical aggregates first occurred as the cesium content was increased, in the presence and in the absence of LBG. The images observed by cryo-TEM in 0.1% KC are depicted in Fig. 3 for some different salt mixtures. For pure KC, no structures could be observed in 0.1 M NaI, which confirms our earlier observation with a different elec-
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tron microscope [37]: non-aggregated KC helices are not visible by cryo-TEM, due to low contrast or low resolution (or both). In XCs =0.2 the large majority of the images were also completely void of fibers but in two images (out of 23) a few and very faint fibers, were observed (Fig. 3b). As also described previously [37], the rod-like fibers of KC (‘the superhelical rods’ Fig. 3c) can be observed first at and above a cesium content of XCs = 0.4. In the LBG preparations, we saw also some fibers, although the LBG is not considered to be a rigid polymer (fiberlike). The appearance of these fibers were different from the KC fibers. The LBG fibers were often thicker, more flexible and had a higher contrast (Fig. 4). The same type of fibers were observed in all salt mixtures investigated (XCs 0 – 0.4). The (projected) length of these fibers was very polydisperse and some very short fibers often occurred. A peculiar feature was the very sharp turns (90°) that occurred occasionally (can be seen for example at the lower left side of Fig. 4). Some fibers ‘dissolved’, the contrast decreased and
Fig. 3. Cryo-TEM micrographs of 0.1% ultrasonically degraded KC in mixed 0.1 M NaI/CsI-salt solutions. a: XCs = 0 (0.1 M NaI); b: XCs =0.2. The dark corners are due to the supporting film; and c: superhelical KC-rods at XCs = 1.0. Scale bar is 200 nm.
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Fig. 4. A cryo-TEM micrograph of 0.1% LBG in 0.1 M NaI, showing the thick and highly contrasted LBG-fibers (scale bar 200 nm). Note the sharp 90°-turn at the lower left part of the image. The dark spots are frost deposits.
the thickness increased as the end of the fiber was approached (also shown in Fig. 4), possibly due that the observed fibers are aggregates of several LBG chains. The amount of these fibers varied between the different LBG preparations which could be caused by minor differences in the sample history (for example storage time before vitrification). LBG has, to our knowledge not been studied by cryo-TEM before. A study of KC/LBG mixtures by the mica sandwich replica technique was reported by Lundin [2,31]. In that work, no microstructure of LBG was observed except for some cases where a structure was probably made visible due to shearing of LBG polymer when the mica plates were put together. We have also examined by cryo-TEM the same LBG as the one used by Lundin (LBG80) and this also displays similar types of fibers as observed in our samples. It is not clear why we see these structures, since LBG is not considered as a rigid, fiberlike polymer. One possible explanation could be that our preparation technique (by cryo-TEM) may also expose the sample to relatively high shear forces leading to visible structures. As well, the history of the samples could be very important if the structures are developing with time. As mentioned earlier, LBG is known to self associate during freezethawing treatment and long ageing. For the mixed systems a structure similar to the pure LBG was observed in 0.1 M NaI (Fig. 5(a)). At XCs = 0.2 many thin and rigid fibers of the KC-type were observed (Fig. 5(b)). Interestingly, also some short and thick LBG fibers could be observed coexisting with the KC fibers under these salt conditions. At XCs =0.4 CsI we observed many KC fibers (Fig. 5(c)). Although the concentrations used for the microscopy studies are relatively low in comparison with those used in the rheological experiments, our previous studies have clearly established that the onset and the magnitude of hysteresis (critical XCs content) is independent on the molecular weight (degree of polymerisation) between 100 000 and 600 000 for the helix form and the KC concentration (0.1–2%) [36–38,41].
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3.2. Effects on 6iscoelastic properties. For a thermoreversible polymer gel, the cross-over point of the storage and loss moduli at some frequency is sometimes (arbitrarily) taken as the ‘gel point’. Fig. 6 shows the cross-over of G% and G%% on cooling at the experimental frequency of 1.6 Hz for different KC/ LBG mixtures. At 1.5% of KC, the cross-over temperatures were very close to the temperatures for the onset of conformational ordering, as detected by rheology. However, at 0.75% KC and at low cesium contents the cross-over temperatures were significantly lower than the onset temperatures (cf. also Fig. 1a), i.e. a substantial proportion of helical KC was required in order for the elastic behavior to dominate. On increasing the cesium content, a sharp increase in the storage modulus was found previously at XCs \ 0.4 for the 1.5% pure KC gels, conditions where the super-
Fig. 5. Cryo-TEM micrographs of mixed 0.1% LBG + 0.1% ultrasonically degraded KC in different NaI/CsI-salt mixtures. In 0.1 M NaI (a), only the LBG-structure is visible (note the sharp turns and the thick, strongly contrasted fibers). The dark spots are frost deposits. When cesium is introduced, the thinner, more rigid superhelical rods of kappa – carrageenan occur at XCs = 0.2 (b), but coexisting with some short and thick LBG-fibers. At XCs = 0.4 (c), the KC fibers are more abundant. The scale bar is 200 nm in all images.
Fig. 6. Changes of cross-over temperatures of G% and G%% at 1.6 Hz, accompanying the formation of mixed gels of KC/LBG vs. cesium content; 0.75/075 ( ); 0.75/1.5 ();1.5/0.75 (); 1.5/0 ( × ).
helical rod formation was identified from cryo-TEM [37,38]. Fig. 7 illustrates (in both logarithmic and linear representations) the variation of G% for KC/LBG mixtures recorded at the end of 30 min isothermal run at 20°C, on completion of the cooling scans. Visual inspection revealed self-supporting networks in all series of mixtures, except for the mixing ratio 0.75/0.75 in 0.1 M NaI. The replacement of KC with LBG (cf. the pure 1.5% KC gels with the 0.75/0.75 mixtures) resulted in a decrease in the modulus except at intermediate cesium contents, where the shift in the onset of aggregation of KC helices gave rise to a true synergism. Addition of LBG generally raised the rigidity of the systems, except for the 0.75 KC/1.5 LBG mixtures at high cesium contents, where problems with slippage were found. At low cesium contents the increase was much lower when the KC concentration was high (1.5%). Two further observations are noteworthy. First, for the weak gels at low cesium, the moduli of the mixtures increased markedly with increasing XCs, although no such strong variation was seen for the respective pure systems. The 1.5% LBG alone showed a solution-like behavior (G%% \G%, from 0.01 to 10 Hz) and a low storage modulus at all ionic compositions. Second, the increase in G% at the ‘new’ critical XCs = 0.25 for the mixtures was rather modest, compared to the increase seen for pure 1.5% KC at XCs = 0.4. Especially from the linear plot, it is apparent that the large increase in G% still occurred around XCs = 0.4 also for the mixtures. As already noted, slippage at high cesium contents was observed repeatedly, and the problems became worse on addition of LBG. This was unexpected, since it is typically observed that galactomannan addition decreases the tendency towards syneresis and contraction. Measurements were impossible even with the
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cross-hatched configuration for the 0.75/1.5 (KC/LBG) mixtures above XCs = 0.7, and for the 1.5/0.75 mixtures at 0.1 M CsI. The frequency dependencies for representative samples at 20°C are presented in Fig. 8a – d. At high cesium contents, the mechanical spectra of the mixed preparations were typical of true gels. Below XCs =0.3, however, the separation between G% and G%% was clearly reduced, and both moduli showed a greater variation across the frequency range (Fig. 8b, c). Nevertheless, a plot of log h* versus log v was still linear, which is one of the characteristic features of a gel-like response [42]. For a true gel, the slope of such a plot equals −1. The mechanical spectra for the ratios 0.75/0.75 and 0.75/1.5 demonstrate that the addition of galactomannan gave a significant enhancement in both moduli, but the viscoelastic character remained unaffected; i.e. the slopes of log h* versus log v were similar, and so were the separation of G% and G%% as well as the frequency dependence. When the concentration of carrageenan was increased (Fig. 8d), the moduli increased and the spectra exhibited a much weaker frequency dependence. Fig. 9 shows the (negative of) the linear slopes of log h* versus log v over the whole range of cesium contents for all polymer concentrations studied. Below XCs B 0.3 the slopes suggest that the systems were entangled networks with weak crosslinks. Permanent elastic networks, as known for conventional polysaccharide gels [43], were formed only at higher cesium contents, where the slopes approached the limiting value of − 1. The effect of the compositional variation and ionic conditions on tan d, measured at a single frequency (1.6
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Hz) at the end of the 30-min isothermal run, reflects the same tendencies of Fig. 9. Generally, tan d was high at low cesium contents, and decreased progressively to level off at a constant value at XCs \ 0.4. This response again implies a development from an entangled network to a weak gel-like character and, finally, a formation of true gels at a large content of the gel-promoting cation. A nearly 40-fold variation in tan d across the salt composition range was seen for the ratio 0.75/0.75, while the transition was less sharp, and the tan d values remained high at high cesium, upon increasing the concentration of galactomannan (the 0.75/1.5 mixture). Most probably this was due to the tendency of slippage observed for this sample. In systems containing 1.5% carrageenan, less elastic systems were obtained at low cesium contents when LBG was added (cf. 1.5% KC with the 1.5/0.75 mixture). This simply suggests an additive effect of LBG on the weak KC network.
3.3. Time dependent effects When the cesium content was around 0.25–0.3, the heating transitions of the mixtures displayed a second melting step at higher temperatures, as illustrated in Fig. 10. The amplitude of the second step became higher as XCs increased and only a single step appeared at a cesium content of about 0.4. A two-step transition ‘on cooling’ was previously observed for KC alone at XCs : 0.5 and a cooling rate of 1°C/min. The latter feature disappeared, however, when the cooling rate was lowered to 0.2°C/min, and the effect was thus attributed to slow kinetics [38]. To explore further the
Fig. 7. Storage modulus G% vs. cesium content for the various KC/LBG mixtures. 0.75/075 ( ); 0.75/1.5 ();1.5/0.75 (); 1.5/0 ( ×); and 1.5% LBG () (1.6 Hz, 0.5% strain).
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Fig. 8. Frequency dependence at 20°C of G% (), G%% () and h* () for KC/LBG mixtures: a: 0.75/0.75 XCs =0.8; b: 0.75/0.75 XCs = 0.2: c: 0.75/1.5 XCs =0.2: and d: 1.5/0.75 XCs = 0.2.
two-step melting curve in the mixtures (Fig. 10a), we adopted the same strategy and measured a heatingcooling cycle at a much slower rate (0.2°C/min instead of 1°C/min). As shown in Fig. 10b, no changes were observed in the transition temperatures, and the twostep melting process still persisted. Thus, there was no evidence to suggest that the origin of the two steps was a kinetic effect. On the other hand, the network modulus increased at the slower cooling rate, as is most easily seen from the value of G% at the lowest temperature (20°C). In fact, for the gels that showed thermal hysteris, i.e. at cesium contents above XCs :0.25, we quite generally observed a significant increase of the storage moduli of the gels during the 30 min isothermal run at 20°C (the difference between cooling and heating data at 20°C in Fig. 1b). We also compared the frequency sweeps accompanying the different cooling rates for the mixture corresponding to Fig. 10. Not only did the storage modulus increase (from 95 to 170 Pa) at the lower cooling rate,
but so did the gel-like character, as shown in Fig. 11a, b. The difference between G% and G%% increased for the slower cooling rate, and the slope of log h* versus log v became steeper (− 0.71 and − 0.82 for 1 and 0.2°C/ min, respectively). Therefore, the viscoelastic properties of the composite, but not the coil–helix–coil transition temperatures, are significantly affected by the cooling rate and the equilibration time.
4. Discussion The results of the present study on KC gels and solutions, with or without LBG present, confirm the trends seen in previous studies: in pure NaI or at low contents of cesium, the coil–helix transition is quite reversible, whereas a thermal hysteresis sets in at a quite well defined value of XCs. The appearance of hysteresis coincides with the development of rigid, superhelical rods of KC, which are visible by cryo-TEM. The onset
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of helix formation on cooling is only weakly sensitive to the presence of LBG, which indicates that the same primary aggregate (the helix) forms both in the presence and in the absence of LBG. Only at higher cesium content does LBG cause a slight increase in the coil-tohelix transition temperatures. Both with and without LBG is there an increase in the sharpness of the cooling transition with increasing cesium content, in line with recent calorimetric measurements on KC alone [41]. The most significant new result of the present study is the lowering of the critical cesium content required for the onset of hysteresis in the conformational transition, and for the formation of superhelical rods of KC, from XCs :0.4 for pure KC to XCs :0.25 in KC/LBG mixtures. In contrast, polarimetric studies in our laboratory (Ramzi, unpublished) have shown that there is no shift in the critical cesium content required for hysteresis when KC is mixed with guar gum, a galactomannan that associates only weakly or not at all with KC. Our task is now to interpret these results in the light of what is currently known about the gelation of KC and the association between KC and LBG. Fig. 12 illustrates the various steps in the association and gelation of KC, according to the picture that has emerged from recent studies in our laboratory. This model differs from previous models in that it explicitly includes the superhelical rod as an intermediate state between the helix and the gel; the gel is formed by the association of the super-helical rods in higher order aggregates. The bulk of previous studies [30,33] have shown that at least no strong aggregates are formed between nonaggregated KC helices and LBG molecules. By strong aggregates, we mean aggregates that give rise to a hysteresis in the coil – helix transition temperature. This
Fig. 9. Composition dependence of linear slopes of (log h* vs. log v) vs. cesium connect for the mixtures of KC with galactomannan. Symbols as in Fig. 6. Values taken at a fixed time of 30 min at 20°C after cooling (1.6 Hz, 0.5% strain, absolute values).
Fig. 10. Thermal change of G% ( —), G%% (---) on cooling and G% ( – – –), G%% (---) on heating at different scan rates: a: 0.75/0.75 XCs =0.25 with 1°C/min; b: 0.75/0.75 XCs =0.25 at 0.2°C/min and; c: 1.5/0.75 XCs =0.3 with 1°C/min (1.6 Hz, 0.5% strain).
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picture is confirmed here. As soon as there is evidence of strong KC–LBG aggregation, there is also evidence of KC self-aggregation; the characteristic images of super-helical rods of KC appear in the cryo-TEM micrographs. The simplest interpretation that is consistent with our results is then that ‘the super-helical rod is the state of KC that is in6ol6ed in the strong aggregation with LBG. When LBG molecules are present, they thus stabilize the super-helical rods, so that these can form already at a lower content of rod-promoting cations, such as cesium’. The association between the KC rods and LBG molecules should have consequences for the higher level association of the KC rods and the gelation process. Previous authors have shown results indicating a more limited aggregation of KC in the presence of LBG [2,31]. This is in line with our interpretation, if it is assumed that LBG to some extent prevents some higher
Fig. 11. Mechanical spectra (20°C, 0.5% strain) shown the frequency dependence of G% (), G%% () and h* () for 0.75/0.75 KC/LBG systems at XCs = 0.25, developed with a different cooling rate. a: 1°C/min; b: 0.2°C/min.
level of association of rods, but not the formation of the rods themselves (which is actually promoted by LBG). However, our studies also clearly demonstrate the strong similarities in the rheological properties between the mixed systems and those containing KC alone. This implies that LBG/KC mixtures must also contain a self-aggregated KC network structure similar to that in pure KC, i.e. we should not think of a mixed gel where all rod–rod junctions have been replaced by rod–LBG junctions. Probably, the role of LBG is more to reinforce the KC network and, possibly, to prevent its collapse. In any case, however, an excess of unbound LBG should eventually develop at sufficiently large LBG/KC ratios, owing to a saturation of the available surface area on the KC rods. A segregation between unbound LBG and saturated mixed aggregates would then be a possibility, which might be the reason for the experimental difficulties (slippage) encountered here for the 0.75/1.5 (KC/LBG) mixtures. More firm conclusions regarding the nature of the mixed network are difficult to draw, however, until there is more detailed knowledge about the structure of the network of KC alone. An interesting finding made here is the two-step melting transition for the mixtures in the region 0.25B XCs B 0.4 (Fig. 10). We note that also Gonc¸alves et al. [33] found a two-step melting in KC/LBG mixtures under salt conditions (NaCl) where KC does not display hysteresis or form super-helical rods on its own. The occurrence of two melting steps indicate that ‘only a fraction’ of the KC helices formed on cooling are converted to rigid rods, the rest remaining as individual helices. We propose the following explanation to why the rod fraction is limited, but increases with increasing XCs, in the interval 0.25BXCs B 0.4. LBG is not a homogeneous polymer, but contains a range of structures with more or less strong tendencies to associate with KC. Close to XCs = 0.25, probably only the fractions with the strongest binding have the capacity to ‘pull over’ KC to the rod state. When this strongly associating fraction is consumed in mixed aggregates, the excess carrageenan undergoes a reversible coil–helix transition as in the absence of LBG. As XCs increases towards 0.4, however, the required binding strength for stabilizing the rods decreases, more mixed aggregates are formed, and the fraction of rods increases. It also seems clear that that the value of G% measured at room temperature should depend on the content of the rod fraction. The gradual build-up of the rod fraction with increasing cesium content in the mixtures then explains our observation that the increase in G% as a function of XCs is not so strongly correlated with the onset of hysteresis for the mixed gels as for the gels of KC alone (Fig. 7). For KC alone, both events occur at XCs : 0.4, whereas no sharp increase in G% occurs at XCs : 0.25 in the mixtures. Similarly, a gel-like vis-
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Fig. 12. Schematic representation of the various steps in the association and gelation of KC. LBG associates to the super-helical rods of KC.
coelastic behavior (as indicated by the tan d values and the slopes of log h* versus log v) also appears only at XCs \0.4 in all systems. A final point to address is the observation of a significant dependence of the moduli and the tan d of the mixed gels on the cesium content also below the onset of hysteresis (XCs B0.25). This is remarkable, since no corresponding dependence was seen for either of the pure systems. We cannot offer a definite explanation of this phenomenon at present. The obvious interpretation in terms of an association, at finite cesium contents, between LBG and ‘individual’ KC helices is not entirely satisfactory, since such an association— contrary to our observation — should increase the stability of the helix and shift the coil – helix transition towards higher temperatures. Since no significant temperature shift was observed, this would imply that the association, if it indeed exists, is quite weak.
Acknowledgements We are grateful to Professor Anne-Marie Hermansson (SIK, Go¨teborg) for valuable discussions and for sending us the LBG80 sample used by L. Lundin.
References [1] Dea ICM, Morrison A. Adv Carbohydr Chem Biochem 1975;31:241 – 312. [2] Lundin L. PhD Thesis, SIK, Sweden, ISBN 91-7197-468-7, 1997. [3] Dea ICM, Morris ER, Rees DA, Welsh EJ, Barnes HA, Price J. Carbohydr Res 1977;57:249–72. [4] Rees DA, Morris ER, Thom D, Madden JK. In: Aspinal GO, editor. The Polysaccharides1. New York: Academic Press, 1982:195 – 290. [5] Rees DA. Adv Carbohydr Chem 1977;24:267–332. [6] Rochas C, Rinaudo M. Biopolymers 1980;19:1675–87. [7] Grasdalen H, Smidsrød O. Macromolecules 1981;14:229– 31. [8] Belton PS, Morris VJ, Tanner SF. Int J Biol Macromol 1985;7:53 – 6.
[9] Piculell L, Nilsson S, Stro¨m P. Carbohydr Res 1989;188:121–35. [10] Nilsson S, Piculell L, Jonsson B. Macromolecules 1989;22:2367– 75. [11] Nilsson S, Piculell L. Macromolecules 1991;24:3804 – 11. [12] Smidsrød O, Grasdalen H. Hydrobiologia 1984;116/117:178–86. [13] Grasdalen H, Smidsrød O. Macromolecules 1981;14:1842–5. [14] Norton IT, Morris ER, Rees DA. Carbohydr Res 1984;134:89– 101. [15] Dea ICM, MacKinnon AA, Rees DA. J Mol Biol 1972;68:153– 72. [16] Dea ICM, Rees DA. Carbohydr Polym 1987;7:183 – 224. [17] Rees DA. Biochem J 1972;126:257 – 73. [18] Dea IC, MacKinnon AA. Adv Carbohydr Chem Biochem 1975;3:241 – 312. [19] Cairns P, Morris VJ, Miles MJ, Browney GJ. Food Hydrocolloids 1986;1:89 – 93. [20] Turquois T, Rochas C, Taravel FR. Carbohydr Polym 1992;17:263 – 8. [21] Miles MJ, Morris VJ, Carroll V. Macromolecules 1984;17:2443– 5. [22] Cairns P, Miles MJ, Morris VJ, Brownsey GJ. Carbohydr Res 1987;160:411 – 23. [23] Cairns P, Atkins EDT, Miles MJ, Morris VJ. Int J Biol Macromol 1991;13:65 – 8. [24] Rochas C, Taravel FR, Turquois T. Int J Biol Macromol 1990;12:353 – 8. [25] Fernandes PB, Goncalves MP, Doublier J-L. Carbohydr Polym 1991;16:253 – 74. [26] Fernandes PB, Goncalves MP, Doublier J-L. Carbohydr Polym 1992;19:261 – 9. [27] Turquois T, Rochas C, Doublier J-L, Taravel FR, Axelos MAV. Biopolymers 1995;36:559 – 67. [28] Tako M, Nakamura S. Agric Biol Chem 1986;50:2817–22. [29] Piculell L, Zhang W, Turquois T, Rochas C, Taravel F-R, Williams PA. Carbohydr Res 1994;265:281 – 90. [30] Goycoolea FM, Richardson RK, Morris ER, Gidley MJ. Biopolymers 1995;36:643 – 58. [31] Lundin L, Hermansson AM. Carbohydr Polym 1995;26:129– 140; 28:91 – 99. [32] Williams PA, Langdon MJ. Biopolymers 1996;38:655 –64. [33] Gonc¸alves MP, Gomes C, Langdon MJ, Viebke C, Williams PA. Biopolymers 1997;41:657 – 71. [34] Viebke C. Carbohydr Polym 1995;28:101 – 5. [35] Parker A, Lelimousin D, Miniou C, Boulenguer P. Carbohydr Res 1995;272:91 – 6. [36] Piculell L, Borgstro¨m J, Chronakis IS, Quist PO, Viebke C. Int J Biol Macromol 1997;21:141 – 53.
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Conformation and association of k-carrageenan in the presence of locust bean gum in mixed NaI/CsI solutions from rheology and cryo-TEM Ioannis S. Chronakis *, Johan Borgstro¨m, Lennart Piculell Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund Uni6ersity, Box 124, S-221 00 Lund, Sweden Received 30 August 1998; accepted 25 January 1999
Abstract Mixtures of locust bean gum (LBG) with k-carrageenan (KC) in 0.1 M aqueous solutions of the mixed salts NaI/CsI were investigated by cryo-transmission electron microscopy (cryo-TEM) and dynamic viscoelastic measurements. Previous studies have shown that as the cesium content is increased in such mixed salt solutions, a transition occurs from molecularly dispersed helices to ‘superhelical rods’ of KC. We now found that LBG stabilises the superhelical rods, shifting the transition to a lower content of Cs for the mixtures than for KC alone. The formation of superhelical rods was evidenced both by cryo-TEM images and by an onset of thermal hysteresis in the coil–helix transition of KC. In the mixtures, the transition temperatures on cooling and heating were insensitive to the proportions of LBG and KC present at all cesium contents. Under conditions where no helix aggregation occurred (no hysteresis) the mixtures showed high tan d values and low storage moduli. Under aggregated conditions, gels formed, and gels with added LBG had enhanced moduli compared to gels with KC alone. On the basis of these results we propose that LBG associates to the super-helical rods of KC. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Coil – helix transitions; k-Carrageenan; LBG; Cryo-TEM; Rheology
1. Introduction Galactomannans consist of (14)-b-D mannopyranosyl main chains to which are attached to a single a-D-galactopyranosyl residues at O(6) on a proportion of the mannose residues. In the case of locust bean gum (LBG), a plant polysaccharide from the seed endosperm of Ceratonia siliqua, the galactose-substituted ‘hairy’ regions occur in blocks of about 25 residues with non-substituted ‘smooth’ mannose regions 60 residues in length [1]. These numbers represent average values since the distribution of galactose along the mannan backbone is non-regular, while such fluctuations in the substitution pattern are important [1,2]. In solution, LBG adopts a conformationaly-disordered random coil structure. LBG is known to self-associate under certain conditions (freeze-thaw treatment, long ageing) [1,3]. * Corresponding author. E-mail address: [email protected] (I.S. Chronakis)
The carrageenans are a family of polymeric sulfated galactans extracted from various species of red seaweed (i.e. Eucheuma cottoni, Eucheuma spinosum, Gigartina acicularis) [4]. The gel-forming members have linear backbones based on a repeating disaccharide sequence of 1,3-linked b-D-galactopyranose and 1,4-linked 3,6anhydro-D-galactopyranose residues. k-Carrageenan (KC) forms thermoreversible gels under certain conditions, strongly influenced by the type and quantity of ions present in the medium. The essential step in the gelation is a coil–helix conformational transition induced by lowering the temperature [5]. It has been shown that ‘specific’ cations such as K + , Cs + , NH4+ and Rb + bind to size selective sites on the double helix, promoting a subsequent aggregation of helices and increasing the gelling ability [6–9]. Other monovalent cations such as Li + , Na + , and (CH3)4N + are less effective since they bind primarily by long-range Coulomb interactions [10]. Not only cations but also anions specifically affect the conformational transition of KC [11–14]. Thus, SCN − and I − anions bind to the helix
0141-8130/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 4 1 - 8 1 3 0 ( 9 9 ) 0 0 0 5 0 - 1
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and promote the helix formation but prevent further aggregation of helices. The nature of the ion-binding sites on the carrageenan helix is not clear. When added to KC, LBG can considerably increase the gel strength, increase the elasticity, counteract syneresis and reduce the minimum concentration for gelation [1,15–20]. There have been numerous studies of the interactions between LBG and carrageenans and the gel structures of the mixtures [1,15 – 27]. A number of recent studies have given strong support to the model for the mixed gels originally proposed by Dea and co-workers with some extensions [28 – 33]. It has been demonstrated that mixed aggregates are formed, where the galactomannan chains take part in some direct association process involving bundles of self-aggregated KC. Mixed heterotypic aggregation has been demonstrated by means of rheology, differential scanning calorimetry, optical rotation, electron spin resonance spectroscopy [30 – 33], NMR [24,29], small angle X-ray scattering [27], static light scattering [34], and swelling experiments [35]. However, much remains to be clarified regarding the nature of the mixed aggregates, the structure of the heterotypic junctions and the conditions for their stability. The mixed aggregation depends not only on the type of galactomannan (galactose substitution) but also on the degree or state of self-aggregation of the carrageenan. A prerequisite for heterotypic aggregation seems to be a self-aggregation of carrageenan helices, as evidenced by a thermal hysteresis in the coil –helix transition. The heterotypic association then shows up most markedly as an increase in the melting temperature of the aggregate, i.e. an additional stability is conferred to these aggregates. There is no evidence of association with i-carrageenan nor for KC in NaI [34,36] where no hysteresis is found. Therefore, the case of KC is interesting since for this carrageenan, the extent of aggregation (as manifested by the degree of thermal hysteresis) can be varied by choice of salt conditions as pointed out above; from none at all (in certain iodide salts) to massive aggregation in salts like KCl and CsCl. Recent experiments in our laboratory for KC alone showed that when the cation content in mixed salts of NaI and CsI is varied (the molar fraction of the cesium salt, XCs = [CsI]/ ([CsI]+ [NaI], was varied between 0 and 1), an onset of aggregate formation occurs at a well-defined Cs content [37,38]. Thus, in 0.1 M NaI/CsI mixtures the aggregate formation in KC appears at XCs =0.4. This onset is evident from a number of observable properties such as a thermal hysteresis in the coil – helix transition (monitored by optical rotation, dynamic oscillation measurements), a substantial increase of the storage modulus and viscosity and an appearance of visible structures in cryo-transmission electron microscopy (cryo-TEM). For the KC samples with XCs =0 – 0.3 no microstruc-
ture was observed by cryo-TEM, while long (300–400 nm) thin (5 nm diameter) and very rigid fibers, ‘superhelical rods’, appeared at XCs = 0.4. On further increase of the cesium content, the superhelical rods did not seem to grow in size, but they aggregated into higherlevel aggregates [37]. The rheological studies showed, furthermore, that at low cesium contents, but at sufficiently high concentrations of KC, ‘weak gels’ (with no thermal hysteresis) were formed. Taken together, this recent evidence suggests that KC alone may form gels or gel-like networks by, at least, two different routes depending on the salt content: 1. coil helixgel (reversible association); and 2. coil helixsuperhelical rod gel (permanent aggregation). The intermediate structures of superhelical rods and aggregates of them, are still not understood. The internal structure of these rods cannot be resolved from the micrographs but no variation in thickness was seen regardless of the degree of aggregation [37]. This suggest that the fibers correspond to an aggregate with a well-defined structure. These rods have been referred to earlier also as ‘superstrands’ consisting of densely packed helices [39]. In the present study we use the same strategy of mixed NaI/CsI salts for the composite KC/LBG, to study to what extent the self-aggregation of KC is affected by added galactomannan. Since the successive steps in the aggregation of KC can be resolved by gradually changing XCs, it should also be possible by this strategy to achieve a more detailed information about the type of KC aggregate that is involved in the mixed aggregates with LBG. Both rheology and cryoTEM measurements are used in the study. We also compare the results on the mixtures with those from the earlier studies on KC alone. To facilitate this comparison we have looked at a few strategic proportions of the mixed systems. A preliminary account of part of this work has been published previously [36].
2. Materials and methods
2.1. Preparation of polysaccharide sample The KC (Envoi No 12869) and LBG (Lygomme 6, B 7756) samples were kindly donated by Sanofi Bio Industries (France), with molecular weights of 3 ×105 (in the coil form) and 106 g/mol, respectively. The sodium salt form of KC was prepared by cation exchange on a Dowex-50W strong acid cation exchange resin from Sigma. The resin was first converted to the H + form by elution with HCl, and then to the Na + form by using appropriate chloride salts. Ion exchange of the polysaccharide (1.5% w/w) was carried out at 95°C to maintain the polymer in the disordered coil form. The resultant
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samples were then freeze-dried and stored. To check for possible degradation of the polysaccharide the elastic modulus was measured before and after the ion exchange and no substantial difference was observed. Aqueous NaI and CsI (AnalaR grade from Sigma) solutions of a total concentration of 0.1 M were prepared and mixed in the desired proportions. KC and LBG were added to the appropriate salt solution at room temperature and then heated at 95°C at least for 20 min while stirring. Millipore® water was used throughout. All concentrations are given in weight percent (w/w).
2.2. Low amplitude oscillatory measurements Rheological measurements under low amplitude oscillatory shear were performed on a controlled stress Carri-Med CSL100 rheometer (TA Instruments, UK) using a parallel plate geometry (40 mm radius; 1 mm separation) at a frequency of 1.6 Hz. All measurements were performed with 0.5% strain since strain sweeps on selected gels demonstrated that the working deformation was well within the linear viscoelastic region. To avoid slippage due to syneresis [38,40], as repeatedly observed when CsI was predominating, a cross-hatched acrylic upper plate (40 mm diameter, 0.5 mm separation) was used at XCs ]0.4. Each sample was loaded as a hot solution on the platen of the rheometer pre-set at 80°C. The sample periphery was coated with silicone oil to minimize loss of solvent or adsorption of atmospheric moisture. The rheological parameters were monitored on cooling at 1°C/min to 20°C. Cooling scans were followed by a 30-min isothermal run and a frequency sweep between 0.01 and 10 Hz (about another 30 min). The rheological routine was completed with a heating run from 20 to 95°C (1°C/min). The above sequence of experimental procedures allowed recording of the storage modulus (G%), the loss modulus (G%%), tan d (=G%%/G%) and the complex viscosity (h*= (G%2 +G%%2)1/2/v) as functions of time, temperature, and frequency of oscillation. The coil–helix and helix – coil transition temperatures reported are ‘helix onset temperatures’, taken as the points where the storage modulus just exceeds the noise (for example see Fig. 1a, b). The increase in G% is frequently so steep that the noise level does not significantly affect the value of the transition temperature.
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imens were stored under liquid nitrogen until they were transferred to the cryo-holder. The cryo-holder was cooled by liquid nitrogen to a temperature lower than − 170°C. The electron microscope used was a Philips CM 120 Bio-twin run at an accelerating voltage of 120 kV equipped by a Gatan multi scan CCD and an Oxford cryo-holder. At least 20 micrographs were recorded of each solution and some solutions were observed several times. To facilitate the formation of thin liquid films, a small amount of C12E5 was added (1.62×10 − 6% w/w), giving a concentration in the solution of approximately 70% of critical micelle concentration (cmc). Three different salt mixtures were investigated by cryo-TEM: XCs = 0, 0.2 and 0.4. Due to the difficulty of preparing viscous solutions with this technique, the polymer concentration was kept low and an ultrasonically degraded KC was used [37]. Our studies
2.3. Cryo-TEM A small drop of the liquid was placed on a perforated carbon film, supported by a 200-mesh copper grid, and most of the liquid was removed by blotting with a filter paper. The thin liquid films formed across the holes were subsequently vitrified by plunging the grid into liquid ethane at its freezing point (− 183°C). The spec-
Fig. 1. Rheology of 0.75% KC and 0.75% LBG in 0.1 M mixture of NaI and CsI. a: Temperature dependence on cooling and heating of storage moduli G% ( — ) and loss moduli G%% (---) in XCs =0.2; and b: variation of G% ( —), G%% (---) on cooling and G% (– – – ), G%% (– – –) on heating for XCs =0.8. (1°C/min, 1.6 Hz, 0.5% strain).
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established that the microstructure of both intact and degraded KC solutions, as observed with cryo-TEM, does not differ substantially [37]. The polymer concentration was 0.1% for the pure polymer systems but for the mixed system (LBG+KC) the concentration of each component was 0.1%, giving a total polymer concentration of 0.2%. All solutions were stored at least over night before any preparations for the cryo-TEM were made.
3. Results The compositions of the mixtures for the rheological studies were chosen using the 1.5% pure KC systems of our previous study as a point of reference. Thus, in the first set of mixtures studied here (0.75% KC/0.75% LBG), half of the KC content of the pure KC systems was replaced by LBG, while, in the second set of mixtures (1.5% KC/0.75% LBG), the same amount of LBG (0.75%) was added. Finally, the third set of mixtures (0.75% KC/1.5% LBG) had the same amount of KC as the first set of mixtures, but twice the amount of LBG. As will be shown below, these compositions demonstrated consistent trends in the rheology of the mixtures. Sometimes, however, small deviations were seen for the 0.75% KC/1.5% LBG mixtures, which possibly may be due to the fact that problems with slippage in the measurements at high cesium were particularly severe for these mixtures.
3.1. Effects on hysteresis and aggregation Fig. 1 shows the temperature dependencies of the storage and loss moduli during conformational ordering and melting for the mixture of 0.75 KC and 0.75 LBG. As for 1.5% KC alone [38], different types of behavior was observed at low and high cesium contents, here represented by at XCs =0.2 and 0.8, respectively. In the presence of low amounts of cesium (Fig. 1a), the helix formation was accompanied by a small increase in rigidity (G%) with no detectable thermal hysteresis on melting. A higher cesium content displaced the conformational transition of the composite to higher temperatures and caused a more co-operative onset of gelation (more steep traces) with a substantial increase in G% (Fig. 1b). When cesium dominated a strong thermal hysteresis appeared in the setting-melting transitions, demonstrating the formation of aggregated helices of KC. Similar traces were obtained for the other proportions of the mixtures. Fig. 2 illustrates the coil –helix formation and melting transition temperatures, as defined in the experimental section, for the different ratios of the mixed polysaccharides, and at different
Fig. 2. Temperature dependence of coil – helix transitions (filled symbols) on cooling and helix – coil on melting (open symbols) vs. cesium content for KC/LBG mixtures; 0.75/075 (squares); 0.75/1.5 (triangles); 1.5/0.75 (circles); 1.5/0 (line, dotted lines on melting); (1°C/min, 1.6 Hz, 0.5% strain).
ratios of NaI and CsI. At any salt content, the observed transition temperature for a mixture on cooling was close to that observed for the algal polysaccharide alone [38,41]. For all systems, the cooling transition temperatures increased smoothly as a function of XCs, and at high cesium content, the transitions for the mixtures occurred a few degrees above the corresponding process for KC alone. The transitions were fully reversible for XCs 5 0.2. However, thermal hysteresis and aggregation was initiated at XCs : 0.25 and further developed with increasing cesium content. Obviously, added LBG caused the KC helices to aggregate at a lower cesium content than for carrageenan alone, i.e. at XCs : 0.25 instead of 0.4 [37,38,41]. In accordance with previous findings [30,32,33], there was also an increase in the extent of the thermal hysteresis when LBG was present. Note that the thermal hysteresis increased with increasing XCs. Thus, the differences in the transition temperatures varied from about 8 to 17°C for the 0.75/0.75 ratio. On the other hand, the melting transition temperatures—and thus, in all probability, the nature of the final aggregated states—in the mixtures were rather independent of the proportions and total concentrations of the two polysaccharides. Our investigation was further continued with cryoTEM studies to detect when the superhelical aggregates first occurred as the cesium content was increased, in the presence and in the absence of LBG. The images observed by cryo-TEM in 0.1% KC are depicted in Fig. 3 for some different salt mixtures. For pure KC, no structures could be observed in 0.1 M NaI, which confirms our earlier observation with a different elec-
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tron microscope [37]: non-aggregated KC helices are not visible by cryo-TEM, due to low contrast or low resolution (or both). In XCs =0.2 the large majority of the images were also completely void of fibers but in two images (out of 23) a few and very faint fibers, were observed (Fig. 3b). As also described previously [37], the rod-like fibers of KC (‘the superhelical rods’ Fig. 3c) can be observed first at and above a cesium content of XCs = 0.4. In the LBG preparations, we saw also some fibers, although the LBG is not considered to be a rigid polymer (fiberlike). The appearance of these fibers were different from the KC fibers. The LBG fibers were often thicker, more flexible and had a higher contrast (Fig. 4). The same type of fibers were observed in all salt mixtures investigated (XCs 0 – 0.4). The (projected) length of these fibers was very polydisperse and some very short fibers often occurred. A peculiar feature was the very sharp turns (90°) that occurred occasionally (can be seen for example at the lower left side of Fig. 4). Some fibers ‘dissolved’, the contrast decreased and
Fig. 3. Cryo-TEM micrographs of 0.1% ultrasonically degraded KC in mixed 0.1 M NaI/CsI-salt solutions. a: XCs = 0 (0.1 M NaI); b: XCs =0.2. The dark corners are due to the supporting film; and c: superhelical KC-rods at XCs = 1.0. Scale bar is 200 nm.
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Fig. 4. A cryo-TEM micrograph of 0.1% LBG in 0.1 M NaI, showing the thick and highly contrasted LBG-fibers (scale bar 200 nm). Note the sharp 90°-turn at the lower left part of the image. The dark spots are frost deposits.
the thickness increased as the end of the fiber was approached (also shown in Fig. 4), possibly due that the observed fibers are aggregates of several LBG chains. The amount of these fibers varied between the different LBG preparations which could be caused by minor differences in the sample history (for example storage time before vitrification). LBG has, to our knowledge not been studied by cryo-TEM before. A study of KC/LBG mixtures by the mica sandwich replica technique was reported by Lundin [2,31]. In that work, no microstructure of LBG was observed except for some cases where a structure was probably made visible due to shearing of LBG polymer when the mica plates were put together. We have also examined by cryo-TEM the same LBG as the one used by Lundin (LBG80) and this also displays similar types of fibers as observed in our samples. It is not clear why we see these structures, since LBG is not considered as a rigid, fiberlike polymer. One possible explanation could be that our preparation technique (by cryo-TEM) may also expose the sample to relatively high shear forces leading to visible structures. As well, the history of the samples could be very important if the structures are developing with time. As mentioned earlier, LBG is known to self associate during freezethawing treatment and long ageing. For the mixed systems a structure similar to the pure LBG was observed in 0.1 M NaI (Fig. 5(a)). At XCs = 0.2 many thin and rigid fibers of the KC-type were observed (Fig. 5(b)). Interestingly, also some short and thick LBG fibers could be observed coexisting with the KC fibers under these salt conditions. At XCs =0.4 CsI we observed many KC fibers (Fig. 5(c)). Although the concentrations used for the microscopy studies are relatively low in comparison with those used in the rheological experiments, our previous studies have clearly established that the onset and the magnitude of hysteresis (critical XCs content) is independent on the molecular weight (degree of polymerisation) between 100 000 and 600 000 for the helix form and the KC concentration (0.1–2%) [36–38,41].
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3.2. Effects on 6iscoelastic properties. For a thermoreversible polymer gel, the cross-over point of the storage and loss moduli at some frequency is sometimes (arbitrarily) taken as the ‘gel point’. Fig. 6 shows the cross-over of G% and G%% on cooling at the experimental frequency of 1.6 Hz for different KC/ LBG mixtures. At 1.5% of KC, the cross-over temperatures were very close to the temperatures for the onset of conformational ordering, as detected by rheology. However, at 0.75% KC and at low cesium contents the cross-over temperatures were significantly lower than the onset temperatures (cf. also Fig. 1a), i.e. a substantial proportion of helical KC was required in order for the elastic behavior to dominate. On increasing the cesium content, a sharp increase in the storage modulus was found previously at XCs \ 0.4 for the 1.5% pure KC gels, conditions where the super-
Fig. 5. Cryo-TEM micrographs of mixed 0.1% LBG + 0.1% ultrasonically degraded KC in different NaI/CsI-salt mixtures. In 0.1 M NaI (a), only the LBG-structure is visible (note the sharp turns and the thick, strongly contrasted fibers). The dark spots are frost deposits. When cesium is introduced, the thinner, more rigid superhelical rods of kappa – carrageenan occur at XCs = 0.2 (b), but coexisting with some short and thick LBG-fibers. At XCs = 0.4 (c), the KC fibers are more abundant. The scale bar is 200 nm in all images.
Fig. 6. Changes of cross-over temperatures of G% and G%% at 1.6 Hz, accompanying the formation of mixed gels of KC/LBG vs. cesium content; 0.75/075 ( ); 0.75/1.5 ();1.5/0.75 (); 1.5/0 ( × ).
helical rod formation was identified from cryo-TEM [37,38]. Fig. 7 illustrates (in both logarithmic and linear representations) the variation of G% for KC/LBG mixtures recorded at the end of 30 min isothermal run at 20°C, on completion of the cooling scans. Visual inspection revealed self-supporting networks in all series of mixtures, except for the mixing ratio 0.75/0.75 in 0.1 M NaI. The replacement of KC with LBG (cf. the pure 1.5% KC gels with the 0.75/0.75 mixtures) resulted in a decrease in the modulus except at intermediate cesium contents, where the shift in the onset of aggregation of KC helices gave rise to a true synergism. Addition of LBG generally raised the rigidity of the systems, except for the 0.75 KC/1.5 LBG mixtures at high cesium contents, where problems with slippage were found. At low cesium contents the increase was much lower when the KC concentration was high (1.5%). Two further observations are noteworthy. First, for the weak gels at low cesium, the moduli of the mixtures increased markedly with increasing XCs, although no such strong variation was seen for the respective pure systems. The 1.5% LBG alone showed a solution-like behavior (G%% \G%, from 0.01 to 10 Hz) and a low storage modulus at all ionic compositions. Second, the increase in G% at the ‘new’ critical XCs = 0.25 for the mixtures was rather modest, compared to the increase seen for pure 1.5% KC at XCs = 0.4. Especially from the linear plot, it is apparent that the large increase in G% still occurred around XCs = 0.4 also for the mixtures. As already noted, slippage at high cesium contents was observed repeatedly, and the problems became worse on addition of LBG. This was unexpected, since it is typically observed that galactomannan addition decreases the tendency towards syneresis and contraction. Measurements were impossible even with the
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cross-hatched configuration for the 0.75/1.5 (KC/LBG) mixtures above XCs = 0.7, and for the 1.5/0.75 mixtures at 0.1 M CsI. The frequency dependencies for representative samples at 20°C are presented in Fig. 8a – d. At high cesium contents, the mechanical spectra of the mixed preparations were typical of true gels. Below XCs =0.3, however, the separation between G% and G%% was clearly reduced, and both moduli showed a greater variation across the frequency range (Fig. 8b, c). Nevertheless, a plot of log h* versus log v was still linear, which is one of the characteristic features of a gel-like response [42]. For a true gel, the slope of such a plot equals −1. The mechanical spectra for the ratios 0.75/0.75 and 0.75/1.5 demonstrate that the addition of galactomannan gave a significant enhancement in both moduli, but the viscoelastic character remained unaffected; i.e. the slopes of log h* versus log v were similar, and so were the separation of G% and G%% as well as the frequency dependence. When the concentration of carrageenan was increased (Fig. 8d), the moduli increased and the spectra exhibited a much weaker frequency dependence. Fig. 9 shows the (negative of) the linear slopes of log h* versus log v over the whole range of cesium contents for all polymer concentrations studied. Below XCs B 0.3 the slopes suggest that the systems were entangled networks with weak crosslinks. Permanent elastic networks, as known for conventional polysaccharide gels [43], were formed only at higher cesium contents, where the slopes approached the limiting value of − 1. The effect of the compositional variation and ionic conditions on tan d, measured at a single frequency (1.6
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Hz) at the end of the 30-min isothermal run, reflects the same tendencies of Fig. 9. Generally, tan d was high at low cesium contents, and decreased progressively to level off at a constant value at XCs \ 0.4. This response again implies a development from an entangled network to a weak gel-like character and, finally, a formation of true gels at a large content of the gel-promoting cation. A nearly 40-fold variation in tan d across the salt composition range was seen for the ratio 0.75/0.75, while the transition was less sharp, and the tan d values remained high at high cesium, upon increasing the concentration of galactomannan (the 0.75/1.5 mixture). Most probably this was due to the tendency of slippage observed for this sample. In systems containing 1.5% carrageenan, less elastic systems were obtained at low cesium contents when LBG was added (cf. 1.5% KC with the 1.5/0.75 mixture). This simply suggests an additive effect of LBG on the weak KC network.
3.3. Time dependent effects When the cesium content was around 0.25–0.3, the heating transitions of the mixtures displayed a second melting step at higher temperatures, as illustrated in Fig. 10. The amplitude of the second step became higher as XCs increased and only a single step appeared at a cesium content of about 0.4. A two-step transition ‘on cooling’ was previously observed for KC alone at XCs : 0.5 and a cooling rate of 1°C/min. The latter feature disappeared, however, when the cooling rate was lowered to 0.2°C/min, and the effect was thus attributed to slow kinetics [38]. To explore further the
Fig. 7. Storage modulus G% vs. cesium content for the various KC/LBG mixtures. 0.75/075 ( ); 0.75/1.5 ();1.5/0.75 (); 1.5/0 ( ×); and 1.5% LBG () (1.6 Hz, 0.5% strain).
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Fig. 8. Frequency dependence at 20°C of G% (), G%% () and h* () for KC/LBG mixtures: a: 0.75/0.75 XCs =0.8; b: 0.75/0.75 XCs = 0.2: c: 0.75/1.5 XCs =0.2: and d: 1.5/0.75 XCs = 0.2.
two-step melting curve in the mixtures (Fig. 10a), we adopted the same strategy and measured a heatingcooling cycle at a much slower rate (0.2°C/min instead of 1°C/min). As shown in Fig. 10b, no changes were observed in the transition temperatures, and the twostep melting process still persisted. Thus, there was no evidence to suggest that the origin of the two steps was a kinetic effect. On the other hand, the network modulus increased at the slower cooling rate, as is most easily seen from the value of G% at the lowest temperature (20°C). In fact, for the gels that showed thermal hysteris, i.e. at cesium contents above XCs :0.25, we quite generally observed a significant increase of the storage moduli of the gels during the 30 min isothermal run at 20°C (the difference between cooling and heating data at 20°C in Fig. 1b). We also compared the frequency sweeps accompanying the different cooling rates for the mixture corresponding to Fig. 10. Not only did the storage modulus increase (from 95 to 170 Pa) at the lower cooling rate,
but so did the gel-like character, as shown in Fig. 11a, b. The difference between G% and G%% increased for the slower cooling rate, and the slope of log h* versus log v became steeper (− 0.71 and − 0.82 for 1 and 0.2°C/ min, respectively). Therefore, the viscoelastic properties of the composite, but not the coil–helix–coil transition temperatures, are significantly affected by the cooling rate and the equilibration time.
4. Discussion The results of the present study on KC gels and solutions, with or without LBG present, confirm the trends seen in previous studies: in pure NaI or at low contents of cesium, the coil–helix transition is quite reversible, whereas a thermal hysteresis sets in at a quite well defined value of XCs. The appearance of hysteresis coincides with the development of rigid, superhelical rods of KC, which are visible by cryo-TEM. The onset
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of helix formation on cooling is only weakly sensitive to the presence of LBG, which indicates that the same primary aggregate (the helix) forms both in the presence and in the absence of LBG. Only at higher cesium content does LBG cause a slight increase in the coil-tohelix transition temperatures. Both with and without LBG is there an increase in the sharpness of the cooling transition with increasing cesium content, in line with recent calorimetric measurements on KC alone [41]. The most significant new result of the present study is the lowering of the critical cesium content required for the onset of hysteresis in the conformational transition, and for the formation of superhelical rods of KC, from XCs :0.4 for pure KC to XCs :0.25 in KC/LBG mixtures. In contrast, polarimetric studies in our laboratory (Ramzi, unpublished) have shown that there is no shift in the critical cesium content required for hysteresis when KC is mixed with guar gum, a galactomannan that associates only weakly or not at all with KC. Our task is now to interpret these results in the light of what is currently known about the gelation of KC and the association between KC and LBG. Fig. 12 illustrates the various steps in the association and gelation of KC, according to the picture that has emerged from recent studies in our laboratory. This model differs from previous models in that it explicitly includes the superhelical rod as an intermediate state between the helix and the gel; the gel is formed by the association of the super-helical rods in higher order aggregates. The bulk of previous studies [30,33] have shown that at least no strong aggregates are formed between nonaggregated KC helices and LBG molecules. By strong aggregates, we mean aggregates that give rise to a hysteresis in the coil – helix transition temperature. This
Fig. 9. Composition dependence of linear slopes of (log h* vs. log v) vs. cesium connect for the mixtures of KC with galactomannan. Symbols as in Fig. 6. Values taken at a fixed time of 30 min at 20°C after cooling (1.6 Hz, 0.5% strain, absolute values).
Fig. 10. Thermal change of G% ( —), G%% (---) on cooling and G% ( – – –), G%% (---) on heating at different scan rates: a: 0.75/0.75 XCs =0.25 with 1°C/min; b: 0.75/0.75 XCs =0.25 at 0.2°C/min and; c: 1.5/0.75 XCs =0.3 with 1°C/min (1.6 Hz, 0.5% strain).
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picture is confirmed here. As soon as there is evidence of strong KC–LBG aggregation, there is also evidence of KC self-aggregation; the characteristic images of super-helical rods of KC appear in the cryo-TEM micrographs. The simplest interpretation that is consistent with our results is then that ‘the super-helical rod is the state of KC that is in6ol6ed in the strong aggregation with LBG. When LBG molecules are present, they thus stabilize the super-helical rods, so that these can form already at a lower content of rod-promoting cations, such as cesium’. The association between the KC rods and LBG molecules should have consequences for the higher level association of the KC rods and the gelation process. Previous authors have shown results indicating a more limited aggregation of KC in the presence of LBG [2,31]. This is in line with our interpretation, if it is assumed that LBG to some extent prevents some higher
Fig. 11. Mechanical spectra (20°C, 0.5% strain) shown the frequency dependence of G% (), G%% () and h* () for 0.75/0.75 KC/LBG systems at XCs = 0.25, developed with a different cooling rate. a: 1°C/min; b: 0.2°C/min.
level of association of rods, but not the formation of the rods themselves (which is actually promoted by LBG). However, our studies also clearly demonstrate the strong similarities in the rheological properties between the mixed systems and those containing KC alone. This implies that LBG/KC mixtures must also contain a self-aggregated KC network structure similar to that in pure KC, i.e. we should not think of a mixed gel where all rod–rod junctions have been replaced by rod–LBG junctions. Probably, the role of LBG is more to reinforce the KC network and, possibly, to prevent its collapse. In any case, however, an excess of unbound LBG should eventually develop at sufficiently large LBG/KC ratios, owing to a saturation of the available surface area on the KC rods. A segregation between unbound LBG and saturated mixed aggregates would then be a possibility, which might be the reason for the experimental difficulties (slippage) encountered here for the 0.75/1.5 (KC/LBG) mixtures. More firm conclusions regarding the nature of the mixed network are difficult to draw, however, until there is more detailed knowledge about the structure of the network of KC alone. An interesting finding made here is the two-step melting transition for the mixtures in the region 0.25B XCs B 0.4 (Fig. 10). We note that also Gonc¸alves et al. [33] found a two-step melting in KC/LBG mixtures under salt conditions (NaCl) where KC does not display hysteresis or form super-helical rods on its own. The occurrence of two melting steps indicate that ‘only a fraction’ of the KC helices formed on cooling are converted to rigid rods, the rest remaining as individual helices. We propose the following explanation to why the rod fraction is limited, but increases with increasing XCs, in the interval 0.25BXCs B 0.4. LBG is not a homogeneous polymer, but contains a range of structures with more or less strong tendencies to associate with KC. Close to XCs = 0.25, probably only the fractions with the strongest binding have the capacity to ‘pull over’ KC to the rod state. When this strongly associating fraction is consumed in mixed aggregates, the excess carrageenan undergoes a reversible coil–helix transition as in the absence of LBG. As XCs increases towards 0.4, however, the required binding strength for stabilizing the rods decreases, more mixed aggregates are formed, and the fraction of rods increases. It also seems clear that that the value of G% measured at room temperature should depend on the content of the rod fraction. The gradual build-up of the rod fraction with increasing cesium content in the mixtures then explains our observation that the increase in G% as a function of XCs is not so strongly correlated with the onset of hysteresis for the mixed gels as for the gels of KC alone (Fig. 7). For KC alone, both events occur at XCs : 0.4, whereas no sharp increase in G% occurs at XCs : 0.25 in the mixtures. Similarly, a gel-like vis-
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Fig. 12. Schematic representation of the various steps in the association and gelation of KC. LBG associates to the super-helical rods of KC.
coelastic behavior (as indicated by the tan d values and the slopes of log h* versus log v) also appears only at XCs \0.4 in all systems. A final point to address is the observation of a significant dependence of the moduli and the tan d of the mixed gels on the cesium content also below the onset of hysteresis (XCs B0.25). This is remarkable, since no corresponding dependence was seen for either of the pure systems. We cannot offer a definite explanation of this phenomenon at present. The obvious interpretation in terms of an association, at finite cesium contents, between LBG and ‘individual’ KC helices is not entirely satisfactory, since such an association— contrary to our observation — should increase the stability of the helix and shift the coil – helix transition towards higher temperatures. Since no significant temperature shift was observed, this would imply that the association, if it indeed exists, is quite weak.
Acknowledgements We are grateful to Professor Anne-Marie Hermansson (SIK, Go¨teborg) for valuable discussions and for sending us the LBG80 sample used by L. Lundin.
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