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Recent advances in the understanding of heat set gelling polysaccharides
Trends in Food Science & Technology 15 (2004) 305–312
Recent advances in the understanding of heat set gelling polysaccharides K. Nishinaria,* and H. Zhangb a
Department of Food and Nutrition, Graduate School of Human Life Science, Osaka City University, Osaka 558-8585, Japan (Tel.: +81-6-6605-2818; fax: +81-6-6605-3086; e-mail: [email protected]) b College of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China Since the mechanism of heat set gelation is not wellunderstood, the recent advances in the understanding of the gelation of Konjac glucomannan and curdlan are described mainly based on rheological and thermal studies. Although both gels formed at sufficiently higher temperatures are thermo-irreversible, hydrogen bonding is believed to play an important role in the network formation. The contribution of hydrophobic interaction should be also studied further. # 2004 Elsevier Ltd. All rights reserved.
Introduction Many polysaccharides can form hydrogels by either heating or cooling although some of them such as pullulan can only form an aqueous solution at any temperatures. In comparison with cold set gels like agarose, carrageenans, gelatins, and gellan (Clark & RossMurphy, 1987; te Nijenhuis, 1997; Nishinari, 2000a), the gelation mechanism of heat set gels is not well understood. The temperature dependence of the elastic
* Corresponding author. 0924-2244/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tifs.2003.05.001
modulus of polysaccharide gels may be classified into four categories (Nishinari, 2000b): (1) so-called cold set gels like agarose, carrageenans and gellan which form a gel on cooling the solution, (2) so-called heat set gels like some cellulose derivatives such as methyl cellulose (MC), hydroxypropyl methyl cellulose (HPMC), curdlan and konjac glucomannan (KGM) which form a gel on heating the solution. Curdlan here seems to be somewhat unique since it can form both a cold-set gel and a heat-set gel, respectively. (3) re-entrant gels like xyloglucan from which some galactose residues are removed. Xyloglucan forms gels at intermediate specific temperature ranges and remains in the sol state at higher and lower temperatures outside of this specific temperature range, and (4) inverse re-entrant gels like a mixed solution of methyl cellulose and gelatin which forms a gel at higher and lower temperatures and stays in a sol state in the intermediate temperature range. Gelatin may be replaced by some polysaccharides which form a gel on cooling. Gels belonging to the first class are mostly thermo-reversible and have been studied extensively although the gelation mechanism has not completely been clarified at the molecular level. The other three classes have not been elucidated so well. Some polysaccharides like MC or HPMC or curdlan forms a gel on heating by itself, however, KGM forms a gel on heating only in the presence of alkali which removes acetyl groups. Some of these heat-set gels are thermo-reversible (MC or HPMC gel, low-set gel of curdlan), whilst others (KGM gel and high-set gel of curdlan) are thermo-irreversible. It is well established that pullulan behaves like a flexible coil in water (Kato, Katsuki, & Takahashi, 1984; Kawahara, Ohta, Miyamoto, & Nakamura, 1984; Nishinari et al., 1991), but it does not form a gel at a reasonable concentration. It has been reported recently that the minimum concentration for gel formation in which hydrogen bonding is the main molecular force decreases with increasing stiffness of polymeric chains by a computer simulation (Okada, Koga, & Tanaka, 2002). The difficulty in the study on the gelation mechanism of polysaccharides lies in the poor solubility of gelling polysaccharides in water. It is almost impossible to obtain the reliable value of the molecular weight of poorly soluble gelling polysaccharides in water, and molecular parameters such as molecular weight and radius of gyration can be
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obtained only in some solvents like dimethylsulfoxide, alkali solutions or cadoxen. However, it is important to understand the gelation mechanism of polysaccharides in water and not in toxic solvents as mentioned above to develop further utilisation of these gelling polysaccharides in the food industry. Here, the recent advances in the understanding the gelation mechanism of konjac glucomannan and curdlan both of which are poorly soluble in water are reviewed.
Gelation of konjac glucomannan Konjac glucomannan is a polysaccharide extracted from the tuber of Amorphophallus konjac C.Koch. It is cultivated in Gunma prefecture and other prefectures in Japan, and grows in China and other Asian countries. It is a main ingredient in konnyaku gels used in Japanese traditional dishes and recently it is also used as a gelling agent mixed with k-carrageenan or some other hydrocolloids in dessert jellies (Nishinari, 1988, 2000c; Nishinari et al., 1992). Konjac glucomannan consists of glucose and mannose in the ratio 1.6–1.0. It contains some acetyl groups which confer the solubility to the konjac polymer. It is said that there are some branching points. It forms a gel on heating in the presence of alkali and the role of alkali is believed to remove the acetyl groups. The peak at
Fig. 1. Time dependence of G0 and G00 for 2% dispersions of konjac gluycomannan. &: KGM1 (MW=2.56105), ^: KGM2 (MW=4.38105), ~: KGM3 (MW=4.44105), : KGM4 (MW=5.96105). The solid lines for time dependence of G0 are first order fits.
1730 cm1 in the FTIR absorbance spectra of KGM films attributed to the acetyl group (Zhang et al., 2001) disappear by alkali treatment. Time dependence of G0 and G00 for 2% aqueous dispersions of KGM with different molecular weights from 2.56 to 5.96105 in the presence of 0.02 mol/l sodium carbonate at 60 C is shown in Figure 1 (Yoshimura & Nishinari, 1999). The time t=0 was defined as a time when the alkali was added. The observed curve for G0 as a function of time was fitted well by an equation for the reaction of the first order. The gelation time t0, at which G0 began to deviate from the baseline, became shorter and the saturated G0 increased with increasing molecular weight (Yoshimura & Nishinari, 1999). Time dependence of G0 and G00 for aqueous dispersions of KGM with a molecular weight of 2.56105 with different concentrations from 1.0 to 3.0% in the presence of 0.02 mol/l sodium carbonate at 65 C fitted the first order equation. The gelation time t0 became shorter and the saturated G0 increased with increasing concentration (Yoshimura & Nishinari, 1999). In the time dependence of G0 and G00 for 2 and 3% aqueous dispersions of KGM with the molecular weight of 4.38105 at different temperatures from 40 to 90 C in the presence of 0.02 mol/l sodium carbonate, the maximum of G0 was observed at higher temperatures above 75 C. To examine whether the maximum of G0 is a real phenomenon or an artifact caused by slippage, the penetration force was measured using a spherical Teflon plunger of radius 5 mm (Zhang et al., 2001). This method has an advantage that it is free from slippage. The normalized penetration force observed at different temperatures from 50 to 90 C did not show any maximum as a function of time, indicating that the maximum observed in G0 by the oscillatory shear mode mentioned above was induced by slippage. The apparent maximum in G0 in the study of gelation is a notorious problem, and has been sometimes reported erroneously (Zhang et al., 2001). The time dependence of G0 and G00 for aqueous dispersions of KGM with molecular weight 1.17106 at different concentrations from 0.5 to 2.0% in the presence of 0.02 mol/l sodium carbonate at 80 C did not show any maximum. The gelation time t0 became shorter and the saturated G0 increased with increasing concentration. The slippage may be due to disentanglement of KGM chains adsorbed on the parallel plates of the rheological apparatus during gelation. When the surface of the parallel plates is occupied by the free water molecules predominantly, catastrophic slippage takes place (Zhang et al., 2001). To understand the role of acetyl groups in the gelation of KGM solutions, several fractions of KGM with different degrees of acetylation were prepared (Long, Takahashi, Kobayashi, Kawase, & Nishinari, 1999). Five fractions with different acetylation levels were
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obtained. Degree of acetylation DA (the weight percent of acetyl substituted residues in KGM backbone) ranged from 1.6 (not treated) to 5.3%. The molecular weight determined by light scattering in cadoxen decreased to about the half value by the deacetylation (Long, Takahashi, Kobayashi, Kawase, & Nishinari, 1999). However, the various acetylation reaction conditions, such as different acetylation temperature and the amount of catalyst, did not lead to a remarkable difference in molecular weight. Therefore, the slight difference in molecular weight between the acetylated samples was not taken into account, and the attention was paid mainly to the influence of DA. The mechanism of the main chain scission should be explored in the future to obtain better samples with different DA without changing the molecular weight. Figure 2(a) shows the time dependence of G0 of 2.0wt.% aqueous dispersions of KGM with different DA in the gelation process at 45 C in the presence of Na2CO3. The concentration of Na2CO3 was fixed as
Fig. 2. (a) Time dependence of G0 of 2.0wt.% KGM aqueous dispersions in the presence of Na2CO3 at 45 C. Concentration ratio of Na2CO3 to KGM was 0.2wt.%. The degree of acetylation DA of each sample is shown in Fig. 3. (b) Plot of the gel time at which G0 =G00 against the degree of acetylation of each sample.
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0.2wt.%. The value of G0 at t=0 for a native fraction Rs was far larger than that for acetylated KGM samples. However, G0 of Rs was overtaken by that of acetylated KGM samples finally with time elapsing. The gelation time tgel defined as the time of the crossover of G0 and G00 , as shown in Figure 2(b), became longer with increasing DA. Although the gelation time determined in this way slightly changed with the frequency, this method was adopted for the simplicity. It is expected that both the deacetylation reaction and further aggregation process for KGM samples with higher DA need a longer time than for KGM with lower DA. G0 of all the samples increased rapidly at the start of gelation and attained plateau values. It took a longer time for KGM with higher DA to reach the saturated value of G0 . G0 of the native KGM, Rs attained maximum values in ca. 245 min (ca. 4 h); while G0 of a KGM fraction with the highest DA, Ac 32 still continued to increase even after 2300 min (ca. 38 h). The reason why Ac32 showed a different behaviour is interpreted as follows: the DA of Ac32 is the highest, and the largest amount of alkali is required. The present concentration of Na2CO3 solution was insufficient for 2wt.% Ac32 dispersion to form a gel in the short time. It was difficult to observe the exact plateau values of G0 for some acetylated KGM samples in these experiments due to the long measuring time. Both storage and loss moduli G0 and G00 of Rs aqueous dispersions in the presence of Na2CO3 at a fixed concentration ratio of Na2CO3 to the degree of acetylation of KGM (0.1) were found to increase monotonically and attained plateau values, the saturated storage modulus G0 sat and loss modulus G00 sat, respectively, after a certain time (data not shown) as was observed previously. tgel increased sharply with decreasing concentration and G0 sat increased with increasing concentration. Since molecular chains are close to each other at higher
Fig. 3. Time dependence of G0 of 2.0wt.% KGM aqueous dispersions in the presence of Na2CO3 at 60 C. The ratio of alkaline concentration to the degree of acetylation was fixed to a constant (0.1).
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concentrations, the probability of the formation of junction zones is higher than that at lower concentrations. Gelation would begin even before the complete loss of acetyl groups at higher concentrations; therefore tgel became shorter with increasing concentration of KGM as expected (Long et al., 2002). If the alkaline concentration (CAl) to DA was fixed to a constant value (0.1), a similar time course of G0 for all the samples except the fraction with the highest DA was observed as shown in Figure 3. It indicates that CAl/DA plays a crucial role in the gelation process. The gelation rate is determined mainly by the ratio of alkaline concentration to the degree of acetylation (Long et al., 2002). However, the fact that Ac32 shows exceptional behaviour should be explored in the near future. In conclusion, it was concluded that deacetylation leads to the aggregation of stiffened molecular chains. In the presence of excessive alkali, gelation proceeds too fast resulting in a gel with smaller elastic modulus. The final elastic modulus of gels depends strongly on the gelation rate. The KGM gel is thermo-irreversible and the rearrangement of network chains does not seem to occur as in cold-set gels such as gellan gels and -carrageenan gels. It was reported recently that the helix-coil transition occurs in a self-supporting gels of gellan induced by temperature change (Nitta, Ikeda, Takaya, & Nishinari, 2001), which also occurs in -carrageenan gels by the immersion in salt solutions (Watase & Nishinari, 1982). In the gelation of KGM, the gelation rate was shown to be governed by the concentration and molecular weight of polymer, temperature as well as by DA and alkaline concentration, and it was also shown that slower gelation leads to the stronger gels. Although molecular forces responsible for gel formation in both cold-set gels and in KGM gels are believed to be hydrogen bonds, tighter stacking occurs in KGM gels than in other thermo-reversible gels such as gellan, carrageenan, agarose etc. Stacking might be tighter in solutions of polymers with longer persistent length. Unfortunately, the persistent length of KGM is not known because of its poor solubility in water. The AFM observation will be useful to elucidate this.
Gelation of curdlan Curdlan was discovered by the late Prof. Harada in 1966, and is a b-1,3 glucan produced by bacteria such as Alcaligenes faecalis var.myxogenes. It is not soluble in water. The aqueous suspension of curdlan forms a gel on heating, and has been used in various foods such as tofu, noodles, and jellies (Nishinari & Zhang, 2001; Funami, 2000). Two types of gels, a high-set gel formed at higher temperatures above Tc 60 C which is thermo-irreversible, and a low-set gel formed below Tc which is thermo-reversible, have been reported. This transition temperature Tc 60 C depends on the concentration and molecular weight of curdlan.
The storage and loss moduli of aqueous suspensions of curdlan as a function of frequency at 40 C and at 70 C show similar behaviour (Nishinari & Zhang, 2001; Funami, 2000); even before gelation at 40 C, G0 is larger than G00 at all the frequencies and hardly dependent on the frequency, typical of weak gel behaviour. This aqueous suspension in a test tube flows when the test tube is tilted. At 70 C, both moduli increase and show plateau values, and the loss tangent becomes far smaller than that for the suspension at 40 C. This is typical behaviour of an elastic gel. When curdlan is dissolved in DMSO, the solution shows an ordinary mechanical spectrum for a solution of flexible polymer chains; G00 predominates G0 at lower frequencies because there is enough time for molecular chains to disentangle whilst G0 is dominant at higher frequencies because there is not enough time for chains to disentangle. Figure 4(a) shows a reheating DSC curve of curdlan aqueous dispersion which has been heated at a certain temperature, as indicated beside each curve, for 60 min and then cooled. The reheating DSC curve for curdlan aqueous dispersion which has been heated below 50 C shows almost the same DSC curve as in the first heating DSC curve, and the reheating DSC curve for curdlan aqueous dispersion which has been heated above 110 C does not show any endothermic peak. The degree of thermo-irreversible gelation dG for a 10% aqueous dispersion of curdlan as a function of heating temperature is shown in Figure 4(b) (Nishinari, Hirashima, Miyoshi, & Takaya, 1998). Figure 5 shows the thermal rheological scanning measurements of G0 and G00 for a 2% curdlan suspension at a scan rate of 1 C /min (Zhang, Nishinari, Williams, Foster, & Norton, 2002). On the first heating run, both G0 and G00 increase slightly with increasing temperature up to about 45 C then decrease slightly up to 55 C, before increasing gradually with temperature. On cooling from 80 C, both G0 and G00 increased slightly and showed a steep increase at 40 C, which may be attributed to the strengthening of the network by hydrogen bonds. Whether this increase is due to the reformation of the triple helices or not should be explored in the near future. Once a gel is formed, both G0 and G00 gradually decrease on heating, suggesting that the hydrogen bonds are responsible for network formation. On cooling the steep increase of G0 and G00 at 40 C was observed again. Over the same temperature range the spin-spin relaxation time T2 in the proton NMR measurements initially shows a marked decrease on heating. This suggests that in this case the exchange rate of protons is low, and shows that the chain mobility of the biopolymer is low and hence that the chains are very rigid. This is consistent with the proposed helical structures. However, during the first heating these large changes in T2 are not reversible implying that it is likely that changes
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Fig. 4. (a) Second run DSC heating curves of 10% aqueous dispersions of curdlan kept at various temperatures for 60 min. Heating rate: 1 C/min. The numbers beside each curve represent the temperature in C at which the dispersion was kept. (b)The degree of thermo-irreversible gelation dG=1H2T/H1 for a 10% aqueous dispersion of curdlan as a function of heating temperature T, where H2T is the endothermic enthalpy determined from the endothermic peak in the second run DSC heating curve for a 10% aqueous dispersion of curdlan kept at temperature T for 60 min, and H1 is the endothermic enthalpy in the first run heating.
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in the fractional population of exchangeable protons on the curdlan molecule are also occurring, as would be expected during the process of solvent ingress and hydration of the dried material. As the temperature is raised further it is interesting to see that in the vicinity of the temperature at which G0 starts to increase again the measured T2 value increases significantly. The large increase is consistent either with a reduction in the number of accessible biopolymer protons or with an increased average mobility of the biopolymer chains that now shifts the dominating factor from the exchange rate to the polymer mobility. This would require the spin-spin relaxation time of the biopolymer protons to change from the order of 100 ms or less to a value approaching 1 ms. This is the sort of change that might be expected in, for example, single helix melting and furthermore, it can be seen in DSC curves that the temperature of increased average mobility coincides well, not only with the temperature at which the modulus starts to change, but also with a DSC endotherm. Furthermore, the continued evolution of the relaxation time, in which the T2 value subsequently begins to fall again, strongly suggests that it is indeed the spin-spin relaxation time of the biopolymer protons that is the contributing factor here, reporting on the reduction of chain mobility incurred by attempts at the reformation of the triple helical structures and formation of a gel network. On cooling from 80 C, no T2 change was observed until 40 C, at which the relaxation time decreased steeply, indicating the reduction in average mobility of biopolymer chains and the strengthening of the network, which appeared as the steep increase in G0 in Figure 5. In the second run the value of T2 did not show any remarkable increase and decrease was observed because the network as already settled, and on cooling no T2 change was observed until 40 C, at which the relaxation time decreased steeply (Zhang et al., 2002). DSC thermal scanning cycles for a 2% aqueous suspension of curdlan are shown in Figure 6. Two exothermic peaks were found in DSC cooling curves (Hirashima, Takaya, & Nishinari, 1997), but the precise origin of these exothermic transitions remains a matter of debate. It is however worth noting that (albeit at low biopolymer concentrations) a strong increase in the optical rotation below 40 C was reported, which suggests that the underlying molecular event here may be the formation of single helices (Tako & Hanashiro, 1997). In such a scenario it is possible that the two peaks originate from single helical transitions either of separate chains or single chain sections of imperfectly formed triplex. Such a scheme is also entirely consistent with the DSC results shown here where the appearance of exothermic peaks, indicating the reformation of bonding and kinetic trapping of the current microstructural state, are observed in concert with the NMR
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Fig. 5. Rheological measurements carried out during thermal scanning cycles for a 2% suspension of curdlan CUD1 (MW=1.92106). Scan rate: 1 C/min. (a) first cycle, (b) second cycle.
detected reduction in chain mobility and the rheologically determined increase in gel modulus (Zhang et al., 2002). In order to further characterize such molecular events the molecular weight dependence of the rheological evolution under temperature cycling was examined. The results obtained for 2% curdlan with lower Mw are shown in Figure 7. It can clearly be seen that the lower molecular weight, the more predominant the rise in G0 on increasing the temperature and correspondingly, the lower the increase observed below 40 C on cooling. Harada and his co-workers have reported that lower molecular weight material can anneal more successfully compared to higher molecular weight counterparts that require more ordering (Harada et al., 1994) and, as such, these observations are consistent with a model in which annealing of triplexes takes place on heating to temperatures above about 58 C and changes occurring below 40 C on cooling originate from un-annealed portions of the sample (Zhang et al., 2002).
Fig. 6. DSC themal scanning cycles for a for a 2% suspension of curdlan CUD1 (MW=192104). Scan rate: 1 C/min.
Cooling DSC curves have been examined after heating curdlan samples to different temperatures and holding for 30 min before cooling, and it is interesting to note that as the temperature of holding is increased the size of the exothermic peak decreases. These results also clearly show that the lower temperature exothermic peak disappears before that at higher temperatures (Zhang et al., 2002). There is then evidence that holding samples at higher temperatures or reducing the molecular weight both produce the same manifest changes in the DSC behaviour, consistent with the annealing of more perfectly formed triple helical structures being the origin of the observed behaviour. NMR relaxation data obtained by cycling 2% curdlan to 40 and 50 C at 1 C min1 showed that the change of the spin-spin relaxation time is not reversible (Zhang et al., 2002). However, this thermal range is entirely below the temperature indicated by the DSC endothermic peak, and thus is associated with time dependent molecular level changes of another kind. It seems reasonable to suggest then, as in the initial description, that these changes are a consequence of the hydration of the dry material (Zhang et al., 2002). While the exact details of the network assembly are complex it is well known that, in general, helical forming polysaccharides are prone to aggregate. A gelation model has been proposed for these glucans that is a three chain analogue of that proposed for Welan (Morris et al., 1996), a polysaccharide that is biosynthesised as perfectly registered double helices, that upon de-naturing and subsequent re-naturing forms a gel with each chain involved in more than one junction zone. Indeed this model has been proposed in order to explain the gelation of schizphyllan in the presence of sorbitol (Maeda et al., 1999). While there may be some involvement of this type, particularly at the helix ends, these flexible joints in curdlan seem unable to prevent a further layer of assembly. Micrographs of the curdlan
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Fig. 7. Rheological measurements carried out during thermal scanning cycles for a 2% suspension of curdlan CUD3 (MW=5.0105). Scan rate: 1 C/min. (a) first cycle, (b) second cycle.
system clearly show the overwhelming domination of mibrofibrils (Harada, 1991) and there can be little doubt that it is the aggregation of helices that largely leads to the gel structure on a macroscopic level. In conclusion, from the comparison of data for curdlan obtained by rheology and DSC, the correspondence of changes in the observed behaviour generated by reducing the molecular weight or holding samples at high temperatures for longer times provides strong evidence that the annealing of triplex structures is the main time dependent phenomenon occurring. This hypothesis is supported by estimates of renaturing kinetics obtained by low resolution NMR. Transitions of the un-annealed portion of the sample below 40 C would seem to play a crucial role in the kinetic trapping of microstructural states. It is clear that the high and low temperature gels of conventional rhetoric are not discrete and do not form a complete set of possible behaviours, but rather are two possibilities of a whole spectrum of possible gels in which the contribution of annealed triple helical elements to the stress bearing properties of the generated network can be varied. Holding a sample closer to the transition temperature, allows a greater number of imperfect triplexes to be melted out than a correspondingly lower temperatures and as such, more annealed samples are more resilient and thermo-irreversible. The formation of longer junction zones of increased thermal stability at temperatures closer to the transition temperature is consistent with previous work on the biopolymers gelatin (Michon et al., 1997) and agarose (Mohammed et al., 1998). However, the recent report on the effect of thermal history on gelation for agarose (Aymard et al., 2001) is contradictory with these conclusions. It is necessary to study this problem further.
Concluding remarks It is necessary to clarify the irreversibility of both KGM and curdlan gels. Factors such as gelation rate,
concentration, may affect the structure of junction zones, and a future study using AFM will reveal the effect of these factors on the gel structure. Interaction of water and polymers has been studied by many groups and this problem is closely related with the gelation process and the properties of resultant gels and the freezing process. Whether the molecular chains can be rearranged or not, whether some molecular chains can be incorporated further after the gel formation in the resultant gels or not should be clarified in the near future. This may have a close relation to flavour release. Flavour release in gel-like foods has been discussed mainly from the viewpoint of the fracture strain (Morris, 1994) or fracture stress (Clark, 1998), however, it should also have a close relation with syneresis or water-holding capacity of gels. Both KGM and curdlan gels have rubber-like texture and since it takes a long time for seasonings like shouyu, soy sauce, to be absorbed in ordinary KGM gels, the study of the diffusion of small molecules in these gels might be useful for the food industry. The diffusion process of these molecules might be influenced strongly by the network structure although it has been reported that the diffusion coefficient of water molecules in gellan gels is not different from that of bulk water by NMR (Ohtsuka & Watanabe, 1996).
Acknowledgements The authors thank Prof. P. A. Williams for improving the English and Drs. M.A.K. Williams, M. Yoshimura, R. Takahashi, H. Long for their precious contribution to the present study.
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Nishinari, K. (2000c). Novel macromolecules in food systems. In G. Doxastakis, & V. Kiosseoglou (pp. 309–330). Amsterdam: Elsevier. Nishinari, K., Hirashima, M., Miyoshi, E., & Takaya, T. (1998). In G. O. Phillips, D. J. Wedlock, & P. A. Williams (Eds.), Gums and stabilisers for the food industry 9 (pp. 26–33). Royal Soc. Chem. Nishinari, K., Kohyama, K., Williams, P. A., Phillips, G. O., Burchard, W., & Ogino, K. (1991). Macromolecules, 24, 5590–5593. Nishinari, K., Williams, P. A., & Phillips, G. O. (1992). Food Hydrocoll., 6, 199–222. Nishinari, K., & Zhang, H. (2001). In G. O. Phillips, & P. A. Williams (Eds.), Handbook of hydrocolloids. Cambridge, UK: Woodhead. Nitta, Y., Ikeda, S., Takaya, T., & Nishinari, K. (2001). Trans. MRS-J., 26, 621–624. Ohtsuka, A., & Watanabe, T. (1996). Carbohydr. Polym., 30, 135–144. Okada, Y., Koga, T., & Tanaka, F. (2002). Polym. Prep. Japan, 51, 3306–3307. Tako, M., & Hanashiro, I. (1997). Polym. Gels & Netw., 5, 241–250. Watase, M., & Nishinari, K. (1982). Colloid Polym. Sci., 260, 971–975. Yoshimura, M., & Nishinari, K. (1999). Food Hydrocoll., 13, 227–233. Zhang, H., Nishinari, K., Williams, M. A. K., Foster, T. J., & Norton, I. T. (2002). Int. J. Biol. Macromol., 30, 7–16. Zhang, H., Yoshimura, M., Nishinari, K., Williams, M. A. K., Foster, T. J., & Norton, I. T. (2001). Biopolymers, 59, 38–50.
Further reading Gao, S., & Nishinari, K. (2004). Effect of degree of acetylation on gelation of konjac glucomannan. Biomacromolecules, 5, 175–185.
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Recent advances in the understanding of heat set gelling polysaccharides K. Nishinaria,* and H. Zhangb a
Department of Food and Nutrition, Graduate School of Human Life Science, Osaka City University, Osaka 558-8585, Japan (Tel.: +81-6-6605-2818; fax: +81-6-6605-3086; e-mail: [email protected]) b College of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China Since the mechanism of heat set gelation is not wellunderstood, the recent advances in the understanding of the gelation of Konjac glucomannan and curdlan are described mainly based on rheological and thermal studies. Although both gels formed at sufficiently higher temperatures are thermo-irreversible, hydrogen bonding is believed to play an important role in the network formation. The contribution of hydrophobic interaction should be also studied further. # 2004 Elsevier Ltd. All rights reserved.
Introduction Many polysaccharides can form hydrogels by either heating or cooling although some of them such as pullulan can only form an aqueous solution at any temperatures. In comparison with cold set gels like agarose, carrageenans, gelatins, and gellan (Clark & RossMurphy, 1987; te Nijenhuis, 1997; Nishinari, 2000a), the gelation mechanism of heat set gels is not well understood. The temperature dependence of the elastic
* Corresponding author. 0924-2244/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tifs.2003.05.001
modulus of polysaccharide gels may be classified into four categories (Nishinari, 2000b): (1) so-called cold set gels like agarose, carrageenans and gellan which form a gel on cooling the solution, (2) so-called heat set gels like some cellulose derivatives such as methyl cellulose (MC), hydroxypropyl methyl cellulose (HPMC), curdlan and konjac glucomannan (KGM) which form a gel on heating the solution. Curdlan here seems to be somewhat unique since it can form both a cold-set gel and a heat-set gel, respectively. (3) re-entrant gels like xyloglucan from which some galactose residues are removed. Xyloglucan forms gels at intermediate specific temperature ranges and remains in the sol state at higher and lower temperatures outside of this specific temperature range, and (4) inverse re-entrant gels like a mixed solution of methyl cellulose and gelatin which forms a gel at higher and lower temperatures and stays in a sol state in the intermediate temperature range. Gelatin may be replaced by some polysaccharides which form a gel on cooling. Gels belonging to the first class are mostly thermo-reversible and have been studied extensively although the gelation mechanism has not completely been clarified at the molecular level. The other three classes have not been elucidated so well. Some polysaccharides like MC or HPMC or curdlan forms a gel on heating by itself, however, KGM forms a gel on heating only in the presence of alkali which removes acetyl groups. Some of these heat-set gels are thermo-reversible (MC or HPMC gel, low-set gel of curdlan), whilst others (KGM gel and high-set gel of curdlan) are thermo-irreversible. It is well established that pullulan behaves like a flexible coil in water (Kato, Katsuki, & Takahashi, 1984; Kawahara, Ohta, Miyamoto, & Nakamura, 1984; Nishinari et al., 1991), but it does not form a gel at a reasonable concentration. It has been reported recently that the minimum concentration for gel formation in which hydrogen bonding is the main molecular force decreases with increasing stiffness of polymeric chains by a computer simulation (Okada, Koga, & Tanaka, 2002). The difficulty in the study on the gelation mechanism of polysaccharides lies in the poor solubility of gelling polysaccharides in water. It is almost impossible to obtain the reliable value of the molecular weight of poorly soluble gelling polysaccharides in water, and molecular parameters such as molecular weight and radius of gyration can be
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obtained only in some solvents like dimethylsulfoxide, alkali solutions or cadoxen. However, it is important to understand the gelation mechanism of polysaccharides in water and not in toxic solvents as mentioned above to develop further utilisation of these gelling polysaccharides in the food industry. Here, the recent advances in the understanding the gelation mechanism of konjac glucomannan and curdlan both of which are poorly soluble in water are reviewed.
Gelation of konjac glucomannan Konjac glucomannan is a polysaccharide extracted from the tuber of Amorphophallus konjac C.Koch. It is cultivated in Gunma prefecture and other prefectures in Japan, and grows in China and other Asian countries. It is a main ingredient in konnyaku gels used in Japanese traditional dishes and recently it is also used as a gelling agent mixed with k-carrageenan or some other hydrocolloids in dessert jellies (Nishinari, 1988, 2000c; Nishinari et al., 1992). Konjac glucomannan consists of glucose and mannose in the ratio 1.6–1.0. It contains some acetyl groups which confer the solubility to the konjac polymer. It is said that there are some branching points. It forms a gel on heating in the presence of alkali and the role of alkali is believed to remove the acetyl groups. The peak at
Fig. 1. Time dependence of G0 and G00 for 2% dispersions of konjac gluycomannan. &: KGM1 (MW=2.56105), ^: KGM2 (MW=4.38105), ~: KGM3 (MW=4.44105), : KGM4 (MW=5.96105). The solid lines for time dependence of G0 are first order fits.
1730 cm1 in the FTIR absorbance spectra of KGM films attributed to the acetyl group (Zhang et al., 2001) disappear by alkali treatment. Time dependence of G0 and G00 for 2% aqueous dispersions of KGM with different molecular weights from 2.56 to 5.96105 in the presence of 0.02 mol/l sodium carbonate at 60 C is shown in Figure 1 (Yoshimura & Nishinari, 1999). The time t=0 was defined as a time when the alkali was added. The observed curve for G0 as a function of time was fitted well by an equation for the reaction of the first order. The gelation time t0, at which G0 began to deviate from the baseline, became shorter and the saturated G0 increased with increasing molecular weight (Yoshimura & Nishinari, 1999). Time dependence of G0 and G00 for aqueous dispersions of KGM with a molecular weight of 2.56105 with different concentrations from 1.0 to 3.0% in the presence of 0.02 mol/l sodium carbonate at 65 C fitted the first order equation. The gelation time t0 became shorter and the saturated G0 increased with increasing concentration (Yoshimura & Nishinari, 1999). In the time dependence of G0 and G00 for 2 and 3% aqueous dispersions of KGM with the molecular weight of 4.38105 at different temperatures from 40 to 90 C in the presence of 0.02 mol/l sodium carbonate, the maximum of G0 was observed at higher temperatures above 75 C. To examine whether the maximum of G0 is a real phenomenon or an artifact caused by slippage, the penetration force was measured using a spherical Teflon plunger of radius 5 mm (Zhang et al., 2001). This method has an advantage that it is free from slippage. The normalized penetration force observed at different temperatures from 50 to 90 C did not show any maximum as a function of time, indicating that the maximum observed in G0 by the oscillatory shear mode mentioned above was induced by slippage. The apparent maximum in G0 in the study of gelation is a notorious problem, and has been sometimes reported erroneously (Zhang et al., 2001). The time dependence of G0 and G00 for aqueous dispersions of KGM with molecular weight 1.17106 at different concentrations from 0.5 to 2.0% in the presence of 0.02 mol/l sodium carbonate at 80 C did not show any maximum. The gelation time t0 became shorter and the saturated G0 increased with increasing concentration. The slippage may be due to disentanglement of KGM chains adsorbed on the parallel plates of the rheological apparatus during gelation. When the surface of the parallel plates is occupied by the free water molecules predominantly, catastrophic slippage takes place (Zhang et al., 2001). To understand the role of acetyl groups in the gelation of KGM solutions, several fractions of KGM with different degrees of acetylation were prepared (Long, Takahashi, Kobayashi, Kawase, & Nishinari, 1999). Five fractions with different acetylation levels were
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obtained. Degree of acetylation DA (the weight percent of acetyl substituted residues in KGM backbone) ranged from 1.6 (not treated) to 5.3%. The molecular weight determined by light scattering in cadoxen decreased to about the half value by the deacetylation (Long, Takahashi, Kobayashi, Kawase, & Nishinari, 1999). However, the various acetylation reaction conditions, such as different acetylation temperature and the amount of catalyst, did not lead to a remarkable difference in molecular weight. Therefore, the slight difference in molecular weight between the acetylated samples was not taken into account, and the attention was paid mainly to the influence of DA. The mechanism of the main chain scission should be explored in the future to obtain better samples with different DA without changing the molecular weight. Figure 2(a) shows the time dependence of G0 of 2.0wt.% aqueous dispersions of KGM with different DA in the gelation process at 45 C in the presence of Na2CO3. The concentration of Na2CO3 was fixed as
Fig. 2. (a) Time dependence of G0 of 2.0wt.% KGM aqueous dispersions in the presence of Na2CO3 at 45 C. Concentration ratio of Na2CO3 to KGM was 0.2wt.%. The degree of acetylation DA of each sample is shown in Fig. 3. (b) Plot of the gel time at which G0 =G00 against the degree of acetylation of each sample.
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0.2wt.%. The value of G0 at t=0 for a native fraction Rs was far larger than that for acetylated KGM samples. However, G0 of Rs was overtaken by that of acetylated KGM samples finally with time elapsing. The gelation time tgel defined as the time of the crossover of G0 and G00 , as shown in Figure 2(b), became longer with increasing DA. Although the gelation time determined in this way slightly changed with the frequency, this method was adopted for the simplicity. It is expected that both the deacetylation reaction and further aggregation process for KGM samples with higher DA need a longer time than for KGM with lower DA. G0 of all the samples increased rapidly at the start of gelation and attained plateau values. It took a longer time for KGM with higher DA to reach the saturated value of G0 . G0 of the native KGM, Rs attained maximum values in ca. 245 min (ca. 4 h); while G0 of a KGM fraction with the highest DA, Ac 32 still continued to increase even after 2300 min (ca. 38 h). The reason why Ac32 showed a different behaviour is interpreted as follows: the DA of Ac32 is the highest, and the largest amount of alkali is required. The present concentration of Na2CO3 solution was insufficient for 2wt.% Ac32 dispersion to form a gel in the short time. It was difficult to observe the exact plateau values of G0 for some acetylated KGM samples in these experiments due to the long measuring time. Both storage and loss moduli G0 and G00 of Rs aqueous dispersions in the presence of Na2CO3 at a fixed concentration ratio of Na2CO3 to the degree of acetylation of KGM (0.1) were found to increase monotonically and attained plateau values, the saturated storage modulus G0 sat and loss modulus G00 sat, respectively, after a certain time (data not shown) as was observed previously. tgel increased sharply with decreasing concentration and G0 sat increased with increasing concentration. Since molecular chains are close to each other at higher
Fig. 3. Time dependence of G0 of 2.0wt.% KGM aqueous dispersions in the presence of Na2CO3 at 60 C. The ratio of alkaline concentration to the degree of acetylation was fixed to a constant (0.1).
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concentrations, the probability of the formation of junction zones is higher than that at lower concentrations. Gelation would begin even before the complete loss of acetyl groups at higher concentrations; therefore tgel became shorter with increasing concentration of KGM as expected (Long et al., 2002). If the alkaline concentration (CAl) to DA was fixed to a constant value (0.1), a similar time course of G0 for all the samples except the fraction with the highest DA was observed as shown in Figure 3. It indicates that CAl/DA plays a crucial role in the gelation process. The gelation rate is determined mainly by the ratio of alkaline concentration to the degree of acetylation (Long et al., 2002). However, the fact that Ac32 shows exceptional behaviour should be explored in the near future. In conclusion, it was concluded that deacetylation leads to the aggregation of stiffened molecular chains. In the presence of excessive alkali, gelation proceeds too fast resulting in a gel with smaller elastic modulus. The final elastic modulus of gels depends strongly on the gelation rate. The KGM gel is thermo-irreversible and the rearrangement of network chains does not seem to occur as in cold-set gels such as gellan gels and -carrageenan gels. It was reported recently that the helix-coil transition occurs in a self-supporting gels of gellan induced by temperature change (Nitta, Ikeda, Takaya, & Nishinari, 2001), which also occurs in -carrageenan gels by the immersion in salt solutions (Watase & Nishinari, 1982). In the gelation of KGM, the gelation rate was shown to be governed by the concentration and molecular weight of polymer, temperature as well as by DA and alkaline concentration, and it was also shown that slower gelation leads to the stronger gels. Although molecular forces responsible for gel formation in both cold-set gels and in KGM gels are believed to be hydrogen bonds, tighter stacking occurs in KGM gels than in other thermo-reversible gels such as gellan, carrageenan, agarose etc. Stacking might be tighter in solutions of polymers with longer persistent length. Unfortunately, the persistent length of KGM is not known because of its poor solubility in water. The AFM observation will be useful to elucidate this.
Gelation of curdlan Curdlan was discovered by the late Prof. Harada in 1966, and is a b-1,3 glucan produced by bacteria such as Alcaligenes faecalis var.myxogenes. It is not soluble in water. The aqueous suspension of curdlan forms a gel on heating, and has been used in various foods such as tofu, noodles, and jellies (Nishinari & Zhang, 2001; Funami, 2000). Two types of gels, a high-set gel formed at higher temperatures above Tc 60 C which is thermo-irreversible, and a low-set gel formed below Tc which is thermo-reversible, have been reported. This transition temperature Tc 60 C depends on the concentration and molecular weight of curdlan.
The storage and loss moduli of aqueous suspensions of curdlan as a function of frequency at 40 C and at 70 C show similar behaviour (Nishinari & Zhang, 2001; Funami, 2000); even before gelation at 40 C, G0 is larger than G00 at all the frequencies and hardly dependent on the frequency, typical of weak gel behaviour. This aqueous suspension in a test tube flows when the test tube is tilted. At 70 C, both moduli increase and show plateau values, and the loss tangent becomes far smaller than that for the suspension at 40 C. This is typical behaviour of an elastic gel. When curdlan is dissolved in DMSO, the solution shows an ordinary mechanical spectrum for a solution of flexible polymer chains; G00 predominates G0 at lower frequencies because there is enough time for molecular chains to disentangle whilst G0 is dominant at higher frequencies because there is not enough time for chains to disentangle. Figure 4(a) shows a reheating DSC curve of curdlan aqueous dispersion which has been heated at a certain temperature, as indicated beside each curve, for 60 min and then cooled. The reheating DSC curve for curdlan aqueous dispersion which has been heated below 50 C shows almost the same DSC curve as in the first heating DSC curve, and the reheating DSC curve for curdlan aqueous dispersion which has been heated above 110 C does not show any endothermic peak. The degree of thermo-irreversible gelation dG for a 10% aqueous dispersion of curdlan as a function of heating temperature is shown in Figure 4(b) (Nishinari, Hirashima, Miyoshi, & Takaya, 1998). Figure 5 shows the thermal rheological scanning measurements of G0 and G00 for a 2% curdlan suspension at a scan rate of 1 C /min (Zhang, Nishinari, Williams, Foster, & Norton, 2002). On the first heating run, both G0 and G00 increase slightly with increasing temperature up to about 45 C then decrease slightly up to 55 C, before increasing gradually with temperature. On cooling from 80 C, both G0 and G00 increased slightly and showed a steep increase at 40 C, which may be attributed to the strengthening of the network by hydrogen bonds. Whether this increase is due to the reformation of the triple helices or not should be explored in the near future. Once a gel is formed, both G0 and G00 gradually decrease on heating, suggesting that the hydrogen bonds are responsible for network formation. On cooling the steep increase of G0 and G00 at 40 C was observed again. Over the same temperature range the spin-spin relaxation time T2 in the proton NMR measurements initially shows a marked decrease on heating. This suggests that in this case the exchange rate of protons is low, and shows that the chain mobility of the biopolymer is low and hence that the chains are very rigid. This is consistent with the proposed helical structures. However, during the first heating these large changes in T2 are not reversible implying that it is likely that changes
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Fig. 4. (a) Second run DSC heating curves of 10% aqueous dispersions of curdlan kept at various temperatures for 60 min. Heating rate: 1 C/min. The numbers beside each curve represent the temperature in C at which the dispersion was kept. (b)The degree of thermo-irreversible gelation dG=1H2T/H1 for a 10% aqueous dispersion of curdlan as a function of heating temperature T, where H2T is the endothermic enthalpy determined from the endothermic peak in the second run DSC heating curve for a 10% aqueous dispersion of curdlan kept at temperature T for 60 min, and H1 is the endothermic enthalpy in the first run heating.
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in the fractional population of exchangeable protons on the curdlan molecule are also occurring, as would be expected during the process of solvent ingress and hydration of the dried material. As the temperature is raised further it is interesting to see that in the vicinity of the temperature at which G0 starts to increase again the measured T2 value increases significantly. The large increase is consistent either with a reduction in the number of accessible biopolymer protons or with an increased average mobility of the biopolymer chains that now shifts the dominating factor from the exchange rate to the polymer mobility. This would require the spin-spin relaxation time of the biopolymer protons to change from the order of 100 ms or less to a value approaching 1 ms. This is the sort of change that might be expected in, for example, single helix melting and furthermore, it can be seen in DSC curves that the temperature of increased average mobility coincides well, not only with the temperature at which the modulus starts to change, but also with a DSC endotherm. Furthermore, the continued evolution of the relaxation time, in which the T2 value subsequently begins to fall again, strongly suggests that it is indeed the spin-spin relaxation time of the biopolymer protons that is the contributing factor here, reporting on the reduction of chain mobility incurred by attempts at the reformation of the triple helical structures and formation of a gel network. On cooling from 80 C, no T2 change was observed until 40 C, at which the relaxation time decreased steeply, indicating the reduction in average mobility of biopolymer chains and the strengthening of the network, which appeared as the steep increase in G0 in Figure 5. In the second run the value of T2 did not show any remarkable increase and decrease was observed because the network as already settled, and on cooling no T2 change was observed until 40 C, at which the relaxation time decreased steeply (Zhang et al., 2002). DSC thermal scanning cycles for a 2% aqueous suspension of curdlan are shown in Figure 6. Two exothermic peaks were found in DSC cooling curves (Hirashima, Takaya, & Nishinari, 1997), but the precise origin of these exothermic transitions remains a matter of debate. It is however worth noting that (albeit at low biopolymer concentrations) a strong increase in the optical rotation below 40 C was reported, which suggests that the underlying molecular event here may be the formation of single helices (Tako & Hanashiro, 1997). In such a scenario it is possible that the two peaks originate from single helical transitions either of separate chains or single chain sections of imperfectly formed triplex. Such a scheme is also entirely consistent with the DSC results shown here where the appearance of exothermic peaks, indicating the reformation of bonding and kinetic trapping of the current microstructural state, are observed in concert with the NMR
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Fig. 5. Rheological measurements carried out during thermal scanning cycles for a 2% suspension of curdlan CUD1 (MW=1.92106). Scan rate: 1 C/min. (a) first cycle, (b) second cycle.
detected reduction in chain mobility and the rheologically determined increase in gel modulus (Zhang et al., 2002). In order to further characterize such molecular events the molecular weight dependence of the rheological evolution under temperature cycling was examined. The results obtained for 2% curdlan with lower Mw are shown in Figure 7. It can clearly be seen that the lower molecular weight, the more predominant the rise in G0 on increasing the temperature and correspondingly, the lower the increase observed below 40 C on cooling. Harada and his co-workers have reported that lower molecular weight material can anneal more successfully compared to higher molecular weight counterparts that require more ordering (Harada et al., 1994) and, as such, these observations are consistent with a model in which annealing of triplexes takes place on heating to temperatures above about 58 C and changes occurring below 40 C on cooling originate from un-annealed portions of the sample (Zhang et al., 2002).
Fig. 6. DSC themal scanning cycles for a for a 2% suspension of curdlan CUD1 (MW=192104). Scan rate: 1 C/min.
Cooling DSC curves have been examined after heating curdlan samples to different temperatures and holding for 30 min before cooling, and it is interesting to note that as the temperature of holding is increased the size of the exothermic peak decreases. These results also clearly show that the lower temperature exothermic peak disappears before that at higher temperatures (Zhang et al., 2002). There is then evidence that holding samples at higher temperatures or reducing the molecular weight both produce the same manifest changes in the DSC behaviour, consistent with the annealing of more perfectly formed triple helical structures being the origin of the observed behaviour. NMR relaxation data obtained by cycling 2% curdlan to 40 and 50 C at 1 C min1 showed that the change of the spin-spin relaxation time is not reversible (Zhang et al., 2002). However, this thermal range is entirely below the temperature indicated by the DSC endothermic peak, and thus is associated with time dependent molecular level changes of another kind. It seems reasonable to suggest then, as in the initial description, that these changes are a consequence of the hydration of the dry material (Zhang et al., 2002). While the exact details of the network assembly are complex it is well known that, in general, helical forming polysaccharides are prone to aggregate. A gelation model has been proposed for these glucans that is a three chain analogue of that proposed for Welan (Morris et al., 1996), a polysaccharide that is biosynthesised as perfectly registered double helices, that upon de-naturing and subsequent re-naturing forms a gel with each chain involved in more than one junction zone. Indeed this model has been proposed in order to explain the gelation of schizphyllan in the presence of sorbitol (Maeda et al., 1999). While there may be some involvement of this type, particularly at the helix ends, these flexible joints in curdlan seem unable to prevent a further layer of assembly. Micrographs of the curdlan
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Fig. 7. Rheological measurements carried out during thermal scanning cycles for a 2% suspension of curdlan CUD3 (MW=5.0105). Scan rate: 1 C/min. (a) first cycle, (b) second cycle.
system clearly show the overwhelming domination of mibrofibrils (Harada, 1991) and there can be little doubt that it is the aggregation of helices that largely leads to the gel structure on a macroscopic level. In conclusion, from the comparison of data for curdlan obtained by rheology and DSC, the correspondence of changes in the observed behaviour generated by reducing the molecular weight or holding samples at high temperatures for longer times provides strong evidence that the annealing of triplex structures is the main time dependent phenomenon occurring. This hypothesis is supported by estimates of renaturing kinetics obtained by low resolution NMR. Transitions of the un-annealed portion of the sample below 40 C would seem to play a crucial role in the kinetic trapping of microstructural states. It is clear that the high and low temperature gels of conventional rhetoric are not discrete and do not form a complete set of possible behaviours, but rather are two possibilities of a whole spectrum of possible gels in which the contribution of annealed triple helical elements to the stress bearing properties of the generated network can be varied. Holding a sample closer to the transition temperature, allows a greater number of imperfect triplexes to be melted out than a correspondingly lower temperatures and as such, more annealed samples are more resilient and thermo-irreversible. The formation of longer junction zones of increased thermal stability at temperatures closer to the transition temperature is consistent with previous work on the biopolymers gelatin (Michon et al., 1997) and agarose (Mohammed et al., 1998). However, the recent report on the effect of thermal history on gelation for agarose (Aymard et al., 2001) is contradictory with these conclusions. It is necessary to study this problem further.
Concluding remarks It is necessary to clarify the irreversibility of both KGM and curdlan gels. Factors such as gelation rate,
concentration, may affect the structure of junction zones, and a future study using AFM will reveal the effect of these factors on the gel structure. Interaction of water and polymers has been studied by many groups and this problem is closely related with the gelation process and the properties of resultant gels and the freezing process. Whether the molecular chains can be rearranged or not, whether some molecular chains can be incorporated further after the gel formation in the resultant gels or not should be clarified in the near future. This may have a close relation to flavour release. Flavour release in gel-like foods has been discussed mainly from the viewpoint of the fracture strain (Morris, 1994) or fracture stress (Clark, 1998), however, it should also have a close relation with syneresis or water-holding capacity of gels. Both KGM and curdlan gels have rubber-like texture and since it takes a long time for seasonings like shouyu, soy sauce, to be absorbed in ordinary KGM gels, the study of the diffusion of small molecules in these gels might be useful for the food industry. The diffusion process of these molecules might be influenced strongly by the network structure although it has been reported that the diffusion coefficient of water molecules in gellan gels is not different from that of bulk water by NMR (Ohtsuka & Watanabe, 1996).
Acknowledgements The authors thank Prof. P. A. Williams for improving the English and Drs. M.A.K. Williams, M. Yoshimura, R. Takahashi, H. Long for their precious contribution to the present study.
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Further reading Gao, S., & Nishinari, K. (2004). Effect of degree of acetylation on gelation of konjac glucomannan. Biomacromolecules, 5, 175–185.
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