A Molecular Description of the Gelation Mechanism of Konjac Mannan

Biomacromolecules 2000, 1, 440-450

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A Molecular Description of the Gelation Mechanism of Konjac Mannan Martin A. K. Williams,* Tim J. Foster, Dave R. Martin, and Ian T. Norton Unilever Research Colworth, Colworth House, Sharnbrook, Bedford MK44 1LQ, U.K.

Miki Yoshimura and Katsuyoshi Nishinari Department of Food and Nutrition, Faculty of Human Life Science, Osaka City University, Sumiyoshi, Osaka 558-8585, Japan Received March 1, 2000; Revised Manuscript Received May 24, 2000

A molecular level description of the time course of the gelation of the polysaccharide konjac mannan (KM) is presented and the role of alkali addition is considered in detail. NMR relaxometry is utilized as a complementary methodology to mechanical spectroscopy in order to probe events occurring as a prelude to network formation, and high-resolution NMR is used to follow the deactetylation process. It is shown that the addition of alkali plays an important solubilizing role in addition to facilitating the deacetylation of the chain. Deacetylation is important both in reducing the inherent aqueous solubility of the polymer and in progressively negating the alkali-induced polyelectrolytic nature of the polysaccharide chain via reaction induced pH changes. It is proposed that observed induction periods following alkali addition (during which the elastic modulus does not rise) are not simply deacetylation delays but are related to the aggregation kinetics of the deacetylated material. Introduction Konjac mannan. Konjac mannan (KM) is an essentially linear polysaccharide that is the main component of the konjac flour produced from the tubers of Amorphophallus konjac C. Koch. It consists of β-1-4-linked glucose and mannose units, and although enzyme digestion work suggests that there is no simple repeating unit, it is known that the glucose:mannose ratio is between 1:1.61 and 1:1.42 and that there are some branches, occurring approximately one in 11 residues, linked at the C-3 position of mannose and typically 11-16 residues in length.1 The glucomannan backbone possesses 5-10% acetyl substituted residues,3,4 and it is widely accepted that the presence of this group confers solubility on the biopolymer in aqueous solution (it should be noted that unsubstituted β-1-4-linked glucomannan is insoluble in water3). Studies of deacetylated KM films have found that the glucomannan packs isomorphously with the crystal structure of mannan II, suggesting that the substitution of some mannose residues with glucose does not disrupt the packing of the hydrated structure.5 The crystal structure of native (acetylated) KM has also been studied in moist fibers and while the mannan II form is again favored, these experiments and molecular modeling work demonstrate that the crystal structure incorporates only acetyl free segments of the glucomannan chain.6,7 NMR data indicate that this mannan II isomorph is also present in KM gels that are routinely formed by the addition of alkali, (and hence consist of deacetylated chains).8,9 However, in contrast to the high solids systems, it has not been reported that native (acetylated) KM can gel simply by forming junction zones incorporating

substituent free stretches of the polymer backbone. It is possible that the reason for this is simply the increased relative importance of the loss of entropy in solution. It is interesting, however, to consider another possibility, that the gelation of native KM solutions may occur without deacetylation, but over extended time periods. Recent work carried out on the galactomannan locust bean gum (LBG), which consists of an exclusively mannose backbone with 20-23% galactose side chains, has found that while gelation does occur from solutions containing in excess of 1% solids, this takes place at ambient temperatures on the time scale of months.10-12 LBG gels are more routinely formed via a freeze/thaw cycle, and have been found to contain mannanmannan associations whenever the polymer backbone is unsubstituted for block lengths of greater than a weightaverage of six monomer units.13 As the backbones of LBG and KM pack isomorphously and KM contains stretches of unsubstituted backbone in excess of six monomer units more commonly than LBG, it might be expected that KM would exhibit the same long time gelation behavior. The rate of this gelation process would be expected to be a function of concentration and indeed it has been reported that 8% KM will gel without the addition of alkali.8 To make a comparison of the respective kinetics of such proposed LBG and KM gelation it is important to bear in mind the branching evident in the KM structure in addition to the presence of acetyl groups. Although the number of branching substituents is reduced compared to the galactose substitution of the galactomannan, branches of 11-16 residues are likely to yield a significantly increased steric hindrance to chain association compared to single galactose units. Further

10.1021/bm005525y CCC: $19.00 © 2000 American Chemical Society Published on Web 07/06/2000

Gelation Mechanism of Konjac Mannan

evidence for commonality in the behavior of these biopolymers is provided by reported similarities in the crystal lattice constants of mannan, KM, LBG and other galactomannans such as guar and tara. It has further been proposed that KM shares a tendency with these galactomannans to hyperentangle in solution; that is, to form chain associations over and above topological entanglements.14,15 Amorphophallus konjac C. Koch is indigenous to Southeast Asia, and the ability of KM to form thermally stable gels by heating in the presence of alkali has long been exploited in the traditional cuisine of the East. It is now the subject of renewed interest in a number of diverse contexts in the West. Initial interest focused upon the role of KM as a texture modifier,16 and it has been classified as indigestible dietary fiber and implicated as a cholesterol absorber.17 From early Japanese stress-relaxation experiments18-20 to more conventional oscillatory measurements,21,22 the rheological behavior of KM gels has also been of particular interest owing to the similarity of many features with covalently cross-linked materials. Its material properties and inherent biodegradability has led to interest in the use of KM in films and packaging23,24 and even as a controlled release matrix.25 There is little doubt that KM is best known owing to its exploitation in synergistic gels, and consequently a great deal of attention has been focused on gelation models for these systems,26 which classically have involved xanthan, LBG,27 or κ-carrageenan28 and more recently deacetylated acetan.29,30 Despite this, work on the gelation of KM alone has not progressed significantly since the early studies of Meakaji,31-34 which while addressing many interesting aspects were often carried out over a narrow temperature range. While empirical models have recently been applied to describe the time course of the increase in its elastic modulus 35 the gelation mechanism of KM has not been fully explained. It is the aim of this paper to elucidate further the role of alkali addition and to provide a molecular level description of the time-course of gelation. This primarily involves the use of NMR relaxometry as a complementary methodology to mechanical spectroscopy in order to probe events occurring as a prelude to network formation. NMR Relaxometry. Biopolymers are normally studied under dilute conditions (e.g., T2b-1 and the mobility of the polymer backbone dominates the observed relaxation behavior through its fluctuating dipolar interactions. That is, eq ii described in the introductory section is appropriate. The temperature dependence of T2a was measured in an independent experiment and has been used with eq ii to analyze the data. Figure 1 also shows a fit that has been obtained using eq ii and assuming T2b follows an Arrhenius relationship with temperature. It can be seen that there is excellent agreement over the entire temperature range at all concentrations. The fit was performed assuming that the value of Pb could be taken as that derived from the biopolymer concentration by a calculation based on the weight and number of exchangeable protons per average KM residue. Adopting this model allows the data to be fitted as a function of temperature and concentration with a common infinite temperature relaxation


Figure 2. Temperature dependence (measured at 1 °C min-1) of G′ and G′′ (measured at 1 Hz and 0.5% strain) for a 1% w/w KM sample. The imposed temperature profiles are also shown for comparison with the rheological response.

time (0.27 ( 0.05 s). The extracted activation energies are 8.3 ( 0.5, 9.0 ( 0.5, and 10.2 ( 0.5 kJ mol-1 for 0.5, 1, and 2% KM, respectively. This suggests that the energy barrier to thermally induced molecular motions may be raised slightly with an increase in concentration, which is eminently reasonable for an entangled polymer solution. This gives T2b values on the order of 10 ms at ambient temperatures, which are reasonable for a system of this type,46 although it should be noted that in polydisperse biopolymer systems this represents an average value. Figure 2 shows the temperature dependence of G′ and G′′ (measured at 1 Hz and 0.5% strain) for a 1% w/w KM sample. The principle of so-called time-temperature superposition has long been recognized in mechanical spectroscopy47 and used to produce master curves in which measurements carried out as a function of temperature are mapped onto an extended frequency range that would have been unobtainable practically if the temperature had been kept fixed and the measurement frequency altered. For a single relaxation process a simple Maxwell model embodies this equivalence by representing the elastic and loss moduli in terms of a spectral density of ωτ where ω is the frequency of measurement and τ is the temperature-dependent relaxation time. (Of course in practice stress relaxation processes in biopolymer systems will involve many molecular level motional modes but for the purposes of discussion we assume that one average mode is dominant.) The simplest assumption for a biopolymer solution is that the dominant relaxation time depends on temperature in an Arrhenius fashion and substitution of this form into the basic Maxwell equations predicts linear behavior for plots of ln(G′) and ln(G′′) vs reciprocal temperature with gradients of (2E/R) and (E/R) respectively, where E is the activation energy of the motion facilitating relaxation and R is the gas constant. Figure 3 shows data from Figure 2 replotted in this fashion and the results of fits to this form, which can be seen to be in good agreement over the range. The extracted values for the activation energy of the motional process governing the relaxation were found by this method to be 10.8 ( 0.4 and 12.5 ( 1.0 kJ mol-1. This is similar to the activation energy obtained from the NMR experiments for the thermally activated motional process responsible for facilitating spinspin relaxation. However, it should be borne in mind that these techniques are looking at motional fluctuations on very different time scales and while it might be tempting to

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Figure 3. Dependence of ln(G′) and ln(G′′) (measured at 1 Hz and 0.5% strain) on reciprocal temperature (measured at 1 °C min-1) for a 1% w/w KM sample. A fit to the data is also shown and is described in detail in the text.

Figure 4. Observed spin-spin relaxation times (T2) as a function of temperature (measured at 1 °C min-1) obtained from KM solutions with and without the addition of alkali.

suggest that the limiting step to these motional modes could be common it is more likely that this agreement is purely coincidental. KM With Added Alkali. Figure 4 shows the effect on T2 of adding alkali to the KM solutions, as described in the Experimental Section (measured at 1 °C min-1 over the temperature range from 30 to 37 °C). For the 1 and 2% solutions, the effect can be seen to be analogous to an increase in temperature, with increased chain mobility being the likely source of such an effect. For the 1% sample, this equivalence is illustrated graphically in the figure by comparing the temperatures at which the samples with and without added alkali exhibit the same T2 value. (Such an observation might be conceived of as evidence for the reduction of hyper-entanglements as found in galactomannans). To assert with confidence that indeed it is this effect that is manifest here it is important to eliminate possibilities other than increasing chain motion that may explain the changes in the observed T2. One such possibility is that a change in the exchange rate has occurred and that this has affected the reported T2 value. However, the addition of alkali to the solution will certainly lead to an increased exchange rate, and merely strengthen the adherence of the system to the criterion determining the use of eq ii, in which the value of k does not feature. Another alternative that cannot be completely discounted is that the alkali has in some way altered the number of exchangeable protons and hence Pb. However, the observed variation could only be explained by a reduction in Pb, which again is unlikely. It seems then that the most reasonable interpretation of the NMR data is that the mobility of the chains has increased upon the addition

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Figure 5. Temperature dependence (measured at 1 °C min-1) of G′ and G′′ (measured at 1 Hz and 0.5% strain) for a 1% w/w KM sample with and without the addition of alkali.

Figure 6. Viscosity data (measured at 20 s-1) from 0.5% and 1% KM solutions as a function of temperature (measured at 1 °C min-1) with and without the addition of alkali.

of alkali. (Discussion of the 0.5% sample is deferred until after the following section.) Figure 5 shows the measured rheological behavior of 1% KM following the addition of alkali, as described in the NMR experiments. It can again be seen that the perturbation is manifest in a way that can be interpreted as a molecular level change equivalent to that which occurs as a consequence of increasing temperature. Again, this equivalence is illustrated graphically in the figure by comparing the temperatures at which the samples with and without added alkali exhibit the same G′ and G′′ values. As discussed previously, this can be considered as an increase in chain mobility that moves ωτ to smaller values and hence reduces the measured moduli values (as long as ωτ < 1). Furthermore, it is interesting to note that for the 1% samples the changes induced in both the rheological and the NMR relaxation behavior are both equivalent to increases in the chain mobility to a degree corresponding to a temperature increase of 8 °C. Still further weight is added to this line of reasoning by measuring the effect of the addition of alkali on the viscosity of a 1% KM solution. Figure 6 shows the results from such an experiment as a function of temperature (measured at 1 °C min-1), and once again it can be seen that, on the addition of the same amount of alkali used in the previous experiments, the 1% KM system behaves initially as if the temperature has been increased by 8 °C. It is equally of interest to observe, in Figures 4 and 6, the behavior of the less concentrated solution of 0.5% KM on the addition of alkali. In this case, while both NMR relaxation measurements and viscosity data show behavior analogous

Figure 7. (a) T2 values obtained from 1% KM samples after the addition of alkali, measured during a series of temperature ramps (at 1 °C min-1) from 20 °C to 50, 60, 70, and 80 °C and subsequent holding periods. (b) Corresponding elastic modulus (measured at 1 Hz and 0.5% strain) for a 1% KM solution under the same thermal regime as the NMR experiments (part a) with identical alkali addition. In both cases, the data are given in the same symbols as the corresponding temperature ramp (also shown) under which they were obtained.

to a 5 °C temperature change, the observed behavior can be seen to be equivalent to a reduction in temperature. This clear difference between the behavior of the 0.5% and 1% KM samples could be explained by the decreasing importance of hyper-entanglements in less concentrated solutions. The induced polyelectrolytic behavior of the individual chains could be more important in the low concentration regime leading to more extended conformations that limit the ability of backbone protons to average their dipolar interactions by molecular tumbling and increase the solution viscosity. This idea is consistent with specific volume increases seen by Nishinari in solutions of concentrations up to ∼0.4%.48 Figure 7a shows T2 values obtained from 1% KM samples after the addition of alkali, measured during a series of temperature ramps (at 1 °C min-1) from 20 °C to 50, 60, 70, and 80 °C and subsequent holding periods. The initial linearlike response has been discussed in detail in the previous section and merely reflects the temperature dependence of the chain mobility, although it should be remembered that at this concentration there is an initial solubilizing effect of alkali. After this initial period (some 25-30 min of ramping at 1 °C min-1), it is clear that the observed T2 begins to drop. The simplest interpretation here is that the chain mobility now begins to decrease owing to the aggregation of material that has undergone alkali induced deacetylation. It is clear from the rheological data shown in Figure 7b that the ultimate


state of these KM samples is indeed that of a self-supporting gel. It is important here to be clear that it is still the T2 of the biopolymer protons that governs the observed T2, even though the chain mobility and consequently T2b is falling (that is, even though the criterion for the application of eq ii is being less strongly satisfied). This is clearly seen by carrying out temperature rescans on a preformed gel. Such systems are the most likely candidates of systems in which k may be important in determining the relaxation behavior (owing to the maximum mobility decrease, therefore the smallest T2b values, and the largest chance that the use of eq ii is no longer valid). However, although changing the temperature of the gelled sample must vary k dramatically, the observed T2 is only slightly perturbed and in a direction consistent with T2b domination of the observed value. The reduced magnitude of the change observed under such circumstances compared with that observed in the sol is a reflection of the decreased temperature dependence of the averaging of the dipolar interaction in the gelled state as expected. The only other molecular level change that could be manifest in a T2 reduction (the behavior of T2a is known, and changes in δω, and k are negligible) would be increases in Pb as the temperature is raised. This hypothesis relies on the fact that not all intrinsically exchangeable protons are exchangeable in the initial state. In fact Pb would have to increase by a factor of about 10 to explain the observed changes in relaxation time, which seems unreasonable. Of course it is more difficult to say that some change in Pb could not be occurring and that this coupled with T2b changes produce the observed effect. There is some evidence to suggest that changes in Pb cannot be very large, notably that no large differences in the integrated high resolution NMR signal are evident. It is therefore most likely that changes in chain mobility are the dominant factor in determining changes in the observed T2 over the whole time course of the experiment. Figure 7 (b) shows the corresponding elastic modulus measured at 1 Hz and 0.5% strain for a 1% KM solution under the same thermal regime as the NMR experiments with identical alkali addition. The initial decrease in the modulus has been discussed previously. With a continued temperature increase the modulus increases with the rate dependent on the holding temperature. There also seems to be some evidence of a delay period at the lower temperature (50 °C) after the final temperature is reached before the elastic modulus is detected to rise. Figure 8, parts a and b, shows the same experiments but carried out for lower holding temperatures, and here delay times in modulus evolution are more clearly seen. It should be noted that corresponding delay times are also observed in G′′, but these are omitted for the sake of brevity and clarity of the figure. In addition, it can be seen in Figures 7 and 8 that there is a correspondence between the time at which G′ begins to be detectable and a particular T2 value being reached. This is highlighted by an indication of the induction periods in the rheological plots and a corresponding graphical interpolation from T2 the relaxation data. This indicates that there needs to be a particular molecular state or degree of


Figure 8. (a) T2 values obtained from 1% KM samples after the addition of alkali, measured during a series of temperature ramps (at 1 °C min-1) from 30 °C to 30, 40, and 45 °C and subsequent holding periods. (b) Corresponding elastic modulus (measured at 1 Hz and 0.5% strain) for a 1% KM solution under the same thermal regime as the NMR experiments (part a) with identical alkali addition. In both cases, the data are given in the same symbols as the corresponding temperature ramp (also shown) under which they were obtained.

aggregation before rheologically detected processes can proceed (i.e., a nucleation site). Such induction periods (delay times after which alkali is added to KM that gel formation can occur) have been observed since the earliest experiments on KM, and it has become accepted dogma that this induction period is equivalent to a period of deacetylation and, by implication, that this is the rate-limiting step. This is largely based on initial work by Meakaji in which activation energies were measured for deacetylation and for the “induction rate” (calculated as the reciprocal of the induction period) and found to be similar (11.8 and 11.6 kcal mol-1, respectively). However, both calculations are based on limited temperature ranges and in the former case on only three measurements. A reappraisal of the data presented in ref 32 gives the activation energy for deacetylation as 12.2 ( 9.4 kcal mol-1 (95% confidence limits), suggesting that caution should be applied to the interpretation of these results. Furthermore, after studies on a peptized gel Meakaji himself concluded that the deacetylation was a “necessary but not sufficient” step in the gelation of KM.31 To examine the relationship between deacetylation and the events detected by NMR and rheology a study of the deacetylation process was undertaken using high-resolution NMR. High-resolution proton NMR spectra of 0.25%, 0.5%, and 1% (w/v in D2O) KM solutions were recorded. All spectra show that there is some evidence for more than one acetate peak (∼2.2 ppm), reflecting the distribution between the two sugar residues on the polymer backbone. Integration of the relative areas of the acetate peak to the anomeric

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Figure 9. Time course of deacetylation as measured by highresolution NMR for various concentrations of KM at 50 °C following the addition of 20 µL of 1 M Na2CO3 solution per 1 g of KM solution. The plot shows the time evolution of the ratio of intensities from the bound and free acetate signals. First-order kinetics are shown as a guide to the eye.

proton peaks (∼4.8 ppm) suggests that there are approximately 1 acetate group per 20 sugar residues, in keeping with the results of previous studies. There is evidence of free acetate (∼1.9 ppm) present in samples prepared without the addition of alkali suggesting that some acetate groups have already been removed from the backbone. These moieties may have been present as contamination in the sample and/or some acetate groups have been removed during sample preparation. A 0.25% sample kept at 60 °C overnight showed a detectable increase in free acetate, but this was deemed insignificant compared to the effects of alkali addition and a quantitative study was not pursued further. In addition to spectra of these starting solutions alkali was added to the KM samples and they were then introduced into the spectrometer at a preset temperature. The signal was acquired for 5 min and the experiment repeated as a function of time in order to observe the time course of deacetylation. The only observable difference upon addition of alkali was a reduction of the acetate group signal at 2.2 ppm, and the corresponding increase in free acetate at 1.9 ppm. No other major changes were detected in any spectra as a function of temperature (in contrast to methylcellulose49) or time after alkali addition, suggesting that no conformational change takes place and that the number of exchangeable sites will not be significantly changing (this has important consequences for the interpretation of the relaxometry data as already mentioned). Integration of the peak intensities corresponding to free and biopolymer acetate was performed after baseline correction. The identity of the free acetate peak was confirmed by spiking the solution with sodium acetate. In the absence of an internal standard the peak areas were arbitrarily referenced to the magnitude of the free acetate signal at the starting measurement temperature. While the lines were observed to broaden with an increase in concentration, as would be expected as a consequence of reduced molecular mobility, the integration of the free and bound acetate peaks was found not to have been be detrimentally affected and while the 0.25% and 0.5% samples were run at 50 °C, the 1% sample was run at 30, 50, and 80 °C. The results are shown in Figures 9 and 10.

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Figure 10. Time course of deacetylation as measured by highresolution NMR for 1% KM at 30, 50, and 80 °C following the addition of 20 µL of 1 M Na2CO3 solution per 1 g of KM solution. The plot shows the evolution of the ratio of intensities from the bound to free acetate signals. First-order kinetics are shown as a guide to the eye.

The procedure of sample mixing, introducing the sample to the NMR tube and subsequently to the spectrometer, and the length of time required for the concatenation of the signal in order to achieve an acceptable signal-to-noise ratio made the measurements difficult. Nevertheless, it is possible to draw some broad conclusions, and it is apparent that the data do give a good indication of the progress of deacetylation. While the speed of the process at 80 °C is problematic, the results obtained at 50 and 30 °C are clearly indicative of a first-order reaction, and this theoretical form is plotted as a guide to the eye. Figure 11, parts a, b, and c, shows the kinetics of deacetylation, T2 change, and elastic modulus evolution for a 1% KM sample at 30 °C following mixing with alkali. It should be noted that in contrast to the measurement of relaxation times and rheological properties the deacetylation work was carried out in D2O and as such it is implicit in the comparison of data that the measured deacetylation kinetics are assumed to be a good approximation of those in H2O, while this is not directly measured. In making a comparison between techniques, it is worthwhile to consider the complimentarity of the NMR relaxometry and rheological methods which stems from the way that the detected signal is perturbed by the presence of small amounts material having differing mobility to the bulk. In the initial stages of an aggregation process, a small amount of material has a reduced reorientational correlation time. Owing to the way in which the NMR signal is averaged, by fast chemical exchange that is capable of observing all distinct sites in the time scale of the experiment, even small amounts of biopolymer material changing their mobility (e.g., in nuclei formation) can be detected. In contrast the mobility of a larger amount of the sample must be perturbed in order to be detected in measurements of elastic moduli. Hence, if the mobility of the entire biopolymer sample is changed, by, for example, alterations of temperature, then NMR and mechanical spectroscopy will detect this equally as well, as already demonstrated. However, if a small fraction of the sample becomes motionally restricted in, for example, the early stages of aggregation, NMR will detect this much more readily than rheological measurement. Hence in Figure 11 we can see with NMR relaxometry that during an induction period after alkali addition (before elastic modulus increase), far from being in a slumbering state diligently


Figure 11. (a) Deacetylation at 30 °C following mixing with alkali. (b) T2 change at 30 °C following mixing with alkali. (c) Elastic modulus evolution at 30 °C following mixing with alkali.

awaiting deacetylation, the biopolymer is already deacetylated and aggregating at a molecular level. It is likely then that it is the kinetics of this aggregation process that is responsible for the delay in modulus evolution. Indeed, at this temperature it is possible to see that long after the deacetylation is complete the elastic modulus still does not rise (in fact, a decrease in G′ is initially observed for the 1 and 2% samples, presumably associated with the kinetics of hyper-entanglement dissolution). Only after a prerequisite number of aggregates are formed is the mobility reduction manifest in an increase in both storage and loss moduli. Subsequently, as further aggregation proceeds, a spacespanning network is established. This point is demonstrated even more clearly by examining the data for 0.5%. Figure 9 clearly shows that the kinetics of deactetylation are similar for 1% and 0.5% systems, and as such, Figure 11a is a reasonable approximation for the 0.5% sample. Figure 11, parts b and c clearly show that while chain aggregation is soon evident, no response in G′ is seen for some considerable time. The measurement of the aggregation behavior may also be amenable to further study by other optical methods, such as light scattering and turbidity measurements, and such investigations form part of the ongoing work.


One aim of this paper has been to consider the wider implications of alkali addition and to reflect on how this might impact on our understanding of the gelation of KM. Alkaline solvents have long been used to solubilize cellulosic materials and polysaccharides in general. Indeed, an alternative method of forming gels from biopolymers such as agar, curdlan, and carrageenan, which are essentially insoluble in aqueous solution at ambient temperatures, is to increase their solvation by the addition of alkali instead of heating. Rather than subsequent cooling in the conventional thermal method, gel formation can be achieved by neutralization of the solution under a CO2 atmosphere. This method has been used extensively by Harada and Kanzawa50,51 in the preparation of gels for EM. In fact KM was included in these studies, and it was commented on that while KM gels could be formed by neutralization of alkali solutions they could not, in contrast to the other biopolymers examined, be formed by heating and subsequent cooling alone. Unfortunately these studies neglected the chemistry of KM and made no comment on deacetylation, which clearly explains the reported observation. It seems however that a combination of these ideas, deacetylation and increased solvation, can be useful in the consideration of the gelation mechanism. It was reported in early work34 that while a 0.8% KM solution gelled after 7.5 min at 22 °C upon the addition of 3.25 mM NaOH, the same sample buffered at pH 11.35 took almost twice as long (13.8 min), clearly implicating alkali in a solubilizing role. In addition, Cadoxen, a traditional cellulose solvator containing cadmium, ethylenediamine, and sodium hydroxide has been used as a mobile phase to run GPC experiments in an attempt to obtain molecular weight information on disaggregated KM.52,53 The strong alkali conditions (pH values of around 13.5) will undoubtedly have led to the deacetylation of the KM chains and yet the resultant glucomannan remained solubilized, owing to the extreme alkali conditions imposing polyelectrolytic nature on the polymer. To understand the behavior of a KM solution upon the addition of alkali a number of factors must be borne in mind. As the pH approaches the hydroxyl pka of around 11-12 the polymer becomes a polyelectrolyte and this has profound consequences for the solubility of the material. The consequences of charging the biopolymer are dependent on the initial molecular state. In more dilute systems individual polymer molecules will take up a more extended conformation. This can clearly be seen in specific volume changes reported by Nishinari.48 Above a certain concentration, however, hyper-entanglements, as discussed previously, may be dissolved (as seen with galactomannans14). There also exists the possibility that depolymerization reactions may ensue, although it has previously been claimed that the aforementioned, strongly alkaline, cadoxen was not considered to have damaged the KM chain.53 Of course there is also the deacetylation reaction itself that does undoubtedly leave the molecule inherently less soluble than its acetylated forbear. In light of the above comments it is clear that the deacetylation process may play a further consequential role by inexorably modifying the solution pH as the reaction

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Figure 12. T2 values measured for a 1% KM solution after the addition of alkali during a thermal ramp (at 1 °C min-1) from 30 to 60 °C and a subsequent rapid temperature jump back to 30 °C compared with samples continually ramped to 60, 70, and 90 °C and held at the final temperatures.

proceeds, transforming the polymer into a less charged species and facilitating aggregation and ultimately gel formation. Taking the T2 data to be a monitor of the aggregation process that ultimately results in gelation and is essentially the rate limiting step, it is interesting to study the relaxation data in more detail. Turning attention back to Figure 7a it appears superficially that at temperatures above about 60 °C the kinetics are invariant to temperature. That is, samples held at 60 °C or those that have undergone continued heating to 70 and 80 °C seem to show the same rates of T2 evolution. However, it can easily be shown that this is merely a consequence of the fact that the aggregation rates over this temperature range are not hugely different, and for samples that have had identical thermal histories on heating from 30 to 60 °C at 1 °C min-1, the process is in any case nearing its end point. Figure 12 shows the resultant behavior following a ramp to 60 °C followed by a rapid temperature jump back to 30 °C which clearly shows that the aggregation rate is, in fact, temperature dependent as expected. To have a chance of obtaining further information on the kinetics of the aggregation process, temperature quenches are much preferable to ramps where the observed behavior is a complicated sum of many rates. Figure 13 shows the results of such T2 measurements carried out on a 1% KM solution. Data are shown as a function of time. Temperature quenches were carried out by introducing the sample tubes directly into a pre-set spectrometer. For experiments shown in which the temperature has been ramped, t ) 0 corresponds to 30 °C. The temperature is then increased at a rate of 1 °C min-1 until the final given value, after which measurements continue to be taken at the holding temperature. A plot is also shown (solid line) obtained from the fit to the pure KM data assuming that the temperature is continually ramped at 1 °C min-1 over the entire displayed time period. A second plot (dotted line) shows the hypothetical predicted form of such a T2 temperature dependence with alkali present but assuming that deacetylation and aggregation do not occur and that the alkali simply solubilizes the KM in a manner equivalent to a temperature change as discussed previously in the context of the initial changes observed. Data from samples quenched as rapidly as possible to final hold temperatures are displayed shifted in time so that the start

Williams et al.

Figure 13. Results of T2 measurements carried out on 1% w/w KM solutions. Data are shown as a function of time for samples submitted to varying thermal regimes. For experiments shown in which the temperature has been ramped, t ) 0 corresponds to 30 °C. The temperature is then increased at a rate of 1 °C min-1 until the final given value, after which measurements continue to be taken at the holding temperature. Quenched samples are displayed shifted in time so that the start time corresponds to the time it would have taken a ramped sample (starting at 30 °C) to reach the final quench temperature. The meaning of the lines is described in detail in the text.

time corresponds to the time it would have taken a ramped sample (starting at 30 °C) to reach the final quench temperature. The purpose of this representation is the following: The T2 value given by the hypothetical temperature ramped variation after, for example, 40 min (i.e., when the temperature would have reached 70 °C) is then the value that would be obtained from an infinitely fast quench to 70 °C (that is, when there has been no time for any deacetylation or aggregation to take place), and as such, this time on the diagram provides a natural “zero time” for the display of the quench data. In fact, it can be seen from the figure that, neglecting the first couple of points (during which the final temperature has no doubt not been reached), an extrapolation of the quenched data to the corresponding “zero time” does give a T2 value that coincides well with the predicted value given by the hypothetical ramped function. In principle, it should be possible to extract a detailed aggregation model from the way in which T2 changes following a temperature quench. Such a model would essentially be based on the time dependence of the parameters in eq ii, that is T2b and Pb. However, in practice this is not trivial even if the time-course is assumed to be dominated by T2b and such a detailed model is elusive owing primarily to the fact that even if the aggregated and unaggregated form are assumed to be the initial and final states, the mobility (the measured parameter) of these states is not well defined. That is, even though the aggregated form is less able to average local dipolar interactions owing to restricted movement compared to its unaggreagated counterpart (and qualitatively this is reflected in the T2 behavior) it is not clear that every chain within each state has the same mobility, so that T2b is highly polydisperse and not uniquely defined for each state. For example, longer-term rearrangement processes will affect mobility but will not relate to the kinetics of the initial aggregation process. In light of this, simple exponential fits have been applied to the quenched data in order to extract a rate that is simply the kinetic evolution of the observed T2. In fact, the data fitted reasonably well to this simple form (r2 > 0.99 in all



of the polymer and in progressively negating the alkali induced polyelectrolytic nature of the polysaccharide chain via reaction induced pH changes. The induction period is not simply a deacetylation delay but relates to the aggregation kinetics of the deacetylated material. Where this is fast, the two processes may appear indistinguishable. Acknowledgment. The authors would like to thank A. H. Clark and M. J. Gidley for helpful discussions, D. Caswell for carrying out the high-resolution NMR experiments, and A. L. Russell for preparing the autoclaved samples. Figure 14. Rate data extracted from the T2 evolution of 0.5, 1 and 2% w/w KM solutions after the addition of alkali, plotted as log(rate) vs reciprocal temperature.

Figure 15. Schematic diagram of the proposed gelation mechanism for KM.

cases) although it should be stressed that this fitting was not carried out with the intention of finding the best empirical description of the data but rather to enable simple rate estimates to be made so that the temperature dependence of these could be examined. (Double-exponential functions were a better fit to the data in a number of cases but these are not so easy to interpret in a simplistic manner.) The work was repeated for 0.5% and 2% KM solutions, and Figure 14 shows the resulting data plotted as log(rate) vs reciprocal temperature. Although a detailed dynamical molecular level picture of the aggregation process is lacking, it is clear that the temperature dependence of these rates is a reasonable fit to an Arrhenius relationship. A linear regression analysis of the 1% data gives a value of 53 ( 5 kJ mol-1 for the activation energy. This is close to the value found by Meakaji based on the length of the induction period at different temperatures and therefore provides further support to a model in which the process responsible for the delay found in the rheological response is the aggregation event evidenced by NMR relaxometry. A summary of the proposed mechanism is given in Figure 15. Conclusions The addition of alkali clearly plays an important solubilizing role in the gelation of konjac mannan in addition to facilitating the deacetylation of the chain. Deacetylation is important both in reducing the inherent aqueous solubility

References and Notes (1) Maeda, M.; Shimahara, H.; Sugiyama, N. Agr. Biol. Chem. 1980, 44 (2), 245-252. (2) Bewley, J. D.; Reid, J. S. G. In Biochemistry of Storage Carbohydrates in Green Plants; Dey, P. M., Dixon, R. A., Eds.; Academic Press: New York, 1985; pp 289-304. (3) Dea, I. C. M.; Morrison, A. AdV. Carbohydr. Chem. Biochem. 1975, 31, 241-312. (4) Maekaji, K. Agr. Biol. Chem. 1978, 42 (1), 177-178. (5) Yui, T.; Ogawa, K.; Sarko, A. Carbohydr. Res. 1992, 229, 41-55. (6) Millane, R. P.; Hendrixson, T. L.;.Morris, V. J.; Cairns, P. In Gums and Stabilizers for the Food Industry 6; Phillips, G. O., Wedlock, D. J., Williams, P. A., Eds.; IRL Press: Oxford, England, 1992; p 531. (7) Millane, R. P.; Hendrixson, T. L. Carbohydr. Polym. 1994, 25, 245251. (8) Nishinari, K.; Williams, P. A.; Phillips, G. O. Food Hydrocolloids 1992, 6, 199-222. (9) Gidley, M. J.; McArthur, A. J.; Underwood, D. R. Food Hydrocolloids 1991, 5, 129-140. (10) Richardson, P. H.; Norton, I. T. Macromolecules 1998, 31, 15751583. (11) Richardson, P. H.; Willmer, J.; Foster, T. J. Food Hydrocolloids 1998, 12, 339-348. (12) Richardson, P. H.; Clark, A. H.; Russell, A. L.; Aymard, P.; Norton, I. T. Macromolecules 1999, 32, 1519-1527. (13) Dea, I. C. M.; Clark, A. H.; McCleary, B. V. Food Hydrocolloids 1986, 1, 129. (14) Goycoolea, F. M.; Richardson, R. K.; Morris, E. R. Macromolecules 1995, 28, 8308-8320. (15) Jacon, S. A.; Rao, M. A.; Cooley, H. J.; Walter, R. H. Carbohydr. Polym. 1993, 20, 35-41. (16) Kohyama, K.; Nishinari, K. JARQ 1997, 31, 301-306. (17) Tye, R. J. Food Technol. 1991, 45 (3), 82-87. (18) Ohta, Y.; Maekaji, K. Nippon Nogeikagaku Kaishi 1980, 54, 741746. (19) Ohta, Y.; Maekaji, K. Nippon Nogeikagaku Kaishi 1981, 55, 11171119. (20) Ohta, Y.; Maekaji, K. Nippon Nogeikagaku Kaishi 1981, 55, 415419. (21) Case, S. E.; Hamann, D. D. Food Hydrocolloids 1994, 8 (2), 147154. (22) Case, S. E.; Knopp, J. A.; Hamann, D. D.; Schwartz, S. J. In Gums and Stabilizers for the Food Industry 6; Phillips, G. O., Wedlock, D. J., Williams, P. A., Eds.; IRL Press: Oxford, England, 1992; p 489. (23) Dave, V.; McCarthy, S. P. J. EnViron. Polym. Degrad. 1997, 5 (4), 237-241. (24) Kohyama, K.; Kim, K. Y.; Shibuya, N.; Nishinari, K. Carbohydr. Polym. 1992, 17, 59-63. (25) Perols, C.; Piffaut, B.; Scher, J.; Ramet, J. P.; Poncelet, D. Enzyme Microbial Technol. 1997, 20, 57-60. (26) Morris, E. R In Biopolymer Mixtures; Harding, S. E., Hill, S. E., Mitchell, J. R., Eds.; Nottingham University Press: Nottingham, England, 1995; pp 247-288. (27) Goycoolea, F. M.; Richardson, R. K.; Morris, E. R.; Gidley, M. J. Macromolecules 1995, 28, 8308-8320. (28) Goycoolea, F. M.; Richardson, R. K.; Morris, E. R.; Gidley, M. J. Macromolecules 1995, 28, 643-658. (29) Ridout, M. J.; Cairns, P.; Brownsey, G. J.; Morris, V. J. Carbohydr. Res. 1998, 309, 375-379. (30) Ojinnaka, C.; Brownsey, G. J.; Morris, E. R.; Morris, V. J. Carbohydr. Res. 1998, 305, 101-108.

450 (31) (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) (45)

Biomacromolecules, Vol. 1, No. 3, 2000 Maekaji, K. Agr. Biol. Chem 1974, 38 (2), 315-321. Maekaji, K. Nippon Nogeikagaku Kaishi 1978, 52, 251-257. Maekaji, K. Nippon Nogeikagaku Kaishi 1978, 52, 485-487. Maekaji, K. Nippon Nogeikagaku Kaishi 1978, 52, 513-517. Yoshimura, M.; Nishinari, K. Food Hydrocolloids 1999, 13 (3), 227233. Hills, B. P.; Takacs, S. F.; Belton, P. S. Mol. Phys. 1989, 67, 903918. Hills, B. P.; Takacs, S. F.; Belton, P. S. Mol. Phys. 1989, 67, 919937. Hills, B. P.; Mol. Phys. 1992, 76, 489-508. Hills, B. P.; Mol. Phys. 1992, 76, 509-523. Carr, H. Y.; Purcell, E. M. Phys. ReV. 1954, 94, 630. Meiboom, S.; Gill, D. ReV. Sci. Instrum. 1958, 29, 688. Carver, J. P.; Richards, R. E. J. Magn. Reson. 1972, 6, 89. Hills, B. P.; Wright, K. M.; Belton, P. S. Mol. Phys. 1989, 67, 13091326. Williams, MAK.; Keenan, R. D.; Halstead, T. K. Magn. Reson. Chem. 1998, 36(3), 163-173. Hedges, N. D.; Derbyshire, W.; Lillford, P. J.; Norton, I. T. In Gums and Stabilizers for the Food Industry 5; Phillips, G. O., Wedlock, D. J., Williams, P. A., Eds.; IRL Press: Oxford, England, 1990; p 521.

Williams et al. (46) Williams M. A. K.; Keenan R. D.; Halstead, T. K. In Biopolymer Mixtures; Harding, S. E., Hill, S. E., Mitchell, J. R., Eds.; Nottingham University Press: Nottingham, England, 1995; pp 117-142. (47) Ferry, J. D. Viscoelastic Properties of Polymers, 3rd ed; Wiley: New York, 1980. (48) Kohyama, K.; Nishinari, K. In Gums and Stabilizers for the Food Industry 5; Phillips, G. O., Wedlock, D. J., Williams, P. A., Eds.; IRL Press: Oxford, England, 1990; p 459. (49) Haque, A.; Jones, A. K.; Richardson, R. K.; Morris, E. R. In Gums and Stabilizers for the Food Industry 7; Phillips, G. O., Wedlock, D. J., Williams, P. A., Eds.; IRL Press: Oxford, England, 1994; p 291. (50) Kanzawa, Y.; Koreeda, A.; Harada, A.; Harada, T. Agric. Biol. Chem 1989, 53, 979-986. (51) Harada, T.; Kanzawa, Y.; Kanenaga, K.; Koreeda, A.; Harada, A. Food Struct. 1991, 10, 1-18. (52) Kohyama, K.; Sano, Y.; Nishinari, K. Food Hydrocolloids 1996, 10 (2), 229-238. (53) Kohyama, K.; Iida, H.; Nishinari, K. Food Hydrocolloids 1993, 7 (3), 213-226.

BM005525Y

A Molecular Description of the Gelation Mechanism of Konjac Mannan

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