Effect of Degree of Acetylation on Gelation of Konjac Glucomannan

Biomacromolecules 2004, 5, 175-185

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Effect of Degree of Acetylation on Gelation of Konjac Glucomannan Shanjun Gao* and Katsuyoshi Nishinari* Department of Food and Human Health Sciences, Graduate School of Human Life Science, Osaka City University, Sugimoto, Sumiyoshi, Osaka 558-8585, Japan Received August 17, 2003; Revised Manuscript Received October 17, 2003

Effect of the degree of acetylation (DA) on the gelation behaviors on addition of sodium carbonate for native and acetylated konjac glucomannan (KGM) samples with a DA range from 1.38 to 10.1 wt % synthesized using acetic anhydride in the presence of pyridine as catalyst was studied by dynamic viscoelastic measurements. At a fixed alkaline concentration (CNa), both the critical gelation times (tcr) and the plateau values of storage moduli (G′sat) of the KGM gels increased with increasing DA, while at a fixed ratio of alkaline concentrations to values of DA (CNa/DA), similar tcr and G′sat values independent of DA were observed. On the whole, increasing KGM concentration or temperature shortened the gelation time and enhanced the elastic modulus for KGM gel. The effect of deacetylation rate related to the CNa/DA on the gelation kinetics of the KGM samples was discussed. Introduction The gelation mechanism of polysaccharides has been extensively studied not only from a scientific viewpoint but also from a demand of the increasing application of polysaccharides as texture modifiers, gelling agents, thickeners, emulsifiers, and stabilizers.1 Konjac glucomannan (KGM) is a neutral polysaccharide isolated from the tubers of Amorphophallus konjac C. Koch. It consists of β-1,4-linked glucose and mannose units, and the glucose/mannose ratio has been reported to be around 1:1.6.2 There are some branches linked to the backbone, but the exact branched position is still in debate.3 The glucomannan backbone of KGM possesses 5-10% acetyl-substituted residues,4,5 and it is widely accepted that the presence of this group confers water solubility on the glucomannan. The stable gel made by heating konjac flour in the presence of alkali has long been used as a noncalorie health-care food classified as indigestible dietary fiber in Japan.6 It is worth noting that KGM also plays an active role in weight-control and modifying the intestinal microbial metabolism,7 lowering plasma cholesterol,8 scavenging the free radical for isolated islets,9 and inhibiting tumor genesis and metastasis.10 The preparation of KGM-related composite materials and their promising application for edible film, coating, and biodegradable film material have also been studied.11-13 KGM is known to form synergistic gels with other polysaccharides involving xanthan,14 κ-carrageenan,15 and acetan or deacetylated acetan.16,17 The synergistic models of these binary mixtures have been presented on the basis of the intermolecular interaction supported by different characterization methods. However, the gelation mechanism of * Corresponding authors. Telephone: +81-6-66052818. Fax: +81-666053086. E-mail addresses: [email protected]; gaosjwhu@ 21cn.com.

KGM alone is still under progress since the early studies of Maekaji,18,19 which demonstrated that the deacetylation generated by the addition of alkali is a crucial step leading to the gelation. It has also been reported that gelation of KGM may also occur under a high KGM concentration over 7%20 or through the hydrophobic interaction in the presence of lyotropic salt.21 An earlier study22 also suggested that KGM dispersions could, under certain circumstances, form a gel without alkali coagulant. On the basis of the result from NMR relaxation in addition to mechanical spectroscopy, Williams et al.23 suggested that the induction period following alkali addition observed in the time course of elastic modulus for KGM dispersion corresponds to both the aggregation kinetics of the deacetylated product and the deacetylation delay. Obviously, there is little doubt that the detail of gelation mechanism for KGM has not been fully comprehended yet. Although the deacetylation is generally believed to be a triggering step for the gelation, the role of acetyl groups is not fully elucidated possibly because of the difficulty in obtaining well-fractionated KGM samples and the very few acetyl groups in refined KGM. In a latest work,24 five acetylated KGM fractions with different degrees of acetylation (1.6-5.3%) were obtained using acetic anhydride in the presence of zinc chloride as catalyst. Regrettably, molecular weights of the acetylated samples were about half of that of the native sample due to the degradation. In the present case, acetylation of KGM was carried out using pyridine as catalyst. We attempted to change the acetylation reaction condition to obtain acetylated products with a wider degree of acetylation (DA) range. The rheological behavior in the gelation process of KGM samples with different DA on addition of Na2CO3 was investigated by dynamic viscoelastic measurements to make further insight into the effect of amount of acetyl groups on the gelation behavior for KGM.

10.1021/bm034302f CCC: $27.50 © 2004 American Chemical Society Published on Web 12/02/2003

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Table 1. Effect of the Amount of Pyridine and Temperature on the Extent of Acetylation and the Results of Viscosity Measurements of the Products sample Rs pyridine (mL) temp (°C) time (h) DA (%) DS [η] (cm3 g-1) Mv × 10-5

Ac1

Ac2

Ac3

Ac4

Ac5

Ac6

Ac-D

0.5 40 2 1.38 4.13 0.05 0.16 557 493 12.0 10.1

1 40 2 4.47 0.18 520 10.9

1.5 40 2 4.82 0.19 486 9.88

2 50 2 5.85 0.23 500 10.3

2.5 50 2 7.40 0.30 524 11.0

2.5 80 2 7.57 0.31 487 9.91

10 40 3 10.15 0.42 480 9.70

Experimental Section Materials. The raw KGM sample (Rs) was a gift from Shimizu Chemical Co. (Hiroshima, Japan) and further purified by mixing with three times weight of 50 and 80 wt % ethanol for 2 h and with waterless ethanol for 4 h and then vacuum-drying at 60 °C for 4 h. All chemicals were reagent pure grade and purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Acetylation. Ten grams of Rs was mixed with 20 mL of 50 vol % acetic acid for 30 min and then dried at 60 °C for 30 min. The pretreated sample was added into a roundbottomed flask equipped with a mechanical stirrer. Fifty milliliters of acetic anhydride was added into the flask and mixed thoroughly with the Rs sample under slow stirring for 30 min. The predetermined amount of pyridine used as catalyst was added, and the reaction was controlled at the required temperature for different times. Then, 50 mL of deionized water was added, and the solution was mixed for 10 min. The precipitate was obtained by adding 100 mL of ethanol and then filtered off by a G3 glass filter. One hundred milliliters of 60% ethanol was added, and then the solution was mixed for 30 min and filtered. This operation was repeated twice. One hundred milliliters of redistilled ethanol was added, and then the mixture was filtered. The final product was obtained by drying at 60 °C for 4 h. By changing the amount of pyridine or reaction temperature, we have obtained a series of acetylated products with different values of DA coded as Ac1, Ac2, Ac3, Ac4, Ac5, and Ac6. To examine the effect of preparation method on the molecular characteristics of product, another route was carried out. Ten grams of KGM was immersed with 100 mL of 50 vol % ethanol for 30 min and filtered with a G3 filter. The sample was transferred to a 300 mL two-necked flask, and then 100 mL of N′,N-dimethyl formamide (DMF) was added. The KGM sample was thoroughly suspended in DMF for 4 h under mechanical stirring at 40 °C. The mixture of 10 mL of pyridine and 10 mL of acetic anhydride was added. The acetylation was controlled at 40 °C for 3 h. Then, the crude product was obtained by filtration with a G3 glass filter, and the other purification processes were the same as above. The acetylated sample thus obtained was coded as Ac-D. The synthesis condition for each acetylated sample is indicated in Table 1. Determination of DA. DA defined as the weight percent of acetyl-substituted residues in the KGM backbone was examined by a modified Eberstadt method including saponi-

fication and successive titration.25 This method is based on ASTM volumetric method and usually used to determine the acetyl content in cellulose acetate.26 The degrees of substitution (DS) were calculated by DS )

162DA 43 - 42DA

The values of DA and DS of the native and acetylated KGM samples are listed in Table 1. Preparation of Cadoxen Solutions of KGM and Viscosity Measurements. About 29 wt % of aqueous solution of ethylenediamine was saturated with cadmium oxide (CdO) in an ice-water bath under vigorous stirring and kept overnight below 5 °C. The solution was centrifuged at 9000g for 30 min, and then the supernatant was filtered through a G3 glass filter and refrigerated until use. The cadmium content in cadoxen was 4.7 wt %. The KGM sample solutions were prepared by mixing with required volume of cadoxen and stirring for 12 h before viscosity measurements. The intrinsic viscosity measurements of KGM cadoxen solutions were carried out at 25 ( 0.02 °C by using an Ubbelohde-type viscometer (Kaburagi Scientific Instruments Co. Ltd., Tokyo, Japan). Extrapolation to infinite dilution was made using both Huggins and Kraemer plots from which the intrinsic viscosity [η] was calculated. The flow time of the cadoxen solvent was 609 s. The viscosity-average molecular weights (Mv) of the KGM samples were calculated according to the Mark-Houwink equation [η] ) (3.55 × 10-2)M0.69 elucidated in a previous work.27 Rheological Measurements. Powders of native and acetylated KGM samples were dispersed in distilled water at room temperature for 1 h and were heated to 80 °C and then maintained at 80 °C for 1 h and cooled to room temperature. The aqueous dispersions with a concentration of 2 wt % were equilibrated at room temperature for 2 days before dynamic viscoelastic measurements. Dynamic viscoelastic measurements were carried out using a Fluids spectrometer RFS II (Rheometrics Co, Ltd.) with a parallel plate geometry (25 mm in diameter and 1.5 mm gap). The strain in all measurements of the present work was set as 1%, which is within a linear viscoelastic regime. One gram of KGM aqueous dispersions was poured onto the plate of the instrument, which had been kept at each measurement temperature. The gelation kinetics was studied at constant temperature of 50 °C other than specification. Forty milligrams of solution with different concentrations was added at time t ) 0 to the KGM dispersion and mixed with the desired amount of alkali, and then the storage shear modulus (G′) and the loss shear modulus G′′ were measured as a function of time at a constant frequency of 1 rad s-1. The alkaline concentration (CNa) was represented by the ratio of the concentration of Na2CO3 to the degree of acetylation of the sample in the investigation of effect of DA and alkaline concentration on gelation. Results and Discussion Effect of CatalystsPyridine. Various homogeneous or heterogeneous methods for the acetylation of carbohydrates

Biomacromolecules, Vol. 5, No. 1, 2004 177

Gelation of Konjac Glucomannan

are realized by using acetic anhydride, acetyl chloride, and ketene as acetylating agents in the presence of either basic or acidic catalysts.28-30 It has been reported that zinc chloride showed high reactivity in the acetylation of methyl R-Dhexopyranosides31 and KGM.32 However, obvious degradation of native KGM occurred after acetylation.24,32 By comparing the effect of p-toluenesulfonyl chloride (p-TsCl) and pyridine (as catalyst) on the homogeneous acetylation of cellulose, Saikia et al.30 found that the molecular chain degraded at temperature higher than 60 °C using p-TsCl as catalyst, but no degradation took place in the case of pyridine as catalyst. Furthermore, the activation energy for esterification using pyridine as catalyst was found to be less than that using p-TsCl as catalyst. It has been accepted that no marked depolymerization occurs during the acetylation of homogalacturonans,33 starch,34 and glucuronoxylan35 when pyridine was used as catalyst. The reaction conditions including the amount of pyridine, temperature, and reaction time for each product and the values of DA and DS are listed in Table 1. Under the same temperature, the DA increased with increasing amount of pyridine by comparing the DA of Ac1 with Ac2 and Ac3 or that of Ac4 with Ac5. Higher temperature is favorable for higher DA at the same amount of pyridine, compare DA of Ac5 to Ac6. Gatenholm et al.35 found that the DS at a certain amount of catalyst reached a maximum value and did not increase any more after 100 min. So, the reaction time was predetermined to be 2 h for Ac1-6 samples in the present study. The formamide/pyridine system has been regarded as the mildest medium without marked degradation for the heterogeneous acylation of carbohydrates.33-35 Furthermore, KGM powder can be well suspended and swollen in the DMF/pyridine mixture or pyridine alone.36 In the present study, the Ac-D product synthesized in DMF/pyridine medium using the highest amount of pyridine showed the highest DA. From Table 1, the intrinsic viscosity of native KGM decreased by about 9% from 557 cm3 g-1 of Rs to 500 ( 20 cm3 g-1 of each acetylated sample. Comparably, the intrinsic viscosity of native KGM decreased by 28% after acetylation by using zinc chloride as catalyst in a latest work.24 A slight decrease in the viscosity-average molecular weight from 12.0 × 105 of native KGM to (9.70-11.0) × 105 of acetylated KGM products was observed. This indicates that pyridine is a milder catalyst than zinc chloride in the acetylation of KGM. Sol-Gel Transition Points. The sol-gel transition is an important physical phenomenon for hydrocolloids with many applications in foods and cosmetics products. It has been reported that the intersection of G′ and G′′ (G′ ) G′′ ) G*) in the time course of shear modulus was suggested to mark the gelation point.37 Figure 1 shows the time evolution of storage modulus (G′) and loss modulus (G′′) for 2 wt % Ac1 aqueous dispersion at different frequencies and at 50 °C. G′ and G′′ increased with time and attained a steadily increased plateau after a period of sharp increase. The intersection of G′ and G′′ was observed at the frequency lower than 4 rad s-1, and the time corresponding to the intersection (t*) shifted to longer times with increasing frequency. This reflected that

Figure 1. Time dependence of G′ (solid symbols) and G′′ (open symbols) of 2.0 wt % Ac1 aqueous dispersion at different frequencies and at 50 °C. The Na2CO3 concentration in the dispersion was 0.8 wt %.

the time of intersection is a function of frequency, as observed in our previous work.24 Winter and Chambon38 have proposed a method to determine the gelation point from mechanical spectra for chemical gels, where the gelation point corresponding to the specific instant, tcr, is defined as a point at which G′(ω) ≈ G′′(ω) ≈ ωn, 0 < n < 1

(1)

tan δ ) G′′/G′ ) tan(nπ/2)

(2)

and

hold simultaneously, where n is the relaxation exponent. A system with n approaching 1 is defined as a purely viscous gel, whereas n approaching 0 indicates a purely elastic gel.39 The tcr corresponds to the point at which tan δ remains as a constant and is independent of frequency. The application of this gelation criterion was proposed originally for chemically cross-linked gels, because it is important to access lowfrequency data to be confident that the interactions being observed are permanent and not physical interactions with some infinite time scale.40 However, it has been proved that this criterion can be applied to some synthetic or natural physical gels including poly(vinyl chloride),41 xanthan,40 and carrageenan.42 The gelation process of Ac1 aqueous dispersion monitored by the change of tan δ with time was shown in Figure 2. The critical gelation time, tcr, was determined to be 35 min, and n was calculated to be 0.24. From the frequency dependence of Ac1 aqueous dispersion at various times at 50 °C as shown in Figure 3, both G′ and G′′ became proportional to ωn at t ) 35 min, where n ) 0.24 for G′ and G′′ and the results simultaneously agreed with the formulas 1 and 2. In the sol state, Ac1 shows a liquidlike behavior typical of a concentrated polymer solution, where G′′ is larger than G′ at lower frequency and both moduli increase with increasing frequency. Whereas an elastic behavior typical of a solidlike network material is observed in the gel state, where G′ is larger than G′′ and the moduli become frequencyindependent at all frequencies. For 2 wt % Rs dispersion, G′ is already larger than G′′ in the initial stage of dynamic time sweep measurements even

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Figure 2. Loss tangent, tan δ, as a function of time at different frequencies at 50 °C for 2.0 wt % Ac1 aqueous dispersion.

Figure 3. Frequency dependence of G′ and G′′ of 2.0 wt % Ac1 at various times at 50 °C. The data are shifted along horizontal and vertical axes by 10a and 10b, respectively, to avoid overlapping.

at the lowest frequency of 1 rad/s (data not shown) and at 50 °C, which also have been found in our previous work,43 indicating the initial concentrated and highly entangled nature of native KGM dispersion. This phenomenon was also found for concentrated locust bean gum/sucrose mixture solution.44 Therefore, determination of gelation time by tracking the intersection of G′ and G′′ is not applicable for native KGM in the present case. However, the Winter-Chambon method was found to be still effective in determination of the critical gelation time. As shown in Figure 4 by the time dependence of loss tangent, tan δ, at different frequencies, the tcr is determined to be 18 min, and the corresponding n was calculated to be 0.15. Winter-Chambon criterion was found to be strictly effective in determination of tcr for all of the samples from the results of time sweep measurements at different frequencies in the present work. Effect of DA on Gelation Kinetics. Figure 5 shows the time course of G′ of 2.0 wt % KGM dispersions in the presence of Na2CO3 at 50 °C, where the concentration ratio of Na2CO3 to KGM was fixed to 0.4. The G′ of Rs in the initial stage was found to be far higher than that of all acetylated samples. However, G′ of Rs was gradually overtaken by that of acetylated samples with increasing time. An obvious delay period in the initial stage, so-called

Gao and Nishinari

Figure 4. Loss tangent, tan δ, as a function of time at different frequencies at 50 °C for 2.0 wt % Rs aqueous dispersion. The CNa was 0.8 wt %.

Figure 5. Time dependence of G′ of 2.0 wt % KGM aqueous dispersions in the presence of Na2CO3 at 50 °C and at a frequency of 1 rad s-1. The concentration ratio of Na2CO3 to KGM was fixed to 0.4.

induction time, was observed for each acetylated KGM sample. The G′ of the KGM aqueous dispersions increased monotonically after the induction time and then attained plateaus (also called saturated value, G′sat). It took relatively longer time for the acetylated samples of Ac1, Ac2, Ac3, and Ac4 to reach the corresponding plateau in comparison with that for Rs, where the G′ of Rs approximately remained as a constant after 200 min. It should be noted that G′ of the acetylated samples with relatively higher DA (Ac5, Ac6, and Ac-D) obviously continued to increase even after 1200 min (20 h). Figures 6 and 7 show the critical gelation time (tcr) determined by Winter-Chambon method (based on the results of time courses of KGM dispersions at different frequencies and at 50 °C, data not shown, and similarly hereinafter) and the G′ at 20 h (G′20), respectively, for each KGM dispersion. The time corresponding to the intersection of G′ and G′′ (t*) at the frequency of 1 rad s-1 and the tcr value for each acetylated sample were plotted against DA in Figure 6. Both tcr and G′20 show an obvious dependence on DA of the KGM samples and increased with increasing DA at a fixed alkaline concentration, and a steep increase


Figure 6. The critical gelation time (tcr) and the t* obtained from Figure 5 as a function of DA.

Figure 7. The G′ at 20 h (G′20) obtained from Figure 5 as a function of DA.

of tcr and G′20 can be observed at higher DA (>6%). The native sample Rs, which can be regarded as the KGM fraction with the lowest DA, has the shortest tcr (18 min). Interestingly, the change of t* as a function of DA showed a similar tendency to that of tcr, exhibiting a sharp increase when DA is above 6%. The G′20 of each acetylated sample is larger than G′sat of Rs. It is difficult to obtain the saturated modulus for the acetylated samples with higher DA at the experimental condition captioned in Figure 5 (alkaline concentration, temperature, and polymer concentration) because there is a very wide range of DA for the samples prepared in this work. Note that the acetylated samples with relatively higher DA (>6%) have very high G′20 typical of strong elastic gels, and it is reasonable to presume that, given enough time, the difference in G′sat between the samples with higher DA (Ac5, Ac6, and Ac-D) and those with lower DA will be enlarged. The relaxation exponents n at the critical gelation point related to the Winter-Chambon criterion (eq 2) for the KGM samples with different DA obtained from Figure 5 were 0.21-24 for the acetylated samples and 0.15 for the native KGM sample Rs. According to previous studies,39,45,46 the value of power law exponent n is related to the physically fractal dimension and reflects the degree of compactness of network structure. In our


present study, the deacetylated gels from acetylated samples with higher DA are more elastic than that from native KGM, and the high values of relaxation exponent n suggested the more compact networks of acetylated samples than that of native KGM. These values of n for KGM are far less than those for gellan gum47 and poly(vinyl chloride),41 indicating a more elastic nature of KGM gels. From above results, it is obvious that the gelation behavior of the KGM dispersions shows strong dependence on the amount of acetyl groups (DA). At the same alkaline concentration and temperature, the higher the DA of KGM sample is, the longer tcr and the larger G′20 the sample has. Maekaji18,19 reported that the absorption band at 1720 cm-1 in the IR absorption spectrum of KGM, which is attributed to carbonyl groups, disappeared or decreased considerably after gelation with alkali. It was recently confirmed by Zhang et al.43 Maekaji found that during the gelation process KGM consumes 0.33 mmol of alkali per 1 g of KGM. Alkaline consumption was proportional to the intensity of the absorption band at 1720 cm-1 observed before gelation. Maekaji48 used an amylograph to monitor the rheological changes accompanying gelation. He showed that gelation by alkaline treatment occurred after a certain induction period. Considering the induction reaction as a chemical reaction, he estimated the activation energy from the Arrhenius plot as 11.6 kcal/ mol. This value was almost constant irrespective of the gelling conditions and agreed fairly well with the activation energy for the deacetylation reaction of 11.8 kcal/mol. Upon the basis of above results, he concluded that the induction reaction corresponded to deacetylation. Considering deacetylation as a saponification reaction, the reaction rate at a fixed alkaline concentration will monotonically decrease with increasing amount of acetyl groups in KGM samples. Therefore, the critical gelation time (tcr) of each acetylated KGM sample is longer than that of native KGM and increased with the increase of DA (Figures 5 and 6). The loss of substituted residues in the branch points of KGM molecular chains generated by the removal of acetyl groups results in the reduction in solubility, as reported in an old work49 and recently confirmed by Williams et al.23 As a result, the physical cross-links through intermolecular interaction including hydrogen bonding and hydrophobic interaction occur, and then junction zones thus formed lead to the change of KGM dispersion from sol to network-structural gel.50 It is reasonable to say that the deacetylation rate, which can be denoted by the ratio of removed acetyl groups to total acetyl groups, decreased with the increase of DA if the concentration of alkaline was fixed. Therefore, it should take a longer time for the acetylated KGM samples with higher DA to obtain enough entanglement, which makes it possible for G′ to overtake G′′, which demonstrated the increase of t* with increasing DA (Figures 5 and 6). The time corresponding to the intersection of G′ and G′′ reflects the relaxation time of the entangled network in polymer solution.51 When preparing KGM dispersions, we found that the acetylated KGM fractions can be thoroughly hydrated to form homogeneous aqueous dispersions in a relatively shorter time than native KGM. Furthermore, the hydration time was gradually shortened with increasing DA, implying that acetyl

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groups control the water solubility of KGM fractions52,53 and their water solubility was improved gradually by the introduction of acetyl groups within the DA range in the present work. This good water solubility also delayed the aggregation of KGM molecular chains after deacetylation because the alkaline concentration was fixed and irrespective of DA. It should be noted that the alkaline concentration in the rheological measurements shown in Figure 5 was far higher than our previous works,24,43 so all of the acetyl groups can be removed if given enough time. Theoretically, the number of junction zones of the KGM fraction gels after a long time should be the same and independent of DA. However, G′sat of KGM gel, which is thought to be directly related to the intermolecular cross-link through junction zone, was significantly enhanced, especially for the gels from the KGM samples with higher DA. This can be interpreted as deacetylation reaction rate (represented by the ratio of removed acetyl groups to total acetyl groups) at a fixed alkaline concentration being lowered because of the increase of DA, which ensures that the KGM dispersions with higher DA have enough time to form more homogeneously distributed junction zones. As a result, the gels thus formed have intimately entangled and homogeneous network structure. Additionally, the improvement of water solubility for the KGM with higher DA is also favorable for the sufficient distribution of junction zones during gelation. Further morphological structure information about the KGM gels from microscopic measurements should be explored in the following work. Effect of Deacetylation Rate on Gelation Behavior. Because the gelation kinetics and the mechanical properties of the gels are both dependent on the deacetylation rate, it is necessary to take into account the effect of deacetylation rate on the gelation behaviors of the native and acetylated KGM sample. In the presence of the same amount of alkali, the initial deacetylation rate of the samples was virtually different because of the different content of acetyl groups in the samples. For the deacetylation reaction, the molar ratio of alkali to acetyl groups reflects the reaction rate. Because the concentration of KGM dispersions was fixed to 2 wt %, the ratio of sodium carbonate concentration to DA (CNa/DA) was chosen to represent the deacetylation rate in the present work. Figure 8 shows the time dependence of G′ of 2.0 wt % KGM aqueous dispersions in the presence of Na2CO3 at 50 °C and at a frequency of 1 rad s-1, where the ratio of CNa/DA was fixed to 0.2. Obviously, the induction periods of all of the samples (within 100 min) were very close to each other, which is markedly different from the observation in the case where the Na2CO3 concentration was fixed to 0.8 wt % (Figure 5). The plateau values of G′ of Rs, Ac5, Ac6, and Ac-D ranged from 5000 to 9000 Pa, far less than those observed in Figure 5. However, the other acetylated samples with lower DA showed different gelation behaviors, where G′ quickly increased in the initial stage and then sharply decreased, namely, initial peaks appeared. The appearance of initial peaks of G′ in the gelation process of KGM at relatively high temperature has been reported previously54 and studied in detail by Zhang et al.43 It was suggested that the main reason was related to the wall slip

Gao and Nishinari

Figure 8. Time dependence of G′ of 2.0 wt % KGM aqueous dispersions in the presence of Na2CO3 at 50 °C and at 1 rad s-1. The ratio of Na2CO3 concentration to DA (CNa/DA) was fixed to 0.2.

between sample and measuring geometry, owing to a rapid gelation process with syneresis or disentanglement of molecular chains adsorbed on the surface of parallel plate from those located in the dispersion or both. For the present case, it should be noted that the alkaline concentration corresponding to a ratio of CNa/DA to 0.2 is very high, which resulted in a rapid gelation process. As a result, syneresis occurred, leading to slippage and the partial rupture of the network structure of the gel. The factors including temperature, applied strain, gap size, molecular weight, and KGM concentration were found to be responsible for the slippage.43 Besides, the degree of acetylation (DA) also showed effect on the slippage in the present work. The reason that the KGM samples with higher DA did not show slippage of G′ can possibly be assigned to the stronger and more elastic gel network (Figure 5), which prevents the exudation of water from the KGM gel. For native KGM (Rs), the reason that there is no initial peak of G′ should be related to the nature of chemical structure. There is strong molecular chain interaction from intramolecular and intermolecular hydrogen bonding. When dispersed in water, the native KGM became highly viscous, which inhibited the mobility of free water molecules. Consequently, the water exuded from the gel is not inclined to form a sliding interface between sample and measuring geometry. The values of tcr of the 2.0 wt % KGM aqueous dispersions in the case of CNa/DA ) 0.2 are plotted as a function of DA as shown in Figure 9 (solid square); tcr of the KGM dispersions obtained from Figure 5 (hollow circle) where the alkaline concentration was fixed at 0.8 wt % (concentration ratio of Na2CO3 to KGM was fixed at 0.4) was also presented for comparison. In contrast, tcr showed a tendency of independence of DA, although a distinctly higher tcr of Ac-D and a slight difference between other samples had been observed. We also examined the time course of G′ of 2.0 wt % KGM aqueous dispersions in the presence of Na2CO3 at 50 °C and a frequency of 1 rad s-1 where the ratio of Na2CO3 concentration to DA (CNa/DA) was fixed to 0.1 (data not shown). All KGM samples except Ac-D showed a close induction period. The KGM samples with lower DA (Rs, Ac1, Ac2, Ac3, Ac4, and Ac5) attained plateau values within



Figure 9. Plots of tcr of the KGM samples as a function of DA in the case of CNa/DA ) 0.2 from Figure 8 (9) and CNa/DA ) 0.1 (b) and in the case of the concentration of sodium carbonate (CNa) as 0.8 wt % from Figure 5, respectively.

the experimental time (600 min). The values of G′sat were in the range of 5000-14 000 Pa, and it seems that there is no regular dependence on DA. However, G′sat of Ac6 and AcD, which have higher DA among the acetylated samples, could not be observed within 600 min, owing to the relatively low alkaline concentration (0.76 wt % and 1.0 wt % for Ac6 and Ac-D, respectively, calculated according to their DA). The DA dependence of tcr of the KGM dispersions determined from the results of the time courses of G′ of 2.0 wt % KGM aqueous dispersions at 50 °C and at different frequencies is shown in Figure 9 (solid circle). Similar to the conclusion from Figure 8, tcr of the KGM samples were closer to each other, compared to that observed in the case that the Na2CO3 concentration was fixed to 0.8 wt %. The discrepancy among the values of tcr in the case of CNa/DA as 0.1 was larger than that observed in the case of CNa/DA ) 0.2; tcr of Ac-D, the acetylated sample with the highest DA, was obviously larger than those of other samples. In conclusion, the deacetylation rate, which is influenced by the ratio of CNa/DA, plays an important role in controlling the gelation behavior of the KGM dispersions. The dependency of tcr on DA disappeared or at least was weakened by changing the alkaline concentration with DA and fixing CNa/ DA to constants. On the whole, the discrepancies among the plateau values of elastic moduli when the CNa/DA ratio was fixed to constants were not so large, in comparison with those in the case that the alkaline concentration was kept as 0.8 wt % and independent of DA. These results further support that, based on the results concluded from Figure 5, the deacetylation rate directly affects the distribution of the junction zones and the formation of network structure in the course of gelation. When the actual concentration of sodium carbonate was increased with the increase of DA, that is, CNa/DA was fixed to a constant, every step in the whole gelation process including appearance of junction zones, growth of the cross-linking points, and final formation of network proceeded at a theoretically identical rate. As a result, the similar gelation behaviors were observed. It also reveals the dynamic nature of the KGM gelation, which is largely influenced by the removal rate of acetyl groups.

Figure 10. Time dependence of G′ of 2 wt % Ac1 dispersion at different ratios of CNa/DA measured at 50 °C and 1 rad s-1. The symbols represent the experimental results, and the solid lines represent the calculated curves from the best-fitting results using the first-order kinetics equation. Table 2. The Critical Gelation Time (tcr) and Parameters of the First-Order Kinetics Model for the Gelation of 2 wt % Ac1 Dispersion from Figure 10

CNa/DA

tcr (min)

k × 103 (min-1)a

G′sat (MPa)b

rc

0.05 0.1 0.14 0.18 0.20

140 66.4 48.1 36.1 32.8

3.25 ( 0.40 7.28 ( 0.38 11.37 ( 1.28 15.21 ( 3.9

12 448 ( 649 13 741 ( 281 11 726 ( 468 10 440 ( 576

0.9927 0.9977 0.9910 0.9679

a k is the rate constant. b G′ sat is the plateau value of G′ correlation coefficient.

c

r is the

Effect of Alkaline Concentration on Gelation. The time course of G′ of Ac1 dispersion at 50 °C and 1 rad/s is shown in Figure 10. The ratios of CNa/DA were changed to investigate the effect of alkaline concentration on gelation. It shows that both the induction time and plateau values of of Ac1 are influenced by CNa/DA. The evolution of G′ against time at constant temperature corresponding to some gelation processes can be well approximated by an equation of firstorder kinetics: G′(t) ) G′sat(1 - e-k(t-t0)) where G′sat is the plateau value of G′ after a long time, k is the rate constant of gelation process, and t0 is the gelation time.54 In the present work, the gelation time t0 is taken as the critical gelation time tcr determined by dynamic time sweep measurements using the Winter-Chambon criterion. In Figure 10, the symbols represent the experimental results and the solid lines represent the best fitting curves. The values tcr, G′sat, and k (determined from the best curve fitting results) are listed in Table 2. With increasing CNa/DA, the critical gelation time tcr became shorter and the rate constant k became larger. The plateau value G′sat of Ac1 seemed to show a maximum (1.37 × 104 Pa) at the ratio CNa/DA of 0.1. Above this ratio, G′sat decreased with the further increase of CNa/DA, indicating that a too rapid gelation rate resulted in a smaller plateau value of G′. We also examined the time dependence of G′ of Ac-D dispersion at 50 °C and a frequency of 1 rad/s measured at

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Table 3. The Critical Gelation Time (tcr) and Parameters of the First-Order Kinetics Model for the Gelation of 2 wt % Ac-D Dispersion

CNa/DA

tcr (min)

k × 103 (min-1)a

G′sat (MPa)b

rc

0.05 0.07 0.1 0.15 0.2 0.25

480 268 155 91.0 55.0 40.0

2.18 ( 0.36 3.38 ( 0.57 9.0 ( 0.3 10.2 ( 1.75

6701 ( 306 7037 ( 351 9157 ( 59 12 402 ( 2363

0.9448 0.9415 0.9982 0.8841


c

r is the

different CNa/DA ratios (data not shown), and the critical gelation time tcr, the plateau value G′sat, and the rate constant k from the best fitting results using first-order kinetics model for 2 wt % Ac-D dispersion are summarized in Table 3. In the case of CNa/DA equal to 0.05 and 0.07, the plateau value of G′ cannot be obtained because it still showed obvious increase even after 20 h (data not shown). Furthermore, it seemed that the plateau values G′sat at these two lower alkaline concentrations were significantly higher than those at other alkaline concentrations, although it seemed that the plateau values G′sat increased with CNa/DA within the CNa/DA range of 0.1-0.25. This indicates that a very low gelation rate at a very low alkaline concentration is favorable for Ac-D, the acetylated KGM sample with a highest DA, to form a particularly elastic gel. The detailed reason is not clear and should be worked out in the near future. From Table 3, tcr decreased monotonically with increasing the alkaline concentration, and the rate constant k became larger gradually. However, there is no maximum of plateau value G′sat in the CNa/DA range of 0.1-0.25, which is different from what is observed for Ac1 (Figure 10). Note that the absolute alkaline added to Ac-D, at the same ratio CNa/DA, is more than 2 times of that added to Ac1. Maekaji48 suggested that the concentration of hydroxide ion [OH-], independent of the kind of alkali, governs the reaction during the induction time. Hata et al.55 found that alkaline compounds play only a partial role in the binding mechanism because KGM gels retained their structure even after removal of alkaline compounds. It was reported that the specific volume of KGM in aqueous solution was almost constant between pH 3 and 11 and then increased steeply at above pH 11 with increasing pH.56 It was also reported that the gelation of native and acetylated KGM occurred at the pH range from 11.3 to 12.6 and 10.0 to 10.8, respectively.24,56 It was suggested that the change in molecular structure is necessary for the gelation of KGM. In the present work, increasing the alkaline concentration improved the contact possibility between acetyl groups in KGM and hydroxide ion, and thus the deacetylation ratio at the same reaction period increased. The critical gelation time tcr of Ac1 (listed in Table 2) and Ac-D (listed in Table 3) are plotted against the ratio CNa/DA, as shown in Figure 11. The solid lines represent the results of linear least-squares fit to the data in a double logarithmic representation. Noticeably, the experimental data can be well approximated using linear fitting. The slopes of the solid lines for Ac1 and Ac-D are -1.047

Figure 11. Double logarithmic representation of CNa/DA dependence of critical gelation time tcr for 2 wt % aqueous dispersion of Ac1 (O) and Ac-D (b) obtained from Tables 2 and 3, respectively. Table 4. The Critical Gelation Time (tcr) and Parameters of the First-Order Kinetics Model for the Gelation of 2 wt % Ac-D Dispersion from Figure 16 temp (°C)

tcr (min)

k × 103 (min-1)a

G′sat (MPa)b

rc

40.0 45.0 50.1 54.6 59.5 64.4

178 107 55.0 34.9 19.8 10.0

2.83 ( 0.52 3.52 ( 0.04 9.0 ( 0.3

8569 ( 247 9839 ( 30 9157 ( 59

0.9912 0.9959 0.9982


c

r is the

( 0.012 (99% confidence) and -1.525 ( 0.029 (98% confidence), respectively. This implies that within the experimental error and in the range of CNa/DA used in the present work tcr ∝ CNa-1.047 for Ac1 and tcr ∝ CNa-1.525 for Ac-D. A larger absolute value of the exponent (1.525) observed in the power law dependence of tcr on CNa/DA for Ac-D implied a stronger alkaline concentration dependence of gelation rate of Ac-D than that of Ac1 with relatively low DA. From the comparative results of time course of G′ between Ac1 (Figure 10) and Ac-D (data not shown), the slippage of G′, which should be only a function of alkaline concentration if other experimental conditions were fixed, showed obvious dependence on DA of KGM. These discrepancies in the ratio CNa/DA dependence of gelation behavior for the KGM samples with different DA also suggested the effect of DA on the gelation process of KGM. Effect of Temperature on Gelation. The gelation processes of 2 wt % Ac-D dispersion in the presence Na2CO3 (the ratio CNa/DA was fixed to 0.2) were observed at the temperature range from 40-65 °C (data not shown), and the critical gelation time, tcr, the gelation rate, k, and the plateau value, G′sat, from the best curve fitting using firstorder kinetics model for the gelation of 2 wt % Ac-D dispersion are listed in Table 4. The plateau value of G′ observed at 45 °C was higher than that at 40 °C, indicating that a more elastic KGM gel can be obtained at a higher temperature. However, G′sat of Ac-D gel tended to decrease gradually when the temperature was further raised. Indeed, a pronounced slippage appeared when the measuring tem-


perature was higher than 50 °C, and the shape of the initial peaks of G′, on the whole, became sharper at relatively higher temperatures (data not shown). As discussed above, this slippage was attributed to syneresis during the gelation process due to a too rapid gelation rate at high temperatures. We have examined the gelation processes of other 2 wt % KGM samples with different values of DA in the presence of Na2CO3 (the ratio CNa/DA was fixed to 0.2) at different temperatures from 35-65 °C (data not shown). Similar to Ac-D, each KGM sample showed a critical temperature at which the maximum G′sat was observed, and this temperature varied with DA of the KGM samples. Below this temperature, G′sat increased with increasing temperature, whereas above it, various degrees of slippage appeared. For all of the KGM samples, tcr values were shortened monotonically with increasing temperature. This can be attributed to increasing temperature promoting the aggregation of molecular chains of KGM and the subsequent formation of junction zones, resulting in a faster gelation rate, as observed in the heat-induced gelation of globular protein.57 For the gelation process of KGM, a relatively high temperature, within a specific temperature range, leads to a more elastic gel, as observed in previous works.24,43,54 Maekaji48 suggested that the deacetylation ratio, that is, acetyl groups removed per total acetyl groups, was almost independent of gelling temperature.. Therefore, the reason for the existence of difference in the plateau value G′sat of the KGM samples at different temperatures should be related to the distribution of junction zones.56,57 In other words, the cross-linking densities of the KGM samples at higher temperature are relatively dense. The increase in saturated modulus with temperature (with a specific temperature range before the appearance of syneresis) was observed for the rubber-like KGM gel, where the molecular chain segments between cross-links became more dynamic with temperature.44,60 The critical gelation time, tcr, of the 2 wt % aqueous dispersions of KGM samples with different DA at different temperatures from 35 to 65 °C are shown in Figure 12 using Arrehenius representations. The ratio CNa/DA was fixed to 0.2. The apparent activation energy (Ea) for each KGM sample was obtained from the slopes of the linear leastsquares fitting to the experimental results of tcr. The solid lines of linear fitting for the KGM samples except Ac-D were not represented in Figure 12 to prevent the overlap of the lines. Obviously, tcr of the KGM samples with different DA, except Ac-D, located at the similar region in Figure 12. This is in agreement with the conclusions from Figure 9 that, at the same temperature and a fixed ratio CNa/DA, the gelation rates of the KGM samples become close to each other and independent of DA. The apparent activation energies, Ea, of the KGM samples were 102-120 kJ mol-1 and were almost independent of DA. The average value of Ea for the gelation processes of the KGM samples was 110.6 ( 1.1 kJ mol-1, close to the Ea value (79-100 kJ mol-1) reported in a previous work.24 Possibly, the difference in the value of Ea comes from the different alkaline concentration used in the KGM gelation process. The Arrehenius law was used to examine the temperature dependence of gelation processes


Figure 12. Arrehenius representations of the temperature dependence of the critical gelation time tcr for the 2 wt % KGM aqueous dispersions. The ratio CNa/DA was fixed to 0.2. The solid line represents the result of a linear least-squares fit for Ac-D.

for some polysaccharides and proteins, such as β-lactoglobulin,57 methylcellulose,59 and κ-carrageenan.61 Generally, the formation and rupture of molecular chain linkage in junction zones during gelation process was considered as the main contributor to the apparent activation energy. The interchain linkages related to the junction zones always include several kinds of intermolecular interactions, such as hydrogen bonding, hydrophobic interactions, and dipoledipole complexation, which depend on the detailed gelation mechanism. KGM forms a thermoirreversible gel in the presence of alkali. The deacetylation reaction promoted by heating on the addition of alkali and the aggregation of KGM molecules through intermolecular hydrogen bonding induced by the reduction of water solubility due to acetylation were proposed to predominantly account for the induction time during KGM gelation.18,19,23,48 As shown in Figure 12, when the ratio CNa/DA was fixed to a constant, which ensures that the deacetylation reaction proceeded at the same rate, little discrepancy in Ea among the KGM samples was observed. This supports the finding reported in previous studies48,50 that hydrogen bonding was the most important intermolecular interaction that plays a main role in the formation of associated network KGM gelation. Effect of KGM Concentration on Gelation. The time evolution of G′ of Rs aqueous dispersions with a concentration range of 0.87-2.91 wt % measured at 50 °C and 1 rad s-1 where the concentration ratio of Na2CO3 to Rs was fixed to 0.2 were examined (data not shown), and the critical gelation times, tcr, determined by the Winter-Chambon criterion38 and the plateau values G′sat obtained by the firstorder kinetic model fitting to the experimental results are plotted as a function of Rs concentration, as shown in Figure 13, panels a and b, respectively. tcr was found to decrease sharply when Rs concentration increased from 0.87 to 1.8 wt % and then to decrease slightly at the Rs concentration above 1.8 wt %. This dependence of gelation rate (a shorter tcr corresponding to a faster gelation rate) on polymer concentration is consistent with that predicted by the mean field Cascade formalism for gelling polymers.44,62 At higher

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lower concentration range (0.80-1.28 wt %) and a smaller n at higher concentration range (1.50-2.0 wt %). These observations are in line with those reported previously.26,56 In conclusion, the wide DA range of the KGM samples in the present work made it possible to investigate the effect of deacetylation rate on gelation kinetics of the KGM samples by changing the ratio of alkaline concentration to the value of DA (CNa/DA). The distinct difference in the DA dependence of gelation times and saturated moduli for the KGM samples when the gelation condition was set at a fixed alkaline concentration independent of DA or a fixed ratio of CNa/DA suggested that deacetylation rate governed the gelation kinetics of KGM samples. At a fixed alkaline concentration, the aqueous dispersions of KGM samples with higher DA formed more elastic gels, at least in the DA range of the present work, suggesting that the relatively slow gelation rate is favorable for the formation of more homogeneous junction points. For the gelation of KGM, temperature, alkaline concentration, and KGM concentration also affected the gelation kinetics and the elastic modulus of KGM gel. A faster gelation rate and a more elastic modulus was realized by raising temperature or increasing KGM (or alkaline) concentration. However, too rapid gelation rates by changing any above condition resulted in the pronounced slippage during the initial gelation stage. The apparent activation energy (Ea) for the gelation of KGM samples was found to be independent of DA, and an average Ea of 110.6 ( 1.1 kJ mol-1 was observed. Figure 13. Rs concentration dependence of the critical gelation time, tcr (a), and the plateau values G′sat (b) for Rs aqueous dispersions.

Rs concentrations, the molecular chains are close and the interactions between them are intimate, so the probability of the formation of junction zones is higher than that at lower concentration.24 Maekaji48 found that the deacetylation ratio during KGM gelation decreased with increasing KGM concentration. This means that the gelation of KGM might begin even before the complete loss of acetyl groups at higher Rs concentration. Therefore, tcr became shorter with increasing Rs concentration as expected. Indeed, an obvious syneresis occurred at a Rs concentration of 2.91 wt %, resulting from a rapid gelation process at such a high concentration and in the presence of alkali. At Rs concentration above 3 wt % and in the presence of alkali, tcr became unexpectedly short because of the very rapid gelation process. Additionally, it has been reported that concentrated KGM aqueous dispersion (>7 wt %) exhibited a certain degree of gelation behavior even in the absence of alkali.22 For gelling polymers, the concentration dependence of elastic modulus of gels has been investigated and analyzed using the gelation theory including cascade model58,62 and modified rubbery elastomer model.63 It was predicted by both theories that a steep slope at lower polymer concentrations and a gradual slope at higher concentrations could be obtained from the results of linear fitting to the experimental results using a double logarithmic scale. From Figure 13b, the power law dependence of plateau modulus on Rs concentration, G′sat ≈ Cn, exhibited a bigger exponent n at

Acknowledgment. Shanjun Gao gratefully acknowledges the Japan Society For the Promotion of Science (JSPS), Japan, for the award of a postdoctoral fellowship (Grant P02391) and the financial support. References and Notes (1) Gallegos, C.; Franco, J. M. Curr. Opin. Colloid Interface Sci. 1999, 4, 288. (2) Kato, K.; Matsuda, K. Agric. Biol. Chem. 1969, 33, 1446. (3) Katsuraya, K.; Okuyama, K.; Hatanaka, K.; Oshima, R.; Sato, T.; Matsuzaki, K. Carbohydr. Polym. 2003, 53, 183. (4) Dea, I. C. M.; Morrison, A. AdV. Carbohydr. Chem. Biochem. 1975, 31, 241. (5) Maekaji, M.; Shimahara, H.; Sugiyama, N. Agric. Biol. Chem. 1980, 44, 245. (6) Nishinari, K. In NoVel Macromolecules in Food Systems; Doxastakis, G., Kiosseoglou, V., Eds.; Elsevier Science: New York, 2000; p 309. (7) Fujiwara, S.; Hirota, T.; Nakazato, H.; Muzutani, T.; Mitsuoka, T. Food Chem. Toxicol. 1991, 29, 601. (8) Levrat-Verny, M. A.; Behr, S.; Mustad, V.; Remesy, C.; Demigne, C. J. Nutr. 2000, 130, 243. (9) Lu, X.; Chen, X.; Fu, D.; Cong, W.; Ouyang, F. Life Sci. 2002, 72, 711. (10) Luo, D.; Li, Y.; Chin. J. Oncol. 1992, 14 (1), 48. (11) Xiao, C.; Gao, S.; Zhang, L.; Wang, H. J. Appl. Polym. Sci. 2000, 76 (4), 509. (12) Gao, S.; Zhang, L. J. Appl. Polym. Sci. 2001, 81 (9), 2076. (13) Gao, S.; Zhang, L. Macromolecules 2001, 34 (7), 2202. (14) Annable, P.; Williams, P. A.; Nishinari, K. Macrmolecules 1994, 27, 4204. (15) Williams, P. A.; Clegg, S. M.; Langdon, M. J.; Nishinari, K.; Piculell, L. Macrmolecules 1993, 26, 5441. (16) Ridout, M. J.; Brownsey, G. J.; Morris, V. J. Macrmolecules 1998, 31, 2539. (17) Ridout, M. J.; Cairns, P.; Brownsey, G. J.; Morris, V. J. Carbohydr. Res. 1998, 309, 375. (18) Maekaji, K. Agric. Biol. Chem. 1978, 42, 177.

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BM034302F

Effect of Degree of Acetylation on Gelation of Konjac Glucomannan

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