Physicochemical Characterization of Konjac Glucomannan - American

Biomacromolecules 2005, 6, 1977-1986

1977

Physicochemical Characterization of Konjac Glucomannan Ian Ratcliffe,† Peter A. Williams,*,† Christer Viebke,† and John Meadows‡ Centre for Water Soluble Polymers, The North East Wales Institute, Mold Road, Wrexham, United Kingdom LL11 2AW, and Maelor Pharmaceuticals Ltd., Riversdale, Cae Gwilym Road, Newbridge, Wrexham, United Kingdom LL14 3JG Received December 8, 2004; Revised Manuscript Received April 4, 2005

Four commercial konjac glucomannan (KGM) samples and a glucomannan derived from yeast were characterized by aqueous gel permeation chromatography coupled with multi angle laser light scattering (GPC-MALLS). Disaggregation of aqueous glucomannan solutions through controlled use of a microwave bomb facilitated reproducible molar mass distribution determination alleviating the need for derivatization of the polymer or the use of aggressive solvents. Further characterization was undertaken by use of capillary viscometry and photon correlation spectroscopy (PCS). The weight average molecular masses (Mw) determined were in the region of 9.0 ( 1.0 × 105 g mol-1 for KGM samples and 1.3 ( 0.4 × 105 g mol-1 for the yeast glucomannan. The values determined for KGM in aqueous solution are in agreement with those reported for KGM in aqueous cadoxen. The degradation of samples observed upon autoclaving has been quantified by GPC-MALLS and intrinsic viscosity determination, allowing comparison with reported Mark-Houwink parameters. Shear flow experiments were undertaken for a range of KGM solutions of concentration 0.05 to 2.0% using a combination of controlled stress and controlled strain rheometers. The concentration dependence of the zero shear specific viscosity was determined by analysis of the data using the Ellis model. The dependence of the zero shear specific viscosity on the coil overlap parameter was defined and interpretation discussed in terms of the Martin and Tuinier equations. Introduction Konjac glucomannan (KGM) is a food storage polysaccharide extracted in high yield from the tubers of Amorphophallus konjac C. Koch.1,2 Konjac has been cultivated for centuries in Japan and the flour used in the production of, for example, noodles and jellies. Only recently KGM has found use in the West as a texture modifier and thickener. KGM is a β-(1f4) linked polysaccharide composed of a D-glucosyl and D-mannosyl backbone lightly branched, possibly through β-(1f6) glucosyl units.3 The M:G ratio is typically reported to be approximately 1.6:1,4-7 and although some authors report a random distribution of these residues,7 others favor a complex nonrandom distribution.3 Unsubstituted linear β-(1f4) mannans and glucans (cellulose) are both insoluble in water owing primarily to interchain association through hydrogen bonding yet KGM is soluble. This solubility may partly be attributed to the long side chains of the glucomannan8 which serve to hinder intermolecular association and enhance solvation9 but predominantly is believed to be associated with the presence of the acetyl substituents. Removal of these groups facilitates gelation. The precise role of the acetyl groups in promoting solubility is still a matter of controversy. Dea et al.10 proposed that solubility was a consequence of the high entropic penalty of chain association owing to the steric effects of the acetyl group. X-ray crystallography studies which revealed crystal* To whom correspondence should be addressed. † The North East Wales Institute. ‡ Maelor Pharmaceuticals Ltd.

line mannan-II lattice in “acetylated”, i.e., native, KGM led Brownsey et al.11 to dispute this, suggesting instead that the presence of the acetyl groups simply suppressed intermolecular hydrogen bonding. Later studies using the same technique,12 however, indicated that the crystalline regions were composed of acetyl-free sections of the chain. Although average molecular masses for glucomannans have been determined by vapor pressure osmometry,13 intrinsic viscosity,14,15 and light scattering,16 very few workers have reported determination of molecular mass distribution. Although this has been undertaken by both GPC coupled with light scattering1,14,17 and by flow field-flow fractionation,18 sample preparation for such techniques frequently involves either modification of the polymer to improve the solubility or else the employment of specialized solvents. Chemical modifications reported for KGM include partial methylation,16 nitration,19 and oxidation.18 Such procedures are, however, time-consuming and may result in the degradation of the polymer.20 Although solvents including isoamyl acetate19 and aqueous cadoxen (CdO/ethylenediamine)17 have been used to solubilize KGM, problems are still encountered using GPC.17 Studies have shown that KGM aggregates resist complete hydration even in 70% aqueous cadoxen.21 Considerable success in producing homogeneous solutions of KGM and similar materials has been reported using “physical” methods whereby supramolecular aggregates are dispersed by increasing the energy of the component polymer chains. Such techniques include sonication, irradiation and the application of heat at elevated pressure. The need to

10.1021/bm0492226 CCC: $30.25 © 2005 American Chemical Society Published on Web 05/12/2005

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overcome such aggregation in order to measure the “true” molecular mass of KGM was recognized by Clegg et al.,14 who employed a sonication technique. Although the sonication regime was not specified beyond reporting the exposure time (5 min), a recent study using guar galactomannan22 reported a 50% reduction in molecular mass following a 5 min sonication treatment, attributed to random chain scission. Other methods of dispersing aggregates include treatment with heat under increased pressure. For example autoclaving, primarily undertaken for the commercial sterilization of polysaccharides, has been employed.23 A similar approach employing a commercial pressure/heating cell features in a comprehensive study of guar, locust bean, tara and detarium gums, and tamarind seed polysaccharide.20,24,25 Bello-Perez et al.26 dissolved starch in water by heating in a sealed vessel (microwave bomb) in a microwave oven. Additionally, it has been suggested that low doses of gamma irradiation bring about dissociation of aggregates of guar and locust bean gum, whereas higher doses were believed to cause backbone cleavage according to a hydrolysis mechanism involving free radicals.27 Recent dynamic light scattering studies of aqueous guar solutions28,29 indicated that they contained both small discrete aggregates and larger aggregates (which may include clusters of loosely bound small aggregates). The present study describes the determination of the molecular mass distribution and radii of gyration of five glucomannans using aqueous GPC-MALLS. Samples have been dissolved by heating in a microwave oven in a commercial microwave bomb. The effects of heat/pressure treatment on the molecular mass distribution have been studied using an autoclave. Intrinsic viscosities have been determined by capillary viscometry, and the hydrodynamic radii have been measured by PCS. The zero shear viscosity has been determined from shear flow measurements of KGM over a range of concentrations. Materials and Methods KGM (commercial grades CHSM, CH, SM, AC) originating from the Gunma Prefecture, Japan and a glucomannan derived from yeast, and referred to hereafter as YGM were gifts from Chesham Chemicals Ltd, Harrow, U.K. All five samples were used as supplied without further purification. Moisture content was determined gravimetrically following oven drying at 105 °C (CHSM 5.90%; CH 8.68%; SM 5.83%; AC 7.74%, YGM 1.20%). Sodium chloride 99.5% “Goldbrand” was purchased from M56 Chemicals, Sutton Weaver, U.K. Determination of Molecular Mass. Solution Preparation. Dissolution of each glucomannan, 0.1 g, was undertaken by addition to the vortex of magnetically stirred distilled water, 99.9 g, at ambient temperature. Following 30 min mixing at ambient temperature, the mixing was continued for 2 h at 80 °C in a loosely covered vessel. After cooling and addition of distilled water to compensate for evaporative loss, the insoluble fraction was removed by centrifugation (2500 rpm, 25 °C, 30 min) using an MSE Mistral 3000i benchtop centrifuge (Measuring and Scientific Equipment Ltd, Craw-

Ratcliffe et al.

ley, U.K.). Samples were heated for varying times in a model 4782 microwave bomb (Parr Instrument Company, Moline, IL) prior to measurement. It was found that the optimum heating time was 30 s, and this was used to undertake Mw measurements. The bomb was charged with 20 g of centrifuged analyte solution and heated in a Powerwave 800 W microwave oven (Proline, Korea) operated at 90% full power. GPC-MALLS Apparatus. The configuration comprised a 3215R degasser (ERC Inc., Saitama, Japan), Constametric 3200 pump (Thermo Separation Products, San Jose, CA), a Rheodyne 7125 injection valve (Rheodyne Inc., Cotati, CA) fitted with a 500 µL loop, a Suprema guard column and Suprema 100, 3000, and 30 000 columns (PSS, Germany) or Hema Bio guard column and Hema Bio linear column (MZ Analyzentechnik, Mainz, Germany), Dawn DSP Laser Photometer (Wyatt Technology, Santa Barbara, CA), and Optilab DSP Interferometric Refractometer (Wyatt Technology). GPC-MALLS Conditions. The eluent employed was sodium chloride, 0.1 M, filtered (0.22 µm) under reduced pressure and delivered at 0.5 mL min-1. Analytes were filtered using a 3µm membrane filter prior to injection. Interpretation of Light Scattering Data. Analyses were processed using the Astra 4.50 software package (Wyatt Technology). The raw data thus acquired in the light scattering experiment comprises values of Rθ, the excess Rayleigh ratio for different values of the scattering angle θ and concentration, c. The software facilitates extrapolation of the data to zero angle, when the weight average molecular mass, Mw and radius of gyration, Rg, can be determined by evaluation of eq 1

[

( )]

K*c θ 1 1 + 16π2 2 〈Rg 〉 sin2 + 2A2c ) Rθ Mw 2 3λ2

(1)

where K* ) “optical” constant, λ ) wavelength of optical light, and A2 ) second virial coefficient. For larger molecules, the extrapolation may be undertaken more successfully using the Berry or Debye plots than the more familiar Zimm plot. The Debye plot is essentially the reciprocal of the latter, such that Rθ/K*c is plotted against sin2(θ/2), while the Berry plot adopts (K*c/Rθ)1/2 as the ordinate function. Determination of the Intrinsic Viscosity, [η]. Samples for intrinsic viscosity determination were made according to the procedure described above. Polymer concentration was determined gravimetrically following oven drying of 30 g aliquots at 105 °C. Measurements were made at 25.0 ( 0.1 °C using a Cannon 75 capillary dilution viscometer (Cannon Instrument Company, PA). Determination of the Hydrodynamic Radius, Rh. KGM CHSM, 0.1% w/w solution, prepared and microwaved as described for the GPC-MALLS experiment, was diluted with appropriate amounts of pre-filtered (0.22 µm) distilled water to achieve concentrations in the range 0.025-0.10% w/w. Photon correlation spectroscopy (PCS) assays were made on each at 25 °C using a Zetasizer 1000HSa instrument (Malvern Instruments, Malvern, U.K.) and analyzed using

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Characterization of Konjac Glucomannan

the CONTIN algorithm. Analytes were filtered using 3µm membrane filters prior to measurement. The procedure was repeated for the same sample using 0.05M sodium chloride as solvent. Determination of the Zero Shear Viscosity, η0. Solution Preparation. KGM CHSM stock solution, 1.0% w/w, was prepared by the addition of the polysaccharide, 4.0 g, to distilled water, 396.0 g. The KGM was added to the vortex created by an overhead electric stirrer motor equipped with a twin-bladed paddle, and mixing was continued at ambient temperature for 30 min. The mixing apparatus was then transferred to a water bath thermostated at 80 °C, and mixing continued for 2 h. Distilled water was added to the cooled solution to compensate for evaporative loss. Adopting the same technique, a 2.0% stock solution was also prepared. To appropriate aliquots of the 1.0% stock solution was added distilled water to 100 g to achieve concentrations of 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, and 0.75% w/w. From the 2.0% stock solution were similarly prepared 1.25, 1.50, and 1.75% w/w solutions. The solutions were mixed on a roller mixer, and sodium azide was added to each at 0.02% w/w as a preservative. The solutions were stored in a refrigerator for no more than 5 days and when required were allowed to equilibrate to ambient temperature for 30 min. Measurement of η0. Shear flow measurements were carried out using a combination of controlled stress and controlled strain rheometers: AR500 and CSL500 (TA Instruments, New Castle, DE) and “Ares” (Rheometrics Scientific, Piscataway, NJ). Geometries employed were double concentric cylinder, single concentric cylinder, 5 cm diameter cone, and 4 cm diameter cone for concentrations of 0.05-0.10, 0.200.50, 0.75-1.75, and 2.0% respectively. Measurements were made at 25 °C, typically in the shear rate range of 0.2-200 s-1. The samples were loaded by pouring an appropriate aliquot onto the lower geometry member. Zero shear viscosity was determined using the Ellis model. This may be defined in the form of eq 2 η - η∞ 1 ) η0 - η∞ 1 + (Kσ)n

(2)

where η ) viscosity, η∞ ) infinite shear viscosity, η0 ) zero shear viscosity, σ ) shear stress, n ) power law exponent, and K ) arbitrary constant. Data in the dilute regime was acquired by calculating viscosities from capillary viscometry measurements made to determine intrinsic viscosity. Effects of Autoclave Treatment on KGM. Effect on Molecular Weight and Intrinsic Viscosity. Aqueous 0.1% w/w KGM CHSM solutions were subjected to up to four 15 min autoclave cycles at 121 °C using a 2100 Classic electric autoclave (Prestige Medical, Blackburn, U.K.). For each sample, the molecular mass distribution and intrinsic viscosity were determined as for the untreated polymer. Effect of the Solution Concentration when AutoclaVing on the Zero Shear Viscosity, η0. Solution Preparation. Dissolution of KGM CHSM was undertaken by addition of the polysaccharide (1.0, 1.5, 2.0, and 2.5 g) to distilled water, 95 g at ambient temperature. The KGM was added to the

vortex created by employment of an overhead electric stirrer motor equipped with a twin-bladed paddle, and mixing was continued at ambient temperature for 30 min. The mixing apparatus was then transferred to a water bath thermostated at 80 °C and mixing continued for 2 h. Following addition of distilled water to the cooled solution to a total mass of 100 g and tumble mixing, the solution was autoclaved at 121 °C for 15 min. The procedure was repeated three times thus obtaining KGM solutions of concentration 1.0, 1.5, 2.0, and 2.5% w/w. The cooled solutions were carefully diluted to 1.0% w/w where appropriate with distilled water. Gradual addition of the water with stirring was required due to the high viscosity of the solutions. After tumbling for 4 h the solutions were allowed to stand overnight in a refrigerator. Measurement of η0. For each KGM solution, shear viscosity measurements were made, and the zero shear viscosity was determined as described in the preceding section. Results and Discussion Choice of Processing Model for MALLS Experiments. Calculation of molecular masses from MALLS raw data necessitates diligence in selection of the most appropriate mathematical treatments. The Zimm, Debye, and Berry formalisms commonly employed each have particular merits and limitations and may not be of equal utility for a given experimental data set. Andersson et al.30 present a rigorous exposition of the applicability of each method. The procedure employed in this study, illustrated in Figure 1, was accordingly to first eliminate data that deviated from a Debye plot of a linear Zimm fit to the data and subsequently extrapolate using a linear Berry fit. Although it was acknowledged that a linear fit would not give the most accurate extrapolation, it was preferred for its robustness, being less affected by remaining error in the selected data. Furthermore, as can be seen in Table 1, the results are comparable to those obtained by a second polynomial Debye treatment. The refractive index increment, dn/dc employed in determination of K* was determined experimentally as 0.140 g mol-1. Microwave Bomb Treatment. The effect of microwave bomb exposure time upon the molecular mass distribution of KGM CHSM was determined in order to ascertain an appropriate treatment regime for subsequent assays. The results are presented graphically, Figure 2, and also in terms of the effect on the weight average molecular mass, Mw, in Table 2. Whereas for exposure times (t) of 30 and 45 s a constant molar mass was recorded (9.2 ( 1.5 × 105 g mol-1), longer exposure times resulted in a progressive molar mass reduction. The reproducibility of repeat assays on each sample was much improved in all cases where microwave bomb treatment was employed. It is suggested that for exposure times 30-45 s the molar mass represents that of the disaggregated polymer without degradation. For t g 60, however, it is believed that the disaggregation is accompanied by degradation of the polymer, i.e., hydrolysis of the glucomannan backbone. To demonstrate that the treatment regime in the former case was not degrading the polymer a sample was subjected

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Figure 2. Variation in the RI elution profile of KGM CHSM following microwave bomb treatment. (s) 30 s exposure, (-‚-‚) 45 s exposure, (-‚‚-) 60 s exposure, (‚‚‚) 75 s exposure. Table 2. Effect of Microwave Bomb Exposure Time on the Molecular Mass of KGM CHSMa microwave regime (exposure time) control - zero exposure

single exposure, 30s

single exposure, 45s single exposure, 60s single exposure, 75s double exposure, 2 × 30s Figure 1. Illustration of Debye plots for konjac glucomannan CHSM constructed according to the Zimm, Debye and Berry methods. (a) preliminary linear Zimm fit - all detectors. (b-d) Fits to selected data points only for Zimm, Debye and Berry formalisms respectively; (- - -) first polynomial, (s) second polynomial. Table 1. Comparison of Data Generated from GPC-MALLS Analysis of KGM CHSM Processed Using the First to Fourth Polynomial Fits of the Zimm, Debye, and Berry Models parameter

Zimm

Debye

Berry

Mn × 105 1st polynomial Mw × 105 1st polynomial Mz × 105 1st polynomial Rg (nm) 1st polynomial Mn × 105 2nd polynomial Mw × 105 2nd polynomial Mz × 105 2nd polynomial Rg (nm) 2nd polynomial Mn × 105 3rd polynomial Mw × 105 3rd polynomial Mz × 105 3rd polynomial Rg (nm) 3rd polynomial Mn × 105 4th polynomial Mw × 105 4th polynomial Mz × 105 4th polynomial Rg (nm) 4th polynomial

9.9 ( 0.3 11.9 ( 0.6 18.5 ( 7.4 115 ( 1 11.5 ( 0.7 14.2 ( 1.3 20.5 ( 7.0 141 ( 5 12.0 ( 1.7 13.4 ( 2.3 16.3 ( 7.9 139 ( 16 11.6 ( 3.4 12.6 ( 4.6 15.5 ( 14.6 135 ( 47

6.7 ( 0.3 7.0 ( 0.3 7.6 ( 0.9 53 ( 3 8.3 ( 0.3 9.0 ( 0.3 10.2 ( 0.9 76 ( 3 9.6 ( 0.4 10.5 ( 0.4 12.2 ( 1.2 93 ( 6 10.7 ( 0.9 11.8 ( 0.9 14.3 ( 2.7 107 ( 13

8.5 ( 0.3 9.4 ( 0.4 11.0 ( 1.2 85 ( 2 10.3 ( 0.4 12.2 ( 0.6 20.0 ( 4.8 113 ( 3 11.5 ( 1.1 13.2 ( 1.7 19.0 ( 9.1 128 ( 12 11.7 ( 2.6 13.6 ( 3.6 19.2 ( 14.9 134 ( 34

to two thirty second exposures, with a cooling period allowed between. The recorded molar mass (Table 2) demonstrated

Mw g mol-1

[% recovery]

(14.4 ( 0.6) × (14.8 ( 0.6) × 105 (10.5 ( 0.3) × 105 (9.4 ( 0.4) × 105 (7.7 ( 0.2) × 105 (9.1 ( 0.5) × 105 (8.8 ( 0.4) × 105 (10.6 ( 0.5) × 105 (5.3 ( 0.4) × 105 (4.9 ( 0.2) × 105 (4.4 ( 0.2) × 105 (4.6 ( 0.2) × 105 (10.2 ( 0.5) × 105 105

[86%] [72%] [51%] [74%] [68%] [75%] [64%] [72%] [81%] [84%] [91%] [88%] [80%]

a The values tabulated for single exposure represent duplicate GPCMALLS measurements of a single sample.

that the second 30s treatment did not further affect the polymer - it is suggested that this is a result of the polymer being already fully disaggregated from the first exposure. Gel Permeation Chromatography of Glucomannans. The initial GPC experiments using the Hema-Bio column were largely unsuccessful for KGM but achieved good separation of the yeast glucomannan (Figure 3) having a much lower molecular mass. Although the nominal operating range of the Hema-Bio column is 100 to 1.0 × 106 Da (suppliers specification), the upper limit may be considerably lower for a relatively rigid β-(1f4) linked polysaccharide. In contrast, the three column Suprema system offered a separation range up to 1.0 × 108 Da and delivered improved separation of KGM. Due to the high viscosity of even dilute aqueous KGM solutions, sample size and concentration had to be minimized, often resulting in poor sensitivity with the interferometric refractometer detector. The GPC-MALLS molar mass distributions measured for the five glucomannans using the Suprema columns are presented in Figure 4a-e. Data determined for the samples by GPC-MALLS: molecular mass, radius of gyration, polydispersity and percentage recovery is combined with that from capillary viscometry: intrinsic viscosity, Huggins



Figure 3. Molar mass distribution of glucomannan YGM using the Hema-Bio column. (9) molar mass, (s) R. I. response.

coefficient, viscosity average molecular mass, and effective frictional radius in Table 3. The viscosity average molecular mass Mv was calculated from the Mark Houwink relationship (eq 3) utilizing K and R values determined by Qi et al.31 [η] ) KMvR

(3)

where K ) 5.06 × 10-4 and R ) 0.754. The effective frictional radius RE was calculated from [η] and Mv according to eq 432 [η] )

3 5 4π NA RE 2 3 100 M

(4)

where NA is the Avogadro number. The data in Table 3 indicates that there are no significant molecular mass differences between the four KGM samples, while the intrinsic viscosity values show a little more variation. The Mws determined from GPC-MALLS i.e.,

approximately 8.5 ( 1.0 × 105 g mol-1 show reasonable agreement with those determined by Kohyama et al.33 at 6.89 × 105 g mol-1, while significantly higher than those of Clegg et al.14 (4.90 × 105 g mol-1) and lower than those of Takigami1 (13.2 × 105 g mol-1). The agreement with the results of Kohyama et al. is despite our measurement being carried out in water as opposed to cadoxen. The lower values of Clegg et al.14 may be expected in light of comments made in the Introduction regarding the use of sonication. The value reported by Takigami1 shows agreement with the “control” measurements of KGM made without microwave bomb treatment (Table 2); the author did not specify the conditions or solvent employed. The polydispersity values are comparable with those reported in the literature for similar polysaccharides, e.g., guar galactomannan (1.285).34 The Mv values determined generally show good agreement with the values reported from GPC-MALLS. They are considerably higher than comparable values reported by Shatwell et al.35 (1.9 × 105) and Clegg et al.14 (∼3.1 × 105). The determined [η] values are in good agreement with values determined by Kishida et al.16 (18.6-19.9 dL g-1) and Maeda et al.4 (12.7-23.6 dL g-1) although again higher than values reported more recently by Clegg et al.14 (7.3-7.6 dL g-1) and Shatwell et al.35 (5.5 dL g-1). The radii of gyration obtained from GPC-MALLS and the effective frictional radii obtained from [η] using eq 4 are considerably lower than those generally reported, i.e., in the region of 130 nm.1,16,36 Where lower values are reported, e.g., Clegg et al.14 (58, 72 nm) and Torigata19 (56.3 nm), they are usually associated with low reported Mws. Variation in the reported values of Mw (and similarly Mv, Rw, [η]) is expected as such values have been shown to be dependent upon botanical strain, cultivation conditions and processing methods.36

Figure 4. (a) Molar mass distribution of konjac glucomannan CHSM using the Suprema columns. (9) molar mass, (s) R. I. response. (b) The molar mass distribution of konjac glucomannan CH using the Suprema columns. (9) molar mass, (s) R. I. response. (c) The molar mass distribution of konjac glucomannan SM using the Suprema columns. (9) molar mass, (s) R. I. response. (d) The molar mass distribution of konjac glucomannan AC using the Suprema columns. (9) molar mass, (s) R. I. response. (e) The molar mass distribution of glucomannan YGM using the Suprema columns. (9) molar mass, (s) R. I. response.

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Table 3. Molecular Mass Distribution, Weight Average Radius of Gyration, and Polydispersity Determined by GPC/MALLS Compared with [η], Huggins Coefficient: kH, Viscosity Average Molecular Weight and Effective Frictional Radius from Capillary Viscometry for Five Glucomannan Samples grade CHSM CH SM AC YGM

Mn × 105, g mol-1

Mw × 105, g mol-1

Mz × 105, g mol-1

Rg, nm

8.5 ( 0.3 6.9 ( 0.2 8.0 ( 0.5 7.0 ( 0.3 8.0 ( 0.4 7.6 ( 0.4 6.9 ( 0.3 7.3 ( 0.3 1.1 ( 0.1 0.8 ( 0.2

9.4 ( 0.4 7.7 ( 0.2 9.5 ( 0.8 7.9 ( 0.5 9.6 ( 0.7 8.9 ( 0.6 8.1 ( 0.4 8.6 ( 0.4 1.6 ( 0.1 1.2 ( 0.2

11.0 ( 1.2 8.8 ( 0.7 13.1 ( 4.0 9.5 ( 1.5 13.6 ( 3.6 11.4 ( 2.1 10.0 ( 1.3 11.3 ( 2.0 2.7 ( 0.5 2.3 ( 0.5

85 ( 2 80 ( 1 95 ( 3 88 ( 3 93 ( 3 95 ( 3 85 ( 3 87 ( 2 34 ( 10 45 ( 15

Figure 5. Determination of D0 for konjac glucomannan CHSM. 0 solvent ) sodium chloride 0.05 M. 9 solvent ) water. For clarity, the error bars have been omitted for the latter.

Rh from Dynamic Light Scattering. The diffusion coefficients were measured for a range of KGM concentrations and extrapolated back to zero concentration (shown for KGM CHSM in both water and 0.05 M sodium chloride in Figure 5). Rh was then calculated from D0 using the Stokes-Einstein equation37 (eq 5) D0 )

kT 6πηRh

(5)

where k ) Boltzmann constant, T ) absolute temperature, and η )viscosity of the solvent. For KGM CHSM, a combined extrapolation of both data sets gave a value for Rh of 144 nm. The ratio Rg/Rh, on occasion referred to as the F parameter,38-41 is indicative of the polymer architecture. Zimm theory predicts values for F of 1.6 for a linear monodisperse polymer in good solvent, through to 0.77 for a hard sphere.42 For KGM CHSM, F is found to be ∼0.5. Similar values have been observed for various polysaccharides including β-D(1,3)(1,4)-glucan and tamarind seed polysaccharide (TSP),38-41 the low value of F being attributed not to a hard sphere architecture but rather an aggregate comprising a core of aggregated chains surrounded by a corona of hairy

kH

Mv × 105, g dl-1

RE, nm

20.0

1.4

12.5

72

16.0

1.5

9.2

60

19.6

1.4

12.2

71

17.2

1.6

10.3

64

polydispersity

recovery, %

[η], dL g-1

1.1 ( 0.1 1.1 ( 0.0 1.2 ( 0.1 1.1 ( 0.1 1.2 ( 0.1 1.2 ( 0.1 1.2 ( 0.1 1.2 ( 0.1 1.4 ( 0.2 1.5 ( 0.4

74 68 80 78 87 86 79 79 79 99

dangling chains. It is prudent, however, in this instance to acknowledge the potential for error in determination of F owing to, for example, the dependence of Rg upon the choice of model used to fit the data, errors associated with the use of algorithms such as CONTIN© in determining Rh42 and the polydispersity of the sample. The latter results in a broad distribution of values for Rg and Rh and this must be contemplated when evaluating the usefulness of F. The effect of polydispersity is illustrated for Rg in Figure 6 and for the relaxation time (from which Rh is ultimately derived) in Figure 7. The Huggins coefficients determined from the capillary viscometry measurements (Table 3) fall outside the “expected” 0.3-0.5. However Huggins coefficients >1 have been recorded for aqueous solutions of locust bean gum (LBG),43,44,45 guar GM,44 cassia and tara gums,43and also KGM.33 The abnormally high values are indicative of a poor solvent and are commonly attributed to presence of unusually strong solute-solute interactions or ‘hyperentanglements’. Critical Overlap Concentration, C*, of KGM. It is useful to combine the zero shear specific viscosities from shear flow measurements and viscosities determined using capillary viscometry in the form of a double logarithmic plot against the coil overlap parameter, c[η]. There is, however, no general agreement as to the most appropriate treatment of such data. Commonly,46-48 the data is considered simply as two power law regimes (Figure 8a). The polymer concentration at the intersection of these lines then defines the critical overlap concentration, C*. For this study C* ) 0.08 gdL-1, well below that reported, e.g., by Murakami and Motozato47 (0.29%). For most random coil polysaccharides, C* coincides with an overlap parameter of approximately 4, whereas values considerably lower (∼2.5) have been reported for galactomannans.49 Close inspection of Figure 8a reveals that for KGM CHSM C*[η] ) approximately 1.6, similar to that reported for a GM from the seeds of Mimosaceae spp.48 Kohyama et al.,50 alhough not reporting a specific value for C* commented that significant coil overlap was apparent when C[η] was greater than unity. The gradient of the lines are 1.5 for the dilute regime and 4.3 for the semidilute regime. The exponent in the dilute regime compares well with others reported for KGM: 1.4,50 1.66,51 and also more generally for random coil polysaccharides.49 For regular



Figure 6. Distribution of the Radius of Gyration of konjac glucomannan CHSM; eluent ) sodium chloride 0.1 M. (9) rms radius of gyration, (s) R. I. response.

Figure 7. Distribution of the Relaxation Time of konjac glucomannan CHSM; solvent ) sodium chloride 0.05 M.

random coil polymers, the exponent in the concentrated regime is reported to fall within the range 3.3 ( 0.3, whereas higher values found for, e.g., guar GM and LBG, have been attributed to “hyperentanglements”.49,52 That is to say that there exists chain associations above those expected from purely physical entanglements. Values for this exponent of 5.0 have been reported for guar GM46 and of 5.147 and 3.9851 for KGM. The reported deviation of KGM solutions from the Cox-Merz relationship51 may also be interpreted in terms of these hyperentanglements. A similar treatment of the zero shear specific viscosity data comprises assignment of two critical concentrations, thus defining three concentration regimes.53,54 The critical concentrations are attributed to the onset at C* of shrinkage of the polymer coils which continues until, at C** the polymer coils attain their limiting size. The data in Figure 8a does not lend itself to treatment by this method as there is limited data associated with the “intermediate” concentration regime. For guar GM and LBG, the scaling exponents associated

Figure 8. (a) Determination of C* from the variation in the zero-shear specific viscosity, ηsp0 of KGM CHSM with the coil overlap parameter, C[η]. (b) Illustration of curve fitting for the variation in the zero-shear specific viscosity, ηsp0 of KGM CHSM with the coil overlap parameter, C[η]. (9) Experimental data, (s) Martin model, (- - -) Tuinier model, (‚‚‚) power law.

with the three concentration regimes were reported to be 1.16, 1.9, and 4.0.54 An arguably more appropriate treatment of the data is to fit it to a curve. Tuinier et al.55 report that for a range of

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Ratcliffe et al.

Figure 9. Variation in the molecular mass distribution of KGM CHSM following autoclave treatment. Symbols represent molar masses: 0, control; O, 1x autoclave; and 4, 2x autoclave; lines represent the R. I. responses: (s) control, (-‚-‚) 1x autoclave, and (‚‚‚) 2x autoclave. Table 4. Effects on Molecular Mass, Polydispersity, and Intrinsic Viscosity of Multiple Autoclave Treatments for KGM CHSM autoclave regime

Mw × 105, g mol-1

[% recovery]

polydispersity*

[η], dL g-1

control/zero exposure

(15.4 ( 1.2)

[76%]

1.2 ( 0.1

20.0

(13.0 ( 1.2) (10.9 ( 0.7) (5.3 ( 0.2)

[92%] [79%] [77%]

1.3 ( 0.2 1.2 ( 0.1 1.3 ( 0.1

9.4

(5.6 ( 0.2) (5.4 ( 0.2) (5.5 ( 0.2) (5.2 ( 0.4) (5.4 ( 0.4) (3.2 ( 0.2)

[86%] [82%] [82%] [100%] [89%] [91%]

1.2 ( 0.1 1.3 ( 0.1 1.2 ( 0.1 1.5 ( 0.2 1.3 ( 0.1 1.2 ( 0.1

(3.3 ( 0.2) (3.4 ( 0.1) (3.5 ( 0.2) (2.7 ( 0.3) (2.0 ( 0.2)

[85%] [86%] [84%] [96%] [102%]

1.2 ( 0.1 1.3 ( 0.1 1.3 ( 0.1 1.4 ( 0.2 1.5 ( 0.2

7.5

(3.1 ( 0.6) (2.1 ( 0.6)

[101%] [108%]

1.4 ( 0.3 1.1 ( 0.5

6.2

(2.5 ( 0.4)

[98%]

1.4 ( 0.3

1 × 15 min exposure

2 × 15 min exposure

3 × 15 min exposure 4 × 15 min exposure

7.7

1 ([η]0c)7/2 25

Table 5. Variation in the Zero Shear Viscosity of KGM CHSM (Autoclaved for 15 min at 121 °C and Diluted to 1.0% w/w) with Change in the Solution Concentration When Autoclaving solution concentration when autoclaved, %w/w

zero shear viscosity of 1% w/w solution, Pa s

1.0 1.5 2.0 2.5

2.6 3.1 1.1 8.1

regime fitted to a simple power law. Similar behavior has been reported for xanthan gum56 ηsp0 ) c[η]0 eKc[η]0

polysaccharides the dependence of the zero shear specific viscosity on the coil overlap parameter can be described by eq 6 ηsp0 ) [η]0c +

Figure 10. Effect of solution concentration when autoclaving on the shear viscosity of 1% KGM CHSM. Autoclaved at 0, 1% w/w; O, 1.5% w/w; 4, 2%w/w; ], 2.5% w/w. Example shown of data fitted to the Ellis model (solid line).

(6)

Such a treatment is indicated by a broken line in Figure 8b for KGM CHSM. Clearly, although this approach is not appropriate for KGM, this is not entirely surprising in light of a similar deviation from the ηsp0 versus c[η] master curve reported for galactomannans.49 This again is attributed to the presence of non specific segment-segment interactions. An alternative curve fit is also illustrated in Figure 8b according to the Martin equation56 (eq 7). A reasonable fit is apparent for c[η] < 5, with the data in more concentrated

(7)

The constant K expresses the balance of polymer-polymer and polymer-solvent interactions.57 The power law exponent in the more concentrated regime is analogous to that reported in the discussion of Figure 8a. Autoclave Treatment. The molecular mass distribution of KGM CHSM following multiple autoclave treatments is presented in Figure 9. The associated trends in weight average molecular mass and polydispersity are tabulated in Table 4, alongside intrinsic viscosity values determined by capillary viscometry. Collectively, these data show a progressive reduction in molecular mass with each subsequent autoclave treatment. The magnitude of molecular mass reduction agrees well with that noted for autoclaving studies utilizing oat β-glucan23 and guar.20 A heat treatment study of guar58 reports a comparable decrease in intrinsic viscosity and the expected increase in low molecular mass fractions and polydispersity. It is noted that both [η] and Mw reductions decrease with each successive treatment. The mechanism of polymer degradation is not certain, but Mitchell et al.59 showed that viscosity loss on autoclaving of certain galactomannans could be reduced by treatment with antioxidants. This suggests that oxidative-reductive depolymerization may play an important role in the hydrolysis.


Figure 11. Comparison of the molar mass dependence of intrinsic viscosity for autoclaved KGM CHSM samples with linear representations of the Mark Houwink parameters reported in the literature. (9) this study, (s) Kishida et al.,16 (‚‚‚‚‚) Qi et al.31

Efforts were made to evaluate the influence of the KGM solution concentration when autoclaving on the observed Mw reduction. Regrettably, difficulty was encountered in achieving moleculary dispersed solutions suitable for GPC-MALLS by diluting concentrated KGM solutions. The solutions were, however, amenable to study by rheological measurement and zero shear viscosities were determined (Table 5, Figure 10). The results indicate that the magnitude of the Mw reduction is greatest when the solution concentration is lowest. The molar mass dependence of the intrinsic viscosity of the autoclaved samples is compared to linear representations of the Mark Houwink equations reported for both native KGM31 and methylated KGM16 (Figure 11). Conclusions A procedure was developed which facilitated reproducible determination of the molecular mass distribution of aqueous solutions of KGM. The method comprised pretreatment of the solutions using a microwave bomb, and selecting an appropriate exposure time in order to prevent degradation of the polymer. It is proposed that such treatment disaggregated material present in simple aqueous solutions. Subsequent GPC-MALLS measurements of commercial KGM samples utilizing a three column Suprema system gave reproducible molar masses in close agreement with those determined using aqueous cadoxen. Measurements were also made on autoclaved material for comparison. Acknowledgment. Financial support from the Engineering and Physical Sciences Research Council (EPSRC) and Maelor Pharmaceuticals, Wrexham, U.K. is gratefully acknowledged. References and Notes (1) Takigami, S. In Handbook of Hydrocolloids; Phillips, G. O., Williams, P. A., Eds.; Woodhead Publishing: Cambridge, U.K., 2000; p 413. (2) Nishinari, K.; Williams, P. A. Food Hydrocolloids 1992, 6, 199. (3) Katsuraya, K.; Okuyama, K.; Hatanaka, K.; Oshima R.; Sato T.; Matsuzaki, K. Carbohydr. Polym. 2003, 53, 183.

Biomacromolecules, Vol. 6, No. 4, 2005 1985 (4) Maeda, M.; Shimahara, H.; Sugiyama, N. Agric. Biol. Chem. 1980, 44, 245. (5) Shimahara, H.; Suzuki, H.; Sugiyama, N.; Nisizawa, K. Agric. Biol. Chem. 1975, 39, 293. (6) Kato, K.; Matsuda, K. Agric. Biol. Chem. 1969, 33, 1446. (7) Cescutti, P.; Campa, C.; Delben, F.; Rizzo, R. Carbohydr. Polym 2002, 337, 2505. (8) Williams, M. A. K.; Foster, T. J.; Martin, D. R.; Norton, I. T.; Yoshimura, M.; Nishinari, K. Biomacromolecules 2000, 1, 440. (9) Hwang, J.; Kokini, J. L. J. Text. Stud. 1991, 22, 123. (10) Dea, I. C. M.; Morris, E. R.; Rees, D. A.; Welsh, E. J., Barnes, H. A.; Price, J. Carbohydr. Res. 1977, 57, 249. (11) Brownsey, G. J.; Cairns, P.; Miles, M. J.; Morris, V. J. Carbohydr. Res. 1988, 176, 329. (12) Millane, R. P.; Hendrixson, T. L.; Morris, V. J.; Cairns, P. In Gums and Stabilisers for the Food Industry 6; Phillips, G. O., Williams, P. A., Wedlock, D. J., Eds.; Pergamon Press: Oxford, U.K., 1992; p 531. (13) Kolarova, N.; Matulova, M.; Capek, P. J. Carbohydr. Chem. 1997, 16, 609. (14) Clegg, S. M.; Phillips, G. O.; Williams, P. A. In Gums and Stabilisers for the Food Industry 5; Phillips, G. O., Williams, P. A., Wedlock, D. J., Eds.; Pergamon Press: Oxford, U.K., 1990; p 463. (15) Smirnova, N. I.; Mestechkina, N. M.; Shcherbukhin, V. D. Appl. Biochem. Microbiol. 2001, 37, 287. (16) Kishida, N.; Okimasu, S.; Kamata, T. Agric. Biol. Chem. 1978, 42, 1645. (17) Kohyama, K.; Sano, Y.; Nishinari, K. Food Hydrocolloids 1996, 10, 229. (18) Benincasa, M. A.; Cartoni, G.; Delle Fratte, C. J. Chromatogr. A 2002, 967, 219. (19) Torigata, H. Nippon Kagaku Zasshi 1951, 72, 166. (20) Picout, D. R.; Ross-Murphy, S. B.; Errington, N.; Harding, S. E. Biomacromolecules 2001, 2, 1301. (21) Wang, Q.; Wood, P. J.; Cui, W.; Ross-Murphy, S. B. In Gums and Stabilisers for the Food Industry 10; Williams, P. A., Phillips, G. O., Ed.; The Royal Society of Chemistry: Cambridge, U.K., 2000; p 82. (22) Tayal, A.; Khan, S. A. Macromolecules 2000, 33, 9488. (23) Wang, Q.; Wood, P. J.; Cui, W.; Ross-Murphy, S. B. Carbohydr. Polym 2001, 45, 355. (24) Picout, D. R.; Ross-Murphy, S. B.; Jumel, K.; Harding, S. E. Biomacromolecules 2002, 3, 761. (25) Picout, D. R.; Ross-Murphy, S. B.; Errington, N.; Harding, S. E. Biomacromolecules 2003, 4, 799. (26) Bello-Perez, L. A.; Roger, P.; Band, B.; Colonna, P. J. Cereal Sci. 1998, 27, 267-278. (27) King, K.; Gray, R. Food Hydrocolloids 1993, 6, 559. (28) Pinder, D. N.; Nash, W.; Hemar, Y.; Singh, H. Food Hydrocolloids 2003, 17, 463. (29) Gittings, M. R.; Cipelletti, L.; Trappe, V.; Weitz, D. A.; In, M.; Marques, C. J. Phys. Chem. B 2000, 104, 4381. (30) Andersson, M.; Wittgren, B.; Wahlund, K.-G. Anal. Chem. 2003, 75, 4279-4291. (31) Qi, L.; Li, G. J.; Zong, M. H. Acta Polym. Sinica 2003, 5, 650-654. (32) Boyd, R. H.; Phillips, P. J. The Science of Polymer Molecules; Cambridge University Press: Cambridge, U.K., 1993; p 74. (33) Kohyama, K.; Iida, H.; Nishinari, K. Food Hydrocolloids 1993, 7, 213. (34) Richardson, P. H.; Norton, I. T. Macromolecules 1998, 31, 15751583. (35) Shatwell, K. P.; Sutherland, I. W.; Ross-Murphy, S. B.; Dea, I. C. M. Carbohydr. Polym 1991, 14, 131. (36) Sugiyama, N.; Shimahara, H.; Andoh, T.; Takemoto, M.; Kamata, T. Agric. Biol. Chem. 1972, 36, 1381. (37) Sun, S. F. Physical Chemistry of Macromolecules: Basic Principles and Issues; John Wiley and Sons: New York, 1994; p 242. (38) Burchard, W. Papier 1994, 48, 755. (39) Lang, P.; Burchard, W. Makromol. Chem. 1993, 194, 3157. (40) Burchard, W.; Schulz, L. Macromol. Symp. 1995, 99, 57. (41) Grimm, A.; Kru¨ger, E.; Burchard, W. Carbohydr. Polym. 1995, 27, 205. (42) Rubinstein, M.; Colby, R. H. Polymer Physics; Oxford University Press: Oxford, U.K., 2003; p 347. (43) Daas, P. J. H.; Grolle, K.; Van Vliet, T.; Schols, H. A.; De Jongh, H. H. J. J. Agric. Food Chem. 2002, 50, 4282. (44) Gaisford, S. E.; Harding, S. E.; Mitchell, J. R.; Bradley, T. D. Carbohydr. Polym 1986, 6, 423. (45) Doublier, J. L.; Launay, B. J. Text. Stud. 1981, 12, 151.

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BM0492226

Physicochemical Characterization of Konjac Glucomannan - American

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