Size and Shape of Inulin in Dimethyl Sulphoxide Solution B.H
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CARP 1214 Carbohydrate Polymers 38 (1999) 231–234 Size and shape of inulin in dimethyl sulphoxide solution B.H. Azisa, B. China, M.P. Deacona, S.E. Hardinga, G.M. Pavlovb,* aNational Centre for Macromolecular Hydrodynamics, University of Nottingham, School of Biology, Sutton Bonington, LE12 5RD UK bInstitute of Physics, St. Petersburg University, Ul’anovskaya ul. 1, 19890 St. Petersburg, Russia Received 3 March 1998; revised 5 April 1998; accepted 7 April 1998 Abstract A comparative hydrodynamic characterization of the solution properties of the fructan polysaccharide inulin extracted from two different sources and solubilized in dimethyl sulphoxide is described. For Jerusalem artichoke inulin a weight average molecular weight M w of 3400 Ϯ 150 Da from sedimentation equilibrium in the analytical ultracentrifuge is obtained, together with an intrinsic viscosity [h] of 9.1 Ϯ ¹1 0.2 ml g and a sedimentation coefficient (corrected to a solvent density and viscosity of that at water at 20ЊC) s20,w of ϳ0.4 S. Chicory ¹1 ¹1 root inulin had somewhat similar properties, with an M w of 6200 Ϯ 200 g mol ,[h] of 10.7 Ϯ 0.2 ml g and s20,w also of ϳ0.7 S. These results appear reasonably consistent with a rather compact model with a relatively large degree of solvent association. ᭧ 1999 Elsevier Science Ltd. All rights reserved. Keywords: Inulin; Fructan; Sedimentation coefficient 1. Introduction Fructans are synthesized by the transfer of a fructose residue from sucrose onto another sucrose receptor Inulins are members of the fructan class of storage poly- molecules (Pollock and Chatterton, 1988). Enzyme systems saccharides and are found in large amounts in only a small capable of doing this in three different ways are known to variety of plants, but are widely distributed. Inulins, in com- exist leading to the main three types of fructans: isokestose mon with the other fructans have considerable biotech- fructans or ‘inulins’, kestose fructans and neokestose fruc- nological importance (see Fuchs (1987), Silver (1996), tan or ‘levans’, although the terms ‘inulin’ is sometimes Schacht (1985)) and for two reasons. The first is that they used to describe all these fructans. The isokestose inulins are a potential source of fructose, for which there is growing are linear chains with b(2 → 1) links of the general form demand in the food industry as a sweetener; the second is → → ( ) → Glc ¹ 1 2 ¹ Fru ¹ 1 2 ¹ Fru ¹ 1 n 2 ¹ Fru they are the only major class of polysaccharide based on the furanose structure (Haworth et al., 1932). Fructans were first with a maximum degree of polymerization (DP) claimed to isolated from Inula helenium, hence the name ‘inulin’ and be in the region of 30 to 40 (Praznik and Beck, 1987). many other Compositae have since been found to make In terms of physical properties early work on Jerusalem them. They occur in all parts of the plant, notably, from artichoke inulin by Phelps (1965) suggested a weight aver- ϳ the biotechnology point of view, in the seeds of the Grami- age molecular weight, Mw of 7000 Da (light scattering neae though only to the extent of 1% or 2% (Meier and Reid, data of P. Johnson, not ultracentrifugation as reported 1982). The best known cultivated dicotyledon is the Jerusa- in their abstract) and an intrinsic viscosity [h]of ϳ ¹1 lem artichoke (Helianthus tuberosus), the tubers of which, 7.1 ml g . The problems of solubility in aqueous solvents together with cereals, are the major source of inulin used as was illustrated. Eigner et al. (1988) obtained a similar result Ϯ a special food for diabetics. Apart from use in diabetic diets for Mw on heated solutions of 7200 100 Da using size- some herbal extracts which are probably high in inulin were exclusion chromatography coupled to fixed low-angle light advocated as diuretics, while inulin itself is used in kidney scattering measurements. Despite the small molecular size clearance tests since it remains intact in the blood after these workers were also able to obtain an estimate for the injection for long periods. diffusion coefficient and this together with a radius of gyra- tion Rg from small angle x-ray scattering suggested a rather elongated helical structure in solution. French (1988) on the * Corresponding author basis of computer modelling suggested possible conformations 0144–8617/98/$ - see front matter ᭧ 1999 Elsevier Science Ltd. All rights reserved. PII: S0144-8617(98)00096-4 232 B.H. Azis et al. / Carbohydrate Polymers 38 (1999) 231–234 Table 1 Properties of Jerusalem artichoke and chicory root inulin in DMSO as a solvent Property Jerusalem artichoke Chicory root Partial specific volume (ml g ¹1) 0.601 Ϯ 0.021 N.D. Molecular Weight (Da) 3400 Ϯ 150 6200 Ϯ 200 Intrinsic viscosity [h] (ml g ¹1) 9.1 Ϯ 0.2 10.7 Ϯ 0.2 Sedimentation coefficient, s 20,w (S) ϳ0.4 ϳ0.7 N.D.: not determined. of the inulin polymer, ranging from compact to more dynamic viscosities (Tanford, 1955) were not significant. extended structures. Intrinsic viscosities [h] were obtained from a Huggins In this paper we report a complementary hydrodynamic (1942) plot of reduced viscosity hred vs concentration c: study on inulins from two different sources: Jersuslam arti- h ¼ [h](1 þ K [h]c) (2) choke and chicory root. To avoid problems of poor solu- red H bility in aqueous solvent media we follow the practice where KH is the Huggins constant. successfully used by workers on starch (see Dickinson et al., 1984) of using dimethyl sulphoxide (DMSO) as the 2.4. Sedimentation velocity solvent (Table 1). An MSE (Crawley, UK) Centriscan ultracentrifuge was employed equipped with scanning Schlieren optics. The top 2. Materials and methods rotor speed of 50000 rpm was chosen because of the small size and expected small sedimentation coefficient for the 2.1. Materials inulin. Sedimentation coefficients, s, obtained at 25.0ЊCin the DMSO solvent were corrected to standard conditions of The inulins were supplied by Sigma (Poole, Dorset, UK) solvent density and viscosity (that of water at 20ЊC) accord- (lot no. I2880, I2255) using an alcohol recrystallization pur- ing to (Van Holde, 1985): ification procedure. Pure-grade dimethyl sulphoxide 1 ¹ vr20, w ho (DMSO) was supplied by Fisons Scientific equipment Ltd s20, w ¼ · ·s (3) 1 ¹ vr h , (Loughborough, UK). o 20 w where ro and ho are the solvent density and viscosity at Њ 2.2. Densimetry 25.0 C and r20,w and h20,w are the corresponding values for water at 20.0ЊC, the assumption is made that v is the same. An Anton Paar (Graz, Austria) digital precision density meter of the mechanical oscillator type (Kratky et al., 1973) 2.5. Sedimentation equilibrium was used. Constant temperature was maintained using a Hetrofrig water bath and recorded using a Comar platinum A Beckman (Palo Alto, USA) Model E ultracentrifuge resistance thermometer. A plot of solution density r(g ml¹1) was used equipped with an RTIC temperature measurement vs concentration c(g ml¹1) yielded the density increment dr/ system and a 5 mW He–Ne laser light source. A rotor speed dc from which the partial specific volume v was evaluated of 40000 rpm was employed at a rotor temperature of according to (Kratky et al., 1973): 21.0ЊC. Equilibrium solute distributions were recorded using Rayleigh Interference Optics and data capture semi- 1 dr v ¼ 1 ¹ (1) automatically from photographic film read into an LKB ro dc Ultroscan densitometer. Fringe distributions were converted where ro is the solvent density. into accurate records of relative concentration vs radial dis- placement using the Fourier cosine series algorithm ANA- 2.3. Viscometry LYSER (see Rowe et al., 1992). Apparent weight average molecular weights Mw,app were obtained using the MSTARI An Ostwald type viscometer of 2 ml capacity was used to routine (Colfen and Harding, 1997) using the M* function determine the intrinsic viscosity of the inulins. The vis- and the identity M*(cell base) ¼ Mw,app for the whole dis- cometer was suspended in a thermostatically controlled tribution of solute in the ultracentrifuge cell. water bath (Schott-Gera¨te, FRG) maintained at 25.00 Ϯ 0.01ЊC and flow times recorded automatically. Relative viscosities were obtained from the ratios of flow times of 3. Results and discussion solution to solvent. Because of the low concentrations used Ͻ 0.5 mg ml¹1) density corrections to convert kinetic to A partial specific volume of 0.601 Ϯ 0.021 ml g¹1 was B.H. Azis et al. / Carbohydrate Polymers 38 (1999) 231–234 233 Fig. 1. Solution density vs concentration of Jerusalem artichoke inulin in DMSO obtained at a temperature of 25.00ЊC?. dr/dc ¼ (0.342 Ϯ 0.012).v ¼ 0.601 ml g ¹1. obtained from the density increment plot (Fig. 1) for the inulin from Jerusalem artichoke. Since the saccharide com- position of the two inulins was the same this value was taken as the same for the chicory root sample. The value obtained is in exact agreement with that of Phelps (1965) whose result was obtained in aqueous solvent. Sedimentation velocity in the ultracentrifuge yielded sedimentation diagrams indicative of homogeneous preparations (see Fig. 2). However because of the very small size and rapid diffusion of the boundaries only an Fig. 3. Plot of M* vs the radial displacement squared function y for: (a) Jerusalem atrtichoke inulin; and (b) chicory root inulin. Loading con- estimate for the sedimentation coefficient was possible for ϳ ¹1 ϳ centration c 0.7 mg ml . Rotor speed ¼ 40000 rpm. Solvent ¼ DMSO. both species, with s20,w values of 0.4 S (at 2.5, 3.4 and Temperature ¼ 21.0ЊC.