Isolation and characteristics

Isolation and characteristics of inulin from topinambur

Studies conducted by assignment of and in cooperation with CHARODEITSI Ltd. within the framework of the “Topinambur in Bulgaria” project

Pantelei P. Denev1, Naiden D. Delchev2, Georgi T. Dobrev, Ivan N. Panchev, Nikolai A. Kirchev

University of Food Technologies – Plovdiv
Department of Organic Chemistry and Microbiology

Abstract

Inulin from four topinambur varieties (Energina, Verona, Topstar, and Spindel) was isolated. The yield, purity and basic physicochemical characteristics (specific optical rotation, molecular mass, melting temperature, viscosity of aqueous solutions) of the obtained polysaccharide were measured. Inulin content and total fructans were comparatively characterized. The results revealed that the amounts of target product obtained from the four varieties are closely similar, the difference being in the range of 1–3 %. Inulin isolated from the Energina variety, cultivated on the biological farm No 086001 of CHARODEITSI Ltd., manifested a slightly higher yield (18.7 %) and purity (91 %). Therefore, this variety of topinambur was regarded as the most promising source of inulin.

Key words: topinambur, inulin, isolation, physicochemical characterization

Introduction

Inulin is a reserve polysaccharide, a member of the fructan group. It is widespread in the plant kingdom, however, the largest amounts are found in the roots, root systems and tubers of the members of the Asteraceae family. From chemical point of view, it is a linear polymer composed of ß-(2?1) bonded units of D-fructose. The biosynthesis of fructans is closely related to sucrose metabolism since they are products of its fructolyzation [2]. That is why each of their molecules contains a remainder of ?-D-glucose.

Over the last years there has been an increasing interest in inulin, mainly because of its health effects (improves calcium absorption, hypolipidemic action, prebiotic effect) and functional properties (gel formation ability, emulsifying properties, foam stabilizing ability, etc.). As an ingredient, it is included in foods to improve certain organoleptic and technological parameters, as well as to enrich them with vegetable fibers [9]. There are several methods for inulin isolation, differing mainly in the type of extragents, extraction conditions, purification and concentration [1,4,5,6]. Industrially, inulin is mainly obtained from chicory (Cichorium intybus L.). Recently, both researchers and manufacturers have directed their attention towards other nontraditional plant species like topinambur (Helianthus tuberosus L.), also known as earth apple or Jerusalem artichoke, whose tubers contain inulin between 7 and 20 % of the green mass [8].

Topinambur is extremely undemanding as far as soil, climate or cultivation are concerned. It gives high yields, thrives and grows well on the territory of the whole country. Nevertheless, the plant is still underestimated in Bulgaria and data about its chemical composition are scarce. There is no information available on the determination, isolation and characterization of inulin obtained from different local topinambur varieties.

The aim of the present work was to isolate inulin from four topinambur varieties, grown in Bulgaria, and to determine its basic physicochemical characteristics.

Materials and Methods
1. Raw materials for inulin

Tubers from the following topinambur varieties were used in the study: Energina (from the region of the town of Parvomai, Plovdiv district), Verona, Topstar and Spindel (from the region of the town of Berkovitsa, district of Montana). The above plant sources were in a phase of technological maturity of the tubers.

2. Method of inulin extraction

An amount of 100 g of ground raw material was hot-water extracted (t°~90°C) for 2-3 min. The volume remaining after the first extraction was topped up with water and the procedure was repeated. The two filtrate portions were mixed together. A Ca(OH)2 solution was added until ?? 8.0, and the mixture was left at room temperature for 1 h. The precipitated residue was filtered. The filtrate was neutralized (at t°=60-65°?) with oxalic acid to ?? 7.0, a small amount of activated carbon was added, and the mixture was then stirred and filtered. The filtrate was left to cool down at 2-5°? for 24 h. The precipitated amorphous mass was filtered through a büchner funnel and rinsed twice with 95 % ethanol and once with acetone. It was dried at 40°?.

3. Analytical methods
3.1 Spectrophotometric method for determination of inulin:

2.0 ?m3 of aqueous solution of inulin were mixed with 2.0 ?m3 of 10 % HCl and heated for 10 min in a boiling water bath. 0.5 ?m3 of Selivanov’s reagent (0.5 % solution of resorcinol in 20 % HCl) was added and further heated for 1 min. Absorption was measured at 520 nm. To plot a standard straight line (Fig. 1), fructose and a spectrophotometer Camspec M107 were used. The reagents were AR grade.

Fig. 1. Standard straight line for quantitative determination of inulin.

3.2 Method of enzyme quantitative determination of fructans.

Fructans were determined with a standard Megazyme test kit (Megazyme: fructan assay procedure, 2008).

3.3 Determination of molecular mass of inulin.

A HPSEC system (Waters) was used on Ultrahydrogel 500 and Ultrahydrogel 120 columns, standardized with Pullulan; the eluent was 0.1 ? NaNO3 at flow velocity of 0.8 cm3/min.

3.4 Determination of specific optical rotation.

An automatic digital polarimeter ?3001RS, KRÜSS (Germany) was used at ?=589 nm.

3.5 Determination of melting temperature.

The melting point was determined on a BÜCHI 510 (Switzerland) apparatus.

3.6 Determination of reducing sugars.

The reducing sugars were determined according to Shoorl’s method [3].

3.7 Moisture and ash evaluation.

The moisture and ash contents were determined as follows:

Moisture content – after drying the sample at 105°? until stable dry weight;

Ash content – after incinerating the sample at 600°? for 8 h.

3.8 Determination of dynamic viscosity

A Ubbelohde type capillary viscometer with a 0.54-nm-diameter capillary was used. The flow rate of the control sample (distilled water) was 90.3 s at 25±0.1°?. The inulin concentrations of the studied samples were 1, 3, 5, 7 and 10 %. Each solution was tested by tenfold replication, and the calculations were conducted by the classical statistical procedure for analysis of test data according to Gauss’ method. The density of the solutions was determined with a Gay-Lussac pycnometer. The dynamic viscosity of the solutions was calculated according to Poiseuille’s formula:

?=?0. ?.t ,

?0.t0

where ?0 is the dynamic viscosity of water, mPa.s;

? and t – density and flow rate of the solution;

?0 and t0 – density and flow rate of water.

Results and Discussion

Table 1 presents data about the inulin content and total inulin-type fructans in the four topinambur varieties subject of this study.

Table 1. Inulin content and total fructans in topinambur tubers.

Sample №	Topi- nambur vari- ety	Dry matter %	Inulin yield %	Inulin purity %	Inulin content % green mass	Inulin content % a.d.m.	Fructan content % a.d.m.
1	Energina	23,0	18,7	91	17,0	73,9	74,4
2	Verona	21,9	17,2	88	15,1	68,9	70,1
3	Topstar	20,8	16,4	89	14,6	70,2	72,5
4	Spindel	23,1	17,8	88	15,7	67,9	69,4

a.d.m. – absolute dry matter
* – Yield of isolated product per amount of green mass.

The presented results show that inulin yields from all four varieties are closely similar, with differences being within 1-3 %. Inulin isolated from the Energina variety gave a higher yield and purity, which makes this topinambur variety the most perspective source of inulin. Compared to inulin, the content of fructans is slightly higher, which is normal, as the applied enzyme method determines the total content of fructans, including the lower molecular weight fructooligosaccharides (sucrose excluded).

Table 2 presents the results from the comparative studies of the main physicochemical parameters of inulin isolated from the four topinambur varieties.

Table 2. Physicochemical characteristics of inulin from topinambur.

СП – степен на полимеризация; Тт – температура на топене; [α]D20 – специфичен ъгъл на оптично въртене.

DP – degree of polymerization; ?m – melting temperature; [?]D20 – specific optical rotation. An important characteristic of inulin which greatly determines its functional properties is its molecular weight. In the four test samples it varies between 4882-5600 Da, which is a reason to define inulin as a medium-chain polysaccharide with DP 28-33. This is also confirmed by the specific optical rotation, which, according to data in literature, is in the range from -32 to -40° depending on DP. The high molecular weight inulin obtained by recrystallization from aqueous solutions has [?] D20=-40° [5]. Apart from the spectrophotometric method applied here, the inulin purity can be determined by its physicochemical characteristics. The results about the moisture and ash content, reducing sugars, melting temperature and ?? of aqueous solutions in this study are similar to those in literature about inulin isolated from wild chicory roots (Radix Cichorii) [7]. The mean values of the dynamic viscosity ? dependent on the concentration of inulin (obtained from the Energina variety) are given in Fig. 2.

The analytical type of the dependency ?=f(c) was established by the regression analysis method, and subordinates to a linear dependence of the type ?=a+b.c, where c is the concentration of the inulin solutions; a and b are regression coefficients. For the studied solutions ?=0,9874+0,0682.c, for linear correlation coefficient R=0,9962. Thus, it is possible to predict the viscosity of the inulin solutions according to the polysaccharide concentration. The results obtained indicate that up to ??10 % of inulin aqueous solutions behave like Newton liquids.

Conclusion

The method used in this study for inulin isolation under selected optimal conditions resulted in comparatively high yields and purity of the target product. The relatively higher inulin content in the Energina variety established it as the most perspective source of inulin. The molecular masses of the studied polysaccharide characterize it as a medium-chain polysaccharide, with DP 28-33, which to a great extent determines its functional properties (water solubility, gel formation ability, etc.). The measurement of the viscosity of the solutions is a standard method, which characterizes their rheological behaviour. The use of inulin in the food technologies would require, among other physicochemical characteristics, familiarity with the numerical values of its viscosity. With the results in Fig. 2 it is possible to determine the viscosity of inulin aqueous solutions of different concentrations. The suggested spectrophotometric method (3.1) can be successfully used both for determination of inulin purity and for its quantitative identification in various food materials and products.