KMITL SCIENCE AND TECHNOLOGY JOURNAL VOL.13, NO.2, 2013 67 Extraction, Purification, and Physicochemical Characterization of Seed Galactomannans Wancheng Sittikijyothin* Department of Chemical Engineering, Faculty of Engineering, Burapha University Chonburi, Thailand ABSTRACT different M/G ratios make galactomannans versatile materials used to modify textural attributes as The galactomannans as endosperm mucilage were thickening, water holding, stabilizing, and emulsifying extracted from seeds of Caesalpinia pulcherrima and agents in the food and other industrial applications Delonix regia. Crude galactomannans were then [1], [2], [5]. In principle, the M/G ratio causes purified by using isopropanol precipitation, the significant changes in the solubility, viscosity, and purified galactomannan yields of 75 and 82% of crude ability of galactomannan to interact with other mass were obtained for Caesalpinia pulcherrima, carbohydrates. For seed galactomannan is classified in Cassia fistula, and Delonix regia, respectively. The weakly branched classical galactomannans [2] due to physicochemical properties of both crude and purified high mannose. For example, guar gum (M/G ~ 2) galactomannans were analyzed. The results showed disperses more rapidly in water than locust bean gum that the crude galactomannans revealed high protein which has lower galactose content (M/G ~ 4) [2], [5]. content which eliminated after purification. Crude Because galactomannans with higher mannose content galactomannans obtained 3.46 and 6.12 of mannose to consist of long blocks of unsubstituted mannose units galactose (M/G) ratio for Caesalpinia pulcherrima that can either covalently interact with or chemically and Delonix regia, respectively. After purification the bind other polysaccharides (cross-linking) [6]. M/G ratio exhibited lower: 2.92 for Caesalpinia In Thailand, there are many plants that belong to pulcherrima and 4.72 for Delonix regia. The intrinsic the leguminous family which obtain seed viscosity and viscosity average molecular mass of galactomannan. Caesalpinia pulcherrima and Delonix galactomannan samples were estimated by using regia are some of them and native Caesalpiniaceae Huggins’ and Kraemer equations and the Mark- species (Leguminosae-Caesalpinioideae) which widely Houwink relationship, respectively. The obtained cultivated as ornamental trees in many regions of results showed that the intrinsic viscosity of purified country. Since, the industry trends demand the galactomannans increased when compared with crude opening of alternative sources of seed galactomannans. It indicated that the purification galactomannans. For that reason, the physicochemical process used alter the chemical structure. Since the properties of seed galactomannans from Caesalpinia intrinsic viscosity directly resulted in chemical pulcherrima and Delonix regia as alternative sources structure as M/G ratio. were interested to characterize and get more information in this work. Keywords: Galactomannan, Extraction, Purification, Intrinsic viscosity, Caesalpinia pulcherrima, Delonix II. METERIAL AND METHODS regia A. Plant Materials I. INTRODUCTION Pods of Caesalpinia pulcherrima and Delonix regia Galactomannans are plant carbohydrates that were collected in Chonburi province, Thailand. occur in the endosperm of the seeds of leguminous Mature seeds were manually separated from the pods plants. They are heteropolysaccharides whose and maintained in a cool dry place until the structural components are two monosaccharides: D- extraction process. mannose and D-galactose. The mannose units form a linear chain consist of (1→4)- β-D-mannopyranosyl B. Extraction and Purification residues with (1→6) linked α-D-galactopyranosyl The sample seeds were crushed before they were residues [1], [2]. In general, galactomannans from soaked. The endosperms were manually removed from seeds of different botanical sources differ by the the germ and the hull. Then, they were dried, milled mannose to galactose (M/G) ratio [1], [3], [4]. The and ground through a 355 micron mesh sieve. The extraction and purification of galactomannans * Corresponding author: E-mail: [email protected] is shown in Fig. 1. The ground powder of crude galactomannan was extracted in water under mechanical stirring at room temperature (time varying from 0.5 to 3 hours). Sodium azide was added 68 W. Sittikijyothin: Extraction, Purification, and Physicochemical Characterization of Seed Galactomannans (5 ppm) to avoid bacterial growth. The solutions were precipitate was washed with acetone and dried in heated for 2 hours at 80 oC. After cooling, the non- vacuum oven at 30 oC. Then it was ground to a fine dissolved material was removed by centrifugation at powder (purified sample) by the 355 micron mesh 6,000 g for 1 hour. The solubilized galactomannan sieve. was precipitated with excess of isopropanol. The Figure 1 Extraction and purification processes of seed galactomannans from Caesalpinia pulcherrima and Delonix regia C. Chemical Composition D. Intrinsic Viscosity Moisture and ash contents were determined The stock galactomannan solution (0.1 wt%) was according to the American Society for Testing and prepared. The galactomannan powder was slowly materials methods (ASTM-D2974-87) and AOAC added to the appropriate amount of distilled water in Official Method 923.03, respectively. Protein content the presence of sodium azide (5 ppm) in order to was obtained from the total nitrogen content (N×5.7) prevent bacterial degradation. The dispersion was by the Kjeldahl method, as described in the AOAC moderately stirred for 1 hour, at room temperature, Official Method of Analysis 981.10. Fat content was and then heated at 80 oC in the water bath for 30 determined according to the AOAC Official Method min, under continuous stirring. After cooling, a clear of Analysis 923.06. solution was, normally, obtained. The stock solution The main monosaccharide component was was centrifuged. Then, supernatants recovered. analyzed by GC-FID (Agilent Technologies 6890N Further dilutions were made to obtain a wide range of Network GC system) fitted with flame ionization solution concentrations. detector, equipped with a 30×0.25 mm. DB-225 Viscosities of dilute solutions were measured at column [7]. 20.0±0.1oC with a Cannon-Fenske Routine Viscometer (9721-A53) (ASTM-D2515, and Series KMITL SCIENCE AND TECHNOLOGY JOURNAL VOL.13, NO.2, 2013 69 100). The limiting viscosity number (“intrinsic of purified galactomannans at different extraction viscosity”), [h], is conventionally obtained by double periods were presented in Fig. 2. The obtained results extrapolation to zero concentration of Huggins’ and show that increasing of solubility period at room Kraemer equations, respectively. temperature enhanced more yields for both galactomannans. sp 2 (1) kC' C 100 90 C. pulcherrima D. regia (ln ) 2 82 rel kC'' (2) 79 79 C 80 75 69 70 62 Where hsp and hrel are the (dimensionless) specific 60 and relative viscosities, k', k'' are the Huggins’ and 50 Kraemer’s coefficients, respectively, and C is the solution concentration. % / yield Purified 40 30 E. Viscosity average molecular mass 20 Viscosity average molecular masses ( ) were Mv 10 calculated using the Mark-Houwink relationship that 0 account the different mannose to galactose ratios of 0.5 1 3 galactomannans [8]. Extraction time / h Figure 2 Purified yields of galactomannans at different 11.55 106 [(1 )M ]0.98 (3) v extraction periods at room temperature and fixed for 2 hours at 80°C where 1 / [(MG / ) 1] and [h] is expressed in dL/g. B. Chemical Composition III. RESULTS AND DISCUSSION In Table 1, the main characteristics of both crude A. Extraction and Purification and purified galactomannans from three legume plants are presented. It can be seen that the seed The crude galactomannans obtained by milling the galactomannans are rich source in polysaccharide with seeds of Caesalpinia pulcherrim and Delonix regia, some amount of protein content. However, the were extracted as in the following Fig. 1. The crude purification process seems efficient to eliminate galactomannan yields of 34 and 27% were obtained protein and other impurities from crude for Caesalpinia pulcherrima and Delonix regia, galactomannans [9-12]. Regarding the impurities, they respectively. According to extraction process, only are the important factor to estimate the purity of extraction period at room temperature was varied gums, particularly in food applications [4] from 0.5 to 3 hours while the extraction time at 80oC was fixed for 2 hours. Then, crude galactomannan was purified by precipitation with isopropanol. Yields TABLE I. CHEMICAL COMPOSITION OF GALACTOMANNANS Caesalpinia pulcherrima Delonix regia Crude Purified Crude Purified Moisture (%) 10.9 12.86 11.17 12.99 Ash (%) 0.06 0.05 0.11 0.10 Protein (%N5.7) 4.62 1.52 2.86 1.62 Fat (%) 1.45 0.18 0.90 0.17 Polysaccharidea (%) 93.87 98.25 96.13 98.11 M/G ratio 3.46 2.92 6.12 4.72 All values (%) on a dried weight basis are mean ± standard deviation of three determinations. aPolysaccharid values (%) were calculated by difference. Then, the mannose to galactose (M/G) ratio of M/G ratios for crude galactomannans from galactomannan samples was analyzed by GC-FID. Caesalpinia pulcherrima and Delonix regia seeds were The obtained result confirms that the M/G ratio about 3.46 and 6.12, respectively, higher than the varied for the different leguminous species. Hence, the values obtained by previous researchers who found 70 W. Sittikijyothin: Extraction, Purification,