Discovery of Unusual Highly Branched Galactomannan from Seeds of Desmanthus illinoensis *

A galactomannan was isolated from seeds of a leguminous plant, Desmanthus illinoensis, which is grown in Okinawa, Japan. D-Galactose (molar ratio, 1.0) and D-mannose (0.82) were identified via High-performance Anion Exchange Chromatography Coupled with a Pulse Amperometric Detector. The molecular mass and specific rotation were estimated to be 1000 kDa and +53.8 ̊, respectively. The infrared spectrum indicated that the galactomannan was involved in both αand β-linkages, and two types of α-linkages were detected at 814 and 830 cm. The Hand C-NMR spectra indicated that the majority of the β-D-mannan main chain was substituted with mono α-D-galactose or α-D-galacto-disaccharide-side chains. Methylation analysis was used to identify 2,3,4,6-tetra-O-methyl-D-galactose (molar ratio, 3.3), 2,3,4-tri-O-methylD-galactose (1.0) and 2,3-di-O-methyl-D-mannose (3.1). Specifically, unique 2,3,4-tri-O-methyl D-galactose residue was identified from mass spectrum. The results suggested that the galactomannan was 1,4-linked-β-D-mannan substituted with α-D-galactose or 1,6-linked-α-D-galacto-disaccharide side chains at C-6 on the main chain. The galactomannan isolated from D. illinoensis was an unusual highly branched polysaccharide, and its chemical structure was proposed. This work is the first to report on the galactomannan involving 1,6-linked α-D-galacto-disaccharide side chains in addition to α-D-galactose mono side chains. *A part of paper presented at the 35th Annual Meeting of the Japanese Society of Carbohydrate Research, Kochi, Japan, September 1-3, 2016; the 19th European Carbohydrate Symposium, Barcelona, Spain, July 2-6, 2017. How to cite this paper: Tako, M., Tamaki, Y. and Teruya, T. (2018) Discovery of Unusual Highly Branched Galactomannan from Seeds of Desmanthus illinoensis. Journal of Biomaterials and Nanobiotechnology, 9, 101-116. https://doi.org/10.4236/jbnb.2018.92009 Received: December 18, 2017 Accepted: April 17, 2018 Published: April 20, 2018 Copyright © 2018 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
Galactomannans are involved in the endosperms of leguminous plant seeds.
Since the polysaccharides have high viscosity [1] and synergistic interactions with xanthan gum [2] [3] [4], κ-carrageenan [5] and agarose [6], they are often used as thickening, stabilizing and gelling agents in the food industry.The structure of the polysaccharides is 1,4-linked β-D-mannopyranosyl main chain with various proportions of single α-D-galactopyranosyl side chains at C-6 of the main chain.The proportions of D-mannose and D-galactose in the galactomannans are dependent on the source and greatly affect the solution properties, including water solubility, viscosity and synergistic interactions.
The association occurred between C-2 of D-mannose of galactomannan, which adopted an axial orientation, and hemiacetal oxygen atom of the inner D-mannose-side chain of xanthan with hydrogen bonding.The pyruvate methyl groups of xanthan also participated in the interaction with galactomannan molecules.The association site contributing OH-2 of D-mannose main chains was suggested from the intra-and/or intermolecular associations of gelling κ-carrageenan [5] [15], ι-carrageenan [16] and agarose [17] molecules.The interaction between galactomannan and xanthan suggested that such D-mannose-specific interaction exist in plant and pathogen recognition processes because xanthan-producing bacterium is one of plant pathogen bacterium [14].Consequently, in principle, basic rules exist in gel-formation processes including water molecules and polysaccharides at the molecular level [18] [19].The principle is also examined in amylose, amylopectin and starch gelatinization as well as retrogradation including water molecules at the molecular Journal of Biomaterials and Nanobiotechnology level [20] [21].
Desmanthus illinoensis is a leguminous plant grown in Okinawa, but the plant is originated in USA.The highly unusual branched chemical structure of galactomannan from seeds of D. illinoensis is presented.

Polysaccharide Preparation
The seeds of Desmanthus illinoensis were collected in campus of University of the Ryukyus, Nishihara, Okinawa, Japan.Seeds were crushed to a powder with a mixer and kept in refrigerator at 4˚C.The powder was soaked in ethanol overnight and soaked again in acetone to remove lipids, then it was dried in vacuo.
The defatted powder (20 g) was suspended in water at 90˚C for 2 h, and filtered through Celite 545 filter aid.Two volumes of ethanol were added to the filtrate and the polysaccharide was dried in vacuo.The crude polysaccharide was dissolved in distilled water at room temperature and the solution was passed through Celite 545 filter aid.Then, the filtrate was precipitated by adding 2 volumes of ethanol and the resulting solid was dried in vacuo.The purified polysaccharide was dissolved in purified water at room temperature and lyophilized.
Locust bean gum obtained from Taiyo Chemical Co.Ltd (Mie, Japan) was prepared with the same methods described above.

Chemical Components Analysis
The polysaccharide carbohydrate content was determined by the phenol-sulfuric acid method [30] using D-mannose as the standard.

High-performance Anion Exchange Chromatography Coupled with a Pulse Amperometric Detector (HPAEC-PAD)
The polysaccharide was dissolved in purified water and sulfuric acid was added to a final concentration of 1.0 M. The solution was heated at 100˚C for 2 h.The hydrolysate was neutralized with BaCO 3 and filtered.The hydrolysate was analyzed by high-performance anion exchange chromatography (HPAEC) on DX 500 (Dionex, Sunnyvale, CA, USA) fitted with a column of CarboPac PAi (4 × 250 mm) and a pulsed amperometric detector.The column was eluted at a flow rate of 1 mL/min at 35˚C with 14 mM NaOH [31] [32] [33].
The HPLC operation was performed at room temperature.The column was developed with 50 mm phosphate buffer, and the same buffer supplemented with 150 mM sodium chloride; fractions (3 mL each) were collected at a flow rate of 0.5 mL/min.The column was conditioned with 0.15 M sodium chloride in 0.05 M sodium phosphate buffer (pH 7.2), and elution was conducted with the same buffer.Standard pullulan (Showa Denko, Tokyo, Japan) with a definite molecular mass were used as molecular mass markers (P5, 5 kDa; P200, 200 kDa; P400, 400 kDa; P800, 800 kDa) [33].

Fourier Transform Infrared (FTIR) Spectroscopy and Specific Rotation
The FTIR spectra of the polysaccharide and locust bean gum as a standard were measured using an FT-IR-8000 spectrophotometer (Jasco Co., Ltd., Japan) in transmittance mode from 4000 to 400 cm −1 in KBr disc.The KBr disc was prepared by dispersing solid sample in the KBr salt [25] [31].
The specific rotation of the polysaccharide was measured at 589 nm on a polarmeter (DIP-180, Jasco Com., Ltd., Tokyo, Japan) with a cell 5 cm in length for a 0.2% (W/V) solution in distilled water at room temperature.

1 H-and 13 C-Nuclear Magnetic Resonance
The polysaccharide was dissolved in D 2 O and then freeze dried.The dried sample was dissolved again in D 2 O (2.0%, W/V) and the solution was examined in 5 mm o.d.tube. 1 H-and 13 C-NMR spectra were recorded on a α500 FT-NMR spectrometer (JEOL Co., Ltd, Japan) at 500.00 and 125.65 MHz, respectively.

Methylation Analysis
Methylation of the polysaccharide was carried out by the Hakomori method [35].The methylated polysaccharide or locust bean gum was extracted with CHCl 3 .The extracted methylated polysaccharide was hydrolyzed with 2 M TFA (2 mL) at 120˚C for 2 h.The hydrolysate was dissolved in 1 M NH 4 OH (0.2 mL).
DMSO (1 mL) containing 20 mg of NaBH 4 was added and the mixture was incubated at 40˚C for 90 min.Subsequently acetic anhydride (0.2 mL) was added to the mixture.Anhydrous 1-methylimidazole (0.2 mL) and acetic anhydride (1 mL) were then added, and the reaction mixture was incubated at ambient temperature for 10 min.After extraction with chloroform and washing with water, partially methylated alditol acetates were obtained [25].
The partially methylated alditol acetates of the polysaccharide were analyzed using a gas chromatograph (GC-14A; Shimadzu Corp., Kyoto, Japan) equipped with a flame ionization detector using a capillary column (DB-1: 40 m × 0.25 mm, J&W Scientific Inc., CA, U.S.A.).The injector and detector temperatures were 210˚C and 270˚C, respectively.After injection, the oven temperature was maintained at 150˚C for 5 min, and then raised at 5˚C /min to 250˚C.This temperature was maintained for 5 min.The identities of the peaks were confirmed using GC-MS (GCMS-QP 1000EX; Shimadzu Corp., Kyoto, Japan) under the same conditions.Locust bean gum was also methylated as a standard.

Identification of the Polysaccharide Sugar Components
The polysaccharide contained 93.5% (w/w) carbohydrate.As shown in Figure 3, the anion exchange high-performance liquid chromatography of the acid hydrolysate of the polysaccharide showed the presence of D-galactose (peak 1) and D-mannose (peak 2), the molar ratio of which was 1.0:0.82.The result indicates that the polysaccharide is a galactomannan.The molar ratio of a galactomannan from D. illinoensis collected in Tucson, Arizona, USA was reported to be 1.0 (D-gal):2.07(D-man) [22].However, the galactomannan collected in Okinawa, Japan had reversed molar ratio.It was reported that a galactomannan from Fenugreek (Trigonella foenum-graecum L.) also involved a reversed molar ratio with more D-galactose than D-mannose (1.0:0.86)[29].

Molecular Mass
The molecular mass of the galactomannan isolated from D. illinoensis was determined by HPLC using a chromatograph on a column of Superdex 200 (Figure 4).According to the standard calibration curve obtained from the definite molecular mass pullulan, the molecular mass of the galactomannan was calculated to be approximately 1000 kDa.

FTIR Spectrum and Specific Rotation
Figure 5 shows spectra of the galactomannan and standard locust bean gum.An absorption at 3450 cm −1 was common to all the polysaccharides due to OH groups and an absorption at 2900 cm −1 was due to C-H stretching.Absorption common to galactomannan was observed in the range of 1245 -1000 cm −1 suggesting that the monosaccharide of the molecules consists of a pyranose ring [25] [31].Absorption at 814 cm −1 was attributed to α-D-galactopyranose [25].Journal of Biomaterials and Nanobiotechnology   Another absorption at 830 cm −1 was observed indicating that a different type of α-glycoside was co-involved.An absorption at 873 cm −1 was attributed to β-D-mannopyranose [25].The galactomannan spectrum was consistent with that of locust bean gum (Figure 5) over wide range of wave numbers, except at 830 cm −1 .
The specific rotation [α] 589 of the galactomannan (0.2%) was measured at 589 nm on a polarimeter at room temperature and estimated to be a value of +53.8˚ (H 2 O).The result suggests that the polysaccharide was substituted with many side chains as in Leucaena gum (+45˚) [23], because few branched locust bean gum [2] and Delonix gum [25] existed, which were at +22˚ and +15˚, respectively.

Methylation Analysis
The galactomannan was methylated according to the procedure described by Journal of Biomaterials and Nanobiotechnology  Hakomori [35].The obtained permethylated polysaccharide was subjected to complete acid hydrolysis to furnish mixtures of methylated sugars, which were analyzed as the corresponding alditol acetates using gas-liquid chromatography (GC) and combined gas-liquid chromatography/mass spectrometry (MS).The Gas chromatogram and mass spectroscopic data are shown in Figure 8 and Figure 9.

Discussion
This study presents an investigation of galactomannan isolated from Desmanthus illinoensis which was grown in Okinawa, Japan.From HPAEC, D-galactose and D-mannose was identified with a molar ratio was 1.0:0.82.A small amount of D-mannose was in contrast with previous studies on galactomannans [22]- [28], except that of fenugreek gum [29].The result suggested that the galactomannan substituted many side chains.The galactomannan dissolved in distilled water even at room temperature (25˚C) due to presence of many side chains.
The signals in the infrared spectrum at 814 and 830 cm −1 , that were the first demonstration ever reported, were attributed to two types of α-D-galactose sidechains.
Furthermore, high specific rotation (+53.8˚) also supported that the galactomannan was substituted with many side chains.The 1 H-and 13 C-NMR spectra of the galactomannan were in good agreement with those fenugreek gum which was a highly branched galactomannan where α-D-galactose residues were involved as mono or 1,4-linked disaccharide side chains [29], The structure of side chains of fenugreek gum was identified by 2D NMR spectra without methylation analysis.However, we identified 1,6-linked α-D-galactose disaccharide side chain via methylation analysis.Indeed, 1 H-and    The galactomannan could be used as a substitute for guar gum [3].Synergistic interaction between xanthan and the galactomannan from D. illinoensis is now in progress.

Conclusion
The combination of infrared spectroscopy, NMR ( 1 H-and 13 C-) spectroscopy, and methylation analysis confirms the polysaccharide isolated from Desmanthus illinoensis is an unusual highly branched galactomannan.As the molar ratio of D-galactose to D-mannose is 1.00:0.82,the chemical structure of the galactomannan, nano-saccharide repeating unit, is illustrated in Figure 10.This study is the first to report on galactomannan involving 1,6-linked α-D-galacto-disaccharide sidechains in addition to α-D-galactose mono side chains at C6 of β-D-mannan mainchain.

Figure 1 .
Figure 1.Synergistic gelation mechanism between galactomannan and xanthan gum: (---) hydrogen bonding and (/////) van der Waals forces of attraction.As the tertiary structure of the xanthan molecule may keep a single stranded helix, its side-chains are inserted into the adjacent, unsubstituted segments of the backbone of the galactomannan molecule.A molecule of xanthan may combine with two or more molecules of galactomannan, the ratio depending on the favored conformation in aqueous solution.As the side-chains of the native and depyruvated xanthan molecules are somewhat rigid because of the intramolecular associations contributed by acetyl group and OH-3 of D-glucosyl residue, an incomplete interaction may exist in part and greater interaction may result from deacetylation (Reproduced with permission from reference 21.Copyright 2014 Scientific Research Publishing).
Desmanthus illinoensisD.illinoensis grows 0.5 to 1.0 m in height.Multiple stems grow from a woody caudex.As shown in Figure2(a), the leaves are a twice pinnately compound.Ten to 15 pinnae have 15 -25 leaflets.The brown pods (Figure2(b)) are oblong and flat; their lengths are 3 to 4 cm longer than their widths.Several dark brown seeds (Figure1(c)), which are 4 to 5 mm in diameter, are contained within a pod and were matured from August -September in Okinawa, Japan.The seeds of D. illinoensis were collected on the campus of the University of the Ryukyus, Nishihara, Okinawa, Japan.The polysaccharide was prepared and purified as described in the Materials and Methods section.The purified polysaccharide was a colorless, fibrous powder, with a yield of 5.5% (w/w) based on the dried powder.Journal of Biomaterials and Nanobiotechnology

Figure 3 .
Figure 3. High-performance anion exchange chromatogram of hydrolysate of the polysaccharide isolated from D. illinoensis.

( 4 .
77 ppm) are involved in the galactomannan molecule.From the proportion of anomeric signals, ratio of D-gal to D-man, 1.0:0.82,was observed, which was in agreement with that chemical analysis results (Figure 3).A signal at H-5 of β-D-mannpyranose without substituting side chain (3.57ppm) was very small, but H-5' (3.76 ppm) with a side chain was very large suggesting that most of the β-D-mannopyranose residues substituted with α-D-galactopyranosyl side chains.The signals at H-6 of α-D-galactopyranose and β-D-mannopyranose were assigned at 3.77 and 3.76 ppm [28] [29], respectively.The ring proton signals (H-2 to H-5) Journal of Biomaterials and Nanobiotechnology

Figure 9 .
Figure 9. Mass spectra patterns (a) and corresponding structure (b) of partially methylated alditol acetates of the galactomannan.*Number in Figure 8.

13 C-
NMR analysis agreed with the results from methylation analysis.The corresponding signals for 1,6-linkage was assigned at 5.09 ppm and 101.07 ppm.

Figure 10 .
Figure 10.Chemical structure of the galactomannan isolated from D. illinoensis.

Table 1 .
Chemical shifts of the 1 H-and13C-NMR spectra of the galactomannan from seeds of D. illinoensis.

Table 2 .
Methylation analysis of the galactomannan isolated from D. illinoensis.