Study Of Fine Structures And Conformational Properties Of Gum Ghatti

Gum Ghatti is versatile translucent exudate (water-soluble gum) from Anogeissus latifolia, a tree that is native to India and Sri Lanka. The excellent emulsifying properties of gum ghatti offer great potential for its use in the food industry. However, information about the characterisation of its molecular structure, conformational properties in solution and the linkage between protein and polysaccharide is very limited. Since the structure information is very important to understand the structure-property relationship of gum ghatti, this research focused on fractionation, physical and chemical investigation of gum ghatti, and elucidation the detailed molecular structure of two fractions of gum ghatti, followed by study the structure of a glycoprotein and conformational analysis of gum ghatti fractions. Numerous techniques were involved, such as, methylation analysis-GC-MS, Maldi-TOF MS and 2D NMR spectroscopy including homonuclear 1H/1H correlations spectroscopy (COSY, TOCSY), heteronuclear 13C/1H multiple-quantum coherence spectroscopy (HMQC) and heteronuclear multiple bond correlation (HMBC) etc.The fractionations, chemical and physical characterization of processed gum Ghatti (Gatifolia SD) were first investigated, and the source of its surface activity was identified. Four fractions were separated using the gradual ethanol precipitation method. With the increase of alcohol concentration, chemical composition of the fractions exhibited a pattern: arabinose content increased, but the galactose, protein and uronic acid contents decreased in the order of: F50 (50% ethanol precipitate), F65 (65% ethanol precipitate), F80 (80% ethanol precipitate) and FS (the supernatant after 80% ethanol precipitation). Rheologically gum ghatti and its fractions exhibited Newtonian flow behaviour until gum concentrations reached to 20% (w/v), at which point gum ghatti showed some shear-thinning flow behavior. At the same shear rate and concentration, the apparent viscosities of these fractions decreased in the order of F50>F65>F80>FS. When compared at same concentration, the FS fraction had the highest surface activity relative to Gatifolia SD, the other fractions and even gum Arabic. Monosaccharide composition and preliminary structural analysis showed that the branching of the polymer increased in the order of F50, F65 and F80. The degree of branching levels, protein and uronic acid content could be responsible for the different solubility of the fractions in alcohol. However, the molecular structure of FS is significantly different from the other fractions. FT-IR spectroscopy revealed no esterified carboxyl group in gum ghatti.The major structure of gum ghatti as represented by F80 was characterized using methylation analysis, 1D and 2D NMR spectroscopy. The detailed structure information about the main polysaccharide of gum ghatti, including linkage patterns, configuration of and the sequences of each sugar unit was presented. Methylation and GC-MS analysis indicated that F80 was a highly branched polysaccharide; the terminal sugar residues were about 40.8% of the total sugars. The majority of the terminal units wereα-L-Araf, with small amounts of t-GlcpA, t-Arap, t-Rhap and t-Galp. About 14.2% of the total sugar residues were�'6)-β-D-Galp-(1�'branched at 3 and 4 positions. The linear portion of the arabinogalactan was composed of�'4)-GlcpA(1�',�'6)-Galp(1�'and�'2)-L-Araf-(1�'linkages. Based on the results from methylation analysis, 1D and 2D spectroscopy, a schemed structure was proposed and summarized as follows: The backbone is composed of 1,6-linked galactopyransyl (Galp) residues substituted at O-3 and O-4 position, which can be called“hairy region”, while the“smooth region”consists of�'2)-Araf-(1�'4)-GlcpA-(1�'6)-Galp-(1�'6)-Galp-(1�'. Side chains are terminated by arabinofuranosyl (Araf) and occasionally by rhamnopyranosyl (Rhap), arabinopyranosyl (Arap), galactopyranosyl (Galp) and glucuronopyranosyl (GlcpA) residues.The structure of a globular gum ghatti fraction (FS) with high surface activity was investigated using the same methods as F80. FS is proposed to be a highly branched polysaccharide with small amount of acetyl substitution. It consists of 1,6-linked galactopyransyl (galp) backbone, branched at O-3 and O-4 position by various of sugar residues, including�'6)-β-D-Galp-(1�',�'2)-α-L-Araf-(1�',�'5)-α-L-Araf-(1�', t-α-L-Araf and�'2,3,5)-α-L-Araf-(1�'. Moreover, the�'2,3,5)-α-L-Araf-(1�'residue on the side chain can have two side chains at O-2 and O-3 position, which gives the multi-branched structure of FS. Most of the side chains have terminal arabinofuranosyl (Araf), and are occasionally terminated by rhamnopyranosyl (Rhap), arabinopyranosyl (Arap), Galp and glucuronopyranosyl (GlcpA) residues. Some of the side chains are as long as five sugar units. Comparing with the main polysaccharide (F80) of gum ghatti, fraction FS is much more branched and has longer side chains. These structural features are consistent with the physical properties such as solubility for the FS fraction (FS is more soluble than F80). This multi-branched structure most likely accounts for the special physical properties including the excellent surface activities exhibited by FS.Since the protein content of gum ghatti was 4.34% (w/w), proteinaceous part was reported to play an important role to the emulsification properties. In this study, enzymatic degradation (α-L-arabinofuranosidase andβ-D-galactosidase) and chemical hydrolysis results indicated that most of the protein was covalently linked to the backbone of the polysaccharide. The amino acid composition and protease hydrolysis results indicated that the linkage between polysaccharide and protein were different from that of gum Arabic, which had a wattle blossom structure.The structure of gum ghatti glycoprotein was investigated by Maldi-TOF MS and 1D&2D NMR spectroscopy. Combined with the polysaccharide structure results, a structure model was proposed: it was a 1,6-linked galactose backbone with numerous of side chains, occasionally, xylose and mannose were also appeared on the backbone. Proteins or polypeptides attached directly to the core part of the polysaccharide. The linkage site of amino acids and polysaccharides was determined as N-linked (Hex)n-GlcNAc-Asn.Conformational properties of original gum ghatti and four fractions (F50, F65, F80 and FS) were investigated by dynamic and static light scattering techniques. For the known structure fractions (F80 and FS), two models were built to simulate structure properties, and the conformational parameters calculated after RMMC simulation were compared with experimental results. Gum ghatti molecules formed aggregates in pure water and 0.2M NaCl solution, with the apparent mean diameter of 360.52 and 217.43 nm, respectively. The aggregates were successfully eliminated by dissolving in 0.5M NaOH solution and the solution was stable in two days at room temperature without visible degradationHPSEC coupled with multiple detectors gave numerous properties including intrinsic viscosity [η], weight average molecular weight (Mw), number average molecular weight (Mn), Mark-houwink equation parameters (α) and logκ., Theαvalues indicated the random coil conformation of four fractions: F50 was much rigid than other fractions, while F65 exhibited loosely extended chain close to the spherical conformation, suggested a highly branched structure.The results from DLS and SLS in aggregate free (0.5M NaOH) solution further confirmed the conformation properties of gum ghatti obtained from the HPSEC. More parameters were derived including Rg, A2 andρ=Rg/Rh. The high positive value of A2 for each fraction suggested that 0.5 M NaOH solution was a good solvent for gum ghatti. The values of Rg decreased in the order of F80, F65, F50 and FS, which is consistent with molecular weight. The hydrodynamic radius Rh had the same trend which was obtained from CONTIN method. The less branched fraction F50 (ρ=1.694) demonstrated a random coil conformation in 0.5M NaOH solution, whereas F65 andF80 exhibited regular stars conformation due to the highly branched structure Combining with the computational method, the structure-functionality relationship for each fraction of gum ghatti was partially established. The 3D molecular model of gum ghatti fractions F80 and FS was created and the regular star chain conformation was first visualized. The conformational properties of F80 and FS calculated by RMMC simulation were in good agreement with experimental results.The structure-function relationship of gum ghatti was established in the present study. The highly branched molecular structure of gum ghatti, to some extend, led to the regular star and globular conformation of molecules. The structural characteristics and the relative compact conformation, as well as the conjugation between polysaccharide and protein contributed to the excellent emulsification and stabilization properties.The elucidation of the fine structure and conformation of gum ghatti can provide guidances for the study of other polysaccharide especially exudates gums. Also, the establishment of the structure-function relationship of gum ghatti will not only expand its applications of gum ghatti, but also provide the theoretical base for physicochemical modification of natural gums for the production of high quality natural emulsifiers and stabilizers.