Methodology for Structural Analysis of Polysaccharides

Polysaccharide molecules are constructed by long chains of monosaccharide units bound together via glycosidic linkages. Naturally occurring polysaccharides can be simply classified into four categories according to the source differences: plant polysaccharides, seaweed polysaccharides, animal polysaccharides and microbial polysaccharides (Table 1.1). Each category has its own specific structural features, e.g. most hemicelluloses as plant polysaccharides contain 6 monosaccharides (rhamnose, arabinose, galactose, glucose, xylose and mannose) while some derivatized monosaccharides, e.g. anhydrogalactose, can be found in some seaweeds polysaccharides. The molecular structure offers the most fundamental knowledge for understanding the functional, conformational and physiological properties of polysaccharides, which in turn facilitate their food and non-food applications. However, structural characterization of polysaccharides is a fairly challenging task due to the molecular complexity in terms of monosaccharide composition, glycosidic bonds (linkage patterns), degree of branching/branching position, α- or β-configurations, functional groups, molecular weight and molecular weight distribution.

Some polysaccharides can be collected directly from the ground endosperm of beans (guar gum) or husks (psyllium). Exudate gums such as gum arabic and gum ghatti can be picked up directly from the tree bark (Kang, Guo, Phillips, & Cui, 2014). However, most of the non-starch polysaccharides need to be extracted by water, mild base or acid solution (Guo et al., 2011, 2015). Assisted methods such as heating, microwave, and sonication were also extensively used to improve the extraction efficiency (Benko et al., 2007). Polysaccharides then can be recovered from the aqueous solution by either dialysis, ethanol precipitation, salt precipitation or directly freeze drying, accordingly.

As a fundamental characteristic, the molecular weight and molecular weight distribution of polysaccharides are important in relation to many physical properties. Average molecular weight and polydispersity index are required to quantify the molecular weight and its distribution of the polysaccharides.

Monosaccharide composition analysis is critical for understanding the structure of polysaccharides, as it could provide the first clue of the structural information. For example, polysaccharides containing arabinose and xylose are mostly assigned to arabinoxylan family; polysaccharides composed of galactose and mannose mostly belong to galactomannan group; pectic polysaccharides are mostly constructed by galacturonic acid and some neutral monosaccharides.

Polysaccharides are generally degraded into oligosaccharides prior to their structural characterization, while the degradation methods can be classified into two big categories, non-specific degradation and controlled specific degradation.

Methylation analysis, a powerful tool for primary structure characterization, has been used for decades and is still widely used nowadays (Chandra et al. in Carbohydrate Research 344(16):2188–2194, 2009; Pereira et al in Phytochemistry 71(17–18), 2132–2139 2010; Wu et al. in Food Hydrocolloids 23(6), 1535–1541 2009). As shown in Fig. 6.1, it includes four main steps: (1) methylation reaction using methyl iodide to convert all free hydroxyl groups of the polysaccharide molecules into methoxy groups; (2) acidic hydrolysis (usually by TFA) to convert the polymer into monomer; (3) reduction (using sodium borodeuteride) and acetylation (by acetic anhydride) to give volatile products: partially methylated alditol acetates (PMAA); (4) GC-MS analysis to identify and quantify the produced PMAAs (Dell in Meth Enzymol 193:647–660, 1990; Liu in Food carbohydrates: chemistry, physical properties, and applications. Taylor and Francis, Boca Raton, pp 309–355, 2005). The linkage patterns for each monomer and the molar ratios can be obtained using this method (Ciucanu & Kerek in Carbohydr Res 131(2):209–217, 1984).

NMR spectroscopy, as a powerful tool for carbohydrate structural analysis, could provide a complete picture of oligosaccharides structure and their behavior in solution. Structural features including linkage patterns, configuration (α- or β-), sequencing as well as the ratio of different sugar residues all could be obtained. In addition, some conformational properties (molecular dynamics) of polysaccharides also are obtainable by this technique.

Mass spectroscopy has been widely applied for carbohydrate analysis in terms of molecular mass, constituent monosaccharides, linkage types, sequencing of sugar residues, branching features, types of modifying groups, and the quantity.

As FTIR spectrometer can simultaneously collect high-resolution data over a wide spectral range in a short time, it has been extensively used for carbohydrate analysis (Guo et al in Carbohydr Polym 86(2):831–836, 2011; Guo et al in Food Hydrocolloids 44:320–327, 2015; Kang et al in Food Hydrocolloids 25(8):1984–1990, 2011; Singthong et al in Food Hydrocolloids 19(5):793–801, 2005). FTIR spectra in the wavenumber between 950 and 1200 cm⁻¹ are considered as the ‘fingerprint’ region for carbohydrates, which allows the identification of major chemical groups in polysaccharides as the position and intensity of the bands are specific for every polysaccharide. The other typical FT-IR spectra for polysaccharides are listed in Table 9.1

Weigh 10 mg polysaccharide sample, adding 1 mL 12 M sulphate acid and keep stirring at room temperature for 30 min until sample dissolved well. Dilute the solution accordingly, normally 10 times and waiting for test.

This book provides a comprehensive review of the state of the art on the structure analysis of complex polysaccharides. Unlike protein and nucleic acid, the structure investigation of polysaccharides can only be termed as structural characterization rather than molecular sequencing due to the complexity and irregularity of the molecular chain. The obtained molecular structure can only be referred as “the proposed structure”.