Non-enzymatic approaches to depolymerize polysaccharides into oligosaccharides for polysaccharides fingerprinting

Plant polysaccharides are the most abundant biomacromolecules found in nature and frequently used in foods. Despite this, detailed characterization of their structures remains challenging. A method often used to characterize and recognize polysaccharides in full detail relies on the enzymatic digestion of polysaccharides to structure-informative oligosaccharides (enzymatic fingerprinting), prior to full analysis of these oligomers with LC and MS approaches. However, the enzyme-polysaccharide specificity hampers the use of this method as universal polysaccharide depolymerization approach for polysaccharides fingerprinting. In this thesis, various chemical-induced polysaccharide depolymerization approaches were investigated to reach a generic fingerprinting of polysaccharides.

A polysaccharide depolymerization method based on TEMPO-oxidation and partial acid-hydrolysis was investigated for the fingerprinting of arabinoxylans (AXs), the main cereal hemicellulose components. TEMPO:NaClO2:NaOCl oxidation of AXs selectively oxidized the arabinose side chains of AXs into arabinuronic acid, resulting in an arabinuronoxylan with a xylan structure having a substitution pattern mostly resembling the parental AX. Subsequently, three structurally different AXs were TEMPO-oxidized, partially acid-hydrolysed and reduced, releasing arabinurono-xylo-oligomer alditols (AUXOS-A). Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) with a stationary phase of porous-graphitized carbon (PGC) allowed the separation and identification of various isomeric AUXOS-A. AX-specific UHPLC-PGC-MS profiles of AUXOS-A were obtained, allowing to distinguish different AXs. Additionally, tandem MS analysis of individual AUXOS-A enabled tentative and conclusive characterization of their structures, which allowed us to substantiate the main structural differences among the AXs investigated.

Periodate oxidation of plant polysaccharides with and without subsequent autoclave (AC) thermal treatment was a second approach investigated to reach a generic fingerprinting of plant polysaccharides. After periodate oxidation and AC treatment, all investigated plant polysaccharides, except xyloglucan, and mixes thereof released oligosaccharides. These oligosaccharides had highly complex structures, comprising intact sugar units, and oxidized sugars in the form of dialdehydes, hemialdals, and remnants of oxidized sugars. This high structural complexity resulted in clusters of oxidized oligosaccharides that were polysaccharide structure-dependent, giving unique ESI-Ion trap-MS and MALDI-TOF MS oligosaccharide profiles per polysaccharide. These findings allowed us to distinguish individual plant polysaccharides, within and between polysaccharide classes, and identify the polysaccharide classes present in a polysaccharide mix by their oligosaccharides fingerprint.

Thus, partial acid-hydrolysis of TEMPO-oxidized AXs, and thermal treatment of periodate-oxidized plant polysaccharides have potential to more generically recognize AXs and plant polysaccharides, respectively, by oligosaccharides fingerprinting. This can be of high interest for the food industry to study the carbohydrate fraction of a food product.