Biochemically modified polysaccharides from leguminous plants with versatile properties for industrial applications

Galactomannans constitute one of the major families of natural polysaccharides. They can be obtained from the endosperm of the seed of some leguminous plants and are widely employed in a number of industrial fields as rheology modifiers. Each galactomannan has a unique mannose-to-galactose ratio (M/G), which strongly influences its physico-chemical properties, including solubility and viscosity. Usually, polysaccharides are characterized by nuclear magnetic resonance (NMR), whereas mass spectrometry (MS) has not seen a wide application in this field. In this thesis, a quantification method for sugar diastereomers in galactomannans was developed, exploiting the typical fragmentation pathways of mannose and galactose in tandem MS analyses. The tandem MS spectra of commercially available galactose and mannose display an accumulation of the same fragments for both monosaccharides, but significant differences in the intensities of the product ions. Mixtures with known concentrations of mannose and galactose have been analysed employing the same procedure in order to evaluate the applicability of this method for quantification aims. The relative intensities over the entire spectrum can be deconvoluted as a linear combination of the intensities of pure mannose and galactose spectra, leading to a reliable and reproducible quantification of these monosaccharides. After validation, the method has been applied to the quantification of chemical hydrolysis products of galactomannans from different leguminous plants, like guar (Cyamopsis tetragonolobus), sesbania (Sesbania bispinosa) and tara (Caesalpina spinosa), to evaluate specific susceptibility to different chemical hydrolysis conditions. Galactomannans are commonly employed not only in their native, but also in their chemically and/or biochemically modified form. In this thesis, gums from guar, sesbania and fenugreek (Trigonella foenum-graecum L.) have been oxidised by a laccase/TEMPO-mediated system. Chemo-enzymatic oxidation of galactomannans in aqueous solution caused a viscosity increase up to fifteen-fold, generating structured and stable hydrogels. Upon lyophilisation of these hydrogels, water-insoluble aerogels were generated, capable of uptaking aqueous solutions several times their own weight. The aerogels have been characterized by scanning electron microscopy, calorimetry and X-ray diffraction. Analyses by stability assays, electrospray ionisation MS, NMR and Fourier-transform infrared spectroscopy demonstrate that the chemo-enzymatic treatment leads to the formation of carbonyl and carboxyl groups from primary hydroxyls on the polymers and subsequent establishment of hemiacetal and ester bonds, cross-linking the gels. Fenugreek-based materials emerged to be more structured and stable compared to guar and sesbania aerogels, which is likely due to the higher amount of oxidable galactose units present in fenugreek gum (i.e., fenugreek has a M/G of 1, whereas guar and sesbania have a M/G between 1.3 and 1.6) and, therefore, to more extensive cross-linking of the resulting elastic gel. Active principles have been absorbed into guar, sesbania and fenugreek aerogels by incubating the materials in aqueous solutions of different actives (polymyxin B, nisin, enzymes). The aerogels were rinsed, blotted on filter paper and re-lyophilised, and the release of the active principles was tested in appropriate media. The release of polymyxin B was evaluated against six Gram-negative bacterial strains, whereas the release of nisin was tested against two Gram-positive bacterial strains. Protease and lipase release was evaluated in solution by monitoring the increase in protein concentration and enzymatic activity. The analyses performed during this project suggest that these aerogels represent versatile bio-compatible delivery systems based on renewable raw materials and could be employed for biomedical and industrial applications.