Ahmed, Hafiz Ibrahim (2019) Production, Isolation and Characterisation of Polysaccharides from Probiotic Bacteria. Doctoral thesis, University of Huddersfield.

The bacterial species L. fermentum, L. salivarius, and L. mucosae have received considerable attention due to their probiotic properties and exopolysaccharide (EPS) production. This research aims to isolate and purify the EPSs synthesised by these probiotic strains with a view to assessing their biological activity.

Under optimised conditions, the bacterial strain L. fermentum LF2 synthesises a mixture of polysaccharides, up to 2 gL-1, during growth. Analysis of the polysaccharide mixture by 1H-NMR spectroscopy and SEC-MALLS analysis indicated the presence of more than one polysaccharide with two populations of molecular masses: high molecular mass (HMw) of 1.23 x 106 gmol-1 and medium molecular mass (MMw) of 8.80 x 104 gmol-1. The first population containing the HMw polysaccharide was successfully isolated from the mixture by employing preparative size exclusion chromatography using a Sephacryl S-500 HR column. Monomer, linkage analysis and NMR spectroscopy revealed that the HMw EPS is a β-D-glucan with a trisaccharide-repeating unit having a terminal glucose, a 1,3-linked glucose in the main chain and a 1,2,3-linked glucose in the main chain bearing the side chain. Interpretation of ROESY and HMBC NMR spectra confirmed the HMw polysaccharide to possess a trisaccharide-repeating unit with the sequence: →3)-β-D-Glcp-(1→3)-[β-D-Glcp-(1→2)]-β-D-Glcp-(1→

The combined results of the 1H-NMR and SEC-MALLS analysis of the second population containing the MMw polysaccharides suggested the presence of two MMw polysaccharides accounting for more than 75 % of the EPS mixture by weight, with average weight molecular mass of 8.80 x 104 gmol-1. The structures of the two MMw polysaccharides were investigated by subjecting the MMw polysaccharide mixture to Smith degradation. The combined results of the NMR, monomer and linkage analysis of the Smith degraded products identified one of the MMw polysaccharides (MMwa) as having a repeating unit with the sequence: →3)-β-D-Glcp-(1→3)-β-D-Araf-(1→6)-β-D-Galf-(1→, whereas the other MMw polysaccharide (MMwb) was identified as having a repeating unit comprising of 1,6-linked-β-D- galactofuranosides. Analysis of the native EPS mixture revealed the structure of the repeating unit contained in MMwa to be →3)-β-D-Glcp-(1→3)-β-D-Galf-(1→6)-[α-D-Glcp-(1→2)]-β-D-Galf-(1→, and that of MMwb to be →6)-β-D-Galf-(1→6)-[α-D-Glcp-(1→2)]-β-D-Galf-(1→. The extent to which the 1,6- linked-β-D-galactofuranoside in MMwb is substituted at the 2-position was very dependent on fermentation conditions. Under optimized fermentation conditions, the 1,6-linked-β-D-galactofuranoside was found to greater than 80% be substituted at the 2-position.

The novel probiotic bacterial strain, L. mucosae VG1, isolated at University of Huddersfield, produces 62 mg/L of EPS, whose weight average molecular mass was determined to be 1.51 × 104 gmol-1. Monomer, linkage and absolute configuration analysis revealed that the EPS is a D-galactan having a non-reducing terminal galactopyranose, a 1,3-linked galactopyranose, a 1,6-linked galactopyranose, a 1,6-linked galactopyranose and a 1,3,6-linked galactopyranose. 1H-NMR spectra recorded for the native EPS revealed it contains a hexasaccharide repeating unit. Further analysis by 2D-NMR spectroscopy revealed the presence of three possible structures for the L. mucosae VG1-EPS. The three similar structures obtained were differentiated by subjecting the L. mucosae VG1-EPS to Smith degradation. Monomer, linkage and NMR analysis of the Smith degraded products eliminated all but one out of the three possible structures and therefore the structure of the L. mucosae VG1-EPS was found to have a repeating unit containing →6)-β-D-Galp-(1→6)-β-D-Galf-(1→3)-[α-D-Galp-(1→6)]-β-D-Galp-(1→6)-β-D- Galf-(1→3)-α-D-Galp(1→

The growth of the strain L. salivarius 702343 was monitored in Huddersfield Broth Media and was found to produce 34 mgL-1 of a capsular polysaccharides (CPSs) composed of repeating unit of a 1,4- linked D-glucose (bacterial glycogen). The low yield 19 mgL-1 of EPS synthesised by the strain was determined to contain more than one polysaccharide. Unfortunately, use of mild acid hydrolysis, preparative size exclusion chromatography (Sephacryl S500 HR column) and Smith degradation, could not separate the polysaccharides present in the EPS mixture. However, monomer analysis performed on the EPS mixture revealed that the repeating units are composed of glucose and galactose residues. Linkage analysis of the EPS mixture revealed the presence of a non-reducing terminal glucose, a non- reducing terminal galactose, a 1,2-linked hexopyranose, a 1,4-linked hexopyarose, a 1,5-linked hexofuranose, a 1,6-linked hexofuranose, 1,6-linked hexopyranose and a 1,2,6-linked hexofuranose.

FINAL THESIS - Ahmed, Hafiz.pdf - Accepted Version
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