Ngehnyuiy, Ngo Hansel (2020) Characterisation of Bacterial Polysaccharides. Doctoral thesis, University of Huddersfield.

A number Gram-positive bacterial strains including Lactobacillus paracasei DG, Lactobacillus salivarius CCUG44481 and Bifidobacteria breve 7017 have been known to possess probiotic properties which has led to their increasing use in commercial probiotic products. A range of exopolysaccharides (EPS) and capsular polysaccharides (CPS) produced by these number of probiotic Gram-positive bacteria were studied in an attempt to determine if the EPS contributed to the biological activity of these cultures. EPSs were isolated, purified and their structures determined. Lactobacillus paracasei DG generated an EPS and a CPS, which were similar. The 1H-NMR spectrum indicated six anomeric signals confirming that the EPS contained six monosaccharides in their repeating units. The molecular weight determination indicated that a single polymer was produced with a narrow polydispersity. The combined GC-MS and HPAEC-PAD results confirmed that the structure was made up of L-rhamnose, D-galactose and D-N-acetylgalactosamine in a molar ratio 4:1:1. The nuclear magnetic resonance (NMR) as well as the linkage analysis indicated that the repeating unit contained 1,2-linked, 1,2,3-linked and 1,3-linked rhamnoses with a terminal hexose and an N-acetyl sugar. This analysis permitted the complete characterisation of the novel EPS structure. The results of this study allowed other research groups to determine the biological activity of the EPS with full knowledge that the immunotolerance observed was generated by a highly pure and well characterised polysaccharide.

The 1H-NMR spectrum of the EPS recovered from Lactobacillus salivarius CCUG44481 identified three anomeric signals and these matched those observed in a commercial dextran. The NMR spectra suggested that the CCUG44481 strain produced a highly branched dextran than the commercial dextran with at least a third of the hexoses being present in the branches. The HPAEC-PAD and GC-MS results all gave a single glucose peak. The linkage analysis showed that the backbone was a repeating unit of α-(1,6)-linked glucose having multiple branches of 1,3-linked glucoses. The NMR spectra and the wet chemical analysis are all consistent with the EPS isolated from L. salivarius CCUG44481 being a highly branched dextran.

Two EPSs (S1 and S2) and two CPSs (C1 and C2) were isolated from Bifidobacteria breve 7017. NMR and chemical analysis of the S1 and C1 fractions were bacterial glycogen with a medium molecular weight. For the S2 fraction, 1H-NMR spectrum indicated the presence of five anomeric peaks with one being identified as an impurity. The NMR spectra recorded for S2 changed with time and these corresponded to the slow loss of a ribitol like moiety on prolonged storage. The structure of the repeat unit of the S2 fraction was determined. The analysis of the C2 fraction identified two homoglycans: a β-D-(1,6)-galactofuranan and a β-D-(1,6)-glucan.

The characterisation of the EPS requires a time-consuming linkage analysis method. As part of this research an attempt was made to develop a more rapid method for linkage analysis using HPAEC-PAD. A range of variously substituted methyl glucose were separated: mono, di and tri-substituted glucoses could be separated. However, different isomers of either mono or di-substituted glucoses could not be separated. This then meant a HPAEC-PAD linkage method was unlikely to succeed.

FINAL THESIS - Ngehnyuiy.pdf - Accepted Version
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