Chadha, Marcus J. (2009) Novel Techniques for the Characterisation of Exopolysaccharides Secreted by Lactic Acid Bacteria. Doctoral thesis, University of Huddersfield.

This project investigated the structures and physical characteristics of exopolysaccharides
(EPSs) secreted by lactic acid bacteria.

The structure of a novel exopolysaccharide (EPS) produced by Lactobacillus acidophilus 5e2 has been characterised. Analysis of the anomeric region of the H-NMR showed that the repeating oligosaccharide contained seven monosaccharides. GC-MS showed the structure to consist of D-glucose, D-galactose and D-N-acetyl-glucosamine in a molar ratio of 3:3:1. The linkage analysis showed that there were two terminal, three di-linked and two tri-linked monosaccharides, and in collaboration with data generated from a series of D-NMR experiments, an overall structure was determined.

The weight-average molecular weight (Mw) of the EPS secreted by Lactobacillus acidophilus 5e2 when grown in skimmed milk was monitored during extended fermentation times. During the exponential growth phase, the increase in Mw closely followed the increase in yield of EPS. Under the fermentation conditions applied in this study, few if any new polysaccharide chains were formed during this growth phase despite a twenty five-fold increase in the cell count; almost the entire increase in yield can be accounted for by an increase in chain length.

These results suggested that synthesis of new EPS chains is switched off during the exponential and stationary phase of fermentation. The increase in
yield observed in this period is a consequence of the bacteria's ability to extend existing chains right up to the mid-stationary phase. These results raise questions about the factors that control EPS production and chain length.
Depolymerisation techniques have been shown to reduce the Mw of the polysaccharide in a controlled manner. The H-NMR results have shown that the physical methods, constant pressure and ultrasonic disruption break the EPS randomly through the repeating oligosaccharide unit; polydispersity data suggests that the breakages were occurring midchain.
A change to the peaks in the anomeric region of the H-NMR spectrum showed that depolymerisation, by acid hydrolysis, was chemically modifying the EPS structure. The approximate intrinsic viscosities of the EPS produced by Lactobacillus acidophilus 5e2 were determined to range between 0.6–2.0 dL g-1 for the Mw range of 1.59x105 – 4.78x105 g mol-1.

A capillary zone electrophoresis method was developed to determine the monosaccharide
composition of two EPS samples. The method successfully determined D-glucose and Dgalactose,
but a peak for D-N-acetyl-glucosamine was not seen. The method was sensitive compared to current techniques, but not as low as using a HP-AEC-PAD.

A novel method using LC-MS was developed for the linkage analysis of EPSs. Methylation, hydrolysis and reductive amination were used to derivatise the polysaccharide, and the fragmentation patterns were examined to determine the different linkage positions. Due to undesirable further fragmentation the method could not unequivocally differentiate between the different linkage positions, but the method was capable of resolving the monosaccharides residues with different linkage positions, at approximately the correct relative ratio.

Marcus_Chadha_-_PhD_Thesis_Final_Version.pdf - Accepted Version
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