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Structure and Function Relationships of Galactosylated Oligosaccharides and Their Anti-adhesive Effects Against Enterotoxigenic Escherichia coli K88
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- Author / Creator
- Yan, Yalu
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β-Galactosidase is used in the production of galacto-oligosaccharides (GOS), lactosucrose, and other oligosaccharides. GOS prevented enteropathogenic Escherichia coli adherence to human epithelial cells, and the anti-adhesive effects of GOS depended on their chemical structures, such as linkage type and degree of polymerization. This research aimed to investigate the relationship between the structures of galactosylated oligosaccharides produced by β-galactosidase and their anti-adhesive effects against enterotoxigenic Escherichia coli (ETEC). ETEC K88 causes diarrhea in weaned piglets, resulting an economic loss in swine industries. ETEC adheres to intestinal mucosa by fimbriae and releases toxins. This thesis investigated the effects of galactosylated chitosan-oligosaccharides (Gal-COS) against ETEC K88 adherence to porcine erythrocytes. Gal-COS were synthesized through transgalactosylation of β-galactosidase with lactose and COS. Fractions of Gal-COS were obtained through cation exchange and size exclusion chromatography. Fractions of acetylated Gal-COS were obtained through chemical acetylation followed by size exclusion fractionation. Hemagglutination assay and ELISA assay were used to measure the anti-adhesive ability of oligosaccharides against ETEC K88. Gal-COS F2 containing the largest oligosaccharides composed of 8 or more monosaccharides had the highest anti-adhesion activity with the minimum inhibotry concentration of 0.22g/L, followed by F3 and F4. Adhesion of ETEC K88 to procine erythrocytes was significantly decreased by Gal-COS F2 to more than 50% when compared to ETEC control. Acetylation of Gal-COS decreased their ability against ETEC K88. The compositions of active oligosaccharides were determined by LC-MS. The structure and functional relationship of oligosaccharides binding to ETEC K88 fimbriae were also studied. β- Or α-galacto-oligosaccharides (βGOS and αGOS), β-galactosylated melibiose, α-galactosylated lactose and β-/ɑ-galactosylated COS were produced with β-/ α-galactosidase using lactose or melibiose as galactosyl donor, respectively. Fructosylated COS were produced by levansucrase with sucrose and COS. Anti-adhesive effect of oligosaccharides against ETEC K88 adhesion was measured with a hemagglutination assay, ELISA with ETEC cells, or with purified K88 fimbriae. High molecular weight β-GalCOS, β galactosylated melibiose, and βGOS had strong anti-adhesion activity. Other oligosaccharides had weak or no anti-adhesive effects. The ability of oligosaccharides to prevent binding of ETEC K88 or purified K88 fimbriae decreased with decreasing molecular weight. β-Linked galactosyl moieties were necessary for preventing ETEC K88 attachment to porcine erythrocytes, the addition of N-glucosamine moieties improved the anti-adhesive ability against ETEC K88. In conclusion, galactosylation of COS produces oligosaccharides which reduce ETEC K88 adhesion; moreover resulting oligosaccharides match the composition of human milk oligosaccharides, which prevent adhesion of multiple pathogens.
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- Subjects / Keywords
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- Graduation date
- Spring 2018
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.