Feasibility study for the production of novel galacto-oligosaccharides based on Bifidobacterial β-galactosidases

Ambrogi, Valentina

Feasibility study for the production of novel galacto-oligosaccharides based on Bifidobacterial β-galactosidases

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Date

2020-04

Authors

Ambrogi, Valentina

Publisher

University College Cork

Abstract

Galacto-oligosaccharides (GOS) are complex carbohydrates that are produced from lactose by means of an enzymatic reaction catalised by a β-galactosidase. Together with fructo-oligosaccharides (FOS) and inulin, GOS have been reported to promote growth of purported beneficial bacteria in the gut, in particular bifidobacteria and lactobacilli. Because of their beneficial properties GOS have been classified as a prebiotic, and details of GOS structure, production and beneficial activities are reviewed in Chapter I of this thesis, representing an overview of relevant scientific literature. Chapter II of this thesis describes a bioinformatic analysis aimed at identifying β-galactosidase-encoding genes belonging to infant-derived bifidobacteria: B. breve, B. longum subsp. longum, B. bifidum and B. longum subsp. infantis. The corresponding enzymes were then expressed and characterized for their ability to utilize a range of saccharidic substrates. In Chapter III seven of these previously (as described in Chapter II) identified β-galactosidases that were shown to be most active towards lactose, were expressed and purified, and then tested for their ability to synthetize GOS through transgalactosylation. In all seven cases this was shown to be successful, while the resulting products were further characterized revealing two distinct GOS chromatography profiles. In Chapter IV, various experimental approaches are described to optimize GOS synthesis employing four of the seven enzymes reported in Chapter III. The GOS preparation obtained for two of the β-galactosidases were shown to be suitable for further purification and compositional evaluation. The ability to support bifidobacterial growth was assessed for each of these (purified) novel GOS preparations, named here as GOS-A and GOS-E, and compared to the commercially available Vivinal GOS. In Chapter V one of the novel GOS preparations, GOS-E, was tested for its potential anti-pathogenic activity employing a cell tissue culture. A pilot experiment aimed at investigating the bifidogenic activity of GOS-E based on faecal fermentation was also carried out. The work described in this thesis represents novel information on bifidobacteria-derived β-galactosidases and their application for galacto-oligosaccharide synthesis. GOS-A and GOS-E represent two such novel GOS preparations, which could be produced and purified in sufficient quantities for compositional characterization and bacterial growth tests. Both novel substrates were shown to be different in composition to each other and to the commercially available Vivinal GOS, and shown to support growth of a selected number of human-derived bifidobacterial strains. Furder functional tests revealed that GOS-E elicits anti-adhesive activity against a particular Escherichia coli strain when employed in combination with the intestinal epithelial cell line C2BBe1. GOS-E and a commercial GOS were also assessed for their bifidogenic activity in a fecal fermentation model. However, this pilot experiment did not reveal a statistically significant bifidogenic effect by any of these GOS preparations, nor on the overall microbiota composition, showing that experimental adjustments will need to be made to this faecal fermentation set-up for future prebiotic activity assessment of this novel GOS preparation.

Keywords

Prebiotics , Bifidobacterium , Gut microbiota , Microbiome , Bifidogenic , Galacto-oligosaccharides , Infant , Oligosaccharides

Citation

Ambrogi, V. 2020. Feasibility study for the production of novel galacto-oligosaccharides based on Bifidobacterial β-galactosidases. PhD Thesis, University College Cork.