Enzymatic production and purification of prebiotic oligosaccharides by chromatography and membrane systems

Malwina Michalak

Research output: Book/ReportPh.D. thesisResearch

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Enzymatic treatment of biomass is an environmentally friendly method to obtain a range of products, such as biofuels, animal feed or food ingredients. The objective of this PhD study was to produce functional food ingredients – oligosaccharides and polysaccharides by means of enzymatic catalysis from biomass: casein glycomacropeptide (cGMP) and potato pulp. These saccharides should possess prebiotic properties, i.e., they should be non-digestable, selectively fermented and allow specific changes, both in the composition and/or activity of the gastrointestinal microbiota that confers benefits upon host well-being. Therefore the obtained compounds were incubated with single bacterial cultures to examine their prebiotic potential.
Different types of oligosaccharides were produced in the present study. The first group of compounds was human milk oligosaccharides (HMOs) containing sialic acid in their structures. These were synthesized from cGMP (a donor of sialic acid) and appropriate glycan in trans-glycosylation reaction catalysed by mutant sialidase from Trypanosama rangeli expressed in Pichia pastoris, Tr6. Production of the model HMO, sialyllactose was enlarged to 5 L scale.
The second type of sialylated oligosaccharides was obtained with the same donor of sialic acid – cGMP and different glycans with a new Trypanosoma rangeli transsialidase, Tr13. Well-documented prebiotics galactooligosaccharides (GOS), isomaltooligosaccharides (IMO) and lactulose, and three other compounds, i.e., melibiose, maltose, and fucose were sialylated with this enzyme resulting in creating novel human milk-like oligosaccharides. Both HMO and human milk- like oligosaccharides were purified by filtration and chromatography.
The last compounds produced during this study were GOS and some galactopolysaccharides. They were generated from isolated galactan and galactan contained in solubilised potato pulp polysaccharides (SPPP). An endo-1,4-ß-galactanase from Emericella nidulans was produced in a recombinant P. pastoris strain to catalyse hydrolysis of galactan and SPPP. This enzyme was purified, characterized and its crystal structure was determined. The products of enzymatic hydrolysis were fractionated according to their molecular weight using membrane filtration.
The results of this work pave the way for development of new functional food ingredients from industrial side-streams and generate value-added products with valuable biological properties and great market potential.
Original languageEnglish
PublisherTechnical University of Denmark, Department of Chemical and Biochemical Engineering
Number of pages125
Publication statusPublished - 2014


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