Publikationsserver der RWTH Aachen University
Synthese von neuartigen Fructoseoligosacchariden mit nativer und immobilisierter Levansucrase aus Zymomonas mobilis
Abstract
dc:descriptionSugars, especially sucrose, were accused bad reputation as obesity and caries promoting thickener for a long time. Meanwhile their favourable physiological and technological properties were newly discovered. Special fructose-oligosaccharides (FOS), which are synthesised from sucrose, have been successfully applied as functional food ingredients, so called “prebiotic oligosaccharides”, in different foods. This soluble fibre are defined as indigestible food components, promoting growth and/or activity of healthful micro-organisms in the intestine. Due to increasing health-conscience of the population there is a growing demand on innovative products with new physiological an technological properties. In the present work the biotechnological production of novel oligosaccharides with prebiotic potential was investigated. For the enzymatic synthesis we used cell-associated levansucrase of two different strains of Z. mobilis. We got a suitable crude enzyme extract from the flocculent strain Z. mobilis ATCC after disruption by ultrasonic treatment and removal of disturbing activity of glucose-fructose-oxidoredcutase (GFOR) by SDS-treatment. The enzyme preparation had an oligosaccharide-forming activity of 4,5 mg 6-kestotriose·mg·h-1. Beside the flocculent mutant we investigated in contrary, the oligosaccharide-forming activity of a crude-enzyme-extract from a GFOR-free mutant (Z. mobilis ACM 3963) which had only an oligosaccharide-forming activity of 1,5 mg 6-kestotriose mgh-1. Levansucrase exhibits a levan-forming and a hydrolytic activity besides the wanted oligosaccharide-forming activity. Therefor a characterisation of the enzyme in regard to the modulation of the enzyme activity in the direction of the synthesis of oligosaccharides was necessary. As optimal reaction-conditions with highest oligosaccharide-forming activity, no levan-forming and minimised hydrolytic side-activity of levansucrase we found the synthesis with 2,5 M sucrose as substrate at 40°C and a pH-value of 6,5 (0,05 M ammonium-acetate-buffer). Furthermore, the high sucrose concentration enables an non-sterile and therefore simple processing. By batch synthesis a FOS-product with about 45% trisaccharides, 25% tetrasaccarides and 30% oligosaccharides with higher degree of polymerisation was obtained. To develop a continuous process, the immobilisation of the crude enzyme extract and the cells was investigated. Stability and immobilisation efficiency were highest in calcium-alginate-beads entrapped crude enzyme extract and cells. Matrix entrapped and native levansucrase showed similar spectra of FOS. For continuous synthesis of fructose-oligosaccharides, a packed bed reactor was shown to be the best reactor form to minimise negative effects of product-inhibition by glucose. Under these conditions, the substrate-conversion was 100%. During work, a scale up from 0,05 L to 1,9 L was successfully established. The system showed operation-stability of over 800 hours. 50 liters of FOS were synthesised with unchanged enzyme activity. Volumetric productivity was about 13,95 g FOS·L·h-1. Resulting crude product contained 424 mg FOS·mL-1. For the removal of the by-products glucose, fructose and sucrose column-chromatography and fermentative purification of FOS was investigated. By-products were eliminated by Ca-Lewatit-chromatography with a yield of 50%. Alternatively the continuous removal of disturbing sugars with H. polymorpha was established. The advantages of the fermentative removal compared to chromatography were a yield of FOS-recovery of 100% and the unchanged composition of FOS after fermentation. In contrast, chromatography resulted in unavoidable losses of FOS with low molecular weight and did not run continuously. Acid stability of newly synthesised FOS was similar to commercial FOS. The purified FOS were shown to posses a high acidic resistance, which was important for their potential use as prebiotic. During incubation under stomach-like conditions only 18% of FOS were hydrolysed indicating their acidic resistance during passage through the upper intestine. Ten FOS were isolated out of the product by a single purification step, using semi-preparative HPAEC. Five of this sugars were identified by comparison with standards and analysis of their monomeric composition. Beside sugars of the inuline-series, like 1-kestotriose and 1-1-kestotetraose, FOS with 2-6-glycosidic linkage, like 6-kestotriose, 6G-kestotriose and 1&6-kestotriose were identified. The result was supported by the co-chromatography of the synthesised FOS with standards of the levan and graminan-series, which demonstrate the existence of FOS up to an DP of 12. To evaluate the prebiotic potential, we investigated the ability of Bifidobacteria to metabolise the synthesised FOS. Batch fermentation without pH-regulation with B. adolescentis, B. infantis, B. pseudocatenulatum and B. breve show, that predominantly FOS with lower degree of polymerisation were metabolised. However, fermentation with pH-control show the FOS to be completely metabolised by Bifodobacteria and demonstrate their suitability as prebiotic oligosaccharides. Batch fermentation with another 32 isolated intestinal bacteria confirmed the suitability of the synthesised FOS as carbon source for the most important Bifidobacteria and Lactobacilli. However, of the 8 tested Clostridia-strains, 4 were able to grow with the synthesised FOS, but they also metabolised commercial available FOS.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2001
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- König, Simone Anna Elisabeth
- Contributors dc:contributor
-
- Hartmeier, Winfried
Subjects
dc:subject × 7Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger