BioMatNet Item: FAIR-CT97-3811 - Development of xylo-oligosaccharides and xylitol for use in pharmaceutical and food industries (XYLOPHONE)

FAIR-CT97-3811
Development of xylo-oligosaccharides and xylitol for use in pharmaceutical and food industries (XYLOPHONE)

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Summary Information

	Proposal No:	FAIR-CT97-3811
	Date Prepared:	July 2001, September 1999
	Source:	Final Report Abstract and Scientific Synthesis Progress Report April 1999

Final Report Scientific Synthesis

Final Report Abstract

Source: Progress Report May 2000

Consortium: The project is co-ordinated by INETI, Department of Biotechnology, Lisboa (Portugal) in partnership with University of Vigo-Ourense, Department of Chemical Engineering, Ourense (Spain), National Technical University of Athens, Department of Chemical Engineering, Athens (Greece), Productos Aditivos, SA, Barcelona (Spain) and the Department of Food Science, The Agricultural University, Wageningen (The Netherlands).

Abstract

Objectives: The present R&D project has the general aims:

to optimise selective processes for lignocellulosic wastes fractionation using hydrothermal-based treatments producing crude xylo-oligosaccharides syrups
to purify different xylo-oligosaccharides as raw material for pharmaceutical and food applications
to resolve the structure of novel oligosaccharides with biological activity obtained from lignocellulosic wastes
to obtain a fast xylo-oligosaccharide degrading recombinant Debaryomyces hansenii
to optimise the fermentation of xylo-oligosaccharides-based and xylose-based media for xylitol production in batch reactors using wild and recombinant yeast strains
to establish the feasibility of novel industrial processes for oligosaccharide and xylitol production, covering technical and economic considerations.

Activities: During the reporting period, most of experimental work carried out under task A was focused on production of xylo-oligosaccharides from the four materials under study. The fractionation, purification and characterisation of oligomers was already attained and it was continued the work concerning the optimisation of operational conditions for xylan release during hydrothermal treatments. A first approach for the simulation and evaluation of the overall hydrothermal treatment was attempted using a chemical kinetic model. The biological screening tests has just started but no important data was obtained until this moment.

In task B research work was mainly focused on the evaluation of various strategies for detoxification the hydrothermal hydrolysates for yeast xylitol fermentation as well as to get clean hydrolysates for biological and technological assays. The biological production of xylitol using hydrothermal hydrolysates from Eucalyptus, corn cobs and spent grains started already using wild yeast strains. The engineering of Debaryomyces hansenii has continued and the work has been focused on the transformation of D. hansenii with yeast shuttles and alternatively, find out a suitable yeast strain exhibiting an already available transformation system for further hydrolases-expressing genes cloning. There is already a transformation system for D. hansenii based on an antibiotic selective marker but the studies are still continuing to find a novel transformation system based on ura gene.

For task C, a start was made in the identification of the main parameters (and variables) necessary to be included in the simulation model to assess the economic and engineering feasibility of a biomass sequential-extraction process (SEP). The products market study was continued during this period and novel data was obtained.

Progress: All the tasks and sub-tasks scheduled for this reporting year were started and are in progress, some of a them well advanced.

Achievements: The operational conditions to maximise xylo-oligosaccharides extraction from all four materials are almost concluded with success and significant progress has been made concerning both the detoxification of the xylo-oligosaccharide hydrolysates and fractionation, separation and purification of the xylo-oligosaccharide mixtures. It must be stressed that all of this research work has been carried out for four different plant materials, each one having different chemical behaviour. The data obtained so far are being subject to a Patent Application according to the rules of the EC.

Future actions: From now on research efforts will be directed to:

conclude the study of the optimal operational conditions for the obtaining different mixtures of soluble oligosaccharides from the feedstock materials.
continue the work on the utilisation of the solid residues which will be produced from the hydrothermal treatments in view of an integrated process of total upgrading of all fractions of the feedstock materials.
evaluate at least one model for the economic assessment of biomass fractionation as well as the evaluation of the overall hydrothermal process.
get recombinant yeast strains for testing on xylo-oligosaccharides-based medium under batch fermentation
obtain pure oligomers from wheat bran hydrothermal hydrolysates
carry out the bio-activity screening of detoxified mixtures of xylo-oligosaccharides
carry out fermentation trials for xylitol production using wild yeast strains with the four raw materials.
carry out fermentation trials of Bifidobacterium sp. grown on mixtures of xylo-oligosaccharides.

Progress Report April 1999

Introduction This project aims to investigate the processing of plant raw materials, such as agro-industrial, agricultural and forest wastes into higher added-value final or intermediate products by using physical, chemical and biotechnological processes. Hemicellulose-rich plant wastes, will be fractionated by physical treatments to selectively release the Dylan fraction, from the remaining plant biomass. The goal is to optimise an integrated process for biomass fractionation that can be used for different biomass wastes producing xylitol and xylo-oligosaccharides, the latter as intermediate products for pharmaceutical and food applications. The xylo-oligosaccharides will be extensively purified, structurally characterised and their biological activity as anti-inflammatory or anti-tumour agent will be assessed.

Xylo-oligosaccharide syrups will be obtained by optimising different hydrothermal treatments. depending of the raw material. The xylo-oligosaccharide mixtures will be separated and isolated by preparative size exclusion (HPAEC) chromatography. Structural analysis to identify the range of substituted oligosaccharides carrying a residual arabinose. acetyl or 4-0-methylglucuronic substituent will be subject of study digesting the oligosaccharides using well characterised enzymes.

For both pharmaceutical and food industrial applications, the research will focus on oligosaccharides from DP range of 2-7. Alternatively, crude xylo-oliigosaccharide syrups will be used as fermentation media for xylitol production. Wild yeast strains do not readily use oligosaccharides as C-source but only after hydrolysis to mono-saccharides. Hence, the cloning of the p-xylanase, p-xylosidase and -arabinofuranosidase genes from fungi into Debaryomyces hansenii or other Candida sp. will be carried out in order to produce xylitol from xylo- or arabino-xylo-oligosaccharides. Another approach is engineering Saccharomyces cerevisiae with the above genes plus the xylose reductase gene for xylitol production.

The agro-industrial and crop raw materials to be used are chosen on the basis that they are a surplus of EU agriculture or an agro-industrial waste and are xylan-rich. An inexpensive and abundant disposable raw material exists in the form of spent grains from brewing. These contain about 34% of hemicellulose. The wastes from wood of Eucalyptus globulus represent another abundant raw material in Portugal and Spain. In Portugal the production of Eucalyptus pulp is around 1400 ton/year. The hemicellulose content is around 22%. Corn cobs and wheat bran, low value plant cell wall residues from the milling industries, occur in abundance within the EU. The hemicellulose content of this material is around 30% on dry weight basis according to the literature.

Objectives This research project has the following general aims:

to optimise selective processes for lignocellulosic wastes fractionation using hydrothermal-based treatments producing crude xylo-oligosaccharides syrups
to purify various xylo-oligosaccharides as raw material for pharmaceutical and food applications
to resolve the structure of novel oligosaccharides with biological activity obtained from lignocellulosic wastes
to obtain a fast xylo-oligosaccharide degrading recombinant Debaryomyces hansenii
to optimise the fermentation of xylo-oligosaccharides-based and xylose-based media for xylitol production in batch reactors using wild and recombinant yeast strains
to establish the feasibility of novel industrial processes for oligosaccharide and xylitol production, covering both technical and economical considerations

These will be achieved through a workplan divided into three main sections:

optimisation of biomass fractionation processes for xylo-oligosaccharides production
bioconversion of xylo-oligosaccharides for xylitol production in one-step process
economic evaluation of the biomass fractionation and production of higher value-added products

Activities During the reporting period, most of experimental work was focused on the chemical characterisation of feedstock materials and selection of the optimal operating conditions for xylan release from these materials during hydrothermolysis producing crude xylo-oligosaccharides mixtures. Experimental work on resolving structures of oligosaccharides has started by identifying some specific xylo-oligosaccharides obtained from hydrothermolysis of Eucalyptus wood.

Further research work was focused on the transformation of Debaryomyces hansenii with yeast shuttles and alternatively, find out a suitable yeast strain exhibiting an already available transformation system for further cloning of hydrolase-expressing genes

A first approach was made in relation to the feasibility and economic assessment of biomass fractionation as well as for xylitol and xylo-oligosaccharide production. However, this task is in the initial phase of activity, therefore there are no significant results to date.

Progress All the tasks and sub-tasks scheduled for the first year were started and are continuing, some of them are well advanced.

The four feedstock materials have been extensively characterised resulting in a technical report Chemical characterisation of feedstock composition supplied to the EC. This chemical characterisation of the feedstocks, including an initial extraction of the xylans followed by enzymatic degradation, resulted in a detailed picture of the types of xylans present. Other activities completed included the establishment of the operating conditions for the reactor for production of xylo-oligosaccharides during hydrothermolysis of Eucalyptus wood, brewery spent grain and wheat bran. The further characterisation of crude oligosaccharide mixtures, identifying the initial oligomer structures obtained after the hydrothermal treatments, was undertaken using advanced analytical techniques such as MALDI-TOF mass spectrometry.

Arabinoxylans from Brewer's spent grain and wheat bran exhibited lower acetylation, as compared to Eucalyptus and corn cobs. The latter feedstock materials showed a higher uronic acids content compared to spent grain and wheat bran. The arabinose/xylose ratio measured after alkaline extraction of hemicellulose, allowed a preliminary classification of the materials in term of structural similarities into two groups.

Future activities Further research efforts will be as follows:

to continue the study of the optimal operational conditions for obtaining different mixtures of soluble oligosaccharides from the feedstock materials
to continue the work on the utilisation of the solid residues which are produced from the hydrothermal treatments in view of an integrated process of total upgrading of all fractions of the feedstock materials
to obtain an initial feasibility and economic assessment of biomass fractionation as well as the evaluation of the overall hydrothermal process
to get recombinant yeast strains for testing on xylo-oligosaccharides-based medium under batch fermentation.
to purify some oligomers present in the various hydrothermal hydrolysates. Limiting structures will be picked out, purified and characterised. In addition more detailed information about the different structures/oligomers present over a time course of hydrothermolysis to obtain information about the mechanism of hydrolysis under specific conditions
to start the bioactivity screening of xylo-oligosaccharides mixtures for further purification and to resolve the structure of the bioactive oligomers
to get information about hindering structures of oligomers in the raw C-medium, in order to facilitate choice of the best recombinant yeast suitable for the simultaneous saccharification and fermentation process
to assess the fermentation of xylo-ohgosaccharides-based and xylose-based hydrothermal media for xylitol production in batch reactors using wild yeast strain