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FAIR-CT97-3811 Development of xylo-oligosaccharides and xylitol for use in pharmaceutical and food industries (XYLOPHONE) |
Source: Consolidated Progress Report of 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).
SCIENTIFIC SYNTHESIS
Introduction
This project is investigating 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 minimising the environmental impact (avoiding acid, alkali or organosolv additions) to selectively release the xylan fraction, from the remaining plant biomass. The goal is to optimise an integrated process for biomass fractionation that can use various biomass wastes to produce food additives or ingredients (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. A model, in a user friendly format, will be developed for process assessment. 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 with pure and well characterised enzymes. For both pharmaceutical and food industrial applications, we foresee to focus the research on oligosaccharides from DP range of 2- 7. One of the partners will focus its research efforts to develop application technologies for some of the xylo-oligosaccharides as additive for healthier foods.
Alternatively, crude xylo-oligosaccharide syrups will be used as fermentation media for xylitol production. As wild yeast strains do not easily use oligosaccharides as C-source but only after hydrolysis to monosaccharides, the cloning of the p-xylanase, O-xylosidase and a-L- arabinofuranosidase genes from fungi into strains of Debaryomyces or Candida will be carried out in order to produce directly xylitol front xylo- or arabino-xylo-oligosaccharides. Another approach is engineering Saccharomyces cerevisiae with the above genes plus xylose reductase gene for xylitol production.
Raw materials: The raw materials to be used in this proposal obey to the two following items:
Brewer's spent grains represent an inexpensive and abundant disposable raw material that exists concentrated in nearly all Brewery's companies in the European Union. The malt and grain residue obtained after the liquefaction and saccharification of the starch known as 'spent grain' is essentially a hemicellulosic material. It contains about 34% of hemicellulose according to the literature. Eucalyptus globulus L. wood wastes represent an important and abundant raw material in Portugal and Spain (Galiza). In the former country, the production of Eucalyptus pulp paper by the mills is above 1 400 ton/year. The hemicellulose content is around 22% according to the literature. Corn cobs and wheat bran are low grade plant cell wall residues being the main residue from the milling industries. Both agricultural residues represent abundant residues inside the EU and the hemicellulose content is around 30% on a dry weight basis according to the literature.
Objectives
The specific aims of this contract are therefore:
Activities
The workplan of this project is divided into three main sections
A - Optimisation of biomass fractionation processes for oligosaccharides production
B - Bioconversion of xylo-oligosaccharides for xylitol production in a one-step process
C - Economic evaluation of the biomass fractionation and production of higher value- added products.
Each of these research areas are divided into various tasks/sub-tasks according to the main scientific aims of the research work. The results obtained during this reporting period are thus presented divided into the above sections.
Part A. During the reporting period, most of experimental work relating to this part of the programme was focused on production of xylo- oligosaccharides from the four materials under study. The fractionation, purification and characterisation of oligomers had already been achieved. Hence, the work concentrated on 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 were started but no significant data has been obtained so far.
Task A2 The main aim of this task is the optimisation of selective processes for lignocellulosic wastes fractionation using hydrothermal based treatments producing crude xylo-oligosaccharides syrups. A considerable amount of data concerning optimisation of hydrothermal treatments both under isothermal and non-isothermal conditions for xylo-oligosaccharide production was obtained for Eucalyptus wood, corn cobs and wheat bran. This part of work was a continuation of the work carried out during the first 12-months of project and it is close to the end. The experimental data already obtained is currently being used as input for the sub-task A2.3 concerning the analysis, simulation and evaluation of the overall hydrothermal treatment. During this period, both non-isothermal hydrothermolysis of Eucalyptus wood and isothermal hydrothermolysis of all four materials has been modelled.
Liquors containing high levels of xylo-oligosaccharides were extensively characterised by HPSEC, HPAEC and MALDI-TOF MS. The xylo-oligosaccharide composition of the plant material hydrolysates were thus obtained. Trials for the development of a pulping process for Eucalyptus wood were continued. The comparative use of organosolv pulping treatment for the different materials is currently being tested.
Task A3 During this period four chromatographic methods were developed for fractionation of xylo-oligosaccharide mixtures. These methods were used for separation and purification of arabinoxylo- and glucoronoxylo-oligomers containing hydrothermal hydrolysates of all four materials. The chromatographic methods were optimised for analytical purposes but are also being scaled-up for preparative purposes. A preparative separation of the hydrolysates from Eucalyptus and Brewery's spent grains has already been achieved. Several series of pentose oligomeric structures were obtained. These included neutral and acidic oligosaccharides of variable structural composition, depending on the raw material used. The purification and fine characterisation by NMR of some of the oligomers will follow. A start was made on the biological assays of selected detoxified oligosaccharide mixtures but no significant results have been obtained so far.
Part B In this area, research work was mainly focused on the evaluation of different 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 was started using wild yeast strains.
The genetic engineering of Debaryomyces hansenii has continued with the work focused on the transformation of D. hansenii with yeast shuttles. An alternative approach, to find a suitable yeast strain with an available transformation system for cloning of hydrolases-expressing genes, has also been taken. There is already a transformation system for D. hansenii based on an antibiotic selective marker but the studies are continuing to find a novel transformation system based on the ura gene.
The transformation of Debaryomyces hansenii was successful using a yeast shuttle plasmid, containing a geneticin resistance gene. However the resulting transformation system is unrealistic from an industrial point view since the use of an antibiotic as selective pressure to maintain the recombinant strain inside the industrial reactors is costly. Although, for the purpose of this project this is not an important problem. Research work is continuing on an alternative transformation system based on ura gene disruption. A genomic library for D. hansenii is almost finished and the disruption of ura gene cassette will follow. A start was made on cloning of hydrolase-expressing genes in the G418 geniticin-resistance gene of the transformed D. hansenii strain. In addition, an auxotrophic mutant of a xylose-assimilating Kluyveromyces marxianus has also been studied as an alternative biological system for xylo-oligosaccharide fermentation to xylitol. Several recombinant yeast strains carrying various hydrolase-expressing genes will be of more use than a single recombinant strain carrying a super-plasmid containing multi-expressed genes.
Task B2 The main objective of this sub-task was the removal of growth-inhibiting compounds from hydrothermal-treated syrups. Different detoxification strategies (liming, solvent extraction, charcoal adsorption and vacuum evaporation) were carried out in order to remove the biological growth inhibitors present in the hydrothermal hydrolysates from Eucalyptus, corn cobs and Brewery's spent grain. Substantial knowledge was attained in this sub-task and the results will be integrated in a patent application already in preparation. A start was made on production of xylitol by yeasts using raw and detoxified hydrolysates. Both individual and sequential fermentations (with cell recycle) were carried out to establish the ability of non-adapted or adapted yeasts for fermentation.
Part 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). A products market study was continued during this period and novel data was obtained.
As Task Al is almost complete there is now enough technical data available to use as an input during the construction and validation of the model. The market study was continued during this period and some conclusions were already obtained. The market for xylo-oligosaccharides does not exist by itself in Europe and will very likely a part of the oligosaccharides market, as well as a share of some other product market (thickeners, bulking agents, etc). So, the whole market for oligosaccharides (OS) in general has been analysed, paying particular attention to the aspects relevant to xylo-oligosaccharide (XOS) product. In Europe the consumption of OS is dominated by inulin derived OS (fructo-oligosaccharides). The role foreseen for XOS will be to penetrate the current market for OS, which will depend on the technical properties of the XOS. However, XOS have a decisive advantage over FOS: their resistance to hydrolysis under acid pH. This is particularly important for food applications under acidic conditions (marmalades, jams, yoghurts, etc).
On the other hand, xylitol has a well established market both in Europe and outside and the identified main threat to our biological process is the price. Xylitol has technological advantages over other available polyhydric alcohols but its price is considerably higher than others such as sorbitol. This fact restricts the present applications in the market that would be wider if the price could fall. The main challenge of this project is to find a novel biological process that could decrease the price of xylitol in the markets.
Discussion
Hydrothermolysis profiles showing maximal of xylo-oligosaccharide (XOS) concentration at different operational conditions (temperature, liquid/solid ratio) had already been obtained for all four materials which clearly demonstrates the project feasibility in terms of xylo-oligosaccharide production from the feedstock materials. The technical and economic evaluation of XOS production by this hydrothermolysis from the plant materials is still dependent on
The use of advanced analytical techniques has enabled information to be obtained concerning the type of xylo-oligosaccharides present in the hydrothermal hydrolysates.
The investigation of alternative means of detoxification of hydrolysates is almost complete. This will allow an improved xylitol fermentation process using yeast strains to be used.
A genetically engineering yeast strain capable of degrading oligosaccharides is expected to be available before the end of the project.
Future actions
Work concerning the relationship hydrothermal treatment and structural characterisation of the oligomers will be continued. Some oligomers present in various hydrolysates will be purified and characterised. The assays of bio-activity of oligosaccharide mixtures and a limited amount of pure oligosaccharides (strictly dependent of the preparative scale of this project) will be continued. The biological production of xylitol from hydrolysates by yeasts will be the subject of greater attention during and it is expected that the first xylitol fermentation using a recombinant yeast strain will be carried out. The economic analysis of hydrothermolysis and the economic evaluation of xylitol and xylo-oligosaccharide production will be completed. The work covering the technical assessment of some mixtures of oligosaccharides will be reduced since after the start of the project it was realised that the pilot plant size necessary for some of the assays would require efforts more suited to a demonstration project and is out of the scope of this project.
From now on research efforts will be directed to:
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