We aim at creating a stable and quality driven research cluster in Biomedicine.

Project 5: Production, Purification, Structural Characterization and Evaluation of Properties of Bioactive Molecules Derived From Biomass. (EQ2, EQ9, QO1)

Description and objectives:

The objectives we are after in this project are:
1. Fractionation of raw materials through new methods (using catalysts derived from biomass, less aggressive with the environment and leading to higher yields on bioactive molecules, hemicellulosic polysaccharides, pectin and lignin.
2. Development of efficient and scalable methods of concentration, refining, purification and separation of the bioactive molecules.
3. Application of advanced methods for identification and quantification of unwanted by-products present in concentrates with biological activity.
4. Structural determination and evaluation of biological activities of selected molecules derived from hemicellulose and pectin (including the effects on intestinal flora, and the bacterial population dynamics in strictly anaerobic conditions, by FISH and / or RT-PCRq).
5. Interrelation between the structure of bioactive molecules and activity related to colon microbiota.
6. Development of improved methods for extraction and generation of natural antioxidants from biomass.
7. Application of advanced methods for identification and quantification of unwanted by-products present in purified fractions with high antioxidant capacity.
8. Identification of products with potential market value present in waste fractions generated in the purification of concentrates to get antioxidants.
9. Structure-function interrelationship in antioxidant activity (assessed in vitro for up to 6 different methods) of model compounds representative of isolated fractions from biomass.
10. Implementation of new techniques for quality control of prebiotic molecules or fractions, or antioxidants derived from biomass.

Development:
We are focused on three families of compounds:

1) hemicelluloses and their derivatives
2) Pectins and their derivatives
3) phenolic compounds.

Work has been carried out in these three lines.

1) Different types of industrial waste were considered as raw materials (rice hulls, barley hulls, pine sawdust) containing xylan-based hemicelluloses (for shells) or glucomannans (case of pine sawdust).

Experimental optimized protocols were followed that allowed the selective conversion of hemicellulosic polysaccharides into mixtures of low molecular weight polymers and/or oligosaccharides, with high yields.
tasks performed were:
- Characterization of structural components, including cellulose, hemicellulose and lignin.
- Optimization of reaction conditions to obtain maximum yields of oligosaccharides.
- Quantification of unwanted impurities in liquors
- Refining of hemicellulosic oligosaccharides, membrane and ion exchange, and material balance
- Processing of xylanase or mannanase refined to reduce the degree of polymerization,
- Isolation and structural analysis (by NMR and mass spectrometry, performed by QO1 group) of the most abundant hemicellulosic oligosaccharides,
- Determination of prebiotic potential of purified products, based on the generation of short-chain fatty acids and the selective growth of probiotic bacteria found in fecal cultures (based on RT-PCR-q)

2) For the second group of compounds, we started from waste materials from the food industries (solid residue obtained from pressed apple of juice processing and cider f actories, orange peels and sugar beet pulp).

We are working on:
- Analysis of pectin content, and fractions that might interfere with the processing of pectin (particularly, extracts and proteins)
- Application of energy efficient and low environmental impact processes for the selective separation of the pectic fraction,
- Enzymatic treatment to reduce the molecular size of pectin oligomers, and purification of mixtures
- Determination of molecular weights of the resulting products, and quantification of major relevant components
- Assessment of prebiotic potential, as probiotic bacteria substrates selected for the production of short chain fatty acids (SCFA), and its dynamics of growth (by FISH) in cultures containing pectin oligosaccharides as sole carbon source, from fecal inocula

3) The worked on phenolic compounds were done by processing brown urchins hydrolysates.

The following experimental work was performed:
- Implementation of fractionation technologies based and acid hydrolysis and autohydrolysis
- Refining of extracts by extractive technologies
- Fractionation of extracts purified by preparative column chromatography with automated fractions collection.
- Evaluation of performance of relevant isolated fractions, and determination of their phenolic content,
- Determination of the equivalent antioxidant capacity of special interest fractions
- Identification of model compounds by chromatographic and spectrometric analysis.