Semana de Engenharia 2010 Guimarães, 11 a 15 de Outubro MICROWAVE-ASSISTED EXTRACTION OF SULFATED FUCANS FROM BROWN SEAWEED AND EVALUATION OF FUNGAL STRAINS FOR ENZYMES ACTIVE PRODUCTION TOWARD THIS CLASS OF POLYSACCHARIDE

Sulphated polysaccharides from brown seaweedss comprise a complex group of macromolecules with a wide range of important biological properties such as anticoagulant, antioxidant, antiproliferative, antitumoral, anticomplementary, anti-inflammatory, antiviral, antipeptic and antiadhesive activities. Fucoidan is one of the main sulphated fucan, mostly interesting for their biological activities specially the potential to inhibit HIV reverse transcriptase and the possible application as active compound in antiretroviral drugs. However, algae remain largely unexploited and seaweeds can be found in sufficient amount for the commercial exploitation. Usually, most of the processes to recover sulfated polysaccharides from natural sources consist in acid extractions during long reaction times. Specific enzymes able to degrade fucoidan matrix (fucoidanases) are important tools to establish structural characteristics and biological functions of this polysaccharide. Such enzymes, have been only isolated from marine organisms. Reports of fungal microorganisms with enzymatic activity over this sulfated-polysaccharide are scarce. The aims of the present work were: 1) to recovery of sulphated polysaccharides (fucoidan) by microwave-assisted extraction under different operational conditions and 2) the identification of fungal strains able to growth over fucoidan-based media and to produce active fucoidanases. MATERIALS AND METHODS Microwave-assisted extraction Fucus vesiculosus brown seaweed from North Portugal was used in the experiments. The extraction reactions were performed in a microwave digestion oven MDS-2000 (CEM Corporation), under different operational conditions: time (1, 11 and 31 min), pressure (30, 75 and 120 psi) and alga/water ratio (1/25, 3/25 and 5/25 g/ml), according to a 2 3 full factorial design. Total sugars content, monosaccharide composition and sulphated content were quantified Screening of fungal strains Aspergillus niger PSH, Penicillium purpurogenum GH2 and Mucor sp. 3P were the screened strains.. Fucoidan of Laminaria japonica and urea were used as carbon and nitrogen source. Radial growth rate (Ur) was kinetically monitored measuring colony diameters. Hyphal length (Lav) and diameter (Dh) were quantified by image analyses measurements. Fermentation for culture media selection Fermentation assays were performed with Aspergillus niger PSH. The tested media were Czapek Dox and Pontecorvo, supplemented with two carbon sources: a primary source composed by fucoidan Laminaria japonica (10 gL -1 ) and a secondary source composed by glucose, sucrose, lactose, fructose or sodium acetate, (5 gL -1 ); urea (5 gL -1 ) was used as nitrogen supply. The experiments were carried out in 100 mL Erlenmeyer flasks at 140 rpm, 30 °C, and 1x10 6 spores·ml -1 of conidial concentration. Biomass production, substrate consumption, extracellular and intracellular activity and protein content were evaluated. Kinetic parameters adjusted to Velhurst-Pearl and Luedeking y Piret models were estimated. RESULTS AND DISCUSSION Sulfated polysaccharide production The percentage of fucoidan recovered and the total sugar yield were highest when using 120 psi, 1 min, and 1 g alga/25 ml water (18.22% and 27.62%, respectively). However, similar values were found when using 30 psi, 31 min, and 1 g alga/25 ml water (15.61% and 24.52%, respectively), which can be associated to hydrolysis effect measured by the