Caracterização e atividade antibiofilme de polissacarídeos sulfatados extraídos da alga marinha verde Enteromorpha sp. cultivada em laboratório

Abstract

Rampant harvesting of algae for extraction of its by products causes a huge environmental problem, one solution to lessening that impact would be to grow it. As green algae became an important source of bioactive compounds, among them the sulfated polysaccharides, the objective was to cultivate the green marine macroalgae Enteromorpha sp. in the laboratory, from the isolation of their spores obtained in nature, and extract, purify, characterize these macromolecules and evaluate their effect against bacterial biofilm formation. The algae were grown in Guillard f / 2 medium and the total sulfated polysaccharides (PST) were extracted by enzymatic digestion with papain and fractionated by ion exchange chromatography DEAE- Cellulose using a stepwise salt gradient with increasing concentrations NaCl (0.5 - 2.0 M). The most metachromatic major fraction was produced in a preparative column, isolated from the others, dialysed and lyophilized. For a better characterization of the extracted polysaccharides, a high efficiency chromatography (HPLC) analysis was performed on a DEAE-Cellulose ion exchange column, eluted by linear NaCl gradient (0 - 3.0M). The PST fractions isolated in the chromatograms were submitted to agarose and polyacrylamide gel electrophoresis. The determination of total sugars, uronic acids and sulfate were by phenol-sulfuric acid, carbazole-sulfuric acid and barium gelatine, respectively. The monosaccharide composition was obtained by gas chromatography coupled to mass spectrometry (GC-MS). The activities of the sulfated polysaccharides on the planktonic growth of bacteria Staphylococcus aureus and Pseudomonas aeruginosa, as well as their action under the formation of biofilms, were analyzed. The algae grew 4 cm high after 70 days of cultivation. The yield of its PST corresponded to 10.8% of its biomass. In the fractionation of PST in DEAE-Cellulose ion exchange chromatography, stepwise, five fractions were obtained (0.7,0.9, 1.2, 1.6, 1.8 M) and the highest metachromatic activity evidenced in the F1.2 M fraction. In the HPLC system, three fractions were isolated from the PST and a single peak from the F1.2 M fraction. The electrophoretic analysis showed a similar mobility, homogenous bands and high molecular weight of samples. The PST and the fractions presented significant amounts of total sugar, varied uronic acid content and high sulfation. The polysaccharide presented monomers of rhamnose (41.49%), xylose (17.35%), glucose (9.51%) and galactose (5.50%), 3-methyl hexauronic acid (6.91%) and 3-methyl hexose (2.81%). In the bacterial assay, a significant decrease in planktonic growth was observed and in relation to biofilm formation the results showed a statistically significant reduction for all concentrations of polysaccharides tested in both bacteria. It is possible to cultivate the green marine macroalgae Enteromorpha sp., in the laboratory from its spores and using colorimetric, turbidimetric, electrophoretic and chromatographic methods, together with mass spectrometry it is possible to fractionate, purify and chemically analyze its structure, in this work it was also concluded. That these molecules are efficient in decreasing the formation of bacterial biofilms.