This study reports the antibacterial activity of an oligosaccharide, prepared by partial acid hydrolysis of a native Paecilomyces sp. exopolysaccharide, and of its aminoglycosylated derivative, prepared by reductive alkylation of the oligosaccharide, against E. coli and S. aureus.
Organism collectionPaecilomyces sp. was cultivated in potato dextrose agar. Stock cultures were maintained on the same medium and transferred to fresh medium at a four-weeks interval. A voucher specimen of the fungus is deposited in the fungi collection of the Departamento de Ciencias Básicas, Universidad del Bío-Bío, Chillán, Chile.
Purifi cation of the exopolysaccharideThe resulting culture fi ltrate was mixed with four volumes of absolute ethanol, stirred vigorously, and kept overnight al -10 °C. The precipitate was centrifuged at 1,006 x g for 15 min and the supernatant was discarded. After repeated precipitation steps, the resulting EPS was dialyzed at room temperature overnight in de-ionized water, lyophilized, and its weight determined.
Partial acid hydrolysis of the exopolysaccharideEPS (3 g) was heated for 1 h at 90 °C with 36 mL of 0.10 M HCl, the mixture was cooled and poured into 100 mL of acetone. The resulting precipitate was separated by centrifugation, washed three times with acetone, dissolved in water, and freeze-dried.
Gel permeation chromatographyAn aqueous solution of the partially hydrolyzed EPS (2 mg/mL) was chromatographed on a Sephadex G-75 (Sigma-Aldrich) column (100 mm x 13 mm) and eluted with 1% (v/v) acetic acid (pH 5.3) (Huber et al., 1984). The column was calibrated with 2 mL of Blue Dextran 2000 (4 mg/mL) and Dglucose (4 mg/mL). Elution was monitored spectrophotometrically at 480 nm with the phenol-sulfuric acid reagent for sugars (Chaplin, 1986) (Fig. 1).
Reductive alkylationPartially hydrolyzed EPS (0.4 g) was suspended in 20 mL of methanol/acetic acid (3:1, v/v), and 1.33 g D-(+)-glucosamine hydrochloride in 15 mL water and 1.0 g sodium cyanoborohydride were added. The mixture was stirred for 6 d at room temperature, fi ltered, and the solid was washed exhaustively with methanol and dried to give a white powder, soluble in water (62% yield).
MicroorganismsStandard strains of Escherichia coli (ATCC 31705) and Staphylococcus aureus (ATCC 6538) were used for determination of the antibacterial activity (Hu et al., 2007).
Antibacterial activitiesA series of tubes containing different concentrations of either the native Paecilomyces sp. EPS, or of the oligosaccharide and of its aminoglycosylated derivative were prepared. Each tube was inoculated with the microorganism and incubated at 37 °C for 18 h. The presence or absence of turbidity suggests the growth of microorganisms, which in turn indicates the bacterial sensitivity to the compounds tested. The lowest concentration that completely inhibited the bacterial growth was designated the minimum inhibitory concentration (MIC) (Hu et al., 2007).
Results and DiscussionThe EPS obtained from a submerged culture of Paecilomyces sp. by means of precipitation wit...