Antibacterial activity of chemically defined chitosans: influence of molecular weight, degree of acetylation and test organism.

Chitosans, polysaccharides obtained from the exoskeleton of crustaceans, have been shown to exert antibacterial activity in vitro and their use as a food preservative is of growing interest. However, beyond a consensus that chitosan appears to disrupt the bacterial cell membrane, published data are inconsistent on the chemical characteristics that confer the antibacterial activity of chitosan. While most authors agree that the net charge density of the polymer (reflected in the fraction of positively charged amino groups at the C-2 position of the glucosamine unit) is an important factor in antibacterial activity, conflicting data have been reported on the effect of molecular weight and on the susceptibility among different bacterial species to chitosan. Therefore, we prepared batches of water-soluble hydrochloride salts of chitosans with weight average molecular weights (M(w)) of 2-224kDa and degree of acetylation of 0.16 and 0.48. Their antibacterial activity was evaluated using tube inhibition assays and membrane integrity assays (N-Phenyl-1-naphthylamine fluorescence and potassium release) against Bacillus cereus, Escherichia coli, Salmonella Typhimurium and three lipopolysaccharide mutants of E. coli and S. Typhimurium. Chitosans with lower degree of acetylation (F(A)=0.16) were more active than the more acetylated chitosans (F(A)=0.48). No trends in antibacterial action related to increasing or decreasing M(w) were observed although one of the chitosans (M(w) 28.4kDa, F(A)=0.16) was more active than the other chitosans, inhibiting growth and permeabilizing the membrane of all the test strains included. The test strains varied in their susceptibility to the different chitosans with wild type S. Typhimurium more resistant than the wild type E. coli. Salmonellae lipopolysaccharide mutants were more susceptible than the matched wild type strain. Our results show that the chitosan preparation details are critically important in identifying the antibacterial features that target different test organisms.

Preparation and characterisation of fluorescent chitosans using 9-anthraldehyde as fluorophore.

Chitosans with chemical composition ranging from a fraction of N-acetylated units (F(A)) of 0.01 to 0.61 were used to prepare fluorescence labelled chitosans by reductive amination with 9-anthraldehyde. Fluorescent chitosans with a low theoretical degree of substitution (DS, 0.001-1%) were prepared, and the actual DS of the products were determined by UV and (1)H NMR spectroscopy. The fluorescence excitation and emission spectra of the chitosan with F(A) of 0.09 and DS 1% showed an excitation maximum at 254 nm and an emission maximum at 413 nm. The intrinsic viscosities ([eta]) of the fluorescent chitosans were compared to those of the original chitosans, showing that the derivatisation procedure lead only to a negligible decrease in [eta]. The conformation of these fluorescent chitosans with very low DS-values is not altered and they can conveniently be directly quantified by UV or fluorescence spectroscopy.

Screening of chitosans and conditions for bacterial flocculation.

Chitosans with different chemical compositions and molecular weights have been evaluated as flocculants of Escherichia coli suspensions. The flocculation performance of chitosans at different conditions (pH, ionic strength) was followed by residual turbidity measurements. For precise comparison, the chitosan concentrations corresponding to 75% flocculated bacteria (x(75)) were calculated from a mathematical function fitted to the measured data. At all conditions, an increase in the fraction of acetylated units (F(A)) resulted in lower x(75) and thereby better flocculation efficiency. Especially the most acetylated chitosans (F(A) 0.49 and F(A) 0.62) were excellent flocculants. An increase in F(A) from 0.002 to 0.6 caused a 10-fold reduction in necessary concentrations, at both pH 5 and 6.8. pH was a rather insignificant factor in the range 4-7.4, further pH increase led to either increase of necessary doses at low F(A) or sudden ceasing of flocculation at high F(A). The chitosans flocculated in a broad range of molecular weights, although an increase in molecular weight was a favorable factor. Increase in ionic strength caused a severalfold reduction in x(75) for all chitosans and considerable broadening of flocculation intervals.

Electrophoretic light scattering studies of chitosans with different degrees of N-acetylation.

Electrostatic properties of three chitosans with fractions of N-acetylated units (F(A)) of 0.01, 0.13 and 0.49 were examined by electrophoretic light-scattering technique (ELS) and (1)H NMR spectroscopy. From the dependency of mean electrophoretic mobilities on pH, the pK(a) values were calculated. Despite their large differences in chemical composition, all chitosans had similar pK(a) values of 6.5-6.6. All chitosans also showed the same polyelectrolyte behavior when apparent pK(a) values were calculated according to Katchalsky and plotted as a function of the degree of ionization alpha. The intrinsic pK(a) values (pK(0)) extrapolated to zero charge were about 9. The results derived from an independent (1)H NMR study of the same chitosan samples showed no effect of F(A) on titration behavior of chitosan, confirming the results obtained by ELS.