Antibacterial Activity of Neat Chitosan Powder and Flakes

Abstract

This study investigates the antibacterial activity of neat chitosan powder and flakes against three different bacterial species, Escherichia coli, Listeria innocua and Staphylococcus aureus, which are frequent causes of food spoilage. The effect of chitosan concentration and purity, as well as the influence of temperature, ionic strength (salt) and impact of a solid physical support in the medium are examined. Results show that the antibacterial activity of neat chitosan: (i) requires partial solubilisation; (ii) can be promoted by environmental factors such as adequate temperature range, ionic strength and the presence of a solid physical support that may facilitate the attachment of bacteria; (iii) depends on bacterial species, with a sensitivity order of E. coli > L. innocua > S. aureus; and (iv) increases with chitosan concentration, up to a critical point above which this effect decreases. The latter may be due to remaining proteins in chitosan acting as nutrients for bacteria therefore limiting its antibacterial activity. These results on the direct use of chitosan powder and flakes as potential antimicrobial agents for food protection at pH values lower than the chitosan pK_a (6.2-6.7) are promising.

Keywords: E. coli; L. innocua; S. aureus; antibacterial activity; chitosan.

Figure 1

Cumulative weight (Mw)/number (Mn) fraction as a function of molar mass for chitosan powder and flakes.

Figure 2

Chitosan in flakes and powder form (A,C) and their particle size distribution (B,D). The symbols a, b and SSA (in B and D) represent the average particle size, thickness and the specific surface area, respectively.

Figure 3

Effect of chitosan concentration in PBS on the number of viable survivors. C_c is the critical concentration above which the AB activity of chitosan decreases. Dashed lines represent the reduction in bacterial concentration after deproteinization. Samples are P-95-57 (powder) and F-90-207 (flakes). The number of viable organisms was the same after 18 and 48 h incubation on the agar plates, suggesting that recovery from sub-lethal injury had not taken place. For each chitosan grade, means that do not share a letter are significantly different with a confidence level of 95% by Tukey Pairwise Comparisons.

Figure 4

FTIR spectra: Peaks at 1345, 1420, 1560, 1655 and 3290 cm⁻¹ confirm the solubility of chitosan powder and flakes in the suspensions during the AB tests.

Figure 5

Recovery of viable bacteria after exposure of chitosan and filtrate from chitosan suspensions to E. coli. The number of viable organisms was the same after 18 and 48 h incubation on the agar plates, suggesting that recovery from sub-lethal injury had not taken place. Means that do not share a letter are significantly different with a confidence level of 95% by Tukey pairwise comparisons.

Figure 6

Recovery of viable bacteria after exposure of CaCO₃ and CS solution (F-90-207) to E. coli. The number of viable organisms was the same after 18 and 48 h incubation on the agar plates, suggesting that recovery from sub-lethal injury had not taken place. Means that do not share a letter are significantly different with a confidence level of 95% by Tukey pairwise comparisons.

Figure 7

Identification of proteins before and after deproteinization of chitosan. Samples are P-95-57 (powder) and F-90-207 (flakes).

Figure 8

Effect of temperature on the antibacterial activity of chitosan (number of viable survivors). Bars with different letter are significantly different (p < 0.05). Samples are F-90-207 (flakes) and P-95-57 (powder). The number of viable organisms was the same after 18 and 48 h incubation on the agar plates, suggesting that recovery from sub-lethal injury had not taken place. Means that do not share a letter are significantly different with a confidence level of 95% by Tukey pairwise comparisons.

Figure 9

Effect of salt concentration and ionic strength (I) on the antibacterial activity of chitosan. Bars with different letters are significantly different (p < 0.05). Sample is F-90-207 (flakes). The number of viable organisms was the same after 18 and 48 h incubation on the agar plates, suggesting that recovery from sub-lethal injury had not taken place. Means that do not share a letter are significantly different with a confidence level of 95% by Tukey pairwise comparisons.

Figure 10

Morphology of intact: (a) E. coli; (b) L. innocua; (c) S. aureus cells. Images were kindly provided by Mounia Arkoun from the Chemical Engineering Department, Polytechnique Montréal.