Preparation And Antimicrobial Properties Of Chitosan Derivatives

Chitosan (CTS), an abundant polysaccharide in nature, has attracted much attention invarious fields in recent years because of its excellent physical and chemical properties. It hasbeen reported that chitosan can be applied as antimicrobial agent, however the application ofchitosan was limited due to its insolubility and low antimicrobial activities in nutralenvironment. Therefore, improvements on solubility and antimicrobial effectiveness ofchitosan were investigated in this study. To improve the solubility of chitosan,N-[(2-hydroxy-3-trimethylammonium) propyl] chloride chitosan (HTCC) was synthesizedusing2,3-epoxypropyl trimethyl ammonium chloride; for enhancing the antimicrobialeffectiveness of chitosan, chitosan metal complexes (CTS-Co, CTS-Ni) were prepared withcobalt and nicke. The chemical structures of these antimicrobial materials were characterizedby FT-IR,1H NMR and TG. The antimicrobial activities of these materials were examinedagainst bacteria (E.coli and S.aureus) and fungi (Cercospora arachidicola、Rhizoctoniasolani、Botryosphaeria berengriana and Colletotrichum coccodes) in different conditions.The antimicrobial mechanism was preliminarily studied by the analyses of cell membraneintegrity, the changes of electrophoreses bands of protein and DNA.(1) Antifungal properties of HTCC: HTCC was successfully synthesized with-NH2replaced by quaternary ammonium groups via analysing of FT-IR and1H NMR spectra.HTCC had better water-solubility than original CTS. The inhibition rates of HTCC againstC.arachidicola、 B.berengriana were74.8%and71.8%, respectively, when HTCCconcentration was2.0mg/mL; pH was an important factor influencing the antifungalproperties; the inhibition rates of HTCC against the test fungi, except B.berengriana,increased by adding metal ions, and the inhibition rate of100%was reached when addingMg2+and Ca2+. The leakage of protein and nucleic acid of the mycelia occurred withtreatment of HTCC. HTCC was able to destroy the integrity of the cell and make theintracellular constituents leakage, and HTCC was able to interact with protein and DNA.(2) Antifungal activities of CTS-Co and CTS-Ni: The-NH2and-OH groups of chitosanparticipated in the synthesis of new materials by the analyses of FTIR spectra, and thethermal stability of CTS-Co and CTS-Ni decreased slightly by TGA. The inhibition rates ofCTS-Co against C.arachidicola and C.coccodes were90.27%and89.02%when CTS-Coconcentrations were0.5mg/mL and1.0mg/mL, respectively; the inhibition rate of CTS-Ni against C.arachidicola was87.78%with CTS-Ni concentration of1.0mg/mL. Antifungalproperties of CTS-Co and CTS-Ni were stronger at lower pH. The integrity of the cell wasdestroyed with the treatment of CTS-Co, which resulted in the leakage of protein and nucleicacid of the mycelia. Furthermore, the results of SDS-PAGE and agarose gel electrophoresisindicated that the interaction of chitosan with the soluble proteins was one of the modes ofantifungal action.(3) Antibacterial activities of CTS-Co and CTS-Ni: The antibacterial activities and theantibacterial mode of action of CTS-Co and CTS-Ni against Gram-negative bacteriaEseherahia coli and Gram-positive bacteria Staphylococcus aureus were investigated.Results showed that CTS-Co and CTS-Ni had stronger antibacterial activities than originalchitosan. The minimum inhibitory concentrations (MICs) of CTS-Co and CTS-Ni rangedfrom0.06to0.1mg/mL against two tested bacteria. pH value affected remarkablyantibacterial activities of chitosan-metal complexes. Growth curves of E.coli and S.aureusshowed that the values of OD treated with samples (added at lag phase or exponential phase)were significantly lower compared to control group, and the lag periods of two bacteria wereextended obviously by adding samples at lag phase. The integrity of cell membranes wasdestroyed and the protein bands of treated sample appeared shallow. Electrophoretic mobilityshift assay of DNA revealed that CTS-Co possessed DNA binding capability.