Antibacterial Activity Of Chitosan Microspheres In Dispersing State

Chitosan is a natural polysaccharide which is a partially N-deacetylated derivative of chitin. Due to the unique polycationic nature, chitosan and its derivatives have been proposed for various applications in biomedical, food, agriculture and especially applied as antimicrobial agent. Investigations about the antibacterial property of chitosan and its derivative prove that chitosan has a natural and broad spectrum of antimicrobial activities. There has been great improvement in the knowledge of the antibacterial mechanism, however, the certain has never been known yet. Additionally, since chitosan is insoluble in water, most of the researches about the antibacterial activity were focused on the solution. Many efforts to prepare functional derivatives by chemical modifications o increase the solubility in water have been reported. In this paper, preparation and physiochemical properties of chitosan icrospheres in solid dispersing state have been studied, in order to investigate the antibacterial activity of chitosan through interfacial effect.chitosan microspheres (CMs) are prepared by emulsification cross-linking method, which have better thermal stability and disperse well at 110 rpm in culture. The CMs are smooth-surface and spherical shape of diameter of about 124μm. FTIR spectrum of the CMs indicates the differences between samples with different degree of deacetylation(DD), and the oleoyl groups are introduced into the CMs as OCMs. The DD of the three samples are 97.3%,83.7%,62.6%, respectively. While the degree of substitution of OCMs are 5% and 10%. The protonation of constant pK a shows changes correlating to the different constructions, which will affect the antibacterial activity of the CMs.CMs show inhibitory effect against E. coli and S. aureus. The antibacterial activities increase with the increment of concentrations of the CMs, and decrease with the the increment of the pH value, as well as exert at neutral condition. Owing to the different strain of the tested bacterium, the antibacterial activities are different. CMs inhibit S. aureus by cooperative effects of protonation and hydrophobic interaction, when the latter plays a greater role. For the E. coli, the inhibitory effect is mostly depended on the free amido on the chitosan molecule, when the hydrophobic interaction plays an important effect but not the primary.The effect of Magnesium on the stability of the bacterial cell wall indicates that the combination of the divalent cations on the cell wall, which are essential to the stability of the wall, influences the antibacterial activity of chitosan. Under acidic condition, the protonation of the surfaces makes CMs compete with the divalent cations to combine with the anion existing on the bacterial surface. At neutral pH, for gram-positive bacterium, the hydrophobic parts change the conformation of the peptidoglycan layer and cause it destabilized by chelating the divalent cations. In the case of gram-negative bacterium, the free amido on the surface of CMs could chelate the divalent cations directly, and result in the destabilization of the outer membrane. The corruption of the cell wall after the interfacial effect between CMs and the bacterium, which causes the phospholipids exposed, is the first step the antibacterial activity.The effect on the membrane proteins shows that CMs could change the construction of the cell wall as well as membrane even, and alter the conformation of the protein existing on the wall and membrane. The scan electronic microscope (SEM) photographs exhibit the CMs combine with the bacterium and inhibit their growth through interfacial interaction on the surface. The photos also offer direct evidence of the depletion of the cell and the leakage of the intracellular substances. The permeability evaluation illuminates CMs increase the bacterial membrane permeability and weaken the protection of the intracellular substances correspondingly. The disruption strengthened with the increase of the hydrophobic effect. The experiment of lecithin influence indicates that phospholipids are the combining spot on the membrane, where CMs interact with the E.coli and S. aureus. The interaction alters the membrane permeability and makes the membrane corrupted, which leads to the death of the bacterium consequently.