Preliminary Study On The Antibacterial Mechanism Of Chitosan From Red Swamp Crayfish

Objective:To master the method to abstract and characterize chitosan(CTS) from Red Swamp Crayfish,we used shells of Red Swamp Crayfish.To investigate the antibacterial activities of CTS from Red Swamp Crayfish and study the antibacterial mechanism,we carried out corresponding experiments.Methods:1) CTS from Red Swamp Crayfish,with particular deacetylated degree and molecular weight,was obtained from shells of Red Swamp Crayfish by the means such as take off calcium,protein and deacetylation et al..2) With Escherichia coli(E.coli) and Staphylococcus aureus(S.aureus) as bacteria tested,CTS from Red Swamp Crayfish was labelled by fluorescent reagent 5(6)-carboxy-tetramethylrhodamine-5-maleimide [5(6)-TAMRA-5-ML].The antibacterial activities of fluorescence labeled CTS,nonfluorescence labelled CTS and free fluorescent reagent were examined to determine the effects of fluorescent labelling on antibacterial activities.3) Bacteria tested were treated with CTS from Red Swamp Crayfish, leakage of cellular contents and synergetic bacteriolysis of sodium dodecyl sulfate(SDS) were determined.Morphological alterations of bacterial cells were examined by transmission electron microscopy (TEM).With fluorescence labeled CTS as fluorescent marker, aggregation position of CTS in bacterial cells was detected by laser scanning confocal microscope(LSCM) to investigate the antibacterial target sites of CTS from Red Swamp Crayfish.Results:1) The contents of CTS from Red Swamp Crayfish abstracted from shells of Red Swamp Crayfish were rather high.The average molecular weight of CTS from Red Swamp Crayfish was related with time for soaked in acid,and degree of deacetylation was related with time for recirculation in sodium ethoxide.2) The assay for antibacterial activities showed that compared with nonfluorescence labeled CTS,antibacterial activities of fluorescence labeled CTS degraded.When mixed with fluorescent reagent according to 9:1(v/v),CTS still had 50%antibacterial activities.3) It was confirmed that CTS from Red Swamp Crayfish could make cellular contents lost,and exert synergistic bacteriolysis effects with SDS.In electron micrographs of cells treated with CTS from Red Swamp Crayfish,the shape of cells became irregular and cellular kytoplasm became sparse.LSCM demenstrated that CTS from Red Swamp Crayfish could not noly stick to the bacterial surface but also enter bacterial cells.Conclusion:In this paper,CTS from Red Swamp Crayfish,with particular deacetylated degree and molecular weight,was abstracted by author. Compared with nonfluorescence labeled CTS,when mixed with fluorescent reagent according to 9:1(v/v),CTS had 50%antibacterial activities and possessed fluorescence visible,thus could be a kind of fluorescent marker to investigate the antibacterial target sites.The antibacterial mechanism of CTS from Red Swamp Crayfish included the damage of barrier function of bacterial surface followed by leakage of cellular contents and morphological alterations,and entering cells to interfere their metabolism.