| Chitosan Oligosaccharides(COS), the physical, chemical and the enzyme hydrolyzed product of chitosan, is a low molecular weight of alkaline amino oligosaccharides that have better biocompatibility, degradability, biological nontoxic, antimicrobial and solubility due to their shorter chain lengths and free amino groups in D-glucosamine units. Besides, the research and development of COS was under the spotlight due to its authentic natural and safe character. However, it has several drawbacks for its utilization in food industry application, including defective antimicrobial activity under physiological conditions. The chemical modification of chitosan oligosaccharides is a widely used strategy that could overcome these limitations and improve the antimicrobial activity of its.In this study, three kinds of novel and efficient water-soluble chitosan oligosaccharides(COS) derivative, were prepared by using the selective partial alkylation. The derivative was characterized by UV-vis spectroscopy, FTIR, 1H NMR, 13 CNMR, TGA, and XRD techniques. The antibacterial activity of them against Staphylococcus aureus(ATCC 25923), Streptococcus pyogenes(ATCC 12344), Bacillus subtilis(ATCC 21332), Escherichia coli(ATCC 25922), and Salmonella typhimurium(CMCC 50013) were tested. The COS-O-MB, was selected to study the mechanism of action, which showed the strongest antibacterial activity. The major findings and results were listed as follows:A novel COS derivative, chitosan oligosaccharide-O-kojic acid derivative assigned as COS-O-KA, was prepared by four-step reaction: Step 1, synthesis of chlorokojic acid(yield: 96.3%). Step 2, synthesis of the schiff’s base of COS(yield: 90.2%). Step 3, the schiff’s base of COS-O-KA derivative was prepared through O-alkylation reaction between chlorokojic acid and the Schiff’s base of COS(yield: 66.3%). Step 4, synthesis of chitosan oligosaccharides-O-kojic acid derivative(COS-O-KA)(yield: 96.2%) in overall yields of 55.4%. The derivative was confirmed by UV-vis spectroscopy, FT-IR, 1HNMR, 13 CNMR, TGA, and XRD techniques, which showed that the O-alkylation reaction took place at the OH positions of COS, and the 5-hydroxypyranone group of Kojic acid was introduced to the chain of COS. The TGA and solubility results indicated that the thermal stability of COS-O-KA decreased compared with COS, which might be attributed to the disruption of crystalline structure, and the product exhibited an excellent solubility in organic solvents and distilled water. The antibacterial results indicated that the antibacterial activity of COS-O-KA was strengthened relative to COS(S.aureus: IC50=6.25 mg/m L, E.coli: IC50=7.29 mg/m L) with the increase of degree of substitution(DS) for above six bacteria. The derivative with the DS of 1.21 showed the best antibacterial activity among the COS and the other derivatives. The IC50 values of S.aureus and E.coli were 1.04 mg/m Land 1.23 mg/m L, respectively, which were 6 to 7 times of COS. Besides, COS-O-KA showed higher activity against Gram positive bacteria than against Gram-negative bacterium.In order to further improve the antibacterial activity of the derivatives, the chlorinated kojic acid-mannich bases(MB) which has strong antibacterial activity was introduced into the C-2 amino of COS molecular. Hence, an efficient procedure, Chitosan oligosaccharide-N-Kojic acid-Mannich bases derivative assigned as COS-N-MB, was prepared by three-step reaction: Step 1, synthesis of chlorokojic acid(yield: 96.3%). Step 2, synthesis of chlorokojic acid-Mannich bases(MB) by chlorokojic acid and N-methyl piperazine(yield: 93.8%). Step 3, COS-N-Kojic acid-Mannich bases derivative(COS-N-MB) was prepared through N-alkylation reaction between MB and COS(yield: 77.4%) in overall yields of 69.9%. X-ray crystal diffraction, thermal stability and solubility experiment results showed that the introduction of 5-hydroxypyranone group of Kojic acid and piperazine to the ring of COS, the crystalline of COS molecular was increased, the molecular crystal structure of derivative was became more neat and orderly, the thermal stability and solubility of derivative was also enhanced. The antibacterial results indicated that the antibacterial activity of COS-N-MB was strengthened relative to COS and COS-O-KA with the increase of DS for above six bacteria. The derivative with the DS of 1.62 showed the best antibacterial activity among the COS and the other derivatives. The IC50 values of S.aureus and E.coli were 0.12 mg/m Land 0.25 mg/m L, respectively. Besides, COS-N-MB showed higher activity against Gram positive bacteria than against Gram-negative bacterium.Another strong antibacterial active substance, MB was introduced into the hydroxy of COS, and the C-2 amino, which has antibacterial activity, was reserved. Hence, an efficient procedure to prepare chitosan oligosaccharide-O-Kojic acid-Mannich bases derivative assigned as COS-O-MB was prepared by five-step reaction. Step 1, synthesis of chlorokojic acid(yield: 96.3%). Step 2, synthesis of chlorokojic acid-Mannich bases(MB) by chlorokojic acid and N-methyl piperazine(yield: 93.8%). Step 3, synthesis of the schiff’s base of COS(yield: 90.2%); Step 4, prepared the schiff’s base of COS-O-Kojic acid-Mannich bases derivative(yield: 85.2%). Step 5, synthesis of chitosan oligosaccharides-O-Kojic acid-Mannich bases derivative(COS-O-MB)(yield: 90.1%) in overall yields of 62.5%. The physical properties of COS-O-MB showed that the crystalline of COS molecular was increased, the molecular crystal structure of derivative was became more neat and orderly than COS, the thermal stability and solubility of derivative was also enhanced. The antibacterial results indicated that the antibacterial activity of COS-O-MB was strengthened relative to COS, COS-O-KA, and COS-N-MB with the increase of DS for above six bacteria. The derivative with the DS of 1.71 showed the best antibacterial activity among the COS and the other derivatives. The IC50 values of S.aureus and E.coli were 0.02 mg/m Land 0.15 mg/m L, respectively. Besides, COS-O-MB showed higher activity against Gram positive bacteria than against Gram-negative bacterium. It was selected to study the structure-activity relationship, for it has potential application value in food preservation.The COS-O-MB derivative was selected to study the the structure-activity relationship for its strongest antibacterial activity, and the integrity of cell membrane, membrane permeability, outer membrane(OM) and inner membrane(IM) permeabilization, the interaction with metal cation, and the change of protein conformation of bacteria were examined. The integrity of the cell membrane could be destroyed by the C-2 amino of COS-O-MB derivative, and small ions such as potassium and phosphate tend to leach out followed by large molecules such as DNA, RNA, and other materials. The ion permeability of bacterial cell membrane was increased followed by intracellular electrolyte exosmosis and cytoplasmic β-galactosidase. The bacteria conductivity, which was treated with COS-O-MB derivative changed much faster than treated with COS and KA. At the same time, COS-O-MB had strongly effect on the configuration of protein. On the other hand, the 5-hydroxypyranone group and N-methyl piperazine were introduced into the molecular chain, which can form complexes with various metal ions, and may interaction with metal cation(Ca2+,Mg2+,Cu2+,Na+), which is necessary for cell normal functions, and change the conformation of proteins.Our study showed that all of thees three COS derivative had good inhibition effect on bacteria tested. Hence, they were improved the antibacterial activity and extent the application scope of COS, which will be of great academic significance and application value for the use of COS as food preservatives in the food industry, and development of environmentally friendly COS derivative... |
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