该文的研究目的用安石榴苷作为绿色还原剂制备壳聚糖/纳米银(CS/AgNPs)溶胶,并对CS/AgNPs表征及抑菌性能进行研究.结果表明:pH值为5,浓度为0.5%~1%的壳聚糖100 mL,硝酸银溶液浓度0.6 mol/L,加入质量浓度1%的安石榴苷溶液,25 ℃还原6 h可得粒径范围较窄,银粒子质量分数较高较的稳定纳米复合材料CS/AgNPs溶胶.经透射电镜和X-射线衍射仪分析溶胶中纳米银粒子粒径为8~11 nm,晶型为面心立方型.傅里叶红外光谱表明壳聚糖中-NH2与Ag+发生了螯合及CS/AgNPs中AgNPs的存在.CS/AgNPs溶胶对大肠杆菌和金黄色葡萄球菌作用24 h后的抑菌率为95.5%,97.6%,刃天青法检测对2种菌的最小抑菌浓度均为0.096 μL/mL.透射电镜扫描及细胞内容物渗漏实验表明, CS/AgNPs溶胶可以吸附菌体于细胞,破坏细胞膜,增加细胞的通透性,抑制菌体生长.安石榴苷原位还原制备的CS/AgNPs溶胶具有良好的抑菌性能,可为食品保鲜或医用材料制备提供理论支撑.%Chitosan is the only natural cation polysaccharides derived from shrimp and crab shell, which displays some bioactive functions and antibacterial properties with a narrow antimicrobial spectrum. Here, we investigated in situ synthesis of nano-silver particles to enhance the antibacterial properties of chitosan. The chitosan/nano-silver particles (CS/AgNPs) sol was prepared by reducing silver nitrate in situ synthesis within chitosan using punicalagin as a green reducing agent, and then its antibacterial properties and mechanism were investigated. The preparation of CS/AgNPs sol was optimized by single factor experiments. The effects of the preparation conditions pH, temperature and reaction time, with concentration of chitosan, silver nitrate and punicalagin solution were evaluated by characteristic ultraviolet-visible spectrophotometry of nano-silver particles. The existence AgNPs in CS/AgNPs sol was confirmed by UV-visible spectra, Turbiscan Lab, XRD diffraction patterns and TEM images. The optimal conditions to prepare CS/AgNPs sol were found as follows. Every 100 mL of chitosan at pH value 5 in concentration of 0.5% - 1% blended well with 250 mL of silver nitrate in concentration of 0.6 mol/L, and then chelated for 30 min at room temperature. The silver nitrate in this mixture was reduced by adding 800 μL of punicalagin solution in concentration of 1%, bathing at 25 and stirring for 6 h. Consequently, a kind of stable CS/AgNPs sol with high content of ℃AgNPs was produced. Transmission electron microscopy (TEM) images indicated that the AgNPs nanoparticles were between 8 and 11 nm in diameter dispersed uniformly in the sol. The X-ray diffraction (XRD) showed that AgNPs formed in a type of face-centered cubic structure reflected by the enhanced diffraction intensity of Ag. And the Fourier transform infrared spectroscopy (FTIR) showed that -NH2 groups of chitosan chelated with Ag+in the CS/AgNPs. We used methods of disc agar diffusion and liquid medium dilution to study the bacteriostasis. For disc agar diffusion, the experiment was modified that the filters would be replaced by the fabric, so as to improve the diffusion ability of chitosan in agar. The results indicated that the CS/AgNPs sol could inhibit the two typical species of bacteria (E.coli and S.aureus) which caused infection. The inhibition zone of the S.aureus was 5.5 mm, slightly higher than the inhibition zone of the E.coli, when soaked in the fabric. The inhibitory rate of CS/AgNPs sol on E. coli and S. aureus was 95.5% and 97.6% after a 24 h-incubation respectively. The antibacterial effect of CS/AgNPs sol was tested by the minimum inhibitory concentrations (MIC). The results showed that the MIC for both E.coli and S.aureus were 0.096 μL/mL detected by resazurin test for the CS/AgNPs sol taking punicalagin as the reducing agent, which was moderately better than that taking sodium borohydride as the reducing agent from which the MIC for both E.coli and S.aureus were 0.19 μL/mL by resazurin test. The bacteriostatic mechanism of this kind of composites was explored. The SEM scanning and cell contents leakage experiments of the cultures after a 24 h-incubation showed that the CS/AgNPs sol can adsorb to bacterial cells, disrupted the cell membrane, and increased the cell permeability and then inhibited the cell growth. The CS/AgNPs sol prepared by the green reducing reagent punicalagin showed a good performance on technical parameters and bacteriostatic. This article provides a theoretical basis for the application in the future, which could be used in food preservative or as the medical material.
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