作者：张瑶心, 刘煜东, 陈曦, 侯文倩, 连小琴, 王兰金, 张宇

作者单位：湖北第二师范学院化学与生命科学学院 植物抗癌活性物质提纯与应用湖北省重点实验室，湖北 武汉 430305

关键词：检测方法;抗菌性能;壳聚糖复合材料;MTT法

摘要：

为了建立评价复合材料抗菌性能的实验分析方法，检测壳聚糖/PVA/ZnO复合膜(CPZ膜)的抗菌活性。通过平板扩散法测量复合膜的抑菌环直径检测其抑菌活性，利用CCK-8法、MTT法和平板菌落计数法检测复合膜在摇瓶振荡条件下的杀菌率。结果表明平板扩散法的灵敏性和重复性均较差，无法反映抑菌活性。摇瓶振荡杀菌实验中，MTT法比CCK-8法和平板菌落计数法的灵敏性和准确性更高，CPZ膜对3种供试菌的杀菌活性均明显强于壳聚糖/PVA复合膜（CP膜）。该研究建立了利用MTT法检测抗菌材料在摇瓶振荡条件下的杀菌活性的方法，该方法简单、快速、准确，定量能力显著，可用于常规批量检测。

Comparative study on detection methods of antibacterial properties of chitosan/PVA/ZnO composite films
ZHANG Yaoxin, LIU Yudong, CHEN Xi, HOU Wenqian, LIAN Xiaoqin, WANG Lanjin, ZHANG Yu
Hubei Key Laboratory of Purification and Application of Plant Anti-Cancer Active Ingredients, School of Chemistry and Life Science, Hubei University of Education, Wuhan 430305, China
Abstract: An experimental analysis method was established to evaluate the antibacterial properties of composite materials, by detecting the antibacterial activity of the chitosan/PVA/ZnO composite film (CPZ film). The inhibitory activity of composite films was detected by measuring the diameters by plate diffusion method. The bactericidal activity of composite films was examined in flask shaking experiment by CCK-8 assay, MTT assay and plate colony-counting assay. The sensitivity and repeatability of the plate diffusion method were low, which cannot accurately represent the inhibition activity of composite films. In the flask shaking experiment, MTT assay is more sensitive and accurate than CCK-8 assay and plate colony counting assay. The MTT assay showed that the sterilization rates of all kinds of CPZ films were higher than that of CP film against all three kinds of bacteria, with significant difference. In this study, MTT method was established to detect the bactericidal activity of antibacterial materials under the condition of shaking flask oscillation. This method is not only simple, rapid and accurate, but also has quantitative ability, which can be applied to routine batch detection.
Keywords: detection method;antibacterial property;chitosan composites;MTT assay
2022, 48(4):53-60  收稿日期: 2021-02-03;收到修改稿日期: 2021-04-27
基金项目: 国家自然科学基金（51903077）；湖北省教育厅科研计划项目中青年人才项目（Q20173008）
作者简介: 张瑶心（1986-），女，湖北武汉市人，讲师，博士，研究方向为微生物生理生化
参考文献
[1] NOSHIRVANI N, GHANBARZADEH B, MOKARRAM R R, et al. Preparation and characterization of active emulsified films based on chitosan-carboxymethyl cellulose containing zinc oxide nano particles[J]. International Journal of Biological Macromolecules, 2017, 99: 530-538
[2] GUTHA Y, PATHAK J L, ZHANG W, et al. Antibacterial and wound healing properties of chitosan/poly (vinyl alcohol)/zinc oxide beads (CS/PVA/ZnO)[J]. International Journal of Biological Macromolecules, 2017, 103: 234-241
[3] YOUSSEF A M, ABOU-YOUSEF H, EL-SAYED S M, et al. Mechanical and antibacterial properties of novel high-performance chitosan/nanocomposite films[J]. International Journal of Biological Macromolecules, 2015, 76: 25-32
[4] PERELSHTEIN I, RUDERMAN E, PERKASN, et al. Chitosan and chitosan–ZnO-based complex nanoparticles: formation, characterization, and antibacterial activity[J]. Journal of Materials Chemistry B, 2013, 1(14): 1968-1976
[5] RAHMAN P M, MURALEEDARAN K, & MUJEEB V A. Applications of chitosan powder with in situ synthesized nano ZnO particles as an antimicrobial agent[J]. International Journal of Biological Macromolecules, 2015, 77: 266-272
[6] MALINI M, THIRUMAVALAVAN M, YANG W Y, et al. A versatile chitosan/ZnO nanocomposite with enhanced antimicrobial properties[J]. International Journal of Biological Macromolecules, 2015, 80: 121-129
[7] 宫雪, 魏银蕴, 李全芳, 等. 壳聚糖-PEG-Ag纳米复合材料的制备及抗菌性能[J]. 兰州大学学报: 自然科学版, 2017, 53(1): 139-142
[8] 段晓杰, 徐丽明, 王春仁. 纳米银医用抗菌敷料抗菌性能检测方法的研究[J]. 药物分析杂志, 2012, 32(1): 167-172
[9] 叶德萍, 周李华, 王智, 等. 抗菌金属材料抗菌性能检测方法研究[J]. 中国测试, 2016, 42(5): 66-69
[10] 朱兰芳, 陈鹏鹏, 毛昌杰. 银纳米线与氧化石墨烯复合物的制备及其抗菌性能研究[J]. 安徽大学学报(自然科学版), 2021, 45(1): 83-91
[11] 彭如群, 谢小保, 李金兰, 等. 抗菌塑料的应用及抗菌性能评价[J]. 塑料工业, 2011, 39(S2): 95-98
[12] 张丹, 南黎, 任玲, 等. CCK-8法和MTT法评价医用抗菌不锈钢细胞毒性的比较研究[J]. 口腔医学, 2012, 21(6): 5-8
[13] 唐静, 谈满良, 赵江林, 等. 多孔板-MTT比色法测定植物抗菌成分对细菌的抑制活性[J]. 天然产物研究与开发, 2008(6): 949-952
[14] 刘浩怀, 刘力飞, 卢嘉明, 等. 石墨烯及其衍生物的抗菌性研究进展[J]. 中国测试, 2015(3): 8-13
[15] LIU Y, HE L, MUSTAPHA A, et al. Antibacterial activities of zinc oxide nanoparticles against Escherichia coli O157: H7[J]. Journal of Applied Microbiology, 2009, 107(4): 1193-1201
[16] XIE Y, HE Y, IRWIN P L, et al. Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni[J]. Applied and Environmental Microbiology, 2011, 77(7): 2325-2331
[17] KUCERKA N, PAPP-SZABO E, NIEH M P, et al. Effect of cations on the structure of bilayers formed by lipopolysaccharides isolated from Pseudomonas aeruginosa PAO1[J]. The Journal of Physical Chemistry B, 2008, 112(27): 8057-8062
[18] NIES D H. Efflux-mediated heavy metal resistance in prokaryotes[J]. FEMS Microbiology Reviews, 2003, 27(2-3): 313-339
[19] STÄ HLER F N, ODENBREIT S, HASS R, et al. The novel Helicobacter pylori CznABC metal efflux pump is required for cadmium, zinc, and nickel resistance, urease modulation, and gastric colonization[J]. Infection and Immunity, 2006, 74(7): 3845-3852