载银氧化壳聚糖的制备与抑菌性能研究

doi:10.13304/j.nykjdb.2022.0769

摘要/Abstract

摘要：

为制备一种高效抑菌剂，以壳聚糖（chitosan，CS）为原料，采用漆酶/2，2，6，6-四甲基哌啶-1-氧自由基（2，2，6，6-tetramethylpiperidine-1-oxyl，TEMPO）体系制备6-羧基壳聚糖（6-carboxyl chitosan，C-COS），然后与硝酸银反应制备载银氧化壳聚糖（silver-carried oxidized chitosan，C-COS-Ag），对其抑菌性能进行研究。结果表明，与CS相比，C-COS-Ag的抑菌效果明显提高，其对大肠杆菌和金黄色葡萄球菌的抑菌圈直径分别为（22.75±1.50）和（13.75±2.50） mm，最小抑菌质量浓度均为6.10 μg·mL^-1。细菌生长曲线试验证实，C-COS-Ag能明显降低大肠杆菌和金黄色葡萄球菌的生长速率；细胞内容物渗透试验表明，C-COS-Ag能够破坏细菌的细胞膜和细胞壁形态，导致核酸、蛋白质等大分子物质渗出，碱性磷酸酶活性上升，β-半乳糖苷酶活性下降，半乳糖合成途径受阻，细菌正常代谢受到影响。以上研究结果为C-COS-Ag的应用提供了理论依据。

关键词: 壳聚糖, 硝酸银, 抑菌性, 大肠杆菌, 金黄色葡萄球菌

Abstract:

In order to prepare a highly effective antibacterial agent， chitosan（CS） was used as raw material to prepare 6-carboxyl chitosan（C-COS） by laccase /2，2，6，6-tetramethylpiperidine-1-oxyl radical （TEMPO） system， and then the silver-carried oxidized chitosan （C-COS-Ag） was prepared by reaction with silver nitrate. The antibacterial activity of C-COS-Ag was studied. The results showed that the antibacterial effect of C-COS-Ag was significantly improved compared with CS. Its inhibitory zone diameters against Escherichia coli and Staphylococcus aureus were （22.75±1.50） and （13.75±2.50） mm， respectively， with a minimum inhibitory concentration of 6.10 μg·mL^-1。The bacterial growth curve test confirmed that C-COS-Ag could significantly reduce the growth rate of Escherichia coli and Staphylococcus aureus； the penetration test of cell contents showed that C-COS-Ag could destroy the cell membrane and cell wall morphology of bacteria， resulting in the exudation of nucleic acids， proteins and other macromolecules， the increase of alkaline phosphatase activity， the decrease of β-galactosidase activity， the obstruction of galactose synthesis pathway， and the normal metabolism of bacteria were affected. Above results provided a theoretical basis for the application of C-COS-Ag.

Key words: chitosan, silver nitrate, antiacterstasis, Escherichia coil, Staphylococcus aureus

中图分类号:

TS201.3

纪蕾, 刘天红, 王颖, 李晓, 李红艳, 孙元芹, 姜晓东. 载银氧化壳聚糖的制备与抑菌性能研究[J]. 中国农业科技导报, 2024, 26(3): 214-222.

Lei JI, Tianhong LIU, Ying WANG, Xiao LI, Hongyan LI, Yuanqin SUN, Xiaodong JIANG. Research on Antibacterial Activity of Silver-Carried Oxidized Chitosan[J]. Journal of Agricultural Science and Technology, 2024, 26(3): 214-222.

图/表 12

图1 红外光谱

Fig. 1 FT-IR spectra

图2 不同样品的超微结构比较A： CS； B： CS； C： C-COS； D： C-COS；E： C-COS-Ag； F： C-COS-Ag

Fig. 2 Ultrastructural comparison of different samples

表1 CS和C-COS-Ag的抑菌效果

Table 1 Antimicrobial effect of CS and C-COS-Ag

样品

Sample

大肠杆菌

Escherichia coli

金黄色葡萄球菌

Staphylococcus aureus

抑菌圈

Bacteriostatic circle/mm

敏感性

Sensibility

最小抑菌浓度MIC/（μg·mL^-1）

抑菌圈 Bacteriostatic circle/mm

敏感性

Sensibility

图3 施加C-COS-Ag后大肠杆菌的生长情况

Fig. 3 Growth of Escherichia coil after application of C-COS-Ag

图4 施加C-COS-Ag后金黄色葡萄球菌的生长情况

Fig. 4 Growth of Staphylococcus aureus after application of C-COS-Ag

图5 施加C-COS-Ag后大肠杆菌胞内的小分子物质泄漏（电导率）

Fig. 5 Leakage （conductivity） of small molecular substances in Escherichia coli cells after application of C-COS-Ag

图6 施加C-COS-Ag后金黄色葡萄球菌胞内的小分子物质的泄漏（电导率）

Fig. 6 Leakage （conductivity） of small molecular substances in Staphylococcus aureuscells after application of C-COS-Ag

图7 施加C-COS-Ag后大肠杆菌胞内核酸类物质的泄漏

Fig. 7 Leakage of nucleic acid substances in Escherichia coli cells after application of C-COS-Ag

图8 施加C-COS-Ag后金黄色葡萄球菌胞内核酸物质的泄漏

Fig. 8 Leakage of nucleic acid substances in Staphylococcus aureus cells after application of C-COS-Ag

图9 施加C-COS-Ag后大肠杆菌胞内蛋白质的泄漏

Fig. 9 Leakage of intracellular protein of Escherichia coli after application of C-COS-Ag

图10 施加C-COS-Ag后金黄色葡萄球菌胞内蛋白质的泄漏

Fig. 10 Leakage of intracellular protein of Staphylococcus aureus after application of C-COS-Ag

表2 施加C-COS-Ag后大肠杆菌和金黄色葡萄球菌胞内酶的活性

Table 2 Intracellular enzyme activity of Escherichia coli and Staphylococcus aureus after C-COS-Ag application

样品 Sample	大肠杆菌Escherichia coli		金黄色葡萄球菌Staphylococcus aureus
样品 Sample	碱性磷酸酶活性 AKP activity/（U·mg^-1 pro）	β-半乳糖苷酶活性 β-GAL activity/（OD₄₀₀）	碱性磷酸酶活性 AKP activity/（U·mg^-1 pro）	β-半乳糖苷酶活性 β-GAL activity/（OD₄₀₀）
对照 Control	0.070±0.015	0.033±0.001	0.076±0.035	0.028±0.001
CS 1×MIC	0.081±0.035	0.031±0.002	0.078±0.041	0.024±0.002
C-COS-Ag 1×MIC	0.130±0.071	0.030±0.001	0.087±0.084	0.023±0.001
C-COS-Ag 2×MIC	0.170±0.068	0.027±0.002	0.180±0.095	0.022±0.002

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