生物合成纳米银/壳聚糖复合抑菌剂的制备与性能

吴承宗; 魏亚楠; 黄婷; 苗雨颖; 张译丹; 刘煜扬; 刘小莉; 赵彦翠; 乔洪金; 王磊

doi:10.13801/j.cnki.fhclxb.20230117.003

生物合成纳米银/壳聚糖复合抑菌剂的制备与性能

doi: 10.13801/j.cnki.fhclxb.20230117.003

鲁东大学　生命科学学院，烟台 264025

基金项目: 山东省自然科学基金项目(ZR2021MC102)；烟台市科技发展计划(2022XDRH010)

详细信息

通讯作者:
王磊，博士，教授，硕士生导师，研究方向为纳米技术在病害防控中的应用　E-mail: wanglei9909@163.com

吴承宗和魏亚楠为共同第一作者，对本文具有同等贡献
中图分类号: TB333
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- 被引次数: 0
出版历程
- 收稿日期: 2022-11-14
- 修回日期: 2022-12-12
- 录用日期: 2023-01-08
- 网络出版日期: 2023-01-18
- 刊出日期: 2023-10-15

Biosynthesis and properties of AgNPs/chitosan composite antibacterial agent

College of Life Sciences, Ludong University, Yantai 264025, China

Funds: Shandong Natural Science Foundation Project (ZR2021MC102); Science and Technology Development Plan of Yantai (2022XDRH010)

摘要

摘要: 为制备安全、稳定、高效的新型纳米银抑菌剂，本文分别以腊梅花瓣提取液和壳聚糖作为还原剂和稳定剂采用一步法生物合成纳米银/壳聚糖(AgNPs/CS)复合抑菌剂。通过单因素实验确定了最佳制备工艺，制备的产品利用紫外-可见吸收光谱、TEM、X 射线衍射等技术进行表征，并对其抑菌活性、抗耐药性、稳定性及生物安全性进行全面的评价。实验结果表明：AgNPs/CS在451 nm处有AgNPs的特征吸收峰，AgNPs呈球形，均匀分散，平均粒径为12.83 nm，为面心立方结构晶体。AgNPs/CS在离体和活体抑菌实验中对水产病原菌均表现出显著的抑菌活性，并表现出优良的抗耐药性、生物安全性和稳定性。因此，AgNPs/CS是一种理想的复合抑菌剂，在水产养殖领域中具有广阔的应用前景。
- 纳米银 /
- 壳聚糖 /
- 生物合成 /
- 抑菌活性 /
- 耐药性 /
- 稳定性
Abstract: In order to prepare a new type of safe, stable and efficient nano silver composite antibacterial agent, silver nanoparticles/chitosan (AgNPs/CS) was biosynthesized by one-step method using Chimonanthus praeco petal extract and chitosan as reducing agent and stabilizing agent, respectively. The optimal preparation procedure was determined by single factor experiment. The products were characterized by UV-vis absorption spectroscopy, TEM and X-ray diffraction, and their antibacterial activity, anti-drug resistance, stability as well as biological safety were comprehensively evaluated. The experimental results show that the AgNPs/CS has the characteristic absorption peak of AgNPs at 451 nm, and the evenly dispersed AgNPs are spherical with average diameter of 12.83 nm, and the crystals have a face-centered cubic structure. The AgNPs/CS show antibacterial activity against aquatic pathogens both in vitro and in vivo, and has excellent anti-drug resistance, biosafety and stability. Therefore, AgNPs/CS is an ideal composite antibacterial agent, which has a prospective application in aquaculture field.
- silver nanoparticles /
- chitosan /
- biosynthesis /
- antibacterial activity /
- drug resistance /
- stability
注释:

1) 吴承宗和魏亚楠为共同第一作者，对本文具有同等贡献

HTML全文

图 1 纳米银反应条件的优化

Figure 1. Optimization of AgNPs reaction conditions

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图 2 腊梅提取液、壳聚糖和纳米银/壳聚糖(AgNPs/CS)溶液的照片(a)及紫外吸收光谱图(b)

Figure 2. Photograph (a) and UV-vis absorption spectra (b) of Chimonanthus praecox extracts, chitosan and silver nanoparticles/chitosan (AgNPs/CS)

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图 3 AgNPs/CS的TEM图像(a)和粒径分布图(b)

Figure 3. TEM image (a) and particle size distribution (b) of AgNPs/CS

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图 4 AgNPs/CS的XRD图谱

Figure 4. XRD pattern of AgNPs/CS

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图 5 AgNPs/CS对6种水产病原菌的抑菌圈实验

Figure 5. Inhibition zone test of AgNPs/CS against 6 aquatic pathogens

1—AgNPs/CS; 2—AgNPs; 3—Chimonanthus praecox extracts; 4—Amp; 5—Normal saline

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图 6 AgNPs/CS不同浓度下灿烂弧菌攻毒处理菲律宾蛤仔的死亡率

Figure 6. Fatality rate of Ruditapes philippinarum challenged with Vibrio splendidus at different concentrations of AgNPs/CS

*—Significant difference; **—Highly significant difference

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图 7 AgNPs/CS 对灿烂弧菌生物膜的影响

Figure 7. Effect of the AgNPs/CS on biofilm formation by Vibrio splendidus

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图 8 不同存放时间 AgNPs/CS (a)和AgNPs (b)紫外吸收光谱图

Figure 8. UV-vis absorption spectra of AgNPs/CS (a) and AgNPs (b) at different storage time

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图 9 不同存放时间AgNPs/CS (a)和AgNPs (b)的Zeta电位图

Figure 9. Zeta potential of the biosynthesized AgNPs/CS (a) and AgNPs (b) at different storage time

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表 1 AgNPs/CS浓度对体外细胞相对增值率和细胞毒性评级的影响

Concentration(μg/mL)	OD value	RGR(%)	cytotoxicity
0	0.896±0.126	100.00	0
0.25	0.847±0.085	94.534	1
0.5	0.832±0.073	92.818	1
1.0	0.768±0.094	85.707	1
5.0	0.639±0.082	71.317	2
10.0	0.568±0.065	63.393	2
20.0	0.492±0.071	54.911	2
40.0	0.061±0.041	6.885	4
80.0	0.046±0.023	5.154	4

下载: 导出CSV

表 1 对6种病原菌的抑菌圈直径(n=3)

Table 1. Diameter of inhibition zone against 6 pathogens (n=3)

Strain	Inhibition zone diameter/mm
Strain	AgNPs/CS	AgNPs	Ampicillin (Amp)
Aeromonas punctata	19.24±0.47	16.73±0.53	16.25±0.86
Vibrio alginolyticus	23.39±1.03	21.07±0.19	15.72±0.28
Vibrio parahaemolyticus	24.52±0.76	23.35±1.08	23.17±0.10
Vibrio anguillarum	22.01±0.37	19.93±0.11	15.35±0.28
Vibrio harveyi	18.11±0.25	17.98±0.66	9.03±0.34
Vibrio splendidus	26.59±0.65	22.93±2.26	21.20±1.47

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表 2 AgNPs对灿烂弧菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC) (n=3)

Table 2. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests of AgNPs against Vibrio splendidus (n=3)

Number	Concentration/(μg·mL⁻¹)	AgNPs/CS		Concentration/(μg·mL⁻¹)	AgNPs
Number	Concentration/(μg·mL⁻¹)	24 h	48 h	Concentration/(μg·mL⁻¹)	24 h	48 h
1	112.50	−	−	33.33	−	−
2	56.30	−	−	16.67	−	−
3	28.10	−	−	8.33	−	−
4	14.10	−	−	4.17	−	−
5	7.03	−	−	2.08	−	−
6	3.51	−	−	1.04	−	−
7	1.75	−	−	0.52	−	+
8	0.87	−	−	0.26	+	+
9	0.43	−	+	0.13	+	+
10	0.21	+	+	0.07	+	+

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表 3 AgNPs/CS对多次传代灿烂弧菌MIC的测试(n=3)

Table 3. MIC tests of AgNPs/CS against multigenerational Vibrio splendidus (n=3)

Passage number	MIC/(μg·mL⁻¹)
Passage number	AgNPs/CS	Amp
1	0.43	9.76
2	0.43	19.53
3	0.43	19.53
4	0.43	39.06
5	0.43	78.13
6	0.87	78.13
7	0.87	312.5
8	0.87	312.5
9	0.87	312.5
10	0.87	625
11	0.87	625
12	0.87	625
13	0.87	625
14	0.87	1250
15	0.87	1250
16	0.87	1250
17	0.87	1250
18	0.87	1250
19	0.87	1250
20	0.87	1250

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表 4 AgNPs/CS浓度对体外细胞相对增值率(RGR)和细胞毒性评级的影响

Table 4. Effect of AgNPs/CS concentration on relative growth rate (RGR) and cytotoxicity rating of cells in vitro

Concentration/(μg·mL⁻¹)	Absorption value	RGR/%	Cytotoxicity
0	0.896±0.126	100.00	0
0.25	0.847±0.085	94.534	1
0.5	0.832±0.073	92.818	1
1.0	0.768±0.094	85.707	1
5.0	0.639±0.082	71.317	2
10.0	0.568±0.065	63.393	2
20.0	0.492±0.071	54.911	2
40.0	0.061±0.041	6.885	4
80.0	0.046±0.023	5.154	4

下载: 导出CSV

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