Principle Investigation and Method Standardization of Inhibition Zone Assay Based on Antimicrobial Peptides Extracted from Black Soldier Fly Larvae

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS

ACS Applied Bio Materials Pub Date : 2024-08-09 DOI:10.3390/biotech13030031

Wenyue Shen, Ranxia Xue, Yanxia Liu, Shibo Sun, Xi Chen, Dongye Sun, Ouyang Han, Yuxin Li, Jianqiang Xu, Xiaoying Dong, Fengyun Ji, Weiping Xu

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引用次数: 0

Abstract

The black soldier fly is a valuable resource insect capable of transforming organic waste while producing antimicrobial peptides (AMPs). The inhibition zone assay (IZA) is a method used to demonstrate the antimicrobial activity of AMPs. This study aimed to examine the experimental principles and establish a standardized IZA method. Results indicated that the AMPs extract consisted of proteins ranging in molecular weights from 0 to 40 kDa. The AMPs diffused radially on an agar plate through an Oxford cup. The diffusion radius was influenced by the concentration and volume of the AMPs but ultimately determined by the mass of the AMPs. The swabbing method was found to be effective for inoculating bacteria on the agar plate. Among the conditions tested, the plate nutrient concentration was the most sensitive factor for the IZA, followed by bacterial concentration and incubation time. Optimal conditions for the IZA included a nutrient plate of 0.5× TSA, a bacterial concentration of 106 CFU/mL, and an incubation time of 12 h, with oxytetracycline (OTC) at 0.01 mg/mL serving as the positive control. The antimicrobial-specific activity of AMPs could be standardized by the ratio of inhibition zone diameters between AMPs and OTC. These findings contribute to the standardization of the IZA method for profiling the antimicrobial activity of AMPs.

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基于从黑剑蝇幼虫中提取的抗菌肽的抑菌区测定原理研究和方法标准化

黑兵蝇是一种宝贵的资源昆虫，能够在转化有机废物的同时产生抗菌肽（AMPs）。抑菌区试验（IZA）是一种用于证明 AMPs 抗菌活性的方法。本研究旨在研究实验原理并建立标准化的 IZA 方法。结果表明，AMPs 提取物由分子量在 0 至 40 kDa 之间的蛋白质组成。AMPs 通过牛津杯在琼脂平板上进行径向扩散。扩散半径受 AMPs 浓度和体积的影响，但最终取决于 AMPs 的质量。结果表明，拭子法可有效地将细菌接种到琼脂平板上。在测试的条件中，平板营养浓度是对 IZA 最敏感的因素，其次是细菌浓度和培养时间。IZA 的最佳条件包括：营养板为 0.5× TSA，细菌浓度为 106 CFU/mL，培养时间为 12 小时，以 0.01 mg/mL 的土霉素（OTC）作为阳性对照。AMPs 的抗菌特异性活性可通过 AMPs 与 OTC 的抑菌区直径之比进行标准化。这些发现有助于标准化 IZA 方法，以分析 AMPs 的抗菌活性。

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来源期刊

ACS Applied Bio Materials Chemistry-Chemistry (all)

CiteScore

9.40

自引率

2.10%

发文量

464

期刊介绍： ACS Applied Bio Materials is an interdisciplinary journal publishing original research covering all aspects of biomaterials and biointerfaces including and beyond the traditional biosensing, biomedical and therapeutic applications. The journal is devoted to reports of new and original experimental and theoretical research of an applied nature that integrates knowledge in the areas of materials, engineering, physics, bioscience, and chemistry into important bio applications. The journal is specifically interested in work that addresses the relationship between structure and function and assesses the stability and degradation of materials under relevant environmental and biological conditions.