The purpose of this paper is to systematically evaluate the antimicrobial peptides from larvae of Protaetia brevitarsis Lewis, a resource insect with important development prospects, in order to provide scientific basis for the efficient utilization of antimicrobial peptides and the development of antibacterial agents for plant pathogenic fungi. Firstly, antimicrobial peptides were extracted using four methods:- DOI:10.3969/j.issn.1000-1565.2025.06.007白星花金龟幼虫抗菌肽提取方法对比及其抑菌活性和稳定性研究陈思彤1,张肖肖1,陈新2,高梦洁2,李静1,白明1,3,4(1.河北农业大学 植物保护学院,河北 保定 071001;2. 沧州师范学院 生命科学学院,河北 沧州 061001;3. 中国科学院 动物研究所,动物多样性保护与有害动物防控重点实验室,北京 100101;4.青海师范大学 高原科学与可持续发展研究院,青海 西宁 810016)摘 要:通过对具有重要开发前景的资源昆虫白星花金龟(Protaetia brevitarsis Lewis)幼虫抗菌肽进行系统评估,为抗菌肽高效利用和植物病原真菌抑菌剂的开发提供科学依据.首先利用醋酸铵缓冲液提取法、醋酸钠缓冲液浸提法、氢氧化钠提取法、醋酸海藻酸法4种方法提取抗菌肽,再将获得的白星花金龟幼虫抗菌肽使用超滤管进行纯化,然后比较得率.评价所获白星花金龟幼虫抗菌肽对6种植物病原真菌的抑菌活性及其在不同情况下的稳定性.结果表明:4种提取法中,氢氧化钠提取法得到的非纯化蛋白占总质量的18.41%,纯化蛋白占总质量的5.34%,技术流程较为简便,建议在白星花金龟幼虫抗菌肽提取中使用;白星花金龟幼虫抗菌肽对梨黑斑病菌(Alternaria kikuchiana)抑菌效率最高为84.78%,有开发为梨黑斑病菌抑菌剂的潜在可能;在反复冻融1~2次,温度80 ℃以下,pH为5.5~7.5时,白星花金龟幼虫抗菌肽的抑菌活力保持率在80%以上,在此区间外容易失去抗菌活性.关键词:白星花金龟;抗菌肽;抑菌活性;稳定性中图分类号:Q969.97 文献标志码:A 文章编号:1000-1565(2025)06-0615-08Comparison of extraction methods of antimicrobial peptides from the larva of the Protaetia brevitarsis Lewis and study on its antibacterial activity and stabilityCHEN Sitong1, ZHANG Xiaoxiao1, CHEN Xin2, GAO Mengjie2, LI Jing1, BAI Ming1,3,4(1. College of Plant Protection, Hebei Agricultural University, Baoding 071001, China;2.College of Life Science, Cangzhou Normal University, Cangzhou 061001, China;3.Key Laboratory of Animal Biodiversity Conservation and Integrated Pest Management, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China;4. Academy of Plateau Science and Sustainability, Qinghai Normal University, Xining 810016, China)Abstract: The purpose of this paper is to systematically evaluate the antimicrobial peptides from larvae of Protaetia brevitarsis Lewis, a resource insect with important development prospects, in order to provide scientific basis for the efficient utilization of antimicrobial peptides and the development of antibacterial agents for plant pathogenic fungi. Firstly, antimicrobial peptides were extracted using four methods:- 收稿日期:2024-10-11;修回日期:2024-12-20 基金项目:国家重点研发计划项目(2022YFC2601200);贵州省科技计划项目(黔科合支撑［2022］一般 173) 第一作者:陈思彤(1994—),男,满族,河北农业大学在读硕士研究生,主要从事抗菌肽方向研究. E-mail:15530233569@163.com 通信作者:李静(1976—),女,河北农业大学教授,博士,主要从事昆虫多样性及系统发育方向研究. E-mail:lijing1976416514@163.com白明(1977—),男,中国科学院研究员,博士,主要从事下一代昆虫分类学方向研究. E-mail: baim@ioz.ac.cn 第6期陈思彤等:白星花金龟幼虫抗菌肽提取方法对比及其抑菌活性和稳定性研究河北大学学报(自然科学版) 第45卷ammonium acetate buffer extraction method, sodium acetate buffer extraction method, sodium hydroxide extraction method, and acetic acid alginate method. The antibacterial peptides obtained from P. brevitarsis larvae were purified using ultrafiltration tubes, followed by yield comparison. Secondly, the antibacterial activity and stability of the antimicrobial peptides obtained from the larva of P. brevitarsis Lewis against six plant pathogenic fungi were evaluated. The results indicated that among the four extraction methods, the sodium hydroxide extraction method was identified as the most effective. It yielded the highest proportion of total non-purified protein at 18.41% of the total mass, and a relatively high content of purified protein at 5.34%. Due to its relatively simple technical procedure, this method is recommended for the extraction of antimicrobial peptides from the larvae of P. brevitarsis. The antibacterial peptides derived from P. brevitarsis larvae showed the highest inhibition rate of 84.78% against Alternaria kikuchiana, suggesting their potential as a biocontrol agent for this disease. Under repeated freeze-thaw cycles of less than 2 times, temperature below 80 ℃, and pH between 5.5—7.5, the antibacterial activity of P. brevitarsis Lewis antibacterial peptide was maintained at over 80%.Outside this range, it is easy to lose antibacterial activity.
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