苦杏仁抑菌肽的筛选富集及结构鉴定

张涛; 马杰; 年国芳; 周芷夷; 张婷; 周建中

doi:10.13386/j.issn1002-0306.2023070042

苦杏仁抑菌肽的筛选富集及结构鉴定

Screening, Enrichment and Structural Identification of Bitter Almond Antibacterial Peptides

摘要

摘要: 为探究苦杏仁抑菌肽的制备、富集及结构分析，本实验以苦杏仁为原料，在脱脂后提蛋白，使用酶法制备苦杏仁蛋白抑菌肽，优化制备工艺，检测抑菌肽的抑菌活性，进一步纯化富集，分离出抑菌活性最强的抑菌肽组分并对其进行结构分析。结果表明，木瓜蛋白酶酶解物对于革兰氏阳性菌尤其是金黄色葡萄球菌的抑菌作用最强。优化苦杏仁抑菌肽的制备工艺最佳条件为：酶解温度为74 ℃，酶底比为2.5%，pH为7。得到抑菌肽的蛋白浓度为19.21%，并测得最低抑菌浓度为3.13 mg/mL。利用超滤和凝胶过滤色谱（Sephadex G-25）对抑菌肽进行分离纯化，从中筛选出肽组分A-II-b对金黄色葡萄球菌的抑制活性最强。将凝胶过滤条件优化为：样品浓度9.38 mg/mL，洗脱速度0.68 mL/min、洗脱剂为纯水。在此条件下测得肽组分A-II-b的峰面积比为13.15%。采用液相色谱-质谱联用技术对肽组分A-II-b进行结构分析，将结果导入Uniprot数据库与APD3数据库进行匹配，筛选出7种分子量在1500~2500 Da的潜在具有抗菌活性的苦杏仁肽序列，分别是ALPDEVLQNAFRIS、ESWNPRDPQFQWAGVA、VAYWSYNNGEQPLVA、FLDLSNDQNQLQLDQVPR、GENDNRNQIIRVR、RNLQGENDNRNQIIRVR和RALPDEVLQNAFRIS。本研究促进了杏仁活性肽的深度开发和资源再利用，同时也为其作为抑菌肽的研究和进一步开发利用提供理论依据。

Abstract: To explore the preparation, enrichment, and structural analysis of bitter almond antimicrobial peptides, in this experiment, the protein was extracted from the defatted bitter almonds, and the protein was hydrolyzed by enzymes to prepare bitter almond protein antibacterial peptides. The preparation process was optimized, and the resulting peptides' antibacterial activity was detected. The peptides were further purified and enriched. The components with the strongest antibacterial activity were isolated, and their structure was analyzed. The results showed that the papain hydrolysate had the strongest bacteriostatic effect on Gram-positive bacteria, especially Staphylococcus aureus. The optimal conditions for the preparation of bitter almond antibacterial peptides were as follows: The enzymatic hydrolysis temperature was 74℃, the enzyme-to-substrate ratio was 2.5%, and the pH was 7. The protein concentration of the antibacterial peptides was 19.21%, and the minimum inhibitory concentration was 3.13 mg/mL. The antibacterial peptides were isolated and purified by ultrafiltration and gel filtration chromatography (Sephadex G-25). Peptide component A-II-b was screened out to have the strongest inhibitory activity against Staphylococcus aureus. The gel filtration conditions were optimized with a sample concentration of 9.38 mg/mL, an elution rate of 0.68 mL/min, and pure water as the eluent. Under these conditions, the peak area ratio of peptide component A-II-b was 13.15%. The structure of peptide component A-II-b was analyzed by liquid chromatography-mass spectrometry, and the results were imported into the UniProt database and the APD3 database for matching. Seven kinds of bitter almond peptides with potential antibacterial activity with molecular weights of 1500~2500 Da were screened out, including ALPDEVLQNAFRIS, ESWNPRDPQFQWAGVA, VAYWSYNNGEQPLVA, FLDLSNDQNQLDQVPR, GENDNRNQIIRVR, RNLQGENDNRNQIIRVR, and RALPDEVLQNAFRIS. This study promoted the in-depth development and resource reuse of almond active peptides and also provided a theoretical basis for their research and further development and utilization as an antibacterial peptide.