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投稿时间：2023-04-25
投稿时间：2023-04-25
中文摘要: 以液压压榨澳洲坚果粕为原料，采用碱性蛋白酶水解制备澳洲坚果多肽（macadamia nut peptide-0，MNP-0），以对金黄色葡萄球菌与白色念珠菌的抑菌活性为跟踪指标，通过超滤、DA201-C 型大孔吸附树脂、交联葡聚糖凝胶Sephadex G-25 对其进行逐级分离纯化，获得抑菌活性较强的抗菌肽，并运用液相色谱串联质谱（liquid chromatography tandem mass spectrometry，LC-MS/MS）技术对其进行肽段组成与氨基酸序列分析。结果表明：超滤分离的不同分子量澳洲坚果多肽在所有多肽中所占质量分数不同，抑菌活性也有所差异。其中，澳洲坚果多肽组分MNP-4 占比最高，为29.55%；抑菌活性也最强，对金黄色葡萄球菌与白色念珠菌的抑菌圈直径分别为10.01 mm 与9.41 mm。大孔吸附树脂纯化多肽组分MNP-4 的技术条件为75%乙醇溶液为解吸剂，静态吸附3 h，动态洗脱体积60 mL。经大孔吸附树脂与交联葡聚糖凝胶Sephadex G-25 分离纯化，获得3 个澳洲坚果纯化多肽（macadamia nut purified peptide，MPP）组分，其中组分MPP-2 的抑菌活性最强，对金黄色葡萄球菌与白色念珠菌的抑菌圈直径分别为18.67 mm 与16.83 mm，优于多肽MNP-0 与超滤分离多肽组分。分离纯化的澳洲坚果抗菌活性多肽MPP-2 含有DDLTDPAPA、VLL、WDY、VPV、WDL、LLW、LWL、FDW 8 个肽段，经预测分析，属于抗菌肽的肽段为WDL 与FDW，其概率得分a（1.00≥a≥0.50）均为1.00，疏水率均为66.67%，均带有1 个负电荷，等电点分别为0.69 与0.67，并具有较好的溶解性。研究结果表明澳洲坚果可通过酶法制备具有抗菌活性的肽类成分。
Abstract:Using hydraulic-pressed macadamia nut cake as raw material,macadamia nut polypeptide MNP-0 was prepared by alkali protease hydrolysis. The antibacterial activity against Stɑphylococcus ɑureus and Cɑndidɑ ɑlbicɑns was employed as the tracking index,and the antimicrobial peptides with strong antibacterial activity were separated and purified through ultrafiltration and DA201-C macroporous resin adsorption followed by cross-linked dextran gel Sephadex G-25. The peptide composition and amino acid sequences of antimicrobial peptide were measured through liquid chromatography tandem mass spectrometry（LC-MS/MS）. The results showed that different molecular weight macadamia nut peptides obtained by ultrafiltration had varying mass fractions and antimicrobial activities. Macadamia nut polypeptide MNP-4 had the highest proportion,accounting for 29.55%,and its antimicrobial activities against Stɑphylococcus ɑureus and Cɑndidɑ ɑlbicɑns were the strongest,with inhibition zone diameters of 10.01 mm and 9.41 mm,respectively.The purification technical conditions of macadamia nut polypeptide MNP-4 by macroporous resin adsorption were as follows:static adsorption time of 3 h,75% ethanol solution as desorption agent,and dynamic elution volume for 60 mL. Three macadamia nut polypeptide fractions were obtained by separation and purification of macroporous resin adsorption and cross-linked dextran gel.Among them,MPP-2 had the strongest antibacterial activity against Stɑphylococcus ɑureus and Cɑndidɑ ɑlbicɑns,with inhibitory zone diameters of 18.67 mm and 16.83 mm,respectively,surpassing the polypeptide MNP-0 and polypeptide fractions separated by ultrafiltration at the same concentration. Macadamia nut polypeptide MPP-2 contained eight peptide segments:DDLTDPAPA,VLL,WDY,VPV,WDL,LLW,LWL and FDW. Predictive analysis indicated that the antimicrobial peptide segments were WDL and FDW,which had probability scores（a）of 1.00（1.00≥a≥0.50）and hydrophobicity of 66.67%,carry one negative charge,and had isoelectric points of 0.69 and 0.67,respectively These peptides also demonstrated good solubility. The results suggest that antimicrobial peptides could be prepared from macadamia nuts by enzymatic method.
keywords: macadamia nut antimicrobial peptide isolation and purification peptide fragment structural identification amino acid sequence analysis
文章编号：202408022 中图分类号： 文献标志码：
基金项目:云南省重大科技专项计划（202202AE090006）；云南省热带作物科技创新体系建设专项（RF2024-6）；云南省科技人才和平台计划项目（202405AD350060）；“兴滇英才支持计划”项目经费支持；西双版纳州“雨林英才支持计划”产业创新人才项目
| 作者 | 单位 |
| 郭刚军1，2，胡小静3，马尚玄1，2，付镓榕1，2，缪福俊4，肖县田3，黄克昌1，2，贺熙勇1，2 | 1.云南省热带作物科学研究所,云南景洪 666100；2.云南省澳洲坚果农业工程研究中心,云南景洪 666100；3.文山学院三七医药学院,云南文山 663099；4.云南省林业和草原科学院,云南昆明 650201 |
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