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梅花鹿鹿角盘活性肽的分离鉴定及其抑菌活性分析
周怡君1, 杨 梅1, 王懿璞,等1
吉林农业大学 生命科学学院
摘要:
【目的】分离和鉴定梅花鹿鹿角盘活性肽,并研究其抑菌活性,为其制备和开发利用提供参考。【方法】利用酸提醇沉法从梅花鹿鹿角盘粉末中提取总蛋白,过0.45 μm滤器后,用凝胶过滤层析和反相高效液相色谱(RP-HPLC)等方法,从鹿角盘总蛋白中分离、纯化天然活性肽,利用液相色谱 质谱联用(LC-MS/MS)方法对其进行鉴定,通过二倍稀释法分析天然活性肽对大肠杆菌(Escherichia coli)和金黄色葡萄球菌(Staphylococcus aureus)的最小抑菌浓度(minimal inhibit concentration,MIC);在此基础上,以不添加活性肽的菌悬液为对照,用不同质量浓度的天然活性肽处理大肠杆菌和金黄色葡萄球菌,检测菌液的碱性磷酸酶(alkaline phosphatase,AKP)活性和电导率,分析天然活性肽对病原菌菌体生长曲线以及细胞壁和细胞膜的影响。【结果】从梅花鹿鹿角盘总蛋白中成功分离出纯度为93.70%的天然活性肽,其相对分子质量为4 543.14,序列与抗菌肽(UNIPORT:A8QJ91)相似,并将其命名为梅花鹿鹿角盘活性肽(sika antler plate bioactive peptide,SAPBP)。SAPBP对大肠杆菌和金黄色葡萄球菌的最小抑菌浓度分别为0.5和1 mg/mL,对以上2种病原菌的生长曲线有明显影响。与对照相比,不同质量浓度SAPBP作用大肠杆菌和金黄色葡萄球菌菌体后,碱性磷酸酶活性和菌液电导率均明显上升,可知SAPBP对病原菌菌体细胞壁和细胞膜造成不同程度的损伤,其中2 MIC SAPBP对病原菌的破坏作用最为明显。【结论】从梅花鹿鹿角盘总蛋白中分离纯化出了天然活性肽,该活性肽对大肠杆菌和金黄色葡萄球菌具有一定的抑制作用。
关键词:  鹿角盘  活性肽  大肠杆菌  金黄色葡萄球菌  抑菌活性
DOI:
分类号:
基金项目:吉林省科技发展计划项目(20200404024YY);吉林省省级产业创新专项资金项目(2018C048-4)
Isolation,identification and antibacterial activity of bioactive peptides from sika antler plate
ZHOU Yijun,YANG Mei,WANG Yipu,et al
Abstract:
【Objective】Active peptides from sika antler plate were isolated and identified and the antibacterial activity was investigated to provide references for its preparation and development.【Method】The total protein was extracted from the powder of sika antler plate by acid extraction and alcohol precipitation.After filtration by 0.45 μm filter,the natural active peptide was purified by ultrafiltration,gel filtration chromatography and reversed-phase high performance liquid chromatography (RP-HPLC).The natural active peptides were identified by liquid chromatography-mass spectrometry (LC-MS/MS) and analyzed by two-fold dilution method for their minimal inhibiting concentration against Escherichia coli and Staphylococcus aureus.Then,E. coli and S. aureus were treated with natural bioactive peptides using no bioactive peptides as control.The growth curve,alkaline phosphatase activity and electrical conductivity were detected,and the effects of natural bioactive peptides on cell wall and cell membrane of pathogenic bacteria were analyzed.【Result】A natural active peptide with purity of 93.70%,relative molecular mass of 4 543.14 and sequence similar to bactenecin was successfully isolated from sika antler plate and named sika antler plate bioactive peptide (SAPBP).SAPBP affected growth curve of E.coli and S.aureus with minimum inhibitory concentrations of 0.5 and 1 mg/mL,respectively.Compared with the control,the alkaline phosphatase activity and liquid conductivity of bacteria treated with different concentrations of SAPBP increased significantly,indicating that SAPBP caused different degrees of damage to cell wall and cell membrane of pathogenic bacteria.The 2 MIC SAPBP had the largest destructive effect on pathogens.【Conclusion】The natural active peptide isolated and purified from antler plate had certain inhibitory effect on E. coli and S. aureus.
Key words:  sika antler plate  active peptide  Escherichia coli  Staphylococcus aureus  antibacterial activity