山羊乳酪蛋白抗菌肽分离纯化及结构鉴定  

Purification and Structural Identification of Antimicrobial Peptides Derived from Goat Milk Casein

作　　者：李志成[1] 苏晋文[1] 任娇[1] 郝洁[1] 付芒娟 

机构地区：[1]西北农林科技大学食品科学与工程学院,陕西杨凌712100

出　　处：《中国食品学报》2014年第7期214-223,共10页Journal of Chinese Institute Of Food Science and Technology

基　　金："十二五"国家科技支撑计划项目(2012BAD12B07);中央高校基本科研业务费专项(QN2011070);陕西省科技统筹创新工程计划项目(2011KT-CL02-11)

摘　　要：为制备山羊乳酪蛋白抗菌肽,阐明抗菌肽的构效关系,采用LS-106大孔吸附树脂、凝胶色谱和高效液相色谱(HPLC)对山羊乳酪蛋白酶解物进行逐级分离纯化,以对大肠杆菌、沙门氏菌和金黄色葡萄球菌的抑菌圈直径、最小抑菌浓度(MIC)和最小杀菌浓度(MBC)为指标,对分离组分进行抑菌试验,采用预测软件、质谱与圆二色谱对分离抗菌肽的一、二级结构进行预测和鉴定。试验结果表明:(1)大孔吸附树脂分离酶解液,其中体积分数为60%的乙醇洗脱组分抑菌作用较强。(2)凝胶色谱将60%乙醇洗脱组分分离成3个色谱峰,峰3抑菌作用较强。(3)HPLC将峰3分离为2个组分F3-1和F3-2,其中F3-1对大肠杆菌、沙门氏菌和金黄色葡萄球菌的MIC均为297.9μg/mL,MBC为1191.6μg/mL;F3-2对大肠杆菌的MIC和MBC分别为309.2,1236.8μg/mL,对沙门氏菌和金黄色葡萄球菌的MIC均为154.6μg/mL,MBC为618.4μg/mL。(4)F3-1和F3-2的一级结构分别为Leu-Gly-Gly-Val-Gln-Glu-Pro-Asp-Pro-Asn-Thr-Asp-Val-Arg(1497.76)和Val-Val-Asn-Leu-Glu-GluLys-Leu-Pro-Gly(1242.66)。F3-1和F3-2均属阴离子抗菌肽,虽然其抑菌作用稍弱于阳离子抗菌肽,但是其二级结构为无毒性的无规则卷曲结构,与α-螺旋结构抗菌肽相比,在食品、药品和化妆品等安全应用方面潜力巨大。In order to prepare antibacterial peptides derived from goat milk casein and clarify their structure-activity relationships. Goat milk casein hydrolyzates were isolated and purified by LS-106 macroporous absorption resin, gel filtration chromatography and high performance liquid chromatogram（HPLC）, the antibacterial activities of the separated fractions were studied by the indexes of inhibition zone diameter, minimum inhibitory concentration（MIC） and minimum bactericidal concentration（MBC） against escherichia coli、salmonella、staphylococcus aureus. The primary and secondary structure of antimicrobial properties were prediction and identification by prediction software, mass spectrometry（MS） and circular dichroism（CD） spectroscopy. The results showed that：（1） the 60% alcohol elution component showed stronger antibacterial effect from goat milk casein hydrolyzates isolated by macroporous absorption resin.（2） The 60% alcohol elution component was purified into there fractions by gel filtration chromatography, which fraction 3（F3） showed stronger antibacterial effect.（3）HPLC was used to separate F3 into two fractions, F3-1 and F3-2. The MIC and MBC of F3-1against escherichia coli、salmonella、staphylococcus aureus were all 297.9 μg/mL and 1191.6 μg/mL. The MIC and MBC of F3-2 against escherichia coli were 309.2 μg/mL and 1236.8 μg/mL respectively. The MIC and MBC of F3-2 against salmonella、staphylococcus aureus were 154.6 μg/mL and 618.4 μg/mL respectively.（4） The primary structure of antibacterial peptides F3-1 and F3-2 were Leu-Gly-Gly-Val-Gln-Glu-Pro-Asp-Pro-Asn-Thr-Asp-Val-Arg（1497.76） and ValVal-Asn-Leu-Glu- Glu-Lys-Leu-Pro-Gly（1242.66）, respectively, which all belonged to anionic antimicrobial peptides.The antibacterial activity of antibacterial peptides F3-1 and F3-2 was slightly weaker than that of cationic antimicrobial peptides, but they belonged to non-toxic random curl structure compared with the alpha helix antibacterial antimicrobial peptide

关 键 词：山羊乳酪蛋白酶解物 分离纯化 抗菌肽 结构鉴定 

分 类 号：TS252.1[轻工技术与工程—农产品加工及贮藏工程]