辣椒籽抗菌肽的抑菌活性及其稳定性研究
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摘要
本试验旨在探究辣椒籽抗菌肽作为新型抗菌物质的开发潜力。试验测定了辣椒籽抗菌肽对23株常见农产品腐败菌、食源性病原菌及植物病原菌的抑菌活性及抑菌谱,并检测了辣椒籽抗菌肽对温度、pH、金属离子、蛋白酶、紫外线和变性剂的稳定性。结果表明:该抗菌肽可以抑制部分细菌,对所选的13株真菌均有不同程度的抑制作用,展现了其广谱的抑菌活性;辣椒籽抗菌肽热稳定性较好,121℃处理15 min后抑菌活性基本不变;酸性条件下辣椒籽抗菌肽活性有所下降,但pH为2的条件下处理4 h,其抑菌活性仍可以保持80.00%;碱性条件下抗菌肽的抑菌活性比对照有所升高,在pH为12的条件下处理4 h,其抑菌活性为135.38%;辣椒籽抗菌肽对Fe2+较敏感,而对金属离子Na+、K+、Ca2+、Mg2+、Mn2+、Cu2+有良好的稳定性;对不同的蛋白酶均不敏感;紫外线照射0～36 h后辣椒籽抗菌肽抑菌活性均大于95%;经0～6 mol/L的尿素处理后,其仍能保持很好的稳定性。综上,辣椒籽抗菌肽是一种抑菌谱广,具有良好热、金属离子、蛋白酶、紫外线和变性剂稳定性,在碱性条件下抑菌活性显著增强,具备开发为饲料添加剂、新型防腐保鲜剂或生物农药的潜力。
This experiment aimed to clarify the potential of antimicrobial peptides(AMPs) derived from pepper seeds as a novel antimicrobial substance. The antimicrobial activity of AMPs derived from pepper seeds against23 common agricultural spoilage bacteria,foodborne pathogens and plant pathogens were studied. The stability of AMPs derived from pepper seeds under different temperatures,pH,metal ions,protease,ultraviolet light and denaturant were carried out. The results showed that the AMPs derived from pepper seeds could inhibit some kinds of bacteria and could inhibit the selected 13 strains of fungi,showing its broad-spectrum antibacterial activity. The AMPs derived from pepper seeds had good thermal stability, the antimicrobial activity was unchanged after 121 ℃ heat treatment for 15 min. Under acidic conditions,the antimicrobial activity of AMPs derived from pepper seeds decreased. However,the antimicrobial activity of AMPs derived from pepper seeds could still be maintained 80% under pH 2 for 4 h. Under alkaline conditions,the antimicrobial activity of AMPs derived from pepper seeds was higher than that of the control. When treated at pH 12 for 4 h,the antimicrobial activity was up to 135.38%. The AMPs derived from pepper seeds was sensitive to Fe2+. It had good stability to other metal ions(Na+,K+,Ca2+,Mg2+,Mn2+ and Cu2+); AMPs derived from pepper seeds was not sensitive to proteases; the antimicrobial activity of AMPs derived from pepper seeds was stable after UV irradiation for 0 to 36 h and 0 to 6 mol/L urea treated. To sum up,the AMPs derived from pepper seeds is a kind of antimicrobial protein active substance with wide antimicrobial spectrum,and has good stability under temperature,metal ion,protease,ultraviolet and denaturants,while alkaline conditions can significantly improve the antimicrobial activity. It has the potential to develop into a novel antiseptic preservative,biomedical or biocontrol agent.[Chinese Journal of Animal Nutrition,2019,31(9) : 4322-4330]
This experiment aimed to clarify the potential of antimicrobial peptides(AMPs) derived from pepper seeds as a novel antimicrobial substance. The antimicrobial activity of AMPs derived from pepper seeds against23 common agricultural spoilage bacteria,foodborne pathogens and plant pathogens were studied. The stability of AMPs derived from pepper seeds under different temperatures,pH,metal ions,protease,ultraviolet light and denaturant were carried out. The results showed that the AMPs derived from pepper seeds could inhibit some kinds of bacteria and could inhibit the selected 13 strains of fungi,showing its broad-spectrum antibacterial activity. The AMPs derived from pepper seeds had good thermal stability, the antimicrobial activity was unchanged after 121 ℃ heat treatment for 15 min. Under acidic conditions,the antimicrobial activity of AMPs derived from pepper seeds decreased. However,the antimicrobial activity of AMPs derived from pepper seeds could still be maintained 80% under pH 2 for 4 h. Under alkaline conditions,the antimicrobial activity of AMPs derived from pepper seeds was higher than that of the control. When treated at pH 12 for 4 h,the antimicrobial activity was up to 135.38%. The AMPs derived from pepper seeds was sensitive to Fe2+. It had good stability to other metal ions(Na+,K+,Ca2+,Mg2+,Mn2+ and Cu2+); AMPs derived from pepper seeds was not sensitive to proteases; the antimicrobial activity of AMPs derived from pepper seeds was stable after UV irradiation for 0 to 36 h and 0 to 6 mol/L urea treated. To sum up,the AMPs derived from pepper seeds is a kind of antimicrobial protein active substance with wide antimicrobial spectrum,and has good stability under temperature,metal ion,protease,ultraviolet and denaturants,while alkaline conditions can significantly improve the antimicrobial activity. It has the potential to develop into a novel antiseptic preservative,biomedical or biocontrol agent.[Chinese Journal of Animal Nutrition,2019,31(9) : 4322-4330]
引文
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[9]马燕，徐贞贞，邹辉，等．8个品种辣椒籽成分分析与比较[J]．食品科学，2017,38(22):178-183.
[10]韩玉竹，李平兰，何琴，等．辣椒籽抗菌肽提取条件优化及分离纯化[J/OL]．食品科学，1-14[2019-01-10]. http://kns. cnki. net/kcms/detail/11. 2206. TS.20190102.1538.147.html.
[11]韩玉竹，赵建军，陈强，等．辣椒籽抗菌肽的安全性评价及其在玉米防霉中的应用[J/OL]．食品与发酵工业，1-10[2019-01-10].https://doi.org/10.13995/j.cnki.11-1802/ts.019235.
[12] HAN Y Z,ZHANG B,SHEN Q,et al.Purification and identification of tw o antifungal cyclic peptides produced by Bacillus amyloliquefaciens L-H15[J]. Applied Biochemistry and Biotechnology,2015,176(8):2202-2212.
[13]韩玉竹，邓钊，张宝，等．解淀粉芽孢杆菌H15产抗菌肽的发酵条件优化和提取方法比较研究[J]．食品科学，2015,36(15):135-141.
[14] QIAO K,XU W F,CHEN H Y,et al.A new antimicrobial peptide SCY2 identified in Scylla paramamosain exerting a potential role of reproductive immunity[J]. Fish&Shellfish Immunology,2016,51:251-262.
[15]李军，李平兰，王顺，等．萝卜籽蛋白提取物对鲟鱼腐败菌抑制作用及其理化性质的研究[J]．食品科学，2018,39(13):41-46.
[16] ZHANG Z H,TAO X Y,SHAH N P,et al.Antagonistics against pathogenic Bacillus cereus in milk fermentation by Lactobacillus plantarum ZDY2013 and its anti-adhesion effect on Caco-2 cells against pathogens[J]. Journal of Dairy Science,2016,99(4). 2666-2674.
[17] XIA Y J,LIU X F,WANG G Q,et al. Characterization and selection of Lactobacillus brevis starter for nitrite degradation of Chinese pickle[J]. Food Control,2017,78:126-131.
[18] ENRIQUE M,MANZANARES P,YUSTE M,et al.Selectivity and antimicrobial action of bovine lactoferrin derived peptides against w ine lactic acid bacteria[J].Food Microbiology,2009,26(3):340-346.
[19]王洁，胡然，姜虹伶，等．腐败葡萄中酵母菌的分离及鉴定[J]．酿酒科技，2015(5):29-33.
[20]胡俊．拮抗性芽孢杆菌的筛选及其抗菌肽的分离和性质分析[D]．硕士学位论文．芜湖:安徽工程大学，2017.
[21]罗文杰，钟亨任，何露露，等．抗菌肽Lc-NKlysin-1a的抗菌稳定性及其抗菌机理[J]．海南大学学报(自然科学版)，2017,35(4):345-351.
[22]魏晓晓，杭柏林，马翠，等．抗菌肽JH-3的抑菌活性及其稳定性分析[J]．畜牧兽医学报，2016,47(2):361-366.
[23]姜太玲，吴红洋，申光辉，等．花椒籽蛋白抗菌肽的抑菌作用及其稳定性研究[J]．现代食品科技，2015,31(8):129-135.
[24]张宝．解淀粉芽孢杆菌抗菌脂肽Bacillomycin L．的纯化鉴定及抑菌机理研究[D]．博士学位论文．北京:中国农业大学，2014.
[25]周世成．小麦蛋白抗菌肽的制备及其特性研究[D]．硕士学位论文．郑州:河南工业大学，2011.
[26]吕燕妮，李平兰，江志杰．乳酸菌31-1菌株产细菌素的初步研究[J]．中国食品学报，2003(增刊):130-133.
[27] SAMELIS J,BEDIE G K,SOFOS J N,et al. Combinations of nisin w ith organic acids or salts to control Listeria monocytogenes on sliced pork bologna stored at 4℃in vacuum packages[J]. LWT-Food Science and Technology,2005,38(1):21-28.
[2] MACHADO R J A,ESTRELA A B,NASCIMENTO A K L,et al. Characterization of TistH,a multifunctional peptide from the scorpion Tityus stigmurus:structure,cytotoxicity and antimicrobial activity[J].Toxicon,2016,119:362-370.
[3]汪吴晶，高金燕，佟平，等．抗菌肽的作用机制、应用及改良策略[J]．动物营养学报，2017,29(11):3885-3892.
[4] BENKO-ISEPPON A M,GALDINO S L,CALSA T J,Jr,et al. Overview on plant antimicrobial peptides[J]. Current Protein and Peptide Science,2010,11(3):181-188.
[5] NAWROT R,BARYLSKI J,NOWICKI G,et al.Plant antimicrobial peptides[J].Folia M icrobiologica,2014,59(3):181-196.
[6] RAUSHER M D.Co-evolution and plant resistance to natural enemies[J]. Nature,2001,411(6839):857-864.
[7] BOLOURI MOGHADDAM M R,VILCINSKAS A,RAHNAM AEIAN M. Cooperative interaction of antimicrobial peptides w ith the interrelated immune pathw ays in plants[J]. M olecular Plant Pathology,2016,17(3):464-471.
[8]宁娜．辣椒籽的油脂和蛋白质研究[D]．硕士学位论文．重庆:西南大学，2011.
[9]马燕，徐贞贞，邹辉，等．8个品种辣椒籽成分分析与比较[J]．食品科学，2017,38(22):178-183.
[10]韩玉竹，李平兰，何琴，等．辣椒籽抗菌肽提取条件优化及分离纯化[J/OL]．食品科学，1-14[2019-01-10]. http://kns. cnki. net/kcms/detail/11. 2206. TS.20190102.1538.147.html.
[11]韩玉竹，赵建军，陈强，等．辣椒籽抗菌肽的安全性评价及其在玉米防霉中的应用[J/OL]．食品与发酵工业，1-10[2019-01-10].https://doi.org/10.13995/j.cnki.11-1802/ts.019235.
[12] HAN Y Z,ZHANG B,SHEN Q,et al.Purification and identification of tw o antifungal cyclic peptides produced by Bacillus amyloliquefaciens L-H15[J]. Applied Biochemistry and Biotechnology,2015,176(8):2202-2212.
[13]韩玉竹，邓钊，张宝，等．解淀粉芽孢杆菌H15产抗菌肽的发酵条件优化和提取方法比较研究[J]．食品科学，2015,36(15):135-141.
[14] QIAO K,XU W F,CHEN H Y,et al.A new antimicrobial peptide SCY2 identified in Scylla paramamosain exerting a potential role of reproductive immunity[J]. Fish&Shellfish Immunology,2016,51:251-262.
[15]李军，李平兰，王顺，等．萝卜籽蛋白提取物对鲟鱼腐败菌抑制作用及其理化性质的研究[J]．食品科学，2018,39(13):41-46.
[16] ZHANG Z H,TAO X Y,SHAH N P,et al.Antagonistics against pathogenic Bacillus cereus in milk fermentation by Lactobacillus plantarum ZDY2013 and its anti-adhesion effect on Caco-2 cells against pathogens[J]. Journal of Dairy Science,2016,99(4). 2666-2674.
[17] XIA Y J,LIU X F,WANG G Q,et al. Characterization and selection of Lactobacillus brevis starter for nitrite degradation of Chinese pickle[J]. Food Control,2017,78:126-131.
[18] ENRIQUE M,MANZANARES P,YUSTE M,et al.Selectivity and antimicrobial action of bovine lactoferrin derived peptides against w ine lactic acid bacteria[J].Food Microbiology,2009,26(3):340-346.
[19]王洁，胡然，姜虹伶，等．腐败葡萄中酵母菌的分离及鉴定[J]．酿酒科技，2015(5):29-33.
[20]胡俊．拮抗性芽孢杆菌的筛选及其抗菌肽的分离和性质分析[D]．硕士学位论文．芜湖:安徽工程大学，2017.
[21]罗文杰，钟亨任，何露露，等．抗菌肽Lc-NKlysin-1a的抗菌稳定性及其抗菌机理[J]．海南大学学报(自然科学版)，2017,35(4):345-351.
[22]魏晓晓，杭柏林，马翠，等．抗菌肽JH-3的抑菌活性及其稳定性分析[J]．畜牧兽医学报，2016,47(2):361-366.
[23]姜太玲，吴红洋，申光辉，等．花椒籽蛋白抗菌肽的抑菌作用及其稳定性研究[J]．现代食品科技，2015,31(8):129-135.
[24]张宝．解淀粉芽孢杆菌抗菌脂肽Bacillomycin L．的纯化鉴定及抑菌机理研究[D]．博士学位论文．北京:中国农业大学，2014.
[25]周世成．小麦蛋白抗菌肽的制备及其特性研究[D]．硕士学位论文．郑州:河南工业大学，2011.
[26]吕燕妮，李平兰，江志杰．乳酸菌31-1菌株产细菌素的初步研究[J]．中国食品学报，2003(增刊):130-133.
[27] SAMELIS J,BEDIE G K,SOFOS J N,et al. Combinations of nisin w ith organic acids or salts to control Listeria monocytogenes on sliced pork bologna stored at 4℃in vacuum packages[J]. LWT-Food Science and Technology,2005,38(1):21-28.
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