以废蚕茧为原料,通过Na2CO3脱胶、溶解、透析、冷冻干燥后得到丝素蛋白粉末。用蒸馏水溶解后,分别采用碱性蛋白酶(alcalase)、木瓜蛋白酶(papain)水解丝素蛋白,85 ℃将酶灭活,得到丝素肽溶液。采用反相高效液相色谱仪对10种丝素肽组分进行分析,并研究丝素肽对大肠杆菌(Escherichia coli)及金黄色葡萄球菌(Staphylococcus aureus)的抗菌效果,以及对人胚肾细胞(HEK293)的毒性。发现P1、A2、A4、A1丝素肽对大肠杆菌生长有抑制作用,P1、P2、P3丝素肽对金黄色葡萄球菌生长有抑制作用,具有抑菌作用的这6种丝素肽对HEK293细胞增殖没有毒性,且A1、A2、A4、P2丝素肽对HEK293细胞存活具有促进作用,为今后丝素肽的产品开发、生产及应用奠定一定的理论基础。
Silk fibroin powder was obtained from waste silkworm cocoon by degumming with Na2CO3, dissolving, dialysing and freeze-drying. The silk fibroin was hydrolyzed by alcalase and papain in distilled water. The silk fibroin peptides were obtained by inactivating the enzyme at 85 ℃. Reverse phase high performance liquid chromatography (RP-HPLC) was used to analyze the components of the fibroin peptides. Analyze the antimicrobial activity of ten silk fibroin peptides and the cytotoxicity to human embryonic kidney (HEK293) cells. It was found that P1, A2, A4, A1 silk fibroin peptides inhibited Escherichia coli and P1, P2, P3 silk fibroin peptides inhibited Staphylococcus aureus. These six silk fibroin peptides had no toxicity to HEK293 cell proliferation, and A1, A2, A4, P2 silk fibroin peptides could promote the survival of HEK293 cells, laying a theoretical foundation for the development, production and application of silk fibroin peptide in the future.
[1] KAEWPRASITA K,KOBAYASHIB T,DAMRONGSAKKUL S.Thai silk fibroin gelation process enhancing by monohydric and polyhydric alcohols[J].International Journal of Biological Macromolecules,2018,118(10):1 726-1 735.
[2] KI C S,PARK Y H,JIN H J.Silk protein as a fascinating biomedical polymer: structural fundamentals and applications[J].Macromolecular Research,2009,17(12):935-942.
[3] MATHUR A B,GUPTA V.Silk fibroin-derived nanoparticles for biomedical applications[J].Nanomedicine,2010,5(5):807-820.
[4] KUNDU B,KURLAND N E,BANO S,et al.Silk proteins for biomedical applications:bioengineering perspectives[J].Progress in Polymer Science,2014,39 (2):251-267.
[5] 李利军,吴启涛,杨峰,等.超声波对酶法水解丝素作用的研究[J].食品工业科技,2010,31(12):276-278;281.
[6] 杨丽晶,赵国静,裴艳秋,等.关于丝肽、丝素和丝胶产品的开发与应用[J].中国保健食品,2004(8):9-10.
[7] 钱坤,刘迪,徐国文.不同分子量丝素肽的开放[J].蚕桑通报,2017,48(4):51-53.
[8] VACHIRAROJ N,RATANAVARAPORN J,DAMRONGSAKKUL S,et al.A comparison of Thai silk fibroin-based and chitosan-based materials on in vitro biocompatibility for bone substitutes[J].International Journal of Biological Macromolecules,2009,45(5):470-477.
[9] KANOKPANONT S,DAMRONGSAKKUL S,RATANAVARAPORN J,et al.An innovative bilayered wound dressing made of silk and gelatin for accelerated wound healing[J].International Journal of Pharmaceutics,2012,436(2):141-153.
[10] 倪莉,王璋,许时婴,等.酶法水解丝素的研究[J].食品与发酵工业,2000,26(1):20-22.
[11] ZASLOFF M. Antimicrobial peptides of multicellular organisms[J]. Nature,2002,415(6 870):389-395.
[12] 李倩,马慧霞,张皓然,等.人工抗菌肽的研究进展[J].药物生物技术,2017,24(4):364-368.
[13] 王青,徐彦召,刘保国,等.抗菌肽的特点及其基因工程研究进展[J].黑龙江畜牧兽医,2017,17(9):5-8.
[14] 乔想金,李文新,白丽娟,等.抗菌肽VIP在毕赤酵母中的高效表达及鉴定[J].生物工程学报,2018,34(6):1 002-1 011.
[15] 胡建平.鱼胶原蛋白的开发与应用[M].成都:四川大学出版社,2014:48.
[16] 马骏,王学英,李群,等.蚕丝丝胶蛋白提取方法的研究[J].安徽农业科学,2005,33(4):674-675.
[17] 王亮亮,雷彩虹,朱海霖,等.2种酶水解制备丝素肽的形貌结构与止血性能[J].蚕业科学,2016,42(6): 1 099-1 105.
[18] ZHOU Fengjuan,XUE Zhaohui,WANG Jiehua.Antihypertensive effects of silk fibroin hydrolysate by alcalase and purification of an ACE inhibitory dipeptide[J].Journal of Agricultural and Food Chemistry,2010,58(11): 6 735-6 740.
[19] 罗美琪.丝素肽的酶法制备及其生物活性的研究[D].广东:华南理工大学,2012.
[20] FERNANDEZ A,KELLY P.pH-stat vs. free-fall pH techniques in the enzymatic hydrolysis of whey proteins[J].Food Chemistry,2016,199(5):409-415.
[21] 倪莉,陶冠军,戴军,等.血管紧张素转化酶活性抑制剂丝素肽的分离、纯化和结构鉴定[J].色谱,2001,19(3):222-225.
[22] 周凤娟,许时婴,杨瑞金,等.丝素肽的制备及其理化性质研究[J].西北农林科技大学学报(自然科学版),2007,35(12):167-171.
[23] 翁佩芳,江华珍,冯凤琴,等.酶标比浊法评价月桂酸单甘油酯对肉葡萄球菌的抑菌活性[J].中国食品学报,2012,12(5):188-194.
[24] 张亚卓,姜思萌,魏颖,等.小麦低聚肽对体外肠上皮细胞氧化应激损伤的保护作用[J].食品与发酵工业,2015,41(1):46-50.
[25] WANG Fei,ZHANG Yuqing.Effects of alkyl polyglycoside(APG) on Bombyx mori silk degumming and the mechanical properties of silk fibroin fiber[J]. Materials Science and Engineering C,2017,74 (5):152-158.
[26] FENG Xiaowu,HUO Lijie,YANG Mingchong,et al.Thymosins participate inantibacterial immunity of kuruma shrimp, Marsupenaeus japonicas[J]. Fish Shellfish Immunology,2018,84(10):244-251.
