还原型谷胱甘肽诱导溶菌酶界面组装成膜机制及膜性能分析  

Formation Mechanism and Performance of Films through Reduced Glutathione-Induced Lysozyme Interface Assembly

作　　者：周彬 李俊娇 郭大山 武璨 胡煜莹 崔冰 ZHOU Bin;LI Junjiao;GUO Dashan;WU Can;HU Yuying;CUI Bing(Cooperative Innovation Center of Industrial Fermentation(Ministry of Education&Hubei Province),Hubei University of Technology,Wuhan 430068,China)

机构地区：[1]湖北工业大学工业发酵省部共建协同创新中心,湖北武汉430068

出　　处：《食品科学》2024年第19期208-215,共8页Food Science

基　　金：国家自然科学基金面上项目(32172354);湖北工业大学绿色工业科技引领计划-自主探索计划项目(XJ2021004401)。

摘　　要：采用光谱学方法等验证了还原型谷胱甘肽诱导溶菌酶界面组装成膜机制,并进一步采用椭偏仪等对膜性能进行分析。N-(1-芘)马来酰亚胺染色实验证明还原性谷胱甘肽打断了溶菌酶的二硫键后释放出了游离巯基。色氨酸荧光光谱和溶菌酶活性检测实验共同验证了溶菌酶的二硫键Cys6-Cys127发生了断裂。另外,通过溶菌酶活性检测发现解折叠的溶菌酶依然具有抗菌活性。该溶菌酶膜无色透明,原子力显微镜和透射电镜的结果显示其由溶菌酶聚集体组成,表面均匀且光滑致密。X射线光电子能谱和激光共聚焦拉曼光谱的结果均表明该溶菌酶纳米膜可能具有界面黏附性。椭偏仪测试结果显示该膜的最大厚度在900 nm左右,且不随孵育时间的延长或反应物质量浓度的增加而无限制增加;其刚度为29.32~34.21μN/nm,弹性模量为24.00~27.93 GPa,硬度为0.44~0.49 GPa,符合蛋白膜的强度范围;膜的接触角在70°左右,较为亲水;其热稳定性较高,200℃条件下不发生降解。该溶菌酶纳米膜的制备为构筑新型可食性涂层提供了新的思路与借鉴。In this study,the mechanism of film formation through glutathione-induced lysozyme interface assembly was verified by spectroscopic methods,and the formed film was characterized by ellipsometry.N-(1-pyrenyl)maleimide(NPM)staining demonstrated that reduced glutathione broken the disulfide bonds of lysozyme leading to release of free sulfhydryl groups.Together,tryptophan fluorescence spectroscopy and lysozyme activity assay verified that the disulfide bond Cys6-Cys127 in lysozyme was broken.In addition,the unfolded lysozyme was found to still have antibacterial activity.The lysozyme film was transparent and colorless.Atom force microscopy(AFM)and transmission electron microscopy(TEM)revealed that it was composed of lysozyme aggregates with a uniform,smooth and dense surface.The results of X-ray photoelectron spectroscopy(XPS)and laser confocal Raman spectroscopy(LCRS)indicated that the lysozyme nanofilm might possess interfacial adhesion.The results of ellipsometry showed that the maximum thickness of the film was approximately 900 nm,and did not increase without limit with the increase in incubation time or the concentration of the reactants.Its stiffness value,elastic modulus and hardness were 29.32–34.21μN/nm,24.00–27.93 GPa,and 0.44–0.49 GPa,respectively,which were in the strength range of the protein film.The three-phase contact angle of the film was around 70°,indicating good hydrophilicity.Its thermal stability was high,and no degradation occurred at 200℃.The preparation of the lysozyme nanofilm provides a good idea and reference for the construction of novel edible coatings.

关 键 词：还原型谷胱甘肽 溶菌酶 解折叠 界面组装 膜 

分 类 号：TS253.4[轻工技术与工程—农产品加工及贮藏工程]