【作者】 陈颖；

【导师】 徐红华； 朱宏；

【作者基本信息】 东北农业大学 ， 工程硕士（专业学位）， 2022， 硕士

【摘要】 纳米纤维聚合结构是蛋白质分子在特定条件下形成具有cross-β-折叠结构的一种独特的有序聚合物,对功能性质的提升是蛋白质常规结构无法达到的,具有优良的凝胶性、乳化性、起泡性和冻融稳定性等诸多性能。这种独特的组装结构主要通过自发组装方式形成,存在用时长、产量低的问题,使其很难广泛应用。以成熟纤维作为纤维核的核诱导方式能缩短纤维生成时间、提高纤维得率,但提升能力有限。因此,本课题采用团队前期摸索的酶解碎化和多次循环诱导手段对纤维核进行结构改造,通过2种手段协同作用以达到进一步提升纳米纤维得率的目的。本研究以乳清分离蛋白（WPI）为原料制备多次循环诱导纤维核,利用碱性蛋白酶对纤维核进行酶解碎化,通过分析耐酶解能力、纤维核结构保留率,观察不同循环次次数获得的纤维核之间酶解能力的差异;从纤维生成量及聚合动力学的角度分析酶解协同多次循环诱导手段对纤维核诱导能力的影响;对具有诱导能力的纤维核进行不同方式的酶解处理,寻找出提高纤维量的最佳制备方法;结合飞行质谱、原子力显微镜、红外光谱分析以及X-射线衍射等方法,系统分析酶解协同循环诱导手段引起纤维核结构的变化规律,在分子水平上探讨核结构改变与诱导能力之间的关系,进而为食品蛋白质纳米纤维组装结构提供新方法和科学依据。具体研究结果如下:（1）酶解对循环诱导纤维核的影响与原料WPI相比,多次循环诱导制备形成的纤维核会表现出一定的耐酶解能力,并且纤维核耐酶解能力、纤维核结构保留率会随着循环次数的增加呈现先升高后降低的趋势。其中第1次循环形成的纤维核N₁的耐酶解能力最强,其cross-β-折叠核心结构保留率可达67.25%,同初始纤维核N₀相比提高了7.86%,水解液粒径最大。同时,观察不同循环次数制备的纤维核质谱图,虽然都具有相同的纤维结构特征峰,但是随着循环次数增加,酶解处理后纤维特征峰逐渐消失。（2）基于酶解协同循环诱导方式的纤维化组装规律酶解处理对纤维核诱导能力的影响取决于酶解前纤维核自身的诱导能力,对于多次循环诱导而言,前3次循环形成的纤维核N₁、N₂、N₃诱导能力增强,后两次循环形成的纤维核N₄、N₅诱导能力丧失。有诱导能力的纤维核,酶解处理会进一步增强纤维核的诱导能力,提高纤维聚合速率和聚合量、缩短滞后期,其中纤维核N₁水解液诱导能力最佳,相比自发组装纤维量提高了29.20%,相比未水解N₁提高17.14%。并且,聚合动力学参数也会发生很大改变,纳米纤维组装形成速度更快,N₁酶解前后滞后期t_lag时间从0.64 h缩短到0.12 h;而循环获得的丧失诱导能力的纤维核,酶解处理会进一步降低聚合速率和聚合量、延长滞后期。飞行时间质谱谱图结果显示,多次循环获得的纤维核无论酶解处理与否,诱导WPI所形成的纳米纤维聚合物都有3个明显的特征峰,具有与自发组装方式相似的结构特征。（3）多次循环纤维核酶解方式及组装条件的优化酶解碎化协同多次循环诱导手段对纤维核进行结构改造,提高纤维量的能力是目前文献报道远远不能达到的。对循环诱导获得的诱导能力最强的纤维核N₁,经过碱性蛋白酶最佳酶解处理4 h（水解度为26.90%）,其水解液诱导WPI形成纳米纤维的提升幅度最高,以常规自发组装方式形成的乳清蛋白纳米纤维量为标准,纤维得率提升至33.70%,并且,酶解协同多次循环的纤维核诱导方式可以将纳米纤维制备条件的p H值提高至3.0,热处理温度降低至75℃。（4）酶解协同循环诱导对纤维核结构的影响酶解碎化和循环诱导手段都会带来纤维核中cross-β-折叠结构层间距离增大,导致纤维结构更松散;增加酶解次数和循环诱导次数都会加剧松散程度,降低纤维形成能力。因此,cross-β-折叠结构层间距离保持在9.89-10.91?之间是保证纤维核具有诱导能力,提升纤维得率的前提。（5）酶解协同循环诱导对纳米纤维结构的影响酶解协同循环诱导手段形成的乳清蛋白纳米纤维结构不同于自发组装方式,形成的纤维结构更松散。酶解协同循环诱导获得的纤维核结构松散,导致诱导形成的纤维结构更松散,纤维周期长度从40-50 nm增加到45-55 nm、纤维直径从2.0-3.5 nm增加到2.5-4.0 nm;晶体片层距离从10.42?增加到10.96?,这种松散的纤维结构可以改变分子刚性特征,有利于提升界面性能。更多还原

【Abstract】 Nanofibrils polymerization structure is a unique aggregation of protein molecules with cross-β-sheet structure under specific conditions,the enhancement of functional properties is not achieved by conventional protein structure,and it has excellent properties such as gelation,emulsification,foaming and freeze-thaw stability.This unique assembly structure is mainly formed by spontaneous assembly,which has the problems of long time and low output,making it difficult to be widely used.The nuclei induction method with mature fibrils as fibrils nuclei can shorten the fibrils formation time and improve the fibrils yield,but the promotion ability is limited.Therefore,in this project,the structural transformation of fibrils nuclei was carried out by means of enzymatic hydrolysis and multiple cycle induction explored by the team in the early stage,and the two means work together to further improve the yield of nanofibrils.In this study,whey protein isolate（WPI）was used as raw material to prepare multi-cycle induced fibrils nuclei,and alkaline protease was used to enzymolize the fibrils nuclei.By analyzing the enzymatic hydrolysis resistance and the retention rate of fibrils nuclei structure,the differences of enzymatic hydrolysis energy between fibrils nuclei obtained by different cycle times were observed;From the point of view of fibrils production and polymerization kinetics,the effect of enzymatic hydrolysis coordinated multiple cycle induction on the ability of fibrils nuclei induction was analyzed;The fibrils nuclei with inducing ability was treated by different ways of enzymatic hydrolysis to find the best preparation method to improve the amount of fibrils;Combined with flight mass spectrometry,atomic force microscopy,infrared spectroscopy and X-ray diffraction,this paper systematically analyzes the change law of fibrils nuclei structure caused by enzymatic hydrolysis synergistic cycle induction,and discusses the relationship between nuclei structure change and induction ability at the molecular level,so as to provide a new method and scientific basis for the assembly structure of food protein nanofibrils.The specific research results are as follows:（1）Effects of enzymatic hydrolysis on multiple circulation fibrils nucleiCompared with the raw material WPI,the fibrils nuclei formed by repeated cycle induction will show a certain resistance to enzymatic hydrolysis,and the resistance to enzymatic hydrolysis and the retention rate of fibrils nuclei structure will first increase and then decrease with the increase of cycle times.Among them,N₁ formed in the first cycle had the strongest resistance to enzymatic hydrolysis,and the retention rate of cross-β-sheet structure reached 67.25%,which was increased by 7.86%compared with the initial N₀,and the particle size of hydrolysate was the largest.At the same time,the mass spectra of fibrils nuclei prepared with different cycle times were observed.Although they all have the same fibrils structure characteristic peak,the fibrils characteristic peak gradually disappeared after enzymatic hydrolysis with the increase of cycle times.（2）Fibrotic assembly based on enzymatic hydrolysis and cyclic inductionThe effect of enzymatic hydrolysis treatment on the induction ability of fibrils nuclei depends on the induction ability of fibrils nuclei before enzymatic hydrolysis.For multiple cycle induction,the induction ability of fibrils nuclei N₁,N₂ and N₃ formed in the first three cycles was enhanced,and the induction ability of fibrils nuclei N₄ and N₅ formed in the last two cycles were lost.For the fibrils nuclei with induction ability,enzymatic hydrolysis treatment will further enhance the induction ability fibrils nuclei,improve the fibrils polymerization rate and amount,and shorten the lag time.Among them,the induction ability of N₁ hydrolysate of fibrils nuclei was the best,which is 29.20%higher than that of spontaneously assembled fibrils and 17.14%higher than that of non hydrolyzed N₁.Moreover,the polymerization kinetic parameters will also change greatly.The assembly and formation speed of nanofibers was faster,and the lag time before and after N₁enzymatic hydrolysis was shortened from 0.64 h to 0.12 h;However,the enzymatic hydrolysis treatment will further reduce the polymerization rate and amount and prolong the lag time of the fibrils nuclei that have lost the ability of induction.The results of time-of-flight mass spectrometry showed that the nanofiber polymer formed by induced WPI had three obvious characteristic peaks,which were similar to the structural characteristics of spontaneous assembly.（3）Optimization of enzymolysis method and assembly conditions of multiple cycle fibrils nucleiThe ability of enzymatic hydrolysis and fragmentation combined with multiple cycle induction to transform the structure of fibrils nuclei and improve the amount of fibrils was far from being achieved in the literature.For the fibrils nuclei N₁ with the strongest induction ability obtained by cyclic induction,after the optimal enzymatic hydrolysis treatment of alkaline protease for 4 hours（the degree of hydrolysis was 26.90%）,the hydrolysate induced WPI to form nanofibrils with the highest increase range.Taking the amount of whey protein nanofibrils formed by conventional spontaneous assembly as the standard,the fibrils yield was increased to 33.70%,The fibrils nuclei induction method of enzymatic hydrolysis and multiple cycles can increase the p H value of the preparation conditions of nanofibrils to 3.0 and reduced the heat treatment temperature to 75℃.（4）Effects of enzymatic hydrolysis and cyclic induction on fibrous nucleus structureBoth enzymolysis and cyclic induction methods will bring cross-β-sheet distance between layers of folded structure increases in fibrils nuclei,resulting in looser fibrils structure;Increasing the times of enzymatic hydrolysis and cycle induction will aggravate the degree of looseness andreduced the ability of fibrils formation.Therefore,the distance between the layers of cross-β-sheet structure was between 9.89-10.91?to ensure that the fibrils nuclei has induction ability.（5）Effects of enzymatic hydrolysis and cyclic induction on the structure of nanofibrilsThe structure of whey protein nanofibrils formed by enzymatic hydrolysis and cyclic induction was different from that of spontaneous assembly,and the structure of the fibrils was looser.The nuclei structure of the fibrils obtained by enzymatic hydrolysis combined with cyclic induction was loose,resulting in a more loose structure of the fibrils.The fibrils cycle length increased from 40-50 nm to 45-55 nm,and the fibrils diameter increased from 2.0-3.5 nm to 2.5-4.0 nm.The lamellar distance increases from 10.42?to 10.96?,and the loose fibrils structure can change the molecular rigidity characteristics,which is beneficial to improve the interface properties.更多还原