碱性蛋白酶控制酶解诱导大豆分离蛋白纳米颗粒的形成机制

Formation and Underlying Mechanism of Soy Protein Nanoparticles via Controlled Alcalase Hydrolysis

以大豆分离蛋白(soy protein isolate,SPI)为原料,利用碱性蛋白酶对其进行酶解处理(0~24 h),探究SPI的结构变化规律,发现碱性蛋白酶控制酶解可诱导SPI自组装形成系列分布均匀(多相分散系数<0.3)、粒径可控(90~200 nm)且具有不同表面特性的大豆蛋白纳米颗粒(soy protein nanoparticles,SPNs),其中水解度(degree of hydrolysis,DH)及亚基解离/降解是影响SPNs形成的关键性因素。酶解初期(10~30 min,DH约3%),SPI中β-伴大豆球蛋白(7S)组分α与α’亚基的部分降解有利于两亲性结构的释放,提高蛋白表面疏水性,降低临界聚集浓度,形成包含相对完整的7S及大豆球蛋白(11S)亚基的I类纳米颗粒(SPNs-DH 3%)。随着酶解时间的延长(1~2 h),α与α’亚基的进一步降解促进了疏水性β亚基与B亚基的暴露,增强的疏水相互作用导致体系浊度增加,其中可溶性聚集体向不溶性疏水聚集的转化使得蛋白表面疏水性急剧下降,形成以A亚基及部分β亚基为主导的II类亲水型纳米颗粒(SPNs-DH 5%)。酶解后期(4~24 h),A亚基的进一步降解则产生更多亲水性多肽,不利于纳米颗粒的形成。进而探究SPNs的形成机制,圆二色光谱结构表明,SPNs的形成与蛋白α-螺旋和无规卷曲结构向β-折叠转化有关。两类SPNs的整体结构均由疏水相互作用维持,而氢键和二硫键分别参与颗粒表面与内部结构的形成。与SPNs-DH 3%相比,SPNs-DH 5%中形成了更多由二硫键与氢键稳定的折叠结构。此外,由于酶解过程中不断释放抗氧化肽段,其所形成SPNs的抗氧化性较原始SPI均有所提升。

In the present study,soybean protein isolate(SPI)was hydrolyzed by alcalase for up to 24 h.It was found that controlled enzymatic hydrolysis could induce the self-assembly of SPI to form a series of homogeneous spherical soy protein nanoparticles(SPNs)with different surface properties that were uniformly distributed(polydispersity index(PDI)<0.3)ranging from 90 to 200 nm,and that the degree of hydrolysis(DH)and the disassociation/degradation of subunits were the key factors affecting the assembly process.At the initial stage of enzymatic hydrolysis(10-30 min,DH~3%),theαandα’subunits ofβ-conglycinin(7S)were partially degraded,thereby being beneficial to release the amphipathic structure,improve the protein surface hydrophobicity(H_(0))and reduce the critical aggregation concentration and ultimately resulting in the formation of type I nanoparticles(SPNs-DH 3%)containing relatively complete 7S and glycinin(11S)subunits.As the hydrolysis time increased from 1 to 2 h,theαandα’subunits were further degraded,promoting the exposure of the hydrophobicβsubunits and the B subunits,enhancing the hydrophobic interactions and consequently the system’s turbidity,and ultimately leading to the formation of soluble aggregates.The soluble aggregates could transform into insoluble hydrophobic ones,leading to a sharp decrease in the protein surface hydrophobicity and the formation of type II hydrophilic nanoparticles(SPNs-DH 5%)dominated by the A subunits and part of theβsubunits.At the late stage of hydrolysis(4-24 h),the A subunits were further degraded to produce more hydrophilic peptides,which was not conducive to the formation of nanoparticles.The circular dichroism spectrum implied that transformation fromα-helix and random coil toβ-sheet might facilitate the formation of SPNs.Moreover,both type I and II SPNs were mainly maintained by hydrophobic interactions,while hydrogen and disulfide bonds were responsible for the surface and internal structure of the particles,respectively.More folded structures were stabilized by disulfide and hydrogen bonds formed in SPNs-DH 5%compared with SPNs-DH 3%.In addition,due to the continuous release of antioxidant peptides during enzymatic hydrolysis,the antioxidant activity of the formed SPNs was higher than that of SPI.