碱性蛋白酶水解对藜麦分离蛋白结构、聚集行为及凝胶性的影响

doi:10.3864/j.issn.0578-1752.2025.01.013

摘要/Abstract

摘要：

【目的】研究碱性蛋白酶对藜麦分离蛋白结构、理化性质及聚集行为的影响，并探究碱性蛋白酶水解对藜麦分离蛋白凝胶性的作用。【方法】通过碱提酸沉法于4 ℃下提取藜麦分离蛋白，使用碱性蛋白酶在不同酶底比条件下对藜麦分离蛋白进行处理。分析藜麦分离蛋白的组成、粒径、Zeta电位、溶解度、表面疏水性（S₀-ANS）的变化及其与蛋白热聚集体的Th T荧光强度、形貌的关联性。进一步提高蛋白浓度，将藜麦分离蛋白加热制成凝胶，通过对凝胶的微观结构、质构及凝胶中蛋白质二级结构的分析，探讨酶水解处理与聚集行为和凝胶性之间的关系，揭示酶水解促进凝胶性改变的原因。【结果】随着酶底比（E/S）的上升，藜麦分离蛋白溶液（2%，w/v）的蛋白粒径逐渐减小，其电负性呈现先上升后下降的趋势，藜麦分离蛋白的表面疏水性逐渐上升。同时，藜麦分离蛋白聚集体的Th T荧光强度在酶底比为0—0.08%时呈现上升趋势，而当酶底比继续升高（0.08%—0.14%）则下降。通过透射电子显微镜（TEM）观察，在酶底比为0.05%和0.08%时分别形成了较短的纤维（145—306 nm）和较长的纤维（217—406 nm），继续增大酶底比则纤维的长度缩短并且无定型聚集增加。另外，藜麦分离蛋白的不同聚集行为对其凝胶性有显著影响（P<0.05）。在有纤维聚集体形成的条件下，藜麦分离蛋白凝胶具有更高的硬度、储能模量以及更致密的凝胶网络结构。与短纤维相比，长纤维的形成对凝胶硬度、储能模量及凝胶网络的增强效果更加明显。此外，碱性蛋白酶水解显著影响藜麦蛋白凝胶中蛋白质的二级结构，β-折叠和无规则卷曲含量随酶底比的增加呈现出先上升后下降的趋势。【结论】限制性碱性蛋白酶水解有助于藜麦分离蛋白形成有序的纤维聚集体，进而改善其凝胶性。当E/S为0.08%时，藜麦分离蛋白热诱导形成的纤维最长，蛋白凝胶硬度最大，黏弹特性最好，凝胶中蛋白质二级结构有序、微观结构致密。但是，更高的水解程度会增加无定型聚集并降低凝胶强度。因此，碱性蛋白酶限制性水解可以通过促进纤维聚集体形成而显著改善藜麦蛋白凝胶性弱的问题。

关键词: 藜麦分离蛋白, 碱性蛋白酶, 限制性水解, 纤维聚集体, 凝胶网络, 蛋白质二级结构

Abstract:

【Objective】This research studied the effects of alcalase hydrolysis on the structure, physicochemical properties and aggregation behavior of quinoa protein isolate (QPI), and explored its effects on the gelling properties of QPI. 【Method】QPI was extracted by alkaline extraction and acid precipitation method at 4 ℃. Alcalase with different enzyme-substrate ratios was added to the QPI solution to hydrolyze the protein. Thereafter, the changes of the composition, particle size, Zeta potential, solubility, and surface hydrophobicity (S₀-ANS) of QPI were analyzed, and the correlation between these changes and the Th T fluorescence intensity and morphology of protein thermal aggregates were discussed. Furthermore, the concentration of QPI dispersion was elevated to form heat-induced QPI gels. The correlation among alcalase hydrolysis, aggregation behavior and gelling properties of QPI was discussed through the analysis of the microstructure, texture and protein secondary structure of QPI gels. Meanwhile, the reasons for the changes in the gelling properties of QPI induced by alcalase hydrolysis were revealed.【Result】With the increase of enzyme substrate ratio (E/S), the protein particle size of QPI dispersion (2%, w/v) gradually decreased, and its electronegativity increased first and then decreased. Meanwhile, the surface hydrophobicity of QPI gradually increased as the E/S increased. At the same time, the Th T fluorescence intensity of QPI aggregates showed an upward trend as E/S rose from 0 to 0.08%, and then declined when the E/S further increased to 0.14%. Through TEM observation, short fibrils (145-306 nm) and long fibrils (217-406 nm) were formed when E/S was 0.05% and 0.08%, respectively. However, with the further increase of E/S ratio, the length of fibrils became shorter, and more amorphous aggregates appeared. In addition, it was found that the aggregation behavior of QPI had a significant effect on its gelling properties (P<0.05). QPI gels showed enhanced hardness, higher storage modulus and a denser network structure, when fibrillar aggregation was dominant. Compared with short fibrils, long fibrils exerted a more significant effect to improve the gelling properties. Furthermore, alcalase showed a significant effect on the protein secondary structure of QPI gels, and the contents of β-sheet and random coil rose first and then decreased with the increase of E/S.【Conclusion】The limited alcalase hydrolysis promoted QPI to form ordered fibrillar aggregates, and further improved its gelling properties. When the E/S was 0.08%, QPI formed the longest fibrils, and QPI gels exhibited the highest hardness as well as the most favorable viscoelastic properties. Meanwhile, the protein secondary structure of QPI gels was ordered, and their microstructure was dense. However, the higher degree of hydrolysis was unfavorable to form fibrillar aggregates and failed to improve the gelling properties. Therefore, limited alcalase hydrolysis could significantly improve the weak gelling properties of QPI through fibrillation.

Key words: quinoa protein isolate (QPI), alcalase, limited hydrolysis, fibrillar aggregates, gel network, protein secondary structure

冯潇, 魏建锋, 付丽霄, 武朝升, 杨玉玲, 汤晓智. 碱性蛋白酶水解对藜麦分离蛋白结构、聚集行为及凝胶性的影响[J]. 中国农业科学, 2025, 58(1): 170-181.

FENG Xiao, WEI JianFeng, FU LiXiao, WU ChaoSheng, YANG YuLing, TANG XiaoZhi. Effects of Alcalase Hydrolysis on the Structure, Aggregation Behavior and Gelling Properties of Quinoa Protein Isolate[J]. Scientia Agricultura Sinica, 2025, 58(1): 170-181.

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https://www.chinaagrisci.com/CN/Y2025/V58/I1/170

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酶底比 Enzyme substrate ratio (%)	条带强度Band intensity (%)
	非还原条件 Non-reducing condition		还原条件 Reducing condition
	7S & 11S	2S	7S	AS	BS	2S
对照 Control	52.59	19.84	19.80	28.31	31.70	19.32
0.05	48.13	18.92	14.14	23.81	26.53	17.64
0.08	46.01	16.19	13.94	22.41	26.17	15.23
0.11	41.25	15.05	13.12	23.45	23.27	13.93
0.14	37.64	16.18	9.25	21.37	22.35	14.23

酶底比 Enzyme substrate ratio (%)	粒径 Particle size (nm)	分散性指数 PDI	Zeta电位 Zeta potential (mV)
对照 Control	1163.60±305.39a	0.59±0.15a	-11.87±0.35a
0.05	904.37±222.62ab	0.51±0.06a	-13.17±0.37b
0.08	603.13±107.06b	0.39±0.11a	-14.93±0.12d
0.11	571.27±40.81b	0.53±0.35a	-14.73±0.67cd
0.14	545.60±82.69b	0.48±0.64a	-13.83±0.81bc

组别Group	E/S 0.05%	E/S 0.08%	E/S 0.11%	E/S 0.14%
长度 Length (nm)	225.98±80.44bc	312.20±94.24a	182.16±47.12c	247.82±87.34ab
直径 Diameter (nm)	10.13±2.27a	5.46±2.34c	7.87±1.50b	9.84±1.61ab

酶底比 E/S (%)	α-螺旋 α-helix (%)	Β-折叠 β-sheet (%)	β-转角 β-turn (%)	无规则卷曲 Random coil (%)
对照 Control	13.74±2.59a	14.45±0.32cd	30.12±0.72a	37.45±0.67bc
0.05	12.39±0.50b	15.81±0.81b	26.95±1.84c	36.69±3.19c
0.08	13.04±0.26ab	16.85±1.39a	27.52±0.41c	39.17±0.45a
0.11	12.77±1.03ab	15.01±0.62c	28.81±1.08b	38.16±0.39ab
0.14	12.97±0.63ab	14.20±0.68d	28.93±0.84b	38.35±0.64ab