食品工业科技

大分子拥挤剂对熔球态大豆11S球蛋白热稳定性的影响

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杨杨 , 张璨 , 马春敏 , 王冰 , 边鑫 , 张光 , 刘晓飞 , 邹灵 , 张娜 ^*

食品工业科技 | 研究与探讨 2026,47(7): 53-60

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食品工业科技 |研究与探讨 2026 , 47 (7) : 53 -60

大分子拥挤剂对熔球态大豆11S球蛋白热稳定性的影响

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杨杨, 张璨, 马春敏, 王冰, 边鑫, 张光, 刘晓飞, 邹灵, 张娜^*

作者信息

哈尔滨商业大学食品工程学院，黑龙江哈尔滨 150028

通讯作者:

张娜（1979−），女，博士，教授，研究方向：谷物化学与粮食高值化利用，E-mail：foodzhangna@163.com。

作者简介:

杨杨（1991−），女，博士，副教授，研究方向：大豆、谷物化学及加工机理研究，E-mail：foodyangyang@163.com

Effect of Macromolecular Crowding Agents on the Thermal Stability of Molten Globule-state Soybean 11S Globulin

Yang YANG, Can ZHANG, Chunmin MA, Bing WANG, Xin BIAN, Guang ZHANG, Xiaofei LIU, Ling ZOU, Na ZHANG^*

Affiliations

College of Food Science and Engineering, Harbin University of Commerce, Harbin 150028, China

出版时间: 2026-04-01 doi: 10.13386/j.issn1002-0306.2025010076

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摘要

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本研究旨在探讨不同浓度大分子拥挤剂聚蔗糖70（Ficoll 70）对熔球态大豆11S球蛋白在100 ℃水浴中加热20 min高温处理下的热稳定性及功能特性的影响。结果表明：与未加入Ficoll 70处理的熔球态11S球蛋白相比，低浓度Ficoll 70（2~40 mg/mL）维持熔球态二级结构，内源荧光强度和表面疏水性降低，且在40 mg/mL Ficoll 70时热处理后熔球态蛋白的结构与未处理熔球态11S球蛋白最相似。同时Ficoll 70的加入减少分子聚集，增强分子柔性，并显著提高其乳化性和起泡性。当Ficoll 70浓度为40 mg/mL时，热处理后熔球态11S球蛋白平均粒径减小，EAI和ESI提高至12.35±0.01 m²/g和199.95%±0.29%，FC和FS分别从54.29%±0.58%增加至63.71%±1.89%，57.81%±0.51%增加至86.53%±0.46%。然而当Ficoll 70浓度达到50 mg/mL时，其对蛋白质的稳定性和功能特性产生负面影响，导致分子聚集和功能特性下降。以上结果表明，添加适量浓度的Ficoll 70可以通过排除体积效应调节熔球态大豆11S球蛋白的结构，从而影响熔球态大豆11S球蛋白的功能特性。研究为利用大分子拥挤剂改善熔球态大豆11S球蛋白的热稳定性和功能特性提供了理论依据。

关键词

大分子拥挤剂 / 熔球态 / 大豆11S球蛋白 / 结构 / 功能特性 / 热稳定性

Abstract

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This study aimed to investigate the effects of different concentrations of macromolecular crowding agent Ficoll 70 on the thermal stability and functional properties of fused globular soybean 11S globulin in a 100 ℃ water bath heated for 20 min. The results showed that low concentrations of Ficoll 70 (2~40 mg/mL) maintained the secondary structure of fused globular 11S globulin, and the intrinsic fluorescence intensity and surface hydrophobicity were reduced, compared with those without Ficoll 70 treatment. The structure of fused globulin after heat treatment was the most similar to that of untreated fused globular 11S globulin at 40 mg/mL Ficoll 70. Meanwhile, the addition of Ficoll 70 reduced molecular aggregation, enhanced molecular flexibility, and significantly improved its emulsifying and foaming properties. When the concentration of Ficoll 70 was 40 mg/mL, the average particle size of fused globular 11S globulin decreased, EAI and ESI increased to 12.35±0.01 m²/g and 199.95%±0.29%, FC and FS increased from 54.29%±0.58% to 63.71%±1.89%, 57.81%±0.51% to 86.53%±0.46%, respectively. However, when the concentration of Ficoll 70 reached 50 mg/mL, it had a negative impact on the stability and functional properties of the protein, resulting in molecular aggregation and a decline in functional properties. The above results showed that the addition of an appropriate concentration of Ficoll 70 could adjust the structure of fused globular soybean 11S globulin by eliminating the volume effect, thus affecting the functional properties of fused globular soybean 11S globulin. This study provides a theoretical basis for improving the thermal stability and functional properties of fused globular soybean 11S globulin by using a macromolecular crowding agent.

Key words

macromolecular crowding / molten globule / soybean 11S globulin / structure / functional properties / thermal stability

引用本文

杨杨, 张璨, 马春敏, 王冰, 边鑫, 张光, 刘晓飞, 邹灵, 张娜. 大分子拥挤剂对熔球态大豆11S球蛋白热稳定性的影响. 食品工业科技, 2026 , 47 (7) : 53 -60 . DOI: 10.13386/j.issn1002-0306.2025010076

Yang YANG, Can ZHANG, Chunmin MA, Bing WANG, Xin BIAN, Guang ZHANG, Xiaofei LIU, Ling ZOU, Na ZHANG. Effect of Macromolecular Crowding Agents on the Thermal Stability of Molten Globule-state Soybean 11S Globulin[J]. Science and Technology of Food Industry, 2026 , 47 (7) : 53 -60 . DOI: 10.13386/j.issn1002-0306.2025010076

正文

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大豆蛋白是一种具有高营养价值和良好功能特性的优质植物蛋白，且价格低廉，其在食品等领域已得到广泛的应用。大豆蛋白根据沉降系数一般可以分为2S、7S、11S和15S，其中，大豆11S球蛋白约占大豆蛋白的50%，并独立构成大豆11S组分，对大豆蛋白的功能性质有重要的影响^[1]。由于大豆11S球蛋白中硫醇和二硫键的存在，且不同加工条件易使蛋白质结构被破坏，导致其疏水作用增加，往往形成聚集体降低其功能特性，限制其在食品加工中的应用^[2]。蛋白质是由肽链连接折叠而成的具有特定二级和三级结构的氨基酸聚合物，其折叠和展开的过程能够影响其功能特性^[3]。研究发现在球蛋白折叠过程中的天然状态和变性状态之间存在一种中间状态—熔球态（MG），部分折叠的熔球态蛋白是一种紧凑构象，其二级结构和黏度特性与天然蛋白相似，由肽侧链决定的三级结构松散^[4]。由于其特定的结构，与未变性态和完全变性态的蛋白相比，它具有良好的功能特性，如乳化性、起泡性、凝胶性等^[5]。但熔球态作为中间状态，吉布斯自由能较高，导致MG分子易发生聚集，降低了其稳定性^[2]。因此，维持MG球蛋白的稳定性对提高大豆11S球蛋白商用价值具有重要意义。

蛋白质稳定性受蛋白质自身结构和外界条件（环境因素、溶液因素和工艺因素）的影响^[6]。MG态是具有不同无序范围的构象环境，具有瞬态寿命，在中性pH下的正常折叠条件下研究比较困难^[7]。已有研究表明，在极端pH条件下，盐溶液中的阳离子和阴离子会与蛋白质发生键合作用，从而稳定MG状态^[8−9]。添加大分子拥挤剂也是稳定蛋白质构象的重要手段。大分子拥挤剂是指在高浓度下通过占据溶液体积，改变其他大分子行为的物质^[10]。研究表明适量浓度的大分子拥挤剂增强了折叠和部分折叠蛋白质的稳定性和结构含量^[11−13]，一般机制是通过硬核排斥和化学相互作用共同作用。在硬核斥力下，大分子拥挤剂减少可用空间并诱导蛋白质的致密性。硬核排斥只考虑分子的排列，它还通过降低蛋白质解折叠的熵提高蛋白质的稳定性^[14]。化学相互作用中在吸引相互作用的情况下，溶质与蛋白质骨架非特异性结合导致渗透物被排除在蛋白质表面，导致蛋白质水合，由于蛋白质未折叠状态的溶剂可及表面积的增加，使折叠平衡向天然状态转移^[15−16]。Mcphie等^[17]发现加入大分子拥挤试剂能够使去折叠态脱辅基肌红蛋白向熔球态转变，并且这种现象与温度无关。Kumari等^[18]研究表明大分子拥挤效应能够促使酸变性铁细胞色素C稳定并重折叠为MG状态，且不同浓度的大分子拥挤剂影响蛋白质的热力学稳定性。目前研究使用的大分子拥挤剂包括聚乙二醇、葡聚糖70（Dextran70）、聚蔗糖70（Ficoll 70）、血红蛋白等。其中，Ficoll 70因其化学惰性最为常用。Ficoll 70是一种高度交联的蔗糖或环氧氯丙烷共聚物，表现为理想的中性半刚性球体^[19−20]。有研究表明Ficoll 70与肌红蛋白的血红素部分结合，化学相互作用在排除体积效应中占主导地位，从而促进其熔球态的形成^[21]。然而，大分子拥挤剂对蛋白质的稳定性影响较为复杂，它会随蛋白质的种类、形状和拥挤剂种类等因素发生变化。同时，大多数关于MG状态的研究都集中在相对低分子量的蛋白质上，针对大分子蛋白如大豆蛋白的研究较少，且关于大分子拥挤现象对熔球态蛋白质结构稳定性的研究较少，结果也不一致。因此，大分子拥挤现象对蛋白质结构的影响规律还需进一步探索。蛋白质稳定性的研究对于揭示蛋白质结构与功能之间的密切关系具有重要意义。目前高温酶催化、高温杀菌（100 ℃加热20 min）等实际应用中的蛋白稳定性问题有待研究^[22]，例如熔球态大豆11S球蛋白的热稳定性的研究。

因此，本研究以Ficoll 70作为大分子拥挤剂，通过远紫外圆二色谱、内源荧光光谱，以及乳化特性和起泡特性等指标探究在食品加工中常规的高温灭菌（100 ℃加热20 min）下大分子拥挤处理对熔球态大豆11S球蛋白的热稳定性及功能特性的影响。本文为通过添加大分子拥挤剂的手段稳定熔球态蛋白方面的研究提供一定理论参考。

1　材料与方法

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1.1　材料与仪器

大豆（黑农48）　黑龙江省农业科学院大豆研究所；九三非转基因一级大豆油（食品级）　九三粮油集团有限公司；聚蔗糖70（Ficoll70）　生工生物工程（上海）股份有限公司；5,5'-二硫双-2-硝基苯甲酸（DTNB）　北京博润莱特科技有限公司；8-苯胺-1-萘磺酸（ANS）　上海阿拉丁生化科技股份有限公司；胰凝乳蛋白酶（800 U/mg）　国药集团化学试剂有限公司；95%乙醇、正己烷（分析纯）　天津市富宇精细化工有限公司；所有指标测定的试剂均为分析纯。

SCIENTZ-12N冷冻干燥机　宁波新芝生物科技股份有限公司；TD5A台式离心机　盐城市凯特仪器有限公司；Chirascan圆二色谱仪　英国应用光物理公司；722分光光度计　上海光谱仪器有限公司；F-7000荧光分光光度计　日本Hitachi公司；Zetasizer Nano S90激光粒度仪　马尔文仪器公司；HH-4数显恒温水浴锅　上海力辰邦西仪器科技有限公司；UV-2600岛津紫外可见分光光度计　岛津企业管理（中国）有限公司；Ultra-Turrax T25高速分散器　德国IKA公司。

1.2　实验方法

1.2.1　大豆11S球蛋白的制备

参考解长远等^[23]的方法稍作修改。将大豆脱皮粉碎后过80目筛，将大豆粉、95%乙醇、正己烷按照比例为1:1:3（w:v:v）混合，置于25 ℃水浴振荡器中振荡脱脂（190 r/min），每2 h抽滤一次，直至锥形瓶中脱脂液呈无色透明状，干燥得到脱脂大豆粉。

将脱脂大豆粉与去离子水按照料液比1:15（m:v）混合，用2 mol/L的NaOH溶液调节pH至7.5，磁力搅拌2 h，在4 ℃下，4000 r/min离心30 min，取上清液，添加亚硫酸钠粉末保持其浓度为0.98 g/L，搅拌提取1 h后用2 mol/L的HCl将溶液的pH调至6.4，于4 ℃冰箱中静置12 h。然后经过4000 r/min离心20 min保留沉淀，用去离子水水洗2~3次后溶解于去离子水中，用2 mol/L的NaOH溶液调节pH至7.0，混合均匀后冷冻干燥，即可得到大豆11S球蛋白冻干粉（纯度92.01%）。

1.2.2　样品的制备

根据李翔鹏^[24]的方法稍作修改，将用pH7.0的磷酸盐缓冲溶液配制一定浓度的大豆11S蛋白溶液在100 ℃水浴中加热3 min，形成MG态的大豆11S球蛋白溶液。添加不同浓度（0、2、4、6、8、10、20、30、40、50 mg/mL）的Ficoll 70配制成Ficoll 70-熔球态11S球蛋白溶液后，在100 ℃水浴中加热20 min，再5000 r/min离心5 min，得上清液为实验样品（以下简称：Ficoll 70-熔球态蛋白溶液）。

1.2.3　紫外圆二色谱测定

参照Yang等^[25]的方法稍作修改。在波长范围为200~250 nm，光程为0.2 cm的条件下，进一步测定二级结构变化。各Ficoll 70-熔球态蛋白溶液中，熔球态大豆11S球蛋白的浓度均为0.1 mg/mL，扫描三次。

1.2.4　内源荧光光谱测定

根据Wang等^[26]的方法稍作修改。各Ficoll 70-熔球态蛋白溶液中，熔球态大豆11S球蛋白的浓度均为1.0 mg/mL。设定狭缝宽度为5 nm，激发波长为290 nm，激发起始波长为300 nm，发射结束波长为400 nm，1 cm石英比色皿样品池，扫描3次，得到各样品300~400 nm的荧光强度。

1.2.5　表面疏水性测定

用8-苯胺基萘-1-磺酸铵盐（ANS）染料作为荧光探针，通过荧光光谱法检测发光强度进行表征。将3.0 mL的Ficoll 70-熔球态蛋白溶液（熔球态大豆11S球蛋白的浓度为0.5 mg/mL）与50 μL的ANS溶液（8.0 mmol/L）混合均匀，测定样品荧光强度。狭缝宽度设为5 nm，激发波长为390 nm，记录波长为400~600 nm下光谱变化，扫描三次^[27]。

1.2.6　平均粒径测定

参照赵飞^[28]的实验方法，各Ficoll 70-熔球态蛋白溶液中，熔球态大豆11S球蛋白浓度均为0.1 mg/mL，以4000 r/min的转速离心15 min，以孔径为0.45 μm水系醋酸纤维素滤膜过滤后通过马尔文激光粒度仪进行粒径的测定。具体测定参数：样品折射率为1.145，分散相折射率为1.33，在室温下平衡时间2 min。

1.2.7　柔性测定

将250 μL 1 mg/L的胰凝乳蛋白酶溶液（0.05 mol/L，pH8.0 Tris-HCl缓冲液溶解）加入4 mL Ficoll 70-熔球态大豆11S球蛋白溶液（熔球态大豆11S球蛋白的浓度为1.0 mg/mL）中混合均匀，立即在38 ℃水浴中反应5 min，然后用4 mL 5% TCA（三氯乙酸）停止酶反应。在4000 r/min下离心30 min，用分光光度计在280 nm处测定上清液的吸光度。

1.2.8　乳化性及乳化稳定性测定

参考Zhou等^[29]的实验方法并稍作修改。配制Ficoll 70-熔球态大豆11S球蛋白溶液（熔球态大豆11S球蛋白浓度为1.0 mg/mL），取其15 mL与5 mL大豆油混合，以12000 r/min均质2 min后，立即在容器底部取100 μL匀浆，与5 mL 0.1%十二烷基硫酸钠（SDS）溶液均匀混合。以0.1% SDS溶液为空白，在500 nm处分别测定其0 min和30 min时吸光度A₀和A₃₀，表征其乳化性和乳化稳定性。乳化性（EAI）和乳化稳定性（ESI）分别由公式（1）和公式（2）计算：

$\rm EAI(m^2/g)=\frac{2\times 2.303 \times {{A}}_{{0}}\times n}{c\times ({1-\varphi })\times 10000} $

(1)

$\rm ESI({\text{%}})=\frac{{{A}}_{{30}}}{{{A}}_{{0}}}\times 100 $

(2)

式中：EAI为乳化活性，m²/g；ESI为乳化稳定性，%；A₀为0 min的吸光度值；n为稀释倍数；c为形成乳状液之前的蛋白溶液的浓度，g/mL；φ为乳状液中油相体积分数；A₃₀为静置30 min后的吸光度值。

1.2.9　起泡性及起泡稳定性测定

参考何兴芬^[30]的实验方法稍作修改。将15 mL 1.0 mg/mL Ficoll 70-熔球态大豆11S球蛋白溶液以12000 r/min均质1 min产生泡沫，均质后立即记录泡沫体积V₁，静置30 min后再次记录泡沫体积V₂。起泡性（FC）与起泡稳定性（FS）分别由公式（3）和公式（4）计算：

$ \text{FC(\text{%})}=\frac{{\text{V}}_{\text{1}}}{{\text{V}}_{\text{0}}}\text{×100} $

(3)

$ \text{FS(\text{%})}=\frac{{\text{V}}_{\text{2}}}{{\text{V}}_{\text{1}}}\text{×100} $

(4)

式中：FC为起泡性，%；FS为起泡稳定性，%；V₀为初始溶液体积，mL；V₁为均质1 min后泡沫体积，mL；V₂为静置30 min后泡沫体积，mL。

1.3　数据处理

所有实验每次进行至少三次平行实验，结果以平均值±标准差表示，采用SPSS 19.0软件进行方差分析，P<0.05表示差异显著，采用Origin Pro 2019b软件作图。

2　结果与分析

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2.1　Ficoll 70-熔球态11S球蛋白远紫外圆二色谱分析

通过远紫外圆二色谱可以反映不同处理条件下的熔球态大豆11S球蛋白二级结构含量的变化。如图1所示，不同处理条件下的熔球态大豆11S球蛋白的远紫外圆二色谱曲线在212 nm附近呈负峰，这表明熔球态蛋白的二级结构与天然态蛋白相似，有部分α-螺旋构象存在^[24]。图1表明在100 ℃水浴中加热20 min时与熔球态大豆11S球蛋白的圆二色谱曲线相比，未添加Ficoll 70的熔球态大豆11S球蛋白经加热后的圆二色谱曲线的负峰位置发生轻微蓝移，但整体曲线向下移动，这表明高温处理会导致熔球态大豆11S球蛋白的二级结构重排，促使α-螺旋解折叠并转化为β-折叠。这是由于高温加热诱导熔球态大豆11S球蛋白的分子间疏水相互作用增强，形成富含β-折叠的熔球态蛋白分子聚集体，使有序二级结构丢失^[31]。相较于未添加Ficoll 70的熔球态大豆11S球蛋白，在加入2~40 mg/mL的Ficoll 70加热后，随着加入的Ficoll 70浓度的增加，其圆二色谱曲线的负峰位置发生红移，且曲线的振幅逐渐减弱，与熔球态大豆11S球蛋白的圆二色谱曲线相似，这表明Ficoll 70的加入使熔球态蛋白分子的α-螺旋构象含量增加，更好地维持熔球态。因为Ficoll 70作为一种大分子拥挤剂，其高浓度通过占据溶液中的有效体积，通过空间限制作用，阻碍蛋白质在加热时完全解折叠，维持其熔球态的二级结构特征，同时通过空间位阻效应抑制分子间接触，阻止聚集体的形成^[12]。而加入50 mg/mL Ficoll 70后，其圆二色谱曲线负峰位置发生蓝移，且振幅增强，说明此时熔球态大豆11S球蛋白的α-螺旋含量减少，这是因为过高浓度的大分子拥挤剂的机械相互作用导致熔球态大豆11S球蛋白折叠状态改变，抑制了熔球态11S球蛋白的移动，迫使蛋白质间紧密靠近，形成聚集体，损害拥挤剂对蛋白质稳定性的有利影响^[32]。

2.2　Ficoll 70-熔球态11S球蛋白内源荧光光谱分析

如图2所示，在加热处理后未加入Ficoll 70的熔球态大豆11S球蛋白与熔球态大豆11S球蛋白相比，荧光强度显著增强，这表明加热处理后的熔球态大豆11S球蛋白结构被破坏，包埋在分子内部的色氨酸基团进一步暴露。当Ficoll 70浓度增加至10 mg/mL后，加热处理后熔球态大豆11S球蛋白的内源荧光强度呈减弱趋势，这是因为Ficoll 70将熔球态蛋白分子包裹在内，使得蛋白分子的色氨酸基团被掩埋，且随着Ficoll 70浓度的增加，包裹得越紧密，使得蛋白分子发生了一定的重折叠。此外，Ficoll 70的排阻体积效应增强，使色氨酸和邻近氨基酸残基之间形成基态络合物，从而通过静态机制发生荧光猝灭^[33]。然而，当Ficoll 70浓度增加至50 mg/mL时，加热处理后熔球态大豆11S球蛋白的内源荧光强度甚至低于熔球态大豆11S球蛋白，这表明过高的Ficoll 70浓度导致蛋白质-拥挤剂相互作用开始产生负面影响^[32]。上述结果表明一定浓度的Ficoll 70有效地抑制高温变性，且具有浓度依赖性，这与Kumar等^[7]研究发现反应介质中存在拥挤剂是碱基变性蛋白质热稳定性增加的原因的结论相似。

2.3　Ficoll 70-熔球态11S球蛋白表面疏水性分析

ANS作为一种疏水探针，在与蛋白质表面的疏水区域结合时会改变荧光特性^[34]，故通过ANS荧光光谱探究体系中的疏水性变化，如图3所示。与熔球态大豆11S球蛋白相比，100 ℃加热处理后未加入Ficoll 70的熔球态大豆11S球蛋白荧光强度发生大幅度增强，这说明高温环境使熔球态大豆11S球蛋白结构伸展，进一步暴露内部疏水基团，促进与ANS荧光探针结合^[23]。与仅热处理的熔球态大豆11S球蛋白相比，随Ficoll 70浓度的增加，热处理的熔球态大豆11S球蛋白的外源荧光强度逐渐减小，且当Ficoll 70浓度达到50 mg/mL时其荧光强度甚至低于未加热处理的熔球态大豆11S球蛋白。这是由于在2~40 mg/mL范围内，Ficoll 70通过空间位阻抑制分子间接触，其物理约束作用阻碍熔球态大豆11S球蛋白疏水区域完全暴露，进而维持熔球态大豆11S球蛋白的结构稳定^[35]。然而，当Ficoll 70浓度达到50 mg/mL时，过度拥挤打破平衡形成致密聚集体，进一步掩埋疏水区域^[32]。

2.4　Ficoll 70-熔球态11S球蛋白平均粒径结果分析

粒度测定是表征蛋白质大小的一种非常有效敏感的技术^[36]。图4所示为100 ℃热处理20 min后添加不同浓度的Ficoll 70的熔球态大豆11S球蛋白平均粒径的变化。与未处理的熔球态大豆11S球蛋白的粒径（11.11±0.20 nm）相比，100 ℃加热20 min后的熔球态大豆11S球蛋白分子的平均粒径增加至253.23±3.82 nm。这是由于加热导致熔球态蛋白结构伸展，增加其表面疏水性，使蛋白间疏水作用增强，促使蛋白间发生聚集，从而增大熔球态大豆11S球蛋白的平均粒径^[37]。与未加Ficoll 70热处理后的熔球态11S球蛋白相比，在加入2~40 mg/mL Ficoll 70热处理后的熔球态11S球蛋白的平均粒径显著减小（P<0.05），且Ficoll 70浓度为40 mg/mL时平均粒径由253.97±3.82 nm降至74.65±0.24 nm，这表明此时熔球态11S球蛋白仍具有MG状态下的相对致密的结构，没有完全延伸，这是由于Ficoll 70将熔球态大豆11S球蛋白包裹，使熔球态大豆11S球蛋白结构稳定，且减少蛋白分子间疏水相互作用，阻碍聚集体的形成^[3]。而Ficoll 70浓度达到50 mg/mL后，热处理后的熔球态11S球蛋白分子的平均粒径显著增大（P<0.05），这说明当Ficoll 70浓度超过某一临界值，其占据的溶剂体积过大，导致蛋白质分子被强制压缩并紧密靠近，反而形成粒径较大的聚集体^[32]。

2.5　Ficoll 70-熔球态11S球蛋白分子柔性的分析

添加不同浓度的Ficoll 70的熔球态11S球蛋白分子的柔性变化如图5所示。图5显示加热处理后未添加Ficoll 70的熔球态11S球蛋白柔性降低，这是由于温度的升高使熔球态大豆11S球蛋白结构改变，β-折叠结构含量增加，发生疏水性聚集，进而限制了其灵活性^[38]，这一结果与2.3相一致。在添加浓度2~40 mg/mL下，随着Ficoll 70浓度的增大，熔球态大豆11S球蛋白分子柔性逐渐增大，这种现象产生的原因是Ficoll 70的添加导致蛋白质的疏水收缩，抑制了蛋白的聚集，使其分子柔性增大^[6]。而加入50 mg/mL Ficoll 70后，熔球态蛋白分子柔性减小，这是因为此时Ficoll 70的空间排除体积效应增强，这可能会损害拥挤剂对蛋白质稳定性的有利影响。

2.6　Ficoll 70-熔球态11S球蛋白乳化性（EAI）及乳化稳定性（ESI）分析

不同浓度Ficoll 70对乳化性及乳化稳定性的影响如图6所示。未加入Ficoll 70的熔球态蛋白经热处理后，与熔球态大豆11S球蛋白（乳化性为13.85 m²/g，乳化稳定性为224.06%）相比，乳化性降低29.03%，乳化稳定性降低166.84%，这是由于经热处理的熔球态蛋白的分子柔性降低，表面疏水性增加，使熔球态蛋白在油水界面吸附能力降低，且聚集导致的较大粒径，使其分子扩散速率降低^[39]，从而降低熔球态11S球蛋白EAI和ESI。然而，随着加入Ficoll 70的浓度的增加，熔球态蛋白的EAI和ESI也增加，当Ficoll 70浓度为40 mg/mL时有最大EAI和ESI，分别为12.35±0.01 m²/g和199.95%±0.29%。这说明Ficoll 70的加入有利于提高熔球态11S球蛋白的EAI和ESI，一方面是因为Ficoll 70包裹熔球态11S球蛋白，在排阻体积效应下促使熔球态蛋白分子结构再折叠，减小表面疏水性，且Ficoll 70浓度的增加增大溶液黏度，有效防止絮凝，与Lee等^[40]结论相似；另一方面是因为Ficoll 70与熔球态蛋白分子发生化学相互作用^[41]，生成的复合物既有蛋白质的表面活性又有Ficoll 70的亲水性，具有良好的乳化活性。然而，当加入的Ficoll 70浓度达到50 mg/mL时，EAI和ESI开始下降，这是由于蛋白体系过度拥挤，拥挤剂的机械相互作用导致折叠状态的局部变形，产生更紧凑结构的蛋白聚集体，进而影响其功能特性^[32]。

2.7　Ficoll 70-熔球态11S球蛋白起泡性（FC）及起泡稳定性（FS）分析

如图7所示，未加入Ficoll 70的熔球态蛋白经热处理后的FC和FS与未经热处理的熔球态大豆11S球蛋白相比，分别减小了28.94%和65.16%。这是因为加热使熔球态11S球蛋白结构被破坏，降低其溶解性，且减小蛋白柔性，从而降低泡沫形成能力。图7显示随Ficoll 70浓度的增加，熔球态11S球蛋白的FC和FS呈现先升高后下降的趋势，当Ficoll 70浓度为40 mg/mL时有最大FC和FS，分别从54.29%±0.58%增加至63.71%±1.89%，57.81%±0.51%增加至86.53%±0.46%，这表明Ficoll 70与熔球态11S球蛋白发生化学相互作用形成的复合物具有两亲性，增加蛋白的溶解性，从而利于泡沫形成。此外，Ficoll 70通过排除体积效应维持熔球态11S球蛋白的部分展开状态，这既保留其一定柔性又避免完全聚集，促进熔球态11S球蛋白形成更致密的气-液界面层，减缓泡沫厚度变薄的速度，从而提高其FS^[3,27]。然而当Ficoll 70浓度超过40 mg/mL时，热处理的熔球态11S球蛋白的FC和FS开始下降，此时蛋白体系过度拥挤，Ficoll 70的空间阻碍作用增强，使蛋白结构进一步再折叠形成致密结构^[18]，阻碍蛋白分子迁移到气-液界面，且形成的界面膜结构缺乏弹性，导致FC和FS下降。

3　结论

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本研究揭示了大分子拥挤剂Ficoll 70对熔球态大豆11S球蛋白在高温处理下的热稳定性、其结构的变化及功能特性的影响。远紫外圆二色谱、荧光光谱以及表面疏水性和平均粒径结果分析表明适量Ficoll 70（2~40 mg/mL）通过分子拥挤效应抑制熔球态大豆11S球蛋白的高温解折叠，二级结构α-螺旋构象增加，维持其二级结构，三级结构折叠，减少疏水区域暴露，并有效抑制聚集（Ficoll 70浓度为40 mg/mL时平均粒径由253.97±3.82 nm降至74.65±0.24 nm），使得蛋白分子的柔韧性增加。其中Ficoll 70为40 mg/mL浓度适宜，低浓度Ficoll 70显著提升乳化性与起泡性，归因于其对蛋白质结构的稳定作用以及对表面疏水性的调节，增强蛋白界面吸附能力及液膜机械强度；而高浓度（50 mg/mL）因致密聚集导致功能劣化。因此，拥挤剂Ficoll 70对高温处理下熔球态大豆11S球蛋白的热稳定性和功能特性影响有一定的浓度依赖性。本研究发现通过调控分子拥挤剂浓度可实现熔球态蛋白热稳定性与功能特性的协同优化。

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2026年第47卷第7期

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doi: 10.13386/j.issn1002-0306.2025010076

接收时间：2025-01-09
首发时间：2026-10-24
出版时间：2026-04-01

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收稿日期：2025-01-09

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哈尔滨商业大学食品工程学院，黑龙江哈尔滨 150028

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张娜（1979−），女，博士，教授，研究方向：谷物化学与粮食高值化利用，E-mail：foodzhangna@163.com。

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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