摘 要
菜籽分离蛋白具有很高的营养价值，氨基酸种类丰富，同时也具有良好的功能结构特性，如溶解性、起泡性、乳化性等。我国菜籽分离蛋白的年产量巨大，但是对其的实际应用却远远不够，如何提升菜籽分离蛋白在食品工业的利用率成为了食品科学的研究重点，同时这也有着巨大的经济效益和现实意义。本文研究了pH值对菜籽分离蛋白功能特性的影响。由于菜籽分离蛋白分散体系的形成和稳定性与其界面特性有着密切关系，故本文接下来研究了pH值、菜籽分离蛋白浓度、离子强度对菜籽分离蛋白分子在气液界面的吸附动力学、体系泡沫特性的影响。研究结果及结论如下：
1. 随着pH值升高，随着碱性环境的增加，菜籽蛋白分离物的电泳带密度降低；表面疏水性也降低；红外光谱特征吸收峰的位置并未发生大的改变，但菜籽蛋白的透射率逐渐增加；通过扫描电子显微镜表征发现，随着pH的升高，蛋白质微观结构逐渐变得分散、紊乱程度逐渐增加。电位分析表明菜籽分离蛋白等电点约为4.2，随着pH值升高，其溶解度逐渐增大，与此同时，蛋白分子的粒径逐渐变小，说明在pH值升高，使蛋白质分子愈加分散；菜籽分离蛋白热重分析表明，随着pH值增加，菜籽分离蛋白的热稳定性逐渐降低。
2. 吸附动力学研究表明，表面压力π随着吸附时间增加而增强，增强过程可以分为三个阶段，诱导期、快速增长期、缓慢增长期。表面压力π小于10 mN/m时，曲线符合Ward-Tordai扩散模型，扩散控制吸附动力学，扩散速率和初始体相蛋白浓度呈正比，在pH值为7和9时扩散速率最大。；表面压力π大于10 mN/m时，曲线符合第一速率方程
，展开和重拍机制控制吸附动力学，展开和重排速率和初始体相蛋白浓度呈正比，但是初始体相蛋白浓度过高，反而抑制了其在气液界面展开和重排的速率。pH值为7和9时，蛋白分子容易在气液界面展开和重排，而在pH值为5时，因位于等电点附近，蛋白分子较为团聚，难以在界面展开和重排。菜籽蛋白的起泡性和起泡稳定性随pH值的增大而上升，在pH值为11时有小幅下降；随蛋白浓度的增加而增大。荧光光谱分析表明随着pH值的增加，蛋白表面色氨酸和酪氨酸暴露量逐渐增多，疏水性增强。
3. 通过研究离子强度对菜籽分离蛋白吸附动力学影响，发现表面压力π小于10 mN/m时，曲线同样符合Ward-Tordai扩散模型，扩散控制吸附动力学，π-t1/2曲线呈直线，扩散速率随着离子强度的增大而加快；表面压力π大于10 mN/m时，曲线符合第一速率方程，展开和重拍机制控制吸附动力学，展开和重排速率受到离子强度的影响，随着离子强度的增大，展开和重排速率也随着变大，在离子强度较低时，蛋白分子较为团聚，难以在界面展开和重排。随着离子强度的增大，菜籽分离蛋白的起泡性逐渐增大，在pH值为11时有小幅下降；在很高的离子强度下，会导致菜籽蛋白的分子结构柔性下降，导致了菜籽分离蛋白溶液的泡沫稳定性降低。荧光光谱分析表明随着离子强度的增加，菜籽蛋白构想先发生团聚，而后又逐渐展开，在谱图上体现为荧光强度先降低在增加，最大吸收峰所处波长先发生蓝移，再发生红移。
关键词：菜籽分离蛋白；功能特性；气液界面吸附动力学；泡沫特性
菜籽分离蛋白具有很高的营养价值，氨基酸种类丰富，同时也具有良好的功能结构特性，如溶解性、起泡性、乳化性等。我国菜籽分离蛋白的年产量巨大，但是对其的实际应用却远远不够，如何提升菜籽分离蛋白在食品工业的利用率成为了食品科学的研究重点，同时这也有着巨大的经济效益和现实意义。本文研究了pH值对菜籽分离蛋白功能特性的影响。由于菜籽分离蛋白分散体系的形成和稳定性与其界面特性有着密切关系，故本文接下来研究了pH值、菜籽分离蛋白浓度、离子强度对菜籽分离蛋白分子在气液界面的吸附动力学、体系泡沫特性的影响。研究结果及结论如下：
1. 随着pH值升高，随着碱性环境的增加，菜籽蛋白分离物的电泳带密度降低；表面疏水性也降低；红外光谱特征吸收峰的位置并未发生大的改变，但菜籽蛋白的透射率逐渐增加；通过扫描电子显微镜表征发现，随着pH的升高，蛋白质微观结构逐渐变得分散、紊乱程度逐渐增加。电位分析表明菜籽分离蛋白等电点约为4.2，随着pH值升高，其溶解度逐渐增大，与此同时，蛋白分子的粒径逐渐变小，说明在pH值升高，使蛋白质分子愈加分散；菜籽分离蛋白热重分析表明，随着pH值增加，菜籽分离蛋白的热稳定性逐渐降低。
2. 吸附动力学研究表明，表面压力π随着吸附时间增加而增强，增强过程可以分为三个阶段，诱导期、快速增长期、缓慢增长期。表面压力π小于10 mN/m时，曲线符合Ward-Tordai扩散模型，扩散控制吸附动力学，扩散速率和初始体相蛋白浓度呈正比，在pH值为7和9时扩散速率最大。；表面压力π大于10 mN/m时，曲线符合第一速率方程
，展开和重拍机制控制吸附动力学，展开和重排速率和初始体相蛋白浓度呈正比，但是初始体相蛋白浓度过高，反而抑制了其在气液界面展开和重排的速率。pH值为7和9时，蛋白分子容易在气液界面展开和重排，而在pH值为5时，因位于等电点附近，蛋白分子较为团聚，难以在界面展开和重排。菜籽蛋白的起泡性和起泡稳定性随pH值的增大而上升，在pH值为11时有小幅下降；随蛋白浓度的增加而增大。荧光光谱分析表明随着pH值的增加，蛋白表面色氨酸和酪氨酸暴露量逐渐增多，疏水性增强。3. 通过研究离子强度对菜籽分离蛋白吸附动力学影响，发现表面压力π小于10 mN/m时，曲线同样符合Ward-Tordai扩散模型，扩散控制吸附动力学，π-t1/2曲线呈直线，扩散速率随着离子强度的增大而加快；表面压力π大于10 mN/m时，曲线符合第一速率方程
，展开和重拍机制控制吸附动力学，展开和重排速率受到离子强度的影响，随着离子强度的增大，展开和重排速率也随着变大，在离子强度较低时，蛋白分子较为团聚，难以在界面展开和重排。随着离子强度的增大，菜籽分离蛋白的起泡性逐渐增大，在pH值为11时有小幅下降；在很高的离子强度下，会导致菜籽蛋白的分子结构柔性下降，导致了菜籽分离蛋白溶液的泡沫稳定性降低。荧光光谱分析表明随着离子强度的增加，菜籽蛋白构想先发生团聚，而后又逐渐展开，在谱图上体现为荧光强度先降低在增加，最大吸收峰所处波长先发生蓝移，再发生红移。 关键词：菜籽分离蛋白；功能特性；气液界面吸附动力学；泡沫特性
ABSTRACT
Rapeseed protein isolate has high nutritional value, rich amino acid species, and also has good functional and structural characteristics, such as solubility, foaming, emulsification, etc. The annual output of rapeseed protein in China is huge, but its practical application is far from enough. How to improve the utilization of rapeseed protein in the food industry has become the focus of food science research. Realistic significance. In this paper, the effect of pH on the functional properties of rapeseed protein isolate was studied. Because the formation and stability of rapeseed protein dispersion system is closely related to its interface characteristics, this paper next studies the adsorption kinetics of rapeseed protein molecules at the gas-liquid interface by pH, rapeseed protein concentration and ionic strength The influence of system foam characteristics. The findings and conclusions are as follows:
1. As the pH value increases, the alkaline environment increases, the density of the electrophoretic band and the surface hydrophobicity of rapeseed protein isolate decreases; the position of the characteristic absorption peak of the infrared spectrum does not change greatly, but the transmittance of rapeseed protein gradually increased; by scanning electron microscopy, it was found that with the increase of pH, the microstructure of the protein gradually became dispersed. Potential analysis shows that the isoelectric point of rapeseed protein isolate is about 4.2, and its solubility gradually increases with increasing pH. At the same time, the particle size of protein molecules gradually decreases, indicating that the increase in pH value causes protein molecules more dispersed; thermogravimetric analysis with the pH value increases, the thermal stability of rapeseed protein isolate gradually decreases.
2.The study of adsorption kinetics shows that the surface pressure π increases with the increase of the adsorption time. The enhancement process can be divided into three stages, the induction period, the rapid growth period, and the slow growth period. When the surface pressure π is less than 10 mN/m, the curve conforms to the Ward-Tordai diffusion model, diffusion controls adsorption kinetics, the diffusion rate is proportional to the initial bulk protein concentration, and the diffusion rate is maximum at pH 7 and 9. ; When the surface pressure π is greater than 10 mN/m, the curve conforms to the first rate equation the expansion and retake mechanism controls the adsorption kinetics, and The discharge rate is directly proportional to the initial body protein concentration, but the initial body protein concentration is too high, which inhibits its rate of expansion and rearrangement at the gas-liquid interface. At pH 7 and 9, protein molecules are easy to expand and rearrange at the gas-liquid interface. At pH 5, protein molecules are clustered near the isoelectric point, making it difficult to expand and rearrange at the interface. The foamability and foaming stability of rapeseed protein increased with the increase of pH value, and decreased slightly at pH 11; it increased with the increase of protein concentration. Fluorescence spectrum analysis showed that as the pH value increased, the exposure of tryptophan and tyrosine on the protein surface gradually increased, and the hydrophobicity increased.
3.By studying the influence of ionic strength on the adsorption kinetics of rapeseed protein isolate, it was found that when the surface pressure π is less than 10 mN / m, the curve also conforms to the Ward-Tordai diffusion model. The diffusion controls the adsorption kinetics. The rate increases with increasing ionic strength; when the surface pressure π is greater than 10 mN/m, the curve conforms to the first-rate equation.The beat mechanism controls the adsorption kinetics. The expansion and rearrangement rates are affected by ionic strength. As the ionic strength increases, the expansion and rearrangement rates also increase. When the ionic strength is low, the protein molecules are more agglomerated. With the increase of ionic strength, the foaming property of rapeseed protein gradually increases, and it decreases slightly at pH value 11. Under high ionic strength, the molecular structure of rapeseed protein flexibility decreases, resulting in decreased foam stability of the rapeseed protein isolate solution. Fluorescence spectrum analysis shows that as the ionic strength increases, the concept of rapeseed protein first agglomerates, and then gradually unfolds. The spectrum shows that the fluorescence intensity decreases first and then increases, and the wavelength is blue shifted first, then red shift.
KEY WORDS：rapeseed protein isolate; functional characteristics; interface adsorption kinetics; foam characteristics
Rapeseed protein isolate has high nutritional value, rich amino acid species, and also has good functional and structural characteristics, such as solubility, foaming, emulsification, etc. The annual output of rapeseed protein in China is huge, but its practical application is far from enough. How to improve the utilization of rapeseed protein in the food industry has become the focus of food science research. Realistic significance. In this paper, the effect of pH on the functional properties of rapeseed protein isolate was studied. Because the formation and stability of rapeseed protein dispersion system is closely related to its interface characteristics, this paper next studies the adsorption kinetics of rapeseed protein molecules at the gas-liquid interface by pH, rapeseed protein concentration and ionic strength The influence of system foam characteristics. The findings and conclusions are as follows:
1. As the pH value increases, the alkaline environment increases, the density of the electrophoretic band and the surface hydrophobicity of rapeseed protein isolate decreases; the position of the characteristic absorption peak of the infrared spectrum does not change greatly, but the transmittance of rapeseed protein gradually increased; by scanning electron microscopy, it was found that with the increase of pH, the microstructure of the protein gradually became dispersed. Potential analysis shows that the isoelectric point of rapeseed protein isolate is about 4.2, and its solubility gradually increases with increasing pH. At the same time, the particle size of protein molecules gradually decreases, indicating that the increase in pH value causes protein molecules more dispersed; thermogravimetric analysis with the pH value increases, the thermal stability of rapeseed protein isolate gradually decreases.
2.The study of adsorption kinetics shows that the surface pressure π increases with the increase of the adsorption time. The enhancement process can be divided into three stages, the induction period, the rapid growth period, and the slow growth period. When the surface pressure π is less than 10 mN/m, the curve conforms to the Ward-Tordai diffusion model, diffusion controls adsorption kinetics, the diffusion rate is proportional to the initial bulk protein concentration, and the diffusion rate is maximum at pH 7 and 9. ; When the surface pressure π is greater than 10 mN/m, the curve conforms to the first rate equation
the expansion and retake mechanism controls the adsorption kinetics, and The discharge rate is directly proportional to the initial body protein concentration, but the initial body protein concentration is too high, which inhibits its rate of expansion and rearrangement at the gas-liquid interface. At pH 7 and 9, protein molecules are easy to expand and rearrange at the gas-liquid interface. At pH 5, protein molecules are clustered near the isoelectric point, making it difficult to expand and rearrange at the interface. The foamability and foaming stability of rapeseed protein increased with the increase of pH value, and decreased slightly at pH 11; it increased with the increase of protein concentration. Fluorescence spectrum analysis showed that as the pH value increased, the exposure of tryptophan and tyrosine on the protein surface gradually increased, and the hydrophobicity increased.3.By studying the influence of ionic strength on the adsorption kinetics of rapeseed protein isolate, it was found that when the surface pressure π is less than 10 mN / m, the curve also conforms to the Ward-Tordai diffusion model. The diffusion controls the adsorption kinetics. The rate increases with increasing ionic strength; when the surface pressure π is greater than 10 mN/m, the curve conforms to the first-rate equation
.The beat mechanism controls the adsorption kinetics. The expansion and rearrangement rates are affected by ionic strength. As the ionic strength increases, the expansion and rearrangement rates also increase. When the ionic strength is low, the protein molecules are more agglomerated. With the increase of ionic strength, the foaming property of rapeseed protein gradually increases, and it decreases slightly at pH value 11. Under high ionic strength, the molecular structure of rapeseed protein flexibility decreases, resulting in decreased foam stability of the rapeseed protein isolate solution. Fluorescence spectrum analysis shows that as the ionic strength increases, the concept of rapeseed protein first agglomerates, and then gradually unfolds. The spectrum shows that the fluorescence intensity decreases first and then increases, and the wavelength is blue shifted first, then red shift. KEY WORDS：rapeseed protein isolate; functional characteristics; interface adsorption kinetics; foam characteristics
目 录
第一章 绪论
1.1 油菜及油菜籽概述
1.1.1 油菜的种类
1.1.2 油菜的分布
1.1.3 油菜籽
1.1.4 双低油菜籽粕
1.2 菜籽蛋白组成结构及功能特性研究
1.2.1 菜籽蛋白组成及结构
1.2.2 菜籽蛋白的功能特性
1.2.2.1 溶解性 4
1.2.2.2 吸水性
1.2.2.3 乳化性 4
1.2.2.4 起泡性 5
1.3 菜籽蛋白提取分离方法研究
1.3.1 菜籽蛋白提取方法
1.3.2 菜籽蛋白纯化方法
1.4菜籽蛋白在食品中的应用
1.5蛋白质界面吸附动力学研究概况
1.5.1 吸附动力学
1.5.2 吸附动力学与泡沫稳定性的关系
1.6课题的立项背景、意义及主要研究内容
1.6.1 立项背景、意义
1.6.2 主要研究内容
第二章 pH值对菜籽分离蛋白功能性质的影响
2.1材料与试剂
2.1.1 实验材料
2.1.2 实验试剂
2.1.3 实验仪器
2.2 实验方法
2.2.1菜籽蛋白结构性质分析方法
2.2.1.1 十二烷基硫酸钠一聚丙烯酰胺凝胶电泳(SDS-PAGE)测定 14
2.2.1.2 表面疏水性测定 1
2.2.1.3 巯基和二硫键含量测定 14
2.2.1.4 红外光谱（FT-IR）测定 15
2.2.1.5 扫描电镜（SEM）测定 15
2.2.2菜籽蛋白结构性质分析方法
2.2.2.1 溶解度测定 15
2.2.2.2 粒度分布和ζ电位的测定 15
2.2.2.3 差示热重扫描(DSC)测定 15
2.3 结果与讨论
2.3.1菜籽蛋白结构性质分析
2.3.1.1 SDS-PAGE分析 15
2.3.1.2表面疏水性分析 16
2.3.1.3巯基和二硫键含量分析 16
2.3.1.4 FT-IR分析 17
2.3.1.5 SEM分析 18
2.3.2菜籽蛋白功能特性分析
2.3.2.1 溶解性、电位图和粒度分析 19
2.3.2.2差示热重扫描(DSC)分析 20
2.4 本章小结 21
第三章 pH值和浓度对菜籽分离蛋白气液界面行为及泡沫特性的影响
3.1材料与试剂
3.1.1实验材料
3.1.2实验试剂
3.1.3实验仪器 22
3.2 实验方法 22
3.2.1界面压力检测 22
3.2.2起泡性及起泡稳定性测定 23
3.2.3荧光光谱测定 23
3.3 结果与讨论 24
3.3.1表面压力随吸附时间的变化 24
3.3.2菜籽蛋白气液界面扩散动力学 25
3.3.3菜籽蛋白气液界面展开和重排的动力学 28
3.3.4菜籽蛋白起泡性及起泡稳定性 30
3.3.5菜籽蛋白荧光光谱测定 32
3.4 本章小结 33
第四章 离子强度对菜籽分离蛋白气液界面行为及泡沫特性的影响
4.1材料与试剂 35
4.1.1实验材料 35
4.1.2实验试剂 35
4.1.3实验仪器 35
4.2 实验方法 35
4.2.1界面压力检测 35
4.2.2起泡性及起泡稳定性测定 35
4.2.3荧光光谱分析 35
4.3 结果与讨论 36
4.3.1表面压力随吸附时间的变化 36
4.3.2离子强度对气液界面扩散动力学的影响 36
4.3.3离子强度对气液界面展开和重排动力学的影响 38
4.3.4离子强度对起泡性及泡沫稳定性的影响 40
4.3.5不同离子强度下的菜籽分离蛋白荧光光谱分析 41
4.4 本章小结 42
第五章 结论
参考文献
致谢 52
攻读硕士学位期间科研成果情况 53
第一章 绪论
1.1 油菜及油菜籽概述
1.1.1 油菜的种类
1.1.2 油菜的分布
1.1.3 油菜籽
1.1.4 双低油菜籽粕
1.2 菜籽蛋白组成结构及功能特性研究
1.2.1 菜籽蛋白组成及结构
1.2.2 菜籽蛋白的功能特性
1.2.2.1 溶解性 4
1.2.2.2 吸水性
1.2.2.3 乳化性 4
1.2.2.4 起泡性 5
1.3 菜籽蛋白提取分离方法研究
1.3.1 菜籽蛋白提取方法
1.3.2 菜籽蛋白纯化方法
1.4菜籽蛋白在食品中的应用
1.5蛋白质界面吸附动力学研究概况
1.5.1 吸附动力学
1.5.2 吸附动力学与泡沫稳定性的关系
1.6课题的立项背景、意义及主要研究内容
1.6.1 立项背景、意义
1.6.2 主要研究内容
第二章 pH值对菜籽分离蛋白功能性质的影响
2.1材料与试剂
2.1.1 实验材料
2.1.2 实验试剂
2.1.3 实验仪器
2.2 实验方法
2.2.1菜籽蛋白结构性质分析方法
2.2.1.1 十二烷基硫酸钠一聚丙烯酰胺凝胶电泳(SDS-PAGE)测定 14
2.2.1.2 表面疏水性测定 1
2.2.1.3 巯基和二硫键含量测定 14
2.2.1.4 红外光谱（FT-IR）测定 15
2.2.1.5 扫描电镜（SEM）测定 15
2.2.2菜籽蛋白结构性质分析方法
2.2.2.1 溶解度测定 15
2.2.2.2 粒度分布和ζ电位的测定 15
2.2.2.3 差示热重扫描(DSC)测定 15
2.3 结果与讨论
2.3.1菜籽蛋白结构性质分析
2.3.1.1 SDS-PAGE分析 15
2.3.1.2表面疏水性分析 16
2.3.1.3巯基和二硫键含量分析 16
2.3.1.4 FT-IR分析 17
2.3.1.5 SEM分析 18
2.3.2菜籽蛋白功能特性分析
2.3.2.1 溶解性、电位图和粒度分析 19
2.3.2.2差示热重扫描(DSC)分析 20
2.4 本章小结 21
第三章 pH值和浓度对菜籽分离蛋白气液界面行为及泡沫特性的影响
3.1材料与试剂
3.1.1实验材料
3.1.2实验试剂
3.1.3实验仪器 22
3.2 实验方法 22
3.2.1界面压力检测 22
3.2.2起泡性及起泡稳定性测定 23
3.2.3荧光光谱测定 23
3.3 结果与讨论 24
3.3.1表面压力随吸附时间的变化 24
3.3.2菜籽蛋白气液界面扩散动力学 25
3.3.3菜籽蛋白气液界面展开和重排的动力学 28
3.3.4菜籽蛋白起泡性及起泡稳定性 30
3.3.5菜籽蛋白荧光光谱测定 32
3.4 本章小结 33
第四章 离子强度对菜籽分离蛋白气液界面行为及泡沫特性的影响
4.1材料与试剂 35
4.1.1实验材料 35
4.1.2实验试剂 35
4.1.3实验仪器 35
4.2 实验方法 35
4.2.1界面压力检测 35
4.2.2起泡性及起泡稳定性测定 35
4.2.3荧光光谱分析 35
4.3 结果与讨论 36
4.3.1表面压力随吸附时间的变化 36
4.3.2离子强度对气液界面扩散动力学的影响 36
4.3.3离子强度对气液界面展开和重排动力学的影响 38
4.3.4离子强度对起泡性及泡沫稳定性的影响 40
4.3.5不同离子强度下的菜籽分离蛋白荧光光谱分析 41
4.4 本章小结 42
第五章 结论
参考文献
致谢 52
攻读硕士学位期间科研成果情况 53
第一章 绪论
1. 油菜及油菜籽概述
油菜在植物学的分类中属于十字花科芸苔类的植物，目前主要分布在中国，欧洲的法国，北美的加拿大和南亚次大陆的印度[]。目前对油菜的发源地主流学术界一般认为有两个：芥菜型油菜和白菜心油菜，亚洲是其发源地；甘蓝型油菜的发源地则在欧洲的地中海地区[]。油菜在我国的栽培历史可谓源远流长，可以追溯到二千多年前的秦朝，古代油菜被称之为“芸苔”或者“胡菜”，根据东汉文献《通俗文》的记载，油菜最早在我国的少数民族的活动区域种植，如古书中记载的胡、羌、陇、氐等，就是现在我国的青海、甘肃、新疆、内蒙古等省份。由于油菜可食用，油菜籽可以提油，慢慢得到了古人的喜爱，逐步种植到黄河流域，进而传播到长江一带广为种植，直到今日[]。最近几十年来，油菜已经成为我国最重要的农作物之一，我国的油菜种植生产显著提高，种植面积和年产量均位居世界前茅，在长江流域的湖北、四川、湖南等省份，油菜是第二大农作物，种植量仅次于水稻[]。油菜可以提供丰富的植物油和饲料蛋白质，也有很大潜力成为未来生物能源作物[]
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