多光谱学与分子模拟技术表征甜菜苷与乳清蛋白的相互作用

胡婷; 耿勤; 付敏; 陈军; 何雪梅; 孙健; 戴涛涛

doi:10.13386/j.issn1002-0306.2022020243

多光谱学与分子模拟技术表征甜菜苷与乳清蛋白的相互作用

doi: 10.13386/j.issn1002-0306.2022020243

胡婷^1,,
耿勤¹,
付敏¹,
陈军¹,
何雪梅²,
孙健²,
戴涛涛^2, ,

1.
南昌大学食品科学与技术国家重点实验室，江西南昌 330047
2.
广西果蔬贮藏与加工新技术重点实验室，广西南宁 530007

基金项目: 广西农业科学院博士后基金项目资助（桂农科博2020037）；中国博士后基金项目资助（2020M6832211）。

详细信息

作者简介:
胡婷（1998−），女，硕士研究生，研究方向：食品组分相互作用，E-mail：huting_1998@163.com

通讯作者:
戴涛涛（1991−），男，博士，研究方向：农副产品综合利用，E-mail：ncubamboo@163.com

中图分类号: Q513
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- 文章访问数: 153
- HTML全文浏览量: 31
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- 被引次数: 0
出版历程
- 收稿日期: 2022-02-28
- 网络出版日期: 2022-11-01
- 刊出日期: 2022-11-23

The Interaction between Betanin and Whey Protein: Based on Multi-spectroscopy and Molecular Simulation

HU Ting^1
,,
GENG Qin¹,
FU Min¹,
CHEN Jun¹,
HE Xuemei²,
SUN Jian²,
DAI Taotao^{2
, ,}

1.
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China
2.
Guangxi Key Laboratory of Fruits and Vegetables Storage-processing Technology, Nanning 530007, China

摘要

摘要: 为研究甜菜苷（Betanin）与乳清分离蛋白（Whey protein isolate, WPI）的相互作用及乳清分离蛋白对甜菜苷热稳定的影响，本文通过自组装法构建了WPI-Betanin复合物，利用紫外可见光谱验证了复合物的形成，并通过荧光光谱和圆二色谱探究了复合物形成的作用机制及蛋白质结构的变化，最后采用分子模拟技术将复合物相互作用可视化。结果表明，甜菜苷与乳清分离蛋白主要通过氢键与范德华力形成复合物，结合位点数约为1。复合物的形成导致蛋白的浊度增加，表面疏水性降低，内源荧光猝灭，且猝灭机制为静态猝灭，常温下的猝灭常数为1.78×10³ L/mol。相互作用改变了乳清分离蛋白中色氨酸、酪氨酸的微环境和二级结构含量，使甜菜苷在80 ℃下加热1 h的保留率从6.17%提高到27.26%。本研究为功能性蛋白色素复合物的应用开发和甜菜苷的护色提供了理论基础。
- 甜菜苷 /
- 乳清蛋白 /
- 相互作用 /
- 光谱学 /
- 分子模拟
Abstract: In order to study the interaction between betanin and whey protein isolate (WPI) and the effect of whey protein isolate on the thermal stability of betanin, WPI-Betanin complex was constructed by self-assembly method. UV-vis spectroscopy was used to verify the formation of complex. The mechanism of complex formation and the changes of protein structure were investigated by fluorescence spectroscopy and circular dichroism, and the complex was visualized by molecular simulation technology. The results showed that betanin and whey protein isolate formed a complex mainly through van der Waals force and hydrogen bond, and the number of binding sites was about 1. The formation of the complex resulted in increased turbidity and decreased surface hydrophobicity of the protein, and the quenching mechanism was static, with a quenching constant of 1.78×10³ L/mol at room temperature. The interaction changed secondary structure contents and the microenvironment of tryptophan and tyrosine in whey protein isolate. Moreover, the thermal stability of betanin was improved by WPI. The retention rate of betanin increased from 6.17% to 27.26% after heating at 80 ℃ for 1 h. This study provides a theoretical basis for the application and development of functional protein pigment complex and the color protection of betanin.
- betanin /
- whey protein /
- interaction /
- spectroscopy /
- molecular simulation.

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图 1 不同比例甜菜苷对乳清分离蛋白浊度的影响

Figure 1. Effect of different proportions of betanin on turbidity of WPI

注：图中不同小写字母表示差异显著（P<0.05）。

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图 2 不同浓度甜菜苷对乳清分离蛋白紫外吸收的影响

Figure 2. Ultraviolet spectra of WPI treated by betanin with different betanin concentrations

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图 3 不同浓度乳清分离蛋白的荧光强度和不同浓度甜菜苷对乳清分离蛋白的荧光猝灭

Figure 3. Fluorescence intensity of WPI at different concentrations and fluorescence quenching of WPI at different concentrations of betanin

注：A：不同浓度乳清分离蛋白的荧光强度；B：298 K；C：304 K；D：310 K。

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图 4 乳清分离蛋白-甜菜苷在298、304、310 K温度下Stern-Volmer拟合（A）和静态猝灭方程的拟合（B）

Figure 4. Stern-Volmer fitting (A) and static quenching equation fitting (B) at 298, 304 and 310 K for WPI and betanin

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图 5 不同浓度甜菜苷对乳清分离蛋白表面疏水性的影响

Figure 5. Effect of different concentrations of betanin on surface hydrophobicity of WPI

注：a、b、c、d、e、f、g、h、i分别表示甜菜苷的浓度为0、0.025、0.05、0.075、0.1、0.125、0.15、0.175、0.2 mg/mL。

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图 6 不同浓度甜菜苷对乳清分离蛋白圆二色谱的影响

Figure 6. Effect of different concentrations of betanin on circular dichroism spectrum of WPI

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7 甜菜苷分别与（A）α-乳白蛋白（1NFZ）、（B）牛血清蛋白（3VO3）、（C）乳铁蛋白（1BLF）、（D）β-乳球蛋白（3NPO）的分子对接图

7. Molecular docking of betanin with (A) α-lactalbumin (1NFZ), (B) bovine serum protein (3VO3), (C) lactoferrin (1BLF), and (D) β -lactoglobulin (3NPO)

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图 8 WPI-Betanin复合物加热过程中甜菜苷保留率的变化

Figure 8. Change of retention rate of betanin in WPI-Betanin complex during heating

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表 1 乳清分离蛋白与甜菜苷的结合参数

Table 1. Binding parameters of WPI and betanin

温度（K）	K_SV （10²·mol⁻¹）	K_q （10¹⁰ L·mol⁻¹·s⁻¹）	n	K_a （L/mol）	ΔH （kJ/mol）	ΔS （J·mol⁻¹·K⁻¹）	ΔG （kJ/mol）
298	2.06	3.32	1.26±0.03	1783.16	−	−	−18.55
304	2.26	3.65	1.13±0.03	672.59	−123.28	−351.42	−16.44
310	2.58	4.16	1.00±0.04	259.81	−	−	−14.34

下载: 导出CSV

表 2 不同浓度甜菜苷对乳清分离蛋白二级结构的影响

Table 2. Effects of different concentrations of betanin on secondary structure of WPI

样品	α-螺旋（%）	β-折叠（%）	β-转角（%）	无规卷曲（%）
空白	12.5	31.1	24.4	31.9
0.1 mg/mL甜菜苷	10.8	33.6	24.0	31.6
0.2 mg/mL 甜菜苷	10.1	34.2	23.8	31.9

下载: 导出CSV

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