pH对肌原纤维蛋白及其热诱导凝胶非共价键作用力与结构的影响

doi:10.3864/j.issn.0578-1752.2017.03.015

摘要/Abstract

摘要： 【目的】研究pH对鸡肉肌原纤维蛋白热诱导凝胶非共价键作用力和结构的影响，揭示凝胶非共价键作用力与结构之间的关系。【方法】活AA鸡宰杀，提取鸡胸肉肌原纤维蛋白，配制不同pH（5.0、5.5、6.0、6.5、7.0）的肌原纤维蛋白溶液并制成热诱导凝胶，运用Zeta电位仪测定肌原纤维蛋白凝胶分子表面的电位值来表征静电相互作用；利用拉曼光谱仪测定肌原纤维蛋白凝胶I760/I1003反映疏水相互作用变化，I850/I830反映凝胶氢键变化，并通过分析酰胺带I最大峰的波数计算蛋白和凝胶的二级结构含量；用粒度仪测定肌原纤维蛋白粒径大小和分布情况；用扫描电镜观察凝胶微观结构。【结果】pH由7.0降至5.0，肌原纤维蛋白热诱导凝胶的Zeta电位值从-17.87显著变化到-0.263（P＜0.05），表明肌原纤维蛋白凝胶分子表面所带负电荷急剧减少，静电斥力显著减弱；归一化强度I760/I1003比值由0.86逐渐增大到0.927，表明肌原纤维蛋白中色氨酸包埋程度增加，凝胶分子间的疏水相互作用增强；归一化强度I850/I830比值从1.039减小至0.987，表明肌原纤维蛋白酪氨酸残基苯环上-OH与水分子生成的氢键逐渐减少、与蛋白质上其他基团生成的氢键逐渐增加，即蛋白分子间的氢键作用增强，蛋白与水的作用减弱。pH由7.0降至6.5，肌原纤维蛋白热诱导凝胶的α-螺旋含量从59.96%降低到55.24%（P＜0.05），β-折叠含量从15.83%显著增加到19.44%（P＜0.05），β-转角和无规则卷曲含量都显著增加（P＜0.05）；pH 在6.5—6.0时，各种结构含量变化都不显著（P＞0.05）；pH 在6.0—5.0时，肌原纤维蛋白热诱导凝胶的α-螺旋含量从51.61%降低到16.76%（P＜0.05），β-折叠含量从22.23%显著增加到48.93%（P＜0.05），β-转角和无规则卷曲含量都显著增加（P＜0.05）。随着pH降低，肌原纤维蛋白α-螺旋的含量逐渐降低，而β-折叠、β-转角和无规则卷曲含量都显著增加（P＜0.05）。pH 在7.0—5.0时，肌原纤维蛋白粒径大小逐渐增大，D₁₀从13.4 μm上升到48.4 μm，D₅₀从38 μm上升到253 μm，D₉₀从236 μm上升到805 μm。pH 7.0时形成的凝胶微观结构有序，孔径最大；随着pH减小，凝胶微观结构有序程度降低，孔径变小；pH 5.0时形成的凝胶微观结构无序、孔径最小。pH与凝胶静电相互作用、疏水相互作用极显著负相关（P＜0.01），与氢键、α-螺旋含量显著正相关（P＜0.05），与β-折叠含量显著负相关（P＜0.05），这表明pH显著影响静电斥力、疏水相互作用、分子间氢键和二级结构含量。静电相互作用、疏水相互作用、氢键与蛋白凝胶二级结构都显著相关（P＜0.05），表明非共价键作用力显著影响二级结构。【结论】肌原纤维蛋白凝胶非共价键作用力、二级结构和微观结构与pH密切相关；pH从7.0降到5.0，静电斥力减小、疏水相互作用增大、分子间氢键增大，是α-螺旋含量减小、β-折叠含量增多以及凝胶微观结构变得无序、孔径减小的原因。

关键词: 肌原纤维蛋白凝胶, 静电, 疏水, 氢键, 二级结构, 粒径, 微观结构

Abstract: 【Objective】The influence of pH on non-covalent forces and structure of myofibrillar protein heat-induced gel was studied. The relationship between gel non-covalent forces and gel structure was revealed. 【Method】 AA type broilers were slaughtered. The myofibrillar proteins were extracted from breast muscle. The myofibrillar protein solution and heat-induced gel with different pH values (5.0, 5.5, 6.0, 6.5, 7.0) were prepared. The potential on myofibrillar protein gel molecule presents the electrostatic interaction was measured by zeta potential instrument. The I760/I1003 showing the hydrophobic interaction of gel, the I850/I830 showing the hydrogen bonding of gel, and the secondary structure contents were calculated by analyzing the amide I Raman spectrum region, these were measured using Raman spectrometer. The particle size distribution was measured by a particle size analyzer. The microstructure was measured using scanning electron microscope.【Result】 From pH 7.0 to 5.0, Zeta potential value of the gel changed from -17.87 to -0.263 (P＜0.05), which show that the surface negative charges and the electrostatic interaction of myofibrillar protein gel had significant decline. The normalized intensity of 760 cm^-1 increased from 0.86 to 0.927, which show more Trytophan were buried and a general increase in hydrophobic interactions of myofibrillar protein gel. The normalized intensity of I850/I830 ratio decreased from 1.039 to 0.927, which indicated hydroxyl groups on the phenyl ring of tyrosine are to form hydrogen bonds with water molecules change to generate hydrogen bonds with other protein molecule residues. The interactions between myofibrillar protein molecules increased, and the interactions between myofibrillar protein and water therefore declined. From pH 7.0 to 6.5, the α-helix content of myofibrillar protein gel abruptly decreased from 59.96% to 55.24% (P＜0.05). The β-sheet content significantly increased from 15.83% to 19.44% (P＜0.05). β-turn and random coil content both significantly increased (P＜0.05). From pH 6.5 to 6.0, all structure content had no obvious change (P＞0.05). From pH 6.0 to 5.0, the α-helix content of myofibrillar protein gel significantly decreased from 51.61% to 16.76% (P＜0.05). The β-sheet content significantly increased from 22.23% to 48.93% (P＜0.05). β-turn and random coil content both significantly increased (P＜0.05). As the pH decrease, the α-helix content of myofibrillar protein gradually decreased, the β-sheet, β-turn and random coil content significantly increased (P＜0.05). From pH 7.0 to 5.0, particle size of myofibrillar protein gradually increased. D₁₀increased from 13.4 μm to 48.4 μm, D₅₀ increased from 38 μm to 253 μm, D₉₀ increased from 236 μm to 805 μm. As the pH far away from neutral condition, the microstructure of gel changed to unordered and had smaller pore. Gel has disordered microstructure in pH 5.0, when has ordered structure at pH 7.0. The largest gel pore ware found at pH 5.0, the least were found at pH 7.0. pH had a highly negative significant correlation with electrostatic interaction and hydrophobic interactions (P＜0.01), and had a positive significant correlation with hydrogen bonding and α-helix content (P＜0.05). pH also led to negative significant change of β-sheet content (P＜0.05). These show that pH had a significant impacts on electrostatic repulsion, hydrophobic interactions, intermolecular hydrogen bonding and secondary structure. Electrostatic interaction, hydrophobic interactions and hydrogen bonding had significant correlation with secondary structure (P＜0.05), which indicated non-covalent forces had significant effects on secondary structure.【Conclusion】 Non-covalent forces and secondary structure content are significantly correlated with the pH valves. The reasons of gel α-helix reduction and β-sheet increases are the decreases of electrostatic interaction, and the increase of the hydrophobic interaction and the intermolecular hydrogen bonding of myofibrillar protein gel, as the pH far away the neutral conditions.

Key words: myofibrillar protein gel, electrostatic, hydrophobic, hydrogen bond, secondary structure, particle size, microstructure

张兴，杨玉玲，马云，王静宇. pH对肌原纤维蛋白及其热诱导凝胶非共价键作用力与结构的影响[J]. 中国农业科学, 2017, 50(3): 564-573.

ZHANG Xing, YANG YuLing, MA Yun, WANG JingYu. Effects of pH on the Non-Covalent Forces and Structure of Myofibrillar Protein and Heat Induced Gel[J]. Scientia Agricultura Sinica, 2017, 50(3): 564-573.

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