基于光谱分析技术的肉桂醛与玉米醇溶蛋白作用机理的研究

通过荧光光谱、紫外光谱、圆二色谱、红外光谱、核磁共振光谱研究肉桂醛与玉米醇溶蛋白间的作用机理，为改善玉米醇溶蛋白膜的机械性能及抗菌性和抗氧化性提供研究依据。通过三维荧光光谱检测，发现肉桂醛对玉米醇溶蛋白有明显的荧光猝灭作用，而且溶剂乙醇对猝灭现象有影响。用紫外差谱观察到玉米醇溶蛋白在紫外区278 nm的吸收强度随肉桂醛浓度增大而加大，但增加的幅度与浓度改变不成比例，氨基酸残基的特征吸收峰的位置没有变化。加入肉桂醛前后，圆二色谱显示的两条曲线近乎重合。利用全反射ATR附件测定傅里叶变换红外光谱，发现加入肉桂醛之后，1 650和1 538 cm-1的吸收峰峰位没有发生明显的变化，但1 625 cm-1处出现了明显的肩峰，体现了肉桂醛碳碳双键的吸收，指纹区879.44 cm-1处的峰消失，973.46 cm-1处出现新峰，显示出肉桂醛反式双键的吸收，说明玉米醇溶蛋白与肉桂醛发生了非键合作用。对酰胺Ⅰ带进行自去卷积计算，发现玉米醇溶蛋白的二级结构中α螺旋结构变化甚微，β转角发生显著改变。通过核磁共振氢谱，分析4个位置的质子H的化学位移，仅改变了0.01，而且加入肉桂醛1和4 h之后，化学位移改变量相等，证明肉桂醛与玉米醇溶蛋白的结合反应发生在蛋白表面。对体系的热力学参数进行计算，可知肉桂醛与玉米醇溶蛋白之间发生的是结合比为1∶1的自发结合反应；在肉桂醛低浓度时，猝灭常数随温度的升高而降低，但变化不显著；结合常数很大，数量级达到105，且随温度的升高而降低。在有无肉桂醛的条件下，对玉米醇溶蛋白荧光寿命的测定，进一步确认二者之间发生的是静态猝灭。综合多种光谱分析结果，说明肉桂醛与玉米醇溶蛋白主要在芳香区的外部发生π-π堆积，以静电力结合，是静态猝灭机制，与作用时间的长短无关。结果表明玉米醇溶蛋白中加入肉桂醛，对其二级结构不会造成明显的影响。

Study on the Mechanism of Interaction between Cinnamaldehyde and Zein Based on Spectral Analysis Technology

The aim of this study was to investigate the mechanism of interaction between cinnamaldehyde and zein by fluorescence spectroscopy, UV spectroscopy, circular dichroism spectroscopy, infrared spectroscopy, and nuclear magnetic resonance spectroscopy, so as to provide research basis for improving the mechanical properties, antibacterial properties, and antioxidant properties of zein films. Through three-dimensional fluorescence spectroscopy, it was found that cinnamaldehyde has obvious fluorescence quenching effect on zein, and solvent ethanol has an effect on quenching. When UV-spectroscopy was used to explore the mechanism, it was observed that the absorption intensity of zein in the UV region at 278 nm increased with the increasing concentration of cinnamaldehyde, but the increasing range was not proportional, and the characteristic absorption peak positions of the amino acid residues was not changed. Before and after addition of cinnamaldehyde, the two curves exhibited by circular dichroism were nearly coincident. Using the attenuated total reflection ATR appendage for Fourier transform infrared spectroscopy, we found that after the addition of cinnamaldehyde, the absorption peak at 1 650 cm-1 indicates C=O stretching vibration, and 1 538 cm-1 indicates the NH bending vibration in plane. The peak positions at these two sites did not change significantly, but there were obvious acromion peaks at 1 625 cm-1, reflecting the absorption of cinnamaldehyde carbon-carbon double bonds. The peak at 879.44 cm-1 of the fingerprint area disappeared, and a new peak appeared at 973.46 cm-1, reflecting the absorption of the cinnamaldehyde trans double bond, indicating that a non-bonding of zein with cinnamaldehyde. The self-deconvolution calculation of the amide Ⅰ band revealed that the α-helix structure of the zein secondary structure changed little and the β-turn changed significantly. By means of NMR, the chemical shift of proton H in 4 positions changed only 0.01, and after 1 hour and 4 hours of cinnamaldehyde addition, the change of chemical displacement was equal, indicating that the binding reaction of cinnamaldehyde to zein occurred on the surface of the protein and did not cause changes in the secondary structure of zein. The thermodynamic parameters of the system were calculated. It was found that the spontaneous binding reaction occurred between cinnamaldehyde and zein, and the binding ratio of them was 1∶1. When the concentration of cinnamaldehyde was low, the quenching constant decreases with the increase of temperature, but the change was not significant; when the binding constant was very large, the order of magnitude reached 105, and decrease with the increase of temperature. The determination of the fluorescence lifetime of zein in the presence or absence of cinnamaldehyde further confirmed that static quenching occurred between cinnamaldehyde and zein. Comprehensive analysis of various spectra showed that cinnamaldehyde and zein was mainly π-π stacked on the outside of the aromatic region, and it was a combination of electrostatic forces, was static quenching mechanism, and was independent of the action time. The results showed that the combination of cinnamaldehyde and zein do not significantly affect the secondary structure of zein.