傅里叶红外结合拉曼分析卡拉胶寡糖对南美白对虾蛋白结构影响

为深入研究卡拉胶寡糖对反复冻融南美白对虾样品品质与蛋白变化影响，采用傅里叶变换中红外（Fourier transform infrared, FTIR）互补结合激光显微拉曼（micro Raman）光谱技术，以不同预处理方式（无菌蒸馏水、三聚磷酸盐溶液与卡拉胶寡糖溶液）下反复冻融南美白对虾肌原纤维蛋白为研究对象，深入研究了卡拉胶寡糖预处理对不同冻融阶段南美白对虾肌肉品质和蛋白的影响机制。分别对三种预处理方式下经0，2，4和6次冻融循环后的南美白对虾肌原纤维蛋白进行FTIR和拉曼光谱分析。FTIR和拉曼一阶谱图中各特征峰强度变化谱图可得出，随冻融次数增加，南美白对虾肌肉的蛋白损失和结构破坏明显加剧。样品肌肉蛋白二级结构的主链构象主要由酰胺Ⅰ带（1 600~1 700 cm-1）表征，FTIR分析显示新鲜虾肉蛋白二级结构以β-转角为主，其次为β-折叠，其弥补了拉曼光谱对β-折叠和β-转角的不敏感。FTIR与拉曼光谱酰胺Ⅰ带高斯拟合后谱图可定性定量显示冻融过程中样品的蛋白二级结构变化主要是α-螺旋结构的减少与无规卷曲结构的增加，而卡拉胶寡糖预处理能明显抑制冻融过程中α-螺旋结构的损失。FTIR对蛋白表面氨基酸变化不敏感，拉曼光谱则可互补显示样品蛋白侧链的构象变化。其表征酪氨酸残基的谱带出现在850和830 cm-1，峰强比表征了样品中酪氨酸的暴露程度在冻融期间呈增长趋势；脂肪族侧链氨基酸残基的C-H弯曲与伸缩振动分别在1 440~1 465和2 800~3 100 cm-1区间，1 448和2 935 cm-1处峰强变化表征了样品侧链氨基酸的疏水相互作用在冻融过程中逐渐增强。拉曼光谱中蛋白侧链的特征谱带变化表明冻融过程使虾肉蛋白中分子内、分子间氢键断裂和侧链酪氨酸、脂肪族氨基酸残基暴露，而卡拉胶寡糖处理后明显延缓了该变化。因此，卡拉胶寡糖可延缓反复冻融虾肉蛋白中氢键断裂、侧链疏水基暴露，进一步稳定蛋白二级结构，维持其蛋白功能特性，从而起到对反复冻融南美白对虾肌肉品质的保护作用。同时，本研究将FTIR与拉曼光谱技术结合应用在卡拉胶寡糖对冻融南美白对虾肌肉品质保护机制研究上，发现FTIR在南美白对虾蛋白二级结构的表征上更敏感，拉曼光谱则能为样品蛋白侧链构象变化提供参考，二者结合可通过提供互补信息，更好表征样品经处理后蛋白的结构变化。

Effects of Carrageenan Oligosaccharides on the Protein Structure of Litopenaeus Vannamei by Fourier Transform Infrared and Micro-Raman Spectroscopy

In order to study the influencing mechanism of carrageenan oligosaccharide on the muscle quality and protein in Litopenaeus vannamei during F-T cycles, the myofibrillar protein of samples during F-T cycles with different pretreatment methods (sterile distilled water, tripolyphosphate and carrageenan oligosaccharide) were measured respectively by Fourier Transform Infrared (FT-IR) and Micro-Raman (M-Raman). The data of FT-IR and M-Raman for myofibrillar protein in Litopenaeus vannamei with different pretreatments after 0, 2, 4, 6 F-T cycles were collected respectively. The results showed that the destructive effect of F-T cycles on the protein structure in Litopenaeus vannamei could be demonstrated by first-order spectrogram, and the structural changes of protein under different pretreatments could be qualitatively and quantitatively analyzed by second order derivation and gaussian curve fitting. The intensity changes of each characteristic peak at first-order spectrogram in FT-IR and M-Raman were indicated that F-T cycles could aggravate the loss and structural damage of muscle protein in shrimp. The primary chain conformation of secondary structure in the shrimp muscle protein was mainly characterized by amide band Ⅰ (1 600～1 700 cm-1). The results of FT-IR showed that the secondary structure of fresh shrimp protein was mainly β-turn, followed by β-sheet. It could compensate for the insensitivity of β-turn and β-sheet by M-Raman. The amide band Ⅰ of FT-IR and M-Raman spectrum after gaussian fitting qualitatively and quantitatively showed the secondary structure changes of protein during F-T cycles mainly made the decrease of α-helix and the increase of random coil, and carrageenan oligosaccharide pretreatment could significantly inhibit the loss of α-helix in shrimp protein during F-T cycles. FT-IR was not sensitive to the changes in the compositions of amino acid exposed to protein surface, while M-Raman spectra could compensate it to show the changes in protein conformation on side chain. The bands represented that tyrosine residues appeared at 850 and 830 cm-1, and the peak intensity ratio indicated the exposure of tyrosine, which showed an increasing trend during F-T cycles. The C-H bending and stretching vibration of aliphatic side chain amino acid residues were in 1 440～1 465 and 1 465～2 800 cm-1 respectively, and the peak strength changes of 1 448 and 2 935 cm-1 were on behalf of hydrophobic interaction strength of amino acid on side chain, which increased during F-T cycles. The changes in characteristic spectral bands of protein on side chains by M-Raman showed that F-T cycles made the hydrogen bond rupture of protein intramolecular or intermolecular, the exposure of tyrosine or amino acid residues on aliphatic side chain, and carrageenan oligosaccharides pretreatment could slow down the change significantly. Therefore, carrageenan oligosaccharides could delay the rupture of hydrogen bond and exposure of side chain hydrophobic group in muscle protein during F-T cycles, the secondary structure of protein could be further stabilized, the function of protein could be maintained and the quality of shrimp could be improved under F-T cycles. In addition, the innovative combination of FT-IR and M-Raman spectra was applied to the research on the mechanism of carrageenan oligosaccharide for improving the muscle quality under F-T cycles. It was found that FT-IR was more sensitive to the characterization of secondary structure in muscle protein, while M-Raman spectra could provide the theoretical references for the conformation changes of protein on side chains. The combination of them could provide the compatible and complementary information to better analyze the structural changes of protein in samples after different pretreatments.