不同解冻方式反复冻融对金乌贼蛋白质分子间作用力的影响

Effects of different thawing ways and multiple freeze–thaw cycles on myofibrillar protein intermolecular force of Sepia esculenta

为研究反复冻融对乌贼肉蛋白质性质的影响,探究冻融过程中蛋白质分子间作用力的变化。以新鲜金乌贼肉为研究对象,3种不同解冻方式(0℃解冻、流水解冻和室温解冻)分别进行9次冻融处理。结果显示,在反复冻融过程中,冻融方式对分子间作用力的影响显著,0℃解冻优于流水解冻优于室温解冻。3种解冻方式解冻乌贼肉时,分子间作用力变化趋势保持一致,离子键和氢键含量降低,离子键分别降低了7.71%、10.64%和13.61%,氢键分别由31.91%、31.97%和31.87%降低到了26.76%、25.53%和23.94%;疏水相互作用、二硫键和非二硫共价键的含量均增加,疏水作用力分别增加了8.86%、12.35%和14.72%,二硫键分别增加了1.43%、1.96%和2.85%,非二硫共价键分别由1.16%、1.28%和1.55%增加到了3.75%、4.05%和5.50%;肌原纤维蛋白表面疏水性分别由30.47、31.31和32.26μg增加至46.10、53.51和58.91μg,与疏水作用力的结果保持一致;巯基与活性巯基含量降低,巯基分别降低了13.07、38.99和40.32 nmol/mg,活性巯基分别降低了6.55、24.26和43.16 nmol/mg,与二硫键生成趋势保持一致。红外光谱分析反复冻融过程中蛋白质中二级结构的变化规律,得到反复冻融使肌原纤维蛋白的空间构象发生改变,9次冻融后,0℃解冻、流水解冻和室温解冻3种冻融方式的光谱带向不同波数方向依次有规律移动,其中酰胺A带和酰胺Ⅲ带向高波数方向微移,酰胺Ⅰ带、酰胺Ⅱ带向低波数方向微移。蛋白质二级结构发生变化,α-螺旋和β-折叠二者含量之和占总比降低,β-转角和无规则卷曲二者含量之和增加。反复冻融过程实则是蛋白质缓慢氧化的过程。

This study explored the effects of multiple freeze-thaw cycles on myofibrillar protein intermolecular force of Sepia esculenta and its protein properties. Fresh S. esculenta meat were defrosted by three different ways which are 0 ℃, running water and normal temperature for 9 freeze-thaw cycles. The results showed that the effects of defrosting ways were significant on intermolecular forces, and thaw at 0 ℃ is better than running water and normal temperature. With the increase of freeze-thaw times, the content of ionic bond and hydrogen bond showed a significant decline, the ionic bond decreased by 7.71%, 10.64% and 13.61%, respectively, and the hydrogen bond decreased from 31.91%, 31.97% and 31.87% to 26.76%, 25.53% and 23.94%. On the contrary the hydrophobic interaction force, disulfide bond and non-disulfide covalent bond tended to increase, the hydrophobic forces increased by 8.86%, 12.35%, and 14.72%, respectively, and the disulfide bonds increased by 1.43%, 1.96%, and2.85%, respectively, the non-disulfide covalent bonds increased from 1.16%, 1.28%, and 1.55% to 3.75%, 4.05%and 5.50%. The surface hydrophobicity of myofibrillar protein increased from 30.47 μg, 31.31 μg and 32.26 μg to46.10 μg, 53.51 μg and 58.91 μg, respectively, consistent with hydrophobic force results. Both sulfhydryl and reactive thiol content were decreased, the sulfhydryl groups were reduced by 13.07 nmol/mg, 38.99 nmol/mg and40.32 nmol/mg, respectively, and the active sulfhydryl groups were decreased by 6.55 nmol/mg, 24.26 nmol/mg and 43.16 nmol/mg, consistent with disulfide bond generation trends. The changes of the secondary structure of myofibrillar protein were analyzed by Infrared spectroscopy(FTIR), indicated that during the freeze-thaw cycles the spatial conformation of myofibrin made a change. Three thawing ways were treatments after 9 freeze-thaw cycles, the spectral band shifted to different wavelength regularly, and the amide A band and the amide Ⅲ band moved slightly toward the high wave number direction, and the amide Ⅰ band and the amide Ⅱ band moved to the low wave number direction. The secondary structure of the protein changed, as the sum of the contents of bothα-helix and β-sheet reduced, and the sum of the contents of β-turn and random curl increased. Repeated freezing and thawing is actually a process of slow oxidation of proteins.