低温等离子体杀菌对虹鳟肌球蛋白降解及相关组织蛋白酶活性的影响

孙协军; 陈笑俏; 李秀霞; 励建荣

doi:10.13386/j.issn1002-0306.2024060181

低温等离子体杀菌对虹鳟肌球蛋白降解及相关组织蛋白酶活性的影响

Effect of Low Temperature Plasma Sterilization on Myosin Degradation and Related Cathepsin Activity of Oncorhynchus mykiss

摘要

摘要: 为明确低温等离子体杀菌对虹鳟肌球蛋白降解及相关组织蛋白酶活性的影响，在经过70 kV 介质阻挡放电低温等离子体（Dielectric barrier discharge，DBD）杀菌处理并冰温贮藏8 d后，分析不同贮藏时间的处理组和对照组虹鳟鱼肉质构特性、TCA-可溶性肽、氨基酸态氮、肌原纤维小片化指数（Myofibrillar fragmentation index，MFI）的变化，并对鱼肉中的组织蛋白酶B、D、L活性以及它们对肌球蛋白的降解作用进行探究，进一步通过分子动力学模拟的方法分析了DBD处理产生的主要活性物质NO₃⁻与组织蛋白酶的结合情况。结果表明，70 kV DBD处理对贮藏初期的鱼肉硬度、弹性无显著影响（P>0.05），能够延缓鱼肉硬度和弹性的降低程度，且延缓了TCA-可溶性肽、肌原纤维小片化指数（MFI）、氨基酸态氮含量的增加，显著抑制组织蛋白酶B、L活性（P<0.05），并减缓肌球蛋白轻链MLC-2片段的降解。DBD处理显著降低了组织蛋白酶B、D和L的活性（P<0.05），分子动力学（Molecular dynamics，MD）模拟结果显示，组织蛋白酶B、D和L与NO₃⁻的结合自由能分别为：−10.937、−5.0555、−19.112 kcal/mol，组织蛋白酶B、D、L与NO₃⁻的氢键出现频率分别为13.56%、15.02%和31.23%，说明NO₃⁻与组织蛋白酶L的氢键作用较强。DBD处理能延缓鱼肉蛋白质降解，降低鱼体内组织蛋白酶的活性，保持鱼肉良好的质地，其产生的活性成分中的NO₃⁻对组织蛋白酶L的作用效果最大，能够在其活性中心中稳定存在且具有较低的结合自由能。

Abstract: To clarify the mechanism of the effect of low temperature plasma sterilization on the degradation of Oncorhynchus mykiss and related cathepsin activity, Oncorhynchus mykiss fillets were treated with 70 kV dielectric barrier discharge low temperature plasma (DBD) sterilization and storage under ice temperature for 8 d. Changes in texture, TCA soluble peptides, amino acid nitrogen, myofibrillar fragmentation index (MFI) of Oncorhynchus mykiss fillets from different storage days were analyzed, and the activities of cathepsin B, D, and L in the fillets and their degradation effects on myosin were explored. The binding of the main active substance NO₃⁻ produced by DBD sterilization with cathepsin was further analyzed through molecular dynamics simulation. The results showed that 70 kV DBD treatment had no significant effect on the hardness and elasticity of Oncorhynchus mykiss fillets during early storage (P>0.05), and the reduction of hardness and elasticity was delayed during the storage. It also delayed the increase of TCA soluble peptides, myofibrillar fragmentation index (MFI), and amino acid nitrogen content. The activity of cathepsins B and L were significantly inhibited (P<0.05), and the degradation of myosin light chain MLC-2 fragment was slowed. The activity of cathepsin B, D and L was significantly reduced after DBD sterilization (P<0.05). Molecular dynamics simulations showed that the binding free energies of cathepsin B, D and L with NO₃⁻ were −10.937, −5.0555, and −19.112 kcal/mol, respectively, and the hydrogen bonding frequencies of cathepsin B, D, and L with NO₃⁻ were 13.56%, 15.02%, and 31.23%, respectively, indicating stronger hydrogen bonding between NO₃⁻ and cathepsin L. The degradation of fish protein and activity of cathepsin could be delayed by DBD sterilization, and the texture of fish fillet was maintained. The greatest effect on cathepsin L was shown by the active ingredient NO₃⁻ produced by DBD sterilization, which could exist in a stable way in its active center and had low binding free energy.

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