虾壳源多肽酶解条件的优化及其抗冻活性和作用机制的探究

Optimization of enzymatic hydrolysis conditions for shrimp shell-derived peptides and investigation of their antifreeze activity and mechanism

中文关键词:  抗冻肽  肽组学  复合酶解  冰晶生长抑制  分子动力学模拟

英文关键词:antifreeze peptide  shrimp shell  peptidomics  complex enzymatic hydrolysis  ice crystal growth inhibition  molecular dynamics simulation

中文摘要:

      目的 优化从虾壳中提取多肽的酶解条件, 并探究虾壳多肽的抗冻活性及作用机制。方法 以小龙虾虾壳为原料, 使用复合酶酶解提取多肽。在单因素实验的基础上, 通过正交实验得到虾壳最佳酶解条件。以面包酵母菌为抗冻测试对象测试其抗冻能力。采用液相色谱-串联质谱法(liquid chromatography-mass spectrometry, LC-MS/MS)对虾壳多肽的组成进行鉴定, 基于多肽的理化性质和序列, 结合抗冻预测平台筛选抗冻肽(antifreeze peptides, AFPs)。使用分子动力学模拟探究了AFPs的作用机制。结果 在酶解温度40℃, 底物质量浓度20 mg/mL, 加酶量12000 U, 酶比0.33的最佳酶解条件下, 多肽提取率达到了70%以上。虾壳多肽中含有1004种肽段, 能明显提升酵母菌存活率。从具有抗冻活性且置信度靠前的20条多肽中进一步筛选出了潜在的抗冻肽AFP1、AFP2。AFP2和冰面之间形成的氢键数量为14, 部分水分子同时与2个氨基酸残基形成氢键, 充当水桥稳定多肽的构象, AFP2与冰之间的结合能为?105.08 kcal/mol, 冰结合模拟效果理想。结论 AFPs与冰结合并抑制冰晶生长可能归因于带电荷的亲水性氨基酸残基与水分子形成的氢键、范德华力等分子间作用力, 可开发为有益的冷冻保护剂, 用于冷冻食品加工, 具备良好的应用前景。

英文摘要:

      Objective To optimize the enzymatic hydrolysis conditions of peptides extracted from shrimp shells, and investigate the antifreeze activity and mechanism of shrimp shell peptides. Methods Firstly, the crayfish shell was used as raw material to extract polypeptide by compound enzyme hydrolysis. Based on the single-factor experiment results, the optimum enzymatic hydrolysis conditions of shrimp shells were determined through orthogonal experiments. Subsequently, the antifreeze capability was tested using bread yeast as the freezing resistance test organism. Liquid chromatography-mass spectrometry (LC-MS/MS) was employed to identify the composition of shrimp shell-derived peptides. Antifreeze peptides (AFPs) were screened by an antifreeze prediction platform based on polypeptide physicochemical properties and sequences. Molecular dynamics simulations were utilized to investigate the mechanism of AFPs. Results The optimal hydrolysis conditions were achieved at a temperature of 40°C, substrate concentration of 20 mg/mL, enzyme dosage of 12000 U, and an enzyme ratio of 0.33, resulting in a peptide extraction yield of over 70%. The analysis revealed 1004 peptide sequences in shrimp shell-derived peptides, significantly enhancing yeast survival rates. From the top 20 peptides with antifreeze activity and high confidence, AFP1 and AFP2 were further screened as potential antifreeze peptides. AFP2 formed 14 hydrogen bonds with the ice surface. Some water molecules concurrently form hydrogen bonds with two amino acid residues, acting as water bridges to stabilize the peptide conformation. The binding energy of AFP2 to ice was ?105.08 kcal/mol, with the favourable ice-binding simulation effect. Conclusion The binding of antifreeze peptides to ice and the inhibition of ice crystal growth may be attributed to the intermolecular forces such as hydrogen bonds and van der Waals forces formed by charged hydrophilic amino acid residues and water molecules. It can be developed as a valuable cryoprotectant for frozen food processing and has a good application prospect.