摘要
目的 以厚壳贻贝(Mytilus coruscus)为原材料,以抗氧化活性为筛选指标,运用酶解技术和现代分离纯化技术,制备抗氧化多肽.方法 以1,1-二苯基-2-三硝基苯肼自由基(DPPH·)、羟自由基(HO·)及2,2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸自由基(ABTS·)清除率为指标,优化酶解条件.通过超滤、凝胶过滤层析和反相高效液相色谱分离抗氧化肽,并评价其抗氧化活性.结果 最佳酶解条件为:木瓜蛋白酶、酶解时间3.0 h、加酶量3.0％、料液比1∶50.通过反相高效液相色谱及测序,分离并鉴定4条多肽分别为HK1:Gln-Thr-Asp(362.34 Da);HK2:His-Gln-Glu-Glu(541.52 Da);HK3:Leu-Glu-Gly-Asp-Thr(533.54 Da);HK4:Thr-Gln-Glu(376.37 Da).其对 DPPH·清除率的半最大效应浓度(EC50)分别为(0.523±0.066)、(0.419±0.042)、(0.467±0.078)、(0.682±0.134)mg/mL;对 O2-·清除率的 EC50 分别为(0.239±0.021)、(0.245±0.019)、(0.611±0.012)、(0.716±0.024)mg/mL;对 HO·清除率的 EC50分别为(0.587±0.150)、(0.866±0.123)、(1.009±0.060)、(1.014±0.122)mg/mL;对 ABTS·清除率的 EC50 分别为(0.218±0.109)、(0.117±0.013)、(0.121±0.019)、(0.415±0.054)mg/mL.结论 木瓜蛋白酶酶解厚壳贻贝蛋白可以产生具有显著自由基清除活性的酶解肽,可为厚壳贻贝抗氧化肽的开发利用提供理论依据和参考.
Abstract
Objective To prepare antioxidant peptides from Mytilus coruscus,using enzymatic hydrolysis and modern separation and purification technology.Methods The scavenging rates of DPPH radical(DPPH·),hydroxyl radical(HO·),and 2,2'-biazo-bis-3-ethylbenzothiazoline-6-sulfonic acid free radical(ABTS·)were measured as the index to optimize the conditions of enzymatic hydrolysis.Antioxidant peptides were isolated by ultrafiltration,gel filtration chromatography,and reverse high-performance liquid chromatography,and their antioxidant activities were evaluated.Results The optimal conditions for enzymatic hydrolysis were as follows:papain,enzymatic hydrolysis time of 3.0 h,enzyme dosage of 3.0％,feed-liquid ratio of 1∶50.Four peptides were isolated and identified as HK1:Gln-Thr-Asp(362.34 Da);HK2:His-Gln-Glu-Glu(541.52 Da);HK3:Leu-Glu-Gly-Asp-Thr(533.54 Da);HK4:Thr-Gln-Glu(376.37 Da)by reverse high-performance liquid chromatography and amino acid N-terminal sequencing.Their EC50 values for DPPH·scavenging were(0.523±0.066),(0.419±0.042),(0.467±0.078),and(0.682±0.134)mg/mL,respectively;and their EC50 values for HO·scavenging were(0.587±0.150),(0.866±0.123),(1.009±0.060),(1.014±0.122)mg/mL,respectively;their EC50 values for O2-·scavenging were(0.239±0.021),(0.245±0.019),(0.611±0.012),(0.716±0.024)mg/mL,respectively;the EC50 values of ABTS·scavenging were(0.218±0.109),(0.117±0.013),(0.121±0.019),and(0.415±0.054)mg/mL,respectively.Conclusion This study concluded that enzymatic hydrolysis of Mytilus coruscus protein by papain could produce peptides with significant free radical scavenging activities which could provide a theoretical basis and reference for the development and utilization of Mytilus coruscus.
基金项目
2023年浙江省中医药科技计划项目(2023ZR057)
舟山市医药卫生科技计划项目(2022YA10)
舟山市医药卫生科技计划项目(2022YA14)
达州市市校合作专项()
国家级大学生创新创业训练计划项目(202110340026)
国家级大学生创新创业训练计划项目(202210340028)
NETL
NSTL
万方数据