厚壳贻贝酶解物的制备及其降血压肽的分离纯化、结构表征
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摘要
贻贝是重要的世界性养殖品种,以新鲜贻贝为主要销售形式,经济效益低；作为传统的滋补食品,贻贝具有较高的营养价值和多种药理功效。本文以厚壳贻贝为原料,采用酶水解技术制备厚壳贻贝蛋白多肽,经超滤、凝胶过滤层析和RP-HPLC、质谱测序等手段对活性肽成分进行分离、纯化、结构表征,并通过体外试验验证,获得厚壳贻贝降血压肽。主要研究结果如下：
分别用碱性蛋白酶(Alcalase 2.4L)、中性蛋白酶(Neutrase)、风味蛋白酶(Flavourzyme)、胰蛋白酶(Trypsin)和复合蛋白酶(Promatex)对厚壳贻贝蛋白进行水解,比较酶解物的水解度DH、蛋白质回收率及ACE抑制率,确定出水解厚壳贻贝蛋白的酶源为碱性蛋白酶；在碱性蛋白酶水解厚壳贻贝蛋白单因素试验的基础上,采用响应面设计进行试验优化,结合各因素对响应值的影响顺序,确定最优酶解条件为[E]/[S]2.18%、pH 8.8,温度55℃,此条件下制备的厚壳贻贝酶解物的ACE抑制率为83.12%。
采用5KD超滤膜对酶解物进行初步分离,收集流出液,所得组分MCP在对ACE抑制的IC50由超滤之前的0.367 mg/mL降为0.308mg/mL。将该初步纯化的组分MCP进一步通过Sephadex G-10凝胶层析分离,以超纯水为洗脱液洗脱,得到三个组分MCP-1、MCP-2和MCP-3,分别测其在对ACE抑制的IC5o为0.739 mg/mL、0.298mg/mL和0.163 mg/mL,反复进样,选择活性最高的峰MCP-3收集。对MCP-3进行活性稳定性实验,结果显示,MCP-3对温度、酸碱变化以及消化酶均有不同程度的耐受性,随着温度或酸碱条件的变化,其对ACE的抑制活性均有良好的稳定性,体外模拟消化实验也表明MCP-3经胃肠道酶系消化后仍会保持较稳定的ACE抑制活性。此外,通过考察不同抑制剂浓度下,底物浓度变化与反应速率的关系,研究得出MCP-3对ACE的抑制类型为竞争性抑制。
对MCP-3利用半制备型RP-HPLC进行进一步的分离纯化,得到5个主要的洗脱峰,分别进行活性测定,选择ACE活性抑制率最高的洗脱峰MCP-3-4进行收集,并利用分析型RP-HPLC对其进行纯度鉴定,结果显示,该组分在220 nm和280 nm的洗脱谱图中,只出现了一个单一的尖峰,说明MCP-3-4是个单一的肽段。通过液质联用进行序列分析,得到MCP-3-4为相对分子质量294.2的二肽,其一级序列结构为Glu-Phe。
Mussel is an important aquaculture species worldwide, but people can't make more profits if only mussel is sold directly. Mussel not only is a traditional tonic food, but also has a high nutritional value and variety of pharmacological effects.
The objectives of this study are to prepare hydrolysates from Mytilus coruscus by enzyme hydrolysis, separate and isolate the activity peptides from Mytilus coruscus hydrolysates (MCHs) by a series of separation and analysis technologies, including ultra-filtration, gel chromatography and reversed-phase high performance liquid chromatography, and identify the active peptides sequences by mass spectrum technology. The antihypertensive effect is evaluated in vitro. The work will be a base for development of antihypertensive functional food production and explore the potential of Mytilus coruscus as a functional Ingredient for value-added foods. The major results obtained in this study are as follows:
Mytilus coruscus protein was hydrolyzed using five different proteases. The results showed that Alcalase 2.4L had the best hydrolyzing capacity, the higher protein recovery and the stronger ACE (AngiotensinⅠ-coverting Enzyme) inhibitory activity. Response surface methodology (RSM) was applied to optimize the hydrolysis conditions using Alcalase 2.4L. The optimum values for enzyme/substrate ratio, pH and temperature were found to be 2.18%,8.8 and 55℃respectively. Under those conditions, the ACE inhibitory rate was 83.12%(1.Omg/mL).
The IC50 of the fraction MCP separated by ultra-filtration was 0.308 mg/mL. Then Sephadex G-10 gel filtration chromatography was used to separate MCP, eluted by H2O. Consequently, three fractions, MCP-1, MCP-2 and MCP-3 were obtained. In Anti-hypertension test, MCP-3 showed best ACE inhibition effects, with its IC50 value of 0.163 mg/mL, which retained activity under various temperature and pH treatments, and showed resistance to in vitro digestion by gastrointestinal proteases. Lineweaver-Burk plots suggested that the MCP-3 acted as competitive inhibitors against ACE.
MCP-3 was further separated by semi-preparative RP-HPLC, and 5 fractions were obtained, among which fractions MCP-3-4 showed the highest ACE inhibition activity. RP-HPLC analysis of MCP-3-4 suggested it was pure. Amino acid sequence of the pure peptide was identified with LC-MS/MS, its structure was Glu-Phe.
分别用碱性蛋白酶(Alcalase 2.4L)、中性蛋白酶(Neutrase)、风味蛋白酶(Flavourzyme)、胰蛋白酶(Trypsin)和复合蛋白酶(Promatex)对厚壳贻贝蛋白进行水解,比较酶解物的水解度DH、蛋白质回收率及ACE抑制率,确定出水解厚壳贻贝蛋白的酶源为碱性蛋白酶；在碱性蛋白酶水解厚壳贻贝蛋白单因素试验的基础上,采用响应面设计进行试验优化,结合各因素对响应值的影响顺序,确定最优酶解条件为[E]/[S]2.18%、pH 8.8,温度55℃,此条件下制备的厚壳贻贝酶解物的ACE抑制率为83.12%。
采用5KD超滤膜对酶解物进行初步分离,收集流出液,所得组分MCP在对ACE抑制的IC50由超滤之前的0.367 mg/mL降为0.308mg/mL。将该初步纯化的组分MCP进一步通过Sephadex G-10凝胶层析分离,以超纯水为洗脱液洗脱,得到三个组分MCP-1、MCP-2和MCP-3,分别测其在对ACE抑制的IC5o为0.739 mg/mL、0.298mg/mL和0.163 mg/mL,反复进样,选择活性最高的峰MCP-3收集。对MCP-3进行活性稳定性实验,结果显示,MCP-3对温度、酸碱变化以及消化酶均有不同程度的耐受性,随着温度或酸碱条件的变化,其对ACE的抑制活性均有良好的稳定性,体外模拟消化实验也表明MCP-3经胃肠道酶系消化后仍会保持较稳定的ACE抑制活性。此外,通过考察不同抑制剂浓度下,底物浓度变化与反应速率的关系,研究得出MCP-3对ACE的抑制类型为竞争性抑制。
对MCP-3利用半制备型RP-HPLC进行进一步的分离纯化,得到5个主要的洗脱峰,分别进行活性测定,选择ACE活性抑制率最高的洗脱峰MCP-3-4进行收集,并利用分析型RP-HPLC对其进行纯度鉴定,结果显示,该组分在220 nm和280 nm的洗脱谱图中,只出现了一个单一的尖峰,说明MCP-3-4是个单一的肽段。通过液质联用进行序列分析,得到MCP-3-4为相对分子质量294.2的二肽,其一级序列结构为Glu-Phe。
Mussel is an important aquaculture species worldwide, but people can't make more profits if only mussel is sold directly. Mussel not only is a traditional tonic food, but also has a high nutritional value and variety of pharmacological effects.
The objectives of this study are to prepare hydrolysates from Mytilus coruscus by enzyme hydrolysis, separate and isolate the activity peptides from Mytilus coruscus hydrolysates (MCHs) by a series of separation and analysis technologies, including ultra-filtration, gel chromatography and reversed-phase high performance liquid chromatography, and identify the active peptides sequences by mass spectrum technology. The antihypertensive effect is evaluated in vitro. The work will be a base for development of antihypertensive functional food production and explore the potential of Mytilus coruscus as a functional Ingredient for value-added foods. The major results obtained in this study are as follows:
Mytilus coruscus protein was hydrolyzed using five different proteases. The results showed that Alcalase 2.4L had the best hydrolyzing capacity, the higher protein recovery and the stronger ACE (AngiotensinⅠ-coverting Enzyme) inhibitory activity. Response surface methodology (RSM) was applied to optimize the hydrolysis conditions using Alcalase 2.4L. The optimum values for enzyme/substrate ratio, pH and temperature were found to be 2.18%,8.8 and 55℃respectively. Under those conditions, the ACE inhibitory rate was 83.12%(1.Omg/mL).
The IC50 of the fraction MCP separated by ultra-filtration was 0.308 mg/mL. Then Sephadex G-10 gel filtration chromatography was used to separate MCP, eluted by H2O. Consequently, three fractions, MCP-1, MCP-2 and MCP-3 were obtained. In Anti-hypertension test, MCP-3 showed best ACE inhibition effects, with its IC50 value of 0.163 mg/mL, which retained activity under various temperature and pH treatments, and showed resistance to in vitro digestion by gastrointestinal proteases. Lineweaver-Burk plots suggested that the MCP-3 acted as competitive inhibitors against ACE.
MCP-3 was further separated by semi-preparative RP-HPLC, and 5 fractions were obtained, among which fractions MCP-3-4 showed the highest ACE inhibition activity. RP-HPLC analysis of MCP-3-4 suggested it was pure. Amino acid sequence of the pure peptide was identified with LC-MS/MS, its structure was Glu-Phe.
引文
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