花椒籽蛋白降血压肽的制备及性质研究

发布时间：2018-09-07 08:50

【摘要】：花椒籽是调味料花椒果皮生产的主要副产物,其中富含蛋白质,且氮基酸组成齐全,是一种优质、无毒的新型植物蛋白质资源。目前对花椒籽的研究主要集中在优化花椒籽油的提取工艺,关于花椒籽蛋白质开发利用的研究报道很少。本研究采用响应面法优化酶解花椒籽蛋白制备降血压肽的工艺,同时采用超滤膜法和Sephadex G-25凝胶层析对其进行初步纯化,并对该降血压肽主要的性质进行了研究,为花椒籽蛋白降血压肽的进一步加工利用奠定了理论基础。本研究主要研究内容及结果如下：1.采用胰蛋白酶、木瓜蛋白酶、中性蛋白酶、碱性蛋白酶和复合蛋白酶5种不同蛋白酶分别水解花椒籽蛋白,以酶解物对血管紧张素转换酶(ACE)抑制率为指标,筛选出制备花椒籽蛋白降血压肽的最佳蛋白酶为木瓜蛋白酶。在单因素实验基础上,根据Box-Benhnken中心组合试验设计原理,考察酶解时间、加酶量、酶解温度和pH值等制备条件对获得的酶解产物ACE抑制率的影响。结果表明：回归模型能较好地反映各因素水平与响应值之间的关系,并获得酶解花椒籽蛋白制备降血压肽的最佳工艺参数为：底物质量浓度3g/100mL,酶解时间4.9 h,加酶量10200 U/g,酶解温度为37℃,pH值为6.9,在此条件下,所得酶解产物的ACE抑制率为68.00%。2.采用截留分子量为5 kDa的超滤膜超滤酶解液,其中超滤液组分ACE抑制率得到显著提高(p0.05),达到78.75%。将该组分真空冷冻干燥后用Sephadex G-25凝胶层析进一步分离纯化。主要分析了洗脱流速、上样量以及样品浓度对分离效果的影响,得到最佳凝胶层析条件为：以超纯水为洗脱溶液,洗脱流速0.6 mL/min,上样量2.0 mL,样品浓度30 mg/mL,在此条件下可收集得到4个组分,其中洗脱峰Ⅱ有较高的ACE抑制效果,ACE抑制率为85%,IC50为0.021 mg/mL,该组分经Tricine-SDS-PAGE测定分子量在3 kDa以下。3. 采用氨基酸自动分析仪测定花椒籽蛋白酶解液超滤前后所得组分的氨基酸组成及其含量,并研究5 kDa的超滤膜截留液组分的功能特性及其稳定性。结果表明：酶解产物、截留液、超滤液和层析液样品中氨基酸组成较齐全,其中谷氨酸含量最高,分别为6.96%、6.58%、4.]5%、0.69%；截留液在较宽的pH值(2.0-10.0)范围内溶解性良好,达到80%以上：有较高的吸油性,在温度30-70℃范围内平均吸油性为4.0mL/g；在pH值2.0-10.0范围内起泡性呈先降低后升高的趋势。但泡沫稳定性正相反：乳化性随着pH值的升高而增大,但乳化稳定性先降低后升高：截留液经过63℃-95℃热处理后ACE抑制率还能维持在48%以上,与原样品(51.03%)差异不显著(p0.05)：在不同酸碱环境下ACE抑制率维持在47%左右；光照72 h后ACE抑制率由44%下降到40%,差异不显著(p=0.12,p0.05)；葡萄糖、乳糖和蔗糖对降血压肽的影响较小,ACE抑制率无显著性差异(p0.05),保持在50%不变；花椒籽蛋白降血压肽经胃蛋白酶和胰蛋白酶消化后,仍能保持较高的ACE抑制活性。
[Abstract]:Zanthoxylum bungeanum seed is the main by-product of the peel production of the seasoning Zanthoxylum bungeanum, which is rich in protein and has a complete composition of nitrogen-based acids. It is a high-quality and non-toxic new plant protein resource. Response surface methodology (RSM) was used to optimize the enzymatic hydrolysis of Zanthoxylum bungeanum seed protein to prepare antihypertensive peptides. Ultrafiltration membrane method and Sephadex G-25 gel chromatography were used to purify the peptides. The main properties of the antihypertensive peptides were studied, which laid a theoretical foundation for the further processing and utilization of Zanthoxylum bungeanum seed protein antihypertensive peptides. The research contents and results are as follows: 1. Papaya protein was hydrolyzed by trypsin, papain, neutral protease, alkaline protease and complex protease, respectively. The best protease for preparing anti-hypertensive peptides of Zanthoxylum bungeanum seed protein was selected as papain protein according to the inhibition rate of the hydrolysate on angiotensin converting enzyme (ACE). Enzyme. On the basis of single factor experiment and according to the design principle of Box-Benhnken central combination experiment, the effects of enzymatic hydrolysis time, enzyme dosage, enzyme hydrolysis temperature and pH value on the inhibition rate of ACE were investigated. The optimum technological parameters for preparing antihypertensive peptides from pepper seed protein were as follows: substrate concentration 3 g/100 mL, enzymatic hydrolysis time 4.9 h, enzyme content 10 200 U/g, enzymatic hydrolysis temperature 37 C and pH 6.9. Under these conditions, the ACE inhibition rate of the hydrolysate was 68.00%. 2. Ultrafiltration membrane enzymatic hydrolysate with 5 kDa intercepted molecular weight was used, and the ACE inhibitor of the ultrafiltration component was found. After vacuum freeze-drying, the component was further separated and purified by Sephadex G-25 gel chromatography. The effects of elution velocity, sample loading and sample concentration on the separation efficiency were analyzed. The optimum conditions of gel chromatography were as follows: the elution solution was ultrapure water, the elution velocity was 0.6 mL/min, and the elution velocity was 0.6 mL/min. The sample size was 2.0 mL and the sample concentration was 30 mg/mL. Four components were collected under this condition. The elution peak II had higher ACE inhibition effect. The inhibition rate of ACE was 85% and the IC50 was 0.021 mg/mL. The molecular weight of the component was below 3 kDa by Tricine-SDS-PAGE. The amino acid automatic analyzer was used to determine the hydrolysate of Zanthoxylum bungeanum protease before and after ultrafiltration. The results showed that the amino acid compositions of enzymatic hydrolysates, interceptors, ultrafilters and chromatographic samples were relatively complete, and the content of glutamic acid was the highest (6.96%, 6.58%, 4.5%, 0.69%) and the intercepted solution had a broader pH value (2.0-10.6%). In the range of pH value 2.0-10.0, the foaming property first decreases and then increases. But the foaming stability is on the contrary: the emulsifying property increases with the increase of pH value, but the emulsifying stability first decreases and then increases: cut off The ACE inhibitory rate of the reserved solution after heat treatment at 63 ~95 C could still be maintained above 48%, and there was no significant difference between the original sample (51.03%) and glucose, lactose and sucrose (p0.05): ACE inhibitory rate maintained at about 47% in different acid-base environments; ACE inhibitory rate decreased from 44% to 40% after 72 hours of illumination, and the difference was not significant (p = 0.12, p0.05); the effect of glucose, lactose and sucrose on antihypertensive peptides There was no significant difference in ACE inhibitory rate between the two groups (p0.05). The antihypertensive peptides of Zanthoxylum bungeanum seed protein could still maintain high ACE inhibitory activity after digested by pepsin and trypsin.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TS201.2

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