【摘要】：花椒籽是調(diào)味料花椒果皮生產(chǎn)的主要副產(chǎn)物,其中富含蛋白質(zhì),且氮基酸組成齊全,是一種優(yōu)質(zhì)、無毒的新型植物蛋白質(zhì)資源。目前對花椒籽的研究主要集中在優(yōu)化花椒籽油的提取工藝,關(guān)于花椒籽蛋白質(zhì)開發(fā)利用的研究報道很少。本研究采用響應(yīng)面法優(yōu)化酶解花椒籽蛋白制備降血壓肽的工藝,同時采用超濾膜法和Sephadex G-25凝膠層析對其進行初步純化,并對該降血壓肽主要的性質(zhì)進行了研究,為花椒籽蛋白降血壓肽的進一步加工利用奠定了理論基礎(chǔ)。本研究主要研究內(nèi)容及結(jié)果如下：1.采用胰蛋白酶、木瓜蛋白酶、中性蛋白酶、堿性蛋白酶和復合蛋白酶5種不同蛋白酶分別水解花椒籽蛋白,以酶解物對血管緊張素轉(zhuǎn)換酶(ACE)抑制率為指標,篩選出制備花椒籽蛋白降血壓肽的最佳蛋白酶為木瓜蛋白酶。在單因素實驗基礎(chǔ)上,根據(jù)Box-Benhnken中心組合試驗設(shè)計原理,考察酶解時間、加酶量、酶解溫度和pH值等制備條件對獲得的酶解產(chǎn)物ACE抑制率的影響。結(jié)果表明：回歸模型能較好地反映各因素水平與響應(yīng)值之間的關(guān)系,并獲得酶解花椒籽蛋白制備降血壓肽的最佳工藝參數(shù)為：底物質(zhì)量濃度3g/100mL,酶解時間4.9 h,加酶量10200 U/g,酶解溫度為37℃,pH值為6.9,在此條件下,所得酶解產(chǎn)物的ACE抑制率為68.00%。2.采用截留分子量為5 kDa的超濾膜超濾酶解液,其中超濾液組分ACE抑制率得到顯著提高(p0.05),達到78.75%。將該組分真空冷凍干燥后用Sephadex G-25凝膠層析進一步分離純化。主要分析了洗脫流速、上樣量以及樣品濃度對分離效果的影響,得到最佳凝膠層析條件為：以超純水為洗脫溶液,洗脫流速0.6 mL/min,上樣量2.0 mL,樣品濃度30 mg/mL,在此條件下可收集得到4個組分,其中洗脫峰Ⅱ有較高的ACE抑制效果,ACE抑制率為85%,IC50為0.021 mg/mL,該組分經(jīng)Tricine-SDS-PAGE測定分子量在3 kDa以下。3. 采用氨基酸自動分析儀測定花椒籽蛋白酶解液超濾前后所得組分的氨基酸組成及其含量,并研究5 kDa的超濾膜截留液組分的功能特性及其穩(wěn)定性。結(jié)果表明：酶解產(chǎn)物、截留液、超濾液和層析液樣品中氨基酸組成較齊全,其中谷氨酸含量最高,分別為6.96%、6.58%、4.]5%、0.69%；截留液在較寬的pH值(2.0-10.0)范圍內(nèi)溶解性良好,達到80%以上：有較高的吸油性,在溫度30-70℃范圍內(nèi)平均吸油性為4.0mL/g；在pH值2.0-10.0范圍內(nèi)起泡性呈先降低后升高的趨勢。但泡沫穩(wěn)定性正相反：乳化性隨著pH值的升高而增大,但乳化穩(wěn)定性先降低后升高：截留液經(jīng)過63℃-95℃熱處理后ACE抑制率還能維持在48%以上,與原樣品(51.03%)差異不顯著(p0.05)：在不同酸堿環(huán)境下ACE抑制率維持在47%左右；光照72 h后ACE抑制率由44%下降到40%,差異不顯著(p=0.12,p0.05)；葡萄糖、乳糖和蔗糖對降血壓肽的影響較小,ACE抑制率無顯著性差異(p0.05),保持在50%不變；花椒籽蛋白降血壓肽經(jīng)胃蛋白酶和胰蛋白酶消化后,仍能保持較高的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.
【學位授予單位】：四川農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：TS201.2

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