魔芋ACE抑制肽制备技术及理化性质研究

发布时间：2018-04-04 04:09

本文选题：魔芋飞粉　切入点：ACE抑制肽　出处：《陕西科技大学》2017年硕士论文

【摘要】：血管紧张素转化酶(Angiotensin I-Converting Enzyme,ACE)抑制肽是一种对ACE活性具有抑制作用的多肽,一般由2~20个氨基酸残基组成。ACE抑制肽通过与ACE发生竞争性结合,能阻断血管紧张素Ⅱ生成、促进舒缓肽和脑啡肽生成,从而起到降压作用。目前,已有从绿豆、大豆、酪蛋白、牛乳、麦胚蛋白、牡蛎等原料中制备ACE抑制肽的报道,而以魔芋为原料制备ACE抑制肽的资料甚少。检测结果发现,富含蛋白的魔芋飞粉经过酶法降解可产生支链氨基酸寡肽、ACE抑制肽、高F值寡肽、甘露聚糖肽等活性肽。对魔芋飞粉这一天然药物资源进行研究开发,有望为ACE抑制肽的制备开辟一条绿色的新途径。本文以魔芋飞粉为原料,采用酶解法制备魔芋ACE抑制肽,依次通过超滤及大孔吸附树脂对制备的ACE抑制肽分离纯化,并对其进行质量检测及理化性质分析。研究结果如下:1.魔芋ACE抑制肽酶解法制备:以ACE抑制率为指标,通过对预实验筛选出的胃蛋白酶酶解制备魔芋ACE抑制肽的工艺进行单因素考察和正交优化,得到最佳酶解工艺条件为:底物浓度2%、酶用量5000U/g、温度37℃、pH2.5、酶解时间210min,此时酶解液的ACE抑制率为92.65%、多肽得率为12.60%。2.超滤法分离魔芋ACE抑制肽:以ACE抑制率为指标,预选出最佳超滤膜的截留分子量为1kDa。单因素考察及正交实验优化得到的最佳超滤条件为:在多肽液浓度2%、p H4.0、温度40℃的条件下,采用1kDa的超滤膜超滤15min,产物的ACE抑制率可达到92.75%。3.大孔吸附树脂分离魔芋ACE抑制肽:以吸附率和解吸率为指标,通过对预选出的DA201-C型大孔吸附树脂进行吸附-解吸工艺实验,确定出最佳吸附工艺条件为:上样液质量浓度8mg/mL、p H2.0、上样量8mL、上样流速0.5mL/min;解吸工艺条件为:洗脱液乙醇体积分数80%、洗脱流速1.0mL/min、洗脱时间2h。在此条件下,得到的魔芋ACE抑制肽的抑制率为95.19%。4.红外光谱分析:对分离纯化后的魔芋ACE抑制肽及ACE抑制肽标准品进行红外光谱扫描,结果表明,二者的峰形和峰值大体一致,均存在O-H、N-H、酰胺键、C-O、芳烃的C-H弯曲振动等。综合分析结果,魔芋ACE抑制肽与标准品红外光谱特征基本一致。5.采用紫外全波长扫描对魔芋ACE抑制肽进行检测,结果在220nm处有最大吸收峰,与ACE抑制肽标准品基本一致。高效液相色谱法分析结果表明,魔芋ACE抑制肽与标准品峰形特征相似。紫外分光光度法检测出纯化后样品中魔芋ACE抑制肽含量为81.37%。凝胶渗透色谱法测定出魔芋ACE抑制肽的峰值分子量(MP)为655Da,分子量范围为500~700Da。6.魔芋ACE抑制肽理化性质分析:分离纯化后得到的魔芋ACE抑制肽为灰白色固体粉末,有腥臭味;室温下pH2.0时在水中的溶解率可达95%左右;30~50℃下稳定性较好,但90℃水浴下5h后其ACE抑制率几乎为零;在pH2.0~4.0范围内室温放置2h ACE抑制活性无明显变化,但随着p H值逐渐增大,ACE抑制活性会逐渐降低;NaCl浓度为0.2~0.6mol/L时室温放置2h ACE抑制率仍能维持在90%左右;37℃下魔芋ACE抑制肽在消化酶中稳定性较好,ACE抑制率基本维持在85%。
[Abstract]:Angiotensin converting enzyme (Angiotensin I-Converting Enzyme, ACE) inhibitory peptide is a polypeptide with inhibitory effect on the activity of ACE, consists of 2~20 amino acid residues of.ACE inhibitory peptides through the combination of competition and ACE, can block angiotensin, promote soothing peptides and enkephalin generated thereby to the hypotensive effect. At present, the existing from mung bean, soybean, casein, milk, wheat germ protein, preparation of ACE inhibitory peptides from oyster and other raw materials reported, with Konjac as raw materials for preparation of ACE inhibitory peptides with little data. The results show that the rich protein containing konjac powder after enzymatic degradation can produce branched amino acid peptide, ACE inhibitory peptide, high F-measure oligopeptide, mannan peptide active peptides. The konjac powder the natural medicine resources for research and development, is expected for the preparation of ACE inhibitory peptides to open up a new way to green. In this paper, konjac powder As raw materials, preparation of ACE inhibitory peptides by enzyme hydrolysis method of konjac, followed by ultrafiltration and macroporous resin on the preparation of ACE inhibitory peptides were isolated and purified, and carries on the analysis and physicochemical properties of quality detection. The results are as follows: 1. konjac ACE inhibitory peptides prepared by enzymatic hydrolysis with ACE inhibition rate as the index. The pepsin of pre experiment screened the solution preparation process of konjac ACE inhibitory peptides of single factor test and orthogonal optimization, the optimal enzymatic hydrolysis conditions: substrate concentration of 2%, enzyme dosage 5000U/g, temperature 37 C, pH2.5, hydrolysis time 210min, the enzymolysis liquid of ACE inhibition rate was 92.65%. The yield of polypeptide 12.60%.2. ultrafiltration separation of konjac ACE inhibitory peptides with ACE inhibitory activity as index, the optimum selection of ultrafiltration membrane MWCO 1kDa. single factor and orthogonal experiment to optimize the best ultrafiltration conditions: in peptide concentration of 2%, P H4.0, temperature 40 degrees C under the condition of using 1kDa ultrafiltration membrane ultrafiltration 15min, the inhibition rate of the product ACE can achieve the separation of konjac ACE inhibitory peptides by 92.75%.3. macroporous resin adsorption rate and desorption rate as the index, the adsorption of DA201-C macroporous resin pre selected desorption experiment, the optimum adsorption conditions: the concentration of sample solution 8mg/mL, P H2.0, sample volume 8mL, sample flow rate 0.5mL/min; desorption conditions: eluent ethanol concentration 80%, elution rate 1.0mL/min, elution time 2h. under these conditions, the inhibition rate of ACE peptide was konjac 95.19%.4. infrared spectroscopy analysis of separation purified konjac ACE inhibitory peptides and ACE inhibitory peptides standard IR scanning results showed that two of the peak and shape of roughly the same, there are O-H, N-H, C-O, aromatic amide bond, the C-H bending vibration. The comprehensive analysis. Fruit, konjac ACE inhibition characteristics of infrared spectra of peptides with the standard is consistent with.5. UV wavelength scanning of konjac ACE inhibitory peptides were detected in the 220nm has the maximum absorption peak, consistent with the ACE inhibitory peptide standard. HPLC analysis showed that konjac ACE inhibitory peptides with standard peak similar characteristics. The ultraviolet spectrophotometry to detect the purified samples of konjac ACE inhibitory peptide content of 81.37%. gel permeation chromatography method for the determination of the peak molecular weight of ACE inhibitory peptide of konjac (MP) for 655Da, the molecular weight range of 500~700Da.6. konjac ACE inhibitory peptide physicochemical properties analysis: purified konjac ACE inhibition the peptide is grey white powder, have xingchouwei; rate of up to 95% solubility in water at room temperature at about 30~50 DEG pH2.0; good stability, but under 90 DEG C water bath after 5h the ACE inhibition rate is almost zero; in the range of 4 pH2.0~ Room temperature placed 2H ACE inhibitory activity had no obvious change, but with the P H increases, ACE inhibitory activity will gradually decrease; when the concentration of 0.2~0.6mol/L NaCl 2H ACE inhibition rate at room temperature can be maintained at about 90% DEG C; 37 konjac ACE inhibitory peptide had better stability of digestive enzymes, the inhibition rate of ACE remained at 85%.

【学位授予单位】：陕西科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R945

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