【作者】 王兴；

【导师】 王敏；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2009， 硕士

【摘要】 苦荞具有很高的营养价值和药用价值。苦荞类黄酮和蛋白质是其最主要的生物活性成分,赋予荞麦抗氧化、降血糖、降血脂和抗肿瘤等多种生理功能。近年来对苦荞蛋白的研究结果表明苦荞蛋白质拥有:降低人体血液胆固醇、抑制脂肪蓄积,促进肌肉增长、抑制胆结石的形成等生理功能,使得苦荞蛋白质越来越受到重视。但其经人体消化后的活性状况变化未见报道。为此本文以苦荞面粉为原料,采用碱萃取工艺提取苦荞蛋白,并对影响其溶解性和乳化性的因素进行了分析,同时模拟体外消化过程及吸收过程,研究苦荞蛋白质消化后产生肽的组成、活性状况及其吸收特性。主要结论如下:1.苦荞蛋白质碱萃取过程中pH值为主要影响因素,此外pH值和料液比的交互作用以及温度和料液比的交互作用对萃取过程也有显著影响。对碱萃取工艺分析,得到回归模型:Y=-1980.08+408.85X₁-1.93X₂+17.23X₃-2.60X₁X₃+0.13X₂X₃-18.52X₁²,提取最佳条件为:pH值10.0,提取温度50℃,料液比1:10,在此条件下苦荞蛋白质得率为138.8±4.16g·kg^-1。最佳酸沉淀pH值为4.2。2. pH、温度和离子强度对其溶解性和乳化性等功能特性有着不同影响。远离等电点的pH值使苦荞蛋白质产生的静电斥力,增加其溶解性和乳化性;低离子强度（<0.5%）的盐溶作用能不同程度的改善苦荞蛋白质的溶解性和乳化性;高离子强度（>0.5）降低其溶解性和乳化性。3.苦荞蛋白质经模拟消化过程10小时时产生多肽对?OH清除率、DPPH清除率、大鼠肝脂质过氧化抑制率和大鼠H₂O₂诱导红细胞溶血抑制率达到最高,O2^-·清除率及总还原能力在10小时时都有较理想效果,但在11小时时肽含量降低,氨基态氮含量升高,各抗氧化指标都有显著下降趋势。4.葡聚糖凝胶G-25与G-15串联柱对荞麦多肽模拟消化过程10小时所产生多肽进行分离,分离出5个组分分子量分别为:峰Ⅰ,5100 Da～5200Da;峰Ⅱ,2700 Da～2800 Da;峰Ⅲ,1300 Da～1400 Da;峰Ⅳ,870 Da～970 Da;峰Ⅴ,570 Da～670Da。其中第4个组分对大鼠肝脂质过氧化抑制率和红细胞溶血抑制率表现出很强的活性,IC50分别0.056mg·mL^-1和0.13 mg·mL^-1,其分子量为870 Da～970 Da。5.利用模拟消化过程不同时间下H2O2诱导大鼠红细胞氧化溶血抑制率与动力学模型所求得的水解度进行非线性回归分析,结果发现单变量抛物线模型决定系数最高（R2=0.9935）。将动力学模型代入相关模型中,最终得到以酶用量（E0）、底物浓度（S0）和消化时间（t）为自变量,抗氧化活性为因变量的相关模型。模型如下:模拟胃消化过程:y= 0. 792- 0.05·{0.189·Ln[1+（0.0169·E₀/S₀-0.0919）·t]-3.333}²模拟肠消化过程: y=0. 792- 0.05·{1.28·[1+（0.002290E₀/S₀-0.0101）·（t-t₀）]-3.333}²式中:E₀为初始酶添加量（U/mL）;S0为初始底物浓度（g/mL）;t为总消化时间（h）;t0为胃消化时间（h）。模型可预测范围为胃消化过程1～8小时;肠消化过程1～8小时。对最终相关模型进行验证,结果表明相关模型所得预测值与试验实测值比较偏差较小,方程拟和度良好,模拟消化抗氧化相关模型能够较好的预测模拟消化过程抗氧化指标变化。6.苦荞蛋白质经模拟人体消化可得到血管紧张素转运酶（Angiotensin I-Converting Enzyme, ACE）抑制活性肽,肠消化过程是ACE抑制活性肽产生的主要过程。在模拟消化过程10小时时,ACE抑制活性最强。荞麦蛋白模拟消化所得多肽分子量范围在870-970Da有最强的ACE抑制活性。经大鼠离体肠道吸收试验表明,回肠是ACE抑制活性肽最佳吸收部位,经消化后所产生的多肽分子量为1300 Da～1400 Da,870 Da～970 Da,570 Da～670Da可被大鼠离体小肠吸收。更多还原

【Abstract】 Tartary buckwheat, a traditional Asian food stuff, is said to be one of the candidates responsible for beneficial health effects. The main active component are the tartary buckwheat flavone and protein. They are endowed with various physiological activity, for example, antioxidant activity, hypoglycemic activity, reducing blood lipid activity, antitumor activity, et al. The tartary buckwheat protein has many physiological activity as follows: lower the cholesterol in human blood, inhibit lipid accumulation, promote muscle growth, inhibit cholelithiasis formation, et al. But there lack of researching on the physiological activity after degistion. The tartary buckwheat protein was extracted from the tartary buckwheat flour, and it was digested by simulating gastrointestinal digestion, and then the antioxidation activity and the ACE-inhibitory properties was determined, the absorption was also determined by perfusing of isolated small intestine in rats. The main results was showed as follow:1. The pH value was the main influence factor of the alkali extraction of the tartary buckwheat protein. It was the significant effect that the interaction of the pH value and the solid-liquid ratio during the alkali extraction. The model was obtained by analysising on the alkali extraction,it was as follow: Y=-1980.08+408.85X₁-1.93X₂+17.23X₃-2.60X₁X₃+0.13X₂X₃-18.52X₁². The optimum conditions of the alkali extraction technology were obtained as follows: pH value 10.0, temperature 50℃, the ratio of material to solvent 1:10. At the optimized condition, the extraction yield of was 138.8g/kg. The best pH value of acid precipitation was 4.2.2. The solubility of tartary buckwheat protein was the lowest around isoelectric point, and the solubility increased with the increase of low ion concentration. Effects of pH and ion concentrations on concentration on emulsibility were similar to those effects on solubility. The higher the concentration of tartary buckwheat protein the better of its emulsibility.3. The clearance rate of·OH, DPPH and the inhibition rate of lipid peroxidation and red blood hemolysis reached the maximum at the 10 hour of the process of the tartary buckwheat protein by simulating gastrointestinal digestion. The clearance rate of O₂^· and the total reduction ability also get the ideal effect at the time point. But the six indexes of the antioxidation activity decreased significantly at the 11 hour of the process of the tartary buckwheat protein by simulating gastrointestinal digestion, dure to the peptides content decreased.4. The digestive production of the 10 hour of the process of the tartary buckwheat protein by simulating gastrointestinal digestion was separated by gel filtration chromatography on series connection of Sephadex G-25 and Sephadex G-15. Five major peptide fractions were present, and were estimated to be 5100-5200, 2700-2800, 1300-1400, 870-970 and 570-670 Da. Peak 4 possessed the strongest inhibition rate of lipid peroxidation and red blood hemolysis (IC50=0.056mg·mL^-1 and 0.13 mg·mL^-1). The molecular weight was estimated to be 870 Da～970 Da.5. the nonlinear regression analyses the inhibition rate of red blood hemolysis and dynamic model of simulating gastrointestinal digestion. The single variable parabola model has the best coefficient of determination（R2=0.9935）. And then the antioxidation activity model was obtained as follow:The process of simulating gastric digestion y= 0. 792- 0.05·{0.189·Ln[1+（0.0169·E₀/S₀-0.0919）·t]-3.333}²The process of simulating intestinal digestion: y=0. 792- 0.05·{1.28·[1+（0.002290E₀/S₀-0.0101）·（t-t₀）]-3.333}²the letters of the model as follow: E₀: addition of initial enzyme（U/mL）;S0: initial substrate concentration（g/mL）;t: total digestion time（h）;t0: gastric digestion time（h）.6. Buckwheat protein has the high ACE inhibitory activity via simulating gastrointestinal digestion. The process of simulating intestinal digestion was the main process of production of ACE inhibitory activity peptides. The buckwheat peptides with Mr 870-970 possessed the strongest ACE inhibitory activity, and the IC50 is 0.07mg/mL. The peptides with molecular weight at 1300 Da～1400 Da, 870 Da～970 Da, 570 Da～670Da can be absorbed by IIR, and the best absorb part was the ileum.更多还原