【作者】 黎金；

【导师】 张国权；

【作者基本信息】 西北农林科技大学 ， 粮食、油脂及植物蛋白工程， 2009， 硕士

【摘要】 本文以荞麦蛋白为材料,系统分析了碱性蛋白酶对荞麦蛋白的酶解特性,并运用对数函数对其水解动力学过程进行描述和拟合,确定不同温度和pH条件下碱性蛋白酶水解荞麦蛋白过程的Km值、最大反应速率和临界底物浓度,并得出特定水解条件下,水解度与水解时间的关系函数;对碱性蛋白酶酶解荞麦蛋白制备抗氧化多肽的工艺条件进行了优化,得出碱性蛋白酶水解荞麦蛋白制备荞麦多肽的最佳工艺条件;以Vc为对照,通过对不同水解条件下荞麦多肽的还原能力以及超氧阴离子自由基、ABTS自由基、DPPH自由基和羟自由基清除能力的分析与比较,明确了荞麦多肽的抗氧化活性;并初步探讨了不同荞麦多肽粉添加量以及原荞麦蛋白粉对弱筋和强筋面粉流变学特性的影响。主要研究结果如下:(1) Km值是荞麦蛋白酶解反应的重要特征常数,米式方程通过Km值部分描述了酶反应的性质。在碱性蛋白酶水解荞麦蛋白的过程中,水解度随水解时间的变化呈对数函数关系,可用公式h=(1/b)ln(1+abt)对其水解动力学过程进行描述;用pH-stat法的特点和Hanes-Woolf作图法求解不同pH和温度下的Km值,结果表明:在65℃,pH 10时,碱性蛋白酶水解荞麦蛋白的Km值为0.35%;在50℃,pH 9.8条件下,碱性蛋白酶水解荞麦蛋白过程的Km值为0.96%,最大反应速率为15.7mg mL-1 min-1。(2)底物具有促进水解反应速率和抑制酶活性的双重作用,在低底物浓度条件下,荞麦蛋白的水解度较高;当底物浓度增加到6%时,荞麦蛋白水解度与底物浓度的关系曲线出现转折,水解度较低且趋于平缓;在酶浓度为0.002 g/mL时,水解反应的临界底物浓度为12.27%;在pH9.8、水解温度为50℃、水解时间30 min的条件下,碱性蛋白酶水解荞麦蛋白的动力学方程为1.2182ln(1 12.6( [ ]0.058) )h Et(3)以水解度为指标,各因素对碱性蛋白酶酶解荞麦蛋白制备抗氧化多肽的影响顺序依次为加酶量、底物浓度、水解时间。在60℃、pH9.5、底物浓度5.21%、水解时间2.7 h、加酶量3999.99 U/g蛋白时,荞麦蛋白的理论水解度为11.982%,实际水解度为12.156%,多肽浓度为22.095 mg/mL。以多肽浓度为指标,各因素对碱性蛋白酶酶解荞麦蛋白制备抗氧化多肽的影响顺序依次为底物浓度、水解时间、加酶量。为得到较高的多肽浓度,在60℃、pH9.5条件下,碱性蛋白酶酶解荞麦蛋白的最佳工艺参数为底物浓度5.77%,水解时间2.72 h,加酶量3999.98 U/g蛋白,此时荞麦蛋白的理论多肽浓度为23.57 mg/mL;实测水解度为11.405%,多肽浓度为24.571 mg/mL。(4)不同水解条件下制备的荞麦多肽均具有较强的清除超氧阴离子自由基、ABTS自由基、DPPH自由基和羟自由基的能力,而未水解荞麦蛋白清除超氧阴离子自由基、ABTS自由基和羟自由基的能力并不明显。未水解荞麦蛋白的还原能力和清除DPPH自由基能力与水解后的荞麦多肽相当,这表明未水解的荞麦蛋白也具有一定的抗氧化性能。相同浓度荞麦蛋白及荞麦多肽的抗氧化能力低于Vc。(5)添加不同量荞麦多肽的面团,吸水率均随荞麦多肽粉添加量的增加而不断降低,当超过8%添加量后,吸水率受到的影响较小;面筋的含量随着荞麦多肽粉添加量的增加呈现先增加后减小的趋势;面团形成时间和稳定时间随荞麦多肽粉添加量的增加均呈先减小后增加的趋势,弱化度呈上升趋势,评价值也呈现一直增加的趋势。从拉伸特性来看,随着荞麦多肽粉添加量的增加,面团的延伸性变好,拉伸阻力在高浓度添加量的情况下比原面粉大,拉伸能量则呈现一直增加的趋势。总体而言,添加荞麦蛋白粉及其多肽粉对面团粉质及拉伸特性均具有促进作用。更多还原

【Abstract】 Buckwheat protein was used as material in this paper, the enzymatic hydrolysis characteristics of buckwheat protein hydrolyzed with alkaline protease was systematically analyzed, and logarithmic equation was used to describe and fit the relationship between hydrolysis degree and time. Km value, Vmax under different pH and temperature and critical substrate concentration was solved. Under certain hydrolyzing condition, the relationship between hydrolysis degree and time was determined. Under this condition, optimized the technology condition of buckwheat protein hydrolyze with alkaline protease for preparing antioxidant peptide. Determined the reducing capacity, clearing superoxide anion radical ability, eliminating ABTS radical, DPPH radical, hydroxyl free radical ability of the peptides getting from different hydrolyzing condition. The effects of buckwheat peptide power on rheological characteristics of strong gluten flour and weak gluten flour was preliminary studied in this paper. The main results were:(1) Km is the main character constant during the hydrolysis process, M-equation can partly described the characteristic of enzyme reaction through Km value. During the process of buckwheat protein hydrolyzed with alkaline protease, the change between hydrolysis degree and time presented logarithmic function relationship, its hydrolytic kinetic process can be described using the formula of h=(1/b)ln(1+abt).Use the feature of pH-stat method and Hanes-Woolf mapping method solving Km value under different pH and temperature. The results showed that, under the condition of 65℃, pH10, the Km value was 0.35%; under the condition of 50℃,pH 9.8, Km value was 0.96%, Vmax was 15.7mg mL-1 min-1.(2) The substrate concentration possessed dual function of accelerating hydrolysis reaction rate and depressing enzyme activation, under lower substrate concentration condition, the hydrolysis degree of buckwheat protein was higher; as the substrate concentration added to 6%, the relationship curve between protein hydrolysis degree and substrate concentration presented a transition, the hydrolysis degree became lower and tended to flat; under the enzyme concentration of 0.002g/mL, the critical substrate concentration was 12.27%. Under the condition of pH 9.8, temperature 50℃, the kinetic equation of alkaline protease catalytic hydrolyze buckwheat protein in the first 30 min can be expressed as (3) Under the condition of 60℃, pH9.5, the optimum technological parameters for preparing antioxidant peptide was 5.21% substrate concentration, hydrolysis time 2.7h, enzyme addition 3999.99U per gram protein , at this time the theoretical hydrolysis degree was 11.982%, close to the measured hydrolysis degree 12.156%, and the measured peptide concentration was 22.095mg/mL. Taking hydrolysis degree as an index, the order of each factor affecting alkaline enzyme hydrolyzing buckwheat protein to prepare antioxidant peptide was: enzyme concentration, substrate concentration, time. In order to get a higher peptide concentration, under the condition of 60℃, pH9.5, the optimum technological parameters for preparing antioxidant peptide was 5.77% substrate concentration, hydrolysis time 2.72h, enzyme addition 3999.98U per gram protein, at this time the measured hydrolysis degree was 11.405%, the theoretical peptide concentration was 23.57mg/mL, close to the measured peptide concentration 23.57mg/mL. Taking peptide concentration as an index, the order of each factor affecting alkaline enzyme hydrolyzing buckwheat protein to prepare antioxidant peptide was: substrate concentration, time, enzyme concentration.(4) Buckwheat peptides all have strong clearing superoxide anion radical ability, eliminating ABTS radical, DPPH radical, hydroxyl free radical ability getting from different hydrolyzing condition, however, the same ability of buckwheat protein without hydrolyzing was not significant, but their reducing capacity and DPPH radical eradication were equivalent, it proved that buckwheat protein without hydrolysis had a certain antioxidant ability. But overall, the antioxidant ability of buckwheat protein and peptide of the same substrate was not as good as Vc.(5) From the whole farinograph properties, the higher buckwheat peptide powder addition level, the lower water absorption rate. However, as the addition level above 8%, the effect of buckwheat peptide powder on water absorption rate was smaller. Dough development time and stability time decreased first and then rose as buckwheat peptide powder addition level rose, weakening level presented increasing tendency, evaluation value as a comprehensive index, showed an increasing trend. From extenograph properties, the higher buckwheat peptide powder addition level, the better extensibility, the higher extension resistance in a high buckwheat peptide concentration, the bigger extension energy. Overall, buckwheat protein or buckwheat peptide has a positive effect on farinograph and extenograph properties.更多还原