| China duck production in the world, duck bone resources were extremely rich, but utilization was very low.At present, using angiotensin-I converting enzyme inhibitors (ACEI) as helicase activity of such drugs to suppress angiotensin-I converting enzyme(ACE) was the important way of clinical treatment of hypertension, and foodborne antihypertensive peptides was also a kind of effective ACEI. It was safe and no side effects, and had become a research hotspot now and the development trend of prevention and treatment of hypertension in future. In this study,we preparated peptides with ACE inhibitory activity by hydrolysis of papain from duck bone,and evaluated the ACE inhibitory activity in vitro of the hydrolysates,then with a series of purified studies, designed to explore the process line and process parameters of preparation of highly active ACE inhibitory peptides with duck bone protein.That provided the experimental evidence and theoretical basis for industrial preparation of ACE inhibitory peptides.1.Using papain to zymolyse duck bone protein for preparation of ACE inhibitory peptides, optimizated hydrolyze process through quaternary quadratic general rotatory orthogonal experiment, and as the index of the degree of hydrolysis and the ACE inhibition rate to establish a mathematical model,to determine the best of enzymatic hydrolysis.The results showed that:the best of enzymatic hydrolysis of papain hydrolysis duck bone protein to preparate the ACE inhibitory peptides was that:the substrate concentration for 11.5g/100mL,the enzyme bottom for 8000U/g, the hydrolysis temperature for 70℃, the hydrolysis time for 5.5h,pH5.5.With these conditions, the ACE inhibition rate of zymolysing liquid was the highest of 85.71%,and the half-inhibitory concentration(ICso) was 1.728 mg/mL,the hydrolysis degree was 20.81%.The influence of the substrate concentration (X1), the enzyme bottom (X2), the hydrolysis temperature (X3) and the hydrolysis time (X4) with the hydrolysis degree was X2> X3> X4> X1. The regression model equation of the hydrolysis degree (Y1) with the four factors was:Y1=23.969+0.175X1+1.455X2+0.959X3+0.426X4-0.053X1X2+0.126X1X3-0.066X1X4 +0.107X2X3+0.003X2X4+0.367X3X4+0.275X12-0.152X22-0.318X32+0.343X42. The influence of the four factors with the ACE inhibition rate was:X4> X0> X3> X2.The regression model equation of the ACE inhibition rate (Y2) with the four factors was: Y2=74.481-5.748X1-2.345X2-5.160X3-7.843X4-3.849XiX4-3.821X12-3.620X32-2.698X42.2.Analysising the correlation of the hydrolysis degree and the ACE inhibition rate, and establishing the mathematical model. The results showed that:the hydrolysis degree (X) and the ACE inhibition rate (Y) were significant correlation (P<0.01), the curve fitting model was:Y=-157.572+21.215X-0.491X2.It explained that the hydrolysis degree and the ACE inhibition rate were positively correlated in a certain range, when the hydrolysis degree of up to a certain value, the hydrolysis degree and the ACE inhibition rate were negatively correlated; And the model was confirmed successful by validation, and could be generalized.3.Using the ion exchange chromatography to separation and purification the zymolysing product, with investigating the influence of the buffer pH, the ionic strength of the starting buffer and the elution, the injection flow rate and the elution flow rate on the effect of separation, to obtain the optimum conditions of separation and purification. The results showed that:when the sample volume for 15mg/150mL (peptide mass/bed volume), the initial buffer for pH=4.8,0.05mol/L sodium acetate, the elution method for the linear gradient elution, the limit buffer for pH=4.8,0.05mol/L sodium acetate with 1.0mol/L NaCl solution, the injection flow rate and the elution flow rate for 1.0mL/min, it had the best separation for ACE inhibitory peptides of duck bone by SP-Sephadex C-25 chromatographic column(Φ2.5×40).It isolated from seven components with higher ACE inhibitory activity, and the ACE inhibition rate of the highest ACE inhibitory activity component was 70.515%,the IC50 alue concentration was 31.14μg/mL4.Using four specifications ultrafiltration centrifugal tubes to separate the higher ACE inhibitory activity component of the products of the ion exchange chromatography,whose intercept molecular weight was 10KDa,5KDa,3KDa,2KDa.The results showed that:after ultrafiltration, the order of the ACE inhibitory activity of each component was:the component of molecular size 2-3KDa> the component of molecular size more than 10KDa> the component of molecular size less than 2KDa> the component of molecular size 3-5KDa> the component of molecular size 5-10KDa.Among them, the component of molecular size 2-3KDa had the highest ACE inhibitory activity of 91.37%, and the IC50 alue concentration was 0.927 mg/mL.5. According to the high ACE inhibitory activity component after ultrafiltration, using gel chromatography for further purification, by investigating the influence of the sample volume,the injection flow rate and the elution flow rate to the separation effect, to obtain the optimum conditions of separation and purification.The results showed that:when the sample concentration for 10mg/mL,the sample size for the bed size of 1.5%(2.25mL), the eluent for ultra-pure water, the injection flow rate and the elution flow rate was 0.5mL/min, it had the best separation for ACE inhibitory peptides of duck bone by Sephadex G-25 (fine) chromatographic column(Φ1.6×80).It isolated from five components with higher ACE inhibitory activity, and the ACE inhibition rate of the highest ACE inhibitory activity component was was 90.33%, the IC50 alue concentration was 20.05μg/mL... |
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