| Rice bran has high nutritional value, which contains about 14% protein. Rice bran can prevent cardiovascular disease, cancer and other diseases. The functional factors in rice bran have some contribution to lowering blood pressure and blood fat. It has four kinds of protein according to the solubility of rice bran protein, which is including albumin, gliadin, gluten and globulin. Rice bran protein is a good raw material for producing bioactive peptides because of some advantages, such as its balanced amino acid composition, highly ratio of hydrophobic amino acids and aromatic amino acid, high bioavailability and low anti-nutritional factors. Low molecular weight peptides of rice bran protein have higher efficacy in antihypertensive and antioxidant. In this study, rice bran protein was as raw material to produce peptide with high activity ACE inhibitory by restriction enzyme technology. Using Origin 8.0 soft ware, the dynamics model of the hydrolysis rate and kinetic model of the degree of hydrolysis-hydrolysis time were fitted based on classical Mie theory equations. Secondary ultrafiltration desalting rating was studied by multifunctional ultrafiltration device of pilot. Parameters of separation technology of simulated moving bed chromatography were determined by single column separation process and then the conditions were optimized for further. The separation technology for producing he industrialized production of high purity, high yield ACE inhibitory peptides was obtained. The amino acid sequence of the purified components, which were separated by gel chromatography and HPLC, was determined by Edman N-terminal sequencing method. The activity of ACE inhibitory peptides separated by simulated moving bed chromatography were studied both in vitro and in vivo.In this study, rice bran protein was as raw material. ACE inhibitory peptide of rice bran protein was produced and separated by the method of enzyme. The main contents and results were as following.1. Rice bran protein was as raw material and was hydrolyzed restrictively by seven kinds of enzyme respectively, including Alkaline protease, Alcalase 2.4L, Neutral protease, Papain, Trypsin, Chymotrypsin and Proteinase K. The ideal protease species was determined by the ACE inhibitory of hydrolyzate. ACE inhibitory of hydrolyzate using different enzyme was detected at different pH though controlling the temperature of hydrolyzation and it was shown that Alcalase 2.4L was the best choice for improving ACE inhibitory. With Alcalase 2.4L, some conditions were detected, including temperature, addition of enzyme, pH, substrate concentration and reaction time. The optimal conditions of hydrolyzation were as following: reaction temperature 50℃, addition level of enzyme 3000U/g, pH 8.0, and protein concentration 3.0%, reaction time 120min. Based on single factor conditions, the interaction between factors were optimized by the method of quadratic orthogonal rotation combination design. Interactions between substrate concentration and temperature, substrate concentration and addition level of enzyme, addition level of enzyme and temperature were significantly. The optimal conditions of hydrolyzate were as following:reaction temperature 62℃, protein concentration 3.6%, addition level of enzyme 3567U/g, and reaction time 106min, pH8.5. Under the optimal conditions, ACE inhibitory of peptide of rice bran protein was reached to 73.30%.2. Enzymatic reaction system was created according to rice bran protein and Alcalase 2.4L. Based on Michaelis-Menten equation, enzyme kinetics model was derived. Relationship between the degree of hydrolysis and hydrolysis equation of time was determined. Kinetic model parameters were determined according to the condition of hydrolysis. The kinetics model of hydrolysis of rice bran protein was obtained through nonlinear fitting between the relationship of the degree of hydrolysis and reaction time using software. Kinetic model of hydrolysis rate was R=(94.754E0/S0-0.0597) exp[-0.157(DH)]and kinetic model of the degree of hydrolysis was DH=6.371n[1+(14.88E0/S0-0.009)t]. a=94.754E0/S0-0.0597, b=0.157, k2=94.754 min-1, and the constant of enzyme inactivation was 16.144 min-1. In actual production, production needs could be achieved faster through analyzing the kinetic model.3. The mixed hydrolysates, which were obtained under the optimal conditions, were classified by inorganic ceramic membrane with preliminary filtration and then using the organic film of 1000Da and 3000 Da. The single factor test and orthogonal test were used to investigate the effect of temperature, stress, time and concentration of separation liquid on flux and recovery rate and desalination rate. To compare ACE inhibitory of hydrolyzate, the variety of ACE inhibitory after ultrafiltration was analyzed. As a result, ACE inhibitory peptide of rice bran protein was reached to maximum under the condition of temperature of 30℃ and separation flow of 7 L/min. When the molecular weight was less than 1000 Da, ACE inhibitory of peptide was reached to 78.16%.4. For industrial production of antihypertensive peptides, the macroporous resins of D-152, 007×1, HPD-400, HY-209, XAD-7HP and XAD-761 were used to compare the effect in the experiment of adsorption rate and desorption ratio. And XAD-761 was the best choice for this study. The condition of separation of single-column chromatographic separation was determined, then ACE inhibitory peptide of rice bran protein was separated by simulated moving bed chromatography. There were 20 columns and four areas. The parameters of simulated moving bed chromatography was:switching time of 6 min, sample concentration of ACE inhibitory peptide 14mg/mL, injection flow rate with 7mL/min, flow rate of washing 1 with 22mL/min, the flow rate of desorption of regeneration alcohol with 18 mL/min, the flow rate of washing 2 with 28 mL/min. Under above conditions, the purity of ACE inhibitory peptide obtained was 90.9% and yield was 88.5%. The rate of ACE inhibitory was 84.4%. The molecular of ACE inhibitory peptide of rice bran protein was 250-510 Da.5. After purification by simulated moving bed, ACE inhibitory peptide was further purified with sephadexG-15 Dextran. Biological Activity of each absorption peak was determined. And then the time of separation was determined. The condition of gel chromatography was that elution flow rate of buffer solution of HAc-NaAc was 0.8mL/min and sample concentration was 100 mg/mL. The rate of ACE inhibitory of component of SP1 with high ACE inhibitory was reached to 89.45%. The molecular of SP1 was 250～520 by HPLC, which was same to the result of MS.6.The component of SP1 was separated further by Molecular exclusion chromatographic. There were six components, wherein the component P3 highest activity. Continue to P3 component by HPLC, select two wavelength to compared separation effect that there are higher absorption peaks polypeptides in 220 nm and 280 nm.There are two component. Peak fractions were collected and measured ACE inhibition rate, CP1 components is strongest activity, Its concentrate was subjected to freeze-drying, in an aqueous solution 0.1 mg/mL, measured theACE inhibition rate of 94.84%. CP2 measured the ACE inhibition rate of 92.15%. The amino acid sequence of CP1, CP2 was tested with Edman degradation. CP1 was Ala-Asn-Tyr, which was not reported previously. CP2 was Tyr-Val.7. After separated by simulated moving be d chromatography, the changes of the rate of ACE inhibitory were detected in different conditions, including temperature, pH, concentration of salt, method of drying, and environment of gastrointestinal digestion simulated in vitro. And then the stability of ACE inhibitory peptide of rice bran protein was determined. When the temperature was 4℃,25℃,37℃ and 50～70℃, the rate of inhibitory was stable. ACE inhibitory was reached to 70.2% at 90℃. It was shown that ACE inhibitory peptide of rice bran protein has good performance at high temperature. The rate of ACE inhibitory was decreased significantly in acid and alkali environment (p<0.05). ACE inhibitory was lowest at pH 3, and it reached highest at pH 8. Yet ACE inhibitory was decreased at pH10, which was 70.6%. When the concentration of NaCl was 0-0.6 mol/L, the rate of ACE inhibitory was stable. When the concentration of NaCl was above 0.8mol/L, the rate of ACE inhibitory was decreased rapidly. The rate of ACE inhibitory was 58% when concentration of NaCl was 1.2mol/L. Hot air drying had significantly effect on the rate of ACE inhibitory otherwise freeze-drying and spray drying could keep ACE inhibitory peptide of rice bran protein stable, which had little effect (P>0.05). The rate of ACE inhibitory was decline when pepsin was added, and the rate of ACE inhibitory was decreased little when both pepsin and trypsin were added in. It was shown that ACE inhibitory peptide could tolerance enzymatic hydrolysis by both pepsin and trypsin.8. The methods of oral and intravenous were used in essential hypertension (SHR) rats with high purity ACE inhibitory peptide, separated by simulated moving bed chromatography, which was compared with the group of captopril and normal rats. There were three groups through gavage, low-dose group of 30mg/kg.bw, middle dose group of 60mg/kg-bw, high-dose group of 90 mg/kg-bw. And dose of intravenous administration was low-dose group of 10mg/kg.bw, middle dose group of 30mg/kg-bw, high-dose group of 45 mg/kg·bw, respectively. The changes in blood pressure were measured every 2h after disposable administered and continuous measurement for 8h. Blood pressure and weight were measured every week after long-term administration (orally once a day, for 5 weeks). As a result, the blood pressure was decreased significantly for SHR rats with ACE inhibitory peptide of rice bran protein. The pressure was reached to the lowest after 2h and the most significant decrease in blood pressure was (31 ± 6.8) mmHg. Otherwise, there was little change in normal rats with ACE inhibitory peptide of rice bran. The blood pressure of rat by gavage using captopril could be decreased significantly. Gavage with ACE inhibitory peptide of rice bran for a long time, the pressure of SHR rats was decreased significantly (p<0.05) and it could be kept stable and the weight was increased significantly. Antihypertensive effect was obviously under dose of 45 mg/kg-bw by intravenous (p<0.05). The dose of intravenous was half of that of gavage and the effect of antihypertensive was better than that of gavage. |
PDF Full Text Request