| Oat bran protein is high-quality protein resources, its development and utilization have important significance. And the exploitation and utilization of bioactive peptides have been the research focus in the present society, which is also the trend in the development of health products in the future. At the same time, the design and operational processe of the enzymatic membrane reactor have incomparable advantages over other reactors, and it will have broad application prospects in the preparation of bioactive peptides. Therefore, the subject decided to prepare ACE inhibitory peptides and antioxidative peptides by hydrolysis of oat protein in an enzymatic membrane reactor and provide basis for the deep processing of oat bran, the preparation of bioactive peptides and the application of the enzymatic membrane reactor.This paper mainly studied the different extraction methods of oat bran protein and their nutritional value, separation of oat bran protein with Hollow Fiber Ultrafiltration Membrane, preparation of ACE inhibitory peptides and antioxidative peptides by hydrolysis of oat bran protein in the enzymatic membrane reactor and isolation and purification of oat bioactive peptides. This paper showed the results as follows:Adopting single factor experiments and orthogonal experimental design, the optimum conditions of alkaline extraction, ultrasonic wave-assisted alkaline extraction and ultrasonic wave-assisted enzymatic and alkaline extraction for extracting oat bran protein from oat bran were determined, and the extraction rate of oat bran protein could amount to 31.96 %, 39.31 % and 61.43 %. According to the study on the solubility and precipitation coefficient of oat bran protein at different pH, the isoelectric point of oat bran protein extracted by alkaline extraction, ultrasonic wave-assisted alkaline extraction and ultrasonic wave-assisted enzymatic and alkaline extraction were discovered to be pI=3.4, 3.6 and 3.8,and the precipitation rate of oat bran protein could amount to 58.15 %, 63.24 % and 72.24 %. Through the analysis of the content and composition of amino acids and nutritive value of oat bran protein extracted by three methods, oat bran protein extracted by three methods were in good agreement with FAO/WHO protein model, and EAAI of oat bran protein extracted by three methods were basically identical, which showed that their nutritive value were equivalent with each other.According to recovery efficiency of oat bran protein, clearance efficiency of total sugar, degree of membrane fouling, concentration efficiency and recovery efficiency of flux of the factors, the optimum conditions for separating oat bran protein with Hollow Fiber Ultrafiltration Membrane and cleaning method were determined. The best operating conditions of the MOF503 were as follows: temperature 35℃, pressure 0.02 MPa, pH=8 and rate of flow 25 L/h. Under these conditions, the results of recovery efficiency of oat bran protein, clearance efficiency of total sugar, degree of membrane fouling and concentration efficiency were 80.3 %, 79.5 %, 41 % and 43.5 L/(m2?h). And the batch ultrafiltration method supplementing with backpressure-flushing was better to separate oat bran protein. Through the experiments of physical and chemical cleaning with MOF503, the optimum conditions of cleaning with isotonic and backpressure-flushing water were determined, and the cleaning effect of backpressure-flushing water was better, and the optimum chemical cleaning agent were HCl(pH=2) and NaOH+H2O2+HCl. If the physical and chemical cleaning were used alternately, recovery efficiency of flux can be up to more than 94.74 %. The work indicated that separating oat bran protein with Hollow Fiber Ultrafiltration Membrane was feasible, and it would be a base for researching into separating protein with membrane.Through the research on the ACE inhibitory activity, antioxidant activity and product yield of products by hydrolysis of oat bran protein with Alcalase, Trypsin and Pepsin respectively, it was determined that Alcalase was identified as the initial enzyme for preparing the bioactive peptides. And by researching the two-stage hydrolysis of Alcalase+Trypsin and Alcalase+Pepsin respectively for the activity of products, it was determined that the process of technological optimization test was not considered the two-stage hydrolysis. Through the study on the velocity of the hydrolysis of oat bran protein with Alcalase, the result illustrated the decrease of velocity was mainly due to the consumption of substrate and the loss of enzyme activity. Meanwhile, by researching the influence for enzyme activity of the enzymatic membrane reactor, it was determined that the enzyme was perfectly confined in the enzymatic membrane reactor, and the destruction of pumping and adsorption of membrane were the main factors of the loss of enzyme activity.By using DPS to design uniform experiment and SPSS to analyze experimental data, the results obtained the optimum conditions of the retention rate of enzyme activity and the productivity of peptides in the process of preparation of the bioactive peptides by hydrolysis of oat bran protein in the enzymatic membrane reactor. The best operating conditions were as follows: substrate concentration 3 %, enzyme concentration (E/S) 3 %, rate of flow 45L/h, pressure 0.06 MPa, temperature 60℃and pH=7. Under these conditions, the results of the retention rate of enzyme activity and the productivity of peptides were 92.86 % and 1896.5 mg/(m2?h). And by using Design Expert 6.0.5 to design response surface methodology of four factors and three levels, the optimum conditions for preparing ACE inhibitory peptides and antioxidative peptides by hydrolysis of oat bran protein in the enzymatic membrane reactor. The best operating conditions were as follows: 1) ACE inhibitory peptides: substrate concentration 3 %, enzyme concentration (E/S) 3.5 %, rate of flow 45 L/h, pressure 0.07 MPa, temperature 61℃and pH=8, under these conditions, the ACE-inhibitory activity of the products was 67.86 %; 2) antioxidative peptides: substrate concentration 3 %, enzyme concentration (E/S) 3.2 %, rate of flow 45 L/h, pressure 0.07MPa, temperature 55℃and pH=7.3, under these conditions, scavenging effect on DPPH of the products was 57.39 %.Adopting orthogonal experiment design, the optimum decoloration conditions of the solution of oat bran protein hydrolysate with YD-303 were as follows: quantity of activated carbon 1.5 %, pH=3.5, temperature 40℃and adsorption time 75 min. Under these conditions, the results of decolorization rate and recovery efficiency of peptides were 85.36 % and 83.79 %. Desalination for oat bran protein hydrolysate with macroporous adsorption resins(MAR) is an effective methord. By the experiments of static adsorption and desorption, DA201-CⅡMAR was selected having the highest capability of adsorption. According to the study, the optimum conditions were as follows: the pH of oat bran protein hydrolysate was 4.0, the concentration of oat peptides was 39 mg/ml and SV=1 BV/h, 75 % ethanol as eluant and SV=1.5 ml/min. Under these conditions, the desalting ratio reached about 93.4 %, whereas the recovery of oat peptides was about 72.3 %. According to the changes of the activity of groups by different concentrations of ethanol eluting DA201 - CⅡ, it was illustrated that it could take advantage of the different polarity eluting agent to achieve the purpose of classification purification. As using DA201 - CⅡto purify ACE inhibitory peptides, the group which was eluted by 75% ethanol has the highest activity, its ACE-inhibitory activity was 91.7 %,while using DA201 - CⅡto purify antioxidative peptides, the group which was eluted by 95 % ethanol has the highest activity, its scavenging effect on DPPH was 35.01%.Although DA201 - CⅡis able to achieve the purpose of purification by the different polarity eluting agent for ACE inhibitory peptides and antioxidative peptides, because of inherent defects of DA201 - CⅡand the difficult control of the polarity of eluting agent and so on, it can not obtain high-purity active peptides. So ACE inhibitory peptides and antioxidative peptides need to be separated and purified furtherly with Sephadex, RP-HPLC, HPLC-MS and so on, to finally isolate single peptides. |
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