Study On The Enzymatic Preparation And The Antioxidant Activity Of Oat Polypeptides

In recently years, bioactive polypeptides with good physiological and biochemical functions, such as soybean polypeptides and corn polypeptides, have received tremendous attention. As one of eight main food corps in the world, oat, being rich in essential amino acids, mineral elements and vitamins,is a kind of excellent raw material for bioactive polypeptides. Oat polypeptides with good antioxidant activity and the function of free radical scavenging, anti-aging and lowering the blood pressure can be applied to health care and cosmetic industry. However, there are few reports regarding the preparation of oat polypeptides. Among the methods for preparation of bioactive polypeptides, the enzymatic hydrolysis of protein is effective, economic and commonly used in industry, with the advantages of mild reaction conditions, environmental friendliness, etc. Alkaline protease is often used for the preparation of oat polypeptides. However, free enzyme is usually fragile and difficult to be reused. Therefore, in this dissertation, amino-functionalized magnetic nanoparticles immobilized alkaline protease(AFMNs-AP) was used for the hydrolysis of oat bran to oat polypeptides, and the antioxidant activity of the resulting oat polypeptides was examined.(1) Preparation of immobilized alkaline protease and investigation of its characteristics.Firstly, amino-functionalized magnetic nanoparticles(AFMNs) were prepared and transmission electron microscopy(TEM), fourier transformed infrared spectroscopy(FTIR), X-ray photoelectron spectroscopy(XPS), X-ray diffraction(XRD), vibrating sample magnetometer(VSM) were applied to characterize the structure of AFMNs. The results indicated that the AFMNs were successfully prepared and had good magnetic performance. Next, alkaline protease was immobilized on AFMNs with glutaraldehyde as cross-linker. Under the optimized immobilization conditions(the ratio of support to enzyme, cross-linker concentration and cross-linking time were 250(w/v, mg/m L), 28.0 mmol/L and 4 h, respectively, other factors including temperature and rotation speed were 25 oC, 200 r/min), the enzyme activity recovery reached 54.2%. N-α-benzoyl-L-arginine ethyl ester hydrochloride(BAEE) was used as a substrate to explore the characteristics of free and immobilized alkaline protease(AFMNs-AP),with the results that the optimum temperatures of AFMNs-AP and free enzyme were 65 oC and 60 oC, respectively and the optimum p H of AFMNs-AP and free enzyme was the same(7.5). The tests of thermal stability and p H stability indicated that AFMNs-AP was superior to the free enzyme in terms of thermal stability and p H tolerance. It was found that the catalytic efficiency(Vmax/Km) of AFMNs-AP was higher than that of free enzyme(37.0 vs. 33.3 min-1), showing that the AFMNs-AP had relatively higher catalytic efficiency.(2)Preparatiron of oat polypeptides with AFMNs-AP catalyzed hydrolysis of oat branAfter the optimization of the reaction conditions with single factor, response surface design included Plackett-Burman design(PB) and Box-Behnken design(BBD), the optimum amount of AFMNs-AP, concentration of oat bran, reaction temperature, buffer p H and reaction time were found to be 0.62 U/m L, 54 mg/m L, 50 oC, p H 7.5, 2.25 h, respectively. Under the above-mentioned conditions, the DPPH radical scavenging rate of obtained oat polypeptides reached 82.3%. In addition, according to the antioxidant analysis of obtained oat polypeptides, the IC50 of DPPH scavenging, hydroxyl radical scavenging or chelating Fe2+ was 2.63 mg/m L, 0.18 mg/m L or 0.81 mg/m L, respectively, clearly indicating the formed oat polypeptides had excellent antioxidant activity.(3)Preliminary study on the amino acid sequence of the antioxidant oat polypeptidesFirstly, the oat polypeptides were purified with Sephadex G-25 gel filtration chromatography, giving peaks A and B with IC50 of DPPH scavenging being 1.10 mg/m L and 1.35 mg/m L, respectively. Compared with the oat polypeptides before purification(IC50 2.63 mg/m L), the antioxidant activities of peaks A and B were significantly improved. Then, peak A was further purified with Sephadex C-15 gelfiltration column and three peaks(A1, A2 and A3) were eluted. The three peaks exhibited lower IC50 values of DPPH radical scavenging(0.20 mg/m L, 0.28 mg/m L and 0.30 mg/m L, respectively) than both peak A and vitamin C(0.44 mg/m L), indicating that they have higher antioxidant activity than both peak A and ascorbic acid. Finally, peak A1 was found to contain the amino acid sequences(Ser-Gly-Trp)3、Leu-Val-Glu-Asp-Lys、Thr-Ser-Glu、Trp-Asn、Ile-His-Asn and Glu-Asp-Lys, with the help of high performance liquid chromatography-electrospray ionization-tandem mass spectrometry(LC-ESI-MS/MS) analysis.This study not only provides a novel and efficient route to oat polypeptides, but also could promote the application of magnetic nanomaterials in the field of enzyme immobilization, and laid the foundation the research of structure activity of oat polypeptides.