The Study Of Preparing Soluble Eggshell Membrane Protein And Peptide & Antioxidative Activity

Some researches has been carried out as following:Firstly, two times extraction technology was first applied to prepare SEP, in addition, the physical and chemical properties of SEP have also been tested in this paper. Secondly, the optimum hydrolysis condition to prepare peptides has been studied. Thirdly, the antioxidant activities of the SEP and its peptides were investigated using a chemiluminescence (CL) method. Lastly, the component with high antioxidant activity has been isolated by Sephadex G-25 method, and average molecular weight of each component has been determined by gel chromatography. The research results were as follows:The preparation of SEP has been studied in this paper. The results showed that the first extraction rate could reach 17.80%. Based on the first extraction, an innovative way for the second extraction introduced, and results showed the second extraction rate could up to 75.82%. The extraction rate of SEP could rise up to 93.62% by two times, basically achieved the purpose of using the whole membrane.In addition, the physical and chemical properties of SEP have also been tested in this paper. The results showed that SEP almost retained the complete secondary structure of protein by UV, FT-IR and CD. It can be also forecasted SEP was a great potential protein resource for development by measuring its water absorption, water retention, emulsifying and foaming ability.The enzymolysis of alkaline protease, papaya protease, trypsin, neutral protease and pepsin had been studied. The results showed that alkaline protease and papain complex had better effect, quadratic orthogonality rotational combination design was used to analyze the results. And the optimum conditions to achieve the maximum hydrolysis rate and recycle rate of nitrogen were as follows:the ratio of alkaline protease and papain 8:2, [E]/[S] 16000 U/g, alkaline protease enzymolysis time 2 h, papain enzymolysis time 1 h, alkaline protease pH 9.0, papain pH 5.5, enzymolysis temperature 50℃. Under these conditions, the hydrolysis rate is 46.12%, and recycle rate of nitrogen is 85.56%. The antioxidant activities of SEP and its peptides were investigated by using a CL method. The scavenging ability of SEP and its peptides on superoxide anion, hydroxide radical, hydrogen peroxide and the effect of DNA damage preventing was determined by the four different CL systems. The comprehensive test results showed that the alkaline protease hydrolysate had better antioxidant activity. Therefore, the optimum enzymolysis technology condition for production the antioxidant activity polypeptides from SEP by alkaline protease has been studied, and the results were: enzyme alkaline protease, time 4h, temperature 55°C, pH 9.0. In addition, the abilities of scavenge O₂^- ,·OH, and the effect of preventing DNA damage in Pyrogallol - Luminol, FeSO₄-Luminol-H₂O₂ and CuSO₄-Phen-Vc-H₂O₂-DNA CL systems respectively has also been investigated The results showed that their half inhibitory concentration (IC₅₀) were 0.157 mg/mL, 0.215 mg/mL and 1.128 mg/mL, respectively. The antioxidant activity was improved greatly than SEP. Compared with other sources peptides, SEP antioxidative peptide increased dozens of times or even hundreds of times.Various peptides existed in the hydrolysate from SEP were fractionated by Sephadex G-25 gel filtration chromatography. Three different components have been obtained, and named SP1, SP2, SP3, their average molecular weight was 10522.14 Da, 618.86 Da, and 300.83 Da, respectively, and the content proportion was about 26.59%, 68.15% and 5.26%. In these peptides the fraction SP2 had stronger antioxidant activity by CL method. The half inhibitory concentration (IC₅0) of scavenge O₂^- ,·OH, and the effect of preventing DNA damage was 0.10,0.18 and 0.95 mg/mL.