| Mulberry leaf has great resource advantage in our country.In China,mulberry leaf is known as a plant source that has applications in both medicine and food.Mulberry leaf contains abundant protein,polysaccharide,alkaloid and phenols.Until now,the phenolic acid,polysaccharide,1-deoxynojirimycin(DNJ),and gamma-aminobutyric acid have been widely studied.Compared with the large protein,the peptide is more easily absorbed by the human body,and the activity is stronger.However,there are few studies on the abundant mulberry leaf protein;the study of mulberry leaf peptide is still rare.To enrich related contents of mulberry leaf protein,and enhance the economic value of mulberry leaves,this paper introduced the Osbone extraction of mulberry leaf protein.Meanwhile,the physicochemical property and antioxidant activity of the four extracted proteins were systematically investigated.To improve the application value of mulberry leaf protein and enhance their antioxidant activity,the mulberry leaf protein with high antioxidant ability was hydrolysed by enzyme.Then,the hydrolysates with higher antioxidant activity were further separated and purified to obtain high purity antioxidant peptides.Due to the strong correlation between anti-oxidative and immunological activity,we analyzed the immunological activity of antioxidant peptide.The main results are as follows:Albumin and glutelin were the major fractions.The antioxidant activity of albumin was significantly higher than the other proteins(p < 0.05).Moreover,the DPPH radical scavenging ability of albumin at 0.6 mg/m L was comparable to that of L-glutathione(GSH).The solubility,water holding ability and emulsification stability of albumin were significantly better than other fractions.Therefore,albumin showed encouraging functional properties and antioxidant activities,and could be considered as potential food ingredients and antioxidants.To improve antioxidant efficiency,mulberry leaf albumin(MP)was hydrolyzed using Alcalase,Protemax,papain,Flavourzyme,Neutrase,and trypsin.Results showed that Neutrase,Alcalase,and Protamex were more suitable for MP digestion and antioxidant peptide preparation.The antioxidant activities of these hydrolysates were significantly higher than that of MP(p < 0.05).These hydrolysates had high essential amino acid content.They were abundant in the 0.3–0.5 and 0.5–6.5 k Da fractions and were mainly composed of disordered coils and ?-folds.Among them,Neutrase hydrolysate(NH)showed the best antioxidant activity.The enzymatic condition of NH was optimized.Results showed that the optimum conditions were: enzymolysis time,2 h;Enzyme/Substrate,1%;substrate concentration,20 mg/m L.To investigate the effect of GI digestion and separate intestinal digestion on the stability of MP and its neutrase hydrolysates(HMP),changes in the secondary structure,molecular weights,amino acids and antioxidant activity were analyzed.Results showed that HMP was more easily hydrolyzed by intestinal digestion than by gastric digestion,whereas the opposite was true for MP.After GI digestion,the antioxidant efficiency of HMP was reduced,while that of MP was improved.The antioxidant activities of continuous GI digests and separated pancreatin digests of MP and HMP were different.The separate intestinal digests of HMP(DHMP)exhibited higher antioxidant abilities than the GI digests of(GHMP).In contrast,the individual intestinal digests of MP(DMP)exhibited higher DPPH.and O2-.scavenging ability,but lower ABTS+.quenching capacity,than the GI digests of MP(GMP).Therefore,special treatment such as microencapsulation may be considered as an efficient method of meeting the practical demands of MP and HMP.In order to describe the antioxidant mechanism of HMP and GHMP at the cellular level,the hemolysis inhibition ability of HMP and GHMP was analyzed.Results showed that both HMP and GHMP could effectively inhibit erythrocyte hemolysis at the concentration of 0.025-1.0 mg/m L.HMP and GHMP could effectively inhibit the production of ROS and reduce the content of MDA as well as maintain the balance between enzymatic and non-enzymatic antioxidant defenses.Compared with HMP,the hemolysis inhibition ability of GHMP was decreased,but it was significantly higher than that of injury group(p < 0.05).The changes of enzyme activity and the content of non-enzymatic substances in the red blood cells were analyzed.Results showed that HMP and GHMP could inhibit the oxidative damage of red blood cells by inhibiting ROS production and scavenging free radicals.HMP was succesively purified with Diethylaminoethyl Sepharose Fast Flow(DEAE Sepharose FF),Sephadex G-15,two fractions,G1 and G2,were obtained.And G1 exhibited significant higher antioxidant activity than G2(p < 0.05).G1 was then purified wirh semi-preparative reverse phase high performance liquid chromatography(RP-HPLC),and two fractions,R1 and R2,were obtained.The antioxidant activity of R1 was significantly better than that of R2.Then,the amino acid sequence analysis of R1 was carried out using high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).Three new antioxidant peptides(P1-P3)were obtained.The sequence(molecular weight)of P1,P2 and P3 is SVL(317.29 Da),EAVQ(445.47 Da)and RDY(452.47 Da),respectively.Then,these three peptides were synthesized.The purity of the synthetic peptides was above 97%.The antioxidant activity of synthetic peptides was compared,and P3 showed the highest antioxidant.The purified antioxidant peptide R1 was chosen to investigate the immunological activity.Results showed that R1 significantly improved the production of NO,IL-6,TNF-?,and ROS(p < 0.01).Meanwhile,R1 enhanced the phagocytosis of neutral red. |
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