Study On The Preparation, Purification, And Identification Of Antioxidant Peptides From Rice Endosperm Protein

Rice endosperm protein (REP) is hypoallergenic and contains good quantity of amino acids, which aroused researchers'great interests all over the world. Simultaneously, as natural antioxidants, antioxidant peptides gained from proteins have attracted increasing interests because of their safety and wide distribution properties in recent years. Actually, there were large numbers of rice for REP in China. Therefore, if REP is further exploited and utilized, it can represent a significant economic and social benefit.In this dissertation, REP was extracted from rice flour by the procedure of extracting, separating, concentrating and drying. Further, REP was hydrolyzed by five types of proteases, and Neutrase was chosen as the optimum enzyme to hydrolyze REP for antioxidant peptides. The optimum conditions for Neutrase hydrolysates (NHREPs) were determined by response surface methodology as follows: [E]/[S] 2.3%; Temperature 37°C; pH7.0 and Time 4.2h. Under the optimum conditions, DH of the NHREP was 13.26%, and its DPPH radical (DPPH·) scavenging activity reached 60.54%.The antioxidant properties of NHREPs at various DH were evaluated using different antioxidant assays in vitro. The results showed that the antioxidant activities of NHREPs were related with its DH, and NHREP at DH 13% had the most powerful antioxidant activities. The molecular weight distribution profile of NHREPs exhibited that the ratio of peptide, with molecular weight between 500 and 1,020, was 54.73% at DH 13%.NHREP (DH=13%) was initially separated using SP-Sephadex C-25, Sephadex G-15 and RP-HPLC. At last, F3b and F3c with higher antioxidative activities were collected. The F3b and F3c were then identified by MALDI-TOF/TOF MS/MS combined with the amino acids composition analysis. Their molecular weights were 959.5 and 1002.5 respectively. The amino acid sequence of F3b was Phe-Arg-Asp-Glu-His-Lys-Lys(that is FRDEHKK), and that of F3c was Lys-His-Asn-Arg-Gly-Asp-Glu-Phe (KHNRGDEF).At last, FRDEHKK was chemically synthesized. And its antioxidant activities were validated using the linoleic acid model system and the DPPH/superoxide/hydroxyl radical scavenging assays. It exhibited higher inhibition of peroxidation in linoleic acid model system than that ofα-tocopherol at the same concentration. The order of its free radical scavenging activities was hydroxyl radical>DPPH radical>superoxide radical. Moreover, this peptide possessed strong Fe²⁺ chelating effect, which was close to EDTA. At the molecular level, we deduced that the amino acid compositions and sequences of the peptides might play an important role in their activities. Donating protons easily to electron deficient radicals and chelating ions effect of the peptides may have also contributed to its higher radical scavenging potential. By capturing peroxide free radicals, peptides developed into more stable intermediates, prevented or diminished the free radicals chain reaction.