Preparation Of Antioxidative Peptides From Round Scad Protein

Round scad (Decapterus maruadsi) belongs to the family of mackerel, it is an important economic fish in China. Processing this low-valued pelagic fish into high market-value products of surimi production and bioactive peptide will pave a way for the full use of round scad. In the study, antioxidative peptide was prepared from round scad protein through enzymatic technology, and purified by a series of separation technology, then matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) was used to identify the amino acid sequence of the purified peptides. Subsequently, the identified peptides was synthesized, antioxidative activities and reaction dynamics of the synthetic peptides were studied. At last, the effects of temperature, pH, and the enzyme in the simulated gastrointestinal digestion on the antioxidative peptide were discussed, a pilot study on the synergistic effects between round scad protein and other antioxidants was also carried out. The major results are as follows:(1) The protein content was determined to be80.16%by using Kjeldahl, lipid content of10.38%was measured through Soxhlet extraction method, OPA-precolumn derivatization and RP-HPLC assembly system were used to analyse amino acids composition. Therefore, Round scad is a kind of food material with high protein, low fat, ideal essential amino acids (46.28g/100g protein) and antioxidative amino acids content (32.59g/100g protein). Five commercial proteases, namely, alcalase, papain, neutral protease, trypsin and pepsin were used to hydrolyze round scad protein. The antioxidative activities of round scad protein hydrolysate (RSH) were evaluated through methods of DPPH· scavenging activity, O2-· scavenging activity and reducing power. Therefore, alcalase-treated RSH was selected for further study after comprehensive comparison.(2) Taking the degree of hydrolysis (DH) as criterion, single factor experiment and response surface methodology (RSM) were used to optimize the hydrolysis conditions. The effects of initial substrate concentration (S0, X1), initial enzyme concentration (E0, X2), pH (X3), and temperature (T, X4) on DH (Y) were obtained by multiple regression analysis using Box-Benhnken Design (BBD):Y=22.23-1.05X1+0.66X2+0.79X3+1.09X4-0.082X,X2+0.11X1X3+0.16X1X4-0.17X2X3-0.067X2X4-0.25X3X4-0.04X12-0.22X22-0.37X32-0.44X42. A theoret-ical DH of22.2%could be obtained after reacted for300min under the optimal conditions:initial substrate concentration of16.3g·L-1, initial enzyme concentration of0.11g·L-1,50.5?and pH8.18, which was validated to be closed to the experimental DH of22.4%. It was also confirmed that the molecular weight distribution, amino acid composition and antioxidative activity of RSH varied with DH. Fractions with the molecular weight under3.0kDa keep increasing with the increase of DH. A slight increase of antioxidative amino acids could be found when DH was ranged from17.0%to23.0%, RSH exhibited the strongest antioxidative activities at23.0%DH.(3) Thermal inactivation of alcalase was found to follow the first-order kinetics in the temperature range from45.0?to65.0?. A series of hydrolytic experiments of round scad protein with alcalase were processed at50.0?and pH9.5. The kinetic parameter, Km, was estimated as29.115g·L-1through Lineweaver-Burk plot. The kinetic equation of DH and hydrolysis rate (r) for the enzymatic hydrolysis of round scad protein by using alcalase were suggested as respectively.(4) Alcalase-treated RSH was purified by ultrafiltration, gel chromatography, and RP-HPLC method, two hydrophilic fractions of B11and B12were obtained. The amino acid sequences of B11and B12were identified as His-Asp-His-Pro-Val-Cys (HDHPVC,707.11Da) and His-Glu-Lys-Val-Cys (HEKVC,615.22Da) by MALDI-TOF/TOF MS, respectively. After being synthesized, antioxidative activities of the two peptides were verified. Results revealed that the two novel peptides exhibited effective antioxidative capacity.(5) Kinetic study of HDHPVC and HEKVC through DPPH-scavenging method indicated that the two peptides could be classified as slow kinetics behaviour. EC50values for HDHPVC and HEKVC at fixed30min reaction time were31.0?M and67.7?M respectively. Whereas, at the steady state, IC50 values for HDHPVC and HEKVC were9.0?M and14.5?M respectively, which were only part of the EC50values at fixed30min. The calculated values of k2for GSH, HDHPVC and HEKVC were2.30×10-5?M-1·s-1,2.00×10-5?M-1·s-1and0.70×10-5?M-1·-s-1, respectively. Thus the antiradical properties could be arranged as GSH?HDHPVC>HEKVC.(6) The effects of temperature, pH and in vitro simulated digestion model system on the DPPH· and O2-· scavenging activities of RSH-III (Mw<5.0kDa) were investigated. RSH-III exhibited good tolerance for heat treatment and acid conditions under pH7.0, but not alkali-resistant. RSH-III could also resist gastrointestinal enzyme digestion, about70%of initial antiradical activity was maintained after being digested by pepsin and trypsin. A pilot study of synergistic effects between RSH-III and other antioxidants was processed. Among all combinations, group of RSH-III and GSH revealed synergist effects when scavenging DPPH· and O2-· at low concentration. For vitamin C, slight synergist effects could be observed for scavenging DPPH· under low concentration, but there was no synergist effect existed for O2-· scavenging activity.