| This study aimed to prepare antioxidant peptides of fish scale gelation extracted from grass carp scales by two-step enzymatic hydrolysis.Two-step enzymatic hydrolysis process was optimized by response surface methodology.Secondly,we invested the possible protective effects of hydrolysates(ATHs)prepared by the optimal process against hydrogen peroxide(H2O2)induced oxidative stress injury in Hep G2 cells.Finally,the structure of peptides in ATHs was identified by Nano-LC-ESI-QOrbitrap-MS/MS and De novo sequences.Peptides with relatively stronger antioxidant ability were selected.This study can provide theoretical guidance for high value utilization of fish by-products and development and application of antioxidant peptides from grass carp scales.(1)Based on the alcalase hydrolysis,the grass carp scales gelatin was subjected to two-step enzymatic hydrolysis with trypsin,flavor protease,papain and pepsin respectively,to prepare antioxidant peptides.Results showed that two-step enzymatic hydrolysis could effectively increase hydrolysis degree and soluble protein content,and decrease the molecular weight of hydrolysates.Alcalase-trypsin hydrolysates exhibited a relatively stronger antioxidant capacity,showed DPPH·,O2-· and ·OH scavenging rate were 16.98%,92.28% and 43.22% respectively,and Fe2+ chelating rate was 63.26% at 1 mg/m L.The ·OH scavenging rate and Fe2+ chelating rate of alcalase-trypsin hydrolysates were higher than BHT at the same concentration(60.48% and 49.57% respectively),which indicated that alcalase-trypsin hydrolysates had a potential replacement of synthetic antioxidant.(2)The effect of time,temperature,substrate concentration and p H value during the trypsin two-step enzymatic hydrolysis on ferrous ion chelating rate and relative content of component IV of alcalase-trypsin hydrolysates were studied in single factor test.Response surface test was used to optimize two-step enzymatic hydrolysis conditions,and the optimal two-step enzymatic hydrolysis conditions were as follows: substrate concentration,100 mg/m L;p H value,7.8;temperature,53°C;and hydrolysis time,50 min.Under these conditions,alcalase-trypsin hydrolysates exhibited a Fe2+ chelating rate of 65.72%,relative content of component IV was 53.36%.The IC50 value of DPPH·?·OH and O2-· of alcalase-trypsin hydrolysates prepared according to this optimal condition were 7.39 mg/m L and 0.68 mg/m L and 1.84 mg/m L,respectively.(3)The oxidative injured Hep G2 cell model was established by incubation with 800 ?M H2O2 for 2 h,and MTT assay and flow cytometry were used to detect the survival rate and apoptosis rate of Hep G2 cells respectively.The results showed that pretreatment of 0.19~3.00 mg/m L ATHs for 24 could significantly increase Hep G2 cells survival rate and decrease its apoptosis rate.The protective effects of ATHs on oxidative injured Hep G2 induced by H2O2 mainly due to it can improve antioxidant enzymes(SOD,CAT and GPX)activity,thereby reducing ROS level and MDA content as well as DNA damage in Hep G2 cells.Within the selected concentration range,0.19 mg/m L ATHs showed the most protective effects on oxidative injured Hep G2 cells induced by H2O2.(4)Totally,31 peptides with ALC>99% was identified through Nano-LC-ESI-QOrbitrap-MS/MS and De novo sequence,and then nine peptides were selected for synthesis among them.YGPR and LLGFDNVR exhibited relatively stronger ·OH scavenging capacity,with ·OH scavenging rate were 34.97% and 33.11% at 2.5 mg/m L.VGPS and FPFLR showed relatively stronger ferrous ion chelating ability,with ferrous ion chelating rate were 16.04% and 25.74% at 2.5 mg/m L.SGLDGAK showed both relatively stronger ·OH scavenging capacity and ferrous ion chelating ability,with ·OH scavenging rate and ferrous ion chelating rate were 33.11% and 14.97% respectively.The ·OH scavenging capacity and ferrous ion chelating ability of nine synthesis peptides were lower than ATHs,which probably due to the interaction between peptides. |
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