Moderate Ultrasound-assisted Enzymolysis To Prepare Antioxidant Peptides From Nannochloropsis Oceanica

Nannochloropsis oceanica after oil extraction（residue）still contains a large amount of protein（about 50%）,crude fiber and other components.which is generally discarded as industrial waste.This not only caused environmental pollution,but also caused a serious waste of protein resources.For this reason,this study used defatted Nannochloropsis oceanica as a raw material to prepare the antioxidant peptides.In addition,to avoid prolonged ultrasonic treatment will cause the reduction of protease activity and aggregation of peptides.Moderate ultrasound-assisted enzymolysis（MUE）was used to replace the traditional whole-process ultrasound-assisted enzymolysis（WUE）and ultrasound pretreatment enzymolysis（UPE）,which proper time ultrasound was used to open the structure of protein macromolecules and improve the activity of protease,followed by conventional enzymolysis（CE）for subsequent enzymolysis.The DPPH（1,1-diphenyl-2-picrylhydrazyl）and OH（hydroxyl）free radicals scavenging rates of the hydrolysates were used as indexes to optimize the preparation of antioxidant peptides from Nannochloropsis oceanica protein by single factor and Response surface methodology design.Objective to compare the antioxidant activity of the hydrolysates of Nannochloropsis oceanica protein（NPHs,which were named as MUH,control group,UPH and WUH,respectively）prepared by MUE,CE,UPE and WUE,their molecular weight distribution and amino acid composition were determined.At the same time,their structures were measured and analyzed to explore the mechanism of the effect of ultrasound on their antioxidant activity.The antioxidant activity of the fractions of MUH was evaluated.Also,the protective effect of MUH fractions against H₂O₂-mediated oxidative damage on L929cell lines was evaluated,which provided certain basic data for the application and development of natural antioxidant peptides from Nannochloropsis oceanica protein.The main conclusions are as follows:（1）The enzymolysis conditions of Nannochloropsis oceanica protein were optimized.The optimal enzymatic hydrolysis conditions of MUE were obtained:ultrasonic power density 0.7 W/m L,ultrasound time 21 min,substrate concentration40 g/L,enzyme dosage 7000 U/g,total enzymolysis time（ultrasonic+coventional）60 min.Under these conditions,the DPPH and OH radicals scavenging rates and yelied of the MUH were 75.33%±0.21%,58.08%±0.26%and 64.51%,respectively.The DPPH,OH radicals scavenging rates and yelied of MUH were increased by15.18%,7.29%and 16.57%than CE,respectively.（2）The effect of moderate ultrasound on the antioxidant activity of NPHs and its characteristics were studied.Moderate ultrasound significantly increased the antioxidant activity of NPHs and changed the results of molecular weight distribution and amino acid composition.The changes of FTIR and UV spectrum indicated that Moderate ultrasound changed the structure of the protein during enzymolysis and caused the structure of MUH to also change,the results of-SH,S-S content and surface hydrophobicity indicated that moderate ultrasound can expose the hydrophobic groups and regions inside the protein,and more hydrophobic amino acid terminal peptides produced during enzymolysis,the result of zeta potential showed that MUH is more stable and the ability to donate electrons is enhanced.At the same time,the results of scanning electron microscopy（SEM）and atomic force microscope（AFM）showed that modearte ultrasound can make MUH looser and increase surface roughness.Thus,MUH had higher antioxidant activity.（3）The antioxidant activity of MUH fractions and their protective effects on L929 cell lines against oxidative damage mediated by H₂O₂were studied.MUH-1（<3k Da）showed the highest antioxidant activity.MUH and its fractions can alleviate cell oxidative damage by reducing the content of malondialdehyde（MDA）in cells and inhibiting the decrease of superoxide dismutase（SOD）activity,and the protective effect of MUH-1 on L929 cell oxidative damage is the best than other sample groups,slightly lower than 0.5 mg/m L glutathione（GSH）.