Study On The Preparation And Antioxidant Function Of Enzymatic Hydrolyzed Peptides Of Chlorella

Chlorella belongs to the class of single-celled green algae.It is one of the highest levels of protein in microalgae and has good health care efficacy.It is a microalga resource with important commercial value.However,there is no relevant research to prove that the main substances that play a high biological activity in chlorella are composed of which components.And what are the biological activities and functions of different components?In this paper,the cell wall of chlorella was broken by a high-pressure homogenizer,cellulase and pectinase to extract chlorella protein.By selecting the optimal proteases and using the response surface method to optimize the preparation of peptides by enzymatic hydrolysis.The multi-stage separation technology of ultrafiltration membranes is used to obtain chlorella polypeptides with different molecular weight segments.The antioxidant activity of chlorella hydrolyzed peptides in different molecular weight regions was explored through three aspects of in vitro antioxidant function experiments,cell experiments,and aging mouse in vivo model.The main findings are as follows:1.To determine the optimal protease and enzymatic conditions for obtaining the best antioxidant zymohydrolyzed peptide fragment,Using the total reduction capacity in vitro as an indicator,According to the strength of the total reducing ability of different proteases,the protease is screened.It was determined that the total reductive ability of the enzymolyzed products of chlorella protein complexed by alkaline protease and trypsin is the highest.Further response surface methodology was used to establish the optimal enzymatic hydrolysis parameters for alkaline protease and trypsin:pH 8.8,Enzymolysis temperature 60?,Addition amount of enzyme 2.2%,Enzymatic hydrolysis time 30.0 min,Substrate concentration 4.0%,Alkaline protease to trypsin ratio 5:1.The actual degree of proteolysis measured under this condition was 71.06%2.The enzymolysis solution was subjected to ultrafiltration membrane separation technology to obtain polypeptides with molecular weight>10 kDa,5-10 kDa,3.5-5 kDa,1-3.5 kDa and<1 kDa.3.Through the detection of antioxidant capacity and total reducing ability in vitro,It was confirmed that the 1-3.5 kDa hydrolyzed polypeptide had the best antioxidative capacity in vitro.When the concentration of 1-3.5 kDa peptide was 10 mg/mL,the DPPH radical scavenging rate was 88.53%.When the concentration of 1-3.5 kDa hydrolyzed peptide was 12.5 mg/mL,the hydroxyl radical scavenging rate was 96.81%.Through regression analysis of SPSS software,The half-inhibitory concentrations(IC50)of 1-3.5 kDa hydrolyzed peptide to DPPH and hydroxyl free radicals were 2.62 mg/mL and 5.74 mg/mL,respectively.Further through the experiment of Bel7402 human hepatoma cells,It was confirmed that 1-3.5 kDa hydrolyzed peptides can exert anti-oxidation effects in cells and reduce intracellular ROS levels.4.Using old mouse experiments,By measuring the in vivo antioxidant capacity of each molecular weight segment of enzymatic peptides was examined.It was confirmed that 1-3.5 kDa hydrolyzed peptides have the most significant ability to reduce the MDA level and increase the activity of antioxidant enzymes SOD and GSH-PX,and it was showed significant dose dependence in reducing the MDA level and increasing the activity of SOD and GSH-PX.The enzymolysis polypeptide group in 1-3.5 kDa section was compared with the control group and other peptide groups.The MDA in mouse serum decreased from 6.81 nmoL/mL to 4.68 nmoL/mL in control group.GSH-PX activity increased from 545.74 U/mL to 950.46 U/mL in control group.SOD activity increased from 173.33 U/mL to 209.28 U/mL in control group.In summary,the enzymatic peptides of chlorella hydrolyzed by alkaline protease and trypsin has antioxidant activity,especially in the molecular weight range of 1-3.5 kDa.This study can provide the necessary technical and theoretical basis for the large-scale enzymatic hydrolysis of chlorella protein to obtain antioxidant peptides and for the further development of chlorella enzymatic peptides.