Study On Biomimetic Microcapsules Of Peruvian Salmon Protein Antioxidant Peptides Based On Ferrous Ions

This research uses Peruvian squid as raw material,firstly prepares antioxidant peptides of carp by means of complex enzymatic hydrolysis,and then separates antioxidant peptides with different molecular weights by membrane separation equipment,and uses Fe²⁺chelate with anti-oxidation peptide,one of the trace elements required by human body.Changing the concentration of peptides during chelation and the pH of the chelation system,exploring the microstructure and particle size distribution and thermal stability of self-assembled microcapsules,and finally determining the activity of the prepared microcapsules by constructing an in vitro simulated digestion model and Identification of changes in microstructure.The main findings are as follows:1)The effects of papain and alkaline protease complex enzymatic hydrolysis on the ferrous chelation ability and antioxidant activity of the enzymatic peptide were studied by using Peruvian carp surimi.Response surface methodology was used to investigate the optimal composite enzymatic hydrolysis process.The results showed that the ferrous chelation rate and DPPH free radicals scavenging rate of the single enzyme enzymatic peptide were lower than that of the complex enzymatic peptide by the ferrous chelating rate and the DPPH free radicals radical scavenging rate.In the study of the effect of pH and the addition of complex enzymes on the antioxidant activity and the ferrous chelating ability of enzymatic hydrolysis peptides,it was found that when papain was first added,the alkaline protease was added and the pH of papain was controlled to be 7 and alkaline.When the protease pH is 8.5,the effect is best.Further,the pH,the total amount of enzyme added and the ratio of enzyme addition ratio,the ferrous chelate ability and the DPPH free radicals scavenging rate are the response values,and the optimal enzymatic hydrolysis process is obtained by the response surface method:papain pH 7.00,The alkaline protease pH was 8.42,the total amount of enzyme added was 11800U/g of subdtrate,and the ratio of the two enzymes was 0.69?papain:alkaline protease=3.41%:4.96%?.Under these conditions,the ferrous chelating ability of the enzymatic peptide was 56.35%,and the DPPH clearance rate was 64.32%,which provided process support for further utilization of squid antioxidant peptides.2)The anti-oxidation peptide of carp surimi was fractionated by ultrafiltration membrane separation method and its separation effect was verified by SDS-polyacrylamide gel electrophoresis.The ferrous chelation ability of self-assembled microcapsule of ferrous chelating squid antioxidant peptide,named Fe???-SAPMC,was studied by using different molecular weights,protein peptide concentrations and chelating pH as variables.Scanning electron microscopy,transmission electron microscopy and Fourier infrared were used.The microcapsules were characterized by spectroscopy and nanopotential particle size analyzer,and the thermal stability was studied by differential calorimetry.The results show that the ultrafiltration membrane has good separation effect from SDS-polyacrylamide gel electrophoresis,and can obtain five peptides?>10 kDa,5?10 kDa,3.5?5 kDa,1?3.5 kDa,<1 kDa?of salmon antioxidant peptides.The antioxidant peptides of different molecular weights were chelated with ferrous ions,and the chelation rate of<1 kDa was found to be the highest.Changing the pH of the chelating system and the peptide concentration found that the maximum chelation rate was obtained when the chelation pH was 7.0 and the peptide concentration was 3%.Further analysis of the structure of the microcapsules shows that the self-assembled microcapsules of different molecular weights have different forms,and many layers are overlapped,and the internal structure is not clear.The chelating peptides with molecular weights of 5?10 kDa and 3.5?5 kDa can be self-assembled into spherical microcapsules,and similar to silkworm pupa,with an average particle size of 300nm.Fourier infrared spectroscopy shows that the ferrous ion is chelated with the peptide.The main chelating sites are chemical bonds in the carboxylic acid group?including C=O bond and—OH bond?and amide bond in the amino group.The secondary structure of Fe???-SAPMC is mostly?-sheet.From the results of differential scanning calorimetry before and after chelation,the denaturation temperature and melting temperature of the Fe???-SAPMC were higher than those of the original peptide,indicating that the microcapsules have better heat resistance.3)By constructing a simulated human oral saliva digestion,gastric fluid digestion and intestinal digestion model,the DPPH free radical scavenging rate,hydroxyl radical scavenging rate and superoxide anion free radical scavenging rate were used to study the Fe???-SAPMC in the simulation.The changes of antioxidant activity before and after gastrointestinal digestion were investigated,and its acid resistance and bile salt resistance were investigated.The microstructure and particle size of microcapsules before and after digestion were characterized.The results showed that the antioxidant activity of each molecular weight microcapsule decreased after digestion in the human gastrointestinal tract,and the smaller the molecular weight,the greater the impact.After incubation for 90 min in a low-pH gastric acid environment,the antioxidant activity decreased significantly,especially for microcapsules with a molecular weight of 5-10 kDa,and microcapsules exhibited a certain sustained release property in the intestinal environment of high bile salts.The results of scanning electron microscopy showed that the microcapsules were uniform and spheroidal after simulated digestion,and the particle size became smaller.The average particle size was 100 nm.It is speculated that during the digestion process,the microcapsules will be carried out under the action of digestive juice.From this we guess the Fe???-SAPMC will be secondary self-assembly,the mechanism of which needs further exploration.