Preparation,Characterization And Cellularabsorption Characteristics Of Peptide Chelatediron From Sika Deer Blood

Deer blood came from red deer（Cervus elaphus Linnaeus）or sika deer（Cervus nipport Temminck）.This paper used sika deer blood as raw material,deer blood peptides（SDBP）was extracted by using enzymatic hydrolysis and optimizing the process.Deer blood peptide chelated iron（SDBPCI）was prepared using inorganic iron chelation technology.The relative molecular weight of SDBP and SDBPCI was determined using molecular exclusion chromatography,and structural changes were analyzed using infrared and ultraviolet spectroscopy.The microstructure was observed using scanning electron microscopy.Using in vitro simulation of Caco-2 cells to investigate the stability,cytotoxicity,and cellular absorption characteristics of SDBPCI.The main conclusions obtained were as follows:（1）Considering fresh sika deer blood as raw material,SDBP was extracted,then screened the best protease for explaining the total protein of deer blood through hydrolysis degree determination method;By conducting single factor experiments to study how different factors affected the hydrolysis degree of SDBP,in order to find the optimal enzymatic hydrolysis process;Using response surface technology to improve the optimal extraction scheme.The conclusion proved that the composite protease（modulating with trypsin,neutral protease,and alkaline protease 1:1:1）had the best degradation effect on deer blood peptides.The optimal enzymatic hydrolysis process for SDBP was as follows:the dosage of composite protease was 4990 U/g,p H was 6.7,enzymatic hydrolysis temperature was 60.3℃,enzymatic hydrolysis time was 5 hours,and hydrolysis degree was 32.42±0.23%.（2）Taking SDBP and Fe Cl₂as raw materials,the complexation reaction was carried out by examining factors such as temperature,p H,and reaction time.Using chelation rate as an indicator,single factor experiments were conducted to investigate the effects of various factors on chelation rate and determine the optimal chelation process for SDBPCI.The final chelation process of SDBPCI was as follows:Fe²⁺concentration was 6 mmol/L,SDBP concentration was 3 mg/m L,p H value was 5.0,and chelation time was 40 minutes.The chelation rate was 74.52±0.17%.（3）The molecular exclusion method measurement showed that the main concentration area of molecular weight size had transferred a portion to 3KD-5KDa;The composition analysis of SDBP and SDBPCI amino acids indicated that they contained 17 amino acids and 8 essential amino acids for the human body.The UV and infrared spectra indicated that iron ions mainly bound to the nitrogen atoms of the amino group and the oxygen atoms of the carboxyl group in SDBP,with significant stretching and displacement of the C=O bond,N-H bond,and-COOH bond.Observing the scanning electron microscope images,it was found that the microstructure characteristics of SDBP and SDBPCI were different,indicating the formation of chelated iron.The stability experiment of SDBPCI showed that under p H 2-10 and 50-100℃conditions,the iron retention rate of SDBPCI could reach over 87%,proving that SDBPCI was an excellent iron nutritional supplement with heat resistance,acid and alkali resistance.（4）MTT toxicity test showed that SDBPCI had an impact on the survival of Caco-2cells,below 200μg/m L.SDBPCI was beneficial for the growth and reproduction of Caco-2 cells,but when the amount of SDBPCI exceeding 200μg/ml,there was a relatively significant decrease in cell activity.Establishing an SDBPCI Caco-2 cell absorption model to evaluate the chelating iron absorption characteristics of deer blood peptides.The experiment showed that the absorption rate of SDBPCI was superior to that of the Fe Cl₂group and the mixed group of Fe Cl₂and SDBP.