Research On Enzymatic Preparation And Functional Characteristics Of Heme-iron Enriched Polypeptide

Heme-iron enriched polypeptide possesses anti-anemia, antioxidant and antimicrobial functions. Its preparation, protection, purification and functional properties were studied in the study.Preparation of heme-iron enriched polypeptide.Neutral protease and flavourzyme were used for hemoglobin hydrolysis. The optimal conditions obtained from single-factor and orthogonal experiments for neutral protease were:4%of substrate concentration,10%of enzyme concentration, pH7.5,1h for hydrolysis. The optimums from sigle-factor and multiple linear regression experiments for flavourzyme were:4%of enzyme concentration, pH7,1h for hydrolysis. Different types of protective agents were added in substrate and hydrolysates to determine the protecting effect on ferrous iron of heme-iron enriched polypeptide, and the hydrolysates were exposed in the air for168h. The results showed that the optimal agents were ascorbic acid/anhydrous sodium sulfite complexes (2:1) for substrate and exposed hydrolysates. Ferrous iron respectively increased24.5%and37.7%against control.Purification of heme-iron enriched polypeptide. Ultrafiltration was used to purify heme-iron enriched polypeptide, the influence of flow rate, solution pH and temperature were studied in the study. Single-factor and ternary quadratic regression design were used to optimize the experiments. The standard curve of iron was work out, the correlation coefficient r2=0.9998, the RSD of precision test was0.11%, which met the precision requirements; the RSDof reproducibility test was0.53%, which meant the standard cure accurate and stabile; the recovery of standard addition recoveries test was95.4-104.07%, which placed within the confidence interval.The optimal results from single factor and regression experiments were:2.67X10-2L/min of flow rate, pH7.5,20℃of temperature. Ferrous iron content reached170.566μg/ml, increased by3.26%than that of unpurified ones, which had certain significance.Preparation of heme-iron enriched polypeptide chelates. Inorganic iron source FeCl2was used for heme-iron enriched polypeptide chelation. Effect of ferrous iron concentration rate of the two sources, chelating pH, and chelating time were studied. The optimum of single-factor and quadratic regression coherent design were:a1:1concentration rate, pH3,30min of chelating time. Verified experiments showed the chelation rate of93%. Na2S experiment determined that the products we prepared were iron chelates. The composition of the chelates was analyzed; the main components were peptide, heme and iron. The content of them were71.21%,8.49%and10.32%respectively. A small amout of sodium, water and other ingredients were found. Protective agents were added in chelating substrate and products to protect ferrous iron, and the products were exposed in the air for168h. Results showed that0.1%of ascorbic acid and ascorbic acid/anhydrous sodium sulfite complexes (2:1) worked best for substrate and exposed products respectively, ferrous iron increased38.9%and58.9%against control.The antimicrobial activity of heme-iron enriched polypeptide was studied. Escherichia coli and Staphylococcus aureus were used for the research. MIC against the two bacteria, pH and temperature were determined. The results for optimal E. coli inhibition were:100μg/ml of MIC, pH6,37℃of temperature; the results for Staphylococcus aureus were:200μg/ml of MIC, pH6,37℃of temperature. Heme-iron enriched polypeptide possessed antimicrobial activity.The antioxidant properties of heme-iron enriched polypeptide chelates were studied. In vitro antioxidant was studied first. DPPH, H2O2clearance rate, reducing power and T-AOC were determined; ascorbic acid was used as control. Results showed that heme-iron enriched polypeptide chelates had the ability to scavenge free radical, possessed reducing power and T-AOC. The activity of GSH-Px, SOD, CAT, the content of MDA and T-AOC were determined for in vivo experiments, FeSO4was used as control. The results proved that heme-iron enriched polypeptide chelates could increased the activity of GSH-Px, SOD, CAT, reduced the content of MDA, improved T-AOC, the antioxidant ability of medium dose group showed the optimum.Anti-anemia experiment and safety regard were studied. After iron was supplied, the growth of rats went well. HGB, RBC content increased until beyond the normal levels, serum iron content increased gradually. Acute toxicity test showed that heme-iron enriched polypeptide was actually non-toxic. Organ coefficient of each group showed no significant difference. Absorption rate was calculated and the highest absorption reached34.1%of the medium dose group. Results showed that the iron chelates was efficient and non-toxic for iron supplement.