Research On Enzymatic Preparation And Functional Characteristics Of Heme Iron

Heme iron processes anti-anemia, free radical scavenging and anti-oxidant functions.We mainly studied its preparation, protection, purification and functionality.We used sodium sulfite reduction and pyridine sodium hydroxide chromogenic method to draw a standard curve. The cure precision RSD was 0.449%, the reproducibility RSD was 0.4813%, colouration stable, and the recovery placed in confidence interval. R² of hemin standard curve was 0.9999, the recovery also placed in confidence interval. HPLC method was used to draw a standard curve, the precision RSD was 0.70%, the reproducibility RSD was 2.02%, and the recovery placed in confidence interval. The results indicated that the standard cure was precise, the method was feasible.We used compound enzyme for hemoglobin enzymolysis. Single factor, orthogonal and regression experiments were selected to optimize hydrolysis conditions which affected the concentration of substrate, enzyme concentration, pH and enzymolysis time of the two enzymes. Single antioxidants, single reductant, composite antioxidants and antioxidant/reductant were added to protect ferrous iron. The optimum conditions were: substrate concentration 7%, Alkaline concentration 1%, pH 8, hydrolysis time 2h; flavor enzyme concentration 2%, pH 6.5, hydrolysis time 2h; the yield of heme achieved 11.18mg/ml. Protective agents anhydrous sodium sulfite(0.03%)/l-ascorbyl palmitate (0.03%) proved to be the optimum for ferrous iron protection, ferroprotoporphyrin yield reached 12.60mg/ml, which raised 11.27%.The enzymatic hydrolysate was exposed 168h then, the yield was 7.44mg/ml, which raised 6.85%.Acid-alkali method and salting-out method was used to purify heme iron. The single factors of acid-alkali method were alkali concentration, solid-liquid ratio, and pH value. We designed the RSM model to obtain the optimal conditions: alkali concentration 0.5%, solid-liquid ratio 1:15, pH value 6, purity 9.63%, yield 3.48mg/ml. The single factors of salting-out method were alkali concentration, solid-liquid ratio, and calcium salt addition. The optimal conditions were: alkali concentration 1%, solid-liquid ratio 1:10, calcium salt addition 1g, purity 60.91%, yield 2.71mg/ml. It was evidenced that salting-out method proved to be better than acid-alkali method.We carried out antioxidation experiment of heme iron. In the in vitro assays, we mainly determined scavenging of radical DPPH anions, H₂O₂ free radical, reduce power and total antioxidant activity. The scavenging rate of DPPH radical was 63.25-65.75% with the rise of heme iron concentration. The scavenging rate of H₂O₂ free radical was 15-85% of ascorbic acid with the rise of heme iron concentration. The reduce power was 12-69% of ascorbic acid with the rise of heme iron concentration. The total antioxidant activity was 5-11% of ascorbic acid with the rise of heme iron concentration. Heme iron also showed strong suppressive effect in vivo assays. The results indicated that intragastric administration of heme iron could increase levels of CAT, SOD, T-AOC, GSH and decrease MDA content. The middle-heme group showed highest T-AOC mean. It indicated that the middle-heme group possessed the highest antioxidant activity.Heme iron absorption effect experiments were also designed in the iron-deficient rats showed high bioavailability of heme iron, compared with the positive control group. We determined body weights, organ coefficient, hemoglobin concentration, RBC hematocrit, average hemoglobin concentration, leukocyte, neutrophil, neutrophile ratio and serum iron. The results showed that each index of middle dose group of heme was higher than control group, middle dose of heme iron improved iron supplement significantly.