Study On The Preparation And Properties Of Porcine Hemoglobin Iron Chelating Peptide

In this thesis, porcine blood was used as the raw material to extraction hemoglobin, enzymatic hydrolysis, bleaching, ultrafiltration separation and chelating technology were used to prepare porcine hemoglobin iron chelating peptide; the stability of hemoglobin iron chelating peptide was studied; in vitro evaluation of anti-oxidative activity method was used to evaluate antioxidant activity of hemoglobin iron chelating peptide; in vitro digestion-Caco-2 cell model method was used to evaluate iron bioavailability; on this basis, porcine hemoglobin iron chelating peptide was used to preliminary applied research. The main conclusions obtained were as follows:Fresh porcine blood was used as raw material to obtain blood cells, the effect of cell disruption was was acted as evaluation index to obtained freezing and thawing combination of ultrasonic as the method of broken blood cells; decolorization effect was acted as evalution index preferred the decolorization techonology as activated carbon 2%, temperature 57 ℃, p H4.25, adsorption time 60 min; the degree of hydrolysis and the and the iron chelating activity of the hydrolyzate was acted as evalution index to obtained: the optimal protease was alkaline protease; combine one-factor-at-a-time(OFAT) with Box-Behnken design(BBD) of response surface methodology(RSM), the optimum process parameters were: substrate concentration7.8%, enzyme dosage 8300 u/g, hydrolysis temperature 55 ℃, p H8.5, reaction time3.3 h.Porcine hemoglobin polypeptides prepared before was used as raw materials;one-factor-at-a-time(OFAT) and the ternary quadratic orthogonal design were used to optimize the experimental design; the iron chelating activity was acted as evalution index; the optimal chelating condition was obtained: peptide iron mass ratio of 3: 1, the optimal value of the peptide concentration 5.13 %, optimalchelation time is 34.0 min.Five kinds of molecular hemoglobin polypeptides were obtained after ultrafiltration, concentrated and lyophilized, respectively were PHP-I(molecular weight> 30 k Da), PHP-II(10 k Da-30 k Da), PHP-III(3 k Da-10 k Da), PHP-IV(1k Da-3k Da) and PHP-V(molecular weight <1 k Da). Wherein, PHP-III showed the highest rate of ferrous ion chelation, each component of peptide has the largest ferrous ion chelating rate at the concentration of 5 mg/m L; the DPPH, ABTS and hydroxyl radical scavenging activity were used as evalution index, comprehensive consideration, iron chelating peptide prepared by PHP-III had the strongest antioxidant activity. Stability tests obtained: in the solid powder and solution state stability size: porcine hemoglobin iron chelating peptide > ferrous sulphate> ferrous chloride; there were no significant effect on the hemoglobin iron chelating peptide oxidation rate of ferrous with sucrose concentration of 0 ~ 10 mg/m L or sodium chloride concentration of 0 ~ 10 mg/m L.In vitro digestive-Caco-2 cell model method was used to evaluate the iron bioavailability of porcine hemoglobin iron chelating peptide; Caco-2 cells were inoculated and cultured for 21 days to build the model Caco-2 cell monolayers,the results showed that: the TEER value of Caco-2 cells monolayer cultured for 21 days exceeded 400 Ωcm2, Caco-2 cell monolayer film structure had been differentiate to form, and maintain stability. Alkaline phosphatase activity of AP side of the cells was 14.20 times of the BL side, there has been polarization distribution; sodium fluorescein permeation rate was 0.49% ? h-1 ? cm-2, less than 0.6% ? h-1 ? cm-2,meeted the amount of standard of the sodium fluorescein penetration markers,Caco-2 cell monolayer model was successfully constructed, it could be used to simulate the intestinal iron absorption; after in vitro digestion, Fe SO4 solution,Fe SO4 solution mix with porcine hemoglobin peptide and hemoglobin iron chelating peptide with the same iron content was added to the Caco-2 cell monolayer model,the iron transport experiments showed that the bioavailability of hemoglobin iron chelating peptide was better than Fe SO4 solution and Fe SO4 solution mix withporcine hemoglobin peptide, could be used as a good iron supplement.To study the preliminary application of porcine hemoglobin iron chelating peptide, a kind of gel candy containing porcine hemoglobin iron chelating peptide was designed, according to the Chinese Dietary DRIs recommended list and sensory index recommended adequate intake of iron to determine 2.0% dosage of porcine hemoglobin iron chelating peptide; gel candy overall sensory evaluation was used as evalution index, the optimal ratio of pectin and carrageenan was 3:2; single factor and orthogonal experiment were used as optimization method to get the best formula were: porcine hemoglobin iron chelating peptide 2.0%, 0.1% citric acid, jujube juice2 g, white sugar 30 g, mixing glue dosage 30 g, starch syrup 40 g, iron content of the resulting gel candy was 2.02 ± 0.38 mg/g.