Controlled Preparation And Property Study Of Heme-Peptide With High Acid Solubility

Porcine blood has a high iron content, and the main existing form of iron is heme which owns high bioavailability. However, as a kind of iron fortifier, heme is unable to be dissolved in acidic and neutral aqueous solution, therefore, its subsequent absorption in gastrointestinal tract is influenced. Heme peptide with corresponding molecular weight can be prepared by one-step enzyme under the control of the degree of hydrolysis(DH), nevertheless, even though the acid solubility was improved, the yield of heme and the ratio of heme to peptide remain low. The combination of heme and peptide will happen spontaneously under certain conditions, which is helpful to improve the acid solubility of heme. Accordingly, on the one hand, preparing peptides with certain molecular weight distribution by hydrolysis with low DH and ultrafiltration, on the other hand, through hydrolysis with high DH to concentrate heme, the heme peptide with both high acid solubility and yield was expected to obtained by the solubilization of peptide on heme. In this study, the conditions of two-step enzyme was optimized, and the protection of antioxidants on ferrous heme in hydrolysis with high DH was studied. Then, the influence of ultrafiltration conditions on the molecular weight distribution of hemoglobin peptide and the relationship between the heme acid solubility and molecular weight distribution of peptide was researched. In addition, the interactions between heme and hemoglobin peptide was analyzed. Finally the physicochemical properties of heme peptide product was studied.With the(ferrous) heme acid solubility,(ferrous) heme content and DH as indices, the conditions of composite enzymatic hydrolysis was optimized. It was found that the acid solubility of total heme and ferrous heme respectively achieved 73% and 71% under the optimal conditions of hydrolysis with low DH: the enzymatic hydrolysis time was 3 h, the additive content of Alcalase protease was 2250 u/g, the initial hydrolysis pH was 8.4 and the stirring speed was 280 r/min. The yield of total heme and ferrous heme respectively achieved 74% and 59% under the optimal conditions of hydrolysis with high DH: redissolution pH was 9.5, the time of hydrolysis was 7 h, the additive content of Alcalase and flavourzyme protease was respectively 2025 u/g and 800 u/g. With the yield of ferrous heme and the dissolved oxygen content as indices, It’s was found that the combination of tea polyphenols and ascorbic acid showed a better ferrous heme protection effect than each of them alone. Reduced iron powder has good protective effect on ferrous heme which increased the yield of heme to 71% with the additive content of 0.75%.Based on high performance liquid phase chromatography and amino acid analysis, with the acid solubility as indices, the hydrolysate with low DH was ultrafiltrated to concentrate target peptide, it was found that serious concentration polarization phenomenon appeared when the protein concentration in hydrolysate with low DH during ultrafiltration was 45.0 mg/m L, but the situation improved significantly when the protein concentration decreased to 22.5 mg/mL or less, and the peptide with relative molecular weight less than 1×103 and 1×103 to 2×103 respectively declined and rose. The acid-soluble percentages of heme achieved 84% with the mass ratios of protein in ultrafiltration liquid to heme in hydrolysate with high DH of 1.15. The second-stage ultrafiltration was uesd to increase the ratio of heme to peptide and acid solubility. The results showed that the 67% mass of amino acids and small peptides can be entrapped by ultrafiltration membrane with relative molecular weight cutoffs of 3×103. As a result, protein content reduced from 46.5 mg/m L to 15.4 mg/m L, the ratio of acid soluble iron in heme to peptide increased from 0.92% to 2.56%.The interaction between heme and peptide was investigated by exogenous and endogenous probes. The addition of heme can not only cause the fluorescence quenching of exogenous probe 1,6-diphenyl-1,3,5-hexatriene and 8-amino-1-naphthalenesulfonic acid, but also quench the internal amino acids(tryptophan and tyrosine) through affecting relative microenvironment. The maximum diffusion constant of more than 2×1010 L/mol/s represented that the quenching type of heme to endogenous amino acid is static quenching, the main interaction force is probably hydrophobic interactions. Forces which can cause enthalpy change such as coordination bond, Van der Waals forces and hydrogen bonding play an supporting role.The relative solubility curve of heme peptide product is a shape of "U", solubility under high acidic conditions was significantly higher than that of pure heme. The results of in vitro digestion showed that the bioavailability of heme peptide decreased from 91% to 70% in simulated gastric fluid after 3 h of incubation, and the simulated intestinal fluid digestion caused a decline of 10%- 15% of bioavailability after 4 h of incubation. The effect of vacuum storage on the protection of ferrous iron is better than that of exposure storage. The ability of heme peptide to scaveng 2,2-diphenyl-1-picrylhydrazyl free radical is weaker than ascorbic acid and tea polyphenols, while the ability of inhibiting O2-? free radical is stronger.