Preparation And Stability Of Bovine Hemoglobin Peptide-ferrous Chelate

Iron deficiency anemia is one of the most common nutritional deficiencies worldwide,which can result from inadequate dietary iron intake or poor absorption of iron in the gastrointestinal tract,leading to various functional abnormalities.However,adding trace elements of iron directly to food faces many physiological and industrial technological challenges.Currently,commonly used iron supplements have low utilization rates and some toxic side effects.In recent years,more and more food-derived peptides with ferrous chelating activity have been discovered,which have good biological availability,high absorbability,stability,and safety.Therefore,using peptide-iron chelates to fortify food has gained widespread attention.In this study,bovine blood was used as raw material,and the process conditions for preparing bovine hemoglobin peptides(BHP)by protease hydrolysis were optimized.Bovine hemoglobin peptide-iron chelate(BHP-Fe)was prepared through decolorization and ferrous salt chelation techniques,and the structure of BHP-Fe and its stability under in vitro conditions were explored.This provides a theoretical reference for the high-value utilization of bovine blood byproducts and future applications.Specific research results are as follows:(1)Fresh bovine blood was used as the raw material to extract bovine hemoglobin.Seven different proteases were used to hydrolyze bovine hemoglobin,with hydrolysis degree and Fe2+ chelation ability as evaluation indexes.It was found that pepsin and alkaline protease were the most suitable proteases for hydrolysis.Under the condition of fixed preliminary hydrolysis by pepsin,a two-step enzymatic hydrolysis process was developed using alkaline protease.The secondary enzymatic hydrolysis process of alkaline protease was optimized using a combination of single-factor and response surface methodology.The results of response surface methodology showed that the optimal conditions for the secondary hydrolysis of alkaline protease were a liquid-to-material ratio of 1:3,hydrolysis p H of 9.8,hydrolysis temperature of 41 °C,enzyme addition amount of 5800 u/g,and hydrolysis time of 2 h.Under these conditions,the Fe2+ binding ability of BHP obtained by hydrolysis was 72.11 ± 0.30%,and the hydrolysis degree of bovine hemoglobin was 35.07 ±1.32%.(2)The preparation process of bovine hemoglobin peptide iron chelate was investigated.Based on the single-factor experiment,a four-factor three-level orthogonal optimization experiment was conducted with the iron chelation rate as the evaluation index to determine the optimal process parameters for preparing the peptide iron chelate: peptide concentration of 2%,peptide to iron mass ratio of 4:1,chelation p H of 7.0,and chelation temperature of30 °C.Under these conditions,the optimal chelation rate of the obtained chelate was53.76%.Qualitative identification of the chelate confirmed that the bovine hemoglobin peptide iron chelate met the formation characteristics of a chelate.(3)The structure and properties of bovine hemoglobin peptide-iron chelate were analyzed.Results from ultraviolet spectroscopy,fluorescence spectroscopy,Fourier transform infrared spectroscopy,X-ray diffraction analysis,thermal gravimetric analysis,amino acid content,and scanning electron microscopy all indicated that the peptide-iron chelate was a new substance with a different structure from the peptide.The UV and IR spectra showed that the ferrous ion underwent a chelation reaction with the carboxyl oxygen and amino nitrogen on the BHP peptide chain to form a new substance.The X-ray diffraction results indicated that a new crystal structure was formed after BHP bound to ferrous ions.Thermal stability tests revealed that BHP was more heat-resistant than BHP-Fe below 300 ℃,while BHP-Fe was more heat-resistant than BHP above 300℃.Scanning electron microscopy showed obvious differences in the microstructure between the two.Changes in amino acid content showed that aspartic acid,glutamic acid,and lysine provided binding sites for the binding of the peptide and metal ions.Stability tests showed that the bovine hemoglobin peptide-iron chelate had good stability in systems with p H 6~10 and ion strengths of 4%~12%,and maintained high stability at different temperatures.In addition,by simulated digestion of the human gastrointestinal tract and in vitro antioxidant activity tests,it was found that BHP-Fe had higher stability than Fe SO4 and ferrous lactate,and its antioxidant ability was significantly increased after the chelation reaction of polypeptides with ferrous ions.In summary,the peptide derived from bovine hemoglobin obtained by double-enzyme stepwise enzymatic hydrolysis,forms a chelate with ferrous ions,possessing the structural characteristics of metal-chelating peptides,mainly by chelating with ferrous ions through carboxyl oxygen atoms and amino nitrogen atoms.BHP-Fe maintains good stability in different exogenous environments.However,its absorption and utilization in the body have not yet been clarified and further research is needed.