Preparation And Chelation Properties Of Chicken Protein Calcium Chelating Peptide

Calcium is one of the most important mineral elements in the body,and calcium nutrient loss and deficiency have become a worldwide health problem.Inorganic calcium supplements have problems of easy precipitation and difficulty in absorption in the body,and peptide bingding calcium has attracted more attention due to its high bioavailability.Chicken meat is an important bulk meat resource in China.As an important category of farmed chicken,spent hens are eliminated after the end of its laying period.At present,the added value of spent hen product is low.In this paper,spent hen protein peptide with calcium chelation activity was prepared by enzymatic hydrolysis method,and the effect of ethanol stepwise separation on the calcium chelation of peptide was studied,and the calcium-binding peptide was analysised and characterized by various means.This study provided theoretical guidance for the deep processing of spent hen protein peptide calcium chelate.The main conclusions are as follows:1.The effects of different enzymatic conditions on the calcium chelation of chicken protein peptides were studied.When spent hen protein was hydrlysated by papain,the calcium chelating ability of hydrolyzate increased first and then decreased with hydrolysis time.Three ways of hydrolysis were compared:papain hydrolysis,papain flavorzyme simultaneous hydrolysis,and two-enzyme-stepwise,which has found that two-enzyme-stepwise hydrolysis product has stronger calcium chelating ability.Under the condition of two enzyme-stepwise-hydrolysis,the effects of four factors:papain hydrolysis time,flavorzyme hydrolysis time,papain addition and flavorzyme addition on the calcium chelating ability were studied,and the optimal enzymatic hydrolysis process was obtained by response surface analysis.The hydrolysis time of papain was 3.5 h,the hydrolysis time of flavorzyme was 2.84 h,and the addition of flavorzyme was 0.18%.2.The chicken protein hydrozate(CPHs)were prepared by the processing above.And the effect of ethanol stepwise separation on peptide's calcium chelating ability was studied:Four fractions were obtained after ethanol separation,among which The S3 component accounted for the highest proportion of total nitrogen in CPHs.As the degree of ethanol separation increased,the calcium chelating ability of the fractions gradually increased,the surface hydrophobicity of the fractions gradually decreased,and the zeta potential gradually decreased.The average particle size gradually increases,and the turbidity in the aqueous solution gradually decreases.Scanning electron microscopy shows that the structure of the fractions changes from a lamellar structure to a spherical structure,and the fractions tend to be more tightly aggregated,and the volume gradually increases.The hydrophobic amino acid content of each fractions gradually decreased,and the average hydrophobic Q gradually decreased;the DPPH·clearing ability of the fractions gradually increased.While the molecular weight of each component was mainly concentrated between 1000 Da-5000 Da,and there was no significant correlation between the calcium chelating ability.3.The peptide calcium chelate S3-Ca was prepared by using the S3,which has the highest chelating ability after ethanol separation.The chelate S3-Ca was characterized and analyzed by various methods:When the mass ratio of S3 to calcium 1:1,there is maximum calcium chelating ability.Fluorescence titration experiments also confirmed that the chelation reaction reached saturation when the peptide calcium mass ratio was 1:1.Fourier infrared spectroscopy showed that after chelation of calcium ions,the position and intensity of the peaks of the-COOH and-NH2 functional groups were changed.The circular dichroism showed that the negative groove of the spectrum at 240 nm was weakened after calcium chelation.Scanning electron microscopy showed that the S3 has a smooth spherical structure,and after chelation of calcium ions,a bridged and lamellar structure is formed.X-ray diffraction pattern shows that the spectrum of the polypeptide has no diffraction peak and exhibits an amorphous state,while the peptide calcium chelate has three new diffraction peaks,indicating that the degree of crystallization of the peptide calcium chelate increased.~1H NMR spectrum showed that after chelation of calcium ions,the ~1H peaks of the spectra changed.The above results can be qualitatively demonstrated that calcium ion chelation changes the structure of the polypeptide to form a peptide calcium chelate,and the binding sites are mainly-COOH and-NH2.Using the isothermal titration calorimetry method,the binding site N(Sites)of S3-Ca chelation reaction was determined to be 10,the binding constant Kd=2.26×10-3;?G=-15.1kJ/mol,?H=-13.3 kJ/mol,-T?S=-1.85,indicating that the reaction is an exothermic reaction and can be carried out spontaneously at all temperatures.