Study On The Extraction,Purification And Properties Of Hemoglobin In Chicken Blood

As an important animal protein resource,chicken blood is rich in nutrients.Hemoglobin(Hb)is the most abundant protein in chicken blood.It is prone to auto-oxidation and also promotes lipid oxidation,which leads to chicken deterioration.In order to determine suitable storage conditions for chicken hemoglobin,this research used hemoglobin extracted from chicken blood as the research object.The best extraction and purification process of hemoglobin was explored.The influence of external conditions on the stability,solubility and oxidation characteristics of chicken blood hemoglobin was explored in the external conditions.The main methods and results are as follows:(1)The extraction method of hemoglobin was screened,and the influence of material-to-liquid ratio,washing times,lysis time,and Na Cl volume on hemoglobin content was studied by single factor experiment.The conditions of hemoglobin extraction were optimized by response surface test:the material-to-liquid ratio is 1:4,washing times are 3times,lysis time is 60 min and 0.10times of Na Cl volume.Under these conditions,the content of hemoglobin extract is 3.5mmol/L.(2)The crude hemoglobin extract was purified by Sephadex G-75,scanned wavelength at 300-600 nm,and identified by SDS-PAGE.The results showed that the purified product had characteristic peaks at wavelengths of 415 nm,540 nm,and 575 nm,and a clear band around 68 KDa.The relative purity of the hemoglobin was 93.77%.(3)Take the absorbance value A₅₄₀ as an indicator,the effects of different storage temperatures,p H,oxidants,metal ions and cryoprotectants on the stability of chicken hemoglobin were studied.The results show that high stable storage conditions of hemoglobin are at-80°C,neutral p H,and adding glycerol or sucrose,have high stability;When the concentration of H₂O₂is 1%,the influence on hemoglobin stability is obviously higher than other concentrations,and it has the obvious influence on hemoglobin stability(P<0.05);Fe³⁺and Cu²⁺increase the absorbance value of hemoglobin,which has an adverse effect on the stability,K⁺?Ca²⁺?Mg²⁺and Na⁺did not obvious influence on the stability of hemoglobin(P>0.05).(4)The effects of temperature,p H,storage time and Na Cl concentration on the oxidation characteristics of chicken hemoglobin were studied with the percentage content of Met Hb as an index.The results showed that the percentage content of Met Hb increased and the oxidation of hemoglobin accelerated with the increase of temperature,storage time and Na Cl concentration;Hemoglobin is more stable alkaline conditions,and the percentage of Met Hb at acidic conditions is significantly higher than that at alkaline conditions(P<0.05).(5)To study the effects of different temperature,p H,Na Cl concentration and sucrose concentration on the functional properties of hemoglobin,the solubility,emulsification and foaming properties of hemoglobin were used as measurement indicators.The results showed that hemoglobin had the maximum solubility when p H deviated from isoelectric point,temperature was 60°C,Na Cl solution and sucrose solution were 0.4 mol/L;The emulsification and solubility curves of hemoglobin were similar;The emulsification stability of hemoglobin can be promoted temperature 60°C,p H5.0 and 0.4 mol/L Na Cl solution;The foamability of hemoglobin increased with the increase of p H and temperature,and the foamability was stronger in 0.4 mol/L Na Cl solution and 0.6 mol/L sucrose solution.The foam stability of hemoglobin decreased with the increase of temperature and Na Cl concentration,and the foam stability was better at low temperature and p H5.0.