The Aqueous Two-phase Extraction、Purification And Identification Of Pig Plasma Albumin

The process of slaughtering pig will produce a lot of blood,which is the most by-products.At present the utilization of pig blood mainly is as the raw material added in food and feed.The added value is low.Especially for the functional protein.The reason mainly lies in that the current methods to isolate and purify blood albumin include cold ethanol precipitation,salting out,chromatography,etc.All of them have the following disadvantages:high price and loss,low recovery rate,complicated operation,high demand for production equipment and the environment,etc.Considering these problems,this project aims to isolate albumin with aqueous two-phase method.Pig blood is as the raw materials.Investigate the influence of different factors on the recovery rate and partition coefficient,identify the structure and properties of extracted albumin,and purify it further with the method of column chromatography.The main results of this project are as followed:1.Several common aqueous solutions ofhydrophilic organic solvents and salts were plotted.Ethanol/potassium phosphate system was selected for further study.In order to study the effects of ethanol and salt concentration,protein handling capacity,etc on the isolation,a response surface experiment was designed.Two binary multiple regression equations are obtained through the analysis about partition coefficient and recovery.Finally,the optimum conditions were determined as follows:ethanol concentration 15%(W/W),dipotassium hydrogen phosphate concentration 23%(W/W),protein concentration 24 mg/mL.The partition coefficient and recovery rate of albumin was 15.71and 96.75%.2.The purity of albumin treated with a two-phase aqueous solution is 97.35%.Compared with the standard albumin,Plasma albumin has an ultraviolet absorption peak of 290.8,which is close to the characteristic absorption peak of standard albumin.The result of SDS-PAGE show that the protein containsubunits of albumin which molecular weight was 66 kDa and immunoglobulin heavy chain and light chain which molecular weight was 50 kDa and 27 kDa.The result from Infrared scanning shown:plasma protein retains the characteristic peaks of amide land amide II with wave number of 1649.45 cm-1 and 1532.65 cm-1,but another characteristic peak appeared near the waves of 1075.55 cm-1,it is due to the formation of protein aggregation.The results from Circular Dichroism(CD)shown that the albumin extracted by the two-phase aqueous method exhibited the same negative peak as the standard albumin,On the wavelengths of 208 nm and 222 nm,indicating that the a-helix structure had not been significantly damaged.The denaturation temperature was 61.1 ℃.The determination of solubility,foaming ability and emulsification under different pH show that two characteristic peaks appeared on pH=4 and pH=8 in the solubility curve,foaming and emulsifying properties.Indicate that the protein powder could be a mixture of albumin and immunoglobulin.3.In order to further purify the albumin extracted by aqueous two-phase extraction,The plasma protein was further purified by phenyl-agarose gel.Study the effects of equilibrium solution pH,the types of salt and salt content on the elution.The elution was the best with types of salt and salt content on the elution.The elution was the best with the phosphate buffer pH 7 and the dipotassium hydrogen phosphate added was 0.8 mol,the peak of the elution volume for 18～24 mL.Compared with the albumins eparated by aqueous two-phase method,The result of SDS-PAGE show that the purified protein contain albumin which molecular weight was 66 kDa.The heavy chain which molecular weight was 50 kDa and light chain which molecular weight was 27 kDa of immune globulinhas been removed.The result from Infrared scanning shown:plasma protein retains the characteristic peaks of amide I and amide II with wave number of 1649.45 cm-1 and 1532.65 cm-1,and the formation of protein aggregation is decreased.The results from Circular Dichroism(CD)shown that the albumin extracted by the two-phase aqueous method exhibited the same negative peak as the standard albumin,On the wavelengths of 208 nm and 222 nm,indicating that the a-helix structure had not been significantly damaged.It was found that the purification procedure could keep the structure of protein to avoid damage.