| Bovine milk,which contains rich,high quality and adequate nutrient components,is a kind of natural food that is easy to absorb and digest by human beings.The protein content of the milk is about 30-35g per litre,of which 80% is casein (26g/L)and other 20% is whey protein.The casein(CN) is a kind of protein which can be precipitated from the skim milk by acid when the pH reaches 4.6.The main component of the casein has 4 kinds of proteins,namelyαs1-CN,αs2-CN,β-CN andκ-CN.There exists remarkable difference in their structures and properties.The casein also has broad applications.For example,it can be used to manufacture formula powder,biologically active peptides,to produce proteins with more powerful functional properties and to optimize the process of production of yogurt and its flavor compounds.Due to the widespread function of casein individuals,their separation and purification work has long been the focus of our attention.Using the whole casein as the raw material,the objective of this study is to investigate the separation method of casein individuals in order to obtain its main components(αs1-CN,αs2-CN,β-CNandκ-CN)separately, so as to expand their application range,improve the practicality and the added value of the products.This is helpful to the deep processing of milk and milk products and its resource utilization.The research is composed of 4 parts.The first part used the SinoChrom 300A C8 as the separation column and established the RP-HPLC method for the quantitation of casein individuals.The result showed,when we treated the whole casein by the 7M urea solution with 2% ME and 1% sodium citrate and used the water and acetonitrile both containing 0.1%TFA(v/v) as the mobile phase A and B respectively to separate casein at the flow rate of 1ml/min with the detection wave of 214nm,the separation of casein individuals can be achieved effectively with a large response value,good linearity,also both the stability(including the peak area and retention time)and the recovery rate was satisfactory.Overall, this method proved to be practicable and feasible.The second part used the Q-Sepharose Fast Flow as the separation medium,whole casein as the raw material and studied the separation conditions.Firstly,we determined the pH of the absorption medium and its absorption capacity.Then we discussed the effect of flow rate and the elution program on separation.The final result was as follows:the mobile phase A is 4M urea in 20mM Tris-HCl buffer containing 0.5%ME with pH 8.0.The mobile phase B was the same as A plus 2M NaCl.Detection was performed at the flow rate of 1ml/min,with the wavelength of 280nm.Step elution method was employed,which can achieve the preliminary enrichment of casein individuals and lay the solid foundation for the further separation.The third part investigated the conditions of urea precipitation for separation of casein individuals and analyzed the effect of urea,casein concentration and the temperature on separation.Firstly single factor experiment was carried out at the basis of which L9(34) orthogonal experiment was conducted.The result showed the optimal condition was as follows:precipitation temperature 40℃,concentration of casein 6% and that of urea 3.2mol/L.Then the method of urea precipitation and ion exchange were combined for the separation.The final result is as follows:forκ-CN,the relative purity and recovery rate were 57.3% and 49.8% respectively;forαs-CN,75.4% and 73.8% correspondently;and that ofβ-CN were 87.9% and 62.0%.The forth part tookαs-CN elution sample for example and used Sephadex G-25 as the gel filtration medium and 40mM ammonium bicarbonate at pH8 as mobile phase for desalting operation.Sample volume and flow rate were discussed.The result showed the optimal sample volume and flow rate were 3ml and 4ml respectively.This process of chromatography can effectively remove the salt introduced by the ion exchange chromatography and proved to be fast and convenient.This was helpful to improve the separation efficiency and save the separation time.
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