| In this paper,a-lactalbumin(?-La)was separated and purified by selective precipitation from concentrated whey protein powders,which contained 11 wt%of ?-La and 52 wt%of P-lactoglobulin(?-Lg).In order to obtain the purified ?-La product,there were three separation units used in the proposed separation process,which were so called as preseparation,crude extraction and purification.During the experimental processing,various influencing factors(the separation temperature,the pH value of the solution,processing time and so on)were determined by single factor experiment based on the separation efficiency of a-La and the optimum separation conditions were obtained.The purity of a-La product was about 96 wt%,meanwhile the recovery of a-La was about 78%.The a-La product in this study met the requirements of infant formula milk powder.The analytical method of RP-HPLC was established.The C8 column was used with a column temperature of 25 °C.Mobile phase A was 0.1%trifluoroacetic acid(TFA)solution,mobile phase B was 0.1%TFA-acetonitrile solution with a flow rate of 1.0 mL/min.The sample volume was 20 ?L with a detection wavelength of 215 nm by gradient elution.The standard deviations of the retention time and peak area of a-La were 0.02%and 0.2%respectively,the(3-Lg was 0.2%and 0.1%respectively.The test method had better reproducibility and accuracy.In the preseparation process,sodium citrate was selected as the chelating agent,and when acetic acid,citric acid,and phosphoric acid were used as pH adjusting agents,the precipitation effect of ?-La was examined.The results showed that the separation effect of citric acid was the best.When the dispersion of WPC were treated with sodium citrate(0.1 mol/L),acidified pH to 3.9 using citric acid and stirred for 1 h at 45?,the separation efficiency of a-La was better,the precipitation yield of?-La reached to 91%.For the process of crude extraction,the effects of NaCl concentration,temperature,solid-liquid ratio and washing times on the separation of a-La in the crude product was investigated using the principle of salting out.When the solid-liquid ratio was 1:5,the crude product was washed twice with 7 wt%NaCl solution at 45 ? with 60 min each time,89%of ?-Lg was removed,and the loss rate of ?-La was minimized.In purification unit,ca-La was purified by reconstitution method.The effects of CaCl2 concentration,pH,temperature,extraction time and solid-liquid ratio on the reconstitution rate of ?-La were investigated.When the the pH was 7.5,the CaCl2(0.2 mol/L)solution with a solid-liquid ratio of 1:3 could reconstitute 88%of the ?-La dissolving 90 min at 50 ?.It was found that the two stage structure of the final ?-La product obtained basically unchanged and no irreversible change occurs analysed by circular dichroism and infrared spectrum.The thermal denaturation temperature of ?-La product tested by DSC was about 67.5 ?,which meant that thermal stability of ?-La product was good.The results of SDS-PAGE showed that the a-La preparation showed only a single band of a-La,indicating that the purity of the a-La preparation prepared by this process was relatively high,and the purity of the a-La preparation was 96 wt%tested by high performance liquid chromatography.The ICP test showed that the content of Ca2+ and Na+ in a-La product met the requirement of infant milk powder.The ultraviolet spectral analysis test showed that the content of tryptophan in the a-La product was high,which indicated that the a-La product had a high nutritional value. |
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