Two-dimensional Liquid Chromatography System For The Separation Of The Protein From Edible Bird’s Nest

In this research, a stepwise method for extracting protein from edible bird’s nest was developed. An off-line SCX/RP two-dimensional liquid chromatography system was established to analyze the extractive of the protein from edible bird’s nest.Compared with another four extraction methods, the optimal one was the stepwise method. The process was determined by the single-factor and orthogonal experiment. The sample was dissolved by solid to liquid ratio1:40,60℃, used ultrasonic extraction for40minutes, centrifuged. Then the supernatant was collected, the residue extracted once more. The twice supernatants were combined. As follows, the residue was used0.5mol/L NaCl,0.1mol/L NaOH and0.5mol/L HC1to extract in turn by above method. At last, each supernatant was dialyzed respectively in de-ionized water for48hours. Four kinds of dialysate were combined and lyophilized to get protein crude.In final extraction of four kinds of edible bird’s nest samples, the glucose content was about3%and the protein extraction rate was6%, which was two times than common methods. Gel electrophoresis experiments showed the molecular weight of protein extracted from35to116kDa. UV absorption spectrum of each sample was also scanned.Each sample was separated ineffectively by one-dimensional SCX or RP chromatography. On the basis of one-dimensional liquid chromatography analysis, an off-line SCX/RP two-dimensional liquid chromatography system was established. In this system, the first dimension used SCX carried out a linear gradient program:the concentration of salt phase was increased from0to100%during30minutes. The SCX elute was cut every5minutes to form6fractions, which were collected in fraction collector between the first and second dimension. Each fraction was lyophilized and re-dissolved to analyze by the second dimension, which used RP chromatography carried out a linear gradient program:the concentration of acetonitrile phase was increased from0to100%during40minutes. Compared to one-dimensional separation, the volume of peaks and system resolution soared greatly, which expressed an effective separation.Four samples were analyzed by the off-line SCX/RP two-dimensional liquid chromatography system. There were7characteristic peaks in the same3fractions. In the reproducibility experiments, the RSDs of these7characteristic peaks were all below6%, which denoted high stability of the system. The results indicated that bird’s nest protein samples could be separated effectively by the off-line SCX/RP two-dimensional liquid chromatography system established in this experiment.