Separation And Purification Of Raffinose And Cottonseed Protein From Cottonseed Meal

Raffinose, the effective proliferation factor of bifidobacteria, is a kind of typical functional oligosaccharides. Raffinose has obvious effects on the improvement of digestion, healing property, anti-oxidation activity, enhancing immunity and so on. For these reasons, raffinose can be used as food and cosmetics additives, and used in organ transplant engineering. Cotton seed as a major resource of raffinose contains around4%the oligosaccharides. Therefore, the research on separation and purification of raffinose from cottonseed meal is necessary.Because of difficulty in separation of raffinose and cottonseed protein, the further application of raffinose is limited. The purpose of this research is to purify raffinose by liquild-liquild extraction, membrane technology and gel permeation chromatography.Firstly, a HPLC method for quantitative analysis of raffinose was established. A C30column was used with pure deionized water as mobile phase at a flow rate of1.0mL·min-1. The analysis was carried out at35℃, and the internal temperature of RI detector was also set at35℃.To optimize the conditions for extraction of raffinose from cottonseed meal, studies on the elution curve of raffinose and the effect of solvent consumption on extraction were investigated. The optima operation conditions were as follows:2.5bed volumes (BV) of percolate were collected after the meal being soaked for about1h at a flow rate of2BV·h-1with aqueous solution of75%(v:v) ethanol at60℃, and solid to liquild ratio was1:7. The purity of raffinose in the extract was36.0%and protein content was0.92%in raffinose extract.The liquild-liquild extraction was used to remove cottonseed protein preliminarily in raffinose extract. The influences of extraction stages, material concentration on the removal of protein were discussed. After the operation of three cross-flow extractions, the purity of raffinose was improved up to46.3%, and the protein content was reduced to0.13%, the recovery of raffinose was96.8%and the removal rate of protein was91.2%. Then the raffinose in raffinate phase was further purified by membrane technique. According to the membrane type, ultrafiltration time and concentration ratio, etc, the optima operating conditions were10000MWCO polysulfone membrane,0.03MPa～0.1MPa, normal temperature, VCR=1.4. Protein rejection coefficient was75.0%, and the purity of raffinose in the permeate was47.2%.At last, gel permeation chromatography was used for separation of oligosaccharide and protein. The type of gel packing, temperature, composition of mobile phase and feed concentration were investigated. The optimized chromatographic conditions were obtained as follows:HW-40C was used as stationary phase and deionized water as mobile phase with flow rate of1.82BV·h-1. Besides, the process was operated at40℃and feed concentration up to100mg·mL-1. Moreover, the attempt was made to remove cottonseed protein from raffinose extract, and a recovery of64.8%and purity of89.1%of raffinose was obtained, the removal ratio of protein was above95%. This proposed method was proved to be simple and effective for removal of plant protein from biology samples.