Study On Isolation And Purification Of Chilgoza Pine Nut And Its Protein Extraction

In this paper, the separation, purification and enzyme properties of a protease from Chilgoza pine nut, and its protein extraction conditions were preliminary studied in order to fully utilize pine nuts resources, meet the needs of the food and pharmaceutical industries for higher quality of pine nuts, and further understanding the deterioration mechanism of Chilgoza pine nut, so as to extend its shelf life. The main results were as follows:(1) The effects of different extraction solvents on the enzyme activity was investigated through the use of distilled water, lactate buffer, phosphate buffer, borate buffer and Tris-HCl buffer, which are commonly used to extracte protease. The experimental results showed that the extraction rate by them from highest to lowest is as follows:borate buffer, Tris-HCl buffer, phosphate buffer, lactate buffer, and distilled water. Therefore, the borate buffer was the best extraction solvent. Single-factor experiments were further conducted to study the effects of pH, extraction time and solid-liquid ratio on exptraction efficiency. Orthogonal test based on above single-factor tests was then carried out to determine the optimum process parameters such as extraction time, buffer pH and solid-liquid ratio. The optimal conditions for protease extraction were obtained as follows:material/liquid ratio1:8, Boric acid-borate buffer (pH9.0), and extraction time60min.(2) The fractional salt of ammonium sulfate coupled with DEAE-Sepharose FF cation exchange column chromatography was used for separating and purifying the protease in Chilgoza pine nut so as to obtain purified enzyme. The results showed that the enzyme activity in supernatant liquid were not much affected when ammonium sulfate saturation is less than20%, while it was not detected when ammonium sulfate saturation reached80%. Thus80%(NH4)2SO4saturation can effectively precipitate the target protease. The purification effect of this cation exchange chromatography was quite obvious, with specificity activity of the purified protease was increased to8.61times with a recovery rate of21.65%.The separation and purification method mentioned above was highly efficient. There’s only one peak during the process of elution.(3) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the protein molecular weight was33kDa. The results of SDS-polyacrylamide gel electrophoresis showed it was a single band, which indicated that the purity of protease had reached electrophoretically pure. The enzyme activity maintained relatively stable between20℃and50℃by measuring the impact of temperature on protease stability. The optimal temperature and pH value for enzyme activity were50℃and9.0respectively. Mn2+exerted a strong activating effect on the enzyme activity, whereas Na+and K+exerted a slight activating effect. Ca2+and Mg2+inhibited the enzyme activity slightly. Cu2+exerted the strongest inhibitory effect. Therefore, the enzyme is a Mn2+activated protease.(4) The protein was extracted from defatted pine nut by borate buffer and assisted by alkali protease, and the effects of pH, extraction time, temperature, enzyme content and solid-to-liquid ratio on the extraction rate of protein were studied. The extraction process was optimized by the response surface methodology (RSM). The results showed that the time, enzyme content and solid-to-liquid ratio had significant influences on the extraction rate. The optimum conditions were as follows:time62.20min, enzyme content0.99%, and solid-to-liquid ratio1:52.30. Under the conditions the extraction rate of pine nut protein was74.13%, and theoretical protein extraction rate was71.44%.