Study On The Extraction And Purification Of Ginger Protease And The Removal Of Tannin From Roxburghi

Ginger（Zingiber officinale Rose）is one of the important characteristic agricultural products in China.It is not only a condiment on people’s daily table,but also a commonly used Chinese herbal medicine in ancient Chinese medicine books.It is also proved by modern medicine to have more and clear pharmacological activities.It is a typical and widely used medicinal and edible plant.Ginger protease,an important bioactive component in ginger,was extracted.The extraction,separation and purification of ginger protease from fresh ginger and ginger residue after supercritical CO₂extraction were studied.The extracted ginger protease was used to remove tannin in Rosa roxburghii juice to reduce the convergence of Rosa roxburghii juicee and improve its quality.The main research contents and conclusions are as follows:1.Study on the extraction process of ginger proteaseIn order to extract ginger protease efficiently,fresh ginger was used as raw material.The crude ginger protease was extracted by aqueous solution extraction and purified by ultrafiltration.Using Na₂HPO₄-Na H₂PO₄(containing 5 mmol?L^-1EDTA-2Na)as extraction solvent,protein extraction rate and protease activity as indexes,the crude extraction process was optimized by single factor experiment and response surface experiment.The results were as follows:The optimum conditions for the extraction of ginger protein were as follows:water bath stirring time 2.78 h,phosphate p H 6.62,solid-liquid ratio 1:4.24 g?m L^-1,water bath temperature 5°C,phosphate ionic strength 0.010 mol?L^-1;The extraction rate of ginger protein was 0.8927%and the activity ratio of ginger protease was 68 U?mg^-1.The protein solution extracted from non-degreased ginger contains many fat-soluble components,and there is a certain emulsification phenomenon,resulting in difficulty in ultrafiltration.A degreasing scheme before protein extraction was proposed.2.Study on the combined extraction process of ginger oil and ginger proteaseIn order to extract the two most important bioactive components in ginger,ginger oil and ginger protease.A series of single factor process optimization were carried out with ginger oil yield,ginger phenolic content,ginger protein extraction rate and enzyme activity as the indexes.The results were as follows:The water content of ginger is positively correlated with the extraction rate and enzyme activity of ginger protease.When the drying temperature is 45°C and the moisture content is 15～20%,the effect on the leaching rate and enzyme activity of protease is small,and it is beneficial to the subsequent supercritical CO₂ginger oil extraction.Placing dried ginger powder at-20℃is beneficial to protecting ginger protease activity.Under the conditions of supercritical CO₂extraction time 1 h,temperature45°C and pressure 15 MPa,the yield of ginger oil and the content of gingerols were 4.02%and 226.85 mg·g^-1,respectively.The ginger protease was extracted from ginger residue after extracting ginger oil.The extraction rate and enzyme activity of ginger protease were（0.70±0.01）%and（47.37±1.2）U·mg^-1,respectively.Compared with the ginger protease extracted from fresh ginger,the extraction rate and enzyme activity are relatively low,but the quality is better,which is conducive to further purification.Adding ascorbic acid to the extraction solution is beneficial to the extraction of ginger protease.3.Purification and molecular weight determination of ginger proteaseThe extracted ginger protease was purified by column chromatography with glucan G-75 as the filler.The purity and molecular weight of the purified protein were determined by reduction electrophoresis and non-reduction electrophoresis.Using C₆H₈O₇-Na₂HPO₄solution、Na₂HPO₄-Na H₂PO₄solution、Na Cl solution and H₂O solution were used as the column chromatography eluent,and the enzyme activity was used as the evaluation index.The results were as follows:The second protein peak could not detect protein content and enzyme activity.The molecular weight of the two elution peaks was determined by reduction electrophoresis and non-reduction electrophoresis.The first elution peak could detect the protein band,and the distribution position was roughly the same.The molecular weight of the protein was approximately 14 KDa and 25 KDa.No protein band was detected in the second elution peak.Combined with protein content and enzyme activity analysis,there was no protein in the second elution peak.4.Study on the process of removing tannins from Rosa roxburghii juice using ginger proteinIn order to effectively remove tannin from Rosa roxburghii juice,reduce astringency and improve taste.In this study,ginger protein extracted from ginger residue after supercritical CO₂extraction of ginger oil was used as a tannin removal agent for Rosa roxburghii juice.The tannin in Rosa roxburghii juice was removed by chemical precipitation method.The removal rate of tannin and the retention rate of VC were used as indexes.The effects of liquid-solid ratio,p H,stirring temperature and stirring time on the index were studied by single factor test.On the basis of single factor experiment,orthogonal experiment was further used to optimize the process.The results were as follows:The optimum conditions for removing tannin from Rosa roxburghii juice by ginger protein were as follows:liquid-solid ratio 30:1.2（m L:g）,p H 3.0,stirring temperature 5°C,stirring time30 min.The removal rate of tannin was（47.451±0.608）%,and the retention rate of VC was（75.904±1.244）%.The transmittance of juice increased from（8.44±0.662）%to（92.47±0.297）%.The astringency of Rosa roxburghii juice was significantly improved,and its flavor was enrich.