Preparation Of Gardenia Blue And Genipin By Genetically Modified

Gniposide is the main functional compent contained in the dried ripe fruit of Gardenia jasminoides Elli of Rubiaceae, with the varying from3%to6%in different areas. Geniposide has many pharmacological activities, such as chologogic and antiinflammatory effects, analgesia, and antihypertensive. Grnipin, which can be obtained from Geniposide by hydrolysis of B-glucosidase,is an excellent natural biological crosslinking agent.Under different conditions,the enzymatic hydrolyzate of geniposide and amino acid can be prepared Gardenia Blue and Gardenia red.Many new active compounds can be developed from geniposide because of its special structure; otherwise, geniposide,which plays a role in promoting plant growth, has great development and utilization prospects. Based on the biotrancformation of geniposide and modern research technology, the different sources and performance filter β-glucosidase were selected through β-glucosidase activity measurem-ent experiment and the transformation conditions and effect of gardenia blue pigment and genipin was studied by different β-glucosidase.In addition, separation purification and stability the gardenia blue pigment has been stuided.The specific experiments are as follows:(1) B-glucosidase were selected from several plant materials (semen armeniacae amarum,beans,soybeans and green beans)、microbial enzymes and Commercial β-glucosi-dase.In experiment,we compared different sources and performance filter β-glucosidase of enzyme activity、stability and the applicability of biotransformation of geniposide thoughr the determination of β-glucosidase enzyme activity of DNS method.The result showed:the enzyme activity of emulsin and the specific activity of commercial β-glucosidase were respectively highest; In addition, A Filamental Fungus has been from the Nanjing Normal University,pre-experimental was suitable for gardenia Blue.Therefore, emulsin、β-glucosidase of filamentous fungi and commercial β-glucosidase can be used as the alternate enzyme source of the transformation of geniposide.(2) B-glucosidase was immobilized with the sodium alginate-chitosan-alginate(ACA) microcapsule, in which the microcapsule was prepared by two-step method. In a single factor experiment,the conditions of the immobilization and the specific activity of the immobilized β-glucosidase were investigated.The optimal conditions of immobilized enzyme were that0.15g enzyme dissolved in3.5%sodium alginate,then slowly dropped in2%calcium chloride, reacted for25min to get microspheres;these microspheres were added to0.4%chitosan solution(dissolved by0.5%ethylic acid),then added to0.2%sodium alginate to overlay; Finally, these microspHeres were treated with1%glutaraldehyde at4℃for4hr to get the immobilized enzyme.The total recovery percentage of the immobilized enzyme specific activity was68.3%,and good stability, high repeat interest rates. The packed bed reactor was made of immobilized β-glucosidase by ACA microcapsule, the flow rate0.5%geniposide (Purity75%)was0.5BV/h when it thoughed the packed bed reactor. The transformation of geniposide to genipin was96%,purity of genipin was85%,and the yield was58.67%.(3)By single factor test,the optimun conditions of enzymolytic of geniposide (Purity≥92%)were as follows:pH=5.0,temperature50℃,enzymolytic time40min and enzyme dosage0.3mL,and the optimun conditions of preparation of gardenia blue pigment were as follows:temperature80℃, reaction time2h and added ratio of sodium glutamate0.3mL,the color value of gardenia blue pigment reached20.A Filamental Fungus could be used in fermentation to transport Geniposide (Purity75%)to gardenia blue. The results induced that the optimum conditions was as follows: culture broth quantity50/250mL flask,fermentation period120h,pH7.5,inoculum4%(v/v) and sodium glutamate2%.The transformation of geniposide to Gardenia blue was95.8%by HPLC. the color value of gardenia blue pigment is greater than90.The selection from some kinds of macroporous resins which can purify gardenia blue had been studied through the static adsorption and dynamic desorption experments.The results showed that D4020macroporous resins was suitable to prepare gardenia blue of high quality. The optimum adsorption and resolve conditions of D4020were as follows: the absorbing velocity of flow was1.5mL/h, the concentration of loading sample was0.885of OD590nm,the maximum sample volume was1800mL, the column was eluted with1BV water and then with2BV60%ethanol at a flow rate of1.5mL/h, after purification of gardenia blue pigment by D4020macroreticular resin, the color value was130.82, and the yield was64.47%.The stability of gardenia blue pigment was studied.The result showed that the pigment was stable to light and heat. It had a certain stability in acid and alkaline solutions,and had higher stability in alkali to alkaline. It was stability to ions Mg2+and Ca2+,the ions Cu2+, Fe3+and Cr2O72-all could cause the pigment solution precipitation,and the effect of these ions to pigment was concern with processing time;the antioxidation activities of gardenia blue pigment was weak.