| Peanut is an important economic crop. Apart from Tibet, Qinghai, and Ningxia province, it is also widely cultivated in the North China Plain, the Bohai Bay coast, South China coast the Sichuan Basin, etc. The Resveratrol (Res) contained in peanuts is a compound with a variety of pharmacological effects and biological activities. It is antibacterial anti-inflammatory, anti-oxidation, heart disease preventing, anticancer, anti-platelet aggregation, protecting the liver, immune system regulating, anti-radiation, estrogen acting, anti-HIV effectiveness, thus reputated as one of the brightest prospects in medicines for preventing human cancer in the 21st century. Res, which has an important value of research and development, is widely used in food, medication, health care products, cosmetics, food additives and other fields. The currently commercially available one and its products are mainly extracted from plants like polygonum cuspidatum, grape, etc. Taking peanut plants as the experimental material, we have studied systematically the biotechnology of peanut plants, and the extraction,the separation and purification technology of secondary metabolic product of Res:conducting the induced culture of callus and the suspension culture of peanut cell in the shake flask, building of a structured kinetic mathematical model, researching the effect of elicitors on cell growth and resveratrol accumulation in root cultures of peanut and macro porous resin purification.The major findings are as follows:1. Media types, explant type and hormone combinations on callus induction and trans-resveratrol had a significant influence in the callus induction culture. The best hormone combination was determined by the quadratic regression general rotation design with three factors, and thus obtain the optimal medium is:MS+2.50 mg/L 2,4-D+1.80 mg/L KT+3.00 mg/L 6-BA. The maximum biomass from peanut cell reached 43.95 g/L, and the highest yield of resveratrol 9.07mg/g.2. The effects of shaker rotational speed, inoculum density, and pH and sucrose concentration were investigated in the course of suspension culture. The conditions of suspension cell culture were determined by the quadratic regression general rotation design with four factors, that is 130 r/min,38.00 g/L inoculum,5.80 pH and 40.00 g/L sucrose. The determination of the protein content, phenylalanine ammonialyase activity and peroxidase activity reacts to the growth of suspension culture cell and the changes of resveratrol content to a certain extent.3. In cell suspension cultures of peanut root system, a structured kinetic model of the growth of Peanut cell suspension was constructed by the way of experiencing the basis of sucrose in the culture medium. The relationship between peanut root cell growth and Res synthetic accumulation and medium consumption were described by use of the structure kinetic model. In order to clearly describe the process of various reactions in peanut cell suspension cultures, the reaction rate expression based on mass balance equation is established in bio-phase and non-living phase of each component.According to the material balance principle, biosynthesis rate of any one component j in Culture system is as follows:The whole model is a set of nonlinear differential equations. Kinetic model parameters were identified by test determination and analysis, documentation and differential equations by computer. The results of model calculated basically coincided with the results of experimental. It indicated that the structural kinetic smodel could be used to describe the peanut root cell suspension culture process, and some phenomena of culture process could be given a reasonable explanation.4. Salicylic acid (SA) and ethephon’s effects on biomass of peanut ot cells and yield of resveratrol were studied. The results showed that:S A had no obvious effects on the biomass accumulation of Peanut root cell. Excess SA could cause cell browning, while low concentration ethrel showed a significant result on the growth of cell (<5 mg/L).SA and ethrel could promote the accumulation for the increase of resveratrol. 5.00mg/L SA improved the yield of reveratrol slightly after 9 d of elicitation(up to 8.00 mg/L), while 5.00mg/L ethrel had a better effect after the treatment of 9 d (up to 96.00 mg/L). 5. Based on the shaker suspension culture,2L air-lift bioreactor for the initial amplification of peanut suspension cell culture experiment was carried out. Res accumulation, substrate consumption, pH, physiological and biochemical indexes had been tracked and detected in the bioreactor culture process, and their trend of changes was similar to shaker culture.6. The type optimized test of macro porous adoption resin, DA-201 resin adsorption kinetics experiments, DA-201 resin adsorption isotherm testing, the loading volume test and the dynamic eluting test and the column chromatography experiments on three factors(loading velocity, eluting velocity and concentration of eluting solvent)lead to this conclusion:DA-201 resin adsorption process for the resveratrol conformed to Freundilch and Langmuir equation, and the process of absorption was heat absorption, but was physical adsorption process; the optimum conditions of DA-201 macro porous resin column chromatography test was determined by these experiments and all kinds of analysis:the concentration of sample is 0.70mg/mL, pH= 3.00, the sample volume is 20.0 mL, elution volume is 15.0mL, macroporous resin purification resveratrol mathematical model can be achieved: Y=75.516-4.790X1-1.549X2+8.914X3-1.612X1X2-2.542X12-2.498X22-8.107X32Through Regression analysis and Variance analysis, the optimum conditions on macroporous resin purification were drawn:the sample velocity is 1.0 mL/min; the elution flow is 1.6 mL/min, the ethanol concentration is 76%, the yield rate of purified resveratrol is 80.43±0.13%, which was measured by HPLC, and the purity of separated resveratrol can reach 39.61%. |
PDF Full Text Request