| Cordyceps militaris is known as the Chinese rare caterpillar fungus, is a kind of traditional tonic Chinese herbal medicine,and has similar, if not superior,pharmacological activities to the well known chinese traditional medicine Cordyceps sinensis in the treatment of certain diseases. Cordyceps militaris is accepted by customers because of that it has high medicinal value and extensive pharmacological effects,such as adjust the immune system function,antitumor,anti-fatigue and so on.There are two methods to cultivate Cordyceps militaris, including liquid medium method and solid medium method.Solid medium method has been implemented widely in the culture field of Cordyceps militarism Link in recent years, there are large amount of myceliums grow in the solid culture medium in cultivating process.The solid culture residue medium is rich of many functional compands such as polysaccharide, cordycepin and so on, but for there no systemic process to extract the active components of the culture medium.At present,with the Cordyceps militaris(L.Fr) Link large-scale cultivating domain, most of the culture medium are being rejected for waste, it lead to the waste of resource, and threat the environment synchronously.The the solid culture residue medium of Cordyceps militaris Link were used as the raw material to study the best technology conditions of flash extraction method for extracting polysaccharide from C. militaris medium.At the same time,to research the optimum technology condition of extracting and purificating of cordycepin from Cordyceps militaris residue medium by ultra high pressure method and high speed counter-current chromatography.The aim of this study was to provide certain theoretical basis about further development and utilization of the solid culture residue medium of Cordyceps Militarism Link.The main results were as follows:1. The starch of solid culture residue medium of Cordyceps militaris Link was removed. Effect of different enzymatic temperatures, enzymatic pH, enzyme dosage and enzymatic time of α-amylase on starch removal were studied. The results showed that the amount of reducing sugar close to stable when enzymatic time of α-amylase is 180 min in pH 6.0, material to α-amylases rate 50:1, temperature 60 ℃. The detection of iodine liquid is non blue appearance under the condition, so the starch of the solid culture residue medium of Cordyceps militaris Link can be removed.2. Polysaccharides from solid culture medium of Cordyceps militaris was extracted by using hydrothermal refluxing extraction. Extract was obtained under hydrothermal refluxing extraction 3h, material to water ratio 1:30, temperature 90 ℃ conditions. Adding α-amylase and non adding α-amylase were compared in extract. The results showed that polysaccharides yield were respectively 8.42% and 3.48% in no adding α-amylase and adding α-amylase extract. Polysaccharides yield of non adding α-amylase extract was 2.42 times of.adding α-amylase extract. When the starch from solid culture medium of Cordyceps militarism was not removed, the polysaccharides yield was falsely high and the result was not accurate. Therefore, determination of polysaccharides of Cordyceps militarism was a barrier in non adding a-amylase condition.3. Polysaccharides from solid culture medium of Cordyceps militaris was extracted by using flash extract technology. Effect of liquid to material ratio, extract time, rotation rate on polysaccharides of Cordyceps militaris were studied. The process parameters of polysaccharides extract was optimized by using orthogonal test method. The results indicated that the influencing order of Cordyceps militaris polysaccharides in the extraction of flash factors were rotation rate influence biggest, next is the extraction time, the impact is the smallest liquid to material ratio. The extract time and rotation rate of flash extract technology has a significant effect on the extraction of polysaccharides from Cordyceps militaris culture medium through the variance analysis. The optimum processing conditions for polysaccharides extraction from cordyceps militarism culture medium were liquid to material ratio 35:1, extract time 12 min, rotation rate 8000 r/min. The polysaccharides yield was 3.52% under the conditions.By using flash extract technology, the polysaccharides yield was higher than hydrothermal refluxing extraction method. Flash extract can effectively avoid the long time and high temperature on the extraction of polysaccharides structure damage at room temperature, and the extraction time was reduced from 3h to 12min. Flash extract can save energy and improve the efficiency.4. Cordycepin from solid culture medium of Cordyceps militaris was extracted by using ultra-high pressure extraction method. Effect of extract pressure, extract time, material to liquid ratio, grinding degree and extract times on cordycepin yield were studied. The significant factors were screened by using Plackett-Burman (PB) experimental design. The results indicated that cordycepin yield was better in extract pressure 400 MPa, extract time 12 min, material to liquid ratio 70:1, grinding degree 20 mush, extract times 2. The fitting of first order regression model was better (R2=88.84%) through Plackett-Burman experimental design, and extract times has a significant effect on the extraction of cordycepin.5. Ultra-high pressure extract was filtered impurity by using ceramic membrane. The results showed that cordycepin content in extract was improved from 44.86% to 49.56%. The removal of the most impurities for high speed counter current chromatography (HSCCC) reduced the difficulty of purification. Cordycepin crude extract was separated and purified by using HSCCC, and separated solvent of cordycepin was screened. The optimum solvent system was ethyl acetate/n-butanol/ water system (2:3:5, V/V). In this solvent system separation effect was better, the stationary phase retention rate was 52.7%, separation time was less than 3 h, and cordycepin purity reached 97.6%. |
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