CN1472330A

CN1472330A - Method for increasing content of cordyceps sinensis

Info

Publication number: CN1472330A
Application number: CNA031352952A
Authority: CN
Inventors: 梁宗琦; 李祝; 刘爱英; 韩燕峰
Original assignee: Guizhou University
Current assignee: Guizhou University
Priority date: 2003-06-23
Filing date: 2003-06-23
Publication date: 2004-02-04

Abstract

A process for increasing the content of cordycepin includes such steps as slant culturing of chosen bacterial strains, activating, getting spore powder, liquid culturing, shaker-flask culturing to obtain liquid mother seeds, adding potassium hydroxide to fermenting culture medium, high-temp sterilizing, cooling, regulating pH value, inoculating, shaker-flask culturing to obtain fermented mycelia, centrifugal separating of liquid, flushing mycelia with distilled water, centrifugal treating, removing culture medium, baking, grinding and sieving. Its advantages are high content of cordycepin (more than 2%), and low cost.

Description

A kind of method that improves cordycepin content

Technical field

The present invention relates to the raw material of medicament preparation, specifically, relate to the medical material that contains biological organic effective constituent, particularly the production method of cordycepin.

Background technology

Cordycepin (Cordycepin) promptly 3 '-Desoxyadenosine (3 '-Deoxyaadenosine) claim again cordycepin, cordycepin to be a kind of cytotoxic substance.The molecular formula of cordycepin is C ₁₀H ₁₃N ₅O ₃, molecular weight 251, alkalescence, needle-like or plate crystal, 230～231 ℃ of fusing points, water-soluble, hot ethanol and methyl alcohol are insoluble to benzene, EC, the anthrone reaction positive, the maximum absorption wavelength of UV-light is 259nm.The structural formula of cordycepin is as follows:

During the biosynthesizing of cordycepin, adenosine directly changes into 3 '-Desoxyadenosine and the fracture of carbon-free-nitrogen glycosidic link, and promptly cordycepin synthetic is to be direct precursor with adenosine.

That cordycepin has is antibacterial, antitumor, suppress virus, kill mosquito, arrestin matter kinases, suppress various biological activity such as nucleic acid methylates.Instrument as different RNA polysaccharase in the discriminate between cells has caused the attention of pharmacy circle.In recent years, people have strengthened extracting the research of cordycepin, have obtained some achievements, typical example such as Chinese patent application part No. 00130376, No. 00134688, No. 01124109, No. 01126910 etc.But because worm lead fungi cellulose content is not high in the natural Cordyceps militaris (L.) Link., be generally 0.1%～0.4%, certainly will cause costing an arm and a leg, be difficult to satisfy being extensive use of clinically with the medicine source of the cordycepin that extracts as Worm grass anticancer one class Chinese medicine; The cycle of the cordycepin of prior art extraction simultaneously is longer.

Summary of the invention

The purpose of this invention is to provide a kind of method that improves cordycepin content in the fermentation mycelium and shorten the high yield cordycepin production time, to overcome the defective of cordycepin prior art.

The contriver is through after secular systematic study and testing, and the method for proposition comprises:

(1) selected bacterial strain is transferred on slant medium, cultivates, activates, obtain that the inclined-plane is female plants;

(2) get the female spore powder of planting in inclined-plane with transfering loop, be connected in the liquid mother culture media, shake-flask culture under the normal temperature obtains the female kind of liquid; (3) in fermention medium, add potassium hydroxide, the high-temperature sterilization postcooling is used the salt acid for adjusting pH value, more female kind of liquid is inserted, and carries out shake-flask culture under the normal temperature, obtains fermentation mycelium; (4) mycelium and fermented liquid are all changed in the centrifuge tube, centrifugation bacterium liquid removes its supernatant liquor, washes mycelia with distilled water, repeated centrifugation behind the basic flush away of substratum, is collected mycelium, dries to constant weight, pulverize then, sieve, make cordycepin content greater than 2% dried hypha powder.

In the female kind of aforesaid liquid was cultivated, the volume fraction of substratum loading amount was 20%.

In the cultivation of above-mentioned fermentation mycelium, used substratum is added peptone 2%, yeast extract paste 3%, maltose 3%, sucrose 4% with the amount that contains adenosine 2mg/ml in the soya-bean milk and is made into; The amount that adds KOH is 0.3mol/L; The concentration of hydrochloric acid that is used for regulating pH is 3mol/L.

In the centrifugal separation processes of above-mentioned mycelium and fermented liquid, used centrifugation rotating speed is 2500r/min, and a centrifugation time is 10min, carries out 3 centrifugations usually.

In above-mentioned mycelial drying course, bake out temperature commonly used is 55 ℃.

The method that the above-mentioned dried hypha powder of making can adopt reference standard curve method and marker method to combine, measure cordycepin content with the uv-spectrophotometric device, concrete measuring method is as follows: (1) precision takes by weighing dried hypha powder and places tool plug test tube, add ethanol, use heating in water bath, add ethanol, double-deck filter paper filtering to scale, accurately get filtrate in tool plug test tube, must supply test agent liquid with alcohol dilution to scale; (2) will be for test agent liquid in 752 type ultraviolet spectrophotometer 259nm places, be blank with ethanol, reference standard curve method and marker method are combined, the absorbancy of working sample liquid records fermentation mycelium cordycepin content thus.

The invention provides a kind of method that improves cordycepin content in the fermentation mycelium, the fermentation mycelium cordycepin content that makes is higher than 2%, and the time that will produce cordycepin shortens to only need 48h, this fermentation mycelium can also be used as the medicine source of Worm grass anticancer one class Chinese medicine directly as protective foods; This method is easy to operate, and is with short production cycle, and its cost is low, is fit to suitability for industrialized production.

Description of drawings

Fig. 1 is the block diagram of the inventive method.

Embodiment

The present invention is described in further detail below by example:

Embodiment: transfer on PDA slant medium (potato, glucose and agar) with the microbial strains 96-0706 that is preserved in fungus resource institute of Guizhou University, cultivate 7d down, activate three times, obtain the female kind in inclined-plane at 27 ℃; Get the female spore powder of planting in 3-4 ring inclined-plane with transfering loop afterwards, be connected to peptone 2%, sucrose 2%, MgSO ₄0.05%, KH ₂PO ₄In 0.1% the liquid mother culture media, the volume fraction of substratum loading amount is 20%, under 27 ℃, with the rotating speed shake-flask culture 84h of 140r/min, obtain the female kind of liquid, mycelium wherein closely tangles the formation particle mutually, be suspended in the nutrient solution equably, mycelium pellet is through visual inspection: tiny and even, be milk yellow, and light fragrance is arranged; Microscopic examination: have fine and close reticulated structure, no ghost occurs; Then the amount that contains adenosine 2mg/ml in the soya-bean milk is added in the fermention medium, this fermention medium is to screen through repeatedly experiment (single factor, orthogonal design more earlier), and filling a prescription is: peptone 2%, yeast extract paste 3%, maltose 3%, sucrose 4%; KOH is added with the amount of 0.3mol/L, 121 ℃, sterilization 30min, cooling, transferring pH with 7N hydrochloric acid is 7.0, the more female amount of planting with 7% of liquid is inserted, under 27 ℃, the rotating speed shake-flask culture 48h with 140r/min obtains fermentation mycelium; At last mycelium and fermented liquid are all changed in the centrifuge tube, centrifugal 10min under 2500r/min carries out bacterium liquid and separates, go its supernatant, wash mycelia with distilled water, centrifugal again, triplicate, behind the basic flush away of substratum, collect mycelium, 55 ℃ dry to constant weight, pulverize then, cross 60 mesh sieves, obtain dried hypha powder.The dried hypha powder 0.2g that during mensuration precision is taken by weighing places 20ml tool plug test tube, add 20% ethanol 1.2ml, with 50 ℃ of heating in water bath 30min, add 20% ethanol to scale, double-deck filter paper filtering, accurately get filtrate 0.5ml in 5ml tool plug test tube, 20% alcohol dilution to scale must supply test agent liquid; To be blank with 20% ethanol for test agent liquid in 752 type ultraviolet spectrophotometer 259nm places again, reference standard curve method and marker method will be combined, the absorbancy of working sample liquid, recording fermentation mycelium cordycepin content thus is 2.36%.

Claims

1 one kinds of methods that improve cordycepin content in the fermentation mycelium, its feature comprises: (1) is transferred selected bacterial strain and is cultivated on slant medium, activates, and obtains the female kind in inclined-plane; (2) get the female spore powder of planting in inclined-plane with transfering loop, be connected in the liquid mother culture media, shake-flask culture under the normal temperature obtains the female kind of liquid; (3) in fermention medium, add potassium hydroxide, the high-temperature sterilization postcooling is used the salt acid for adjusting pH value, more female kind of liquid is inserted, and carries out shake-flask culture under the normal temperature, obtains fermentation mycelium; (4) mycelium and fermented liquid are all changed in the centrifuge tube, centrifugation bacterium liquid removes its supernatant liquor, washes mycelia with distilled water, repeated centrifugation behind the basic flush away of substratum, is collected mycelium, dries to constant weight, pulverize then, sieve, obtain cordycepin content greater than 2% dried hypha powder.

The method of cordycepin content in the 2 raising fermentation myceliums as claimed in claim 1, it is characterized in that in the cultivation of described fermentation mycelium used substratum is added peptone 2%, yeast extract paste 3%, maltose 3%, sucrose 4% with the amount that contains adenosine 2mg/ml in the soya-bean milk and is made into; The amount that adds KOH is 0.3mol/L; The concentration of hydrochloric acid that is used for regulating pH is 3mol/L.

The method of cordycepin content in the 3 raising fermentation myceliums as claimed in claim 1, it is characterized in that in the centrifugal separation processes of described mycelium and fermented liquid, used centrifugation rotating speed is 2500r/min, and a centrifugation time is 10min, carries out 3 centrifugations usually.