CN1212387C - Fine rod bundle spore SX-1 separated and cultured from cordyceps and its saparation, culture process and use - Google Patents
Fine rod bundle spore SX-1 separated and cultured from cordyceps and its saparation, culture process and use Download PDFInfo
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- CN1212387C CN1212387C CN 02103669 CN02103669A CN1212387C CN 1212387 C CN1212387 C CN 1212387C CN 02103669 CN02103669 CN 02103669 CN 02103669 A CN02103669 A CN 02103669A CN 1212387 C CN1212387 C CN 1212387C
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Abstract
The present invention discloses a novel strain isolated and cultured from natural aweto, an artificial isolating and culturing method and a purpose thereof. The preservation number of the strain is CGMCC NO. 0706 and is characterized in that circular colonies are laid out with limits, annular patterns and wrinkles, are white at first and hoary late, have the diameter of 1.5 to 2.2cm and have taupe backs to purple brown backs after cultured for 10 days on a PDA culture medium under 25 DEG C; colorless hypha with branches has 1 to 2.5 mum of membranes; white spore stalk bundles with branches have the height of 0.5 to 3.5cm and the diameter of 0.2 to 1.5mm. The dry object of the present invention has a tumor inhibiting effect and can be used for preparing anti-tumor drugs. The present invention provides a novel strain which can be produced in industrialization for people, and the strain has the advantages of stability and anti-tumor activity and contains active components similar to the natural aweto. The strain provides a novel selectable species for people and also provides a novel raw material for anti-cancer drugs.
Description
Technical field
The present invention relates to Cordyceps and artificial culture method and purposes, specifically belong to new bacterial strain and the artificial culture method and the purposes of separation and Culture from natural cordyceps.
Background technology
Cordyceps sinensis (be called for short Chinese caterpillar fungus) be the mixture of the larva corpse of the stroma of Clavicipitaceae plant Cordyceps fungus (Cordyceps sincesis (Berk.) Sace.) and host's Hepialidae insect Chinese caterpillar fungus bat moth (Hepialusarmoricanus Oberthur) thereof, and it mainly contains amino acid, polyose, cordycepin, cordycepic acid, ergosterol, adenosine class, super oxygen disproportionation glycosides element and micro-.The height of adenosine content wherein is as the main foundation (see " Pharmacopoeia of People's Republic of China (2000 editions) " P499: JINSHUIBAO glue is assisted, the quality control standard of fermented Cordyceps mycopowder) of drug quality control.Modern pharmacology studies show that, all multiactions such as Cordyceps sinensis has calmness, anticonvulsion, cooling, eliminating phlegm and relieving asthma, resists myocardial ischemia, anti-arrhythmia, anti-stress, anti-ageing, anti-inflammatory, antibiotic, antitumor and enhance immunity power.Natural cordyceps is to infect bat moth larvae with spore by Cordyceps fungus in the fall the time, follow the bat moth larvae to pierce in the soil, the Chinese caterpillar fungus spore utilize polypide nutrition, grow up to mycelium, and form sclerotium in the winter time, cause the death (being complete polypide external form this moment) of polypide, when waiting until summer, the stroma of Cordyceps sinensis is promptly emerged from the head of polypide, as grass, and form bacterium worm complex body, be Cordyceps sporophore.Because the bat moth is quite harsh to the requirement of ecotope, per 500 bat moth larvaes produce 2 Cordyceps sinensis only.So natural cordyceps output is extremely low, it can not satisfy people's actual demand far away.The artificial now extensive Cordyceps sinensis beyond example still of cultivating, its major cause is a bat moth larvae breeding difficulty, and also has many problems that are difficult to overcome at aspects such as inoculation technique and ecological artificial technology.For satisfying human actual demand to Cordyceps sinensis as early as possible, people begin imagination and utilize artificial fermentation's mode to carry out suitability for industrialized production to solve the problem of natural cs inadequate resource.For example disclosed cordyceps species 814 seed selections of CN85102231B and zymotechnique thereof are people a kind of Cordyceps sinensis surrogate are provided.
Summary of the invention
One of purpose of the present invention is to provide a kind of new bacterial strain from the cordycep separation and Culture for fully satisfying human demand to Cordyceps sinensis, with the surrogate as Cordyceps sinensis.Two of purpose of the present invention provides a kind of corresponding strain culturing method.Three of purpose of the present invention provides a kind of purposes of this bacterial strain.
The object of the present invention is achieved like this:
Thin Isaria provided by the invention (Isaria felina) but SX-1 is the new bacterial strain of a kind of suitability for industrialized production of cultivate obtaining through strain separating from natural cordyceps, it is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on January 30th, 2002, and preserving number is CGMCC NO.0706.
The morphological specificity of fine rod bundle spore SX-1 of the present invention be on the PDA substratum 25 ℃ cultivated 10 days, the bacterium colony circle, tiling, limitation has ring grain and fold, initial white, back canescence, diameter 1.5～2.2cm, back side beige is to puce; Hyphae colorless, branch, tool barrier film, wide 1～2.5 μ m; Coremium white, branch, high 0.5～3.5cm, diameter 0.2-1.5mm; The curved neck doleiform of conidiogenous cell is cylindrical, colourless, bending, 4.8～8.5 * 1～2.2 μ m; The conidium ellipse is to oval, monospore, colourless, level and smooth, 2.5～4.3 * 2～3 μ m.
Identify the following file of method reference of fine rod bundle spore SX-1 morphological specificity of the present invention:
Petch，Trans.Brit.Mycol.Soc.19：34，1934
Hoog，Stud.Mycol.I.1972
Samson，Stud.Mycol.VI.1974
The cultural method of fine rod bundle spore SX-1 of the present invention is: culture presevation number is placed peptone, glucose, yeast extract substratum for the mycelia of CGMCC NO.0706, under 23-27 ℃ of condition, cultivated 1.5-50 days, get bacterium liquid or mycelia kind in purified substratum, operation moves in circles.
In the aforesaid method, substratum can be selected liquid nutrient medium for use, also can select solid medium for use.Select liquid substratum for use, the condition when it is cultivated preferably is controlled at 23-27 ℃, and 180～200 rev/mins, incubation time 36-72 hour.The prescription of liquid substratum is 6 parts of peptones, 12 parts of yeast extracts, 24 parts of sucrose, 0.5 part in sal epsom, 1 part of potassium primary phosphate preferably, 1000 parts of distilled water, pH value 6.0-7.0; The prescription of liquid substratum also can be selected 6 parts of peptones, 10 parts of yeast powders, 20 parts of sucrose, 0.5 part in sal epsom, 1 part of potassium primary phosphate, 1000 parts of distilled water, pH value 6.5-7.0 for use.
When carrying out liquid culture, necessary strict controlled temperature can cause mycelia death when temperature is too high, crosses when hanging down to cause that mycelial growth is slow, influences yield.PH value also will be strict controlled in the scope of 6.0-7.0, to guarantee the homogeneity of mycelial growth.
Selecting for use its culture condition of solid medium preferably to be controlled at temperature is 23-25 ℃, incubation time 40-50 days, the prescription of solid medium is 20 parts in rice (rice, millet all can), 1 part of sucrose, 0.025 part in sal epsom, 0.05 part of potassium primary phosphate preferably, 50 parts of distilled water, pH value 6.0-7.0;
The another screening formulation of solid medium is: 5 parts of peptones, 10 parts of yeast extracts, 20 parts of sucrose, 0.5 part in sal epsom, 1 part of potassium primary phosphate, 20 parts in agar, 1000 parts of distilled water, pH value 6-7.
The culture of strains method makes the present invention realize that suitability for industrialized production becomes possibility.
When the present invention was used for suitability for industrialized production, its liquid fermentation process flow process was:
Slant strains (the female kind) at 23-27 ℃, was cultivated 6-9 days; Insert first order seed, shake in the bottle, cultivated 2-3 days at 26-27 ℃; Insert secondary seed,, cultivated 2-3 days at 26-27 ℃; Insert seeding tank at 26-27 ℃, enlarge seed culture 2-3 days; Insert fermentor tank,, cultivated 2-3 days at 26-27 ℃; Quality inspection.
For making the bacterial classification after going down to posterity possess good growing way, preferably carrying out the rejuvenation of bacterial classification in bacterial classification goes down to posterity handles, it is a small amount of promptly to get the mycelia for the treatment of rejuvenation, direct inoculation is on grain, sucrose medium (identical with go down to posterity used grain, sucrose medium of bacterial classification), under 20-23 ℃, relative humidity 40-50% condition, cultivated 9-13 days, and observed every day when treating mycelia length, select sturdy person to collect frozen getting final product to about 1cm left and right sides.Sturdy inadequately as mycelia, can repeat to cultivate till rejuvenation.
Dry goods of the present invention has the effect that suppresses tumour, therefore can be used for preparing antitumor drug.
Dry goods of the present invention has the active ingredient close with natural cs, it includes sweet dew alcohols material 〉=8%, polyose 16-18%, adenosine 2.5～3.5mg/g wherein measures the sweet dew alcohols and adopts sodium thiosulfate titration, adopt sulfuric acid-phynol method to measure when measuring polysaccharide, the adenosine class adopts high performance liquid chromatography.
Toxicity of the present invention is lower, and its toxicity test fails to measure LD
50, the characteristics that its toxicity is little are further confirmed through the determination experiment of SX-1 gastric infusion maximum tolerated dose.Its experiment and result are as follows:
Animal is selected in experiment for use: with 1 grade of Kunming healthy mice, and 6 ages in week, body weight 18-22g.(laboratory animal portion of Beijing Medical University provides.Certification of fitness: the moving word of doctor 01-3049 number).
Be administered twice gastric infusion on 1st.Observe animal subject and gave the toxic reaction and the death condition that are produced behind heavy dose of medicine in one day.The small white mouse body weight is at 18-22g.Water is can't help in small white mouse fasting (12-16 hour), and experimental group is given SX-1, press the calculating of 25g/kg body weight, is 150 times of human body recommended amounts.Control group is given isopyknic distilled water.After animal gives the SX-1 of 150 times of clinical consumptions of people, observed continuously 7, its behavioral activity, the mental status, stool and urine, fur etc. are all normal, and body weight does not have influence, does not have animal dead in 7 days.
The result shows that SX-1 does not have acute toxicity.
The anti-tumor activity that dry goods of the present invention had to three kinds of transplanted tumor experiments of mouse, has obtained confirmation by SX-1.
The SX-1 dry goods is tested three kinds of transplanted tumors of mouse:
Experiment material is:
Animal
Kunming mouse, body weight 20 ± 2g, male and female dual-purpose.Doctor laboratory animal portion provides by north.
The test product source;
SX-1 dry goods Hebei Shenxing Sea-buckthorn Inst
Endoxan Shanxi Tai Sheng pharmaceutical Co. Ltd lot number: 000909
Physiological saline Datong District curry favour reaches pharmaceutcal corporation, Ltd's lot number: 000692
The knurl source
S
180Sarcoma, Lewis lung cancer, H
22The strain of liver cancer knurl, the institute of materia medica provides by Beijing the Temple of Heaven.
Dosage
Experimental group adopts the SX-1 dry goods, and medication and dosage are:
Experimental group is irritated stomach once 1 every day, each 0.5ml/20g (body weight), and concentration is 30mg/ml.
Experimental group is irritated stomach once 2 every days, each 0.5ml/20g (body weight), and concentration is 60mg/ml.
Experimental group is irritated stomach once 3 every days, each 0.5ml/20g (body weight), and concentration is 120mg/ml.
Positive controls
Positive controls adopts endoxan.Used dosage is 30mg/kg.Each stomach 0.5ml/20g body weight of irritating.
Blank group: isopyknic physiological saline.
Experimental technique:
With well-grown mouse S
180Lewis lung cancer, liver cancer tissue knurl piece, under aseptic condition, add physiological saline, make desired concn and cell suspension with the disinfectant glass grinding, it is subcutaneous to be inoculated in mouse bottom right armpit after the filtration respectively, every 0.2ml, and at random be divided into five group by body weight next day, the blank group, positive controls, experimental group 1, experimental group 2, experimental group 3, the male and female dual-purpose, every group 10, in inoculation beginning in 24 hours gastric infusion, successive administration 10 days is put to death mouse in the last administration and weigh next day, peel off the knurl piece and claim weight in wet base, the gained data are learned processing by statistics, obtain the heavy inhibiting rate (%) of knurl, and experimental result sees Table 1-3.
Experimental result:
The SX-1 dry goods is to S
180The effect table 1 of mouse
The group number of animals
Body weight (g) Knurl heavy (g)Tumour inhibiting rate
Begin to finish X ± SD
Blank group 10 21.86 ± 1.59 27.41 ± 2.16 2.87 ± 0.61
Positive controls 10 21.25 ± 1.39 24.01 ± 3.67 0.75 ± 0.40 73.86%
*
Experimental group 1 10 21.35 ± 1.94 26.16 ± 2.68 2.80 ± 0.57 2.43%
Experimental group 2 10 22.29 ± 1.51 27.07 ± 1.95 1.87 ± 0.54 34.84%
*
Experimental group 3 10 20.65 ± 1.83 26.47 ± 0.97 1.52 ± 0.29 47.03%
*
Compare with the blank group,
*Expression P＜0.05,
*Expression P＜0.01
The SX-1 dry goods is to the effect table 2 of Lewis mouse
The group number of animals
Body weight (g) Knurl heavy (g)Tumour inhibiting rate
Begin to finish X ± SD
Blank group 10 20.49 ± 1.43 28.20 ± 2.15 1.18 ± 0.15
Positive controls 10 20.36 ± 0.92 26.26 ± 2.41 0.37 ± 0.12 68.64%
*
Experimental group 1 10 20.73 ± 2.35 27.29 ± 1.27 0.89 ± 0.16 24.57%
Experimental group 2 10 22.09 ± 1.40 28.33 ± 2.41 0.75 ± 0.14 36.44%
*
Experimental group 3 10 22.10 ± 1.63 27.83 ± 1.48 0.67 ± 0.21 43.22%
*
Compare with the blank group,
*Expression P＜0.05,
*Expression P＜0.01
The SX-1 dry goods is to liver cancer (H
22) the effect table 3 of mouse
The group number of animals
Body weight (g) Knurl heavy (g)Tumour inhibiting rate
Begin to finish X ± SD
Blank group 10 20.13 ± 2.55 29.28 ± 4.94 1.96 ± 0.48
Positive controls 10 20.62 ± 1.64 25.86 ± 3.42 0.68 ± 0.33 65.31%
*
Experimental group 1 10 19.76 ± 1.17 28.92 ± 2.91 1.72 ± 0.62 12.24%
Experimental group 2 10 20.80 ± 1.17 30.98 ± 2.80 1.41 ± 0.46 28.06%
Experimental group 3 10 19.45 ± 1.42 29.73 ± 3.26 1.25 ± 0.27 36.22%
*
Compare with the blank group,
*Expression P＜0.05,
*Expression P＜0.01
Experiment shows: the experimental study by three kinds of mice transplanted tumors proves that SX-1 dry goods of the present invention has function of tumor inhibition, to S
180Malignant tumour, lung cancer, liver cancer etc. all have significant curative effect.
The present invention passed 30 generations through 9 years, did not find the bacterial strain variation, can carry out suitability for industrialized production fully and use.
But the present invention provides the bacterial strain of a new suitability for industrialized production for people, and its bacterial strain is stable, has anti-tumor activity, has the activeconstituents close with natural cordyceps simultaneously.It provides a kind of available new variety for people, also provides a kind of new raw material for cancer therapy drug.
Fine rod bundle spore SX-1 adaptability of the present invention is strong, can go up growth at most fungi culture mediums (as PDM substratum, Cha Shi substratum, MDA substratum etc.), and its maximum tolerable temperature is 28 ℃, and bacterial strain can be dead in a few days when surpassing this temperature.This bacteria growing later stage such as nutrition exhaust, and mycelia and spore all can disintegrate voluntarily, and this moment, the substratum pH value can be raised to more than 8.This bacterium generates needs oxygen, and anoxic can cause the death of this bacterial classification.
Embodiment
Embodiment 1:
This bacterial classification (fine rod bundle spore SX-1) on the PDA substratum 25 ℃ cultivated 10 days, the bacterium colony circle, tiling, limitation has ring grain and fold, initial white, back canescence, diameter 1.5～2.2cm, back side beige is to puce; Hyphae colorless, branch, tool barrier film, wide 1～2.5 μ m; Coremium white, branch, high 0.5～3.5cm diameter 0.2-1.5mm: the curved neck doleiform of conidiogenous cell is cylindrical, colourless, bending, 4.8～8.5 * 1～2.2 μ m; The conidium ellipse is to oval, monospore, colourless, level and smooth, 2.5～4.3 * 2～3 μ m.
Embodiment 2:
Female separation and Culture of planting
Preparation PSA substratum:
Potato (peeling) 200g, agar 18g, sucrose 20g, distilled water 1000ml, pH value 6-6.5;
Get potato, clean the peeling stripping and slicing, add water 1200ml, slow fire boiled 20 minutes, crossed leaching juice, supplied the water yield to 1000ml, after adding agar, sucrose, treating fully dissolving, transfer pH value to 6-6.5,121 ℃ of steam sterilizings 30 minutes are poured in aseptic plate or the test tube while hot after the taking-up, after the cooled and solidified, put under the 25-28 ℃ of condition, the blank cultivation 1-2 days checked pollution-free can the use.
Selecting Sichuan Aba area Cordyceps sinensis for use is provenance, to the Chinese caterpillar fungus surface with 75% alcohol rub 3 times, each 0.8min, usefulness aseptic water washing 4 times places the aseptic plate of lid; Remove surface-moisture with the aseptic filter paper suction, tear from polypide front end back with aseptic nipper, organize several piece, be inoculated on the PSA substratum with aseptic inoculation pin picking, put in 20 ± 1 ℃ the incubator and cultivate, humidity is controlled at 85%, regularly observes and in time superseded assorted bacterium, after 4 days, grow white hypha on the tissue block, white hypha is transplanted to cultivated again on the slant tube 10 days, receive inclined-plane mycelia (promptly female kind) and put into aseptic freezing pipe, directly put into refrigerator below-30 ℃ or liquid nitrogen and preserve.This mother plants and is fine rod bundle spore SX-1, and culture presevation number is CGMCCNO.0706.
Embodiment 3:
Preparation fine rod bundle spore SX-1 dry goods.
Place substratum to be prepared into first order seed mother's kind, the prescription of substratum is that peptone 36 restrains, yeast extract 72 restrains, sucrose 144 restrains, sal epsom 3 restrains, potassium primary phosphate 6 restrains, 6000 milliliters of distilled water, accent pH value 6.5.After substratum is deployed, in 130 ℃ of steam sterilizings 20 minutes, inoculation when being cooled to 26 ± 0.5 ℃.
Inoculation: under the aseptic condition above-mentioned substratum is respectively charged into 6 capacity and is in 3000 milliliters the triangular flask, insert one-level kind 50ml in every bottle.Place rotary shaking table to cultivate triangular flask, 26 ± 0.5 ℃ of temperature, 180 rev/mins of rotating speeds were cultivated 48 hours.
Results: the outward appearance nutrient solution is dense thick, and surveying the substratum pH value is 6, and microscopy does not have assorted bacterium, in 2000 rev/mins centrifugal 20 minutes, collect supernatant and throw out (mycelia) respectively and put 80 degrees centigrade of oven dry, dry goods 207.2 restrains.
Dry thing can be used for preparing antitumor medicine.
Embodiment 4:
Preparation fine rod bundle spore SX-1 dry goods
Mother's kind is placed substratum, and the prescription of substratum is: rice 200 grams, sucrose 10 grams, 250 milligrams in sal epsom, 500 milligrams of potassium primary phosphates, 500 milliliters of distilled water, pH value 6.0-7.0.
The culture medium preparation method:
Various batchings that will be except that rice are soluble in water, adjust pH value to 6.0-7.0, put into rice after the heated and boiled, continue heating and constantly stir, treat into solid state after, stop to heat, be respectively charged in the wide-necked bottle of 10 500ml capacity, seal with one deck kraft paper.In the autoclaving pot 130 ℃, sterilized 20 minutes, take out cool to below 27 ℃, standby.
Inoculation culture:
Under aseptic condition, open culturing bottle and seal, mother is planted choose, be inoculated on the substratum, again the bottleneck former state is sealed, add the suitable process disinfectant plastics film of lid layer size above, (hole of 1 square centimeter of size is cut at the plastics film center, in order to gaseous interchange).Postvaccinal substratum is placed 24-25 ℃, and relative humidity is 40-50%, cultivates 45 days in the draughty environment, and long good culture is taken out, and stirs, and is dry under 60-80 ℃ of condition immediately, obtains dry goods 130 grams.
Embodiment 5:
The prescription of substratum and preparation method are identical with embodiment 4.
Take out the mycelia of cultivating among the embodiment 4 40 days for the treatment of rejuvenation in substratum, be inoculated into above-mentioned media surface, at 22 ℃, under relative humidity 45% condition, cultivated 10 days, when mycelia length arrived 1cm, it was frozen to select sturdy person to collect.
Claims (9)
1, a kind of fine rod bundle spore SX-1 from the cordycep separation and Culture, culture presevation number is CGMCC NO.0706, its morphological specificity is: cultivated 10 days bacterium colony circle, tiling on the PDA substratum for 25 ℃, limitation, ring grain and fold are arranged, initial white, back canescence, diameter 1.5～2.2cm, back side beige is to puce; Hyphae colorless, branch, tool barrier film, wide 1～2.5 μ m; Coremium white, branch, high 0.5～3.5cm diameter 0.2-1.5mm; The curved neck doleiform of conidiogenous cell is cylindrical, colourless, bending, 4.8～8.5 * 1～2.2 μ m; The conidium ellipse is to oval, monospore, colourless, level and smooth, 2.5～4.3 * 2～3 μ m.
2, a kind of cultural method of fine rod bundle spore SX-1, it is characterized in that culture presevation number is placed peptone, yeast, sucrose medium or grain, sucrose medium for the mycelia of CGMCCNO.0706, under 23-27 ℃ of temperature, cultivated 1.5-50 days, get bacterium liquid or mycelium inoculation on pure substratum, operation moves in circles.
3, the cultural method of fine rod bundle spore SX-1 according to claim 2, it is characterized in that this method includes rejuvenation of spawn, promptly get the mycelia for the treatment of rejuvenation, direct inoculation is on grain, sucrose medium, under 20-23 ℃ of temperature, relative humidity 40%-50% cultivated 9-13 days, when treating that mycelia grows to the 1cm left and right sides, it is frozen to select sturdy person to collect.
4, the cultural method of fine rod bundle spore SX-1 according to claim 2 is characterized in that said peptone, yeast, sucrose medium are liquid substratum, and its culture condition is temperature 23-27 ℃, 180-200 rev/min, and time 36-72 hour.
5, the cultural method of fine rod bundle spore SX-1 according to claim 4, it is characterized in that said liquid nutrient media components weight proportion is 6 parts of peptones, 12 parts of yeast extracts, 24 parts of sucrose, 0.5 part in sal epsom, 1 part of potassium primary phosphate, 1000 parts of distilled water, pH value 6-7.
6, the cultural method of fine rod bundle spore SX-1 according to claim 2 is characterized in that peptone, yeast, the sucrose medium of said bacterial classification in going down to posterity is solid medium, and culture condition is that temperature is controlled at 23-25 ℃, time 40-50 days.
7, the cultural method of fine rod bundle spore SX-1 according to claim 6, the composition weight proportioning that it is characterized in that said solid medium is 20 parts in rice, 1 part of sucrose, 0.025 part in sal epsom, 0.05 part of potassium primary phosphate, 50 parts of distilled water, pH value 6.0-7.0.
8, the cultural method of fine rod bundle spore SX-1 according to claim 6, the composition weight proportioning that it is characterized in that said solid medium is 5 parts of peptones, 10 parts of yeast extracts, 20 parts of sucrose, 0.5 part in sal epsom, 1 part of potassium primary phosphate, 20 parts in agar, 1000 parts of distilled water, pH value 6.0-7.0.
9, a kind of purposes of the fine rod bundle spore SX-1 from the cordycep separation and Culture is characterized in that culture presevation number is used to prepare antitumor drug for the microorganism of CGMCC NO.0706.
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| CN102228471B (en) * | 2011-06-27 | 2012-10-17 | 山西大学 | Use of cat isaria mycelium |
| CN102813669A (en) * | 2012-08-18 | 2012-12-12 | 山西省肿瘤医院 | Application of Isaria felina crude polysaccharide to preparation of anti-tumor drug |
| CN107118967B (en) * | 2017-07-12 | 2018-03-02 | 西藏藏草宜生生物科技有限公司 | Hirsutella hepiali Chen et Shen filament quick separating rejuvenation technique |
| CN109266557A (en) * | 2018-10-17 | 2019-01-25 | 上海市农业科学院 | The preparation method of a kind of thin foot Isaria cordyceps sinensis aggregate species strain with health role |
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