WO2019223287A1 - 用于蝙蝠蛾拟青霉Cs-4的培养基及其制备方法 - Google Patents

用于蝙蝠蛾拟青霉Cs-4的培养基及其制备方法 Download PDF

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WO2019223287A1
WO2019223287A1 PCT/CN2018/119031 CN2018119031W WO2019223287A1 WO 2019223287 A1 WO2019223287 A1 WO 2019223287A1 CN 2018119031 W CN2018119031 W CN 2018119031W WO 2019223287 A1 WO2019223287 A1 WO 2019223287A1
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medium
culture medium
glucose
powder
tank
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French (fr)
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吴彪
万义斌
葛友群
左飞鸿
杨明
何琳
李进进
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江西国药有限责任公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor

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  • the invention belongs to the field of microbiology, and particularly relates to a culture medium used for Paecilomyces hepiali Cs-4 and a preparation method thereof.
  • Cordyceps sinensis is a medicinal name that is a combination of animals and fungi. It is a complex of the germ of the ergot family of Cordyceps sinensis and the host larvae of bat moths, insects, crickets, bat moths, etc.
  • Cordyceps sinensis is a complex composed of two parts: the constellation (that is, the grass part) and the sclerotium (that is, the carcass part of the insect). Winter larvae dwell in the soil, fungi parasitize them, absorb nutrients, and the larvae are filled with mycelium and die; in summer, seedlings are formed from the larvae's carcass, shaped like grass, and collected around the summer solstice.
  • Cordyceps sinensis contains protein, fat, crude fiber, carbohydrates, multivitamins, cordycepin, cordycepin, various amino acids, and iron, phosphorus, calcium, zinc, manganese, selenium and other essential elements of the human body.
  • Cordyceps sinensis has the effects of regulating the body's immunity, enhancing physical fitness, delaying aging, enhancing the ability to resist hypoxia, resisting kidney damage, resisting pathogenic microorganisms, asthma and expectorant, and increasing white blood cells.
  • Cordyceps also has a good sedative and analgesic effect, which can alleviate the pain and adverse reactions of chemotherapy during the onset of tumors.
  • Cordyceps powder came into being.
  • Cordyceps powder was based on Pacilomyces hepiali Cs-4, which was obtained by expanding the culture medium and then deep fermentation.
  • the quality of Cordyceps powder mainly depends on the quality of Cordyceps mycelium. Mycelium is the product of strain expansion and fermentation culture. The medium is very important for the growth of Cs-4 strain.
  • the purpose of the present invention is to provide a culture medium for Paecilomyces hepiali Cs-4, which provides rich nutrition for the growth of Cs-4 strains, promotes the formation of Cs-4 Cordyceps mycelia, and enhances mycelia.
  • the content of active ingredients such as adenosine and ergosterol.
  • the Cs-4 mycelium obtained not only has the medicinal effects of insect grasslands, but also has the pharmacological use of notoginsenosides, especially ginsenosides.
  • the present invention provides the following technical solutions:
  • a culture medium for Paecilomyces hepiali Cs-4 comprising a seed medium and a liquid fermentation medium, the seed medium comprising: glucose, peptone, bran, inorganic salts and amino acids
  • the liquid fermentation culture Bases include: hot pressed bean cake powder, glucose, sucrose, inorganic salts, amino acids, vitamin B 1 and adenine.
  • Adding amino acids to the seed medium promotes the growth of the strain and ensures the stability of the passage.
  • the composition of the fermentation medium at each level is slightly different to meet the nutritional requirements of the strains at different growth stages.
  • Adenine was added to the medium, and the Cs-4 strain was fermented and cultured.
  • the content of adenosine in the cordyceps powder was significantly increased, and the content of ergosterol, total amino acids and mannitol was also increased.
  • the inorganic salt is selected from the group consisting of potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, zinc sulfate, zinc chloride, sodium chloride, and calcium chloride.
  • One or more amino acids are selected from one or more of histidine, arginine, glycine and L-glutamic acid.
  • the seed medium includes a slanted medium and a shake flask medium, wherein, based on weight / volume g / mL, the slanted medium includes: 2-4% glucose, 0.2-0.4% peptone, and 0.4-0.8 bran.
  • shake flask medium includes: glucose 1-5%, peptone 0.2 -0.4%, bran 0.4-0.6%, fish bone meal 0.2-0.3%, potassium dihydrogen phosphate 0.1-0.3%, magnesium sulfate 0.03-0.05%, zinc sulfate 0.03-0.05%, histidine 0.1-0.3%, water margin.
  • the liquid fermentation medium further contains panax notoginseng powder.
  • the panax notoginseng powder After the panax notoginseng powder is added, the cordyceps fungus powder obtained by fermentation and cultivation has the effects of notoginseng saponins and ginsenosides.
  • the content of ergosterol was significantly increased, and the content of panax notoginsenoside was also increased compared with the addition of panax notoginseng powder and no adenine.
  • the liquid fermentation medium includes a seed tank medium, a propagation tank medium, and a fermentation tank medium, wherein, based on the weight / volume g / mL, the seed tank medium includes: hot pressed bean cake powder 2-4%, Glucose 2-4%, sucrose 2-4%, potassium dihydrogen phosphate 0.2-0.4%, magnesium sulfate 0.05-0.1%, zinc sulfate 0.05-0.1%, histidine 0.1-0.2%, vitamin B 1 0.02-0.3% , Adenine 0.05-0.2% and soybean oil 0.1-0.2%, the balance of water.
  • the seed tank medium includes: hot pressed bean cake powder 2-4%, Glucose 2-4%, sucrose 2-4%, potassium dihydrogen phosphate 0.2-0.4%, magnesium sulfate 0.05-0.1%, zinc sulfate 0.05-0.1%, histidine 0.1-0.2%, vitamin B 1 0.02-0.3% , Adenine 0.05-0.2% and soybean oil 0.1-0.2%, the balance of water.
  • the liquid fermentation medium includes a seed tank culture medium, a propagation tank culture medium and a fermentation tank culture medium, wherein, based on the weight / volume g / mL, the propagation tank culture medium includes: hot pressed bean cake powder 2-4%, Glucose 2-4%, sucrose 2-4%, potassium dihydrogen phosphate 0.1-0.2%, magnesium sulfate 0.04-0.06%, zinc sulfate 0.03-0.05%, histidine 0.1-0.2%, glycine 0.1-0.2%, vitamins B 1 0.02-0.3%, adenine 0.05-0.2% and soybean oil 0.2-0.3%, water balance.
  • the propagation tank culture medium includes: hot pressed bean cake powder 2-4%, Glucose 2-4%, sucrose 2-4%, potassium dihydrogen phosphate 0.1-0.2%, magnesium sulfate 0.04-0.06%, zinc sulfate 0.03-0.05%, histidine 0.1-0.2%, glycine 0.1-0.2%, vitamins B 1 0.02-0.3%, a
  • the liquid fermentation medium includes a seed tank medium, a propagation tank medium, and a fermentation tank medium, wherein, based on the weight / volume g / mL, the fermentation tank medium includes: 3-4% of hot-pressed soybean cake powder, Glucose 1-2%, sucrose 1-2%, potassium dihydrogen phosphate 0.1-0.2%, magnesium sulfate 0.04-0.06%, zinc sulfate 0.03-0.05%, histidine 0.1-0.2%, glycine 0.1-0.2%, vitamins B 1 0.02-0.3%, adenine 0.1-0.2% and soybean oil 0.2-0.3%, the balance of water.
  • the liquid fermentation medium contains panax notoginseng powder, wherein the composition of the fermentation tank culture medium is 4% of hot-pressed soybean cake powder, 2% of glucose, 1% of sucrose, 0.1% of potassium dihydrogen phosphate, and 0.05% of magnesium sulfate. %, Zinc sulfate 0.03%, histidine 0.1%, glycine 0.1%, vitamin B 1 0.05%, adenine 0.15%, soybean oil 0.2% and panax notoginseng powder 1%, water balance.
  • the invention also provides a method for preparing a culture medium, wherein the method for preparing a slanted medium includes the following steps:
  • the difference between the preparation method of the shake flask medium and the preparation method of the slanted medium is that there is no process of adding agar in step (4);
  • a liquid fermentation medium is prepared by measuring 50% of water, adding each medium component thereto, dissolving it by heating, adding water to make a volume, and then sterilizing.
  • the bran is sieved through a 40-mesh sieve before weighing the bran.
  • the sterilization is high-pressure steam sterilization, the pressure is 0.09-0.12 MPa, the temperature is 120 ⁇ 1 ° C., and the time is 30-40 minutes.
  • the composition of the seed medium and the fermentation medium is different.
  • the composition of the fermentation medium at each level is also slightly different, which ensures the nutritional supply of the strains at different stages of growth, and the resulting mycelium has a high yield and high nutritional value.
  • Cs-4 mycelium not only has the medicinal effects of insect pests, but also has the pharmacological use of monomer saponins, especially ginsenosides.
  • the content of saponin in cordyceps powder was higher than that of panax notoginseng alone, and the content of ergosterol was higher than that of adenine alone.
  • the invention provides a culture medium for Paecilomyces hepiali Cs-4, which includes a seed medium and a liquid fermentation medium, wherein the seed medium is further divided into a slanted medium and a shake flask medium. It is a seed tank medium, a propagation tank medium, and a fermentation tank medium.
  • the slant culture medium includes: 2-4% glucose, 0.2-0.4% peptone, 0.4-0.8% bran, 0.1-0.3% potassium dihydrogen phosphate, 0.03-0.05% magnesium sulfate, and histamine. Acid 0.1-0.3%, agar 2-4%, make up to volume with water, pH is natural.
  • the shake flask culture medium includes: 1-5% of glucose, 0.2-0.4% of peptone, 0.4-0.6% of bran, 0.2-0.3% of fish bone meal, 0.1-0.3% of potassium dihydrogen phosphate, and sulfuric acid Magnesium 0.03-0.05%, zinc sulfate 0.03-0.05%, histidine 0.1-0.3%, make up to volume with water, pH is natural.
  • the seed tank culture medium includes: hot pressed bean cake powder 2-4%, glucose 2-4%, sucrose 2-4%, potassium dihydrogen phosphate 0.2-0.4%, magnesium sulfate 0.05-0.1% , Zinc sulfate 0.05-0.1%, histidine 0.1-0.2%, vitamin B 1 0.02-0.3%, adenine 0.05-0.2% and soybean oil 0.1-0.2%, make up to volume with water, the pH is natural.
  • the culture medium of the breeding tank includes: 2-4% of hot-pressed soybean cake powder, 2-4% of glucose, 2-4% of sucrose, 0.1-0.2% of potassium dihydrogen phosphate, and 0.04-0.06% of magnesium sulfate. , Zinc sulfate 0.03-0.05%, histidine 0.1-0.2%, glycine 0.1-0.2%, vitamin B 1 0.02-0.3%, adenine 0.05-0.2% and soybean oil 0.2-0.3%, add water to make up the volume, the pH is natural.
  • the fermentation tank culture medium includes: hot pressed soybean cake powder 3-4%, glucose 1-2%, sucrose 1-2%, potassium dihydrogen phosphate 0.1-0.2%, magnesium sulfate 0.04-0.06% , Zinc sulfate 0.03-0.05%, histidine 0.1-0.2%, glycine 0.1-0.2%, vitamin B 1 0.02-0.3%, adenine 0.1-0.2% and soybean oil 0.2-0.3%, make up to volume with water, the pH is natural.
  • the liquid fermentation medium further contains panax notoginseng powder in an amount of 0.5-1%.
  • the general method for preparing the seed medium according to the present invention is: the bran is boiled with water and filtered to obtain a filtrate; under heating conditions, peptone is added first, then other components are sequentially added, and after heating or adding water to dissolve, add water to make up the volume, and finally sterilize .
  • a liquid fermentation medium is prepared by measuring 50% of water, adding each medium component thereto, dissolving it by heating, adding water to make a volume, and then sterilizing.
  • the bran is sieved through a 40-mesh sieve, it is weighed, and then 6 g of bran is added per 100 mL of water for feeding, heated to boiling, maintained for 30-40 minutes, and then filtered to obtain the filtrate.
  • the filtrate contains a variety of trace elements and vitamins, combined with amino acids in the seed medium, the strains grow quickly and are stable for passage.
  • composition of the slanting medium is: glucose 2%, peptone 0.2%, bran 0.4%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.03%, histidine 0.1%, agar 2%, and the volume is adjusted to 1000 mL, and the pH is natural. .
  • the preparation method is as follows: the bran that has passed through a 40 mesh sieve is weighed according to the calculated amount, and the corresponding volume of water is added (calculated by adding 6 g of bran per 100 mL of water), heated to boiling, maintained for 30 minutes, and filtered to obtain a filtrate. Add the peptone, heat and stir to dissolve the peptone, then add other components except agar, heat and replenish the water to dissolve all the components, and add water to make up the volume.
  • the composition of the shake flask medium is: glucose 1%, peptone 0.2%, bran 0.4%, fish bone meal 0.2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.03%, zinc sulfate 0.03%, histidine 0.1%, water margin.
  • the difference between the preparation method of the shake flask culture medium and the preparation method of the slanted culture medium is that the agar is not added. After adding water to make up the volume, it is divided into 750 mL conical flasks and sterilized under the same conditions. Under aseptic conditions, a 1 cm 2 slant culture colony was inoculated with a spatula into a shake flask medium, placed on a shaker, and cultured at 15 ° C. for 7 days. After the test is qualified, the divided bacterial cultures of the shake flasks are combined into an inoculation steel bottle, and then transferred to a seed tank for cultivation.
  • the composition of the seed tank culture medium is: hot pressed bean cake powder 2%, glucose 2%, sucrose 2%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.05%, zinc sulfate 0.05%, histidine 0.1%, vitamin B 1 0.02 %, Adenine 0.05%, soybean oil 0.1%, water balance.
  • the preparation method is to first measure 50% water, add each medium component, heat and dissolve, add water to make up the volume, and the pH is natural.
  • the prepared culture medium is put into a sterilization cabinet under high pressure steam sterilization, the steam pressure is 0.10Mpa, the temperature is 121 ° C, and the sterilization time is 35 minutes. After 4 days of seed tank culture, the seed tanks were cultured.
  • the composition of the breeding tank culture medium is: hot pressed bean cake powder 2%, glucose 2%, sucrose 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.04%, zinc sulfate 0.03%, histidine 0.1%, glycine 0.1%, Vitamin B 1 0.02%, adenine 0.05%, soybean oil 0.2%, water balance.
  • the medium preparation method and sterilization conditions are the same as those of the seed tank medium. After 5 days of breeding tank culture, it was transferred to a fermentation tank for cultivation.
  • the composition of the fermenter culture medium is: hot pressed soybean cake powder 3%, glucose 1%, sucrose 1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.04%, zinc sulfate 0.03%, histidine 0.1%, glycine 0.1%, Vitamin B 1 0.02%, adenine 0.1%, soybean oil 0.2%, water balance.
  • the medium preparation method and sterilization conditions are the same as those of the seed tank medium.
  • the fermenter was cultured for 4 days and then discharged out of the tank.
  • the fermentation liquid was solid-liquid separated. The solid was dried and crushed to obtain Cs-4 Cordyceps powder.
  • the composition of the slanted medium is: glucose 3%, peptone 0.3%, bran 0.6%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.04%, histidine 0.2%, agar 3%, and the volume is adjusted to 1000 mL, and the pH is natural. .
  • the composition of the shake flask medium is: glucose 3%, peptone 0.3%, bran 0.5%, fish bone meal 0.2%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.04%, zinc sulfate 0.04%, histidine 0.2%, water margin.
  • the preparation method of the slant and shake flask culture medium, the total preparation amount, and other conditions such as inoculation and culture were the same as those in Example 1.
  • the bran boiling time is 40 minutes.
  • composition of the seed tank culture medium is: hot pressed bean cake powder 3%, glucose 3%, sucrose 3%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.08%, zinc sulfate 0.06%, histidine 0.15%, vitamin B 1 0.15 %, Adenine 0.1%, soybean oil 0.1%, water balance.
  • composition of the breeding tank culture medium is: hot pressed bean cake powder 3%, glucose 3%, sucrose 3%, potassium dihydrogen phosphate 0.15%, magnesium sulfate 0.05%, zinc sulfate 0.04%, histidine 0.1%, glycine 0.1%, Vitamin B 1 0.15%, adenine 0.1%, soybean oil 0.25%, water balance.
  • the composition of the fermenter culture medium is: hot pressed bean cake powder 3%, glucose 1%, sucrose 1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, zinc sulfate 0.04%, histidine 0.15%, glycine 0.1%, Vitamin B 1 0.2%, adenine 0.15%, soybean oil 0.25%, water balance.
  • the preparation method and total amount of the culture medium of the seed tank, the propagation tank and the fermentation tank are the same as those in Example 1.
  • the sterilization pressure of the culture medium is 0.12 MPa
  • the temperature is 119 ° C
  • the sterilization is performed for 40 minutes.
  • the inoculation method, fermentation and culture conditions, and treatment method after the tank were all the same as in Example 1, and finally Cs-4 Cordyceps powder was obtained.
  • the composition of the slanted medium is: glucose 4%, peptone 0.4%, bran 0.8%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.05%, histidine 0.3%, agar 4%, and the volume is adjusted to 1000 mL.
  • the pH is natural. .
  • the composition of the shake flask culture medium is: glucose 5%, peptone 0.4%, bran 0.6%, fish bone meal 0.3%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.05%, zinc sulfate 0.05%, histidine 0.3%, water margin.
  • the preparation method of the slant and shake flask culture medium, the total preparation amount, and other conditions such as inoculation and culture were the same as those in Example 1.
  • composition of the seed tank culture medium is: 4% hot-pressed soybean cake powder, 4% glucose, 4% sucrose, 0.4% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 0.1% zinc sulfate, 0.2% histidine, and vitamin B 1 0.3. %, Adenine 0.2%, soybean oil 0.2%, water balance.
  • composition of the breeding tank culture medium is: hot pressed soybean cake powder 4%, glucose 4%, sucrose 4%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.06%, zinc sulfate 0.05%, histidine 0.2%, glycine 0.2%, Vitamin B 1 0.3%, adenine 0.2%, soybean oil 0.3%, water balance.
  • the composition of the fermenter culture medium is 4% hot-pressed soybean cake powder, 2% glucose, 2% sucrose, 0.2% potassium dihydrogen phosphate, 0.06% magnesium sulfate, 0.05% zinc sulfate, 0.2% histidine, 0.2% glycine, Vitamin B 1 0.3%, adenine 0.2%, soybean oil 0.3%, water balance.
  • the preparation method and total amount of the culture medium of the seed tank, the breeding tank and the fermentation tank are the same as those in Example 1.
  • the inoculation method, fermentation culture conditions, and the treatment method after the tank are the same as in Example 1, and finally Cs-4 Cordyceps powder.
  • the seed tank culture medium also contains 0.6% of panax notoginseng powder
  • the breeding tank medium also contains 0.8% of panax notoginseng powder
  • the composition of the fermentation tank culture medium contains hot pressed soybean cake powder 4 %, Glucose 2%, sucrose 1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, zinc sulfate 0.03%, histidine 0.1%, glycine 0.1%, vitamin B 1 0.05%, adenine 0.15%, soybean oil 0.2 % And Panax notoginseng powder 1%, water balance.
  • the Cs-4 strain was fermented and cultured in the same way, and the Cs-4 Cordyceps powder was finally obtained.
  • Example 2 The difference between this embodiment and Example 2 is that the seed tank culture medium also contains 0.5% of panax notoginseng powder, the breeding tank medium also contains 0.8% of panax notoginseng powder, and the fermentation tank medium also contains 1% of panax notoginseng powder. .
  • the Cs-4 strain was fermented and cultured in the same way, and the Cs-4 Cordyceps powder was finally obtained.
  • This comparative example is different from the composition of the culture medium of Example 1.
  • the preparation method of the culture medium and other steps of culturing the strain are the same as those of Embodiment 1.
  • the composition of the slanted medium is: glucose 2%, peptone 0.2%, bran 0.4%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.03%, agar 2%, and the volume is adjusted to 1000 mL with natural pH.
  • composition and amount of the shake flask medium are the same as those of the slanted medium, except that there is no agar in the medium.
  • the medium composition of the seed tank, the breeding tank and the fermentation tank in the liquid fermentation medium are the same, including: 2% of soybean cake powder, 2% of glucose, 2% of sucrose, 0.2% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, and soybean oil 0.1%, water balance.
  • the Cs-4 strain was fermented and cultured in the same way, and the Cs-4 Cordyceps powder was finally obtained.
  • the slanted surface and shake flask culture medium of this embodiment are the same as those of Embodiment 1, and the culture medium of the seed tank, breeding tank and fermentation tank is different from that of Embodiment 1 in that it does not contain adenine.
  • the Cs-4 strain was fermented and cultured in the same manner, and the Cs-4 Cordyceps powder was finally obtained.
  • This comparative example differs from Example 1 in that the histidine content in the slanted and shake flask culture medium is 0.05%, the histidine in the seed tank culture medium is 0.06%, and the adenine is 0.02%; in the propagation tank culture medium Histidine 0.03%, glycine 0.06%, adenine 0.03%; histidine 0.04%, glycine 0.05%, and adenine 0.04% in the fermentation tank culture medium.
  • the Cs-4 strain was fermented and cultured in the same way, and the Cs-4 Cordyceps powder was finally obtained.
  • This comparative example differs from Example 4 in that adenine is not added to the liquid fermentation medium.
  • the content of saponin in Cordyceps powder prepared in Comparative Example 4 is lower than the content of saponin in Cordyceps powder prepared in Example 4, which indicates that the addition of adenine can also help the strain to use the Panax notoginseng powder, and can increase the saponin in the powder. content.

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Abstract

一种用于蝙蝠蛾拟青霉Cs-4的培养基及其制备方法，包括种子培养基和液体发酵培养基，种子培养基的组分有葡萄糖、蛋白胨、麸皮、无机盐和氨基酸，液体发酵培养基的组分有热榨豆饼粉、葡萄糖、蔗糖、无机盐、氨基酸、维生素B1，腺嘌呤和三七粉。该培养基的制备方法简单，适合工业化大生产。在不同阶段，使用不同培养基，有利于菌株的生长。培养基中添加氨基酸、维生素B1和腺嘌呤，Cs-4的菌丝体生长旺盛，制备得到的虫草菌粉中的腺苷、总氨基酸、麦角甾醇及甘露醇的含量高。三七粉加入培养基后，能被菌株利用，在菌粉中能检测到三七皂苷和人参皂苷，同时菌粉中原有活性成分的含量也提高，虫草菌粉的药用价值明显提高。

Description

用于蝙蝠蛾拟青霉Cs-4的培养基及其制备方法 技术领域
本发明属于微生物学领域，尤其涉及用于蝙蝠蛾拟青霉Cs-4的培养基及其制备方法。
背景技术
冬虫夏草是药用名，是动物和真菌类的综合体，为麦角菌科植物冬虫夏草菌的子座及其寄主蝙蝠蛾科昆虫虫萆蝙蝠蛾等的幼虫尸体的复合体。冬虫夏草由子座(即草部分)与菌核(即虫的尸体部分)两部分组成的复合体。冬季幼虫蛰居土里，菌类寄生其中，吸取营养，幼虫体内充满菌丝而死；到了夏季，自幼虫尸体之上生出幼苗，形似草，夏至前后采集而得。冬虫夏草主要活性成分是虫草素、虫草腺苷，具有免疫调节、抗肿瘤、抗疲劳等多种功效。现代研究发现，冬虫夏草含有蛋白质、脂肪、粗纤维、碳水化合物、多种维生素、虫草素、虫草酸、多种氨基酸及铁、磷、钙、锌、锰、硒等人体必需的元素。药理试验证实，冬虫夏草具有调节机体免疫力、增强身体素质、延缓衰老、增强耐缺氧能力、抗肾损伤、抗病原微生物、平喘祛痰、升高白细胞等作用。同时，虫草还有较好的镇静、镇痛作用，能够减轻肿瘤发作时的疼痛和化疗的不良反应。
随着人民生活水平的提高，对于大健康产业的关注度越来越高，对于冬虫夏草的药理功效也有了广泛的认知，对其需求量也达到供不应求的状态。作为冬虫夏草的替代品虫草菌粉也应运而生，虫草菌粉是以蝙蝠蛾拟青霉Paccilomyces hepiali Cs-4为菌种，经过培养基的扩大培养，再经深层发酵而得。虫草菌粉的质量主要取决于虫草菌丝体的质量，菌丝体是菌株扩大培养和发酵培养后的产物，培养基对Cs-4菌株的生长至关重要。
发明内容
本发明的目的在于提供一种用于蝙蝠蛾拟青霉Cs-4的培养基，为Cs-4菌株的生长提供丰富的营养，促进Cs-4虫草菌丝体的形成，提高菌丝体中腺苷和麦角甾醇等活性成分的含量。同时，培养基中添加三七粉，得到的Cs-4菌丝体不仅具备虫草原有的药用功效，还具备三七皂苷尤其是人参皂苷的药理用 途。
具体来说，针对现有技术的不足，本发明提供了如下技术方案：
一种用于蝙蝠蛾拟青霉Cs-4的培养基，包括种子培养基和液体发酵培养基，所述种子培养基包括：葡萄糖、蛋白胨、麸皮、无机盐和氨基酸，所述液体发酵培养基包括：热榨豆饼粉、葡萄糖、蔗糖、无机盐、氨基酸、维生素B 1和腺嘌呤。
种子培养基中添加氨基酸，促进菌株生长，保证了传代的稳定性。各级发酵培养基的组成略有不同，满足菌株在不同生长阶段的营养需求。培养基中添加腺嘌呤，发酵培养Cs-4菌株，制备得到的虫草菌粉中的腺苷含量明显提高，麦角甾醇、总氨基酸和甘露醇的含量也提高。
优选地，所述无机盐选自磷酸二氢钾、磷酸二氢钠、磷酸氢二钾、磷酸氢二钠、硫酸镁、氯化镁、硫酸锌、氯化锌、氯化钠和氯化钙中的一种或几种，氨基酸选自组氨酸、精氨酸、甘氨酸和L-谷氨酸中的一种或几种。
优选地，所述种子培养基包括斜面培养基和摇瓶培养基，其中，按重量体积g/mL计，斜面培养基包括：葡萄糖2-4％、蛋白胨0.2-0.4％、麸皮0.4-0.8％、磷酸二氢钾0.1-0.3％、硫酸镁0.03-0.05％、组氨酸0.1-0.3％、琼脂2-4％，水余量；摇瓶培养基包括：葡萄糖1-5％、蛋白胨0.2-0.4％、麸皮0.4-0.6％、鱼骨粉0.2-0.3％、磷酸二氢钾0.1-0.3％、硫酸镁0.03-0.05％、硫酸锌0.03-0.05％、组氨酸0.1-0.3％，水余量。
优选地，所述液体发酵培养基中还含有三七粉。添加三七粉后，发酵培养得到的虫草菌粉在具备原有虫草功效的同时，还具备三七皂苷、人参皂苷的功效。此外，发酵培养基中同时添加腺嘌呤和三七粉后，麦角甾醇的含量明显提高，三七皂苷的含量相比单独加三七粉、不加腺嘌呤时，也得到提高。
优选地，所述液体发酵培养基包括种子罐培养基、繁殖罐培养基和发酵罐培养基，其中，按重量体积g/mL计，种子罐培养基包括：热榨豆饼粉2-4％、葡萄糖2-4％、蔗糖2-4％、磷酸二氢钾0.2-0.4％、硫酸镁0.05-0.1％、硫酸锌0.05-0.1％、组氨酸0.1-0.2％、维生素B 1 0.02-0.3％、腺嘌呤0.05-0.2％和豆油0.1-0.2％，水余量。
优选地，所述液体发酵培养基包括种子罐培养基、繁殖罐培养基和发酵罐 培养基，其中，按重量体积g/mL计，繁殖罐培养基包括：热榨豆饼粉2-4％、葡萄糖2-4％、蔗糖2-4％、磷酸二氢钾0.1-0.2％、硫酸镁0.04-0.06％、硫酸锌0.03-0.05％、组氨酸0.1-0.2％、甘氨酸0.1-0.2％、维生素B 1 0.02-0.3％、腺嘌呤0.05-0.2％和豆油0.2-0.3％，水余量。
优选地，所述液体发酵培养基包括种子罐培养基、繁殖罐培养基和发酵罐培养基，其中，按重量体积g/mL计，发酵罐培养基包括：热榨豆饼粉3-4％、葡萄糖1-2％、蔗糖1-2％、磷酸二氢钾0.1-0.2％、硫酸镁0.04-0.06％、硫酸锌0.03-0.05％、组氨酸0.1-0.2％、甘氨酸0.1-0.2％、维生素B 1 0.02-0.3％、腺嘌呤0.1-0.2％和豆油0.2-0.3％，水余量。
优选地，所述液体发酵培养基中含有三七粉，其中，发酵罐培养基的组成为：热榨豆饼粉4％、葡萄糖2％、蔗糖1％、磷酸二氢钾0.1％、硫酸镁0.05％、硫酸锌0.03％、组氨酸0.1％、甘氨酸0.1％、维生素B 1 0.05％、腺嘌呤0.15％、豆油0.2％和三七粉1％，水余量。
本发明还提供了培养基的制备方法，其中，斜面培养基的制备方法包括以下步骤：
(1)称量麸皮，加水煮沸，过滤得到滤液；
(2)将蛋白胨加入滤液中，加热、搅拌，直至溶解；
(3)加入除琼脂外的其他培养基组分，加热和/或加水溶解后，加水定容；
(4)加热条件下，往步骤(3)配制好的溶液中加入琼脂；
(5)分装配制好的培养基，然后灭菌；
摇瓶培养基的制备方法与斜面培养基的制备方法的区别在于没有步骤(4)加入琼脂的过程；
液体发酵培养基的制备方法为量取50％的水，往其中加入各培养基组分，加热溶解后，加水定容，然后灭菌。
优选地，在称量麸皮之前，先将麸皮过40目筛，所述灭菌为高压蒸汽灭菌，压力为0.09-0.12MPa，温度为120±1℃，时间为30-40分钟。
与现有技术相比，本发明的效果和益处在于：
(1)种子培养基和发酵培养基的组成不同，试验证明斜面培养基适合 Cs-4菌株生长，保证了传代的稳定性，为之后的发酵培养提供优良种子。各级发酵培养基的组成也略有不同，保证了不同阶段菌株生长的营养供应，得到的菌丝体产量高，营养价值高。
(2)相比普通的豆饼粉，热榨豆饼粉中的脂质含量少，Cs-4菌株能够更好地利用热榨豆饼粉中的蛋白质，原料利用率高。液体发酵培养基中除基础营养外，还添加了氨基酸、维生素B 1和腺嘌呤，发酵培养的Cs-4菌株的菌丝体生长旺盛，收率比普通培养基培养的菌丝体收率高。由于腺嘌呤是腺苷生物合成的前体物质，因此利用添加腺嘌呤的培养基发酵得到的虫草菌丝体中的腺苷含量明显增加。检测结果显示，发酵培养得到的虫草菌丝体中的麦角甾醇、甘露醇和氨基酸总含量也提高。
(3)培养基中添加三七粉后，Cs-4菌株生长过程中对三七粉中的三七总皂苷进行了生物转化，发酵结束后，在发酵产物中能检测到单体皂苷。因此，Cs-4菌丝体不仅具备虫草原有的药用功效，还具备单体皂苷尤其是人参皂苷的药理用途。发酵培养基中同时添加腺嘌呤和三七粉后，虫草菌粉中的皂苷含量相比单独添加三七粉更高，麦角甾醇含量相比单独添加腺嘌呤的更高。
具体实施方式
本发明提供了用于蝙蝠蛾拟青霉Cs-4的培养基，包括种子培养基和液体发酵培养基，其中，种子培养基又分为斜面培养基和摇瓶培养基，液体发酵培养基分为种子罐培养基、繁殖罐培养基和发酵罐培养基。
按重量体积g/mL计，斜面培养基包括：葡萄糖2-4％、蛋白胨0.2-0.4％、麸皮0.4-0.8％、磷酸二氢钾0.1-0.3％、硫酸镁0.03-0.05％、组氨酸0.1-0.3％、琼脂2-4％，加水定容，pH自然。
按重量体积g/mL计，摇瓶培养基包括：葡萄糖1-5％、蛋白胨0.2-0.4％、麸皮0.4-0.6％、鱼骨粉0.2-0.3％、磷酸二氢钾0.1-0.3％、硫酸镁0.03-0.05％、硫酸锌0.03-0.05％、组氨酸0.1-0.3％，加水定容，pH自然。
按重量体积g/mL计，种子罐培养基包括：热榨豆饼粉2-4％、葡萄糖2-4％、蔗糖2-4％、磷酸二氢钾0.2-0.4％、硫酸镁0.05-0.1％、硫酸锌0.05-0.1％、组氨酸0.1-0.2％、维生素B 1 0.02-0.3％、腺嘌呤0.05-0.2％和豆油0.1-0.2％，加水定容，pH自然。
按重量体积g/mL计，繁殖罐培养基包括：热榨豆饼粉2-4％、葡萄糖2-4％、蔗糖2-4％、磷酸二氢钾0.1-0.2％、硫酸镁0.04-0.06％、硫酸锌0.03-0.05％、组氨酸0.1-0.2％、甘氨酸0.1-0.2％、维生素B 1 0.02-0.3％、腺嘌呤0.05-0.2％和豆油0.2-0.3％，加水定容，pH自然。
按重量体积g/mL计，发酵罐培养基包括：热榨豆饼粉3-4％、葡萄糖1-2％、蔗糖1-2％、磷酸二氢钾0.1-0.2％、硫酸镁0.04-0.06％、硫酸锌0.03-0.05％、组氨酸0.1-0.2％、甘氨酸0.1-0.2％、维生素B 1 0.02-0.3％、腺嘌呤0.1-0.2％和豆油0.2-0.3％，加水定容，pH自然。
在一优选实施方式中，液体发酵培养基中还含有三七粉，其用量为0.5-1％。
本发明所述种子培养基配制的一般方法为：麸皮加水煮沸，过滤得到滤液；加热条件下，先加入蛋白胨，然后依次加入其它组分，加热或加水溶解后，加水定容，最后灭菌。液体发酵培养基的制备方法为量取50％的水，往其中加入各培养基组分，加热溶解后，加水定容，然后灭菌。
麸皮过40目筛后，称量，然后按照每100mL水中加6g麸皮进行投料，加热至沸腾，维持沸腾30-40分钟，然后过滤取滤液。滤液中含有多种微量元素和维生素，结合种子培养基中的氨基酸，菌株生长速度快，传代稳定。
下面通过具体的实施例对本发明进行进一步介绍。
需说明的是，本发明涉及的材料和设备均为市售，蝙蝠蛾拟青霉Cs-4菌株于1997年10月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC NO.0327。菌株Cs-4分离自青海新鲜冬虫夏草，也可以通过商业途径获取。
实施例1
斜面培养基的组成为：葡萄糖2％、蛋白胨0.2％、麸皮0.4％、磷酸二氢钾0.1％、硫酸镁0.03％、组氨酸0.1％、琼脂2％，加水定容至1000mL，pH自然。
制备方法为：按计算量称量过40目筛的麸皮，加入相应体积的水(按照每100mL水中加6g麸皮进行投料计算)，加热至沸腾，维持沸腾30分钟，过滤得滤液。加入蛋白胨，加热、搅拌使蛋白胨溶解，然后加入除琼脂外的其它组分，加热并补充水使组分全部溶解，加水定容。加热条件下，加入琼脂，琼脂融化，然后将配制好的培养基分装入茄形瓶内，塞上棉塞，包扎好，放入灭菌柜内高 压蒸汽灭菌，蒸汽压力0.09Mpa，温度120℃，灭菌时间30分钟，冷却后按要求制成斜面。将Cs-4菌株接种于斜面，在15℃培养7天。菌株在斜面生长旺盛，孢子多。
摇瓶培养基的组成为：葡萄糖1％、蛋白胨0.2％、麸皮0.4％、鱼骨粉0.2％、磷酸二氢钾0.1％、硫酸镁0.03％、硫酸锌0.03％、组氨酸0.1％，水余量。
摇瓶培养基的制备方法与斜面培养基制备方法的区别在于不用加入琼脂，加水定容后，分装入750mL的锥形瓶内，按照同样的条件进行灭菌。无菌条件下，用接种铲子取1cm 2斜面培养菌落接种至摇瓶培养基内，置于摇床上，15℃培养7天。检测合格后，将分装的各摇瓶培养后的菌液合并至接种钢瓶，然后接入种子罐培养。
种子罐培养基的组成为：热榨豆饼粉2％、葡萄糖2％、蔗糖2％、磷酸二氢钾0.2％、硫酸镁0.05％、硫酸锌0.05％、组氨酸0.1％、维生素B 1 0.02％、腺嘌呤0.05％，豆油0.1％，水余量。制备方法为先量取50％水，加入各培养基组分，加热溶解后，加水定容，pH自然。配制好的培养基放入灭菌柜内高压蒸汽灭菌，蒸汽压力0.10Mpa，温度121℃，灭菌时间35分钟。种子罐培养4天后，接入繁殖罐培养。
繁殖罐培养基的组成为：热榨豆饼粉2％、葡萄糖2％、蔗糖2％、磷酸二氢钾0.1％、硫酸镁0.04％、硫酸锌0.03％、组氨酸0.1％、甘氨酸0.1％、维生素B 10.02％、腺嘌呤0.05％，豆油0.2％，水余量。培养基配制方法和灭菌条件与种子罐培养基相同。繁殖罐培养5天后，接入发酵罐培养。
发酵罐培养基的组成为：热榨豆饼粉3％、葡萄糖1％、蔗糖1％、磷酸二氢钾0.1％、硫酸镁0.04％、硫酸锌0.03％、组氨酸0.1％、甘氨酸0.1％、维生素B 10.02％、腺嘌呤0.1％，豆油0.2％，水余量。培养基配制方法和灭菌条件与种子罐培养基相同。发酵罐培养4天后出罐，发酵液固液分离，将固体烘干、粉碎得到Cs-4虫草菌粉。
实施例2
斜面培养基的组成为：葡萄糖3％、蛋白胨0.3％、麸皮0.6％、磷酸二氢钾0.2％、硫酸镁0.04％、组氨酸0.2％、琼脂3％，加水定容至1000mL，pH自然。
摇瓶培养基的组成为：葡萄糖3％、蛋白胨0.3％、麸皮0.5％、鱼骨粉0.2％、 磷酸二氢钾0.2％、硫酸镁0.04％、硫酸锌0.04％、组氨酸0.2％，水余量。
斜面和摇瓶培养基的配制方法、配制总量以及接种和培养等其他条件均与实施例1相同。但是，麸皮沸煮的时间为40分钟。
种子罐培养基的组成为：热榨豆饼粉3％、葡萄糖3％、蔗糖3％、磷酸二氢钾0.3％、硫酸镁0.08％、硫酸锌0.06％、组氨酸0.15％、维生素B 1 0.15％、腺嘌呤0.1％，豆油0.1％，水余量。
繁殖罐培养基的组成为：热榨豆饼粉3％、葡萄糖3％、蔗糖3％、磷酸二氢钾0.15％、硫酸镁0.05％、硫酸锌0.04％、组氨酸0.1％、甘氨酸0.1％、维生素B 10.15％、腺嘌呤0.1％，豆油0.25％，水余量。
发酵罐培养基的组成为：热榨豆饼粉3％、葡萄糖1％、蔗糖1％、磷酸二氢钾0.1％、硫酸镁0.05％、硫酸锌0.04％、组氨酸0.15％、甘氨酸0.1％、维生素B 10.2％、腺嘌呤0.15％，豆油0.25％，水余量。
种子罐、繁殖罐和发酵罐培养基的配制方法及配制总量与实施例1相同，培养基的灭菌压力为0.12MPa，温度为119℃，灭菌40分钟。接种方法、发酵培养条件以及出罐后的处理方法均与实施例1相同，最终得到Cs-4虫草菌粉。
实施例3
斜面培养基的组成为：葡萄糖4％、蛋白胨0.4％、麸皮0.8％、磷酸二氢钾0.3％、硫酸镁0.05％、组氨酸0.3％、琼脂4％，加水定容至1000mL，pH自然。
摇瓶培养基的组成为：葡萄糖5％、蛋白胨0.4％、麸皮0.6％、鱼骨粉0.3％、磷酸二氢钾0.3％、硫酸镁0.05％、硫酸锌0.05％、组氨酸0.3％，水余量。
斜面和摇瓶培养基的配制方法、配制总量以及接种和培养等其他条件均与实施例1相同。
种子罐培养基的组成为：热榨豆饼粉4％、葡萄糖4％、蔗糖4％、磷酸二氢钾0.4％、硫酸镁0.1％、硫酸锌0.1％、组氨酸0.2％、维生素B 1 0.3％、腺嘌呤0.2％，豆油0.2％，水余量。
繁殖罐培养基的组成为：热榨豆饼粉4％、葡萄糖4％、蔗糖4％、磷酸二氢钾0.2％、硫酸镁0.06％、硫酸锌0.05％、组氨酸0.2％、甘氨酸0.2％、维生素B 10.3％、腺嘌呤0.2％，豆油0.3％，水余量。
发酵罐培养基的组成为：热榨豆饼粉4％、葡萄糖2％、蔗糖2％、磷酸二氢 钾0.2％、硫酸镁0.06％、硫酸锌0.05％、组氨酸0.2％、甘氨酸0.2％、维生素B 10.3％、腺嘌呤0.2％，豆油0.3％，水余量。
种子罐、繁殖罐和发酵罐培养基的配制方法及配制总量与实施例1相同，接种方法、发酵培养条件以及出罐后的处理方法均与实施例1相同，最终得到Cs-4虫草菌粉。
实施例4
本实施例与实施例1的区别在于：种子罐培养基中还含有三七粉0.6％，繁殖罐培养基中还含有三七粉0.8％，发酵罐培养基的组成为含有热榨豆饼粉4％、葡萄糖2％、蔗糖1％、磷酸二氢钾0.1％、硫酸镁0.05％、硫酸锌0.03％、组氨酸0.1％、甘氨酸0.1％、维生素B 1 0.05％、腺嘌呤0.15％、豆油0.2％和三七粉1％，水余量。按照相同的方法发酵培养Cs-4菌株，最终得到Cs-4虫草菌粉。
实施例5
本实施例与实施例2的区别仅在于，种子罐培养基中还含有三七粉0.5％，繁殖罐培养基中还含有三七粉0.8％，发酵罐培养基中还含有三七粉1％。按照相同的方法发酵培养Cs-4菌株，最终得到Cs-4虫草菌粉。
对比例1
(斜面培养基不含氨基酸，液体发酵培养基不含氨基酸、VB1和腺嘌呤)
本对比例与实施例1的培养基组成不同，培养基的制备方法及菌株培养等其他步骤均与实施例1相同。
斜面培养基的组成为：葡萄糖2％、蛋白胨0.2％、麸皮0.4％、磷酸二氢钾0.1％、硫酸镁0.03％、琼脂2％，加水定容至1000mL，pH自然。
摇瓶培养基的组分及用量与斜面培养基相同，只是培养基中没有琼脂。
液体发酵培养基中的种子罐、繁殖罐和发酵罐内的培养基组成相同，均包括：豆饼粉2％、葡萄糖2％、蔗糖2％、磷酸二氢钾0.2％、硫酸镁0.05％、豆油0.1％，水余量。按照相同的方法发酵培养Cs-4菌株，最终得到Cs-4虫草菌粉。
对比例2
(液体发酵培养基中不含腺嘌呤)
本实施例的斜面和摇瓶培养基与实施例1相同，种子罐、繁殖罐和发酵罐培养基与实施例1的区别在于不含有腺嘌呤。按照相同的方法发酵培养Cs-4菌 株，最终得到Cs-4虫草菌粉。
对比例3
(培养基中氨基酸和腺嘌呤的含量过低)
本对比例与实施例1的区别在于：斜面和摇瓶培养基中的组氨酸含量为0.05％，种子罐培养基中的组氨酸为0.06％，腺嘌呤0.02％；繁殖罐培养基中组氨酸0.03％，甘氨酸0.06％，腺嘌呤0.03％；发酵罐培养基中组氨酸0.04％，甘氨酸0.05％，腺嘌呤0.04％。按照相同的方法发酵培养Cs-4菌株，最终得到Cs-4虫草菌粉。
对比例4
(液体发酵培养基中不加腺嘌呤，含三七粉)
本对比例与实施例4的区别在于，液体发酵培养基中不添加腺嘌呤。
实施例6
从实施例1-5和对比例1-4制备得到的Cs-4虫草菌粉取样，测定其中的总氨基酸、甘露醇、腺苷、麦角甾醇和皂苷的含量。检测结果见表1。
从表1可以看到，实施例1-3发酵得到的虫草菌粉中的总氨基酸、甘露醇、腺苷和麦角甾醇含量高，尤其腺苷含量相比使用常规培养基的对比例1制备的虫草菌粉高很多。实施例4和5的液体发酵培养基中添加三七粉后，虫草菌粉中的总氨基酸、甘露醇、腺苷和麦角甾醇含量依旧很高，尤其麦角甾醇的含量相比实施例1-3也提高了很多，此外，虫草菌粉中还能检测到三七皂苷和人参皂苷。对比例2使用的液体发酵培养基中没有添加腺嘌呤，但是各种培养基中都添加了氨基酸，检测结果表明，相比实施例1-3，制得的虫草菌粉中仅腺苷含量明显下降，其他成分含量比实施例1略低。对比例3的检测数据表明，培养基中的氨基酸和腺嘌呤需要达到一定量，才能明显提高虫草菌粉的药用价值。对比例4制备的虫草菌粉中皂苷的含量低于实施例4制备的虫草菌粉中的皂苷含量，说明添加腺嘌呤还有助于菌株对三七粉的利用，能提高菌粉中的皂苷含量。
Figure PCTCN2018119031-appb-000001
表1.Cs-4虫草菌粉中的总氨基酸、甘露醇、腺苷、麦角甾醇和皂苷含量
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。

Claims (10)

  1. 用于蝙蝠蛾拟青霉Cs-4的培养基，包括种子培养基和液体发酵培养基，其特征在于，所述种子培养基包括：葡萄糖、蛋白胨、麸皮、无机盐和氨基酸，所述液体发酵培养基包括：热榨豆饼粉、葡萄糖、蔗糖、无机盐、氨基酸、维生素B 1和腺嘌呤。
  2. 根据权利要求1所述的培养基，其特征在于，所述无机盐选自磷酸二氢钾、磷酸二氢钠、磷酸氢二钾、磷酸氢二钠、硫酸镁、氯化镁、硫酸锌、氯化锌、氯化钠和氯化钙中的一种或几种，氨基酸选自组氨酸、精氨酸、甘氨酸和L-谷氨酸中的一种或几种。
  3. 根据权利要求2所述的培养基，其特征在于，所述种子培养基包括斜面培养基和摇瓶培养基，其中，按重量体积g/mL计，斜面培养基包括：葡萄糖2-4％、蛋白胨0.2-0.4％、麸皮0.4-0.8％、磷酸二氢钾0.1-0.3％、硫酸镁0.03-0.05％、组氨酸0.1-0.3％、琼脂2-4％，水余量；摇瓶培养基包括：葡萄糖1-5％、蛋白胨0.2-0.4％、麸皮0.4-0.6％、鱼骨粉0.2-0.3％、磷酸二氢钾0.1-0.3％、硫酸镁0.03-0.05％、硫酸锌0.03-0.05％、组氨酸0.1-0.3％，水余量。
  4. 根据权利要求1-3任一项所述的培养基，其特征在于，所述液体发酵培养基中还含有三七粉。
  5. 根据权利要求1-3任一项所述的培养基，其特征在于，所述液体发酵培养基包括种子罐培养基、繁殖罐培养基和发酵罐培养基，其中，按重量体积g/mL计，种子罐培养基包括：热榨豆饼粉2-4％、葡萄糖2-4％、蔗糖2-4％、磷酸二氢钾0.2-0.4％、硫酸镁0.05-0.1％、硫酸锌0.05-0.1％、组氨酸0.1-0.2％、维生素B 1 0.02-0.3％、腺嘌呤0.05-0.2％和豆油0.1-0.2％，水余量。
  6. 根据权利要求1-3任一项所述的培养基，其特征在于，所述液体发酵培养基包括种子罐培养基、繁殖罐培养基和发酵罐培养基，其中，按重量体积g/mL计，繁殖罐培养基包括：热榨豆饼粉2-4％、葡萄糖2-4％、蔗糖2-4％、磷酸二氢钾0.1-0.2％、硫酸镁0.04-0.06％、硫酸锌0.03-0.05％、组氨酸0.1-0.2％、甘氨酸0.1-0.2％、维生素B 1 0.02-0.3％、腺嘌呤0.05-0.2％和豆油0.2-0.3％，水余量。
  7. 根据权利要求1-3任一项所述的培养基，其特征在于，所述液体发酵培养基包括种子罐培养基、繁殖罐培养基和发酵罐培养基，其中，按重量体积 g/mL计，发酵罐培养基包括：热榨豆饼粉3-4％、葡萄糖1-2％、蔗糖1-2％、磷酸二氢钾0.1-0.2％、硫酸镁0.04-0.06％、硫酸锌0.03-0.05％、组氨酸0.1-0.2％、甘氨酸0.1-0.2％、维生素B 1 0.02-0.3％、腺嘌呤0.1-0.2％和豆油0.2-0.3％，水余量。
  8. 根据权利要求7所述的培养基，其特征在于，所述液体发酵培养基中含有三七粉，其中，发酵罐培养基的组成为：热榨豆饼粉4％、葡萄糖2％、蔗糖1％、磷酸二氢钾0.1％、硫酸镁0.05％、硫酸锌0.03％、组氨酸0.1％、甘氨酸0.1％、维生素B 1 0.05％、腺嘌呤0.15％、豆油0.2％和三七粉1％，水余量。
  9. 权利要求3所述培养基的制备方法，其中，斜面培养基的制备方法包括以下步骤：
    (1)称量麸皮，加水煮沸，过滤得到滤液；
    (2)将蛋白胨加入滤液中，加热、搅拌，直至溶解；
    (3)加入除琼脂外的其他培养基组分，加热和/或加水溶解后，加水定容；
    (4)加热条件下，往步骤(3)配制好的溶液中加入琼脂；
    (5)分装配制好的培养基，然后灭菌；
    摇瓶培养基的制备方法与斜面培养基的制备方法的区别在于没有步骤(4)加入琼脂的过程；
    液体发酵培养基的制备方法为量取50％的水，往其中加入各培养基组分，加热溶解后，加水定容，然后灭菌。
  10. 根据权利要求9所述的培养基制备方法，其特征在于，在称量麸皮之前，先将麸皮过40目筛，所述灭菌为高压蒸汽灭菌，压力为0.09-0.12MPa，温度为120±1℃，时间为30-40分钟。
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CN105779299A (zh) * 2016-01-07 2016-07-20 江苏苏中药业集团股份有限公司 一株高产腺苷及甘露醇类物质的蝙蝠蛾拟青霉菌株及应用
CN106520562A (zh) * 2016-10-14 2017-03-22 锬酃藏虫草生物科技(深圳)有限公司 一种利用参类提取物培养冬虫夏草菌的方法及其应用
CN108504621A (zh) * 2018-05-25 2018-09-07 江西国药有限责任公司 用于蝙蝠蛾拟青霉Cs-4的培养基及其制备方法
CN108676730A (zh) * 2018-05-25 2018-10-19 江西国药有限责任公司 一种蝙蝠蛾拟青霉Cs-4的发酵生产工艺

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CN117467725A (zh) * 2023-12-28 2024-01-30 翔宇药业股份有限公司 一种高核苷含量的蝙蝠蛾拟青霉菌丝体粉发酵生产工艺
CN117467725B (zh) * 2023-12-28 2024-03-22 翔宇药业股份有限公司 一种高核苷含量的蝙蝠蛾拟青霉菌丝体粉发酵生产工艺

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