WO2015180520A1 - 一种高产虫草酸蛹虫草的培养方法 - Google Patents

一种高产虫草酸蛹虫草的培养方法 Download PDF

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WO2015180520A1
WO2015180520A1 PCT/CN2015/073782 CN2015073782W WO2015180520A1 WO 2015180520 A1 WO2015180520 A1 WO 2015180520A1 CN 2015073782 W CN2015073782 W CN 2015073782W WO 2015180520 A1 WO2015180520 A1 WO 2015180520A1
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culture
parts
cordyceps militaris
fruit body
cultivating
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熊艳
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熊艳
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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
    • C12N1/14—Fungi; Culture media therefor
    • 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
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to the field of edible fungi cultivation, and in particular to a method for cultivating a high-yield Cordyceps militaris.
  • Cordyceps militaris also known as Cordyceps militaris and Cordyceps militaris, is a worm-like complex formed by fungi that is parasitic on insects such as Lepidoptera. It is an important nutrient for nourishing action.
  • Cordyceps militaris has many anti-tumor and anti-inflammatory effects. It has been widely used as a tonic and medicinal fungus in East Asia. The Ministry of Health officially listed it as a new resource food on March 16, 2009.
  • Cordycepic acid is one of the main active ingredients of Cordyceps militaris.
  • the content of Cordycepic acid is one of the main criteria for measuring the quality of Cordyceps. It is generally considered that Cordyceps sinensis has a high medicinal value.
  • Cordycepic acid can prevent and treat cerebral thrombosis, cerebral hemorrhage, myocardial infarction, long-term failure; anti-liver tissue fibrosis, anti-lipid peroxidation, and enhance the detoxification of liver by enhancing the immune function of Cordyceps, thereby effectively protecting liver cells .
  • the content of oxalic acid in Cordyceps militaris is mainly concentrated by deep fermentation of liquid.
  • the mycelium of Cordyceps militaris obtained by deep fermentation has a content of 3-4.5%, and the fermentation is obtained.
  • the culture includes not only Cordyceps itself, but also some media residues that affect the purity of Cordyceps acid.
  • the object of the present invention is to provide a method for cultivating Cordyceps militaris seed culture medium, Cordyceps militaris growth medium and a highly productive Cordyceous Cordyceps militaris to solve the above problems.
  • a Cordyceps militaris seed culture medium which comprises the following components by weight: 8-12 parts of glucose, 2-4 parts of peptone, 4-6 parts of yeast powder, 0.02- 0.06 parts of potassium dihydrogen phosphate, 0.02-0.06 parts of magnesium sulfate, 4-6 parts of sodium chloride, 0.05-0.15 parts of vitamin B1, 8-12 parts of sorghum powder, and 800-1200 parts of water.
  • a Cordyceps militaris growth medium comprising, by weight, 24-26 parts of sorghum powder, 4-6 parts of ginkgo leaf powder, 0.04-0.06 peptone, 0.04-0.08 parts of mannose, 0.02-0.04 parts of potassium dihydrogen phosphate, 0.02-0.03 parts of magnesium sulfate, 0.04-0.06 parts of glucose, 0.0005-0.001 parts of vitamin B1, 30-40 parts of water.
  • a method for cultivating a high-yield Cordyceps militaris comprises the following steps:
  • the culture conditions are: 25-30 ° C, shaking culture at 240-260 rpm / min.
  • the seed liquid is obtained by culturing for 3-5 days.
  • the seed liquid is diluted 20-30 times, it is inoculated at a volume percentage of 5-8%.
  • the mycelial growth culture is cultured in a sterilized culture under the following conditions: a temperature of 20-25 ° C, a light of 2-4 h per day, and an illumination intensity of 250-300 Lux. Humidity 60-70%, culture 5-7d.
  • the culture condition of the hyphae color culture is: temperature 14-18 ° C, humidity 70-80%, ventilation 1.5-2.5 h per day, illumination 8-12 h, light intensity For 250-300 Lux, culture for 3-5d.
  • the culture condition of the fruit body culture is: gas permeable culture, temperature is 20 ° C ⁇ 2 ° C, humidity is 80-90%, ventilation is 1.5-2.5 h per day, blue light illumination 12 -14h, the light intensity is 250-300 Lux, and the culture is carried out until the fruit body length is 7-8 cm, and the Cordyceps militaris fruit body is harvested.
  • the disinfection is: the container containing the peracetic acid solution is placed in a water bath at 75-85 ° C for fumigation to disinfect the culture chamber, the fumigation time is 90-100 min, the peroxyacetic acid per cubic meter space
  • the dosage is 3-3.2 g; during the sterilization period, the room temperature is not lower than 20 ° C and the humidity is 60% - 80%.
  • the method for cultivating the high-yield Cordyceps militaris provided by the embodiment of the present invention the spore suspension is inoculated into the seed culture medium of the Cordyceps militaris provided in the present invention to obtain a seed liquid; and the seed liquid is inoculated to the growth of the Cordyceps militaris provided by the present invention.
  • the mycelial growth culture, the hyphal color culture and the fruit body culture are sequentially carried out in the culture medium, the culture method is simple, the culture period is short, the obtained Cordyceps militaris fruit liquid has high oxalic acid content, and the cordycepic acid extracted by the Cordyceps militaris fruit body is used. High purity.
  • a Cordyceps militaris seed culture medium which comprises the following components by weight: 8-12 parts of glucose, 2-4 parts of peptone, 4-6 parts of yeast powder, 0.02- 0.06 parts of potassium dihydrogen phosphate, 0.02-0.06 parts of magnesium sulfate, 4-6 parts of sodium chloride, 0.05-0.15 parts of vitamin B1, 8-12 parts of sorghum powder, and 800-1200 parts of water.
  • the following components are included in parts by weight: 9-11 parts of glucose, 3-4 parts of peptone, 4-5 parts of yeast powder, 0.04-0.05 parts of potassium dihydrogen phosphate, 0.04-0.05 parts of sulfuric acid Magnesium, 4-5 parts of sodium chloride, 0.10-0.15 parts of vitamin B1, 9-10 parts of sorghum powder, and 1000 parts of water.
  • the obtained seed culture medium of Cordyceps militaris is comprehensive in nutrition, and the concentration and ratio of the medium are appropriate; the spore suspension of Cordyceps militaris is inoculated into the culture medium, and some spores grow into mycelium, the spores have strong vitality, and the spore number is expanded rapidly. And a part of the mycelium is obtained, and the obtained seed liquid contains spores and mycelium, which is favorable for subsequent growth.
  • a Cordyceps militaris growth medium comprising, by weight, 24-26 parts of sorghum powder, 4-6 parts of ginkgo leaf powder, 0.04-0.06 peptone, 0.04-0.08 parts of mannose, 0.02-0.04 parts of potassium dihydrogen phosphate, 0.02-0.03 parts of magnesium sulfate, 0.04-0.06 parts of glucose, 0.0005-0.001 parts of vitamin B1, 30-40 parts of water.
  • sorghum powder 25-26 parts of sorghum powder, 4-5 parts of ginkgo leaf powder, 0.04-0.05 peptone, 0.06-0.07 parts of mannose, 0.03-0.04 parts of phosphoric acid Potassium hydrogen, 0.02-0.03 parts of magnesium sulfate, 0.04-0.05 parts of glucose, 0.0005-0.001 parts of vitamin B1, 30-40 parts of water.
  • the obtained growth medium of Cordyceps militaris is comprehensive in nutrition, and the concentration and ratio of the medium are suitable; the seed liquid of Cordyceps militaris is inoculated into the medium to facilitate the growth of spores into mycelium, and more mycelium is obtained, which is beneficial to obtain more
  • the sorghum powder involved in the seed culture medium of Cordyceps militaris and the growth medium of Cordyceps militaris is obtained by passing through a 20-30 mesh sieve, and the ginkgo leaf powder is pulverized into a flaky size. 0.5-1.5 mm x 0.5-1.5 mm.
  • the use of the sorghum powder and the ginkgo leaf powder of this particle size can be evenly distributed during the sterilization process, and the components in the medium obtained after sterilization are uniformly distributed.
  • the ingredients contained in the Cordyceps militaris seed culture medium and the Cordyceps militaris growth medium were weighed and mixed, and were all autoclaved at a temperature of 121 ° C, sterilized for 20-30 min, and cooled to room temperature after sterilization. The medium used to culture the strain.
  • a method for cultivating a high-yield Cordyceps militaris comprises the following steps:
  • the Cordyceps militaris seed culture medium and the Cordyceps militaris growth medium provided by the present invention are used for the cultivation of Cordyceps militaris, the culture method is simple, the culture period is short, the cost is low, and the obtained Cordyceps militaris fruit body oxalic acid content is high; in addition, in the prior art
  • the Cordyceps militaris is cultured by liquid fermentation, and most of the Cordyceps militaris is mixed in the fermentation broth, and the secretions produced by itself are mixed in the fermentation broth, and thus the obtained Cordyceps militaris contains the components of the fermentation broth, and the growth and cultivation of the Cordyceps militaris provided by the present invention.
  • the base is a solid medium
  • the solid Cordyceps militaris is used to extract the Cordyceps militaris fruit body, thereby avoiding the defects in the culture medium when the impurities in the medium are mixed into the Cordyceps militaris fruit body, and the obtained Cordyceps militaris has less impurities, so The purity of the cordycepic acid extracted by the subsequent Cordyceps militaris is higher.
  • the Cordyceps militaris strain was activated and cultured.
  • the Cordyceps militaris strain was purchased from the China Center for Type Culture Collection under the accession number CCTCCM2013056.
  • the Cordyceps militaris strain was inoculated into potato slant medium and cultured at 27 °C ⁇ 2 °C for 6-8 days.
  • the spores of Cordyceps militaris were washed with sterile water containing 0.05% Tween 80 to prepare a spore suspension having a concentration of 1-3 ⁇ 10 7 CFU/ml.
  • the potato slant medium component comprises: 100 g of potato, 10 g of glucose, 1 g of potassium dihydrogen phosphate, 16 g of agar, 1000 ml of water; the preparation method is: taking 100 g of peeled potato, cutting into small pieces, adding water 1000m1, boil for 20min, filter with 4-6 layers of gauze, then make up the water to 1000m1, add 16g of agar to dissolve, then add 10g of glucose, 1g of potassium dihydrogen phosphate, dispense test tubes, autoclave at 121 °C 25 Potato slant medium was prepared at -30 min. The use of potato slant medium to activate Cordyceps militaris strains is simple and easy, and many spores are obtained.
  • Spores were prepared into a spore suspension with a concentration of 1-3 ⁇ 10 7 CFU/ml using sterile water of 0.05% Tween 80. The spore suspension spores were evenly distributed; then 3-8% by volume. The ratio was inoculated to the seed culture medium of Cordyceps militaris, and the inoculum amount was moderate, which facilitated the rapid growth of the seed culture medium of Cordyceps militaris.
  • the culture conditions are: 25-30 ° C, shaking culture at 240-260 rpm / min. It has been verified that the culture temperature is 25-30 °C, and the growth of Cordyceps militaris in the seed culture medium of Cordyceps militaris is rapid; the rotation speed is 240-260 rpm/min, and the amount of dissolved oxygen is increased without harming the spores of Cordyceps militaris, which is beneficial to spores and hyphae.
  • the growth of the body can also mix the Cordyceps militaris seed culture medium to prevent the growth of spores and mycelia by the nutrient components of the local Cordyceps militaris seed culture medium. Therefore, the spore growth of Cordyceps militaris under this culture condition is fast. The formation of mycelium is more, saving cultivation time.
  • the seed liquid is obtained by culturing for 3-5 days. After culturing for 3-5 days, the obtained seed liquid has a high bacterial content and is vigorously grown.
  • the above seed liquid is diluted 20-30 times, it is inoculated at a volume percentage of 5-8%. Inoculate an appropriate amount of seed liquid in the growth medium of Cordyceps militaris to obtain the fruit body of Cordyceps militaris; in order to prevent the difference in inoculum size and the uneven distribution after inoculation, the seed liquid is first diluted, After inoculation with 5-8% by volume, a growth medium of Cordyceps militaris with uniform distribution of seed liquid can be obtained, which is beneficial to the growth of the cells.
  • the hyphal growth culture is cultured in the sterilized culture
  • the culture condition is: temperature 20-25 ° C, day light 2-4 h, light intensity is 250-300 Lux, humidity 60 -70%, cultured for 5-7 days.
  • the growth of the cells is rapid, the culture vessel is full of hyphae, and the culture period is short.
  • the culture condition of the hyphae color culture is: temperature 14-18 ° C, humidity 70-80%, ventilation 1.5-2.5 h per day, illumination 8-12 h, light intensity 250 -300 Lux, cultured for 3-5 days.
  • the cells grow rapidly, and a millet-like primordium is formed on the surface of the culture medium, and the mycelium has a good color conversion effect and a short culture period.
  • the culture condition of the above-mentioned fruit body culture is: gas permeable culture, temperature is 20 ° C ⁇ 2 ° C, humidity is 80-90%, ventilation is 1.5-2.5 h per day, and blue light illumination is 12-14 h.
  • the light intensity is 250-300 Lux, and the length of the fruit body is 7-8 cm, and the above-mentioned Cordyceps militaris fruit body is harvested.
  • Breathing culture 35-45ml of Cordyceps militaris growth medium is placed in a triangular culture flask (capacity: 200-250ml), the mouth is sealed with a sealing film, and then cultured, by 3-5 holes on the sealing film, Breathable culture to facilitate air circulation, which is conducive to the growth of culture. Moreover, under the culture condition, the growth of the cells is rapid, the culture period is short, and the obtained Cordyceps militaris has a high content of oxalic acid.
  • the Cordyceps militaris obtained by solid culture can be directly cut, and the impurities in the culture medium are prevented from being mixed into the obtained Cordyceps militaris fruit body, and the obtained Cordyceps militaris has less impurities. Therefore, the purity of the cordycepic acid extracted by the subsequent Cordyceps militaris is higher.
  • the disinfection is: the container containing the peracetic acid solution is placed in a water bath at 75-85 ° C for fumigation to disinfect the above culture chamber, the fumigation time is 90-100 min, and the peroxyacetic acid per cubic meter space is 3 -3.2g.
  • the space in which the Cordyceps militaris grows is disinfected to prevent it from being infected and to prevent contamination by other microorganisms; the peracetic acid is used to sterilize the sterilizing and disinfection of the culture, and the culture of the Cordyceps militaris itself is harmless.
  • the Cordyceps militaris culture grows well, and the obtained Cordyceps militaris fruit body is non-polluting and the obtained Cordyceps militaris fruit body has high oxalic acid content.
  • the method for cultivating the high-yield Cordyceps militaris provided by the embodiment of the present invention the spore suspension is inoculated into the seed culture medium of the Cordyceps militaris provided in the present invention to obtain a seed liquid; and the seed liquid is inoculated to the growth of the Cordyceps militaris provided by the present invention.
  • the mycelial growth culture, the hyphal color culture and the fruit body culture are sequentially carried out in the culture medium, the culture method is simple, the culture period is short, the obtained Cordyceps militaris fruit liquid has high oxalic acid content, and the cordycepic acid extracted by the Cordyceps militaris fruit body is used. High purity.
  • the following ingredients were weighed by weight: 8 parts of glucose, 2 parts of peptone, 4 parts of yeast powder, 0.02 parts of potassium dihydrogen phosphate, 0.02 parts of magnesium sulfate, 4 parts of sodium chloride, 0.05 parts Vitamin B1, 8 parts of sorghum powder, 800 parts of water, then sterilized at 121 ° C for 25 min, sterilized and cooled to room temperature to obtain Cordyceps militaris seed culture medium;
  • the following ingredients were weighed by weight: 24 parts of sorghum powder, 4 parts of ginkgo leaf powder, 0.04 peptone, 0.04 parts of mannose, 0.02 parts of potassium dihydrogen phosphate, 0.02 parts of magnesium sulfate, 0.04 parts Glucose, 0.0005 parts of vitamin B1, 30 parts of water, then sterilized at 121 ° C for 25 min, sterilized and cooled to room temperature to obtain a growth medium of Cordyceps militaris;
  • the method for cultivating high-yield Cordyceps militaris comprises the following steps:
  • the mycelial growth culture is carried out in a sterilized culture, and the culture condition is: temperature 20 ° C, light every day for 4 h, light intensity is 250 Lux, humidity 60%, culture for 7 d;
  • the culture conditions for mycelial color change culture are: temperature 14 ° C, humidity 70%, ventilation for 1.5 h per day, illumination for 12 h, light intensity of 250 Lux, culture for 3 d;
  • the culture is cultured in a fruit body culture condition: gas culture, temperature 20 ° C ⁇ 2 ° C, humidity 80%, ventilation 1.5 h per day, blue light illumination 14 h, light intensity 250 Lux, Incubating to a fruiting body length of 7-8 cm, harvesting the Cordyceps militaris fruit body;
  • the disinfection is carried out in the following manner: the container containing the peracetic acid solution is placed in a water bath at 75 ° C for fumigation to disinfect the above culture chamber, the fumigation time is 100 min, and the peroxyacetic acid per cubic meter space is 3 -3.2 g; during the sterilization, the room temperature is not lower than 20 ° C and the humidity is 60% - 80%.
  • the obtained Cordyceps militaris fruit body was golden yellow, and the content of cordycepic acid was determined by colorimetry, and the content of cordycepic acid was 18%.
  • the method for cultivating high-yield Cordyceps militaris comprises the following steps:
  • the mycelial growth culture is carried out in a sterilized culture, and the culture condition is: temperature 22 ° C, light for 3 h per day, light intensity is 280 Lux, humidity 65%, culture for 6 d;
  • the culture conditions of mycelial color change culture are: temperature 16 ° C, humidity 75%, ventilation for 2.0 h per day, illumination for 10 h, light intensity of 280 Lux, culture for 4 d;
  • the culture is cultured in a fruit body culture condition: gas culture, temperature 20 ° C ⁇ 2 ° C, humidity 85%, daily ventilation 2.0 h, blue light illumination 13 h, light intensity 280 Lux, Incubating to a fruiting body length of 7-8 cm, harvesting the above-mentioned Cordyceps militaris fruit body;
  • the disinfection is carried out in the following manner: the container containing the peracetic acid solution is placed in a water bath at 80 ° C for fumigation to disinfect the above culture chamber, the fumigation time is 95 min, and the peroxyacetic acid per cubic meter space is 3 -3.2g.
  • the obtained Cordyceps militaris fruit body was golden yellow, and the content of cordycepic acid was determined by colorimetry, and the content of cordycepic acid was 20%.
  • the following ingredients were weighed by weight: 26 parts of sorghum powder, 6 parts of ginkgo leaf powder, 0.06 parts of peptone, 0.08 parts of mannose, 0.04 parts of potassium dihydrogen phosphate, 0.03 parts of magnesium sulfate, 0.06 parts Glucose, 0.001 parts of vitamin B1, 40 parts of water, then sterilized at 121 ° C for 35 min, sterilized and cooled to room temperature to obtain a growth medium of Cordyceps militaris;
  • the method for cultivating high-yield Cordyceps militaris comprises the following steps:
  • the mycelial growth culture is carried out in the sterilized culture, the culture condition is: temperature 25 ° C, day light 2 h, light intensity is 300 Lux, humidity 70%, culture 5d;
  • the cultivation conditions of mycelial color change culture are: temperature 14-18 ° C, humidity 70-80%, ventilation for 2.5 h per day, illumination for 8 h, light intensity of 300 Lux, culture for 5 d;
  • the culture is cultured in a fruit body culture condition: gas culture, temperature is 20 ° C ⁇ 2 ° C, humidity is 90%, ventilation is 2.5 h per day, blue light is 12 h, and light intensity is 300 Lux. Incubating to a fruiting body length of 7-8 cm, harvesting the Cordyceps militaris fruit body;
  • the disinfection is carried out in the following manner: the container containing the peracetic acid solution is placed in a water bath at 85 ° C for fumigation to disinfect the culture chamber, the fumigation time is 90 min, and the peroxyacetic acid per cubic meter of space is used. It is 3-3.2g.
  • the obtained Cordyceps militaris fruit body was golden yellow, and the content of cordycepic acid was determined by colorimetry, and the content of cordycepic acid was 25%.
  • the method for cultivating the high-yield Cordyceous Cordyceps militaris provided by the invention has simple cultivation method and is cultured in different culture stages in different culture stages to ensure the vigor of the strain and the needs of adapting to the cultivation target; the culture conditions and environment of each culture stage The conditions are strictly controlled, which is conducive to the standardization and standardization of the cultivation and management of Cordyceps militaris; the obtained oxalic acid of Cordyceps militaris has a high content of oxalic acid of 18-25%, and the purity of Cordyceps acid extracted from the fruit body of Cordyceps militaris is high.

Abstract

本发明提供了蛹虫草种子培养基、蛹虫草生长培养基、以及蛹虫草的培养方法。所述培养方法得到的蛹虫草子实体的虫草酸含量高。

Description

一种高产虫草酸蛹虫草的培养方法 技术领域
本发明涉及食用菌栽培领域，具体而言，涉及一种高产虫草酸蛹虫草的培养方法。
背景技术
蛹虫草又称北虫草、北冬虫夏草，是真菌寄生于鳞翅目等昆虫的虫体上形成的虫菌复合体，是一种具有滋补作用的重要的营养品。蛹虫草具有抗肿瘤、抗炎等多种药力功效，它已经在东亚地区被广泛作为滋补品及药用菌使用，我国卫生部于2009年3月16日正式将其列为新资源食品。
虫草酸是蛹虫草的主要活性成分之一，虫草酸含量的高低是衡量虫草质量的主要标准之一，一般认为虫草酸含量高的虫草的药用价值高。虫草酸可预防与治疗脑血栓、脑出血、心肌梗塞、长期衰竭；抗肝组织纤维化，抗脂质过氧化，同时由于虫草的增强免疫功能使肝脏的解毒作用增强，从而能够有效保护肝细胞。
目前，蛹虫草虫草酸的含量主要集中于通过液体深层发酵来提高，但是，深层发酵得到的为蛹虫草的菌丝体，其产生的虫草酸的含量一般在3-4.5％左右，并且发酵得到的培养物不仅包括蛹虫草自身，还含有一些培养基残渣，影响虫草酸的纯度。
发明内容
本发明的目的在于提供蛹虫草种子培养基、蛹虫草生长培养基以及一种高产虫草酸蛹虫草的培养方法，以解决上述的问题。
在本发明的实施例中提供了一种蛹虫草种子培养基，按重量份计，包括以下成分：8-12份的葡萄糖、2-4份的蛋白胨、4-6份的酵母粉、0.02-0.06份的磷酸二氢钾、0.02-0.06份的硫酸镁、4-6份的氯化钠、0.05-0.15份的维生素B1、8-12份的高粱粉、800-1200份的水。
在本发明的实施例中还提供了一种蛹虫草生长培养基，按重量份计，包括以下成分：24-26份的高粱粉、4-6份的银杏叶粉、0.04-0.06的蛋白胨、0.04-0.08份的甘露糖、0.02-0.04份的磷酸二氢钾、0.02-0.03份的硫酸镁、0.04-0.06份的葡萄糖、0.0005-0.001份的维生素B1、30-40份的水。
一种高产虫草酸蛹虫草的培养方法，包括以下步骤：
(a)将蛹虫草菌种活化培养，制得浓度为1-3×107CFU/ml的孢子悬液；
(b)将所述孢子悬液以体积百分数为3-8％的比例接种于权利要求1所述的蛹虫草种子培养基中培养，得到种子液；
(c)将所述种子液接种于权利要求2所述的蛹虫草生长培养基中依次进行菌丝生长培养、菌丝转色培养和子实体培养，得到蛹虫草子实体。
优选地，在所述步骤(b)中，培养条件为：25-30℃，240-260rpm/min振荡培养。
优选地，在所述步骤(b)中，培养3-5d，得到所述种子液。
优选地，将所述种子液稀释20-30倍后，以体积百分数为5-8％进行接种。
优选地，在所述步骤(c)中，所述菌丝生长培养在已消毒的培养间进行培养，培养条件为：温度20-25℃，每天光照2-4h，光照强度为250-300Lux，湿度60-70％，培养5-7d。
优选地，在所述步骤(c)中，所述菌丝转色培养的培养条件为：温度14-18℃，湿度70-80％，每天通风1.5-2.5h，光照8-12h，光照强度为250-300Lux，培养3-5d。
优选地，在所述步骤(c)中，所述子实体培养的培养条件为：透气培养，温度为20℃±2℃，湿度为80-90％，每天通风1.5-2.5h，蓝光光照12-14h，光照强度为250-300Lux，培养至子实体长度为7-8cm，采收得到所述蛹虫草子实体。
优选地，所述消毒为：将装有过氧乙酸溶液的容器置于75-85℃的水浴中熏蒸对所述培养间消毒，熏蒸时间为90-100min，所述过氧乙酸每立方米空间用量为3-3.2g；消毒期间，培养间室温不低于20℃，湿度为60％-80％。
本发明实施例提供的高产虫草酸蛹虫草的培养方法，将孢子悬液接种于本发明中提供的蛹虫草种子培养基中培养，得到种子液；将种子液接种到本发明提供的蛹虫草生长培养基中依次进行菌丝生长培养、菌丝转色培养和子实体培养，培养方法简单，培养周期短，得到的蛹虫草子实体虫草酸含量高，且用该蛹虫草子实体提取的虫草酸的纯度高。
具体实施方式
下面通过具体的实施例子对本发明做进一步的详细描述。
在本发明的实施例中提供了一种蛹虫草种子培养基，按重量份计，包括以下成分：8-12份的葡萄糖、2-4份的蛋白胨、4-6份的酵母粉、0.02-0.06份的磷酸二氢钾、0.02-0.06份的硫酸镁、4-6份的氯化钠、0.05-0.15份的维生素B1、8-12份的高粱粉、800-1200份的水。
进一步地，按重量份计，包括以下成分：9-11份的葡萄糖、3-4份的蛋白胨、4-5份的酵母粉、0.04-0.05份的磷酸二氢钾、0.04-0.05份的硫酸镁、4-5份的氯化钠、0.10-0.15份的维生素B1、9-10份的高粱粉、1000份的水。
得到的蛹虫草种子培养基营养全面，培养基成分浓度及配比合适；将蛹虫草的孢子悬液接种至本培养基，部分孢子生长成菌丝体，孢子生命力旺盛，孢子数扩增快，并且得到部分菌丝体，得到的种子液含有孢子和菌丝体，利于后续生长。
在本发明的实施例中还提供了一种蛹虫草生长培养基，按重量份计，包括以下成分：24-26份的高粱粉、4-6份的银杏叶粉、0.04-0.06的蛋白胨、0.04-0.08份的甘露糖、0.02-0.04份的磷酸二氢钾、0.02-0.03份的硫酸镁、0.04-0.06份的葡萄糖、0.0005-0.001份的维生素B1、30-40份的水。
进一步地，按重量份计，包括以下成分：25-26份的高粱粉、4-5份的银杏叶粉、0.04-0.05的蛋白胨、0.06-0.07份的甘露糖、0.03-0.04份的磷酸二氢钾、0.02-0.03份的硫酸镁、0.04-0.05份的葡萄糖、0.0005-0.001份的维生素B1、30-40份的水。
得到的蛹虫草生长培养基营养全面，培养基成分浓度及配比合适；将蛹虫草的种子液接种至本培养基，利于孢子生长为菌丝体，得到较多的菌丝体，利于获得更多的蛹虫草子实体，且得到的蛹虫草子实体虫草酸含量高。
其中，蛹虫草种子培养基和蛹虫草生长培养基中涉及的高粱粉是过20-30目筛得到的，银杏叶粉为将干银杏叶粉粉碎为片状大小为 0.5-1.5mm×0.5-1.5mm。使用这种粒度的高粱粉和银杏叶粉，其在灭菌过程中能分布均匀，灭菌后得到的培养基中的成分分布均一。
分别将蛹虫草种子培养基和蛹虫草生长培养基各自含有的成分称取后混合，均采用高温高压灭菌，具体为121℃，灭菌20-30min，灭菌后冷却至室温，得到可直接用于培养菌株的培养基。
一种高产虫草酸蛹虫草的培养方法，包括以下步骤：
(a)将蛹虫草菌种活化培养，制得浓度为1-3×107CFU/ml的孢子悬液；
(b)将上述孢子悬液以体积百分数为3-8％的比例接种于本发明提供的蛹虫草种子培养基中培养，得到种子液；
(c)将所述种子液接种于本发明提供的蛹虫草生长培养基中依次进行菌丝生长培养、菌丝转色培养和子实体培养，得到蛹虫草子实体。
采用本发明提供的蛹虫草种子培养基和蛹虫草生长培养基进行蛹虫草的培养，培养方法简单，培养周期短，成本低，得到的蛹虫草子实体虫草酸含量高；另外，现有技术中采用液体发酵培养蛹虫草，蛹虫草的大部分在发酵液中，自身产生的分泌物等物质混杂在发酵液中，因而得到的蛹虫草含有发酵液的成分，而本发明提供的蛹虫草生长培养基为固体培养基，采用固体培养蛹虫草，采摘得到的蛹虫草子实体，避免了采用液体发酵培养时培养基中的杂质混入蛹虫草子实体中的缺陷，得到的蛹虫草杂质更少，因此，后续蛹虫草提取的虫草酸的纯度更高。
蛹虫草菌种活化培养，使用的蛹虫草菌种购自中国典型培养物保藏中心，保藏号为CCTCCM2013056，将蛹虫草菌种接种于马铃薯斜面培养基，27℃±2℃培养6-8d，得到蛹虫草的孢子，用含有0.05％的吐温80的无菌水 洗下孢子，制得浓度为1-3×107CFU/ml的孢子悬液。其中，马铃薯斜面培养基成分包括：100g的马铃薯、10g的葡萄糖、1g的磷酸二氢钾、16g的琼脂、1000ml的水；配制方法为：取已去皮的马铃薯100g，切成小块，加水1000m1，煮沸20min，用4-6层纱布过滤，然后补足失水至1000m1，加入16g的琼脂溶化，然后再加入10g的葡萄糖，1g的磷酸二氢钾，分装试管，121℃高压灭菌25-30min，制备得到马铃薯斜面培养基。采用马铃薯斜面培养基活化蛹虫草菌种，方法简单易行，且得到的孢子多。采用0.05％的吐温80的无菌水将孢子制成浓度为1-3×107CFU/ml的孢子悬液，制得的孢子悬液孢子分布均匀；然后以体积百分数为3-8％的比例接种至蛹虫草种子培养基，接种量适中，利于在蛹虫草种子培养基快速生长。
优选地，在上述步骤(b)中，培养条件为：25-30℃，240-260rpm/min振荡培养。经验证，培养温度为25-30℃，蛹虫草在蛹虫草种子培养基中生长快速；转速240-260rpm/min，在不损害蛹虫草孢子的情况下，增加溶氧量，利于孢子和菌丝体的生长，同时可以将蛹虫草种子培养基混合均匀，以防止局部蛹虫草种子培养基营养成分不足而影响孢子和菌丝体的生长，因此，这种培养条件下蛹虫草的孢子生长速度快，形成的菌丝体较多，节约培养时间。
优选地，在上述步骤(b)中，培养3-5d，得到上述种子液。培养3-5d，得到的种子液菌体含量高，且生长旺盛。
优选地，将上述种子液稀释20-30倍后，以体积百分数为5-8％进行接种。在蛹虫草生长培养基中接种适量的种子液，利于得到蛹虫草子实体；为了防止接种量差异大以及接种后分布不均一，先将种子液进行稀释，然 后以体积百分数为5-8％进行接种，可以得到种子液分布均匀的蛹虫草生长培养基，利于菌体的生长。
优选地，在上述步骤(c)中，上述菌丝生长培养在已消毒的培养间进行培养，培养条件为：温度20-25℃，每天光照2-4h，光照强度为250-300Lux，湿度60-70％，培养5-7d。该培养条件下，菌体生长快速，培养容器长满菌丝，培养周期短。
优选地，在上述步骤(c)中，上述菌丝转色培养的培养条件为：温度14-18℃，湿度70-80％，每天通风1.5-2.5h，光照8-12h，光照强度为250-300Lux，培养3-5d。该培养条件下，菌体生长快速，开始在培养基表面形成小米状原基，菌丝体转色效果好，且培养周期短。
优选地，在上述步骤(c)中，上述子实体培养的培养条件为：透气培养，温度为20℃±2℃，湿度为80-90％，每天通风1.5-2.5h，蓝光光照12-14h，光照强度为250-300Lux，培养至子实体长度为7-8cm，采收得到上述蛹虫草子实体。
透气培养为：蛹虫草生长培养基35-45ml放入三角培养瓶(容量为200-250ml)中，其口用封口薄膜封住，然后培养，通过在封口薄膜上扎3-5个孔，进行透气培养，以利于空气流通，利于培养物生长。并且在该培养条件下，菌体生长快速，培养周期短，得到的蛹虫草子实体虫草酸含量高。
此外，固体培养得到的蛹虫草，直接切割即可得到，防止了培养基中的杂质混入得到的蛹虫草子实体中，得到的蛹虫草杂质更少。因此，后续蛹虫草提取的虫草酸的纯度更高。
优选地，上述消毒为：将装有过氧乙酸溶液的容器置于75-85℃的水浴中熏蒸对上述培养间消毒，熏蒸时间为90-100min，上述过氧乙酸每立方米空间用量为3-3.2g。对蛹虫草生长的空间进行消毒，以防止其受病，且防止其他微生物的污染；采用过氧乙酸对熏蒸对培养间消毒，消毒彻底，且对蛹虫草培养物本身无伤害，在培养期间，蛹虫草培养物生长状态好，得到的蛹虫草子实体无污染且得到的蛹虫草子实体虫草酸含量高。
本发明实施例提供的高产虫草酸蛹虫草的培养方法，将孢子悬液接种于本发明中提供的蛹虫草种子培养基中培养，得到种子液；将种子液接种到本发明提供的蛹虫草生长培养基中依次进行菌丝生长培养、菌丝转色培养和子实体培养，培养方法简单，培养周期短，得到的蛹虫草子实体虫草酸含量高，且用该蛹虫草子实体提取的虫草酸的纯度高。
实施例1
按重量份计，称取以下成分：8份的葡萄糖、2份的蛋白胨、4份的酵母粉、0.02份的磷酸二氢钾、0.02份的硫酸镁、4份的氯化钠、0.05份的维生素B1、8份的高粱粉、800份的水，然后121℃，灭菌25min，灭菌后冷却至室温，得到蛹虫草种子培养基；
按重量份计，称取以下成分：24份的高粱粉、4份的银杏叶粉、0.04的蛋白胨、0.04份的甘露糖、0.02份的磷酸二氢钾、0.02份的硫酸镁、0.04份的葡萄糖、0.0005份的维生素B1、30份的水，然后121℃，灭菌25min，灭菌后冷却至室温，得到蛹虫草生长培养基；
高产虫草酸蛹虫草的培养方法，包括以下步骤：
(a)将蛹虫草菌种活化培养，制得浓度为1×107CFU/ml的孢子悬液；
(b)将孢子悬液以体积百分数为8％的比例接种于蛹虫草种子培养基中培养，培养条件为：25℃，240rpm/min振荡培养，培养3d，得到种子液；
(c)将种子液稀释20倍后，以体积百分数为5％将种子液接种于蛹虫草生长培养基中依次进行菌丝生长培养、菌丝转色培养和子实体培养；
菌丝生长培养在已消毒的培养间进行培养，培养条件为：温度20℃，每天光照4h，光照强度为250Lux，湿度60％，培养7d；
菌丝转色培养的培养条件为：温度14℃，湿度70％，每天通风1.5h，光照12h，光照强度为250Lux，培养3d；
菌丝转色培养完成后，对培养物进行子实体培养，培养条件为：透气培养，温度为20℃±2℃，湿度为80％，每天通风1.5h，蓝光光照14h，光照强度为250Lux，培养至子实体长度为7-8cm，采收得到所述蛹虫草子实体；
其中，对培养间采用以下方式进行消毒：将装有过氧乙酸溶液的容器置于75℃的水浴中熏蒸对上述培养间消毒，熏蒸时间为100min，上述过氧乙酸每立方米空间用量为3-3.2g；消毒期间，培养间室温不低于20℃，湿度为60％-80％。
得到的蛹虫草子实体呈金黄色，比色法测定虫草酸的含量，得到虫草酸含量为18％。
实施例2
按重量份计，称取以下成分：10份的葡萄糖、3份的蛋白胨、5份的酵母粉、0.04份的磷酸二氢钾、0.04份的硫酸镁、5份的氯化钠、0.10份的维 生素B1、10份的高粱粉、1000份的水，然后121℃，灭菌30min，灭菌后冷却至室温，得到蛹虫草种子培养基；
按重量份计，称取以下成分：25份的高粱粉、5份的银杏叶粉、0.05份的蛋白胨、0.06份的甘露糖、0.03份的磷酸二氢钾、0.03份的硫酸镁、0.05份的葡萄糖、0.001份的维生素B1、35份的水，然后121℃，灭菌30min，灭菌后冷却至室温，得到蛹虫草生长培养基；
高产虫草酸蛹虫草的培养方法，包括以下步骤：
(a)将蛹虫草菌种活化培养，制得浓度为2×107CFU/ml的孢子悬液；
(b)将孢子悬液以体积百分数为5％的比例接种于蛹虫草种子培养基中培养，培养条件为：27℃，250rpm/min振荡培养，培养4d，得到种子液；
(c)将种子液稀释25倍后，以体积百分数为6％将种子液接种于蛹虫草生长培养基中依次进行菌丝生长培养、菌丝转色培养和子实体培养；
菌丝生长培养在已消毒的培养间进行培养，培养条件为：温度22℃，每天光照3h，光照强度为280Lux，湿度65％，培养6d；
菌丝转色培养的培养条件为：温度16℃，湿度75％，每天通风2.0h，光照10h，光照强度为280Lux，培养4d；
菌丝转色培养完成后，对培养物进行子实体培养，培养条件为：透气培养，温度为20℃±2℃，湿度为85％，每天通风2.0h，蓝光光照13h，光照强度为280Lux，培养至子实体长度为7-8cm，采收得到上述蛹虫草子实体；
其中，对培养间采用以下方式进行消毒：将装有过氧乙酸溶液的容器置于80℃的水浴中熏蒸对上述培养间消毒，熏蒸时间为95min，上述过氧乙酸每立方米空间用量为3-3.2g。
得到的蛹虫草子实体呈金黄色，比色法测定虫草酸的含量，得到虫草酸含量为20％。
实施例3
按重量份计，称取以下成分：12份的葡萄糖、4份的蛋白胨、6份的酵母粉、0.06份的磷酸二氢钾、0.06份的硫酸镁、6份的氯化钠、0.15份的维生素B1、12份的高粱粉、1200份的水，然后121℃，灭菌35min，灭菌后冷却至室温，得到蛹虫草种子培养基；
按重量份计，称取以下成分：26份的高粱粉、6份的银杏叶粉、0.06份的蛋白胨、0.08份的甘露糖、0.04份的磷酸二氢钾、0.03份的硫酸镁、0.06份的葡萄糖、0.001份的维生素B1、40份的水，然后121℃，灭菌35min，灭菌后冷却至室温，得到蛹虫草生长培养基；
高产虫草酸蛹虫草的培养方法，包括以下步骤：
(a)将蛹虫草菌种活化培养，制得浓度为3×107CFU/ml的孢子悬液；
(b)将孢子悬液以体积百分数为3％的比例接种于蛹虫草种子培养基中培养，培养条件为：30℃，260rpm/min振荡培养，培养5d，得到种子液；
(c)将种子液稀释30倍后，以体积百分数为8％将种子液接种于蛹虫草生长培养基中依次进行菌丝生长培养、菌丝转色培养和子实体培养；
菌丝生长培养在已消毒的培养间进行培养，培养条件为：温度25℃，每天光照2h，光照强度为300Lux，湿度70％，培养5d；
菌丝转色培养的培养条件为：温度14-18℃，湿度70-80％，每天通风2.5h，光照8h，光照强度为300Lux，培养5d；
菌丝转色培养完成后，对培养物进行子实体培养，培养条件为：透气培养，温度为20℃±2℃，湿度为90％，每天通风2.5h，蓝光光照12h，光照强度为300Lux，培养至子实体长度为7-8cm，采收得到所述蛹虫草子实体；
其中，对培养间采用以下方式进行消毒：将装有过氧乙酸溶液的容器置于85℃的水浴中熏蒸对所述培养间消毒，熏蒸时间为90min，所述过氧乙酸每立方米空间用量为3-3.2g。
得到的蛹虫草子实体呈金黄色，比色法测定虫草酸的含量，得到虫草酸含量为25％。
本发明提供的高产虫草酸蛹虫草的培养方法，培养方法简单，在不同培养阶段采用不同的培养基进行培养，以确保菌种的活力和适应栽培目标的需求；各个培养阶段的培养条件、环境条件严格控制，利于蛹虫草培养和管理的规范化和标准化；得到的蛹虫草子实体蛹虫草虫草酸含量高，为18-25％，且用该蛹虫草子实体提取的虫草酸的纯度高。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 蛹虫草种子培养基，其特征在于，按重量份计，包括以下成分：8-12份的葡萄糖、2-4份的蛋白胨、4-6份的酵母粉、0.02-0.06份的磷酸二氢钾、0.02-0.06份的硫酸镁、4-6份的氯化钠、0.05-0.15份的维生素B1、8-12份的高粱粉、800-1200份的水。
  2. 蛹虫草生长培养基，其特征在于，按重量份计，包括以下成分：24-26份的高粱粉、4-6份的银杏叶粉、0.04-0.06的蛋白胨、0.04-0.08份的甘露糖、0.02-0.04份的磷酸二氢钾、0.02-0.03份的硫酸镁、0.04-0.06份的葡萄糖、0.0005-0.001份的维生素B1、30-40份的水。
  3. 一种高产虫草酸蛹虫草的培养方法，其特征在于，包括以下步骤：
    (a)将蛹虫草菌种活化培养，制得浓度为1-3×107CFU/ml的孢子悬液；
    (b)将所述孢子悬液以体积百分数为3-8％的比例接种于权利要求1所述的蛹虫草种子培养基中培养，得到种子液；
    (c)将所述种子液接种于权利要求2所述的蛹虫草生长培养基中依次进行菌丝生长培养、菌丝转色培养和子实体培养，得到蛹虫草子实体。
  4. 根据权利要求3所述的高产虫草酸蛹虫草的培养方法，其特征在于，在所述步骤(b)中，培养条件为：25-30℃，240-260rpm/min振荡培养。
  5. 根据权利要求4所述的高产虫草酸蛹虫草的培养方法，其特征在于，在所述步骤(b)中，培养3-5d，得到所述种子液。
  6. 根据权利要求5所述的高产虫草酸蛹虫草的培养方法，其特征在于，在所述步骤(c)中，将所述种子液稀释20-30倍后，以体积百分数为5-8％进行接种。
  7. 根据权利要求6所述的高产虫草酸蛹虫草的培养方法，其特征在于，在所述步骤(c)中，所述菌丝生长培养在已消毒的培养间进行培养，培养条件为：温度20-25℃，每天光照2-4h，光照强度为250-300Lux，湿度60-70％，培养5-7d。
  8. 根据权利要求7所述的高产虫草酸蛹虫草的培养方法，其特征在于，在所述步骤(c)中，所述菌丝转色培养的培养条件为：温度14-18℃，湿度70-80％，每天通风1.5-2.5h，光照8-12h，光照强度为250-300Lux，培养3-5d。
  9. 根据权利要求8所述的高产虫草酸蛹虫草的培养方法，其特征在于，在所述步骤(c)中，所述子实体培养的培养条件为：透气培养，温度为20℃±2℃，湿度为80-90％，每天通风1.5-2.5h，蓝光光照12-14h，光照强度为250-300Lux，培养至子实体长度为7-8cm，采收得到所述蛹虫草子实体。
  10. 根据权利要求7所述的高产虫草酸蛹虫草的培养方法，其特征在于，所述消毒为：将装有过氧乙酸溶液的容器置于75-85℃的水浴中熏蒸对所述培养间消毒，熏蒸时间为90-100min，所述过氧乙酸每立方米空间用量为3-3.2g；消毒期间，培养间室温不低于20℃，湿度为60％-80％。
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