WO2015180519A1 - 一种高产虫草多糖蛹虫草的培养方法 - Google Patents

一种高产虫草多糖蛹虫草的培养方法 Download PDF

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WO2015180519A1
WO2015180519A1 PCT/CN2015/073774 CN2015073774W WO2015180519A1 WO 2015180519 A1 WO2015180519 A1 WO 2015180519A1 CN 2015073774 W CN2015073774 W CN 2015073774W WO 2015180519 A1 WO2015180519 A1 WO 2015180519A1
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cordyceps militaris
parts
culture
cordyceps
cultivating
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熊艳
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熊艳
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity

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  • the present invention relates to the field of edible fungi cultivation, and in particular to a method for cultivating a high-yield Cordyceps polysaccharide polysaccharide 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.
  • polysaccharides in Cordyceps militaris are mainly polysaccharides composed of mannose, cordycepin, adenosine, galactose, arabinose, xylose, glucose, and fucose.
  • Cordyceps polysaccharide can improve human immune function, increase white blood cells, improve respiratory system, increase the content of cholesterol in adrenal gland, plasma cortisol, aldosterone and adrenal gland, promote adrenal gland, inhibit tumor growth, and have anti-tumor, Anti-radiation, hypoglycemic and lipoprotein, cough, phlegm, lung and anti-aging pharmacological effects, clinically used to treat malignant tumors, therefore, Cordyceps polysaccharide has important value.
  • Cordyceps militaris the polysaccharide content of Cordyceps militaris is mainly concentrated in deep fermentation by liquid.
  • the deep fermentation results in the mycelium of Cordyceps militaris.
  • 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-producing Cordyceps polysaccharide polysaccharide 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 by weight 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 yam powder, 800-1200 parts of water.
  • a Cordyceps militaris growth medium which comprises the following components by weight: 12-18 parts of corn flour, 5-10 parts of wheat flour, 3-8 parts of yam powder, 2 - 5 parts of dried thorn powder, 0.02-0.05 parts of peptone, 0.02-0.04 parts of potassium dihydrogen phosphate, 0.02-0.03 parts of magnesium sulfate, 0.02-0.08 parts of glucose, 0.0005-0.001 parts of vitamin B1, 30 - 40 parts of water.
  • a method for cultivating a highly-producing Cordyceps polysaccharide polysaccharide 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 dark culture is cultured in a sterilized culture under the conditions of a temperature of 20-25 ° C, a humidity of 60-70%, and a culture period of 6-8 days.
  • the culture condition of the hyphae color culture is: temperature 15-20 ° C, humidity 60-70%, ventilation 1.5-2.5 h per day, illumination 8-12 h, light intensity It is 250-300 Lux and cultured for 5-8 days.
  • 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, and illumination is 6- 8h, the light intensity is 250-300 Lux, and the culture is carried out until the fruit body length is 6-10 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 polysaccharide 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 culture medium is sequentially subjected to dark culture, hyphal color culture and fruit body culture, the culture method is simple, the culture period is short, the obtained Cordyceps militaris fruit body Cordyceps militaris polysaccharide content is high, and the Cordyceps militaris polysaccharide 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 by weight 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 yam powder, 800-1200 parts of water.
  • the following components are included in parts by weight: 9-10 parts of glucose, 3-4 parts of peptone, 5-6 parts of yeast powder, 0.03-0.05 parts of potassium dihydrogen phosphate, 0.03-0.05 parts of sulfuric acid Magnesium, 4-5 parts of sodium chloride, 0.05-0.15 parts of vitamin B1, 9-10 parts of yam 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 which comprises the following components by weight: 12-18 parts of corn flour, 5-10 parts of wheat flour, 3-8 parts of yam powder, 2 - 5 parts of dried thorn powder, 0.02-0.05 parts of peptone, 0.02-0.04 parts of potassium dihydrogen phosphate, 0.02-0.03 parts of magnesium sulfate, 0.02-0.08 parts of glucose, 0.0005-0.001 parts of vitamin B1, 30- 40 parts of water.
  • the following components are included in parts by weight: 14-16 parts of corn flour, 6-8 parts of wheat flour, 4-6 parts of yam powder, 3-4 parts of dried thorn powder, 0.03-0.04 parts.
  • 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 A large number of Cordyceps militaris fruit bodies, and the obtained Cordyceps militaris fruit body Cordyceps polysaccharide content is high.
  • the corn flour and wheat flour involved in the seed culture medium of Cordyceps militaris and the growth medium of Cordyceps militaris are both 1-3 mm, and the yam powder and the dried thorn powder are prepared in the following manner: the fresh yam is dried and then pulverized. The yam powder having a particle size of 0.8-1.5 mm is obtained; the fresh thorn pear is dried, and the seed is pulverized to obtain a dried thorn pear powder having a particle size of 0.8-1.5 mm. Corn flour, wheat flour, yam powder and dried thorn powder of this size can be used for uniform distribution during sterilization, and the composition of the medium obtained after sterilization is uniform.
  • 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.
  • the method for cultivating the Cordyceps militaris polysaccharide Cordyceps militaris comprises the following steps:
  • the Cordyceps militaris seed culture medium and the Cordyceps militaris growth medium provided by the invention are used for cultivating Cordyceps militaris, the culture method is simple, the culture period is short, the cost is low, and the obtained Cordyceps militaris fruit body Cordyceps militaris polysaccharide content is high;
  • the liquid fermentation is used to culture Cordyceps militaris. Most of the Cordyceps militaris is mixed in the fermentation broth, and the secretions produced by itself are mixed in the fermentation broth. Therefore, the obtained Cordyceps militaris contains the components of the fermentation broth, and the Cordyceps militaris provided by the present invention grows.
  • the medium is a solid medium, and the Cordyceps militaris is cultured in solid form, and the obtained Cordyceps militaris fruit body is avoided.
  • the impurities in the culture medium are mixed with the defects in the fruit body of the Cordyceps militaris, the obtained Cordyceps militaris has less impurities, and therefore, the polysaccharide extracted by the subsequent Cordyceps militaris is higher in purity.
  • the Cordyceps militaris strain was purchased 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 to obtain Cordyceps militaris.
  • the spores were washed with sterile water containing 0.05% Tween 80 to prepare a spore suspension having a concentration of 1-3 x 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- At 30 min, a potato slant medium was prepared.
  • 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%.
  • the dark culture is cultured in the sterilized culture under the conditions of a temperature of 20-25 ° C, a humidity of 60-70%, and a culture for 6-8 days.
  • the growth of the cells is rapid, the culture is filled with hyphae, and the culture period is short.
  • the culture condition of the hyphae color culture is: temperature 15-20 ° C, humidity 60-70%, ventilation 1.5-2.5 h per day, illumination 8-12 h, light intensity 250 -300 Lux, cultured for 5-8 days.
  • the cells grow rapidly, and the hyphae begin to form a millet-like primordium 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 fruit body culture is: gas permeable culture, the temperature is 20 ° C ⁇ 2 ° C, the humidity is 80-90%, the ventilation is 1.5-2.5 h per day, the illumination is 6-8 h, The light intensity is 250-300 Lux, and the length of the fruit body is 6-10 cm, and the above-mentioned Cordyceps militaris fruit body is harvested.
  • the gas permeable culture is: a few holes are placed on the film of the culture container mouth to facilitate air circulation and facilitate the growth of the culture.
  • the growth of the cells is rapid, the culture period is short, and the obtained polysaccharide of the Cordyceps militaris has a high polysaccharide content.
  • 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, the peroxyacetic acid per cubic meter
  • the amount of space 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 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 Cordyceps militaris polysaccharide content is high.
  • the method for cultivating the high-yield Cordyceps polysaccharide 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 culture medium is sequentially subjected to dark culture, hyphal color culture and fruit body culture, the culture method is simple, the culture period is short, the obtained Cordyceps militaris fruit body Cordyceps militaris polysaccharide content is high, and the Cordyceps militaris polysaccharide 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 yam 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 method for cultivating the Cordyceps militaris polysaccharide Cordyceps militaris comprises the following steps:
  • the culture conditions are: temperature 20 ° C, humidity 60%, culture for 6d;
  • the culture conditions of mycelial color change culture are: temperature 15 ° C, humidity 60%, ventilation for 1.5 h per day, illumination for 8 h, light intensity of 250 Lux, culture for 5 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, light 6 h, light intensity 250 Lux, culture
  • the length of the fruiting body is 6-10cm, and the fruit body of the cordyceps militaris is harvested;
  • 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 amount of peroxyacetic acid per cubic meter is 3- 3.2g; during 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 total sugar content of the Cordyceps militaris fruit body was determined by anthrone colorimetric method, and the obtained polysaccharide content of Cordyceps militaris was 18%.
  • the following ingredients were weighed by weight: 10 parts of glucose, 3 parts of peptone, 5 parts of yeast powder, 0.04 parts of potassium dihydrogen phosphate, 0.04 parts of magnesium sulfate, 5 parts of sodium chloride, 0.10 parts Vitamin B1, 10 parts of yam powder, 1000 parts of water, then sterilized at 121 ° C for 30 min, sterilized and cooled to room temperature to obtain Cordyceps militaris seed culture medium;
  • the method for cultivating the Cordyceps militaris polysaccharide Cordyceps militaris comprises the following steps:
  • the dark culture is cultured in the sterilized culture, and the culture conditions are: temperature 22 ° C, humidity 65%, culture for 7 days;
  • the culture conditions of mycelial color change culture are: temperature 18 ° C, humidity 65%, ventilation for 2.0 h per day, illumination for 10 h, light intensity of 280 Lux, culture for 7 d;
  • the culture is cultured in a fruit body culture condition: gas culture, temperature 20 ° C ⁇ 2 ° C, humidity 85%, daily ventilation for 2.0 h, light 7 h, light intensity 280 Lux, culture
  • the length of the fruiting body is 6-10cm, and the fruit body of the cordyceps militaris is harvested;
  • 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 -3.2 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 total sugar content of the Cordyceps militaris fruit body was determined by anthrone colorimetric method, and the obtained polysaccharide content of Cordyceps militaris was 20%.
  • the method for cultivating the Cordyceps militaris polysaccharide Cordyceps militaris comprises the following steps:
  • the dark culture is cultured in the sterilized culture, and the culture conditions are: temperature 25 ° C, humidity 70%, culture for 8 days;
  • the culture conditions of mycelial color change culture are: temperature 20 ° C, humidity 70%, ventilation for 2.5 h per day, illumination for 12 h, light intensity of 300 Lux, culture for 8 d;
  • the culture is cultured in a fruit body culture condition: gas culture, temperature 20 ° C ⁇ 2 ° C, humidity 90%, ventilation for 2.5 h per day, illumination for 8 h, light intensity of 300 Lux, culture
  • the length of the fruiting body is 6-10cm, and the fruit body of the cordyceps militaris is harvested;
  • 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 above culture chamber, the fumigation time is 90 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 total sugar content of the Cordyceps militaris fruit body was determined by anthrone colorimetric method, and the obtained polysaccharide content of Cordyceps militaris was 25%.
  • the method for cultivating the high-yield Cordyceps polysaccharide polysaccharide Cordyceps militaris provided by the invention has simple cultivation method, and is cultured in different culture stages in different culture stages to ensure the vitality 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 polysaccharide of Cordyceps militaris has a high polysaccharide content of 18-25%, and the purity of Cordyceps militaris polysaccharide extracted with the Cordyceps militaris fruit body is high.

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Abstract

一种蛹虫草种子培养基、生长培养基及高产多糖蛹虫草的培养方法，该方法包括以下步骤：将蛹虫草菌种活化培养，制得孢子悬液；将孢子悬液以体积百分数为3-8％的比例接种于蛹虫草种子培养基中培养，得到种子液；将种子液接种于蛹虫草生长培养基中依次进行暗培养、菌丝转色培养和子实体培养，得到蛹虫草子实体。本培养方法简单，培养周期短，得到的蛹虫草子实体蛹虫草多糖含量高、纯度高。

Description

一种高产虫草多糖蛹虫草的培养方法 技术领域
本发明涉及食用菌栽培领域，具体而言，涉及一种高产虫草多糖蛹虫草的培养方法。
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。
其中蛹虫草中的多糖，主要是由甘露糖、虫草素、腺苷、半乳糖、阿拉伯糖、木糖精、葡萄糖、岩藻糖组成的多聚糖。实验证明，虫草多糖可提高人体免疫功能，升高白细胞，改善呼吸系统，可使肾上腺、血浆皮质醇、醛固酮及肾上腺内胆固醇含量增加，有促进肾上腺的作用，抑制肿瘤生长，并具有抗肿瘤、抗辐射、降血糖和脂蛋白、止咳、化痰、润肺和延缓衰老等药理作用，临床已用于治疗恶性肿瘤，因此，虫草多糖具有重要的利用价值。
目前，蛹虫草多糖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菌丝体，除包括蛹虫草自身外，还有一些培养基残渣，影响蛹虫草多糖的纯度。
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多糖蛹虫草的培养方法，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份的山药粉、800-1200份的水。
在本发明的实施例中还提供了蛹虫草生长培养基，按重量份计，包括以下成分：12-18份的玉米粉、5-10份的麦子粉、3-8份的山药粉、2-5份的干刺梨粉、0.02-0.05份的蛋白胨、0.02-0.04份的磷酸二氢钾、0.02-0.03份的硫酸镁、0.02-0.08份的葡萄糖、0.0005-0.001份的维生素B1、30-40份的水。
一种高产虫草多糖蛹虫草的培养方法，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暗培养、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，所述暗培养在已消毒的培养间进行培养，培养条件为：温度20-25℃，湿度60-70％，培养6-8d。
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15-20℃，湿度60-70％，每天通风1.5-2.5h，光照8-12h，光照强度为250-300Lux，培养5-8d。
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，光照6-8h，光照强度为250-300Lux，培养至子实体长度为6-10cm，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多糖蛹虫草的培养方法，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暗培养、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蛹虫草多糖含量高，且用该蛹虫草子实体提取的蛹虫草多糖的纯度高。
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份的山药粉、800-1200份的水。
进一步地，按重量份计，包括以下成分：9-10份的葡萄糖、3-4份的蛋白胨、5-6份的酵母粉、0.03-0.05份的磷酸二氢钾、0.03-0.05份的硫酸镁、4-5份的氯化钠、0.05-0.15份的维生素B1、9-10份的山药粉、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蛹虫草生长培养基，按重量份计，包括以下成分：12-18份的玉米粉、5-10份的麦子粉、3-8份的山药粉、2-5份的干刺梨粉、0.02-0.05份的蛋白胨、0.02-0.04份的磷酸二氢钾、0.02-0.03份的硫酸镁、0.02-0.08份的葡萄糖、0.0005-0.001份维生素B1、30-40份的水。
进一步地，按重量份计，包括以下成分：14-16份的玉米粉、6-8份的麦子粉、4-6份的山药粉、3-4份的干刺梨粉、0.03-0.04份的蛋白胨、0.02-0.04份的磷酸二氢钾、0.02-0.03份的硫酸镁、0.03-0.06份的葡萄糖、0.0005-0.001份维生素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蛹虫草多糖含量高。
其中，蛹虫草种子培养基和蛹虫草生长培养基中涉及的玉米粉和麦子粉的粒度均为1-3mm，山药粉和干刺梨粉采用以下方式制备：将鲜山药烘干，然后粉碎，得到粒度为0.8-1.5mm的山药粉；将鲜刺梨烘干，去籽后粉碎，得到粒度为0.8-1.5mm的干刺梨粉。使用这种粒度的玉米粉、麦子粉、山药粉以及干刺梨粉，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。
高产虫草多糖蛹虫草的培养方法，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)将上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暗培养、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蛹虫草多糖含量高；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，后续蛹虫草提取的多糖的纯度更高。
蛹虫草菌种活化培养，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，加入琼脂16g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，上述暗培养在已消毒的培养间进行培养，培养条件为：温度20-25℃，湿度60-70％，培养6-8d。该培养条件下，菌体生长快速，培养器中长满菌丝，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15-20℃，湿度60-70％，每天通风1.5-2.5h，光照8-12h，光照强度为250-300Lux，培养5-8d。该培养条件下，菌体生长快速，菌丝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，光照6-8h，光照强度为250-300Lux，培养至子实体长度为6-10cm，采收得到上述蛹虫草子实体。透气培养为：在培养容器口的薄膜上扎几个孔，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蛹虫草多糖含量高。
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；消毒期间，培养间室温不低于20℃，湿度为60-80％。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蛹虫草多糖含量高。
本发明实施例提供的高产虫草多糖蛹虫草的培养方法，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暗培养、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蛹虫草多糖含量高，且用该蛹虫草子实体提取的蛹虫草多糖的纯度高。
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份的山药粉、800份水，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：12份的玉米粉、5份的麦子粉、3份的山药粉、2份的干刺梨粉、0.02份的蛋白胨、0.02份的磷酸二氢钾、0.02份的硫酸镁、0.02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；
高产虫草多糖蛹虫草的培养方法，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暗培养、菌丝转色培养和子实体培养；
暗培养在已消毒的培养间进行培养，培养条件为：温度20℃，湿度60％，培养6d；
菌丝转色培养的培养条件为：温度15℃，湿度60％，每天通风1.5h，光照8h，光照强度为250Lux，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80％，每天通风1.5h，光照6h，光照强度为250Lux，培养至子实体长度为6-10cm，采收得到蛹虫草子实体；
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，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，蒽酮比色法测定蛹虫草子实体总糖含量，得到的蛹虫草多糖含量为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份的山药粉、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：15份的玉米粉、8份的麦子粉、5份的山药粉、4份的干刺梨粉、0.04份的蛋白胨、0.03份的磷酸二氢钾、0.02份的硫酸镁、0.05份的葡萄糖、0.0008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；
高产虫草多糖蛹虫草的培养方法，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暗培养、菌丝转色培养和子实体培养；
暗培养在已消毒的培养间进行培养，培养条件为：温度22℃，湿度65％，培养7d；
菌丝转色培养的培养条件为：温度18℃，湿度65％，每天通风2.0h，光照10h，光照强度为280Lux，培养7d；
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，光照7h，光照强度为280Lux，培养至子实体长度为6-10cm，采收得到蛹虫草子实体；
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；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，蒽酮比色法测定蛹虫草子实体总糖含量，得到的蛹虫草多糖含量为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份的山药粉、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：18份的玉米粉、10份的麦子粉、8份的山药粉、5份的干刺梨粉、0.05份的蛋白胨、0.04份的磷酸二氢钾、0.03份的硫酸镁、0.08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；
高产虫草多糖蛹虫草的培养方法，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暗培养、菌丝转色培养和子实体培养；
暗培养在已消毒的培养间进行培养，培养条件为：温度25℃，湿度70％，培养8d；
菌丝转色培养的培养条件为：温度20℃，湿度70％，每天通风2.5h，光照12h，光照强度为300Lux，培养8d；
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，光照8h，光照强度为300Lux，培养至子实体长度为6-10cm，采收得到蛹虫草子实体；
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℃的水浴中熏蒸对上述培养间消毒，熏蒸时间为90min，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，蒽酮比色法测定蛹虫草子实体总糖含量，得到的蛹虫草多糖含量为25％。
本发明提供的高产虫草多糖蛹虫草的培养方法，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多糖含量高，为18-25％，且用该蛹虫草子实体提取的蛹虫草多糖的纯度高。
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份的山药粉、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：12-18份的玉米粉、5-10份的麦子粉、3-8份的山药粉、2-5份的干刺梨粉、0.02-0.05份的蛋白胨、0.02-0.04份的磷酸二氢钾、0.02-0.03份的硫酸镁、0.02-0.08份的葡萄糖、0.0005-0.001份的维生素B1、30-40份的水。
  3. 一种高产虫草多糖蛹虫草的培养方法，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暗培养、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所述的高产虫草多糖蛹虫草的培养方法，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所述的高产虫草多糖蛹虫草的培养方法，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所述的高产虫草多糖蛹虫草的培养方法，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所述的高产虫草多糖蛹虫草的培养方法，其特征在于，在所述步骤(c)中，所述暗培养在已消毒的培养间进行培养，培养条件为：温度20-25℃，湿度60-70％，培养6-8d。
  8. 根据权利要求7所述的高产虫草多糖蛹虫草的培养方法，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15-20℃，湿度60-70％，每天通风1.5-2.5h，光照8-12h，光照强度为250-300Lux，培养5-8d。
  9. 根据权利要求8所述的高产虫草多糖蛹虫草的培养方法，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，光照6-8h，光照强度为250-300Lux，培养至子实体长度为6-10cm，采收得到所述蛹虫草子实体。
  10. 根据权利要求7所述的高产虫草多糖蛹虫草的培养方法，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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