WO2022100701A1 - 一种罗伯茨虫草的培养方法 - Google Patents

一种罗伯茨虫草的培养方法 Download PDF

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WO2022100701A1
WO2022100701A1 PCT/CN2021/130405 CN2021130405W WO2022100701A1 WO 2022100701 A1 WO2022100701 A1 WO 2022100701A1 CN 2021130405 W CN2021130405 W CN 2021130405W WO 2022100701 A1 WO2022100701 A1 WO 2022100701A1
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culture
culturing
solid
medium
temperature
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PCT/CN2021/130405
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French (fr)
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谭悠久
董建飞
孙长胜
海沃尔-琼斯奈杰尔
李增智
朱冰舟
江可
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浙江泛亚生物医药股份有限公司
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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
    • 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
    • A01G18/20—Culture media, e.g. compost

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  • the invention relates to a method for culturing Cordyceps sinensis, belonging to the field of artificial cultivation of Cordyceps sinensis.
  • Cordyceps is an important fungal resource, and is known as a treasure trove of natural compounds because of its ability to form various secondary metabolites.
  • Cordyceps fungi have a variety of physiological activities such as protecting kidney, anti-tumor, anti-pathogen, anti-oxidation, anti-radiation, lowering blood sugar, regulating lipid metabolism, and enhancing human immune function, so they are widely used in medicine, food and health food. of great economic value.
  • Ophiocordyceps robertsii also known as Cordyceps robertsii
  • Cordyceps robertsii Ophiocordyceps robertsii, also known as Cordyceps robertsii
  • the M ⁇ ori use the pigment produced by Cordyceps robots for tattoos, and the fruiting bodies of Cordyceps robots are also used to treat stomach upsets.
  • Cordyceps roberts is distributed in Guizhou and Yunnan. At present, there are few research reports on Cordyceps robots in China, and there is no report on the cultivation of fruiting bodies of Cordyceps robots.
  • the present invention provides a method for culturing Cordyceps robots, which adopts specific medium and cultivation conditions in a solid cultivation stage to obtain fruiting bodies of Cordyceps robots, which lays a foundation for the development and application of Cordyceps robots.
  • the present invention provides a method for culturing Cordyceps roberts, the method comprising the processes of liquid culture, solid culture and harvesting;
  • the solid medium includes grains and animal protein.
  • the grain is selected from any one or more of rice, millet, brown rice, oat, wheat, barley, and sorghum; the solid medium has a material-water ratio of 1:(1.5-1.8).
  • the animal protein is beef meal or silkworm pupae powder; the added amount of the animal protein is 7.5% to 25% of the grain; further preferably, the added amount of the animal protein is 8% to 22% of the grain or 9% to 20% or 10% or 19%.
  • the solid culturing process is aimed at culturing and producing fruit bodies, including a mycelium growth stage and a fruit body growth stage that are carried out successively; °C; in the growth stage of the fruit body, light cultivation is adopted, and the cultivation temperature is 17 °C to 19 °C, preferably 18 °C. Further preferably, the relative humidity of the solid culture is 70% to 80%.
  • the inoculum amount of the solid culture is 7% to 22%; more preferably, the inoculum amount is 10%, 15% or 20%.
  • a culture box or a culture flask is used as a culture vessel for the solid culture.
  • the liquid culture process of the present invention is aimed at the expansion of bacterial species, and conventional bacterial species expansion methods can be adopted, including any one or a combination of several methods among shaking flask culture, seed tank culture, and fermenter culture.
  • the “material-water ratio” in the present invention refers to the weight/volume ratio (kg/L) of grain to water during the preparation of the solid medium.
  • the "inoculation amount is XX%" in the present invention refers to the percentage of the inoculated seed liquid volume (L) in the weight (kg) of the solid medium; the weight of the solid medium is the weight of the grain.
  • the operations are preferably performed under substantially aseptic conditions, and various tools and materials used are
  • the sterilized, for example, solid medium is sterilized neatly at 110°C to 130°C and a pressure of 0.1 to 0.2 MPa for 40 to 50 minutes.
  • the Cordyceps robots of the present invention originates from New Zealand, and there is no patent or literature on artificial cultivation of Cordyceps robots produced in New Zealand in the prior art.
  • the research of the present invention found that, unlike other Cordyceps sinensis, the conventional grain medium and conventional fruiting body culture temperature for Cordyceps robots could not produce fruiting bodies at all. Oat, wheat, barley, rice, brown rice and millet were used to prepare solid medium, and the results showed that none of them could produce fruit bodies or only a very small amount of small fruit bodies; the inventor also tried to add other nutrients, such as yeast powder, phosphoric acid Potassium dihydrogen and magnesium sulfate can only produce a very small amount of small fruiting bodies. The inventor accidentally found that only adding silkworm pupa powder or beef powder to the solid medium can produce fruiting bodies, and the fruiting bodies are sturdy and the biotransformation rate can reach 15%.
  • Fig. 2 Cordyceps roberts cultivated in Example 5.
  • Fig. 3 Effect of different solid medium on the growth of Cordyceps robots fruiting body, wherein: A is adding silkworm pupae powder to the solid medium, and B is not adding silkworm pupae powder to the solid medium.
  • Fig. 4 The effect of different culture temperatures on the growth of Cordyceps robots fruiting bodies in the growth stage of fruiting bodies, where: A is the culture temperature of 18°C, B is the culture temperature of 15°C, and C is the culture temperature of 22°C.
  • the fermented seed medium was loaded into the fermenter, sterilized under high temperature and high pressure at 121°C for 30 minutes, cooled to room temperature naturally, and the shake flask seed liquid was inoculated onto the fermented seed medium at 5%, at a temperature of 22 ⁇ 1°C, stirring Under the condition of the speed of 150r/min, the aeration rate of 0.5vvm is fermented and cultured, and the fermentation time is 3 days to obtain the fermentor seed liquor.
  • Inclined tube medium potato 200g boiled juice, sucrose 20g, tryptone 10g/L, magnesium sulfate 1.0g/L, potassium dihydrogen phosphate 1.0g/L, agar 15g/L, pH 6.5;
  • Liquid shake flask seed medium sucrose 20g/L, soybean peptone 10g/L, yeast powder 10g/L, magnesium sulfate 0.5g/L, potassium dihydrogen phosphate 1g/L;
  • Fermentation tank seed medium glucose 20g/L, soybean peptone 10g/L, yeast powder 10g/L, magnesium sulfate 0.5g/L, potassium dihydrogen phosphate 1g/L.
  • the fermentation seed medium into the fermenter, sterilize it under high temperature and high pressure at 121°C for 30min, naturally cool to room temperature, inoculate 5% of the shake flask seed liquid on the medium, at a temperature of 22 ⁇ 1°C and a stirring rate of 150r Under the condition of /min, the aeration rate was 0.5vvm for fermentation and culture, and the fermentation time was 3 days to obtain the fermentor seed liquid.
  • Inclined tube medium sucrose 20g, tryptone 10g/L, yeast powder 10g/L, magnesium sulfate 1.0g/L, potassium dihydrogen phosphate 2.0g/L, agar 20g/L, pH 6.5;
  • Liquid shake flask seed medium glucose 20g/L, tryptone 5g/L, yeast powder 10g/L, magnesium sulfate 1g/L, potassium dihydrogen phosphate 2g/L;
  • Fermentation tank seed medium glucose 20g/L, tryptone 10g/L, yeast powder 10g/L, magnesium sulfate 1g/L, potassium dihydrogen phosphate 2g/L.
  • Solid culture medium add 50 g of solid culture medium to a culture flask with a diameter of 10 cm and a height of 15 cm, add drinking water at a ratio of 1:1.5 to the ratio of material to water, pH is 6 to 6.5, mix well, cover the lid, Sterilize at 126°C ⁇ 2°C for 40-50min, cool naturally to obtain a solid medium;
  • the solid medium consists of: 32g of rice, 10g of millet, 8g of silkworm chrysalis powder, and 75ml of drinking water;
  • Example 1 under sterile conditions, the fermentor seeds prepared in Example 1 were inoculated onto the surface of the solid medium with a spray gun, and the inoculation amount in each bottle of the solid medium was 5ml;
  • Mycelium growth stage place the culture bottle in an artificial climate room at 20°C, keep the dark conditions, and the relative humidity at 70% to 80%, and cultivate for 10 to 15 days, so that the mycelium covers the surface of the medium;
  • Fruiting body growth stage turn on the light source to ensure sufficient light, the light intensity is 50-100 lux, the relative humidity is 70%-80%, the temperature is 18 °C, and the air is changed every 4-5 hours; after 10 days, the primordium of the fruiting body begins to form ; Cultivate for 50 to 60 days, the fruit body is full of the bottle, and the artificially cultivated Roberts Cordyceps fruit body is obtained when it no longer grows; Harvest.
  • the cultivated fruit bodies are shown in Figure 1.
  • the yield of fruit bodies obtained by culturing the solid medium That is, with 1 kg of medium, 150 grams of dry fruit bodies can be cultured).
  • Solid culture medium add 50 g of solid culture medium to a culture flask with a diameter of 10 cm and a height of 15 cm, add drinking water at a ratio of 1:1.8 to water, pH is 6 to 6.5, mix evenly, cover the lid, Sterilize at 126°C ⁇ 2°C for 40-50 min, cool naturally to obtain a solid medium; the solid medium consists of: 40 g of rice, 5 g of beef powder, and 80 ml of drinking water.
  • Example 1 under sterile conditions, the fermentor seeds prepared in Example 1 were inoculated onto the surface of the solid medium with a spray gun, and the inoculation amount in each bottle of the solid medium was 10ml;
  • Mycelium growth stage place the culture bottle in an artificial climate chamber at 22°C to keep the dark conditions; the relative humidity is 70% to 80%, and cultivate for 10 to 15 days, so that the mycelium covers the surface of the medium;
  • Fruiting body growth stage turn on the light source to ensure sufficient light, the light intensity is 50 ⁇ 100lux, the relative humidity is 70% ⁇ 80%, the temperature is 19°C, and the ventilation is every 4 ⁇ 5 hours.
  • the primordium of the fruiting body begins to form; after culturing for 50 to 60 days, the fruiting body fills the bottle, and the artificially cultivated fruiting body of Cordyceps roberts is obtained when it no longer grows; harvesting.
  • Solid culture medium add 500 g of solid culture medium to an incubator with a length, width and height of 30cm x 30cm x 25cm, add drinking water at a ratio of 1:1.7 to water, pH is 6 to 6.5, and mix well , covered with a culture bag with a breathable film, tied the bag mouth with a nylon rope, sterilized at a high temperature of 121 ° C for 40 min, and cooled naturally to obtain a solid medium; the solid medium consists of: oat 450g, silkworm chrysalis powder 50g, drinking water 850ml;
  • Example 2 under aseptic conditions, the fermentor seeds prepared in Example 2 were inoculated onto the surface of the solid medium with a spray gun, and the inoculation amount in each box of the solid medium was 100ml;
  • Mycelium growth stage place the culture box in an artificial climate room at 20°C to keep the dark conditions; the relative humidity is 70% to 80%, and cultivate for 10 to 15 days, so that the mycelium covers the surface of the medium;
  • Fruiting body growth stage turn on the light source to ensure sufficient light, the light intensity is 50 ⁇ 200lux, the relative humidity is 70% ⁇ 80%, the temperature is 18°C, and the ventilation is every 4 ⁇ 5 hours.
  • the primordium of the fruiting body begins to form; after culturing for 50 to 60 days, the fruiting body fills the box, and the artificially cultivated fruiting body of Cordyceps roberts is obtained when it no longer grows; harvesting.
  • the cultivated fruit bodies are shown in Figure 2.
  • Preparation of solid culture medium add 500g of solid culture medium to an incubator with a length, width and height of 30cm x 30cm x 25cm, add drinking water at a ratio of 1:1.6 to water, pH is 6 to 6.5, and mix well , covered with a culture bag with a breathable membrane, tied the bag mouth with a nylon rope, sterilized at a high temperature of 121 ° C for 40 min, and cooled naturally to obtain a solid medium; the solid medium was composed of: 450 g of rice, 50 g of silkworm chrysalis powder, and 50 g of drinking water. 800ml;
  • Example 2 under aseptic conditions, the fermentor seeds prepared in Example 2 were inoculated onto the surface of the solid medium with a spray gun, and the inoculation amount in each box of the solid medium was 100ml;
  • Mycelium growth stage place the culture box in an artificial climate chamber at 22°C to keep the dark conditions; the relative humidity is 50% to 60%, and cultivate for 10 to 15 days, so that the mycelium covers the surface of the medium;
  • Fruiting body growth stage turn on the light source to ensure sufficient light, the light intensity is 50 ⁇ 200lux, the relative humidity is 70% ⁇ 80%, the temperature is 19°C, and the ventilation is every 4 ⁇ 5 hours.
  • the primordium of the fruiting body begins to form; after culturing for 50 to 60 days, the fruiting body fills the box, and the artificially cultivated fruiting body of Cordyceps roberts is obtained when it no longer grows; harvesting.
  • Example 4-6 Artificially cultured Cordyceps robots fruiting body linear, erect or curved, light brown to brown, solitary, very few simply bifurcated, 10-15 cm long and 0.5-3 mm wide.
  • Embodiment 7 Solid medium investigation
  • Preparation of seed liquor Refer to Example 1 to prepare a fermentor seed liquor.
  • the solid medium of the experimental group was composed of: 32 g of rice, 10 g of millet, 8 g of silkworm chrysalis powder, and 75 ml of drinking water;
  • the solid medium of the control group consisted of: 32 g of rice, 10 g of millet, and 63 ml of drinking water.
  • the culture conditions of the inoculum amount, the growth stage of mycelium and the growth stage of fruit body are the same as those in Example 3.
  • the cultivated fruit body is shown in Figure 3, wherein A is adding silkworm chrysalis powder to the culture medium, and B is not adding silkworm chrysalis powder to the culture medium.
  • Preparation of seed liquor Refer to Example 1 to prepare a fermentor seed liquor.
  • Fruiting body growth stage turn on the light source to ensure sufficient light, the light intensity is 50 ⁇ 100lux, the relative humidity is 70% ⁇ 80%, and three culture temperatures are set respectively: 15°C, 18°C and 22°C, every 4 ⁇ Aeration every 5 hours; cultured for 50 to 60 days.
  • the cultivated fruit bodies are shown in Figure 4, where A is the culture temperature of 18°C, B is the culture temperature of 15°C, and C is the culture temperature of 22°C.

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Abstract

一种罗伯茨虫草的培养方法，包括液体培养、固体培养和采收的过程；所述固体培养的培养基包括谷物和动物蛋白；固体培养过程的子实体生长阶段培养温度为17℃～19℃。该培养方法能显著缩短培养周期并提高罗伯茨虫草的产量和质量。

Description

一种罗伯茨虫草的培养方法 技术领域
本发明涉及一种罗伯茨虫草的培养方法，属于虫草人工培养领域。
背景技术
虫草是一类重要的真菌资源，由于它们能形成各类次生代谢产物，被而被誉为天然化合物宝库。虫草类多种真菌具有护肾、抗肿瘤、抗病原、抗氧化、抗辐射、降血糖、调节脂类代谢、增强人体免疫功能等多种生理活性，因此在医药、食品和保健食品等方面有的巨大经济价值。
罗伯茨虫草(Ophiocordyceps robertsii)也称为罗伯茨线虫草(Cordyceps robertsii)，最早报道于澳洲，主要分布于新西兰、澳大利亚地区。毛利人利用罗伯茨虫草产生的色素进行纹身，也将罗伯茨虫草子实体用于治疗胃部不适。
在我国，罗伯茨虫草在贵州、云南地区有分布。目前国内关于罗伯茨虫草的研究报道较少，未见关于罗伯茨虫草子实体培养的报道。
发明内容
本发明提供一种罗伯茨虫草的培养方法，该方法在固体培养阶段采用特定的培养基和培养条件，获得罗伯茨虫草子实体，为罗伯茨虫草的开发应用奠定了基础。
作为本发明的一个方面，本发明提供一种罗伯茨虫草的培养方法，该方法包括液体培养、固体培养和采收的过程；
其中，所述固体培养过程中，固体培养基包括谷物和动物蛋白。
优选的，所述谷物选自大米、小米、糙米、燕麦、小麦、大麦、高粱中的任意一种或几种；所述固体培养基的料水比为1：(1.5～1.8)。
优选的，所述动物蛋白为牛肉粉或蚕蛹粉；所述动物蛋白的添加量为谷物的7.5％～25％；进一步优选的，所述动物蛋白的添加量为谷物的8％～22％或9％～20％或10％或19％。
优选的，所述固体培养过程以培养产生子实体为目的，包括先后进行的菌丝体生长阶段和子实体生长阶段；优选的，菌丝体生长阶段采用避光培养，培养温度为20℃～22℃；子实体生长阶段采用光照培养，培养温度为17℃～19℃，优选18℃。进一步优选的，所述固体培养的相对湿度为70％～80％。
优选的，所述固体培养的接种量为7％～22％；进一步优选的，所述接种量为10％、15％或20％。
优选的，所述固体培养采用培养盒或者培养瓶作为培养容器。
本发明所述液体培养过程以菌种扩增为目的，可采用常规的菌种扩培方法，包括摇瓶培养、种子罐培养、发酵罐培养中的任意一种或几种方法的组合。
本发明所述“料水比”为固体培养基配制过程中谷物与水的重量/体积比(kg/L)。
本发明所述“接种量为XX％”是指接种的种子液体积(L)占固体培养基重量(kg)的百分比；所述固体培养基重量为谷物的重量。
在本发明中，未作相反说明的情况下，如本领域公知的，为了避免杂菌污染，所进行的操作均优选在基本上无菌的条件下进行，所用的各种工具和材料均为已经消毒过的，例如固体培养基在110℃～130℃、压力0.1～0.2MPa下进行整齐灭菌40～50min。
本发明罗伯茨虫草来源于新西兰，现有技术未见针对新西兰产罗伯茨虫草人工培养的专利或文献。本发明研究发现，与其他虫草不同，对于罗伯茨虫草采用常规的谷物培养基和常规子实体培养温度根本无法产生子实体，发明人尝试将培养温度降低，并变换固体培养基的主料，分别以燕麦、小麦、大麦、大米、糙米、小米制备固体培养基，结果表明，依然均无法产生子实体或只能产生极少量细小的子实体；发明人又尝试加入其它营养成分，如酵母粉、磷酸二氢钾、硫酸镁，也均只能产生极少量细小的子实体。发明人无意中发现只有在固体培养基中加入蚕蛹粉或牛肉粉才能产生子实体，且子实体粗壮，生物转化率可以达到15％。
附图说明
图1实施例3培养的罗伯茨虫草。
图2实施例5培养的罗伯茨虫草。
图3不同固体培养基对罗伯茨虫草子实体生长的影响，其中：A为固体培养基中加入蚕蛹粉，B为固体培养基中不加入蚕蛹粉。
图4子实体生长阶段不同培养温度对罗伯茨虫草子实体生长的影响，其中：A为培养温度为18℃，B为培养温度为15℃，C为培养温度为22℃。
具体实施方式
以下实施例和对照实施例均采用分离于新西兰的罗伯茨虫草Ophiocordyceps robertsii，该菌株保藏在位于我国北京的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:20275。
实施例1 液体培养
A、斜面试管菌种活化培养
将罗伯茨虫草菌种接种于斜面试管培养基，温度22±1℃培养20天，待菌丝长满试管，得到斜面菌种；
B、液体摇瓶种子培养
在500ml三角瓶中装入200ml摇瓶种子培养基，在121℃高温高压下灭菌30min，自然冷却至室温，将斜面菌种接种到液体摇瓶种子培养基上，在温度22±1℃、150r/min条件下恒温振荡培养，培养时间为4天，得到液体摇瓶种子；
C、发酵罐种子培养
在发酵罐中装入发酵种子培养基，在121℃高温高压下灭菌30min，自然冷却至室温，将摇瓶种子液按照5％接种到发酵种子培养基上，在温度22±1℃、搅拌速率150r/min条件下，通气量0.5vvm发酵培养，发酵时间为3天，得到发酵罐种子液。
液体培养阶段的培养基：
斜面试管培养基：土豆200g煮汁，蔗糖20g、胰蛋白胨10g/L、硫酸镁1.0g/L、磷酸二氢钾1.0g/L，琼脂15g/L，pH6.5；
液体摇瓶种子培养基：蔗糖20g/L、大豆蛋白胨10g/L、酵母粉10g/L、 硫酸镁0.5g/L、磷酸二氢钾1g/L；
发酵罐种子培养基：葡萄糖20g/L、大豆蛋白胨10g/L、酵母粉10g/L、硫酸镁0.5g/L、磷酸二氢钾1g/L。
实施例2 液体培养
A、斜面试管菌种活化培养
将罗伯茨虫草菌种接种于斜面试管培养基，温度22±1℃培养15天，待菌丝长满试管，得到斜面菌种；
B、液体摇瓶种子培养
在500ml三角瓶中装入200ml摇瓶种子培养基，在121℃高温高压下灭菌30min，自然冷却至室温，将斜面菌种接种到液体摇瓶种子培养基上，在温度22±1℃、150r/min条件下恒温振荡培养，培养时间为4天，得到液体摇瓶种子；
C、发酵罐种子培养
在发酵罐中装入发酵种子培养基，在121℃高温高压下灭菌30min，自然冷却至室温，将摇瓶种子液按照5％接种到培养基上，在温度22±1℃、搅拌速率150r/min条件下，通气量0.5vvm发酵培养，发酵时间为3天，得到发酵罐种子液。
液体培养阶段的培养基：
斜面试管培养基：蔗糖20gg、胰蛋白胨10g/L、酵母粉10g/L、硫酸镁1.0g/L、磷酸二氢钾2.0g/L，琼脂20g/L，pH6.5；
液体摇瓶种子培养基：葡萄糖20g/L、胰蛋白胨5g/L、酵母粉10g/L、硫酸镁1g/L、磷酸二氢钾2g/L；
发酵罐种子培养基：葡萄糖20g/L、胰蛋白胨10g/L、酵母粉10g/L、硫酸镁1g/L、磷酸二氢钾2g/L。
实施例3 固体培养
固体培养基的制备：在直径10厘米，高为15cm培养瓶中加入50g固体培养料，按料水比1:1.5的比例加入饮用水，pH值为6～6.5，混合均匀，盖上盖子，在126℃±2℃灭菌40～50min，自然冷却，得到固体培养基；固体培养基组成为：大米32g，小米10g，蚕蛹粉8g，饮用水75ml；
接种：在无菌条件下，将实施例1制备的发酵罐种子用喷枪接种到固体培养基表面，每瓶固体培养基中的接种量为5ml；
菌丝体生长阶段：将培养瓶置于20℃的人工气候室中，保持黑暗条件，相对湿度在70％～80％，培养10～15天，使菌丝长满培养基表面；
子实体生长阶段：打开光源，保证有充足的光照，光照强度为50～100lux，相对湿度70％～80％、温度18℃，每4～5小时换气一次；10天后子实体原基开始形成；培养50～60天，子实体长满瓶子，不再生长时即得到人工培养的罗伯茨虫草子实体；采收。
培养的子实体见图1。子实体线型，直立或弯曲，浅褐色至褐色，单生，极少数简单分叉，长10～15cm，宽0.5～3mm；生物转化率为15％(所述“生物转化率”为单位重量的固体培养基培养得到的子实体产量。即用1公斤培养基，可以培养出干的子实体150克)。
实施例4
固体培养基的制备：在直径10厘米，高为15cm培养瓶中加入50g固体培养料，按料水比1:1.8的比例加入饮用水，pH值为6～6.5，混合均匀，盖上盖子，在126℃±2℃灭菌40～50min，自然冷却，得到固体培养基；固体培养基组成为：大米40g，牛肉粉5g，饮用水80ml。
接种：在无菌条件下，将实施例1制备的发酵罐种子用喷枪接种到固体培养基表面，每瓶固体培养基中的接种量为10ml；
菌丝体生长阶段：将培养瓶置于22℃的人工气候室中，保持黑暗条件；相对湿度在70％～80％，培养10～15天，使菌丝长满培养基表面；
子实体生长阶段：打开光源，保证有充足的光照，光照强度为50～100lux，相对湿度70％～80％、温度19℃，每4～5小时换气一次。10天后子实体原基开始形成；培养50～60天，子实体长满瓶子，不再生长时即得到人工培养的罗伯茨虫草子实体；采收。
子实体线型，直立或弯曲，浅褐色至褐色，单生，极少数简单分叉，长7～10cm，宽0.5～2.5mm；生物转化率为10％。
实施例5
固体培养基的制备：在长、宽、高分别为30cm×30cm×25cm培养盒中加 入500g固体培养料，按料水比1:1.7的比例加入饮用水，pH值为6～6.5，混合均匀，用带透气膜的培养袋罩住，用尼龙绳扎紧袋口，在121℃高温灭菌40min，自然冷却，得到固体培养基；固体培养基组成为：燕麦450g，蚕蛹粉50g，饮用水850ml；
接种：在无菌条件下，将实施例2制备的发酵罐种子用喷枪接种到固体培养基表面，每盒固体培养基中的接种量为100ml；
菌丝体生长阶段：将培养盒置于20℃的人工气候室中，保持黑暗条件；相对湿度在70％～80％，培养10～15天，使菌丝长满培养基表面；
子实体生长阶段：打开光源，保证有充足的光照，光照强度为50～200lux，相对湿度70％～80％、温度18℃，每4～5小时换气一次。10天后子实体原基开始形成；培养50～60天，子实体长满盒子，不再生长时即得到人工培养的罗伯茨虫草子实体；采收。培养的子实体见图2。
实施例6
固体培养基的制备：在长、宽、高分别为30cm×30cm×25cm培养盒中加入500g固体培养料，按料水比1:1.6的比例加入饮用水，pH值为6～6.5，混合均匀，用带透气膜的培养袋罩住，用尼龙绳扎紧袋口，在121℃高温灭菌40min，自然冷却，得到固体培养基；固体培养基组成为：大米450g，蚕蛹粉50g，饮用水800ml；
接种：在无菌条件下，将实施例2制备的发酵罐种子用喷枪接种到固体培养基表面，每盒固体培养基中的接种量为100ml；
菌丝体生长阶段：将培养盒置于22℃的人工气候室中，保持黑暗条件；相对湿度在50％～60％，培养10～15天，使菌丝长满培养基表面；
子实体生长阶段：打开光源，保证有充足的光照，光照强度为50～200lux，相对湿度70％～80％、温度19℃，每4～5小时换气一次。10天后子实体原基开始形成；培养50～60天，子实体长满盒子，不再生长时即得到人工培养的罗伯茨虫草子实体；采收。
实施例4-6人工培养的罗伯茨虫草子实体线型，直立或弯曲，浅褐色至褐色，单生，极少数简单分叉，长10～15cm，宽0.5～3mm。
实施例7 固体培养基考察
实验目的：考察固体培养基中加入蚕蛹粉与不加入蚕蛹粉对子实体生长的影响。
实验方法：
1、种子液的制备：参照实施例1制备发酵罐种子液。
2、固体培养基的制备：在直径10厘米，高为15cm培养瓶中加入50g固体培养料，按料水比1:1.5的比例加入饮用水，pH值为6～6.5，混合均匀，盖上盖子，在126℃±2℃灭菌40～50min，自然冷却，得到固体培养基；
实验组固体培养基组成为：大米32g，小米10g，蚕蛹粉8g，饮用水75ml；
对照组固体培养基组成为：大米32g，小米10g，饮用水63ml。
3、接种量、菌丝体生长阶段和子实体生长阶段的培养条件与实施例3相同。
培养的子实体见图3，其中A为培养基中加入蚕蛹粉，B为培养基中不加入蚕蛹粉。
由图3可见，加入蚕蛹粉的实验组子实体粗壮、旺盛，而不加入蚕蛹粉的对照组仅产生稀疏的极少量子实体。发明人又尝试变换固体培养基的主料，分别以燕麦、小麦、大麦、糙米制备固体培养基，并尝试加入营养成分，如酵母粉、磷酸二氢钾、硫酸镁，也均只能产生极少量细小的子实体。结果也均只能产生极少量细小的子实体。
实施例8 培养温度的考察
实验目的：考察子实体生长阶段培养温度对子实体生长的影响。
实验方法：
1、种子液的制备：参照实施例1制备发酵罐种子液。
2、固体培养基的制备、接种量以及菌丝体生长阶段的培养条件同实施例3。
3、子实体生长阶段：打开光源，保证有充足的光照，光照强度为50～100lux，相对湿度70％～80％、分别设置三个培养温度：15℃、18℃和22℃，每4～5小时换气一次；培养50～60天。
培养的子实体见图4，其中A为培养温度为18℃，B为培养温度为15℃，C为培养温度为22℃。
由图4可见，子实体生长阶段培养温度为22℃时，根本无子实体产生；培养温度为15℃时，子实体短小、较细(长3～3.5cm，宽1～2mm，生物转化率为6.2％)，发明人尝试延长培养时间，但对子实体的生长无明显改变；而当培养温度为18℃时，子实体粗壮、旺盛(长10～15cm，宽0.5～3mm；生物转化率为15％)。可见，对于罗伯茨虫草，最佳的子实体生长温度为18℃。

Claims (10)

  1. 一种罗伯茨虫草的培养方法，该方法包括液体培养、固体培养和采收的过程；其特征在于，所述固体培养的培养基包括谷物和动物蛋白。
  2. 如权利要求1所述的培养方法，其特征在于，所述谷物选自大米、小米、糙米、燕麦、小麦、大麦、高粱中的任意一种或几种。
  3. 如权利要求1所述的培养方法，其特征在于，所述动物蛋白为牛肉粉或蚕蛹粉。
  4. 如权利要求3所述的培养方法，其特征在于，所述动物蛋白的添加量为谷物的7.5％～25％。
  5. 如权利要求4所述的培养方法，其特征在于，所述动物蛋白的添加量为谷物的8％～22％或9％～20％或10％或19％。
  6. 如权利要求1所述的培养方法，其特征在于，所述固体培养过程的子实体生长阶段采用光照培养，培养温度为17℃～19℃。
  7. 如权利要求6所述的培养方法，其特征在于，所述培养温度为18℃。
  8. 如权利要求6所述的培养方法，其特征在于，所述固体培养过程的菌丝体生长阶段采用避光培养，培养温度为20℃～22℃。
  9. 如权利要求6所述的培养方法，其特征在于，所述固体培养过程的接种量为7％～22％。
  10. 如权利要求9所述的培养方法，其特征在于，所述固体培养过程的接种量为10％、15％或20％。
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