WO2018018476A1 - 一种牛樟芝皿式培养方法 - Google Patents

一种牛樟芝皿式培养方法 Download PDF

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WO2018018476A1
WO2018018476A1 PCT/CN2016/091960 CN2016091960W WO2018018476A1 WO 2018018476 A1 WO2018018476 A1 WO 2018018476A1 CN 2016091960 W CN2016091960 W CN 2016091960W WO 2018018476 A1 WO2018018476 A1 WO 2018018476A1
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culture
medium
antrodia camphorata
mycelium
dish
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PCT/CN2016/091960
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English (en)
French (fr)
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赵宗杰
张向阳
方晓
柴行
刘家飞
张康
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深圳市仁泰生物科技有限公司
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Priority to PCT/CN2016/091960 priority Critical patent/WO2018018476A1/zh
Publication of WO2018018476A1 publication Critical patent/WO2018018476A1/zh

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  • the invention belongs to the field of cultivation of Antrodia camphorata, and in particular relates to a method for cultivating a burdock.
  • Antrodia camphorata also known as burdock mushroom, belongs to the genus Basidiomycetes, Polyporaceae, and Phaeoporus. Perennial fungi are rare medicinal fungi that are parasitic on the decaying inner wall of the unique tree species of Burdock in Taiwan, known as the ruby of the forest. Antrodia camphorata contains a variety of physiologically active ingredients, such as polysaccharides, triterpenoids, superoxide dismutase (SOD) adenosine, protein (including immune proteins), multivitamins, trace elements, nucleic acids, lectins, amino acids, cholesterol, Lignin, blood pressure stabilizing substances, etc.
  • physiologically active ingredients such as polysaccharides, triterpenoids, superoxide dismutase (SOD) adenosine, protein (including immune proteins), multivitamins, trace elements, nucleic acids, lectins, amino acids, cholesterol, Lignin, blood pressure stabilizing substances, etc
  • physiologically active functions include anti-tumor, immune-enhancing, anti-viral, anti-allergic, anti-hypertensive, anti-platelet aggregation, blood pressure lowering, cholesterol lowering, anti-bacterial, liver protection, etc.
  • triterpenoids are medicinal fungi.
  • the core pharmacological component has strong physiological activity
  • the triterpenoid compound is a main component of medicinal fungi exerting anti-inflammatory, analgesic, poisoning and killing tumor cells, and inducing apoptosis and anti-oxidation of tumor cells;
  • the role of immunity is to promote lymphocyte proliferation, improve the phagocytic capacity and lethality of macrophages, NK cells and T cells; and effectively promote hepatocyte regeneration, repair and improve liver function, effectively reducing patients during chemotherapy Liver and kidney damage.
  • there are more than 280 species of triterpenoids in Antrodia camphorata there are more than 280 species of triterpenoids in Antrodia camphorata, and its potential value is immeasurable.
  • CN 104322279A A method for cultivating Antrodia camphorata by arborvitae is to first culture the burdock strain in a culture dish, and then prepare the burdock bacillus liquid solution, and then immerse the eucalyptus in the burdock bacillus liquid for 50-60 hours, and remove it after no The whole environment is monitored and cultured in a bacterial environment, and the temperature, humidity, air quality and illumination required for the growth of the body of the Antrodia camphorata are given. The measured triterpenoid content of the Antrodia camphorata fruit body reached 35%, which is close to 37% of the wild Antrodia camphorata.
  • CN 105483160 A a culture composition of Antrodia camphorata and a preparation method thereof, the seed of Antrodia camphorata is added to a solid medium containing money lotus and bright grass, and cultured in an alternating magnetic field environment, and the obtained fermentation product of Antrodia camphorata is subjected to subcritical extraction.
  • the obtained extract was determined to have a maximum content of 1.61% by the content of triterpenoids.
  • CN105145112A A method for cultivating the fruit body of Antrodia camphorata using the cerebral palsy section, which is based on the wood of the cerebral palsy, is immersed in water, and is sterilized by high temperature sterilization, and then cultured with the genus Antrodia camphorata, the content of the scorpion is not Less than 80mg/100g.
  • CN104855137A A method for improving the yield and biologically active substance content of Antrodia camphorata, wherein the roots, stems and leaves of the pine are mixed in different proportions, subjected to ultrasonication treatment, and supercritical fluid extraction is used to extract the five-leaf turpentine to obtain different pH. Extract.
  • the pH of the growth environment of A. angustifolia mycelium is maintained between 5 and 7 during the cultivation of Antrodia camphorata by adding the corresponding pH of the extract of A. sinensis, and the concentration of carbon dioxide is controlled to be 1 to 5%.
  • the oxygen concentration is 5 to 15%, and the content of triterpenoids in the mycelium obtained by the final fermentation is 8000 ppm.
  • the current artificial cultivation of Antrodia camphorata is divided into three methods, 1) liquid fermentation method, 2) solid cultivation method, and 3) eucalyptus cultivation method.
  • the eucalyptus cultivation method is the closest culture method for the cultured burdock and the wild burdock triterpenoids, but it also has certain defects, such as long cultivation time, high cost and less eucalyptus resources.
  • the technical problem to be solved by the present invention is to provide a method for cultivating a burdock dish, which aims to quickly obtain the fruit body of Antrodia camphorata and increase the content of triterpenoids in Antrodia camphorata.
  • the present invention is achieved by a method for cultivating a burdock dish, the culturing method comprising:
  • Medium preparation step weigh 5-40 g/L of bran, 5-40 g/L of corn starch, 5-40 g/L of agar, 1-3 g/L of peptone, 0.5 ⁇ according to the mass to volume ratio of water. 4.0 g/L of magnesium sulfate and 0.5-10 ml/L of olive oil to prepare a culture medium for the strain of Antrodia camphorata;
  • Inoculation step inoculation of the A. serrata strain on the medium in a sterile environment
  • Culture step the cultured medium is cultured in a constant temperature and humidity environment for 3 to 12 months, and the temperature, humidity and illumination time are adjusted according to the growth characteristics of Antrodia camphorata.
  • the invention has the beneficial effects that the bovine oyster dish culture method provided by the invention combines different physiological characteristics of the fermentation stage and the culture stage of the genus Antrodia camphorata, and adopts a unique medium formula to provide sufficient nutrient demand. And help to improve the content of triterpenoids in Antrodia camphorata; coating inoculation with liquid Mycelium mycelium helps to rapid growth of Antrodia camphorata in the early stage, greatly shortening the maturity cycle of fruiting bodies, and the shortest growth cycle is reduced to 3 month. Further, according to the different growth stages of O.
  • the corresponding temperature, humidity and illumination time are adjusted to promote the growth of the mycelium of Antrodia camphorata and the formation of triterpenoids of the active ingredient, higher temperature, high humidity and longer illumination time. It helps the rapid growth of mycelium of Antrodia camphorata. The lower temperature, medium humidity and dark environment are more conducive to the growth of Antrodia camphorata fruit body and accelerate the secondary metabolism of Antrodia camphorata, accumulating more triterpenoids.
  • the present invention further optimizes the preparation process and the inoculation process of the strain of Antrodia camphorata, which all contribute to the maximum effect of the technical effect of the present invention.
  • the invention solves the big problem that the content of triterpenoids in the artificial culture of Antrodia camphorata is low, and is a breakthrough in the research of artificial culture of Antrodia camphorata, and has great significance for the research and future development of the triterpenoids of Antrodia camphorata.
  • Dish culture as the most primitive fungal culture method, can quickly obtain the fruiting body of Antrodia camphorata, such as the development of a high-yield triterpenoid compound Antrodia camphora culture method, which will solve the problem of the lack of Burdock tree and improve the application range of Antrodia camphorata. And application value.
  • the invention provides a method for cultivating a burdock dish, comprising:
  • Medium preparation step weigh 5-40 g/L of bran, 5-40 g/L of corn starch, 5-40 g/L of agar, 1-3 g/L of peptone, 0.5 ⁇ according to the mass to volume ratio of water. 4.0 g/L of magnesium sulfate and 0.5-10 ml/L of olive oil to prepare a culture medium for the strain of Antrodia camphorata;
  • Inoculation step inoculating the Antrodia camphorata strain on the culture medium under a sterile environment
  • Culture step the cultured medium is cultured in a constant temperature and humidity environment for 3 to 12 months.
  • the present invention not only improves the specific formulation, but also optimizes the amount of each raw material in the formulation, so that the content of the triterpenoids in the obtained Antrodia camphorata is significantly improved.
  • the culture condition in the culturing step is: before the mycelium is not filled with the culture dish, the temperature is 25 to 30 ° C, the humidity is 85% to 95%, preferably 95%, and the daily illumination time is 4 to 6 Hours, the light intensity is 0 ⁇ 400 lux (lux); after the mycelium is overgrown with the culture dish, the temperature is 18 ⁇ 22 ° C, the humidity is 65% ⁇ 75%, preferably 65%, the daily light time is 2 ⁇ 4 hours, light The strength is 0 to 400 lux.
  • the method for cultivating the burdock cultivar provided by the present embodiment combined with the different physiological characteristics of the fermentation stage and the culture stage of the genus Antrodia camphorata, adopts a unique medium formula, can provide sufficient nutrient requirements, and contributes to the improvement of the triterpenoids of Antrodia camphorata. content.
  • the parameters of the medium culture process were improved, and the strains of A. annuum were better grown.
  • the content of triterpenoids in A. annuum was further improved after culture.
  • the preparation process of the medium is specifically:
  • the filtrate is filtered; corn starch is added to the filtrate, and the gelatinization is carried out; agar, peptone, magnesium sulfate, olive oil are sequentially added, and the mixture is stirred to obtain a volume of the mixed liquid.
  • the columnar medium has a thickness of 1 to 3 cm.
  • the A. serrata strain is inoculated on the culture dish medium in the form of a culture medium, and the preparation process of the culture medium is:
  • the culture condition of the Antrodia camphorata strain in the liquid seed culture medium is: a temperature of 28 ° C, a rotation speed of 160 rpm, and a time of 15 days.
  • the inoculation process is specifically: adding 2 to 5 mL of the culture solution of the strain on the dish medium, and coating the culture solution of the strain into a circle having a diameter of 5 to 8 cm by a coating inoculation method. .
  • the liquid inoculum is coated with liquid mycelium, which helps the rapid growth of Antrodia camphorata in the initial stage, greatly shortens the maturity cycle of the fruiting body, and the shortest growth period is reduced to 3 months.
  • the formula and related parameters of the culture medium of Antrodia camphorata By optimizing the formula and related parameters of the culture medium of Antrodia camphorata, the effect of the strain of Antrodia camphorata was improved.
  • the culturing step further comprises a drying step comprising: separating the mycelium from the culture dish, and drying at a temperature of 40 to 50 ° C until the moisture content is less than 1.8% to obtain a sample of Antrodia camphorata.
  • the strain of Antrodia camphorata is ATCC200183.
  • the invention finally determines the culture condition of the high-yield triterpenoid compound Antrodia camphorata by using a special medium formula and a large number of repeated comparison experiments on the medium dosage, temperature, humidity, culture time and the like.
  • Preparation of medium Weigh 20g/L of bran and add water for 40min. After filtering, take the filtrate, then add 20g/L of corn starch into the filtrate of bran. Stir well and heat paste. After gelatinization is completed. Add agar 20g / L, peptone 1.5g / L, magnesium sulfate 2.0g / L olive oil 2ml / L, stir to dissolve completely, then hydrated to 1 L. It is autoclaved by a sterilizer for 20 minutes, and poured into a Petri dish with a thickness of about 2 cm.
  • liquid seed medium composition glucose: 20.0g / L, malt extract 20.0g / L, peptone 1.0g / L, magnesium sulfate 0.5 g / L, pH 4.0, distilled water to volume, autoclaved for 20min.
  • the spores of Antrodia camphorata were collected aseptically and placed in a shake flask for liquid culture.
  • the culture conditions were as follows: temperature 28 ° C, rotation speed 160 rpm, and culture for 15 days, to obtain a bacterial liquid which did not form an obvious bacterial ball.
  • Inoculation step inoculation by coating inoculation method, in a sterile environment, adding 2 to 5 mL of the culture solution of the shake flask to each culture dish, and then coating the bacterial solution with a coating stick to a diameter of about 5 ⁇ A circle of about 8cm.
  • Preparation of the medium Weigh the bran 10g/L and add water to cook for 40min. After filtering, take the filtrate, then add the corn starch 10g/L into the filtrate of the bran, stir evenly and then heat the gelatinization. After the gelatinization is completed. Add agar 15g / L, peptone 1.0g / L, magnesium sulfate 1.0g / L olive oil 1ml / L, stir to dissolve completely, hydrated to 1 L. It is autoclaved by a sterilizer for 20 minutes, and poured into a Petri dish with a thickness of about 2 cm.
  • liquid seed medium composition glucose: 20.0g / L, malt extract 20.0g / L, peptone 1.0g / L, magnesium sulfate 0.5 g / L, pH 4.0, distilled water to volume, autoclaved for 20min.
  • the spores of Antrodia camphorata were collected aseptically and placed in a shake flask for liquid culture.
  • the culture conditions were as follows: temperature 28 ° C, rotation speed 160 rpm, and culture for 15 days, to obtain a bacterial liquid which did not form an obvious bacterial ball.
  • Inoculation step inoculation by coating inoculation method, in a sterile environment, adding 2 to 5 mL of the culture solution of the shake flask to each culture dish, and then coating the bacterial solution with a coating stick to a diameter of about 5 ⁇ A circle of about 8cm.
  • Preparation of medium Weigh 20g/L of bran and add water for 40min. After filtering, take the filtrate, then add 20g/L of corn starch into the filtrate of bran. Stir well and heat paste. After gelatinization is completed. Add agar 20g / L, peptone 1.5g / L, magnesium sulfate 2.0g / L olive oil 2ml / L, stir to dissolve completely, then hydrated to 1 L. It is autoclaved by a sterilizer for 20 minutes, and poured into a Petri dish with a thickness of about 2 cm.
  • liquid seed medium composition glucose: 20.0g / L, malt extract 20.0g / L, peptone 1.0g / L, magnesium sulfate 0.5 g / L, pH 4.0, distilled water to volume, autoclaved for 20min.
  • the spores of Antrodia camphorata were collected aseptically and placed in a shake flask for liquid culture.
  • the culture conditions were as follows: temperature 28 ° C, rotation speed 160 rpm, and culture for 15 days, to obtain a bacterial liquid which did not form an obvious bacterial ball.
  • Inoculation step inoculation by coating inoculation method, in a sterile environment, adding 2 to 5 mL of the culture solution of the shake flask to each culture dish, and then coating the bacterial solution with a coating stick to a diameter of about 5 ⁇ A circle of about 8cm.
  • Preparation of medium Weigh 20g/L of bran and add water for 40min. After filtering, take the filtrate, then add 20g/L of corn starch into the filtrate of bran. Stir well and heat paste. After gelatinization is completed. Add agar 20g / L, peptone 1.5g / L, magnesium sulfate 2.0g / L olive oil 2ml / L, stir to dissolve completely, then hydrated to 1 L. The tube was sterilized by autoclaving for 20 minutes, and poured into a Petri dish with a thickness of about 1 cm.
  • liquid seed medium composition glucose: 20.0g / L, malt extract 20.0g / L, peptone 1.0g / L, magnesium sulfate 0.5 g / L, pH 4.0, distilled water to volume, autoclaved for 20min.
  • the spores of Antrodia camphorata were collected aseptically and placed in a shake flask for liquid culture.
  • the culture conditions were as follows: temperature 28 ° C, rotation speed 160 rpm, and culture for 15 days, to obtain a bacterial liquid which did not form an obvious bacterial ball.
  • Inoculation step inoculation by coating inoculation method, in a sterile environment, adding 2 to 5 mL of the culture solution of the shake flask to each culture dish, and then coating the bacterial solution with a coating stick to a diameter of about 5 ⁇ A circle of about 8cm.
  • Preparation of medium Weigh 20g/L of bran and add water for 40min. After filtering, take the filtrate, then add 20g/L of corn starch into the filtrate of bran. Stir well and heat paste. After gelatinization is completed. Add agar 20g / L, peptone 1.5g / L, magnesium sulfate 2.0g / L olive oil 2ml / L, stir to dissolve completely, then hydrated to 1 L. It is autoclaved by a sterilizer for 20 minutes, and poured into a Petri dish with a thickness of about 2 cm.
  • liquid seed medium composition glucose: 20.0g / L, malt extract 20.0g / L, peptone 1.0g / L, magnesium sulfate 0.5 g / L, pH 4.0, distilled water to volume, autoclaved for 20min.
  • the spores of Antrodia camphorata were collected aseptically and placed in a shake flask for liquid culture.
  • the culture conditions were as follows: temperature 28 ° C, rotation speed 160 rpm, and culture for 15 days, to obtain a bacterial liquid which did not form an obvious bacterial ball.
  • Inoculation step inoculation by coating inoculation method, in a sterile environment, adding 2 to 5 mL of the culture solution of the shake flask to each culture dish, and then coating the bacterial solution with a coating stick to a diameter of about 5 ⁇ A circle of about 8cm.
  • Preparation of medium Weigh 20g/L of bran and add water for 40min. After filtering, take the filtrate, then add 20g/L of corn starch into the filtrate of bran. Stir well and heat paste. After gelatinization is completed. Add agar 20g / L, peptone 1.5g / L, magnesium sulfate 2.0g / L olive oil 2ml / L, stir to dissolve completely, then hydrated to 1 L. It is autoclaved by a sterilizer for 20 minutes, and poured into a Petri dish with a thickness of about 2 cm.
  • liquid seed medium composition glucose: 20.0g / L, malt extract 20.0g / L, peptone 1.0g / L, magnesium sulfate 0.5 g / L, pH 4.0, distilled water to volume, autoclaved for 20min.
  • the spores of Antrodia camphorata were collected aseptically and placed in a shake flask for liquid culture.
  • the culture conditions were as follows: temperature 28 ° C, rotation speed 160 rpm, and culture for 15 days, to obtain a bacterial liquid which did not form an obvious bacterial ball.
  • Inoculation step inoculation by coating inoculation method, in a sterile environment, adding 2 to 5 mL of the culture solution of the shake flask to each culture dish, and then coating the bacterial solution with a coating stick to a diameter of about 5 ⁇ A circle of about 8cm.
  • Preparation of medium Weigh 20g/L of bran and add water for 40min. After filtering, take the filtrate, then add 20g/L of corn starch into the filtrate of bran. Stir well and heat paste. After gelatinization is completed. Add agar 20g / L, peptone 1.5g / L, magnesium sulfate 2.0g / L olive oil 2ml / L, stir to dissolve completely, then hydrated to 1 L. It is autoclaved by a sterilizer for 20 minutes, and poured into a Petri dish with a thickness of about 2 cm.
  • liquid seed medium composition glucose: 20.0g / L, malt extract 20.0g / L, peptone 1.0g / L, magnesium sulfate 0.5 g / L, pH 4.0, distilled water to volume, autoclaved for 20min.
  • the spores of Antrodia camphorata were collected aseptically and placed in a shake flask for liquid culture.
  • the culture conditions were as follows: temperature 28 ° C, rotation speed 160 rpm, and culture for 15 days, to obtain a bacterial liquid which did not form an obvious bacterial ball.
  • Inoculation step inoculation by coating inoculation method, in a sterile environment, adding 2 to 5 mL of the culture solution of the shake flask to each culture dish, and then coating the bacterial solution with a coating stick to a diameter of about 5 ⁇ A circle of about 8cm.

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Abstract

一种牛樟芝皿式培养方法，包括：培养基制备步骤：按照与水的质量体积比称取5～40g/L的麸皮、5～40g/L的玉米淀粉、5～40g/L的琼脂、1～3g/L的蛋白胨、0.5～4.0g/L的硫酸镁以及0.5～10ml/L的橄榄油，制备牛樟芝菌种的皿式培养基；接种步骤：在无菌环境下，将牛樟芝菌种接种于所述培养基上；培养步骤：将接种后的培养基置于恒温恒湿环境中培养3～12个月。该培养方法通过改进培养基所用配方和优化培养步骤，使所得的牛樟芝中三萜类化合物含量显著提高。

Description

一种牛樟芝皿式培养方法 技术领域
本发明属于牛樟芝栽培领域，尤其涉及一种牛樟芝皿式培养方法。
背景技术
牛樟芝(Antrodia camphorata)又名牛樟菇，属担子菌纲、多孔菌科、薄孔菌属、 多年生蕈菌类，是寄生于我国台湾特有树种牛樟树腐朽内壁的珍稀药用真菌，被称为森林的红宝石。牛樟芝中含有多种生理活性成分，如多糖、三萜类化合物、超氧歧化酶（SOD）腺苷、蛋白质（含免疫蛋白）、多种维生素、微量元素、核酸、凝集素、氨基酸、胆固醇、木质素、血压稳定物质等。其具备的生理活性功能有抗肿瘤、增强免疫力、抗病毒、抗过敏、抗高血压、抑制血小板凝集、降血压、降胆固醇、抗细菌、保护肝脏等；其中三萜类化合物是药用真菌的核心药理成分，具有强烈的生理活性，三萜类化合物是药用真菌发挥抗炎、镇痛、毒杀肿瘤细胞及诱发肿瘤细胞凋亡、抗氧化等作用的主要成分；而且还具有提高机体免疫力的作用，表现在促进淋巴细胞增生，提高巨噬细胞、NK细胞、T细胞的吞噬能力和杀伤力；并能够有效地促进肝细胞再生、修复和提升肝脏机能，有效地减少化疗期间病人肝脏和肾脏的损伤。据国内外的报道，牛樟芝中三萜类化合物的种类多达280种以上，其潜在的价值不可估量。
国内专利中已有很多相关于牛樟芝三萜含量的报道，如：
CN 104322279A：一种椴木培养牛樟芝的方法，是先将牛樟芝菌种在培养皿中培养，再制取牛樟芝菌液，然后将椴木置于牛樟芝菌液中浸泡50—60小时，取出后在无菌环境中进行全天候监控培养，给予牛樟芝子实体成长所需的温度、湿度、空气质量和光照。通过测量得到的牛樟芝子实体的三萜含量，达到35%，跟野生的牛樟芝的37%很接近。
CN 105483160 A：一种牛樟芝培养组合物及其制备方法，是将牛樟芝种子接入含有金钱莲和明月草的固体培养基中，置于交变磁场环境中培养，得到的牛樟芝发酵产物经过亚临界萃取，得到的萃取物中通过三萜含量的测定，其含量最高为1.61%。
CN105145112A：一种得用龙脑樟段木培育牛樟芝子实体的方法，是以龙脑樟段木为基质，经水浸泡处理，高温灭菌后接上牛樟芝菌种培养，其三萜的含量不少于80mg/100g。
CN104855137A：一种提高牛樟芝产量及其生物活性物质含量的方法，是将五叶松的根、茎、叶以不同的比例混合，经过超声破碎处理，超临界流体萃取提取五叶松脂，得到不同pH的提取液。采用谷类作为培养基，在牛樟芝的培养过程中通过添加相应pH的五叶松提取液来控制牛樟芝菌丝体生长环境的pH值保持在5～7之间，同时控制二氧化碳浓度为1～5%、氧气浓度为5～15%，最终发酵得到的菌丝体中三萜的含量为8000ppm。
总结来说，目前牛樟芝人工培育分为三种方法，1）液体发酵法，2）固体栽培法，3）椴木栽培法。椴木栽培法是目前培养出来的牛樟芝与野生牛樟芝三萜成份最接近的一种培养方法，但是其也存在一定的缺陷，如：培养时间长、成本花费高、椴木资源少。
 技术问题
本发明所要解决的技术问题在于提供一种牛樟芝皿式培养方法，旨在快速得到牛樟芝的子实体，同时提高牛樟芝中三萜类化合物的含量。
 技术解决方案
本发明是这样实现的，一种牛樟芝皿式培养方法，所述培养方法包括：
培养基制备步骤：按照与水的质量体积比称取5～40g/L的麸皮、5～40g/L的玉米淀粉、5～40g/L的琼脂、1～3g/L的蛋白胨、0.5～4.0g/L的硫酸镁以及0.5～10ml/L的橄榄油，制备牛樟芝菌种的培养皿式培养基；
接种步骤：在无菌环境下，采用涂布法将牛樟芝菌种接种于所述培养基上；
培养步骤：将接种后的培养基置于恒温恒湿环境中培养3～12个月，并根据牛樟芝的生长特性调控温度、湿度、光照时间。
 有益效果
本发明与现有技术相比，有益效果在于：本发明提供的牛樟芝皿式培养方法，结合牛樟芝菌种发酵阶段和培养阶段的不同生理特性，采用独特的培养基配方，能提供充足的营养需求，并且有助于提高牛樟芝三萜类化合的含量；采用液态樟芝菌丝体涂布接种，有助于在初期阶段牛樟芝快速生长，大大缩短子实体的成熟周期，最短生长周期降为3个月。进一步地，根据牛樟芝不同生长阶段调控相对应的温度、湿度、光照时间，促进樟芝菌丝体的生长和活性成分三萜类化合物的生成，较高温度、高湿度和较长的光照时间，有助于樟芝菌丝体的快速生长，较低温度中等湿度和阴暗环境更有助于樟芝子实体的生长和加速樟芝的次代谢，积累更多的三萜类化合物。此外，本发明还进一步优化了牛樟芝菌种的制备过程和接种过程，这些都有助于牛樟芝菌种最大限度发挥本发明的技术作用。
本发明解决了牛樟芝人工培养三萜类化合物含量低的大难题，是人工培养牛樟芝研究上的一大突破，对牛樟芝三萜类化合物的研究和今后的发展具有重大的意义。
 本发明的实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
皿式培养，作为最原始的真菌培养方法，能快速得到牛樟芝的子实体，如能开发出一种高产三萜类化合物牛樟芝皿式培养方法，将解决牛樟树缺乏等问题，提高牛樟芝的应用范围及应用价值。
本发明提供了一种牛樟芝皿式培养方法，包括：
培养基制备步骤：按照与水的质量体积比称取5～40g/L的麸皮、5～40g/L的玉米淀粉、5～40g/L的琼脂、1～3g/L的蛋白胨、0.5～4.0g/L的硫酸镁以及0.5～10ml/L的橄榄油，制备牛樟芝菌种的培养皿式培养基；
接种步骤：在无菌环境下，将牛樟芝菌种接种于所述培培养基上；
培养步骤：将接种后的培养基置于恒温恒湿环境中培养3～12个月。
在培养基制备步骤中，本发明不仅改进了具体配方，且配方中各原料的用量也进行了优化，使所得的牛樟芝中三萜类化合物含量显著提高。
具体地，所述培养步骤中的培养条件为：在菌丝体未长满培养皿之前，温度为25～30℃、湿度为85%～95%，优选95%，每天光照时间为4～6小时，光照强度为0～400lux（勒克斯）；菌丝体长满培养皿后，温度为18～22℃、湿度为65%～75%，优选65%，每天光照时间为2～4小时，光照强度为0～400lux。
本实施例提供的牛樟芝皿式培养方法，结合牛樟芝菌种发酵阶段和培养阶段的不同生理特性，采用独特的培养基配方，能提供充足的营养需求，并且有助于提高牛樟芝三萜类化合的含量。对培养基培养过程各参数均进行了改进，使牛樟芝菌种更好地生长，经培养后所得的牛樟芝中三萜类化合物含量进一步提高。
具体地，所述培养基制备过程具体为：
将麸皮加入水中煮40min后，过滤得滤液；将玉米淀粉加入所述滤液中，加热糊化；依次加入琼脂、蛋白胨、硫酸镁、橄榄油，搅拌后得混合液，将所得混合液的体积加水补至水的初始体积；高压灭菌20min，趁热倒至培养皿中，冷却凝固得柱状培养基。所述柱状培养基的厚度为1～3cm。
具体地，所述接种步骤中，所述牛樟芝菌种是以菌种培养液的形式接种于所述培养皿式培养基上，所述菌种培养液的制备过程为：
按照与水的质量体积比称取20.0g/L的葡萄糖、20.0g/L的麦芽浸膏、1.0 g/L的蛋白胨以及0.5 g/L的硫酸镁，加入水中得混合液，调节所得混合液的pH值至4.0；高压蒸汽灭菌20min后得液体种子培养基；无菌条件下将牛樟芝菌种接入液体种子培养基中进行培养，获得菌种培养液。
具体地，所述牛樟芝菌种在所述液体种子培养基中的培养条件为：温度28℃，转速160rpm，时间15天。
具体地，所述接种过程具体为：在所述皿式培养基上加入2~5mL所述菌种培养液，采用涂布接种法将所述菌种培养液涂成直径为5～8cm的圆圈。
本实施例采用液态樟芝菌丝体涂布接种，有助于在初期阶段牛樟芝快速生长，大大缩短子实体的成熟周期，最短生长周期降为3个月。通过优化牛樟芝菌种培养基的配方及相关参数，提高了牛樟芝菌种的作用效果。
所述培养步骤后还包括干燥步骤，所述干燥步骤包括：将菌丝体从所述培养皿中分离出来，置于40～50℃温度下干燥至水分含量低于1.8%，获得牛樟芝样品。
具体地，所述牛樟芝菌菌种为ATCC200183。
本发明通过运用特殊的培养基配方，对培养基用量、温度、湿度、培养时间等条件进行大量反复比对实验的基础上，最终确定了高产三萜类化合物牛樟芝培养条件。
实施例1
（1）培养基制备步骤：称取麸皮20g/L加水后煮40min，过滤后取滤液，再加入玉米淀粉20g/L进麸皮的滤液中，搅拌均匀后加热糊化，糊化完成后依次加入琼脂20g/L、蛋白胨1.5g/L、硫酸镁2.0g/L橄榄油2ml/L，搅拌使其溶解完全后，补水至1 L。通过灭菌锅高温高压灭菌20min， 趁热倒至培养皿中，厚度在2cm左右，放冷使其凝固。
（2）菌种准备：液体种子培养基组成：葡萄糖:20.0g/L，麦芽浸膏20.0g/L，蛋白胨1.0g/L，硫酸镁0.5 g/L，pH 4.0，蒸馏水定容，高压蒸汽灭菌20min。无菌收集牛樟芝孢子，接入摇瓶进行液体培养，培养的条件为温度28℃，转速160rpm，培养15天，得到未形成明显菌球的菌液。
（3）接种步骤：采用涂布接种法接种，在无菌的环境下，每个培养皿加入2~5mL摇瓶培养出来的菌液，再用涂布棒将菌液涂成直径大约5～8cm左右的圆圈。
（4）培养步骤：将接种后的培养皿倒置放在恒温恒湿环境中培养，在菌丝体未长满培养皿的2个月内，环境温度为28℃、湿度为90%，每天光照时间为6小时，光照强度为0～400lux，菌丝体在培养基上生长，慢慢长满整个平板，菌丝体的颜色从白色变成橘红色；2个月后，改变环境条件，温度为20℃、湿度为70%，每天光照时间为2小时，光照强度为0～400lux，菌丝体逐渐长厚，10个月后生长十分缓慢，此时樟芝呈橘红色，约有0.8cm厚。
（5）牛樟芝子实体的采收：将生长出来的菌丝体从培养皿中分出来，将带出来的培养基刮除干净，再用45℃水冲洗菌丝体的底部，再把得到的菌丝体放置烘箱中烘干至水分低于1.8%，温度控制在40～50℃之间。烘干粉碎后取1.0g样品，通过分光光度法测其三萜含量为：28.2%。
实施例2
（1）培养基制备步骤：称取麸皮10g/L加水后煮40min，过滤后取滤液，再加入玉米淀粉10g/L进麸皮的滤液中，搅拌均匀后加热糊化，糊化完成后依次加入琼脂15g/L、蛋白胨1.0g/L、硫酸镁1.0g/L橄榄油1ml/L，搅拌使其溶解完全后，补水至1 L。通过灭菌锅高温高压灭菌20min， 趁热倒至培养皿中，厚度在2cm左右，放冷使其凝固。
（2）菌种准备：液体种子培养基组成：葡萄糖:20.0g/L，麦芽浸膏20.0g/L，蛋白胨1.0g/L，硫酸镁0.5 g/L，pH 4.0，蒸馏水定容，高压蒸汽灭菌20min。无菌收集牛樟芝孢子，接入摇瓶进行液体培养，培养的条件为温度28℃，转速160rpm，培养15天，得到未形成明显菌球的菌液。
（3）接种步骤：采用涂布接种法接种，在无菌的环境下，每个培养皿加入2~5mL摇瓶培养出来的菌液，再用涂布棒将菌液涂成直径大约5～8cm左右的圆圈。
（4）培养步骤：将接种后的培养皿倒置放在恒温恒湿的环境中培养，前2个月，在菌丝体未长满培养皿之前，温度为25℃、湿度为90%，每天光照时间为6小时，光照强度为0～400lux，菌丝体在培养基上生长，慢慢长满整个平板，菌丝体的颜色从白色变成橘红色；菌丝体长至10个月，改变环境条件，温度为20℃、湿度为70%，每天光照时间为2小时，光照强度为0～400lux，橘红色菌丝体逐渐长厚，最后菌丝体约有0.8cm厚。
（5）牛樟芝子实体的采收：将生长出来的菌丝体从培养皿中分出来，将带出来的培养基刮除干净，再用45℃水冲洗菌丝体的底部，再把得到的菌丝体放置烘箱中烘干至水分低于1.8%，温度控制在40～50℃之间。烘干粉碎后取1.0g样品，通过分光光度法测其三萜含量为：21.4%。
实施例3
（1）培养基制备步骤：称取麸皮20g/L加水后煮40min，过滤后取滤液，再加入玉米淀粉20g/L进麸皮的滤液中，搅拌均匀后加热糊化，糊化完成后依次加入琼脂20g/L、蛋白胨1.5g/L、硫酸镁2.0g/L橄榄油2ml/L，搅拌使其溶解完全后，补水至1 L。通过灭菌锅高温高压灭菌20min， 趁热倒至培养皿中，厚度在2cm左右，放冷使其凝固。
（2）菌种准备：液体种子培养基组成：葡萄糖:20.0g/L，麦芽浸膏20.0g/L，蛋白胨1.0g/L，硫酸镁0.5 g/L，pH 4.0，蒸馏水定容，高压蒸汽灭菌20min。无菌收集牛樟芝孢子，接入摇瓶进行液体培养，培养的条件为温度28℃，转速160rpm，培养15天，得到未形成明显菌球的菌液。
（3）接种步骤：采用涂布接种法接种，在无菌的环境下，每个培养皿加入2~5mL摇瓶培养出来的菌液，再用涂布棒将菌液涂成直径大约5～8cm左右的圆圈。
（4）培养步骤：将接种后的培养皿倒置放在恒温恒湿的环境中培养，前2个月，在菌丝体未长满培养皿之前，温度为25℃、湿度为85%，每天光照时间为6小时，光照强度为0～400lux，菌丝体在培养基上生长，慢慢长满整个平板，菌丝体的颜色从白色变成橘红色；2个月后改变生长环境，温度为20℃、湿度为70%，每天光照时间为2小时，光照强度为0～400lux，菌丝体逐渐长厚，生长至3个月时，樟芝呈橘红色，约有0.2cm厚。
（5）牛樟芝子实体的采收：将生长出来的菌丝体从培养皿中分出来，将带出来的培养基刮除干净，再用45℃水冲洗菌丝体的底部，再把得到的菌丝体放置烘箱中烘干至水分低于1.8%，温度控制在40～50℃之间。烘干粉碎后取1.0g样品，通过分光光度法测其三萜含量为：8.4%。
实施例4
（1）培养基制备步骤：称取麸皮20g/L加水后煮40min，过滤后取滤液，再加入玉米淀粉20g/L进麸皮的滤液中，搅拌均匀后加热糊化，糊化完成后依次加入琼脂20g/L、蛋白胨1.5g/L、硫酸镁2.0g/L橄榄油2ml/L，搅拌使其溶解完全后，补水至1 L。通过灭菌锅高温高压灭菌20min， 趁热倒至培养皿中，厚度在1cm左右，放冷使其凝固。
（2）菌种准备：液体种子培养基组成：葡萄糖:20.0g/L，麦芽浸膏20.0g/L，蛋白胨1.0g/L，硫酸镁0.5 g/L，pH 4.0，蒸馏水定容，高压蒸汽灭菌20min。无菌收集牛樟芝孢子，接入摇瓶进行液体培养，培养的条件为温度28℃，转速160rpm，培养15天，得到未形成明显菌球的菌液。
（3）接种步骤：采用涂布接种法接种，在无菌的环境下，每个培养皿加入2~5mL摇瓶培养出来的菌液，再用涂布棒将菌液涂成直径大约5～8cm左右的圆圈。
（4）培养步骤：将接种后的培养皿倒置放在恒温恒湿的环境中培养，前2个月，在菌丝体未长满培养皿之前，温度为28℃、湿度为85%，每天光照时间为6小时，光照强度为0～400lux，菌丝体在培养基上生长，慢慢长满整个平板，菌丝体的颜色从白色变成橘红色；菌丝体长满培养皿直至5个月，环境温度为20℃、湿度为70%，每天光照时间为2小时，光照强度为0～400lux，菌丝体逐渐长厚，呈橘红色，约有0.3cm厚。
（5）牛樟芝子实体的采收：将生长出来的菌丝体从培养皿中分出来，将带出来的培养基刮除干净，再用45℃水冲洗菌丝体的底部，再把得到的菌丝体放置烘箱中烘干至水分低于1.8%，温度控制在40～50℃之间。烘干粉碎后取1.0g样品，通过分光光度法测其三萜含量为：16.1%。
实施例5
（1）培养基制备步骤：称取麸皮20g/L加水后煮40min，过滤后取滤液，再加入玉米淀粉20g/L进麸皮的滤液中，搅拌均匀后加热糊化，糊化完成后依次加入琼脂20g/L、蛋白胨1.5g/L、硫酸镁2.0g/L橄榄油2ml/L，搅拌使其溶解完全后，补水至1 L。通过灭菌锅高温高压灭菌20min， 趁热倒至培养皿中，厚度在2cm左右，放冷使其凝固。
（2）菌种准备：液体种子培养基组成：葡糖:20.0g/L，麦芽浸膏20.0g/L，蛋白胨1.0g/L，硫酸镁0.5 g/L，pH 4.0，蒸馏水定容，高压蒸汽灭菌20min。无菌收集牛樟芝孢子，接入摇瓶进行液体培养，培养的条件为温度28℃，转速160rpm，培养15天，得到未形成明显菌球的菌液。
（3）接种步骤：采用涂布接种法接种，在无菌的环境下，每个培养皿加入2~5mL摇瓶培养出来的菌液，再用涂布棒将菌液涂成直径大约5～8cm左右的圆圈。
（4）培养步骤：将接种后的培养皿倒置放在恒温恒湿的环境中培养，设定的生长条件是：温度为25℃、湿度为85%，每天光照时间为6小时，光照强度为0～400lux，菌丝体在30天左右长满整个平板。菌丝体长满培养皿后的2个月后，改变生长环境，温度为20℃、湿度为65%，每天光照时间为2小时，光照强度为0～400lux，菌丝体逐渐长厚，呈橘红色，长到6个月约有0.4cm厚，此时菌丝体生长缓慢，开始采摘。
（5）牛樟芝子实体的采收：将生长出来的菌丝体从培养皿中分出来，将带出来的培养基刮除干净，再用45℃水冲洗菌丝体的底部，再把得到的菌丝体放置烘箱中烘干至水分低于1.8%，温度控制在40～50℃之间。烘干粉碎后取1.0g样品，通过分光光度法测其三萜含量为：18.2%。
实施例6
（1）培养基制备步骤：称取麸皮20g/L加水后煮40min，过滤后取滤液，再加入玉米淀粉20g/L进麸皮的滤液中，搅拌均匀后加热糊化，糊化完成后依次加入琼脂20g/L、蛋白胨1.5g/L、硫酸镁2.0g/L橄榄油2ml/L，搅拌使其溶解完全后，补水至1 L。通过灭菌锅高温高压灭菌20min， 趁热倒至培养皿中，厚度在2cm左右，放冷使其凝固。
（2）菌种准备：液体种子培养基组成：葡糖:20.0g/L，麦芽浸膏20.0g/L，蛋白胨1.0g/L，硫酸镁0.5 g/L，pH 4.0，蒸馏水定容，高压蒸汽灭菌20min。无菌收集牛樟芝孢子，接入摇瓶进行液体培养，培养的条件为温度28℃，转速160rpm，培养15天，得到未形成明显菌球的菌液。
（3）接种步骤：采用涂布接种法接种，在无菌的环境下，每个培养皿加入2~5mL摇瓶培养出来的菌液，再用涂布棒将菌液涂成直径大约5～8cm左右的圆圈。
（4）培养步骤：将接种后的培养皿倒置放在恒温恒湿的生化培养箱中培养，设定的生长条件是：温度为28℃、湿度为85%，每天光照时间为5小时，光照强度为0～400lux，菌丝体在2个月长满整个平板。菌丝体长满培养皿后，变换生长环境，温度为22℃、湿度为65%，每天光照时间为3小时，光照强度为0～400lux，菌丝体逐渐长厚，长到第8个月的时候菌丝体生长十分缓慢，呈此时采摘，此时厚度为0.6cm。
（5）牛樟芝子实体的采收：将生长出来的菌丝体从培养皿中分出来，将带出来的培养基刮除干净，再用45℃水冲洗菌丝体的底部，再把得到的菌丝体放置烘箱中烘干至水分低于1.8%，温度控制在40～50℃之间。烘干粉碎后取1.0g样品，通过分光光度法测其三萜含量为：23.4%。
实施例7
（1）培养基制备步骤：称取麸皮20g/L加水后煮40min，过滤后取滤液，再加入玉米淀粉20g/L进麸皮的滤液中，搅拌均匀后加热糊化，糊化完成后依次加入琼脂20g/L、蛋白胨1.5g/L、硫酸镁2.0g/L橄榄油2ml/L，搅拌使其溶解完全后，补水至1 L。通过灭菌锅高温高压灭菌20min， 趁热倒至培养皿中，厚度在2cm左右，放冷使其凝固。
（2）菌种准备：液体种子培养基组成：葡萄糖:20.0g/L，麦芽浸膏20.0g/L，蛋白胨1.0g/L，硫酸镁0.5 g/L，pH 4.0，蒸馏水定容，高压蒸汽灭菌20min。无菌收集牛樟芝孢子，接入摇瓶进行液体培养，培养的条件为温度28℃，转速160rpm，培养15天，得到未形成明显菌球的菌液。
（3）接种步骤：采用涂布接种法接种，在无菌的环境下，每个培养皿加入2~5mL摇瓶培养出来的菌液，再用涂布棒将菌液涂成直径大约5～8cm左右的圆圈。
（4）培养步骤：将接种后的培养皿倒置放在恒温恒湿的生化培养箱中培养，前2个月，在菌丝体未长满培养皿之前，温度为28℃、湿度为95%，每天光照时间为6小时，光照强度为0～400lux，菌丝体在培养基上生长，慢慢长满整个平板，菌丝体的颜色在长满整个平板时只有淡淡的黄色；菌丝体长满培养皿后的12个月，温度为20℃、湿度为65%，每天光照时间为2小时，光照强度为0～400lux，菌丝体逐渐长厚，呈橘红色，菌体厚度约有0.95cm。
（5）牛樟芝子实体的采收：将生长出来的菌丝体从培养皿中分出来，将带出来的培养基刮除干净，再用45℃水冲洗菌丝体的底部，再把得到的菌丝体放置烘箱中烘干至水分低于1.8%，温度控制在40～50℃之间。烘干粉碎后取1.0g样品，通过分光光度法测其三萜含量为35.0%。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (9)

  1. 一种牛樟芝皿式培养方法，其特征在于，所述培养方法包括：
    培养基制备步骤：按照与水的质量体积比称取5～40g/L的麸皮、5～40g/L的玉米淀粉、5～40g/L的琼脂、1～3g/L的蛋白胨、0.5～4.0g/L的硫酸镁以及0.5～10ml/L的橄榄油，制备牛樟芝菌种的培养皿式培养基；
    接种步骤：在无菌环境下，将牛樟芝菌种接种于所述培养基上；
    培养步骤：将接种后的培养基置于恒温恒湿环境中培养3～12个月。
  2. 如权利要求1所述的培养方法，其特征在于，所述培养步骤中的培养条件为：在菌丝体未长满培养皿之前，温度为25～30℃、湿度为85%～95%，每天光照时间为4～6小时，光照强度为0～400lux；菌丝体长满培养皿后，温度为18～22℃、湿度为65%～75%，每天光照时间为2～4小时，光照强度为0～400lux。
  3. 如权利要求1所述的培养方法，其特征在于，所述培养基制备过程具体为：
    将麸皮加入水中煮40min后，过滤得滤液；将玉米淀粉加入所述滤液中，加热糊化；依次加入琼脂、蛋白胨、硫酸镁、橄榄油，搅拌后得混合液，将所得混合液的体积加水补至水的初始体积；高压灭菌20min，趁热倒至培养皿中，冷却凝固得柱状培养基。
  4. 如权利要求3所述的培养方法，其特征在于，所述柱状培养基的厚度为1～3cm。
  5. 如权利要求1所述的培养方法，其特征在于，所述接种步骤中，所述牛樟芝菌种是以菌种培养液的形式接种于所述培养皿式培养基上，所述菌种培养液的制备过程为：
    按照与水的质量体积比称取20.0g/L的葡萄糖、20.0g/L的麦芽浸膏、1.0 g/L的蛋白胨以及0.5 g/L的硫酸镁，加入水中得混合液，调节所得混合液的pH值至4.0；高压蒸汽灭菌20min后得液体种子培养基；无菌条件下将牛樟芝菌种接入液体种子培养基中进行培养，获得菌种培养液。
  6. 如权利要求5所述的培养方法，其特征在于，所述牛樟芝菌种在所述液体种子培养基中的培养条件为：温度28℃，转速160rpm，时间15天。
  7. 如权利要求5所述的培养方法，其特征在于，所述接种过程具体为：在所述培养皿式培养基上加入2~5mL所述菌种培养液，采用涂布接种法将所述菌种培养液涂成直径为5～8cm的圆圈。
  8. 如权利要求1所述的培养方法，其特征在于，所述培养步骤后还包括干燥步骤，所述干燥步骤包括：将菌丝体从所述培养皿中分离出来，置于40～50℃温度下干燥至水分含量低于1.8%，获得牛樟芝样品。
  9. 如权利要求1所述的培养方法，其特征在于，所述牛樟芝菌菌种为ATCC200183。
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