WO2014131200A1 - 一种高产三萜类化合物的樟芝菌丝体发酵方法 - Google Patents

一种高产三萜类化合物的樟芝菌丝体发酵方法 Download PDF

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WO2014131200A1
WO2014131200A1 PCT/CN2013/072094 CN2013072094W WO2014131200A1 WO 2014131200 A1 WO2014131200 A1 WO 2014131200A1 CN 2013072094 W CN2013072094 W CN 2013072094W WO 2014131200 A1 WO2014131200 A1 WO 2014131200A1
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culture
liquid
seed
mycelium
yield
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PCT/CN2013/072094
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French (fr)
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赵宗杰
谢海涛
杨如钢
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深圳市仁泰生物科技有限公司
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Priority to PCT/CN2013/072094 priority Critical patent/WO2014131200A1/zh
Publication of WO2014131200A1 publication Critical patent/WO2014131200A1/zh

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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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes

Definitions

  • the invention relates to the field of fermentation engineering, in particular to a method for fermenting an Antrodia camphorata mycelium with high yield of triterpenoids.
  • Enzyme (SOD) adenosine, protein (including immune protein), multivitamins, trace elements, nucleic acids, lectins, amino acids, cholesterol, lignin, blood pressure stabilizing substances.
  • SOD enzyme
  • Its physiologically active functions include anti-tumor, immunity, anti-virus, anti-allergy, anti-hypertension, inhibition of platelet aggregation, blood pressure lowering, cholesterol lowering, anti-bacteria, and protection of the liver.
  • triterpenoids are the core pharmacological components of medicinal fungi and have strong physiological activity.
  • Triterpenoids are medicinal fungi that exert anti-inflammatory, analgesic, poisonous tumor cells, induce tumor cell apoptosis, anti-hypoxia, etc.
  • the main component of the action but also has the effect of improving the body's immunity, which is manifested in promoting lymphocyte proliferation and improving the phagocytic ability and lethality of macrophages, NK cells and T cells. It can effectively promote liver cell regeneration, repair and improve liver function, and effectively reduce liver and kidney damage during chemotherapy.
  • Antrodia sinensis has all the advantages of Ganoderma lucidum, and its efficacy is better than that of Ganoderma lucidum. Its active ingredient triterpenoid content is even more than ten times that of Ganoderma lucidum, so it is known as the "King of Ganoderma lucidum". According to the research data, there are only 20-50 kinds of triterpenoids in a single variety of Ganoderma lucidum, and the total number of triterpenoids in different varieties of Ganoderma lucidum is more than 200, and a single variety of Antrodia lucidum has more than 200 kinds of triterpenoids, and No fatty acids and other impurities, the anti-cancer effect is more outstanding.
  • the object of the present invention is to provide a method for fermenting the mycelium of the Antrodia camphorata with high yield of triterpenoids, not only to increase the yield of mycelium, but more importantly to increase the content of triterpenoids in the mycelium.
  • a method for fermenting an Antrodia camphorata mycelium with high yield of triterpenoids comprising the following steps,
  • the activity of the slanted species was transferred to the plate medium by the stalk of the A. sinensis.
  • the diameter of the A. sinensis colony was more than 2 cm, it was transferred to a fresh plate medium again to make the mycelium grow over the plate and form an orange color. spore;
  • the liquid seed is expanded, and the shake flask seed is transferred to the seed tank to expand the culture.
  • the seed tank is transferred to the next stage seed tank, and the liquid seed for fermentation production is expanded step by step;
  • the three-head condition fermentation the above liquid seeds were transferred to the next-stage fermenter according to the 10% inoculum, and the fermentation conditions were: 30-30 ° C, pH 3.0-5.0, ventilation ratio 0.3-2.0 vvm,
  • the culture time is 2-3 days, and the composition of the triterpene condition fermentation medium is: corn starch 10-60 g/L, bran 10-60 g/L, magnesium sulfate 0.5-2.0 g/L, and olive oil 2-10 ml/L.
  • the fermentation conditions of the triterpenoids are: 28 ° C, pH 3.0, aeration ratio of 0.35 vvm, culture time of 2 days, the composition of the triterpene fermentation medium is: corn starch 20-25 g / L, bran 20-25 g / L , magnesium sulfate 1.5-2.0g / L, olive oil 2-5ml / L.
  • the seed tank inoculation amount is 10%, and the seed tank culture condition is: temperature 25-30 ° C, pH 3.0-5.0, aeration ratio 0.5-2.0 vvm, culture time 3-5 days; seed
  • the composition of the tank medium glucose 10-20 g / L, malt extract 10-20 g / L, peptone 1-5 g / L, magnesium sulfate 0.1-1.0 g / L.
  • the seed tank culture conditions are: temperature 28 ° C, pH 4.0, aeration ratio of 1.0 vvm, culture time of 3 days; seed tank medium composition: glucose 18.8 g / L, malt extract 18.8 g / L, peptone 3.3 g / L, magnesium sulfate 0.5g / L.
  • the conditions of the shake flask liquid culture are: temperature 25-30 ° C, pH 5.0-7.0, rotation speed 100-150 rpm, culture time 3-7 days; the composition of the liquid seed culture medium is: Glucose 10-20 g/L, malt extract 10-20 g/L, peptone 1-5 g/L, magnesium sulfate 0.1-1.0 g/L.
  • the condition of the shake flask liquid culture is: temperature 28 ° C, pH value 4.0, rotation speed 120 rpm, culture time 5 days; liquid seed culture medium composition: glucose 18.8 g / L, malt extract 18.8 g / L, peptone 3.3 g/L, magnesium sulfate 0.5 g/L.
  • the bran was boiled for 30-60 min on a light fire, and the filtrate was filtered.
  • the corn starch is subjected to gelatinization treatment before use, and the gelatinization condition is that the corn starch solution is maintained at a temperature of 100 ° C for 5 to 60 minutes.
  • the plate medium is an EMA plate medium
  • the culture temperature is 25-30 ° C
  • the culture time is 10-20 days.
  • the plate medium is an EMA plate medium
  • the culture temperature is 28 ° C
  • the culture time is 15 days.
  • EMA plate medium malt extract 20g, glucose 20g, peptone 1g, agar powder 20g, pH 5.0,
  • the method for fermenting the Antrodia camphorata mycelium of the invention greatly improves the yield of triterpenoids in the liquid fermentation product of Antrodia camphorata, the content of triterpenoids in the mycelium of Antrodia camphorata produced by the method of cultivation of Eucalyptus and the cultivation method Quite, but the cost is reduced by a thousand times, even if compared with the existing liquid fermentation process, the production of triterpenoids is significantly improved, and the cost reduction is very obvious.
  • the fermentation time of Sancha condition was greatly shortened, and the shortest time was 2 days, which further reduced the production cost and laid a solid foundation for the industrialization of Antrodia.
  • the invention adopts the strain of Antrodia camphorata, ATCC 200183 and CCRC35396
  • the content of the triterpenoids in each of the examples of the present invention is determined by the following method:
  • Oleanolic acid is a pentacyclic triterpenoid which reacts with various developers to develop a colorimetric assay. Oleic acid reacts with vanillin-perchloric acid reagent, vanillin-sulfuric acid reagent to develop a purple color. There is a linear relationship between the concentration of oleanolic acid and the absorbance, which is consistent with Lambert Beer's law, so colorimetric determination can be performed.
  • oleanolic acid standard accurately weigh 10mg oleanolic acid standard, and make 100mL of oleanolic acid ethanol solution with 0.1mg/mL as absolute solvent.
  • Sample determination accurately take 0.1mL of sample, heat to evaporate solvent, then add 0.4mL of freshly prepared 5% vanillin-glacial acetic acid solution and 1.5mL perchloric acid, heated in a constant temperature water bath at 70 °C for 15min, running water to room temperature, Further, 5 mL of ethyl acetate was added and diluted to shake, and the absorbance value was measured at a wavelength of 560 nm.
  • the A. sinensis (ATCC200183) slant strain was transferred to the EMA plate medium to carry out the activation culture of the slanted species.
  • the diameter of the A. sinensis colony was greater than 2 cm, it was transferred to fresh EMA plate medium again, and cultured at 25 ° C.
  • the mycelium grew over the plate and formed orange-colored spores.
  • the scorpion spores were aseptically collected and placed in a shake flask for liquid culture.
  • the culture temperature was 25 ° C
  • the rotation speed was 100 rpm
  • the culture was carried out for 6 days.
  • the seed liquid appeared turbid and no obvious hyphae were formed.
  • Liquid seed medium composition glucose 10g / L, malt extract 10g / L, peptone 1.0g / L, magnesium sulfate 0.5 g/L, pH 5.0, distilled water to volume, 121 ° C, autoclaved for 20 min.
  • the medium composition was 60 g/L corn starch, 60 g/L bran, and 0.5 g/L magnesium sulfate.
  • the defoamer olive oil was added in an amount of 5 ml/L.
  • the culture temperature was 28 ° C, the initial pH was 5.0, the aeration ratio was 1.0 vvm, and the rotation speed was 100 rpm, and the culture was continued for 5 days under the conditions.
  • the bran was boiled for 30 min on a light fire, filtered, and the filtrate was taken.
  • the corn starch was subjected to gelatinization before use, and the gelatinization condition was that the corn starch solution was maintained at a temperature of 100 ° C for 5 min.
  • the mycelium was collected by centrifugation and dried at 40 ° C to obtain 18.65 g/L of mycelium.
  • the content of triterpenoids in the mycelium was 783 mg/L, accounting for 4.2% of the dry weight of the mycelium.
  • the A. sinensis (ATCC200183) slanted strain was transferred to the EMA plate medium to carry out the activation culture of the slanted species.
  • the diameter of the A. sinensis colony was greater than 2 cm, it was transferred to fresh EMA plate medium again, and cultured at 28 ° C. After 15 days of culture, the mycelium grew over the plate and formed orange-colored spores.
  • Liquid seed medium composition glucose 20.0g / L, malt extract 20.0g / L, peptone 5.0g / L, magnesium sulfate 0.5 g/L, pH 4.0, distilled water to volume, 121 ° C, autoclaved for 20 min.
  • the scorpion spores were aseptically collected and placed in a shake flask for liquid culture.
  • the culture temperature was 28 ° C
  • the rotation speed was 120 rpm
  • the culture was carried out for 5 days.
  • the seed liquid appeared turbid and no obvious hyphae were formed.
  • the shake flask seeds were transferred to a 5 L fermentor at a 10% inoculum (the liquid volume in the tank after inoculation was about 3.8 L), and the three-dimensional fermentation was carried out.
  • the medium composition is corn starch 30g/L, bran 30 g/L, magnesium sulfate 1.85 g/L.
  • the defoamer olive oil was added in an amount of 2 ml/L.
  • the culture temperature was 28 ° C, the initial pH was 3.0, the aeration ratio was 0.35 vvm, and the number of rotations was 80 rpm, and the culture was continued for 3 days under the conditions.
  • the bran was boiled for 40 min on a light fire, filtered, and the filtrate was taken.
  • the corn starch was subjected to gelatinization before use, and the gelatinization condition was that the corn starch solution was maintained at a temperature of 100 ° C for 30 min.
  • the mycelium was collected by centrifugation, and 12.27 g/L of the mycelium of Antrodia camphorata was obtained after drying at 40 ° C.
  • the content of triterpenoids in the mycelium was determined to be 983 mg/L, accounting for 8.0% of the dry weight of the mycelium.
  • the A. sinensis (ATCC200183) slanted strain was transferred to the EMA plate medium to carry out the activation culture of the slanted species.
  • the diameter of the A. sinensis colony was greater than 2 cm, it was transferred to fresh EMA plate medium again, and cultured at 28 ° C. After 15 days of culture, the mycelium grew over the plate and formed orange-colored spores.
  • Liquid seed medium composition glucose 18.8g / L, malt extract 18.8.g / L, peptone 3.3g / L, magnesium sulfate 0.5 g/L, pH 4.0, distilled water to volume, 121 ° C, autoclaved for 20 min.
  • the scorpion spores were aseptically collected and placed in a shake flask for liquid culture.
  • the culture temperature was 28 ° C
  • the rotation speed was 120 rpm
  • the culture was carried out for 5 days.
  • the seed liquid appeared turbid and no obvious hyphae were formed.
  • the shake flask seeds were transferred to a 5 L fermentor at a 10% inoculum (the liquid volume in the tank after inoculation was about 3.8 L), and the three-dimensional fermentation was carried out.
  • the medium composition is corn starch 10g/L, bran 10 g/L, magnesium sulfate 1.85 g/L.
  • the defoamer olive oil was added in an amount of 5 ml/L.
  • the culture temperature was 26 ° C, the initial pH was 3.0, the aeration ratio was 0.35 vvm, and the number of rotations was 90 rpm, and the culture was continued for 3 days under the conditions.
  • the bran was boiled for 60 min on a light fire, filtered, and the filtrate was taken.
  • the corn starch was subjected to gelatinization treatment before use, and the gelatinization condition was that the corn starch solution was maintained at a temperature of 100 ° C for 60 minutes.
  • the mycelium was collected by centrifugation, and dried at 40 ° C to obtain 7.07 g/L of the mycelium of Antrodia camphorata.
  • the content of triterpenoids in the mycelium was determined to be 998 mg/L, accounting for 14.1% of the dry weight of the mycelium.
  • the A. sinensis (ATCC200183) slanted strain was transferred to the EMA plate medium to carry out the activation culture of the slanted species.
  • the diameter of the A. sinensis colony was greater than 2 cm, it was transferred to fresh EMA plate medium again, and cultured at 28 ° C. After 15 days of culture, the mycelium grew over the plate and formed orange-colored spores.
  • Liquid seed medium composition glucose 18.8g / L, malt extract 18.8.g / L, peptone 3.3g / L, magnesium sulfate 0.5 g/L, pH 4.0, distilled water to volume, 121 ° C, autoclaved for 20 min.
  • the scorpion spores were aseptically collected and placed in a shake flask for liquid culture.
  • the culture temperature was 28 ° C
  • the rotation speed was 120 rpm
  • the culture was carried out for 5 days.
  • the seed liquid appeared turbid and no obvious hyphae were formed.
  • the shake flask seeds were transferred to a 50L seed tank for strain expansion. (The liquid volume of the fermenter after transfer was about 38L.)
  • the composition of the seed tank medium glucose 18.8g/L, malt extract 18.8.g/L, peptone 3.3g /L, magnesium sulfate 0.5 g/L, pH 4.0, distilled water to volume, 121 ° C, autoclaved for 20 min.
  • the seed tank culture conditions were: temperature 28 ° C, aeration ratio of 1.0 vvm, rotation speed of 120 rpm, and the seed tank appeared turbid when cultured for 3 days.
  • the seed tank seeds were transferred to a 500 L fermentor at a 10% inoculum for three-dimensional fermentation, and the final liquid volume of the ferment tank was 380 L.
  • the medium composition was corn starch 22.2g/L, bran 22.2 g/L, 1.85 g/L of magnesium sulfate, and the amount of defoamer added to olive oil was 10 g/L.
  • the culture temperature was 26 ° C, the initial pH was 3.0, the aeration ratio was 0.35 vvm, and the number of rotations was 90 rpm.
  • the fermentation was stopped under the conditions of 65 hours.
  • the bran was boiled for 60 min on a light fire, filtered, and the filtrate was taken.
  • the corn starch was subjected to gelatinization before use, and the gelatinization condition was that the corn starch solution was maintained at a temperature of 100 ° C for 30 min.
  • the mycelium was collected by centrifugation and dried at 40 °C to obtain 12.05 g/L of mycelium.
  • the content of triterpenoids in the mycelium was determined to be 1269 mg/L, accounting for 10.5% of the dry weight of mycelium. .
  • the A. sinensis (ATCC200183) slanted strain was transferred to the EMA plate medium to carry out the activation culture of the slanted species.
  • the diameter of the A. sinensis colony was greater than 2 cm, it was transferred to fresh EMA plate medium again, and cultured at 28 ° C. After 15 days of culture, the mycelium grew over the plate and formed orange-colored spores.
  • Liquid seed medium composition glucose 18.8g / L, malt extract 18.8.g / L, peptone 3.3g / L, magnesium sulfate 0.5 g/L, pH 4.0, distilled water to volume, 121 ° C, autoclaved for 20 min.
  • the scorpion spores were aseptically collected and placed in a shake flask for liquid culture.
  • the culture temperature was 28 ° C
  • the rotation speed was 120 rpm
  • the culture was carried out for 5 days.
  • the seed liquid appeared turbid and no obvious hyphae were formed.
  • the shake flask seeds were transferred to a 50L seed tank for strain expansion. (The liquid volume of the fermenter after transfer was about 38L.)
  • the composition of the seed tank medium glucose 18.8g/L, malt extract 18.8.g/L, peptone 3.3g /L, magnesium sulfate 0.5 g/L, pH 4.0, distilled water to volume, 121 ° C, autoclaved for 20 min.
  • the seed tank culture conditions were as follows: temperature 28 ° C, aeration ratio of 1.0 vvm, rotation speed 120 rpm, and the seed tank appeared turbid when cultured for 3 days.
  • the 500L seed tank was transferred (the liquid volume of the fermenter after transfer was about 380L), and the seed was expanded twice.
  • the medium composition and culture conditions were the same as the 50L seed tank.
  • the 10% inoculum of 360L seeds was transferred to a 5000L fermenter for three-dimensional fermentation, and the final liquid volume of the fermenter was 3750L.
  • the medium composition was corn starch 22.2g/L, bran 22.2 g/L, 1.85 g/L of magnesium sulfate, and the amount of defoamer olive oil added was 3.3 ml/L.
  • the culture temperature was 26 ° C, the initial pH was 3.0, the aeration ratio was 0.35 vvm, and the number of rotations was 90 rpm.
  • the fermentation was stopped under the conditions of 48 hours.
  • the bran was boiled for 45 min on a light fire, filtered, and the filtrate was taken.
  • the corn starch was subjected to gelatinization treatment before use, and the gelatinization condition was that the corn starch solution was maintained at a temperature of 100 ° C for 35 minutes.
  • the mycelium was collected by centrifugation and dried at 40 °C to obtain 11.68 g/L of mycelium.
  • the content of triterpenoids in the mycelium was 1472 mg/L, accounting for 12.6% of the dry weight of mycelium. .
  • Liquid seed medium composition glucose 18.8g / L, malt extract 18.8.g / L, peptone 3.3g / L, magnesium sulfate 0.5 g/L, pH 4.0, distilled water to volume, 121 ° C, autoclaved for 20 min.
  • the scorpion spores were aseptically collected and placed in a shake flask for liquid culture.
  • the culture temperature was 28 ° C
  • the rotation speed was 120 rpm
  • the culture was carried out for 5 days.
  • the seed liquid appeared turbid and no obvious hyphae were formed.
  • the shake flask seeds were transferred to a 50L seed tank for strain expansion. (The liquid volume of the fermenter after transfer was about 38L.)
  • the composition of the seed tank medium glucose 18.8g/L, malt extract 18.8.g/L, peptone 3.3g /L, magnesium sulfate 0.5 g/L, pH 4.0, distilled water to volume, 121 ° C, autoclaved for 20 min.
  • the seed tank culture conditions were as follows: temperature 28 ° C, aeration ratio of 1.0 vvm, rotation speed 120 rpm, and the seed tank appeared turbid when cultured for 3 days.
  • the 500L seed tank was transferred (the liquid volume of the fermenter after transfer was about 380L), and the seed was expanded twice.
  • the medium composition and culture conditions were the same as the 50L seed tank.
  • the 10% inoculum of 380L seeds was transferred to a 5000L fermenter for three-dimensional fermentation, and the final liquid volume of the fermenter was 3700L.
  • the medium composition was corn starch 22.2g/L, bran 22.2 g/L, 1.85 g/L of magnesium sulfate, and the amount of defoamer olive oil added was 3.3 ml/L.
  • the culture temperature was 26 ° C, the initial pH was 3.0, the aeration ratio was 0.35 vvm, and the number of rotations was 90 rpm.
  • the fermentation was stopped under the conditions of 48 hours.
  • the bran was boiled for 45 min on a light fire, filtered, and the filtrate was taken.
  • the corn starch was subjected to gelatinization treatment before use, and the gelatinization condition was that the corn starch solution was maintained at a temperature of 100 ° C for 35 minutes.
  • the mycelium was collected by centrifugation and dried at 40 °C to obtain 12.02 g/L of mycelium.
  • the content of triterpenoids in the mycelium was 1442 mg/L, accounting for 12.0% of the dry weight of mycelium. .
  • the strain used in Examples 1-5 was ATCC200183, and the strain used in Example 6 was CCRC35396.
  • Examples 4-5 increase the liquid seed expansion step, and the liquid seed expansion increases the vigor of the seeds, and improves the fermentation mycelium.
  • the content of terpenoids has a promoting effect.
  • Example 4-5 The main difference between Examples 4-5 and Examples 1-2 is that the content of bran and corn starch in the fermentation conditions of Example 4-5 is lower than that of Examples 1-2; Example 4-5 Compared with Example 3, the content of bran and corn starch in the triterpene condition fermentation medium was higher than that in Example 3; it can be seen that controlling the content of nutrients in the fermentation medium can effectively control the vegetative growth of the mycelium, and The invention effectively promotes the production of the mycelium of the mycelium.
  • the present invention has repeatedly and repeatedly explored, and on the basis of a large number of experiments, found a high-yield triterpenoid compound of the mycelium fermentation medium and culture conditions.
  • the present invention is the first to add olive oil to the mycelium fermentation medium of Antrodia camphorata.
  • the olive oil can be used as both a defoaming agent and a triterpenoid-promoting factor. It is verified by the present invention that 2-10 ml is added per liter of fermentation medium. Olive oil can effectively increase the content of triterpenoids in mycelium.
  • the method of the invention is particularly suitable for industrial large-scale production of anthrax mycelium and its secondary metabolite, a triterpenoid compound, which has the remarkable advantages of high yield, low time consumption and low cost.

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Abstract

本发明提供了一种高产三萜类化合物的樟芝菌丝体发酵方法，包括以下步骤，斜面菌种活化，摇瓶液体种子制备，液体种子扩培，三萜条件发酵，三萜条件发酵时间最短为2天，三萜条件发酵培养基的成分为：玉米淀粉10-60g/L，麸皮10-60g/L，硫酸镁0.5-2.0g/L，橄榄油2-10ml/L。所述樟芝菌丝体发酵方法提高了樟芝液体发酵产物中三萜类化合物的产量，降低了成本。

Description

一种高产三萜类化合物的樟芝菌丝体发酵方法 技术领域
本发明涉及发酵工程领域，尤其涉及高产三萜类化合物的樟芝菌丝体发酵方法。
背景技术
樟芝属担子菌纲，多孔菌目，多孔菌科，薄孔菌属，是一种珍稀的药用真菌，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具有抗肿瘤、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、抗缺氧等作用的主要成分；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。
樟芝具有灵芝所有优点，功效更胜于灵芝，其有效成分三萜类化合物含量甚至是灵芝的十倍以上，因此素有“灵芝之王”的美誉。研究数据表明，单一品种的灵芝只有20-50种三萜类化合物，不同品种灵芝的三萜类化合物总和才达到200多种，而单一品种的樟芝即有200多种三萜类化合物，而且无脂肪酸等杂质，抗癌效果更加出众。
樟芝人工培育可以分为三种方法，1）液体发酵法，2）固体栽培法，3）椴木栽培法。深层液体发酵是目前深度开发药用菌活性成分的最有效方法，可以快速、高效的获得樟芝菌丝体，并已在香菇、灰树花、巴西蘑菇、虫草拟青霉上实现规模化生产。
国内外已有文献报道樟芝液体发酵工艺，但研究主要集中在提高生物量和多糖，以提高樟芝三萜类化合物为目的的研究较少。台湾部分研究者研究了樟芝液体培养工艺，但是三萜类化合物产率较低。江南大学真菌研究实验室对樟芝液体发酵进行了研究，主要是以提高樟芝菌丝体产量为目标，樟芝三萜类化合物产量在65mg/L左右。
 技术问题
本发明的目的在于提供一种高产三萜类化合物的樟芝菌丝体发酵方法，不仅仅提高菌丝体的产量，更重要的是提高菌丝体中三萜类化合物的含量。
 技术解决方案
为达到上述目的，本发明的技术方案为，
一种高产三萜类化合物的樟芝菌丝体发酵方法，包括以下步骤，
斜面菌种活化，将樟芝斜面菌种转接至平板培养基上，待樟芝菌落直径大于2cm时，再次转接至新鲜平板培养基上培养，使菌丝体长满平板并形成橘红色孢子；
摇瓶液体种子制备，无菌收集樟芝孢子，接入摇瓶进行液体培养，待液体培养基出现浑浊而未形成菌丝球时停止培养，得到摇瓶种子；
液体种子扩培，将摇瓶种子转入种子罐进行菌种扩培，待发酵液出现浑浊而未形成菌丝球时，转入下一级种子罐，逐级扩大制备发酵生产用液体种子；
三萜条件发酵，将上述液体种子按10%接种量转入下一级发酵罐，进行三萜条件发酵，发酵条件为：25-30℃，pH值3.0-5.0，通气比0.3-2.0vvm，培养时间2-3天，三萜条件发酵培养基的成分为：玉米淀粉10-60g/L，麸皮10-60g/L，硫酸镁0.5-2.0g/L，橄榄油2-10ml/L。
所述三萜发酵条件为：28℃，pH值3.0，通气比0.35vvm，培养时间2天，三萜条件发酵培养基的成分为：玉米淀粉20-25g/L，麸皮20-25g/L，硫酸镁1.5-2.0g/L，橄榄油2-5ml/L。
所述液体种子扩培步骤中，种子罐接种量为10%，种子罐培养条件为：温度25-30℃，pH值3.0-5.0，通气比0.5-2.0vvm，培养时间3-5天；种子罐培养基组成：葡萄糖10-20g/L，麦芽浸膏10-20g/L，蛋白胨1-5g/L，硫酸镁0.1-1.0g/L。
所述种子罐培养条件为：温度28℃，pH值4.0，通气比1.0vvm，培养时间3天；种子罐培养基组成：葡萄糖18.8g/L，麦芽浸膏18.8g/L，蛋白胨3.3g/L，硫酸镁0.5g/L。
所述摇瓶液体种子制备步骤中，摇瓶液体培养的条件为：温度25-30℃，pH值5.0-7.0，转速100-150rpm，培养时间3-7天；液体种子培养基的成分为：葡萄糖10-20g/L，麦芽浸膏10-20g/L，蛋白胨1-5g/L，硫酸镁0.1-1.0g/L。
所述摇瓶液体培养的条件为：温度28℃，pH值4.0，转速120rpm，培养时间5天；液体种子培养基的成分为：葡萄糖18.8g/L，麦芽浸膏18.8g/L，蛋白胨3.3g/L，硫酸镁0.5g/L。
所述麸皮用微火煮沸30-60min，过滤取滤液。
所述玉米淀粉用前先进行糊化处理，糊化条件是玉米淀粉溶液在温度100℃维持5-60min。
所述斜面菌种活化步骤中，所述平板培养基为EMA平板培养基，培养温度25-30℃，培养时间10-20天。
所述斜面菌种活化步骤中，所述平板培养基为EMA平板培养基，培养温度28℃，培养时间15天。
EMA平板培养基：麦芽浸膏20g，葡萄糖20g，蛋白胨1g，琼脂粉20g，PH5.0，
 有益效果
本发明所述樟芝菌丝体发酵方法大大提高了樟芝液体发酵产物中三萜类化合物的产量，三萜类化合物含量与椴木培植方法生产的樟芝菌丝体中三萜类化合物含量相当，但成本降低千倍，即使与现有液体发酵工艺相比，三萜类化合物产量也是显著提高，成本降低十分明显。同时，三萜条件发酵时间大大缩短，最短为2天，进一步降低了生产成本，为樟芝产业化奠定了坚实的基础。
本发明的实施方式
本发明采用樟芝菌菌种，ATCC 200183和CCRC35396
本发明每个实施例中三萜类化合物含量测定采用如下方法：
三萜类物质含量的分光光度法测定
一、 原理
齐墩果酸是五环三萜类化合物，它可与多种显色剂发生显色反应，从而可进行比色测定。齐墩果酸与香草醛-高氯酸试剂，香草醛-硫酸试剂发生显色反应呈现紫色。齐墩果酸的浓度与吸光度存在一定的线性关系，符合朗伯比尔定律，因此可进行比色测定
二、 仪器和试剂
1． 仪器：752-型紫外分光光度计，电热恒温水浴锅，分析天平，电热恒温干燥箱，具塞试管
2． 试剂：香草醛，冰醋酸，无水乙醇，乙酸乙酯和高氯酸（以上均为分析纯），齐墩果酸标准品
三、 操作步骤
1．齐墩果酸标准品的制备：精密称取10mg齐墩果酸标准品，以无水乙醇为溶剂，配制成100mL浓度为0.1mg/mL的齐墩果酸乙醇溶液。
2．香草醛冰醋酸溶液的制备：准确快速称取香草醛0.552g，迅速用适量的冰醋酸溶解并立即倒入10mL的容量瓶，迅速用冰醋酸稀释至刻度，以备当日之用。
3．标准曲线的绘制：准确吸取齐墩果酸标准液0.1,0.2，0.3,0.3,0.4,0.5mL分别置于具塞试管中，加热挥发去溶剂，再加入0.4mL新配制的5%香草醛-冰醋酸溶液及1.5mL高氯酸，在70℃恒温水浴中加热15min，流水冷却至室温，再加入乙酸乙酯5mL稀释摇匀，在560nm波长处测定吸光度值。
4．样品提取：取1g的样品粉末，加入30mL无水乙醇90℃恒温水浴回流1.5h，回流三次，合并滤液并定容100mL。
5．样品测定：准确取样品0.1mL，加热挥发去溶剂，再加入0.4mL新配制的5%香草醛-冰醋酸溶液及1.5mL高氯酸，在70℃恒温水浴中加热15min，流水冷却至室温，再加入乙酸乙酯5mL稀释摇匀，在560nm波长处测定吸光度值。
6．其中空白以最大0.5mL无水乙醇为参比液，根据步骤3绘制的标准曲线来计算样品中的齐墩果酸含量，并换算为百分含量。
实施例1 高产三萜类化合物的樟芝菌丝体发酵方法
（1）斜面菌种活化
将樟芝(ATCC200183)斜面菌种转接至EMA平板培养基上，进行斜面菌种的活化培养，待樟芝菌落直径大于2cm时，再次转接至新鲜EMA平板培养基上培养，25℃，培养18天时菌丝体长满平板并形成橘黄色孢子。
（2）摇瓶液体种子制备
无菌收集樟芝孢子，接入摇瓶进行液体培养，培养温度25℃，转速100rpm，培养6天，种子液出现浑浊而未形成明显的菌丝球。
液体种子培养基组成：葡萄糖10g/L，麦芽浸膏10g/L，蛋白胨1.0g/L，硫酸镁0.5 g/L，pH值5.0，蒸馏水定容，121℃，高压蒸汽灭菌20min。
（3）三萜条件发酵，将摇瓶种子按10%接种量转入5L发酵罐(接种后罐内液体体积约3.8L)，进行三萜条件发酵。培养基组成为玉米淀粉60g/L，麸皮60g/L，硫酸镁0.5g/L。消泡剂橄榄油添加量为5ml/L。培养温度28℃，初始pH值5.0，通气比1.0vvm，转速100rpm，在此条件下连续培养5天。所述麸皮用微火煮沸30min，过滤，取滤液。所述玉米淀粉用前先进行糊化处理，糊化条件是玉米淀粉溶液在温度100℃维持5min。
（4）离心收集菌丝体，40℃干燥后获得樟芝菌丝体18.65g/L，经测定菌丝体中三萜类化合物含量为783mg/L，占菌丝体干重的4.2%。
实施例2 高产三萜类化合物的樟芝菌丝体发酵方法
（1）斜面菌种活化
将樟芝(ATCC200183)斜面菌种转接至EMA平板培养基上，进行斜面菌种的活化培养，待樟芝菌落直径大于2cm时，再次转接至新鲜EMA平板培养基上培养，28℃，培养15天时菌丝体长满平板并形成橘黄色孢子。
（2）摇瓶液体种子制备
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5.0g/L，硫酸镁0.5 g/L，pH值4.0，蒸馏水定容，121℃，高压蒸汽灭菌20min。
无菌收集樟芝孢子，接入摇瓶进行液体培养，培养温度28℃，转速120rpm，培养5天，种子液出现浑浊而未形成明显的菌丝球。
（3）三萜条件发酵：将摇瓶种子按10%接种量转入5L发酵罐(接种后罐内液体体积约3.8L)，进行三萜条件发酵。培养基组成为玉米淀粉30g/L，麸皮30 g/L，硫酸镁1.85g/L。消泡剂橄榄油添加量为2ml/L。培养温度28℃，初始pH值3.0，通气比0.35vvm，转速80rpm，在此条件下连续培养3天。所述麸皮用微火煮沸40min，过滤，取滤液。所述玉米淀粉用前先进行糊化处理，糊化条件是玉米淀粉溶液在温度100℃维持30min。
（4）离心收集菌丝体，40℃干燥后获得樟芝菌丝体12.27g/L，经测定菌丝体中三萜类化合物含量为983mg/L，占菌丝体干重的8.0%。
实施例3 高产三萜类化合物的樟芝菌丝体发酵方法
（1）斜面菌种活化
将樟芝(ATCC200183)斜面菌种转接至EMA平板培养基上，进行斜面菌种的活化培养，待樟芝菌落直径大于2cm时，再次转接至新鲜EMA平板培养基上培养，28℃，培养15天时菌丝体长满平板并形成橘黄色孢子。
（2）摇瓶液体种子制备
液体种子培养基组成：葡萄糖18.8g/L，麦芽浸膏18.8.g/L，蛋白胨3.3g/L，硫酸镁0.5 g/L，pH值4.0，蒸馏水定容，121℃，高压蒸汽灭菌20min。
无菌收集樟芝孢子，接入摇瓶进行液体培养，培养温度28℃，转速120rpm，培养5天，种子液出现浑浊而未形成明显的菌丝球。
（3）三萜条件发酵：将摇瓶种子按10%接种量转入5L发酵罐(接种后罐内液体体积约3.8L)，进行三萜条件发酵。培养基组成为玉米淀粉10g/L，麸皮10 g/L，硫酸镁1.85g/L。消泡剂橄榄油添加量为5ml/L。培养温度26℃，初始pH值3.0，通气比0.35vvm，转速90rpm，在此条件下连续培养3天。所述麸皮用微火煮沸60min，过滤，取滤液。所述玉米淀粉用前先进行糊化处理，糊化条件是玉米淀粉溶液在温度100℃维持60min。
（4）离心收集菌丝体，40℃干燥后获得樟芝菌丝体7.07g/L，经测定菌丝体中三萜类化合物含量为998mg/L，占菌丝体干重的14.1%。
实施例4 高产三萜类化合物的樟芝菌丝体发酵方法
（1）斜面菌种活化
将樟芝(ATCC200183)斜面菌种转接至EMA平板培养基上，进行斜面菌种的活化培养，待樟芝菌落直径大于2cm时，再次转接至新鲜EMA平板培养基上培养，28℃，培养15天时菌丝体长满平板并形成橘黄色孢子。
（2）摇瓶液体种子制备
液体种子培养基组成：葡萄糖18.8g/L，麦芽浸膏18.8.g/L，蛋白胨3.3g/L，硫酸镁0.5 g/L，pH值4.0，蒸馏水定容，121℃，高压蒸汽灭菌20min。
无菌收集樟芝孢子，接入摇瓶进行液体培养，培养温度28℃，转速120rpm，培养5天，种子液出现浑浊而未形成明显的菌丝球。
（3）液体种子扩培
将摇瓶种子转入50L种子罐进行菌种扩培，（转接后发酵罐液体体积约38L）种子罐培养基组成：葡萄糖18.8g/L，麦芽浸膏18.8.g/L，蛋白胨3.3g/L，硫酸镁0.5 g/L，pH值4.0，蒸馏水定容，121℃，高压蒸汽灭菌20min。
种子罐培养条件为：温度28℃，通气比1.0vvm，转速为120rpm，培养3天时种子罐出现浑浊。
（4）三萜条件发酵
将种子罐种子按10%接种量转接入500L发酵罐进行三萜条件发酵，发酵罐最终液体体积为380L。培养基组成为玉米淀粉22.2g/L，麸皮22.2 g/L，硫酸镁1.85g/L，消泡剂橄榄油添加量为10g/L。培养温度26℃，初始pH值3.0，通气比0.35vvm，转速90rpm，在此条件下培养65小时停止发酵。所述麸皮用微火煮沸60min，过滤，取滤液。所述玉米淀粉用前先进行糊化处理，糊化条件是玉米淀粉溶液在温度100℃维持30min。
（5）离心收集菌丝体，40℃低温干燥后得樟芝菌丝体12.05g/L，经测定菌丝体中三萜类化合物含量为1269mg/L，占菌丝体干重的10.5%。
实施例5 高产三萜类化合物的樟芝菌丝体发酵方法
（1）斜面菌种活化
将樟芝(ATCC200183)斜面菌种转接至EMA平板培养基上，进行斜面菌种的活化培养，待樟芝菌落直径大于2cm时，再次转接至新鲜EMA平板培养基上培养，28℃，培养15天时菌丝体长满平板并形成橘黄色孢子。
（2）摇瓶液体种子制备
液体种子培养基组成：葡萄糖18.8g/L，麦芽浸膏18.8.g/L，蛋白胨3.3g/L，硫酸镁0.5 g/L，pH值4.0，蒸馏水定容，121℃，高压蒸汽灭菌20min。
无菌收集樟芝孢子，接入摇瓶进行液体培养，培养温度28℃，转速120rpm，培养5天，种子液出现浑浊而未形成明显的菌丝球。
（3）液体种子扩培
将摇瓶种子转入50L种子罐进行菌种扩培，（转接后发酵罐液体体积约38L）种子罐培养基组成：葡萄糖18.8g/L，麦芽浸膏18.8.g/L，蛋白胨3.3g/L，硫酸镁0.5 g/L，pH值4.0，蒸馏水定容，121℃，高压蒸汽灭菌20min。
种子罐培养条件为：温度28℃，通气比1.0vvm，转速120rmp，培养3天时种子罐出现浑浊。
按10%接种量转接入500L种子罐（（转接后发酵罐液体体积约380L）进行种子二次扩培，培养基组成和培养条件同50L种子罐。
（4）三萜条件发酵
将360L种子10%接种量转接入5000L发酵罐进行三萜条件发酵，发酵罐最终液体体积为3750L。培养基组成为玉米淀粉22.2g/L，麸皮22.2 g/L，硫酸镁1.85g/L，消泡剂橄榄油添加量为3.3ml/L。培养温度26℃，初始pH值3.0，通气比0.35vvm，转速90rpm，在此条件下培养48小时停止发酵。所述麸皮用微火煮沸45min，过滤，取滤液。所述玉米淀粉用前先进行糊化处理，糊化条件是玉米淀粉溶液在温度100℃维持35min。
（5）离心收集菌丝体，40℃低温干燥后得樟芝菌丝体11.68g/L，经测定菌丝体中三萜类化合物含量为1472mg/L，占菌丝体干重的12.6%。
实施例6 高产三萜类化合物的樟芝菌丝体发酵方法
（1）斜面菌种活化
将樟芝(CCRC 35396)斜面菌种转接至EMA平板培养基上，进行斜面菌种的活化培养，待樟芝菌落直径大于2cm时，再次转接至新鲜EMA平板培养基上培养，28℃，培养15天时菌丝体长满平板并形成橘黄色孢子。
（2）摇瓶液体种子制备
液体种子培养基组成：葡萄糖18.8g/L，麦芽浸膏18.8.g/L，蛋白胨3.3g/L，硫酸镁0.5 g/L，pH值4.0，蒸馏水定容，121℃，高压蒸汽灭菌20min。
无菌收集樟芝孢子，接入摇瓶进行液体培养，培养温度28℃，转速120rpm，培养5天，种子液出现浑浊而未形成明显的菌丝球。
（3）液体种子扩培
将摇瓶种子转入50L种子罐进行菌种扩培，（转接后发酵罐液体体积约38L）种子罐培养基组成：葡萄糖18.8g/L，麦芽浸膏18.8.g/L，蛋白胨3.3g/L，硫酸镁0.5 g/L，pH值4.0，蒸馏水定容，121℃，高压蒸汽灭菌20min。
种子罐培养条件为：温度28℃，通气比1.0vvm，转速120rmp，培养3天时种子罐出现浑浊。
按10%接种量转接入500L种子罐（（转接后发酵罐液体体积约380L）进行种子二次扩培，培养基组成和培养条件同50L种子罐。
（4）三萜条件发酵
将380L种子10%接种量转接入5000L发酵罐进行三萜条件发酵，发酵罐最终液体体积为3700L。培养基组成为玉米淀粉22.2g/L，麸皮22.2 g/L，硫酸镁1.85g/L，消泡剂橄榄油添加量为3.3ml/L。培养温度26℃，初始pH值3.0，通气比0.35vvm，转速90rpm，在此条件下培养48小时停止发酵。所述麸皮用微火煮沸45min，过滤，取滤液。所述玉米淀粉用前先进行糊化处理，糊化条件是玉米淀粉溶液在温度100℃维持35min。
（5）离心收集菌丝体，40℃低温干燥后得樟芝菌丝体12.02g/L，经测定菌丝体中三萜类化合物含量为1442mg/L，占菌丝体干重的12.0%。
实施例1-5所用菌株为ATCC200183，实施例6所用菌株为CCRC35396
实施例4-5与实施例1-3相比，主要不同点在于：实施例4-5增加了液体种子扩培步骤，液体种子扩培增加了种子的活力，对提高发酵菌丝体中三萜类物质含量具有促进作用。
实施例4-5与实施例1-2相比，主要不同点在于：实施例4-5三萜条件发酵培养基中麸皮和玉米淀粉含量低于实施例1-2；实施例4-5与实施例3相比，三萜条件发酵培养基中麸皮和玉米淀粉含量高于实施例3；可见控制发酵培养基中营养成分的含量，可以达到既有效控制菌丝体的营养生长，又有效促进菌丝体此生代谢物产生的目的，本发明经过多次，反复探索，在大量实验的基础上找到了高产三萜类化合物樟芝菌丝体发酵培养基及培养条件。
本发明首次在樟芝菌丝体发酵培养基中添加橄榄油，橄榄油既可作为消泡剂，又可作为三萜类化合物生成促进因子，经本发明验证每升发酵培养基添加2-10ml橄榄油可有效提高菌丝体中三萜类化合物的含量。
本发明所述方法尤其适合工业化大规模生产樟芝菌丝体及其次生代谢物-三萜类化合物，具有产量高，耗时少，成本低的显著优点。

Claims (10)

  1. 一种高产三萜类化合物的樟芝菌丝体发酵方法，包括以下步骤，
    斜面菌种活化，将樟芝斜面菌种转接至平板培养基上，待樟芝菌落直径大于2cm时，再次转接至新鲜平板培养基上培养，使菌丝体长满平板并形成橘红色孢子；
    摇瓶液体种子制备，无菌收集樟芝孢子，接入摇瓶进行液体培养，待液体培养基出现浑浊而未形成菌丝球时停止培养，得到摇瓶种子；
    液体种子扩培，将摇瓶种子转入种子罐进行菌种扩培，待发酵液出现浑浊而未形成菌丝球时，转入下一级种子罐，逐级扩大制备发酵生产用液体种子；
    三萜条件发酵，将上述液体种子按10%接种量转入下一级发酵罐，进行三萜条件发酵，发酵条件为：25-30℃，pH值3.0-5.0，通气比0.3-2.0vvm，培养时间2-3天，三萜条件发酵培养基的成分为：玉米淀粉10-60g/L，麸皮10-60g/L，硫酸镁0.5-2.0g/L，橄榄油2-10ml/L。
  2. 根据权利要求1所述的一种高产三萜类化合物的樟芝菌丝体发酵方法，其特征在于，所述三萜发酵条件为：28℃，pH值3.0，通气比0.35vvm，培养时间2天，三萜条件发酵培养基的成分为：玉米淀粉20-25g/L，麸皮20-25g/L，硫酸镁1.5-2.0g/L，橄榄油2-5ml/L。
  3. 根据权利要求1或2所述的一种高产三萜类化合物的樟芝菌丝体发酵方法，其特征在于，所述液体种子扩培步骤中，种子罐接种量为10%，种子罐培养条件为：温度25-30℃，pH值3.0-5.0，通气比0.5-2.0vvm，培养时间3-5天；种子罐培养基组成：葡萄糖10-20g/L，麦芽浸膏10-20g/L，蛋白胨1-5g/L，硫酸镁0.1-1.0g/L。
  4. 根据权利要求3所述的一种高产三萜类化合物的樟芝菌丝体发酵方法，其特征在于，所述种子罐培养条件为：温度28℃，pH值4.0，通气比1.0vvm，培养时间3天；种子罐培养基组成：葡萄糖18.8g/L，麦芽浸膏18.8g/L，蛋白胨3.3g/L，硫酸镁0.5g/L。
  5. 根据权利要求1或2所述的一种高产三萜类化合物的樟芝菌丝体发酵方法，其特征在于，所述摇瓶液体种子制备步骤中，摇瓶液体培养的条件为：温度25-30℃，pH值5.0-7.0，转速100-150rpm，培养时间3-7天；液体种子培养基的成分为：葡萄糖10-20g/L，麦芽浸膏10-20g/L，蛋白胨1-5g/L，硫酸镁0.1-1.0g/L。
  6. 根据权利要求5所述的一种高产三萜类化合物的樟芝菌丝体发酵方法，其特征在于，所述摇瓶液体培养的条件为：温度28℃，pH值4.0，转速120rpm，培养时间5天；液体种子培养基的成分为：葡萄糖18.8g/L，麦芽浸膏18.8g/L，蛋白胨3.3g/L，硫酸镁0.5g/L。
  7. 根据权利要求1，2或4中任一项所述的一种高产三萜类化合物的樟芝菌丝体发酵方法，其特征在于，所述麸皮用微火煮沸30-60min，过滤取滤液。
  8. 根据权利要求1，2或4中任一项所述的一种高产三萜类化合物的樟芝菌丝体发酵方法，其特征在于，所述玉米淀粉用前先进行糊化处理，糊化条件是玉米淀粉溶液在温度100℃维持5-60min。
  9. 根据权利要求1所述的一种高产三萜类化合物的樟芝菌丝体发酵方法，其特征在于，所述斜面菌种活化步骤中，所述平板培养基为EMA平板培养基，培养温度25-30℃，培养时间10-20天。
  10. 根据权利要求9所述的一种高产三萜类化合物的樟芝菌丝体发酵方法，其特征在于，所述斜面菌种活化步骤中，所述平板培养基为EMA平板培养基，培养温度28℃，培养时间15天。
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