WO2015024190A1 - 樟芝高产三萜菌株及其应用 - Google Patents

樟芝高产三萜菌株及其应用 Download PDF

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WO2015024190A1
WO2015024190A1 PCT/CN2013/081863 CN2013081863W WO2015024190A1 WO 2015024190 A1 WO2015024190 A1 WO 2015024190A1 CN 2013081863 W CN2013081863 W CN 2013081863W WO 2015024190 A1 WO2015024190 A1 WO 2015024190A1
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strain
yield
triterpenoid
mycelium
fermentation
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PCT/CN2013/081863
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French (fr)
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赵宗杰
谢海涛
蔡青松
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深圳市仁泰生物科技有限公司
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Priority to US14/413,420 priority Critical patent/US20160230192A1/en
Priority to PCT/CN2013/081863 priority patent/WO2015024190A1/zh
Publication of WO2015024190A1 publication Critical patent/WO2015024190A1/zh

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the invention relates to the field of bioengineering, in particular to a new strain of Antrodia camphorata with high yield of triterpenoids selected by ultraviolet mutagen protoplasts and application thereof.
  • Antrodia is also called Antrodia Cinnamomea) is a rare medicinal fungus native to Taiwan. It has great medicinal value and is known as the “anti-cancer holy medicine”, “king of the liver” and “king of detoxification”. It is also the most expensive. Medicinal fungi. Modern medical research has confirmed that the main active ingredient of Antrodia camphorata is triterpenoids. However, the amount of wild Antrodia camphora is rare, and the artificially cultivated Antrodia camphorata fruit body has a similar triterpenoid component to wild Antrodia camphorata, but the fruiting body grows slowly and is far from meeting the market demand. At present, the artificial fermentation using modern biotechnology is the best way to obtain the triterpenoids of Antrodia camphorata.
  • the artificial fermentation technology is mainly divided into solid fermentation and deep liquid fermentation.
  • Anthraquinone solid fermentation cycle is longer ( 90-180 days), the total triterpenoid content in the solid fermentation product is about 5%, which is far lower than the total triterpenoid content of 10%-45% in the wild fruit body; the liquid fermentation cycle of Antrodia camphorata is relatively short. (7-14 days), but the product is essentially free of triterpenoids. Therefore, both artificial fermentation methods have the disadvantages of long fermentation cycle and low production of triterpenoids.
  • the strain is an intrinsic factor that determines the level of fermentation. Many medicinal fungi have significantly increased the yield of medicinal active ingredients through mutagenic breeding, and realized the industrial production of medicinal fungi.
  • Li Gang (2001), Li Yingying et al (2011) obtained the excellent strains of high-yield polysaccharides and triterpenoids of Ganoderma lucidum by conventional mutagenesis breeding; Zhang Rijun (2004) screened the high-yield proteases of Bacillus by chemical and physical combination mutagenesis; Ying Hanjie Et al. (2007) obtained a high yield of nuclease P1 from Penicillium citrinum by injecting spores into a low energy particle beam. Similar studies are numerous, and it can be seen that it is an effective means to increase the active yield of edible and medicinal fungi by mutation breeding.
  • Gg is a high-yield strain of triterpenoids, the yield of triterpenoids is 57.86% higher than that of the original strain, the yield of total triterpenoids in liquid fermentation is 18mg/L, and the total triterpenoid content of the dried mycelium is 0.47%, but in fact the content of triterpenoids is Not too high, there is still a big gap from the requirements of industrialization.
  • the present invention aims to provide a strain of Astragalus membranaceus high-yield and its application, and aims to solve the problem of low yield of triterpenoids in the artificial fermentation of the existing A. camphor strain.
  • Mutagenic breeding includes physical mutagenesis and chemical mutagenesis.
  • Common physical mutagenesis methods include UV mutagenesis, microwave mutagenesis, space mutagenesis, 60 Co- ⁇ ray irradiation mutagenesis, X-ray mutagenesis, and ion implantation.
  • Mutagenesis, etc. chemical mutagens mainly include alkylating agents, base analogs, hydroxylamine, acridine pigments and the like.
  • UV mutagenesis has the advantages of simple operation, high mutagenic efficiency, strong reproducibility, and easy availability of mutagenic sources, and has become a routine means for mutagenic breeding of many biological materials.
  • Antrodia camphorata belongs to large fungi, and its spore shell is tough. If spores are used directly as mutagenic materials, not only high-intensity mutagenic treatment is required, but also the probability of success is low. Even if the mutagenesis is successful, it is difficult to germinate under artificial conditions. However, mycelium of A. philippinarum is mostly a chain-like composite cell body, and it is difficult to screen a single cell for mutagenesis. Therefore, mycelium is not suitable as a mutagenic material. At present, the more advanced method is indirect mutagenesis of protoplasts, that is, the protoplasts are prepared by removing the cell wall by biological enzymatic method.
  • the protoplasts are sensitive to the external environment, and are easily mutated after mutagenesis treatment, and the regenerated colonies are developed from single cells. Easy to screen, the regenerated strain can maintain the main biological characteristics of the original strain, the breeding cycle is greatly shortened, and the biological characteristics of the mutant strain are relatively stable and not easy to degrade.
  • the starting strains used in mutagenesis breeding are also critical. At present, there are more than a dozen species of Antrodia camphorata in the world's various species collection institutions, most of which are isolated from the fruit bodies of wild A. sinensis or the burdock tree hole. The mycelium of these strains is weaker and stronger. Slow growth in plates and liquid media.
  • the invention adopts the Antrodia camphor strain with the strain number ATCC200183 as the starting strain, and the mycelium of the strain is relatively thick and strong, and the growth speed is relatively fast in the plate and the liquid medium, and it is beneficial to screen out the ideal cockroach by using it as the starting strain. Excellent strain of Zhizhi.
  • the object of the present invention is to obtain a high-yield triterpenoid strain of A. angustifolia which produces a triterpenoid compound under liquid culture conditions.
  • the time required for the mycelium of A. camphorata to grow over the plate and the total yield of the three cells of the shake flask fermented mycelium were taken as indicators, and the protoplasts of Antrodia camphorata were repeatedly mutagenized by ultraviolet rays to obtain high-yield Sancha.
  • a new strain of Antrodia camphorata was obtained.
  • the present invention discloses a method for breeding a high-yield triterpenoid compound Antrodia camphorata by ultraviolet mutagen protoplasts and a strain of Astragalus membranaceus high-yield, which is selected by the method, and the strain collection number is CCTCC. M2013359.
  • the mutagenesis step comprises: preparing the protoplasts by shaking the cultured mycelium with ATCC200183 as the starting strain, and performing ultraviolet mutagenesis; coating on the MEA plate, selecting a single colony for shaking flask culture; and producing mycelium And the intracellular total triterpenoid content is an index, and finally a strain of Antrodia camphorata provided by the present invention is obtained.
  • the mycelium of the strain has a fast growth rate, high yield of triterpenoids, stable genetic property, and is suitable for industrial fermentation.
  • the mycelial yield increased by more than 1.5 times in liquid culture conditions, the intracellular triterpenoid yield increased by 2-3 times, and the fermentation cycle was shortened from the original 10 days to 65 hours; under solid culture conditions The total triterpenoid yield increased by 1-2 times, and the fermentation cycle was shortened from the original 90 days to 5 days.
  • the invention also provides the application of the A. sinensis high-yield triterpenoid strain, which is used for fermenting a triterpenoid compound.
  • the present invention discloses a method for breeding a high-yield triterpenoid compound Antrodia camphorata by ultraviolet mutagen protoplasts and a strain of Astragalus membranaceus high-yield selected by the method, and the strain collection number is CCTCC. M2013359.
  • the mutagenesis step includes preparing the protoplasts by shaking the cultured mycelium with ATCC200183 as the starting strain, performing ultraviolet mutagenesis, coating on the MEA plate, selecting a single colony for shake flask culture, and producing the mycelium.
  • the intracellular total triterpenoid content is an index, and finally a strain of the high-yield triterpenoid strain of the anthraquinone provided by the invention is obtained.
  • the mycelium of the strain has a fast growth rate, high triterpenoid yield, stable genetic performance, and is suitable for industrial fermentation.
  • Example 1 is a dry weight of a mycelium and a yield of triterpenes per liter of fermentation broth of each group of mycelium dried after completion of fermentation in Example 2 of the present invention.
  • Fig. 2 is a graph showing the comparison of the results of the determination of the dry weight of the mycelium in Example 2 of the present invention.
  • Fig. 3 is a graph showing comparison of data of three sputum production measurement results in Example 2 of the present invention.
  • Fig. 4 is a graph showing the dry weight of the mycelium and the yield of the triterpenes per liter of the fermentation broth of the two groups of mycelium after the completion of the fermentation in Example 3 of the present invention.
  • Fig. 5 is a graph showing the comparison of the measured results of the dry weight of the mycelium and the content of the triterpene in Example 3 of the present invention.
  • Fig. 6 is a graph showing the content of triterpene detected after drying at 40 ° C and sampling and pulverizing after completion of fermentation in Example 4 of the present invention.
  • Fig. 7 is a comparison diagram of data of determination results of triterpene content in Example 4 of the present invention.
  • the present invention provides a strain of high-yield triterpenoids of Antrodia camphorata and application thereof, and the present invention will be further described in detail below in order to make the objects, technical solutions and effects of the present invention more clear and clear. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
  • the method for breeding a high-yield triterpenoid compound Antrodia camphorata by the ultraviolet mutagen protoplast includes the following steps:
  • the ATCC200183 Antrodia camphorata strain preserved in this laboratory was inoculated on the MEA inclined surface, and placed in a constant temperature incubator at 26 ° C for 8-10 days in the dark to stop the culture.
  • the puncher was aseptically sampled and placed in a shake flask, and cultured at 120 rpm, and kept at a constant temperature of 26 ° C for 3-5 days to obtain a mycelium of the starting strain.
  • the shake flask mycelium in step 1) was centrifuged, the supernatant was removed, 10 volumes of osmotic pressure stabilizer was added, and the mixture was shaken well and centrifuged twice. After the supernatant was removed, the complex enzyme solution was added to the mycelial precipitate according to the ratio of the enzymatic hydrolysate: mycelial body weight of 3:1 (m/V), and the mixture was shaken well, and cultured at 30 ° C for 3 hours to carry out enzymatic hydrolysis.
  • the enzymatic hydrolysate was dispensed into a 50 ml sterile centrifuge tube, centrifuged at 3000 rpm for 10 min at 4 ° C, and the pellet was washed 3 times with 10 volumes of osmotic stabilizer to completely remove the enzyme solution.
  • the stabilizer was resuspended and centrifuged at 1800 rpm for 10 min, and the precipitate was discarded.
  • the supernatant was centrifuged at 600 rpm for another 10 min, and the supernatant was transferred to a supernatant to discard the precipitate. Finally, the supernatant was centrifuged at 3500 rpm for 10 min to obtain high-purity protoplasts of Antrodia camphorata.
  • Step 2 The obtained precipitate was dissolved with an osmotic pressure stabilizer, counted on a hemocytometer, and the cell concentration was adjusted to 1-5 ⁇ 10 5 /ml, which was used on the same day. Take 5 9cm sterile culture dishes, pour 3-5ml of protoplast suspension into each plate, and irradiate them with ultraviolet light for 30′′, 60′′, 90′′, 120′′ and 150′′ respectively. The lamp power is 15-30W, the irradiation distance is 25-35cm; immediately after irradiation, it is applied to the regeneration plate for regeneration, and the primary screening is carried out according to the size of the regenerated colony. Selecting 10-30 single colonies with faster growth of mycelium is carried out. The shake flask was rescreened, the total triterpenoid content of the fermented mycelium was determined by the oleanolic acid method, and 5-10 strains of high yield triacetate RT00X were selected.
  • the MEA solid medium in step 1) and step 3) is: 1-3% glucose, 1-3% malt extract, 0.1-0.3% peptone, agar 1.5 -2.0%, the balance is water, the pH is natural, and the heat is sterilized for 20 minutes.
  • the liquid medium in step 1) is: 1-3% glucose, 1-3% malt extract, 0.1-0.3% peptone, the balance is water, the pH is natural, and the heat is sterilized for 20 min.
  • the osmotic pressure stabilizer described in the steps 2) and 3) was 0.6 mol/L of mannitol, which was sterilized by a disposable needle filter.
  • the compound enzyme solution in the step 2) contains a lysozyme having a mass concentration of 2.0%, a snail enzyme of 0.5%, and a cellulase of 0.5%, and the solid enzyme is scaled and dissolved in 0.6 mol/L of nectar.
  • the filter is stored at -20 ° C after sterilization.
  • the conditions for the primary screening in the step 3) are as follows: on the MEA medium, the culture is carried out at 26 ° C for 8-10 days, and the single colony having a larger diameter is selected as the re-screening seed.
  • the medium composition was: 3.0% bran, 2.5% corn flour, 0.05% magnesium sulfate, 0.3% (m/v) cod liver oil, the balance being water, pH 2.9-3.1.
  • the preparation method of the culture medium is as follows: the bran is boiled for 30 minutes, the filtrate is taken, the corn flour is gelatinized at 100 ° C for 10 min, and then mixed with the bran filtrate, magnesium sulfate and cod liver oil are added, and the pH of the solution is adjusted to 2.9 by concentrated hydrochloric acid. 3.1, the liquid volume 80ml / 200ml, after the package is 121 ° C wet heat sterilization for 20min.
  • the culture condition was 26 ° C, the rotation speed was 90 rpm, the seed was a log phase liquid seed, the inoculum amount was 5%, and the culture time was 4 days.
  • the MEA solid medium used in step 4) is the same as step 1), and the liquid medium used is the same as the rescreen medium in step 3).
  • the total triterpenoid content of the fermented mycelium is determined by the oleanolic acid method, and the principle is that the oleanolic acid is a pentacyclic triterpenoid compound, which can develop color with various color developing agents.
  • the reaction is carried out so that colorimetric determination can be carried out.
  • Oleic acid is purple in the color reaction of vanillin-perchloric acid reagent and vanillin-sulfuric acid reagent.
  • concentration of oleanolic acid and the absorbance which is consistent with Lambert Beer's law, so colorimetric determination can be performed.
  • the instruments and reagents used are as follows:
  • Instrument 752-type UV spectrophotometer, electric thermostatic water bath, analytical balance, electric thermostat drying oven, with stoppered test tube.
  • Reagents vanillin, glacial acetic acid, absolute ethanol, ethyl acetate and perchloric acid (all of which are of analytical grade), oleanolic acid standard.
  • oleanolic acid standard 10 mg of oleanolic acid standard was accurately weighed, and 100 ml of oleanolic acid ethanol solution with a concentration of 0.1 mg/ml was prepared using anhydrous ethanol as a solvent.
  • Sample determination accurately measure the sample 0.1ml, heat to evaporate the 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 for 70min, cooled to room temperature Then, it was diluted with 5 ml of ethyl acetate and shaken, and the absorbance value was measured at a wavelength of 560 nm.
  • the blank is made up of 0.5 ml absolute ethanol as a reference solution, and the oleanolic acid content in the sample is calculated according to the standard curve drawn in step 3, and converted into a percentage.
  • the new strain of high-yield triterpenoids of Antrodia camphorata obtained by the above mutagenesis breeding method is classified as: Antrodia Cinnamomea, a new strain of Antrodia camphorata deposited in the China Center for Type Culture Collection, deposited on August 2, 2013, and deposited under CTCCC M2013359.
  • mutagenesis breeding method provided by the present invention, a new strain of Antrodia camphora which can produce a triterpenoid compound with high yield is selectively mutagenized.
  • the mutagenesis steps are as follows:
  • the ATCC200183 strain of Aconite Take the ATCC200183 strain of Aconite and transfer it to the MEA inclined surface, and incubate it in the incubator at 26 °C for 8 days in the dark.
  • the sample is placed in a shake flask by aseptic sampling with a puncher, 120 rpm, and cultured at 26 ° C for 5 days at constant temperature to obtain the starting strain.
  • the mycelium fermentation broth was 300 ml in total.
  • the MEA slant medium is: 2% glucose, 2% malt extract, 0.1% peptone, agar 1.5%, the balance is water, the pH is natural, moist heat sterilization for 20 min; the liquid medium is: 2% glucose, 2 % malt extract, 0.1% peptone, the balance is water, the pH is natural, and the heat is sterilized for 20 min.
  • the 1.1 fermentation broth was centrifuged, and the supernatant was removed to obtain a hyphal ball pellet of about 25 g, and 250 ml was added.
  • Mannitol at a concentration of 0.6 mol/L was centrifuged at 5000 rpm for 10 min after sufficient shaking, and repeated twice. After the supernatant was removed, about 80 ml of the complex enzyme solution was added to the mycelial precipitate, and the mixture was shaken well, and cultured at 30 ° C for 3 hours to carry out enzymatic hydrolysis.
  • the enzymatic hydrolysate was dispensed into a 50 ml sterile centrifuge tube, centrifuged at 3000 rpm for 10 min at 4 ° C, and the precipitate was washed 3 times with a sufficient amount of 0.6 mol/L mannitol to completely remove the enzyme solution.
  • the mannitol was resuspended and centrifuged at 1800 rpm for 10 min, and the precipitate was discarded.
  • the supernatant was centrifuged at 600 rpm for another 10 min, and the supernatant was transferred to a supernatant to discard the precipitate. Finally, the supernatant was centrifuged at 3500 rpm for 10 min to obtain a high-purity protoplast of Antrodia camphorata.
  • the complex enzyme solution contains a lysozyme having a mass concentration of 2.0%, a snail enzyme of 0.5%, and a cellulase of 0.5%.
  • the solid enzyme is weighed and dissolved in 0.6 mol/L of mannitol, and filtered. Store at -20 °C after sterilization.
  • the resulting pellet was dissolved with an osmotic pressure stabilizer, counted on a hemocytometer, adjusted to a cell concentration of 5.0 ⁇ 10 5 /ml, and used on the same day.
  • the power is 18W
  • the irradiation distance is 32cm; immediately after irradiation, it is applied to the regeneration plate for regeneration, and cultured at 26°C for 8 days, and the primary colony is selected according to the size of the regenerated colony.
  • RT001 The 28 single colonies with faster growth of the mycelium are selected and labeled as RT001, RT002, RT003...RT0027, RT0028 were shaken and sieved.
  • the total triterpenoid content of fermented mycelium was determined by oleanolic acid method.
  • Six strains with high yield of triterpenoids were selected, which were labeled RT002 and RT009. Strains of RT0017, RT0023, RT0024 and RT0027.
  • the conditions of the shake flask rescreening include the composition of the rescreening medium, the preparation method of the medium, and the culture conditions, as follows:
  • the medium composition was: 3.0% bran, 2.5% corn flour, 0.05% magnesium sulfate, 0.5% (m/v) cod liver oil, the balance being water, pH 2.95.
  • the preparation method of the culture medium is as follows: the bran is boiled for 30 minutes, the filtrate is taken, the corn flour is gelatinized at 100 ° C for 10 min, and then mixed with the bran filtrate, magnesium sulfate and cod liver oil are added, and the pH of the solution is adjusted to 2.95 by concentrated hydrochloric acid.
  • the liquid volume was 50ml/200ml, and it was sterilized by damp heat at 121 °C for 20 min after dispensing.
  • the fermentation conditions were 26 ° C, the rotation speed was 90 rpm, the seed was a log phase liquid seed, the inoculum amount was 5%, and the fermentation time was 4 days.
  • the MEA plate medium and shake flask fermentation conditions for the passage were the same as the screening conditions.
  • strain RT0023 The characteristics of strain RT0023 are described as follows:
  • the strain of Antrodia camphorata needs to be cultured in the dark, and the mycelium has a lock-like combination to produce spores, and a large amount of triterpenoids can be produced under suitable liquid culture conditions and solid culture conditions.
  • the strain grew slower on PDA plates and grew faster on MEA solid plates.
  • the suitable growth temperature is 22-28 ° C, and the optimum growth temperature is 26 ° C.
  • the growth is extremely slow below 10 ° C, and basically does not grow above 32 ° C.
  • On the MEA plate the entire plate was evenly spread around the inoculation point.
  • the time required to fill the plate on a 9 cm diameter plate at 26 ° C was about 20-22 days.
  • the mycelium color began to be white, and then slowly changed.
  • the mycelium produces volatile substances during the growth process, and has a special fruity and fragrant taste.
  • the strain Under the condition of shake flask liquid shaking culture, the strain can produce mycelial spheres of different sizes, and the fermentation liquid has a special peach peach flavor; when the culture is static, the liquid surface is covered with orange yellow mycelium.
  • conditional fermentation of the shake flask is carried out in three conditions:
  • the A. sinensis strain A and the mutagenized strain B slanted species were transferred to the MEA plate medium, and the slanted species were activated and cultured at a culture temperature of 26 ° C for 15 days.
  • the hyphae of the strain A and the mutagenized strain B were respectively taken out by a puncher, and transferred to a liquid shake flask for liquid seed culture, the culture temperature was 26 ° C, the rotation speed was 100 rpm, and the culture was carried out for 6 days. .
  • the composition of the liquid seed medium glucose 1.0%, malt extract 1.0%, peptone 0.1%, magnesium sulfate 0.05%, the balance is water, the pH is natural, 121 ° C, and moist heat sterilization for 20 min.
  • the shake flask seeds of the two strains obtained in 2.2 were transferred to a triangular flask according to 5% inoculation amount, and the volume of the triangular flask was 500 ml.
  • the fermentation was carried out, and each strain was made into 5 parallels, which were labeled as A1, A2, A3, and A4. , A5, B1, B2, B3, B4, B5, wherein Ax corresponds to the starting strain, and Bx corresponds to the mutagenized strain.
  • the fermentation medium consisted of: 3.0% bran, 2.5% corn flour, 0.05% magnesium sulfate, 0.5% (m/v) cod liver oil, the balance being water, pH 2.95.
  • the preparation method of the culture medium is as follows: the bran is boiled for 30 minutes, the filtrate is taken, the corn flour is gelatinized at 100 ° C for 10 min, and then mixed with the bran filtrate, magnesium sulfate and cod liver oil are added, and the pH of the solution is adjusted to 3.1 by concentrated hydrochloric acid.
  • the liquid volume was 500ml/2000ml, and it was sterilized by damp heat at 121 °C for 20 min after dispensing.
  • the culture conditions were as follows: culture temperature 26 ° C, initial pH 3.1, rotation speed 90 rpm, and continuous culture under these conditions for 4 days.
  • the mycelium is collected by centrifugation according to the number, dried at 40 ° C, and the dry weight and triterpenoid content of the mycelium of each group of fermentation broth are determined, and then converted according to the dry weight of the mycelium and the content of triterpenoids.
  • the dry weight of the mycelium in the fermentation broth and the yield of the triterpenoids are shown in Figure 1.
  • the dry weight of the bacteria is shown in Figure 2, and the yield of the three is shown in Figure 3.
  • the upper 500L fermentor is subjected to fermentation culture, and the specific implementation is as follows:
  • the A. sinensis strain A and the mutagenized strain B slanted species were transferred to the MEA plate medium, and the slanted species were activated and cultured at a culture temperature of 26 ° C for 15 days.
  • the composition of the liquid seed medium glucose 1.0%, malt extract 1.0%, peptone 0.1%, magnesium sulfate 0.05%, the balance is water, the pH is natural, 121 ° C, and moist heat sterilization for 20 min.
  • the shake flask seeds A and B obtained in 3.2 were transferred to a 50 L fermentor at a 10% inoculum, and seed expansion culture was carried out.
  • the seed tank medium consisted of: 5.0% bran, 4.5% corn flour, 0.05% magnesium sulfate, and the balance was water.
  • the culture conditions were as follows: the culture temperature was 26 ° C, the initial pH value was natural, the rotation speed was 110 rpm, and the aeration ratio was 1. 0 vvm, continuous culture under these conditions for 5 days.
  • the preparation method of the culture medium is as follows: the bran is boiled for 30 minutes, the filtrate is taken, the corn flour is gelatinized at 100 ° C for 10 min, and then mixed with the bran filtrate, and the magnesium sulfate is added, the pH value is natural, and the liquid volume is 35 L/50 L, 121. °C moist heat sterilization for 20min.
  • the seeded seed obtained from 3.3 was transferred to a 500 L fermentor at a 10% inoculum and subjected to a three-inch process.
  • the fermenter medium consisted of: 2.2% bran, 2.0% corn flour, 0.05% magnesium sulfate, 0.5% (m/v) cod liver oil, the balance being water, pH 3.0; culture conditions: culture temperature 26 ° C, initial The pH was 3.0, the rotation speed was 90 rpm, and the aeration ratio was 0.6 vvm, and the culture was continued for 65 hours under the conditions.
  • the preparation method of the culture medium is as follows: the bran is boiled for 30 minutes, the filtrate is taken, the corn flour is gelatinized at 100 ° C for 10 min, and then mixed with the bran filtrate, magnesium sulfate and cod liver oil are added, and the pH of the solution is adjusted to 3.0 by concentrated hydrochloric acid.
  • the liquid volume was 350L/500L, and it was sterilized by wet heat at 121 °C for 20 min.
  • the mycelium is collected by plate and frame filtration, dried at 40 ° C, and the total dry weight and triterpenoid content of the mycelium are determined, and then converted into fermentation broth according to the obtained dry weight of the mycelium and the content of triterpenoids.
  • the dry weight of the mycelium and the yield of the triterpenoids are shown in Fig. 4 and Fig. 5.
  • the A. sinensis strain A and the mutagenized strain B slanted species were transferred to the MEA plate medium, and the slanted species were activated and cultured at a culture temperature of 26 ° C for 15 days.
  • the composition of the liquid seed medium glucose 1.0%, malt extract 1.0%, peptone 0.1%, magnesium sulfate 0.05%, the balance is water, the pH is natural, 121 ° C, and moist heat sterilization for 20 min.
  • the shake flask seeds obtained in 4.2 were separately transferred into solid medium, shaken, and subjected to solid fermentation. Each strain was made in 5 parallels, labeled as A1, A2, A3, A4, A5 and B1, B2, B3. , B4, B5.
  • the solid medium composition was: 85% bran, 14.5% corn flour, 0.1% magnesium sulfate, 0.4% olive oil, and a mixture of diluted hydrochloric acid having a pH of 3.0, and a ratio of material to liquid of 1.05:1.
  • the prepared medium was placed in a 2 L flask, and each flask was charged at 450 g.
  • the fermentation conditions were: 5% inoculation (5 ml of liquid seed per 100 g of wet material), and cultured at 25 ° C for 10 days.
  • the strain (CCTCC) M2013359) has a high growth rate of mycelium, high yield of triterpenoids, stable genetic performance, and is suitable for industrial fermentation.
  • the mycelial yield increased by more than 1.5 times in liquid culture conditions
  • the intracellular triterpenoid yield increased by 2-3 times
  • the fermentation cycle was shortened from the original 10 days to 65 hours; under solid culture conditions
  • the total triterpenoid yield increased by 1-2 times, and the fermentation cycle was shortened from the original 90 days to 10 days.

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Abstract

提供了一种樟芝菌株及其应用，该菌株的保藏编号为CCTCC M2013359，且其产生的三萜类化合物产量高。该菌株是以保藏号为ATCC 200183的樟芝菌株作为出发菌株，经紫外诱变后筛选获得。与出发菌株相比，其菌丝体生长速度快，三萜类化合物产量高，遗传性能稳定，适合工业化发酵。

Description

樟芝高产三萜菌株及其应用 技术领域
本发明涉及生物工程领域，尤其涉及一种经紫外诱变原生质体所选育得到的三萜类化合物产量高的樟芝新菌株及其应用。
 背景技术
　　　樟芝又叫牛樟芝（Antrodia cinnamomea），是原产于台湾的珍稀药用真菌，具有极大的药用价值，被誉为“抗癌圣药”、“保肝之王”、“解毒之王”，也是目前价格最贵的药用真菌。现代医学研究证实，樟芝的主要活性成分为三萜类化合物。而野生樟芝数量稀少，人工培植的樟芝子实体虽然具有与野生樟芝相似的三萜成分，但子实体生长缓慢，远远不能满足市场需求。目前，采用现代生物技术进行人工发酵是获得樟芝三萜类化合物的最佳途径，人工发酵技术主要分为固体发酵和深层液体发酵。樟芝固体发酵周期较长( 90-180天)，固体发酵产物中总三萜含量约5%左右，远低于野生子实体中10%-45%的总三萜含量；樟芝液体发酵周期相对较短 (7-14天)，但产物中基本不含三萜类化合物。因此，这两种人工发酵方式都存在发酵周期长、三萜产量低的缺点。菌种是决定发酵水平的内在因素，多种药用真菌通过诱变育种均显著提升了药用活性成分的产量，实现了药用真菌的产业化生产。李刚（2001）、李颖颖等（2011）通过常规诱变育种分别获得了灵芝高产多糖和三萜的优良菌株；张日俊（2004）通过化学和物理组合诱变筛选到了高产蛋白酶的芽孢杆菌；应汉杰等（2007）通过低能粒子束注入孢子的方式获得了高产核酸酶P1的桔青霉菌。类似的研究不胜枚举，可见，通过诱变育种提高食药用真菌的活性物产量不失为一种有效的手段。
　　　截止目前，有关樟芝的诱变育种研究鲜有报道，仅有的文献资料显示，青岛农业大学山东省应用真菌重点实验室的田雪梅等人（2012）通过物理诱变获得了5株樟芝诱变株，其中编号为327的菌株为多糖和蛋白质高产菌株，多糖和蛋白质的产量分别比出发菌株提高238.2%和10.33%。Gg为三萜高产菌株，三萜产量比出发菌株提高57.86%，液体发酵总三萜产量为18mg/L，折合成干燥菌丝体总三萜含量为0.47%，但是事实上这个三萜含量并不算高，离产业化要求尚有很大差距。
因此，现有技术还有待于改进和发展。
 技术问题
鉴于上述现有技术的不足，本发明的目的在于提供一种樟芝高产三萜菌株及其应用，旨在解决现有樟芝菌株人工发酵时三萜类化合物产量低的问题。
技术解决方案
　　　在本发明方案中，采用的是紫外诱变原生质体的方法。菌种诱变育种包括物理诱变和化学诱变，常见的物理诱变方法有紫外诱变、微波诱变、空间诱变、60Co-γ射线辐照诱变、X射线诱变、离子注入诱变等，化学诱变剂主要有烷化剂、碱基类似物、羟胺、吖啶色素等。其中，紫外诱变具有操作简单、诱变效率高、可重复性强、诱变源易于获得等优点，成为很多生物材料诱变育种的常规手段。
　　　樟芝属于大型真菌，它的孢子外壳坚韧，如果直接用孢子作为诱变材料不仅需要高强度诱变处理，且成功概率低，即使诱变成功也难以在人工条件下萌发。而樟芝菌丝体多为链状复合细胞体，单个细胞发生诱变难以筛选出来，因此菌丝体也不宜作为诱变材料。目前比较先进的做法是原生质体间接诱变，即利用生物酶法去除细胞壁制备原生质体，原生质体对外界环境较为敏感、经诱变处理后很容易发生突变，且再生菌落由单细胞发育而来，容易筛选，再生菌株可以保持原有菌种的主要生物学特点，育种周期大大缩短，突变株生物学性状比较稳定，不易退化。
　　　关于出发菌株的选择，诱变育种所采用的出发菌株也十分关键。目前全世界各菌种保藏机构的樟芝菌种有十几种之多，多数从自然界野生樟芝的子实体或牛樟树树洞中分离而来，这些菌株的菌丝体长势较弱、在平板和液体培养基中生长缓慢。本发明采用菌种编号为ATCC200183的樟芝菌种作为出发菌株，该菌株菌丝体相对粗壮，在平板和液体培养基中生长速度相对较快，用它作为出发菌株有利于筛选出理想的樟芝优良菌种。
　　　本发明的目的是获得在液体培养条件下高产三萜类化合物的樟芝高产三萜菌株。在筛选过程中，以樟芝菌丝体长满平板所需的时间和摇瓶发酵菌丝体总三萜产量为考察指标，利用紫外线对樟芝原生质体进行反复诱变，来获得高产三萜类化合物的樟芝新菌株。
　　　综上，本发明中公开了一种紫外诱变原生质体选育高产三萜类化合物樟芝新菌株的方法及采用此方法所选育得到的樟芝高产三萜菌株，菌株保藏编号为CCTCC M2013359。其诱变步骤包括，以ATCC200183樟芝菌株为出发菌株，摇瓶培养菌丝体制备原生质体，进行紫外诱变；涂布于MEA平板上，挑选单菌落进行摇瓶培养；以菌丝体产量和胞内总三萜含量为指标，最后得到一株本发明所提供的樟芝菌株。该菌株菌丝体生长速度快，三萜产量高，遗传性能稳定，适合工业化发酵。与出发菌株ATCC200183相比，液体培养条件下菌丝体产量提高了1.5倍以上，胞内三萜产量提高了2-3倍，发酵周期从原来的10天以上缩短为65小时；固体培养条件下总三萜产量提高了1-2倍，发酵周期从原来的90天以上缩短至5天。
　　　本发明中还提供所述樟芝高产三萜菌株的应用，将所述樟芝高产三萜菌株用于发酵生产三萜类化合物。
 有益效果
有益效果：本发明中公开了一种紫外诱变原生质体选育高产三萜类化合物樟芝新菌株的方法及采用此方法所选育得到的樟芝高产三萜菌株，菌株保藏编号为CCTCC M2013359。其诱变步骤包括，以ATCC200183樟芝菌株为出发菌株，摇瓶培养菌丝体制备原生质体，进行紫外诱变，涂布于MEA平板上，挑选单菌落进行摇瓶培养，以菌丝体产量和胞内总三萜含量为指标，最终得到一株本发明所提供的樟芝高产三萜菌株。该菌株菌丝体生长速度快、三萜产量高、遗传性能稳定、适合工业化发酵。
 附图说明
　　　图1是本发明实施例2中发酵完成后，40℃烘干，各组菌丝体每升发酵液中的菌丝体干重和三萜产量。
　　　图2为本发明实施例2中菌丝体干重测定结果数据对比图。
　　　图3为本发明实施例2中三萜产量测定结果数据对比图。
　　　图4是本发明实施例3中发酵完成后，40℃烘干，两组菌丝体每升发酵液中的菌丝体干重和三萜产量。
　　　图5为本发明实施例3中菌丝体干重和三萜含量的测定结果数据对比图。
　　　图6为本发明实施例4中发酵完成后，40℃烘干，取样粉碎后检测的三萜含量。
图7为本发明实施例4中三萜含量测定结果数据对比图。
 本发明的实施方式
　　　本发明提供一种樟芝高产三萜菌株及其应用，为使本发明的目的、技术方案及效果更加清楚、明确，以下对本发明进一步详细说明。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。
　　　具体地，所述紫外诱变原生质体选育高产三萜类化合物樟芝新菌株的方法，包括以下步骤：
　　　1）出发菌株菌丝体制备
　　　将本实验室保存的ATCC200183樟芝菌种接种到MEA斜面上，置于26℃恒温培养箱中避光培养8-10天停止培养。打孔器无菌取样置于摇瓶中，120rpm，26℃恒温培养3-5天获得出发菌株的菌丝体。
　　　2）出发菌株原生质体制备
　　　将步骤1）中的摇瓶菌丝体进行离心，去上清，加入10倍体积的渗透压稳定剂，充分震荡后离心，重复2次。去上清后，按照酶解液：菌丝体重3:1（m/V）的比例，在菌丝体沉淀中加入复合酶液，充分振荡，30℃培养3小时进行酶解。酶解完成后，立即将酶解液分装到50ml无菌离心管中，4℃，3000rpm离心10min，沉淀用10倍体积的渗透压稳定剂清洗3次，彻底去除酶液。稳定剂重悬后1800rpm离心10min，弃沉淀。上清600rpm再离心10min，转移上清弃沉淀。最后将上清3500rpm离心10min，即可获得高纯度樟芝原生质体。
　　　3）原生质体诱变
　　　步骤2）所得沉淀用渗透压稳定剂溶解，血球计数板计数，调整细胞浓度至1-5×105个/ml，当天配置当天使用。取5个9cm的无菌培养皿，每个平皿倒入3-5ml原生质体悬液，在电磁搅拌下，用紫外灯分别照射处理30″、60″、90″、120″和150″，紫外灯功率为15-30W，照射距离为25-35cm；照射后立即涂布至再生平板上再生，根据再生菌落的大小进行初筛。挑选10-30个菌丝体生长较快的单菌落，进行摇瓶复筛，齐墩果酸法测定发酵菌丝体的总三萜含量，选择5-10株三萜产量高的菌株RT00X。
　　　4） 遗传性状稳定性验证
　　　将上述5-10株诱变筛选过的高产菌株连续传代20-30代，考察其在MEA平板上的菌丝体生长状况和摇瓶发酵菌丝体的总三萜产量，最终筛选出一株生长速度快、高产三萜类化合物、性状稳定的新菌株。
　　　其中，步骤1）和步骤3）中的MEA固体培养基为：1-3%葡萄糖，1-3%麦芽浸膏，0.1-0.3%蛋白胨，琼脂1.5 -2.0%，余量为水，pH值自然，湿热灭菌20min。
　　　步骤1）中的液体培养基为：1-3%葡萄糖，1-3%麦芽浸膏，0.1-0.3%蛋白胨，余量为水，pH值自然，湿热灭菌20min。
　　　步骤2）和3）中所述的渗透压稳定剂为0.6mol/L的甘露醇，经一次性针头过滤器除菌。
　　　步骤2）中所述复合酶液中含有质量浓度为2.0%的溶壁酶、0.5%的蜗牛酶、0.5%的纤维素酶，将固体酶按比例称好后溶于0.6mol/L的甘露醇中，过滤除菌后-20℃保存。
　　　步骤3）中所述初筛的条件为：在MEA培养基上，26℃培养8-10天，挑选单菌落直径较大的作为复筛种子。
　　　步骤3）中所述摇瓶复筛的条件为：
　　　培养基组成为：3.0%麸皮，2.5%玉米粉，0.05%硫酸镁，0.3%（m/v）鱼肝油，余量为水，pH值2.9-3.1。
　　　所述培养基配制方式为：麸皮微火煮沸30min，取滤液，玉米粉100℃糊化10min后同麸皮滤出液混合，加入硫酸镁和鱼肝油，浓盐酸调节溶液的pH值至2.9-3.1，装液量80ml/200ml，分装后121℃湿热灭菌20min。
　　　所述的培养条件为26℃，转速90rpm，种子为对数期液体种子，接种量为5%，培养时间4天。
　　　步骤4）中所用的MEA固体培养基同步骤1），所用的液态培养基同步骤3）中的复筛培养基。
　　　步骤3）中，是采用齐墩果酸法测定发酵菌丝体的总三萜含量，其原理为：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。齐墩果酸于香草醛-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。所采用的仪器和试剂如下：
　　　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。
　　　采用齐墩果酸法测定三萜类化合物含量的操作步骤如下：
　　　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、0.4、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，合并滤液并定容至100ml。
　　　5．样品测定：准确量取样品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。
　　　采用上述诱变选育方法得到的樟芝高产三萜类化合物新菌株，其分类命名为：樟芝Antrodia cinnamomea，此樟芝新菌株保藏于中国典型培养物保藏中心，保藏日期为2013年8月2日，保藏号为CCTCC M2013359。
　　　以下通过实施例对本发明做进一步的说明。
　　　实施例1
　　　按照本发明所提供的诱变育种方法，诱变选育可高产三萜类化合物的樟芝新菌株。诱变步骤如下：
　　　1.1出发菌株菌丝体制备
　　　取樟芝出发菌株ATCC200183转接到MEA斜面上，置26℃恒温培养箱中避光培养8天，用打孔器无菌取样置于摇瓶中，120rpm，26℃恒温培养5天获得出发菌株的菌丝体发酵液共300ml。
　　　所述MEA斜面培养基为：2%葡萄糖，2%麦芽浸膏，0.1%蛋白胨，琼脂1.5%，余量为水，pH值自然，湿热灭菌20min；液体培养基为：2%葡萄糖，2%麦芽浸膏，0.1%蛋白胨，余量为水，pH值自然，湿热灭菌20min。
　　　1.2出发菌株原生质体制备
　　　将1.1发酵液离心，去上清，获得菌丝球沉淀约25g，加入250ml 浓度为0.6mol/L的甘露醇，充分震荡后5000rpm离心10min，重复2次。去上清后，在菌丝体沉淀中加入复合酶液约80ml，充分振荡，30℃培养3小时进行酶解。酶解完成后，立即将酶解液分装到50ml无菌离心管中，4℃，3000rpm离心10min，沉淀用足量0.6mol/L的甘露醇清洗3次，彻底去除酶液。甘露醇重悬后1800rpm离心10min，弃沉淀。上清600rpm再离心10min，转移上清弃沉淀。最后将上清3500rpm离心10min，获得高纯度樟芝原生质体。
　　　所述复合酶液中含有质量浓度为2.0%的溶壁酶、0.5%的蜗牛酶、0.5%的纤维素酶，将固体酶按比例称好后溶于0.6mol/L的甘露醇中，过滤除菌后-20℃保存。
　　　1.3原生质体诱变
　　　1.2所得沉淀用渗透压稳定剂溶解，血球计数板计数，调整细胞浓度至5.0×105个/ml，当天配置当天使用。取5个直径9cm的无菌培养皿，每个平皿倒入5ml原生质体悬液，在电磁搅拌下，用紫外灯分别照射处理30″、60″、90″、120″和150″，紫外灯功率为18W，照射距离为32cm；照射后立即涂布至再生平板上再生，26℃培养8天，根据再生菌落大小进行初筛。挑选28个菌丝体生长较快的单菌落，分别标记为RT001、 RT002、RT003……RT0027、RT0028进行摇瓶复筛，齐墩果酸法测定发酵菌丝体的总三萜含量，选择6株三萜产量高的菌株，分别为标记是RT002、RT009、RT0017、RT0023、RT0024和RT0027的菌株。
　　　所述摇瓶复筛的条件包括复筛培养基组成、培养基配制方式、培养条件，具体如下：
　　　培养基组成为：3.0%麸皮，2.5%玉米粉，0.05%硫酸镁，0.5%（m/v）鱼肝油，余量为水，pH值2.95。
　　　所述培养基配制方式为：麸皮微火煮沸30min，取滤液，玉米粉100℃糊化10min后同麸皮滤出液混合，加入硫酸镁和鱼肝油，浓盐酸调节溶液的pH值至2.95，装液量50ml/200ml，分装后121℃湿热灭菌20min。
　　　发酵条件为26℃，转速为90rpm，种子为对数期液体种子，接种量为5%，发酵时间4天。
　　　
　　　1.4 遗传性状稳定性验证
　　　将上述6株诱变筛选过的高产菌株连续传代30代，根据其在MEA平板上的菌丝体生长状况和摇瓶发酵菌丝体的总三萜产量，最终筛选出一株生长速度快，高产三萜类化合物，性状稳定的新菌株RT0023。此菌株RT0023即为保藏编号为CCTCC M2013359的菌株。
　　　所述传代用的MEA平板培养基和摇瓶发酵条件同复筛条件。
　　　菌株RT0023特征描述如下：
　　　该樟芝菌株需避光培养，显微镜观察菌丝体具有锁状联合，可产生孢子，在合适的液体培养条件和固体培养条件下可产生大量的三萜类化合物。该菌株在PDA平板上生长较慢，在MEA固体平板上生长速度较快。适宜生长温度为22-28℃，最适生长温度为26℃，低于10℃生长极其缓慢，高于32℃基本不生长。在MEA平板上，以接种点为中心均匀铺满整个平板，在26℃，直径为9cm平皿上长满平板所需的时间约20-22天，菌丝体颜色开始为白色，后慢慢变为浅黄色至橘黄色。菌丝体生长过程中产生挥发性物质，具有特殊的水果味和香樟味。摇瓶液体震荡培养条件下，该菌株可产生大小不一的菌丝球，发酵液有特殊的水蜜桃香味；静置培养时会在液面长满橘黄色菌丝体。
　　　实施例2
　　　分别采用出发菌株A（ATCC200183）和诱变菌株B（CCTCC M2013359）
作为发酵菌种，进行摇瓶樟芝三萜条件发酵，具体实施如下：
　　　2.1斜面菌种活化
　　　分别将樟芝出发菌株A和诱变菌株B斜面菌种转接至MEA平板培养基上，进行斜面菌种的活化培养，培养温度26℃，培养时间15天。
　　　2.2摇瓶液体种子制备
　　　无菌条件下，分别用打孔器取出发菌株A和诱变菌株B的菌丝块各5个，转入液体摇瓶进行液体种子培养，培养温度为26℃，转速为100rpm，培养6天。
　　　液体种子培养基组成：葡萄糖1.0%，麦芽浸膏1.0%，蛋白胨0.1%，硫酸镁0.05%，余量为水，pH值自然，121℃，湿热灭菌20min。
　　　2.3 发酵培养
　　　分别将2.2所得两株菌株的摇瓶种子按5%接种量转入三角瓶，三角瓶装液量为500ml，进行发酵培养，每个菌株做5个平行，分别标记为A1、A2、A3、A4、A5，B1、B2、B3、B4、B5，其中Ax对应出发菌株，Bx对应诱变菌株。
　　　所述发酵培养基组成为：3.0%麸皮，2.5%玉米粉，0.05%硫酸镁，0.5%（m/v）鱼肝油，余量为水，pH值2.95。
　　　所述培养基配制方式为：麸皮微火煮沸30min，取滤液，玉米粉100℃糊化10min后同麸皮滤出液混合，加入硫酸镁和鱼肝油，浓盐酸调节溶液的pH值至3.1，装液量500ml/2000ml，分装后121℃湿热灭菌20min。
　　　所述培养条件为：培养温度26℃，初始pH值3.1，转速90rpm，在此条件下连续培养4天。
　　　2.4 菌丝体和三萜产量测定
　　　发酵完成后，按照编号分别离心收集菌丝体，40℃烘干，分别测定各组发酵液的菌丝体干重和三萜含量，再根据所得菌丝体干重和三萜含量换算为每升发酵液中的菌丝体干重和三萜产量，具体结果见图1，菌体丝干重图如图2，三萜产量图如图3所示。
　　　实施例3
　　　分别采用出发菌株A（ATCC200183）和诱变菌株B（CCTCC M2013359）
作为发酵菌种，上500L发酵罐进行发酵培养，具体实施如下：
　　　3.1斜面菌种活化
　　　分别将樟芝出发菌株A和诱变菌株B斜面菌种转接至MEA平板培养基上，进行斜面菌种的活化培养，培养温度26℃，培养时间15天。
　　　3.2摇瓶液体种子制备
　　　无菌条件下，分别用打孔器取出发菌株A和诱变菌株B的菌丝块各5个，转入液体摇瓶进行液体种子培养，培养温度26℃，转速100rpm，培养6天。
　　　液体种子培养基组成：葡萄糖1.0%，麦芽浸膏1.0%，蛋白胨0.1%，硫酸镁0.05%，余量为水，pH值自然，121℃，湿热灭菌20min。
　　　3.3 种子扩培
　　　分别将3.2所得摇瓶种子A和B按10%接种量转接入50L发酵罐中，进行种子扩大培养。种子罐培养基组成为：5.0%麸皮，4.5%玉米粉，0.05%硫酸镁，余量为水。培养条件为：培养温度26℃，初始pH值自然，转速110rpm，通气比1. 0vvm，在此条件下连续培养5天。
　　　所述培养基配制方式为：麸皮微火煮沸30min，取滤液，玉米粉100℃糊化10min后同麸皮滤出液混合，加入硫酸镁，pH值自然，装液量35L/50L，121℃湿热灭菌20min。
　　　3.4发酵培养
　　　分别将3.3所得种子罐种子按10%接种量转入500L发酵罐，进行三萜工艺培养。发酵罐培养基组成为：2.2%麸皮，2.0%玉米粉，0.05%硫酸镁，0.5%（m/v）鱼肝油，余量为水，pH值3.0；培养条件为：培养温度26℃，初始pH值3.0，转速90rpm，通气比0.6vvm，在此条件下连续培养65小时。
　　　所述培养基配制方式为：麸皮微火煮沸30min，取滤液，玉米粉100℃糊化10min后同麸皮滤出液混合，加入硫酸镁和鱼肝油，浓盐酸调节溶液的pH值至3.0，装液量350L/500L，121℃湿热灭菌20min。
　　　3.5 菌丝体和三萜产量测定
　　　发酵完成后，分别板框过滤收集菌丝体，40℃烘干，测定两组菌丝体总干重和三萜含量，再根据所得菌丝体干重和三萜含量换算为每升发酵液中的菌丝体干重和三萜产量，具体结果见图4和图5。
　　　实施例4
　　　分别采用出发菌株A（ATCC No 200183）和诱变菌株B(CCTCC M2013359)作为发酵菌种，上500L发酵罐进行发酵培养，具体实施如下：
　　　4.1斜面菌种活化
　　　分别将樟芝出发菌株A和诱变菌株B斜面菌种转接至MEA平板培养基上，进行斜面菌种的活化培养，培养温度26℃，培养时间15天。
　　　4.2摇瓶液体种子制备
　　　无菌条件下，分别用打孔器取出发菌株A和诱变菌株B的菌丝块各5个，转入液体摇瓶进行液体种子培养，培养温度26℃，转速100rpm，培养6天。
　　　液体种子培养基组成：葡萄糖1.0%，麦芽浸膏1.0%，蛋白胨0.1%，硫酸镁0.05%，余量为水，pH值自然，121℃，湿热灭菌20min。
　　　4.3 固体发酵
　　　将4.2所得的摇瓶种子分别转接入固体培养基中，摇匀，进行固体发酵，每个菌种做5个平行，分别标记为A1、A2、A3、A4、A5和B1、B2、B3、B4、B5。固体培养基组成为：85%麸皮，14.5%玉米粉，0.1%硫酸镁，0.4%橄榄油，用pH值为3.0的稀盐酸拌料，料液比为1.05:1。将配好的培养基装入2L三角瓶中，每个三角瓶装料量为450g。发酵条件为：接种量为5%（每100g湿料接种5ml液体种子），25℃，培养10天。
　　　发酵完成后，分别将每个三角瓶的培养物进行40℃低温烘干，取样粉碎后检测三萜含量，具体结果见图6和图7。
　　　综上所述，从实施例2~4可以看出，该菌株(CCTCC M2013359)的菌丝体生长速度快、三萜产量高、遗传性能稳定、适合工业化发酵。与出发菌株ATCC200183相比，液体培养条件下菌丝体产量提高了1.5倍以上，胞内三萜产量提高了2-3倍，发酵周期从原来的10天以上缩短为65小时；固体培养条件下总三萜产量提高了1-2倍，发酵周期从原来的90天以上缩短至10天。
应当理解的是，本发明的应用不限于上述的举例，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进和变换都应属于本发明所附权利要求的保护范围。

Claims (2)

  1. 樟芝高产三萜菌株，其特征在于，所述樟芝高产三萜菌株的保藏编号为CCTCC M2013359。
  2. 一种如权利要求1所述的樟芝高产三萜菌株的应用，其特征在于，将所述樟芝高产三萜菌株用于发酵生产三萜类化合物。
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