CN103125270A - High-yield antrodia cinnamomea mycelium fermentation method for triterpenoids - Google Patents
High-yield antrodia cinnamomea mycelium fermentation method for triterpenoids Download PDFInfo
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Abstract
The invention relates to the field of fungus liquid fermentation culturing and provides a high-yield antrodia cinnamomea mycelium fermentation method for triterpenoids. The method includes following steps: cant strain activation, shake flask liquid seed preparation, liquid seed cultivation and fermentation under terpene conditions, fermentation time under the terpene conditions is greatly reduced to shortest 2 days, and culture medium for fermentation under terpene conditions is composed of 10-60g/L of corn starch, 10-60g/L of bran, 0.5-2.0g/L of magnesium sulfate and 2-10ml/L olive oil. By the method, yield of triterpenoids in antrodia cinnamomea liquid fermentation product is greatly increased, the content of triterpenoids is equivalent to that in antrodia cinnamomea mycelium produced by the basswood cultivation method while cost is lowered by thousand times, and even compared with existing liquid fermentation processes, the method has the advantages that the yield of triterpenoids is substantially increased, cost is lowered quite obviously, and a solid foundation is laid for industrialization of antrodia cinnamomea.
Description
Technical field
The present invention relates to field of fermentation engineering, relate in particular to the Antrodia camphorata mycelium fermented method of high yield triterpene compound.
Background technology
The camphor tree sesame belongs to Basidiomycetes, Aphyllophorales, Polyporaceae, Antrodia belongs to, it is a kind of rare medicinal fungi, contain multiple physiologically active ingredient in the camphor tree sesame, as polysaccharide, triterpene compound, Sudismase (SOD) adenosine, protein (containing immune protein), multivitamin, trace element, nucleic acid, agglutinin, amino acid, cholesterol, lignin, blood pressure stabilization material etc.That it physiological active functions that possesses has is antitumor, strengthen immunity, antiviral, antiallergy, anti-hypertension, inhibition platelet aggregation, hypotensive, norcholesterol, antibacterium, protection liver etc.Wherein triterpene compound is the core pharmaceutical component of medicinal fungi, has strong physiologically active, and triterpene compound is the main component of the effects such as medicinal fungi performance anti-inflammatory, analgesia, poisoning tumour cell and induced tumor Apoptosis, anti-anoxic; But also have the effect that improves immunity of organisms, and show the promotion lymphocytosis, improve phagocytic activity and the lethality of macrophage, NK cell, T cell.And can effectively promote liver cell regeneration, reparation and lifting liver function, effectively reduce the damage of chemotherapeutic period patient liver and kidney.
Therefore the camphor tree sesame has all advantages of glossy ganoderma, and effect more is better than glossy ganoderma, and more than ten times of its active ingredient triterpene compound content or even glossy ganoderma have the good reputation of " king of glossy ganoderma ".Data shows, the glossy ganoderma of single variety only has 50 kinds of triterpene compounds of 20 ?, and the triterpene compound summation of different cultivars glossy ganoderma just reaches kind more than 200, and the camphor tree sesame of single variety namely has more than 200 kind of triterpene compound, and the impurity such as FAF, anticancer effect is more outstanding.
Camphor tree sesame artificial culture can be divided into three kinds of methods, 1) solution fermentation, 2) the solid state cultivation method, 3) the cultivation basswood method.Deep fermentation is the most effectual way of present deep development medicinal fungus active component, can obtain fast and efficiently Antrodia camphorata mycelium, and has accomplished scale production on mushroom, grifola frondosus, Brazilian mushroom, paecilomyces hepiali Chen.
Existing bibliographical information camphor tree sesame liquid fermentation process, improve biomass and polysaccharide but study mainly to concentrate on both at home and abroad, and is less take raising camphor tree sesame triterpene compound as the research of purpose.Taiwan part Study person has studied camphor tree sesame liquid culture technique, but the triterpene compound productive rate is lower.Southern Yangtze University's fungal studies laboratory is studied camphor tree sesame liquid fermentation, is mainly that to improve Antrodia camphorata mycelium output be target, and camphor tree sesame triterpene compound output is in the 65mg/L left and right.
Summary of the invention
The object of the present invention is to provide a kind of Antrodia camphorata mycelium fermented method of high yield triterpene compound, not only improve mycelial output, the more important thing is the content that improves triterpene compound in mycelium.
For achieving the above object, technical scheme of the present invention is,
A kind of Antrodia camphorata mycelium fermented method of high yield triterpene compound comprises the following steps,
Slant strains activation is forwarded to camphor tree sesame slant strains on plating medium,, again is forwarded on fresh plating medium and cultivates during greater than 2cm until camphor tree sesame colony diameter, makes mycelium cover with flat board and forms the Chinese red spore;
The preparation of shaking flask liquid seeds, aseptic collection camphor tree Ganoderma lucidum spore, the access shaking flask is carried out liquid culture, does not stop cultivating when forming mycelium pellet until liquid nutrient medium appearance muddiness, obtains shake-flask seed;
Liquid seeds spreads cultivation, and changes shake-flask seed over to seeding tank and carries out bacterial classification and spread cultivation, and occurs muddy and when not forming mycelium pellet, changes the next stage seeding tank over to until zymotic fluid, enlarges step by step preparation fermenting and producing liquid seeds;
The fermentation of triterpene condition, change the aforesaid liquid seed over to the next stage fermentation tank by 10% inoculum concentration, carry out the fermentation of triterpene condition, fermentation condition is: 25-30 ℃, and pH value 3.0-5.0, ventilation ratio 0.3-2.0vvm, incubation time 2-3 days, the composition of triterpene condition fermentation medium was: corn starch 10-60g/L, wheat bran 10-60g/L, magnesium sulfate 0.5-2.0g/L, olive oil 2-10ml/L.
Described triterpene fermentation condition is: 28 ℃, and pH value 3.0, ventilation ratio 0.35vvm, incubation time 2 days, the composition of triterpene condition fermentation medium is: corn starch 20-25g/L, wheat bran 20-25g/L, magnesium sulfate 1.5-2.0g/L, olive oil 2-5ml/L.
Described liquid seeds spreads cultivation in step, and the seeding tank inoculum concentration is 10%, and the seed tank culture condition is: temperature 25-30 ℃, and pH value 3.0-5.0, ventilation ratio 0.5-2.0vvm, incubation time 3-5 days; The seed tank culture base forms: glucose 10-20g/L, malt extract 10-20g/L, peptone 1-5g/L, magnesium sulfate 0.1-1.0g/L.
Described seed tank culture condition is: 28 ℃ of temperature, pH value 4.0, ventilation ratio 1.0vvm, incubation time 3 days; The seed tank culture base forms: glucose 18.8g/L, malt extract 18.8g/L, peptone 3.3g/L, magnesium sulfate 0.5g/L.
In described shaking flask liquid seeds preparation process, the condition of shaking flask liquid culture is: temperature 25-30 ℃, and pH value 5.0-7.0, rotating speed 100-150rpm, incubation time 3-7 days; The composition of liquid seed culture medium is: glucose 10-20g/L, malt extract 10-20g/L, peptone 1-5g/L, magnesium sulfate 0.1-1.0g/L.
The condition of described shaking flask liquid culture is: 28 ℃ of temperature, pH value 4.0, rotating speed 120rpm, incubation time 5 days; The composition of liquid seed culture medium is: glucose 18.8g/L, malt extract 18.8g/L, peptone 3.3g/L, magnesium sulfate 0.5g/L.
Described wheat bran boils 30-60min with low baking temperature, crosses leaching filtrate.
Described corn starch first carries out gelatinization and processes with front, and the gelatinization condition is that corn starch solution is kept 5-60min 100 ℃ of temperature.
In described slant strains activation step, described plating medium is the EMA plating medium, cultivation temperature 25-30 ℃, and incubation time 10-20 days.
In described slant strains activation step, described plating medium is the EMA plating medium, 28 ℃ of cultivation temperature, incubation time 15 days.
The EMA plating medium: malt extract 20g, glucose 20g, peptone 1g, agar powder 20g, PH5.0,
Antrodia camphorata mycelium fermented method of the present invention has improved the output of triterpene compound in camphor tree sesame liquid fermentation production greatly, in the Antrodia camphorata mycelium that triterpene compound content and linden cultivating method are produced, triterpene compound content is suitable, but thousand times of costs, even compare with existing liquid fermentation process, triterpene compound output also significantly improves, and cost is fairly obvious.Simultaneously, triterpene condition fermentation time shortens greatly, and the shortest is 2 days, has further reduced production cost, for solid foundation has been established in the industrialization of camphor tree sesame.
Embodiment
The present invention adopts camphor tree sesame bacterium bacterial classification, ATCC200183 and CCRC35396
In each embodiment of the present invention, the triterpene compound assay adopts following method:
The spectrophotometry of triterpenes content
One, principle
Oleanolic acid is pentacyclic triterpenoid, it can with multiple developer generation chromogenic reaction, thereby can carry out colorimetric estimation.Oleanolic acid and Xiang Cao Quan ?perchloric acid reagent, Xiang Cao Quan ?sulphate reagent generation chromogenic reaction present purple.There are certain linear relation in the concentration of oleanolic acid and absorbance, meet langbobier law, therefore can carry out colorimetric estimation
Two, instrument and reagent
1. instrument: 752 ?the type ultraviolet specrophotometer, electric-heated thermostatic water bath, analytical balance, Constant Temp. Oven, tool plug test tube
2. reagent: vanillin, glacial acetic acid, absolute ethyl alcohol, ethyl acetate and perchloric acid (more than be analyze pure), oleanolic acid standard items
Three, operating procedure
1. the preparation of oleanolic acid standard items: precision takes 10mg oleanolic acid standard items, and take absolute ethyl alcohol as solvent, being mixed with 100mL concentration is the oleanolic acid ethanolic solution of 0.1mg/mL.
2. the preparation of vanillin glacial acetic acid solution: accurately take fast vanillin 0.552g, also pour immediately the volumetric flask of 10mL into appropriate glacial acetic acid dissolving rapidly, be diluted to scale with glacial acetic acid rapidly, in order to the use on the same day.
3. the drafting of calibration curve: accurately draw oleanolic acid titer 0.1,0.2,0.3,0.3,0.4,0.5mL be placed in respectively tool plug test tube, the heated volatile desolventizing, then add the new preparation of 0.4mL 5% Xiang Cao Quan ?glacial acetic acid solution and 1.5mL perchloric acid, heat 15min in 70 ℃ of waters bath with thermostatic control, flowing water is cooled to room temperature, then adds ethyl acetate 5mL dilution to shake up, and the place measures absorbance at the 560nm wavelength.
4. sample extraction: get the sample powder of 1g, add 90 ℃ of water bath with thermostatic control backflow 1.5h of 30mL absolute ethyl alcohol, reflux three times, merging filtrate and constant volume 100mL.
5. sample determination: accurate sample thief 0.1mL, the heated volatile desolventizing, add again the new preparation of 0.4mL 5% Xiang Cao Quan ?glacial acetic acid solution and 1.5mL perchloric acid, heat 15min in 70 ℃ of waters bath with thermostatic control, flowing water is cooled to room temperature, add ethyl acetate 5mL dilution to shake up, the place measures absorbance at the 560nm wavelength again.
6. its empty is take maximum 0.5mL absolute ethyl alcohol as reference liquid, and the calibration curve of drawing according to step 3 comes the content of oleanolic acid in calculation sample, and is scaled percentage composition.
The Antrodia camphorata mycelium fermented method of embodiment 1 high yield triterpene compound
(1) slant strains activation
Camphor tree sesame (ATCC200183) slant strains is forwarded on the EMA plating medium, carry out the activation culture of slant strains, until camphor tree sesame colony diameter during greater than 2cm, again be forwarded on fresh EMA plating medium and cultivate, 25 ℃, when cultivating 18 days, mycelium covers with flat board and forms the crocus spore.
(2) shaking flask liquid seeds preparation
Aseptic collection camphor tree Ganoderma lucidum spore, the access shaking flask is carried out liquid culture, 25 ℃ of cultivation temperature, rotating speed 100rpm cultivated 6 days, and seed liquor occurs muddy and does not form obvious mycelium pellet.
Liquid seed culture medium forms: glucose 10g/L, malt extract 10g/L, peptone 1.0g/L, magnesium sulfate 0.5g/L, pH value 5.0, distilled water constant volume, 121 ℃, high pressure steam sterilization 20min.
(3) triterpene condition fermentation changes shake-flask seed over to 5L fermentation tank (the rear pot liquid volume of inoculation is 3.8L approximately) by 10% inoculum concentration, carries out the fermentation of triterpene condition.Medium consists of corn starch 60g/L, wheat bran 60g/L, magnesium sulfate 0.5g/L.Defoamer olive oil addition is 5ml/L.28 ℃ of cultivation temperature, initial pH value 5.0, ventilation ratio 1.0vvm, rotating speed 100rpm, continuous culture is 5 days with this understanding.Described wheat bran boils 30min with low baking temperature, filters, and gets filtrate.Described corn starch first carries out gelatinization and processes with front, and the gelatinization condition is that corn starch solution is kept 5min 100 ℃ of temperature.
(4) centrifugal collection mycelium obtains Antrodia camphorata mycelium 18.65g/L after 40 ℃ of dryings, and in mycelium, triterpene compound content is 783mg/L after measured, accounts for 4.2% of mycelium dry weight.
The Antrodia camphorata mycelium fermented method of embodiment 2 high yield triterpene compounds
(1) slant strains activation
Camphor tree sesame (ATCC200183) slant strains is forwarded on the EMA plating medium, carry out the activation culture of slant strains, until camphor tree sesame colony diameter during greater than 2cm, again be forwarded on fresh EMA plating medium and cultivate, 28 ℃, when cultivating 15 days, mycelium covers with flat board and forms the crocus spore.
(2) shaking flask liquid seeds preparation
Liquid seed culture medium forms: glucose 20.0g/L, malt extract 20.0g/L, peptone 5.0g/L, magnesium sulfate 0.5g/L, pH value 4.0, distilled water constant volume, 121 ℃, high pressure steam sterilization 20min.
Aseptic collection camphor tree Ganoderma lucidum spore, the access shaking flask is carried out liquid culture, 28 ℃ of cultivation temperature, rotating speed 120rpm cultivated 5 days, and seed liquor occurs muddy and does not form obvious mycelium pellet.
(3) triterpene condition fermentation: change shake-flask seed over to 5L fermentation tank (the rear pot liquid volume of inoculation is 3.8L approximately) by 10% inoculum concentration, carry out the fermentation of triterpene condition.Medium consists of corn starch 30g/L, wheat bran 30g/L, magnesium sulfate 1.85g/L.Defoamer olive oil addition is 2ml/L.28 ℃ of cultivation temperature, initial pH value 3.0, ventilation ratio 0.35vvm, rotating speed 80rpm, continuous culture is 3 days with this understanding.Described wheat bran boils 40min with low baking temperature, filters, and gets filtrate.Described corn starch first carries out gelatinization and processes with front, and the gelatinization condition is that corn starch solution is kept 30min 100 ℃ of temperature.
(4) centrifugal collection mycelium obtains Antrodia camphorata mycelium 12.27g/L after 40 ℃ of dryings, and in mycelium, triterpene compound content is 983mg/L after measured, accounts for 8.0% of mycelium dry weight.
The Antrodia camphorata mycelium fermented method of embodiment 3 high yield triterpene compounds
(1) slant strains activation
Camphor tree sesame (ATCC200183) slant strains is forwarded on the EMA plating medium, carry out the activation culture of slant strains, until camphor tree sesame colony diameter during greater than 2cm, again be forwarded on fresh EMA plating medium and cultivate, 28 ℃, when cultivating 15 days, mycelium covers with flat board and forms the crocus spore.
(2) shaking flask liquid seeds preparation
Liquid seed culture medium forms: glucose 18.8g/L, malt extract 18.8.g/L, peptone 3.3g/L, magnesium sulfate 0.5g/L, pH value 4.0, distilled water constant volume, 121 ℃, high pressure steam sterilization 20min.
Aseptic collection camphor tree Ganoderma lucidum spore, the access shaking flask is carried out liquid culture, 28 ℃ of cultivation temperature, rotating speed 120rpm cultivated 5 days, and seed liquor occurs muddy and does not form obvious mycelium pellet.
(3) triterpene condition fermentation: change shake-flask seed over to 5L fermentation tank (the rear pot liquid volume of inoculation is 3.8L approximately) by 10% inoculum concentration, carry out the fermentation of triterpene condition.Medium consists of corn starch 10g/L, wheat bran 10g/L, magnesium sulfate 1.85g/L.Defoamer olive oil addition is 5ml/L.26 ℃ of cultivation temperature, initial pH value 3.0, ventilation ratio 0.35vvm, rotating speed 90rpm, continuous culture is 3 days with this understanding.Described wheat bran boils 60min with low baking temperature, filters, and gets filtrate.Described corn starch first carries out gelatinization and processes with front, and the gelatinization condition is that corn starch solution is kept 60min 100 ℃ of temperature.
(4) centrifugal collection mycelium obtains Antrodia camphorata mycelium 7.07g/L after 40 ℃ of dryings, and in mycelium, triterpene compound content is 998mg/L after measured, accounts for 14.1% of mycelium dry weight.
The Antrodia camphorata mycelium fermented method of embodiment 4 high yield triterpene compounds
(1) slant strains activation
Camphor tree sesame (ATCC200183) slant strains is forwarded on the EMA plating medium, carry out the activation culture of slant strains, until camphor tree sesame colony diameter during greater than 2cm, again be forwarded on fresh EMA plating medium and cultivate, 28 ℃, when cultivating 15 days, mycelium covers with flat board and forms the crocus spore.
(2) shaking flask liquid seeds preparation
Liquid seed culture medium forms: glucose 18.8g/L, malt extract 18.8.g/L, peptone 3.3g/L, magnesium sulfate 0.5g/L, pH value 4.0, distilled water constant volume, 121 ℃, high pressure steam sterilization 20min.
Aseptic collection camphor tree Ganoderma lucidum spore, the access shaking flask is carried out liquid culture, 28 ℃ of cultivation temperature, rotating speed 120rpm cultivated 5 days, and seed liquor occurs muddy and does not form obvious mycelium pellet.
(3) liquid seeds spreads cultivation
Changing shake-flask seed over to the 50L seeding tank carries out bacterial classification and spreads cultivation, (switching after fermentation tank liquid volume is 38L approximately) seed tank culture base forms: glucose 18.8g/L, malt extract 18.8.g/L, peptone 3.3g/L, magnesium sulfate 0.5g/L, pH value 4.0, distilled water constant volume, 121 ℃, high pressure steam sterilization 20min.
The seed tank culture condition is: 28 ℃ of temperature, and ventilation ratio 1.0vvm, rotating speed are 120rpm, when cultivating 3 days, seeding tank occurs muddy.
(4) triterpene condition fermentation
The seeding tank seed is transferred by 10% inoculum concentration carry out the fermentation of triterpene condition into the 500L fermentation tank, the final liquid volume of fermentation tank is 380L.Medium consists of corn starch 22.2g/L, wheat bran 22.2g/L, and magnesium sulfate 1.85g/L, defoamer olive oil addition is 10g/L.26 ℃ of cultivation temperature, initial pH value 3.0, ventilation ratio 0.35vvm, rotating speed 90rpm, cultivate with this understanding stopped in 65 hours the fermentation.Described wheat bran boils 60min with low baking temperature, filters, and gets filtrate.Described corn starch first carries out gelatinization and processes with front, and the gelatinization condition is that corn starch solution is kept 30min 100 ℃ of temperature.
(5) centrifugal collection mycelium gets Antrodia camphorata mycelium 12.05g/L after 40 ℃ of low temperature dryings, in mycelium, triterpene compound content is 1269mg/L after measured, accounts for 10.5% of mycelium dry weight.
The Antrodia camphorata mycelium fermented method of embodiment 5 high yield triterpene compounds
(1) slant strains activation
Camphor tree sesame (ATCC200183) slant strains is forwarded on the EMA plating medium, carry out the activation culture of slant strains, until camphor tree sesame colony diameter during greater than 2cm, again be forwarded on fresh EMA plating medium and cultivate, 28 ℃, when cultivating 15 days, mycelium covers with flat board and forms the crocus spore.
(2) shaking flask liquid seeds preparation
Liquid seed culture medium forms: glucose 18.8g/L, malt extract 18.8.g/L, peptone 3.3g/L, magnesium sulfate 0.5g/L, pH value 4.0, distilled water constant volume, 121 ℃, high pressure steam sterilization 20min.
Aseptic collection camphor tree Ganoderma lucidum spore, the access shaking flask is carried out liquid culture, 28 ℃ of cultivation temperature, rotating speed 120rpm cultivated 5 days, and seed liquor occurs muddy and does not form obvious mycelium pellet.
(3) liquid seeds spreads cultivation
Changing shake-flask seed over to the 50L seeding tank carries out bacterial classification and spreads cultivation, (switching after fermentation tank liquid volume is 38L approximately) seed tank culture base forms: glucose 18.8g/L, malt extract 18.8.g/L, peptone 3.3g/L, magnesium sulfate 0.5g/L, pH value 4.0, distilled water constant volume, 121 ℃, high pressure steam sterilization 20min.
The seed tank culture condition is: 28 ℃ of temperature, and ventilation ratio 1.0vvm, rotating speed 120rmp, when cultivating 3 days, seeding tank occurs muddy.
Transferring by 10% inoculum concentration, ((switching after fermentation tank liquid volume is 380L approximately) carries out the seed secondary and spreads cultivation, and medium composition and condition of culture are with the 50L seeding tank into the 500L seeding tank.
(4) triterpene condition fermentation
360L seed 10% inoculum concentration is transferred carry out the fermentation of triterpene condition into the 5000L fermentation tank, the final liquid volume of fermentation tank is 3750L.Medium consists of corn starch 22.2g/L, wheat bran 22.2g/L, and magnesium sulfate 1.85g/L, defoamer olive oil addition is 3.3ml/L.26 ℃ of cultivation temperature, initial pH value 3.0, ventilation ratio 0.35vvm, rotating speed 90rpm, cultivate with this understanding stopped in 48 hours the fermentation.Described wheat bran boils 45min with low baking temperature, filters, and gets filtrate.Described corn starch first carries out gelatinization and processes with front, and the gelatinization condition is that corn starch solution is kept 35min 100 ℃ of temperature.
(5) centrifugal collection mycelium gets Antrodia camphorata mycelium 11.68g/L after 40 ℃ of low temperature dryings, in mycelium, triterpene compound content is 1472mg/L after measured, accounts for 12.6% of mycelium dry weight.
The Antrodia camphorata mycelium fermented method of embodiment 6 high yield triterpene compounds
(1) slant strains activation
Camphor tree sesame (CCRC35396) slant strains is forwarded on the EMA plating medium, carry out the activation culture of slant strains, until camphor tree sesame colony diameter during greater than 2cm, again be forwarded on fresh EMA plating medium and cultivate, 28 ℃, when cultivating 15 days, mycelium covers with flat board and forms the crocus spore.
(2) shaking flask liquid seeds preparation
Liquid seed culture medium forms: glucose 18.8g/L, malt extract 18.8.g/L, peptone 3.3g/L, magnesium sulfate 0.5g/L, pH value 4.0, distilled water constant volume, 121 ℃, high pressure steam sterilization 20min.
Aseptic collection camphor tree Ganoderma lucidum spore, the access shaking flask is carried out liquid culture, 28 ℃ of cultivation temperature, rotating speed 120rpm cultivated 5 days, and seed liquor occurs muddy and does not form obvious mycelium pellet.
(3) liquid seeds spreads cultivation
Changing shake-flask seed over to the 50L seeding tank carries out bacterial classification and spreads cultivation, (switching after fermentation tank liquid volume is 38L approximately) seed tank culture base forms: glucose 18.8g/L, malt extract 18.8.g/L, peptone 3.3g/L, magnesium sulfate 0.5g/L, pH value 4.0, distilled water constant volume, 121 ℃, high pressure steam sterilization 20min.
The seed tank culture condition is: 28 ℃ of temperature, and ventilation ratio 1.0vvm, rotating speed 120rmp, when cultivating 3 days, seeding tank occurs muddy.
Transferring by 10% inoculum concentration, ((switching after fermentation tank liquid volume is 380L approximately) carries out the seed secondary and spreads cultivation, and medium composition and condition of culture are with the 50L seeding tank into the 500L seeding tank.
(4) triterpene condition fermentation
380L seed 10% inoculum concentration is transferred carry out the fermentation of triterpene condition into the 5000L fermentation tank, the final liquid volume of fermentation tank is 3700L.Medium consists of corn starch 22.2g/L, wheat bran 22.2g/L, and magnesium sulfate 1.85g/L, defoamer olive oil addition is 3.3ml/L.26 ℃ of cultivation temperature, initial pH value 3.0, ventilation ratio 0.35vvm, rotating speed 90rpm, cultivate with this understanding stopped in 48 hours the fermentation.Described wheat bran boils 45min with low baking temperature, filters, and gets filtrate.Described corn starch first carries out gelatinization and processes with front, and the gelatinization condition is that corn starch solution is kept 35min 100 ℃ of temperature.
(5) centrifugal collection mycelium gets Antrodia camphorata mycelium 12.02g/L after 40 ℃ of low temperature dryings, in mycelium, triterpene compound content is 1442mg/L after measured, accounts for 12.0% of mycelium dry weight.
Embodiment 1-5 bacterial strain uses therefor is ATCC200183, and embodiment 6 bacterial strain uses therefors are CCRC35396
Embodiment 4-5 compares with embodiment 1-3, and the main difference point is: embodiment 4-5 has increased the liquid seeds step that spreads cultivation, and liquid seeds spreads cultivation has increased the vigor of seed, and in fermentation mycelium, triterpenes content has facilitation to improving.
Embodiment 4-5 compares with embodiment 1-2, and the main difference point is: in embodiment 4-5 triterpene condition fermentation medium, wheat bran and Maize Starch Content are lower than embodiment 1-2; Embodiment 4-5 compares with embodiment 3, and in triterpene condition fermentation medium, wheat bran and Maize Starch Content are higher than embodiment 3; As seen the content of nutrient component in the controlled fermentation medium, can reach and both effectively control mycelial nourishing and growing, this gives birth to the purpose that metabolite produces effectively to promote again mycelium, the present invention is through repeatedly, repeatedly explore, found the Antrodia camphorata mycelium fermented medium of high yield triterpene compound and condition of culture on the basis of great many of experiments.
The present invention adds olive oil first in Antrodia camphorata mycelium fermented medium, olive oil both can be used as defoamer, can be used as again triterpene compound and generate the promotion factor, verify that through the present invention every liter of fermentation medium adds the content that the 2-10ml olive oil can effectively improve triterpene compound in mycelium.
The method of the invention especially is fit to large-scale industrialization and produces Antrodia camphorata mycelium and secondary metabolites-triterpene compound thereof, has output high, and is consuming time few, the remarkable advantage that cost is low.
Claims (10)
1. the Antrodia camphorata mycelium fermented method of a high yield triterpene compound, comprise the following steps,
Slant strains activation is forwarded to camphor tree sesame slant strains on plating medium,, again is forwarded on fresh plating medium and cultivates during greater than 2cm until camphor tree sesame colony diameter, makes mycelium cover with flat board and forms the Chinese red spore;
The preparation of shaking flask liquid seeds, aseptic collection camphor tree Ganoderma lucidum spore, the access shaking flask is carried out liquid culture, does not stop cultivating when forming mycelium pellet until liquid nutrient medium appearance muddiness, obtains shake-flask seed;
Liquid seeds spreads cultivation, and changes shake-flask seed over to seeding tank and carries out bacterial classification and spread cultivation, and occurs muddy and when not forming mycelium pellet, changes the next stage seeding tank over to until zymotic fluid, enlarges step by step preparation fermenting and producing liquid seeds;
The fermentation of triterpene condition, change the aforesaid liquid seed over to the next stage fermentation tank by 10% inoculum concentration, carry out the fermentation of triterpene condition, fermentation condition is: 25-30 ℃, and pH value 3.0-5.0, ventilation ratio 0.3-2.0vvm, incubation time 2-3 days, the composition of triterpene condition fermentation medium was: corn starch 10-60g/L, wheat bran 10-60g/L, magnesium sulfate 0.5-2.0g/L, olive oil 2-10ml/L.
2. the Antrodia camphorata mycelium fermented method of a kind of high yield triterpene compound according to claim 1, it is characterized in that, described triterpene fermentation condition is: 28 ℃, pH value 3.0, ventilation ratio 0.35vvm, incubation time 2 days, the composition of triterpene condition fermentation medium is: corn starch 20-25g/L, wheat bran 20-25g/L, magnesium sulfate 1.5-2.0g/L, olive oil 2-5ml/L.
3. the Antrodia camphorata mycelium fermented method of a kind of high yield triterpene compound according to claim 1 and 2, it is characterized in that, described liquid seeds spreads cultivation in step, the seeding tank inoculum concentration is 10%, the seed tank culture condition is: temperature 25-30 ℃, pH value 3.0-5.0, ventilation ratio 0.5-2.0vvm, incubation time 3-5 days; The seed tank culture base forms: glucose 10-20g/L, malt extract 10-20g/L, peptone 1-5g/L, magnesium sulfate 0.1-1.0g/L.
4. the Antrodia camphorata mycelium fermented method of a kind of high yield triterpene compound according to claim 3, is characterized in that, described seed tank culture condition is: 28 ℃ of temperature, pH value 4.0, ventilation ratio 1.0vvm, incubation time 3 days; The seed tank culture base forms: glucose 18.8g/L, malt extract 18.8g/L, peptone 3.3g/L, magnesium sulfate 0.5g/L.
5. the Antrodia camphorata mycelium fermented method of a kind of high yield triterpene compound according to claim 1 and 2, is characterized in that, in described shaking flask liquid seeds preparation process, the condition of shaking flask liquid culture is: temperature 25-30 ℃, pH value 5.0-7.0, rotating speed 100-150rpm, incubation time 3-7 days; The composition of liquid seed culture medium is: glucose 10-20g/L, malt extract 10-20g/L, peptone 1-5g/L, magnesium sulfate 0.1-1.0g/L.
6. the Antrodia camphorata mycelium fermented method of a kind of high yield triterpene compound according to claim 5, is characterized in that, the condition of described shaking flask liquid culture is: 28 ℃ of temperature, pH value 4.0, rotating speed 120rpm, incubation time 5 days; The composition of liquid seed culture medium is: glucose 18.8g/L, malt extract 18.8g/L, peptone 3.3g/L, magnesium sulfate 0.5g/L.
7. according to claim 1, the Antrodia camphorata mycelium fermented method of the described a kind of high yield triterpene compound of any one, is characterized in that in 2 or 4, and described wheat bran boils 30-60min with low baking temperature, crosses leaching filtrate.
8. according to claim 1, the Antrodia camphorata mycelium fermented method of the described a kind of high yield triterpene compound of any one in 2 or 4, it is characterized in that, described corn starch first carries out gelatinization and processes with front, and the gelatinization condition is that corn starch solution is kept 5-60min 100 ℃ of temperature.
9. the Antrodia camphorata mycelium fermented method of a kind of high yield triterpene compound according to claim 1, is characterized in that, in described slant strains activation step, described plating medium is the EMA plating medium, cultivation temperature 25-30 ℃, and incubation time 10-20 days.
10. the Antrodia camphorata mycelium fermented method of a kind of high yield triterpene compound according to claim 9, is characterized in that, in described slant strains activation step, described plating medium is the EMA plating medium, 28 ℃ of cultivation temperature, incubation time 15 days.
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