CN102356728A - Solid culturing method for increasing active product yield of antrodia camphorata - Google Patents
Solid culturing method for increasing active product yield of antrodia camphorata Download PDFInfo
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- CN102356728A CN102356728A CN2011102318689A CN201110231868A CN102356728A CN 102356728 A CN102356728 A CN 102356728A CN 2011102318689 A CN2011102318689 A CN 2011102318689A CN 201110231868 A CN201110231868 A CN 201110231868A CN 102356728 A CN102356728 A CN 102356728A
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- camphor tree
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- tree sesame
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- antrodia camphorata
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- 238000012258 culturing Methods 0.000 title abstract 7
- 241000723346 Cinnamomum camphora Species 0.000 claims description 59
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- Medicines Containing Plant Substances (AREA)
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Abstract
The invention discloses a solid culturing method for increasing the active product yield of antrodia camphorata by adopting a two-stage temperature control technology. The method comprises the following steps of: preparing an antrodia camphorata slant strain and a liquid seed, and inoculating the liquid seed onto a solid culture medium for culturing; controlling the culturing temperature at 26-37 DEG C at the first culturing stage (0-20 days); and controlling the culturing temperature at 18-26 DEG C at the second culturing stage (20-40 days). During solid culture of active products of antrodia camphorata with a two-stage temperature control process, the yield of Antrodin C is up to 5.0-7.0 mg/g, the yield of AntrodinB is up to 1.0-2.0 mg/g, and the yield of Antroquinonol is up to 1.2-2.5 mg/g. The two-stage temperature control technology for the active products of antrodia camphorata provided by the invention is simple and efficient and has an important industrial application value; and meanwhile, the production of other active metabolic products of antrodia camphorata have certain enlightening meanings.
Description
Technical field
The present invention relates to a kind of cultural method that improves camphor tree sesame activated product output, especially a kind of method of passing through two phase temperature control solid state fermentation raising camphor tree sesame metabolite Antrodins and Antroquinonol.
Technical background
The camphor tree sesame (
Antrodia camphorata) be the distinctive rare medicinal fungi in China Taiwan, have another name called Cinnamomum kanahirai hay mushroom, Antrodia camphorata, belong to Basidiomycetes, Aphyllophorales, porous section, perennial basidiomycetes, the Cinnamomum kanahirai hay that is grown in the Taiwan is set the inwall of rotten trunk.Camphor tree sesame complicated component contains various active compositions such as triterpene, sterol, adenosine, polysaccharide, aldehydes matter.Among the people, the camphor tree sesame is used as the traditional medicine that protects the liver of rescue for a long time, is used to treat liver diseases, caused poisoning symptom of food and medicine and diarrhoea, and stomachache, hypertension, skin is itched and tumor disease etc.In recent years, many scholar's research show the camphor tree sesames have anticancer, protect the liver, physiological active functionses such as anti-inflammatory, anti-DNA damnification.On market, the wild fruit body price of camphor tree sesame is up to 15000 dollars/kilogram, and far above traditional medicinal materials such as glossy ganoderma, genseng, Cordyceps sinensis, the camphor tree sesame more and more receives popular concern.
Modern study shows, camphor tree sesame active component has very good effect the aspect such as protecting the liver, anticancer.Among the peoplely look the camphor tree sesame for protecting the liver good medicine, legend Taiwan aboriginal is higher because of the hepatopathy ratio of being addicted to drink, take camphor tree sesame decocting liquid after the liver health situation then be greatly improved.The ethanolic extract of Antrodia camphorata mycelium all shows good effect to wild type and Lamivudine resistant mutant strain HBV virus.Still experiment in vitro in the body no matter, camphor tree sesame fruit body and mycelium all show good anti-cancer activity.Research such as Madamanchi shows the CHCl of camphor tree sesame fruit body
3/ MeOH extract has significant cytotoxin effect to P-388 mouse cell, and EC50 is 4.1 μ g/mL.The acetic acid ethyl ester extract of camphor tree sesame fruit body demonstrates good withered effect to cancerous cell line Hep G2, Hep 3B, PLC/PRF/5.
Experiment shows that the characteristic active component that camphor tree sesame solid state fermentation thalline is contained is Antrodins and Antroquinonol.People such as Nakamura separate from camphor tree sesame liquid state fermentation thalline and obtain five kinds of new maleic acids and succinic acid derivative (Antrodin A-E); It is the main active in the liquid state fermentation camphor tree sesame thalline; Have significant antihepatitic activity, wherein Antrodin B and Antrodin C have good inhibition effect to the LLC cancer cell.The Phuong result of study shows that the Antrodin A-E that contains in the Antrodia camphorata mycelium has the good restraining activity to HCV virus, and wherein Antrodin A inhibition ability is the highest; Research finds that simultaneously Antrodin C can be converted into Antrodin A in vivo, thereby represents good liver-protecting activity.Antrodins compounds in the camphor tree sesame thalline is after nineteen sixty-eight is found to fly the water silibin, the natural products that liver-protecting activity is best.Human n-hexane extraction camphor tree sesame solid state fermentation powder such as Lee; Obtain a noval chemical compound (Antroquinonol); Belong to the ubiquinone compounds; Experiment shows that multiple cancer cells such as breast cancer, liver cancer and prostate cancer are had good anticancer effect, has simultaneously to suppress HBsAg and the synthetic potentiality of HBeAg, thereby suppresses duplicating of HBV.
Publication number is a CN137908 (denomination of invention: the solid culture method of camphor tree sesame; Gained solid culture and products thereof and purposes), the extensive solid culture method of a kind of camphor tree sesame) etc. (denomination of invention: patent documentation discloses the solid-state cultured method of a series of camphor tree sesames to CN101803534A; But the used technology of above-mentioned document is space bag plate method; The product that obtains is a camphor tree sesame fruit body; Cultivation cycle is longer, does not have the report about camphor tree sesame solid state fermentation characteristic medicinal ingredient Antrodins and Antroquinonol production method.。
At CN101151957, people such as Huang disclose a kind of method of cultivating the camphor tree sesame with solid medium, at first on the PDA slant medium, cultivate camphor tree sesame thalline in advance, take out camphor tree sesame that a part turns out and it is seeded in the liquid medium.Afterwards, the camphor tree sesame in the liquid medium is inoculated in the solid medium cultivates.Solid medium mainly is made up of rice, agar, a kind of carbon source, a kind of nitrogenous source, mineral salt etc.At CN101151957, the method can obtain camphor tree sesame thalline mixture on a large scale, and has and the similar physiologically active of wild camphor tree sesame fruit body.But the disclosed product of this method is a camphor tree sesame solid state fermentation thalline mixture, does not have the report about camphor tree sesame solid state fermentation characteristic medicinal ingredient Antrodins and Antroquinonol production method.
Because being rich in the camphor tree sesame product demand of physiological activator grows with each passing day; Be badly in need of the method that a kind of special mass artificial is cultivated the camphor tree sesame; This method can improve the content of camphor tree Zhi Tezheng activated product Antrodins and Antroquinonol compounds targetedly; To improve camphor tree sesame product quality, satisfy the continual renovation development in market.And show according to our novelty assessment report, at present not about the cultural method of camphor tree Zhi Tezheng activated product Antrodins and Antroquinonol and the report of process optimization.
The solid state fermentation of camphor tree sesame bacterium, temperature has important effect to the biosynthesis of its bioactivator.Its dominant mechanism is that the best use of temperature of biosynthetic enzyme is different in the different production stage.The time of megathermal period and middle temperature phase also is to have certain rules governed.Therefore in the regular hour, adopt different fermentation temperatures, can promote the biosynthesis of bioactivator.So best fermentation time and fermentation temperature need be confirmed through a large amount of experiments.The preferable operation scheme that the key technology of this patent draws with regard to being based on long-term test and big quantitative analysis.
Summary of the invention
Technical problem to be solved by this invention provides a kind of process for solid culture that improves camphor tree sesame activated product output, to significantly improve the output of camphor tree Zhi Tezheng activated product Antrodins and Antroquinonol compounds.
For solving the problems of the technologies described above, technical scheme of the present invention is:
At first prepare camphor tree sesame slant strains and liquid seed, liquid seed is inoculated on the solid medium cultivates afterwards.In the phase I (0-20d) of cultivating, the control cultivation temperature is between 26-32 ℃; In the second stage (20-40d) of cultivating, the control cultivation temperature is between 18-26 ℃.
Phase I mainly is the vegetative stage of Antrodia camphorata mycelium; Second stage is the biosynthesis stage of camphor tree sesame physiological activator.
The used camphor tree sesame bacterial strain of the present invention (
Antrodia camphorata) derive from Fumao Edible Mushroom Co., Ltd., Shanghai of Taiwan-owned enterprise.Bacterial classification source can be with reference to Chinese patent: 201010164142.3, and open.
The inoculum concentration of said camphor tree sesame is between the 10%-50%, and it is that the gross weight in solid medium is 100%.
Solid medium comprises matrix, nitrogenous source and more than one mineral salt, and said matrix is corn, seed of Job's tears, Chinese sorghum, rice, glutinous rice, millet and combination thereof; Said nitrogenous source is NH
4Cl, peptone, analysis for soybean powder, corn starch, Fructus Hordei Germinatus extract, yeast extract and combination thereof, the interpolation percentage by weight of nitrogenous source is 0.3-6%; Said mineral salt example be phosphate, magnesium sulfate, citrate, sodium chloride and combination thereof wherein; Phosphate is K
2HPO
4, KH
2PO
4, NaH
2PO
4, Na
2HPO
4And combination; The interpolation percentage by weight of said mineral salt is 0.02-2 %; 121 ℃ of sterilization 10-40min after solid medium preparation is accomplished, be cooled to then 15-25 ℃ for use.
Antrodin B, Antrodin C and Antroquinonol adopt high performance liquid chromatography (HPLC) to analyze.Get the camphor tree sesame solid state fermentation powder 0.5g of pulverizing, add 60ml ethanol, 50 ℃, oscillation extraction 1.5h gets supernatant through 0.22 μ m filter membrane micro-filtration after leaving standstill, and carries out HPLC and analyzes.Analysis condition is following: chromatographic column: Sepax Amethyst C18 (4.6 mm * 250 mm); Flow velocity: 1 mL/min; Detect wavelength: 254 nm; Mobile phase A: water/acetate=100/0.5, Mobile phase B: acetonitrile; Gradient is following: 0-10min, Mobile phase B 35%-50%; 10-35min, Mobile phase B 50%-60%; 35-50min, Mobile phase B 60%-80%; 50-60min, Mobile phase B 80%-100%.
Utilize the present invention can significantly improve the output of camphor tree sesame activated product Antrodins and Antroquinonol compounds.With the solid-state cultivation of this two phase temperature control technology camphor tree sesame activated product, wherein Antrodin C output reaches 5.0-7.0mg/g, and Antrodin B output reaches 1.0-2.0mg/g, and Antroquinonol output reaches 1.2-2.5mg/g.Camphor tree sesame activated product two phase temperature control technology technologies provided by the present invention are simple, efficient, and important industrial application value is arranged, and the production of other active metabolites of camphor tree sesame simultaneously has certain inspiration meaning.
Embodiment
Embodiment 1
Camphor tree sesame bacterial classification changes the inclined-plane earlier and in 28 ℃ of incubators, cultivates 12d, inserts then in the liquid medium and cultivates.Liquid medium (g/L): glucose 20, Na
2HPO
40.5, MgSO
40.5, citric acid 0.5, analysis for soybean powder 4, corn starch 2.
The liquid culture condition is: 28 ℃ of cultivation temperature, and shaking speed 100 r/min, pH 4.0, incubation time 4d.Solid medium: adopt food-grade cereal (corn, millet, Chinese sorghum etc.) fermentation.NH
4Cl 0.3%, Na
2HPO
40.02%, be 100% in the gross weight of solid medium.Nitrogenous source and salt are mixed with nutrient solution, and the ratio of cereal and nutrient solution is 4:1,3,121 ℃ of sterilizations of pH 20min.
Two phase temperature control technologys: in the phase I of solid-state cultivation (0-20d) control cultivation temperature is 26-32 ℃, to obtain the faster growing of thalline.Second stage (20-40d) control cultivation temperature in solid-state cultivation is 18 ℃.With the solid-state cultivation of this two phase temperature control strategy camphor tree sesame production characteristic activated product, Antrodin C output reaches 5.0mg/g, and Antrodin B output reaches 1.0mg/g, and Antroquinonol output reaches 1.2mg/g.
Embodiment 2
Camphor tree sesame bacterial classification changes the inclined-plane earlier and in 28 ℃ of incubators, cultivates 12d, inserts then in the liquid medium and cultivates.Liquid medium (g/L): glucose 20, Na
2HPO
40.5, MgSO
40.5, citric acid 0.5, analysis for soybean powder 4, corn starch 2.
The liquid culture condition is: 28 ℃ of cultivation temperature, and shaking speed 100 r/min, pH 4.0, incubation time 4d.Solid medium: adopt food-grade cereal (corn, millet, Chinese sorghum etc.) fermentation.Na
2HPO
41%, MgSO
40.02%, corn starch 3% is 100% in the gross weight of solid medium.Nitrogenous source and salt are mixed with nutrient solution, and the ratio of cereal and nutrient solution is 2:1,5.0,121 ℃ of sterilizations of pH 20min.
Adopt two phase temperature control solid medium cultivation, all the other conditions are with embodiment 1.In the phase I of solid-state cultivation (0-20d) control cultivation temperature is 26-32 ℃, to obtain the faster growing of thalline.Second stage (20-40d) control cultivation temperature in solid-state cultivation is 22 ℃.With the solid-state cultivation of this two phase temperature control strategy camphor tree sesame production characteristic activated product, Antrodin C output reaches 7.0mg/g, and Antrodin B output reaches 2.0mg/g, and Antroquinonol output reaches 2.5mg/g.
Embodiment 3
Camphor tree sesame bacterial classification changes the inclined-plane earlier and in 28 ℃ of incubators, cultivates 12d, inserts then in the liquid medium and cultivates.Liquid medium (g/L): glucose 20, Na
2HPO
40.5, MgSO
40.5, citric acid 0.5, analysis for soybean powder 4, corn starch 2.
The liquid culture condition is: 28 ℃ of cultivation temperature, and shaking speed 100 r/min, pH 4.0, incubation time 4d.Solid medium: adopt food-grade cereal (corn, millet, Chinese sorghum etc.) fermentation.KH
2PO
42%, analysis for soybean powder 6% is 100% in the gross weight of solid medium.Nitrogenous source and salt are mixed with nutrient solution, and the ratio of cereal and nutrient solution is 1:1, and pH 7.0.121 ℃ of sterilization 20min.
Adopt two phase temperature control solid medium cultivation, all the other conditions are with embodiment 1.In the phase I of solid-state cultivation (0-20d) control cultivation temperature is 26-32 ℃, to obtain the faster growing of thalline.Second stage (20-40d) control cultivation temperature in solid-state cultivation is 26 ℃.With the solid-state cultivation of this two phase temperature control strategy camphor tree sesame production characteristic activated product, Antrodin C output reaches 6.5mg/g, and Antrodin B output reaches 1.7mg/g, and Antroquinonol output reaches 2.0mg/g.
Though the present invention with preferred embodiment openly as above; But it is not in order to limiting the present invention, anyly is familiar with this technological people, is not breaking away from the spirit and scope of the present invention; All can do various changes and modification, so protection scope of the present invention should be as the criterion with what claims were defined.
Claims (8)
1. a process for solid culture that improves camphor tree sesame activated product output is characterized in that camphor tree sesame seed liquor inoculation back employing stage cultivation, and the phase I cultivation temperature is 26-32 ℃, and the second stage cultivation temperature is 18-26 ℃.
2. method according to claim 1, the incubation time that it is characterized in that the phase I is 0-20 days.
3. method according to claim 1, the incubation time that it is characterized in that second stage is 20-40 days.
4. method according to claim 1 is characterized in that the medium of solid-state cultivation comprises matrix, nitrogenous source and more than one mineral salt, pH3.0-7.0.
5. method according to claim 4 is characterized in that said matrix is corn, seed of Job's tears, Chinese sorghum, rice, glutinous rice, millet and combination thereof.
6. method according to claim 4 is characterized in that said nitrogenous source is NH
4Cl, peptone, analysis for soybean powder, corn starch, Fructus Hordei Germinatus extract, yeast extract and combination thereof, the interpolation percentage by weight of nitrogenous source is 0.3-6%.
7. method according to claim 4, it is characterized in that said mineral salt example for phosphate, magnesium sulfate, citrate, sodium chloride and combination thereof wherein; Phosphate is K
2HPO
4, KH
2PO
4, NaH
2PO
4, Na
2HPO
4And combination; The interpolation percentage by weight of said mineral salt is 0.02-2 %; 121 ℃ of sterilization 10-40min after solid medium preparation is accomplished, be cooled to then 15-25 ℃ for use.
8. method according to claim 1, the inoculum concentration that it is characterized in that said camphor tree sesame is between the 10%-50%, it is that the gross weight in solid medium is 100%.
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