CN102356728B - 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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- CN102356728B CN102356728B CN 201110231868 CN201110231868A CN102356728B CN 102356728 B CN102356728 B CN 102356728B CN 201110231868 CN201110231868 CN 201110231868 CN 201110231868 A CN201110231868 A CN 201110231868A CN 102356728 B CN102356728 B CN 102356728B
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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 two phase temperature of passing through are controlled the method that solid state fermentation improves 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, be grown in the inwall of the rotten trunk of Cinnamomum kanahirai hay tree of Taiwan.Camphor tree sesame complicated component contains the various active compositions such as triterpene, sterol, adenosine, polysaccharide, aldehydes matter.Among the people, the camphor tree sesame is used as saving the traditional medicine that protects the liver for a long time, is used for the treatment of liver diseases, the 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, the 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 medicines such as glossy ganoderma, ginseng, Cordyceps sinensis, the camphor tree sesame more and more is subjected to 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 be greatly improved.The ethanolic extract of Antrodia camphorata mycelium all shows good effect to wild type and Lamivudine resistant mutant strain HBV virus.No matter in body or experiment in vitro, camphor tree sesame fruit body and mycelium all show good active anticancer.Madamanchi etc. studies show that 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, the contained feature active component of camphor tree sesame solid state fermentation thalline is Antrodins and Antroquinonol.The 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 liquid state fermentation camphor tree sesame thalline, have significant antihepatitic activity, wherein Antrodin B and Antrodin C have good inhibition to the LLC cancer cell.The Phuong result of study shows that the Antrodin A-E that contains in Antrodia camphorata mycelium has good inhibition 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 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.The human n-hexane extraction camphor tree sesame solid state fermentation powder such as Lee, obtain a noval chemical compound (Antroquinonol), belong to Studies on Ubiquinone Derivatives, experiment shows that the multiple cancer cells such as breast cancer, liver cancer and prostate cancer are had good anticancer effect, have simultaneously and suppress HBsAg and the synthetic potentiality of HBeAg, thereby suppress copying of HBV.
Publication number is the CN137908(denomination of invention: the solid culture method of camphor tree sesame, gained solid culture and products thereof and purposes), the CN101803534A(denomination of invention: the extensive solid culture method of a kind of camphor tree sesame) etc. patent documentation discloses the method for the solid-state cultivation of a series of camphor tree sesame, but above-mentioned document technology used is space bag plate method, the product that obtains is camphor tree sesame fruit body, cultivation cycle is longer, does not have the report about camphor tree sesame solid state fermentation feature medicinal ingredient Antrodins and Antroquinonol production method.。
At CN101151957, the yellow people of grade discloses a kind of method of cultivating the camphor tree sesame with solid medium, and at first preculture camphor tree sesame thalline on the PDA slant medium, take out a part of camphor tree sesame of turning out and it is seeded in a liquid culture medium.Afterwards, the camphor tree sesame in liquid culture medium is inoculated in solid medium cultivates.Solid medium mainly is comprised 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 the method is camphor tree sesame solid state fermentation thalline mixture, does not have the report about camphor tree sesame solid state fermentation feature medicinal ingredient Antrodins and Antroquinonol production method.
Because the demand of the camphor tree sesame product that is rich in physiological activator grows with each passing day, be badly in need of a kind of special extensive artificial method of cultivating the camphor tree sesame, the method can improve the content of camphor tree Zhi Tezheng activated product Antrodins and Antroquinonol compounds targetedly, to improve camphor tree sesame product quality, the continuous renewal development of satisfying the 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, the biosynthesis of temperature to its bioactivator has very important effect.Its dominant mechanism is that the best use of temperature of biosynthetic enzyme is different in the different production phases.The time of megathermal period and middle temperature phase is also to have certain rule governed.Therefore adopt different fermentation temperatures within the regular hour, can promote the biosynthesis of bioactivator.Therefore best fermentation time and fermentation temperature need to be determined by a large amount of experiments.The better operation scheme that the key technology of this patent draws with regard to being based on long-term test and large quantitative analysis.
Summary of the invention
Technical problem to be solved by this invention is to provide 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 solid medium cultivates afterwards.In the phase I (0-20d) of cultivating, control cultivation temperature between 26-32 ℃; In the second stage (20-40d) of cultivating, control cultivation temperature between 18-26 ℃.
Phase I is mainly the vegetative stage of Antrodia camphorata mycelium; Second stage is the biosynthesis stage of camphor tree sesame physiological activator.
The present invention's camphor tree sesame bacterial strain used (
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 described camphor tree sesame is between 10%-50%, and it is that gross weight take solid medium is as 100%.
Solid medium comprises matrix, nitrogenous source and more than one mineral salt, and described matrix is corn, seed of Job's tears, Chinese sorghum, rice, glutinous rice, millet and combination thereof; Described 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%; Described 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 described mineral salt is 0.02-2 %; 121 ℃ of sterilization 10-40min after solid medium preparation is completed, then be cooled to 15-25 ℃ stand-by.
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 standing, carries out HPLC and analyzes.Analysis condition is as follows: chromatographic column: Sepax Amethyst C18 (4.6 mm * 250 mm); Flow velocity: 1 mL/min; Detect wavelength: 254 nm; Mobile phase A: water/acetic acid=100/0.5, Mobile phase B: acetonitrile; Gradient is as follows: 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.Control the solid-state cultivation of technique camphor tree sesame activated product with this two phase temperature, 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 techniques 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 first turns the inclined-plane and cultivate 12d in 28 ℃ of incubator, then accesses in liquid culture medium and cultivates.Liquid culture 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%, take the gross weight of solid medium as 100%.Nitrogenous source and salt are mixed with nutrient solution, and the ratio of cereal and nutrient solution is 4:1,3,121 ℃ of sterilization 20min of pH.
Two phase temperature control technologys: controlling cultivation temperature in the phase I of solid-state cultivation (0-20d) is 26-32 ℃, to obtain the faster growing of thalline.Controlling cultivation temperature in the second stage (20-40d) of solid-state cultivation is 18 ℃.With the solid-state cultivation of this two phase temperature control strategy camphor tree sesame production feature 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 first turns the inclined-plane and cultivate 12d in 28 ℃ of incubator, then accesses in liquid culture medium and cultivates.Liquid culture 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 take the gross weight of solid medium as 100%.Nitrogenous source and salt are mixed with nutrient solution, and the ratio of cereal and nutrient solution is 2:1,5.0,121 ℃ of sterilization 20min of pH.
Adopt two phase temperature to control solid medium cultivation, all the other conditions are with embodiment 1.Controlling cultivation temperature in the phase I of solid-state cultivation (0-20d) is 26-32 ℃, to obtain the faster growing of thalline.Controlling cultivation temperature in the second stage (20-40d) of solid-state cultivation is 22 ℃.With the solid-state cultivation of this two phase temperature control strategy camphor tree sesame production feature 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 first turns the inclined-plane and cultivate 12d in 28 ℃ of incubator, then accesses in liquid culture medium and cultivates.Liquid culture 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 take the gross weight of solid medium as 100%.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 to control solid medium cultivation, all the other conditions are with embodiment 1.Controlling cultivation temperature in the phase I of solid-state cultivation (0-20d) is 26-32 ℃, to obtain the faster growing of thalline.Controlling cultivation temperature in the second stage (20-40d) of solid-state cultivation is 26 ℃.With the solid-state cultivation of this two phase temperature control strategy camphor tree sesame production feature activated product, Antrodin C output reaches 6.5mg/g, and Antrodin B output reaches 1.7mg/g, and Antroquinonol output reaches 2.0mg/g.
Although the present invention with preferred embodiment openly as above; but it is not to limit the present invention, any person skilled in the art, without departing 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 with being as the criterion that claims were defined.
Claims (5)
1. process for solid culture that improves camphor tree sesame Antrodins and Antroquinonol compounds output, it is characterized in that the rear employing stage cultivation of camphor tree sesame seed liquor inoculation, the phase I cultivation temperature is 26-32 ℃, incubation time is 20 days, the second stage cultivation temperature is 22 ℃, and incubation time is 20-40 days.
2. 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; Described matrix is corn, seed of Job's tears, Chinese sorghum, rice, glutinous rice, millet and combination thereof.
3. method according to claim 2, is characterized in that described 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%.
4. method according to claim 2, is characterized in that described mineral salt are phosphate, magnesium sulfate, citrate, sodium chloride and combination thereof, and wherein, phosphate is K
2HPO
4, KH
2PO
4, NaH
2PO
4, Na
2HPO
4And combination; The interpolation percentage by weight of described mineral salt is 0.02-2%; 121 ℃ of sterilization 10-40min after solid medium preparation is completed, then be cooled to 15-25 ℃ stand-by.
5. method according to claim 1, the inoculum concentration that it is characterized in that described camphor tree sesame is between 10%-50%, it is that gross weight take solid medium is as 100%.
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Cited By (1)
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| CN105463038A (en) * | 2016-01-28 | 2016-04-06 | 厦门北化生物产业研究院有限公司 | Method for producing 4-Acetylantroquinonol B through fermentation |
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| TWI596210B (en) * | 2013-12-02 | 2017-08-21 | Medium for culture of Antrodia camphorata and culture method | |
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| TWI372049B (en) * | 2004-02-27 | 2012-09-11 | Simpson Biotech Co Ltd | Novel mixture and compounds from mycelia of antrodia camphorata having hepatoprotection, anti-inflammatory and anti-tumor activities |
| CN101151957A (en) * | 2006-09-26 | 2008-04-02 | 国鼎生物科技股份有限公司 | Culture method and uses of antrodia cinnamomea |
| TW200944120A (en) * | 2008-04-23 | 2009-11-01 | Qi-Xi Lian | Artificial cultivation method for antrodia camphorata |
| TWI384992B (en) * | 2008-11-21 | 2013-02-11 | Well Shine Biotechnology Dev Co Ltd | Novel compounds from antrodia camphorata |
| CN101785402B (en) * | 2009-01-23 | 2012-05-30 | 伟诚生物科技有限公司 | Method for preparing antradiacomphora extract with high contents of polysaccharides and triterpenoids |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105463038A (en) * | 2016-01-28 | 2016-04-06 | 厦门北化生物产业研究院有限公司 | Method for producing 4-Acetylantroquinonol B through fermentation |
| CN105463038B (en) * | 2016-01-28 | 2018-10-02 | 厦门北化生物产业研究院有限公司 | A kind of method of fermenting and producing 4-Acetylantroquinonol B |
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