CN107304407A - Medical grade anticancer novelty Antrodia camphorata bacterial strain - Google Patents
Medical grade anticancer novelty Antrodia camphorata bacterial strain Download PDFInfo
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- CN107304407A CN107304407A CN201610246546.4A CN201610246546A CN107304407A CN 107304407 A CN107304407 A CN 107304407A CN 201610246546 A CN201610246546 A CN 201610246546A CN 107304407 A CN107304407 A CN 107304407A
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- antrodia camphorata
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- deea
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Classifications
-
- 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
-
- 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
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
A large amount and novelty Antrodia camphorata (Antrodia cinnamomea) separation strains of peculiar anticancer triterpene compound are produced the present disclosure generally relates to a kind of.The present invention separately provides the method that triterpene compound is prepared using the separation strains.
Description
Technical field
The present invention be on a kind of Antrodia camphorata (Antrodia cinnamomea) novel separation strains, cultivate the separation strains to obtain containing a large amount of triterpene compounds (such as hydrogen sulfurenic acid (Dehydrosulphurenic acid,) and/or dehydrogenation perforation bacterium acid (Dehydroeburicoic acid DeSA, DeEA the method for cultured products)), with the composition comprising the cultured products.The present invention further relates to the method by preparing triterpene compound in the cultured products.
Background technology
Antrodia camphorata (Antrodia cinnamomea) (T.T. (Chang, T.T.) et al., 1995. mycology researchs (Mycological Research) 99:756-758) it is the peculiar fungi in Taiwan that is only grow on cinnamomum kanehirai, due to containing adenosine (adenosine), Polysaccharides (polysaccharides) (old J.J. (Chen, J.J.) et al., 2007. natural products magazines (Journal of Natural Products) 70:989-992), triterpene compound (triterpenoids) (old C.H. (Chen, C.H.) et al., 1995. natural products magazines 58:1655-1661;Into I.H. (Cherng, I.H.) et al., 1995. natural products magazines 58:365-371;With into I.H. et al., 1996. Phytochemistry (Phytochemistry) 41:263-267), steroid (Lee I.H. (Lee, I.H.) et al., 2002. federation of European Microbiological Societies's microbiology bulletins (FEMS Microbiology Letters) 209:63-67), diterpenoids (Lee Z.H. (Lee, Z.H.) et al., 2007. medicinal plants (Planta Med.) 73:1412-1415), sesquiterpenoids (sharp grace H.M. (Lien, H.M.) et al., 2011. evidence-baseds supplement alternative medicine (Evid.Based Complementary Altern.Med.) 2011:1-10), phenyl ring class (middle village N. (Nakamura, N.) et al., 2004. natural products magazines 67:46-48) and the tool physiologically active such as phenols composition; it is considered to have function (Lu M.C. (Lu such as antitumor, increase immunity, antihypertensive, hypoglycemic, suppression platelet aggregation, antiviral, antibacterium, anti-inflammatory, anti-oxidant, immunological regulation, reducing blood lipid, protection nerve and protection liver; M.C.) et al., 2013. pharmacology and cure (Pharmacology&Therapeutics) 139:124-156).
TW I417380B disclose evolutionary taxonomy, kenel feature, physiologically active and the Strain identification method of Antrodia camphorata, and its entire contents is hereby expressly incorporated by reference.
Antrodia camphorata has a variety of physiologically actives, and Related product is favored by consumers, but because wild Antrodia camphorata is slow-growing, it is impossible to the market demand is met, therefore the Antrodia camphorata product that exploitation is manually cultivated has its necessity.Presently commercially available Antrodia camphorata product can divide into the classifications such as wild Antrodia camphorata fructification, artificial cultivation Antrodia camphorata fructification, Antrodia camphorata space bag and Cinnamomum kanahirai hay camphorata mycelium (solid or liquid fermentation) according to the difference of raw material.It is up to 18 months to 36 months the time required to artificial cultivation production Antrodia camphorata fructification, the mycelium production time of solid-liquid fermented and cultured then only needs 1-3 months.Therefore the short time can the solid-liquid fermentation mycelium product of harvest have become in the market main flow gradually.
Antrodia camphorata bacterial strain is the core emphasis for producing Antrodia camphorata product, but effect activity difference of different bacterial strains is very big, therefore the excellent Antrodia camphorata bacterial strain of screening acquirement is particularly important.
Old Y.J. (Chen, Y.J.) et al. finds that DeSA has activity (the in vitro toxicology (Toxicity in Vitro) 23 for suppressing human leukemia and cancer of pancreas:418-424,2009).Du Y.C. (Du, Y.C.) et al. has found that DeEA has antileukemie active (plant medical science (Phytomedicine) 19:788-796,2012), even H.M. (Lien, H.M.) et al. has found that DeEA has the activity (molecule (Molecules) 19 (7) of anti-hepatocellular carcinoma:9033-9050,2014), and Deng J.Y. (Deng, J.Y.) et al. has found that DeEA has activity (chemical research (Chemical Research in Toxicology) 22 (11) in toxicology of anti-glioblast cancer:1817-1826,2009).Therefore, the Antrodia camphorata bacterial strain that can prepare high yield DeSA and/or DeEA is sought, and epochmaking value is respectively provided with commercial in the application of suppression cancer and anticancer.
The content of the invention
The purpose of the present invention is to be to provide a kind of Antrodia camphorata separation strains, and it can prepare the DeSA and/or DeEA of high yield simultaneously.
It is another object of the present invention to be to provide a kind of method for cultivating the Antrodia camphorata separation strains to obtain the cultured products containing a large amount DeSA and/or DeEA.
It is another object of the present invention to be to provide a kind of by the above method to obtain the cultured products containing a large amount DeSA and/or DeEA.
It is another object of the present invention to be to provide a kind of composition for including the Antrodia camphorata cultured products for containing a large amount DeSA and/or DeEA.
It is another object of the present invention to be to provide a kind of method for preparing DeSA and/or DeEA by the Antrodia camphorata cultured products for containing a large amount DeSA and/or DeEA.
It is another object of the present invention to be that offer is a kind of to be somebody's turn to do the Antrodia camphorata cultured products containing a large amount DeSA and/or DeEA in the purposes in suppression cancer and anticancer.
The present invention is described in detail in following part.The other features, objects and advantages of the present invention can be easily seen in embodiments of the present invention and claims.
Brief description of the drawings
Fig. 1 is shown 18 plants of Antrodia camphorata bacterial strain rDNA ITS sequence and LSU D1-D3 sequences are merged after, the relationship constructed with maximum brief several methods (Maximun Parsimony Method, MP) analyses, which develops, to be set, and BCRC36937 perfume China fir sesames are outer group.
Embodiment
The present invention can be understood by the various inventive aspects disclosed in following embodiments, embodiment narration related to Biao Lie's.Unless separately defined herein, the implication that the term used (comprising technology and scientific terminology) there should be those skilled in the art in the invention to be understood otherwise is associated with the present invention.And it should will be seen that, unless definition provided herein is otherwise indicated, otherwise in the case of any potential ambiguity, the definition of term should be consistent with the term (as defined in dictionary) generally used.It can be further appreciated that, term used in this case is used only as the purpose in terms of description particular implementation, not for restriction.
Must be noted that unless clearly indicated to the contrary, the singular forms " one kind " otherwise used in specification or claims " and it is " described " comprising complex representation.Therefore, unless the context requires otherwise, otherwise singular references should also include odd number by plural term comprising plural number.
The scope of the present invention is represented with " from " about " special value and/or to another " about " special value ".When scope is represented by aforesaid way, its include from a special value and/or to another special value scope.Similarly, when numerical value can be by term " about " to represent approximation, it will be appreciated that it is the other side of a particular value.It can further appreciate that, when carrying and itself, the two-end-point of each scope is all meaningful about other end points and other end points.± 10% is represented according to " about " of the invention.
In the present invention, term " through separation " or " separation " mean to remove material from its primal environment (for example, being natural surroundings if naturally occurring).It is purified that term " through separation " or " separation ", which are not necessarily referring to material,.
The purpose of the present invention one is to provide a kind of Antrodia camphorata separation strains, and it includes such as SEQ ID NO:The ITS sequence of nucleotide sequence shown in 1 and such as SEQ ID NO:The LSU D1-D3 sequences of nucleotide sequence shown in 2.
One in the present invention is preferable to carry out in aspect, Antrodia camphorata (Antrodia cinnamomea) separation strains are to be deposited at Germany Microbiological Culture Collection Center, Ma Xuluode streets 1b, D38124, Brunswick, Germany (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D38124, Braunschweig, Germany), deposit numbering is DSM 32199 bacterial strain, or there is the mutant strain of substantial identical feature with being deposited at Germany Microbiological Culture Collection Center and depositing the bacterial strain that numbering is DSM 32199.
Above-mentioned " variant " means that cover total cells genetic constitution is changed by such as chemical mutagenesis, spontaneous mutation, genetic engineering, conversion or transfection, so that influenceing any Antrodia camphorata bacterial strain of its physics or biochemical characteristic.However, the mutant strain should have to deposit all identification features that numbering is the bacterial strain that DSM 32199 is deposited at Germany Microbiological Culture Collection Center.
It is another object of the present invention to provide a kind of method for preparing Antrodia camphorata cultured products, it, which is included, is inoculated in the Antrodia camphorata separation strains of the present invention in suitable culture medium, and is cultivated to obtain the cultured products under proper condition.The present invention also provides the cultured products obtained by the above method.
Heretofore described " the suitable culture medium " for being used to cultivate Antrodia camphorata separation strains can be any known fluid nutrient medium or solid medium for being used to cultivate Antrodia camphorata.According to the present invention, training method can be any known solid fermentation or liquid fermentation (such as Batch fermentation, feedback are expected Batch fermentation and continuously fermented).
Can be suitable for the fluid nutrient medium of the present invention, but it is not limited to potato fluid nutrient medium (Potato Dextrose Broth, PDB) (U.S. Di Fuke (Difco, )) and malt extract culture medium (Malt Extract Broth, MEB) (U.S. Di Fuke) USA.It can be suitable for the solid medium of the present invention, but be not limited to the solid medium based on cereal or the solid medium based on food industry accessory substance.
The solid medium of " based on cereal " of the present invention or " based on food industry accessory substance " refers to that the main component (weight ratio about more than 90%) in the solid medium is cereal or food industry accessory substance, and wherein still with including any known group composition for being used to cultivate Antrodia camphorata, such as nitrogen source (such as yeast extract, peptone, ammonium chloride, ammonium nitrate, ammonium nilrite and amino acid);Carbon source (such as glucose and glycerine, starch, sucrose, fructose);Salt (such as sodium salt, sylvite and magnesium salts) and buffer (such as phosphoric acid buffer agent).In the present invention, the cereal includes, but is not limited to rice, barley, buckwheat, sorghum, adlay, wheat, rye, oat or its combination;And the food industry accessory substance includes, but it is not limited to bagasse, starch, wheat bran, rice bran, cornstalk, coffee grounds or its combination.
And be used to cultivate the conditions such as " felicity condition " intention such as temperature and incubation time of Antrodia camphorata separation strains in the present invention, its tolerable described Antrodia camphorata separation strains grows, breeds and manufacture DeSA and/or DeEA.The composition and condition of culture that those skilled in the art scholar can be directed to culture medium according to existing knowledge are adjusted.In general, the culture cultivation temperature of Antrodia camphorata separation strains is about 15 DEG C-about 35 DEG C, preferably about 22 DEG C-about 28 DEG C, more preferably approximately 25 DEG C;And incubation time can be greater than 20 days, preferably about 20 days-about 180 days, more preferably approximately 30 days-about 120 days.
The method of the present invention can be optionally further comprising the step that the Antrodia camphorata cultured products are sterilized, heated and/or dried.
It is another object of the present invention to provide a kind of composition of the Antrodia camphorata cultured products of the present invention comprising effective dose.Due to DeSA and DeEA containing a large amount in the Antrodia camphorata cultured products of the present invention, therefore it may be used as the active component (old Y.J. (Chen of antitumor (including cancer of pancreas, hepatocellular carcinoma, glioblast cancer and/or anti-leukocythemia), Y.J.) et al., 2009. in vitro toxicology (Toxicity in Vitro) 23:418-424;Shut out Y.C. (Du, Y.C.) et al., 2012. plant medical science (Phytomedicine) 19:788-796), it is possible to be prepared into medical composition or food compositions.Antrodia camphorata cultured products prepared by the inventive method can be allocated and obtained by the composition of the present invention by any known techniques with other known additives.The additive includes, but is not limited to supporting agent, excipient, adhesive, extender, disintegrant, diluent, lubricant and/or sweetener or flavor enhancement etc..
It is another object of the present invention to provide a kind of method for preparing DeSA and/or DeEA, it is included isolates DeSA and/or DeEA from the Antrodia camphorata cultured products of the present invention.The separation method can be any known method for separating DeSA and/or DeEA, such as fluid extraction, HPLC and ion exchange resin (US 2009/0318400A;Shen C.C. (Shen, C.C.) et al., 2003. Chinese medical periodicals (Journal of Chinese Medicine), 14:247–258).
All publication as described herein, patent and patent document are incorporated herein in entirety by reference.
Following instance is provided to aid in those skilled in the art to implement the present invention.Even so, the limitation of the present invention is examples should not be construed as, because the modifications and variations that those skilled in the art in the invention are carried out in the case of without departing substantially from the spirit or scope of the present invention to embodiments described herein, and the scope of the present invention is still fallen within.
Example
The solid fermentation thing culture of embodiment one, Antrodia camphorata bacterial strain
Antrodia camphorata bacterial strain GW01, GW03, GW04 and GW05 are the Antrodia camphorata bacterial strains that applicant voluntarily separates.Applicant separately buys 3 kinds of Antrodia camphorata bacterial strain products from market, compares for experiment.
After above-mentioned 4 plants of bacterial strains are taken out and activated from -80 DEG C of freezer glycerol tubes, potato dextrose agar (PDA) (U.S. Di Fuke) and malt extract agar (MEB) (U.S. Di Fuke) flat board are inoculated in.After 25 DEG C are cultivated 14-21 days, sterilized water is added, after being smashed with homogenizer, is inoculated in the triangle shaking flask for including 50 grams of cereal matrix sterilized in advance, after being cultivated 30 days to 120 days at 25 DEG C, with 50 DEG C of drying, that is, obtained containing mycelial solid fermentation thing.
The analysis of DeSA and DeEA contents in embodiment two, Antrodia camphorata solid fermentation thing
(1) analysis method
1 gram of Antrodia camphorata solid fermentation thing is weighed to be placed in centrifuge tube, 5ml methanol is added in centrifuge tube, shaken 15 minutes in ultrasonic vibrating machine, methanol solution is drawn with injection needle tube, and after being filtered with 0.45 μm of filtration module, filtrate is placed in liquid chromatograph dedicated pipe.The content of the Antrodia camphorata active metabolites such as DeSA and DeEA is analyzed using HPLC.
HPLC analysis conditions are as follows:
Chromatograph tubing string:MN, UNCLEODUR C18FTEC, 5 μm,
Internal diameter 4.6mm × 25cm
Fluorescent detector:PDA (photodiode array, light diode array) UV180~300nm
Mobile phase solution:Acetonitrile is with water with 90:10 (v/v) proportion grading starting, gradient is system in 40 minutes increase acetonitriles to 100%
Sample analysis volume:15μl
Mobile phase flow velocity:1ml/min
(2) DeSA and DeEA of the Antrodia camphorata grain of rice fermentate content
Different Antrodia camphorata bacterial strains are in after grain of rice solid fermentation 90 days, and the content of contained DeSA and DeEA isoreactivity compounds is as shown in table 1 in fermentate.In addition to not containing DeSA except commercially available prod 2, other strain fermentation things and commercially available prod can contain DeSA and DeEA.DeSA and DeEA total amounts are up to 31,030ppm in bacterial strain GW01 fermentate, are 1.4-15.3 times of other bacterial strains, and DeSA yield (6,119ppm) and DeEA yield (24,911ppm) rank the first in 7 samples.Show that bacterial strain GW01 has the ability for producing a large amount DeSA and DeEA isoreactivity compound simultaneously.
The different Antrodia camphorata bacterial strains of table 1. are in DeSA the and DeEA contents after grain of rice solid fermentation
(3) in the fermentate of Antrodia camphorata GW01 different number of days DeSA and DeEA content
Antrodia camphorata bacterial strain GW01 is incubated in grain of rice matrix, DeSA and DeEA content can be observed to be increased with the increase of fermentation number of days.This experiment ferments Antrodia camphorata strain culturing the different times, fermentation 60 is analyzed respectively, after 90 and 120 days, the content of contained DeSA and DeEA isoreactivity compounds in fermentate, as a result as shown in table 2.In bacterial strain GW01 grain of rice fermentate, 120 days are fermented compared to fermentation 60 days, DeSA content improves about 2.15 times, and DeEA content improves about 2.67 times, DeSA and DeEA total content improves about 2.56 times, the bacterial strain GW01 Extending culture times are shown, the yield of DeSA and DeEA isoreactivity compounds can be improved.
The Antrodia camphorata bacterial strain GW01 of table 2. is in DeSA the and DeEA contents of grain of rice solid fermentation different number of days
Result above is shown, DeSA the and DeEA isoreactivities compound production of the Antrodia camphorata bacterial strain of the present invention is the champion of all test strains, and DeSA and DeEA total amount is about 31,030ppm, far above other bacterial strains (about 2,029-22,843ppm), it is 1.4-15.3 times of other bacterial strains.In addition, DeSA and DeEA specific yield and total output all can further be improved with longer fermentation times, show that the bacterial strain can produce three kinds of active ingredients such as DMB, SY-1 and MBDD of a large amount simultaneously.
Embodiment three, bacterial strain GW01 form and Molecular Identification
(1) morphologic observation:
Bacterial strain GW01 is inoculated in PDA (U.S. Di Fuke) and MEA (U.S. Di Fuke) culture medium, in being cultivated 35 days at 25 DEG C, can all produce the rotten fragrance of honey peach fruit;35 days colony diameter averagely about 45-50mm on MEA flat boards, bacterium colony is in light orange to orange, and aerial hyphae is by villiform, powdery, cotton-wool to ulotrichy, and the bacterium colony back side is in light crocus;35 days colony diameter averagely about 40-45mm on PDA plate, bacterium colony is in light crocus to orange, and aerial hyphae is by villiform, powdery, cotton-wool to ulotrichy, and the bacterium colony back side is in light orange to light crocus.
Bacterial strain GW01 is observed under an optical microscope, it is observed that a large amount of conidiums.Conidium is oval, shaft-like, column to long column shape, and size is about (2.5-) 3~6 (- 9.5) × (1.5-) 2~3.5 (- 4) μm.The mycelia of fresh cultured bacterium colony is about (1.5-) 2~3.5 (- 4.5) μm wide, tool clasp body (clamp connection), more ripe mycelia outer wall visible crocus crystal once in a while.
(2) Molecular Identification:
1. gene order compares analysis result
GW01 bacterial strains are subjected to DNA extractions, carry out transcription interval (internal transcribed spacer in ribosomes, ITS) partial sequence is analyzed, sequence overall length is 823bp, it is compared with known Antrodia camphorata sequence on NCBI GenBank, GW01 the sequencing results are closest to Antrodia camphorata (Antrodia camphorata) (AY378095) ﹙=Antrodia camphorata (Antrodia cinnamomea) ﹚, the coverage rate of sequence is 100% (823/823), and similarity is 100% (823/823);It is additionally carried out the big sub-cell fragment of ribosomes nucleic acid (the Large subunit ribosomal DNA of GW01 bacterial strains, LSU rDNA) analysis of D1-D3 partial sequences, sequence overall length is 956bp, it is compared with the known array on NCBI GenBank, GW01 the sequencing results are closest to Antrodia camphorata (Taiwanofungus camphoratus) (AY333841) ﹙=Antrodia camphorata (Antrodia cinnamomea) ﹚, the coverage rate of sequence is 100% (956/956), and similarity is 99% (955/956).
According to morphological features such as colonial morphology, color, conidium and clasp bodies, and according to its special odor, with ITS the and LSU D1-D3 fragment sequence analysis results of rDNA genes, it is Antrodia camphorata (Antrodia cinnamomea) mycelium to identify the sample (Antrodia camphorata and Taiwanofungus camphoratus are all Antrodia cinnamomea synonyms).
Bacterial strain GW01 ITS sequence:
SEQ ID NO:1
TTTGTACACACCGCCCGTCGCTACTACCGATTGAATGGCTTAGTGAGGTCTTGGGATTGGCTTCGGGGAGCCGGCAACGGCATCCTGTTGCTGAGAACTTGATCAAACTTGGTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATTGTATTTGAAAGGGGTTGTAGCTGACCTCCTCTTGAAAAGGGGGGAGGTATGTGCACACCTCTGTTCATTCATATTCTCTCACACCTGTGCATGCTTTGTAGGTTGGTTTTGAATGGTTGTCTTCTCTGATGGAGACAGCTGTTTTGACCTTCCTATGTTTTTTAAATTGACTCCGTATCAGTTACAGAATGTATGTTGCGTGTAACGCATATTGTATAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACACGGCGAAATGTGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTTGGTATTCTGAGGAGCATGCCTGTTTGAGTGTCATGGAATTATCAACCCTTTTGACTTTTTGTTGAATGGGCTTGGATTTGGAGGGTTAAATTGCTGGCTCTTTTTTTTGATTCAGCTCCTCTTGAATGCATTAGCTTGAACCCTTGTGGATTGACCTTATCGGTGTGATAGTCATCTATGCCGTGGTTGTCTGAGGTGGGATCGGCTTCTAATGGTGCAAGTCCCTTCAGGGGGATGATTTTCTAATGACCTTCTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAATAAGCGGA
Bacterial strain GW01 LSU (D1-D3) sequence:
SEQ ID NO:2
AGCATATCAATAAGCGGAGGAAAAGAAACTAACAAGGATTCCCCTAGTAACTGCGAGTGAAGCGGGATAAGCTCAAATTTAAAATCTGGTGGGTGTTTGGCCTGCCCGAGTTGTAGTCTGGAGAAGTGCTTTCTGTGCTGAACCGTGTACAAGTCTCTTGGAACAGAGCGTCATAGAGGGTGAGAATCCCGTCTTTGACACGGACTATCAGTGCTTTGTGATGCGCTCTCAATGAGTCGAGTTGTTTGGGAATGCAGCTCAAAATGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGTGAACAAGTACCGTGAGGGAAAGATGAAAAGCACTTTGGAAAGAGAGTTAAACAGTACGTGAAATTGCTGAAAGGGAAACACTTGAAGTCAGTCGCGTTGACCGGAGCTCAACCTTGCTTTTTTGGGCTTGGTGCACTTTCTGGTTGACGGGTCAGCATCAATTTTGACTGTTGGAGAAGGGTTGGGGAAATGTGGCACCTTCGGGTGTGTTATAGCCCCCAGTCACATACAATGGTTGGGATTGAGGAACTCAGCACACCTTTATGGTCAGGGTTTTTTTTAACCTATGTTATTGTGCTTA
GGATGCTGGCGTAATGGCTTTAAATGACCCGTCTTGAAACACGGACCAAGGAGTCTAACATGCCTGCGAGTGTTTGGGTGGCAAACCCGAGTGCGTAATGAAAGTGAAAGTTGGGACCTCTGTTGTGGGAGGGCACCGATGCCCGGACCAGACCTTTTGTGATGGATCCGCGGTAGAGCATGTATGTTGGGACCCGAAAGATGGTGAACTATGCCTGAATAGGGTGAAGCCAGAGGAAACTCTGGTGGAGGCTCGTAGCGATTCTGACGTGCAAATCGATCGTCAAATTTGGGTATAGGGGCGAAAGACTAATCGAACCATCTAGTAGCTGGTTCCTGCCGA
2. the sequence alignment analysis result with known Antrodia camphorata kind
The another open bacterial strain BCRC35396 for being preserved GW01 and Foodstuff Industrial Development Inst. of Financial Group Legal Persons's living resources preservation and research center (BCRC), BCRC35398, BCRC35716, BCRC36401, BCRC36711, BCRC36795, BCRC37609, BCRC37616, BCRC37848, BCRC37849, BCRC37850, BCRC37889, BCRC37890, BCRC37891, BCRC37893, BCRC37894, BCRC37941 counts 17 plants of bacterial strains progress ITS partial sequences and compared with LSU D1-D3 partial sequences.
It was found that ITS sequence, which has 7 core candy body DNA sites, has otherness, wherein GW01 has the sequence difference in 0-2 site with above-mentioned other 17 plants of bacterial strains respectively, and similarity is 99.7%-100%.LSU D1-D3 segment portion gene orders also have 10 core candy body DNA sites to have otherness, and wherein GW01 has the sequence difference in 1-4 site with above-mentioned other 17 plants of bacterial strains respectively, and similarity is 99.5%-99.9%.
Then this two sections of gene orders are together in series by all bacterial strain rDNA gene orders (ITS and LSU D1-D3) with Notepad softwares again, and shelves are turned with DAMBE softwares, it is analysis to be done relationship using the softwares of Mega 4 and closed, with brief several method (the Maximun Parsimony Method of maximum, MP) set to construct to develop, and it is to set the holding strength on each node to be calculated relationship with the 1000 duplicate sampling estimations technique (Bootstrap) repetitions and closed, the evolution tree of last gained is represented with synthesizing tree (consensus tree), it is tree to obtain Antrodia camphorata bacterial strain relationship as shown in Figure 1 and close.
Closed by two sections of genetic fragment Analysis of Partial results of ITS and LSU D1-D3 and relationship is that tree shows the sequence of all disclosed bacterial strains of GW01 and BCRC and differed.
GW01 Antrodia camphoratas bacterial strain is deposited at Foodstuff Industrial Development Inst. of Financial Group Legal Persons's living resources on the 8th in September in 2015 and preserved and research center, and deposit numbering is BCRC 930178;And Antrodia camphorata (Antrodia cinnamomea) bacterial strain has also been deposited at Germany Microbiological Culture Collection Center according to budapest treaty (Budapest Treaty) on October 23rd, 2015, Ma Xuluode streets 1b, D38124, Brunswick, Germany (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D38124, Braunschweig, Germany), deposit numbering is DSM 32199.
Claims (12)
1. a kind of Antrodia camphorata separation strains, the Antrodia camphorata separation strains be deposited at Germany Microbiological Culture Collection Center and
The bacterial strain that deposit numbering is DSM 32199.
2. a kind of method for preparing Antrodia camphorata cultured products, it is included Antrodia camphorata according to claim 1
Separation strains are inoculated in suitable culture medium, and are cultivated to obtain the culture production under proper condition
Thing.
3. method according to claim 2, wherein the culture medium is solid medium or fluid nutrient medium.
4. method according to claim 3, wherein the culture medium is the solid culture based on cereal
Base or the solid medium based on food industry accessory substance.
5. method according to claim 4, wherein the cereal is rice, barley, buckwheat, sorghum, the heart of a lotus seed
Benevolence, wheat, rye, oat or its combination.
6. method according to claim 4, wherein the food industry accessory substance is bagasse, starch, bran
Skin, rice bran, cornstalk, coffee grounds or its combination.
7. the method according to any claim in claim 2 to 6, wherein the temperature of the culture is
About 20 DEG C-about 35 DEG C.
8. the method according to any claim in claim 2 to 6, wherein the time of the culture is
More than 20 days.
9. a kind of method for preparing DeSA and/or DeEA, it is included from according in claim 2 to 8
Isolated in Antrodia camphorata cultured products obtained by method described in any claim DeSA and/or
DeEA。
10. the Antrodia camphorata culture obtained by a kind of method according to any claim in claim 2 to 8
Product is in preparation to the purposes for the composition for pressing down cancer and anticancer.
11. purposes according to claim 10, wherein the composition is medical composition or food compositions.
12. purposes according to claim 10, wherein the cancer be leukaemia, cancer of pancreas, hepatocellular carcinoma and/
Or glioblast cancer.
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CN107858292A (en) * | 2017-09-18 | 2018-03-30 | 刘伯雅 | A kind of Antrodia camphorata bacterial strain and its method for preserving |
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BE1029918B1 (en) * | 2022-05-25 | 2023-06-07 | Greenyn Biotechnology Co Ltd | PROCESS FOR MANUFACTURING AND USE OF ANTRODIA CINNAMOMEA MYCELIUM IN THE TREATMENT OF CANCER |
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Address after: 1, floor 15, Lane 2, Lane 161, paddy field street, Hsinchu, Taiwan Applicant after: Agricultural biotechnology Limited by Share Ltd Address before: No. 447, Rong min Road, Zhongli District, Taoyuan City, Taiwan, China Applicant before: Agricultural biotechnology Limited by Share Ltd |
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WD01 | Invention patent application deemed withdrawn after publication | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20171031 |