CN105132288A - Novel Antrodia cinnamomea isolate and culture product thereof - Google Patents
Novel Antrodia cinnamomea isolate and culture product thereof Download PDFInfo
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Abstract
The invention relates to a Antrodia cinnamomea isolate and a method for preparing an Antrodia cinnamomea culture product. The invention also provides a method for preparing 2,3-dimethoxy-5-methyl-p-benzoquinone (DMB), 4,7-dimethoxy-5-methyl-1,3-benzodioxole (SY-1) and/or 5-methyl-benzo[1,3]dioxole-4,7-diol (MBDD).
Description
Technical field
The present invention relates to the novel strain isolated of a kind of Antrodia camphorata (Antrodiacinnamomea), cultivate described strain isolated to obtain containing a large amount of 2,3-dimethoxy-5-methyl-p-benzoquinone (DMB), 4,7-dimethoxy-5-methyl isophthalic acid, 3-benzodioxole (SY-1) and 5-methyl-benzo [1,3] dioxole-4, the method of the cultured products of 7-glycol (MBDD), and comprise the composition of described cultured products.The method of the present invention also about preparing DMB, SY-1 and/or MBDD from described cultured products.
Background technology
Antrodia camphorata (Antrodiacinnamomea) (T.T. (Chang, the people such as T.T.), 1995. mycology research (MycologicalResearch) 99:756-758) be the peculiar fungi in Taiwan being only grown on Cinnamomum kanahirai hay tree, due to containing adenosine (adenosine), Polysaccharides (polysaccharides) (old J.J. (Chen, the people such as J.J.), 2007. natural product magazine (JournalofNaturalProducts) 70:989-992), triterpene compound (triterpenoids) (old C.H. (Chen, the people such as C.H.), 1995. natural product magazine 58:1655-1661, become the people such as I.H. (Cherng, I.H.), 1995. natural product magazine 58:365-371, with become the people such as I.H., 1996. vegetable chemistry (Phytochemistry) 41:263-267), steroid (Lee I.H. (Lee, the people such as I.H.), 2002. federation of European Microbiological Societies microbiology bulletin (FEMSMicrobiologyLetters) 209:63-67), diterpenoids (Lee Z.H. (Lee, the people such as Z.H.), 2007. medicinal plants (PlantaMed.) 73:1412-1415), sesquiterpenoids (sharp grace H.M. (Lien, the people such as H.M.), 2011. follow card supplements alternative medicine (Evid.BasedComplementaryAltern.Med.) 2011:1-10.), phenyl ring class (middle village N. (Nakamura, the people such as N.), 2004. natural product magazine 67:46-48) and the composition of the tool physiologically active such as phenols, be considered to have antitumor, increase immunizing power, antihypertensive, hypoglycemic, suppress platelet aggregation, antiviral, antibacterium, the functions such as protection liver.
Prior document TWI417380B discloses the evolutionary taxonomy of Antrodia camphorata, kenel feature, physiologically active and Strain identification method, and its entire contents is incorporated to for reference herein.
Antrodia camphorata has multiple physiologically active, and related products deeply by consumers, but due to wild Antrodia camphorata poor growth, cannot be met the need of market, and the Antrodia camphorata product therefore developing artificial culture has its necessity.Current commercially available Antrodia camphorata product, according to the difference of raw material, can divide into the classifications such as wild Antrodia camphorata sporophore, artificial culture Antrodia camphorata sporophore, Antrodia camphorata space bag and Antrodia camphorata mycelium (solid or liquid fermenting).Artificial culture is produced Antrodia camphorata sporophore required time and is reached 18 months to 36 months, and the mycelium production time of solid-liquid fermentation culture then only needs 1-3 month.Therefore the short period of time solid-liquid fermentation mycelium product of harvest can become main flow on market gradually.
Antrodia camphorata bacterial strain is the core emphasis producing Antrodia camphorata product, but effect activity difference of different strains is very big, and therefore to obtain excellent Antrodia camphorata bacterial strain very important in screening.In the active metabolite that Antrodia salmonea can produce, DMB has activity (the elementary people of Chen Jing of angiogenesis inhibiting, 2012, Taiwan Patent I369201), SY-1 has the activity (people such as sharp grace H.M. of the propagation suppressing human colon cell, 2011. follow card supplements alternative medicine 2011:1-10) and the effect (painting S.H. (Tu for the treatment of intestinal cancer, the people such as S.H.), 2012. agricultural food product the Chemicalss (JAgricFoodChem.) 60 (14): 3612-8), and MBDD then has anti-oxidant activity (Wu M.D. (Wu, the people such as M.D.), 2011. Natural products researchs (NaturalProductResearch), 25 (16): 1488-1496).Therefore, the seeking of Antrodia camphorata bacterial strain of high yield DMB, SY-1 and/or MBDD can be prepared, commercially there is epochmaking value.
summary of the invention
One object of the present invention is to provide a kind of Antrodia camphorata strain isolated, and it can prepare high yield DMB, SY-1 and MBDD simultaneously.
Another object of the present invention is that providing a kind of cultivates described Antrodia camphorata strain isolated to obtain the method for the cultured products containing a large amount DMB, SY-1 and MBDD.
Another object of the present invention is to provide a kind of cultured products containing a large amount DMB, SY-1 and MBDD obtained by aforesaid method.
Another object of the present invention is to provide a kind of composition comprising the described Antrodia camphorata cultured products containing a large amount DMB, SY-1 and MBDD.
Another object of the present invention is to provide a kind of method by described Antrodia camphorata cultured products preparative separation DMB, SY-1 and/or MBDD containing a large amount DMB, SY-1 and MBDD.
The present invention describes in detail in lower part.Other features, objects and advantages of the present invention can easily see in embodiments of the present invention and claims.
Accompanying drawing explanation
After the ITS sequence of 18 strain Antrodia camphorata bacterial strain rDNA and LSUD1-D3 sequence merge by Fig. 1 display, the relationship of constructing with the analysis of MP method develops and sets, and BCRC36937 Antrodia salmonea is outer group.
Embodiment
The present invention can be understood by relevant the describing of the various inventive aspects, embodiment and the tabular that disclose in following embodiment.Unless separately defined in this article, otherwise the term (comprising technology and scientific terminology) associating use with the present invention should have the implication that those skilled in the art in the invention understand.And should understand, unless the definition provided herein separately explains, otherwise when any potential ambiguity, term that the definition of term generally should use with described (as in dictionary define) consistent.Can understanding person further, the term that this case uses only is used as the object describing particular implementation aspect, but not for limiting.
Must be noted that unless clearly indicated to the contrary, otherwise in the singular forms " one " that specification sheets or claims use " and " described " comprise complex representation.Unless the context requires otherwise, otherwise singular references should comprise plural number and plural term also comprises odd number therefore.
Scope of the present invention represents with " from " about " special value and/or to another " about " special value ".When scope is represented by aforesaid way, it comprises from a special value and/or the scope to another special value.Similarly, when numerical value by term " about " to represent approximation, can realize it is another aspect of a particular value.Can understand further, when mention about other end points and other end points itself, the two-end-point of each scope is all significant.± 10% is represented according to the present invention " about ".
In the present invention, term " through being separated " or " separation " mean shifts out material from its primal environment (if natural existence, being such as, natural surroundings).Term " through being separated " or " separation " might not refer to that material is purified.
Object one of the present invention is to provide a kind of Antrodia camphorata strain isolated, its comprise nucleotide sequence as shown in SEQIDNO:1 ITS sequence and as shown in SEQIDNO:2 the LSUD1-D3 sequence of nucleotide sequence.
In in a preferred enforcement of the present invention, described Antrodia camphorata strain isolated is deposited at Foodstuff Industrial Development Inst. of Financial Group Legal Persons and deposits the bacterial strain being numbered BCRC930164, or be deposited at Foodstuff Industrial Development Inst. of Financial Group Legal Persons and deposit the mutant strain that the bacterial strain being numbered BCRC930164 has identical in fact feature.
Above-mentioned " variant " means that containing total cells genetic composition has passed through to change as chemical mutagenesis, spontaneous mutation, genetic engineering, conversion or transfection, so that affects any Antrodia camphorata bacterial strain of its physics or biochemical characteristic.But described mutant strain should have to deposit and is numbered all recognition features that BCRC930164 is deposited at the bacterial strain of Foodstuff Industrial Development Inst. of Financial Group Legal Persons.
Another object of the present invention is to provide a kind of method preparing Antrodia camphorata cultured products, and it comprises and is inoculated in applicable substratum by Antrodia camphorata strain isolated of the present invention, and carries out under proper condition cultivating to obtain described cultured products.The present invention also provides the cultured products obtained by aforesaid method.
Can be any known liquid nutrient medium for cultivating Antrodia camphorata or solid medium for cultivating " be applicable to substratum " of Antrodia camphorata strain isolated described in the present invention.And " felicity condition " for cultivating Antrodia camphorata strain isolated in the present invention refers to as the condition such as temperature and incubation time, it can be allowed the growth of described Antrodia camphorata strain isolated, breeds and manufacture DMB, SY-1 and MBDD.Those skilled in the art can adjust for the composition of substratum and culture condition according to existing knowledge.According to the present invention, training method can be any known solid fermentation or liquid fermenting (such as Batch fermentation, feedback are expected Batch fermentation and continuously ferment).
Being applicable to liquid nutrient medium of the present invention can be, but be not defined as potato liquid nutrient medium (PotatoDextroseBroth, PDB) (U.S. Di Fuke (Difco,) and Fructus Hordei Germinatus extract substratum (MaltExtractBroth, MEB) (U.S. Di Fuke) USA).Being applicable to solid medium of the present invention can be, but the solid medium be not defined as based on cereal or the solid medium based on foodstuffs industry by product.
The solid medium of " based on cereal " of the present invention or " based on foodstuffs industry by product " refers to that the main component (weight ratio about more than 90%) in described solid medium is cereal or foodstuffs industry by product, and wherein still can comprise any known composition for cultivating Antrodia camphorata to divide, such as nitrogenous source (as yeast extract, peptone, ammonium chloride, ammonium nitrate, ammonium nitrite and amino acid); Carbon source (as glucose and glycerine, starch, sucrose, fructose); Salt (as sodium salt, sylvite and magnesium salts) and buffer reagent (as phosphoric acid buffer agent).In the present invention, described cereal comprises, but is not defined as rice, barley, buckwheat, Chinese sorghum, heart of a lotus seed benevolence, wheat, rye, oat or its combination; And described foodstuffs industry by product comprises, but be not defined as bagasse, starch, wheat bran, rice bran, corn stalk, coffee grounds or its combination.
In in an enforcement of the present invention, cultivate in prepared Antrodia camphorata cultured products in the solid medium based on rice, with the total weight of described Antrodia camphorata cultured products, 2, 3-dimethoxy-5-methyl-p-benzoquinone (DMB), 4, 7-dimethoxy-5-methyl isophthalic acid, 3-benzodioxole (SY-1) and 5-methyl-benzo [1, 3] dioxole-4, the ultimate production of 7-glycol (MBDD) is greater than about 1, 200mg/kg, be preferably about 1, 200mg/kg is to about 1, 700mg/kg, be more preferably about 1, 200mg/kg is to about 1, 600mg/kg, most preferably be about 1, 200mg/kg is to about 1, 500mg/kg.
In in another enforcement of the present invention, cultivate in prepared Antrodia camphorata cultured products in the solid medium based on buckwheat, with the total weight of described Antrodia camphorata cultured products, 2, 3-dimethoxy-5-methyl-p-benzoquinone (DMB), 4, 7-dimethoxy-5-methyl isophthalic acid, 3-benzodioxole (SY-1) and 5-methyl-benzo [1, 3] dioxole-4, the ultimate production of 7-glycol (MBDD) is greater than about 1, 500mg/kg, be preferably about 1, 500mg/kg is to about 2, 000mg/kg, be more preferably about 1, 500mg/kg is to about 1, 900mg/kg, most preferably be about 1, 500mg/kg is to about 1, 800mg/kg.
Method of the present invention optionally can comprise the step of described Antrodia camphorata cultured products being carried out sterilizing, heating and/or drying further.
Another object of the present invention is to provide a kind of composition including the Antrodia camphorata cultured products of the present invention of effective amount.Due to DMB, SY-1 and MBDD containing a large amount in Antrodia camphorata cultured products of the present invention, therefore it can be used as, activeconstituents (land M.C. (the Lu of such as antitumor, angiogenesis inhibiting and/or anti-oxidant activity, the people such as M.C.), 2013. pharmacology and therapeutics (Pharmacology & Therapeutics) 139:124-156), and medical composition or food compositions can be prepared into.Antrodia camphorata cultured products prepared by the inventive method and other known additive can be allocated and obtain by any known techniques by composition of the present invention.Described additive comprises, but is not defined as supporting agent, vehicle, tackiness agent, extender, disintegrating agent, thinner, lubricant and/or sweeting agent or seasonings etc.
Another object of the present invention is to provide a kind of method preparing DMB, SY-1 and/or MBDD, and it comprises isolates DMB, SY-1 and/or MBDD in Antrodia camphorata cultured products of the present invention.Described separation method can be any known method for separating of DMB, SY-1 and MBDD, such as HPLC, ion exchange resin (Wu M.D. (Wu, the people such as M.D.), 2011. Natural products researchs, 25 (16): 1488-1496).
The mode that all publication as herein described, patent and patent documentation are quoted all is in full incorporated herein.
Following instance is provided to implement the present invention with auxiliary those skilled in the art.Even so, described example should be considered as restriction of the present invention, because those skilled in the art in the invention to the modifications and variations that embodiment discussed in this article is carried out, and still belong to scope of the present invention when not deviating from the spirit or scope of the present invention.
Example
The solid fermentation thing of embodiment one, Antrodia camphorata bacterial strain is cultivated
Antrodia camphorata bacterial strain BCRC35398, BCRC36711 are purchased from Foodstuff Industrial Development Inst. of Financial Group Legal Persons.Antrodia camphorata bacterial strain GW01, GW02, GW03, GW04 and GW05 are the Antrodia camphorata bacterial strains that applicant is separated voluntarily.
Above-mentioned 7 strain bacterial strains to be taken out from-80 DEG C of freezer glycerine pipes and after activating, be inoculated in potato dextrose agar (PDA) (U.S. Di Fuke) and Fructus Hordei Germinatus extract agar (MEB) (U.S. Di Fuke) is dull and stereotyped.Cultivate after 14-21 days in 25 DEG C, add sterilized water, after smashing with clarifixator, what be inoculated in prior sterilizing includes in the triangle shaking flask of 50 grams of cereal matrix, cultivates after 30 days, with 50 DEG C of oven dry, namely obtain and contain mycelial solid fermentation thing at 25 DEG C.
In embodiment two, Antrodia camphorata solid fermentation thing, DMB, SY-1 and MBDD are containing quantitative analysis
(1) analytical procedure
Taking 1 gram of Antrodia camphorata solid fermentation thing is placed in centrifuge tube, 5ml methyl alcohol is added in centrifuge tube, shakes 15 minutes in ultrasonic vibrating machine, draw methanol solution with injection needle tube, and after filtering with 0.45 μm of filtration module, filtrate is placed in liquid chromatograph dedicated pipe.HPLC is utilized to analyze the content of 3 kinds of Antrodia camphorata active metabolites such as DMB, SY-1 and MBDD.
HPLC analysis condition is as follows:
Chromatography tubing string: MN, UNCLEODURC18FTEC, 5 μm,
Internal diameter 4.6mm × 25em
Fluorescent analyzer: PDA (photodiodearray, light diode array) UV180 ~ 300nm
Mobile phase solution: acetonitrile and water initial with the proportion grading of 90: 10 (v/v), gradient system 40 minutes increase acetonitrile to 100%
Sample analysis volume: 15 μ l
Mobile phase flow velocity: 1ml/min
(2) content of DMB, SY-1 and MBDD of Antrodia camphorata grain of rice fermented product
Different Antrodia camphorata bacterial strain is in grain of rice solid fermentation after 30 days, and the content of three kinds of active compounds such as DMB, SY-1 and MBDD contained by fermented product is as shown in table 1.Except bacterial strain GW01 does not produce after 30 days except the third-class compound such as DMB, SY-1 and MBDD in grain of rice substrate fermentation, other bacterial strain all can produce DMB, SY-1 and MBDD.In the fermented product of bacterial strain GW03, DMB, SY-1 and MBDD total amount is up to 1214.2mg/kg, be 1.7-7.5 times of other bacterial strain, and the output (552.6mg/kg) of the output of SY-1 (503.8mg/kg) and MBDD all rank the first in 8 samples.The output (157.8mg/kg) of DMB is number two name in 8 samples.Display bacterial strain GW03 has the ability simultaneously producing three kinds of active compounds such as a large amount DMB, SY-1 and MBDD.
DMB, SY-1 and MBDD content of the different Antrodia camphorata bacterial strain of table 1. after grain of rice solid fermentation
Unit (mg/kg)
(3) content of DMB, SY-1 and MBDD of Antrodia camphorata Semen Fagopyri Esculenti fermented product
Antrodia camphorata bacterial strain GW03 can be incubated at the cereal matrix beyond the grain of rice, and DMB, SY-1 and MBDD output after fermenting 30 days is also higher than other bacterial strain.Different Antrodia camphorata strain culturing in buckwheat, is analyzed fermentation after 30 days by this experiment, and the content of three kinds of active compounds such as DMB, SY-1 and MBDD contained by fermented product, result is as shown in table 2.The grain of rice fermented product of bacterial strain GW03 is compared, SY-1 contained in the Semen Fagopyri Esculenti fermented product of bacterial strain GW03 reduces, but the content of DMB and MBDD increases, and DMB, SY-1 and MBDD total amount is up to 1555.1mg/kgDMB, SY-1 and MBDD, the 2.3-45.5 being other strain fermentation sample doubly, also shows bacterial strain GW03 and has the ability simultaneously producing three kinds of active compounds such as a large amount DMB, SY-1 and MBDD.
DMB, SY-1 and MBDD content of the different Antrodia camphorata bacterial strain of table 2. after buckwheat solid fermentation
Unit (mg/kg)
Above result display, three kinds of active compound output such as DMB, SY-1 and MBDD of Antrodia camphorata bacterial strain of the present invention are all front three of all test strain, and the total amount of three kinds of compounds is about 1,214-1,555mg/kg, far above other bacterial strain (about 34-690mg/kg), be 1.7-45 times of other bacterial strain.Show three kinds of active ingredients such as DMB, SY-1 and MBDD that described bacterial strain can produce a large amount simultaneously.
The form of embodiment three, bacterial strain GW03 and Molecular Identification
(1) morphologic observation:
Bacterial strain GW03 is inoculated in PDA (U.S. Di Fuke) and MEA (U.S. Di Fuke) substratum, cultivates 35 days at 25 DEG C, all can produce the rotten fragrance of honey peach fruit; At MEA flat board colony diameter upper 35 day on average about 55-60mm, bacterium colony is from the inside to the outside in white, light orange extremely light safran, and aerial hyphae is by fine hair shape, powdery, cotton-wool to ulotrichy, and the bacterium colony back side is light safran; At PDA flat board colony diameter upper 35 day on average about 45-50mm, bacterium colony is from the inside to the outside in faint yellow, safran extremely light safran, and aerial hyphae is by fine hair shape, powdery, cotton-wool to ulotrichy, and the bacterium colony back side is faint yellow.
Bacterial strain GW03 observes under an optical microscope, can observe a large amount of conidium.Conidium ovalize, shaft-like, column to long column shape, size is about (4-) 5 ~ 15 (-27) × (1.5-) 2 ~ 4.5 μm.Mycelia about (1.5-) 2 ~ 3.5 (-4) μm of fresh culture bacterium colony wide, tool clasp body (clampconnection), and more ripe mycelia outer wall is visible safran crystallisate once in a while.
(2) Molecular Identification:
1. gene order compare of analysis result
Bacterial strain GW03 carries out the analysis of rDNAITS1-5.8S-ITS2 partial sequence, sequence overall length is 819bD, sequence has the situation (R=A or G) of the sudden change of 3 bases, with the sequencing results on NCBI gene pool (GenBank) closest to Antrodia camphorata (Antrodiacamphorata) (AY378095) (=Antrodia camphorata (Antrodiacinnamomea)), the fraction of coverage of sequence is 100% (819/819), and similarity is 99% (816/819); Carry out the analysis of LSUrDNAD1-D3 partial sequence in addition, sequence overall length is 955bp, sequence has the situation (Y=T or C) of the sudden change of 1 base, with the sequencing results on NCBI gene pool closest to Antrodia camphorata (Taiwanofunguscamphoratus) (AY333841) (=Antrodia camphorata (Antrodiacinnamomea)), the fraction of coverage of sequence is 100% (955/955), and similarity is 99% (954/955).
According to morphological specificitys such as colonial morphology, color, conidium and clasp bodies, and according to its special odor, with rDNAITS1-5.8S-ITS2 and LSUrDNAD1-D3 the sequencing results, identify that described sample is the mycelium of Antrodia camphorata (Antrodiacinnamomea).
The ITS1/ITS2 sequence of bacterial strain GW03:
SEQIDNO：1
TTGTACACACCGCCCGTCGCTACTACCGATTGAATGGCTTAGTGAGGTCTTGGGAT TGGCTTCGGGGAGCCGGCAACGGCATCCTGTTGCTGAGAACTTGATCAAACTTGGT CATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGA TCATTATTGTATTTGAAAGGGGTTGTAGCTG
rcCTCCTCTTGAAAAGGGGGGAGGTATGTGCACACCTCTGTTCATTCATATTCTCTC ACACCTGTGCATGCTTTGTAGGTTGGTTTTGAATGGTTGTCTTCTCTGATGGAGAC AGCTGTTTTGACCTTCCTATGTTTTTTAAATTGACTCCGTATCAGTTACAGAATGT ATGTTGCGTGTAACGCATATTGTATAACTTTCAGCAACGGATCTCTTGGCTCTCGC ATCGATGAAGAACAC
rgCGAAATGTGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGA ACGCACCTTGCGCTCCTTGGTATTCTGAGGAGCATGCCTGTTTGAGTGTCATG
rlSU (D1-D3) sequence of AATTATCAACCCTTTTGACTTTTTGTTGAATGGGCTTGGATTTGGAGGGTTAAATT GCTGGCTCTTTTTTTTGATTCAGCTCCTCTTGAATGCATTAGCTTGAACCCTTGTG GATTGACCTTATCGGTGTGATAGTCATCTATGCCGTGGTTGTCTGAGGTGGGATCG GCTTCTAATGGTGCAAGTCCCTTCAGGGGGATGATTTTCTAATGACCTTCTGACCT CAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAATAAGC bacterial strain GW03:
SEQIDNO：2
AGCATATCAATAAGCGGAGGAAAAGAAACTAACAAGGATTCCCCTAGTAACTGCGAGTGAAGCGGGATAAGCTCAAATTTAAAATCTGGTGGGTGTTTGGCCTGCCCGAGTTGTAGTCTGGAGAAGTGCTTTCTGTGCTGAACCGTGTACAAGTCTCTTGGAACAGAGCGTCATAGAGGGTGAGAATCCCGTCTTTGACACGGACTATCAGTGCTTTGTGATGCGCTCTCAATGAGTCGAGTTGTTTGGGAATGCAGCTCAAAATGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGTGAACAAGTACCGTGAGGGAAAGATGAAAAGCACTTTGGAAAGAGAGTTAAACAGTACGTGAAATTGCTGAAAGGGAAACACTTGAAGTCAGTCGCGTTGA
YCGGAGCTCAACCTTGCTTTTTTGGGCTTGGTGCACTTTCTGGTTGACGGGTCAGCATCAATTTTGACTGTTGGAGAAGGGTTGGGGAAATGTGGCACCTTCGGGTGTGTTATAGCCCCCAGTCACATACAATGGTTGGGATTGAGGAACTCAGCACACCTTTATGGTCAGGGTTTTTTTAACCTATGTTATTGTGCTTAGGATGCTGGCGTAATGGCTTTAAATGACCCGTCTTGAAACACGGACCAAGGAGTCTAACATGCCTGCGAGTGTTTGGGTGGCAAACCCGAGTGCGTAATGAAAGTGAAAGTTGGGACCTCTGTTGTGGGAGGGCACCGATGCCCGGACCAGACCTTTTGTGATGGATCCGCGGTAGAGCATGTATGTTGGGACCCGAAAGATGGTGAACTATGCCTGAATAGGGTGAAGCCAGAGGAAACTCTGGTGGAGGCTCGTAGCGATTCTGACGTGCAAATCGATCGTCAAATTTGGGTATAGGGGCGAAAGACTAATCGAACCATCTAGTAGCTGGTTCCTGCCGA
2. with the sequence alignment analysis result of known Antrodia camphorata kind
Separately by GW03 and the open bacterial strain BCRC35396 that Foodstuff Industrial Development Inst. of Financial Group Legal Persons's Biological resources are preserved and research centre (BCRC) preserves, BCRC35398, BCRC35716, BCRC36401, BCRC36711, BCRC36795, BCRC37609, BCRC37616, BCRC37848, BCRC37849, BCRC37850, BCRC37889, BCRC37890, BCRC37891, BCRC37893, BCRC37894, BCRC37941 counts 17 strain bacterial strains and carries out ITS partial sequence and the comparison of LSUD1-D3 partial sequence.
Find that ITS sequence has 8 core candy body deoxyribose nucleic acid sequences and has otherness, wherein GW03 and other 17 strain bacterial strain above-mentioned have the sequence difference in 3-5 site respectively, and similarity is 99.1%-99.4%.LSUD1-D3 segment portion gene order also has 10 core candy body deoxyribose nucleic acid sequences to have otherness, and wherein GW03 and other 17 strain bacterial strain above-mentioned have the sequence difference in 1-4 site respectively, and similarity is 99.4%-99.8%.
Then this two fragment genes sequence is together in series with Notepad software by all bacterial strain rDNA gene orders (ITS and LSUD1-D3) again, and turn shelves with DAMBE software, Mega4 software is used to do Genetic relationship, with maximum brief several method (MaximunParsimonyMethod, MP) tree that develops is constructed, and repeat to calculate sibship with 1000 duplicate sampling estimations technique (Bootstrap) and set holding strength on each node, the evolution tree of last gained represents with synthesizing tree (consensustree), obtain Antrodia camphorata bacterial strain sibship tree as shown in Figure 1.
The sequence being set all openly bacterial strains of display GW03 and BCRC by ITS with LSUD1-D3 two fragment gene segment portion the sequencing results and sibship is not identical.
GW03 Antrodia camphorata bacterial strain is deposited at Foodstuff Industrial Development Inst. of Financial Group Legal Persons's Biological resources on November 14th, 2013 and preserves and research centre, and depositing numbering is BCRC930164; And described Antrodia camphorata bacterial strain is also deposited at German Culture Collection (DeutscheSammlungvonMikroorganismenundZellkulturenGmbH (DSMZ) according to budapest treaty (BudapestTreaty) on February 24th, 2014, MascheroderWeg1b, D38124, Braunschweig, Germany), depositing numbering is DSM28462.
Claims (13)
1. an Antrodia camphorata strain isolated, its comprise nucleotide sequence as shown in SEQIDNO:1 ITS sequence and as shown in SEQIDNO:2 the LSUD1-D3 sequence of nucleotide sequence.
2. Antrodia camphorata strain isolated according to claim 1, described Antrodia camphorata strain isolated is deposited at German Culture Collection and deposits the bacterial strain being numbered DSM28462, or be deposited at German Culture Collection and deposit the mutant strain that the bacterial strain being numbered DSM28462 has identical in fact feature.
3. prepare a method for Antrodia camphorata cultured products, it comprises and is inoculated in applicable substratum by Antrodia camphorata strain isolated according to claim 1 and 2, and carries out under proper condition cultivating to obtain described cultured products.
4. method according to claim 3, wherein said substratum is solid medium or liquid nutrient medium.
5. method according to claim 4, wherein said substratum is the solid medium based on cereal or the solid medium based on foodstuffs industry by product.
6. method according to claim 5, wherein said cereal is rice, barley, buckwheat, Chinese sorghum, heart of a lotus seed benevolence, wheat, rye, oat or its combination.
7. method according to claim 6, wherein cultivate in the solid medium based on rice in prepared Antrodia camphorata cultured products, with the total weight of described Antrodia camphorata cultured products, 2,3-dimethoxy-5-methyl-p-benzoquinone (DMB), 4,7-dimethoxy-5-methyl isophthalic acid, 3-benzodioxole (SY-1) and 5-methyl-benzo [1,3] dioxole-4, the ultimate production of 7-glycol (MBDD) is greater than about 1,200mg/kg.
8. method according to claim 6, wherein cultivates in prepared Antrodia camphorata cultured products in the solid medium based on buckwheat, and with the total weight of described Antrodia camphorata cultured products, the ultimate production of DMB, SY-1 and MBDD is greater than about 1,500mg/kg.
9. method according to claim 5, wherein said foodstuffs industry by product is bagasse, starch, wheat bran, rice bran, corn stalk, coffee grounds or its combination.
10., for the preparation of a method of DMB, SY-1 and/or MBDD, it comprises isolates DMB, SY-1 and/or MBDD from the Antrodia camphorata cultured products prepared by the method according to claim arbitrary claim 3 to 9.
11. 1 kinds of Antrodia camphorata cultured products, it is prepared by the method according to claim arbitrary in claim 3 to 9.
12. 1 kinds of compositions, it includes the Antrodia camphorata cultured products according to claim 11 of effective amount.
13. compositions according to claim 12, it is medical composition or food compositions.
Priority Applications (1)
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Cited By (6)
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| CN107304407A (en) * | 2016-04-20 | 2017-10-31 | 观文农业生技股份有限公司 | Medical grade anticancer novelty Antrodia camphorata bacterial strain |
| CN108611268A (en) * | 2018-05-08 | 2018-10-02 | 浙江海正药业股份有限公司 | A kind of production spore device for simulating wild environment and its application in Antrodia camphorata |
| CN108785303A (en) * | 2017-05-02 | 2018-11-13 | 科捷生物科技股份有限公司 | Prevent and/or treat the medical composition and application thereof of liver fibrosis |
| CN109757300A (en) * | 2019-03-14 | 2019-05-17 | 安佑生物科技集团股份有限公司 | A kind of breeding method of Antrodia camphorata and its application in animal feed |
| US10945991B2 (en) | 2018-10-29 | 2021-03-16 | Cojet Biotech Inc. | Use and pharmaceutical composition for liver fibrosis prevention and/or treatment |
| CN114214205A (en) * | 2021-11-10 | 2022-03-22 | 王毅 | Antrodia cinnamomea symbiotic fungus AcEF007 and separation method thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107304407A (en) * | 2016-04-20 | 2017-10-31 | 观文农业生技股份有限公司 | Medical grade anticancer novelty Antrodia camphorata bacterial strain |
| CN108785303A (en) * | 2017-05-02 | 2018-11-13 | 科捷生物科技股份有限公司 | Prevent and/or treat the medical composition and application thereof of liver fibrosis |
| CN108785303B (en) * | 2017-05-02 | 2021-03-12 | 科捷生物科技股份有限公司 | Pharmaceutical composition for preventing and/or treating hepatic fibrosis and application thereof |
| CN108611268A (en) * | 2018-05-08 | 2018-10-02 | 浙江海正药业股份有限公司 | A kind of production spore device for simulating wild environment and its application in Antrodia camphorata |
| CN108611268B (en) * | 2018-05-08 | 2022-02-22 | 浙江海正药业股份有限公司 | Spore production device for simulating wild environment and application of spore production device in antrodia camphorata |
| US10945991B2 (en) | 2018-10-29 | 2021-03-16 | Cojet Biotech Inc. | Use and pharmaceutical composition for liver fibrosis prevention and/or treatment |
| CN109757300A (en) * | 2019-03-14 | 2019-05-17 | 安佑生物科技集团股份有限公司 | A kind of breeding method of Antrodia camphorata and its application in animal feed |
| CN114214205A (en) * | 2021-11-10 | 2022-03-22 | 王毅 | Antrodia cinnamomea symbiotic fungus AcEF007 and separation method thereof |
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| CN105132288B (en) | 2018-08-10 |
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