CN104830716B - Microbacterium and application thereof - Google Patents
Microbacterium and application thereof Download PDFInfo
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- CN104830716B CN104830716B CN201510178094.6A CN201510178094A CN104830716B CN 104830716 B CN104830716 B CN 104830716B CN 201510178094 A CN201510178094 A CN 201510178094A CN 104830716 B CN104830716 B CN 104830716B
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- microbacterium
- salvia miltiorrhiza
- hairy root
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- 241001467578 Microbacterium Species 0.000 title claims abstract description 19
- DOUMFZQKYFQNTF-WUTVXBCWSA-N (R)-rosmarinic acid Chemical compound C([C@H](C(=O)O)OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-WUTVXBCWSA-N 0.000 claims abstract description 30
- 241001467579 Microbacterium arborescens Species 0.000 claims abstract description 28
- 241000304195 Salvia miltiorrhiza Species 0.000 claims abstract description 24
- 235000011135 Salvia miltiorrhiza Nutrition 0.000 claims abstract description 23
- 239000002253 acid Substances 0.000 claims abstract description 17
- ZZAFFYPNLYCDEP-HNNXBMFYSA-N Rosmarinsaeure Natural products OC(=O)[C@H](Cc1cccc(O)c1O)OC(=O)C=Cc2ccc(O)c(O)c2 ZZAFFYPNLYCDEP-HNNXBMFYSA-N 0.000 claims abstract description 15
- DOUMFZQKYFQNTF-MRXNPFEDSA-N rosemarinic acid Natural products C([C@H](C(=O)O)OC(=O)C=CC=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-MRXNPFEDSA-N 0.000 claims abstract description 15
- TVHVQJFBWRLYOD-UHFFFAOYSA-N rosmarinic acid Natural products OC(=O)C(Cc1ccc(O)c(O)c1)OC(=Cc2ccc(O)c(O)c2)C=O TVHVQJFBWRLYOD-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000009825 accumulation Methods 0.000 claims abstract description 8
- 239000006228 supernatant Substances 0.000 claims description 7
- 235000015097 nutrients Nutrition 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 2
- 241000196324 Embryophyta Species 0.000 abstract description 6
- 240000007164 Salvia officinalis Species 0.000 description 18
- 239000005712 elicitor Substances 0.000 description 18
- 235000005412 red sage Nutrition 0.000 description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 241000894006 Bacteria Species 0.000 description 15
- 239000006916 nutrient agar Substances 0.000 description 10
- 238000004321 preservation Methods 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 9
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000000463 material Substances 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000009629 microbiological culture Methods 0.000 description 5
- 150000007965 phenolic acids Chemical class 0.000 description 5
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 235000015278 beef Nutrition 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000004224 protection Effects 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- AIGAZQPHXLWMOJ-UHFFFAOYSA-N Tanshinone I Chemical compound C1=CC2=C(C)C=CC=C2C(C(=O)C2=O)=C1C1=C2C(C)=CO1 AIGAZQPHXLWMOJ-UHFFFAOYSA-N 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
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- 238000002360 preparation method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229930000044 secondary metabolite Natural products 0.000 description 2
- QAIPRVGONGVQAS-DUXPYHPUSA-N trans-caffeic acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-DUXPYHPUSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- PAFLSMZLRSPALU-MRVPVSSYSA-N (2R)-3-(3,4-dihydroxyphenyl)lactic acid Chemical compound OC(=O)[C@H](O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-MRVPVSSYSA-N 0.000 description 1
- ACEAELOMUCBPJP-UHFFFAOYSA-N (E)-3,4,5-trihydroxycinnamic acid Natural products OC(=O)C=CC1=CC(O)=C(O)C(O)=C1 ACEAELOMUCBPJP-UHFFFAOYSA-N 0.000 description 1
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 description 1
- 235000013479 Amaranthus retroflexus Nutrition 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 206010007247 Carbuncle Diseases 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 240000005674 Ceanothus americanus Species 0.000 description 1
- 235000014224 Ceanothus americanus Nutrition 0.000 description 1
- 235000001904 Ceanothus herbaceus Nutrition 0.000 description 1
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 description 1
- PAFLSMZLRSPALU-QMMMGPOBSA-N Danshensu Natural products OC(=O)[C@@H](O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-QMMMGPOBSA-N 0.000 description 1
- 206010013786 Dry skin Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 235000002789 Panax ginseng Nutrition 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 208000001431 Psychomotor Agitation Diseases 0.000 description 1
- 206010038743 Restlessness Diseases 0.000 description 1
- 241001529742 Rosmarinus Species 0.000 description 1
- 235000017276 Salvia Nutrition 0.000 description 1
- PAFLSMZLRSPALU-UHFFFAOYSA-N Salvianic acid A Natural products OC(=O)C(O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-UHFFFAOYSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 229930183118 Tanshinone Natural products 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 244000144987 brood Species 0.000 description 1
- 235000004883 caffeic acid Nutrition 0.000 description 1
- 229940074360 caffeic acid Drugs 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000005961 cardioprotection Effects 0.000 description 1
- 229930016911 cinnamic acid Natural products 0.000 description 1
- 235000013985 cinnamic acid Nutrition 0.000 description 1
- QAIPRVGONGVQAS-UHFFFAOYSA-N cis-caffeic acid Natural products OC(=O)C=CC1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-UHFFFAOYSA-N 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 150000004141 diterpene derivatives Chemical class 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000009229 glucose formation Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 230000004112 neuroprotection Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000001053 orange pigment Substances 0.000 description 1
- 230000003119 painkilling effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 150000003648 triterpenes Chemical class 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
- 238000004073 vulcanization Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
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- 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
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Pest Control & Pesticides (AREA)
- Dentistry (AREA)
- Plant Pathology (AREA)
- Agronomy & Crop Science (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to one plant of microbacterium and its application in Hairy Root Cultures of Salvia miltiorrhiza cultivating system.Specifically：It is Microbacterium arborescens LG2 the invention discloses a kind of microbacterium, its preserving number is：CCTCC NO:M 2015083.The purposes of the microbacterium is：Promote the accumulation of Rosmarinic acid and tanshin polyphenolic acid B in Hairy Root Cultures of Salvia miltiorrhiza；And promote Hairy Root Cultures of Salvia miltiorrhiza weightening.
Description
Technical field
The present invention relates to one plant of microbacterium and its application in Hairy Root Cultures of Salvia miltiorrhiza cultivating system.
Background technology
The red sage root (Salvia miltiorrhiza Bunge) also known as red ginseng, radix salviae miltiorrhizae, red root etc., it is labiate, its
Dry root and rhizome can be used as medicine.Its record used as medicinal material, sees earliest《Sheng Nong's herbal classic》, then beam court, the Tang Dynasty,
Also there is the record being used as medicine to the red sage root in the traditional Chinese medicine works of five generations and the Ming Dynasty.Be distributed in we Hebei, Shanxi, Shaanxi,
, also there is the distribution of the red sage root on the ground such as Shandong, Henan, Jiangsu, Zhejiang, Anhui, Jiangxi and Hunan in Japan.There is the red sage root promoting blood circulation to adjust
Through, stasis-dispelling and pain-killing, cool blood to disappear carbuncle, relieving restlessness that clears away heart-fire, the effect such as nourishing blood and tranquilization.Red sage root mainly containing fat-soluble Diterpenoids from bulbus and
Water miscible phenolic acid components, also containing flavonoids, triterpenes, the other active components such as sterol.Originally people are to fat-soluble tanshinone
The pharmacological research of class material is relatively more, but water miscible liposoluble ingredient is also increasingly valued by people now.It is water-soluble
The phenolic acid of property mainly has caffeic acid, Rosmarinic acid, salviandic acid A, tanshin polyphenolic acid B, cinnamic acid, danshensu etc..Research shows
Water soluble ingredient have different from liposoluble constituent activity (including anti-oxidant, neuroprotection, cardioprotection, protection blood vessel, control
Treat type-II diabetes and anti-apoptotic etc.).Therefore the market demand of the red sage root increasingly increases, and the red sage root about 80% is wild money
Source, and effective active composition is unstable, growth cycle is long, far can not meet the demand in market.Again because people are excessive for a long time
Excavation causes resource increasingly atrophy；Artificial cultivation face again no breeding, quality deterioration, production cycle length, seed in spite of illness with agricultural chemicals
Residual is exceeded, grows under various circumstances, and its effective component composition and content have very big difference, unstable etc. on clinical treatment asked
Topic.The culture technique of hairy root can largely produce red rooted salvia resource, improvement red sage root quality and greatly improve red sage root drug effect into
Divide Yield and quality and stability.But the Hairy Root Cultures of Salvia miltiorrhiza culture systems set up at present are low there are still active yield
Problem, so as to influence the further amplification and industrialization of culture.
Therefore, screening, which can more effectively improve the elicitor of Hairy Root Cultures of Salvia miltiorrhiza active component content, has very important meaning
Justice.The material that plant can be induced to produce secondary metabolites is called elicitor (Elicitor), and such as yeast extract biology is lured
The abiotic inducible factor such as inducement and metal ion.Under the induction of biological or abiotic component, the resistance meeting of plant
Enhancing, while promoting the accumulation of botanical secondary metabolite.Microbial elicitors are accessed into hairy root, are to improve and obtain the red sage root
The brand-new route of hairy root active constituent content, so that what the quality Control and the red sage root that solve the current red sage root faced in cultivating
Problem.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of microbacterium, the bacterial strain can promote rosemary in Hairy Root Cultures of Salvia miltiorrhiza
The accumulation of acid and tanshin polyphenolic acid B.
In order to solve the above-mentioned technical problem, the present invention provides a kind of microbacterium, is Microbacterium
Arborescens LG2, its preserving number is：CCTCC NO:M 2015083.
Present invention also offers the purposes of above-mentioned microbacterium：Promote the product of Rosmarinic acid and tanshin polyphenolic acid B in Hairy Root Cultures of Salvia miltiorrhiza
It is tired.
It is used as the improvement of the purposes of the microbacterium of the present invention：Promote Hairy Root Cultures of Salvia miltiorrhiza weightening.
Microbacterium Microbacterium arborescens LG2 are that inventor is dilute using flat board from red sage root plant
One plant of isolated endogenetic bacteria of interpretation of the law, identified its zymotic fluid and thalline, which have, promotes Hairy Root Cultures of Salvia miltiorrhiza Rosmarinic acid and pellet
The effect of phenolic acid B Composition accumulation.
Separation method is：
1) after, the root of the red sage root is handled through surface sterilization, add sterilized water and be ground dilution, be coated on nutrient agar
On, cultivated 1~3 day in 28~32 DEG C；
Nutrient agar is formulated：Peptone 10g, beef extract 3g, NaCl 5g, agar 18g and distilled water 1000mL, pH=7.2
~7.4.
2), picking single bacterium falls within the line purifying of identical nutrient agar panel, is cultivated 1~3 day in 28~32 DEG C；
3), repeat step 2), until obtaining pure culture.
The preservation information of the microbacterium of the present invention is specific as follows：Microbacterium arborescens LG2, preservation
Unit：China typical culture collection center, preservation address：Wuhan, China Wuhan University, deposit number：CCTCC NO:M
2015083, March 4 2015 preservation time.
Microbacterium -- Microbacterium arborescens LG2 (the CCTCC NO of the present invention:M 2015083)
Bacterium colony is rounded after nutrient agar culture 24h, shows smooth, bacterium colony is smaller, neat in edge, produces orange pigment.
The bacterial strain Gram-positive, aerobic, thin rod shape does not produce brood cell；Catalase positive, oxidase negative；
The well-grown in the environment that growth temperature is 20~40 DEG C, pH value is 6.0~8.0；Energy metabolism glucose production acid；Produce vulcanization
Hydrogen, energy gelatin hydrolysate, it is impossible to hydrolysis starch.
Microbacterium arborescens LG2 (the CCTCC NO:M 2015083) 16S rDNA sequences point
Analysis shows that the similitude of the bacterial strain Microbacterium arborescens with being recorded in Genbank international data centers is
99%.
Microbacterium arborescens LG2(CCTCC NO:M 2015083) in Hairy Root Cultures of Salvia miltiorrhiza culture body
Promote the effect of Rosmarinic acid and tanshin polyphenolic acid B accumulation in system as elicitor.
Microbacterium arborescens LG2 (the CCTCC NO of the present invention:M 2015083), the induction of its thalline
Son can promote hairy root rosmarinic acid contents to improve 183%, and content of danshinolic acid B improves 69%；Bacterium solution elicitor can promote hairy
Root rosmarinic acid contents improve 7%, and content of danshinolic acid B improves 105%, and fresh weight increases by 20%.It is nontoxic to people and animals, to ring
Border is not polluted.It can be applied in Hairy Root Cultures of Salvia miltiorrhiza culture, produce certain economic benefit.
The present invention microbacterium for promote Hairy Root Cultures of Salvia miltiorrhiza phenolic acid accumulate when, only need by
Microbacterium arborescens LG2(CCTCC NO:M 2015083) in nutrient broth medium cultivate after, from
The supernatant and thalline that the heart is collected can be connected in Hairy Root Cultures of Salvia miltiorrhiza culture medium respectively as elicitor, you can promote Hairy Root Cultures of Salvia miltiorrhiza
The increase of middle Rosmarinic acid and content of danshinolic acid B.
The beneficial effects are mainly as follows：The Microbacterium arborescens provided using the present invention
LG2(CCTCC NO:M 2015083) fermented supernatant fluid and thalline elicitor, be added in Hairy Root Cultures of Salvia miltiorrhiza cultivating system i.e.
The content of Rosmarinic acid and tanshin polyphenolic acid B can be improved, and then improves the content of total phenolics class, the Hairy Root Cultures of Salvia miltiorrhiza induced can be used for
The extraction of Rosmarinic acid and tanshin polyphenolic acid B list product.
Embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This.
Embodiment 1, Microbacterium arborescens LG2 (CCTCC NO:M 2015083) side of being separately cultured
Method is as follows：
1st, wild Salvia miltiorrhiza plant is gathered in Sichuan Zhong Jiang, 25KHz ultrasonications 5 minutes after root is cleaned；
2nd, aseptically, the material obtained by step 1 is rinsed 3 times with sterile deionized water, and respectively with 75% (v/
V) alcohol and 3% (m/V) liquor natrii hypochloritis's surface sterilization, so as to kill material to be tested surface microorganism；
3rd, in gnotobasis, by the aseptic water washing 3 times of the material to be tested obtained by step 2, last 1 punching of 100 μ l is taken
The aseptic water washing liquid coating washed is inoculated on nutrient agar, after 30 DEG C of culture 24h, carries out sterile checking.
Such as produced without bacterium colony, then continue follow-up step 4；Produced if any bacterium colony, then return to step 2 continues at surface sterilization
Reason.
Nutrient agar is：Peptone 10g, beef extract 3g, NaCl 5g, agar 18g and distilled water 1000mL, pH
=7.2~7.4.
4th, in super-clean bench, the red sage root 1~2g of sample handled well is taken, is fully ground in sterile mortar, and add 5mL
Sterilized water, is mixed, and is stood 15min, is taken 100 μ l supernatant dilution spreads in nutrient agar, put 28~32 DEG C (preferable 30
DEG C) cultivate 1~3 day；
5th, the single bacterium kind produced in picking step 4 is put in same medium (nutrient agar) line purifies and separates
Cultivated 1~3 day in 28~32 DEG C (preferable 30 DEG C), continue picking single bacterium colony；
Aforesaid operations are repeated untill pure culture is obtained.
6th, single bacterium colony resulting in step 5 is subjected to 16s rDNA sequencings, through sequence alignment, determines bacterial strain, and
Preservation.Obtain Microbacterium arborescens LG2 (CCTCC NO:M 2015083).
Embodiment 2, Microbacterium arborescens LG2 (CCTCC NO:M 2015083) elicitor preparation
Method, is followed the steps below successively：
1、Microbacterium arborescens LG2(CCTCC NO:M 2015083) transfer it is oblique in nutrient agar
Face, 30 DEG C are cultivated 2 days；
Nutrient agar slopes are：Peptone 10g, beef extract 3g, NaCl 5g, agar 18g and distilled water 1000mL, pH=
7.2~7.4.
2nd, with the slant strains obtained by the ring step 1 of oese picking one, it is inoculated in the nutrient broth medium containing 50ml
In 250ml triangular flasks, 72h is cultivated with 220rpm in 28 DEG C, Microbacterium arborescens LG2CCTCC M are obtained
2015083 fermenation raw liquids；
Nutrient broth medium is：Peptone 10g, beef extract 3g, NaCl 5g and distilled water 1000mL, pH=7.2~
7.4。
Above-mentioned steps 1 and step 2 are cultivated under the conditions of natural light.
3rd, step 2 gained fermenation raw liquid is taken, in 4 DEG C, 12000rpm centrifuges 2min, takes supernatant to cross 0.22 μm of miillpore filter,
Gained filtrate (is also known as bacterium for Microbacterium arborescens LG2 CCTCC M 2015083 supernatant elicitor
Liquid elicitor).
Bacterial sediment deionized water obtained by above-mentioned centrifugation is cleaned 3 times, is resuspended in 50mL deionized waters, 121 DEG C, high pressure is gone out
Bacterium 20min.Thallus suspension liquid after sterilizing is filtered by vacuum with three layers of 0.22 μm of filter membrane, and gained filtrate is thalline elicitor.
Embodiment 3, Microbacterium arborescens LG2 (CCTCC NO:M 2015083) to red sage root hairy
The promotion experiment of root Rosmarinic acid accumulation：
1st, Hairy Root Cultures of Salvia miltiorrhiza (female root) 0.3g is inoculated in the 250ml triangular flasks in the culture mediums of 6.7-V containing 100ml, 25
DEG C, 100rpm lucifuges culture 18 days；Based on thing；
Experimental group 1：1.5ml Microbacterium arborescens LG2 (CCTCC are added in every bottle of base
NO:M2015083) supernatant elicitor (gained of embodiment 2, bacterium solution elicitor), continuing the same terms, (in 25 DEG C, 100rpm is kept away
Optical culture) cultivate 6 days；It is used as experimental group 1；
Experimental group 2：1.5ml Microbacterium arborescens LG2 (CCTCC are added in every bottle of base
NO:M2015083) thalline elicitor (gained of embodiment 2), continues the same terms (25 DEG C, the culture of 100rpm lucifuges) and cultivates 6 days；
It is used as experimental group 2；
Negative control：1.5ml sterile vegetative broth bouillon is added in every bottle of base, continues the same terms (25
DEG C, the culture of 100rpm lucifuges) cultivate 6 days；It is used as negative control.
Above-mentioned every kind of experimental group and negative control set 5 repetitions respectively；
Hairy Root Cultures of Salvia miltiorrhiza obtained by every kind of experimental group and negative control is proceeded as follows respectively：
2nd, culture is cleaned the hairy root that step 1 is obtained three times with distilled water after terminating, and with blotting paper suck dry moisture, is claimed
Fresh weight (fresh weight, FW)；The hairy root for having claimed fresh weight is put into baking oven afterwards, 55 DEG C of dryings to constant weight determine dry weight
(dry weight, DW).
3rd, the red sage root dried to constant weight in right amount, grind into powder are taken.0.5g dry powder is taken to add 2.5mL 70% (v/v) first
Ultrasonic extraction 60min in alcohol, 120W supersonic wave cleaning machines (water temperature can not be too high in ultrasonic procedure, can be suitably added ice cube, i.e.
Control temperature≤20 DEG C), extract crosses 0.45 μm of miillpore filter, and Rosmarinic acid is carried out with the method for high performance liquid chromatography (HPLC)
With the measure of content of danshinolic acid B.
Measurement result is as follows：
Microbacterium arborescens LG2 (CCTCC NO are added in experimental group:M 2015083) thalline lures
Guide can promote hairy root rosmarinic acid contents to improve 183%, and content of danshinolic acid B improves 69%；Adding bacterium solution elicitor can promote
Enter hairy root rosmarinic acid contents and improve 7%, content of danshinolic acid B improves 105%, fresh weight increase by 20%.It is specific as described in Table 1.
Comparative example, the bacterial strain work closely planted from following Microbacterium arborescens bacterial strains and this symbolic animal of the birth year respectively
For comparative example, specific bacterial strain is as follows：
The Microbacterium that A, the Code Number of Germany Microbiological Culture Collection Center DSMZ preservations are DSM 20754
arborescens；
B, the Code Number CICC 23772 of Chinese industrial Microbiological Culture Collection administrative center CICC preservations
Microbacterium arborescens；
C, the Code Number CICC 22616 of Chinese industrial Microbiological Culture Collection administrative center CICC preservations
Microbacterium arborescens；
D, the Code Number CICC 20196 of Chinese industrial Microbiological Culture Collection administrative center CICC preservations
Microbacterium arborescens；
The Microbacterium that E, the Code Number of Germany Microbiological Culture Collection Center DSMZ preservations are DSM 8607
dextranolyticum；
By Microbacterium arborescens LG2 (the CCTCC NO in above-mentioned bacterial strains alternate embodiment 2:M
2015083) elicitor preparation is carried out, the bacterium solution of gained, thalline elicitor are detected according to embodiment 3, acquired results such as table 1
It is described.
Table 1.Microbacterium arborescens LG2CCTCC M 2015083 and comparative example are to Hairy Root Cultures of Salvia miltiorrhiza
The influence of phenolic acid accumulation
It is laboratory mean values ± standard deviation in table.
Finally, in addition it is also necessary to it is noted that listed above is only several specific embodiments of the invention.Obviously, this hair
It is bright to be not limited to above example, there can also be many deformations.One of ordinary skill in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (3)
1. microbacterium, it is characterized in that：For Microbacterium arborescens LG2, its preserving number is：CCTCC NO:
M2015083。
2. the purposes of microbacterium as claimed in claim 1, it is characterized in that：Promote Rosmarinic acid and danshinolic acid in Hairy Root Cultures of Salvia miltiorrhiza
B accumulation.
3. the purposes of microbacterium according to claim 2, it is characterized in that：The microbacterium is cultivated in nutrient broth medium
The supernatant being collected by centrifugation afterwards promotes Hairy Root Cultures of Salvia miltiorrhiza weightening.
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| CN104224811A (en) * | 2013-06-17 | 2014-12-24 | 天士力制药集团股份有限公司 | Salvia miltiorrhiza medicine and preparation method thereof |
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| CN103834685A (en) * | 2013-11-29 | 2014-06-04 | 陕西师范大学 | Carrier capable of simultaneously improving rosmarinic acid and salvianolic acid B content of red sage root and use thereof |
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| "丹参毛状根中多种丹酚酸类化合物的鉴定与生物合成";赵淑娟等;《药学学报》;20111112;第46卷(第11期);全文 * |
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