CN102154185A - Eleutherococcous senticosus endosymbiotic bacteria capable of producing coffeic acid - Google Patents
Eleutherococcous senticosus endosymbiotic bacteria capable of producing coffeic acid Download PDFInfo
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- CN102154185A CN102154185A CN 201110080188 CN201110080188A CN102154185A CN 102154185 A CN102154185 A CN 102154185A CN 201110080188 CN201110080188 CN 201110080188 CN 201110080188 A CN201110080188 A CN 201110080188A CN 102154185 A CN102154185 A CN 102154185A
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- eleutherococcous
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Abstract
The invention discloses eleutherococcous senticosus endosymbiotic bacteria capable of producing coffeic acid and relates to endosymbiotic bacteria. The eleutherococcous senticosus endosymbiotic bacteria capable of producing the coffeic acid are Bacillus subtilis CX33, belongs to the Bacillus, is preserved in the China center for type culture collection (CCTCC) and has a preservation number of CCTCC NO:M2011078, wherein the preservation address is Wuhan University in Wuhan city; and the preservation date is March 22nd in 2011. Metabolites of the eleutherococcous senticosus endosymbiotic bacteria capable of producing the coffeic acid, disclosed by the invention, have better bacteriostatic activity and can provide reference for screening of a novel antibacterial agent. Meanwhile, from the metabolites of the eleutherococcous senticosus endosymbiotic bacteria, the eleutherococcous senticosus endosymbiotic bacteria are found to contain the coffeic acid. The coffeic acid is a medicament for stanching and increasing white blood cells and has effects of constricting and reinforcing capillaries, improving the functions of blood coagulation factors and increasing the white blood cells and blood platelets. The eleutherococcous senticosus endosymbiotic bacteria have a guide significance of producing the coffeic acid by adopting the eleutherococcous senticosus endosymbiotic bacteria CX33.
Description
Technical field
The present invention relates to a strain endogenetic bacteria.
Background technology
Endophyte of plant is a microbe groups that diversity is very abundant, they are lived in the cell of the various tissues of health plant and organ inside or the intercellular substance and are not caused the plant pathology, are the quasi-microorganisms that the harmony of setting up reciprocal coexistence, mutual restriction with host plant in secular common evolutionary process is united relation.In recent years, endophyte of plant has caused extensively as a kind of new Microbial resources.
Endophytic bacterium is the potential resources that produces active substance and novel chemical substance, not only can help to safeguard human health, also can safeguard the health of animal and plant.Medicinal plant itself just can produce the various active material, experimental study shows, plant and endogenetic bacteria are in interactional process, there is endogenetic bacteria can produce and the same or analogous active substance of plant, therefore from medicinal plant, is separated to the research tendency that the endogenetic bacteria that produces active substance has become current scholar.
Summary of the invention
The invention provides a strain and produce caffeinic Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria.
Produce caffeinic Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria, it is subtilis (Bacillus subtilis) CX33, belong to bacillus (Bacillus), in China's typical culture collection center preservation, deposit number is CCTCC No:M2011078, the preservation address is a Wuhan City Wuhan University, and preservation date is on March 22nd, 2011; It is a Gram-positive bacillus, and length is 2.0～3.0 μ m, and wide is 0.7～0.8 μ m, and gemma, flagellum and mycoderm are arranged, and forms white circular bacterium colony on beef-protein medium.
Produce caffeinic Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria among the present invention, it is subtilis (Bacillus subtilis) CX33, its starch hydrolysis is positive, gelatine liquefication is positive, catalase test is positive, lipase test is negative, methyl red is negative, the acetyl methyl carbinol reaction is positive, growth temperature is 10～50 ℃, optimum growth temperature is 40 ℃, the growth tolerance of salinity is 0.5%～20%, and the growth tolerance of salinity is 2%, and the suitableeest growth pH value is 6.8～7.6.
Produce caffeinic Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria among the present invention, it is subtilis (Bacillus subtilis) CX33, its 16S rDNA sequence is by the GENBANK sequence alignment, CX33 and sequence number are that the subtilis TCCC11021 bacterial strain sequence similarity of AB199317.1 reaches 99%, in conjunction with ne ar feature, growth conditions, Physiology and biochemistry qualification result, determine that subtilis (Bacillus subtilis) CX33 is the novel bacterial of bacillus.
Produce caffeinic Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria among the present invention, it is subtilis (Bacillus subtilis) CX33, belong to bacillus (Bacillus), in China's typical culture collection center preservation, deposit number is CCTCC No:M2011078, the preservation address is a Wuhan City Wuhan University, and preservation date is on March 22nd, 2011.
The present invention utilizes the endogenetic bacteria of microbiological pharmacy means extraction separation Radix Et Caulis Acanthopanacis Senticosi healthy plant, with the bacteriostatic activity is index, adopt the filter paper method to carry out bacteriostatic experiment, screening obtains the stronger purpose bacterial strain of some bacteriostatic activity, and with thin layer chromatography, the HPLC method is an analysis means, from the Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria, find the bacterial strain that can produce activeconstituents, and from its fermented liquid the extraction separation active substance, measure its active metabolite, at last the purpose bacterial strain that obtains is passed through morphological observation, its kind is identified in Physiology and biochemistry measuring and 16S rDNA sequential analysis.
Produce caffeinic Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria among the present invention, it is subtilis (Bacillus subtilis) CX33, and its metabolite has bacteriostatic activity preferably, and the screening that can be the novel antibacterial medicine provides reference; Find from its metabolite that simultaneously it contains coffic acid, coffic acid is the hemostasis drug for increasing white cells, have function, leukocyte increasing and the hematoblastic effect that increases solid capillary blood vessel, improves thrombin of shrinking, hemorrhage or the hemostasis of prevention when being used for surgical operation, and the hemostasis of hemorrhagic diseasess such as internal medicine, Obstetric and Gynecologic Department.Also be used for leukopenia, thrombocytopenia that a variety of causes causes.The present invention has directive significance to adopting Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria CX33 to produce coffic acid.
Description of drawings
Fig. 1 is the HPLC color atlas of coffic acid reference substance in the embodiment one, and wherein a represents coffic acid; Fig. 2 is the HPLC color atlas of trial-product in the embodiment one, and wherein a represents trial-product CX33; Fig. 3 is the HPLC color atlas that adds trial-product behind the reference substance in the embodiment one, and wherein a represents to add trial-product CX33a behind the coffic acid.
Embodiment
Embodiment one: present embodiment is produced caffeinic Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria, it is subtilis (Bacillus subtilis) CX33, belong to bacillus (Bacillus), in China's typical culture collection center preservation, deposit number is CCTCC No:M2011078, the preservation address is a Wuhan City Wuhan University, and preservation date is on March 22nd, 2011; It is a Gram-positive bacillus, and length is 2.0～3.0 μ m, and wide is 0.7～0.8 μ m, and gemma, flagellum and mycoderm are arranged, and forms white circular bacterium colony on beef-protein medium.
Subtilis in the present embodiment (Bacillus subtilis) CX33, it carries out conventional Physiology and biochemistry and identifies that experimental result is: the starch hydrolysis is positive, gelatine liquefication is positive, catalase test is positive, lipase test is negative, methyl red is negative, the acetyl methyl carbinol reaction is positive.
Subtilis in the present embodiment (Bacillus subtilis) CX33, its growth temperature is 10～50 ℃, and optimum growth temperature is 40 ℃, and the growth tolerance of salinity is 0.5%～20%, and the growth tolerance of salinity is 2%, the suitableeest growth pH value is 6.8～7.6.
Produce caffeinic Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria in the present embodiment, it is subtilis (Bacillus subtilis) CX33, this strains separation is from healthy wild Radix Et Caulis Acanthopanacis Senticosi plant, pick up from area, cap mountain, Heilongjiang Province, be accredited as Araliaceae Radix Et Caulis Acanthopanacis Senticosi Acanthopanax senticosus (Rupr.et Maxim) Harms through the Fu Kezhi researcher of Heilongjiang Province Academy of Traditional Chinese Medicine Pharmaceutical Manufacturing Plant, its sample is existing in bio-pharmaceuticals specialized laboratory of life science institute of Heilongjiang University; It carries out separation and Culture according to the following steps:
(1) material pre-treatment: choose healthy Radix Et Caulis Acanthopanacis Senticosi rhizome and rinse the back well with the aseptic filter paper suck dry moisture, be cut into the 1cm segment with tap water, standby;
(2) surface sterilization: in aseptic super clean bench, the Radix Et Caulis Acanthopanacis Senticosi rhizome is carried out surface sterilization as follows: alcohol-pickled 30s-aseptic water washing of 95% alcohol-pickled 1min-10% clorox immersion 5min-95% 3 times or the alcohol-pickled 1min-aseptic water washing of 75% alcohol-pickled 1min-10% hydrogen peroxide dipping 15min-75% 3 times;
(3) negative control: get last aseptic water washing liquid coating culture plate or get an amount of this washing fluid and pour in the beef extract-peptone liquid separation culture medium and compare, experiment material after will sterilizing is in addition rolled on the beef extract-peptone solid separation culture medium and is compared in a week, cultivates under identical condition;
(4) yeast culture: the experiment material after will sterilizing places liquid and solid separation culture medium respectively, in 37 ℃ of permanent shaking baths and warm incubator, cultivate 3～5d, treat to grow thalline around the experiment material, the picking thalline changes the flat board separation and purification of repeatedly ruling over to, bacterial strain behind the purifying is placed the inclined-plane solid medium, and 4 ℃ of preservations are standby;
Wherein the every L of beef extract-peptone solid separation culture medium is by the agar powder of NaCl, the 16.0g of the peptone of the extractum carnis of 3.0g, 10.0g, 5.0g and the H of surplus
2O forms, and the pH value is 7.0～7.2,121 ℃ of following autoclaving 30min;
The every L of beef extract-peptone liquid separation culture medium is by the extractum carnis of 3.0g, the peptone of 10.0g, the NaCl of 5.0g and the H of surplus
2O forms, and the pH value is 7.0～7.2,121 ℃ of following autoclaving 30min;
The inclined-plane solid medium is loaded in the test tube for getting 5mL beef extract-peptone solid separation culture medium, behind 121 ℃ of autoclaving 30min, is paved into the inclined-plane cooling, in order to preserving bacterial classification.
The result: from the Radix Et Caulis Acanthopanacis Senticosi rhizome, isolate endogenetic bacteria, and all do not have any bacterium in the dull and stereotyped and contrast liquid nutrient medium of contrast in the negative control and grow, repeatedly repeat all so, prove that the bacterium of being assigned to is an endophytic bacterium, rather than surperficial epiphyte.The caffeinic Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria of the product that filters out is carried out conventional Physiology and biochemistry to be identified; Molecular Identification is used international Bacteria Identification universal primer and is carried out PCR, obtain the amplified fragments of about 1600bp, being connected into the T carrier behind the purifying checks order, the recon that carries out that transforms detects, show that the purpose fragment has changed the T carrier over to, and successfully in intestinal bacteria, increase, bacterium liquid is sent to the order-checking of order-checking company; Its 16S rDNA sequence is by the GENBANK sequence alignment, with sequence number be that the subtilis TCCC11021 bacterial strain sequence similarity of AB199317.1 reaches 99%, in conjunction with ne ar feature, growth conditions, Physiology and biochemistry qualification result, determine that the caffeinic Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria of the product that filters out is the novel bacterial of bacillus, called after subtilis (Bacillus subtilis) CX33.
Produce caffeinic Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria in the present embodiment, it is subtilis (Bacillus subtilis) CX33, the bacteriostatic activity experiment of its fermented liquid, the result is as shown in table 1, the bacteriostatic activity experimental result of Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria fermented liquid shows, CX33 is to faecium, enterococcus faecalis, streptococcus pyogenes, Klebsiella Pneumoniae all have stronger restraining effect.
Table 1CX33 fermented liquid is to the bacteriostatic activity The selection result of 12 kinds of test organismss
Annotate: A: subtilis; B: bacillus pumilus; C: streptococcus aureus; D: enterococcus faecalis; E: faecium; F: Listeria monocytogenes; G: streptococcus pyogenes; H: intestinal bacteria; I: pseudomonas aeruginosa; J: Acinetobacter bauamnnii; K: Klebsiella Pneumoniae; L: Candida albicans; Str: Streptomycin sulphate; Nys: nysfungin;-non-activity; + activity a little less than, antibacterial circle diameter＜11mm; ++ active medium, antibacterial circle diameter 11～15mm; +++activity is stronger, antibacterial circle diameter＞15mm.
Produce caffeinic Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria in the present embodiment, it is subtilis (Bacillus subtilis) CX33, and the detection of its active metabolite is as follows:
The preparation of a, each component of CX33 fermented liquid:
The silica gel G plate activates 1h in 120 ℃ of baking ovens, put in the moisture eliminator standby, draw each 5 μ L of CX33 trial-product liquid respectively, put respectively on silica-gel plate (silica gel G+0.25%CMC, relative humidity 90%) with quantitative kapillary, with chloroform: methyl alcohol=9: 1 (v/v) is developping agent, launch, take out, dry, put under the ultraviolet lamp (254nm) and inspect; Trial-product liquid has corresponding clear bright blackening point, and negative controls is no any spot on the corresponding position;
A large amount of trial-product liquid is carried out the thin-layer chromatography experiment, and the blackening of getting each position is scraped in the position of each component after ultraviolet lamp (254nm) writes down chromatography down, be dissolved in the methyl alcohol, ultrasonic 60min is with 0.45 μ m filtering with microporous membrane, get filtrate, filtrate is placed room temperature, volatilize methyl alcohol, obtain small amount of solid,, obtain each higher component crystal of purity through recrystallization repeatedly, called after: CX33a successively, CX33b places 4 ℃ of refrigerators to preserve, and is standby;
B, employing filter paper method are carried out bacteriostatic experiment to the active ingredient of CX33, and the result is as shown in table 2, and the component CX33a of CX33, CX33b have certain bacteriostatic activity;
Each component of table 2CX33 is to the bacteriostatic activity The selection result of 12 kinds of test organismss
Annotate: A: subtilis; B: bacillus pumilus; C: streptococcus aureus; D: enterococcus faecalis; E: faecium; F: Listeria monocytogenes; G: streptococcus pyogenes; H: intestinal bacteria; I: pseudomonas aeruginosa; J: Acinetobacter bauamnnii; K: Klebsiella Pneumoniae; L: Candida albicans; Str: Streptomycin sulphate; Nys: nysfungin;-non-activity; + activity a little less than, antibacterial circle diameter＜11mm; ++ active medium, antibacterial circle diameter 11～15mm; +++activity is stronger, antibacterial circle diameter＞15mm.
C, active metabolite detected result
Chromatographic condition
Chromatographic column: VenusiL XBP-C
18Post (4.6mm * 250mm, 5 μ m);
Moving phase: methanol-water-phosphoric acid (30: 70: 0.2);
Flow velocity: 1mLmin
-1
Detect wavelength: 254nm;
Column temperature: 25 ℃;
Sample size: 10 μ L;
Under chromatographic condition, in the bacterium CX33 fermented liquid component CX33a trial-product consistent with the chromatographic peak of coffic acid reference substance corresponding position, and chromatographic peak has obtained confirming preferably through the reference substance addition method, the results are shown in Figure 1,2 and 3, conclusion: Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria CX33 is subtilis Bacillus subtilis, its metabolite has bacteriostatic activity preferably, find from its metabolite that simultaneously it contains coffic acid, have directive significance adopting Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria CX33 to produce coffic acid.
Claims (1)
1. caffeinic Radix Et Caulis Acanthopanacis Senticosi endogenetic bacteria is produced in a strain, it is characterized in that it is subtilis (Bacillus subtilis) CX33, belong to bacillus (Bacillus), in China's typical culture collection center preservation, deposit number is CCTCC No:M2011078, the preservation address is a Wuhan City Wuhan University, and preservation date is on March 22nd, 2011.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104605139A (en) * | 2015-01-08 | 2015-05-13 | 河北农业大学 | Bacillus subtilis-acanthopanax senticosus polysaccharide synbiotic, as well as preparation method and application thereof as feed additive |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101392224A (en) * | 2008-09-19 | 2009-03-25 | 南京工业大学 | Aspergillus niger strain for high yield of chlorogenic acid hydrolase and use thereof |
| CN101565694A (en) * | 2008-04-25 | 2009-10-28 | 天津天士力制药股份有限公司 | Salvianolic acid enzyme and mixed enzyme of same and ginsenoside and method for converting same into medicinal materials |
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| CN101565694A (en) * | 2008-04-25 | 2009-10-28 | 天津天士力制药股份有限公司 | Salvianolic acid enzyme and mixed enzyme of same and ginsenoside and method for converting same into medicinal materials |
| CN101392224A (en) * | 2008-09-19 | 2009-03-25 | 南京工业大学 | Aspergillus niger strain for high yield of chlorogenic acid hydrolase and use thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104605139A (en) * | 2015-01-08 | 2015-05-13 | 河北农业大学 | Bacillus subtilis-acanthopanax senticosus polysaccharide synbiotic, as well as preparation method and application thereof as feed additive |
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