CN112369435B - Salvia miltiorrhiza growth regulating bacteria suspension and preparation method and application thereof - Google Patents
Salvia miltiorrhiza growth regulating bacteria suspension and preparation method and application thereof Download PDFInfo
- Publication number
- CN112369435B CN112369435B CN202011418459.5A CN202011418459A CN112369435B CN 112369435 B CN112369435 B CN 112369435B CN 202011418459 A CN202011418459 A CN 202011418459A CN 112369435 B CN112369435 B CN 112369435B
- Authority
- CN
- China
- Prior art keywords
- salvia miltiorrhiza
- parts
- culture medium
- growth regulating
- growth
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- 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
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
- A01N37/04—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof polybasic
-
- 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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
-
- 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
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Environmental Sciences (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Organic Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dentistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Ecology (AREA)
- Forests & Forestry (AREA)
- Botany (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to the technical field of microorganisms, in particular to a salvia miltiorrhiza growth regulating bacterial suspension as well as a preparation method and an application thereof, wherein the salvia miltiorrhiza growth regulating bacterial suspension is prepared from the following raw materials in parts by weight: 10-15 parts of water-soluble protein; 3-9 parts of salvia miltiorrhiza endophytic fungi agent; 3-6 parts of an actinomycete agent; 12-18 parts of oligomeric chitosan oligosaccharide; 40-60 parts of MS liquid culture medium; 0.1-0.3 part of oxalic acid. Under the combined action of the endophytic fungi agent of the salvia miltiorrhiza, the actinomycete agent, the oligochitosan oligosaccharide and the oxalic acid, the growth of the salvia miltiorrhiza is promoted and the quality of the salvia miltiorrhiza is improved through the mutual cooperation of the microorganisms, the growth regulator and the induction factor.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a salvia miltiorrhiza growth regulating bacterial suspension and a preparation method and application thereof.
Background
Natural danshensu (tanshinol) is the main water-soluble component of Salvia miltiorrhiza Bge (Salvia militari Bge) of Labiatae, and has chemical name of D- (+) -beta- (3, 4-dihydroxyphenyl) lactic acid. Salvianic acid A, phenolic aromatic acid compounds, brown yellow powder or yellow powder, is the main water-soluble component in traditional Chinese medicine salvia miltiorrhiza, and is suitable for chest stuffiness and blood stasis symptoms, such as chest distress, palpitation, angina, acute and chronic myocardial infarction, ischemic cerebral apoplexy, cerebral infarction or apoplexy sequela. The tanshinol has effects of dilating coronary artery, inhibiting platelet aggregation, reducing blood plasma viscosity, and accelerating erythrocyte flow rate, and is beneficial for improving microcirculation and preventing thrombosis.
Tanshinone is also known as total tanshinone. Is a fat-soluble phenanthrenequinone compound with bacteriostatic action extracted from traditional Chinese medicine Salvia miltiorrhiza (root of Salvia miltiorrhizae Bunge of Labiatae), 10 tanshinone monomers such as tanshinone I, tanshinone IIA, tanshinone IIB, cryptotanshinone, iso-cryptotanshinone and the like are obtained from the fat-soluble phenanthrenequinone compound, wherein 5 tanshinone monomers such as cryptotanshinone, dihydrotanshinone II, hydroxy tanshinone, methyl danshenate and tanshinone IIB have antibacterial action and also have anti-inflammatory and cooling effects. The tanshinone IIA sodium sulfonate which is a sulfonated product of tanshinone IIA can be dissolved in water, and clinical trials prove that the tanshinone IIA sodium sulfonate has remarkable effect of treating angina and small side effect, and is a new medicament for treating coronary heart disease; the total tanshinone has the effects of resisting bacteria, diminishing inflammation, promoting blood circulation to disperse blood clots, promoting wound healing, etc. and has no obvious side effect after long term taking.
In the prior art, the salvia miltiorrhiza is often researched by adopting plant growth regulating bacterial suspension, the research focuses on how to promote the rapid propagation of the salvia miltiorrhiza, and the result shows that the yield of the salvia miltiorrhiza can only be increased and the quality of the salvia miltiorrhiza cannot be improved by adopting the conventional plant growth regulating bacterial suspension; in addition, the adoption of the plant growth regulating bacterial suspension often causes side effects on the quality of the salvia miltiorrhiza, for example, the antioxidant activity of the salvia miltiorrhiza is easy to be obviously reduced under the combined action of the Zhuanggen and the paclobutrazol, so that the growth regulating bacterial suspension applied to the salvia miltiorrhiza is required to replace the existing plant growth regulating bacterial suspension, the growth of the salvia miltiorrhiza can be promoted, the content of tanshinol and tanshinone components in the salvia miltiorrhiza can be increased, and the quality and medicinal value of the salvia miltiorrhiza are further improved.
Disclosure of Invention
Aiming at the technical defects, the invention aims to provide a salvia miltiorrhiza growth regulating bacteria suspension, a preparation method and application thereofActinomycetesUnder the combined action of the microbial inoculum, the oligochitosan oligosaccharide and the oxalic acid, the growth of the salvia miltiorrhiza is promoted and the quality of the salvia miltiorrhiza is effectively improved through the mutual cooperation of the microorganisms, the growth regulator and the induction factor.
In order to solve the technical problems, the invention adopts the following technical scheme:
a salvia miltiorrhiza growth regulating bacteria suspension is prepared from the following raw materials in parts by weight:
10-15 parts of water-soluble protein; 3-9 parts of salvia miltiorrhiza endophytic fungi agent;actinomycetes3-6 parts of a microbial inoculum; 12-18 parts of oligomeric chitosan oligosaccharide; 40-60 parts of MS liquid culture medium; 0.1-0.3 part of oxalic acid.
Preferably, the method comprises the following steps:
(1) weighing the following raw materials in parts by weight: 10-15 parts of water-soluble protein; 3-9 parts of salvia miltiorrhiza endophytic fungi agent;actinomycetes3-6 parts of a microbial inoculum; 12-18 parts of oligomeric chitosan oligosaccharide; 40-60 parts of MS liquid culture medium; 0.1-0.3 part of oxalic acid for later use;
(2) sequentially dissolving water-soluble protein and oligochitosan oligosaccharide in an MS liquid culture medium, uniformly mixing, sterilizing, cooling to room temperature, and adding oxalic acid to obtain a culture base solution;
(3) inoculating radix Salviae Miltiorrhizae endophytic fungi agent in culture basic solutionActinomycetesAnd performing activation culture on the microbial inoculum under the aseptic condition at the temperature of 25-28 ℃ for 8-10 days to prepare the salvia miltiorrhiza growth regulating bacterial suspension.
Preferably, the sterilization conditions of the step (2) are as follows: sterilizing at 110-120 deg.C for 30-60s or sterilizing with high pressure steam at 121 deg.C for 15 min.
Preferably, the concentration of the thallus in the salvia miltiorrhiza endophytic fungi agent in the step (3) is 106-107CFU·mL-1，Paying-off device BacteriaThe thallus concentration in the microbial inoculumDegree of 109-1011CFU·mL-1。
The invention also protects the application of the salvia miltiorrhiza growth regulating bacteria suspension in preparing the salvia miltiorrhiza growth and tanshinol and tanshinone component synthesis accelerant.
Preferably, the application method comprises the following steps: and (2) placing the detoxified seedlings of the stem tips of the salvia miltiorrhiza bunge in a culture medium, inoculating the salvia miltiorrhiza bunge growth regulating bacterial suspension in the culture medium, irradiating for 14-16 h/day under the illumination condition of 2000-3000lx, and culturing for 65-75d at the room temperature of 25-28 ℃.
Preferably, the culture medium is a mixture of humus soil and zeolite, and the mass ratio of the humus soil to the zeolite is 60-70: 30-40.
Preferably, the culture medium is a mixture of humus soil and zeolite, and the mass ratio of the humus soil to the zeolite is 65: 35.
Compared with the prior art, the invention has the beneficial effects that:
1. the actinomycete can obviously promote the growth of salvia miltiorrhiza, improve the yield, greatly improve the quality of salvia miltiorrhiza medicinal materials, and especially improve the content of tanshinol; oxalic acid induces the increase of tanshinone and salvianolic acid content in the salvia miltiorrhiza bunge, and regulates the distribution of calcium ions inside and outside cells; the endophytic fungi of the salvia miltiorrhiza achieves the coexistence of the endophytic fungi and the stems and leaves of the salvia miltiorrhiza so as to promote the growth of the salvia miltiorrhiza; the oligochitosan oligosaccharide can promote the growth of salvia miltiorrhiza plants and root systems and promote the generation of tanshinone; according to the invention, water-soluble protein is used as a carbon source and a nitrogen source, an MS liquid culture medium provides nutrients and inorganic salts for plant growth, the growth of salvia miltiorrhiza is promoted through the combined action of an endophytic fungi agent of salvia miltiorrhiza, actinomycetes and oligochitosan oligosaccharide, the generation of the tanshinone component is promoted by the oligochitosan oligosaccharide, and the generation of the tanshinol and the tanshinone component is induced and promoted by oxalic acid.
2. Root is adopted for the salvia miltiorrhiza, chemical pesticide is applied to prevention and control of diseases and insect pests of the salvia miltiorrhiza and repair of continuous cropping obstacles in the prior art, so that the quality of the salvia miltiorrhiza is reduced.
Detailed Description
The following description is provided for the best mode of carrying out the invention.
The preservation name of the salvia miltiorrhiza endophytic fungus is phoma herbarum (Phoma herbarum) D603, the preservation unit is the China general microbiological culture Collection center of the culture Collection management Committee of microorganisms, the preservation time is 11 months and 26 days in 2018, and the preservation number is CGMCC No 16884.
The above-mentionedActinomycetesThe microbial inoculum is commercially availableActinomycetesThe fungus powder was purchased from Cangzhou Wang Producer technology research institute, Inc.
Separation of salvia miltiorrhiza endophytic fungi:
(1) soaking Saviae Miltiorrhizae radix seed with 75% ethanol for 1min, treating with 5 wt% NaClO for 1min, washing with sterile water for 3 times, each for 1min, sterilizing with 2.5 wt% PBS-Tween20 solution, and soaking the seed overnight;
(2) continuously washing the test material obtained in the step 1 with sterile water for 3 times in a sterile environment, coating 100 mu l of the sterile water washing liquid washed for the last 1 time on a PDA (personal digital assistant) flat plate, culturing at 30 ℃ for 24 hours, and performing sterility verification;
(3) if the sterile colony is generated, continuing the subsequent step 4; if the bacterial colony is generated, returning to the step 1 to continue the surface disinfection treatment;
(4) placing the seeds subjected to surface sterilization treatment in a sterile mortar in a super clean bench, adding sterilized quartz sand, grinding into powder, adding 5mL of sterile water, mixing uniformly, standing for 15min, diluting 100 μ l of supernatant, coating the diluted supernatant in an improved Martin medium, and culturing at 28-32 deg.C for 1-3 days;
(5) and (3) selecting the single strain in the step (4) to perform streak purification separation on the improved Martin medium, and culturing for 1-3 days at 28-32 ℃ to finally obtain the salvia miltiorrhiza endophytic fungus strain with the preservation name of phoma herbarum (P.herbarum) D603 and the preservation number of CGMCC No. 16884.
Example 1
A preparation method of a salvia miltiorrhiza growth regulating bacterial suspension comprises the following steps:
(1) according to the following weightWeighing the following raw materials in parts: 13 parts of water-soluble protein; 3 parts of salvia miltiorrhiza endophytic fungi agent;put Linesless fungi6 parts of a microbial inoculum; 12 parts of oligochitosan; 40 parts of MS liquid culture medium; 0.2 part of oxalic acid for later use;
(2) sequentially dissolving water-soluble protein and oligo-chitosan oligosaccharide in an MS liquid culture medium, uniformly mixing, sterilizing at 110 ℃ for 60s, cooling to room temperature, and adding oxalic acid to obtain a culture base solution;
(3) inoculating radix Salviae Miltiorrhizae endophytic fungi agent in culture basic solutionActinomycetesActivating and culturing the bacterial agent at 25 ℃ for 10 days under the aseptic condition, wherein the concentration of the bacterial body in the salvia miltiorrhiza endophytic fungi bacterial agent is 106CFU·mL-1SaidActinomycetesThe concentration of thallus in the microbial inoculum is 109CFU·mL-1Preparing a salvia miltiorrhiza growth regulating bacteria suspension;
the application method of the salvia miltiorrhiza growth regulating bacteria suspension in promoting the growth of salvia miltiorrhiza and the synthesis of the tanshinol and tanshinone components comprises the following steps:
putting the stem tip detoxified seedlings of the salvia miltiorrhiza into a culture medium, inoculating the salvia miltiorrhiza growth regulating bacterial suspension into the culture medium, wherein the inoculation mass percentage of the culture medium and the bacterial suspension is 5 percent, irradiating for 16 h/day under 2000x illumination condition, and culturing for 65d at 28 ℃ room temperature environment;
the culture medium is a mixture of humus soil and zeolite, and the mass ratio of the humus soil to the zeolite is 60: 40.
Example 2
A preparation method of a salvia miltiorrhiza growth regulating bacterial suspension comprises the following steps:
(1) weighing the following raw materials in parts by weight: 10 parts of water-soluble protein; 8 parts of salvia miltiorrhiza endophytic fungi agent;put Linesless fungi6 parts of a microbial inoculum; 15 parts of oligochitosan; 50 parts of MS liquid culture medium; 0.1 part of oxalic acid for later use;
(2) sequentially dissolving water-soluble protein and oligo-chitosan oligosaccharide in an MS liquid culture medium, uniformly mixing, sterilizing at 115 ℃ for 45s, cooling to room temperature, and adding oxalic acid to obtain a culture base solution;
(3) inoculating radix Salviae Miltiorrhizae endophytic fungi in culture basic solutionAndactinomycetesActivating and culturing the bacterial agent at 27 ℃ for 9 days under the aseptic condition, wherein the concentration of the bacterial body in the salvia miltiorrhiza endophytic fungi bacterial agent is 107CFU·mL-1SaidActinomycetesThe concentration of the medium thallus is 1010CFU·mL-1Preparing a salvia miltiorrhiza growth regulating bacteria suspension;
the application method of the salvia miltiorrhiza growth regulating bacteria suspension in promoting the growth of salvia miltiorrhiza and the synthesis of the tanshinol and tanshinone components comprises the following steps:
putting the stem tip detoxified seedlings of the salvia miltiorrhiza into a culture medium, inoculating the salvia miltiorrhiza growth regulating bacterial suspension into the culture medium, wherein the inoculation mass percentage of the culture medium and the bacterial suspension is 8 percent, irradiating for 15 h/day under the illumination condition of 2500lx, and culturing for 70d under the room temperature environment of 27 ℃;
the culture medium is a mixture of humus soil and zeolite, and the mass ratio of the humus soil to the zeolite is 65: 35.
Example 3
A preparation method of a salvia miltiorrhiza growth regulating bacterial suspension comprises the following steps:
(1) weighing the following raw materials in parts by weight: 15 parts of water-soluble protein; 9 parts of salvia miltiorrhiza endophytic fungi agent;put Linesless fungi5 parts of a microbial inoculum; 18 parts of oligochitosan; 60 parts of MS liquid culture medium; 0.3 part of oxalic acid for later use;
(2) sequentially dissolving water-soluble protein and oligo-chitosan oligosaccharide in an MS liquid culture medium, uniformly mixing, sterilizing at 120 ℃ for 30s, cooling to room temperature, and adding oxalic acid to obtain a culture base solution;
(3) inoculating radix Salviae Miltiorrhizae endophytic fungi agent in culture basic solutionActinomycetesActivating and culturing the bacterial agent at 25-28 ℃ for 8 days under the aseptic condition, wherein the concentration of the bacterial in the salvia miltiorrhiza endophytic fungi bacterial agent is 106CFU·mL-1SaidActinomycetesThe concentration of thallus in the microbial inoculum is 1011CFU·mL-1Preparing a salvia miltiorrhiza growth regulating bacteria suspension;
the application method of the salvia miltiorrhiza growth regulating bacteria suspension in promoting the growth of salvia miltiorrhiza and the synthesis of the tanshinol and tanshinone components comprises the following steps:
putting the stem tip detoxified seedlings of the salvia miltiorrhiza into a culture medium, inoculating the salvia miltiorrhiza growth regulating bacterial suspension into the culture medium, wherein the inoculation mass percentage of the culture medium and the bacterial suspension is 10 percent, irradiating for 14 h/day under the illumination condition of 3000lx, and culturing for 75d at the room temperature of 25 ℃;
the culture medium is a mixture of humus soil and zeolite, and the mass ratio of the humus soil to the zeolite is 70: 30.
Comparative example 1
Putting the stem tip detoxified seedlings of the salvia miltiorrhiza into a culture medium, adding 10 parts of water-soluble protein and 50 parts of MS liquid culture medium into the culture medium, irradiating for 15 h/day under the illumination condition of 2500lx, and culturing for 70d at the room temperature of 27 ℃;
the culture medium is a mixture of humus soil and zeolite, and the mass ratio of the humus soil to the zeolite is 65: 35.
Comparative example 2
The preparation method is the same as the preparation method of the comparative example 1, except that 8 parts of salvia miltiorrhiza endophytic fungus agent is added.
Comparative example 3
The same procedure as in comparative example 1 was followed except thatActinomycetes6 parts of a microbial inoculum.
Comparative example 4
The same procedure as in comparative example 1 was followed except that 15 parts of oligochitosan was further added.
Comparative example 5
The same procedure as in comparative example 1 was followed except that 0.1 part of oxalic acid was further added.
In examples 1 to 3 of the present invention, high quality salvia miltiorrhiza rich in tanshinone and tanshinol is prepared, and the phenotype and quality are similar, in the following example 2, compared with comparative examples 1 to 5, comparative example 1 is a blank control group, and only contains the same amount of water-soluble protein and MS liquid culture medium as example 2, and the specific comparison method and results are as follows:
the samples of comparative examples 1-5 and example 2 were randomly selected, 10 plants per group, and the growth status of the plants after 7 days of culture was determined, with the specific results shown in table 1:
TABLE 1 comparative examples 1 to 5 and comparative example 2
| Item | Dry weight (g) | Fresh weight (g) | Plant height (cm) | Root length (cm) |
| Example 2 | 185.57 | 988.42 | 69.69 | 3.58 |
| Comparative example 1 | 58.2 | 749.1 | 46.3 | 2.82 |
| Comparative example 2 | 80.15 | 799.2 | 49.81 | 2.96 |
| Comparative example 3 | 100.39 | 821.81 | 54.27 | 3.08 |
| Comparative example 4 | 91.27 | 814.37 | 51.72 | 3.06 |
| Comparative example 5 | 179.63 | 984.18 | 68.72 | 3.51 |
Comparative example 1 compared to comparative example 2, comparative example 3, comparative example 4 and example 2, respectively, the dry weight increase was: 21.95g, 42.19g, 33.07g and 127.38g, the sum of the dry weight increases of comparative example 2, comparative example 3 and comparative example 4 being less than the dry weight increase of example 2; the fresh weight increases were: 50.1g, 72.71g, 65.27g and 239.32g, the sum of the fresh weight increases of comparative example 2, comparative example 3 and comparative example 4 being less than the fresh weight increase of example 2; the plant height increases are respectively: 3.51cm, 7.97cm, 5.42cm and 20.69cm, and the sum of the plant height increases of comparative example 2, comparative example 3 and comparative example 4 is less than the plant height increase of example 2; the root length growth is respectively: 0.14cm, 0.26cm, 0.24cm and 0.76cm, the sum of the root length increases of comparative example 2, comparative example 3 and comparative example 4 being less than the root length increase of example 2; the growth vigor of the salvia miltiorrhiza is greatly improved, and the salvia miltiorrhiza endophytic fungi microbial inoculum,Paying-off device BacteriaThe microbial inoculum and the oligochitosan oligosaccharide can act synergistically and promote the growth of the salvia miltiorrhiza; example 2 is comparable to comparative example 5, and it can be seen that oxalic acid does not act to modulate the phenotypic growth of Salvia miltiorrhiza.
The method for measuring the active ingredients in the salvia miltiorrhiza comprises the following steps:
after the samples of example 2 and comparative examples 1-5 were dried and ground, respectively, the salvia miltiorrhiza roots were extracted with methanol by the specific method: dissolving 20mg of salvia miltiorrhiza powder in 2ml of 70% methanol solution by volume fraction, carrying out ultrasonic treatment for 60 minutes, separating and analyzing danshensu and tanshinone by using High Performance Liquid Chromatography (HPLC), comparing with a blank control group, and respectively researching the content increase rate of the danshensu and the content increase rate of the tanshinone, wherein the results are shown in table 2:
TABLE 2 results of the content increase rate of tanshinol and tanshinone
The result shows that compared with the comparative example 1, the comparative example 1 does not contain actinomycete microbial inoculum, chitosan oligosaccharide salvia miltiorrhiza endophytic fungus microbial inoculum and oxalic acid, so that the contents of the tanshinol and the tanshinone are greatly different from the example 2; compared with the comparative example 2, the comparative example 2 contains the salvia miltiorrhiza endophytic fungus agent, so that the growth of the salvia miltiorrhiza is promoted, and the influence on the contents of tanshinol and tanshinone is smaller and is close to that of the comparative example 1; example 2 compared with comparative example 3, comparative example 3 containsActinomycetesA bacterial agent, a microbial agent,actinomycetesThe microbial inoculum promotes the growth of the tanshinol, so that the tanshinol is greatly improved compared with the tanshinol in the comparative example 1; compared with the comparative example 4, the comparative example 4 contains the oligochitosan oligosaccharide which promotes the growth of tanshinone, so that the tanshinone is greatly improved compared with the comparative example 1; compared with the comparative example 5, the comparative example 5 contains oxalic acid, which plays a role in inducing the generation of the tanshinol and the tanshinone, and compared with the tanshinol and the tanshinone in the comparative example 1, the generation of the tanshinol and the tanshinone in the comparative example 5 is greatly improved; the improvement rates of the tanshinol in the example 2, the comparative example 3 and the comparative example 5 are 71%, 39% and 28% respectively compared with the comparative example 1, and the improvement rate of the tanshinol in the example 2 is higher than the sum of the improvement rates of the comparative example 3 and the comparative example 5, which shows thatActinomycetesThe combined action of the microbial inoculum and the oxalic acid is more beneficial to the generation of the tanshinol; example 2, comparative example 4 and comparative example 5 the increase rates of tanshinone were 97%, 56% and35 percent, the increase rate of the tanshinone in the example 2 is higher than the sum of the increase rates of the comparative example 4 and the comparative example 5, which shows that the combined action of the oligochitosan oligosaccharide and the oxalic acid is more beneficial to the generation of the tanshinone; in summary, inActinomycetesUnder the combined action of the microbial inoculum, the oligochitosan oligosaccharide and the oxalic acid, the generation of the tanshinol and the tanshinone is more facilitated, and the quality of the salvia miltiorrhiza is improved.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (8)
1. A salvia miltiorrhiza growth regulating bacteria suspension is characterized by being prepared from the following raw materials in parts by weight:
10-15 parts of water-soluble protein; 3-9 parts of salvia miltiorrhiza endophytic fungi agent; 3-6 parts of an actinomycete agent; 12-18 parts of oligomeric chitosan oligosaccharide; 40-60 parts of MS liquid culture medium; 0.1-0.3 part of oxalic acid;
the preservation name of the salvia miltiorrhiza endophytic fungus is Phoma herbarum (Phoma herbarum) D603, the preservation unit is the China general microbiological culture Collection center of the culture Collection management Committee of microorganisms, the preservation time is 11 months and 26 days in 2018, and the preservation number is CGMCC No 16884.
2. The method for preparing a suspension of Salvia miltiorrhiza Bunge growth regulating bacteria according to claim 1, comprising the following steps:
(1) weighing the following raw materials in parts by weight: 10-15 parts of water-soluble protein; 3-9 parts of salvia miltiorrhiza endophytic fungi agent; 3-6 parts of an actinomycete agent; 12-18 parts of oligomeric chitosan oligosaccharide; 40-60 parts of MS liquid culture medium; 0.1-0.3 part of oxalic acid for later use;
(2) sequentially dissolving water-soluble protein and oligochitosan oligosaccharide in an MS liquid culture medium, uniformly mixing, sterilizing, cooling to room temperature, and adding oxalic acid to obtain a culture base solution;
(3) inoculating endophytic fungi agent and actinomycete agent of Saviae Miltiorrhizae radix in the culture base solution, and activating and culturing at 25-28 deg.C under aseptic condition for 8-10 days to obtain Saviae Miltiorrhizae radix growth regulating bacteria suspension.
3. The method for preparing a salvia miltiorrhiza growth regulating bacterial suspension according to claim 2, wherein the sterilization conditions in the step (2) are as follows: sterilizing at 110-120 deg.C for 30-60s or sterilizing with high pressure steam at 121 deg.C for 15 min.
4. The method for preparing a suspension of Salvia miltiorrhiza Bunge growth regulating bacteria according to claim 2, wherein the thallus concentration in the endophytic fungi preparation of Salvia miltiorrhiza Bunge in the step (3) is 106-107CFU·mL-1The concentration of the bacteria in the actinomycete agent is 109-1011CFU·mL-1。
5. The use of a suspension of growth-regulating bacteria of Salvia miltiorrhiza Bunge according to claim 1 in the preparation of a promoter for the growth of Salvia miltiorrhiza Bunge, the synthesis of tanshinol and tanshinone.
6. The application according to claim 5, wherein the application method is as follows: and (2) placing the detoxified seedlings of the stem tips of the salvia miltiorrhiza bunge in a culture medium, inoculating the salvia miltiorrhiza bunge growth regulating bacterial suspension in the culture medium, irradiating for 14-16 h/day under the illumination condition of 2000-3000lx, and culturing for 65-75d at the room temperature of 25-28 ℃.
7. The use according to claim 6, wherein the culture medium is a mixture of humus soil and zeolite, and the mass ratio of the humus soil to the zeolite is 60-70: 30-40.
8. The use according to claim 7, characterized in that the culture medium is a mixture of humus soil and zeolite, the mass ratio of humus soil to zeolite being 65: 35.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011418459.5A CN112369435B (en) | 2020-12-07 | 2020-12-07 | Salvia miltiorrhiza growth regulating bacteria suspension and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011418459.5A CN112369435B (en) | 2020-12-07 | 2020-12-07 | Salvia miltiorrhiza growth regulating bacteria suspension and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112369435A CN112369435A (en) | 2021-02-19 |
| CN112369435B true CN112369435B (en) | 2021-10-08 |
Family
ID=74590622
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011418459.5A Active CN112369435B (en) | 2020-12-07 | 2020-12-07 | Salvia miltiorrhiza growth regulating bacteria suspension and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112369435B (en) |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102577941A (en) * | 2011-01-06 | 2012-07-18 | 开国银 | Method for improving tanshinone content in salvia miltiorrhiza hair roots through methyl jasmonic acid treatment |
| CN102676392A (en) * | 2011-04-08 | 2012-09-19 | 中国人民解放军第二军医大学 | Endophytic fungus in salvia miltiorrhiza bunge and application thereof |
| CN103733829A (en) * | 2013-12-05 | 2014-04-23 | 陕西师范大学 | Salviae miltiorrhizae artificial cultivation method |
| CN103798293A (en) * | 2012-11-07 | 2014-05-21 | 中国人民解放军第二军医大学 | Preparation and applications of salvia miltiorrhiza endophytic fungi hypha water-soluble extract and fractions of salvia miltiorrhiza endophytic fungi hypha water-soluble extract |
| CN103992961A (en) * | 2013-02-16 | 2014-08-20 | 中国医学科学院药用植物研究所 | Paecilomyces sp. fungi improving salvia miltiorrhiza output and total phenolic-acid content |
| CN104170558A (en) * | 2014-08-12 | 2014-12-03 | 安徽永兴岛生态农业科技有限公司 | Salvia miltiorrhiza planting method |
| CN105505798A (en) * | 2016-01-15 | 2016-04-20 | 中国人民解放军第二军医大学 | Endophytic fungus for generating ergosterol and application of endophytic fungus |
| CN106801014A (en) * | 2016-12-02 | 2017-06-06 | 中国人民解放军第二军医大学 | A kind of endogenetic fungus and its application for improving red sage root yield and its active constituent content |
| CN107142218A (en) * | 2017-06-20 | 2017-09-08 | 泰山医学院 | One plant of Wite red-rooted salvia root endogenetic fungus BDF09 and its application |
| CN109706084A (en) * | 2018-12-13 | 2019-05-03 | 浙江理工大学 | A kind of Radix Salviae Miltiorrhizae endogenetic fungus and its application in promotion Salvia miltiorrhiza Growth/or effective component synthesis |
| CN109749939A (en) * | 2018-12-13 | 2019-05-14 | 浙江理工大学 | A kind of microbial inoculum containing Radix Salviae Miltiorrhizae endogenetic fungus and its application |
| CN110438011A (en) * | 2019-07-30 | 2019-11-12 | 浙江理工大学 | Superfine branch spore is mould and its promotes the purposes of Radix Salviae Miltiorrhizae root system effective component synthesis |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102960156A (en) * | 2012-11-28 | 2013-03-13 | 西安福安创意咨询有限责任公司 | Planting method for producing agricultural products containing Salvia miltiorrhiza |
| CN103992952B (en) * | 2013-02-16 | 2016-12-28 | 中国医学科学院药用植物研究所 | One strain improves Radix Salviae Miltiorrhizae yield and the Cladosporium fungus of total phenolics content |
| CN107711386A (en) * | 2017-12-07 | 2018-02-23 | 泰顺县韦中家庭农场 | A kind of red sage root implantation methods for being advantageous to red sage root healthy growth |
-
2020
- 2020-12-07 CN CN202011418459.5A patent/CN112369435B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102577941A (en) * | 2011-01-06 | 2012-07-18 | 开国银 | Method for improving tanshinone content in salvia miltiorrhiza hair roots through methyl jasmonic acid treatment |
| CN102676392A (en) * | 2011-04-08 | 2012-09-19 | 中国人民解放军第二军医大学 | Endophytic fungus in salvia miltiorrhiza bunge and application thereof |
| CN103798293A (en) * | 2012-11-07 | 2014-05-21 | 中国人民解放军第二军医大学 | Preparation and applications of salvia miltiorrhiza endophytic fungi hypha water-soluble extract and fractions of salvia miltiorrhiza endophytic fungi hypha water-soluble extract |
| CN103992961A (en) * | 2013-02-16 | 2014-08-20 | 中国医学科学院药用植物研究所 | Paecilomyces sp. fungi improving salvia miltiorrhiza output and total phenolic-acid content |
| CN103733829A (en) * | 2013-12-05 | 2014-04-23 | 陕西师范大学 | Salviae miltiorrhizae artificial cultivation method |
| CN104170558A (en) * | 2014-08-12 | 2014-12-03 | 安徽永兴岛生态农业科技有限公司 | Salvia miltiorrhiza planting method |
| CN105505798A (en) * | 2016-01-15 | 2016-04-20 | 中国人民解放军第二军医大学 | Endophytic fungus for generating ergosterol and application of endophytic fungus |
| CN106801014A (en) * | 2016-12-02 | 2017-06-06 | 中国人民解放军第二军医大学 | A kind of endogenetic fungus and its application for improving red sage root yield and its active constituent content |
| CN107142218A (en) * | 2017-06-20 | 2017-09-08 | 泰山医学院 | One plant of Wite red-rooted salvia root endogenetic fungus BDF09 and its application |
| CN109706084A (en) * | 2018-12-13 | 2019-05-03 | 浙江理工大学 | A kind of Radix Salviae Miltiorrhizae endogenetic fungus and its application in promotion Salvia miltiorrhiza Growth/or effective component synthesis |
| CN109749939A (en) * | 2018-12-13 | 2019-05-14 | 浙江理工大学 | A kind of microbial inoculum containing Radix Salviae Miltiorrhizae endogenetic fungus and its application |
| CN110438011A (en) * | 2019-07-30 | 2019-11-12 | 浙江理工大学 | Superfine branch spore is mould and its promotes the purposes of Radix Salviae Miltiorrhizae root system effective component synthesis |
Non-Patent Citations (4)
| Title |
|---|
| Effects of Streptomyces pactum Act12 on Salvia miltiorrhiza Hairy Root Growth and Tanshinone Synthesis and Its Mechanisms;Yan Yan,Shuncang Zhang,Dongfeng Yang,Jiayi Zhang,Zongsuo Liang;《Appl Biochem Biotechnol》;20140415;第173卷(第4期);摘要 * |
| 低聚壳寡糖植物生长调节剂对丹参生长及次生代谢产物的影响;张元，林强;《安徽农业科学》;20110325;第38卷(第34期);19317-19318 * |
| 放线菌剂对丹参生长及有效成分的影响;段佳丽，舒志明，孙群，魏良柱，傅亮亮，薛泉宏，于妍华;《西北农林科技大学学报（自然科学版）》;20120109;第40卷(第2期);197-198 * |
| 草酸促进丹参悬浮细胞生长和丹参酮、酚酸的积累;张宗申，田鹏玮，刘同祥，杨芳;《中国中药杂志》;20090401;第34卷(第7期);920-921 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112369435A (en) | 2021-02-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Powthong et al. | Screening of Antimicrobial Activities of the Endoph ytic Fungi Isolated from Sesbania grandiflora (L.) Pers. | |
| CN108642018B (en) | Lytic bacteriophage capable of preventing and controlling tomato bacterial wilt and application thereof | |
| WO2022135328A1 (en) | Application of 2-amino-3-methylhexanoic acid as plant immunity inducer | |
| US20180220608A1 (en) | A Method for Increasing the Content of Notoginsenoside R1 in Panax Notoginseng | |
| CN113661998B (en) | Energy metabolism repressor for resisting gray mold | |
| CN105779302B (en) | Hypocrea virens composite microbial inoculum for preventing and treating cucumber fusarium wilt and preparation method thereof | |
| CN115536446B (en) | Fertilizer for promoting plant growth and application thereof | |
| CN106148221B (en) | Stenotrophomonas bacteria for inducing peanuts to resist root-knot nematode disease | |
| CN112574892B (en) | Mucor circinelloides for promoting root system development of red sage root and tanshinone synthesis and its use | |
| CN112369435B (en) | Salvia miltiorrhiza growth regulating bacteria suspension and preparation method and application thereof | |
| CN109749939B (en) | Microbial inoculum containing salvia miltiorrhiza endophytic fungi and application thereof | |
| CN109749938B (en) | Endophytic fungus for reducing incidence rate of panax notoginseng root rot and microbial inoculum thereof | |
| CN113892484B (en) | Application of alpha-pinene in promoting growth and inducing resistance of panax notoginseng | |
| CN114644986A (en) | Trichoderma atroviride and application thereof in preventing and treating panax notoginseng diseases | |
| CN111213586B (en) | Anoectochilus roxburghii tissue culture medium and preparation method thereof | |
| CN108849901A (en) | It is a kind of for preventing and treating the biological pesticide preparation and its preparation and application of the late blight of potato | |
| CN111296448B (en) | Application of lentinan in preventing and treating fungal diseases of tea trees | |
| CN115886028B (en) | Compound medicament and application thereof in preventing and treating bacterial spot of tomatoes | |
| Rizk et al. | Biological Control of Fusarium Oxysporum and Verticillium dahliaeBy Trichoderma harzianum and Gliocladium virens of Two Mint Species | |
| CN116584506B (en) | Pesticide for fruit trees and preparation method and application thereof | |
| CN114128729B (en) | Application of chinaberry leaf and fructus evodiae volatile oil in prevention and treatment of phytophthora blight of capsicum | |
| CN117247976B (en) | Preparation of kuh-seng fermentation extract and application of kuh-seng fermentation extract in efficacy type oral care products | |
| JP3065589B2 (en) | Enterococcal antibacterial agent | |
| Maslova et al. | Anti-Bacterial Activity of the Vallus Tissue Ballota Nigral L in Vitro. | |
| Rasi-Bonab et al. | Evaluation of silver nanoparticles and alcoholic extract of Amaranthus retroflexus on pathogenic bacteria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |