CN115399343B - Plant composite antibacterial agent containing peony extract and preparation method and application thereof - Google Patents
Plant composite antibacterial agent containing peony extract and preparation method and application thereof Download PDFInfo
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- 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
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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- 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
- A01N63/22—Bacillus
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- 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
- A01N63/28—Streptomyces
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- 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
- A01N63/32—Yeast
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- 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
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
- A01N65/12—Asteraceae or Compositae [Aster or Sunflower family], e.g. daisy, pyrethrum, artichoke, lettuce, sunflower, wormwood or tarragon
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- 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
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
- A01N65/32—Ranunculaceae [Buttercup family], e.g. hepatica, hydrastis or goldenseal
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a plant composite antibacterial agent containing a peony extract, and a preparation method and application thereof, wherein the plant composite antibacterial agent comprises the following components in parts by mass: 20-30 parts of peony extract, 10-20 parts of jerusalem artichoke extract, 10-15 parts of coptis extract, 1-3 parts of streptomyces griseus, 1-3 parts of dead bacillus vallismortis and 1-3 parts of saccharomyces cerevisiae. According to the invention, multiple antibacterial substances are compounded, and the plant antibacterial source and the microorganism antibacterial source have synergistic effect, so that the antibacterial and bactericidal composition has good antibacterial and bactericidal effects, overcomes the limitation of a single antibacterial source, and has the characteristics of broad spectrum and high efficiency.
Description
Technical Field
The invention relates to the technical field of plant antibacterial agents, and particularly relates to a plant composite antibacterial agent containing a peony extract, and a preparation method and application thereof.
Background
Phytopathogenic fungi can affect crop growth. The application of chemical pesticide can cause residue in crops, water and soil, which causes the problems of pesticide residue exceeding standard and environmental pollution. Meanwhile, the chemical pesticide can also inhibit and even kill other beneficial microorganisms in the soil, so that the organic matter content of the soil is reduced, the soil fertility is reduced, and a plurality of social, economic and ecological problems are caused.
The antibacterial agent is a medicament for preventing and treating plant diseases caused by various pathogenic microorganisms. An agent which kills or inhibits the growth of pathogenic microorganisms without interfering with the normal growth of plants. Antibacterial agents are generally classified into inorganic antibacterial agents, organic antibacterial agents and natural antibacterial agents. The inorganic antibacterial agent is prepared by fixing metals (or ions thereof) such as silver, copper, zinc and the like on the surface of porous materials such as fluorite, silica gel and the like by using the antibacterial capacity of the metals such as silver, copper, zinc and the like through a physical adsorption ion exchange method and the like, and then adding the antibacterial agent into a corresponding product to obtain the material with the antibacterial capacity. Metals such as mercury, cadmium, lead and the like also have antibacterial capacity, but are harmful to human bodies; ions such as copper, nickel, lead and the like have colors and affect the appearance of products, and zinc has certain antibacterial property, but the antibacterial strength of the zinc is only 1/1000 of that of silver ions. The main varieties of the organic antibacterial agent comprise vanillin or ethyl vanillin compounds, acylaniline compounds, imidazoles, thiazoles, isothiazolone derivatives, quaternary ammonium salts, biguanidine compounds, phenols and the like. Generally, organic antibacterial agents have poor heat resistance, are easily hydrolyzed, and have a short pot life. Natural antibacterial agents are mainly extracted from natural substances and are the earliest antibacterial agents used by people. Natural antibacterial agents are classified into three categories according to their origin: the antibacterial agent is a botanical antibacterial agent, and the main active ingredients of the antibacterial agent are terpenoids, derivatives thereof and the like; secondly, animal-derived antibacterial agents, the most commonly used antibacterial agents are chitosan, and the antibacterial activity of the antibacterial agents is enhanced along with the increase of the activity of metal ions; and the microbial antibacterial agent comprises microorganisms and antagonistic metabolites of the microorganisms, and has an antibacterial effect by secreting antibiotics and inducing hosts to generate anti-pathogens and the like. The prior natural antibacterial agent has limited antibacterial action, low bactericidal rate, no broad-spectrum long-acting use and small quantity.
As an ideal antibacterial agent, it should have immediate, broad-spectrum, long-lasting, stable and safe antibacterial effects. However, none of the existing antimicrobial agents, whether inorganic, organic, or natural biological, meet the desired requirements.
Peony is a plant belonging to Ranunculaceae and Paeonia. Is perennial deciduous shrub, the stem is up to 2 meters; the branches are short and thick. The leaves are usually two-time-three-time-multiple leaves, the surface is green and unhairing, the back is light green, sometimes white, the petioles are 5-11 cm long, and the axes of the leaves are unhairing. The flower single grows on the top of the branch, the bract 5 is in an oblong shape; sepals 5, green, wide ovoid, petals 5 or heavy petals, rose, magenta, pink to white, generally with a large variation, inverted ovoid, with an irregular wave-like top; the anther is long round and 4 mm long; floral pansy, cupped, purplish red; the heart-skin, dense and soft hair. Gu whole-length long circle, dense raw yellow brown bristle. The flowering phase is 5 months; the fruit period is 6 months. The peony comprises the following main components: paeonol, gallic acid, galloformic acid, theaflavin, tannic acid, quercetin, etc. The peony flower can be made into scented tea, and is a good product for women. The peony root bark extract also has a great effect on the antibacterial aspect. The moutan bark is proved to contain active ingredients with the functions of tranquilizing and easing pain, clearing heat and reducing blood pressure, resisting bacteria and inflammation, resisting oxidation, resisting allergy and stopping bleeding in various pharmacological experimental reports. At present, most of peony extracts are applied to cosmetic preservation, and no antibacterial agent applied to plant diseases is reported.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a plant composite antibacterial agent containing a peony extract and a preparation method thereof.
In order to realize the purpose, the invention adopts the following technical scheme:
the invention provides a first aspect of a plant composite antibacterial agent containing a peony extract, which comprises the following components in parts by mass:
20-30 parts of peony extract, 10-20 parts of jerusalem artichoke extract, 10-15 parts of coptis extract, 1-3 parts of streptomyces griseus, 1-3 parts of killed millet bacillus and 1-3 parts of saccharomyces cerevisiae.
Preferably, the preparation method of the peony extract comprises the following steps: the method comprises the steps of drying whole peony plants, crushing the whole peony plants to 100 meshes to obtain peony materials, adding a mixed enzyme solution into the peony materials for enzymolysis, wherein the adding amount of the mixed enzyme solution is 4-6 times of the weight of the peony materials, the mass concentration of cellulase in the mixed enzyme solution is 0.3mg/mL, the mass concentration of pectinase is 0.3mg/mL, the enzymolysis time is 2-4 hours, inactivating enzyme at 85-90 ℃, performing ultrasonic extraction on the solution after enzyme inactivation, performing ultrasonic extraction at the ultrasonic frequency of 80-100kHz and the ultrasonic time of 0.5-1 hour for three times, and separating and combining extracting solutions to obtain a peony extract.
Preferably, the preparation method of the jerusalem artichoke extract comprises the following steps: oven drying folium Helianthi and rhizome of Helianthus tuberosus L.to obtain mixture, adding ethanol with concentration of 85% and ethanol amount of 3 times of the mixture, heating and extracting at 95 deg.C for 1 hr, and separating extractive solution to obtain Helianthus tuberosus L.extract.
Preferably, the preparation method of the coptis chinensis extract comprises the following steps: pulverizing Coptidis rhizoma, adding ethanol with concentration of 85% and the amount of ethanol 3 times of Coptidis rhizoma, heating and extracting at 90 deg.C for 1 hr, and separating the extractive solution to obtain Coptidis rhizoma extract.
Preferably, the effective viable count of the streptomyces griseus in the antibacterial agent is more than or equal to 1 x 10 7 cfu/g, the effective viable count of dead bacillus vallismortis in the antibacterial agent is more than or equal to 1 multiplied by 10 7 cfu/g, the effective viable count of the saccharomyces cerevisiae in the antibacterial agent is more than or equal to 1 multiplied by 10 7 cfu/g。
More preferably, the Streptomyces griseus has a deposit number of CICC11002, the killed Bacillus vallismortis has a deposit number of CICC21334, and the Saccharomyces cerevisiae has a deposit number of CICC1288.
The second aspect of the invention provides a preparation method of the plant composite antibacterial agent containing the peony extract, which comprises the following steps:
mixing peony extract, jerusalem artichoke extract and Coptidis rhizoma extract, adding Streptomyces griseus, dead Bacillus cereus and Saccharomyces cerevisiae, and stirring to obtain antibacterial agent.
The third aspect of the invention provides the application of the plant composite antibacterial agent containing the peony extract in preventing and treating plant diseases.
Preferably, the plant disease is caused by one or more of fusarium oxysporum, aspergillus niger, xanthomonas campestris.
Preferably, the antimicrobial agent is diluted 200-500 times at the time of use and applied to the plants by spraying or irrigation.
The peony extract, the jerusalem artichoke extract and the coptis extract are used as plant antibacterial sources, and the plant antibacterial sources mainly have the effects of destroying or degrading cell walls, destroying cytoplasmic membranes and destroying cell membrane protein structures, so that cell contents are leaked, cytoplasm is condensed, and proton motility is weakened. However, these mechanisms of action are not independent and may affect each other. Streptomyces griseus, killed glutamic bacillus and saccharomyces cerevisiae are used as microbial antibacterial sources, and the antibacterial mechanism of the microbial antibacterial sources is mainly as follows: (1) Through secretion of antibiotics, synthesis of bacterial cell walls is inhibited, permeability of plasma membranes is changed, synthesis of proteins is inhibited, and metabolism of nucleic acid and folic acid is influenced; (2) Weakening by occupying living space, consuming oxygen and the like so as to eliminate certain pathogens in the same living environment; (3) Inducing host to produce defense reaction or directly parasitize pathogenic bacteria to inhibit pathogenic bacteria; (4) Parasitic on pathogenic bacteria, secrete enzymes that destroy the fungal cell wall.
The invention has the beneficial effects that:
the invention compounds a plurality of antibacterial substances, and the plant antibacterial source and the microorganism antibacterial source have synergistic effect, thereby having good antibacterial and bactericidal effects, overcoming the limitation of a single antibacterial source, and having the characteristics of broad spectrum and high efficiency.
The plant antibacterial source in the invention destroys cell walls and cell membranes, and makes the cell contents leak; the microbial antibacterial source can secrete antibiotics, inhibit pathogenic bacteria and secrete enzymes for destroying the cell wall of fungi; the substances have synergistic effect, and the sterilization and bacteriostasis capability of the antibacterial agent is improved.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background art, the invention provides a plant composite antibacterial agent containing a peony extract, which comprises the following components in parts by mass:
20-30 parts of peony extract, 10-20 parts of jerusalem artichoke extract, 10-15 parts of coptis extract, 1-3 parts of streptomyces griseus, 1-3 parts of killed millet bacillus and 1-3 parts of saccharomyces cerevisiae.
The preparation method of the peony extract comprises the following steps: drying and crushing the whole peony plant to 100 meshes to obtain a peony material, adding a mixed enzyme solution into the peony material for enzymolysis, wherein the addition amount of the mixed enzyme solution is 4-6 times of the weight of the peony material, the mass concentration of cellulase in the mixed enzyme solution is 0.3mg/mL, the mass concentration of pectinase is 0.3mg/mL, the enzymolysis time is 2-4h, inactivating enzyme at 85-90 ℃, performing ultrasonic extraction on the solution after enzyme inactivation, wherein the ultrasonic frequency is 80-100kHz, the ultrasonic time is 0.5-1h, repeating for three times, and separating and combining the extracting solution to obtain a peony extract.
The preparation method of the jerusalem artichoke extract comprises the following steps: oven drying folium Helianthi and rhizome of Helianthus tuberosus L.to obtain mixture, adding ethanol with concentration of 85% and ethanol amount of 3 times of the mixture, heating and extracting at 95 deg.C for 1 hr, and separating extractive solution to obtain Helianthus tuberosus L.extract.
The preparation method of the coptis extract comprises the following steps: pulverizing Coptidis rhizoma, adding ethanol with concentration of 85% and the amount of ethanol 3 times of Coptidis rhizoma, heating and leaching at 90 deg.C for 1 hr, and separating extractive solution to obtain Coptidis rhizoma extract.
The effective viable count of the streptomyces griseus in the antibacterial agent is more than or equal to 1 multiplied by 10 7 cfu/g, the effective viable count of dead bacillus vallismortis in the antibacterial agent is more than or equal to 1 multiplied by 10 7 cfu/g, the effective viable count of the saccharomyces cerevisiae in the antibacterial agent is more than or equal to 1 multiplied by 10 7 cfu/g. The preservation number of the streptomyces griseus is CICC11002, the preservation number of the killed bacillus vallismortis is CICC21334, and the preservation number of the saccharomyces cerevisiae is CICC1288.
The preparation method of the plant composite antibacterial agent containing the peony extract comprises the following steps:
mixing peony extract, jerusalem artichoke extract and Coptidis rhizoma extract, adding Streptomyces griseus, dead Bacillus cereus and Saccharomyces cerevisiae, and stirring to obtain antibacterial agent.
The plant composite antibacterial agent containing the peony extract is applied to preventing and treating plant diseases, wherein the plant diseases are caused by one or more of fusarium oxysporum, aspergillus niger and xanthomonas campestris; the antibacterial agent is diluted 200-500 times at application time, and is applied to plants by spraying or irrigating.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available.
Streptomyces griseus (used in the following examples and comparative examples of the present invention: (S. Griseus) (S. Griseus))Streptomyces griseus) Purchased from China center for culture collection and management of industrial microorganisms, and the strain collection number is CICC11002; dead Bacillus vallismortis: (Bacillus vallismortis) Purchased from China center for culture collection and management of industrial microorganisms, and the strain preservation number is CICC21334; saccharomyces cerevisiae (Saccharomyces cerevisiae) Purchased from China center for culture collection and management of industrial microorganisms, and the strain preservation number is CICC1288; fusarium oxysporum (F.), (Fusarium oxysporum Schltdl) Aspergillus nigerAspergillus niger) All purchased from Huang Yan Brevibacterium (Brevibacterium paradise, inc.)Xanthomonas campestris) Purchased from Ningbo Test Biotechnology, inc.
Example 1
25 parts of peony extract, 15 parts of jerusalem artichoke extract, 12 parts of coptis extract, 2 parts of streptomyces griseus, 2 parts of dead bacillus vallismortis and 2 parts of saccharomyces cerevisiae.
(1) The method comprises the steps of drying and crushing the whole peony plant to 100 meshes to obtain a peony material, adding a mixed enzyme solution into the peony material for enzymolysis, wherein the adding amount of the mixed enzyme solution is 6 times of the weight of the peony material, the mass concentration of cellulase in the mixed enzyme solution is 0.3mg/mL, the mass concentration of pectinase is 0.3mg/mL, the enzymolysis time is 4 hours, inactivating enzyme at 90 ℃, performing ultrasonic extraction on the solution after enzyme inactivation, repeating the steps for three times, separating and combining extracting solution to obtain a peony extract.
(2) Oven drying folium Jerusalem artichoke and rhizome of Jerusalem artichoke, pulverizing to obtain Jerusalem artichoke mixture, adding ethanol with concentration of 85% and ethanol content 3 times of the Jerusalem artichoke mixture, heating and extracting at 95 deg.C for 1 hr, and separating the extractive solution to obtain Jerusalem artichoke extract.
(3) Pulverizing Coptidis rhizoma, adding ethanol with concentration of 85% and the amount of ethanol 3 times of Coptidis rhizoma, heating and extracting at 90 deg.C for 1 hr, and separating the extractive solution to obtain Coptidis rhizoma extract.
(4) Mixing peony extract, jerusalem artichoke extract and Coptidis rhizoma extract, adding Streptomyces griseus, dead Bacillus cereus and Saccharomyces cerevisiae, and stirring to obtain antibacterial agent.
Example 2
20 parts of peony extract, 10 parts of jerusalem artichoke extract, 10 parts of coptis extract, 1 part of streptomyces griseus, 1 part of dead bacillus vallismortis and 1 part of saccharomyces cerevisiae.
(1) The method comprises the steps of drying and crushing the whole peony plant to 100 meshes to obtain a peony material, adding a mixed enzyme solution into the peony material for enzymolysis, wherein the adding amount of the mixed enzyme solution is 6 times of the weight of the peony material, the mass concentration of cellulase in the mixed enzyme solution is 0.3mg/mL, the mass concentration of pectinase is 0.3mg/mL, the enzymolysis time is 4 hours, inactivating enzyme at 90 ℃, performing ultrasonic extraction on the solution after enzyme inactivation, repeating the steps for three times, separating and combining extracting solution to obtain a peony extract.
(2) Oven drying folium Helianthi and rhizome of Helianthus tuberosus L.to obtain mixture, adding ethanol with concentration of 85% and ethanol amount of 3 times of the mixture, heating and extracting at 95 deg.C for 1 hr, and separating extractive solution to obtain Helianthus tuberosus L.extract.
(3) Pulverizing Coptidis rhizoma, adding ethanol with concentration of 85% and the amount of ethanol 3 times of Coptidis rhizoma, heating and extracting at 90 deg.C for 1 hr, and separating the extractive solution to obtain Coptidis rhizoma extract.
(4) Mixing peony extract, jerusalem artichoke extract and Coptidis rhizoma extract, adding Streptomyces griseus, dead Bacillus cereus and Saccharomyces cerevisiae, and stirring to obtain antibacterial agent.
Example 3
30 parts of peony extract, 20 parts of jerusalem artichoke extract, 15 parts of coptis extract, 3 parts of streptomyces griseus, 3 parts of dead bacillus vallismortis and 3 parts of saccharomyces cerevisiae.
(1) The method comprises the steps of drying and crushing the whole peony plant to 100 meshes to obtain a peony material, adding a mixed enzyme solution into the peony material for enzymolysis, wherein the adding amount of the mixed enzyme solution is 6 times of the weight of the peony material, the mass concentration of cellulase in the mixed enzyme solution is 0.3mg/mL, the mass concentration of pectinase is 0.3mg/mL, the enzymolysis time is 4 hours, inactivating enzyme at 90 ℃, performing ultrasonic extraction on the solution after enzyme inactivation, repeating the steps for three times, separating and combining extracting solution to obtain a peony extract.
(2) Oven drying folium Helianthi and rhizome of Helianthus tuberosus L.to obtain mixture, adding ethanol with concentration of 85% and ethanol amount of 3 times of the mixture, heating and extracting at 95 deg.C for 1 hr, and separating extractive solution to obtain Helianthus tuberosus L.extract.
(3) Pulverizing Coptidis rhizoma, adding ethanol with concentration of 85% and the amount of ethanol 3 times of Coptidis rhizoma, heating and extracting at 90 deg.C for 1 hr, and separating the extractive solution to obtain Coptidis rhizoma extract.
(4) Mixing peony extract, jerusalem artichoke extract and Coptidis rhizoma extract, adding Streptomyces griseus, dead Bacillus cereus and Saccharomyces cerevisiae, and stirring to obtain antibacterial agent.
Comparative example 1
The raw materials of the antibacterial agent in the comparative example are 30 parts of peony extract, 20 parts of jerusalem artichoke extract and 15 parts of coptis extract, and the rest is the same as that in the example 3.
Comparative example 2
The raw materials of the antibacterial agent in the comparative example are 3 parts of streptomyces griseus, 3 parts of dead bacillus cereus and 3 parts of saccharomyces cerevisiae, and the rest is the same as that of the example 3.
Examples of the experiments
Antibacterial activity determination, minimum inhibitory concentration determination and minimum lethal concentration determination of the antibacterial agent are carried out by adopting the steps in the literature, "plant extract antibacterial activity and antibacterial mechanism" (Zhou Yun winter, south Jiangnan university, 2019.).
1. Determination of antibacterial Activity of antibacterial Agents
The bacteria were cultured in LB liquid medium at 37 ℃ for 12 h, washed twice with physiological saline and prepared to have a cell density of 1.0X 10 8 CFU·mL -1 The cell suspension of (3). Culturing mould PDA slant at 28 deg.C for 48 h, adding 5 mL sterile normal saline, gently scraping off spores on agar surface, scattering sterile glass beads, filtering with sterilized absorbent cotton to remove mycelium, washing filter residue with normal saline for 3 times,final dilution to a concentration of 1.0X 10 8 CFU·mL -1 The spore suspension of (1).
Prepared by diffusion method using agar filter paper sheet, each sterile culture dish (90 mm) with 20 mL LB, YEPD, PDA culture medium, after coagulation, 100 μ L of thallus concentration is 1 × 10 8 CFU·mL -1 The suspension was spread evenly on the surface of the medium and after the surface was slightly dry, a 6 mm sterile filter paper disk impregnated with 10 μ L of the antimicrobial agent was attached to the top layer and sterile saline was used as a negative control. The antibacterial activity of the antibacterial agents of examples 1-3 and comparative examples 1-3 was evaluated by measuring the diameter of the zone of inhibition after 24 h were cultured with bacteria at 37 ℃ and 48 h was cultured with mold at 30 ℃, and the results are shown in table 1. Bacteriostatic rate (%) = (control diameter-treated diameter)/(control diameter-6) × 100%.
TABLE 1
In the inhibition experiment of the xanthomonas campestris, the examples 1-3 use the plant antibacterial source and the microorganism antibacterial source, and the inhibition rate is more than 98 percent; comparative example 1, a plant antibacterial source is used alone, and the bacteriostasis rate is 35%; comparative example 2, the microbial antibacterial source is separated, and the bacteriostatic rate is 41%; the sum of the bacteriostatic effects of the plant antibacterial source and the microorganism antibacterial source which are used independently is 76 percent, which is lower than that of the embodiment 3 which uses the plant antibacterial source and the microorganism antibacterial source simultaneously, so that the plant antibacterial source and the microorganism antibacterial source which are used simultaneously have the synergistic promotion effect on the aspect of inhibiting and killing bacteria.
In the inhibition experiment of the aspergillus niger, the plant antibacterial source and the microorganism antibacterial source are used in the examples 1-3, and the bacteriostasis rate is more than 98 percent; comparative example 1, a plant antibacterial source is used alone, and the bacteriostasis rate is 31 percent; comparative example 2 a microbial antibacterial source is separated, and the bacteriostasis rate is 46%; the sum of the bacteriostatic effects of the plant antibacterial source and the microorganism antibacterial source which are used independently is 77 percent, which is lower than that of the embodiment 3 which uses the plant antibacterial source and the microorganism antibacterial source simultaneously, therefore, the invention uses the plant antibacterial source and the microorganism antibacterial source simultaneously to have the synergistic promotion function in the aspect of inhibiting and killing fungi.
In conclusion, the antibacterial agent of the present invention has a synergistic effect in inhibiting and killing bacteria and fungi.
2. Minimum inhibitory and minimum lethal concentration determination
With reference to the American Clinical Laboratory Standards Institute (CLSI) microdilution protocol, 100. Mu.L of diluted antimicrobial agent (final concentrations 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, and 0 mg. ML) were added to sterile 96-well flat-bottomed microplates -1 ) With the cell concentration of 1X 10 8 CFU·mL -1 The bacterial suspensions were mixed and cultured at 37 ℃ for 24 h, and the optical density was measured using a multifunctional microplate reader 600 nm. An optical density change of less than 0.05 was considered as the lowest concentration for inhibiting the growth of the cells and was defined as the Minimum Inhibitory Concentration (MIC). The minimal lethal concentration (MBC) was defined as the number of colonies per plate less than 5 after 100. Mu.L of bacterial suspension was pipetted from wells greater than the minimal inhibitory concentration and spread evenly on fresh LB agar plates and subcultured at 37 ℃ for 24 h. Referring to the American Clinical Laboratory Standards Institute (CLSI) microdilution method, in sterile 96-well flat-bottom micro culture, the experimental results are shown in Table 2.
TABLE 2
The minimum inhibitory concentration and the minimum lethal concentration of the antibacterial agents of examples 1-3 of the present invention are much smaller than those of comparative examples 1-2, indicating that the antibacterial efficacy of examples 1-3 is much higher than that of comparative examples 1-3.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (3)
1. The plant composite antibacterial agent containing the peony extract is characterized by comprising the following components in parts by mass: 20-30 parts of peony extract, 10-20 parts of jerusalem artichoke extract, 10-15 parts of coptis extract, 1-3 parts of streptomyces griseus, 1-3 parts of dead bacillus cereus and 1-3 parts of saccharomyces cerevisiae;
the preparation method of the peony extract comprises the following steps: drying whole peony plants, crushing to 100 meshes to obtain peony materials, adding a mixed enzyme solution into the peony materials for enzymolysis, wherein the addition amount of the mixed enzyme solution is 4-6 times of the weight of the peony materials, the mass concentration of cellulase in the mixed enzyme solution is 0.3mg/mL, the mass concentration of pectinase is 0.3mg/mL, the enzymolysis time is 2-4h, inactivating enzyme at 85-90 ℃, performing ultrasonic extraction on the solution after enzyme inactivation, wherein the ultrasonic frequency is 80-100kHz, the ultrasonic time is 0.5-1h, repeating for three times, separating and combining the extracting solution to obtain a peony extract;
the preparation method of the jerusalem artichoke extract comprises the following steps: oven drying folium Jerusalem artichoke and rhizome of Jerusalem artichoke, pulverizing to obtain Jerusalem artichoke mixture, adding ethanol with concentration of 85% and ethanol content 3 times of the Jerusalem artichoke mixture, heating and extracting at 95 deg.C for 1 hr, and separating extractive solution to obtain Jerusalem artichoke extract;
the preparation method of the coptis extract comprises the following steps: pulverizing Coptidis rhizoma, adding ethanol with concentration of 85% and the amount of ethanol 3 times of Coptidis rhizoma, heating and extracting at 90 deg.C for 1 hr, and separating the extractive solution to obtain Coptidis rhizoma extract;
the effective viable count of the streptomyces griseus in the antibacterial agent is more than or equal to 1 multiplied by 10 7 cfu/g, the effective viable count of dead bacillus vallismortis in the antibacterial agent is more than or equal to 1 multiplied by 10 7 cfu/g, the effective viable count of the saccharomyces cerevisiae in the antibacterial agent is more than or equal to 1 multiplied by 10 7 cfu/g;
The preservation number of the streptomyces griseus is CICC11002, the preservation number of the killed bacillus vallismortis is CICC21334, and the preservation number of the saccharomyces cerevisiae is CICC1288.
2. The method for preparing a composite plant antibacterial agent containing peony extract as claimed in claim 1, comprising the steps of:
mixing peony extract, jerusalem artichoke extract and Coptidis rhizoma extract, adding Streptomyces griseus, dead Bacillus cereus and Saccharomyces cerevisiae, and stirring to obtain antibacterial agent.
3. The use of the composite plant antibacterial agent containing peony extract according to claim 1 for controlling plant diseases, wherein said plant diseases are caused by one or more of fusarium oxysporum, aspergillus niger and xanthomonas campestris;
the antibacterial agent is diluted 200-500 times at application time, and is applied to plants by spraying or irrigating.
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