CN112575048A - Development method of pine tree immunity inducing agent - Google Patents
Development method of pine tree immunity inducing agent Download PDFInfo
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- CN112575048A CN112575048A CN202011470817.7A CN202011470817A CN112575048A CN 112575048 A CN112575048 A CN 112575048A CN 202011470817 A CN202011470817 A CN 202011470817A CN 112575048 A CN112575048 A CN 112575048A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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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
- 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
- A01N37/44—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 containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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Abstract
The invention discloses a method for developing a pine tree immunity inducer, and relates to the technical field of plant immunity inducers. The method comprises the following steps of degrading soybean polypeptide powder, and filtering to obtain a first raw material; dissolving 1-n-dodecyl nitrogen heterocyclic cycloheptane-2-ketone by using absolute ethyl alcohol to obtain a second raw material; preparing fatty alcohol-polyoxyethylene ether into an aqueous solution to obtain a third raw material, sequentially adding the first raw material, the second raw material and the third raw material into water for mixing, and heating the mixed solution in a water bath at 35-50 ℃ for 10-12 hours under the assistance of ultrasonic waves to obtain the pine tree immune resistance inducer. The soybean polypeptide powder is degraded and filtered, and then is mixed with the second raw material and the third raw material, so that the soybean polypeptide powder can conveniently permeate through xylem of the plant to be efficiently absorbed and utilized, the preparation cost of the soybean polypeptide powder is reduced by preparing the soybean polypeptide powder through soybean protein, and meanwhile, the plant immunity inducer prepared by taking the soybean polypeptide powder as the key raw material can obviously improve the disease resistance, stress resistance and yield increasing capability of the plant.
Description
Technical Field
The invention belongs to the technical field of plant immunity inducer, and particularly relates to a method for developing pine immunity inducer.
Background
The plant immunity inducer is also called plant vaccine, is a new field of vaccine engineering technology after human vaccine and animal vaccine, and is a popular research direction for creating biological pesticides internationally. When the plant is stimulated by the outside or is in a stress condition, the plant can generate immune reaction by regulating the defense and metabolic systems of the plant, the defense reaction or immune resistance reaction of the plant can delay or relieve the occurrence and development of diseases of the plant, reduce the use amount of chemical pesticides and chemical fertilizers and reduce the residue of agricultural products, and the plant immune inducer has no direct killing effect on crop diseases and insect pests, but generates resistance substances by inducing or activating the plant by exogenous organisms or molecules to generate resistance on certain pathogenic substances or inhibit the growth of pathogenic bacteria. After the pesticide composition is applied to crops, the crop is induced to generate substances for resisting or preventing and controlling crop diseases and insect pests, so that the aim of preventing and controlling the diseases and the insect pests is fulfilled.
The research shows that the active peptide has the characteristics of high permeability, high conductivity and good absorption, can obviously improve the disease resistance, stress resistance and yield increase of plants, and can be used as a key raw material for preparing the plant immunity inducer.
Disclosure of Invention
The invention aims to provide a method for developing a pine immune inducer, which is characterized in that soybean polypeptide powder is degraded and filtered, then the soybean polypeptide powder is mixed with a second raw material and a third raw material of the soybean polypeptide powder, the soybean polypeptide powder conveniently permeates through the cuticle of a plant and is efficiently absorbed and utilized, the soybean polypeptide powder is prepared through soybean protein, the preparation cost is reduced, meanwhile, an immune induction factor, namely an organic active small molecule (active peptide), required by the pine immune inducer prepared by taking the soybean polypeptide powder as a key raw material can be rapidly permeated and conducted to each part of a tree body, the defense reaction of the pine can be activated, and the tree body is induced to generate substances for resisting nematodes. Can obviously improve the disease resistance and stress resistance of pine trees and solve the problems of difficult absorption, low efficacy and high preparation cost of the existing polypeptide.
In order to solve the technical problems, the invention is realized by the following technical scheme:
the invention relates to a method for developing a pine immune resistance inducer, which comprises the following steps:
the method comprises the following steps: degrading soybean polypeptide powder, and filtering to obtain a first raw material;
step two: dissolving 1-n-dodecyl azepin-2-ketone in absolute ethyl alcohol to obtain a second raw material, wherein the weight ratio of the 1-n-dodecyl azepin-2-ketone to the absolute ethyl alcohol is 1: 3-1: 5;
step three: preparing fatty alcohol-polyoxyethylene ether into an aqueous solution to obtain a third raw material, wherein the weight ratio of the fatty alcohol-polyoxyethylene ether to water is 1: 4-1: 6;
step four: sequentially adding the first raw material, the second raw material and the third raw material into water, mixing, and heating the mixed solution in a water bath at 35-50 ℃ for 10-12 hours under the assistance of ultrasonic waves to obtain the pine tree immune resistance inducer.
Preferably, the soybean polypeptide powder is prepared by adopting the following method:
putting the soybean hydrolysate into a microwave reactor, adding 200-4000 ml of distilled water or purified water, uniformly stirring, adjusting the pH value of the soybean hydrolysate to 7.0-9.0 by using potassium hydroxide, keeping the soybean hydrolysate for 10-30 minutes under the microwave condition with the microwave power of 500-1000 w, performing denaturation treatment on the soybean hydrolysate, and cooling to obtain a soybean protein isolate aqueous solution;
adding alkaline protease into the obtained soybean protein aqueous solution, starting a microwave generator to increase the temperature to 30-70 ℃ for enzymolysis, wherein the microwave power is 100-1000 w, the microwave energy is continuously supplied during the enzymolysis, and the microwave enzymolysis time is maintained for 5-30 minutes to obtain soybean protein isolate enzymolysis liquid;
increasing the microwave power of the obtained soybean protein isolate enzymolysis liquid to 500-2000 w, keeping the soybean protein isolate enzymolysis liquid for 2-10 minutes at the temperature of more than 90 ℃, inactivating enzyme, cooling the soybean protein isolate enzymolysis liquid to 30-50 ℃, filtering the soybean protein isolate enzymolysis liquid by using a microfiltration membrane, and removing filter residues to obtain soybean protein isolate enzymolysis filtrate;
adding activated carbon powder accounting for 1-3% of the volume of the soybean protein isolate enzymolysis filtrate into the soybean protein isolate enzymolysis filtrate, adjusting the pH value of the soybean protein isolate enzymolysis filtrate to 3.0-7.0 by using oxalic acid, maintaining the temperature of the soybean protein isolate enzymolysis filtrate at 30-80 ℃, carrying out decoloration and debitterizing treatment for 1-3 hours, then filtering the soybean protein isolate enzymolysis filtrate, and removing waste activated carbon residues to obtain a soybean polypeptide fine filtrate;
and carrying out low-temperature vacuum concentration and spray drying treatment on the obtained soybean polypeptide fine filtrate to obtain soybean polypeptide powder.
Preferably, the buffer solution is acetic acid-sodium acetate solution, and the pH value of the buffer solution is between 4 and 6.
Preferably, the soy hydrolysate is prepared by the following method:
adding cathepsin L into soybean protein, adjusting the pH value to 5.5-8.5, stirring at room temperature for 20-60 minutes, and reacting in a water bath at 35-42 ℃ for 2-24 hours to obtain the soybean protein cathepsin L, wherein the weight ratio of the soybean protein to the cathepsin L is 100-1000: 1.
Preferably, the usage amount of each raw material is as follows: the weight parts of the first raw material, the second raw material, the third raw material and the water are respectively 4-6, 2-5, 0.5-2 and 85-95.
The invention has the following beneficial effects:
1. the soybean polypeptide powder is degraded and filtered, and then is mixed with the second raw material and the third raw material, so that the soybean polypeptide powder can conveniently permeate the plant cuticle to be efficiently absorbed and utilized.
2. The soybean polypeptide powder is prepared from the soybean protein, so that the preparation cost is reduced;
3. the plant immunity inducer prepared by taking the soybean polypeptide powder as the key raw material can obviously improve the disease resistance, stress resistance and yield increasing capability of plants.
Of course, it is not necessary for any product in which the invention is practiced to achieve all of the above-described advantages at the same time.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
FIG. 1 is a MALDI mass spectrum of molecular weight of the soybean peptide of the present invention.
The first embodiment is as follows:
the invention relates to a method for developing a pine immune resistance inducer, which comprises the following steps:
the method comprises the following steps: degrading soybean polypeptide powder, and filtering to obtain a first raw material;
step two: dissolving 1-n-dodecyl azepin-2-ketone in absolute ethyl alcohol to obtain a second raw material, wherein the weight ratio of the 1-n-dodecyl azepin-2-ketone to the absolute ethyl alcohol is 1: 3-1: 5;
step three: preparing fatty alcohol-polyoxyethylene ether into an aqueous solution to obtain a third raw material, wherein the weight ratio of the fatty alcohol-polyoxyethylene ether to water is 1: 4-1: 6;
step four: sequentially adding the first raw material, the second raw material and the third raw material into water, mixing, and heating the mixed solution in a water bath at 35-50 ℃ for 10-12 hours under the assistance of ultrasonic waves to obtain the pine tree immune resistance inducer.
The soybean polypeptide powder is prepared by the following method:
putting the soybean hydrolysate into a microwave reactor, adding 200-4000 ml of distilled water or purified water, uniformly stirring, adjusting the pH value of the soybean hydrolysate to 7.0-9.0 by using potassium hydroxide, keeping the soybean hydrolysate for 10-30 minutes under the microwave condition with the microwave power of 500-1000 w, performing denaturation treatment on the soybean hydrolysate, and cooling to obtain a soybean protein isolate aqueous solution;
adding alkaline protease into the obtained soybean protein aqueous solution, starting a microwave generator to increase the temperature to 30-70 ℃ for enzymolysis, wherein the microwave power is 100-1000 w, the microwave energy is continuously supplied during the enzymolysis, and the microwave enzymolysis time is maintained for 5-30 minutes to obtain soybean protein isolate enzymolysis liquid;
increasing the microwave power of the obtained soybean protein isolate enzymolysis liquid to 500-2000 w, keeping the soybean protein isolate enzymolysis liquid for 2-10 minutes at the temperature of more than 90 ℃, inactivating enzyme, cooling the soybean protein isolate enzymolysis liquid to 30-50 ℃, filtering the soybean protein isolate enzymolysis liquid by using a microfiltration membrane, and removing filter residues to obtain soybean protein isolate enzymolysis filtrate;
adding activated carbon powder accounting for 1-3% of the volume of the soybean protein isolate enzymolysis filtrate into the soybean protein isolate enzymolysis filtrate, adjusting the pH value of the soybean protein isolate enzymolysis filtrate to 3.0-7.0 by using oxalic acid, maintaining the temperature of the soybean protein isolate enzymolysis filtrate at 30-80 ℃, carrying out decoloration and debitterizing treatment for 1-3 hours, then filtering the soybean protein isolate enzymolysis filtrate, and removing waste activated carbon residues to obtain a soybean polypeptide fine filtrate;
and carrying out low-temperature vacuum concentration and spray drying treatment on the obtained soybean polypeptide fine filtrate to obtain soybean polypeptide powder.
The buffer solution is acetic acid-sodium acetate solution, and the pH value of the buffer solution is between 4 and 6.
The soybean hydrolysate is prepared by the following method:
adding cathepsin L into soybean protein, adjusting the pH value to 5.5-8.5, stirring at room temperature for 20-60 minutes, and reacting in a water bath at 35-42 ℃ for 2-24 hours to obtain the soybean protein cathepsin L, wherein the weight ratio of the soybean protein to the cathepsin L is 100-1000: 1.
The usage amount of each raw material is as follows: 4-6 parts of first raw material, 2-5 parts of second raw material, 0.5-2 parts of third raw material and 85-95 parts of water.
Example two:
indoor pine tree immunity elicitor permeability test: taking two groups of pine samples, one group of which is 3-5 parts, selecting one group of the pine samples to be injected with the pine immune resistance-inducing agent prepared by the invention, injecting other immune resistance-inducing agents in the market into the other group of the pine samples in the same time, standing for 24 hours, observing the medicament absorption conditions of the two groups of the pine samples through an optical microscope, and obtaining a conclusion by observing the permeation conditions of the medicaments of the two groups of the pine samples, wherein the permeability of the pine immune resistance-inducing agent prepared by the invention is higher than 85 percent, and the permeability of the common pine immune resistance-inducing agent is 60 percent, so the pine immune resistance-inducing agent disclosed by the invention can be absorbed and utilized efficiently by penetrating through the xylem of plants.
Through the research, the liquid is cut into the micromolecular peptide with the molecular weight of between 500 and 2000 under the high-speed shearing machine after the soybean enzyme is hydrolyzed into the polypeptide, the better permeation, conduction and absorption effects are achieved, the polypeptide is generated after the soybean enzyme is hydrolyzed, and the polypeptide is cut into the proper molecular weight through the high-speed shearing machine, and the polypeptide is called as the active peptide. The active peptide has the characteristics of high permeability, high conductivity and good absorption, and can improve the disease resistance and stress resistance of pine trees.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (5)
1. A method for developing a pine tree immunity inducer is characterized by comprising the following steps: the method comprises the following steps:
the method comprises the following steps: degrading soybean polypeptide powder, and filtering to obtain a first raw material;
step two: dissolving 1-n-dodecyl azepin-2-ketone in absolute ethyl alcohol to obtain a second raw material, wherein the weight ratio of the 1-n-dodecyl azepin-2-ketone to the absolute ethyl alcohol is 1: 3-1: 5;
step three: preparing fatty alcohol-polyoxyethylene ether into an aqueous solution to obtain a third raw material, wherein the weight ratio of the fatty alcohol-polyoxyethylene ether to water is 1: 4-1: 6;
step four: sequentially adding the first raw material, the second raw material and the third raw material into water, mixing, and heating the mixed solution in a water bath at 35-50 ℃ for 10-12 hours under the assistance of ultrasonic waves to obtain the pine tree immune resistance inducer.
2. The method for developing the pine tree immunity inducer according to claim 1, wherein the soybean polypeptide powder is prepared by the following method:
putting the soybean hydrolysate into a microwave reactor, adding 200-4000 ml of distilled water or purified water, uniformly stirring, adjusting the pH value of the soybean hydrolysate to 7.0-9.0 by using potassium hydroxide, keeping the soybean hydrolysate for 10-30 minutes under the microwave condition with the microwave power of 500-1000 w, performing denaturation treatment on the soybean hydrolysate, and cooling to obtain a soybean protein isolate aqueous solution;
adding alkaline protease into the obtained soybean protein aqueous solution, starting a microwave generator to increase the temperature to 30-70 ℃ for enzymolysis, wherein the microwave power is 100-1000 w, the microwave energy is continuously supplied during the enzymolysis, and the microwave enzymolysis time is maintained for 5-30 minutes to obtain soybean protein isolate enzymolysis liquid;
increasing the microwave power of the obtained soybean protein isolate enzymolysis liquid to 500-2000 w, keeping the soybean protein isolate enzymolysis liquid for 2-10 minutes at the temperature of more than 90 ℃, inactivating enzyme, cooling the soybean protein isolate enzymolysis liquid to 30-50 ℃, filtering the soybean protein isolate enzymolysis liquid by using a microfiltration membrane, and removing filter residues to obtain soybean protein isolate enzymolysis filtrate;
adding activated carbon powder accounting for 1-3% of the volume of the soybean protein isolate enzymolysis filtrate into the soybean protein isolate enzymolysis filtrate, adjusting the pH value of the soybean protein isolate enzymolysis filtrate to 3.0-7.0 by using oxalic acid, maintaining the temperature of the soybean protein isolate enzymolysis filtrate at 30-80 ℃, carrying out decoloration and debitterizing treatment for 1-3 hours, then filtering the soybean protein isolate enzymolysis filtrate, and removing waste activated carbon residues to obtain a soybean polypeptide fine filtrate;
and carrying out low-temperature vacuum concentration and spray drying treatment on the obtained soybean polypeptide fine filtrate to obtain soybean polypeptide powder.
3. The method for developing an immune resistance-inducing agent for pine trees according to claim 1, wherein the buffer solution is acetic acid-sodium acetate solution, and the pH value of the buffer solution is between 4 and 6.
4. The method for developing a pine tree immunity inducer according to claim 2, wherein the soybean hydrolysate is prepared by the following method:
adding cathepsin L into soybean protein, adjusting the pH value to 5.5-8.5, stirring at room temperature for 20-60 minutes, and reacting in a water bath at 35-42 ℃ for 2-24 hours to obtain the soybean protein cathepsin L, wherein the weight ratio of the soybean protein to the cathepsin L is 100-1000: 1.
5. The method for developing a pine tree immunity inducer according to claim 1, wherein the usage amount of each raw material is as follows: the weight parts of the first raw material, the second raw material, the third raw material and the water are respectively 4-6, 2-5, 0.5-2 and 85-95.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114907441A (en) * | 2022-05-05 | 2022-08-16 | 德州谷神蛋白科技有限公司 | Preparation method for improving functional characteristics of soybean protein isolate through microwave-assisted pH shift treatment |
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| US5888501A (en) * | 1994-07-26 | 1999-03-30 | Auburn University | Induced systemic resistance of plants to pathogenic microorganisms |
| CN102094059A (en) * | 2009-12-10 | 2011-06-15 | 陈栋梁 | Method for preparing soybean polypeptide by microwave-promoted hydrolysis of soybean protein |
| CN108041026A (en) * | 2017-12-15 | 2018-05-18 | 浙江海洋大学 | A kind of preparation method of high osmosis plant immune inducer |
| CN110037056A (en) * | 2019-03-15 | 2019-07-23 | 甘肃农业大学 | A kind of synergistic complex agent and its application |
| CN110063364A (en) * | 2019-05-15 | 2019-07-30 | 福建省亚热带植物研究所 | A kind of plant immune inducer and its application |
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2020
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5888501A (en) * | 1994-07-26 | 1999-03-30 | Auburn University | Induced systemic resistance of plants to pathogenic microorganisms |
| CN102094059A (en) * | 2009-12-10 | 2011-06-15 | 陈栋梁 | Method for preparing soybean polypeptide by microwave-promoted hydrolysis of soybean protein |
| CN108041026A (en) * | 2017-12-15 | 2018-05-18 | 浙江海洋大学 | A kind of preparation method of high osmosis plant immune inducer |
| CN110037056A (en) * | 2019-03-15 | 2019-07-23 | 甘肃农业大学 | A kind of synergistic complex agent and its application |
| CN110063364A (en) * | 2019-05-15 | 2019-07-30 | 福建省亚热带植物研究所 | A kind of plant immune inducer and its application |
Non-Patent Citations (3)
| Title |
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| 杨波等: "植物免疫诱抗剂的应用研究进展", 《中国植保导刊》 * |
| 邱德文: "我国植物免疫诱导技术的研究现状与趋势分析", 《植物保护》 * |
| 陈捷等: "《现代植物病理学研究方法》", 30 November 2007, 中国农业出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114907441A (en) * | 2022-05-05 | 2022-08-16 | 德州谷神蛋白科技有限公司 | Preparation method for improving functional characteristics of soybean protein isolate through microwave-assisted pH shift treatment |
| CN114907441B (en) * | 2022-05-05 | 2023-08-01 | 德州谷神蛋白科技有限公司 | Preparation method for improving functional characteristics of soy isolate protein by microwave-assisted pH shift treatment |
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