CN113261567A - Preparation method and application of earthworm polypeptide for promoting crop growth and resisting diseases - Google Patents
Preparation method and application of earthworm polypeptide for promoting crop growth and resisting diseases 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
- 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
- A01N63/50—Isolated enzymes; Isolated proteins
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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
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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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
- A01N65/40—Liliopsida [monocotyledons]
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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 provides a preparation method and application of earthworm polypeptide for promoting crop growth and resisting diseases, which comprises the following steps: (1) cleaning and crushing earthworms to obtain earthworm meat paste, adding 1-10 times of distilled water, adjusting the pH value to 6-8, and reacting for 12-48 hours; (2) inoculating bacillus subtilis in the reacted feed liquid for fermentation culture, and inactivating the obtained fermentation product; (3) ultrafiltering the inactivated feed liquid, and intercepting to small molecular filtrate of 1-10 ten thousand daltons; (4) adding rhizoma Acori Graminei extract into the filtrate, and mixing under magnetic stirring. The earthworm polypeptide for promoting the growth of crops and resisting diseases is beneficial to the growth of crops, and improves the disease resistance of crops.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a preparation method and application of earthworm polypeptide for promoting crop growth and resisting diseases.
Background
Earthworms are invertebrate soil animals with the largest biomass in the terrestrial ecosystem, and the organisms of the earthworms can provide nutrients needed by animals such as proteins, amino acids, vitamins, mineral elements and the like, and bioactive components such as antioxidant enzymes, thrombolytic enzymes, hemolysins, antitumor components and the like. The Lumbricus has various pharmacological effects in treating cardiovascular diseases, cancer, asthma, etc., and also has effects in enhancing immunity, and promoting wound healing. The earthworm contains rich nutrient substances required by human body, including protein, nucleic acid, trace elements and vitamins, in which the total content of protein is 45.90-68.11% of dry mass. The earthworm can obtain micromolecular polypeptide and amino acid through enzymatic hydrolysis, not only can provide balanced and easily absorbed nutrient substances, but also has physiological activities of oxidation resistance, immunity enhancement, antibiosis and the like, and has potential application prospect in the field of health food. Earthworms are rich in protease and are easily subjected to autolytic enzymolysis under certain conditions. The current process research on preparing amino acid by animal and plant protein shows that the enzymatic hydrolysis process is often the first choice, because the enzymatic hydrolysis reaction condition is mild, the reaction efficiency is high, the amino acid is not damaged, and the racemization effect is not generated; meanwhile, part of small molecular peptides generated by enzymatic hydrolysis have biological activity, and can promote the immunity and the production performance of animals to be improved.
As a common animal medicinal material, the earthworm peptide has various chemical components and complex structures, is mostly a high-molecular organic compound, has been widely applied due to obvious pharmacological activity and rich resources, but has relatively weak basic research on chemical components, pharmacology and the like, so that the deep clinical application of the earthworm peptide is greatly limited, and the earthworm peptide as a natural source active peptide has the advantages of small molecular weight, relatively easy preparation, high purity, weak antigenicity, relatively clear function, strong specificity, small toxic and side effects, easy multi-way absorption and the like which are incomparable with macromolecular proteins. At present, no report is found about the application and specific pharmacological action research of earthworm polypeptides.
Disclosure of Invention
In view of the above, the present invention provides a preparation method and application of earthworm polypeptide for promoting crop growth and resisting diseases, which aims to overcome the defects in the prior art.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
a preparation method of earthworm polypeptide for promoting crop growth and resisting diseases comprises the following steps:
(1) cleaning and crushing earthworms to obtain earthworm meat paste, adding 1-10 times of distilled water, adjusting the pH value to 6-8, and reacting for 12-48 hours;
(2) inoculating bacillus subtilis in the reacted feed liquid for fermentation culture, and inactivating the obtained fermentation product;
(3) ultrafiltering the inactivated feed liquid, and intercepting to small molecular filtrate of 1-10 ten thousand daltons;
(4) adding rhizoma Acori Graminei extract into the filtrate, and mixing under magnetic stirring.
Further, the time of the fermentation step in the step (2) is 12-48 hours, and the temperature is 32-36 ℃.
Further, the grassleaf sweelflag rhizome extract in the step (4) is prepared by the following steps: cleaning rhizoma Acori Graminei, oven drying, pulverizing, adding 10-50 times of ethanol solution into the obtained rhizoma Acori Graminei powder, performing first ultrasonic extraction, filtering after extraction, adding 10-50 times of ethanol solution into the residue, performing second ultrasonic extraction, filtering after extraction, mixing filtrates, and concentrating to obtain the final product.
Further, the concentration of the ethanol solution is 40-80%; the temperature and the time of the first ultrasonic extraction and the second ultrasonic extraction are the same or different and are the same or different; the temperature of the first ultrasonic extraction is 20-50 ℃, and the time is 20-120 min; the temperature of the second ultrasonic extraction is 20-50 deg.C, and the time is 20-120 min.
Further, the mass ratio of the earthworms to the grassleaf sweelflag rhizome is 1: 1-100.
Preferably, the mass ratio of the earthworms to the grassleaf sweelflag rhizome is 1: 30-80.
Preferably, the mass ratio of the earthworms to the grassleaf sweelflag rhizome is 1: 42-65.
An earthworm polypeptide for promoting the growth of crops and preventing diseases is prepared from earthworm through dissolving, fermenting and mixing with the extract of grass-leaved sweetflag rhizome.
Further, bacillus subtilis is adopted in the fermentation step for inoculation culture; the time of the fermentation step is 12-48 hours, and the temperature is 32-36 ℃.
Further, the grassleaf sweelflag rhizome extract is prepared by the following steps: cleaning rhizoma Acori Graminei, oven drying, pulverizing, adding 10-50 times of ethanol solution into the obtained rhizoma Acori Graminei powder, performing first ultrasonic extraction, filtering after extraction, adding 10-50 times of ethanol solution into the residue, performing second ultrasonic extraction, filtering after extraction, mixing filtrates, and concentrating to obtain the final product.
Further, the mass ratio of the earthworms to the grassleaf sweelflag rhizome is 1: 1-100.
An application of earthworm polypeptide for promoting the growth of crops and preventing diseases is disclosed.
The rhizoma acori graminei is the rhizome of the acori graminei of the Araceae family, is pungent and bitter in taste and warm in nature, has the functions of inducing resuscitation and eliminating phlegm, refreshing and benefiting intelligence, and resolving dampness and stimulating appetite, and is widely used for treating diseases such as apoplectic aphasia coma, epilepsy, phlegm syncope, amnesia, senile dementia and other intelligence disorders in clinic. The medicinal part and chemical components of rhizoma Acori Graminei include volatile oil, flavones, quinones, alkaloids, triterpenoid saponins, phenylpropanoids, organic acids, amino acids and saccharides. Rhizoma Acori Graminei has effects of tranquilizing, resisting depression, resisting dementia, protecting cardiac muscle cell, invigorating stomach, resisting oxidation, relieving cough and asthma, resisting bacteria and inflammation, and relieving fatigue.
Compared with the prior art, the invention has the following advantages:
the earthworm polypeptide for promoting the growth of crops and resisting diseases is prepared by mixing earthworms which are dissolved and fermented with a grassleaf sweelflag rhizome extract, a large amount of micromolecule peptide and amino acid are obtained by ultrafiltration after the earthworms are dissolved and fermented, the grassleaf sweelflag rhizome extract contains a large amount of substances such as flavone, volatile oil and the like, and the micromolecule peptide and the amino acid obtained after the earthworms are dissolved and fermented act on the crops together, so that the growth of the crops is facilitated, and the disease resistance of the crops is improved.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The bacillus subtilis adopts a commercial product.
The present invention will be described in detail with reference to examples.
Example 1
Cleaning 100g of earthworm, crushing into meat paste, adding 5 times of distilled water, uniformly mixing, adjusting the pH value of the mixture to about 7, and dissolving for 24 hours.
Activating and culturing bacillus subtilis, inoculating the bacillus subtilis into the dissolved mixture, fermenting for 24 hours at 35 ℃, centrifuging for 10 minutes after the fermentation is finished, taking supernate for inactivation treatment, and then performing ultrafiltration through an ultrafiltration membrane of 100000D.
Weighing 5000g of rhizoma acori graminei, cleaning, drying, crushing, adding 20 times of 60% ethanol solution into the obtained rhizoma acori graminei powder, carrying out first ultrasonic extraction for 1 hour at 30 ℃, filtering after extraction, adding 20 times of ethanol solution into filter residues, carrying out second ultrasonic extraction for 1 hour at 30 ℃, filtering after extraction, combining filtrates, and concentrating the filtrate to obtain the rhizoma acori graminei extract.
Adding rhizoma Acori Graminei extract into the filtrate after ultrafiltration, and magnetically stirring at 35 deg.C for 2 hr to obtain Lumbricus polypeptide.
Example 2
The only difference from example 1 is that the grass-leaved sweetflag was 1000 g.
Example 3
The only difference from example 1 is that the grass-leaved sweetflag is 50 g.
Example 4
Cleaning 100g of earthworm, crushing into meat paste, adding 5 times of distilled water, uniformly mixing, adjusting the pH value of the mixture to about 7, and dissolving for 24 hours.
Centrifuging the mixture after dissolving for 10 minutes, taking supernatant for inactivation treatment, and then performing ultrafiltration through an ultrafiltration membrane of 100000D.
Weighing 5000g of rhizoma acori graminei, cleaning, drying, crushing, adding 20 times of 60% ethanol solution into the obtained rhizoma acori graminei powder, carrying out first ultrasonic extraction for 1 hour at 30 ℃, filtering after extraction, adding 20 times of ethanol solution into filter residues, carrying out second ultrasonic extraction for 1 hour at 30 ℃, filtering after extraction, combining filtrates, and concentrating the filtrate to obtain the rhizoma acori graminei extract.
Adding rhizoma Acori Graminei extract into the filtrate after ultrafiltration, and magnetically stirring at 35 deg.C for 2 hr to obtain Lumbricus polypeptide.
Example 5
Cleaning 100g of earthworm, crushing into meat paste, adding 5 times of distilled water, uniformly mixing, adjusting the pH value of the mixture to about 7, and dissolving for 24 hours.
Activating and culturing bacillus subtilis, inoculating the bacillus subtilis into the dissolved mixture, fermenting for 24 hours at 35 ℃, centrifuging for 10 minutes after fermentation is finished, taking supernate, inactivating, and ultrafiltering by using an ultrafiltration membrane of 100000D to obtain the earthworm polypeptide.
Example 6
Cleaning 100g of earthworm, crushing into meat paste, adding 5 times of distilled water, uniformly mixing, adjusting the pH value of the mixture to about 7, and dissolving for 24 hours.
Activating and culturing bacillus subtilis, inoculating the bacillus subtilis into the dissolved mixture, fermenting for 24 hours at 35 ℃, centrifuging for 10 minutes after the fermentation is finished, and filtering to obtain a supernatant.
Weighing 5000g of rhizoma acori graminei, cleaning, drying, crushing, adding 20 times of 60% ethanol solution into the obtained rhizoma acori graminei powder, carrying out first ultrasonic extraction for 1 hour at 30 ℃, filtering after extraction, adding 20 times of ethanol solution into filter residues, carrying out second ultrasonic extraction for 1 hour at 30 ℃, filtering after extraction, combining filtrates, and concentrating the filtrate to obtain the rhizoma acori graminei extract.
Adding rhizoma Acori Graminei extract into the filtrate after ultrafiltration, and magnetically stirring at 35 deg.C for 2 hr to obtain Lumbricus polypeptide.
Example 7 determination of crop growth promoting Effect of earthworm Polypeptides
70 tomato plants with consistent growth vigor are selected for testing, the tomato plants are divided into seven groups, the earthworm polypeptides prepared in the examples 1-6 are applied to the first six groups respectively, the last group is used as a blank control, water and fertilizer are applied by a conventional method, and disease management is enhanced in the testing period. The tomato index was measured after 30 days and is shown in table 1.
TABLE 1 tomato growth results
| Plant height (cm) | Fresh weight of underground (g) | Fresh weight of overground part (g) | |
| Example 1 | 52.29±2.51 | 35.06±1.63 | 5.12±0.41 |
| Example 2 | 49.57±6.45 | 33.12±2.41 | 4.93±0.28 |
| Example 3 | 45.31±4.16 | 27.34±2.70 | 4.35±0.73 |
| Example 4 | 42.35±5.74 | 26.56±3.62 | 3.84±0.54 |
| Example 5 | 40.12±3.02 | 24.16±1.40 | 3.50±0.39 |
| Example 6 | 46.23±1.85 | 29.41±3.05 | 4.49±0.71 |
| Control group | 38.71±5.36 | 22.59±1.67 | 3.07±0.34 |
As can be seen from Table 1, the earthworm polypeptide of the present invention has a significant effect of promoting the growth of tomato, the mass ratio of earthworm to Acorus tatarinowii Schott is 1:42-65, and the growth rate of tomato plant decreases with the decrease of Acorus tatarinowii Schott, while the growth state of the tomato plant in example 5 without Acorus tatarinowii Schott is significantly different from that in example 1; in example 4, the effect on the growth rate of tomato plants is reduced without fermentation culture of the self-solubilized earthworm liquid, indicating that fermentation is critical for earthworm polypeptides.
Example 8 determination of the disease-resistant Effect of earthworm Polypeptides
70 tomato plants with consistent growth vigor are selected for testing and divided into seven groups, the earthworm polypeptides prepared in the examples 1-6 are respectively applied to the first six groups every day for 2 weeks, the last group is used as a blank control, each test group is applied by a conventional water-fertilizer method, and disease management is enhanced during the test period. After 2 weeks, each tomato plant was inoculated with tobacco mosaic virus, and then the first six groups were continued to be administered the earthworm polypeptides prepared in examples 1-6 daily for 2 weeks.
The specific symptoms of tobacco mosaic virus infection are: bright vein symptoms appear on tender leaves, namely semitransparent phenomena appear on side veins and branch vein tissues of the leaves; the leaf has the symptom of uneven thickness, the leaf has spots, and different yellow-green areas are presented; necrosis of leaf tissue, large area of brown necrotic spots on tobacco leaves, distortion and shrinkage of leaf shape; the heavily diseased lobes form bubbles with inwardly curved edges.
The results of the above observations of tomato plants are shown in Table 2.
TABLE 2 tomato disease resistance results
As can be seen from Table 2, the earthworm polypeptide prepared by the invention has obvious disease-resistant effect and certain disease-resistant effect on tomato plants infected with tobacco mosaic virus. Under the condition of containing the earthworm micromolecule peptide alone or simultaneously containing the earthworm micromolecule peptide and the grassleaf sweelflag rhizome extract, the tomato extract has the disease-resistant function on tomato plants infected with tobacco mosaic virus; under the condition of simultaneously containing the earthworm small molecular peptide and the grassleaf sweelflag rhizome extract, the earthworm polypeptide has obvious disease prevention effect, and the effect is not obvious when the earthworm small molecular peptide is singly used for preventing diseases.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A preparation method of earthworm polypeptide for promoting crop growth and resisting diseases is characterized in that: the method comprises the following steps:
(1) cleaning and crushing earthworms to obtain earthworm meat paste, adding 1-10 times of distilled water, adjusting the pH value to 6-8, and reacting for 12-48 hours;
(2) inoculating bacillus subtilis in the reacted feed liquid for fermentation culture, and inactivating the obtained fermentation product;
(3) ultrafiltering the inactivated feed liquid, and intercepting to small molecular filtrate of 1-10 ten thousand daltons;
(4) adding rhizoma Acori Graminei extract into the filtrate, and mixing under magnetic stirring.
2. The method for preparing the earthworm polypeptide for promoting crop growth and resisting diseases according to claim 1, wherein the earthworm polypeptide comprises the following components: the time of the fermentation step in the step (2) is 12-48 hours, and the temperature is 32-36 ℃.
3. The method for preparing the earthworm polypeptide for promoting crop growth and resisting diseases according to claim 1, wherein the earthworm polypeptide comprises the following components: the rhizoma acori graminei extract in the step (4) is prepared by the following steps: cleaning rhizoma Acori Graminei, oven drying, pulverizing, adding 10-50 times of ethanol solution into the obtained rhizoma Acori Graminei powder, performing first ultrasonic extraction, filtering after extraction, adding 10-50 times of ethanol solution into the residue, performing second ultrasonic extraction, filtering after extraction, mixing filtrates, and concentrating to obtain the final product.
4. The method for preparing the earthworm polypeptide for promoting crop growth and resisting diseases according to claim 3, wherein the earthworm polypeptide comprises the following components: the concentration of the ethanol solution is 40-80%; the temperature and the time of the first ultrasonic extraction and the second ultrasonic extraction are the same or different and are the same or different; the temperature of the first ultrasonic extraction is 20-50 ℃, and the time is 20-120 min; the temperature of the second ultrasonic extraction is 20-50 deg.C, and the time is 20-120 min.
5. The method for preparing the earthworm polypeptide for promoting crop growth and resisting diseases according to claim 3, wherein the earthworm polypeptide comprises the following components: the mass ratio of the earthworms to the grassleaf sweelflag rhizome is 1: 1-100.
6. The method for preparing the earthworm polypeptide for promoting crop growth and resisting diseases according to claim 5, wherein the earthworm polypeptide comprises the following components: the mass ratio of the earthworms to the grassleaf sweelflag rhizome is 1: 30-80.
7. The method for preparing the earthworm polypeptide for promoting crop growth and resisting diseases according to claim 5, wherein the earthworm polypeptide comprises the following components: the mass ratio of the earthworms to the grassleaf sweelflag rhizome is 1: 42-65.
8. An earthworm polypeptide for promoting crop growth and resisting diseases, which is characterized in that: the earthworm polypeptide is prepared by mixing earthworm self-dissolving, fermented and rhizoma acori graminei extract.
9. The application of earthworm polypeptide for promoting crop growth and resisting diseases is characterized in that: the application of the earthworm polypeptide in promoting the growth of crops and resisting diseases.
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