Earthworm protein extract as well as extraction method and application thereof

Abstract

The invention provides an earthworm protein extract as well as an extraction method and application thereof. The earthworm protein extract can be applied to agricultural production. The extraction method comprises the following steps: preparing cleaned earthworms into earthworm homogenate; adding water into the earthworm homogenate, and carrying out solid-liquid separation to obtain an extracting solution containing earthworm protein; then protease is added for enzymolysis; and performing purification treatment on the extracting solution after enzymolysis to obtain the earthworm protein extract. The extraction method of the earthworm protein extract is simple, extraction parameters are controllable, and the extracted earthworm protein is high in extraction rate and purity. The earthworm protein extract is rich in eighteen amino acids, various nucleotides, FA proteins and other components, can provide essential raw materials for plant metabolism, is beneficial to plant tissues to coordinate cell division and differentiation activities, can effectively improve the yield, quality and resistance of crops, and has the effects of improving the soil structure, improving the soil fertility and the like.

Description

Technical Field

[0001] The invention belongs to the field of earthworm protein extraction, and specifically relates to an earthworm protein extract, an extraction method thereof and application in agricultural production. Background Art

[0002] Earthworm protein is extracted from earthworms and contains collagenase, plasmin, lumbrokinase, fibrinolytic activating protein (FA protein), nucleic acids, trace elements and other components. Its molecular weight is between 5000 and 10000 and it is a short-chain small molecule substance.

[0003] Earthworms are also called earthworms. Earthworms are rich in protein. The protein content of earthworms is 56-66% on a dry basis, which is 1.5 times that of soybeans and 6 times that of rice. Earthworm protein contains a variety of free amino acids, which are the main nutrients of earthworms. Its protein is composed of 18 kinds of amino acids, among which leucine and glutamic acid have the highest content, with 22.58 grams of leucine and 5.35 grams of glutamic acid per 100 grams of sample, respectively.

[0004] Pheretima also contains more nucleotide components such as hypoxanthine, xanthine, guanine, uracil, etc., which can provide essential raw materials for plant metabolism. It is one of the precursors for the synthesis of various cytokinins. Various cytokinin molecules contain adenine, which is provided to plant tissues, which is beneficial for plant tissues to coordinate their own cell division and differentiation activities. The purpose of adding cytokinins to the culture medium is mainly to promote cell division and differentiation of adventitious buds from callus tissue or organs.

[0005] Earthworms are rich in trace elements such as strontium, selenium, magnesium, zinc, copper, molybdenum, nickel, cobalt, etc. These elements are essential to ensure plant health. For example, nitrogen is the basis of important biological molecules such as proteins and nucleic acids, and is crucial for the growth and development of crops, leaf growth and photosynthesis; phosphorus participates in the energy transfer of plant cells, nucleic acid synthesis and other processes, and plays an important role in the development of the root system and the differentiation of flower buds; potassium helps to improve the resistance of crops to stress, such as lodging resistance, disease and pest resistance, etc.

[0006] Earthworms can provide rich nutrients for microorganisms in the soil, thereby promoting the growth and reproduction of microorganisms. Experimental results show that the use of fertilizers containing earthworm protein can reduce the use of chemical fertilizers by 22.6%.

[0007] The aggregate structure can also enhance soil stability and reduce soil erosion and loss. In addition, earthworms secrete some mucus and substances when they move in the soil. These substances work together with earthworm protein to make the soil looser and reduce soil compaction, thereby increasing the growth rate and yield of crops.

[0008] Earthworm protein contains some substances that can stimulate the growth of plant roots, such as auxin and other plant growth regulators. These substances can promote the division and elongation of root cells, increase the length and density of the root system, and enable crops to better absorb nutrients and water from the soil. Earthworm protein can also enhance the stress resistance of crop roots, enabling them to better cope with adverse environmental conditions such as drought, floods, pests and diseases.

[0009] After using earthworm protein, the immunity of crops will be improved, and the plants can produce more resistance substances, such as phytoalexins and polyphenols, which can enhance the resistance of plants to diseases and pests and make crops grow more healthily. Earthworm protein can also promote the photosynthesis and nutrient absorption of crops, allowing crops to accumulate more organic substances, such as sugars, proteins, vitamins, etc. For some fruit crops, earthworm protein can promote the ripening of fruits, so that the fruits can be put on the market earlier and increase farmers' income.

[0010] The combination of earthworm protein with amino acids, trace elements, macroelements, calcium and magnesium has the following effects on agriculture: earthworm protein combined with these nutrients can play a synergistic role. Earthworm protein contains rich biologically active substances, such as enzymes and growth regulators, which can promote the absorption and utilization of nutrients by plants. For example, the enzymes in earthworm protein can decompose organic matter in the soil and release more nutrients for plants to absorb; growth regulators can regulate the growth and development of plants and improve their ability to adapt to adversity.

[0011] At the same time, earthworm protein can also promote the activity of soil microorganisms, regulate soil pH, and create a suitable growth environment for plants. The combination of earthworm protein and nutrients can also improve plant resistance to pests and diseases, improve plant growth conditions, enhance stress resistance, promote plant growth and development, improve photosynthesis efficiency, and promote flower bud differentiation and fruit development.

[0012] In short, the combination of earthworm protein with amino acids, trace elements, macroelements, calcium and magnesium can provide comprehensive nutrition for plants, improve the soil environment, enhance the stress resistance of plants, promote the growth and development of plants, and improve the yield and quality of agricultural products. However, during use, the fertilization plan should be reasonably adjusted according to the needs of different plants and soil conditions to achieve the best effect.

[0013] It is precisely because earthworms contain rich nutrients that are beneficial to plant growth that the extraction of nutrients from earthworms has become one of the current research hotspots.

[0014] For example, the patent application with application number: 201710255679.2 discloses a method for extracting earthworm protein, comprising the following steps: 1) taking living earthworms, removing impurities, and crushing after treating with liquid nitrogen; 2) adding anhydrous ethanol, soaking, centrifuging, and removing the supernatant; 3) adding perchloric acid solution, leaching, centrifuging, and extracting the supernatant; 4) adding activated carbon, stirring and standing, centrifuging, and extracting the supernatant; 5) adjusting the pH value of the supernatant to 5-6 with a phosphate buffer, adding sodium sulfate powder, precipitating, and obtaining a crude extract of earthworm protein; 6) dissolving the crude extract of earthworm protein with a pH7.2 20mM Tris-HCl buffer, purifying with a DEAE-Sephadex ion exchange method, collecting the eluted components, and dialyzing, concentrating, and freeze-drying the eluted components to obtain the earthworm protein. The preparation method of the present invention is simple, short in cycle, good in extraction effect, and high in purity. The obtained earthworm protein can prolong the thrombin time and effectively reduce the total number of EOS in the BALF of mice. Summary of the invention

[0015] Based on the above technical background, the main purpose of the present invention is to provide a earthworm protein extract, an extraction method and application thereof, so as to overcome the deficiencies in the prior art.

[0016] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0017] The first aspect of the present invention is to provide a method for extracting a protein extract of earthworm, the method comprising the following steps:

[0018] Step 1, adding earthworms into physiological saline, stirring and washing, adding the washed earthworms into water, stirring, and obtaining earthworm homogenate;

[0019] Step 2, adding water to the earthworm homogenate, heating and stirring, and then performing solid-liquid separation to obtain an extract containing earthworm protein;

[0020] Step 3, adding protease to the extract containing earthworm protein, heating, adjusting the pH to perform enzymolysis, and obtaining an extract after enzymolysis;

[0021] Step 4: ultrafiltration the extract after enzymatic hydrolysis, and then purify, concentrate and dry to obtain the earthworm protein extract.

[0022] In step 1,

[0023] Preferably, water is added to the cleaned earthworm, the amount of water added is 9 to 12 times the mass of the earthworm, and the mixture is stirred at a stirring speed of 8000 to 12000 rpm for 15 to 30 minutes.

[0024] In step 2,

[0025] Preferably, water is added to the earthworm homogenate, and the volume ratio of the earthworm homogenate to water is 1:(1.5-3).

[0026] Preferably, after adding water, the mixture is heated to 35-45° C. and stirred at a stirring speed of 9000-11000 rpm for 1-3 hours.

[0027] In step 3,

[0028] Preferably, the protease is selected from one or more of Bacillus subtilis neutral protease and Bacillus polymyxa neutral protease.

[0029] Preferably, the added amount of the protease is 0.15-0.2% of the total mass of the extract containing earthworm protein.

[0030] Preferably, after the protease is added, the mixture is heated to 40-50° C., the pH is adjusted to 6.8-7.3, and the enzymolysis is carried out under this condition for 4-7 hours.

[0031] In step 4,

[0032] Preferably, the extract after enzymatic hydrolysis is first subjected to macromolecular ultrafiltration using an ultrafiltration membrane of 9000 to 10000 Daltons, and then subjected to small molecule ultrafiltration using an ultrafiltration membrane of 5000 to 6000 Daltons, the ultrafiltration flow rate is 10 to 50 ml / min, the ultrafiltration pressure is 0.1 to 0.5 MPa, and the retained earthworm protein molecular weight is 5000 to 10000 earthworm protein liquid.

[0033] Preferably, the concentration condition is: concentration at a temperature of 35 to 45° C. for 2 to 4 hours.

[0034] The second aspect of the present invention is to provide an earthworm protein extract obtained by extracting the earthworm protein extract according to the method for extracting the earthworm protein extract according to the first aspect of the present invention.

[0035] The third aspect of the present invention is to provide an application of the earthworm protein extract described in the second aspect of the present invention in agricultural production.

[0036] The beneficial effects of the present invention are:

[0037] (1) The extraction method of the present invention first uses physiological saline and water to clean the earthworm, then adds water, heats and stirs to promote the full release of protein components in the earthworm, so that the effective ingredients in the earthworm are gradually dissolved in the water; then adds protease, heats and adjusts the pH to perform enzymatic hydrolysis, the enzymatic hydrolysis process can promote the further release and decomposition of earthworm protein, and heating enzymatic hydrolysis can make the earthworm protein molecules smaller and improve its bioavailability; finally, the extract after enzymatic hydrolysis is subjected to ultrafiltration treatment, purification, concentration and drying in sequence, purification can effectively separate the earthworm protein from other impurities, and concentration can increase the concentration of the protein.

[0038] (2) The extraction method of the earthworm protein extract of the present invention is simple, and the parameters in the extraction process are controllable. The earthworm protein extract obtained has a high extraction rate and high purity. The extraction rate can reach 86.6% or more, and the purity can reach 93.2% or more.

[0039] The earthworm protein extracted by the extraction method can improve soil fertility, improve soil structure, promote root growth, enhance stress resistance, improve the quality of agricultural products, promote photosynthesis and nutrient absorption, and increase the content of organic matter in agricultural products.

[0040] (3) The earthworm protein extract contains aspartic acid, valine, lysine, arginine, alanine, glycine, methionine, serine, threonine, phenylalanine, proline, histidine and tyrosine. The earthworm protein extract is beneficial for plant tissues to coordinate their own cell division and differentiation activities, and can also promote the expansion and quality improvement of fruits. Applying earthworm protein to the soil can provide a continuous and stable nutrient supply for crops, promote the growth and development of crops, and a large amount of experimental data shows that it can improve the fertilizer efficiency of conventional fertilizers by 17.73%.

[0041] Earthworm protein extract can promote the aggregation of soil particles and form more aggregate structures. It has good air permeability and water retention, allowing air and water to flow freely in the soil, providing sufficient oxygen and water for the roots of crops.

[0042] The protein extract of earthworm contains some antibacterial and antiviral substances, which can inhibit the growth and reproduction of pathogens and viruses in the soil, reduce the chance of crops being infected by diseases and pests, and improve the immunity of crops.

[0043] The combined use of earthworm protein extract and nutrients can increase the organic matter content in the soil, improve soil structure and increase soil fertility.

[0044] (4) The earthworm protein extract has broad application prospects in agricultural production. Specifically, the earthworm protein extract combined with microorganisms has the following effects on agriculture: (1) It can enhance the stability of soil aggregate structure. A good aggregate structure can improve the air permeability, water retention and fertilizer retention of the soil, creating a suitable environment for the growth of plant roots. (2) Reduce the degree of soil compaction: After combining earthworm protein liquid with microbial agents, the problem of soil compaction can be effectively improved, the growth of fruit tree roots can be promoted, and the yield and quality of fruit trees can be improved. (3) Increase the nutrient content of the soil: Earthworm protein liquid contains rich nutrients such as nitrogen, phosphorus, and potassium, which can directly provide nutrients for plants. The beneficial microorganisms in the microbial agent can decompose organic matter in the soil and convert it into effective nutrients that can be absorbed and utilized by plants. The combination of the two can significantly increase the nutrient content in the soil and meet the needs of plant growth. (4) Promote nutrient recycling: The microorganisms in the microbial agent can participate in the recycling and transformation process of nitrogen, phosphorus, potassium and other elements in the soil. The active ingredients in earthworm protein liquid can stimulate the growth and metabolic activities of microorganisms, promote the recycling of nutrients, and improve the sustainability of soil fertility. (5) Stimulate the growth of plant roots: Earthworm protein liquid contains some growth regulators, such as auxins, cytokinins, etc., which can stimulate the growth and development of plant roots. The beneficial microorganisms in microbial agents can form a symbiotic relationship with plant roots, such as the symbiosis between mycorrhizal fungi and plant roots, which can expand the absorption area of ​​plant roots and improve the root system's ability to absorb nutrients and water. The combination of the two can significantly promote the growth of plant roots and enhance plant growth potential. (6) Enhance plant resistance to stress: The beneficial microorganisms in microbial agents can produce some stress resistance substances, such as antibiotics, chitinase, etc., which can enhance the plant's disease resistance, insect resistance, and stress resistance. The combination of the two can significantly improve the plant's resistance to stress, reduce the occurrence of diseases and pests, reduce the use of pesticides, and improve the quality and safety of vegetables. (7) Reduce the use of chemical fertilizers and pesticides: The combination of earthworm protein liquid and microbial agents can improve soil fertility, promote plant growth, and reduce dependence on chemical fertilizers. It can achieve sustainable agricultural development and protect the ecological environment. (8) Promote the balance of soil ecosystems: The combination of earthworm protein liquid and microbial agents can increase the number of beneficial microorganisms in the soil, improve the structure of soil microbial communities, promote the balance of soil ecosystems, and improve the sustainability of agricultural production. DETAILED DESCRIPTION

[0045] The present invention will be described in detail below, and the characteristics and advantages of the present invention will become clearer and more distinct with the description.

[0046] The first aspect of the present invention is to provide a method for extracting a protein extract of earthworm, the method comprising the following steps:

[0047] Step 1, adding earthworms into physiological saline, stirring and washing, adding the washed earthworms into water, stirring, and obtaining earthworm homogenate;

[0048] Step 2, adding water to the earthworm homogenate, heating and stirring, and then performing solid-liquid separation to obtain an extract containing earthworm protein;

[0049] Step 3, adding protease to the extract containing earthworm protein, heating, adjusting the pH to perform enzymolysis, and obtaining an extract after enzymolysis;

[0050] Step 4: ultrafiltration the extract after enzymatic hydrolysis, and then purify, concentrate and dry to obtain the earthworm protein extract.

[0051] The above steps are described in detail below.

[0052] In step 1, add the earthworms to saline solution and stir to remove dirt and impurities on the surface of the earthworms, then rinse the earthworms repeatedly with clean water until the cleaning solution is clear.

[0053] The stirring is preferably carried out in a mixer, and water is added to the cleaned earthworm, the amount of water added is 9 to 12 times the mass of the earthworm, and the stirring is carried out at a stirring speed of 8000 to 12000 rpm for 15 to 30 minutes.

[0054] Preferably, water is added to the cleaned earthworm, the amount of water added is 11 times the mass of the earthworm, and stirring is carried out at a stirring speed of 12000 rpm for 20 minutes.

[0055] In step 2, water is added to the earthworm homogenate, and the volume ratio of the earthworm homogenate to water is 1:(1.5-3).

[0056] Preferably, the volume ratio of earthworm homogenate to water is 1:2.

[0057] After adding water, heat to 35-45°C and stir at a stirring speed of 9000-11000 rpm for 1-3 hours to gradually dissolve the active ingredients in the earthworm into the water. During this process, continuous stirring is required to promote the full release of the ingredients.

[0058] Preferably, after adding water, the mixture is heated to 40° C. and stirred at a stirring speed of 10,000 rpm for 2 hours.

[0059] After the extraction is completed by heating and stirring, solid-liquid separation is performed, preferably by filtering or the like, to obtain an extract containing earthworm protein.

[0060] In step 3, the protease is selected from one or more of Bacillus subtilis neutral protease and Bacillus polymyxa neutral protease.

[0061] Preferably, the protease is Bacillus subtilis neutral protease, so as to promote the further release and decomposition of earthworm protein.

[0062] The added amount of the protease is 0.15-0.2% of the total mass of the extract containing earthworm protein.

[0063] Preferably, the added amount of the protease is 0.18% of the total mass of the extract containing earthworm protein.

[0064] After adding protease, heat to 40-50°C, adjust the pH to 6.8-7.3, and perform enzymolysis under this condition for 4-7 hours. The heating enzymolysis process can make the earthworm protein molecules smaller and improve its bioavailability.

[0065] Preferably, after the protease is added, the mixture is heated to 43° C., the pH is adjusted to 6.8, and the enzymolysis is carried out under this condition for 6 hours.

[0066] In step 4, the extract after enzymolysis is first subjected to macromolecular ultrafiltration using an ultrafiltration membrane of 9000 to 10000 Daltons, and then subjected to small molecule ultrafiltration using an ultrafiltration membrane of 5000 to 6000 Daltons. The ultrafiltration flow rate is 10 to 50 ml / min, and the ultrafiltration pressure is 0.1 to 0.5 MPa to ensure effective separation of earthworm protein, retaining earthworm protein liquid with a molecular weight of 5000 to 10000, and macromolecular impurities and small molecule ineffective components can be removed by ultrafiltration.

[0067] Preferably, the extract after enzymatic hydrolysis is first subjected to macromolecular ultrafiltration using a 9000 Dalton ultrafiltration membrane, and then subjected to small molecule ultrafiltration using a 5000 Dalton ultrafiltration membrane. The ultrafiltration flow rate is 35 ml / min and the ultrafiltration pressure is 0.25 MPa.

[0068] After ultrafiltration, the earthworm protein is purified by gel filtration chromatography to effectively separate the earthworm protein from other impurities.

[0069] The purified earthworm protein solution is concentrated to increase the protein concentration by using a reverse osmosis method.

[0070] The concentration conditions are: concentration at a temperature of 35 to 45° C. for 2 to 4 hours to avoid protein denaturation and loss.

[0071] Preferably, the concentration condition is: concentration at 40° C. for 3 h.

[0072] The concentrated earthworm protein is frozen and ground into powder using dry ice freeze-drying method.

[0073] The second aspect of the present invention is to provide an earthworm protein extract obtained by extracting the earthworm protein extract according to the method for extracting the earthworm protein extract according to the first aspect of the present invention.

[0074] The third aspect of the present invention is to provide an application of the earthworm protein extract described in the second aspect of the present invention in agricultural production.

[0075] Example

[0076] The present invention is further described below by means of specific examples. These embodiments are only intended to illustrate the present invention but are not intended to limit the scope of the present invention.

[0077] Example 1

[0078] Select fresh, disease-free earthworms, and perform preliminary cleaning on the earthworms to remove dirt and impurities on the surface. Put the fresh earthworms into a container, add physiological saline, stir to remove dirt and impurities on the surface of the earthworms, and then rinse the earthworms repeatedly with clean water until the cleaning solution is clear. Put the cleaned earthworms into a blender, add water, the amount of water added is 11 times the mass of the earthworms, and stir at a stirring speed of 12000rpm for 20 minutes to obtain earthworm homogenate.

[0079] Place the earthworm homogenate into an extraction container, then add water, the volume ratio of the earthworm homogenate to water is 1:2, then heat to 40°C, stir at a stirring speed of 10,000 rpm for 2 hours, and after the heating and stirring extraction is completed, use filtration to separate the solid and liquid to obtain an extract containing earthworm protein.

[0080] Bacillus subtilis neutral protease is added to the extract containing earthworm protein, the added amount of the protease is 0.18% of the total mass of the extract containing earthworm protein, and then heated to 43° C., adjusted to pH 7, and enzymolysis is carried out under this condition for 6 hours.

[0081] The extract after enzymolysis was first subjected to ultrafiltration of macromolecules using an ultrafiltration membrane of 9000 Daltons, and then subjected to ultrafiltration of small molecules using an ultrafiltration membrane of 5000 Daltons. The flow rate of ultrafiltration was 35 ml / min, and the pressure of ultrafiltration was 0.25 MPa to ensure the effective separation of earth dragon protein, retaining earth dragon protein with a molecular weight of 5000-10000 earth dragon protein liquid, and ultrafiltration could remove macromolecular impurities and small molecule ineffective components. After ultrafiltration, the earth dragon protein was purified by gel filtration chromatography to effectively separate the earth dragon protein from other impurities. The purified earth dragon protein solution was concentrated at 40 ° C for 3 hours. Reverse osmosis was used for concentration. After concentration, dry ice freeze drying was used for drying, and the concentrated earth dragon protein was frozen and ground into powder to obtain earth dragon protein extract. The extraction rate of the earth dragon protein extract was measured to be 95.8% and the purity was 99.0%.

[0082] Example 2

[0083] Select fresh, disease-free earthworms, perform preliminary cleaning on the earthworms, and remove the dirt and impurities on the surface. Put the fresh earthworms into a container, add physiological saline, stir to remove the dirt and impurities on the surface of the earthworms, and then rinse the earthworms repeatedly with clean water until the cleaning solution is clear. Put the cleaned earthworms into a stirrer, add water, the amount of water added is 9 times the mass of the earthworms, stir at a stirring speed of 8000rpm for 15 minutes to obtain earthworm homogenate.

[0084] Place the earthworm homogenate into an extraction container, then add water, the volume ratio of the earthworm homogenate to water is 1:1.5, then heat to 35°C, stir at a stirring speed of 9000rpm for 3 hours, and after the heating and stirring extraction is completed, use filtration to separate the solid and liquid to obtain an extract containing earthworm protein.

[0085] Bacillus subtilis neutral protease is added to the extract containing earthworm protein, wherein the amount of the protease added is 0.15% of the total mass of the extract containing earthworm protein, and then the extract is heated to 40° C., the pH is adjusted to 6.8, and enzymolysis is performed under this condition for 7 hours.

[0086] The extract after enzymatic hydrolysis was first subjected to macromolecular ultrafiltration using a 9000 Dalton ultrafiltration membrane, and then subjected to small molecule ultrafiltration using a 5000 Dalton ultrafiltration membrane. The flow rate of ultrafiltration was 10 ml / min, and the pressure of ultrafiltration was 0.1 MPa to ensure the effective separation of earth dragon protein, retaining earth dragon protein with a molecular weight of 5000 to 10000. The earth dragon protein liquid can remove macromolecular impurities and small molecule ineffective components by ultrafiltration. After ultrafiltration, the earth dragon protein was purified by gel filtration chromatography to effectively separate the earth dragon protein from other impurities. The purified earth dragon protein solution was concentrated at 35 ° C for 4 hours. Reverse osmosis was used for concentration. After concentration, dry ice freeze drying was used for drying, and the concentrated earth dragon protein was frozen and ground into powder to obtain earth dragon protein extract. The extraction rate of the earth dragon protein extract was measured to be 86.6% and the purity was 96.5%.

[0087] Example 3

[0088] Select fresh, disease-free earthworms, perform preliminary cleaning on the earthworms, and remove the dirt and impurities on the surface. Put the fresh earthworms into a container, add physiological saline, stir to remove the dirt and impurities on the surface of the earthworms, and then rinse the earthworms repeatedly with clean water until the cleaning solution is clear. Put the cleaned earthworms into a blender, add water, the amount of water added is 12 times the mass of the earthworms, and stir at a stirring speed of 11000rpm for 30min to obtain earthworm homogenate.

[0089] Place the earthworm homogenate into an extraction container, then add water, the volume ratio of the earthworm homogenate to water is 1:3, then heat to 45°C, stir at a stirring speed of 11000rpm for 1h, after the heating and stirring extraction is completed, use filtration to separate the solid and liquid to obtain an extract containing earthworm protein.

[0090] Bacillus subtilis neutral protease is added to the extract containing earthworm protein, the added amount of the protease is 0.2% of the total mass of the extract containing earthworm protein, and then heated to 50° C., adjusted to pH 7.3, and enzymolysis is carried out under this condition for 4 hours.

[0091] The extract after enzymolysis was first subjected to macromolecular ultrafiltration using an ultrafiltration membrane of 10,000 Daltons, and then subjected to micromolecular ultrafiltration using an ultrafiltration membrane of 6,000 Daltons. The flow rate of ultrafiltration was 50 ml / min, and the pressure of ultrafiltration was 0.5 MPa to ensure the effective separation of earth dragon protein, retaining earth dragon protein with a molecular weight of 5,000-10,000. The macromolecular impurities and small molecular ineffective components can be removed by ultrafiltration. After ultrafiltration, the earth dragon protein was purified by gel filtration chromatography to effectively separate the earth dragon protein from other impurities. The purified earth dragon protein solution was concentrated at 45 ° C for 2 hours. Reverse osmosis was used for concentration. After concentration, dry ice freeze drying was used for drying, and the concentrated earth dragon protein was frozen and ground into powder to obtain earth dragon protein extract. The extraction rate of the earth dragon protein extract was measured to be 91.4% and the purity was 93.2%.

[0092] The present invention has been described in detail above in conjunction with specific implementations and exemplary examples, but these descriptions cannot be understood as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, a variety of equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its implementation methods, all of which fall within the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims.

Claims

1. A method for extracting earthworm protein extract, characterized in that: The extraction method comprises the following steps: Step 1, adding earthworms into physiological saline, stirring and washing, adding the washed earthworms into water, stirring, and obtaining earthworm homogenate; Step 2, adding water to the earthworm homogenate, heating and stirring, and then performing solid-liquid separation to obtain an extract containing earthworm protein; Step 3, adding protease to the extract containing earthworm protein, heating, adjusting the pH to perform enzymolysis, and obtaining an extract after enzymolysis; Step 4: ultrafiltration the extract after enzymatic hydrolysis, and then purify, concentrate and dry to obtain the earthworm protein extract.

2. The extraction method according to claim 1, characterized in that In step 1, Add water to the cleaned earthworms, the amount of water added is 9 to 12 times the mass of the earthworms, and stir at a stirring speed of 8000 to 12000 rpm for 15 to 30 minutes.

3. The extraction method according to claim 1, characterized in that In step 2, Add water to the earthworm homogenate, the volume ratio of the earthworm homogenate to water is 1: (1.5-3).

4. The extraction method according to claim 1, characterized in that In step 2, After adding water, heat to 35-45°C and stir at a stirring speed of 9000-11000 rpm for 1-3 hours.

5. The extraction method according to claim 1, characterized in that In step 3, The protease is selected from one or more of Bacillus subtilis neutral protease and Bacillus polymyxa neutral protease.

6. The extraction method according to claim 1, characterized in that In step 3, The added amount of the protease is 0.15-0.2% of the total mass of the extract containing earthworm protein.

7. The extraction method according to claim 1, characterized in that In step 3, After adding protease, heat to 40-50°C, adjust the pH to 6.8-7.3, and perform enzymolysis under this condition for 4-7 hours.

8. The extraction method according to claim 1, characterized in that In step 4, The extract after enzymatic hydrolysis is firstly subjected to macromolecular ultrafiltration using an ultrafiltration membrane of 9000-10000 Daltons, and then subjected to micromolecular ultrafiltration using an ultrafiltration membrane of 5000-6000 Daltons, the flow rate of ultrafiltration is 10-50 ml / min, the pressure of ultrafiltration is 0.1-0.5 MPa, and the earthworm protein liquid with a molecular weight of 5000-10000 is retained; The concentration conditions are: concentration at a temperature of 35 to 45° C. for 2 to 4 hours.

9. A Pheretima protein extract obtained by the extraction method according to any one of claims 1 to 8.

10. Use of the earthworm protein extract according to claim 9 in agricultural production.