Method and equipment for extracting high-content earthworm protein

Abstract

The invention relates to the technical field of protein extraction, in particular to a high-content earthworm protein extraction method and device.The extraction method comprises the following steps that S1, a fluid-state protein protective agent and a granular-state protein protective agent are sequentially added into smashed live earthworms, and mixed powder is obtained; s2, water is added into the mixed powder for centrifugation, and supernate is obtained; s3, performing enzymolysis and filtration on the supernate; s4, the filtrate is subjected to activated carbon adsorption and freeze drying, and earthworm protein is obtained; the equipment sequentially comprises a pretreatment chamber, a centrifugal cylinder, an enzymolysis adsorption chamber and a freeze-drying chamber from left to right, according to the extraction method, the protein protective agent is immediately added after the earthworms are crushed, and protein degradation is prevented and protein integrity and biological activity are maintained by inhibiting endogenous protease activity and chelating metal ions; and the protein protective agent maintains the structural stability of the substrate, so that the subsequent enzymolysis is more sufficient, and the protein yield is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein extraction, in particular to a high-content earthworm protein extraction method and device. BACKGROUND

[0002] Earthworm protein is extracted from earthworms and contains collagenase, fibrinolysin, acaenase, fibrinolysis activating protein (FA protein), nucleic acid, trace elements and other components, has a molecular weight of 5000-10000 and belongs to short-chain small molecular substances. Earthworm protein has a wide influence on the blood clotting system and fibrinolysis system in the body, can significantly reduce the platelet adhesion rate of rats, prolong the formation of blood clots in the body and dissolve blood clots in the body, increase cerebral blood flow, reduce cerebral vascular resistance, improve microcirculation and reduce the harm of three highs to the human body.

[0003] Since proteins are prone to inactivation or degradation during extraction, the existing extraction amount of earthworm protein is usually not high. Therefore, the present application decides to design a high-content earthworm protein extraction method and device to optimize this problem. SUMMARY

[0004] In order to solve the above problems, the present application provides a high-content earthworm protein extraction method and device.

[0005] A high-content earthworm protein extraction method, comprising the following steps:

[0006] S1, after the live earthworm is removed, grinding and crushing are carried out at 0-4℃, and mixed with a fluid-state protein protective agent at a solid-liquid ratio of 1g:6-8ml, and an ultrasonic wave of 250-280W is applied for 10-12min, and after mixing is completed, vacuum drying is carried out to obtain pretreated powder with a particle size of 70-80μm; the obtained pretreated powder is mixed with a particle-state protein protective agent with a particle size of 80-100μm at a mass ratio of 1:0.4-0.6, and an ultrasonic wave of 420-460W is applied for 3-6min to obtain a mixed powder;

[0007] S2, water is added to the mixed powder obtained in S1 to obtain slurry with a water content of 80-85%, and the slurry is centrifuged at 10000-11000rpm for 10-15min to obtain supernatant;

[0008] S3, proteinase is added to the supernatant obtained in S2 for enzymolysis at a liquid-solid ratio of 1ml:0.03-0.05g, the temperature is 40-43℃, the time is 2.5-3h, after the enzymolysis is completed, the enzyme is inactivated at 95-115℃ for 1.5-2.5h to obtain an enzymolysis liquid, and the enzymolysis liquid is subjected to ultrafiltration using a first ultrafiltration membrane to obtain a filtrate;

[0009] S4, adding activated carbon into the filtrate obtained from S3 at a liquid-solid ratio of 1ml: 0.08-0.1g, stirring at 220-270rpm for 15-20min, standing for 5-10min, centrifuging at 9000-9500rpm for 15-20min to obtain supernatant, and then performing ultrafiltration using a second ultrafiltration membrane to obtain a retentate, and freeze-drying the retentate to obtain the earthworm protein.

[0010] Further, in S1, the preparation method of the fluid protein protective agent is as follows: mixing modified soybean fiber, sucrose and sorbitol solution with a mass concentration of 50-60% at a ratio of 5-7g: 3g: 4ml, performing homogenization treatment, magnetic stirring for 0.5-1.5h, and pressure of 65-75MPa to obtain a homogenate; then adding Tween 80 into the homogenate at a solid-liquid ratio of 1g: 40-45ml, emulsifying and dispersing for 40-50min to obtain the fluid protein protective agent.

[0011] The preparation method of the granular protein protective agent is as follows: mixing modified soybean fiber, genipin solution with a mass concentration of 0.8-1%, reduced glutathione, trehalose and vitamin C at a liquid-solid ratio of 4g: 10-12ml: 2g: 5g: 1g, mixing at 350-450rpm for 15-25min, and then performing freeze-drying to obtain the granular protein protective agent.

[0012] Description: Sorbitol in the fluid protein protective agent inhibits protease autolysis and reduces the generation of bitter peptides; sucrose maintains isotonic environment and reduces the dissolution of nucleic acid when cells are broken; Tween 80 and sucrose-sorbitol form an amphiphilic complex to wrap the hydrophobic groups of proteins, reduce hydrophobic aggregation in ultrasonic treatment, and improve solubility; in the granular protein protective agent, reduced glutathione contains free sulfhydryl groups, which can remove free radicals and reduce oxidized protein sulfhydryl groups, thereby enhancing the antioxidant capacity of the protein protection system and preventing the structural damage of earthworm protein caused by oxidation; genipin solution contains a large number of amino and hydroxyl groups, which can form hydrogen bonds with the hydroxyl groups in the modified soybean fiber, thereby enhancing the stability of the protein protection system and improving the yield of earthworm protein.

[0013] Further, the preparation method of the modified soybean fiber is as follows:

[0014] The soybean meal is crushed through a 400-mesh screen to obtain soybean residue, 0.7-0.9wt% of a composite enzyme is added into the soybean residue, and enzymolysis is performed at 40-45℃ and pH=6-8 for 2-3h; after the enzymolysis is completed, enzyme inactivation is performed at 95-105℃ for 5-10min, pressure treatment is performed at 0.4-0.5MPa for 2.5-3h, homogenization is performed at 50-55MPa for 1-2min, and finally drying is performed at -60--40℃ for 12-24h to obtain the soybean fiber.

[0015] The soybean fiber is immersed in an ethanol solution containing 70-80wt% birch bark extract at a solid-liquid ratio of 1g:15-25ml, the immersion temperature is 50-60℃, and the immersion time is 2.5-3.5h, and then vacuum drying is performed after the immersion is completed to obtain the modified soybean fiber.

[0016] Description: The soybean fiber formed by enzymatic hydrolysis of soybean meal has a porous structure and can adsorb earthworm proteins and buffer ultrasonic cavitation impact. The natural antioxidants (such as betulinol) in the ethanol solution containing 70-80wt% birch bark extract are embedded in the fiber network to inhibit protein oxidation and denaturation, and maintain the active conformation of heat-sensitive proteins such as plasmin.

[0017] Further, the complex enzyme is composed of cellulase, ginger protease and phytase at a mass ratio of 1:0.3-0.5:0.6.

[0018] Description: Cellulase degrades the cellulose skeleton in the soybean cell wall, destroys the fiber structure, and releases soluble dietary fiber. Ginger protease selectively hydrolyzes soybean antigenic proteins to reduce allergenicity. Phytase decomposes phytate-mineral complexes to release minerals such as calcium and phosphorus, thereby improving the nutritional value of the fiber. At the same time, phytase decomposes phytic acid molecules to remove the inhibition of protease.

[0019] Further, in S3, the protease is composed of bacillus licheniformis protease, trypsin and papain at a mass ratio of 1:1:0.6-0.8.

[0020] Description: Bacillus licheniformis protease efficiently cleaves fibrous proteins and collagen in earthworm tissues, destroys the intercellular matrix structure, and releases bound target proteins. Trypsin specifically cleaves the peptide bond on the carboxyl side of lysine and arginine, avoiding excessive hydrolysis that leads to loss of small fragments. Papain hydrolyzes hydrophobic antigenic peptides to reduce the allergenicity of earthworm proteins.

[0021] Further, in S3, the first ultrafiltration membrane has a molecular weight cut-off of 15000-20000Da.

[0022] Description: First, the first ultrafiltration membrane is used to remove macromolecular impurities.

[0023] Further, in S4, the second ultrafiltration membrane has a molecular weight cut-off of 5000-10000Da.

[0024] Description: Then, the second ultrafiltration membrane is used to remove proteins.

[0025] Further, in S4, the freeze-drying method is as follows: first, drying at -40 to -35℃ for 2-3h, then drying at -60 to -50℃ for 15-18h, and finally drying at 25-30℃ for 2-3h.

[0026] Description: First, low-temperature rapid freezing makes the sample water form small ice crystals, reduces mechanical damage to protein structure, blocks protease autolysis reaction, and maintains the natural conformation of heat-sensitive proteins such as plasmin, lumbriconase, etc.; then, ultra-low temperature combined with long-time vacuum drying makes the ice crystals directly sublimate, avoids protein aggregation caused by liquid phase, maintains the stability of proteins such as collagenase, superoxide dismutase, etc., and finally gradually warms up to room temperature to completely evaporate the bound water.

[0027] The device for the high-content earthworm protein extraction method according to any one of the preceding items comprises, from left to right, a pretreatment chamber, a centrifuge cylinder, an enzymatic adsorption chamber, and a freeze-drying chamber, and temperature controllers are arranged on the pretreatment chamber, the enzymatic adsorption chamber, and the freeze-drying chamber.

[0028] The inside of the pretreatment chamber is divided into a crushing cavity and a treatment cavity by a first partition plate, an ultrasonic generator, a storage box, and a feeding pipe communicating with the crushing cavity are arranged on the pretreatment chamber, a first discharge pipe for communicating the crushing cavity and the treatment cavity is arranged on the first partition plate, a filter screen is arranged in the first discharge pipe, the inside of the storage box is divided into a fluid-state protein protective agent storage cavity communicating with the inside of the crushing cavity and a granular-state protein protective agent storage cavity communicating with the inside of the treatment cavity, a water tank communicating with the inside of the treatment cavity is arranged on the treatment cavity, and a second discharge pipe communicating with the centrifuge cylinder is arranged on the treatment cavity.

[0029] The centrifuge cylinder is provided with a first liquid outlet pipe communicating with the enzymatic adsorption chamber, a second liquid outlet pipe communicating with the freeze-drying chamber, and a recovery box for recovering centrifugal solids and waste liquid.

[0030] The inside of the enzymatic adsorption chamber is divided into an enzymatic cavity and an adsorption cavity by a second partition plate, a protease storage box communicating with the inside of the enzymatic cavity is arranged on the enzymatic cavity, an activated carbon storage box communicating with the inside of the adsorption cavity is arranged on the adsorption cavity, a third discharge pipe for communicating the enzymatic cavity and the adsorption cavity is arranged on the second partition plate, a first ultrafiltration membrane is arranged in the third discharge pipe, and a fourth discharge pipe communicating with the centrifuge cylinder is arranged on the adsorption cavity.

[0031] The freeze-drying chamber is provided with a third liquid outlet pipe communicating with the recovery box, and a second ultrafiltration membrane is arranged on the third liquid outlet pipe.

[0032] Stirrers are arranged in the treatment cavity, the enzymatic cavity, and the adsorption cavity.

[0033] Further, a grinding roller connected to the side wall of the crushing cavity through a telescopic rod is arranged in the crushing cavity, the grinding roller is rotationally connected to the telescopic rod, gears are arranged at both ends of the grinding roller, and a rack meshing and driving the gears is arranged on the inner wall of the crushing cavity.

[0034] The grinding roller rotating and grinding in the crushing cavity improves the crushing efficiency of fresh earthworms.

[0035] Compared with the existing earthworm protein extraction method, the present application has the following advantages:

[0036] (1) In the extraction method of the present application, after the earthworm is crushed, the internal protein of the earthworm is exposed and is prone to denaturation due to oxidation, enzymatic hydrolysis or mechanical shearing. At this time, the fluid-state protein protective agent can quickly penetrate into the crushed tissue fragments and fully contact with the exposed protein molecules. The protein is wrapped by the fluid protective agent through hydrogen bonding, hydrophobic interaction and other ways. When dried, the fluid protective agent forms an amorphous glass state, which wraps the protein molecules, inhibiting the structural denaturation of the protein during the drying process. The protein is prone to structural instability during subsequent extraction and release, and the granular protein protective agent mixed with the pretreated powder can play a slow-release role, avoiding local supersaturation aggregation caused by protein burst release. Proteases hydrolyze non-target proteins in S3 stage to release small molecule active peptides, improve the content of earthworm protein, and remove macromolecular impurities by using the first ultrafiltration membrane. After adsorbing pigments and small molecular impurities by activated carbon in S4, the second ultrafiltration membrane is used to accurately intercept target proteins, so that the purity is improved.

[0037] (2) The fluid-state protein protective agent used in the present application has a porous structure formed by enzymatic hydrolysis of soybean meal, which can adsorb earthworm protein and buffer the impact of ultrasonic cavitation. The natural antioxidants (such as betulinol) in the 70-80wt% ethanol solution of birch bark extract embedded in the fiber network inhibit protein oxidation and denaturation, maintain the active conformation of heat-sensitive proteins such as plasmin, and the sorbitol in the protective agent inhibits protease autolysis and reduces the production of bitter peptides. Sucrose maintains isotonic environment, reduces the dissolution of nucleic acids when cells are broken, and Tween 80 forms an amphiphilic complex with sucrose-sorbitol to wrap the hydrophobic groups of proteins, reduce hydrophobic aggregation during ultrasonic treatment, and improve solubility. The reduced glutathione in the granular protein protective agent used in the present application contains free sulfhydryl groups, which can remove free radicals and reduce oxidized protein sulfhydryl groups, enhance the antioxidant capacity of the protein protection system, and prevent the structural damage of earthworm protein caused by oxidation. The genipin solution contains a large number of amino and hydroxyl groups, which can form hydrogen bonds with the hydroxyl groups in the soybean fiber and hydrophobic interactions with the polyphenols in the birch bark extract, thereby enhancing the stability of the protein protection system and improving the yield of earthworm protein.

[0038] (3) The extraction equipment of the present application can effectively extract earthworm protein in combination with the extraction method. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is the overall appearance diagram of the extraction equipment of the present application;

[0040] Figure 2 is the overall top view of the extraction equipment of the present application;

[0041] Figure 3 is the internal structure diagram of the extraction equipment of the present application;

[0042] Figure 4 is Figure 3 is an enlarged schematic view of I in the middle of the figure;

[0043] Figure 5 is a comparison chart of the results of the present invention research 1;

[0044] Figure 6 is a comparison chart of the results of the present invention research 2;

[0045] Figure 7 is a comparison chart of the results of the present invention research 3.

[0046] In the figure: 1-preprocessing chamber, 11-feeding pipe, 12-ultrasonic generator, 13-storage box, 14-water tank, 15-first partition, 151-first discharging pipe, 152-filter screen, 16-pulverizing cavity, 161-grinding roller, 162-telescopic rod, 163-gear, 164-rack, 17-treatment cavity, 171-second discharging pipe, 172-stirrer, 2-centrifugal cylinder, 21-first liquid outlet pipe, 22-second liquid outlet pipe, 23-recovery tank, 3-enzymatic adsorption chamber, 31-second partition, 311-third discharging pipe, 312-first ultrafiltration membrane, 32-enzymatic cavity, 321-protease storage box, 33-adsorption cavity, 221-fourth discharging pipe, 332-activated carbon storage box, 4-freeze-drying, 41-third liquid outlet pipe, 411-second ultrafiltration membrane. DETAILED DESCRIPTION

[0047] In order to further illustrate the manner of carrying out the present invention and the effects achieved, the technical solutions of the present invention will be described clearly and completely below in conjunction with experiments.

[0048] Example 1: A high-content earthworm protein extraction method, comprising the following steps:

[0049] S1, after removing impurities from the live earthworm, grinding and pulverizing at 2℃, mixing with fluid-state protein protectant at a solid-liquid ratio of 1g:7ml, and applying ultrasonic waves of 265W for 11min, vacuum drying after mixing is completed, the temperature of vacuum drying is 30℃, the vacuum degree is 10Pa, the time is 18h, obtaining pretreated powder with particle size of 73~77μm; mixing the obtained pretreated powder with granular protein protectant with particle size of 85~95μm at a mass ratio of 1:0.5, and applying ultrasonic waves of 440W for 5min, obtaining mixed powder;

[0050] The preparation method of the fluid protein protective agent is that modified soybean fiber, sucrose and sorbitol are mixed at 6 g:3 g:4 ml, homogenized, magnetic stirring for 1.5 h, and the pressure is 70 MPa to obtain a homogenate; then Tween 80 is added to the homogenate at a solid-liquid ratio of 1 g:43 ml, emulsified and dispersed for 45 min to obtain the fluid protein protective agent;

[0051] The preparation method of the granular protein protective agent is that modified soybean fiber, 0.9% genipin solution, reduced glutathione, trehalose and vitamin C are mixed at a liquid-solid ratio of 4 g:11 ml:2 g:5 g:1 g at 400 rpm for 20 min, and then freeze-dried, the freeze-drying temperature is-40℃, and the freeze-drying time is 12 h to obtain the granular protein protective agent;

[0052] The preparation method of the modified soybean fiber is that soybean meal is crushed through a 400-mesh sieve to obtain soybean residue, 0.8wt% of a composite enzyme is added to the soybean residue, and enzyme hydrolysis is carried out at 43℃ and pH=7 for 2.5 h; after the enzyme hydrolysis is completed, the enzyme is inactivated at 100℃ for 8 min, then pressure treatment is carried out at 0.45 MPa for 2.8 h, and then homogenization is carried out at 53 MPa for 1.5 min; finally, the soybean fiber is dried at-50℃ for 18 h; the composite enzyme is composed of cellulase, ginger protease and phytase at a mass ratio of 1:0.4:0.6;

[0053] The soybean fiber is immersed in an ethanol solution containing 75wt% white birch bark extract at a solid-liquid ratio of 1 g:20 ml, the immersion temperature is 55℃, and the immersion time is 3 h; after the immersion is completed, vacuum drying is carried out at 60℃ and 10 Pa for 1 h to obtain the modified soybean fiber;

[0054] S2, water is added to the mixed powder obtained in S1 to obtain a slurry with a water content of 83%, and then the slurry is centrifuged at 10500 rpm for 13 min to obtain a supernatant;

[0055] S3, a protease is added to the supernatant obtained in S2 at a liquid-solid ratio of 1 ml:0.04 g for enzyme hydrolysis, the protease is composed of bacillus licheniformis protease, trypsin and papain at a mass ratio of 1:1:0.7, the temperature is 42℃, the time is 2.8 h, and the pH is 7.5; after the enzyme hydrolysis is completed, the enzyme is inactivated at 105℃ for 2 h to obtain an enzyme hydrolysate; the enzyme hydrolysate is subjected to ultrafiltration using a first ultrafiltration membrane with a molecular weight cut-off of 15000-20000 Da to obtain a filtrate;

[0056] S4, adding activated carbon into the filtrate obtained from S3 at a liquid-solid ratio of 1ml:0.09g, stirring at 250rpm for 18min, standing for 8min, centrifuging at 9300rpm for 18min to obtain supernatant, and then performing ultrafiltration on the supernatant using a second ultrafiltration membrane with a molecular weight cut-off of 5000-10000Da to obtain a cut-off liquid, and then freeze-drying the cut-off liquid, wherein the freeze-drying method comprises: first drying at -38℃ for 2.5h, then drying at -55℃ for 16.5h, and finally drying at 28℃ for 2.5h, to obtain the pumilopus protein.

[0057] Example 2: The equipment used in the high-content pumilopus protein extraction method described in Example 1, as shown in Figure 1 and Figure 2 from left to right, sequentially comprising a pretreatment chamber 1, a centrifugal cylinder 2, an enzymatic adsorption chamber 3, and a freeze-drying chamber 4, wherein the pretreatment chamber 1, the enzymatic adsorption chamber 3, and the freeze-drying chamber 4 are each provided with a temperature controller 5;

[0058] as shown in Figure 3 and Figure 4 the inside of the pretreatment chamber 1 is divided into a crushing cavity 16 and a treatment cavity 17 by a first partition plate 15, the pretreatment chamber 1 is provided with an ultrasonic generator 12, a storage box 13, and a feeding pipe 11 communicating with the crushing cavity 16, the first partition plate 15 is provided with a first discharging pipe 151 for communicating the crushing cavity 16 and the treatment cavity 17, and the first discharging pipe 151 is provided with a filter screen 152, the inside of the storage box 13 is divided into a fluid-state protein protective agent storage cavity communicating with the inside of the crushing cavity 16 and a granular-state protein protective agent storage cavity communicating with the inside of the treatment cavity 17, the treatment cavity 17 is provided with a water tank 14 communicating with the inside thereof, and the treatment cavity 17 is provided with a second discharging pipe 171 communicating with the centrifugal cylinder 2; the crushing cavity 16 is provided with a grinding roller 161 connected to the side wall of the crushing cavity 16 through an extension rod 162, the grinding roller 161 is rotationally connected to the extension rod 162, both ends of the grinding roller 161 are provided with gears 163, and the inner wall of the crushing cavity 16 is provided with a rack 164 in meshing transmission with the gears 163;

[0059] as shown in Figure 3 the centrifugal cylinder 2 is provided with a first liquid outlet pipe 21 communicating with the enzymatic adsorption chamber 3, a second liquid outlet pipe 22 communicating with the freeze-drying chamber 4, and a recovery tank 23 for recovering centrifugal solids and waste liquid; the recovery tank 23 is provided with three; the centrifugal cylinder 2 is composed of a top cover, a cylinder portion rotationally and sealingly connected to the top cover, and a bottom plate communicating with the bottom of the cylinder portion and rotationally and sealingly connected to the top cover, the bottom plate connects the inside of the cylinder portion and the inside of the recovery tank 23 through a communication pipe, and the top cover is provided with a rotating motor connected to the inner bottom of the cylinder portion;

[0060] as shown in Figure 3As shown, the inside of the enzymatic desorption chamber 3 is divided into an enzymatic lysis cavity 32 and a desorption cavity 33 by the second partition plate 31, the upper part of the enzymatic lysis cavity 32 is provided with a protease storage box 321 in communication with the inside thereof, the upper part of the desorption cavity 33 is provided with an activated carbon storage box 332 in communication with the inside thereof, the second partition plate 31 is provided with a third discharge pipe 311 for communicating the enzymatic lysis cavity 32 and the desorption cavity 33, the third discharge pipe 311 is provided with a first ultrafiltration membrane 312, and the desorption cavity 33 is provided with a fourth discharge pipe 331 in communication with the centrifugal cylinder 2;

[0061] As shown in the figure, Figure 3 The freeze-drying chamber 4 is provided with a third liquid outlet pipe 41 in communication with the recovery tank 23, and the third liquid outlet pipe 41 is provided with a second ultrafiltration membrane 411.

[0062] As shown in the figure, Figure 3 The inside of the processing cavity 17, the enzymatic lysis cavity 32 and the desorption cavity 33 are all provided with a stirrer 172, the driving motor of the stirrer 172 can be embedded in the inside of the corresponding device side wall, and the first discharge pipe 151, the second discharge pipe 171, the third discharge pipe 311, the fourth discharge pipe 331, the first liquid outlet pipe 21, the second liquid outlet pipe 22 and the third liquid outlet pipe 41 are all provided with a switch valve, and all the electric control devices used in the present application adopt existing devices.

[0063] The method for extracting by using the device is as follows: S1, the crushed and impurity-removed live earthworms are added into the crushing cavity 16 from the feeding pipe 11, the telescopic rod 161 is started to drive the grinding roller 161 to grind and crush the live earthworms on the surface of the first partition plate 15, and in the reciprocating translation process of the grinding roller 161, the rack 164 drives the gear 163 and the grinding roller 16 to rotate, thereby enhancing the grinding effect;

[0064] S2, after the grinding and crushing are completed, the first discharge pipe 151 is opened, the earthworm powder is filtered into the processing cavity 17, then according to the operation of S2, the single use amount of the protein protective agent, the single use power and time of the ultrasonic wave are adjusted, after multiple adjustments are completed, an appropriate amount of water is added into the processing cavity 17 to reach the water content, and then the slurry is obtained after stirring and mixing uniformly, the slurry is introduced into the centrifugal cylinder 2 from the second discharge pipe 171 for centrifugation, and after the centrifugation is completed, the supernatant is transferred to the enzymatic lysis cavity 32 through the first liquid outlet pipe 21;

[0065] Example 3: The difference between this example and example 1 is that the grinding and crushing are carried out at 0℃, the solid-liquid ratio of 1g:6ml is used to mix with the fluid state protein protective agent, the ultrasonic wave of 250W is applied, and the mixing is carried out for 10min, and then vacuum drying is carried out to obtain the pretreated powder with a particle size of 70-73μm.

[0066] Example 4: The difference between this example and Example 1 is that the grinding pulverization is performed at 4°C, mixed with the fluid state protein protective agent at a solid-liquid ratio of 1 g:8 ml, and ultrasonic waves of 280 W are applied for 12 min, and after the mixing is completed, vacuum drying is performed to obtain a pretreated powder having a particle size of 77-80 μm.

[0067] Example 5: The difference between this example and Example 1 is that the obtained pretreated powder is mixed with the granular state protein protective agent having a particle size of 80-85 μm at a mass ratio of 1:0.4, and ultrasonic waves of 420 W are applied for 3 min to obtain a mixed powder.

[0068] Example 6: The difference between this example and Example 1 is that the obtained pretreated powder is mixed with the granular state protein protective agent having a particle size of 95-100 μm at a mass ratio of 1:0.6, and ultrasonic waves of 460 W are applied for 6 min to obtain a mixed powder.

[0069] Example 7: The difference between this example and Example 1 is that water is added to the mixed powder obtained in S1 to obtain a slurry having a water content of 85%, and the slurry is centrifuged at 10,000 rpm for 10 min to obtain a supernatant, and a protease is added to the supernatant at a liquid-solid ratio of 1 ml:0.03 g to perform enzymatic hydrolysis at a temperature of 40°C for 2.5 h, and the protease is composed of bacillus licheniformis protease, trypsin, and papain at a mass ratio of 1:1:0.6.

[0070] Example 8: The difference between this example and Example 1 is that water is added to the mixed powder obtained in S1 to obtain a slurry having a water content of 80%, and the slurry is centrifuged at 11,000 rpm for 15 min to obtain a supernatant, and a protease is added to the supernatant at a liquid-solid ratio of 1 ml:0.05 g to perform enzymatic hydrolysis at a temperature of 43°C for 3 h, and the protease is composed of bacillus licheniformis protease, trypsin, and papain at a mass ratio of 1:1:0.8.

[0071] Example 9: The difference between this example and Example 1 is that after the enzymatic hydrolysis is completed, the enzyme is inactivated at 95°C for 1.5 h to obtain an enzymatic hydrolysate, and the enzymatic hydrolysate is subjected to ultrafiltration using a first ultrafiltration membrane having a molecular weight cut-off of 15,000-18,000 Da.

[0072] Example 10: The difference between this example and Example 1 is that after the enzymatic hydrolysis is completed, the enzyme is inactivated at 115°C for 2.5 h to obtain an enzymatic hydrolysate, and the enzymatic hydrolysate is subjected to ultrafiltration using a first ultrafiltration membrane having a molecular weight cut-off of 18,000-20,000 Da.

[0073] Example 11: The difference between this example and Example 1 is that activated carbon is added to the filtrate obtained in S3 at a liquid-solid ratio of 1 ml:0.08 g, stirred at 220 rpm for 15 min, and then left to stand for 5 min.

[0074] Example 12: The difference between this example and Example 1 is that activated carbon is added to the filtrate obtained in S3 at a liquid-solid ratio of 1 ml:0.1 g, stirred at 270 rpm for 20 min, and then left to stand for 10 min.

[0075] Example 13: The difference between this example and Example 1 is that the supernatant is obtained by centrifugation at 9000 rpm for 15 min, and then ultrafiltration is performed using a second ultrafiltration membrane with a molecular weight cut-off of 5000-7000 Da.

[0076] Example 14: The difference between this example and Example 1 is that the supernatant is obtained by centrifugation at 9500 rpm for 20 min, and then ultrafiltration is performed using a second ultrafiltration membrane with a molecular weight cut-off of 7000-10000 Da.

[0077] Example 15: The difference between this example and Example 1 is that drying is first performed at -40℃ for 3 h, then at -60℃ for 18 h, and finally at 25℃ for 2 h.

[0078] Example 16: The difference between this example and Example 1 is that drying is first performed at -35℃ for 2 h, then at -50℃ for 15 h, and finally at 30℃ for 3 h.

[0079] Example 17: The difference between this example and Example 1 is that modified soybean fiber, a genipin solution with a mass concentration of 0.8%, reduced glutathione, trehalose, and vitamin C are mixed at a liquid-solid ratio of 4 g:10 ml:2 g:5 g:1 g at 350 rpm for 15 min, and then freeze-drying is performed after mixing is completed to obtain a granular protein protective agent.

[0080] Example 18: The difference between this example and Example 1 is that modified soybean fiber, a genipin solution with a mass concentration of 1%, reduced glutathione, trehalose, and vitamin C are mixed at a liquid-solid ratio of 4 g:12 ml:2 g:5 g:1 g at 450 rpm for 25 min, and then freeze-drying is performed after mixing is completed to obtain a granular protein protective agent.

[0081] Example 19: The difference between this example and Example 1 is that soybean meal is crushed through a 400-mesh sieve to obtain soybean residue, and then 0.7 wt% of a composite enzyme is added to the soybean residue, which is enzymatically hydrolyzed at 40℃ and pH=6 for 2 h, and the composite enzyme is composed of cellulase, ginger protease, and phytase at a mass ratio of 1:0.3:0.6.

[0082] Example 20: The difference between this example and Example 1 is that the soybean meal is crushed to obtain soybean residue through a 400-mesh sieve, and then 0.9wt% of the composite enzyme consisting of cellulase, ginger protease and phytase in a mass ratio of 1:0.5:0.6 is added to the soybean residue for enzymolysis at 45℃ and pH=8 for 3h.

[0083] Example 21: The difference between this example and Example 1 is that after the end of the enzymolysis, the enzyme is inactivated at 95℃ for 5min, then pressure treated at 0.4MPa for 2.5h, then homogenized at 50MPa for 1min, and finally dried at-40℃ for 12h.

[0084] Example 22: The difference between this example and Example 1 is that after the end of the enzymolysis, the enzyme is inactivated at 105℃ for 10min, then pressure treated at 0.5MPa for 3h, then homogenized at 55MPa for 2min, and finally dried at-60℃ for 24h.

[0085] Example 23: The difference between this example and Example 1 is that the soybean fiber is immersed in an ethanol solution containing 70wt% birch bark extract at a solid-liquid ratio of 1g:15ml, the immersion temperature is 50℃, and the immersion time is 2.5h.

[0086] Example 24: The difference between this example and Example 1 is that the soybean fiber is immersed in an ethanol solution containing 80wt% birch bark extract at a solid-liquid ratio of 1g:25ml, the immersion temperature is 60℃, and the immersion time is 3.5h.

[0087] Example 25: The difference between this example and Example 1 is that the modified soybean fiber, sucrose and sorbitol solution with a mass concentration of 50% are mixed at 5g:3g:4ml for homogenization treatment, the magnetic stirring time is 0.5h, and the pressure is 65MPa to obtain a homogenate; then Tween 80 is added to the homogenate at a solid-liquid ratio of 1g:45ml for emulsification and dispersion for 40min to obtain a fluid protein protective agent.

[0088] Example 26: The difference between this example and Example 1 is that the modified soybean fiber, sucrose and sorbitol solution with a mass concentration of 60% are mixed at 7g:3g:4ml for homogenization treatment, the magnetic stirring time is 1.5h, and the pressure is 75MPa to obtain a homogenate; then Tween 80 is added to the homogenate at a solid-liquid ratio of 1g:40ml for emulsification and dispersion for 50min to obtain a fluid protein protective agent.

[0089] Experimental Example: The description of this experimental example is based on the description of Example 1, and is intended to illustrate the actual application effect of the present application.

[0090] The water content of live earthworms was determined according to the method in the Chinese Pharmacopoeia. The yield of earthworm protein was calculated as follows: Earthworm protein yield = total amount of earthworm protein obtained / [mass of live earthworms × (1-r)]; where r is the water content of fresh earthworms.

[0091] Investigation 1: Investigate the effect of S1 parameters on the yield of earthworm protein.

[0092] The difference between Comparative Example 1 and Example 1 is that in S1, only fluid protein protectant is added, and no particulate protein protectant is added;

[0093] The difference between Comparative Example 2 and Example 1 is that in S1, no fluid protein protectant is added, only particulate protein protectant is added;

[0094] The difference between Comparative Example 3 and Example 1 is that in S1, the particulate protein protectant is added first, followed by the fluid protein protectant;

[0095] Depend on Figure 5 The results show that, in Control Example 1, the lack of sustained release and rigid support from particulate protein protectants resulted in a loose structure of the pretreated powder after drying. During subsequent extraction, the protein was easily exposed to oxygen and temperature changes, leading to oxidation or thermal denaturation. In Control Example 2, the particulate matter, unlike the fluid form, could not penetrate the internal gaps of the broken earthworm tissue before drying, resulting in a significant reduction in the contact area between the protein protectant and the protein. In Control Example 3, the protein surface was overprotected due to particulate aggregation, while the interior lacked protection due to insufficient fluid penetration. During ultrasound, the internal protein was prone to denaturation due to the lack of protection, leading to localized inactivation of the extracted protein. Therefore, the yield of earthworm protein in Control Examples 1-3 was lower than that in Examples 1 and 3-6.

[0096] Comparing Examples 1 and 3 to 6, it can be seen that if the preparation parameters of the pretreated powder are too small or too large, or the preparation parameters of the mixed powder are too small or too large, the yield of earthworm protein will be reduced. Therefore, from a comprehensive perspective, the parameters of Example 1 are relatively better.

[0097] Investigation 2: Investigate the effects of parameters S2, S3, and S4 on the yield of earthworm protein.

[0098] Depend on Figure 6 The results of comparing Examples 1 and 7-16 show that excessively small or large centrifugation parameters, excessively small or large enzyme inactivation parameters, excessively small or large activated carbon adsorption parameters, excessively small or large centrifugation parameters, and excessively small or large freeze-drying parameters all reduce the yield of earthworm protein. Therefore, from a comprehensive perspective, the parameters in Example 1 are relatively better.

[0099] Investigation 3: Investigate the effects of preparation parameters of fluid and particulate protein protectants on the yield of earthworm protein.

[0100] The difference between Comparative Example 4 and Example 1 is that the soybean fiber was not soaked;

[0101] Depend on Figure 7 The results show that, in Control Example 4, the birch bark extract is rich in flavonoids and phenolic acids, and its antioxidant activity is significantly better than that of conventional synthetic antioxidants. Soybean fiber without soaking lacks these components, which leads to a significant reduction in the antioxidant capacity of the fluid and particulate protein protectants prepared from it. They cannot effectively scavenge free radicals, which will accelerate protein oxidation and denaturation, resulting in loss of protein activity. Therefore, the yield of earthworm protein in Control Example 4 is lower than that in Examples 1 and 3 to 6.

[0102] Comparing Examples 1 and 17-26, it can be seen that excessively small or large parameters in the preparation of particulate protein protectant, excessively small or large parameters in soybean meal enzymatic hydrolysis, excessively small or large parameters in homogenization and drying, excessively small or large parameters in impregnation, and excessively small or large parameters in the preparation of fluid protein protectant will all reduce the yield of earthworm protein. Therefore, from a comprehensive perspective, the parameters in Example 1 are relatively better.

Claims

1. A method for extracting high-content earthworm protein, characterized in that, Includes the following steps: S1. After removing impurities from the live earthworm, grind and pulverize it at 0-4℃ and mix it with a fluid protein protectant at a solid-liquid ratio of 1g:6-8ml. Apply ultrasound at 250-280W for 10-12min. After mixing, vacuum dry to obtain a pretreated powder with a particle size of 70-80μm. Mix the obtained pretreated powder with a particulate protein protectant with a particle size of 80-100μm at a mass ratio of 1:0.4-0.6 and apply ultrasound at 420-460W for 3-6min to obtain a mixed powder. S2. Add water to the mixed powder obtained in S1 to obtain a slurry with a water content of 80-85%. Then centrifuge the slurry at 10000-11000 rpm for 10-15 min to obtain the supernatant. S3. Add protease to the supernatant obtained in S2 at a liquid-to-solid ratio of 1 ml: 0.03-0.05 g for enzymatic hydrolysis at a temperature of 40-43°C for 2.5-3 h. After enzymatic hydrolysis, inactivate the enzyme at 95-115°C for 1.5-2.5 h to obtain the enzymatic hydrolysate. Then, use the first ultrafiltration membrane to ultrafilter the enzymatic hydrolysate to obtain the filtrate. S4. Add activated carbon to the filtrate obtained in S3 at a liquid-to-solid ratio of 1 ml: 0.08-0.1 g, stir at 220-270 rpm for 15-20 min, let stand for 5-10 min, then centrifuge at 9000-9500 rpm for 15-20 min to obtain the supernatant, then use a second ultrafiltration membrane to perform ultrafiltration to obtain the retentate, freeze-dry the retentate to obtain earthworm protein.

2. The method for extracting high-content earthworm protein as described in claim 1, characterized in that, In S1, the preparation method of the fluid protein protectant is as follows: 5-7 g of modified soybean fiber, sucrose, and sorbitol solution with a mass concentration of 50-60% are added. Mix 3g:4ml and homogenize the mixture. Stir magnetically for 0.5-1.5h at a pressure of 65-75MPa to obtain a homogenate. Then add Tween 80 to the homogenate at a solid-liquid ratio of 1g:40-45ml and emulsify and disperse for 40-50min to obtain a fluid protein protectant. The particulate protein protectant is prepared by mixing modified soybean fiber, genipin solution with a mass concentration of 0.8-1%, reduced glutathione, trehalose, and vitamin C at a liquid-solid ratio of 4g:10-12ml:2g:5g:1g at 350-450rpm for 15-25min. After mixing, the mixture is freeze-dried to obtain the particulate protein protectant.

3. The method for extracting high-content earthworm protein as described in claim 1, characterized in that, The method for preparing the modified soybean fiber is as follows: Soybean meal is pulverized and passed through a 400-mesh sieve to obtain soybean residue. Then, 0.7-0.9 wt% of a compound enzyme is added to the soybean residue, and enzymatic hydrolysis is carried out at 40-45℃ and pH=6-8 for 2-3 hours. After enzymatic hydrolysis, the enzyme is inactivated at 95-105℃ for 5-10 minutes, and then pressure treated at 0.4-0.5MPa for 2.5-3 hours. Then, it is homogenized at 50-55MPa for 1-2 minutes. Finally, it is dried at -60 to -40℃ for 12-24 hours to obtain soybean fiber. Soybean fiber was impregnated in an ethanol solution containing 70-80 wt% birch bark extract at a solid-liquid ratio of 1 g: 15-25 ml. The impregnation temperature was 50-60 °C and the time was 2.5-3.5 h. After impregnation, the fiber was vacuum dried to obtain modified soybean fiber.

4. The method for extracting high-content earthworm protein as described in claim 3, characterized in that, The complex enzyme is composed of cellulase, ginger protease and phytase in a mass ratio of 1:0.3 to 0.5:0.

6.

5. The method for extracting high-content earthworm protein as described in claim 1, characterized in that, In S3, the protease is composed of Bacillus licheniformis protease, trypsin, and papain in a mass ratio of 1:1:0.6 to 0.

8.

6. The method for extracting high-content earthworm protein as described in claim 1, characterized in that, In S3, the molecular weight cutoff of the first ultrafiltration membrane is 15,000 to 20,000 Da.

7. The method for extracting high-content earthworm protein as described in claim 1, characterized in that, In S4, the molecular weight cutoff of the second ultrafiltration membrane is 5000-10000 Da.

8. The method for extracting high-content earthworm protein as described in claim 1, characterized in that, In S4, the freeze-drying method is as follows: first, dry at -40 to -35°C for 2 to 3 hours, then dry at -60 to -50°C for 15 to 18 hours, and finally dry at 25 to 30°C for 2 to 3 hours.

9. The equipment used in the high-content earthworm protein extraction method according to any one of claims 1 to 8, characterized in that, From left to right, the chamber includes a pretreatment chamber (1), a centrifuge tube (2), an enzymatic hydrolysis and adsorption chamber (3), and a freeze-drying chamber (4). Each of the pretreatment chamber (1), the enzymatic hydrolysis and adsorption chamber (3), and the freeze-drying chamber (4) is equipped with a temperature controller (5). The pretreatment chamber (1) is divided into a crushing chamber (16) and a processing chamber (17) by a first partition (15). The pretreatment chamber (1) is equipped with an ultrasonic generator (12), a storage box (13) and a feed pipe (11) connected to the crushing chamber (16). The first partition (15) is equipped with a first discharge pipe (151) for connecting the crushing chamber (16) and the processing chamber (17). The first discharge pipe (151) is equipped with a filter screen (152). The storage box (13) is divided into a fluid protein protectant storage chamber connected to the crushing chamber (16) and a particulate protein protectant storage chamber connected to the processing chamber (17). The processing chamber (17) is equipped with a water tank (14) connected to its interior and a second discharge pipe (171) connected to the centrifuge cylinder (2). The centrifuge tube (2) is provided with a first outlet pipe (21) connected to the enzymatic hydrolysis and adsorption chamber (3), a second outlet pipe (22) connected to the freeze-drying chamber (4), and a recovery box (23) for recovering centrifuged solids and waste liquid; The enzymatic hydrolysis and adsorption chamber (3) is divided into an enzymatic hydrolysis chamber (32) and an adsorption chamber (33) by a second partition (31). The enzymatic hydrolysis chamber (32) is equipped with a protease storage box (321) communicating with its interior, and the adsorption chamber (33) is equipped with an activated carbon storage box (332) communicating with its interior. The second partition (31) is equipped with a third discharge pipe (311) for connecting the enzymatic hydrolysis chamber (32) and the adsorption chamber (33). The third discharge pipe (311) is equipped with a first ultrafiltration membrane (312), and the adsorption chamber (33) is equipped with a fourth discharge pipe (331) communicating with the centrifuge cylinder (2). The freeze-drying chamber (4) is provided with a third liquid outlet pipe (41) that is connected to the recovery box (23), and the third liquid outlet pipe (41) is provided with a second ultrafiltration membrane (411); The processing chamber (17), the enzymatic hydrolysis chamber (32), and the adsorption chamber (33) are all equipped with stirrers (172).

10. The equipment used in the high-content earthworm protein extraction method as described in claim 9, characterized in that, The grinding chamber (16) is provided with a grinding roller (161) connected to the side wall of the grinding chamber (16) via a telescopic rod (162). The grinding roller (161) is rotatably connected to the telescopic rod (162). Gears (163) are provided at both ends of the grinding roller (161). A rack (164) that meshes with the gears (163) is provided on the inner wall of the grinding chamber (16).

Images