Silkworm chrysalis protein capable of resisting fatigue and improving athletic ability

Abstract

The invention discloses silkworm pupa protein capable of resisting fatigue and improving exercise ability, and relates to the technical field of silkworm pupa protein preparation, and a preparation method of the silkworm pupa protein comprises the following steps: S1, raw material pretreatment: removing heads and feet of fresh silkworm pupa, washing the silkworm pupa with clear water, soaking the silkworm pupa in sterile water for 1-2 hours, stirring at a speed of 30-40 rpm, and taking out the silkworm pupa; the silkworm chrysalis is washed with cold boiled water at the temperature of 20-30 DEG C, the washed silkworm chrysalis is dried at the temperature of 40-50 DEG C, the drying time is 10-30 min, and S2, degreasing treatment is conducted, specifically, grease components in the silkworm chrysalis are removed through multiple degreasing processes. The silkworm chrysalis protein is extracted from silkworm chrysalis in a superfine grinding-ultrasonic wave-enzyme method combined extraction mode, and through the synergistic effect of the three methods, the extraction efficiency of the silkworm chrysalis protein is greatly improved, the silkworm chrysalis protein with higher purity is obtained, the product quality is improved, and meanwhile, the biological activity of the silkworm chrysalis protein can be better reserved; and the functionality of the silkworm pupa protein can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of silkworm pupa protein preparation, in particular to silkworm pupa protein with the functions of resisting fatigue and improving exercise ability. Background Art

[0002] Silkworm pupae are rich in protein, including a full range of essential amino acids for the human body. They are a medicine and food that nourishes the body, soothes the spleen and stomach, and grows muscles. The silkworm pupa protein extracted from silkworm pupae has anti-fatigue, antioxidant, and anti-aging functions. The existing silkworm pupa protein is difficult to improve the extraction purity of silkworm pupa protein while better retaining the biological activity of silkworm pupa protein.

[0003] The defects of existing silkworm pupa protein are: 1. Patent document CN105348369B discloses a method for extracting and separating silkworm pupa protein from silkworm pupae using ionic liquids. The method mainly considers how to avoid the corrosion of the traditional alkali dissolution and acid precipitation equipment, and the fat content of the obtained silkworm pupa protein is low. It does not consider how to improve the purity of silkworm pupa protein extraction while better retaining the biological activity of silkworm pupa protein; 2. Application document CN116693600A discloses a method for extracting and producing silkworm pupa protein, which mainly considers how to effectively improve the efficiency of protein extraction and protein extraction, and effectively avoid the destruction of protein molecules, but does not consider how to obtain silkworm pupa protein with higher purity and active peptides that can resist fatigue and improve exercise ability; 3. Application document CN117958346A discloses a silkworm pupa protein granule and a preparation method thereof, which mainly considers how to effectively improve the preparation efficiency of silkworm pupa protein peptides without polluting the silkworm pupa protein peptides, and can prepare silkworm pupa protein peptides with higher purity, but does not consider how to further improve the anti-fatigue and exercise ability of silkworm pupa protein; 4. The existing silkworm pupa protein has limited drying efficiency during the preparation process, and it is difficult to retain the active ingredients and functional properties of the active peptides in the silkworm pupa protein. Summary of the invention

[0004] The purpose of the present invention is to provide a silkworm pupa protein having the functions of resisting fatigue and improving exercise ability, so as to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a silkworm pupa protein having the functions of anti-fatigue and improving exercise ability, and a method for preparing the silkworm pupa protein, comprising the following steps: Step S1: Raw material pretreatment: remove the head and feet of fresh silkworm pupae, rinse them with clean water, then soak them in sterile water for 1-2 hours, and stir them at a speed of 30-40 rpm, then rinse them with cold boiled water at 20-30° C., and dry the washed silkworm pupae at a temperature of 40-50° C. for 10-30 minutes; Step S2: Degreasing treatment: removing the oil components in the silkworm pupae through multiple degreasing processes; Step S3: pulverizing and extracting: extracting silkworm pupa protein from the defatted silkworm pupae by combining ultrafine pulverization, ultrasonic wave and enzyme method; Step S4: multi-stage separation: separating the protein extracted in step S3 to obtain a silkworm pupa protein solution; Step S5: Refining and purification: decolorizing and deodorizing the separated silkworm pupa protein; Step S6: Drying: Drying the silkworm pupa protein after the treatment in step S5 to obtain powdered silkworm pupa protein; Step S7: Packaging and storage: vacuum-packing the silkworm pupa protein prepared with a plastic composite film, and storing it in a dry, light-proof, low-temperature environment below 4°C; Step S8: The prepared silkworm pupa protein is tested.

[0006] Preferably, in step S2, the following steps are also included: Step S21: Preliminary defatting: adding the washed silkworm pupae to petroleum ether, stirring at a speed of 150-180 rpm for 3-10 min, then centrifuging at 1000-1100 r / min for 5-10 min, and moving the separated silkworm pupae to a drying oven, drying at 45° C. for 2.5 h to obtain preliminarily defatted silkworm pupae; Step S22: Secondary degreasing: Put the silkworm pupae after preliminary degreasing in step S21 into new petroleum ether, stir at a speed of 240-280rpm for 3.5-6h, then centrifuge at 1000-1100r / min for 5-10min, and then put the separated silkworm pupae into a drying oven and dry at 50°C for 3h.

[0007] Preferably, in step S3, the following steps are also included: Step S31: pre-crushing: using a crusher to pre-crush the defatted silkworm pupae, wherein the speed of the crusher is 1000-2000 rpm and the crushing time is 2-5 min; Step S32: ultrafine grinding: adding the pre-crushed silkworm pupae to an ultrafine grinding device for ultrafine grinding, wherein the rotation speed of the device is 2200-3000 rpm, the grinding time is 10-40 min, and fine powder of 1000-3000 mesh is obtained; Step S33: Ultrasonic extraction: Then, the ultrafine powder of silkworm pupa is prepared into a suspension with a mass fraction of 2% to 6% with distilled water, and the suspension is placed in an ultrasonic device for treatment at an ultrasonic frequency of 20 to 30 kHz and a power of 300 to 400 W for 0.5 to 1 h, with intermittent ultrasonic operation for 3 seconds and rest for 3 seconds; Step S34: Enzymatic hydrolysis: The ultrasonically treated silkworm pupa protein suspension is then adjusted to a pH value of 6-7, and then a composite enzyme is added to the suspension, and the suspension is placed in a constant temperature water bath at 45-50° C. for enzymatic hydrolysis, and the reaction time is 4-5 hours, wherein the mass-volume ratio of the composite enzyme to the suspension is (2-4):100; Step S35: Inactivate enzyme: After the enzymatic reaction is completed, heat the reaction solution to 90-100°C and maintain for 20-30 minutes; Step S36: secondary enzymolysis: first adjust the pH of the solution after enzyme inactivation to 6-7, place the suspension in a constant temperature water bath at 50-60°C, and add the complex enzyme to perform a secondary enzymolysis reaction, wherein a magnetic stirrer is used to stir at a speed of 220-240 rpm for 2-4 hours, and the mass-volume ratio of the complex enzyme to the solution is (1-5):100. After the secondary enzymolysis is completed, repeat the enzyme inactivation operation of step S35.

[0008] Preferably, in step S4, the following steps are also included: Step S41: initial centrifugation: centrifuge the silkworm pupa protein reaction solution after enzyme inactivation at a speed of 5000 rpm for 20-25 min; Step S42: Secondary centrifugation: The supernatant obtained by the initial centrifugation is then subjected to secondary centrifugation using a centrifuge, wherein the speed is 7000-8000 r / min, the time is 25-40 min, and the supernatant is collected; Step S43: ultrafiltration membrane separation: then use an ultrafiltration membrane with a molecular weight cutoff of 1000-2000Da to filter and separate the supernatant after centrifugation; Step S44: ion exchange chromatography: soak the cation exchange resin in 4% hydrochloric acid solution for 2-3h, then rinse with deionized water until neutral, pour the treated ion exchange resin suspension into the chromatographic column, and knock the column to make the resin evenly filled, pass Tris-HCl buffer through the chromatographic column at a flow rate of 0.5-1ml / min until the pH value and ionic strength of the effluent are the same as those of the actual buffer, then filter the silkworm pupa protein reaction solution, and then place the treated reaction solution into the chromatographic column at a flow rate of 0.3-0.4ml / min, and finally elute with eluent at an elution flow rate of 1-2ml / min, and the elution time is 20-60min. Finally, the ion exchange resin in the chromatographic column is regenerated.

[0009] Preferably, in step S6, the following steps are also included: Step S61: pre-drying: placing the silkworm pupa protein solution in a vacuum drying oven at a temperature of 50-60° C. and a wind speed of 1 m / s for 3-5 hours; Step S62: Freeze drying: Then place the pre-dried silkworm pupa protein material in a freeze dryer, reduce the temperature to -30℃~-40℃ within 1-2 hours, then turn on the vacuum system, reduce the pressure in the freeze drying chamber to a vacuum degree of 20-30 Pa, then start the heating system to heat the material to -20℃~-30℃.

[0010] Preferably, in step S5, the following steps are also included: Step S51: decolorization treatment: mix the silkworm pupa protein solution with powdered activated carbon, stir at a speed of 100-150 rpm for 5-20 min, and then filter out the activated carbon; Step S52: Deodorization treatment: ozone gas is then introduced into the silkworm pupa protein solution at a rate of 20-30 mg / L and stirred at a speed of 60-80 rpm for 1.5-2.5 hours, and then the tail gas is treated by activated carbon adsorption.

[0011] Preferably, in step S8, the following steps are also included: Step S81: Nutritional value detection: the yield of protein content is determined according to the ratio of the mass of silkworm pupae before and after silkworm pupae preparation to the mass of the prepared silkworm pupae protein, and the fat content is determined according to GB5009.168-2016 "National Food Safety Standard Determination of Fatty Acids in Foods"; Step S82: Functional property detection: The silkworm pupa protein is taken into the experimental mice to measure the anti-fatigue and exercise capacity, and the storage stability of the silkworm pupa protein is detected by referring to the determination method in GB / T31617-2014 "National Food Safety Standard Food Nutrition Fortifier Casein Phosphopeptide".

[0012] Preferably, the composite enzyme is a mixture of neutral protease, papain and flavor protease in a mass ratio of 1:(1-3):1.

[0013] Preferably, the cation exchange resin is one of a carboxylic acid-based resin, a sulfonic acid-based resin and a phenolic hydroxyl resin.

[0014] Preferably, the mass-volume ratio of silkworm pupae to petroleum ether in step S21 is 1:(4-7), and the mass-volume ratio of silkworm pupae to petroleum ether in step S22 is 1:(5-6).

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention extracts silkworm pupa protein from silkworm pupae by an extraction method combining ultrafine grinding, ultrasonic wave and enzyme method. Silkworm pupae are firstly crushed into micrometer or even nanometer level by ultrafine grinding to form silkworm pupa particles with smaller particles, thereby increasing the specific surface area of ​​silkworm pupa protein, making it convenient for subsequent enzymes and solvents to contact with silkworm pupa protein more fully. Then, cavitation effect, mechanical vibration and other effects generated by ultrasonic wave can further break silkworm pupa cells and increase the dissolution of silkworm pupa protein. Then, enzyme is allowed to contact with silkworm pupa protein to further accelerate the release of protein. Through the synergistic effect of the three, the extraction efficiency of silkworm pupa protein is greatly improved, silkworm pupa protein with higher purity is obtained, product quality is improved, and the biological activity of silkworm pupa protein can be better retained, and the functionality of silkworm pupa protein, including fatigue resistance, motor ability and storage stability, is improved.

[0016] 2. The present invention separates and purifies the protein extracted by multi-stage separation technology. First, large impurity particles such as cell fragments and macromolecular polymers are quickly removed by two centrifugations to achieve preliminary separation and concentration, which reduces the burden on subsequent ultrafiltration membrane separation and ion exchange chromatography. Then, ultrafiltration membrane separation is used to further intercept and separate impurities, colloids and some proteins with larger molecular weight, and protein peptides with anti-fatigue and improved athletic ability are further concentrated. Finally, ion exchange chromatography is used to further separate the protein peptides according to their charge properties, thereby obtaining higher-purity silkworm pupa protein with active peptides that have anti-fatigue and improved athletic ability.

[0017] 3. The present invention uses a composite enzyme to perform multiple enzymatic hydrolysis on silkworm pupa protein so as to produce more peptides with specific physiological functions through the synergistic action of different enzymes, and precisely controls the enzymatic hydrolysis conditions so that the silkworm pupa protein hydrolyzes to produce more active peptides with anti-fatigue and motor ability improvement, thereby contributing to the improvement of the anti-fatigue and motor ability of silkworm pupa protein, and adds some enzyme auxiliary factors during the enzymatic hydrolysis process to further increase the activity of the enzyme, thereby enhancing the enzymatic hydrolysis effect and allowing the silkworm pupa protein to produce more high-quality protein peptides.

[0018] 4. The present invention adopts a combined drying method, first pre-drying, and then post-drying by freeze-drying, which can retain the active ingredients and functional characteristics of the active peptides in the silkworm pupa protein to the greatest extent, while improving the drying efficiency and achieving efficient drying. The silkworm pupa protein after combined drying has an extremely low water content, and due to the protective effect of freeze-drying, the stability of the active peptides and enzyme components in the silkworm pupa protein product is enhanced, and its biological activity and chemical stability can be maintained for a long time, reducing deterioration, which is beneficial to the long-term storage and application of the product. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] The present invention provides a silkworm pupa protein having the functions of resisting fatigue and improving exercise ability. The preparation method of the silkworm pupa protein is as follows: Step S1: Raw material pretreatment: remove the head and feet of fresh silkworm pupae, rinse them with clean water, then soak them in sterile water for 2 hours, stir them at 40 rpm, then rinse them with 25°C cold boiled water, and dry the washed silkworm pupae at 40°C for 30 minutes; Step S2: degreasing treatment: adding the washed silkworm pupae to petroleum ether, stirring at a speed of 160 rpm for 8 minutes, and then centrifuging at 1100 r / min for 7 minutes, and moving the separated silkworm pupae to a drying oven, drying at 45° C. for 2.5 hours to obtain preliminarily defatted silkworm pupae, and then placing the preliminarily defatted silkworm pupae into new petroleum ether, stirring at a speed of 260 rpm for 5 hours, and then centrifuging at 1000 r / min for 10 minutes, and then placing the separated silkworm pupae into a drying oven, and drying at 50° C. for 3 hours; Step S3: crushing and extraction: the defatted silkworm pupae are pre-crushed by a crusher, wherein the speed of the crusher is 1500 rpm, and the crushing time is 4 minutes, and then the pre-crushed silkworm pupae are added to the ultrafine crushing equipment for ultrafine crushing, wherein the speed of the equipment is 2200-3000 rpm, and the crushing time is 10-40 minutes, to obtain fine powder of 1000-3000 mesh, and then the ultrafine crushed silkworm pupae fine powder is prepared into a suspension with a mass fraction of 4% by distilled water, and the suspension is placed in an ultrasonic device for treatment, and the ultrasonic frequency is 20-30kHz, the power is 300-400W, and the treatment is carried out for 0.5-1h, and the intermittent ultrasonic method is used to work for 3s, and the interval is 3s. The silkworm pupa protein suspension treated with ultrasound is then adjusted to a pH value of 6, and then a composite enzyme is added to the suspension, and the suspension is placed in a 50°C constant temperature water bath for enzymatic hydrolysis, the reaction time is 4-5 hours, wherein the mass-volume ratio of the composite enzyme to the suspension is (2-4):100, after the enzymatic hydrolysis reaction is completed, the reaction solution is heated to 100°C and maintained for 250 minutes, and then the pH of the solution after enzyme inactivation is adjusted to 7, the suspension is placed in a 60°C constant temperature water bath, and the composite enzyme is added to perform a secondary enzymatic hydrolysis reaction, wherein a magnetic stirrer is used to stir at a speed of 240 rpm for 3 hours, and the mass-volume ratio of the composite enzyme to the solution is (1-5):100, and after the secondary enzymatic hydrolysis is completed, the enzyme inactivation treatment is performed again; Step S4: multi-stage separation: centrifuge the silkworm pupa protein reaction solution after enzyme inactivation at a speed of 5000 rpm for 20 min, then centrifuge the supernatant obtained by the initial centrifugation at a speed of 8000 r / min for 30 min, collect the supernatant, and then filter and separate the supernatant using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, soak the cation exchange resin in 4% hydrochloric acid solution for 3 h, then rinse with deionized water until neutral, and treat The treated ion exchange resin suspension is poured into the chromatographic column, and the column is knocked to make the resin fill evenly, and Tris-HCl buffer is passed through the chromatographic column at a flow rate of 1 ml / min until the pH value and ionic strength of the effluent are the same as those of the actual buffer, and then the silkworm pupa protein reaction liquid is filtered, and then the treated reaction liquid is placed in the chromatographic column at a flow rate of 0.4 ml / min, and finally eluted with an eluent at an elution flow rate of 1 ml / min, and the elution time is 40 minutes, and finally the ion exchange resin in the chromatographic column is regenerated; Step S5: Refining and purification: the silkworm pupa protein solution was mixed with powdered activated carbon, stirred at a speed of 120 rpm for 10 min, and then the activated carbon was filtered out, and then ozone gas was passed into the silkworm pupa protein solution at a rate of 25 mg / L and stirred at a speed of 70 rpm for 2 h, and then the tail gas was treated by activated carbon adsorption; Step S6: Drying treatment: placing the silkworm pupa protein solution in a vacuum drying oven at a temperature of 60° C. and a wind speed of 1 m / s for 3 hours, then placing the pre-dried silkworm pupa protein material in a freeze dryer, reducing the temperature to -40° C. within 2 hours, then starting the vacuum system, reducing the pressure in the freeze drying chamber to a vacuum degree of 30 Pa, and then starting the heating system to heat the material to -20° C.; Step S7: Packaging and storage: vacuum-packing the silkworm pupa protein prepared with a plastic composite film, and storing it in a dry, light-proof, low-temperature environment below 4°C; Step S8: The prepared silkworm pupa protein is tested for nutritional value and functional properties. When measuring anti-fatigue and exercise capacity, ICR mice are selected as experimental subjects, with an age of 6-8 weeks and a body weight of 18-22g. The mice are randomly divided into a normal control group, a silkworm pupa protein low-dose group, a silkworm pupa protein medium-dose group and a silkworm pupa protein high-dose group, with 10 mice in each group. The silkworm pupa protein is taken by gavage, with a low dose of 0.1g / kg body weight, a medium dose of 0.3g / kg body weight, and a high dose of 0.5g / kg body weight. The drugs are administered regularly every day. This was continued for 3 weeks. After 2 weeks, the mice were subjected to adaptive training in a swimming tank at 26°C. The training was generally conducted for 5 days, 15 minutes a day. After the administration was completed on the 3rd day, a lead block weighing 5% of the mouse's body weight was attached to the mouse's tail. The mouse was placed in the tank to swim and the time from the start of swimming to exhaustion was recorded to comprehensively evaluate the exercise capacity. After the administration was completed, the mouse was placed in an experimental device with a running wheel. The speed of the running wheel was adjusted to allow the mouse to exercise continuously at an intensity of 20 rpm. The time the animal continued to exercise on the running wheel was recorded until it fell off the running wheel due to fatigue, and the fatigue resistance was comprehensively evaluated.

[0021] Further, the value in step S3 is changed, and steps S1-2 and S4-8 are performed, and the prepared silkworm pupa protein is tested for nutritional value and functional properties.

[0022] Embodiment 1: Step S3: crushing and extraction: the defatted silkworm pupae are pre-crushed by a crusher, wherein the speed of the crusher is 1500 rpm, and the crushing time is 4 min. Then, the pre-crushed silkworm pupae are added to an ultrafine crushing device for ultrafine crushing, wherein the speed of the device is 2400 rpm, and the crushing time is 30 min to obtain 1200 mesh fine powder. Then, the ultrafine crushed silkworm pupae fine powder is prepared into a suspension with a mass fraction of 4% by distilled water, and the suspension is placed in an ultrasonic device for treatment. The ultrasonic frequency is 20 kHz and the power is 300 W. The treatment is carried out for 1 h, and the intermittent ultrasonic method is used to work for 3 s, and the ultrasonic treatment is continued for 3 s. The silkworm pupa protein suspension is adjusted to a pH value of 6, and then a composite enzyme is added to the suspension, and the suspension is placed in a constant temperature water bath at 50°C for enzymolysis reaction, the reaction time is 4-5 hours, wherein the mass-volume ratio of the composite enzyme to the suspension is 2:100, after the enzymolysis reaction is completed, the reaction solution is heated to 100°C and maintained for 250 minutes, and then the pH of the solution after enzyme inactivation is adjusted to 7, the suspension is placed in a constant temperature water bath at 60°C, and the composite enzyme is added for secondary enzymolysis reaction, wherein a magnetic stirrer is used to stir at a speed of 240rpm for 3 hours, and the mass-volume ratio of the composite enzyme to the solution is 2:100, and after the secondary enzymolysis is completed, the enzyme inactivation treatment is performed again.

[0023] Further, the protein yield of silkworm pupa protein was measured to be 64.1%, the fat content was 0.21%, the storage stability evaluation was: good, the swimming time of the normal control group was: 11.21±3.73min, the wheel time was: 28.25±4.22min, the swimming time of the silkworm pupa protein low-dose group was: 19.21±4.38min, the wheel time was: 34.56±4.25min, the swimming time of the silkworm pupa protein medium-dose group was: 20.49±5.72min, the wheel time was: 35.86±5.42min, the swimming time of the silkworm pupa protein high-dose group was: 23.29±6.28min, the wheel time was: 38.21±6.72min, the comprehensive anti-fatigue evaluation was: good, and the comprehensive exercise ability evaluation was: good.

[0024] Embodiment 2: Step S3: crushing and extraction: the defatted silkworm pupae are pre-crushed by a crusher, wherein the speed of the crusher is 1500 rpm, and the crushing time is 4 min. Then, the pre-crushed silkworm pupae are added to an ultrafine crushing device for ultrafine crushing, wherein the speed of the device is 2600 rpm, and the crushing time is 30 min to obtain 2000 mesh fine powder. Then, the ultrafine crushed silkworm pupae fine powder is prepared into a suspension with a mass fraction of 4% by distilled water, and the suspension is placed in an ultrasonic device for treatment. The ultrasonic frequency is 20 kHz and the power is 300 W. The treatment is carried out for 1 h, and the intermittent ultrasonic method is used to work for 3 s, and the ultrasonic treatment is continued for 3 s. The silkworm pupa protein suspension is adjusted to a pH value of 6, and then a composite enzyme is added to the suspension, and the suspension is placed in a constant temperature water bath at 50°C for enzymolysis reaction, the reaction time is 4-5 hours, wherein the mass-volume ratio of the composite enzyme to the suspension is 3:100, after the enzymolysis reaction is completed, the reaction solution is heated to 100°C and maintained for 250 minutes, and then the pH of the solution after enzyme inactivation is adjusted to 7, the suspension is placed in a constant temperature water bath at 60°C, and the composite enzyme is added to perform a secondary enzymolysis reaction, wherein a magnetic stirrer is used to stir at a speed of 240rpm for 3 hours, and the mass-volume ratio of the composite enzyme to the solution is 3:100, and after the secondary enzymolysis is completed, the enzyme inactivation treatment is performed again.

[0025] Further, the protein yield of silkworm pupa protein was measured to be 66.5%, the fat content was 0.12%, the storage stability evaluation was: good, the swimming time of the normal control group was: 11.21±3.73min, the wheel time was: 28.25±4.22min, the swimming time of the silkworm pupa protein low-dose group was: 19.93±4.67min, the wheel time was: 35.23±4.31min, the swimming time of the silkworm pupa protein medium-dose group was: 21.21±6.12min, the wheel time was: 37.12±5.87min, the swimming time of the silkworm pupa protein high-dose group was: 24.64±6.28min, the wheel time was: 38.54±6.85min, the comprehensive anti-fatigue evaluation was: good, and the comprehensive exercise ability evaluation was: good.

[0026] Embodiment three: Step S3: crushing and extraction: the defatted silkworm pupae are pre-crushed by a crusher, wherein the speed of the crusher is 1500 rpm, and the crushing time is 4 min. Then, the pre-crushed silkworm pupae are added to an ultrafine crushing device for ultrafine crushing, wherein the speed of the device is 2800 rpm, and the crushing time is 30 min to obtain 2400 mesh fine powder. Then, the ultrafine crushed silkworm pupae fine powder is prepared into a suspension with a mass fraction of 4% by distilled water, and the suspension is placed in an ultrasonic device for treatment. The ultrasonic frequency is 20 kHz and the power is 300 W. The treatment is carried out for 1 h, and the intermittent ultrasonic method is used to work for 3 s, and the ultrasonic treatment is continued for 3 s. The silkworm pupa protein suspension is adjusted to a pH value of 6, and then a composite enzyme is added to the suspension, and the suspension is placed in a constant temperature water bath at 50°C for enzymolysis reaction, the reaction time is 4-5 hours, wherein the mass-volume ratio of the composite enzyme to the suspension is 4:100, after the enzymolysis reaction is completed, the reaction solution is heated to 100°C and maintained for 250 minutes, and then the pH of the solution after enzyme inactivation is adjusted to 7, the suspension is placed in a constant temperature water bath at 60°C, and the composite enzyme is added to perform a secondary enzymolysis reaction, wherein a magnetic stirrer is used to stir at a speed of 240rpm for 3 hours, and the mass-volume ratio of the composite enzyme to the solution is 3:100, and after the secondary enzymolysis is completed, the enzyme inactivation treatment is performed again.

[0027] Further, the protein yield of silkworm pupa protein was measured to be 63.4%, the fat content was 0.17%, the storage stability evaluation was: good, the swimming time of the normal control group was: 11.21±3.73min, the wheel time was: 28.25±4.22min, the swimming time of the silkworm pupa protein low-dose group was: 19.31±4.37min, the wheel time was: 34.94±4.21min, the swimming time of the silkworm pupa protein medium-dose group was: 20.82±5.93min, the wheel time was: 36.53±5.11min, the swimming time of the silkworm pupa protein high-dose group was: 22.11±6.12min, the wheel time was: 38.12±6.73min, the comprehensive anti-fatigue evaluation was: good, and the comprehensive exercise ability evaluation was: good.

[0028] The performance test results of silkworm pupa protein are as follows:

[0029] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A silkworm pupa protein having the functions of resisting fatigue and improving exercise ability, characterized in that: The method for preparing silkworm pupa protein comprises the following steps: Step S1: Raw material pretreatment: remove the head and feet of fresh silkworm pupae, rinse them with clean water, then soak them in sterile water for 1-2 hours, and stir them at a speed of 30-40 rpm, then rinse them with cold boiled water at 20-30° C., and dry the washed silkworm pupae at a temperature of 40-50° C. for 10-30 minutes; Step S2: Degreasing treatment: removing the oil components in the silkworm pupae through multiple degreasing processes; Step S3: pulverizing and extracting: extracting silkworm pupa protein from the defatted silkworm pupae by combining ultrafine pulverization, ultrasonic wave and enzyme method; Step S4: multi-stage separation: separating the protein extracted in step S3 to obtain a silkworm pupa protein solution; Step S5: Refining and purification: decolorizing and deodorizing the separated silkworm pupa protein; Step S6: Drying: Drying the silkworm pupa protein after the treatment in step S5 to obtain powdered silkworm pupa protein; Step S7: Packaging and storage: vacuum-packing the silkworm pupa protein prepared with a plastic composite film, and storing it in a dry, light-proof, low-temperature environment below 4°C; Step S8: The prepared silkworm pupa protein is tested.

2. The silkworm pupa protein having the function of resisting fatigue and improving exercise ability according to claim 1, characterized in that: In step S2, the following steps are also included: Step S21: Preliminary defatting: adding the washed silkworm pupae to petroleum ether, stirring at a speed of 150-180 rpm for 3-10 min, then centrifuging at 1000-1100 r / min for 5-10 min, and moving the separated silkworm pupae to a drying oven, drying at 45° C. for 2.5 h to obtain preliminarily defatted silkworm pupae; Step S22: Secondary degreasing: Put the silkworm pupae after preliminary degreasing in step S21 into new petroleum ether, stir at a speed of 240-280rpm for 3.5-6h, then centrifuge at 1000-1100r / min for 5-10min, and then put the separated silkworm pupae into a drying oven and dry at 50°C for 3h.

3. The silkworm pupa protein having the function of resisting fatigue and improving exercise ability according to claim 1, characterized in that: In step S3, the following steps are also included: Step S31: pre-crushing: using a crusher to pre-crush the defatted silkworm pupae, wherein the speed of the crusher is 1000-2000 rpm and the crushing time is 2-5 min; Step S32: ultrafine grinding: adding the pre-crushed silkworm pupae to an ultrafine grinding device for ultrafine grinding, wherein the rotation speed of the device is 2200-3000 rpm, the grinding time is 10-40 min, and fine powder of 1000-3000 mesh is obtained; Step S33: Ultrasonic extraction: Then, the ultrafine powder of silkworm pupa is prepared into a suspension with a mass fraction of 2% to 6% with distilled water, and the suspension is placed in an ultrasonic device for treatment at an ultrasonic frequency of 20 to 30 kHz and a power of 300 to 400 W for 0.5 to 1 h, with intermittent ultrasonic operation for 3 seconds and rest for 3 seconds; Step S34: Enzymatic hydrolysis: The ultrasonically treated silkworm pupa protein suspension is then adjusted to a pH value of 6-7, and then a composite enzyme is added to the suspension, and the suspension is placed in a constant temperature water bath at 45-50° C. for enzymatic hydrolysis, and the reaction time is 4-5 hours, wherein the mass-volume ratio of the composite enzyme to the suspension is (2-4):100; Step S35: Inactivate the enzyme: After the enzymatic reaction is completed, heat the reaction solution to 90-100°C and maintain for 20-30 minutes; Step S36: secondary enzymolysis: first adjust the pH of the solution after enzyme inactivation to 6-7, place the suspension in a constant temperature water bath at 50-60°C, and add the complex enzyme to perform a secondary enzymolysis reaction, wherein a magnetic stirrer is used to stir at a speed of 220-240 rpm for 2-4 hours, and the mass-volume ratio of the complex enzyme to the solution is (1-5):

100. After the secondary enzymolysis is completed, repeat the enzyme inactivation operation of step S35.

4. The silkworm pupa protein having the functions of resisting fatigue and improving exercise ability according to claim 1, characterized in that: In step S4, the following steps are also included: Step S41: initial centrifugation: centrifuge the silkworm pupa protein reaction solution after enzyme inactivation at a speed of 5000 rpm for 20-25 min; Step S42: Secondary centrifugation: The supernatant obtained by the initial centrifugation is then subjected to secondary centrifugation using a centrifuge, wherein the speed is 7000-8000 r / min, the time is 25-40 min, and the supernatant is collected; Step S43: ultrafiltration membrane separation: then use an ultrafiltration membrane with a molecular weight cutoff of 1000-2000Da to filter and separate the supernatant after centrifugation; Step S44: ion exchange chromatography: soak the cation exchange resin in 4% hydrochloric acid solution for 2-3h, then rinse with deionized water until neutral, pour the treated ion exchange resin suspension into the chromatographic column, and knock the column to make the resin evenly filled, pass Tris-HCl buffer through the chromatographic column at a flow rate of 0.5-1ml / min until the pH value and ionic strength of the effluent are the same as those of the actual buffer, then filter the silkworm pupa protein reaction solution, and then place the treated reaction solution into the chromatographic column at a flow rate of 0.3-0.4ml / min, and finally elute with eluent at an elution flow rate of 1-2ml / min, and the elution time is 20-60min. Finally, the ion exchange resin in the chromatographic column is regenerated.

5. The silkworm pupa protein having the functions of resisting fatigue and improving exercise ability according to claim 1, characterized in that: In step S6, the following steps are also included: Step S61: Pre-drying: placing the silkworm pupa protein solution in a vacuum drying oven at a temperature of 50-60° C. and a wind speed of 1 m / s for 3-5 hours; Step S62: Freeze drying: Then place the pre-dried silkworm pupa protein material in a freeze dryer, reduce the temperature to -30℃~-40℃ within 1-2 hours, then turn on the vacuum system, reduce the pressure in the freeze drying chamber to a vacuum degree of 20-30 Pa, then start the heating system to heat the material to -20℃~-30℃.

6. The silkworm pupa protein having the functions of resisting fatigue and improving exercise ability according to claim 1, characterized in that: In step S5, the following steps are also included: Step S51: decolorization treatment: mixing the silkworm pupa protein solution with powdered activated carbon, stirring at a speed of 100-150 rpm for 5-20 min, and then filtering out the activated carbon; Step S52: Deodorization treatment: ozone gas is then introduced into the silkworm pupa protein solution at a rate of 20-30 mg / L and stirred at a speed of 60-80 rpm for 1.5-2.5 hours, and then the tail gas is treated by activated carbon adsorption.

7. The silkworm pupa protein having the functions of resisting fatigue and improving exercise ability according to claim 1, characterized in that: In step S8, the following steps are also included: Step S81: Nutritional value detection: the yield of protein content is determined according to the ratio of the mass of silkworm pupae before and after silkworm pupae preparation to the mass of the prepared silkworm pupae protein, and the fat content is determined according to GB5009.168-2016 "National Food Safety Standard Determination of Fatty Acids in Foods"; Step S82: Functional property detection: The silkworm pupa protein is taken into the experimental mice to measure the anti-fatigue and exercise capacity, and the storage stability of the silkworm pupa protein is detected by referring to the determination method in GB / T31617-2014 "National Food Safety Standard Food Nutrition Fortifier Casein Phosphopeptide".

8. The silkworm pupa protein having the functions of resisting fatigue and improving exercise ability according to claim 3, characterized in that: The composite enzyme is prepared by mixing neutral protease, papain and flavor protease in a mass ratio of 1:(1-3):

1.

9. The silkworm pupa protein having the function of resisting fatigue and improving exercise ability according to claim 4, characterized in that: The cation exchange resin is one of a carboxylic acid resin, a sulfonic acid resin and a phenolic hydroxyl resin.

10. The silkworm pupa protein having the functions of resisting fatigue and improving exercise ability according to claim 2, characterized in that: The mass-volume ratio of silkworm pupae to petroleum ether in step S21 is 1:(4-7), and the mass-volume ratio of silkworm pupae to petroleum ether in step S22 is 1:(5-6).

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