Preparation method and application of hermetia illucens protein peptide

Abstract

The invention discloses a preparation method and application of hermetia illucens protein peptide. According to the preparation method, protein in hermetia illucens pulp is hydrolyzed based on high-pressure carbon dioxide autolysis and hydrolytic enzyme enzymolysis coupling. According to the method, hermetia illucens pulp is subjected to enzymolysis in a high-pressure carbon dioxide environment, high-pressure carbon dioxide is dissolved to form carbonic acid, the pH of the whole system can be reduced, then the integrity of microbial cell membranes in larva bodies is destroyed, meanwhile, an anaerobic environment is created, low-temperature efficient sterilization is achieved, and no chemical preservative is needed; besides, a high-pressure enzymolysis environment promotes spatial structure expansion of the hermetia illucens protein and exposes more enzyme cutting sites, meanwhile, the acidification effect of carbon dioxide can also simulate the animal digestion environment and enhance the hydrolytic activity of pancreatin on specific peptide bonds, the hydrolytic efficiency of the hermetia illucens protein is remarkably improved, and the antioxidant activity is high. When the hermetia illucens protein peptide prepared by the method is used for breeding laying hens, the quality of eggs can be improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of deep processing of resource insects, and particularly relates to a preparation method of protein peptides of black soldier fly and application thereof. BACKGROUND

[0002] Black soldier fly (Hermetia illucens L.) belongs to the family of Diptera and the genus of Hermetia, and its Chinese name is bright spot flat angle water fly. Hermetia illucens L. It is a kind of saprophytic insects. At present, black soldier fly larvae have the characteristics of wide food spectrum, large food intake, low nutritional requirement and high safety, and are widely used in the treatment of solid organic waste such as kitchen waste, poultry manure, wet waste and crop straw. Among them, black soldier fly larvae are rich in protein, fat and antibacterial peptides and other substances, and the crude protein content in the dry matter of black soldier fly larvae is as high as 47%-50%, which is equivalent to aquatic and livestock animals, and can be used as a good substitute for fish meal and soybean meal in the field of aquatic and livestock breeding. However, due to the complex composition of black soldier fly slurry, the protein is mainly in the form of complex protein, and the large molecular structure of the protein may not be effectively decomposed by digestive enzymes in the animal intestinal tract, resulting in low protein utilization rate and affecting the growth and development of animals. Therefore, black soldier fly slurry and black soldier fly powder cannot be directly used as animal breeding feed.

[0003] In order to improve the utilization rate of black soldier fly protein, the black soldier fly protein is usually decomposed into small molecular protein peptides or amino acids. At present, the hydrolysis of black soldier fly protein mainly has microbial fermentation method and enzymatic hydrolysis method. The biological fermentation method is to use microorganisms to ferment and hydrolyze to obtain black soldier fly protein, but the microorganisms will convert the black soldier fly protein into bacterial protein during the fermentation process, which consumes the black soldier fly protein to a certain extent and affects the effective utilization rate of the black soldier fly protein. In addition, the metabolites produced in the microbial metabolism process usually affect the yield and quality of the hydrolysis product. The enzymatic hydrolysis method mainly depends on exogenous protease hydrolysis, and the black soldier fly protein hydrolysis rate is low, and due to the breeding environment and saprophytic nature of black soldier fly, chemical preservatives need to be added during the hydrolysis process. It can be seen that how to improve the hydrolysis rate of black soldier fly protein and obtain small molecular protein peptides is still a technical problem to be solved in the industry. SUMMARY

[0004] Therefore, the present application provides a preparation method of black soldier fly protein peptides and application thereof.

[0005] To achieve the above-mentioned purpose, the present application adopts the following technical scheme: The preparation method of black soldier fly protein peptides provided by the present application is based on high-pressure carbon dioxide autolysis and enzyme hydrolysis to hydrolyze the protein in black soldier fly slurry, and specifically includes the following contents: Firstly, a certain volume of water is added to washed and dried black soldier fly bodies, and then the black soldier fly bodies are broken and homogenized to obtain black soldier fly slurry; wherein the weight ratio of black soldier fly bodies to water is 1:(0.4-1.6); In the second step, the pH of the black soldier fly paste is adjusted to 7.0-9.0, and the paste is preheated at 80-95℃; In the third step, after high-temperature pre-treatment, the black soldier fly paste is cooled to 50℃ or below, and a hydrolytic enzyme is added, and the enzyme is stirred in a high-pressure carbon dioxide environment; wherein the pressure of the high-pressure carbon dioxide environment is 0.5-1.0 MPa, the enzyme hydrolysis temperature is 40-50℃, the enzyme hydrolysis time is 6-12 h, and the stirring speed is 150-300 rpm; In the fourth step, after enzyme hydrolysis, the enzyme hydrolysis liquid is filtered under sterile conditions to remove impurities, and the hydrolytic enzyme is inactivated to obtain a black soldier fly protein peptide complex; wherein the hydrolytic enzyme inactivation treatment is high-temperature spray drying or high-temperature heating.

[0006] The beneficial effects are: the black soldier fly paste is hydrolyzed in a high-pressure carbon dioxide environment, carbon dioxide is dissolved to form carbonic acid, which can reduce the pH of the whole system, thereby destroying the integrity of the microbial cell membrane in the insect body, and at the same time creating an anaerobic environment to achieve low-temperature and high-efficiency sterilization without the need for chemical preservatives; the high-pressure enzyme hydrolysis environment of the present application promotes the unfolding of the spatial structure of black soldier fly protein, exposing more enzyme cutting sites, and the acidification effect of carbon dioxide can also simulate the animal digestive environment, enhancing the hydrolysis activity of pancreatic enzymes on specific peptide bonds, significantly improving the hydrolysis efficiency of black soldier fly protein, and also improving the antioxidant activity of the target substance.

[0007] Preferably, the hydrolytic enzyme in the third step is an industrial-grade pancreatic enzyme or a food-grade pancreatic enzyme; when the hydrolytic enzyme is an industrial-grade pancreatic enzyme, the amount of industrial-grade pancreatic enzyme is 2%-5% of the mass of the black soldier fly paste; when the hydrolytic enzyme is a food-grade pancreatic enzyme, the amount of food-grade pancreatic enzyme is 2%-4% of the mass of the black soldier fly paste. More preferably, the enzyme activity of the industrial-grade pancreatic enzyme is 4000 U / g, and the enzyme activity of the food-grade pancreatic enzyme is 20000 U / g.

[0008] Preferably, the hydrolytic enzyme in the third step is a food-grade trypsin with an enzyme activity of ≥200000 U / g, and the amount of food-grade trypsin is 1.5%-2.5% of the mass of the black soldier fly paste.

[0009] The heating pre-treatment time in the second step is 20-40 min; the reaction vessel in the third step is a reaction kettle. Through experiments, when the pressure in the reaction kettle is 0.5 MPa, the hydrolysis rate of black soldier fly protein is as high as 98%.

[0010] Preferably, the black soldier fly bodies in the first step are five instar black soldier fly fresh insects, five instar black soldier fly frozen insects, pre-pupa black soldier fly fresh insects, and / or pre-pupa black soldier fly frozen insects.

[0011] The application also provides application of the black soldier fly protein peptide in feed. The black soldier fly protein peptide powder prepared based on the high-pressure carbon dioxide and exogenous enzyme coupling hydrolysis technology is used as chicken feed, so that the T-SOD activity in egg white of the chicken egg is significantly improved, and the antioxidant capacity of the chicken egg is improved. In the egg chicken breeding, the addition amount of the black soldier fly protein peptide powder is preferably 0.3%-0.6% of the feed.

[0012] Compared with the prior art, the black soldier fly insect pulp is enzymolyzed in the high-pressure carbon dioxide environment, the carbon dioxide is dissolved to form carbonic acid, the pH of the whole system is reduced, the integrity of the microbial cell membrane in the insect body is destroyed, an anaerobic environment is created, low-temperature and high-efficiency sterilization is realized, and no chemical preservative is needed; in addition, the high-pressure enzymolysis environment promotes the unfolding of the spatial structure of the black soldier fly protein, more enzyme cutting sites are exposed, the acidification effect of the carbon dioxide can also simulate the animal digestion environment, the hydrolysis activity of pancreatic enzymes on specific peptide bonds is enhanced, the hydrolysis efficiency of the black soldier fly protein is significantly improved (up to 96%), and the antioxidant activity is high. The black soldier fly protein peptide prepared by the application is used for egg chicken breeding, and the quality of the chicken egg is improved. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is a process flowchart of the application. DETAILED DESCRIPTION

[0014] The preparation method of the application will be described in detail below with reference to the drawings. The embodiments are implemented on the premise of the process of the application, and detailed implementation modes and specific operation processes are given. It should be pointed out that the addition amount of the hydrolytic enzyme in each embodiment of the application is the mass fraction relative to the black soldier fly insect pulp.

[0015] The application provides a preparation method of a black soldier fly protein peptide, which hydrolyzes black soldier fly insect pulp based on high-pressure carbon dioxide autolysis and enzyme coupling hydrolysis technology. Figure 1 It can be known that the specific process flow is as follows: Firstly, a certain volume of water is added into washed and dried black soldier fly insect bodies, and the black soldier fly insect bodies are crushed and homogenized to obtain black soldier fly insect pulp; wherein the weight ratio of the black soldier fly insect bodies to water is 1:(0.4-1.2); the black soldier fly insect bodies can be five instar black soldier fly fresh insects, five instar black soldier fly frozen insects, pre-pupa black soldier fly fresh insects and / or pre-pupa black soldier fly frozen insects, and the frozen insects are thawed before being crushed and homogenized if the frozen insects are used; In the second step, the pH of the black soldier fly paste is adjusted to 7.0-9.0 and heated at 80-95 DEG C for 20-40 min. Before hydrolysis, the black soldier fly paste is adjusted to weak alkaline to promote the unfolding of the spatial structure of the black soldier fly protein, destroy the hydrogen bonds and hydrophobic interactions in the higher structure, thereby exposing more enzyme cutting sites and enhancing the catalytic efficiency of hydrolytic enzymes (such as trypsin or trypsin). In addition, alkaline conditions can also preliminarily inhibit the growth of microorganisms, reduce the consumption of proteins by microorganisms and the interference of metabolic products in the subsequent enzymatic hydrolysis process, and provide a cleaner and more efficient reaction system for the subsequent high-pressure CO2 enzymatic hydrolysis. In the present application, the black soldier fly paste is first subjected to high-temperature pretreatment before hydrolysis. On the one hand, the pretreatment can kill part of the microorganisms in the paste, reduce the initial bacterial count, reduce the influence on the hydrolysis process of the black soldier fly protein, and reduce the influence of chemical preservatives. On the other hand, the high-temperature pretreatment can further promote the denaturation and unfolding of the black soldier fly protein, destroy its natural structure, especially the chitin and other difficult-to-degrade components, and improve the accessibility and enzymatic sensitivity of the protein. In the third step, the black soldier fly paste is added to the reaction kettle, hydrolytic enzymes are added to the reaction kettle (the hydrolytic enzymes are cooled to below 50 DEG C before being added to ensure the activity of the hydrolytic enzymes), and carbon dioxide is introduced to make the black soldier fly paste undergo stirring enzymatic hydrolysis in a high-pressure carbon dioxide environment. The pressure of the reaction kettle is 0.5-1.0 MPa, the enzymatic hydrolysis temperature is 40-50 DEG C, the enzymatic hydrolysis time is 6-12 h, and the stirring speed is 150-300 rpm. The hydrolytic enzymes in the present application can be industrial-grade trypsin, food-grade trypsin, and food-grade trypsin, etc. When the hydrolytic enzyme is industrial-grade trypsin (enzyme activity 4000 U / g), its dosage is 2%-5% of the mass of the black soldier fly paste. When the hydrolytic enzyme is food-grade trypsin (enzyme activity 20000 U / g), its dosage is 2%-4% of the mass of the black soldier fly paste. When the hydrolytic enzyme is food-grade trypsin (enzyme activity 200000 U / g), its dosage is 1.5%-2.5% of the mass of the black soldier fly paste, to ensure the hydrolysis effect of the black soldier fly protein. In the fourth step, after the enzymatic hydrolysis is completed, the enzymatic hydrolysate is filtered under sterile conditions (the filtering mesh is preferably more than 60 meshes), the hydrolytic enzymes are inactivated, and a black soldier fly protein peptide complex is obtained. The inactivation treatment of the hydrolytic enzymes is high-temperature spraying drying or high-temperature heating. The high-temperature spraying drying is high-temperature spraying at 140-190 DEG C, which can not only achieve inactivation treatment, but also obtain a powdery product (i.e. black soldier fly protein peptide powder). The high-temperature heating inactivation is inactivation at 90 DEG C for 20-40 min, to obtain a liquid black soldier fly protein peptide product. In actual production, the inactivation method can be selected according to actual needs.

[0016] The application enzymolyzes black soldier fly larvae pulp in a high-pressure carbon dioxide environment, carbon dioxide is dissolved to form carbonic acid, which can reduce the pH of the whole system, and further destroy the integrity of microbial cell membranes in the larvae, and create an anaerobic environment to achieve low-temperature and high-efficiency sterilization without chemical preservatives; the high-pressure enzymolysis environment of the application promotes the unfolding of the spatial structure of black soldier fly protein, exposes more enzyme cutting sites, and the acidification effect of carbon dioxide can also simulate the animal digestive environment to enhance the hydrolysis activity of pancreatic enzymes on specific peptide bonds, significantly improve the hydrolysis efficiency of black soldier fly protein, and also improve the antioxidant performance of protein peptide products, and further improve the absorption rate of animals.

[0017] In actual production, the internal pressure of the reaction kettle is preferably controlled at 0.5-0.7 MPa, and most preferably at 0.5 MPa, which can significantly improve the hydrolysis rate of black soldier fly protein on the basis of ensuring the viable count of hydrolytic enzymes.

[0018] The method of the application will be described in more detail below in combination with specific examples. It should be pointed out that the protein hydrolysis rate of black soldier fly larvae pulp is used to evaluate the hydrolysis capacity in the application, and the protein hydrolysis rate of black soldier fly larvae pulp in the application = (initial acid-soluble protein content of black soldier fly larvae pulp - acid-soluble protein content of enzymatic hydrolysate) / initial acid-soluble protein content of black soldier fly larvae pulp x 100%. D 2 - D 1 )x 100% / A-D 1 , in the formula, A is the crude protein content of black soldier fly larvae pulp, D 1 is the initial acid-soluble protein content before hydrolysis, D 2 is the acid-soluble protein content in the enzymatic hydrolysate after hydrolysis; The crude protein content is determined by Kjeldahl method; the specific determination method of acid-soluble protein is as follows: take the enzymatic hydrolysate, centrifuge at 4000 r / min for 10 min, accurately take 5 mL of supernatant, add 20% TCA solution, mix and oscillate uniformly, then stand for 10 min, filter, centrifuge at 4000 r / min for 10 min, accurately take 10 mL of supernatant in a digestion tube, and then the acid-soluble protein content can be obtained by Kjeldahl method.

[0019] Example 1: Effect of different process parameters on the protein hydrolysis rate of black soldier fly 1. Effect of weight ratio of larvae to water on black soldier fly protein hydrolysis, the specific content is as follows: Firstly, a certain amount of water is added to multiple portions of thawed five instar black soldier fly larvae to obtain black soldier fly larvae pulp by crushing and homogenization; wherein the weight ratio of black soldier fly larvae to water is 1:0.4, 1:0.6, 1:0.8, 1:0.8 and 1:1.2. ​Second step, adjust the pH of black soldier fly insect paste to 8.0, heat at 80-95℃ for 20 min-40 min; Third step, cool the high-temperature pretreated black soldier fly insect paste to 40℃-50℃, add 4% industrial-grade trypsin (4000U / g) to the black soldier fly insect paste, hydrolyze under 0.5 MPa carbon dioxide for 10h, and the hydrolysis temperature is 45℃. Among them, the protein hydrolysis rate corresponding to different insect-water ratios is shown in Table 1.

[0020] 2. The effect of hydrolysis temperature on black soldier fly protein hydrolysis, the specific content is as follows: First step, add a certain amount of water to the thawed five instar black soldier fly larvae, and break and homogenize to obtain black soldier fly insect paste; wherein the weight ratio of black soldier fly larvae to water is 1:0.6; Second step, adjust the pH of black soldier fly insect paste to 8.0, heat at 80-95℃ for 20 min-40 min; Third step, cool the high-temperature pretreated black soldier fly insect paste to 40℃-50℃, add 4% industrial-grade trypsin (4000U / g) to the black soldier fly insect paste, hydrolyze under 0.5 MPa carbon dioxide for 10h, and the hydrolysis temperature is 45℃. Among them, the protein hydrolysis rate corresponding to different insect-water ratios is shown in Table 1.

[0021] 3. The effect of hydrolysis time on black soldier fly protein hydrolysis, the specific content is as follows: First step, add a certain amount of water to the thawed five instar black soldier fly larvae, and break and homogenize to obtain black soldier fly insect paste; wherein the weight ratio of black soldier fly larvae to water is 1:0.6; Second step, adjust the pH of black soldier fly insect paste to 8.0, heat at 80-95℃ for 20 min-40 min; Third step, cool the high-temperature pretreated black soldier fly insect paste to 40℃-50℃, add 4% industrial-grade trypsin (4000U / g) to the black soldier fly insect paste, hydrolyze under 0.5 MPa carbon dioxide for 10h, and the hydrolysis temperature is 45℃. Among them, the protein hydrolysis rate corresponding to different insect-water ratios is shown in Table 1.

[0022] 4. The effect of initial pH on black soldier fly protein hydrolysis, the specific content is as follows: First step, add a certain amount of water to the thawed five instar black soldier fly larvae, and break and homogenize to obtain black soldier fly insect paste; wherein the weight ratio of black soldier fly larvae to water is 1:0.6; Second step, the pH of the black soldier fly larvae slurry was adjusted to 6.0, 7.0, 8.0, 9.0 and 10.0, respectively, and heated at 80-95℃ for 20-40 min; third step, the high-temperature pretreated black soldier fly larvae slurry was cooled to 40-50℃, and industrial-grade trypsin (4000 U / g) was added to the black soldier fly larvae slurry, and hydrolyzed under 0.5 MPa carbon dioxide for 10 h, with a hydrolysis temperature of 45℃. Among them, the protein hydrolysis rate is shown in Table 1.

[0023] 5. The effect of industrial-grade trypsin addition on black soldier fly protein hydrolysis, the specific content is as follows: First step, add a certain amount of water to the thawed five instar black soldier fly larvae, and break and homogenize to obtain black soldier fly larvae slurry; wherein the weight ratio of black soldier fly larvae to water is 1:0.6; Second step, the pH of the black soldier fly larvae slurry was adjusted to 8.0, and heated at 80-95℃ for 20-40 min; Third step, the high-temperature pretreated black soldier fly larvae slurry was cooled to 40-50℃, and industrial-grade trypsin (4000 U / g) was added to the black soldier fly larvae slurry, and hydrolyzed under 0.5 MPa carbon dioxide for 10 h, with a hydrolysis temperature of 45℃, the amount of industrial-grade trypsin was 1%, 2%, 3%, 4% and 5%, respectively, and the hydrolysis rate is shown in Table 1.

[0024] 6. The effect of food-grade trypsin dosage on black soldier fly protein hydrolysis, the specific content is as follows: First step, add a certain amount of water to the thawed five instar black soldier fly larvae, and break and homogenize to obtain black soldier fly larvae slurry; wherein the weight ratio of black soldier fly larvae to water is 1:0.6; Second step, the pH of the black soldier fly larvae slurry was adjusted to 8.0, and heated at 80-95℃ for 20-40 min; Third step, the high-temperature pretreated black soldier fly larvae slurry was cooled to 40-50℃, and food-grade trypsin (20000 U / g) was added to the black soldier fly larvae slurry, and hydrolyzed under 0.5 MPa carbon dioxide at 45℃ for 10 h, with an enzyme dosage of 1%, 1.5%, 2%, 2.5% and 3%. Among them, the protein hydrolysis rate is shown in Table 1.

[0025] Table 1 Effect of different process parameters on black soldier fly protein hydrolysis rate From table 1, by investigating the ratio of insects to water, hydrolysis temperature, hydrolysis time, initial pH and enzyme addition amount, the results show that the optimal hydrolysis process of the application is that the ratio of insects to water is 1:0.6, the hydrolysis temperature is 45 DEG C, the hydrolysis time is 10 h, and the initial pH before hydrolysis is 8.0. Among them, when the hydrolysis enzyme is industrial grade trypsin (4000 U / g), the addition amount is greater than or equal to 4%; when the hydrolysis enzyme is food grade trypsin (enzyme activity 200000 U / g), the addition amount is greater than or equal to 2%. In addition, food grade trypsin is preferably used in hydrolysis.

[0026] Example 2 Influence of pressure in the reaction kettle on the hydrolysis rate of black soldier fly protein In the first step, a certain volume of water is added to the thawed five instar black soldier fly larvae, and the black soldier fly slurry is obtained by crushing and homogenizing; wherein the weight ratio of black soldier fly body to water is 1:0.6; In the second step, the pH of the black soldier fly slurry is adjusted to 8.0, and the black soldier fly slurry is heated at 80-95 DEG C for 20-40 min; In the third step, the black soldier fly slurry after high temperature pretreatment is cooled to 40-50 DEG C (whether cooling is needed), the black soldier fly slurry is added to the reaction kettle, the hydrolysis enzyme (food grade trypsin, the amount is 2% of the weight of the black soldier fly slurry) is added to the reaction kettle, and carbon dioxide is introduced, so that the black soldier fly slurry is stirred and enzymatically hydrolyzed in a high pressure carbon dioxide environment, the enzymatic hydrolysis temperature is 45 DEG C, the enzymatic hydrolysis time is 10 h, and the stirring speed is 150-300 rpm; In order to investigate the influence of pressure in the kettle on the hydrolysis rate of black soldier fly protein and hydrolysis enzyme, multiple pressures are designed in this step, which are 0 MPa, 0.3 MPa, 0.5 MPa, 0.7 MPa, 1.0 MPa; In the fourth step, after the enzymatic hydrolysis is completed, the enzyme solution is filtered under sterile conditions to remove impurities, and inactivated at 90 DEG C for 30 min, and the hydrolysis rate and viable bacterial count under each pressure condition are detected by sampling, and the results are shown in table 2.

[0027] Table 2 Influence of carbon dioxide pressure on hydrolysis rate and hydrolysis enzyme From the above table, when the carbon dioxide pressure is 0.3 MPa-0.7 MPa, the preparation process of the application shows high hydrolysis capacity, which is mainly because under a certain pressure, carbon dioxide can penetrate into the inside of protein molecules, destroy hydrogen bonds and hydrophobic interaction, promote the unfolding of protein spatial structure, and expose more enzyme cutting sites, so as to improve the catalytic efficiency of protease; but too high pressure will increase the rigidity of enzyme and substrate, and hinder the "induced fitting" combination process between enzyme and substrate, and finally inhibit the hydrolysis rate.

[0028] From the above table, with the increase of carbon dioxide pressure, the number of viable bacteria in the hydrolysis system first decreases and then maintains at 10 4 CFU / mL. This is mainly because high pressure carbon dioxide can penetrate the phospholipid bilayer of the cell membrane, dissolve the membrane lipid, destroy its integrity, and thus inhibit its growth. Therefore, considering the hydrolysis rate and the number of viable bacteria, the pressure of high pressure carbon dioxide in the present application is preferably controlled at 0.5 MPa-0.7 MPa, and most preferably at 0.5 MPa.

[0029] Example 3 Hydrolysis of five instar black soldier fly frozen larvae by industrial grade trypsin (4000 U / g) In the first step, a certain volume of water is added to the thawed five instar black soldier fly larvae, and the homogenate is broken to obtain black soldier fly slurry; wherein the weight ratio of black soldier fly body to water is 1:0.6; In the second step, the pH of the black soldier fly slurry is adjusted to 8.0, and heated at 80-95℃ for 20 min-40 min; In the third step, the black soldier fly slurry after high temperature pretreatment is cooled to 40℃-50℃, 4% industrial grade trypsin (4000 U / g) is added to the black soldier fly slurry, and hydrolysis is carried out under the condition of 0.5 MPa carbon dioxide for 10 h, with a hydrolysis temperature of 45℃; After hydrolysis, filter with plate and frame filter, filter mesh is 80 mesh; Spray drying at 190℃ to obtain black soldier fly protein peptide powder. It is determined that the protein hydrolysis rate in this embodiment is 94.56%, the ratio of acid-soluble protein to crude protein is 89.27%, and the protein and nutrient distribution in black soldier fly enzymatic hydrolysate is shown in Tables 3-4.

[0030] Table 3 Protein molecular weight distribution in black soldier fly protein peptide powder in this embodiment As can be seen from Table 3, after hydrolysis of black soldier fly protein, the content of protein peptide with molecular weight less than 3000 Da is as high as 93.3%, of which protein peptide within 1300 Da accounts for nearly 85%, and especially protein small molecules within 403 Da account for as high as 69.28%. The results show that the present application can hydrolyze macromolecular protein into small molecular protein peptide, which is beneficial to the absorption of animals and can improve the utilization rate of black soldier fly protein.

[0031] Table 4 Nutritional ingredients of black soldier fly protein peptide powder in this embodiment As can be seen from Table 4, the protein content of the black soldier fly protein peptide powder after enzymolysis is as high as 38.13%, which is high enough to efficiently support poultry muscle development, tissue repair and egg production needs; the moderate fat content in the black soldier fly protein peptide powder plays the role of energy core, and its high metabolic energy characteristics provide intensive and economical energy supply for the daily activities, body temperature maintenance and growth production of poultry, effectively reducing the metabolic burden caused by relying on a large amount of grain energy supply; more importantly, this fat, as a carrier of fat-soluble vitamins, greatly promotes the absorption and utilization of these key nutrients in the body, thereby collectively strengthening the immune barrier, bone health and antioxidant defense system of poultry. Furthermore, the microbial level and heavy metal content in the black soldier fly protein peptide powder of the present application meet the relevant requirements of GB 13078-2017 Feed Hygiene Standard, and the volatile base nitrogen content in the black soldier fly protein peptide powder is 20.8 mg / 100g, indicating that the protein in the product has not been spoiled and the product smell is normal.

[0032] Example 4 Hydrolysis process of food-grade trypsin (200000 U / g) for five-age black soldier fly frozen larvae In the first step, a certain volume of water is added to the thawed five-age black soldier fly larvae, and the black soldier fly larvae slurry is obtained by crushing and homogenizing; wherein the weight ratio of black soldier fly larvae to water is 1:0.6; In the second step, the pH of the black soldier fly larvae slurry is adjusted to 8.0, and the slurry is heated at 80-95℃ for 20-40 min; In the third step, the high-temperature pretreated black soldier fly larvae slurry is cooled to 40-50℃, 2% food-grade trypsin (200000 U / g) is added to the black soldier fly larvae slurry, and the slurry is hydrolyzed at 45℃ in a 0.5 MPa carbon dioxide bar environment for 10 h. After hydrolysis, the slurry is filtered with a plate and frame filter (80 mesh), and spray dried at 190℃ to obtain black soldier fly protein peptide powder. It is determined that the protein hydrolysis rate in this embodiment is 89.56%, and the ratio of acid-soluble protein to crude protein is 81.27%.

[0033] Example 5 Comparison analysis of different hydrolysis processes In this embodiment, the black soldier fly larvae slurry of each comparison group and test group is obtained by crushing and homogenizing the thawed five-age black soldier fly larvae and water at a ratio of 1:0.6, and the pH is adjusted to 8.0.

[0034] Comparison group 1 (i.e. high-temperature inactivation group): the black soldier fly larvae slurry is inactivated at 90℃ for 30 min, and then 4% food-grade trypsin is added and stirred at 45℃ for 10 h.

[0035] Comparison group 2 (with preservative): 4% food-grade trypsin and 0.42% kathon (as preservative) are added to the black soldier fly larvae slurry, and the slurry is stirred and hydrolyzed at 45℃ for 10 h.

[0036] The test group (i.e., the present application): 4% food-grade pancreatin is added to the black soldier fly worm paste, and the worm paste is hydrolyzed at 45℃ for 10h under stirring in a carbon dioxide environment at 0.5 MPa.

[0037] After the hydrolysis is completed, the protein hydrolysis rate, the ratio of acid-soluble protein to crude protein, the number of viable bacteria, the small peptide content (<3000 Da), and the DPPH free radical scavenging capacity are determined by sampling, and the results are shown in Table 5.

[0038] Table 5 Hydrolysis performance of the comparison group and the test group As shown in Table 5, the protein hydrolysis rate of the black soldier fly protease hydrolysis technology based on high-pressure carbon dioxide in the present application is as high as 96.46%, which is significantly better than that of the traditional high-temperature inactivation hydrolysis group (i.e., the comparison group 1) and the preservative group (i.e., the comparison group 2); the ratio of acid-soluble protein to crude protein in the hydrolyzate is as high as 87.56%, which is increased by 9.3% and 17.8% compared with the same period, the small molecule peptide content in the hydrolyzate is as high as 93.3%, and the hydrolyzate has high antioxidant capacity. The test results show that the hydrolysis technology based on high-pressure carbon dioxide in the present application not only can promote the hydrolysis of protein, but also can improve the antioxidant capacity, which is conducive to the absorption of animals in the breeding industry and improves the effective utilization rate of black soldier fly protein.

[0039] In summary, the enzymatic hydrolysis method based on high-pressure carbon dioxide in the present application does not need to add chemical preservatives, the hydrolysis rate of black soldier fly protein can reach 96%, the small peptide yield can reach 93%, and the antioxidant capacity is strong, which provides an innovative path for the green industrialization development of black soldier fly protein and lays a foundation for the large-scale application of black soldier fly in breeding.

[0040] Application Example The application of the black soldier fly protein peptide powder prepared in the present application in the breeding of laying hens The black soldier fly protein peptide powder is obtained by spraying the black soldier fly hydrolysate corresponding to 0.5 MPa in Example 3 of the present application, and the black soldier fly protein peptide powder is mixed with the basic feed to feed the laying hens. Among them, the laying hens are selected from the laying hens with good health and similar production performance in a certain chicken farm, a total of 240, which are randomly divided into 4 groups, each group has three repeats, and each repeat has 20 laying hens; one of the four groups of laying hens is a blank group, and the remaining three groups are test groups, and the black soldier fly protein peptide powder addition amount in the test groups corresponds to 0.3%, 0.6% and 1.2% of the daily diet of the laying hens in turn, the daily feed intake and egg production rate of the chickens are recorded, and the egg quality is determined during the peak egg production period, and the results are shown in Tables 6 and 7. In addition, after 42 days of feeding, the serum and liver of the four groups of laying hens are subjected to antioxidant detection, and the results are shown in Tables 8-9.

[0041] Table 6 Effect of black soldier fly protein peptide powder of the present application on the production performance of laying hens Table 7 Effect of black soldier fly protein peptide powder of the application on egg quality of chicken From Tables 6-7, it can be seen that adding 0.3% of the black soldier fly protein peptide powder to the basic feed can significantly improve the egg laying rate and egg weight of laying hens, and can also significantly improve the egg white height, Haugh unit and egg shape index. The results show that the black soldier fly protein peptide powder (addition amount 0.3%) of the application can improve the production performance and egg quality of laying hens.

[0042] Table 8 Antioxidant indexes of serum of laying hens after adding the black soldier fly protein peptide powder of the application Table 9 Antioxidant indexes of liver of laying hens after adding the black soldier fly protein peptide powder of the application From Tables 8 and 9, it can be seen that adding the black soldier fly protein peptide powder to the basic diet can significantly improve the T-SOD activity in the serum of laying hens, and the T-SOD activity of the 1.2% test group is increased by 60.13 U / mgprot compared with the blank group, indicating that the overall antioxidant capacity of the laying hens is enhanced; the MDA in the serum of the three test groups is significantly lower than that of the blank group, indicating that the degree of oxidative stress of the laying hens may be reduced, thereby improving the quality of eggs and the like; GSH can eliminate free radicals and protect cells from oxidative damage, and CAT can protect cells and tissues from oxidative stress damage, and adding the black soldier fly protein peptide powder can improve the GSH content and CAT activity of the laying hens.

[0043] In summary, the black soldier fly protein peptide prepared by the application can improve the serum biochemical indexes of laying hens, improve the antioxidant capacity of laying hens, and also improve the quality of eggs. For the breeding of laying hens, the optimal addition amount of the black soldier fly protein peptide powder of the application is 0.3%-0.6% of the basic diet of laying hens.

Claims

1. A method for preparing black soldier fly protein peptides, characterized in that: The preparation method is based on the coupled hydrolysis of proteins in black soldier fly larvae plasma using high-pressure carbon dioxide autolysis and enzymatic hydrolysis, and specifically includes the following: The first step is to add a certain volume of water to the washed and dried black soldier fly bodies, crush and homogenize them to obtain black soldier fly slurry; wherein, the weight ratio of black soldier fly bodies to water is 1:(0.4~1.2). The second step is to adjust the pH of the black soldier fly larvae slurry to 7.0–9.0 and pretreat it at a high temperature of 80–95°C. The third step involves cooling the black soldier fly larvae slurry to below 50°C after high-temperature pretreatment, adding hydrolytic enzymes, and then stirring and hydrolyzing it in a high-pressure carbon dioxide environment. The high-pressure carbon dioxide environment pressure is 0.5–1.0 MPa, the hydrolysis temperature is 40°C–50°C, the hydrolysis time is 6–12 h, and the stirring speed is 150–300 rpm. The fourth step is to filter the enzymatic hydrolysate to remove impurities under sterile conditions and inactivate the hydrolytic enzyme to obtain the black soldier fly protein peptide complex. The hydrolytic enzyme inactivation treatment is carried out by high-temperature spray drying or high-temperature heating.

2. The method for preparing black soldier fly protein peptides according to claim 1, characterized in that: The hydrolytic enzyme in the third step is industrial-grade pancreatic enzyme or food-grade pancreatic enzyme. When the hydrolytic enzyme is industrial-grade pancreatic enzyme, the amount of industrial-grade pancreatic enzyme used is 2% to 5% of the mass of black soldier fly larvae plasma; when the hydrolytic enzyme is food-grade pancreatic enzyme, the amount of food-grade pancreatic enzyme used is 2% to 4% of the mass of black soldier fly larvae plasma.

3. The method for preparing black soldier fly protein peptides according to claim 2, characterized in that: The industrial-grade pancreatic enzyme has an enzyme activity of 4000 U / g, while the food-grade pancreatic enzyme has an enzyme activity of 20000 U / g.

4. The method for preparing black soldier fly protein peptides according to claim 1, characterized in that: The hydrolytic enzyme in the third step is food-grade trypsin with an enzyme activity ≥200,000 U / g. The amount of food-grade trypsin used is 1.5% to 2.5% of the mass of black soldier fly larvae pulp.

5. The method for preparing black soldier fly protein peptides according to claim 1, characterized in that: The heating pretreatment time in the second step is 20-40 minutes; the reaction vessel in the third step is a reaction kettle.

6. The method for preparing black soldier fly protein peptides according to claim 1, characterized in that: The black soldier fly larvae in the first step are fifth-instar fresh black soldier fly larvae, fifth-instar frozen black soldier fly larvae, prepupal stage fresh black soldier fly larvae, and / or prepupal stage frozen black soldier fly larvae.

7. The use of black soldier fly protein peptides prepared according to any one of claims 1-6 in feed.

8. The application according to claim 7, characterized in that: The amount of black soldier fly protein peptide added is 0.3% to 0.6% of the feed mass.

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