Acidification hydrolysis method for improving yield of hermetia illucens small molecule peptide and application

Abstract

The invention discloses an acidification hydrolysis method for improving the yield of hermetia illucens small molecule peptides and application. The acidification hydrolysis method comprises the following steps: firstly, carrying out acidification pretreatment on hermetia illucens bodies, and adding protease for hydrolysis treatment under an acidic condition; by means of acidic hydrolysis, on one hand, bacterial cell membrane proton gradient imbalance can be achieved, membrane protein of bacterial cell membranes is inactivated, organic acid can penetrate through bacterial cell membranes, intracellular dissociation is achieved, intracellular pH is reduced, ATP synthesis is inhibited, then microbial activity is inhibited, and sterilization of bacteria in hermetia illucens is achieved; on the other hand, hydrogen bonds and salt bridges in the hermetia illucens protein can be destroyed by the acid environment, so that a compact spherical structure of the protein is expanded, more enzyme cutting sites (especially hydrophobic residues) are exposed, and more small molecule peptide fragments are obtained. Through tests, small peptides with the molecular weight of 729 Da to 246 Da in the hydrolyzed product account for 73.03% of the total content.

Description

Technical Field

[0001] This invention relates to the field of deep processing of resource-based insects, and in particular to an acid hydrolysis method and its application for improving the yield of small molecule peptides from black soldier fly larvae. Background Technology

[0002] Black soldier fly ( Hermetia illucens L. Black soldier fly larvae, belonging to the genus *Bufo scintillans* of the family Bandidae in the order Diptera, are saprophytic insects. Currently, black soldier fly larvae are widely used for the treatment of solid organic waste such as kitchen waste, poultry manure, wet waste, and crop straw due to their broad diet, large food intake, low nutritional requirements, and high safety. Black soldier fly larvae are rich in protein, fat, and antimicrobial peptides; the crude protein content in their dry matter is as high as 47%-50%, comparable to that of aquatic and livestock animals, making them a good substitute for fishmeal and soybean meal in aquaculture and livestock farming. However, if black soldier fly powder is directly fed to animals as a feed additive, the large molecular structure of its protein may not be effectively broken down by digestive enzymes in the animal's intestines, leading to reduced protein utilization and affecting animal growth and development. In addition, black soldier flies mainly feed on kitchen waste and livestock manure, which leads to them carrying pathogenic bacteria such as Bacillus and Salmonella. The metabolism of these bacteria produces biogenic amines such as putrescine and cadaverine, which cause foul odor and toxicity, resulting in the deterioration of the insect powder or insect paste.

[0003] To improve the utilization rate of black soldier fly protein, it is typically broken down into small-molecule protein peptides or amino acids. Currently, there are two main methods for hydrolyzing black soldier fly protein: microbial fermentation and enzymatic hydrolysis. Microbial fermentation utilizes microorganisms to hydrolyze black soldier fly protein, but during fermentation, microorganisms convert the protein into cell protein, consuming some of the protein and affecting its effective utilization rate. Furthermore, metabolic byproducts produced during microbial metabolism often affect the yield and quality of the hydrolysate. Enzymatic hydrolysis mainly relies on exogenous proteases, resulting in a lower hydrolysis rate. Moreover, due to the breeding environment and scavenging nature of black soldier flies, chemical preservatives need to be added during the hydrolysis process. Therefore, improving the hydrolysis rate of black soldier fly protein and obtaining small-molecule protein peptides remains a key technical challenge that the industry needs to overcome. Summary of the Invention

[0004] In view of this, the first objective of this invention is to propose an acid hydrolysis method to improve the yield of small molecule peptides from black soldier fly larvae, and the second objective of this invention is to propose the application of black soldier fly protein peptides.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The acid hydrolysis method for improving the yield of small molecule peptides from black soldier fly larvae, as described in this invention, involves first pretreating the black soldier fly larvae with acid before hydrolysis. Specifically, the method includes the following steps: The first step is to add a certain volume of organic acid solution to the washed and dried black soldier fly bodies, and then crush and homogenize them to obtain black soldier fly slurry; wherein the weight ratio of black soldier fly bodies to organic acid is 1:(0.4~1.2). The second step involves adjusting the pH of the black soldier fly larvae slurry to 3.0–5.5, and then adding an exogenous enzyme to the acidified black soldier fly larvae slurry and stirring to hydrolyze it; wherein the exogenous enzyme is an acidic protease or pepsin. The third step is to filter out impurities after enzymatic hydrolysis and inactivate the hydrolysate to obtain black soldier fly protein peptides. The inactivation of the hydrolysate includes heat inactivation and spray inactivation. Heat inactivation is performed at 90°C for 20 to 30 minutes, and spray inactivation is performed by spray drying at 140 to 190°C.

[0006] The beneficial effects are as follows: This invention utilizes an acid solution and a homogenized black soldier fly body, followed by hydrolysis under acidic conditions. On the one hand, the acidic environment can cause an imbalance in the proton gradient of the bacterial cell membrane, inactivating its membrane proteins. On the other hand, the organic acid can penetrate the bacterial cell membrane, dissociate inside the cell, lower the intracellular pH, inhibit ATP synthesis, and thus inhibit microbial activity, thereby sterilizing the bacteria in the black soldier fly and simplifying the entire hydrolysis process.

[0007] On the other hand, an acidic environment can disrupt the hydrogen bonds and salt bridges in black soldier fly proteins, allowing the protein's tight globular structure to unfold and exposing more cleavage sites (especially hydrophobic residues). Exogenous enzymes can then effectively cleave aromatic amino acids (Phe, Trp, Tyr) and leucine, yielding small peptides. Experiments have shown that small peptides with molecular weights between 729 Da and 246 Da account for 73.03% of the total content in the hydrolysis product of this invention.

[0008] Furthermore, the acidic environment of this invention can effectively inhibit the generation of microbial decarboxylases (such as lysine decarboxylase and ornithine decarboxylase), thereby blocking the generation of toxic biogenic amines such as cadaverine and putrescine from the source and improving the safety of drinking black soldier fly hydrolysates. Experiments have shown that the volatile basic nitrogen (VBN) in the hydrolysates obtained using this invention is only 41.5 mg / 100g, proving that biogenic amines are effectively controlled.

[0009] Preferably, the organic acid in the first step is any one of citric acid solution, propionic acid solution, or lactic acid solution, wherein the mass concentration of citric acid is 6%–8%, the mass concentration of propionic acid is 0.5%–2%, and the mass concentration of lactic acid is 0.5%–2%. Citric acid, lactic acid, and propionic acid are all aqueous solutions.

[0010] Preferably, in the second step, the acidic protease has an enzyme activity of 200,000 U / g, and its dosage is 0.07%–0.3% of the mass of the black soldier fly larvae plasma; the pepsin has an enzyme activity of 200,000 U / g, and its dosage is 0.1%–0.3% of the mass of the black soldier fly larvae plasma. In actual hydrolysis, either acidic protease or pepsin can be used.

[0011] Preferably, the hydrolysis temperature in the second step is 40℃~50℃, and the hydrolysis time is 60~72 h.

[0012] The application of black soldier fly protein peptides prepared by this invention in feed. Specifically, the amount of black soldier fly protein peptides accounts for 12.5% ​​of the total shrimp feed. Experiments have shown that the black soldier fly protein peptides of this invention, as a shrimp feed additive, can significantly improve the survival rate and growth performance of shrimp, and significantly enhance the activity of antioxidant enzymes in shrimp serum, indicating that adding the hydrolysate of this invention can improve the health status and disease resistance of shrimp. Attached Figure Description

[0013] Figure 1 This is a process flow diagram of the present invention. Detailed Implementation

[0014] The preparation method of the present invention will be described in detail below with reference to the accompanying drawings. This embodiment is implemented based on the process of the present invention, and provides detailed implementation methods and specific operating procedures. It should be noted that the amount of hydrolytic enzyme added in each embodiment of the present invention is a relative mass fraction of black soldier fly larvae slurry.

[0015] This invention proposes an acidification and hydrolysis method for black soldier fly protein. The method involves first pretreating the black soldier fly body with acid, then hydrolyzing it in an acidic environment to obtain black soldier fly protein peptide products. Specifically, the method includes the following steps: The first step involves adding a certain volume of organic acid solution to the washed and dried black soldier fly larvae, then crushing and homogenizing them to obtain black soldier fly larvae slurry. The weight ratio of black soldier fly larvae to organic acid solution is 1:(0.4–1.2). The organic acid can be any one of citric acid, propionic acid, or lactic acid, and is prepared using ultrapure water. The mass concentration of citric acid is 6%–10%, the mass concentration of propionic acid is 0.5%–2%, and the mass concentration of lactic acid is 0.5%–2%. The black soldier fly larvae include fresh fifth-instar black soldier fly larvae, frozen fifth-instar black soldier fly larvae, fresh prepupal black soldier fly larvae, and / or frozen prepupal black soldier fly larvae. For frozen larvae, they should be thawed before crushing and homogenizing. The second step involves adjusting the pH of the black soldier fly larvae slurry to 3.0–5.5, adding exogenous enzymes to the acidified slurry, and hydrolyzing at 40–50°C for 60–72 hours. The exogenous enzymes used are acidic protease or pepsin. The acidic protease has an activity of 200,000 U / g, and its dosage is 0.07–0.3% of the mass of the black soldier fly larvae slurry. The pepsin has an activity of 200,000 U / g, and its dosage is 0.1%–0.3% of the mass of the black soldier fly larvae slurry. The third step, after enzymatic hydrolysis, is to filter (with a mesh size of 60 mesh or higher) to remove impurities and inactivate the hydrolysate to obtain black soldier fly protein peptides. The inactivation of the hydrolysate includes heat inactivation and spray inactivation. Heat inactivation is performed at 90°C for 20 to 30 minutes to obtain black soldier fly protein peptide hydrolysate. Spray inactivation is performed by spray drying at 140 to 190°C to obtain black soldier fly protein peptide powder.

[0016] This invention utilizes an acid solution and a homogenized black soldier fly larvae, followed by hydrolysis under acidic conditions. On one hand, the acidic environment can cause an imbalance in the proton gradient of the bacterial cell membrane, inactivating its membrane proteins. On the other hand, the organic acids can penetrate the bacterial cell membrane, dissociate inside the cell, lower the intracellular pH, inhibit ATP synthesis, and thus inhibit microbial activity, thereby sterilizing the bacteria in the black soldier fly larvae and simplifying the entire hydrolysis process.

[0017] On the other hand, an acidic environment can disrupt the hydrogen bonds and salt bridges in black soldier fly proteins, allowing the protein's tight globular structure to unfold and exposing more cleavage sites (especially hydrophobic residues). Exogenous enzymes can then effectively cleave aromatic amino acids (Phe, Trp, Tyr) and leucine, yielding small peptides. Experiments have shown that small peptides with molecular weights between 729 Da and 246 Da account for 73.03% of the total content in the hydrolysis product of this invention.

[0018] Furthermore, the acidic environment of this invention can effectively inhibit the generation of microbial decarboxylases (such as lysine decarboxylase and ornithine decarboxylase), thereby blocking the generation of toxic biogenic amines such as cadaverine and putrescine from the source and improving the safety of drinking black soldier fly hydrolysates. Experiments have shown that the volatile basic nitrogen (VBN) in the hydrolysates obtained using this invention is only 41.5 mg / 100g, proving that biogenic amines are effectively controlled.

[0019] The method of the present invention will be described in more detail below with reference to specific embodiments. It should be noted that the present invention evaluates the hydrolysis ability using the protein hydrolysis rate of black soldier fly larvae slurry. In the present invention, the protein hydrolysis rate of black soldier fly larvae slurry = ( D 2 - D 1 )×100% / (A- D 1In the formula, A represents the crude protein content of black soldier fly larvae slurry. D 1 This represents the initial acid-soluble protein content before hydrolysis. D 2 The content of acid-soluble proteins in the enzymatic hydrolysate after hydrolysis; The crude protein content was determined by the Kjeldahl method. The specific method for determining acid-soluble protein was as follows: take the enzymatic hydrolysate, centrifuge at 4000 r / min for 10 min, accurately measure 5 mL of the supernatant, add 20% TCA solution, mix and shake evenly, then let stand for 10 min, filter, centrifuge at 4000 r / min for 10 min, accurately measure 10 mL of the supernatant into a digestion tube, and then determine the acid-soluble protein content by the Kjeldahl method.

[0020] Example 1: Effects of various process parameters on the hydrolysis of black soldier fly protein 1. Effect of acidic protein dosage on black soldier fly protein hydrolysis The first step is to add a certain volume of 1% propionic acid solution to the thawed fifth-instar black soldier fly larvae, and then crush and homogenize them to obtain black soldier fly larvae pulp; wherein the weight ratio of black soldier fly larvae body to 1% propionic acid solution is 1:0.6. The second step involved adjusting the pH of the black soldier fly larvae slurry to 4.5, dividing the slurry into multiple portions, and adding acidic protease (enzyme activity 200,000 U / g) to each portion. The portions were then hydrolyzed at 45°C with stirring for 72 hours. The amounts of acidic protease added were 0%, 0.03%, 0.05%, 0.07%, 0.1%, and 0.15% of the mass of the black soldier fly larvae slurry, respectively. The third step, after enzymatic hydrolysis, is to heat at 90℃ for 20 to 30 minutes to inactivate the enzymes, centrifuge to obtain the supernatant, and determine the protein content and viable bacteria count of the supernatant to calculate the protein hydrolysis rate, as detailed in Table 1.

[0021] Table 1. Effects of acidic protease dosage on protein hydrolysis and microbial activity in black soldier fly larvae. As shown in Table 1, the protein hydrolysis rate steadily increased with the increase of acidic protease content. When the protease content reached 0.1%, the rate of increase in the protein hydrolysis rate slowed significantly and no longer showed statistical significance. Therefore, the optimal amount of acidic protease added is below 0.15%, more preferably 0.1%. The viable cell count after adding protease can be suppressed to below 10⁻⁶. 5 The concentration of CFU / mL indicates that the addition of acidic protease can effectively inhibit the growth of harmful bacteria in black soldier flies.

[0022] 2. Effect of propionic acid solution concentration on the hydrolysis of black soldier fly protein The first step involves adding a certain volume and concentration of propionic acid solution (with mass concentrations of 0%, 0.5%, 1%, 1.5%, and 2%) to the thawed fifth-instar black soldier fly larvae, then crushing and homogenizing the larvae to obtain black soldier fly larvae pulp; wherein the weight ratio of fifth-instar black soldier fly larvae to 1% propionic acid solution is 1:0.6. The second step involves adjusting the pH of the black soldier fly larvae slurry to 4.5, adding acidic protease (enzyme activity 200,000 U / g), and hydrolyzing at 45°C for 72 hours with stirring. The amount of acidic protease added is 0.1% of the mass of the black soldier fly larvae slurry. The third step, after enzymatic hydrolysis, is to heat at 90℃ for 20 to 30 minutes to inactivate the enzymes, centrifuge to obtain the supernatant, and determine the protein content and viable bacteria count of the supernatant to calculate the protein hydrolysis rate, as detailed in Table 2.

[0023] Table 2. Effects of different concentrations of propionic acid solution on protein hydrolysis and microorganisms in black soldier fly larvae. Table 2 shows that as the propionic acid concentration increased from 0% to 2%, the pH of the black soldier fly larvae slurry gradually decreased, from 6.9 to approximately 4.5. Without propionic acid, the protein content was only 37.82%, but after adding 0.5% propionic acid, it significantly increased to 76.43%, reaching a peak of 81.42% at a concentration of 1.5%, after which it tended to stabilize. Simultaneously, the viable bacterial count decreased significantly with increasing propionic acid concentration. The results indicate that the addition of propionic acid not only effectively lowered the pH of the system, creating a more suitable acidic environment for the action of acidic proteases, thus significantly improving the protein hydrolysis rate; but also, as an organic acid, propionic acid has a strong antibacterial effect, significantly reducing the number of microorganisms and minimizing their interference with the protein hydrolysis process, further promoting efficient protein hydrolysis. Therefore, propionic acid in this experiment has a dual role in regulating pH and inhibiting microorganisms, making it a key factor in improving the efficiency of black soldier fly protein hydrolysis and the yield of small molecule protein peptides. In this invention, the propionic acid concentration was 0.5–2%.

[0024] 3. Effect of insect-to-solid ratio on protein hydrolysis in black soldier fly larvae A certain volume of 1% propionic acid solution was added to the thawed fifth-instar black soldier fly larvae, and the mixture was crushed and homogenized to obtain black soldier fly larvae slurry. The weight ratio of black soldier fly larvae to 1% propionic acid solution was 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1, and 1:1.2. The pH of the black soldier fly larvae slurry was adjusted to 4.5, and 0.1% acidic protease (enzyme activity 200,000 U / g) was added. The mixture was stirred and hydrolyzed at 45℃ for 72 h. After the enzymatic hydrolysis was completed, the larvae were heated at 90℃ for 20 min to 30 min to obtain the supernatant. The protein content and viable bacterial count of the supernatant were determined, as shown in Table 3.

[0025] Table 3. Effects of different insect-to-solid ratios on protein hydrolysis and microorganisms in black soldier fly larvae. Table 3 shows that the protein hydrolysis rate initially increases and then decreases with increasing insect-to-solid ratio. The highest hydrolysis rate of 79.97% is achieved at an insect-to-solid ratio of 1:0.6. This indicates that when the propionic acid solution ratio is low, the system's water content is insufficient, potentially leading to inadequate contact between the enzyme and substrate, poor fluidity, and thus limiting enzymatic hydrolysis efficiency. As the liquid ratio increases, the system gradually dilutes, resulting in more sufficient contact between the enzyme and protein, and an increased hydrolysis rate. However, when the liquid ratio is too high, the substrate concentration per unit volume in the system decreases significantly, leading to a relatively insufficient enzyme concentration and weakened reaction driving force, thus causing a decrease in the protein hydrolysis rate. Therefore, an insect-to-solid ratio of 1:0.6 may be the optimal hydrolysis system ratio under the conditions of this experiment.

[0026] 4. Effect of hydrolysis time on the hydrolysis of black soldier fly protein.

[0027] A certain volume of 1% propionic acid solution was added to the thawed fifth-instar black soldier fly larvae, and the mixture was crushed and homogenized to obtain black soldier fly larvae slurry. The weight ratio of black soldier fly larvae to 1% propionic acid solution was 1:0.6. The pH of the black soldier fly larvae slurry was adjusted to 4.5, and acidic protease (enzyme activity 200,000 U / g) was added. The mixture was stirred and hydrolyzed at 45°C for 36 h, 48 h, 60 h, 72 h, 84 h, and 96 h, respectively. After the enzymatic hydrolysis was completed, the larvae were heated at 90°C for 20 min to 30 min to inactivate the enzymes. After centrifugation, the protein content and viable bacterial count of the supernatant were determined, as shown in Table 4.

[0028] Table 4. Effects of different hydrolysis times on black soldier fly protein hydrolysis and microorganisms. As shown in Table 4, the hydrolysis rate of black soldier fly protein showed a continuous upward trend as the hydrolysis time increased from 36 h to 96 h, significantly increasing from 43.55% at 36 h to 81.94% at 72 h. However, after 72 h, the increase in the hydrolysis rate decreased sharply, with hydrolysis rates of 82.08% and 82.41% at 84 h and 96 h, respectively, indicating that the hydrolysis process gradually stabilized. Considering all factors, 72 h can be regarded as the optimal hydrolysis time in this invention, balancing efficiency and economy.

[0029] 3. Effect of hydrolysis temperature on the hydrolysis of black soldier fly protein.

[0030] A certain volume of 1% propionic acid solution was added to the thawed fifth-instar black soldier fly larvae, and the mixture was crushed and homogenized to obtain black soldier fly larvae slurry. The weight ratio of black soldier fly larvae to 1% propionic acid solution was 1:0.6. The pH of the black soldier fly larvae slurry was adjusted to 4.5, and acidic protease (enzyme activity 200,000 U / g) was added. The mixture was stirred and hydrolyzed for 72 h at a certain temperature. The hydrolysis temperatures were 30℃, 35℃, 40℃, 45℃, 50℃, and 55℃. After the enzymatic hydrolysis was completed, the larvae were heated at 90℃ for 20-30 min to inactivate the enzymes. The supernatant was obtained by centrifugation, and the protein content and viable cell count of the supernatant were determined. The protein hydrolysis rate was calculated, as shown in Table 5.

[0031] Table 5. Effects of different hydrolysis temperatures on protein hydrolysis and microbial activity in black soldier fly larvae. As shown in Table 5, the hydrolysis rate of black soldier fly protein initially increases and then decreases with increasing hydrolysis temperature, reaching a maximum of 80.37% at 45℃. Specifically, between 30℃ and 45℃, the kinetic energy of enzyme molecules increases with rising temperature, leading to increased collision efficiency between the enzyme and substrate molecules and a faster reaction rate, thus resulting in a continuous increase in the hydrolysis rate. However, when the temperature exceeds 45℃, the high temperature begins to damage the structure of the enzyme protein, causing enzyme denaturation and inactivation. Therefore, the optimal hydrolysis temperature for the acidic protease in this invention is preferably 45℃.

[0032] Example 2 This embodiment analyzes the prepared product, specifically including the following: a certain volume of 1% propionic acid solution was added to the thawed fifth-instar black soldier fly larvae, and the mixture was crushed and homogenized to obtain black soldier fly larvae pulp; wherein, the weight ratio of the thawed fifth-instar black soldier fly larvae to 1% propionic acid was 1:0.6. The pH of the black soldier fly larvae slurry was adjusted to 4.5, and 0.1% acidic protease was added. The mixture was stirred and hydrolyzed at 45°C for 72 h. After hydrolysis, the mixture was filtered (100 mesh) and spray-dried at 190°C to obtain black soldier fly protein peptide powder. In this example, the protein hydrolysis rate was 79.3%, and the ratio of acid-soluble protein to crude protein was 81.49%. The black soldier fly protein peptide powder obtained in this example was analyzed, and the results are shown in Tables 6-7.

[0033] Table 6. Protein molecular weight distribution in black soldier fly protein peptide powder Table 7 shows that after hydrolysis, the molecular weight of black soldier fly protein is concentrated in the range of 729-246 Da, with a content as high as 73.03%, and the content of small molecule peptides (2000 Da) is as high as 95.49%. The results indicate that the present invention can hydrolyze large molecule proteins into small molecule protein peptides with a molecular weight of less than 2000 Da, thereby improving the utilization rate of black soldier fly protein.

[0034] Table 7 Nutritional composition of black soldier fly protein peptide powder As shown in Table 7, the protein content of the enzymatically hydrolyzed black soldier fly protein peptide powder is as high as 40.13%, which is sufficient to efficiently support the muscle development, tissue repair, and egg production needs of poultry, fish, and shrimp. Furthermore, the microbial level and heavy metal content of the black soldier fly protein peptide powder of this invention meet the requirements of GB 13078-2017, and the volatile basic nitrogen content in the black soldier fly protein peptide powder is 41.5 mg / 100g, indicating that biogenic amines are effectively controlled.

[0035] Example 3 This embodiment uses citric acid solution as the acid solution for acidification treatment to investigate its effect on the hydrolysis of black soldier fly protein. Specifically, the following steps are included: adding a certain concentration of citric acid solution (with citric acid mass concentrations of 4%, 5%, 6%, 7%, and 8%) to thawed fifth-instar black soldier fly larvae, crushing and homogenizing to obtain black soldier fly larvae slurry; wherein the weight ratio of frozen larvae to citric acid solution is 1:0.8; adding 0.2% pepsin to the black soldier fly larvae slurry, adjusting the pH to 3.5, and hydrolyzing at 45°C for 72 hours; after enzymatic hydrolysis, filtering (80 mesh), and spray drying at 180°C to obtain black soldier fly protein peptide powder.

[0036] Table 8. Effects of different concentrations of citric acid solution on protein hydrolysis and microorganisms in black soldier fly larvae. Table 8 shows that with the increase of citric acid solution concentration, the hydrolysis rate of black soldier fly protein initially increased significantly and then tended to stabilize, while the number of viable bacteria in the sample decreased significantly. The results indicate that when the citric acid concentration is ≥6.0%, it effectively lowers the pH of the system, creating a more suitable acidic environment for pepsin activity, thereby promoting protein hydrolysis. Furthermore, as an organic acid, citric acid has strong antibacterial ability, significantly reducing the number of viable bacteria by disrupting the microbial cell membrane structure and interfering with metabolic processes.

[0037] Example 4 This embodiment uses lactic acid as an organic acid to investigate its effect on the hydrolysis of black soldier fly protein. Specifically, the following steps were taken: a certain concentration of lactic acid solution (with citric acid concentrations of 0.1%, 0.5%, 1%, 1.5%, and 2%) was added to thawed prepupal black soldier fly larvae, and the mixture was crushed and homogenized to obtain black soldier fly larvae slurry; the weight ratio of prepupal black soldier fly larvae to lactic acid solution was 1:1; 0.2% pepsin was added to the black soldier fly larvae slurry, the pH was adjusted to 3.5, and hydrolysis was carried out at 45°C with stirring for 72 h; after enzymatic hydrolysis, the mixture was filtered (80 mesh) and spray-dried at 180°C to obtain black soldier fly protein peptide powder. The protein hydrolysis rate and the number of viable bacteria in the hydrolysate are shown in Table 9.

[0038] Table 9. Effects of different concentrations of lactic acid solution on protein hydrolysis and microorganisms in black soldier fly larvae. As shown in Table 9, within the concentration range of 0.1% to 2%, the hydrolysis rate of black soldier fly protein significantly increased and gradually stabilized, while the viable cell count fluctuated slightly at low concentrations before significantly decreasing. The increase in lactic acid concentration effectively lowered the pH of the system, providing a more suitable environment for pepsin, thereby promoting protein decomposition. Secondly, lactic acid, as an organic acid, has strong antibacterial properties and can significantly reduce the viable cell count by disrupting microbial cell membranes and inhibiting enzyme activity. Therefore, in this invention, the lactic acid concentration is preferably controlled at 0.5% to 2% to promote the generation of small molecule protein peptides from black soldier fly larvae.

[0039] Example 5 In this embodiment, 1% propionic acid was used as an organic acid to investigate its effect on the hydrolysis of black soldier fly protein. Specifically, the following steps were taken: 1% propionic acid solution was added to washed and dried prepupal black soldier fly larvae, and the larvae were crushed and homogenized to obtain black soldier fly larvae pulp; wherein, the weight ratio of prepupal black soldier fly larvae to 1% propionic acid solution was 1:1. 0.1% acidic protease was added to black soldier fly larvae slurry to adjust the pH to 4.5, and the mixture was stirred and hydrolyzed at 45°C for 72 h. After enzymatic hydrolysis, the mixture was filtered (100 mesh) and spray-dried at 190°C to obtain black soldier fly protein peptide powder. In this example, the protein hydrolysis rate was 77.3%, and the ratio of acid-soluble protein to crude protein was 80.49%.

[0040] Example 6 Comparison of Hydrolysis Processes Control group 1 (i.e. microbial fermentation group): thawed fifth-instar black soldier fly larvae and distilled water were homogenized at a mass ratio of 1:0.6, inoculated with 4% Aspergillus niger solution, fermented at 37℃ for 72h, and then filtered.

[0041] Control group 2 (i.e. alkaline hydrolysis group): The thawed fifth-instar black soldier fly larvae and distilled water were homogenized at a mass ratio of 1:0.6, 3% Kathon preservative (mass ratio, effective concentration of 14%) and 0.1% food-grade trypsin were added, the pH was adjusted to 8.0, and hydrolysis was carried out at 45℃ for 72 hours.

[0042] Control group 3 (inorganic acid treatment group): The thawed fifth-instar black soldier fly larvae and distilled water were homogenized at a mass ratio of 1:0.6, the pH was adjusted to 4.5 with 6 mol / L hydrochloric acid, and 0.1% acidic protease was added. The mixture was hydrolyzed at 45℃ for 72 h.

[0043] Experimental group (i.e., the acidification and hydrolysis group of the present invention): thawed fifth-instar black soldier fly larvae and 1% propionic acid solution were homogenized at a mass ratio of 1:0.6, 0.1% acidic protease was added, the pH was adjusted to 4.5, and hydrolysis was carried out at 45°C for 72 hours.

[0044] After the above four groups of hydrolysis were completed, the supernatant was obtained by centrifugation and filtration. The supernatant was then used for analysis, and the results are shown in Table 10-11.

[0045] Table 10 Comparative Analysis of Hydrolysis Results of Four Groups of Hydrolysis Processes Table 11 Comparative Analysis of Peptide Distribution in Hydrolysis Products from Four Hydrolysis Processes As shown in Tables 10 and 11, compared with control groups 1-3, the acid-soluble protein / crude protein ratio in the experimental group was as high as 82.56%, significantly better than control groups 1-3. The content of small peptides with molecular weights in the range of 3000-300D in the experimental group was as high as 78.42%, significantly higher than control groups 1-3. This indicates that acidic protease hydrolysis under suitable conditions can better avoid excessive hydrolysis of proteins into free amino acids or inactive small peptides, thus retaining more bioactive intermediate peptides. The results show that the acidification pretreatment synergistically with acidic protease in this invention can efficiently cleave the large molecular weight proteins of black soldier fly larvae, generating more easily absorbed bioactive small peptides. Furthermore, the volatile basic nitrogen content in the experimental group was significantly lower than that in control groups 1-3, indicating that the protein degradation process of the present invention is more controllable, effectively inhibiting the formation of putrefactive amines and thus improving product stability. Moreover, compared to traditional alkaline hydrolysis and inorganic acid hydrolysis, the propionic acid system of the present invention, while efficiently releasing small peptides, maintains the stability of the hydrolysis environment through self-buffering antibacterial action, avoiding the introduction of exogenous preservatives, and providing an optimized path for the green preparation of high-purity peptide products.

[0046] Application Example: Application of the black soldier fly larvae protein peptide powder of the present invention as a feed additive in shrimp farming. The black soldier fly protein peptide powder obtained in this embodiment of the invention was mixed with the basic feed for shrimp farming of Litopenaeus vannamei. The Litopenaeus vannamei shrimp were sourced from the Henan Agricultural University Science and Technology Park farming base. 1200 uniformly sized and healthy shrimp (initial weight 1.20±0.02g) were selected and randomly divided into four groups, with three replicates per group and 100 shrimp per replicate. The four groups were randomly assigned to 12 1.0m×1.0m×1.2m net cages, which were then randomly placed in indoor farming ponds for 8 weeks. One group served as the control group (without the black soldier fly protein peptide powder of this invention), while the other three groups were experimental groups. The black soldier fly protein peptide powder was used at 6.25%, 12.5%, and 18.75% of the total feed, respectively. Specific feed ratios are shown in Table 12.

[0047] Table 12 Feed composition of four groups of shrimp After the aquaculture was completed, 24 hours after feeding was stopped, the shrimp in each net cage were counted and weighed, and the survival rate, weight gain rate and feed conversion ratio of the shrimp were calculated. The results are shown in Table 13. Five shrimp were randomly selected from each net cage, and hemolymph was extracted from the pericardial cavity. The samples were centrifuged at 4℃ and 4000 r / min for 10 min. The supernatant was used to determine the immunoenzyme activity (if it could not be determined in time, the supernatant was stored at -80℃ for later use). The results are shown in Table 14.

[0048] Table 13 Growth of four groups of shrimp Table 14 Antioxidant properties of four groups of shrimp Tables 13 and 14 show that the final average weight, weight gain rate, and survival rate of experimental group B showed an increasing trend, and the feed conversion ratio of experimental group B was significantly lower than that of the control group, saving shrimp feed costs. Furthermore, the total antioxidant capacity, peroxidase, and superoxide dismutase levels in the serum of Litopenaeus vannamei in experimental groups B and C were significantly increased, especially in experimental group B. In Litopenaeus vannamei farming, the optimal addition level of black soldier fly larvae protein peptide powder is 12.5% ​​of the shrimp diet, which can replace part of the fishmeal, reduce feed consumption, improve shrimp survival rate and antioxidant capacity, and is beneficial to the commercial development of black soldier fly larvae protein.

[0049] In summary, this invention utilizes organic acid to pre-acidify black soldier fly larvae before enzymatic hydrolysis. The combination of acidification and protease can inhibit the growth of miscellaneous bacteria in the hydrolysate and promote the hydrolysis of black soldier fly protein into small molecule peptides, thereby increasing the yield of small molecule peptides and facilitating the commercial development of black soldier fly larvae.

Claims

1. An acid hydrolysis method for improving the yield of small molecule peptides from black soldier fly larvae, characterized in that: The acidification and hydrolysis method involves first pretreating the black soldier fly body with acid before hydrolysis, specifically including the following steps: The first step is to add a certain volume of organic acid solution to the washed and dried black soldier fly bodies, and then crush and homogenize them to obtain black soldier fly slurry; wherein the weight ratio of black soldier fly bodies to organic acid is 1:(0.4~1.2). The second step involves adjusting the pH of the black soldier fly larvae slurry to 3.0–5.5, and then adding an exogenous enzyme to the acidified black soldier fly larvae slurry and stirring to hydrolyze it; wherein the exogenous enzyme is an acidic protease or pepsin. The third step is to filter out impurities after enzymatic hydrolysis and inactivate the hydrolysate to obtain black soldier fly protein peptides. The inactivation of the hydrolysate includes heat inactivation and spray inactivation. Heat inactivation is performed at 90°C for 20 to 30 minutes, and spray inactivation is performed by spray drying at 140 to 190°C.

2. The acid hydrolysis method for improving the yield of small molecule peptides from black soldier fly larvae according to claim 1, characterized in that: The organic acid in the first step is any one of citric acid solution, propionic acid solution, or lactic acid solution, with a mass concentration of 6% to 8% for citric acid, 0.5% to 2% for propionic acid, and 0.5% to 2% for lactic acid.

3. The acid hydrolysis method for improving the yield of small molecule peptides from black soldier fly larvae according to claim 1, characterized in that: In the second step, the acidic protease has an enzyme activity of 200,000 U / g and is used at a rate of 0.07 to 0.2% of the mass of black soldier fly larvae slurry. The pepsin has an enzyme activity of 200,000 U / g and is used at a rate of 0.1% to 0.3% of the mass of black soldier fly larvae slurry.

4. The acid hydrolysis method for improving the yield of small molecule peptides from black soldier fly larvae according to claim 1, characterized in that: The hydrolysis temperature in the second step is 40℃~50℃, and the hydrolysis time is 60~72 h.

5. The application of the black soldier fly protein peptide obtained according to any one of claims 1-4 in feed.

6. The application according to claim 1, characterized in that: The amount of black soldier fly protein peptides used accounts for 12.5% ​​of the total shrimp feed.

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