Preparation method of high-purity insect protein enzymatic hydrolysate

Abstract

The invention discloses a preparation method of a high-purity insect protein enzymatic hydrolysate, and belongs to the technical field of insect processing.The preparation method comprises the steps that before protein is extracted, most chitin and lipid are removed through pretreatment, and the influence of the chitin and lipid on the turbidity of an extracting solution is reduced; according to the fly maggot protein dynamic isoelectric point regulation and control method and system based on the support vector machine, Zeta potential and turbidity data are fused on line, the optimal isoelectric point is predicted in real time, pH is dynamically adjusted, and high-precipitation-efficiency and high-purity protein is obtained; the polyethersulfone membrane is modified until the water contact angle is less than or equal to 65 degrees and the Zeta potential is less than or equal to-35mV, so that the pollution of protein to the membrane is reduced, and the purification efficiency of a proteolysis product is improved.

Description

Technical Field

[0001] This invention belongs to the field of insect processing technology, specifically relating to a method for preparing high-purity insect protease hydrolysate. Background Technology

[0002] Fly larvae are not only high in protein, but also rich in polysaccharides, vitamins, and fatty acids. Early utilization of fly larvae was mainly for treating feces and garbage to address environmental pollution problems, and they were also used as animal feed. This wasted fly larvae resources and failed to meet hygiene standards. Currently, many countries have done extensive work on large-scale fly larvae farming and developed related fly larvae products for use as animal feed and additives. However, research on fully extracting fly larvae protein and enzymatically hydrolyzing it into small molecule peptides with specific functional activities is still limited.

[0003] Existing fly larvae enzymatic hydrolysis processes typically involve steps such as raw material pretreatment, protein extraction, enzymatic hydrolysis, and product purification. The purpose of fly larvae protein extraction is to efficiently separate proteins from a complex matrix (10-15% chitin, 20-30% lipids) to obtain a high-purity protein concentrate. Currently, the main methods for protein extraction and preparation include alkaline extraction and acid precipitation, ultrasonic / microwave-assisted extraction, water extraction, and enzymatic extraction. Among these, alkaline extraction and acid precipitation mainly suffer from the following problems: 1. The isoelectric point range of fly larvae protein is 4.0-5.0, and traditional fixed pH precipitation methods cannot accurately match the optimal isoelectric point, resulting in low precipitation efficiency; 2. Fly larvae contain 10-15% chitin, which increases the turbidity of the extract, interfering with Zeta potential and turbidity measurements and reducing accuracy; 3. The high content of hydrophobic proteins easily forms emulsions, affecting precipitation and subsequent enzymatic hydrolysis; 4. Traditional offline Zeta potential detection is time-consuming and cannot adapt to dynamic pH control in real time, limiting industrial-scale efficiency. Polyethersulfone (PES) membranes possess excellent acid and alkali resistance, pressure resistance, stable mechanical and thermodynamic properties, and good film-forming properties, and have been widely used in purification applications. However, the hydrophobicity of PES membranes significantly limits their application in purifying fly larvae enzymatic hydrolysates. They readily adsorb hydrophobic and negatively charged large protein molecules, leading to membrane fouling and a decrease in filtration performance. Furthermore, fly larvae enzymatic hydrolysates contain a high proportion of hydrophobic and acidic amino acids, which can easily adsorb onto PES membranes through hydrophobic interactions, reducing the final product yield. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this invention provides a method for preparing high-purity insect protease hydrolysates. Before protein extraction, pretreatment removes most of the chitin and lipids, reducing their impact on the turbidity of the extract. A support vector machine-based dynamic isoelectric point control method and system for fly larvae proteins is used to predict the optimal isoelectric point in real time by fusing zeta potential and turbidity data online, dynamically adjusting the pH to obtain proteins with high precipitation efficiency and high purity. By modifying the polyethersulfone membrane to achieve a water contact angle ≤65° and a zeta potential ≤-35mV, protein contamination of the membrane is reduced, improving the purification efficiency of the protease hydrolysates.

[0005] The technical solution for achieving the objective of this invention is as follows: A method for preparing high-purity insect protease hydrolysate, comprising the following steps:

[0006] S1. Fresh fly larvae were collected, cleaned, frozen to inactivate microorganisms, thawed, dispersed in water and pulverized. The resulting slurry was mixed with choline chloride, glycerol and urea, placed in a pulsed microwave reactor for processing, cooled and then β-cyclodextrin was added and stirred. After centrifugation, the solid was collected and washed with deionized water. The collected solid was crude protein concentrate.

[0007] S2. Dissolve the crude protein concentrate in the extract, and collect the Zeta potential, turbidity, and pH data of the fly maggot protein extract in real time. Perform normalization preprocessing on the Zeta potential, turbidity, and pH data, and use a support vector regression model to fuse the data to predict the optimal isoelectric point pH value. Based on the predicted pH value, dynamically adjust the reactor pH to control the Zeta potential to -0.5 to 0.5 mV and the turbidity to reach the peak value. Precipitation is carried out at the optimal pH to recover high-purity fly maggot protein.

[0008] S3. Disperse high-purity fly larvae protein in a buffer solution with a pH of 2.0-3.0, add acidic protease for enzymatic hydrolysis, adjust the pH to 8.0-9.0 and add alkaline protease for enzymatic hydrolysis, adjust the pH to 6.5-7.5 and add neutral protease for enzymatic hydrolysis, inactivate the enzyme after enzymatic hydrolysis, centrifuge and take the supernatant to obtain fly larvae enzymatic hydrolysate;

[0009] S4. Under ultrasound assistance, the fly maggot enzymatic hydrolysate was loaded onto a modified polyethersulfone membrane, purified by ultrafiltration, and the filtrate was collected, concentrated, and evaporated to obtain a high-purity insect enzymatic hydrolysate.

[0010] Preferably, in step S1, the solid-liquid ratio of fresh fly larvae to water is 1 g:(3 mL to 5 mL); the molar ratio of choline chloride, glycerol, and urea is 2:2:1, and the preparation method is to heat to 70-80°C and stir until a transparent liquid is formed, then cool to 40°C for later use; the volume ratio of the mixture of choline chloride, glycerol, and urea to water in the slurry is 1:(3-5); the power of the pulsed microwave reactor is 300-350 W, the frequency is 5-10 Hz, and the time is 6-8 min; the amount of β-cyclodextrin added is 0.8-1.2 wt.% of the total mass of the system.

[0011] Preferably, the extraction solution in step S2 is an aqueous solution prepared by dissolving 0.15–0.25 mol of sodium chloride and 0.14–0.16 mol of potassium dihydrogen phosphate in 1 L of water, and the solid-liquid ratio of the protein concentrate to the extraction solution is 1 g:(4 mL–6 mL); the normalization process involves normalizing the Zeta potential to [-1, 1], the turbidity to [0, 1], and removing chitin interference outliers based on the Z-Score (>3σ); the vector regression model uses a radial basis function kernel, with a regularization parameter C = 1.0, a kernel width γ = 0.1, a channel width ε = 0.05, and a prediction error... The pH difference was ±0.1; the vector regression model was based on training data obtained from the analysis of amino acid content of crude high-purity insect protease hydrolysate from step S4, wherein lysine content was 6.5–8.5 wt.%, glutamic acid content was 19.0–21.0 wt.%, acidic amino acids were 25.0–27.5 wt.%, and basic amino acids were 12.0–14.0 wt.%; the pH adjustment was achieved by a peristaltic pump driven by a PLC controller, with a step size of 0.05 and a rate of 0.1–0.2 pH / min, verifying a zeta potential of -0.5–0.5 mV and turbidity >120 NTU.

[0012] Preferably, in step S3, the solid-liquid ratio of the high-purity fly larvae protein to the buffer solution is 1 g:(15 mL to 25 mL), and the buffer solution is citrate buffer; the mass ratio of the high-purity fly larvae protein to acidic protease is 15:1, the mass ratio of the high-purity fly larvae protein to alkaline protease is 25:1, and the mass ratio of the high-purity fly larvae protein to neutral protease is 20:1; the enzymatic hydrolysis temperature of the acidic protease is 35–40°C, and the time is 1–2 h; the enzymatic hydrolysis temperature of the alkaline protease is 45–50°C, and the time is 1–2 h; the enzymatic hydrolysis temperature of the neutral protease is 45–50°C, and the time is 1–2 h.

[0013] Preferably, the ultrasonic power in step S4 is 200-250W and the frequency is 15-18kHz; the pressure during ultrafiltration is 0.15-0.20MPa and the flow rate is 1.0-1.3L / min.

[0014] Preferably, the water contact angle of the modified polyethersulfone membrane is ≤65°, and the zeta potential of the modified polyethersulfone membrane is ≤-35mV.

[0015] Preferably, the method for preparing the modified polyethersulfone film in step S4 includes the following steps:

[0016] A sulfonic acid compound containing double bonds is dispersed in a mixed solvent of ethanol and water. Then, a polyethersulfone membrane is immersed in the solution. After the air in the reaction flask is replaced with an inert gas and sealed, it is irradiated at room temperature at 15–25 kGy for 1–3 h. The grafted polyethersulfone membrane is then immersed in deionized water for 20–30 h and finally freeze-dried to obtain a modified polyethersulfone membrane.

[0017] Preferably, the MWCO of the polyethersulfone film is one or more of 5kDa to 10kDa.

[0018] Preferably, the sulfonic acid compound containing a double bond has the following structure:

[0019] In the formula, R is independently at least one of a hydroxyl group and a sulfonic acid group.

[0020] Beneficial effects

[0021] This invention offers the following advantages: It provides a method for preparing high-purity insect protein hydrolysates. By optimizing the isoelectric point (pH) prediction of fly maggot proteins and subsequent enzymatic hydrolysis and purification steps, it achieves efficient and economical protein extraction and processing, offering significant advantages over existing technologies:

[0022] (1) The fly maggot slurry was treated by pulsed microwave reactor to retain protein activity. The green solvents of choline chloride, glycerol and urea and β-cyclodextrin effectively removed most of the chitin and lipids, reducing the influence of chitin and lipids on the turbidity of the extract.

[0023] (2) A method and system for dynamic isoelectric point regulation of fly maggot protein based on support vector machine, which predicts the optimal isoelectric point in real time by online fusion of Zeta potential and turbidity data, and dynamically adjusts pH to obtain protein with high precipitation efficiency and high purity.

[0024] (3) By modifying the polyethersulfone membrane to a water contact angle ≤65° and a zeta potential ≤-35mV, protein contamination of the membrane is reduced, and the purification and recovery efficiency of protease hydrolysis products is improved. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the dynamic isoelectric point system of fly maggot proteins based on support vector machines according to the present invention;

[0026] Figure 2This is a schematic diagram of the synthesis steps of the sulfonic acid compound containing a double bond according to the present invention;

[0027] Figure 3 This is the 1H NMR spectrum of the sulfonic acid compound 2 containing double bonds of the present invention. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0030] The raw materials and equipment used in the embodiments and comparative examples are described below:

[0031] Polyethersulfone membrane: grade UPS010, MWCO10 kD purchased from Unisole membrane;

[0032] Phosphorus tribromide: Anaiji Chemicals;

[0033] Sodium sulfite: Shanghai Aladdin;

[0034] N-(2,3-dihydroxypropyl)carbamate tert-butyl ester: Bide Pharmaceuticals;

[0035] 3-Bromopropene: Anage Chemicals;

[0036] Hydrogen peroxide: Sinopharm Chemical Reagent Co., Ltd.;

[0037] Sodium styrene sulfonate: Anaiji Chemical;

[0038] Maleic anhydride: Shanghai Aladdin;

[0039] Tetrabutylammonium bromide: Shanghai Aladdin.

[0040] Polyhydroxy compounds

[0041] (1) Under a nitrogen atmosphere and ice bath conditions, 3 eq of N-(2,3-dihydroxypropyl)carbamate tert-butyl ester and 1.5 eq of tetrabutylammonium bromide (TBAB) were dissolved in dichloromethane and added to the reaction vessel. Then, 3.2 eq of 3-bromopropene and 15 eq of 10 mol / L sodium hydroxide aqueous solution were added. After heating to 30 °C and stirring for 12 h, water was added to dilute the mixture. The mixture was extracted three times with dichloromethane, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The product a was purified by column chromatography.

[0042] (2) Under a nitrogen atmosphere and ice bath conditions, 3.0 eq of hydrogen peroxide was slowly added dropwise to 1 eq of acetic acid and stirred for 1 h to obtain a peroxide compound; 1 eq of product a obtained in step (1) was dissolved in dichloromethane and 1.5 eq of the obtained peroxide compound was slowly added dropwise at a temperature of 0-5℃, the temperature was raised to 30℃ and stirred for 10 h, an excess of saturated sodium bicarbonate solution was added, and stirring was continued for 6 h. The product was extracted three times with dichloromethane, dried with anhydrous magnesium sulfate, the solvent was removed by rotary evaporation, and purified by column chromatography to obtain product b;

[0043] (3) After product b continues steps (1) and (2) once, it is dissolved in a mixed solution of dichloromethane and trifluoroacetic acid under ice bath conditions, wherein the volume ratio of dichloromethane to trifluoroacetic acid is 1:1. The temperature is raised to 35°C, and after stirring for 1 hour, excess saturated sodium bicarbonate solution is added. The product is extracted three times with dichloromethane, dried with anhydrous magnesium sulfate, the solvent is removed by rotary evaporation, and purified by column chromatography to obtain product c.

[0044] (4) Under a nitrogen atmosphere, 2.0 eq of maleic anhydride, 1.0 eq of product c, and 10 eq of triethylamine were dispersed in toluene, heated to 120°C, stirred for 24 h, and after the solution cooled, the solvent was removed and dichloromethane was added. The solution was washed successively with 2 mol / L hydrochloric acid and saturated sodium carbonate solution, the organic phase was collected, dried with anhydrous magnesium sulfate and filtered, and excess solvent was removed by rotary evaporation to obtain polyhydroxy compounds.

[0045] 1. Sulfonic acid compounds containing double bonds

[0046] A stirred solution of 1 eq of a polyhydroxy compound dispersed in tetrahydrofuran was cooled to 0°C in an ice bath. A feeding funnel containing 15 eq of phosphorus tribromide dissolved in tetrahydrofuran was fixed on the reaction vessel, and the solution was added dropwise to the stirred solution over 30 min. The reaction mixture was stirred for 15 h, and the ice bath was gradually restored to room temperature. After removing the solvent, dichloromethane was added, and the mixture was washed with saturated sodium carbonate solution. The organic phase was collected, and excess solvent was removed by rotary evaporation. No further purification was required. 4 eq of sodium sulfite was dissolved in a water / ethanol (4:1) solution, and 1 eq of the product obtained above was added. The reaction mixture was heated under reflux for 24 h, cooled to 0°C, and the white crystals were collected by filtration and vacuum drying to obtain a sulfonic acid compound containing double bonds.

[0047] 2 sulfonic acid compounds containing double bonds

[0048] A stirred solution of 1 eq of a polyhydroxy compound dispersed in tetrahydrofuran was cooled to 0°C in an ice bath. A feeding funnel containing 15 eq of phosphorus tribromide dissolved in tetrahydrofuran was fixed on the reaction vessel, and the solution was added dropwise to the stirred solution over 30 min. The reaction mixture was stirred for 15 h, and the ice bath was gradually restored to room temperature. After removing the solvent, dichloromethane was added, and the mixture was washed with saturated sodium carbonate solution. The organic phase was collected, and excess solvent was removed by rotary evaporation. No further purification was required. 8 eq of sodium sulfite was dissolved in a water / ethanol (4:1) solution, and 1 eq of the product obtained above was added. The reaction mixture was heated under reflux for 24 h, cooled to 0°C, and the white crystals were collected by filtration and dried under vacuum to obtain a sulfonic acid compound containing double bonds.

[0049] 3 sulfonic acid compounds containing double bonds

[0050] A stirred solution of 1 eq of a polyhydroxy compound dispersed in tetrahydrofuran was cooled to 0°C in an ice bath. A feeding funnel containing a solution of 15 eq of phosphorus tribromide dissolved in tetrahydrofuran was fixed on the reaction vessel, and the solution was added dropwise to the stirred solution over 30 min. The reaction mixture was stirred for 15 h, and the ice bath was gradually restored to room temperature. After removing the solvent, dichloromethane was added, and the mixture was washed with saturated sodium carbonate solution. The organic phase was collected, and excess solvent was removed by rotary evaporation. No further purification was required. 16 eq of sodium sulfite was dissolved in a water / ethanol (4:1) solution, and 1 eq of the product obtained above was added. The reaction mixture was heated under reflux for 24 h, cooled to 0°C, and the white crystals were collected by filtration and dried under vacuum to obtain a sulfonic acid compound containing double bonds.

[0051] 4 sulfonic acid compounds containing double bonds

[0052] Sodium styrene sulfonate was selected as the sulfonic acid compound containing a double bond.

[0053] Modified polyethersulfone membrane 1

[0054] 15g of sulfonic acid compound 1 containing double bonds was dispersed in a mixed solvent of 50mL ethanol and 50mL water. Then, the polyethersulfone membrane was immersed in the solution. After the air in the reaction flask was replaced with an inert gas and sealed, it was irradiated at 15kGy for 1h at room temperature. The grafted polyethersulfone membrane was then immersed in deionized water for 24h and then freeze-dried to obtain modified polyethersulfone membrane 1.

[0055] Modified polyethersulfone membrane 2

[0056] The difference between the preparation method of the modified polyethersulfone film 1 and the method of the modified polyethersulfone film 1 is that the sulfonic acid compound 1 containing double bonds is replaced with the sulfonic acid compound 2 containing double bonds.

[0057] Modified polyethersulfone membrane 3

[0058] The difference between the preparation method of the modified polyethersulfone film 1 and the method of the modified polyethersulfone film 1 is that the sulfonic acid compound 1 containing double bonds is replaced with the sulfonic acid compound 3 containing double bonds.

[0059] Modified polyethersulfone membrane 4

[0060] The difference between the preparation method of the modified polyethersulfone film 1 and the method of the modified polyethersulfone film 1 is that the sulfonic acid compound 1 containing double bonds is replaced with the sulfonic acid compound 4 containing double bonds.

[0061] The following are the test methods for performance parameters involved in this invention:

[0062] Characterization of modified polyethersulfone membranes: The hydrophilicity of different modified membranes was characterized using a water contact angle meter, and the zeta potential of the membrane surface was analyzed using a zeta potential analyzer. The grafting rate of the membrane was calculated by comparing the quality of the membrane before and after modification.

[0063] Protein content determination: The Kjeldahl method was used for determination. The main steps are as follows: Accurately weigh 0.2 g of the well-mixed solid sample or 10 g of the liquid sample into a digestion tube, accurate to 0.001 g. Then, add 5.5 g of catalyst (K₂SO₄:GuSO₄ = 9:1) and 10 mL of concentrated sulfuric acid to the digestion tube, and digest in a digestion furnace for 40 min. After digestion, allow the digestion tube to cool to room temperature, place it on a Kjeldahl nitrogen analyzer, add 60 mL of 40% NaOH, and distill. Titrate with 0.1 mol / L HCl to the endpoint, and then the instrument automatically calculates and displays the protein content (mg / mL).

[0064] Protein extraction rate:

[0065] Amino acid composition analysis of enzymatic hydrolysis products: Take 3 mL of the fly larvae enzymatic hydrolysate before ultrafiltration or the filtrate after ultrafiltration, add 3 mL of 6 mol / L HCl, blow with nitrogen for 15 min, seal the tube, and place it in an oven for hydrolysis (110℃, 24 h). After hydrolysis, remove and cool, and bring the volume to 25 mL. Accurately pipette 2 mL of the final volume sample, deacidify using a rotary evaporator (60℃) until dry, add 2 mL of 0.02 mol / L HCl, shake to mix, filter through a 0.22 μm aqueous filter membrane, and determine the amino acid content using a fully automated amino acid analyzer.

[0066] Amino acid recovery rate of ultrafiltration membrane:

[0067]

[0068] Table 1 Characterization of modified polyethersulfone films 1-4

[0069] Water contact angle, ° Zeta potential, mV Grafting rate, % Modified polyethersulfone membrane 1 53.4 -41.2 22.4 Modified polyethersulfone membrane 2 51.7 -46.7 22.5 Modified polyethersulfone membrane 3 55.2 -51.5 22.7 Modified polyethersulfone membrane 4 64.1 -30.6 21.5 polyethersulfone membrane 81.2 -19.4 -

[0070] As shown in Table 1, the water contact angle and Zeta potential of modified polyethersulfone films 1-4 are all smaller than those of the unmodified polyethersulfone film. Among them, the performance of modified polyethersulfone films 1-3 is better than that of modified polyethersulfone film 4. The Zeta potential of modified polyethersulfone films 1-3 gradually increases, which is due to the increased proportion of sulfonic acid groups in the grafted sulfonic acid compounds containing double bonds.

[0071] Example 1

[0072] S1. Fresh fly larvae were collected, cleaned, and frozen to inactivate microorganisms. After thawing, 1000g of the larvae were weighed and dispersed in 4000mL of water. The larvae were then pulverized to obtain a slurry. Choline chloride, glycerol, and urea were weighed in a molar ratio of 2:2:1 and heated to 80℃ and stirred until a transparent liquid was formed. After cooling to 40℃, 1000mL of the slurry was measured and mixed with the slurry. The mixture was placed in a pulsed microwave reactor and treated at 300W and 10Hz for 8 minutes. After cooling, 1.0wt.% β-cyclodextrin was added to the total mass of the system and stirred for 30 minutes. After centrifugation, the solid was collected and washed with deionized water. The collected solid was the crude protein concentrate.

[0073] S2. Prepare a 1L aqueous solution of 0.2mol sodium chloride and 0.15mol potassium dihydrogen phosphate as the extraction solution. Dissolve 200g of crude protein concentrate in the 1L extraction solution. Collect the zeta potential, turbidity, and pH data of the fly maggot protein extract in real time at 10s intervals. Normalize and preprocess the zeta potential, turbidity, and pH data to construct a support vector regression model. The training data comes from the amino acid content obtained by analyzing the crude high-purity insect protein hydrolysate in step S4, which contains 8.2wt.% lysine, 20.2wt.% glutamic acid, 26.4wt.% acidic amino acids, and 13.1wt.% basic amino acids. The model is dynamically updated through online learning and predicts the optimal isoelectric point pH to be 4.6. Based on the predicted pH value, dynamically adjust the reactor pH with a step size of 0.05 and a rate of 0.1pH / min. Stop the adjustment when the zeta potential reaches -0.2mV and the turbidity reaches its peak value, and precipitate and recover the high-purity fly maggot protein.

[0074] S3. Disperse high-purity fly larvae protein in disodium hydrogen phosphate-citric acid buffer at pH 2.5 (solid-liquid ratio 1g:20mL), add acidic protease (mass ratio 15:1), and enzymatically hydrolyze at 37℃ for 1.5h. Adjust the pH to 8.5, add alkaline protease (mass ratio 25:1), and enzymatically hydrolyze at 45℃ for 1.5h. Adjust the pH to 7.0, add neutral protease (mass ratio 20:1), and enzymatically hydrolyze at 45℃ for 1.5h. Inactivate the enzyme (90℃, 10min), centrifuge, and collect the supernatant to obtain fly larvae enzymatic hydrolysate.

[0075] S4. Under ultrasonic assistance (power 220W, frequency 16kHz), the fly larvae enzymatic hydrolysate was loaded onto the modified polyethersulfone membrane 1, ultrafiltered (pressure 0.18MPa, flow rate 1.2L / min), the filtrate was collected, concentrated and evaporated to obtain high-purity insect enzymatic hydrolysate.

[0076] Example 2

[0077] Compared with the preparation method of Example 1, the difference is that the modified polyethersulfone membrane 1 in step S4 is replaced with the modified polyethersulfone membrane 2.

[0078] Example 3

[0079] Compared with the preparation method of Example 1, the difference is that the modified polyethersulfone membrane 1 in step S4 is replaced with the modified polyethersulfone membrane 3.

[0080] Example 4

[0081] Compared with the preparation method of Example 1, the difference is that the modified polyethersulfone membrane 1 in step S4 is replaced with the modified polyethersulfone membrane 4.

[0082] Comparative Example 1

[0083] Compared with the preparation method in Example 1, the difference is that step S1 is replaced by collecting fresh fly larvae, cleaning them, freezing and inactivating the microorganisms, thawing them, weighing 1000g and dispersing them in 5000mL of water, pulverizing them to obtain a slurry, centrifuging them, collecting the solid and washing them with deionized water. The collected solid is a crude protein concentrate, that is, without the treatment of alkali chloride, glycerol and urea slurry and β-cyclodextrin.

[0084] Comparative Example 2

[0085] Compared with the preparation method in Example 1, the difference is that in step S2, the pH is adjusted by manual titration, the Zeta potential is recorded, the optimal pH is determined to be 4.6 by visually observing the turbidity peak, and high-purity fly larvae protein is recovered by precipitation.

[0086] Comparative Example 3

[0087] Compared with the preparation method of Example 1, the difference is that the modified polyethersulfone membrane 1 in step S4 is replaced with a polyethersulfone membrane.

[0088] Table 2 Protein analysis in examples and comparative examples.

[0089] Protein extraction rate, % Amino acid recovery rate of ultrafiltration membrane, % Example 1 15.03 91.5 Example 2 14.93 92.3 Example 3 15.27 91.7 Example 4 15.11 89.8 Comparative Example 1 13.51 91.2 Comparative Example 2 12.97 90.8 Comparative Example 3 15.06 85.1

[0090] As shown in Table 2, the protein extraction rates of fresh fly larvae (wet weight) were all above 14.0%, with the amino acid recovery rates of the ultrafiltration membranes in Examples 1-3 being slightly higher than that in Example 4. The data from Comparative Example 1 showed that the protein extraction rate was lower when chitin and lipid treatment was not performed. This may be because the presence of chitin and lipids affected the turbidity of the extract, leading to a decrease in the accuracy of the prediction in step S3 and consequently a lower protein extraction rate. The data from Comparative Example 2 showed that the protein extraction rate recovered by manual titration to adjust pH and precipitate was lower. The data from Comparative Example 3 showed that the amino acid recovery rate of the proteolytic enzyme hydrolysate from fresh fly larvae obtained using an unmodified polyethersulfone membrane was lower than that in the examples.

[0091] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for the preparation of a high purity insect proteolysate, characterized in that, The method comprises the following steps: S1. Fresh fly larvae are collected, and microorganisms are inactivated by freezing. After thawing, the fly larvae are dispersed in water and pulverized. The obtained slurry is mixed with choline chloride, glycerol and urea, and then placed in a pulsed microwave reactor for treatment. After cooling, β-cyclodextrin is added and stirred. The solid is collected by centrifugation and washed with deionized water. The collected solid is a crude protein concentrate; S2. The crude protein concentrate is dissolved in an extraction solution. The Zeta potential, turbidity and pH data of the fly larva protein extraction solution are collected in real time. The Zeta potential, turbidity and pH data are normalized and pretreated. A support vector regression model is used to fuse the data and predict the optimal isoelectric point pH value. According to the predicted pH value, the pH of the reactor is dynamically adjusted to control the Zeta potential to be -0.5-0.5 mV and the turbidity to reach a peak. Precipitation is carried out at the optimal pH, and high-purity fly larva protein is recovered; S3. The high-purity fly larva protein is dispersed in a buffer solution with a pH of 2.0-3.0, and acid protease is added for enzymatic hydrolysis. Then, the pH is adjusted to 8.0-9.0, and alkaline protease is added for enzymatic hydrolysis. Finally, the pH is adjusted to 6.5-7.5, and neutral protease is added for enzymatic hydrolysis. After enzyme inactivation, the supernatant is obtained by centrifugation to obtain a fly larva enzymatic hydrolysate; S4. Under the assistance of ultrasonic, the fly larva enzymatic hydrolysate is loaded onto a modified polyether sulfone membrane, and then purified by ultrafiltration. The filtrate is collected and concentrated and evaporated to obtain a high-purity insect protein hydrolysate.

2. A process for the preparation of a high purity insect proteolysate according to claim 1, characterized in that, The solid-liquid ratio of fresh fly larvae to water in step S1 is 1g:(3mL-5mL). The molar ratio of choline chloride, glycerol and urea is 2:2:

1. The preparation method is to heat to 70-80℃ and stir until a transparent liquid is formed, and then cool to 40℃ for use. The volume ratio of the choline chloride, glycerol and urea mixture to water in the slurry is 1:(3-5). The power of the pulsed microwave reactor is 300-350W, the frequency is 5-10Hz, and the time is 6-8min. The addition amount of β-cyclodextrin is 0.8-1.2wt.% of the total mass of the system.

3. A process for the preparation of a high purity insect proteolysate according to claim 1, characterized in that, The extraction solution in step S2 is 0.15-0.25 mol of sodium chloride and 0.14-0.16 mol of potassium dihydrogen phosphate configured as a 1L aqueous solution, the solid-liquid ratio of the protein concentrate and the extraction solution is 1g:(4mL-6mL), the normalization processing is to normalize the Zeta potential to [-1, 1] and the turbidity to [0, 1], and to remove chitin interference outliers based on Z-Score (>3σ); the vector regression model adopts a radial basis function kernel, a regularization parameter C=1.0, a kernel width γ=0.1, a pipe width ε=0.05, and a prediction error ±0.1 pH; the vector regression model is based on training data derived from the amino acid content obtained by analyzing the step S4 crude high-purity insect protease product, wherein the lysine content is 6.5-8.5 wt.%, the glutamic acid content is 19.0-21.0 wt.%, the acidic amino acid content is 25.0-27.5 wt.%, and the basic amino acid content is 12.0-14.0 wt.%; the pH adjustment is driven by a PLC controller to drive a peristaltic pump, with a step size of 0.05 and a rate of 0.1-0.2 pH / min, and the verification Zeta potential is -0.5-0.5 mV and the turbidity is >120 NTU.

4. A process for the preparation of a high purity insect proteolysate according to claim 1, characterized in that, The solid-liquid ratio of the high-purity fly maggot protein and the buffer solution in step S3 is 1g:(15mL-25mL), and the buffer solution is a citric acid buffer solution; the mass ratio of the high-purity fly maggot protein to the acid protease is 15:1, the mass ratio of the high-purity fly maggot protein to the alkaline protease is 25:1, and the mass ratio of the high-purity fly maggot protein to the neutral protease is 20:1; the temperature for the acid protease to perform enzymolysis is 35-40℃, and the time is 1-2h; the temperature for the alkaline protease to perform enzymolysis is 45-50℃, and the time is 1-2h; the temperature for the neutral protease to perform enzymolysis is 45-50℃, and the time is 1-2h.

5. A process for the preparation of a high purity insect proteolysate according to claim 1, characterized in that, The power of the ultrasonic assistance in step S4 is 200-250W, and the frequency is 15-18kHz; the pressure during the ultrafiltration is 0.15-0.20Mpa, and the flow rate is 1.0-1.3L / min.

6. A process for the preparation of a high purity insect proteolysate according to claim 1, characterized in that, The water contact angle of the modified polyether sulfone membrane is ≤65°, and the Zeta potential of the modified polyether sulfone membrane is ≤-35mV.

7. A process for the preparation of a high purity insect proteolysate according to claim 1, characterized in that, The preparation method of the modified polyether sulfone membrane in step S4 has the following steps: The double-bond-containing sulfonic acid-based compound is dispersed in a mixed solvent of ethanol and water, then the polyether sulfone membrane is immersed in the solution, the air in the reaction bottle is replaced by inert gas and sealed, after irradiation at room temperature for 1-3h at 15-25kGy, the grafted polyether sulfone membrane is placed in deionized water for 20-30h, and then the membrane is freeze-dried to obtain the modified polyether sulfone membrane.

8. A process for the preparation of a high purity insect proteolysate according to claim 7, characterized in that, The MWCO of the polyether sulfone membrane is one or several of 5kDa-10kDa.

9. A process for the preparation of a high purity insect proteolysate according to claim 7, characterized in that, The double-bond-containing sulfonic acid-based compound has the following formula structure: wherein R is independently at least one of a hydroxyl group and a sulfonic acid group.

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