A method for large-scale preparation of high-quality euphausia superba peptide based on complex enzyme enzymolysis

Abstract

This invention discloses a method for large-scale preparation of high-quality Antarctic krill peptides based on compound enzyme hydrolysis, comprising the following steps: S1, pretreatment of defatted Antarctic krill powder; S2, compound enzyme synergistic hydrolysis; S3, graded filtration and slag removal; S4, defluorination and impurity removal; S5, concentration and drying. Through the optimized compound enzyme synergistic hydrolysis process, the spatial structure of Antarctic krill protein can be fully cleaved, retaining more physiologically active small molecule peptides. The resulting Antarctic krill peptides have a molecular weight ≤1000Da ≥94%, protein purity ≥85%, fluorine content ≤39ppm, selenium retention rate ≥90%, and DPPH free radical scavenging rate ≥67%. Furthermore, it can significantly enhance the antioxidant enzyme activity of aging model animals and alleviate liver and kidney tissue damage. The use of a proprietary defluorination and impurity removal technology can effectively remove residual fluoride ions, soluble salts, pigments, and other impurities from the raw materials, ensuring that the product meets relevant national food safety standards and avoiding the food safety risks caused by excessive fluorine content.

Description

Technical Field

[0001] This invention belongs to the field of industrial preparation technology of marine bioactive peptides, specifically relating to a method for large-scale preparation of high-quality Antarctic krill peptides based on enzymatic hydrolysis of compound enzymes. Background Technology

[0002] Antarctic krill is one of the world's largest marine biological resources in terms of biomass. Its muscle dry sample contains 64.44% crude protein, includes all eight essential amino acids, and has a high selenium content of 2.80 mg / kg, making it a high-quality selenium-rich protein resource. Antarctic krill peptides, as hydrolyzed products of Antarctic krill protein, possess various physiological activities such as easy absorption, antioxidant properties, and anti-aging effects, and have broad application prospects in functional foods and health products.

[0003] Currently, the preparation of Antarctic krill peptides mostly adopts a single-enzyme hydrolysis process. However, when hydrolyzing with a single alkaline protease or neutral protease, the spatial structure of krill protein cannot be fully broken down, resulting in a small molecule active peptide (molecular weight ≤1000Da) accounting for only about 75%, and the recovery rate of active ingredients (such as antioxidant peptides and anti-aging peptides) is less than 60%, which is difficult to meet the needs of high-end products. In addition, the fluoride residue in Antarctic krill shells can easily transfer to peptide products. Products prepared by traditional processes often have a fluoride content exceeding 60ppm and an ash content (mainly sodium and calcium) of >12%, which not only affects the taste of the product but also poses a risk to food safety and does not meet the relevant requirements of GB2762-2022 "National Food Safety Standard Limits of Contaminants in Food". Summary of the Invention

[0004] The purpose of this invention is to provide a method for large-scale preparation of high-quality Antarctic krill peptides based on enzymatic hydrolysis of a complex enzyme, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for large-scale preparation of high-quality Antarctic krill peptides based on enzymatic hydrolysis using a complex enzyme, comprising the following steps:

[0006] S1. Pretreatment of defatted Antarctic krill powder: Take defatted Antarctic krill powder with residual oil rate ≤1.5% and protein content ≥60%, add 0.5-1.5 times the volume of water each time, stir and soak for 0.5-2 hours, then filter. Repeat this operation three times. After ultra-fine grinding and passing through a 100-mesh sieve, add deionized water at a material-to-liquid ratio of 1:4.5 (kg:L), stir and rehydrate for 30 minutes, adjust the pH of the system to 7.5-8.5, and heat to 50-55℃ to obtain a krill protein suspension.

[0007] S2. Synergistic Enzymatic Hydrolysis with Compound Enzymes: A compound enzyme preparation is added to the krill protein suspension. The compound enzyme consists of an alkaline protease with an activity of 20,000 U / g, a flavor protease with an activity of 15,000 U / g, and a neutral protease with an activity of 18,000 U / g, in a mass ratio of 3:1:1. Enzymatic hydrolysis is carried out at 55-60℃ and a stirring rate of 120 rpm for 4-4.5 hours, during which the pH is monitored every hour and kept stable. After enzymatic hydrolysis, the temperature is raised to 80-85℃ and held for 15 minutes to inactivate the enzyme, and then cooled to room temperature.

[0008] S3. Graded filtration and residue removal: The enzyme hydrolysate after enzyme inactivation is sequentially filtered through a 20μm filter screen and a 0.45μm microfiltration membrane to remove shrimp shell residue and unhydrolyzed protein, and the clarified enzyme hydrolysate is collected.

[0009] S4. Defluorination and impurity removal: The clarified enzymatic hydrolysate is passed through 0.1% activated carbon and 0.2% chelating agent and reacted at 20-40℃ for 3 hours to remove pigment impurities and most of the fluoride ions by adsorption. The purified solution is then obtained by filtration.

[0010] S5. Concentration and Drying: The refined liquid is concentrated to a solid content of 50%-55% using a vacuum concentration device, and then freeze-dried or spray-dried to obtain the Antarctic krill peptide product. The finished product is then screened, packaged, and stored.

[0011] Preferably, in step S1, the defatted Antarctic krill powder has a residual oil rate of 1.0%-1.5% and a protein content of 59%-62%, clearly defining the key indicator range of the pretreated raw materials to ensure stable raw material quality and guarantee the uniformity of subsequent product quality.

[0012] Preferably, in step S2, the total amount of the compound enzyme added is 0.22%-0.25% of the mass of defatted Antarctic krill powder. Limiting the amount of compound enzyme added ensures the enzymatic hydrolysis efficiency while avoiding excessive enzyme addition that increases costs or insufficient enzyme addition that affects the hydrolysis effect.

[0013] Preferably, in step S3, during the graded filtration, degreased cotton and diatomaceous earth can be added to avoid filter clogging, optimize the filtration process, prevent filter clogging, and ensure the continuity and efficiency of large-scale production.

[0014] Preferably, in step S4, the chelating agent is a silicon-based chelating agent that can specifically bind fluoride ions without affecting the retention of selenium. This clarifies the type and advantages of the chelating agent, achieving the dual effect of efficient removal of fluoride ions and retention of beneficial selenium.

[0015] Preferably, in step S4, after obtaining the purified liquid through filtration, a membrane separation purification step is further included: the filtered purified liquid is pumped into a 400-500 Da nanofiltration membrane system and treated under conditions of 1.5-2.0 MPa and 30-35°C, 0.1%-0.2% silicon chelating agent is added, and the retentate is collected for subsequent concentration and drying to further remove small molecule impurities and residual fluoride ions, thereby significantly improving the purity and quality of the product.

[0016] Preferably, in step S5, the freeze-drying conditions are -50℃ and a vacuum degree of 510Pa, and the spray-drying conditions are an inlet air temperature of 160-165℃ and an outlet air temperature of 78-80℃. By specifying the specific parameters of the two drying methods, the quality of the finished product is ensured to be stable and adaptable to different product needs.

[0017] In the preferred embodiment of S5, the finished Antarctic krill peptide product has the following specifications: protein purity ≥85%, moisture ≤3.3%, ash content ≤9.98%, fluorine content ≤39ppm, selenium content ≥2.5mg / kg, molecular weight ≤1000Da ≥94%, and DPPH free radical scavenging rate ≥67%.

[0018] Preferably, in step S1, the pH regulator used to maintain pH stability during enzymatic hydrolysis is a sodium hydroxide solution or a sodium bicarbonate solution, with a concentration of 0.1-0.5 mol / L. This avoids introducing impurities that could affect product purity. By clearly defining the type and concentration of the pH regulator, the enzymatic hydrolysis efficiency can be maintained while preventing the introduction of impurities and ensuring product purity.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] (1) Through the optimized compound enzyme synergistic enzymatic hydrolysis process, the spatial structure of Antarctic krill protein can be fully cleaved, and more physiologically active small molecule peptides can be retained, so that the active function of the product can be effectively enhanced. The resulting Antarctic krill peptides have a molecular weight of ≤1000Da and a proportion of ≥94%, protein purity ≥85%, fluorine content ≤39ppm, selenium retention rate ≥90%, DPPH free radical scavenging rate ≥67%, and can significantly improve the antioxidant enzyme activity of aging model animals and alleviate liver and kidney tissue damage. The activity is far superior to that of products made by traditional processes.

[0021] (2) The exclusive defluorination and impurity removal technology can effectively remove residual fluoride ions, soluble salts, pigments and other impurities from the raw materials, ensuring that the products meet the relevant national food safety standards and avoiding the food safety risks caused by excessive fluoride content. At the same time, the technology can retain beneficial trace elements such as natural selenium in the raw materials to the maximum extent, achieving the unity of product safety, nutritional value and active function, and broadening the application scope of the products.

[0022] (3) The combination of graded filtration and chelating agent can effectively avoid equipment blockage in large-scale production. The dual solution of freeze / spray drying takes into account both high-end products and mass production needs. The parameters of each step are adapted to large-scale production. The unit product cost is reduced by 15%~20% compared with the traditional process, and continuous production can be achieved. Attached Figure Description

[0023] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "sleeved with," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be a connection within two components. For those skilled in the art, the specific meaning of the above terms in this invention can be understood according to the specific circumstances.

[0026] Please see Figure 1 The present invention provides a technical solution:

[0027] Example 1

[0028] Pretreatment: Take 100 kg of defatted Antarctic krill powder (residual oil rate 1.2%, protein content 62%, purchased from an Antarctic krill deep processing enterprise, processed by low-temperature defatting process, odorless and lumpy), add 1.0 volume (i.e., 100 L) of deionized water each time, and stir with an electric stirrer at a speed of 80 rpm for 10 min to fully mix the krill powder with the water. Then soak at room temperature for 1.0 h, stirring once every 20 min (stirring speed 50 rpm). After soaking, filter with a plate and frame filter press (filtration pressure 0.3 MPa, filter cloth pore size 5 μm) to remove water-soluble impurities, trace residual oil and small shrimp shell fragments. Repeat the soaking-filtration operation three times to ensure that impurities are fully removed. Place the filtered krill powder in a vacuum drying oven and dry it at 60℃ and vacuum degree 0.08 MPa until the moisture content is ≤5.0%, and then send it to... The shrimp powder was pulverized in an ultra-fine pulverizer (pulverization speed 28000 rpm). After pulverization, it was sieved through a 100-mesh standard sieve (aperture 0.154 mm). The material on the sieve (about 3.2 kg, accounting for 3.2%) was pulverized a second time (pulverization speed 28000 rpm, pulverization time 5 min) and sieved again until all of it passed through the 100-mesh sieve. The sieved shrimp powder was taken and 450 L of deionized water was added precisely at a material-to-liquid ratio of 1:4.5 (kg:L). The mixture was placed in a stainless steel reactor and stirred at 100 rpm for 30 min to rehydrate. During this period, the pH of the system was monitored in real time with a pH meter and the pH was adjusted to 8.2 with 0.3 mol / L sodium hydroxide solution. Then, the mixture was heated through a water bath in the reactor jacket and slowly heated to 52 °C (heating rate 2 °C / min). The mixture was stirred at a constant temperature for 10 min to obtain a uniformly dispersed krill protein suspension without obvious precipitation.

[0029] Synergistic enzymatic hydrolysis with compound enzymes: A compound enzyme preparation was precisely added to the above-mentioned krill protein suspension at 0.22% (i.e., 0.22 kg) of defatted Antarctic krill powder. The compound enzyme consisted of an alkaline protease with an activity of 20,000 U / g (purchased from a biotechnology company, model Alcalase 2.4L), a flavor protease with an activity of 15,000 U / g (model Flavorzyme 500MG), and a neutral protease with an activity of 18,000 U / g (model Neutrase). Mix 0.8L of the enzyme preparation at a mass ratio of 3:1:1 until homogeneous. After adding the enzyme preparation, stir at 120 rpm for 15 minutes to ensure sufficient contact between the enzyme and the suspension. Stabilize the reactor temperature at 58℃ and maintain a stirring rate of 120 rpm for enzymatic hydrolysis. The hydrolysis time is 4.2 hours. During this period, monitor the system pH every hour with a pH meter. If the pH deviates from 8.2±0.1, finely adjust it to a stable range using 0.3 mol / L sodium hydroxide solution or 0.2 mol / L hydrochloric acid solution to avoid pH fluctuations affecting enzyme activity. After the enzymatic hydrolysis is completed, rapidly heat the mixture to 82℃ using a jacketed water bath and hold it at this temperature for 18 minutes to inactivate the enzyme (completely terminate the enzymatic hydrolysis reaction and prevent over-hydrolysis from affecting product quality). After enzyme inactivation, rapidly cool the mixture to room temperature (25℃) using an ice-water bath while maintaining a low stirring speed of 50 rpm during the cooling process to prevent protein precipitation.

[0030] Staged filtration and slag removal: The cooled enzymatic hydrolysate is transported to the staged filtration system through pipelines. First, it passes through a 20μm stainless steel filter screen (filtration pressure 0.2MPa) to remove shrimp shell residue, incompletely crushed solid particles, and other large impurities. During the filtration process, the filter screen is rinsed with deionized water every 30 minutes to prevent clogging. Then, the coarse filtrate is sent to a 0.45μm ceramic microfiltration membrane device (operating pressure 0.4MPa, membrane flux 150L / (m²·h)) to remove unhydrolyzed macromolecular proteins, colloidal impurities, and some microorganisms. During the filtration process, the feed liquid temperature is controlled at 25±2℃. The clarified enzymatic hydrolysate (420L in total) is collected. After testing, the transmittance of the clarified enzymatic hydrolysate is ≥95%, and there are no visible suspended solids.

[0031] Defluorination and impurity removal: The clarified enzymatic hydrolysate was fed into a defluorination reaction vessel. Food-grade activated carbon (100 mesh particle size, specific surface area 800 m² / g) was added at 0.1% (0.42 kg) of the enzymatic hydrolysate volume, and silicon chelating agent (model FS-01, fluoride ion adsorption capacity ≥15 mg / g) was added at 0.2% (0.84 kg) of the enzymatic hydrolysate volume. The reaction temperature was adjusted to 30℃, and the reaction was stirred at 80 rpm for 3 hours. During the reaction, the fluoride ion concentration was sampled every 30 minutes to ensure the fluoride ion removal effect. After the reaction was completed, the solution was filtered through a 0.22 μm filter membrane to remove the activated carbon and chelating agent residues, resulting in a clear and transparent krill peptide refined solution. The fluoride content of the refined solution was ≤40 ppm, the pigment removal rate was ≥85%, and there was no obvious odor.

[0032] Concentration and Drying: The refined liquid was sent to a vacuum concentration tank and concentrated under vacuum conditions of 0.09 MPa and 60°C. During the concentration process, the liquid was continuously stirred (stirring speed 60 rpm), and the solid content was checked every 15 minutes. When the solid content reached 52%, the concentration was stopped, and a concentrated liquid (about 88 L) was obtained. The concentrated liquid was sent to a freeze dryer and the freeze drying conditions were set as follows: pre-freezing temperature -50°C, pre-freezing time 2 h, sublimation drying temperature -30°C, desorption drying temperature 25°C, vacuum degree 510 Pa, and drying time 12 h. After drying, 18.5 kg of loose, milky white Antarctic krill peptide product was obtained. A small amount of agglomerates were removed by sieving through a 100-mesh sieve. Then, the product was vacuum-packed in aluminum foil (500 g per bag) and stored in a dry, cool, and ventilated warehouse (temperature 15-25°C, relative humidity ≤60%).

[0033] Product performance testing: Protein content was 86.8% using the Kjeldahl method, moisture content was 3.1% using the Karl Fischer method, ash content was 9.8% using the gravimetric method, fluorine content was 38 ppm using the ion-selective electrode method, selenium content was 2.65 mg / kg using hydride generation-atomic fluorescence spectrometry, the percentage of molecules with a molecular weight ≤1000 Da was 94.5% using gel filtration chromatography (GPC), and free radical scavenging rate was 68.2% using the DPPH method. Anti-aging activity testing: Using a D-galactose-induced aging mouse model, after continuous gavage administration of this product (dose 200 mg / kg·d) for 30 days, the SOD activity in the mouse liver tissue increased by 32% and the MDA content decreased by 28% compared to the model group, indicating that the product has significant anti-aging activity.

[0034] Example 2

[0035] Pretreatment: Take 200 kg of defatted Antarctic krill powder (residual oil rate 1.0%, protein content 61%, same source as in Example 1), add 1.2 times the volume (i.e., 240 L) of deionized water each time, stir with an electric stirrer at 85 rpm for 12 min, soak at room temperature for 1.5 h, stirring once every 30 min (speed 55 rpm), after soaking, filter with a plate and frame filter press (filtration pressure 0.35 MPa, filter cloth pore size 5 μm), repeat the soaking-filtration operation three times; put the filtered krill powder into a vacuum drying oven, dry at 65℃ and vacuum degree 0.085 MPa until the moisture content is ≤4.8%, and then send it to an ultrafine... The shrimp powder was pulverized in a pulverizer (28,000 rpm) and sieved through a 100-mesh sieve. The material remaining on the sieve (approximately 5.8 kg, accounting for 2.9%) was pulverized again and sieved again to ensure that all of it passed through. The sieved shrimp powder was then added to 1000 L of deionized water at a material-to-liquid ratio of 1:5 (kg:L) and placed in a large stainless steel reactor (1500 L capacity). The mixture was stirred at 105 rpm for 30 min to rehydrate the water. The pH was adjusted to 8.5 with 0.3 mol / L sodium hydroxide solution. The mixture was then heated to 55 °C at a rate of 2 °C / min through a jacketed water bath and stirred at a constant temperature for 15 min to obtain a uniform krill protein suspension with no obvious stratification.

[0036] Enzymatic hydrolysis with compound enzyme: Add compound enzyme preparation (enzyme type and ratio as in Example 1) to the krill protein suspension at 0.25% (i.e., 0.5 kg) of defatted Antarctic krill powder. Stir at 120 rpm for 20 min to ensure uniform dispersion of the enzyme preparation. Stabilize the reactor temperature at 60℃ and maintain the stirring rate at 120 rpm for 4.5 h. During this period, monitor the pH every 1 h and finely adjust it to 8.5 ± 0.1 with 0.3 mol / L sodium hydroxide solution or 0.2 mol / L hydrochloric acid solution to ensure stable enzymatic hydrolysis. After enzymatic hydrolysis, rapidly heat the jacketed water bath to 85℃ and keep it at this temperature for 20 min to inactivate the enzyme. After inactivation, cool the system to room temperature (25℃) using an ice-water bath. During the cooling process, maintain low-speed stirring at 55 rpm to prevent precipitation.

[0037] Staged filtration: The cooled enzymatic hydrolysate is fed into a staged filtration system. First, it is filtered through a 20μm stainless steel filter screen (filtration pressure 0.25MPa) to remove large particulate impurities. During filtration, the filter screen is rinsed with deionized water every 25 minutes to avoid clogging. Then, the coarse filtrate is fed into a 0.45μm ceramic microfiltration membrane device (operating pressure 0.45MPa, membrane flux 145L / (m²·h)). The feed temperature is controlled at 25±2℃. Unhydrolyzed proteins and colloidal impurities are removed by filtration. 920L of clear enzymatic hydrolysate is collected. The transmittance is ≥96%, and no visible impurities are found.

[0038] Defluorination and impurity removal: The clarified enzymatic hydrolysate was fed into a defluorination reaction vessel, and 0.1% food-grade activated carbon (0.92 kg, same as in Example 1) and 0.2% silicon chelating agent (1.84 kg) were added. The reaction temperature was adjusted to 35°C, and the mixture was stirred at 85 rpm for 3 hours. During this period, the fluoride ion concentration was sampled every 30 minutes to ensure the defluorination effect. After the reaction, the mixture was filtered through a 0.22 μm filter membrane to remove the activated carbon and chelating agent residues, resulting in a clarified krill peptide purified solution with a fluoride content ≤38 ppm and an odor removal rate ≥90%.

[0039] Membrane separation purification: The purified solution is pumped into a 400-500Da nanofiltration membrane system (membrane material is polyamide, operating pressure is 2.0MPa, membrane flux is 80L / (m²·h)) using a high-pressure pump. The feed temperature is controlled at 35℃. At the same time, a silicon chelating agent is added at 0.2% (i.e., 1.84kg) of the purified solution volume to further remove small molecule impurities (such as small molecule salts and residual fluoride ions). During operation, the membrane surface is rinsed once every 1 hour to prevent membrane fouling. 320L of nanofiltration retentate is collected. After testing, the retentate fluoride content is ≤37ppm, the ash content is ≤10.0%, and the protein purity is ≥87%.

[0040] Concentration and Drying: The retentate was fed into a vacuum concentrator and concentrated under a vacuum of 0.095 MPa and a temperature of 62°C. The stirring rate was 65 rpm, and the solid content was checked every 15 minutes. Concentration was stopped when the solid content reached 55%, yielding a concentrated liquid (approximately 128 L). The concentrated liquid was then fed into a spray dryer with an inlet air temperature of 160°C, an outlet air temperature of 80°C, a feed rate of 15 L / h, and an atomization pressure of 0.3 MPa. After drying, 38.2 kg of Antarctic krill peptide product was obtained. The product was a light yellow, loose powder, free of lumps and odor. After sieving through a 100-mesh sieve, it was vacuum-packed in aluminum foil (1 kg per bag) and stored in a warehouse (warehouse conditions were the same as in Example 1).

[0041] Product performance indicators: Protein content 87.5% (Kjeldahl method), moisture 3.0% (Karl Fischer method), ash 9.6% (gravimetric method), fluorine content 36ppm (ion-selective electrode method), selenium content 2.70mg / kg (hydride generation-atomic fluorescence spectrometry), molecular weight ≤1000Da 95.1% (GPC method), DPPH free radical scavenging rate 69.5%; Anti-aging activity test: After D-galactose-induced aging mice were administered this product by gavage (dose 200mg / kg·d) for 30 days, the SOD activity in liver tissue increased by 34% and the MDA content decreased by 30% compared with the model group, and the antioxidant and anti-aging activities were superior to the product in Example 1.

[0042] Example 3

[0043] Pretreatment: Take 50 kg of defatted Antarctic krill powder (residual oil rate 1.5%, protein content 59%, same source as in Example 1), add 0.8 times the volume (i.e., 40 L) of deionized water each time, stir with an electric stirrer at 75 rpm for 8 min, soak at room temperature for 0.8 h, stirring once every 20 min (speed 45 rpm). After soaking, filter with a plate and frame filter press (filtration pressure 0.25 MPa, filter cloth pore size 5 μm), repeat the soaking-filtration operation three times; place the filtered krill powder in a vacuum drying oven and dry at 58℃ and vacuum degree 0.075 MPa until the moisture content is ≤5.2%. The shrimp powder was fed into an ultrafine pulverizer (28,000 rpm) for pulverization. After sieving through a 100-mesh sieve, the material remaining on the sieve (approximately 1.6 kg, accounting for 3.2%) was pulverized again and sieved again to ensure that all of it passed through. The sieved shrimp powder was then added to 200 L of deionized water at a material-to-liquid ratio of 1:4 (kg:L) and placed in a stainless steel reactor (300 L capacity). The mixture was stirred at 95 rpm for 30 min to rehydrate the water. The pH was adjusted to 7.5 with 0.3 mol / L sodium hydroxide solution. The mixture was then heated to 50 °C at a rate of 2 °C / min through a jacketed water bath and stirred at a constant temperature for 10 min to obtain a uniform krill protein suspension.

[0044] Compound enzyme hydrolysis: Add compound enzyme preparation (enzyme type and ratio as in Example 1) to the krill protein suspension at 0.20% (i.e., 0.1 kg) of defatted Antarctic krill powder. Stir at 120 rpm for 15 min to ensure thorough mixing of enzyme and suspension. Stabilize the reactor temperature at 55℃ and maintain a stirring rate of 120 rpm for 4 h of hydrolysis. Monitor the pH every 1 h and finely adjust it to 7.5 ± 0.1 with 0.3 mol / L sodium hydroxide solution or 0.2 mol / L hydrochloric acid solution to ensure hydrolysis efficiency. After hydrolysis, heat the jacketed water bath to 80℃ and keep it at that temperature for 15 min to inactivate the enzyme. After inactivation, cool to room temperature (25℃) in an ice-water bath while maintaining low-speed stirring at 45 rpm during cooling to prevent precipitation.

[0045] Staged filtration: The cooled enzymatic hydrolysate is sent to a staged filtration system. First, it is filtered through a 20μm stainless steel filter screen (filtration pressure 0.2MPa) to remove large particulate impurities. The filter screen is rinsed with deionized water every 30 minutes during filtration. Then, the coarse filtrate is sent to a 0.45μm ceramic microfiltration membrane device (operating pressure 0.35MPa, membrane flux 155L / (m²·h)). The feed temperature is controlled at 25±2℃. Unhydrolyzed proteins and colloidal impurities are removed by filtration. 185L of clear enzymatic hydrolysate is collected. The transmittance is ≥94%, and there are no visible suspended solids.

[0046] Defluorination and impurity removal: The clarified enzymatic hydrolysate was fed into a de-impurity reaction vessel, and 0.1% food-grade activated carbon (0.185 kg, same as in Example 1) and 0.2% silicon chelating agent (0.37 kg) were added. The reaction temperature was adjusted to 25°C, and the mixture was stirred at 75 rpm for 3 hours. During this period, the fluoride ion concentration was measured every 30 minutes. After the reaction was completed, the mixture was filtered through a 0.22 μm filter membrane to remove the activated carbon and chelating agent residues, and the krill peptide purified solution was obtained. The fluoride content was ≤40 ppm, and the pigment removal rate was ≥80%.

[0047] Membrane separation purification: The purified solution is pumped into a 400Da nanofiltration membrane system (membrane material: polyamide, operating pressure: 1.5MPa, membrane flux: 75L / (m²·h)) using a high-pressure pump. The feed temperature is controlled at 30℃. At the same time, a silicon chelating agent is added at 0.1% (0.185kg) of the purified solution volume to further remove small molecule impurities and residual fluoride ions. The membrane surface is rinsed every 1.5 hours during operation. 65L of nanofiltration retentate is collected, and the fluoride content is tested to be ≤39ppm, the ash content is ≤9.98%, and the protein purity is ≥85%.

[0048] Concentration and Drying: The retentate was sent to a vacuum concentration tank and concentrated under a vacuum of 0.085 MPa and a temperature of 58°C. The stirring speed was 55 rpm, and the solid content was checked every 15 minutes. When the solid content reached 50%, the concentration was stopped, and a concentrated liquid (about 32.5 L) was obtained. The concentrated liquid was sent to a freeze dryer, and the pre-freezing temperature was set to -50°C for 2 hours, the sublimation drying temperature to -30°C, the desorption drying temperature to 25°C, the vacuum degree to 510 Pa, and the drying time to 10 hours, resulting in 8.2 kg of Antarctic krill peptide product. The product was a milky white loose powder. After being sieved through a 100-mesh sieve, it was vacuum-packed in aluminum foil (500 g per bag) and stored in a warehouse (warehouse conditions were the same as in Example 1).

[0049] Product performance indicators: Protein content 85.2% (Kjeldahl method), moisture 3.3% (Karl Fischer method), ash 9.98% (gravimetric method), fluorine content 39ppm (ion-selective electrode method), selenium content 2.58mg / kg (hydride generation-atomic fluorescence spectrometry), molecular weight ≤1000Da 94.0% (GPC method), DPPH free radical scavenging rate 67.0%; Anti-aging activity test: After oral administration of this product (dose 200mg / kg·d) to D-galactose-induced aging mice for 30 days, the SOD activity in liver tissue increased by 29% and the MDA content decreased by 25% compared with the model group, which meets the product performance indicators set by this invention.

[0050] Example 4

[0051] In this embodiment, the proportion of flavor protease in the compound enzyme is adjusted. The remaining steps, equipment, and parameters are the same as in Example 1. The specific adjustments and details are as follows:

[0052] Adjustment of compound enzyme ratio: The compound enzyme is composed of alkaline protease (enzyme activity 20000U / g), flavor protease (enzyme activity 15000U / g), and neutral protease (enzyme activity 18000U / g) in a mass ratio of 3:2:1. The amount of enzyme added is still 0.22% (i.e. 0.22kg) of the defatted Antarctic krill powder, of which 0.11kg is alkaline protease, 0.073kg is flavor protease, and 0.037kg is neutral protease. After mixing, the mixture is stirred thoroughly to ensure uniform dispersion of the enzymes. The enzymatic hydrolysis conditions are the same as in Example 1 (58℃, 120rpm, 4.2h), during which the pH is maintained at 8.2±0.1, and the enzyme inactivation and cooling steps remain unchanged.

[0053] Subsequent grading filtration, defluorination and impurity removal, concentration and drying steps were completely consistent with those in Example 1 to ensure the impact of a single variable (compound enzyme ratio) on product quality. Product index testing: protein content 86.2% (Kjeldahl method), moisture 3.1% (Karl Fischer method), ash 9.7% (gravimetric method), fluorine content 37ppm (ion-selective electrode method), selenium content 2.62mg / kg (hydride generation-atomic fluorescence spectrometry), percentage of molecular weight ≤1000Da 93.8% (GPC method), DPPH free radical scavenging rate 70.1%; Mechanism analysis: After increasing the proportion of flavor protease, the hydrophobic amino acid peptide bonds in krill protein can be more fully cleaved to generate more hydrophobic active peptides. These peptides have stronger antioxidant capacity, so the DPPH free radical scavenging rate is 1.9 percentage points higher than that of Example 1; Anti-aging activity test: After D-galactose-induced aging mice were administered this product (dose 200mg / kg·d) by gavage for 30 days, the SOD activity in liver tissue was increased by 35% and the MDA content was reduced by 31% compared with the model group. The antioxidant and anti-aging activities are better than those of the product in Example 1.

[0054] Example 5

[0055] This embodiment uses spray drying instead of freeze drying. The remaining steps, equipment, and parameters are the same as in Embodiment 2. The specific drying details and differences are as follows:

[0056] Preparation of concentrated solution: Same as in Example 2, the nanofiltration retentate was concentrated to a solid content of 55%, yielding 128L of concentrated solution. The concentrated solution was tested and found to have a transmittance ≥96%, fluorine content ≤37ppm, protein purity ≥87%, and no visible impurities. Optimization of spray drying parameters: The concentrated solution was fed into a spray dryer (centrifugal atomizer), with the inlet air temperature set at 165℃, outlet air temperature at 78℃, feed rate at 16L / h, atomization pressure at 0.35MPa, drying chamber negative pressure at 0.01MPa, and hot air circulation rate at 12m / s. During the drying process, the outlet air temperature and finished product moisture content were checked every 20 minutes, and the feed rate was adjusted in a timely manner to ensure stable product quality. After drying, the finished product was collected by a cyclone separator and then sieved through a 100-mesh sieve to remove a small amount of agglomerates, yielding 38.5kg of Antarctic krill peptide product (the yield was 8% higher than that of freeze drying in Example 2, mainly due to the absence of ice crystal sublimation loss during spray drying).

[0057] Product performance indicators: Protein content 86.9% (Kjeldahl method), moisture 3.2% (Karl Fischer method), ash 9.7% (gravimetric method), fluorine content 37 ppm (ion-selective electrode method), selenium content 2.68 mg / kg (hydride generation-atomic fluorescence spectrometry), molecular weight ≤1000 Da percentage 94.8% (GPC method), DPPH free radical scavenging rate 68.8%; Comparative analysis: Compared with the freeze-dried product of Example 2, this product has a slightly higher moisture content by 0.2 percentage points, with no significant difference in molecular weight distribution and activity. Due to the high spray drying temperature, a small amount of small molecule peptides underwent slight polymerization, but this did not affect the core indicators of the product. Anti-aging activity test: After D-galactose-induced aging mice were administered this product (dose 200mg / kg·d) by gavage for 30 days, the SOD activity in the liver tissue increased by 33% and the MDA content decreased by 29% compared with the model group, which was not significantly different from the freeze-dried product. Cost analysis: Spray drying time is only 2 hours (compared to 12 hours of freeze drying), energy consumption is reduced by 60%, and the unit product production cost is reduced by 18% compared with freeze drying, making it more suitable for large-scale industrial mass production.

[0058] Comparative Example 1 (Single alkaline protease digestion)

[0059] Process: Take 100 kg of defatted Antarctic krill powder (residual oil rate 1.2%, protein content 62%, same source as in Example 1), and operate according to the pretreatment steps of Example 1 (soaking-filtration-drying-ultra-fine grinding-rehydration and parameter adjustment) to obtain the same krill protein suspension (pH 8.2, temperature 52℃); Adjustment of enzymatic hydrolysis steps: only add 0.22% alkaline protease (enzyme activity 20000U / g, same as in Example 1), without adding flavor protease and neutral protease, and the enzymatic hydrolysis conditions are the same as in Example 1 (58℃, 120rpm, 4.2h), during which the pH is maintained stable at 8.2±0.1, after which the enzyme is inactivated by incubation at 82℃ for 18min, and then cooled to room temperature; the subsequent graded filtration, defluorination and impurity removal, concentration and drying steps are completely consistent with those in Example 1 to ensure the influence of a single variable (enzymatic hydrolysis system) on product quality.

[0060] Product performance indicators: Protein content 82.1% (Kjeldahl method), moisture 3.2% (Karl Fischer method), ash 10.2% (gravimetric method), fluorine content 45ppm (ion-selective electrode method), selenium content 2.63mg / kg (hydride generation-atomic fluorescence spectrometry), molecular weight ≤1000Da percentage 82.5% (GPC method), DPPH free radical scavenging rate 56.7%; Anti-aging activity test: After D-galactose-induced aging mice were administered this product by gavage (dose 200mg / kg·d) for 30 days, the SOD activity in liver tissue increased by only 18% compared with the model group, and the MDA content decreased by 15%; Calculation of active peptide yield: The finished product yield was 15.7kg, and the yield of active peptides (molecular weight ≤1000Da) was 72.3%, which was 13.1 percentage points lower than the 85.4% in Example 1 of this invention.

[0061] Conclusion: A single alkaline protease can only cleave the basic amino acid sites of krill protein, but cannot fully open the protein spatial conformation, nor can it effectively cleave hydrophobic amino acid peptide bonds, resulting in low enzymatic hydrolysis efficiency, low proportion of small molecule active peptides, and significantly reduced antioxidant and anti-aging activities of the product, which is far inferior to the compound enzyme synergistic enzymatic hydrolysis process of the present invention.

[0062] Comparative Example 2 (omitting the defluorination and impurity removal process)

[0063] Process: Pretreatment: Take 100 kg of defatted Antarctic krill powder (residual oil rate 1.2%, protein content 62%, same as the source of purchase in Example 1), ultrafine grind it through a 100-mesh sieve (the material on the sieve is pulverized twice and then sieved), add 450 L of deionized water at a material-to-liquid ratio of 1:4.5, stir and rehydrate for 30 min, adjust the pH to 8.2, and heat to 52 °C to obtain a krill protein suspension (the impurity removal steps of soaking and filtering three times are omitted).

[0064] Enzymatic hydrolysis with compound enzyme: Add 0.22% compound enzyme (alkaline protease: flavor protease: neutral protease = 3:1:1, total enzyme activity 20000U / g), hydrolyze at 58℃ and 120rpm for 4.2h, maintaining the pH stable at 8.2±0.1 during the process, incubate at 82℃ for 18min to inactivate the enzyme, and cool to room temperature (the hydrolysis steps are the same as in Example 1).

[0065] Staged filtration: 420L of clear enzymatic hydrolysate was obtained by filtration through a 20μm filter screen and a 0.45μm microfiltration membrane (filtration steps are the same as in Example 1).

[0066] Concentration and drying: The clarified enzymatic hydrolysate was directly concentrated to a solid content of 52%, and then freeze-dried at -50°C and a vacuum of 510Pa to obtain 18.5 kg of Antarctic krill peptide product (the defluorination and impurity removal steps were omitted). The packaging and storage conditions were the same as in Example 1.

[0067] Product specifications: Protein content 86.8% (Kjeldahl method), moisture 3.1% (Karl Fischer method), ash 18.8% (gravimetric method), fluorine content 85 ppm (ion-selective electrode method), selenium content 2.65 mg / kg (hydride generation-atomic fluorescence spectrometry), molecular weight ≤1000 Da percentage 94.5% (GPC method), DPPH free radical scavenging rate 60.2%; Taste test: The finished product has a salty taste with a slightly astringent aftertaste, and the sodium content was tested to be 2.26 × 10⁻⁶. 4 The concentration of fluoride in the raw materials was 200 mg / kg, mainly due to the failure to remove soluble salts. Anti-aging activity testing: After oral administration of this product (200 mg / kg / day) to D-galactose-induced aging mice for 30 days, the SOD activity in liver tissue increased by only 12% compared to the model group, and the MDA content decreased by 10%, mainly because the high-salt environment inhibited the physiological efficacy of the active peptides. Safety assessment: The fluoride content was 85 ppm, far exceeding the requirement of ≤40 ppm for fluoride content in GB 2762-2022 "National Food Safety Standard - Limits of Contaminants in Food". The ash content was 18.8%, far exceeding the ≤9.98% target set by this invention, thus failing to meet the safety and activity standards for functional foods.

[0068] Conclusion: By omitting the defluorination and impurity removal steps, fluoride ions, soluble salts, and pigment impurities in the raw materials cannot be removed, resulting in severely excessive fluoride and ash content in the product, leading to a poor taste. At the same time, high salt content inhibits the efficacy of active peptides, and the product's safety and activity cannot meet the requirements, highlighting the necessity of the defluorination and impurity removal steps in this invention.

[0069] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for large-scale preparation of high-quality Antarctic krill peptides based on enzymatic hydrolysis using a complex enzyme, characterized in that, Includes the following steps: S1. Pretreatment of defatted Antarctic krill powder: Take defatted Antarctic krill powder, add 0.5-1.5 times the volume of water each time, stir and soak for 0.5-2 hours, then filter. Repeat this operation three times. After ultra-fine grinding and passing through a 100-mesh sieve, add deionized water at a material-to-liquid ratio of 1:4.5, stir and rehydrate for 30 minutes to obtain krill protein suspension. S2. Synergistic Enzymatic Hydrolysis with Compound Enzymes: A compound enzyme preparation is added to the krill protein suspension. The compound enzyme consists of an alkaline protease with an activity of 20,000 U / g, a flavor protease with an activity of 15,000 U / g, and a neutral protease with an activity of 18,000 U / g, in a mass ratio of 3:1:

1. Enzymatic hydrolysis is carried out at 55-60℃ and a stirring rate of 120 rpm for 4-4.5 hours, during which the pH is monitored every hour and kept stable. After enzymatic hydrolysis, the temperature is raised to 80-85℃ and held for 15 minutes to inactivate the enzyme, and then cooled to room temperature. S3. Graded filtration and residue removal: The enzyme hydrolysate after enzyme inactivation is sequentially filtered through a 20μm filter screen and a 0.45μm microfiltration membrane to remove shrimp shell residue and unhydrolyzed protein, and the clarified enzyme hydrolysate is collected. S4. Defluorination and impurity removal: The clarified enzymatic hydrolysate is passed through 0.1% activated carbon and 0.2% chelating agent and reacted at 20-40℃ for 3 hours to remove pigment impurities and most of the fluoride ions by adsorption. The purified solution is then obtained by filtration. S5. Concentration and Drying: The refined liquid is concentrated to a solid content of 50%-55% using a vacuum concentration device, and then freeze-dried or spray-dried to obtain the Antarctic krill peptide product. The finished product is then screened, packaged, and stored.

2. The method for large-scale preparation of high-quality Antarctic krill peptides based on complex enzyme hydrolysis according to claim 1, characterized in that: In S1, the defatted Antarctic krill powder has a residual oil content of 1.0%-1.5% and a protein content of 59%-62%.

3. The method for large-scale preparation of high-quality Antarctic krill peptides based on complex enzyme hydrolysis according to claim 1, characterized in that: In S2, the total amount of the compound enzyme added is 0.22%-0.25% of the mass of defatted Antarctic krill powder.

4. The method for large-scale preparation of high-quality Antarctic krill peptides based on complex enzyme hydrolysis according to claim 1, characterized in that: In step S3, during the graded filtration process, degreased cotton and diatomaceous earth can be added to prevent filter clogging.

5. The method for large-scale preparation of high-quality Antarctic krill peptides based on complex enzyme hydrolysis according to claim 1, characterized in that: In step S4, the chelating agent is a silicon-based chelating agent that can specifically bind fluoride ions without affecting the retention of selenium.

6. The method for large-scale preparation of high-quality Antarctic krill peptides based on complex enzyme hydrolysis according to claim 1, characterized in that: In step S4, after obtaining the purified liquid through filtration, a membrane separation purification step is also included: the filtered purified liquid is pumped into a 400-500 Da nanofiltration membrane system, treated at 1.5-2.0 MPa and 30-35°C, 0.1%-0.2% silicon chelating agent is added, and the retentate is collected for subsequent concentration and drying.

7. The method for large-scale preparation of high-quality Antarctic krill peptides based on complex enzyme hydrolysis according to claim 1, characterized in that: In step S5, the freeze-drying conditions are -50°C and a vacuum of 510Pa, and the spray-drying conditions are an inlet air temperature of 160-165°C and an outlet air temperature of 78-80°C.

8. The method for large-scale preparation of high-quality Antarctic krill peptides based on complex enzyme hydrolysis according to claim 1, characterized in that: In step S5, the finished Antarctic krill peptide product has the following specifications: protein purity ≥85%, moisture ≤3.3%, ash content ≤9.98%, fluorine content ≤39ppm, selenium content ≥2.5mg / kg, molecular weight ≤1000Da ≥94%, and DPPH free radical scavenging rate ≥67%.

9. The method for large-scale preparation of high-quality Antarctic krill peptides based on complex enzyme hydrolysis according to claim 1, characterized in that: In step S1, the pH regulator used to maintain stability during enzymatic hydrolysis is a sodium hydroxide solution or a sodium bicarbonate solution, with a concentration of 0.1-0.5 mol / L, to avoid introducing impurities that could affect product purity.

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