Preparation method of high-activity immune-regulating fish peptide by directional enzymolysis

Abstract

The application discloses a high-activity immunoregulation paddlefish peptide directional enzymolysis preparation method, which simultaneously removes lipoxygenase and blocks the oxidative degradation of the immunopeptide from the source by supermicro crushing coupled with low-temperature supercritical CO2 degreasing; the immunopeptide is subjected to targeted quaternary fractional enzymolysis + 0.5 mol / L phosphate buffer online continuous flow addition and pressure stabilization system, the standard of four-protein enzyme exclusive enzyme activity is limited, macromolecules are layered and sheared, and the immunologically active oligopeptide specific to paddlefish is reserved at a fixed point, so that the problems of random destruction of the immunopeptide in traditional hydrolysis and large activity fluctuation of batches are solved; low-temperature activated carbon adsorption coupled with beer yeast short-time fermentation phase separation deodorization and oxidation inhibition greatly reduces the deodorization loss of the immunopeptide; a complete set of activity protection procedures including oxygen isolation low-temperature vacuum concentration and instantaneous temperature control spray drying are matched; the proportion of the product immunologically active peptide with a molecular weight of less than 1000 Da is greater than or equal to 92%, the cell immunity and humoral immunity regulation activity is increased by more than 40% than that of a general single enzyme process on the market, and the finished product is free of earthy and bitter tastes.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide preparation technology, and particularly relates to a method for the targeted enzymatic hydrolysis preparation of highly active immunomodulatory turtle peptides. Background Technology

[0002] Soft-shelled turtles are rich in collagen and lymphocyte immune-active proteins. They contain unique immune-active peptide precursor proteins. The small-molecule oligopeptides produced after hydrolysis of these proteins can stimulate the proliferation of macrophages and splenic lymphocytes, thereby enhancing the body's cellular and humoral immunity. They also have antioxidant and bone-repairing effects. However, the amino acid sequences of immune-active peptides are highly specific, making them easily destroyed by indiscriminate hydrolysis and acid-base oxidation during processing. The enzymatic hydrolysis process directly determines the absorption rate and immune function activity of the product, which is a core technological bottleneck in the industry.

[0003] Currently, the industrialization of turtle peptides generally suffers from the following technical defects: The hydrolysis system lacks a targeted design for preserving immune peptides: Industrial-scale hydrolysis uses only trypsin or papain, both enzymes recognizing only single amino acid carboxyl sites. This results in intact turtle immunoglobulins and macromolecular collagen folding to encapsulate immunoactive peptides, with hydrolysis cycles exceeding 6 hours. Prolonged random hydrolysis directly cleaves the characteristic amino acid sequences of immune peptides, resulting in less than 50% of the product containing short peptides with molecular weights <1000 Da and immunomodulatory activity. In vitro spleen cell proliferation activity is extremely low, and direct intestinal absorption is very poor. Existing limited aquatic protein compound enzymes only use random binary or ternary combinations, with different enzyme catalytic sites competing and antagonizing each other. This prevents the formation of a graded hydrolysis logic of "macromolecular pre-cleavage - targeted protection of immune peptides - removal of bitter residue ends," failing to specifically preserve turtle-specific immunoactive peptides and resulting in very low product immune enhancement. Furthermore, existing compound systems lack dedicated dynamic pH buffering, leading to significant pH fluctuations during hydrolysis. This directly damages the surface active charge structure of immune peptides, causing batch-to-batch fluctuations in immune activity exceeding 25% after process scale-up, making it difficult to meet the high requirements of end-product production such as special diets and post-operative immune nutrition oral solutions.

[0004] Raw material pretreatment without endogenous oxidase removal steps: Traditional hot water defatting temperature is ≥80℃, and the high temperature directly causes denaturation and inactivation of immune proteins; ordinary pulverization is only 40-60 mesh, the cross-linked network of connective tissue is not broken, and the dissolution rate of intracellular immune proteins is less than 75%; the fat layer of raw materials is rich in lipoxygenase, and the immune peptides are continuously oxidized and degraded throughout the hydrolysis process, resulting in rapid decline of immune activity during the storage period of the finished product.

[0005] Deodorization and immune activity protection are incompatible: existing single activated carbon high-temperature adsorption will retain a large amount of hydrophobic immune oligopeptides, and single yeast fermentation for a long time will accumulate organic acids, reduce the pH of the system and destroy the spatial conformation of immune peptides; both types of processes have the industry contradiction of "deodorization will greatly reduce immune activity, while preservation will result in a strong fishy smell", and the finished product needs a lot of flavoring and sweeteners to mask the taste, making it unsuitable for children and postoperative patients without added immune special diets. Summary of the Invention

[0006] To address the problems in the prior art, the present invention proposes the following technical solution: A method for the targeted enzymatic hydrolysis preparation of highly active immunomodulatory turtle peptides includes the following steps: S1. Raw material pretreatment: Turtle skirt, internal organs, minced meat and skeleton are mixed in a mass ratio of 2:1:3:4. After removing impurities, the mixture is coarsely crushed to 1-3 cm and then ultra-finely pulverized to 80-120 mesh. Supercritical CO2 defatting is performed at a temperature of 32-42℃ and a pressure of 18-28 MPa for 1-2 hours. Supercritical low-temperature simultaneous extraction is used to remove lipoxygenase and block the oxidative degradation pathway of immune peptides. S2. Water-soluble extraction: The mass ratio of defatted turtle powder to purified water is 1:(9-13), the pH is adjusted to 7.5-9.5, and the extraction is carried out at a constant temperature of 42-58℃ for 2-4 hours. The weakly alkaline environment maintains the negative charge of the immunoprotein, avoids flocculation and precipitation, and maximizes the dissolution of immunogenic precursor proteins. S3. Targeted four-stage controllable enzymatic hydrolysis of immune peptides: Adjust the pH of the extract to 6.5–8.0 and the temperature to 45–55℃. Add a compound enzyme at 1%–3% of the dried turtle powder. The compound enzyme is: alkaline protease: neutral protease: trypsin: flavor protease = (2–3):(3–4):1:1. Hydrolyze at a constant temperature for 3–6 hours. The pH is controlled online using a 0.5 mol / L potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution, with a pH fluctuation of ≤±0.2. After hydrolysis, inactivate the enzyme at 90–100℃ for 10–15 minutes. The activity standards for each enzyme in the composite enzyme are as follows: alkaline protease ≥200,000 U / g, neutral protease ≥100,000 U / g, trypsin ≥50,000 U / g, and flavor protease ≥30,000 U / g. The four-level enzymatic digestion and layering logic avoids the characteristic sites of immunologically active peptides, preserving the complete short peptide sequences with macrophage activation function. The phosphate buffer system stabilizes the charge structure of immunopeptides, preventing irreversible loss of immune activity caused by acid-base fluctuations. S4. Phase-separated differential synchronous deodorization and oxidation inhibition coupled treatment: Adjust the pH of the enzymatic hydrolysate to 5.0-7.0, add 0.8%-2.5% food-grade activated carbon by weight of the enzymatic hydrolysate, and filter at a low temperature of 25-30℃ for 40-80 minutes; inoculate the filtrate with brewer's yeast at 0.1%-0.6% by volume, ferment at 25-30℃ for 1-3 hours, and inactivate the yeast at 80℃ for 10 minutes; the low-temperature activated carbon does not retain hydrophobic immune oligopeptides, and the yeast decomposes and oxidizes the substrate to avoid the oxidative loss of immune peptides; S5. Multi-stage precision filtration: The deodorizing solution is centrifuged at 3000-5000 r / min for 10-20 min, and then filtered sequentially through a 200-400 mesh plate and frame filter and a 0.22-0.45 μm polyethersulfone membrane; metal ions and colloidal oxidation catalysts are removed, and the oxidation rate of stored immune peptides is reduced. S6. Vacuum oxygen-free concentration: Dual-effect equipment at 50-70℃ and vacuum of -0.06 to -0.09MPa, with trace amounts of nitrogen to isolate oxygen throughout the process, concentrating to 30%-50% solids; the oxygen-free low-temperature environment avoids the high-temperature oxidation and inactivation of immune peptides. S7. Spray drying: air inlet 160~190℃, air outlet 70~90℃, atomizing disc 15000~20000r / min, finished product obtained by sieving through 80~100 mesh in Class 10,000 cleanroom.

[0007] As a preferred embodiment of the above technical solution, S1 is ultra-finely pulverized to 100 mesh and supercritically degreased at 38℃, 22MPa, and 1.5h. According to the methylene blue spectrophotometric method, the lipoxygenase removal rate after degreasing is ≥93%, and the extraction rate of raw material immune protein is ≥90%.

[0008] As the preferred option of the above technical solution, the S2 material-to-water ratio is 1:11, the extraction pH is 8.5, the temperature is 50℃, and the extraction time is 3h, which increases the amount of immune protein soluble by 22% compared with the hot water degreasing process.

[0009] As a preferred embodiment of the above technical solution, the S3 compound enzyme is added at 2% of the dry powder mass, with a ratio of 2.5:3.5:1:1. The enzymatic hydrolysis is carried out at pH 7.5, 50℃, and for 4 hours, with phosphate buffer added online throughout the process. The pH fluctuation is ≤0.2, and the enzyme is inactivated at 95℃ for 12 minutes. Under this ratio, the proportion of immunologically active peptides with a molecular weight <1000Da is increased by 25%, and the proliferation activity of spleen lymphocytes is increased by 40% compared with the random compounding process without buffer.

[0010] As a preferred embodiment of the above technical solution, the addition amount of S4 activated carbon is 1.5%, adsorption is performed at 28℃ for 60 minutes; yeast inoculation is 0.4%, fermentation is performed at 28℃ for 2 hours; the loss of immune peptides in the entire deodorization process is ≤4.5%, while the loss of immune peptides in traditional single activated carbon deodorization can reach more than 27%.

[0011] As a preferred embodiment of the above technical solution, the S5 centrifuge speed is 4000 r / min and the time is 15 min; the plate and frame are made of 300 mesh filter cloth and the microporous membrane has a pore size of 0.22 μm.

[0012] As a preferred embodiment of the above technical solution, S6 is concentrated at 60℃, vacuum -0.08MPa, and nitrogen protection to 40% solids; S7 has an inlet air temperature of 180℃, an outlet air temperature of 80℃, an atomizing disc speed of 18000r / min, and passes through a 100-mesh sieve.

[0013] A highly active immunomodulatory turtle peptide product, prepared by any of the above methods, meets the following specifications: moisture ≤ 6.0%, crude protein ≥ 92.0%, molecular weight < 1000 Da, and proportion of immunomodulatory characteristic peptides ≥ 92%; compared with a single trypsin hydrolysate as a control, it increases the proliferation activity of mouse spleen lymphocytes by more than 40%, macrophage phagocytosis rate by 38%, and DPPH free radical scavenging rate by more than 35%; the loss of immunomodulatory peptides in the deodorization process is ≤ 4.5%, the sensory odor value is ≤ Grade 1, and the total bacterial count is ≤ 1000 CFU / g.

[0014] As a preferred embodiment of the above technical solution, the finished product is a milky white fine powder with excellent water solubility, no earthy or bitter taste, and no need to add any flavoring or masking excipients. In vitro cell experiments have confirmed that at a dose of 0.5 mg / mL, it can significantly promote the proliferation of splenic lymphocytes and has clear dual regulatory activity of humoral immunity and cellular immunity.

[0015] As a preferred embodiment of the above technical solution, it is used to prepare immune-enhancing, antioxidant, bone-repairing, and intestinal-repairing products, selected from adult solid immune beverages, children's growth immune-enhancing oral liquids, postoperative immune recovery nutritional special diets, middle-aged and elderly antioxidant immune capsules, canine and cat pet immune-enhancing additives, aquatic fish and shrimp anti-stress immune premixes, immune-regulating compressed candies, functional meal replacement powders, and gentle skin-care antioxidant active ingredients.

[0016] The beneficial effects of this invention are as follows: 1. Targeted four-stage enzymatic hydrolysis of immune peptides + pH buffering and stabilization throughout the process Existing aquatic peptide complex enzymes lack targeted protection logic for immune peptides and real-time pH buffer control, representing a common, crude hydrolysis approach in the industry. This invention features a unique four-stage progressive targeted enzymatic digestion mechanism, coupled with a proprietary online phosphate buffering and stabilization process, specifically designed for turtle immune peptides, possessing a unique technological barrier. The four-stage enzymatic digestion and activation logic (targeted protection of immune-specific peptides): A high proportion of neutral protease acts as the primary backbone cleavage enzyme, gently breaking down large immunoglobulin and collagen chains, opening only the outer fold structure without contacting the internal immune-active peptide sequences; the secondary alkaline protease degrades only non-immunologically active hydrophobic peptide segments; the tertiary trypsin site-specifically recognizes lysine and arginine sites, precisely bypassing the characteristic amino acid segments of turtle immune peptides, completely preserving oligopeptides with immune-activating functions; the quaternary flavor protease only removes bitter hydrophobic residues at the ends of peptide chains, without destroying the mid-chain immune-active sequences. The ratio of these four enzymes is screened for immune cell proliferation activity, eliminating catalytic antagonism and directionally enriching immune-active small peptides; Innovation in online continuous flow and pressure stabilization of phosphate: For the first time, a 0.5 mol / L potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer system is introduced into the turtle peptide hydrolysis system, coupled with online pH-linked feeding, to control pH fluctuations ≤ ±0.2 throughout the process; solving the industry pain point that pH fluctuations in traditional hydrolysis destroy the charge structure of immune peptides and cause significant activity reduction; compared with the unbuffered process, the retention rate of immune active peptides is increased by 23%, and the batch activity fluctuation is reduced from 25% to less than 6%; By standardizing the minimum enzyme activity of four proteases, the problem of process non-reproducibility caused by enzyme preparations from different manufacturers is eliminated, and the stability of industrial scale-up is greatly improved.

[0017] 2. Ultrafine grinding coupled with supercritical CO2 defatting simultaneously removes lipoxygenase. Traditional processes involve simple pulverization followed by hot water degreasing. High temperatures cause denaturation of immune proteins, and lipoxygenases continuously oxidize and degrade immune peptides. This invention uses 80-120 mesh ultra-micro shearing to break down the cross-linked network of connective tissue, fully releasing intracellular immune proteins. Simultaneous supercritical removal of oils and lipoxygenases at 32-42℃ achieves a lipoxygenase removal rate of ≥93%, cutting off the immune peptide oxidation pathway at the source. Protein extraction rate remains stable at ≥90%, and the amount of immune proteins dissolved is 22% higher than traditional hot water degreasing. This unique synergistic innovation in pretreatment is not a common industry practice.

[0018] 3. Phase-separated coupled low-temperature deodorization and oxidation inhibition process Existing common deodorization methods involve either single activated carbon or single yeast fermentation, both of which result in significant loss of immune peptides. This invention employs a two-phase differentiated treatment: low-temperature activated carbon selectively adsorbs fat-soluble odor substrates without retaining water-soluble immune oligopeptides; short-term fermentation with brewer's yeast decomposes water-soluble odor amines and free radical precursors, rapidly inactivating and preventing the degradation of immune peptides by organic acids; the loss of immune peptides in the deodorization process is controlled to within 4.5%, far lower than the loss of over 27% in traditional processes, achieving both odorlessness and high immune activity simultaneously, thus overcoming the limitations of existing common deodorization technologies. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments.

[0020] Unified and complete testing and judgment standards Protein extraction rate: GB5009.5-2016 Kjeldahl method, protein conversion factor 6.25; Lipoxygenase activity: methylene blue spectrophotometry, detection wavelength 665 nm, calculate enzyme activity removal rate before and after defatting; Molecular weight distribution: TSKgelG2000SWXL gel filtration chromatography, standards: cytochrome C, aprotinin, bacitracin, glycine dipeptide, integral calculation of the proportion of immunologically active peptides <1000Da. Immunological activity assays (core indicators): MTT assay of mouse spleen lymphocytes and phagocytosis assay of neutral red by macrophages; Splenic lymphocyte proliferation: Lymphocyte suspensions were prepared from mouse spleens aseptically, with sample concentration gradients of 0.2–1.6 mg / mL, and cultured in 5% CO2 for 48 h. MTT staining was performed, and absorbance was measured at 570 nm. Higher absorbance indicates stronger immunoproliferative activity. The activity enhancement was calculated using a single trypsin hydrolysis product as a blank control. Macrophage phagocytosis rate: RAW264.7 macrophages were plated, and after 24 h of sample treatment, neutral red staining solution was added. After lysis, absorbance was measured at 540 nm to characterize non-specific immune enhancement ability. DPPH free radical scavenging rate: 0.2 mmol / L DPPH ethanol solution, reaction in the dark for 30 min, measured at 517 nm, IC50 was calculated. 50 With clearance rate; Blind sensory test of fishy smell: 10 evaluators were trained with graded standard samples, and linear scores were given from 0 to 10. Extreme values ​​were removed and the average was taken. Product yield = (mass of dried finished product / mass of fresh turtle mixed by-product raw material) × 100%; Microbiological indicators: Total bacterial count GB4789.2; Moisture content GB5009.3.

[0021] Example 1

[0022] S1 Raw Material Pretreatment: 100kg of mixed turtle by-products (skirt: viscera: minced meat: skeleton = 2:1:3:4), coarsely crushed to 2cm, ultrafine crushed to 100 mesh; supercritical defatting at 38℃, 22MPa, for 1.5h; lipoxygenase activity before defatting was 128.6U / g, and after defatting it was 7.1U / g, with a removal rate of 94.5% and an immunoglobulin extraction rate of 91%.

[0023] S2 extraction: material-to-water ratio 1:11, pH 8.5, extraction at 50℃ for 3 hours, the amount of immunoproteins dissolved increased by 22% compared with hot water defatting.

[0024] S3 Immunotargeted Enzymatic Hydrolysis: Extraction solution pH 7.5, 50℃; 2% dry powder of compound enzyme added, ratio 2.5:3.5:1:1, enzyme activity of four enzymes uniformly calibrated: alkaline protease 205000U / g, neutral protease 108000U / g, trypsin 56000U / g, flavor protease 32000U / g; continuous online addition of 0.5mol / L pH 7.2 potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer throughout the process, pH fluctuation ≤0.2; enzymatic hydrolysis for 4h, enzyme inactivation at 95℃ for 12min.

[0025] S4 coupling deodorization and oxidation inhibition: pH 6.0, add 1.5% activated carbon for adsorption at 28℃ for 60 min; 0.4% brewer's yeast for fermentation at 28℃ for 2 h; yeast sterilization at 80℃, fishy odor grade 0.5, immune active peptide loss 4.1%.

[0026] S5 filtration: centrifuge at 4000 r / min for 15 min, 300 mesh plate and frame filter + 0.22 μm PES membrane.

[0027] S6 Oxygen-free Concentration: Concentrate to 40% solids under trace nitrogen protection at 60℃ and -0.08MPa.

[0028] S7 spray drying: air inlet 180℃, air outlet 80℃, atomizing disc 18000r / min; sieve through 100 mesh, finished product 22.3kg, yield 22.3%.

[0029] Test indicators: moisture 5.1%, crude protein 92.6%, <1000Da immunologically active peptides 93%; spleen lymphocyte proliferation activity increased by 42% compared with the single trypsin control, macrophage phagocytosis rate increased by 39%; DPPH clearance rate increased by 38%, total bacterial count 620 CFU / g.

[0030] Applicable scenarios: Adult immune solid beverages, middle-aged and elderly antioxidant compound immune health foods.

[0031] Example 2

[0032] S1 Pretreatment: 200kg of mixed by-products, coarsely crushed to 3cm, ultrafine to 120 mesh; defatted at 40℃, 25MPa, for 1.2h; after defatting, the lipoxygenase activity was 8.0U / g, the removal rate was 93.8%, and the extraction rate was 92%.

[0033] S2 extraction: material-to-water ratio 1:12, pH 9.0, extraction at 55℃ for 3.5h.

[0034] S3 enzymatic hydrolysis: pH 7.0, 45℃, add 2.5% compound enzyme 3:4:1:1, standardize enzyme activity; continuously add buffer solution with stable pressure, enzymatic hydrolysis for 5h, enzyme inactivation at 100℃ for 10min.

[0035] S4 deodorization: pH 5.5, 2% activated carbon adsorption at 26℃ for 80 min; 0.5% yeast fermentation at 26℃ for 2.5 h, fishy odor grade 0.3, immune peptide loss 3.8%.

[0036] S5 filtration: centrifuge at 4500 r / min for 12 min, filter cloth with 400 mesh + 0.45 μm membrane.

[0037] S6 Concentration: 65℃, -0.07MPa, nitrogen protection, 45% solids.

[0038] S7 Drying: Inlet air 190℃, outlet air 85℃, atomizing disc 20000r / min; 120 mesh sieve, finished product 45.2kg, yield 22.6%.

[0039] Test results: Moisture 4.8%, protein 93.0%, immunologically active peptides 94%; spleen cell proliferation activity increased by 45%, macrophage phagocytosis increased by 41%, DPPH increased by 40%.

[0040] Applicable scenarios: Children's growth and immune-boosting special diet, transparent immune oral liquid, fruit and vegetable compound functional beverage.

[0041] Example 3

[0042] S1 Pretreatment: 100kg of turtle by-products, coarsely crushed to 1cm, ultrafine 80 mesh; defatted at 32℃, 18MPa, for 2h; lipoxygenase removal rate 93.3%, extraction rate 90.2%.

[0043] S2 extraction: material-to-water ratio 1:9, pH 7.5, extraction at 42℃ for 2 hours.

[0044] S3 enzymatic hydrolysis: pH 6.5, 45℃, add 1% compound enzyme 2:3:1:1, buffer and stabilize the pressure for 3h enzymatic hydrolysis, and inactivate the enzyme at 90℃ for 15min.

[0045] S4 deodorization: pH 5.0, 0.8% activated carbon adsorption at 25℃ for 40 min; 0.1% yeast fermentation at 25℃ for 1 h, fishy odor grade 0.8.

[0046] S5 filtration: Centrifuge at 3000 r / min for 20 min, then filter through a 200 mesh + 0.45 μm membrane.

[0047] S6 Concentration: 50℃, -0.06MPa, 30% solids.

[0048] S7 Drying: Inlet air 160℃, outlet air 70℃, 15000r / min; 80 mesh sieve, finished product 21.1kg, yield 21.1%.

[0049] Detection: 92.1% of immune-active peptides were detected, and spleen cell proliferation activity was increased by 40%.

[0050] Applicable scenarios: immune nutritional paste for dogs and cats, functional immune additives in pet staple food.

[0051] Example 4

[0052] S1 Pretreatment: 100kg of mixed by-products, coarsely crushed to 2cm, 100-mesh ultrafine; defatted at 42℃, 28MPa, for 1h; lipoxygenase removal rate 93.9%, extraction rate 90.8%.

[0053] S2 extraction: material-to-water ratio 1:13, pH 9.5, extraction at 58℃ for 4 hours.

[0054] S3 enzymatic hydrolysis: pH 8.0, 55℃, add 3% compound enzyme 3:3:1:1, buffer and stabilize the pressure for 6 hours of enzymatic hydrolysis, and inactivate the enzyme at 100℃ for 10 minutes.

[0055] S4 deodorization: pH 7.0, 2.5% activated carbon adsorption at 30℃ for 80 min; 0.6% yeast fermentation at 30℃ for 3 h, fishy odor grade 0.4.

[0056] S5 filtration: Centrifuge at 5000 r / min for 10 min, then filter through a 300 mesh + 0.22 μm membrane.

[0057] S6 Concentration: 70℃, -0.09MPa, nitrogen protection, 50% solids.

[0058] S7 Drying: Inlet air 185℃, outlet air 82℃, 19000r / min; 100 mesh sieve, finished product 22.8kg, yield 22.8%.

[0059] Detection: 93.6% of immune-active peptides were detected, and spleen cell proliferation activity was increased by 44%.

[0060] Applicable scenarios: Postoperative bone repair + immune recovery special diet, raw material for antioxidant immune capsules.

[0061] Example 5

[0062] S1 Pretreatment: 150kg of turtle by-products, coarsely crushed to 2cm, 100 mesh; defatted at 36℃, 23MPa, for 1.4h; lipoxygenase removal rate 94.2%, extraction rate 90.5%.

[0063] S2 extraction: material-to-water ratio 1:10, pH 8.2, extraction at 49℃ for 2.8h.

[0064] S3 enzymatic hydrolysis: pH 7.3, 51℃, add 2.2% compound enzyme 2.8:3.2:1:1, buffer and stabilize the enzymatic hydrolysis for 4.2h, and inactivate the enzyme at 94℃ for 13min.

[0065] S4 deodorization: pH 6.3, 1.6% activated carbon adsorption at 27℃ for 55 min; 0.38% yeast fermentation at 27℃ for 1.8 h, fishy odor grade 0.4.

[0066] S5 filtration: Centrifuge at 4300 r / min for 13 min, then filter through a 300 mesh + 0.22 μm membrane.

[0067] S6 Concentration: 63℃, -0.075MPa, nitrogen protection, solids 43%.

[0068] S7 Drying: Inlet air 178℃, outlet air 79℃, 17800r / min; 100 mesh sieve, finished product 33.4kg, yield 22.3%.

[0069] Detection: 92.5% of immune-active peptides were detected, and spleen cell proliferation activity was increased by 41%.

[0070] Applicable scenarios: Stress-resistant and immune-boosting feed additives for aquatic fish and shrimp.

[0071] Example 6

[0072] S1 Pretreatment: 100kg of by-product, coarsely crushed to 2cm, 100 mesh; defatted at 35℃, 21MPa, for 1.6h; lipoxygenase removal rate 93.5%, extraction rate 90.1%.

[0073] S2 extraction: material-to-water ratio 1:10.5, pH 8.3, extraction at 47℃ for 2.6 h.

[0074] S3 enzymatic hydrolysis: pH 7.4, 48℃, add 1.9% compound enzyme 2.4:3.6:1:1, buffer and stabilize the enzymatic hydrolysis for 3.6h, and inactivate the enzyme at 96℃ for 11min.

[0075] S4 deodorization: pH 6.1, 1.0% activated carbon adsorption at 28℃ for 42 min; 0.29% yeast fermentation at 28℃ for 1.5 h, fishy odor grade 0.6.

[0076] S5 filtration: Centrifuge at 3900 r / min for 16 min, then filter through a 300 mesh + 0.22 μm membrane.

[0077] S6 Concentration: 59℃, -0.08MPa, solids 39%.

[0078] S7 Drying: Inlet air 174℃, outlet air 77℃, 17300r / min; 100 mesh sieve, finished product 22.0kg, yield 22.0%.

[0079] Detection: 91.8% of immune-active peptides were detected, and spleen cell proliferation activity was increased by 39%.

[0080] Applicable scenarios: Intestinal repair and immune-boosting meal replacement powder, probiotic compound immune raw materials.

[0081] Example 7

[0082] S1 Pretreatment: 100kg of mixed by-products, coarsely crushed to 2cm and 100 mesh; defatted at 37℃, 24MPa, for 1.3h; lipoxygenase removal rate 93.8%, extraction rate 91.2%.

[0083] S2 extraction: material-to-water ratio 1:11.5, pH 8.8, extraction at 52℃ for 3.2h.

[0084] S3 enzymatic hydrolysis: pH 7.1, 52℃, add 2.4% compound enzyme 2.6:3.4:1:1, buffer and stabilize the enzymatic hydrolysis for 4.5h, and inactivate the enzyme at 93℃ for 14min.

[0085] S4 deodorization: pH 5.9, 1.4% activated carbon adsorption at 27℃ for 52 min; 0.34% yeast fermentation at 27℃ for 1.9 h, fishy odor grade 0.4.

[0086] S5 filtration: Centrifuge at 4400 r / min for 12 min, 300 mesh + 0.22 μm membrane.

[0087] S6 Concentration: 66℃, -0.07MPa, nitrogen protection, solids 44%.

[0088] S7 Drying: Inlet air 184℃, outlet air 83℃, 18600r / min; 100 mesh sieve, finished product 22.5kg, yield 22.5%.

[0089] Detection: 93.2% of immune-active peptides were detected, and spleen cell proliferation activity was increased by 43%.

[0090] Applicable scenarios: immune-modulating compressed candies, effervescent tablets, and general-purpose non-odorous functional foods.

[0091] Comparative Example 1 (Traditional general process: single trypsin, hot water defatting, activated charcoal deodorization only, no pH buffer) The raw material is 100kg of turtle skirt, a single raw material, which is crushed into ordinary 40-mesh powder and degreased with 80℃ hot water; only 2% trypsin hydrolysis is performed, without quaternary complex enzymes or phosphate pH buffer; only activated carbon is used for deodorization, without yeast coupling fermentation; the remaining concentration and drying parameters are the same as in Example 1, which belongs to the common aquatic peptide process on the market.

[0092] The finished product weighed 16.1 kg with a yield of 16.1%. The content of immunologically active peptides <1000 Da was only 48.7%, and the fishy smell was grade 6.8. The splenic lymphocyte proliferation activity was only 65% ​​of that in Example 1 of this invention, the loss of immunological peptides was 28.3%, and the batch activity fluctuation was 24%.

[0093] Application restrictions: Only suitable for low-priced basic aquatic feeds; absolutely not suitable for use in immune-boosting diets, oral beverages, or health foods.

[0094] Comparative Example 2 (Binary complex enzyme, no pH buffer, single activated carbon deodorization, normal pressure high temperature concentration, general improved process) Pretreatment and extraction were the same as in Example 1; only neutral + trypsin binary compound was used, without quadruple targeted enzyme digestion or online phosphate stabilization; only activated charcoal was used for deodorization, without yeast simultaneous degradation of oxidizing substrates; concentration was carried out at 95°C under normal pressure without oxygen isolation; the rest of the drying was the same, which is a standard industry improvement scheme, without immune peptide targeted protection design.

[0095] The finished product weighed 17.5 kg with a yield of 17.5%. The small molecule immune peptides accounted for 76.4% of the total, and the fishy odor was grade 4.6. High temperature and acid-base fluctuations destroyed the immune peptide sequence, and the spleen cell proliferation activity was only 62% of that in Example 1, with a batch activity fluctuation of 23%.

[0096] Application restrictions: Only for dark-colored, heavily flavored oral liquids; prohibited for use in children's immune-boosting diets, transparent powders, and postoperative nutritional products.

[0097] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it.

Claims

1. A method for the targeted enzymatic hydrolysis preparation of highly active immunomodulatory turtle peptides, characterized in that, Includes the following steps: S1. Raw material pretreatment: Turtle skirt, internal organs, minced meat, and skeleton are mixed in a mass ratio of 2:1:3:

4. After removing impurities, the mixture is coarsely crushed to 1-3 cm and then ultra-finely pulverized to 80-120 mesh. Supercritical CO2 degreasing is performed at a temperature of 32-42℃ and a pressure of 18-28 MPa for 1-2 hours. Low-temperature supercritical simultaneous removal of lipoxygenase is also performed, with an overall lipoxygenase removal rate ≥93%. S2. Water-soluble extraction: The mass ratio of defatted turtle powder to purified water is 1:(9-13), the pH is adjusted to 7.5-9.5, and the extraction is carried out at a constant temperature of 42-58℃ for 2-4 hours. The weakly alkaline system is used to directionally dissolve the immune-active precursor protein of turtle. S3. Targeted four-stage controllable enzymatic hydrolysis of immune peptides: Adjust the pH of the extract to 6.5–8.0 and the temperature to 45–55℃. Add a compound enzyme at 1%–3% of the dried turtle powder. The compound enzyme is alkaline protease: neutral protease: trypsin: flavor protease = (2–3):(3–4):1:

1. Hydrolyze at a constant temperature for 3–6 hours. During the entire hydrolysis process, continuously add 0.5 mol / L potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer online to adjust the pH in real time. The pH fluctuation of the system is ≤±0.

2. After the enzymatic hydrolysis is completed, inactivate the enzyme at 90–100℃ for 10–15 minutes. The alkaline protease has an enzyme activity ≥200,000 U / g, the neutral protease has an enzyme activity ≥100,000 U / g, the trypsin has an enzyme activity ≥50,000 U / g, and the flavor protease has an enzyme activity ≥30,000 U / g; the four-stage enzymatic digestion and layering process selectively preserves short, immunologically active peptides with functions of splenic lymphocyte proliferation and macrophage activation. S4. Phase-separated differentiated synchronous deodorization and oxidation inhibition coupled treatment: Adjust the pH of the enzymatic hydrolysate to 5.0–7.0, add 0.8%–2.5% activated carbon by weight of the enzymatic hydrolysate, and filter at a low temperature of 25–30℃ for 40–80 min; inoculate the filtrate with brewer's yeast at 0.1%–0.6% by volume, ferment briefly at 25–30℃ for 1–3 h, and inactivate the yeast at 80℃ for 10 min; the low-temperature activated carbon does not retain hydrophobic immune oligopeptides, and the yeast simultaneously decomposes the oxidized substrate, with a total loss of immune peptides ≤4.5%; S5. Multi-stage filtration: Centrifuge the deodorized liquid at 3000-5000 r / min for 10-20 min, and then filter it sequentially through a 200-400 mesh plate and frame filter and a 0.22-0.45 μm polyethersulfone membrane. S6. Vacuum oxygen-free concentration: Double-effect equipment at 50-70℃ and vacuum of -0.06 to -0.09 MPa, with trace amounts of nitrogen used to isolate oxygen throughout the process, concentrating to 30%-50% solids. S7. Spray drying: air inlet 160~190℃, air outlet 70~90℃, atomizing disc 15000~20000r / min, finished product obtained by sieving through a Class 10,000 clean 80~100 mesh screen.

2. The method for preparing highly active immunomodulatory turtle peptides by targeted enzymatic hydrolysis according to claim 1, characterized in that, S1 ultrafine pulverization at 100 mesh, supercritical defatting at 38℃, 22MPa, and 1.5h, resulted in lipoxygenase activity ≤7.5U / g and immunoprotein extraction rate ≥90%.

3. The method for preparing highly active immunomodulatory turtle peptides by targeted enzymatic hydrolysis according to claim 1, characterized in that, S2 material-to-water ratio 1:11, extraction pH 8.5, temperature 50℃, time 3h.

4. The method for preparing highly active immunomodulatory turtle peptides by targeted enzymatic hydrolysis according to claim 1, characterized in that, The S3 compound enzyme was added at 2% of the dry powder mass, with a ratio of 2.5:3.5:1:

1. Enzymatic hydrolysis was performed at pH 7.5, 50℃, and for 4 hours. The pH fluctuation was controlled to be ≤0.2 by online addition of buffer solution throughout the process. The enzyme was inactivated at 95℃ for 12 minutes. The enzyme activities of the four proteases were calibrated as follows: alkaline protease 205000 U / g, neutral protease 108000 U / g, trypsin 56000 U / g, and flavor protease 32000 U / g.

5. The method for preparing highly active immunomodulatory turtle peptides by targeted enzymatic hydrolysis according to claim 1, characterized in that, S4 activated carbon addition of 1.5%, adsorption at 28℃ for 60 min; yeast inoculation of 0.4%, fermentation at 28℃ for 2 h.

6. The method for preparing highly active immunomodulatory turtle peptides by targeted enzymatic hydrolysis according to claim 1, characterized in that, The S5 centrifuge operates at 4000 r / min for 15 min; the plate and frame are made of 300 mesh filter cloth, and the microporous membrane has a pore size of 0.22 μm.

7. The method for preparing highly active immunomodulatory turtle peptides by targeted enzymatic hydrolysis according to claim 1, characterized in that, S6 concentration temperature 60℃, vacuum -0.08MPa, nitrogen protection concentration to 40% solids; S7 inlet air 180℃, outlet air 80℃, atomizing disc 18000r / min, passed through 100 mesh sieve.

8. A highly active immunomodulatory turtle peptide product, characterized in that, The product is prepared by the targeted enzymatic hydrolysis method of the highly active immunomodulatory turtle peptide according to any one of claims 1 to 7, and the indicators meet the following requirements: moisture content ≤ 6.0%, crude protein ≥ 92.0%, molecular weight < 1000 Da, and proportion of immunomodulatory characteristic peptides ≥ 92%; compared with the single trypsin hydrolysis product as a control, the proliferation activity of mouse spleen lymphocytes is increased by more than 40%, the phagocytic rate of macrophages is increased by 38%, and the DPPH free radical scavenging rate is increased by more than 35%; the loss of immunomodulatory peptides in the deodorization process is ≤ 4.5%, the sensory fishy smell value is ≤ grade 1, and the total bacterial count is ≤ 1000 CFU / g.

9. The turtle peptide product according to claim 8, characterized in that, The finished product is a milky white, fine powder with excellent water solubility. It has no earthy or bitter taste and requires no additional flavoring or masking additives. In vitro cell experiments have confirmed that it can significantly promote lymphocyte proliferation at a concentration of 0.5 mg / mL, and has dual regulatory effects on cellular and humoral immunity.

10. The application of the highly active immunomodulatory turtle peptide according to claim 8, characterized in that, Used to prepare immune-enhancing, antioxidant, bone-repairing, and intestinal-repairing products, selected from adult solid immune beverages, children's growth immune-enhancing oral liquids, postoperative immune recovery nutritional special diets, middle-aged and elderly antioxidant immune capsules, pet immune-enhancing additives, aquatic fish and shrimp anti-stress immune premixes, immune-regulating compressed candies, functional meal replacement powders, and active raw materials for gentle skin care cosmetics.