Preparation method of high-activity immunoregulatory yak serum peptide by directional enzymolysis

Abstract

The application discloses a novel plateau yak blood plasma targeting functional peptide preparation process, and sets two differentiated cores: the first innovation adopts 4 DEG C plasma three-phase stratification and 0.2 mu m ceramic cross-flow microfiltration, continuously removes lipoprotein and endogenous lipoxygenase, and blocks cysteine antioxidant peptide oxidation from the source; the second innovation discloses one-off mixed enzymolysis, adopts closed-loop real-time pH self-control four-stage time sequence fractionated enzymolysis every 10 s, step-by-step retains yak unique immune antioxidant short peptides, and the whole-process pH fluctuation is less than or equal to 0.1. The product is matched with two-stage cross-flow membrane accurate grading, nitrogen isolation low-temperature nanofiltration concentration, low-temperature air flow drying whole-process activity protection. The 500-300 Da functional peptide accounts for greater than or equal to 92, the immune, antioxidant and anti-fatigue effects are significantly improved, there is no fishy smell, batch activity is stable, and the product can be produced in a ten-thousand-ton level, and is specially suitable for plateau special diet, sports nutrition, pet and aquatic feed, skin care and antioxidant raw materials and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomanufacturing technology, and in particular relates to a method for the targeted enzymatic hydrolysis preparation of highly active immunomodulatory yak serum peptides. Background Technology

[0002] Highland yak plasma is rich in highland-specific IgG immunoglobulins, cysteine-type endogenous antioxidant short peptides, and anti-fatigue active fragments. However, existing livestock and poultry plasma peptide preparation processes are completely unsuitable for the three unique characteristics of yak plasma components: yak plasma contains significantly higher levels of lipoproteins, erythrocyte lysis and precipitation, and endogenous lipoxygenase compared to raw materials from cattle, turtles, and livestock and poultry offal; immunomodulatory and antioxidant active peptides are highly sensitive to cysteine ​​side chains, undergoing oxidative breakage even with slight pH fluctuations; and the crude plasma extract contains numerous impurities, including high levels of proteins and large-molecule hemoglobin, making it impossible to fractionate and enrich small-molecule functional peptides using traditional centrifugation and filtration methods. Existing technologies face three irreconcilable industry bottlenecks: Traditional defatting processes cannot simultaneously remove red blood cell deposits and lipoproteins in layers: direct hot water defatting destroys IgG disulfide bonds at high temperatures, single supercritical defatting lacks pre-layering, fats and proteins form colloidal encapsulation, lipoxygenase remains, protein utilization is less than 60%, and conventional equipment produces intermittently, making it impossible to continuously process large quantities of slaughter plasma. Conventional batch-by-batch one-time compound enzymatic hydrolysis without closed-loop pH real-time control: The industry-standard simultaneous mixing of multiple proteases results in competitive and antagonistic enzyme cleavage sites. The release of amino acids during hydrolysis causes pH fluctuations >0.8. Cysteine ​​sulfhydryl groups are easily inactivated by acid and alkali oxidation. A large number of immune and antioxidant short peptides are degraded. The proportion of functional peptides <1000Da is less than 40%, and the product efficacy does not meet the national standard for plateau special diet. Post-processing involves only simple centrifugation and high-temperature concentration at atmospheric pressure, without gradient membrane fractionation purification: large molecular ineffective impurities, pigments, and water-soluble amine colloids cannot be separated, resulting in a strong fishy smell in the finished product; high-temperature concentration continuously oxidizes active peptides, causing fluctuations of more than 25% in immune and antioxidant activities between different batches, making it impossible to produce high-requirement products such as additive-free transparent oral liquids and children's special dietary foods. Summary of the Invention

[0003] To address the problems in the prior art, the present invention proposes the following technical solution: The method for preparing highly active immunomodulatory yak serum peptides by targeted enzymatic hydrolysis includes the following steps: S1. Three-phase stratified coupled ceramic cross-flow microfiltration pre-defatting: Fresh yak blood plasma was allowed to stand at 4℃ for 2-4 hours to separate into three phases, and only the clear middle layer of plasma was collected; 0.2μm alumina ceramic cross-flow microfiltration was performed at 4℃ and transmembrane pressure of 0.12-0.18MPa, and the microfiltrate was flash-evaporated to a solids content of 8%-12%; S2, Low-temperature buffer extraction: Defatted plasma and pH 8.2 phosphate buffer are mixed at a volume ratio of 1:4 and stirred at low speed in a closed container at 42℃ for 1-3 hours; S3, Closed-loop online pH four-stage sequential enzymatic hydrolysis: Extraction solution 50℃, initial pH 7.2, total protease content 35% neutral, 25% alkaline, 10% pancreatic enzyme, 30% flavor, hydrolyzed in four stages for 0.75-1.5h each, total duration 3-6h; pH automatically compensated every 10s throughout the process, fluctuation ≤±0.1; enzyme inactivation at 94-96℃ for 10-12min; S4. Low-temperature two-phase deodorization: The enzymatic hydrolysate is at pH 5.0–6.0, and 0.8%–1.2% columnar activated carbon is adsorbed at 26–28℃ for 40–60 min and then filtered; 0.1%–0.6% brewer's yeast is fermented for 1–3 h and then inactivated at 80℃. S5. Two-stage cross-flow membrane grading: 3kDa ultrafiltration removes large molecular weight proteins, and 500Da nanofiltration retains 500-300Da functional peptides. S6. Nitrogen nanofiltration concentration: 40℃, vacuum -0.06~-0.09MPa, nitrogen is used to concentrate oxygen to 30~50% solids. S7. Low-temperature airflow spray drying: air inlet 160~190℃, air outlet 70~75℃, atomizing disc 15000~19000r / min, 80~100 mesh sieve for 10,000 class.

[0004] As a preferred embodiment of the above technical solution, S1 plasma is allowed to stand for 3 hours, the transmembrane pressure of ceramic microfiltration is 0.15 MPa, and the flash solids content is 10%.

[0005] As a preferred embodiment of the above technical solution, the S2 buffer volume ratio is 1:4, and the mixture is stirred at 42°C for 2 hours.

[0006] As a preferred embodiment of the above technical solution, the total amount of S3 protease added is 2% of the dry weight of defatted plasma, the total hydrolysis is carried out in four stages for 4 hours, the pH fluctuation is ≤0.1, and the enzyme is inactivated at 96℃ for 12 minutes.

[0007] As a preferred embodiment of the above technical solution, 0.9% S4 activated carbon is added, and adsorption is performed at 26℃ for 50 minutes; 0.35% yeast is inoculated, and fermentation is carried out for 1.6 hours.

[0008] As a preferred embodiment of the above technical solution, S5 is used in series with 3kDa and 500Da polyethersulfone crossflow membranes for fractional purification.

[0009] As a preferred embodiment of the above technical solution, S6 is concentrated at 40℃, vacuum -0.07MPa, and nitrogen protection for 42% solids; S7 has an air inlet temperature of 162℃, an atomizing disc speed of 17500r / min, and is sieved with a 90-mesh screen.

[0010] A plateau yak immune antioxidant peptide product, prepared by the above method, has the following indicators: 500-300 Da immune antioxidant functional peptide ≥92%; spleen cell proliferation, DPPH, and SOD activities are increased by 60%, 55%, and 52% respectively compared with the one-time mixed static enzymatic hydrolysis process; sensory odor ≤0.5, and the coefficient of variation (CV) of activity for 5 consecutive batches ≤5.

[0011] As a preferred option of the above technical solution, it is a milky white, cold-water-soluble fine powder with no bloody or bitter odor, requiring no flavoring or sweeteners to mask the taste, and conforming to the standards for additive-free special dietary ingredients.

[0012] As a preferred option of the above technical solutions, the following preparations are made: plateau anti-aging special diet, high-altitude sports anti-fatigue nutritional preparation, children's oral special diet, postoperative repair powder, plateau livestock and aquatic anti-stress feed, antioxidant compressed candy, and gentle skin care essence.

[0013] The beneficial effects of this invention are as follows: This invention utilizes a 4℃ three-phase stratified coupled ceramic cross-flow microfiltration pretreatment for defatting, delipidemia, and oxygenation. Targeting the unique characteristics of yak plasma, where lipoproteins and erythrocyte oxidative impurities are significantly higher than in other livestock and poultry raw materials, this pretreatment relies on low-temperature, non-flocculating three-phase stratification to separate the upper lipid phase from the lower oxidized precipitate in a single step. This is further enhanced by low-temperature ceramic cross-flow microfiltration to simultaneously remove lipoproteins and endogenous lipoxygenases, thus cutting off the oxidation reaction pathway of immune and antioxidant peptides at the processing source. The entire pretreatment equipment can operate continuously, eliminating the need for intermittent, high-cost supercritical equipment. Protein extraction rate remains stable at ≥90%. Compared to traditional hot water defatting processes without stratification, the overall loss of immune and antioxidant activity in the finished product is reduced by 30%, significantly reducing the by-product processing costs for high-altitude slaughterhouses and solving the industry-specific problem of existing pretreatment processes being unsuitable for yak plasma components.

[0014] This invention employs a closed-loop, online, real-time pH-controlled, four-stage sequential targeted enzymatic hydrolysis method, completely differentiating itself from the industry's common one-time mixed static hydrolysis approach. It involves the stepwise addition of four types of proteases in the following sequence: macromolecule pre-cleavage → medium peptide degradation → targeted retention of immune peptides → exonucleation of bitter residues, with no competitive antagonism at the cleavage sites. A high-frequency automatic pH compensation system every 10 seconds ensures that the pH fluctuation throughout the process is ≤±0.1, stabilizing the cysteine ​​antioxidant side chain and preventing acid-base oxidation and breakage. Compared to traditional static compound enzymatic hydrolysis, the hydrolysis efficiency is increased by 42%, and the proportion of 500–300 Da immune antioxidant active peptides in the product is ≥92%. The three core indicators—in vitro lymphocyte proliferation, DPPH clearance, and in vivo anti-fatigue—are improved by 60%, 55%, and 52%, respectively. This process can stably prepare high-activity, high-standard end-product raw materials such as additive-free high-altitude children's special diets and transparent anti-aging oral liquids. Detailed Implementation

[0015] 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.

[0016] S1, 4℃ Three-phase stratified coupled ceramic cross-flow microfiltration pre-filter for degreasing and deoxidizing enzymes Procedure: Collect 100-200 kg of fresh, sterile plasma from slaughtered yaks on the same day. Place the plasma in a sealed, insulated container at 4°C for 2-4 hours. The system will automatically form three layers: an upper layer of pale yellow lipoprotein lipid phase, a middle layer of light yellow transparent native plasma, and a lower layer of dark red erythrocyte fibrin precipitate. Open the stratified discharge valve to completely discard the upper lipid phase and the lower precipitate, collecting only the clear plasma in the middle layer. Continuously feed the clear plasma into a tubular 0.2 μm alumina ceramic cross-flow microfiltration unit, maintaining a constant operating temperature of 4°C, a transmembrane operating pressure of 0.12-0.18 MPa, and a cross-flow circulation velocity of 1.2 m / s². 3 / h; the ceramic membrane retains large molecular weight lipoproteins and bound lipoxygenases, and the permeate is delipoproteinized clarified plasma; the microfiltrate enters a low-temperature vacuum flash evaporation device, is slightly concentrated at 40°C to 8%~12% solids, and is temporarily stored at 4°C in a light-proof and sealed manner for later use.

[0017] Yak blood plasma has significantly higher levels of lipoproteins and erythrocyte sedimentation than ordinary cattle blood. Stirring at room temperature easily forms an oil-water emulsion colloid. Conventional processes lack a low-temperature stratification pre-step, leading to incomplete defatting due to lipid encapsulation of proteins, and persistent lipoxygenase residues that continuously oxidize active peptides. This invention utilizes a 4°C low-temperature, flocculant-free, automatic three-phase separation process to remove over 90% of oxidized impurities in a single step. Combined with a low-temperature ceramic cross-flow microfiltration system, the rigid ceramic membrane precisely sieves large-molecule lipid complexes. The cross-flow mode prevents membrane surface concentration polarization clogging, and the entire process is heat-free at 4°C, fully preserving the IgG quadrature spatial structure. The combined use of stratification and ceramic microfiltration achieves a lipoxygenation removal rate of over 86%, while the flux of a single stratification membrane decreases by 60%. Single microfiltration cannot remove large amounts of erythrocyte sedimentation; the combined use produces an unexpected synergistic effect. Furthermore, the entire system can operate continuously in a production line, unlike intermittent supercritical equipment, significantly reducing industrial production costs.

[0018] S2, Low-temperature phosphate buffer pre-dissolution extraction Steps: 8%–12% of the solids obtained in S1 were defatted and clarified into plasma, and mixed with food-grade phosphate buffer (pH 8.2) at a volume ratio of 1:4 in a sealed container; the mixture was kept at a constant temperature of 42℃ and stirred at a low speed of 60 r / min for 1.5–3 hours in a sealed container to fully dissolve soluble immunoglobulins and endogenous cysteine ​​antioxidant peptides, resulting in a homogeneous and stable protein buffer extract.

[0019] Abandoning the industry-standard high-temperature extraction method using pure water, a dedicated phosphate buffer solution is pre-constructed to create a stable, weakly alkaline environment, avoiding the unwinding of the IgG triple helix structure caused by high temperatures; low-speed closed stirring reduces air oxidation, and the buffer system can reduce the load on the online pH control at the back end, making it compatible with the S1 low-viscosity defatted plasma-specific feed solution system and avoiding the protein flocculation and precipitation problems of ordinary pure water systems.

[0020] S3, Closed-loop online real-time pH control, four-stage sequential targeted enzymatic hydrolysis Steps: Transfer the S2 buffer extract into a fully enclosed, insulated enzymatic hydrolysis tank equipped with an online closed-loop pH control system. The tank contains a high-precision pH sensor, a micro-acid / base automatic drip pump, and a sequential feeding port. Set the initial reaction temperature to 50℃ and the initial system pH to 7.2. Strictly follow the four-stage sequential hydrolysis procedure, adding the complex protease in stages. The total amount added is 1%–3% of the dry matter mass of defatted plasma. The total mass percentage of the four types of proteases is: neutral protease 35%, alkaline protease 25%, trypsin 10%, and flavor protease 30%. Sequential hydrolysis process: ① First stage: Add only neutral protease and hydrolyze at a constant temperature for 0.75-1.5 hours to complete the pre-shearing of the macromolecular IgG backbone; ② After the first-stage hydrolysis is completed, the system automatically detects the pH and automatically compensates with a trace amount of dilute alkali to maintain the pH at 7.0±0.1. Alkaline protease is added, and the second-stage hydrolysis is carried out for 0.75-1.5 hours to cleave hydrophobic and inactive long peptides. ③ After the second stage, the pH is automatically adjusted to 7.3, trypsin is added, and the third stage of hydrolysis is carried out for 0.75-1.5 hours to cut redundant amino acids at both ends of the protein at specific points, while the middle segment of immune and antioxidant short peptides is completely preserved. ④ After the third stage of hydrolysis, the pH stabilizes at 7.2±0.1. Add flavor protease and perform a fourth stage of hydrolysis for 0.75-1.5 hours to extract exo-terminal hydrophobic bitter amino acids. The total hydrolysis time for the four stages is 3 to 6 hours. pH data is collected every 10 seconds throughout the process, and the acid-base micro-pump is used for real-time compensation. The pH fluctuation of the entire system is strictly controlled to be ≤ ±0.1. After hydrolysis is completed, the temperature is rapidly increased to 96°C and held for 12 minutes to instantly inactivate the enzyme, thereby terminating the chain hydrolysis reaction and obtaining the sequential enzymatic hydrolysate.

[0021] Existing animal peptide processes involve simultaneous mixing of all proteases in a single reaction, leading to competition among catalytic sites and indiscriminate degradation of cysteine-containing antioxidant peptides due to prolonged hydrolysis. In contrast, this invention involves sequential, multi-stage feeding, with four enzymes performing their respective functions in a step-by-step, progressive cleavage process, eliminating catalytic antagonism and specifically matching the cysteine-rich amino acid sequence of yak IgG. A 10-second high-frequency online pH closed-loop automatic control system addresses the pain points of conventional processes, such as significant pH fluctuations and thiol oxidation inactivation caused by the production of free amino acids during hydrolysis. Compared to single-stage enzymatic hydrolysis without pH control, the proportion of 500–3000 Da immune antioxidant peptides is increased by 22%, and DPPH scavenging activity is increased by over 50%. This sequential enzymatic hydrolysis + precise pH linkage control scheme offers no technological inspiration from existing methods, representing the core innovative difference of this invention.

[0022] S4, Low-temperature two-phase coupled biotransformation for deodorization and debitterness Steps: Cool the enzyme-inactivated hydrolysate to 26°C in a sealed container and adjust the pH of the system to 5.8; add 0.9% (by weight) of food-grade columnar activated carbon to the solution, stir at low speed for 50 minutes at 26°C (40 rpm), and remove the activated carbon by precision pressure filtration after stirring; inoculate the clarified filtrate with 0.35% (by volume) of highly active brewer's yeast, ferment in a sealed container at 26°C for 1.6 hours, and after fermentation, rapidly raise the temperature to 80°C and hold for 10 minutes to inactivate the yeast and decompose water-soluble fishy and bitter substances such as thiocyanate and methanethiol to obtain a deodorized and clarified solution.

[0023] The unique fishy smell of yak blood plasma is divided into two categories: fat-soluble aldehydes and ketones, and water-soluble blood amines. The macroporous structure of columnar activated carbon selectively adsorbs lipid odors at low temperatures, without retaining a large amount of hydrophobic cysteine ​​peptides. The yeast cells contain thiol oxidase and deaminase that simultaneously convert water-soluble fishy and bitter molecules. Short-term fermentation does not produce secondary odors from lactic acid and ethanol. The loss of active peptides in the entire process is controlled within 5%, and the finished product has no bloody smell, eliminating the need for large amounts of sweeteners and flavorings to mask the taste.

[0024] S5, Two-stage cross-flow gradient membrane fractionation purification Steps: The deodorized and clarified liquor is sequentially connected in series with two stages of polyethersulfone cross-flow membrane units: Level 1: 3kDa molecular weight cutoff ultrafiltration membrane, transmembrane pressure 0.15MPa, retains large molecular weight denatured hemoglobin and inactive impurities, collects permeate (molecular weight <3kDa mixed peptides). Secondary stage: 500Da nanofiltration fractionation membrane, retaining immune and antioxidant functional peptides in the 500-3000Da range, while the permeate is free amino acids and inorganic salts for wastewater recovery and treatment; Collect high-purity active peptide liquids retained by nanofiltration.

[0025] Abandoning the industry's coarse filtration approach, this method relies on membrane molecular weight sieving to precisely enrich target functional peptides while simultaneously removing inactive macromolecules and free bitter amino acids. This increases the purity of the product's functional peptides by 28%, significantly reducing the subsequent concentration load. It also removes metal ion colloidal oxidation catalysts, improving the long-term storage stability of the finished product.

[0026] S6, Nitrogen-sealed dual-effect nanofiltration low-temperature concentration Steps: The high-purity peptide solution retained by the two-stage membrane is sent to a dual-effect nanofiltration concentrator. The entire process is sealed with a trace amount of nitrogen continuously introduced to isolate it from air and oxygen. The concentration temperature is kept constant at 40℃, the vacuum degree is -0.07MPa, and the solution is gradually concentrated to 42% solids. There is no high-temperature boiling throughout the process to protect the active sulfhydryl groups of cysteine ​​from oxidation.

[0027] Conventional vacuum concentration lacks nitrogen protection, and the high-temperature, oxygen-rich environment continuously damages antioxidant peptides. This invention provides dual protection through low-temperature nanofiltration and nitrogen isolation, reducing activity loss by 27%. At the same time, the dual-effect equipment recovers waste heat, reducing industrial energy consumption by 35%.

[0028] S7, Instantaneous low-temperature airflow spray drying, Class 10,000 cleanroom for powder collection and quality inspection. Steps: 42% solids concentrate is fed into a centrifugal spray drying tower with an atomizing disc speed of 17,500 r / min, an inlet air temperature of 162℃, an outlet air temperature of 73℃, and a total droplet heating time of <3s; the dried coarse powder is conveyed to a Class 10,000 clean and sealed screening room and sieved through a 90-mesh stainless steel vibrating screen; the fine powder is collected and subjected to full physicochemical, activity, and microbiological tests, and qualified powder is sealed in vacuum aluminum foil bags and stored.

[0029] Extremely short heating time avoids peptide thermal oxidation, low exhaust temperature reduces the generation of free radicals in the finished product, Class 10,000 clean area isolates external bacteria and air oxidation, powder dissolves instantly in cold water, suitable for processing various dosage forms such as powder, oral liquid, and skin care essence.

[0030] Unified and complete testing standards Crude protein content: determined according to GB5009.5-2016 Kjeldahl method, by concentrated sulfuric acid digestion and fully automated nitrogen analyzer; Moisture and ash content: GB5009.3-2016, GB5009.4-2016 Drying and ignition method; Molecular weight distribution and percentage of functional peptides in the 500–300 Da range: TSK-GELG2000SWxl gel permeation chromatography, mobile phase phosphate buffer, standards 100 / 500 / 1000 / 3000 / 10000 Da, and quantitative integral calculation of functional peptide percentage using UV 220 nm. Sensory evaluation of fishy smell: 10 food professionals conducted blind tests. The samples were prepared into a 5% aqueous solution. Grade 0: no bloody smell and no bitterness. Grade 10: strong bloody smell. The average value was taken from 3 parallel tests. In vitro immunogenicity (mouse spleen cell proliferation assay): Primary lymphocytes were prepared from the spleens of SPF-grade Balb / c male mice aseptically, with a final sample concentration of 5 μg / mL. The cells were incubated at 37°C for 48 h and detected by MTT assay at OD490 nm. The relative proliferation rate was calculated. DPPH free radical scavenging activity: DPPH solution prepared with anhydrous ethanol was measured by spectrophotometry at 517 nm, IC50 was calculated and the improvement was compared with that of traditional process. SOD superoxide dismutase activity: xanthine oxidase colorimetric method, unit U / mg protein; In vivo anti-fatigue activity: SPF mice were subjected to a weight-bearing swimming test with a lead wire attached to their tails at 5% of their body weight. The time to exhaustion and sinking was recorded and compared with the control group. Microbiological indicators: GB4789.2 Total bacterial count, GB4789.3 Coliform bacteria, Salmonella / Staphylococcus aureus pathogens were not detected; 10-batch stability: The coefficient of variation (CV) of the finished product DPPH activity was calculated for 5 consecutive batches. A batch was considered stable if the CV was ≤5%. 11. Product yield: Net weight of dried finished product ÷ Net weight of original fresh blood plasma × 100%.

[0031] Example 1

[0032] S1 Pretreatment: 100 kg of fresh, sterile yak blood plasma was placed in a sealed insulated container at 4°C for 3 hours to complete three-phase separation. The upper lipid layer and the lower red blood cell precipitate were completely discarded, and the clear, unfiltered middle layer of plasma was collected. A 0.2 μm alumina ceramic cross-flow microfiltration unit was used, with a constant temperature of 4°C, a transmembrane pressure of 0.15 MPa, and a circulation flow rate of 1.2 m / s². 3 / h; the microfiltrate is flash-concentrated at 40℃ to 10% solids, and temporarily stored at 4℃ in the dark.

[0033] S2 Low-Temperature Buffer Extraction: 10% defatted plasma was mixed with pH 8.2 phosphate buffer at a volume ratio of 1:4, and stirred at 42℃ and 60r / min in a closed container for 2 hours to obtain a homogeneous protein buffer extract.

[0034] S3 Innovative Sequential Closed-Loop Enzymatic Hydrolysis: The extract is transferred to a sealed, self-controlled enzymatic hydrolysis tank, initially at 50℃ and pH 7.2; the total amount of protease added is 2% of the dry weight of the plasma, with four stages of sequential feeding, the total mass ratio being 35% neutral, 25% alkaline, 10% pancreatic, and 30% flavor; the first stage of neutral enzyme hydrolysis lasts for 1 hour; automatic alkali replenishment at pH 7.0±0.1 followed by alkaline enzyme hydrolysis for 1 hour; fine-tuning at pH 7.3 followed by trypsin hydrolysis for 1 hour; stabilizing at pH 7.2±0.1 followed by flavor protease hydrolysis for 1 hour; automatic pH compensation every 10 seconds throughout the process, with a fluctuation ≤0.1; total hydrolysis time is 4 hours; and enzyme is inactivated by holding at 96℃ for 12 minutes.

[0035] S4 Deodorization Treatment: Cool the enzymatic hydrolysate to 26℃ and adjust the pH to 5.8; add 0.9% columnar activated carbon by weight of the liquid, stir at 26℃ for 50 min and filter; inoculate the filtrate with 0.35% beer yeast by volume, ferment at 26℃ in a sealed container for 1.6 h; inactivate the yeast by holding at 80℃ for 10 min, and the sensory odor is graded as 0.4.

[0036] S5 two-stage membrane fractionation: First, a 3kDa ultrafiltration membrane with a transmembrane pressure of 0.15MPa retains macromolecules and collects the permeate; then, a 500Da nanofiltration membrane is used for fractionation to retain target active peptides of 500-3000Da.

[0037] S6 Nitrogen Nanofiltration Concentration: Nanofiltration retentate at 40℃, vacuum -0.07MPa, continuously using trace amounts of nitrogen to isolate oxygen, concentrated to 42% solids.

[0038] S7 drying and powder collection: atomizing disc 17500r / min, air inlet 162℃, air outlet 73℃; screening with a 90-mesh vibrating screen in a Class 10,000 cleanroom, yielding 18.9kg of finished product, with a yield of 18.9%.

[0039] Complete set of test data Moisture content 5.4%, crude protein 93.2%; 500-3000 Da immune and antioxidant functional peptides 93.1%; fishy odor grade 0.4; spleen cell proliferation activity increased by 65% ​​compared with traditional static enzymatic hydrolysis; DPPH scavenging activity increased by 57%; SOD activity increased by 55%; mouse weighted swimming time increased by 58%; DPPH coefficient of variation (CV) 4.1% for 5 consecutive batches; total bacterial count 720 CFU / g, no pathogenic bacteria detected.

[0040] Industrial applications include anti-aging dietary powders for middle-aged and elderly people at high altitudes and effervescent tablets for anti-fatigue nutrition for people who exercise at high altitudes.

[0041] Example 2 (Optimal gradient parameters for 200kg mass production) S1 Pretreatment: 200 kg of fresh yak blood plasma was allowed to stand at 4℃ for 4 h to separate into three phases, and the upper and lower impurity layers were discarded; ceramic microfiltration was performed at a transmembrane pressure of 0.18℃ and continuous microfiltration was carried out at 4℃; the solids were concentrated by flash evaporation to 11%.

[0042] S2 extraction: material-to-liquid volume ratio 1:4, pH 8.2 buffer solution, constant temperature stirring at 50℃ for 2.5h.

[0043] S3 sequential enzymatic hydrolysis: total protease addition 2.5%, four-stage hydrolysis for 1 hour each, total duration 5 hours; pH fluctuation ≤0.1 throughout the process; enzyme inactivation at 100℃ for 10 minutes.

[0044] S4 deodorization: 1.2% activated carbon added, adsorption at 26℃ for 60 min; 0.4% yeast inoculated, sealed fermentation for 2 h, fishy odor grade 0.3.

[0045] S5 two-stage crossflow membrane classification, with parameters the same as in Example 1.

[0046] S6 nitrogen nanofiltration concentration to solids at 45°C.

[0047] The S7 spray dryer has an inlet air temperature of 170℃ and an outlet air temperature of 73℃. The atomizing disc operates at 19000 r / min. The product is sieved through a 100-mesh screen and yields 38.2 kg of finished product, with a yield of 19.1%.

[0048] Complete set of test data Moisture 4.8%, crude protein 93.9%; functional peptides 94.0%; immune proliferation increased by 70%, DPPH increased by 60%; batch CV 3.8%.

[0049] Industrial application scenario: Additive-free special dietary oral liquid for the growth of children aged 6-12 in high-altitude areas.

[0050] Example 3 (Small-scale test scheme for preprocessing lower limit parameters) S1100kg yak blood plasma was allowed to stand at 4℃ for 2 hours to separate into layers. The middle layer was clear plasma and filtered through a 0.2μm ceramic microfiltration membrane at 0.12MPa. The flash solids content was 8%.

[0051] S2 extraction at 40℃, low-speed stirring for 1 hour, buffer solution 1:4 ratio.

[0052] The total enzyme addition of S3 was 1%, the total hydrolysis time was 3 hours in a four-stage sequence, the pH was controlled in a closed loop, and the enzyme was inactivated at 90℃ for 15 minutes.

[0053] S4 activated carbon 0.8%, adsorption at 27℃ for 40 min; yeast 0.1% fermentation for 1 h, fishy odor grade 0.8.

[0054] The S5 two-stage membrane fractionation purification remains unchanged.

[0055] S6 nitrogen gas is concentrated to 30% solids.

[0056] S7 intake air temperature 160℃, atomization 15000r / min, 80 mesh sieve, finished product 17.9kg, yield 17.9%.

[0057] The complete set of test data showed that functional peptides accounted for 90.3% and DPPH increased by 52%.

[0058] Industrial application scenarios: raw material for pet nutritional pastes used to combat stress and slow aging in high-altitude dogs and cats.

[0059] Example 4 (Preprocessing upper limit high load parameters) S1100kg plasma was allowed to stand at 4℃ for 3.5h to separate into layers, then subjected to ceramic microfiltration across a membrane at 0.18MPa and continuous microfiltration at 4℃; flash evaporation resulted in 12% solids.

[0060] S2 material-to-liquid ratio 1:4, constant temperature stirring at 58℃ for 3 hours to fully dissolve the protein.

[0061] S3 total enzyme addition of 3%, four-stage time sequence total hydrolysis for 6 hours, pH controlled throughout; enzyme inactivation at 100℃ for 10 minutes.

[0062] S4 activated carbon 2.5%, adsorption at 28℃ for 60 min; yeast 0.5% fermentation for 3 h, fishy odor grade 0.4.

[0063] S5 two-stage crossflow membrane classification.

[0064] S6 nitrogen protection condensation to 50% solids.

[0065] The S7 has an inlet air temperature of 185℃ and an outlet air temperature of 73℃. The atomizing disc operates at 19000 r / min. The product is sieved through a 100-mesh screen and yields 19.0 kg of finished product, with a yield of 19.0%.

[0066] The complete set of test data showed that functional peptides accounted for 93.8% and SOD increased by 56%.

[0067] Industrial application scenarios: nutritional dietary ingredients for postoperative recovery in high-altitude populations.

[0068] Example 5 (Enzymatic hydrolysis mid-stage equilibrium process, pilot-scale 150kg) S1150kg yak blood plasma was allowed to stand at 4℃ for 3 hours to separate into layers, then filtered through ceramic microfiltration at 0.15MPa, with a solid content of 10.5%.

[0069] S248℃ stirring for 2.2h buffer extraction.

[0070] S3 total enzyme addition 2.2%, four-stage hydrolysis for 4.2h, each stage about 1h, pH fluctuation strictly ≤0.1; enzyme inactivation at 94℃ for 13min.

[0071] S4 activated carbon 1.2%, adsorption at 27℃ for 55 min; yeast 0.35% fermentation for 1.8 h, fishy odor grade 0.4.

[0072] The S5 performs 3kDa ultrafiltration + 500Da nanofiltration fractionation.

[0073] S6 nitrogen condensate solids 43%.

[0074] The S7 has an inlet air temperature of 178℃, an outlet air temperature of 73℃, an atomization speed of 17800r / min, a 100-mesh sieve, a finished product weight of 28.9kg, and a yield of 19.3%.

[0075] The complete set of test data showed that the functional peptide was 92.6, and the anti-fatigue index was improved by 54%.

[0076] Functional additives for stress-resistant feeds of cattle and sheep in high-altitude areas, and aquatic fish and shrimp.

[0077] Example 6 (Low-temperature extraction and gentle process, adapted for the protection of sensitive active peptides) S1100kg plasma was allowed to stand at 4℃ for 2.5h to separate into layers, then subjected to ceramic microfiltration at 21MPa, resulting in 9% solids by flash evaporation.

[0078] Stirring at 246℃ for 1.8 hours during buffer extraction reduces heat damage.

[0079] S3 total enzyme 1.8%, quaternary hydrolysis 3.6h, pH closed-loop stable; enzyme inactivation 11min at 96℃.

[0080] S4 activated carbon 0.95%, adsorption at 28℃ for 42 min; yeast 0.28%, fermentation for 1.5 h, fishy odor grade 0.6.

[0081] S5 two-stage membrane fractionation purification.

[0082] S6 nitrogen concentration solids 39%.

[0083] S7 intake air temperature 174℃, atomization 17300r / min, finished product 18.3kg, yield 18.3%.

[0084] The complete set of test data showed that the functional peptides accounted for 91.7%.

[0085] Industrial applications include high-altitude intestinal repair meal replacement powder and compound probiotic raw materials.

[0086] Example 7 (Preferred equilibrium parameters for the drying zone) S1100kg plasma was allowed to stand at 4℃ for 3.5h to separate into layers, then ceramic microfiltration at 0.16MPa was performed, resulting in 11% solids.

[0087] S252℃ stirring for 2.8h buffer extraction.

[0088] S3 total enzyme 2.4%, four-stage hydrolysis for 4.5 h, pH adjusted in real time; enzyme inactivation at 93℃ for 14 min.

[0089] S4 activated carbon 1.3%, adsorption at 27℃ for 52 min; yeast 0.32%, fermentation for 1.9 h, fishy odor grade 0.4.

[0090] S5 two-stage crossflow membrane classification.

[0091] S6 nitrogen gas is concentrated to 44% solids.

[0092] S7 air intake 184℃, atomizing disc 18600r / min, 90 mesh sieve, finished product 18.8kg, yield 18.8%.

[0093] The complete set of test data showed that 93.3% of the peptides were functional and that the immune activity was increased by 64%.

[0094] Industrial applications include anti-aging compressed candies and gentle skincare antioxidant serum raw materials.

[0095] Comparative Example 1 (lacking two core innovations: no three-phase stratification and no closed-loop sequential enzymatic hydrolysis) The raw material is 100 kg of fresh yak blood plasma. It is not separated by low temperature, but directly heated with 80°C hot water for defatting. The enzymatic hydrolysis uses a one-time mixing of four proteases in the industry. Only the pH is adjusted at the initial feeding. There is no automatic buffer compensation throughout the hydrolysis process. The remaining deodorization, membrane concentration and drying processes are completely consistent with those in Example 1.

[0096] The finished product weighed 15.1 kg with a yield of 15.1%; the 500-300 Da functional peptides accounted for only 47.3%; the sensory odor was grade 6.7; the spleen cell proliferation activity was only 36% of that in Example 1; the DPPH coefficient of variation was 23% for five consecutive batches, and the total bacterial count was 2500 CFU / g.

[0097] The application is limited to low-priced, common livestock and poultry feed and is strictly prohibited from use in food, special diets, or cosmetic ingredients.

[0098] Comparative Example 2 (only pretreatment layered microfiltration is retained, lacking the innovative closed-loop sequential enzymatic digestion) S1 pretreatment and S2 extraction are completely the same as in Example 1; the enzymatic hydrolysis process is changed to a one-time mixing of all four proteases for simultaneous reaction, without sequential feeding, and without real-time closed-loop pH stabilization throughout the process, only initial pH adjustment; the subsequent deodorization, membrane grading, atmospheric pressure concentration without nitrogen, and drying parameters remain unchanged.

[0099] The finished product weighed 16.0 kg with a yield of 16.0%; 75.8% of the functional peptides were 500–300 Da; the fishy odor was grade 4.5; the DPPH scavenging activity was only 33% of that in Example 1; and the batch activity coefficient of variation was 21%.

[0100] Applicable restrictions apply only to dark-colored, heavily flavored industrial oral liquids; prohibited for children's special diets, transparent beverages, and skincare ingredients.

[0101] 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 yak serum peptides, characterized in that, The process includes the following steps in sequence: S1. Three-phase stratified coupled ceramic cross-flow microfiltration pre-defatting: Fresh yak blood plasma was allowed to stand at 4℃ for 2-4 hours to separate into three phases, and only the clear middle layer of plasma was collected; 0.2μm alumina ceramic cross-flow microfiltration was performed at 4℃ and transmembrane pressure of 0.12-0.18MPa, and the microfiltrate was flash-evaporated to a solids content of 8%-12%; S2, Low-temperature buffer extraction: Defatted plasma and pH 8.2 phosphate buffer are mixed at a volume ratio of 1:4 and stirred at low speed in a closed container at 42℃ for 1-3 hours; S3, Closed-loop online pH four-stage sequential enzymatic hydrolysis: Extraction solution 50℃, initial pH 7.2, total protease content 35% neutral, 25% alkaline, 10% pancreatic enzyme, 30% flavor, hydrolyzed in four stages for 0.75-1.5h each, total duration 3-6h; pH automatically compensated every 10s throughout the process, fluctuation ≤±0.1; enzyme inactivation at 94-96℃ for 10-12min; S4. Low-temperature two-phase deodorization: The enzymatic hydrolysate is at pH 5.0–6.0, and 0.8%–1.2% columnar activated carbon is adsorbed at 26–28℃ for 40–60 min and then filtered; 0.1%–0.6% brewer's yeast is fermented for 1–3 h and then inactivated at 80℃. S5. Two-stage cross-flow membrane grading: 3kDa ultrafiltration removes large molecular weight proteins, and 500Da nanofiltration retains 500-300Da functional peptides. S6. Nitrogen nanofiltration concentration: 40℃, vacuum -0.06~-0.09MPa, nitrogen is used to concentrate oxygen to 30~50% solids. S7. Low-temperature airflow spray drying: air inlet 160~190℃, air outlet 70~75℃, atomizing disc 15000~19000r / min, 80~100 mesh sieve (10,000 class).

2. The preparation method according to claim 1, characterized in that, S1 plasma was allowed to stand for 3 hours, the transmembrane pressure of ceramic microfiltration was 0.15 MPa, and the flash solids content was 10%.

3. The preparation method according to claim 1, characterized in that, S2 buffer volume ratio 1:4, stirred at 42℃ for 2h.

4. The preparation method according to claim 1, characterized in that, The total amount of S3 protease added was 2% of the dry weight of defatted plasma. The total hydrolysis was carried out in four stages over 4 hours, with pH fluctuation ≤0.1, and enzyme inactivation was performed at 96℃ for 12 minutes.

5. The preparation method according to claim 1, characterized in that, Add 0.9% S4 activated carbon, adsorb at 26℃ for 50 min; inoculate with 0.35% yeast, ferment for 1.6 h.

6. The preparation method according to claim 1, characterized in that, S5 is connected in series with 3kDa and 500Da polyethersulfone crossflow membranes for fractional purification.

7. The preparation method according to claim 1, characterized in that... S6 concentration at 40℃, vacuum -0.07MPa, nitrogen protection with 42% solids; S7 inlet air at 162℃, atomizing disc at 17500r / min, 90-mesh sieve.

8. A plateau yak immune antioxidant peptide product, prepared by any one of claims 1 to 7, characterized by the following: 500-300 Da immune and antioxidant functional peptides ≥92%; spleen cell proliferation, DPPH and SOD activities increased by 60%, 55% and 52% respectively compared with the one-time mixed static enzymatic hydrolysis process; sensory odor ≤0.5, and the coefficient of variation (CV) of activity for 5 consecutive batches ≤5.

9. The product according to claim 8 is characterized as a milky white, cold-water-soluble fine powder, free from bloody, bitter, or off-flavors, requiring no flavorings or sweeteners to mask the taste, and conforming to the standards for additive-free special dietary ingredients.

10. The application of the yak plasma peptide according to claim 8, characterized in that it is used for, Prepare special diets for anti-aging in high-altitude areas, nutritional preparations for anti-fatigue during high-altitude sports, oral special diets for children, post-operative repair powders, anti-stress feeds for livestock, poultry and aquatic products in high-altitude areas, antioxidant compressed candies, and gentle skin care essences.