Deer whole blood small molecule peptide and preparation method thereof

Abstract

The application discloses a preparation method of deer whole blood small molecule peptides, and solves the technical problem of poor accessibility of enzyme cutting sites caused by the quaternary structure of hemoglobin and the combination characteristics of hematin through specific recognition of functional domain sequences, layered construction of special composite enzyme systems and distributed enzymolysis; the obtained deer whole blood small molecule peptides have good color, high yield of 500-3000 Da peptide segments, and also retain characteristic trace elements such as iron and zinc.

Description

Technical Field

[0001] This invention belongs to the field of deer whole blood peptide technology, specifically relating to deer whole blood small molecule peptides and their preparation methods. Background Technology

[0002] Deer blood, as a precious food and medicine ingredient, has a complex protein composition and rich bioactivity. Existing blood protein peptide preparation technology has significant technical bottlenecks in the processing of deer blood: (1) Traditional enzymatic hydrolysis technology is insufficient in handling the differences between plasma proteins and blood cell proteins in deer blood, resulting in low enzymatic hydrolysis efficiency; (2) The quaternary structure of hemoglobin and the binding characteristics of heme lead to poor accessibility of enzyme cleavage sites; (3) Heme release results in a dark product color and a strong metallic taste, while existing decolorization technology causes a large loss of deer blood peptide activity; (4) The ability to directionally cleave specific bioactive sequences is weak; (5) Characteristic trace elements such as iron and zinc are easily lost in large quantities.

[0003] Chinese patent CN103484516A discloses a method for preparing hemoglobin peptides. This method involves pretreatment, enzymatic hydrolysis, and decolorization of porcine hemoglobin to essentially remove heme, improve product color and odor, and simultaneously achieve controllable degradation of hemoglobin to obtain easily digestible and absorbable oligopeptide products. While this technique achieves some success in heme removal and sensory property improvement, it uses only hemoglobin as a raw material and does not consider the differences in the synergistic composition of plasma proteins and blood cell proteins in deer whole blood. Furthermore, it does not conduct sequence-level targeted screening and enrichment design for the unique antioxidant and anti-fatigue functional peptides in deer blood. Therefore, it still falls short in achieving efficient enzymatic hydrolysis and fine fractionation while maintaining the unique biological activity of deer whole blood.

[0004] Chinese patent CN101550178B discloses a method for preparing blood polypeptides. After obtaining animal blood proteins through ethanol precipitation, the proteins are enzymatically hydrolyzed using a single trypsin at 35–40°C, and then dried to obtain the blood polypeptide product. This method uses "animal blood proteins" as the whole raw material, failing to distinguish the differences in composition and structure between plasma proteins and blood cell proteins. It also fails to design specific enzymatic hydrolysis strategies tailored to the hemoglobin tetramer structure and heme binding characteristics, thus remaining a conventional single-enzyme hydrolysis process.

[0005] Existing research indicates that deer whole blood proteins possess unique compositional and functional characteristics: hemoglobin content reaches 30%-35%, serum albumin 15%-20%, immunoglobulins 8%-12%, and they are rich in trace elements such as iron and zinc. Their amino acid sequences contain abundant antioxidant sequences (Val-Leu-Ser, Glu-Cys-Asp) and ACE-inhibiting sequences (Ile-Pro-Pro, Val-Pro-Pro), but current enzymatic hydrolysis techniques have failed to develop specific hydrolysis schemes targeting these characteristic functional domains. Given the unique composition and diverse functional domains of deer whole blood proteins, there is a need to develop methods for preparing small molecule peptides from deer whole blood that can overcome the aforementioned technical bottlenecks. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing small molecule peptides from deer whole blood, thereby solving the technical problems of low efficiency and easy loss of trace elements in the enzymatic hydrolysis preparation of deer whole blood peptides in the prior art.

[0007] According to a first aspect of the present invention, a method for preparing small molecule peptides from deer whole blood is provided, comprising the following steps:

[0008] (1) After adding sodium citrate to deer whole blood, centrifuge to obtain blood cells and plasma. Add deionized water to the blood cells, then adjust the pH to 3.0, and then combine with plasma. Adjust the solid content to 8%-10% to obtain pretreated deer whole blood.

[0009] (2) Add pepsin or acidic protease to pretreated deer whole blood at 45℃±1℃ and pH 2.9-4.0 for 2.0h±0.2h.

[0010] (3) Adjust the temperature of the product from step (2) to 50℃±1℃ and the pH to 8.0±0.1, then add alkaline protease and the first complex enzyme, react for 3.0h±0.2h and control the degree of hydrolysis to 28%-30%;

[0011] (4) Adjust the temperature of the product from step (3) to 55℃±1℃ and the pH to 7.5±0.1, then add the second complex enzyme and the bioactive protectant, react for 1.5h±0.2h and control the degree of hydrolysis to 35%-38%;

[0012] (5) Inactivate the enzyme in the product of step (4) to obtain an enzyme-inactivated solution;

[0013] (6) Separate the enzyme-inactivating solution through a membrane and collect the nanofiltration retentate with a molecular weight of 1000 Da-5000 Da to obtain the solution;

[0014] The bioactive protective agent is composed of antioxidants and sodium phytate.

[0015] The first complex enzyme consists of trypsin, chymotrypsin and complex enzyme;

[0016] The second complex enzyme consists of flavor protease and catalase.

[0017] The method for preparing small molecule peptides from deer whole blood of the present invention employs a stepwise time-controlled process of acidic pre-depolymerization, alkaline deep enzymatic hydrolysis, and neutral color and flavor regulation. This allows different functional enzyme systems to function in stages, under their respective optimal conditions, thereby avoiding inter-enzyme interference and improving overall enzymatic hydrolysis efficiency and the yield of target active peptides. Specifically, pepsin and alkaline protease work synergistically to increase the release rate of heme. Pepsin is added first, followed by alkaline protease for hydrolysis. Pepsin initially depolymerizes the quaternary structure of hemoglobin; at this stage, the functional domains are not fully exposed, and a large number of 500-3000 Da target peptides are not formed. Then, with the hemoglobin structure already opened, alkaline protease and the first complex enzyme are added. The functional domains are precisely identified and cleaved, achieving deep cleavage. This fully releases heme, opens the hemoglobin structure, and sufficiently exposes the cleavage sites, preventing subsequent broad-spectrum proteases from preferentially cleaving easily accessible sites and "preempting" the sites of functional peptides to be enriched, thus causing excessive fragmentation of the target peptides and affecting functional enrichment.

[0018] Furthermore, by controlling the degree of hydrolysis, this invention avoids both incomplete enzymatic hydrolysis leading to a large number of large peptides with poor functionality, and excessive hydrolysis preventing the over-hydrolysis of peptides into amino acids, thus avoiding the loss of active structural domains (such as the antioxidant sequence Val-Leu-Ser). Additionally, if the heme, metal ions, and some oxidation intermediates released during the acidic stage coexist with the later enzyme system, they can easily inhibit the activity of alkaline proteases, trypsin, etc., through metal complexation and oxidation. Specifically, metal ions (Fe...) 2+ Cu 2+ The Fenton reaction can catalyze the complexation of oxidized thiol groups with enzyme molecules, thereby inhibiting the activity of trypsin and alkaline protease, leading to a decrease in the efficiency of subsequent deep enzymatic hydrolysis. This invention combines an antioxidant with sodium phytate as a bioactive protectant, which reduces the participation of free metal ions in the oxidation reaction by chelating metal ions (such as iron and zinc) and inhibiting oxidation. This not only reduces the loss of trace elements in small molecule peptides from deer whole blood, but also avoids a decrease in the efficiency and rate of subsequent deep enzymatic hydrolysis.

[0019] In some embodiments, the mass ratio of pepsin, alkaline protease, first complex enzyme, second complex enzyme and bioactive protective agent is 1:2:5:1:1.

[0020] In some embodiments, the complex protease comprises a neutral protease and an alkaline protease. The mass ratio of the neutral protease to the alkaline protease is 1:(0.5-2). The proportions of each component enzyme in the complex protease are usually preset by the manufacturer, and the content and activity of the alkaline protease are difficult to precisely control based on the specific characteristics of the raw materials. Using only the complex protease is insufficient to meet the requirements of this invention for the hydrolytic strength of the alkaline protease. This invention, by supplementing with alkaline protease, can further enhance the cleavage ability of collagen's hydrophobic regions and cross-linked structures, thereby improving the generation efficiency of small molecule peptides, especially 3-8 peptides, and improving the molecular weight distribution and biological activity of the product.

[0021] In some embodiments, the amount of the first complex enzyme is 0.4%-0.6% of the weight of deer whole blood. Preferably, the amount of the first complex enzyme is 0.5% ± 0.05% of the weight of deer whole blood.

[0022] In some embodiments, the amount of the second complex enzyme is 0.15%-0.25% of the weight of deer whole blood. Preferably, the amount of the second complex enzyme is 0.2% ± 0.02% of the weight of deer whole blood.

[0023] In some embodiments, in step (1), the mass of sodium citrate is 0.1% of the mass of deer whole blood.

[0024] In some embodiments, in step (1), the centrifugation process involves centrifuging at 4~10°C and 4000~6000 rpm to separate blood cells and plasma.

[0025] In some implementations, in step (1), the pH is adjusted to 3.0 with 0.1 mol / L HCl.

[0026] In some embodiments, in step (2), pepsin is added to the pretreated deer whole blood at 45℃±1℃ and pH 3.0±0.1.

[0027] In other embodiments, in step (2), acidic protease is added to the pretreated deer whole blood at 45℃±1℃ and pH 3.5-4.0.

[0028] In some embodiments, the mass ratio of pepsin to alkaline protease is 0.9-1.1:1.8-2.2. Preferably, the mass ratio of pepsin to alkaline protease is 1:2.

[0029] First, pepsin is used to cleave the Phe and Leu sites of hemoglobin under acidic conditions to depolymerize hemoglobin. Then, alkaline protease is used to destroy the quaternary structure of hemoglobin under alkaline conditions. The newly exposed functional domains are then targeted and enzymatically digested by other enzymes, thus overcoming the enzymatic cleavage barrier caused by heme encapsulation.

[0030] In some embodiments, the activity of pepsin is 3000-5000 U / g; the activity of alkaline protease is 200000-250000 U / g.

[0031] In some embodiments, the mass ratio of trypsin, chymotrypsin, and complex protease in the first complex enzyme is 1.8-2.2:0.9-1.1:1.8-2.2. Preferably, the mass ratio of trypsin, chymotrypsin, and complex protease is 2:1:2.

[0032] In some embodiments, the first complex enzyme has the following enzyme activities: trypsin has an activity of 2500-3000 USP U / g; chymotrypsin has an activity of 1000-1500 U / g; and the complex protease has an activity of 50000-60000 U / g.

[0033] In the first complex enzyme, alkaline protease opens the hemoglobin structure; trypsin cleaves the Lys / Arg sites to rapidly build the product backbone; and chymotrypsin specifically cleaves Tyr / Trp / Phe to promote the release of aromatic domains to obtain antioxidant peptides. When all three are present, structural depolymerization and sequence recognition occur simultaneously, achieving a dual-dimensional structure-sequence approach and synergistically and significantly improving the release efficiency of functional peptides.

[0034] In some embodiments, the mass ratio of flavor protease to catalase in the second complex enzyme is 2.7-3.3:0.9-1.1. Preferably, the mass ratio of flavor protease to catalase is 3:1.

[0035] In some embodiments, the second complex enzyme contains a flavor protease with an activity of 500-600 LAPU / g and a catalase with an activity of 200,000-300,000 U / g.

[0036] In some embodiments, the antioxidant is selected from at least one of L-cysteine, glutathione, and vitamin C. Preferably, the antioxidant is L-cysteine. The purpose of the antioxidant is to protect the active sulfhydryl groups in deer whole blood.

[0037] In some embodiments, the mass ratio of antioxidant to sodium phytate is 2.7-3.2:4.5-5.5. Preferably, the mass ratio of antioxidant to sodium phytate is 3:5.

[0038] In some embodiments, the enzyme inactivation method is to inactivate the product of step (4) at 80℃±1℃ for 12s±2s.

[0039] In some embodiments, in step (6), the membrane separation process is as follows: the product obtained in step (5) is first filtered through a 0.45 μm ceramic membrane, and then the filtrate is sequentially passed through a 30 kDa ultrafiltration membrane, a 5 kDa ultrafiltration membrane, and a 1000 Da nanofiltration membrane to collect a nanofiltration retentate of 1000 Da-5000 Da.

[0040] According to a second aspect of the present invention, a deer whole blood small molecule peptide prepared by the above preparation method is provided, wherein the yield of the 500-3000 Da peptide is 72.5%±3.5%; the iron retention rate is 80%-83%; and the zinc retention rate is 78.2%±2.3%.

[0041] The reason why the nanofiltration retentate of the deer whole blood small molecule peptides prepared by the above method contains peptides with a molecule size less than 1000 Da is that some peptides with a molecule size less than 500 Da may be retained due to membrane adsorption or charge action. Based on the main distribution range of antioxidant, ACE inhibitory, and mineral-bound peptides, this invention selects 500-3000 Da as the target peptide range for deer whole blood small molecule peptides, and the results show that the yield of the target peptide range is relatively high.

[0042] The beneficial effects of this invention are as follows:

[0043] (1) This invention improves the overall degree of hydrolysis of complex protein components in deer whole blood to 38%±2% through layered enzymatic hydrolysis, and increases the enzymatic hydrolysis efficiency by more than 40%.

[0044] (2) This invention solves the structural accessibility problem in an acidic environment and achieves directional cleavage and protection in an alkaline environment by pretreatment with pepsin and synergistic action of alkaline protease. The heme release rate reaches 85%±5%, and the hemoglobin structure is significantly opened. This solves the technical problem of poor accessibility of enzyme cleavage sites caused by the quaternary structure of hemoglobin and the binding characteristics of heme. The product color is also significantly improved. Furthermore, by adding alkaline protease and the first complex enzyme to deer whole blood at the same time, the hemoglobin structure is significantly opened and can be targeted by the first complex enzyme for enzymatic hydrolysis, avoiding secondary oxidation and excessive cleavage of the target peptide (500-3000 Da), thereby achieving a high active peptide yield and a high iron retention rate.

[0045] (3) By specifically recognizing functional domain sequences, this invention increases the content of characteristic active peptides (antioxidant, ACE inhibitory peptides) from 15%-20% in traditional methods to 45%±5%, significantly enhancing the functionality of the product;

[0046] (4) The present invention adds bioactive protective agents and specific enzymes to deer whole blood, so that the iron retention rate is ≥80% and the zinc retention rate is ≥75%, thus maximizing the preservation of the nutritional components of deer whole blood;

[0047] (5) The present invention removes bitterness by enzymatic debittering and catalase to remove off-flavors, reducing the bitterness value of the product by 85%±5%, basically eliminating the metallic and bloody taste, and achieving the sensory quality requirements of food grade.

[0048] (6) The targeted enrichment of functional peptides increases the product’s antioxidant, ACE inhibitory and anti-fatigue activities by 50%, 45% and 35% respectively, demonstrating significant functional properties. Attached Figure Description

[0049] Figure 1 This is a technical roadmap of the deer whole blood small molecule peptide of the present invention;

[0050] Figure 2 This is the SEC-HPLC chromatogram of small molecule peptides from deer whole blood in Example 1. Detailed Implementation

[0051] The present invention will now be described in further detail with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto. The raw materials and reagents involved in the following embodiments are all commercially available.

[0052] The technical route diagram for preparing small molecule peptides from deer whole blood in this invention is as follows: Figure 1 As shown. Through functional domain analysis of complex protein components in deer whole blood, deer whole blood was subjected to fractional pretreatment and a specific, highly adaptable complex enzyme system was constructed. Stepwise enzymatic hydrolysis and four-stage membrane separation purification conditions were designed to enrich functional components. Finally, the molecular weight distribution verified that the above process yielded high-quality deer whole blood small molecule peptides.

[0053] I. Protein Composition Characteristics and Functional Domain Identification in Deer Whole Blood

[0054] A systematic analysis of the structure and functional domains of the major protein components (hemoglobin, serum albumin, immunoglobulins, and transferrin) in deer whole blood was conducted. The following key features were identified:

[0055] 1. Hemoglobin: Tetrameric spatial structure, heme binding domain, rich in Pro, His, and Phe.

[0056] 2. Serum albumin: Three-domain model, metal ion binding site, rich in Cys, Glu, and Asp.

[0057] 3. Immunoglobulins: Fab / Fc functional domain, antigen binding site, rich in Gly, Pro, and Tyr.

[0058] 4. Characteristic functional sequence:

[0059] (1) Antioxidant peptides: Val-Leu-Ser, Glu-Cys-Asp, Tyr-Pro-Tyr;

[0060] (2) ACE inhibitory peptides: Ile-Pro-Pro, Val-Pro-Pro, Leu-Pro-Pro;

[0061] (3) Immunomodulatory peptides: Thr-Pro-Gly, Ser-Pro-Tyr.

[0062] Based on the above analysis and considering the metal-binding characteristics of transferrin, a layered enzymatic digestion strategy targeting different protein components and functional domains was designed. Specifically, for hemoglobin, pepsin and alkaline protease were used for depolymerization; for serum albumin, a complex protease was used for broad-spectrum cleavage; for immunoglobulins, trypsin and chymotrypsin were used for targeted cleavage; and for transferrin, considering its metal-binding structural characteristics, under overall mild enzymatic digestion conditions, sodium phytate was used to chelate metal ions to reduce oxidation and enzyme activity inhibition. This strategy serves as the theoretical basis for the design.

[0063] II. Construction of a specific, highly adaptable, dedicated complex enzyme system

[0064] 1. Ratio of dedicated compound enzyme system

[0065] Using L9(3) 4 An orthogonal experiment was conducted to examine four factors (enzyme type, ratio, temperature, and time), with three levels for each factor.

[0066] A: Hemoglobin depolymerase group (mass ratio of alkaline protease to pepsin)

[0067] A1 is 1:1; A2 is 2:1; A3 is 3:1;

[0068] B: Functional domain cleavage enzyme group (mass ratio of trypsin, chymotrypsin, and complex protease)

[0069] B1 is 1:1:1; B2 is 2:1:2; B3 is 3:2:1;

[0070] C: Enzymatic hydrolysis temperature of the enzyme group that regulates color and flavor

[0071] C1 is 45℃; C2 is 50℃; C3 is 55℃;

[0072] D: Total enzymatic hydrolysis time

[0073] D1 is 5 hours; D2 is 6.5 hours; D3 is 8 hours;

[0074] In L9(3) 4 Based on orthogonal experiments, the proportion of active peptides in the 500-3000 Da range and the ACE inhibitory activity (IC50) were used to determine the optimal levels of activity. 50The iron retention rate was the primary indicator, combined with secondary indicators such as degree of hydrolysis (DH) and bitterness value for comprehensive evaluation. The results showed that the order of influence of factors was: functional domain cleavage enzyme group (B) > hemoglobin depolymerization enzyme group (A) > temperature (C) > time (D), and the optimal enzyme system combination was A2B2C3D1.

[0075] Under these combined conditions (alkaline protease: pepsin = 2:1 (w / w); trypsin: chymotrypsin: complex protease = 2:1:2 (w / w); third stage at 55℃; total enzymatic hydrolysis time 6.5 h), the key evaluation results are as follows: the proportion of active peptides in the 500-3000 Da range is 72.8% ± 1.2%; the ACE inhibitory activity IC50 is 72.8% ± 1.2%; and the ACE inhibitory activity IC50 is 72.8% ± 1.2%. 50 The concentration was 0.42 ± 0.05 mg / mL; iron retention was 80.3% ± 2.1%; degree of hydrolysis (DH) was 21.6% ± 0.4%; sensory and color characteristics: L * ≥75, the metallic and bloody tastes are basically eliminated, and the taste score is ≥4.2 (out of 5).

[0076] 2. Selection of dedicated complex enzyme systems

[0077] The dedicated complex enzyme system constructed in this invention comprises four functional modules, the composition and mechanism of action of each module are as follows:

[0078] 2.1 Hemoglobin structural depolymerase group

[0079] (1) Alkaline protease (enzyme activity specification: 200,000-250,000 U / g): destroys the quaternary structure of hemoglobin and releases the enzyme cleavage site;

[0080] (2) Pepsin (enzyme activity specification: 3000-5000 U / g): specifically cleaves Phe and Leu sites under acidic conditions;

[0081] (3) Addition ratio: alkaline protease: pepsin = 2:1 (w / w).

[0082] 2.2 Functional Domain Directed Cleavage Enzyme Mechanism

[0083] (1) Trypsin (enzyme activity specification: 2500-3000 USP U / g): specifically cleaves Lys and Arg sites, targeting functional domains;

[0084] (2) Chymotrypsin (enzyme activity specification: 1000-1500 U / g): specifically cleaves Tyr, Trp, and Phe sites;

[0085] (3) Complex protease (enzyme activity specification: 50,000-60,000 U / g): broad-spectrum cleavage, which increases the degree of hydrolysis and promotes the formation of peptides in the target molecular weight range after the hemoglobin structure is opened;

[0086] Source / Commercial Type: Commonly used compound protease preparations in the food industry (can be selected from Novozymes, Genentech, Angel Enzyme Preparations or equivalent domestic food-grade manufacturers);

[0087] Enzyme activity specification: 50,000–60,000 U / g;

[0088] Main components: neutral protease (mainly acts on hydrophobic amino acid residues), alkaline protease (enhances the ability to deeply hydrolyze large protein molecules), and a small amount of exoprotease (promotes the generation of small peptide molecules).

[0089] (4) Addition ratio: trypsin: chymotrypsin: complex protease = 2:1:2 (w / w).

[0090] 2.3 Color and flavor regulating enzyme group

[0091] (1) Flavor protease (enzyme activity specification: 500-600 LAPU / g): eliminates bitterness and improves taste;

[0092] (2) Catalase (enzyme activity specification: 200,000-300,000 U / g): degrades peroxides and prevents oxidative odors;

[0093] (3) Addition ratio: Flavor protease: catalase = 3:1 (w / w).

[0094] 2.4 Bioactive Protective Agent Group

[0095] (1) L-cysteine: The amount added is 0.03% (w / w) of the weight of whole deer blood, to protect the active sulfhydryl groups;

[0096] (2) Sodium phytate: The amount added is 0.05% (w / w) of the weight of whole deer blood, which integrates metal ions and prevents oxidation;

[0097] (3) Addition ratio: L-cysteine: sodium phytate = 3:5 (w / w).

[0098] 2.5 Total enzyme system ratio

[0099] Hemoglobin structural depolymerization enzyme group: functional domain directional cleavage enzyme group: color and flavor regulating enzyme group: bioactive protective agent group = 3:5:1:1 (w / w).

[0100] The layered action mechanism of the special complex enzyme system is as follows: First, pepsin opens the quaternary structure of hemoglobin through enzymatic hydrolysis in an acidic environment. Then, under alkaline conditions, functional domain cleaving enzymes and alkaline proteases perform targeted cleavage of the exposed active sequences. Finally, through color and flavor regulation enzymes and bioactive protectants, oxidation reactions are inhibited and sensory quality is improved, thereby achieving a balance between high peptide yield and high trace element retention.

[0101] III. Time-Controlled Stepwise Enzymatic Hydrolysis Process Based on Molecular Directed Shearing

[0102] 1. A stepwise enzymatic hydrolysis strategy with layered time-controlled processing is adopted, and the specific process parameters are as follows:

[0103] Phase 1: Directed depolymerization of hemoglobin structure

[0104] (1) Reaction conditions: temperature 45℃±1℃, pH 3.0±0.1, reaction time 2.0h±0.2h;

[0105] (2) Enzyme addition: Add pepsin;

[0106] (3) Environmental control: nitrogen protection, dissolved oxygen concentration (DO) ≤ 0.3 ppm;

[0107] (4) Process monitoring: Real-time monitoring of heme release rate, with a target release rate of ≥85%.

[0108] Phase 2: Deep enzymatic digestion of protein components

[0109] (1) Condition adjustment: Adjust the temperature to 50℃±1℃ and the pH to 8.0±0.1;

[0110] (2) Enzyme addition: Add alkaline protease and functional domain-directed cleavage enzyme group (trypsin, chymotrypsin and complex protease).

[0111] (3) Reaction time: 3.0h ± 0.2h;

[0112] (4) Process monitoring: The target degree of hydrolysis (DH) is controlled at 28-30%.

[0113] Phase 3: Refining of Functional Peptides

[0114] (1) Condition adjustment: temperature 55℃±1℃, pH 7.5±0.1;

[0115] (2) Enzyme addition: Add color and flavor regulating enzyme group (flavor protease and catalase) and bioactive protectant group (L-cysteine ​​and sodium phytate).

[0116] (3) Reaction time: 1.5h ± 0.2h;

[0117] (4) Process monitoring: The target degree of hydrolysis (DH) is controlled at 35%-38%.

[0118] Phase 4: Mild Termination of Reaction

[0119] (1) Inactivation conditions: instantaneous inactivation at 85℃±1℃ / 15s±2s;

[0120] (2) Cooling treatment: rapidly cool to 35℃±2℃.

[0121] By mass percentage, the percentages of each ingredient added to the total weight of deer whole blood were as follows: pepsin 0.2% ± 0.02%; alkaline protease 0.4% ± 0.04%; trypsin 0.2% ± 0.02%; chymotrypsin 0.1% ± 0.01%; complex protease 0.2% ± 0.02%; flavor protease 0.15% ± 0.015%; and catalase 0.05% ± 0.005%.

[0122] 2. Preparation of a dedicated complex enzyme system

[0123] Prepare a specialized compound enzyme system according to the following proportions:

[0124] Pepsin (4000 U / g): 10g;

[0125] Alkaline protease (220,000 U / g): 20g;

[0126] Trypsin (2500 USP U / g): 20g;

[0127] chymotrypsin (1000 U / g): 10g;

[0128] Complex protease (55000 U / g): 20g;

[0129] Flavor protease (550 LAPU / g): 15g;

[0130] Catalase (250,000 U / g): 5g;

[0131] L-cysteine: 3g;

[0132] Sodium phytate: 5g;

[0133] Total weight 108g. Add approximately 1.1% of the substrate (whole deer blood) by weight when using.

[0134] IV. A four-stage membrane separation and purification system with precise molecular weight control

[0135] A four-stage membrane separation system was established to achieve precise separation of target active peptides and protection of trace elements.

[0136] 1. Membrane type in solid-liquid separation system

[0137] (1) 0.45 μm ceramic membrane;

[0138] (2) Function: Removes unhydrolyzed cell debris and impurities.

[0139] 2. Membrane types for macromolecular removal systems

[0140] (1) 30 kDa ultrafiltration membrane;

[0141] (2) Operating parameters: operating pressure 0.4-0.6 MPa, temperature 40℃±2℃;

[0142] (3) Function: Removes large protein molecules that have not been enzymatically digested.

[0143] 3. Target peptide screening system

[0144] (1) Membrane type: 5 kDa ultrafiltration membrane;

[0145] (2) Operating parameters: operating pressure 0.6-0.8 MPa, temperature 42℃±2℃;

[0146] (3) Function: Remove medium molecular weight peptides and screen for target small molecule peptides.

[0147] 4. Active peptide enrichment system

[0148] (1) Membrane type: 1 kDa nanofiltration membrane;

[0149] (2) Operating parameters: operating pressure 1.0-1.2 MPa, temperature 38℃±2℃;

[0150] (3) Function: Precisely retains 500-3000 Da target active peptides while protecting trace elements.

[0151] Although the target peptide range is 500-3000 Da, considering the distribution of active peptides, membrane flux and industrial feasibility, this invention selects a 1 kDa nanofiltration membrane as the enrichment limit to avoid excessive loss of active peptides in the 500-1000 Da range.

[0152] V. Product Quality Evaluation

[0153] A complete quality evaluation system for deer blood peptides was established, based on small molecule peptides from deer whole blood, including the following tests:

[0154] 1. Verification of molecular directional shearing effect

[0155] (1) MALDI-TOF MS

[0156] Used to analyze peptide molecular weight distribution and directional shearing effects.

[0157] After desalting, the sample was mixed with α-cyano-4-hydroxycinnamic acid (CHCA) matrix at a 1:1 (v / v) ratio, spotted onto a target plate, and detected in positive ion reflectance mode with a mass scan range of 300-5000 Da. The molecular weight distribution characteristics of the example and comparative samples were compared to evaluate the targeted shearing effect of layered enzymatic hydrolysis on the target peptide (500-3000 Da).

[0158] (2) Amino acid composition

[0159] The amino acid composition was determined using an automated amino acid analyzer.

[0160] The samples were hydrolyzed in 6 mol / L HCl solution at 110℃ for 24 h under sealed conditions, then evaporated to dryness under reduced pressure, reconstituted, and filtered. The contents of each amino acid were quantitatively analyzed by ion exchange chromatography-post-column derivatization. The changes in the proportions of hydrophobic amino acids, acidic amino acids, and sulfur-containing amino acids were compared to evaluate the structural characteristics of functional peptides.

[0161] (3) RP-HPLC

[0162] Used to detect the proportion of characteristic functional peptides.

[0163] A C18 reversed-phase column was used with gradient elution of 0.1% trifluoroacetic acid aqueous solution (A) / acetonitrile (B) as the mobile phase, and detection was performed at 220 nm. Retention time and peak area distribution were used as evaluation indicators to compare the relative abundance of moderately hydrophobic functional peptides in different samples.

[0164] (4) SEC-HPLC

[0165] Used to determine the molecular weight distribution of peptides.

[0166] Size exclusion chromatography (SEC-HPLC) was performed using a gel size exclusion column with phosphate buffer as the mobile phase at a flow rate of 0.5 mL / min, and detection was performed at 220 nm. Molecular weight calibration was performed using standard peptides, and the relative proportions of peptides in each molecular weight range (e.g., <500 Da, 500-3000 Da, >3000 Da) were calculated.

[0167] 2. Evaluation of color and flavor effects

[0168] (1) Colorimetric detection

[0169] The color of the samples was measured using a colorimeter. The samples were spread evenly under the same concentration conditions, and the measurements were repeated three times. The average value was used to evaluate the changes in product brightness and reddish-brown hue.

[0170] (2) Sensory evaluation

[0171] Sensory evaluation was conducted using a blind 5-point scale. An evaluation panel of 10-12 trained judges scored the samples for metallic taste, bloody taste, bitterness, and overall acceptability under randomized coding conditions. A score of 5 indicates no metallic, bloody, or bitter taste, i.e., no unpleasant flavor or excellent acceptability; a score of 4 indicates a slight off-flavor, acceptable; a score of 3 indicates a noticeable off-flavor, barely acceptable; a score of 2 indicates a noticeable off-flavor, not easily acceptable; and a score of 1 indicates a strong off-flavor, unacceptable.

[0172] (3) Volatile substances

[0173] The composition of volatile compounds was analyzed by GC-MS. The samples were treated with headspace solid-phase microextraction (HS-SPME) before injection. The volatile components related to bloody and metallic odors, such as aldehydes, ketones, pyrazines, and sulfur-containing compounds, were analyzed. Semi-quantitative comparisons were performed by peak area normalization.

[0174] 3. Bioactivity identification

[0175] (1) DPPH free radical scavenging ability

[0176] The standard DPPH free radical scavenging method was used. The sample solution was mixed with DPPH and ethanol solution and reacted in the dark for 30 min. The absorbance was measured at 517 nm, the free radical scavenging rate was calculated, and the result was compared with the control sample.

[0177] (2) ACE inhibitory activity

[0178] Reaction system construction: Hippuryl-His-Leu (HHL) was used as the substrate, and angiotensin-converting enzyme (ACE) solution and sample solutions of different concentrations were added. The reaction was carried out at 37℃ for 60 min.

[0179] Reaction termination and detection: Hydrochloric acid was added to terminate the reaction. The hippuric acid produced by extraction with ethyl acetate was evaporated to dryness and dissolved in ultrapure water. The absorbance was measured at a wavelength of 228 nm.

[0180] Data processing: The ACE inhibition rate was calculated, and a nonlinear regression was performed with sample concentration on the x-axis and inhibition rate on the y-axis to calculate IC. 50 value.

[0181] (3) Iron ion binding capacity

[0182] The determination was performed using a combination of dialysis and ICP-MS.

[0183] The sample was mixed with Fe 2+ After the solution was incubated under certain conditions, it was placed in a dialysis bag for dialysis. The iron ion content before and after dialysis was determined by ICP-MS. The iron ion binding capacity of the sample was calculated by combining the changes in iron content before and after dialysis.

[0184] (4) Zinc content

[0185] Sample pretreatment: Accurately weigh 1.0 g of deer blood peptide powder and digest it using a microwave digester (Mars6, CEM) with a nitric acid-hydrogen peroxide system.

[0186] Instrumental analysis: Zinc content was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7900).

[0187] Calculation formula: Zinc retention rate (%) = (Zinc content in the final product / Zinc content in the raw deer blood) × 100%.

[0188] It should be noted that the molecular weight of the "active peptide" in this invention is 500-3000 Da (SEC-HPLC statistics). The "characteristic functional peptide" refers to an active peptide that further satisfies the following conditions: RP-HPLC retention time of 15-35 min; rich in Tyr / Phe / Trp (the total content of aromatic amino acids Tyr, Phe, and Trp accounts for no less than 10% of the total amino acid content); and its content is obtained by calculating the peak area ratio using RP-HPLC.

[0189] The characteristic functional peptides exhibited significant antioxidant and ACE-inhibiting activities in in vitro functional evaluations, and met at least one of the following criteria:

[0190] (1) In the DPPH free radical scavenging experiment, IC 50 ≤1.0 mg / mL, or clearance rate of not less than 50% at a concentration of 1 mg / mL;

[0191] (2) In the ACE inhibition experiment, IC 50 ≤0.5 mg / mL, or an inhibition rate of not less than 60% at a concentration of 1 mg / mL.

[0192] 4. LC-MS / MS analysis revealed that representative amino acid sequences of some characteristic functional peptides include, but are not limited to: Gly-Pro-Hyp-Tyr, Pro-Hyp-Phe, Gly-Leu-Trp, and Ala-Pro-Phe-Tyr. All of these sequences exhibited good antioxidant and ACE inhibitory activities.

[0193] Example 1

[0194] This embodiment provides a method for preparing small molecule peptides from deer whole blood, including the following steps:

[0195] (1) Raw material pretreatment

[0196] Take 100 kg of fresh deer whole blood and add 0.1% sodium citrate (by weight of the whole blood) as an anticoagulant. Centrifuge at 4℃ and 5000 rpm to separate blood cells and plasma. For the blood cell fraction, add twice the volume of deionized water for hemolysis, then adjust the pH to 3.0 with 0.1 mol / L HCl. The plasma fraction is used directly for subsequent enzymatic hydrolysis. After separate processing, combine the two fractions and adjust the solids content to 8%-10% to obtain pretreated deer whole blood.

[0197] (2) The pretreated deer whole blood was pumped into the enzymatic hydrolysis tank and nitrogen protection was turned on (DO≤0.3ppm). The temperature was adjusted to 45℃ and the pH to 3.0. Pepsin was added to the enzymatic hydrolysis tank and the reaction was carried out for 2.0h. At this time, the heme release rate reached 87%.

[0198] (3) Adjust the temperature to 50℃ and pH to 8.0, add alkaline protease, trypsin, chymotrypsin and complex protease, and react for 3.0h. At this time, the degree of hydrolysis DH reaches 29.5%.

[0199] (4) Adjust the temperature to 55℃ and pH to 7.5, add flavor protease, catalase and bioactive protectant group, and react for 1.5h. At this time, the degree of hydrolysis DH reaches 36.8%. Immediately perform enzyme inactivation at 85℃ for 15s, and quickly cool to 35℃ to obtain enzyme-inactivated solution.

[0200] (5) The enzyme-inactivating solution was first pretreated with a 0.45 μm ceramic membrane, and then passed sequentially through a 30 kDa ultrafiltration system (operating pressure 0.5 MPa, temperature 40℃), a 5 kDa ultrafiltration system (operating pressure 0.7 MPa, temperature 42℃), and a 1 kDa nanofiltration system (operating pressure 1.1 MPa, temperature 38℃), collecting the nanofiltration retentate. The nanofiltration retentate was vacuum concentrated at 45℃ and -0.08 MPa until the solid content was 35%, obtaining the concentrate.

[0201] (6) Spray drying

[0202] The concentrate was centrifugally spray-dried at an inlet air temperature of 160°C and an outlet air temperature of 68°C to obtain deer whole blood small molecule peptide powder. The product was immediately packaged in nitrogen under conditions of <25% humidity, with a residual oxygen content of <0.3%.

[0203] Example 1: A special compound enzyme system was prepared according to the proportions in "Preparation of Special Compound Enzyme System". The total mass of the special compound enzyme system was 1080g, of which 100g of pepsin was added.

[0204] It should be noted that in this invention, 0.1 mol / L HCl solution or 0.1 mol / L NaOH solution is used to adjust the pH value, and the pH is monitored by an online pH electrode.

[0205] Adding water reduces the solids content, while concentration increases the solids content; the solids content can be adjusted through these methods.

[0206] The deer whole blood small molecule peptides prepared in Example 1 were evaluated for product quality. The yield of the deer whole blood small molecule peptides was 76.8%. RP-HPLC results showed that the content of characteristic functional peptides was 46.8%, and the content of branched-chain amino acids was 19.2%. The DPPH free radical scavenging rate was 88.5%, and the ACE inhibitory activity IC50 was [missing value]. 50 The concentration was 0.42 mg / mL, with an iron retention rate of 82.5%. The product was light yellow in color, with no bloody or metallic taste, and a taste score of 4.3.

[0207] SEC-HPLC results are as follows Figure 2 As shown in Table 1, the molecular weight distribution of peptides is shown in Table 2.

[0208] Table 1 SEC-HPLC Chromatographic Results

[0209]

[0210] Table 2. Peptide molecular weight distribution

[0211]

[0212] The significant increase in the yield of active peptides in Example 1 was mainly due to the generation of a higher proportion of 500-3000 Da peptides during enzymatic hydrolysis through molecular directional shearing technology. The role of the four-stage membrane separation system was to "precisely enrich" these target peptides that had already been generated in large quantities, rather than to relatively increase the yield by removing a large number of them.

[0213] MALDI-TOF MS analysis showed that the target active peptide accounted for 72.5% ± 3.5%, and RP-HPLC results showed that the content of characteristic functional peptides (antioxidant and ACE inhibitory peptides) was ≥45%; the content of branched-chain amino acids was ≥18%.

[0214] From the perspective of color and flavor evaluation, the colorimetric test results showed an L value ≥ 75 and a b value ≤ 15. An L value ≥ 75 indicates a significant increase in product brightness; a b value ≤ 15 indicates a reduction in yellow / brown discoloration, indicating that heme oxidation and Maillard reaction were effectively inhibited. The metallic and bloody taste of the deer whole blood small molecule peptides were significantly reduced, with a taste score of 4.2-4.5 (out of 5). The aldehyde and pyrazine off-flavor substances were reduced by 90%-95% compared to the deer whole blood small molecule peptides in Comparative Example 1.

[0215] Bioactivity assays showed that the DPPH free radical scavenging rate of deer whole blood small molecule peptides was 88.5% ± 3.2% (1 mg / mL); the ACE inhibitory activity IC50 was [missing value]. 50 The value was 0.42±0.05 mg / mL; the iron ion binding capacity was 85%±5%.

[0216] Amino acid analysis results showed that, compared with the small molecule peptides from deer whole blood in the comparative example, the hydrophobic amino acids (Leu, Val, Ile), aromatic amino acids (Tyr, Phe, Trp), and sulfur-containing amino acids in the example samples showed an enrichment trend in relative composition, indicating that the layered enzymatic hydrolysis and membrane separation process of the present invention is beneficial for enriching functional peptides with antioxidant and ACE inhibitory activities.

[0217] Example 2

[0218] This embodiment provides a method for preparing small molecule peptides from deer whole blood. The difference from Embodiment 1 is that, after the protein content is detected, each raw material is added according to the "dedicated complex enzyme system".

[0219] The process parameters used in this embodiment are:

[0220] (1) Processing capacity: 2000 L / batch;

[0221] (2) Enzymatic hydrolysis tank volume: 10 m³ 3 It is equipped with an online pH, temperature, and DO precision monitoring system;

[0222] (3) Membrane system specifications: 30 kDa ultrafiltration membrane with an area of ​​400 m² 2 5 kDa ultrafiltration membrane with a surface area of ​​300 m² 2 1 kDa nanofiltration membrane area 250 m² 2 ;

[0223] (4) Drying system: centrifugal spray drying tower with an evaporation rate of 400 kg / h.

[0224] Example 3

[0225] This embodiment provides a method for preparing small molecule peptides from deer whole blood. The difference from Embodiment 1 is that in step (2), the temperature is adjusted to 45°C and the pH is 3.5, and acidic protease is added to the enzymatic hydrolysis tank.

[0226] The yield of small molecule peptides from deer whole blood was 75.2%, the proportion of active peptides was 70.8%, and the content of characteristic functional peptides (molecular weight of 500-3000 Da, rich in hydrophobic amino acids; containing Tyr, Phe, and Trp; with ACE inhibition and antioxidant capacity) was 44.5%.

[0227] Example 4

[0228] This embodiment provides a method for preparing small molecule peptides from deer whole blood. The difference from Example 1 is that in step (4), the 1 kDa nanofiltration membrane is replaced with a 0.45 μm ceramic membrane, and the operating pressure is adjusted to 0.9-1.1 MPa.

[0229] Testing revealed that the yield of small molecule peptides from deer whole blood was 77.5%, with an active peptide ratio of 72.8%, and the membrane lifespan was extended by approximately 35% compared to Example 1. However, the cost and energy consumption of the ceramic membrane were higher than those of the preparation method in Example 1, which is not conducive to industrial promotion.

[0230] Example 5

[0231] This embodiment provides a method for preparing small molecule peptides from deer whole blood, which differs from Example 1 in that...

[0232] In step (2), the temperature is adjusted to 45°C and the pH to 7.0, and microbial protease is added to the enzymatic hydrolysis tank.

[0233] The microbial protease is a food-grade microbial alkaline protease (derived from Bacillus subtilis, purchased from Nanning Pangbo Biotechnology Co., Ltd. (China), with the main components being neutral protease and alkaline protease, and an enzyme activity of 50,000–60,000 U / g).

[0234] Testing revealed that the deer whole blood small molecule peptides prepared in Example 5, compared to the product in Example 1, exhibited a broader molecular weight distribution, a lower peak concentration in the 500-3000 Da range, and lower ACE IC50. 50 A slight increase indicates a decrease in its directionality, and the proportion of active peptides is slightly lower than in Example 1.

[0235] Comparative Example 1

[0236] This comparative example provides a method for preparing small molecule peptides from deer whole blood, including the following steps:

[0237] Take 100 kg of fresh deer whole blood, adjust the pH to 8.0, add alkaline protease (200,000 U / g, 1% w / w), and enzymatically hydrolyze at 50℃ for 4 h. Then immediately inactivate the enzyme at 85℃ for 15 s, and rapidly cool to 35℃ to obtain an inactivated enzyme solution. The inactivated enzyme solution is then spray-dried to obtain deer whole blood small molecule peptide powder.

[0238] Comparative Example 2

[0239] This comparative example provides a method for preparing small molecule peptides from deer whole blood, including the following steps:

[0240] Take 100 kg of fresh deer whole blood and add 0.1% sodium citrate as an anticoagulant. Centrifuge at 5000 rpm to remove coarse clots and impurities, then add 1000 g of complex protease and 500 g of flavor protease, react at pH 7.5 for 4 h, and then pass through a 10 kDa ultrafiltration membrane to retain liquid with a molecular weight less than 10 kDa, which is the final product.

[0241] Comparative Example 3

[0242] This comparative example provides a method for preparing small molecule peptides from deer whole blood, including the following steps:

[0243] (1) Raw material pretreatment

[0244] Take 100 kg of fresh deer whole blood and add 0.1% sodium citrate as an anticoagulant. Centrifuge at 4℃ and 5000 rpm to separate blood cells and plasma. Add twice the volume of deionized water to the blood cell fraction for hemolysis, then adjust the pH to 3.0 with 0.1 mol / L HCl. Use the plasma fraction directly for subsequent enzymatic hydrolysis. Combine the two fractions after separate treatment, and adjust the solids content to 8%-10% to obtain pretreated deer whole blood.

[0245] (2) The pretreated deer whole blood was pumped into the enzymatic hydrolysis tank and nitrogen protection was turned on (DO≤0.3ppm). The temperature was adjusted to 45℃ and the pH to 3.0. Pepsin was added to the enzymatic hydrolysis tank and the reaction was carried out for 2.0h. At this time, the heme release rate reached 87%.

[0246] (3) Adjust the temperature to 50℃ and pH to 8.0, add alkaline protease, and react for 3.0 h;

[0247] (4) Add trypsin, chymotrypsin and complex protease, and react for 3.0 h;

[0248] (5) Adjust the temperature to 55℃ and pH to 7.5, add flavor protease, catalase and bioactive protectant group, and react for 1.5h. At this time, the degree of hydrolysis DH reaches 36.8%. Immediately perform enzyme inactivation at 85℃ for 15s, and quickly cool to 35℃ to obtain enzyme-inactivated solution.

[0249] (6) The enzyme-inactivating solution was first pretreated with a 0.45 μm ceramic membrane, and then sequentially passed through a 30 kDa ultrafiltration system (operating pressure 0.5 MPa, temperature 40℃), a 5 kDa ultrafiltration system (operating pressure 0.7 MPa, temperature 42℃), and a 1 kDa nanofiltration system (operating pressure 1.1 MPa, temperature 38℃), and the nanofiltration retentate was collected. The nanofiltration retentate was vacuum concentrated at 45℃ and -0.08 MPa until the solid content was 35%, to obtain a concentrated solution.

[0250] (7) Spray drying

[0251] The concentrate was centrifugally spray-dried at an inlet air temperature of 160°C and an outlet air temperature of 68°C to obtain deer whole blood small molecule peptide powder. The product was immediately packaged in nitrogen under conditions of <25% humidity, with a residual oxygen content of <0.3%.

[0252] Testing revealed that, compared to Example 1, the yields of antioxidant and anti-fatigue peptides from deer whole blood prepared in Comparative Example 3 decreased, as reflected in IC50. 50 The values ​​are higher than in Example 1. This is because alkaline protease first acts alone on deer whole blood, causing excessive structural fragmentation. Functional domains (such as His–Val–Leu, Tyr–Phe–His, etc.) are randomly cleaved, and the subsequently added trypsin and chymotrypsin cannot regain their sequence recognition advantage. In contrast, Example 1 adds alkaline protease, trypsin, chymotrypsin, and a complex protease simultaneously. Through synergistic action, the functional domains of hemoglobin that have just been exposed are immediately and precisely cleaved by the corresponding enzymes, thereby avoiding secondary oxidation or degradation of the functional domains. The resulting functional peptides have a more concentrated molecular weight range.

[0253] Comparative Example 4

[0254] This comparative example provides a method for preparing small molecule peptides from deer whole blood. The difference between this method and Example 1 is that the comparative example does not include the bioactive protective agents (L-cysteine ​​and sodium phytate).

[0255] Upon testing, compared to Example 1, the small molecule peptides prepared in Comparative Example 4 showed significantly lower iron and zinc retention rates due to the subsequent loss of free iron and zinc elements; enzyme activity decreased, oxidation intensified, and the color darkened.

[0256] The products obtained from Comparative Examples 1 and 2 were tested, and the results were compared with those of the deer whole blood small molecule peptide from Example 1. The power consumption (kW·h) of the entire preparation process—including the enzymatic hydrolysis tank (online monitoring system), membrane system (ultrafiltration / nanofiltration), and spray drying tower—was recorded. The energy cost was calculated by dividing the energy consumption by the product weight (kg), with the result of Comparative Example 1 as the baseline. Each test was performed in triplicate, and the results are shown in Table 3. In Table 3, "Performance Improvement" is the median value (calculated as the mean) of the result of Example 1 relative to the result of Comparative Example 1.

[0257] As shown in Table 3, the yield of the deer whole blood small molecule peptides prepared in Example 1 (500-3000 Da), the content of characteristic functional peptides, and the iron retention rate were significantly improved compared to Comparative Example 1. Furthermore, the L value of the product color was higher than that of Comparative Example 1 and Comparative Example 2, indicating that the product prepared by the method of this invention has significantly improved color, solving the problem of dark product color caused by heme release. The reason why the deer whole blood small molecule peptides in Comparative Example 1 had a higher content of characteristic functional peptides and a lower iron retention rate is that it used a single protease for enzymatic hydrolysis, leading to excessive protein hydrolysis or structural damage, causing trace elements such as iron and zinc to be released from their bound state and lost during subsequent separation.

[0258] Although Comparative Example 2 is close to Example 1 in terms of the total proportion of small molecule peptides, it lacks molecularly directed cleavage and protective separation, resulting in a significantly lower content of characteristic functional peptides and a lower retention rate of trace elements compared to Example 1.

[0259] Table 3. Properties of small molecule peptides from deer whole blood

[0260]

[0261] The content of active peptide fragments (500-3000 Da) in deer whole blood small molecule peptides in Example 1 and Comparative Example 1 was significantly higher than that in Comparative Example 1. This is the result of the combined action of membrane separation and a dedicated complex enzyme system.

[0262] The zinc content of the deer whole blood small molecule peptides prepared in Example 1, Comparative Example 1, and Comparative Example 2 was determined, and the results are shown in Table 4. Table 4 shows that Comparative Example 1, using a single enzymatic hydrolysis, had a zinc retention rate of 55.4% ± 2.5%, while the zinc retention rate of the deer whole blood small molecule peptides obtained in Example 1 was 78.2% ± 2.3%, significantly higher than the two comparative examples (p < 0.01). This is mainly attributed to the protective separation process related to membrane separation and the effective effect of the bioactive protective agent.

[0263] Table 4 Zinc content test results

[0264]

[0265] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for preparing small molecule peptides from deer whole blood, characterized in that, Includes the following steps: (1) Take whole blood from deer, add sodium citrate and centrifuge to obtain blood cells and plasma. Add deionized water to the blood cells and adjust the pH to 3.

0. Then combine with plasma and adjust the solid content to 8%-10% to obtain pretreated whole blood from deer. (2) Add pepsin or acidic protease to pretreated deer whole blood at 45℃±1℃ and pH 2.9-4.0 for 2.0h±0.2h. (3) Adjust the temperature of the product from step (2) to 50℃±1℃ and the pH to 8.0±0.1, then add alkaline protease and the first complex enzyme, react for 3.0h±0.2h and control the degree of hydrolysis to 28%-30%; (4) Adjust the temperature of the product from step (3) to 55℃±1℃ and the pH to 7.5±0.1, then add the second complex enzyme and the bioactive protectant, react for 1.5h±0.2h and control the degree of hydrolysis to 35%-38%; (5) Inactivate the enzyme in the product of step (4) to obtain an enzyme-inactivated solution; (6) Separate the enzyme-inactivating solution through a membrane and collect the nanofiltration retentate with a molecular weight of 1000 Da-5000 Da to obtain the solution; The bioactive protective agent is composed of an antioxidant and sodium phytate; the antioxidant is selected from L-cysteine; the mass ratio of the antioxidant to sodium phytate is 2.7-3.2:4.5-5.

5. The first complex enzyme consists of trypsin, chymotrypsin and complex enzyme; The second complex enzyme consists of flavor protease and catalase; in the first complex enzyme, the mass ratio of trypsin, chymotrypsin and complex protease is 1.8-2.2:0.9-1.1:1.8-2.2; in the second complex enzyme, the mass ratio of flavor protease and catalase is 2.7-3.3:0.9-1.

1. The mass ratio of pepsin, alkaline protease, first complex enzyme, second complex enzyme and bioactive protective agent is 1:2:5:1:

1.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass of sodium citrate is 0.1% of the mass of deer whole blood; the pH is adjusted to 3.0 with 0.1 mol / L HCl solution.

3. The preparation method according to claim 1, characterized in that, In step (6), the membrane separation process is as follows: the product obtained in step (5) is first filtered through a 0.45 μm ceramic membrane, and then the filtrate is passed through a 30 kDa ultrafiltration membrane, a 5 kDa ultrafiltration membrane and a 1000 Da nanofiltration membrane in sequence, and the nanofiltration retentate of 1000 Da-5000 Da is collected.

4. Deer whole blood small molecule peptides, characterized in that, The deer whole blood small molecule peptides were prepared by the preparation method according to any one of claims 1 to 3. The yield of peptides with a size of 500-3000 Da was 72.5% ± 3.5%; the iron retention rate was 80%-83%; and the zinc retention rate was 78.2% ± 2.3%.

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