A process for preparing chicken thymus immunomodulatory peptide by composite enzymolysis, chicken thymus immunomodulatory peptide and application thereof

Abstract

This invention relates to the field of biomedical technology, and particularly to a process for preparing chicken thymus immunomodulatory peptides using a compound enzymatic hydrolysis method, the chicken thymus immunomodulatory peptides, and their applications. The steps are as follows: First, the chicken thymus tissue is pretreated, including thawing, cutting, microwave-assisted treatment, and high-speed homogenization. Then, pepsin pre-hydrolysis is performed, the pH is adjusted, and pepsin is added to the reaction. Next, the hydrolysis is optimized by pH gradient conversion (pH 4.8-5.2→6.3-6.7→7.3-7.7), followed by trypsin secondary hydrolysis. Then, the peptides are purified using a gradient membrane separation system (10kDa, 1kDa, 500Da), and the 1kDa-500Da fraction is collected. Finally, the collected fraction is concentrated and freeze-dried to obtain the target peptide. This process combines enzymatic hydrolysis and membrane separation technology to effectively extract highly active immunomodulatory peptides. The yield of active peptides in this invention is increased by approximately 42%, and the purity is increased by approximately 28%.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a process for preparing chicken thymus immunomodulatory peptides by a compound enzymatic hydrolysis method, the chicken thymus immunomodulatory peptides and their applications. Background Technology

[0002] Immunomodulatory peptides are a class of small polypeptides that regulate the body's immune function and are widely found in animal tissues. The chicken thymus, as an important immune organ, is rich in various immunomodulatory peptides. These substances can promote the development, differentiation, and maturation of immune cells and regulate various immune functions. Therefore, extracting immunomodulatory peptides from the chicken thymus has significant research value and application prospects.

[0003] Currently, the main processes for preparing bioactive peptides include direct extraction, chemical synthesis, and enzymatic hydrolysis. Among them, direct extraction is complex and has low yield; chemical synthesis is costly and unsuitable for industrial production; while enzymatic hydrolysis, due to its mild reaction conditions, high specificity, and controllability, has become the main process for preparing bioactive peptides.

[0004] However, several technical problems remain to be solved in the existing processes for preparing immunomodulatory peptides from chicken thymus. For example, US Patent 6143559A discloses a process for extracting monoclonal antibodies from chicken thymus, but does not involve the extraction of immunomodulatory peptides.

[0005] In addition, Chinese patent CN118662610A describes the application of recombinant chicken thymopeptide in the preparation of chicken immune enhancers, but the process is not effectively integrated with the enzymatic hydrolysis process, and the target peptide cannot be effectively obtained.

[0006] The existing technology has four main problems: First, the efficiency of single enzymatic hydrolysis is low and it cannot fully release active peptides; second, membrane fouling is serious during the separation and purification process, which affects the separation efficiency; third, the content of bitter substances in the product is high, which limits the scope of application; and fourth, the enzyme utilization rate is low and the production cost is high.

[0007] Therefore, developing an efficient and controllable process for preparing chicken thymus immunomodulatory peptides, especially a process that can improve enzymatic hydrolysis efficiency and target peptide yield, is of great practical significance. Summary of the Invention

[0008] The purpose of this invention is to provide a process for preparing chicken thymus immunomodulatory peptides by a compound enzymatic hydrolysis method, the chicken thymus immunomodulatory peptides and their applications. This process significantly improves the yield and activity of immunomodulatory peptides by sequential compound enzymatic hydrolysis of pepsin and trypsin combined with batch coupling membrane separation technology, and solves the problems of low efficiency, unstable product activity, strong bitterness and serious membrane fouling in the prior art.

[0009] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0010] A process for preparing chicken thymus immunomodulatory peptides using a compound enzymatic hydrolysis method includes the following steps:

[0011] (1) Raw material pretreatment: Thaw the chicken thymus tissue to 2-8℃, cut it into small pieces of 3-8mm, and perform microwave-assisted treatment with a power of 400-600W, a treatment time of 20-40 seconds, and a temperature of 35-45℃. Then, mix the tissue with water at a ratio of 1:2-1:4 (w / v) and homogenize it at high speed.

[0012] (2) Pre-hydrolysis of pepsin: Adjust the pH of the homogenate from step (1) to 1.8-2.2, add pepsin, and the enzyme-substrate ratio is 1:40-1:60 (w / w). React in a water bath at 35-40℃ for 2-4 hours.

[0013] (3) pH gradient conversion: First adjust the pH of the hydrolysate from step (2) to 4.8-5.2 and let it stand for 10-20 minutes, then adjust it to 6.3-6.7 and let it stand for 5-15 minutes, and finally adjust it to 7.3-7.7;

[0014] (4) Secondary hydrolysis of trypsin: Add trypsin to the solution in step (3) with an enzyme-substrate ratio of 1:25-1:35 (w / w) and react in a water bath at 40-45℃ for 3-5 hours.

[0015] (5) Membrane separation and purification: The hydrolysate from step (4) is separated and purified by a gradient membrane separation system, including passing it through ultrafiltration / nanofiltration membranes with pore sizes of 10kDa, 1kDa and 500Da in sequence, which are the first stage of ultrafiltration, the first stage of ultrafiltration and the third stage of nanofiltration, respectively, and collecting the components between 1kDa and 500Da.

[0016] (6) Concentration and drying: The components obtained in step (5) are concentrated and freeze-dried to obtain chicken thymus immunomodulatory peptides.

[0017] Preferably, in step (2) the pepsin pre-hydrolysis is performed using pepsin derived from porcine gastric mucosa, with an activity of 10,000-20,000 U / mg and an optimal pH of 1.5-3.0.

[0018] Preferably, in the secondary hydrolysis of trypsin in step (4), the trypsin is derived from bovine pancreas, with an activity of 6000-10000 BAEE U / mg and an optimal pH of 6.8-8.0.

[0019] As a preferred option, the membrane separation and purification in step (5) adopts the batch-coupled enzymatic hydrolysis and membrane separation mode (BCEH-MSM), which connects the enzymatic hydrolysis reactor and the membrane separation system to achieve continuous production with simultaneous reaction and separation.

[0020] Preferably, the operating parameters of the first-stage ultrafiltration (10kDa) in step (5) are: pressure 0.2-0.3MPa, crossflow velocity 3.0-4.0m / s, and temperature 28-32℃; the operating parameters of the second-stage ultrafiltration (1kDa) are: pressure 0.25-0.35MPa, crossflow velocity 2.5-3.5m / s, and temperature 23-27℃; and the operating parameters of the third-stage nanofiltration (500Da) are: pressure 0.7-0.9MPa, crossflow velocity 2.0-3.0m / s, and temperature 18-22℃.

[0021] A chicken thymus immunomodulatory peptide prepared by the aforementioned process, wherein the main molecular weight of the immunomodulatory peptide is distributed in the range of 800-1200 Da, the peptide content is ≥85% (w / w), the moisture content is ≤8.0%, and the ash content is ≤5.0%.

[0022] Preferably, the immunomodulatory peptide can enhance the phagocytic activity of macrophages, with a phagocytic rate ≥60% and a phagocytic intensity ≥45% higher than the negative control; and promote IL-6 secretion, with the secretion amount ≥180% higher than the control group.

[0023] Preferably, the chicken thymus immunomodulatory peptide is used in the preparation of functional foods that enhance immunity.

[0024] Preferably, the chicken thymus immunomodulatory peptide is used in the preparation of immunomodulators, which are used to enhance the body's resistance to viral and bacterial infections.

[0025] Preferably, the use of the chicken thymus immunomodulatory peptide in the preparation of a pharmaceutical composition for promoting postoperative recovery or improving immunodeficiency is described, wherein the dosage of the pharmaceutical composition is 50-200 mg / kg / day.

[0026] The core innovations of this invention are: 1) It adopts a sequential complex enzymatic hydrolysis process using pepsin and trypsin to fully leverage the synergistic effect of the two enzymes; 2) It designs a precise pH gradient conversion process to ensure that the protein structure is properly opened after the first round of enzymatic hydrolysis without severe denaturation, creating favorable conditions for the second round of enzymatic hydrolysis; 3) It creatively proposes a batch-coupled enzymatic hydrolysis and membrane separation mode (BCEH-MSM), which realizes the organic combination of enzymatic hydrolysis reaction and membrane separation process, separating while reacting, effectively reducing product inhibition, improving enzymatic hydrolysis efficiency, and reducing membrane fouling.

[0027] The present invention has the following beneficial effects:

[0028] 1. Significantly improved yield: Compared with the traditional single enzymatic hydrolysis method, the process of this invention increases the yield of active peptides by about 42% and the purity by about 28%.

[0029] 2. Significantly enhanced activity: The immunomodulatory peptides prepared in this invention can significantly enhance the phagocytic activity of macrophages (increase by 56%) and promote IL-6 secretion (increase by 3.2 times).

[0030] 3. Significantly reduced costs: By using a batch-coupled enzymatic hydrolysis and membrane separation mode, the production cost of the process of this invention is reduced by about 30% compared with the prior art.

[0031] 4. Significantly improved stability: The bitterness of the product is significantly reduced, and the stability at 25°C has increased from 6 months to 18 months.

[0032] 5. Extended equipment lifespan: The lifespan of membrane modules is extended from approximately 20 batches to approximately 50 batches, an increase of approximately 150%. Attached Figure Description

[0033] Figure 1 The figure shows the flow cytometry results of the effect of the chicken thymus immunomodulatory peptide prepared in this embodiment of the invention on the phagocytic activity of macrophages. In the figure, A is the scatter plot of FSC-A / SSC-A and P1 gate selection of RAW264.7 cells in each group, B is the comparison of the superimposed histograms of FITC channels in each group, C is the statistical bar chart of phagocytic rate in each group, and D is the statistical bar chart of phagocytic intensity (MFI) in each group.

[0034] Figure 2 The image shows the fluorescence microscopy results (200×) of the effect of the chicken thymus immunomodulatory peptide prepared in the embodiments of the present invention on the phagocytosis of FITC-labeled yeast by macrophages. A is the negative control group, B is the positive control group (LPS), C is the group of Example 1, and D is the comparative example 1. Blue represents the cell nuclei stained with DAPI, and green represents the phagocytosed FITC-yeast. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0036] Chicken thymus immunomodulatory peptides are a class of small molecule polypeptides with immunomodulatory effects extracted from chicken thymus tissue. Their molecular weight is typically between 2-5 kDa, and they are rich in amino acids such as lysine, glutamic acid, and alanine. As members of the thymic peptide family, these peptides play an important role in the development and functional regulation of the immune system, exhibiting biological activities such as promoting T cell differentiation and maturation, regulating the function of various immune cells, and promoting cytokine production.

[0037] In the embodiments of this invention, the main raw material used is the thymus of healthy 6-8 week old white-feathered broiler chickens, with a thymus index >3.0 and intact, undamaged tissue. Auxiliary materials include deionized water (resistivity >18 MΩ·cm) and food-grade hydrochloric acid / sodium hydroxide for pH adjustment.

[0038] Regarding enzyme preparations, pepsin is derived from porcine gastric mucosa, with an activity ≥15000 U / mg, purity ≥95%, optimal pH 1.5-3.0, and optimal temperature 37-42℃; trypsin is derived from bovine pancreas, with an activity ≥8000 BAEE U / mg, purity ≥98%, optimal pH 6.8-8.0, and optimal temperature 40-45℃.

[0039] The membrane material used is polyethersulfone (PES) membrane with a molecular weight cutoff (MWCO) of 10 kDa and 1 kDa, and an effective area of ​​0.5 m² / sheet. The nanofiltration membrane uses a polysulfone-modified membrane with an MWCO of 500 Da and an effective area of ​​0.3 m² / sheet. The use of PES-modified membrane material and pulsed cross-flow technology, along with regular online cleaning, reduces membrane fouling.

[0040] The buffer system includes: acidic buffer (0.2M glycine-hydrochloric acid buffer, pH 2.0), neutral buffer (0.2M phosphate buffer, pH 7.5), and transition buffer (0.1M acetate-sodium acetate buffer, pH 5.0).

[0041] Example 1: A process for preparing chicken thymus immunomodulatory peptides using a sequential complex enzymatic hydrolysis method:

[0042] The specific steps of this embodiment are as follows:

[0043] (1) Raw material pretreatment: Chicken thymus tissue was thawed from -20℃ to 4℃, and after removing connective tissue and blood vessels, it was cut into 5mm×5mm×5mm pieces and washed three times with PBS buffer. Then, it was subjected to microwave-assisted treatment at 500W for 30 seconds, with the temperature controlled at 40±2℃. Subsequently, the treated tissue was mixed with water at a ratio of 1:3 (w / v) and homogenized at 10,000rpm for 3 minutes.

[0044] (2) Pre-hydrolysis with pepsin: The pH of the homogenate from step (1) was adjusted to 2.0±0.1 using hydrochloric acid solution, and pepsin was added at an enzyme-substrate ratio (E / S) of 1:50 (w / w). The reaction was carried out in a water bath at 37±1℃ for 3 hours with a stirring speed of 300 rpm. The pH and degree of hydrolysis (DH) were measured every 30 minutes during the reaction.

[0045] (3) pH gradient conversion: First, slowly add transition buffer to pH 5.0±0.1 and let stand for 15 minutes; then slowly add neutral buffer to pH 6.5±0.1 and let stand for 10 minutes; finally adjust to pH 7.5±0.1.

[0046] (4) Secondary hydrolysis with trypsin: Add trypsin to the solution from step (3) at an enzyme-to-substrate ratio (E / S) of 1:30 (w / w). Incubate the reaction in a water bath at 42±1℃ for 4 hours with a stirring speed of 250 rpm. After the reaction is complete, heat the solution in a water bath at 95℃ for 10 minutes to inactivate the enzyme.

[0047] (5) Membrane separation purification: Batch-coupled enzymatic hydrolysis and membrane separation mode (BCEH-MSM) is adopted, connecting the hydrolysis reactor to the membrane separation system. First-stage ultrafiltration (10kDa): operating pressure 0.25MPa, cross-flow rate 3.5m / s, temperature 30±2℃; Second-stage ultrafiltration (1kDa): operating pressure 0.30MPa, cross-flow rate 3.0m / s, temperature 25±2℃; Third-stage nanofiltration (500Da): operating pressure 0.8MPa, cross-flow rate 2.5m / s, temperature 20±2℃, conductivity monitoring ≤50μS / cm to ensure desalination effect.

[0048] (6) Concentration and Drying: The components obtained in step (5) are concentrated by vacuum rotary evaporation at a temperature not exceeding 40°C, with a final concentration ≥20% (w / v). Subsequently, freeze-drying is performed: pre-freezing at -40°C for 4 hours, primary drying at -30°C for 24 hours, and secondary drying at 20°C for 12 hours. Finally, the dried product is pulverized through a 60-mesh sieve, sealed in packaging, and stored at 4°C.

[0049] Trypsin and pepsin differ significantly in their cleavage specificity and optimal reaction conditions:

[0050] Pepsin: Optimal pH 1.5-3.0, mainly cleaves peptide bonds of aromatic amino acid residues;

[0051] Trypsin: Optimal pH 6.8-8.0, specifically cleaves peptide bonds at the carboxyl terminus of lysine and arginine;

[0052] Synergistic effect principle: Pre-hydrolyzing pepsin under acidic conditions disrupts the tertiary structure of proteins, exposing more sites recognizable by trypsin. Subsequently, trypsin further hydrolyzes the proteins under neutral conditions, forming more low-molecular-weight immunomodulatory peptides. Studies have shown that this synergistic effect can increase hydrolysis efficiency by nearly 5 times, with the degree of hydrolysis increasing by up to 77%, significantly higher than single-enzyme hydrolysis or non-sequential complex enzymatic hydrolysis processes.

[0053] First stage: Using a 10kDa MWCO polyethersulfone ultrafiltration membrane to remove unhydrolyzed proteins and macromolecular impurities;

[0054] Second stage: Using a 1kDa MWCO polysulfone ultrafiltration membrane to concentrate the target small molecule peptides;

[0055] Third stage: using a 500Da MWCO nanofiltration membrane for further separation and desalting purification;

[0056] Compared with traditional single-stage membrane separation, this gradient system can improve the recovery rate of target peptides by 32% and the purity by 41%, and effectively reduce the impact of membrane fouling on long-term operation.

[0057] This innovative solution employs a batch-coupled enzymatic hydrolysis and membrane separation mode, linking the enzymatic reaction and membrane separation process within the same system to achieve continuous production with simultaneous reaction and separation. Compared to traditional offline membrane separation, this mode offers three major advantages:

[0058] 1. Reduced product inhibition: Timely separation of small molecule peptides from the reaction system reduces the inhibition of enzyme reactions by the products;

[0059] 2. Improved enzymatic hydrolysis efficiency: By continuously removing the product, the enzymatic hydrolysis reaction is always maintained at a high efficiency.

[0060] 3. Product quality improvement: Avoid excessive hydrolysis and bitterness caused by prolonged reaction;

[0061] Experimental results show that the BCEH-MSM mode improves the enzymatic hydrolysis efficiency by 28% and the functional activity of the product by 35% compared with the traditional enzymatic hydrolysis and offline membrane separation mode (TEH-OMSM).

[0062] Testing revealed that the chicken thymus immunomodulatory peptides prepared in this embodiment have a molecular weight distribution in the range of 800-1200 Da, with the peptides around 1000 Da accounting for the highest proportion, reaching approximately 40%. The peptide content is 92.3% (w / w), the moisture content is 4.2%, and the ash content is 2.8%.

[0063] Example 2: A process for preparing chicken thymus immunomodulatory peptides using a low-temperature compound enzymatic hydrolysis method:

[0064] The difference between this embodiment and Embodiment 1 is that:

[0065] (1) Raw material pretreatment: The microwave-assisted processing power was reduced to 400W, the processing time was extended to 40 seconds, and the temperature was controlled at 35±2℃. The ratio of tissue to water was adjusted to 1:2 (w / v).

[0066] (2) Pre-hydrolysis with pepsin: pH value was adjusted to 1.8±0.1, reaction temperature was reduced to 35±1℃, and reaction time was extended to 4 hours.

[0067] (3) pH gradient conversion: In the first stage, adjust to pH 4.8±0.1 and let stand for 20 minutes; in the second stage, adjust to pH 6.3±0.1 and let stand for 15 minutes; finally, adjust to pH 7.3±0.1.

[0068] (4) Secondary hydrolysis of trypsin: The enzyme-substrate ratio was adjusted to 1:25 (w / w), the reaction temperature was reduced to 40±1℃, and the reaction time was extended to 5 hours.

[0069] (5) Membrane separation and purification: The operating pressure of the first-stage ultrafiltration (10kDa) was reduced to 0.20MPa and the crossflow rate was reduced to 3.0m / s; the operating parameters of the second and third stages remained unchanged.

[0070] The core innovation of this approach lies in introducing a strictly controlled pH gradient transition process to achieve optimal sequential activity of pepsin and trypsin. Microwave-assisted pretreatment further enhances the disruption of raw material tissues, thereby increasing the efficiency of subsequent enzymatic hydrolysis. The pH transition from pepsin hydrolysis (pH 2.0) to trypsin hydrolysis (pH 7.5) requires precise control; otherwise, activity instability may result. An automated pH control system is designed to achieve smooth pH transitions through buffer gradient addition, adding an intermediate stabilization step to prevent protein denaturation.

[0071] The test results showed that the main molecular weight of the chicken thymus immunomodulatory peptide prepared in this embodiment was distributed in the range of 850-1250 Da, the peptide content was 94.1% (w / w), the moisture content was 3.8%, and the ash content was 2.5%.

[0072] Example 3: A process for preparing chicken thymus immunomodulatory peptides using a high-temperature rapid complex enzymatic hydrolysis method:

[0073] The difference between this embodiment and Embodiment 1 is that:

[0074] (1) Raw material pretreatment: The microwave-assisted processing power was increased to 600W, the processing time was shortened to 20 seconds, and the temperature was controlled at 45±2℃. The ratio of tissue to water was adjusted to 1:4 (w / v).

[0075] (2) Pre-hydrolysis with pepsin: pH value was adjusted to 2.2±0.1, reaction temperature was increased to 40±1℃, and reaction time was shortened to 2 hours.

[0076] (3) pH gradient conversion: In the first stage, adjust to pH 5.2±0.1 and let stand for 10 minutes; in the second stage, adjust to pH 6.7±0.1 and let stand for 5 minutes; finally, adjust to pH 7.7±0.1.

[0077] (4) Secondary hydrolysis of trypsin: The enzyme-substrate ratio was adjusted to 1:35 (w / w), the reaction temperature was increased to 45±1℃, and the reaction time was shortened to 3 hours.

[0078] (5) Membrane separation and purification: The operating pressure of the first-stage ultrafiltration (10kDa) was increased to 0.30MPa and the cross-flow rate was increased to 4.0m / s; the operating pressure of the second-stage ultrafiltration (1kDa) was increased to 0.35MPa and the cross-flow rate was increased to 3.5m / s; the operating pressure of the third-stage nanofiltration (500Da) was increased to 0.9MPa and the cross-flow rate was increased to 3.0m / s.

[0079] The test results showed that the main molecular weight of the chicken thymus immunomodulatory peptide prepared in this embodiment was distributed in the range of 750-1150 Da, the peptide content was 89.7% (w / w), the moisture content was 5.0%, and the ash content was 3.2%.

[0080] Example 4: A process for preparing chicken thymus immunomodulatory peptides using a moderate-condition complex enzymatic hydrolysis method:

[0081] The specific parameters of this embodiment are between those of embodiments 1, 2, and 3, and are as follows:

[0082] (1) Raw material pretreatment: The microwave-assisted treatment power was 550W, the treatment time was 25 seconds, and the temperature was controlled at 42±2℃. The ratio of tissue to water was 1:3.5 (w / v).

[0083] (2) Pre-hydrolysis of pepsin: pH value was 2.1±0.1, reaction temperature was 38±1℃, reaction time was 2.5 hours, and enzyme-substrate ratio was 1:45 (w / w).

[0084] (3) pH gradient conversion: In the first stage, adjust to pH 5.1±0.1 and let stand for 12 minutes; in the second stage, adjust to pH 6.6±0.1 and let stand for 8 minutes; finally, adjust to pH 7.6±0.1.

[0085] (4) Secondary hydrolysis of trypsin: the enzyme-substrate ratio was 1:28 (w / w), the reaction temperature was 43±1℃, and the reaction time was 3.5 hours.

[0086] (5) Membrane separation and purification: The first stage ultrafiltration (10kDa) operates at a pressure of 0.28MPa and a crossflow rate of 3.8m / s; the second stage ultrafiltration (1kDa) operates at a pressure of 0.32MPa and a crossflow rate of 3.2m / s; the third stage nanofiltration (500Da) operates at a pressure of 0.85MPa and a crossflow rate of 2.8m / s.

[0087] The test results showed that the main molecular weight of the chicken thymus immunomodulatory peptide prepared in this embodiment was distributed in the range of 780-1180 Da, the peptide content was 91.5% (w / w), the moisture content was 4.5%, and the ash content was 2.9%.

[0088] Example 5: A process for preparing chicken thymus immunomodulatory peptides using a composite enzymatic hydrolysis method employing magnetic nanoparticle-immobilized enzymes:

[0089] This embodiment is an improvement upon Embodiment 1, with the innovation lying in the use of magnetic nanoparticle immobilization technology to achieve efficient enzyme recovery and reuse. The specific steps are as follows:

[0090] (1) Raw material pretreatment: Same as in Example 1.

[0091] (2) Pre-hydrolysis of pepsin: using magnetic nanoparticles ( Immobilized pepsin replaced free pepsin, and other conditions were the same as in Example 1.

[0092] (3) pH gradient conversion: Same as in Example 1. After the reaction was completed, the immobilized pepsin was separated and recovered using an external magnetic field.

[0093] (4) Secondary hydrolysis of trypsin: Trypsin immobilized with magnetic nanoparticles was used instead of free trypsin, and other conditions were the same as in Example 1. After the reaction, the immobilized trypsin was separated and recovered using an external magnetic field.

[0094] (5) Membrane separation and purification: Same as in Example 1.

[0095] (6) Concentration and drying: Same as in Example 1.

[0096] Enzyme activity assays showed that the recovered immobilized pepsin and trypsin could be reused more than 15 times, with an enzyme activity decrease of no more than 5% after each reuse. Analysis revealed that the chicken thymus immunomodulatory peptides prepared in this embodiment had a main molecular weight distribution in the range of 820-1220 Da, a peptide content of 90.8% (w / w), a moisture content of 4.3%, and an ash content of 3.0%. This embodiment achieved efficient enzyme recovery and reuse through immobilized enzyme technology, further reducing production costs by approximately 25%.

[0097] Example 6: A process for preparing chicken thymus immunomodulatory peptides using a composite enzymatic hydrolysis method employing pulsed cross-flow technology:

[0098] This embodiment is an improvement upon Embodiment 1. The innovation lies in the use of pulsed cross-flow technology during membrane separation, which reduces membrane fouling and extends membrane lifespan. The specific implementation method is as follows:

[0099] (1) Raw material pretreatment: Same as in Example 1.

[0100] (2) Pre-hydrolysis with pepsin: Same as in Example 1.

[0101] (3) pH gradient conversion: Same as in Example 1.

[0102] (4) Secondary hydrolysis with trypsin: Same as in Example 1.

[0103] (5) Membrane separation and purification: Pulsed cross-flow technology is employed, specifically: the first-stage ultrafiltration (10kDa) operates at a pressure of 0.25MPa, with a basic cross-flow rate of 3.5m / s, and a 10-second high-speed pulse (5.0m / s) is applied every 60 seconds; the second-stage ultrafiltration (1kDa) operates at a pressure of 0.30MPa, with a basic cross-flow rate of 3.0m / s, and a 10-second high-speed pulse (4.5m / s) is applied every 90 seconds; the third-stage nanofiltration (500Da) operates at a pressure of 0.8MPa, with a basic cross-flow rate of 2.5m / s, and a 10-second high-speed pulse (4.0m / s) is applied every 120 seconds. Simultaneously, online cleaning is performed every 8 hours using a 0.1% NaOH solution.

[0104] (6) Concentration and drying: Same as in Example 1.

[0105] Through long-term operation and testing, the service life of the membrane module in this embodiment was extended from approximately 50 batches to approximately 80 batches, an improvement of approximately 60%. Testing revealed that the chicken thymus immunomodulatory peptides prepared in this embodiment have a main molecular weight distribution in the range of 810-1210 Da, a peptide content of 92.0% (w / w), a moisture content of 4.4%, and an ash content of 2.7%.

[0106] Example 7: A process for preparing chicken thymus immunomodulatory peptides using a compound enzymatic hydrolysis method with added antioxidants:

[0107] This embodiment is an improvement upon Example 1. The innovation lies in adding an antioxidant during the enzymatic hydrolysis process to prevent the active peptides from being oxidized during preparation, thereby improving product stability. The specific implementation method is as follows:

[0108] (1) Raw material pretreatment: Same as in Example 1, but 0.05% (w / v) of ascorbic acid was added to the tissue as an antioxidant before homogenization.

[0109] (2) Pre-hydrolysis with pepsin: Same as in Example 1.

[0110] (3) pH gradient conversion: Same as in Example 1.

[0111] (4) Secondary hydrolysis of trypsin: Same as in Example 1, but 0.02% (w / v) sodium ascorbate was added to the solution as an antioxidant before the reaction started.

[0112] (5) Membrane separation and purification: Same as in Example 1.

[0113] (6) Concentration and drying: Same as in Example 1, but 0.1% (w / v) of ascorbic acid is added to the solution as an antioxidant before concentration.

[0114] Accelerated stability testing (stored at 40°C / 75%RH for 3 months) showed that the peptide content of the product prepared in this example decreased by approximately 40% compared to Example 1, demonstrating better storage stability. Analysis revealed that the chicken thymus immunomodulatory peptides prepared in this example had a main molecular weight distribution in the range of 800-1200 Da, a peptide content of 93.1% (w / w), a moisture content of 4.0%, and an ash content of 2.6%.

[0115] Example 8: A process for preparing chicken thymus immunomodulatory peptides using a combined enzymatic hydrolysis method involving ion exchange chromatography:

[0116] This embodiment is an improvement upon Example 1. The innovation lies in adding an ion exchange chromatography purification step after membrane separation, further improving product purity. The specific implementation method is as follows:

[0117] (1) Raw material pretreatment: Same as in Example 1.

[0118] (2) Pre-hydrolysis with pepsin: Same as in Example 1.

[0119] (3) pH gradient conversion: Same as in Example 1.

[0120] (4) Secondary hydrolysis with trypsin: Same as in Example 1.

[0121] (5) Membrane separation and purification: Same as in Example 1.

[0122] (6) Ion exchange chromatography: The fraction obtained in step (5) was further purified using a weak cation exchange resin (CM Sepharose Fast Flow). The specific operation was as follows: after packing the resin into the column, it was equilibrated with 5 column volumes of equilibration buffer (20 mM sodium acetate buffer, pH 5.0); the sample to be purified was adjusted to pH 5.0 and then loaded; non-specific adsorbed substances were eluted with 5 column volumes of equilibration buffer; then the target peptide was eluted with linear gradient elution buffer (20 mM sodium acetate + 0-1.0 M NaCl, pH 5.0); the main peak eluted fraction was collected.

[0123] (7) Concentration and drying: The components obtained in step (6) are concentrated and freeze-dried, with other conditions the same as in Example 1.

[0124] Testing revealed that the chicken thymus immunomodulatory peptides prepared in this embodiment had a main molecular weight distribution in the range of 800-1200 Da, with a significantly increased peptide content of 97.2% (w / w), a moisture content of 3.5%, and an ash content of 1.8%.

[0125] Comparative Example 1

[0126] Process for preparing chicken thymus immunomodulatory peptides by single pepsin hydrolysis:

[0127] This comparative example uses only pepsin for single enzymatic hydrolysis, and the specific steps are as follows:

[0128] (1) Raw material pretreatment: Same as in Example 1.

[0129] (2) Pepsin hydrolysis: Adjust the pH of the homogenate from step (1) to 2.0±0.1, add pepsin, and the enzyme-substrate ratio (E / S) is 1:50 (w / w). React at a constant temperature of 37±1℃ in a water bath for 7 hours with a stirring speed of 300 rpm. After the reaction is completed, heat the solution in a 95℃ water bath for 10 minutes to inactivate the enzyme.

[0130] (3) Membrane separation and purification: The hydrolysate was sequentially passed through ultrafiltration / nanofiltration membranes with pore sizes of 10kDa, 1kDa, and 500Da, and the fraction between 1kDa and 500Da was collected. Membrane separation was performed in a conventional offline mode, not a batch-coupled mode.

[0131] (4) Concentration and drying: Same as in Example 1.

[0132] Testing revealed that the product prepared in this comparative example had a wide molecular weight distribution, ranging from 500 to 5000 Da, with a peptide content of 68.5% (w / w), a moisture content of 6.2%, and an ash content of 4.5%.

[0133] Comparative Example 2

[0134] Process for preparing chicken thymus immunomodulatory peptides by single trypsin hydrolysis:

[0135] This comparative example uses only trypsin for single enzymatic hydrolysis, and the specific steps are as follows:

[0136] (1) Raw material pretreatment: Same as in Example 1.

[0137] (2) Trypsin hydrolysis: Adjust the pH of the homogenate from step (1) to 7.5±0.1, add trypsin, and the enzyme-substrate ratio (E / S) is 1:30 (w / w). React at a constant temperature in a water bath at 42±1℃ for 7 hours with a stirring speed of 250 rpm. After the reaction is complete, heat the solution in a 95℃ water bath for 10 minutes to inactivate the enzyme.

[0138] (3) Membrane separation and purification: Same as Comparative Example 1.

[0139] (4) Concentration and drying: Same as in Example 1.

[0140] The comparative sample was tested and found that the main molecular weight of the product was distributed in the range of 1000-8000 Da, the peptide content was 72.3% (w / w), the moisture content was 5.8%, and the ash content was 4.2%.

[0141] Comparative Example 3

[0142] A process for preparing chicken thymus immunomodulatory peptides using a non-sequential complex enzymatic hydrolysis method:

[0143] This comparative example uses pepsin and trypsin for simultaneous hydrolysis, rather than sequential hydrolysis. The specific steps are as follows:

[0144] (1) Raw material pretreatment: Same as in Example 1.

[0145] (2) Compound enzymatic hydrolysis: The pH of the homogenate from step (1) was adjusted to 5.0±0.1, and pepsin and trypsin were added simultaneously, with an enzyme-substrate ratio (E / S) of 1:50 (w / w). The reaction was carried out at a constant temperature of 40±1℃ in a water bath for 6 hours with a stirring speed of 300 rpm. After the reaction was completed, the solution was heated in a 95℃ water bath for 10 minutes to inactivate the enzyme.

[0146] (3) Membrane separation and purification: Same as Comparative Example 1.

[0147] (4) Concentration and drying: Same as in Example 1.

[0148] The comparative sample was tested and found that the main molecular weight of the product was distributed in the range of 700-3000 Da, the peptide content was 78.6% (w / w), the moisture content was 5.5%, and the ash content was 3.8%.

[0149] Comparative Example 4

[0150] A process for preparing chicken thymus immunomodulatory peptides using a sequential enzymatic hydrolysis method without pH gradient switching:

[0151] This comparative example uses sequential enzymatic hydrolysis but does not involve pH gradient switching. The specific steps are as follows:

[0152] (1) Raw material pretreatment: Same as in Example 1.

[0153] (2) Pre-hydrolysis with pepsin: Same as in Example 1.

[0154] (3) pH adjustment: The hydrolysate from step (2) is directly adjusted to pH 7.5±0.1 without setting a transition stage.

[0155] (4) Secondary hydrolysis with trypsin: Same as in Example 1.

[0156] (5) Membrane separation and purification: Same as Comparative Example 1.

[0157] (6) Concentration and drying: Same as in Example 1.

[0158] The comparative sample was tested and found that the main molecular weight of the product was distributed in the range of 800-2500 Da, the peptide content was 81.2% (w / w), the moisture content was 5.0%, and the ash content was 3.5%.

[0159] Comparative Example 5

[0160] A process for preparing chicken thymus immunomodulatory peptides using a compound enzymatic hydrolysis method without microwave-assisted pretreatment:

[0161] This comparative example did not use microwave-assisted preprocessing. The specific steps are as follows:

[0162] (1) Raw material pretreatment: Chicken thymus tissue was thawed from -20℃ to 4℃, and after removing connective tissue and blood vessels, it was cut into 5mm×5mm×5mm pieces and washed 3 times with PBS buffer. Then the tissue was mixed with water at a ratio of 1:3 (w / v) and homogenized at 10,000 rpm for 3 minutes.

[0163] (2) Pre-hydrolysis with pepsin: Same as in Example 1.

[0164] (3) pH gradient conversion: Same as in Example 1.

[0165] (4) Secondary hydrolysis with trypsin: Same as in Example 1.

[0166] (5) Membrane separation and purification: Same as Comparative Example 1.

[0167] (6) Concentration and drying: Same as in Example 1.

[0168] The comparative sample was tested and found that the main molecular weight of the product was distributed in the range of 850-1350 Da, the peptide content was 85.7% (w / w), the moisture content was 4.8%, and the ash content was 3.2%.

[0169] The chicken thymus immunomodulatory peptide prepared by this invention has a molecular weight distribution in the range of 800-1200 Da. This molecular weight range differs from that of traditional thymosin (2000-5000 Da), but has clear scientific basis and unique advantages.

[0170] From the perspective of immunomodulatory mechanisms, the efficacy of immunomodulatory peptides depends primarily on their specific amino acid sequences and spatial conformations, rather than simply their molecular weight. Studies have shown that many small peptides with high immunomodulatory activity often have molecular weights in the range of 500-1500 Da. For example, thymopentin (TP-5), with a molecular weight of only 679.77 Da, is a widely used immunomodulator in clinical practice; glutamine dipeptide, with a molecular weight of approximately 217 Da, also exhibits significant immunomodulatory functions.

[0171] This invention employs a sequential pepsin-trypsin enzymatic hydrolysis technique, which allows for precise control of the degree of hydrolysis while preserving key immunomodulatory sequences. Pepsin primarily cleaves the peptide bonds of aromatic amino acid residues (phenylalanine, tyrosine, and tryptophan), while trypsin specifically cleaves the peptide bonds at the carboxyl terms of lysine and arginine. This sequential enzymatic hydrolysis strategy generates small peptides rich in basic amino acids, which have been shown to be closely related to the recognition and binding of receptors on the surface of immune cells.

[0172] In addition, small molecule peptides (800-1200 Da) have the following advantages over large molecule peptides (2000-5000 Da): First, they have a higher intestinal absorption rate. Small molecule peptides can be directly absorbed through the peptide transporter (PepT1) of intestinal epithelial cells, which significantly improves bioavailability. Second, they have better stability. Smaller peptides have simpler spatial structures and are less prone to aggregation or degradation. Third, they have lower bitterness. By precisely controlling the degree of enzymatic hydrolysis, bitter peptides produced by excessive hydrolysis are avoided.

[0173] Peptide product performance evaluation:

[0174] 1. Molecular weight distribution analysis

[0175] The molecular weight distribution of the products in each example and comparative example was analyzed by gel filtration chromatography (GFC), and the results are shown in Table 1:

[0176] Table 1. Molecular weight distribution of products from each example and comparative example

[0177]

[0178] As can be seen from Table 1, in the products of the embodiments of the present invention, the proportion of peptides in the 800-1200 Da range is significantly higher than that in the comparative examples. Peptides in this molecular weight range generally have better bioactivity and absorption. In particular, in Example 8, by adding an ion exchange chromatography purification step, the proportion of peptides in the target molecular weight range reached 72.6%, which is significantly higher than that of other samples.

[0179] 2. Macrophage phagocytic activity assay:

[0180] 2.1 Experimental Materials

[0181] Cell line: RAW264.7 mouse macrophage cell line, passaged to the 3rd-8th generation for experiments.

[0182] Culture media and reagents: DMEM high glucose medium (Gibco), fetal bovine serum (Gibco), penicillin-streptomycin solution (100×) (Gibco), FITC-labeled yeast particles (Sigma), lipopolysaccharide LPS (Sigma), trypan blue solution (0.4%) (Solarbio), PBS buffer (pH 7.4), 0.25% trypsin-EDTA solution.

[0183] Main instruments: flow cytometer, inverted fluorescence microscope, CO2 incubator, clean bench, low-speed centrifuge, enzyme-linked immunosorbent assay (ELISA) reader.

[0184] 2.2 Cell Culture

[0185] RAW264.7 cells were cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and incubated at 37°C with 5% [unspecified medium]. Culture in a saturated humidity incubator. When the cell confluence reaches 80-90%, gently scrape adherent cells with a cell scraper for passage at a ratio of 1:3-1:4.

[0186] 2.3 FITC-Yeast Phagocytosis Assay Method

[0187] Step 1: Cell plating

[0188] RAW264.7 cells in logarithmic growth phase were digested with 0.25% trypsin, and the cell density was adjusted to [a specific concentration] using complete culture medium. 500 μL / well was inoculated into 24-well plates (i.e., 1 / mL). (each cell), and a sterile coverslip is pre-placed in the well for subsequent microscopic observation. 37℃, 5% Cultured under the specified conditions for 24 hours to allow the cells to fully adhere to the culture vessel.

[0189] Step 2: Sample processing

[0190] Aspirate the culture supernatant and wash the cells twice with sterile PBS. Add 500 μL of fresh culture medium per well containing different concentrations of the sample, according to the experimental groups. The groups included: negative control group (complete culture medium only), positive control group (LPS added, final concentration 100 ng / mL), Example 1 group (chicken thymus immunomodulatory peptide final concentration 50 μg / mL), Example 4 group, Example 8 group, Comparative Example 1 group, and Comparative Example 3 group. Each group had 6 replicates. Pretreatment was performed by incubating at 37°C for 24 hours.

[0191] Step 3: Add FITC-yeast

[0192] Take the FITC-labeled yeast particle suspension and adjust the concentration to the appropriate level using serum-free DMEM medium. Count / mL. Aspirate the culture medium from each well, add 500 μL of FITC-yeast suspension (yeast:macrophage ≈ 100: 1) to each well, and continue culturing at 37°C for 2 hours to induce phagocytosis.

[0193] Step 4: Stop the engulfing and washing

[0194] After the culture was completed, the culture medium containing FITC-yeast was aspirated, and the cells were washed three times with cold PBS (4°C), gently shaking for 30 seconds each time to remove unphagocytosed free yeast particles and yeast adsorbed on the cell surface.

[0195] Step 5: Flow cytometry detection

[0196] Cells were digested with 0.25% trypsin and collected into 1.5 mL centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. Cells were resuspended in 500 μL of cold PBS and transferred to flow cytometry tubes. Detection was performed using a BD FACSCanto II flow cytometer with excitation wavelength of 488 nm and emission wavelength of 530 nm (FITC channel). 10,000 cells were analyzed for each sample, and data were analyzed using FlowJo V10 software.

[0197] Flow cytometry parameters: FSC voltage 250V, SSC voltage 280V, FITC voltage 400V, acquisition speed medium (approximately 1000 events / second), FSC threshold set to exclude cell debris.

[0198] Data analysis method: Macrophage populations (P1 gate) were selected based on the FSC-A / SSC-A scatter plot, excluding cell debris and dead cells. In the FITC histogram, a positive threshold M1 gate was set based on the fluorescence signal of the negative control group, and the proportion of FITC-positive cells (i.e., phagocytosis rate) in each group was calculated. Phagocytosis intensity was expressed as the mean fluorescence intensity (MFI) of the FITC channels, reflecting the average number of yeast cells phagocytosed by a single cell.

[0199] Calculation formula: Phagocytosis rate (%) = Number of FITC positive cells / Total number of cells detected × 100%; Phagocytosis intensity improvement rate (%) = (Experimental group MFI - Negative control group MFI) / Negative control group MFI × 100%.

[0200] Step 6: Observation under a fluorescence microscope

[0201] Cells were fixed in the wells of pre-placed coverslips with 4% paraformaldehyde for 15 minutes and washed three times with PBS. Cell nuclei were stained with DAPI (1 μg / mL) for 5 minutes and washed with PBS to remove excess dye. The coverslips were then inverted onto the slide and mounted with an anti-fluorescence quenching mounting medium. Representative fields of view (200× and 400× magnification) were photographed under an inverted fluorescence microscope in both the blue channel (DAPI, Ex 358nm / Em 461nm) and the green channel (FITC, Ex 494nm / Em 518nm). Five fields of view were randomly selected from each sample, and the number of macrophages phagocytosing ≥1 yeast cell in 100 macrophages was counted.

[0202] 2.4 Experimental Results

[0203] 2.4.1 Flow cytometry results

[0204] like Figure 1 As shown, the scatter plots and histograms obtained by flow cytometry clearly demonstrate the differences in phagocytic activity among the macrophages in each group. The negative control group, RAW264.7 cells, showed weak FITC fluorescence signal and a phagocytic rate of 32.6%, reflecting the baseline phagocytic level. The positive control group (LPS 100 ng / mL) showed a significantly increased proportion of FITC-positive cells, with a phagocytic rate reaching 71.5% and a significantly increased MFI value, confirming the effectiveness of the experimental system.

[0205] The phagocytosis rate of the chicken thymus immunomodulatory peptide (50 μg / mL) treatment group prepared in Example 1 of this invention was 65.3%, which was about 100% higher than that of the negative control group, and the MFI value was 56.4% higher than that of the negative control group. The phagocytosis rates of Examples 4 and 8 were 65.9% and 68.9%, respectively, both significantly higher than those of the control groups.

[0206] The phagocytosis rate of Comparative Example 1 (single pepsin hydrolysate) was only 46.2%, with a phagocytosis intensity improvement rate of 30.5%; the phagocytosis rate of Comparative Example 3 (non-sequential complex enzymatic hydrolysate) was 53.8%, with a phagocytosis intensity improvement rate of 38.6%. Statistical analysis showed that there were significant differences between the embodiments of the present invention and the comparative examples (P<0.01), confirming the importance of sequential complex enzymatic hydrolysis technology in improving the immunomodulatory activity of the products.

[0207] 2.4.2 Results of fluorescence microscopy observation

[0208] like Figure 2As shown in the fluorescence microscopy images, the phagocytosis of FITC-labeled yeast by macrophages is clearly demonstrated. In the merged image, blue fluorescence represents DAPI-stained cell nuclei, and green fluorescence represents phagocytosed FITC-labeled yeast. The negative control group showed sparse green fluorescence signals in the cytoplasm, with only a few cells phagocytosing 1-2 yeast particles. The macrophages in the Example 1 treatment group showed abundant and dense green fluorescence signals in the cytoplasm, indicating that the cells had phagocytosed a larger number of yeast particles. Five to ten phagocytosed yeast particles were clearly identifiable in some cells, and the cell morphology exhibited a typical activated state with clearly extended pseudopodia. The green fluorescence signal in the Comparative Example 1 treatment group was between that of the negative control group and the Example 1 group, and the number of phagocytosed yeast particles was significantly less than that in the Example 1 group of this invention, further confirming that the small molecule immunomodulatory peptide prepared in this invention has a significant activity in enhancing the phagocytic function of macrophages.

[0209] 2.5 Statistical Analysis

[0210] All experiments were independently repeated three times, and data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons between groups, and LSD-t tests were used for pairwise comparisons. P < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 9.0 software.

[0211] The results are shown in Table 2:

[0212] Table 2. Effects of each example and comparative product (50 μg / mL) on macrophage phagocytic function

[0213]

[0214] *The positive control was the lipopolysaccharide (LPS, 100 ng / mL) treatment group.

[0215] The negative control group consisted of untreated RAW264.7 mouse macrophages. As shown in Table 2, the immunomodulatory peptides prepared in the embodiments of this invention significantly enhanced the phagocytic function of macrophages. In particular, Example 8 showed an increase in phagocytic rate and intensity that was close to the level of the positive control LPS-treated group, and significantly superior to all other control groups.

[0216] 3. Assay for the effect of IL-6 in promoting secretion:

[0217] The human peripheral blood mononuclear cell (PBMC) model was used to determine the promoting effect of various samples on IL-6 secretion. After treating cells with different concentrations (5, 10, 25, 50 μg / mL) for 12 hours, the IL-6 content in the culture supernatant was measured by ELISA. The results are shown in Table 3.

[0218] Table 3. Effects of each example and comparative product (50 μg / mL) on IL-6 secretion by PBMCs

[0219]

[0220] The control group consisted of untreated human peripheral blood mononuclear cells (PBMCs). As shown in Table 3, the immunomodulatory peptides prepared in the embodiments of this invention significantly promoted IL-6 secretion by PBMCs, with an increase rate exceeding 300%, which was significantly better than the control groups. In particular, in Example 8, the IL-6 secretion increase rate reached 350.9%.

[0221] 4. Product stability evaluation:

[0222] Accelerated stability tests (stored at 40℃ / 75%RH for 3 months) and long-term stability tests (stored at 4℃ for 24 months) were conducted on each sample. The peptide content, macrophage phagocytic activity, and IL-6 secretion-promoting effect were measured before and after storage, respectively. The results are shown in Table 4.

[0223] Table 4. Stability evaluation of products from each embodiment and comparative example

[0224]

[0225] *Activity retention rate is the ratio of macrophage phagocytic activity after storage to that before storage.

[0226] As shown in Table 4, the products of the embodiments of the present invention have better stability. After accelerated stability testing and long-term stability testing, the decrease in peptide content and the loss of activity are significantly lower than those of the comparative examples. In particular, Example 7, due to the addition of antioxidants during the preparation process, exhibits the best stability, with an activity retention rate of 91.8% after a 24-month long-term stability test.

[0227] The technical mechanism of this invention can be analyzed from the following aspects:

[0228] 1. Mechanism of sequential synergistic action of pepsin and trypsin:

[0229] Pepsin and trypsin have different cleavage specificities and optimal reaction conditions. Pepsin has the highest activity in an acidic environment (pH 1.5-3.0) and mainly cleaves the peptide bonds of aromatic amino acid residues (such as phenylalanine, tyrosine, and tryptophan) in proteins; while trypsin has the highest activity in a neutral environment (pH 6.8-8.0) and specifically cleaves the peptide bonds at the carboxyl terminus of lysine and arginine.

[0230] In the sequential enzymatic hydrolysis process of this invention, pepsin first pre-hydrolyzes chicken thymus protein under acidic conditions. This process opens up the higher-order structure of the protein, exposing more internal peptide bonds, especially more sites recognizable by trypsin. Subsequently, trypsin performs a secondary hydrolysis under neutral conditions, further cleaving those sites exposed in the primary hydrolysis to generate more small peptide fragments.

[0231] The results of Comparative Examples 1 and 2 show that the peptides produced by a single enzymatic hydrolysis method have a wide molecular weight distribution, with a low proportion of peptides in the 800-1200 Da range. The results of Comparative Example 3 show that the non-sequential enzymatic hydrolysis method (using two enzymes simultaneously) is also not ideal, mainly because the two enzymes cannot reach their optimal activity state simultaneously under the same pH conditions.

[0232] 2. The importance of pH gradient transition:

[0233] This invention designs a precise three-step pH gradient transition process: first, adjusting the pH to 4.8-5.2 and allowing it to stand for 10-20 minutes, starting from the acidic environment of pepsin hydrolysis (pH 1.8-2.2); then adjusting it to pH 6.3-6.7 and allowing it to stand for 5-15 minutes; finally, adjusting it to the neutral environment of trypsin hydrolysis (pH 7.3-7.7). The importance of this process is reflected in:

[0234] First, proteins and peptides are prone to denaturation or aggregation when subjected to rapid pH changes, affecting the efficiency of subsequent enzymatic digestion. By setting a pH transition phase, the protein structure can remain relatively stable during pH changes, reducing the occurrence of denaturation or aggregation.

[0235] Secondly, pH 5.0 is the critical point where the activity of acidic proteases (such as pepsin) decreases while the activity of alkaline proteases (such as trypsin) has not fully recovered. Allowing the system to stand at this pH for a period of time can keep the protein structure in the system in a state of being appropriately open but not completely denatured, creating favorable conditions for subsequent trypsin hydrolysis.

[0236] The results of Comparative Example 4 show that the proportion of peptides in the 800-1200 Da range generated by the sequential enzymatic hydrolysis process without pH gradient conversion is significantly lower than that in the embodiments of the present invention, which confirms the importance of pH gradient conversion in the sequential enzymatic hydrolysis process.

[0237] 3. Advantages of batch-coupled enzymatic hydrolysis and membrane separation mode (BCEH-MSM):

[0238] Traditionally, enzymatic hydrolysis and membrane separation are performed separately; that is, the enzymatic hydrolysis is completed first, followed by membrane separation and purification. This offline approach has the following problems: First, small peptides generated during enzymatic hydrolysis accumulate in the reaction system, inhibiting enzyme activity; second, prolonged enzymatic hydrolysis may lead to over-hydrolysis, producing bitter substances; and third, membrane fouling is easily caused by the presence of large molecules in the feed during membrane separation.

[0239] This invention creatively proposes a batch-coupled enzymatic hydrolysis and membrane separation mode (BCEH-MSM), which connects the enzymatic hydrolysis reactor to a membrane separation system to achieve continuous production with simultaneous reaction and separation. This mode has the following advantages:

[0240] First, by continuously removing the enzymatic hydrolysis products (small molecule peptides), the inhibition of enzyme activity by the products is reduced, so that the enzymatic hydrolysis reaction is always kept in a highly efficient state.

[0241] Secondly, by removing peptides within the target molecular weight range in a timely manner, excessive hydrolysis that may occur during prolonged reactions is avoided, thereby reducing the production of bitter substances.

[0242] Third, because macromolecular proteins and intermediates are retained in the reactor, contaminant deposition on the membrane surface is reduced, extending the service life of the membrane module.

[0243] The test results of each example and comparative example show that the immunomodulatory peptides prepared by the BCEH-MSM mode are significantly superior to those prepared by the traditional offline mode in terms of yield, activity and stability.

[0244] 4. The role of microwave-assisted pretreatment:

[0245] This invention employs microwave-assisted treatment in the raw material pretreatment stage, a step that plays a crucial role in improving the efficiency of subsequent enzymatic hydrolysis. Microwave energy can directly act on the interior of molecules, causing them to rotate and vibrate rapidly, generating heat. This unique heating method can quickly break down the tight structure of proteins, increasing the contact opportunity between the enzyme and the substrate, and may also weaken some peptide bonds, creating favorable conditions for subsequent enzymatic hydrolysis.

[0246] The results of Comparative Example 5 show that the product prepared without microwave-assisted pretreatment is inferior to that of the embodiments of the present invention in terms of molecular weight distribution, activity and stability, which confirms the importance of microwave-assisted pretreatment in improving enzymatic hydrolysis efficiency and product quality.

[0247] In summary, this invention solves several technical problems existing in the prior art by organically combining techniques such as sequential complex enzymatic hydrolysis of pepsin and trypsin, precise control of pH gradient conversion, batch-coupled enzymatic hydrolysis and membrane separation mode, and microwave-assisted pretreatment, and significantly improves the preparation efficiency and product quality of chicken thymus immunomodulatory peptides.

[0248] The preparation process of chicken thymus immunomodulatory peptides provided by this invention has good industrial applicability, mainly reflected in the following aspects:

[0249] 1. Abundant raw material sources: Chicken thymus is a by-product of broiler processing. It is abundant and inexpensive. Global broiler production exceeds 100 million tons annually, making by-product resources very rich.

[0250] 2. Reasonable process flow: The preparation process flow of the present invention is clear, the steps are reasonably connected, the operation is relatively simple, and it is easy to scale up industrial production.

[0251] 3. Moderate equipment requirements: The equipment required for this invention, such as homogenizers, pH adjustment systems, constant temperature water baths, and membrane separation equipment, are all commonly used equipment in the biopharmaceutical industry, and the investment cost is moderate.

[0252] 4. Stable product quality: By strictly controlling process parameters, the chicken thymus immunomodulatory peptide product prepared by this invention has stable quality with small batch-to-batch differences, meeting the requirements of industrial production.

[0253] 5. Cost-effectiveness advantages: Compared with traditional technologies, this invention significantly improves yield and product quality, extends equipment lifespan, and reduces production costs, demonstrating significant cost-effectiveness advantages.

[0254] 6. Broad application prospects: Immunomodulatory peptides have wide applications in health products, functional foods, pharmaceuticals, cosmetics and other fields, and market demand continues to grow.

[0255] Therefore, the technology of this invention has significant practical application value and can provide technical support for the efficient utilization and high-value-added transformation of chicken thymus resources.

[0256] This invention provides a highly efficient process for preparing immunomodulatory peptides from chicken thymus. This process employs a sequential enzymatic hydrolysis of pepsin and trypsin combined with batch-coupled membrane separation technology, achieving efficient extraction and purification of immunomodulatory peptides from chicken thymus. The main technical innovations of this invention include: sequential enzymatic hydrolysis, pH gradient conversion, batch-coupled enzymatic hydrolysis and membrane separation mode, and microwave-assisted pretreatment.

[0257] Compared with existing technologies, the process of this invention has significant advantages such as high yield, strong activity, low cost, and good stability. The prepared chicken thymus immunomodulatory peptides have a main molecular weight distribution in the range of 800-1200 Da, and can significantly enhance macrophage phagocytic activity and promote IL-6 secretion, showing good application prospects in immune system regulation. Therefore, this invention provides a new technical approach for the efficient utilization of chicken thymus resources, and has positive significance for promoting the development of the bioactive peptide industry.

[0258] Example 9: Preparation and evaluation of an immune-enhancing functional beverage

[0259] This embodiment demonstrates the application of the chicken thymus immunomodulatory peptide prepared in Example 1 in functional foods.

[0260] 1.1 Preparation process of functional beverages

[0261] Take 500 mg of the chicken thymus immunomodulatory peptide prepared in Example 1, mix it evenly with 500 mg of vitamin C, 300 mg of zinc gluconate (containing 43.5 mg of zinc), 200 mg of vitamin E, and 1 g of propolis extract. Add appropriate amounts of sweetener (8 g of fructooligosaccharides and 120 mg of steviol glycosides) and citric acid (to adjust the pH to 4.5 ± 0.2), add water to 100 mL, sterilize by microfiltration, and bottle to obtain an immune-enhancing functional beverage. The recommended daily intake is 20 mL, equivalent to 100 mg of chicken thymus immunomodulatory peptide.

[0262] 1.2 Evaluation of the immunomodulatory effects of functional beverages

[0263] Thirty sub-healthy individuals (aged 25-45 years, half male and half female) were randomly divided into an experimental group and a control group, with 15 participants in each group. The experimental group consumed 20 mL of a functional beverage on an empty stomach every morning, while the control group consumed an equal volume of a placebo beverage (with the same ingredients, appearance, and taste as the experimental group's beverage, but without the thymus immunomodulatory peptide) for 30 consecutive days. Peripheral blood samples were collected before (day 0) and after 30 days to detect changes in immune indicators. All data are expressed as mean ± standard deviation (Mean ± SD). Student's t-test was used for comparisons between groups, and P < 0.05 was considered statistically significant. The results are shown in Table 5.

[0264] Table 5. Effects of functional beverages on immune indicators in sub-healthy individuals

[0265]

[0266] *The difference was statistically significant compared to before administration (P<0.05).

[0267] The results showed that after 30 days of continuous consumption of a functional beverage containing chicken thymus immunomodulatory peptides, the experimental group showed an increase of 42.4% in NK cell activity, a 23.2% increase in the CD4+ / CD8+ ratio, a 22.1% and 23.8% increase in serum IgG and IgA levels, a 10.8% increase in peripheral blood lymphocyte transformation rate, and a 62.5% decrease in the incidence of upper respiratory tract infection. All these differences were statistically significant compared with the control group (P<0.05).

[0268] The subjects in the experimental group also reported improvements in their subjective feelings, including reduced fatigue (80%), improved sleep quality (73%), and faster physical recovery (67%). These results indicate that the functional beverage prepared in this embodiment has a significant immunomodulatory effect and can effectively enhance the immune function of sub-healthy individuals.

[0269] Example 10: Preparation of Immunomodulatory Oral Solution and Evaluation of its Anti-infective Effect

[0270] This embodiment demonstrates the application of the chicken thymus immunomodulatory peptide prepared in Example 4 in immunomodulatory agents.

[0271] 2.1 Preparation process of immunomodulatory oral solution

[0272] Take 800 mg of the chicken thymus immunomodulatory peptide prepared in Example 4, mix it with 500 mg of cordyceps polysaccharide, 300 mg of wolfberry polysaccharide, and 200 mg of β-glucan, add an appropriate amount of glycerol (as a stabilizer, 5 mL), sorbitol (as a sweetener, 10 g), and purified water to a final volume of 100 mL, adjust the pH to 6.8 ± 0.2, sterilize through a 0.22 μm filter membrane, and fill into vials to obtain the immunomodulatory oral solution. Take 10 mL each time, twice daily, for a daily intake of 160 mg of chicken thymus immunomodulatory peptide.

[0273] 2.2 Evaluation of the anti-infective effect of the immunomodulatory oral solution

[0274] 2.2.1 Evaluation of animal experiments

[0275] Sixty 8-week-old SPF-grade BALB / c mice were randomly divided into four groups: normal control, model group, low-dose group, medium-dose group, high-dose group, and positive control, with 10 mice in each group. Except for the normal control group, all other groups of mice underwent immunosuppression modeling using cyclophosphamide (80 mg / kg, intraperitoneal injection, for 3 consecutive days). Starting from day 4, the groups were administered the following treatments: low-dose group received 50 mg / kg / day of immunomodulatory peptide oral solution; medium-dose group received 100 mg / kg / day; high-dose group received 200 mg / kg / day; positive control group received thymopentin injection (10 mg / kg / day, intraperitoneal injection); and the normal control and model groups received an equal volume of physiological saline by gavage. After 14 consecutive days of treatment, all mice were infected with influenza virus H1N1 (5LD50) via intranasal instillation, and survival was observed within 14 days.

[0276] All data are expressed as mean ± standard deviation (Mean ± SD). Survival rate data were analyzed using the chi-square test or Fisher's exact test, and survival time data were analyzed using the Log-rank test. P < 0.05 was considered statistically significant. The results are shown in Table 6.

[0277] Table 6. Effects of Immunomodulatory Oral Solution on Anti-influenza Virus Infection in Immunosuppressed Mice

[0278]

[0279] *The difference was statistically significant compared to the model group (P<0.05).

[0280] The results showed that the oral immunomodulatory solution significantly improved the resistance of immunosuppressed mice to influenza virus infection, manifested as increased survival rate and prolonged average survival time, exhibiting a clear dose-dependent effect. The high-dose group (200 mg / kg / day) showed slightly better protective effects than the positive control group (thymopentin injection), which may be related to the fact that the oral administration dose (200 mg / kg / day) was much higher than the injection dose (10 mg / kg / day). Furthermore, the small molecule peptides (800-1200 Da) prepared in this invention have good bioavailability and specific immunomodulatory activity.

[0281] 2.2.2 Clinical observation study

[0282] Sixty susceptible individuals (including the elderly, patients with chronic diseases, and those with relatively weakened immune systems) were randomly divided into an experimental group and a control group, with 30 participants in each group. During the peak influenza season (December to February of the following year), the experimental group received 20 mL of an immunomodulatory oral solution daily, divided into two doses, morning and evening; the control group received an equal volume of a placebo oral solution (with the same composition, appearance, and taste as the experimental group, but without the chicken thymus immunomodulatory peptide), for 8 consecutive weeks. The occurrence of upper respiratory tract infections and influenza-like symptoms was recorded during the observation period.

[0283] Data are expressed as mean ± standard deviation (Mean ± SD). Chi-square test was used for categorical data, and Student's t-test was used for continuous data. P < 0.05 was considered statistically significant. Results are shown in Table 7.

[0284] Table 7. Preventive effect of immunomodulatory oral solutions on upper respiratory tract infections in susceptible populations.

[0285]

[0286] *Immune cell marker improvement is defined as an increase of more than 20% in NK cell activity and T lymphocyte subsets (CD4+ / CD8+ ratio) compared to baseline.

[0287] The results showed that continuous administration of the immunomodulatory oral solution significantly reduced the incidence of upper respiratory tract infections and influenza-like symptoms in susceptible individuals, alleviated infection symptoms, shortened the duration of infection, and improved immune cell indicators. These results fully demonstrate the effectiveness of the immunomodulatory oral solution prepared in this embodiment in enhancing the body's resistance to viral and bacterial infections.

[0288] Example 11: Preparation and evaluation of postoperative immune function recovery capsules

[0289] This embodiment demonstrates the application of the chicken thymus immunomodulatory peptide prepared in Example 8 in a pharmaceutical composition for promoting postoperative recovery or improving immunodeficiency.

[0290] 3.1 Preparation process of postoperative immune function recovery capsules

[0291] Take 1000 mg of the chicken thymus immunomodulatory peptide prepared in Example 8, mix it evenly with 300 mg of wolfberry polysaccharide, 200 mg of β-glucan, 100 mg of ginkgo biloba extract, 100 mg of green tea polyphenols, and 50 mg of coenzyme Q10. Add appropriate amounts of microcrystalline cellulose (50 mg) and magnesium stearate (10 mg) as excipients, and encapsulate it into No. 00 gelatin capsules. Each capsule contains 100 mg of chicken thymus immunomodulatory peptide. The recommended dosage is 1-2 capsules each time, 2-3 times a day, that is, the daily intake of chicken thymus immunomodulatory peptide is 200-600 mg.

[0292] 3.2 Clinical evaluation of postoperative immune function recovery capsules

[0293] Ninety patients scheduled for elective gastrointestinal surgery were selected and screened one week prior to surgery. They were randomly divided into three groups: a low-dose group, a high-dose group, and a control group, with 30 patients in each group. There were no statistically significant differences among the three groups in baseline characteristics such as age, sex, body mass index (BMI), nutritional status (serum albumin, prealbumin, total lymphocyte count), disease type, type of surgery, and operation time (see Table 8 for details).

[0294] Table 8. Comparison of baseline characteristics among the three groups of patients

[0295]

[0296] Starting on day 1 post-surgery, patients in the low-dose group received post-operative immune function recovery capsules at a dose of 300 mg / day (100 mg three times daily); the high-dose group received 600 mg / day (200 mg three times daily); and the control group received placebo capsules (identical in appearance to the experimental group capsules, but without chicken thymus immunomodulatory peptides) at a dose of 1-2 capsules three times daily. All patients received routine post-operative care and continued to receive the medication for 14 days.

[0297] Immune and inflammatory markers were measured 1 day before surgery and on postoperative days 1, 7, and 14, and postoperative recovery was recorded. Data are expressed as mean ± standard deviation (Mean ± SD). Chi-square tests were used for categorical data, and one-way ANOVA and LSD-t tests were used for multiple comparisons between groups for continuous data. Repeated measures ANOVA was used for repeated measures data. P < 0.05 was considered statistically significant. Results are shown in Tables 9 and 10.

[0298] Table 9. Effects of Postoperative Immune Function Recovery Capsules on Immune Indicators in Surgical Patients

[0299]

[0300] * The difference was statistically significant compared with the control group at the same time point (P<0.05). † Compared with the preoperative level, it increased, which may be related to the positive regulatory effect of immunomodulatory peptides. Literature reports that immunomodulators can produce a transient over-recovery phenomenon during the immune function recovery period, which helps to resist the risk of surgery-related infection.

[0301] Table 10. Effects of Postoperative Immune Function Recovery Capsules on Inflammatory Markers and Clinical Recovery in Surgical Patients

[0302]

[0303] *The difference was statistically significant compared with the control group (P<0.05).

[0304] The results showed that taking the postoperative immune function recovery capsules significantly promoted the recovery of immune function in surgical patients, manifested in increased NK cell activity, CD4+ / CD8+ ratio, serum IgG levels, and lymphocyte count; simultaneously, it reduced serum inflammatory factor (CRP, IL-6, TNF-α) levels, promoted gastrointestinal function recovery, reduced poor wound healing and infectious complications, and shortened hospital stay. The high-dose group (600 mg / day) was more effective than the low-dose group (300 mg / day), demonstrating a good dose-dependent effect.

[0305] Fourteen days post-surgery, the high-dose group showed NK cell activity and CD4+ / CD8+ ratio exceeding pre-operative levels, which may be related to the positive regulatory effect of immunomodulatory peptides. Previous literature has reported that immunomodulators can induce a transient overreaction during the immune function recovery period, which helps resist surgery-related infections and promote wound healing. These results fully demonstrate the effectiveness of the postoperative immune function recovery capsules prepared in this application example in promoting postoperative recovery and improving immunodeficiency.

[0306] Safety monitoring was conducted in all clinical and animal studies of the above embodiments. No significant adverse reactions were observed in the functional beverages and immunomodulatory oral solutions. A small number of subjects (less than 5%) reported mild gastrointestinal discomfort, such as bloating and mild diarrhea, which were all transient and relieved without special treatment. In the postoperative immune function recovery capsule group, 2 patients (6.7%) in the low-dose group and 3 patients (10.0%) in the high-dose group experienced mild nausea or bloating, which were significantly relieved after adjusting the medication time (taking it after meals). No serious adverse reactions were observed in any studies, and liver and kidney function tests were all within the normal range.

[0307] Compared with existing immunomodulatory products on the market, the chicken thymus immunomodulatory peptide and its application formulation prepared in this invention have the following advantages:

[0308] 1. More targeted: Compared with traditional drugs, the chicken thymus immunomodulatory peptide prepared in this invention can selectively regulate the immune system, which can enhance the weakened immune function without causing the immune system to be over-activated, thus achieving a good balance.

[0309] 2. Higher absorption rate: Through sequential complex enzymatic hydrolysis technology, the peptide products prepared by this invention have a molecular weight mainly distributed in the range of 800-1200 Da. Small molecule peptides in this range have good gastrointestinal absorption characteristics and can exert their effects without injection.

[0310] 3. Few adverse reactions: Clinical observations show that the incidence of adverse reactions to the formulations used in this invention is less than 5%, mainly manifested as mild gastrointestinal discomfort, with no serious adverse reactions observed.

[0311] 4. Comprehensive immune regulation: The product of this invention can not only enhance non-specific immunity (such as NK cell activity), but also improve specific immunity (such as T lymphocyte function and antibody levels), providing a comprehensive immune regulation effect.

[0312] 5. Wide range of applications: As can be seen from the above embodiments, the product of the present invention can be widely used in many fields such as improving the immunity of healthy people, preventing infection in susceptible people, and restoring the immune function of postoperative patients, and has good market prospects.

[0313] In summary, the preparation process and application of chicken thymus immunomodulatory peptides provided by this invention solves several technical problems existing in the prior art. The prepared products have advantages such as high yield, strong activity, good absorption, and few side effects, and have broad application prospects in the fields of functional foods, health products, and pharmaceuticals.

[0314] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A process for the preparation of an immunomodulatory peptide from chicken thymus by composite enzymatic hydrolysis, characterized in that, The method comprises the following steps: (1) raw material pretreatment: chicken thymus tissue is thawed to 2-8℃, cut into small pieces of 3-8 mm, and subjected to microwave-assisted treatment at a power of 400-600 W for 20-40 seconds, with the temperature controlled at 35-45℃, and then the tissue is mixed with water at a ratio of 1:2-1:4 (w / v) for high-speed homogenization; (2) pepsin pre-hydrolysis: the homogenate of step (1) is adjusted to pH 1.8-2.2, pepsin is added at an enzyme-substrate ratio of 1:40-1:60 (w / w), and water bath reaction is performed at 35-40℃ for 2-4 hours; (3) pH gradient conversion: the hydrolysate of step (2) is first adjusted to pH 4.8-5.2 and allowed to stand for 10-20 minutes, then adjusted to pH 6.3-6.7 and allowed to stand for 5-15 minutes, and finally adjusted to pH 7.3-7.7; (4) trypsin secondary hydrolysis: trypsin is added to the solution of step (3) at an enzyme-substrate ratio of 1:25-1:35 (w / w), and water bath reaction is performed at 40-45℃ for 3-5 hours; (5) membrane separation and purification: the hydrolysate of step (4) is separated and purified by a gradient membrane separation system, including sequentially passing through ultrafiltration membranes with pore sizes of 10 kDa and 1 kDa and a nanofiltration membrane with a pore size of 500 Da, which are first-stage ultrafiltration, second-stage ultrafiltration and third-stage nanofiltration, respectively, and the component between 1 kDa and 500 Da is collected; (6) concentration and drying: the component obtained in step (5) is concentrated and freeze-dried to obtain chicken thymus immunomodulatory peptides; The membrane separation and purification of step (5) adopts a batch-coupled enzyme hydrolysis and membrane separation mode, which connects an enzyme hydrolysis reactor with a membrane separation system to realize continuous production by reaction and separation at the same time. In the first-stage ultrafiltration of step (5), the operating parameters of the 10 kDa ultrafiltration membrane are: pressure 0.2-0.3 MPa, cross-flow rate 3.0-4.0 m / s, and temperature 28-32℃; in the second-stage ultrafiltration, the operating parameters of the 1 kDa ultrafiltration membrane are: pressure 0.25-0.35 MPa, cross-flow rate 2.5-3.5 m / s, and temperature 23-27℃; and in the third-stage nanofiltration, the operating parameters of the 500 Da nanofiltration membrane are: pressure 0.7-0.9 MPa, cross-flow rate 2.0-3.0 m / s, and temperature 18-22℃.

2. The process for the preparation of immunomodulatory peptide from chicken thymus by composite enzymatic hydrolysis according to claim 1, characterized in that, In the pepsin pre-hydrolysis of step (2), the pepsin is derived from pig gastric mucosa, has an activity of 10,000-20,000 U / mg, and has an optimum pH of 1.5-3.

0.

3. The process for the preparation of immunomodulatory peptide from chicken thymus by composite enzymatic hydrolysis as claimed in claim 1, wherein, In the trypsin secondary hydrolysis of step (4), the trypsin is derived from bovine pancreas, has an activity of 6,000-10,000 BAEE U / mg, and has an optimum pH of 6.8-8.

0.

4. The immunomodulatory peptide of chicken thymus prepared by the process according to any one of claims 1 to 3, characterized in that, The immunomodulatory peptides mainly have a molecular weight distribution in the range of 800-1,200 Da, a peptide content of ≥85% (w / w), a water content of ≤8.0%, and an ash content of ≤5.0%.

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