Immune-regulatory short peptides released from bovine colostrum powder and application thereof

Abstract

The application discloses an immune-regulating short peptide released by digestion of bovine colostrum powder and application thereof, and the amino acid sequence of the immune-regulating short peptide is LPLMI. The application adopts simulated digestion to prepare a bovine colostrum short peptide library, and completes immune target point targeting screening based on AlphaFold3 structure prediction, and verifies that the bovine colostrum can effectively assist in enhancing the immune function of the body by combining in-vivo experiments on mice and in-vitro experiments on RAW264.7 cells. The LI5 simulated digestion short peptide screened has good safety, can significantly inhibit the release of inflammatory factors and clear active oxygen, has excellent immune-regulating and anti-inflammatory and anti-oxidation functions, and can be used for preparation of immune-enhancing products.

Description

Technical Field

[0001] This application relates to the field of bioactive peptide technology, specifically to an immunomodulatory short peptide released from bovine colostrum powder after digestion and its application. Background Technology

[0002] Immune function is the core foundation for the body to resist external stress and maintain its own physiological homeostasis. Naturally derived immunomodulatory substances have attracted much attention in the fields of food and bioactive substances research due to their high safety and suitability for daily health maintenance needs. Bovine colostrum, the milk secreted by cows within 3 days after calving, is rich in various active substances such as immunoglobulins, lactoferrin, growth factors, and small molecule bioactive peptides. It is a recognized natural immunomodulatory raw material that can help enhance the body's immune function, maintain immune cell homeostasis, and improve the body's anti-inflammatory and antioxidant status, making it extremely valuable in immunomodulatory research and applications.

[0003] Current research on bovine colostrum primarily focuses on crude extracts, which suffers from issues such as unclear active components, weak targeting, and a lack of efficient preparation and screening systems. Bovine colostrum, through simulated digestion or in vitro proteolytic hydrolysis, can release small-molecule short peptides, which possess advantages such as easy absorption, good stability, and outstanding bioactivity, making them a high-quality raw material for exploring immune-enhancing functional components. However, existing technologies lack integrated solutions for targeted preparation via complex enzymatic hydrolysis, precise enrichment of short peptides, and targeted screening based on structural biology. This makes it difficult to efficiently identify core immune-enhancing short peptides in bovine colostrum, limiting the functional exploration and mechanistic research of short peptides from simulated digestion of bovine colostrum.

[0004] In the evaluation system of immunomodulatory activity, macrophages (such as the RAW264.7 cell line) are the most widely used classic model cells. Their nitric oxide (NO) production and reactive oxygen species (ROS) levels are core markers linking antioxidant capacity and immune function. Their expression levels can directly reflect the degree of immune cell activation, the intensity of inflammatory response, and the ability to maintain immune homeostasis. As a key immune messenger molecule, NO is mainly synthesized by inducible nitric oxide synthase (iNOS). Within the physiological concentration range, NO can effectively mediate pathogen clearance, participate in immune signal transduction, and finely regulate the secretion of inflammatory factors (TNF-α, IL-6, IL-1β). However, when NO is produced in excess, it can induce oxidative stress in the body, exacerbate inflammatory damage, and disrupt tissue microenvironment homeostasis. ROS (mainly including superoxide anion, hydrogen peroxide, etc.) are core metabolites of macrophages during oxidative stress, and their level changes are closely related to the regulation of immune function: low levels of ROS can promote macrophage polarization towards the M1 type and enhance the body's immune defense capabilities by activating key signaling pathways such as TLR / NF-κB and MAPK; however, if ROS continues to accumulate, it will trigger lipid peroxidation, cause DNA damage and cell dysfunction, thereby inhibiting the immune response and amplifying the inflammatory response.

[0005] Numerous basic studies have confirmed that inhibiting excessive NO release and abnormal ROS accumulation is a key in vitro evaluation criterion for assessing whether bioactive substances can exert anti-inflammatory, antioxidant, and immunomodulatory functions. Their mechanism of action is primarily related to the inhibition and balance of the MAPK / NF-κB signaling pathway. Several studies have confirmed that NO and ROS levels in RAW264.7 cells are closely related to NF-κB, MAPK, and other signaling pathways and inflammatory responses. These studies provide strong support for using NO and ROS as core indicators for evaluating macrophage immune activity and lay a theoretical foundation for the evaluation and mechanistic study of bovine colostrum peptides. Summary of the Invention

[0006] This application provides an immunomodulatory short peptide released from bovine colostrum powder after digestion and its application. This application clarifies the immune-enhancing effects of bovine colostrum and the short peptides obtained from simulated digestion. A bovine colostrum short peptide library is constructed through simulated digestion, and targeted screening of immune targets is completed based on AlphaFold3 structure prediction. Functional verification is achieved through in vivo experiments in mice and in vitro experiments in RAW264.7 cells, providing a scientific basis and technical support for the research and evaluation of immunomodulatory short peptides derived from bovine colostrum.

[0007] An immunomodulatory short peptide, wherein the amino acid sequence of the immunomodulatory short peptide is TVFP or LPLMI.

[0008] This application also provides a bovine colostrum powder, which produces the immunomodulatory short peptide after digestion or in vitro simulated digestion.

[0009] This application also provides the use of the immunomodulatory short peptide or the bovine colostrum powder in the preparation of health foods for enhancing immunity.

[0010] This application also provides a health food for enhancing immunity, comprising an active ingredient and a food science-acceptable carrier or excipient, wherein the active ingredient is the immunomodulatory short peptide or the bovine colostrum powder.

[0011] The carrier or excipients include solvents, binders, fillers, sugars, and sweeteners, which can be selected according to different needs. The type of health food can be any of the following: tablets, capsules, oral liquids, compressed candies, solid beverages, pills, granules, decoctions, pastes, syrups, drop pills, or tea bags. The amount and proportion of related excipients added can be adjusted according to actual needs.

[0012] Optionally, the enhanced immunity includes at least one of the following: (1) Maintain stable body weight and improve weight fluctuations caused by abnormal immune status; (2) Increase the indices of immune organs such as the spleen and thymus, and maintain the normal development and morphology of immune organs; (3) Improves cyclophosphamide-induced immunosuppression and spleen tissue atrophy, and maintains the integrity of spleen tissue structure and normal morphology; (4) Increase the number of peripheral blood leukocytes, lymphocytes, monocytes and neutrophils, and improve the level of immune cells in the body; (5) Significantly increases the levels of serum IgG, IgA, and IgM immunoglobulins, enhancing the body's humoral immune function.

[0013] This application also provides the use of the immunomodulatory short peptide in the preparation of a formulation for enhancing the antioxidant capacity of RAW264.7 cells.

[0014] Optionally, the antioxidant capacity includes: inhibiting LPS-induced excessive NO production in RAW264.7 cells; and scavenging intracellular ROS to reduce ROS levels.

[0015] This application also provides a method for preparing the bovine colostrum powder, comprising: (1) Collect bovine colostrum within 24 hours after calving and freeze it at -20 ℃ to -18 ℃ within 10 min; (2) The bovine colostrum frozen in step (1) is slowed down at low temperature; (3) The slowed bovine colostrum after step (2) is mixed with food production water for hydration. The amount of food production water added is 0.5 to 2.5 times the volume of bovine colostrum. The hydration treatment temperature is controlled at 0 to 10 ℃ and the hydration time is 30 to 90 min. (4) After the bovine colostrum was hydrated in step (3), it was successively degreased at low temperature, sterilized at low temperature, concentrated at low temperature and freeze-dried to obtain the bovine colostrum powder.

[0016] The bovine colostrum powder of this application is prepared from bovine colostrum collected within 24 hours after calving by cows through a specific processing technology, and has better immune-enhancing effects compared with similar products.

[0017] Optionally, in step (1), before collection, the milking equipment, milk collection containers, and operators are cleaned and disinfected. During the collection process, one cow per bucket or separate collection is used, and the colostrum from different cows is not mixed to avoid dilution or interference between the compositional differences of colostrum from different individuals.

[0018] During collection, milk containing mastitis, blood, abnormal color, and abnormal odor is removed. The collected bovine colostrum is then graded for quality, with initial screening preferably conducted using a colostrum refractometer or equivalent detection method.

[0019] After collection, bovine colostrum is transferred to a frozen environment for storage within a short period of time. Preferably, it is stored in a frozen environment below ~20 °C within 10 minutes after collection. Rapid freezing can reduce the uncontrolled degradation of colostrum proteins by endogenous proteases and microbial enzymes, and reduce changes in protein composition caused by temporary storage at room temperature, thereby helping to retain protein precursors that can be digested and released into target peptides.

[0020] The bovine colostrum collected in this step must have a protein content of no less than 5.0%, an immunoglobulin G content of no less than 20.0 mg / mL, a fat content of no less than 4.5%, an acidity of no more than 40 °T, and pass the antibiotic residue test. It must then be frozen and stored.

[0021] Optionally, in step (2), the low-temperature slowing process adopts a jacketed temperature control method, with the jacketed water temperature controlled at 45~60℃, while the temperature of the bovine colostrum material is controlled not to exceed 4℃, and the slowing time is 12~24 h.

[0022] Further preferably, the retardation time is 12-24 h, more preferably 16-20 h.

[0023] By using the above-mentioned low-temperature thawing method, frozen bovine colostrum can be fully thawed while avoiding local overheating and prolonged exposure to room temperature, reducing protein denaturation, aggregation, and uncontrolled degradation, which is beneficial to maintaining the integrity of milk-derived proteins and immunoglobulin precursors in colostrum.

[0024] Optionally, in step (3), the amount of water added for food production is 1.0 to 2.0 times the volume of bovine colostrum, the hydration treatment temperature is controlled at 2 to 8 ℃, and the hydration time is 45 to 75 min.

[0025] More preferably, the amount of water added for food production is 2.0 times the volume of bovine colostrum, the hydration treatment temperature is controlled at 2~8 ℃, and the hydration time is 60 min.

[0026] Hydrated bovine colostrum systems exhibit reduced viscosity and improved protein dispersibility. During defatting, the sedimentation and entrainment loss of casein micelles, denatured protein aggregates, and protein-fat complexes are reduced, which is beneficial for retaining the milk protein precursors required for the subsequent release of characteristic peptides through simulated in vitro digestion.

[0027] Optionally, the low-temperature degreasing includes: The slowed bovine colostrum is preheated before entering the centrifugation process. The preheating temperature is controlled at 37~43 ℃, preferably 38~42 ℃. The centrifugation speed is controlled at 6000~8000 r / min, preferably 7000~7500 r / min. Through the above-mentioned low-temperature centrifugation method, some fat, impurities, or unstable components can be removed.

[0028] Optionally, the low-temperature membrane sterilization includes: After centrifugation, the bovine colostrum enters the membrane sterilization step. During membrane sterilization, the material temperature is controlled at 35~45℃, preferably 38~42℃; the inlet pressure is controlled at 0.35~0.50 MPa, the outlet pressure at 0.12~0.25 MPa, and the permeate-side pressure at 0.10~0.20 MPa. This low-temperature membrane sterilization method reduces the microbial load in the bovine colostrum material and minimizes the thermal damage to immunoglobulins, whey proteins, casein, and other milk-derived bioactive protein precursors caused by traditional high-temperature sterilization.

[0029] Optionally, the low-temperature concentration includes: After membrane sterilization, the bovine colostrum enters the low-temperature concentration step. During concentration, the feed temperature is controlled at 38~43 ℃, preferably 40~42 ℃; the inlet pressure is controlled at 0.35~0.50 MPa, the outlet pressure at 0.12~0.25 MPa, and the feed pressure at 0.12~0.22 MPa. The multi-stage concentration pressure is controlled at 0.30~0.40 MPa. The final concentration endpoint, based on total solids content, results in a total solids content of 16%~20%, preferably 17%~19%. This low-temperature concentration method reduces the moisture content of the material, improves subsequent freeze-drying efficiency, reduces heat load, and minimizes protein thermal denaturation and irreversible aggregation.

[0030] Optionally, the freeze-drying includes: Concentrated bovine colostrum was pre-frozen and then freeze-dried under vacuum. The pre-freezing temperature was controlled between -32°C and -40°C. During freeze-drying, the material temperature was controlled below 45°C, preferably below 40°C; the vacuum degree inside the freeze-drying chamber was controlled below 30 Pa; and the freeze-drying time was 36–48 hours. Freeze-drying bovine colostrum powder avoids the thermal damage to colostrum proteins caused by the high inlet air temperature during spray drying. The freeze-dried bovine colostrum powder retains a high degree of milk-derived proteins, immunoglobulins, and other protein precursors.

[0031] Compared with the prior art, this application has at least one of the following beneficial effects: (1) This invention uses simulated digestion to prepare a bovine colostrum short peptide library. Based on AlphaFold3 structure prediction, immune target screening is completed. Combined with in vivo experiments in mice and in vitro experiments in RAW264.7 cells, it is confirmed that bovine colostrum can effectively help enhance the body's immune function. The TP4 and LI5 simulated digestion short peptides obtained by screening have good safety, can significantly inhibit the release of inflammatory factors and clear reactive oxygen species, and have excellent immunomodulatory, anti-inflammatory and antioxidant functions.

[0032] (2) The short peptides in this application are derived from bovine colostrum powder collected within 24 hours. Through improvements in the collection process, the bovine colostrum powder retains a high degree of milk protein, immunoglobulin and other protein precursors. After in vivo digestion or simulated digestion, it is decomposed into an active peptide spectrum mainly composed of small molecule peptides of 3 to 10 amino acids. Compared with other bovine colostrum powders on the market, it has a significant immune enhancement effect. Attached Figure Description

[0033] Figure 1 This is the experimental planning diagram for the mouse experiment in Example 2; Figure 2 The figure shows the in vivo verification results of the immune enhancement effect of bovine colostrum on mice in Example 2 (A in the figure is the change in mouse body weight, B is the result of mouse spleen index, and C is the result of mouse thymus index). Figure 3 shows the morphological diagram of spleen tissue in each group of mice after modeling in Example 2; Figure 4 shows the effect of bovine colostrum on the count of five blood cells in mouse serum in Example 2 (A represents the number of white blood cells, B represents the number of neutrophils, C represents the number of lymphocytes, D represents the number of monocytes, and E represents the number of eosinophils). Figure 5 The figure shows the effect of bovine colostrum on the serum immunoglobulin content of mice in Example 2 (A in the figure represents IgM content, B represents IgG content, and C represents IgA content). Figure 6 shows the length distribution of short peptides in bovine colostrum after simulated digestion in Example 3. Figure 7 shows the molecular weight and abundance distribution of peptides with different amino acid numbers in Example 3; Figure 8 This is the first-order mass spectrum of TP4; Figure 9 This is a diagram showing the molecular docking of TP4 with the target protein TRAF6. Figure 10 This is the secondary mass spectrum of TP4; Figure 11 This is the first-order mass spectrum of LI5; Figure 12 This is a diagram showing the molecular docking of LI5 with the target protein TRAF6. Figure 13 The mass spectrum of LI5 is shown below. Figure 14 shows the effect of different bovine colostrum-simulated digestion short peptides on the viability of RAW264.7 cells in Example 4; Figure 15 shows the effect of different bovine colostrum-simulated digestion short peptides on LPS-induced NO production in RAW264.7 cells in Example 4; Figure 16 shows the effect of bovine colostrum-simulated digestion of short peptides TP4 and LI5 on intracellular ROS levels in RAW264.7 cells in Example 4. Detailed Implementation

[0034] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0036] Example 1: Preparation of bovine colostrum powder (1) Bovine colostrum collection Colostrum should be collected from cows within 24 hours of calving. Before collection, milking equipment, milk collection containers, and personnel should be cleaned and disinfected. During collection, one cow per bucket or separate collection methods should be used; colostrum from different cows should not be mixed to avoid dilution or interference due to differences in composition between individual cows.

[0037] During collection, milk containing mastitis, blood, abnormal color, and abnormal odor is removed. The collected bovine colostrum is then graded for quality.

[0038] After collection, the samples should be stored in a freezer at ~20 °C or below within 10 minutes. Rapid freezing can reduce the uncontrolled degradation of colostrum proteins by endogenous proteases and microbial enzymes, and reduce changes in protein composition caused by temporary storage at room temperature, thus helping to preserve protein precursors that can be digested and released into target peptides.

[0039] The final collected bovine colostrum must have a protein content of no less than 5.0%, an immunoglobulin G content of no less than 20.0 mg / mL, a fat content of no less than 4.5%, an acidity of no more than 40 °T, and pass the antibiotic residue test. It must then be frozen for storage.

[0040] (2) Low temperature slowing Frozen bovine colostrum was placed in a thawing tank for thawing. The thawing process employed a jacketed temperature control method, with the jacket water temperature maintained at 50 ℃ and the bovine colostrum material temperature controlled to not exceed 4 ℃. The thawing time was 20 h.

[0041] (3) Hydration The slowed bovine colostrum was mixed with food production water, with the amount of water added being 2.0 times the volume of the bovine colostrum. The hydration treatment temperature was controlled at 5±2℃; the hydration time was 60 min.

[0042] (4) Low temperature degreasing After slowing down, the bovine colostrum is preheated before centrifugation. The preheating temperature is controlled at 40±2 ℃, and the centrifugation speed is controlled at 7000 r / min. Low-temperature centrifugation can remove some fat, impurities, or unstable components.

[0043] (5) Low temperature membrane sterilization After centrifugation, the bovine colostrum enters the membrane sterilization process. During membrane sterilization, the material temperature is controlled at 40±2℃; the inlet pressure is controlled at 0.4 MPa, the outlet pressure is controlled at 0.2 MPa, and the permeate side pressure is controlled at 0.15 MPa.

[0044] (6) Low-temperature concentration After membrane sterilization, the bovine colostrum enters the low-temperature concentration stage. During concentration, the feed temperature is controlled at 40±2 ℃; the inlet pressure is controlled at 0.4 MPa, the outlet pressure at 0.2 MPa, and the feed pressure at 0.2 MPa. The multi-stage concentration pressure is controlled at 0.35 MPa. The concentration endpoint, based on total solids content, shows that the total solids content of the concentrated material is 18%.

[0045] (7) Freeze-drying The concentrated bovine colostrum material was pre-frozen and then freeze-dried under vacuum. The pre-freezing temperature was controlled between -32 ℃ and -40 ℃. During the freeze-drying process, the material temperature was controlled below 40 ℃; the vacuum degree inside the freeze-drying chamber was controlled below 30 Pa; and the freeze-drying time was 40 hours to obtain bovine colostrum powder.

[0046] Example 2: In vivo validation of the immune-enhancing effect of bovine colostrum This embodiment first verified the immune-enhancing effect of bovine colostrum in mice using five core indicators: mouse body weight, immune organ index, spleen tissue morphology, peripheral blood cell count, and serum immunoglobulin content. This provides an in vivo basis for subsequent in vitro screening of simulated digestive short peptides.

[0047] The specific implementation is as follows: 1. Experimental materials and animals SPF-grade C57BL / 6 male mice, weighing 18-22 g, were selected and acclimatized for one week. The bovine colostrum powder used in the experiment was prepared in Example 1, and other physiological saline, cyclophosphamide, and levamisole were all commercially available compliant products. The experimental instruments included an electronic analytical balance, a high-speed refrigerated centrifuge, a fully automated hematology analyzer, an enzyme-linked immunosorbent assay (ELISA) reader, and a stereomicroscope.

[0048] 2. Experimental grouping and treatment See also Figure 1 The treatment procedure shown involves randomly dividing mice into 4 groups of 10 mice each, with the following grouping and treatment: (1) Normal control group: Daily gavage with normal saline, followed by intraperitoneal injection of normal saline for three consecutive days after 14 days of intervention; (2) Model group: Daily oral administration of normal saline, followed by intraperitoneal injection of cyclophosphamide (80mg / kg) for three days after 14 days of continuous intervention. (3) Levamisole group (positive drug group): daily oral administration of levamisole (25mg / kg), followed by intraperitoneal injection of cyclophosphamide (80mg / kg) for three consecutive days after 14 days of continuous intervention. (4) Bovine colostrum group: Bovine colostrum powder (150 mg / kg, prepared in Example 1) was administered by gavage daily, followed by intraperitoneal injection of cyclophosphamide (80 mg / kg) for three consecutive days after 14 days of continuous intervention.

[0049] 3. Detection Indicators and Methods (1) Weight monitoring: Weigh and record the weight of mice at fixed times every day, and record and analyze the trend and fluctuation of weight changes in each group of mice.

[0050] (2) Determination of immune organ index: On day 24 of the experiment, mice were euthanized by dislocation under anesthesia. The spleen and thymus were aseptically separated, rinsed with pre-cooled physiological saline, and dried with filter paper. The wet weight was measured using an electronic balance. The immune organ index was calculated according to the formula: Immune organ index (mg / g) = Immune organ wet weight (mg) / Mouse body weight (g).

[0051] (3) Observation of spleen tissue morphology: Spleen tissue of mice in each group was taken and observed under a stereomicroscope. The size, color, texture and edge morphology of the spleen were recorded, and photos were taken and the differences between the groups were compared.

[0052] (4) Peripheral blood cell count: Blood was collected from the orbital venous plexus of mice and placed in an EDTA-K2 anticoagulant tube. The tube was gently inverted and mixed. The absolute number of white blood cells (WBC), lymphocytes (LY), monocytes (MONO), and neutrophils (NEUT) was detected using a fully automated blood cell analyzer.

[0053] (5) Serum immunoglobulin detection: Mouse whole blood was left to stand at room temperature for 30 min, centrifuged at 3000 r / min for 15 min, and the upper serum layer was separated; the ELISA kit was used strictly in accordance with the instructions to detect the content of IgG, IgA and IgM in the serum. The absorbance at 450 nm was read by the enzyme-linked immunosorbent assay (ELISA) reader, and the corresponding concentrations were calculated according to the standard curve.

[0054] 4. Experimental Results The results of mouse body weight, spleen index, and thymus index are as follows: Figure 2 As shown in the figure, A represents the detection results of mouse body weight change, B represents the detection results of mouse spleen index, and C represents the detection results of mouse thymus index. express P <0.01 (the difference is highly significant) express P <0.0001 (the difference is extremely significant). Figure 2 The results from the study showed that, compared with the model group, the weight of mice in the bovine colostrum intervention group remained stable throughout the process, with a significantly reduced fluctuation range, effectively improving the abnormal weight loss caused by cyclophosphamide. Figure 2 The results of B and C showed that the spleen index and thymus index of mice in the bovine colostrum intervention group were significantly increased, approaching the level of the positive control group, which can effectively maintain the normal development of immune organs.

[0055] Spleen volume of mice in each treatment group compared to the following figures: Figure 3 As shown, the results indicated that the spleen volume of mice in the model group was significantly reduced, the texture became softer, and the color became dull, exhibiting typical immunosuppressive atrophy; the spleen volume, morphology, and color of the bovine colostrum group showed significant improvement compared to the model group, and significantly reversed cyclophosphamide-induced spleen tissue damage.

[0056] The numbers of leukocytes, neutrophils, lymphocytes, monocytes, and eosinophils in the peripheral blood of mice in each treatment group are as follows: Figure 4 As shown in the figure, A represents the number of white blood cells, B represents the number of neutrophils, C represents the number of lymphocytes, D represents the number of monocytes, and E represents the number of eosinophils. express P <0.1 (the difference is statistically significant) express P <0.01 (the difference is highly significant) express P <0.0001 (the difference was extremely significant). The results showed that, compared to the model group, the number of peripheral blood leukocytes in the bovine colostrum group mice ( Figure 4 (A) Neutrophils ( Figure 4 B), lymphocytes ( Figure 4 (C) and eosinophils ( Figure 4 The number of E cells in the mice was significantly increased, with effects comparable to those in the levamisole positive control group, effectively repairing the deficiency in the number of immune cells in immunosuppressed mice.

[0057] Comparison of serum IgM, IgG, and IgA levels in mice of different treatment groups (e.g.) Figure 5 As shown in the figure, A represents the serum IgM content, B represents the serum IgG content, and C represents the serum IgA content. express P <0.1 (the difference is statistically significant) express P <0.01 (the difference is highly significant) express P <0.0001 (the difference was extremely significant). The results showed that serum IgM in mice in the bovine colostrum group ( Figure 5 Medium A), IgG ( Figure 5 middle B), IgA ( Figure 5 The content of C in the medium was significantly increased compared with the model group, and was comparable to or even better than the positive drug control group, indicating a significant enhancement of humoral immune function.

[0058] In summary, the bovine colostrum powder prepared in this application can exert a significant immune-enhancing effect from multiple dimensions, including the morphology and function of immune organs, the number of immune cells, and humoral immune responses.

[0059] Example 3: Bovine colostrum simulated digestion and immune enhancement short peptide targeted screening This embodiment establishes a complete process for simulated digestion of bovine colostrum, short peptide enrichment, and structural biology targeted screening. Based on the in vivo immune-enhancing effect of bovine colostrum in Example 2, it further explores its core active short peptides. Details are as follows: 1. In vitro simulated digestion of bovine colostrum A quantitative sample of bovine colostrum was taken, and a static in vitro digestion model was strictly followed according to the INFOGEST 2.0 standard. Simulated saliva (SSF), simulated gastric juice (SGF), and simulated intestinal juice (SIF) were added sequentially to carry out a three-step continuous in vitro simulated digestion from the mouth to the stomach to the intestine. The reaction temperature was kept constant at 37 ℃ throughout the process.

[0060] First, bovine colostrum powder sample (prepared in Example 1) was mixed with simulated saliva at a 1:1 (mass ratio). CaCl2 was added to a final concentration of 1.5 mmol / L, and salivary amylase was added to a final activity of 75 U / mL. The pH of the system was adjusted to 7.0, and the mixture was incubated at a constant temperature with shaking for 2 min to complete oral phase digestion. Next, the oral digested bolus was mixed with simulated gastric juice at a 1:1 (volume ratio) equal volume. CaCl2 was added to a final concentration of 0.15 mmol / L, pepsin to a final activity of 2000 U / mL, and gastric lipase to a final activity of 60 U / mL. The pH of the system was adjusted to 3.0 using hydrochloric acid, and the mixture was incubated at 37 ℃ with continuous shaking for 2 h to complete gastric phase digestion. Then, the gastric digested chyme was mixed with simulated intestinal juice at a 1:1 (volume ratio). CaCl2 was added to a final concentration of 0.6 mmol / L, bile salts to a final concentration of 10 mmol / L, and trypsin was added to a final concentration of 100 U / mL based on trypsin activity. The pH of the system was adjusted to 7.0 using sodium hydroxide, and the mixture was incubated at 37 ℃ with shaking for 2 h to complete gastric phase digestion. Intestinal phase digestion was completed by constant temperature shaking incubation for 2 h. After digestion, the digestive enzymes were immediately inactivated, and the supernatant was collected by centrifugation. Small short peptides with 2-10 amino acid residues were screened and enriched using ultrafiltration membrane separation technology, while large proteins and long-chain polypeptides were removed. The obtained short peptide samples were stored at low temperature for later use.

[0061] 2. Short peptide enrichment and sequence purification This study pretreated peptides with molecular weights less than 10 kDa by reduction, alkylation, and desalting. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) in data-dependent acquisition mode was used for sample detection. The raw mass spectrometry data were then analyzed using de novo sequencing with PEAKS software. Details are as follows: In this experiment, 2 mg of bovine colostrum in vitro digest sample was weighed, dissolved thoroughly in pure water, and then centrifuged at 12,000 rpm for 10 min using a 10 kDa ultrafiltration tube for washing and fractionation. Peptide fractions with a molecular weight less than 10 kDa were collected, and the peptide concentration was measured using a Nanodrop instrument. Subsequently, 100 μg of low molecular weight peptide sample was accurately taken, and dithiothreitol was added to adjust the final concentration of the system to 10 mmol / L. The sample was then subjected to reduction treatment at 56 ℃ for 1 h, followed by the addition of iodoacetamide to achieve a final concentration of 20 mmol / L. The alkylation reaction was carried out at room temperature in the dark for 40 min, and an equal amount of dithiothreitol was added again to neutralize the unreacted iodoacetamide in the system. The modified sample was desalted using a C18 stage-tip and then vacuum dried at 45 ℃ for later use.

[0062] For liquid chromatography analysis of samples, a pre-column with an inner diameter of 150 μm and a length of 50 mm, filled with 3 μm Reprosil-Pur120C18-AQ packing material, and an analytical column with an inner diameter of 150 μm and a length of 170 mm, filled with 1.9 μm Reprosil-Pur120C18-AQ packing material were used. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 80% acetonitrile solution containing 0.1% formic acid. The flow rate was set at 600 nL / min, and the overall analysis time for a single sample was 66 min. The gradient elution program was as follows: 0 min to maintain 4% B, 2 min to adjust to 8% B, 35 min to increase to 28% B, 55 min to increase to 40% B, 56 min to rapidly increase to 95% B and maintain it until 66 min.

[0063] Mass spectrometry detection employs a data-dependent acquisition mode, with the first-stage mass spectrometer resolution set to 70,000 and the automatic gain control target value set to 3 × 10⁻⁶. 6 Maximum ion implantation time: 100 ms; mass-to-charge ratio scan range: 100–1500 m / z; secondary mass spectrometry resolution: 17500 m / z; automatic gain control target value: 1 × 10⁻⁶ m / z. 5 The maximum ion implantation time was 50 ms, and 20 dominant precursor ions were selected for fragmentation detection in a single run. The normalized collision energy was set to 28. Data retrieval and analysis were performed using cysteine ​​carbamoyl methylation as a fixed modification, and methionine oxidation and N-terminal acetylation of peptides as variable modifications. A non-specific enzyme digestion mode was used. The mass deviation of the primary mass spectrometry was 20 ppm, and the mass deviation of the secondary fragment ions was 0.02 Da.

[0064] The experiment employed ultrafiltration combined with liquid chromatography to screen for short peptides with 2-10 amino acids, while removing large polypeptides and incompletely hydrolyzed proteins. Invalid sequences with modification tags were also removed, ultimately constructing a pure short peptide library with 11,300 valid short peptide sequences. The distribution of short peptides of different lengths within this library is shown in the figure below. Figure 6 As shown, by Figure 6 The results of short peptide sequence distribution show that the short peptides obtained after in vitro simulated digestion of bovine colostrum are mainly small molecule peptides composed of 3 to 10 amino acids. The peptide length distribution is relatively concentrated, which can meet the requirements of subsequent experiments related to immune target screening and bioactivity verification.

[0065] 3. Immune target screening based on AlphaFold3 Five key immune regulatory proteins—IKBKB, JAK1, CD247, BTK, and TRAF6—were selected. Target sequences and MSA information were downloaded from AlphaFold DB, and a JSON input file was constructed. AlphaFold3 was used to predict the three-dimensional structure of the peptide-target complex. A threshold of ipTM > 0.75 was set, and 573 high-confidence models were selected. Following the de novo binder scoring process, the structure was evaluated using Python pandas and Bio.PDB tools.

[0066] The molecular weight and abundance distribution of peptides with different amino acid numbers are shown in Figure 7. The results show that there are significant differences in molecular weight and mass spectrometry abundance among peptides of different lengths, and high abundance peptides are highly consistent with peptides with high target binding activity. Based on the results of mass spectrometry abundance, structure scoring and molecular docking evaluation, nine candidate short peptides with both high abundance and excellent immunomodulatory potential were finally screened: HK8 (SEQ ID NO: 1), AT4 (SEQ ID NO: 2), TP9 (SEQ ID NO: 3), NP5 (SEQ ID NO: 4), MP4 (SEQ ID NO: 5), RE8 (SEQ ID NO: 6), LI5 (SEQ ID NO: 7), TP4 (SEQ ID NO: 8), and IP4 (SEQ ID NO: 9) (Table 1).

[0067] Table 1. Structures and docking characterization parameters of nine candidate short peptides

[0068] Figure 8~ Figure 13 The sequence identification and binding mode characterization results of the preferred short peptides TP-4 and LI-5 are presented. Figure 8 and... Figure 11 The images show the first-order mass spectra of TP-4 and LI-5, respectively. Figure 10 and Figure 13 The secondary mass spectra of TP-4 and LI-5 are shown in Figure 9, used to confirm the molecular weight and amino acid sequence of the peptides. The results show that the molecular weight of TP-4 is 463.25 and the amino acid sequence is TVFP, while the molecular weight of LI-5 is 586.45 and the amino acid sequence is LPLMI. Figure 12 The diagram shows the molecular docking structure of the two peptides with the target protein TRAF6, which is used to characterize the spatial binding mode between the short peptide and the target protein.

[0069] Nine candidate short peptides obtained through screening were prepared using the Fmoc solid-phase synthesis method: CTC-2cl resin was used as the solid-phase support, and a TBTU+DIEA condensation system was employed. The process involved resin swelling, amino acid cyclic coupling, piperidine deprotection, TFA cleavage, and ether precipitation to obtain crude peptides, which were then purified by high-performance liquid chromatography to obtain high-purity polypeptides. Alternatively, the synthesis can be directly commissioned to a synthetic institution to provide samples for subsequent immunofunctional validation experiments.

[0070] Example 4: In vitro evaluation of the immune-enhancing effect of short peptides obtained from bovine colostrum-mimicked digestion Based on the in vivo effects of Example 2 and the screening results of Example 3, this example uses RAW264.7 mouse macrophages as a model to complete the in vitro functional verification of candidate short peptides through three indicators: cell viability, NO production rate, and ROS level.

[0071] Specifically as follows: 1. Cell Culture RAW264.7 mouse macrophage cell line was purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee. It was cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. Cells were passaged daily, and cells in the logarithmic growth phase and in good condition were used for subsequent experiments.

[0072] 2. Cell viability assay (CCK-8 assay) RAW264.7 cells were cultured at a rate of 1×10⁻⁶. 4 Cells were seeded at a density of 10 cells / well in 96-well cell culture plates, with 6 replicates per group, and cultured adherently in an incubator for 12 hours. The following groups were included: a normal control group, an LPS model group, a dexamethasone positive control group, and 9 candidate short peptide intervention groups (10, 30, 100, and 200 μM). Except for the normal control group, all other groups received 1 μg / mL LPS (lipopolysaccharide) to induce an inflammation model, while the short peptide intervention groups received the corresponding concentration of short peptide. Cultures were continued for 24 hours. 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C in the dark for 30 minutes. The absorbance (OD value) at 450 nm was measured using a microplate reader.

[0073] Calculate cell viability using the formula: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0074] 3. Detection of NO production in cells Following the above method of inoculation and grouping, after 4 hours of intervention, 1 μg / mL LPS was added, and the cells were cultured for another 24 hours. The cell culture supernatant was then collected. Using a NO detection kit, strictly following the instructions, the supernatant and chromogenic agent were mixed thoroughly, and the reaction was carried out at room temperature in the dark for 30 minutes. The absorbance at 540 nm was measured using a microplate reader, and the relative NO production level was calculated based on the standard curve.

[0075] 4. Intracellular ROS detection (fluorescent probe method) RAW264.7 cells were seeded in confocal culture dishes and cultured adherently for 12 h before being divided into groups for intervention. After group treatment, the culture medium was discarded, and DCFH~DA fluorescent probe at a final concentration of 10 μmol / L was added. The cells were incubated at 37°C in the dark for 30 min. The cells were washed three times with serum-free culture medium to remove unloaded probes. Fluorescence images were observed and captured using a laser confocal microscope to reflect intracellular ROS levels.

[0076] 5. Experimental Results The effects of various bovine colostrum-mimicking digestive peptides on RAW264.7 cell viability are as follows: Figure 14 As shown, the results indicated that the cell viability of all nine candidate short peptides remained above 80% within the concentration range of 0–200 μM, and they showed no significant toxicity to RAW264.7 cells, demonstrating good biosafety.

[0077] The effects of various bovine colostrum-simulated digestion short peptides on LPS-induced NO production in RAW264.7 cells are shown in Figure 15 (A~I in the figure represent short peptides HK-8, AT-4, LI-5, MP-4, IP-4, TP-4, NP-5, TP-9, and PE-8, respectively). The results showed that TP4 and LI5 short peptides could inhibit LPS-induced excessive NO production in RAW264.7 cells in a dose-dependent manner. At a concentration of 200 μM, the inhibitory effect was close to that of the dexamethasone positive control group, and the anti-inflammatory effect was significant.

[0078] Figure 16 shows the effects of bovine colostrum-mimicked digestion peptides TP4 and LI5 on intracellular ROS levels in RAW264.7 cells. The results show that LI5 peptides at concentrations of 10, 100, and 200 μM can effectively scavenge intracellular ROS; TP4 peptides at concentrations of 100 and 200 μM can significantly reduce ROS levels, but have no significant effect at a concentration of 10 μM. Both peptides have excellent antioxidant capacity.

[0079] In summary, in vivo experiments have confirmed that the bovine colostrum of this application can stabilize mouse body weight, increase immune organ indices, repair spleen morphology, increase the number of peripheral blood immune cells, and enhance serum immunoglobulin levels, exhibiting a comprehensive immune-enhancing effect. In vitro screening and validation show that TP4 and LI5 are the core short peptides with the best immunomodulatory, anti-inflammatory, and antioxidant activities among the short peptides mimicking bovine colostrum digestion, and can be used as novel immunomodulatory functional peptides in the fields of biopharmaceuticals and health foods.

[0080] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. An immunomodulatory short peptide, characterized in that, The amino acid sequence of the immunomodulatory short peptide is LPLMI.

2. The use of the immunomodulatory short peptide as described in claim 1 in the preparation of health food products that help enhance immunity.

3. A health food product that helps enhance immunity, characterized in that, It includes an active ingredient and a food-grade acceptable carrier or excipient, wherein the active ingredient is the immunomodulatory short peptide as described in claim 1.

4. The use of the immunomodulatory short peptide as described in claim 1 in the preparation of a formulation for enhancing the antioxidant capacity of RAW264.7 cells.

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