Enzymolysis preparation method of bovine colostrum peptide with high immunocompetence, active peptide and application of active peptide

Abstract

The invention discloses an enzymolysis preparation method of high-immunocompetence bovine coloctrum peptide, active peptide and application thereof, and belongs to the field of biotechnology and functional foods.The enzymolysis preparation method is characterized by comprising the steps that bovine coloctrum is degreased, trehalose is added into obtained supernate, first stirring is conducted, and first mixed liquid is obtained; wherein the supernate comprises whey protein and Ig; adding medium protease into the first mixed solution for hydrolysis to obtain enzymatic hydrolysate; wherein the enzymatic hydrolysate comprises a mixture of bovine colostrum hydrolyzed peptides, the Ig activity retention rate is larger than or equal to 90% through the targeted protection and directional enzymolysis technology, the beta-lactoglobulin peptide with the synergistic immune enhancement effect is obtained, the Ig and small molecule peptides in the product have the synergistic effect, the body immune regulation capacity and the intestinal immune function are remarkably improved, the immunity of the human body is improved, and the immunity of the human body is improved. The method can be widely applied to the fields of infant formulas, postoperative nutrition, functional drinks and the like.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and functional food processing, specifically to an enzymatic hydrolysis preparation method for highly immunomodulatory bovine colostrum peptides and its application, particularly to the preparation of bovine colostrum products that simultaneously retain highly active Ig and functional peptides through targeted enzymatic hydrolysis and activity protection technology. Background Technology

[0002] Bovine colostrum is the milk secreted by cows within 72 hours after calving. It is rich in bioactive components such as immunoglobulins (especially Ig) and whey proteins (such as β-lactoglobulin and α-lactalbumin). It has important functions such as immune regulation and promoting growth and development, and has broad application prospects in the field of functional foods and nutritional supplements.

[0003] However, existing technologies for preparing bovine colostrum peptides have the following key problems:

[0004] Ig activity is easily compromised: Traditional enzymatic hydrolysis processes often employ broad-spectrum proteases such as neutral and alkaline proteases. While hydrolyzing whey protein, these enzymes non-specifically cleave the Fc region of Ig, causing Ig to lose its antibody activity and immunomodulatory function, with an activity retention rate typically ≤40%. Studies have confirmed that the immunomodulatory activity of Ig depends on the intact Fc region structure; its binding to receptors on the surface of immune cells and subsequent immune activation both require the structural integrity of the Fc region.

[0005] Separation and compounding are costly and have poor compatibility: Some existing technologies (such as Chinese invention patent application CN118648641A) involve preparing bovine colostrum peptide powder and bovine colostrum powder enriched with immunoglobulins separately, and then mixing them together through a dry mixing process to obtain a product with immune regulation and intestinal health improvement functions. However, this separate preparation process is not only complex and cumbersome, increasing production costs, but also leads to a significant loss of Ig activity during the separation and purification process. In addition, the physical mixing of dry powders results in poor compatibility and uneven dispersion due to charge repulsion and density differences between different components, making it difficult to achieve the synergistic effect of Ig and peptides.

[0006] Lack of targeted preparation and application of specific functional peptides: Existing technologies mostly focus on the activity of the whole enzymatic hydrolysis products, while there is insufficient research on the isolation, identification and mechanism of action of specific peptides in bovine colostrum that have the ability to synergistically enhance immune regulation with Ig (such as β-lactoglobulin-derived peptides), which limits the efficient utilization of bovine colostrum resources.

[0007] Therefore, developing a simple and low-cost method for preparing bovine colostrum peptides that can efficiently retain Ig activity while obtaining functional peptides with synergistic effects has become an urgent technical problem to be solved in this field. Summary of the Invention

[0008] To address the problems of low Ig activity retention in existing technologies, and to further resolve issues such as high separation and compounding costs, poor compatibility, or lack of specific functional peptide applications, this invention provides an enzymatic preparation method for highly immunomodulatory bovine colostrum peptides and its applications. This invention achieves efficient retention of Ig activity and synergistic enhancement of functional peptides through specific pretreatment protection, targeted enzymatic hydrolysis, and recombinant activity techniques, while simultaneously isolating a β-lactoglobulin peptide with significant immunomodulatory effects.

[0009] In a first aspect, the method for preparing the bovine colostrum peptide product according to the embodiments of this application includes...

[0010] Bovine colostrum is defatted, and the pH of the resulting supernatant is adjusted to 5.2-5.6. Trehalose is added, and the mixture is stirred for the first time to obtain a first mixture. The supernatant contains whey protein and 1g.

[0011] Add protease to the first mixture for hydrolysis to obtain an enzymatic hydrolysate;

[0012] The enzymatic hydrolysate includes a mixture of hydrolyzed bovine colostrum peptides.

[0013] According to the preparation method of bovine colostrum peptide product in the embodiments of this application, bovine colostrum is defatted to remove casein, the pH of the resulting supernatant is adjusted to 5.2-5.6, trehalose and metal ion chelating agent are added, and the mixture is stirred for the first time to obtain a first mixture; wherein the supernatant includes whey protein and Ig.

[0014] In this process, bromelain and flavor protease are added to the first mixture for hydrolysis to obtain an enzymatic hydrolysate.

[0015] Chitosan oligosaccharide is added to the enzymatic hydrolysate, and the mixture is stirred a second time to obtain a second mixture, wherein the second mixture includes a mixture of bovine colostrum hydrolysate peptides.

[0016] According to the preparation method of bovine colostrum peptide product in the embodiments of this application, the second mixture is dried to obtain the dried bovine colostrum peptide product.

[0017] According to the preparation method of bovine colostrum peptide product in the embodiments of this application, the final concentration of trehalose in the first mixture is 0.1~0.5% (w / v), and the final concentration of metal ion chelating agent in the first mixture is 5µM.

[0018] Among them, the metal ion chelating agent includes EDTA-2Na.

[0019] According to the preparation method of bovine colostrum peptide product in the embodiments of this application, the first stirring is to stir in a water bath at 30~50℃ for 5~15 minutes.

[0020] According to the preparation method of bovine colostrum peptide product in the embodiments of this application, the mass ratio of bromelain to flavor protease in the protease is 1:9~10, and the mass-volume ratio of enzyme to first mixture is 0.9~1.1g / L.

[0021] Enzymatic hydrolysis includes a first stage and a second stage;

[0022] In the first stage, bromelain is added, and the enzymatic hydrolysis is carried out at a pH of 5.2-5.6, a temperature of 40-45℃, and a time of 2-3 hours.

[0023] In the second stage, flavor protease is added, and the enzymatic hydrolysis is carried out at a pH of 5.2-5.6, a temperature of 37-40℃, and a time of 1-2 hours.

[0024] According to the method for preparing bovine colostrum peptide products in the embodiments of this application, the final concentration of chitosan oligosaccharide in the enzymatic hydrolysate is 0.05% (w / v).

[0025] According to the preparation method of bovine colostrum peptide product in the embodiments of this application, the second stirring is carried out at 4~20℃ for 15~25 min.

[0026] In a second aspect, the method for preparing β-lactoglobulin peptide from bovine colostrum according to the embodiments of this application includes...

[0027] Filter the second mixture from any of the above-mentioned methods through a filter membrane;

[0028] The filtrate was initially separated using a 20 mm × 100 mm Sephadex G-15 column, with a mobile phase of 0.1% (v / v) trifluoroacetic acid aqueous solution, a flow rate of 0.5 mL / min, a detection wavelength of 220 nm, and a collection time of 18–20 min.

[0029] The initially separated filtrate was finely purified using a 250 mm × 4.6 mm, 5 μm C18 column. The mobile phase A was 0.1% (v / v) trifluoroacetic acid aqueous solution, and the mobile phase B was acetonitrile. Gradient elution was performed under the following conditions: 0–20 min, 5–30% B; 20–25 min, 30–95% B; 25–30 min, 95% B; flow rate 1 mL / min; detection wavelength 220 nm; and collection time 12.5 min for the main peak fraction to obtain the β-lactoglobulin peptide.

[0030] In a third aspect, the active peptide according to the embodiments of this application has the amino acid sequence shown in SEQ ID NO:1.

[0031] In a fourth aspect, the composition according to the embodiments of this application includes the above-described active peptide and Ig.

[0032] In a fifth aspect, the active peptides or compositions described in the embodiments of this application are used in the preparation of immune-enhancing drugs, food or beverages, or milk powder.

[0033] Beneficial effects:

[0034] 1. Targeted protection and high activity retention of Ig

[0035] This invention achieves specific protection of Ig through the synergistic effect of trehalose and EDTA-2Na: by adjusting the pH to 5.2-5.6 (preferably 5.5) to create protein charge differentiation, trehalose is added to selectively protect Ig through electrostatic binding. Since whey protein and trehalose have no electrostatic interaction, their surface cleavage sites are fully exposed, ensuring that the protease can effectively hydrolyze whey protein, achieving the dual goals of retaining Ig and increasing peptide yield.

[0036] EDTA-2Na specifically chelates free Fe 2 ⁺ / Cu 2 ⁺ This method blocks the catalytic oxidation of Ig at oxidation-sensitive sites (Met residues in the CH2 / CH3 domain) by these metal ions, while the key hydrolysis sites of β-lactoglobulin (such as Leu-Leu and Val-Leu) are unaffected by metal ions. Therefore, the hydrolysis rate of whey protein remains unaffected. Experimental results in the examples show that, using the protection system of this invention, the Ig activity retention rate can reach over 90%.

[0037] 2. Directional and efficient enzymatic hydrolysis process

[0038] Bromelain and flavor protease, which still have good activity under the above pH environment (5.2~5.6 (preferably 5.5)), were selected to hydrolyze small molecule whey proteins (such as β-lactoglobulin and α-lactalbumin). Ig, due to its large molecular weight (150kDa) and steric hindrance, and its surface covered by a protective agent, had a significantly reduced hydrolysis rate, thus achieving selective protection of Ig and efficient hydrolysis of whey proteins.

[0039] The segmented temperature control strategy further optimizes enzymatic hydrolysis efficiency: the first stage at 45℃ promotes whey protein denaturation and accelerates hydrolysis; the second stage at 7℃ reduces the heat sensitivity of Ig and prevents its denaturation. The final peptide yield can reach over 60%, and the content of active peptides (such as LDIQKVAGTTE) is significantly increased.

[0040] 3. Significant synergistic effect

[0041] In the product prepared by this invention, the Fc fragment of Ig can bind to the Fcγ receptor on the surface of immune cells, activating the immune response; small molecule peptides (such as β-lactoglobulin peptide LDIQKVAGTTE) can activate Toll-like receptor (TLR4), synergistically enhancing the phagocytic capacity of macrophages and the level of cytokine secretion; the addition of chitosan oligosaccharide not only enhances the intestinal barrier function, but also promotes the absorption and action of Ig and peptide fragments in the intestine, further amplifying the immunomodulatory effect.

[0042] The experimental results in the examples confirm that the product can increase the NO secretion of macrophages by more than 40%, significantly increase the content of sIgA in the intestine, and has a better overall immune regulation effect than a single component or physical mixture.

[0043] 4. Low cost and easy industrialization

[0044] This invention eliminates the need for separate purification of Ig. It directly combines Ig with enzymatic hydrolysis and simple separation, saving expensive purification steps. The process is simple and suitable for large-scale production.

[0045] 5. Discovery and application of specific functional peptides

[0046] The β-lactoglobulin peptide isolated in this invention has the amino acid sequence LDIQKVAGTTE, which has a clear amino acid sequence and a synergistic effect with Ig to enhance immune regulation. Moreover, it is a newly discovered active peptide segment after database retrieval, providing a new material basis and theoretical basis for further development of highly active immunomodulatory peptide products. Detailed Implementation

[0047] The present invention will be further described below with reference to the embodiments. It should be noted that the embodiments do not constitute a limitation on the scope of protection of the present invention.

[0048] Example: This disclosure discloses a method for preparing a mixture of highly immunomodulatory bovine colostrum peptides, which belongs to the category of bovine colostrum hydrolyzed peptide products and is a mixture of peptides. Its preparation includes the following steps:

[0049] S1. Bovine colostrum pretreatment and Ig stabilization

[0050] (1) Raw material processing: Fresh bovine colostrum within 72 hours after calving was collected and defatted by centrifugation at 4,000×g for 15 minutes at 4℃. The pH of the defatted milk was adjusted to 4.6 with 1M HCl. pH 4.6 is the isoelectric point of casein to remove casein. After standing at 4℃ for 30 minutes, the milk was centrifuged at 8,000×g for 20 minutes and the supernatant was collected. The supernatant contained whey protein and immunoglobulins (Ig). The whey protein included α-lactalbumin, β-lactoglobulin, serum albumin, bovine lactoferrin, etc.

[0051] (2) Ig protection: 1M citric acid was added to the supernatant to adjust the pH to 5.5, followed by the addition of trehalose and EDTA-2Na. The final concentration of trehalose was 0.1-0.5% (w / v), and the final concentration of EDTA-2Na was 5µM. The mixture was stirred in a 40℃ water bath for 10 min to form an Ig protection system, resulting in the first mixture. By adjusting the pH of the supernatant to 5.5, the surface of Ig molecules exhibits a net positive charge, while whey proteins (such as β-lactoglobulin and α-lactalbumin) exhibit a net negative charge or neutrality at pH 5.5 (β-lactoglobulin has an isoelectric point of approximately 5.1, at which point it carries a slight negative charge; α-lactalbumin has an isoelectric point of approximately 4.8, exhibiting a negative charge), thus creating a differential charge distribution between Ig and whey proteins. The negatively charged trehalose binds to the positively charged Ig molecules through electrostatic attraction, rather than interacting with the negatively charged / neutral whey proteins. Electrostatic binding allows trehalose to form a hydrated protective shell on the surface of Ig molecules, preventing proteases from contacting the Fab / Fc functional regions of Ig and avoiding enzymatic cleavage. EDTA-2Na is a metal ion chelating agent that chelates free Fe ions. 2 ⁺ / Cu 2 The presence of metal ions such as ⁺ specifically blocks the oxidation pathway of Ig, and stirring also allows EDTA-2Na to fully chelate the free metal ions. This invention interrupts the hydrolysis and oxidation pathways of Ig through these two methods, resulting in more stable Ig retention.

[0052] S2. Targeted enzymatic hydrolysis

[0053] (1) Enzyme selection and compounding: At a mass-to-volume ratio of 0.9~1.1 g / L, bromelain (enzyme activity 2.0 × 10⁻⁶ g / L) was added to the supernatant after step 1 in the first stage. 5 The main enzyme is (U / g), and flavor protease (enzyme activity 1.0 × 10⁻⁶ U / g) is added in the second stage. 5 Bromelain (U / g) acts as a coenzyme. Bromelain cleaves the Gly-X and Ala-X peptide bonds of whey protein, effectively hydrolyzing whey protein and avoiding the main Fab / Fc functional regions of Ig, resulting in lower hydrolytic activity towards Ig. Flavor protease, as a coenzyme, supplements the enzymatic specificity of the main enzyme, further hydrolyzing the peptides obtained from hydrolyzing whey protein into small-molecule active peptides without affecting the protective effect of Ig, and can further enhance the immune activity of the peptides.

[0054] (2) Segmented temperature-controlled enzymatic hydrolysis: In the first stage, bromelain was added and enzymatically hydrolyzed at pH 5.5 and 45℃ for 2 hours. This temperature is the suitable enzymatic hydrolysis temperature for bromelain. This temperature causes moderate denaturation of whey protein, exposing the cleavage sites and accelerating hydrolysis into peptides. Under the protection of the protective layer formed by the electrostatic binding of trehalose and the protection of free metal ions chelated by EDTA-2Na, coupled with the low hydrolytic activity of bromelain itself on Ig, Ig can be kept stable. In the second stage, flavor protease was added and the temperature was lowered to 37℃ for 1 hour of enzymatic hydrolysis. 37℃ is close to the physiological temperature of the human body and is the temperature range in which the conformation of Ig is most stable. This avoids the potential denaturation effect of prolonged high temperature on the Fab / Fc functional region of Ig, that is, reduces the denaturation risk of Ig. At the same time, under the protection of the trehalose protective layer and the protection of free metal ions chelated by EDTA-2Na, coupled with the low hydrolytic activity of bromelain itself on Ig, Ig can continue to be kept stable. Furthermore, this temperature reaches the optimal hydrolysis temperature for flavor proteases, further enhancing the hydrolytic effect of flavor proteases to complete the hydrolysis of whey protein.

[0055] (3) Hydrolysis degree control: The degree of hydrolysis was monitored in real time using the pH-stat method. The second-stage enzymatic hydrolysis time was set to 1-2 hours. At this time, the degree of hydrolysis could reach 15-20%, at which point the enzymatic hydrolysis was terminated to avoid excessive hydrolysis and damage to the Ig structure. When the degree of hydrolysis reached 15-20%, the enzymatic hydrolysis was terminated. This degree of hydrolysis corresponds to the critical value at which whey protein is basically completely hydrolyzed and Ig is not significantly hydrolyzed. This avoids the non-specific cleavage of the edge peptide bonds of the Ig functional region by the protease when the hydrolysis is too high.

[0056] S3. Active recombination

[0057] Add chitosan oligosaccharide (molecular weight 3~5kDa) to the enzymatic hydrolysate obtained in step 2, wherein the final concentration of chitosan oligosaccharide is 0.05% (w / v), and stir at 4~20℃ for 20 min to promote the formation of chitosan oligosaccharide-Ig-peptide ternary complex, so that chitosan oligosaccharide has a protective effect on Ig-peptide, enhances intestinal barrier function and immune regulation synergistic effect.

[0058] S4. Drying and Finished Product Preparation

[0059] The mixture from step 3 is spray-dried at an inlet temperature of 160℃, an outlet temperature of 80℃, and a feed rate of 15mL / min. Alternatively, it can be freeze-dried at -50℃ and a vacuum of 10Pa. After drying, the powdered product is collected to obtain the peptide product of highly immunologically active bovine colostrum peptides.

[0060] The present invention also relates to a method for isolating β-lactoglobulin peptide from the above-mentioned enzymatic hydrolysate. The amino acid sequence of the β-lactoglobulin peptide is shown in SEQ ID NO:1. This peptide has the ability to enhance immunomodulation and can synergistically enhance immunomodulation with Ig.

[0061] SEQ ID NO:1: LDIQKVAGTTE.

[0062] The isolation and purification steps of the β-lactoglobulin peptide are as follows:

[0063] (1) The enzymatic hydrolysate obtained in step 2 is filtered through a 0.22 μm filter membrane for sterilization. The 0.22 μm is a commonly used pore size for sterilization, and the user obtains sterile and clear filtrate.

[0064] (2) Preliminary separation was performed using a Sephadex G-15 gel chromatography column with a diameter of 20 mm × 100 mm (length and inner diameter): 0.1% trifluoroacetic acid aqueous solution was used as the mobile phase, the flow rate was 0.5 mL / min, the detection wavelength was 220 nm, and the fractions with a retention time of 18 to 20 min were collected.

[0065] (3) Fine purification was performed using a C18 reversed-phase high-performance liquid chromatography column (250 mm × 4.6 mm, 5 μm, length, inner diameter, and packing particle size): gradient elution was performed using 0.1% trifluoroacetic acid aqueous solution (phase A) and acetonitrile (phase B) as the mobile phase. The gradient elution program was 0~20 min, 5~30% B; 20~25 min, 30~95% B; 25~30 min, 95% B; flow rate 1 mL / min, detection wavelength 220 nm, and the main peak component with a retention time of 12.5 min was collected. After freeze-drying, high-purity β-lactoglobulin peptide was obtained.

[0066] Example 1: Preparation of a highly immunologically active bovine colostrum peptide product

[0067] S1. Pretreatment: Take 10L of fresh bovine colostrum, centrifuge at 4℃ and 4,000×g for 15min to defatted the milk, and collect the defatted milk; adjust the pH of the defatted milk to 4.6 with 1MHCl, let it stand at 4℃ for 30min, and then centrifuge at 8,000×g for 20min.

[0068] S2.Ig Stabilization: Adjust the pH of the supernatant to 5.5, add 0.3% (w / v) trehalose and 5µM EDTA-2Na, stir at 40℃ for 10 min to obtain approximately 8 L of the first mixture.

[0069] S3. Targeted enzymatic hydrolysis: Add 7.2g of bromelain (2.0×10⁻⁶ mg / L) at a mass-to-volume ratio of enzyme to the first mixture. 5(U / g), under pH 5.5 conditions, enzymatic hydrolysis was carried out at 45℃ for 2 hours, followed by cooling to 37℃, and then flavor protease (0.8g, 1.0×10) was added. 5 (U / g), continue enzymatic hydrolysis for 1 hour, and monitor using pH-stat method. Terminate enzymatic hydrolysis when DH reaches 18.5%.

[0070] S4. Active recombination: Add chitosan oligosaccharide to the enzymatic hydrolysate, with a final concentration of 0.05% (w / v), and stir at 30°C for 20 min.

[0071] S5. Drying: Spray drying is used, with an inlet temperature of 160℃ and an outlet temperature of 80℃, yielding approximately 560g of finished product.

[0072] S6. Isolation and purification of β-lactoglobulin peptide:

[0073] (1) The enzymatic hydrolysate containing chitosan oligosaccharide obtained in step S4 is filtered through a 0.22 μm filter membrane for sterilization. The 0.22 μm is a commonly used pore size for sterilization, and the user obtains sterile and clear filtrate.

[0074] (2) Preliminary separation was performed using a Sephadex G-15 gel chromatography column with a diameter of 20 mm × 100 mm (length and inner diameter): 0.1% trifluoroacetic acid aqueous solution was used as the mobile phase, the flow rate was 0.5 mL / min, the detection wavelength was 220 nm, and the fractions with a retention time of 18 to 20 min were collected.

[0075] (3) Fine purification was performed using a C18 reversed-phase high-performance liquid chromatography column (250 mm × 4.6 mm, 5 μm, length, inner diameter, and packing particle size): gradient elution was performed using 0.1% trifluoroacetic acid aqueous solution (phase A) and acetonitrile (phase B) as the mobile phase. The gradient elution program was 0~20 min, 5~30% B; 20~25 min, 30~95% B; 25~30 min, 95% B; flow rate 1 mL / min, detection wavelength 220 nm, and the main peak component with a retention time of 12.5 min was collected. After freeze-drying, high-purity β-lactoglobulin peptide was obtained.

[0076] Experiment Example 1: Effect of Protectant on Ig Activity and Enzymatic Hydrolysis Efficiency

[0077] Control group: Same as Example 1, except that it does not have step S2. After 1 hour of enzymatic hydrolysis in step S3, the degree of hydrolysis reaches 20.3%.

[0078] Experimental group: Example 1;

[0079] Blank group: Same as Example 1, except that it does not have step S3.

[0080] Detection indicators and methods:

[0081] Ig activity retention rate: Using the Fcγ receptor binding assay, recombinant human FcγRIIIa is immobilized on an ELISA plate and specifically binds to the Fc fragment of Ig in the sample. The binding amount is detected by HRP-labeled secondary antibody, reflecting the functional activity of Ig. Activity retention rate = (OD value after enzyme digestion / OD value before enzyme digestion) × 100%.

[0082] β-lactoglobulin residual rate: determined by HPLC;

[0083] Peptide yield: The peptide yield was determined according to the method described in GB 5009.5 "National Food Safety Standard - Determination of Protein in Food";

[0084] Degree of hydrolysis (DH): determined by pH-stat method.

[0085] Experimental results:

[0086]

[0087] Results analysis: The Ig activity retention rate in the experimental group (91.6%) was significantly higher than that in the control group (38.2%), indicating that the synergistic effect of trehalose and EDTA-2Na can effectively protect Ig activity. The β-lactoglobulin residual rate in the experimental group (3.9%) was not significantly different from that in the control group (4.1%), while the peptide yield (63.4%) was lower than that in the control group (72.7%), indicating that trehalose and EDTA-2Na do not affect the hydrolysis efficiency of whey protein. The peptide yield in the experimental group decreased to some extent because Ig was not over-hydrolyzed. The results of the blank group showed that trehalose and EDTA-2Na themselves did not have a destructive effect on the structure of Ig and whey protein.

[0088] Experimental Example 2: Activity Experiment of β-lactoglobulin peptide LDIQKVAGTTE

[0089] Experimental materials: β-lactoglobulin peptide LDIQKVAGTTE isolated and purified in Example 1, and mouse RAW264.7 macrophages.

[0090] Experimental Groups:

[0091] Blank control group: cell culture medium;

[0092] LPS control group: cell culture medium containing 1 μg / mLLPS;

[0093] Low-dose peptide group: cell culture medium containing 10 μg / mβ-lactoglobulin peptide;

[0094] High-dose peptide group: cell culture medium containing 50 μg / mL β-lactoglobulin peptide;

[0095] Peptide + Ig group: Cell culture medium containing 50 μg / mL β-lactoglobulin peptide and 100 μg / mL Ig.

[0096] Detection indicators: After 24 hours of culture, the amount of NO secreted by macrophages was detected by the Griess method, and the amount of IL-6 secreted was detected by ELISA.

[0097] Experimental results:

[0098]

[0099] Results analysis: β-lactoglobulin peptide can dose-dependently promote the secretion of NO and IL-6 by macrophages, indicating that it has an immune-activating effect; when used in combination with Ig, the secretion of NO and IL-6 is significantly higher than that of the peptide alone or the LPS control group, indicating that the peptide has a significant synergistic effect with Ig.

[0100] Experiment Example 3: Activity Experiment of Highly Immunologically Active Bovine Colostrum Peptide Products

[0101] Experimental animals: SPF-grade ICR mice, half male and half female, weighing 20±2g.

[0102] Experimental Groups:

[0103] Normal control group: basal diet;

[0104] Model control group: basal diet + cyclophosphamide (intraperitoneal injection, 50 mg / kg, once a week, to establish an immunodeficiency model);

[0105] Commercially available product group: basic feed + cyclophosphamide + commercially available bovine colostrum powder (addition amount 1g / kg body weight);

[0106] The low-dose group of this invention consists of: basal feed + cyclophosphamide + the product from step S5 of this invention (addition amount 0.5g / kg body weight).

[0107] The high-dose group of this invention consists of: basic feed + cyclophosphamide + the product from step S5 of this invention (addition amount 1g / kg body weight).

[0108] Experiment duration: 30 days.

[0109] Testing indicators:

[0110] (1) Indicators of immune regulation in the body: mouse spleen index, serum Ig content, and peritoneal macrophage phagocytosis rate;

[0111] (2) Intestinal immune function indicators: intestinal mucosal sIgA content and ileal tissue IL-10 content.

[0112] Experimental results:

[0113]

[0114] Results Analysis: Compared with the model control group, the product of this invention significantly increased the spleen index, serum Ig content, and macrophage phagocytic rate in immunocompromised mice, indicating that it can enhance both non-specific and specific immune functions. Simultaneously, the product significantly increased intestinal sIgA content and ileum IL-10 content, indicating that it can enhance intestinal immune function and mucosal barrier function. Furthermore, the effect of the product of this invention is superior to commercially available products, and the effect of the high-dose group is close to that of the normal control group, demonstrating its excellent immunomodulatory effect.

[0115] The highly immunomodulatory bovine colostrum peptide product prepared by this invention can be widely used in infant immune formula powder: it helps enhance infant immunity and promote growth and development; it can also be used as a postoperative nutritional supplement to promote the recovery of immune function in postoperative patients and accelerate wound healing; and it can be used as a functional beverage suitable for people with low immunity.

[0116] This invention solves the problems of low Ig activity, poor synergistic effect, and high cost in existing technologies by innovating the enzymatic hydrolysis process, applying active protectants, and synergistic design of functional components. The product has significant immunomodulatory function, and the process is simple and easy to industrialize, which has important economic value and social significance.

[0117] This invention belongs to the field of biotechnology and functional foods, and discloses an enzymatic hydrolysis method for highly immunomodulatory bovine colostrum peptides and its application. Through targeted protection and directed enzymatic hydrolysis technology, the Ig activity retention rate is ≥90%, yielding a β-lactoglobulin peptide with synergistic immune-enhancing effects. In this product, Ig and small molecule peptides work synergistically to significantly improve the body's immune regulation capacity and intestinal immune function, and can be widely used in infant formula, postoperative nutrition, and functional beverages. Finally, it should be noted that the above are only some specific embodiments of this invention. All derivatives that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A method of preparing a bovine colostrum peptide product, characterized by, Comprising The bovine colostrum is defatted, and the obtained supernatant is adjusted to pH 5.2-5.6, and trehalose is added to perform first stirring to obtain a first mixed solution; wherein the supernatant comprises whey protein and Ig; A protease is added to the first mixed solution to perform hydrolysis to obtain an enzymatic hydrolysate; The enzymatic hydrolysate comprises a mixture of bovine colostrum hydrolysate peptides.

2. The method of claim 1, wherein the bovine colostrum peptide product is prepared by the steps of, The bovine colostrum is defatted, and the obtained supernatant is adjusted to pH 5.2-5.6, and trehalose is added to perform first stirring to obtain a first mixed solution; wherein the supernatant comprises whey protein and Ig; The first mixed solution is added with bromelain and flavor protease to perform hydrolysis to obtain an enzymatic hydrolysate; Chitosan is added to the enzymatic hydrolysate to perform second stirring to obtain a second mixed solution, wherein the second mixed solution comprises a mixture of bovine colostrum hydrolysate peptides. The second mixed solution is dried to obtain the dried bovine colostrum peptide product.

3. The method of claim 2, wherein the bovine colostrum peptide product is prepared by the steps of: The bovine colostrum is defatted, and the obtained supernatant is adjusted to pH 5.2-5.6, and trehalose is added to perform first stirring to obtain a first mixed solution; wherein the supernatant comprises whey protein and Ig; 4. The method of claim any one of claims 1 to 3, c h a r a c t e r i z e d i n t h a t, The final concentration of trehalose in the first mixed solution is 0.1-0.5% (w / v), and the final concentration of metal ion chelator in the first mixed solution is 5 µM; The metal ion chelator comprises EDTA-2Na. The first stirring is stirring in a water bath at 30-50 °C for 5-15 min.

5. The method of claim any one of claims 1-3, wherein the bovine colostrum peptide product is prepared by, The first stirring is stirring in a water bath at 30-50 °C for 5-15 min. The mass ratio of bromelain to flavor protease in the protease is 1:9-10, and the mass-volume ratio of the enzyme to the first mixed solution is 0.9-1.1 g / L; 6. The method of claim any one of claims 1-3, wherein the bovine colostrum peptide product is prepared by, The hydrolysis comprises a first stage and a second stage; The first stage adds bromelain, the hydrolysis pH is 5.2-5.6, the hydrolysis temperature is 40-45 °C, and the hydrolysis time is 2-3 h; The second stage adds flavor protease, the hydrolysis pH is 5.2-5.6, the hydrolysis temperature is 37-40 °C, and the hydrolysis time is 1-2 h. The final concentration of chitosan in the enzymatic hydrolysate is 0.05% (w / v). The final concentration of chitosan in the enzymatic hydrolysate is 0.05% (w / v).

7. The method of claim any one of claims 2-3, wherein the bovine colostrum peptide product is prepared by, The second stirring is stirring at 4-20 °C for 15-25 min. The second stirring is stirring at 4-20 °C for 15-25 min.

8. The method of claim any one of claims 2-3, wherein the bovine colostrum peptide product is prepared by, The second mixed solution of any one of claims 1-7 is filtered through a filter membrane; The filtrate is preliminarily separated by a Sephadex G-15 chromatography column with a size of 20 mm x 100 mm, wherein the mobile phase is 0.1% (v / v) trifluoroacetic acid aqueous solution, the flow rate is 0.5 mL / min, the detection wavelength is 220 nm, and the collected component is 18-20 min; 9. A method for preparing a beta-lactoglobulin peptide from bovine colostrum, characterized by, The preliminarily separated filtrate is finely purified by a C18 chromatography column with a size of 250 mm x 4.6 mm, 5 µm, wherein the mobile phase A is 0.1% (v / v) trifluoroacetic acid aqueous solution, the mobile phase B is acetonitrile, gradient elution, the gradient elution condition is 5-30% B for 0-20 min, 30-95% B for 20-25 min, 95% B for 25-30 min, the flow rate is 1 mL / min, the detection wavelength is 220 nm, the collected component is the main peak component for 12.5 min, and the β-lactoglobulin peptide is obtained. The amino acid sequence is shown in SEQ ID NO:

1. ​ ​ 10. An active peptide, characterized in that, ​ 11. A composition comprising the active peptide of claim 10 and Ig.

12. Use of the active peptide of claim 10 or the composition of claim 11 for the preparation of an immune-enhancing medicament, food or beverage or milk powder.

Images