Albumin postbiotic peptide and its use in the preparation of a medicament for the relief or treatment of food allergy

Abstract

The application discloses an albumin postbiotic peptide and application thereof in preparation of a medicine for relieving or treating food allergy, and belongs to the technical field of bioactive peptides. The albumin postbiotic peptide is prepared from egg white protein powder through enzymolysis, inoculation of fermentation lactobacillus mucus IOB802, fermentation and inactivation again. The polysaccharide content in the albumin postbiotic peptide is as high as 1860.74 mg / 100g, the albumin postbiotic peptide can regulate immune balance, reduce allergy-related factors such as IgE and IL-4 and histamine level, increase IgG2a antibody level, reduce infiltration of eosinophils and mast cells in jejunum tissue, down-regulate expression of proinflammatory factors such as IL-25 and TSLP, up-regulate tight junction proteins such as ZO-1 and Occludin, repair intestinal barrier, block penetration of allergens and relieve food allergy symptoms.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to an albumin post-genetic peptide and its application in the preparation of drugs for relieving or treating food allergies. Background Technology

[0002] Food allergies have become an increasingly serious public health challenge worldwide, affecting approximately 10% of the global population, and the incidence rate continues to rise. IgE-mediated immediate anaphylactic reactions are the main type. Traditional anti-allergy medications, such as antihistamines and corticosteroids, primarily treat symptoms and are insufficient to regulate the underlying immune balance; furthermore, long-term use carries the risk of side effects. Therefore, developing safe and long-term usable functional anti-allergy ingredients has become a research hotspot. Bioactive peptides, due to their high safety, good biocompatibility, and diverse target mechanisms, have received widespread attention in the field of anti-allergy treatment.

[0003] Albumin peptides are a class of small-molecule bioactive peptides prepared from ovalbumin in egg white through enzymatic hydrolysis. Ovalbumin is the main protein component of egg white, accounting for approximately 65% ​​of the total protein. Its amino acid composition closely resembles human requirements, and its biological value is over 95%, making it a recognized high-quality protein source. Besides being used as a nutritional supplement, ovalbumin and its enzymatic hydrolysates have been reported to possess various biological activities, such as antioxidant, immunomodulatory, and blood pressure-lowering effects, showing broad application prospects in the food and pharmaceutical fields.

[0004] Currently, the preparation of bioactive peptides mainly includes three methods: enzymatic hydrolysis, acid-base hydrolysis, and microbial fermentation. Among them, enzymatic hydrolysis is widely used in the research on the preparation of bioactive peptides from food proteins.

[0005] The anti-allergic effects of existing enzymatically hydrolyzed albumin peptides mainly rely on the direct regulation of immune cells or hyaluronidase by specific peptide segments. However, food allergies are complex processes involving multiple stages, including intestinal epithelial barrier function, mucosal immunity, effector cell activation, and tissue inflammation. Simple immune regulation is insufficient to fundamentally block allergies. Although some studies have attempted to introduce probiotics or their enzymes into protein hydrolysate systems, current technologies mainly manifest in two modes: one is to use probiotic-derived enzymes to perform secondary enzymatic hydrolysis of the hydrolysate (as an enzyme source tool); the other is to directly add live probiotics to the hydrolysate formulation (as an additional component). These two modes are essentially a physical combination or instrumental use of "hydrolysate + probiotics," failing to achieve deep integration and biotransformation of probiotics and hydrolysates. Probiotics play only a limited role in the system, and their metabolites and bacterial components are not fully utilized, making it difficult to form a multi-component synergistic functional complex system.

[0006] However, existing technologies, whether using albumin peptides prepared solely through enzymatic hydrolysis or probiotic-derived metabiotics, each have their limitations. The anti-allergic effect of albumin peptides prepared solely through enzymatic hydrolysis mainly relies on the direct regulation of immune cells or hyaluronidase by specific peptide segments, with a weak effect on intestinal barrier repair, making it difficult to physically block the continuous penetration of allergens. More importantly, even when enzymatically hydrolyzed peptides are physically mixed with probiotic metabiotics, existing technologies cannot achieve the functional synergy and material transformation effects resulting from the metabolic transformation of probiotics using enzymatic hydrolysis products during fermentation. Therefore, there is an urgent need to develop a novel functional product that can systematically intervene in the pathological process of food allergies and possesses both immune regulation and barrier repair functions. Summary of the Invention

[0007] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0008] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0009] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide an albumin post-genetic peptide, wherein the mass ratio of peptides with a relative molecular mass ≤1000 Da is ≥85% and the polysaccharide content is ≥1500 mg / 100g.

[0010] As a preferred embodiment of the albumin post-biotic peptide of the present invention, the peptide is obtained by fermentation of denatured enzymatically hydrolyzed egg white protein by lactobacillus.

[0011] As a preferred embodiment of the albumin post-genetic peptide of the present invention, the lactobacillus is Limosilactobacillus fermentum IOB802, with accession number CGMCC No.23120.

[0012] As a preferred embodiment of the albumin post-genetic peptide of the present invention, the enzymatic hydrolysis employs a complex protease, which includes at least one of alkaline protease, neutral protease, and flavor protease.

[0013] As a preferred embodiment of the albumin post-biotic peptide of the present invention, the inoculation amount of the lactobacillus is 1%-2% v / v of the enzymatic hydrolysate, and the fermentation time is 10-15h.

[0014] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing albumin post-genic peptides. Egg white protein powder is dissolved in water and subjected to protein heat denaturation treatment to obtain a protein denaturation solution; a complex protease is added, followed by enzymatic hydrolysis and high-temperature inactivation to obtain an enzymatic hydrolysate; the enzymatic hydrolysate is fermented in a sealed environment using *Lactobacillus mucinus* IOB802 and then post-treated to obtain albumin post-genic peptides; wherein, the preservation number of *Lactobacillus mucinus* IOB802 is CGMCC No. 23120.

[0015] In a preferred embodiment of the method for preparing albumin post-genetic peptides according to the present invention, the complex protease includes at least one of alkaline protease, neutral protease, and flavor protease.

[0016] In a preferred embodiment of the method for preparing albumin post-genetic peptides according to the present invention, the fermentation process involves inoculating Lactobacillus mucinus IOB802 at a concentration of 1%-2% v / v of the enzymatic hydrolysate for 10-15 hours.

[0017] As a preferred embodiment of the preparation method of albumin post-genetic peptide of the present invention, the post-processing includes filtration, concentration, sterilization and drying.

[0018] Another objective of this invention is to overcome the shortcomings of the prior art and provide an albumin post-genetic peptide prepared by the aforementioned preparation method.

[0019] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of albumin post-biotic peptide in the preparation of a drug for relieving or treating food allergies.

[0020] As a preferred embodiment of the use of the albumin post-genetic peptide described in this invention in the preparation of a medicament for relieving or treating food allergies, wherein the food allergy includes IgE-mediated food allergies.

[0021] As a preferred embodiment of the use of the albumin post-genetic peptide described in this invention in the preparation of a medicament for relieving or treating food allergies, wherein: the accompanying symptoms of the food allergy include skin urticaria, gastrointestinal spasms, diarrhea, respiratory cough or asthma, mast cell degranulation, eosinophil infiltration or impaired intestinal barrier function.

[0022] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of albumin post-biotic peptide in the preparation of drugs that regulate food allergy-related immune imbalances.

[0023] Beneficial effects of this invention: (1) The enzymatic hydrolysis step breaks down egg white protein into small peptides and free amino acids, providing a nitrogen source that probiotics can directly absorb and utilize, thus ensuring the efficient fermentation process. The enzyme inactivation step ensures that probiotics are not destroyed by residual proteases. During fermentation, the fermenting *Lactobacillus mucinus* IOB802 strain actively secretes various active metabolites such as polysaccharides. Component analysis results show that, compared with albumin peptides, the polysaccharide content of albumin post-fermentative peptides increased to 1860.74 mg / 100g, an increase of 31%. In contrast, the polysaccharide content of the physical mixture of albumin peptides and fermented *Lactobacillus mucinus* IOB802 bacterial powder was only 1041.61 mg / 100g, confirming that simple physical mixing cannot produce the polysaccharide enrichment resulting from fermentation. This invention achieves a qualitative change from traditional enzymatically hydrolyzed mixtures to post-fermentative complexes, ensuring both the nutritional supply for efficient probiotic growth and avoiding the problems of low efficiency and uncontrollability associated with direct fermentation, clearly distinguishing the essential differences between "fermentation" and "mixing".

[0024] (2) The animal experimental results of the present invention show that albumin post-epigenetic peptides can significantly downregulate the gene expression of pro-inflammatory factors IL-25, IL-33 and TSLP in intestinal epithelial cells by 45.8%, 51.3% and 42.6%, respectively. In contrast, albumin peptide groups downregulate the gene expression of pro-inflammatory factors IL-25, IL-33 and TSLP in intestinal epithelial cells by 22.1%-25.9%, respectively, thus cutting off the initiation signal of Th2 immune activation from the upstream of the allergic inflammatory chain.

[0025] (3) Animal experiments of this invention show that the albumin post-biotic peptide of this invention can comprehensively alleviate the physical symptoms and abnormal levels of specific antibodies in serum caused by food allergies. Compared with the model group, the albumin post-biotic peptide reduced IgE, IgG, IgG1, and histamine by 43.1%, 59.0%, 42.8%, and 31.5%, respectively, while increasing IgG2a antibody levels by 193.9%; Th2 cytokines IL-4, IL-5, and IL-13 decreased by 51.1%, 59.1%, and 66.5%, respectively. The albumin peptide group reduced IgE, IgG, IgG1, and histamine levels by 18.4%, 24.1%, 21.1%, and 13.8%, respectively, while increasing IgG2a antibody levels by 74.6%. This indicates that the albumin post-biotic peptide can systematically regulate food allergy-related immune imbalances, and its effects are more comprehensive than those of albumin peptide alone.

[0026] (4) Animal experiments showed that albumin post-biotic peptides have a significant repair effect on intestinal barrier damage caused by food allergies. Compared with the model group, albumin post-biotic peptides upregulated the expression of tight junction proteins ZO-1, Occludin, and Claudin-1 in jejunal tissue by 325.3%, 398.4%, and 403.2%, respectively, effectively repairing intestinal barrier damage and physically blocking the penetration of allergens across the intestinal wall. In contrast, the albumin peptide group only upregulated the expression of tight junction proteins ZO-1, Occludin, and Claudin-1 by 162.6%-186.3%, indicating that the metabolites produced by albumin post-biotic peptides through fermentation, especially macromolecules such as polysaccharides, have a unique role in intestinal barrier repair.

[0027] (5) Animal experiments showed that albumin post-biotic peptides can effectively reduce the infiltration of eosinophils and mast cells in jejunal tissue, reduce MCPT-1 expression by 48.7%, and albumin peptide group by 22.9%, thus inhibiting the effector pathway of allergic reactions. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 To investigate the effect of different drug administration groups on the expression level of the intestinal epithelial cell cytokine IL-25 gene; Figure 2 To investigate the effect of different drug administration groups on the expression level of the intestinal epithelial cell cytokine IL-33 gene; Figure 3 To investigate the effect of different drug administration groups on the expression level of the intestinal epithelial cytokine Tslp gene; Figure 4 The effect of different drug administration groups on the level of specific IgE antibodies in allergic mice; Figure 5 The effect of different drug administration groups on the level of specific IgG antibodies in allergic mice; Figure 6 The effect of different drug administration groups on the level of specific IgG1 antibodies in allergic mice; Figure 7 The effect of different drug administration groups on the level of specific IgG2a antibodies in allergic mice; Figure 8 The effect of different drug administration groups on histamine levels in allergic mice; Figure 9The effect of different drug administration groups on the level of cellular inflammatory factor IL-4; Figure 10 The effect of different drug administration groups on the level of the cellular inflammatory factor IL-5; Figure 11 The effect of different drug administration groups on the level of the cellular inflammatory factor IL-13; Figure 12 This represents the relative expression level of ZO-1 in allergic mice. Figure 13 The relative expression level of Occludin in allergic mice; Figure 14 The relative expression level of Claudin-1 in allergic mice; Figure 15 Semi-quantitative analysis of the number of eosinophils infiltrating mouse jejunal tissue; Figure 16 Semi-quantitative analysis of the number of mast cells in mouse jejunal tissue; Figure 17 The effect of MCPT-1 on serum levels in mice; Figure 18 Changes in rectal temperature before and after stimulation in allergic mice; Figure 19 Scoring of allergic symptoms in allergic mice after provocation; Figure 20 The effect of different drug administration groups on the spleen index, an immune organ, in allergic mice; Figure 21 To investigate the effects of different drug administration groups on the thymus index of the immune organ in allergic mice. Detailed Implementation

[0029] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0030] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0031] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0032] Unless otherwise specified, all raw materials used in this invention are commercially available. All test results in this invention are the average values ​​of three parallel tests.

[0033] The Limosilactobacillus fermentum strain IOB802 provided by this invention was deposited on August 5, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 23120.

[0034] The *Limosilactobacillus reuteri* IOB423 used in this invention was deposited on June 29, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16023.

[0035] Before use, the strain should be activated: Inoculate the Lactobacillus fermentans IOB802 from the cryopreservation tube into MRS medium at an inoculation rate of 2%±1%, and incubate in a sealed environment at 37℃±2℃ for 22h±2h to obtain seed culture; take the seed culture and inoculate it into secondary medium at an inoculation rate of 2%±1%, and incubate in a sealed environment at 37℃±2℃ for 22h±2h to obtain Lactobacillus fermentans IOB802 fermentation broth; centrifuge the Lactobacillus fermentans IOB802 fermentation broth, discard the supernatant, and dry the bacterial sludge to obtain Lactobacillus fermentans IOB802 bacterial powder.

[0036] Example 1

[0037] This embodiment provides a method for preparing albumin post-genic peptides, specifically as follows: S1 Preparation: Mix egg white protein powder with water at a ratio of 8%±2% to obtain an egg white protein powder solution; adjust the pH of the egg white protein powder solution to 10.5±0.5 using calcium hydroxide solution; S2 heat treatment: The mixed egg white protein powder solution is heated at 90℃±5℃ for 15±5min to denature the protein, and a protein denaturation solution is obtained. S3 enzymatic hydrolysis: After cooling the protein denaturation solution to 53℃±2℃, add 2%-6% (m / m) of complex protease and enzymatically hydrolyze at 53℃ for 6h±0.5h. After the hydrolysis is completed, the enzyme is inactivated by high temperature to obtain the enzymatic hydrolysate. The complex protease is composed of alkaline protease, neutral protease and flavor protease in a mass ratio of 1:0.3:0.2.

[0038] S4 fermentation: Fermentation orthogonal experiment was conducted by inoculating fermentation Lactobacillus mucinus IOB802 bacterial powder according to the conditions listed in Table 1; Table 1. Fermentation Orthogonal Factor Level Table

[0039] S5 filtration: The enzymatic hydrolysate is filtered, and the feed rate is controlled during the filtration process; S6 Concentration: Concentrate the filtrate to obtain a concentrated solution; S7 Drying: After sterilizing the concentrate, it is dried to obtain a total of 9 albumin-derived post-genetic peptide powders.

[0040] Comparative Example 1

[0041] This comparative example prepared post-biotics from fermented Lactobacillus mucinus IOB802, specifically as follows: The bacterial sludge obtained by centrifuging the fermentation broth of Lactobacillus mucinus IOB802 was heat-inactivated at 85℃±5℃ for 20min±5min, and then dried to obtain the post-biotic of Lactobacillus mucinus IOB802.

[0042] Comparative Example 2

[0043] The albumin peptide was prepared in this comparative example as follows: S1 Pretreatment: Weigh the egg white powder, add water to make a uniform suspension, and slowly pour it into the enzymatic hydrolysis tank.

[0044] S2 enzymatic hydrolysis: Weigh out food-grade protease, add an appropriate amount of water to a clean container, and stir well to prepare an enzyme solution. Slowly add the prepared enzyme solution to the enzymatic hydrolysis vessel and mix thoroughly. Maintain the temperature inside the vessel at 55±2℃ and treat for 4.5 hours.

[0045] S3 Inactivation: After enzymatic hydrolysis, heat the solution to above 95°C and keep it at that temperature for 17 min ± 3 min to completely inactivate the protease.

[0046] S4 filtration: The enzymatic hydrolysate is filtered, and the feed rate is controlled during the filtration process.

[0047] S5 Concentration: Concentrate the filtrate.

[0048] S6 Drying: After sterilization, the concentrated liquid is dried to obtain albumin peptides.

[0049] Comparative Example 3

[0050] The comparative example of preparing fermented albumin peptides differs from Example 1 in that the fermentation conditions are selected and sterilization in the S7 drying step is omitted. Specifically: S1-S3 are the same as in Example 1; S4 fermentation: After cooling the enzymatic hydrolysate to 37°C, aseptically inoculate with Lactobacillus mucinus IOB802 at 2% (v / v). After inoculation, ferment in a sealed container at 37°C for 12 hours. S5-S6 are the same as in Example 1; S7 Drying: Dry the concentrate to obtain fermented albumin peptides.

[0051] Comparative Example 4

[0052] The comparative preparation of albumin fermentation peptides in this example differs from that in Example 1 in that the S3 enzymatic hydrolysis step is omitted. Specifically: S1-S2 are the same as in Example 1; S4 fermentation: After cooling the protein denaturation treatment solution to 37°C, aseptically inoculate with fermenting Lactobacillus mucinus IOB802 at 2% (v / v). After the inoculation is completed, ferment in a closed system at 37°C for 12 hours. S5-S7 are the same as in Example 1, yielding albumin fermentation peptides.

[0053] Comparative Example 5

[0054] In this comparative example, a mixed sample of albumin peptide and IOB802 bacterial powder was prepared. The albumin peptide and IOB802 bacterial powder prepared in Comparative Example 2 were mixed and shaken at a mass ratio of 1:1.

[0055] Comparative Example 6

[0056] In this comparative example, a mixed sample of albumin peptide and IOB802 postbiotic was prepared. The albumin peptide prepared in Comparative Example 2 and the IOB802 postbiotic prepared in Comparative Example 1 were mixed and shaken at a mass ratio of 1:1.

[0057] Comparative Example 7

[0058] This comparative example uses *Lactobacillus reuteri*, a congener of the same genus, to prepare albumin post-genetic peptides. The preparation method is the same as in Example 1, except that *Lactobacillus reuteri* IOB423 is used in the S4 fermentation step.

[0059] Example 2: Detection of polysaccharide content

[0060] Nine types of albumin post-biotic peptides, albumin peptides, albumin fermentation peptides, fermented albumin peptides, and albumin peptides + IOB802 bacterial powder prepared by orthogonal experiments in Example 1 were mixed (bacterial activity 10 before inactivation). 8 CFU / g, ratio 1:1), albumin peptide + IOB802 post-biotic mixture (bacterial activity 10 before inactivation) 8 The polysaccharide content of the sample (CFU / g, ratio 1:1) was determined according to the method described in GB / T 40632-2021.

[0061] The detection method is as follows: 1. Sample processing

[0062] Five g of each of the nine albumin post-biotic peptides, albumin peptides, albumin fermentation peptides, albumin peptide + IOB802 bacterial powder mixture, and albumin peptide + IOB802 post-biotic mixture samples prepared by orthogonal experiments in Example 1 were taken. The precipitates were extracted in a 95°C water bath for 3 hours at a material-to-liquid ratio of 1:40. The filtrate was collected by centrifugation, and four times the volume of anhydrous ethanol was added to precipitate the precipitate. After standing for 24 hours, the solution and precipitate were poured into centrifuge tubes, centrifuged for 20 minutes, and the supernatant was removed. The remaining precipitate was then combined after centrifugation. The precipitate was dissolved in ultrapure water and then freeze-dried under vacuum to constant weight.

[0063] 2. Standard curve and sample testing

[0064] Take 0.3 mL of 80% phenol solution and add 3.7 mL of water to prepare a 6% phenol solution.

[0065] Add 100 mg of glucose (dried to constant weight) to a small amount of distilled water, dissolve, and then dilute to volume in a 100 mL volumetric flask to prepare a 1.0 mg / mL glucose standard stock solution.

[0066] Glucose standard solutions with concentrations of 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, and 0.4 mg / mL were prepared, with a quality control concentration of 0.05 mg / mL. 0.03 mL of the above glucose standard solution, 0.03 mL of 6% phenol solution, and 0.15 mL of 98% concentrated sulfuric acid were taken sequentially, shaken well, and allowed to stand at room temperature for 30 min. The absorbance of the solutions was measured at 490 nm, and a standard curve was plotted.

[0067] Take 10 mg of lyophilized crude polysaccharide and add 50 mL and 20 mL of pure water respectively to prepare sample solutions of 0.2 mg / mL and 0.5 mg / mL. Take 0.03 mL of the prepared sample solution, 0.03 mL of 6% phenol solution, and 0.15 mL of 98% concentrated sulfuric acid sequentially, shake well, and let stand at room temperature for 30 min. Measure the absorbance of the solution at 490 nm. Substitute these values ​​into the standard curve regression equation. The standard curve formula is: y = 3.7179x + 0.0251, R0 2 = 0.9996, calculate the polysaccharide content.

[0068] Result Calculation and Representation The polysaccharide content in the sample is expressed as a mass fraction X, in milligrams per 100 grams (mg / 100g). , In the formula: c -- The concentration of polysaccharides in the sample solution obtained from the standard curve, in milligrams per milliliter (mg / mL); V -- The final volume of the sample polysaccharide solution, expressed in milliliters (mL); m1 -- Total crude polysaccharide mass extracted from the sample, in milligrams (mg); m2 -- The initial mass of the sample, expressed in grams (g); m3 -- The mass of crude polysaccharide required to prepare the sample polysaccharide solution, in milligrams (mg); f -- Moisture content (%) in the sample; 0.9 -- Correction factor for converting glucose to dextran.

[0069] The calculation result is rounded to 3 decimal places.

[0070] The experimental results are shown in Table 2. The polysaccharide content detection results show that the albumin post-genic peptide obtained under the following conditions: fermentation temperature 37℃, inoculum size 2% (v / v), and fermentation time 12h. Furthermore, compared with the product prepared in the comparative example, the polysaccharide content of the albumin post-genic peptide was significantly increased.

[0071] Table 2. Results of polysaccharide content detection

[0072] Example 3: Detection of peptide molecular weight distribution

[0073] The albumin post-genic peptides from Example 1 were analyzed for peptide molecular weight distribution according to the detection method of national standard GB / T 22492-2008. The results are shown in Table 3. The proportion of peptides with a molecular weight below 1000 Da in the albumin post-genic peptides prepared by this invention was 88.97%, indicating that the preparation process of this invention has a good hydrolysis effect, which can decompose large protein molecules into small peptide molecules, providing a certain material basis for anti-allergic effects.

[0074] Table 3. Peptide molecular weight distribution table

[0075] Example 4: Establishment of an allergy animal model

[0076] Six-week-old female BALB / c mice were pre-fed for 7 days at room temperature and with a humidity level maintained at approximately 50%. The mice were randomly divided into eight groups: control group, model group, experimental group 1, experimental group 2, experimental group 3, experimental group 4, experimental group 5, and experimental group 6. The model group and experimental groups 1-6 were intraperitoneally injected with 100 μg ovalbumin (OVA) + 20 μg cholera toxin (CT) on days 1, 7, and 14, respectively, to establish an allergic reaction model. The control group was injected with an equal volume of physiological saline.

[0077] After the model was constructed, from day 21 to 24, the control group and the model group were intraperitoneally injected with 10 mL / kg body weight (bw), while the remaining groups were administered their respective solutions by gavage at a dose of 0.45 g / kg. The specific dosing regimen was as follows: Experimental Group 1: IOB802 post-genetic agent prepared in Comparative Example 1 Experimental Group 2: Fermented albumin peptides prepared in Comparative Example 3 Experimental Group 3: Albumin fermentation peptides prepared in Comparative Example 4 Experimental Group 4: Albumin peptide prepared in Comparative Example 6 + IOB802 post-biotic mixture Experiment 5: Albumin peptides prepared in Comparative Example 2 Experiment 6: Albumin post-genetic peptides prepared in Example 1 On days 21-24, one hour after the end of gavage, the model group, experimental group 1, experimental group 2, experimental group 3, experimental group 4, experimental group 5 and experimental group 6 were gavaged with 50mg of ovalbumin, while the blank group had an equal volume of physiological saline in the peritoneal cavity to induce allergic stimulation.

[0078] Example 5: Effect of albumin-derived post-genic peptide on gene expression levels of intestinal epithelial cells

[0079] In the allergy model, the gene expression levels of IL-25, IL-33, and TSLP significantly drove the immune inflammatory response, mainly by activating the Th2 pathway, recruiting inflammatory cells, and disrupting tissue barriers, thereby exacerbating the allergic pathological process. Total RNA was extracted using the TRIzol method according to the kit instructions. Results are as follows... Figure 1 , Figure 2 and Figure 3 As shown, compared with the control group, the expression levels of IL-25, IL-33, and TSLP in the model group were significantly increased, indicating that the allergy model successfully induced the upregulation of epithelial pro-inflammatory signals. After gavage administration of albumin peptides, the expression of the above cytokines decreased by 22.5%, 25.9%, and 22.1%, respectively. After gavage administration of albumin post-biotic peptides, the expression of the above cytokines decreased by 45.8%, 51.3%, and 42.6%, respectively, with significantly better downregulation than the albumin peptide group. This indicates that albumin post-biotic peptides can more efficiently inhibit the secretion of pro-inflammatory factors from intestinal epithelial cells, interrupting the initiation signal of Th2 immune activation at the upstream of the allergic inflammatory chain, and the effect is significantly stronger than that of albumin peptides alone.

[0080] Example 6: Albumin-derived post-evolutionary peptides can improve Th1 / Th2 immune balance.

[0081] (1) Detection of serum specific antibodies and histamine levels

[0082] The immune response is a complex process involving multiple immune cells and molecules, and changes in antibody levels in the body directly affect the intensity of allergic reactions. Overactivation of the Th2 immune response leads to the production of large amounts of allergy-related antibodies and histamine by B cells.

[0083] Blood was collected from the orbital rim of mice in Example 3, and serum was separated. The levels of OVA-specific IgE, IgG, IgG1, IgG2a, and histamine in the serum were detected using an indirect ELISA method according to the kit instructions.

[0084] like Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 8 As shown, compared with the control group, the model group mice had significantly increased levels of IgE, IgG, IgG1, and histamine, and significantly decreased levels of IgG2a, indicating that food allergy leads to excessive activation of Th2 immunity. Compared with the model group, the albumin peptide group showed a 18.4%, 24.1%, 21.1%, and 13.8% decrease in IgE, IgG, IgG1, and histamine, respectively, and a 74.6% increase in IgG2a; the albumin post-genic peptide group showed a 43.1%, 59.0%, 42.8%, and 31.5% decrease in IgE, IgG, IgG1, and histamine, respectively, and a 193.9% increase in IgG2a, with all indicators showing significantly better improvement than the albumin peptide group. This indicates that albumin post-genic peptides can more comprehensively reshape the Th1 / Th2 immune balance, significantly reduce the levels of sensitizing antibodies and inflammatory mediators, and are far more effective than albumin peptides.

[0085] (2) Detection of inflammatory factors in spleen cells

[0086] In allergy models, IL-4 initiates Th2 immunity and drives IgE production, IL-5 mediates eosinophilic inflammation, and IL-13 dominates mucus secretion and tissue remodeling. These three constitute a synergistic pathway between the IL-4 / IL-13 signaling center and the amplified effect of IL-5, which is a key target for blocking the pathological process of allergies.

[0087] The spleen of the mouse in Example 3 was used to prepare a single-cell suspension. After 72 h of restimulation with OVA (50 ng / mL), the supernatant was collected and the concentrations of IL-4, IL-5 and IL-13 were detected using an ELISA kit.

[0088] The experimental results are shown in Figure 9 , Figure 10 and Figure 11Compared with the control group, the concentrations of IL-4, IL-5, and IL-13 in the supernatant of the model group were significantly increased, indicating that food allergy can directly lead to the release of Th2 immune-related factors. Compared with the model group, the albumin peptide group showed a decrease of 25.3%, 24.8%, and 30.0% in IL-4, IL-5, and IL-13, respectively. The albumin post-biotic peptide group showed a decrease of 51.1%, 59.1%, and 66.5% in IL-4, IL-5, and IL-13, respectively, with a significantly greater decrease than the albumin peptide group. This indicates that the albumin post-biotic peptide has a much stronger inhibitory effect on Th2 cytokines than albumin peptide and can effectively block the effector pathway of allergic reactions.

[0089] Example 7: Albumin-derived post-genic peptides can improve intestinal barrier damage caused by food allergies.

[0090] The integrity of the tight junctions of the intestinal epithelium is crucial for preventing the penetration of allergens. Alterations in intestinal barrier integrity during food allergies are associated with decreased expression levels of Occludin, Claudin-1, and ZO-1. Total protein was extracted from mouse jejunal tissue obtained in Example 3. Western blot analysis was used to detect the protein expression levels of ZO-1, Occludin, and Claudin-1.

[0091] The experimental results are shown in Figure 12 , Figure 13 , Figure 14 Compared with the control group, the protein expression levels of the three tight junction proteins in the model group were significantly decreased, indicating severe damage to the intestinal barrier. Compared with the model group, the protein expression levels of ZO-1, Occludin, and Claudin-1 in the albumin peptide group were upregulated by 162.6%, 175.3%, and 186.3%, respectively. The protein expression levels of ZO-1, Occludin, and Claudin-1 in the albumin post-genic peptide group were upregulated by 325.3%, 398.4%, and 403.2%, respectively. This indicates that the upregulation effect of albumin post-genic peptides on intestinal tight junction proteins is significantly stronger than that of albumin peptides, and can effectively repair the intestinal barrier at the physical level, blocking the transintestinal wall penetration of allergens.

[0092] Example 8: Effector cells of albumin-derived post-albumin peptides inhibiting allergic reactions

[0093] Eosinophils and mast cells are key effector cells in allergic reactions, and their infiltration level directly reflects tissue inflammation and damage. This example examines the effects of albumin-derived post-evolutionary peptides on the infiltration of these two cell types and the release of MCPT-1.

[0094] Jejunal tissue from mice in Example 3 was used for HE staining (to count eosinophils) and toluidine blue staining (to count mast cells). The serum MCPT-1 (mast proteinase-1) level was also detected using an ELISA kit.

[0095] The experimental results are shown in Figure 15 , Figure 16 and Figure 17 In the model group, a large number of eosinophils infiltrated the submucosa of the jejunum, and toluidine blue staining showed a significant increase in the number of mast cells, indicating that food allergy led to tissue inflammation and damage. The infiltration level was reduced in the albumin peptide group, while it was significantly decreased in the albumin post-genic peptide group, approaching the level of the control group. Serum MCPT-1 levels showed a significant increase compared to the control group. The albumin peptide group showed a 22.9% decrease, and the albumin post-genic peptide group showed a 48.7% decrease, with significant differences. This indicates that albumin post-genic peptides can more effectively inhibit the infiltration of effector cells into the intestine, reduce the release of allergic mediators, and thus alleviate tissue damage.

[0096] Example 9: Albumin-derived post-albumin peptides can improve the overall signs of food allergies.

[0097] (1) Improvement of allergy symptoms and body temperature

[0098] Hypothermia is a key and objectively measurable indicator of systemic allergic reactions in mice, and mice exhibit clinical reactions related to food allergies, such as itching, diarrhea, and rapid breathing, after being stimulated.

[0099] Within 30 minutes after each OVA challenge, the allergic reaction of mice was assessed in a double-blind manner (0-20 points) according to the criteria in Table 4. The temperature within 1 cm of the anus was recorded using a temperature detector at 25-30 minutes after challenge in each group of mice and recorded as body temperature.

[0100] Table 4 Scoring criteria for signs and symptoms of allergic reactions

[0101] The results are as follows Figure 18 , 19 As shown, at the last challenge, the average score of the model group was 4 points, indicating that egg white protein sensitization leads to a decrease in anal temperature and a significant increase in diarrhea scores. After gavage, the albumin peptide group's score dropped to 3 points, and the albumin post-biotic peptide group's score dropped to 1.3 points, indicating a significant reduction in symptoms.

[0102] (2) Regulatory effect on immune organ indices

[0103] As key immune organs, elevated levels of the thymus and spleen indices typically reflect abnormal activation of the immune system and excessive proliferation of lymphocytes. Mice were euthanized after the experiment, and the spleen and thymus were weighed. The spleen index (spleen mg / body weight g) and thymus index (thymus mg / body weight g) were calculated, respectively.

[0104] Depend on Figure 20 and 21 As shown, compared with the control group, the spleen index and thymus index in the model group increased by 50.6% and 43.6%, respectively, indicating that the immune system in the model group was overactivated. The albumin peptide group decreased by 10.9% and 11%, respectively, while the albumin post-genic peptide group decreased to an increase of 21.8% and 23.0%, respectively, close to the level of the control group, and the effect was significantly better than that of the albumin peptide group.

[0105] The above results indicate that albumin post-biotic peptides can significantly alleviate the clinical symptoms of food allergies and restore the abnormal enlargement of immune organs caused by allergies to near normal levels, with an overall protective effect superior to albumin peptides.

[0106] In summary, both albumin peptides and albumin post-biotic peptides can alleviate food allergy-related symptoms to some extent. However, albumin post-biotic peptides prepared by fermentation with *Lactobacillus mucinus* IOB802 show significantly better anti-allergy effects than unfermented albumin peptides. Specifically, both can alleviate allergic reactions, reduce serum levels of specific antibodies IgE, IgG, IgG1, and histamine, increase IgG2a antibody levels, and reduce levels of inflammatory factors such as IL-4, IL-5, and IL-13 to alleviate immune damage. They also reduce eosinophil and mast cell infiltration in jejunal tissue, decrease MCPT-1 expression, and upregulate the expression of tight junction proteins ZO-1 and Occludin to repair the intestinal barrier. However, albumin peptides only significantly improve some indicators, while albumin post-biotic peptides significantly regulate most of the above indicators. In addition, albumin post-biotic peptides can more efficiently downregulate the gene expression of inflammatory factors IL-25, IL-33 and TSLP, with a regulatory strength significantly superior to albumin peptides, thereby more comprehensively blocking the initiation and amplification pathways of allergic inflammation.

[0107] Furthermore, albumin-derived post-biotic peptides can more effectively reverse the vicious cycle of food allergies—epithelial pro-inflammatory Th2 imbalance, inflammatory infiltration, and barrier damage—into a virtuous cycle of immune balance, inflammation reduction, barrier repair, and feedback inhibition, thereby systematically achieving a better relief effect for food allergies.

[0108] This invention provides an albumin post-biotic peptide, which is obtained by enzymatically hydrolyzing and inactivating egg white protein powder, followed by fermentation with *Lactobacillus fermentum* IOB802 and subsequent inactivation. The core innovation of this process is that the enzymatic hydrolysis step breaks down egg white protein into small peptides and free amino acids, providing a directly absorbable nitrogen source for probiotics and ensuring efficient fermentation. The enzyme inactivation step ensures that the probiotics are not destroyed by residual proteases. During fermentation, the *Lactobacillus fermentum* IOB802 strain actively secretes various active metabolites, including polysaccharides. Component analysis results show that the polysaccharide content of the albumin peptide is 1419.96 mg / 100g, while the polysaccharide content of the albumin post-biotic peptide increases to 1860.74 mg / 100g, an increase of 31%. In stark contrast, the polysaccharide content of the physical mixture of albumin peptide and *Lactobacillus fermentum* IOB802 powder is only 1041.61 mg / 100g, confirming that simple physical mixing cannot produce the polysaccharide enrichment resulting from fermentation. This application achieves a qualitative change from traditional enzymatic hydrolysis of mixtures to postbiotic complexes, which not only ensures the nutritional supply for the efficient growth of probiotics, but also avoids the problems of low efficiency and uncontrollability of direct fermentation, clearly distinguishing the essential difference between "fermentation" and "mixing".

[0109] Bioactive peptides are highly safe and biocompatible, attracting significant attention in the field of immune regulation. Epigenetics, as a mixture of inactivated probiotics, offer multiple health benefits, including regulating gut microbiota and enhancing the immune barrier. Albumin epigenetic peptides combine the advantages of both bioactive peptides and epigenetics, and are functional products obtained from egg white protein powder through enzymatic hydrolysis, probiotic fermentation, and inactivation.

[0110] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An albumin-derived metagenic peptide, characterized in that: The albumin post-genetic peptides contain peptides with a relative molecular mass ≤1000 Da, accounting for ≥85% of the total mass and polysaccharide content ≥1500 mg / 100g.

2. The albumin post-genic peptide as described in claim 1, characterized in that: The peptide was obtained by fermentation of denatured enzymatically hydrolyzed egg white protein with Lactobacillus.

3. The albumin post-genic peptide as described in claim 2, characterized in that: The lactobacillus mentioned is Limosilactobacillus fermentum IOB802, with accession number CGMCC No. 23120.

4. The albumin post-genic peptide as described in claim 2, characterized in that: The enzymatic hydrolysis uses a complex protease, which includes at least one of alkaline protease, neutral protease, and flavor protease.

5. The albumin post-genic peptide as described in claim 2, characterized in that: The inoculation amount of the lactobacillus is 1%-2% v / v of the enzymatic hydrolysate, and the fermentation time is 10-15h.

6. The use of the albumin post-biotic peptide as described in any one of claims 1-5 in the preparation of a medicament for relieving or treating food allergies.

7. The use of the albumin post-genic peptide as described in claim 6 in the preparation of a medicament for relieving or treating food allergies, characterized in that: The food allergies mentioned include IgE-mediated food allergies.

8. The use of the albumin post-genic peptide as described in claim 7 in the preparation of a medicament for relieving or treating food allergies, characterized in that: Accompanying symptoms of the food allergy include skin hives, gastrointestinal spasms, diarrhea, respiratory cough or asthma, mast cell degranulation, eosinophil infiltration, or impaired intestinal barrier function.

9. The use of the albumin post-biotic peptide as described in any one of claims 1-5 in the preparation of a medicament for regulating food allergy-related immune imbalance.

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