Antigen epitope peptide of beta-lactoglobulin and method for reducing BLG sensitization by using antigen epitope peptide

Abstract

The invention discloses an antigen epitope peptide of beta-lactoglobulin and a method for reducing BLG sensitization by using the antigen epitope peptide, belongs to the technical field of protein engineering, and provides the antigen epitope peptide of which the amino acid sequence is shown as SEQ ID NO.1-SEQ ID NO.7. The invention also discloses a method for reducing BLG sensitization by using the antigen epitope peptide. The method for reducing BLG sensitization by using the antigen epitope peptide of beta-lactoglobulin comprises the following steps: simulating infant gastrointestinal digestion by using a beta-lactoglobulin solution to obtain an in-vitro digestion product; identifying digestion-resistant polypeptide in the in-vitro digestion product, screening to obtain beta-lactoglobulin antigen epitope peptide, and determining an IgE combined amino acid site; by taking the amino acid site as a cutting target, screening, determining and selecting alkaline protease; and adding the beta-lactoglobulin into an in-vitro digestion product, and carrying out enzymolysis to obtain the low-sensitization beta-lactoglobulin. According to the invention, the problem that the specificity of a beta-lactoglobulin desensitization method is not strong is solved, and the antigen epitope peptide is provided for specifically reducing the sensitization of beta-lactoglobulin.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering, and in particular relates to an antigenic epitope peptide of beta-lactoglobulin and a method for reducing BLG sensitization by using the same. Background Art

[0002] As a functional protein, milk protein is rich in nutrition and is widely used in the food industry, especially in infant formula.

[0003] However, during the intake of cow's milk, some infants and young children develop cow's milk protein allergy (CMPA) due to factors such as genetic allergic constitution, imperfect development of the digestive system, immature development of the immune system, and imperfect intestinal barrier function, resulting in symptoms such as vomiting, diarrhea, respiratory asthma, and skin allergies. Worldwide, the prevalence of CMPA in infants and young children is 2% to 7.5%, while that in adults is less than 1%. Due to changes in living habits and social structure in recent years, infant formula has become the best substitute and complementary food for breastfeeding. At the same time, the risk of CMPA in infants and young children has also increased. In order to ensure the food safety of infants and young children, it is crucial to reduce the allergenicity of infant formula. At present, the extensively hydrolyzed infant formula (eHF) available on the market can be consumed by CMPA infants and young children, but studies have shown that the weight of infants and young children who consume eHF before the age of one is significantly lower than that of normal infants and young children, indicating that eHF has lost the original nutritional function of cow's milk protein to a certain extent, and the deeper the degree of hydrolysis, the more bitter the milk powder tastes. In view of this, partially hydrolyzed infant formula with relatively less allergenic residues is an ideal milk powder for CMPA infants.

[0004] Whey protein is an important ingredient in infant formula milk powder, of which β-lactoglobulin (BLG) accounts for more than 50% of the total whey protein. β-lactoglobulin is resistant to gastric acid and is not easily hydrolyzed by proteases. It can pass through the gastrointestinal tract without being digested and directly enter the blood circulation. It belongs to the family of strong allergens. Since human milk does not contain BLG, BLG is considered to be the most important allergen in whey protein.

[0005] After milk allergens are ingested by the body and digested by the gastrointestinal tract, the normal body will develop oral tolerance to the antigen, but when oral tolerance is broken or lost, patients with milk allergies will have abnormal IgE reactions to allergen proteins. Since allergenic proteins generally have strong resistance to the digestive function of the gastrointestinal tract, in vitro enzymatic hydrolysis can quickly destroy the spatial structure of conformational epitopes and cleave linear epitopes. Therefore, pre-enzymatic hydrolysis of proteins in vitro is one of the effective methods to eliminate some allergenicity. At present, there is a lack of effective technical methods to significantly reduce the allergenicity of BLG based on the degree of reduction of allergic epitopes, resulting in the continued use of a large number of ingredients for deep hydrolysis of proteins that are not conducive to the growth and development of infants. Therefore, there is an increasing call for methods that can accurately eliminate whey protein antigen epitopes while retaining other functional structures.

[0006] In summary, no reports have been found on using simulated gastrointestinal digestion to pre-treat BLG, screen for digestion-resistant IgE binding antigen epitopes and key amino acids, and select appropriate proteases based on this to improve the specific reduction of BLG sensitization. Therefore, the development of a method for reducing the sensitization of β-lactoglobulin based on in vitro simulated digestion combined with specific enzymatic hydrolysis is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0007] In order to solve the above technical problems, the present invention proposes an antigenic epitope peptide of β-lactoglobulin and a method for reducing BLG sensitization using the same, screens out the digestion-resistant IgE antigenic epitope and key amino acid sites of β-lactoglobulin, and solves the problem of low specificity of current β-lactoglobulin desensitization methods.

[0008] To achieve the above object, the present invention provides an antigenic epitope peptide of β-lactoglobulin, the amino acid sequence of the antigenic epitope peptide being one or more of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQID NO.6 or SEQ ID NO.7.

[0009] The present invention also provides the use of the antigenic epitope peptide of beta-lactoglobulin in the preparation of a low-allergenic milk protein product.

[0010] The present invention also provides the use of the antigenic epitope peptide of beta-lactoglobulin in preparing a low-allergenic whey protein product.

[0011] The present invention also provides a method for reducing BLG sensitization by using the antigenic epitope peptide of β-lactoglobulin, comprising the following steps:

[0012] (1) The β-lactoglobulin solution is mixed with simulated infant gastric juice to simulate gastric digestion, and the enzyme is inactivated to terminate the gastric digestion reaction to obtain the final product of gastric digestion;

[0013] (2) mixing the gastric digestion end product obtained in step (1) with simulated infant duodenal fluid to simulate intestinal digestion, inactivating enzymes to terminate the intestinal digestion reaction, and obtaining an in vitro digestion product;

[0014] (3) identifying a digestion-resistant polypeptide in the in vitro digestion product obtained in step (2), performing molecular docking of the digestion-resistant polypeptide with an antigen-binding fragment of immunoglobulin IgE, screening out β-lactoglobulin antigen epitope peptides with high binding energy, and determining the amino acid site where the β-lactoglobulin antigen epitope peptide binds to IgE;

[0015] (4) using the amino acid site where the β-lactoglobulin antigen epitope peptide binds to IgE in step (3) as the cleavage target, screening and selecting an alkaline protease;

[0016] (5) adding the alkaline protease described in step (4) to the in vitro digestion product obtained in step (2), performing enzymatic hydrolysis, centrifuging, taking the supernatant, and freeze-drying to obtain low-allergenic β-lactoglobulin.

[0017] Preferably, the concentration of the β-lactoglobulin solution in step (1) is 3-5% w / v; the simulated infant gastric juice in step (1) contains pepsin; the mixing ratio of the β-lactoglobulin solution and the simulated infant gastric juice in step (1) is calculated according to β-lactoglobulin: pepsin of 1 mg: 22.75 U; the pH of the simulated gastric digestion in step (1) is 3.00, the temperature of the simulated gastric digestion is 37° C., the rotation speed of the simulated gastric digestion is 500 rpm, and the time of the simulated gastric digestion is 2 hours.

[0018] Preferably, the simulated infant duodenal fluid described in step (2) contains trypsin and α-chymotrypsin; the ratio of the gastric digestion end product and the simulated infant duodenal fluid in step (2) is calculated according to β-lactoglobulin: trypsin: α-chymotrypsin of 1 mg: 3.45 U: 0.04 U; the pH of the simulated intestinal digestion in step (2) is 6.50, the temperature of the simulated intestinal digestion is 37°C, the rotation speed of the simulated intestinal digestion is 500 rpm, and the time of the simulated intestinal digestion is 2 hours.

[0019] Preferably, the amount of alkaline protease added in step (5) is calculated based on an enzyme-substrate ratio of 4% w / v; the pH of the enzymatic hydrolysis in step (5) is 10.0, the temperature of the enzymatic hydrolysis is 60° C., and the time of the enzymatic hydrolysis is 50 min.

[0020] The present invention also provides the method for reducing the allergenicity of BLG to prepare the obtained low-allergenic β-lactoglobulin.

[0021] The present invention also provides the method for reducing the allergenicity of BLG to prepare the obtained low-allergenic β-lactoglobulin or the use of the low-allergenic β-lactoglobulin in preparing low-allergenic cow's milk products.

[0022] The present invention also provides the method for reducing the allergenicity of BLG to prepare the obtained low-allergenic β-lactoglobulin or the use of the low-allergenic β-lactoglobulin in preparing a low-allergenic infant formula milk powder product.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects:

[0024] In the present invention, β-lactoglobulin is used as a raw material, and molecular docking is performed after in vitro simulation of infant gastrointestinal digestion, and the main allergenic epitopes and key amino acids that bind to IgE are screened, and a food-grade commercial protease with a targeted cleavage site is selected accordingly to further enzymolyze the digestion product and optimize the conditions, so as to achieve the purpose of specifically reducing the allergenicity of β-lactoglobulin. Among them, it is preferred to prepare an alkaline protease with low allergenicity, more target amino acid cleavage, and relatively intact enzymolysis products of the remaining peptide segments. The optimal enzymolysis conditions are pH 10.0, time 50min, and temperature 60°C. After various verifications, the allergenicity of the enzymolysis product obtained under this condition is reduced by more than 85%, and the degree of hydrolysis is not higher than 20%.

[0025] The 7 antigen epitopes screened by the present invention are obtained by molecular docking screening based on the digestion products of β-lactoglobulin infants and young children, supplementing the digestion-resistant antigen epitope library of β-lactoglobulin, and the number of amino acids in the peptide segment is 10 to 15, the sequence is relatively short, the structure is simple, the mechanism of action is clear, and it has good application prospects.

[0026] The present invention establishes a new method for reducing the allergenicity of β-lactoglobulin. Compared with the prior art, the method reduces the degradation of polypeptide chains while specifically cleaving antigen epitopes and key amino acids, thereby improving the effect of targeted allergenicity reduction to a certain extent. The method can be widely used in low-allergenic cow's milk, infant formula milk powder and special medical foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 To simulate the distribution of peptides of gastrointestinal digestion products for β-lactoglobulin;

[0029] Figure 2The binding sites and modes of the 7 screened β-lactoglobulin antigen epitope peptides to IgE-Fab, wherein a is the antigen epitope peptide shown in SEQ ID NO.1, b is the antigen epitope peptide shown in SEQ ID NO.2, c is the antigen epitope peptide shown in SEQ ID NO.3, d is the antigen epitope peptide shown in SEQ ID NO.4, e is the antigen epitope peptide shown in SEQ ID NO.5, f is the antigen epitope peptide shown in SEQ ID NO.6, and g is the antigen epitope peptide shown in SEQ ID NO.7;

[0030] Figure 3 To investigate the residual allergenicity of the in vitro digestion products of β-lactoglobulin hydrolyzed by the four screened proteases;

[0031] Figure 4 The residual allergenicity of the in vitro digestion products of β-lactoglobulin hydrolyzed by alkaline protease and papain;

[0032] Figure 5 is the degree of hydrolysis of the in vitro digestion product of β-lactoglobulin by alkaline protease and papain;

[0033] Figure 6 The peptide distribution of the in vitro digestion product of β-lactoglobulin hydrolyzed by alkaline protease, where the peptides marked in black font are the key antigen epitopes identified by screening, and each arrow represents a key amino acid;

[0034] Figure 7 The content of each component of the secondary structure of β-lactoglobulin before and after alkaline protease hydrolysis was determined by circular dichroism. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0038] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention description, which will be apparent to those skilled in the art. Other embodiments obtained from the present invention description will be apparent to the technician. The present invention description and examples are only exemplary. The experimental methods, detection methods, etc. designed in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods already available in the prior art.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Sources of materials used in the present invention: β-lactoglobulin was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai); trypsin inhibitor was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai).

[0041] Preparation of gastric electrolyte solution: 1L of freshly prepared gastric electrolyte solution contains 6.9mM KCl, 0.9mM KH2PO4, 25mM NaHCO3, 47.2mM NaCl, 0.1mM MgCl2(H2O)6 and 0.5mM (NH4)2CO3.

[0042] The formula of simulated infant gastric juice is as follows: 45.5 mg of pepsin with an enzyme activity of 250 U / mg is dissolved in 100 mL of gastric electrolyte solution, and the pH is adjusted to 3.00 using 1 M HCl to prepare simulated infant gastric juice.

[0043] Preparation of duodenal electrolyte solution: 1L of freshly prepared duodenal electrolyte solution contains 6.8mM KCl, 0.8mM KH2PO4, 85mM NaHCO3, 38.4mM NaCl, and 0.33mM MgCl2(H2O)6.

[0044] The formula of simulated infant duodenal fluid is as follows: 3.5 mg of trypsin with an enzyme activity of 250 U / mg, 18 μL of α-chymotrypsin solution, 0.43 g of sodium taurocholate, 0.30 g of deoxyglycocholic acid and 8.3 mg of CaCl2 are weighed into 100 mL of duodenal electrolyte solution, and the pH is adjusted to 6.50 with 1 M NaHCO3 to prepare simulated infant duodenal fluid.

[0045] Example 1

[0046] 1. In vitro simulation of infant gastrointestinal digestion

[0047] The pH of the β-lactoglobulin solution (concentration of 3-5% w / v) was adjusted to 3.00 using 1M HCl; then, simulated infant gastric juice was added at a volume ratio of 1:1, and the mixture was placed in a constant temperature oscillating mixer at 37°C and 500rpm for 2h to simulate gastric digestion; finally, 1M NaHCO3 was added to adjust the pH to neutral to terminate the gastric digestion reaction and obtain the final product of gastric digestion. The pH of the final product of gastric digestion was adjusted to 6.50 using 1M NaHCO3, and then simulated infant duodenal juice was added at a volume ratio of 1:1, and the mixture was placed in a constant temperature oscillating mixer at 37°C and 500rpm for 2h to simulate intestinal digestion; after the end, 10μL of trypsin inhibitor was added to terminate the enzymatic reaction, and the in vitro digestion product was obtained and stored at 4°C for testing. The distribution of the in vitro digestion product in intact β-lactoglobulin was detected by high performance liquid chromatography-tandem mass spectrometry.

[0048] The results are as follows Figure 1 As shown, most of the resistant peptides in the in vitro digestion products are located in the relatively stable β-fold cavity of β-lactoglobulin, and another part is located on the α-helical structure.

[0049] 2. Prediction of β-lactoglobulin IgE antigen binding epitopes

[0050] The three-dimensional structure of the in vitro digestion product of β-lactoglobulin identified by LC-MS / MS was constructed using ChemDraw and Chem3D systems, and energy minimization was performed. The constructed structure was stored in pdb format as a ligand library. The heavy chain H and light chain L of the IgE antibody binding fragment (antigen-binding fragment, IgE-Fab, ID: 2r56) were downloaded from the RCSB Protein DataBank protein database as receptors and imported into MOE 2020 software for further optimization, including structure correction, protonation, removal of unbound water molecules, energy optimization, etc. The processed IgE protein was stored in pdb format as a receptor protein. The Dock program in MOE software was used to perform molecular docking between the ligand and the receptor. Semi-flexible docking was used for docking, that is, the receptor protein structure remained rigid and the ligand protein remained flexible during the docking process. Scoring was performed by London dG and GBVI / WSAdG functions, and the top five of the 30 search results were retained for each peptide segment. The affinity between the peptide and the receptor protein is analyzed by the scoring function, and the interaction force and the ligand binding site are visualized by Ligand Interaction to reveal the interaction mechanism, and the amino acid residues that contribute the most to the interaction force are screened as key amino acid residues.

[0051] Table 1 Antigenic epitope peptides and key amino acids

[0052]

[0053]

[0054] The representative epitope peptides and key amino acids screened are shown in Table 1 above. The bold amino acids in the table are the amino acids with the strongest binding energy. The conformation and interaction of the peptide-IgE complex system were visualized using PyMOL. The results are shown in Figure 2 Middle a, Figure 2 Middle b, Figure 2 Middle c, Figure 2 Middle d, Figure 2 Middle e, Figure 2 Medium f and Figure 2 As shown in g.

[0055] 3. Screening of proteases that specifically destroy β-lactoglobulin allergenicity

[0056] ExPASy-Peptide_Cutter was used as an analytical tool for screening proteases, and the key amino acids in the screened antigen epitope peptides shown in Table 1 were used as cleavage targets to analyze the damage of the proteases to the target amino acids.

[0057] Table 2 Destruction of key amino acids in antigen epitope peptides by different proteases

[0058] Enzyme type System Name Theoretical damage degree / % Alkaline protease Alcalase 58.99 Papain Papain 58.99 Neutral protease Neutrase 49.99 Bromelain Bromelain 49.99 Endoaspartate Asp-Nendopeptidase 41.67 Proteinase K Proteinase K 41.67 Thermolysin Thermolysin 33.33 Lysinase N LysC 24.99 Chymotrypsin Chymotrypsin 16.67 Glutathione endopeptidase Glutamylendopeptidase 16.67 Staphylococcal peptidase Staphylococcalpeptidase 16.67 Lysyl endo-C LysN 8.33

[0059] The theoretical destruction degree is shown in Table 2 above. Then, alkaline protease, neutral protease, papain and bromelain with relatively high theoretical destruction degrees shown in Table 2 were used to enzymatically hydrolyze the in vitro digestion products of β-lactoglobulin after simulated gastrointestinal digestion, respectively. The specific operations are as follows.

[0060] Alkaline protease: Take 50 mL of β-lactoglobulin in vitro digestion product, adjust the pH to 10.0 and the temperature to 60°C, add alkaline protease according to the enzyme-substrate ratio of 4% (w / v), heat in a water bath for 1 hour, and after the hydrolysis is completed, quickly place the solution at 95°C to inactivate the enzyme for 5 minutes, then quickly cool with ice water, centrifuge the obtained enzymatic hydrolyzate at 6000r / min for 15 minutes, take the supernatant and freeze-dry it at -50°C for 40 hours, which is the alkaline protease hydrolyzate.

[0061] Neutral protease: Take 50mL of β-lactoglobulin in vitro digestion product, adjust the pH to 7.0 and the temperature to 50°C, add neutral protease according to the enzyme-substrate ratio of 4% (w / v), heat in a water bath for 1h, and after the hydrolysis is completed, quickly place the solution at 95°C to inactivate the enzyme for 5min, then quickly cool with ice water, centrifuge the obtained enzymatic hydrolyzate at 6000r / min for 15min, take the supernatant and freeze-dry it at -50°C for 40h, which is the neutral protease hydrolyzate.

[0062] Papain: Take 50mL of β-lactoglobulin in vitro digestion product, adjust the pH to 7.2, the temperature to 50℃, add papain according to the enzyme-substrate ratio of 4% (w / v), heat in a water bath for 1h, and after the hydrolysis is completed, quickly place the solution at 95℃ to inactivate the enzyme for 5min, then quickly cool with ice water, centrifuge the obtained enzymatic hydrolyzate at 6000r / min for 15min, take the supernatant, and freeze-dry at -50℃ for 40h to obtain the papain hydrolyzate.

[0063] Bromelain: Take 50mL of β-lactoglobulin in vitro digestion product, adjust the pH to 6.5, the temperature to 45°C, add bromelain according to the enzyme-substrate ratio of 4% (w / v), heat in a water bath for 1h, and after the hydrolysis is completed, quickly place the solution at 95°C to inactivate the enzyme for 5min, then quickly cool with ice water, centrifuge the obtained enzymatic solution at 6000r / min for 15min, take the supernatant, and freeze-dry at -50°C for 40h to obtain the bromelain hydrolysate.

[0064] The residual allergenicity of each enzymatic hydrolysate was determined by indirect competitive ELISA. The IgE serum of patients allergic to whey protein was used as the competitive primary antibody to evaluate the IgE binding ability of the enzymatic hydrolysate. The lower the binding ability, the smaller the residual allergenicity.

[0065] The results are as follows Figure 3 As shown, it can be seen that the residual allergenicity of β-lactoglobulin decreased after the above enzymatic hydrolysis, among which the effects of alkaline protease and papain were more obvious, which were 13.49% and 15.67%, respectively, compared with the allergenicity of the in vitro digestion product before enzymatic hydrolysis (54.99%), which were reduced by 75.47% and 71.50%, respectively, indicating that the enzymatic hydrolysis effects of alkaline protease and papain were better than those of neutral protease and bromelain, and further conditions optimization can be carried out.

[0066] 4. Optimize enzymatic hydrolysis conditions

[0067] The enzymatic conditions of the in vitro digestion products of β-lactoglobulin after simulated gastrointestinal digestion were optimized using alkaline protease and papain, which have a relatively high degree of actual allergenicity reduction. The specific operations are as follows:

[0068] Alkaline protease: Take 60 mL of β-lactoglobulin in vitro digestion product, adjust the pH to 10.0 and the temperature to 60°C, add alkaline protease according to the enzyme-substrate ratio of 4% (w / v), heat in a water bath for 0-60 min, take out 5 mL of enzymatic hydrolysate every 5 min, and after the hydrolysis is completed, quickly place the solution at 95°C to inactivate the enzyme for 5 min, then quickly cool with ice water, centrifuge the obtained enzymatic hydrolysate at 6000 r / min for 15 min, take the supernatant, and freeze-dry at -50°C for 40 h to obtain the alkaline protease hydrolysate.

[0069] Papain: Take 60 mL of β-lactoglobulin in vitro digestion product, adjust the pH to 7.2, the temperature to 50°C, add papain according to the enzyme-substrate ratio of 4% (w / v), heat in a water bath for 0-60 min, take out 5 mL of enzymatic hydrolysate every 5 min, and after the hydrolysis is completed, quickly place the solution at 95°C to inactivate the enzyme for 5 min, then quickly cool with ice water, centrifuge the obtained enzymatic hydrolysate at 6000 r / min for 15 min, take the supernatant, and freeze-dry at -50°C for 40 h to obtain the papain hydrolysate.

[0070] The residual allergenicity of each enzymatic hydrolysis product was determined by indirect competitive ELISA. Figure 4 As shown in the figure, with the increase of enzymatic hydrolysis time, the allergenicity of the enzymatic hydrolysis products showed a downward trend, among which the residual allergenicity of the alkaline protease product decreased significantly.

[0071] The degree of hydrolysis of the enzymatic hydrolysis product was determined by the OPA method. Figure 5 As shown in the figure, with the increase of enzymatic hydrolysis time, the hydrolysis degree of the enzymatic hydrolysis product gradually increased, indicating that the long peptide segment was gradually hydrolyzed into short peptides and amino acids.

[0072] The peptide distribution of alkaline protease products was analyzed by LC-MS / MS. Figure 6As shown in the figure, with the increase of enzymatic hydrolysis time, the number of peptides with more amino acids gradually decreased. According to the reduction of antigenic epitopes and key binding amino acids, it was found that when the enzymatic hydrolysis time was 50 minutes, the key amino acids on the antigenic epitopes could be basically eliminated, and the remaining peptides were relatively intact, that is, the purpose of minimizing the degradation of polypeptide chains could be achieved. The hydrolysis degree was moderate, at 16.43%; the residual allergenicity was low, at 14.29%, which was 74.01% lower than the allergenicity of the digestion product before enzymatic hydrolysis (54.99%). It shows that in vitro simulated digestion pretreatment combined with alkaline protease hydrolysis for 50 minutes can achieve the purpose of specifically reducing the allergenicity of BLG, and the obtained enzymatic hydrolyzate is a BLG hydrolyzate with low allergenicity.

[0073] Circular dichroism can be used to detect the secondary structure of proteins and analyze the effect of alkaline protease hydrolysis on the secondary structure of BLG. Figure 7 The influence of the content of each component of the secondary structure of BLG before and after enzymatic hydrolysis was determined by circular dichroism spectrometry. It can be seen that in vitro simulated gastrointestinal digestion and alkaline protease hydrolysis have a significant effect on the secondary structure content of β-lactoglobulin. BLG is a highly structured globular protein dominated by β-folding, which contains 8 to 10 antiparallel β-folding structures and an α-helical structure in its three-dimensional structure. When digested in vitro, pepsin and trypsin unfold the protein structure, break the peptides, and reduce the content of α-helix and β-folding. When further treated with alkaline protease, the number of amino acids on the polypeptide chain gradually decreases, the content of α-helix and β-folding continues to decrease, and the content of irregular coiling increases, making the previously dense and stable protein structure unfold.

[0074] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An antigenic epitope peptide of β-lactoglobulin, characterized in that: The amino acid sequence of the antigen epitope peptide is one or more of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.

7.

2. Use of the antigenic epitope peptide of β-lactoglobulin as claimed in claim 1 in the preparation of a low-allergenic milk protein product.

3. Use of the antigenic epitope peptide of β-lactoglobulin as claimed in claim 1 in the preparation of a low-allergenic whey protein product.

4. A method for reducing BLG sensitization using the antigenic epitope peptide of β-lactoglobulin according to claim 1, characterized in that: The following steps are involved: (1) The β-lactoglobulin solution is mixed with simulated infant gastric juice to simulate gastric digestion, and the enzyme is inactivated to terminate the gastric digestion reaction to obtain the final product of gastric digestion; (2) mixing the gastric digestion end product obtained in step (1) with simulated infant duodenal fluid to simulate intestinal digestion, inactivating enzymes to terminate the intestinal digestion reaction, and obtaining an in vitro digestion product; (3) identifying a digestion-resistant polypeptide in the in vitro digestion product obtained in step (2), performing molecular docking of the digestion-resistant polypeptide with an antigen-binding fragment of immunoglobulin IgE, screening out β-lactoglobulin antigen epitope peptides with high binding energy, and determining the amino acid site where the β-lactoglobulin antigen epitope peptide binds to IgE; (4) using the amino acid site where the β-lactoglobulin antigen epitope peptide binds to IgE in step (3) as the cleavage target, screening and selecting an alkaline protease; (5) adding the alkaline protease described in step (4) to the in vitro digestion product obtained in step (2), performing enzymatic hydrolysis, centrifuging, taking the supernatant, and freeze-drying to obtain low-allergenic β-lactoglobulin.

5. The method for reducing BLG sensitization according to claim 4, characterized in that: The concentration of the β-lactoglobulin solution in step (1) is 3-5% w / v; the simulated infant gastric juice in step (1) contains pepsin; the mixing ratio of the β-lactoglobulin solution and the simulated infant gastric juice in step (1) is calculated according to β-lactoglobulin: pepsin of 1 mg: 22.75 U; the pH of the simulated gastric digestion in step (1) is 3.00, the temperature of the simulated gastric digestion is 37° C., the rotation speed of the simulated gastric digestion is 500 rpm, and the time of the simulated gastric digestion is 2 hours.

6. The method for reducing BLG sensitization according to claim 4, characterized in that: The simulated infant duodenal fluid described in step (2) contains trypsin and α-chymotrypsin; the ratio of the gastric digestion end product and the simulated infant duodenal fluid in step (2) is calculated according to β-lactoglobulin: trypsin: α-chymotrypsin of 1 mg: 3.45 U: 0.04 U; the pH of the simulated intestinal digestion in step (2) is 6.50, the temperature of the simulated intestinal digestion is 37° C., the rotation speed of the simulated intestinal digestion is 500 rpm, and the time of the simulated intestinal digestion is 2 hours.

7. The method for reducing BLG sensitization according to claim 4, characterized in that: The amount of alkaline protease added in step (5) is calculated based on an enzyme-substrate ratio of 4% w / v; the pH of the enzymatic hydrolysis in step (5) is 10.0, the temperature of the enzymatic hydrolysis is 60° C., and the time of the enzymatic hydrolysis is 50 min.

8. The low-allergenic β-lactoglobulin prepared by the method for reducing the allergenicity of BLG according to any one of claims 4 to 7.

9. Use of the low-allergenic β-lactoglobulin prepared by the method for reducing BLG allergenicity according to any one of claims 4 to 7 or the low-allergenic β-lactoglobulin according to claim 8 in preparing low-allergenic milk products.

10. Use of the low-allergenic β-lactoglobulin prepared by the method for reducing BLG allergenicity according to any one of claims 4 to 7 or the low-allergenic β-lactoglobulin according to claim 8 in the preparation of a low-allergenic infant formula milk powder product.

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