An antigenic epitope peptide of alpha-lactalbumin and a method for reducing ala sensitization by using the same

Abstract

The application discloses an antigen epitope peptide of alpha-lactalbumin and a method for reducing ALA sensitization by using the antigen epitope peptide, and belongs to the technical field of protein engineering, and provides the antigen epitope peptide, and the amino acid sequence is shown in SEQ ID NO.1-SEQ ID NO.5. The method for reducing ALA sensitization by using the antigen epitope peptide of alpha-lactalbumin comprises the following steps: performing simulation infant gastrointestinal digestion on an alpha-lactalbumin solution to obtain in-vitro digestion products; identifying digestion-resistant polypeptides in the in-vitro digestion products to screen alpha-lactalbumin antigen epitope peptides and determine IgE binding amino acid sites; taking the amino acid sites as cutting targets to screen and determine selected alkaline proteases; adding the in-vitro digestion products to perform enzymolysis to obtain low-sensitization alpha-lactalbumin. The application solves the problem that the alpha-lactalbumin desensitization method is not specific enough, and provides the antigen epitope peptide to specifically reduce the sensitization of alpha-lactalbumin.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein engineering, and in particular relates to an antigenic epitope peptide of alpha-lactalbumin and a method for reducing the allergenicity of ALA by utilizing the same. Background Art

[0002] Cow's milk protein, a functional protein with rich nutritional value, is widely used in the food industry, especially in infant formula. However, due to genetic allergies, imperfect digestive and immune system development, and imperfect intestinal barrier function, some infants and young children may develop cow's milk protein allergy (CMPA) during cow's milk consumption. This can lead to symptoms such as vomiting, diarrhea, respiratory asthma, and skin allergies. Globally, the prevalence of CMPA in infants and young children ranges from 2% to 7.5%, while in adults it is less than 1%. Recent changes in lifestyle and social structure have made infant formula the preferred alternative and complementary food for breastfeeding. However, this has also increased the risk of CMPA in infants and young children. To ensure the food safety of infants and young children, it is crucial to reduce the allergenic potential of infant formula. Currently, extensively hydrolyzed infant formula (eHF) is commercially available for infants with CMPA. However, studies have shown that infants consuming eHF weigh significantly less than normal infants before their first birthday, indicating that eHF loses some of the nutritional benefits of milk protein. Furthermore, the deeper the hydrolysis, the bitterer the taste. Therefore, partially hydrolyzed infant formula, with relatively few allergenic residues, is an ideal milk powder for infants with CMPA.

[0003] Whey protein is currently a key ingredient in infant formula, with α-lactalbumin (ALA) being one of the most prominent allergens in milk protein, present in milk at concentrations of 1 to 1.5 g / L. ALA's spatial structure relies on four disulfide bonds to form a stable globular protein, consisting of four α-helices and three antiparallel β-sheets. This stable structure makes ALA a significant allergen in milk protein. However, ALA plays an important regulatory role in the galactosyltransferase system during lactose synthesis and can also interact with lipid membranes and stearic acid. Therefore, effectively mitigating the allergenicity of ALA is crucial.

[0004] After milk allergens are ingested by the body and digested through the gastrointestinal tract, the normal body will develop oral tolerance to the antigen. However, when oral tolerance is broken or lost, patients with milk allergies will develop abnormal IgE reactions to the allergen protein. Since proteins that can cause allergies are generally resistant 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 ALA based on the degree of reduction of allergic epitopes. As a result, ingredients of deep hydrolysis of proteins that are not conducive to infant growth and development are still widely used. Therefore, there is an increasing call for methods that can accurately eliminate whey protein antigen epitopes while retaining other functional structures.

[0005] In summary, no reports have been found using simulated gastrointestinal digestion to pre-treat ALA, screen for digestion-resistant IgE-binding epitopes and key amino acids, and then select appropriate proteases based on these pre-treatments to improve the specific reduction of ALA allergenicity. Therefore, developing a method for reducing the allergenicity of α-lactalbumin based on in vitro simulated digestion combined with specific enzymatic hydrolysis is an urgent issue for those skilled in the art. Summary of the Invention

[0006] To solve the above technical problems, the present invention proposes an antigenic epitope peptide of α-lactalbumin and a method for reducing the allergenicity of ALA using the same, which solves the problem of low specificity of the α-lactalbumin desensitization method. α-lactalbumin is first subjected to simulated in vitro digestion, and the antigenic epitope peptide and amino acid binding site that play a key role in allergies are determined using the molecular docking method. Then, alkaline protease is used to cleave the binding site, thereby specifically reducing the allergenicity of α-lactalbumin.

[0007] To achieve the above object, the present invention provides an α-lactalbumin epitope peptide, the amino acid sequence of the 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 or SEQ ID NO.5.

[0008] The present invention also provides the use of the antigenic epitope peptide of α-lactalbumin in the preparation of a low-allergenic milk protein product.

[0009] The present invention also provides the use of the antigenic epitope peptide of α-lactalbumin in the preparation of a low-allergenic whey protein product.

[0010] The present invention also provides a method for reducing ALA allergenicity by using the antigenic epitope peptide of α-lactalbumin, comprising the following steps:

[0011] (1) α-lactalbumin 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 gastric digestion product;

[0012] (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;

[0013] (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 for digestion-stable α-lactalbumin antigen epitope peptides, and determining the amino acid site at which the α-lactalbumin antigen epitope peptide binds to IgE;

[0014] (4) using the amino acid site where the α-lactalbumin antigen epitope peptide binds to IgE in step (3) as the cleavage target, and screening and selecting an alkaline protease;

[0015] (5) adding the alkaline protease described in step (4) to the in vitro digestion product obtained in step (2), performing enzymatic hydrolysis, centrifuging, collecting the supernatant, and freeze-drying to obtain hypoallergenic α-lactalbumin.

[0016] Preferably, the concentration of the α-lactalbumin solution in step (1) is 3-5% w / v; the simulated infant gastric juice in step (1) contains pepsin; the ratio of the α-lactalbumin solution and the simulated infant gastric juice in step (1) is calculated according to 1 mg α-lactalbumin: 22.75 U pepsin; the pH of the simulated gastric digestion in step (1) is 3.00, the temperature of the simulated gastric digestion is 37° C., the speed of the simulated gastric digestion is 500 rpm, and the time of the simulated gastric digestion is 2 hours.

[0017] Preferably, the simulated infant duodenal fluid 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 α-lactalbumin: 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 h.

[0018] 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.

[0019] The present invention also provides the method for reducing the allergenicity of ALA and the resulting low-allergenic α-lactalbumin.

[0020] The present invention also provides the method for reducing the allergenicity of ALA to prepare the obtained low-allergenic α-lactalbumin or the use of the low-allergenic α-lactalbumin in preparing low-allergenic milk products.

[0021] The present invention also provides the method for reducing the allergenicity of ALA to prepare the obtained low-allergenic α-lactalbumin or the use of the low-allergenic α-lactalbumin in preparing a low-allergenic infant formula milk powder product.

[0022] Compared with the prior art, the present invention has the following advantages and technical effects:

[0023] The present invention uses α-lactalbumin as raw material, performs molecular docking after in vitro simulated infant gastrointestinal digestion, screens out the main allergenic antigen epitope peptides and key amino acid sites that bind to IgE, and selects a food-grade commercial alkaline protease with targeted cleavage sites to further enzymolyze the digestion product, thereby achieving the purpose of specifically reducing the allergenicity of α-lactalbumin. Alkaline protease is selected for further enzymolysis to prepare an enzymolyzate with low allergenicity, with more target amino acid cleavage and relatively intact remaining peptide segments. The optimal enzymolysis conditions are pH 10.0, time 50 minutes, and temperature 60°C. After various verifications, the enzymolyzate product obtained under these conditions, the allergenicity of the low-allergenic α-lactalbumin, is reduced by more than 85%, and the degree of hydrolysis is not higher than 20%.

[0024] The present invention screened out 5 antigen epitope peptides, which were obtained based on the digestion products of α-lactalbumin infants and young children through molecular docking screening, supplementing the digestion-resistant antigen epitope library of α-lactalbumin. The peptide segments have 8-15 amino acids, are relatively short in sequence, simple in structure, and have a clear mechanism of action, thus having good application prospects.

[0025] The present invention establishes a new method for reducing the allergenicity of α-lactalbumin. Compared with the existing technology, it reduces the degradation of the polypeptide chain while specifically cleaving the antigen epitope 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

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0027] Figure 1 To simulate the peptide distribution of gastrointestinal digestion products of α-lactalbumin;

[0028] Figure 2 The binding sites and modes of the five screened antigenic epitope peptides α-lactalbumin and IgE-Fab are shown, wherein a is the antigenic epitope peptide shown in SEQ ID NO.1, b is the antigenic epitope peptide shown in SEQ ID NO.2, c is the antigenic epitope peptide shown in SEQ ID NO.3, d is the antigenic epitope peptide shown in SEQ ID NO.4, and e is the antigenic epitope peptide shown in SEQ ID NO.5;

[0029] Figure 3 The residual allergenicity of the in vitro digestion products of α-lactalbumin hydrolyzed by four proteases;

[0030] Figure 4 The residual allergenicity of the in vitro digestion products of α-lactalbumin hydrolyzed by alkaline protease and papain;

[0031] Figure 5 is the degree of hydrolysis of the in vitro digestion product of α-lactalbumin by alkaline protease and papain;

[0032] Figure 6 The peptide distribution of the in vitro digestion product of α-lactalbumin hydrolyzed by alkaline protease. The black-font peptides in the figure are the key antigen epitopes identified by screening, and each arrow represents a key amino acid.

[0033] Figure 7 The contents of the secondary structure components of α-lactalbumin before and after alkaline protease hydrolysis were determined by circular dichroism. DETAILED DESCRIPTION

[0034] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] Sources of materials used in the present invention: α-lactalbumin was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai); trypsin inhibitor was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai).

[0040] Preparation of gastric electrolyte solution: 1L of freshly prepared gastric electrolyte solution contains 6.9mM KCl, 0.9mMKH2PO4, 25mM NaHCO3, 47.2mMNaCl, 0.1mM MgCl2(H2O)6 and 0.5mM (NH4)2CO3.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] Example 1

[0045] 1. In vitro simulation of infant gastrointestinal digestion

[0046] The pH of the α-lactalbumin solution (3-5% w / v) was adjusted to 3.00 using 1M HCl. Simulated infant gastric fluid was then added in a 1:1 volume ratio and the mixture was incubated in a thermostatic shaker at 37°C and 500 rpm for 2 hours to simulate gastric digestion. Finally, 1M NaHCO₃ was added to neutral pH to terminate gastric digestion, yielding the final gastric digestion product. The pH of the final gastric digestion product was adjusted to 6.50 using 1M NaHCO₃. Simulated infant duodenal fluid was then added in a 1:1 volume ratio and the mixture was incubated in a thermostatic shaker at 37°C and 500 rpm for 2 hours to simulate intestinal digestion. 10 μL of trypsin inhibitor was added to terminate the enzymatic digestion reaction, yielding the in vitro digestion product, which was stored at 4°C until further analysis. The distribution of the in vitro digestion product within intact α-lactalbumin was determined by high-performance liquid chromatography-tandem mass spectrometry.

[0047] The results are as follows Figure 1 As shown in the figure, most of the resistant peptides in the in vitro digestion products are located in the relatively stable β-pleated cavity of α-lactalbumin, and another part is located on the α-helical structure.

[0048] 2. Prediction of α-lactalbumin IgE antigen binding epitopes

[0049] The three-dimensional structure of an in vitro digest of α-lactalbumin, identified by LC-MS / MS, was constructed using ChemDraw and Chem3D systems. Energy minimization was performed, and the constructed structure was stored in pdb format as a ligand library. The heavy chain H and light chain L of an IgE antibody-binding fragment (IgE-Fab, ID: 2r56) were downloaded from the RCSB Protein DataBank as a receptor and imported into MOE 2020 software for further optimization, including structural modification, protonation, removal of unbound water molecules, and energy optimization. The processed IgE protein was stored in pdb format as a receptor protein. Molecular docking of the ligand and receptor was performed using the Dock program in MOE software. Semi-flexible docking was used, meaning that the receptor protein structure remained rigid and the ligand protein remained flexible during the docking process. Scoring was performed using the London dG and GBVI / WSAdG functions, with the top five peptides from each of the 30 search results retained. The affinity between the peptide and the receptor protein is analyzed by the scoring function, and the interaction force and 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.

[0050] Table 1 Antigen epitope peptides and key amino acids

[0051]

[0052]

[0053] 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 as follows: Figure 2 Middle a, Figure 2 Middle b, Figure 2 Middle C, Figure 2 Zhongd and Figure 2 As shown in e.

[0054] 3. Screening of proteases that specifically destroy α-lactalbumin allergenicity

[0055] 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.

[0056] Table 2 Destruction of key amino acids in antigenic epitope peptides by different proteases

[0057] Enzyme type System Name Theoretical damage degree / % Alkaline protease Alcalase 55.56 Papain Papain 55.56 Neutral protease Neutrase 50.00 Bromelain Bromelain 50.00 Endoaspartate Asp-Nendopeptidase 44.44 Proteinase K Proteinase K 44.44 Thermolysin Thermolysin 38.89 Lysinyl endo-N LysC 33.33 Chymotrypsin Chymotrypsin 27.78 Glutamyl endopeptidase Glutamylendopeptidase 22.22 Staphylococcal peptidase Staphylococcalpeptidase 22.22 Lysinyl endo-C LysN 11.11

[0058] 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 α-lactalbumin after simulated gastrointestinal digestion. The specific operations are as follows.

[0059] Alkaline protease: Take 50 mL of the in vitro digestion product of α-lactalbumin, adjust the pH to 10.0 and the temperature to 60°C, add alkaline protease at an enzyme-substrate ratio of 4% (w / v), and heat in a water bath for 1 hour. 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 resulting enzymatic hydrolyzate at 6000 rpm for 15 minutes, and take the supernatant and freeze-dry it at -50°C for 40 hours to obtain the alkaline protease hydrolyzate.

[0060] Neutral protease: Take 50 mL of α-lactalbumin in vitro digestion product, adjust the pH to 7.0 and the temperature to 50°C, add neutral protease at an enzyme-substrate ratio of 4% (w / v), and heat in a water bath for 1 hour. 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 resulting enzymatic hydrolyzate at 6000 rpm for 15 minutes, and take the supernatant and freeze-dry it at -50°C for 40 hours. This is the neutral protease hydrolyzate.

[0061] Papain: Take 50 mL of the in vitro digestion product of α-lactalbumin, adjust the pH to 7.2 and the temperature to 50°C, add papain at an enzyme-substrate ratio of 4% (w / v), and heat in a water bath for 1 hour. 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 resulting enzymatic hydrolyzate at 6000 rpm for 15 minutes, collect the supernatant, and freeze-dry at -50°C for 40 hours to obtain the papain hydrolyzate.

[0062] Bromelain: Take 50 mL of α-lactalbumin in vitro digestion product, adjust the pH to 6.5 and the temperature to 45°C, add bromelain according to the enzyme-substrate ratio of 4% (w / v), and heat in a water bath for 1 hour. 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 resulting enzymatic solution at 6000 rpm for 15 minutes, take the supernatant, and freeze-dry at -50°C for 40 hours to obtain the bromelain hydrolyzate.

[0063] The residual allergenicity of each enzymatic hydrolysis product was determined by indirect competitive ELISA. The IgE serum of patients allergic to whey protein was used as the competing primary antibody to evaluate the IgE binding ability of the enzymatic hydrolysis product. The lower the binding ability, the lower the residual allergenicity.

[0064] The results are as follows Figure 3As shown in the figure, it can be seen that the residual allergenicity of α-lactalbumin decreased after the above enzymatic hydrolysis, among which the effects of alkaline protease and papain were more obvious, which were 9.13% and 12.47% respectively. Compared with the allergenicity of the in vitro digestion product before enzymatic hydrolysis (46.33%), they were reduced by 80.29% and 73.08% respectively, indicating that the enzymatic hydrolysis effects of alkaline protease and papain were better than those of neutral protease and bromelain, and further condition optimization can be carried out.

[0065] 4. Optimize enzymatic hydrolysis conditions

[0066] The enzymatic hydrolysis conditions of the in vitro digestion products of α-lactalbumin after simulated gastrointestinal digestion were optimized using alkaline protease and papain, which have relatively high actual allergenicity reduction. The specific operation is as follows:

[0067] Alkaline protease: Take 60 mL of the in vitro digestion product of α-lactalbumin, adjust the pH to 10.0 and the temperature to 60°C, add alkaline protease at an enzyme-substrate ratio of 4% (w / v), heat in a water bath for 0-60 minutes, remove 5 mL of the enzymatic hydrolysate every 5 minutes, 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 resulting enzymatic hydrolysate at 6000 rpm for 15 minutes, take the supernatant, and freeze-dry at -50°C for 40 hours to obtain the alkaline protease hydrolysate.

[0068] Papain: Take 60 mL of the in vitro digestion product of α-lactalbumin, adjust the pH to 7.2 and the temperature to 50°C, add papain at an enzyme-substrate ratio of 4% (w / v), and heat in a water bath for 0-60 minutes. Remove 5 mL of the hydrolyzate every 5 minutes. After the hydrolysis is completed, quickly incubate the solution at 95°C for 5 minutes to inactivate the enzyme, then quickly cool with ice water. Centrifuge the resulting hydrolyzate at 6000 rpm for 15 minutes, take the supernatant, and freeze-dry at -50°C for 40 hours to obtain the papain hydrolyzate.

[0069] 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.

[0070] 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.

[0071] The peptide distribution of alkaline protease products was analyzed by LC-MS / MS. Figure 6As shown, with increasing enzymatic hydrolysis time, the number of peptides with a large number of amino acids gradually decreased. Based on 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 epitope were essentially eliminated, while the remaining peptides remained relatively intact, thus minimizing the degradation of the polypeptide chain. Furthermore, the degree of hydrolysis was moderate, at 16.27%, and the residual allergenicity was low, at 11.91%, a 74.29% reduction compared to the allergenicity of the digestion product before enzymatic hydrolysis (46.33%). This indicates that in vitro simulated digestion pretreatment combined with alkaline protease hydrolysis for 50 minutes can achieve the goal of specifically reducing the allergenicity of ALA, and the resulting enzymatic hydrolyzate is an ALA hydrolyzate with low allergenicity.

[0072] 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 ALA. Figure 7 Figure 3 shows the effects of ALA secondary structure components before and after enzymatic digestion, as measured by circular dichroism. It can be seen that in vitro simulated gastrointestinal digestion and alkaline protease hydrolysis significantly affect the secondary structure of α-lactalbumin. During in vitro digestion, pepsin and trypsin disrupt the stable globular structure of ALA, causing peptide fragmentation and a decrease in α-helical and β-sheet content. Further treatment with alkaline protease gradually reduces the number of amino acids in the polypeptide chain, further decreasing the α-helical and β-sheet content, while increasing the random coil content, causing the previously compact and stable protein structure to unfold.

[0073] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for reducing the allergenicity of ALA, characterized in that: The following steps are involved: (1) α-lactalbumin solution is mixed with simulated infant gastric juice to simulate gastric digestion, and the enzyme is inactivated to terminate the gastric digestion reaction and obtain the final gastric digestion product; (2) The gastric digestion end product obtained in step (1) is mixed with simulated infant duodenal fluid to simulate intestinal digestion, and the enzyme is inactivated to terminate the intestinal digestion reaction to obtain an in vitro digestion product; (3) identifying the digestion-resistant polypeptides in the in vitro digestion product obtained in step (2), performing molecular docking of the digestion-resistant polypeptides with the antigen-binding fragment of immunoglobulin IgE, screening to obtain digestion-stable α-lactalbumin antigen epitope peptides, and determining the amino acid sites at which the α-lactalbumin antigen epitope peptides bind to IgE; (4) using the amino acid site where the α-lactalbumin 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 hypoallergenic α-lactalbumin; The simulated infant gastric juice described in step (1) contains pepsin; The simulated infant duodenal fluid described in step (2) contains trypsin and α-chymotrypsin.

2. The method for reducing ALA allergenicity according to claim 1, characterized in that: The concentration of the α-lactalbumin solution in step (1) is 3% to 5% w / v; the ratio of the α-lactalbumin solution and the simulated infant gastric juice in step (1) is calculated according to α-lactalbumin: 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 speed of the simulated gastric digestion is 500 rpm, and the time of the simulated gastric digestion is 2 h.

3. The method for reducing ALA allergenicity according to claim 1, characterized in that: The ratio of the final product of gastric digestion and the simulated infant duodenal fluid in step (2) is calculated according to 1 mg of α-lactalbumin: trypsin: α-chymotrypsin: 1 mg of α-lactalbumin: 3.45 U of α-chymotrypsin: 0.04 U of α-chymotrypsin; 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 h.

4. The method for reducing ALA allergenicity according to claim 1, 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.

5. Hypoallergenic α-lactalbumin prepared by the method for reducing the allergenicity of ALA according to any one of claims 1 to 4.

6. Use of the hypoallergenic α-lactalbumin according to claim 5 in the preparation of a hypoallergenic milk product.

7. Use of the hypoallergenic α-lactalbumin according to claim 5 in the preparation of a hypoallergenic infant formula milk powder product.

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