Method for reducing sensitization of beta-lactoglobulin and hypoallergenic milk product

Abstract

The invention discloses a method for reducing sensitization of beta-lactoglobulin and a hypoallergenic milk product, the hypoallergenic milk product is prepared through interaction reaction of beta-lactoglobulin and a natural anti-allergic active substance, and the natural anti-allergic active substance is myricetin. The method has the beneficial effects that the allergenicity of beta-Lg can be effectively reduced, a theoretical basis is provided for mechanism research of infant food allergy, and a data support is provided for development of functional infant food; the histamine release rate of KU812 and the expression quantity of IL-4, IL-6 and the like can be remarkably reduced, the food anaphylaxis is regulated and controlled on the gene expression level, and a feasible way is provided for achieving the anti-allergic effect to a certain degree.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for reducing the allergenicity of beta-lactoglobulin and a low-allergenic milk product prepared by the method. Background Art

[0002] With improved living standards and a more diverse food supply, the incidence of food allergies has increased significantly. Dubbed the "second wave" of allergic diseases after asthma, it has become a major global issue for food safety and public health. For infants and young children, milk plays a crucial role in their diet, making it impractical to avoid it. Therefore, reducing the allergenic potential of milk has become a critical scientific challenge requiring urgent attention in the field of food safety.

[0003] Approximately 25 allergenic proteins have been identified in milk, of which β-lactoglobulin (β-Lg) is considered the major allergenic protein. Various approaches have been developed to reduce the allergenicity of β-Lg, including heating, glycosylation, ultrahigh pressure treatment (UHP), and hydrolysis. However, these methods have several significant drawbacks. Heating and UHP treatment may increase the antigenicity of products. Furthermore, glycosylation and hydrolysis not only alter flavor and texture but may also produce harmful byproducts, leaving significant potential for exploration in this area. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for reducing the allergenicity of beta-lactoglobulin and a low-allergy milk product.

[0005] To solve the above technical problems, the present invention provides the following technical solution: a method for reducing the allergenicity of β-lactoglobulin, wherein a natural anti-allergic active substance is added to a β-lactoglobulin solution, and the two interact with each other to reduce the allergenicity.

[0006] Preferably, the natural anti-allergic active substance is myricetin.

[0007] Preferably, the myricitrin solution is added to the β-lactoglobulin solution and mixed evenly, so that the structure of the allergen is changed through the non-covalent binding of β-lactoglobulin and myricitrin, thereby reducing the allergenicity.

[0008] Preferably, the molar ratio of the beta lactoglobulin solution to the myricetin solution is in the range of 1:10-1:40.

[0009] Preferably, the molar ratio of the β-lactoglobulin solution to the myricetin solution is 1:40.

[0010] Preferably, the non-covalently bound complex of β-lactoglobulin and myricetin is β-Lg-MYR.

[0011] A low-allergy milk product comprises beta-lactoglobulin in milk, and the allergenicity of the beta-lactoglobulin is reduced by the above-mentioned method.

[0012] Preferably, myricetin is added to milk, β-Lg non-covalently binds to the myricetin to change the structure of the β-Lg, and the resulting complex in the milk is β-Lg-MYR.

[0013] Preferably, myricetin is added to milk, and the molar ratio of the β-Lg to the myricetin is 1:40.

[0014] Preferably, the components in milk include β-Lg, MYR and β-Lg-MYR.

[0015] The beneficial effects of the present invention are as follows: the proposed method for reducing the allergenicity of β-lactoglobulin can effectively reduce the allergenicity of β-Lg, providing a theoretical basis for the study of the mechanism of food allergies in infants and young children, and providing data support for the development of functional infant foods; it can significantly reduce the histamine release rate of KU812 and the expression levels of IL-4, IL-6, etc., regulate food allergic reactions at the gene expression level, and provide a feasible way to achieve a certain degree of anti-allergic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a schematic diagram of the binding pattern of the β-Lg-MYR complex of the present invention; Figure 2 Schematic diagram of the fluorescence emission spectra of the β-Lg-MYR complex at different temperatures (296 K, 303 K and 310 K) of the present invention; Figure 3 Schematic diagram of the determination of the binding ability of the β-Lg-MYR complex of the present invention to IgE; Figure 4 Schematic diagram of the determination of histamine release rate of KU812 cells of the present invention; Figure 5 Schematic diagram of the relative expression levels of mRNA of KU812 cell-related genes IL-4, IL-6 and TNF-α in the present invention. DETAILED DESCRIPTION

[0017] The following describes the specific embodiments of the present invention in detail with reference to the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. It should be noted that the experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. Example

[0018] The purpose of this invention is to achieve efficient targeted development and utilization of natural products, provide a theoretical basis for the study of the mechanism of infant food allergies, provide data support for the development of functional infant foods, and provide a new technical method and idea for related research and applications in fields such as life sciences and biotechnology.

[0019] The present invention provides a method for reducing the allergenicity of beta-lactoglobulin. The method is prepared by the interaction between beta-lactoglobulin and a natural anti-allergic active substance. The natural anti-allergic active substance is myricetin. Myricetin is a natural flavonoid compound belonging to the flavonol subgroup. It is abundant in nature, has a relatively simple extraction process, and has multiple effects such as anti-inflammatory.

[0020] Reference Figure 1 Schematic diagram showing the binding pattern of the β-Lg-MYR complex. Specifically, the method for reducing the allergenicity of β-lactoglobulin comprises the following steps: (1) Weigh a certain amount of myricitrin and β-lactoglobulin on an electronic balance to obtain a mother solution; (2) Mix β-lactoglobulin and myricetin solution in a certain proportion and place in the dark for 2 hours; (3) The molar ratio of β-lactoglobulin to myricetin solution is 1:40.

[0021] Reference Figure 2 As shown in the figure, β-Lg exhibits significant fluorescence intensity with a peak emission wavelength of around 330 nm, which is mainly due to the fluorescence generated by its aromatic amino acid residues. Figure 2 The figure shows the fluorescence emission spectra of the β-Lg-MYR complex at different temperatures (296 K, 303 K, and 310 K). The lines shown at each temperature correspond to MYR concentrations from 0 to 35 μmol / L, respectively. As MYR concentration increases, the maximum emission peak of β-Lg remains relatively constant, while the fluorescence intensity gradually decreases, showing a consistent trend across different temperatures. The results show that the interaction between MYR and β-Lg effectively suppresses the intrinsic fluorescence of β-Lg. The quenching results indicate significant binding between MYR and β-Lg. Example

[0022] Methods for reducing the allergenicity of beta-lactoglobulin in vitro sensitization evaluation - ELISA.

[0023] (1) β-Lg and the β-Lg-MYR complex formed after adding MYR were pre-coated in a 96-well plate and incubated at 4°C overnight.

[0024] (2) After washing, add 250 μL of gelatin blocking solution to each well, block at 37°C for 1 hour, add human serum sample, incubate at 37°C for 2 hours, add goat anti-human IgE-HRP diluent, and incubate at 37°C for 1 hour.

[0025] (3) After washing, add TMB colorimetric solution and incubate in the dark for 30 minutes. Then add 50 μL of stop solution and measure the OD value of each well at 450 nm.

[0026] Reference Figure 3 As shown, the schematic diagram shows the determination of the binding capacity of β-Lg to IgE after adding MYR to β-Lg. As the MYR concentration increases, the IgE binding capacity of β-Lg decreases significantly, indicating that the interaction between the two reduces the allergenic potential of β-Lg. In particular, when the molar ratio of β-Lg to MYR is 1:40, the specific IgE binding capacity of the protein reaches the lowest level. Studies have shown that the ability of polyphenols to reduce the antigenicity of certain allergenic proteins is closely related to their ability to bind to linear epitopes and conformational epitopes of allergens. Therefore, this example can prove that the binding site between MYR and β-L can overlap with the binding site between IgE and β-L, that is, the binding sites between β-Lg-MYR and β-Lg-IgE completely overlap. In the case of such a variety of binding sites, because MYR can accurately and completely overlap with the binding site of IgE, it prevents the binding of epitope-specific IgE antibodies, resulting in a reduction in the allergenicity of β-Lg.

[0027] At the same time, in this embodiment, too high a MYR concentration will affect the binding effect of MYR and β-L, resulting in less β-Lg-MYR complex formed, affecting the formation of the complex, and the concentration of free β-Lg cannot be effectively reduced, thereby directly affecting the effect of reducing the sensitization to the allergen β-Lg, and causing various side effects and increasing the risk. Example

[0028] The method of reducing the allergenicity of β-lactoglobulin can reduce food allergy indicators in establishing a KU812 cell allergy model.

[0029] (1) Take out the frozen human peripheral blood basophilic leukemia cell line, resuscitate, activate and passage the cells, and culture them in a cell culture incubator (37°C, 5% CO2).

[0030] (2) The cells described above were seeded in 24-well cell culture plates. The cells were divided into a blank group, β-Lg, and β-Lg:MYR groups at different molar ratios with three biological replicates per group, and cultured in a cell culture incubator (37°C, 5% CO2).

[0031] (3) The cell supernatant was used to detect the histamine release rate. The cell pellet was used to extract total RNA, and the RNA concentration was detected using a nucleic acid protein concentration meter for subsequent experiments. The extracted RNA was immediately reverse transcribed to synthesize cDNA, and then RT-qPCR was performed, using GAPDH as the internal reference gene, according to 2 -ΔΔCt The relative expression level of the target gene was calculated by this method.

[0032] Reference Figure 4 As shown, it shows the determination of the histamine release rate of KU812 cells. The KU812 cell sensitization model was used to further evaluate the sensitization potential of pure proteins and their complexes. After being activated by allergens, IgE-activated KU812 cells are stimulated by allergens, resulting in degranulation and the release of bioactive substances, which can promote the occurrence of allergic reactions in the body. Histamine is an important indicator reflecting the degree of cell degranulation, and its content is positively correlated with the severity of allergic reactions. Compared with pure β-Lg, the histamine release rate of β-Lg-MYR complexes with different molar ratios is lower and has a certain concentration dependence, from Figure 4 It can be seen that with the increase of MYR concentration, the histamine release rate of KU812 cells gradually stabilized.

[0033] Reference Figure 5 Figure 2 shows the relative mRNA expression levels of the KU812 cell-related genes IL-4, IL-6, and TNF-α, from left to right. β-Lg significantly increased the expression levels of IL-4, TNF-α, and IL-6 in KU812 cells. Compared with β-Lg, the β-Lg-MYR complex at various molar ratios significantly reduced the expression levels of these cytokines (IL-4, IL-6, and TNF-α). These results indicate that the β-Lg-MYR complex inhibits the allergic response in KU812 cells.

[0034] In summary, the results of ELISA and cell experiments showed that β-Lg binds to MYR through non-covalent interaction, accurately coinciding with the binding site of IgE, thereby reducing the IgE binding activity of KU812 cells and inhibiting the allergic response of KU812 cells. Figure 3-5 In the table, one asterisk (*) indicates that the P value is less than 0.05, that is, the result is significant at the 0.05 level. Two asterisks (**) indicate that the P value is less than 0.01, that is, the result is significant at the 0.01 level. Three asterisks (***) indicate that the P value is less than 0.001, that is, the result is very significant at the 0.001 level. Example

[0035] This embodiment proposes a low-allergy milk product. The milk includes β-lactoglobulin. The method for reducing the allergenicity of β-lactoglobulin described in the above embodiment is used to reduce the allergenicity of β-lactoglobulin in milk, thereby obtaining a low-allergy milk product.

[0036] Specifically, myricitrin is added to milk, and β-Lg non-covalently binds to myricitrin, changing the structure of β-Lg and forming a complex in the milk called β-Lg-MYR. Therefore, the milk product in this embodiment includes free, unbound β-Lg, MYR, and β-Lg-MYR.

[0037] In actual application, myricitrin is added to milk, and the content of β-Lg in different types of milk products is detected or calculated. Therefore, even if the concentration of MYR in different types of milk products will be different, it is only necessary to add MYR according to the measured β-Lg content to ensure that the molar ratio of β-Lg to myricitrin (MYR) is 1:40, thus forming different hypoallergenic milk products with different MYR concentrations.

[0038] The natural bioactive substance that non-covalently binds to β-lactoglobulin in the present invention is myricetin, and the anti-allergic activity of the method for reducing the allergenicity of β-lactoglobulin is applied to a cell model. The potential allergenicity to β-lactoglobulin was evaluated using the KU812 cell model and the indirect ELISA method, and the changes in the relative expression levels of related genes were detected by RT q-PCR. The mechanism of action of the method for reducing the allergenicity of β-lactoglobulin was explored at the gene level. The purpose of the present invention is to achieve efficient targeted development and utilization of natural active products, provide a theoretical basis for the study of the mechanism of infant food allergies, provide data support for the development of functional infant foods, and provide a new technical method and idea for related research and applications in the fields of life sciences and biotechnology.

[0039] It should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will appreciate that other variations or modifications can be made based on the above descriptions and ideas, and it is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the technical solutions of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A method for reducing the allergenicity of β-lactoglobulin, characterized by: Natural anti-allergic active substances are added to the β-lactoglobulin solution, and the two interact with each other to reduce allergenicity.

2. The method for reducing β-lactoglobulin allergenicity according to claim 1, characterized in that: The natural anti-allergic active substance is myricetin.

3. The method for reducing β-lactoglobulin allergenicity according to claim 1, characterized in that: Myricetin solution is added to the β-lactoglobulin solution and mixed evenly. The non-covalent binding of β-lactoglobulin and myricetin changes the structure of the allergen and reduces the allergenicity.

4. The method for reducing β-lactoglobulin allergenicity according to claim 1, wherein: The molar ratio of the beta lactoglobulin solution to the myricetin solution is 1:10-1:

40.

5. The method for reducing β-lactoglobulin allergenicity according to claim 1, characterized in that: The molar ratio of the beta lactoglobulin solution to the myricetin solution is 1:

40.

6. The method for reducing β-lactoglobulin allergenicity according to claim 3, characterized in that: The complex formed by non-covalent binding of β-lactoglobulin and myricetin is β-Lg-MYR.

7. A hypoallergenic milk product, characterized by: Milk contains β-lactoglobulin, and the method according to claims 1 to 6 is used to reduce the allergenicity of β-lactoglobulin.

8. The hypoallergenic milk product according to claim 7, characterized in that: Myricetin is added to milk, and β-Lg non-covalently binds to the myricetin to change the structure of the β-Lg. The resulting complex in the milk is β-Lg-MYR.

9. The hypoallergenic milk product according to claim 8, characterized in that: Myricetin is added to milk, and the molar ratio of the β-Lg to the myricetin is 1:

40.

10. The hypoallergenic milk product according to claim 7, characterized in that: The components in milk include β-Lg, MYR and β-Lg-MYR.

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