Method for reducing sensitization of beta-lactoglobulin through cooperation of ultrasound and myricetin

Abstract

The invention discloses a method for cooperatively reducing sensitization of beta-lactoglobulin by ultrasound and myricetin, which comprises the following steps: uniformly mixing beta-lactoglobulin with pure water, dissolving myricetin with methanol, mixing the beta-lactoglobulin and the myricetin in proportion, carrying out ultrasonic treatment together, and finally freeze-drying the beta-lactoglobulin-myricetin mixed solution subjected to ultrasonic treatment to obtain the beta-lactoglobulin-myricetin. Therefore, the hypoallergenic beta-lactoglobulin-myricetin powder is obtained, and the allergenicity of beta-lactoglobulin is reduced by more than 40%. According to the method disclosed by the invention, the sensitization of the beta-lactoglobulin can be effectively reduced under the condition that the primary structure of the beta-lactoglobulin is not changed, and the oxidation resistance of the product is improved.

Description

Technical Field

[0001] This invention belongs to the field of food processing, and specifically relates to a method for synergistically reducing the sensitization of β-lactoglobulin by ultrasound and myricetin. Background Technology

[0002] Cow's milk is one of the most important sources of high-quality protein in modern society and one of the most important supplementary foods for infants and young children. Cow's milk protein mainly consists of casein and whey protein, with the former accounting for approximately 80% and the latter approximately 20%. Whey protein is a high-quality protein extracted from whey; it can be used directly as a high-protein supplement and is also an important food processing raw material with high utilization value. However, milk proteins (including whey protein) are also one of the eight major allergens recognized by the World Health Organization (milk, eggs, fish, shellfish, nuts, peanuts, wheat, and soybeans). The main allergens in whey protein include β-lactoglobulin, α-lactalbumin, bovine serum albumin, immunoglobulins, and lactoferrin. β-lactoglobulin is the core component of whey protein in fresh milk, accounting for more than 50% of whey protein and approximately 7% to 12% of the total protein in fresh milk. Because β-lactoglobulin is completely absent in human breast milk, it is one of the most significant allergens in cow's milk. Milk protein allergy can trigger symptoms such as allergic rhinitis, asthma, eczema, diarrhea, and gastrointestinal bleeding, threatening the health of allergy sufferers and even leading to death. Therefore, the research and development of hypoallergenic functional milk proteins has attracted significant attention from the medical and nutritional communities.

[0003] Studies have found that methods such as heating, hydrolysis, high pressure, glycosylation, and methylation can reduce the allergenicity of milk. However, improper handling of these methods can destroy nutrients in milk, significantly reduce its taste, and even cause bitterness. In recent years, technologies have been developed to combine plant polyphenols with milk proteins to reduce the allergenicity of the latter. For example, patent 201811150233.4 discloses a method for changing the conformational and linear epitopes of milk proteins by reacting tea polyphenols with milk proteins under alkaline conditions; patent 202411028410.7 discloses a method for a low-allergenic whey protein hydrolysate-polyphenol complex, in which whey protein is first subjected to ultrasonic treatment, then hydrolyzed using a complex enzyme composed of endopeptidases and exopeptidases, and finally at least one of epigallocatechin gallate, chlorogenic acid, caffeic acid, gallic acid, and quercetin is added for stirring reaction. However, these technologies either fail to reduce the allergenicity of milk proteins effectively or cause excessive damage to the structure of milk proteins, thus affecting their flavor and functional properties. Therefore, it is essential to develop a technology that reduces the allergenicity of milk proteins without affecting their primary structure. Summary of the Invention

[0004] The purpose of this invention is to provide a method for synergistically reducing the sensitization of β-lactoglobulin using ultrasound and myricetin. This method can enhance the sensitization effect of myricetin on β-lactoglobulin without affecting the primary structure of β-lactoglobulin.

[0005] The above-mentioned objective of the present invention can be achieved by the following technical solution: a method for synergistically reducing the sensitization of β-lactoglobulin by ultrasound and myricetin, comprising the following steps:

[0006] (1) Dissolve β-lactoglobulin in purified water to prepare an aqueous solution of β-lactoglobulin, dissolve myricetin in methanol to prepare a methanol solution of myricetin, and mix the aqueous solution of β-lactoglobulin and the methanol solution of myricetin in proportion to prepare a non-covalent mixture of β-lactoglobulin and myricetin.

[0007] (2) The β-lactoglobulin-myricetin non-covalent mixture obtained in step (1) was subjected to ultrasonic treatment;

[0008] (3) Freeze-dry the product after ultrasonic treatment to obtain low-allergenic β-lactoglobulin-myricetin powder.

[0009] In the above-mentioned method of synergistic reduction of β-lactoglobulin sensitization by ultrasound and myricetin:

[0010] Preferably, the concentration of the β-lactoglobulin aqueous solution in step (1) is 1 to 10 mg / mL.

[0011] Preferably, the concentration of the myricetin methanol solution in step (1) is 1 to 10 mg / mL.

[0012] Preferably, the molar ratio of β-lactoglobulin to myricetin in step (1) is 1:8 to 12.

[0013] Preferably, in step (1), the β-lactoglobulin aqueous solution and the myricetin methanol solution are mixed in a shaker at room temperature for 1 to 3 hours; more preferably, for 2 hours.

[0014] Preferably, in step (2), the ultrasonic treatment is carried out in an ice bath with an ultrasonic intensity of 240W. Intermittent ultrasonic treatment is used, that is, each ultrasonic treatment lasts for 7 seconds and is paused for 3 seconds, for a total of 5 to 30 minutes.

[0015] More preferably, in step (2), the ultrasonic treatment is carried out in an ice bath, with the ultrasonic probe immersed in the sample about 1 cm from the bottom, the ultrasonic intensity is 240 W, and intermittent ultrasonic treatment is used, that is, each ultrasonic treatment lasts 7 seconds and stops for 3 seconds, for a total of 10 to 30 minutes.

[0016] Preferably, in step (3), the product after ultrasonic treatment is first dialyzed in ultrapure water for 48 hours and then freeze-dried.

[0017] Preferably, in step (3), during freeze drying, the freeze is first pre-frozen at -20°C and then freeze-dried at -40 to -50°C until dry.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) Excellent effect in reducing the allergenicity of milk protein: When β-milk protein and myricetin are mixed and ultrasonically treated together, the allergenic epitopes hidden in β-milk protein can be bound by myricetin at the same time as they are exposed, thus preventing the allergenic epitopes from hiding again after ultrasonic treatment is stopped, thereby further enhancing the effect of myricetin in reducing the allergenicity of milk protein.

[0020] (2) It does not damage the primary structure of milk protein: SDS-PAGE electrophoresis analysis showed that the prepared low-allergenic β-lactoglobulin-myricetin sample did not change the primary structure of β-lactoglobulin (molecular weight remained unchanged);

[0021] (3) Simple operation: The main operations involved in this invention include dissolving milk protein and myricetin, mixing the two solutions, ultrasonic treatment of the mixed solution and freeze drying, etc., which are relatively simple to operate. Attached Figure Description

[0022] Figure 1 This represents the effect of each group of samples in Example 1 on reducing β-LG sensitization. The vertical axis represents the IgE binding inhibition rate (%).

[0023] Figure 2 These are the SDS-PAGE analysis results of each group of samples in Example 1, where bands 1-5 represent β-LG, U-β-LG, β-LG-M, U(β-LG-M) and U(β-LG)-M, respectively.

[0024] Figure 3 This is the effect of ultrasound-assisted non-covalent binding of myricetin on the secondary structure of β-LG in Example 1. (a) is the circular dichroism chromatogram of each group, with the vertical axis Circular Dichroism (mdeg) representing the circular dichroism chromatogram. (b) is the secondary structure composition, with the vertical axis Secondary structure content (%) representing the secondary structure composition. Detailed Implementation

[0025] The following embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention. Those skilled in the art can achieve the objectives of the present invention based on the above-disclosed scope.

[0026] Unless otherwise specified in the following implementation plan, standard test conditions or the test conditions recommended by the instrument company are generally followed. Unless otherwise specified, all materials and reagents used are commercially available.

[0027] Example 1

[0028] (I) Screening of polyphenol myricetin:

[0029] Dissolve 50 mg β-LG in 25 mL of purified water, stir at room temperature for 2 h, then place in a 4 °C refrigerator overnight until fully dissolved, and determine the protein concentration using a BCA kit.

[0030] Weigh 5 mg of different polyphenols and prepare a 2 mg / mL solution with methanol. Store the solution at -20°C in the dark.

[0031] β-LG aqueous solution and different polyphenol solutions were mixed at a molar ratio of 1:16 and reacted in a shaker at room temperature for 2 hours.

[0032] After mixing, each group of sample solutions was dialyzed in ultrapure water for 48 hours, with the dialysate changed every 6 hours. After the dialysate was completed, the solution was freeze-dried until dry.

[0033] The IgE binding capacity of different polyphenols to β-LG complexes was evaluated by ELISA (the same below), and the results are shown in Table 1. Thus, the flavonoid myricetin was selected as having the best effect in reducing the sensitization of β-LG.

[0034] Table 1. Effects of 30 polyphenols on the binding capacity of β-LG IgE

[0035]

[0036]

[0037] (II) A method for synergistic reduction of β-lactoglobulin sensitization by ultrasound and myricetin

[0038] (1) β-lactoglobulin dissolution: 50 mg β-LG was dissolved in 25 mL of purified water, stirred at room temperature for 2 h, and then placed in a 4 °C refrigerator overnight to fully dissolve. The protein concentration was determined using a BCA kit.

[0039] Myricetin dissolution: Weigh 5 mg of myricetin and prepare a 2 mg / mL solution with methanol. Store in the dark at -20°C.

[0040] Add myricetin methanol solution dropwise to β-LG aqueous solution at a molar ratio of 1:8 (β-LG: myricetin), and react in a shaker at room temperature for 2 hours.

[0041] (2) After the two are mixed evenly, ultrasonic treatment is performed using an ultrasonic cell disruptor. The ultrasonic probe with a diameter of 6 mm is immersed in the sample, 1 cm from the bottom, and the ultrasonic intensity is 240 W. Intermittent ultrasonic treatment is used, that is, each ultrasonic treatment is 7 seconds and then stopped for 3 seconds, for a total of 15 minutes. The ultrasonic treatment is carried out in an ice bath to prevent the solution from heating during the ultrasonic process.

[0042] The groups are divided into the following 5 groups:

[0043] The untreated β-LG solution is classified as the β-LG group.

[0044] The β-LG solution treated with ultrasound belongs to the U-β-LG group;

[0045] Untreated β-LG was mixed with myricetin, forming the β-LG-M group;

[0046] After ultrasonic pretreatment, β-LG was mixed with myricetin, resulting in the U(β-LG)-M group.

[0047] β-LG was mixed with myricetin and then subjected to ultrasound treatment, resulting in the U(β-LG-M) group.

[0048] (3) After dialyzing each treatment group's sample solution in ultrapure water for 48 hours, it was freeze-dried. The effect of each group's sample on reducing β-LG sensitization is shown in the figure. Figure 1 The study found that mixing the two substances and then using ultrasound to reduce sensitization yielded the best results, with β-LG showing an IgE binding inhibition rate of 46.0%, significantly higher than other groups. SDS-PAGE analysis showed... Figure 2 The molecular weights of the two molecules did not change significantly, indicating that myricetin and ultrasonic treatment had no effect on the primary structure of β-LG. Figure 3 The effect of ultrasound-assisted non-covalent binding of myricetin on the secondary structure of β-LG is shown, including circular dichroism chromatograms (a) and secondary structure composition (b) for each group. Figure 3 It can be seen that myricetin and ultrasonic treatment affect the secondary structure of β-LG, thereby reducing its sensitization by influencing its linear epitopes and conformational epitopes.

[0049] Example 2

[0050] (i) Same as in Example 1, myricetin was selected as the polyphenol with the best ability to reduce the sensitization of β-LG;

[0051] (II) A method for synergistically reducing the sensitization of β-lactoglobulin using ultrasound and myricetin, comprising the following steps:

[0052] (1) Dissolve 1000 mg β-LG in 500 mL of purified water, stir at room temperature for 2 h, and then place in a 4 °C refrigerator overnight to fully dissolve. Determine the protein concentration using a BCA kit.

[0053] Weigh 100 mg of myricetin and prepare a 2 mg / mL solution with methanol. Store at -20°C in the dark.

[0054] Myricetin methanol solution was added dropwise to β-LG aqueous solution at a molar ratio of 1:10, and the mixture was reacted in a shaker at room temperature for 2 hours.

[0055] (2) After the two are mixed evenly, ultrasonic treatment is performed using an ultrasonic cell disruptor. The ultrasonic probe with a diameter of 6 mm is immersed in the sample, 1 cm from the bottom, and the ultrasonic intensity is 240 W. Intermittent ultrasonic treatment is used, that is, each ultrasonic treatment is 7 seconds and then stopped for 3 seconds, for a total of 15 minutes. The ultrasonic treatment is carried out in an ice bath to prevent the solution from heating during the ultrasonic process.

[0056] (3) Finally, the sample solution was dialyzed in ultrapure water for 48 h and then freeze-dried. The IgE binding inhibition rate of β-LG was found to be 47.20%.

[0057] Example 3

[0058] (i) Same as in Example 1, myricetin was selected as the polyphenol with the best ability to reduce the sensitization of β-LG;

[0059] (II) A method for synergistically reducing the sensitization of β-lactoglobulin using ultrasound and myricetin, comprising the following steps:

[0060] (1) Dissolve 1000 mg β-LG in 500 mL of purified water, stir at room temperature for 2 h, and then place in a 4 °C refrigerator overnight to fully dissolve. Determine the protein concentration using a BCA kit.

[0061] Weigh 100 mg of myricetin and prepare a 2 mg / mL solution with methanol. Store at -20°C in the dark.

[0062] Myricetin methanol solution was added dropwise to β-LG aqueous solution at a molar ratio of 1:12, and the mixture was reacted in a shaker at room temperature for 2 hours.

[0063] (2) After the two are mixed evenly, ultrasonic treatment is performed using an ultrasonic cell disruptor. The ultrasonic probe with a diameter of 6 mm is immersed in the sample, 1 cm from the bottom, and the ultrasonic intensity is 240 W. Intermittent ultrasonic treatment is used, that is, each ultrasonic treatment is 7 seconds and then stopped for 3 seconds, for a total of 15 minutes. The ultrasonic treatment is carried out in an ice bath to prevent the solution from heating during the ultrasonic process.

[0064] (3) Finally, the sample solution was dialyzed in ultrapure water for 48 hours and then freeze-dried.

[0065] The IgE binding inhibition rate of β-LG was measured to be 47.50%.

[0066] The above embodiments are merely examples illustrating the technical solutions of the present invention and are not intended to limit the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A method for synergistically reducing the sensitization of β-lactoglobulin using ultrasound and myricetin, characterized in that, Includes the following steps: (1) Dissolve β-lactoglobulin in purified water to prepare an aqueous solution of β-lactoglobulin, dissolve myricetin in methanol to prepare a methanol solution of myricetin, and mix the aqueous solution of β-lactoglobulin and the methanol solution of myricetin to prepare a non-covalent mixture of β-lactoglobulin and myricetin. (2) The β-lactoglobulin-myricetin non-covalent mixture obtained in step (1) was subjected to ultrasonic treatment; (3) Freeze-dry the product after ultrasonic treatment to obtain low-allergenic β-lactoglobulin-myricetin powder.

2. The method for synergistically reducing β-lactoglobulin sensitization by ultrasound and myricetin according to claim 1, characterized in that, The concentration of the β-lactoglobulin aqueous solution in step (1) is 1 to 10 mg / mL.

3. The method for synergistically reducing β-lactoglobulin sensitization by ultrasound and myricetin according to claim 1, characterized in that, The concentration of the myricetin methanol solution mentioned in step (1) is 1 to 10 mg / mL.

4. The method for synergistically reducing β-lactoglobulin sensitization by ultrasound and myricetin according to claim 1, characterized in that, The molar ratio of β-lactoglobulin to myricetin in step (1) is 1:8 to 12.

5. The method for synergistically reducing β-lactoglobulin sensitization by ultrasound and myricetin according to claim 1, characterized in that, In step (1), the aqueous solution of β-lactoglobulin and the methanol solution of myricetin are mixed and reacted on a shaker at room temperature for 1 to 3 hours.

6. The method for synergistically reducing β-lactoglobulin sensitization by ultrasound and myricetin according to claim 1, characterized in that, In step (2), the ultrasonic treatment is carried out in an ice bath with an ultrasonic intensity of 240W. Intermittent ultrasonic treatment is used, that is, each ultrasonic treatment lasts for 7 seconds and then stops for 3 seconds, for a total of 5 to 30 minutes.

7. The method for synergistically reducing β-lactoglobulin sensitization by ultrasound and myricetin according to claim 1, characterized in that, In step (3), the ultrasonically treated product is first dialyzed in ultrapure water for 48 hours and then freeze-dried.

8. The method for synergistically reducing β-lactoglobulin sensitization by ultrasound and myricetin according to claim 1, characterized in that, In step (3), during freeze drying, the freeze is first pre-frozen at -20℃ and then freeze-dried at -40 to -50℃ until dry.

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