A method for producing a hypoallergenic casein

Abstract

The present application relates to a kind of low sensitization casein preparation method and low sensitization casein obtained using the preparation method, belong to food biotechnology field;The inventor of the present application accidentally found that when using the combination of "sodium dodecyl sulfate and dithiothreitol" specific proportion is handled to casein, the sensitization of linear casein after development is significantly reduced;Specifically, after treatment, the globular protein structure of casein is not only developed into linear protein structure, but also unexpectedly found that aggregation exists between protein molecules, and the effects of development and aggregation together lead to most of the sensitization sites on casein being covered, thereby reducing the sensitization of casein;The method of the present application is simple and low in cost, can significantly reduce the sensitization of casein without destroying the nutritional quality of casein itself.

Description

Technical Field

[0001] This invention relates to a method for preparing low-allergenic casein, belonging to the field of food biotechnology. Background Technology

[0002] Food allergy, also known as food hypersensitivity, refers to an abnormal immune response of the body to food allergens (usually proteins) that enter the body. It usually leads to the disruption of physiological functions or tissue damage and triggers a series of clinical symptoms, such as hives on the skin, respiratory asthma, cough, gastrointestinal nausea, vomiting, diarrhea and other clinical symptoms. In severe cases, it can lead to shock or even death.

[0003] Milk is one of the eight major allergens, primarily affecting infants and young children, and is often the first allergen most infants encounter in their early years. Milk protein is mainly composed of two components: casein (CN) and whey protein (WP); casein accounts for 80% of the total protein in milk and is the most significant allergen.

[0004] Currently, common methods for reducing the allergenicity of milk proteins include heat treatment, fermentation, and enzymatic hydrolysis. Heat treatment is the most widely used, but its effect on reducing milk allergenicity is not significant. In recent years, some non-thermal processing technologies, including fermentation and enzymatic hydrolysis, have shown less impact on milk nutrition and can effectively eliminate and destroy allergenic epitopes on casein, attracting considerable attention from the academic community. However, the products of these enzymatic hydrolysates and fermentations typically have a slightly bitter taste and poor palatability, and are accompanied by high production costs, making their application and promotion difficult in foods such as infant formula and hypoallergenic milk products.

[0005] Some scholars in this field have also attempted to modify casein allergic epitopes through chemical methods, including the Maillard reaction, to reduce their sensitization. However, the Maillard reaction cannot be precisely controlled and can also form other chemical substances, including late glycosylation products, which not only reduce glycosylation efficiency but are also harmful to human health.

[0006] Therefore, there is a desire in the art to develop new methods to reduce the allergenicity of casein, especially methods to reduce its allergenicity without compromising the nutritional quality of casein itself. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention provides a method for preparing low-allergenic casein, wherein the preparation method comprises the following steps performed sequentially:

[0008] Step 1): Obtain casein solids;

[0009] Step 2): Dissolve the casein solid with the aid of an emulsifier to obtain a casein solution;

[0010] Step 3): Dissolve sodium dodecyl sulfate and dithiothreitol in the casein solution obtained in step 2) and heat to 95-120°C to obtain the treated casein solution;

[0011] Furthermore, in step 3), based on the mass of casein solids contained in the casein solution, 1.2–1.5 g of sodium dodecyl sulfate and 0.025–0.032 g of dithiothreitol are added per 1 g of casein solids.

[0012] Step 4): Cool the treated casein solution obtained in Step 3) to room temperature, separate and purify it to obtain the low-allergenic casein.

[0013] Preferably, in step 3), 1.4g of sodium dodecyl sulfate and 0.03g of dithiothreitol are added for every 1g of casein solids.

[0014] Preferably, in step 2), the casein solid is dissolved in PBS solution with the assistance of Tween-80 or soybean lecithin to obtain a casein solution.

[0015] Preferably, in step 2), each 1g of casein solid is mixed with 0.5-1.5ml of Tween-80 and dissolved in PBS solution; or, in step 2), each 1g of casein solid is mixed with 1-2g of soybean lecithin and dissolved in PBS solution.

[0016] Preferably, in step 2), the obtained casein solution is incubated at a temperature of 30–50°C for at least 30 minutes.

[0017] Preferably, in step 4), the separation and purification method is protein dialysis.

[0018] Preferably, the protein dialysis conditions are a dialysis temperature of 2-8°C and at least 4 dialysis cycles.

[0019] Preferably, the casein solid obtained in step 1) is casein solid extracted from milk.

[0020] Preferably, the steps for extracting the casein solids from milk are as follows:

[0021] Step a: Take fresh milk, centrifuge it, and collect the supernatant; the centrifugation speed is 2500-3000 x g, and the centrifugation time is 15-20 minutes;

[0022] Step b: Take the supernatant obtained in step a, adjust the pH to 4.5-4.7, centrifuge and collect the precipitate;

[0023] Step c: Wash the precipitate obtained in step b with 75% ethanol;

[0024] Step d: Wash the precipitate obtained in step c with deionized water;

[0025] Step e: After dialysis of the precipitate obtained from washing in step d, freeze-dry it to obtain the casein solid from step 1).

[0026] In another aspect, the present invention provides a casein with low allergenicity, wherein the casein with low allergenicity is prepared by the preparation method described above.

[0027] This invention provides a method for preparing casein with low allergenicity. The method is simple to operate and low in cost, and can significantly reduce its allergenicity without destroying the nutritional quality of casein itself. Attached Figure Description

[0028] Figure 1 This is an SDS-PAGE electrophoresis image of the purified casein solid obtained in Example 1;

[0029] Figure 2 SDS-PAGE electrophoresis images of the treated casein solids obtained in Examples 2-4 and Comparative Examples 2a and 2b;

[0030] Figure 3 Scanning electron microscope images of "untreated" casein in Example 1 and treated casein in Example 2;

[0031] Figure 4 Native PAGE electrophoresis images of the "untreated" casein solid obtained in Example 1, and the treated casein solids obtained in Example 2, Comparative Example 2a, and Comparative Example 2b.

[0032] Figure 5 The results show the binding capacity of the treated casein solids obtained in Examples 2-4 to IgE in the serum of patients with milk allergy.

[0033] Figure 6 Results of β-hexosamine release rate assay in LAD2 cell sensitization experiment;

[0034] Figure 7 The results of histamine content determination in the LAD2 cell sensitization experiment. Detailed Implementation

[0035] The research institution of the inventors of this application is dedicated to the study of food allergenicity; among them, "reducing the allergenicity of casein", especially how to reduce its allergenicity without destroying the nutritional quality of casein itself, is the research topic of the inventors of this application.

[0036] The inventors of this application tried various protein unfolding reducing agents in their research. Many of these reducing agents could unfold casein to a certain extent. However, in the verification experiment on sensitization, it was found that the sensitization of the unfolded casein was not significantly reduced. This indicates that the sensitization sites of the unfolded casein did not change.

[0037] During their research, the inventors unexpectedly discovered that when casein was treated with a specific combination of sodium dodecyl sulfate and dithiothreitol, the sensitization of the developed linear casein was significantly reduced. Specifically, the IgE binding capacity of the serum of patients with milk allergy was significantly reduced, and the release rates of β-hexosamine and histamine were significantly reduced in the LAD2 cell sensitization experiment.

[0038] Scanning electron microscopy (SEM) results and Native PAGE electrophoresis results of linear casein treated with the method described in this application show that the treated casein not only undergoes structural changes (the globular protein structure unfolds into a linear protein structure), but also exhibits aggregation between protein molecules. Based on these experimental results, the inventors hypothesize that treating casein with a specific combination of sodium dodecyl sulfate and dithiothreitol in a specific ratio results in the unfolding of the casein structure and the aggregation between protein molecules. These two effects combined lead to the coverage of most of the sensitizing sites on the casein, thereby reducing the sensitizing properties of casein.

[0039] The present invention will be further illustrated by the following examples, but the present invention is not limited to these specific embodiments.

[0040] Example 1: Extraction of casein solids

[0041] In this embodiment, a method for extracting casein solids from milk is used, which includes the following steps:

[0042] Step a: Take fresh milk, centrifuge it, and collect the supernatant; the centrifugation speed is 2500-3000 x g, and the centrifugation time is 15-20 minutes; the supernatant obtained in step a is skim milk;

[0043] Step b: Take the supernatant obtained in step a, adjust the pH to 4.5-4.7, centrifuge and collect the precipitate;

[0044] Since casein is an acidic protein with an isoelectric point of approximately 4.6, it can be separated using isoelectric point precipitation. Specifically, in this embodiment, the supernatant (skim milk) is acidified with a 1 mol / L solution, and the precipitate obtained by centrifugation is the crude extract of casein.

[0045] Step c: Wash the precipitate obtained in step b with 75% ethanol;

[0046] The crude casein extract obtained in step b can be washed with 75% ethanol. Specifically, in this embodiment, the precipitate obtained in step b is mixed with 75% ethanol at a ratio of 3-5 ml of ethanol per 1 g of precipitate, thoroughly shaken, centrifuged (1500 x g, 20 min), and the precipitate is collected; the washing is performed 1-3 times.

[0047] Step d: Wash the precipitate obtained in step c with deionized water;

[0048] Specifically in this embodiment, the precipitate obtained in step c is mixed with deionized water at a ratio of 1g precipitate to 3-5ml deionized water, shaken thoroughly, centrifuged (1500xg, 15min), and the precipitate is collected; the washing is performed 2-4 times.

[0049] Step e: After dialysis of the precipitate obtained from washing in step d, freeze-dry it to obtain casein solids.

[0050] Specifically, in this embodiment, the dialysis temperature is 2-8°C, and the number of dialysis cycles is 3-5. Specifically, in this embodiment, the dialysis fluid is changed approximately every 6-8 hours.

[0051] The purified casein solid obtained in Example 1 was analyzed by SDS-PAGE. The specific procedure was as follows: The casein solid was dissolved in deionized water and diluted to 1 mg / mL, and mixed thoroughly with loading buffer at a ratio of 4:1. A 15% separating gel and a 5% stacking gel were prepared. The electrophoresis apparatus was prepared, and electrode buffer was poured in. The protein loading volume for each well was 10 μL. During gel running, the gel was first run at 80V for 15 min. When the protein bands were compressed into a single line, the voltage was increased to 150V. The gel running was stopped when the bromophenol blue indicator reached the glass plate. Then, the gel was removed and placed in staining solution, and stained on a shaker for about 45 min. The staining solution was discarded, and destaining solution was added for destaining, changing the destaining solution during the process until the protein bands were clearly visible. The gel bands were scanned using the Alpha gel imaging system and analyzed using Alpha View SA3.4.0 electrophoresis image analysis software.

[0052] The SDS-PAGE electrophoresis image of the purified casein solid obtained in Example 1 is shown below. Figure 1 ,from Figure 1As can be seen from the electrophoresis pattern, four bands of different molecular weights were observed, representing the four subunits of casein (αS1, αS2, β, and κ), respectively. The bands were clear and without tails. The molecular weights corresponding to the four bands (from top to bottom) were 30.7 kDa (αS2-CN), 27.4 kDa (β-CN), 24.8 kDa (αS1-CN), and 17.5 kDa (κ-CN). By comparing with the electrophoresis bands of casein standards, it can be confirmed that the solid casein obtained in Example 1 was purified.

[0053] Example 2: Method for processing casein

[0054] The preparation method includes the following steps performed sequentially:

[0055] Step 1): Obtain casein solids;

[0056] In one specific embodiment of this application, the casein solid can be an isolated and extracted casein solid, such as casein extracted from dairy products; or it can be a casein solid obtained by direct purchase.

[0057] Specifically, in this embodiment, the casein solid obtained in Example 1 above is used.

[0058] Step 2): Dissolve the casein solids with the help of an emulsifier to obtain a casein solution.

[0059] In one specific embodiment of this application, in step 2), casein solids are dissolved in PBS solution with the assistance of Tween-80 or soy lecithin to obtain a casein solution.

[0060] In one preferred embodiment of this application, in step 2), 1g of casein solids is mixed with 0.5-1.5ml of Tween-80 and dissolved in PBS solution. In another preferred embodiment of this application, in step 2), 1g of casein solids is mixed with 1-2g of soy lecithin and dissolved in PBS solution.

[0061] In a preferred embodiment of this application, in step 2), the obtained casein solution is incubated at a temperature of 30–50°C for at least 30 minutes.

[0062] Specifically in this embodiment, in step 2), 100 mg of casein solid is mixed with 1 ml of Tween-80 and dissolved in 100 ml of PBS solution to obtain a casein solution, and the casein solution is incubated at 40°C for at least 30 minutes.

[0063] Step 3): Dissolve sodium dodecyl sulfate and dithiothreitol in the casein solution obtained in step 2) and heat to 95-120°C to obtain a treated casein solution; and in step 3), based on the mass of casein solids contained in the casein solution, add 1.2-1.5g of sodium dodecyl sulfate and 0.025-0.032g of dithiothreitol for every 1g of casein solids.

[0064] Specifically in this embodiment, in step 3), 0.14g of sodium dodecyl sulfate and 0.003g of dithiothreitol are added to the casein solution obtained in step 2) (i.e., 1.4g of sodium dodecyl sulfate and 0.03g of dithiothreitol are added for every 1g of casein solid).

[0065] Specifically, in this embodiment, after adding sodium dodecyl sulfate and dithiothreitol, the entire container is placed in a water bath at 95-100°C and heated for about 5 minutes.

[0066] Step 4): Cool the treated casein solution obtained in Step 3) to room temperature, separate and purify it to obtain the low-allergenic casein.

[0067] In one specific embodiment of this application, in step 4), the separation and purification method is protein dialysis. In a preferred embodiment of this application, the protein dialysis conditions are a dialysis temperature of 2-8°C and at least 4 dialysis cycles.

[0068] Specifically, in this embodiment, after cooling the treated casein solution obtained in step 3) to room temperature, protein dialysis is performed using a dialysis bag with a molecular weight cutoff of 8 kDa. The dialysis temperature is 2°C, and the dialysis solution is replaced every 6-8 hours for a total of 5 dialysis cycles. After dialysis, the treated casein solid is obtained by freeze-drying.

[0069] Example 3

[0070] The preparation method of Example 3 differs from that of Example 2 as follows:

[0071] In step 2), 100 mg of casein solids were mixed with 0.5 ml of Tween-80 and dissolved in 100 ml of PBS solution to obtain a casein solution, which was then incubated at 40°C for at least 30 minutes.

[0072] In step 3), 0.12 g of sodium dodecyl sulfate and 0.0025 g of dithiothreitol are added to the casein solution obtained in step 2) (i.e., 1.2 g of sodium dodecyl sulfate and 0.025 g of dithiothreitol are added for every 1 g of casein solid).

[0073] The other operations of the preparation method in Example 3 are the same as those in Example 2, and will not be repeated here.

[0074] Example 4

[0075] The preparation method of Example 4 differs from that of Example 2 as follows:

[0076] In step 2), 100 mg of casein solids are mixed with 200 mg of soybean lecithin and dissolved in 100 ml of PBS solution to obtain a casein solution, which is then incubated at 40°C for at least 30 minutes.

[0077] In step 3), 0.15 g of sodium dodecyl sulfate and 0.0032 g of dithiothreitol are added to the casein solution obtained in step 2) (i.e., 1.5 g of sodium dodecyl sulfate and 0.032 g of dithiothreitol are added for every 1 g of casein solid).

[0078] The other operations of the preparation method in Example 4 are the same as in Example 2, and will not be repeated here.

[0079] Comparative Examples 2a and 2b

[0080] The preparation method of Comparative Example 2a differs from that of Example 2 as follows: In step 3), only 0.14g of sodium dodecyl sulfate (i.e., 1.4g of sodium dodecyl sulfate per 1g of casein solid) was added to the casein solution obtained in step 2), and dithiothreitol was not added.

[0081] The preparation method of Comparative Example 2b differs from that of Example 2 as follows: In step 3), only 0.003 g of dithiothreitol (i.e., 0.03 g of dithiothreitol per 1 g of casein solid) was added to the casein solution obtained in step 2), and sodium dodecyl sulfate was not added.

[0082] Results data

[0083] 1) SDS-PAGE analysis

[0084] SDS-PAGE analysis was performed on the treated casein solids obtained in Examples 2-4 and Comparative Examples 2a and 2b (see the corresponding content in Example 1 for specific procedures); the electrophoresis results are shown below. Figure 2 .

[0085] from Figure 2As can be seen, the linear casein bands obtained in Examples 2-4 and Comparative Examples 2a and 2b are basically the same as those obtained in Example 1, all consisting of four bands with different molecular weights. The protein bands are clear without tails and without obvious impurities. From top to bottom, the molecular weights corresponding to the bands are: 30.9 kDa (αS2-CN), 28.1 kDa (β-CN), 25.1 kDa (αS1-CN), and 17.3 kDa (κ-CN). Furthermore, according to AlphaView SA3.4.0 electrophoresis image analysis software, the processed casein solids obtained in Examples 2-4 have a purity of approximately 95%, which is relatively high.

[0086] 2) Scanning electron microscopy imaging analysis

[0087] Take appropriate amounts of protein samples from the "untreated" casein solid obtained in Example 1 and the treated casein solid obtained in Example 2, respectively.

[0088] Protein samples were dropped onto the sample stage with 20 μL of conductive gel, dried, and coated with gold for 1 min. Then, they were observed and photographed using a scanning electron microscope at magnifications of 5000× and 20000×, respectively. The results are shown below. Figure 3 As shown; Figure 3 The upper half of the image is an electron microscope photograph of the untreated casein solid obtained in Example 1; Figure 3 The lower half of the image is an electron microscope image of the processed casein solid obtained in Example 2; the upper right corner of each image is a 20,000x magnified image.

[0089] Depend on Figure 3 As can be seen, under a scanning electron microscope, the untreated casein solid obtained in Example 1 is in the form of dispersed particles. Individual spherical particles can be seen at 20,000× magnification. The surface is rough and uneven.

[0090] The treated casein solid obtained in Example 2 showed a denser and less rough surface at higher magnification (5000×) compared to untreated casein; at lower magnification (20000×), it could be determined that the structure of the treated casein expanded, transforming from irregular spherical or blocky structures of varying sizes into slender, flat fibrous structures, with obvious stacking and aggregation.

[0091] 3) Native PAGE experiment

[0092] The untreated casein solids obtained in Example 1, and the treated casein solids obtained in Examples 2, 2a, and 2b, were dissolved in deionized water and diluted to 1 mg / mL, respectively. They were then mixed thoroughly with loading buffer at a 1:1 ratio. A 12% separating gel and a 5% stacking gel were prepared. The electrophoresis apparatus was prepared, and electrode buffer was added. The protein loading volume for each well was 20 μL. During gel running, the gel was initially run at 80 V for 20 min. When the protein bands were compressed into a single line, the voltage was increased to 180 V and run for 2 h. The gel was then removed and placed in staining solution, stained on a shaker for approximately 60 min. The staining solution was discarded, and destaining solution was added, changing the destaining solution as needed, until the protein bands were clearly visible.

[0093] The gel bands were scanned using the Alpha gel imaging system and analyzed using AlphaView SA3.4.0 electrophoresis image analysis software. The results are shown in [link to relevant documentation]. Figure 4 .

[0094] from Figure 4 The results show that the treated casein obtained in Example 2 has clear bands without tailing. Compared with the untreated casein obtained in Example 1, the treated casein has a larger molecular weight (89.1 kDa and 128.4 kDa), indicating that there is aggregation between the treated casein molecules. In contrast, the bands of Comparative Examples 2a and 2b show obvious tailing, and the molecular weight changes are not significant. The above results indicate that the synergistic use of sodium dodecyl sulfate and dithiothreitol can more effectively promote the aggregation between linear casein molecules.

[0095] Based on the above SDS-PAGE electrophoresis analysis results, scanning electron microscopy results, and Native PAGE electrophoresis analysis results, the casein processed by the method of this application unfolds from an amorphous, irregular spherical structure into a relatively smooth, flat linear structure, and there are stacking and aggregation phenomena between different linear casein molecules.

[0096] 4) Determination of IgE binding capacity in serum of patients with milk allergy

[0097] The "untreated" casein solids obtained in Example 1 and the treated casein solids obtained in Examples 2-4 were mixed with serum from milk-allergic patients (serum samples from 12 different patients obtained from Plasmalab International, USA) and their IgE binding capacity was measured.

[0098] The specific steps are as follows:

[0099] Add 10 μL of protein sample (1 mg / mL) (10 μL of 0.01 mol / L PBS solution was used as the negative control) and 100 μL of carbonate coating solution to a 96-well microplate, and incubate overnight at 4°C. Add 100 μL / well blocking buffer and incubate at 37°C for 2 h. Wash 5 times with washing buffer and blot dry. Add 100 μL of serum from a milk-allergic patient (1:50 dilution), incubate at 37°C for 1 h, wash 5 times with washing buffer and blot dry. Add 100 μL of HRP-labeled goat anti-human IgE secondary antibody (1:5000 dilution) and incubate at 37°C for 1 h. Wash 5 times with washing buffer, blot dry, add 100 μL of TMB chromogenic solution, and incubate in the dark for 30 min. Add 2M H2SO4 stop solution (50 μL / well) and measure absorbance at 450 nm.

[0100] Test results as follows Figure 5 As shown. From Figure 5 As can be seen, the untreated casein obtained in Example 1 showed significantly higher IgE binding capacity in 58.33% of patient serum samples, while the treated linear casein obtained in Examples 2-4 only showed high IgE binding capacity in 16.7%–25% of patient serum samples, indicating that the linear casein obtained in Examples 2-4 had a lower probability of causing milk allergies in patients. Compared with Comparative Examples 2a and 2b, Examples 2-4 also showed a lower sensitization probability, indicating that the linear casein obtained after synergistic treatment with sodium dodecyl sulfate and dithiothreitol had a better desensitizing effect than the linear casein obtained after treatment with these two additives alone.

[0101] 5) LAD2 cell sensitization assay

[0102] During the food allergy effect period, the allergen binds to specific IgE on the surface of mast cells, triggering cell degranulation and releasing allergy mediators (such as β-hexosaminease and histamine), causing allergy symptoms in a short period of time.

[0103] In this experiment, the LAD2 human mast cell line was used to evaluate the ability of the "untreated" casein obtained in Example 1 and the treated linear casein obtained in Examples 2-4 to induce mast cell degranulation by measuring its β-hexosaminease release rate and histamine content. The specific experimental details are as follows:

[0104] Thawed human mast cells (LAD2, derived from Plasmalab International, USA) were transferred to XGI-1640 medium and activated at 37°C with 5% CO2. The cell suspension concentration was adjusted to 1×10⁻⁶. 6Cells were seeded at a density of 100 μL / mL into 96-well plates; 20 μL of serum from milk-allergic patients (1:50 dilution) was added, and the plates were incubated at 37°C with 5% CO2 for 24 h; the cells were centrifuged at 500 x g for 5 min, the supernatant was discarded, and the cells were washed three times with PBS; the cells were resuspended in 100 μL of HEPES buffer, and 20 μL of protein sample was added, and the plates were incubated at 37°C for 2 h. The cells were then centrifuged at 500 x g for 5 min, and the supernatant was used to perform the following experiments to determine the β-hexosamine release rate and histamine content.

[0105] β-Hexosaminease release rate assay: Transfer 30 μL of supernatant to a new 96-well plate, add 50 μL of 4-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranose solution (pH 4.5), and incubate at 37℃ with 5% CO2 for 2 h. Simultaneously, add 1% Triton X 100 to the original 96-well plate to lyse cells, 10 μL per well. Centrifuge at 500 x g for 5 min, discard the supernatant, and transfer 30 μL of supernatant to a new microplate. Add 50 μL of PANG solution and incubate at 37℃ with 5% CO2 for 2 h. Terminate the reaction with 0.2 M glycine solution and measure the absorbance at 450 nm.

[0106] Histamine content determination: Take 30 μL of supernatant and centrifuge at 4000 x g for 20 min at 4℃. According to the kit instructions (Mouse Histamine Kit purchased from Thermo Fisher Scientific, USA), prepare blank wells, standard curve wells, and sample wells, 50 μL per well. Add 40 μL of sample diluent to the sample wells, followed by 10 μL of the centrifuged cell supernatant. Incubate at 37℃ for 30 min, then wash. Add 100 μL of HRP-labeled secondary antibody, incubate at 37℃ for 30 min, then wash. Add 50 μL of chromogenic reagent A and 50 μL of chromogenic reagent B sequentially, and incubate at 37℃ in the dark for 15 min. Add 50 μL of stop solution, and measure the OD value at 450 nm within 15 min.

[0107] The results of the β-hexosamine enzyme release rate assay are shown below. Figure 6 For histamine content determination results, please refer to [link / reference]. Figure 7 .

[0108] from Figure 6 and Figure 7The results show that, compared with the untreated casein obtained in Example 1, the linear casein obtained in Examples 2-4 exhibited significantly lower β-hexosamine release rates and histamine release rates, with decreases of 33.7–38.4% and 65.5–66.4%, respectively. Compared with Comparative Examples 2a and 2b, Examples 2-4 also showed significantly lower β-hexosamine release rates and histamine release rates, indicating that the linear casein prepared by this method not only has a significantly lower ability to induce food allergic reactions than natural casein, but also exhibits a better desensitizing effect than casein prepared by treatment with sodium dodecyl sulfate or dithiothreitol alone.

[0109] It should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0110] The detailed descriptions listed above are merely specific descriptions of feasible embodiments of the present invention, and are not intended to limit the scope of protection of the present invention. All equivalent embodiments or modifications made without departing from the spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for producing a hypoallergenic casein protein, characterized by: The preparation method comprises the following steps in sequence: Step 1): obtaining casein solid; Step 2): dissolving the casein solid in the presence of an emulsifier to obtain a casein solution; Step 3): dissolving sodium dodecyl sulfate and dithiothreitol in the casein solution obtained in step 2) and heating to 95-120 DEG C to obtain a treated casein solution; In step 3), 1.2-1.5 g of sodium dodecyl sulfate and 0.025-0.032 g of dithiothreitol are added per 1 g of casein solid; Step 4): cooling the treated casein solution obtained in step 3) to room temperature, and obtaining the low-sensitivity casein after separation and purification.

2. The production method according to claim 1, characterized by: In step 3), 1.4 g of sodium dodecyl sulfate and 0.03 g of dithiothreitol are added per 1 g of casein solid.

3. The production method according to claim 1, wherein: In step 2), the casein solid is dissolved in a PBS solution in the presence of Tween-80 or soybean lecithin to obtain a casein solution.

4. The production method according to claim 3, characterized by: In step 2), 0.5-1.5 ml of Tween-80 is mixed with per 1 g of the casein solid and dissolved in a PBS solution; or in step 2), 1-2 g of soybean lecithin is mixed with per 1 g of the casein solid and dissolved in a PBS solution.

5. The production method according to claim 4, characterized by: In step 2), the obtained casein solution is incubated at a temperature of 30-50 DEG C for at least 30 minutes.

6. The production method according to claim 1, wherein: In step 4), the method for separation and purification is protein dialysis.

7. The production method according to claim 6, characterized by: The conditions for protein dialysis are a dialysis temperature of 2-8 DEG C and at least 4 times of dialysis.

8. The production method according to any one of claims 1 to 7, characterized by: The casein solid obtained in step 1) is a casein solid extracted from milk.

9. The production method according to claim 8, characterized by: The steps for extracting the casein solid from milk are as follows: Step a: taking fresh milk, centrifuging and taking supernatant; wherein the centrifugal speed is 2500-3000xg and the centrifugal time is 15-20 minutes; Step b: taking the supernatant obtained in step a, adjusting the pH to 4.5-4.7, and centrifuging to obtain a precipitate; Step c: washing the precipitate obtained in step b with 75% ethanol; Step d: washing the precipitate obtained in step c with deionized water; Step e: freezing and drying the precipitate obtained in step d after protein dialysis to obtain the casein solid in step 1).

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