Walnut hypoallergenic complex protein, preparation method and application

Abstract

The invention belongs to the technical field of protein preparation, and discloses a walnut hypoallergenic complex protein, a preparation method and an application, wherein shelled walnuts are ground to obtain walnut powder, acetone is added to defat and then a protein extract is added to extract crude protein, isoelectric precipitation, ammonium sulfate graded precipitation, dialysis desalination, and freeze drying are performed to obtain walnut allergenic protein; the walnut allergenic protein is dissolved in ultrapure water, a free radical initiator is added, polyphenols are added to form a walnut protein-polyphenol complex, the sample solution is dialyzed to ensure that unreacted polyphenols are completely removed, and a hypoallergenic complex is obtained after freeze drying. The first seven kinds of walnut proteins extracted by the present invention are all allergenic proteins, and the complex prepared by the present invention adopts a free radical grafting method, which is simple to operate and does not produce toxic byproducts. The complex prepared by the present invention belongs to a hypoallergenic protein, which can reduce allergenicity by changing the spatial structure of walnut protein.

Description

Technical Field

[0001] The invention belongs to the technical field of protein preparation, and in particular relates to a walnut low-allergy complex protein, a preparation method and an application thereof. Background Art

[0002] Food allergy is an adverse reaction to food mediated by immune mechanisms, accompanied by a series of rapid symptoms, including gastrointestinal, skin, respiratory and even systemic allergic reactions, mostly IgE-mediated type I hypersensitivity reactions. In recent years, the incidence of food allergies has been increasing year by year, and has become a food safety and public health issue of global concern. According to epidemiological surveys, the incidence of food allergies in adults is 5%, and the incidence in children is as high as 8%. Nut allergies currently account for 0.5% of adults and 1.1% of children in the United States. Among them, walnut allergy is the most common nut allergy in the United States, accounting for 37-48%, and it is almost a lifelong allergy. The main allergens are Jug r1, Jug r 2 and Jug r 4.

[0003] Common desensitization methods include physical methods such as heat treatment, chemical methods such as enzymatic hydrolysis and glycosylation, and biological methods such as genetic engineering. However, the problems and defects of the existing technologies are as follows: the existing methods for preparing hypoallergenic proteins have limitations, such as long-term heat treatment and enzymatic hydrolysis, which will affect the quality of aquatic products, change the flavor, and cause the loss of nutrients and active ingredients; the safety of irradiation technology is still controversial, etc.

[0004] Many studies have shown that covalent binding of polyphenols to allergenic proteins is an effective method to reduce protein allergenicity. Covalent binding does not require special equipment and is relatively simple and safe in the production of hypoallergenic protein products. It can also reduce protein allergenicity by changing the structure and epitopes of allergenic proteins. There are currently no studies on the hypoallergenic effect between walnuts and polyphenols. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a walnut low-allergy complex protein, a preparation method and an application thereof.

[0006] The present invention is achieved by extracting walnut allergenic protein by adopting ammonium sulfate precipitation method, isoelectric precipitation method, dialysis desalination and other steps, identifying the extracted protein, and confirming the allergenic protein component. The polyphenol and walnut protein are covalently bound by free radical binding method, the walnut allergenic protein is dissolved in ultrapure water, ascorbic acid and hydrogen peroxide are added, and the walnut allergenic protein is allowed to stand at 25°C, polyphenol is added, and after 24 hours, the walnut protein is dialyzed at 4°C for 48 hours to form a stable polyphenol-walnut protein complex.

[0007] The preparation method of the walnut hypoallergenic complex protein provided by the present invention comprises:

[0008] Step 1: Grind the shelled walnuts to obtain walnut powder, add acetone to defat, then add protein extract to extract crude protein, perform isoelectric precipitation, ammonium sulfate graded precipitation, dialysis desalination, and freeze-drying to obtain walnut allergenic protein;

[0009] Step 2: dissolve the walnut allergenic protein in ultrapure water, add a free radical initiator, add polyphenols to form a walnut protein-polyphenol complex, dialyze the sample solution to ensure that unreacted polyphenols are completely removed, and obtain a low-sensitivity complex after freeze-drying.

[0010] Furthermore, the method for extracting walnut allergenic protein in step 1 specifically comprises:

[0011] Step 1) walnuts are shelled and ground into fine powder, weighed and then added with 10 times acetone for degreasing, degreased under magnetic stirring at 4°C for 2h, and then centrifuged at 6000rpm / min for 30min at 4°C to obtain defatted walnut powder;

[0012] Step 2) mixing the defatted walnut powder obtained in step 1) with the protein extract, stirring, centrifuging, and taking the supernatant to obtain a first walnut protein crude extract;

[0013] Step 3) The first walnut protein crude extract obtained in step 2) is slowly lowered with HCl under magnetic stirring until the isoelectric point pH reaches 5.6, and the stirring is continued for 30 minutes, and then transferred to a 4°C refrigerator and allowed to stand for 12 hours. After the supernatant and the protein layer are clearly separated, centrifuged at 6000 rpm / min for 15 minutes at 4°C to obtain a second walnut protein crude extract;

[0014] Step 4) dissolving the second walnut protein crude extract obtained in step 3) in 10 times the volume of PBS, adjusting the pH to 7.5 with NaOH, adding ammonium sulfate to a saturation of 70%, and standing at 4°C for 2 hours; centrifuging at 4°C, 6500 rpm / min for 30 minutes, collecting the precipitate, and obtaining a third walnut protein crude extract;

[0015] Step 5) The protein precipitate in step 4) is dissolved in ultrapure water, placed in a 3.5 kDa dialysis bag, dialyzed in distilled water for 48 hours to remove salt, and then freeze-dried to obtain walnut allergenic protein.

[0016] Further, the protein extract in step 2) is a Tris-HCl salt buffer. Weigh 3.02g Tris, add 900mL distilled water to dissolve, adjust the pH to 7.5 with hydrochloric acid, and finally dilute to 1L with distilled water to prepare a Tris-HCl extraction buffer (25mM, pH 7.5); add 0.5M NaCl (i.e., 29.25g) to the above Tris-HCl extraction buffer to prepare a Tris-HCl salt buffer.

[0017] Furthermore, in the step 2), the defatted walnut powder and the protein extract are mixed at a mass: volume ratio of 1:10, stirred magnetically for 12 hours at 4° C., and then centrifuged at 6500 rpm / min for 20 minutes at 4° C.

[0018] Furthermore, the final concentration of polyphenols in step 2 is 0.2-2 g / L.

[0019] Furthermore, in step 2, the free radical initiators are hydrogen peroxide and ascorbic acid, the final concentration of hydrogen peroxide is 0.02-0.2 mol / L, and the final concentration of ascorbic acid is 0.25-5 g / L.

[0020] Furthermore, in the step 2, after adding the free radical initiator, the reaction conditions are to stand at 25° C. for 2 hours, and to react for 24 hours after adding the polyphenol.

[0021] Furthermore, the polyphenols in step 2 are four polyphenols including epigallocatechin gallate, resveratrol, quercetin and trans-ferulic acid.

[0022] Another object of the present invention is to provide a walnut protein-polyphenol low-allergy complex prepared by using the preparation method of walnut low-allergy complex protein.

[0023] Another object of the present invention is to provide an application of the walnut protein-polyphenol complex in the preparation of walnut hypoallergenic food / medicine / health product.

[0024] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0025] First, in view of the technical problems existing in the above-mentioned prior art and the difficulty of solving the problems, closely combining the technical solutions to be protected by the present invention and the results and data in the research and development process, the technical problems solved by the technical solutions of the present invention are analyzed in detail and in depth, and the types of protein extracts and the isoelectric points of allergenic proteins are determined, and the concentration of ammonium sulfate added is calculated based on the saturation. Some creative technical effects are brought about after solving the problems. The specific description is as follows:

[0026] (1) The first seven walnut proteins extracted by the present invention are all allergenic proteins, including bean B protein (Dietal.2020), Jug r 2, Jug r 4, Jug r 6, 11S globulin seed storage protein, Jug r 1, and globulin-like seed storage protein.

[0027] (2) The composite prepared by the present invention adopts a free radical grafting method, which is simple to operate and does not produce toxic byproducts.

[0028] (3) The complex prepared by the present invention is a low-sensitivity protein and can reduce the allergenicity by changing the spatial structure of walnut protein.

[0029] Second, considering the technical solution as a whole or from the perspective of the product, the technical effects and advantages of the technical solution to be protected by the present invention are described in detail as follows:

[0030] The invention uses the extracted walnut allergenic protein as a raw material, adds ascorbic acid and hydrogen peroxide as free radical initiators, and makes them interact with each other to form a stable complex protein, and the protein has low sensitivity.

[0031] Third, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0032] (1) The commercial value of the technical solution of the present invention after transformation is: providing a method for extracting walnut allergenic protein, providing basic raw materials for studying walnut allergenic protein. At the same time, the covalent binding of walnut allergenic protein with plant polyphenols can reduce the binding ability of protein with IgE, which has the potential application value of reducing protein allergenicity, and can improve some functional characteristics of protein, which can provide new ideas and theoretical basis for the development of new low-allergenic protein.

[0033] (2) The technical solution of the present invention fills the technical gap in the industry at home and abroad: Walnut allergy is the most common nut allergy in the United States, accounting for 37-48% of nut allergies, and is almost a lifelong allergy, but the methods for reducing walnut allergies are currently only heating baking and ultra-high pressure methods. This invention uses the interaction between natural active substances polyphenols and walnut protein to understand the interaction characteristics and structural changes between the two, and uses mouse experiments to study the effect of polyphenols on walnut protein sensitization, which is conducive to enriching the research data on the application of polyphenols in food allergies and providing more theoretical basis for finding plant polyphenols with desensitization effects to solve allergy problems caused by walnuts or other nuts. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a flow chart of the preparation method of walnut hypoallergenic complex protein provided by an embodiment of the present invention;

[0035] Figure 2 is the SDS-PAGE electrophoresis band diagram of the walnut allergenic protein extracted in Example 1 of the present invention;

[0036] Figure 3 This is a graph showing the effect of walnut allergenic protein and low-sensitivity complex protein on IgE inhibition rate in an embodiment of the present invention;

[0037] Figure 4A This is a food allergy symptom scoring diagram after mice were fed with the walnut allergenic protein and the hypoallergenic complex protein provided in the embodiments of the present invention;

[0038] Figure 4B This is a scoring diagram showing the effect of the walnut allergenic protein and the low-allergenic complex protein provided in the embodiment of the present invention on the diarrheal reaction of mice after feeding mice with the walnut allergenic protein and the low-allergenic complex protein;

[0039] Figure 4C This is a graph showing the effect of feeding mice with walnut allergenic protein and low-allergenic complex protein provided in an embodiment of the present invention on the rectal temperature of mice after stimulation;

[0040] Figure 4D This is a graph showing the effect of feeding mice with the walnut allergenic protein and the low-allergenic complex protein provided in the embodiments of the present invention on their weight growth rate;

[0041] Figure 5 This is a graph showing the effects of the walnut allergenic protein and the hypoallergenic complex protein provided in the embodiments of the present invention on the thymus and spleen indexes of mice after feeding them;

[0042] Figure 6 This is a graph showing the effect of the walnut allergenic protein and the hypoallergenic complex protein provided in the embodiments of the present invention on the IgE-specific antibodies in the serum of mice after feeding the mice;

[0043] Figure 7 This is a graph showing the effect of the walnut allergenic protein and the hypoallergenic complex protein provided in the embodiments of the present invention on the IgG1 specific antibody in the serum of mice after feeding the mice;

[0044] Figure 8 This is a graph showing the effect of the walnut allergenic protein and the low-sensitivity complex protein provided in the embodiments of the present invention on the IgG2a specific antibody in the mouse serum after feeding the mice;

[0045] Fig. 9 This is a graph showing the effect of feeding mice with the walnut allergenic protein and the hypoallergenic complex protein provided in the embodiments of the present invention on the serum histamine HIS content of mice;

[0046] Fig.10 This is a graph showing the effect of feeding mice with the walnut allergenic protein and the low-sensitivity complex protein provided in the embodiments of the present invention on the IL-4 cytokine concentration in mice;

[0047] Fig.11 This is a graph showing the effect of the walnut allergenic protein and the low-allergenic complex protein provided in the embodiments of the present invention on the INF-γ cytokine concentration in mice after feeding the mice. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0049] like Figure 1 As shown, the preparation method of walnut hypoallergenic complex protein provided by the embodiment of the present invention comprises:

[0050] S101, grinding the shelled walnuts to obtain walnut powder, adding acetone to defat, and then adding protein extract to extract crude protein, performing isoelectric precipitation, ammonium sulfate graded precipitation, dialysis desalination, and freeze drying to obtain walnut allergenic protein;

[0051] S102, dissolving walnut allergenic protein in ultrapure water, adding a free radical initiator, adding polyphenols to form a walnut protein-polyphenol complex, dialyzing the sample solution to ensure that unreacted polyphenols are completely removed, and freeze-drying to obtain a low-sensitivity complex.

[0052] Example 1: Extraction of walnut allergenic protein

[0053] Step 1) Walnut defatting: Peel the walnuts, remove the outer hard shell of the walnuts and grind them into fine powder. Weigh the obtained walnut powder, add a 10-fold acetone solution that has been pre-cooled at -20°C overnight, defatting at 4°C for 2 hours with magnetic stirring, then centrifuge at 4°C and 6000 rpm / min for 30 minutes, discard the supernatant, and repeat the above method for defatting the obtained precipitate twice, and fully dry it to obtain defatted walnut powder.

[0054] Step 2) Extraction of walnut crude protein: The defatted walnut powder of step 1 is mixed with the protein extract at a mass: volume ratio of 1:10, stirred magnetically at 4°C for 12 hours, then centrifuged at 4°C and 6500 rpm / min for 20 minutes, the supernatant is taken to obtain the crude protein, which is placed at 4°C for later use, and the precipitate is taken and repeated once.

[0055] Step 3) Isoelectric precipitation: The crude protein in step 2 was added dropwise with 1 mol / L HCl under magnetic stirring to slowly reduce the pH of the solution until the isoelectric point of pH 5.6 was reached. Stirring was continued for 30 min, and then transferred to a 4°C refrigerator and allowed to stand for 12 h. After the supernatant and the protein layer were clearly separated, the solution was centrifuged at 4°C and 6000 rpm / min for 15 min to collect the protein precipitate. The precipitate was dissolved again with 10 times the volume of protein buffer, the pH was adjusted to 7.5, and then the isoelectric precipitation was performed again according to the above steps to collect the precipitate.

[0056] Step 4) ammonium sulfate fractional precipitation: the protein precipitate obtained in step 3 was dissolved in 10 volumes of PBS (pH 7.4), the pH was adjusted to 7.5 with 1 mol / L NaOH, ammonium sulfate was slowly added to make the saturation reach 70%, and the mixture was allowed to stand at 4°C for 2 h; the mixture was centrifuged at 4°C, 6500 rpm / min for 30 min, and the precipitate was collected.

[0057] Step 5) Dialysis desalination: Dissolve the protein precipitate with ultrapure water, put it into a 3.5 kDa dialysis bag, and place the dialysis bag in distilled water for 48 hours to desalinate. Change the water every 4-8 hours.

[0058] Example 2: Preparation of walnut protein-epigallocatechin gallate hypoallergenic complex protein

[0059] Dissolve 0.25 g walnut protein in 50 mL ultrapure water, add 0.25 g ascorbic acid and 1.0 mL 5.0 M hydrogen peroxide, let stand at 25 °C for 2 h, then add 0.35 mmol epigallocatechin gallate. After 24 h, dialyze at 4 °C for 48 h, and lyophilize into powder for later use.

[0060] Example 3: Preparation of walnut protein-resveratrol hypoallergenic complex protein

[0061] Dissolve 0.25 g walnut protein in 50 mL ultrapure water, add 0.25 g ascorbic acid and 1.0 mL 5.0 M hydrogen peroxide, let stand at 25 °C for 2 h, then add 0.35 mmol resveratrol. After 24 h, dialyze at 4 °C for 48 h, and lyophilize into powder for later use.

[0062] Example 4: Preparation of walnut protein-quercetin hypoallergenic complex protein

[0063] Dissolve 0.25 g walnut protein in 50 mL ultrapure water, add 0.25 g ascorbic acid and 1.0 mL 5.0 M hydrogen peroxide, let stand at 25 °C for 2 h, then add 0.35 mmol quercetin. After 24 h, dialyze at 4 °C for 48 h, and lyophilize into powder for later use.

[0064] Example 5: Preparation of walnut protein-resveratrol hypoallergenic complex protein

[0065] Dissolve 0.25 g walnut protein in 50 mL ultrapure water, add 0.25 g ascorbic acid and 1.0 mL 5.0 M hydrogen peroxide, let stand at 25 °C for 2 h, then add 0.35 mmol trans-ferulic acid. After 24 h, dialyze at 4 °C for 48 h, and lyophilize into powder for later use.

[0066] In order to prove the creativity and technical value of the technical solution of the present invention, this section provides application examples of the claimed technical solution on specific products or related technologies.

[0067] (1) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

[0068] The walnut allergenic protein finally obtained in Example 1 of the present invention was subjected to SDS-PAGE analysis. The results are as follows: Figure 2 As shown, lane "1" is the original walnut protein lane, and lane "2" is the purified walnut allergenic protein, showing darker proteins at around 180KDa and 52KDa ​​and proteins at around 38KDa, 34KDa and 13KDa. This shows that the present invention can effectively extract walnut allergenic proteins through the multi-stage precipitation method.

[0069] Liquid chromatography / tandem mass spectrometry was further used to identify walnut allergenic proteins, and Mascot was used to search for matching proteins. The results showed that the top 7 were all walnut allergenic proteins, including legume B protein (Di et al. 2020), Jug r 2, Jug r 4, Jug r 6, 11S globulin seed storage protein, Jug r 1, and globulin-like seed storage protein. The results are shown in Table 1:

[0070] Table 1 The top seven proteins matched using Mascot search

[0071]

[0072] (2) Enzyme-linked immunosorbent assay (ELISA)

[0073] The IgE binding capacity of Example 1, Example 2, Example 3, Example 4, and Example 5 of the present invention was analyzed by indirect competitive ELISA. 100 μL of walnut protein solution (1 mg / mL) was added to a 96-well plate, placed in a 4°C refrigerator for 16 hours, and washed 3 times with PBS-Tween-20 (PBST), 300 μL per well, 5 minutes. 200 μL of 3.0% bovine serum albumin (BSA) was added to the well, blocked with blocking buffer at 37°C for 2 hours, the blocking buffer was removed, and the sample was washed 3 times with PBST, 5 minutes each time. 50 μL of polyphenol-protein complex solution was mixed with 50 μL of walnut allergy patient serum (diluted 1:100) and incubated for 1 hour. The mixture was added to each well, incubated at 37°C for 1 hour, and the sample was washed 3 times with PBST, and then 100 μL of horseradish peroxidase-labeled goat anti-human IgE antibody at 1:2000 was added to the 96-well plate and incubated at 37°C for 1 hour. Remove the antibody solution and wash the sample 3 times with PBST, 5 min each time. Add 50 μL TMB colorimetric solution to each well and incubate at 37°C in the dark for 10 min. Finally, add 50 μL stop solution (H2SO4, 2M) to terminate the enzyme reaction. Measure the absorbance at 450 nm.

[0074] Wherein, B indicates the use of inhibitors, and B0 indicates the absorbance when no inhibitors are used. Figure 3 shown.

[0075] Animal in vivo experimental applications:

[0076] The walnut protein-polyphenol complex hypoallergenic protein prepared in this experimental example was applied to animal in vivo experiments, and the results are summarized as follows:

[0077] 6.1 Using the Balb / c mouse sensitization animal model, the effects of oral administration of original walnut allergenic protein and four different complex proteins on mouse allergic symptoms, spleen cell index, immunoglobulins (IgE and IgG), histamine (HIS), interleukin 4 (IL-4) and other indicators were studied.

[0078] 6.2 Experimental materials: walnut allergenic protein, walnut protein-polyphenol complex protein (Example 2, Example 3, Example 4, Example 5), test kit: including serum IgE, IgG, HIS, IL-4 and other test kits

[0079] 6.3 Experimental Animal Treatment

[0080] Experimental animal grouping: 60 6-week-old female Balb / c mice were randomly divided into seven groups, namely, a blank control group, a walnut protein sensitization group, an Example 2 sensitization test group, an Example 3 sensitization test group, an Example 4 sensitization test group, and an Example 5 sensitization test group, with 10 mice in each group.

[0081] Experimental animal feeding and sensitization: After one week of acclimation, the mice in the group were used to establish the mouse allergy model. The experimental group was orally sensitized with 2 mg / g weight of walnut protein and polyphenol-walnut protein complex plus 10 μg / g weight of cholera toxin (CT) in PBS on days 0, 7, and 14, and with 5 mg / g weight of walnut protein and polyphenol-walnut protein complex plus 10 μg / g weight of CT in PBS on days 21 and 28. The PBS blank control group was orally administered with only PBS and CT. On the 35th day, 50 mg of walnut protein and polyphenol-walnut protein complex were used for gavage stimulation, and the symptoms of the mice were observed within 30-60 minutes and the scores were recorded.

[0082] Experimental animal sacrifice and sampling: After the feeding was completed, the mice in each group were fasted overnight, and on the 36th day, the mice were anesthetized and sacrificed by cervical dislocation. Sample collection: blood, spleen tissue, jejunum, feces. Mouse serum was separated from whole blood by centrifugation at 3000g for 10 minutes (4°C) and stored at -80°C for subsequent experimental detection.

[0083] 6.4 Indicator Monitoring

[0084] 6.4.1 Evaluation of allergic symptoms

[0085] The symptoms of mice were observed and scored within 30 minutes after gavage challenge. The diarrhea score and systemic score criteria are shown in Table 2. Body temperature was measured using a rectal thermometer. The body weight of mice in all groups was monitored once a week during the experiment.

[0086] Table 2 Behavioral scores of allergic symptoms in mice

[0087]

[0088] 6.4.2 Determination of mouse thymus index and spleen index

[0089] After 24 hours of protein stimulation, the body weight of each group of mice was weighed, the thymus and spleen were removed, and the fat and connective tissue were removed and washed with saline. The weight of the thymus and spleen of the mice was recorded. The thymus and spleen index of each group of mice was calculated using the following formula:

[0090] Thymus index (mg / g) = thymus mass (mg) / mouse body mass (g);

[0091] Spleen index (mg / g) = spleen weight (mg) / mouse body weight (g).

[0092] 6.4.3 Determination of mouse serum IgG1, IgG2a, IgE and histamine levels

[0093] Detect IgG, IgG1, and IgE in serum by indirect ELISA. WP protein was applied to the plate. Then, mouse serum (specific IgE diluted 1:5, specific IgG1 and IgG2a diluted 1:200) was added as the primary antibody, and HRP-conjugated goat anti-mouse IgE, IgG1, and IgG2a (IgE 1:1000, IgG1 1:2000, IgG2a 1:5000) antibodies were added as the secondary antibody. Finally, the absorbance was measured at 450nm.

[0094] Serum histamine levels were measured using a histamine ELISA kit.

[0095] 6.4.4 Detection of cytokines in mouse serum

[0096] IL-4 and IFN-γ levels were measured from serum using ELISA kits.

[0097] 6.5 Data Analysis

[0098] All data are expressed as mean ± standard deviation (SD). SPSS 20.0 statistical software was used for statistical analysis of the data. One-way analysis of variance (ANOVA) was used to analyze the significant differences in the results, and LSD test was used for multiple comparisons. The differences between the means were statistically significant when p < 0.05.

[0099] 6.6 Results and Analysis

[0100] 6.6.1 Food allergy symptoms, diarrhea, rectal temperature and body weight changes in mice

[0101] After the stimulation, the diarrhea, allergic reaction and rectal temperature of mice in the walnut protein sensitization group and the low-sensitivity complex group were measured and evaluated within half an hour. After stimulation, severe symptoms were observed in the walnut protein sensitization model group compared with the blank control group (allergic symptoms: 3.10±0.74 points; diarrhea: 1.60±0.52 points). The sensitization test group of Example 2 (allergic symptoms: 1.60±0.70 points; diarrhea: 0.50±0.53 points), the sensitization test group of Example 3 (allergic symptoms: 1.22±0.83 points; diarrhea: 0.78±0.44 points), the sensitization test group of Example 4 (allergic symptoms: 1.00±0.82 points; diarrhea: 0.40±0.52 points) and the sensitization test group of Example 5 (allergic symptoms: 1.20±0.92 points; diarrhea: 0.60±0.69 points) were able to significantly inhibit the reaction after stimulation (P<0.05) (such as Figure 4A and Figure 4B The rectal temperature of the walnut protein sensitization group was significantly lower than that of the blank control group, and the rectal temperature of the low-sensitivity complex group was higher than that of the walnut protein sensitization group. The sensitization test group of Example 3 and the sensitization test group of Example 4 could significantly improve the rectal temperature of mice (P<0.05) (e.g. Figure 4C ). These results indicate that hypoallergenic walnut protein can reduce walnut allergy by inhibiting the allergic reaction in mice. The weight growth rate of mice before and after sensitization was calculated. Figure 4D It can be seen that the growth rates of the walnut protein sensitized model group and the low-sensitivity complex group were lower than those of the blank control group.

[0102] 6.6.2 Spleen and thymus index

[0103] Compared with the blank control group, the thymus and spleen indexes of the walnut protein sensitization model group were significantly increased (P<0.05), while the thymus and spleen indexes of the walnut protein-polyphenol complex groups in different groups were significantly lower than those of the walnut protein sensitization group (P<0.05). These findings suggest that the complex has a protective effect on the lymphoid tissue of allergic mice.

[0104] 6.6.3 Determination of serum IgE, IgG1, IgG2a and histamine levels in mice

[0105] The sensitization ability of the walnut protein sensitization group and the complex group was evaluated by detecting the specific IgE, IgG1, IgG2a and histamine levels in the mouse serum. Compared with the blank control group, the IgE, IgG1, IgG2a and histamine levels in the mice in the walnut protein sensitization group were significantly increased (P<0.05), and IgG1 / IgG2a>1, indicating that the Th2 type immune response was dominant in the mice in the walnut protein sensitization group, and the mice had a humoral immune response and were sensitized. The walnut protein-polyphenol complex embodiment group can significantly reduce IgE, IgG1, IgG2a and histamine in mice. (As Figure 5 , Figure 6 , Figure 7 , Figure 8 ).

[0106] 6.6.4 Detection of cytokines in mouse serum

[0107] The balance between Th1 / Th2 cell immunity is crucial for immune stability. IFN-γ is a Th1 cytokine, and IL-4 is a Th2 cytokine. Figures 9 to 11 As shown in the figure, compared with the blank control group, the concentration of Th2-related cytokine IL-4 in the serum of the walnut protein sensitized group increased significantly, while the level of IFN-γ was inhibited (P<0.05), indicating that walnut protein allergy protein breaks the Th1 / Th2 immune balance of mice, upregulates the number of Th2 cytokines, promotes B cell differentiation and produces a large amount of IgE, thereby producing food allergy symptoms. The concentration of IL-4 factor in the serum of the four complex groups decreased significantly (P<005), and the concentration of INF-γ increased significantly (P<005).

[0108] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a walnut hypoallergenic complex protein, characterized in that: The following steps are involved: Step 1: Grind the shelled walnuts to obtain walnut powder, add acetone to defat, then add protein extract to extract crude protein, perform isoelectric precipitation, ammonium sulfate graded precipitation, dialysis desalination, and freeze-drying to obtain walnut allergenic protein; Step 2, dissolving the walnut allergenic protein in ultrapure water, adding a free radical initiator, adding polyphenols to form a walnut protein-polyphenol complex, dialyzing for 48 hours at 4°C, and freeze-drying to obtain a low-sensitivity complex; The method for extracting walnut allergenic protein in step 1 specifically comprises: Step 1), walnut defatting: walnuts are shelled and ground into fine powder, weighed and then added with 10 times acetone for defatting, and degreased under magnetic stirring at 4°C for 2h, and then centrifuged at 6000rpm for 30min at 4°C to obtain defatted walnut powder; Step 2), extraction of walnut crude protein: the defatted walnut powder obtained in step 1) is mixed with the protein extract at a mass: volume ratio of 1:10, stirred magnetically for 12 hours at 4°C, and then centrifuged at 6500 rpm for 20 minutes at 4°C, and the supernatant is taken to obtain a first walnut protein crude extract; Step 3), isoelectric precipitation: the first walnut protein crude extract obtained in step 2) is slowly lowered with HCl under magnetic stirring until the isoelectric point pH 5.6 is reached, stirring is continued for 30 minutes, and then transferred to a 4°C refrigerator and allowed to stand for 12 hours. After the supernatant and the protein layer are clearly separated, centrifuged at 6000 rpm for 15 minutes at 4°C to obtain a second walnut protein crude extract; Step 4), ammonium sulfate graded precipitation: dissolve the second walnut protein crude extract obtained in step 3) in 10 volumes of PBS, adjust the pH to 7.5 with NaOH, add ammonium sulfate to 70% saturation, and stand at 4°C for 2 hours; centrifuge at 4°C, 6500 rpm for 30 minutes, collect the precipitate, and obtain a third walnut protein crude extract; Step 5), dialysis desalination: the protein precipitate in step 4) is dissolved in ultrapure water, placed in a 3.5 kDa dialysis bag, dialyzed in distilled water for 48 hours to desalinate, and then freeze-dried to obtain walnut allergenic protein; The protein extract in step 2) is a Tris-HCl buffer solution; the preparation method of the Tris-HCl buffer solution is: weigh 3.02 g Tris, add 900 mL distilled water to dissolve, adjust the pH to 7.5 with hydrochloric acid, and finally dilute to 1 L with distilled water to prepare a 25 mM Tris-HCl extraction buffer solution with a pH of 7.5; add 0.5 M NaCl to the Tris-HCl extraction buffer solution to prepare a Tris-HCl buffer solution; In the step 2, the free radical initiators are hydrogen peroxide and ascorbic acid, the final concentration of hydrogen peroxide is 0.02-0.2 mol / L, and the final concentration of ascorbic acid is 0.25-5 g / L; The polyphenol in step 2 is any one of epigallocatechin gallate, resveratrol, quercetin, and trans-ferulic acid, and the final concentration of the polyphenol is 0.2-2 g / L; In the step 2, after adding the free radical initiator, the reaction conditions are to stand at 25° C. for 2 hours, and after adding the polyphenol, the reaction is carried out for 24 hours.

2. A walnut protein-polyphenol low-allergy complex prepared by the preparation method of the walnut low-allergy complex protein according to claim 1.

3. Use of the walnut protein-polyphenol complex as claimed in claim 2 in the preparation of walnut hypoallergenic food.

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