A low-sensitization penaeus vannamei, and a preparation method and application thereof

Abstract

The application provides a kind of low sensitization of penaeus vannamei and its preparation method and application, belong to food biotechnology field.The application uses fucoidan to treat penaeus vannamei by covalent modification, after treatment, protein spatial conformation has changed significantly, allergen epitope is hidden.After BALB / c mouse sensitization model and RBL-2H3 cell degranulation model test shows, the sensitization of covalent modification penaeus vannamei decreases by 72.88%, low sensitization penaeus vannamei can inhibit mast cell degranulation and increase the threshold of sensitization, but still has immunogenicity.Through the construction of BALB / c mouse oral tolerance model, it is found that low sensitization penaeus vannamei can significantly alleviate the multiple immunoglobulin-mediated hypersensitivity caused by mouse allergy, which proves that low sensitization penaeus vannamei has auxiliary effect on oral tolerance, can promote oral tolerance ability, can be used as an effective immune tolerance agent for shrimp allergy patients.

Description

TECHNICAL FIELD

[0001] The application provides a penaeus vannamei with low allergenicity and a preparation method and application thereof, and belongs to the technical field of food biotechnology. BACKGROUND

[0002] Food allergy has become a food safety problem that the food industry urgently needs to face and solve. When food allergy occurs, it can involve multiple systems in the body, and can cause anaphylactic shock and even endanger life in severe cases. According to statistical data, shrimp allergy has the highest proportion of food allergy incidence, with 26% of adult and infant populations allergic to shrimp and its products. Penaeus vannamei is widely loved by consumers because of its delicious and nutritious meat, and has become the highest yield of shrimp in China. The food allergy problem caused by it shows a growing trend and has attracted widespread attention.

[0003] At present, the main allergens reported in penaeus vannamei include tropomyosin (TM), arginine kinase (AK), sarcoplasmic calcium-binding protein (SCP), hemocyanin subunits (HCS), myosin light chain (MLC), and pyruvate kinase (PK). Among them, the research on tropomyosin and arginine kinase is relatively mature, and high-temperature and high-pressure treatment, irradiation treatment, enzyme cross-linking, and glycosylation reaction in food processing technology can reduce the allergenicity. Covalent modification can use Maillard reaction to form a covalent bond between the amino group of the amino acid side chain of the protein and the carbonyl group at the reducing end of the carbohydrate, which is considered as a potential allergenicity reduction technology. Domestic and foreign scholars often use monosaccharides such as glucose, fructose, and ribose to modify allergens, but monosaccharides are prone to browning and difficult to control, and can generate more by-products, which have potential safety risks. Covalent modification of proteins with polysaccharides can integrate the functional properties of natural proteins and polysaccharides, and is considered as a component for processing new foods, with safety and health characteristics. However, current research focuses on reducing the allergenicity of purified allergens through processing, and in specific foods, the composition of allergens is often complex, so how to better improve the allergenicity of food has become a problem to be solved. SUMMARY

[0004] In view of the above problems, the purpose of the present application is to provide a penaeus vannamei with low allergenicity, which is treated with fucoidan to improve the allergenicity of penaeus vannamei.

[0005] In order to achieve the above object, the present application provides a method for preparing low-sensitization Penaeus vannamei, comprising the following steps:

[0006] 1) washing: washing Penaeus vannamei and removing head, shell and intestinal tract;

[0007] 2) soaking: soaking the washed Penaeus vannamei in a fucoidan solution;

[0008] 3) boiling: boiling the Penaeus vannamei in the fucoidan solution in step (2) to obtain low-sensitization Penaeus vannamei.

[0009] In an embodiment of the present application, the mass concentration of the fucoidan solution is 1.25-2.5%.

[0010] In an embodiment of the present application, in step (2), the mass ratio of the Penaeus vannamei to the fucoidan solution is 1:3-4.

[0011] In an embodiment of the present application, in step (2), the soaking is carried out at low temperature, the soaking temperature is 0-4℃, and the soaking time is 3-4h.

[0012] In an embodiment of the present application, in step (3), the boiling temperature is 90-95℃, and the boiling time is 45-50min.

[0013] The present application also provides low-sensitization Penaeus vannamei prepared by the above method.

[0014] The present application also provides the use of the above low-sensitization Penaeus vannamei in preparing an oral immunotolerance agent.

[0015] The present application has the following advantages:

[0016] (1) The present application uses fucoidan to treat Penaeus vannamei by covalent modification reaction, so that the spatial conformation of protein is changed significantly, the allergen epitope is hidden, masked or destroyed, the intestinal barrier of the body is improved, thereby reducing the sensitization of Penaeus vannamei allergen, and the low-sensitization Penaeus vannamei has an auxiliary effect on oral tolerance, can promote the oral tolerance ability, and can be used as an effective immunotolerance agent for shrimp allergy patients. The method is simple in operation, low in cost and easy to realize large-scale production.

[0017] (2) The test by BALB / c mouse sensitization model shows that the Penaeus vannamei treated by the method of the present application can be used as an effective immunotolerance agent for shrimp allergy patients, can be applied in the field of immunotherapy, and the sensitization of the Penaeus vannamei treated by the method of the present application is reduced by 72.88%, which can be applied in the field of low-sensitization food.

[0018] (3) The test by using RBL-2H3 cell degranulation model shows that the Penaeus vannamei treated by the method provided in the application can significantly inhibit cell degranulation and increase the sensitization threshold, and the inhibition rate of 10 ng / mL of the covalently modified Penaeus vannamei on the release of β-hexosaminidase is 26.06%.

[0019] (4) The Penaeus vannamei provided in the application not only has low allergenicity, but also is simple to operate, and can be used for immunotherapy of patients allergic to Penaeus vannamei, and also provides a reference value for the treatment of other related allergic diseases. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The browning degree data of Penaeus vannamei protein before and after covalent modification in Example 2;

[0021] Figure 2 The grafting degree data of Penaeus vannamei protein before and after covalent modification in Example 2;

[0022] Figure 3 The infrared spectrum of Penaeus vannamei protein before and after covalent modification in Example 2;

[0023] Figure 4 The scanning electron microscope image of Penaeus vannamei protein before and after covalent modification in Example 2;

[0024] Figure 5 The specific implementation scheme diagram of the allergic mouse model in Example 2;

[0025] Figure 6 The change of allergen of Penaeus vannamei before and after covalent modification and the binding ability of specific IgE of mouse serum in Example 2;

[0026] Figure 7 The change of cell degranulation of RBL-2H3 (hereinafter referred to as RBL-2H3 mast cell) after Penaeus vannamei protein before and after covalent modification in Example 2 induced sensitized rat basophilic leukemia (RBL) cell strain RBL-2H3;

[0027] Figure 8 The linear antigen epitope masking situation of Penaeus vannamei allergen after covalent modification in Example 2;

[0028] Figure 9 The change of intestinal morphology of mice caused by Penaeus vannamei allergen before and after covalent modification in Example 2;

[0029] Figure 10 The specific implementation scheme diagram of the oral tolerance mouse model in Example 2;

[0030] Figure 11 For the change of the IgE binding capacity of the covalently modified Penaeus vannamei protein with fucoidan as an immunological tolerance agent in Example 2, different letters indicate significant differences between groups (P < 0.05); the asterisk indicates significant differences between groups: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001.

[0031] Figure 12 For the change of the IgG1 binding capacity of the covalently modified Penaeus vannamei protein with fucoidan as an immunological tolerance agent in Example 2, different letters indicate significant differences between groups (P < 0.05); the asterisk indicates significant differences between groups: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. DETAILED DESCRIPTION

[0032] In order to better understand the technical scheme of the present application, the exemplary embodiments of the present application will be described in more detail below. Although exemplary embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided so that the present application can be more thoroughly understood and the scope of the present application can be fully conveyed to those skilled in the art.

[0033] The materials used in the embodiments of the present application are all ordinary commercially available products and can be purchased in the market.

[0034] The present application will be further described below in conjunction with specific examples, but the implementation and protection scope of the present application is not limited thereto. For process parameters not specifically indicated, conventional techniques can be referred to.

[0035] The sensitization of Penaeus vannamei is evaluated by a BALB / c mouse sensitization model and a RBL-2H3 cell degranulation model in the embodiments of the present application.

[0036] The effectiveness of the low-sensitization Penaeus vannamei as an immunological tolerance agent is demonstrated by a BALB / c mouse oral tolerance model in the embodiments of the present application.

[0037] Example 1

[0038] A preparation method of a low-sensitization Penaeus vannamei, comprising the following steps:

[0039] 1) Washing: washing the Penaeus vannamei and removing the head, shell and shrimp line;

[0040] 2) Immersion: placing 100 g of the washed Penaeus vannamei into 400 g of a fucoidan solution with a concentration of 1.25% and immersing for 3 h at 4℃;

[0041] 3) Cooking: the Penaeus vannamei in step (2) is boiled with fucoidan solution at 95℃ for 45 min to prepare low-sensitization Penaeus vannamei.

[0042] Example 2

[0043] 1. Degree of covalent modification of Penaeus vannamei with fucoidan

[0044] The degree of covalent modification is achieved by extracting proteins from Penaeus vannamei and analyzing the proteins.

[0045] Extraction of total proteins from Penaeus vannamei: Take Penaeus vannamei before and after fucoidan modification, respectively, and place them in 4℃, 10 times volume (v / v) PBS buffer containing 0.3 mol / L NaCl (0.2 mol / L NaH2PO4·2H2O, 0.2 mol / L Na2HPO4·12H2O), crush the tissue and stand, extract at 4℃ for 3h, centrifuge (8000r / min, 4℃, 30min), and take the supernatant. Then dialyze the supernatant in ultrapure water at 4℃ for 48h, and freeze-dry the proteins obtained after dialysis at -50℃ for 48h, i.e. obtain total protein powders of Penaeus vannamei before and after fucoidan modification. The preparation method of Penaeus vannamei after fucoidan modification is different from that of Example 1, in that the cleaned Penaeus vannamei is placed in fucoidan solutions with concentrations of 1.25%, 2.5%, 5% and 10%, respectively.

[0046] (1) Analyzing the degree of covalent modification of Penaeus vannamei proteins with fucoidan by browning degree.

[0047] S1. Determination of browning degree

[0048] Take total protein powders of Penaeus vannamei before and after fucoidan modification, dissolve them in deionized water, and prepare 1mg / mL protein solutions, respectively, and measure the absorbance at 294nm and 420nm, with ultrapure water without any sample as a blank control.

[0049] S2. Determination of grafting degree

[0050] Prepare o-phthalaldehyde (OPA) solution: accurately weigh 80mg OPA into 2mL methanol solution; then mix 50mL 0.1mol / L sodium tetraborate solution, 5mL 200mg / mL SDS solution, 200μL β-mercaptoethanol thoroughly, add OPA methanol solution, mix to 100mL in a brown bottle. When measuring, accurately weigh 4.00mL OPA solution, add 200μL sample solution, place in a 35℃ water bath for 2min, then measure the absorbance at 340nm. Deionized water is used as a blank control group. The grafting degree is calculated according to the following formula:

[0051] Grafting degree (%) = {(A0-A1) / A0} x 100%

[0052] Wherein: A0 is the absorbance value of the unmodified protein solution; A1 is the absorbance value of the sample solution after covalent modification.

[0053] The results: The glycosylated intermediates such as hydroxymethyl furan in the initial stage of covalent modification reaction were determined by absorbance at 294 nm. The formation of brown chromophore was characterized by the absorbance of the browning reaction at 420 nm. Combined with A294 and A420, the progress and speed of the covalent modification reaction can be tracked. From Figure 1 It can be seen that the value of A294 gradually increases with the increase of fucoidan concentration, while A420 reaches the maximum when the fucoidan concentration is 2.50%, and then decreases, and when the fucoidan concentration increases to 5% and above, the value of A420 has no significant change and is not significantly different from the control group (Control). In addition, from Figure 1 It can also be seen that the increase of A294 is higher than that of A420, indicating that the early covalent modification reaction is dominant.

[0054] The free amino groups of the protein are consumed by the covalent modification reaction with the reducing end carbonyl of the saccharide, and some non-reducing polysaccharides consume the hydroxyl group to react with the free amino group of the protein, so the progress of the covalent modification reaction can be shown by determining the content of free amino acids, and the decrease of free amino acid content is calculated as the grafting degree. From Figure 2 It can be seen that the grafting degree of the sample increases with the increase of the concentration of fucoidan, and reaches the maximum when the concentration of fucoidan is 5.00%, and then shows a slow downward trend. Throughout the reaction process, the grafting degree increases with the increase of the concentration of polysaccharide, because the increase of the number of sugar molecules increases the collision probability between them and the free amino groups of the protein, which can improve the reactivity and increase the grafting degree; however, as the sugar content continues to increase, the viscosity of the solution increases and the steric hindrance effect occupies the main reason. These all show that the South American white shrimp is covalently modified by fucoidan and a certain degree of browning occurs. However, the covalent modification reaction process shows a trend of first increasing and then decreasing, and the appropriate concentration is needed to maximize the reaction degree.

[0055] (2) Use infrared spectroscopy to determine the changes of the molecular structure of the covalent modification of South American white shrimp protein and fucoidan.

[0056] Mix the South American white shrimp whole protein powder before and after fucoidan modification with potassium bromide in a mass ratio of 1:100 and press into tablets. Use a Fourier infrared spectrometer to scan 32 times at a resolution of 4 cm -1 in the range of 4000-400 cm -1 to obtain the infrared spectrum of the sample.

[0057] The experimental results: by infrared spectrum( Figure 3 ) can be known, because of the existence of multiple hydroxyl in the sugar chain of fucoidan, the number of hydroxyl in the modified fucoidan of white shrimp protein is increased, which is embodied in 3700-3200cm -1 The peak width of white shrimp protein modified by different concentrations of fucoidan is widened, and the spectral intensity is increased. At the same time, the sample after covalent modification appears red shift at 1100cm -1 , and the absorption intensity increases, which may be due to the increase of hydroxyl group introducing-OH bending vibration. In addition, the spectral intensity of covalent modification product is further increased at 3000-2800cm -1 , which indicates that the covalent combination of sugar molecules causes-CH stretching vibration.

[0058] (3) the change of microstructure of white shrimp protein and fucoidan covalent modification was determined by scanning electron microscopy.

[0059] The sample was fixed on the metal sample table with conductive double-sided adhesive, and then placed in the ion sputtering instrument to evaporate a layer of 10-20nm thick platinum film on the sample surface. The electron microscope was adjusted to the best shooting field of view, magnification, observation and photography.

[0060] It can be seen from Figure 4 that the uncovalently modified white shrimp presents a loose flaky structure with many pores on the surface, while the white shrimp protein after covalent modification with different concentrations appears a reticular structure formed by the crosslinking of protein fibers, and there is obvious layering and aggregation phenomenon. The change of protein conformation caused by covalent modification may produce certain steric hindrance, mask the antigen epitope, and further lead to the decrease of IgE binding capacity.

[0061] 2, the evaluation of the allergenicity of white shrimp and fucoidan covalent modification

[0062] The specific implementation of the allergic mouse model scheme is as Figure 5The BALB / c mice were used as the allergic model, and all the mice were randomly divided into 6 groups (10 mice in each group): PBS group, unmodified group, and covalent modification group with four different fucoidan concentrations. On the 0th, 7th, 14th, 21st, and 28th day of the feeding period, the mice in the PBS group, the unmodified group, and the covalent modification group were respectively given 0.5 mL of PBS, 0.5 mL of unmodified protein solution (5 mg of unmodified white shrimp protein dissolved in PBS), and 0.5 mL of covalently modified protein solution (5 mg of covalently modified white shrimp protein dissolved in PBS) by gavage. On the 35th day of the feeding period, the mice were subjected to blood collection from the inner canthus vein, and the serum was obtained by centrifugation at 5000 r / min and 4°C for 10 min, and then was aliquoted into 50 μL and stored at -20°C for subsequent determination of IgE binding activity and RBL-2H3 cell degranulation experiment.

[0063] (1) Analysis of the change in IgE binding activity of covalently modified white shrimp protein with fucoidan by using a BALB / c mouse sensitization model

[0064] The change in the specific IgE binding capacity of white shrimp before and after processing was determined by using an enzyme-linked immunosorbent assay.

[0065] TBST buffer was prepared by dissolving 8.755 g of NaCl, 2.422 g of tris-hydroxymethyl aminomethane, and 0.5 mL of Tween-20 in 800 mL of ultrapure water, and then adjusting the pH to 7.5 with 1 mol / L hydrochloric acid, and then diluting to 1000 mL.

[0066] The unmodified and modified white shrimp proteins were dissolved in coating solution (0.05 mol / L sodium carbonate buffer), and then were added to the enzyme-labeled plate (10 μg / mL, 100 μL / well) for overnight incubation at 4°C. After washing the plate with washing solution (TBST containing 0.1% bovine serum albumin, 300 μL / well) for 5 times, blocking solution (TBST containing 1% bovine serum albumin, 150 μL / well) was added for blocking at 37°C for 2 h. After blocking, the plate was washed for 5 times, and then the serum sample of the corresponding sensitized group of mice diluted with blocking solution at a ratio of 1:10 (v / v) (150 μL / well) was added for incubation at 37°C for 2 h. After washing the plate for 6 times, biotin-labeled rat anti-mouse IgE diluted with blocking solution at a ratio of 1:1000 (150 μL / well) and HRP-labeled streptavidin were sequentially added for incubation at 37°C for 1 h. After washing the plate for 6 times, TMB color developing solution (100 μL / well) was added for incubation at 37°C for 15 min. Finally, 2 mol / L H2SO4 (50 μL / well) was added to terminate the reaction, and the absorbance value (OD 450nm) at 450 nm was determined by using an enzyme-labeled instrument.

[0067] The experimental results are shown in Table 1. Figure 6As shown, the IgE binding activity of the unmodified group (Control) was significantly higher than that of the PBS group, indicating that the allergic model of the BALB / c mice to P. vannamei was successfully constructed. The specific IgE antibody level of the group covalently modified with 2.5% fucoidan was reduced by 72.88% compared with the unprocessed group, but the IgE antibody content of the group covalently modified with 5.00% fucoidan and the group covalently modified with 10.00% fucoidan was increased. Therefore, when the fucoidan concentration is 1.25-2.5%, the P. vannamei prepared thereby can significantly reduce the allergenicity but still retain the immunogenicity.

[0068] (2) Analysis of the change in cell degranulation of P. vannamei protein covalently modified with fucoidan by sensitizing RBL-2H3 cells with mouse sensitized serum

[0069] RBL-2H3 cells were inoculated into a 96-well cell culture plate at a concentration of 5×10 5 mL (200 μL / well) and cultured for 24 h. Subsequently, the cells were washed with PBS buffer, 100 μL / well of mouse allergic serum (diluted with DMEM medium without fetal bovine serum) was added, and the cells were cultured for 24 h. After washing with Tyrode's buffer for 3 times, 0.01, 0.1, 1, 10, 100, 1000 ng / mL protein solution (diluted with Tyrode's buffer, 50 μL / well) was added to each group in turn, and the reaction was carried out at 37°C for 45 min. After the reaction was completed, the reaction was terminated by placing the cells in an ice box. 30 μL of the supernatant of the cells after the reaction was taken to a 96-well plate, 50 μL of a solution of 1 mmol / L 4-nitrophenyl Nacetyl-β-D-glucosaminide (PNAG) in citric acid was added, and the reaction was carried out at 37°C for 1 h. Then, 200 μL of sodium carbonate buffer was added, and the absorbance value of each well at 450 nm was measured. The total release group was the absorbance value obtained after the cells were treated with 1% Trition X-100 instead of the protein sample, and the negative control group was the absorbance value obtained after the cells were treated with Tyrdes's buffer instead of the protein sample.

[0070] The release rate of β-amino hexosidase = {(absorbance of the sample group - absorbance of the negative control group) / (absorbance of the total release group - absorbance of the negative control group)}

[0071] The inhibition rate (%) of the release of β-amino hexosidase = {(1 - (release rate of the covalently modified group / release rate of the control group)) × 100%

[0072] The experimental results are shown in Table 1. Figure 7As shown, the covalent modification of fucoidan can significantly inhibit the degranulation of RBL-2H3 cells to release β-hexosaminidase, and increase the sensitization threshold to significantly reduce the allergenicity of shrimp proteins. Among them, the maximum release rate of β-hexosaminidase of G-5.00% is reduced by 53.62% compared with shrimp without fucoidan treatment. The release rate of β-hexosaminidase of G-2.50% is higher than that of proteins without fucoidan treatment at the protein concentration of 100-1000 ng / mL, but it has a significant inhibitory effect on the degranulation of RBL-2H3 cells to release β-hexosaminidase at the protein concentration of 0.1-10 ng / mL. Among them, the inhibition rate of 10 ng / mL G-2.50% protein sample on the release of β-hexosaminidase is 26.06%. Combined with the degree of browning, it is speculated that covalent modification can mask the antigenic epitopes of Penaeus vannamei, inhibit mast cell degranulation, and increase the sensitization threshold to reduce its allergenicity. Combined with the mouse allergy model, it is known that the Penaeus vannamei prepared in this embodiment can significantly reduce its allergenicity but still retain immunogenicity. Among them, when the concentration of fucoidan is 2.5% for covalent modification, the animal model shows the most significant effect, but there is a certain discrepancy in the cell model, which indicates that in addition to the masking of linear antigenic epitopes of allergens, fucoidan also affects the allergenicity.

[0073] 3. Mechanism of covalent modification of Penaeus vannamei with fucoidan to reduce allergenicity

[0074] (1) Mass spectrometry combined with bioinformatics to analyze the masking of linear epitopes of Penaeus vannamei allergens after covalent modification with fucoidan at a concentration of 2.5%

[0075] S1, 100 μg of fucoidan covalently modified Penaeus vannamei protein was taken and made up to 100 μL with 8M urea (90 μL with 8M urea, 10 μL of 1M triethylammonium bicarbonate buffer (TEAB) was added to make the PH = 8, and the total volume was 100 μL). 2 μL of 0.5M trichloroethyl phosphate (TCEP) was added and reacted at 37°C for 1 hour, followed by the addition of 4 μL of 1M iodoacetamide (IAM), and reacted at room temperature for 40 min in the dark. Then, -20°C pre-cooled acetone was added at a sample:acetone (volume ratio) of 1:5, and precipitated at -20°C overnight. Then, high-speed centrifugation (12000g, 20 min, 4°C) was performed to discard the supernatant. 1 mL of -20°C pre-cooled 90% acetone solution was added, vortexed to mix well, and then high-speed centrifugation (12000g, 20 min, 4°C) was performed to discard the supernatant. The washing step was repeated twice. After the acetone on the surface of the precipitate was completely dried at room temperature, it was resuspended in 100 μL of 100 mM triethylammonium bicarbonate buffer (TEAB), and trypsin (Promega Trypsin) was added at an enzyme:protein (mass ratio) of 1:50, and enzymolyzed at 37°C overnight. Desalination was performed using a C18 desalination column, and then freeze-dried. Subsequently, LC-MS / MS mass spectrometer was used for identification, the accurate molecular weight of the peptide was obtained, the shift of the protein mass-to-charge ratio was observed, and a series of shielded sites were obtained.

[0076] S2, the sequences of Penaeus vannamei tropomyosin (TM), arginine kinase (AK), parvalbumin (SCP), myosin light chain (MLC), pyruvate kinase (PK), and hemocyanin (HCS) were obtained from the NCBI database, and the corresponding peptide segments and shielded sites of the covalently modified proteins were located in the protein sequences.

[0077] S3, five bioinformatics software DNAStar, AntheProt, ABCpred, Immunomedicine Group, and BepiPred 1.0 were used to predict the linear antigen epitopes of Penaeus vannamei tropomyosin (TM), arginine kinase (AK), parvalbumin (SCP), myosin light chain (MLC), pyruvate kinase (PK), and hemocyanin (HCS), and the predicted epitopes of ≥3 software were taken as the linear antigen epitopes of Penaeus vannamei allergens, and the predicted linear antigen epitopes of Penaeus vannamei were located in the corresponding allergen sequences of Penaeus vannamei described in step S2.

[0078] S4, the shielded sites of Penaeus vannamei allergens described in step S2 were compared with the predicted linear antigen epitopes of Penaeus vannamei described in step S3 to analyze the masking of the allergen epitopes during the covalent modification reaction.

[0079] S5, using SWISS-MODEL software, the highest similarity protein as a template, homologous modeling to establish the three-dimensional model of the allergen of Penaeus vannamei;

[0080] S6, using Pymol software, the allergen epitope masking site in the covalent modification reaction process is located in the three-dimensional model corresponding to Penaeus vannamei described in step S5.

[0081] The experimental results are shown in Figure 8 and Tables 1-3, and the Penaeus vannamei is covalently modified at a concentration of 2.5% fucoidan. The masked sites of tropomyosin (TM), arginine kinase (AK), sarcoplasmic calcium binding protein (SCP), myosin light chain (MLC), pyruvate kinase (PK), and hemocyanin (HCS) contained in the Penaeus vannamei are 210, of which 49 are located on the predicted linear epitopes, distributed in 34 linear epitope polypeptides. Among them, the linear antigen epitope polypeptide of tropomyosin (TM) is 2, respectively 19 DRADTLEQQNKEANNRAEKTEEE 42 、 97 NDLDQV 102 ; the linear antigen epitope polypeptide of arginine kinase (AK) is 7, respectively 92 GFKQTDKHPNKD 103 、 112 NVDPEGK 118 、 131 LQGYPF 136 、 139 CLTESQYKE 147 、 173 MSKEVQ 178 、 295 EKLEE 299 、 307 QVRGTRGEHTE 317 ; the linear antigen epitope polypeptide of sarcoplasmic calcium binding protein (SCP) is 4, respectively 75 CQGKKYG 81 、 104 GKVGLD 109 、 132 NKLTTEDDRK 141 、 159 SNPDESC 165 ; the linear antigen epitope polypeptide of myosin light chain (MLC) is 2, respectively 74 QVKKDKDSGS 83 、 119 EKSEL 123; 13 linear epitope polypeptides of myosin light chain (MLC) are shielded, respectively 11 AADTHTQVDHMAAL 24 、 70 GTHEYHSE 77 、 89 YSDKIGHS 96 、 138 SYYEKCSEDVL 148 、 177 KDVGSDSIDCE 187 、 195 GSKKGVNL 202 、 211 AVSEKD 216 、 244 EIRDVLGEKG 253 、 304 MIAKCNKVGKPVI 316 、 325 MVKKPRP 331 、 488 GKECGFIKPG 497 、 507 QKGAG 511 、 564 IPWPSFLAHMGPYHLC 579 ; 4 linear epitope polypeptides of hemocyanin (HCS) are shielded, respectively 48 ANSFDPVGNLGSYSDGGAA 66 、 277 KYGGQFPARPDN 288 、 311 RDAIAHGYIVDS 322 、 569 ATGLPNRFLLP 579 . Therefore, the covalent modification can shield the epitopes of P. vannamei, thereby reducing the sensitization.

[0082] (2) Analysis of the effect of covalent modification of P. vannamei protein with 2.5% fucoidan on the intestinal barrier using a BALB / c mouse sensitization model.

[0083] The establishment process of the mouse sensitization model is the same as that in the sensitization evaluation, except that only 2.5% fucoidan is used as the covalent modification group when evaluating the effect on the intestinal barrier.

[0084] S1. In the BALB / c mouse sensitization model, collect the intestinal tissue of the mouse 30 min after gavage challenge.

[0085] ​​S2, immerse the small intestine tissue in embedding agent, and then place in liquid nitrogen for 10-20 s to rapidly freeze the tissue into a block. After the block is prepared, place in a cryostat microtome to slice the round cavity into slices of 5-10 μm.

[0086] S3, air dry the frozen slices at room temperature for 15 min, and then immerse in PBS solution for 15 min to remove the embedding agent.

[0087] S4, immerse the slices in hematoxylin staining solution for 5 min, and then repeatedly rinse in PBS solution for 5 times after taking out. Then immerse in 0.5% hydrochloric acid ethanol solution for 5 s, and then repeatedly rinse in PBS solution for 5 times after taking out.

[0088] S5, immerse the slices in eosin staining solution for 5 min, and then repeatedly rinse in PBS solution for 5 times after taking out.

[0089] S6, immerse the slices in 80% ethanol and 95% ethanol for 30 s, respectively, and then immerse in anhydrous ethanol I and II solution for 2 min, and then immerse in xylene I and II solution for 2 min. Finally, seal with gum.

[0090] S7, observe and take photos under a microscope.

[0091] The experimental results are shown in Table 1. Figure 9 As shown in Table 1, the villi of the PBS group mice are arranged in order and the structure is complete, and the cells are arranged uniformly. Compared with the blank group, the villi of the unprocessed group are arranged in disorder, and a large area of the intestinal villi is shed, the intestinal villi is eroded, and there is obvious damage. The intestinal villi of the covalently modified group is significantly relieved, and there is no obvious inflammatory cell infiltration, and the tissue morphology is regular. This shows that the Penaeus vannamei covalently modified has a certain effect of relieving the damage of the intestinal tract caused by sensitization, and can improve the intestinal barrier function.

[0092] 4. Effectiveness evaluation of Penaeus vannamei covalently modified with fucoidan as an immune tolerance agent

[0093] Establishment of oral tolerance mouse model: the specific implementation of the oral tolerance mouse model scheme is shown in Table 2. Figure 10The BALB / c mice were used as the oral tolerance model, and all the mice were randomly divided into three groups (10 mice in each group): PBS intervention group, unprocessed intervention group, and covalent modification intervention group with 2.5% fucoidan concentration. On the 0th, 7th, 14th, 21st, and 28th days of the feeding period, 0.5 mL of unprocessed protein solution (5 mg of unprocessed white shrimp protein dissolved in PBS per mouse) was administered to the three groups by gavage. On the 35th, 38th, 42nd, 45th, 49th, 52nd, 56th, 59th, 63rd, and 66th days of the culture period, 0.5 mL of PBS, 0.5 mL of unprocessed protein solution (5 mg of unprocessed white shrimp protein dissolved in PBS per mouse), and 0.5 mL of covalently modified protein solution (5 mg of covalently modified white shrimp protein dissolved in PBS per mouse) were administered to the mice in the PBS group, the unprocessed group, and the covalently modified group, respectively. On the 73rd day of the feeding period, the mice were subjected to eye internal angular vein blood collection, 5000 r / min, 4°C centrifugation for 10 min, serum collection, 50 μL aliquot freezing at -20°C, and subsequent IgE, IgG1 binding activity determination.

[0094] (1) Analysis of the change in the IgE binding capacity of white shrimp protein covalently modified with fucoidan as an immune tolerance agent using a BALB / c mouse oral tolerance model

[0095] Analysis of the change in the IgE binding capacity of white shrimp protein covalently modified with fucoidan as an immune tolerance agent using a BALB / c mouse oral tolerance model

[0096] TBST buffer preparation: 8.755 g of NaCl, 2.422 g of tris-hydroxymethyl aminomethane, 0.5 mL of Tween-20, dissolved in 800 mL of ultrapure water, and then adjusted to pH 7.5 with 1 mol / L hydrochloric acid, and then diluted to 1000 mL.

[0097] Unprocessed P. vannamei protein was dissolved in coating solution (0.05 mol / L sodium carbonate buffer solution), and then added to an enzyme-labeled plate (10 μg / mL, 100 μL / well), and coated at 4°C overnight. After washing the plate 5 times with washing solution (TBST containing 0.1% bovine serum albumin, 300 μL / well), blocking solution (TBST containing 1% bovine serum albumin, 150 μL / well) was added, and blocked at 37°C for 2 h. After blocking was completed, the plate was washed 5 times, and the serum sample of the orally tolerant mouse diluted with blocking solution at 1:10 (v / v) (150 μL / well) was added, and incubated at 37°C for 2 h. After washing the plate 6 times, biotin-labeled rat anti-mouse IgE and HRP-labeled streptavidin diluted at 1:1000 (150 μL / well) were sequentially added, and incubated at 37°C for 1 h. After washing the plate 6 times, TMB color developing solution (100 μL / well) was added, and incubated at 37°C for 15 min. Finally, 2 mol / L H2SO4 (50 μL / well) was added to terminate the reaction, and the absorbance value (OD 450nm) at 450 nm was determined by using an enzyme-labeled instrument.

[0098] The experimental results are shown in Table 1. Figure 11 As shown in Table 1, the IgE binding activity of the unprocessed intervention group was significantly lower than that of the PBS intervention group, indicating that the BALB / c mouse P. vannamei oral tolerance model was successfully constructed, and the tolerance phenomenon was not caused by self-tolerance. The IgE binding capacity of the covalent modification intervention group decreased by 40.57% compared with the PBS intervention group. The results show that the P. vannamei prepared by the processing method of the present application has an auxiliary effect on oral tolerance, and can become an effective immune tolerance agent, and can be used for immunotherapy of patients allergic to P. vannamei.

[0099] (2) Analysis of the change of the IgG1 binding capacity of P. vannamei protein covalently modified with fucoidan as an immune tolerance agent by using a BALB / c mouse oral tolerance model

[0100] The change of the IgG1 binding capacity of P. vannamei as an immune tolerance agent after processing was determined by using an enzyme-linked immunosorbent assay.

[0101] TBST buffer solution: 8.755 g NaCl, 2.422 g tris-hydroxymethyl aminomethane, 0.5 mL Tween-20, dissolved in 800 mL ultrapure water, and then adjusted to pH 7.5 with 1 mol / L hydrochloric acid, and then diluted to 1000 mL.

[0102] Unprocessed P. vannamei protein was dissolved in coating solution (0.05 mol / L sodium carbonate buffer solution), and then added to an enzyme-labeled plate (10 μg / mL, 100 μL / well), and coated at 4°C overnight. After washing the plate with washing solution (TBST containing 0.1% bovine serum albumin, 300 μL / well) for 5 times, blocking solution (TBST containing 1% bovine serum albumin, 150 μL / well) was added, and blocked at 37°C for 2 h. After the blocking was completed, the plate was washed for 5 times, and the serum sample of the orally tolerant mouse diluted with the blocking solution at 1:10 (v / v) (150 μL / well) was added, and incubated at 37°C for 2 h. After washing the plate for 6 times, HRP-labeled goat anti-mouse IgG1 diluted with the blocking solution (IgG1 diluted at 1:2000, 150 μL / well) was added, and incubated at 37°C for 1 h. After washing the plate for 6 times, TMB color developing solution (100 μL / well) was added, and incubated at 37°C for 15 min. Finally, 2 mol / L H2SO4 (50 μL / well) was added to terminate the reaction, and the absorbance value (OD450nm) at 450 nm was determined by using an enzyme-labeled instrument.

[0103] The experimental results are shown in Table 1. Figure 12 As shown in Table 1, compared with the PBS intervention group, the concentration of IgG1 in the serum of the unprocessed intervention group was significantly reduced. Therefore, the oral tolerance model was successfully established. Compared with the unprocessed intervention group, the covalent modification intervention group showed a lower specific IgG1 response. The results show that the P. vannamei prepared by the processing method of the present application can promote the oral tolerance ability, can significantly reduce the response value of various immunoglobulin-mediated hypersensitivity reactions, and can become an effective immunological tolerance agent.

[0104] Table 1: Shielding sites of P. vannamei after covalent modification

[0105]

[0106]

[0107]

[0108] The shadow in the table represents the shielding site.

[0109] Table 2: Linear epitopes of P. vannamei allergen predicted by five biological information software

[0110]

[0111] Table 3: Shielding linear antigen epitopes of P. vannamei after covalent modification

[0112]

[0113] The shadow in the table represents the linear antigen epitope shielding site.

[0114] The above embodiments are not intended to limit the scope of the present application, and the described steps are not intended to limit the order of execution. Those skilled in the art will make obvious modifications to the present application in light of the prior art, and such modifications fall within the scope of the claims of the present application.

Claims

1. A process for the preparation of hypoallergenic Penaeus vannamei, characterized by, The method comprises the following steps: 1) washing: washing and deheading, deshelling and removing the intestinal tract of the white shrimp; 2) soaking: soaking the washed white shrimp in a fucoidan solution, the mass concentration of the fucoidan solution being 1.25-2.5%, the mass ratio of the white shrimp to the fucoidan solution being 1:3-4, the temperature of the soaking being 0-4℃, and the soaking time being 3-4h; 3) boiling: boiling the white shrimp in the fucoidan solution to obtain the low-sensitization white shrimp.

2. The production method according to claim 1, characterized by, In step (3), the boiling temperature is 90-95℃, and the boiling time is 45-50min.

3. The low-sensitization white shrimp prepared by the method of claim 1 or 2.

4. The use of the low-sensitization white shrimp of claim 3 in the preparation of low-sensitization food and medicine, which can improve the intestinal barrier.

5. Use according to claim 4, characterized in that, The low-sensitization medicine includes an oral immunological tolerance agent.

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