Method for reducing allergenicity of tropomyosin through curcumin-mediated photodynamic and application

Abstract

According to the method for reducing the allergenicity of tropomyosin through curcumin-mediated photodynamic and the application, a curcumin photosensitizer and a sensitizing factor are mixed, incubation is conducted in the dark, and then light treatment is conducted. The method for reducing the allergenicity of the aquatic products is low in cost, easy to operate and high in safety, a brand new channel for reducing the allergenicity of the shrimps is opened up, and a positive strategy is provided for transformation of allergen prevention and control in the food industry from passive response to active guarantee. Compared with the prior art, the method has the advantages that the operation is extremely simple, high-temperature or other treatment modes are not needed, the applicability is good, the selected curcumin is a food-grade photosensitizer, and the technology can be widely popularized and applied in the food industry.

Description

Technical Field

[0001] The invention belongs to the field of food, and specifically relates to a method and application of curcumin-mediated photodynamic reduction of tropomyosin sensitization. Background Art

[0002] Food allergy is an epidemic that causes abnormal reactions in the body's immune system, which can easily lead to symptoms such as diarrhea, vomiting, asthma, urticaria and gastrointestinal pain, and even anaphylactic shock or death. It has become a world-recognized food safety and public safety issue. Aquatic products, mainly shrimp, are identified by FAO and WHO as one of the main allergens that cause food allergies. In Asia, about 40% of children and 33% of adults are allergic to crustaceans such as shrimp. It is worth noting that the proportion of people in my country who are allergic to such aquatic products is as high as 40.3%.

[0003] Current research has found that tropomyosin (TM) is the most representative allergenic factor in shrimp, and the allergic reactions caused by it account for more than 80% of shrimp allergic reactions. TM is composed of two identical α-helical chains entangled with each other, and contains rich amino acid residues such as glutamic acid, tyrosine, phenylalanine, histidine and serine. The allergic immune response caused by TM is mainly determined by its epitope, that is, the part of the protein recognized by the antibody. The epitope is a special chemical group in the TM molecule, located on the surface of the protein, which can be recognized by antibodies or antigen receptors. It is composed of 5 to 17 amino acid residues and can specifically bind to the IgE antibody produced by B lymphocytes, playing a key role in the body's entire allergic reaction.

[0004] In the prior art, the prevention of shrimp food allergies is mainly through effective prevention and processing to reduce the food allergy rate. Generally, there are two ways: (1) early identification and management of food allergic reactions, accurate labeling, reminders and warnings to people who are susceptible to allergies, such as the United States, EU member states, Japan and other countries have mandatory labeling of food containing crustaceans; (2) using specific processing methods for foods containing allergens to achieve the purpose of reducing the allergenicity of allergenic foods.

[0005] Among them, heat treatment is one of the most important and widely used technologies in the food industry. Heat treatment of food is accompanied by different modification reactions, such as denaturation, hydrolysis of peptide bonds, reorganization of disulfide bonds, and interaction with other components including carbohydrates and lipids. Heat treatment can lead to the loss of secondary and tertiary structures and non-covalent interactions. Therefore, these treatments can reduce the allergenicity by destroying the conformational epitopes of shrimp TM. However, due to the acid and alkali resistance, high temperature resistance and digestion resistance of shrimp TM protein, conventional heat treatment methods cannot destroy its antigenic epitopes and achieve the purpose of reducing allergenicity. In addition, shrimp TM protein contains a large amount of lysine, and heat processing will accelerate its reaction rate with reducing sugars to form new antigenic epitopes. In addition, chemical methods and enzymes are also commonly used to reduce the allergenicity of shrimp TM. Glycosylation modification can change the spatial conformation, surface hydrophobicity and amino acid content of shrimp TM, reduce the binding ability of TM's structural epitopes with IgE, and thus reduce the allergenicity of shrimp TM.

[0006] Although existing research methods can reduce the allergenicity of shrimp to a certain extent, long-term processing will bring some negative effects. For example, chemical methods will cause changes in food flavor and texture and produce harmful substances, and the process cannot be precisely controlled; the degree of reduction produced by physical methods varies, and long-term physical fields will have a certain impact on food texture.

[0007] Therefore, conducting research on green, controllable, and targeted allergenic epitope desensitization technology for shrimp will be of great theoretical significance and practical application value. Summary of the invention

[0008] The purpose of the present invention is to provide a method for reducing the sensitization of tropomyosin by photodynamic therapy mediated by curcumin, which comprises mixing a curcumin photosensitizer with a sensitizing factor, incubating in a dark place, and then treating with light. The method for reducing the sensitization of aquatic products of the present invention is low in cost, simple in operation, and highly safe, which opens up a new channel for reducing the sensitization of shrimps, and also provides a positive strategy for the transformation of allergen prevention and control in the food industry from "passive response" to "active protection".

[0009] Another object of the present invention is to provide an application of a method for reducing the allergenicity of tropomyosin by photodynamic therapy mediated by curcumin, which is used to reduce the allergenicity of aquatic products, especially to reduce the allergenicity of shrimps.

[0010] The specific technical solutions of the present invention are as follows:

[0011] The present invention provides a method for reducing tropomyosin sensitization by curcumin-mediated photodynamic therapy, comprising the following steps:

[0012] 1) mixing curcumin and sensitizing factors, and incubating the resulting mixture with shaking in a dark environment;

[0013] 2) Treating the incubated material in step 1) with light.

[0014] In step 1), the concentration of curcumin in the mixture is 10 to 100 μM, preferably, the concentration of curcumin in the mixture is 15 to 80 μM, and more preferably, the concentration of curcumin in the mixture is 25 to 60 μM.

[0015] In step 1), the concentration of the sensitizing factor in the mixture is 1-20 g / L.

[0016] In step 1), the sensitizing factor is tropomyosin, preferably shrimp tropomyosin;

[0017] The present invention has found through experiments that with the increase of curcumin concentration, the sensitization of tropomyosin, the main allergenic factor of shrimp, is significantly reduced. When the concentration of curcumin in the treatment system is increased to 30 μM, the best effect of reducing shrimp allergenicity is achieved. Therefore, this study found that the optimal curcumin concentration for curcumin-mediated photodynamic reduction of shrimp allergenicity is 30 μM. In addition, the protection scope of the photosensitizer curcumin of the present invention is not limited to this. In the actual operation process of this application field, the amount of curcumin is appropriately added according to the specific operation. Here, only a specific feasible solution is provided.

[0018] In step 2), the shaking incubation time is 10-30 minutes, preferably, the shaking incubation time is 20 minutes. At the same time, the incubation temperature of the present invention has little effect on the results, and there is no need to specifically limit the temperature. For example, the method of the present invention can work normally at 0-5°C, and the present invention is carried out and completed at room temperature (25°C ± 4°C).

[0019] In step 2), the oscillator speed is 10-30 r / min. Preferably, the oscillator speed is 15-25 r / min. In addition, the protection scope of the oscillator speed of the present invention is not limited thereto. In the actual operation process of this application field, the speed of the oscillator during the incubation process is adjusted according to the specific operation.

[0020] Step 3) The illumination treatment time is 10-50min. Preferably, the illumination treatment time is 15-40min. The present invention has found through experiments that with the increase of the illumination time of the LED, the sensitization of tropomyosin, the main allergenic factor of shrimp, is significantly reduced. When the illumination time of the LED is 30min, the effect of reducing the sensitization of shrimp is achieved. Therefore, the present invention finds that the illumination time of the LED is 10-50min. In addition, the protection scope of the illumination time of the present invention is not limited to this. In the actual operation process of this application field, the illumination time of the LED is appropriately changed according to the specific operation. Only a specific feasible solution is provided here.

[0021] In the light treatment in step 3), the light power density of the light source is 5 to 300 mW / cm 2 Preferably, the light power density of the light source is 10 to 250 mW / cm 2 More preferably, the present invention uses 150mW / cm 2 The light power density of the LED has the advantage of low energy consumption. The protection scope of the light power density of the present invention is not limited to this. In the actual operation process of this application field, the light power density of the LED is appropriately changed according to the specific operation. Here, only a specific feasible solution is provided.

[0022] In step 3), during the light treatment, the distance between the light source and the sample is 5-20 cm.

[0023] In step 3), the light treatment is performed using an LED light source. More importantly, the excitation light source used in the present invention is not limited to a blue LED light source, and any light source that can excite the photosensitizer to produce ROS, such as a red LED, can also be used to implement the present invention.

[0024] The invention provides an application of a method for reducing the allergenicity of tropomyosin by photodynamic therapy mediated by curcumin, which is used for treating aquatic products to reduce the allergenicity.

[0025] The specific application method is:

[0026] After removing the heads and shells of aquatic products, add water to make minced meat. Mix the minced meat with curcumin, stir in a dark place, and then pat into thin slices and expose to light.

[0027] The minced meat is mixed with curcumin, wherein the concentration of curcumin is 10-100 μM / L, preferably 30 μM / L; the moisture content of the minced meat is 80%;

[0028] The stirring time is 10-30min;

[0029] The thickness of the sheet is 1-3 mm;

[0030] The light treatment time is 10-50 minutes.

[0031] The light power density of the light source is 10-250 mW / cm 2 More preferably, the present invention uses 150mW / cm 2 Light power density; for illumination treatment, the distance between the light source and the sample is 5-20cm.

[0032] The aquatic products include but are not limited to whiteleg shrimp, Chinese shrimp and Procambarus clarkii (crayfish).

[0033] Photodynamic therapy refers to the process in which photosensitizers are stimulated by light of a specific wavelength and then produce reactive oxygen species (ROS) through type I and type II reactions, such as singlet oxygen ( 1 O2), superoxide anion (·O2 - ), hydroxyl radical (·OH) and hydrogen peroxide (H2O2), etc. 1 The electrophilic property of O2 has the ability to react with organic compounds containing heteroatoms (N, S, etc.), so aromatic amino acids (tryptophan and lysine) and sulfur-containing amino acids (cysteine, etc.) on proteins are 1 O2 is an important oxidation target. It is worth noting that aromatic amino acids are the key amino acids that cause shrimp tropomyosin allergenicity; at the same time 1 O2 also has the ability to cut peptide bonds and destroy the primary structure of allergenic proteins. In addition, the ·OH and ·O2 produced by PDI through type I reaction - Free radicals such as PDI also have the ability to modify and oxidize amino acid residues, such as methionine and cysteine ​​sulfhydryl groups are easily oxidized by OH to form disulfide cross-links. Arginine and glutamic acid (key amino acids that cause allergenicity in shrimp tropomyosin) are easily oxidized to glutamine semialdehyde, and some binding sites in the protein backbone release oxidation products when attacked by free radicals, such as aspartic acid residues, which can cause peptide chain breakage after binding with ROS. These research results show that ROS generated by curcumin-mediated PDI can effectively modify amino acid residues on proteins, especially key amino acids that cause shrimp tropomyosin allergenicity, such as arginine, glutamic acid and aromatic amino acids. Therefore, the method of establishing a photodynamic technology to reduce shrimp allergenicity in the present invention is of great practical significance to the development of the food industry.

[0034] The present invention utilizes curcumin as a photosensitizer and applies it to photodynamic reduction of aquatic product allergenicity, especially shrimp allergenicity. The chemical structure of curcumin contains multiple phenolic hydroxyl groups, which have the ability to complex with abundant polar amino acids on shrimp allergenic tropomyosin through intermolecular hydrogen bonds, and can achieve targeted enrichment of antigenic epitopes of curcumin on the main allergenic factor of shrimp (tropomyosin), thereby achieving the effect of strongly reducing shrimp allergenicity. Through computer simulation molecular docking with tropomyosin of white shrimp, it was found that curcumin can produce a strong binding potential with 5 regions of tropomyosin (L1:88-101; L2:129-143; L3:166-173; L4:183-197; L5:235-243) ( Figure 1), these five regions just overlap with the predicted epitopes of tropomyosin in white shrimp to a large extent. Therefore, during the incubation process, curcumin has the ability to target and enrich the epitopes of tropomyosin. Under the induction of LED light, a large amount of ROS can be instantly generated at the epitopes of tropomyosin, achieving the effect of targeted attack on the sensitizing sites of shrimp tropomyosin and achieving the purpose of reducing shrimp sensitization.

[0035] The beneficial effects of the present invention are as follows:

[0036] (1) The raw material curcumin used in the method of using curcumin-mediated photodynamic therapy to reduce the allergenicity of aquatic products in the present invention is a polyphenolic substance extracted from the genus Curcuma, which is safe and non-toxic, has no side effects on the human body, and is a food additive approved for use by the state.

[0037] (2) The photodynamic technology used in the curcumin-mediated photodynamic method for reducing the allergenicity of aquatic products adopted by the present invention is widely used in research in the fields of food preservation and sterilization. It is a non-thermal processing technology and has little effect on the nutritional components and tissue structure of food.

[0038] (3) The curcumin chemical structure used in the curcumin-mediated photodynamic method for reducing the allergenicity of aquatic products used in the present invention contains multiple phenolic hydroxyl groups, which have the ability to recombine with the abundant polar amino acids on the shrimp allergenic tropomyosin through intermolecular hydrogen bonds. During the incubation process, curcumin has the ability to target and enrich the antigenic epitopes of the tropomyosin of shrimp. Under the induction of LED light, curcumin can instantly generate a large amount of ROS at the shrimp antigenic epitopes, target and attack the antigenic epitopes of tropomyosin, and effectively change the allergenicity of the antigenic epitopes.

[0039] (4) The curcumin-mediated photodynamic therapy method used in the present invention to reduce the allergenicity of aquatic products does not produce metabolites during the treatment process. Compared with chemical methods (such as succinylation of proteins to reduce the allergenicity of aquatic products, especially shrimp allergenicity), it can have a better protective effect on the environment.

[0040] (5) Compared with traditional methods such as heat processing and enzymatic hydrolysis to reduce the allergenicity of aquatic products, especially shrimp, the curcumin-mediated photodynamic method of the present invention causes less damage to shrimp and is more conducive to the subsequent processing of reconstituted food.

[0041] Compared with the prior art, the operation of the present invention is extremely simple, does not require high temperature or other treatment methods, has good applicability, and the curcumin selected in the present invention is a food-grade photosensitizer. The technology can be widely promoted and applied in the food industry, reducing the allergenicity of allergenic factors in aquatic products, perfectly meeting the needs of allergic people for food safety, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the simulation diagram of the docking of curcumin and shrimp tropomyosin protein molecules;

[0043] Figure 2 The effect of different concentrations of curcumin-mediated photodynamic therapy on the binding ability of crayfish tropomyosin to IgE;

[0044] Figure 3 The immunoblot images of crayfish tropomyosin mediated by photodynamic therapy with different concentrations of curcumin are shown in Figure 1. 1: control; 2: 5 μM; 3: 10 μM; 4: 20 μM; 5: 30 μM;

[0045] Figure 4 The effect of different concentrations of curcumin-mediated photodynamic therapy on the binding ability of Antarctic krill tropomyosin to IgE;

[0046] Figure 5 The immunoblot images of Antarctic krill tropomyosin mediated by photodynamic therapy with curcumin at different concentrations, where 1: control; 2: 5 μM; 3: 10 μM; 4: 20 μM; 5: 30 μM;

[0047] Figure 6 The effect of curcumin-mediated photodynamic therapy on tropomyosin on cell morphology, where A: photodynamic therapy group, B: control group;

[0048] Figure 7 Effect of curcumin-mediated photodynamic treatment of shrimp paste on the hydrophobicity of protein. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0051] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0052] In the present invention, in v / m, the unit of v is ml, and the unit of m is g; in v / v, the unit of v is ml; in w / w, the unit of m is g.

[0053] Example 1

[0054] A method for reducing the allergenicity of tropomyosin in aquatic products by using curcumin-mediated photodynamic therapy, specifically a method for reducing the allergenicity of tropomyosin in lobster, comprising the following steps:

[0055] 1) -10°C precooled acetone and crayfish meat were mixed and minced at a ratio of 3:1 (v / m), stirred for 8 hours and centrifuged for 10 minutes, repeated three times, and the obtained mixture was mixed with PBS buffer at a ratio of 1:1 (v / v) and centrifuged for 10 minutes to obtain a crude protein solution. After the crude protein solution was heated in a 60°C water bath for 6 minutes, an equal volume of 35% ammonium sulfate aqueous solution was added and centrifuged for 15 minutes. The obtained precipitate was tropomyosin, and the extracted tropomyosin was redissolved in 0.02M Tris-HCl at a ratio of 10:1 (v / m), and the above tropomyosin solution was purified using an anion column to obtain crayfish tropomyosin.

[0056] 2) Curcumin-mediated photodynamic treatment of tropomyosin: Curcumin and crayfish tropomyosin were incubated in the dark for 30 min, where the concentration of crayfish tropomyosin was 10 g / L, and the concentrations of curcumin were 5, 10, 20, 30, and 40 μM, respectively. During the incubation, the speed of the oscillator was 25 r / min, and then a blue LED light with a wavelength of 455-460 nm was used for irradiation for 30 min, where the light intensity was 100 mW / cm 2 The distance between the light source and the sample was 15 cm. The sample without any treatment (no curcumin added and no light) was used as the blank control group.

[0057] 3) Sensitization analysis: ELISA was used to measure the changes in the binding ability of crayfish tropomyosin and immunoglobulin IgE mediated by curcumin at different concentrations of photodynamic therapy. The purified / treated TM was diluted to 5μg / mL with coating solution and added to a 96-well plate. The tropomyosin solutions mediated by curcumin at different concentrations of photodynamic therapy were coated and incubated at 37°C for 2 hours, washed 3 times, and then incubated in blocking solution for 4 hours; the serum of shrimp allergic patients was used as the primary antibody and incubated at 37°C for 2 hours; HRP-IgE antibody was used as the secondary antibody and incubated for 2 hours. After adding TMB colorimetric solution and reacting in the dark for 10 minutes, 2 mol / L sulfuric acid stop solution was added and the absorbance value was measured with an enzyme reader. The results are as follows Figure 2 As shown: In the control group, the binding capacity of untreated tropomyosin to immunoglobulin IgE was 1.21 (OD 450nm ), after curcumin-mediated photodynamic treatment, the binding capacity of tropomyosin to immunoglobulin IgE was significantly reduced. When the concentration of curcumin reached 30 μM, the binding capacity of tropomyosin to immunoglobulin IgE was 0.28 (OD 450nm), the sensitization was reduced by 76.9% compared with the control group.

[0058] Further Western Blot experiments were performed, and the curcumin-mediated photodynamic treatment of tropomyosin solution was coated and incubated at 37°C for 2 hours, washed with PBS solution three times, and incubated with 200μL blocking solution for 4 hours; the serum of shrimp allergy patients (purchased from Shanghai Shenggong Biological Co., Ltd.) was used as the primary antibody and incubated at 37°C for 2 hours; HRP-IgE antibody was used as the secondary antibody for incubation for 2 hours, and the untreated tropomyosin was stained with TMB single-component colorimetric solution and scanned and analyzed, and the untreated tropomyosin was used as the control. The results are shown in Figure 2. Figure 3 As shown: the color of the sensitized protein band of tropomyosin in the control group was darker between the molecular weight of 30 and 40 ku. The color of the bands of tropomyosin sensitized by curcumin-mediated photodynamic therapy at the molecular weight of 30 to 40 ku of crayfish became lighter to varying degrees, among which the sensitized protein complex bands treated with curcumin at a concentration of 30 μM changed more significantly. The results of the Western Blot experiment further verified and supported the experimental results of the above ELISA, and also showed that curcumin-mediated photodynamic therapy can effectively reduce the sensitization of crayfish tropomyosin.

[0059] Example 2

[0060] A method for reducing the allergenicity of tropomyosin in aquatic products by using curcumin-mediated photodynamic therapy, specifically a method for reducing the allergenicity of tropomyosin in Antarctic krill. Antarctic krill is known as the last protein pool for humans. The protein of Antarctic krill is rich in all essential amino acids for the human body, and its biological value is significantly higher than that of other animal and plant proteins. It is a high-quality protein source for the development of future food. However, the allergenicity of krill protein has greatly hindered its application in food. This case uses curcumin-mediated photodynamic therapy to reduce the allergenicity of tropomyosin in Antarctic krill, including the following steps:

[0061] 1) Extraction of Antarctic krill tropomyosin: -10°C precooled acetone and Antarctic krill meat were mixed and minced at a ratio of 3:1 (v / m), stirred overnight and centrifuged for 8 minutes, the obtained mixture was mixed with PBS buffer at a ratio of 1:1 (v / v) and centrifuged for 10 minutes to obtain a crude protein solution. After heating the crude extract for 10 minutes, an equal volume of 35% ammonium sulfate solution was added and centrifuged for 15 minutes to obtain Antarctic krill tropomyosin, and the extracted tropomyosin was redissolved in 0.02M Tris-HCl at a ratio of 10:1 (v / m), and the Antarctic krill tropomyosin solution was purified using an anion column.

[0062] 2) Curcumin-mediated photodynamic treatment of Antarctic krill tropomyosin: Curcumin of different concentrations was incubated with Antarctic krill tropomyosin for 25 min in the dark. The concentration of Antarctic krill tropomyosin was 15 g / L, and the concentrations of curcumin were 5, 10, 20, 30, and 40 μM, respectively. During the incubation, the speed of the oscillator was 20 r / min, and then a blue LED light with a wavelength of 455-460 nm was used for irradiation for 25 min, and the light intensity was 150 mW / cm 2 The samples without any treatment (no curcumin added and no light irradiation) served as the blank control group.

[0063] 3) Analysis of the sensitization of Antarctic krill tropomyosin: ELISA was used to measure the changes in the binding ability of tropomyosin to immunoglobulin IgE after photodynamic treatment with curcumin at different concentrations (protein concentration 15g / L). The specific method is the same as that in Case 1. The results of the binding ability of tropomyosin to immunoglobulin IgE are as follows: Figure 4 As shown: In the control group, the binding capacity of untreated tropomyosin to immunoglobulin IgE was 1.36 (OD 450nm ), after curcumin-mediated photodynamic treatment, the binding capacity of krill tropomyosin to immunoglobulin IgE was significantly reduced. When the concentration of curcumin reached 30 μM, the binding capacity of krill tropomyosin to immunoglobulin IgE was 0.31 (OD 450nm ), the sensitization was reduced by 77.2% compared with the control group.

[0064] Western Blot results Figure 5 As shown: The color of the allergenic protein band of krill tropomyosin in the control group was darker at the molecular weight of 30-40 ku. The color of the band of Antarctic krill tropomyosin at the molecular weight of 30-40 ku treated with curcumin-mediated photodynamic therapy was lighter to varying degrees. The results of ELISA and Western Blot experiments showed that curcumin-mediated photodynamic therapy can effectively reduce the allergenicity of Antarctic krill tropomyosin.

[0065] In addition, after photodynamic treatment of Antarctic krill tropomyosin, a cell safety experiment was conducted. The cells were seeded in a 96-well plate, and curcumin-mediated photodynamic treatment of tropomyosin was added to the cultured cells. The final concentration of krill tropomyosin was 5 mg / mL, and the concentration of curcumin was 25 μM. The untreated protein was used as the blank group. After 24 hours of culture, the culture medium was discarded, and 100 μL of CCK-8 (10%) culture medium was added. After incubation for 30 minutes, the absorbance at 450 nm was measured, and the cell survival rate was calculated. The cell morphology was observed under a microscope. The study found that there was no significant difference in the cell morphology and cell survival rate of HAEC cells between curcumin-mediated photodynamic treatment of tropomyosin and untreated group proteins (see Figure 6 ), indicating that krill protein after curcumin-mediated photodynamic treatment is non-toxic to cells and has high safety.

[0066] Example 3

[0067] A method for reducing the allergenicity of tropomyosin in aquatic products by using curcumin-mediated photodynamics, specifically a method for reducing the allergenicity of shrimp paste of Penaeus vannamei by using curcumin-mediated photodynamics. Shrimp paste and shrimp paste products are important processed products of Penaeus vannamei. They have the advantages of delicious taste and high nutritional value, and are therefore favored by consumers. However, they still have the problem of allergenicity. In this example, curcumin-mediated photodynamics is used to reduce the allergenicity of shrimp paste of Penaeus vannamei. The specific method is:

[0068] 1) Preparation of shrimp paste: fresh white shrimps were shelled and decapitated to obtain shrimp meat. The shrimp meat was stirred in a meat grinder for 1 minute, then pounded for 10 minutes, the moisture content was adjusted to 80%, 3% (w / w) salt and 1% TG enzyme were added, and the shrimp paste was obtained by further pounding for 5 minutes.

[0069] 2) Curcumin-mediated photodynamic treatment of shrimp paste: The shrimp paste was mixed with curcumin to a final concentration of 30 μM, then stirred in the dark for 20 min, and then cut into 1-3 mm slices and placed under LED blue light with a light density of 300 mW / cm 2 The sample was irradiated for 50 min, and the distance between the light source and the sample was 15 cm.

[0070] 3) Analysis of shrimp paste allergenicity: ELISA and Western Blot experiments were performed. The specific experimental methods were the same as those in Example 1. The allergenicity of shrimp paste treated with curcumin-mediated photodynamic therapy was analyzed. The results showed that the allergenicity of shrimp paste treated with curcumin-mediated photodynamic therapy was reduced by 43.2% compared with the control group.

[0071] The present invention further analyzes the secondary structure and hydrophobicity changes of tropomyosin in shrimp paste. The tropomyosin in the shrimp paste is extracted (the extraction method is according to Example 1), and the freeze-dried tropomyosin is mixed with potassium bromide at a ratio of 1:100 (m / m, all units are g) and then ground and tableted. The tableted sample is then placed in an infrared spectrometer to collect infrared data of the sample. Finally, PeakFit software 4.12 is used to analyze the amide I band (1600-1700cm -1 ) and calculated the content of different secondary structures. The results are shown in Table 1: Compared with the untreated, the α-helix and β-fold of the protein in the shrimp paste after photodynamic treatment were significantly reduced, and the random coil was significantly increased. The change in the secondary structure content was mainly due to the destruction of the protein spatial structure caused by the singlet oxygen induced by photodynamic treatment, which in turn destroyed its allergenicity.

[0072] Table 1 Effect of curcumin treatment on the secondary structure of shrimp paste protein

[0073]

[0074] Surface hydrophobicity is one of the important indicators that reflect the spatial structure of proteins, and is of great significance to protein structure and function. The present invention uses 8-anilino-1-naphthalenesulfonic acid (ANS) probe to determine the surface hydrophobicity of proteins in shrimp paste. ANS (8.0mM, 0.01M PBS) was added to the gradient dilutions (25, 50, 100, 200, 400, 800 μg / mL) of tropomyosin in shrimp paste obtained after curcumin-mediated photodynamic treatment according to the method of Example 3. The fluorescence intensity of the protein aqueous solution at 36nm (excitation wavelength) and 475nm (emission wavelength) was analyzed using a spectrophotometer F7100. The slope obtained by plotting the protein concentration as the horizontal coordinate and the absorbance value as the vertical coordinate is the hydrophobicity of the treated group protein. According to the method of Example 3, the protein was treated with light for 10min, 20min, 30min and 40min, and the surface hydrophobicity results of the protein are as shown in the figure. Figure 7 As shown in the figure: the hydrophobicity of the protein in the control group was 234.6, the protein in the photodynamic treatment group increased significantly, and the hydrophobicity of the shrimp paste protein in the 30-min illumination group increased significantly to 682.5. The above results show that curcumin-mediated photodynamic treatment can effectively change the spatial structure of shrimp paste protein, and thus effectively change the binding ability of allergenic protein with immunoglobulin IgE.

[0075] Therefore, this technology is expected to develop into a new and effective means to reduce the allergenicity of shrimp and its products in the future, providing a new technical approach for the processing of low-allergenic foods.

[0076] The description of the above embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A method for reducing tropomyosin sensitization by curcumin-mediated photodynamic therapy, characterized in that: The method comprises the following steps: 1) mixing curcumin and sensitizing factors, and incubating the resulting mixture with shaking in a dark environment; 2) Treating the incubated material in step 1) with light.

2. The method according to claim 1, characterized in that In step 1), the concentration of curcumin in the mixture is 10-100 μM.

3. The method according to claim 1 or 2, characterized in that: In step 1), the concentration of the sensitizing factor in the mixture is 1-20 g / L.

4. The method according to claim 1, characterized in that: In step 2), the shaking incubation time is 10-30 min, and the shaking speed is 10-30 r / min.

5. The method according to claim 1, characterized in that Step 3) The illumination treatment time is 10-50 min; the light power density of the light source is 5-300 mW / cm 2 .

6. An application of the method according to any one of claims 1 to 5, characterized in that: Used to treat aquatic products to reduce allergenicity.

7. The use according to claim 6, characterized in that: The application method is: After removing the heads and shells of aquatic products, add water to make minced meat. Mix the minced meat with curcumin, stir in a dark place, and then pat into thin slices and expose to light.

8. The use according to claim 7, characterized in that: The minced meat is mixed with curcumin, wherein the concentration of curcumin is 10-100 μM; and the water content of the minced meat is 80%.

9. The use according to claim 7 or 8, characterized in that: The stirring time is 10-30 min.

10. The use according to claim 7 or 8, characterized in that: The light treatment time is 10-50 minutes, and the light power density of the light source is 10-300 mW / cm 2 .

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