CN115005358A

CN115005358A - Method for reducing sensitization of tropomyosin

Info

Publication number: CN115005358A
Application number: CN202210486465.7A
Authority: CN
Inventors: 刘书成; 魏帅; 王新; 孙钦秀; 夏秋瑜; 韩宗元; 王泽富; 邱惠; 刘阳; 吉宏武
Original assignee: Guangdong Ocean University
Current assignee: Guangdong Ocean University
Priority date: 2022-05-06
Filing date: 2022-05-06
Publication date: 2022-09-06

Abstract

The invention provides a method for reducing the sensitization of tropomyosin. The method comprises the following steps of carrying out high-density carbon dioxide treatment on tropomyosin, wherein the conditions of the high-density carbon dioxide treatment are as follows: the temperature is 40-70 ℃, the time is 15-60 min, and the pressure is 5-40 MPa. The invention adopts the high-density carbon dioxide technology with specific conditions to treat the TM protein, not only obviously changes the spatial configuration of the TM protein after DPCD treatment, but also realizes the effective reduction of the sensitization of the TM protein.

Description

Method for reducing sensitization of tropomyosin

Technical Field

The invention belongs to the technical field of food safety control. And more particularly to a method of reducing tropomyosin sensitization.

Background

Food allergy refers to an abnormal reaction caused by immunoglobulin e (ige) after the body is exposed to a certain food, which repeatedly occurs, and is manifested by some adverse reactions in skin, respiratory tract and gastrointestinal tract, and in severe cases, anaphylactic shock and even death may be caused. At present, no good cure method for food allergy exists, but effective prevention can be realized by avoiding the intake of allergic food, such as early identification and management of allergic reaction, accurate identification, and reminding and warning of allergic people; sensitization of foods that may cause allergies may also be eliminated or reduced by processing techniques. Tropomyosin (TM) is a main allergen of various crustacean aquatic products, such as shrimps, crabs, clams and the like, has a molecular weight of 32-39kDa, is in a rod-shaped structure formed by mutually winding two same alpha-helical chains, has salt solubility and thermal stability, and is difficult to reduce allergenicity.

CO ₂ The acid gas is an acid gas, widely exists in atmospheric environment, and can form high-pressure, acid, anaerobic and other environments under the action of external force due to the characteristics of no toxicity, low price, no residue and the like, so the acid gas is commonly used for sterilizing or inactivating enzymes of food, and can prolong the shelf life of the food. High density CO ₂ The technique (DPCD) is CO Phase Carbon Dioxide ₂ DPCD, a food processing technology using a high-pressure medium, can maintain the qualities of color, nutrition, and the like of food to a greater extent than conventional thermal processing, and has received much attention from researchers in recent years. Zhou scholar et al disclose high density CO ₂ The technology can change the structure of protein (Zhou Xue Fu, Zheng Yuan Rong, Liu Zheng Min, Wang Danfeng, Deng Yun, high-density carbon dioxide has influence on the research progress of protein structure and its processing characteristics in food [ J]Science and technology of dairy industry2020,43(01):39-44.DOI 10.15922/j. cnki. jdst.2020.01.008.), but changing the structure of a protein does not necessarily reduce the sensitization of the protein, and no use of high density CO has been made so far ₂ The related reports of techniques to abrogate protein sensitization.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for utilizing high-density CO ₂ The method for reducing protein allergenicity is used for reducing the probability of food allergy and effectively preventing food allergy.

It is a first object of the present invention to provide a method of reducing the sensitization of tropomyosin.

The second purpose of the invention is to provide a method for reducing the sensitization of the crustacean aquatic products.

The above purpose of the invention is realized by the following technical scheme:

the invention provides a method for reducing sensitization of tropomyosin, which specifically comprises the following steps of carrying out high-density carbon dioxide treatment on the tropomyosin, wherein the conditions of the high-density carbon dioxide treatment are as follows: the temperature is 40-70 ℃, the time is 15-60 min, and the pressure is 5-40 MPa.

The invention adopts the high-density carbon dioxide technology (DPCD), and specially controls the conditions (temperature, pressure and time) of the high-density carbon dioxide technology according to the characteristics of the TM protein, so that the ultraviolet absorption peak of the TM protein treated by the DPCD generates blue shift, the fluorescence spectrum generates red shift, the alpha-helix content is obviously reduced, the random coiling and the beta-folding are obviously improved, the surface hydrophobicity is obviously changed, and the free amino and sulfydryl content are reduced, thereby showing that the space configuration of the TM protein treated by the DPCD is obviously changed and even denatured; also makes the TM protein in CO ₂ The sensitization is obviously reduced under the effect and the pH effect, and the effective reduction of the sensitization of the TM protein is realized.

Preferably, the temperature is 50-60 ℃. Most preferably 55 deg.c.

Preferably, the time is 15-30 min. Most preferably 15 min.

Preferably, the pressure is 30-40 MPa. Most preferably 30 MPa.

Preferably, the high density carbon dioxide treatment is preceded by a pre-treatment of tropomyosin.

Further preferably, the pretreatment is: and diluting tropomyosin to 0.4-0.6 mg/mL by using ultrapure water, and then placing the diluted tropomyosin in a treatment kettle. Most preferably 0.5 mg/mL.

The invention also provides a method for reducing the allergenicity of the crustacean aquatic products, and particularly relates to a method for treating tropomyosin separated from the crustacean aquatic products.

Preferably, the crustacean aquatic products comprise one or more of shrimp products, crab products, and clam products.

The invention has the following beneficial effects:

Drawings

FIG. 1 shows the result of SDS-PAGE gel electrophoresis. FIG. 1A shows the SDS-PAGE gel electrophoresis results of the TM protein under different DPCD pressures (wherein 1 and 11 are Marker groups, 2 is an untreated group, and 3-10 are DPCD treated groups under pressures of 5, 10, 15, 20, 25, 30, 35, and 40MPa, respectively); FIG. 1B shows the SDS-PAGE gel electrophoresis results of the TM protein at different DPCD times (wherein 1 is Marker group, 2 is untreated group, and 3-8 are DPCD treatment groups at 15, 30, 45, 60, 75, and 90min, respectively); FIG. 1C shows the SDS-PAGE gel electrophoresis results of the TM proteins at different temperatures of DPCD (wherein 1 is Marker group, 2 is untreated group, and 3-9 are DPCD treated groups at temperatures of 40, 45, 50, 55, 60, 65, and 70 ℃ respectively).

FIG. 2 is a photograph of a TM protein solution. FIG. 2a is a photograph of the TM protein solution under different DPCD pressures (wherein 1 is an untreated group, and 2-9 are DPCD treated groups under pressures of 5, 10, 15, 20, 25, 30, 35, and 40MPa, respectively); FIG. 2b is a photograph of the TM protein solution at different times in DPCD (wherein 1 is the untreated group, and 2-7 are the DPCD treated groups at

times

15, 30, 45, 60, 75, and 90min, respectively); FIG. 2c is a photograph of the TM protein solution at different temperatures of DPCD (1 is untreated group, 2-8 are DPCD treated groups at 40, 45, 50, 55, 60, 65, 70 ℃ C.).

FIG. 3 shows the results of the Western blot assay for TM proteins. FIG. 3A shows the results of IgG binding activity assay under different DPCD pressures (wherein 1 is untreated group, and 2-9 are DPCD treated groups under pressures of 5, 10, 15, 20, 25, 30, 35, and 40MPa, respectively); FIG. 3a shows the results of the IgE binding activity test under different DPCD pressures (wherein 1 is an untreated group, and 2-9 are DPCD treated groups under pressures of 5, 10, 15, 20, 25, 30, 35, and 40MPa, respectively); FIG. 3B shows the results of the IgG binding activity assay at different DPCD times (wherein 1 is the untreated group, and 2-7 are the DPCD treated groups at

times

15, 30, 45, 60, 75, and 90min, respectively); FIG. 3b shows the results of the IgE binding activity test of DPCD at different times (wherein 1 is the untreated group, and 2-7 are the DPCD treated groups at

times

15, 30, 45, 60, 75 and 90min, respectively); FIG. 3C shows the results of IgG binding activity assay at different temperatures for DPCD (1 is untreated group, and 2-8 are DPCD treated groups at 40, 45, 50, 55, 60, 65, and 70 ℃ respectively); FIG. 3c shows the results of the IgE binding activity test at different temperatures for DPCD (1 is untreated group, and 2-8 are DPCD treated groups at 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, and 70 deg.C).

FIG. 4 shows the result of indirect ELISA using TM protein. FIG. 4A shows the results of IgG binding activity assay at various pressures in DPCD; FIG. 4a shows the results of the IgE binding activity assay at various pressures in DPCD; FIG. 4B shows the results of the IgG binding activity assay at various times in DPCD; FIG. 4b shows the results of the IgE binding activity assay at different times for DPCD; FIG. 4C shows the results of the DPCD temperature-dependent IgG binding activity assay; FIG. 4c shows the results of the IgE binding activity assay at various temperatures for DPCD.

FIG. 5 shows the result of SDS-PAGE gel electrophoresis after digestion with gastric juice or intestinal juice. FIG. 5A shows the results of SDS-PAGE gel electrophoresis of the TM protein samples after gastric digestion (wherein 1 is marker group, 2-10 are untreated group after digestion for 0, 1, 2, 5, 10, 15, 20, 30 and 60min, respectively, and 11 is control group); FIG. 5B shows the results of SDS-PAGE gel electrophoresis of the treated TM protein samples after gastric juice digestion (wherein 1 is marker group, 2-10 are treated groups digested for 0, 1, 2, 5, 10, 15, 20, 30 and 60min, respectively, and 11 is control group); FIG. 5C shows the results of SDS-PAGE gel electrophoresis of the TM protein samples after intestinal digestion (wherein 1 is marker group, 2-11 are untreated groups digested for 0, 1, 5, 7, 15, 30, 60, 120, 150 and 180min, respectively, and 12 is control group); FIG. 5D shows the results of SDS-PAGE gel electrophoresis of treated TM protein samples after intestinal digestion (wherein 1 is marker group, 2-11 are treated groups after digestion for 0, 1, 5, 7, 15, 30, 60, 120, 150 and 180min, respectively, and 12 is control group).

FIG. 6 shows the result of Western blot analysis of a TM protein sample after digestion with gastric juice. FIG. 6a shows the results of IgG binding activity test after digestion of the TM protein sample in the untreated group with gastric juice (wherein 1-9 are untreated groups digested for 0, 1, 2, 5, 10, 15, 20, 30 and 60min, respectively, and 10 is a control group); FIG. 6b is the result of the IgE binding activity test of the TM protein sample after gastric juice digestion in the untreated group (wherein 1-9 are the untreated groups at digestion time of 0, 1, 2, 5, 10, 15, 20, 30 and 60min, respectively, and 10 is the control group); FIG. 6c shows the results of IgG binding activity test after gastric juice digestion of TM protein samples in treated groups (wherein 1-9 are treated groups digested for 0, 1, 2, 5, 10, 15, 20, 30 and 60min, respectively, and 10 is a control group); FIG. 6d shows the results of the IgE binding activity test of the TM protein samples after gastric juice digestion in the treated group (wherein 1-9 are treated groups at digestion time of 0, 1, 2, 5, 10, 15, 20, 30 and 60min, respectively, and 10 is a control group).

FIG. 7 shows the result of Western blot analysis of the TM protein sample after intestinal juice digestion. FIG. 7a shows the results of the IgG binding activity assay after intestinal fluid digestion of the TM protein sample of the untreated group (wherein 1-10 are untreated groups digested for 0, 1, 5, 7, 15, 30, 60, 120, 150 and 180min, respectively, and 11 is a control group); FIG. 7b shows the results of the IgE binding activity test of the TM protein sample in the untreated group after intestinal fluid digestion (wherein, 1-10 are the untreated groups at 0, 1, 5, 7, 15, 30, 60, 120, 150 and 180min of digestion, respectively, and 11 is the control group); FIG. 7c shows the results of IgG binding activity assay after intestinal digestion of treated group TM protein samples (wherein 1-10 are treated groups digested for 0, 1, 5, 7, 15, 30, 60, 120, 150 and 180min, respectively, and 11 is a control group); FIG. 7d shows the results of the IgE binding activity test of the treated TM protein samples after intestinal juice digestion (wherein 1-10 are treated groups at 0, 1, 5, 7, 15, 30, 60, 120, 150 and 180min of digestion, respectively, and 11 is a control group).

FIG. 8 shows the results of indirect ELISA. FIG. 8a shows the result of an IgG binding activity assay after gastric fluid digestion of a TM protein sample; FIG. 8b shows the result of IgE binding activity test after digestion of TM protein sample by gastric juice; FIG. 8c shows the results of an IgG binding activity assay after intestinal fluid digestion of a TM protein sample; FIG. 8d shows the results of the IgE binding activity assay after intestinal fluid digestion of TM protein samples.

Fig. 9 shows the results of uv spectroscopy. FIG. 9a shows the results of UV spectroscopy analysis of DPCD under different pressures; FIG. 9b shows the UV spectrum analysis results of DPCD at different times; FIG. 9c shows the results of UV spectroscopy at different temperatures of DPCD.

FIG. 10 shows the results of circular dichroism analysis. FIG. 10a shows the results of circular dichroism analysis of DPCD under different pressures; FIG. 10b shows the results of circular dichroism analysis of DPCD at different times; FIG. 10c shows the results of circular dichroism analysis of DPCD at different temperatures.

FIG. 11 shows the secondary structure content calculated by the CDNN software. FIG. 11a shows the content of each secondary structure at different times in DPCD; FIG. 11b shows the content of each secondary structure at different temperatures of DPCD; FIG. 11c shows the secondary structure content of DPCD at different pressures.

FIG. 12 shows the results of fluorescence spectrum analysis. FIG. 12a shows the results of fluorescence spectroscopy analysis of DPCD at different pressures; FIG. 12b shows the results of fluorescence spectrum analysis of DPCD at different times; FIG. 12c shows the results of fluorescence spectrum analysis of DPCD at different temperatures.

Fig. 13 shows the results of surface hydrophobicity analysis. FIG. 13a shows the results of surface hydrophobicity analysis under different pressures of DPCD; FIG. 13b shows the results of surface hydrophobicity analysis at different times in DPCD; FIG. 13c shows the results of surface hydrophobicity analysis at different temperatures of DPCD.

FIG. 14 shows the results of analysis of free amino groups. FIG. 14a shows the analysis results of free amino groups at different pressures in DPCD; FIG. 14b shows the analysis results of free amino groups at different times in DPCD; FIG. 14c shows the analysis results of free amino groups at different temperatures of DPCD.

FIG. 15 shows the results of total thiol analysis. FIG. 15a shows the results of total thiol analysis of DPCD under different pressures; FIG. 15b shows the total thiol analysis of DPCD at different times; FIG. 15c shows the total thiol analysis of DPCD at different temperatures.

Detailed Description

The invention is further described with reference to the drawings and specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 method for reducing Promyosin sensitization

First, experimental material

(1) Test materials

Litopenaeus vannamei (Litopenaeus vannamei) with the specification of 30 tails/Kg is purchased in the Dongfeng aquatic product market in Zhanjiang.

(2) Experimental reagent

The skimmed milk powder is purchased from Shanghai workers; 10 XPBST, 10 XPBS (pH 7.4, 0.1M), Coomassie brilliant blue dye from Kulaibo technologies, Inc. of Beijing; DEAE FF is available from GE, USA; rabbit anti-shrimp IgG was purchased from INDOR, USA; horseradish peroxidase (HRP) -labeled goat anti-rabbit IgG purchased from Cell Signaling Technology; goat anti-human IgE labeled with horseradish peroxidase was purchased from KPL corporation, USA; prestained protein marker was purchased from smobi corporation; the 12% prefabricated glue, 0.22 μm PVDF membrane, transfer filter paper, membrane transfer liquid, ECL hypersensitive luminescence kit, TMB color solution, stop solution and 5 Xprotein sample buffer solution are purchased from Shanghai Binyan biotechnology limited company; artificial gastric juice and artificial intestinal juice (sterile and enzyme-free) are purchased from Shanghai-sourced leaf Biotechnology GmbH; pepsin and trypsin were purchased from beijing solilebao scientific ltd.

(3) Serum for allergic patients

The shrimp-specific allergic serum is purchased from Chongqing NuoYinfei biotechnology limited, the skin prick test is positive, the IgE level is higher than 0.35ku/L, and the clinical and serum characteristic detailed information of shrimp allergic patients is shown in Table 1. All the sera were mixed well and stored at-80 ℃ for future use.

TABLE 1 clinical and serum characteristics of shrimp allergic patients

(4) Separation and purification of TM protein

Mincing shrimp meat of Litopenaeus vannamei, adding 4 times volume of pre-cooled acetone, stirring for 4h, and centrifuging at 8500r/min for 15 min; taking the precipitate, repeating the steps until the shrimp meat is colorless, spreading the precipitate on filter paper for natural air drying, adding PBS (0.1M pH7) with the volume of 6 times of the shrimp meat powder after air drying, stirring for 6h at 4 ℃, centrifuging for 15min at 8500r/min, carrying out 6min boiling water bath on the supernatant to remove thermolabile protein, centrifuging for 10min at 8500r/min, and obtaining centrifugal supernatant and centrifugal precipitate; then adding 35% (M/v) ammonium sulfate into the centrifugal supernatant, centrifuging at 8500r/min for 15min, dissolving the centrifugal precipitate in Tris-HCl (0.02M, pH7), purifying TM protein by adopting a DEAEFF chromatographic column, performing linear elution by adopting Tris-HCl containing 1M sodium chloride, determining the eluate containing the TM protein by SDS-PAGE, dialyzing the eluate at the temperature of 4 ℃ for 72h, and finally freezing, drying and storing.

Second, high density carbon dioxide treatment

The TM protein obtained by the separation and purification is diluted to 0.5mg/mL by ultrapure water, and is loaded in 25mL sample cups (each sample cup is loaded with 5mL of the TM protein), and then the sample cups are respectively placed in a treatment kettle and are divided into a treatment group and an untreated group.

The treatment group is treated according to the following steps:

(1) DPCD treatment groups at different pressures: randomly taking 8 sample cups containing 5ml of LTM protein, and carrying out high-density carbon dioxide treatment under the following conditions: the temperature is fixed at 50 ℃, the time is fixed at 30min, the pressure is respectively set to be 5, 10, 15, 20, 25, 30, 35 and 40MPa, and the pressure is released at the rate of 5 MPa/min;

(2) DPCD treatment groups at different times: randomly taking 6 sample cups containing 5ml of the protein, and carrying out high-density carbon dioxide treatment under the conditions that: the pressure is fixed at 30MPa, the temperature is fixed at 50 ℃, the time is respectively set to be 15, 30, 45, 60, 75 and 90min, and the pressure is released at the speed of 5 MPa/min;

(3) DPCD treatment groups at different temperatures: randomly taking 7 sample cups containing 5ml of the protein, and carrying out high-density carbon dioxide treatment under the conditions that: the pressure is fixed at 30MPa, the time is fixed at 15min, the temperature is respectively set at 40, 45, 50, 55, 60, 65 and 70 ℃, and the pressure is relieved at the speed of 5 MPa/min;

untreated group: 5ml of LTM protein without any treatment.

Respectively transferring the TM proteins of the treated group and the untreated group into a centrifuge tube, standing in a refrigerator at 4 ℃ for 12h, centrifuging at 4 ℃ and 3500r/min for 15min, and taking the supernatant obtained by centrifugation at 4 ℃ for later use.

Example 2 analysis of TM protein sensitization following high Density carbon dioxide (DPCD) treatment

First, SDS-PAGE gel electrophoresis analysis

1. Experimental methods

The treated and untreated groups were analyzed by SDS-PAGE using a boiling water bath for 6min after mixing 40. mu.L of the LTM protein sample with 10. mu.L of the 5 Xprotein loading buffer. The TM protein was loaded at 10. mu.L and marker at 8. mu.L for 1h in constant voltage 120V and constant current 40 mA. After electrophoresis, the Gel was stained with Coomassie Brilliant blue stain for 2h, destained until the Gel bands were clear, and analyzed by SDS-PAGE Gel electrophoresis using a Biorad Gel Doc XR imaging system.

2. Results of the experiment

The SDS-PAGE gel electrophoresis results are shown in FIG. 1, and FIG. 1A shows the SDS-PAGE gel electrophoresis results of TM protein under different DPCD pressures (wherein, 1 and 11 are Marker groups, 2 is an untreated group, and 3-10 are DPCD treated groups under pressures of 5, 10, 15, 20, 25, 30, 35, and 40MPa, respectively); FIG. 1B shows the SDS-PAGE gel electrophoresis results of the TM protein at different DPCD times (wherein 1 is Marker group, 2 is untreated group, and 3-8 are DPCD treatment groups at

times

15, 30, 45, 60, 75, and 90min, respectively); FIG. 1C shows the SDS-PAGE gel electrophoresis results of the TM proteins at different temperatures of DPCD (wherein 1 is Marker group, 2 is untreated group, and 3-9 are DPCD treated groups at temperatures of 40, 45, 50, 55, 60, 65, and 70 ℃ respectively). It can be seen that no new bands were generated in the DPCD treated group compared with the untreated group, but the bands were lighter in gray, indicating that the DPCD action did not change the TM protein molecular weight (the native TM protein molecular weight is 36kDa), degrade or polymerize the TM protein, and the primary structure of the TM protein was not changed.

Taking pictures of TM protein of treated group and untreated group, as shown in FIG. 2, FIG. 2a is a photograph of TM protein liquid under DPCD different pressures (wherein, 1 is untreated group, 2-9 are DPCD treated groups under pressure of 5, 10, 15, 20, 25, 30, 35, 40MPa, respectively); FIG. 2b is the photograph of the TM protein solution in DPCD at different times (1 is the untreated group, 2-7 are the DPCD treated groups at the time of 15, 30, 45, 60, 75, 90min, respectively); FIG. 2c is a photograph of the TM protein solution at different temperatures of DPCD (1 is untreated group, 2-8 are DPCD treated groups at 40, 45, 50, 55, 60, 65, 70 ℃ respectively). It can be seen that the TM protein fluid of the untreated group was clear and transparent, and the TM protein fluid of the treated group precipitated to different degrees with the changes in pressure, time, and temperature of the DPCD treatment, indicating that the DPCD treatment denatured the TM protein.

Second, Western blot analysis

1. Experimental methods

The IgG/IgE binding capacity of the DPCD-treated TM protein was analyzed by Western blotting: respectively diluting TM protein samples of the treated group and the untreated group to 0.1mg/mL, separating proteins by SDS-PAGE, transferring to a pre-activated PVDF membrane, sealing for 30min, and washing the membrane for 5 times (3 min each time) by TBST. Respectively taking rabbit anti-shrimp IgG (diluted by a volume ratio of 1: 2000) and human serum IgE (diluted by a volume ratio of 1: 100) as primary antibodies, incubating for 12h at 4 ℃, washing the membrane, respectively taking goat anti-rabbit IgG (diluted by a volume ratio of 1: 10000) marked by HRP and goat anti-human IgE (diluted by a volume ratio of 1: 5000) as secondary antibodies, incubating for 2h at 25 ℃, and washing the membrane for 5 times by TBST (3 min each time). Photographic analysis was performed by a day 5200 chemiluminescence system using an enhanced ECL luminescence solution.

2. Results of the experiment

The TM protein immunoblot assay results are shown in fig. 3, and fig. 3A is the IgG binding activity assay results under DPCD different pressures (wherein 1 is untreated group, and 2-9 are DPCD treated groups under pressures of 5, 10, 15, 20, 25, 30, 35, and 40MPa, respectively); FIG. 3a shows the results of the IgE binding activity test under different DPCD pressures (wherein 1 is an untreated group, and 2-9 are DPCD treated groups under pressures of 5, 10, 15, 20, 25, 30, 35, and 40MPa, respectively); FIG. 3B shows the results of the IgG binding activity assay at different DPCD times (wherein 1 is the untreated group, and 2-7 are the DPCD treated groups at

times

As can be seen from FIGS. 3A and 3A, at 5-30 MPa, the band becomes weaker gradually with the increase of pressure, indicating that the increase of pressure can affect the activity of TM protein; under high pressure, a large amount of CO ₂ The water solubility makes the system present an acidic environment, which is considered to be one of the causes of the decrease in the sensitization of TM protein.

As can be seen from FIGS. 3B and 3B, in the IgG binding activity test results, the gray value of the band is reduced at 0-15 min, the gray value is increased at 15-45 min, the gray value is reduced at 45-90 min, wherein the gray value of the band is the minimum at 15 min; in the results of the IgE binding activity assay, the band gray values showed a tendency to decrease first and then increase, with the band gray value being the smallest at 15 min. Therefore, when the DPCD treatment time is 15min, the sensitization of the TM protein is the weakest, and the sensitization reduction effect of tropomyosin is the best. In addition, the fact that the gray value is increased at 15-45 min is considered that along with the increase of time, the epitope buried in the TM protein is exposed, and the sensitization is increased; and with further time extension (45-90 min), the spatial conformation of the protein is changed, so that the newly exposed epitope is buried or destroyed, and the sensitization of the TM protein is reduced. The gray values of the bands for the 75min and 90min treated groups in the IgE binding activity assay are close to those of the untreated group, whereas the gray values for the 60min treated group in the IgG assay are reduced due to the difference in epitope binding sites between the different antibodies.

As is clear from FIGS. 3C and 3C, the bands gradually decreased before 55 ℃ and were the lowest at 55 ℃ indicating that the sensitization of the DPCD-treated group was the weakest and the sensitization-reducing effect of tropomyosin was the best at 55 ℃.

Three, indirect ELISA

1. Experimental methods

The TM protein samples of the treated and untreated groups were diluted to 5. mu.g/mL with coating buffer and coated at 4 ℃ for 12h in a 96-well plate at 100. mu.L/well. The plates were washed 5 times with TBST, 200. mu.L of 5% skim milk was added to each well, blocked at 37 ℃ for 2h, and the plates were washed. Respectively taking 100 mu L/well of human serum IgE (diluted by a volume ratio of 1: 50) and rabbit anti-shrimp IgG (diluted by a volume ratio of 1: 5000) as primary antibodies, and incubating for 3h at 37 ℃; the plate was washed and incubated with HRP-labeled goat anti-human IgE (diluted 1: 5000 by volume) and goat anti-rabbit IgG (diluted 1: 10000 by volume) as secondary antibodies at 37 ℃ for 1.5 h. And (3) respectively adding 100 mu L of TMB color development solution after washing the plate twice, reacting for 15min in a dark place, adding 100 mu L of stop solution, uniformly mixing, and measuring the absorbance value at 450nm of an enzyme-labeling instrument.

2. Results of the experiment

The results of indirect ELISA using TM protein are shown in FIG. 4, and FIG. 4A shows the results of IgG binding activity assay under different DPCD pressures; FIG. 4a shows the results of the IgE binding activity assay at various pressures in DPCD; FIG. 4B shows the results of the IgG binding activity assay at various times in DPCD; FIG. 4b shows the results of the IgE binding activity assay at different times for DPCD; FIG. 4C shows the results of the IgG binding activity assay at different temperatures of DPCD; FIG. 4c shows the results of the IgE binding activity assay at different temperatures of DPCD.

As can be seen from FIGS. 4A and 4A, the IgG/IgE immunoreactivity was significantly reduced in the DPCD-treated group at a pressure of more than 25MPa, while the IgG and IgE immunoreactivity was the lowest at 30MPa and reduced by 13.9% and 26.5%, respectively, indicating that the sensitization in the DPCD-treated group was the weakest and the sensitization-reducing effect of tropomyosin was the best at a pressure of 30 MPa.

As can be seen from fig. 4B and 4B, the IgG binding activity tended to decrease first and then increase and then decrease while the IgE binding activity tended to decrease first and then increase, which corresponds to the results of the immunoblot assay, and the binding activities of both IgG and IgE reached the lowest at 15min, which were decreased by 18.4% and 27.1%, respectively, indicating that the DPCD treatment group had the weakest sensitization and the best sensitization reduction of tropomyosin at 15 min.

As can be seen from FIGS. 4C and 4C, the IgG and IgE activities were gradually decreased before 55 ℃ and were the lowest at 55 ℃ and were decreased by 19% and 54.2%, respectively, indicating that DPCD significantly decreased the IgG and IgE activity of TM protein, and decreased the sensitization, and that at 55 ℃, the sensitization of DPCD treated group was the weakest and the sensitization of tropomyosin was the best.

Four, in vitro simulated digestion

1. Experimental method

According to the method of United states pharmacopeia, digestive enzyme and the like are added into artificial gastric juice and artificial intestinal juice (sterile and enzyme-free) to prepare the artificial gastric juice and the artificial intestinal juice.

Simulating gastric juice digestion: preheating artificial gastric juice at 37 ℃ for 10min, and adding TM protein samples of a treated group (30MPa, 15min, 55 ℃) and an untreated group into two artificial gastric juice respectively to ensure that the total volume of each reaction solution is 2mL, and the mass ratio of pepsin to TM protein samples is 1: 50, digesting with shaking at 37 deg.C, sampling 100 μ L at 0, 1, 2, 5, 10, 15, 20, 30 and 60min, respectively, and transferring to 30 μ L of 0.2M Na ₂ CO ₃ Mixing the mixture in a centrifuge tube to stop digestion; in addition, the control group was set to be added in the artificial gastric juice firstInto 0.2M Na ₂ CO ₃ Then, the TM protein samples (treated and untreated) were added and digested at 37 ℃ for 60min with shaking.

Simulating intestinal fluid digestion: preheating the artificial intestinal juice at 37 ℃ for 10min, adding TM protein samples of a treated group (30MPa, 15min and 55 ℃) and an untreated group into two artificial intestinal juice respectively, wherein the total volume of each reaction solution is 2mL, and the mass ratio of trypsin to the TM protein sample is 1: 100, digesting by shaking at 37 ℃, sampling 100 mu L of the mixture at 0, 1, 5, 7, 15, 30, 60, 120, 150 and 180min respectively, transferring the sample into a centrifuge tube, and immediately carrying out boiling water bath for 10min to inactivate enzyme so as to stop digesting; in addition, the settings of the control group were: the enzyme was inactivated and the TM protein samples (treated and untreated) were added for digestion.

Samples digested by gastric juice and intestinal juice were analyzed by SDS-PAGE gel electrophoresis, Western blot, and indirect ELISA.

2. Results of the experiment

(1) SDS-PAGE gel electrophoretic analysis

The results are shown in fig. 5, and fig. 5A shows the results of SDS-PAGE gel electrophoresis of the TM protein sample of the untreated group after gastric juice digestion (wherein 1 is the marker group, 2-10 are the untreated groups digested for 0, 1, 2, 5, 10, 15, 20, 30 and 60min, respectively, and 11 is the control group); FIG. 5B shows the results of SDS-PAGE gel electrophoresis of the treated TM protein samples after gastric juice digestion (wherein 1 is marker group, 2-10 are treated groups digested for 0, 1, 2, 5, 10, 15, 20, 30 and 60min, respectively, and 11 is control group); FIG. 5C shows the results of SDS-PAGE gel electrophoresis of the TM protein samples after intestinal digestion (wherein 1 is marker group, 2-11 are untreated groups digested for 0, 1, 5, 7, 15, 30, 60, 120, 150 and 180min, respectively, and 12 is control group); FIG. 5D shows the results of SDS-PAGE gel electrophoresis of treated TM protein samples after intestinal digestion (wherein 1 is marker group, 2-11 are treated groups after digestion for 0, 1, 5, 7, 15, 30, 60, 120, 150 and 180min, respectively, and 12 is control group).

It can be seen that the TM protein samples of the treated and untreated groups still existed after gastric digestion, the band gray levels did not change significantly, indicating that native TM protein is resistant to pepsin digestion, and that DPCD treatment did not alter the pepsin digestion sites of TM protein; in simulated intestinal digestion, new bands are generated at 15kDa and 34kDa of proteins of a treated group and an untreated group at 1min, the TM protein band becomes shallow gradually along with the extension of digestion time, the TM protein of the untreated group is almost completely reduced after 120min, and the treated TM protein is completely reduced at 60min, which shows that DPCD changes the spatial structure of the TM protein, exposes a trypsin digestion site and improves the intestinal digestion rate of the TM protein.

(2) Western blot analysis

The Western blot analysis result of the TM protein sample after gastric juice digestion is shown in fig. 6, fig. 6a is the IgG binding activity test result of the TM protein sample of the untreated group after gastric juice digestion (wherein 1-9 are the untreated group at digestion time of 0, 1, 2, 5, 10, 15, 20, 30 and 60min, respectively, and 10 is the control group); FIG. 6b is the result of the IgE binding activity test of the TM protein sample after gastric juice digestion in the untreated group (wherein 1-9 are the untreated groups at digestion time of 0, 1, 2, 5, 10, 15, 20, 30 and 60min, respectively, and 10 is the control group); FIG. 6c shows the results of IgG binding activity test after gastric juice digestion of TM protein samples in treated groups (wherein 1-9 are treated groups digested for 0, 1, 2, 5, 10, 15, 20, 30 and 60min, respectively, and 10 is a control group); FIG. 6d shows the results of the IgE binding activity test of the TM protein samples after gastric juice digestion in the treated group (wherein 1-9 are treated groups at digestion time of 0, 1, 2, 5, 10, 15, 20, 30 and 60min, respectively, and 10 is a control group).

Western blot analysis results of the TM protein samples digested by intestinal juice are shown in FIG. 7, FIG. 7a is IgG binding activity test results of the TM protein samples digested by intestinal juice in an untreated group (wherein, 1-10 are untreated groups digested for 0, 1, 5, 7, 15, 30, 60, 120, 150 and 180min, respectively, and 11 is a control group); FIG. 7b shows the results of the IgE binding activity test of the TM protein sample after intestinal digestion (wherein 1-10 are untreated groups digested for 0, 1, 5, 7, 15, 30, 60, 120, 150 and 180min, respectively, and 11 is a control group); FIG. 7c shows the results of the IgG binding activity assay after intestinal fluid digestion of the TM protein samples of the treated group (wherein 1-10 are the treated groups at

digestion time

0, 1, 5, 7, 15, 30, 60, 120, 150 and 180min, respectively, and 11 is the control group); FIG. 7d shows the results of the IgE binding activity test of the treated TM protein samples after intestinal juice digestion (wherein 1-10 are treated groups at 0, 1, 5, 7, 15, 30, 60, 120, 150 and 180min of digestion, respectively, and 11 is a control group).

It can be seen that the activity of the TM protein in the treated and untreated groups remained unchanged throughout the gastric digestion process without significant change. In intestinal digestion, untreated TM protein begins to produce small bands after 1min, with almost no positive reaction after 120 min; the treated TM protein had no positive reaction after 60min (especially after 60min in FIG. 7c, the band was completely obscured because the TM protein sample under the treatment condition was completely digested and failed to bind to the antibody, indicating reduced sensitization), which is a conclusion that the DPCD treated TM protein is more favorable to trypsin hydrolysis, corresponding to the SDS-PAGE gel electrophoresis analysis.

(3) Indirect ELISA

The indirect ELISA results are shown in FIG. 8, in which FIG. 8a is the result of IgG binding activity test after digestion of TM protein sample by gastric juice; FIG. 8b shows the results of the IgE binding activity assay of TM protein samples after digestion with gastric juice; FIG. 8c shows the results of an IgG binding activity assay after intestinal fluid digestion of a TM protein sample; FIG. 8d shows the results of the IgE binding activity assay after intestinal fluid digestion of TM protein samples.

It can be seen that the OD is digested in the stomach with the digestion time being prolonged ₄₅₀ There was no significant change, indicating that the TM protein conformation or linear epitope was not hydrolyzed by pepsin. In the intestinal digestion test, OD is measured as the digestion test proceeds ₄₅₀ The reduction is rapid, and the digestion rate of trypsin is improved after the DPCD treatment, namely most of linear tables of TM can be hydrolyzed by trypsin, which shows that the space conformation of TM protein is changed after the DPCD treatment, more trypsin digestion sites are exposed, and the degree of trypsin hydrolysis is improved.

Example 3 stability analysis of TM protein conformation after high Density carbon dioxide treatment (DPCD) — UV Spectroscopy

1. Experimental methods

The TM protein solutions of the treated group and the untreated group were diluted to 0.1mg/mL with 0.1M PBS, placed in a 10mm micro quartz cuvette, zeroed with 0.1M PBS, scanned three times at 25 ℃ over a scanning range of 190-400 nm, UV absorption spectra were recorded and averaged.

2. Results of the experiment

The uv spectrum analysis results are shown in fig. 9, wherein fig. 9a is the uv spectrum analysis results of DPCD under different pressures; FIG. 9b shows the UV spectrum analysis of DPCD at different times; FIG. 9c shows the results of UV spectroscopy at different temperatures of DPCD.

As can be seen, the maximum absorption peak of the TM protein in the untreated group is at 260nm, and the ultraviolet absorption intensity and the maximum absorption wavelength of the TM protein are obviously changed in the DPCD treated group.

As shown in FIG. 9a, the UV absorption peak blue shifts with increasing DPCD pressure at a fixed time and temperature, the absorption peaks decrease at 5 to 20MPa and 40MPa, the intensity of the absorption peak increases at 25 to 35MPa, and the maximum absorption wavelength of TM protein blue shifts from 260nm to 250nm at 25 MPa.

As shown in FIG. 9b, the UV absorption intensity of the TM protein was the maximum after DPCD was applied for 15min, and the maximum absorption wavelength was blue-shifted from 260nm to 253nm, indicating that the sensitization of the TM protein was the lowest when the DPCD was applied for 15 min; the ultraviolet absorption intensity of the TM protein after DPCD is acted for 90min is minimum, and the maximum absorption wavelength is blue-shifted to 256 nm.

As shown in FIG. 9c, the spatial configuration of the TM protein is significantly changed by the temperature difference of DPCD, the UV absorption intensity is maximum at 55 deg.C, and the absorption wavelength is blue-shifted from 260nm to 253nm, indicating that the sensitization of the TM protein is lowest when the temperature of DPCD is 55 deg.C; the absorption intensity was minimal at 45 ℃ and the absorption peak was blue shifted to 297 nm.

Aromatic amino acid residues (tryptophan Trp and tyrosine Tyr) in the protein determine the ultraviolet characteristic absorption peak, and the TM protein does not contain tryptophan groups, so the reason that the spectral intensity of the TM protein is changed is considered to be that the Tyr of the TM protein is changed under the action of DPCD.

Two, round two chromatography

1. Experimental method

The TM proteins of the treated group and the untreated group are respectively diluted to 0.1mg/mL by 0.1M PBS (pH 7.4), circular spectrum scanning is carried out in the spectrum range of 190-260 nm at 25 ℃, the sample light pool is 2mm, the scanning speed is 50nm/min, and the bandwidth is 1 nm. Three scans were averaged. CDNN software was used to analyze protein secondary structure.

2. Results of the experiment

The results of circular dichroism analysis are shown in FIG. 10, wherein FIG. 10a is the results of circular dichroism analysis of DPCD under different pressures; FIG. 10b shows the results of circular dichroism analysis of DPCD at different times; FIG. 10c shows the results of circular dichroism analysis of DPCD at different temperatures.

As can be seen, the TM protein has two negative peaks at 222nm and 208nm, and a positive peak at 192nm, indicating that the TM protein is a typical alpha-helical structure. The negative and positive peaks of the CD spectrum changed with changes in pressure, temperature, time, indicating that DPCD treatment altered the secondary structure of the TM protein.

The secondary structure content calculated by the CDNN software is shown in fig. 11, where fig. 11a is the secondary structure content at different time of DPCD; FIG. 11b shows the content of each secondary structure at different temperatures of DPCD; FIG. 11c shows the secondary structure content of DPCD at different pressures.

As can be seen from FIG. 11a, the alpha-helix content of the TM protein fluctuates with time, and at 75min, the alpha-helix content is at its lowest (24.03%), while the beta-sheet and random coil contents are 26.69% and 31.78%, respectively.

As can be seen from FIG. 11b, the contents of the secondary structures of the TM proteins at 50 ℃ and 55 ℃ are almost the same, the contents of α -helices are 48.29% and 48.84%, respectively, the contents of β -sheets are 10.61% and 10.42%, respectively, the contents of β -turns are 16.40% and 16.37%, respectively, and the contents of random coils are 24.70% and 24.37%, respectively.

As can be seen in FIG. 11c, the α -helix content of the TM protein was minimized at a pressure of 35MPa, decreasing from 79.00% to 24.11%, the β -sheet increasing from 2.49% to 30.53%, and the random coil increasing from 7.73% to 27.45%.

Third, fluorescence spectrum analysis

1. Experimental methods

Respectively diluting TM protein samples of a treated group and a non-treated group to 0.1mg/mL, performing fluorescence spectroscopy in a spectral range of 290-600 nm at 25 ℃, and setting the maximum excitation wavelength lambda ex to 280; the scanning speed is 240nm/min, and the gaps of the excitation path and the emission path are respectively 10nm and 5 nm. Three scans were averaged.

2. Results of the experiment

The results of fluorescence spectrum analysis are shown in FIG. 12 and Table 2, in which FIG. 12a shows the results of fluorescence spectrum analysis at different pressures of DPCD; FIG. 12b shows the results of fluorescence spectrum analysis of DPCD at different times; FIG. 12c shows the results of fluorescence spectrum analysis of DPCD at different temperatures.

TABLE 2 wavelength of maximum emission peak of TM protein under different conditions

As can be seen from FIG. 12a and Table 2, the fluorescence intensity of the TM protein under different pressures of DPCD is significantly changed, and the maximum emission peak is red-shifted. The TM protein fluorescence intensity of the treated groups of 10MPa, 25MPa, 30MPa and 35MPa is greater than that of the untreated group, the TM protein fluorescence of the treated groups of 5MPa, 15MPa, 20MPa and 40MPa is quenched, the fluorescence intensity of the treated groups of 25MPa is 365 maximum, and the fluorescence intensity of the treated groups of 40MPa is 216 minimum.

As can be seen from FIG. 12b and Table 2, the maximum emission peak of the TM protein fluorescence at different time intervals in DPCD is red-shifted, the fluorescence absorption intensity is increased at 60min, and the fluorescence intensity is decreased at 15min, 30min, 45min, 75min and 90min, and the minimum fluorescence intensity at 75min and 90min is 140.

As can be seen from FIG. 12c and Table 2, the peak position of the maximum emission peak of the TM protein fluorescence at different temperatures of DPCD is red-shifted, the fluorescence intensity is reduced at 40-55 deg.C and increased at 60-70 deg.C.

CO ₂ With Trp and Tyr residuesThe interaction results in a significant change in fluorescence spectrum, and the TM protein does not contain Trp group, so the change in spatial conformation of the TM protein is considered to be caused by CO ₂ And the protein reacts with Tyr in the TM protein, so that the microenvironment of groups in the protein is changed.

Fourth, surface hydrophobicity analysis

1. Experimental method

The surface hydrophobicity is an important index for reflecting the space structure of the protein, is an index for reflecting the number of surface hydrophobic groups when the protein molecules are contacted with the environment, and has important significance for the stability and the functions of the protein. The surface hydrophobicity of TM protein samples of a treated group and an untreated group is measured by using an ANS fluorescent probe, the TM protein concentration is diluted to 50-200 mu g/mL by using 0.1M PBS buffer solution (pH 7.0), 10 mu L of 5mM ANS solution is added into 1mL of sample, the mixture is uniformly mixed and is subjected to dark reaction for 10min, and the fluorescence intensity is measured at the excitation wavelength of 370nm and the emission wavelength of 490nm of a fluorescence spectrometer. Sample surface hydrophobicity (S) ₀ ANS) is defined as the initial slope of fluorescence intensity versus protein concentration.

2. Results of the experiment

The results of the surface hydrophobicity analysis are shown in fig. 13, wherein fig. 13a is the results of the surface hydrophobicity analysis under different pressures of DPCD; FIG. 13b shows the results of surface hydrophobicity analysis at different times in DPCD; FIG. 13c shows the results of surface hydrophobicity analysis at different temperatures of DPCD.

FIG. 13a shows that the hydrophobicity of the TM protein surface tends to increase at 5-15 MPa, decrease at 15-30 MPa, and increase at 30-40 MPa; the surface hydrophobicity reaches 363 at 15MPa, which is increased by 4.1 times compared with the natural TM protein; at 30MPa 40.8, reaching a minimum, a 45.84% reduction compared to the native TM protein.

As shown in FIG. 13b, the surface hydrophobicity of the TM protein changed significantly with time, and at 75min, the surface hydrophobicity was 329 maximal, which was increased by 3.7 times compared to the untreated group.

As can be seen from FIG. 13c, the surface hydrophobicity of the DPCD-treated group of TM proteins gradually increased with the increase of temperature.

Penta, free amino and Total thiol analysis

1. Experimental methods

Adding 100 μ L TM protein samples of treated group and untreated group into 1mL OPA reagent (80mg o-phthalaldehyde and 2mL absolute ethanol dissolved in the dark, adding 0.1g SDS, 1.9068g sodium tetraborate, 0.2mL beta-mercaptoethanol, and fixing volume to 100mL brown volumetric flask) to perform dark reaction for 5min, and placing in an ultraviolet spectrophotometer OD ₃₄₀ The absorbance was measured, and the result was calculated based on the free amino group content of the untreated group as 100% based on L-leucine as a standard control. The reaction solution for measuring the total sulfydryl content is as follows: 4.5mL of a mixture of 0.2M Tris-HCl buffer (containing 8M urea, 2% SDS and 10mM EDTA) and 100. mu.L of 0.1% 2-nitrobenzoic acid (in 0.1M PBS). Taking 500 mu L of TM protein samples of the treated group and the untreated group, respectively mixing the samples with the reaction solution uniformly, then carrying out a dark reaction for 30min, taking the reaction solution as a blank sample, respectively measuring absorbance at 412nm, and calculating according to the following formula:

wherein m is the mass of the TM protein sample, A is the absorbance of the TM protein sample, A ₀ Absorbance of blank sample.

2. Results of the experiment

The results of free amino group analysis are shown in FIG. 14, in which FIG. 14a shows the results of free amino group analysis under different pressures of DPCD; FIG. 14b shows the results of free amino analysis of DPCD at different times; FIG. 14c shows the analysis results of free amino groups at different temperatures of DPCD. It can be seen that with increasing pressure the free amino content decreases significantly, by 13.38% at 40MPa compared to the untreated group; when the time reached 90min, a reduction of 24.43% compared to the untreated group; at 55 ℃ the temperature was reduced by 14.33% compared to the untreated group.

The total thiol analysis results are shown in FIG. 15, in which FIG. 15a shows the total thiol analysis results of DPCD under different pressures; FIG. 15b shows the total thiol analysis of DPCD at different times; FIG. 15c shows the total thiol analysis of DPCD at different temperatures.

As can be seen, the treated group of TM proteinsThe mercapto content was significantly lower than that of the untreated group, but there was no significant difference between the different treated groups with changes in pressure, temperature, and time. The decrease in total thiol content of the TM protein in the treated group was considered to be at pH and CO ₂ Under the combined action, the space structure of the TM protein is damaged, so that groups buried in the interior of the TM protein are exposed and oxidized to form disulfide bonds.

In conclusion, the invention adopts the high-density carbon dioxide technology (DPCD), and specially controls the conditions (temperature, pressure and time) of the high-density carbon dioxide technology according to the characteristics of the TM protein, so that the ultraviolet absorption peak of the DPCD-treated TM protein generates blue shift, the fluorescence spectrum generates red shift, the alpha-helix content is obviously reduced, the random coiling and the beta-folding are obviously improved, the surface hydrophobicity is obviously changed, and the free amino group and the sulfhydryl group content are reduced, which indicates that the space configuration of the DPCD-treated TM protein is obviously changed and even denatured; also makes the TM protein in CO ₂ The sensitization is obviously reduced under the effect and the pH effect, and the effective reduction of the sensitization of the TM protein is realized. Under the optimal DPCD condition (pressure of 30MPa, temperature of 55 ℃ and time of 15min), the IgG/IgE binding activity of the TM protein is respectively reduced by 19 percent and 54.2 percent, and compared with an untreated group, the SIF digestion rate is also obviously improved, which indicates that the sensitization of the TM protein is effectively reduced.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A method of reducing sensitization in tropomyosin comprising subjecting tropomyosin to high density carbon dioxide treatment under conditions such that: the temperature is 40-70 ℃, the time is 15-60 min, and the pressure is 5-40 MPa.

2. The method according to claim 1, wherein the temperature is 50 to 60 ℃.

3. The method of claim 2, wherein the temperature is 55 ℃.

4. The method according to claim 1, wherein the time is 15-30 min.

5. The method of claim 4, wherein the time period is 15 min.

6. The method according to claim 1, wherein the pressure is 30 to 40 MPa.

7. The method of claim 6, wherein the pressure is 30 MPa.

8. The method of claim 1, wherein the high density carbon dioxide treatment is preceded by a pre-treatment of tropomyosin.

9. A method of reducing the allergenicity of crustacean aquatic products, comprising treating tropomyosin isolated from crustacean aquatic products by the method of any one of claims 1 to 8.

10. The method of claim 9, wherein the crustacean aquatic product comprises one or more of shrimp products, crab products, clam products.