WO2022210004A1

WO2022210004A1 - Reagent for extracting protein in foods and extraction method

Info

Publication number: WO2022210004A1
Application number: PCT/JP2022/012492
Authority: WO
Inventors: 直樹森下; 久美子神谷; 昂士郎橋本
Original assignee: 日本ハム株式会社
Priority date: 2021-03-31
Filing date: 2022-03-18
Publication date: 2022-10-06

Abstract

The present invention provides an improved means for extracting a protein in foods, which is capable of solving problems such as safety, speed, and reliability. The extraction reagent of the present invention is a reagent for extracting a protein in foods that includes a reducing agent and a surfactant. The reducing agent is a mercaptoalkanol and/or a sulfite, the surfactant is an alkyl sulfate, and the extraction reagent is for treatment by a shake method for from 30 minutes to 8 hours. The test kit of the present invention includes the extraction reagent and a specific antibody to a target protein for quantitatively or qualitatively detecting a target protein by an immunological measurement method.

Description

Reagents and extraction methods for the extraction of proteins in foods

The present invention relates to technology for extracting proteins contained in foods (for example, food allergens).

In food allergies, the body recognizes specific proteins (food allergens) contained in ingested foods as foreign substances, and various symptoms such as itching of the skin, hives, and coughing are induced through hypersensitivity immunological mechanisms. It is a symptom, and in severe cases, it becomes life-threatening, such as loss of consciousness, hypotension, and shock symptoms. Food allergies are estimated to affect 1-2% of the general population (about 10% if limited to infants). In order to prevent food allergy patients from ingesting foods containing food allergens, especially processed foods that are difficult to distinguish from their appearance, it is necessary to label foods that contain raw materials that contain food allergens. Mandatory or recommended by law. At present, labeling is obligatory for 7 items: "egg, milk, wheat, shrimp, and crab" (those with many cases) and "buckwheat and peanuts" (those that often cause severe symptoms and are life-threatening). It is designated as a "specified raw material" and includes "abalone, squid, salmon roe, oranges, cashew nuts, kiwifruit, beef, walnuts, sesame, salmon, mackerel, soybeans, chicken, bananas, pork, matsutake mushrooms, peaches, yams, apples. , gelatin, and almonds” (those whose onset has been reported with a certain frequency in the past) are designated as “compliance with specified raw materials” for which labeling is recommended. The Consumer Affairs Agency (formerly the Ministry of Health, Labor and Welfare) conducts surveys every three years to grasp the nationwide situation of food allergies, and based on the results, it is reviewing specified raw materials.

In order to operate the system related to food allergens as described above, food manufacturers and public inspection institutions, etc., check whether raw materials containing food allergens (specified raw materials, etc.) are contained in food (processed products). etc. have been used. ELISA methods using antigen-antibody reactions as screening tests and PCR methods using DNA amplification reactions are generally used as techniques for detecting specific raw materials contained in processed products. Among these methods, the ELISA method extracts food allergens and other proteins contained in foods, and then uses antibodies that recognize the proteins to be detected. to detect Immunochromatography, which also uses antigen-antibody reaction, is also used as a qualitative but rapid measurement method, instead of quantitative measurement such as the ELISA method and the PCR method.

When detecting proteins in foods such as ELISA and immunochromatography, it is first necessary to extract the proteins contained in the foods. Foods produced through the process tend to have insolubilized proteins such as food allergens and are difficult to extract. For example, in bread and noodles, it is believed that heating in the manufacturing process denatures proteins, and gluten and egg proteins bond together via SH groups, resulting in insolubilization of these proteins. Therefore, there is a problem that it is difficult to obtain accurate inspection results for processed products due to such poor extraction efficiency.

Conventionally, surfactants such as SDS, denaturants such as urea, reducing agents such as 2-mercaptoethanol, dithiothreitol, and sulfites have been used to extract proteins from processed products that are difficult to extract. It has been used as a solubilizer. A solution containing such a solubilizer (extraction reagent) is used to process foods such as processed products to extract proteins. A step of detecting the target protein by immunochromatography was carried out.

In the conventional protein extraction process, for example, after adding an extraction reagent to food in a container, the container is immersed in boiling water to heat (hereinafter referred to as "heating method"). A method of shaking (hereinafter referred to as "shaking method"), a method of shearing and stirring with a miller (hereinafter referred to as "shearing and stirring method"), and the like have been adopted.

In Patent Document 1, an aqueous Extraction and/or solubilization of poorly water-soluble/poorly-extractable proteins in samples such as foods using a solvent is described. In Patent Document 1, as a preferred embodiment using the above aqueous solvent, a poorly water-soluble/poorly-extractable protein is extracted with an aqueous solvent containing a high-concentration ionic surfactant, and then the resulting protein solution is boiled, That is, it describes heating at a temperature of at least 80° C. or higher for 5 minutes or longer.

Patent Document 2 describes a food extract (reagent) containing thioglycerol as a reducing agent and other solubilizers (for example, urea and/or alkyl sulfates such as SDS). In the patent document, as a method for treating such a food extract, conventionally used appropriate methods such as pulverization, emulsification, stirring, shaking, or boiling can be used alone or in combination. The extraction process can be shortened by using a "high-speed stirring and shearing method" using a mixer, etc., and by adopting thioglycerol as a reducing agent, it can be denatured and insoluble without boiling. It is possible to extract even a certain protein, and an extraction method by the “shaking method” can also be preferably used. It describes mixing with a liquid and shaking for 12 hours or more, preferably 16 hours (for example, overnight) using a shaker.

Japanese Patent Application Laid-Open No. 2005-106629 (Patent No. 3600231) WO2013/179663 (Patent No. 6129832)

The heating method used in the conventional protein extraction process has the problem of being dangerous because it requires boiling water. The conventional shaking method takes a long time, generally 16 hours or more, and takes two days or more from extraction to measurement, leaving room for improvement in the process time. The agitated shear method may cause contamination with other specimens.

An object of the present invention is to provide an improved means for extracting proteins in foods, which can solve the problems of safety, speed, reliability, etc. of conventional extraction processes as described above. .

The present inventors found that when using specific reducing agents and surfactants for extracting proteins contained in foods (various food allergens, etc.), unexpectedly, the treatment in the shaking method The inventors have found that the target protein can be extracted and detected in the same degree as the conventional processing time even if the time is shortened compared to the conventional method, and have completed the present invention. In other words, the inventors have found that the inclusion of certain types of reducing agents and surfactants are intended for certain short-time agitation treatments, which have not been commonly practiced in the past. We have found a reagent for extracting proteins in food.

Specifically, in one aspect, the present invention provides the following inventions.
[1]
A reagent for the extraction of proteins in foods comprising a reducing agent and a surfactant, comprising:
the reducing agent is a mercaptoalkanol and/or a sulfite;
the surfactant is an alkyl sulfate,
An extraction reagent for treatment by a shaking method for 30 minutes or more and 8 hours or less.
[2]
Item 2. The extraction reagent according to Item 1, wherein the mercaptoalkanol is thioglycerol, 2-mercaptoethanol or dithiothreitol.
[3]
Item 3. The extraction reagent according to

Item

1 or 2, wherein the reducing agent is thioglycerol and has a concentration of 0.5% by weight or more relative to the total amount of the extraction reagent.
[4]
Item 2. The extraction reagent according to Item 1, wherein the reducing agent is a sulfite and has a concentration of 100 mM or more relative to the total amount of the extraction reagent.
[5]
Item 5. The extraction reagent according to any one of Items 1 to 4, wherein the surfactant has a concentration of 0.5% by weight or more relative to the total amount of the extraction reagent.
[6]
Item 6. The extraction reagent according to any one of Items 1 to 5, wherein the surfactant has a concentration of less than 8.0% by weight with respect to the total amount of the extraction reagent.
[7]
Item 7. The extraction reagent according to any one of Items 1 to 6, further comprising at least one selected from the group consisting of nonionic surfactants, chelating agents and preservatives.
[8]
A kit for testing food allergens in foods,
The extraction reagent according to any one of Items 1 to 7,
A test kit comprising a target protein-specific detection molecule for quantitatively or qualitatively detecting the target protein by an immunoassay method.
[9]
Item 9. The test kit according to Item 8, wherein the immunoassay method is ELISA or immunochromatography.
[10]
1. A method of extracting protein in a food product comprising treating the food product with an extraction reagent comprising a reducing agent and a surfactant, the method comprising:
the reducing agent is a mercaptoalkanol and/or a sulfite;
the surfactant is an alkyl sulfate,
The extraction method, wherein the treatment is by a shaking method for 30 minutes or more and 8 hours or less.
[11]
Item 11. The extraction method according to Item 10, wherein the mercaptoalkanol is thioglycerol, 2-mercaptoethanol or dithiothreitol.
[12]
Item 12. The extraction method according to Item 10 or 11, wherein the reducing agent is thioglycerol and its concentration is 0.5% by weight or more relative to the total amount of the extraction reagent.
[13]
Item 11. The extraction method according to Item 10, wherein the reducing agent is a sulfite and has a concentration of 100 mM or more relative to the total amount of the extraction reagent.
[14]
Item 14. The extraction method according to any one of Items 10 to 13, wherein the surfactant has a concentration of 0.5% by weight or more relative to the total amount of the extraction reagent.
[15]
Item 15. The extraction method according to any one of Items 10 to 14, wherein the surfactant has a concentration of less than 8.0% by weight with respect to the total amount of the extraction reagent.
[16]
Item 16. The extraction method according to any one of Items 10 to 15, wherein the extraction reagent further contains at least one selected from the group consisting of nonionic surfactants, chelating agents and preservatives.
[17]
A method for testing food allergens in food, comprising:
In the step, the target protein in the food extracted by the extraction method according to any one of Items 10 to 16 is quantified by an immunoassay method using a detection molecule specific to the target protein. An inspection method, which is a process of detecting either objectively or qualitatively.
[18]
Item 18. The test method according to Item 17, wherein the immunoassay is ELISA or immunochromatography.

A person skilled in the art can convert the matters related to the invention described in each of the above items into matters related to the invention of other categories based on the technical idea of the present invention, the matters described in the specification and common general technical knowledge ( It is possible to change the reading). For example, Section 1, "A reagent for extracting proteins in foods, comprising a reducing agent and a surfactant, wherein the reducing agent is a mercaptoalkanol and/or a sulfite, and the surfactant is an alkyl sulfate. and for treatment by a shaking method for 30 minutes or more and 8 hours or less." wherein said reducing agent is a mercaptoalkanol and/or sulfite, said surfactant is an alkyl sulfate, and said extraction is carried out by a shaking method treatment for 30 minutes or more and 8 hours or less. It can be converted into inventions such as

According to the present invention, an extraction reagent is provided that can extract proteins in foods even with shaking treatment at room temperature for a relatively short period of time (up to about 8 hours). By using such an extraction reagent of the present invention, it becomes possible to implement an extraction method and an inspection method that are excellent in speed and safety. In particular, compared with the conventional shaking method, which has required treatment for 16 hours or longer, the shaking method of the present invention can extract and detect proteins at the same level as the conventional method in about 8 hours at the longest. As a result, the inspection process from extraction to measurement, which previously took two days or more, can now be completed in half a day at the shortest. In other words, the shaking method of the present invention compares to conventional extraction methods specialized for rapidity, such as heating methods that require heating in boiling water but require a processing time of about 10 minutes. yields much higher amounts of target protein extracted and detected.

FIG. 1 shows the results of extraction of wheat protein contained in each specimen in Test Example 1 using each extraction reagent and extraction method shown in Table 1 and quantification by ELISA. The vertical axis is a relative value when the measured value of Test Group 1-A is set to 1. FIG. 2 shows the results of extraction of wheat protein contained in each specimen in Test Example 2 using each extraction reagent and extraction method shown in Table 2 and quantification by ELISA. The vertical axis is a relative value when the measured value of Test Group 2-A is set to 1. FIG. 3 shows the results of extraction of wheat protein contained in each sample in Test Example 3 using each extraction reagent and extraction method shown in Table 3 and quantification by ELISA. The vertical axis is a relative value when the measured value of Test Group 3-A is set to 1. 4(A) and (B) show the results of quantifying the total protein concentration after extracting proteins contained in each sample using each extraction reagent and extraction method shown in Table 4 in Test Example 4. FIG. 4(C) and (D) show the results of quantifying the egg allergen concentration by the ELISA method after extracting the protein contained in each sample with each extraction reagent and extraction method shown in Table 4 in Test Example 4. represents FIG. 5 shows the results of quantifying the total protein concentration by extracting the protein contained in each sample using each of the extraction reagents and the extraction method shown in Table 5 in Test Example 5. FIG. FIG. 6 shows the results of quantifying the total protein concentration by extracting the protein contained in each specimen using each of the extraction reagents and the extraction method shown in Table 6 in Test Example 6. FIG. FIG. 7 shows the results of quantifying the total protein concentration by extracting proteins contained in each sample using each extraction reagent and extraction method shown in Table 7 in Test Example 7. FIG.

―Extraction Reagents―
The "extraction reagent" of the present invention is a reagent for extracting proteins in foods containing a reducing agent and a surfactant, wherein the reducing agent is a mercaptoalkanol and/or a sulfite, and the surfactant is It is an alkyl sulfate and is treated by a shaking method for 30 minutes or more and 8 hours or less. It should be noted that the details of "treating by the shaking method for 30 minutes or more and 8 hours or less", which will be described later in relation to the "examination kit", are similarly applicable to the relation to the "extraction reagent".

The use of the extraction reagent of the present invention is not particularly limited. The extraction reagent of the present invention is typically used in kits for inspecting foods (for example, processed products) for specific raw materials containing food allergens for extracting proteins in foods. used. However, the extraction reagent of the present invention can be used in other aspects, for example, in food ( It may also be used to extract proteins in, for example, processed products.

An extraction reagent is generally a solution prepared by adding an agent for extracting various proteins in food, that is, a solubilizer, to a buffer solution with an appropriate pH. In the present invention, at least specific reducing agents and surfactants are used as solubilizers.

Examples of the "buffer" for preparing the extraction reagent include Tris buffer, phosphate buffer, citrate buffer, EDTA buffer, HEPES buffer, and acetate buffer. Preferred buffers in the present invention are, for example, Tris buffers and phosphate buffers. The pH of the buffer solution is generally 4.5 to 8.0 (the range of pH that can occur in vivo), for example 6.0 to 8.0. A person skilled in the art can prepare a buffer solution having a desired concentration and a desired pH by using an appropriate amount of an appropriate compound (adding an appropriate amount of each of a plurality of compounds to water and dissolving them). .

In the present invention, at least "alkyl sulfate" is used as the "surfactant". "Alkyl" in alkyl sulfates is, for example, dodecyl, decyl, nonyl, octyl. "Salts" of alkyl sulfates are, for example, sodium salts, potassium salts, ammonium salts. Specific examples of alkyl sulfates include sodium dodecyl sulfate (SDS).

The concentration of the alkyl sulfate as a surfactant in the extraction reagent of the present invention depends on the type of alkyl sulfate and its effect (influence on the effect of the present invention), other components in the extraction reagent (reduction It can be appropriately adjusted in consideration of the type and concentration of the agent and other components used as necessary). In particular, in the present invention, it is appropriate to adjust the concentration of the alkylsulfate as a surfactant so as to exhibit the effect of an extraction reagent "for treatment by a shaking method for 30 minutes or more and 8 hours or less". . For example, in the shaking method, target protein extraction and / or detection in the conventional treatment time (e.g., 16 hours) compared to the measured value of target protein in "30 minutes or more and 8 hours or less" Efficiency of extraction and / or detection (measured value as an index thereof) is the same or increased, or is reduced within a range that is acceptable for use (for example, practicality as a test kit does not pose a problem) The concentration of alkyl sulfate can be set. In addition, a method other than the shaking method, for example, a heating method (e.g., treatment temperature 100 ° C.) with a treatment time of "30 minutes or more and 8 hours or less" (or shorter, for example, 10 minutes) to extract the target protein and / Or efficiency of target protein extraction and / or detection when the treatment time is "30 minutes or more and 8 hours or less" in the shaking method of the present invention compared to the detection efficiency (measured value as its index) ( The concentration of the alkyl sulfate can be set such that the measured value as its index) is equal or increased.

The concentrations of alkyl sulfates such as SDS in the extraction reagent of the present invention are, for example, 0.1% by weight, 0.5% by weight, 1.0% by weight, 1.5% by weight, 2.0% by weight, and 5.0% by weight. It can be 0% by weight, 8.0% by weight or 10.0% by weight, and these numbers can optionally be lower or upper limits (in any combination). For example, the lower limit is selected from 0.1 wt%, 0.5 wt%, 1.0 wt%, 1.5 wt% or 2.0 wt%, and the upper limit is 10.0 wt%, 8.0 wt%. It can be selected from weight % or 5.0 weight %, for example the lower limit is 0.5 weight % and the upper limit is not set or can be 8.0 weight % as required. For example, when the concentration of alkyl sulfate such as SDS in the extraction reagent of the present invention is relatively high, the amount of target protein detected or the ratio of the amount of target protein detected to the total amount of protein tends to decrease. A possible reason for this is that the reaction system (antigen-antibody reaction, etc.) is inhibited in the ELISA method or other immunoassay methods.

In the present invention, a surfactant other than the alkylsulfate may be further used (in combination with the alkylsulfate) as needed and within the allowable range in relation to the effects of the present invention. Examples of surfactants other than alkyl sulfates include anionic (anionic) surfactants other than alkyl sulfates, cationic (cationic) surfactants and nonionic (nonionic) surfactants. are mentioned. Examples of anionic surfactants other than alkylsulfates include alkylbenzenesulfonates. "Alkyl" in alkylbenzenesulfonates is, for example, dodecyl, decyl, nonyl, octyl. "Salts" of alkylbenzenesulfonates are, for example, sodium salts, potassium salts and ammonium salts. Specific examples of alkylbenzenesulfonates include sodium dodecylbenzenesulfonate. Specific examples of cationic surfactants include hexadecylpyridinium chloride, hexadecyltrimethylammonium bromide, and hexadecyltrimethylammonium chloride. Specific examples of nonionic surfactants include "Tween 20" (polyoxyethylene sorbitan monolaurate), "Tween 40" (polyoxyethylene sorbitan monopalmitate), "Tween 60" (polyoxyethylene sorbitan monostearate), "Tween 80" (polyoxyethylene sorbitan monooleate).

When the extraction reagent of the present invention contains a surfactant other than an alkyl sulfate, It can be appropriately adjusted in consideration of the types and concentrations of other components (reducing agents and other components used as necessary) in the reagent. The lower limit of the concentration of surfactants other than alkyl sulfuric acid in the extraction reagent of the present invention may be, for example, 0.005% by weight, 0.01% by weight, or 0.02% by weight (w/v%). The upper limit can be, for example, 1.0% by weight, 0.5% by weight or 0.1% by weight (w/v%), and these upper and lower limits can be arbitrarily combined. can.

In the present invention, mercaptoalkanols and/or sulfites are used as "reducing agents". "Mercaptoalkanol" includes, for example, thioglycerol, 2-mercaptoethanol, dithiothreitol (sometimes referred to as "dithiothreitol"), and compounds or derivatives having structures similar thereto. In addition, the "mercaptoalkanol" in the present invention means not only compounds in which one end of the main chain of a linear or branched alkanol is substituted with a mercapto group, such as 2-mercaptoethanol, but also compounds such as thioglycerol. In addition, straight-chain or branched-chain alkanols, such as dithiothreitol, a compound in which one end of the main chain of a straight-chain or branched-chain alkanol is substituted with a mercapto group and the other site is substituted with a hydroxyl group. A term encompassing compounds in which both ends of the alkanol main chain are substituted with mercapto groups and substituted with hydroxyl groups at other sites, etc. In other words, linear or branched alkanols such as 2-mercaptoethanol is a term encompassing derivatives of compounds in which one end of the main chain of is substituted with a mercapto group. It is clear that sodium cyanoborohydride, dimethylaminoborane, and sodium borohydride, which have been used as reducing agents in conventional extraction reagents, do not correspond to the "mercaptoalkanol" in the present invention. Sulfites include, for example, alkali metal sulfites and other metal sulfites, and examples of alkali metals include sodium and potassium. Specific examples of sulfites include sodium sulfite and potassium disulfite.

The concentration of mercaptoalkanol and/or sulfite as a reducing agent in the extraction reagent of the present invention depends on the type of reducing agent and its effect (influence on the effect of the present invention), and other components in the extraction reagent. It can be appropriately adjusted in consideration of the type and concentration of (surfactant and other components used as necessary). In particular, in the present invention, it is appropriate to adjust the concentration of the alkylsulfate as a reducing agent so as to exhibit the effect of an extraction reagent for "treatment by a shaking method for 30 minutes or more and 8 hours or less". For example, in the shaking method, target protein extraction and / or detection in the conventional treatment time (e.g., 16 hours) compared to the measured value of target protein in "30 minutes or more and 8 hours or less" Extraction and / or detection efficiency (measured value as an index thereof) is the same or increased, or is within a range that is acceptable for use (for example, practicality as a test kit does not pose a problem). The concentration of reducing agent can be set. In addition, a method other than the shaking method, for example, a heating method (e.g., treatment temperature 100 ° C.) with a treatment time of "30 minutes or more and 8 hours or less" (or shorter, for example, 10 minutes) to extract the target protein and / Or efficiency of target protein extraction and / or detection when the treatment time is "30 minutes or more and 8 hours or less" in the shaking method of the present invention compared to the detection efficiency (measured value as its index) The concentration of the reducing agent can be set such that the measured value as its index) is the same or increased.

The concentration of the mercaptoalkanol (eg, thioglycerol) in the extraction reagent of the present invention is, for example, 0.1% by weight, 0.5% by weight, 1.0% by weight, 1.5% by weight, 2.0% by weight, 5% by weight, 0% by weight, 8.0% by weight or 10.0% by weight, and these figures can optionally be lower or upper limits (any combination). For example, the lower limit is selected from 0.1 wt%, 0.5 wt%, 1.0 wt%, 1.5 wt% or 2.0 wt%, and the upper limit is 10.0 wt%, 8.0 wt%. It can be selected from weight % or 5.0 weight %, for example the lower limit is 0.5 weight % and the upper limit is not provided or can be 10.0 weight % as required.

The concentration of sulfite (e.g., sodium sulfite) in the extraction reagent of the present invention can be, for example, 10 mM, 50 mM, 100 mM, 200 mM, 500 mM, and 1000 mM, and these numerical values are arbitrarily set as lower or upper limits. (arbitrarily combined). For example, the lower limit is selected from 10 mM, 50 mM or 100 mM, the upper limit is selected from 10.0, 8.0 or 5.0% by weight, and as an example the lower limit is 0.5% by weight and the upper limit is set. or 10.0% by weight, if desired.

The solubilizing agent of the present invention may contain components other than the reducing agent and the surfactant, if necessary and to the extent permitted in relation to the effects of the present invention. Such ingredients include, for example, chaotropic agents and chelating agents. Examples of chaotropic agents include urea, formamide, and salts containing anions or cations that have a salt-solubilizing effect (e.g., guanidinium hydrochloride containing guanidinium ions, sodium chloride containing sodium ions, potassium chloride containing potassium ions, etc.). are mentioned. Chelating agents include, for example, EDTA. The concentrations of these components can be appropriately set according to the concentrations of conventional solubilizers, taking into consideration the effects of the present invention, if necessary.

The extraction reagent of the present invention may contain components other than the solubilizer as necessary and within the range permitted in relation to the effects of the present invention. Examples of such components include preservatives (microbial growth inhibitors) such as ProClin (trademark, Merck & Co.) and Na azide. Concentrations of these components can be appropriately set according to concentrations in conventional extraction reagents and, if necessary, in consideration of the effects of the present invention.

―Examination kit―
The "test kit" of the present invention is a kit for testing food allergens, comprising the extraction reagent of the present invention and the and a detection molecule specific for the target protein.

The "target protein" is not particularly limited, so long as it is a protein that can be used to test for food allergens, even if it is the food allergen protein itself, Proteins other than allergens may be used.

The "detection molecule" may be any molecule that can recognize and bind to the target protein and can be modified for detection with the necessary specificity according to its use. The detection molecule should not bind to various proteins in the food, such as proteins other than those in the specific raw materials to be inspected (food allergens or other proteins in raw materials not to be inspected); It is preferable that the binding be limited to a degree that does not affect the results of the test method of the invention. Non-specific adsorption to specific proteins or other substances is acceptable.

The detection molecule in the present invention is not particularly limited, and can be selected from various known molecules capable of binding to proteins. etc.), aptamers, receptors, antimicrobial peptides or other peptides.

The "antibody" may be a polyclonal antibody or a monoclonal antibody depending on the application and embodiment of the test kit. For polyclonal antibodies and monoclonal antibodies, specificity (whether it reacts only with the target protein and does not react (cross) with other proteins), reproducibility (whether there is a difference in test results depending on the production lot), and stability (whether or not the binding to the antigen is lost due to the immobilization treatment, labeling treatment, etc. of the antibody in the kit), etc., so the preferred antibody may vary depending on the application and embodiment of the test kit. With that in mind, one can choose to use polyclonal antibodies, monoclonal antibodies, or a combination of both. "Antigen-binding fragments" include, for example, Fab, Fab', F(ab') ₂ , Fv, scFv, dsFv, diabodies (bispecific antibodies), nanobodies, and the like.

The detection molecule can be in a form suitable for quantitatively or qualitatively detecting the target protein by immunoassay.

"Immunological assay" is a method that makes it possible to quantitatively or qualitatively detect a target protein by utilizing a specific reaction between the target protein and a detection molecule (typically an antibody). It is not particularly limited as long as it is, and various known methods can be used. In addition, test kits compatible with various immunoassay methods are known and commercially available, and the test kit of the present invention can also be modified to use the specific one of the present invention as an extraction reagent. Other than that, it can be manufactured according to a known test kit.

In one embodiment of the present invention, the immunoassay method is ELISA (Enzyme-Linked Immuno Sorbent Assay). ELISA is capable of quantitatively detecting target proteins with a high degree of accuracy, and is a suitable measurement method for detecting specific raw materials containing food allergens from processed foods that have been heated. is doing.

Detection molecules for ELISA (typically antibodies, etc.) include, for example, detection molecules for immobilization and detection molecules for enzyme labeling. The detection molecule for immobilization is intended to be immobilized on the surface of the member on which ELISA is performed, for example, the bottom of a plate (well). The detection molecule for enzyme labeling binds to the target protein captured by the immobilized detection molecule, and then reacts with the substrate to develop a color with an intensity corresponding to the amount of the target protein. The detection molecule for enzyme labeling may be a directly enzyme-labeled detection molecule, i.e. an anti-target protein with an enzyme already attached covalently (e.g. via a linker) to the detection molecule itself. , a detection molecule that will be indirectly labeled with an enzyme, for example, an enzyme-labeled detection molecule by further reaction with an enzyme bound to streptavidin, which is exemplified in the procedure described below in connection with the "test method" Such as biotin-conjugated detection molecules can be completed.

In one embodiment of the present invention, the immunoassay method is immunochromatography. Immunochromatography is a measurement method capable of qualitatively detecting target proteins in a relatively short period of time with simple operations.

The detection molecules (typically antibodies and the like) for immunochromatography include, for example, detection molecules for immobilization and detection molecules for chromogenic labels (eg, colloidal gold label, latex particle label, platinum particle label). The detection molecule for the color-developing label is generally a detection molecule that is preliminarily bound to the color-developing label and is contained in a predetermined position (sample drop portion) of the test strip, and in a state of forming a complex with the target protein. A detection molecule that, after being captured by an immobilized detection molecule, reacts with a substrate to develop a color indicating the presence of the target protein. A detection molecule for immobilization is generally a detection molecule that is contained in a predetermined position (test line) of a test strip, and a complex of a target protein and a detection substance for color-developing label that have migrated by capillary action is detected. A detection molecule for capturing. Instead of visible chromogenic labels, detection molecules bound with fluorescent labels for fluorescence observation, such as quantum dots made of nanoparticles such as silica, can also be used.

The test kit of the present invention contains at least a reducing agent and a surfactant as extraction reagents, and can contain further components as necessary, but each of these components is divided in the test kit. It may also be an embodiment such that it is mixed with a buffer or other suitable solvent (diluent) when used.

The test kit of the present invention contains at least the extraction reagent and the detection molecule of the present invention, but if necessary, can contain arbitrary contents such as other reagents and members according to the embodiment. In the case of an ELISA kit, for example, a plate with wells, a diluent for preparing a target protein solution, a washing solution for washing the plate (well) after each step, and an enzymatic reaction stop in ELISA Liquid, BSA to protect the extracted protein from degradation, an instruction manual describing the procedure of the test method by ELISA, etc. can be included in the contents of the kit along with the extraction reagents and detection molecules of the present invention. . In the case of immunochromatographic kits, for example, test strips that can develop various solutions and reagents by capillary action, diluents for preparing target protein solutions, and procedures for testing methods by immunochromatography are described. Instructions and the like can be included in the kit contents along with the extraction reagents and detection molecules of the invention.

―Extraction method―
The "extraction method" of the present invention is a method for extracting proteins in food, using the extraction reagent of the present invention, that is, an extraction reagent containing a predetermined reducing agent and a surfactant as described above. (referred to herein as an “extraction step”).

The application of the extraction method of the present invention is not particularly limited. The extraction method of the present invention is typically used to extract proteins in foods in methods for inspecting foods (e.g., processed foods) for specific raw materials containing food allergens. be done. However, the extraction method of the present invention can be used in other aspects, for example, to prepare detection molecules (such as antibodies) against various target proteins (food allergens or other proteins) contained in foods (such as It may be used to extract proteins in processed products).

"Foods" may be foods that contain food allergens that can be specified raw materials, etc., or processed products that contain specified raw materials, etc. (subject to inspection). Although the form of the food (processed product) is not particularly limited, for example, the protein is generally insolubilized and difficult to extract by being produced through a process performed under heating and / or pressure conditions. It may be a processed product that tends to be The food may be, for example, solid, semi-solid, jelly, liquid, or emulsion.

・Extraction process In the "extraction process", the proteins in the food are extracted using conventional extraction reagents, except that the extraction reagent containing the specific reducing agent and surfactant according to the present invention is used for extraction. It is the same as the step of performing the treatment to be performed, and can be modified as appropriate so as to be adapted to the present invention as necessary.

The food to be subjected to the extraction process can be subjected to the shaking method as it is, but if necessary according to the form of the food, high-speed shearing and stirring using a food cutter, miller, mixer, homogenizer, etc. It can also be finely divided or emulsified into a homogeneous state by Such a treatment such as micronization may be performed on the food or the like in advance before the extraction step, and the obtained micronized food and the extraction reagent of the present invention may be mixed, A mixture of the food and the extraction reagent of the present invention may be subjected to the extraction treatment at the same time as the treatment such as miniaturization. Processing conditions such as micronization (time, temperature, number of revolutions (rpm) or centrifugal force (x g), etc.) are determined according to the selected processing method such as micronization and the processing apparatus used, and the effects of the present invention. It can be adjusted accordingly. In addition, the food to be subjected to the extraction step may be further subjected to treatment, such as defatting treatment, in consideration of extraction of the target protein and/or detection after extraction, if necessary.

The extraction process can be performed by mixing the food as it is or the food that has been micronized as described above with the extraction reagent of the present invention, and then shaking the mixture. Shaking treatment conditions (time, temperature, shaking speed or rotation speed (rpm), etc.) depend on the type and concentration of the selected reducing agent and surfactant, the type and concentration of other components contained in the extraction reagent, and Depending on the shaking treatment method and the treatment equipment to be used, it can be appropriately adjusted while considering the effects of the present invention.

In the present invention, the shaking treatment time is 30 minutes or more and less than 8 hours at room temperature (under conditions not heated or cooled, usually 10 to 40 ° C., for example, 20 to 30 ° C.). The time can be adjusted within the above time range, taking into consideration the embodiment of the invention, such as the type and concentration of the reducing agent and surfactant, the degree of effect exhibited, and the like. The duration of the shaking treatment can be, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours, and any of these numerical values can be the lower limit or the upper limit (optionally combined). be able to. For example, the lower limit can be selected from 1 hour, 2 hours, or 3 hours, and the upper limit can be selected from 6 hours, 5 hours, or 4 hours. can.

The temperature of the shaking treatment is usually room temperature, but may be at elevated temperature if necessary and if the extraction method embodiment permits, e.g. depending on the local conditions to be carried out. . The temperature of the shaking treatment can be, for example, 40° C., 50° C., 60° C., 70° C., 80° C., 90° C. or 100° C. Any of these numerical values can be the lower limit or the upper limit (optional can be combined with each other). For example, the lower limit can be selected from 40°C or 50°C, and the upper limit can be selected from 80°C or 70°C. From the viewpoint of protein solubilization, the upper limit of temperature may not necessarily be set (for example, the upper limit may be 100 ° C.), but in consideration of work safety, etc., intense heating conditions such as boiling are necessary. The upper limit is preferably less than 100°C so as not to The above-described shaking treatment time is set assuming an embodiment at room temperature, but when shaking treatment is performed under heating, it can be shorter than the above shaking treatment time (the lower limit can be lower). There is a tendency. For example, the duration of the shaking treatment under heating can be 5 minutes or longer, 10 minutes or longer, 20 minutes or longer, or 30 minutes or longer. It is possible to adjust the time range of the shaking treatment under desired heating conditions so that the effect equivalent to the time range of the shaking treatment at room temperature of "30 minutes or more and less than 8 hours" is exhibited.

Recovery step In the extraction method of the present invention, after performing a shaking treatment using a predetermined extraction reagent, the obtained treated product is further centrifuged to recover the supernatant or filtered. A step of recovering the filtrate (referred to herein as a “recovery step”) can be performed. The supernatant or filtrate obtained from the recovery step contains the target protein and other proteins extracted from the food. The treatment conditions (time, temperature, centrifugal rotation speed (rpm) or centrifugal force (x g), filtration filter pore size, etc.) for recovery treatment by centrifugation, filtration, etc. are determined by the selected recovery treatment method and the treatment equipment used. etc., it can be adjusted as appropriate.

Purification step In the present invention, a step of purifying the protein contained in the obtained recovery solution (referred to herein as a “purification step”) can be performed after the extraction step and the recovery step. For example, by using an appropriate means such as gel filtration chromatography typified by SDS-PAGE or ion exchange chromatography, the target protein is extracted from the mixture of the target protein and other proteins contained in the recovery solution. can be isolated and purified. The treatment conditions for purification treatments such as gel filtration chromatography and ion exchange chromatography can be appropriately adjusted according to the selected purification treatment method, the treatment apparatus used, and the like.

In addition, the target protein contained in the recovery liquid obtained by the recovery process depends on what kind of food it is extracted from, for example, what kind of manufacturing process or storage process (kneading, molding, pressurization, heating , drying, enzymatic treatment, freezing, thawing, etc., and processing conditions) that are physically and/or chemically different from the natural target protein, i.e., denatured to some extent. It may be a protein that Alternatively, the recovered target proteins may be a mixture of target proteins with varying degrees of denaturation.

-extracted product-
In another aspect of the present invention, the extraction reagent of the present invention obtained by the extraction method of the present invention, or a predetermined reducing agent and surfactant that play an essential function thereof, and a food-derived solubilizer Provided is a processed extract containing proteins that are

The extraction product of the present invention contains substances other than a predetermined reducing agent, a surfactant, and solubilized protein, such as food as extraction residue, before the supernatant is separated as an extraction solution by filtration. You can

-Inspection methods-
The "testing method" of the present invention is a food allergen testing method, comprising (1) a step of extracting protein in food by the extraction method of the present invention (corresponding to the above-described "extraction step"), and ( 2) A step of quantitatively or qualitatively detecting the target protein in the extracted food by an immunoassay method using a detection molecule specific to the target protein (herein referred to as the "detection step ).

The "immunological measurement method" is not particularly limited as long as it can detect proteins such as food allergens extracted from foods, and can be selected from various known methods.

In one embodiment of the invention, the immunoassay in the detecting step is ELISA. ELISA can be performed using a target protein in a specimen and an antibody that binds thereto, for example, according to the procedure described below.
1) A target protein solution is added to the wells of a plate equipped with an immobilized antibody, the detection molecule for immobilization (typically an antibody, etc.) is brought into contact with the target protein, and a specific reaction ( A first complex is formed by binding (typically by an antigen-antibody reaction).
2) After washing the wells, a solution of a biotin-conjugated detection molecule (typically an antibody or the like) is added, the target protein in the first complex is brought into contact with the biotin-labeled detection molecule, and a specific reaction ( A second complex is formed by binding (typically by an antigen-antibody reaction).
3) After washing the wells, a streptavidin-binding enzyme solution is added, and the biotin-binding detection molecules in the second complex are brought into contact with the streptavidin-binding enzyme to bind by a biotin-streptavidin reaction. A third complex is formed.
4) After washing the wells, a substrate solution (coloring agent) is added to allow the enzyme in the third complex to react with the substrate to develop color.
5) After stopping the coloring reaction, measure the absorbance at a predetermined wavelength with a plate reader.
6) Separately prepare a standard curve graph from the absorbance obtained by measuring the standard solution, read the amount of target protein (solution concentration) from the absorbance measured in 5) and the standard curve graph, and dilute the solution. Multiply the scale factor to calculate the amount of target protein in the sample.

In one embodiment of the invention, the immunoassay in the detecting step is immunochromatography. Immunochromatography can be performed using a target protein in a sample and an antibody that binds thereto, for example, according to the procedure described below.
1) A sample solution containing a target protein is applied to a predetermined site (sample drop portion) on a test strip containing detection molecules for color-developing labels (typically antibodies labeled with colloidal gold or the like). A first complex is formed by dripping, contacting the detection molecule for color development label with the target protein, and binding through a specific reaction (typically an antigen-antibody reaction).
2) The sample solution containing the first complex, unreacted color-developing label detection molecule, etc. develops at a predetermined site (development part) on the test strip, and along with this, the first complex etc. Moves by capillary action.
3) When the first complex reaches a predetermined site (test line portion) containing a detection molecule for immobilization (typically an antibody or the like) and is captured by the immobilized antibody, it develops color. A line colored by the label (for example, a reddish-purple line by gold colloid) appears. Appearance of the test line indicates that the sample solution contained the target protein.
4) A predetermined site (control line section, test downstream of the line), a line colored by the chromogenic label appears. If the control line does not appear, it is suggested that there was an abnormality in the development of the sample solution, regardless of whether the sample contained the target protein (requires retesting).

Hereinafter, the embodiments of the present invention will be disclosed more specifically through examples, but the technical scope of the present invention is not limited to the embodiments disclosed as examples. A person skilled in the art can fully consider the technical idea of the present invention and the contents of the specification and drawings, and the embodiments disclosed as examples, so as to adapt to the intended use and effect of the present invention. can be expanded, modified into various other embodiments, or, if necessary, technical features of the prior art (known inventions) can be further combined. can. Matters necessary for carrying out the present invention other than those described in this specification can appropriately refer to common general technical knowledge and prior art in the technical field to which the present invention belongs.

[Test Example 1]
According to a conventional method, the base buffer shown in Table 1 and various components (% in the table represents w / v% unless otherwise specified. The same applies to the tables of other test examples in Examples.) For detection containing Reagents were prepared. 19 mL of each extraction reagent was added to 1 g of a sample (dried noodles, chocolate snacks or baumkuchen) and processed by the extraction method shown in Table 1. The resulting extract was centrifuged at room temperature (3000×g, 20 minutes, 20° C.), and the supernatant was filtered through 5A filter paper to obtain a filtrate (undiluted extract solution).

Wheat protein in the undiluted extraction solution was extracted according to the protocol of the product "FASTKIT Eliza Ver.III Wheat" (Nippon Ham Co., Ltd.), except that the reagent prepared according to Table 1 was used as the detection reagent and the extraction method was changed. was measured by the ELISA method. The formulations of test groups A to C correspond to the standard detection reagents included in the above product, and the extraction method for test group A follows the standard protocol for the above product. be. A specific procedure for measurement by the ELISA method is as follows.

　Into each well of the microtiter plate, 100 μL of the diluted standard solution and the measurement solution (extract stock solution) were added and allowed to stand at room temperature (20-25°C) for 1 hour. The solution in the wells was discarded, and the wells were washed 5 times with a washing solution. 100 μL of the prepared biotin-conjugated antibody solution was added to each well and allowed to stand at room temperature for 1 hour. The wells were washed 5 times and 100 μL of enzyme-streptavidin conjugate solution was added and allowed to stand at room temperature for 30 minutes. After washing each well 5 times, 100 μL of a coloring agent was added and allowed to stand at room temperature (20 to 25° C.) for 20 minutes. 100 μL of a reaction stop solution was added to each well to stop color development, and absorbance at a dominant wavelength of 450 nm and a minor wavelength of 600 to 650 nm was measured using a plate reader.

Fig. 1 shows the measurement results of the amount of wheat protein (relative value with test area 1-A as 1). Test plot 1-G treated for 1 hour by shaking method using the detection reagent of the present invention is roughly equivalent to test plot 1-A treated for 16 hours by shaking method using the standard detection reagent of the product used. More than the amount of wheat protein was extracted/detected, and the same or more amount of wheat protein was extracted/detected than the test group 1-H treated for 10 minutes by the heating method (100 ° C), so it is safe. It can be evaluated that it corresponds to the extraction method of the present invention with flexibility and speed. Test group 1-B, in which the treatment time by the shaking method was shortened to 1 hour while using the standard detection reagent of the product used, was the standard 16-hour test of the product in which the treatment time by the shaking method was also used. Compared to Section 1-A, the amount of wheat protein extracted/detected is approximately the same or slightly increased depending on the food, and the detection reagent of the present invention can shorten the processing time depending on the embodiment. It can be evaluated as applicable. On the other hand, Test Group 1-F, which was treated for 1 hour by the shaking method using a detection reagent that does not contain a surfactant (SDS), was treated for 16 hours by the shaking method using the same detection reagent. With D and plot 1-E treated with the shear agitation method, the amount of wheat protein extracted/detected was not sufficient.

[Test Example 2]
Except for changing the composition of the detection reagent and the extraction method as shown in Table 2, the extraction stock solution was obtained in the same manner as in Test Example 1, and then the amount of wheat protein was measured by ELISA. The results are shown in FIG. When SDS and thioglycerol are used in combination as a surfactant and a reducing agent, if the concentration of SDS is 2% (w/v), the concentration of thioglycerol is 0.5% or more. -E (test group 1-G) can extract / detect the same amount of wheat protein, so it can be evaluated that any test group in Test Example 2 corresponds to the extraction method of the present invention.

[Test Example 3]
Except for changing the composition of the detection reagent and the extraction method as shown in Table 3, the extraction stock solution was obtained in the same manner as in Test Example 1, and then the amount of wheat protein was measured by ELISA. The results are shown in FIG. When using a combination of SDS and thioglycerol as a surfactant and a reducing agent, if the concentration of thioglycerol is 2% (w/v), the concentration of SDS is 0.5% or more. -E (test group 1-G) can extract / detect the same amount of wheat protein, so it can be evaluated that any test group of Test Example 3 corresponds to the extraction method of the present invention.

[Test Example 4]
Bread and noodles were used as food samples, and the composition of the detection reagent and the extraction method were changed as shown in Table 4 (extraction time by shaking method was 1, 3, 6, or 16 hours). An extract stock solution was obtained in the same manner as in Example 1. After that, the total protein concentration in the undiluted extract was measured using "Quant Kit" (Global Life Science Technologies Japan Co., Ltd.). In addition, the concentrations of wheat protein (allergen) and egg protein (allergen) in the extract stock solution were measured by ELISA using "FASTKIT Eliza Ver.III Wheat" and "FASTKIT Eliza Ver.III Egg" (Nippon Ham Co., Ltd.). It was measured.

The results are shown in Figures 4-1 and 4-2. The extraction reagent for test group 4-A is the total protein concentration in the bread, the total protein concentration in the noodles, and the egg allergen concentration in the bread. Although the concentration was about the same as when the time was set to 16 hours, the concentration of the egg allergen in the noodles gradually decreased as the treatment time in the shaking method was shortened compared to when the treatment time was set to 16 hours. A trend was seen. It was shown that the extraction reagent of Test Group 4-A can be used as the extraction reagent of the present invention by adjusting the extraction time and target food or protein. On the other hand, the extraction reagent for test group 4-B was 16 hours even if the treatment time in the shaking method was 1, 3 or 6 hours for both bread and noodles, the concentration of total protein, and the concentration of egg allergen protein. The concentration was almost the same as when the time was changed. In addition, the concentration of each protein tended to be higher in the extraction reagent of test group 4-B than in the extraction reagent of test group 4-A. It has been shown that the extraction reagent of test group 4-B can be a preferred extraction reagent of the present invention, which can shorten the time of extraction processing and can target a wider range of foods or proteins. rice field.

[Test Example 5]
The food to be tested was changed to pasta (not dried noodles), chocolate snacks or Baumkuchen, and the composition of the detection reagent and extraction method were changed as shown in Table 5 (extraction time by shaking method was 30 minutes or 1 hour). A stock extract was obtained in the same manner as in Test Example 1, except that After that, the total protein concentration in the undiluted extract was measured using "Quant Kit" (Global Life Science Technologies Japan Co., Ltd.).

The results are shown in Figure 5. Pasta, chocolate snacks, and baumkuchen yielded a good amount of protein when extracted by the shaking method for 30 minutes (test group 5-A) and 1 hour (5-B). Pasta (not dry noodles), chocolate snacks, and baumkuchen are foods from which protein extraction is difficult.

[Test Example 6]
The same procedure as in Test Example 1 was performed except that the food sample was changed to chocolate snacks, and the composition of the detection reagent and the extraction method were changed as shown in Table 6 (extraction time by shaking method was 1 hour). to obtain an extract stock solution. After that, the total protein concentration in the undiluted extract was measured using "Quant Kit" (Global Life Science Technologies Japan Co., Ltd.).

The results are shown in Figure 6. When mercaptoalkanols other than thioglycerol (dithiothreitol and 2-mercaptoethanol) or sodium sulfite are used as reducing agents, the same amount of protein contained in the sample as when thioglycerol is used can be extracted and extracted. could be detected.

[Test Example 7]
The food to be tested was changed to pasta (not dried noodles), chocolate snacks or Baumkuchen, and the composition of the detection reagent and extraction method were changed as shown in Table 7 (extraction time by shaking method was 1 hour). A stock extract was obtained in the same manner as in Test Example 1 except for the above. After that, the total protein concentration in the undiluted extract was measured using "Quant Kit" (Global Life Science Technologies Japan Co., Ltd.).

The results are shown in Figure 7. It was shown that the concentrations of thioglycerol and SDS in the extraction reagent are both 0.1 w/v % or more, so that the embodiment can exhibit the effects of the present invention.

Claims

A reagent for the extraction of proteins in foods comprising a reducing agent and a surfactant, comprising:
the reducing agent is a mercaptoalkanol and/or a sulfite;
the surfactant is an alkyl sulfate,
An extraction reagent for treatment by a shaking method for 30 minutes or more and 8 hours or less.
The extraction reagent according to claim 1, wherein the mercaptoalkanol is thioglycerol, 2-mercaptoethanol or dithiothreitol.
The extraction reagent according to claim 1 or 2, wherein the reducing agent is thioglycerol and its concentration is 0.5% by weight or more relative to the total amount of the extraction reagent.
The extraction reagent according to claim 1, wherein the reducing agent is a sulfite and has a concentration of 100 mM or more relative to the total amount of the extraction reagent.
The extraction reagent according to any one of claims 1 to 4, wherein the surfactant has a concentration of 0.5% by weight or more with respect to the total amount of the extraction reagent.
The extraction reagent according to any one of claims 1 to 5, wherein the surfactant has a concentration of less than 8.0% by weight with respect to the total amount of the extraction reagent.
The extraction reagent according to any one of claims 1 to 6, further comprising at least one selected from the group consisting of nonionic surfactants, chelating agents and preservatives.
A kit for testing food allergens in foods,
The extraction reagent according to any one of claims 1 to 7,
A test kit comprising a target protein-specific detection molecule for quantitatively or qualitatively detecting the target protein by an immunoassay method.
The test kit according to claim 8, wherein the immunoassay method is ELISA or immunochromatography.
1. A method of extracting protein in a food product comprising treating the food product with an extraction reagent comprising a reducing agent and a surfactant, the method comprising:
the reducing agent is a mercaptoalkanol and/or a sulfite;
the surfactant is an alkyl sulfate,
The extraction method, wherein the treatment is by a shaking method for 30 minutes or more and 8 hours or less.
The extraction method according to claim 10, wherein the mercaptoalkanol is thioglycerol, 2-mercaptoethanol or dithiothreitol.
The extraction method according to claim 10 or 11, wherein the reducing agent is thioglycerol and its concentration is 0.5% by weight or more relative to the total amount of the extraction reagent.
The extraction method according to claim 10, wherein the reducing agent is sulfite, and its concentration is 100 mM or more relative to the total amount of the extraction reagent.
The extraction method according to any one of claims 10 to 13, wherein the surfactant has a concentration of 0.5% by weight or more with respect to the total amount of the extraction reagent.
The extraction method according to any one of claims 10 to 14, wherein the surfactant has a concentration of less than 8.0% by weight with respect to the total amount of the extraction reagent.
The extraction method according to any one of claims 10 to 15, wherein said extraction reagent further contains at least one selected from the group consisting of nonionic surfactants, chelating agents and preservatives.
A method for testing food allergens in food, comprising:
In the step, the target protein in the food extracted by the extraction method according to any one of claims 10 to 16 is detected by an immunoassay method using a detection molecule specific to the target protein. An inspection method comprising a step of quantitatively or qualitatively detecting.
18. The test method according to claim 17, wherein said immunoassay is ELISA or immunochromatography.