JPWO2013179663A1 - Food ingredient extract and extraction method - Google Patents

Abstract

The present invention is a food extract that can be extracted even when the food allergen in food is cooked and becomes difficult to extract, and an allergen test method and kit using the extract, which are irritating and toxic It is intended to provide a simple test method and kit that uses a food extract that does not contain 2-mercaptoethanol (2-ME) and that does not require a boiling step and that can provide accurate test results in a short time. is there. The present invention is characterized by using a food extract containing thioglycerol and a solubilizing agent in order to extract food allergens from food. As a result, various allergen proteins from various processed foods, including egg proteins that exist in a hardly soluble state such as baked goods and dry noodles, can be detected efficiently and with high sensitivity. Moreover, even if it is applied to a simple immunochromatography method or an ELISA method with high inspection accuracy, it can be detected accurately without showing false positives.

Description

  The present invention relates to a food extract for extracting a food allergen in food, and a test method and kit for food allergen using the extract.

  In recent years, patients with food allergies who develop food allergies are increasing mainly in younger age groups. Symptoms of food allergies are diverse and high in risk, including itching and inflammation of the skin, and anaphylactic shock that results in shock and death. Therefore, in 2001, “Ministerial Ordinance (Ministry of Health, Labor and Welfare Ordinance No. 23) to revise a part of the Ministerial Ordinance on Food Sanitation Law Enforcement Regulations and Milk and Dairy Product Component Standards” was established. As for “eggs, wheat, milk” and “soba, peanuts” with severe symptoms, the labeling of raw materials is obligatory as a specified raw material. Nineteen items (abalone, squid, how much, shrimp, orange, crab, kiwi fruit, beef, walnut, salmon, mackerel, chicken, pork, matsutake, peach, yam, apple, gelatin) follow the specified raw materials Display as recommended. Furthermore, in December 2004, banana was added to the recommended display items. In June 2008, the recommended display item “Shrimp, Crab” became a required display item. A total of 7 items. In other countries, the issue of food allergies is regarded as important as in Japan, and many countries have their own food allergy labeling items. For example, in the United States, `` gluten-containing grains, crustaceans, eggs, fish, “Peanuts, soybeans, milk, nuts” in the EU are “gluten-containing cereals, shellfish, eggs, fish, peanuts, soybeans, milk, nuts, celery, mustard, sesame, 10 mg / kg or 10 mg / kg or more anhydrous “Sulphite and sulfite, lupine, mollusc” are mandatory labeling items. In China, labeling for gluten-containing cereals, crustaceans, fish, eggs, peanuts, soybeans, milk and nuts is recommended.

  In processed foods produced by mixing various raw materials, it is complicated to trace the history of individual raw materials, and even if they are not blended as raw materials, they may be mixed in the production line. Therefore, it is necessary to inspect the obtained product directly to determine whether the processed food contains seven specified raw materials of “egg, wheat, milk, buckwheat, peanut, shrimp, crab” that are required to be labeled. There is. And since allergens that cause severe allergic symptoms are proteins derived from specific raw materials, it is essential to have a detection method that can accurately detect whether or not specific raw material-derived proteins are contained in processed foods. A measurement kit that can be measured quickly and easily is desired.

  However, many processed foods are manufactured under harsh conditions such as heating and pressurization, and such foods have the problem that the allergen protein to be tested is insoluble and difficult to extract by conventional extraction methods. It was. For example, in the manufacturing process of bread and noodles, there is a report that gluten and egg protein are bound via SH groups and insolubilized by heating. In addition, some proteins are thought to be denatured and insolubilized by heating. Due to these poor extraction efficiencies, there were problems such as inaccurate inspection results.

Highly concentrated (0.5-10%) sodium dodecyl sulfate (SDS) and a reducing agent 2-mercaptoethanol (as a method for efficiently extracting poorly water-soluble or difficultly extracted proteins from processed foods 2-ME) or an aqueous solvent containing dithiothreitol (DTT) is added, and a method of solubilizing and extracting in a boiling step of heating for 5 minutes is known (Patent Documents 1 and 2). The boiling operation of an aqueous solvent containing 2-ME or DTT having an irritating odor is difficult to carry out at the food production site. (% Is w / v% unless otherwise specified, and v is the volume of the extract. The same applies hereinafter.)
On the other hand, it is widely used because it can be extracted without boiling operation by performing a shaking operation overnight, but it takes time to obtain a test result is a problem from the viewpoint of speed .

  Previously, the present applicant has strongly denatured heat by using a food extract that combines a reducing agent such as 2-ME, DTT, or tris (2-carboxyethyl) phosphine (TCEP) and a solubilizing agent such as SDS or urea. We have developed a food ingredient extract that can be extracted with higher efficiency than the conventional protein (Patent Document 3). In that case, the extraction / solubilization process can be extracted for a long time, such as shaking for 12 hours or more. Solubilization work was necessary, and it could not be said to be a rapid measurement method. Moreover, the problem of using 2-ME with an irritating odor still remained. DTT is the same in that it has an irritating odor, and TCEP or its related reducing agent tris (3-hydroxypropyl) phosphine (THP) is a relatively expensive reagent and disadvantageous in terms of cost. There is also.

  2-ME has been designated as a poison by the “Decree to Revise the Order for Designation of Poisonous and Deleterious Substances (2008 Decree No. 199)” that came into effect on July 1, 2008. It is not desirable to handle such toxic odors at food production sites or factories, and the demerit that it is necessary to ensure the safety of disposal of residual waste after inspection. large.

On the other hand, as a method for measuring allergen protein, there is a method in which a specific region in DNA encoding the target protein is amplified and detected, but in general, antigen-antibody reaction is performed from the viewpoint of rapidity and simplicity. Utilized immunological measurement methods are used. Of the immunological measurement methods, the ELISA method is suitable for accurately quantifying the amount of the target allergen, but if it is only to qualitatively confirm the presence or absence of the allergen, it can be conveniently performed. An immunochromatography method that can be detected visually with high sensitivity is suitable.
However, it is known that 2-ME, a reducing agent, frequently produces false positive reactions when applied to immunochromatography. As the cause, it was thought that the free thiol group which 2-ME has aggregates particles, such as a metal colloid and latex which labeled the antibody, and causes a false positive reaction (patent documents 4-6). In order to suppress the influence of this false positive reaction, a method of measuring by reducing the 2-ME concentration in the extract to 1.54 ppm or less at the time of immunochromatography (Patent Document 4) has also been reported, but the number of dilution steps increases. As a result, the detection sensitivity is lowered.

As described above, although 2-ME is an excellent reducing agent, in addition to the difficulty in handling in the field of food production based on the above-mentioned irritating odor and toxicity, false positive results in an immunochromatography method suitable for a simple test. Due to the large amount of research, search for a reducing agent in place of 2-ME and research and development of an extraction method that enables efficient extraction without using 2-ME have been actively promoted.
Although a method of extracting by shaking for 12 hours or more using sulfite or THP instead of 2-ME as a reducing agent has been proposed (Patent Documents 5 and 6), the problem is that it takes a long time for extraction. was there. As an improved method, by performing extraction using sulfite and SDS in a boiling step, it has become possible to extract denatured proteins in processed foods that are difficult to extract in a short time (Patent Document 7). Further, paying attention to cysteine as a reducing agent, a food extract (Patent Document 8) containing an alkyl sulfate, urea and cysteine, or DTT at a certain ratio has been proposed. However, these methods require boiling for 10 to 60 minutes in the extraction operation, and it is necessary to provide a dedicated boiling device, so that it is difficult to use at the food manufacturing site. In addition, some foods such as raw noodles can be lumped by boiling, and internal protein extraction may be incomplete.

  From the above, in the processed food manufacturing field, a food extract that does not contain an irritating and toxic reducing agent such as 2-ME is used, and a boiling step is not required. There has been a strong demand for the development of a test method and test kit for an allergen protein that can provide a test result and that can be used for detecting denatured proteins in difficult-to-extract processed foods.

JP-A-2005-106629 JP 2005-106811 A JP 2006-71509 A JP 2008-298664 A JP 2009-133712 A JP 2010-2369 A JP 2010-286480 A JP 2010-78448 A

  The present invention can be extracted even when food allergens in food are cooked and difficult to extract, does not contain 2-mercaptoethanol (2-ME) having irritation and toxicity, and does not require a boiling step It is an object to provide a simple food extract and extraction method. Furthermore, it aims at providing the simple and accurate test | inspection method and test | inspection kit of the food allergen extracted using the said food extract.

  In general, in order to efficiently extract food components, a method is often used in which food is pulverized and extracted with a solvent for extraction using an apparatus such as a miller, a mixer, a homogenizer, or a stomacher. However, even if these means are used, it is difficult to extract poorly soluble proteins in processed foods including proteins contained in baked confectionery and dried noodles, and sufficient sensitivity cannot be obtained.

Accordingly, as a result of intensive efforts, the present inventors have achieved a highly efficient food extract combined with a solubilizer using “thioglycerol (HOCH ₂ CH (OH) CH ₂ SH)” as a reducing agent. The present inventors have found that various allergen proteins can be extracted from various processed foods. It has been confirmed that an allergen protein in a hardly extractable state in a cooked food, which has been a problem in the past, for example, an egg protein existing in a hardly soluble state such as baked confectionery and dry noodles can be efficiently extracted. And at the time of extraction, it can be used at a lower concentration compared to the case where 2-ME, which is a toxic substance commonly used in food extracts and has a strong irritating odor, is used as a reducing agent. The extraction efficiency was equal to or higher than when 2-ME was used.
Moreover, in the conventional knowledge (patent document 5 etc.), when the food extract containing 2-ME is used, it is because of the free SH group which 2-ME has as a reason why a false positive occurs in the immunochromatography method. However, there was a concern that the immunochromatographic method would cause false positives even with “thioglycerol” having the same free SH group, but when the immunochromatographic results were verified by the ELISA method, It was confirmed that a result highly correlated with the ELISA method was obtained.
By obtaining the above knowledge, the present invention has been completed.

That is, the present invention is as follows.
[1] A food extract for extracting a food allergen in food, which is a food extract containing a reducing agent and a solubilizing agent, wherein thioglycerol is used as the reducing agent. Here, a solubilizer is a well-known solubilizer for those skilled in the art, and it is also possible to use a solubilizer together.
[2] The food extract according to [1], wherein the solubilizer is urea and / or an alkyl sulfate. Here, SDS is a preferred alkyl sulfate.
[3] The concentration of thioglycerol in the extract is 0.001 to 10 (w / v)%, preferably 0.005 to 5%, more preferably 0.01 to 5%, most preferably with respect to the total amount of the extract The food extract according to [1] or [2], preferably 0.02 to 1%.
[4] The food extract according to any one of [1] to [3], wherein the food allergen is an allergen protein that is difficult to extract in a heat-cooked food.
[5] A method for extracting food allergens from foods, wherein a food extract containing thioglycerol and a solubilizing agent is used in the method for extracting food allergens from foods. The solubilizer used in the extraction method is a well-known solubilizer for those skilled in the art, and a solubilizer can be used in combination. Preferred solubilizers are urea or alkyl sulfates (preferred alkyl sulfates include SDS). As the extraction method, conventional pulverization, emulsification, stirring, shaking and boiling methods can be used alone or in combination. A preferable extraction method is a “high-speed stirring and shearing method” or a “shaking method”.
[6] The presence or absence of the food allergen extracted from the food using the food extract according to any one of [1] to [4] above is detected by immunoassay or the amount of the food allergen is measured. A method for testing food allergens, comprising: Here, as a preferred immunological assay, immunochromatography or ELISA is used. As the ELISA, the sandwich method is preferable.
[7] A test kit for detecting the presence or absence of a food allergen in food or measuring the amount thereof, together with thioglycerol and a solubilizer for addition to a food extract A food allergen test kit comprising an antibody that specifically recognizes an allergen. Here, the thioglycerol and the solubilizing agent to be added to the food extract may be packaged as a food extract previously dissolved in a buffer solution (PBS or the like). The solubilizer used in the test kit is a well-known solubilizer for those skilled in the art, and a plurality of solubilizers may be used in combination. Preferred solubilizers are urea and / or alkyl sulfates (preferred alkyl sulfates include SDS). As an antibody specifically recognizing a food allergen used in the test kit, it is preferable to use two kinds of antibodies, an antibody immobilized on a support and a labeled antibody.

  By using the food extract containing the thioglycerol and the solubilizing agent of the present invention, it is highly modified and hardly soluble from various processed foods without using 2-ME having irritation and toxicity. Extraction including food allergen protein was possible with high sensitivity and efficiency. For example, egg protein can be extracted from dry noodles or bread without a boiling step. Therefore, simple and accurate inspection is possible even at the food manufacturing site. Further, even when applied to a simple immunochromatography method or an ELISA method with high inspection accuracy, it can be detected accurately without showing false positives.

1. Food component extract (1-1) Food and food allergen protein targeted by the present invention The food targeted by the present invention is a food containing a protein serving as a food allergen. In particular, the protein of food that can be efficiently extracted by the food extract of the present invention is an allergen protein in a difficult-to-extract state in heat-cooked food. Specifically, it is a processed food produced under heat and pressure conditions, in which allergen protein is insolubilized and difficult to extract, such as dried noodles (instant ramen etc.), egg protein contained in bread and the like. Any food allergen protein of the food extract of the present invention can be efficiently extracted, and a measurement solution that can be detected with high sensitivity by a simple operation such as dilution can be prepared.
The form of the food targeted by the present invention may be any of solid, semi-solid, jelly, liquid, and emulsified liquid.

(1-2) Thioglycerol The food extract of the present invention contains a reducing agent and a solubilizing agent, and thioglycerol (HOCH ₂ CH (OH) CH ₂ SH) is used as the reducing agent. To do. The concentration in the extract can be appropriately adjusted according to the type of food to be tested and the type of food allergen protein to be tested. For example, 0.001 to 10 (w / v)%, preferably 0.005 to 5%, more preferably 0.01 to 5%, and most preferably 0.02 to 1%.
When the amount of thioglycerol is less than the lower limit of the above range, the extraction efficiency of the denatured protein is lowered. Moreover, extraction efficiency falls also when exceeding an upper limit.

(1-3) Solubilizer used in the present invention As the solubilizer used in the present invention, a conventional solubilizer can be used. For example, ionic surfactants such as urea and alkyl sulfates can be exemplified, and two or more of these solubilizers may be used in combination. It is preferable to use urea and / or alkyl sulfates. Examples of the alkyl group of the alkyl sulfate include octyl, nonyl, decyl, dodecyl and the like, and the salt is not particularly limited as long as it is a water-soluble salt, but examples thereof include sodium salt, potassium salt, ammonium salt and the like. The most preferred alkyl sulfate is sodium dodecyl sulfate (hereinafter referred to as SDS).
Other ionic surfactants include anionic surfactants such as alkylbenzene sulfonates such as sodium dodecylbenzenesulfonate, cations such as hexadecylpyridinium chloride, hexadecyltrimethylammonium bromide, and hexadecyltrimethylammonium chloride. A surfactant can also be used.
When a miller, a mixer, a homogenizer or the like is used for the extraction method operation, urea is most preferable as a solubilizer because it does not cause foaming, but alkyl sulfates such as SDS do not have any problem.
The concentration of the solubilizer in the extract can be appropriately adjusted according to the type of food to be tested and the type of food allergen protein to be tested, and according to the concentration of the reducing agent thioglycerol. For example, when urea is used as a solubilizer, it is usually 0.01 to 10 (w / v)%, preferably 0.05 to 5.0%, more preferably 0.1 to 3.0%. When an alkyl sulfate such as SDS or other ionic surfactant is used, it is usually 0.005 to 5.0 (w / v)%, preferably 0.01 to 3.0%. More preferably, it is adjusted to about 0.05 to 2.0%.
When the solubilizer is less than the lower limit of the above range, the protein extraction efficiency may be reduced due to insufficient protein solubilization. If the upper limit is exceeded, there is no problem in extraction efficiency, but up to 5.0% is sufficiently effective, and no further concentration is required.

(1-4) Aqueous medium in extract As the aqueous medium used for the extract of the present invention, for example, water, buffer (phosphate buffer, Tris-HCl buffer), saline, and the like can be exemplified. . Although pH in an extract can be suitably adjusted for every processed food used as extraction object, it is normally adjusted to about pH 6-8.

2. Extraction Method As the extraction method of the present invention, any conventional method such as pulverization, emulsification, stirring, shaking, or boiling can be used alone or in combination. By using the “method”, the extraction process can be shortened.
Although the temperature at the time of extraction is normally performed at room temperature, the extraction operation may be performed by heating or heating the extract to 40 to 100 ° C.
In the present invention, the “high-speed stirring and shearing method” used for shortening the extraction time uses a miller, a mixer, a homogenizer or the like for the extraction operation, and high-speed stirring and shearing to pulverize food in a short time. Emulsify. By using the extract containing the thioglycerol and the solubilizing agent of the present invention in this step, even if there is no boiling step, even a protein that is denatured and hardly soluble can be extracted.
When “Milcer” is used as a specific example of the extraction operation, it is as follows.
For example, 38 mL of the above-described thioglycerol and an extract containing a solubilizer are added per 2 g of the target food, and high-speed cutting is performed 30 seconds × 3 times using a miller. Then, after normal centrifugation such as “3000 × g for 20 minutes”, the mixture is filtered to obtain a food component extract.
Further, as the extraction method of the present invention, an extraction method by a “shaking method” can also be preferably used. Specifically, the target food is directly or after being pulverized, mixed with an extract containing thioglycerol and a solubilizing agent, and shaken using a shaker for 12 hours or more, preferably 16 hours (for example, overnight). Then, it is centrifuged and filtered to obtain a food component extract.
The “shaking” process is performed, for example, under conditions of “90 to 110 rpm, one reciprocation is one rotation, and 90 to 110 reciprocations per minute” at room temperature.
The liquid extracted with the extraction liquid of the present invention is diluted as it is or with PBS or the like, adjusted to near neutrality (pH 6.0 to 8.0) if necessary, and a test solution such as immunochromatography or ELISA is used. By applying to an immunological measurement method, it can be used for detection or measurement of protein content.
In addition, for the detection of allergens other than proteins (histamine, etc.) using the extract obtained by the extraction method of the present invention, measurement with a spectrophotometer or the like may be performed, and detection of food allergen genes with PCR reagents It is also possible to perform.

3. Food Inspection Method The extract obtained by the extraction method of the present invention can be applied to a detection method using a spectrophotometer or a detection method using a PCR method. However, when the food allergen is a protein, In view of accuracy, it is preferable to apply an immunoassay.
As a preferred food inspection method of the present invention, a well-known immunological measurement method such as immunochromatography, ELISA, or Western blotting is used.
(3-1) Antibody Since each measurement kit corresponding to a food allergen is commercially available as a food component test kit or the like, an antibody associated with the kit can be used, but an antibody against a specific food allergen protein is actually used. Or a commercially available antibody may be used.

(3-2) Examination method by immunochromatography The immunochromatography method is a simple, rapid and sensitive immunological measurement method, and is widely used as a simple food component testing method that does not require a measuring instrument. The principle is as follows.
In the immunochromatography, a first antibody that specifically recognizes an allergen to be examined is fixed in a band at a specific position on a thin film support such as a cellulose membrane, and a second antibody labeled near the sample dropping part is labeled. Antibodies are held movably. When the extract of the food to be analyzed is dropped onto the sample dropping part, if it contains “allergens for test purposes”, it binds to the labeled second antibody and moves through the membrane, and is fixed in a band shape. When the position reaches the position of the first antibody, it also binds to the first antibody, so that the label gathers in a band at the position where the first antibody is immobilized and can be confirmed with the naked eye. If the food to be analyzed does not contain “allergen for testing” or is below the detection limit, no band appears. After dropping the extract into the sample dropping part, the result can be judged in about 10 to 15 minutes. It is a simple, rapid and sensitive method and is widely used as a food component inspection kit.

As the label, in addition to a metal colloid such as a gold colloid, an enzyme label, a colored latex particle, or the like can be used.
Such immunochromatography for food inspection can be prepared by itself, but it is more convenient if immunochromatography for various allergens already on the market is used.

(3-3) ELISA Method The ELISA method is also an immunochromatography method in that it is an immunoassay method for detecting an antigen-antibody reaction. In terms of simplicity, it is not as good as immunochromatography, but it is a method with good sensitivity and excellent quantification.
There are mainly a direct method and a sandwich method, but a calibration curve is generally prepared by a sandwich method and quantified.
In the “sandwich method”, two types of antibodies that recognize different epitopes are prepared in advance as antibodies specific to the target allergen (if there are multiple identical epitopes in the target substance, one type of antibody is also possible). The first antibody is adsorbed on a solid phase (such as a microtiter plate), and after the food component extract is allowed to act, the labeled second antibody is reacted to detect the target allergen in the same manner. Can be quantified.
As labeling means used in the ELISA method, usually, a chromogenic enzyme (for example, peroxidase, alkaline phosphatase), a fluorescent substance (for example, fluorescein isothiocyanate), a bioluminescent substance (for example, luciferin-luciferase), a chemiluminescent substance (for example, Luminol, an acridine derivative, or an adamantane derivative). Colloidal gold or radioactive material (eg ³² P) can also be used.

4). Test kit A food extract containing thioglycerol and a solubilizer along with an antibody that specifically recognizes each food allergen to be tested in order to detect the presence or absence of various food allergens in food. Can be used as a food allergen test kit. At that time, there may be a plurality of types of food allergens to be examined, and it is preferable that all of the specific raw materials are available.
The antibody specifically recognizing each food allergen is preferably immobilized on a thin film support in the same or different immunochromatography, and eggs, wheat, milk, buckwheat, Immunochromatography for detecting peanut and soy food allergens can also be used. For example, a test reagent that combines a support (fixed on the judgment line near the center of the support) with a polyclonal antibody corresponding to each food allergen and a colloidal gold labeled antibody together with a buffer solution for dilution. Have already been marketed as “FASTKIT Slim” manufactured by Nippon Ham Co., Ltd. Regarding egg protein, “FASTKIT slim egg made by Nippon Ham Co., Ltd.”, about milk protein, “FASTKIT slim milk made by Nippon Ham Co., Ltd.”, and about wheat protein, “FASTKIT slim wheat made by Nippon Ham Co., Ltd.” As for buckwheat protein, “FASTKIT slim buckwheat” manufactured by Nippon Ham Co., Ltd. For “peanut protein” “FASTKIT slim peanut manufactured by Nippon Ham Co., Ltd.”, and as for soy protein, “FASTKIT slim soybean manufactured by Nippon Ham Co., Ltd.” Can be used.
Also for ELISA, microtiter plates adsorbing each first antibody and enzyme-labeled second antibody (polyclonal antibody) solution etc. were set with dilution buffer solution etc. corresponding to various food allergens Measurement kits for ELISA are already commercially available, for example, as “FASTKIT Elizer Ver.II” series manufactured by Nippon Ham Co., Ltd.
The food allergen test kit of the present invention is a test kit for detecting the presence or absence of a food allergen in a food or measuring the amount thereof, which is added to a food extract. And a solubilizing agent and an antibody specifically recognizing the food allergen, which can be expressed as “a food allergen test kit.” As the thioglycerol and the solubilizer, those packed in a container as a food extract previously dissolved in water or a buffer solution (such as PBS) may be used. The solubilizer used in the test kit is a well-known solubilizer for those skilled in the art, and a plurality of solubilizers may be used in combination. Preferred solubilizers are urea and / or alkyl sulfates (preferred alkyl sulfates include SDS). As an antibody specifically recognizing a food allergen used in the test kit, it is preferable to use two kinds of antibodies, an antibody immobilized on a support and a labeled antibody. In the food allergen test kit of the present invention, as in the case of the above-described commercially available immunochromatography or ELISA kit, a dilution buffer used in the test, an instruction manual, etc. are used as a set. For ELISA, a standard solution, a color developing solution, a stop solution, and the like may be further set. At that time, the thioglycerol and solubilizing agent for the food extract, which are one element of the test kit, may be individually packaged, but are used together in a buffer solution dissolved in a buffer solution. May be. Moreover, you may package packaging bodies, such as a bag and a container with which each element of the test kit was enclosed.

EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.
Other terms and concepts in the present invention are based on the meanings of terms conventionally used in the field, and various techniques used for carrying out the present invention include those that clearly indicate the source. Except for this, it can be easily and reliably carried out by those skilled in the art based on known documents and the like. In addition, various analyzes were performed by applying the methods described in the analytical instruments or reagents used, kit instruction manuals, catalogs, and the like.
In addition, the description content in the technical literature, the patent gazette, and the patent application specification cited in this specification shall be referred to as the description content of the present invention.

(Example 1) Determination of composition of food extract (1-1) Examination of various reducing agents In this example, protein extraction experiments in processed foods were performed using various reducing substances.
Whether dry noodles (ramen) is selected as the processed food to be tested, and whether it can be detected by immunochromatography by applying an extraction method using Mirther to egg proteins that are insoluble in the cooking process Verified. The immunochromatography used is a commercial kit (FASTKIT slim series) manufactured by Nippon Ham.

Specifically, it was performed as follows.
38 mL of various extract solutions shown below were added to 2 g of dry noodles (ramen), cut at high speed for 30 seconds × 3 times with a miller (Iwatani Sangyo Co., Ltd., IFM720G), centrifuged at 3000 × g for 20 minutes, and filtered. To do. Next, 100 μL of each of the obtained extracts was diluted 10-fold with PBS, dropped onto “FASTKIT slim egg manufactured by Nippon Ham Co., Ltd.”, and the presence or absence of a positive line was visually determined after 15 minutes.
The criteria for the results were as follows. (The same applies to the following examples.)
+: Positive ±: Weak positive-: Negative NT: Not implemented

<Extract composition>
・ None 1% urea, PBS
・ Others 1% urea, 0.2% of each reducing agent in Table 1 above in PBS
Addition

As a result, “thioglycerol (HOCH ₂ CH (OH) CH ₂ SH)” was the only compound that showed an egg protein extraction effect comparable to that of 2-ME (Table 1). “Thioglycerol” is structurally similar to 2-ME but has a low irritating odor and is not designated as a government-designated poison. It was.

(1-2) Examination of reducing agent concentration Next, using a thioglycerol-containing extract (1% urea, PBS), under the same conditions as in the above-mentioned experiment (1-1), Egg protein was extracted and the optimal thioglycerol concentration as an extract was examined.

  As a result, it is difficult to mix 0.01 to 10.0%, preferably 0.01 to 5.0%, more preferably 0.02 to 1.0% of thioglycerol in the food extract. It was confirmed that soluble allergen proteins can be extracted with high sensitivity. Thus, the ability to extract efficiently over a wide concentration range by using thioglycerol as a reducing agent means that there is a greater possibility that a wider range of samples can be examined than the conventional method. High extraction efficiency can be achieved even at a low concentration of 02%. Even if the toxicity is very low compared to 2-Me, it is possible to suppress the use of thioglycerol, which is known to cause eye irritation, as much as possible. This means that there is a big merit from the viewpoint of the safety of the engineers who handle it.

(Example 2) Detection sensitivity of egg protein from various processed foods (2-1) Comparison of sensitivity with various extracts and extraction methods Detection sensitivity from foods that are difficult to detect by conventional methods among various processed foods Verified. At the same time, comparison with the extracts of Patent Documents 7 and 8 was also made.
As for the specific procedure, in the extraction method using Milcer (manufactured by Iwatani Corporation), as in Example 1, 38 mL of the following various extracts were added to 2 g of the target food, and 30 seconds × 3 with Milser. High-speed cutting, and centrifugation at 3000 × g for 20 minutes, followed by filtration. On the other hand, in the case of “boiling extraction method”, 19 mL of the following extract is added to 1 g of the target food, and after boiling for 10 minutes (method of Patent Document 7) or 60 minutes (method of Patent Document 8), 3000 × g Centrifuge for 20 minutes and filter.
Each obtained extract was diluted 10 times with PBS to obtain a measurement solution.
As in Example 1, 100 μL of the measurement solution was dropped onto “Nippon Ham Co., Ltd. FASTKIT slim egg”, and the presence or absence of a positive line was visually determined after 15 minutes (the determination criteria are the same as in Example 1). .

<Extract composition>
-This extract 0.05% thioglycerol, 1% urea, PBS
・ Control 1% urea, PBS
-Extract of patent document 7 0.5% SDS, 0.1M sodium sulfite,
PBS
-Extract of patent document 8 0.25% SDS, 2% urea,
0.2% cysteine, PBS

As a result, when thioglycerol was selected as the reducing agent, especially when Myrcer extraction was performed, the egg protein in a poorly soluble state was successfully extracted from processed food, which had been considered difficult in the past.
The thioglycerol concentration in this extract was increased to 0.5% and this experiment was followed up with the same results. (Data not shown)

(2-2) Detection of egg protein from various processed foods A measurement solution obtained by diluting a thioglycerol-containing extract derived from various processed foods prepared in the same manner as in (2-1) 10 times with PBS, and as required Accordingly, using a measurement solution further diluted serially with PBS, the determination was made by “FASTKIT slim egg manufactured by Nippon Ham Co., Ltd.” in the same manner as in (2-1).

<Extract composition>
-This extract 0.05% thioglycerol, 1% urea, PBS
・ Control 1% urea, PBS

  As a result, compared to the control, the detection sensitivity of egg protein in commercial processed foods using this extract was significantly increased, demonstrating that egg protein can be detected from any processed food with high sensitivity. .

(Example 3) Examination of solubilizing agent In this example, in order to show that the same excellent extraction results can be obtained even if a general solubilizing agent other than urea is applied as the solubilizing agent. At the same time when urea (1%) was used as a solubilizer, an experiment was conducted in which SDS (sodium dodecyl sulfate) was used as a solubilizer instead of urea. For comparison, a control experiment in which no solubilizer was used was also conducted.
<Extract solution composition>
-Extract 1-0.05% thioglycerol, 1% urea, PBS
-Extract-2-0.05% thioglycerol, 0.05% SDS,
PBS
Control 0.05% thioglycerol, PBS
Specifically, as in Example 2, 38 mL of the following various extracts were added to 2 g of the target food, cut at high speed for 30 seconds × 3 times with a miller, and centrifuged after centrifugation at 3000 × g for 20 minutes. Then, the obtained extract was diluted 10 times with PBS to obtain a measurement solution.
The measurement method was the same as in Examples 1 and 2, in which 100 μL of the measurement solution was dropped onto “Nippon Ham Co., Ltd. FASTKIT Slim Egg”, and the presence or absence of a positive line was visually determined after 15 minutes (the determination criteria are also examples). Same as 1.)

  As a result, the solubilizing agent used in combination with the reducing agent thioglycerol is not limited to urea, and even when SDS is used, the extraction rate is improved, and egg proteins can be detected with high sensitivity even from commercially processed foods that are difficult to detect. I was able to confirm that I could do it.

(Example 4) Detection experiment for various food allergens This example is for verifying whether this extract containing thioglycerol is effective in detecting various food allergens other than egg protein.
As the target processed foods, model processed foods to which various food allergens (egg protein, milk protein, wheat protein, buckwheat protein, peanut protein, crustacean protein and soy protein) were added in an amount of 10 ppm each were prepared. Specific food names are kamaboko, sausage, sweet potato, and chicken dumpling. It was verified whether each allergen protein added to each food could be extracted. At that time, the same measurement sample as that in the immunochromatography method was additionally tested in the ELISA method, and the accuracy of the immunochromatography method was verified.
The specific procedure for preparing the food extract is the same as the above (Example 1), 38 mL of the following various extracts are added to 2 g of the target food, and high-speed cutting is performed 30 seconds × 3 times with a miller. After centrifugation at 3000 × g for 20 minutes, filtration was performed. Subsequently, it diluted 10 times with PBS and was set as the measurement solution.

  The measurement by immunochromatography uses the same “FASTKIT slim egg manufactured by Nippon Ham Co., Ltd.” as in Example 1 for egg protein, and “FASTKIT slim milk manufactured by Nippon Ham Co., Ltd.” for wheat protein and wheat protein. “Nippon Ham Co., Ltd. FASTKIT Slim Wheat”, for buckwheat protein, “Nippon Ham Co., Ltd. FASTKIT Slim Soba”, for peanut protein, “Nippon Ham Co., Ltd. FASTKIT Slim Peanut” About protein, it determined using "prototype kit" and soybean protein using "Nippon Ham Co., Ltd. FASTKIT slim soybean." The specific procedure was the same as in Example 1, in which 100 μL of the measurement solution was dropped into each kit, and the presence or absence of a positive line was visually determined after 15 minutes (the determination criteria are the same as in Example 1).

<Extract solution composition>
-This extract 0.05% thioglycerol, 1% urea, PBS
・ Control 1% urea, PBS

  By using this extract, various food allergens (eggs, milk, wheat, buckwheat, peanuts, shellfish, soybeans) in various forms of processed foods could be detected with a high detection rate.

(Example 5) Measurement by ELISA In this example, when thioglycerol was used as a reducing agent and extracted in combination with a solubilizing agent, conventional 2-mercaptoethanol (2-ME) having high toxicity was used. An experiment was conducted to confirm that the performance is inferior to that of the case by the ELISA method. For comparison, a control experiment in which no reducing agent was used was also performed.
<Extract composition>
-This extract 0.05% thioglycerol, 0.5% SDS,
PBS
-Extract containing 2-ME 2.0% 2-ME, 0.5% SDS, PBS
Control 0.5% SDS, PBS
Specifically, 2 g of the pulverized target food was mixed with 38 ml of the above extract in a centrifuge tube and mixed overnight (about 16 hours) at room temperature (about 20 ° C.) in a shaker (manufactured by Tokyo Rika Kikai Co., Ltd.). Shake with. The “shaking” process was performed under the conditions of “90 to 110 rpm, one reciprocation is one rotation, and 90 to 110 reciprocations per minute”. Next, the mixture was centrifuged at 3000 × g for 20 minutes, filtered, and diluted 10 times with PBS to obtain a measurement solution.
The measuring method measured the egg protein by ELISA method using "Nippon Ham Co., Ltd. FASTKIT Elizer Ver.II egg" as follows.
In each well of the microtiter plate, 100 μL of diluted standard solution and measurement solution were added and allowed to stand at room temperature (20 to 25 ° C.) for 1 hour. The solution in the well was discarded and the well was washed 5 times. 100 μL of the prepared biotin-conjugated antibody solution was added to each well and left at room temperature for 1 hour. The well was washed 5 times, 100 μL of enzyme-streptavidin conjugate solution was added, and allowed to stand at room temperature for 30 minutes. After each well was washed 5 times, 100 μL of color former was added and allowed to stand at room temperature (20-25 ° C.) for 20 minutes. 100 μL of a reaction stop solution was added to each well to stop color development, and the absorbance at a main wavelength of 450 nm and a sub wavelength of 600 to 650 nm was measured with a plate reader.

  As a result, even when thioglycerol was used as a reducing agent and extracted overnight by shaking, egg protein was detected from commercial processed foods that were difficult to detect, with the same sensitivity as when the toxic mercaptoethanol (2-Me) was used. It was confirmed that it could be detected. Moreover, it is a great merit that a sufficient extraction effect can be obtained even at a concentration of 1/40 compared to 2-Me.

Claims (7)

  A food extract containing a reducing agent and a solubilizing agent, wherein thioglycerol is used as the reducing agent, and the food extract for extracting food allergen in food.   The food extract according to claim 1, wherein the solubilizer is urea and / or an alkyl sulfate.   The food extract according to claim 1 or 2, wherein the thioglycerol concentration in the extract is 0.001 to 10 w / v% with respect to the total amount of the extract.   The food extract according to any one of claims 1 to 3, wherein the food allergen is an allergen protein that is difficult to extract in a heat-cooked food.   A method for extracting a food allergen from food, wherein a food extract containing thioglycerol and a solubilizing agent is used in the method for extracting a food allergen from food.   A food allergen extracted from a food using the food extract according to any one of claims 1 to 4, wherein the presence or absence of the food allergen is detected by an immunoassay or the amount of the food allergen is measured. Allergen testing method.   A test kit for detecting the presence or presence of a food allergen in food or measuring the abundance thereof, wherein the food allergen is specifically added together with thioglycerol and a solubilizer to be added to a food extract. A food allergen test kit comprising an antibody to be recognized.