JP2015078875A - Allergen measurement method in food product - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring an allergen amount existing in a food product, especially a powder state food product in a dry state, and confirm that the food product is safe by measuring the allergen easily.SOLUTION: An allergen simple measurement kid is used for measuring an allergen amount included in a food product, especially a powder state food product in a dry state such as wheat flour, a powder for thin cake with various ingredients, and a powder for tempura. In addition for quality management in a food factory, measurement whether there is allergen or not in food products is simply performed.

Description

The present invention relates to a method for easily measuring the amount of allergen contained in a food, particularly a food such as flour in a dry state.

Allergic diseases such as asthma, atopic dermatitis, and allergic rhinitis are plagued by many people, and in recent years, the tendency has been increasing. The causes of these allergic diseases are various allergens present in the environment. Among them, mites, pet animal dander, pollen, and mold that inhabit indoors are typical inhalable allergens. Well known. In particular, leopard mites, which are dust mites that live in the house, are a major problem as a source of allergens. Leopard mites are a hotbed for growing textile products in the house such as tatami mats, carpets, bedding, curtains, etc., or seat sheet fabrics of moving vehicles such as trains and cars outdoors.

Among the leopard mites, Dermatophagoides farinae and Dermatophagoides pteronysinsinus are typical species, and the dead bodies and feces of these mites are strong allergen substances.

As mentioned above, these ticks breed in the environment such as indoors, but ticks live everywhere as long as food exists, so if they are mixed with stored food, they explode. Potentially proliferate. There is a risk of serious health damage if foods contaminated with ticks are eaten without knowing that they are contaminated.

For example, recently, ticks are mixed in unsealed okonomiyaki that has been opened and stored, and ticks grow over time, resulting in okonomiyaki being contaminated with a large amount of mite allergens. An accident has been reported that causes anaphylactic shock because of eating. For example, at the 62nd Annual Meeting of the Japanese Society of Allergology, a case was reported in which an immediate allergic attack was caused by dust mites that had propagated on okonomiyaki powder. In this report, 1 g of okonomiyaki powder contains mite allergens Der1 and Der2. A large amount is detected.

In this way, when mixed with dry powdered foods such as wheat flour, okonomiyaki flour, and tempura flour, ticks are in a very suitable environment for tick breeding because they are rich in nutrients. There is a possibility of breeding and multiplying explosively. When a large amount of ticks are propagated in such a powdered food, both Der 2 which is an allergen derived from mite insect bodies and allergen Der 1 which is derived from mite feces are mixed in the food in a large amount. Cooking and eating such contaminated foods can cause serious health problems. Therefore, if it is possible to easily measure the propagation of ticks due to mite contamination in foods, the risk of health damage can be reduced.

In addition, even in powdered food manufacturing factories such as wheat flour, okonomiyaki flour, and tempura flour, it cannot be said that there is no risk of mite contamination, and that there is no contamination with mites. Can be included as one of the quality control tests. When such quality control is performed, the purpose is to confirm that mite allergen is not mixed, so a simple mite allergen measurement method is used rather than using an enzyme immunoassay method that requires complicated operations. It is easier to use.

A method using a simple measurement kit for measuring the amount of allergen in the environment has been known so far, and several methods are known. However, it has not been done to measure the amount of allergen in food using a simple measurement kit and manage the amount of allergen.

JP 2001-305134 A proposes a kit for simply measuring the amount of mite allergen in the environment using an antigen-antibody reaction with a monoclonal antibody. There has been proposed a method for measuring mite allergens using this kit by collecting samples from carpets, tatami mats, bedding, curtains, stuffed animals, flooring, sofas, cushions, car seats, and the like. Japanese Patent Application Laid-Open No. 2006-345801 proposes a method for measuring allergens in the environment using protease activity of allergens, and Japanese Patent Application Laid-Open No. 08-285841 discloses aromatic diazo compounds in house dusts. There has been proposed a method of detecting by a color reaction. However, there is no description about measuring allergens in foods using these allergen measuring methods. On the other hand, Japanese Patent Application Laid-Open No. 2013-33062 proposes a method for measuring a specific component in food using an ELISA method using an antibody, but it has not been performed for measuring allergens.

JP 2001-305134 A JP 2006-345801 A JP 2013-33062 A

It is an object of the present invention to provide a method for easily measuring or analyzing the amount of allergens in order to determine whether foods, particularly powdered foods in a dry state, are contaminated with allergens due to reproduction of mites, etc. It is.

As a result of diligent research to solve such problems, the present inventors have used a simple measurement kit for measuring allergens present in the environment, and used foods, in particular, powders in a dry state. It has been found that the amount of allergen in food can be measured.

That is, the present invention is (1) an allergen measurement method for measuring the amount of allergen present in food using a simple measurement kit, and (2) an allergen measurement method in which the food is in a powder state in a dry state. (3) An allergen measuring method in which the allergen is a mite allergen, and (4) the allergen measuring method is an allergen measuring method using an enzyme immunoassay using a monoclonal antibody against the allergen.

By using the test method of the present invention, it is possible to easily measure allergens in foods, and it is possible to easily confirm whether or not allergens that are harmful to products in food factories are mixed, Quality management can be easily performed.

The food to be measured for allergen is not particularly limited, but powdered food in a dry state is suitable. Such powdery food materials include wheat flour, rice flour, oat flour, barley flour, rye flour, corn flour, corn grits, straw flour, bread crumbs, starch, corn flour, mung bean flour, soy flour, potato flour, cassava Examples thereof include powdery food materials such as powder, and a premix obtained by mixing two or more of these may be used. Further, it may be a powder product prepared by blending auxiliary materials such as sugars, salts, starches, emulsifiers, enzyme agents and the like as necessary. Examples of such flour products include wheat flour mix products such as dried fried powder, okonomiyaki flour, tempura flour, takoyaki flour, bakery mix, and confectionery mix.

As flour, it is a thing normally used for foodstuffs, for example, a strong flour, a semi-strong flour, a medium flour, and a thin flour, and a mixture thereof may be used. Although it does not specifically limit as starch, For example, corn starch, wheat starch, rice starch, potato starch, tapioca starch, sweet potato starch, sago palm starch, etc. are mentioned, These starches may be individual or mixed. Processed starch such as roasted dextrin, highly branched cyclic dextrin, enzyme-modified starch, acid-degraded starch, oxidized starch, esterified starch, etherified starch, cross-linked starch, pregelatinized starch or wet heat-treated starch, etc. It is also possible to use it.

Examples of premixes include fried powder, tempura powder, okonomiyaki powder, bakery mix, hot cake mix, etc. For example, a mix for steamed products such as white sauce and a sauce mix for producing sauces such as white sauce.

In these powdered foods, sugars, seasonings, spices, pigments, sweeteners, thickeners, fats and oils, swelling agents, emulsifiers, fragrances, vitamins, etc. may be used as various additives. There is no problem.

Examples of the saccharide include monosaccharides, disaccharides, oligosaccharides, dextrins, sugar alcohols, and the like. Seasonings include inorganic salts such as sodium chloride, potassium chloride, and tripotassium phosphate, sodium L-aspartate, and DL-alanine. , Sodium glutamate, disodium 5'-inosinate, calcium citrate, trisodium citrate, soy sauce, fish sauce, vinegar, sauce, amino acid, chemical seasoning, natural extract, etc. , Musk, Chen skin, yam, pepper, pepper, garlic, ginger, onion, green onion, etc., as the pigment, paprika pigment, gardenia yellow pigment, gardenia red pigment, gardenia blue pigment, red pepper pigment, spirulina pigment, caramel pigment, Examples include cacao dyes, bemoji dyes, and rack dyes.

Sweeteners include dextrin, oligosaccharide, sugar, xylose, glucose, sugar alcohol, reduced syrup, sodium saccharin, xylitol, aspartame, sucralose, sorbitol, stevia, etc., and thickeners include gelatin, gellan gum, carrageenan, Examples include xanthan gum, locust bean gum, glucomannan, agar, guar gum, alginic acid, sodium alginate, tamarind gum, tara gum, gum arabic, tragacanth gum, caraya gum, pullulan, carboxymethylcellulose, microfibrous cellulose, and far celerin.

Regarding fats and oils, examples of vegetable oils include rice bran oil, rice germ oil, palm oil, soybean oil, rapeseed oil, corn oil, cottonseed oil, sunflower oil, coconut oil, perilla oil, etc. Examples thereof include beef tallow, pork tallow, fish oil, milk fat, and the like, and it is also possible to use oils and the like that have been subjected to processing such as partial hydrogenation, hydrogenation fractionation, fractionation, and transesterification.

Further, examples of the expanding agent include sodium hydrogen carbonate, ammonium hydrogen carbonate, ammonium carbonate, DL-potassium hydrogen tartrate, potassium L-tartrate, adipic acid, L-ascorbic acid, ammonium chloride, baking powder, etc. Fatty acid esters, sucrose fatty acid esters, and the like are listed. Examples of the flavor include ethyl acetoacetate, acetophenone, and natural flavors. Examples of vitamins include L-ascorbic acid, vitamin E, and β-carotene.

Regarding allergen measurement, it is usually difficult for ordinary people who have no knowledge of allergen measurement by enzyme immunoassay to easily measure allergen levels in foods, and this can only be done using a commercially available simple measurement kit. it can. Even in a factory that manufactures foods, a simple allergen measurement kit can be used as a quality control method for confirming that allergens are not mixed in foods as products. The simple measurement kit for allergen refers to a tool that is sold as a tool that can easily measure allergen using the principle of enzyme immune reaction or the like.

The type of allergen to be measured is not particularly limited, but mite allergen, cedar pollen allergen, cat allergen, dog allergen, cavia allergen, cockroach allergen, and the like can be targeted. In the case of mite allergens, common allergens are often caused by mite mites and mite mites, and examples of allergens include Der f1 derived from feces of mite mites and Der f2 derived from insect bodies of mite mites. Can be mentioned. A typical example of the pollen allergen is a cedar pollen allergen, and Cry j1 mainly present in the outer layer of the cedar pollen is mentioned as an allergen protein. In addition, cypress, mugwort, ragweed, hurghaya, camo, etc. are mentioned as plant species from which pollen causing allergens is derived. Sources other than mite allergens and pollen allergens include molds, dog and cat dander, insects such as cockroaches and chironomids. For example, these allergen proteins include Blag1 derived from cockroaches, Can f1 derived from dog dandruff, Fel d1 derived from cat dandruff, Alt a1 derived from mold, and the like. Of these allergens, mite allergens for which simple measurement kits are generally sold are preferred.

There are several commercially available allergen simple measurement kits for measuring the amount of allergen in food, such as Mighty Checker (registered trademark) (manufactured by Sumika Enviro Science Co., Ltd.), Taniscan (registered trademark). (Asahi Food and Healthcare Co., Ltd.) can be used.

The measurement principle of these allergen simple measurement kits is that using a monoclonal antibody utilizing an antigen-antibody reaction, but a method utilizing other principles may be used. Examples of such a simple allergen measurement kit include a method of detecting with a coloring reagent or the like by utilizing the properties of allergen as a protein or peptide.

At present, simple measurement kits are not widely used for allergens other than mite allergens, but if simple measurement kits are developed in the future, such products can be applied to the measurement of allergens in foods. .

In order to measure allergens in food, it is necessary to extract allergens, and an extract can be used. For this extract, a buffer having a neutral pH range can be used. As the buffer, phosphates, salts of organic acids such as citric acid, tartaric acid, and acetic acid can be used.

Next, after allergen is recovered using a buffer solution, the amount of allergen in the allergen extract is measured. Although the measurement principle of allergen is not particularly limited, a method using a commercially available allergen simple measurement kit utilizing an enzyme immunization method (ELISA method) or the like as described above can be considered. The ELISA method is a method for measuring an antigen using an antigen-antibody reaction, and corresponds to an allergen measured by the antigen in the present invention.

When measuring allergens in foods, especially powdered foods, foods are likely to contain various additives such as high concentrations of protein, fat and sugar, so calibration for allergen measurement In order to prepare a line, it is preferable to prepare these foods to which a known amount of measurement allergen is added.

The amount of allergen added to food for preparing a calibration curve depends on the specifications of the simplified measurement kit for allergen used, but the protein amount is 0.1 to 100 μg / mL as a crude allergen in the allergen aqueous solution, preferably 1 ˜25 μg / mL.

The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. In addition, all% shown below are weight%.

The following powdered commercial products were used as food samples.
Thin wheat flour (Nisshin Flour Milling Co., Ltd.)
Okonomiyaki (Nisshin Foods Co., Ltd.)
Tempura flour (Nisshin Foods Co., Ltd.)
Fried powder from Nissin Foods Co., Ltd.

Mite Extract-Df (manufactured by LSL) as a standard allergen was dissolved in a phosphate buffer of pH 7.2, and a mite allergen solution was prepared so that the Der f2 concentration was 2.0 μg / mL as the amount of crude allergen protein. The allergen level of the mite allergen solution was measured using a mighty checker (manufactured by Sumika Enviro Science Co., Ltd.) as a mite allergen simple measurement kit. The criteria shown in Table 1 were applied and confirmed to be +.

1 g of a food sample was placed in a 15 mL plastic tube, and 10 mL of mite allergen solution was added and stirred thoroughly. Next, centrifugation was performed at 12000 rpm for 5 minutes using a centrifuge, and the supernatant produced in the plastic tube was used as an analysis sample. In addition, a control analysis sample was prepared by performing the same operation using a phosphate buffer solution having a pH of 7.2 instead of the mite allergen solution.

Mite allergen measurement (measurement using a simple measurement kit)
The mighty checker was immersed in the analysis sample for 3 seconds, and after 10 minutes, the degree of color development was evaluated.

Mite allergen measurement by ELISA (enzyme immunoassay) Enzyme immunoassay (Der f2 sandwich ELISA)
First, Der f2 monoclonal antibody 15E11 diluted to 2 μg / mL with a phosphate buffer (pH 7.4, containing 0.1 wt% NaN ₃ ) was added per well of F16 MAXISORP NUNC-IMMUNO MODULE plate (manufactured by NUNC). 200 μL each was added and sensitized at 4 ° C. for 3 days or more. After sensitization, the solution was discarded, and a blocking reagent {1% by weight bovine serum albumin + phosphate buffer (pH 7.2, containing 0.1% by weight NaN ₃ )} was added in an amount of 200 μL per well. Reacted for 1 minute. After the reaction, the plate was washed with a phosphate buffer (pH 7.2, containing 0.1 wt% polyoxyethylene (20) sorbitan monolaurate).

Next, 100 μL of mite allergen measurement sample solution extracted from the food sample was dropped into one well and reacted at 37 ° C. for 60 minutes. After the reaction, the plate was washed with a phosphate buffer (pH 7.2, containing 0.1 wt% polyoxyethylene (20) sorbitan monolaurate). The peroxidase-labeled Der f2 monoclonal antibody was dissolved in distilled water at 200 μg / mL, and was dissolved in phosphate buffer {pH 7.2, 1% by weight bovine serum albumin and 0.1% by weight polyoxyethylene (20) sorbitan monolaur. A solution diluted 1000 times with a rate containing} was added at 100 μL per well. After reacting at 37 ° C. for 60 minutes, the plate was washed with a phosphate buffer (pH 7.2, containing 0.1 wt% polyoxyethylene (20) sorbitan monolaurate) and then with distilled water. Add 100 μL per well of 13 mol of 0.1 mol / L phosphate buffer (pH 6.2) plus ortho-phenylenediamine dihydrochloride (manufactured by SIGMA CHEMICAL CO .: 26 mg Table) and 13 μL of hydrogen peroxide. And allowed to react at 37 ° C. for 5 minutes. Immediately thereafter, 50 μL of ₂ mol / L H ₂ SO ₄ was added to stop the reaction, and the absorbance (OD 490 nm) was measured with a spectrophotometer for microplate (Tecan Japan Co., Ltd.). The Der f2 concentration of the measurement sample was determined from a calibration curve similarly measured using a Der f2 aqueous solution having a known concentration.

Table 2 shows the allergen concentration measurement results by the simple measurement kit and the ELISA method. The measurement result by the simple measurement kit showed a good correlation with the measurement result by the ELISA method, and the amount of allergen in the food sample could be easily measured.

According to the present invention, it is possible to easily measure the amount of allergen in a food, particularly a powdered food in a dry state, and it is possible to easily check for allergen contamination in a food factory.

Claims (4)

  A method for measuring allergen, characterized by measuring the amount of allergen present in food using a simple measurement kit   The method for measuring allergen according to claim 1, wherein the food is in a powder form in a dry state. The allergen measuring method according to claim 1 or 2, wherein the allergen is a mite allergen. The allergen measuring method according to any one of claims 1 to 3, wherein the allergen measuring method utilizes an enzyme immunoassay using a monoclonal antibody against the allergen.