Food Allergen Composition Reference Materials

Abstract

To provide a dietary allergen composition standard substance that contains two or more proteins extracted from the food causing the food allergy and has excellent storage stability and bottle-to-bottle homogeneity.SOLUTION: A dietary allergen composition standard substance for use in a mass spectrometer is characterized that it contains proteins extracted from a food allergenic food by using a phase-transfer solubilizer (PTS) and not acetone precipitated and the PTS and it contains two or more types of proteins. It may be dispensed and stored in containers in volumes of 10 to 1000 micro L.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a food allergen composition standard for use in mass spectrometry. [Background technology]

[0002] Regarding food allergen analysis methods, the Consumer Affairs Agency has designated ELISA as a quantitative testing method, and PCR and Western blotting as qualitative testing methods. However, the ELISA method has the potential to produce false positive or false negative results, commercially available kits are only available for specific raw materials and a few other items, and each test is limited to one allergen. Therefore, there is a need to establish a method that can quickly and accurately test for multiple allergens contained in food.

[0003] Meanwhile, allergen analysis using mass spectrometry has attracted attention, with SCIEX proposing a method using a liquid chromatography mass spectrometer (LC-MS / MS) that has received AOAC First Action Official Method classification. Compared to conventional analytical methods, this method offers advantages such as high selectivity, high sensitivity, and the ability to simultaneously analyze multiple allergens. Use of this analytical method, for example, makes it possible to distinguish between barley, which would give a false positive using ELISA, significantly reduces processing time (in principle, it would be possible to analyze specified allergens and similar substances at the same time), and enables the early detection of unintentional contamination in foods from which specific proteins, such as food additives, have been removed. Proteins (such as casein) contained in specific raw materials are commercially available as standard products, but this analysis method is not widely used in Japan because there are few types in general circulation and the analysis method has not been established.

[0004] The Notification of the Deputy Director General of the Consumer Affairs Agency, March 30, 2015, No. 139 "Regarding Food Labeling Standards" (hereinafter referred to as the Notification Act) describes the preparation method of a standard sample for the ELISA method. However, this standard sample contains a surfactant, and if a sample containing this surfactant is subjected to a mass spectrometer, it may have adverse effects such as damaging the mass spectrometer, so it is recommended that it not be used in the above analysis method.

[0005] Furthermore, the notification law stipulates that it is desirable for the reference standard to contain "total protein." Here, "total protein" refers to all proteins contained in milk, not a specific protein such as casein, for example. The type of each protein constituting the total protein contained in the reference standard is specified for each specific raw material. For example, when a milk reference standard is subjected to electrophoresis, the notification law specifies three bands in the range of 40 to 23 kDa (presumed to be αs1-casein, αs2-casein, and β-casein) and one band around 16 kDa (presumed to be β-lactoglobulin). Since casein and β-lactoglobulin together account for approximately 77% of the total protein, the reference standard contains a variety of proteins that make up approximately 77% of the total protein.

[0006] In contrast, the standards for mass spectrometers that have been commercially available to date have been very simplified, containing only one type of protein, and cannot be said to contain multiple types of proteins like total protein, and therefore are not standards that comply with the notification method.

[0007] Therefore, at the 25th Academic Conference of the Japanese Society of Food Chemistry (held on June 6th and 7th, 2019), the present inventors attempted to prepare standard solutions by extracting proteins from standard samples such as commercially available milk, eggs, wheat, peanuts, buckwheat, shrimp, and soybeans using PTS (phase transfer solubilizer), which has excellent protein solubilizing ability, and then performing acetone precipitation and ultrafiltration (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Abstracts of the 25th General Meeting and Academic Conference of the Japanese Society of Food Chemistry, Eri Inagaki, Yoshiko Yamashita, and Ushio Tateda, "Development of a simultaneous analysis method for food allergens using LC-MS / MS," p. 95 (published June 6, 2019, by the Japanese Society of Food Chemistry) Summary of the Invention [Problem to be solved by the invention]

[0009] However, in the method described in Non-Patent Document 1, not only does it take a long time to redissolve the protein after acetone precipitation, but some precipitates cannot be dissolved, which suggests that the number of proteins in the acetone precipitated sample may be reduced compared to the total protein content. Furthermore, the completed standard solution was highly viscous due to concentration, and did not fully satisfy the storage stability and bottle-to-bottle homogeneity required for a standard product.

[0010] Therefore, the object of the present invention is to provide a food allergen composition reference material that contains two or more types of proteins extracted from foods that cause food allergies and has excellent storage stability and bottle-to-bottle uniformity. [Means for solving the problem]

[0011] As a result of extensive efforts to solve the above-mentioned problems, the inventors discovered that a food allergen composition standard that satisfies excellent storage stability and bottle-to-bottle uniformity can be produced by extracting proteins from foods that cause food allergies without acetone precipitation and using a composition containing these proteins, thereby completing the present invention.

[0012] The gist of the present invention is [1] A food allergen composition standard substance to be subjected to a mass spectrometer, comprising: a food allergen composition standard material, which contains proteins extracted from food causing food allergies using a phase transfer solubilizer (PTS) and not subjected to acetone precipitation, and the PTS, and is characterized in that the proteins are of two or more types; [2] The food allergen composition reference material according to [1], which is dispensed and stored in a container in a volume of 10 to 1000 μL. [3] The food allergen composition reference material according to [1] or [2], wherein the food causing the food allergy is one or more selected from wheat, eggs, milk, peanuts, buckwheat, shellfish, and soybeans. [4] At least one of the proteins Food The food allergen composition reference material according to any one of [1] to [3] above, which is an allergenic protein; Regarding. [Effects of the Invention]

[0013] The food allergen composition standard material of the present invention contains two or more types of proteins extracted from foods that cause food allergies, and the proteins constitute the total protein of the foods that cause food allergies.In addition, it has excellent storage stability and bottle-to-bottle homogeneity, and therefore can be suitably used as a standard material for mass spectrometers. [Brief explanation of the drawings]

[0014]

Figure 1

Figure 2

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Figure 4

Embodiments for Carrying Out the Invention

[0015] Hereinafter, specific embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be implemented with appropriate modifications within the scope of the object of the present invention. Note that, for parts where the description is repetitive, the description may be omitted as appropriate, but the present invention is not limited thereby.

[0016] In the present invention, a reference material refers to a substance that is sufficiently homogeneous and stable with respect to one or more specified characteristics and is prepared to be suitable for the purpose of use in the measurement process, as described in Japanese Industrial Standard JIS Q 0030:2019. In addition, a food allergen composition reference material refers to a reference material that can characterize the causative foods of food allergies, and refers to a mixture (including total protein) containing food allergenic proteins and other proteins extracted from the causative foods of food allergies. The food allergen composition reference material also refers to a mixed food allergen composition reference material in which a plurality of food allergen composition reference materials extracted from different causative foods of food allergies, such as wheat and eggs, are mixed. The fact that there are two or more types of proteins means that there are two or more types of proteins extracted from each causative food, for example, two or more types in wheat and two or more types in eggs.

[0017] The food allergen composition reference material of the present invention is for use in a mass spectrometer. Examples of the mass spectrometer include a liquid chromatography mass spectrometer (LC-MS / MS), a time-of-flight mass spectrometer (TOF / MS), and the like. For example, the liquid chromatograph mass spectrometer (LC-MS / MS) is a type of high-performance liquid chromatograph (HPLC) that separates components in a liquid using the difference in the interaction between the stationary phase and the mobile phase and detects them with a mass detector. The mass detector is connected to the LC-MS / MS to fragment and detect only specific masses. A time-of-flight mass spectrometer (TOF / MS) is an analyzer that measures the mass of an object by measuring the time of flight of accelerated charged particles (ions or electrons). The mass spectrometer may be a commercially available product, and there is no particular limitation on the type.

[0018] The food allergen composition standard of the present invention is a composition containing a protein extracted from a food causing food allergy using a phase transfer solubilizer (PTS) and not subjected to acetone precipitation, and the PTS.

[0019] The food causing the food allergy is a food that causes food allergies, and for example, seven items, namely wheat, eggs, milk, peanuts, buckwheat, shrimp, and crab, are listed as foods that are required to be labeled in Japan. In addition, 21 items are listed as foods that are equivalent to the above-mentioned specified ingredients: abalone, squid, salmon roe, oranges, cashew nuts, kiwi fruit, beef, walnuts, sesame, salmon, mackerel, soybeans, chicken, bananas, pork, matsutake mushrooms, peaches, yams, apples, gelatin, and almonds. In addition to the above, other fruits include cherries, melons, mangoes, etc.

[0020] In the present invention, the proteins extracted from the food causing the food allergy using PTS and not subjected to acetone precipitation refer to proteins that constitute the total protein derived from the food causing the food allergy. The total protein refers to all proteins contained in milk, rather than specific proteins such as casein and whey protein in the case of milk, for example. The total protein may be, for example, a protein obtained by the method described in the notification method.

[0021] The food allergen composition reference substance of the present invention contains two or more of the proteins that make up the total protein. For example, as the proteins that make up milk protein, according to P. Walstra & R. Jenness, Dairy Chemistry and Physics, it is about 80% casein and about 19% whey protein. And the casein is divided into αs1-casein 30.6%, αs2-casein 8.0%, β-casein 28.4%, γ-casein 2.4%, κ-casein 10.1%. The whey protein is divided into serum albumin 1.2%, β-lactoglobulin 9.8%, α-lactalbumin 3.7%, immunoglobulin 2.1%, and other whey proteins 2.4%. Therefore, examples of the food allergen composition reference substance derived from milk include those containing two or more of the proteins that make up the casein and the whey protein. Among them, it is preferable to contain casein, β-lactoglobulin, and α-lactalbumin.

[0022] In addition, from the viewpoint of the effectiveness as a reference substance, at least one of the proteins contained in the food allergen composition reference substance of the present invention is Food preferably an allergenic protein. The Food allergenic protein refers to a protein that causes an allergic reaction when ingested. The Food allergenic proteins are registered, for example, in the database of WHO-IUIS (World Health organization / International Union of Immunological Society).

[0023] For example, wheat-derived food allergen composition reference materials include those containing gliadin, glutenin, etc. Gliadin and glutenin account for approximately 85% of the total wheat protein (they are present in roughly equal amounts), and are considered to be the major allergens in wheat.

[0024] Examples of egg-derived food allergen composition standards include those containing two or more of ovalbumin, ovotransferrin, ovomucoid, lysozyme, etc. Ovalbumin, ovotransferrin, ovomucoid, and lysozyme are allergens found in egg white, with egg white proteins (but not total egg proteins) accounting for approximately 54% ovalbumin, 12% ovotransferrin, 11% ovomucoid, and 3% lysozyme. The major allergens are ovalbumin and ovomucoid.

[0025] Milk-derived food allergen composition standards include those containing two or more of casein, β-lactoglobulin, and α-lactalbumin. Total milk protein comprises approximately 80% casein, 10% beta-lactoglobulin, and 4% alpha-lactalbumin. The major allergens are casein and beta-lactoglobulin.

[0026] Examples of peanut-derived food allergen composition standards include those containing globulin, albumin, etc. Globulins account for approximately 87% of the total protein in peanuts, and both globulins and albumin are considered to be major allergens.

[0027] Examples of buckwheat-derived food allergen composition reference materials include those containing two or more of globulin, albumin, glutenin, etc. Buckwheat contains approximately 45-40% globulin, 25-20% albumin, and 10-13% glutenin in total protein. The main allergens are globulin and albumin.

[0028] Crustacean-derived food allergen composition standards include those containing two or more of tropomyosin, arginine kinase, myosin, actin, etc. Myosin and actin are proteins found in large amounts in crustaceans, with tropomyosin being the major allergen.

[0029] Soybean-derived food allergen composition standards include those containing two or more of globulin, albumin, hydrophobic seed protein (LTP), etc. Approximately 80% of the total protein in soybeans is globulin. Major allergens include globulin, albumin, and LTP.

[0030] Furthermore, it is preferable that the proteins contained in the food allergen composition standard material of the present invention account for 50% or more of the proteins constituting the total proteins derived from the food causing the food allergy. For example, if the milk-derived food allergen composition standard material contains the αs1-casein (approximately 31%) and β-casein (approximately 28%), it will contain types of proteins that account for 50% or more of the total protein content derived from foods that cause food allergies. The type of the protein can be confirmed by electrophoresis as described below.

[0031] In addition, in the case of known standard products, for example, in the case of an egg standard product, it is thought that a type with a composition ratio of approximately 34 to 42% of the total protein is specified, so the food allergen composition standard substance of the present invention can fully serve as a standard product.

[0032] Furthermore, when an allergic food is extracted using PTS and then subjected to acetone precipitation, the precipitated proteins do not dissolve easily again in PTS, and some of the precipitates cannot be dissolved. This suggests that the acetone-precipitated proteins contain fewer types of proteins than the total proteins.

[0033] Furthermore, the protein content of the food allergen composition reference material of the present invention is preferably, for example, 5 to 50 mg / mL in a liquid state, since the protein concentration at the time of preparation can be maintained even when stored for a long period of time, and this is preferable from the viewpoint of the stability of the quality of the reference material. Furthermore, the reference material prepared in a liquid state can also be made into a solid state by lyophilization or the like.

[0034] In addition to the proteins, the food allergen composition standard of the present invention also contains a phase transfer solubilizer (PTS), which is a component used to extract proteins from foods that cause food allergies, as described below.

[0035] Examples of the PTS include SDC (sodium deoxycholate) and SLS (sodium N-lauroylsarcosinate). The food allergen composition standard of the present invention can also contain highly hydrophobic proteins by using PTS. Furthermore, although PTS is a type of surfactant, its hydrophobicity increases under acidic conditions. By performing liquid-liquid partitioning using ethyl acetate and trifluoroacetic acid (TFA), PTS can be removed from food allergen composition standards, which has the advantage of enabling measurement using the mass spectrometer.

[0036] The food allergen composition standard of the present invention contains water. The amount of water contained is not particularly limited as long as it is adjusted so that the protein has a desired concentration.

[0037] The food allergen composition standard of the present invention may also contain water-soluble components other than proteins. There are no particular limitations on the type or concentration of these water-soluble components, as long as they do not cause adverse effects such as errors when the food allergen composition standard of the present invention is subjected to mass spectrometry.

[0038] The food allergen composition reference material of the present invention can be produced, for example, as follows.

[0039] (1)Mixing process The food causing the food allergy is mixed with the PTS solution. The state of the food causing food allergy used as a raw material is not particularly limited, and may be in liquid or solid form. If it is solid, it is preferably pulverized before use. For example, milk may be in a liquid state or may be freeze-dried and then made into a powder state. The PTS solution can be prepared by mixing a surfactant such as SDC or SLS with a solvent such as Tris-HCl. There is no particular limitation on the concentration of PTS.

[0040] The resulting mixture is stirred or shaken, for example, at a temperature of 60 to 80°C for 1 to 4 hours. The conditions for efficient extraction vary depending on the type of protein, so extraction time, temperature, etc. may be changed.

[0041] The protein extraction method using the PTS can be carried out based on the procedure described in, for example, Masuda et al., Journal of Proteome Research 2008.

[0042] (2) Centrifugal separation process The protein extract obtained as described above is centrifuged, for example, at 5,000 to 8,000 rpm for 10 to 30 minutes, a solvent such as hexane is added to the supernatant, and the mixture is centrifuged at 10,000 to 20,000 rpm for 10 to 30 minutes. If a hexane layer is present, it is removed, and the supernatant is recovered. Note that since the conditions for efficient centrifugation vary depending on the type of protein, the rotation speed, time, number of times, etc. may be changed.

[0043] As a method for concentrating proteins, acetone precipitation treatment is known. However, in the production of the food allergen composition reference substance of the present invention, when this method is used, some proteins may not dissolve during redissolution, resulting in a decrease in the types of proteins constituting the total protein, and there is a possibility that the concentration may be excessive and the viscosity may increase. Therefore, when obtaining the food allergen composition reference substance of the present invention, acetone precipitation treatment is not performed.

[0044] (3) Ultrafiltration step The supernatant recovered as described above is subjected to ultrafiltration. By performing ultrafiltration, the proteins in the food allergen composition reference substance can be concentrated. For the ultrafiltration, from the viewpoint of working efficiency, it is preferable to use an ultrafiltration filter of a centrifugal separation method. Regarding the conditions of the ultrafiltration, they may be appropriately determined according to the type of food causing food allergy. For example, it may be 5000 to 8000 rpm for 5 to 20 minutes. The solution after ultrafiltration can be recovered to obtain a food allergen composition reference substance.

[0045] Regarding the concentration of the protein contained in the food allergen composition reference substance, for example, it can be measured using a BCA kit, Bradford kit, 2-D Quant kit.

[0046] Regarding the types of proteins contained in the food allergen composition reference substance of the present invention, for example, using a commercially available reference substance for the food causing food allergy or a reference substance extracted from the food causing food allergy according to the above-mentioned notification method as a control, and subjecting these reference substances and the food allergen composition reference substance of the present invention to electrophoresis and comparing the positions of the detected bands, it can be confirmed. In the electrophoresis, there is almost no influence from PTS contained in the food allergen composition reference material or surfactants contained in commercially available reference products. As the conditions for electrophoresis, those same as the conditions for ordinary proteins may be used, and there are no particular limitations.

[0047] Among these, it is preferable that the electrophoresis image obtained using the food allergen composition reference material of the present invention conforms to the reference product specifications in Attachment 3 of the Inspection Methods for Foods Containing Allergens, Notification No. 139, issued by the Deputy Director General of the Consumer Affairs Agency on March 30, 2015, because it will become a reference product conforming to the notification law.

[0048] Further, the food allergen composition reference material of the present invention has inter-bottle homogeneity with a standard error (RSD) of 10% or less by the homogeneity test method. Because it has the above-described inter-bottle homogeneity, for example, when the food allergen composition reference material of the present invention is dispensed and stored in a container, reference products with substantially the same protein concentration can be used regardless of the position from which it is dispensed and stored (for example, which position among the upper, middle, and lower positions), and there is an advantage that the stability is improved and the long-term storage property becomes good. The homogeneity test method is a known method, and the RSD can also be measured by a known technique.

[0049] Further, from the viewpoint of storage stability, it is desirable that the food allergen composition reference material of the present invention be dispensed and stored in a concentrated state by ultrafiltration or the like. As the conditions for the dispensing and storage, it is preferable that it be dispensed and stored in a container with a volume of 10 to 1000 μL. As the container for the storage, there are no particular limitations on the size, but it is preferable to select a material to which the protein is less likely to adsorb to the container.

[0050] The food allergen composition reference material of the present invention has stability with a decrease in protein concentration of 20% or less by the stability test method. As well-known standard substances, for example, standard substances composed of peptides that constitute a part of the protein contained in the causative food of food allergy, such as the milk allergen-derived peptide described in Patent No. 6903661, are known. Since such peptides are low molecular weight, they have the characteristic of being less likely to deteriorate even when stored for a long time. On the other hand, proteins are high molecular compounds, and when the storage period becomes long, they tend to be decomposed into a low molecular state, and the RSD also tends to exceed 20%. Therefore, the food allergen composition standard substance of the present invention can suppress the concentration fluctuation of the protein, although the detailed mechanism is unknown, by dispensing and storing it in the concentrated state as described above, and can maintain the state where the RSD is within 20% for a long time. Since the protein concentration is pre-assigned and dispensed for storage, there is no need to weigh and prepare the concentration, the error during weighing can be minimized, and since there is no concentration fluctuation between bottles, standard substances of the same concentration can be used at any time. In addition, since repeated freezing and thawing are not performed, it is an advantage that quality deterioration can be suppressed. Examples of the stability test method include the method described in the examples below.

[0051] The conditions for storing the food allergen composition standard substance of the present invention may be the same as those of well-known standard substances, and there is no particular limitation.

[0052] When the food allergen composition standard substance of the present invention is subjected to a mass spectrometer, for example, it can be carried out by the following procedure. The food allergen composition standard substance is appropriately diluted, reductively alkylated using DTT (dithiothreitol) and IAA (iodoacetamide), then the protein is cleaved into peptides by trypsin digestion, and then the PTS in the food allergen composition standard substance is removed by liquid-liquid partitioning using ethyl acetate and TFA, and then analyzed by a mass spectrometer.

[0053] In addition, by using the food allergen composition standard substance of the present invention, an addition recovery test can be performed. The spike recovery test is one of the methods for confirming the accuracy of an analytical method. By adding a pure product of the target component with a known concentration to a sample and performing an analysis, if the obtained analysis result matches the actual added amount, it can be determined that the analytical method is accurate. The food allergen composition reference material of the present invention contains two or more types of the proteins constituting the total protein, and since the protein concentration thereof is valued, by using it instead of the pure product, for example, it becomes possible to confirm the accuracy of allergen analysis by a mass spectrometer.

[0054] In addition, although allergen analysis by a mass spectrometer generally targets peptides rather than proteins, in the reference standard containing the peptides, the spike recovery test can only be performed after trypsin digestion, and an error due to the digestion efficiency of trypsin occurs, so the accuracy of the analytical method cannot be confirmed.

[0055] In addition, the food allergen composition reference material of the present invention can be prepared promptly even when a new allergenic food is detected. In the case of commercially available reference standards, it takes time to establish the protein extraction and purification methods, but this reference material can also be applied to unknown allergenic foods. In addition, food allergen analysis by a mass spectrometer such as LC-MS / MS does not involve an antigen-antibody reaction like the ELISA method, so plate preparation is not required, and it can respond promptly when the protein sequence is elucidated.

[0056] Next, the present invention will be described in detail based on examples, but the present invention is not limited only to such examples.

Example

[0057] (Example 1) Seven items of specific raw materials etc. (wheat, egg, crustaceans (shrimp, crab), buckwheat, milk, peanut, and soybean) were used as target allergens, and food allergen-causing raw materials were prepared with reference to the method for preparing reference standards described in the said notification method. Incidentally, all of the said raw materials were made into powder form. Next, 0.5 g of the raw material was weighed and placed in a 50 mL centrifugal tube, and then 9 mL of a PTS solution (12 mM SDC, 12 mM SLS, 100 mM Tris-HCl (pH 9.0)) was added. The centrifugal tube was vigorously shaken using a vortex and by hand, then subjected to shaking extraction in a water bath at 70°C for 2 hours, followed by ultrasonic treatment at 55°C for 15 minutes. Next, after centrifugation at 7200 rpm for 20 minutes (25°C), 1 mL of hexane was added and the mixture was shaken by hand for 1 minute, and then centrifuged again at 15000 rpm for 20 minutes (4°C).

[0058] The supernatant was collected with a pipette and placed in a centrifugal ultrafiltration filter (volume 15 mL).

[0059] The resulting liquid was used as a food allergen composition standard, and 40 μL aliquots were dispensed into 2 mL tubes and stored.

[0060] Next, protein quantification was performed using a commercially available BCA kit. Wheat 7.6~8.5mg / mL Eggs 30.6~44.2mg / mL Crustaceans: 30.3-31.0 mg / mL Buckwheat 23.3-28.3mg / mL Milk 17.5~18.0mg / mL Peanuts 31.0-43.6mg / mL Soybean 33.2~35.8mg / mL It was.

[0061] (Test Example 1: Mass spectrometry (LC-MS / MS analysis)) Mass spectrometry was performed using the seven food allergen composition standards obtained in Example 1. The food allergen composition standard was prepared at 10 mg / kg. The mass spectrometer used was the LC-MS / MS analyzer "QTRAP5500" (manufactured by AB Sciex), and the HPLC was the "LC-30AD" (manufactured by Shimadzu Corporation). The applications used were vMethod Application for Multiple Allergen Screening in Food Matrices using LC-MS / MS and vMethod Application for Gluten Quantitation in Food Matrices using LC-MS / MS (manufactured by AB Sciex). The mobile phase used was 0.1% formic acid in water and 0.1% formic acid in acetonitrile.

[0062] The results obtained are shown in FIG. As a result, it was found that all seven food allergen composition standard substances had a sensitivity of S / N ratio of 10 or more, meeting the target concentration of 10 mg / kg, the standard for labeling food allergies.

[0063] (Test Example 2: Electrophoresis) Electrophoresis was performed using the seven types of food allergen composition standard substances obtained in Example 1 and compared with the standard products specified in the notified method. Electrophoresis was performed using 10 μg / lane, 10% Bolt gel, and MOPS buffer. In wheat, one clear band was confirmed at around 17 kDa, one at around 35 kDa, and four or more clear bands at around 40 to 120 kDa. In the eggs, clear bands were observed at around 40, 75, 130, and 200 kDa. In crustaceans, one clear band was observed at around 41 kDa, one at around 37 kDa, and four clear bands at around 16 to 20 kDa. For buckwheat, one clear band was confirmed around 16-18 kDa, one around 22 kDa, and four or more clear bands around 32-83 kDa. In milk, one clear band was observed around 16 kDa and three clear bands around 25 to 40 kDa. In peanut, three to four clear bands were observed around 15 to 30 kDa and one clear band around 70 kDa. In soybean, one clear band each was confirmed at around 12-17 kDa and 76 kDa, two at around 25-35 kDa, and two or more clear bands at around 48-65 kDa. The results are shown in Table 1.

[0064] [Table 1]

[0065] As a result, the bands observed by electrophoresis matched those of standard products conforming to the notified method for five types of food, except for crustaceans and soybeans: wheat, eggs, buckwheat, milk, and peanuts.

[0066] For crustaceans, actin (around 41 kDa), tropomyosin (around 37 kDa), troponin, and myosin light chain (four bands around 16-20 kDa) were confirmed compared with the standard sample conforming to the notification method, but the myosin heavy chain band (around 160 kDa) was missing. When confirmed using a known ELISA method, it was found that the myosin heavy chains were extracted with only partial breakage. This revealed that the protein composition covers almost all major muscle-constituting proteins, and includes proteins that account for more than 50% of the total protein.

[0067] As for soybeans, as there is no mention of this in the notification law, we investigated the molecular weights of the proteins that make up the majority of the components, and found that they are 11S globulin (approximately 40%, around 52-61 kDa) and 7S globulin (approximately 20%, around 48-65 kDa). Both bands were confirmed by electrophoresis, making it clear that soybeans contain types that make up more than 50% of the total protein.

[0068] (Comparative Example 1) To 1 g of the powdery raw material of wheat prepared in the same manner as in Example 1, 20 mL of a PTS solution (10 mM SDC, 12 mM SLS, Tris-HCl (pH 9.0)) was mixed, shaken at 70 °C for 20 minutes, and then sonicated at 55 °C for 15 minutes. Next, 15 mL of the supernatant obtained by centrifuging at 7000 g for 30 minutes (room temperature) was placed in a 50 mL centrifuge tube, added with TFA to a concentration of 0.5%, and then centrifuged at 2270 g for 10 minutes (room temperature). The ethyl acetate phase in the upper layer was removed, 25 mL of refrigerated acetone was added, and then centrifuged at 5000 g for 10 minutes (room temperature) for acetone precipitation treatment. The acetone in the supernatant was removed, and the residue was air-dried. When 8 mL of the PTS solution was added to the residue, it did not dissolve easily, and not all of the precipitate could be dissolved. Also, the supernatant obtained by centrifuging at 7000 g for 10 minutes (room temperature) had viscosity and was difficult to take out, but it was centrifuged again under the same conditions. The obtained supernatant still had viscosity, but was ultrafiltered (centrifugation conditions: 5000 g, 10 minutes (room temperature)), and the resulting solution part was used as the standard solution.

[0069] When the above standard solution was electrophoresed according to Test Example 2, deletions occurred in the bands around 17 kDa (12 - 17 kDa is presumed to be α-amylase / trypsin inhibitor) and around 35 kDa (30 - 45 kDa is the location of ω-1,2 gliadin, α / β gliadin, γ-gliadin, and low molecular weight glutenin, so it is presumed to be any of them). Since a method for extracting many proteins is desirable as a standard substance, it became clear that this operation is not suitable for the present invention.

[0070] (Comparative Example 2) Also, acetone precipitation treatment was performed on the supernatant after protein extraction in Example 1 to prepare a standard solution using wheat, egg, crustacean, buckwheat, milk, peanut, and soybean.

[0071] The protein concentrations of the acetone-precipitated standard solutions prepared in Comparative Example 2 were examined and found to be 31 mg / mL for wheat, 64 mg / mL for eggs, 97 mg / mL for crustaceans, 93 mg / mL for buckwheat, 47 mg / mL for milk, 169 mg / mL for peanuts, and 107 mg / mL for soybeans, which were higher concentrations than the food allergen composition standard substance in Example 1. Furthermore, since the sample did not redissolve after being stored in a freezer, it became clear that excessive concentration by acetone precipitation would increase the viscosity, and would not be able to fully satisfy the storage stability and bottle-to-bottle homogeneity required for a reference standard.

[0072] (Test Example 3: ELISA method) The band for myosin heavy chain (160 kDa), a major protein in crustaceans, could not be confirmed by electrophoresis. We suspect that this is not because the myosin heavy chain was not extracted, but because it was extracted but partially broken down. In that case, if we compare the difference between the food allergen composition standard material in which the myosin heavy chain band cannot be confirmed and the notified standard standard in which the band can be confirmed using an ELISA kit (targeting a specific protein and quantifying protein) and a 2-D Quant kit (targeting an unspecified protein and quantifying protein), the difference should be small. To confirm this, a newly prepared crustacean standard (notified standard) according to the method described in the notified method was compared with the crustacean food allergen composition standard prepared in Example 1 using ELISA and a 2-D Quant kit. The ELISA kit used was the FA Test EIA Crustacean II "Nissui" manufactured by Nissui Pharmaceutical Co., Ltd., and the FA Test II extraction reagent, and the 2-D Quant kit used was the Cytiva 2-D Quant Kit manufactured by Global Life Science Technologies Japan Co., Ltd. The results are shown in Figure 2. As a result, the difference between the Crustacean food allergen composition standard substance and the notified standard substance was 3.6%. Therefore, it was considered that the Crustacean food allergen composition standard substance prepared in Example 1 had little effect on the quantitative values.

[0073] (Test Example 4: Allergen confirmation test using kamaboko) The seven types of food allergen composition standard substances prepared in Example 1 were added to commercially available kamaboko at 10 mg / kg, and a spike test was carried out using LC-MS / MS analysis. The results obtained are shown in FIG. As shown in the results, six food allergen composition standards - wheat, buckwheat, shellfish, milk, peanuts, and soybeans - were able to be identified even in the presence of a food matrix (because kamaboko contains eggs as an ingredient, the egg result may be a peak of unknown origin).

[0074] (Test Example 5: Homogeneity test, stability test) The protein concentrations of the seven food allergen reference materials obtained in Example 1 were measured by the BCA method to confirm that they were homogeneous regardless of which part was sampled and dispensed in 40 μL aliquots into 2 mL tubes (homogeneity test).Furthermore, after dispensing 40 μL aliquots into 2 mL tubes, the samples were stored at -20°C and periodically measured by the BCA method to confirm whether the protein concentrations had changed (stability test).

[0075] Specifically, the protein concentration of each food allergen composition standard was measured immediately after production for the homogeneity test, and immediately after production, one month later, and six months later for the stability test. For the measurements, three batches of each food allergen composition standard substance were prepared, each divided into upper, middle, and lower layers, and then 40 μL aliquots were dispensed into 2 mL tubes, and the protein concentration was measured using a BCA kit.

[0076] As a result, for all seven types of ingredients (wheat, eggs, buckwheat, shellfish, milk, peanuts, and soybeans), the protein concentrations were nearly identical between the top, middle, and bottom layers, with the standard error (RSD) within 10% (homogeneity test).In addition, although there were slight changes in protein concentration after one month and six months compared to immediately after production, no decrease of 20% or more was confirmed (stability test). Therefore, it can be seen that among the seven types of food allergen composition reference materials obtained in Example 1, proteins have excellent bottle-to-bottle homogeneity and long-term storage stability.

[0077] (Test Example 6: Evaluation of the validity of the simultaneous analysis method for food allergens using LC-MS / MS) In Japan, the ELISA method has been approved as an allergen analysis method, but it cannot analyze multiple allergens at once and has issues such as the possibility of false positives. Therefore, the validity of the food allergen simultaneous analysis method was evaluated using the food allergen composition standard substances of the type obtained in Example 1.

[0078] Proteins in a commercially available stew roux (containing no specific ingredients) were extracted using PTS. The extraction method was the same as in Example 1. Next, the protein was reductively alkylated using DTT and IAA, then digested with trypsin to cleave the protein into peptides, and then PTS in the standard sample was removed by liquid-liquid partitioning using ethyl acetate and TFA, followed by purification on a solid-phase column to prepare a sample containing the protein contained in commercially available stew roux. The obtained sample was analyzed using LC-MS / MS in the same manner as in Test Example 1. Two analysts performed five parallel branching tests per day, and evaluated the selectivity, repeatability, intra-laboratory reproducibility, trueness, linearity of the calibration curve, and the lower limit of quantitation.

[0079] The target values for validity evaluation were set as follows: Selectivity: Less than 1 / 10 of the peak area corresponding to 10 mg / kg Repeatability: RSD 20% or less Indoor reproduction accuracy: RSD25% or less Accuracy: 70% or more and 120% or less Linearity of calibration curve: correlation coefficient 0.98 or higher Lower limit of quantification: 10mg / kg or less

[0080] In addition, a calibration curve was prepared using the standard addition method (1, 5, 10, 20, and 50 mg / kg). The results are shown in Figure 4. In the figure, the upper line indicates ion 1 (quantitative ion), and the lower line indicates ion 2 (qualitative ion).

[0081] As a result, selectivity was such that no peaks interfering with quantification were observed from the blank solution. The repeatability was 4-11%, the intra-laboratory reproducibility was 4-10%, the accuracy was 90-104%, the linearity of the calibration curve was 0.983-0.998, and the lower limit of quantitation was 1-10 mg / kg, meeting the target values for all allergens. Since it was possible to determine a concentration of 10 mg / kg, which is the target concentration for the food allergy labeling standard, this analytical method is considered to be useful as a method for confirming food allergens.

Claims

**Claim 1** A food allergen composition reference material for use in a mass spectrometer, containing a protein constituting the total protein derived from the causative food of food allergy and a phase transfer solubilizer (PTS), wherein the number of types of proteins constituting the total protein derived from the causative food of food allergy is two or more, and at least one type is a food allergic protein, and the food allergic protein is 50% or more of the proteins constituting the total protein derived from the causative food of food allergy, characterized food allergen composition reference material. **Claim 2** The food allergen composition reference material according to claim 1, dispensed and stored in a container in a volume of 10 to 1000 μL. **Claim 3** The food allergen composition reference material according to claim 1 or 2, wherein the causative food of food allergy is one or more selected from wheat, egg, milk, peanut, buckwheat, crustacean or soybean.

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