JP7715992B2 - Food allergen composition reference materials - Google Patents

Description

Article 30, Paragraph 2 of the Patent Act applies. Date of website publication: October 15, 2021. Website address: http://shokuhineisei.or.jp/ https://chat.lincbiz.jp/a006007/

Article 30, Paragraph 2 of the Patent Act applies. Date of website publication: November 24, 2020. Website address: http://shokuhineisei.or.jp/ https://conference.iap-jp.org/jsfhs/conference/

The present invention relates to a food allergen composition standard for use in mass spectrometry.

Regarding food allergen analysis methods, the Consumer Affairs Agency has designated the ELISA method as a quantitative testing method, and the PCR method and Western blotting method 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.

Meanwhile, allergen analysis using mass spectrometry has attracted attention, with SCIEX proposing a method using a liquid chromatograph mass spectrometer (LC-MS/MS) that has been accepted for 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 otherwise give a false positive using ELISA, significantly shortens the analysis time (in principle, it is possible to analyze specified allergens and similar substances simultaneously), 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.

The Notice from the Deputy Director-General of the Consumer Affairs Agency, dated March 30, 2015, No. 139, "Regarding Food Labeling Standards" (hereinafter referred to as the "Notification"), describes the preparation method for 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 with this analytical method.

The notification law also stipulates that it is desirable for the reference standard to contain "total protein." Here, total protein refers to all proteins contained in milk, rather than a specific protein such as casein, for example. The type of each protein that makes up 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 40-23 kDa range (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 protein types that make up approximately 77% of the total protein.

In contrast, the standard 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 protein like total protein, and therefore are not standard standards that comply with the aforementioned notification method.

At the 25th Annual Meeting of the Japanese Society of Food Chemistry (held June 6th and 7th, 2019), the present inventors attempted to prepare standard solutions by extracting proteins from commercially available standard samples such as milk, eggs, wheat, peanuts, buckwheat, shrimp, and soybeans using a phase transfer solubilizer (PTS), which has excellent protein solubilizing properties, followed by acetone precipitation and ultrafiltration (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)

However, with 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 of the precipitates cannot be dissolved, which suggests that the number of types of protein in the acetone precipitated sample may be reduced compared to the total protein content. Furthermore, the completed standard solution was highly viscous due to its concentration, and did not fully satisfy the storage stability and bottle-to-bottle homogeneity required for a standard product.

The present invention therefore aims 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.

As a result of extensive efforts to solve the above-mentioned problems, the inventors discovered that by extracting proteins from foods that cause food allergies without acetone precipitation and using a composition containing these proteins, it is possible to prepare a food allergen composition reference material that exhibits excellent storage stability and bottle-to-bottle uniformity, thereby completing the present invention.

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 comprising a protein extracted from a food causing food allergy using a phase transfer solubilizer (PTS) and not subjected to acetone precipitation, and the PTS, wherein the protein contains two or more types of proteins;
[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] The food allergen composition reference material according to any one of [1] to [3], wherein at least one of the proteins is a food allergen protein.
Regarding.

The food allergen composition standard of the present invention contains two or more types of proteins extracted from foods that cause food allergies, and these proteins make up the total protein content of the foods that cause food allergies. Furthermore, it has excellent storage stability and bottle-to-bottle homogeneity, making it suitable for use as a standard for mass spectrometry.

FIG. 1 shows the results of mass spectrometry performed in Test Example 1 using the seven food allergen composition standard substances obtained in Example 1. Figure 2 is a graph showing the difference between the Crustacean food allergen composition standard substance prepared in Example 1 and the notified standard, measured in Test Example 3, using an ELISA kit and a 2-D Quant kit. FIG. 3 shows the results of the allergen confirmation test for kamaboko carried out in Test Example 4. FIG. 4 shows the calibration curve results obtained in Test Example 6.

Specific embodiments of the present invention are described in detail below, but the present invention is not limited to the following embodiments and can be implemented with appropriate modifications within the scope of the invention. Note that redundant explanations may be omitted where appropriate, but this does not limit the present invention.

In the present invention, a reference material refers to a material that is sufficiently homogeneous and stable with respect to one or more specified properties, and that has been prepared to be suitable for its intended use in a measurement process, as described in Japanese Industrial Standard JIS Q 0030:2019.
Furthermore, a food allergen composition reference material is a reference material that can characterize the food that causes a food allergy, and refers to a mixture (including total protein) containing food allergen proteins and other proteins extracted from the food that causes the food allergy. A food allergen composition reference material also refers to a mixed food allergen composition reference material that is a mixture of multiple food allergen composition reference materials extracted from different foods that cause food allergies, such as wheat and eggs. "Two or more types of proteins" means that two or more types of proteins are extracted from each of the causative foods, for example, two or more types of wheat and two or more types of eggs.

The food allergen composition standard of the present invention is intended to be subjected to mass spectrometry.
Examples of the mass spectrometer include a liquid chromatograph mass spectrometer (LC-MS/MS) and a time-of-flight mass spectrometer (TOF/MS).
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 a stationary phase and a mobile phase and detects them with a mass detector. The mass detector is connected to the instrument, and only specific masses are fragmented and detected. Furthermore, 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.

The food allergen composition standard of the present invention is a composition containing a protein extracted from a food that causes food allergies using a phase transfer solubilizer (PTS) and that has not been subjected to acetone precipitation, and the PTS.

The food causing the food allergy is a food that causes food allergy, 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, including 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, are listed as foods similar to the above-mentioned specified ingredients.
In addition to the above, other fruits include cherries, melons, mangoes, etc.

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 does not refer to specific proteins such as casein and whey protein in the case of milk, but refers to all proteins contained in milk.
The total protein may be, for example, a protein obtained by the method described in the notification method.

The food allergen composition standard of the present invention contains two or more types of proteins that constitute the total protein.
For example, according to P. Walstra & R. Jness, "Dairy Chemistry and Physics," milk proteins consist of approximately 80% casein and approximately 19% whey protein. The casein is divided into 30.6% αs1-casein, 8.0% αs2-casein, 28.4% β-casein, 2.4% γ-casein, and 10.1% κ-casein. The whey proteins are divided into 1.2% serum albumin, 9.8% β-lactoglobulin, 3.7% α-lactalbumin, 2.1% immunoglobulins, and 2.4% other whey proteins.
Therefore, examples of a standard substance for a milk-derived food allergen composition include those containing two or more of the proteins that constitute the casein and whey protein.
Among these, it is preferable to contain casein, β-lactoglobulin, and α-lactalbumin.

Furthermore, from the viewpoint of effectiveness as a standard substance, it is preferable that at least one of the proteins contained in the food allergen composition standard substance of the present invention is a food allergenic protein.
The food allergenic protein refers to a protein that causes an allergic reaction when ingested. Examples of the food allergenic protein are registered in the database of the World Health Organization/International Union of Immunological Society (WHO-IUIS).

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 protein in wheat (the two are present in roughly equal amounts), and are considered to be the major allergens in wheat.

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.

Examples of milk-derived food allergen composition standards include those containing two or more of casein, β-lactoglobulin, and α-lactalbumin.
Casein accounts for approximately 80% of total milk protein, β-lactoglobulin for approximately 10%, and α-lactalbumin for approximately 4%. The main allergens are casein and β-lactoglobulin.

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 albumins are considered to be major allergens.

Examples of buckwheat-derived food allergen composition standards 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. Major allergens include globulin and albumin.

Examples of standard materials for food allergen compositions derived from crustaceans include those containing two or more of tropomyosin, arginine kinase, myosin, actin, and the like.
Myosin and actin are proteins found in large amounts in crustaceans, with tropomyosin being the major allergen.

Examples of 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.

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 in which the constituent ratio of the total protein derived from the food that causes food allergies is 50% or more.
The type of the protein can be confirmed by electrophoresis as described below.

In addition, in the case of publicly known standard products, for example, egg standard products are thought to be designated as having a composition ratio of approximately 34-42% of total protein. Therefore, the food allergen composition standard substance of the present invention is fully capable of fulfilling the role of a standard product.

Furthermore, when acetone precipitation is performed after extraction using PTS from foods that cause food allergies, the precipitated proteins do not dissolve easily when attempts are made to redissolve them in PTS, and some of the precipitate cannot be dissolved. This suggests that the acetone-precipitated proteins contain fewer types of proteins than the total proteins.

Furthermore, the protein content contained in 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 perspective of the stability of the quality of the reference material. Furthermore, a reference material prepared in a liquid state can also be made into a solid state by freeze-drying or other methods.

The food allergen composition standard of the present invention contains, in addition to the protein, a phase transfer solubilizer (PTS). As described below, PTS is a component used to extract proteins from foods that cause food allergies.

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 utilizing this, PTS can be removed from the food allergen composition standard by performing liquid-liquid partitioning using ethyl acetate and trifluoroacetic acid (TFA), which has the advantage of enabling measurement using the mass spectrometer.

The food allergen composition standard of the present invention contains water. The water content is not particularly limited, as long as it is adjusted so that the protein has the desired concentration.

Furthermore, the food allergen composition standard substance 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 have adverse effects such as causing errors when the food allergen composition standard substance of the present invention is subjected to a mass spectrometer.

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

(1) Mixing Step 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 are no particular limitations on the concentration of PTS.

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 the extraction time, temperature, etc. may be changed.

Protein extraction using the PTS can be performed, for example, based on the procedures described in Masuda et al., Journal of Proteome Research 2008.

(2) Centrifugation Step The protein extract obtained as described above is centrifuged, for example, at 5,000 to 8,000 rpm for 10 to 30 minutes, and a solvent such as hexane is added to the supernatant, followed by centrifugation 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.
Since the conditions for efficient centrifugation vary depending on the type of protein, the rotation speed, time, number of times, etc. may be changed.

Acetone precipitation is known as a method for concentrating proteins, but when this method is used to produce the food allergen composition standard substance of the present invention, there is a possibility that some proteins will not dissolve when reconstituted, resulting in a decrease in the number of protein types that make up the total protein, or that the substance will be too concentrated and become more viscous. Therefore, acetone precipitation is not performed when obtaining the food allergen composition standard substance of the present invention.

(3) Ultrafiltration Step The supernatant collected as described above is subjected to ultrafiltration.
Ultrafiltration can be used to concentrate proteins in food allergen composition standards.
From the viewpoint of work efficiency, it is preferable to use a centrifugal separator type ultrafiltration filter for the ultrafiltration.
The conditions for the ultrafiltration may be determined appropriately depending on the type of food causing the food allergy, and may be, for example, 5,000 to 8,000 rpm for 5 to 20 minutes.
The solution after ultrafiltration can be collected and used as a food allergen composition standard.

The protein concentration contained in the food allergen composition standard can be measured using, for example, a BCA kit, Bradford kit, or 2-D Quant kit.

Furthermore, the types of proteins contained in the food allergen composition standard material of the present invention can be confirmed by, for example, using a commercially available standard product for foods that cause food allergies or a standard product extracted from foods that cause food allergies in accordance with the aforementioned notified method as a control, electrophoresing these standard products with the food allergen composition standard material of the present invention, and comparing the positions of the detected bands.
The electrophoresis is hardly affected by PTS contained in the food allergen composition standard substance or surfactants contained in commercially available standard products.
The electrophoresis conditions are not particularly limited as long as they are the same as those for ordinary proteins.

In particular, if the electrophoretic image obtained using the food allergen composition standard substance of the present invention conforms to the standard product specifications in Appendix 3 of the Testing Method for Foods Containing Allergens, Notification No. 139 of the Deputy Director-General of the Consumer Affairs Agency, dated March 30, 2015, it is preferable because it will be a standard product that complies with the notified law.

Furthermore, the food allergen composition reference material of the present invention has inter-bottle homogeneity with a standard deviation (RSD) of 10% or less as determined by the homogeneity test method.
Because of the above-mentioned bottle-to-bottle homogeneity, for example, when the food allergen composition standard substance of the present invention is dispensed and stored in a container, a standard product with approximately the same protein concentration can be used regardless of the position (e.g., top, middle, or bottom) from which it is dispensed and stored, which has the advantage of improved stability and good long-term storage properties.
The homogeneity test method is a known method, and the RSD can also be measured by a known technique.

From the viewpoint of storage stability, it is desirable that the food allergen composition reference material of the present invention be stored in aliquots after being concentrated by ultrafiltration or the like.
As for the conditions for dispensing and storing the solution, it is preferable that the solution is dispensed and stored in a volume of 10 to 1000 μL in a container.
The size of the storage container is not particularly limited, but it is preferable to select a material that does not easily adsorb proteins to the container.

The food allergen composition reference material of the present invention is stable, with a decrease in protein concentration of no more than 20% as determined by a stability test method.
Known examples of known standards include peptides that constitute part of proteins contained in foods that cause food allergies, such as the peptides derived from milk allergens described in Japanese Patent No. 6903661. Such peptides have the characteristic of being low in molecular weight and therefore resistant to deterioration even when stored for long periods of time.
On the other hand, proteins are high molecular weight compounds, and when stored for a long period of time, they tend to decompose into low molecular weight compounds, causing the RSD to easily exceed 20%.
Therefore, by storing the food allergen composition standard substance of the present invention in aliquots in a concentrated state as described above, fluctuations in protein concentration can be suppressed, and the RSD can be maintained at 20% or less for a long period of time, although the detailed mechanism is unknown.
Since the protein concentration is pre-assigned and stored in aliquots, there is no need to weigh out the sample to adjust the concentration, minimizing weighing errors. Furthermore, since there is no variation in concentration between bottles, the same concentration of standard material can be used at any time. Another advantage is that quality deterioration can be suppressed because there is no need to repeatedly freeze and thaw the sample.
The stability test method may be the method described in the Examples below.

The conditions for storing the food allergen composition standard of the present invention are not particularly limited, as long as they are the same as those for known standard products.

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

Furthermore, by using the food allergen composition standard substance of the present invention, a recovery test can be carried out.
A recovery test is a method for verifying the accuracy of an analytical method. A known concentration of the target component is added to a sample and analyzed. If the analysis results match the actual amount added, the analytical method can be judged to be accurate.
The food allergen composition standard material of the present invention contains two or more types of proteins that make up the total protein, and the protein concentrations are valued. Therefore, by using it in place of the pure product, it is possible to confirm the accuracy of allergen analysis, for example, by a mass spectrometer.

Allergen analysis using mass spectrometry generally targets peptides rather than proteins, but with standard samples containing these peptides, the spike recovery test can only be performed after trypsin digestion, which results in errors due to the efficiency of trypsin digestion, making it impossible to confirm the accuracy of the analytical method.

Furthermore, the food allergen composition standard of the present invention can be quickly prepared even when a new allergen food is detected.
In the case of commercially available standards, it takes time to establish methods for extracting and purifying the proteins, but this standard material can also be applied to foods with unknown allergens.
Furthermore, food allergen analysis using mass spectrometers such as LC-MS/MS does not involve an antigen-antibody reaction like the ELISA method, so there is no need to prepare plates, and analysis can be carried out quickly once the protein sequence is elucidated.

Next, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

Example 1
Seven specific raw materials (wheat, eggs, crustaceans (shrimp, crab), buckwheat, milk, peanuts, and soybeans) were selected as target allergens, and food ingredients causing food allergies were prepared using the standard preparation method described in the notification law. All of the ingredients were in powder form.
Next, 0.5 g of the raw material was weighed and placed in a 50 mL centrifuge 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, followed by shaking by hand for 1 minute, and then centrifuging again at 15000 rpm for 20 minutes (4°C).

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

The resulting liquid was used as a food allergen composition standard and was dispensed in 40 μL aliquots into 2 mL tubes and stored.

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 23.3-28.3 mg/mL
Milk 17.5-18.0mg/mL
Peanuts 31.0-43.6 mg/mL
Soybean 33.2-35.8mg/mL
It was.

(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 an LC-MS/MS analyzer "QTRAP5500" (manufactured by AB Sciex Corporation), and the HPLC used was an "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 Corporation). The mobile phase used was 0.1% formic acid in water and 0.1% formic acid in acetonitrile.

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

(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 carried out at 10 μg/lane using 10% Bolt gel in MOPS buffer.
In wheat, one clear band was observed around 17 kDa, one around 35 kDa, and four or more clear bands around 40 to 120 kDa.
In the eggs, clear bands were confirmed 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 observed 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 observed at around 12-17 kDa and 76 kDa, two at around 25-35 Da, and two or more clear bands at around 48-65 kDa.
The results are shown in Table 1.

As a result, the bands observed in electrophoresis matched those of standard samples compliant with the notified law for five types of food - wheat, eggs, buckwheat, milk, and peanuts - except for crustaceans and soybeans.

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 a standard sample conforming to the notified 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 therefore includes types that account for more than 50% of the total protein.

As soybeans are not listed in the notification law, the molecular weights of the proteins that make up the majority of the soybean content were examined, and were found to be 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 the soybeans contain types that make up more than 50% of the total protein.

(Comparative Example 1)
1 g of wheat powder raw material prepared in the same manner as in Example 1 was mixed with 20 mL of PTS solution (10 mM SDC, 12 mM SLS, Tris-HCl (pH 9.0)), shaken at 70°C for 20 minutes, and then ultrasonicated at 55°C for 15 minutes.
Next, 15 mL of the supernatant obtained by centrifugation at 7000 g for 30 minutes (room temperature) was placed in a 50 mL centrifuge tube, and TFA was added to a concentration of 0.5%, followed by centrifugation at 2270 g for 10 minutes (room temperature).
The upper layer, ethyl acetate, was removed, and 25 mL of chilled acetone was added, followed by centrifugation at 5000 g for 10 minutes (room temperature) to perform acetone precipitation.
The acetone in the supernatant was removed and the residue was air-dried.
When 8 mL of PTS solution was added to the residue, it did not dissolve easily, and it was not possible to dissolve all of the precipitate. Furthermore, the supernatant obtained after centrifugation at 7000 g for 10 minutes (room temperature) was viscous and difficult to remove, but it was centrifuged again under the same conditions.
The resulting supernatant was still viscous, but was subjected to ultrafiltration (centrifugation conditions: 5000 g, 10 minutes (room temperature)), and the resulting solution was used as a standard solution.

When the above standard solution was subjected to electrophoresis in the same manner as in Test Example 2, bands were missing at around 17 kDa (12-17 kDa is presumed to be α-amylase/trypsin inhibitor) and around 35 kDa (30-45 kDa is presumed to be one of ω-1,2 gliadin, α/β gliadin, γ-gliadin, or low molecular weight glutenin). Since a method that extracts a large amount of protein is desirable for use as a standard substance, it became clear that this procedure was not suitable for the present invention.

(Comparative Example 2)
Furthermore, the supernatant after protein extraction in Example 1 was subjected to acetone precipitation to prepare standard solutions for wheat, eggs, crustaceans, buckwheat, milk, peanuts, and soybeans.

The protein concentrations of the acetone-precipitated standard solutions prepared in Comparative Example 2 were examined to find that they were 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 of 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.

(Test Example 3: ELISA method)
The band for myosin heavy chain (160 kDa), a major protein in crustaceans, could not be confirmed by electrophoresis. This was presumed to be due to the fact that the myosin heavy chain was extracted but partially broken, rather than being unable to be extracted. In this case, if the difference between a food allergen composition standard in which the myosin heavy chain band cannot be confirmed and a notified standard standard in which the band can be confirmed is compared using an ELISA kit (targeting a specific protein and quantifying the protein) and a 2-D Quant kit (targeting an unspecified protein and quantifying the protein), the difference should be small.
To confirm this, a new Crustacean standard (notified standard) prepared according to the method described in the notified method was compared with the Crustacean food allergen composition standard prepared in Example 1 using the ELISA method and the 2-D Quant kit.
The ELISA kit used was FA Test EIA Crustacean II "Nissui" manufactured by Nissui Pharmaceutical Co., Ltd., and FA Test II extraction reagent, and the 2-D Quant kit was 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.

(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 an addition 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 (kamaboko contains eggs as an ingredient, so the egg result may be a peak of unknown origin).

(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).

Specifically, the protein concentration of each food allergen composition standard was measured immediately after production in the homogeneity test, and immediately after production, one month later, and six months later in 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.

As a result, for all seven types of ingredients (wheat, egg, buckwheat, shellfish, milk, peanut, and soybean), the protein concentrations in the upper, middle, and lower layers were nearly identical, with the standard error (RSD) within 10% (homogeneity test). Furthermore, 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 standard materials obtained in Example 1, proteins have excellent bottle-to-bottle homogeneity and long-term storage stability.

(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.

Proteins in a commercially available stew roux (containing no specific ingredients) were extracted using PTS in the same manner 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 the PTS in the standard 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 by 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 quantification.

The target values for validity evaluation were set as follows:
Selectivity: 1/10 or less of the peak area corresponding to 10 mg/kg Repeatability: RSD 20% or less Intra-laboratory reproducibility: RSD 25% or less Accuracy: 70% to 120% or less Linearity of calibration curve: Correlation coefficient 0.98 or more Lower limit of quantitation: 10 mg/kg or less

A calibration curve was also 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 represents ion 1 (quantitative ion), and the lower line represents ion 2 (qualitative ion).

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 (3)

A food allergen composition standard for use in a mass spectrometer, comprising:
It contains proteins that constitute the total proteins derived from foods that cause food allergies and a phase transfer solubilizer (PTS),
the total protein derived from the food causing the food allergy is composed of two or more types of proteins, and at least one of the types is a food allergy protein;
A food allergen composition standard material , characterized in that the food allergy protein accounts for 50% or more of the total proteins derived from the food causing the food allergy. The food allergen composition reference material according to claim 1, stored in a container in a volume of 10 to 1000 μL. 3. The food allergen composition reference material according to claim 1, wherein the food causing the food allergy is one or more selected from the group consisting of wheat, eggs, milk, peanuts, buckwheat, shellfish, and soybeans.