Method for detecting cashew nuts using a mass spectrometer

Abstract

When cashew nuts, which may cause allergies, are contained in food ingredients or products, the system detects them with high sensitivity even in trace amounts. [Solution] A cashew nut detection method that includes the steps of extracting protein from a sample, obtaining an enzymatic digest of the extracted protein using a proteolytic enzyme, analyzing the enzymatic digest, and detecting any of the peptides of SEQ ID NOs: 1 to 3 using a mass spectrometer to qualitatively or quantitatively determine whether or not cashew nut protein is present in the sample.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting cashew nuts using a mass spectrometer, which enables high-sensitivity detection of even trace amounts of cashew nuts, which may cause food allergies, when they are contained in food ingredients or products. [Background technology]

[0002] Cashew nuts (Anacardium occidentale) are a plant of the Anacardiaceae family. In Japan, they are designated as "equivalent to specified allergens," which are recommended for labeling as substances that cause food allergies (Food Labeling Standards, Food Labeling Standards, March 30, 2015, Food Labeling Standards, Notice No. 139). According to a survey conducted to examine the appropriateness and need for revision of specified allergens, the top three foods causing immediate-type food allergies were previously chicken eggs, milk, and wheat. However, in recent years, cases of tree nuts have been increasing, surpassing wheat to become the third most common (Report on the FY2021 Research Project on Food Labeling Related to Food Allergies). Among tree nuts, cashews are the second most common after walnuts, accounting for approximately 25% of all tree nuts.

[0003] Foods that may cause allergies may be contaminated unintentionally in minute amounts during the production, distribution, or processing stages, so it is important for providers of food ingredients or products to carry out quality control to determine whether or not such contaminants are present.

[0004] Methods for testing for the presence or absence of contamination of specific foods include methods that use antigen-antibody reactions such as ELISA, Western blotting, and immunochromatography to detect characteristic proteins, and methods that use PCR to detect characteristic DNA base sequences.

[0005] Recently, a method has been reported that uses mass spectrometry to detect peptides derived from proteins that are characteristic of specific foods. This technology can quantify the protein in the target raw material, and has the advantages of reducing false positive reactions that tend to occur when using antigen-antibody reactions, and of enabling simultaneous detection of multiple items.

[0006] As prior art relating to the detection of cashew nuts, for example, the following prior art has been disclosed by the applicants. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 6660504

[0008] On the other hand, the patent document is directed to detecting genes, and it is assumed that other methods may also be possible. DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0009] Therefore, an object of the present invention is to provide an analytical method using a mass spectrometer that can specifically and highly sensitively detect cashew nuts, which may cause allergies, in food ingredients and products. [Means for solving the problem]

[0010] To achieve the above object, the present inventors focused on the amino acid sequences of the allergen proteins of cashew nuts to be detected and conducted extensive research into a method for specifically and sensitively detecting cashew nuts. As a result, they discovered amino acid sequences characteristic of cashew nuts and found that detecting these amino acid sequences enables specific and highly sensitive detection of cashew nuts, leading to the completion of the present invention. That is, the present invention first relates to the following items.

[0011] Section 1. A cashew nut detection method comprising the steps of extracting protein from a sample, obtaining an enzymatic digest of the extracted protein using a protease, analyzing the enzymatic digest, and detecting at least one peptide selected from the group consisting of SEQ ID NOs: 1 to 3 using a mass spectrometer to qualitatively or quantitatively determine whether or not cashew nut protein is present in the sample.

[0012] Next, as a method for detecting at least one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 3, a method is preferred in which the peptides are analyzed by liquid chromatography tandem mass spectrometry (LC-MS / MS) to monitor at least one or more precursor-product ion pair transitions having specific m / z values ​​associated with specific amino acid sequences. That is, the present invention relates to the following item 2.

[0013] Section 2. A step of extracting proteins from a sample, a step of obtaining an enzymatic digest of the extracted proteins using a protease, and a step of analyzing the enzymatic digest by liquid chromatography tandem mass spectrometry (LC-MS / MS) to obtain the following: i) m / z values ​​of SEQ ID NO: 1, about 452 / 304, or 452 / 391 ii) SEQ ID NO: 2, m / z values ​​of about 445 / 346, 445 / 512 or 445 / 722 iii) SEQ ID NO: 3, m / z values ​​of about 765 / 215, 765 / 362 or 765 / 490 and determining whether or not a cashew nut protein is present in a sample by monitoring at least one precursor-product ion pair transition having a specific m / z value associated with a specific amino acid sequence selected from the group consisting of:

[0014] Next, it is preferable to monitor two or more of the precursor-product ion pair transitions. That is, the present invention relates to the following item 3. Section 3. and a step of qualitatively or quantitatively determining whether or not cashew nut protein is present in the sample by monitoring at least two or more precursor-product ion pair transitions having specific m / z values ​​associated with the specific amino acid sequence. [Effects of the Invention]

[0015] The present invention enables the detection of cashew nut protein-derived peptides by LC-MS / MS analysis, thereby enabling quality control testing to determine whether the above-mentioned cashew nuts are mixed into or used in test food ingredients or test foods. It can also contribute to the prevention of allergies and the investigation of causative substances when allergic symptoms occur. [Brief explanation of the drawings]

[0016] [Figure 1] Peaks of cashew nut protein-derived peptides in the chromatogram obtained from a standard sample with known concentrations of cashew nut [Figure 2] A calibration curve was prepared by plotting the area of ​​the cashew nut protein-derived peptides in the chromatogram obtained from a standard sample with a known cashew nut concentration against the known cashew nut protein concentration in the standard sample. [Figure 3] Exemplary chromatogram of instant noodle sample without cashew nuts [Figure 4] An exemplary chromatogram containing cashew protein-derived peptides obtained from an instant noodle sample spiked with cashew protein. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides a method for detecting trace amounts of cashew nut protein in a test sample, such as a food ingredient or processed food. Specifically, the method includes the steps of extracting protein from the test sample, obtaining an enzymatic digest of the extracted protein with a protease, and analyzing the enzymatic digest by LC-MS / MS to obtain a chromatogram of the target peptides. A preferred embodiment of the method according to this embodiment is described below.

[0018] Proteins can be extracted from test samples using a buffer solution containing a surfactant or a commercially available protein analysis kit.

[0019] Preferably, the protein extract from the test sample is further reduced and alkylated to block thiol groups.

[0020] The sample prepared as described above is treated with a protease. Examples of protease used in the method of the present invention include trypsin and chymotrypsin, with trypsin being preferred. Treatment conditions may be appropriately selected depending on the type of enzyme. The enzyme treatment degrades the target protein to generate multiple peptides.

[0021] The resulting enzymatic digests are preferably analyzed by LC-MS / MS after removing the surfactant and purifying using a reversed-phase solid-phase column.

[0022] The peptide sequences analyzed in LC-MS / MS are as follows: Sequence number 1 GSESEEESEDEK Sequence number 2 GQVQVVDNFGNR Sequence number 3 DVFQQQQQHQSR Although various methods can be used to detect these cashew nut-derived peptides, the present invention utilizes a mass spectrometer. Among these, methods using liquid chromatography are particularly preferred, such as methods using LC-MS and LC-MS / MS. In particular, it is preferable to analyze the obtained enzymatic digest by LC-MS / MS after removing the surfactant and purifying it using a reversed-phase solid-phase column.

[0023] In addition, a standard sample with a known concentration of cashew nut-derived protein can be treated in the same way as the test sample, analyzed by LC-MS / MS, and a calibration curve can be created to quantitatively analyze cashew nut protein.

[0024] The type of test sample to be used in the cashew nut detection method of the present invention is not particularly limited. Examples of test samples include food ingredients and processed foods. Food ingredients include food ingredients that are intentionally produced without cashew nuts at a food ingredient production factory that handles cashew nuts. Processed foods include confectioneries, noodles, powdered soups, liquid soups, hot-air-dried or freeze-dried ingredients, and various prepared foods containing these processed foods. Other examples include processed foods that are intentionally produced without cashew nuts at a food production factory that handles cashew nuts. Furthermore, when producing processed foods that do not contain cashew nuts after producing processed foods containing cashew nuts, careful cleaning of the food production equipment is essential, with the removal of cashew nut residue in mind. To verify the effectiveness of this cleaning method and the presence or absence of cashew nut residue in the food production equipment, wiped samples from the production equipment can also be used as test samples.

[0025] Example The present invention will be described in more detail below using examples, but the present invention should not be construed as being limited to these examples. Furthermore, the present invention can be modified appropriately without departing from the gist of the present invention.

[0026] Example 1 Analysis of standard samples with known protein concentrations in cashew nuts In order to verify the quantitative capability of the cashew nut detection method by LC-MS / MS of the present invention, standard samples with known concentrations of cashew nut protein were analyzed and a calibration curve was created.

[0027] Proteins were extracted from cashew nuts purchased from a store using MPEX PTS Reagents (60 mM SDC SLS / 50 mM TEAB) (GL Sciences) and analyzed using the Pierce BCA Protein Assay Kit. The total protein concentration was determined by a standard standard (Thermo Fisher Scientific).

[0028] Of the prepared standard sample, 50 μg of protein was placed in a 2.0 mL low-adsorption polypropylene tube, and 1000 μg of egg-derived ovalbumin and 100 μg of bovine albumin were added to bring the total solution volume to 700 μL.

[0029] 70 μL of 1M TEAB and 28 μL of 1M DTT were added, and the mixture was left to stand at 75°C for 15 minutes, then at room temperature for 30 minutes. Next, 56 μL of 1M iodoacetamide solution prepared with distilled water was added, and the mixture was left to stand at room temperature in the dark for 45 minutes, after which 28 μL of 1M DTT was added (reduction / alkylation).

[0030] 10 μL of a trypsin solution derived from bovine pancreas, adjusted to 20 mg / mL with 0.1% formic acid, was added and the mixture was left standing overnight at 37°C to perform enzymatic digestion of the cashew nut standard sample.

[0031] Formic acid was added to the resulting enzyme digest to make it acidic, and then ethyl acetate was added to remove the surfactant contained in the extract solution by liquid-liquid partitioning. This removal procedure was repeated three times.

[0032] The solution after removing the surfactant was concentrated using a centrifugal evaporator, and after adding 0.1% formic acid, the residue was purified using a C18 reversed-phase solid-phase extraction centrifugal column and a silica gel-based anion exchange solid phase.

[0033] The refined solution was dried using a centrifugal evaporator, dissolved in 0.1% formic acid containing 5% acetonitrile, and a dilution series with a total cashew nut protein concentration of 1.25 - 10 μg / mL was prepared for analysis by LC-MS / MS.

[0034] <LC-MS / MS device> LC section: ExionLC AD system (SCIEX) MS / MS section: QTRAP (registered trademark) 6500+ system (SCIEX) <LC conditions> Analysis column: YMC-Triart C18, particle size 3 μm, 100 x 2.1 mm id. (YMC) Column temperature: 40 °C Column flow rate: 0.3 mL / min Eluent A: 0.1% formic acid; Eluent B: acetonitrile containing 0.1% formic acid Gradient: 0 min (B: 5%) → 16 min (B: 40%) → 18 min (B: 95%) → 23 min (B: 95%) → 23.1 min (B: 5%) → initialization <Mass spectrometry conditions> Ionization: electrospray ionization method Polarity: positive Spray voltage: 5500 V

[0035] The sequences and MRM transitions of the cashew nut protein-derived peptide fragments targeted for detection are shown in Table 1.

[0036]

Table 1

[0037] The chromatogram when analyzing a standard sample with a total cashew nut protein concentration of 1.25 μg / mL is illustrated in Figure 1 (peptide sequence: GQVQVVDNFGNR (SEQ ID NO: 2), Q1: 444.9, Q3: 722.3).

[0038] A calibration curve under the same detection conditions as in Figure 1 is shown in Figure 2. A good calibration curve with R2: 0.994 was obtained in the cashew nut total protein concentration range of 1.25 to 10 μg / mL.

[0039] Example 2 Cashew nut protein spike test on crushed instant noodles To examine the applicability of the cashew nut detection method of the present invention using LC-MS / MS to processed foods, a cashew nut-free instant noodle powder sample was spiked with a cashew nut protein standard sample to achieve a product content of 10 ppm, and the analysis was carried out, and the recovery rate was calculated.

[0040] 1 g of a sample of ground instant noodles containing no cashew nuts was weighed into a 50 mL polypropylene centrifuge tube, and the cashew nut protein standard sample used in Example 1 was added to give a total cashew nut protein concentration of 10 ppm.

[0041] 30 μL of ethylenediaminetetraacetic acid (EDTA) prepared at 100 mg / mL with 1N sodium hydroxide solution was added.

[0042] 9 mL of the extraction solution used in Example 1 was added, and the mixture was shaken at 90 to 110 rpm overnight to extract the protein.

[0043] The mixture was centrifuged at 10,000×g at 4° C. for 30 minutes, and 700 μL of the supernatant was collected in a 2.0 mL low-adsorption polypropylene tube.

[0044] The subsequent procedures were carried out in the same manner as in Example 1, and the final undiluted solution was analyzed by LC-MS / MS.

[0045] Figure 3 shows a chromatogram of a sample of ground instant noodles that does not contain cashew nuts, and Figure 4 shows a chromatogram of a sample to which a cashew nut protein standard sample was added to achieve a product content of 10 ppm (peptide sequence: GQVQVVDNFGNR (SEQ ID NO: 2), Q1: 444.9, Q3: 722.3).

[0046] The target peak was observed only when the cashew nut protein standard sample was added.

[0047] This application is based on Japanese application No. 2023-073864 filed on April 27, 2023, the contents of which are incorporated herein by reference.

Claims

1. A cashew nut detection method comprising the steps of extracting protein from a sample, treating the extracted protein with a protease to obtain an enzymatic digest, analyzing the enzymatic digest, and detecting the peptide of sequence number 2 using a mass spectrometer to qualitatively or quantitatively determine whether or not cashew nut protein is present in the sample.

2. A cashew nut detection method comprising the steps of extracting protein from a sample, treating the extracted protein with a protease to obtain an enzymatic digest, analyzing the enzymatic digest, and detecting the peptide of sequence number 1 using a mass spectrometer to qualitatively or quantitatively determine whether or not cashew nut protein is present in the sample.

3. A cashew nut detection method comprising the steps of extracting protein from a sample, treating the extracted protein with a protease to obtain an enzymatic digest, analyzing the enzymatic digest, and detecting the peptide of sequence number 3 using a mass spectrometer to qualitatively or quantitatively determine whether or not cashew nut protein is present in the sample.

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