JP2020187019A - Method for detecting emetic poison of bacillus cereus - Google Patents

Abstract

To provide a method for detecting an emetic poison of Bacillus cereus, capable of simply and quickly detecting the emetic poison produced by the Bacillus cereus.SOLUTION: A method for detecting an emetic poison of Bacillus cereus includes: a sample preparation step of preparing a sample by mixing an analyte, an aqueous solution of formic acid or trifluoroacetic acid, and matrix liquid; a mass spectrum acquisition step of performing mass spectrometry on the sample to acquire a mass spectrum; and a detection step of detecting whether or not the sample contains the emetic poison of the Bacillus cereus on the basis of a peak of a potassium ion addition molecule derived from the emetic poison of the Bacillus cereus.SELECTED DRAWING: None

Description

The present invention relates to a method for detecting Bacillus cereus vomiting toxin.

Bacillus cereus (hereinafter referred to as B. cereus or Bacillus cereus) is known as a causative bacterium of vomiting-type food poisoning. Vomiting-type food poisoning caused by Bacillus cereus is caused by ingesting the vomiting poison called cereulide produced by this bacterium. Cereulide is a cyclic depsipeptide consisting of amino acids and oxyacids. By detecting such cereulide in addition to detecting Bacillus cereus, it is possible to identify that Bacillus cereus is the causative agent of vomiting-type food poisoning.

As a cereulide detection method, a method for confirming cytotoxicity and an analysis method by LC / MS (liquid chromatography / mass spectrometer) are known. For example, in Non-Patent Document 1, cereulide is extracted by adding methanol to the supernatant of the culture solution of the cereulide strain or a food sample, and the vacuum-dried product of the cereulide extract is purified by a solid-phase extraction column to prepare a sample. Then, a method of LC / MS measurement of the sample and detecting cereulide from whether or not an ammonium ion-added molecule peak is detected in the spectrum is disclosed. Further, Non-Patent Document 1 discloses a method for determining the vacuolarization denaturing activity of cereulide on HEp-2 cells and detecting cereulide. Specifically, the supernatant obtained by centrifuging the culture solution of the Seleus strain is mixed with the suspension solution of HEp-2 cells, cultured at 37 ° C. for 24 hours, and then cell vacuoling degeneration (cytotoxicity) under an inverted microscope. ) Is present.

In addition, some Bacillus cereus do not produce cereulide. Therefore, when Bacillus cereus is detected to identify the causative agent of vomiting-type food poisoning, not only is Bacillus cereus detected in foods, but also the presence or absence of cereulide-producing ability and production conditions of Bacillus cereus isolated from foods, etc. Confirmation is important. For example, Patent Document 1 discloses a method for discriminating between a cereulide-producing strain and a non-cereulide-producing strain by confirming the expression of a vomiting venom synthase gene in Bacillus cereus. Specifically, mRNA transcribed from vomiting toxin synthase is amplified by RT-PCR (reverse transcription polymerase PCR reaction) using reverse transcriptase and a specific primer set, and the test target strain and standard strain (seleuride-producing strain) are used. Based on the production curve of the amount of nucleic acid of (and non-producing strains), it is identified whether or not the strain to be tested is a seleurid-producing strain. When the strain to be tested is determined to be a cereulide-producing strain, Bacillus cereus is determined to be the causative agent of vomiting-type food poisoning.

"Food Hygiene Inspection Guideline Microorganisms Revised 2nd Edition 2018" by Japan Food Hygiene Association Published by Japan Food Hygiene Association March 25, 2018

Japanese Patent No. 5146978

However, the method using LC / MS disclosed in Non-Patent Document 1 lacks convenience and speed because pretreatment and LC / MS measurement are complicated and time-consuming. In addition, the method for confirming the presence or absence of cytotoxicity disclosed in Non-Patent Document 1 tends to cause individual differences in the determination of cell vacuolation degeneration and lacks objectivity. Furthermore, since it takes days for pre-culture of cells and confirmation culture for vacuolar denaturation, it lacks rapidity.

In the method disclosed in Patent Document 1, the work of identifying whether or not the strain to be tested is a cereulide-producing strain is complicated, and the RT-PCR reaction takes time, so that it lacks convenience and speed. Further, depending on the Bacillus cereus strain, there is a strain that does not produce cereulide even though it has a vomiting toxin synthase gene. Therefore, in the method disclosed in Patent Document 1, cereulide does not actually exist. Nevertheless, cereulide can be mistakenly determined to be present (ie, the cause of food poisoning). Therefore, there is a demand for a method capable of detecting cereulide easily and quickly.

Therefore, the present invention has been made in view of the above problems, and an object of the present invention is to provide a method for detecting Bacillus cereus vomiting poison, which can easily and quickly detect the vomiting poison produced by Bacillus cereus.

The method for detecting Bacillus cereus vomiting toxin according to the present invention includes a sample preparation step of mixing a sample, an aqueous solution of formic acid or trifluoroacetic acid, and a matrix solution to prepare a sample, and mass spectrometry of the sample to obtain a mass spectrum. A step of acquiring a mass spectrum to be acquired, and a detection step of detecting whether or not the sample contains Bacillus cereus vomiting poison based on the peak of a potassium ion addition molecule derived from Bacillus cereus vomiting poison in the mass spectrum. Has.

According to the present invention, cereulide, which is a vomiting venom of Bacillus cereus, can be detected easily and quickly. In this way, since Bacillus cereus is detected by detecting cereulide, false positives can be suppressed, and rapid identification of Bacillus cereus having a cereulide-producing ability and rapid evaluation of cereulide production conditions become possible. Therefore, it is possible to appropriately respond to the cause investigation when food poisoning occurs or when a microbial contamination accident occurs in a product.

FIG. 1 is a diagram showing the culture temperature dependence of the mass spectrum of a sample (cereulide standard product) prepared from a cereulide standard solution and the mass spectrum of a food-derived Bacillus cereus strain (Cereulide-producing Bacillus cereus strain MEP1901204). In the mass spectrum of the cereulide standard product, [M + H] ⁺ is a hydrogen ion-added molecule peak (M is a cereulide molecule), [M + Na] ⁺ is a sodium ion-added molecule peak, and [M + K] ⁺ is a potassium ion-added molecule peak.

(1) Outline of the present invention As a result of intensive studies, the present inventor suspended a sample collected from a colony of bacterial cells such as Bacillus cereus cultured under various culture conditions in an aqueous solution of formic acid or trifluoroacetic acid. Cereus is measured by using matrix-assisted laser desorption / ionization flight time mass spectrometry (called MALDI-TOF MS) to measure a sample prepared by mixing a matrix solution in which a matrix for mass spectrometry is dissolved. We have found that cereulide as a bacterial vomiting poison can be easily and quickly detected as a potassium ion-added molecule, and have reached the present invention. The invention described below is based on this finding. It should be noted that the following embodiments are merely examples for explanation, and the present invention is not limited to the following embodiments, and the present invention also includes those embodiments to which a person skilled in the art appropriately modifies the design. As long as it has the characteristics of, it is included in the scope of the present invention.

(2) Method for detecting Bacillus cereus vomiting toxin according to the embodiment of the present invention In the method for detecting Bacillus cereus vomiting toxin according to the embodiment of the present invention, a sample, an aqueous solution of formic acid or trifluoroacetic acid, and a matrix solution are mixed. A sample preparation step for preparing a sample, a mass spectrum acquisition step for mass-analyzing the prepared sample to acquire a mass spectrum, and a sample based on the peak of potassium ion-added molecule derived from cereulide in the obtained mass spectrum. It has a detection step of detecting whether or not Bacillus cereus vomiting toxin is contained.

First, a sample to be tested by the method for detecting Bacillus cereus vomiting toxin of the present embodiment will be described. The sample is a culture of a food to be inspected for whether or not it contains Bacillus cereus having a cereulide-producing ability and cells collected from the surrounding environment (manufacturing factory, processing place, etc.) of the food. In this embodiment, a sample obtained by collecting colonies formed on the medium is used.

Next, the sample preparation process will be described. In the sample preparation step, first, an aqueous solution of formic acid or trifluoroacetic acid having a predetermined concentration is prepared. Next, the sample is put into the prepared aqueous solution of formic acid or trifluoroacetic acid and suspended to prepare a suspension. Next, the matrix solution in which the matrix for mass spectrometry is dissolved is mixed with the suspension. Finally, the suspension mixed with the matrix solution is dropped onto the plate, and the droplets of the suspension on the plate are dried to prepare a sample.

In this way, by pretreating the sample with an aqueous solution of formic acid or trifluoroacetic acid in the sample preparation step, the shape of the potassium ion-added molecule peak derived from cereulide appearing in the mass spectrum described later becomes sharp and the peak intensity becomes high. , It becomes easier to detect the peak. The concentration of the aqueous solution is not particularly limited, but the concentration of the trifluoroacetic acid aqueous solution is preferably 1 volume (V / V)%. The concentration of the formic acid aqueous solution is preferably 30% by volume. By using a formic acid aqueous solution having a concentration of 30% by volume, the shape of the peak becomes sharper, the intensity becomes higher, and the peak becomes easier to detect. The concentration of the formic acid aqueous solution can tolerate an error of about ± 10%, and if it is in the range of 27% by volume to 33% by volume, the same effect as that of the formic acid aqueous solution having a concentration of 30% by volume is obtained.

As the matrix for mass spectrometry, cinapinic acid or HCCA: α-cyano-4-hydroxycinnamic acid (α-cyano-4-hydroxycinnamic acid) can be used. Sinapinic acid or HCCA is preferably used as the matrix for mass spectrometry, but other matrix reagents generally used for sample preparation of MALDI-TOF MS may be used. The matrix liquid is a solution of such a matrix reagent in an organic solvent containing an acid or the like. The matrix solution is prepared, for example, by dissolving 10 mg of sinapic acid in 1 mL of an acetonitrile solution containing trifluoroacetic acid at a concentration of 1% and a concentration of 50%.

Subsequently, the mass spectrum acquisition step will be described. In the mass spectrum acquisition step, the sample prepared in the sample preparation step is mass-analyzed by MALDI-TOF MS to acquire a mass spectrum. Specifically, the sample is mass-analyzed using a MALDI-TOF MS device to acquire the mass spectrum of the sample. The measurement conditions of the MALDI-TOF MS device are appropriately set according to the device used for the measurement.

Finally, the detection process will be described. In the detection step, whether the sample contains cereulide based on the peak of the potassium ion-added molecule derived from cereulide (hereinafter, simply referred to as the potassium ion-added molecule peak) in the mass spectrum of the sample acquired in the mass spectrum acquisition step. Detect whether or not. Specifically, if a potassium ion-added molecule peak appears in the mass spectrum, it is detected that the sample contains cereulide, and if a potassium ion-added molecule peak does not appear, the sample contains cereulide. Detect that it is not. In this way, in the detection step, the presence or absence of cereulide in the sample is detected.

The potassium ion-added molecular peak is the peak corresponding to the cereulide to which potassium ion is added and ionized. The peak has a mass-to-charge ratio (m / z) corresponding to the mass of cereulide ionized by the addition of potassium ions, and appears in the mass spectrum in the vicinity of the mass-to-charge ratio m / z 1190.780. In the present embodiment, when a peak appears in the region where the mass-to-charge ratio m / z is 1187.370 to 1194.190 in the mass spectrum, the peak is regarded as a potassium ion-added molecule peak and cereulide is detected. I have to. By doing so, confusion with peaks derived from other substances can be suppressed, and cereulide can be rapidly detected as a potassium ion addition molecule.

In this way, when it is detected that the sample contains cereulide, it is possible to identify that Bacillus cereus having the ability to produce cereulide exists in the test target from which the cells to be cultured are collected. The route of Bacillus cereus contamination can be identified from the sampled material or location of the cells, and appropriate measures such as eradication can be taken.

(3) Action and effect The method for detecting Bacillus cereus vomiting toxin in the embodiment includes a sample preparation step of mixing a sample, an aqueous solution of formic acid or trifluoroacetic acid, and a matrix solution to prepare a sample, and MALDI- Whether the sample contains cereulide based on the mass spectrometric acquisition step of mass spectrometric analysis by TOF MS and the peak of potassium ion addition molecule derived from cereulide as Bacillus cereus vomiting toxin in the mass spectrum. It has a detection step of detecting whether or not it is present.

Therefore, the method for detecting Bacillus cereus vomiting toxin of the embodiment is based on the peak of potassium ion-added molecule derived from cereulide by preparing a sample by mixing a sample, an aqueous solution of formic acid or trifluoroacetic acid, and a matrix solution. Since the presence or absence of cereulide is detected, it is not necessary to perform treatments such as centrifugation and fractionation, and filtering for extracting cereulide from the sample and separating cereulide from the cereulide extract. In addition, since a cleaning process for removing impurities such as lipids and proteins is not required, the operation in the sample preparation step is very simple. Further, the method for detecting Bacillus cereus vomiting toxin in the embodiment does not require complicated measurement such as LC / MS or RT-PCR, and simply mass spectrometrically analyzes the sample by MALDI-TOF MS. Therefore, the method for detecting Bacillus cereus vomiting toxin of the embodiment can detect cereulide easily and quickly.

Further, in the method for detecting Bacillus cereus vomiting toxin of the embodiment, since the presence of Bacillus cereus is identified by detecting cereulide, false positives due to detecting Bacillus cereus having no ability to produce cereulide can be suppressed, and cereulide can be suppressed. It enables rapid identification of Bacillus cereus having a producing ability. In addition, since the method for detecting Bacillus cereus vomiting toxin of the embodiment can rapidly test the sample, the sample collected from the colony of Bacillus cereus cultured under various culture conditions can be tested to accelerate the cereulide production condition. Evaluation becomes possible. Therefore, it is possible to appropriately respond to the cause investigation when food poisoning occurs or when a microbial contamination accident occurs in a product.

(4) Verification experiment (4-1) Confirmation of cereulide-derived peaks First, in order to confirm the cereulide-derived peaks appearing in the mass spectrum obtained by MALDI-TOF MS, the cereulide standard solution was measured by MALDI-TOF MS. First, a cereulide standard solution having a concentration of 5 μg / mL (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd .: prepared by diluting a commercially available product (methanol solution) with acetonitrile) to 3.0 μL and 12.0 μL of a sinapic acid matrix solution (composition is (Shown in Table 1) was mixed, 1.5 μL of which was added dropwise to a MALDI plate and air-dried to prepare a sample of a cereulide standard solution. The air-dried sample was mass-analyzed with a MALDI-TOF MS device. The measuring device and measuring conditions are as shown in Table 2.

The mass spectrum obtained by measuring a sample of the cereulide standard solution (cereulide standard product) is shown at the top of FIG. The horizontal axis of FIG. 1 is the mass-to-charge ratio (m / z), and the vertical axis is the intensity (arbitrary unit). In the mass spectrum, as cereulide-derived peaks, a hydrogen ion-added molecular peak in which hydrogen ions are added to cereulide ([M + H] ⁺ , M is a cereulide molecule) and a sodium ion-added molecular peak in which sodium ions are added to cereulide ([M + Na] ] ⁺ ) And a potassium ion-added molecular peak ([M + K] ⁺ ) in which potassium ions were added to cereulide were observed. Of these, the intensity of the potassium ion-added molecule peak was the highest. Therefore, the present invention detects the presence or absence of cereulide in a sample by using the potassium ion-added molecule peak as an index.

(4-2) Verification of Detection Method of Bacillus Cereus Vomiting Poison of the Present Invention Next, it was confirmed whether or not the presence or absence of cereulide can be detected by the detection method of Bacillus cereus vomiting poison of the present invention. Specifically, 7 strains of Bacillus cereus isolated from food, 1 strain of Bacillus cereus not carrying the vomiting toxin synthase (CRS) gene (reference strain) as a negative control, B. thuringiensis, B. mycoides and B. mycoides closely related to Bacillus cereus. B. Weihenstephanensis Specimens were collected from colonies obtained by culturing a total of 11 strains of 1 strain each (each reference strain), and it was confirmed whether or not the presence or absence of cereulide in the collected specimens could be detected by the method of the present invention. ..

First, the presence or absence of the CRS gene in the above 11 strains was confirmed using the CycleavePCR Bacillus cereus (CRS gene) Detection Kit (manufactured by Takara Bio Inc.). This kit detects cereulide-producing Bacillus cereus by detecting the CRS gene. Here, using this kit, the presence or absence of the CRS gene was determined. The results are shown in Table 3. As shown in Table 3, 6 of the 7 strains of Bacillus cereus isolated from food (MEP1901201 to MEP1901206) carried the CRS gene.

Next, six food-derived Bacillus cereus strains (MEP1901201 to MEP1901206) confirmed to carry the CRS gene and a negative control CRS gene-non-carrying Bacillus cereus strain (reference strain) actually have the ability to produce cereulide. In order to confirm whether or not it was present, the cereulide concentration in the culture supernatant of Bacillus cereus was measured using LC / MS. Specifically, 6 strains of food-derived Bacillus cereus (MEP1901201 to MEP1901206) and a reference strain are inoculated into 50 mL of 10% skim milk powder medium (manufactured by Wako Pure Chemical Industries, Ltd.), shake-cultured at 30 ° C. for 24 hours, and then the culture solution is prepared. Collected in a centrifuge tube. The culture solution collected in the centrifuge tube was centrifuged (1,600 × g, 10 minutes) to collect the culture supernatant, which was heated at 100 ° C. for 10 minutes. Then, 2.5 mL of the culture supernatant was collected in a centrifuge tube, 2.5 mL of acetonitrile and 2.5 mL of n-hexane were added, and the mixture was shaken for 10 minutes. The n-hexane layer was removed, and 2.5 mL each of acetonitrile and n-hexane were added again to the remaining aqueous layer and shaken for 10 minutes. The n-hexane layers were combined and concentrated using a nitrogen concentrator. The concentrate was dissolved in 500 μL of acetonitrile and measured by LC / MS (Agilent SL, manufactured by Agilent Technologies) through a 0.22 μm filter. The measuring device and measuring conditions are as shown in Table 4. The measured cereulide concentration in the culture supernatant of 7 strains of Bacillus cereus is the cereulide standard solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd .: a commercially available product (methanol solution) is diluted with acetonitrile to prepare multiple standard solutions with cereulide concentration). It was calculated from the calibration curve created based on the measurement result of. The results are shown in Table 3 above. In addition, n. d. Means that cereulide was not detected (detection limit 5 ppb). t. Means that the cereulide concentration in the culture supernatant has not been measured.

As shown in Table 3, it was confirmed that cereulide was detected in 5 of the 6 strains of Bacillus cereus carrying the food-derived CRS gene (MEP1901201 to MEP1901205) and that they had the ability to produce cereulide. In addition, it was confirmed that cereulide was not detected in the negative control CRS gene-free Bacillus cereus strain (reference strain) and one of the six food-derived CRS gene-bearing Bacillus cereus strains (MEP1901206), and that they had no ability to produce cereulide. It was. JCM in Table 3 means that the strain is obtained from the Japan Collection of Microorganisms, JCM, RIKEN BioResource Center, and NBRC is the source of the strain. Means that is the Biotechnology Center of the Product Evaluation Technology Infrastructure Organization.

It was confirmed that the food-derived CRS gene-bearing Bacillus cereus strain MEP1901206 and the reference strain did not have the ability to produce cereulide as described above, and the food-derived CRS gene-non-carrying Bacillus cereus strain (MEP1901207) and the CRS gene closely related to Bacillus cereus were confirmed. The three non-carrying Bacillus cerei strains do not carry the CRS gene and therefore do not have the ability to produce cereulide. Therefore, Bacillus cereus strains 6 including 5 strains of Bacillus cereus (MEP1901201 to MEP1901205) confirmed to have the ability to produce cereulide and Bacillus cereus strain (MEP1901206) having the CRS gene derived from foods confirmed to have no ability to produce cereulide. With respect to the strain, it was verified whether or not the presence or absence of cereulide could be accurately identified by the method for detecting Bacillus cereus vomiting toxin of the present invention.

First, the above 11 strains were smeared on different standard agar plates (SMA: Standard Method Agar, manufactured by Eiken Chemical Co., Ltd.) and cultured at 30 ° C. for 16 hours. The colonies formed on the agar plate were collected as a sample, the sample was suspended in a 3.0 μL aqueous solution of formic acid (concentration: 30% by volume, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and the composition shown in Table 1 was further prepared. 12.0 μL of the cinapinic acid matrix solution was added and mixed. A sample was prepared by dropping 1.5 μL of this mixed solution onto a MALDI plate and air-drying. This sample was prepared for each strain. Further, the matrix solution was changed to the HCCA matrix solution having the composition shown in Table 1, and a sample was prepared for each strain in the same manner. In addition to the above, 5 strains of Bacillus cereus (MEP1901201 to MEP1901205) having the ability to produce cereulide were cultured three times in total on different days, and samples were prepared in the same manner using a sinapic acid matrix solution.

Each of the prepared samples was mass-analyzed with a MALDI-TOF MS device to obtain a mass spectrum. The measuring device and measuring conditions are as shown in Table 2. After that, in the acquired mass spectrum, the mass-to-charge ratio of the observed peak was compared with the mass-to-charge ratio of the potassium ion-added molecule peak observed in the cereulide standard solution, and the potassium ion-added molecule peak derived from cereulide was identified. did. This work was performed on all samples prepared. The results are shown in Table 3.

As shown in Table 3, for all five strains of Bacillus cereus (MEP1901201 to MEP1901205) capable of producing cereulide, peaks of potassium ion-added molecules derived from cereulide were observed. On the other hand, no potassium ion addition molecule peak derived from cereulide was observed in all 6 strains of Bacillus spp. That do not have the ability to produce cereulide, including 2 strains of food-derived Bacillus cereus (MEP1901206, MEP1901207). As described above, it was confirmed that the presence or absence of cereulide in the sample can be detected by the method of the present invention. The mass-to-charge ratio m / z of the peak of the observed potassium ion-added molecule was 119.780 on average and 0.341 standard deviation. Therefore, even if the measurement error is taken into consideration, the peak of the potassium ion-added molecule of seleuride has a mass-to-charge ratio m / z of 1187.370 to 1194.190 (mean value of mass-to-charge ratio m / z of 119.780 ± 10SD (standard). It is considered that it is detected in the range of 10 times the deviation)).

(4-3) Confirmation of cereulide production under various culture conditions In order to confirm whether or not cereulide can be detected regardless of the medium, assuming culture conditions such as the Bacillus cereus test method listed in the Food Hygiene Inspection Guideline. , 5 strains of cereulide-producing Bacillus cereus (MEP1901201 to MEP1901205) and 3 strains of non-cereulide-producing Bacillus cereus (reference strains, MEP1901206, MEP1901207) were cultured using various media to accurately determine the cereulide-producing Bacillus cereus strain and the cereulide-non-producing Bacillus cereus strain. It was confirmed whether it could be identified. The media used were LB: Lysogeny Broth medium (preparation medium), SMA medium (manufactured by Eiken Kagaku Co., Ltd.), MYP: Mannitol Yolk Polymixin medium (manufactured by Nissui Pharmaceutical Co., Ltd.), MYP medium (BD: manufactured by Becton Deckonson). , NGKG: NaCl Glycine Kim Goepfect agar medium (manufactured by Eiken Kagaku Co., Ltd.), all of which are flat plates. The culture conditions are as shown in Table 5, and the sample preparation conditions (using the sinapic acid matrix solution), the measuring device for MALDI-TOF MS, and the measuring conditions are the same as in the above verification experiment. For media other than LB medium, samples were collected from the medium immediately after culturing and the medium stored in a refrigerator for 1 week after culturing. The results are shown in Table 5.

As shown in Table 5, peaks of potassium ion-added molecules derived from cereulide were observed in all cereulide-producing Bacillus cereus strains (MEP1901201 to MEP1901205) immediately after culturing and after refrigeration for 1 week, and all cereulide-non-producing Bacillus cereus strains (reference). No peak of potassium ion addition molecule was observed in the strains, MEP1901206, MEP1901207). Therefore, it was confirmed that cereulide production can be detected from the colonies formed on various media (Bacillus cereus test plate) by the method of the present invention. In addition, it was confirmed that the medium in which the colonies were formed could be detected without any problem even when stored in a refrigerator.

(4-4) Use of Bacillus cereus as a method for confirming cereulide production conditions The method for detecting Bacillus cereus vomiting toxin of the present invention was used for confirming the conditions for confirming the conditions for Bacillus cereus production. Bacillus cereus was cultivated at different culture temperatures, and the presence or absence of cereulide was detected in the collected sample by the method for detecting Bacillus cereus vomiting toxin, and the relationship between the culture temperature and cereulide production was investigated.

Specifically, the cereulide-producing Bacillus cereus strain (Food-derived CRS gene-bearing Bacillus cereus strain MEP1901204 in Table 3) was smeared on four standard agar plates (SMA, Eiken Kagaku), and each standard agar plate was smeared at a different temperature (12). Culturing at ° C., 15 ° C., 30 ° C., 42 ° C. for 4 days. A sample was collected from the colonies formed on each agar plate, a sample was prepared using a sinapic acid matrix solution as a matrix solution, and the sample was mass-analyzed by a MALDI-TOF MS apparatus. The preparation conditions, measuring device, and measuring conditions of other samples are the same as those in the above verification experiment. Then, in the obtained mass spectrum, it was determined whether or not cereulide was produced based on the presence or absence of a peak of the potassium ion addition molecule derived from cereulide. The mass spectrum obtained by MALDI-TOF MS measurement is shown in FIG.

As shown in FIG. 1, when cultured at 12 ° C., 15 ° C., and 30 ° C., a peak was observed in the mass-to-charge ratio m / z range of 1187.370 to 1194.190, and potassium derived from cereulide was observed in the mass spectrum. There was a peak of ion-added molecules, and it was confirmed that cereulide was produced. On the other hand, when cultured at 42 ° C., no peak was observed in the mass-to-charge ratio m / z range of 1187.370 to 1194.190, there was no peak of potassium ion-added molecule in the mass spectrum, and cereulide was produced. I was able to confirm that there was no such thing. The results of this experiment show that cereulide production is not observed at culture temperatures above 40 ° C. (Kranzler et al. “Temperature exerts control of Bacillus cereus emetic toxin production on post-transcriptional levels.” Front Microbiol. 7: 1640 2016.) was matched. As described above, the method of the present invention was also useful as a method for confirming the cereulide production conditions easily and quickly.

Claims (8)

A sample preparation step of mixing a sample, an aqueous solution of formic acid or trifluoroacetic acid, and a matrix solution to prepare a sample.
A mass spectrum acquisition step of mass spectrometrically analyzing the sample to acquire a mass spectrum,
Detection of Bacillus cereus vomiting toxin having a detection step of detecting whether or not the sample contains Bacillus cereus vomiting toxin based on the peak of a potassium ion addition molecule derived from Bacillus cereus vomiting toxin in the mass spectrum. Method. The method for detecting Bacillus cereus vomiting poison according to claim 1, wherein the concentration of the aqueous formic acid solution is 27 to 33% by volume. The method for detecting Bacillus cereus vomiting poison according to claim 1 or 2, wherein the concentration of the aqueous formic acid solution is 30% by volume. The sample preparation step is
A step of suspending the sample in the aqueous formic acid solution to prepare a suspension, and
The step of mixing the matrix liquid with the suspension and
The Bacillus cereus vomiting poison according to any one of claims 1 to 3, which comprises a step of dropping the suspension mixed with the matrix solution onto a plate and drying the suspension on the plate. Detection method. The method for detecting Bacillus cereus vomiting toxin according to any one of claims 1 to 4, wherein the mass spectrum acquisition step acquires the mass spectrum by matrix-assisted laser desorption / ionization time-of-flight mass analysis. The Bacillus cereus vomiting poison according to any one of claims 1 to 5, wherein the detection step detects Bacillus cereus vomiting poison based on the peak in which the mass-to-charge ratio appears in the range of 1187.370 to 1194.190. Detection method. The method for detecting Bacillus cereus vomiting toxin according to any one of claims 1 to 6, wherein the matrix solution contains sinapic acid or α-cyano-4-hydroxycinnamic acid. The method for detecting Bacillus cereus vomiting toxin according to any one of claims 1 to 7, wherein the Bacillus cereus vomiting toxin is cereulide.

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