JP2013223438A - Detection method and detection kit for cereulide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new cereulide detection method capable of obtaining a result conveniently and promptly and not needing an expensive measuring instrument.SOLUTION: A detection method of cereulide includes a process for heating a sample with a possibility that cereulide exists therein, a process for culturing cereulide sensitive bacteria in the presence of the sample obtained by the heating process, and a process for detecting, in the culture obtained by the culturing process, cereulide in the sample based on the antibacterial action of cereulide to the cereulide sensitive bacteria.

Description

  The present invention relates to a detection method and a detection kit for cereulide, an emetic toxin produced by Bacillus cereus (hereinafter also referred to as “Cereus bacterium”).

  Bacillus cereus is a spore-forming rod that is widely distributed from the natural environment such as soil to the intestines of feed and livestock, and is known to have many opportunities to contaminate food. There are two types of food poisoning due to Bacillus cereus: diarrhea type due to enterotoxin toxin and vomiting type due to cereulide toxin. Most food poisoning due to Bacillus cereus occurring in Japan is vomiting type. Bacillus cereus is known to form heat-resistant spores, and the surviving spores germinate and multiply in the cooked food, causing food poisoning.

  Cereulide, an emetic toxin, is a cyclic peptide with a molecular weight of 1,153 that is biosynthesized by cereulide synthase from Bacillus cereus. This cereulide itself is also very resistant to heat and is resistant to strong acids and bases, as well as to proteolytic enzymes. Therefore, even if Bacillus cereus is killed in food, food poisoning can occur if cereulide is present in a certain amount.

  In the inspection of food poisoning by Bacillus cereus, the number of bacteria in foods suspected of being contaminated by the bacteria is calculated by a plate culture method, and the cause of food poisoning has been determined based on the result. On the other hand, among the Cereus strains, there are many non-pathogenic strains that do not produce cereulide (non-cereulide producing strains) in the natural environment. It cannot be distinguished from a cereulide-producing strain of Bacillus cereus. Moreover, although the direct cause of food poisoning is cereulide, the presence of Bacillus cereus itself does not provide evidence that there is a sufficient amount of cereulide to cause food poisoning. There is therefore a need to detect and quantify cereulide itself.

  For the detection of cereulide, a biological test based on observation of vacuolar degeneration of HEp-2 cells derived from human laryngeal cancer has been conventionally used as a standard test method. However, in this test, it is necessary to maintain HEp-2 cells at all times, and the skill of the examiner is also required, so it cannot be said that the test is simple. In addition, this test lacks rapidity because it takes about 4 days to obtain a result.

  In recent years, cereulide is also detected by physicochemical tests using liquid chromatography and mass spectrometry. However, such a physicochemical test can obtain a result quickly, but requires an expensive measuring instrument.

  Recently, a method for detecting whether or not Bacillus cereus has a cereulide synthase gene by the PCR method has also been developed (see Patent Document 1). However, this method is a method for examining whether or not Bacillus cereus is a cereulide-producing strain, and is not a method for detecting cereulide itself.

International Publication No. 2003/097821

  As described above, various tests are used for the detection of cereulide. However, it is desired to develop a test method that can obtain a result easily and quickly and does not require an expensive measuring instrument. In view of such circumstances, the present inventor aimed to provide a novel detection method and detection kit for cereulide.

  The present inventor has discovered that cereulide, an emetic toxin, exhibits an antibacterial action against a predetermined bacterial strain different from Bacillus cereus. The present inventor found that cereulide can be detected by using the antibacterial action of cereulide against the bacterial strain as an index, and completed the present invention.

Thus, the present invention
Heating a sample in which cereulide may be present;
Culturing cereulide-sensitive bacteria in the presence of the sample obtained in the heating step, and
And a step of detecting cereulide in the sample based on the antibacterial action of cereulide against the cereulide-susceptible bacteria in the culture obtained in the culturing step.

  The present invention also provides a cereulide detection kit containing a cereulide-sensitive bacterium.

  According to the cereulide detection method of the present invention, cereulide in a sample can be easily and rapidly detected. Moreover, the cereulide detection kit of the present invention can be suitably used for the cereulide detection method of the present invention.

It is a photograph which shows the inhibition circle | round | yen which arose on the solid medium which culture | cultivated a cereulide sensitive microbe in presence of the concentrate of the culture supernatant of a Bacillus cereus BC1 (+) strain | stump | stock.

(1) Method for detecting cereulide [heating step]
In the cereulide detection method of the present invention (hereinafter also simply referred to as “detection method”), a sample in which cereulide may be present is first heated.

  In the embodiment of the present invention, the sample in which cereulide may be present is not particularly limited. For example, food and feed suspected of being contaminated with Bacillus cereus, a sample collected from a living body suspected of food poisoning due to Bacillus cereus And culture supernatant of Bacillus cereus. Examples of specimens collected from a living body include vomit, excrement, saliva, digestive juice and the like. In a particularly preferred embodiment, a liquid sample that can contain cereulide obtained from the above sample by a method known in the art is used. Examples of such a liquid sample include a supernatant obtained by mixing the above sample and an aqueous medium and centrifuging the obtained mixed liquid. Examples of the aqueous medium include water, a buffer solution, and the like, and preferably a phosphate buffered saline for food dilution defined in the food hygiene inspection guidelines.

  Furthermore, in order to improve the detection sensitivity of cereulide, cereulide in the liquid sample may be concentrated by a known method. That is, in the embodiment of the present invention, the detection method may further include a step of concentrating cereulide from a sample in which cereulide may be present. The solvent for such concentration is not particularly limited as long as it is a solvent capable of dissolving cereulide. For example, lower alcohols having 1 to 3 carbon atoms, ethyl acetate and the like are preferably used.

  As described above, in the detection method of the present invention, cereulide in a sample is detected based on the antibacterial action of cereulide against a cereulide-sensitive bacterium described later. However, cereulide does not exhibit antibacterial activity against Bacillus cereus that produces it. Therefore, when Bacillus cereus remains in the sample, accurate detection is hindered. Therefore, in the detection method of the present invention, the above sample is heated under conditions that allow sterilization of Bacillus cereus.

In the embodiment of the present invention, the heating temperature is not particularly limited as long as it is a temperature at which Bacillus cereus can be sterilized, but is usually 75 to 130 ° C, preferably 100 to 121 ° C. Moreover, the heating time at this heating temperature is not particularly limited as long as the Bacillus cereus dies, but it is usually 10 to 60 minutes, preferably 15 to 20 minutes.
In the embodiment of the present invention, the sample heating method is not particularly limited, but it is preferable to heat using a device capable of setting the heating temperature and time. An example of such an apparatus is an autoclave.

  Here, it is known that there are strains of Cereus strains that produce polypeptides that exhibit antibacterial activity against bacterial strains different from self, called bacteriocin, in addition to cereulide, an emetic toxin. ing. Selein is known as a bacteriocin of Bacillus cereus, but selein may have an antibacterial action against cereulide-sensitive bacteria described later. On the other hand, it is known that bacteriocins such as selein are easily inactivated by heating, unlike cereulide. Therefore, according to said heating process, the effect which inactivates antibacterial substances other than cereulide like bacteriocin, and prevents the false positive by these antibacterial substances is also acquired. In addition, bacteriocin can be sufficiently inactivated by heating at a temperature at which the Bacillus cereus can be sterilized.

[Culture process]
Next, in the detection method of the present invention, cereulide-sensitive bacteria are cultured in the presence of the sample obtained in the heating step (hereinafter also referred to as “heated sample”).

  In the present specification, the “cereulide-susceptible bacterium” refers to a bacterium having the property that its survival and / or growth is inhibited by cereulide. Therefore, in this culturing step, when cereulide is present in the heated sample, the survival and / or growth of the cereulide-sensitive bacterium is inhibited.

  In the embodiment of the present invention, the cereulide-susceptible bacterium is not particularly limited as long as it is a bacterium having the above properties, but is preferably a non-pathogenic bacterium from the viewpoint of safety. Such a cereulide-susceptible bacterium is preferably selected from bacteria belonging to the genus Bacillus excluding the cereulide-producing strain Bacillus cereus, and examples thereof include Bacillus sporothermodurans. Further, the cereulide-susceptible bacterium may be a naturally occurring bacterium or a mutant mutated by a known method so that the cereulide sensitivity of the bacterium is enhanced. In addition, about the acquisition means of a cereulide sensitive microbe, when this bacterium itself is well-known, you may isolate | separate and obtain from the environment in which this bacterium inhabits, or you may obtain it from a microbial strain preservation organization.

  In the present invention, since antibacterial activity has been found in cereulide, the presence or absence of cereulide sensitivity of a certain bacterial strain can be easily determined by subjecting the bacterial strain to a known proliferation assay using a cereulide standard. Can be confirmed. Here, the cereulide standard product can be obtained by heat-treating commercially available purified cereulide or cereulide extracted and purified from a culture supernatant of a cereulide-producing strain of Bacillus cereus by a known method as described above.

  The medium used in the detection method of the present invention may be appropriately selected from bacterial media known in the art according to the type of cereulide-sensitive bacteria. In the embodiment of the present invention, the medium may be a liquid medium or a solid medium such as a plate medium, but a solid medium is preferable from the viewpoint of ease of operation. In addition, as an example of a culture medium, when a cereulide sensitive bacterium is a Bacillus sporothermodurance, a brain heart infusion culture medium, tryptosoya agar culture medium, etc. are mentioned.

  The present inventor has found that by adding a potassium salt to the medium, the details of the mechanism are unknown, but the detection sensitivity of cereulide is improved. Therefore, in the embodiment of the present invention, it is preferable to use a medium containing potassium salt (particularly a solid medium). In the embodiment of the present invention, the potassium salt is not particularly limited as long as it does not affect the culture of cereulide-sensitive bacteria, and can be appropriately selected from salts of potassium and inorganic acid or organic acid. For example, potassium chloride, potassium sulfate, potassium dihydrogen phosphate, potassium hydrogen tartrate and the like can be mentioned, among which potassium chloride is particularly preferable. Although the density | concentration of the potassium salt in a culture medium can be suitably determined according to the kind of potassium salt, culture | cultivation conditions, etc., when using potassium chloride, it is 25 mM-1M normally, Preferably it is 100-200 mM.

  In the culturing step, the culturing method is not particularly limited, and may be appropriately selected from known bacterial culturing methods according to the type of cereulide-sensitive bacteria and the antibacterial action evaluation means described below. For example, when a solid medium is used, cereulide-sensitive bacteria are cultured on the surface of the solid medium to which the heated sample is applied, or are cultured while the heated sample is in contact with the surface of the solid medium to which the cereulide-sensitive bacteria are applied. Examples of the method of bringing the heated sample into contact with the surface of the solid medium include dropping or applying the heated sample. Alternatively, a circular disk containing a heated sample may be disposed on the surface of the solid medium. In this case, a concentration gradient of the heated sample is formed around the circular disk on the solid medium.

  Since cereulide is a heat-resistant molecule as described above, a solid medium raw material and a heated sample are mixed, a solid medium is prepared from the obtained mixture, and cereulide-sensitive bacteria may be cultured on this solid medium. Alternatively, when using bacteria that form heat-resistant spores, such as Bacillus sporothermomodulus, as cereulide-susceptible bacteria, a solid medium that has been solated by heating and cereulide-susceptible bacteria are mixed, and the resulting mixture is gelled. A solid medium may be prepared and cultured by bringing a heated sample into contact with the surface of the solid medium. The mixing ratio between the solid medium and the cereulide-sensitive bacteria or the heated sample is not particularly limited as long as the bacteria can be cultured, and can be set as appropriate.

  In an embodiment of the present invention, a method of culturing cereulide-susceptible bacteria by dropping a heated sample on a solid medium or placing a circular disc containing a heated sample, wherein cereulide was present in the sample It is particularly preferred because sometimes a blocking circle is formed on the solid medium.

  Moreover, when using a liquid culture medium, this liquid culture medium and a heating sample are mixed and a cereulide sensitive microbe is culture | cultivated in the obtained mixture. In this case, the mixing ratio of the liquid medium and the heated sample is not particularly limited, but it is preferable to mix at various ratios in order to easily evaluate the antibacterial action of cereulide in the subsequent detection step.

  The culture conditions can be appropriately set according to the type of cereulide-sensitive bacteria. For example, the culture temperature is usually 25 to 35 ° C., preferably 28 to 32 ° C. when the cereulide-susceptible bacterium is Bacillus sporothermomodulus. The culture time is not particularly limited and may be cultured until the antibacterial action of cereulide can be evaluated, but 18 to 24 hours is usually sufficient.

[Detection process]
In the detection method of the present invention, cereulide in a sample is detected based on the antibacterial action of cereulide against cereulide-susceptible bacteria in the culture obtained in the culturing step.

  As used herein, “detecting cereulide” means either or both of finding the presence of cereulide and measuring the amount of cereulide.

  In the embodiment of the present invention, the antibacterial action of cereulide can be evaluated using the degree of survival and / or proliferation of cereulide-susceptible bacteria in the culture obtained in the culturing step as an index. As a specific evaluation means, when a solid medium is used, for example, confirmation of the presence or absence of a region (for example, a blocking circle) on which the bacteria on the solid medium did not propagate, measurement of the size thereof, and the like can be given. It is done. When it can be confirmed that such a region is generated, it can be determined that cereulide is present in the sample. Further, the amount of cereulide in the sample can be estimated based on the size of the region.

  In the case of using a liquid medium, for example, measurement of the number of cells (total number of bacteria and / or viable cells) in the culture, measurement of the absorbance of the culture, and the like can be mentioned. When the number of cells or the value of absorbance is lower than that of the culture in the absence of the heated sample, it can be determined that cereulide was present in the sample.

  In the embodiment of the present invention, confirmation of the inhibition circle generated on the solid medium and measurement of the size thereof are particularly preferable as a means for evaluating the antibacterial action of cereulide because the operation is simple and no measuring instrument is required.

  In addition, what is necessary is just to perform as follows when quantifying cereulide in a sample. First, in the above culturing step, cereulide-susceptible bacteria are cultured in the presence of a cereulide standard having a known concentration. Subsequently, the antibacterial action of cereulide is evaluated in the same manner as described above, and a calibration curve is prepared based on the result. Then, using the obtained calibration curve, the amount of cereulide in the sample can be determined from the result of the sample to be inspected.

(2) Cereulide Detection Kit The cereulide detection kit of the present invention (hereinafter also simply referred to as “kit”) is a kit that is preferably used in the detection method of the present invention, and includes the cereulide-sensitive bacteria described above.

  The cereulide sensitive bacterium contained in the kit of the present invention is the same as described above. In the embodiment of the present invention, the form of cereulide-susceptible bacteria as a component of the kit is not particularly limited, and may be, for example, a liquid culture medium or a frozen form thereof. In addition, when a spore-forming bacterium such as Bacillus sporothermomodulus is employed, it may be in the form of a dried product of the spore.

  The kit of the present invention may further contain a medium containing a potassium salt. The medium is the same as described above. From the viewpoint of ease of operation, the kit of the present invention preferably contains a solid medium. In the embodiment of the present invention, the kit may include a cereulide-sensitive bacterium and a medium separately according to the type of the cereulide-sensitive bacterium, or a structure including the cereulide-sensitive bacterium previously seeded in the medium. It is good.

The method for using the kit of the present invention is the same as that described above for the detection method of the present invention, but the kit of the present invention may contain instructions describing the method of use.
In an embodiment of the present invention, the kit may further contain a solvent for extracting and concentrating cereulide from the sample (for example, a C1-C3 lower alcohol, ethyl acetate, etc.), a cereulide standard as a positive control, and the like. Good. In addition, when the cereulide-sensitive bacterium is a spore dried product, the kit may further contain an aqueous solvent (for example, water, physiological saline, buffer solution, etc.) for suspending it.

  EXAMPLES The present invention will be described in detail below by examples, but the present invention is not limited to these examples.

Example 1: Examination of antibacterial action of cereulide (1) Materials (1-1) Test strains BC1 (+) and 08-151-3 strains were used as Bacillus cereus as cereulide producing strains. The 09-112-7 strain was used as Bacillus cereus, a non-cereulide-producing strain. These Cereus strains are strains obtained by the inventor during isolation of food poisoning cases. In addition, Bacillus sporothermodurance was used as a cereulide-susceptible bacterium. This sensitive bacterium is a bacterium obtained by isolation of the present inventor.

(1-2) Medium / Brain Heart Infusion (BHI) Liquid Medium Brain Heart Infusion (37 g: Fluka) is dissolved in distilled water (1 L) and sterilized in an autoclave at 121 ° C. for 15 minutes. BHI liquid medium was obtained.

・ BHI-SM liquid medium Brain Heart Infusion (37 g: Fluka) and skim milk (30 g) are dissolved in distilled water (1 L), sterilized in an autoclave at 121 ° C for 15 minutes, and BHI-SM liquid A medium was obtained.

・ Tryptosa agar medium (TSA)
Trypsawaya agar medium (40 g: Nissui Pharmaceutical) is dissolved in distilled water (1 L) and sterilized in an autoclave at 121 ° C for 15 minutes. A medium was obtained.

-TSA-KCl overlay medium Tryptosa agar medium (40 g: Nissui Pharmaceutical) and potassium chloride (Nacalai Tesque) are dissolved in distilled water (1 L) to a predetermined final concentration, and this is autoclaved at 121 ° C. Sterilized for 15 minutes to obtain a TSA-KCl overlay medium. The final concentration of potassium chloride was 200 mM, 400 mM, 600 mM, 800 mM and 1M.

(1-3) Seleulide Standard Product Purified cereulide (Biocontrol Laboratories) was used as a cereulide standard product. In addition, the cereulide standard goods as a positive control prepared 3 types, the untreated refined cereulide and the refined cereulide heat-processed for 15 minutes at 100 degreeC and 121 degreeC.

(2) Concentration of cereulide from cereulide culture supernatant
Each cereulide producing strain (300 μL) pre-cultured in BHI medium was added to BHI-SM medium (150 mL), and cultured with shaking at 30 ° C. and 120 rpm for 20 hours. The culture was centrifuged at room temperature at 8000 rpm for 15 minutes, and the resulting supernatant was filtered through a membrane filter (pore size 0.45 μm). A portion of the obtained filtrate was taken and stored, 1.5 times the amount of ethyl acetate (Wako Pure Chemical Industries) was added to the remaining filtrate, and homogenized for 1 minute using a homogenizer (Mitsui Denki Seisakusho). This was centrifuged at 12000 rpm for 10 minutes at room temperature, and the ethyl acetate fraction was collected and concentrated to dryness with an evaporator. 75% methanol (150 μL) was added to the resulting solid and dissolved by sonication to obtain a culture supernatant concentrate. The culture supernatant and concentrate of each bacterium were equally divided, one was not heated and not treated, and the other was heat treated at 100 ° C. for 15 minutes.

(3) Confirmation of cereulide by vacuolar degeneration test using HEp-2 cells Whether or not cereulide is present in the culture supernatant and concentrate obtained in (2) above (hereinafter also simply referred to as “sample”) This was examined by the vacuolar degeneration test. First, each sample was diluted with phosphate buffered saline (PBS) to prepare a 2-fold serial dilution series in a 96-well plate. Moreover, a cereulide standard product was used as a positive control. HEp-2 cells cultured to confluence are detached by trypsin treatment and suspended in 1% fetal bovine serum-containing EMEM (10 mL). Each cell suspension (approximately 10 ⁵ cells / mL) is added to each well. 100 μL each was added. This was cultured in a CO ₂ incubator at 37 ° C. for 48 hours. Thereafter, the supernatant was removed with an aspirator, and the cells were fixed with methanol. The fixed cells were stained with 10% Giemsa solution, and vacuoles were observed with a phase contrast microscope. A sample in which a cell having 10 or more vacuoles in the cytoplasm of one cell was added to a well occupying 30% or more of all cells was determined to be cereulide positive, and the others were determined to be negative. A similar test was performed using a cereulide standard as a positive control. The results are shown in Table 1 below. In the table, “+” indicates positive and “−” indicates negative.

  From Table 1, it was found that the cereulide standard product still retains the activity of inducing vacuolation of HEp-2 cells even when heated at 100 ° C and 121 ° C. In addition, the BC1 (+) strain culture supernatant and concentrate, and the 08-151-3 strain culture supernatant concentrate showed vacuolation of HEp-2 cells. Found that cereulide was present. On the other hand, in the culture supernatant and concentrate of strain 09-112-7, which is a non-cereulide-producing strain, vacuole degeneration was not observed in HEp-2 cells. I understood. These extracts and concentrates were used for the following tests.

(4) Examination of antibacterial action of cereulide It was investigated whether cereulide has an antibacterial action against Bacillus sporothermomodulus which is a cereulide sensitive bacterium.
Bacillus sporothermurance (1 mL, 1 × 10 ⁸ CFU) pre-cultured in Brainhart infusion medium was added to TSA-KCl multi-layer medium (5 mL) dissolved by heating, and the resulting mixture was mixed. Pour onto a TSA plate and harden. After the medium solidified, the above sample (10 μL) was dropped onto the obtained solid medium and cultured at 30 ° C. overnight. After the culture, it was confirmed whether or not a transparent inhibition circle was generated in the solid medium. A similar test was performed using a cereulide standard as a positive control.

  The results are shown in Table 2 below. In the table, “+” indicates that formation of a blocking circle is recognized, and “−” indicates that formation of a blocking circle is not recognized. Moreover, the photograph of the inhibition circle formed in the solid culture medium which dripped the cereulide standard goods or the culture supernatant concentrate of BC1 (+) strain heat-processed at 100 degreeC is shown in FIG.

Even when the cereulide standard product was heated at 100 ° C. and 121 ° C., a circle of inhibition occurred in the solid medium in which Bacillus sporothermomodulus was cultured. Therefore, it was found that cereulide has an antibacterial action against Bacillus sporothermomodulus. In addition, the inhibition circle was observed in the medium having any KCl concentration of 200 mM to 1 M.
On the other hand, no inhibition circle was formed in the culture supernatant and concentrate of the 09-112-7 strain, which is a non-cereulide producing strain. This result is consistent with the results of the vacuolar degeneration test described above.

  Regarding the culture supernatant of the BC1 (+) strain, an inhibition circle was formed in the non-heated sample, but no inhibition circle was produced in the heat-treated sample. On the other hand, vacuolar denaturation activity was observed in the culture supernatant of the heat-treated BC1 (+) strain. . In addition, the BC1 (+) strain culture supernatant concentrate produced a blocking circle even after heat treatment, so the amount of cereulide in the dropped culture supernatant was considered insufficient to form the blocking circle. It is done. In addition, the inhibition circle was observed in the medium having any KCl concentration of 200 mM to 1 M.

  For the culture supernatant of the 08-151-3 strain, no inhibition circle was formed in either the unheated sample or the heat-treated sample. On the other hand, for the concentrate, formation of blocking circles was observed in both the unheated sample and the heat-treated sample.

  From the above results, it was shown that cereulide can be detected by using the antibacterial action of cereulide against cereulide-sensitive bacteria as an index.

Example 2: Detection of cereulide in food (1) Material (1-1) Test strain 03-137-1 strain was used as Bacillus cereus as a cereulide-producing strain. This Cereus strain is a strain obtained by the present inventor when it was isolated during a food poisoning case. Moreover, the same Bacillus sporothermodurance as Example 1 was used as a cereulide sensitive bacterium.
(1-2) Food As a food, commercially available aseptic packaged cooked rice (200 g: Sato Food Industry) was used.
(1-3) PBS for food dilution
Dissolve potassium dihydrogen phosphate (anhydrous) (34 g: Wako Pure Chemical Industries, Ltd.) in distilled water (50 mL), add 1N sodium hydroxide (about 175 mL: Wako Pure Chemical Industries, Ltd.) to adjust the pH to 7.2 Adjusted to 1 L with distilled water. The resulting solution was sterilized by autoclaving at 121 ° C. for 20 minutes to obtain a stock solution. This stock solution was diluted 1: 800 with distilled water and sterilized by autoclaving at 121 ° C. for 20 minutes to obtain a food dilution PBS.

(2) Extraction of cereulide from food
The 03-137-1 strain was seeded in a BHI liquid medium and cultured at 30 ° C. for 12 hours. The culture was diluted 1: 1 × 10 ⁶ with PBS for food dilution to prepare a bacterial solution for inoculation (1.9 cfu / g). Aseptically wrapped cooked rice was peeled off, and the bacterial solution for inoculation (5 mL) was evenly spread on the cooked rice. After spraying, the cooked rice was sealed with a lid and incubated at 30 ° C. At 0, 12, 24, 48 and 72 hours from the start of incubation, 25 g of cooked rice was collected. The collected cooked rice was diluted 10 times with PBS for food dilution and homogenized using a stomacher. This was centrifuged at 12000 rpm for 10 minutes at room temperature, and the supernatant was recovered. The obtained supernatant was heated at 121 ° C. for 10 minutes to obtain a sample.

(3) Examination of antibacterial action Bacillus sporothermomodulance (1 mL, 1 × 10 ⁸ CFU) pre-cultured in brain heart infusion medium is added to TSA-KCl multilayer medium (5 mL) dissolved by heating and mixed. The obtained mixed solution was poured onto a TSA plate medium and solidified. After the medium solidified, the above sample (10 μL) was dropped onto the obtained solid medium and cultured at 30 ° C. overnight. After the culture, it was confirmed whether or not a transparent inhibition circle was generated in the solid medium. As a result, an inhibition circle was formed in the solid medium to which a sample obtained from cooked rice incubated for 48 and 72 hours was dropped.
Therefore, it was shown that cereulide in food contaminated with Bacillus cereus can be detected by the detection method of the present invention.

Claims (11)

Heating a sample in which cereulide may be present;
Culturing cereulide-susceptible bacteria in the presence of the sample obtained in the heating step;
And a step of detecting cereulide in the sample based on the antibacterial action of cereulide against the cereulide-sensitive bacteria in the culture obtained in the culturing step.   The detection method according to claim 1, wherein the cereulide-susceptible bacterium is selected from bacteria belonging to the genus Bacillus excluding the cereulide-producing strain Bacillus cereus and mutants thereof.   The detection method according to claim 1 or 2, wherein the cereulide-susceptible bacterium is selected from Bacillus sporothermodurans and variants thereof.   The detection method according to any one of claims 1 to 3, wherein the heating step is performed under conditions capable of sterilizing Bacillus cereus.   The detection method according to any one of claims 1 to 4, wherein the conditions under which Bacillus cereus can be sterilized are heating conditions at 75 to 130 ° C for 10 to 60 minutes.   The detection method according to claim 1, wherein the culturing step is performed using a medium containing a potassium salt.   The detection method according to claim 6, wherein the medium containing potassium salt is a solid medium.   The detection method according to claim 7, wherein the detecting step is a step of detecting cereulide in the sample based on the size of the inhibition circle generated in the solid medium.   A kit for detecting cereulide containing cereulide-sensitive bacteria.   The cereulide detection kit according to claim 9, further comprising a medium containing a potassium salt.   The kit for cereulide detection according to claim 10, wherein the medium containing potassium salt is a solid medium.