Bacillus cereus group detection medium

Abstract

Provided are: a medium for Bacillus cereus group detection that can be produced easily, allows excellent growth of Bacillus cereus irrespective of temperature conditions, and has exceptional selectivity; and a method for detecting Bacillus cereus using the medium.　A medium for Bacillus cereus group detection that contains a phosphatidylinositol-specific phospholipase C substrate having a detectable chromogenic or fluorescent free radical, and trimethoprim.

Description

[Technical Field]

[0001] The present invention relates to a medium for detecting Bacillus cereus group bacteria. [Background technology]

[0002] Bacillus cereus is a gram-positive, spore-forming bacillus that is generally widely distributed in nature, such as soil and rivers. This bacterium contaminates a wide range of foods, including grains and spices that are closely related to soil, as well as cross-contaminated foods such as noodles like yakisoba and spaghetti, cooked rice dishes like rice balls and fried rice, gratin, pizza, seafood and processed seafood products, meat and processed meat products, food ingredients, confectionery, the environment, and clinical materials. Contamination by this bacterium can sometimes cause spoilage and deterioration of food. This bacterium is also known to produce emetic and diarrheal toxins, which can cause food poisoning. Therefore, control of this bacterium is important from the standpoint of food hygiene and safety (Non-Patent Document 1).

[0003] Known media commonly used for detecting Bacillus cereus include NGKG agar medium and MYP agar medium (Non-Patent Documents 1 to 3, etc.). These media contain egg yolk because they utilize the egg yolk reaction, which is one of the properties of Bacillus cereus, as one of the detection principles. The preparation method for these egg yolk-containing media consists of the following steps: sterilizing and dissolving the medium ingredients other than egg yolk, such as agar; cooling and incubating the medium to about 50°C; adding and mixing collected egg yolk to the medium in a sterile manner; and dispensing the mixed medium ingredients into petri dishes and allowing them to solidify. As described above, adding egg yolk requires at least three steps, making the procedure complicated. This is done to prevent thermal denaturation of egg yolk components. In particular, during the cooling and incubation process, if the medium temperature when adding the harvested egg yolk is too high, the egg yolk components will denature, while if the medium temperature is too low, the agar will solidify, etc. Therefore, temperature control is important and requires skilled experience. Furthermore, the condition of the harvested egg yolk used in the detection principle is prone to significant variation depending on the species and individual differences of the laying hens, the rearing environment, etc., and therefore the performance of the medium is also affected by the condition of the harvested egg yolk. Therefore, an empirical method is required to distinguish colonies that have an egg yolk reaction after culture.

[0004] Therefore, the present applicant has reported a medium for detecting Bacillus cereus group bacteria without using the egg yolk reaction, which contains four components: polymyxin B, trimethoprim, lincomycin antibiotics, and 5-bromo-4-chloro-3-indoxyl-α-D-glucopyranoside, which serves as a substrate for α-glucosidase (Patent Document 1). In addition, a medium for selective detection of Bacillus cereus and Bacillus thuringiensis has been reported, which contains lithium chloride, ceftazidime, polymyxin B sulfate, etc. in addition to a phosphatidylinositol-specific phospholipase C substrate having a detectable color-producing or fluorescent free radical and nutritional components (Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2011-4712 [Patent Document 2] U.S. Patent No. 6,284,517 [Non-patent literature]

[0006] [Non-Patent Document 1] Food Sanitation Inspection Guidelines, Microbiology, 2004, Supervised by the Ministry of Health, Labor and Welfare, Japan Food Sanitation Association, pp. 266-282 [Non-patent document 2] ISO11133 Microbiology of food, animal feed and water-Preparation, production, storage and performance testing of culture media(2014) [Non-patent document 3] ISO7932 Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of presumptive Bacillus cereus - Colony-count technique at 30℃(2004) Summary of the Invention [Problem to be solved by the invention]

[0007] However, it was found that the medium described in Patent Document 1 has problems such as insufficient growth of Bacillus cereus in 24 hours at 30°C, which is the same culture condition as that of the MYP medium of ISO 7932, making it impossible to detect clear colonies, and giving false positives for some Staphylococcus species.Furthermore, it was found that the medium described in Patent Document 2 gives positive results not only for the Bacillus cereus group but also for Listeria monocytogenes and the like. Therefore, an object of the present invention is to provide a medium for detecting Bacillus cereus group bacteria that can be easily produced, allows good growth of Bacillus cereus regardless of temperature conditions, and has excellent selectivity, and a method for detecting Bacillus cereus group bacteria using the same. [Means for solving the problem]

[0008] In view of the above circumstances, the present inventors have investigated various culture media and detection methods specialized for detecting the Bacillus cereus group, and have found that by using a phosphatidylinositol-specific phospholipase C substrate having a detectable chromogenic or fluorescent free radical as a chromogenic or fluorescent substrate and by adding trimethoprim to the medium, it is possible to improve the growth of the Bacillus cereus group while inhibiting the growth of microorganisms other than the Bacillus cereus group, without using the egg yolk reaction, and have completed the present invention.

[0009] That is, the present invention provides the following inventions [1] to [7]. [1] A medium for detecting Bacillus cereus group bacteria, containing a phosphatidylinositol-specific phospholipase C substrate with a detectable chromogenic or fluorescent free radical, and trimethoprim. [2] The medium for detecting the Bacillus cereus group according to [1], further comprising a β-lactam antibiotic. [3] The medium for detecting the Bacillus cereus group according to [1] or [2], further comprising an antifungal agent. [4] The medium for detecting a Bacillus cereus group according to any one of [1] to [3], wherein the phosphatidylinositol-specific phospholipase C substrate having a detectable chromogenic or fluorescent free group is selected from 5-bromo-4-chloro-3-indoxyl myo-inositol-1-phosphate, 5-bromo-6-chloro-3-indoxyl myo-inositol-1-phosphate, 6-chloro-3-indoxyl myo-inositol-1-phosphate, 4-methylumbelliferone myo-inositol-1-phosphate, 4-nitrophenyl-myo-inositol-1-phosphate, luciferin-myo-inositol-1-phosphate, and salts thereof. [5] The medium for detecting the Bacillus cereus group according to any one of [2] to [4], wherein the β-lactam antibiotic is selected from penicillin, cephem, carbapenem, monobactam and penem antibiotics. [6] The medium for detecting the Bacillus cereus group according to any one of [1] to [5], wherein the Bacillus cereus group is a bacterium selected from Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, Bacillus cytotoxicus, and Bacillus toyonensis. [7] A method for detecting the Bacillus cereus group, comprising inoculating a specimen into the medium according to any one of [1] to [6], culturing the specimen, and then determining the colonies detectable on the medium. [Effects of the Invention]

[0010] The medium for detecting the Bacillus cereus group of the present invention enables accurate, efficient, and simple differentiation of the presence of the Bacillus cereus group in a sample containing a mixture of various microorganisms, and the medium is inexpensive and easy to prepare. Therefore, the medium of the present invention can be widely used in testing general foods, beverages, water, etc., as well as in testing manufacturing processes. [Brief explanation of the drawings]

[0011] [Figure 1] This figure compares the growth ability of food samples inoculated with B. cereus on N-BC medium and EX-BC medium, respectively, with MYP medium as the standard. [Figure 2] FIG. 1 shows the state of colonies after culturing in N-BC medium and EX-BC medium at 30° C. for 22 hours. DETAILED DESCRIPTION OF THE INVENTION

[0012] The Bacillus cereus group to be detected in the present invention includes Bacillus cereus and bacteria genetically closely related to Bacillus cereus and having similar biochemical properties. Examples of the Bacillus cereus group include bacteria selected from Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, Bacillus cytotoxicus, and Bacillus toyonensis. Of these, the present invention is preferably used to detect Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus weihenstephanensis, and more preferably to detect Bacillus cereus and Bacillus thuringiensis.

[0013] In the culture medium of the present invention, a phosphatidylinositol-specific phospholipase C substrate having a detectable chromogenic or fluorescent free radical is used as the chromogenic or fluorescent substrate. This substrate is decomposed by phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Bacillus cereus, which is the detection target of the present invention, to liberate a detectable chromogenic or fluorescent free radical. Examples of such chromogenic or fluorescent substrates for PI-PLC include 5-bromo-4-chloro-3-indoxyl myo-inositol-1-phosphate, 5-bromo-6-chloro-3-indoxyl myo-inositol-1-phosphate, 6-chloro-3-indoxyl myo-inositol-1-phosphate, 4-methylumbelliferone myo-inositol-1-phosphate, 4-nitrophenyl-myo-inositol-1-phosphate, luciferin-myo-inositol-1-phosphate, and salts thereof. Among these, 5-bromo-4-chloro-3-indoxyl myo-inositol-1-phosphate (hereinafter sometimes referred to as X-IP) is particularly preferred in terms of color development and ease of colony discrimination. In the present invention, by using a chromogenic or fluorescent substrate of PI-PLC as the chromogenic substrate, trimethoprim is the only growth inhibitor for microorganisms other than the Bacillus cereus group (hereinafter also referred to as "other microorganisms"), and colonies of the Bacillus cereus group can be clearly distinguished regardless of temperature conditions without inhibiting the growth of the Bacillus cereus group. Therefore, it is also advantageous in that it does not require the addition of polymyxin B and lincomycin, which are required in Patent Document 1. The content of the chromogenic or fluorescent substrate of PI-PLC in the culture medium is preferably 0.001 to 10 g / L, more preferably 0.01 to 5 g / L, and even more preferably 0.1 to 1 g / L, as the concentration at the time of detection, from the viewpoint of good color development and determination.

[0014] Trimethoprim used in the medium of the present invention acts as a growth inhibitor of other microorganisms. The addition of trimethoprim can inhibit the growth of many gram-positive bacteria (especially those of the genera Staphylococcus and Enterococcus) and gram-negative bacteria other than the Bacillus cereus group. Trimethoprim has a particularly strong inhibitory effect on gram-negative bacteria, and also acts on gram-positive bacteria other than those of the Bacillus cereus group.

[0015] Specific examples of trimethoprim include trimethoprim and its lactate salt. The content of trimethoprim in the medium is not particularly limited, but the concentration at the time of detection is preferably 0.01 to 500 mg / L, more preferably 0.1 to 50 mg / L, and even more preferably 1 to 5 mg / L.

[0016] In addition to the above two components, the medium of the present invention preferably contains a β-lactam antibiotic for the purpose of enhancing the growth inhibition of Gram-negative bacteria. As the β-lactam antibiotic, one or more antibiotics selected from penicillin, cephem, carbapenem, monobactam, and penem antibiotics are used. Of these, cephem antibiotics or monobactam antibiotics are more preferred in terms of enhancing the growth inhibition of Gram-negative bacteria. Cephem antibiotics include third-generation cephalosporins such as ceftriaxone, cefotaxime, ceftizoxime, ceftazidime, cefoperazone, cefsulodin, ceftibuten, and cefetamet. Monobactam antibiotics include aztreonam, tigemonam, carumonam, nocardicin, and tabtoxin. Of these, ceftazidime or aztreonam is preferably used. The content of the β-lactam antibiotic in the medium is preferably 0.001 to 100 mg / L, more preferably 0.01 to 25 mg / L, and even more preferably 0.5 to 5 mg / L, as the concentration at the time of detection.

[0017] The medium of the present invention preferably further contains an antifungal agent. As the antifungal agent, one or more selected from polyenes (e.g., amphotericin B, etc.), echinocandins (e.g., micafungin, caspofungin, etc.), azoles (e.g., fluconazole, voriconazole, itraconazole, etc.), allylamines (e.g., terbinafine, etc.), and fluoropyrimidines (e.g., flucytosine, etc.) are used. Among these, amphotericin B is preferably contained for the purpose of inhibiting the growth of fungi. The concentration of the antifungal agent in the medium is not particularly limited, but the concentration at the time of detection is preferably 0.001 to 100 mg / L, more preferably 0.1 to 10 mg / L, and even more preferably 1 to 5 mg / L.

[0018] Furthermore, the medium of the present invention preferably contains sugar alcohols and / or inorganic salts. Examples of sugar alcohols include monosaccharide and oligosaccharide sugar alcohols, such as erythritol, xylitol, sorbitol, mannitol, and maltitol. Among these, mannitol is preferred because it facilitates identification of the Bacillus cereus group. These may be used alone or in combination of two or more. The content of sugar alcohols in the medium is not particularly limited, but the concentration at the time of detection is preferably 1 to 50 g / L, more preferably 5 to 30 g / L, and particularly preferably 5 to 20 g / L. Examples of inorganic salts include inorganic acid metal salts such as sodium chloride and sodium thiosulfate; and organic acid metal salts such as ammonium iron citrate and sodium citrate. Among these, the inorganic acid metal salts are preferred, and sodium chloride is preferred because it facilitates identification of the Bacillus cereus group. These may be used alone or in combination of two or more. The content of inorganic salts in the medium is not particularly limited, but the concentration at the time of detection is preferably 0.1 to 20 g / L, more preferably 1 to 10 g / L, and particularly preferably 3 to 8 g / L.

[0019] The medium of the present invention may contain, in addition to the above medium components, other medium components such as carbon sources, nitrogen sources, minerals, vitamins and other nutrients for the bacterial cells, and pH adjusters. For example, the carbon source may be one or more selected from glucose, fructose, lactose, saccharose, etc.; the nitrogen source may be one or more selected from protein hydrolysates (casein peptone, soybean peptone, meat peptone, etc.), yeast extract, meat extract, fish extract, etc.; the mineral source may be one or more selected from copper, zinc, magnesium, cobalt, etc.; and the vitamin may be one or more selected from nicotinic acid, pantothenate, biotin, riboflavin, folic acid, etc.

[0020] Examples of components used in pH adjusters include organic acid salts such as oxalic acid, acetic acid, fumaric acid, malic acid, lactic acid, gluconic acid, and tartaric acid; inorganic salts such as phosphoric acid, hydrochloric acid, and sulfuric acid; carbonates such as sodium carbonate and sodium bicarbonate; hydroxides such as sodium hydroxide; ammonia or aqueous ammonia; amine citrates; lower alkanolamines; and basic amino acids such as arginine and lysine. These may be used alone or in combination of two or more. In this case, the pH of the medium is adjusted to preferably 5 to 8, more preferably 6.5 to 7.7, and even more preferably 6.8 to 7.4.

[0021] Furthermore, to make the medium of the present invention a solid or semi-solid medium, a solidifying or gelling component such as natural components such as gelatin, agar, xanthan gum, locust bean gum, guar gum, carrageenan, or synthetic components such as hydroxyethyl cellulose may be added. These components may be used alone or in combination of two or more.

[0022] Furthermore, the culture medium of the present invention may be used as a simple culture medium by using a fibrous liquid-absorbing material such as a fibrous water-absorbent sheet to absorb the liquid culture medium. Examples of such fibers include natural fibers derived from plants or animals, chemically synthesized fibers, and synthetic fibers derived from glass fibers, and nonwoven fabrics made from fibers in sheet form are also preferred. Examples of simple media include those prepared by the preparation methods described in JP-A Nos. 57-502200, 3-15379, 2-65798, 6-181741, 9-19282, and 2000-325072. Specific examples of simple media include a simple medium in which a medium composition containing (a) an adhesive soluble in water and alcohol, (b) a gelling agent soluble in water but insoluble in alcohol, and (c) bacterial nutrient components is supported on a fibrous absorbent sheet with a mesh larger than the gelling agent (Japanese Patent Laid-Open No. 9-19282); and a simple medium in which an alcohol suspension containing (a) 0.01 to 0.4 wt % of an adhesive soluble in water and alcohol, (b) a gelling agent soluble in water but insoluble in alcohol, and (c) bacterial nutrient components is impregnated into a fibrous absorbent sheet with a mesh larger than the particle size of the gelling agent placed on a waterproof flat plate, and the sheet is dried while suppressing rapid evaporation of the alcohol, thereby adhering the absorbent sheet to the waterproof flat plate (Japanese Patent Laid-Open No. 2000-325072). Examples of the water- and alcohol-soluble adhesive include hydroxypropyl cellulose and polyvinylpyrrolidone. Examples of the water-soluble, alcohol-insoluble gelling agent include the above-mentioned examples of the solidifying component and gelling component. The average particle size of the gelling agent is preferably 0.5 to 50 μm.

[0023] The form of the medium of the present invention is not particularly limited, and examples include liquid medium, agar medium, and sheet-type simple medium. Examples of preparation methods include adding purified water or the like to the above-mentioned medium components, mixing and stirring, followed by sterilization in an autoclave or the like, dispensing the mixture into sterile petri dishes, and cooling or allowing it to cool (in this case, heat-sensitive components such as enzyme substrates and antibiotics may be added separately after autoclave sterilization and before dispensing); or adding alcohol, purified water, or the like to the above-mentioned medium components, mixing and stirring, dispensing the mixture into containers (plastic, glass, etc.) containing a fibrous liquid absorbent material, and sterilizing it by gamma irradiation. Examples of alcohol include ethanol and 2-propanol.

[0024] In the method for detecting Bacillus cereus group bacteria of the present invention, a specimen is inoculated onto the detection medium obtained above, and cultured under predetermined conditions, and then detectable colonies on the medium are assessed. Here, "detectable colonies" refer to formed colonies that have a specific color tone that can be confirmed visually or in the presence of fluorescence, and the colonies are identified to determine the presence of the Bacillus cereus group.

[0025] The specimen is not particularly limited, but may include food suspensions, environmental swabs from kitchens and cooking utensils, culture fluids cultured in enrichment media, soil suspensions, river water, drinking water, etc. The specimen may be inoculated into the medium of the present invention as it is, or after being concentrated or diluted, and then cultured. -10 It is preferable to concentrate or dilute the specimen by about 1:1 from the viewpoint of colony number measurement. The inoculation method is not particularly limited, but the plate smear method, streak smear method, and membrane filter method (a method in which a filter after filtration of a sample is placed on a medium for culture) are preferred.

[0026] The culture temperature is not particularly limited, but is preferably 10 to 48°C, particularly 30 to 35°C, which is a temperature at which the Bacillus cereus group can grow. The culture time is also not particularly limited, but is preferably 18 to 48 hours, particularly 22 to 26 hours. The culture is preferably carried out aerobically in a static state.

[0027] In the present invention, the color or fluorescence of colonies (including their surroundings) of the Bacillus cereus group formed in the culture medium is determined by the presence of color or fluorescence from the substrate, and colonies are identified as Bacillus cereus group colonies based on the presence of color or fluorescence from the substrate. However, the absence of such color or fluorescence determines that the colonies are not Bacillus cereus group colonies. In particular, when X-IP is used as the enzyme substrate, the determination is based on the change in blue color. In this way, the presence or absence of Bacillus cereus group colonies and the number of colonies in the sample are determined. [Example]

[0028] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0029] Test Example 1: Agar medium [Preparation of medium] The composition of the medium based on standard agar medium (SMA) is shown in Table 1, and the composition of the medium based on trypto-soya agar medium (TSA) is shown in Table 2. Preparation method: The medium composition shown in Table 1 or Table 2 (excluding X-IP) was added to 1 L of purified water, heated at 121°C for 15 minutes to dissolve, and after thorough stirring, X-IP was added. The mixture was then stirred and dispensed in 20 mL portions into plastic dishes (90 mm diameter) and allowed to stand until the medium solidified, to prepare the SMA-based medium of the present invention (SMA-BC) and the TSA-based medium of the present invention (TSA-BC).

[0030] [Testing of strains] B. cereus ATCC11778 was pre-cultured in TSA for 24 hours, and then diluted to 1 × 10 with sterilized 0.86% NaCl solution (sterile physiological saline). 2 ~1×10 60.05 mL of the bacterial solution adjusted to cfu / mL was inoculated into each medium.

[0031] [Culture results] As shown in Table 3, when each strain was tested and cultured at 30°C for 22 hours, good growth of B. cereus and blue color development were observed in SMA-BC and TSA-BC. These results demonstrate that B. cereus can be detected by combining a phosphatidylinositol-specific phospholipase C substrate with a detectable chromogenic free radical, such as X-IP, with trimethoprim.

[0032] [Table 1]

[0033] [Table 2]

[0034] [Table 3]

[0035] Test Example 2: Growth test [Preparation of medium] The medium of the present invention (N-BC medium) was prepared by adding a hydroxypropyl cellulose (HPC) solution of the composition shown in Table 5 to the medium composition shown in Table 4, suspending the mixture with stirring, and then dispensing 0.9 mL of the suspension into a container (50 mm diameter) containing a cotton sheet (50 mm diameter). The sheets were stacked in two layers and slowly dried overnight in a closed space, then the lid was closed. The medium was sealed in an aluminum package together with a desiccant and sterilized by gamma ray irradiation at a surface dose of 10 to 20 kGy.

[0036] [Testing of strains] The test strain was pre-cultured in TSA for 24 hours and diluted to 1 × 10 1 ~1×10 41 mL of the bacterial solution adjusted to cfu / mL was inoculated into N-BC medium.

[0037] [Culture results] As shown in Table 6, when each strain was tested and cultured at 30°C for 24 hours, good growth of B. cereus and blue color development were observed on N-BC medium. The results showed that the detection of B. cereus was improved by combining a phosphatidylinositol-specific phospholipase C substrate with a detectable chromogenic free radical, such as X-IP, with a β-lactam antibiotic, such as aztreonam, in addition to trimethoprim. It was also found that the addition of the antifungal agent, amphotericin B, was also effective.

[0038] [Table 4]

[0039] [Table 5]

[0040] [Table 6]

[0041] Test Example 3: Multi-strain test (Bacillus cereus group) [Preparation of medium] The N-BC medium had the composition shown in Table 4, and the EX-BC medium shown in Patent Document 1 (the EX-BC medium) had the composition shown in Table 7. The HPC solution shown in Table 5 was added to each medium and suspended under stirring. 0.9 mL of the suspension was dispensed into a container (50 mm diameter) containing a cotton sheet (50 mm diameter). The two layers were stacked and allowed to dry slowly overnight in a closed space, after which the lid was placed on top. The medium was sealed in an aluminum package together with a desiccant and sterilized by gamma ray irradiation at a surface dose of 10 to 20 kGy. In addition, TSA, MYP agar medium (MYP), and NGKG agar medium (NGKG) were used as control media.

[0042] [Testing of strains] The test strain was pre-cultured in TSA for 24 hours and diluted to 1 × 10 1 ~1×10 4 The bacterial solution was adjusted to a concentration of cfu / mL, and 1 mL of the bacterial solution was inoculated into each medium.

[0043] [Culture results] As shown in Table 8, each strain was cultured at 30°C for 22 hours, and the inoculated bacterial concentration was calculated from the number of colonies that grew. In this case, only EX-BC was cultured at 35°C. These results demonstrate that all B. cereus strains in N-BC medium have growth potential equivalent to that of TSA, MYP, and NGKG.

[0044] [Table 7]

[0045] [Table 8]

[0046] Test Example 4: Trimethoprim concentration change test [Preparation of culture medium] N-BC medium was prepared in the same manner as in Test Example 2. At this time, the test was performed by changing the trimethoprim concentration to 0, 0.1, 5.0, or 50 mg / L, and changing X-IP to 0.4 g / L.

[0047] [Test strains] The strains shown in Table 9 were pre-cultured in TSA for 24 hours and diluted to 1 × 10 1 ~1×10 4 The bacterial solution was adjusted to a concentration of cfu / mL, and 1 mL of the bacterial solution was inoculated into each medium.

[0048] [Culture results] As shown in Table 9, when the trimethoprim concentration in N-BC medium was 0 mg / L, growth of the Listeria and Bacillus cereus groups was confirmed after 24 hours of culture. On the other hand, when the trimethoprim concentration in N-BC medium was 0.1 mg / L, 5 mg / L, or 50 mg / L, growth of the Listeria genus was suppressed after 24 hours of culture, while growth of the Bacillus cereus group was confirmed.

[0049] [Table 9]

[0050] Test Example 5: Multi-strain test (non-Bacillus cereus group) [Preparation of medium] N-BC medium and EX-BC medium were prepared in the same manner as in Test Example 3.

[0051] [Testing of strains] The test strain was pre-cultured in TSA for 24 to 48 hours and diluted to 1 × 10 3 ~1×10 7 The bacterial solution was adjusted to a concentration of cfu / mL, and 1 mL of the bacterial solution was inoculated into each medium.

[0052] [Culture results] As shown in Table 10, each strain was tested, and N-BC was cultured at 30°C for 22 hours, and EX-BC was cultured at 35°C for 22 hours, and the developed colonies were confirmed. Although blue coloration was confirmed in N-BC medium for one strain of Listeria monocytogenes, false positives for the genera Corynebacterium, Enterococcus, and Staphylococcus, which were observed in the control medium, EX-BC medium, were no longer observed. Therefore, it was found that N-BC medium had fewer false positives than EX-BC medium.

[0053] [Table 10]

[0054] Test Example 6: Study using ingredients [Preparation of medium] N-BC medium, EX-BC medium and MYP were prepared in the same manner as in Test Example 3.

[0055] [Testing of strains] B. cereus ATCC 11778, ATCC 14579, and ATCC 19637 were precultured in TSA for 24 hours, and then diluted to 1 × 10 4 , 1×10 6 or 1 x 10 8 0.1 mL of the bacterial solution adjusted to cfu / mL was added to 10 g of a given food material, and the material was left to stand at room temperature, refrigerated, or frozen for 48 hours to 2 weeks. 90 mL of sterile saline was then added to prepare a stomached food material liquid sample solution. 1 mL of the sample solution was inoculated into N-BC medium, EX-BC medium, and MYP medium.

[0056] [Culture results] The strains were tested, and N-BC and MYP were cultured at 30°C for 22 hours, and EX-BC was cultured at 35°C for 22 hours, and the number of bacteria grown on each medium was calculated. As shown in Figure 1, the difference in growth between N-BC medium and the control medium, MYP, was 1 x 10 -0.03 (93% of MYP), whereas EX-BC was 1 × 10 -0.49 (32% of MYP), demonstrating a clear improvement in development.

[0057] Test Example 7: Colony formation ability after 22 hours of culture [Preparation of medium] N-BC medium and EX-BC medium were prepared in the same manner as in Test Example 3.

[0058] [Testing of strains] B. cereus ATCC11778 was cultured in TSA for 24 hours and diluted to 1 × 10 2 The bacterial solution was adjusted to a concentration of cfu / mL, and 1 mL of the bacterial solution was inoculated into each medium.

[0059] [Culture results] As shown in Figure 2, each strain was tested and cultured at 30°C for 22 hours, and the colonies that developed were confirmed. N-BC medium formed larger colonies than the control medium, EX-BC medium, demonstrating faster growth.

[0060] Test Example 8: Comparison with the medium (US medium) described in Patent Document 2 [Preparation of culture medium] The composition of the US medium is shown in Table 11. Preparation method: The medium compositions 1 to 10 in Table 11 were weighed out and added to 1 L of purified water, heated at 121°C for 15 minutes to dissolve, and the medium compositions 11 to 14 in Table 11 were added. After stirring well, 20 mL of the mixture was dispensed into plastic dishes (90 mm diameter) and allowed to stand until the medium solidified to prepare US medium. N-BC medium was prepared in the same manner as in Test Example 2.

[0061] [Table 11]

[0062] [Test strains] The strains shown in Table 12 were pre-cultured in TSA for 24 hours and then diluted with sterile saline to the specified concentration (10 3 ~10 7 The bacterial solution was inoculated into each medium, 100 μL for US medium and 1 mL for N-BC medium.

[0063] [Culture results] As shown in Table 12, when each strain was tested and cultured at 35°C for 22 hours, clear blue colonies were formed for Listeria and clear white colonies were confirmed for Candida on US medium. On the other hand, when each strain was tested and cultured at 30°C for 22 hours on N-BC medium, one strain of Listeria, Listeria monocytogenes, showed a very faint blue coloration over the entire medium, but no colonies were formed for Candida.

[0064] [Table 12]

[0065] Test Example 9: Amphotericin B Concentration [Preparation of medium] The invention medium (N-BC medium) was prepared by adding hydroxypropyl cellulose (HPC) solution of the composition shown in Table 5 to the medium composition shown in Table 13 and suspending with stirring. 0.9 mL of the suspension was dispensed into a container (50 mm diameter) containing a cotton sheet (50 mm diameter). The sheets were stacked in two layers and slowly dried overnight in a closed space, then the lid was closed. The medium was sealed in an aluminum foil bag with a desiccant and sterilized by gamma irradiation at a surface dose of 10 to 20 kGy. Amphotericin B was adjusted to 0 mg, 0.1 mg, 1 mg, or 10 mg in each medium.

[0066] [Table 13]

[0067] [Testing of strains] The test strain was pre-cultured in TSA for 24 to 48 hours and diluted to 1 × 10 2 ~1×10 5 The bacterial solution was adjusted to a concentration of cfu / mL, and 1 mL of the bacterial solution was inoculated into each medium.

[0068] [Culture results] As shown in Table 14, each strain was tested and cultured at 30°C for 24 to 44 hours, and the colonies that had grown were confirmed. After 24 hours of culture, blue colonies were confirmed for the three strains of B. cereus and B. thuringiensis on N-BC medium, but no growth was observed for the two strains of C. albicans. On the other hand, after 44 hours of culture, the results for the three strains of B. cereus and B. thuringiensis were similar to those for the 24-hour culture, but for the two strains of C. albicans, when the amphotericin B was 0 mg or 0.1 mg, the bacterial concentration was 10 5 or 10 4 Growth or weak coloration was observed in cfu / mL.

[0069] [Table 14]

[0070] Test Example 10: Agar medium [Preparation of medium] The TSA-based medium composition is shown in Table 15. Preparation method: The medium composition in Table 15 (excluding X-IP, aztreonam, and ceftazidime) was added to 1 L of purified water and dissolved by heating at 121°C for 15 minutes. After thorough stirring, X-IP and, if necessary, aztreonam or ceftazidime were added, followed by stirring. 20 mL of the medium was dispensed into plastic dishes (90 mm diameter) and allowed to stand until solidified to prepare TSA-BC.

[0071] [Table 15]

[0072] [Testing of strains] The strains shown in Table 16 were pre-cultured in TSA for 24 hours, and then diluted to 1 × 10 1 ~1×10 5 0.05 mL of the bacterial solution adjusted to cfu / mL was inoculated into each medium.

[0073] [Culture results] As shown in Table 16, when each bacterial strain was tested and cultured at 30°C for 22 hours, TSA-BC showed good growth of B. cereus and blue color development. On the other hand, the addition of aztreonam or ceftazidime inhibited the formation of white colonies of E. coli or E. amnigenus.

[0074] [Table 16]

[0075] From the above, the medium of the present invention can accurately, efficiently, and simply distinguish the presence or absence of Bacillus cereus group in test samples. Moreover, the medium is inexpensive and easy to prepare. Therefore, the medium can be widely used for testing general foods, beverages, water, etc., as well as for testing manufacturing processes.

Claims

1. A medium for detecting Bacillus cereus group bacteria, comprising a phosphatidylinositol-specific phospholipase C substrate having a detectable chromogenic or fluorescent free radical, trimethoprim, aztreonam or ceftazidime, and amphotericin B, wherein the Bacillus cereus group bacteria is a bacterium selected from Bacillus cereus and Bacillus thuringiensis.

2. 2. The medium for detecting a Bacillus cereus group according to claim 1, wherein the phosphatidylinositol-specific phospholipase C substrate having a detectable chromogenic or fluorescent free group is selected from the group consisting of 5-bromo-4-chloro-3-indoxyl myo-inositol-1-phosphate, 5-bromo-6-chloro-3-indoxyl myo-inositol-1-phosphate, 6-chloro-3-indoxyl myo-inositol-1-phosphate, 4-methylumbelliferone myo-inositol-1-phosphate, 4-nitrophenyl-myo-inositol-1-phosphate, luciferin-myo-inositol-1-phosphate, and salts thereof.

3. 3. The medium for detecting a Bacillus cereus group according to claim 1 or 2, comprising a phosphatidylinositol-specific phospholipase C substrate having a detectable chromogenic or fluorescent free radical at a detection concentration of 0.001 to 10 g / L, trimethoprim at a detection concentration of 0.01 to 500 mg / L, aztreonam or ceftazidime at a detection concentration of 0.001 to 100 mg / L, and amphotericin B at a detection concentration of 0.001 to 100 mg / L.

4. The medium for detecting a Bacillus cereus group strain according to any one of claims 1 to 3, further comprising sugar alcohols and / or inorganic salts.

5. A method for detecting a Bacillus cereus group, comprising inoculating a specimen into the medium according to any one of claims 1 to 4, culturing the specimen, and then determining the number of detectable colonies on the medium.

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