Culture medium for detection of Bacillus cereus

Abstract

The present invention provides a medium for detecting Bacillus cereus groups, which can be easily produced, has good growth of Bacillus cereus regardless of temperature conditions, and has excellent selectivity, and a method for detecting Bacillus cereus groups using the medium. The medium for detecting Bacillus cereus groups contains a phosphatidylinositol-specific phospholipase C substrate and trimethoprim, and the phosphatidylinositol-specific phospholipase C substrate has a color-developing or fluorescent free radical that can be detected.

Description

Technical Field

[0001] The invention relates to a culture medium for detecting Bacillus cereus groups. Background Art

[0002] Bacillus cereus is a Gram-positive bacillus that is widely distributed in the natural world such as soil and rivers. The scope of contamination caused by this bacterium includes cereals and spices that are closely related to soil, and foods that are cross-contaminated by the use of these foods, such as noodles such as fried noodles and pasta, rice balls and fried rice, gratins, pizza, seafood and processed products, edible meat and processed products, food raw materials, snacks, etc., the environment, clinical materials, and many other types.

[0003] The contamination caused by this bacterium sometimes causes decay and deterioration. In addition, this bacterium is known to produce vomitoxin and diarrheal toxin, and sometimes causes food poisoning.

[0004] Therefore, from the viewpoint of food hygiene and safety, control of this bacterium is also important (Non-Patent Document 1).

[0005] Generally, as a culture medium for detecting Bacillus cereus, NGKG agar medium or MYP agar medium (non-patent documents 1 to 3, etc.) are known. These culture media use the egg yolk reaction, which is one of the characteristics of Bacillus cereus, as one of the detection principles, and therefore contain egg yolk. The preparation method of these culture media containing egg yolk consists of the following steps: a step of sterilizing and dissolving the culture medium raw materials such as agar except egg yolk in advance; a step of cooling to about 50°C and keeping warm; a step of further adding and mixing the collected egg yolk to the culture medium in an aseptic manner; and a step of dispensing the culture medium raw materials thus mixed into a culture dish and solidifying it.

[0006] As mentioned above, at least 3 processes are required when adding egg yolk, and the steps are complicated. This is to prevent the yolk component from thermal denaturation. Especially in the cooling and heat preservation process, if the temperature of the culture medium when adding the collected yolk is too high, the yolk component will be denatured, and if the culture medium temperature is too low, it will cause the solidification of agar, etc., so the management of this temperature is important and requires skilled experience. In addition, since the state of the collected yolk utilized by the detection principle is easily changed significantly due to the difference in breed or individual difference of egg-laying chickens, the feeding environment, etc., and then the performance of the culture medium is also controlled by the state of the collected yolk, therefore, for the identification of the bacterium colony with yolk reaction after cultivation, it is necessary to have certain empirical rules.

[0007] Therefore, as a culture medium for detecting Bacillus cereus groups that does not utilize the egg yolk reaction, the inventors of the present application proposed a culture medium containing four components: polymyxin B, trimethoprim, lincomycin antibiotics, and 5-bromo-4-chloro-3-indolyl-α-D-pyranoglucoside, which is a substrate for α-glucosidase (Patent Document 1).

[0008] In addition, a culture medium for selectively detecting Bacillus cereus and Bacillus thuringiensis has been proposed, which, in addition to a phosphatidylinositol-specific phospholipase C substrate and nutrients having a detectable colorimetric or fluorescent free radical, also contains lithium chloride, ceftazidime, polymyxin B sulfate, etc. (Patent Document 2).

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: Japanese Patent Application Publication No. 2011-4712

[0012] Patent Document 2: U.S. Patent No. 6,284,517

[0013] Non-patent literature

[0014] Non-patent document 1: Food Hygiene Index Microorganism Edition 2004 Ministry of Health and Welfare Supervisory Corporation Japan Food Hygiene Association pages 266-282

[0015] Non-patent document 2: ISO11133 Microbiology of food, animal feed and water-Preparation, production, storage and performance testing of culture media (2014)

[0016] 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

[0017] Technical Problems to be Solved by the Invention

[0018] However, it is known that the culture medium described in Patent Document 1 has problems such as insufficient growth of Bacillus cereus for 24 hours at 30°C, which is the same culture condition as the MYP culture medium of ISO7932, and no clear colonies can be detected, and some Staphylococcus species may cause false positives. In addition, it is known that the culture medium described in Patent Document 2 is positive not only for Bacillus cereus but also for Listeria and the like.

[0019] Therefore, the technical problem of the present invention is to provide a medium for detecting Bacillus cereus groups which can be easily produced, has good growth of Bacillus cereus regardless of temperature conditions, and has excellent selectivity, and a method for detecting Bacillus cereus groups using the medium.

[0020] Technical means to solve technical problems

[0021] Therefore, in view of the above-mentioned actual situation, the inventors of the present application have conducted various studies on a culture medium specifically used for detecting Bacillus cereus groups and a detection method thereof. As a result, it was found that if a phosphatidylinositol-specific phospholipase C substrate having a detectable chromogenic or fluorescent free radical is used as a chromogenic or fluorescent substrate, and trimethoprim is contained in the culture medium, then even without utilizing the egg yolk reaction, the growth of the Bacillus cereus group can be improved while suppressing the reproduction of microorganisms other than the Bacillus cereus group, thereby completing the present invention.

[0022] That is, the present invention provides the following inventions [1] to [7].

[0023] [1] A culture medium for detecting Bacillus cereus, comprising a phosphatidylinositol-specific phospholipase C substrate and trimethoprim, wherein the phosphatidylinositol-specific phospholipase C substrate has a colorimetric or fluorescent free radical that can be detected.

[0024] [2] The culture medium for detecting Bacillus cereus groups according to [1], further comprising a β-lactam antibiotic.

[0025] [3] The culture medium for detecting Bacillus cereus groups according to [1] or [2], further comprising an antifungal agent.

[0026] [4] The culture medium for detecting Bacillus cereus according to any one of [1] to [3], wherein the phosphatidylinositol-specific phospholipase C substrate having a detectable colorimetric or fluorescent free radical is selected from 5-bromo-4-chloro-3-indolyl-inositol-1-phosphate, 5-bromo-6-chloro-3-indolyl-inositol-1-phosphate, 6-chloro-3-indolyl-inositol-1-phosphate, 4-methylumbelliferyl-inositol-1-phosphate, 4-nitrophenyl-inositol-1-phosphate, fluorescein-inositol-1-phosphate and salts thereof.

[0027] [5] The culture medium for detecting Bacillus cereus according to any one of [2] to [4], wherein the β-lactam antibiotic is selected from the group consisting of penicillins, cephems, carbapenems, monocyclines, and penems.

[0028] [6] The culture 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 the group consisting of Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, Bacillus cytotoxicus and Bacillus toyonensis.

[0029] [7] A method for detecting a Bacillus cereus group, characterized in that a specimen is inoculated into the culture medium described in any one of [1] to [6] and cultured, and then the detectable colonies on the culture medium are identified.

[0030] Effects of the Invention

[0031] If the culture medium for detecting Bacillus cereus group of the present invention is used, the presence of Bacillus cereus group in a specimen in which various microorganisms are mixed can be accurately, efficiently and simply identified, and the culture medium is inexpensive and easy to prepare. Therefore, the culture medium of the present invention can also be widely used in the inspection of conventional food, beverage, water, etc. or the inspection of the manufacturing process. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This graph compares the growth performance of food samples inoculated with Bacillus cereus in N-BC medium and EX-BC medium, respectively, using MYP medium as the benchmark.

[0033] Figure 2This is a graph showing the state of colonies after culturing at 30°C for 22 hours using N-BC medium and EX-BC medium. DETAILED DESCRIPTION

[0034] The Bacillus cereus group as the detection object in the present invention is Bacillus cereus and bacteria that are genetically related to it and have similar biochemical characteristics. As the Bacillus cereus group, for example, bacteria selected from Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephensis, Bacillus cytotoxicus and Bacillus toyota can be listed. The present invention is preferably used to detect Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides and Bacillus weihenstephensis, and is further preferably used to detect Bacillus cereus and Bacillus thuringiensis.

[0035] In the culture medium of the present invention, a phosphatidylinositol-specific phospholipase C substrate having a chromogenic or fluorescent free radical that can be detected is used as a chromogenic or fluorescent substrate. The substrate is decomposed by the phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Bacillus cereus, which is the detection object of the present invention, thereby releasing a chromogenic or fluorescent free radical that can be detected.

[0036] As the color developing property of this PI-PLC or fluorescent substrate, 5-bromo-4-chloro-3-indolyl inositol-1-phosphate, 5-bromo-6-chloro-3-indolyl inositol-1-phosphate, 6-chloro-3-indolyl inositol-1-phosphate, 4-methylumbelliferyl inositol 1-phosphate, 4-nitrophenyl-inositol-1-phosphate, fluorescein-inositol-1-phosphate and their salt can be listed.Wherein, with regard to color developing property, easy identification bacterium colony this point, particularly preferably 5-bromo-4-chloro-3-indolyl inositol-1-phosphate (hereinafter, sometimes also referred to as X-IP).

[0037] In the present invention, by using a chromogenic or fluorescent substrate of PI-PLC as a chromogenic substrate, it is possible to clearly identify colonies of the Bacillus cereus group independently of temperature conditions, using only trimethoprim, which is a growth inhibitor for microorganisms other than the Bacillus cereus group (hereinafter, also referred to as "other microorganisms"), without inhibiting the growth of the Bacillus cereus group. Therefore, it is also advantageous in that it is not necessary to add polymyxin B and lincomycin required in Patent Document 1.

[0038] From the perspective of good color development and identification, the content of the color development 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 further preferably 0.1 to 1 g / L, in terms of concentration during detection.

[0039] The trimethoprim used in the culture medium of the present invention acts as a growth inhibitor of other microorganisms. By adding trimethoprim, the growth of many Gram-positive bacteria (especially Staphylococcus and Enterococcus) and Gram-negative bacteria other than the Bacillus cereus group can be inhibited. In particular, the growth inhibitory effect on Gram-negative bacteria is strong, and it also acts on Gram-positive bacteria other than the Bacillus cereus group.

[0040] Specific examples of trimethoprim include trimethoprim and its lactate.

[0041] The content of trimethoprim in the culture medium is not particularly limited, but 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 in terms of concentration during detection.

[0042] In the culture medium of the present invention, in addition to the above two components, it is preferred to contain a β-lactam antibiotic for the purpose of enhancing the inhibition of the growth of Gram-negative bacteria. As the β-lactam antibiotic, one or more selected from penicillins, cephems, carbapenems, monocyclines and penems can be used. Among them, from the point of view of enhancing the inhibition of the growth of Gram-negative bacteria, cephem antibiotics or monocycline antibiotics are more preferred.

[0043] As cephem antibiotics, there are third generation cephalosporins, for example, ceftriaxone, ceftriaxone, ceftazidime, cefoperazone, cefsulodin, ceftibuten, ceftriaxone, etc. can be listed. In addition, as monocycline antibiotics, aztreonam, tigemonam, carumonam, nocardin, tabtoxin can be listed. Among them, ceftazidime or aztreonam is preferably used. Regarding the content of β-lactam antibiotics in the culture medium, in terms of the concentration at the time of detection, it is preferably 0.001 to 100 mg / L, more preferably 0.01 to 25 mg / L, and further preferably 0.5 to 5 mg / L.

[0044] In addition, it is preferred that the culture medium of the present invention further contains an antifungal agent. As the antifungal agent, one or more selected from polyenes (such as amphotericin B, etc.), candins (such as micafungin, caspofungin, etc.), azoles (such as fluconazole, voriconazole, itraconazole, etc.), allylamines (such as terbinafine, etc.), fluoropyridines (such as flucytosine, etc.) can be used. Among them, for the purpose of inhibiting the reproduction of fungi, amphotericin B is preferably contained. The concentration of the antifungal agent in the culture medium is not particularly limited, and is preferably 0.001 to 100 mg / L, more preferably 0.1 to 10 mg / L, and further preferably 1 to 5 mg / L, based on the concentration at the time of detection.

[0045] Furthermore, it is preferred that the culture medium of the present invention contain sugar alcohols and / or inorganic salts.

[0046] Here, as sugar alcohols, for example, sugar alcohols of monosaccharides or oligosaccharides can be listed, and erythritol, xylitol, sorbitol, mannitol, maltitol, etc. can be listed. Among them, because it is easy to identify the Bacillus cereus group when using mannitol, it is preferred. These sugar alcohols can be used alone or in combination of two or more.

[0047] The content of sugar alcohols in the culture medium is not particularly limited, but is preferably 1 to 50 g / L, more preferably 5 to 30 g / L, and particularly preferably 5 to 20 g / L in terms of concentration during detection.

[0048] In addition, inorganic salts include inorganic acid metal salts such as sodium chloride and sodium thiosulfate; organic acid metal salts such as ammonium ferric citrate and sodium citrate, etc. Among them, the inorganic acid metal salts are preferred, and sodium chloride is preferred because it is easy to identify the Bacillus cereus group. These inorganic salts can be used alone or in combination of two or more.

[0049] The content of the inorganic salts in the culture medium is not particularly limited, but is preferably 0.1 to 20 g / L, more preferably 1 to 10 g / L, and particularly preferably 3 to 8 g / L in terms of concentration during detection.

[0050] In addition to the above-mentioned medium components, the medium of the present invention may contain medium components such as carbon sources, nitrogen sources, minerals, vitamins and other bacterial nutrients and pH adjusters.

[0051] For example, as the carbon source, one or more substances selected from glucose, fructose, lactose, sucrose, etc. can be listed; as the nitrogen source, one or more substances selected from protein decomposition products (casein peptone, soy peptone, meat peptone, etc.), yeast extract, meat extract, fish extract, etc. can be listed; as the mineral source, one or more substances selected from copper, zinc, magnesium, cobalt, etc. can be listed; as vitamins, one or more substances selected from niacin, pantothenate, biotin, riboflavin, folic acid, etc. can be listed.

[0052] In addition, as components used for pH adjusters, for example, organic acid salts of oxalic acid, acetic acid, fumaric acid, malic acid, lactic acid, gluconic acid, tartaric acid, etc.; inorganic salts of phosphoric acid, hydrochloric acid, sulfuric acid, etc.; carbonates such as sodium carbonate and sodium bicarbonate; hydroxides such as sodium hydroxide; ammonia gas or ammonia water; amine citrates; lower alkanolamines; basic amino acids such as arginine and lysine, etc. These components used for pH adjusters can be used alone or in combination of two or more. At this time, the pH of the culture medium is preferably adjusted to 5 to 8, more preferably 6.5 to 7.7, and further preferably 6.8 to 7.4.

[0053] Furthermore, in order to prepare a solid or semi-solid culture medium, the culture medium of the present invention may contain a solidifying component or a gelling component such as a natural substance such as gelatin, agar, xanthan gum, locust bean gum, guar gum, carrageenan, or a synthetic substance such as hydroxyethyl cellulose. These components may be used alone or in combination of two or more.

[0054] In addition, the culture medium of the present invention can also be used as a simple culture medium by using a fibrous liquid absorbing material such as a fibrous water-absorbing sheet to absorb the liquid culture medium. Examples of the fiber include natural fibers derived from plants, animals, etc.; chemical fibers derived from chemical synthesis, glass fibers, etc.; and nonwoven fabrics formed by making the fibers into sheets are preferred.

[0055] Examples of the simplified culture medium include simplified culture media prepared by the preparation methods described in Japanese Patent Application Laid-Open Nos. 57-502200, 3-15379, 2-65798, 6-181741, 9-19282, and 2000-325072.

[0056] As a specific example of a simplified culture medium, the following simplified culture medium can be cited: a simplified culture medium in which a culture medium composition containing (a) a binder soluble in water and alcohol, (b) a gelling agent soluble in water but insoluble in alcohol, and (c) nutrients for bacterial cells is supported on a fibrous absorbent sheet having meshes larger than those of the gelling agent (Japanese Patent Application Laid-Open No. 9-19282); a simplified culture medium in which an alcohol suspension containing (a) 0.01 to 0.4 wt % of a binder soluble in water and alcohol, (b) a gelling agent soluble in water but insoluble in alcohol, and (c) nutrients for bacterial cells is impregnated in a fibrous absorbent sheet having meshes 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 alcohol, and the absorbent sheet is fixed to the waterproof flat plate (Japanese Patent Application Laid-Open No. 2000-325072).

[0057] Examples of the water- and alcohol-soluble binder include hydroxypropylcellulose and polyvinyl pyrrolidone. Examples of the water-soluble but alcohol-insoluble gelling agent include the above-mentioned solidifying components or gelling components. The average particle size of the gelling agent is preferably 0.5 to 50 μm.

[0058] The form of the culture medium of the present invention is not particularly limited, and examples thereof include liquid culture medium, agar culture medium, and sheet-shaped simple culture medium. As a preparation method at this time, for example, the following methods can be cited: after adding purified water and the like to the above-mentioned culture medium components and mixing and stirring, sterilizing by autoclave and the like, and dispensing it into a sterilized culture dish and the like, cooling or cooling (in this case, heat-sensitive components such as enzyme substrates and antibiotics can be added after sterilization by autoclave and before dispensing.); after adding alcohol or purified water and the like to the above-mentioned culture medium components and mixing and stirring, dispensing it into a container (plastic, glass, etc.) containing a fibrous liquid absorbent material, and sterilizing it by gamma ray irradiation. As alcohol, for example, ethanol, 2-propanol, etc. can be cited.

[0059] In the method for detecting the Bacillus cereus group of the present invention, a specimen is inoculated into the detection medium obtained above and cultured under predetermined conditions, and then the detectable colonies on the medium are identified.

[0060] Here, the term "detectable colonies" refers to colonies formed with a specific color tone that can be observed with the naked eye or in the presence of fluorescence, and the presence of the Bacillus cereus group is identified by recognizing the colonies.

[0061] The specimen is not particularly limited, and a food suspension, a specimen collected by swabbing in an environment such as a kitchen or kitchenware, a culture solution obtained by culturing in a culture medium for bacterial enrichment, a soil suspension, river water, drinking water, etc. can be used.

[0062] The sample can be directly or concentrated or diluted and inoculated in the culture medium of the present invention and cultured. In this case, from the perspective of the determination of the number of colonies, it is preferred to concentrate or dilute the sample to 10 to 10 at the time of inoculation. -10 About times.

[0063] The inoculation method is not particularly limited, but a spread plate method, a streak coating method, and a filter method (a method in which a filter membrane after filtering a specimen is placed on a culture medium and culture is performed) are preferred.

[0064] The culture temperature is not particularly limited, but is preferably 10 to 48° C., a temperature at which Bacillus cereus can grow, and is particularly preferably 30 to 35° C. The culture time is not particularly limited, but is preferably 18 to 48 hours, and particularly preferably 22 to 26 hours. The culture is preferably performed aerobically under static conditions.

[0065] Regarding the color or fluorescence of the colonies (including the periphery) of the Bacillus cereus group formed in the culture medium, since the present invention uses an enzyme substrate that can develop color or emit fluorescence, the colonies of the Bacillus cereus group can be identified by the presence of the color development or fluorescence of the substrate, and when the presence of the color tone or fluorescence is not confirmed, it is identified as a colony that is not a Bacillus cereus group. In particular, when X-IP is used as an enzyme substrate, it can be judged by the change in the blue color tone. In this way, it can be identified whether there is a Bacillus cereus group in the specimen and its colony number.

[0066] Example

[0067] The present invention will be specifically described below with reference to Examples, but the present invention is not limited to these Examples.

[0068] Test Example 1: Agar medium

[0069] [Preparation of culture medium]

[0070] Table 1 shows the composition of the medium based on standard agar medium (SMA), and Table 2 shows the composition of the medium based on tryptone soy agar medium (TSA).

[0071] Preparation method: Add 1L of purified water to the culture medium composition of Table 1 or Table 2 (excluding X-IP), heat and dissolve at 121°C for 15 minutes, and stir carefully, then add X-IP, stir, and dispense 20mL each into plastic culture dishes (90Φmm), and let stand until the culture medium solidifies to prepare the SMA-based culture medium of the present invention (SMA-BC) and the TSA-based culture medium of the present invention (TSA-BC).

[0072] [Strains for testing]

[0073] Bacillus cereus ATCC11778 was pre-cultured in TSA for 24 hours and the concentration was adjusted to 1×10 2 ~1×10 6 cfu / mL, and 0.05 mL of the bacterial solution thus obtained was inoculated into each culture medium.

[0074] [Cultivation results]

[0075] As shown in Table 3, after each strain was subjected to the test and cultured at 30°C for 22 hours, good growth of Bacillus cereus and blue color development were confirmed in SMA-BC and TSA-BC.

[0076] This result shows that Bacillus cereus can be detected by combining X-IP, a phosphatidylinositol-specific phospholipase C substrate having a detectable color-developing free radical, with trimethoprim.

[0077] [Table 1]

[0078] serial number Raw material name composition 1 Casein Peptone 5.0g 2 Yeast Extract 2.5g 3 glucose 1.0g 4 Agar 15.0g 5 X-IP 0.6g 6 Trimethoprim 0.005g

[0079] [Table 2]

[0080] serial number Raw material name composition 1 Peptone 15.0g 2 Soybean Peptone 5.0g 3 Sodium chloride 5.0g 4 Agar 15.0g 5 X-IP 0.6g 6 Trimethoprim 0.005g

[0081] [Table 3]

[0082]

[0083] Test Example 2: Growth Test

[0084] [Preparation of culture medium]

[0085] For the medium (N-BC medium) of the present invention, a hydroxypropyl cellulose (HPC) solution of the composition shown in Table 5 was added to the medium composition shown in Table 4, and after being suspended while stirring, 0.9 mL of the suspension was dispensed into a container (50 Φ mm) containing a cotton sheet (50 Φ mm), and two pieces of the cotton sheet were stacked, and the medium was slowly dried overnight in a non-open space and then covered. After the medium was sealed and packaged together with the desiccant using an aluminum packaging material, it was sterilized by gamma ray irradiation with a surface dose of 10 to 20 kGy.

[0086] [Strains for testing]

[0087] The test strain was pre-cultured in TSA for 24 hours and adjusted to 1×10 1 ~1×10 4 cfu / mL, and 1 mL of the bacterial solution obtained was inoculated into N-BC medium.

[0088] [Cultivation results]

[0089] As shown in Table 6, each strain was subjected to the test and cultured at 30°C for 24 hours. Good growth of Bacillus cereus and development of blue color were confirmed in the N-BC medium.

[0090] According to the results, it is known that if a phosphatidylinositol-specific phospholipase C substrate having a detectable color-developing free radical such as X-IP is combined with β-lactam antibiotics such as trimethoprim and aztreonam, Bacillus cereus can be detected more effectively. In addition, it is also known that amphotericin B can be further added as an antifungal agent.

[0091] [Table 4]

[0092] serial number Raw material name composition 1 Casein Peptone 10g 2 Meat Extract 5g 3 Sodium chloride 5g 4 Mannitol 10g 5 Sodium pyruvate 1g 6 <![CDATA[Trimethoprim ※ > 0.005g 7 <![CDATA[Amphotericin B ※ > 0.00117g 8 Aztreonam 0.0005g 9 X-IP 0.6g 10 Anhydrous disodium hydrogen phosphate 4g 11 Sodium dihydrogen phosphate anhydrous 1g 12 Xanthan gum 45g 13 Anhydrous sodium carbonate Correction amount

[0093] ※ Add to the composition only when "+" is shown in Table 6

[0094] [Table 5]

[0095] serial number Raw material name composition 1 Hydroxypropyl Cellulose 1g 2 Ethanol 0.9L

[0096]

[0097] Test Example 3: Multi-strain test (Bacillus cereus group)

[0098] [Preparation of culture medium]

[0099] For N-BC medium, the HPC solution shown in Table 4 was added to the medium composition shown in Table 7, and for the medium shown in Patent Document 1 (EX-BC medium), the HPC solution shown in Table 5 was added to the medium composition shown in Table 7, and after being suspended while stirring, 0.9 mL of the suspension was dispensed into a container (50 Φ mm) containing a cotton sheet (50 Φ mm), and two cotton sheets were stacked, and slowly dried overnight in a non-open space, and then covered. After the medium was sealed and packaged together with the desiccant using aluminum packaging material, it was sterilized by gamma ray irradiation with a surface dose of 10 to 20 kGy.

[0100] In addition, TSA, MYP agar medium (MYP), and NGKG agar medium (NGKG) were used as control media.

[0101] [Strains for testing]

[0102] The test strain was pre-cultured in TSA for 24 hours and adjusted to 1×10 1 ~1×10 4 1 mL of the bacterial solution was inoculated into each culture medium.

[0103] [Cultivation results]

[0104] As shown in Table 8, each strain was subjected to the test and cultured at 30°C for 22 hours, and the inoculated bacterial concentration was calculated from the number of colonies grown. At this time, only EX-BC was cultured at 35°C. According to the results, the growth performance of any Bacillus cereus in N-BC medium is equivalent to its growth performance in TSA, MYP and NGKG.

[0105] [Table 7]

[0106] serial number Raw material name composition 1 Casein Peptone 10g 2 Meat Peptone 10g 3 Beef Extract Powder (Lab-Lemco Powder) 5g 4 Sodium chloride 5g 5 Glycine 10g 6 Mannitol 10g 7 Sodium pyruvate 1g 8 Trimethoprim 0.005g 9 Amphotericin B 0.00117g 10 Polymyxin B 500,000 u 11 Lincomycin 0.0025g 12 5-Bromo-4-chloro-3-indolyl-α-D-glucopyranoside 0.15g 13 Xanthan gum 20g 14 Anhydrous sodium carbonate Correction amount

[0107] [Table 8]

[0108]

[0109] (Unit: Log 10 cfu / mL)

[0110] Test Example 4: Trimethoprim concentration change test

[0111] [Preparation of culture medium]

[0112] N-BC medium was prepared in the same manner as in Test Example 2. At this time, the test was carried out by changing the concentration of trimethoprim to 0 mg / L, 0.1 mg / L, 5.0 mg / L, and 50 mg / L, and changing the concentration of X-IP to 0.4 g / L.

[0113] [Strains for testing]

[0114] The strains shown in Table 9 were pre-cultured in TSA for 24 hours and the concentration was adjusted to 1×10 1 ~1×10 4 1 mL of the bacterial solution was inoculated into each culture medium.

[0115] [Cultivation results]

[0116] As shown in Table 9, when the trimethoprim concentration of the N-BC medium was 0 mg / L, the growth of Listeria and Bacillus cereus was confirmed after 24 hours of culture. However, when the trimethoprim concentration of the N-BC medium was 0.1 mg / L, 5 mg / L, and 50 mg / L, although the growth of Listeria was suppressed after 24 hours of culture, the growth of Bacillus cereus was confirmed.

[0117] [Table 9]

[0118]

[0119] Test Example 5: Multi-strain test (non-Bacillus cereus group)

[0120] [Preparation of culture medium]

[0121] In the same manner as in Test Example 3, N-BC medium and EX-BC medium were prepared.

[0122] [Strains for testing]

[0123] The test strains were pre-cultured in TSA for 24 to 48 hours and adjusted to 1×10 3 ~1×10 7 1 mL of the bacterial solution was inoculated into each culture medium.

[0124] [Cultivation results]

[0125] As shown in Table 10, each strain was subjected to the test, cultured at 30°C for 22 hours in N-BC, and cultured at 35°C for 22 hours in EX-BC, and growing colonies were confirmed. Although one strain of Listeria monocytogenes was confirmed to be blue in N-BC medium, false positives of Corynebacterium, Enterococcus, and Staphylococcus observed in EX-BC medium as a control medium were not confirmed. Therefore, it can be seen that N-BC medium has fewer false positives than EX-BC medium.

[0126] [Table 10]

[0127]

[0128] Test Example 6: Research using food materials

[0129] [Preparation of culture medium]

[0130] In the same manner as in Test Example 3, N-BC medium, EX-BC medium and MYP were prepared.

[0131] [Strains for testing]

[0132] Bacillus cereus ATCC 11778, ATCC14579 and ATCC19637 were pre-cultured in TSA for 24 hours and adjusted to 1×10 4 , 1×10 6 or 1×10 8cfu / mL, add 0.1 mL of the bacterial solution obtained in this way to 10 g of the specified food, let it stand at room temperature, refrigerated or frozen for 48 hours to 2 weeks, add 90 mL of sterile saline to prepare the digested food liquid specimen. 1 mL of the specimen was inoculated into N-BC medium, EX-BC medium and MYP.

[0133] [Cultivation results]

[0134] The strains were used for the test, cultured in N-BC and MYP at 30°C for 22 hours, and cultured in EX-BC at 35°C for 22 hours, and the number of bacteria grown in each culture medium was calculated. Figure 1 As shown, the growth difference between N-BC medium and MYP as control medium was 1×10 -0.03 (93% relative to MYP), while the growth difference between EX-BC and MYP as the control medium was 1×10 -0.49 (32% relative to MYP), it can be seen that the growth of the strain has been significantly improved.

[0135] Test Example 7: Colony formation ability after 22 hours of culture

[0136] [Preparation of culture medium]

[0137] In the same manner as in Test Example 3, N-BC medium and EX-BC medium were prepared.

[0138] [Strains for testing]

[0139] The strain prepared by pre-culturing Bacillus cereus ATCC11778 in TSA for 24 hours was used, and the concentration was adjusted to 1×10 2 1 mL of the bacterial solution was inoculated into each culture medium.

[0140] [Cultivation results]

[0141] like Figure 2 As shown, each strain was used for the test and cultured at 30°C for 22 hours, and the grown colonies were confirmed. It was found that N-BC medium formed larger colonies and grew faster than EX-BC medium as a control medium.

[0142] Test Example 8: Comparison with the culture medium described in Patent Document 2 (US culture medium)

[0143] [Preparation of culture medium]

[0144] The medium composition of US medium is shown in Table 11.

[0145] Preparation method: Add 1L of purified water to the weighed medium composition of 1 to 10 in Table 11, heat and dissolve at 121°C for 15 minutes, add the medium composition of 11 to 14 in Table 11, and stir carefully, then dispense 20mL each into a plastic culture dish (90Φmm), and let it stand until the medium solidifies to prepare US medium. N-BC medium was prepared in the same manner as in Test Example 2.

[0146] [Table 11]

[0147]

[0148] [Strains for testing]

[0149] The strains shown in Table 12 were pre-cultured in TSA for 24 hours and adjusted to a specified concentration (10 3 ~10 7 cfu / mL). 100 μL of the bacterial solution was inoculated into US medium, and 1 mL of the bacterial solution was inoculated into N-BC medium.

[0150] [Cultivation results]

[0151] As shown in Table 12, after each strain was subjected to the test and cultured at 35°C for 22 hours, it was confirmed that Listeria formed clear blue colonies in the US medium, and Candida formed clear white colonies. On the other hand, after each strain was subjected to the test and cultured at 30°C for 22 hours, in the N-BC medium, although the entire culture medium of one strain of Listeria monocytogenes showed extremely weak blue coloration for Listeria, Candida did not form colonies.

[0152] [Table 12]

[0153]

[0154] Test Example 9: Concentration of Amphotericin B

[0155] [Preparation of culture medium]

[0156] For the culture medium (N-BC culture medium) of the present invention, add the hydroxypropyl cellulose (HPC) solution of the composition shown in Table 5 to the culture medium composition shown in Table 13, and suspend it while stirring, then dispense 0.9 mL of the suspension into a container (50 Φ mm) containing a cotton sheet (50 Φ mm), and stack two pieces of the cotton sheet, slowly dry overnight in a non-open space, and then cover. After sealing and packaging this culture medium together with the desiccant with aluminum packaging material, sterilize it by gamma ray irradiation with a surface dose of 10 to 20 kGy. At this time, each culture medium was adjusted in such a way that amphotericin B was 0 mg, 0.1 mg, 1 mg, and 10 mg, respectively.

[0157] [Table 13]

[0158]

[0159] [Strains for testing]

[0160] The test strains were pre-cultured in TSA for 24 to 48 hours and adjusted to 1×10 2 ~1×10 5 1 mL of the bacterial solution was inoculated into each culture medium.

[0161] [Cultivation results]

[0162] As shown in Table 14, each strain was subjected to the test and cultured at 30°C for 24 to 44 hours, and the growth of colonies was confirmed. In the 24-hour culture, blue colonies were confirmed for the three strains of Bacillus cereus and Bacillus thuringiensis in the N-BC medium, but no growth was observed for the two strains of Candida albicans. On the other hand, in the case of 44-hour culture, the three strains of Bacillus cereus and Bacillus thuringiensis showed the same results as in the 24-hour culture, but the two strains of Candida albicans showed no growth at the concentration of 10 at 0 mg or 0.1 mg of amphotericin B. 5 or 10 4 cfu / mL, growth or weak staining was observed.

[0163]

[0164] Test Example 10: Agar medium

[0165] [Preparation of culture medium]

[0166] The composition of TSA-based medium is shown in Table 15.

[0167] Preparation method: Add 1L of purified water to the culture medium composition in Table 15 (excluding X-IP, aztreonam, and ceftazidime), heat and dissolve at 121°C for 15 minutes, stir carefully, add X-IP and aztreonam or ceftazidime as needed, then stir, dispense 20mL each into plastic culture dishes (90Φmm), and let stand until the culture medium solidifies to prepare TSA-BC.

[0168] [Table 15]

[0169] serial number Raw material name composition 1 Peptone 15.0g 2 Soybean Peptone 5.0g 3 Sodium chloride 5.0g 4 Agar 15.0g 5 X-IP 0.4g 6 Trimethoprim 5mg 7 Amphotericin B 1.12mg 8 Aztreonam or ceftazidime 0, 0.05, 0.5, 5 mg

[0170] [Strains for testing]

[0171] The strains shown in Table 16 were pre-cultured in TSA for 24 hours and adjusted to 1×10 1 ~1×10 5 cfu / mL, and 0.05 mL of the bacterial solution thus obtained was inoculated into each culture medium.

[0172] [Cultivation results]

[0173] As shown in Table 16, after each strain was subjected to the test and cultured at 30°C for 22 hours, good growth of Bacillus cereus and blue coloration were observed in TSA-BC. However, the addition of aztreonam or ceftazidime suppressed the formation of white colonies of Escherichia coli or Enterobacter aerogenes.

[0174] [Table 16]

[0175]

[0176] In summary, if the culture medium of the present invention is used, it is possible to accurately, efficiently and simply identify whether the Bacillus cereus group exists in the test sample. In addition, the culture medium is inexpensive and easy to prepare. Therefore, the culture medium can be widely used in conventional food, beverage, water, etc. inspection or manufacturing process inspection.

Claims

1. A culture medium for detecting Bacillus cereus, comprising (A) a phosphatidylinositol-specific phospholipase C substrate, (B) trimethoprim, and (C) a monocycline antibiotic or a cephem antibiotic, wherein the phosphatidylinositol-specific phospholipase C substrate has a colorimetric or fluorescent free radical that can be detected. in, The monocycline antibiotic is aztreonam, and the cephem antibiotic is ceftazidime. Wherein, the culture medium for detecting Bacillus cereus group further contains a polyene antifungal agent, and the polyene antifungal agent is amphotericin B.

2. The culture medium for detecting Bacillus cereus group according to claim 1, wherein The phosphatidylinositol-specific phospholipase C substrate having a detectable colorimetric or fluorescent free radical is selected from 5-bromo-4-chloro-3-indolyl-inositol-1-phosphate, 5-bromo-6-chloro-3-indolyl-inositol-1-phosphate, 6-chloro-3-indolyl-inositol-1-phosphate, 4-methylumbelliferyl-inositol-1-phosphate, 4-nitrophenyl-inositol-1-phosphate, fluorescein-inositol-1-phosphate and salts thereof.

3. The culture medium for detecting Bacillus cereus group according to claim 1, wherein The Bacillus cereus group is selected from Bacillus cereus ( Bacillus cereus ), Bacillus anthracis ( Bacillus anthracis ), Bacillus thuringiensis ( Bacillus thuringiensis ), Bacillus mycoides ( Bacillus mycoides ), Bacillus pseudomycoides ( Bacillus pseudomycoides ), Bacillus weihenstephensis ( Bacillus weihenstephanensis ), Cytotoxic Bacillus ( Bacillus cytotoxicus ) and Bacillus japonensis ( Bacillus toyonensis ) in bacteria.

4. A method for detecting a Bacillus cereus group, the method not being used for the treatment or diagnosis of a disease, the method being characterized in that: After inoculating a specimen into a culture medium according to any one of claims 1 to 3 and culturing the specimen, the detectable bacterial colonies on the culture medium are identified.

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