Culture medium and method for selectively detecting or screening xerophilic bacteria using the same

Abstract

To provide a medium that is capable of selectively growing xerophilic bacteria, specifically, a medium suitable for selective growth of xerophilic bacteria.SOLUTION: Provided is a medium comprising: at least one selected from the group consisting of sugar, malt extract, and yeast extract; and water, where the medium has a sugar content of 61 to 66 mass% and a water activity of 0.78 to 0.82.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a culture medium, preferably a culture medium suitable for the growth of xerophilic bacteria, and to a method for selectively, or selectively and rapidly, detecting or screening xerophilic bacteria using the culture medium of the present invention. [Background technology]

[0002] Fungi such as filamentous molds and yeasts are often found in food as foreign bodies, as their colonies can be observed with the naked eye and their conidia are highly visible due to their various colors. Mold, in particular, can occur in multiple products, and there have been many recent cases where mold has appeared in food products, leading to product recalls. Mold problems have a significant impact not only on consumers but also on companies. Once mold has occurred, it not only requires a great deal of effort and cost for product recalls, but also leads to a damage to the company's image.

[0003] Among fungi, xerophilic fungi grow in foods with relatively low water activity (hereinafter also referred to as "Aw"), such as semi-perishable sweets, and are one of the causes of food complaints. While the optimum Aw for xerophilic filamentous fungi is 0.95, they can grow even at Aw levels of 0.80 or lower, and examples of such fungi include some species of the Aspergillus genus, some species of the Eurotium genus, the Wallemia genus, the Chrysosporium genus, the Xeromyces genus, and the Baspetospora genus. These filamentous fungi grow poorly or not at all on common fungal media, such as potato dextrose agar, malt extract agar, and Czapek yeast extract agar. Therefore, for testing, a xerophilic fungal medium with a low Aw must be used. Known media for xerophilic fungi include dichloran-glycerin 18 agar (DG18) (Aw: 0.95), 25% glycerol-nitrate agar (G25N) (Aw: 0.93), 20% glucose-malt extract-yeast extract agar (MY20G) (Aw: 0.97), 30% glucose-malt extract-yeast extract agar (MY30G) (Aw: 0.95), 50% glucose-malt extract-yeast extract agar (MY50G) (Aw: 0.89), and 70% glucose / fructose-malt extract-yeast extract agar (MY70GF) (Aw: 0.76). It has also been reported that a medium prepared to have a low Aw by adding sucrose to a commercially available malt extract agar medium can also be used as a medium for growing xerophilic filamentous fungi (see Non-Patent Documents 1 to 3).

[0004] Other known media suitable for the growth of xerophilic filamentous fungi include semi-solid media containing at least 18% (w / w) glycerol, at least about 30% (w / w) glucose, or at least 20% (w / w) sucrose (Patent Document 1). Furthermore, Patent Document 2 describes that 40% sucrose-malt extract-yeast extract agar medium (M40Y), 10% glucose-malt extract-yeast extract agar medium (MY10G), and 30% glucose-malt extract-yeast extract agar medium (MY30G) can be used to culture xerophilic filamentous fungi, but also describes that these media can also efficiently culture hygrophilic filamentous fungi. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2015-519077 [Patent Document 2] International Publication WO2012 / 169099 [Non-patent literature]

[0006] [Non-Patent Document 1] Shimizu, S. et al. (2011), Food contamination cases of xerophilic molds, Rep. Hokkaido Inst. Pub. Health, 61, 47-51 [Non-patent document 2] Yoshinami, Makoto (2014), Investigation of the causes and countermeasures for mold contamination in food manufacturing sites, Journal of the Japanese Society for Food Microbiology, Jpn. J. Food Microbiol., 31(1), 13-19 [Non-patent document 3] Sugiura, Y. (2020), Food fungi and their testing, JSM Mycotoxins 70(2), 95-104 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a medium capable of selectively growing xerophilic bacteria, i.e., a medium suitable for the selective growth of xerophilic bacteria. More preferably, an object of the present invention is to provide a medium capable of selectively and rapidly growing xerophilic bacteria, i.e., a medium suitable for the selective and rapid growth of xerophilic bacteria. Another object of the present invention is to provide a method for selectively, preferably selectively and rapidly detecting xerophilic bacteria using the medium, and a screening method. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above-mentioned problems, and have found that a medium suitable for the selective growth of xerophilic bacteria can be obtained by adjusting the sugar content of a medium containing at least one selected from the group consisting of sugar, malt extract, and yeast extract, and water, to 61 to 66 mass % and a water activity of 0.78 to 0.82, thereby solving the above-mentioned problems. The present invention was completed through further investigation based on this finding, and includes the following embodiments.

[0009] (I) Culture medium (I-1) A medium containing sugar; at least one selected from the group consisting of malt extract and yeast extract; and water, The medium is characterized in that the sugar content in the medium is 61 to 66% by mass and the water activity is 0.78 to 0.82. (I-2) The medium according to (I-1), wherein the sugar is glucose. (I-3) The medium according to (I-1) or (I-2), which does not contain antibiotics. (I-4) A medium according to any one of (I-1) to (I-3), which contains malt extract and yeast extract. (I-5) The medium according to (I-4), wherein the malt extract content in the medium is 0.66 to 0.72% by mass and the yeast extract content is 0.16 to 0.18% by mass. (I-6) The medium according to any one of (I-1) to (I-5), having a pH of 4.5 to 5.6. (I-7) The medium according to any one of (I-1) to (I-6), further comprising a gelling agent. (I-8) The medium according to (I-7), wherein the gelling agent is agar. (I-9) The medium according to any one of (I-1) to (I-8), which consists essentially of glucose, malt extract, yeast extract, a gelling agent, and water. (I-10) The medium according to any one of (I-1) to (I-9), which is a medium for selectively culturing xerophilic bacteria or a medium for detecting xerophilic bacteria. (I-11) The medium according to (I-10), wherein the xerophilic fungus is a xerophilic filamentous fungus. (I-12) The medium according to (I-11), wherein the xerophilic filamentous fungus is at least one filamentous fungus selected from Aspergillus chevalieri and Wallemia sebi. (I-13) A culture medium according to any one of (I-1) to (I-12), which is used for a falling bacteria test, an air sampler test, or a surface-attached bacteria test.

[0010] (II) Method for detecting xerophilic bacteria (II-1) A method for detecting xerophilic bacteria in a sample, comprising the following steps: (1) A step of applying a sample onto a medium described in any one of (I-1) to (I-13); (2) culturing the medium at 25±1°C; (3) A step of confirming the presence or absence of mycelia, spores, or colonies on the medium within 14 days from the start of culture. (II-2) A detection method according to (II-1), in which, if mycelia, spores, or colony formation is confirmed on the medium in the step (3), it is determined that xerophilic fungi have been detected in the sample. (II-3) A detection method according to (II-1) or (II-2), wherein the specimen is a microbial testing sample derived from a food product having a water activity of 0.80 or less, or an environmental monitoring sample.

[0011] (III) Screening method for xerophilic bacteria (III-1) A method for screening xerophilic bacteria, comprising the following steps: (1) A step of applying a sample onto a medium described in any one of (I-1) to (I-13); (2) culturing the medium at 25±1°C; (3) A step of collecting mycelia, spores, or colonies formed on the medium as xerophilic fungi within 14 days from the start of culture. [Effects of the Invention]

[0012] According to the present invention, it is possible to provide a medium capable of selectively growing xerophilic bacteria, i.e., a medium suitable for the selective growth of xerophilic bacteria. More preferably, it is possible to provide a medium capable of selectively and rapidly growing xerophilic bacteria, i.e., a medium suitable for the selective and rapid growth of xerophilic bacteria. Furthermore, it is possible to provide a method for selectively, preferably selectively and rapidly detecting xerophilic bacteria and a screening method using the medium. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a diagram illustrating the colony size measurement method ([(a+b) / 2] cm) employed in Experimental Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0014] (I) Culture medium, its preparation method, and uses The medium of the present invention (hereinafter referred to as "this medium") contains at least one selected from the group consisting of sugar, malt extract, and yeast extract; and water, and is characterized in that the sugar content of the medium is 61 to 66% by mass and the water activity is 0.78 to 0.82. Each component will be described below.

[0015] (1) Sugar In this medium, sugar is preferably the only exogenous carbohydrate (carbon source) other than the carbohydrates (carbon sources) contained in the malt extract and yeast extract. Examples of sugars include glucose, sucrose, fructose, lactose, xylose, cellobiose, galactose, isomaltol, trehalose, sorbose, allose, tagatose, psicose, and maltose, with glucose, sucrose, and fructose being preferred, and glucose being more preferred. These can be used alone or in combination of two or more. A more preferred embodiment is one in which glucose is used as the only externally added sugar or carbohydrate (carbon source).

[0016] As described above, the sugar content in 100% by mass of the medium is 61 to 66% by mass, preferably 61 to 65.5% by mass, more preferably 61 to 65.1% by mass, and particularly preferably 61 to 65% by mass.

[0017] (2) Malt extract and yeast extract Although not limited thereto, in this medium, malt extract and yeast extract serve as a nitrogen source, a carbon source, or nutritional components (vitamins, minerals, etc.). Malt extract and yeast extract for medium preparation are both commercially available, and these commercially available products can be used conveniently. The medium can be prepared using at least one of malt extract and yeast extract, but is preferably prepared using both malt extract and yeast extract.

[0018] When malt extract is used without yeast extract, the content of malt extract in 100% by mass of the medium is not limited as long as it does not impair the effect of the present invention (selective growth of xerophilic bacteria), and can be selected from the range of 0.66 to 0.72% by mass.Furthermore, when yeast extract is used without malt extract, the content of malt extract in 100% by mass of the medium is not limited as long as it does not impair the effect of the present invention, and can be selected from the range of 0.16 to 0.18% by mass.

[0019] The medium preferably contains both malt extract and yeast extract. In this case, the content of malt extract in 100% by mass of the medium is not limited, but is 0.60 to 0.75% by mass, preferably 0.66 to 0.72% by mass, and the content of yeast extract is not limited, but is 0.10 to 0.20% by mass, preferably 0.16 to 0.18% by mass.

[0020] There is no particular restriction on the incorporation of other nitrogen sources and / or nutritional components (vitamins, minerals, etc.) as long as the effects of the present invention are not impaired. Examples of such components include peptones (casein peptone, meat peptone, myocardial peptone, gelatin peptone, soybean peptone, etc.), meat extract, fish meat extract, heart infusion, potato extract, tomato juice, and inorganic salts (e.g., sodium chloride, sodium thiosulfate, sodium pyruvate, ammonium ferrous phosphate, dipotassium phosphate, dipotassium hydrogen phosphate, ammonium ferrous citrate, sodium citrate, etc.). However, a more preferred embodiment contains only malt extract and yeast extract as nitrogen sources and / or nutritional components, with no other components present.

[0021] (3)Water In addition to the above ingredients, the medium contains water. The water content is not particularly limited as long as it is a proportion that results in a water activity (Aw) of the sterilized medium measured after sterilization of the medium being in the range of 0.78 to 0.82. Aw is preferably 0.78 to 0.81, and more preferably 0.79 to 0.80.

[0022] The water activity (Aw) of this medium can be measured by the dew point method. Specifically, as explained in the Examples, it can be measured using a water activity measuring device (AquaLab 4TE, manufactured by Meter Japan Co., Ltd.). Specifically, after sterilization, 5 g of medium dispensed into a Petri dish (in the case of a solid medium, the medium is dispensed into a Petri dish in its fluid state and then solidified) is placed in the sample holder of the water activity measuring device and measured in single measurement mode (set temperature 25.0°C). In single measurement mode, the measurement is stopped when the fluctuation in water activity value falls within 0.0005aw.

[0023] (4) Gelling agents, thickeners The medium of the present invention includes media in any form (liquid medium, semi-solid medium, and solid medium), with solid medium being preferred.

[0024] A solid medium can be prepared by adding a gelling agent to the above-mentioned components. The gelling agent is used to solidify the medium and may be any agent that does not impair the effects of the present invention, and may be selected alone or in combination from agar, pectin, gelatin, carrageenan, xanthan gum, methylcellulose, HPMC, guar gum, tara gum, locust bean gum, glucomannan, gum arabic, gellan gum, etc. If necessary, metal salts such as calcium salts and magnesium salts may also be used in combination. Agar is preferred as the gelling agent. When preparing a solid medium, the content of the gelling agent in 100% by mass of the solid medium may be an amount that solidifies the medium, and can be selected, for example, from the range of 0.2 to 4.0% by mass. Specifically, when agar is used as the gelling agent, the content is in the range of 0.2 to 3.0% by mass, preferably 0.2 to 1.5% by mass.

[0025] Semi-solid media can be prepared by adding a thickener to the above-mentioned components. The thickener is used to thicken the medium to make it semi-solid, and any thickener that does not interfere with the effects of the present invention can be selected from the following: cellulose derivatives such as methylcellulose, carboxymethylcellulose, and hydroxyalkylcellulose; thickening polysaccharides such as carrageenan, xanthan gum, guar gum, tara gum, locust bean gum, glucomannan, gum arabic, and gellan gum; acrylic acid derivatives such as polyacrylic acid, polyacrylates, and acrylic acid-vinyl alcohol copolymers; and polyethers such as polyethylene glycol and polypropylene glycol. When preparing a semi-solid medium, the content of the thickener in 100% by mass of the semi-solid medium need only be an amount that thickens the medium to make it semi-solid, and can be selected, for example, from the range of 0.2 to 0.4% by mass.

[0026] (5) Other ingredients This medium contains the above ingredients and is adjusted so that the pH after sterilization is in the range of 3.4 to 5.6. To promote the growth of xerophilic bacteria, a preferred pH range is 5.1 to 5.6, more preferably 5.4 to 5.6. As shown in the experimental examples described below, when the pH of the medium is 5.1 or higher, mycelium, spores, or colonies are formed 14 days after the start of cultivation. The pH of the medium can be measured using a glass electrode hydrogen ion concentration meter. Measurements can be performed by inserting an electrode into the liquid medium (25°C) before sterilization, or into the fluid medium (50°C) after sterilization. Alternatively, the pH of the sterilized medium diluted 10-fold with distilled water (10-fold diluted medium) can be measured. In the case of a solid medium, the solidified medium after sterilization can be pulverized and diluted 10 times with distilled water to prepare a "10-fold diluted medium." Preferably, the pH of the 10-fold diluted medium thus prepared is adjusted to be in the range of 3.4 to 5.6, preferably 5.1 to 5.6, and more preferably 5.4 to 5.6.

[0027] In addition to the above-mentioned components, the medium may contain a pH adjuster as needed to adjust the pH to the above range. Any pH adjuster may be used as long as it does not interfere with the effects of the present invention, and examples thereof include sodium carbonate, sodium bicarbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium monohydrogen phosphate, sodium dihydrogen phosphate, acetic acid, formic acid, carbonic acid, potassium carbonate, phosphoric acid, hydrochloric acid, lactic acid, potassium lactate, sodium lactate, carbon dioxide, nitric acid, sulfuric acid, citric acid, malic acid, oxalic acid, polyphosphates, fumaric acid, succinic acid, monosodium succinate, disodium succinate, tartaric acid, potassium hydrogen tartrate, sodium tartrate, and disodium dihydrogen pyrophosphate.

[0028] If necessary, the medium may contain osmotic regulators such as alkali metal salts and polyhydric alcohols to adjust and maintain osmotic pressure. Examples of alkali metal salts include alkali metal chlorides such as sodium chloride and potassium chloride, and examples of polyhydric alcohols include glycerin. While not limited to these, the osmotic pressure of the medium is preferably adjusted to a range of 4700 to 5600 mOsm / kgH2O, more preferably 4750 to 5570 mOsm / kgH2O. Osmotic pressure can be measured using an osmometer such as the Advanced Osmometer-3250 (manufactured by Rico Techno Co., Ltd.).

[0029] It is preferable that the medium does not contain antibiotics or antibacterial substances. Examples of antibiotics include, but are not limited to, antibiotics against Gram-positive bacteria such as erythromycin and clindamycin; and antibiotics against Gram-negative bacteria such as chloramphenicol and streptomycin sulfate. Examples of antibacterial substances include amphotericin B, polylysine, protamine sulfate, glycine, and sorbic acid.

[0030] (6) Preparation method This medium may be prepared by mixing all ingredients and then sterilizing them, or by mixing pre-sterilized ingredients under sterile conditions. Alternatively, it may be prepared by mixing some ingredients and then sterilizing them, and then adding the remaining pre-sterilized ingredients under sterile conditions. Sterilization may be performed by filtration sterilization when the sterilization target is a liquid, or by heat sterilization using an autoclave or other device. In the case of a semi-solid or solid medium, all ingredients are mixed and then heat sterilized (sterilized), and the resulting mixture is poured into a sterilization container (a petri dish, a test tube, etc.) under sterile conditions and cooled to solidify. Alternatively, agar may be dissolved in water and then heat sterilized, and other pre-sterilized ingredients are added to the mixture under sterile conditions, followed by pouring into a sterilization container and cooling to solidify. The water activity of the medium (sterilized medium) thus prepared is, as described above, in the range of 0.78 to 0.82, preferably 0.78 to 0.81, and more preferably 0.79 to 0.80, and the pH is in the range of 4.6 to 5.6, preferably 5.1 to 5.6, and more preferably 5.4 to 5.6.

[0031] (7) Use of this medium This medium allows the selective growth of xerophilic bacteria. In other words, this medium is suitable for the selective growth of xerophilic bacteria. Specifically, as will be explained in the Examples below, when culturing using this medium, xerophilic bacteria can be grown in at least 14 days of culture, whereas bacteria other than xerophilic bacteria (hygrotrophic bacteria, mesophilic bacteria) cannot grow. Therefore, this medium is useful as a selective medium for xerophilic bacteria and as a medium for detecting xerophilic bacteria.

[0032] The xerophilic fungi are preferably xerophilic fungi such as osmotolerant yeasts and xerophilic filamentous fungi. Osmotolerant yeasts include sugar-tolerant yeasts and salt-tolerant yeasts, and specific examples thereof include Zygosaccharomyces rouxii, Z. bailii, and Z. bisporus. Xerophilic filamentous fungi include Aspergillus chevalieri; some filamentous fungi belonging to the Aspergillus genus, such as Aspergillus conicus, Aspergillus penicillioides, and Aspergillus herbariorum; some filamentous fungi belonging to the Eurotium genus, such as Eurotium repens; Wallemia sebi; some filamentous fungi belonging to the Penicillium genus; and filamentous fungi belonging to the Xeromyces genus.

[0033] When the target xerophilic fungus is a filamentous fungus, one method for selectively growing xerophilic fungi using this medium is to inoculate the fungus on this medium and culture it statically under conditions of 70-80% relative humidity, 24-26°C temperature, and 0-98% CO2 concentration. If mycelial elongation or spore formation is observed within 14 days of culture under these conditions, it can be determined that xerophilic filamentous fungi have grown. On the other hand, as will be shown in the Examples below, under such conditions, hygrotrophic and mesophilic filamentous fungi cannot grow within 20 days, preferably within 14 days. Therefore, by adopting such culture conditions, xerophilic fungi can be selectively grown using this medium.

[0034] The growth pattern (growth steps) of filamentous fungi is as follows: Spore → germination → hyphal elongation (hyphae present) → coloration appears at the tip of the hyphae and spore formation (spore formation). Therefore, by visually inspecting the appearance (using a stereomicroscope for spore formation as needed), if hyphae are visible due to hyphal elongation (hyphae present) or if spore formation is visible, the bacteria can be judged as "growth possible." Details of this judgment will be explained in the Examples below.

[0035] When the target xerophilic bacteria is yeast, the method for selectively growing osmotolerant yeast using this medium is as follows. One method involves inoculating the target bacteria onto this medium and culturing them statically under conditions of 70-80% relative humidity, 24-26°C temperature, and 0-98% CO2 concentration. If circular colonies are formed within 14 days of culturing under these conditions, it can be determined that osmotolerant yeast has grown. More specifically, the target bacteria are cultured in M40Y liquid medium (liquid medium) at 25°C for 24 hours, the resulting culture solution is diluted with a glycerin-added aqueous solution to a concentration of 1 cfu or more per 10 μL, the diluted bacterial solution is seeded in the center of the liquid medium, and static culture is performed under the above conditions. The glycerin concentration in the glycerin-added aqueous solution is adjusted so that the Aw of the diluted bacterial solution seeded in the liquid medium does not exceed the Aw of the liquid medium.

[0036] (II) Methods for detecting and screening xerophilic bacteria By using the above-described medium, xerophilic bacteria in a specimen can be selectively detected. The method can be carried out using the medium by the following steps: (1) applying a sample onto the medium; (2) culturing the medium at 25±1°C; (3) A step of confirming the presence or absence of mycelia, spores, or colonies on the medium within 14 days from the start of culture. In the step (3), if mycelia, spores, or colonies are confirmed on the medium, it can be determined that xerophilic fungi have been detected in the sample. In the confirmation step (3), the pH of the medium used is 4.5 to 5.6, preferably 5.1 to 5.6.

[0037] The culturing method in step (2) varies depending on the type of xerophilic fungus being used, but if the xerophilic fungus being used is a filamentous fungus, it can be cultured by inoculating the fungus on the medium and then statically culturing it under conditions of a relative humidity of 70-80%, a temperature of 24-26°C, and a CO2 concentration of 0-98%. Similarly, if the xerophilic fungus being used is yeast, it can be cultured by statically culturing it under conditions of a relative humidity of 70-80%, a temperature of 24-26°C, and a CO2 concentration of 0-98%.

[0038] The aforementioned xerophilic bacteria are widely distributed in the air and can grow in semi-perishable sweets with low water activity (for example, Aw 0.8 or less), such as gummy candy, fresh chocolate, yokan, daifuku, and castella. Xerophilic bacteria can also be found adhering to ceilings, walls, production lines, etc. in food factories. Therefore, the specimens targeted by the method of the present invention can be foods with a water activity of 0.80 or less, or microbial test samples derived from environmental monitoring samples. Environmental monitoring is a management technique for periodically monitoring and measuring the natural environment and biological elements of a specific region or location, with the aim of collecting information on environmental changes and contamination status and evaluating the health of the environment. Examples of environmental monitoring samples include air, water, and wipes taken from the surface of manufacturing equipment. In the present application, air and wipes taken from the surface of manufacturing equipment are preferred from the perspective of Aw. For example, xerophilic bacteria floating in the air can be efficiently collected and subjected to testing by combining airborne bacteria testing using an air sampler with falling bacteria testing using solid plate media (Koch's method). Xerophilic bacteria occurring in or attached to manufacturing equipment such as facilities or production lines can be effectively collected and subjected to testing by combining this with swabbing testing using cotton swabs. Contaminated air flowing in from air conditioning vents or gaps can be effectively collected and subjected to testing by spraying the target air onto solid plate media.

[0039] In step (3), if the formation of hyphae or spores is observed on the medium within 14 days from the start of culture, it can be determined that xerophilic filamentous fungi have been detected in the sample. Furthermore, in step (3), if the formation of circular colonies of 1 cm or less, rather than the formation of hyphae or spores, is observed on the medium within 14 days from the start of culture, it can be determined that osmotolerant yeast have been detected in the sample. The genera and species of xerophilic filamentous fungi and osmotolerant yeast can be identified by standard methods, such as sequence analysis of the 28S rRNA gene, biochemical methods, and morphological methods such as Gram staining.

[0040] As will be shown in the examples below, in the test method performed under the above-mentioned conditions, non-xerophilic fungi such as hygrotrophic and mesophilic fungi cannot grow within 14 days. Therefore, by carrying out the test method of the present invention described above, xerophilic fungi can be selectively screened. In other words, the test method is also useful as a method for selectively screening xerophilic fungi.

[0041] As described above, in this specification, the terms "comprise" and "contain" encompass the meanings of "consist of" and "consist essentially of." [Example]

[0042] The present invention will be explained below using experimental examples to aid in understanding the configuration and effects of the present invention. However, the present invention is not limited by these experimental examples. Unless otherwise specified, the following experiments were carried out at room temperature (25±5°C) and atmospheric pressure. Unless otherwise specified, "%" and "parts" in the following descriptions mean "% by mass" and "parts by mass," respectively.

[0043] The materials (fungi, culture medium) and evaluation methods used in the experimental examples described below will be explained. (1) Fungi Paecilomyces sp: Wild strain No. M101 (classification: mesohumid filamentous fungus) Penicillium sp. #1: Wild strain No. M108 (Classification: Xerophilic filamentous fungi) Penicillium sp. #2: Wild strain No. M109 (Classification: Mesophilic filamentous fungi) Penicillium sp. #3: Wild strain No. M101 (Classification: Mesophilic filamentous fungi) Penicillium sp. #4: Wild strain No. M5 (Classification: Mesophilic filamentous fungi) Penicillium sp. #5: Wild strain No. M16 (Classification: Mesophilic filamentous fungi) Aspergillus sp. #1: Wild strain No. M102 (Classification: Mesophilic filamentous fungi) Aspergillus sp. #2: Wild strain No. M110 (Classification: Xerophilic filamentous fungi) Aspergillus sp. #3: Wild strain No. M30 (Classification: Mesophilic filamentous fungi) Aspergillus sp. #4: Wild strain No. P110 (Classification: Mesophilic filamentous fungi) Aspergillus chevalieri: NBRC4364 (Classification: Xerophilic filamentous fungi) Wallemia sebi: NBRC5992 (Classification: xerophilic filamentous fungi) Rhizopus microsporus #1: Wild strain No. M104 (classification: hygrophilic filamentous fungus) Rhizopus microsporus #2: Wild strain No. M103 (classification: hygrophilic filamentous fungus) Rhizopus microsporus #3: Wild strain No. P108 (classification: hygrophilic filamentous fungus) Rhizopus microsporus #4: Wild strain No. P103 (classification: hygrophilic fungus) Rhizopus microsporus #5: Wild strain No. P104 (classification: hygrophilic filamentous fungus) Mucor hiemalis:NBRC9405 (Classification: Hygrophilic filamentous fungus) Rhizopus oryzae: NBRC4716 (Classification: Hygrophilic filamentous fungi) Cladosporium cladosporioides #1: NBRC6368 (Classification: Mesophilic filamentous fungi) Cladosporium cladosporioides #2: NBRC4459 (Classification: Mesophilic filamentous fungi) Paecilomyces variotii: NBRC33284 (classification: mesohumid filamentous fungus) Eurotium repens:JCM1580 (classification: xerophilic fungi) Rhodotorula sp.: Wild strain (classification: osmotolerant yeast) Cryptococcus saitoi: Wild strain (classification: osmotolerant yeast)

[0044] Here, the wild-type strains are environmentally derived strains that have been identified and stored (No. indicates the in-house strain number); the NBRC strains are filamentous fungal strains purchased from the National Institute of Technology and Evaluation (NITE); and the JCM strains are filamentous fungal strains purchased from the Microbial Materials Development Laboratory of the RIKEN BioResource Research Center.

[0045] Aspergillus chevalieri and Wallemia sebi are harmful microorganisms found in semi-perishable sweets such as gummy candies and fresh chocolate, and Cladosporium cladosporioides is a type of environmental filamentous fungus commonly found in the environment.

[0046] (2) Culture medium (2-1) Source of medium components Malt extract: Bacto TM Malt Extract (manufactured by Thermo Fisher Scientific) Yeast extract: Bacto TM Yeast Extract (manufactured by Thermo Fisher Scientific) Sucrose: Wako Grade 1 Sucrose (Shonan Wako Pure Chemical Industries, Ltd.) Glucose: Glucose Wako Grade 1 (Shonan Wako Pure Chemical Industries, Ltd.) Fructose: Wako Special Grade Fructose (Shonan Wako Pure Chemical Industries, Ltd.) Agar: Bacto TM Agar (manufactured by BD)

[0047] (2-2) Medium composition, heat sterilization conditions, and pH after heat sterilization The pH of the medium was measured using a glass electrode hydrogen ion concentration meter (DK Toa Corporation). In the pH values ​​below, the number before " / " indicates the pH of the medium after heat sterilization, and the number after " / " indicates the pH measured after pulverizing the solid medium and diluting it 10 times with distilled water. (a) M40Y agar medium (pH: 5.8 / 5.9) 20g malt extract Yeast extract 5g 400g sucrose 20g agar 1000ml distilled water Heat sterilization conditions: 121°C for 15 minutes

[0048] (b) MY50G agar medium (pH: 4.8 / 4.8) 10g malt extract Yeast extract 3g 500g glucose 10g agar 500ml distilled water Heat sterilization conditions: 121°C for 15 minutes

[0049] (c) MY60G agar medium (pH: 5.4 / 5.9) 10g malt extract Yeast extract 2.5g 600g glucose 10g agar 500ml distilled water Heat sterilization conditions: 121°C for 15 minutes

[0050] (d) MY70GF agar medium (pH: 4.5 / 4.6) Malt extract 6.0g Yeast extract 1.5g Fructose 350.0g Glucose 350.0g Agar 6.0g 286.5ml distilled water Heat sterilization conditions: 100°C for 30 minutes

[0051] (e) Potato dextrose agar (PDA) 200g potatoes 20g glucose 20g agar 1000ml distilled water Peel the potatoes and cut them into cubes, then add 1000 mL of distilled water and boil for 1 hour, then filter through a cloth. Add distilled water to bring the total volume to 1000 mL, then add glucose and agar and dissolve.

[0052] (3) Method for measuring water activity (Aw) of culture medium The water activity of the medium was measured by the dew point method using a water activity measuring device (AquaLab 4TE, manufactured by Meter Japan Co., Ltd.). Specifically, 5 g of medium dispensed into a petri dish and solidified before or after sterilization was placed in the sample holder of the water activity measuring device and measured in single measurement mode (set temperature 25.0°C). In single measurement mode, the measurement is stopped when the fluctuation in water activity value falls within 0.0005aw.

[0053] Table 1 shows the water activity (Aw) measured for each of the above media before and after sterilization. [Table 1]

[0054] Experimental Example 1: Evaluation of the growth of xerophilic filamentous fungi The growth rates of two environmental filamentous fungi (Paecilomyces sp., Penicillium sp.) and two xerophilic filamentous fungi (Aspergillus chevalieri, Wallemia sebi) (hereinafter collectively referred to as "test fungi") were evaluated using M40Y agar medium, MY50G agar medium, MY60G agar medium, and MY70GF agar medium, respectively. Evaluations were performed by checking the presence and size of mycelium or spore formation over time while storing at 25°C. After heat sterilization, 5 ml of each agar medium was placed in a sterilized polystyrene dish (diameter 35.4 (±0.2) mm) and allowed to solidify at room temperature before use (solid agar medium).

[0055] The test bacteria were activated by culturing them on an activation medium placed in a petri dish at 25±2°C for 7 to 10 days. Potato dextrose agar (PDA) was used for the activation of Paecilomyces sp., and M40Y agar was used for the activation of the other three species. The incubation period was extended until spore formation was observed. Spore formation can be determined visually, using a stereomicroscope (in the petri dish), or by optical microscopy (observing the filamentous fungal tissue on a slide at 200-400x magnification).

[0056] The formed spores (dried spores) were picked up with the tip of a sterilized bamboo skewer (until the tip of the skewer became visibly colored) and aseptically smeared onto the center of each of the aforementioned solid agar media (Petri dishes 35.4 (±0.2) mm in diameter). The spores were then cultured at 25°C + 1°C (without humidity control) and the colony formation status was observed during the culture period. Ten repeated experiments confirmed that the amount of smeared onto the media when the spores were picked up enough to color the tip of the skewer was nearly consistent. The colony size evaluated during the culture period was determined by measuring the longest diameter (a: cm) of the circular colony that formed and the diameter (b: cm) of the section at a 90-degree angle to that diameter with a ruler. The average (cm) of a and b was calculated and evaluated as the "colony size" (see Figure 1).

[0057] The growth pattern (growth steps) of filamentous fungi is as follows: [Proliferation step] Spore → germination → hyphal elongation (hyphae present) → coloration appears at the tip of the hyphae and spore formation (spore formation). Based on this growth step, the growth status (presence of mycelia or spore formation) was evaluated by visually inspecting the appearance (spore formation was evaluated using a stereomicroscope as necessary). Colony formation was considered to have occurred when the colony size reached 0.2 cm or more (rounded to the nearest 1 cm). The evaluation results are shown in Table 2. A photograph was taken on the first day of inoculation, and if there was no change when compared with the photograph, it was recorded as "no growth."

[0058] [Table 2]

[0059] As shown in Table 2, the experimental results showed that on M40Y agar medium, MY50G agar medium, and MY60G agar medium, the formation of hyphae or spores was observed for all filamentous fungi on day 13 from the start of cultivation. On MY70GF agar medium, growth of Paecilomyces sp., a mesophilic filamentous fungus, was not observed. On MY70GF agar medium, growth (hyphae, spores, or colony formation) of Penicillium sp., a xerophilic filamentous fungus, on day 13 from the start of cultivation, of Aspergillus chevalieri on day 24, and of Wallemia sebi on day 33 from the start of cultivation was observed on MY70GF agar medium. Thus, it was confirmed that although MY70GF agar medium can selectively grow xerophilic filamentous fungi, it is not suitable as a medium for rapid growth.

[0060] These results indicate that M40Y agar medium (Aw: 0.978 after sterilization), MY50G agar medium (Aw: 0.883 after sterilization), and MY60G agar medium (Aw: 0.824 after sterilization) are all unsuitable as selective media for xerophilic filamentous fungi, and that MY70GF agar medium (Aw: 0.736 after sterilization) is a selective medium for xerophilic filamentous fungi but is not suitable for early growth.

[0061] Experimental Example 2: Adjustment of water activity of agar medium and evaluation of xerophilic bacteria growth (1) Adjustment of water activity (Aw) of agar medium As mentioned above, the Aw of MY70GF agar medium after sterilization is 0.74 or less, while the Aw of M40Y agar medium, MY50G agar medium, and MY60G agar medium after sterilization is 0.88 or more. Therefore, based on the compositions of M40Y agar medium, MY50G agar medium, and MY50G agar medium, the sugar content was adjusted to prepare media so that Aw was in the range of 0.78 to 0.82. The composition, pH, and Aw (before and after sterilization) of the prepared agar media are shown in Tables 3 and 4, respectively.

[0062] [Table 3]

[0063] [Table 4]

[0064] (2) Growth evaluation of xerophilic bacteria (part 1) The growth rates of two environmental filamentous fungi (Paecilomyces sp., Penicillium sp.), two xerophilic filamentous fungi (Aspergillus chevalieri, Wallemia sebi), and two yeasts (Rhodotorula sp., Cryptococcus saitoi) (collectively referred to as "test fungi") were evaluated using the MY64G agar medium and MY66G agar medium prepared as described above. M40Y agar medium was also used for comparison. Potato dextrose agar (PDA) was used as the activation medium for Paecilomyces sp. and yeast, while M40Y agar was used for the activation of the remaining three fungi. Evaluations were performed over time at 25°C, as in Experimental Example 1, by observing the presence and size of mycelia, spores, or colonies. After heat sterilization, 5 ml of each agar medium was placed in a sterilized polystyrene dish (diameter 35.4 (±0.2) mm) and allowed to solidify at room temperature before use (solid agar medium). The evaluation results are shown in Table 5.

[0065] [Table 5]

[0066] As shown in Table 5, when MY64G agar medium and MY66G agar medium with Aw in the range of 0.78 to 0.81 were used, the xerophilic filamentous fungi Aspergillus chevalieri and Wallemia sebi both showed hyphal elongation (initiation of colony formation) on the 7th day of culture. For the other mesophilic filamentous fungi and yeast, no hyphae, spores, or colony formation was observed even after the 17th day of culture. On the other hand, on M40Y agar medium with Aw of 0.978, all fungi showed hyphae, spores, or colony formation on the 7th day of culture. These results demonstrate that the use of media with Aw in the range of 0.78 to 0.81 allows selective growth of xerophilic filamentous fungi (hyphae, spores, or colony formation).

[0067] (3) Growth evaluation of xerophilic filamentous fungi (part 2) Similar to the above experiment, the growth rates of various filamentous fungi were evaluated using M40Y agar medium, MY64G agar medium, and MY66G agar medium. Potato dextrose agar medium (PDA) was used for activating Rhizopus microsporus, and M40Y agar medium was used for activating other filamentous fungi. After heat sterilization, each agar medium was placed in a sterilized polystyrene dish (diameter 35.4 (±0.2) mm) in an amount of 5 ml and allowed to solidify at room temperature before use (solid agar medium). The evaluation results are shown in Table 6.

[0068] [Table 6]

[0069] As shown in Table 6, on MY64G agar medium and MY66G agar medium, the xerophilic filamentous fungus Aspergillus chevalieri showed hyphal elongation on the 7th day of culture, and clear spore formation was observed on the 14th day of culture. Similarly, the xerophilic filamentous fungus Aspergillus sp. #2 showed hyphal elongation on the 14th day of culture. On the other hand, no growth was observed after the 14th day (up to the 28th day) for any of the other filamentous fungi (hygrophilic and mesophilic fungi).

[0070] (4) Growth evaluation of xerophilic filamentous fungi (part 3) Similar to the above experiment, the growth rates of various filamentous fungi (hygrophilic, mesophilic, and xerophilic fungi) were evaluated using M40Y agar medium, MY50G agar medium, MY62G agar medium, MY64G agar medium, MY66G agar medium, and MY70GF agar medium, respectively. Potato dextrose agar medium (PDA) was used for activating hygrophilic and mesophilic fungi, and M40Y agar medium was used for activating xerophilic fungi. After heat sterilization, 5 ml of each agar medium was placed in a sterilized polystyrene dish (inner diameter 35.4 (±0.2) mm) and allowed to solidify at room temperature before use (solid agar medium). The evaluation results are shown in Table 7.

[0071] [Table 7]

[0072] As shown in Table 7, on MY62G agar medium, MY64G agar medium, and MY66G agar medium, which have Aw in the range of 0.78 to 0.82, the xerophilic filamentous fungi Aspergillus chevalieri, Eurotium repens, and Wallemia sebi all showed hyphal elongation or spore formation on the 14th day of culture. On the other hand, no growth was observed for any of the other filamentous fungi (hygrophilic filamentous fungi and mesophilic filamentous fungi) even on the 21st day. This selective growth of xerophilic filamentous fungi was not observed on other agar media (M40Y agar medium or MY50G agar medium).

[0073] Experimental Example 3: Evaluation of growth of xerophilic filamentous fungi (effect of medium pH) The MY64G agar medium (pH 5.4 after heat sterilization) and the MY66G agar medium (pH 5.5 after heat sterilization) prepared and used in Experimental Example 2 were adjusted using 6N hydrochloric acid so that the pH after heat sterilization was 3.4 and 4.5, respectively.

[0074] The growth rates of two environmental filamentous fungi (Rhizopus microsporus #3 and Aspergillus sp. #4) and two xerophilic filamentous fungi (Aspergillus chevalieri and Wallemia sebi) (hereinafter collectively referred to as "test fungi") were evaluated using the thus-prepared MY64G agar medium (pH 3.4 and 4.5 after heat sterilization) (Aw: 0.804) and MY66G agar medium (pH 3.4 and 4.5 after heat sterilization) (Aw: 0.785). Potato dextrose agar (PDA) was used for the activation of Aspergillus sp. #4, and M40Y agar was used for the activation of the remaining three fungi. Evaluation was performed by observing the presence and size of mycelium or spore formation over time during storage at 25°C, as in Experimental Example 1. After heat sterilization, 5 ml of each agar medium was placed in a sterilized polystyrene dish (diameter 35.4 (±0.2) mm) and allowed to solidify at room temperature before use (solid agar medium). The evaluation results are shown in Table 8.

[0075] [Table 8]

[0076] As shown in Table 8, hygrotrophic and mesophilic filamentous fungi did not grow on either MY64G agar medium (pH 3.4 and 4.5 after heat sterilization) or MY66G agar medium (pH 3.4 and 4.5 after heat sterilization). In contrast, xerophilic filamentous fungi grew on both MY64G agar medium (pH 4.5 after heat sterilization) and MY66G agar medium (pH 4.5 after heat sterilization). However, the growth rate tended to be slower compared to media with a pH of 5.1 or higher. Therefore, the conditions for growing xerophilic filamentous fungi on MY64G agar medium within 14 days are pH 4.5 or higher, preferably pH 5.1 or higher, and particularly pH 5.1 to 5.5; and the conditions for growing xerophilic filamentous fungi on MY66G agar medium within 14 days are pH 5.1 or higher, and particularly pH 5.1 to 5.6.

Claims

1. A solid medium comprising sugar, malt extract, yeast extract, and water, The sugar content in the medium is 61 to 66% by mass, characterized in that the water activity is 0.78 to 0.82; The solid medium.

2. 2. The solid medium of claim 1, wherein the sugar is glucose.

3. 3. The solid medium according to claim 1, which does not contain an antibiotic.

4. 3. The solid medium according to claim 1, wherein the malt extract content in the medium is 0.66 to 0.72% by mass and the yeast extract content is 0.16 to 0.18% by mass.

5. The solid medium according to claim 1 or 2, having a pH of 4.5 to 5.

6.

6. The solid medium according to claim 1 or 2, further comprising a gelling agent.

7. 7. The solid medium of claim 6, wherein the gelling agent is agar.

8. 3. The solid medium according to claim 1, which is a medium for selectively culturing xerophilic bacteria or a medium for detecting xerophilic bacteria.

9. 9. The solid medium according to claim 8, wherein the xerophilic fungus is a xerophilic filamentous fungus.

10. 3. The solid medium according to claim 1, which is used in a falling bacteria test, an air sampler test, or a surface-attached bacteria test.

11. A method for detecting xerophilic bacteria in a specimen, comprising the steps of: (1) a step of applying a specimen onto the solid medium according to claim 1; (2) culturing the medium at 25±1°C; (3) A step of confirming the presence or absence of mycelia, spores, or colonies on the medium within 14 days from the start of culture.

12. 12. The detection method according to claim 11, wherein when formation of mycelia, spores, or colonies is confirmed on the medium in the step (3), it is determined that xerophilic fungi have been detected in the sample.

13. 13. The detection method according to claim 11 or 12, wherein the specimen is a microbial test sample derived from a food product having a water activity of 0.80 or less, or a sample collected for environmental monitoring.

14. A method for screening xerophilic bacteria, comprising the steps of: (1) a step of applying a specimen onto the solid medium according to claim 1; (2) culturing the solid medium at 25±1°C; (3) A step of collecting mycelia, spores, or colonies formed on the medium as xerophilic fungi within 14 days from the start of culture.

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