Medium, and method for selective detection or screening of 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 for xerophilic bacteria using the culture medium of the present invention. [Background technology]

[0002] Fungi such as filamentous molds and yeasts are often found as food foreign bodies because 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 of mold occurring in food products, leading to product recalls. Mold problems have a significant impact not only on consumers but also on companies. Once mold occurs, it not only requires a great deal of effort and cost for product recalls, but also leads to a decline in the company's image.

[0003] Among fungi, xerophilic fungi occur in foods with relatively low water activity (hereinafter also referred to as "Aw"), such as semi-perishable confectionery, and are one of the causes of food complaints. Among them, xerophilic filamentous fungi have an optimum Aw of 0.95, but can grow even with an Aw of 0.80 or less, and examples of such filamentous 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 medium, malt extract agar medium, and Czapek yeast extract agar medium, so that a medium with a low Aw for xerophilic fungi must be used for their testing. Known media for such xerophilic fungi include dicloran-glycerin 18 agar medium (DG18) (Aw: 0.95), 25% glycerol-nitrate agar medium (G25N) (Aw: 0.93), 20% glucose-malt extract-yeast extract agar medium (MY20G) (Aw: 0.97), 30% glucose-malt extract-yeast extract agar medium (MY30G) (Aw: 0.95), 50% glucose-malt extract-yeast extract agar medium (MY50G) (Aw: 0.89), and 70% glucose / fructose-malt extract-yeast extract agar medium (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 at the same time. [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] Shunichi Shimizu et al. (2011), Food contamination cases of xerophilic fungi, 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, Jpn. J. Food Microbiol., 31(1), 13-19 [Non-Patent Document 3] Yoshitsugu Sugiura (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 said medium, and a screening method. [Means for solving the problem]

[0008] The present inventors have conducted intensive 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 these findings, and includes the following embodiments.

[0009] (I) Culture medium (I-1) A medium comprising: 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 an antibiotic. (I-4) The 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 in the medium 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), 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 steps of: (1) A step of applying a sample onto a medium according to 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 mycelium, spores, or colonies on the medium within 14 days from the start of culture. (II-2) The detection method described in (II-1), in which, when formation of mycelium, spores or colonies is confirmed on the medium in the step (3), it is determined that a xerophilic fungus has been detected in the sample. (II-3) The detection method according to (II-1) or (II-2), wherein the specimen is a microbial testing sample derived from a food 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 steps of: (1) A step of applying a sample onto a medium according to any one of (I-1) to (I-13); (2) culturing the medium at 25±1° C.; (3) A step of harvesting mycelia, spores or colonies formed on the medium as xerophilic fungi within 14 days from the start of culture. Effect 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, according to the present invention, 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, according to the present invention, it is possible to provide a method for selectively, preferably selectively and rapidly detecting xerophilic bacteria using the medium, and a screening method. [Brief description of the drawings]

[0013] [Figure 1] FIG. 2 is a diagram for explaining the method for measuring colony size ([(a+b) / 2] cm) employed in Experimental Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] (I) Culture medium, its preparation method, and uses The medium of the present invention (hereinafter, abbreviated 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 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 being preferred. These can be used alone or in combination of two or more. More preferred is an embodiment in which glucose is used as the only sugar or carbohydrate (carbon source) to be added externally.

[0016] The sugar content in 100% by mass of the medium is 61 to 66% by mass as described above, 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, the 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 being combined with yeast extract, the content of the malt extract in the present medium (100% by mass) is not limited as long as it does not impede 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. When yeast extract is used without being combined with malt extract, the content of the malt extract in the present medium (100% by mass) is not limited as long as it does not impede 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] In addition, as long as the effects of the present invention are not hindered, other nitrogen sources and / or nutritional components (vitamins, minerals, etc.) may be added without particular limitation. Examples of such components include peptone (casein peptone, meat peptone, myocardial peptone, gelatin peptone, soybean peptone, etc.), meat extract, fish extract, heart infusion, potato extract, tomato juice, and inorganic salts (e.g., sodium chloride, sodium thiosulfate, sodium pyruvate, ferric ammonium phosphate, dipotassium phosphate, dipotassium hydrogen phosphate, ferric ammonium citrate, sodium citrate, etc.). However, more preferably, the nitrogen source and / or nutritional component contains only malt extract and yeast extract, and does not contain other components.

[0021] (3)Water This medium contains water in addition to the above ingredients. The water content is not particularly limited as long as the water activity (Aw) measured after sterilization of the medium (water activity of the sterilized medium) is 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 described 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 a fluid state and then solidified) is placed in the sample holder of the water activity measuring device, and the water activity can be obtained by measuring in single measurement mode (set temperature 25.0°C). In addition, according to the single measurement mode, the measurement is terminated when the fluctuation value of the water activity value is within 0.0005aw.

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

[0024] The solid medium can be prepared by adding a gelling agent in addition to the above-mentioned components. The gelling agent is used to solidify the medium and may be any agent that does not impede the effects of the present invention, and may be selected from, for example, agar, pectin, gelatin, carrageenan, xanthan gum, methylcellulose, HPMC, guar gum, tara gum, locust bean gum, glucomannan, gum arabic, gellan gum, etc., either alone or in combination. Metal salts such as calcium salts and magnesium salts may also be used in combination as necessary. 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 sufficient to solidify the medium, and may 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] The semi-solid medium can be prepared by adding a thickener in addition to the above components. The thickener is used to thicken the medium to make it semi-solid, and may be any one that does not impair the effects of the present invention, and may be selected from, for example, 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 may be an amount that thickens the medium to make it semi-solid, and may be selected from, for example, a range of 0.2 to 0.4% by mass.

[0026] (5) Other ingredients The medium is prepared by mixing the above-mentioned components and adjusting the pH after sterilization to a range of 3.4 to 5.6. In terms of promoting the growth of xerophilic bacteria, the preferred pH range is 5.1 to 5.6, and more preferably 5.4 to 5.6. As shown in the experimental examples described later, when the pH of the medium is 5.1 or higher, the formation of mycelium, spores or colonies is observed on the 14th day after the start of culture. The pH of the medium can be measured using a glass electrode type hydrogen ion concentration meter. The measurement may be performed by inserting an electrode into the liquid medium (25°C) before sterilization, or may be performed by inserting an electrode into the fluid medium (50°C) after sterilization, or the pH of the sterilized medium diluted 10 times with distilled water (10-fold diluted medium) may be measured. In the case of a solid medium, the medium solidified after sterilization is 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, more preferably 5.4 to 5.6.

[0027] In addition to the above-mentioned components, the medium may contain a pH adjuster as necessary to adjust the pH to the above range. The pH adjuster may be any one that 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, disodium dihydrogen pyrophosphate, and the like.

[0028] If necessary, the medium may contain an osmotic regulator such as an alkali metal salt or polyhydric alcohol as a component for adjusting and maintaining the osmotic pressure. Examples of the alkali metal salt include chlorides of alkali metals such as sodium chloride and potassium chloride, and examples of the polyhydric alcohol include glycerin. Although not limited, the osmotic pressure of the medium is preferably adjusted to be in the range of 4700 to 5600 mOsm / kgH2O. More preferably, the range is 4750 to 5570 mOsm / kgH2O. The osmotic pressure can be measured with an osmometer such as Advanced Osmometer-3250 (manufactured by Ricoh 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, sorbic acid, etc.

[0030] (6) Preparation method The medium may be prepared by mixing all the components to be mixed and then sterilizing them, or by mixing each component that has been sterilized in advance under aseptic conditions. Alternatively, some of the components may be mixed and then sterilized, and the remaining components that have been sterilized in advance are added and mixed under aseptic conditions. When the object to be sterilized is liquid, the sterilization may be performed by filtration sterilization, or by heat sterilization using an autoclave or the like. In the case of a semi-solid medium or solid medium, all the components are mixed and then heat sterilized (sterilized), and the mixture is poured into a sterilization container (a petri dish, a test tube, etc.) under aseptic conditions and cooled to solidify. Alternatively, the medium may be prepared by dissolving agar in water and then heat sterilizing it, adding and mixing other components that have been sterilized in advance under aseptic conditions, and then pouring the mixture into a sterilization container and cooling to solidify. The water activity of the medium thus prepared (sterilized medium) is in the range of 0.78 to 0.82, preferably 0.78 to 0.81, and more preferably 0.79 to 0.80, as described above, 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 xerophilic bacteria to grow selectively. In other words, this medium is suitable for the selective growth of xerophilic bacteria. Specifically, as will be described in the Examples below, in culture using this medium, xerophilic bacteria can be grown in at least 14 days of culture, whereas bacteria other than xerophilic bacteria (hygroscopic bacteria, mesophilic bacteria) cannot grow. For this reason, this medium is useful as a selective medium for xerophilic bacteria and as a medium for detecting xerophilic bacteria.

[0032] The xerophilic fungus is preferably an osmotolerant yeast or a xerophilic fungus such as a xerophilic filamentous fungus. 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 Aspergillus such as Aspergillus conicus, Aspergillus penicillioides, and Aspergillus herbariorum, some filamentous fungi belonging to Eurotium such as Eurotium repens, Wallemia sebi, some filamentous fungi belonging to Penicillium, and filamentous fungi belonging to Xeromyces.

[0033] When the target xerophilic fungus is a filamentous fungus, an example of a method for selectively growing xerophilic fungus using this medium is to inoculate the target fungus on this medium and culture it statically under conditions of a relative humidity of 70-80%, a temperature of 24-26°C, and a CO2 concentration of 0-98%. If hyphal elongation or spore formation is observed within 14 days of culture under these conditions, it can be determined that the xerophilic filamentous fungus has grown. On the other hand, as shown in the Examples described later, 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 tips of the hyphae and spore formation (spore formation). Therefore, by visually judging the appearance (for spore formation, a stereomicroscope may be used in combination as necessary), if hyphae are observed due to hyphae extension (hyphae present) or if spore formation is observed, it can be judged as "growable". Details of this judgment will be explained in the examples below.

[0035] When the xerophilic bacteria of interest is yeast, the method for selectively growing osmotolerant yeast using this medium is as follows. One method is to inoculate the target bacteria on this medium and culture it 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 culture under these conditions, it can be determined that osmotolerant yeast has grown. More specifically, the target bacteria is 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 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-mentioned medium, xerophilic bacteria in a sample 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 mycelium, spores, or colonies on the medium within 14 days from the start of culture. In the step (3) above, when the formation of mycelia, spores or colonies is 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] Although the cultivation method in step (2) varies depending on the type of the xerophilic fungus to be cultivated, when the xerophilic fungus to be cultivated is a filamentous fungus, it can be cultured by static cultivation under conditions of a relative humidity of 70-80%, a temperature of 24-26°C, and a CO2 concentration of 0-98% after inoculation into the medium. Similarly, when the xerophilic fungus to be cultivated is a yeast, it can be cultured by static cultivation 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 confections with low water activity (for example, Aw 0.8 or less), such as gummy candies, fresh chocolate, yokan, daifuku, castella, etc. Xerophilic bacteria can also be found adhering to ceilings, walls, production lines, etc. in food factories. Therefore, the specimen targeted by the method of the present invention can be a food having a water activity of 0.80 or less, or a microbial test sample derived from an environmental monitoring sample. Here, 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 pollution conditions, and evaluating the health of the environment. Examples of environmental monitoring samples include air, water, and wipes of the surface of manufacturing equipment. In the present application, air and wipes of the surface of manufacturing equipment are preferred from the viewpoint of Aw. For example, xerophilic bacteria suspended in the air can be efficiently collected and used for testing by combining airborne bacteria testing using an air sampler and falling bacteria testing using solid plate media (Koch method). Xerophilic bacteria occurring or adhering to manufacturing equipment such as facilities and manufacturing lines can be effectively collected and used for testing by combining wiping testing using a cotton swab. Contaminated air flowing in from air conditioning outlets or gaps can be effectively collected and used for testing by blowing 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. In addition, 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 has been detected in the sample. Identification of the genera and species of xerophilic filamentous fungi and osmotolerant yeast can be performed by standard methods, such as sequence analysis of the 28S rRNA gene, biochemical methods, and morphological methods such as Gram staining.

[0040] As shown in the examples described below, in the test method performed under the above-mentioned conditions, non-xerophilic fungi such as hygroscopic filamentous fungi and mesophilic filamentous 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 selective screening method for xerophilic fungi.

[0041] As described above, in this specification, the terms "comprise" and "contain" include the meanings of "consisting of" and "consisting essentially of". EXAMPLES

[0042] In the following, the present invention will be described 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. The following experiments were carried out at room temperature (25±5°C) and atmospheric pressure conditions unless otherwise specified. In the following, "%" means "% by mass" and "parts" means "parts by mass" unless otherwise specified.

[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 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 strain is an environmentally-derived strain that was identified and stored (No. is an internal strain number); the NBRC strain is a filamentous fungal strain purchased from the National Institute of Technology and Evaluation (NITE); and the JCM strain is a filamentous fungal strain 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 an environmental fungus that is commonly observed 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 (manufactured by Shonan Wako Pure Chemical Industries, Ltd.) Glucose: Glucose Wako Grade 1 (Shonan Wako Pure Chemical Industries, Ltd.) Fructose: Wako Special Grade Fructose (manufactured by Shonan Wako Pure Chemical Industries, Ltd.) Agar: Bacto TM Agar (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 type hydrogen ion concentration meter (DK-TOA Corporation). In the pH descriptions below, the number before the " / " indicates the pH of the medium after heat sterilization, and the number after the " / " indicates the pH measured by crushing the solid medium and diluting it 10 times with distilled water. (a) M40Y agar medium (pH: 5.8 / 5.9) Malt extract 20g Yeast extract 5g 400g sucrose 20g agar Distilled water, 1000ml Heat sterilization conditions: 121℃ for 15 minutes

[0048] (b) MY50G agar medium (pH: 4.8 / 4.8) Malt extract 10g Yeast extract 3g Glucose 500g Agar 10g 500ml distilled water Heat sterilization conditions: 121℃ for 15 minutes

[0049] (c) MY60G agar medium (pH: 5.4 / 5.9) Malt extract 10g Yeast extract 2.5g Glucose 600g Agar 10g 500ml distilled water Heat sterilization conditions: 121℃ 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℃ for 30 minutes

[0051] (e) Potato dextrose agar (PDA) 200g potatoes Glucose 20g 20g agar Distilled water, 1000ml Peel the potatoes and cut them into cubes, then add 1000mL of distilled water and boil for 1 hour, then filter through a cloth. Add distilled water to bring the total volume to 1000mL, 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 the single measurement mode, the measurement is terminated when the fluctuation value of the water activity value falls within 0.0005aw.

[0053] Table 1 shows the water activity (Aw) of each of the media measured 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) (collectively referred to as "test fungi") were evaluated using M40Y agar medium, MY50G agar medium, MY60G agar medium, and MY70GF agar medium, respectively. The evaluation was performed by checking the presence or absence of mycelium or spore formation and their size over time under storage at 25°C. After heat sterilization (sterilization), each agar medium was placed in a sterilized polystyrene petri dish (diameter 35.4 (±0.2) mm) in an amount of 5 ml and solidified at room temperature before use (solid agar medium).

[0055] The test bacteria were activated by culturing them for 7 to 10 days on the activation medium placed in a petri dish at 25±2℃. Potato dextrose agar medium (PDA) was used for the activation of Paecilomyces sp., and M40Y agar medium was used for the activation of the remaining three types. The incubation period was until spore formation was observed. Spore formation can be determined by visual inspection or a stereomicroscope (in the petri dish), or by an optical microscope (the filamentous fungal tissue is placed on a preparation and observed at 200 to 400 times magnification).

[0056] The formed spores (dried spores) were picked up with the tip of a sterilized bamboo skewer (until the tip of the bamboo skewer was visibly colored), aseptically smeared on the center of each of the above-mentioned solid agar media (petri dish diameter 35.4 (±0.2) mm), and cultured at 25 + 1 °C (without humidity control), and the state of colony formation during the culture period was observed. It was confirmed in advance that the amount of smeared on the medium when the spores were picked up to the extent that the tip of the bamboo skewer was colored was almost constant through 10 repeated experiments. The colony size evaluated during the culture period was measured by measuring the longest diameter (a: cm) of the formed circular colony and the diameter of a piece at 90 degrees to that diameter (b: cm) with a ruler, and the average value (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 tips of the hyphae and spore formation (spore formation). Based on this growth step, the growth state (whether there is mycelium or there is spore formation) was evaluated by visually judging the appearance (for spore formation, a stereomicroscope was used as necessary). In addition, colony formation was considered to have occurred when the colony size reached 0.2 cm or more (rounded off to the nearest 1 decimal place). 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 comparing with the photograph, it was recorded as "no growth."

[0058] [Table 2]

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

[0060] These results indicate that M40Y agar medium (Aw after sterilization: 0.978), MY50G agar medium (Aw after sterilization: 0.883), and MY60G agar medium (Aw after sterilization: 0.824) are all unsuitable as selective media for xerophilic filamentous fungi, and that MY70GF agar medium (Aw after sterilization: 0.736) 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 described above, the Aw of the MY70GF agar medium after sterilization is 0.74 or less, and the Aw of the M40Y agar medium, the MY50G agar medium, and the MY60G agar medium after sterilization is 0.88 or more. Based on the compositions of the M40Y agar medium, the MY50G agar medium, and the MY50G agar medium, the sugar content was adjusted to prepare a medium so that the Aw was in the range of 0.78 to 0.82. The composition, pH, and Aw (before and after sterilization) of the prepared agar medium are shown in Tables 3 and 4, respectively.

[0062] [Table 3]

[0063] [Table 4]

[0064] (2) Growth evaluation of xerophilic bacteria (part 1) Using the MY64G agar medium and MY66G agar medium prepared above, 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. In addition, M40Y agar medium was used for comparison experiments. As the activation medium, potato dextrose agar medium (PDA) was used for the activation of Paecilomyces sp. and yeast, and M40Y agar medium was used for the activation of the remaining three. The evaluation was performed by confirming the presence or absence of mycelium, spores, or colony formation and their size over time under 25°C conditions, as in Experimental Example 1. After heat sterilization, 5 ml of each agar medium was placed in a sterilized polystyrene petri dish (diameter 35.4 (±0.2) mm) and allowed to solidify at room temperature before use (agar solid 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, both xerophilic filamentous fungi Aspergillus chevalieri and Wallemia sebi were observed to elongate their hyphae (start of colony formation) on the 7th day of culture. For other mesophilic filamentous fungi and yeasts, no hyphae, spores, or colony formation was observed even after the 17th day of culture. On the other hand, in M40Y agar medium with Aw of 0.978, hyphae, spores, or colony formation was observed for all fungi on the 7th day of culture. From these results, it was found that by using a medium with Aw in the range of 0.78 to 0.81, xerophilic filamentous fungi can be selectively grown (hyphae, spores, or colony formation).

[0067] (3) Evaluation of growth of xerophilic filamentous fungi (part 2) In the same manner as in the above experiment, the growth rates of various filamentous fungi were evaluated using M40Y agar medium, MY64G agar medium, and MY66G agar medium, respectively. As the activation 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 petri dish (diameter 35.4 (±0.2) mm) in an amount of 5 ml and solidified 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, in MY64G agar medium and MY66G agar medium, the xerophilic filamentous fungus Aspergillus chevalieri was observed to have elongated its hyphal structure on the 7th day of culture, and obvious spore formation was observed on the 14th day of culture. Similarly, the xerophilic filamentous fungus Aspergillus sp.#2 was observed to have elongated its hyphal structure on the 14th day of culture. On the other hand, no growth was observed in any of the other filamentous fungi (hygrophilic filamentous fungi, mesophilic filamentous fungi) even after the 14th day (up to the 28th day).

[0070] (4) Growth evaluation of xerophilic filamentous fungi (part 3) In the same manner as in the above experiment, the growth rates of various filamentous fungi (hygrophilic, mesophytic, 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. As the activation medium, potato dextrose agar medium (PDA) was used for activation of hygrophilic and mesophytic fungi, and M40Y agar medium was used for activation of xerophilic fungi. After heat sterilization (sterilization), 5 ml of each agar medium was placed in a sterilized polystyrene petri dish (inner diameter 35.4 (±0.2) mm) and solidified at room temperature before use (agar solid medium). The evaluation results are shown in Table 7.

[0071] [Table 7]

[0072] As shown in Table 7, in 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 were all observed to have hyphal elongation or spore formation on the 14th day of culture. On the other hand, no growth was observed in any of the other filamentous fungi (hygrophilic filamentous fungi, mesophilic filamentous fungi) even on the 21st day. Such selective growth of xerophilic filamentous fungi was not observed in other agar media (M40Y agar medium and MY50G agar medium).

[0073] Experimental Example 3: Evaluation of the 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 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) were used to evaluate the growth rates of two environmental filamentous fungi (Rhizopus microsporus#3, Aspergillus sp.#4) and two xerophilic filamentous fungi (Aspergillus chevalieri, Wallemia sebi) (collectively referred to as "test fungi" below). As the activation medium, potato dextrose agar medium (PDA) was used for the activation of Aspergillus sp.#4, and M40Y agar medium was used for the activation of the remaining three. The evaluation was performed by checking the presence or absence of mycelium or spore formation and their size over time under storage at 25°C, as in Experimental Example 1. After heat sterilization, 5 ml of each agar medium was placed in a sterilized polystyrene petri dish (diameter 35.4 (±0.2) mm) and allowed to solidify at room temperature before use (agar solid medium). The evaluation results are shown in Table 8.

[0075] [Table 8]

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

Claims

1. A medium comprising: sugar; at least one selected from the group consisting of malt extract and 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 medium.

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

3. 3. The medium according to claim 1 or 2, which does not contain an antibiotic.

4. 3. The medium according to claim 1 or 2, which contains malt extract and yeast extract.

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

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

6.

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

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

9. 8. The medium according to claim 7, which consists essentially of glucose, malt extract, yeast extract, a gelling agent, and water.

10. 3. The medium according to claim 1 or 2, which is a medium for selectively culturing xerophilic bacteria or a medium for detecting xerophilic bacteria.

11. The medium according to claim 10, wherein the xerophilic fungus is a xerophilic filamentous fungus.

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

13. A method for detecting xerophilic bacteria in a sample, comprising the steps of: (1) a step of applying a sample onto the medium according to any one of claims 1 to 12; (2) culturing the medium at 25±1° C.; (3) A step of confirming the presence or absence of mycelium, spores, or colonies on the medium within 14 days from the start of culture.

14. The detection method according to claim 13, wherein when formation of mycelia, spores or colonies is confirmed on the medium in the step (3), it is determined that a xerophilic fungus has been detected in the sample.

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

16. A method for screening xerophilic bacteria, comprising the steps of: (1) a step of applying a sample onto the medium according to any one of claims 1 to 12; (2) culturing the medium at 25±1° C.; (3) A step of harvesting mycelia, spores or colonies formed on the medium as xerophilic fungi within 14 days from the start of culture.

Images