Culture medium for detecting lactic acid bacteria

Abstract

To develop a culture medium that can determine a positive result if lactic acid bacteria are proliferating, but a negative result if lactic acid bacteria are not proliferating. [Solution] When acetate is added to the culture medium, the growth of lactic acid bacteria can be detected, but the detection of Escherichia coli and Staphylococcus aureus is suppressed.

Description

[Technical Field]

[0001] This invention relates to a culture medium for detecting lactic acid bacteria, a method for detecting lactic acid bacteria, and a kit for detecting lactic acid bacteria. The above-mentioned culture medium for detecting lactic acid bacteria refers to a culture medium whose use is limited to that of detecting lactic acid bacteria. [Background technology]

[0002] Lactic acid bacteria are recognized as major beneficial bacteria in the gut microbiota that influence the maintenance of human health and are used in dairy products such as yogurt. However, in the sake brewing process, they are recognized as harmful bacteria that cause spoilage, and in processed meat products such as ham, they are recognized as bacteria that cause stickiness and spoilage.

[0003] Conventionally, in order to effectively eliminate lactic acid bacteria contamination, a method for detecting lactic acid bacteria that allows for early, accurate, and efficient visual determination of the presence or absence of lactic acid bacteria in a test sample, and that can be widely used for general food, beverage, and water testing as well as manufacturing process testing, is known. This method involves adding and mixing the test sample to a semi-solid medium for detecting lactic acid bacteria containing glucose, a pH indicator such as bromocresol purple, and agar, culturing it at 25°C for two days, and visually determining the change in the color of the medium before and after culturing (see, for example, Patent Document 1).

[0004] Furthermore, as a testing tool that is convenient to transport and carry and easy to operate, a testing tool is known that comprises a reagent solution that exhibits a color reaction, a sampling device for collecting a sample and adding it to the reagent solution, and a transparent test container, wherein the test container is a sealed bag made of a flexible material, filled with the amount of reagent solution required for one test, and furthermore, a sampling device is removablely housed inside, separated from the reagent solution by a temporary adhesive joint formed by the inner surfaces of the flexible materials being detachably bonded (see, for example, Patent Document 2). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-136272 [Patent Document 2] Japanese Patent Application Laid-Open No. 2001-041954 [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] In order to prevent contamination by lactic acid bacteria in food production sites and products during the storage period, the inventors of the present invention performed swabbing with a cotton swab, and as a means for determining the presence or absence of the growth of lactic acid bacteria with high accuracy in a short time, a method for detecting lactic acid bacteria using a lactic acid bacteria detection kit containing a colorimetric pH indicator liquid medium capable of detecting lactic acid bacteria was developed (Japanese Patent Application No. 2024-011950). Although the above liquid medium has received favorable reviews from food manufacturers and the like who use such kits because lactic acid bacteria can be easily detected, when a swab test is performed on a location where no lactic acid bacteria are present but other bacteria such as Escherichia coli are present, a false positive reaction indicating the presence of lactic acid bacteria (lactic acid bacteria present) may occur, and there have been pointed out cases where misjudgment may occur regarding the presence or absence of lactic acid bacteria.

[0007] An object of the present invention is to develop a medium that can be determined to be positive when lactic acid bacteria are growing, but can be determined to be negative when no lactic acid bacteria are growing even when other bacteria such as Escherichia coli are growing. [Means for Solving the Problems]

[0008] The inventors considered improving the conventional liquid culture medium containing a color-changing pH indicator, which is contained in the aforementioned lactic acid bacteria detection kit, namely the LA medium for lactic acid bacteria detection shown in Table 1 below (manufactured by Prima Ham Co., Ltd., hereinafter referred to as "LA medium"). Therefore, they selected Escherichia coli and Staphylococcus aureus as other bacteria that can grow in LA medium, and conducted further studies to improve the components of the LA medium. As a result, they found that when acetate was added to LA medium, the growth of lactic acid bacteria could be detected, but the growth of Escherichia coli and Staphylococcus aureus was suppressed. Furthermore, they confirmed that this effect was similarly observed in other lactic acid bacteria culture media, thus completing the present invention.

[0009] [Table 1]

[0010] In other words, the present invention is defined as follows: [1] A culture medium for detecting lactic acid bacteria, comprising an acetate and a color pH indicator, wherein the concentration of the acetate is 10 to 25 g / L in terms of sodium acetate. [2] The culture medium for detecting lactic acid bacteria according to [1] above, further comprising a lactate. [3] The culture medium for detecting lactic acid bacteria according to [2] above, characterized in that the concentrations of acetate and lactate are, when converted to sodium acetate and sodium lactate, 1.5-2.5% sodium acetate and 0.5-2.5% sodium lactate. [4] A culture medium for detecting lactic acid bacteria according to any of the above [1] to [3], characterized in that the culture medium is a liquid culture medium. [5] A kit for detecting lactic acid bacteria, characterized in that a stick-shaped film sheet forming body comprises a section for storing a cotton swab for wiping and a section for storing the culture medium for detecting lactic acid bacteria described in [4] above, connected via a partition that can be opened by inserting the cotton tip of a cotton swab. [6] A method for detecting lactic acid bacteria, characterized by using the lactic acid bacteria detection kit described in [5] above. [7] The method for detecting lactic acid bacteria as described in [6] above, comprising the following steps (a) to (f) in order. (a) The step of preparing the lactic acid bacteria detection kit described in [5] above; (b) The step of opening the upper edge of the cotton swab storage compartment and removing the cotton swab from the cotton swab storage compartment; (c) The process of performing a swab test using the cotton tip of the removed cotton swab; (d) Inserting the cotton swab that has been used for the swab test through the partition from the cotton swab housing, bringing the cotton ball portion into contact with the liquid culture medium containing the color pH indicator, and drawing the liquid culture medium into the surface and interior of the cotton ball portion by capillary action; (e) A culture step in which lactic acid bacteria are grown on the surface and inside of a cotton ball that is not immersed in a liquid culture medium; (f) A step to detect the presence or absence of lactic acid bacteria by checking whether or not there is a color change on the surface of the cotton ball; [Effects of the Invention]

[0011] By using the lactic acid bacteria detection medium of the present invention, lactic acid bacteria present in food manufacturing equipment and processed meat products can be detected more accurately and specifically. [Brief explanation of the drawing]

[0012] [Figure 1] These are photographs of the culture media after 24 hours when LA medium and LA medium with seven different sodium acetate supplements were inoculated with six different concentrations of Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus, respectively. [Figure 2] These are photographs of the culture media after 24 hours when LA medium and LA medium with seven types of sodium lactate supplements were inoculated with six different concentrations of Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus, respectively. [Figure 3] These are photographs of the culture media after 24 hours, when four types of LA medium supplemented with sodium acetate and sodium lactate were inoculated with six different concentrations of Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus, respectively. [Figure 4] These are photographs of the culture media after 24 hours, when 12 different sodium acetate + sodium lactate-added LA mediums were inoculated with six different concentrations of Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus, respectively. [Figure 5]These are photographs of the culture media after 24 hours, when five different concentrations of Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus were inoculated into LA medium or MRS broth supplemented with sodium acetate and sodium acetate + sodium lactate, respectively. [Figure 6] This is a schematic diagram showing the components of a swab test kit. [Figure 7] These photographs show the test results after 24 hours of incubation, assuming a swab test using a cotton swab, under three conditions: (a) incubation at 35°C, (b) incubation at 30°C, and (c) incubation at 25°C. [Figure 8] These photographs show the test results after 48 hours of incubation, assuming a swab test using a cotton swab, under three conditions: (a) incubation at 35°C, (b) incubation at 30°C, and (c) incubation at 25°C. [Modes for carrying out the invention]

[0013] The culture medium of the present invention is not particularly limited as long as it contains an acetate and a color pH indicator, and the concentration of the acetate is 10 to 25 g / L in terms of sodium acetate, and is a culture medium for detecting lactic acid bacteria in which lactic acid bacteria can grow. It is desirable that such a culture medium can be used as a kit in which it is contained in the culture medium section of a stick-shaped film sheet forming body in which a cotton swab section and a culture medium section are connected via a partition.

[0014] The lactic acid bacteria detected in this invention are a general term for bacteria that produce lactic acid, and in some cases other acids, through fermentation (metabolism) using sugars, etc., thereby causing a neutral culture medium to become acidic. For convenience, this term includes not only lactic acid bacteria in the narrow sense but also bifidobacteria.

[0015] Specific examples of the above lactic acid bacteria include: Lactobacillus species such as Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, Lactobacillus delbruickii, Lactobacillus fermentum, Lactobacillus sakei, Lactobacillus rhamnosus, and Lactobacillus acidophilus; Lactococcus species such as Lactococcus lactis; Carnobacterium species such as Carnobacterium divergens and Carnobacterium picicola; Leuconostoc species such as Leuconostoc mesenteroides and Leuconostoc citreum; Enterococcus caseriflabus, Enterococcus sulfureus, and En Examples of lactic acid bacteria include Enterococcus species such as Thelococcus faecalis; Weissella species such as Weissella confusa; Atopovium species such as Atopovium minitum and Atopovium purbulus; Bagococcus species such as Bagococcus fluvialis and Bagococcus thermoninarum; Pediococcus species such as Pediococcus acidilactici, Pediococcus damnosus and Pediococcus pentosaceus; Streptococcus species such as Streptococcus thermophilus; Oenococcus species such as Oenococcus oeni; and Tetragenococcus species such as Tetragenococcus halophyllus.

[0016] Examples of the above-mentioned bifidobacteria include bacteria classified under the genus Bifidobacterium, such as Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum.

[0017] Examples of acetate salts in the present invention include alkali metal salts or alkaline earth metal salts of acetic acid, such as sodium acetate, potassium acetate, magnesium acetate, and calcium acetate. These acetate salts can be used individually or in combination of two or more, but sodium acetate is particularly preferred.

[0018] When the above-mentioned lactic acid bacteria can be detected and the detection of other bacteria such as E. coli can be suppressed, the concentration of acetate added alone in the culture medium can be exemplified as 1.0 to 2.5% in terms of sodium acetate, preferably 1.5 to 2.2%, and more preferably 1.5 to 2.0%.

[0019] Examples of color pH indicators used in this invention include bromocresol purple and phenol red. Bromocresol purple is preferred because its purple and yellow color changes are clearly distinguishable by visual inspection, resulting in good visibility. In the case of phenol red, it is red in neutral solutions and yellow in acidic solutions, but it may be difficult to distinguish the color change from red to yellow by visual inspection.

[0020] In the culture medium for detecting lactic acid bacteria containing the above-mentioned acetate and color pH indicator, in light of the objective of the present invention, which is to detect lactic acid bacteria, it is preferable to improve the growth and detection effect of lactic acid bacteria by setting the concentration of glucose, for example, to 15-25 g / L, focusing on the growth-promoting effect of glucose as a nutrient source. Furthermore, it is preferable to add a carbon source such as citric acid, a nitrogen source such as tryptone peptone or yeast extract, minerals such as sodium, magnesium, manganese, or iron, or vitamins such as thiamine.

[0021] An example of the composition of the culture medium for detecting lactic acid bacteria of the present invention is a medium consisting of or containing 10-15 g / L of tryptone peptone, 5-10 g / L of yeast extract, 15-25 g / L of glucose, 2-8 g / L of sodium chloride, 10-25 g / L of sodium acetate, 2-8 g / L of 1.5-2.0% trisodium citrate dihydrate, 0.5-1.2 g / L of magnesium sulfate, 0.1-0.2 g / L of manganese chloride, 0.01-0.08 g / L of iron(II) sulfate, 0.0005-0.002 g / L of thiamine hydrochloride, 0.2-0.9 g / L of agar, 0.06-0.09 g / L of bromocresol purple, and 0.1-0.3 g / L of Tween 800. Specifically, LA medium for detecting lactic acid bacteria with added sodium acetate (manufactured by Prima Ham Co., Ltd.) can be preferred.

[0022] In addition, the culture medium of the present invention can be prepared by adding acetate and a color pH indicator to known lactic acid bacteria culture media suitable for the growth of lactic acid bacteria, such as MRS medium and APT medium.

[0023] The culture medium of the present invention may also be further enriched with lactates. Examples of lactates include alkali metal salts or alkaline earth metal salts of lactic acid, such as sodium lactate, potassium lactate, magnesium lactate, and calcium lactate. These lactates may be used alone or in combination of two or more, but sodium lactate is particularly preferred.

[0024] When lactate is further added to the culture medium containing acetate of the present invention, the concentrations of acetate and lactate in the culture medium can be expressed as follows, when converted to sodium acetate and sodium lactate, 1.5-2.5% sodium acetate and 0.5-2.5% sodium lactate are preferred, 1.5-2.3% sodium acetate and 0.8-2.2% sodium lactate are preferred, 1.6-2.2% sodium acetate and 0.8-2.0% sodium lactate are preferred, 1.6-2.0% sodium acetate and 0.8-1.8% sodium lactate are preferred, 1.7-2.0% sodium acetate and 0.8-1.5% sodium lactate are more preferred, 1.7-2.0% sodium acetate and 0.8-1.3% sodium lactate are even more preferred, and 1.7-1.9% sodium acetate and 0.9-1.1% sodium lactate are particularly preferred.

[0025] The lactic acid bacteria detection kit of the present invention is not particularly limited as long as it comprises a stick-shaped film sheet forming body in which a swab storage section and a culture medium storage section containing the lactic acid bacteria detection culture medium of the present invention are connected via a partition that can be opened by inserting the cotton tip of a swab. A method for producing such a kit can be, for example, a method comprising: (1) the step of producing a vertically elongated stick bag with the bottom heat-sealed; (2) the step of filling the bottom of the vertically elongated stick bag with liquid culture medium to form a culture medium storage section; (3) the step of forming a partition that can be opened by inserting the cotton tip of a swab at the top of the culture medium storage section; (4) the step of storing a swab in a swab storage section formed at the top of the partition of the vertically elongated stick bag; and (5) the step of closing the upper edge of the swab storage section by heat sealing.

[0026] The volume of the liquid culture medium contained in the culture medium container is preferably an amount sufficient for lactic acid bacteria to grow in a solid culture medium with fibers forming the cotton ball as a support. The liquid culture medium can be said to be an amount that wets 50% to 145% of the cotton ball, preferably an amount that wets 52% to 130%, preferably an amount that wets 55% to 125%, more preferably an amount that wets 60% to 120%, and preferably an amount that wets 70% to 115%. Here, 100% wetting of the cotton ball by the liquid culture medium means that when the cotton ball is immersed in the liquid culture medium and the cotton ball is pulled up, the liquid culture medium does not drip from the cotton ball. 50% means 50% of the volume of the liquid culture medium in the state of 100% wetting. Furthermore, if the concentration is set to 150% or higher, the cotton ball will be immersed in the liquid culture medium, and if the growth of lactic acid bacteria is insufficient in the liquid culture medium that is not absorbed by the cotton ball, the color of the liquid culture medium may make it difficult to see the color change of the cotton ball.

[0027] For example, if the diameter of the cotton ball is 8 mm, the amount of liquid culture medium pre-stored in the culture medium storage section is preferably 0.3 to 0.7 mL.

[0028] The above-described bag-shaped stick-shaped film sheet molded body can be formed into a bag shape by overlapping the left and right edges of a single elongated sheet and sealing them vertically, thereby creating an elongated tubular film, and then sealing it horizontally. Heat sealing can be shown as a common means of sealing, and for example, the bag-shaped film sheet molded body can be manufactured by pressing it with a hot mold.

[0029] The partition connecting the cotton swab storage section and the culture medium storage section is formed at the top of the culture medium storage section. While there are no particular restrictions on the configuration of the connection between the cotton swab storage section and the culture medium storage section via the partition, it is preferable that they be connected in a rectangular shape in the vertical direction for convenience of storage, etc. However, a protruding portion or a V-shaped partition can also be formed that protrudes from the culture medium storage section, and such a shape allows the partition to be opened by pressure.

[0030] The material of the sheet described above is preferably one that is impermeable to liquid culture media and bacterial cells, does not leak during transfer from the prepared kit, and maintains flexibility to minimize the space required for storage. Examples include synthetic resins of polymers such as polyethylene, polypropylene, polyethylene terephthalate, polyvinyl chloride, polyvinylidene chloride, polyacrylonitrile, polyamide, and polystyrene; metals such as aluminum foil; or film sheets made of laminates thereof. Furthermore, it is preferable that the sheet be at least transparent or semi-transparent so that the color change of the culture medium due to the pH color indicator can be clearly observed.

[0031] The cotton swab described above is not particularly limited as long as it is a known cotton swab having a shaft and a cotton ball, but it is preferable that it has been sterilized beforehand, and that the cotton ball is housed in the cotton swab housing facing the partition. The material of the shaft is not particularly limited, but it is preferable that it be a water-resistant material such as plastic so that the cotton ball can be impregnated with the liquid culture medium. The cotton ball is not particularly limited as long as it can absorb and hold the liquid culture medium, and examples include aggregates made by conventional cotton swab manufacturing methods, such as cotton fibers that have been flocked, but it is not necessarily limited to cotton fibers as long as it has excellent lactic acid bacteria collection and culture performance. The shape of the cotton ball can be spherical, elliptical, etc., but it may also be a special shape such as a spiral, as long as it is a shape that makes it easy to wipe the target area.

[0032] The size of the cotton ball portion is not particularly limited, as long as it is large enough to capture the target bacteria on the surface of the cotton ball when wiping the surface of food manufacturing equipment or containers, or the surface of processed meat products such as ham and bacon. For example, the diameter of the widest part of the cotton ball portion can be 3 to 15 mm, preferably 5 to 13 mm, more preferably 6 to 10 mm, and even more preferably 7 to 9 mm.

[0033] The method for detecting lactic acid bacteria according to the present invention is not particularly limited as long as it uses the lactic acid bacteria detection kit of the present invention. For example, one example is a method of detecting lactic acid bacteria in which the presence or absence of lactic acid bacteria is determined by whether or not there is a color change from purple to yellow after 24 hours, after wiping the target test area with a cotton swab, immersing and stirring the cotton ball in a culture medium, and culturing it at, for example, 30°C. More specifically, in light of the embodiment in which the culture medium is pre-contained in the culture medium storage section of the lactic acid bacteria detection kit and used as a culture medium in which lactic acid bacteria wiped with a cotton ball can grow, the lactic acid bacteria detection medium of the present invention is preferably a liquid medium, and a semi-solid medium to which a gelling agent such as agar is added may also be included in the liquid medium. A semi-solid medium is one that exhibits fluidity when physical force such as stirring or shaking is applied from the outside, and has less convection than a liquid medium when left standing. In this invention, a gelling agent can be added to maintain fluidity sufficient for the cotton tip of a cotton swab to easily absorb the liquid medium when it comes into contact with the liquid medium, and to give the medium sufficient viscosity to hold the absorbed liquid medium. The agar concentration can be 0.025 to 0.2%, preferably 0.05 to 0.15%, which is the concentration necessary to maintain a semi-solid medium at a typical culture temperature of 20 to 40°C. However, this concentration is the agar concentration necessary to maintain a semi-solid state at a typical culture temperature of about 20 to 40°C, and it is preferable to use a smaller amount of agar at temperatures below this range, and a larger amount of agar at high temperatures.

[0034] The above semi-fluid state can also be achieved with gelling agents other than agar. In addition to agar, other components that contribute to gel strength include gelatin, silica gel, acrylamide, glucomannan, methylcellulose, duran gum, gum arabic, starch, sodium alginate, carrageenan, bentonite, arginate, collagen, fused quartz, water-soluble starch, polyacrylate, cellulose, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, dextran, and polysaccharides. These gelling agents can be used in place of agar or in combination with agar.

[0035] The above liquid culture medium may be a medium with reduced or removed buffering capacity compared to normal. Generally, in many culture media, including those for lactic acid bacteria, buffering capacity is maintained by including phosphate buffers, citrate buffers, acetate buffers, lactic acid buffers, tartaric acid buffers, malic acid buffers, Tris buffers, MOPS buffers, and MES buffers in the medium to reduce damage to the bacterial cells by acids, which are metabolic products of lactic acid bacteria, and to promote growth and protect the grown bacterial cells. However, in the present invention, the purpose is to determine whether or not lactic acid bacteria are contaminated in food, beverages, or food manufacturing processes. In order to quickly and efficiently determine whether or not lactic acid bacteria have grown, it is not necessarily required to use buffers commonly used in culture media, such as dipotassium hydrogen phosphate, and they may or may not be used.

[0036] The culture medium of the present invention changes from purple to yellow when the pH decreases. Therefore, in the culture medium of the present invention, for example, if the entire medium changes to yellow (sometimes represented by "+"), if the entire medium changes to orange (sometimes represented by "+w"), or if the medium changes slightly to orange (sometimes represented by "±"), it can be determined that the growth and / or proliferation of lactic acid bacteria has been observed, and the result is positive (reaction). On the other hand, if lactic acid bacteria cannot be detected, and the color of the medium does not change and remains purple (sometimes represented by "-"), it can be determined that the growth and / or proliferation of lactic acid bacteria has not been observed, and the result is negative (reaction).

[0037] Furthermore, when using the culture medium of the present invention contained in a kit, a swab test is performed using the cotton tip of a cotton swab. After the swab test is completed, the cotton swab is inserted through the partition from the cotton swab housing, bringing the liquid culture medium containing the color pH indicator into contact with the cotton tip. The liquid culture medium is then drawn up to the surface and interior of the cotton tip by capillary action, and lactic acid bacteria are grown and / or proliferated for a predetermined time. If the cotton tip turns entirely yellow (sometimes represented by "+"), if about half of the cotton tip turns yellow (sometimes represented by "+w"), or if a part of the cotton tip turns yellow (sometimes represented by "±"), it can be determined to be positive (reactive). If there is no change in the color of the cotton tip and it remains purple (sometimes represented by "-"), it can be determined to be negative (reactive).

[0038] Features of the culture medium of the present invention include its ability to accurately detect lactic acid bacteria, while suppressing the growth and proliferation of bacteria other than lactic acid bacteria, and / or reducing the detection sensitivity.

[0039] The lactic acid bacteria that can be detected by the culture medium of the present invention include 1.0 × 10⁶ 2 It can be CFU / mL or higher, preferably detectable at 1.0 × 10 CFU / mL or higher, and more preferably detectable at 1 CFU / mL or higher.

[0040] Other bacteria besides the lactic acid bacteria mentioned above include bacteria that may be present in food manufacturing facilities, such as Escherichia coli, Staphylococcus aureus, Bacillus species, Pseudomonas species, Listeria species, and Clostridium perfringens. However, it is preferable to suppress the detection of Escherichia coli, which is frequently present in food manufacturing facilities and has relatively low selectivity for growth on culture media compared to lactic acid bacteria. The concentration at which the detection of Escherichia coli is suppressed when swabbing is performed is 1.0 × 10⁻⁶. 3 Examples include values ​​below CFU / mL, such as 1.0 × 10 4 Preferably CFU / mL or less, 1.0 × 10 5 A concentration of CFU / mL or less is more preferable.

[0041] One step-by-step embodiment of the method for detecting lactic acid bacteria using the lactic acid bacteria detection kit of the present invention is as follows: (i) Opening the upper edge of the cotton swab storage compartment and removing the cotton swab from the cotton swab storage compartment; (ii) The process of performing a swab test using the cotton tip of the removed cotton swab; (iii) Inserting the cotton swab that has been used for the swab test through the partition from the cotton swab housing, bringing the cotton ball portion into contact with the liquid culture medium containing the color pH indicator, and drawing the liquid culture medium into the surface and interior of the cotton ball portion by capillary action; (iv) A culture step in which lactic acid bacteria are grown on the surface and inside of a cotton ball that is not immersed in liquid culture medium; (v) A step to detect the presence or absence of lactic acid bacteria by checking whether or not there is a color change on the surface of the cotton ball; An example can be given of a method that sequentially implements each of these processes.

[0042] The above step i) is not particularly limited as long as it is a step of opening the upper edge of the cotton swab storage section in the lactic acid bacteria detection kit and removing the cotton swab from the cotton swab storage section. The upper edge of the cotton swab storage section must be joined with a seal strength that does not peel off with normal external force in order to prevent leakage or damage of the culture medium during storage, transport, etc. However, when using the kit, it is preferable that the seal on the upper edge of the cotton swab storage section be easily peeled off. Therefore, it is preferable that the upper edge of the cotton swab storage section is weakly bonded with a weak seal or the like so that the inner surfaces of the sheets can be easily peeled off from each other, so that the cotton swab can be removed by pulling one or both of the sheets around the upper edge of the cotton swab storage section with your fingers. It is also possible to provide known opening means such as having a small notch in the opening that allows the upper edge to be easily opened by simply tearing the notch.

[0043] As for the weak seal strength that allows peeling by pulling with the fingers as described above, it can be said that it is 10% to 90% of the strength of the peripheral seal portion, preferably 20% to 80%, and more preferably 30% to 60%.

[0044] The above process (ii) is not particularly limited as long as it involves performing a swab test with the cotton tip of the cotton swab removed above, and the object to which the swab test is performed is not particularly limited as long as it is a place that may be contaminated with lactic acid bacteria, but examples include the surface of food products such as ham and bacon, and the surfaces of various parts of food manufacturing equipment and food containers.

[0045] The above-mentioned swab test can be performed with the cotton tip of the cotton swab removed from the cotton swab housing in a dry or wet state. If the object to be wiped is moist, such as the surface of processed meat products like ham or bacon, the swab test can be performed with a dry cotton tip. If the object to be wiped is a dry metal surface such as food manufacturing equipment, it is preferable to wet the cotton tip with water or saline solution as needed before performing the swab test.

[0046] The above step (iii) is not particularly limited as long as it is a step in which the cotton swab used for the wipe test is inserted from the cotton swab housing through the partition wall to bring the liquid culture medium containing the color pH indicator into contact with the cotton ball portion, and the liquid culture medium is drawn up to the surface and interior of the cotton ball portion by capillary action. Preferably, the seal strength of the partition wall through which the cotton swab is inserted from the cotton swab housing is weak enough to be peeled off by pressure, such that the cotton ball portion can peel off the partition wall by pressing the cotton swab directly with a finger or through a sheet.

[0047] The weak seal strength that can be peeled off by the above pressing can be said to be 10% to 90% of the strength of the peripheral seal portion, preferably 20% to 80%, and more preferably 30% to 60%.

[0048] Step (iv) described above is a culture step in which lactic acid bacteria are grown on the surface and inside of the cotton ball portion that is not immersed in the liquid culture medium, and this is a major feature of the present invention. In the present invention, steps (iii) to (v) described above are performed, for example, 1.0 × 10 2A preferred embodiment involves inoculating with lactic acid bacteria at a concentration of CFU / mL or higher and culturing at 28-32°C, preferably 29-31°C, more preferably 30°C, for 22-26 hours, preferably 23-25 ​​hours, and more preferably 24 hours. Under these culture conditions, lactic acid bacteria are detectable, but Escherichia coli, Staphylococcus aureus, and Bacillus cereus are not detected. The instruction manual may include the above temperature settings and culture time as part of the usage method.

[0049] Generally, lactic acid bacteria are cultured at 30°C for 24 to 48 hours, and Bifidobacterium at 37°C for 72 hours (Food Hygiene Inspection Guidelines, Microbiology Section, supervised by the Ministry of Health, Labour and Welfare, 2004). The culture medium of the present invention is excellent in that it can determine the presence or absence of lactic acid bacteria with greater accuracy in about 24 hours after the cotton swab used to wipe the target area comes into contact with the liquid culture medium.

[0050] The process may also include a step (vi) in which the cotton swab's lower limit indicator portion, which is the upper part of the cotton swab's upper end, is heat-sealed after the above step (iv). Lactic acid bacteria are anaerobic bacteria that do not require oxygen to grow. Anaerobic bacteria can be further classified into facultative anaerobic bacteria, which can grow even in the presence of oxygen, and obligate anaerobic bacteria, which die when exposed to oxygen at atmospheric levels. By sealing the cotton swab's lower limit indicator portion, an environment is provided in which oxygen is not supplied to the solid culture medium that constitutes the cotton ball portion, thereby providing an environment suitable for the growth and proliferation of both facultative anaerobic lactic acid bacteria and obligate anaerobic lactic acid bacteria. For this reason, it is preferable to include the above step (vi). The sealing means at the cotton swab's lower limit indicator portion can preferably be heat-sealed, but known adhesive means such as pre-applied double-sided tape may also be used. When sealing the cotton swab's lower limit indicator portion, it is desirable to remove air from the film sheet forming body while doing so.

[0051] The above step (v) is not particularly limited as long as it is a step of detecting the presence or absence of lactic acid bacteria by the presence or absence of a color change on the surface of the cotton ball. As an example of the above color change, if the color pH indicator is the above bromocresol purple, the liquid medium is neutral at the start of cultivation so it will be purple, and if lactic acid bacteria are not present in the sample that has been tested, the cotton ball will remain purple, but if lactic acid bacteria are present, the solid medium supported by the fibers that make up the cotton ball will turn yellow when the pH falls below 5.2 due to the proliferation of lactic acid bacteria.

[0052] The present invention will be described more specifically below with reference to examples, but the technical scope of the present invention is not limited to these examples. [Examples]

[0053] [Reference example] [Investigation of liquid culture media for detecting lactic acid bacteria] (Preparation of LA medium) As shown in Table 1, a total of 51.991 g of the raw materials for the LA medium for lactic acid bacteria detection (Prima Ham Co., Ltd.) was weighed into an Erlenmeyer flask, dissolved in pure water, and a 1 L medium solution was prepared. This medium solution was autoclaved at 121°C for 15 minutes to obtain the control medium liquid medium (LA medium (a)).

[0054] [Example 1] [Inhibitory effect of sodium acetate-added culture medium on bacterial growth] (Preparation of LA medium with sodium acetate) A predetermined amount of sodium acetate was added to the above LA medium, measured into Erlenmeyer flasks, and dissolved in pure water to prepare 1 L portions. The liquid media, which were autoclaved at 121°C for 15 minutes, were used as the test medium, and seven types of sodium acetate-added LA mediums were prepared as shown in Table 2 below.

[0055] [Table 2]

[0056] Sterilized LA medium (a) and the above seven types of LA media (b) to (h) with sodium acetate added were each dispensed in 0.5 mL amounts into a plastic transparent tube with a lid for 2 mL (manufactured by Biorama: AS ONE Corporation).

[0057] Verification was carried out using three types of bacteria: Enterococcus faecalis as lactic acid bacteria (Enterococcus faecalis, NBRC100480 (hereinafter also simply referred to as "faecalis bacteria")); Escherichia coli as gram-negative bacteria; and Staphylococcus aureus as gram-positive bacteria.

[0058] The precultures of each of the above bacteria were appropriately diluted with physiological saline, and 1.0×10 5 CFU / mL (represented as "10 5 " in the figure); 1.0×10 4 CFU / mL (represented as "10 4 " in the figure); 1.0×10 3 CFU / mL (represented as "10 3 " in the figure); 1.0×10 2 CFU / mL (represented as "10 2 " in the figure); 1.0×10 CFU / mL (represented as "10" in the figure); and 1 CFU / mL (represented as "1" in the figure); Each bacterium was seeded so as to have a concentration of the number of bacteria at six levels. Note that CFU is an abbreviation for Colony Forming Unit, which is the colony forming unit, that is, the number of viable bacteria (number of bacteria). The media seeded with each bacterium were each covered and stored in an incubator set at 35°C, and the media were visually confirmed after 24 hours.

[0059] (Visual confirmation criteria) Visual confirmation of detected bacteria was judged according to the following criteria. LA medium is characterized by its color change from purple to yellow when, for example, lactic acid bacteria proliferate and the pH decreases. During the process of the pH in the medium becoming acidic, the medium changes from orange to yellow. Therefore, a state where the medium color is orange was judged as a positive reaction, indicating that bacterial growth and / or proliferation had been confirmed. On the other hand, "-" indicates that there was no change in the color of the medium, and the purple color was maintained, and it was judged that no bacterial growth and / or proliferation was observed (negative reaction). The results are shown in Figure 1. + = The entire culture medium has turned yellow. +w = The entire culture medium has turned orange. ± = The culture medium has turned slightly orange. - = No change in the color of the culture medium (purple)

[0060] (result) As is clear from Figure 1, in the above LA medium (a) and LA medium supplemented with 0.01% to 0.50% sodium acetate (mediums (b) to (e)), the medium turned yellow regardless of the concentration at which *Enterococcus faecalis* or *Escherichia coli* was inoculated. Therefore, no detection inhibitory effect against *Escherichia coli* was observed. Furthermore, *Staphylococcus aureus* also showed a positive reaction except when inoculated at a concentration of 1 CFU / mL in the above LA medium (a) and LA medium supplemented with 0.01% to 0.1% sodium acetate (mediums (b) to (d)), and when inoculated at a concentration of 1.0 × 10 CFU / mL or less in LA medium supplemented with 0.50% sodium acetate (medium (e)). Therefore, almost no detection inhibitory effect against *Escherichia coli* or *Staphylococcus aureus* was observed.

[0061] In a culture medium supplemented with 1.0% sodium acetate (Culture medium (f)), *Enterococcus faecalis* was detected at all concentrations when inoculated, but *Escherichia coli* was not detected when inoculated at concentrations of 1.0 × 10 CFU / mL or less, and *Staphylococcus aureus* was detected at 1.0 × 10 CFU / mL. 2 When seeding was performed at concentrations below CFU / mL, no detection was observed, and a detection inhibitory effect against Escherichia coli and Staphylococcus aureus was observed when the number of viable bacteria was relatively low.

[0062] In a culture medium (g) supplemented with 2.00% sodium acetate, *Enterococcus faecalis* was detected at concentrations of 1.0 × 10 CFU / mL or higher, while *Escherichia coli* and *Staphylococcus aureus* were detected at concentrations of 1.0 × 10 CFU / mL or higher. 4 Since no bacteria were detected even when CFU / mL was seeded, it became clear that the inhibitory effect on bacteria other than lactic acid bacteria was even greater. However, in the medium with 4.00% sodium acetate added (medium (h)), Escherichia coli and Staphylococcus aureus were not detected, but Enterococcus faecalis was also detected at 1.0 × 10⁶. 2 It was confirmed that no lactic acid bacteria were detected when seeding was performed at concentrations below CFU / mL, and that the detection of lactic acid bacteria was also suppressed.

[0063] From the above, it was found that when sodium acetate is added to LA medium alone, a concentration of 1.00% to 2.00% is appropriate for detecting lactic acid bacteria and suppressing the detection of bacteria other than lactic acid bacteria.

[0064] [Example 2] [Inhibitory effect of sodium lactate-added culture medium on bacterial growth] (Preparation of LA medium with added sodium lactate) A predetermined amount of sodium lactate was added to the above LA medium, measured into Erlenmeyer flasks, and dissolved in pure water to prepare 1 L portions. The liquid media, which were autoclaved at 121°C for 15 minutes, were used as the test medium, and seven types of sodium lactate-added LA mediums were prepared as shown in Table 3 below.

[0065] [Table 3]

[0066] For the 2 mL transparent plastic tubes with lids mentioned above, 0.5 mL each of sterile LA medium (a) and the seven types of sodium lactate-added LA medium (b') to (h') were dispensed.

[0067] Similar to Example 1, the verification was performed using three types of bacteria: Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus. The pre-culture solutions of each of the above bacteria were appropriately diluted with physiological saline and added to the above LA medium (a) and the above seven types of sodium lactate-added LA mediums (b') to (h') in 1.0 × 10⁻¹⁴ units, respectively. 5 CFU / mL; 1.0 × 10 4 CFU / mL; 1.0 × 10 3 CFU / mL; 1.0 × 10 2 Each bacterium was inoculated at six different concentrations: CFU / mL, 1.0 × 10 CFU / mL, and 1 CFU / mL. Each culture medium containing the inoculated bacteria was covered and stored in an incubator set to 35°C. After 24 hours, the culture medium was visually inspected. The visual inspection criteria were as described in Example 1. The results are shown in Figure 2.

[0068] (result) As is clear from Figure 2, in both the LA medium (a) and the LA medium supplemented with 0.01% to 2.0% sodium lactate (mediums (b') to (g')), the medium almost always turned yellow when *Enterococcus faecalis* or *Escherichia coli* were seeded at any of the six concentrations mentioned above. Therefore, little to no inhibitory effect on the growth of *Escherichia coli* was observed.

[0069] Staphylococcus aureus also showed a positive reaction except when seeded at a concentration of 1 CFU / mL or 1.0 × 10 CFU / mL in the above-mentioned LA medium (a) and LA medium supplemented with 0.01-0.1% sodium lactate (mediums (b')-(f')). In the medium supplemented with 2.00% sodium lactate (medium (g')), the result was 1.0 × 10 CFU / mL. 2 It was not detected when seeded at concentrations below CFU / mL, and was detected at 1.0 × 10 in a medium (medium (h')) supplemented with 4.00% sodium lactate. 5 It was not detected even when seeded at a concentration of CFU / mL.

[0070] It was revealed that the detection of *Enterococcus faecalis*, a type of lactic acid bacterium, was also suppressed in a culture medium (culture (h')) to which 4.0% sodium lactate was added. As described above, it was confirmed that lactate alone has little effect in suppressing the growth of bacteria other than lactic acid bacteria, especially *Escherichia coli*.

[0071] [Example 3] [Investigation of the inhibitory effect on bacterial growth in culture media supplemented with sodium acetate and sodium lactate] (Preparation of LA medium with sodium acetate / sodium lactate) Sodium acetate at a concentration of 2.0% or 1.0% and sodium lactate at a concentration of 2.0% or 1.0% were added to the above LA medium, measured into Erlenmeyer flasks, dissolved in pure water, and prepared in 1 L quantities. Each of these was autoclaved at 121°C for 15 minutes to prepare the test mediums, and LA mediums with the four types of sodium acetate and sodium lactate shown in Table 4 below were prepared.

[0072] [Table 4]

[0073] 0.5 mL each of LA medium (A), (F), (G), and (L), to which the four types of sterile sodium acetate and sodium lactate were added, was dispensed into the above-mentioned 2 mL transparent plastic tubes with lids.

[0074] Similar to Example 1, the verification was performed using three types of bacteria: Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus. The pre-culture solutions of each of the above bacteria were appropriately diluted with physiological saline and added to the four types of sodium acetate / sodium lactate-added LA medium (A), (F), (G), and (L), respectively, in a dose of 1.0 × 10⁻⁶. 5 CFU / mL; 1.0 × 10 4 CFU / mL; 1.0 × 10 3 CFU / mL; 1.0 × 10 2Each bacterium was inoculated at six different concentrations: CFU / mL, 1.0 × 10 CFU / mL, and 1 CFU / mL. Each culture medium containing the inoculated bacteria was covered and stored in an incubator set to 35°C. After 24 hours, the culture medium was visually inspected. The visual inspection criteria were as described in Example 1. The results are shown in Figure 3.

[0075] As is clear from Figure 3, in both the medium (A) supplemented with a combination of 2.0% sodium acetate and 2.0% sodium lactate, and the medium (G) supplemented with a combination of 2.0% sodium acetate and 1.0% sodium lactate, Escherichia coli and Staphylococcus aureus showed a negative reaction (-) at all six concentrations listed above when inoculated, indicating the highest detection inhibition effect. For Enterococcus faecalis, in medium (G), Enterococcus faecalis inoculated at concentrations of 1.0 × 10 CFU or higher was detectable, but in medium (A), at 1.0 × 10 CFU... 2 Enterococcus faecalis was not detected unless the seeds were inoculated at a concentration of CFU / mL or higher.

[0076] For reference, Table 5 below summarizes the results of the visual inspection conducted so far.

[0077] [Table 5]

[0078] The results for adding 2.0% or 1.0% sodium acetate from Example 1 and 2.0% or 1.0% sodium lactate from Example 2 to LA medium individually are also shown. Compared to media with sodium acetate or sodium lactate added individually, such as media with sodium acetate (media (g) and (f)) and media with sodium lactate (media (g') and (f')), the media combining sodium acetate and sodium lactate (media (A), (F), (G), and (L)) showed a very high detection inhibitory effect against Escherichia coli and Staphylococcus aureus, indicating that combining sodium acetate and sodium lactate produces a synergistic effect in inhibiting the growth of Escherichia coli and Staphylococcus aureus. From these results, it was considered that a media with approximately 2.0% sodium acetate and approximately 1.0-2.0% sodium lactate combined is suitable.

[0079] [Example 4] [Investigation of the optimal addition concentration of sodium acetate and sodium lactate combinations] Sodium acetate was added to the above LA medium at concentrations of 2.0%, 1.8%, 1.6%, 1.4%, 1.2%, or 1.0%, and sodium lactate at concentrations of 2.0% or 1.0%. The mixture was then measured into Erlenmeyer flasks, dissolved in pure water, and prepared in 1 L portions. Each of these was autoclaved at 121°C for 15 minutes to prepare the test mediums. Twelve different types of LA mediums were prepared by adding sodium acetate and sodium lactate, as shown in Tables 6-1 and 6-2 below.

[0080] [Table 6-1]

[0081] [Table 6-2]

[0082] 0.5 mL of LA medium, containing the 12 types of sterile sodium acetate and sodium lactate mentioned above, was dispensed into 2 mL transparent plastic tubes with lids.

[0083] Similar to Example 1, the verification was performed using three types of bacteria: Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus. The pre-culture solutions of each of the above bacteria were appropriately diluted with physiological saline and added to the 12 types of sodium acetate / sodium lactate-added LA medium, each at a concentration of 1.0 × 10⁶. 5 CFU / mL; 1.0 × 10 4 CFU / mL; 1.0 × 10 3 CFU / mL; 1.0 × 10 2 Each bacterium was inoculated at six different concentrations: CFU / mL, 1.0 × 10 CFU / mL, and 1 CFU / mL. Each culture medium containing the inoculated bacteria was covered and stored in an incubator set to 35°C. After 24 hours, the culture medium was visually inspected. The visual inspection criteria were as described in Example 1. The results are shown in Figure 4.

[0084] (result) As is clear from Figure 4, against Escherichia coli and Staphylococcus aureus, Culture medium (Medium (A)) containing 2.0% sodium acetate and 2.0% sodium lactate; Culture medium (Medium (B)) containing 1.8% sodium acetate and 2.0% sodium lactate; Culture medium containing 1.6% sodium acetate and 2.0% sodium lactate (Culture medium (C)); In a culture medium containing 2.0% sodium acetate and 1.0% sodium lactate (Culture Medium (G)), and a culture medium containing 1.8% sodium acetate and 1.0% sodium lactate (Culture Medium (H)), Escherichia coli and Staphylococcus aureus were found to be present in 1.0 × 10⁶ cells. 4 When seeded at concentrations below CFU / mL, it was not detected, indicating a very high level of detection suppression.

[0085] On the other hand, for Enterococcus faecalis, a negative reaction (-) was observed when 1.0 × 10 CFU / mL was inoculated in medium (A), and when 1 CFU / mL was inoculated in mediums (B), (C), and (G). However, it was detected in medium (H) even when 1 CFU / mL was inoculated.

[0086] Furthermore, when sodium acetate and sodium lactate were added to the culture medium, the color of the *Enterococcus faecalis* bacteria as they proliferated and turned yellow appeared slightly duller. A comparison of visibility revealed that the yellow discoloration of *Enterococcus faecalis* was clearly recognizable in the culture medium (Medium (H)) containing 1.8% sodium acetate and 1.0% sodium lactate. Therefore, for practical application, the culture medium containing 1.8% sodium acetate and 1.0% sodium lactate was considered more suitable. Accordingly, subsequent studies will be conducted using the culture medium containing 1.8% sodium acetate and 1.0% sodium lactate.

[0087] [Example 5] (Study on lactic acid bacteria detection media other than LA medium) We investigated whether using a culture medium other than the LA medium described above, with sodium acetate and / or sodium lactate added, would also have an inhibitory effect on detecting bacteria other than lactic acid bacteria. Based on previous verification results, Escherichia coli was selected as the bacterium that is easily detected, and continued the verification process.

[0088] (Preparation of culture medium for detecting lactic acid bacteria) As a medium for detecting lactic acid bacteria other than LA medium, MRS (de Man, Rogosa, Sharpe) broth (DIFCO Lactobacillus MRS broth, manufactured by Becton Dickinson) was selected. As shown in Table 7(a), MRS broth (medium a) contains 0.5% sodium acetate. Therefore, a medium with 0.5% sodium acetate added to LA medium was prepared and compared. Specifically, as shown in Tables 7 and 8 below, four types of medium components were measured into Erlenmeyer flasks, dissolved in pure water to prepare 1 L each, and then autoclaved at 121°C for 15 minutes. These were: MRS broth (medium a); MRS broth with an additional 1.3% sodium acetate added to bring the total to 1.8%, and then a medium with 1.0% sodium lactate added (medium b); LA medium with 0.5% sodium acetate added (medium c); and LA medium with 1.8% sodium acetate and 1.0% sodium lactate added (medium d). These four types of lactic acid bacteria detection media were examined as test media.

[0089] [Table 7]

[0090] [Table 8]

[0091] 0.5 mL of each sterile test medium was dispensed into the above 2 mL transparent plastic tubes with lids. 1.0 × 10⁶ of each of the four test media was added to each. 5 CFU / mL; 1.0 × 10 4 CFU / mL; 1.0 × 10 3 CFU / mL; 1.0 × 10 2 Enterococcus faecalis and Escherichia coli were inoculated at five different concentrations ranging from CFU / mL to 1.0 × 10 CFU / mL. The culture media were covered and stored in a 35°C incubator, and the media were visually inspected after 24 hours. The visual inspection criteria were as described in Example 1.

[0092] (result) As is clear from Figure 5, in MRS broth medium A and medium C, which is LA medium with 0.5% sodium acetate added, not only Enterococcus faecalis but also Escherichia coli was detected at all concentrations when seeded. In the medium containing 0.5% sodium acetate, the detection of Escherichia coli could not be suppressed, confirming low selectivity between lactic acid bacteria and Escherichia coli. On the other hand, in medium B, which is MRS broth with a final sodium acetate concentration of 1.8% with 1.0% sodium lactate added, and medium D, which is LA medium with 1.8% sodium acetate and 1.0% sodium lactate added, Enterococcus faecalis was detected at all concentrations, but Escherichia coli was detected at 1.0 × 10⁶ 4 Concentration of CFU / mL or less (Culture Medium A), or 1.0 × 10 3 No lactic acid bacteria were detected at concentrations of CFU / mL or less (Culture Medium E). From these results, it was confirmed that adding 1.8% sodium acetate and 1.0% sodium lactate to any of the lactic acid bacteria detection media can significantly suppress the detection of bacteria other than lactic acid bacteria.

[0093] [Example 6] [Verification in a format simulating a swab test] (Contents of the swab test kit) A bag-shaped film sheet was filled with 0.5 mL of liquid culture medium for lactic acid bacteria detection at the bottom to form a culture medium container. To prevent splashing or leakage of the liquid culture medium, the top of the culture medium container was heat-sealed using a heat-sealing machine to form a partition wall, thereby sealing the culture medium container. Furthermore, a cotton swab for wiping was inserted into the top of the partition wall, and the cotton tip of the swab was positioned so that it was in contact with the heat-sealed partition wall at the top of the culture medium container, thus creating a lactic acid bacteria detection kit for swab testing in which the liquid culture medium and the cotton swab were integrated. The cotton swab used was a plastic swab with a longitudinal length of 90 mm and a cotton tip that was 15 mm long and had a diameter of 8 mm at its widest point.

[0094] In Example 4, 1.8% sodium acetate and 1.0% sodium lactate were added to the LA medium (Medium (H)), and the resulting medium was measured into an Erlenmeyer flask. Pure water was added to dissolve it, and 1 L was prepared. This medium was autoclaved at 121°C for 15 minutes and used as the test medium. 0.5 mL of this medium was packed into a film sheet.

[0095] The following bacteria were used to examine the detection accuracy using the swab test kit. <Lactic acid bacteria> (1) Lactobacillus sakei NBRC107868 (hereinafter also simply referred to as "sakei fungus") (2) Leuconostoc mesenteroides NBRC100496 (hereinafter also simply referred to as "Leuconostoc") (3) Enterococcus faecalis <Bacteria other than lactic acid bacteria> (1) Escherichia coli (2) Staphylococcus aureus (3) Bacillus cereus (hereinafter also simply referred to as "Bacillus cereus")

[0096] For the six types of bacteria listed above, the pre-culture solution was diluted with physiological saline and then cultured in the culture medium at a rate of 1.0 × 10⁶. 5 CFU / mL, 1.0 × 10 4 CFU / mL, 1.0 × 10 3 CFU / mL, 1.0 × 10 2 Solutions were prepared at CFU / mL and 1.0 × 10 CFU / mL, and each solution was inoculated onto the tip of a cotton swab using a micropipette. Each cotton swab, containing the cotton tip inoculated with the five different concentrations of bacteria, was inserted through the partition of a film sheet forming body, and the top was clipped to prepare a sample. The samples were stored in incubators at 35°C, 30°C, or 25°C, and the color change was visually observed after 24 hours and 48 hours. The results are shown in Figures 7 and 8.

[0097] (Visual inspection criteria) If detected, visual confirmation was judged according to the following criteria. + = The cotton ball has turned entirely yellow. +w = Approximately half of the cotton ball has turned yellow. ± = A portion of the cotton ball has turned yellow. - = No change in the color of the cotton ball (purple)

[0098] (Results after 24 hours of incubation) When cultured at 35°C (Figure 7(a)), the number of *Mycobacterium sieboldii* was 1.0 × 10⁶. 2 When inoculated at CFU / mL or higher, Leuconostoc and Enterococcus faecalis were detected at inoculation levels of 1.0 × 10 CFU / mL or higher. Escherichia coli was detected at 1.0 × 10 CFU / mL or higher. 3 It was not detected in cases where the dose was below CFU / mL, but 1.0 × 10 4 CFU / mL ~ 1.0 × 10 5 It was detected when inoculated with CFU / mL. Staphylococcus aureus was detected at 1.0 × 10⁻⁶. 4 In inoculations below CFU / mL, Bacillus cereus was not detected, and 1.0 × 10⁶ Bacillus cereus was found. 5 It was not detected in cases where the vaccination level was below CFU / mL.

[0099] When cultured at 30°C (Figure 7(b)), the number of *Mycobacterium sieboldiana* and *Leuconostoc* was 1.0 × 10⁶. 2 Bacillus faecalis was detected when inoculated at CFU / mL or higher, and at 1.0 × 10 CFU / mL or higher. Escherichia coli, Staphylococcus aureus, and Bacillus cereus were not detected in any of these cases.

[0100] When cultured at 25°C (Figure 7(c)), the number of *Lactobacillus sieboldiana* and *Enterococcus faecalis* was 1.0 × 10⁶. 3 When inoculated at CFU / mL or less, Leuconostoc bacteria are 1.0 × 10⁶ 2 No bacteria were detected when inoculated at CFU / mL or lower. Escherichia coli, Staphylococcus aureus, and Bacillus cereus were not detected in any of the cases.

[0101] From these results, it was confirmed that when cultured at 30°C, the detection sensitivity of lactic acid bacteria after 24 hours was superior, and the detection of bacteria other than lactic acid bacteria could be suppressed. In culture at 30°C, the degree of growth of lactic acid bacteria was almost the same as at 35°C, while the inhibitory effect on E. coli was higher compared to 35°C.

[0102] (Results after 48 hours of incubation) When cultured at 35°C (Figure 8(a)), *Lactobacillus saccharinus*, *Leuconostoc*, *Enterococcus faecalis*, and *Escherichia coli* were detected at all inoculation concentrations. *Staphylococcus aureus* was detected at 1.0 × 10⁻⁶. 2 CFU / mL or higher, Bacillus cereus 1.0 × 10 3 The bacteria were detected when inoculated at CFU / mL or higher. Compared to 24 hours, it became clear that bacteria other than lactic acid bacteria also proliferated significantly and could be detected.

[0103] When cultured at 30°C (Figure 8(b)), *Lactobacillus sieboldii*, *Leuconostoc*, and *Enterococcus faecalis* were detected at all inoculation concentrations. *Escherichia coli* was detected at 1.0 × 10⁶. 2 CFU / mL or higher, Staphylococcus aureus 1.0 × 10 3Although detected when inoculated at CFU / mL or higher, Bacillus cereus was not detected. It became clear that bacteria other than lactic acid bacteria proliferated and were detected compared to 24 hours.

[0104] When cultured at 25°C (Figure 8(c)), *Lactobacillus sieboldii*, *Leuconostoc*, and *Enterococcus faecalis* were detected at all inoculation concentrations. *Escherichia coli* was detected at 1.0 × 10⁶. 4 While detected in inoculations of CFU / mL or higher, Staphylococcus aureus and Bacillus cereus were not detected.

[0105] Based on these results, when culturing for 48 hours at 25°C, some other bacteria were detected, but lactic acid bacteria were detectable. [Industrial applicability]

[0106] This invention is useful in the field of food manufacturing.

Claims

1. A culture medium for detecting lactic acid bacteria, characterized in that it contains an acetate and a color pH indicator, wherein the concentration of the acetate is 10 to 25 g / L in terms of sodium acetate.

2. A culture medium for detecting lactic acid bacteria according to claim 1, further comprising lactate.

3. The culture medium for detecting lactic acid bacteria according to claim 2, characterized in that the concentrations of acetate and lactate, when converted to sodium acetate and sodium lactate, are 1.5-2.5% sodium acetate and 0.5-2.5% sodium lactate.

4. A culture medium for detecting lactic acid bacteria according to any one of claims 1 to 3, characterized in that the culture medium is a liquid culture medium.

5. A kit for detecting lactic acid bacteria, characterized in that it comprises a stick-shaped film sheet forming body, wherein a section for storing a cotton swab for wiping and a section for storing a culture medium for detecting lactic acid bacteria as described in claim 4 are connected via a partition that can be opened by inserting the cotton tip of a cotton swab.

6. A method for detecting lactic acid bacteria, characterized by using the lactic acid bacteria detection kit described in claim 5.

7. A method for detecting lactic acid bacteria according to claim 6, comprising the following steps (a) to (f) in sequence. (a) A step of preparing the lactic acid bacteria detection kit according to claim 5; (b) The step of opening the upper edge of the cotton swab storage section and removing the cotton swab from the cotton swab storage section; (c) The process of performing a swab test using the cotton tip of the removed cotton swab; (d) Inserting the cotton swab used for the swab test through the partition from the cotton swab housing, bringing the cotton ball portion into contact with the liquid culture medium containing the color pH indicator, and drawing the liquid culture medium into the surface and interior of the cotton ball portion by capillary action; (e) A culture step in which lactic acid bacteria are grown on the surface and inside of a cotton ball that is not immersed in a liquid culture medium; (f) A step to detect the presence or absence of lactic acid bacteria by checking whether or not there is a color change on the surface of the cotton ball;

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