Method for detecting lactic acid bacteria

Abstract

To provide means for determining the presence or absence of lactic acid bacterial growth by swab wiping, in a shorter time, with higher precision, and in a simplified manner, for preventing contamination by lactic acid bacteria at food manufacturing sites or in products during preservation.SOLUTION: By employing a kit in which a film sheet formation body having a swab housing part and a medium housing part connected through a partition wall part contains, in the medium housing part, a liquid medium including a chromogenic pH indicator capable of detecting lactic acid bacteria, it is possible to detect lactic acid bacteria more quickly and sensitively.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for detecting lactic acid bacteria, and more particularly to a method for detecting lactic acid bacteria using a lactic acid bacteria detection kit containing a liquid medium capable of detecting lactic acid bacteria. [Background technology]

[0002] Lactic acid bacteria are recognized as major beneficial bacteria in the intestinal flora that affect the maintenance of human health, and are used in dairy products such as yogurt. However, they are also recognized as harmful bacteria that cause hiochi in the sake production process, and as bacteria that cause spoilage in processed meat products such as ham, resulting in stickiness.

[0003] Conventionally, in order to effectively eliminate lactic acid bacteria contamination, a method for detecting lactic acid bacteria that can visually determine the presence or absence of lactic acid bacteria in a test sample quickly, accurately, and efficiently and that can be widely used for testing general foods, beverages, water, etc., as well as for testing manufacturing processes, is known in which the test sample is added and mixed to a semi-liquid medium for detecting lactic acid bacteria containing glucose, a pH indicator such as bromocresol purple, and agar, and the medium is cultured at 25°C for two days, for example, and the change in color of the medium before and after culture is visually determined (see, for example, Patent Document 1).

[0004] Furthermore, a known test tool is one that is convenient to transport and carry, and easy to operate, and includes a reagent liquid that exhibits a color reaction, a sampling tool for sampling a sample and adding it to the reagent liquid, and a transparent test container. The test container is a sealed bag made of a flexible material, and is filled with the amount of reagent liquid required for one test. Furthermore, one sampling tool is removably housed in the bag, isolated from the reagent liquid by a temporary adhesive that releasably bonds the inner surfaces of the flexible material together (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 [Problem to be solved by the invention]

[0006] The object of the present invention is to provide a means for determining the presence or absence of lactic acid bacteria proliferation in a shorter time with higher accuracy by wiping with a cotton swab in order to prevent contamination with lactic acid bacteria in food manufacturing sites and products during storage. [Means for solving the problem]

[0007] Previously, kits existed that allow users to easily check for residual proteins on cleaned cooking utensils or production lines by wiping with a cotton swab. When a target area is wiped with the cotton swab included in the kit and then placed in a test solution, if proteins are present, the color of the surface of the cotton swab changes, clearly showing a difference in color from the test solution, allowing the presence of proteins to be detected instantly (Patent Document 2). However, even with such kits, lactic acid bacteria cannot be detected simply and efficiently.

[0008] The present inventors decided to investigate the conditions under which lactic acid bacteria could be efficiently detected by wiping a target area with a cotton swab using a conventional lactic acid bacteria detection medium. First, bromocresol purple was used as a pH indicator. A sufficient amount of conventional lactic acid bacteria detection liquid medium containing this pH indicator was placed in a plastic tube, and a cotton swab containing lactic acid bacteria was placed in the tube for incubation. However, even when the surface of the cotton swab turned yellow, indicating acidity, due to the growth and proliferation of lactic acid bacteria, if the lactic acid bacteria were not sufficiently proliferating in the liquid medium outside the cotton swab, the purple color of the neutral liquid medium sometimes prevented the recognition of the yellow color of the cotton swab.

[0009] On the other hand, when 0.5 mL of liquid medium for detecting lactic acid bacteria was placed in a plastic tube, the liquid medium permeated the entire cotton ball, leaving almost no liquid medium outside the cotton ball, making it easy to recognize the change in production in the cotton ball itself, i.e., the color change from purple to yellow. Based on the recognition that lactic acid bacteria can be detected efficiently when there is no or almost no liquid medium outside the cotton ball, the inventors employed a film sheet molded body as a container more suitable for detection, and created a kit in which a color-changing pH indicator-containing liquid medium capable of detecting lactic acid bacteria was contained in the medium-containing portion of the film sheet molded body, in which the cotton swab-containing portion and the medium-containing portion were connected via a partition. The inventors then confirmed the conditions for more rapid and sensitive detection of lactic acid bacteria, leading to the completion of the present invention.

[0010] That is, the present invention is as follows. [1] A method for detecting lactic acid bacteria, comprising the following steps (a) to (f) in order: (a) preparing a lactic acid bacteria detection kit, in which a liquid medium containing a color pH indicator capable of detecting lactic acid bacteria is accommodated in a medium accommodating section of a film sheet formed body in which a cotton swab accommodating section and a medium accommodating section are connected via a partition; (b) opening the top edge of the swab storage compartment and removing the swab from the swab storage compartment; (c) A process of performing a swab test using the cotton ball of the removed cotton swab; (d) inserting the cotton swab after the swab test from the cotton swab storage section through the partition wall, bringing the color pH indicator-containing liquid medium into contact with the cotton ball, and sucking the liquid medium up onto the surface and inside of the cotton ball by capillary action; (e) a culturing step of growing lactic acid bacteria on the surface and inside of the cotton ball that is not immersed in the liquid medium; (f) detecting the presence or absence of lactic acid bacteria based on the presence or absence of a color change on the surface of the cotton ball; [2] The method for detecting lactic acid bacteria according to [1] above, wherein steps (d) to (f) can be carried out within 24 hours. [3] The method for detecting lactic acid bacteria according to [1] above, wherein the culturing step (e) is carried out at 24 to 35°C. [4] The method for detecting lactic acid bacteria according to [1] above, wherein the color pH indicator is bromocresol purple. [5] The method for detecting lactic acid bacteria according to [1] above, characterized in that the swab test is performed using a dry cotton swab or a cotton swab moistened with water or physiological saline. [6] The method for detecting lactic acid bacteria according to [1] above, characterized in that after the culturing step (e), a step (g) of heat-sealing the cotton swab at the upper end of the cotton swab, which is the cotton swab lowering limit indicator, is provided.

[0011] The present invention is also as follows. [7] A lactic acid bacteria detection kit used in the lactic acid bacteria detection method according to any one of [1] to [6] above, in which a swab storage section and a culture medium storage section are connected in a rectangular shape via a partition section. [8] The lactic acid bacteria detection kit according to [7] above, characterized in that the swab storage section is provided with a swab lowering limit indicator. [Effects of the Invention]

[0012] According to the present invention, lactic acid bacteria present in food manufacturing equipment and processed meat products can be determined more simply, in a shorter time, and with higher accuracy than conventional methods. [Brief explanation of the drawings]

[0013] [Figure 1] Cotton swabs inoculated with five different concentrations of lactic acid bacteria were placed in plastic tubes containing various volumes of liquid culture medium, capped, and stored in an incubator at 25°C. The color change of the cotton swabs was visually observed after 16 hours. (a) shows the results when the culture medium volume was 10 mL, (b) when the culture medium volume was 7 mL, (c) when the culture medium volume was 4 mL, (d) when the culture medium volume was 2 mL, and (e) when the culture medium volume was 0.5 mL. [Figure 2] 1 shows a schematic diagram of a lactic acid bacteria detection kit. [Figure 3]Each cotton swab containing a cotton ball inoculated with five different concentrations of lactic acid bacteria was cultured (a) in a plastic tube containing 0.5 mL of liquid culture medium, and (b) in a film sheet molded body containing 0.5 mL of liquid culture medium. The results are shown below, with the cotton ball inspected visually after 16 hours. [Figure 4] When a cotton swab is placed in a plastic tube or a film sheet molded product, (a) in the case of a plastic tube, a large amount of air is present around the cotton ball, and (b) in the case of a film sheet molded product, the cotton ball containing the liquid medium comes into contact with the film, but the amount of air present around the cotton ball is reduced. [Figure 5] The results of culturing cotton balls inoculated with lactic acid bacteria at five different concentrations were placed in film sheet molded bodies filled with eight different volumes of liquid culture medium: (a) 0.05 mL, (b) 0.1 mL, (c) 0.2 mL, (d) 0.3 mL, (e) 0.4 mL, (f) 0.5 mL, (g) 0.6 mL, or (h) 0.7 mL. [Figure 6] The results of culturing cotton balls inoculated with lactic acid bacteria to five different concentrations were placed in film sheet molded bodies filled with nine different media compositions, with the liquid media concentrations being (a) 0.1 times, (b) 0.25 times, (c) 0.5 times, (d) 0.75 times, (e) 1 time, (f) 1.25 times, (g) 1.5 times, (h) 2 times, and (i) 3 times the concentration of the basal medium. [Figure 7] Various concentrations of lactic acid bacteria were inoculated onto a stainless steel plate and wiped with a cotton ball. (a) The color change after 17 hours of incubation and (b) after 24 hours of incubation is shown. [Figure 8] Various concentrations of lactic acid bacteria were inoculated onto the surface of ham manufactured by Company A and wiped off with a cotton ball. (a) The color change after 17 hours of incubation and (b) after 24 hours of incubation is shown. [Figure 9] Various concentrations of lactic acid bacteria were inoculated onto the surface of ham manufactured by Company B and wiped off with a cotton ball. (a) The color change after 17 hours of incubation and (b) after 24 hours of incubation is shown. [Figure 10] Various concentrations of lactic acid bacteria were inoculated onto the surface of sausages manufactured by Company A and wiped with a cotton ball. (a) The color change after 17 hours of incubation and (b) after 24 hours of incubation is shown. [Figure 11] Lactic acid bacteria of various concentrations inoculated onto the surface of sausages manufactured by Company B were wiped off with a cotton ball, and the color change after (a) 17 hours of incubation and (b) 24 hours of incubation is shown. [Figure 12] Various concentrations of Lactobacillus sakei were inoculated onto the cotton ball, and the color change after (a) 17 hours of incubation and (b) 24 hours of incubation is shown. [Figure 13] Various concentrations of Leuconostoc mesenteroides were inoculated onto the cotton ball, and the color change after (a) 17 hours of incubation and (b) 24 hours of incubation is shown. [Figure 14] Various concentrations of Lactococcus lactis were inoculated onto the cotton ball, and the color change after (a) 17 hours of incubation and (b) 24 hours of incubation is shown. [Figure 15] Various concentrations of Pediococcus acidilactici were inoculated onto the cotton ball, and the color change after (a) 17 hours of incubation and (b) 24 hours of incubation is shown. DETAILED DESCRIPTION OF THE INVENTION

[0014] The method for detecting lactic acid bacteria of the present invention includes the steps of: (a) preparing a lactic acid bacteria detection kit, in which a liquid medium containing a color pH indicator capable of detecting lactic acid bacteria is enclosed in a medium-containing portion of a film sheet formed body in which a swab-containing portion and a medium-containing portion are connected via a partition; (b) opening the top edge of the swab storage compartment and removing the swab from the swab storage compartment; (c) performing a swab test using the removed swab; (d) inserting the swab after the swabbing test through the partition to contact the liquid culture medium, and wicking the liquid culture medium onto the surface and inside of the swab by capillary action; (e) a culturing step of growing lactic acid bacteria on the surface and inside of the cotton ball that is not immersed in the liquid medium; (f) detecting the presence or absence of lactic acid bacteria based on the presence or absence of a color change on the surface of the cotton ball; There are no particular limitations on the method as long as it includes the steps above in order, and after the culturing step (e) above, a step (g) of heat-sealing the cotton swab at the cotton swab lowering limit indicator, which is the upper part of the upper end of the cotton swab, may be provided.

[0015] The lactic acid bacteria detected in the present invention are a general term for bacteria that produce acids such as lactic acid through fermentation using sugars, and thereby cause a neutral medium to become acidic as they grow, and include bifidobacteria.

[0016] Specific examples of the lactic acid bacteria include Lactobacillus genus lactic acid bacteria such as Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus sakei, and Lactobacillus rhamnosus; Lactococcus genus lactic acid bacteria such as Lactococcus lactis; Carnobacterium genus lactic acid bacteria such as Carnobacterium divergens and Carnobacterium pisicola; Leuconostoc genus lactic acid bacteria such as Leuconostoc mesenteroides and Leuconostoc citreum; Enterococcus caeseriflavus, Enterococcus sulphureus, and Enterococcus sulphureus. Examples of lactic acid bacteria include Enterococcus faecalis, Weissella confusa, and other lactic acid bacteria; Atopobium minutum, Atopobium parbulus, and other lactic acid bacteria; Vagococcus fluvialis, Vagococcus thermoninarum, and other lactic acid bacteria; Pediococcus acidilactici, Pediococcus damnosus, and Pediococcus pentosaceus, and other lactic acid bacteria; Streptococcus thermophilus, and other lactic acid bacteria; Oenococcus oeni, and other lactic acid bacteria; and Tetragenococcus halophilus, and other lactic acid bacteria.

[0017] The bifidobacteria is a general name for bacteria classified in the genus Bifidobacterium, and examples thereof include Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum.

[0018] The lactic acid bacteria detection kit prepared in the above step (a) is not particularly limited as long as it is a kit in which a color-changing pH indicator-containing liquid medium capable of detecting lactic acid bacteria is enclosed in the culture medium storage section of a film sheet molded body in which a swab storage section and a culture medium storage section are connected via a partition section. Examples of methods for producing such a kit include: (1) creating a bag-shaped film sheet molded body; (2) filling the bottom of the bag-shaped film sheet molded body with liquid medium to form a culture medium storage section; (3) forming a partition section at the boundary between the culture medium storage section and the swab storage section; and (4) storing a cotton swab in the swab storage section and closing the upper edge of the swab storage section.

[0019] The bag-shaped film sheet molding can be made into a bag shape by overlapping two sheets and sealing the peripheral edges to form a non-adhesive region, or by sealing the open end of a sheet-like cylindrical body to make a bag-shaped container. Heat sealing is a common sealing method, and for example, a bag-shaped film sheet molding can be made by pressing with a hot mold.

[0020] The partition connecting the swab containing section and the culture medium containing section can be arranged so that the swab containing section and the culture medium containing section face each other, and the manner in which the swab containing section and the culture medium containing section are connected via the partition is not particularly limited, but considering convenience of storage, it is preferable that they are connected in a rectangular shape, but it is also possible to form a protruding section that protrudes from the culture medium containing section or a V-shaped partition, and by having such a shape, the partition can be more effectively peeled off by pressing.

[0021] The material of the sheet is preferably impermeable to liquid culture media and bacteria, does not leak during transport from the prepared kit, and maintains flexibility so as to reduce the space required for storage. Examples include synthetic resins such as polymers of polyethylene, polypropylene, polyethylene terephthalate, polyvinyl chloride, polyvinylidene chloride, polyacrylonitrile, polyamide, and polystyrene; metals such as aluminum foil; or film sheets made of laminates thereof. The materials of the two sheets may be the same or different. Furthermore, it is preferable that at least one sheet is transparent or nearly transparent so that the color change of the culture medium due to the pH color indicator can be clearly seen.

[0022] The cotton swab is not particularly limited as long as it has a known shaft and a cotton ball, but it is preferably pre-sterilized and is preferably placed in the swab container with the cotton ball facing the partition. The material of the shaft is not particularly limited, but a water-resistant material such as plastic is preferred, as the cotton ball is immersed in liquid culture medium. The cotton ball is not particularly limited as long as it can absorb and retain liquid culture medium. Examples include aggregates made by conventional cotton swab manufacturing methods, such as flocked cotton fibers, but are not necessarily limited to cotton fibers as long as they are excellent in collecting and culturing lactic acid bacteria. The shape of the cotton ball can be spherical, elliptical, or the like, but may also be special shapes such as spiral, as long as it is easy to wipe the target area.

[0023] The size of the cotton ball is not particularly limited, as long as it is large enough to capture the target bacteria on the surface of the cotton ball by 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 can be 3 to 15 mm, with 5 to 13 mm being preferred, 6 to 10 mm being more preferred, and 7 to 9 mm being even more preferred.

[0024] Examples of the color pH indicator include bromocresol purple and phenol red, and bromocresol purple is preferred because the color change from purple to yellow can be clearly distinguished visually and has good visibility. Phenol red is red in neutral conditions and yellow in acidic conditions, but it may be difficult to distinguish the color change from red to yellow visually.

[0025] Step (b) is not particularly limited as long as it involves opening the top edge of the swab housing compartment in the lactic acid bacteria detection kit and removing the swab from the compartment. The top edge of the swab housing compartment must be sealed with a seal strong enough to withstand normal external forces to prevent leakage or damage to the culture medium during storage, transportation, etc. However, it is preferable that the seal on the top edge of the swab housing compartment be easily peeled when the kit is in use. Therefore, the top edge of the swab housing compartment is preferably weakly adhered with a weak seal or the like, such that the inner surfaces of the sheets can be easily peeled apart, so that the swab can be removed by pulling one or both of the sheets around the top edge of the swab housing compartment with the fingers. Alternatively, a known opening method, such as a small notch at the opening that allows the top edge to be easily opened by simply tearing the notch, can also be used.

[0026] The weak seal strength that allows peeling by pulling with the fingers can be 10% to 90% of the strength of the peripheral seal portion, preferably 20% to 80%, and more preferably 30% to 60%.

[0027] The step (c) is not particularly limited as long as it is a step of conducting a swab test with the cotton ball portion of the removed cotton swab, and the object on which the swab test is conducted is not particularly limited as long as it is a place that may be contaminated with lactic acid bacteria, and examples include the surface of food such as processed meat products such as ham and bacon, and the surfaces of various parts of food manufacturing equipment and food containers.

[0028] The swab test can be performed with the cotton ball of the swab removed from the swab storage unit in a dry or moistened state. If the object to be wiped with the cotton ball is moist, such as the surface of processed meat products such as ham or bacon, the swab test can be performed with a dry cotton ball. If the object to be wiped with the cotton ball is a dry metal surface of food manufacturing equipment, for example, it is preferable to moisten the cotton ball with water or physiological saline before performing the swab test.

[0029] The step (d) is not particularly limited as long as it is a step in which the cotton swab used in the wipe test is inserted from the cotton swab storage section through the partition wall, thereby bringing the color pH indicator-containing liquid medium into contact with the cotton ball section, and the liquid medium is sucked up into the surface and interior of the cotton ball section by capillary action.The seal strength of the partition wall when the cotton swab is inserted from the cotton swab storage section through the partition wall is preferably weak enough to be peeled off by pressure, so that the cotton ball section can peel off the partition wall by pressing the cotton swab with fingers directly or through a sheet.

[0030] The weak seal strength that allows peeling by pressing can be 10% to 90% of the strength of the peripheral seal portion, preferably 20% to 80%, and more preferably 30% to 60%.

[0031] The volume of liquid medium contained in the medium container is preferably an amount that allows lactic acid bacteria to grow in a solid medium using the fibers that form the cotton ball as a support. Examples of such an amount include an amount that wets the cotton ball 50% to 145%, 52% to 130%, preferably 55% to 125%, more preferably 60% to 120%, and most preferably 70% to 115%. Here, "100% wetness of the cotton ball with the liquid medium" refers to a state in which the cotton ball is immersed in liquid medium and the liquid medium does not drip from the cotton ball when the cotton ball is lifted up. "50%" refers to 50% of the volume of the liquid medium when 100% wet. Furthermore, if the concentration is 150% or more, the cotton ball will be immersed in the liquid culture medium, and if lactic acid bacteria are not growing sufficiently in the liquid culture medium that is not absorbed into the cotton ball, the color of the liquid culture medium may make it difficult to visually recognize the color change in the cotton ball.

[0032] For example, when the diameter of the cotton ball is 8 mm, the amount of liquid medium previously stored in the medium storage section is preferably 0.3 to 0.7 mL.

[0033] The liquid medium used in the present invention is not particularly limited as long as it allows the growth of lactic acid bacteria wiped with a cotton ball. However, in light of the objective of the present invention, which is to detect lactic acid bacteria, it is preferable to focus on the growth-promoting effect of glucose, which is often used as a nutrient source in medium components, and to set its concentration at 15 to 25 g / L to improve the growth and detection effect of lactic acid bacteria. It is also preferable to further add a carbon source such as citric acid, a nitrogen source such as tryptone peptone or yeast extract, minerals such as sodium, magnesium, manganese, and iron, and vitamins such as thiamine. An example of the composition of the liquid medium includes 10 to 15 g / L of tryptone peptone, 5 to 10 g / L of yeast extract, 15 to 25 g / L of glucose, 2 to 8 g / L of sodium chloride, 2 to 8 g / L of trisodium citrate dihydrate, 0.5 to 1.1 g / L of magnesium sulfate, 0.1 to 0.2 g / L of manganese chloride, 0.01 to 0.08 g / L of ferrous sulfate (II), 0.0005 to 0.0015 g / L of thiamine hydrochloride, 0.2 to 0.9 g / L of agar, 0.06 to 0.09 g / L of bromocresol purple, and 0.1 to 0.3 g / L of Tween 80.

[0034] The liquid medium can also be made semi-fluid by adding a gelling agent such as agar. Semi-fluid media exhibit fluidity when subjected to external physical forces such as stirring or shaking, but exhibit less convection than liquid media when left stationary. In the present invention, a gelling agent can be added to the liquid medium so that the cotton ball at the tip of the cotton swab can maintain a fluidity sufficient to easily absorb the liquid medium when in contact with the liquid medium, and to provide a viscosity sufficient to retain the absorbed liquid medium. Agar can be added at a concentration of 0.025-0.2%, preferably 0.05-0.15%, necessary to maintain a semi-fluid medium at typical culture temperatures of 20-40°C. However, this concentration is the agar concentration required to maintain a semi-fluid state at typical culture temperatures of approximately 20-40°C. It is preferable to use a smaller amount of agar at temperatures below this range, and a larger amount at higher temperatures.

[0035] The semi-fluid state can also be achieved by using a gelling agent other than agar. In addition to agar, components that provide gel strength include gelatin, silica gel, acrylamide, glucomannan, methylcellulose, gellan gum, gum arabic, starch, sodium alginate, carrageenan, bentonite, alginate, collagen, fused silica, water-soluble starch, polyacrylate, cellulose, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, dextran, polysaccharides, etc. These gelling agents can be used in place of agar or in combination with agar.

[0036] The liquid medium may have a reduced or eliminated buffering capacity compared to ordinary culture media. Generally, in many culture media, including those for lactic acid bacteria, buffering capacity is maintained by adding a phosphate buffer, a citrate buffer, an acetate buffer, a lactate buffer, a tartrate buffer, a malate buffer, a Tris buffer, a MOPS buffer, or an MES buffer to reduce damage to bacterial cells caused by acid, a metabolic product of lactic acid bacteria, and to promote growth and protect the grown bacterial cells. However, since the purpose of the present invention is to determine whether or not foods and beverages or food production processes are contaminated with lactic acid bacteria, it is not necessarily necessary to use a buffer such as dipotassium hydrogen phosphate that is commonly used in ordinary culture media in order to quickly and efficiently determine whether or not lactic acid bacteria are growing.

[0037] The step (e) is a culture step in which lactic acid bacteria are grown on the surface and inside of the cotton ball that is not immersed in the liquid medium, and this step is a major feature of the present invention. This culture step can be carried out, for example, at 24 to 35°C, and preferably at 25 to 32°C.

[0038] The viable cell count of lactic acid bacteria that can be detected by the method of the present invention is 1.0 × 10 2 CFU or more, preferably 1.0 × 10 3 CFU or more.

[0039] After step (e), step (g) can be included, in which the cotton swab lowering limit indicator, located at the upper end of the cotton swab, is heat-sealed. Lactic acid bacteria are anaerobic bacteria that do not require oxygen for growth. Anaerobic bacteria can be further classified into facultative anaerobes, which can grow in the presence of oxygen, and obligate anaerobes, which die when exposed to atmospheric levels of oxygen. Sealing at the cotton swab lowering limit indicator provides an oxygen-free environment for the solid medium comprising the cotton ball, providing an environment suitable for the growth and proliferation of both facultative anaerobic lactic acid bacteria and obligate anaerobic lactic acid bacteria. The method preferably includes step (g) for sealing the cotton swab lowering limit indicator. Heat sealing is a suitable method for sealing the cotton swab lowering limit indicator, but known adhesive means such as pre-installed double-sided tape may also be used. It is desirable to remove air from the film sheet formed body when sealing the cotton swab lowering limit indicator.

[0040] The above step (f) is not particularly limited as long as it is a step of detecting the presence or absence of lactic acid bacteria based on the presence or absence of a color change on the surface of the cotton ball. An example of the color change is that when the color pH indicator is the above bromocresol purple, the liquid medium is neutral at the start of cultivation and therefore appears purple; if lactic acid bacteria are not present in the subject subjected to the swab test, the cotton ball will remain purple even after 24 hours have passed; however, if lactic acid bacteria are present, the solid medium, which is supported by the fibers that make up the cotton ball, will turn yellow when the pH becomes less than 5.2 due to the proliferation of lactic acid bacteria.

[0041] In the present invention, it is preferable that the above steps (d) to (f) can be performed within 24 hours. Generally, lactic acid bacteria are cultured at 30°C for 24 to 48 hours, and Bifidobacteria (bifidobacteria) are cultured at 37°C for 72 hours (Food Sanitation Inspection Guidelines: Microbiology, edited by the Ministry of Health, Labour and Welfare, 2004). The lactic acid bacteria detection method of the present invention is an excellent method in that it can determine the presence or absence of lactic acid bacteria within 24 hours after contacting a cotton swab used to wipe the target area with a liquid medium.

[0042] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. [Example]

[0043] [Example 1] [Consideration of the volume of liquid medium for detecting lactic acid bacteria] (Preparation of liquid medium for detecting lactic acid bacteria) A total of 51.991 g of the ingredients for the LA medium for lactic acid bacteria detection (Prima Meat Packers Co., Ltd.) shown in Table 1 was weighed into an Erlenmeyer flask and dissolved in pure water to prepare 1 L of medium solution. This medium solution was sterilized by high-pressure steam at 121°C for 15 minutes to prepare a liquid medium for lactic acid bacteria detection. The liquid medium for lactic acid bacteria detection was dispensed into five transparent plastic tubes with lids (ThermoScientific: SAMCO) at volumes of 10 mL, 7 mL, 4 mL, 2 mL, and 0.5 mL each. LA medium is characterized by its color changing from purple to yellow when lactic acid bacteria grow.

[0044] [Table 1]

[0045] The pre-culture solution of lactic acid bacteria (Enterococcus faecalis NBRC100480 (hereinafter simply referred to as "faecalis bacteria") was diluted with saline to a concentration of 5.0 × 10 6 CFU / mL, 5.0 × 10 5 CFU / mL, 5.0 × 10 4 CFU / mL, 5.0 × 10 3 The solutions were prepared to have a concentration of 1.0 × 10 CFU / mL and 0 CFU / mL, and 20 μL of each solution was dropped onto the tip of the cotton ball with a micropipette. 5 CFU, 1.0 × 10 4 CFU, 1.0 × 10 3 CFU, 1.0 × 10 2 Five sets of cotton swabs were prepared, each with the above lactic acid bacteria inoculated onto the cotton ball portion to give five different concentrations: 0 CFU, 0 CFU, and 0 CFU.

[0046] Twenty-five samples were prepared by adding one cotton swab containing a cotton ball inoculated with the five concentrations of lactic acid bacteria to a plastic tube containing one of the five volumes of liquid culture medium for lactic acid bacteria detection. Each of the 25 samples was capped and stored in an incubator at 25°C, and the color change was visually observed after 16 hours. The results are shown in Figure 1.

[0047] The CFU stands for Colony Forming Unit, which represents the number of reproducible microorganisms (cells) contained in a microorganism. The cotton swab used was a cotton swab with a plastic shaft length of 90 mm, a vertical length of 15 mm, and a diameter of 8 mm at the widest part of the cotton swab.

[0048] (Visual inspection criteria) The visual confirmation criteria for detecting lactic acid bacteria were as follows: ++ = Medium turns yellow overall + = About half of the medium has turned yellow +w = Part of the medium has turned yellow ± = A small portion of the medium turned yellow - = No change in color of the medium

[0049] (result) As is clear from Figure 1, when the liquid medium volume was 10 mL (Figure 1(a)), 7 mL (Figure 1(b)), and 4 mL (Figure 1(c)), the fertility was 1.0 × 10 5 CFU, and 1.0 × 10 4 Although the cotton ball was confirmed to have a yellowish tint when measuring CFU, only a small portion of the liquid medium changed from purple to yellow. 5 CFU, 1.0 × 10 4 CFU, and 1.0 × 10 3Although a yellowish tint was observed at the CFU level, only a portion of the liquid medium changed from purple to yellow. Therefore, as the amount of liquid medium increased, it became more difficult to determine the color change.

[0050] On the other hand, in the case of 0.5 mL in Figure 1(e), it is 1.0 × 10 5 CFU, 1.0 × 10 4 CFU, and 1.0 × 10 3 When the CFU was measured, the liquid medium was absorbed into the cotton ball and was barely present outside the cotton ball. Therefore, it was clearly confirmed that the entire cotton ball had changed from purple to yellow, indicating the presence of lactic acid bacteria. In addition, when the inoculation amount of lactic acid bacteria was small, 1.0 × 10 3 The presence of lactic acid bacteria was confirmed by CFU, and one of the reasons for this is thought to be that the liquid medium spread throughout the entire cotton ball, and the cotton ball functioned as a solid medium, using the cotton ball as a support, allowing lactic acid bacteria to grow efficiently in the cotton ball.

[0051] As mentioned above, when the volume of liquid medium in the plastic tube is 0.5 mL, the cotton ball turns yellow, which can be clearly seen, and it was confirmed that the presence or absence of lactic acid bacteria can be clearly determined.

[0052] [Example 2] [Consideration of container shape for culture medium suitable for swab testing] (Preliminary consideration) To provide a lactic acid bacteria detection medium as part of a practical swab test kit for detecting lactic acid bacteria, it would be desirable to place 0.5 mL of sterilized liquid medium in a container so that testing could be performed simply by adding a swabbed cotton swab. Assuming the container used to place the swab is a plastic tube or test tube, a container with a height of at least 100 mm and a diameter of at least 15 mm would be required. If the number of test samples is large, a storage location for the samples and a considerable number of incubators with temperature settings would be required. Furthermore, placing only a small amount of medium (0.5 mL) in a test tube or similar would pose the risk of leakage of the liquid medium due to damage during transport, making stable transport difficult.

[0053] However, since it was thought that the packaging form might affect the growth of lactic acid bacteria, we decided to create a kit equipped with a film sheet formed from flexible film, as shown in Figure 2, which can store a liquid culture medium for lactic acid bacteria detection in a bag-like portion, and compare it with a plastic tube.

[0054] (Lactic acid bacteria detection kit) The bottom of the bag-shaped film sheet was filled with 0.5 mL of liquid medium for detecting lactic acid bacteria to form a medium storage section, and the top of the medium storage section was heat-sealed using a heat sealer to form a partition wall to prevent the liquid medium from splashing or leaking. A cotton swab for wiping was inserted into the top of the partition wall, and the cotton ball of the swab was positioned so that it abutted against the heat-sealed partition wall at the top of the medium storage section, thereby producing a lactic acid bacteria detection kit in which the liquid medium and the cotton swab were integrated.

[0055] (Preparation of liquid medium for detecting lactic acid bacteria) The liquid medium for detecting lactic acid bacteria prepared in Example 1 was dispensed into five transparent plastic tubes with lids and five film sheet bodies at a volume of 0.5 mL each. The pre-culture solution of Lactobacillus faecalis was diluted with physiological saline to give a concentration of 5.0 × 10 6 CFU / mL, 5.0 × 10 5 CFU / mL, 5.0 × 10 4 CFU / mL, 5.0 × 10 3 The solutions were prepared so that the bacterial counts were 1.0 × 10 CFU / mL and 0 CFU / mL, and 20 μL of each solution was dropped onto the tip of the cotton swab with a micropipette. 5 CFU, 1.0 × 10 4 CFU, 1.0 × 10 3 CFU, 1.0 × 10 2 Two sets of cotton swabs were prepared, each with the lactic acid bacteria inoculated into the cotton ball to achieve five different concentrations: 0 CFU, 1 CFU, and 0 CFU. The same cotton swabs as those used in Example 1 were used.

[0056] The cotton swabs containing the cotton balls inoculated with the above five concentrations of lactic acid bacteria were placed one by one in a plastic tube and the lid was closed. The cotton swabs containing the cotton balls inoculated with the above five concentrations of lactic acid bacteria were then inserted one by one through the partition of the film sheet formed body and the top was clamped with a clip to prepare 10 types of samples. The above 10 types of samples were each stored in an incubator at 25°C, and color change was visually confirmed after 16 hours. Visual determination of lactic acid bacteria detection was performed as in Example 1. The results are shown in Figure 3.

[0057] (result) As is clear from Figure 3(a), the plastic tube has a melting point of 1.0 × 10 5 CFU ~ 1.0 x 10 3 In the case of CFU, lactic acid bacteria could be detected by visual inspection. On the other hand, as is clear from Figure 3(b), in the film sheet formed body, 1.0 × 10 5 CFU ~ 1.0 x 10 2 Therefore, it was confirmed that the film sheet formed product has a higher sensitivity for detecting lactic acid bacteria by visual inspection than the plastic tube.

[0058] (Consideration) Many lactic acid bacteria are facultative anaerobes and are known to grow under low-oxygen conditions, such as those found in nitrogen-gas-substituted packaging or vacuum packaging. Therefore, the difference in the results above is likely due to the fact that, in the case of the plastic tube, as shown in Figure 4(a), there is a large amount of air around the cotton ball, meaning that the cotton ball containing the liquid medium comes into contact with the air in many places. In contrast, in the case of the film sheet-formed container, as shown in Figure 4(b), there is a small amount of air around the cotton ball, meaning that the cotton ball containing the culture medium comes into contact with the film in many places, meaning that it does not come into contact with the air, creating an environment conducive to the growth of lactic acid bacteria. Based on these findings, we decided to use a kit containing a liquid medium filled in a film sheet-formed container and conducted the following study.

[0059] [Example 3] [Consideration of the volume of liquid culture medium suitable for swab testing to detect lactobacilli] Although the results of Example 1 confirmed that lactic acid bacteria could be detected visually when the volume of the liquid medium was 0.5 mL, we decided to confirm the appropriate range of the liquid medium when using a film sheet formed body. The liquid medium prepared in Example 1 was filled into each film sheet formed body in eight different volumes: 0.05 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, and 0.7 mL.

[0060] The pre-culture solution of Lactobacillus faecalis was diluted with saline to 5.0 × 10 6 CFU / mL, 5.0 × 10 5 CFU / mL, 5.0 × 10 4 CFU / mL, 5.0 × 10 3 The solutions were prepared so that the bacterial counts were 1.0 × 10 CFU / mL and 0 CFU / mL, and 20 μL of each solution was dropped onto the tip of the cotton swab with a micropipette. 5 CFU, 1.0 × 10 4 CFU, 1.0 × 10 3 CFU, 1.0 × 10 2 Eight sets of cotton swabs were prepared, each with the lactic acid bacteria inoculated into the cotton ball to achieve five different concentrations: 0 CFU, 0 CFU, and 0 CFU.

[0061] A cotton swab containing a cotton ball inoculated with the above five concentrations of Lactobacillus faecalis was inserted through the partition of a film sheet containing the above eight volumes of liquid culture medium for lactic acid bacteria detection, and the top was clamped with a clip to prepare 40 types of samples. Each of the 40 types of samples was stored in an incubator at 25°C, and color change was visually confirmed after 16 hours. The results are shown in Figures 5(a) to 5(h).

[0062] The visual evaluation criteria were as follows: ++ = The cotton ball has turned yellow overall + = About half of the cotton ball has turned yellow +w = Part of the cotton ball has turned yellow ± = A small portion of the cotton ball turned yellow - = No change in color of the medium contained in the cotton ball

[0063] (result) As is clear from the results in Figures 5(a)-(h), visual inspection confirmed a yellow color for all medium volumes, even with a very small amount of medium (0.05 mL). However, to detect lactic acid bacteria, the culture medium must come into contact with the surface wiped with the cotton swab. If the amount of culture medium is very small, the culture medium may not come into contact with the surface, resulting in a false negative. To prevent false negatives (oversights), we considered it desirable for the swab's swab to contain more than half the culture medium. Therefore, we determined that the ideal culture medium volume was 0.3 mL to 0.7 mL, which covers more than half of the swab's surface. Ten cotton swabs with a diameter of 8 mm at the widest point were immersed in pure water for 5 seconds and then lifted. The increase in volume of the swab without dripping liquid medium from the swab ranged from 0.4951 to 0.5849 mL, with an average of 0.544 mL. Therefore, applying the above 0.3 mL to 0.7 mL range, the desired range for the liquid medium volume was confirmed to be 51% to 141% of the volume of the swab used. In particular, the 0.5 mL level, which most frequently resulted in a visual ++ result, contained all of the culture medium, turning the entire swab yellow and facilitating evaluation. Based on these results, we decided to use 0.5 mL of liquid medium for lactic acid bacteria detection in the film sheet-formed swab test container for subsequent studies.

[0064] [Example 4] [Concentration of culture medium suitable for swab testing to detect lactobacilli] The following test was carried out to confirm the range of liquid medium concentrations suitable for swabbing tests using a cotton swab. As shown in Table 2, the liquid medium prepared in Example 1 was used as the concentration of the basal medium (1x), and 1L of each of nine media compositions was prepared at concentrations of 0.1x, 0.25x, 0.5x, 0.75x, 1x (basal medium composition), 1.25x, 1.5x, 2x, and 3x the concentration of the basal medium. The reagent formulation per 1L is shown in Table 2 below. Each test medium was prepared by high-pressure steam sterilization at 121°C for 15 minutes, and 0.5mL was filled into a film sheet. The above-mentioned pre-culture solution of Bacillus faecalis was diluted with physiological saline to obtain 5.0 x 10 6 CFU / mL, 5.0 × 10 5 CFU / mL, 5.0 × 10 4 CFU / mL, 5.0 × 10 3 The solutions were prepared so that the bacterial counts were 1.0 × 10 CFU / mL and 0 CFU / mL, and 20 μL of each solution was dropped onto the tip of the cotton swab with a micropipette. 5 CFU, 1.0 × 10 4 CFU, 1.0 × 10 3 CFU, 1.0 × 10 2 Nine sets of cotton swabs were prepared, each containing five different concentrations of the lactic acid bacteria inoculated into the cotton balls: 0 CFU, 1 CFU, and 0 CFU. The same cotton swabs as those used in Example 1 were used.

[0065] A cotton swab containing a cotton ball inoculated with the above five concentrations of lactic acid bacteria was inserted through the partition of a film sheet formed body containing nine concentrations of liquid culture medium, and the top was clamped with a clip to prepare 45 types of samples. Each of the above 45 types of samples was stored in an incubator at 25°C, and after 16 hours, color change was visually confirmed. The visual judgment criteria for detecting lactic acid bacteria were the same as in Example 3.

[0066] [Table 2]

[0067] (result) The results are shown in Figure 6(a) to (i). From the visual observation results, the concentration range from (a) 0.1 times to (g) 1.5 times was 1.0 × 10 5 ~1.0×10 2 Although it was possible to detect CFU, the concentrations that provided the highest visibility and allowed for easy judgment were (c) 0.5x, (d) 0.75x, and (e) 1x. On the other hand, in the liquid medium with (h) 2x or (i) 3x concentrations, only a small portion turned yellow when a large amount of lactic acid bacteria was present, and as the number of bacteria decreased, they became undetectable. Based on this, the concentration of the lactic acid bacteria detection liquid medium in the film sheet forming body for swab testing was set to the concentration of the basic liquid medium composition, and a film sheet forming body filled with 0.5 mL of the lactic acid bacteria detection medium with the above basic medium composition was used as a lactic acid bacteria detection kit for the following studies.

[0068] [Example 5] [Swab test using a lactic acid bacteria detection kit] A test was conducted simulating actual swabbing tests at food manufacturing sites in food factories. Stainless steel plates were used to simulate the equipment used in food factories. Since swabbing tests are also conducted on the products themselves at food factories, one type of ham and one type of sausage were obtained from Company A and one type of sausage from Company B, and a total of four types of processed meat products were subjected to the swabbing test.

[0069] A 0.5 mL portion of the medium containing the above-mentioned basic medium composition, which had been sterilized by high-pressure steam at 121°C for 15 minutes, was filled into the medium storage section of the film sheet formed body, and a lactic acid bacteria detection kit equipped with a sterilized cotton swab was prepared. The pre-culture solution of Lactobacillus faecalis was appropriately diluted with physiological saline to prepare 5.0 x 10 6 CFU / mL, 5.0 × 10 5 CFU / mL, 5.0 × 10 4 CFU / mL, 5.0 × 10 3 The culture medium prepared for each of the above bacterial counts was spotted at four locations (20 μL in total) with a micropipette on the surface of the stainless steel plate, ham, or sausage. In other words, the bacterial count per surface of the stainless steel plate, ham, or sausage was 1.0 × 10 5 CFU, 1.0 × 104 CFU, 1.0 × 10 3 CFU, 1.0 × 10 2 The samples were inoculated to obtain 0 CFU and 0 CFU. Using a cotton swab removed from the pouch of the lactic acid bacteria detection kit, the entire surface of the cotton ball was rubbed against the surface of the stainless steel plate, ham, or sausage on which the lactic acid bacteria had been dropped. Each of the cotton swabs was inserted into the medium-receiving portion of the film sheet formed body and stored in an incubator at 25°C. The condition of the cotton ball was checked after 17 hours and 24 hours. The results are shown in Figures 7 to 11.

[0070] (result) The incubation time was 17 hours, and the results for the stainless steel plate (Fig. 7(a)), the ham from Company A (Fig. 8(a)), the ham from Company B (Fig. 9(a)), the sausage from Company A (Fig. 10(a)), and the sausage from Company B (Fig. 11(a)) all showed a 1.0 × 10 2 The result of the CFU determination was ±. On the other hand, the results for the stainless steel plate in Figure 7(b), Company A's ham in Figure 8(b), Company B's ham in Figure 9(b), Company A's sausage in Figure 10(b), and Company B's sausage in Figure 11(b), which were cultured for 24 hours, were (+w) or (+), meaning that the results were clearer for the 24-hour culture. Furthermore, since the results for the stainless steel plate, ham, and sausage were similar, it was confirmed that lactic acid bacteria can be detected with the same sensitivity whether wiping the metal plate used in the device or wiping the surface of food.

[0071] [Example 6] [Study on detection of various lactic acid bacteria using lactic acid bacteria detection kits] A 0.5 mL portion of the medium containing the above-mentioned basic medium composition, which had been sterilized by high-pressure steam at 121°C for 15 minutes, was filled into the medium containing portion of the film sheet formed body, and a cotton swab was placed in the cotton swab containing portion to prepare a lactic acid bacteria detection kit. The preculture solutions of the four types of lactic acid bacteria shown below were appropriately diluted with physiological saline, and 5.0 x 10 6 CFU / mL, 5.0 × 10 5 CFU / mL, 5.0 × 10 4 CFU / mL, 5.0 × 10 3The solutions were prepared so that the bacterial count was 1.0 × 10 CFU / mL and 0 CFU / mL. 20 μL of each solution was dropped onto the tip of the cotton swab with a micropipette. 5 CFU, 1.0 × 10 4 CFU, 1.0 × 10 3 CFU, 1.0 × 10 2 The inoculated cotton swab was inserted into the medium storage portion of the film sheet formed body and stored in an incubator at 25°C. The condition of the cotton swab was checked after 17 hours and 24 hours. The results are shown in Figures 12 to 15.

[0072] (Various lactic acid bacteria) (1) Lactobacillus sakei NBRC107868 (2) Leuconostoc mesenteroides NBRC100496 (3) Lactococcus lactis NBRC100933 (4) Pediococcus acidilacti NBRC3076

[0073] (result) As is clear from Figures 12 to 15, the visual evaluation results showed that the number of lactic acid bacteria in all four test groups was 1 × 10 2 It was confirmed that the medium turned yellow up to 1 × 10 CFU / mL. It was clear that all types of lactic acid bacteria could be detected after 24 hours. On the other hand, at 17 hours, Lactococcus lactis and Pediococcus acidilactici were detected at 1 × 10 CFU / mL. 2 The result of CFU / mL was (-). [Industrial Applicability]

[0074] The present invention is particularly useful in the food manufacturing field.

Claims

1. A method for detecting lactic acid bacteria, comprising the following steps (a) to (f) in order: (a) preparing a lactic acid bacteria detection kit, in which a color pH indicator-containing liquid medium capable of detecting lactic acid bacteria is accommodated in a medium accommodating section of a film sheet formed body in which a swab accommodating section and a medium accommodating section are connected via a partition; (b) opening the top edge of the swab housing portion and removing the swab from the swab housing portion; (c) performing a swab test using the cotton ball portion of the removed cotton swab; (d) inserting the cotton swab after the swabbing test from the cotton swab storage section through the partition wall, bringing the color pH indicator-containing liquid medium into contact with the cotton ball, and sucking the liquid medium into the surface and interior of the cotton ball by capillary action; (e) a culturing step of growing lactic acid bacteria on the surface and inside of the cotton ball that is not immersed in the liquid medium; (f) detecting the presence or absence of lactic acid bacteria based on the presence or absence of a color change on the surface of the cotton ball;

2. 2. The method for detecting lactic acid bacteria according to claim 1, wherein steps (d) to (f) can be carried out within 24 hours.

3. 2. The method for detecting lactic acid bacteria according to claim 1, wherein the culturing step (e) is carried out at 24 to 35°C.

4. 2. The method for detecting lactic acid bacteria according to claim 1, wherein the color pH indicator is bromocresol purple.

5. 2. The method for detecting lactic acid bacteria according to claim 1, wherein the swab test is performed using a dry cotton swab or a cotton swab moistened with water or physiological saline.

6. 2. The method for detecting lactic acid bacteria according to claim 1, further comprising the step (g) of heat-sealing the cotton swab at the upper end of the cotton swab, which indicates the lowering limit of the cotton swab, after the culturing step (e).

7. A lactic acid bacteria detection kit used in the lactic acid bacteria detection method according to any one of claims 1 to 6, wherein a cotton swab storage section and a culture medium storage section are connected in a rectangular shape via a partition section.

8. 8. The lactic acid bacteria detection kit according to claim 7, wherein the swab storage section is provided with a swab lowering limit indicator.

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