WO2021005813A1

WO2021005813A1 - Culture medium for selectively separating lactic acid bacterium

Info

Publication number: WO2021005813A1
Application number: PCT/JP2019/050396
Authority: WO
Inventors: 寺村　哉; 彩小椋; 翠藤原
Original assignee: Jnc株式会社
Priority date: 2019-07-05
Filing date: 2019-12-23
Publication date: 2021-01-14
Also published as: JP2021010322A

Abstract

Heretofore, for the separation and detection of lactic acid bacteria, a BCP-supplemented plate count agar culture medium, an MRS agar culture medium, an APT agar culture medium and the like have been used. These culture media have poor selectivity for lactic acid bacteria. Therefore, when the culture media are used in specimens not containing bacteria other than lactic acid bacteria in the testing on the number of cells of lactic acid bacteria in a fermented food, any particular problem does not occur. However, when the culture media are used in specimens also containing bacteria other than lactic acid bacteria, the bacteria other than lactic acid bacteria also grow along with the lactic acid bacteria, and it is sometimes impossible to distinguish between the lactic acid bacteria and the bacteria other than the lactic acid bacteria. In this case, the accurate detection of the lactic acid bacteria may become difficult and the number of cells of the lactic acid bacteria may not be counted accurately. In these situations, the present invention provides a culture medium for selectively separating a lactic acid bacterium, which can be prepared in a simple manner and can exclude bacteria other than lactic acid bacteria. A combination of an inorganic metal salt of an organic acid and glycine is added to an existing lactic acid bacterium culture medium such as an MRS agar culture medium. By using the culture medium, it becomes possible to culture only a lactic acid bacterium selectively while excluding bacteria other than the lactic acid bacterium.

Description

Lactic acid bacteria selective separation medium

The present invention relates to a lactic acid bacterium selective separation medium having good selectivity and a method for producing the same.

Lactic acid bacteria are gram-positive bacilli or cocci that produce energy by metabolism of sugars and produce lactic acid in the metabolic process. They are catalase-negative, do not form endoplasmic spores, and consume 50% or more of glucose consumed. Bacteria that convert to lactic acid. Examples of lactic acid bacterium rods include the genus Lactobacillus, and examples of lactic acid bacteria include the genus Lactococcus, the genus Leuconostoc, and the genus Streptococcus. Lactic acid bacteria have traditionally been used as useful bacteria in the production and processing of fermented foods, but on the other hand, they are also known to cause spoilage and putrefaction such as net production, gas generation, and discoloration. (Non-Patent Document 1).

It is extremely rare for lactic acid bacteria to have an adverse effect on human health, but in the food manufacturing industry, the occurrence of food deterioration by lactic acid bacteria is a major problem in quality control, and such quality control The economic loss in the event of the above problems is immeasurable. Therefore, it is extremely important in the food manufacturing industry to control quality control problems such as deterioration caused by lactic acid bacteria.

BCP (Bromocresol Purple) -added plate count agar medium, MRS agar medium, APT agar medium, etc. have been conventionally used for the isolation or detection of lactic acid bacteria (Non-Patent Documents 1 and 2).

However, these media that have been used conventionally have a problem of low selectivity for lactic acid bacteria. That is, there is no particular problem when using these media in a sample in which bacteria other than lactic acid bacteria do not exist, such as a test for the number of lactic acid bacteria in fermented foods, but when these media are used in a sample containing bacteria other than lactic acid bacteria. In some cases, bacteria other than lactic acid bacteria grew in the same manner as lactic acid bacteria, and it was not possible to distinguish between lactic acid bacteria and bacteria other than lactic acid bacteria. As described above, when the sample contains bacteria other than lactic acid bacteria, it is difficult to accurately detect the lactic acid bacteria, so that the accurate number of lactic acid bacteria cannot be measured.

There are many existing improved lactic acid bacteria media, and all of them can sufficiently grow lactic acid bacteria. However, the selectivity of lactic acid bacteria is low. This is because the lactic acid bacterium selectivity of such an existing improved lactic acid bacterium medium often depends only on the low pH (Patent Document 1), and the growth of bacteria other than lactic acid bacteria cannot be reliably suppressed. Is. Further, as one of the selective substances, there is also an existing lactic acid bacterium medium containing an azide of an alkali metal or an alkaline earth metal as an essential component (Patent Document 2).

Japanese Unexamined Patent Publication No. 2011-000045 Japanese Unexamined Patent Publication No. 2006-081536

That is, a selective separation medium having better selectivity for the measurement of lactic acid bacteria was required for accurate measurement of the number of lactic acid bacteria. In view of such a situation, the present invention can be easily prepared.
Another object of the present invention is to provide a lactic acid bacterium selective separation medium capable of eliminating bacteria other than lactic acid bacteria.

After diligent research to solve the above problems, the present inventors eliminated bacteria other than lactic acid bacteria by adding an inorganic metal salt of an organic acid and glycine in combination to an existing lactic acid bacterium medium such as MRS agar medium. While doing so, he found that only lactic acid bacteria could be selectively cultured, and completed the present invention.
That is, the present invention is as follows.
[1] A lactic acid bacterium selective separation medium having a pH of 5.5 to 7.0 and containing peptone, yeast extract, glucose, polysorbate 80, an inorganic metal salt of an organic acid and glycine.
When the inorganic metal salt of the organic acid is sodium acetate, the concentration of sodium acetate is 5.1 to 15 g / L.
When the inorganic metal salt of the organic acid is an inorganic metal salt of an organic acid other than sodium acetate, the concentration of the inorganic metal salt of the organic acid other than sodium acetate is 0.1 to 10 g / L, and the alkali metal or alkaline soil. The medium, which is characterized by not containing an azide of a similar metal. [2] The lactic acid bacterium selective separation medium according to [1], wherein the inorganic metal salt of the organic acid is an inorganic metal salt of at least one organic acid selected from the group consisting of sodium acetate and sodium propionate.
[3] The lactic acid bacterium isolation medium according to [1] or [2], wherein the concentration of the glycine is 0.1 to 10 g / L.
[4] The lactic acid bacterium selective separation medium according to any one of [1] to [3], which further contains a polypeptide antibiotic.
[5] The lactic acid bacterium selective isolation medium according to [4], wherein the polypeptide antibiotic is at least one antibiotic selected from the group consisting of polymyxin B and colistin.
[6] The lactic acid bacterium selective separation medium according to any one of [1] to [5], which further contains L-cysteine hydrochloride.
[7] The lactic acid bacterium selective isolation medium according to any one of [1] to [6], wherein the lactic acid bacterium isolation medium contains a medium component of an MRS agar medium.
[8] A step of adjusting the pH to 5.5 to 7.0, including a step of mixing the components of the lactic acid bacterium selective separation medium according to any one of [1] to [7] in water to prepare a mixed solution. A method for producing a selective isolation medium for lactic acid bacteria.
[9] The method for producing a lactic acid bacterium selective separation medium according to [8], which further comprises a step of solidifying the mixed solution.
[10] A step of inoculating a sample into the lactic acid bacterium selective separation medium according to any one of [1] to [7], a step of culturing the lactic acid bacteria contained in the sample on the medium, and detecting colonies of the lactic acid bacteria. Lactic acid bacteria detection method including steps.

The selective separation medium for lactic acid bacteria of the present invention suppresses the growth of bacteria other than lactic acid bacteria even when a sample containing bacteria other than lactic acid bacteria is used, so that accurate detection of lactic acid bacteria becomes possible and the number of lactic acid bacteria is accurate. Will be able to measure.

The lactic acid bacterium selective separation medium of the present invention is a medium having a pH of 5.5 to 7.0 and containing peptone, yeast extract, glucose, polysorbate 80, an inorganic metal salt of an organic acid and glycine, and is an alkali metal or alkaline earth. It is a medium characterized by containing no metal azide (hereinafter, may be simply referred to as azide).

Lactic acid bacteria can be grown by containing peptone, yeast extract, glucose, and polysorbate 80 as the minimum components in the medium component that is the basis of the medium of the present invention.
The medium component which is the basis of the medium of the present invention is not particularly limited as long as it is a medium capable of culturing lactic acid bacteria, and is, for example, a medium of MRS agar medium, BCP-added plate count agar medium, APT agar medium or BL agar medium. Ingredients can be used. From the viewpoint of high viability and availability of lactic acid bacteria, it is preferable to use a medium component of a commercially available MRS agar medium.

With the lactic acid bacterium selective separation medium of the present invention, lactic acid bacteria can be selectively separated from the sample.
The lactic acid bacteria that can be selectively separated by the medium of the present invention are gram-positive bacilli or bacilli that produce energy by metabolism of saccharides and produce lactic acid in the metabolic process, and are catalase-negative and produce endogenous spores. It is not particularly limited as long as it is a bacterium that does not form and converts 50% or more of consumed glucose into lactic acid, but for example, lactic acid bacteria belonging to the genus Lactobacillus, Lactococcus, Leuconostock or Streptococcus are preferable. Lactobacillus acidophilus, L. brevis, L. delbrueckii subsp. Lactis, L. delbrueckii subsp. Lactis, which belong to the genus Lactobacillus. . helveticus) or Lactobacillus delbrueckii subsp. Bulgaricus, Lactococcus lactis subsp. Cremoris, Lactococcus lactis subsp. Cremoris, Lactococcus lactis subsp. Leuconostoc mesenteroides subsp. Mesenteroides, which belongs to the genus, or Streptococcus thermophilus, which belongs to the genus Streptococcus, is particularly preferable.

The standard components and amounts of MRS agar medium are as follows.
Peptone: 10 g, meat extract: 10 g, yeast extract: 5 g, glucose: 20 g, polysorbate 80: 1 g, ammonium citrate: 2 g, sodium acetate: 5 g, magnesium sulfate (hepatohydrate): 0.1 g, manganese sulfate: 0.05 g, dipotassium hydrogen phosphate: 2 g, agar: 15 g, purified water: 1,000 mL.

The standard components and amounts of BCP-added plate count agar medium are as follows. Peptone: 5 g, yeast extract: 2.5 g, glucose: 1 g, polysorbate 80: 1 g, L-cysteine hydrochloride (monohydrate): 0.1 g, bromcresol purple (BCP): 0.04 g, agar: 15 g , Purified water: 1,000 mL.

The standard components and amounts of APT agar medium are as follows.
Yeast extract: 7.5 g, pancreatic digest of casein: 12.5 g, dextrose: 10 g, sodium citrate: 5 g, thiamine hydrochloride: 1 mg, sodium chloride: 5 g, dipotassium hydrogen phosphate: 5 g, magnesium chloride: 0 .14 g, magnesium sulfate: 0.8 g, ferrous sulfate: 0.04 g, polysorbate 80: 0.2 g, agar: 15 g, purified water: 1,000 mL.

The standard components and amounts of BL agar medium are as follows.
Meat extract: 2.4 g, Proteose peptone: 10 g, Peptone: 5 g, Soybean peptone: 3 g, Yeast extract: 5 g, Liver extract: 3.2 g, Glucose: 10 g, Soluble starch: 0.5 g, Potassium dihydrogen phosphate 1 g, potassium monohydrogen phosphate: 1 g, magnesium sulfate (7 hydrate): 0.2 g, ferrous sulfate (heptahydrate): 0.01 g, manganese sulfate: 0.007 g, antifoaming agent ( Silicon): 0.2 g, polysorbate 80: 1 g, L-cysteine hydrochloride (monohydrate): 0.5 g, agar: 15 g, purified water: 1,000 mL.

The MRS agar medium, BCP-added plate count agar medium, APT agar medium or BL agar medium may be a medium having standard composition components, or may contain other components and / or may not contain some of the standard components. It may be a modified medium. The amount of each component added to the medium may be appropriately changed.

The medium of the present invention can improve the viability and selectivity of lactic acid bacteria when the pH at the time of using the medium is 5.5 to 7.0.

The medium of the present invention has the following advantages because it does not contain azide.
When the azide is contained in the powder medium, if the content of the azide in the powder medium exceeds 0.1% by weight, it is designated as a poison and storage becomes complicated. On the other hand, since the medium of the present invention does not contain azide, it is not designated as a toxic substance and is easy to store.
In addition, when the medium contains azide, if the used medium is passed through a metal pipe at the time of disposal, metal azide is generated and there is a risk of explosion. On the other hand, since the medium of the present invention does not contain azide, there is no risk of metal azide being generated at the time of disposal, and the medium can be safely disposed of.

The lactic acid bacterium selective separation medium of the present invention contains an inorganic metal salt of an organic acid. The inorganic metal salt of the organic acid is not particularly limited. As the organic acid, for example, acetic acid and propionic acid can be used, and as the inorganic metal salt, for example, a sodium salt can be used.
The inorganic metal salt of the organic acid is, for example, sodium acetate or sodium propionate. As the inorganic metal salt of the organic acid, only one kind may be used, or a plurality of kinds may be used.
In particular, sodium acetate is preferably used because it has little effect on the growth of lactic acid bacteria and has high selectivity for Gram-positive bacteria, and the concentration of sodium acetate at the time of use is 5.1 to 15 g / L. Is preferable, 6 to 10 g / L is more preferable, and the concentration in the above range can further improve the accuracy of selection and isolation of lactic acid bacteria.
When the medium of the present invention is a medium containing the medium component of the MRS agar medium, sodium acetate is originally contained in the medium component of the MRS agar medium at a concentration of 5 g / L, so that it is added. The sodium acetate to be added may be added so as to have a total concentration of 5.1 to 15 g / L (more preferably 6 to 10 g / L) with the sodium acetate originally contained in the MRS agar medium.
The concentration of sodium propionate at the time of use is preferably 0.1 to 10 g / L, more preferably 1 to 5 g / L, and the concentration in the above range further enhances the accuracy of selection and isolation of lactic acid bacteria. be able to.

The lactic acid bacterium selective separation medium of the present invention contains glycine. Glycine is used to suppress the growth of gram-positive spore-forming bacteria, which are Bacillus bacteria, and the concentration of glycine at the time of use is preferably 0.1 to 10 g / L, more preferably 1 to 5 g / L, and is in the above range. The accuracy of selection and isolation of lactic acid bacteria can be further improved by the concentration of.

According to the present invention, lactic acid bacteria can be selected and isolated with high accuracy as a lactic acid bacterium selective separation medium by combining the above two types of lactic acid bacterium selective substances (inorganic metal salt of organic acid and glycine).

Furthermore, the lactic acid bacterium selective separation medium of the present invention may contain the following additional components in addition to the above two types of lactic acid bacterium selective substances (inorganic metal salt of organic acid and glycine).

The lactic acid bacterium selective separation medium of the present invention preferably further contains a polypeptide antibiotic. This is for improving the selectivity of lactic acid bacteria, specifically, for suppressing the growth of Gram-negative bacteria.
In the lactic acid bacterium selective separation medium of the present invention, the polypeptide antibiotic is not particularly limited, and is, for example, polymyxin B or colistin. As the polypeptide antibiotic, only one kind may be used, or a plurality of kinds may be used.
As the polypeptide antibiotic, it is preferable to use polymyxin B from the viewpoint of suppressing the growth of Gram-negative bacteria, and the concentration of polymyxin B or colistin at the time of use is preferably 0.1 to 10 mg / L. 1 to 5 mg / L is more preferable, and the concentration in the above range can further improve the accuracy of selection and isolation of lactic acid bacteria. As for polymyxin B, it can be converted to 0.129 μg per unit as standard.

The lactic acid bacterium selective separation medium of the present invention preferably further contains L-cysteine. This is for improving the growth of lactic acid bacteria, and is preferable for improving the growth of a wider range of lactic acid bacteria. The concentration at the time of use is preferably 0.01 to 5 g / L, more preferably 0.1 to 0.5 g / L.

The lactic acid bacterium selective separation medium of the present invention may further contain a reducing agent. Although not particularly limited, for example, ferrous sulfate, ascorbic acid or thioglycolic acid. As the reducing agent, only one kind may be used, or a plurality of kinds may be used.
This is for lowering the redox potential of the medium, and is preferable for improving the growth of a wider range of lactic acid bacteria even in aerobic culture. The concentration of ferrous sulfate, ascorbic acid or thioglycolic acid at the time of use is preferably 0.1 to 10 g / L, more preferably 0.5 to 5 g / L.

The lactic acid bacterium selective separation medium of the present invention may further contain an antifungal agent. This is for further improvement of selectivity. Although not particularly limited, for example, amphotericin B or cycloheximide. As the antifungal agent, only one kind may be used, or a plurality of kinds may be used.
The concentration of amphotericin B or sictoheximide at the time of use is preferably 0.1 to 10 mg / L, more preferably 0.5 to 5 mg / L.

The lactic acid bacterium selective separation medium of the present invention may further contain a color former. This is to form the grown lactic acid bacteria as colored colonies and facilitate the detection. As the color former, only one kind may be used, or a plurality of kinds may be used.
The color former is usually a color-developing enzyme substrate in which a colored chromogen compound is released by a phosphatase or esterase or a redox indicator possessed by lactic acid bacteria in general.
The chromogenic enzyme substrate capable of liberating the chromogen by phosphatase is not particularly limited, but is limited to 5-bromo-4-chloro-3-indoxyl phosphate, 5-bromo-6-chloro-3-indoxyl phosphate, 6-Chloro-3-indoxyl phosphate or 1- (2- (4-dimethylaminobenzoyl) phenyl) -1H-indole-3-yl phosphate disodium salt (1- {2- [4- (Dimethylamino)) benzoyl] phenyl} -1H-indol-3-yl phosphate, disodium salt; manufactured by Biosynth, Aldol 515-phospahte) and the like are preferably mentioned.
The chromogenic enzyme substrate capable of liberating the chromogen by esterase is not particularly limited, but is limited to 5-bromo-4-chloro-3-indoxyl acetic acid, 5-bromo-6-chloro-3-indoxyl acetic acid, 6-. Chloro-3-indoxyl phosphorus acetic acid or 1- (2- (4-dimethylaminobenzoyl) phenyl) -1H-indole-3-ylacetic acid (1- {2- [4- (Dimethylamino) benzoyl] phenyl} -1H -indol-3-yl acetate; manufactured by Biosynth, Aldol 515-acetate) and the like are preferably mentioned.
The content of the phosphatase or esterase substrate in the medium of the present invention is preferably 0.01 to 0.5 g / L, preferably 0.01 to 0.15 g / L, as the substrate concentration at the time of use. Is more preferable.

The lactic acid bacterium selective separation medium of the present invention may contain a redox indicator and is not particularly limited, but is tetrazolium violet, 2,3,5-triphenyltetrazolium chloride, p-iodonitrotetrazolium violet, p-nitroblue chloride. Preferable examples include tetrazolium, nitroblue tetrazolium chloride, 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide, and the like.
The content of the redox indicator in the medium of the present invention is preferably 0.001 to 0.1 g / L, more preferably 0.001 to 0.05 g / L as the concentration at the time of use. ..

As one aspect of the present invention, the lactic acid bacterium selective separation medium does not contain a chromogenic enzyme substrate capable of liberating the chromogen with β-galactosidase.

As another aspect of the present invention, the lactic acid bacterium selective separation medium may contain a chromogenic enzyme substrate capable of liberating the chromogen with β-galactosidase. This is because many lactic acid bacteria carry β-galactosidase, which is useful when selectively detecting only such lactic acid bacteria. Such chromogen compounds are not particularly limited, but are, for example, 5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside, 5-bromo-6-chloro-3-indole. Xyl-β-D-galactopyranoside, 6-chloro-3-indoxyl-β-D-galactopyranoside or 1- (2- (4-dimethylaminobenzoyl) phenyl) -1H-indole-3- Il-β-D-galactopyranoside (1- {2- [4- (Dimethylamino) benzoyl] phenyl} -1H-indol-3-yl β-D-galactopyranoside; Biosynth, Aldol 518-β-D -galactopyranoside) is preferably mentioned.

The lactic acid bacterium selective separation medium of the present invention may be either a solid medium or a liquid medium.

In the case of a solid medium, it further contains a gelling agent. The gelling agent retains the water content of the medium to support the medium and prepares a solid medium, which facilitates the operation of the method for detecting the target bacteria described later. The gelling agent is not particularly limited, and is, for example, cellulose derivatives such as agar, polyvinyl alcohol, methyl cellulose, carboxymethyl cellulose, and hydroxyalkyl cellulose, starch and its derivatives, polysaccharides such as hyaluronic acid, guar gum, and xanthan gum, and polyacrylic acid. , Acrylic acid derivatives such as polyacrylic acid salt, acrylic acid-vinyl alcohol copolymer, polyether such as polyethylene glycol and polypropylene glycol, and proteins such as collagen.
In addition, the content of the gelling agent can be arbitrarily adjusted as long as it is an amount usually used for a solid medium.
Further, the medium of the present invention may be, for example, a sheet-shaped medium (International Publication No. 97/24432) in addition to the usual gel-like mode.

The lactic acid bacterium selective separation medium of the present invention can further optionally contain components used in ordinary microbial media such as water, other selective substances, other nutritional components, inorganic salts, and pH adjusters.
The other selective substance is not particularly limited, and examples thereof include vancomycin. By containing vancomycin in the medium of the present invention, only a specific bacterial species (Leuconostoc genus) among lactic acid bacteria having vancomycin resistance can be selectively isolated.
The other nutritional components are not particularly limited, and preferably include soybean peptone, animal meat extract, fish meat extract, sucrose, lactose and the like.
The inorganic salts are not particularly limited, and preferred examples thereof include inorganic acid metal salts such as sodium chloride and inorganic acid metal salts such as sodium pyruvate.

Another aspect of the present invention is a method for producing any of the above-mentioned lactic acid bacteria selective isolation media.
That is, it includes a step of mixing peptone, yeast extract, glucose, polysorbate 80, an inorganic metal salt of an organic acid and glycine in water to prepare a mixed solution, and includes a step of adjusting the pH to 5.5 to 7.0. , A method for producing a selective separation medium for lactic acid bacteria.

Further, the above-mentioned additional components may be added in the step of preparing the mixed solution.
The step of preparing the mixed solution is not particularly limited as long as each component is uniformly mixed in water, but in order to completely dissolve the agar which is a medium component, a stirring operation, a heating operation, and / or an operation during mixing are performed. It is preferable to perform a sterilization operation. The order in which each component is added to water is not particularly limited. In the stirring operation, the stirring speed, the stirring temperature, and the stirring time are not particularly limited. In the heating operation, the temperature and time are not particularly limited. The sterilization operation is not limited as long as it can be sterilized, but for example, sterilization by autoclave at 121 ° C. for 15 minutes can be performed.

The method for producing a lactic acid bacterium selective separation medium of the present invention may further include a step of solidifying the mixed solution.

In the step of solidifying the mixed solution, the mixture is not limited as long as it can be solidified and used as a culture medium, but for example, the sterilized mixed solution is dispensed into a culture plate and left at room temperature for several hours or more. The mixed solution can be solidified with.

Another aspect of the present invention is a method for detecting lactic acid bacteria using any of the above-mentioned selective separation media for lactic acid bacteria. That is, a step of inoculating a sample into a lactic acid bacterium selective separation medium containing peptone, yeast extract, glucose, polysorbate 80, an inorganic metal salt of an organic acid and glycine, a step of culturing the lactic acid bacterium contained in the sample on the medium, and the above-mentioned step. It is a lactic acid bacterium detection method including a step of detecting a lactic acid bacterium colony.

The step of inoculating the sample into the lactic acid bacterium selective separation medium is not particularly limited. It can be carried out by a method known to those skilled in the art.

In the step of culturing lactic acid bacteria, the culture conditions are preferably 30 to 37 ° C. for 3 days. The culture conditions may be either aerobic or anaerobic, but anaerobic culture is preferable in terms of growth and selectivity of lactic acid bacteria.

The step of detecting a colony of lactic acid bacteria is not particularly limited. It can be carried out by a method known to those skilled in the art.
The medium of the present invention has a color tone that depends on the lactic acid bacterium medium component that is the basis of the lactic acid bacterium selective separation medium, but particularly when a medium that is colored and contains a pigment such as BCP-added plate count agar medium is used as the basal medium. By adding a chromogen compound other than the pigment such as BCP without adding a pigment such as BCP in advance, the colonies of lactic acid bacteria grown on the medium can be detected as colored colonies. Can be easily detected.

Examples of the sample applied to the present invention include environmental samples such as general foods, fermented foods, yogurt, lactic acid bacteria beverages or water, clinical samples such as blood, tissues, sputum or feces, and wiped samples from hospitals and the like. Bacteria other than lactic acid bacteria may be present. In addition, a culture solution obtained by culturing these samples in a culture medium for enrichment in advance can also be used as a sample.
Bacteria other than Lactobacillus include, but are not limited to, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and the like.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

<Examples 1 to 3> Preparation of medium The medium components shown in Table 1 were mixed with purified water, and the agar in the medium components was dissolved by high-pressure steam sterilization at 121 ° C. for 15 minutes. After cooling to about 50 ° C. and mixing well with the medium in which the agar was dissolved, 20 mL each was dispensed into a plastic petri dish (90φ mm). The medium of the present invention was prepared by allowing the dispensed medium to stand until it solidified. The medium of the present invention is brown and transparent.

<Comparative Examples 1 to 3> Preparation of Medium The medium components shown in Table 1 were mixed with purified water, and the agar in the medium components was dissolved by high-pressure steam sterilization at 121 ° C. for 15 minutes. After cooling to about 50 ° C. and mixing well with the medium in which the agar was dissolved, 20 mL each was dispensed into a plastic petri dish (90φ mm). Comparative Examples 1 to 3 were prepared by allowing the dispensed medium to stand until it solidified.
As a comparative medium, a medium to which sodium acetate was not additionally added (Comparative Example 1), a medium to which glycine was not added (Comparative Example 2), and a general lactic acid bacterium medium, Lactobacillus agar medium (manufactured by Japan BD) (comparative). Example 3) was used.

<Example 4> Test of strain As lactic acid bacteria, Lactobacillus acidophilus NBRC 13951, L. brevis NBRC 3960, L. delbrueckii subsp. Lactis NBRC 3376, L. helveticus NBRC 15019, L. delbrueckii subsp. Subsp. Cremoris NBRC 100676, Leuconostoc mesenteroides subsp. Mesenteroides NBRC 100496 and Streptococcus thermophilus NBRC 111149 were used as test strains. As bacteria other than lactic acid bacteria, Bacillus subtilis NBRC 3134, Staphylococcus aureus NBRC 100910, Escherichia coli ATCC 8739 and Pseudomonas aeruginosa NBRC 12689 were used as test strains. Of each test strain, lactic acid bacteria were cultured on MRS agar medium at 30 ° C. for 48 hours, and test strains other than lactic acid bacteria were cultured at 35 ° C. for 24 hours, and then McFarland turbidity standard solution 1 was used using a sterile cotton swab. It was suspended in turn corresponds (approximately ^{3.0 × 10 8 CFU / mL)} 0.05% agar pressurized sterile saline so as to turbidity and the strains stock solution.
Each bacterial stock solution is repeatedly diluted 10-fold with 0.05% agar-sterilized physiological saline until the concentration of each bacterial stock solution reaches ^10-7 , and each bacterial solution is subjected to the Milles-Misra method (new bacterial medium science course-). Above- <Second sales> pp. 182-192 Tested by Modern Publishing Co., Ltd. (1986). After culturing all the tested media under aerobic or anaerobic conditions at 30 ° C. for 72 hours, the number of growing bacteria and the color tone of the colonies were confirmed.
Table 2 shows the results of the number of growing bacteria in the aerobic culture, and Table 3 shows the results of the number of growing bacteria in the anaerobic culture. In Tables 2 and 3, + indicates good growth,-indicates non-growth, numbers indicate the number of growing colonies, and × 2 to × 7 indicate the number of dilutions with respect to the bacterial stock solution, each of which is 10 ^{− of the} bacterial stock solution. corresponding to the bacterial diluent at a concentration of ^{^2-10 -7.}

<Example 5> Confirmation of the number of growing bacteria in aerobic culture

In the medium of the present invention, under the culture conditions of aerobic culture, lactic acid bacteria (Lactobacillus acidophilus NBRC 13951, L. brevis NBRC 3960, L. delbrueckii subsp. Lactis NBRC 3376, L. helveticus NBRC 15019, L. delbrueckii subsp. Bulgaricus NBRC 13953, Lactococcus lactis subsp. Cremoris NBRC 100676, Leuconostoc mesenteroides subsp. Mesenteroides NBRC 100496 and Streptococcus thermophilus NBRC 111149 It was found that the growth of 3134, Staphylococcus aureus NBRC 100910, Escherichia coli ATCC 8739 and Pseudomonas aeruginosa NBRC 12689) was effectively suppressed.
It was shown that Bacillus subtilis and Staphylococcus aureus could not be suppressed when glycine or additional sodium acetate was not added under the culture conditions of aerobic culture. It was also shown that even when the amount of glycine added was reduced, Bacillus subtilis and Staphylococcus aureus could not be sufficiently suppressed.

<Example 6> Confirmation of the number of growing bacteria in anaerobic culture

In the medium of the present invention, even under the culture conditions of anaerobic culture, lactic acid bacteria (Lactobacillus acidophilus NBRC 13951, L. brevis NBRC 3960, L. delbrueckii subsp. Lactis NBRC 3376, L. helveticus NBRC 15019, L. delbrueckii subsp. Bulgaricus NBRC 13953, Lactococcus lactis subsp. Cremoris NBRC 100676, Leuconostoc mesenteroides subsp. Mesenteroides NBRC 100496 and Streptococcus thermophilus NBRC 111149 It was found that the growth of 3134, Staphylococcus aureus NBRC 100910, Escherichia coli ATCC 8739 and Pseudomonas aeruginosa NBRC 12689) was effectively suppressed.
It was shown that Staphylococcus aureus could not be suppressed without the addition of glycine or additional sodium acetate under the culture conditions of anaerobic culture. It was also shown that even when the amount of glycine added was reduced, Staphylococcus aureus could not be sufficiently suppressed.

That is, by using the medium of the present invention, bacteria other than lactic acid bacteria can be maintained under the conditions of both aerobic culture and anaerobic culture while maintaining the same growth of lactic acid bacteria as the MRS agar medium which is a growth medium for lactic acid bacteria. It was found that the growth of lactic acid bacteria can be effectively suppressed.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a selective separation medium having better selectivity for the measurement of lactic acid bacteria. In addition, when used for detecting lactic acid bacteria in a sample containing bacteria other than lactic acid bacteria in the sample, the medium of the present invention capable of reliably suppressing the growth of bacteria other than lactic acid bacteria can be provided as an accurate method for measuring the number of lactic acid bacteria, which is industrially very difficult. It is useful for.

Claims

A lactic acid bacterium selective separation medium having a pH of 5.5 to 7.0 and containing peptone, yeast extract, glucose, polysorbate 80, an inorganic metal salt of an organic acid and glycine.
When the inorganic metal salt of the organic acid is sodium acetate, the concentration of sodium acetate is 5.1 to 15 g / L.
When the inorganic metal salt of the organic acid is an inorganic metal salt of an organic acid other than sodium acetate, the concentration of the inorganic metal salt of the organic acid other than sodium acetate is 0.1 to 10 g / L, and the alkali metal or alkaline soil. The medium, which is characterized by not containing an azide of a similar metal.
The lactic acid bacterium selective separation medium according to claim 1, wherein the inorganic metal salt of the organic acid is an inorganic metal salt of at least one organic acid selected from the group consisting of sodium acetate and sodium propionate.
The lactic acid bacterium isolation medium according to claim 1 or 2, wherein the concentration of the glycine is 0.1 to 10 g / L.
The lactic acid bacterium selective separation medium according to any one of claims 1 to 3, further comprising a polypeptide antibiotic.
The lactic acid bacterium selective isolation medium according to claim 4, wherein the polypeptide-based antibiotic is at least one antibiotic selected from the group consisting of polymyxin B and colistin.
The lactic acid bacterium selective separation medium according to any one of claims 1 to 5, further comprising L-cysteine hydrochloride.
The lactic acid bacterium selective separation medium according to any one of claims 1 to 6, wherein the lactic acid bacterium separation medium contains a medium component of an MRS agar medium.
A step of mixing the components of the lactic acid bacterium selective separation medium according to any one of claims 1 to 7 in water to prepare a mixed solution, and a step of adjusting the pH to 5.5 to 7.0 is included. A method for producing a selective separation medium for lactic acid bacteria.
The method for producing a lactic acid bacterium selective separation medium according to claim 8, further comprising a step of solidifying the mixed solution.
A step of inoculating a sample into the lactic acid bacterium selective separation medium according to any one of claims 1 to 7, a step of culturing the lactic acid bacterium contained in the sample on the medium, and a step of detecting a colony of the lactic acid bacterium are included. , Lactic acid bacteria detection method.