WO2019215873A1 - Method for measuring viable cell count - Google Patents

Abstract

To provide a method for measuring the viable cell count of a specific Bifidobacteria from a subject including one or a plurality of types of bacteria, the method having good precision while also being convenient. A method for measuring the viable cell count of Bifidobacterium breve from a subject including one or a plurality of types of bacteria, the method including a culturing step for culturing the subject using a hyperosmotic medium, and a determination step for identifying a colony formed on the medium as Bifidobacterium breve.

Description

Measuring method of viable count

This technique relates to a method for measuring the viable count of Bifidobacterium, and a medium used for the method.

Bifidobacterium genus bacteria (hereinafter also referred to as “bifidobacteria”) live a lot in the intestinal tract before weaning, and contribute to maintaining the intestinal environment of the infant well. It is considered. Known physiological functions of bifidobacteria include an intestinal infection-protecting action on a host, an immune function-enhancing action, nutrition, an intestinal decay-inhibiting action, and the like. Focusing on the usefulness of bifidobacteria as described above, adding bifidobacteria to infant formula and administering the formulated bifidobacteria to humans prevents infections or promotes treatment of allergic diseases It has been devised. In addition to bifidobacteria, lactic acid bacteria are also expected to have a probiotic effect.

When probiotic effects are expected for bifidobacteria and / or lactic acid bacteria, the probiotic effects often depend on the bacterial species or strain used. For this reason, probiotic products (for example, bacterial powders, foods and drinks, feeds, etc.) added by selecting one or more bacterial species or strains capable of expressing the expected effect from bifidobacteria and / or lactic acid bacteria are added. It is manufactured and commercially available (see Table 1 of Non-Patent Document 1). For example, products with Bifidobacterium longum (Bifidobacterium longum subspecies longum, Bifidobacterium longum subspecies Infantis) added to food as Bifidobacterium are often marketed. There are also products that use Bifidobacterium breve.

In recent years, there is an increasing demand for consumers to directly confirm product components. For this reason, there is a movement to display the number of viable bacteria on the product for each type of useful bacteria contained in the product. Moreover, it is obliged or recommended to display the number of viable bacteria for each type of useful bacteria contained in the product depending on the country.
On the other hand, products containing multiple types of bifidobacteria are increasing. For this reason, there is a need for a simple and accurate method for identifying and measuring the number of viable bacteria for each species of bifidobacteria.

For example, a method is known in which a medium in which sterile defibrinated blood is added to a BL agar medium is used to measure Bifidobacteria belonging to multiple bacterial species from the shape of colonies formed by smear culture. .
In addition, by using a medium containing only L-arabinose as a sugar source, the viable count of Bifidobacterium longum, subspecies longong only from a test sample containing a microorganism belonging to the genus Bifidobacterium Has been proposed (Patent Document 1).
In addition, it is known that Bifidobacterium longum subspecies Infantis M-63 has high streptomycin resistance (Non-patent Document 1).

WO2011 / 118765

As a measuring method for each species of bifidobacteria, there is known a method of distinguishing by colony shape using a BL agar medium supplemented with sterile defibrinated blood. However, specialized knowledge and high skill level are required to distinguish colonies of each bacterial species. For this reason, those who have a low level of skill may find it difficult to distinguish between these colony shapes, and variations are likely to occur among measurers.

Regarding the difference in colony formation between Bifidobacterium breve, Bifidobacterium longum sub-species Infatis and Bifidobacterium longum sub-species longum in BL agar medium supplemented with sterile defibrinated blood, Specifically described below. Bifidobacterium longum sub-species longum has a yellowish brown, hemispherical bulge with a smooth colony shape in a sterile defibrinated blood-added BL agar medium; in Bifidobacterium breve , Raised in a perfect circle, hemisphere, smooth on the surface and periphery, milky brown with a central brownish brown to brownish color; Bifidobacterium longum In Subspecies Infantis, it has a round shape, a hemispherical shape, a milky brown colony shape with a light brown color in the center.
However, from the viewpoint of quality control of the bacterial species and the number of bacteria, a more uniform measurement result is desired without being influenced by the experience of the measurer or individual differences.

In BL agar, aseptic defibrosis distinguishes Bifidobacterium longum subspecies longum, Bifidobacterium longum subspecies infantis and Bifidobacterium breve from colony shape There are countries and regions that are necessary ingredients for this, but aseptic defibrinated blood is not readily available.

From such a situation, there is a need for a method that can easily measure the viable count of a specific bifidobacteria from a subject containing a plurality of types of cells without using aseptic defibrinated blood.

Therefore, the main object of the present technology is to provide a method for measuring the viable count of a specific bifidobacteria that is simple and accurate from a specimen containing one or more types of bacteria.

As a result of intensive studies, the present inventors have used a medium in which the basal medium composition is adjusted to a high osmotic pressure. It was found that the viable count of only breve (hereinafter also referred to as “Bifidobacterium breve” or “breve”) can be measured.
Furthermore, the present inventors use a medium containing streptomycin in the basal medium composition, so that even if the subject has multiple types of Bifidobacteria, Bifidobacterium longum subspecies -It was found that the viable count of only Infantis (hereinafter also referred to as "Bifidobacterium longum subspecies Infantis" or "Infantis") can be measured.

By using the above-described hyperosmotic medium and / or streptomycin-containing medium, the present inventors can increase the number of viable Bifidobacterium breve and The inventors have found that the viable count of Bifidobacterium longum subspecies infantis can be selectively and easily measured with high accuracy, and have completed the present invention as follows.

[1]
A method for measuring the viable count of Bifidobacterium breve from a specimen containing one or more types of bacteria,
A culture step of culturing the subject using a hyperosmotic medium;
A determination step of identifying a colony formed on the medium as a Bifidobacterium breve.
[2]
The method according to [1] above, wherein the osmotic pressure of the high osmotic pressure medium is 890 mOsm or more.
[3]
The method according to [1] or [2] above, wherein the hyperosmotic medium contains at least salts and / or saccharides.
[4]
The method according to any one of [1] to [3], wherein the determining step further includes measuring the formed colony as a Bifidobacterium breve and calculating the number of viable bacteria by a dilution rate. .
[5]
The method according to any one of [1] to [4], wherein the subject contains at least Bifidobacterium breve and Bifidobacterium longum bacteria.
[6]
The viable count of Bifidobacterium breve and Bifidobacterium longum subspices infantis is measured from a specimen containing one or more types of bacteria by the following (A) and (B) And how to
(A) a step of measuring the viable count of Bifidobacterium breve by the method of measuring the viable count of Bifidobacterium breve according to any one of [1] to [5];
(B) Using a streptomycin-containing medium for a specimen containing one or more types of bacteria, the colonies formed are counted as Bifidobacterium longum subspecies infants, and the number of viable bacteria is determined by the dilution factor. A step of determining the number of viable bacteria of the Infantis by including a determination step.
[7]
The method according to [6] above, wherein the streptomycin-containing medium is a streptomycin of 60 mg / L or more.
[8]
One or more kinds of bacteria contained in the subject are a group consisting of Bifidobacterium breve, Bifidobacterium longum subspecies infantitis and Bifidobacterium longum subspecies longum The method according to [6] or [7] above, which is one or more selected from the group consisting of:
[9]
A method for producing a composition comprising one or more bacteria,
Measuring the viable count of each bacterium in the composition by the method for measuring the viable count of the bacterium in the composition according to any one of the above [1] to [8],
Based on the measured viable count of each bacterium in the composition, including the step of adjusting the blending amount of each bacterium so as to be the target viable count for the composition,
The manufacturing method of the said composition which obtains the composition containing the said one or several types of bacteria based on the design of the compounding quantity of each said bacteria.
[10]
A hyperosmotic medium composition for measuring the viable count of Bifidobacterium breve.
[11]
The medium composition according to [10] above, wherein the osmotic pressure of the hyperosmotic medium composition is 890 mOsm or more.

According to the present technology, it is possible to provide a method for measuring the number of viable bacteria of a specific bifidobacteria that is simple and accurate from a specimen containing one or more types of bacteria.
In addition, the effect described here is not necessarily limited, and may be any effect described in the present technology.

Next, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely changed within the scope of the present invention. In the present specification, percentages are expressed by mass unless otherwise specified.

<1. Method for measuring the viable count of Bifidobacterium breve>
This technique is a method for measuring the viable count of Bifidobacterium breve from a specimen containing one or more types of bacteria,
A culture step of culturing the subject using a hyperosmotic medium;
And a determination step for identifying colonies formed in the medium as Bifidobacterium breve.

[Subject]
The specimen used in the present technology is not particularly limited as long as it contains one or more types of bacteria (test bacteria). For example, food and drink (for example, fermented milk, bacterial powder, confectionery, beverage, health food, functional food, functional display food, food for specified health use, infant formula, etc.), products such as pharmaceuticals and livestock feed, Examples include products obtained by subjecting products to processing such as pulverization and dilution with a diluent. From the viewpoint of the necessity of quality control, a product in which the number of viable bacteria is prepared is preferable, for example, a food or drink or a pharmaceutical product is preferable.

Products such as foods and health foods to which the method for measuring the viable cell count of the present technology can be applied are not particularly limited as long as they are bacteria-containing products. For example, useful bacteria such as bifidobacteria and / or lactic acid bacteria are mixed. Examples include foods and health foods. More specific examples of products containing the useful bacteria include infant formula, infant formula, fermented milk, beverages and foods that contain or do not contain milk components, bacterial powder, chocolate, tablets, Capsules, sachets and the like can be mentioned.

[Bacteria in the specimen]
Gram positive bacteria are suitable for one or more types of bacteria to which the viable cell count according to the present technology can be applied. The present technology is preferably a useful bacterium that can be used for food and drink from the viewpoint that it can be used for the presence or absence of Bifidobacterium breve in the product or for measuring and displaying the number of viable bacteria. Such useful bacteria are found in commercially available products, useful bacteria that are preserved as culture collections at microorganism preservation institutions, useful bacteria that are preserved as trustees at international depositories under the Budapest Treaty, and will be found in the future Useful bacteria etc. are mentioned.
Furthermore, useful gram positive bacteria are preferable among the bacteria used as the test bacteria of the present technology, and among these, bifidobacteria and / or lactic acid bacteria are preferable from the viewpoint of display recommendation or display obligation of viable bacteria. Further, it is preferable that the bacterium that is the test bacterium of the present technology is Bifidobacterium, from the viewpoint that the present technology can easily determine the presence or absence of breve and can accurately measure the number of viable bacteria.

The Bifidobacterium genus bacteria (hereinafter also referred to as “Bifidobacterium”) to which the viable cell count measurement according to the present technology can be applied are not particularly limited, but a more specific example of the Bifidobacterium is Bifidobacterium. Bifidobacterium longum subsp. Longum; Bifidobacterium longum subsp. Infantis (hereinafter also referred to as “infantis”); Bifidobacterium Bifidobacterium breve (hereinafter also referred to as “breve”); Bifidobacterium bifidum; Bifidobacterium animalis; Bifidobacterium adolescentis, etc. One of these The bifidus bacteria including a plurality of types may be measured as test bacteria, desirable.
When using a high osmotic pressure medium in the present technology, Bifidobacterium longum (hereinafter also referred to as “Bifidobacterium longum”) and Bifidobacterium containing Bifidobacterium breve are to be measured as test bacteria. It is desirable from the viewpoint of simplicity of measurement and accuracy.
Examples of Bifidobacterium longum include Bifidobacterium longum sub-species longum and Bifidobacterium longum sub-species Infantis.

In this technology, Bifidobacterium longum sub-species longum BB-536, Bifidobacterium longum sub-species longum NCC2705 (CNCM I-2618), Bifidobacterium longum sub-species longum ATCC 51870 , Bifidobacterium breve CC ATCC15700, Bifidobacterium breve -16 M-16V, Bifidobacterium breve ATCC15700, Bifidobacterium breve BP-11175, Bifidobacterium longum subspices infantis It is particularly preferable to use M-63 or the like.

Although the lactic acid bacteria to which the viable cell count measurement according to the present technology can be applied are not particularly limited, bacteria of the genus Lactobacillus, Lactococcus, and Streptococcus are preferable. More specific examples of the lactic acid bacteria include Lactobacillus casei, Lactobacillus paracasei, Lactobacillus obagaserri, Lactobacillus rhamnosus, Lactobacillus rhamnosus, Lactobacillus rhamnosus, Bacillus acidophilus (Lactobacillus acidophilus), Lactobacillus cilbulgaricus (Lactobacillus bulgaricus), Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus fermentum (Lactobacillus fermentum), (Lactobacillus jensenii), Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus reuteri Lactobacillus helveticus, Lactococcus lactis subsp. Lactis, Lactococcus cremoris (Lactococcus lactis subsp.

Furthermore, in the present technology, when using the hyperosmotic medium and / or the streptomycin-containing medium of the present technology, (1) Bifidobacterium breve and Bifidobacterium longum subspices infantis covered. Or (2) Bifidobacterium breve, Bifidobacterium longum subspecies Infantis and Bifidobacterium longum subspecies longum as test bacteria It is preferable from the viewpoint of simplicity of measurement and accuracy. The present technology has an advantage that the number of each of these two or three types of viable bacteria can be measured more easily and accurately.

Among the bifidobacteria to which the viable cell count according to the present technology can be applied, for example, [1] Bifidobacterium longum NITE BP-02621 (also known as BB536 or Bifidobacterium longum subsp. Longum ATCC [2] Bifidobacterium infantis NITE BP-02623 (also known as M-63 or Bifidobacterium longum subsp. Infantis BCCM LMG 23728); [3] Bifidobacterium breve NITE BP-02622 (Alternative name: M-16V); These three types of bacteria [1] to [3] are used as useful bacteria in products, and if this technology is used, each bacteria can be easily and accurately distinguished. From the viewpoint of measurement, it is preferable.

BB536, M-63, and M-16V are included in products sold by Morinaga Milk Industry Co., Ltd. It is desirable to use the measurement method of the present technology for a product containing one or more such bacteria. It is also possible to collect BB536, M-63, and M-16V from commercially available products.

Further, in the present technology, [1] Bifidobacterium longum NITE BP-02621, [2] Bifidobacterium infantis NITE BP-02623, and [3] Bifidobacterium breve NITE BP One or more selected from the group consisting of -02622 (more preferably these three types of bifidobacteria) are those that are used as useful bacteria in the product and using this technology, it is simple and accurate. From the viewpoint of measuring each viable count, it is preferable.

[1] Bifidobacterium longum BB536 (NITE BP-02621) is an independent administrative agency, National Institute for Product Evaluation Technology, Patent Microorganisms Depositary Center (NPMD) -5-8 Room 122) was deposited on January 26, 2018 under the deposit number NITE BP-02621 under the Budapest Treaty. Bifidobacterium longum subsp. Longum ATCC BAA-999 (No .: ATCC BAA-999), which is the same bacterium, is available as ATCC BAA-999 from the American Type Culture Collection (ATCC) (see, for example, JP-A 2012 -223134 etc.).
[2] Bifidobacterium infantis M-63 (NITE BP-02623)
On January 26, 2018, at the National Institute of Technology and Evaluation (NPMD) (address: 2-5-8 122, Kazusa-Kamashita, Kisarazu, Chiba Prefecture 292-0818, Japan) NITE BP-02623 accession number, which was deposited internationally based on the Budapest Treaty. Bifidobacterium longum subsp. Infantis BCCM LMG 23728 (number: BCCM / LMG 23728), which is the same bacterium, is available as BCCM / LMG 23728 from BELGIAN CO-ORDINATED COLLECTIONS OF MICRO-ORGANISMS (BCCM) (for example, JP, 2012-223134, etc.).
[3] Bifidobacterium breve M-16V (NITE BP-02622 is an independent administrative agency, National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (NPMD) (Address: Kazusa Kamaji, Kisarazu City, Chiba Prefecture, Japan 292-0818) No. 2-5-8 122) was deposited on January 26, 2018 under the deposit number of NITE BP-02622 under the Budapest Treaty.

<Culture process>
The present technology includes a culture process in which the above-described subject is cultured using a medium containing a hyperosmotic medium composition.

<Medium>
[High Osmotic Medium]
By using the hyperosmotic medium of the present technology, it is possible to easily and accurately distinguish a breve from other bacterial species even when a specimen containing one or more types of bacteria is used. And only a breve can be identified based on the number of colonies formed in the high osmotic pressure culture medium, and the viable count of a breve can be measured more accurately and simply.
The “osmotic pressure” of the “high osmotic pressure medium” used in the present technology is the osmotic pressure at the time of [medium composition / 1 L of water] before heating and dissolving the agar powder in the medium.
The “high osmotic pressure medium” used in the present technology is a medium medium composition / weight ratio when compared with the osmotic pressure of [medium composition / water 1 L] before heating and dissolving agar powder of a general basal medium. The osmotic pressure at the time of 1 L of water is high. In this technology, this high osmotic pressure is defined as “high osmotic pressure”.
When measuring the “osmotic pressure” of the present technology, a medium composition containing agar and water are mixed to prepare [medium composition / 1 L of water] in which the agar powder is not dissolved by heating; The osmotic pressure of [medium composition / 1 L of water] in which the powder is not dissolved by heating is measured with an osmotic pressure measuring device. The water temperature at the time of preparation may be a temperature at which the agar powder is not dissolved by heating, and is, for example, about 5 to 50 ° C.
Examples of the osmotic pressure device include an advance osmometer 3250 (freezing point osmometer: Advance).
As shown in [Examples] below, the osmotic pressure of a general basal medium is 400 to 600 mOsm when the agar powder is prepared in [medium composition / 1 L of water] without dissolving it by heating.

The high osmotic pressure medium of the present technology is preferably 890 mOsm or more when the osmotic pressure is [medium composition / water 1 L]. By setting the osmotic pressure to 890 mOsm or more when [medium composition / water 1 L], the breve and other bacterial species can be easily and accurately distinguished according to the state of colonies formed in the medium. Thereby, the viable count of breve can be measured more accurately.
The lower limit when the osmotic pressure is [medium composition / water 1 L] is more preferably 950 mOsm or more, and further preferably 1050 mOsm or more. The upper limit when the osmotic pressure is [medium composition / water 1 L] is preferably 1450 mOsm or less, more preferably 1350 mOsm or less. Further, the range for the osmotic pressure [medium composition / water 1 L] is preferably 890 to 1350 mOsm from the viewpoint that colonies of only breve can be formed and the survival rate of the breve is high.

The high osmotic pressure adjusting component used in the high osmotic pressure medium composition of the present technology is not particularly limited as long as it is a substance that can increase the osmotic pressure. For example, salts (for example, inorganic salts, organic salts, etc.), saccharides (for example, Monosaccharides / oligosaccharides, reduced products thereof), amino acids / peptides, organic acid salts, and the like. In the present technology, one or more of these can be used. The high osmotic pressure adjusting component of the present technology is preferable because a water-soluble substance is easy to work, dissolve and adjust the osmotic pressure.

Although it does not specifically limit as salts used as a high osmotic pressure adjustment component by this technique, The salts generally used for a culture medium are preferable.
As the salts, for example, an anion salt formed with any acidic group (for example, halogen, carboxyl) or a cation salt formed with any basic group (for example, alkali metal, amino) may be used.
The salt may be either an inorganic salt or an organic salt. Examples of the salts include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, and salts with organic acids. .

More specifically, examples of the metal salt include alkali metals (for example, sodium and potassium), alkaline earth metals (for example, calcium and magnesium), zinc, copper, iron and aluminum. More specific examples of the halogen include chlorine, bromine, and fluorine.
In the present technology, alkali metal salts and alkaline earth salts are preferable from the viewpoint of easy adjustment of osmotic pressure, and alkali metal halide salts (for example, sodium chloride and potassium chloride) are preferable from the viewpoint of water solubility.
One or more types can be selected from the group of these salts.

Although it does not specifically limit as an organic acid used as a high osmotic pressure adjustment component by this technique, For example, the organic acid etc. of a citric acid circuit system are mentioned. Examples of the organic acid include pyruvic acid, lactic acid, acetic acid, oxaloacetic acid, citric acid, isocitric acid, oxalosuccinic acid, α-ketoglutaric acid, succinic acid, fumaric acid, malic acid, and the like.
The inorganic acid used in the present technology is not particularly limited. Examples of the inorganic acid include nitric acid, sulfuric acid, and phosphoric acid.
One or more kinds can be selected from the group of these organic acids and / or inorganic acids, and these organic acids and / or inorganic acids may form a cation salt or an anion salt by adjusting the pH.

The amino acids and peptides used as the high osmotic pressure adjusting component in the present technology are not particularly limited. Examples of the amino acids include alanine, arginine, asparagine, glutamine, glutamic acid, glycine, histidine, phenylalanine, tyrosine and the like. Peptides include those in which these amino acid residues are the same or different and have about 2 to 10 peptide bonds.
One or more types can be selected from the group of these amino acids and peptides, and these may form a cation salt or an anion salt by adjusting the pH.

The saccharide used as the high osmotic pressure adjusting component in the present technology is not particularly limited, and examples thereof include monosaccharides and oligosaccharides, and reduced products thereof (such as sugar alcohols).
The monosaccharide may be either aldose or ketose, and may be an amino sugar, deoxy sugar, or uronic acid in which the OH group of the sugar is an amino group, deoxy group, or carbonyl group in addition to a neutral sugar. Examples of neutral sugars include aldose such as glyceraldehyde, erythrose, threitol, xylose, ribose, arabinose, glucose, galactose, mannose, and ketose such as fructose. Examples of amino sugars include glucosamine and galactosamine; deoxy sugars such as fucose and rhamnose; and uronic acids such as glucuronic acid and galacturonic acid. Furthermore, sugar alcohols of these reduced products may be used, and examples thereof include sorbitol (glucositol), galactitol, mannitol, gluconic acid and the like.
Examples of the oligosaccharide include maltooligosaccharide (for example, maltose), isomaltoligosaccharide, fructooligosaccharide, galactooligosaccharide, mannan oligosaccharide, sucrose, lactose, raffinose and the like. Disaccharides to tetrasaccharides are preferred. Furthermore, reducing oligosaccharides of these reduced products may be used, and examples thereof include reduced maltose (maltitol).
Moreover, glucose-containing products (for example, isomerized sugar, starch syrup, starch hydrolyzate, etc.) containing monosaccharides and oligosaccharides may be used as saccharides.
One or more types can be selected from the group of these saccharides.

In the present technology, when a combination of a plurality of hyperosmotic pressure adjusting components such as the above-described salts is used, water is added to the medium composition so that the agar powder is not dissolved by heating, so that the high osmotic pressure is within the above range. Can be adjusted with an osmotic pressure measuring device while blending a high osmotic pressure adjusting component. In this technique, since the high osmotic pressure can be adjusted before the heat sterilization, the work is easy, so that the measurement results can be easily homogenized.
The high osmotic pressure medium of the present technology preferably contains at least salts and / or saccharides from the viewpoint of easy adjustment of the osmotic pressure. Since salts and saccharides have many readily water-soluble substances, there is an advantage that high osmotic pressure can be easily adjusted without heating and dissolving agar powder.
In the present technology, the salt content in the medium is preferably 11 to 30 g / medium 1 L, and more preferably 15 to 25 g / medium 1 L.
In the high osmotic pressure medium composition of the present technology, the saccharide content in the medium is preferably 130 to 220 g / L of medium, and more preferably 140 to 190 g / L of medium.
In the high osmotic pressure medium composition of the present technology, when a plurality of salts and saccharides in the medium are contained, these amounts can be adjusted so as to achieve the above-described suitable high osmotic pressure.

The production method of the hyperosmotic medium composition used in the hyperosmotic medium of the present technology is not particularly limited. For example, the osmotic pressure may be increased by adding the above-described hyperosmotic pressure adjusting component to the basic medium composition. It is preferable to obtain a hyperosmotic medium composition by adjusting.

In addition, the basal medium composition used by this technique should just be generally used as a medium composition in which bacteria can grow.
As the basal medium composition, a basal medium composition for lactic acid bacteria and / or bifidobacteria in which lactic acid bacteria and / or bifidobacteria can grow is preferable, and commercially available products can be used as the basal medium composition.
In addition, as an arbitrary medium component of the basal medium composition, an arbitrary antibiotic may be added within a range that does not affect the number of colonies and the number of viable bacteria of the target bacterial species.

Among the basal medium compositions, a basal medium composition for bifidobacteria is further preferable. Since it is for bifidobacteria, the viable count of breve of this technology can be measured more accurately.
In general, components that can be used in a basic medium composition for Bifidobacterium include nitrogen sources such as yeast extract, meat extract, pefton; sodium salts such as sodium chloride, sodium acetate, sodium propionate, and L-cysteine. Salts such as hydrochloride, phosphate, sulfate and the like; sugar sources such as glucose, starch, sucrose, raffinose, galactose, and the like can be used, and one or more of these can be used.
Moreover, you may use the product or culture medium composition marketed as a basic medium composition for bifidobacteria. Examples of the basal medium composition include TOS propionate agar medium composition (hereinafter also referred to as “TOS agar medium composition”), enhanced Clostridium agar medium composition (hereinafter also referred to as “RCM agar medium composition”), and the like. Is mentioned. Among these, the TOS agar medium composition and the RCM agar medium composition are preferable. In the present technology, the “medium composition” may be a medium component before solidification or a solidified medium, but unless otherwise specified, is a medium component before solidification.

And in this technique, components, such as agar powder, are melt | dissolved by heat-sterilizing the culture medium composition containing the high osmotic pressure adjustment component and basal medium composition mentioned above. By pouring this agar powder lysate into a container such as a petri dish and cooling, a hyperosmotic medium for determination of each species of Bifidobacterium and the number of viable bacteria can be obtained.
Moreover, in this technique, you may adjust the osmotic pressure of the said composition by mix | blending a high osmotic pressure adjusting component with the basal medium composition before agar powder melt | dissolution. Then, the adjusted high osmotic pressure medium composition can be sterilized by heating and injected into a petri dish or the like to prepare a high osmotic pressure medium.
In the case of using the solid culture method, it is preferable that the agar powder is blended in the medium so that it becomes 1 to 3% (about 1.5%) in the finally obtained medium.

As another aspect of the present technology, a hyperosmotic medium composition for measuring the viable count of Bifidobacterium breve can be provided.
The high osmotic pressure medium composition of the present technology is preferably 890 mOsm or more from the viewpoint of easily measuring the viable count of Bifidobacterium breve. The osmotic pressure is preferably set in the range of mOsm described above.
The hyperosmotic medium composition of the present technology preferably uses each component of the hyperosmotic medium composition described above, and preferably contains at least salts and / or saccharides.

The culture process of this technique can be implemented using the culture method (solid culture method) performed with an agar medium.
In the culturing step of the present technology, it is desirable to appropriately dilute the subject before culturing in the medium from the viewpoint of measuring the more accurate viable count.
Specifically, the method includes: diluting the suspension with a stepwise dilution to obtain each dilution. Incubate each dilution using agar medium. The number of colonies formed in this agar medium is counted. The number of colonies formed at this time corresponds to the number of viable microorganisms contained in the dilution.
Therefore, the number of viable bacteria contained in the diluted solution is obtained from the value of the number of colonies and the dilution rate. It is preferable to adjust the dilution ratio of the diluted solution so that the total number of colonies is 30 to 300 / medium and the total area is 60 cm ² .
From the number of viable bacteria in the suspension thus obtained and the amount of the sample contained in the suspension, the number of viable bacteria contained in the sample (CFU / g) is obtained.

In the above method, a commonly used diluent can be used as a solution for diluting the suspension.
Examples of the general diluent include 0.85% physiological saline, 0.1% peptone-added physiological saline, Mitsuoka buffer, buffered peptone water, and the like.
In the method recommended by the Japan Fermented Milk Lactobacillus Beverage Association, an anaerobic specimen diluent described in the Food Sanitation Inspection Guidelines is recommended for the diluent. The anaerobic specimen dilution solution was KH ₂ PO ₄ : 4.5 g, Na ₂ HPO ₄ : 6.0 g, L-cysteine HCl · H ₂ O: 0.5 g, Tween 80: 0.5 g, agar: 1.0 g Purified water: a liquid consisting of 1,000 mL and widely used for detecting anaerobic bacteria present in the intestinal flora. The anaerobic specimen diluent may be used as a primary diluent and 0.85% saline may be used as a secondary diluent.

Specific examples of the culture method include a pour method, a plate smear method, and a spiral method. The pour method is a method in which an appropriately diluted suspension is mixed with an agar medium that has been dissolved by heating, solidified by cooling, and cultured. The plate smearing method is a method in which an appropriately diluted suspension is smeared on an agar medium and cultured. The spiral method is a method in which a test sample is plated with a concentration gradient using an instrument or the like.
Of these, the plate smearing method and the pour method are preferable, and the pour method is more preferable.
Regarding the type of agar medium used for the culture and the culture conditions (culture temperature, culture time, etc.), known culture conditions can be used according to the microorganism to be measured. For example, the microorganism to be measured can grow as the agar medium. It may be anything. In the case of bifidobacteria, the colony count is mainly performed after anaerobic culture at 37 ° C. for 72 hours.

<Judgment process>
In the determination step of the present technology, after culturing as described above, a colony formed in or on the hyperosmotic medium is identified as a breve.
When cultured using the hyperosmotic medium of the present technology, the breve can be preferentially colonized. For this reason, this technique can measure the presence or absence (that is, identification) and presence of viable bacteria from the number of colonies formed.

Conventional viable count methods may observe a special colony form (for example, special color development, three-dimensional shape, planar size, shape, etc.). However, in such a case, a high level of skill is required, and even the same person has data variations.
On the other hand, this technique can determine the colony formed using the high osmotic pressure culture medium and the number thereof as a breve and the number of viable bacteria without performing the special colony morphology observation. For this reason, even when the degree of skill is small, the identification of the breve and the number of viable bacteria can be measured easily and accurately. In addition, since the present technology is less influenced by the skill level of the measurer and individual differences, the measurement results can be further homogenized.
Also, with this technology, it is only necessary to count the number of colonies formed, and it is not necessary to determine the colony form based on advanced experience, so it is also possible to perform image analysis using a camera (CCD, etc.) It is. Use of image analysis or the like is advantageous from the viewpoint that colony determination can be automated and calculation of the number of viable bacteria in a subject can be automated.

As a method for measuring the number of viable bacteria according to the present technology, a known measurement method can be used except that the medium according to the present technology is used.
In the present technology, it is preferable to calculate as follows.
Total viable count (CFU / product 1 g) = dilution ratio × total number of colonies formed in or on the medium / ml of application / measured weight of product.
The number of colonies is preferably 30 to 300 per total area of 60 cm ^{2 of} medium.
Alternatively, a partial area of the medium may be arbitrarily set, and the total number of colonies in the total area of the medium may be calculated based on the total number of colonies in the area and the area ratio of (partial area / total area).

<2. Method for measuring the number of viable bacteria of Bifidobacterium longum, Subspecies Infantis>
In this technique, a specimen containing one or more types of bacteria is measured using a streptomycin-containing medium, and the formed colonies are measured as Bifidobacterium longum subspecies infantis, and the viable bacteria are determined by the dilution factor. By including the determination step of calculating the number, it is possible to provide a method for measuring the number of viable bacteria of Infantis.
By using the streptomycin-containing medium of the present technology, even when using a specimen containing one or more types of bacteria, Bifidobacterium longum subspecies Infantis and other bacterial species Can be easily and accurately distinguished. Based on the number of colonies formed in the streptomycin-containing medium, only Infantis can be identified, and the viable count of Bifidobacterium longum subspecies Infantis can be measured more accurately and easily. .
The above <1. The description of the configuration common to the method for measuring the viable count of Bifidobacterium, Bifidobacterium breve> (for example, the specimen and the basal medium composition) is omitted.

[Streptomycin-containing medium]
The streptomycin-containing medium used in the present technology is preferably a mixture of the above-described basal medium composition and streptomycin. Thereby, the viable count of Bifidobacterium longum subspecies infantis can be selectively measured preferentially.

The streptomycin content in the streptomycin-containing medium is preferably 50 mg / medium 1 L or more, more preferably 60 mg / medium 1 L or more, as a lower limit, in order to form colonies of only Infantis with high accuracy. Further, as the upper limit value of the streptomycin content, in order to easily form a colony of Bifidobacterium longum subspices infantis, it is preferably 2000 mg / medium 1 L or less, more preferably 1700 mg / medium 1 L or less, More preferably, it is 1500 mg / liter of culture medium or less. The range of the streptomycin content is more preferably 50 to 2000 mg / medium 1 L, further preferably 60 to 1700 mg / medium 1 L, and more preferably 60 to 1500 mg / medium 1 L.
Thus, the present inventors have found that Infantis can colonize even in a wide range of streptomycin concentrations. For this reason, in the present technology, it is easy to adjust the concentration of streptomycin in the medium, so that the measurement results can be easily homogenized.

The method for measuring viable bacteria of Bifidobacterium longum subspecies infantis of the present technology is the same as the above-described method for measuring the viable count of breve except that a medium composition containing streptomycin is used. In the same manner as in the determination step, the culturing step and the determination step are performed, and the viable cell count of Bifidobacterium longum subspices infantis can be measured.

This technique can determine the number of colonies formed using a streptomycin-containing medium as the number of Bifidobacterium longum subspecies infantis and the number of viable bacteria without observing the morphology of special colonies. For this reason, even when the skill level is low, identification of Infantis and the number of viable bacteria can be measured easily and accurately. Moreover, since this technique has little influence by a measurement person's skill level or an individual difference, it can also make a measurement result more uniform. Furthermore, as described later, it is preferable to perform a method using a hyperosmotic medium and a method using a streptomycin-containing medium in the same subject because the number of each type of viable bacteria can be measured easily and accurately.
Also, with this technology, it is only necessary to count the number of colonies formed, and it is not necessary to determine the colony form based on advanced experience, so it is also possible to perform image analysis using a camera (CCD, etc.) It is. Use of image analysis or the like is advantageous from the viewpoint that colony determination can be automated and calculation of the number of viable bacteria in a subject can be automated.
As another aspect of the present technology, a streptomycin-containing medium composition for measuring the viable count of Bifidobacterium longum subspecies infantis can be provided.

<3. Method for measuring the number of viable bacteria of Bifidobacterium breve and Bifidobacterium longum subspices infantis>
In the present technology, a subject containing one or more types of bacteria is subjected to the following measuring methods or measuring steps (1) and (2);
(1) Using a hyperosmotic medium, count the formed colonies as Bifidobacterium breve and measure the number of viable bacteria; and (2) Use the streptomycin-containing medium to Each of Bifidobacterium breve and Bifidobacterium longum subspecies infantis by measuring as viable bacteria longum subspecies infantis and measuring viable counts It is possible to provide a method for measuring the number of viable bacteria.
The above <1. Method for measuring the viable count of Bifidobacterium breve> and <2. The description of the configuration common to the method for measuring the viable count of Bifidobacterium longum, subspecies, and infantis> (for example, the subject and the basal medium composition) is omitted.

By using the hyperosmotic medium of the present technology, only Bifidobacterium breve can be preferentially identified even when using a specimen containing multiple or multiple types of bacteria. The viable count of the Bifidobacterium breve can be easily and accurately measured.
Furthermore, by using the streptomycin-containing medium of the present technology, only Bifidobacterium longum subspices infantis is given priority even when using a specimen containing one or more types of bacteria. And the viable cell count of the Bifidobacterium longum subspecies infantis can be easily and accurately measured.
In the case of this combination, it is preferable to use the same subject because the number of each type of viable bacteria can be measured easily and accurately.
This combination allows the technology to specifically distinguish Bifidobacterium breve and / or Bifidobacterium longum subspices infantis and more accurately count each viable cell count. It can be measured easily.

By this combination, this technology is less affected by the skill level of the measurer and individual differences, so the measurement results of Bifidobacterium breve and Bifidobacterium longum subspices infantis can be homogenized. . In addition, the use of image analysis or the like is advantageous from the standpoint that colony determination can be automated and calculation of the number of viable bacteria in a subject can be automated.

<4. Method for measuring the viable count of Bifidobacterium breve, Bifidobacterium longum subspecies Infantitis and Bifidobacterium longum subspecies longum>
In the present technology, a subject containing one or more types of bacteria is measured using the following measuring steps or measuring methods (1) to (3);
(1) Using a high osmotic pressure medium, measuring the formed colonies as a breve and measuring the number of viable bacteria;
(2) Using a streptomycin-containing medium, the formed colonies are counted as Bifidobacterium longum subspecies Infantis to determine the number of viable bacteria;
(3) (i) Using a BL agar medium without the addition of sterile defibrinated blood, the Bifidobacterium longum subspecies longum colony formed is designated as Bifidobacterium longum subspecies longum. Measuring and counting the number of viable bacteria, and / or (ii) using the basal medium described above (preferably a basal medium for bifidobacteria), measuring the total number of colonies formed as all bacteria, Measuring the number of viable bacteria in
Including breve, Bifidobacterium longum subspecies Infantitis and Bifidobacterium longum subspecies longum can provide a method for measuring the number of viable bacteria.

Note that <1. Method for measuring the viable count of Bifidobacterium breve>, <2. Method for measuring the viable count of Bifidobacterium longum subspecies Infantis>, <3. How to measure the number of viable bacteria of Bifidobacterium breve and Bifidobacterium longum, subspecies, and infantis >> (for example, subject and basal medium composition) Omitted.

The types of bacteria contained in the subject are preferably two types, Bifidobacterium breve and Bifidobacterium longum. Among the Bifidobacterium longum, Bifidobacterium longum subspecies infants and Bifidobacterium longum subspecies longum are preferable.
In the present technology, three types of Bifidobacterium breve, Bifidobacterium longum subspecies Infantis and Bifidobacterium longum subspecies longum are more preferable.

(3) (i) Using the BL agar medium without the addition of sterile defibrinated blood, the formed colonies are counted as Bifidobacterium longum, subspecies longum, and the number of viable bacteria is measured. By morphological observation in this medium, it is possible to distinguish Bifidobacterium longum subspecies longum.

Using (3) (ii) basal medium (preferably basal medium for bifidobacteria), the total number of colonies formed is counted as all bacteria, and the number of viable bacteria of all bacterial species is measured. Furthermore, the number of viable bacteria of Bifidobacterium longum, subspices and longum is calculated by subtracting the result of the number of viable bacteria using each medium of (1) and (2) from the total number of colonies. can do.
In the present technology, the number of colonies formed using a normal medium can be determined as the number of viable bacteria of all bacterial species without observing the morphology of special colonies. Therefore, by using the hyperosmotic medium and the streptomycin-containing medium of the present technology, finally, even when the skill level is low, the identification and production of Bifidobacterium longum, subspices, longum can be performed easily and accurately. The number of bacteria can be measured. Moreover, since this technique has little influence by a measurement person's skill level or an individual difference, it can also make a measurement result more uniform.
Moreover, you may use each culture medium of said (1) and (2), and also the culture medium of (3) (i). Considering each of these results, the number of viable bacteria of Bifidobacterium breve, Bifidobacterium longum sub-species Infantis and Bifidobacterium longum sub-species longum can be determined. Good.
In addition, since a general basic medium is used in the present technology, it is easy to obtain and a determination medium can be easily produced, so that the measurement results can be easily homogenized.

Conventionally, the number of each viable cell is measured in a subject containing Bifidobacterium breve, Bifidobacterium longum subspecies Infatis and Bifidobacterium longum subspecies longum. In some cases, BL agar medium supplemented with sterile defibrinated blood was used. However, according to the present technology, it is not necessary to use aseptic defibrinated blood, and the number of each viable bacteria can be measured more easily and accurately.

<5. Method for producing a composition containing one or more types of bacteria according to the present technology>
As another aspect of the present technology, the above <1. Method for measuring the viable count of Bifidobacterium breve>, <2. Method for measuring the viable count of Bifidobacterium longum subspecies Infantis>, <3. Method for measuring the number of viable bacteria of Bifidobacterium breve and Bifidobacterium longum subspices infantis>, or <4. Using Bifidobacterium breve, Bifidobacterium longum sub-species Infantitis and Bifidobacterium longum sub-species longum, the number of viable bacteria> It is also possible to provide a method for producing a composition containing the bacterium.
The above <1. Method for measuring the viable count of Bifidobacterium breve> to <4. Method for measuring the number of viable bacteria of Bifidobacterium breve, Bifidobacterium longum sub-species Infantitis and Bifidobacterium longum sub-species longong> Omitted.

The “composition” in the production method of the composition of the present technology is not particularly limited as long as it is a composition that can be used for the subject of the present technology, and may be a product or a prototype. For example, any of food and beverage composition, pharmaceutical composition, feed composition, etc. may be used, but the above <1. A method described in “Method of measuring viable count of breve”> [subject] is preferable.

The method for producing a composition of the present technology is a method for producing a composition containing one or more types of bacteria, and (1) <1. Method for measuring the viable count of Bifidobacterium breve>, <2 Method for measuring the viable count of Bifidobacterium longum subspices infantis>, <3. Method for measuring the number of viable bacteria of Bifidobacterium breve and Bifidobacterium longum subspices infantis>, or <4. Method for measuring the number of viable bacteria of Bifidobacterium breve, Bifidobacterium longum sub-species Infantitis and Bifidobacterium longum sub-species longong> Viable count of at least one of Measuring the number of viable bacteria of each bacterial group (may be each bacterial group) in the composition to be tested using the measurement method according to the above; the viable bacteria of each bacteria in the measured composition Based on the number, including the step of adjusting and designing the amount of each bacterium in the composition to achieve the target viable count,
Based on the design of the amount of each bacterium, it is preferable to obtain a composition containing the one or more types of bacteria.
In addition, the measuring step of the viable cell count of (1) above is <1. Method for measuring the viable count of Bifidobacterium breve> to <4. Viable Bacteria longum subspecies breve, Bifidobacterium longum subspecies Infantis and Bifidobacterium longum subspecies longum Two or more measuring methods may be appropriately combined.

In the method for producing a composition of the present technology, the viable cell count measurement step of (1) described above <1. Method for measuring the viable count of Bifidobacterium breve> to <4. How to measure the number of viable bacteria in Bifidobacterium breve, Bifidobacterium longum subspecies Infantitis and Bifidobacterium longum subspecies longong> Omitted.
In the method for producing a composition according to the present technology, the composition to be the subject is one type or a plurality of types, and in the case of a plurality of types, it is preferable that the same lot, the production time are the same period, and the production raw materials are the same. .
The target viable count may be one type or a plurality of types. In addition, the target viable cell count is not particularly limited, and can be set arbitrarily by the manufacturer, and is the viable cell count added to the composition at the time of manufacture. For example, as the target viable cell count, the viable cell count set as the standard at the time of production; the viable cell count immediately after production minus the viable cell count at the time of production storage is corrected to compensate for the decrease in the viable cell count. Examples include the number of bacteria.
The test bacteria of the present technology are not particularly limited, but are preferably Bifidobacterium, more preferably Bifidobacterium breve and Bifidobacterium longum, still more preferably Bifidobacterium breve, There are two or three types selected from Fidobacterium longum subspecies infantis and Bifidobacterium longum subspecies longum.

Although one or more types of bacteria are contained in the composition, the survival rate of one type of bacteria or each of the plurality of types of bacteria in the composition changes with time. For this reason, in the quality control of the composition containing bacteria, it is also necessary to take measures to adjust the compounding amount and the bacteria ratio of each bacteria, assuming this change when stored for a long period of time. In the present technology, it is possible to more accurately adjust the blending amount and blending ratio of such bacteria.

In addition, since the viability of bacteria tends to decrease when bacteria are usually stored for a long period of time, the method of measuring the number of viable bacteria using a special component causes a lower survival rate than the true survival rate due to the influence of the special component. The remaining rate is easy to calculate and there is a risk of lack of accuracy. However, the medium composition of the present technology can reduce the risk.

In addition, according to the present technology, changes in the number of viable bacteria and the percentage of bacteria in the composition due to storage can be monitored and accurately grasped. In the present technology, the monitoring result is fed back to the manufacturing process, and the number of viable bacteria and the proportion of bacteria in the composition in the manufacturing process are adjusted (increased or decreased), whereby the composition after storage for a certain period of time. The number of viable bacteria and the ratio of bacteria can be achieved.

In the case of this technology, as shown in Examples (especially Example 4) described later, even when stored for a long period of time, if the medium composition of this technology and the method for measuring the number of viable bacteria of this technology are used, The number of bacteria can be measured accurately while being simple.
Moreover, if it is this technique, if it is a storage period equivalent to this at 25 degreeC for 2 years, the target viable count can be measured with sufficient precision, although it is simple.
In the present technology, a comparison between the number of viable bacteria at the time of production and the number of viable bacteria at the time of storage for a certain period is preferable. From this comparison result, it is preferable to adjust (increase / decrease) the composition of the bacteria at the time of manufacture so that the number of live bacteria at the time of storage becomes the target number of live bacteria. Thereby, it can control at the time of manufacture so that it may become a fixed number of living bacteria in the assumed preservation | save period.

The method of the present technology and the result thereof and / or the method of producing the result and the composition, the control unit including the CPU of the apparatus, the storage medium (USB memory, HDD, CD, network server, etc.), etc. It can also be stored as a program in the hardware resources provided and realized by the control unit.
Further, the present technology may be a program for causing a computer to function as the method of the present technology and / or the result and the method of manufacturing the composition.

Hereinafter, the present technology will be described in more detail based on examples and the like. In addition, the Example etc. which are demonstrated below show an example of the typical Example of this technique, and, thereby, the range of this technique is not interpreted narrowly.

[Used bacteria]
B. longum subsp. Longum BB536 (hereinafter also referred to as “Longham BB536”), B. breve M-16V (hereinafter also referred to as “Breve M-16V”), B. longum subsp. Infantis used in this example. M-63 (hereinafter also referred to as “Infantis M-63”) can be collected from products commercially available from Morinaga Milk Industry Co., Ltd.
[1] Bifidobacterium longum NITE BP-02621, which is the same bacterium as longum BB536, and [2] Bifidobacterium breve NITE BP-02622, which is the same bacterium, as [2] Breve M-16V, [3] Bifidobacterium infantis NITE BP-02623, which is the same bacterium as Infantis M-63, may be used.

[Measurement of osmotic pressure]
The osmotic pressure of each medium composition is a value obtained by measuring [medium composition / water 1 L] before dissolving the agar powder with an advanced osmometer 3250 (freezing point osmometer: Advance). “Before dissolution of agar powder [medium composition / water 1 L]” is a mixture of a medium composition containing agar powder before heat sterilization (autoclave sterilization at 100 ° C. or more for 15 minutes) with 1 L of water.
After the osmotic pressure measurement, “before dissolution of agar powder [medium composition / 1 L of water]” is sterilized by heating in an autoclave. The medium dissolved by heating in an autoclave is poured into a petri dish (1 petri dish; diameter 9 cm) according to the pour method or flat plate smearing method, and solidified by cooling. Culture is performed after solidification, and the number of colonies formed in the petri dish is measured.

[Example 1: High osmotic pressure medium composition and its adjustment component]
[Example 1-1: Measurement of viable count of breve M-16V (high osmotic pressure component: saccharide)]
As a test sample, powder containing 1.1 × 10 ⁷ cfu / g of longum BB536, powder containing 1.2 × 10 ⁷ cfu / g of Breve M-16V, 6.4 × 10 ⁶ of Infantis M-63 The powder containing cfu / g was prepared and diluted appropriately.
A medium (1 L of liquid before dissolution of agar powder) containing a TOS agar medium composition in which sucrose was added at 160.0 g / L and the osmotic pressure was increased to 1084 mOsm was prepared.
Using the TOS agar medium supplemented with 160.0 g / L of sucrose and the TOS agar medium without sucrose as a control, the cells were cultured on the plate. For each test sample of each bacterium, pour culture was performed using each of these media.
After culturing at 37 ° C. for 72 hours in an anaerobic state, the number of colonies formed was measured, and the number of bacteria of the bifidobacteria-containing powder was calculated from the dilution rate.
Table 1 shows the number of bacteria per 1 g of a test sample of longum BB536, Breve M-16V, Infantis M-63 obtained using a TOS agar medium supplemented with 160.0 g / L of sucrose, and sucrose. The survival rate was shown in comparison with the number of bacteria determined using a non-TOS agar medium.

As shown in Table 1, in the hyperosmotic medium supplemented with 160.0 g / L sucrose, the breve M-16V survived, but the longum BB536 and Infantis M-63 lived less than 0.2%. No colonies were detected. Only the breve M-16V could be detected on the same plate.
In addition, if this method is used, a product having a high ratio of breve among Bifidobacteria such as Longum BB536, Brave M-16V, Infantis M-63, such as 1: 1000: 1. Even in this case, only the breve and the number of viable bacteria can be detected accurately.

 

[Example 1-2: Measurement of viable count of breve M-16V (high osmotic pressure component: alkali metal halide salt)]
As a test sample, powder containing 1.0 × 10 ⁷ cfu / g of longum BB536, powder containing 1.1 × 10 ⁷ cfu / g of Brave M-16V, 9.2 × 10 ⁶ of Infantis M-63 The powder containing cfu / g was prepared and diluted appropriately.
TOS agar medium with 20.0 g / L of potassium chloride added to increase osmotic pressure to 1058 mOsm, medium containing TOS agar medium composition with 20.0 g / L of sodium chloride added to increase osmotic pressure to 1208 mOsm (dissolved in agar powder) The previous liquid 1L) was prepared.
Plates using the TOS agar medium supplemented with 20.0 g / L of potassium chloride, the TOS agar medium supplemented with 20.0 g / L of sodium chloride, and the TOS agar medium supplemented with neither potassium chloride nor sodium chloride as controls The pouch was cultured above. For each test sample of each bacterium, pour culture was performed using each of these media.
After culturing at 37 ° C. for 72 hours in an anaerobic state, the number of colonies formed was measured, and the number of bacteria of the bifidobacteria-containing powder was calculated from the dilution rate.
Table 2 shows the number of bacteria per 1 g of the test sample of longum BB536, Breve M-16V, Infantis M-63 obtained using TOS agar medium supplemented with 20.0 g / L of potassium chloride or sodium chloride, and The survival rate compared with the number of bacteria calculated | required using the TOS agar medium to which neither potassium chloride nor sodium chloride was added was shown.

As shown in Table 1, in the medium supplemented with 20.0 g / L potassium chloride or sodium chloride, Brave M-16V survived, but Longham BB536 and Infantis M-63 were less than 0.5%. There was no survival and no colonies were detected. Only breve M-16V could be detected on the same plate.
Therefore, if a method using a high osmotic pressure medium composition is used, only the breve can be accurately detected even when the breve is increased in the bifidobacteria.

 

From the results of Example 1-1 and Example 1-2, if a method using a hyperosmotic medium composition is used, the breve can be easily and selectively identified, and the viable count of the breve can be measured. . Furthermore, if the method using the hyperosmotic medium composition is used, Breve and Bifidobacterium longum sub-species longum, Bifidobacterium longum sub-species Infantis can be easily distinguished, and The viable count of only breve can be measured.
Moreover, since it is made into the high osmotic pressure by making it contain highly, even if it is saccharide | sugar utilized, there is little influence by utilization. That is, the high osmotic pressure adjusting component is not particularly limited as long as it is a substance capable of adjusting the high osmotic pressure, and may be a saccharide or a salt.
In the case of a water-soluble component, the state of the osmotic pressure of the medium can also be understood by appropriately measuring the content of the water-soluble high osmotic pressure component contained in the agar medium. For example, it is conceivable to measure the osmotic pressure with a freezing point depression osmometer after pulverizing and stirring with 1 L of agar medium / water.

[Example 2: Adjustment of osmotic pressure]
[Example 2-1: Measurement of viable count of breve M-16V (range of osmotic pressure)]
As a test sample, powder containing 1.1 × 10 ⁷ cfu / g of longum BB536, powder containing 1.4 × 10 ⁷ cfu / g of Brave M-16V, 6.4 × 10 ⁶ of Infantis M-63 The powder containing cfu / g was prepared and diluted appropriately.
The mixture was cultured on a plate using a TOS agar medium added with 220.0 g / L sucrose and an osmotic pressure increased to 1310 mOsm, and a TOS agar medium without sucrose as a control. The bacterial test sample was subjected to pour culture using each of these media.
After culturing at 37 ° C. for 72 hours in an anaerobic state, the number of colonies formed was measured, and the number of bacteria of the bifidobacteria-containing powder was calculated from the dilution rate.
Table 3 shows the number of bacteria per 1 g of the test sample of longum BB536, Breve M-16V, Infantis M-63 obtained using TOS agar medium supplemented with 220.0 g / L of sucrose, and sucrose. The survival rate was shown in comparison with the number of bacteria determined using a non-TOS agar medium.

As shown in Table 3, in the high osmotic pressure medium with the osmotic pressure increased to 1310 mOsm, Brave M-16V survived, but Longham BB536 and Infantis M-63 survived less than 0.2%. No colonies were detected. Only breve M-16V could be detected on the same plate.

 

[Example 2-2: Measurement of breve M-16V viable count (osmotic pressure range)]
As a test sample, a powder containing 1.2 × 10 ¹¹ cfu / g of longum BB536, a powder containing 2.0 × 10 ¹¹ cfu / g of Brave M-16V, and 8.9 × 10 ¹⁰ of Infantis M-63 The powder containing cfu / g was prepared and diluted appropriately.
The mixture was cultured on a plate using an RCM agar medium with sucrose added at 150.0 g / L and an osmotic pressure increased to 895 mOsm, and an RCM agar medium without sucrose added as a control. For each test sample of each bacterium, pour culture was performed using each of these media.
After culturing at 37 ° C. for 72 hours in an anaerobic state, the number of colonies formed was measured, and the number of bacteria of the bifidobacteria-containing powder was calculated from the dilution rate.
Table 4 shows the number of bacteria per 1 g of a test sample of longum BB536, Breve M-16V, Infantis M-63 obtained using RCM agar medium supplemented with 150.0 g / L of sucrose, and sucrose. The survival rate was shown in comparison with the number of bacteria determined using a non-RCM agar medium.

As shown in Table 4, in the high osmotic pressure medium in which the osmotic pressure was increased to 895 mOsm, Brave M-16V survived, but Longham BB536 and Infantis M-63 survived less than 1.2%. No colonies were detected. Only breve M-16V could be detected on the same plate.

From the results of Example 2-1 and Example 2-2, if the osmotic pressure of the medium containing the hyperosmotic medium composition is at least 895 mOSm, Bifidobacterium longum subspecies longum, Bifidobacterium -Since a colony can make undetectable longum subspecies infantitis, a breve can be identified selectively and the viable count of a breve can be measured. In addition, if the osmotic pressure of the medium containing the hyperosmotic medium composition is up to 1310 mOsm or less, the survival rate of the breve can be ensured, so that the breve can be easily and selectively identified, Numbers can be measured.
That is, if the osmotic pressure is within the range of 895 to 1310 mOsm, the brebe and Bifidobacterium longum subspecies longum, Bifidobacterium longum subspecies infantis can be easily distinguished, Furthermore, the viable count of only the breve can be measured.
Furthermore, from Example 1 and Example 2, if the basal medium composition used for the hyperosmotic medium composition is a commercially available product capable of growing Bifidobacterium, it is easy to use Breve, Bifidobacterium longum subspecies, Longum and Bifidobacterium longum subspecies infantis can be easily distinguished, and the viable count of only breve can be measured.

[Example 3: Streptomycin-containing medium composition and amount thereof]
[Example 3-1: Measurement of viable count of Infantis M-63]
As a test sample, powder containing 9.0 × 10 ⁶ cfu / g of longum BB536, powder containing 1.1 × 10 ⁷ cfu / g of Brave M-16V, and 7.3 × 10 ⁶ of Infantis M-63 The powder containing cfu / g was prepared and diluted appropriately.
PTO culture was performed on a plate using a TOS agar medium supplemented with 60.0 mg / L of streptomycin and a TOS agar medium supplemented with no streptomycin as a control. For each test sample of each bacterium, pour culture was performed using each of these media.
After culturing at 37 ° C. for 72 hours in an anaerobic state, the number of colonies formed was measured, and the number of bacteria of the bifidobacteria-containing powder was calculated from the dilution rate.
Table 5 shows the number of bacteria per 1 g of the test sample of longum BB536, Breve M-16V, Infantis M-63 and streptomycin obtained using TOS agar medium supplemented with 60.0 mg / L of streptomycin. The survival rate compared to the number of bacteria determined using no TOS agar medium was shown.

As shown in Table 5, Infantis M-63 survived 100% in the medium supplemented with 60.0 mg / L of streptomycin, while Longham BB536 and Breve M-16V survived less than 0.4%. It was found that only Infantis M-63 could be detected on the same plate.
In addition, if this method is used, Infantis M-63 is contained in Bifidobacteria, such as longum BB536, Brave M-16V, and Infantis M-63. Even in the case of a large proportion of products, only Infantis M-63 and its viable count can be detected accurately.

　

 

[Example 3-2: Measurement of number of viable bacteria of Infantis M-63]
To longum BB536 1.2 × ¹⁰ 11 cfu / g containing a test sample powder, powder of 2.5 × ¹⁰ 11 cfu / g containing breve M-16V, infantis M-63 to 1.2 × ^{10 11} The powder containing cfu / g was prepared and diluted appropriately.
Pour-culture was performed on plates using an RCM agar medium supplemented with 1500.0 mg / L of streptomycin and an RCM agar medium supplemented with no streptomycin as a control. For each test sample of each bacterium, pour culture was performed using each of these media.
After culturing at 37 ° C. for 72 hours in an anaerobic state, the number of colonies formed was measured, and the number of bacteria of the bifidobacteria-containing powder was calculated from the dilution rate.
Table 6 shows the number of bacteria per 1 g of the test sample of longum BB536, Breve M-16V, Infantis M-63 and streptomycin obtained using RCM agar medium supplemented with 1500.0 mg / L of streptomycin. The survival rate compared to the number of bacteria determined using no RCM agar medium was shown.

As shown in Table 6, in the medium supplemented with 1500.0 mg / L of streptomycin, Infantis M-63 survived 51%, while Longham BB536 and Breve M-16V survived less than 1.0%. It was found that only Infantis M-63 could be detected on the same plate.

 

From the results of Example 2-1 and Example 2-2, when a medium containing streptomycin was used, Bifidobacterium longum subspecies infantis was easily and selectively identified, and Bifidobacterium・ The viable count of Longham Subspecies Infantis can be measured. Further, if the method using a medium composition containing streptomycin is used, Bifidobacterium longum subspecies Infantis, Bifidobacterium breve and Bifidobacterium longum subspecies longum and Can be easily distinguished, and the viable count of only Bifidobacterium longum, Subspecies Infantis can be measured.
Furthermore, if the streptomycin concentration of the medium composition is at least 60.0 mg / L, breve and Bifidobacterium longum sub-species longum can be made undetected in colonies. Longum subspecies infantis can be selectively identified and the viable count of Bifidobacterium longum subspecies infantis can be measured. In addition, if the streptomycin concentration of the medium composition is up to 1500.0 mg / L or less, the survival rate of infantis can be ensured, so that Bifidobacterium longum subspecies Infantis can be easily obtained. Can be selectively identified, and the viable count of Bifidobacterium longum subspecies infantis can be measured.

[Example 4: Storage period in which viable count can be measured]
[Example 4-1: Measurement of viable count of Brave M-16V stored at 25 ° C for 2 years]
As a test sample, a powder containing 1.7 × 10 ¹¹ cfu / g of Brave M-16V stored at 25 ° C. for 2 years was prepared and diluted appropriately.
The mixture was cultured on a plate using a TOS agar medium added with 160.0 g / L of sucrose and an osmotic pressure increased to 1084 mOsm, and an RCM agar medium without sucrose as a control. For each test sample of each bacterium, pour culture was performed using each of these media.
After culturing at 37 ° C. for 72 hours in an anaerobic state, the number of colonies formed was measured, and the number of bacteria of the bifidobacteria-containing powder was calculated from the dilution rate.
Table 7 shows the number of bacteria per gram of the Brave M-16V obtained using the TOS agar medium supplemented with 160.0 g / L of sucrose, and the RCM agar medium supplemented with no sucrose. The survival rate compared with the number of bacteria was shown.
As shown in Table 7, M-16V could be accurately detected even in the brave M-16V stored at 25 ° C. for 2 years.

 

[Example 4-2: Measurement of number of viable bacteria of Infantis M-63 stored at 25 ° C. for two and a half years]
As a test sample, a powder containing 6.7 × 10 ¹⁰ cfu / g of Infantis M-63 stored at 25 ° C. for two and a half years was prepared and diluted appropriately. PTO culture was carried out on a plate using a TOS agar medium supplemented with 80.0 mg / L of streptomycin and a TOS agar medium supplemented with no streptomycin as a control. For each test sample of each bacterium, pour culture was performed using each of these media.
After culturing at 37 ° C. for 72 hours in an anaerobic state, the number of colonies formed was measured, and the number of bacteria of the bifidobacteria-containing powder was calculated from the dilution rate.
Table 8 shows the number of bacteria per gram of Infantis M-63 obtained using a TOS agar medium supplemented with 80.0 mg / L of streptomycin, and a TOS agar medium supplemented with no streptomycin. The survival rate compared with the obtained number of bacteria was shown.
As shown in Table 8, Infantis M-63 was also accurately detected in Infantis M-63 stored at 25 ° C. for two and a half years.

 

From the results of Example 4-1, when the storage period of the product containing the breve is several years, the viable count of the breve can be accurately measured even using the high-pressure osmotic medium of the present technology.
From the results of Example 4-2, when the storage period of the product containing Bifidobacterium longum sub-species Infantis extends for several years, the medium containing streptomycin of the present technology can be used even when the medium containing the streptomycin is used. The viable count of Umm Longham Subspecies Infantis can be accurately measured.
Therefore, even when the storage period of a product containing Breve and / or Bifidobacterium longum subspecies Infantis is several years long, using a medium containing a hyperosmotic medium composition and a medium containing streptomycin Can accurately measure the viable count of Breve and Bifidobacterium longum, Subspecies Infantis

From the results of Examples 1 to 4, breve and Bifidobacterium longum subspices infantis are selectively identified by solid culture using a hyperosmotic medium and a streptomycin-containing medium, respectively. The viable count of each of Breve and Bifidobacterium longum subspecies infantis can be measured.
In addition, using the basic culture medium for the growth of Bifidobacteria, measure the total number of viable bacteria consisting of Breve, Bifidobacterium longum subspecies Infantis and Bifidobacterium longum subspecies longum. By virtue of (total viable count-viable count of Bleve and Bifidobacterium longum subspices infantis mentioned above), the viable count of Bifidobacterium longum subspices longum It can be measured.

[Example 5: Measurement of individual viable count in powder containing 3 fungi]
A powder containing Longum BB536, Infantis M-63, and Brave M-16V was used as a test sample and diluted as appropriate.
Table 9 shows the number of bacteria of Longham BB536, Infantis M-63, and Brave M-16V per 1 g of the test sample obtained by the following culture methods.

The above dilution was subjected to pour culture on a plate using a BL agar medium supplemented with sterile defibrinated blood. Cultured at 37 ° C. for 72 hours under anaerobic conditions, and counted the number of colonies for each bacterial species formed. From the dilution rate, Longham BB536, Breve M-16V, Infantis M-63, The number of bacteria was calculated. The results are shown in Table 9.

(1) Measurement of the total number of bacteria The dilution was cultured on a plate using an RCM agar medium. Cultivate in anaerobic condition at 37 ° C for 72 hours, count the number of colonies formed, and calculate the total number of bacteria of Longham BB536, Breve M-16V and Infantis M-63 per gram of the test sample from the dilution rate. Calculations were made using a basal medium for fungal growth. The results are shown in Table 9.

(2) Measurement of the number of viable bacteria of Longum BB536 The above dilution is mixed-cultured on a plate using a BL agar medium to which sterile defibrinated blood is added and a BL agar medium to which sterile defibrinated blood is not added as a control. did. The cells were cultured in an anaerobic state at 37 ° C. for 72 hours, and only colonies specific to longum BB536 were counted from the formed colonies, and the number of bacteria per 1 g of the test sample was calculated from the dilution rate. The results are shown in Table 9.
As shown in Table 9, in the BL agar medium to which sterile defibrinated blood was not added, the number of bacteria equivalent to that of the BL agar medium to which sterile defibrinated blood was added could be confirmed. From this result, it was found that longum BB536 can be measured even in a BL agar medium without the addition of sterile defibrinated blood.

(3) Measurement of number of viable bacteria of Infantis M-63 The dilution was cultured on a plate using an RCM agar medium supplemented with streptomycin at a concentration of 80 mg / L. The cells were cultured in anaerobic conditions at 37 ° C. for 72 hours, the number of colonies formed was counted, and the number of bacteria per 1 g of the test sample was calculated from the dilution rate. The results are shown in Table 9.
As shown in Table 9, when a medium containing streptomycin was used, the number of Infantis M-63 bacteria equivalent to the BL agar medium supplemented with sterile defibrinated blood could be confirmed.

(4) Measurement of viable cell count of breve M-16V The dilution was cultured on a plate using a TOS agar medium supplemented with sucrose at a concentration of 160 g / L. The cells were cultured in anaerobic conditions at 37 ° C. for 72 hours, the number of colonies formed was counted, and the number of bacteria per 1 g of the test sample was calculated from the dilution rate. The results are shown in Table 9.
When a medium containing a hyperosmotic medium composition was used, the number of breve bacteria equivalent to the BL agar medium supplemented with sterile defibrinated blood could be confirmed.

(5) Measurement of viable count of Brave M-16V (subtraction method)
(1) From the total number of bacteria determined using the above-mentioned basic culture medium for bifidobacteria growth, (2) the number of Longgam BB536 bacteria determined using the BL agar medium without the addition of sterile defibrinated blood, and the above (3) The number of breve M-16V bacteria was determined by subtracting the number of Infantis M-63 bacteria determined using a medium containing streptomycin.
As shown in Table 9, the number of breve bacteria obtained using a combination of a basic basal medium for the growth of bifidobacteria, a BL agar medium without addition of sterile defibrinated blood, and a medium containing streptomycin is as follows. It was confirmed that the number of bacteria was the same as the number of bacteria detected in the added BL medium.

 

From the above, one or more selected from a basic basal medium for growth of bifidobacteria, a BL medium without addition of sterile defibrinated blood, a medium containing streptomycin, and a medium containing a hyperosmotic medium composition are used. By virtue of this, it is possible to measure the viable counts of breve, Bifidobacterium longum subspecies infants and Bifidobacterium longum subspices longum. It was confirmed that the number of bacteria was equivalent to that detected in the BL medium supplemented with defibrinated blood.
In the method using the above (1), (3), (4) and (5), since the number of colonies formed in the medium can be counted, the number of each viable cell can be accurately measured even if the skill level is low. . In addition, since the number of colonies formed in the medium can be counted, individual differences are unlikely to occur, so that the measurement results can be homogenized. This allows the number of viable bacteria in the product (particularly the number of viable bacteria in the brebe, Bifidobacterium longum sub-species Infantis and Bifidobacterium longum sub-species longum, regardless of human ability) ) Information can be displayed or reported more accurately.
Further, even when the mediums (1) to (5) are used, the number of viable bacteria in a product stored for a long time can be measured, so that changes in the number of viable bacteria over time can be monitored. It is also possible to design by adjusting the ratio of the number of viable bacteria at the time of production so as to be the ratio of the number of each viable cell at the time of preservation by monitoring.

(1) Bifidobacterium longum NITE BP-02621 (Accession number: NITE BP-02621) (Accession date: January 26, 2018), Contractor: Kazusa Kama feet, Kisarazu City, Chiba Prefecture, Japan 292-0818 2-5-8 Room 122, National Institute for Product Evaluation Technology (NPMD).
(2) Bifidobacterium breve NITE BP-02622 (Accession number: NITE BP-02622) (Accession date: January 26, 2018), Contractor: Kazusa Kama feet, Kisarazu City, Chiba Prefecture, Japan 292-0818 2-5-8 Room 122, National Institute for Product Evaluation Technology (NPMD).
(3) Bifidobacterium infantis NITE BP-02623 (Accession number: NITE BP-02623) (Accession date: January 26, 2018), Contractor: Kazusa, Kisarazu City, Chiba Prefecture, Japan 292-0818 2-5-8, Kamafoot Room 122, National Institute for Product Evaluation Technology Patent Microorganism Depositary Center (NPMD).

Claims (11)

1. A method for measuring the viable count of Bifidobacterium breve from a specimen containing one or more types of bacteria,
   A culture step of culturing the subject using a hyperosmotic medium;
   A determination step of identifying colonies formed in the medium as Bifidobacterium breve.
2. The method according to claim 1, wherein the osmotic pressure of the high osmotic pressure medium is 890 mOsm or more.
3. The method according to claim 1 or 2, wherein the hyperosmotic medium contains at least salts and / or sugars.
4. The method according to any one of claims 1 to 3, wherein the determination step further includes measuring the formed colonies as Bifidobacterium breve and calculating the number of viable bacteria by a dilution rate.
5. The method according to any one of claims 1 to 4, wherein the subject comprises at least Bifidobacterium breve and Bifidobacterium longum bacteria.
6. The viable count of Bifidobacterium breve and Bifidobacterium longum subspices infantis is measured from a specimen containing one or more types of bacteria by the following (A) and (B) And how to
   (A) a step of measuring the viable count of Bifidobacterium breve by the method of measuring the viable count of Bifidobacterium breve according to any one of claims 1 to 5;
   (B) Using a streptomycin-containing medium for a specimen containing one or more types of bacteria, the colonies formed are counted as Bifidobacterium longum subspecies infants, and the number of viable bacteria is determined by the dilution factor. A step of determining the number of viable bacteria of the infantis by including a determination step of calculating.
7. The method according to claim 6, wherein the streptomycin-containing medium is a streptomycin of 60 mg / L or more.
8. The group of one or more bacteria contained in the subject is composed of Bifidobacterium breve, Bifidobacterium longum subspecies infantitis and Bifidobacterium longum subspecies longum The method of Claim 6 or 7 which is 1 type (s) or 2 or more types selected from.
9. A method for producing a composition comprising one or more bacteria,
   Measuring the viable count of each bacterium in the composition by the method for measuring the viable count of the bacterium in the composition according to any one of claims 1 to 8,
   Based on the measured viable count of each bacterium in the composition, including the step of adjusting the blending amount of each bacterium so as to be the target viable count for the composition,
   The manufacturing method of the said composition which obtains the composition containing the said one or several types of bacteria based on the design of the compounding quantity of each said bacteria.
10. A hyperosmotic medium composition for measuring the viable count of Bifidobacterium breve.
11. The medium composition according to claim 10, wherein the osmotic pressure of the high osmotic pressure medium composition is 890 mOsm or more.