Method for detecting histamine-producing lactic acid bacteria

Abstract

An object of the present invention is to provide a method for simply and reliably detecting the presence or absence of histamine-producing lactic acid bacteria in a fermented food product. [Solution] The present invention relates to a method for detecting histamine-producing lactic acid bacteria contained in a fermented food product, which comprises the steps of contacting the fermented food product with a histamine-producing lactic acid bacteria detection medium containing MRS agar medium, L-histidine, and a pH indicator to culture the histamine-producing lactic acid bacteria contained in the fermented food product, and staining the histamine-producing lactic acid bacteria.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting histamine-producing lactic acid bacteria. [Background technology]

[0002] Soy sauce, miso, and other fermented condiments are essential to the Japanese diet. Microorganisms play a role in their production. For example, soy sauce is produced by three types of microorganisms: koji mold, lactic acid bacteria, and yeast, which work in turn to produce the sauce. Specifically, soy sauce koji is made by growing koji mold on soybeans and wheat, and then the soy sauce koji is mixed with cooled salt water to make moromi. Lactic acid fermentation by lactic acid bacteria occurs in the moromi, followed by alcoholic fermentation by yeast. The fermented and matured moromi is then wrapped in filter cloth and squeezed to obtain kiage soy sauce. The squeezed kiage soy sauce is then pasteurized (heat sterilized) to inactivate the microorganisms and enzymes, and filtered to complete the soy sauce.

[0003] In recent years, the accumulation of histamine, a food poisoning hazard that can cause allergy-like symptoms, has become a problem in fermented foods such as soy sauce and miso during the manufacturing process. Histamine is produced during the soy sauce manufacturing process when histidine, an amino acid contained in the soy sauce raw material, is decomposed by histidine decarboxylase in some wild-type soy sauce lactic acid bacteria. Therefore, in soy sauce production, it is important to suppress the production of this histamine, and research into this is being actively conducted.

[0004] For example, Patent Document 1 discloses a method for suppressing histamine production during soy sauce production using specific soy sauce lactic acid bacteria. Furthermore, Patent Document 2 discloses a lactic acid bacterium that does not decarboxylate carboxylic acids in histidine and does not produce histamine.

[0005] Modern soy sauce production methods can be broadly divided into two types: one is to use large-capacity metal tanks and add microorganisms, and the other is to use traditional wooden barrels and utilize the lactic acid bacteria and other microorganisms that live in the barrels. Because the wooden barrel method of soy sauce production produces soy sauce with the umami and unique aroma that is unique to each brewery, this traditional soy sauce production is practiced in some areas of Japan, including Shodoshima in Kagawa Prefecture. However, this traditional soy sauce manufacturing method has the problem that the quality of soy sauce varies because the microorganisms such as lactic acid bacteria that live in the wooden barrels are not of the same type. In particular, in soy sauce production, the problem of histamine production is a constant issue as long as the amino acid histidine is included in the soy sauce ingredients.

[0006] On the other hand, the amount of histamine contained in the produced soy sauce is measured using a measuring device. However, measuring histamine requires expensive equipment such as high performance liquid chromatography (HPLC), and the maintenance of such equipment is not easy. The halo method is commonly used to confirm the presence of bacteria, but this method has the problem that even when streaking fermented foods containing many impurities such as soy sauce moromi, it is difficult to clearly distinguish between histamine-producing and non-histamine-producing lactic acid bacteria based on the presence or absence of halo. Therefore, there is a need for a method for easily and reliably detecting the presence or absence of histamine-producing lactic acid bacteria in fermented food products such as soy sauce. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2020-025506 [Patent Document 2] Japanese Patent Application Laid-Open No. 2001-238666 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for simply detecting the presence or absence of histamine-producing lactic acid bacteria in a fermented food product without using expensive measuring equipment such as HPLC. [Means for solving the problem]

[0009] As a result of extensive research to solve the above-mentioned problems, the present inventors have found that a method for detecting histamine-producing lactic acid bacteria, which comprises the steps of contacting a fermented food product with an MRS agar medium, L-histidine, and a histamine-producing lactic acid bacteria detection medium containing a pH indicator to culture the histamine-producing lactic acid bacteria contained in the fermented food product, and staining the histamine-producing lactic acid bacteria, makes it possible to simply detect the presence or absence of histamine-producing lactic acid bacteria in a fermented food product without using expensive equipment such as HPLC. The present invention was completed based on these findings.

[0010] The present invention relates to the following histamine testing method, etc. Section 1. A method for detecting histamine-producing lactic acid bacteria contained in a fermented food product, comprising: Fermented foods and a step of contacting the fermented food product with a histamine-producing lactic acid bacteria detection medium containing an MRS agar medium, L-histidine, and a pH indicator to culture the histamine-producing lactic acid bacteria contained in the fermented food product; and A method for detecting histamine-producing lactic acid bacteria, comprising a step of staining the histamine-producing lactic acid bacteria. Section 2. Item 2. The method for detecting histamine-producing lactic acid bacteria according to Item 1, wherein the color change range of the pH indicator is a pH range of 4 or higher but lower than 5. Section 3. Item 3. The method for detecting histamine-producing lactic acid bacteria according to Item 1 or 2, wherein the pH indicator is bromocresol green, 2,6-dinitrophenol, 2,4-dinitrophenol, methyl yellow, ethyl orange, bromophenol blue, Congo red, methyl orange, naphthyl red hydrochloride, alizarin red S, 2,5-dinitrophenol, methyl red, lacmoid, ethyl red, p-nitrophenol, chlorophenol red, or o-nitrophenol. Section 4. Item 4. The method for detecting histamine-producing lactic acid bacteria according to any one of Items 1 to 3, wherein the pH indicator is bromocresol green. Section 5. Item 5. The method for detecting histamine-producing lactic acid bacteria according to any one of Items 1 to 4, wherein the fermented food product is unpasteurized soy sauce, soy sauce moromi mash, or a culture solution thereof. Section 6. Item 6. The method for detecting histamine-producing lactic acid bacteria according to any one of Items 1 to 5, wherein the histamine-producing lactic acid bacteria detection medium further contains a growth inhibitor for contaminating bacteria. Section 7. Item 7. The method for detecting histamine-producing lactic acid bacteria according to any one of Items 1 to 6, wherein the bacterial cells are stained even when histamine-producing lactic acid bacteria and non-histamine-producing lactic acid bacteria are mixed in the fermented food product. Section 8. A medium for detecting histamine-producing lactic acid bacteria contained in a fermented food product, comprising: MRS agar medium, L-histidine and A medium for detecting histamine-producing lactic acid bacteria, comprising a pH indicator. Section 9. Item 9. The medium for detecting histamine-producing lactic acid bacteria according to Item 8, wherein the pH indicator changes color in a range of pH 4 or higher but lower than 5. Section 10. A method for easily identifying histamine-producing lactic acid bacteria in a fermented food product, comprising: (1) preparing a medium in which a model lactic acid bacterium is stained; (2) preparing a medium stained with histamine-producing lactic acid bacteria in a food fermentation product; and (3) comparing a medium stained with the model lactic acid bacteria with a medium stained with histamine-producing lactic acid bacteria in a fermented food product, and easily distinguishing the histamine-producing lactic acid bacteria in the fermented food product based on the stained model lactic acid bacteria; The model lactic acid bacteria include two types of lactic acid bacteria: non-histamine-producing lactic acid bacteria and histamine-producing lactic acid bacteria; The step (1) of preparing a medium in which a model lactic acid bacterium is stained includes: (1-1) contacting the two types of model lactic acid bacteria with a histamine-producing lactic acid bacteria detection medium containing an MRS agar medium, L-histidine, and a pH indicator, and culturing the two types of model lactic acid bacteria; and (1-2) staining the two types of model lactic acid bacteria; The step (2) of preparing a medium in which histamine-producing lactic acid bacteria in a fermented food product are stained includes: (2-1) contacting the fermented food product with a histamine-producing lactic acid bacteria detection medium containing MRS agar medium, L-histidine, and a pH indicator to culture the histamine-producing lactic acid bacteria contained in the fermented food product; and (2-2) A method for easily identifying histamine-producing lactic acid bacteria in a fermented food product, comprising the step of staining the histamine-producing lactic acid bacteria contained in the fermented food product. Section 11. Item 10. The medium for detecting histamine-producing lactic acid bacteria according to Item 8 or 9, wherein the pH indicator is bromocresol green, 2,6-dinitrophenol, 2,4-dinitrophenol, methyl yellow, ethyl orange, bromophenol blue, Congo red, methyl orange, naphthyl red hydrochloride, alizarin red S, 2,5-dinitrophenol, methyl red, lacmoid, ethyl red, p-nitrophenol, chlorophenol red, or o-nitrophenol. Section 12. Item 12. The medium for detecting histamine-producing lactic acid bacteria according to Item 8, 9, or 11, wherein the pH indicator is bromocresol green. Section 13. Item 13. The medium for detecting histamine-producing lactic acid bacteria according to Item 8, 9, 11, or 12, wherein the fermented food product is soy sauce, soy sauce moromi, or a culture solution thereof before pasteurization. Section 14. Item 14. The histamine-detecting medium for detecting histamine-producing lactic acid bacteria according to Item 8, 9, 11, 12, or 13, further comprising a growth inhibitor for contaminating bacteria. Section 15. Item 15. The medium for detecting histamine-producing lactic acid bacteria according to Item 8, 9, 11, 12, 13, or 14, wherein the bacterial cells are stained even when histamine-producing lactic acid bacteria and non-histamine-producing lactic acid bacteria are mixed in the fermented food product. Section 16. A simple discriminant medium that can easily determine the presence or absence of histamine-producing lactic acid bacteria contained in a fermented food product using a model lactic acid bacterium as a standard, MRS agar medium, L-histidine and A simple discrimination medium containing a pH indicator. [Effects of the Invention]

[0011] According to the present invention, a method for simply detecting the presence or absence of histamine-producing lactic acid bacteria in a fermented food product can be provided without using expensive equipment such as HPLC. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows photographs of M17 media containing a pH indicator (bromocresol green, bromothymol blue, or bromocresol purple) (M17 media 1 to 3) and MRS media containing a pH indicator (bromocresol green, bromothymol blue, or bromocresol purple) (MRS media 1 to 3). [Figure 2]Figure 2 shows photographs of Reference Example 1 (MRS medium 1 containing bromocresol green), Reference Example 2 (M17 medium 1 containing bromocresol green), Reference Example 3 (MRS medium 2 containing bromothymol blue), Reference Example 4 (M17 medium 2 containing bromothymol blue), Reference Example 5 (MRS medium 3 containing bromocresol purple), and Reference Example 6 (M17 medium 3 containing bromocresol purple) after streaking and culturing model lactic acid bacteria (non-histamine-producing lactic acid bacteria and histamine-producing lactic acid bacteria). [Figure 3] FIG. 3 shows photographs of Example 1 (MRS medium 1 containing bromocresol green) after streaking and culturing a culture solution containing moromi A and moromi B, and Comparative Example 1 (MRS medium 3 containing bromocresol purple) after streaking and culturing a culture solution containing moromi A and moromi B. [Figure 4] FIG. 4 shows photographs of Reference Example 1 (MRS medium 1 containing bromocresol green) after streaking and culturing model lactic acid bacteria (non-histamine-producing lactic acid bacteria and histamine-producing lactic acid bacteria), Example 1 (MRS medium 1 containing bromocresol green) after streaking and culturing a culture solution containing moromi A and moromi B, Reference Example 5 (MRS medium 3 containing bromocresol purple) after streaking and culturing model lactic acid bacteria (non-histamine-producing lactic acid bacteria and histamine-producing lactic acid bacteria), and Comparative Example 1 (MRS medium 3 containing bromocresol purple) after streaking and culturing a culture solution containing moromi A and moromi B. DETAILED DESCRIPTION OF THE INVENTION

[0013] Method for detecting histamine-producing lactic acid bacteria in fermented food products The method for detecting histamine-producing lactic acid bacteria contained in a fermented food product of the present invention comprises the steps of contacting the fermented food product with a histamine-producing lactic acid bacteria detection medium containing MRS agar medium, L-histidine, and a pH indicator to culture the histamine-producing lactic acid bacteria contained in the fermented food product, and staining the histamine-producing lactic acid bacteria.

[0014] fermented food The fermented food product is not particularly limited as long as it is a part of a food or an extract thereof suspected of being infected or contaminated with histamine-producing lactic acid bacteria, and examples thereof include soy sauce moromi mash, pressed soy sauce moromi mash (squeezed juice), miso, and culture solutions thereof. These culture solutions refer to the culture solutions of the fermented food products. Culture solutions can be produced by known methods.

[0015] Examples of soy sauce (also called shoyu or soy sauce) include those listed in the Ministry of Agriculture, Forestry and Fisheries' "Soy Sauce Quality Labeling Standards" and "Japanese Agricultural Standards for Soy Sauce." Specific examples include dark soy sauce, light soy sauce, white soy sauce, tamari soy sauce, and re-brewed soy sauce. Other examples include raw soy sauce, dashi soy sauce, teriyaki soy sauce, namagaeshi soy sauce, quick-brewed soy sauce, amino acid-mixed soy sauce, reduced-salt soy sauce, and low-salt soy sauce. The soy sauce may be one of the above-mentioned soy sauces alone or a combination of two or more of them. Furthermore, in the fermented food product of the present invention, soy sauce also includes products obtained in the soy sauce production process, such as soy sauce moromi mash, pressed soy sauce moromi mash (squeezed juice), and soy sauce before pasteurization. The method for producing soy sauce moromi mash is not particularly limited as long as it is a commonly known method for producing soy sauce and includes the steps up to obtaining soy sauce moromi mash. The soy sauce moromi mash is preferably one that has been left for one to three months after preparation, and is preferably one that has not yet allowed yeast to grow. The soy sauce referred to in the present invention includes not only the soy sauce using soybeans as a raw material as described above, but also meat sauce, fish sauce, herbal sauce, grain sauce, chili bean sauce, sweet bean sauce, sesame sauce, fried sauce, XO sauce, etc. These soy sauces may be used alone or in combination of two or more.

[0016] There are no particular limitations on the method for producing soy sauce. For example, in the case of the traditional brewing method for soy sauce, a mixture of a protein raw material such as heat-denatured soybeans and a starchy raw material such as wheat that has been gelatinized by heating is inoculated with koji mold containing koji mold and cultured to produce koji, soy sauce koji is obtained, the soy sauce koji obtained is then placed in brine and subjected to lactic acid fermentation and aging to obtain soy sauce moromi, the soy sauce moromi obtained is then subjected to yeast fermentation and aging to obtain matured moromi, the matured moromi obtained is then subjected to a compression treatment or a filtration treatment to obtain raw soy sauce, and the raw soy sauce obtained is then pasteurized, etc.

[0017] There are no particular limitations on the tanks (containers) used when producing soy sauce, but examples include FRP (Fiber Reinforced Plastics) tanks, metal tanks, wooden barrels, and the like. Since most FRP and metal tanks are equipped with cooling and heating equipment, they are maintained at a low temperature of around 10-15°C for 20 days, and then the temperature begins to rise. As the moromi temperature rises, lactic acid fermentation begins and the moromi pH drops. After about a month, the moromi pH usually drops to around 5.1-5.3, so yeast is added and the moromi temperature is maintained at 30°C to allow alcoholic fermentation to occur. On the other hand, wooden barrels cannot be kept at low temperatures, so some breweries cool the brine to keep the moromi temperature below 15°C, but since it is difficult to cool the moromi temperature after brewing, the moromi temperature is affected by the air temperature. Since wooden barrels are difficult to clean thoroughly, there is a possibility that miscellaneous bacteria such as histamine-producing lactic acid bacteria will appear. Note that even in places other than wooden barrels, there is a possibility that histamine-producing lactic acid bacteria will appear due to insufficient cleaning of the pathways (pipes).

[0018] One embodiment of the process for obtaining soy sauce moromi mash includes, for example, a process in which starter koji is inoculated into a soy sauce raw material, which is a mixture of steamed and denatured soybeans, roasted and crushed barley, etc., and the soy sauce koji is obtained by aeration koji-making at 20 to 40°C for about 2 to 4 days, and then the soy sauce koji is added to a salt solution having a salt concentration of 20 to 30% (w / v), and soy sauce lactic acid bacteria is optionally added to the mixture, and the mixture is subjected to lactic acid fermentation, alcoholic fermentation by yeast, and aging for 90 to 365 days at 15 to 35°C with appropriate stirring to obtain soy sauce moromi mash.

[0019] The soy sauce raw material is not particularly limited, but examples thereof include soybeans such as whole soybeans and defatted soybeans, wheat, barley, naked barley, adlay and other wheat, wheat gluten, rice, corn and the like.

[0020] The koji starter is not particularly limited as long as it is a koji mold that is normally used in the production of soy sauce, and examples thereof include Aspergillus oryzae and Aspergillus sojae. The soy sauce lactic acid bacteria are not particularly limited as long as they are soy sauce lactic acid bacteria that are normally used in the production of soy sauce, and examples thereof include halotolerant lactic acid bacteria such as Tetragenococcus halophilus.

[0021] In the process of obtaining soy sauce moromi mash, if the amount of starchy raw materials such as wheat and rice among the soy sauce raw materials is small, the reducing sugar content will be low, and there is a possibility that a soy sauce moromi mash capable of undergoing appropriate alcoholic fermentation by yeast will not be obtained. Therefore, it is preferable that the amount of starchy raw materials such as wheat and rice among the soy sauce raw materials is an amount that enables a soy sauce moromi mash with a high reducing sugar content to be obtained. However, this does not apply when a soy sauce moromi mash with a high reducing sugar content is obtained by adding a reducing sugar component such as glucose, fructose, or maltose to the soy sauce moromi mash.

[0022] In the process of obtaining soy sauce moromi broth, the soy sauce moromi broth is obtained by removing the insoluble solids from soy sauce moromi broth (alcohol fermentation product) containing insoluble solids derived from soy sauce raw materials such as soybeans and wheat. The method for removing the insoluble solids from soy sauce moromi broth to obtain the soy sauce moromi broth is not particularly limited, and examples thereof include commonly known solid-liquid separation methods, specifically, squeezing and filtration processes commonly used in soy sauce manufacturing methods, and more specifically, squeezing filtration processes using a press machine with a filter cloth; membrane filtration processes using various permeable membranes such as UF membranes and MF membranes;

[0023] In the step of carrying out alcoholic fermentation with yeast, the soy sauce moromi mash after lactic acid fermentation is subjected to conventional alcoholic fermentation with yeast using soy sauce yeast commonly used in the production of soy sauce under conditions depending on the type of soy sauce yeast, the number of bacteria, etc. The soy sauce yeast is not particularly limited as long as it is a yeast commonly used in the production of soy sauce, and examples thereof include salt-tolerant yeasts such as Zygosaccharomyces rouxii, Zygosaccharomyces bailli, Candida etchellsii, and Candida versatilis.

[0024] The miso is not particularly limited, and examples thereof include red miso, white miso, Sendai miso, Hatcho miso, barley miso, rice miso, etc. These miso may be used alone or in combination of two or more kinds. A typical method for producing miso consists of a koji-making process in which koji mold is added to steamed grains or beans such as rice or barley and cultured to obtain various types of koji, such as rice koji, barley koji, and soybean koji; a preparation process in which steamed grains or beans such as rice or barley, the various types of koji obtained in the koji-making process, salt, water, and other optional ingredients are mixed in desired proportions to obtain a koji mixture; and a fermentation and maturation process in which the koji mixture is fermented and maturated to obtain a brewed product.

[0025] The method for producing a fermented food product used in the present invention is as described above, but for conditions not explicitly stated in each step, conventionally known conditions may be used, and it goes without saying that the conditions can be changed as appropriate as long as the effects obtained by the treatment in each step are achieved.

[0026] The method for detecting histamine-producing lactic acid bacteria contained in a fermented food product of the present invention includes the step of contacting the fermented food product with a histamine-producing lactic acid bacteria detection medium containing MRS agar medium, L-histidine, and a pH indicator, and culturing the histamine-producing lactic acid bacteria contained in the fermented food product. The fermented food product is preferably soy sauce, soy sauce moromi mash, or a culture solution thereof before pasteurization. The contact is not particularly limited as long as the fermented food product comes into contact with the histamine-producing lactic acid bacteria detection medium, and examples thereof include a method in which the fermented food product is streaked on the histamine-producing lactic acid bacteria detection medium. The amount used when contacting (streaking) a fermented food product with an agar medium is not particularly limited, and is, for example, usually 0.01 to 20 parts by mass, preferably 0.1 to 10 parts by mass, and more preferably 1 to 5 parts by mass, per 100 parts by mass of agar. The amount used when contacting a liquid medium is also not particularly limited, and is, for example, usually 0.1 to 1000 parts by mass, preferably 1 to 500 parts by mass, and more preferably 10 to 250 parts by mass, per 100 parts by mass of glucose.

[0027] In the culturing step, the culturing conditions are not particularly limited as long as they allow the culturing of the lactic acid bacteria contained in the fermented food product or the model lactic acid bacteria. For example, the culture temperature differs depending on the type of lactic acid bacteria, but is preferably 0 to 40° C., more preferably 10 to 35° C., and even more preferably 25 to 30° C. There are no particular limitations on the culture humidity. The culture period varies depending on the strain used, but can be, for example, 1 to 7 days. The atmospheric pressure inside and outside the culture vessel and the composition of the gas phase can be, for example, normal air. Furthermore, the culture can be carried out under either anaerobic or aerobic conditions depending on the type of lactic acid bacteria. The pH in the culture step is preferably 3-8, more preferably 4-7, and even more preferably 5-6.

[0028] Histamine-producing lactic acid bacteria detection medium The histamine-producing lactic acid bacteria detection medium contains MRS agar medium, L-histidine, and a pH indicator.

[0029] agar medium Examples of agar media include MRS agar medium, M17 agar medium, and BCP agar medium. MRS agar (de Man, Rogosa, Sharpe) is a non-selective medium that supports the growth of all lactic acid bacteria. This medium is used to quantify mesophilic lactic acid bacteria. M17 agar medium is a medium primarily used for the growth and counting of Lactococcus (lactic streptococci) in dairy products. BCP agar (also known as BCP plate count agar) is an official medium specified in the "Ministry Ordinance on the Compositional Standards of Milk and Dairy Products" based on the Food Sanitation Act. This medium is generally used to measure the number of lactic acid bacteria in fermented milk or lactic acid bacteria drinks. Examples of media that can be used to produce an agar medium for lactic acid bacteria include MRS powder, M17 powder, and BCP powder. The agar medium used in the present invention is MRS agar medium.

[0030] L-histidine L-histidine is an amino acid called 2-amino-3-(1H-imidazol-4-yl)propionic acid. Its abbreviation is His or H. Histidine is classified as an essential amino acid and cannot be synthesized in the body, so it must be ingested through diet. Histidine is found in large amounts in blue fish, and after a period of time has passed since the fish died, it can be converted into histamine by enzymes in the fish flesh. This can cause scombroid food poisoning (histamine food poisoning). The amount of L-histidine to be added is not particularly limited, and is, for example, usually 0.01 to 500 parts by mass, preferably 1 to 300 parts by mass, and more preferably 50 to 100 parts by mass, per 100 parts by mass of agar.

[0031] pH indicators The pH indicator used in the present invention is not particularly limited in type, as long as the color change range of the pH indicator overlaps the range of pH 4 or more and less than 5. (References: Fujifilm Wako Pure Chemical Corporation website, [online], [searched July 10, 2024], Internet<URL:https: / / labchem-wako.fujifilm.com / jp / question / 013389.html> ) Examples of such pH indicators include: Bromocresol Green (color change range: (yellow) pH 3.8 to 5.4 (blue)), 2,6-dinitrophenol (discoloration range: (slight yellow) pH 2.4 to 4.0 (yellow)), 2,4-dinitrophenol (discoloration range: (very pale yellow) pH 2.6-4.0 (yellow)), Methyl yellow (discoloration range: (light reddish red) pH 2.9 to 4.0 (light reddish yellow)), Ethyl orange (color change range: (light red) pH 3.0 to 4.5 (light orange)), Bromophenol blue (color change range: (yellow) pH 3.0 to 5.0 (blue-purple)), Congo Red (color change range: (purple) pH 3.0-5.0 (dark reddish orange)), Methyl orange (discoloration range: (yellowish red) pH 3.1 to 4.4 (reddish yellow)), Naphthyl Red Hydrochloride (color change range: (red) pH 3.7 to 5.0 (yellow-orange)), Alizarin Red S (color change range: (yellow) pH 3.7 to 5.2 (yellowish red)), 2,5-dinitrophenol (discoloration range: (very pale yellow) pH 4.0 to 5.8 (yellow)), Methyl red (color change range: (purplish red) pH 4.2 to 6.2 (yellow)), Lacmoid (color change range: (red) pH 4.4~6.6 (blue)), Ethyl red (color change range: (red) pH 4.5 to 6.5 (orange)), p-Nitrophenol (discoloration range: (very pale yellow) pH 4.8-7.6 (yellow)), Chlorophenol red (discoloration range: (reddish yellow) pH 5.0 to 6.6 (reddish purple)), o-Nitrophenol (Color change range: (slightly pale yellow) pH 5.0-7.0 (blue)) etc. The pH indicator is preferably bromocresol green.

[0032] The amount of pH indicator to be added is not particularly limited, and is, for example, usually 0.0001 to 1 part by mass, preferably 0.001 to 0.1 part by mass, and more preferably 0.01 to 0.05 part by mass, per 100 parts by mass of agar.

[0033] In the present invention, the step of staining the histamine-producing lactic acid bacteria refers to the step of culturing the fermented food product in contact with the histamine detection medium, since the pH indicator is contained in the histamine detection medium, thereby staining the histamine-producing lactic acid bacteria (the bacterial cells themselves) contained in the fermented food product. Furthermore, when histamine or lactic acid is produced, the medium may also be stained depending on the pH of the product. Here, the histamine-producing lactic acid bacteria can also be referred to as histamine-producing lactic acid bacteria cells. The term "cells" refers to the entire bacteria. By using the method for detecting histamine-producing lactic acid bacteria of the present invention, it is possible to stain the bacterial cells even if histamine-producing lactic acid bacteria and non-histamine-producing lactic acid bacteria are mixed in the fermented food product.

[0034] The pH of the MRS agar medium can be adjusted using a pH adjuster such as sodium hydroxide or hydrochloric acid. The amount of pH adjuster to be added is not particularly limited, and is, for example, usually 0.1 to 600 parts by mass, preferably 0.5 to 300 parts by mass, and more preferably 1 to 100 parts by mass, per 100 parts by mass of agar.

[0035] The histamine-producing lactic acid bacteria detection medium preferably further contains a growth inhibitor for contaminating bacteria. The bacterial growth inhibitor is not particularly limited, and examples thereof include inorganic metal salts of organic acids, antifungal agents, antibacterial agents, tellurite salts, azides, bile salts, sodium chloride, and the like. Examples of inorganic metal salts of organic acids include inorganic metal salts of acetic acid such as sodium acetate, inorganic metal salts of propionic acid, inorganic metal salts of citric acid such as ammonium citrate, etc. Only one type of inorganic metal salt of organic acid may be used, or multiple types may be used. Examples of antifungal agents include glutarimides, polyene macrolides, pyrimidine derivatives, imidazoles, triazoles, candins, allylamines, thiocarbamates, benzylamines, and morphomines. Specific examples include glutarimides such as cycloheximide; polyene macrolides such as amphotericin B and nystatin; pyrimidine derivatives such as flucytosine; imidazoles such as miconazole, ketoconazole, clotrimazole, econazole, isoconazole, sulconazole, oxiconazole, cloconazole, bifonazole, neticonazole, and lanoconazole; triazoles such as fluconazole, itraconazole, and voriconazole; candins such as micafungin; allylamines such as terbinafine hydrochloride; thiocarbamates such as liranaftate; benzylamines such as butenafine and amorolfine; and morphomines such as griseofulvin. Among these, preferred antifungal agents are glutarimides, imidazoles, and triazoles, and more preferred are cycloheximide, miconazole, ketoconazole, clotrimazole, econazole, isoconazole, sulconazole, oxiconazole, cloconazole, bifonazole, neticonazole, lanoconazole, fluconazole, itraconazole, and voriconazole. Antibacterial agents include, for example, streptomycin, tetracycline, kanamycin, ampicillin, and the like. Examples of azides include alkali metal salts such as sodium azide, lithium azide, potassium azide, rubidium azide, cesium azide, and francium azide; and alkaline earth metal salts such as calcium azide and barium azide. Examples of tellurite include potassium tellurite (KHTeO3) and sodium tellurite (NaHTeO3). The amount of inorganic metal salt of organic acid, antifungal agent, antibacterial agent, tellurite, azide, or bile salt to be added is not particularly limited, and is, for example, usually 0.0001 to 50 parts by mass, preferably 0.01 to 25 parts by mass, and more preferably 0.05 to 10 parts by mass, per 100 parts by mass of agar. The amount of sodium chloride added is, for example, usually 0.01 to 2000 parts by mass, preferably 1 to 1500 parts by mass, and more preferably 10 to 700 parts by mass, per 100 parts by mass of agar.

[0036] When the antifungal or antibacterial agent is in powder form, it can be used as is, or it can be dissolved or dispersed in water or an aqueous medium before use. Dissolving or dispersing it in water or an aqueous medium allows the antifungal or antibacterial agent to act effectively on cells. Examples of water for dissolving or dispersing the antifungal or antibacterial agent include tap water, ion-exchanged water, distilled water, and ultrapure water. Examples of aqueous media include alcohols such as ethanol, methanol, and isopropyl alcohol; ketones such as acetone and ethyl methyl ketone; and dimethyl sulfoxide.

[0037] In addition, any additives used in MRS agar medium can be further blended in. Examples of additives include nutrients such as sugars. The amount of additives added is not particularly limited, and is, for example, usually 0.1 to 1500 parts by mass, preferably 0.5 to 1000 parts by mass, and more preferably 1 to 200 parts by mass, per 100 parts by mass of agar.

[0038] The histamine-producing lactic acid bacteria detection medium of the present invention can increase the bacterial density by culturing even when the bacterial density in a food (fermented food product) is extremely low, making it possible to reliably detect the presence of bacteria. Furthermore, the method for detecting histamine-producing lactic acid bacteria contained in a fermented food product of the present invention can detect only live histamine-producing lactic acid bacteria that are capable of proliferation, which poses a risk of infection from histamine-producing lactic acid bacteria. In other words, the method for detecting histamine-producing lactic acid bacteria contained in a fermented food product of the present invention can avoid detecting dead bacteria that are unable to proliferate and remain in the food.

[0039] Method for preparing a medium for detecting histamine-producing lactic acid bacteria The histamine-producing lactic acid bacteria detection medium of the present invention can be produced by mixing MRS agar medium, L-histidine, and a pH indicator, as described above. The histamine-producing lactic acid bacteria detection medium of the present invention can also be mixed with the above-mentioned bacterial growth inhibitor and any additives. The histamine-producing lactic acid bacteria detection medium is preferably in a sterilized form. The sterilization method is not particularly limited, and known methods can be used. After sterilization, the histamine-producing lactic acid bacteria detection medium of the present invention may be contained in a container capable of culturing bacteria, such as a petri dish, a test tube, a tube, or a microtiter plate.

[0040] The histamine-producing lactic acid bacteria detection medium of the present invention can be prepared according to a conventionally known method. For example, it can be prepared by dissolving the medium components other than the bacterial growth inhibitor in water, sterilizing the mixture, adding the bacterial growth inhibitor, and then dispensing the mixture into containers for culturing the bacteria. Examples of water used to dissolve the medium components include tap water, ion-exchanged water, distilled water, and ultrapure water. Furthermore, methods for detecting the presence of histamine-producing lactic acid bacteria contained in the fermented food product of the present invention include a method for culturing the fermented food product on a histamine-producing lactic acid bacteria detection medium, a method for detecting the presence of histamine-producing lactic acid bacteria on a histamine-producing lactic acid bacteria detection medium, and the like.

[0041] Method for culturing food fermentation products in a medium for detecting histamine-producing lactic acid bacteria The method for culturing a food fermentation product on the histamine-producing lactic acid bacteria detection medium involves inoculating the food fermentation product (sample) onto the medium, and then culturing it, for example, at 15 to 35°C for two days or more, for example, one week or more.

[0042] Method for detecting the presence of histamine-producing lactic acid bacteria on a histamine-producing lactic acid bacteria detection medium The method for detecting the presence of histamine-producing lactic acid bacteria on a histamine-producing lactic acid bacteria detection medium involves detecting whether or not histamine-producing lactic acid bacteria are present on the histamine-producing lactic acid bacteria detection medium. The presence of histamine-producing lactic acid bacteria can be detected, for example, by visual inspection or observation under an electron microscope.

[0043] kit The present invention can provide a kit for detecting histamine-producing lactic acid bacteria. The kit of the present invention includes the above-mentioned histamine-producing lactic acid bacteria detection medium, i.e., a medium for culturing histamine-producing lactic acid bacteria. The kit of the present invention may further include an instruction manual. The kit of the present invention enables simple and rapid testing for the presence or absence of histamine-producing lactic acid bacteria.

[0044] Agar medium for model lactic acid bacteria The agar medium for model lactic acid bacteria (also referred to as model lactic acid bacteria detection medium) used in the present invention contains MRS agar medium, L-histidine, and a pH indicator.

[0045] Model lactic acid bacteria <Non-histamine-producing lactic acid bacteria> The non-histamine-producing lactic acid bacteria (Tetragenococcus halophilus) used was a strain distributed by the Kagawa Prefectural Fermented Food Research Institute.

[0046] <Histamine-producing lactic acid bacteria> The histamine-producing lactic acid bacteria (Tetragenococcus halophilus) used was a strain distributed by Morita Corporation's Shodoshima Factory.

[0047] The amount of model lactic acid bacteria used when streaking a medium is not particularly limited, and is, for example, typically 0.1 to 20 parts by mass, preferably 0.5 to 10 parts by mass, and more preferably 1 to 5 parts by mass, per 100 parts by mass of agar.

[0048] When converting MRS liquid medium into a solid medium, the medium can be solidified using a solidifying agent, which is not particularly limited and includes, for example, agar, gellan gum, agarose, Gelrite, gelatin, silica gel, etc.

[0049] In the case of a solid medium, the concentration of the solidifying agent in the medium is preferably 20 parts by mass or more, more preferably 80 parts by mass or more, per 100 parts by mass of glucose. The concentration of the solidifying agent is preferably 150 parts by mass or less, more preferably 100 parts by mass or less.

[0050] A simple medium for differentiation of histamine-producing lactic acid bacteria in fermented food products The simple discriminant medium is a medium that can easily determine the presence or absence of histamine-producing lactic acid bacteria in a fermented food product using a model lactic acid bacterium as a standard. This simple discriminant medium contains MRS agar medium, L-histidine, and a pH indicator. The simple discriminant medium described here can be blended with other ingredients, similar to those described for the histamine-producing lactic acid bacteria detection medium described above.

[0051] Method for distinguishing histamine-producing lactic acid bacteria in fermented food products The method for distinguishing histamine-producing lactic acid bacteria in fermented food products is a method for easily distinguishing histamine-producing lactic acid bacteria in fermented food products using model lactic acid bacteria as a standard. A simple method for identifying histamine-producing lactic acid bacteria in fermented food products is as follows: (1) preparing a medium in which a model lactic acid bacterium is stained; (2) preparing a medium stained with histamine-producing lactic acid bacteria in a food fermentation product; and (3) A process is provided in which a culture medium in which the model lactic acid bacteria has been stained is compared with a culture medium in which histamine-producing lactic acid bacteria in a fermented food product have been stained, and the histamine-producing lactic acid bacteria in the fermented food product are easily identified based on the stained model lactic acid bacteria. The model lactic acid bacteria are of two types: non-histamine-producing lactic acid bacteria and histamine-producing lactic acid bacteria. The step (1) of preparing a medium in which a model lactic acid bacterium is stained includes: (1-1) contacting the two types of model lactic acid bacteria with a histamine-producing lactic acid bacteria detection medium containing an MRS agar medium, L-histidine, and a pH indicator, and culturing the two types of model lactic acid bacteria; and (1-2) The method includes a step of staining the two types of model lactic acid bacteria. The step (2) of preparing a medium in which histamine-producing lactic acid bacteria in a fermented food product are stained includes: (2-1) contacting the fermented food product with a histamine-producing lactic acid bacteria detection medium containing MRS agar medium, L-histidine, and a pH indicator to culture the histamine-producing lactic acid bacteria contained in the fermented food product; and (2-2) The method includes a step of staining the histamine-producing lactic acid bacteria contained in the fermented food product. [Example]

[0052] The detection method of the present invention will be specifically described below, but the technical scope of the present invention is not limited to these examples. In the following, "parts" simply means "parts by mass." [Preparation of medium] Each medium component listed below was weighed using a balance. Each medium component was placed in a beaker containing approximately 50% of the desired medium volume of water and dissolved using a stirrer. If necessary, the pH was adjusted once each component was dissolved. Additional water was added to the mixed solution of dissolved medium components, and the resulting solution was measured using a measuring cylinder to adjust to the final volume (100 mL in this case). The prepared medium was sterilized in an autoclave at 121°C for 15 minutes. Then, specified amounts of cycloheximide and pH indicator were added using a micropipette in a clean bench at 70°C or below, and the mixture was stirred. The mixture was then dispensed into sterilized petri dishes. Since cycloheximide decomposes at pH 7.0 or higher, the pH of the medium was adjusted to less than 7.0 to prevent decomposition of cycloheximide. For liquid media, the medium components, excluding agar, were prepared in the same manner as above, placed in a lidded container such as a Corning tube or a lidded tube such as a silicone stopper, and sterilized in an autoclave at 121°C for 15 minutes. Cycloheximide was then added using the same procedure as for preparing the agar medium. Aqueous solutions of sodium azide and bromocresol green were prepared in advance at concentrations 100 to 1000 times higher than the concentrations used, and these solutions were used. Furthermore, 95% ethanol solutions of cycloheximide and bromocresol purple, each having a concentration 100 to 1000 times higher than the concentration used, were prepared and used.

[0053] [Culture medium] MRS agar medium 1: Histidine-Bromocresol Green-MRS agar medium As described above in the preparation of the medium, the following medium components were added in the amounts shown below to obtain histidine-bromocresol green-MRS agar medium (100 mL) (MRS agar medium 1). (Culture components) MRS powder (Merck, hereinafter the same): 5.22g L-histidine: 1g NaCl: 10g Sodium azide: final concentration 10 ppm Cycloheximide: final concentration 10 ppm Bromocresol Green (Special Grade, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.; the same applies below): Final concentration: 0.003% Agar (for bacterial culture, Fujifilm Wako Pure Chemical Industries, Ltd.) 1.5g

[0054] M17 agar medium 1: Histidine-bromocresol green-M17 agar medium As described in the preparation of the medium above, the following medium components were added in the amounts shown below to obtain histidine-bromocresol green-M17 agar medium (100 mL) (M17 agar medium 1). (Culture components) ·M17 powder: 5.22g NaCl: 10g L-histidine: 1g D-glucose: 2g Sodium azide: final concentration 10 ppm Cycloheximide: final concentration 10 ppm Bromocresol Green: Final concentration 0.003% Agar: 1.5g

[0055] MRS agar medium 2: Histidine-bromothymol blue-MRS agar medium As described in the preparation of the medium above, the following medium components were added in the amounts shown below to obtain histidine-bromothymol blue-MRS agar medium (100 mL) (MRS agar medium 2). (Culture components) ·MRS powder: 5.22g NaCl: 10g L-histidine: 1g Sodium azide: final concentration 10 ppm Cycloheximide: final concentration 10 ppm Bromothymol blue (BTB, Fujifilm Wako Pure Chemical Industries, Ltd.): final concentration 0.004% Agar: 1.5g

[0056] M17 agar medium 2: Histidine-bromothymol blue-M17 agar medium As described in the preparation of the medium above, the following medium components were added in the amounts shown below to obtain histidine-bromothymol blue-M17 agar medium (100 mL) (M17 agar medium 2). (Culture components) ·M17 powder: 5.22g NaCl: 10g L-histidine: 1g D-glucose: 2g Sodium azide: final concentration 10 ppm Cycloheximide: final concentration 10 ppm Bromothymol blue (BTB, Fujifilm Wako Pure Chemical Industries, Ltd.): final concentration 0.004% Agar: 1.5g

[0057] MRS agar 3: Histidine-Bromocresol Purple-MRS agar As described in the preparation of the medium above, the following medium components were added in the amounts shown below to obtain histidine-bromocresol purple-MRS agar medium (100 mL) (MRS agar medium 3). (Culture components) ·MRS powder: 5.22g NaCl: 10g L-histidine: 1g Sodium azide: final concentration 10 ppm Cycloheximide: final concentration 10 ppm Bromocresol Purple (Special Grade, manufactured by Kanto Chemical Co., Ltd.; the same applies below): Final concentration 0.004% Agar: 1.5g

[0058] M17 agar medium 3: Histidine-Bromocresol Purple-M17 agar medium As described in the preparation of the medium above, the following medium components were added in the amounts shown below to obtain histidine-bromocresol purple-M17 agar medium (100 mL) (M17 agar medium 3). (Culture components) ·M17 powder: 5.22g NaCl: 10g L-histidine: 1g D-glucose: 2g Sodium azide: final concentration 10 ppm Cycloheximide: final concentration 10 ppm Bromocresol Purple: Final concentration 0.004% Agar: 1.5g Photographs of the six culture media obtained above are shown in FIG.

[0059] [Bacterial strain used] Non-histamine-producing lactic acid bacteria The culture medium of the non-histamine-producing lactic acid bacterium (Tetragenococcus halophilus) strain distributed by the Kagawa Prefectural Fermented Food Research Institute was used. Histamine-producing lactic acid bacteria The histamine-producing lactic acid bacteria (Tetragenococcus halophilus) used was a culture medium of a strain sold by Morita Corporation's Shodoshima factory.

[0060] <Reference Example 1 (Identification experiment of model lactic acid bacteria)> (1) As shown in the lower left of Figure 2, the upper half of the above-mentioned MRS agar medium 1 (histidine-bromocresol green-MRS agar medium) was streaked with non-histamine-producing lactic acid bacteria (Tetragenococcus halophilus), and the lower half was streaked with histamine-producing lactic acid bacteria (Tetragenococcus halophilus). The medium was then cultured at 30°C in an Anaeropack square jar containing Anaeropack (registered trademark, hereafter omitted) Kenki (manufactured by Mitsubishi Gas Chemical Company, Inc.) for 4 days. The photograph in the lower left of Figure 2 is of MRS agar medium 1 after culture. (2) The presence or absence of histamine production was identified by the color of the medium and the color of the bacteria, which changed as a result of the reaction of the pH indicator in the medium with histamine and lactic acid. Note that histamine indicates alkalinity, while lactic acid indicates acidity. (3) If the pH indicator shows alkaline, histamine is being produced. If the pH indicator shows acidity, lactic acid is being produced.

[0061] [result] As a result, in the MRS agar medium 1 (histidine-bromocresol green-MRS agar medium) used in Reference Example 1, the histamine-producing lactic acid bacteria did not change color because the medium was alkaline due to histamine, and the bacterial cells themselves were stained in a color in the alkaline pH range. On the other hand, non-histamine-producing lactic acid bacteria stain the medium in the acidic pH range due to lactic acid, and the bacterial cells themselves are difficult to stain. Therefore, by utilizing this property, it was possible to identify histamine-producing lactic acid bacteria in soy sauce moromi mash. Details are shown in Table 1 below.

[0062] <Reference Example 2 (Identification experiment of model lactic acid bacteria)> Except for changing the medium to M17 agar medium 1 (histidine-bromocresol green-M17 agar medium), an experiment for identifying model lactic acid bacteria was carried out in the same manner as in Reference Example 1. A photograph of M17 agar medium 1 after cultivation is shown in the upper left of Figure 2.

[0063] [result] As a result, in the M17 agar medium 1 (histidine-bromocresol green-M17 agar medium) used in Reference Example 2, neither histamine-producing nor histamine-non-producing lactic acid bacteria caused any change in the color of the medium, and the bacterial cells themselves were stained in a color in the alkaline pH range, making it impossible to identify the histamine-producing lactic acid bacteria in the soy sauce moromi mash. Details are shown in Table 1 below.

[0064] <Reference Example 3 (Identification experiment of model lactic acid bacteria)> Except for changing the medium to MRS agar medium 2 (histidine-bromothymol blue-MRS agar medium), an experiment to identify model lactic acid bacteria was carried out in the same manner as in Reference Example 1. A photograph of MRS agar medium 2 after cultivation is shown in the lower center of Figure 2.

[0065] [result] As a result, in the MRS agar medium 2 (histidine-bromothymol blue-MRS agar medium) used in Reference Example 3, neither histamine-producing nor histamine-non-producing lactic acid bacteria caused any change in the color of the medium, and the bacterial cells themselves were stained in a color in the alkaline pH range, making it impossible to identify the histamine-producing lactic acid bacteria in the soy sauce moromi mash. Details are shown in Table 1 below.

[0066] <Reference Example 4 (Identification experiment of model lactic acid bacteria)> Except for changing the medium to M17 agar medium 2 (histidine-bromothymol blue-M17 agar medium), an experiment to identify model lactic acid bacteria was carried out in the same manner as in Reference Example 1. A photograph of M17 agar medium 2 after cultivation is shown in the upper center of Figure 2.

[0067] [result] As a result, in the M17 agar medium 2 (histidine-bromothymol blue-M17 agar medium) used in Reference Example 4, neither histamine-producing nor histamine-non-producing lactic acid bacteria caused any change in the color of the medium, and the bacterial cells themselves were stained in a color in the alkaline pH range, making it impossible to identify the histamine-producing lactic acid bacteria in the soy sauce moromi mash. Details are shown in Table 1 below.

[0068] <Reference Example 5 (Identification experiment of model lactic acid bacteria)> Except for changing the medium to MRS agar medium 3 (histidine-cresol purple-MRS agar medium), an identification experiment for model lactic acid bacteria was carried out in the same manner as in Reference Example 1. A photograph of MRS agar medium 3 after cultivation is shown in the lower right of Figure 2.

[0069] [result] As a result, in the MRS agar medium 3 (histidine-cresol purple-MRS agar medium) used in Reference Example 5, the color of the bacterial cells was clearly different depending on whether histamine was produced or not, for both histamine-producing and non-histamine-producing lactic acid bacteria. Furthermore, the presence or absence of histamine production could be clearly determined by the color of the medium. Details are shown in Table 1 below.

[0070] <Reference Example 6 (Identification experiment of model lactic acid bacteria)> Except for changing the medium to M17 agar medium 3 (histidine-cresol purple-M17 agar medium), an experiment to identify model lactic acid bacteria was carried out in the same manner as in Reference Example 1. A photograph of M17 agar medium 3 after cultivation is shown in the upper right corner of Figure 2.

[0071] [result] As a result, in the M17 agar medium 3 (histidine-cresol purple-M17 agar medium) used in Reference Example 6, neither histamine-producing nor histamine-non-producing lactic acid bacteria caused any change in the color of the medium, and the bacterial cells themselves were stained in a color in the alkaline pH range, making it impossible to identify the histamine-producing lactic acid bacteria in the soy sauce moromi mash. Details are shown in Table 1 below.

[0072] The above-mentioned Reference Examples 1 to 6 were evaluated for the distinguishability of the bacterial cells and the stainability of the medium, and the results are shown in Table 1 below.

[0073] [evaluation] Identification of bacterial cells The distinguishability (visibility) of the bacterial cells (model lactic acid bacteria) themselves was evaluated using the following five-point scale. <5-point rating> 5: The difference in bacterial color depending on whether histamine is produced or not is clearly visible. 4: The difference in the color of the bacteria depending on whether or not histamine is produced is visible. 3: The difference in the color of the bacteria depending on whether or not histamine is produced is slightly visible. 2. The difference in bacterial color due to the presence or absence of histamine production is difficult to see. 1: There is no visible difference in the color of the bacteria depending on whether or not histamine is produced.

[0074] Staining of the medium The staining properties (appearance) of the medium were evaluated using the following five-point scale. <5-point rating> 5: The presence or absence of histamine production can be clearly determined by the difference in the color of the medium. 4: The presence or absence of histamine production can be determined by the color of the medium. 3: The presence or absence of histamine production can be somewhat determined by the color of the medium. 2: It is difficult to determine whether histamine is produced or not due to differences in the color of the medium. 1: The presence or absence of histamine production cannot be determined by the color of the medium.

[0075] [comprehensive evaluation] The scores for the distinguishability of the bacterial cells (model lactic acid bacteria: histamine-producing and non-histamine-producing lactic acid bacteria) and the score for the stainability of the medium were summed up to form an overall evaluation. A total score of 10 or more was considered a passing grade.

[0076] [Table 1]

[0077] <Example 1 (Experiment using soy sauce moromi mash)> (I) [Preparing the soy sauce moromi] Procedure for detecting histamine-producing lactic acid bacteria from soy sauce moromi mash (1) Soy sauce moromi mash (Moromi A) that had been prepared three months earlier and soy sauce moromi mash (Moromi B) that had been prepared six months earlier were added to the raw liquid medium in an amount of 1 to 10% (W / V), and static culture was carried out at 30°C for four days. At the same time, moromi was added to the liquid medium containing L-histidine, and the production of histamine was confirmed using Kikkoman's Check Color Histamine. (2) The culture was streaked onto the above-mentioned MRS agar medium 1 (histidine-bromocresol green-MRS agar medium) and then cultured for 4 days in an Anaeropack square jar containing Anaeropack Kenki (manufactured by Mitsubishi Gas Chemical Co., Inc.) or in a sealable pouch containing AnaeroPouch® Kenki (manufactured by Mitsubishi Gas Chemical Co., Inc.). (3) The presence or absence of histamine production was identified by the color of the medium and the color of the bacterial cells, which changed as a result of the reaction of the pH indicator in the medium with histamine and lactic acid. (4) If the pH indicator shows alkaline, histamine is being produced. If the pH indicator shows acidity, lactic acid is being produced.

[0078] <Result> Histamine production was measured using Kikkoman's Check Color Histamine test, and the histamine concentration was 370 ppm in the soy sauce moromi mash (Moromi A) that had been prepared for three months. The histamine concentration was also detected at 1311 ppm in the soy sauce moromi mash (Moromi B) that had been prepared for six months.

[0079] (II) [Preparation of histidine-rich liquid medium (100 mL)] As described in the preparation of the medium above, the following components were added in the amounts shown below and dissolved to obtain a histidine-enriched liquid medium (100 mL) (sample 1). 20mL of soy sauce (TN (total nitrogen): 1.7, NaCl: 17.1%, pH 4.7) (nutrient source for lactic acid bacteria) L-histidine (Special Grade, Fujifilm Wako Pure Chemical Industries, Ltd.) 1g (added if needed) NaCl (special grade, manufactured by Hayashi Pure Chemical Industries, Ltd.) 10g (to isolate only salt-tolerant bacteria) D(+)-glucose (special grade, Fujifilm Wako Pure Chemical Industries, Ltd.): 2g (nutrient source for lactic acid bacteria) Sodium azide (special grade, Nacalai Tesque, Inc.: the same applies below): final concentration 10 ppm (10 ppm to 500 ppm) (inhibits the growth of aerobic microorganisms, antibacterial agent) Cycloheximide (for cell biology, Fujifilm Wako Pure Chemical Industries, Ltd.): Final concentration 10 ppm (10 ppm to 500 ppm) (antibacterial agent, glutarimide antibiotic)

[0080] The soy sauce used had a TN of 1.68 to 1.75, an NaCl content of 16.5 to 17.5%, and a pH of 4.6 to 5.0, and was not pasteurized (heat sterilized). Cycloheximide was added to the medium after autoclaving (121°C, 15 minutes) and when the temperature of the medium had dropped to 70°C or below.

[0081] The methods for measuring the TN amount (total nitrogen amount), NaCl amount, and pH are as follows. [Method for measuring TN (total nitrogen)] The TN amount (total nitrogen amount) was measured by a combustion method using a Sumigraph NC-220F (manufactured by Sumika Chemical Analysis Center, Ltd.).

[0082] [Method for measuring NaCl content] The amount of NaCl (amount of sodium chloride) was measured using a potentiometric titrator (AT-700 (manufactured by Kyoto Electronics Co., Ltd.)).

[0083] When making an agar medium, adjust the salt concentration to a final concentration of 10%. If it is more than 10%, the agar will not solidify easily.

[0084] (III) Experiments using soy sauce moromi The above-mentioned moromi A and moromi B were cultured in the above-mentioned histidine fermentation liquid medium, respectively, to obtain a culture solution 1 containing moromi A and a culture solution 2 containing moromi B. An appropriate amount of culture solution 1 was streaked onto the upper half of MRS agar medium 1 (histidine-bromocresol green-MRS agar medium), and an appropriate amount of culture solution 2 was streaked onto the lower half of MRS agar medium 1 (histidine-bromocresol green-MRS agar medium). Each was then cultured at 30°C for 4 days, and the presence or absence of histamine production was confirmed. Details are shown in Table 2 below. A photograph of MRS agar medium 1 after culture is shown on the left in Figure 3.

[0085] <Comparative Example 1 (Experiment using soy sauce moromi mash)> Except for changing the medium to MRS agar medium 3 (histidine-bromocresol purple-MRS agar medium), an experiment using soy sauce moromi mash was carried out in the same manner as in Example 1. A photograph of MRS agar medium 3 after cultivation is shown on the right side of Figure 3.

[0086] [result] Example 1 and Comparative Example 1 (Figure 3) In FIG. 3, Example 1 (left side of FIG. 3) and Comparative Example 1 (right side of FIG. 3) were again evaluated side by side. As a result, for MRS agar medium 1 (histidine-bromocresol green-MRS agar medium) used in Example 1, in Moromi A, where histamine was detected at 370 ppm, the medium itself became acidic due to lactic acid and turned yellow, but the bacterial cells themselves turned green and showed alkalinity.In addition, in Moromi B, where histamine was detected at 1311 ppm, the medium itself turned light green, and it was confirmed that the bacterial cells themselves were also clearly stained green. In contrast, for MRS agar medium 3 (histidine-bromocresol purple-MRS agar medium) used in Comparative Example 1, it was confirmed that moromi B, in which histamine was detected at 1311 ppm, turned purple, but this was not clearly distinguishable for moromi A, in which histamine was detected at 370 ppm. Therefore, histamine production could be more easily detected in the medium of Example 1 than in the medium of Comparative Example 1. It was also found that the medium of Example 1 had higher sensitivity for detecting histamine-producing lactic acid bacteria than the medium of Comparative Example 1. In the medium of Example 1, the color change range of the pH indicator bromocresol green is on the acidic side, and therefore the color tone on the alkaline side due to histamine is more distinct. In contrast, in the medium of Comparative Example 1, the color change range of the pH indicator bromocresol purple is closer to the neutral range, so it is thought that the acidity is strong but the alkaline side is difficult to distinguish. Therefore, it can be said that the medium of Example 1, which contains bromocresol green as a pH indicator, is superior for identifying histamine-producing bacteria in samples containing a large amount of contaminating bacteria, such as moromi. Details are shown in Table 2 below. The evaluation results for Example 1 and Comparative Example 1 are shown in Table 2 below, similar to the evaluation results for Reference Examples 1 to 6 above.

[0087] [Table 2]

[0088] Evaluation of Example 1, Reference Example 1, Comparative Example 1 and Reference Example 5 (FIG. 4) In FIG. 4, Example 1 (upper right in FIG. 4), Reference Example 1 (upper left in FIG. 4), Comparative Example 1 (lower right in FIG. 4), and Reference Example 5 (lower left in FIG. 4) were again evaluated side by side. As a result, Example 1 revealed that histamine-producing bacteria were present in Moromi A, even when compared with the model lactic acid bacteria (control) of Reference Example 1. This supported the results of the histamine concentration measurement using the Check Color Histamine test described above. In contrast, the MRS agar medium 3 (histidine-bromocresol purple-MRS agar medium) used in Comparative Example 1 was confirmed to have turned purple in Moromi B, in which 1311 ppm of histamine was detected, but was not clearly distinguishable in Moromi A, even though 370 ppm of histamine was detected. Furthermore, when compared with the model lactic acid bacteria (control) of Reference Example 5 as a standard, it was not easy to identify the presence of histamine-producing bacteria in both Moromi A and B. Since moromi contains a mixture of non-histamine-producing and histamine-producing bacteria, when there are a lot of non-histamine-producing bacteria, bromocresol purple is not suitable as a pH indicator for qualitative testing, and bromocresol green is the most suitable.

Claims

1. A method for detecting histamine-producing lactic acid bacteria contained in a fermented food product, comprising: Fermented foods and a step of culturing the histamine-producing lactic acid bacteria contained in the fermented food product by contacting the fermented food product with a histamine-producing lactic acid bacteria detection medium containing an MRS agar medium, L-histidine, and a pH indicator; and A method for detecting histamine-producing lactic acid bacteria, comprising a step of staining the histamine-producing lactic acid bacteria.

2. The method for detecting histamine-producing lactic acid bacteria according to claim 1, wherein the color change range of the pH indicator is a pH range of 4 or more and less than 5.

3. The method for detecting histamine-producing lactic acid bacteria according to claim 1, wherein the pH indicator is bromocresol green, 2,6-dinitrophenol, 2,4-dinitrophenol, methyl yellow, ethyl orange, bromophenol blue, Congo red, methyl orange, naphthyl red hydrochloride, alizarin red S, 2,5-dinitrophenol, methyl red, lacmoid, ethyl red, p-nitrophenol, chlorophenol red, or o-nitrophenol.

4. The method for detecting histamine-producing lactic acid bacteria according to claim 1, wherein the pH indicator is bromocresol green.

5. 2. The method for detecting histamine-producing lactic acid bacteria according to claim 1, wherein the fermented food product is unpasteurized soy sauce, soy sauce moromi, or a culture solution thereof.

6. 2. The method for detecting histamine-producing lactic acid bacteria according to claim 1, wherein the histamine-producing lactic acid bacteria detection medium further contains a growth inhibitor for contaminating bacteria.

7. 2. The method for detecting histamine-producing lactic acid bacteria according to claim 1, wherein the bacterial cells are stained even when histamine-producing lactic acid bacteria and non-histamine-producing lactic acid bacteria are present together in the fermented food product.

8. A medium for detecting histamine-producing lactic acid bacteria contained in a fermented food product, comprising: MRS agar medium; L-histidine, A medium for detecting histamine-producing lactic acid bacteria, comprising a pH indicator.

9. The medium for detecting histamine-producing lactic acid bacteria according to claim 8 , wherein the pH indicator changes color in a range of pH 4 or more but less than 5.

10. A method for easily identifying histamine-producing lactic acid bacteria in a fermented food product, comprising: (1) preparing a medium in which a model lactic acid bacterium is stained; (2) preparing a medium stained with histamine-producing lactic acid bacteria in a fermented food product; and (3) comparing a medium in which the model lactic acid bacteria has been stained with a medium in which histamine-producing lactic acid bacteria in a fermented food product have been stained, and simply distinguishing the histamine-producing lactic acid bacteria in the fermented food product based on the stained model lactic acid bacteria; The model lactic acid bacteria include two types of lactic acid bacteria: non-histamine-producing lactic acid bacteria and histamine-producing lactic acid bacteria; The step (1) of preparing a medium in which a model lactic acid bacterium has been stained includes: (1-1) contacting the two types of model lactic acid bacteria with a histamine-producing lactic acid bacteria detection medium containing an MRS agar medium, L-histidine, and a pH indicator, and culturing the two types of model lactic acid bacteria; and (1-2) staining the two types of model lactic acid bacteria; The step (2) of preparing a medium in which histamine-producing lactic acid bacteria in a fermented food product are stained includes: (2-1) contacting the fermented food product with a histamine-producing lactic acid bacteria detection medium containing MRS agar medium, L-histidine, and a pH indicator to culture the histamine-producing lactic acid bacteria contained in the fermented food product; and (2-2) A method for easily identifying histamine-producing lactic acid bacteria in a fermented food product, comprising a step of staining the histamine-producing lactic acid bacteria contained in the fermented food product.

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