Culture medium for beer-turbidity lactic acid bacteria, and method for detecting beer-turbidity lactic acid bacteria

Abstract

To provide a culture medium suitable for culturing and detecting lactic acid bacteria that are responsible for the cloudiness in a beer-like carbonated beverage, and to provide a method for using the culture medium to detect beer-clouding lactic acid bacteria in a beer-like carbonated beverage.SOLUTION: The present invention provides: a culture medium which is used to detect beer-clouding lactic acid bacteria and comprises carbon and nitrogen sources, mevalonic acid, and an oleic acid which is at least one selected from the group consisting of oleic acid esters and oleic acid salts; the culture medium, wherein the oleic acid is at least one selected from the group consisting of sodium oleate, polyoxyethylene sorbitan monooleate, and polyoxyethylene sorbitan trioleate; and a method for detecting beer-clouding lactic acid bacteria includes inoculating a beer-like carbonated beverage into any of the culture media described above, culturing it, and detecting beer-clouding lactic acid bacteria.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a culture medium suitable for culturing lactic acid bacteria that cause turbidity in beer-like sparkling beverages, and a method for detecting beer-turbidity-causing lactic acid bacteria in beer-like sparkling beverages using the culture medium.

Background Art

[0002] In processed foods and beverages, it is necessary to maintain quality from the time of production until the products reach general consumers. In particular, since microorganisms not only cause quality deterioration but also cause food poisoning, prevention of microbial contamination is extremely important from the perspective of food safety. As a method for microbiological testing of foods and beverages, a culture method is generally used in which microorganisms are isolated and cultured from a test sample of the food or beverage using an appropriate plate medium. Microbial species can be identified by analyzing mycological properties, genetic information and the like of the microorganisms isolated and cultured by the culture method.

[0003] Beer-like sparkling beverages such as beer may become turbid due to microbial contamination, and there is a demand for a method that can more accurately detect the presence or absence of contamination by microorganisms that cause turbidity. Such microorganisms mainly include beer-turbidity-causing lactic acid bacteria.

[0004] Lactic acid bacteria are microorganisms that spoil various foods and beverages, not just beer-like sparkling drinks. Therefore, in the food and beverage manufacturing industry, the detection of contaminating lactic acid bacteria in food and beverages is widely practiced. Many lactic acid bacteria can be detected using common culture media for lactic acid bacteria, such as MRS (de Man-Rogosa-Sharpe) medium. However, some lactic acid bacteria spoil only specific foods and beverages, and some species and strains cannot be detected with common culture media. In recent years, Lactobacillus acetotolerans has been reported as one of the beer-turbidity lactic acid bacteria in beer-like sparkling beverages (Non-Patent Literature 1). Lactobacillus acetotolerans is difficult to culture on MRS medium, and requires the addition of catalase for cultivation (Non-Patent Literature 2), or BLB (Briggs liver broth) medium containing tomato juice (Non-Patent Literature 3). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Riedl et al, BrewingScience, 2017, vol.70, p.39-50. [Non-Patent Document 2] Deng et al, Journal of the Institute of Brewing, 2014, vol.120, p.127-132. [Non-Patent Document 3] Entani et al, International Journal of Systematic Bacteriology, 1986, p.544-549. [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to provide a culture medium suitable for culturing and detecting lactic acid bacteria that cause turbidity in beer-like sparkling beverages, and a method for detecting beer-turbidity-causing lactic acid bacteria in beer-like sparkling beverages using the culture medium. [Means for solving the problem]

[0007] The inventors of the present invention have discovered that Lactobacillus acetotolerance can be cultured by including sufficient amounts of mevalonic acid and oleic acid ester or oleate salt in the culture medium, and have completed the present invention.

[0008] The culture medium and the method for detecting beer turbidity-causing lactic acid bacteria according to the present invention are as follows. [1] This is a culture medium used for detecting lactic acid bacteria that cause turbidity in beer. It contains a carbon source, a nitrogen source, mevalonic acid, and one or more oleic acids selected from the group consisting of oleic acid esters and oleates. The oleic acid compounds are one or more selected from the group consisting of sodium oleate, polyoxyethylene sorbitan monooleate, and polyoxyethylene sorbitan trioleate. The aforementioned beer-turbidity lactic acid bacterium is Lactobacillus acetotolerance. Culture medium. [2] The culture medium according to [1], wherein the mevalonic acid concentration is 0.1 ppm by mass or more. [3] The culture medium according to [2], wherein the mevalonic acid concentration is 0.4 ppm by mass or more. [ 4 The concentration of the oleic acid compounds is 0.5 g / L or more, as described in [1]~[ 3 One of the culture media. [ 5 ] pH is 7.5 or less, as described in [1]~[ 4 One of the culture media. [ 6 ] is a plate culture medium, as described above [1]~[ 5 One of the culture media. [ 7 ] Beer-like sparkling beverage, as described in [1]~[ 6 Inoculate into one of the culture media and culture to detect beer-turbidity lactic acid bacteria. death, The aforementioned beer-turbidity lactic acid bacterium is Lactobacillus acetotolerance.A method for detecting lactic acid bacteria that cause turbidity in beer. [Effects of the Invention]

[0009] The culture medium according to the present invention is a medium that can culture not only beer-turbidity lactic acid bacteria that can be cultured in the conventional MRS medium, but also Lactobacillus acetotolerance, which is difficult to culture in MRS medium. Therefore, by using this culture medium, beer-turbidity lactic acid bacteria, including Lactobacillus acetotolerance, can be accurately detected in beer-like sparkling beverages. [Modes for carrying out the invention]

[0010] In the present invention and this specification, "beer-like sparkling beverage" means a beverage containing carbon dioxide that has characteristics of beer. Furthermore, "beer-like" means a taste that evokes beer in terms of aroma and flavor, regardless of the product name or labeling. In other words, a beer-like sparkling beverage is a sparkling beverage that has a flavor, taste, and texture equivalent to or similar to beer, and has high thirst-quenching properties and drinkability, regardless of alcohol content, the presence or absence of malt and hops, or whether or not it is fermented.

[0011] The beer-like effervescent beverage of the present invention may be an alcoholic beverage, or a so-called non-alcoholic beverage or low-alcoholic beverage having an alcohol content of less than 1% by volume. It may also be a beverage produced using malt as a raw material, a beverage produced without using malt as a raw material, a beverage manufactured through a fermentation step, or a beverage manufactured without going through a fermentation step. Specific examples include beer, low-malt beer produced using malt as a raw material, effervescent alcoholic beverages not using malt, low-alcohol effervescent beverages, non-alcoholic beer, and the like. In addition, it may be liqueurs or spirits obtained by mixing a beverage produced using malt as a raw material through a fermentation step with an alcohol-containing distillate. The alcohol-containing distillate is an alcohol-containing solution obtained by a distillation operation, and for example, it may be raw material alcohol, or distilled spirits such as spirits, whiskey, brandy, vodka, rum, tequila, gin, and shochu can be used.

[0012] The culture medium according to the present invention comprises a carbon source, a nitrogen source, mevalonic acid, and one or more oleic acids selected from the group consisting of oleic acid esters and oleic acid salts, and is characterized by being used for detecting beer-turbulent lactic acid bacteria. Since both mevalonic acid and oleic acids are essential for culturing Lactobacillus acetotolerans among beer-turbulent lactic acid bacteria, by using the culture medium according to the present invention when detecting beer-turbulent lactic acid bacteria by a culture method, Lactobacillus acetotolerans contained in a test sample can be proliferated and detected as colonies.

[0013] The concentration of mevalonic acid in the entire culture medium according to the present invention is not particularly limited as long as it is a concentration sufficient to enable the culture of Lactobacillus acetotolerans. The mevalonic acid concentration of the culture medium according to the present invention is preferably 0.1 mass ppm or more, more preferably 0.4 mass ppm or more, and even more preferably 5 mass ppm or more. In addition, the mevalonic acid concentration of the culture medium according to the present invention is preferably 100 mass ppm or less.

[0014] The oleic acids contained in the culture medium according to the present invention consist of one or more selected from the group consisting of oleic acid esters and oleates. The oleic acids contained in the culture medium according to the present invention may be of only one type, or may contain two or more types in combination. For example, the culture medium according to the present invention may contain both one type of oleic acid ester and one type of oleate.

[0015] The oleic acid ester contained in the culture medium according to the present invention is not particularly limited, and for example, can be appropriately selected and used from various oleic acid esters commonly used as emulsifiers (surfactants). The oleic acid ester is preferably an oleic acid ester of polyoxyethylene sorbitan from the viewpoint of good culturing properties of Lactobacillus acetotolerans, and more preferably one or more selected from the group consisting of polyoxyethylene sorbitan monooleate (Tween 80) and polyoxyethylene sorbitan trioleate (Tween 85). The oleic acid ester contained in the culture medium according to the present invention may be of one type, or two or more types.

[0016] The oleate contained in the culture medium according to the present invention may be an inorganic salt or an organic salt. Examples of the inorganic salt include sodium oleate and potassium oleate. Examples of the organic salt include monoethanolamine oleate, triethanolamine oleate and the like. The oleate contained in the culture medium according to the present invention may be of one type, or two or more types.

[0017] The concentration of oleic acid compounds in the culture medium according to the present invention is not particularly limited, as long as it is a sufficient concentration to enable the cultivation of Lactobacillus acetotolerance. The oleic acid compound concentration in the culture medium according to the present invention is preferably 0.1 g / L or more, and more preferably 0.5 g / L or more. Furthermore, the oleic acid compound concentration in the culture medium according to the present invention is preferably 5 g / L or less, more preferably 2.5 g / L or less, and even more preferably 1.5 g / L or less.

[0018] When the oleic acid ester contained in the culture medium according to the present invention is Tween 80, the concentration of Tween 80 relative to the total culture medium according to the present invention is not particularly limited, as long as it is a sufficient concentration to enable the cultivation of Lactobacillus acetotolerance. The Tween 80 concentration of the culture medium according to the present invention is preferably 0.1 g / L or more, and more preferably 0.5 g / L or more. Furthermore, the Tween 80 concentration of the culture medium according to the present invention is preferably 5 g / L or less, more preferably 2.5 g / L or less, and even more preferably 1.5 g / L or less.

[0019] When the oleic acid ester contained in the culture medium according to the present invention is Tween 85, the concentration of Tween 85 in the total culture medium according to the present invention is not particularly limited, as long as it is a sufficient concentration to enable the cultivation of Lactobacillus acetotolerance. The Tween 85 concentration of the culture medium according to the present invention is preferably 0.1 g / L or more, and more preferably 0.5 g / L or more. Furthermore, the Tween 80 concentration of the culture medium according to the present invention is preferably 5 g / L or less, more preferably 2.5 g / L or less, and even more preferably 1.5 g / L or less.

[0020] The culture medium according to the present invention may contain both Tween 80 and Tween 85. When Tween 80 and Tween 85 are contained, the concentrations of Tween 80 and Tween 85 in the culture medium according to the present invention should be sufficient to enable the cultivation of Lactobacillus acetotolerance. For example, the total concentration of Tween 80 and Tween 85 relative to the entire medium is preferably 0.1 g / L or more, and more preferably 0.5 g / L or more. Furthermore, the total concentration of Tween 80 and Tween 85 in the culture medium according to the present invention is preferably 5 g / L or less, more preferably 2.5 g / L or less, and even more preferably 1.5 g / L or less.

[0021] The carbon source contained in the culture medium according to the present invention can be appropriately selected from various carbon sources commonly used in lactic acid bacteria culture. Examples of such carbon sources include sugars, carbohydrates, and fermentable sugar alcohols. Examples of sugars include monosaccharides such as glucose, galactose, and fructose; disaccharides such as sucrose, lactose, maltose, cellobiose, and trehalose; and oligosaccharides consisting of 3 to 10 sugars such as maltotriose, cellotriose, fucosyl lactose, sialyl lactose, gentianose, and raffinose. Examples of carbohydrates include dextrin, starch, and cellulose. Examples of fermentable sugar alcohols include glycerin, mannitol, xylitol, and ribitol. The carbon source contained in the culture medium according to the present invention may be one type or two or more types. Since various lactic acid bacteria can assimilate them, glucose and sucrose are particularly preferred as carbon sources contained in the culture medium according to the present invention.

[0022] The carbon source concentration in the culture medium according to the present invention is not particularly limited, as long as it is sufficient to allow the growth of lactic acid bacteria. The carbon source concentration relative to the total culture medium according to the present invention can be 1 to 100 g / L, preferably 5 to 50 g / L, and more preferably 10 to 30 g / L.

[0023] The nitrogen source contained in the culture medium according to the present invention can be appropriately selected from various nitrogen sources commonly used in lactic acid bacteria culture. Examples of such nitrogen sources include amino acids, protein hydrolysates (peptones), extracts, urea, ammonia, ammonium salts, and nitrates. Examples of amino acids include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolicin, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, and tyrosine. Examples of peptones include casein peptone, soybean peptone, and meat peptone. Examples of extracts include yeast extract, meat extract, malt extract, and potato extract. Examples of ammonium salts include ammonium nitrate, ammonium sulfate, ammonium acetate, ammonium chloride, and their hydrates. Examples of nitrates include sodium nitrate and its hydrate. The nitrogen source contained in the culture medium according to the present invention may be one type or two or more types. Peptone or extract is preferred as the nitrogen source contained in the culture medium according to the present invention because it can be assimilated by various lactic acid bacteria and can also supply vitamins and minerals.

[0024] The nitrogen source concentration in the culture medium according to the present invention is not particularly limited, as long as it is sufficient to allow the growth of lactic acid bacteria. The nitrogen source concentration relative to the total culture medium according to the present invention can be 1 to 100 g / L, preferably 5 to 50 g / L, and more preferably 10 to 30 g / L.

[0025] The culture medium according to the present invention preferably contains vitamins and minerals in addition to a carbon source, a nitrogen source, mevalonic acid, and polyoxyethylene sorbitan monooleate or polyoxyethylene sorbitan trioleate. The vitamins and minerals allow for more efficient cultivation of beer-turbidity lactic acid bacteria.

[0026] Examples of vitamins include biotin, choline, cyanocobalamin, folic acid, inositol, nicotinic acid, 4-aminobenzoic acid, pantothenic acid, pyridoxine, riboflavin, thiamine, and thymidine. These vitamins may also be contained as salts. When the culture medium according to the present invention contains vitamins, the vitamins in the culture medium may be one type or two or more types.

[0027] Examples of minerals include sodium, potassium, magnesium, calcium, phosphorus, sulfur, chlorine, iodine, zinc, manganese, cobalt, nickel, iron, copper, selenium, chromium, and molybdenum. These may be contained as salts such as nitrates, acetates, citrates, chlorides, phosphates, and sulfates, or as hydrates thereof. When the culture medium according to the present invention contains minerals, the minerals in the culture medium may be one type or two or more types.

[0028] The culture medium according to the present invention can also be prepared by adding mevalonic acid and oleic acid compounds to known culture media or modified culture media used for culturing lactic acid bacteria. Examples of known culture media used for culturing lactic acid bacteria include MRS medium, M17 medium, Logosa agar medium, and MSE (MAYEUX SANDINE ELLIKER) agar medium.

[0029] The culture medium according to the present invention may be a liquid medium or a plate medium. In the case of a plate medium, the culture medium according to the present invention further contains agar in addition to a carbon source, etc. The agar concentration of the culture medium according to the present invention can be about the same as that of a plate medium used in general microbial culture, for example, it can be 0.5 to 3% by mass.

[0030] The culture medium according to the present invention can be prepared by dissolving all of its components, such as a carbon source, in raw water. Because lactic acid bacteria grow well, the pH of the culture medium according to the present invention is preferably 7.5 or less, more preferably 7.0 or less, even more preferably 6.5 or less, and even more preferably 6.0 or less. Furthermore, because the pH decreases due to the lactic acid produced by the growth of lactic acid bacteria, the pH of the culture medium according to the present invention before culturing lactic acid bacteria is preferably 5.0 or higher, and more preferably 5.5 or higher. The culture medium according to the present invention may contain buffers or pH adjusters to adjust the pH within a desired range.

[0031] The culture medium according to the present invention is preferably sterilized by various methods after all components, such as the carbon source, have been dissolved in the raw water. This sterilization can be carried out by various methods commonly used for sterilizing culture media. Preferred sterilization methods for the culture medium according to the present invention include high-pressure steam sterilization and filtration sterilization using a sterilization filter.

[0032] The culture medium according to the present invention can be used as a culture medium for detecting lactic acid bacteria by a culture method. Specifically, a test sample to be investigated for the presence or absence of lactic acid bacteria is inoculated into the culture medium according to the present invention and cultured. The culture conditions are not particularly limited and can be carried out at the same culture temperature and time as general lactic acid bacteria culture. For example, the culture temperature can be 15 to 45°C, preferably 20 to 40°C, and more preferably 20 to 38°C. The culture time can be, for example, 72 to 360 hours, preferably 96 to 300 hours, more preferably 96 to 288 hours, and even more preferably 120 to 240 hours.

[0033] If lactic acid bacteria are present in the test sample, the culture obtained after inoculating it in the culture medium according to the present invention and culturing it for a predetermined time will contain the proliferated lactic acid bacteria. In the case of a liquid medium, the presence or absence of lactic acid bacteria growth can be determined by the absorbance of the medium after culturing (e.g., OD). 600) can be detected by, and in the case of agar plates, detection can be performed by the presence or absence of colony formation. Since lactic acid bacteria in the test sample can be easily isolated and cultured, the culture medium used in the culture according to the present invention is preferably agar plate.

[0034] Since the culture medium according to the present invention enables the cultivation of Lactobacillus acetotolerance, which is difficult to culture, it is preferable to use a beer-like sparkling beverage as a test sample to detect beer-like sparkling beverage turbidity lactic acid bacteria in the beverage. By spreading the beer-like sparkling beverage onto a plate of the culture medium according to the present invention and inoculating it, and culturing it for a predetermined time, beer-like sparkling beverage turbidity lactic acid bacteria, including Lactobacillus acetotolerance, can be detected in the beverage. The beer-like sparkling beverage to be inoculated into the culture medium may be concentrated beforehand by a method that does not damage the lactic acid bacteria. Alternatively, the beer-like sparkling beverage may be filtered, the microorganisms contained in the beverage may be collected on the filter, and the filter may be attached to a plate of the culture medium according to the present invention to inoculate the microorganisms in the beer-like sparkling beverage. [Examples]

[0035] The present invention will now be described in more detail with reference to examples and reference examples, but the present invention is not limited to the following examples.

[0036] [Example 1] (1) Identification of essential components for culturing Lactobacillus acetotolerance We modified MRS medium to investigate the components necessary for culturing Lactobacillus acetotolerance. The MRS medium used was Merck's "GranuCult® MRS broth (de MAN, ROGOSA and SHARPE) acc. ISO 15214". The composition of this MRS medium is shown in Table 1. For plate cultures, agar was added at a concentration of 1.5% by mass.

[0037] [Table 1]

[0038] The feasibility of culturing Lactobacillus acetotolerance was investigated using MRS medium, MRS medium with Tween 80 removed (MRSw / oTW80 medium), MRSw / oTW80 medium with 5 ppm by mass of mevalonic acid added (MRSw / oTW80+MVA(5) medium), MRSw / oTW80+MVA(5) medium with 1 g / L of Tween 80 added (MRSw / oTW80+TW80(1)+MVA(5) medium), and MRSw / oTW80+MVA(5) medium with 0.5 g / L of Tween 80 added (MRSw / oTW80+TW80(0.5)+MVA(5) medium). As Lactobacillus acetotolerance strains, Lactobacillus acetotolerance strain ABBC671 and Lactobacillus acetotolerance strain HC146 were used.

[0039] <Pre-culture and bacterial suspension preparation of lactic acid bacteria> Each Lactobacillus acetotolerance strain was inoculated into MRS liquid medium (MRS plate medium with agar removed) to which mevalonic acid was added at a concentration of 5 ppm by mass, and each strain was cultured at 30°C for 7 days. The pre-cultured bacterial cells had approximately 10 bacteria per 1 mL. 5 The bacteria were suspended in sterile physiological saline solution to form individual cells, and this was used as the bacterial suspension.

[0040] <Culturing lactic acid bacteria> Each bacterial suspension was inoculated onto a 10 cm dish of agar plate by spreading 100 μL of each suspension, and the plates were incubated under anaerobic conditions at 30°C for 10 days. The number of colonies formed on the agar plates was then counted. The results of the colony count (n=3) are shown in Table 2.

[0041] [Table 2]

[0042] As shown in Table 2, colonies were detected in media containing both Tween 80 and mevalonic acid. These results indicate that both mevalonic acid and Tween 80 are necessary for culturing Lactobacillus acetotolerance. Furthermore, in both the MRSw / oTW80+TW80(0.5)+MVA(5) medium and the MRSw / oTW80+TW80(1)+MVA(5) medium, the number of colonies detected in Lactobacillus acetotolerance strain ABBC671 was approximately 2.5 to 4 times greater than that of Lactobacillus acetotolerance strain HC146.

[0043] (2) Effective concentration of mevalonic acid Next, to confirm the effective concentration of mevalonic acid, a medium (MRS+MVA(x) medium) was prepared by adding X mass ppm of mevalonic acid to MRS medium. In the same manner, Lactobacillus acetotolerance strain ABBC671 and Lactobacillus acetotolerance strain HC146 were streaked and inoculated into this medium, and then incubated under anaerobic conditions at 30°C for 10 days. The number of colonies formed on the agar plates was counted. The colony count results (n=3) are shown in Table 3.

[0044] [Table 3]

[0045] In MRS medium to which mevalonic acid was added at a concentration of 0.1 ppm by mass or higher, the number of colonies counted increased in a concentration-dependent manner. In particular, when the mevalonic acid concentration was 0.4 ppm by mass or higher, a sufficient number of colonies were counted. Therefore, from the viewpoint of detection accuracy of Lactobacillus acetotolerance, the mevalonic acid concentration in the culture medium is preferably 0.4 ppm by mass or higher, and more preferably 5 ppm by mass.

[0046] Furthermore, culture media were prepared by adding 5 to 100 ppm by mass of mevalonic acid to MRS medium, and Lactobacillus acetotolerance was similarly inoculated and cultured. The number of colonies formed on the agar plates was then counted. The results of the colony count (n=3) are shown in Table 4.

[0047] [Table 4]

[0048] The results shown in Table 4 demonstrate that Lactobacillus acetotolerance can be cultured using a medium containing Tween 80, a carbon source (glucose), and a nitrogen source (peptone and extract) at a mevalonic acid concentration of 5–100 ppm by mass.

[0049] (3) Evaluation of various fatty acid esters The effects of Tween 20 (polyoxyethylene sorbitan monolaurate), Tween 40 (polyoxyethylene sorbitan monopalmitate), Tween 60 (polyoxyethylene sorbitan monostearate), Tween 85 (polyoxyethylene sorbitan trioleate), and Triton 100 (polyoxyethylene octylphenyl ether), which are structural analogs of Tween 80, on the culture of Lactobacillus acetotolerance were investigated.

[0050] Specifically, in addition to MRS medium, the following media were prepared: MRSw / oTW80+MVA(5) medium with 1.0 g / L of Tween 80 added (MRSw / oTW80+TW80(1.0)+MVA(5) medium), MRSw / oTW80+MVA(5) medium with 0.5 g / L of Tween 20 added (MRSw / oTW80+TW20(0.5)+MVA(5) medium), MRSw / oTW80+MVA(5) medium with 0.5 g / L of Tween 20 added (MRSw / oTW80+TW20(1.0)+MVA(5) medium), and MRSw / oTW80+MVA(5) medium with 0.5 g / L of Tween Medium with 40 added (MRSw / oTW80+TW40(0.5)+MVA(5) medium), Medium with 0.5g / L of Tween 40 added to MRSw / oTW80+MVA(5) medium (MRSw / oTW80+TW40(1.0)+MVA(5) medium), Medium with 0.5g / L of Tween 60 added to MRSw / oTW80+MVA(5) medium (MRSw / oTW80+TW60(0.5)+MVA(5) medium), Medium with 0.5g / L of Tween 60 added to MRSw / oTW80+MVA(5) medium (MRSw / oTW80+TW60(1.0)+MVA(5) medium), Medium with 0.5g / L of Tween Mediums with Tween 85 added (MRSw / oTW80+TW85(0.5)+MVA(5) medium), mediums with 0.5 g / L of Tween 85 added to MRSw / oTW80+MVA(5) medium (MRSw / oTW80+TW85(1.0)+MVA(5) medium), mediums with 0.5 g / L of Triton 100 added to MRSw / oTW80+MVA(5) medium (MRSw / oTW80+TRX100(0.5)+MVA(5) medium), and mediums with 0.5 g / L of Triton 100 added to MRSw / oTW80+MVA(5) medium (MRSw / oTW80+TRX100(1.0)+MVA(5) medium) were prepared. Similarly, Lactobacillus acetotolerance strains ABBC671 and HC146 were smeared and inoculated, respectively, and then cultured under anaerobic conditions at 30°C for 10 days. The number of colonies formed on each agar plate after culturing was counted. The results of the colony count (n=3) are shown in Table 5.

[0051] [Table 5]

[0052] As shown in Table 5, unlike the medium containing Tween 80, no colonies were formed in media containing Tween 20, Tween 40, Tween 60, and Triton 100, confirming that these fatty acid esters do not affect the growth of Lactobacillus acetotolerance. On the other hand, in media containing Tween 85, which is the same oleic acid ester as Tween 80, no colonies of Lactobacillus acetotolerance strain HC146 were detected, but colonies of the more proliferative Lactobacillus acetotolerance strain ABBC671 were detected. A comparison of the number of colonies detected in MRSw / oTW80+TW80(1.0)+MVA(5) medium and MRSw / oTW80+TW85(1.0)+MVA(5) medium for Lactobacillus acetotolerance strain ABBC671 suggests that increasing the amount of inoculated bacterial suspension (test sample) allows for the detection of Lactobacillus acetotolerance strain HC146 colonies in both MRSw / oTW80+TW85(1.0)+MVA(5) medium and MRSw / oTW80+TW85TW85(0.5)+MVA(5) medium.

[0053] (4) Evaluation of oleic acids We investigated whether Lactobacillus acetotolerance could be cultured in media containing oleate salts or oleic acid instead of oleate esters. Specifically, we examined MRSw / oTW80+TW80(1)+MVA(5) medium, MRSw / oTW80+MVA(5) medium with 0.12 g / L of sodium oleate added (MRSw / oTW80+OleNa(0.12)+MVA(5) medium), and MRSw / oTW80+MVA(5) medium with 0.24 g / L of sodium oleate added (MRSw / oTW80+OleNa(0.24)+MV The feasibility of culturing Lactobacillus acetotolerance was investigated using medium A(5), medium MRSw / oTW80+MVA(5) with 0.12 g / L of oleic acid added (MRSw / oTW80+OleA(0.12)+MVA(5) medium), and medium MRSw / oTW80+MVA(5) with 0.24 g / L of oleic acid added (MRSw / oTW80+OleA(0.24)+MVA(5) medium).

[0054] Specifically, in the same manner as described above, Lactobacillus acetotolerance strain ABBC671 was inoculated onto agar plates of each culture medium, and then incubated under anaerobic conditions at 30°C for 10 days. The number of colonies formed on each agar plate after incubation was counted. The results of the colony count (n=3) are shown in Table 6.

[0055] [Table 6]

[0056] As shown in Table 6, colonies were detected in the medium containing sodium oleate instead of Tween 80, but not in the medium containing oleic acid. These results indicate that oleic acid esters or oleates, along with mevalonic acid, are necessary for culturing Lactobacillus acetotolerance.

Claims

1. A culture medium for use in detecting lactic acid bacteria that cause turbidity in beer, It contains a carbon source, a nitrogen source, mevalonic acid, and one or more oleic acids selected from the group consisting of oleic acid esters and oleates. The oleic acid compounds are one or more selected from the group consisting of sodium oleate, polyoxyethylene sorbitan monooleate, and polyoxyethylene sorbitan trioleate. A culture medium in which the aforementioned beer-turbidity lactic acid bacteria is Lactobacillus acetotolerance.

2. The culture medium according to claim 1, wherein the mevalonic acid concentration is 0.1 ppm by mass or more.

3. The culture medium according to claim 2, wherein the mevalonic acid concentration is 0.4 ppm by mass or more.

4. The culture medium according to claim 1, wherein the concentration of the oleic acid compounds is 0.5 g / L or more.

5. The culture medium according to claim 1, wherein the pH is 6.0 or less.

6. The culture medium according to claim 1, which is a plate culture medium.

7. A beer-like effervescent beverage is inoculated into a culture medium according to any one of claims 1 to 6 and cultured, and beer-turbidity lactic acid bacteria are detected. A method for detecting beer-turbidity lactic acid bacteria, wherein the beer-turbidity lactic acid bacteria is Lactobacillus acetotolerance.

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