Culture medium for beer-spoilage lactic acid bacteria and method for detecting beer-spoilage lactic acid bacteria

Abstract

To provide a culture medium suitable for culturing and detecting lactic acid bacteria causing deterioration of a beer-like sparkling beverage, and to provide a method for detecting beer-spoilage lactic acid bacteria of the beer-like sparkling beverage by using the culture medium.SOLUTION: The culture medium and the beer-like sparkling beverage are used for detecting beer-spoilage lactic acid bacteria, and the beer-spoilage lactic acid bacteria are detected by inoculating the beer-spoilage lactic acid bacteria into the culture medium and culturing the beer-spoilage lactic acid bacteria in the culture medium, and Lactobacillus paracoinoides or Lactobacillus lindneri is detected by inoculating the beer-like sparkling beverage into the culture medium and culturing the beer-spoilage lactic acid bacteria in the culture medium. K1, To provide a method for detecting beer-spoilage 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 spoilage of beer-like sparkling beverages, and a method for detecting beer-spoilage-causing lactic acid bacteria in beer-like sparkling beverages using the culture medium. [Background technology]

[0002] The quality of processed foods and beverages must be maintained from the time they are produced until they reach the consumer's mouth. In particular, since microorganisms not only cause quality deterioration but also food poisoning, preventing microbial contamination is extremely important from the perspective of food safety. A common method for microbial testing of foods and beverages is the culture method, in which microorganisms are isolated and cultured on appropriate plate media from the food or beverage sample. The microorganisms isolated and cultured by the culture method can be analyzed for their bacteriological properties and genetic information to identify the microbial species.

[0003] Beer and other beer-like sparkling beverages can be subject to spoilage due to microbial contamination, and there is a need for a method for more accurately detecting the presence or absence of contamination by microorganisms that cause spoilage, such as beer-spoilage-causing lactic acid bacteria.

[0004] Lactic acid bacteria are one of the microorganisms that cause spoilage in various foods and beverages, in addition to beer-like sparkling beverages. Therefore, detection of contaminating lactic acid bacteria in foods and beverages is widely practiced in the food and beverage manufacturing industry. Many lactic acid bacteria can be detected using common media for detecting lactic acid bacteria, such as MRS (de Man-Rogosa-Sharpe) medium. However, some lactic acid bacteria are only susceptible to spoilage in specific foods and beverages, and some species and strains have been confirmed to be undetectable using common media. For example, to cultivate beer spoilage bacteria that are difficult to culture in MRS medium, BMRS medium is used, prepared by dissolving MRS medium in beer instead of water (Non-Patent Document 1). Furthermore, a medium containing sufficient amounts of mevalonic acid and oleic acid ester or oleate salts has been reported as a medium for culturing Lactobacillus acetotolerans, a type of beer spoilage bacterium, without using beer (Patent Document 1). Patent Document 1 states: [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2024-000703 [Non-patent literature]

[0006] [Non-Patent Document 1] Suzuki et al, Journal of Applied Microbiology, 2008, vol.104, p.1458-1470. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to provide a culture medium suitable for culturing and detecting lactic acid bacteria that cause spoilage of beer-like sparkling beverages, and a method for detecting beer-spoilage-causing lactic acid bacteria in beer-like sparkling beverages using the culture medium. [Means for solving the problem]

[0008] The present inventors have discovered that by adding sufficient amounts of maltose and one or more members selected from the group consisting of thioglycolic acid, glutathione, hemin, vitamin K1, and L-cysteine ​​hydrochloride to a culture medium, it is possible to cultivate Lactobacillus paracollinoides and Lactobacillus lindneri, which are responsible for spoilage of beer, and have completed the present invention.

[0009] The culture medium and the method for detecting beer spoilage-causing lactic acid bacteria according to the present invention are as follows. [1] A culture medium characterized by containing a carbon source, a nitrogen source, one or more substances selected from the group consisting of thioglycolic acid, glutathione, hemin, vitamin K1, and L-cysteine ​​hydrochloride, and maltose, and being used for detecting beer spoilage-causing lactic acid bacteria. [2] The culture medium according to [1], containing a carbon source, a nitrogen source, thioglycolic acid, and maltose. [3] The culture medium according to [2] above, wherein the thioglycolic acid concentration is 0.05 g / L or more. [4] The culture medium according to any one of [1] to [3] above, wherein the maltose concentration is 0.05 g / L or more. [5] The culture medium according to any one of [1] to [4] above, which does not contain beer. [6] The culture medium according to any one of [1] to [5] above, having a pH of 6.0 or less. [7] The culture medium according to any one of [1] to [6] above, wherein the beer spoilage-causing lactic acid bacterium is Lactobacillus paracoinoides or Lactobacillus lindneri. [8] The culture medium according to any one of [1] to [7] above, which is a plate medium. [9] A method for detecting beer-spoilage-causing lactic acid bacteria, comprising inoculating a beer-like sparkling beverage into a culture medium according to any one of [1] to [8] above, culturing the beverage, and detecting beer-spoilage-causing lactic acid bacteria.

[10] A method for detecting beer spoilage-causing lactic acid bacteria, comprising inoculating a beer-like sparkling beverage into a culture medium according to any one of [1] to [8] above, culturing the beverage, and detecting Lactobacillus paracoinoides or Lactobacillus lindneri. [Effects of the Invention]

[0010] The culture medium of the present invention is capable of culturing not only beer-spoilage lactic acid bacteria that can be cultured in MRS medium, a conventional lactic acid bacteria culture medium, but also Lactobacillus paracoinoides and Lactobacillus lindneri, which are difficult to culture in MRS medium. Therefore, a culture method using this culture medium can accurately detect beer-spoilage lactic acid bacteria, including Lactobacillus paracoinoides and Lactobacillus lindneri, in beer-like sparkling beverages without using beer. DETAILED DESCRIPTION OF THE INVENTION

[0011] In this invention and this specification, "beer-like sparkling beverage" refers to a beverage containing carbon dioxide that has a beer-like flavor. Furthermore, "beer-like" refers to a flavor or taste that is reminiscent of beer, regardless of the product name or labeling. In other words, a beer-like sparkling beverage refers to a sparkling beverage that has a flavor, taste, and texture equivalent to or similar to that of beer, and that has high thirst quenching properties and drinkability, regardless of the alcohol content, the use of malt and hops, or the presence or absence of fermentation.

[0012] The beer-like sparkling beverage of the present invention may be an alcoholic beverage or a so-called non-alcoholic or low-alcoholic beverage having an alcohol content of less than 1% by volume. It may also be a beverage made from malt or a beverage not made from malt, a beverage produced through a fermentation process, or a beverage produced without a fermentation process. Specific examples include beer, malt-based happoshu, malt-free sparkling alcoholic beverages, low-alcohol sparkling beverages, and non-alcoholic beer. Other examples include liqueurs or spirits obtained by blending a beverage made from malt through a fermentation process with an alcohol-containing distillate. The alcohol-containing distillate is a solution containing alcohol obtained by distillation, and may be, for example, raw alcohol, such as spirits, whiskey, brandy, vodka, rum, tequila, gin, or shochu.

[0013] The culture medium of the present invention contains a carbon source, a nitrogen source, one or more substances selected from the group consisting of thioglycolic acid (CAS No.: 68-11-1), glutathione (CAS No.: 70-18-8), hemin (CAS No.: 16009-13-5), vitamin K1 (CAS No.: 12001-79-5), and L-cysteine ​​hydrochloride (CAS No.: 52-89-1), and maltose (CAS No.: 69-79-4), and is characterized in that it is used for detecting beer spoilage-causing lactic acid bacteria. Among the beer spoilage lactic acid bacteria, Lactobacillus paracoinoides and Lactobacillus lindneri require both thioglycolic acid and maltose for cultivation. Therefore, when detecting beer spoilage lactic acid bacteria by culture, the culture medium of the present invention can be used to grow Lactobacillus paracoinoides and Lactobacillus lindneri contained in the test sample and detect them as colonies.

[0014] The culture medium of the present invention may contain one or more compounds selected from the group consisting of thioglycolic acid, glutathione, hemin, and vitamin K1, and may, for example, contain all of thioglycolic acid, glutathione, hemin, and vitamin K1, or may contain glutathione, hemin, and vitamin K1, or may contain thioglycolic acid, hemin, and vitamin K1. From the viewpoint of achieving a higher culture efficiency for beer spoilage-causing lactic acid bacteria, the culture medium of the present invention is preferably a culture medium containing a carbon source, a nitrogen source, thioglycolic acid, and maltose.

[0015] The concentration of one or more selected from the group consisting of thioglycolic acid, glutathione, hemin, vitamin K1, and L-cysteine ​​hydrochloride in the culture medium of the present invention is not particularly limited, as long as it is sufficient to enable the cultivation of Lactobacillus parachoinoides and Lactobacillus lindneri. When the culture medium of the present invention contains thioglycolic acid, the thioglycolic acid concentration in the culture medium of the present invention is preferably 0.05 g / L or more, more preferably 0.10 g / L or more, even more preferably 0.20 g / L or more, and even more preferably 0.25 g / L or more. Furthermore, the thioglycolic acid concentration in the culture medium of the present invention is preferably 1.00 g / L or less.

[0016] The maltose concentration in the culture medium of the present invention is not particularly limited, as long as it is sufficient to allow the cultivation of Lactobacillus parachoinoides and Lactobacillus lindneri. The maltose concentration in the culture medium of the present invention is preferably 0.05 g / L or more, more preferably 0.10 g / L or more, even more preferably 0.25 g / L or more, and even more preferably 0.50 g / L or more. The maltose concentration in the culture medium of the present invention is preferably 20.0 g / L or less, more preferably 15.0 g / L or less, and even more preferably 10.0 g / L or less.

[0017] The carbon source contained in the culture medium of 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 (cane sugar), lactose, maltose, cellobiose, and trehalose; and oligosaccharides consisting of approximately 3 to 10 sugar units such as maltotriose, cellotriose, fucosyllactose, sialyllactose, 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 of the present invention may be one type or two or more types. Glucose and sucrose are particularly preferred carbon sources because they can be assimilated by a variety of lactic acid bacteria.

[0018] 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 in the culture medium according to the present invention relative to the total culture medium can be 1 to 100 g / L, preferably 5 to 50 g / L, and more preferably 10 to 30 g / L.

[0019] The nitrogen source contained in the culture medium of 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, pyrrolysine, 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 hydrates thereof. Examples of nitrates include sodium nitrate and its hydrates. The culture medium according to the present invention may contain one or more nitrogen sources, but peptone and extract are preferred as nitrogen sources because they can be assimilated by various lactic acid bacteria and can also provide vitamins, minerals, and the like.

[0020] The nitrogen source concentration in the culture medium of 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 in the culture medium of the present invention relative to the total culture medium can be 1 to 100 g / L, preferably 5 to 50 g / L, and more preferably 10 to 30 g / L.

[0021] The culture medium of the present invention preferably contains a carbon source, a nitrogen source, one or more selected from the group consisting of thioglycolic acid, glutathione, hemin, vitamin K1, and L-cysteine ​​hydrochloride, maltose, and further contains vitamins (excluding vitamin K1) and minerals. The vitamins and minerals enable beer spoilage-causing lactic acid bacteria to be cultured more efficiently.

[0022] 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 be contained in the form of a salt. When the culture medium according to the present invention contains vitamins, the culture medium may contain one type of vitamin or two or more types of vitamins.

[0023] 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 culture medium may contain one type of mineral or two or more types of minerals.

[0024] The culture medium of the present invention can also be prepared by adding maltose and one or more selected from the group consisting of thioglycolic acid, glutathione, hemin, vitamin K1, and L-cysteine ​​hydrochloride to a known medium or a modified medium thereof used for culturing lactic acid bacteria. Examples of known media used for culturing lactic acid bacteria include MRS medium, M17 medium, Rogosa agar, and MSE (MAYEUX SANDINE ELLIKER) agar. A particularly preferred culture medium of the present invention is a medium containing maltose and one or more selected from the group consisting of thioglycolic acid, glutathione, hemin, vitamin K1, and L-cysteine ​​hydrochloride in a small amount of MRS medium (e.g., 5 to 25% by mass of MRS medium), and more preferably a medium containing 0.05 g / L or more of thioglycolic acid and 0.05 g / L or more of maltose in 5 to 25% by mass of MRS medium.

[0025] The culture medium of the present invention may be a liquid medium or a plate medium. In the case of a plate medium, the culture medium of the present invention further contains agar in addition to a carbon source, etc. The agar concentration of the culture medium of the present invention can be approximately the same as that of plate media used for general microbial culture, for example, 0.5 to 3 mass%.

[0026] The culture medium of the present invention can be prepared by dissolving all of the constituents, such as a carbon source, in raw water. To ensure good growth of lactic acid bacteria, the pH of the culture medium of 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, the pH of the culture medium of the present invention before culturing lactic acid bacteria is preferably 4.5 or more, more preferably 5.0 or more. The culture medium of the present invention may contain a buffer or pH adjuster to adjust the pH within a desired range.

[0027] The culture medium according to the present invention is preferably sterilized by various methods after dissolving all of the constituents, such as the carbon source, in raw water. The 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 sterilizing filter.

[0028] The culture medium of 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 examined for the presence or absence of lactic acid bacteria is inoculated into the culture medium of the present invention and cultured. The culture conditions are not particularly limited, and the culture can be performed at the same culture temperature and time as those used for 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.

[0029] When the test sample contains lactic acid bacteria, the culture medium according to the present invention is inoculated and cultured for a predetermined period of time, and the culture contains the proliferated lactic acid bacteria. In the case of a liquid medium, the presence or absence of proliferation of lactic acid bacteria can be determined by measuring the absorbance (e.g., OD 600 ) and in the case of a plate medium, detection can be made by the presence or absence of colony formation. The culture medium according to the present invention used for culture is preferably a plate medium, since lactic acid bacteria in a test sample can be easily isolated and cultured.

[0030] Since the culture medium of the present invention is capable of culturing the difficult-to-cultivate Lactobacillus paracoinoides and Lactobacillus lindneri, it is preferable to use a beer-like sparkling beverage as a test sample to detect beer-spoilage lactic acid bacteria in the beer-like sparkling beverage. The beer-like sparkling beverage can be inoculated by smearing it onto a plate of the culture medium of the present invention and culturing it for a predetermined period of time, thereby detecting beer-spoilage lactic acid bacteria, including Lactobacillus paracoinoides and Lactobacillus lindneri, in the beer-like sparkling beverage. The beer-like sparkling beverage to be inoculated into the culture medium may be concentrated in advance by a method that does not harm the lactic acid bacteria. Alternatively, the beer-like sparkling beverage may be filtered to collect the microorganisms contained in the beverage onto the filter, and the filter may be attached to a plate of the culture medium of the present invention to inoculate the microorganisms in the beer-like sparkling beverage. [Example]

[0031] 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.

[0032] [Example 1] (1) Identification of essential components for the cultivation of Lactobacillus paracoinoides and Lactobacillus lindneri The MRS medium was modified to investigate the components necessary for the cultivation of Lactobacillus paracoinoides and Lactobacillus lindneri. Merck's GranuCult® MRS broth (de MAN, ROGOSA, and SHARPE) acc. ISO 15214 was used as the MRS medium. The composition of the MRS medium is shown in Table 1. Agar was added to the plate medium at 1.5% by mass. The pH of each medium was 5.0 to 5.4.

[0033] [Table 1]

[0034] Ten types of culture media were prepared according to the formulations shown in Tables 2 to 5. In the tables, "ABD" is a medium prepared by adding sodium acetate to a 5% concentration medium of MRS medium prepared with beer, and "ABD w / o beer" is a medium prepared by adding sodium acetate to a 5% concentration medium of MRS medium prepared with water.

[0035] [Table 2]

[0036] [Table 3]

[0037] [Table 4]

[0038] [Table 5]

[0039] <Pre-cultivation of lactic acid bacteria and preparation of bacterial solution> The lactic acid bacteria used were Lactobacillus paracoinoides ABBC74 VN5 strain, Lactobacillus paracoinoides ABBC90 VN13 strain, and Lactobacillus lindneri ABBC278 VN22 strain. Each lactic acid bacterium was inoculated into ABD liquid medium (ABD plate medium without the agar) and cultured at 30°C for 7 days. The pre-cultured bacteria were cultured at a density of approximately 10 3 The cells were suspended in sterile physiological saline to give a single suspension, which was used as the bacterial solution.

[0040] <Cultivation of lactic acid bacteria> 100 μL of each bacterial solution was smeared and inoculated onto a 9 cm petri dish plate medium and cultured at 25°C under anaerobic conditions for 14 days. The number of colonies formed on the plate medium was then counted (n=3, except for ABD medium, where n=2). The results of the colony counts (mean and standard deviation) are shown in Table 6.

[0041] [Table 6]

[0042] As shown in Table 6, none of the strains grew in ABD w / o beer medium, in which the beer in ABD medium was replaced with water, suggesting that beer was necessary for detecting these lactic acid bacteria. In contrast, in medium 1, in which sodium thioglycolate and maltose were added to MRS, growth of all lactic acid bacteria was confirmed to be comparable to that in ABD medium. These results confirmed that by adding appropriate amounts of sodium thioglycolate and maltose to a medium supplemented with 25% MRS, it is possible to cultivate Lactobacillus parachoinoides and Lactobacillus lindneri without using beer.

[0043] (2) Effective concentration of thioglycolic acid Next, to confirm the effective concentration of thioglycolic acid, media containing 25% MRS and 0.01, 0.05, or 0.25 g / L of sodium thioglycolate (mediums 1–3) and ABD medium were prepared (Tables 2–3). Lactobacillus parachoinoides and Lactobacillus lindneri were inoculated onto each medium as described in (1) above, and then cultured at 25°C for 14 days under anaerobic conditions. The number of colonies formed on the plates was counted (n = 3, except for ABD medium, where n = 2). The results (mean and standard deviation) of colony counts are shown in Table 7.

[0044] [Table 7]

[0045] As shown in Table 7, for all lactic acid bacteria, the number of colonies increased depending on the thioglycolic acid concentration, and culture was favorable in media with a thioglycolic acid concentration of 0.05 g / L or higher.

[0046] (3) Effective concentration of maltose Next, to confirm the effective concentration of maltose, media containing 25% MRS supplemented with 0, 0.05, 0.10, or 0.50 g / L of maltose (media 4–7) and ABD media were prepared (Tables 2–4). Lactobacillus paracoinoides and Lactobacillus lindneri were inoculated onto each medium as described in (1) above, and then cultured at 25°C for 14 days under anaerobic conditions. The number of colonies formed on the plates was counted (n = 3, except for ABD media, where n = 2). The colony count results (mean and standard deviation) are shown in Table 8.

[0047] [Table 8]

[0048] As shown in Table 8, a tendency for the number of colonies to increase depending on the maltose concentration was observed for all lactic acid bacteria. In particular, culturing was favorable in media with a maltose concentration of 0.10 g / L or higher.

[0049] (4) Substitute for sodium thioglycolate Next, to confirm the effectiveness of substitution for sodium thioglycolate, we prepared a medium (medium 8) containing 25% MRS supplemented with reducing agents such as glutathione, hemin solution, vitamin K1 solution, and L-cysteine ​​hydrochloride instead of sodium thioglycolate, and prepared ABD medium (Table 9). Hemin solution was prepared by dissolving 20 mg of hemin in 40 mL of 1 N NaOH. Vitamin K1 solution was prepared by dissolving 5 μL of vitamin K1 in 1 mL of ethanol. Lactobacillus parachoinoides and Lactobacillus lindneri were inoculated onto each medium as described above in (1) and then cultured at 25°C for 14 days under anaerobic conditions. The number of colonies formed on the plates was counted (n = 3 for medium 8, n = 2 for ABD medium). The colony counts (mean and standard deviation) are shown in Table 9.

[0050] [Table 9]

[0051] As shown in Table 9, both Lactobacillus parachoinoides and Lactobacillus lindneri could be cultured in a medium containing a mixture of glutathione, hemin, vitamin K1, and L-cysteine ​​hydrochloride. This suggests that sodium thioglycolate can be substituted with reducing agents such as glutathione, hemin, vitamin K1, and L-cysteine ​​hydrochloride.

Claims

1. A culture medium characterized by containing a carbon source, a nitrogen source, one or more substances selected from the group consisting of thioglycolic acid, glutathione, hemin, vitamin K1, and L-cysteine ​​hydrochloride, and maltose, and being used for detecting beer spoilage-causing lactic acid bacteria.

2. 2. The culture medium according to claim 1, comprising a carbon source, a nitrogen source, thioglycolic acid, and maltose.

3. 3. The culture medium according to claim 2, wherein the concentration of thioglycolic acid is 0.05 g / L or more.

4. 2. The culture medium according to claim 1, wherein the maltose concentration is 0.05 g / L or more.

5. 10. The culture medium of claim 1, which does not contain beer.

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

7. 2. The culture medium according to claim 1, wherein the beer spoilage-causing lactic acid bacterium is Lactobacillus paracoinoides or Lactobacillus lindneri.

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

9. A method for detecting beer-spoilage-causing lactic acid bacteria, comprising inoculating a beer-like sparkling beverage into the culture medium according to any one of claims 1 to 8, culturing the beverage, and detecting beer-spoilage-causing lactic acid bacteria.

10. A method for detecting beer spoilage-causing lactic acid bacteria, comprising inoculating a beer-like sparkling beverage into the culture medium according to any one of claims 1 to 8, culturing the beverage, and detecting Lactobacillus paracoinoides or Lactobacillus lindneri.

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