JP2006136272A - Semifluid medium for detecting lactobacillus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting lactobacillus, enabling the presence/absence of lactobacillus in test samples to be exactly and efficiently judged visually at early stages in order to effectively excluding lactobacillus contamination, thus also widely applicable to testing general food/drink, water, etc. and their production process testing, and to provide a medium for detecting lactobacillus, advantageously usable in the above method. <P>SOLUTION: The medium is a semifluid medium for detecting lactobacillus comprises 15-25 g/L of glucose, a pH indicator such as Bromocresol Purple and 0.025-0.200% of agar-agar. The method for detecting lactobacillus comprises admixing the semifluid medium with a test sample followed by making a culture e.g. at 25°C for 2 days and visually judging the color change of the medium before and after culture. It is preferable that the semifluid medium be free from dipotassium hydrogenphosphate and low in buffering ability, or be 6.0-7.5 in pH level after undergoing sterilization and prior to undergoing culture. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a semi-fluid medium for detecting lactic acid bacteria that can visually determine the presence or absence of lactic acid bacteria in a test sample, and a method for detecting lactic acid bacteria using the same.

  Lactic acid bacteria are generic names for bacteria that ferment sugar and produce large amounts of lactic acid, and are currently classified into 12 genera (Lactobacillus, Pediococcus, Tetragenococcus, Carnobacterium, Vagococcus, Leuconostoc, Weissella, Oenococcus, Atopobium, Streptococcus, Enterococcus, Lactococcus) Two hundred and ten species have been reported. Lactic acid bacteria have long been used in many food processing such as miso, soy sauce, sake, wine, yogurt, cheese, lactic acid bacteria beverages, pickles, fermented sausages, fermented tea, and unfamiliar. In addition, the health function of lactic acid bacteria has been elucidated, and foods for specified health use using probiotic lactic acid bacteria are establishing a new food market. However, lactic acid bacteria used as useful bacteria, mainly in the food processing field, cause various deterioration such as acidity, acid odor, and production of neto, even if the same bacterial species performs the same function. . For example, roasted moromi that tastes sour in sake brewing, fire that causes bottled products to deteriorate, and the taste and turbidity of unheated beer, or acidity in marine products and low-salt pickles are mainly lactic acid bacteria. Is the cause. Meat products such as ham and wiener sausage and processed foods such as side dishes, hamburgers and fried foods are also involved in the processing stage and product quality abnormalities.

  Lactic acid bacteria rarely cause health hazards, but the occurrence of deterioration is a significant economic loss, and its control is an important issue for food processing manufacturers. In order to prevent food quality abnormalities caused by lactic acid bacteria in various foods as well as meat products, prioritizing elimination of lactic acid bacteria leads to the elimination of many pathogenic bacteria and other harmful bacteria. Moreover, since the inspection of lactic acid bacteria in such a case is part of the quality control of mass-produced products at the manufacturer, it is necessary not only to be easy to operate but also to obtain results quickly. For these reasons, the following medium for lactic acid bacteria has been used.

1) BCP-added plate count agar medium It is described in the official method for measuring the number of bacteria (Food Hygiene Inspection Guidelines Microorganism, supervised by the Ministry of Health, Labor and Welfare, 2004) A colony in which the periphery of the colony grown on the agar medium becomes yellow is measured as lactic acid bacteria (BCP changes from blue to yellow when it becomes acidic due to lactic acid production).

2) MRS agar medium, BL agar medium These two mediums are both described in the official method for measuring the number of bacteria (Food Hygiene Inspection Guidelines Microorganism, supervised by the Ministry of Health, Labor and Welfare, 2004). Although effective for the growth of lactic acid bacteria, there is no selectivity, and microorganisms other than lactic acid bacteria grow actively. Both have buffer capacity. It may be used as a liquid medium without agar.

3) APT Agar Developed for lactic acid bacteria (Sharpe, MEand Fryer, TF, Laboratory Practice, June, 1965). Contains 10 g / L of glucose, has buffer capacity, and does not contain an indicator. It may be used as a liquid medium without agar.

4) APT agar + BCP
BPT, a pH indicator, added to APT agar (Compendium of methods for the microbiological examination of foods, Second edition, Marvin L. Speck, Editor, 1984).

5) LBS agar medium Developed as a selective medium for lactobacilli and reported to have higher selectivity for lactobacilli than previously used tomato juice medium (Rogosa, Mitchell and Wiseman, J. Bacteriol, 62: 132) , 1951). Agar plates are cultured at 35 ° C. in a carbon dioxide atmosphere. Contains glucose 20 g / L, has buffering capacity, and does not contain an indicator. It may be used as a liquid medium without agar.

  As a related technique, a culture medium for lactic acid bacteria culture containing a component obtained from a barley shochu distillation residue has been proposed (see Patent Document 1). It relates to the effective use of shochu distillation residue, and is a medium intended to increase the yield of lactic acid bacteria (increase the amount of bacterial cells by culture), and not to detect lactic acid bacteria. In addition, buttermilk was added to an existing liquid medium (MRS medium, LB medium) or a lactic acid bacteria growth promoting medium (see Patent Document 2) containing calcium salt in an existing liquid medium (MRS medium, LB medium). In addition to a medium for promoting lactic acid bacteria growth (see Patent Document 3), a method of adding a fluorescent substance (acridine orange-10-dodecyl bromide) to the medium and measuring the number of viable bacteria by fluorescence intensity (see Patent Document 4), By containing whiskey distillation residue in an existing medium such as Modified NBB medium, a medium for measuring the number of bacteria that rapidly grows lactic acid bacteria (see Patent Document 5), and as a growth promoting substance for lactic acid bacteria, cytidine, thymidine, and wort Contains fermentation medium, medium for detection and count of lactic acid bacteria harmful to beer brewing (see Patent Document 6), sake lees or sake lees extract Growth promoting medium for lactic acid bacteria (see Patent Document 7) it has been proposed that.

  In addition, in order to maintain the anaerobic state, a medium containing a gelling agent (Constarch, alginic acid, agar) is used. Due to the viscosity of the gelling agent, the dissolution of oxygen in the medium is prevented and the anaerobic state is maintained. A method for promoting the growth of bifidobacteria and lactic acid bacteria (see Patent Document 8), which are sex-resistant bacteria, and when culturing using an agar medium, the oxygen partial pressure is adjusted to separate and culture Lactobacillus acidophilus from a stool sample Method (see Patent Document 9) and No. of Lactobacillus genus containing cheese whey. In addition to 14 culture media for separation culture (see Patent Document 10), free bases such as adenine and guanine, ribonucleosides such as adenosine, and deoxyribonucleosides such as 2′-deoxyadenosine, a carbon source for promoting growth, Medium for culturing lactic acid bacteria belonging to the genus Lactobacillus and Bifidobacteria containing buffer, nitrogen source, trace element, antioxidant, vitamin, propionic acid or its salt in addition to oligosaccharide A medium for selective / viable cell count measurement for the purpose of preferential growth of Bifidobacterium out of products containing Bifidobacterium and lactic acid bacteria, and measuring the number of cells (patent document) 12) and for detection of lactic acid bacteria belonging to the genus Bifidobacterium containing lithium chloride and galactose as growth promoters in an agar medium. A medium for measuring the number of bacteria (see Patent Document 13), an agar medium for measuring the number of viable bacteria selected for culturing lactic acid bacteria belonging to the genus Bifidobacterium containing oligosaccharides (see Patent Document 14), pH A liquid selective medium (see Patent Document 15) for culturing lactic acid bacteria belonging to the genus Bifidobacterium using an indicator has been proposed.

  Moreover, in order to promote growth, the nutrient medium of lactic acid bacteria for sourdough for improving the flavor of bread making using yeast extract, manganese and acetic acid and using a buffer with acetic acid and sodium acetate (Patent Document 16) And a method in which lactic acid bacteria producing nisin (Lacrtobacillus lactis) are effectively grown in a medium to which components obtained from shochu distillation residue are added to obtain nisin as a product (see Patent Document 17) Or a method of separating low-turbidity soy lactic acid bacteria on an agar medium that is cultured in a liquid medium containing sodium benzoate and 0.1 M potassium phosphate, and contains an antibacterial substance, cobalt chloride, and does not contain salt (see Patent Document 18) ), Or orthophosphate (contributing to buffer capacity) added to the skim milk solution, boiled, centrifuged to remove some of the calcium in the milk components, and then added the organic acid salt Contains a medium for lactic acid bacteria starter (see Patent Document 19), a medium for promoting the growth of lactic acid bacteria for soy sauce with increased citrate or malate buffer capacity (see Patent Document 20), and L-cysteine A method of cultivating Lactobacillus cerevisiae, particularly Lactobacillus samplan ciscisco used for bread production, to increase the cell recovery effect (see Patent Document 21) has been proposed.

  In addition, 5-20 g / L glucose, methyl red, bromocresol purple, chlorophenol red, bromothymol blue, bromocresol blue, alizarin red S monohydrate and other pH indicators, used to prevent diffusion in the pH indicator zone A medium (see Patent Document 22) for detecting and measuring enteric bacteria, which contains a buffer to be produced, has been proposed.

Japanese Patent No. 3527661 Japanese Patent No. 2673333 JP 2000-102380 A Japanese Patent Laid-Open No. 6-113887 Japanese Patent Laid-Open No. 03-91493 Japanese Patent Laid-Open No. 03-130071 JP-A-3-172171 JP 2000-23663 A Japanese Patent No. 3071128 Japanese Patent No. 3184572 JP 2000-279166 A Japanese Patent Laid-Open No. 11-28098 JP-A-6-343491 JP-A-4-20283 Japanese Patent Laid-Open No. 7-99995 Japanese Patent No. 3118761 Japanese Patent Laid-Open No. 2004-168 JP 2003-79363 A Japanese Unexamined Patent Publication No. 60-105489 JP-A-57-102184 JP 54-143585 A Japanese National Patent Publication No. 10-504464

  An object of the present invention is to effectively and efficiently eliminate contamination of lactic acid bacteria at an early stage and accurately, and can visually determine the presence or absence of lactic acid bacteria in a test sample. It is an object to provide a method for detecting lactic acid bacteria that can be widely used in inspections and manufacturing process inspections, and a medium for detecting lactic acid bacteria that is advantageously used in the detection method.

  The present inventors have intensively studied to solve the above problems, promote the growth of lactic acid bacteria present in the test sample, increase the amount of lactic acid produced, and generate a change in the color of the medium due to the pH indicator, We began to study the basic concept of visually amplifying the generated color change.

  First, in order to promote the growth of lactic acid bacteria present in the test sample, paying attention to the growth promotion effect of glucose often used as a medium component as a nutrient source, the concentration is increased to 15 to 25 g / L. (The glucose concentration of the existing medium for lactic acid bacteria is 5 to 20 g / L). In addition, by utilizing the characteristics of lactic acid bacteria that utilize sugar to produce lactic acid, by increasing the concentration of glucose, the amount of lactic acid produced by lactic acid bacteria is increased, and a pH indicator (acid The color changes when the pH decreases due to the decrease in color) or the change in the color becomes clear, so that it is possible to determine at a glance whether or not the lactic acid bacteria have grown.

  Next, bromocresol purple (BCP) was used as a medium component as a pH indicator that changes color when the pH of the medium becomes acidic due to the production of lactic acid. A pH indicator is generally used for confirming the growth of a target bacterium by color change, such as a BCP-added plate count agar medium (agar medium for measuring the number of lactic acid bacteria) and LIM medium (for intestinal bacteria confirmation). ing. Moreover, in order to change the color of a pH indicator rapidly and vividly, the buffer capacity of the medium was lowered. In many culture media including lactic acid bacteria culture media, the culture media have a buffering capacity for promoting growth and protecting the grown cells. This is because the buffering capacity reduces damage to the cells by the acid that is a metabolite of lactic acid bacteria. For the purpose of detecting lactic acid bacteria, that is, for the purpose of determining whether lactic acid bacteria are contaminated in the food / beverage or food production process, buffer capacity is not required, but rather, buffer capacity is reduced or eliminated. In addition, it was found that the presence or absence of lactic acid bacteria can be more clearly determined by combining with a pH indicator.

  Then, in order to amplify the generated color change visually to facilitate the determination, the inventors made a special study and found a semi-fluid liquid medium. Usually, semi-fluid medium is used to maintain the anaerobic state by preventing the dissolution of oxygen in the medium due to its viscosity when culturing anaerobic bacteria, or to determine whether microorganisms are motile. (Such as LIM medium for Salmonella). However, using a semi-flowing viscosity, it was discovered that the color change of the pH indicator was significantly sharper evenly throughout the medium in the test tube.

  In order to detect a small amount of lactic acid bacteria that cannot be measured as the number of bacteria per gram by integrating the above knowledge, liquid media rich in nutrients such as carbon source and nitrogen source, or iron, magnesium, manganese, etc. In a liquid medium containing trace elements and vitamins, we found that the above problem can be solved by increasing the glucose concentration, adding a pH indicator, reducing or removing the buffer capacity, and making a semi-fluid medium. The invention has been completed.

  That is, the present invention is (1) adding and mixing a test sample to a semi-fluid medium for detecting lactic acid bacteria containing 15 to 25 g / L glucose and a pH indicator, culturing, and changing the color of the medium before and after the culture. (2) The method for detecting lactic acid bacteria, wherein the semi-fluid medium is a low buffer capacity semi-fluid medium containing no dipotassium hydrogen phosphate. The method for detecting lactic acid bacteria according to (1) or (2) above, wherein (3) the semi-fluid medium has a pH of 6.0 to 7.5 after sterilization and before culturing. Detection method and (4) pH indicator is from bromocresol purple, bromocresol green, methyl red, litmus, methyl purple, p-nitrophenol, chlorophenol red, bromothymol blue, alizarin red S The method for detecting lactic acid bacteria according to any one of (1) to (3) above, wherein (5) the semi-fluid medium contains 0.025 to 0.200% agar. The method for detecting a lactic acid bacterium according to any one of the above (1) to (4), which is a semi-fluid medium, or (6) a semi-fluid medium comprising 12.5 g / L tryptone, 7.5 g / L Yeast extract, 20 g / L glucose, 5 g / L trisodium citrate dihydrate, 5 g / L sodium chloride, 0.8 g / L magnesium sulfate, 0.14 g / L manganese chloride; 04 g / L iron (II) sulfate primary, 0.001 g / L thiamine hydrochloride, 0.2 g / L Tween 80, 0.75 g / L agar, 0.06 g / L bromocresol purple (sterile Later pH 6.8 ± 0.2) The method for detecting lactic acid bacteria according to any one of (1) to (5) above.

  Further, the present invention is (7) the method for detecting lactic acid bacteria according to any one of (1) to (6) above, wherein the change in color of the medium after culturing at 25 ° C. for 2 days is visually determined, (8) A semi-fluid medium for detecting lactic acid bacteria, which contains 15 to 25 g / L glucose and a pH indicator, and can detect lactic acid bacteria by visually determining the color change of the medium before and after the culture; A semi-fluid medium for detecting lactic acid bacteria according to the above (8), which is a low buffer capacity semi-fluid medium free of dipotassium hydrogen phosphate, and (10) the pH after sterilization and before culturing is 6. The semi-fluid medium for detecting lactic acid bacteria described in (8) or (9) above, wherein (11) the pH indicator is bromocresol purple, bromocresol green, methyl red, litmus, Methyl purple, p-ni A semi-fluid medium for detecting lactic acid bacteria according to any one of (8) to (10) above, which is selected from rophenol, chlorophenol red, bromothymol blue, and alizarin red S; (12) 0.025 A semi-fluid medium for detecting lactic acid bacteria according to any one of (8) to (11) above, which is a semi-fluid medium containing 0.200% agar, and (13) 12.5 g / L tryptone 7.5 g / L yeast extract, 20 g / L glucose, 5 g / L trisodium citrate dihydrate, 5 g / L sodium chloride, 0.8 g / L magnesium sulfate, 0.14 g / L Manganese chloride, 0.04 g / L iron (II) sulfate primary, 0.001 g / L thiamine hydrochloride, 0.2 g / L Tween 80, 0.75 g / L agar, 0.06 g / L Bromocle Rupapuru about semifluid medium for lactic acid bacteria detection according to any one of the above which is characterized by being composed of (pH 6.8 ± 0.2 after sterilization) (8) - (12).

  When a test sample such as food or beverage is added and mixed in the semi-fluid culture medium for detecting lactic acid bacteria of the present invention and cultured, the color of the medium changes due to the growth of lactic acid bacteria in the test sample. And qualitatively (for example, positive / negative can be discriminated based on the presence or absence of lactic acid bacteria per 10 ml of the test sample solution).

  As a method for detecting lactic acid bacteria according to the present invention, a test sample is added to and mixed with a semi-fluid medium for detecting lactic acid bacteria containing 15 to 25 g / L glucose and a pH indicator, and the color of the medium before and after the cultivation is cultured. The semi-fluid medium for detecting lactic acid bacteria of the present invention contains 15 to 25 g / L glucose and a pH indicator, and the medium before and after the culture is used. It is not particularly limited as long as it is a medium that can detect lactic acid bacteria by visually judging the color change, and as a lactic acid bacterium to be detected, Lactobacillus, Pediococcus, Tetragenococcus, Carnobacterium, Vagococcus, Leuconostoc, Weissella, Examples include lactic acid bacteria belonging to the genera of Oenococcus, Atopobium, Streptococcus, Enterococcus, and Lactococcus. Moreover, the said semi-fluid culture medium means the culture medium with low content of solid components, such as agar, and a certain fluidity | liquidity compared with a solid culture medium.

  The semi-fluid medium for detecting lactic acid bacteria is preferably one having a reduced or removed buffer capacity. In order to maintain the buffer capacity, the medium usually contains phosphate buffer, citrate buffer, acetate buffer, lactate buffer, tartrate buffer, malate buffer, Tris buffer, MOPS buffer, MES buffer. The semi-fluid medium for detecting lactic acid bacteria is preferably one having reduced or eliminated buffering capacity not containing dipotassium hydrogen phosphate or the like. In addition, in this invention, low buffer capacity means having a buffer capacity lower than the buffer capacity in the basic medium 2 in Example 2 mentioned later.

  The semi-fluid medium for detecting lactic acid bacteria preferably has a pH after sterilization and before culturing of 6.0 to 7.5, particularly 6.5 to 7.2, particularly 6.8. In this case, by using a pH indicator that changes color at a pH of 5 to 6, a medium having a lowered pH due to the growth of lactic acid bacteria can be easily and visually found after cultivation. Preferred examples of indicators that change color at pH 5 to 6 include bromocresol purple, bromocresol green, methyl red, litmus, methyl purple, p-nitrophenol, chlorophenol red, bromothymol blue, and alizarin red S. .

  Examples of other acidic range pH indicators that can be used in the present invention include thymol blue; acidic, bromophenol blue, methyl orange, and the like, and alkaline range pH indicators include neutral red, phenol red, and o-cresol. Red, thymol blue; alkaline, phenolphthalein, orthocresolphthalein, thymolphthalein and the like can be mentioned, and examples of the reaction zone pH indicator at pH 6 to 10 include fluorescein, coumarin, xylenol blue and the like.

  The semi-fluid medium exhibits fluidity when a physical force such as agitation and shaking is applied from the outside, but exhibits a property such that convection is less than that of a liquid medium when left standing. The solid medium is used at 1.0 to 2.0% in the case of agar, but the semi-fluid medium is used at 0.025 to 1.000%. The semi-fluid medium for detecting lactic acid bacteria of the present invention is intended to be semi-fluid for easy determination, and is preferably a medium that retains fluidity so that stirring can be easily performed. It is used at 0.025 to 0.200%, preferably 0.05 to 0.15%. However, this concentration is an agar concentration necessary to maintain a semi-fluid state at a general culture temperature of about 20 to 40 ° C. If it is lower than this temperature range, a smaller amount and a higher temperature It is preferable to use a larger amount of agar in the culture.

  The semi-fluid state can also be realized by a gelling agent other than agar. In addition to agar, gelatin, silica gel, acrylamide, glucomannan, methylcellulose, duran gum, gum arabic, starch, sodium alginate, carrageenan, bentonite, alginate, collagen, fused quartz, water-soluble starch, polyacrylate, Examples thereof include cellulose, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, dextran, and polysaccharide. These gelling agents can be used in place of agar or in combination with agar.

  As a semi-fluid medium for detecting lactic acid bacteria of the present invention, 12.5 g / L tryptone, 7.5 g / L yeast extract, 20 g / L glucose, 5 g / L trisodium citrate dihydrate, 5 g / L sodium chloride, 0.8 g / L magnesium sulfate, 0.14 g / L manganese chloride, 0.04 g / L iron (II) sulfate first, 0.001 g / L thiamine hydrochloride, 0.2 g The most preferable example is a medium composed of / L Tween 80, 0.75 g / L agar, 0.06 g / L bromocresol purple (pH 6.8 ± 0.2 after sterilization). it can. This most suitable medium has a buffering capacity (in order to double the glucose concentration and sharpen the color change of the indicator as compared with the medium in which the pH indicator BCP is added to the APT agar of 4) above. Except for dipotassium phosphate), it is not used as an agar medium, but on the premise that it is used as a liquid medium, it contains a small amount of agar and makes it semi-fluid so that the color change caused by the pH indicator is remarkably clear. It is a thing.

  In general, lactic acid bacteria are cultured for 24 to 48 hours at 30 ° C., and for Bifidobacterium (bifidobacteria) at 72 ° C. for 72 hours (food hygiene inspection guidelines, edited by microorganisms, supervised by the Ministry of Health, Labor and Welfare, 2004). In addition, it is known that culturing at a low temperature such as 25 ° C. or less facilitates the growth of low-temperature-producing lactic acid bacteria. However, when culturing at a low temperature, a culture time of 4 to 10 days is required. In the method for detecting a lactic acid bacterium according to the present invention, the culture by adding and mixing a test sample is preferably performed under culture conditions that allow the lactic acid bacterium to suitably grow. For example, in the semi-fluid culture medium for detecting lactic acid bacteria of the present invention, the growth of lactic acid bacteria and increase of acid production by increasing glucose, the use of indicator and removal of buffer capacity and the semi-fluid state can be used to determine the presence or absence of lactic acid bacteria growth. Since it can be easily discriminated, it can be visually determined in a short time. By culturing at 25 ° C, low-temperature-growing lactic acid bacteria are added to general lactic acid bacteria. Whole lactic acid bacteria can be detected.

  According to the method for detecting lactic acid bacteria of the present invention, for example, lactic acid bacteria can be detected as follows. When measuring the presence or absence of lactic acid bacteria in food, add 225 g of sterilized diluent to 25 g of food and pulverize and mix 1 ml of the test sample solution to 10 ml of sterilized semi-fluidic medium for detecting lactic acid bacteria of the present invention, Alternatively, 10 ml of the test sample solution is added to 10 ml of the semi-fluid medium for detecting lactic acid bacteria having a double concentration, and these liquids are mixed. After the culture, the pH of the medium is lowered due to acid production by the lactic acid bacteria, and the color of the medium changes. Thus, the presence or absence of lactic acid bacteria in 1 ml of the test sample solution or 10 ml is visually determined. In addition, when obtaining a very small number of bacteria, a test tube 3 containing a semi-fluid culture medium for detecting lactic acid bacteria according to the present invention is used for each of three-stage test sample solutions diluted 10-fold, such as 10 ml, 1 ml, and 0.1 ml. Add to 5 or 5 and calculate the number of bacteria from the number of positive test tubes by the most probable number (MPN: Most Probable Number) (MPN value is usually displayed per 100 ml or 100 g). In addition, for example, when measuring lactic acid bacteria adhering to the surface of food production process devices and instruments, the surface of the object is wiped off with a cut cotton, cotton swab, gauze tampon, etc. and then eluted directly or with sterilized diluted water The solution is added to the semi-fluid culture medium for detecting lactic acid bacteria of the present invention, and the presence or absence of lactic acid bacteria and a very small number of bacteria are measured.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

(Influence of glucose concentration in the medium)
33 strains of lactic acid bacteria shown in Table 1 were used as test bacteria. Each test bacterium was inoculated into 10 ml of APT broth (Difco), cultured at 30 ° C. for 2 to 3 days, and used as a preculture solution.

Basic medium 1 was prepared with the composition shown below.
Composition of basic medium 1 (g / L)
Tryptone Peptone (Difco) 12.5
Yeast extract (Difco) 7.5
Sodium chloride 5
Trisodium citrate dihydrate 5
Dipotassium hydrogen phosphate 5
Magnesium sulfate 0.8
Manganese chloride 0.14
Iron (II) sulfate first 0.04
Thiamine hydrochloride 0.001
Tween 80 0.2
Bromocresol purple 0.06
PH after sterilization 6.8 ± 0.2

    Glucose was added to this basic medium 1 at 0% (0 g / L), 1.0% (10 g / L), 1.5% (15 g / L), 2.0% (20 g / L), 2.5% ( 25 g / L), 10 ml each was added to a test tube, and then autoclaved at 121 ° C. for 15 minutes to prepare a test medium. This test medium was appropriately inoculated with a preculture solution of each lactic acid strain and inoculated to 10 CFU / ml, followed by culturing at 30 ° C. After 48 hours, detection of lactic acid bacteria due to yellowing of the medium was confirmed. The color of the medium was not changed and the color remained purple,-was slightly yellowed, and + was clearly yellowed. The results are shown in Table 1. As a result, there were many strains in which yellowing of the medium was observed at a glucose concentration of 1.5% to 2.5%. In the following tests, 2.0% glucose was added.

(Influence of the buffer capacity of the medium)
A basic medium 2 was prepared with the composition shown below.
Composition of basic medium 2 (g / L)
Tryptone Peptone (Difco) 12.5
Yeast extract (Difco) 7.5
Glucose 20
Sodium chloride 5
Trisodium citrate dihydrate 5
Magnesium sulfate 0.8
Manganese chloride 0.14
Iron (II) sulfate first 0.04
Thiamine hydrochloride 0.001
Tween 80 0.2
Bromocresol purple 0.06
PH after sterilization 6.8 ± 0.2

  To this basic medium 2, dipotassium hydrogen phosphate 0 g / L, 5 g / L was added and autoclaved at 121 ° C. for 15 minutes to prepare a test medium. A 0.9% lactic acid solution was added dropwise to 100 ml of each medium, and when the pH was changed by 0.25, the change in the color of the medium and the amount of the 0.9% lactic acid solution added were measured. A medium that did not change in color and remained purple,-was slightly yellowed, and + was clearly yellowed. The results are shown in Table 2. As a result, the 0.9% lactic acid solution necessary for the pH 6.5 medium to turn yellow was 18.1 ml / 100 ml for dipotassium hydrogenphosphate 5 g / L and 11.1 for dipotassium hydrogenphosphate 0 g / L. It was 0 ml / 100 ml, and 0 g / L was yellowed with a small amount of lactic acid solution rather than 5 g / L of dipotassium hydrogen phosphate. The following tests were conducted with dipotassium hydrogen phosphate 0 g / L.

(Effect of medium agar concentration)
Lactobacillus sp.-39 was used as a test bacterium, and 0% (0g / L), 0.025% (0.25g / L), 0.050% (0. 50 g / L), 0.075% (0.75 g / L), 0.150% (1.50 g / L), 0.200% (2.00 g / L), 0.250% (2.50 g / L) L), 0.300% (3.00 g / L), 0.600% (6.00 g / L) were added, and autoclaved at 121 ° C. for 15 minutes to prepare a test medium. Each test medium is stored at 25 ° C., and it is confirmed whether it can be easily stirred as a medium for lactic acid bacteria. For those that can be stirred, 10 CFU / ml of the test bacteria is inoculated, and easy to determine after culturing at 25 ° C. for 48 hours. I confirmed. The results are shown in Table 3 and FIG. As a result, the agar concentration that can be stirred at 25 ° C. is 0% to 0.200%, and a part or the whole of the medium is clearly yellowed when the agar is 0.025% or more, particularly 0.075% or more, It can be judged at a glance with the naked eye, and it was found that the judgment was easy. From the above, it was confirmed that by adding 0.025 to 0.200% of agar to the liquid medium, it becomes a semi-fluid medium that can be easily determined.

    Further, Lactobacillus acidophilus and Lactobacillus sp.-6 were used as test bacteria, and 0% (0 g / L) and 0.075% (0.75 g / L) of agar were added to the basic medium 2 shown in Example 2, 121 Sterilized at high pressure for 15 minutes to prepare a test medium. Each test medium was inoculated with 10 CFU / ml of the test bacteria, and the ease of determination was confirmed after culturing at 25 ° C. for 48 hours. The results are shown in FIGS. As a result, it was found that 0.075% compared to 0% agar was more or less yellow in the culture medium and was easier to judge. In the following test, 0.075% of agar was added.

(Detection test for lactic acid bacteria)
As test bacteria, 16 strains of standard strains of lactic acid bacteria, 214 strains isolated from processed foods, meat products, production processes, human feces, and the like were used. The classification is Lactobacillus genus 142 strain, Leuconostoc genus 49 strain, Lactococcus genus 11 strain, Pediococcus genus 6 strain, Streptococcus genus 12 strain, Enterococcus genus 9 strain, Tetragenococcus genus 1 strain. These were inoculated into 10 ml of APT broth (Difco), cultured at 20 ° C. to 35 ° C. for 1 to 7 days, and grown and used as a preculture.

The culture medium for lactic acid bacteria of this invention was produced with the composition shown below.
Composition of lactic acid bacteria medium (g / L)
Tryptone Peptone (Difco) 12.5
Yeast extract (Difco) 7.5
Glucose 20
Sodium chloride 5
Trisodium citrate dihydrate 5
Magnesium sulfate 0.8
Manganese chloride 0.14
Iron (II) sulfate first 0.04
Thiamine hydrochloride 0.001
Tween 80 0.2
Agar 0.75
Bromocresol purple 0.06
PH after sterilization 6.8 ± 0.2

For comparison, APT broth (Difco) added with 0.06 g / L bromocresol purple (Comparative medium 1), MRS broth (Difco) added with 0.06 g / L bromocresol purple (Comparative medium) 2) A BCP-added plate count agar medium obtained by removing agar (comparative medium 3) was used. Comparative media were prepared according to the designated methods. The composition of Comparative Medium 3 is shown below.
Medium composition (g / L)
Peptone (Difco) 5.0
Yeast extract (Difco) 2.5
Glucose 1.0
Tween 80 1.0
L-cysteine 0.1
Bromocresol purple 0.06
PH after sterilization 7.0 ± 0.2

  After 10 ml of the lactic acid bacteria culture medium and comparative culture mediums 1 to 3 of the present invention were dispensed into a test tube, autoclaving at 121 ° C. for 15 minutes was performed to obtain a test medium. In this test medium, the preculture solution of each lactic acid strain is appropriately diluted and inoculated to 10 CFU / ml, and then stored at 20 ° C., 25 ° C., 30 ° C., and 35 ° C., and the color changes over time. Observed. The results are shown in Tables 4-10. As can be seen from Tables 4 to 10 and Table 11 which summarizes the results of Tables 4 to 10, the medium for lactic acid bacteria can detect lactic acid bacteria with high sensitivity at any temperature as compared with the comparative medium, particularly at 25 ° C. It was confirmed that the detection rate was high.

It is a figure which shows the growth of Lactobacillus sp.-39 in the basic culture medium 2 which added each agar density | concentration. 1: Medium before culture, 2-5: Medium after culture 2: Agar 0% (0 g / L) 3: Agar 0.025% (0.25 g / L) 4: Agar 0.050% (0.50 g) / L) 5: Agar 0.075% (0.75 g / L) It is a figure which shows the growth of Lactobacillus acidophilus in the basic culture medium 2 which added each agar density | concentration. 1: Medium before culture, 2-3: Medium after culture 2: Agar 0.075% (0.75 g / L) 3: Agar 0% (0 g / L) It is a figure which shows the growth of Lactobacillus sp.-6 in the basic culture medium 2 which added each agar density | concentration. 1: Medium before culture, 2-3: Medium after culture 2: Agar 0.075% (0.75 g / L) 3: Agar 0% (0 g / L)

Claims (13)

  A test sample is added to and mixed with a semi-fluid medium for detecting lactic acid bacteria containing 15 to 25 g / L glucose and a pH indicator, and the color change of the medium before and after the culture is visually determined. A method for detecting lactic acid bacteria.   The method for detecting lactic acid bacteria according to claim 1, wherein the semi-fluid medium is a low-buffering semi-fluid medium that does not contain dipotassium hydrogen phosphate.   The method for detecting lactic acid bacteria according to claim 1 or 2, wherein the semi-fluid medium has a pH of 6.0 to 7.5 after sterilization and before culturing.   The pH indicator is a pH indicator selected from bromocresol purple, bromocresol green, methyl red, litmus, methyl purple, p-nitrophenol, chlorophenol red, bromothymol blue, and alizarin red S. The detection method of lactic acid bacteria in any one of 1-3.   The method for detecting lactic acid bacteria according to any one of claims 1 to 4, wherein the semi-fluid medium is a semi-fluid medium containing 0.025 to 0.200% agar.   Semi-fluid medium is 12.5 g / L tryptone, 7.5 g / L yeast extract, 20 g / L glucose, 5 g / L trisodium citrate dihydrate, 5 g / L sodium chloride, 0. 8 g / L magnesium sulfate, 0.14 g / L manganese chloride, 0.04 g / L iron (II) sulfate primary, 0.001 g / L thiamine hydrochloride, 0.2 g / L Tween 80, 0 The lactic acid bacterium according to any one of claims 1 to 5, which comprises 0.75 g / L agar and 0.06 g / L bromocresol purple (pH 6.8 ± 0.2 after sterilization). Detection method.   The method for detecting lactic acid bacteria according to any one of claims 1 to 6, wherein the color change of the medium after culturing at 25 ° C for 2 days is visually determined.   A semi-fluid medium for detecting lactic acid bacteria, which contains 15 to 25 g / L glucose and a pH indicator, and can detect lactic acid bacteria by visually determining a change in color of the medium before and after the culture.   The semi-fluid medium for detecting lactic acid bacteria according to claim 8, which is a semi-fluid medium having a low buffer capacity and not containing dipotassium hydrogen phosphate.   The semi-fluid medium for detecting lactic acid bacteria according to claim 8 or 9, wherein the pH after sterilization and before culture is 6.0 to 7.5.   The pH indicator is selected from bromocresol purple, bromocresol green, methyl red, litmus, methyl purple, p-nitrophenol, chlorophenol red, bromothymol blue, and alizarin red S. The semi-fluid culture medium for lactic acid bacteria detection in any one.   The semi-fluidic medium for detecting lactic acid bacteria according to any one of claims 8 to 11, which is a semi-fluidic medium containing 0.025 to 0.200% agar.   12.5 g / L tryptone, 7.5 g / L yeast extract, 20 g / L glucose, 5 g / L trisodium citrate dihydrate, 5 g / L sodium chloride, 0.8 g / L sulfuric acid Magnesium, 0.14 g / L manganese chloride, 0.04 g / L iron (II) sulfate primary, 0.001 g / L thiamine hydrochloride, 0.2 g / L Tween 80, 0.75 g / L The semi-fluid medium for detecting lactic acid bacteria according to any one of claims 8 to 12, characterized in that it is composed of agar and 0.06 g / L bromocresol purple (pH 6.8 ± 0.2 after sterilization). .