JP6557199B2 - Selective medium for lactic acid bacteria - Google Patents

Description

  The present invention relates to a selective medium for lactic acid bacteria, and more specifically to a selective medium for lactic acid bacteria that allows specific lactic acid bacteria to be selectively grown.

  In recent years, as the health consciousness grows, there are many products related to so-called probiotics that contain useful bacteria alive, such as lactic acid bacteria beverages and yogurt. In these products, the importance of the quality of the quality of the product, including the number of live bacteria contained in the product and its stability, and how much it reaches the intestines when ingested is important. There is a case. As a method for measuring the number of viable bacteria contained in a product, a selective medium technique is known. Specifically, a sample is applied on an agar plate medium made of a selective medium, and a colony formed by viable bacteria is counted to determine the number of viable bacteria. For example, a diluted sample is added, cultured under predetermined conditions, and applied to a pre-established most probable number table to estimate the number of viable bacteria (most probable number method). According to the selective medium, the number of viable bacteria can be measured relatively accurately and conveniently, which is convenient. For example, Patent Document 1 discloses a selective medium for detecting Bifidobacterium. Patent Document 2 discloses a selective medium for detecting Lactobacillus delbruecki subspecies bulgaricus.

  Lactobacillus acidophilus, on the other hand, was found as a facultative anaerobic lactic acid bacterium that is abundant in the intestines of infants and is distinct from bifidobacteria. Since then, it has been found that it exists not only in the intestines of infants but also in the intestines, oral cavity and vagina of adults and animals. There are reports that Lactobacillus acidophilus has beneficial effects such as intestinal regulation and infection prevention, and there are many products such as lactic acid bacteria beverages, yogurt, and supplements that contain Lactobacillus acidophilus.

  Conventionally, as a selective medium for detecting Lactobacillus acidophilus, antibiotics ciprofloxacin and clindamycin are added to MRS medium, which is a medium for general lactic acid bacteria, as a selective medium. Yes. For example, an MRS agar medium supplemented with 10 μg / mL ciprofloxacin and 0.1 μg / mL clindamycin is known as an IDF / ISO standard method for measuring the viable count of Lactobacillus acidophilus in dairy products. (Non-Patent Document 1). According to this standard method, in addition to suppressing the growth of other species other than lactic acid bacteria, Lactobacillus reuteri, Lactobacillus delbrueckii subspices bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus) , Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium, and the like can be selectively detected to suppress the detection of Lactobacillus acidophilus It is considered possible.

Japanese Patent Laid-Open No. 11-28098 JP 2007-28942 A

"Milk products-Enumeration of presumptive Lactobacillus acidophilus -Colony count technique at 37 ℃" ISO (International Organization for Standardization) / DIS (Draft International Standard) 20128 | IDF (International Dairy Federation) 192. 2005.

  However, according to the researches of the present inventors, for bacterial species such as Lactobacillus johnsonii, Lactobacillus gasseri, and Lactobacillus crispatus, conventional selection media However, since these are intestinal bacteria without being able to suppress their growth, there has been a problem of causing false positives particularly when trying to detect live bacteria of Lactobacillus acidophilus from stool-derived samples.

  Therefore, an object of the present invention is to provide a selective medium for lactic acid bacteria with improved selectivity.

  In order to achieve the above object, the present inventors have conducted intensive research and found that the selectivity for Lactobacillus acidophilus and the like can be further increased by adding cephalothin as an antibiotic to a conventional selective medium. The present invention has been completed.

  That is, the first of the present invention is a selective medium for selectively growing one or more Lactobacillus bacteria selected from the group consisting of Lactobacillus acidophilus, Lactobacillus amyloboraus, and Lactobacillus brevis. The invention provides a selective medium characterized by containing ciprofloxacin, clindamycin, and cephalothin in a basal medium.

  The selective medium according to the present invention is preferably used as a selective medium for Lactobacillus acidophilus.

  In the selective medium according to the present invention, the cephalothin content is preferably 0.25 mg or more and 1 mg or less per liter of the basal medium.

  Further, in the selective medium according to the present invention, the contents of ciprofloxacin, clindamycin, and cephalothin are 5 mg to 20 mg, 0.05 mg to 0.2 mg, respectively, per liter of the basal medium. , 0.25 mg to 1 mg is preferable.

  In the selective medium according to the present invention, the basal medium is preferably one selected from the group consisting of an MRS medium, an LBS medium, and a BCP medium.

  On the other hand, in the second aspect of the present invention, after the sample is inoculated and cultured in the selective medium, the grown bacteria are selected from the group consisting of Lactobacillus acidophilus, Lactobacillus amyloboras, and Lactobacillus brevis. The present invention provides a method for detecting a Lactobacillus bacterium, which is detected as one or more Lactobacillus bacteria.

  In the method for detecting a Lactobacillus bacterium according to the present invention, the sample is preferably prepared from a food or a pharmaceutical product.

  In the method for detecting Lactobacillus bacteria according to the present invention, the sample is preferably prepared from human or animal feces.

  In the method for detecting Lactobacillus bacteria according to the present invention, it is preferable to measure the number of living bacteria of the Lactobacillus bacteria.

  According to the present invention, in addition to ciprofloxacin and clindamycin in the selective medium for lactic acid bacteria, cephalothin was further used, so that from the group consisting of Lactobacillus acidophilus, Lactobacillus amyloboras, and Lactobacillus brevis One or two or more selected Lactobacillus bacteria can be more selectively grown while suppressing the growth of bacterial species closely related to them. Thereby, the appearance of false positives is reduced as compared with the conventional method, and the number of living bacteria of the genus Lactobacillus can be measured more accurately and easily even from a sample derived from feces, for example.

  The selective medium according to the present invention includes one or more lactobacillus selected from the group consisting of Lactobacillus acidophilus, Lactobacillus amylovorus, and Lactobacillus brevis. It is a selective medium for selectively growing a genus bacterium. Here, “selective” does not mean that microorganisms of other genus species other than the target fungus cannot grow at all, and the target fungus grows and propagates. In comparison with other microorganisms of other genus species, the microorganisms of the other genus species can be distinguished from microorganisms of other genus species according to any desired purpose by the propagation of microorganisms of other genus species not occurring, slow or insufficient. It is sufficient that the selectivity is such that it can be detected. For example, as shown in the examples below, fermented milk products in which the above target bacteria are preferentially contained in the entire flora, stool samples ingesting them, other lactic acid bacteria, Escherichia coli, etc. It is sufficient that only the target bacteria can be detected without becoming incapable of measurement due to a false positive problem due to the above, and if there is such selectivity, the utility value is sufficiently high.

  The selective medium according to the present invention can detect one or more Lactobacillus bacteria selected from the group consisting of Lactobacillus acidophilus, Lactobacillus amyloboraus, and Lactobacillus brevis, based on the selectivity described above. In particular, it is preferably used, but is not limited to its use, and can also be used as a medium for maintaining and preserving the aforementioned Lactobacillus bacteria.

  The selective medium according to the present invention contains ciprofloxacin, clindamycin, and cefalotin in the basal medium in order to give selectivity to the above-mentioned target bacteria. When these three agents are contained in a basal medium and an arbitrary desired sample is cultured in the medium for a predetermined period, if the target fungus is alive in the sample, it grows, Since the growth of other microorganisms does not occur or is insufficient, the presence of the target bacteria can be confirmed even visually after the culture period. For example, it can be confirmed by the fact that viable bacteria form colonies by culturing on an agar plate medium comprising a selective medium. In addition, it can be confirmed by culturing in a liquid medium composed of a selective medium such that the culture solution becomes a suspended state due to propagation of viable bacteria from a clear state.

  In the selective medium according to the present invention, the content of ciprofloxacin is not particularly limited as long as it does not affect the growth and selectivity of the fungus targeted by the present invention. It is preferably 1 mg or more and 20 mg or less, and more preferably 5 mg or more and 20 mg or less. The content of clindamycin is not particularly limited as long as it does not affect the growth and selectivity of the target bacterium of the present invention, but is 0.01 mg or more per liter of the basal medium. It is preferably 2 mg or less, more preferably 0.05 mg or more and 0.2 mg or less. Further, the content of cephalothin is not particularly limited as long as it does not affect the growth and selectivity of the target fungus of the present invention, but is 0.05 mg or more and 1 mg or less per liter of the basal medium. It is preferably 0.25 mg or more and 1 mg or less. There are no particular restrictions on how to prepare these antibiotics in the basal medium. For example, according to a conventional method, antibiotics are dissolved in a relatively high concentration in an appropriate solvent in advance, and sterilized by filter refrigeration or frozen and stored in a basic medium separately sterilized by an autoclave or the like. A mode of adding and mixing so as to achieve a desired final concentration aseptically in a state where the temperature is not high is exemplified. According to such preparation, deterioration of the antibiotic is prevented, and the antibiotic can be present in the medium at a more uniform concentration. Of course, these antibiotics may be used in the form of their acceptable salts or solvates as long as they do not affect the growth and selectivity of the bacteria targeted by the present invention.

  The basal medium that can be used for the selective medium according to the present invention is not particularly limited as long as it is generally a medium having a medium composition suitable for growth of Lactobacillus bacteria. Examples thereof include MRS medium, LBS medium, BCP medium, etc., which are so-called lactic acid bacteria mediums. The typical medium composition is given below. In addition, these media may be reduced or increased for each composition component within the range that does not affect the growth and selectivity of the bacteria targeted by the present invention, or some of the composition components may be removed. Of course, it may be used with an allowable modification such as addition of components.

(MRS medium)
Peptone hydrolyzate 10g
10g beef extract
Yeast extract 5g
Glucose 20g
Polysorbate 80 1g
2g of ammonium citrate
Sodium acetate 5g
Magnesium sulfate 0.1g
Manganese sulfate 0.05g
2g dipotassium phosphate
1000mL water
(In the case of an agar medium, add 15 g of agar)

(LBS medium)
Casein pancreatin degradation product 10g
Yeast extract 5g
Monopotassium phosphate 6g
2g of ammonium citrate
Glucose 20g
Polysorbate 80 1g
Sodium acetate trihydrate 25g
Magnesium sulfate 0.575g
Manganese sulfate 0.12g
Ferrous sulfate 0.034g
Acetic acid 1.32mL
1000mL water
(In the case of an agar medium, add 15 g of agar)

(BCP medium)
Yeast extract 2.5g
Peptone 5g
Glucose 1g
Polysorbate 80 1g
L-cysteine 0.1g
Bromcresol purple 0.06g
1000mL water
(In the case of an agar medium, add 15 g of agar)

  On the other hand, the method for detecting a bacterium belonging to the genus Lactobacillus according to the present invention comprises using the selective medium described above, Lactobacillus acidophilus, Lactobacillus amylovorus contained in a sample, and This is a detection method for detecting one or more Lactobacillus bacteria selected from the group consisting of Lactobacillus brevis. In general, even if microorganisms such as Lactobacillus are present in the sample, the amount of the microorganisms is overwhelmingly less than other components, or other components may interfere with detection and It cannot be recognized. Therefore, the selectivity of the selective medium described above is used. That is, as described above, when a sample is inoculated into a selective medium and cultured for a predetermined period, if the target fungus is alive in the sample, it will proliferate, while other microorganisms Since the growth does not occur or is insufficient, the presence of the target bacteria can be confirmed even visually after the culture period. In the present invention, “detection” not only means the presence of the Lactobacillus bacterium in the sample or the number of bacteria but also the absence of the Lactobacillus bacterium in the sample. It also means to confirm that the amount is small even if it exists.

  The culture can be performed according to a general method for culturing microorganisms. That is, examples of the form of the selective medium include an agar plate medium and a liquid medium. Regarding the inoculation of the sample to the selective medium and the culture conditions, there is no particular limitation on the form thereof, and the sample is appropriately diluted as necessary, and if it is an agar plate medium, about 100 parts by mass of the agar medium is approximately If it is applied on an agar plate medium as a sample liquid of 0.25 to 0.5 parts by mass, or if it is a liquid medium, about 0.1 to 1 part by mass of the sample liquid with respect to 100 parts by mass of the liquid medium What is necessary is just to add and mix in a liquid culture medium. The culture may be performed under an aerobic condition or an anaerobic condition. Although the temperature at the time of culture | cultivation should just be set arbitrarily, it is preferable that it is the temperature suitable for growth of lactic acid bacteria, for example, 36-38 degreeC.

  The method for detecting Lactobacillus bacteria according to the present invention is particularly suitable as a method for detecting the Lactobacillus bacteria contained in foods and pharmaceuticals. That is, the quality as a product containing useful bacteria alive can be directly confirmed by the detection. Here, food means not only food for human consumption, but also includes supplements and health foods intended for nutritional support, foods for specified health use and functional labeling foods for specific health functions, etc. It is. Or it also includes animal food, feed for maintaining health, pet food, and the like. Or it is the meaning also including the additive raw material etc. which were made into the objective to add to those foodstuffs. On the other hand, the drug means not only for diagnosis, treatment and prevention of human diseases and diseases but also for animals. In addition, it is meant to include additive materials intended to be added to these pharmaceuticals. In this case, as the form of the sample for inoculating the selective medium, a part of the food or medicine is collected and used as it is, or when the shape of the food or medicine is liquid or paste, Collect a portion and dilute it with a solvent such as water to make a portion of the sample. If the shape of the food or medicine is solid or powder, collect a portion of the sample. A method of suspending it in a solvent such as water and further using a part of the sample as a sample can be mentioned, but other forms may be used and there is no particular limitation.

  The method for detecting Lactobacillus bacteria according to the present invention is particularly suitable as a method for detecting the Lactobacillus bacteria contained in human or animal feces. That is, by the detection, when a human or an animal ingests a product containing useful bacteria alive, it can be directly confirmed how much the useful bacteria have reached the intestines alive. In this case, as a form of the sample for inoculating the selective medium, a part of human or animal stool is collected and used as it is, or it is suspended in a solvent such as water and a part thereof is used as a sample. However, other forms may be used without any particular limitation.

  The method for detecting Lactobacillus bacteria according to the present invention is particularly suitable as a method for measuring the number of living Lactobacillus bacteria contained in a sample. Specifically, for example, as described above, when a sample is appropriately diluted on an agar plate medium made of a selective medium, and is applied and cultured for a predetermined period of time, viable bacteria are exposed on the surface of the agar plate medium. Since colonies are formed, the number of colonies is counted and converted from the number of colonies, the amount of coating solution and the dilution rate, and the number of viable bacteria contained in the original sample can be determined. Alternatively, a method in which a sample diluted in stages is added to a liquid medium composed of a selective medium, cultured under predetermined conditions, and applied to a pre-established most probable number table to estimate the number of viable bacteria (most probable number method) The number of viable bacteria can be obtained using such as.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these examples do not limit the scope of the present invention.

[Test Example 1]
L. acidophilus YIT 0198 strain was prepared as Lactobacillus acidophilus and examined for resistance to antibiotics (combined use of ciprofloxacin, clindamycin, and cephalothin).

  For this purpose, MRS medium is used as a basal medium, and ciprofloxacin and clindamycin are added at predetermined concentrations as antibiotics in accordance with the IDF / ISO standard method (see Non-Patent Document 1). Cephalotin was added as an agent to prepare an agar plate medium. Specifically, 55 g of MRS medium material (“Lactobacilli MRS broth”, manufactured by Nippon Becton Dickinson), 15 g of agar (“Bacto agar”, manufactured by Nippon Becton, Dickinson) and 992 mL of pure water (Milli-Q water) In addition, after autoclaving at 121 ° C. for 15 minutes and cooling to 45 ° C. in a thermostatic bath, ciprofloxacin (manufactured by Sigma Aldrich Japan) was added thereto to a concentration of 10 μg / mL, and clindamycin was added. (Sigma Aldrich Japan) was added to a concentration of 0.1 μg / mL, and cephalotin (Sigma) was further adjusted to a concentration of 0, 0.25, 0.5, 1, 2, or 4 μg / mL. Aldrich Japan) was added to prepare MRS agar plates containing antibiotics at various concentrations. On this MRS agar plate medium, a bacterial solution (preliminary culture solution) obtained by previously culturing the above strain under aerobic conditions for 1 to 2 days using an MRS liquid medium is appropriately diluted with PBS, and each diluted solution is 50 μl. Each was applied and cultured for 3-4 days under aerobic conditions. The number of colonies that appeared on the agar medium was counted in the range of 30 to 300, and the number of bacteria per 1 mL of the precultured solution was calculated from the number of colonies, the amount of coating solution, and the dilution rate. The test was performed using three agar plate media for each antibiotic concentration, and the average value of the measured number of bacteria was determined. The number of bacteria was expressed as a logarithmic value. The results are shown in Table 1.

  As a result, Lactobacillus acidophilus (L. acidophilus YIT 0198 strain) was obtained by adding ciprofloxacin as an antibiotic at a concentration of 10 μg / mL and adding clindamycin at a concentration of 0.1 μg / mL. There was no effect of inhibiting growth. Furthermore, even when cephalotin was added as an antibiotic, when the concentration was 0.25 or 0.5 μg / mL, the effect of suppressing growth was not observed. On the other hand, in addition to the above-mentioned concentrations of ciprofloxacin and clindamycin as an antibiotic, cephalothin was further added to the MRS medium at a concentration of 2 μg / mL or more, and the growth was remarkably suppressed. From these results, it is possible to combine these three drugs in the MRS medium with ciprofloxacin at a concentration of 10 μg / mL, clindamycin at a concentration of 0.1 μg / mL, and cephalotin at a concentration of 0.5 μg / mL. On the other hand, for the bacterial species closely related to Lactobacillus acidophilus, its growth is expected to be suppressed, while the growth of Lactobacillus acidophilus is not inhibited, but as a selective medium for Lactobacillus acidophilus, It was suggested that the selectivity is improved.

[Test Example 2]
It was verified whether the selectivity was further improved as a selective medium for Lactobacillus acidophilus by combining three drugs of ciprofloxacin, clindamycin, and cephalothin. Hereinafter, the antibiotic-containing MRS medium in which these three agents are used in combination may be represented by “MRS-CCC”.

  Specifically, a total of 22 bacterial strains and 43 strains of 12 bacterial species classified into the L. gasseri subgroup including Lactobacillus acidophilus, 9 other Lactobacillus species, and 1 Streptococcus species are prepared. Then, in the same manner as in Test Example 1, resistance to an antibiotic (a combination of three drugs of ciprofloxacin, clindamycin, and cephalothin) was examined. As for the concentration of antibiotics, ciprofloxacin 10 μg / mL, clindamycin 0.1 μg / mL, and cephalothin 0.5 μg / mL were adopted from the results of Test Example 1, and these three agents were used in combination to contain antibiotics. An MRS agar plate medium was prepared and used for testing. On the other hand, the MRS agar plate medium without antibiotics was used as a control, and the ratio of the number of bacteria when using the MRS-CCC medium to the number of bacteria when using the control medium was defined as the growth rate. When the growth rate was 1/1000 or less, it was evaluated as having no resistance to antibiotics (indicated by “x” in the table), and the others were evaluated as having resistance (indicated by “◯” in the table). .) The results are shown in Table 2.

  As a result, in the L. acidophilus YIT 0198 strain, there was no difference between the number of bacteria when the control medium was used and the number of bacteria when the MRS-CCC medium was used. It was confirmed that the growth was not inhibited. As for other strains of Lactobacillus acidophilus, there are three strains whose growth rate relative to the control medium is 1/10 or more, and two strains within the range of 1/10 to 1/100. It was.

  In addition, for Lactobacillus amylovorus, there are two strains having a growth rate of 1/10 or more with respect to the control medium, and for Lactobacillus brevis, the growth rate with respect to the control medium is 1 /. There were 4 strains that were 10 or more.

  On the other hand, for Lactobacillus crispatus, both of the two strains are below the lower detection limit, and for Lactobacillus delbrueckii ss bulgaricus, both of the two strains are For Lactobacillus delbrueckii ss delbrueckii, both strains were less than the lower detection limit, and Lactobacillus delbruecki subspecies lactis (Lactobacillus delbrueckii ss delbrueckii) For Lactobacillus delbrueckii ss lactis), two strains are both below the lower detection limit, and for Lactobacillus gasseri, all three strains have a growth rate of less than 1/1000 relative to the control medium. Bacillus Ham For teri (Lactobacillus hamsteri), one strain is below the lower limit of detection, and for Lactobacillus helveticus, one of the two strains is below the lower limit of detection, and one has a growth rate relative to the control medium. Less than 1/1000, Lactobacillus intestinalis is less than the lower limit of detection for one strain, and Lactobacillus jensenii is less than the lower limit of detection for both strains For Lactobacillus johnsonii, both 2 strains are less than the lower detection limit, for Lactobacillus reuteri, both 2 strains are less than the lower detection limit, For Lactobacillus casei, one strain was tested. For Lactobacillus rhamnosus, two strains are both below the lower limit of detection, and for Lactobacillus fermentum, one strain is below the lower limit of detection For Lactobacillus fructivorans, one strain is less than the lower limit of detection, and for Lactobacillus plantarum, one strain is less than the lower limit of detection, and Lactobacillus luminis (Lactobacillus ruminis) is less than the lower limit of detection in one strain, and Lactobacillus sakei ss sakei (Lactobacillus sakei ss sakei) is less than the lower limit of detection in one strain, and Streptococcus thermophilus ( For Streptococcus thermophilus), two strains Deviation was also less than the detection limit.

  As mentioned above, according to this MRS-CCC culture medium, it becomes clear that more selective detection of Lactobacillus acidophilus is possible by suppressing the growth of bacterial species closely related to Lactobacillus acidophilus. It was. In particular, since the growth of bacterial species such as Lactobacillus johnsonii, Lactobacillus gasseri, and Lactobacillus crispatus known as enteric bacteria is suppressed, For example, when it was attempted to detect Lactobacillus acidophilus from a stool-derived sample, it became clear that the problem of false positives was reduced and that it could be suitably used.

  In addition to Lactobacillus acidophilus, it was also revealed that it can be used as a selective medium for Lactobacillus amyloboraus or Lactobacillus brevis.

[Test Example 3]
It was verified as follows whether Lactobacillus acidophilus mixed in human feces could be effectively detected by MRS-CCC medium.

Fresh feces from 3 healthy adults (A, B, and C) as stool samples were collected in a glass tube containing 6 mL of anaerobic transport medium (shown below for medium composition) and thoroughly homogenized. Anaerobic transport medium was added to achieve dilution. One volume of L. acidophilus YIT 0198 strain was added to 9 volumes of this stool dilution so that the final number of bacteria was about 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ / mL, respectively. After appropriately diluting with PBS, 50 μL of the diluted solution was applied to each of 3 plates of agar plate medium (medium 1, medium 2, medium 3) of MRS-CCC medium and cultured for 3 days under aerobic or anaerobic conditions. . From the number of colonies appearing on the agar medium, the amount of coating solution, and the dilution rate, the number of bacteria per mL of the stool dilution was calculated. As a control for measurement, the final bacterial count of L. acidophilus YIT 0198 strain was about 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ cells / mL against 9 volumes of anaerobic transport medium without feces. 1 volume each was added, diluted appropriately with PBS, 50 μL of the diluted solution was applied to each of 3 MRS agar plates, and cultured under aerobic conditions for 3 days. And the number of the bacteria calculated from the number of colonies which appeared by this, the amount of coating liquids, and the dilution rate was used as a control. The results are shown in Table 3.

(Anaerobic transport medium)
Salt Solution I * 18.8mL
Salt Solution II ** 18.8mL
0.1% Resazurin 0.25mL
Lab lemco powder 2.5g
L-cysteine 0.125g
Glycerol 25mL
(The whole amount is made up to 250 mL with water.)
* Salt Solution I: KH ₂ PO ₄ 0.3%, NaCl 0.6%, (NH ₄ ) ₂ SO ₄ 0.3%, CaCl ₂ 0.03%, MgSO ₄ 0.03%
** Salt Solution II: K ₂ HPO ₄ 0.3%

  As a result, any number of bacteria as measured in the MRS-CCC medium was applied to a MRS agar plate culture medium in which stool was not mixed, and the number of bacteria in the control was measured for the number of L. acidophilus YIT 0198 strains. It was revealed that Lactobacillus acidophilus mixed in human feces can be effectively detected by MRS-CCC medium. In addition, some of the colonies collected after the measurement were randomly collected and identified by the RAPD method, and it was confirmed that all were L. acidophilus YIT 0198 strains.

[Test Example 4]
It was verified as follows whether Lactobacillus acidophilus can be effectively detected by MRS-CCC medium from the stool of humans who ingested a product containing Lactobacillus acidophilus.

As a product containing Lactobacillus acidophilus, a fermented milk product (“Mill Mill” manufactured by Yakult Honsha Co., Ltd.) containing L. acidophilus YIT 0198 strain as a viable cell at a concentration of 10 ⁷ cells / mL or more was used. The test period will be a total of 5 weeks, 2 weeks pre-drink observation period, 2 weeks drink period, and 1 week post-drink observation period. I asked him to drink a total of 5 stools, once a week.

  Preparation of a stool sample and measurement of the number of bacteria using MRS-CCC medium were carried out in the same manner as in Test Example 3, and the number of colonies appearing on the agar medium after culturing for 3 days under aerobic conditions, the amount of coating solution, The number of bacteria per 1 g of the stool sample was calculated from the dilution factor and the collected stool weight. The results are shown in Table 4.

As a result, no bacteria were detected significantly in the stool samples in the pre-drinking observation period and the post-drinking observation period, while in the stool samples in the first drinking period (3 weeks from the start of the test) The concentration of 6.49 ± 0.92 (Log ₁₀ cfu / g stool) and the average of 6.32 ± 1.00 (Log ₁₀ cfu) from stool samples in the 2nd week of drinking (4 weeks from the start of the study) Lactobacillus acidophilus viable bacteria could be detected at a concentration of (/ g stool). Therefore, it was considered that Lactobacillus acidophilus can be effectively detected by MRS-CCC medium from human feces ingesting fermented milk products containing L. acidophilus YIT 0198 strain. In addition, some of the colonies collected after the measurement were randomly collected and identified by the RAPD method, and it was confirmed that all were L. acidophilus YIT 0198 strains.

Claims (9)

  A selective medium for selectively growing one or more Lactobacillus bacteria selected from the group consisting of Lactobacillus acidophilus, Lactobacillus amyloboraus, and Lactobacillus brevis, A selective medium comprising ciprofloxacin, clindamycin, and cephalothin.   The selective medium according to claim 1, which is used as a selective medium for Lactobacillus acidophilus.   The selective medium according to claim 1 or 2, wherein a content of the cephalothin is 0.25 mg to 1 mg per liter of the basal medium.   Content of the ciprofloxacin, the clindamycin, and the cephalothin is 5 mg to 20 mg, 0.05 mg to 0.2 mg, and 0.25 mg to 1 mg, respectively, per liter of the basal medium. Item 4. The selective medium according to any one of Items 1 to 3.   The selective medium according to any one of claims 1 to 4, wherein the basal medium is one selected from the group consisting of an MRS medium, an LBS medium, and a BCP medium.   After inoculating and culturing the sample on the selective medium according to any one of claims 1 to 5, the grown bacteria are selected from the group consisting of Lactobacillus acidophilus, Lactobacillus amyloboras, and Lactobacillus brevis A method for detecting a bacterium belonging to the genus Lactobacillus, comprising detecting as one or more bacterium belonging to the genus Lactobacillus.   The method for detecting a Lactobacillus bacterium according to claim 6, wherein the sample is prepared from a food or a medicine.   The method for detecting a Lactobacillus bacterium according to claim 6, wherein the sample is prepared from human or animal feces.   The method for detecting a Lactobacillus bacterium according to any one of claims 6 to 8, wherein the viable cell count of the Lactobacillus bacterium is measured.