JP5219058B2 - Selection medium for lactic acid bacteria - Google Patents

Description

  The present invention relates to a selective medium for lactic acid bacteria, specifically, a medium for selectively culturing so-called Bulgarian bacteria.

Lactobacillus delbrueckii subsp. Bulgaricus, a so-called Bulgarian bacterium, is a type of lactic acid bacterium that has been traditionally used for yogurt production. Until now, Bulgarian bacteria could not be detected in the stool of humans eating yogurt, and Bulgarian bacteria were thought to die in the human digestive tract.

  In general, in order to detect a specific bacterium from a sample containing various bacterium, a selective medium for preferentially growing the bacterium is used. As typical selection media for lactic acid bacteria, for example, MRS medium, LBS medium, Rogosa medium and the like are known.

Incidentally, the microorganisms used in the production of lactic acid fermentation products such yogurt, Lactobacillus and Lactococcus (Lactococcus) or Streptococcus (Streptococcus) genus Enterococcus (Enterococcus) or Lactobacillus genus, such as, Bifidobacterium ( There are various bacterial species belonging to Bifidobacterium). For example, not only one bacterial species but also two or three bacterial species are usually used for the production of yogurt. Among these lactic acid bacteria, Rogosa medium or LBS medium is used as a medium for selectively detecting the genus Lactobacillus at the genus level, but it was difficult to select only Bulgarian bacteria at the bacterial species level. It is also conceivable that colonies of Lactobacillus lactic acid bacteria are formed using Rogosa medium or LBS medium, and only Bulgarian bacteria are selected from the shape and color of the colonies. However, it seems that the growth of Bulgaria has been suppressed by other species of the same genus, and in a recent report (Non-patent document 1: Appl. Environ. Microbiol. 71 (1), 547-549, 2005) When trying to detect Bulgaria bacteria using MRS medium, it is said that Bulgaria bacteria could not be detected in feces.

  Under such circumstances, for example, Patent Document 1 (JP-A-6-343491), Patent Document 2 (JP-A-6-38795), Patent Document 3 (JP-A-7-999995), and the like include lactic acid bacteria. A medium for selectively detecting Bifidobacteria from a plurality of bacterial species is disclosed. These media are mainly supplemented with specific nutrients to grow only Bifidobacteria, but no media that can selectively culture only Bulgarian bacteria has yet been found.

Non-Patent Document 2 (Journal of Japanese Society of Nutrition and Food, 46 (2), 139-145, 1993) describes that Bulgarian and Thermophilus were pre-cultured in a medium supplemented with 10% skim milk powder. ing. However, this medium is a liquid medium in which about 0.1 w / v of yeast extract is added to the medium in addition to skim milk powder. This medium is used as a culture medium for subculture or storage of lactic acid bacteria, has no bacteria selectivity, and most lactic acid bacteria grow.
JP-A-6-343491 JP-A-6-38795 Japanese Patent Laid-Open No. 7-99995 Appl. Environ. Microbiol. 71 (1), 547-549,2005 Journal of Japanese Society of Nutrition and Food, 46 (2), 139-145, 1993

  The present invention has been made in view of the above background art, and an object thereof is to provide a selective medium for detecting so-called Bulgarian bacteria in feces.

  The selective medium of the present invention is a selective medium capable of selectively cultivating Lactobacillus delbruecki subspecies bulgaricus, comprising skim milk powder and acetic acid as main components, and containing no nitrogen source other than skim milk powder. Characterized by adding sodium acetate, ammonium citrate and agar as needed.

  Further, the detection method of the present invention is a method for selectively detecting Lactobacillus delbruecki subspecies bulgaricus from a sample containing Lactobacillus delbruecki subspecies bulgaricus and other bacteria. It is characterized by anaerobic culture for 24 to 48 hours under a constant temperature of 48 ± 2 ° C. using a medium containing nonfat dry milk and acetic acid as main components and containing no nitrogen source other than nonfat dry milk.

  According to the present invention, Lactobacillus delbrucky subspecies bulgaricus can be selectively cultured and can be detected from feces. As a result, it is possible to investigate the survival of the bacterium in yogurt in the human digestive tract, or to select Lactobacillus delbruecki, Subspecies bulgaricus having a high survival in the digestive tract.

  The selective medium of the present invention is basically composed of skim milk powder and acetic acid, and uses skim milk powder as a nitrogen source and a carbon source. In the present invention, skim milk powder refers to a product obtained by removing almost all the water from a material obtained by removing milk fat from raw material milk and drying it into a powder form. As the raw milk, milk obtained from various mammals such as cows, sheep, goats, and humans is used. The raw material milk is not particularly limited, but from the viewpoint that a certain quality is easily secured and a large amount of milk can be obtained, the raw material milk is preferable. Moreover, the manufacturing process of skim milk powder should just be a normal process, and does not require a special process. As an example of the process, there is a method in which raw milk is concentrated by heating under reduced pressure and then spray-dried. In the present invention, it is not necessary to adjust the specific component amount. The composition of typical skim milk powder is as follows according to the 5th amendment Japanese Food Standard Composition Table. 100 g of skim milk powder contains 34.0 g of protein, 1.0 g of lipid, 53.3 g of carbohydrates, 7.9 g of ash, 2 mg of vitamin B group, and about 5 g of inorganic substances such as Na, K, Mg, P, and Ca. Of course, there are some fluctuations depending on the type of raw material milk, etc., but the fluctuations are permissible unless the ingredients are extremely adjusted. The variation is preferably about ± 20% of the above component table, desirably about 10%.

  In the medium of the present invention, at least acetic acid is used as a component other than skim milk powder. This is because the growth of other bacteria can be suppressed by adding acetic acid. The amount of acetic acid used is 1.5 to 2.3 ml as glacial acetic acid (acetic acid concentration of 98 w / v% or more, the same shall apply hereinafter) with respect to 100 parts by mass of skim milk powder.

  In addition, sodium acetate is preferably added to the medium. Sodium acetate is not an essential component, but when sodium acetate and acetic acid are combined, the buffering action of the medium is enhanced and the colonies can be enlarged. Sodium acetate may be either anhydrous or hydrated, but trihydrate is preferably used from the viewpoint of stability. The amount used is 9 to 13 parts by weight per 1 ml of glacial acetic acid, and 15 to 30 parts by weight, preferably 18 to 24 parts by weight, as sodium acetate trihydrate with respect to 100 parts by weight of skim milk powder. is there.

  The selective medium of the present invention can be used as either a liquid medium or an agar medium, but is usually used as an agar medium. The amount of agar used may be an amount of agar that can produce a plate medium, but the amount is 10 to 20 parts by mass with respect to 1000 ml of the medium.

  The selective medium of the present invention is adjusted so that the concentration of skim milk powder is 3 to 9 w / v%, preferably 5 to 9 w / v% with respect to water. And sodium acetate is added. Therefore, the final standard is 30 to 90 g of skim milk powder, 1.1 to 1.6 ml of acetic acid, and 10 to 20 parts by mass of sodium acetate with respect to 1000 ml of the medium (water content).

  In order to detect so-called Bulgarian bacteria using the selective medium of the present invention, conditions suitable for the selective medium of the present invention are selected instead of general culture conditions. For detection of lactic acid bacteria and Escherichia coli, culture conditions of about 37 ° C. and 48 hours are usually selected, but when the selective medium of the present invention is used, anaerobic culture at a relatively high temperature of 48 ± 2 ° C. is performed. On the other hand, the culture time is 24 hours to a maximum of 48 hours, preferably about 24 hours. In the culture at 37 ° C. or for more than 48 hours, there is a strong possibility that bacteria other than the Bulgarian bacteria will proliferate and cannot be sufficiently discriminated.

  As a method for sterilizing a medium, any method such as a method using radiation, a method using filter filtration, or a method using an autoclave can be adopted. Moreover, when sterilizing by an autoclave, general sterilization conditions (for example, 121 degreeC 15 minutes) are employ | adopted. However, since the culture medium of the present invention contains skim milk powder, sterilization using heating, depending on the set temperature, heating time, content of skim milk powder, etc., may lead to coloring of the medium and the like, resulting in an inappropriate medium. is there. Therefore, for example, it is desirable to avoid sterilization at high temperatures such as heating at 100 ° C. for about 10 minutes (an autoclave may be used) or sterilization by filter filtration.

  The operation method for detecting Bulgaria bacteria is the same as a general method for handling bacteria, and does not require any special operation. That is, a test solution in which a sample is diluted stepwise is smeared on a flat agar medium prepared under aseptic conditions, or the sample is added to the medium to form a flat medium and cultured. And after culture | cultivation, what is necessary is just to count the colonies on a culture medium. Bulgarian bacteria form white, convex circular colonies. A small number of colonies may be formed on the medium by lactic acid bacteria other than Bulgaria bacteria, such as Streptococcus thermophilus, but can be clearly distinguished from the colonies of Bulgaria bacteria.

  In the selective medium of the present invention, ammonium citrate is added in order to enlarge the colonies of lactic acid bacteria. It is a known fact that lactic acid bacteria cannot use ammonium citrate as a nitrogen source. Ammonium citrate is not an essential component, and 0 to 3.5 parts by mass are used for 100 parts by mass of skim milk powder.

  The selective medium of the present invention was developed for the purpose of detecting Bulgaria bacteria present in feces, and fecal samples are suitable as test samples, but the samples are not limited to feces, Needless to say, dairy products such as yogurt and other products can be used as samples. In addition, it goes without saying that the selective medium of the present invention is not only used for the purpose of detecting viable bacteria in a sample, but can also be used as a culture medium or a storage medium for culturing Bulgaria bacteria.

  The most reliable method for determining the viable cell count is to use it as an agar plate medium and count the number of colonies formed. In addition to this method, the selective medium of the present invention is used as a liquid medium, and can be applied to the bacterial count measurement by the so-called most probable number method. The most probable number method is generally used as a test method for the number of coliforms. In this method, dilution of the sample to be tested is added step by step to multiple culture media, and as a result, the number of positive samples (the media becomes opaque) is determined from the most probable number table. Estimate the number of bacteria in the sample. The details are described in, for example, the hygiene test method (edited by the Japan Pharmaceutical Association). However, since the selective medium of the present invention is originally opaque, for example, when the medium is solidified, it is determined to be positive, and the culture conditions are 24 hours at a constant temperature of 48 ± 2 ° C. and a maximum of 48 hours. Is preferred. It is also possible to determine the absorbance at a specific wavelength as an index. However, even though the medium of the present invention is a selective medium, it cannot be denied that lactic acid bacteria other than Bulgarian bacteria can grow as described above. It is also conceivable that the medium is observed as if the medium had been coagulated by these bacteria. However, by setting culture conditions of 48 ± 2 ° C. and 24 hours, coagulation due to the growth of Bulgarian bacteria and coagulation due to the growth of other lactic acid bacteria can be clearly distinguished, and it is considered that there is little misjudgment of positive / negative. Also, there are various most probable number tables, but there is no big difference in the results even if any most probable number table is used.

  Hereinafter, although it demonstrates still in detail based on the Example of this invention, it cannot be overemphasized that this invention is not limited to the following Example.

First, a selective medium according to an example of the present invention was prepared, and culture of Lactobacillus delbrucky subspecies bulgaricus (hereinafter referred to as “LB bacteria”) in feces was attempted. For comparison, MRS agar medium (pH 6.7 and pH 5.4), LBS medium, and Rogosa medium were used. LB bacteria (2038 strain) was added to a solution obtained by diluting human stool 10 times with physiological saline and physiological saline so that the concentration was about 10 ³ cfu / ml, and 100 μl of the bacterial solution was applied to each medium. Then, it culture | cultivated for 24 to 48 hours in a 37 degreeC and a 48 degreeC thermostat. Thereafter, the total number of colonies that appeared in the medium was counted. The results are shown in Table 1. Moreover, the composition of the used culture medium is as follows. The selective medium of the present invention was heated at 100 ° C. for 10 minutes, the LBS medium and Rogosa medium were heated at 100 ° C. for 30 minutes, and the MRS agar medium was subjected to autoclaving at 121 ° C. for 15 minutes. .

(Selection medium (1) of the present invention)
Nonfat dry milk (Meiji Milk Industry Co., Ltd., Maru Q) 70g
Sodium acetate trihydrate 15g
Triammonium citrate 2g
Glacial acetic acid 1.32ml
Agar 15g
1000ml of water

(MRS agar medium: Lactobacilli MRS Agar, manufactured by Difco)
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
Agar 15g
1000ml of water

(Rogosa medium: Rogosa SL Agar, manufactured by Difco)
Tryptone 10g
Yeast extract 5g
Glucose 10g
Arabinose 5g
5g sucrose
Sodium acetate 15g
2g of ammonium citrate
Monopotassium phosphate 6g
Magnesium sulfate 0.57g
Manganese sulfate 0.12g
Ferrous sulfate 0.03g
1g sorbitan monooleate
Agar 15g
Acetic acid 1.32ml
1000ml of water

(Modified LBS medium)
Casein pancreatin degradation product 10g
Yeast extract 5g
Glucose 20g
Monopotassium phosphate 6g
2g of ammonium citrate
Polysorbate 80 1g
Sodium acetate trihydrate 25g
Magnesium sulfate 0.575g
Manganese sulfate 0.12g
Ferrous sulfate 0.034g
Agar 15g
Acetic acid 1.32ml
1000ml of water

  According to this result, the LBS medium, which is a selective medium for lactic acid bacteria belonging to the genus Lactobacillus, could not grow at all even with LB bacteria diluted in saline, and the growth was remarkably suppressed in the Rogasa medium, which is also a selective medium. In addition, in the MRS medium which is a non-selective medium, the growth was remarkably suppressed at 48 ° C. even if it was able to grow at 37 ° C. under any conditions of pH 6.5 and 5.4.

  On the other hand, when mixed with stool, colonies were formed so that they could not be counted in any medium of Comparative Example, 37 ° C or 48 ° C (1000 or more / plate), and colonies of LB bacteria were discriminated. Could not count. However, when cultured under the condition of 48 ° C. using the medium of the present invention, the number of observed colonies when mixed with stool and the number of observed colonies when diluted in saline are almost the same. In addition, the number of bacteria was almost 100 / plate, and the number of bacteria almost the same as the amount of smear could be measured. The colonies when diluted in saline were white and large convex circular colonies, and the majority of similar colonies were present when mixed with feces. Further, when mixed with feces, a small amount of microcolonies were also observed, but it was clearly distinguishable from white and large convex circular colonies, and it was determined that they were not colonies of LB bacteria. The observed colonies were confirmed to be LB by identification using a bacterial species-specific primer, and confirmed to be the same strain by detecting the genotype using pulse field gel electrophoresis. Has been.

Next, the effect of adding sodium acetate or ammonium citrate was confirmed.
LB bacteria (2038 strain) was added to a solution obtained by diluting human stool 10 times with physiological saline and physiological saline so as to be about 10 ³ cfu / ml, and the bacterial solution was added to each medium shown in Table 2. After applying 100 μl, the cells were cultured in a constant temperature bath at 37 ° C. and 48 ° C. for 24 hours. Thereafter, the total number of colonies of LB bacteria that appeared in the medium was counted. The results are shown in Table 2.

  The number of colonies in the medium (2) and the medium (4) to which sodium acetate was not added was smaller than that of the medium (1) or (3) to which sodium acetate was added. Moreover, the number of colonies in the medium (3) to which sodium acetate was added was slightly larger than that in the medium (1) to which triammonium citrate was further added. When sodium acetate or triammonium citrate was added, the colonies were large and clear. Moreover, in any culture medium, colonies derived from LB bacteria and other bacteria could be distinguished, and LB bacteria in feces could be measured almost accurately. In particular, the culture media of (1) to (3) were easier to distinguish from other bacteria than the culture media (4) in which both were not added.

Furthermore, the fungal selectivity of the medium of the present invention was examined.
Among the genus Lactobacillus that are considered to be resident in the human intestine, three strains belonging to Lactobacillus gasseri (JCM1131 ^T strain, JCM1017 strain, JCM1025 strain) that are detected most frequently ) Was cultured (48 ° C., anaerobic culture for 24 hours) in the medium of the above example, and no colonies could be observed for any of the bacteria.

Subsequently, the optimum amount of skim milk powder was examined.
The amount of skim milk powder used was changed to 3 w / v%, 5 w / v%, 7 w / v%, and 9 w / v%, and the degree of growth of LB bacteria was examined. The amount of other components in the medium was not changed, and the culture conditions were 48 ° C. and 24 hours. In addition, LB bacteria (2038 strain) was added to physiological saline so as to be about 3 × 10 ² cfu / ml, and 100 μl of the bacterial solution was applied to each medium. Thereafter, the total number of colonies of LB bacteria that appeared in the medium was counted. The results are shown in Table 3.

  As a result, the largest number of colonies were detected at a use amount of 7%. Moreover, a large colony was formed in the usage amount of 5 to 9%, and discrimination was easy. Thereby, the density | concentration of skim milk powder was judged to be suitable for the density | concentration of 5-9%.

Finally, the feces of yogurt intakers containing LB bacteria were also examined. 100 g of homemade yogurt containing LB bacteria (LB bacteria: adjusted to contain 4.5 × 10 ⁷ cfu / ml) was taken by 2 healthy adults once a day for 6 days, and the stool from day 7 was collected Then, detection of LB bacteria in feces was attempted by culturing using the selective medium (1) of the present invention. After isolating colonies that appear to be due to LB among the colonies that appeared on the medium, DNA was extracted and the bacterial species was identified using specific primers of Bulgarian bacteria ( L. delbrueckii subsp. Bulgaricus ). went. Furthermore, in order to confirm that the detected bacteria were the same as the ingested bacteria, the strains were also identified by pulse field gel electrophoresis.

Of the colonies that appeared on the medium, all of the selected colonies were confirmed to be Bulgarian bacteria using the Bulgarian specific primers. LB bacteria were detected in all subjects, and the number of bacteria per gram of stool was 1.3 × 10 ⁶ cfu / ml and 1.0 × 10 ² cfu / ml. In addition, LB bacteria were not detected from the stool before ingesting yogurt. Moreover, when the strain was identified by pulse field gel electrophoresis, it was confirmed that the detected bacteria were the same as the ingested bacteria.

  Thus, by using the selective medium of the present invention, Lactobacillus delbruecki subspecies bulgaricus (LB bacteria) could be detected in human feces. As a result, it is considered that the investigation of the action of LB bacteria in the body and the development of more useful yogurt using the new LB bacteria can be performed.

According to the international food standards of WHO / FAO, “Yogurt” is a fermentation using Lactobacillus delbrueckii subsp. Bulgaricus ( L. delbrueckii subsp. Bulgaricus ) and Streptococcus thermophilus. It is defined as milk and Bulgaria is an industrially important species. According to the present invention, it is possible to examine the viability of Bulgarian bacteria in yogurt in the human digestive tract, or to select Bulgarian bacteria having a high survival ability in the digestive tract, and the opportunity for further effective use of yogurt. Become.

Claims (9)

A selective medium for selectively cultivating Lactobacillus delbruecki subspecies bulgaricus,
A selective medium comprising skim milk powder and acetic acid as main components, wherein only skim milk powder is a nitrogen source and a carbon source .   2. The selective medium according to claim 1, further comprising sodium acetate as a constituent component.   The selective medium according to claim 2, comprising 18 to 24 parts by mass of sodium acetate trihydrate and 1.5 to 2.3 ml of acetic acid with respect to 100 parts by mass of skim milk powder. The selective medium according to any one of claims 1 to 3, further comprising 0 to 3.5 parts by mass of triammonium citrate with respect to 100 parts by mass of skim milk powder. The selective medium according to any one of claims 1 to 4, further comprising agar. A method for selectively detecting Lactobacillus delbruxii subspecies bulgaricus from a sample containing Lactobacillus delbruxii subspecies bulgaricus and other bacteria, comprising:
A detection method comprising anaerobic culture at a constant temperature of 48 ± 2 ° C. for 24 to 48 hours using a medium comprising skim milk powder and acetic acid as main components, and only skim milk powder is a nitrogen source and a carbon source .   Furthermore, the detection method of Claim 6 using the culture medium to which sodium acetate was added.   The culture medium containing 18 to 24 parts by mass of sodium acetate trihydrate, 1.5 to 2.3 ml of acetic acid, and 18 to 24 parts by weight of agar is used for 100 parts by mass of skim milk powder. The detection method described.   The detection method according to claim 8, wherein a medium further containing 0 to 3.5 parts by mass of triammonium citrate is used with respect to 100 parts by mass of skim milk powder.