JPH08332085A - Selection culture medium for measuring bifidus cell number - Google Patents

Abstract

PURPOSE: To obtain the subject culture medium, containing acetic acid or sodium acetate and lithium chloride in an agar culture medium component for proliferation of bifidus bacteria and capable of rapidly and accurately measuring the bifidus bacteria in samples such as foods and drinks, medicines or feces. CONSTITUTION: This selection culture medium for measuring bifidus bacteria is obtained by dissolving trypticase, phytone-peptone, a yeast extract, L-cysteine, glucose, K2 HPO4 , KH2 PO4 , MgCl2 .6H2 O, FeSO4 .7H2 O, etc., as culture medium components for proliferation of bifidus bacteria in purified water, adding agar thereto, adding acetic acid or sodium acetate and lithium chloride to the resultant solution so as to provide the acetic acid or sodium acetate at 50-200ppm concentration and the lithium chloride at 5-50mM concentration, then regulating the pH to 5.0-5.8, subsequently sterilizing the mixture at 121 deg.C for 15min in an autoclave and then distributing the sterilized mixture into Petri dishes. The resultant culture medium is used for selectively measuring the bifidus bacterial cell number in foods and drinks, medicines and intestinal floras, capable of simply performing the measuring operations and has a high selectivity and detection ratio of the bifidus bacteria.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a selective medium for bifidobacteria, which is used for selective measurement of the number of bifidobacteria in foods and drinks, pharmaceuticals, and intestinal flora.

[0002]

2. Description of the Related Art With recent achievements in basic research on bifidobacteria and advances in culture technology, yogurt using bifidobacteria has come to be marketed in Japan and other countries around the world, and the consumption thereof has been increasing year by year. There is. It is known that the intestinal action of Bifidobacteria is achieved by ingesting a certain amount of viable Bifidobacteria orally, but Bifidobacterium is generally weak in oxygen and acid (low pH), and in yogurt The viable cell count tends to decrease. Therefore, it is very important in terms of quality control to detect bifidobacteria in yogurt and accurately count the number of the bacteria. However, some types of lactic acid bacteria are generally contained in yogurt, and it is difficult to accurately detect and count only bifidobacteria among them.

The Bifidobacteria test method in fermented milk, which was established by the National Fermented Milk Lactic Acid Beverages Association as a voluntary test method in 1984, is based on the BL agar medium (Glucose blood liver aga).
r) is used as the medium. However, in this method, both lactic acid bacteria of the genus Streptococcus (Streptococcus) and Lactobacillus (Lactobacillus), and bifidobacteria are grown on the BL agar plate,
The number of Bifidobacteria was determined by counting the number of colonies of various shapes that appeared on the plate and observing the colonies of each shape with a Gram-stained specimen under a microscope. From this, it is difficult to detect the colonies of Bifidobacterium by this test method when the number of colonies of Bifidobacterium is 1/100 or less of the number of colonies of lactic acid bacteria of the genus Streptococcus or Lactobacillus.

As a medium capable of selectively growing Bifidobacteria, a medium to which an antibiotic is added as a main selective agent to suppress the growth of Streptococcus or Lactobacillus (Journal of the Japanese Society of Bacteria, 33 (6) ),
753 (1978), Journal of Food Sanitation, 18, 537.
(1977)), and a selective medium (JP-A-60-114186) in which a sugar source (eg, galacto-oligosaccharide) specifically used in bifidobacteria is combined with trypticase peptone and a protein degradation product. is there. However, the former is not suitable for accurate measurement of the number of bifidobacteria, since the growth of lactic acid bacteria may be suppressed and the growth of bifidobacteria may be suppressed at the same time. In particular, since bifidobacteria in yogurt are stressed by oxygen and acid (low pH), their susceptibility to these antibiotics may increase and their growth may be significantly suppressed. Although the latter has a high detection rate of Bifidobacterium, some lactic acid bacteria other than Bifidobacterium also grow and form colonies,
There is a problem that the sugar source used is very expensive.

[0005]

An object of the present invention is to provide a selective medium capable of rapidly and accurately measuring the number of Bifidobacteria in various samples such as foods and drinks, pharmaceuticals, feces and the like.

[0006]

Means for Solving the Problems As a result of intensive studies on selective agents other than antibiotics, the present inventors have found that acetic acid, which is a short-chain fatty acid, does not suppress the growth of Bifidobacterium at a certain concentration. , And found that it works suppressively against other bacteria, and as a result of further research, it was found that 50 to 200 mM of acetic acid or sodium acetate and 5 to 50 mM of chloride were added to a non-selective medium in which bifidobacteria vigorously grow. It was found that only bifidobacteria can be selectively grown by adding lithium and adjusting the pH to 5.0 to 5.8, and the present invention has been completed.

Hereinafter, the present invention will be described in detail. In the present invention, “Bifidobacterium” means Bifidobacterium longum, Bifidobacterium infantis (Bifidobacterium).
infantis), Bifidobacterium breve (Bifido)
bacterium breve), Bifidobacterium bifidum, Bifidobacterium adolescent
is) etc. can be specifically mentioned, but is not limited to these species.

In the present invention, the "agar medium component for growing Bifidobacteria" means a nitrogen source, a carbon source, inorganic salts, agar for solidifying the medium, etc. suitable for growing Bifidobacteria. Examples of the nitrogen source include protein hydrolysates, yeast extracts, fish meat extracts and the like.
Here, the term "protein hydrolyzate" includes both a hydrolyzate of animal protein such as casein and a hydrolyzate of plant protein such as soybean protein. When the protein hydrolyzate is added to the medium, the animal protein or the vegetable protein can be added alone, or both can be added in combination. Further, the protein hydrolyzate can be added up to about 10% (W / V), but the preferable addition amount is 0.5 to 2.0% (W / V). Yeast extract and meat extract are 0.1-5.0% (W
/ V), but the preferable addition amount is 0.1 to 0.5% (W / V). The above nitrogen sources can be used in appropriate combination depending on the intended detection and measurement.

Any carbon source may be used as long as it can be fermented vigorously by Bifidobacterium, and examples thereof include glucose, lactose, fructose and galactose. These can be used in an appropriate combination depending on the intended detection and measurement.
Further, if necessary, monovalent or divalent inorganic salts (excluding lithium chloride) can be added. Among the salts, monovalent salts include sodium chloride, potassium chloride and the like, and divalent salts include magnesium chloride, calcium chloride, manganese sulfate and the like. The addition amount of these salts is 0.0 for monovalent and divalent salts.
It is suitable to add to the medium in the range of 1 to 0.5% (W / V).

The inventors of the present invention firstly examined TPYG medium (trypticase (BBL) 8 g, phytonpeptone (BBL)).
3 g, yeast extract 5 g, sodium chloride 5 g, L-cysteine hydrochloride 0.5 g, glucose 20 g, K ₂ HPO
_{4 2g, KH 2 PO 4 3g} , MgCl 2 · 6H 2 O0.
_{5g, FeSO 4 · 7H 2 O10mg} , H 2 O1000
ml) as a basal medium, to which formic acid, acetic acid, propionic acid, butyric acid or lactic acid was added in an amount of 50 to 400 mM, and the effect on the growth of various intestinal bacteria, lactic acid bacteria and bifidobacteria was examined. Of these, formic acid and butyric acid were more potent in inhibiting the growth of Bifidobacterium than bacteria other than Bifidobacterium. Propionic acid had a weak growth inhibitory effect on some bacteria other than Bifidobacteria. After all, acetic acid did not suppress the growth of Bifidobacteria, and was the most excellent in the selectivity of suppressing the growth of bacteria other than Bifidobacteria. The acetic acid concentration that does not suppress the growth of Bifidobacterium and suppresses the growth of most bacteria other than Bifidobacterium is 50 to 200 mM.
Moreover, this growth inhibitory effect increases as the pH of the medium decreases. The optimum pH range for suppressing the growth of most other bacteria without inhibiting the growth of Bifidobacteria is 5.0 to 5.8, and the growth of Bifidobacterium is suppressed at a pH of less than 5.0, When it is higher than 5.8, proliferation of various kinds of bacteria other than Bifidobacterium can be observed. However, even in the above pH range, most of the lactobacilli show proliferative properties.

Therefore, a specific concentration of lithium chloride is added as an essential component as a growth inhibitor for these lactobacilli to the selective medium for measuring the number of Bifidobacteria of the present invention. Specifically, it does not suppress the growth of Bifidobacterium, but it is used for Lactobacillus, especially yogurt, and is a subspecies of Lactobacillus delbrux.
ekii subsp. bulgaricus) and Lactobacillus acidophilus (Lactobacillus acidophilus) are ideally added at a concentration that suppresses the growth. In the present invention, lithium chloride is added to the medium in the range of 5 to 50 mM for the purpose. And it is preferably added in the range of 10 to 25 mM. The concentration does not suppress the growth of Bifidobacterium, but suppresses the growth of Lactobacillus delbrueckii subsp. Bulgaricus and Lactobacillus acidophilus. If more than 50 mM lithium chloride is added, the growth of Bifidobacterium itself is also suppressed and
If it is less than M, the growth suppression of Lactobacillus delbrueckii subsp. Bulgaricus and Lactobacillus acidophilus will be insufficient, and it will be difficult to accurately measure the number of Bifidobacteria. In the medium of the present invention, as described above, antibiotics were not added, and acetic acid, which is a major metabolite of bifidobacteria, as a selective agent and 1 /
About 5 lithium chloride is added. Therefore, it is possible to accurately measure even the bifidobacteria that have been stressed by oxygen or acid (low pH) in yogurt. Also, 50-2
By adding 00 mM acetic acid to make the pH acidic to 5.0 to 5.8, Eubacterium (Eubacterium) that forms the main flora in the human intestine as well as Gram-negative bacteria
), Peptococcus, Clostridium, and other Gram-positive bacteria can also be suppressed from growing.

In preparing the medium of the present invention, the agar medium components for growing Bifidobacterium (excluding agar) are added to purified water and adjusted to a predetermined pH with hydrochloric acid, and then about 2% of agar for solidifying the medium is added. Is added, and autoclave sterilization is performed.
Bifidobacteria-containing sample diluted appropriately is smeared on the medium of the present invention which is dispensed and solidified in a petri dish, and this is replaced with carbon dioxide gas or a mixed gas of carbon dioxide gas and nitrogen gas under anaerobic conditions at 37 ° C for 48 to Incubate for 72 hours. By counting the colonies that appear after culturing, it is possible to perform the selective counting of bifidobacteria, which is the intended purpose of the present invention.

As typical examples of the sample to which the medium of the present invention is applied, there are bifidobacteria-containing yogurt and human feces. However, not limited to the sample, if the sample containing Bifidobacterium,
In any case, the medium of the present invention can be applied.

[0014]

EXAMPLES The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the invention thereto. Example 1 trypticase (BBL) 8 (g) phytone peptone (BBL) 3 Yeast extract 5 L-cysteine 0.5 glucose _{20 K 2 HPO 4 2 KH 2} PO 4 3 MgCl 2 · 6H 2 O 0. 5 FeSO ₄ .7H ₂ O 0.01 Sodium acetate 8.2 Lithium chloride 1 Agar 22.5 Purified water 1000 ml The above components except for agar were dissolved in purified water and adjusted to pH 5.4 with 1N hydrochloric acid. Add agar to this, 121 ℃
After sterilizing by autoclaving for 15 minutes, the mixture was dispensed into a petri dish to prepare a plate. The agar plate was inoculated with 0.1 ml of a diluted culture solution of various bacteria including bifidobacteria, and the number of colonies formed when anaerobically culturing at 37 ° C. for 48 hours was measured by the gas pack method.

The results are shown in Table 1.

[0016]

[Table 1]

All the tested Bifidobacteria grew well on this medium, and almost the same number of colonies as those appearing on the BL agar medium used as a control were detected. On the other hand, none of the test bacteria other than Bifidobacterium, except Saccharomyces cerevisiae, formed colonies on this medium. Furthermore, the colonies of Saccharomyces cerevisiae that appeared on this medium were extremely small pinpoint colonies, and this medium showed strong selectivity. [Example 2] Fresh feces were collected from 6 healthy adult males (25 to 45 years old), and 9 times the amount of the above anaerobic diluent (World of Enterobacteriaceae, Mitsuoka Tomohashi, 322 (1980), Koubunsha). In addition, it was thoroughly homogenized using a homogenizer under anaerobic conditions. This was further diluted appropriately with the above anaerobic diluent, and then 0.1% was added on the same medium as used in Example 1.
The cells were inoculated by smearing each ml and anaerobically cultured at 37 ° C. for 48 hours.
After culturing, the number of colonies appearing on the medium was counted, and the number of colonies of Bifidobacteria and the number of contaminants were examined by microscopic examination. In order to compare the number of Bifidobacterium, BL
The number of Bifidobacterium colonies was measured when an agar medium was used.

The results are shown in Table 2.

[0019]

[Table 2]

The selection ratio of bifidobacteria (the ratio of the number of bifidobacteria to the total number of bacteria) in this medium was 100% in 4 out of 6 samples, and 80% or more in the remaining 2 samples. Selectivity was confirmed. The contaminants other than Bifidobacteria were of the genus Yubacterium.
On the other hand, the detection rate of bifidobacteria in this medium (the ratio of the number of bifidobacteria in this medium to the number of bifidobacteria in BL agar medium) was 81.0 to 146.7%, which is about the same as the BL agar medium in the non-selective medium. The number of Bifidobacteria was able to be measured. [Example 3] Using the same medium as used in Example 1, the number of Bifidobacterium in yogurt (5 varieties) containing Bifidobacterium marketed in Japan was measured. That is,
These yogurts are appropriately diluted with sterile physiological saline, 0.1 ml each is spread on this medium, and the total number of colonies observed when anaerobically cultured at 37 ° C for 48 hours is measured. The number of bacterial colonies and the number of contaminants were examined. In order to compare the number of Bifidobacteria, the number of Bifidobacteria colonies was measured when a BL agar medium was used.

The results are shown in Table 3.

[0022]

[Table 3]

The bifidobacteria selectivity in this medium was 100% in all cases, and formation of other lactic acid bacteria colonies was not observed at all, confirming the strong selectivity of this medium. In addition, the detection rate of Bifidobacterium in this medium is 90.9 to
It was 111.1%, and the number of Bifidobacterium, which was almost the same as that of the BL agar medium, could be measured.

[0024]

EFFECTS OF THE INVENTION Conventional methods for selecting and measuring the number of Bifidobacterium, which consist of BL agar medium, selective medium using antibiotics, or medium utilizing the difference in the assimilation ability of sugar, require a complicated and skillful method, and the detection rate is high. Was not always stable. The medium of the present invention, which compensates for these drawbacks, has a simple measurement operation, does not require skill, and has extremely high selectivity and detection rate. Therefore,
INDUSTRIAL APPLICABILITY The present invention can be widely used as a method for measuring the number of bifidobacteria containing various foods, beverages, pharmaceuticals, feeds, feces, etc. containing bifidobacteria.

Claims (4)

[Claims] 1. An agar medium component for growing Bifidobacterium,
A selective medium for measuring the number of Bifidobacterium, which comprises acetic acid or sodium acetate and lithium chloride. 2. Acetic acid or sodium acetate in an amount of 50 to 200
The selective medium for measuring the number of Bifidobacterium according to claim 1, wherein the selective medium contains mM. 3. The selective medium for counting the number of Bifidobacterium according to claim 1, which contains 5 to 50 mM of lithium chloride. 4. The selective medium for measuring the number of Bifidobacterium according to claim 1, which has a pH of 5.0 to 5.8.