JPS6366518B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a selective medium for Bifidobacterium. If you want to extract and culture only the Bifidobacterium bacteria from biological materials such as intestinal contents and feces, or food and beverages containing Bifidobacterium bacteria for the purpose of testing or research, select only this bacterium. In many cases, a selective medium is used, which allows for the growth of other bacteria while preventing or significantly suppressing the growth of other bacteria. However, since Bifidobacterium is originally relatively difficult to culture, enhancing selectivity often inhibits the growth of this bacterium. The medium had the disadvantage of being somewhat unsatisfactory in selectivity. An object of the present invention is to provide a superior selective medium for Bifidobacterium that has the ability to actively propagate various Bifidobacterium bacteria while strongly suppressing the growth of other bacteria. . The present invention has succeeded in achieving this objective.
A selective medium for Bifidobacterium containing a sugar selected from the group consisting of glucose, lactose, fructose, ammonium salt, biotin, bantothenic acid, riboflavin, pyruvate, polyoxyethylene sorbitan monofatty acid ester, and nalidixic acid. It is characterized by the selection of a synthetic medium. Each of the above-mentioned medium components is an essential component for the selective growth of Bifidobacterium, and a good selective medium cannot be obtained without any of them.
In addition to these, the selective medium of the present invention preferably contains reducing agents (such as ascorbic acid and cysteine), as well as inorganic nutrient salts that are normally added to Bifidobacterium cultures. Substances effective for promoting proliferation, such as amino acids such as leucine, valine, methionine, and cysteine, nucleobases such as adenine, guanine, xanthine, and uracil, oligosaccharides containing glucosamine,
For example, β-ethyl-N-acetyl-D-glucosaminide and the like may be contained within a range that does not cause any inconvenience in inhibiting the growth of other bacteria. On the other hand, examples of substances that should not be added to the selective medium of the present invention because they cause the growth of bacteria other than Bifidobacterium include:
There are animal milk, meat juice, yeast extract, trypticase, or an amino acid mixture with an amino acid composition similar to these. The content of the essential components in the selective medium of the present invention is preferably within the following range per 100 ml of medium. Sugars (total) 1.0-3.5g Ammonium salt (as ammonium sulfate) 0.5-2.0g Biotin 0.01-0.10mg Pantothenic acid 0.2-1.0mg Riboflavin 0.1-1.0mg Pyruvate 5-20mg Polyoxyethylene sorbitan monofatty acid ster (as Tween 80) ) 0.5 to 0.1 g Nalidixic acid 5 to 10 mg The contents of other medium components may be appropriately selected within a range that does not cause any inconvenience in suppressing the growth of other bacteria. Specific examples include 10 to 50 mg of nucleic acid bases, 10 to 50 mg of amino acids, and 1 to 2 g of various oligosaccharides. Although not related to bacterial growth, the medium may contain resazurin, an indicator for the anaerobic state of the medium, bromcresol purple, a PH indicator for colony identification, and the like. To prepare the selective medium of the present invention, a predetermined amount of saline, resazurin, sugar, ammonium salt,
After dissolving Tween 80 and agar in water, vitamins, pyruvic acid, cysteine, and nalidixic acid are sequentially added. Finally, soda carbonate is added and carbon dioxide gas is bubbled by a gas injection method (anaerobic roll tube method) to replace dissolved oxygen. When the medium is reduced (resazurin decolorizes and riboflavin becomes bright yellow), add bromcresol purple, a PH indicator, and dispense into test tubes under carbon dioxide gas. Since it is a completely anaerobic and simple synthetic medium, sterilization at 100°C for 30 minutes is sufficient. After cooling, store in a cool dark place. In the case of the normal anaerobic jar method (when used as a plate culture medium), carbon dioxide gas is not bubbled, and resazurin and soda carbonate are removed from the above composition, dissolved in the above order, and dispensed after sterilization. It is desirable to always have a fresh medium available for use. For selective culture using the selective culture medium of the present invention, both the anaerobic roll tube method and the anaerobic jar method are usually used.
℃, 5 days of culture is required. However, in the anaerobic jar method, the anaerobic conditions are poorer than in the roll tube method, so depending on the bacterial species, growth may be slightly delayed. The present invention will be explained below with reference to Examples and Experimental Examples. Example 1

[Table] Dissolve the above medium components in distilled water while blowing oxygen-free carbon dioxide gas, then adjust the pH to 6.8 and make the total volume 100 ml. Transfer 5 ml of this into a roll tube while blowing carbon dioxide gas again, sterilize it at 100°C for 30 minutes, cool it to 5°C, and store at the same temperature until use. Experimental Example 1 The feces of eight healthy adults were anaerobically diluted 10 ⁸ to 10 ⁹ times using a diluent having the composition shown below, and 0.5 ml of the obtained diluted solution was inoculated into the selective medium of Example 1. ,
The cells were cultured at 37°C for 5 days. After culturing, isolate the colonies that appeared on the anaerobic roll tube and follow the method of Mitsuoka et al. (Am.J.Clin.Nutrition, 30 , 1799, 1977).
It was confirmed that it was an anaerobic bacterium, and the morphology and
The bacterial species was identified based on Gram staining, the type of acid produced from glucose, and the ability to ferment various sugars. The results are shown in Table 1. In the table, the number of colonies is 8
This is the total value for the sample. Of the 75 strains that appeared, 71 (95%) were Bifidobacterium, confirming the strong selectivity of this medium.

[Diluent composition]

0.1% K ₂ HPO ₄ solution 7.5 ml Solution ^* 7.5 ml 0.1 Resazurin solution 0.1 ml 10% Na ₂ CO ₃ solution 3 ml 5% Cysteine hydrochloride solution 1 ml Distilled water 81 ml (PH 6.8) * Solution composition KH ₂ PO ₄ 0.6% ( NH ₄ ) ₂ SO ₄ 1.2% NaCl 1.2% MgSO ₄・7H ₂ O 0.12% CaCl ₂・2H ₂ O 0.12% Experimental Example 2 The same diluted feces as that prepared in Experimental Example 1
0.5 ml was inoculated into the same selective medium used in Experimental Example 1 and a non-selective medium with the composition shown below for control, and the mixture was incubated at 37°C.
The cells were cultured for 5 days. After the culture was completed, Bifidobacterium colonies formed in each medium were confirmed and counted in the same manner as in Experimental Example 1, and the number was converted to the number of bacteria per 1 g of feces. The results are shown in Table 2. There is no significant difference in the results between using the selective medium of the present invention and when using a non-selective medium, which indicates that the selective medium of the present invention is suitable for quantitative isolation and measurement of Bifidobacterium in samples. I know it can be used.

[Non-selective medium composition]

Glucose 0.2g Beef Extract (BBL) 0.2g Yeast Extract (Difco) 0.5g Tribute Case (BBL) 1g 0.1% K ₂ HPO ₄ Solution 7.5 ml Solution I ^* 7.5 ml 0.1% Resazurin Solution 0.1 ml 8% Na ₂ CO ₃ Solution 5ml 3% cysteine hydrochloride solution 1ml Agar 2g Distilled water 79ml (PH 6.8) *Solution: Same as the one used for the diluted solution above.

Claims (1)

[Claims] 1 Glucose, lactose,
one or more sugars selected from the group consisting of fructose, ammonium salt, biotin, pantothenic acid,
A selective medium for Bifidobacterium, which is a completely synthetic medium containing riboflavin, pyruvate, polyoxyethylene sorbitan monofatty acid ester, and nalidixic acid.