JP3532226B2 - Bifidobacteria growth-promoting substance produced by intestinal bacteria and use thereof - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a bifidobacteria growth-promoting substance produced by enterobacteria inside and outside the cells, a process for producing the same, and use thereof.

[0002]

2. Description of the Related Art Regarding substances useful for promoting the growth of useful bifidobacteria, so-called bifidus factors, several substances have been studied and reported so far. For example, N-acetylglucosamine (Proc.
Soc. Exp. Biol. Med. , 90 , 219
(1955)), peptide-related substances (Am. J. Cli
n. Nutr. , 32 , 1428 (1974); Agr.
ic. Biol. Chem. , 48 , 2159 (198
4)), carrot extract (Nippon Kaika, 55 , 499 (198)
1); Chem. Pharm. Bull. , (Tokyo
o) 14 , 1191 (1966)), sugar-related substances (Tohoku Welfare Bulletin, 10 , 313 (1986)), etc. However, preparation of these growth-promoting substances for Bifidobacterium is complicated, and there is a point that the effect of selectively growing only Bifidobacterium is not always sufficient.

On the other hand, conventionally, in the search for the number of Bifidobacteria in fermented milk or in the intestinal flora, a method of using an antibiotic as a selective agent for a selective medium for measuring the viable cell count (Journal of the Journal, 18 , 537 (1977). ); J. Bacteria, 33 , 75
3 (1973)), the number of Bifidobacteria significantly decreases when the amount of antibiotics used is large, and when the amount of antibiotics used is small, many microorganisms other than Bifidobacterium grow and There was a problem that it could not be distinguished. In addition, a method of adding blood to a non-selective medium and selecting and measuring colonies specific to Bifidobacterium after culturing (milk co-operation materials, 32 , 15 (1983)) is used when the number of bacteria other than coexisting Bifidobacterium is low. Is effective, but when the number of Bifidobacteria is significantly lower than the number of contaminating bacteria, there is a problem that measurement becomes difficult.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides a novel substance capable of selectively and rapidly growing only bifidobacteria, and obtaining a selective medium for analysis such as bifidobacteria count measurement using the substance. Is the purpose.

[0005]

Means for Solving the Problems The present invention has been made in order to achieve the above-mentioned object, and the present inventors have conducted extensive studies on metabolites of microorganisms that promote the growth of bifidobacteria. As a result, enterobacteria (especially Bactero)
idaceae, Enterobacteriacea
e) and other various bifidobacteria (Bifido)
Bacterium longum, B. breve,
B. adolescentis, B. bifidum,
B. infantis, B. animalis, B .; p
It has been found that it produces a growth-promoting substance with high activity against Seudolongum, etc.). The present invention is based on this new finding.

That is, the present invention has as its basic technical idea a bifidobacteria growth-promoting substance derived from intestinal bacteria (hereinafter sometimes referred to as a bifidus factor).

The bifidobacteria growth-promoting substance according to the present invention is an enterobacteria (particularly Bacteroideaceae, E
(Nerobacteriaceae, etc.), the culture itself and / or
Alternatively, the treated product is used as the substance according to the present invention. As the treated product, a culture containing bacterial cells, a culture supernatant, a culture filtrate, a sterilized liquid of the culture, a concentrate of these,
It includes all types of processed products obtained by processing the culture, such as dried products and, if necessary, diluted solutions and diluted products.

As an intestinal bacterium, Bacteroida
ceae, Enterobacteriaceae, P
In addition to bacteria belonging to each family of Ropionobacteriaceae, other bacteria found in the digestive tract of humans or animals are used as appropriate, non-limiting examples of which include:

Bacteroides aceae bacteria include Bacteroides vulgatus,
B. Fragilis ATCC 23745 and other Bacteroides bacteria and others. Enterobac
As a bacterium of the teriaceae family, Escheric
hia coli ATCC 9637 and the like Escherichia bacterium; Enterobacter cloacae and the like Enterobacter genus; Citrobacter fre
undii ATCC 8090 and other genus Citrobacter; Klebsiella pneumoniae A
Klebsiella sp. And others such as TCC 12658 are exemplified. Also, Propionibacterium
As e family bacteria, Propionibacterium
m freudenreichii IFO 1242
4 etc. Propionibacterium genus bacteria etc. are illustrated.

As a growth medium for these enterobacteria, any one can be used as long as it can grow. Moreover, although anaerobic conditions are required for culturing intestinal bacteria, some bacteria do not necessarily have to be anaerobically cultivated and grow sufficiently even under aerobic conditions to produce a growth promoting substance for bifidobacteria.

On the other hand, the growth-promoting substances for bifidobacteria produced by these intestinal bacteria have a growth-promoting activity for bifidobacteria even after heat treatment at 121 ° C. for 5 minutes, and have high heat resistance. Admitted. Further, the substance has a growth promoting activity only in Bifidobacterium, has little or no action on other intestinal bacteria, and also has remarkable properties of extremely high selectivity and specificity ( Example 1).

Furthermore, according to the present invention, the growth of bifidobacteria, the growth of which is suppressed by the presence of antibiotics, etc., by adding the growth-promoting substance (culture containing cells) produced by enterobacteria The ability is restored (Examples 2 to 4).
Therefore, in addition to the use of increasing the number of Bifidobacteria in the digestive tract by directly administering the growth-promoting substance produced by the intestinal bacterium to the human or animal as the culture itself and / or its processed product, food, large intestine It can be used as a selective medium for analyzing Bifidobacterium in contents and fecal samples.

That is, as one of the systems for measuring the number of Bifidobacteria in a sample containing Bifidobacteria, an agar medium containing a selective agent that suppresses the growth of contaminants other than Bifidobacteria was used, and the sample was placed in the medium. A method is known in which only bifidobacteria are proliferated specifically or selectively by inoculating and incubating with, and the presence or absence of bifidobacteria and the viable cell count are measured. However,
When a particularly high concentration of the selective agent is used in the selective medium to which the selective agent is added, not only the growth of contaminants other than Bifidobacterium is suppressed, but the growth of Bifidobacterium itself may also be suppressed, It was difficult to know the exact number of Bifidobacteria.

On the other hand, when the bifidobacteria growth-promoting substance produced by the intestinal bacterium of the present invention (a culture containing bacterial cells and / or a treated product thereof) is added to the selective medium, the growth of bifidobacteria can be made specific. It is possible to promote colonies to form colonies and to accurately measure the number of Bifidobacteria. Moreover, this effect can be obtained by using antibiotics such as paromomycin sulfate and neomycin sulfate as well as propionate, lithium chloride and any other known selective agent as a selective agent. The selective medium for Bifidobacterium assay according to the present invention can be effectively used for a wide range of assay applications.

Examples of the present invention will be described below.

[0016]

Example 1 TPYG medium (trypticase (BBL)
8 g, phytonpeptone (BBL) 3 g, yeast extract 5
g, L-cysteine hydrochloride 0.5 g, glucose 20
g, K ₂ HPO ₄ 2 g, KH ₂ PO ₄ 3 g, MgCl _{2
· 6H 2 O 0.5g, FeSO 4} · 7H 2 O 10m
g, H ₂ O 1000 ml, pH 6.5)
roads vulgatus JCM 5826,
Enterobacter cloacae ATCC
961 was inoculated and anaerobically cultured at 37 ° C. for 48 hours by the gas pack method. The obtained culture solution was treated with 5000 r.p.m.
p. m. The mixture was centrifuged for 15 minutes, and the supernatant was filtered with a Millipore filter having a pore size of 0.45 μm. 1% of these filtrates were added to TPYG medium, and various intestinal bacteria such as bifidobacteria were inoculated at 37 ° C.
Anaerobic culture was carried out for 20 hours by the gas pack method, and the OD ₅₈₀ (absorbance at a wavelength of 580 nm) of each culture solution was measured. Bacteroides vulga as a control
tus JCM 5826, Enterobacter
A TPYG medium not inoculated with the cloacae ATCC 961 culture supernatant was inoculated with each intestinal bacterium. The results are shown in Table 1 below.

[0017]

[Table 1]

As is clear from the above-mentioned test results regarding the effect of the addition of the culture supernatant of intestinal bacteria on the growth of Bifidobacterium, Bacteroides vulga
tus JCM 5826 or Enterobacter
It was found that the addition of the culture supernatant of r cloacae ATCC 961 promotes the growth of Bifidobacterium, but has a low or no effect on the growth of other intestinal bacteria.

[0019]

Example 2 BL sterilized by heating at 121 ° C. for 15 minutes
A selection medium (0.1 g of paromomycin sulfate, 0.4 g of neomycin sulfate, 6 g of lithium chloride, 30 g of sodium propionate) was dissolved in 100 ml of distilled water in an agar medium (Eiken Chemical Co., Ltd.), and a pore size 0.45 μm Millipore filter was used. 1) and 1% and 3% were added to prepare respective agar plate media. On the other hand, Propionibacterium freude was added to TPYG medium.
Inoculation with nreichii IFO 12424 and 37 ℃
Aerobically cultivated for 5 days at room temperature, the culture medium containing the bacterial cells was concentrated 10 times on a rotary evaporator, and 5% of this concentrated solution was BL.
After adding to the agar medium, adjust the pH to 7.2 and
It was heat-sterilized for 5 minutes. 1% of the above-mentioned selective agent
And 3% were added and each agar plate medium was similarly prepared. Controls are selection agents and Propionibacte
ium freudenreichii IFO 1
A BL agar plate medium to which none of 2424 culture concentrates was added was used.

Bif. long
um ATCC 15707 and Bif. adoles
centis ATCC 15703 bacterial solution 0.1 ml
The solution was applied to the surface and anaerobically cultured at 37 ° C. for 2 days by the gas pack method, and the number of bifidobacteria was counted. The results are shown in Table 2 below.

[0021]

[Table 2]

The above-mentioned Propionibacterium
ium freudenreichii IFO 12
As is clear from the test results regarding the effect of the B. 424 culture concentrate on the measured values of Bifidobacterium, Propi
onibacteriumfreudenreichi
i IFO 12424 culture concentrate 5% as selective agent 1 to 3
It was confirmed that the growth inhibitory effect of the selective agent on the growth of Bifidobacterium was canceled by the addition to the BL agar medium containing 10%. When 5% of 10-fold concentrated TPYG medium was added to BL agar medium containing 1 to 3% of selective agent, no increase in the number of Bifidobacterium was observed.

[0023]

Example 3 BL heat-sterilized at 121 ° C. for 15 minutes
A selective medium (0.1 g of paromomycin sulfate, 0.4 g of neomycin sulfate, 6 g of lithium chloride, 30 g of sodium propionate) was dissolved in 100 ml of distilled water in an agar medium (Eiken Chemical Co., Ltd.), and a Millipore filter having a pore size of 0.45 μm was dissolved. 1) and 1% and 3% were added to prepare respective agar plate media. Meanwhile, Bacteroides vulgatus JCM was added to TPYG medium.
5826 was inoculated and anaerobically cultivated at 37 ° C for 2 days, the culture solution containing the bacterial cells was concentrated 10 times by a rotary evaporator, and 5% of this concentrated solution was added to BL agar medium, and the pH was adjusted to 7.2. Then, it was heat-sterilized at 121 ° C. for 15 minutes.
1% and 3% of the above-mentioned selective agent was added thereto, and each agar plate medium was similarly prepared. The control is a selective agent and Bacteroides vulgatus JCM
A BL agar plate medium to which none of the culture concentrates of 5826 was added was used.

Bif. long
um ATCC 15707 and Bif. adoles
centis ATCC 15703 bacterial solution 0.1 ml
The solution was applied to the surface and anaerobically cultured at 37 ° C. for 2 days by the gas pack method, and the number of bifidobacteria was counted. The results are shown in Table 3 below.

[0025]

[Table 3]

The above-mentioned Bacteroides vul
As can be seen from the test results regarding the effect of the culture concentrate of Gatas JCM 5826 on the measured value of the Bifidobacterium count, Bacteroides vulgatus
By adding 5% of JCM 5826 culture concentrate to the BL agar medium containing 1 to 3% of the selective agent, it was confirmed that the inhibition of the growth of Bifidobacterium by the selective agent was released.

[0027]

Example 4 BL heat-sterilized at 121 ° C. for 15 minutes
A selective medium (0.1 g of paromomycin sulfate, 0.4 g of neomycin sulfate, 6 g of lithium chloride, 30 g of sodium propionate) was dissolved in 100 ml of distilled water in an agar medium (Eiken Chemical Co., Ltd.), and a Millipore filter having a pore size of 0.45 μm was dissolved. 1) and 1% and 3% were added to prepare respective agar plate media. Meanwhile, Enterobacter cloacae ATC was added to the TPYG medium.
C 961 was inoculated and cultivated aerobically at 37 ° C. for 2 days, the culture solution containing the cells was concentrated 10 times by a rotary evaporator, and 5% of the concentrate was added to BL agar medium, and then the pH was adjusted to 7.2. And heat sterilized at 121 ° C. for 15 minutes.
1% and 3% of the above-mentioned selective agent was added thereto, and each agar plate medium was similarly prepared. Controls are selection agents and Enterobacter cloacae ATCC
A BL agar plate medium to which none of the 961 culture concentrate was added was used.

Bif. long
um ATCC 15707 and Bif. adoles
centis ATCC 15703 bacterial solution 0.1 ml
The solution was applied to the surface and anaerobically cultured at 37 ° C. for 2 days by the gas pack method, and the number of bifidobacteria was counted. The results are shown in Table 4 below.

[0029]

[Table 4]

The above described Enterobacter c
As is apparent from the test results regarding the effect of the concentrate of L. acaea ATCC 961 culture on the measurement of the number of Bifidobacterium, Enterobacter cloac
ae ATCC 961 culture concentrate 5% with selective agents 1-3
It was confirmed that the growth inhibitory effect of the selective agent on the growth of Bifidobacterium was canceled by the addition to the BL agar medium containing 10%.

[0031]

EFFECTS OF THE INVENTION The bifidous factor according to the present invention has an excellent effect of promoting the growth of bifidobacteria. Therefore, it can be directly administered or ingested to humans or animals as a food or drink or an orally-administered drug to remove bifidobacteria in the digestive tract. It is extremely effective in increasing.

Further, the bifidous factor according to the present invention is
Not only is it excellent in the growth-promoting effect of bifidobacteria, but it has strong selectivity or specificity and no growth effect on other contaminants.Therefore, when this bifidus factor is added to the bifidobacterial selection medium, A colony consisting of only bacteria is rapidly formed, which can be advantageously used for measuring the viable cell count of Bifidobacterium and various other assays.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI // (C12N 1/20 C12N 1/20 C12R 1:01) C12R 1:01 (C12N 1/38 C12R 1:01) (72 ) Inventor Sachiko Meguro 1-21-3 Sakaemachi, Higashimurayama, Tokyo Inside Meiji Dairy Co., Ltd. (72) Inventor Masako Yajima 1-21-3 Sakaemachi, Higashimurayama, Tokyo Meiji Dairy Co., Ltd. In the laboratory (72) Inventor Hideki Suzuki 1-21-3 Sakaemachi, Higashimurayama, Tokyo Meiji Dairy Co., Ltd. In the central laboratory (56) Reference JP-A-5-41995 (JP, A) JP-A-5- 344882 (JP, A) JP 40-27307 (JP, B1) JP 59-39287 (JP, B2) Pharmacology and treatment (1991), Vol. 19, No. 12, p. 4835-4844 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 1/00 A23L 1/29 A61K 35/74 C12P 1/00 CA (STN) BIOSIS / CA / MEDLINE / W PIDS (STN) JSTPlus (JOIS)

Claims (3)

(57) [Claims] 1. A culture of enterobacteria selected from the group consisting of Bacteroides and Enterobacter, and / or
Alternatively, a bifidobacteria growth-promoting substance comprising the treated product as an active ingredient. 2. A method for producing a bifidobacteria growth-promoting substance, which comprises culturing an intestinal bacterium selected from the group consisting of Bacteroides and Enterobacter. 3. A culture of enterobacteria selected from the group consisting of Bacteroides and Enterobacter, and / or
Or a selective medium for analysis of Bifidobacteria, which is obtained by adding the treated product to a selective agent-containing medium.