JP3322663B2 - Bifidobacterium bifidum - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a Bifidobacterium having a specific ability to assimilate ammonia-type nitrogen.
It relates to bifidum bacteria.

[0002]

2. Description of the Related Art Japanese Patent Publication No. 31487/1989 discloses a blood ammonia lowering agent utilizing Bifidobacterium, that is, a general formula Gal- (Gal) n-Glc (where Gal is a galactose residue, Glc Represents a glucose residue, and n represents an integer of 1 to 4.) and a blood ammonia lowering agent comprising an active ingredient of a bacterium belonging to the genus Bifidobacterium.

It is known that when urea and amino acids excreted in the intestinal tract are decomposed into ammonia by intestinal bacteria and absorbed from the intestine, most of them enter the liver and cause various disorders. The blood ammonia lowering agent suppresses the activity of ammonia-producing bacteria by lowering the pH in the intestinal tract, in which the Bifidobacterium bacteria proliferate in the intestine to produce organic acids, and reduce the activity of the Bifidobacterium. It is thought that the bacteria themselves prevent intestinal ammonia-type nitrogen derived from ammonia-producing bacteria as part of the nutrient source, thereby preventing an increase in blood ammonia concentration.

[0004] However, Bifidobacterium assimilates not only ammonia-type inorganic nitrogen compounds, but also assimilates many organic nitrogen compounds such as amino acids and peptides, and also contains amino acids essential for growth.
When an organic nitrogen compound and an ammonium salt coexist in a medium, many bacteria preferentially utilize the organic nitrogen compound.

[0005] There are rarely known Bifidobacterium bacteria that preferentially utilize ammonia-type nitrogen. The ratio derived from type nitrogen) was at most 70% (actual measurement example by the present inventors). Therefore, a search has been made for a bifidobacterium having a stronger ability to take up ammonia-type nitrogen, actively functioning in the intestinal tract rich in organic nitrogen compounds, and exhibiting a strong blood ammonia lowering effect.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a bifidobacterium having a high ability to take up ammonia-type nitrogen, which is expected to have a better action as a constituent of a blood ammonia lowering agent.

[0007]

The inventors of the present invention have examined the ability of many Bifidobacterium bacteria to take up ammonia-type nitrogen, and found that ammonium salts and nitrogen compounds other than ammonium salts coexist as assimilable nitrogen compounds. Synthetic medium X
Bifidobacterium (FERM P-11788 and FERM) having the ability to preferentially utilize ammonia-type nitrogen at an incorporation rate of 75% or more from
P-111791) was found to be present.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Here, the uptake rate of ammonia-type nitrogen is a value measured by the following method. [Ammonia-Type Nitrogen Uptake Measurement Method] A stable isotope of nitrogen having a mass number of 15 which is ¹⁵ N
At 37 ° C. using a synthetic medium X having the following composition containing ammonium acetate as the only ammonium compound.
The Bifidobacterium is cultured for 4 hours.

Medium X composition: D-(+) lactose 97.10
mM, ¹⁵ N-ammonium acetate 25.95 mM, dipotassium phosphate 14.35 mM, riboflavin 1.33 μM, D-pantethine 90 nM, biotin 2.05 nM, potassium pyruvate
27.25 mM, L-cysteine 5.25 mM, magnesium chloride 0.5 mM, ferrous chloride 1 mM, manganese chloride 0.5 μM,
Calcium chloride 50 μM, resazurin 4.36 μM, triplicase 0.5 g, yeast extract 0.5 g; Dissolve the above in distilled water to make the total volume 1 liter. pH 6.6.

After completion of the culture, the culture is centrifuged to collect cells, and the obtained cells are washed twice with a phosphate buffer. To 1.0 g of the washed cells, 4 ml of 4% (w / v) K ₂ S ₂ O ₈ is added and reacted at 120 ° C. for 30 minutes to convert nitrogen in the cells to nitrate nitrogen. After cooling the reaction solution to room temperature, the reaction mixture was cooled to 0.
After adding 7 ml of 14% silver sulfate (sulfuric acid solution) and sufficiently cooling, 0.1 ml of a 5% ethanol solution of 2-sec-butylphenol is added and shaken at room temperature for 15 minutes to react. The reaction solution is transferred to a 50-ml separating funnel, 10 ml of chloroform is added, and the mixture is shaken for 5 minutes and allowed to stand.
The chloroform layer is collected, washed twice with water, dehydrated with sodium sulfate, and the solvent is distilled off with a rotary evaporator. The residue is dissolved in 1 ml of acetone, 50 μl of N, O-bistrimethylsilylacetamide is added, and the mixture is reacted by vigorously shaking for 15 minutes.

By the above-mentioned treatment, nitric acid type nitrogen is converted to 4-NO
It is fixed as ₂ -2-sec-butylphenol trimethylsilyl ester. The obtained reaction solution was analyzed by the GCMF method (mass fragmentography) under the following conditions, and the total nitrogen was determined from the ratio of the peak of m / z 238 indicating the amount of ¹⁴ N to the peak of m / z 239 indicating the amount of ¹⁵ N. ¹⁵ N determined proportions (%) of in to it with ammonia type nitrogen incorporation rate.

[Deposited Bacteria] The Bifidobacterium according to the present invention, which has a specifically high ammonia-type nitrogen uptake rate, comprises a large number of Bifidobacterium strains isolated from feces of neonates, infants, infants, adults and the like. Isolated from inside.
Two strains of Bifidobacterium belonging to Bifidobacterium bifidum (YIT4069 and YIT4070), which have an ammonia type nitrogen uptake rate of 75% or more, have been found. These strains have been deposited with the Research Institute of Microbial Industry as Microbial Deposit Nos. 11788 and 11791.

[Ammonia-type uptake rate] Table 1 shows the results of measuring the ammonia-type uptake rate of the above two strains and representative known Bifidobacterium strains.

[0014]

[Table 1]

In a culture in a medium containing a sufficient amount of an organic nitrogen compound, the uptake rate of ammonia-type nitrogen is 75%.
The fact that selective assimilation is carried out beyond this is surprising from the conventional common knowledge regarding the nutritional requirements of Bifidobacterium. The isolated strain was identified based on the sugar fermentation test method of Mitsuoka et al. (World of enterobacteria-Isolation and identification of anaerobic bacteria: Sobunsha).

[Bacteriological properties] Both of the above two strains have the following bacteriological properties, whereby they were identified as strains belonging to Bifidobacterium bifidum.
Gram positive spore-free bacilli. Microscopic observation shows that rod-shaped short rods often branch off. When cultured in VL-G liquid medium, acetic acid and lactic acid are mainly produced. Growth under aerobic conditions (-), catalase (-), milk coagulation (+), gelatin liquefaction (-), hydrogen sulfide production (-),
Indole production (-), nitrate reduction (-), carbon dioxide production (-). Proliferating conditions are temperature 25-45 ° C, pH 5
The optimal growth conditions are 36-38 ° C., pH 6-7.
It is.

[Sugar fermentability] (ILS base: liquid paraffin overlay method) Arabinose (-), xylose (-), fructose (+), galactose (+), maltose (-), cellobiose (-), lactose (+) ), Raffinose (−), glucose (+), sugar-free condition (−).

[Composition of Produced Fatty Acid]
2 minutes in the supernatant obtained by centrifugation at 000 rpm for 20 minutes.
The protein was deproteinized by adding 10% of 5% metaphosphoric acid (in 5N-sulfuric acid), centrifuged, and the composition of volatile fatty acids in the supernatant was analyzed by gas chromatography.
As a result, it was confirmed that only acetic acid was produced as a volatile fatty acid.

The oligosaccharides constituting the blood ammonia lowering agent together with the Bifidobacterium as described above include:
Examples include galactooligosaccharides, fructooligosaccharides, konjac mannan oligosaccharides, isomaltooligosaccharides, acetylglucosamine oligosaccharides, and sugar alcohol derivatives thereof. Among them, oligosaccharides of the general formula Gal- (Gal) n-Glc (hereinafter referred to as galacto-oligosaccharides: see Japanese Patent Publication No. 31487/1989 and Japanese Patent Publication No. 58-20266) are used to grow Bifidobacterium in the intestinal tract. It is excellent in the action of accelerating and is preferred as the oligosaccharide used in the present invention.

Galactooligosaccharides have the effect of promoting the growth of intestinal indigenous bifidobacteria. Therefore, they are combined with the above-mentioned bifidobacterium of the present invention having a specifically high ammonia-type nitrogen uptake rate. When taken together, the intestinal growth of the Bifidobacterium is promoted and organic acid production is increased, and the activity of ammonia-producing bacteria is suppressed more effectively by lowering the pH in the intestinal tract. The uptake of ammonia-type nitrogen by Pseudomonas bacilli also increases significantly.

[Formulation] In the case of formulation, the oligosaccharide and the freeze-dried bacterial powder of Bifidobacterium are preferably mixed at a ratio of preferably about 5 to 30 parts by weight per 100 parts by weight of the former, and if necessary, Excipients such as starch and hydroxypropylcellulose are also mixed and molded. However, it is not always necessary to formulate a mixture of the oligosaccharide and the Bifidobacterium, and the single preparation may be taken separately. It is desirable to take this blood ammonia lowering agent as a Bifidobacterium bacterium so as to be about 10 ⁸ to ¹⁰ 10 per adult day. The safety of this blood ammonia lowering agent consisting of Bifidobacterium and galactooligosaccharide has been confirmed by a sub-acute toxicity test in rats.

[0022]

Example 1 The effects of bifidobacteria and galactooligosaccharides (4'-β-galactosyl lactose) on the amount of ammonia generated from the contents of the cecum taken out of adult Fischer male rats were examined by the following method.
Rats used to collect cecal contents were fed a diet containing 20% egg white powder in NMF powder diet for 10 days. The rat was sacrificed by decapitation and the cecum was removed. The removed cecum was replaced with a 4 times volume of bicarbonate buffer (artificial saliva from McDougall: Bioche).
mJ 43.99-108.1948) to disperse the cecal contents in the buffer. 0.5 ml of the obtained cecal content suspension
Was collected and adjusted to a total volume of 1 ml with a buffer, with or without additives as described below, and cultured at 37 ° C. The handling of the contents of the cecum was performed anaerobically under carbon dioxide gas.

Additives in the test plot (the amount in parentheses is the amount added per 1 ml of the culture solution) I Galacto-oligosaccharide (10 mg) II Galacto-oligosaccharide (10 mg) + B. bifidum YIT4069 (10 ⁹ ) III Galacto-oligosaccharide (10 mg) + B. breve YIT4006 (10 ⁹ ) No IV

During the culture, a portion of the culture solution is sometimes taken,
Ammonia concentration and pH were measured. FIG. 1 shows the change over time in the ammonia concentration of the culture solution. In group I to which only galactooligosaccharide was added, the concentration remained almost at the initial ammonia concentration, and the production of ammonia was suppressed. II Combined Use of Galactooligosaccharide and Bifidobacterium of the Present Invention
In the group, the ammonia concentration in the culture solution was significantly reduced. In comparison, the control bacteria B. In the case of group III in which brevet was used in combination with galactooligosaccharide, the decrease in ammonia concentration was slight.

The pH change during the cultivation was as shown in FIG. A remarkable decrease in pH was observed in groups II and III,
In these groups, it can be seen that almost the same amount of organic acid was produced by the growth of Bifidobacterium. From these results, it was concluded that in group II, the activity of ammonia-producing bacteria was suppressed by the production of organic acid, and at the same time, the uptake of ammonia by bifidobacteria was actively performed, resulting in a decrease in ammonia concentration.

[0026]

Example 2 Mature Fischer male rats (6 rats per group)
A test was conducted in which the blood ammonia-lowering agent was directly administered into the cecum of the mouse. Rats used in the test were fed a diet containing 20% egg white powder in an NMF powder diet for 16 days. The rat was laparotomized under ether anesthesia, the entrance and exit of the cecum were closed, and 2 ml of the following administration solution was injected from the digestive tract side.

Test Group Administration Solution I Physiological saline II Galactooligosaccharide solution (concentration 0.125 g / ml) III Galactooligosaccharide solution (concentration 0.125 g / ml) + B. Bifidum YIT4069 (5 × 10 ⁹ bacteria)

The laparotomy site was closed and dissected 4 hours later, portal blood was collected and the ammonia concentration was measured. The results of the measurement are as follows. Although the administration of galactooligosaccharide alone compared to the control group I (group II) causes a decrease in blood ammonia concentration, the administration of the Bifidobacterium bacterium of the present invention is further (Group III), the blood ammonia concentration further decreases.

Blood ammonia concentration (μg / dl) in test section I 429.3 ± 109.1 II 301.4 ± 66.0 III 274.7 ± 38.4

[0030]

Example 3 Mature Fischer male rats (6 rats per group)
Then, a test was conducted in which this blood ammonia lowering agent was orally administered. The rats used in the test were fed a diet in which 20% of egg white powder was added to the NMF powder feed for 12 days, and at the same time as the start of the test, the diets containing 10% of galactooligosaccharide were added to the groups II and III. Gave. Group II further includes Bifidobacterium bifidum YIT406
9 were administered at a rate of 5 × 10 ⁹ / day using an intragastric sonte. In addition, a control group I to which neither galactooligosaccharide nor Bifidobacterium was administered was provided.

After the above administration was continued for 4 days, all rats were dissected, abdominal aortic blood and cecal contents were collected, and ammonia concentration of the collected sample was measured. The measurement results are as follows. In group II, the ammonia concentration in blood was significantly reduced by administering the Bifidobacterium bacterium and galactooligosaccharide of the present invention, which was based on the decrease in intestinal ammonia concentration. Was confirmed.

Test arterial blood concentration (μg / dl) Cecal concentration (μg / g) I 149.8 ± 13.9 388.7 ± 81.1 II 109.5 ± 24.0 313.1 ± 42. 7 III 116.5 ± 26.6 371.1 ± 81.3

[0033]

As described above, the Bifidobacterium bacterium of the present invention, which has a specifically high ammonia-type nitrogen uptake ability, is more remarkable when taken with galacto-oligosaccharide than when ordinary Bifidobacterium is used. Intestinal ammonia concentration is reduced, and blood ammonia concentration is significantly reduced accordingly.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of Example 1 according to the present invention, showing the change over time in the ammonia concentration of a solution.

FIG. 2 is a graph showing the results of Example 1 according to the present invention, showing the change over time in the pH of the culture solution.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Akira Iwabuchi 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha (72) Inventor Masaaki Watanuki 1-1-19, Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha (72) Inventor Ryuichiro Tanaka 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha (56) References Tokiko Hei 1-31487 (JP, B2) (58) Fields surveyed (Int .Cl. ⁷ , DB name) C12N 1/20 A61K 35/74 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. An ammonium as an assimilable nitrogen compound.
Synthesis in which salt and nitrogen compound other than ammonium salt coexist
Ammonia-type nitrogen with an uptake rate of 75% or more
Bifidobacterium bifidum FERM P-11788 strain having the ability to preferentially assimilate B. 2. An ammonium compound as an assimilable nitrogen compound.
Synthesis in which salt and nitrogen compound other than ammonium salt coexist
Ammonia-type nitrogen with an uptake rate of 75% or more
Bifidobacterium bifidum FERM P-111791 having the ability to preferentially assimilate B.