JPWO2012005240A1 - Bifidobacteria with an action to reduce intestinal spoilage substances - Google Patents

Abstract

The first problem is to effectively reduce intestinal spoilage substances by effectively using useful microorganisms with high safety. The inventor of the present application has been screened by paying attention to bifidobacteria generally dominant in the human body (intestine), and has a strong indole reducing action, and has also been confirmed to reduce indoxyl sulfate in serum. The selected strains were selected to complete the present invention.

Description

  The present invention relates to a method for improving the state of enteric bacteria. More specifically, the present invention relates to selection of useful microorganisms that reduce indole compounds that cause rot odor caused by intestinal bacteria, and the selected microorganisms.

  An increasing number of people complain of symptoms such as repeated constipation and diarrhea, stomach up, and outgassing due to disordered eating habits, irregular lifestyles, and stress. These symptoms are thought to be due to the influence of the intestinal environment, in other words, the prevalence of spoilage bacteria in the intestinal flora. The intestinal microflora produces spoiled odor components such as indole and its derivatives from tryptophan under anaerobic conditions. Several studies have been conducted on the relationship between such intestinal flora and the production of spoilage products (Non-patent Document 1).

  Indole is thought to be produced from tryptophan by intestinal spoilage bacteria and absorbed from the intestinal tract, then metabolized to indoxyl in the liver, and further combined with sulfuric acid to become indoxyl sulfate. In healthy humans, indoxyl sulfate is excreted in the urine, but it is considered to cause complications such as uremia in dialysis patients and the like (Non-patent Document 2).

  Regarding the removal of indole, which is a harmful substance in the intestine, for example, Patent Document 1 uses a bifidobacterum / thermophilum having an indole-reducing ability. Patent Document 2 describes that an extract or finely ground product of a Salacia plant is used in combination with lactic acid bacteria and / or bifidobacteria to improve the intestinal environment. Furthermore, Patent Document 3 describes that a composition containing propionic acid bacteria promotes the growth of bifidobacteria in the intestine and prevents the release of malodorous gases. It has also been reported that inulin and oligosaccharides are useful for improving the intestinal bacterial flora (Non-patent Document 3).

JP2006-158216 JP2007-031345 JP2007-186529

Intestinal Bacteriology 19: 169-177 (2005) Dialysis Society Vol. 24, No. 3, 312-316 (1991) The Journal of Nutrition 129 (7Suppl): 1438S-1441S (1999)

  It is known that spoilage substances produced in the human body (intestines) promote carcinogenesis and aging, and if the spoilage substances in the body can be actively reduced, it can contribute to human health. It is done. Therefore, first, the present invention has an object to effectively reduce enteric spoilage substances by effectively using highly safe and useful microorganisms.

  The above-mentioned spoilage substances are mainly produced in the large intestine, and examples of microorganisms (bacteria species) that are dominant in the large intestine include lactic acid bacteria and bifidobacteria (genus Bifidobacterium). This bifidobacteria has been used for food since ancient times as a microorganism that exhibits useful functions in vivo, and has a track record of eating experience. Therefore, it is a first object of the present invention to select microorganisms (strains) that can effectively reduce spoilage substances from bifidobacteria predominate in the human body (intestines).

  By the way, conventionally, substances or microorganisms that reduce spoilage substances produced in vivo (in the intestine) have been mainly searched for in an in vitro system. Since the intestinal microflora has not yet been reproduced in vitro, there was a question whether the experiment could reflect the actual state of the intestine. Accordingly, the present invention provides a second in vitro and in vivo system suitable as a verification or search system for the action effect of a substance or microorganism that reduces spoilage substances generated in vivo (in the intestine). Let it be an issue.

  Furthermore, this invention makes it a 3rd subject to provide the microorganisms which have the reduction effect in the density | concentration in the serum of the indoxyl sulfate which causes a complication in a dialysis patient etc.

  The present inventor completed the present invention by isolating strains that effectively reduce intestinal spoilage substances from lactic acid bacteria and bifidobacteria as highly safe and useful microorganisms based on the screening method of the present invention. It was.

  The inventor of the present application uses a buffer solution to proliferate bacterial cells when screening in vitro as a verification system for the action effect of substances or microorganisms that reduce spoilage substances produced in vivo (in the intestine). We examined a method to reproduce difficult conditions and evaluate the reduction effect (reduction ability) of spoilage substances. The inventor of the present application is characterized in that the reaction solution is set (designed) to the amount of glucose that is the minimum energy necessary for the reduction of spoilage substances under conditions where lactic acid bacteria and bifidobacteria hardly grow. An in vitro selection method and / or evaluation method for microorganisms (such as lactic acid bacteria and bifidobacteria) that reduce spoilage substances produced by intestinal bacteria was established.

  In addition, the inventor of the present application uses a mouse for screening in vivo as a verification system for the action effect of a substance or microorganism that reduces spoilage substances produced in vivo (in the intestine), and from serum. We studied to measure indoxyl sulfate. Then, the present inventor added fructooligosaccharide (FOS) and / or galactooligosaccharide (GOS) and tryptophan (L-Tryptophan) to the diet (powder diet), raised the mouse, and indoxyl sulfate in the serum. An in vivo selection method and / or evaluation method for microorganisms (such as lactic acid bacteria and bifidobacteria) that reduce the production of indoxyl sulfate in serum, characterized by evaluating the concentration, was established.

  Furthermore, in order to select microorganisms (strains) that can effectively reduce spoilage substances, the present inventor first selects bifidos that are generally dominant in the human body (intestines) as highly safe and useful microorganisms. Focusing on the fungus, a total of 196 strains, 32 strains of Bifidobacterium breve, 92 strains of Bifidobacterium longum and 72 strains of Bifidobacterium adolescenti, were screened and evaluated in vitro, and 9 strains were preliminarily selected. Next, for the purpose of verifying the action and effect of reducing the amount of spoilage substances in vivo, the selected nine strains were subjected to animal experiments and evaluated in vivo. Bifidobacterium breve OLB6129, which was confirmed to have an action of reducing xylsulfate, was selected to complete the present invention.

  In addition, the invention of the present application includes medicines and foods containing Bifidobacteria as an active ingredient, which has a strong effect of reducing the selected indole and exhibits an action of reducing indoxyl sulfate in serum.

  This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2010-152962, which is the basis for the priority of the present application.

  Bifidobacterium breve (B. breve) of the present invention is a human-derived strain, and has an excellent effect of significantly reducing indole and indoxyl sulfate in serum in a system in which the strain itself does not grow. B. breve, which has long been edible, has high safety, and the bifidobacteria OLB6129 of the present invention can be widely applied and applied in the food and pharmaceutical fields.

Effects of Bifidobacterium breve (live bacteria) administration on serum concentration of serum indoxyl sulfate Effects of Bifidobacterium longum (live bacteria) administration on the serum concentration of indoxyl sulfate in mice Effect of B. breve OLB6129 (live bacteria) concentration on serum indoxyl sulfate concentration after oral administration of B. breve OLB6129 (live bacteria) to mice Number of colonies of B. breve OLB6129 in feces (Fecal) and caecal contents (cecal) detected on TOS propionate agar medium Effects of oral administration of B. breve OLB6129 (viable bacteria) to mice on the concentration of indoxyl sulfate in the serum with and without the addition of FOS Relationship between the number of colonies of B. breve OLB6129 in feces (Fecal) detected on TOS propionate agar and the presence or absence of FOS addition

1. INTRODUCTION It is known that spoilage substances produced in the human body (intestines) promote carcinogenesis and aging, and if the spoilage substances in the body can be actively reduced, it can contribute to human health. It is done. Phenol, p-cresol, indole, skatole, ammonia, etc. can be listed as spoilage substances produced mainly in the large intestine. Among these spoilage substances, indole and skatole are particularly malodorous components and components that may adversely affect health, and therefore reduction of indole and skatole is desired.

  Examples of microorganisms (bacteria species) that predominate in the large intestine include Lactobacillus and Bifidobacterium (genus Bifidobacterium). Bifidobacteria have been used in food since ancient times as microorganisms that exhibit useful functions in vivo. In addition, bifidobacteria are said to have an intestinal regulating action, and are also used for intestinal preparations, etc., which also helps improve the intestinal flora, through the improvement of the intestinal environment, diarrhea suppression, prevention of rough skin, hay fever improvement, It is also said to be effective for immunity Taoism.

2. 2. Evaluation system for verification and search of action effects of microorganisms that reduce intestinal spoilage substances 2-1. In vitro screening or preliminary screening for microorganisms that reduce intestinal spoilage substances Microorganisms are used to reduce intestinal spoilage substances (substances that are produced in the gut and generate malodorous components) in the intestinal environment. In order to achieve this, it is important that the specific microorganism is preferentially present in the intestine. In other words, as a property that such microorganisms should have, resistance to conditions adapted to the intestinal environment is required.

  Therefore, it is desirable to apply a screening method or an evaluation method in accordance with the state (actual state) of the microorganism (mycelium) present in the intestinal environment. Therefore, when screening in vitro for substances or microorganisms that reduce intestinal spoilage substances, the present inventor first uses a buffer solution to reproduce conditions under which microorganisms are difficult to grow, and The evaluation method of the reduction effect (reduction ability) was examined. By adopting such conditions, it is possible to select excellent microorganisms under conditions closer to the intestinal environment, in which spoilage substances can be removed even if the microorganisms do not grow. For example, the components and concentration of the reaction solution can be determined under conditions in which the target microorganism hardly grows and under conditions that set the minimum energy necessary for reducing spoilage substances.

  More specifically, the amount of the medium component serving as an energy source can lower the concentration of glucose, lactose, fructose, etc., compared to the normal culture conditions of the target microorganism, and remove intestinal spoilage substances. A degree of 0.3 to 0.7 (w / v)% can be applied.

  In the screening (evaluation / selection) method (in vitro test), the results of examining the concentration of the reaction solution of a carbon source such as glucose are shown as an example. Can be determined.

2-2. In vivo screening for microorganisms that reduce intestinal spoilage substance Initially, animal experiments were performed and the in vivo effect of reducing indoxyl sulfate in serum could not be fully confirmed. The effect of reducing indoxyl sulfate in the serum is expected to be exerted in the intestine, but even if microorganisms (cells) are administered in the body, most of them are reached before reaching the intestine. There was a possibility that it was dead or that it was not stably maintained (retained) after reaching the intestine. Therefore, in the present invention, after adding microorganisms into the living body, in the hope of contributing to the improvement of its survival and stability, fructooligosaccharide (FOS) was added to the diet. The reduction effect of indoxyl sulfate was confirmed.

  In addition, since a mouse generally used in animal experiments is a young healthy individual, the amount of spoilage substances in the living body is suppressed to a very small amount and often falls below the detection limit. Therefore, in the present invention, in an animal experiment using a mouse, tryptophan (L-Tryptophan), which is a precursor of indole in vivo, is added to the diet (powder), and the baseline of indole generated in vivo is determined. Attempts to increase showed a significant increase in the indole baseline. In other words, the concentration of indoxyl sulfate in serum, which is an index (marker) of spoilage substances, can be evaluated stably and reliably. And the reduction | decrease effect of the spoilage substance about a specific strain came to be able to be evaluated and confirmed as a significant difference compared with the control group.

  More specifically, fructooligosaccharide (FOS) and / or galactooligosaccharide (GOS) and tryptophan (L-Tryptophan) are added to a diet (powder) at a predetermined concentration, and a general mouse (for animal experiments) is added. It can be reared and the concentration of indoxyl sulfate in serum can be evaluated. At this time, the concentration of the total amount of fructooligosaccharide (FOS) and / or galactooligosaccharide (GOS) is 0.5 to 20 (w / v)%, preferably 0.5 to 15 (w / v)%, more preferably , 0.5 to 10 (w / v)%. Although specific experimental results are omitted, if the fructooligosaccharide (FOS) concentration is set to 0.5 (w / v)%, the viability of B. breve OLB6129 is significant as the number of viable bacteria in feces. Has been confirmed to improve. The concentration of tryptophan is set to 1 to 4 (w / v)%, preferably 1.5 to 3.5 (w / v)%, and more preferably 2 to 3 (w / v)%. Thereby, microorganisms (such as lactic acid bacteria and bifidobacteria) that reduce the production of indoxyl sulfate in serum, particularly bifidobacteria (microorganisms of the genus Bifidobacterium) can be selected and / or evaluated and confirmed in vivo.

3. Microorganisms that reduce intestinal spoilage substances The inventor of the present application generally chooses microorganisms (strains) that can effectively reduce spoilage substances as microorganisms that reduce intestinal spoilage substances. The study was conducted on lactic acid bacteria and bifidobacteria, particularly bifidobacteria, which are dominant in (1). As a bifidobacteria, a total of 196 strains including 32 strains of B. breve, 92 strains of B. longum, and 72 strains of B. adolescentis were screened and evaluated in vitro. As a result, a total of 9 strains of 6 strains of B. breve and 3 strains of B. longum were selected.

  Furthermore, for the purpose of verifying the action and effect of reducing the amount of spoilage substances in vivo, an animal experiment was conducted on the selected nine strains and evaluated in vivo. As a result, a strain that exhibits the effect of reducing indole in the intestine was selected.

  The screened Bifidobacterium breve OLB6129 (B. breve OLB6129) is the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Contact: Postal Code 305-8566 Tsukuba, 1-1-1 Tsukuba City, Japan) Deposited at Center Center No. 6) on May 26, 2010, and this microorganism is deposited under the accession number as FERM BP-11257.

4). 4. Food or medicine containing microorganisms that reduce intestinal spoilage substances 4-1. Food containing microorganisms that reduce intestinal spoilage substances The microorganisms (such as lactic acid bacteria and bifidobacteria, preferably B. breve OLB6129) that reduce the intestinal spoilage substances of the present invention can be added to foods. That is, as the food of the present invention, any food to which microorganisms that reduce intestinal spoilage substances are added can be mentioned, and preferably fermented milk products or fermented milk drinks to which microorganisms that reduce intestinal spoilage substances are added. More preferably, a microorganism (B. breve OLB6129) that reduces intestinal spoilage substances, and a fermented milk product or fermented milk drink to which fructooligosaccharide (FOS) and / or galactooligosaccharide (GOS) is added Can be mentioned. It is known that GOS can be used in the same way as FOS (http://www.nyusankin.or.jp/scientific/hosono_3.html). The addition amount of fructooligosaccharide (FOS) and / or galactooligosaccharide (GOS) is 0.5 to 20 (w / v)%, preferably 0.5 to 15 (w / v)%, as the total concentration. Preferably, it can be set to 0.5 to 10 (w / v)%.

  In addition, the food containing a microorganism (Lactic acid bacteria, bifidobacteria, etc., preferably B. breve OLB6129) that reduces intestinal spoilage substances of the present invention is a health health for preventing or improving dialysis complications for renal dialysis patients. It can be applied as food (functional food).

4-2.
The medicament containing a microorganism (Lactic acid bacteria, bifidobacteria, etc., preferably B. breve OLB6129) that reduces the intestinal spoilage substance of the present invention is a reducing agent for intestinal spoilage substance or prevention of dialysis complications for renal dialysis patients. It can be applied as an agent or an improving agent. Furthermore, the pharmaceutical product of the present invention can be applied as a stomach tension improving agent.

  The pharmaceutical of the present invention can be in an appropriate dosage form such as powder, granule, tablet, capsule, liquid and the like. The pharmaceutical of the present invention may contain fructooligosaccharide (FOS) and / or galactooligosaccharide (GOS). The content of fructooligosaccharide (FOS) and / or galactooligosaccharide (GOS) is 0.5 to 20 (w / v)%, preferably 0.5 to 15 (w / v)%, as the total concentration. Preferably, it can be set to 0.5 to 10 (w / v)%. For formulation, adjuvants commonly used for formulation such as excipients, binders, disintegrants, lubricants and the like can be added. Examples of excipients include starch, lactose, sucrose, methylcellulose, carboxymethylcellulose, sodium alginate, calcium hydrogen phosphate, synthetic aluminum silicate, microcrystalline cellulose, polyvinylpyrrolidone (PVP), and hydroxypropyl starch (HPS). . Examples of the binder include starch, microcrystalline cellulose, sodium carboxymethylcellulose, polyvinyl pyrrolidone (PVP), gum arabic powder, gelatin, glucose, sucrose, and water / ethanol solutions thereof. Examples of the disintegrant include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, microcrystalline cellulose, hydroxypropyl starch, and calcium phosphate. Further, as lubricants, for example, carnauba wax, light anhydrous silicic acid, synthetic aluminum silicate, natural aluminum silicate, synthetic magnesium silicate, hardened oil, hardened vegetable oil derivative (Sterotex HM), sesame oil, white beeswax, titanium oxide , Dry aluminum hydroxide / gel stearic acid, calcium stearate, magnesium stearate, talc, calcium hydrogen phosphate, and sodium lauryl sulfate.

  The pharmaceutical product of the present invention is desirably administered orally, and bifidobacteria have established safety, so that any amount can be administered. For example, the dosage can be as much as 1 billion bifidobacteria per day. The number can be from 500 to 500 billion, and preferably from 10 to 500 billion.

[Reference Example 1] Setting the conditions of the in vitro test for selecting microorganisms that have the effect of reducing spoilage substances First, in the composition of the reaction solution, Bifidobacteria hardly grow, and indole (an index of intestinal spoilage substances) The composition of the reaction solution was determined so that the reduction effect was exhibited and the effect could be clearly evaluated.

  In the reaction solution used in this experiment, for example, when glucose was set to 1.0 (w / v)%, bifidobacteria grew excessively and the environment in the intestine could not be sufficiently reproduced. That is, since microorganisms (cells) such as bifidobacteria hardly grow in the intestine, in order to evaluate the effect of reducing the spoilage substances by bifidobacteria in the state close to the intestinal environment, the bifidobacteria The conditions should be set so that they hardly grow, and such an environment should be reproduced. Therefore, for example, glucose is set to 0.7 (w / v)% or less, preferably 0.6 (w / v)% or less, and more preferably 0.55 (w / v)% or less.

  In addition, in the reaction solution used in this experiment, bifidobacteria hardly grow and conditions should be set to provide the minimum energy necessary to reduce spoilage substances. It seems that it is not appropriate to mix.

  On the other hand, in the reaction solution used in this experiment, for example, when glucose is set to 0.1 (w / v)%, the activity (activity) of bifidobacteria is reduced, and the indole reduction effect is hardly exhibited. In this case, if a difference appears in the indole reduction effect or the like for each species of bifidobacteria, the difference is less likely to appear and it is difficult to clearly evaluate the effect. Therefore, glucose is set to, for example, 0.3 (w / v)% or more, preferably 0.4 (w / v)% or more, and more preferably 0.45 (w / v)% or more. Similarly, the composition of the reaction solution was determined for the other components.

The composition of the reaction solution should be 20 μg each of phenol, p-cresol, indole, skatole, 0.1 M Bis-Tris, 0.75 mM magnesium sulfate (MgSO ₄ ), and 0.5 (w / v)% glucose. PH is about 6.5.

[Example 1] Selection of microorganisms capable of reducing spoilage substances "in vitro test"
A total of 199 B. breve strains, 93 B. longum strains, and 72 B. adolescentis strains were cultivated statically. As a result, two B. breve strains and one B. longum strain grew extremely. It was late. Therefore, these three strains were excluded from the experiment. Finally, a total of 196 strains (evaluation / selection) of 32 strains of B. breve, 92 strains of B. longum and 72 strains of B. adolescentis were selected. did.

1 mg each of the above strains (live bacteria), the reaction solution determined in Reference Example 1 (pH: 6.5, phenol / p-cresol / indole / skatole: 20 μg each, Bis-Tris: 0.1M, magnesium sulfate (MgSO ₄ ): 0.75 mM, glucose: 0.5 (w / v)%). The suspension (reaction solution) was allowed to react at 37 ° C. for 24 hours, and then centrifuged to remove bifidobacteria (bacteria), and the pH of the supernatant was adjusted to 7. .

  Next, this supernatant was extracted twice with diethyl ether, and the extract was made up to 5 ml, and the solution was subjected to gas chromatography (GC) to indole, phenol, p-cresol, The concentration of skatole was measured (detected).

  The analysis conditions for gas chromatography (GC) are as follows. Detector: GC-2014 (Shimadzu) equipped with flame ionization detector, column (size): TC-1 (GL Sciences Inc.), ID = 0.25 mm, L = 30 m, dF = 0.25 mm, column temperature: 60 -170 ℃ (increased by 5 ℃ / min), 170-250 ℃ (increased by 20 ℃ / min), carrier gas: nitrogen, flow rate: 71.3 ml / min, injector temperature: 240 ℃, detector temperature: 250 ℃, injection volume: 1 μl.

  The results are shown in Table 1 (32 strains of B. breve), Table 2 (92 strains of B. longum), and Table 3 (72 strains of B. adolescentis).

  In addition, strains whose reference numbers begin with “JCM” are stored in the following storage organizations.

Preservation Institution: RIKEN BioResource Center, Microbial Materials Development Office (Contact: Postal Code 351-0198, 2-1 Hirosawa, Wako City, Saitama, Japan, Phone Number 048-467-9560)

  In the above results, when a strain with a reduced concentration of indole per gram of microorganism (bacteria) of 13 mg / g-cell or more was selected, a total of 6 strains of B. breve and 3 strains of B. longum Nine strains corresponded. The indole reduction concentration of 13 mg / g-cell or more corresponds to an indole reduction rate of 65% or more.

[Example 2] Selection of microorganisms capable of reducing spoilage substances "in vivo test (animal experiment)"
Nine strains selected in Example 1 above were screened (evaluated and selected) by animal experiments (in vivo tests).

After purchasing Balb / c mice, feed (powder CRF-1 / FOS (Wako Pure Chemical Industries, Ltd.): 10 (w / v)%, L-Tryptophan: 2.5 (w / v)%) for 1 week , Free to eat and acclimatized. After the acclimatization period was over, a predetermined strain (live bacteria) was suspended in physiological saline and administered continuously once a day for 2 weeks in the morning. At this time, the administration volume of physiological saline, the administration volume of each strain, and the administration concentration of each strain were 10 ¹⁰ cfu / ml, 0.2 ml / body / day, and 2 × 10 ⁹ cfu / body / day, respectively. At the end of this administration period (2 weeks), whole blood was collected and the concentration of indoxyl sulfate in the serum was measured.

  In addition, the strain (live bacteria) to be administered to mice was selected (screened) by an in vitro test. Specifically, 6 strains of B. breve (MEP22023004, OLB6129, MEP22023007, MEP22023012, MEP22023015, MEP22023016) and 3 strains of B. longum (MEP22023039, MEP22023061, MEP22023078) were selected. Then, an equal volume of physiological saline was used as a control group, and the administration group of a predetermined strain (live bacteria) was compared with the effect of reducing spoilage substances. The results are shown in FIG. 1 (6 strains of B. breve) and FIG. 2 (3 strains of B. longum).

  According to the above results, 6 strains of B. breve (Fig. 1) and 3 strains of B. longum (Fig. 2) were administered to mice with viable bacteria, and when this administration period was over, When the concentration of indoxyl sulfate was measured, it was confirmed that the concentration was significantly decreased in the B. breve OLB6129 administration group compared to the control group. That is, in the group administered with B. breve OLB6129 (FERM BP-11257), a significant decrease was observed in the concentration of indoxyl sulfate in the serum compared to the control group. This result suggests that B. breve OLB6129 may reduce spoilage substances in vivo.

[Example 3] Effect of B. breve OLB6129 concentration administered to living body on reduction effect of spoilage substances [Experimental procedure]
After acclimatizing Balb / c mice (SLC, ♀, 6-week-old) in one week, as shown in Table 4, divided into 4 groups based on body weight. For each group, saline and saline A suspension of B. breve OLB6129 (viable bacteria) was orally administered in the morning for 2 consecutive weeks. During the habituation period, all groups were allowed to freely feed (powder CRF-1 / FOS (Wako Pure Chemical Industries, Ltd.): 10 (w / v)%, L-Tryptophan: 2.5 (w / v)%) .

(1) Two weeks after the administration period of B. breve OLB6129, blood was collected from the tail vein and the concentration of serum indoxyl sulfate was measured. Serum was diluted 100-fold with ultrapure water (Milli-Q), then filtered through SYRINGELESS FILTER DEVICE 0.2 μm (Whatman), and a previous report (Takayama et al., Am J Kidney Dis. 2003 Mar; 41 (3 Suppl 1): Measured by high performance liquid chromatography (HPLC) according to S142-5).

(2) Feces were collected at the end of the administration period of B. breve OLB6129, and cecal contents were collected at the time of dissection, and the number of B. breve OLB6129 (administered bacteria) contained therein was measured.

[Experimental result]
At the end of the administration period of B. breve OLB6129, the concentration of serum indoxyl sulfate was confirmed. Regarding the effect of reducing the concentration of indoxyl sulfate, 2 x 10 ⁸ cfu / body / day administration group (III), The 2 x 10 ⁹ cfu / body / day administration group (IV) was significantly different from the saline administration group (control) (I), but 2 x 10 ⁷ cfu / body / day administration There was no significant difference in the group (II) cfu / body compared to the physiological saline administration group (control) (I) (FIG. 3).

  In addition, B. breve OLB6129 (administered bacteria) in stool and cecal contents was detected in a dose-dependent manner (FIG. 4).

[Example 4] Effect of presence or absence of FOS administered to living body on reduction effect of spoilage substances [Experimental procedure]
After acclimatization of Balb / c mice (SLC, ♀, 6-week-old) in one week, as shown in Table 5, the mice were divided into 4 groups based on body weight. Suspension of B. breve OLB6129 (viable bacteria) in water and physiological saline, and suspension of B. breve OLB6129 (viable bacteria) in saline and physiological saline with FOS added in 2 weeks Orally administered in the morning. During the habituation period, all groups were allowed to freely ingest feed (powder CRF-1 / L-Tryptophan: 2.5 (w / v)%). The concentration of B. breve OLB6129 (viable bacteria) was set to 2 × 10 ⁸ cfu / body / day, which is the minimum dose indicated in Example 3 regarding the need to reduce spoilage substances. .

(1) Two weeks after the FOS administration period, blood was collected from the tail vein and the concentration of serum indoxyl sulfate was measured. Serum was diluted 100-fold with ultrapure water (Milli-Q), then filtered through SYRINGELESS FILTER DEVICE 0.2 μm (Whatman), and a previous report (Takayama et al., Am J Kidney Dis. 2003 Mar; 41 (3 Suppl 1): Measured by high performance liquid chromatography (HPLC) according to S142-5).

(2) At the end of the FOS administration period, stool from the B. breve OLB6129 administration group was collected and the number of B. breve OLB6129 (administration bacteria) that passed through the living body was measured.

[Experimental result]
When the concentration of serum indoxyl sulfate was confirmed at the end of the FOS administration period, the effect of reducing the concentration of indoxyl sulfate was small in the administration group of B. breve OLB6129 alone (FIG. 5). In addition, regarding the effect of reducing the concentration of indoxyl sulfate, there was no significant difference between the FOS-only administration group and the physiological saline administration group (I) (FIG. 5). On the other hand, the effect of reducing the concentration of indoxyl sulfate was significantly different in the B. breve OLB6129 and FOS administration groups compared to the saline administration group (I) and the like (FIG. 5). The number of B. breve OLB6129 (administered bacteria) in the stool was measured in two groups (OLB6129 +) of the B. breve OLB6129 only administration group and B. breve OLB6129 and FOS administration groups. In the administration group, the number of viable bacteria was observed to be significantly higher (FIG. 6).

FERM BP-11257
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (14)

  A novel Bifidobacterium breve OLB6129 (FERM BP-11257) that reduces intestinal spoilage.   An agent for reducing intestinal spoilage substance comprising the bifidobacteria according to claim 1 as an active ingredient.   The agent for reducing intestinal spoilage substance according to claim 2, further comprising fructooligosaccharide (FOS) and / or galactooligosaccharide (GOS).   A reducing agent for indoxyl sulfate in serum, comprising the bifidobacteria according to claim 1 as an active ingredient.   The agent for reducing indoxyl sulfate in serum according to claim 4, further comprising fructooligosaccharide (FOS) and / or galactooligosaccharide (GOS).   A food having an action of reducing intestinal spoilage substances comprising the bifidobacteria according to claim 1 as an active ingredient.   A food having an action of reducing indoxyl sulfate comprising the bifidobacteria according to claim 1 as an active ingredient.   The foodstuff which uses the foodstuff of Claim 6 or 7 as fermented milk product or fermented milk drink.   The food according to any one of claims 6 to 8, further comprising fructooligosaccharide (FOS) and / or galactooligosaccharide (GOS).   In an in vitro screening method for microorganisms that reduce intestinal spoilage substances, the concentration of glucose, which is the minimum energy necessary for reducing spoilage substances, is added to the reaction solution under the condition that the microorganisms hardly grow. And said method.   The method according to claim 10, wherein the glucose concentration is 0.3 to 0.7 (w / v)%.   An in vivo screening method for microorganisms that reduces intestinal spoilage substances, wherein fructooligosaccharide (FOS) and / or galactooligosaccharide (GOS) and tryptophan (L-Tryptophan) are added to the diet, and the mouse is raised. The method is characterized in that the concentration of indoxyl sulfate in serum is evaluated.   The method according to claim 12, wherein the concentration of fructooligosaccharide (FOS) and / or the concentration of galactooligosaccharide (GOS) is 0.5 to 20 (w / v)% and the concentration of tryptophan is 1 to 4 (w / v)%. .   The method according to any one of claims 10 to 13, wherein the microorganism is a lactic acid bacterium and / or a bifidobacteria.

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