WO2016103699A1 - Organic-acid-production promoter, and agent for preventing and/or ameliorate inflammatory bowel disease - Google Patents

Abstract

The present invention enhances the organic-acid-production efficiency of bifidobacteria and lactic acid bacteria regardless of the numbers of these beneficial bacteria. The problem is overcome by an organic-acid-production promoter for promoting the production of organic acids through the use of bifidobacteria and lactic acid bacteria containing polyphenol as an active ingredient. Quercetin, taxifolin, phloretin, epigallocatechin gallate, or procyanidin is preferably used as the polyphenol.

Description

Organic acid production promoter and preventive and / or ameliorating agent for inflammatory bowel disease

The present invention relates to an organic acid production promoter that promotes the production of an organic acid by bifidobacteria or lactic acid bacteria, and an agent for preventing and / or improving inflammatory bowel disease.

It is known that the gut microbiota is closely related to human health and disease. It is said that there are 500 to 1000 types of intestinal bacteria inhabiting the human intestine. Generally, useful microorganisms (useful bacteria) such as bifidobacteria and lactic acid bacteria, and Clostridium perfringens Hazardous microorganisms (harmful bacteria) such as In order to maintain human health, it is important that useful bacteria exist as dominant species in the large intestine.
In other words, bifidobacteria and lactic acid bacteria have the function of producing organic acids such as lactic acid and acetic acid in vivo (in the intestine), and these organic acids suppress the growth of harmful bacteria in the intestinal flora. It is possible to prevent constipation and diarrhea from occurring. It has also been reported that these produced lactic acid and acetic acid are converted into butyric acid by intestinal bacteria, and the inflammatory bowel disease is improved by butyric acid.

On the other hand, in recent years, the number of patients with constipation, diarrhea, inflammatory bowel disease and the like has been increasing, and it is required to effectively improve human intestinal microflora. For this reason, various methods for increasing the number of Bifidobacteria and lactic acid bacteria in the intestine have been proposed.
For example, Patent Document 1 describes that poly-γ-glutamic acid is used as an active ingredient to promote the growth of intestinal bifidobacteria. Patent Document 2 describes that the development of intestinal bacterial flora is promoted by using a lipid composition containing a plant-derived fat source.
In addition, by ingesting foods, beverages, supplements and the like containing lactic acid bacteria and oligosaccharides, bifidobacteria and lactic acid bacteria are proliferated in the intestine.
According to these conventional techniques, it is possible to increase the production amount of the organic acid by growing bifidobacteria and lactic acid bacteria in the intestine.

JP 2011-178864 A Special table 2013-525421 gazette JP-A-6-166618 JP-A-6-248267 JP 2012-92138 A

However, these conventional techniques increase the production of organic acids by increasing the number of useful bacteria in the intestines, and do not improve the organic acid production efficiency of useful bacteria (microorganisms) themselves. It was.
In addition, some foods containing lactic acid bacteria and oligosaccharides have problems with the survival of lactic acid bacteria, and others have diarrhea due to excessive intake.
For this reason, it is desirable if it becomes possible to improve the production efficiency of organic acids of useful bacteria themselves, regardless of the quantity (number of bacteria) of bifidobacteria and lactic acid bacteria. If it is possible to improve the organic acid production efficiency of these useful bacteria themselves, even if it is difficult to ingest large quantities of useful bacteria or it is difficult to grow useful bacteria in the intestines, the intestinal flora Can be improved, and the preventive effect and / or the improvement effect of inflammatory bowel disease can be expected.

Therefore, the present inventors have intensively studied the effect of various phytochemicals on the organic acid production function by bifidobacteria, etc., and as a result, the use of polyphenols can provide an excellent organic acid production promoting effect. I found. Such polyphenols include in particular quercetin, taxifolin, phloretin, epigallocatechin gallate, and procyanidins.

Here, the composition for intestinal regulation of patent document 3 is mentioned as a technique relevant to improving intestinal microflora using polyphenol. It is described that this composition for regulating intestine contains epigallocatechin gallate and the like, and can reduce the pH in the human intestine to improve intestinal flora and constipation.
However, the composition for intestinal regulation described in Patent Document 3 increases the number of bifidobacteria and lactic acid bacteria in the intestine, and promotes the production of organic acids by the bifidobacteria and lactic acid bacteria regardless of the quantity. It was not possible.

Patent Document 4 describes food additives using quercetin, taxifolin and the like as active ingredients. However, this food additive is used as an antioxidant and is not related to the promotion of organic acid production.
Furthermore, Patent Document 5 describes skin cosmetics containing phloretin as an active ingredient. However, these skin cosmetics are used as anti-inflammatory agents and the like, and are not related to the promotion of organic acid production.
In addition, there was no prior literature related to a composition using various polyphenols as an active ingredient and capable of promoting the production of organic acid by Bifidobacteria and lactic acid bacteria.

The present invention has been made in view of the above circumstances, and an organic acid production promoter capable of increasing the amount of organic acid produced by these useful bacteria, regardless of the quantity of bifidobacteria and lactic acid bacteria, and The object is to provide an agent for preventing and / or improving inflammatory bowel disease.

In order to achieve the above object, the present invention is an organic acid production promoter for promoting the production of an organic acid by bifidobacteria and / or lactic acid bacteria, which contains polyphenol as an active ingredient.
In the organic acid production promoter of the present invention, Bifidobacteria are Bifidobacterium longum OLB6290 (NITE P-75), Bifidobacterium longum No. 7 (FERM BP-11242) and Bifidobacterium adolescentis JCM1275 ^T are preferably used. The lactic acid bacterium is preferably a lactic acid bacterium belonging to the genus Lactobacillus, preferably Lactobacillus delbrueckii subsp. Bulgaricus OLL 1181 (FERM BP-11269).

In the organic acid production promoter of the present invention, lactic acid and acetic acid may be mentioned as organic acids produced by bifidobacteria and / or lactic acid bacteria. Also, lactic acid and acetic acid are produced by bifidobacteria and / or lactic acid bacteria, and these are converted to butyric acid by other intestinal bacteria. Therefore, it is possible to increase the amount of butyric acid in the intestine by ingesting the organic acid production promoter of the present invention.

In the organic acid production promoter of the present invention, the polyphenol preferably contains any one or more of flavonoids, phloretins, and procyanidins. At this time, it is preferable to use quercetin, taxifolin, or epigallocatechin gallate as the flavonoid. Moreover, it is preferable to use cacao polyphenol as procyanine.

In the organic acid production promoter of the present invention, the polyphenol and the bifidobacteria are preferably contained as active ingredients, and the polyphenol and the lactic acid bacteria are preferably contained as active ingredients.
Furthermore, the preventive and / or ameliorating agent for inflammatory bowel disease of the present invention contains the above-mentioned organic acid production promoter.

According to the present invention, regardless of the quantity of bifidobacteria and lactic acid bacteria, the organic acid production promoter capable of improving the organic acid production efficiency of these useful bacteria themselves, and the preventive and / or ameliorating agent for inflammatory bowel disease Can be provided.

It is a figure which shows the density | concentration showing the density | concentration of the organic acid produced by the bifidobacteria culture | cultivated using the culture medium each containing various polyphenols. It is a figure which shows the graph showing the density | concentration of the organic acid produced by the bifidobacteria culture | cultivated using the culture medium containing cacao polyphenol as procyanidins. It is a figure which shows the graph showing the density | concentration of the organic acid produced by the lactic acid bacteria cultured using the culture medium which respectively contained various polyphenols.

Hereinafter, preferred embodiments of the present invention will be described in detail.
[First embodiment]
First, the organic acid production promoter according to the first embodiment of the present invention will be described. The organic acid production promoter of this embodiment is an organic acid production promoter for promoting the production of organic acids by bifidobacteria or lactic acid bacteria, which contains polyphenol as an active ingredient.

Bifidobacteria are a kind of Gram-positive obligate anaerobic bacilli and are widely inhabited by animals. Bifidobacteria are microorganisms that decompose saccharides and produce lactic acid and acetic acid. In order to maintain human health, it is desirable that bifidobacteria be present as a dominant species in the intestine for a lifetime.

In the organic acid production promoter of this embodiment, Bifidobacterium is not particularly limited, and examples thereof include Bifidobacterium longum OLB6290 (NITE P-75) and Bifidobacterium longum No. 7 (FERM BP-11242) or Bifidobacterium adolescentis JCM1275 ^T is preferred.

Bifidobacterium longum OLB6290 (Bifidobacteria OLB6290) was deposited on February 3, 2005 under the accession number: NITE P-75 at the National Institute for Product Evaluation and Technology Patent Microorganism Depositary. Microorganisms.

Bifidobacterium longum No. 7 (Bifidobacterium sp. No. 7) is a microorganism deposited on April 20, 1993, under the accession number: FERM BP-11242, in the independent administrative institute Product Evaluation Technology Foundation Patent Microorganism Depositary Center.

Lactic acid bacteria are microorganisms that decompose saccharides and produce lactic acid, and are classified into homolactic acid bacteria that produce only lactic acid as a final product, and heterolactic acid bacteria that produce other than lactic acid at the same time. Examples of lactic acid bacteria include Lactobacillus lactic acid bacteria, Bifidobacterium lactic acid bacteria, Enterococcus lactic acid bacteria, Lactococcus lactic acid bacteria, Pediococcus lactic acid bacteria, and Leuconostoc lactic acid bacteria. In this specification, lactic acid bacteria mean microorganisms that produce lactic acid other than so-called bifidobacteria.

In the organic acid production promoter of the present embodiment, the lactic acid bacterium is not particularly limited, but a lactic acid bacterium belonging to the genus Lactobacillus which is a Gram-positive rod is preferable. For example, Lactobacillus delbrueckii subsp. Bulgaricus is preferable, and specifically, Lactobacillus delbrueckii subsp. Bulgaricus OLL1181 (FERM BP-11269) is preferable.

Lactobacillus delbrueckii subsp. Bulgaricus OLL1181 (Bulgaria bacillus OLL1181) is registered with the independent micro-organisms of the National Institute of Technology and Product Evaluation Technology Bases as the Patent Microorganism Depositary Center as FERM BP-11269, July 16, 2010 Microorganisms deposited every day.

In the organic acid production promoter of the present embodiment, examples of the organic acid produced by bifidobacteria and lactic acid bacteria include lactic acid and acetic acid. Moreover, since lactic acid and acetic acid are converted into butyric acid by intestinal bacteria, it is possible to increase the amount of butyric acid in the intestine by ingesting the organic acid production promoter of this embodiment.
Lactic acid and acetic acid produced by bifidobacteria and lactic acid bacteria suppress the growth of harmful bacteria such as Clostridium perfringens in the intestines, improve the intestinal flora, and suppress or prevent the occurrence of constipation and diarrhea. Is possible. In addition, it is known that butyric acid has an excellent preventive effect and / or ameliorating effect on inflammatory bowel disease.

According to the organic acid production promoter of this embodiment, the amount of lactic acid and acetic acid produced by bifidobacteria and lactic acid bacteria can be increased without increasing the number (number of bacteria) of bifidobacteria and lactic acid bacteria. It is. For this reason, even if a person who has difficulty in growing Bifidobacteria and lactic acid bacteria in the intestine ingests the organic acid production promoter of this embodiment, the amount of lactic acid and acetic acid is increased in the intestine. Is possible. In the intestine, the amount of lactic acid and acetic acid is increased, whereby the intestinal bacterial flora is improved, and a preventive effect and / or an improved effect on inflammatory bowel disease can be obtained.

In the organic acid production promoter of this embodiment, the polyphenol preferably contains a flavonoid, and the flavonoid preferably contains taxifolin, which is a kind of dihydroquercetin.
In the organic acid production promoter of this embodiment, the content of taxifolin is preferably 0.001 to 5.0% by weight, more preferably 0.01 to 4.0% by weight, and still more preferably 0.1 to 3.0% by weight.

In the organic acid production promoter of this embodiment, the effective amount (intake amount, dose) of taxifolin per day for humans is preferably 0.1 to 100 mg, more preferably 0.5 to 50 mg, More preferably, it is 1 to 10 mg.
In addition, the effective amount per day for humans may be ingested (administered) at one time, or may be ingested in two or more times. The same applies to the following embodiments.

Moreover, in the organic acid production promoter of this embodiment, it is also preferable that phloretin is included as polyphenol.
In the organic acid production promoter of this embodiment, the content of phloretin is preferably 0.0001 to 50% by weight, more preferably 0.001 to 30% by weight, and still more preferably 0.01 to 15% by weight. .
In the organic acid production promoter of this embodiment, the effective amount (intake amount, dose) of phloretin per day for humans is preferably 0.001 to 50 mg, more preferably 0.01 to 30 mg. More preferably, it is 0.1 to 15 mg.

Furthermore, in the organic acid production promoter of the present embodiment, the polyphenol preferably contains any one or more of quercetin, epigallocatechin gallate, and procyanidin, and the procyanidin is particularly cacao polyphenol. It is preferable to contain.
In the organic acid production promoter of this embodiment, the quercetin content is preferably 0.0001 to 50% by weight, more preferably 0.001 to 30% by weight, and still more preferably 0.01 to 15% by weight. is there. In the organic acid production promoter of this embodiment, the content of epigallocatechin gallate is preferably 0.0001 to 50% by weight, more preferably 0.001 to 30% by weight, and still more preferably 0.01. ~ 15 wt%. In the organic acid production promoter of this embodiment, the cocoa polyphenol content is preferably 0.0001 to 50% by weight, more preferably 0.001 to 30% by weight, and still more preferably 0.01 to 15%. % By weight.

In addition, in the organic acid production promoter of this embodiment, it is also preferable that the polyphenol contains one or more of quercetin, taxifolin, phloretin, epigallocatechin gallate, and procyanidin in combination. At this time, a synergistic effect can be expected for promotion of production of organic acid by combining two or more kinds of polyphenols.

The organic acid production promoter of this embodiment can be composed of a unit packaging form per serving, and can also be composed of an effective amount of polyphenol per unit packaging. .
In the organic acid production promoter of this embodiment, a known packaging (packaging material) can be used as a packaging form, and is not particularly limited. For example, paper, plastic, glass, nylon, stainless steel, aluminum Iron, copper, silver, bamboo, etc. can be used. The same applies to the following embodiments.
However, since the organic acid production promoter of this embodiment contains bifidobacteria or lactic acid bacteria which are anaerobic bacteria, it is preferable that the packaging form does not come into contact with air or oxygen. For example, in the production process and packaging process of the organic acid production promoter, it is preferable to provide a process for removing the possibility of exposure to air and oxygen, and in storage after packaging the organic acid production promoter, air It is preferable to select a packaging (wrapping material) that does not allow oxygen or oxygen to penetrate inside.

In the organic acid production promoter of this embodiment, the method of ingesting (administering) it is not particularly limited, and known ingestion (oral, tube, enteral, vascular injection, paint, suppository, etc.) All forms of administration) can be applied, preferably oral, tube, enteral, particularly preferably oral. In addition, in the organic acid production promoter of the present embodiment, as components other than polyphenol, other ingestible components, various additives, raw materials for pharmaceuticals, and the like may be contained. The same applies to the following embodiments.

[Second Embodiment]
The organic acid production promoter according to the second embodiment of the present invention is for accelerating the production of organic acid by bifidobacteria, and is the first in that not only polyphenols but also bifidobacteria are active ingredients. Different from one embodiment. Other points can be the same as those in the first embodiment.
If the organic acid production promoter is configured as in the second embodiment, it is possible to simultaneously ingest Bifidobacteria whose organic acid production has been promoted by polyphenol together with polyphenol. It is possible to produce an organic acid.

In the organic acid production promoter of this embodiment, the effective amount (intake amount, dose) of bifidobacteria per day for humans is preferably 2 × 10 ⁷ to 5 × 10 ¹⁰ , more preferably 1 ×. 10 ⁸ to 5 × 10 ¹⁰ , more preferably 5 × 10 ⁸ to 2 × 10 ¹⁰ .

Moreover, in the organic acid production promoter of this embodiment, when the bifidobacteria count is 10 ⁷ or more per 1 g of the bifidobacteria culture, the effectiveness of the bifidobacteria culture per day for humans The amount (intake, dose) is preferably 5 to 1000 g, more preferably 50 to 500 g, still more preferably 80 to 200 g.

At this time, in the production promoter of the organic acids of the present embodiment, per 1g of the culture of Bifidobacterium the number of bacteria Bifidobacterium 10 ⁷ or more, 10 ⁸ or more, it is contained in such 10 ⁹ or more Alternatively, 10 ⁷ , 10 ⁸ , 10 ⁹ or the like may be included. In the organic acid production promoter of this embodiment, if the number of bifidobacteria per gram of the bifidobacteria culture is increased, the bifidobacteria culture can be contained while containing an effective amount of the bifidobacteria. It is possible to reduce the effective amount of Bifidobacteria, and by taking a small amount of the bifidobacteria culture, the effect of promoting the production of organic acids and the effect of preventing and / or improving inflammatory bowel disease can be sufficiently obtained. It becomes possible. The same applies to lactic acid bacteria in the third embodiment.

In the organic acid production promoter of this embodiment, the bifidobacteria culture can be obtained by culturing (growing) the bifidobacteria with known medium components. The number of Bifidobacteria per unit weight of the culture solution can be increased by, for example, centrifuging the obtained Bifidobacterium culture solution. In the organic acid production promoter of the present embodiment, the bifidobacteria may be in a state of being cultured (proliferated), in a state of being mixed with a cryoprotectant or the like, or in a state of being freeze-dried. But you can. The same applies to lactic acid bacteria in the third embodiment.

In the organic acid production promoter of the present embodiment, the temperature when ingesting it is preferably −30 to 50 ° C., more preferably −20 to 45 ° C., still more preferably 0 to 45 ° C. The temperature is preferably 0 to 30 ° C, particularly preferably 0 to 20 ° C, and most preferably 0 to 10 ° C. The same applies to lactic acid bacteria in the third embodiment.

[Third embodiment]
The organic acid production promoter according to the third embodiment of the present invention is for accelerating the production of organic acid by lactic acid bacteria, and is a second embodiment in that lactic acid bacteria are used as an active ingredient instead of bifidobacteria. It is different from the form. Other points can be the same as those in the second embodiment.
If the organic acid production promoter is configured as in the third embodiment, it is possible to simultaneously ingest lactic acid bacteria whose organic acid production has been promoted by polyphenols together with polyphenols. It becomes possible to produce an acid.

In the organic acid production promoter of the present embodiment, the effective amount (intake amount, dose) of lactic acid bacteria per day for humans is preferably 2 × 10 ⁷ to 5 × 10 ¹⁰ , more preferably 1 × 10. ⁸ to 5 × 10 ¹⁰ , more preferably 5 × 10 ⁸ to 2 × 10 ¹⁰ .

In addition, in the organic acid production promoter of this embodiment, when the number of lactic acid bacteria is 10 ⁷ or more per 1 g of the lactic acid bacteria culture, an effective amount of the lactic acid bacteria culture per day for humans (ingestion) The amount is preferably 5 to 1000 g, more preferably 50 to 500 g, and still more preferably 80 to 200 g.

[Fourth embodiment]
The preventive and / or ameliorating agent for inflammatory bowel disease according to the fourth embodiment of the present invention preferably contains the organic acid production promoter of any one of the first to third embodiments. .
By taking such a preventive and / or ameliorating agent for inflammatory bowel disease, it is possible to improve the production efficiency of organic acids by bifidobacteria and lactic acid bacteria in the intestine. For this reason, the organic acid actually produced suppresses the growth of harmful bacteria in the intestine, and obtains an excellent preventive and / or ameliorating effect on inflammatory bowel disease through the effect of improving the intestinal flora. Is possible.

[Fifth embodiment]
Next, a method for producing lactic acid according to the fifth embodiment of the present invention will be described. In the method for producing lactic acid according to this embodiment, lactic acid is produced by bifidobacteria and / or lactic acid bacteria using a composition containing polyphenol as an active ingredient and promoting the production of organic acids by bifidobacteria and / or lactic acid bacteria. Is the method. In addition, the method for producing lactic acid according to the present embodiment uses a composition that promotes the production of organic acid by bifidobacteria and / or lactic acid bacteria, which contains polyphenol and bifidobacteria and / or lactic acid bacteria as active ingredients. It is also preferable to use a method for producing lactic acid with bacteria or lactic acid bacteria.
In the lactic acid production method of the present embodiment, the concentration of lactic acid produced per number of Bifidobacteria and lactic acid bacteria is preferably 0.01 pM / cfu or more (by absolute value), more preferably 0.1 pM / cfu. More preferably, it is 1 pM / cfu or more, and particularly preferably 0.01 nM / cfu or more. In addition, when compared with a method for producing lactic acid by bifidobacteria or lactic acid bacteria without adding polyphenol, in the method for producing lactic acid of this embodiment, the concentration of lactic acid produced per the number of bifidobacteria or lactic acid bacteria Increases (promoted) preferably (in relative value) at 1% or more, more preferably at least 2%, even more preferably at least 5%, particularly preferably at least 10%.

In the lactic acid production method of the present embodiment, the concentration of lactic acid produced per 10 ⁸ bifidobacteria and lactic acid bacteria is preferably (in absolute value) preferably 1 nM / 10 ⁸ cfu or more, more preferably 0.8. 01mM ^/ 10 8 cfu or more, more preferably 0.1 mM ^/ 10 8 cfu or more, and particularly preferably 1 mM ^/ 10 8 cfu or more. In addition, when compared with a method for producing lactic acid by bifidobacteria or lactic acid bacteria without adding polyphenol, in the method for producing lactic acid of this embodiment, the concentration of lactic acid produced per the number of bifidobacteria or lactic acid bacteria Increases (promoted) preferably (in relative value) at 1% or more, more preferably at least 2%, even more preferably at least 5%, particularly preferably at least 10%.
In addition, as this embodiment, it can also be expressed as the use of polyphenols for producing lactic acid by bifidobacteria and / or lactic acid bacteria, the use of polyphenols for promoting the production of lactic acid by bifidobacteria and / or lactic acid bacteria, and the like. it can.

[Sixth embodiment]
Next, a method for producing acetic acid according to the fifth embodiment of the present invention will be described. In the method for producing acetic acid according to the present embodiment, acetic acid is produced by bifidobacteria and / or lactic acid bacteria using a composition containing polyphenol as an active ingredient and promoting the production of organic acids by bifidobacteria and / or lactic acid bacteria. Is the method. In addition, the method for producing acetic acid according to this embodiment uses a composition that promotes the production of organic acid by bifidobacteria and / or lactic acid bacteria, which contains polyphenol and bifidobacteria and / or lactic acid bacteria as active ingredients. It is also preferable to use a method for producing acetic acid by using bacteria or lactic acid bacteria.
In the method for producing acetic acid of the present embodiment, the concentration of acetic acid produced per the number of bifidobacteria and lactic acid bacteria is preferably (in absolute value) preferably 1 fM / cfu or more, more preferably 0.01 pM / cfu or more, More preferably, it is 0.1 pM / cfu or more, and particularly preferably 1 pM / cfu or more. In addition, when compared to the method of producing acetic acid by bifidobacteria or lactic acid bacteria without addition of polyphenol, the concentration of acetic acid produced per the number of bifidobacteria and lactic acid bacteria in the acetic acid production method of the present embodiment Increases (promoted) preferably (in relative value) at 1% or more, more preferably at least 2%, even more preferably at least 5%, particularly preferably at least 10%.

In the method for producing acetic acid of the present embodiment, the concentration of acetic acid produced per 10 ⁸ bifidobacteria and lactic acid bacteria is preferably (in absolute value) preferably 0.1 nM / 10 ⁸ cfu or more, more preferably 1 nM ^/ 10 8 cfu or more, more preferably 0.01 nM ^/ 10 8 cfu or more, and particularly preferably 0.1 nM ^/ 10 8 cfu or more. In addition, when compared to the method of producing acetic acid by bifidobacteria or lactic acid bacteria without addition of polyphenol, the concentration of acetic acid produced per the number of bifidobacteria and lactic acid bacteria in the acetic acid production method of the present embodiment Increases (promoted) preferably (in relative value) at 1% or more, more preferably at least 2%, even more preferably at least 5%, particularly preferably at least 10%.
In addition, as this embodiment, it can also be expressed as the use of polyphenols for producing acetic acid by bifidobacteria and / or lactic acid bacteria, the use of polyphenols for promoting the production of acetic acid by bifidobacteria and / or lactic acid bacteria, and the like. it can.

Hereinafter, the test conducted in order to confirm the effect of the organic acid production promoter according to the embodiment of the present invention will be described in detail. However, the present invention is not limited to the following configuration.

<Test 1>
(Test method)
Four types of polyphenols, quercetin, taxifolin, phloretin, and epigallocatechin gallate were prepared.
Specifically, quercetin (Cayman Chemical Company, Inc.) was dissolved in DMSO (Dimethyl sulfoxide) to prepare 20 mM. Taxifolin ((+)-Taxifolin 1036 Extrasynthese, Funakoshi Co., Ltd.) was dissolved in DMSO to prepare 40 mM. Further, phloretin (Phloretin, Sigma-Aldrich Japan) was dissolved in DMSO to prepare 40 mM. Epigallocatechin gallate (Cayman Chemical Company, Inc.) was dissolved in DMSO to prepare 20 mM. Then, each prepared DMSO solution of quercetin, taxifolin, phloretin, and epigallocatechin gallate was added to DMEM medium (DMEM, Sigma-Aldrich Japan) at 0.01%. As a control, DMSO (Dimethyl sulfoxide) to which polyphenol was not added was added to a DMEM medium at 0.01%.

Each DMEM medium prepared above was inoculated with 10 ⁸ cfu / ml of Bifidobacterium adolescentis JCM1275 ^T as bifidobacteria and anaerobically cultured at 37 ° C. for 3 hours. After labeling lactic acid and acetic acid in the culture supernatant with a labeling reagent (YMC Corporation) for analysis of long and short chain fatty acids, high performance liquid chromatography (HPLC) is used. The concentration of lactic acid and acetic acid in each medium after culture was measured. As HPLC conditions, an Ascentis RP-amide column (100 × 30 mm, 3 μm, Sigma-Aldrich Japan) was used, and the mobile phase was A phase (0.01 M hydrochloric acid, pH 4.5): B phase (acetonitrile: methanol = 2: 1) was 13: 7 and was 0.4 ml / min. The column temperature was 50 ° C., the injection was 4 μl, and the detection wavelength was 400 nm. The result is shown in FIG.

In addition, the number of bifidobacteria in each medium before and after the culture was calculated from the turbidity (absorbance at 650 nm) of each medium before and after the culture and a calibration curve prepared in advance.
As a specific method for preparing a calibration curve, turbidity (absorbance at 650 nm) in a solution of Bifidobacterium count was measured, and then Bifidobacterium was anaerobically cultured on a BCP-added plate count agar medium at 37 ° C. for 72 hours. And the number of colonies was counted. Then, a calibration curve of the number of bifidobacteria (cfu / ml) and turbidity was prepared.

(Test results)
The concentration of the organic acid in the medium not added with polyphenol (DMSO) was 3.46 mM for lactic acid and 4.86 mM for acetic acid. Moreover, as a density | concentration of the organic acid in the culture medium (QUE) which added quercetin, the lactic acid was 3.88 mM and the acetic acid was 5.41 mM. Moreover, as a density | concentration of the organic acid in the culture medium (TAX) which added taxifolin, lactic acid was 5.88 mM and acetic acid was 7.44 mM. Moreover, as a density | concentration of the organic acid in the culture medium (PHL) which added the phloretin, lactic acid was 4.91 mM and acetic acid was 6.26 mM. Moreover, as a density | concentration of the organic acid in the culture medium (EGCG) which added epigallocatechin gallate, lactic acid was 5.25 mM and acetic acid was 7.81 mM.

That is, with the addition of quercetin, lactic acid increased by 12% and acetic acid increased by 11%. In addition, the addition of taxifolin increased lactic acid at 70% and acetic acid at 53%. In addition, addition of phloretin increased lactic acid at 42% and acetic acid at 29%. Moreover, the addition of epigallocatechin gallate increased lactic acid at 42% and acetic acid at 29%.

Moreover, as a result of calculating the number of bifidobacteria in each medium before and after the culture, the number of bifidobacteria in any medium was about 10 ⁸ cfu / ml. This confirmed that the number of bifidobacteria in the culture medium before and after the culture did not change.
Therefore, it is clear from the results of Test 1 that quercetin, taxifolin, phloretin, and epigallocatechin gallate can improve the production efficiency of lactic acid and acetic acid by Bifidobacterium without changing the number of Bifidobacterium. It was.

<Test 2>
(Test method)
As procyanidins, cacao polyphenols were prepared.
Specifically, cacao bean polyphenol extract produced as described below was dissolved in DMSO to prepare 100 mg / ml. As a control, DMSO (Dimethyl sulfoxide) to which polyphenol was not added was added to a DMEM medium at 0.01%.

Cocoa polyphenol was produced as follows.
First, 150 kg of cocoa beans were heated at 80 ° C. for 1 hour. Next, this heated cocoa bean was defatted using an expeller to obtain 80 kg of cocoa powder. 800 kg of ethanol (50%) was added to the defatted cocoa powder and then heated. And after raising this to 50 degreeC, after carrying out stirring extraction in 1 hour, this extract was left to stand at 4 degreeC overnight. The extract was filtered using a screw decanter and a filter press, the filtrate was concentrated under reduced pressure to 27 kg, and 13 kg of ethanol (95%) was added. The concentrated solution was stirred at 40 ° C. for 1 hour, and then refrigerated overnight. The concentrated solution was centrifuged (25 ° C., 12000 rpm) using a sharp press centrifuge. Then, 35 kg of acid clay (Mizuka Ace # 20) was added to the supernatant of this centrifugation, followed by stirring for 1 hour. This was filtered using a Nutsche, and the filtrate was concentrated under reduced pressure and lyophilized to obtain 6.3 kg of powder. And 0.5 L of this powder was charged with 25 L of resin and 50 L of water, and stirred for 30 minutes or more. After the resin was recovered, 75 L of ethanol (80%) was added and stirred for 20-30 minutes. This was filtered using a Nutsche, and the filtrate was concentrated under reduced pressure and lyophilized to obtain cacao polyphenol.

Each DMEM medium prepared above was inoculated with 10 ⁸ cfu / ml of Bifidobacterium adolescentis JCM1275 ^T as bifidobacteria and anaerobically cultured at 37 ° C. for 3 hours. Then, in the same manner as in Test 1, the concentrations of lactic acid and acetic acid in the cultured medium were measured using HPLC. The result is shown in FIG.
Further, the number of bifidobacteria in each medium before and after the culture was calculated in the same manner as in Test 1 using the turbidity (absorbance at 650 nm) of each medium before and after the culture and a calibration curve prepared in advance.

(Test results)
The concentration of the organic acid in the medium (DMSO) to which polyphenol was not added was 5.70 mM for lactic acid and 11.80 mM for acetic acid. Moreover, as a density | concentration of the organic acid in the culture medium (CBP) which added cacao polyphenol, lactic acid was 6.34 mM and acetic acid was 13.18 mM.

That is, with the addition of cacao polyphenol, lactic acid increased by 11% and acetic acid increased by 12%.

Moreover, as a result of calculating the number of bifidobacteria in each medium before and after the culture, the number of bifidobacteria in any medium was about 10 ⁸ cfu / ml. This confirmed that the number of bifidobacteria in the culture medium before and after the culture did not change.
Therefore, from the results of Test 2, it was revealed that cocoa polyphenol can improve the production efficiency of lactic acid and acetic acid by Bifidobacterium without changing the number of Bifidobacterium.

<Test 3>
(Test method)
Four types of polyphenols, quercetin, taxifolin, phloretin, and epigallocatechin gallate were prepared.
Specifically, quercetin (Cayman Chemical Company, Inc.) was dissolved in DMSO (Dimethyl sulfoxide) to prepare 200 mM. Taxifolin ((+)-Taxifolin 1036 Extrasynthese, Funakoshi Co., Ltd.) was dissolved in DMSO to prepare 400 mM. Further, phloretin (Phloretin, Sigma-Aldrich Japan) was dissolved in DMSO to prepare 400 mM. Epigallocatechin gallate (Cayman Chemical Company, Inc.) was dissolved in DMSO to prepare 200 mM. Then, each prepared DMSO solution of quercetin, taxifolin, phloretin, and epigallocatechin gallate was added to DMEM medium (DMEM, Sigma-Aldrich Japan) at 0.01%. As a control, DMSO without polyphenol added to DMEM medium at 0.01% was prepared.

Each DMEM medium prepared above was inoculated with 10 ⁸ cfu / ml of Lactobacillus delbrueckii subsp. Bulgaricus OLL1181 (FERM BP-11269) as a lactic acid bacterium at 37 ° C. for 3 hours. Cultured. Then, in the same manner as in Test 1, the concentration of acetic acid in the cultured medium was measured using HPLC. The result is shown in FIG.
In addition, the number of lactic acid bacteria in each medium before and after the culture was calculated in the same manner as in Test 1 using the turbidity (absorbance at 650 nm) of each medium before and after the culture and a calibration curve prepared in advance.

(Test results)
Acetic acid was 0.63 mM as the concentration of the organic acid in the medium not added with polyphenol (DMSO). Moreover, as a density | concentration of the organic acid in the culture medium (QUE) which added quercetin, the acetic acid was 0.61 mM. Further, acetic acid was 0.97 mM as the concentration of the organic acid in the medium (TAX) to which taxifolin was added. Moreover, as a density | concentration of the organic acid in the culture medium (PHL) which added the phloretin, the acetic acid was 0.72 mM. Moreover, as a density | concentration of the organic acid in the culture medium (EGCG) which added epigallocatechin gallate, the acetic acid was 0.90 mM.

That is, acetic acid decreased by 3% by the addition of quercetin. Moreover, acetic acid increased by 54% by the addition of taxifolin. Moreover, acetic acid increased by 14% by the addition of phloretin. Moreover, acetic acid increased by 43% by addition of epigallocatechin gallate.

In addition, as a result of calculating the number of lactic acid bacteria in each medium before and after the culture, the number of lactic acid bacteria in any medium was about 10 ⁸ cfu / ml. From this, it was confirmed that the number of lactic acid bacteria in the culture medium before and after the culture did not change.
Therefore, the results of Test 3 revealed that quercetin, taxifolin, phloretin, and epigallocatechin gallate can improve the production efficiency of acetic acid by lactic acid bacteria without changing the number of lactic acid bacteria.

As described above, according to the organic acid production promoter of this embodiment, it is possible to increase the amount of organic acid produced by these microorganisms regardless of the number (quantity) of bifidobacteria and lactic acid bacteria. It is. According to the preventive and / or ameliorating agent for inflammatory bowel disease of this embodiment containing such an organic acid production promoter, the growth of harmful bacteria is suppressed in the intestine, and the intestinal flora Through the improvement effect, it is possible to obtain an excellent preventive effect and / or improvement effect against inflammatory bowel disease.

The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the present invention. For example, although quercetin, taxifolin, phloretin, epigallocatechin gallate, and procyanidin are used as the polyphenol in the above-described embodiments and examples, other polyphenols that exhibit the same effects as these may be used. Of course, microorganisms other than the strains shown in the embodiments may be used as the bifidobacteria and lactic acid bacteria. Furthermore, you may contain polyphenol, bifidobacteria, and lactic acid bacteria as an active ingredient combining 2nd embodiment and 3rd embodiment.

The present invention can be suitably used to prevent and / or improve diarrhea, constipation, inflammatory bowel disease, and the like.

Claims (20)

1. An organic acid production promoter for promoting the production of organic acids by bifidobacteria or lactic acid bacteria, containing polyphenol as an active ingredient.
2. The bifidobacteria include Bifidobacterium longum OLB6290 (NITE P-75), Bifidobacterium longum no. 7 (FERM BP-11242), and Bifidobacterium address Sen infantis (Bifidobacterium adolescentis) production promoter of the organic acid according to claim 1, wherein either one or two or more JCM1275 ^T.
3. The organic acid production promoter according to claim 1 or 2, wherein the lactic acid bacterium is a lactic acid bacterium of the genus Lactobacillus.
4. The organic acid production promoter according to claim 3, wherein the lactic acid bacterium of the genus Lactobacillus is Lactobacillus delbrueckii subsp. Bulgaricus OLL1181 (FERM BP-11269).
5. The organic acid production promoter according to any one of claims 1 to 4, wherein the organic acid is lactic acid and / or acetic acid.
6. The organic acid production promoter according to any one of claims 1 to 5, wherein the polyphenol is a flavonoid.
7. The organic acid production promoter according to claim 6, wherein the polyphenol is taxifolin.
8. The organic acid production promoter according to claim 7, wherein the content of taxifolin is 0.001 to 5.0% by weight.
9. The organic acid production promoter according to claim 7, wherein the daily dose of taxifolin for humans is 0.1 to 100 mg.
10. 6. The organic acid production promoter according to claim 1, wherein the polyphenol is phloretin.
11. The organic acid production promoter according to claim 10, wherein the content of phloretin is 0.0001 to 50% by weight.
12. 11. The organic acid production promoter according to claim 10, wherein the daily dose of phloretin to a human is 0.001 to 50 mg.
13. The organic acid production promoter according to any one of claims 1 to 5, wherein the polyphenol is one or more of quercetin, epigallocatechin gallate, and procyanidin.
14. 14. The organic acid production promoter according to claim 1, which contains the polyphenol and the bifidobacteria as active ingredients for promoting the production of an organic acid by the bifidobacteria.
15. 15. The organic acid production promoter according to claim 14, wherein the daily dose of the bifidobacteria to a human is 2 × 10 ⁷ to 5 × 10 ¹⁰ .
16. When 10 ⁷ or more bifidobacteria are contained per 1 g of the bifidobacteria culture contained in the organic acid production promoter according to claim 14, the daily dose of the bifidobacteria culture to humans Is an organic acid production promoter of 5 to 1000 g.
17. 14. The organic acid production promoter according to claim 1, which contains the polyphenol and the lactic acid bacterium as active ingredients and promotes the production of the organic acid by the lactic acid bacterium.
18. The organic acid production promoter according to claim 17, wherein the daily dose of the lactic acid bacteria to a human is 2 × 10 ⁷ to 5 × 10 ¹⁰ .
19. When 10 ⁷ or more lactic acid bacteria are contained per 1 g of the culture of lactic acid bacteria contained in the organic acid production promoter according to claim 17, the daily dose of the culture of lactic acid bacteria to a human is 5 Organic acid production promoter of ˜1000 g.
20. An agent for preventing and / or improving inflammatory bowel disease, comprising the organic acid production promoter according to any one of claims 1 to 19.

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