JP6810610B2 - Organic acid production promoters and preventive and / or ameliorating agents for inflammatory bowel disease - Google Patents

Description

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

It is known that the intestinal flora is closely related to human health and diseases. It is said that more than 100 trillion intestinal bacteria of 500 to 1000 types inhabit the human intestine, and generally, useful microorganisms (useful bacteria) such as bifidobacteria and lactic acid bacteria and Clostridium perfringens Harmful microorganisms (harmful bacteria) such as are inhabited together. In order to maintain human health, it is important that useful bacteria are present in the large intestine as a dominant species.
That is, bifidobacteria and lactic acid bacteria have a function of producing organic acids such as lactic acid and acetic acid in the living body (intestine), and these organic acids suppress the growth of harmful bacteria in the intestinal flora. It is possible to prevent it, and it is possible to suppress the occurrence of constipation and diarrhea. It has also been reported that these produced lactic acid and acetic acid are converted to butyric acid by intestinal bacteria, and that butyric acid improves inflammatory bowel disease and the like.

On the other hand, in recent years, the number of patients with constipation, diarrhea, inflammatory bowel disease, etc. has been increasing, and it is required to effectively improve the human intestinal flora. Therefore, 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 the growth of intestinal bifidobacteria is promoted by using poly-γ-glutamic acid as an active ingredient. Further, Patent Document 2 describes that the development of the intestinal bacterial flora is promoted by using a lipid composition containing a plant-derived fat source.
In addition, it is widely practiced to grow bifidobacteria and lactic acid bacteria in the intestine by ingesting foods, beverages, supplements and the like containing lactic acid bacteria and oligosaccharides.
According to these conventional techniques, it is possible to increase the amount of organic acid produced by growing bifidobacteria and lactic acid bacteria in the intestine.

Japanese Unexamined Patent Publication No. 2011-178764 Special Table 2013-525421 Japanese Unexamined Patent Publication No. 6-166618 Japanese Unexamined Patent Publication No. 6-248267 Japanese Unexamined Patent Publication No. 2012-922138

However, these conventional techniques increase the production of organic acids by increasing the number of useful bacteria in the intestine, and do not improve the production efficiency of organic acids of useful bacteria (microorganisms) themselves. It was.
In addition, some foods containing lactic acid bacteria and oligosaccharides have a problem with the survival of lactic acid bacteria, and some foods cause diarrhea due to excessive intake.
Therefore, it is desirable that it is possible to improve the production efficiency of organic acids of useful bacteria themselves regardless of the number (number of bacteria) of bifidobacteria and lactic acid bacteria. If it becomes possible to improve the production efficiency of organic acids of these useful bacteria themselves, the intestinal flora even when it is difficult to ingest a large amount of useful bacteria or when it is difficult to grow useful bacteria in the intestine. It becomes possible to expect a preventive effect and / or an ameliorating effect on inflammatory bowel disease.

Therefore, as a result of diligent research on the effects of various phytochemical substances on the organic acid production function by bifidobacteria and the like, the present inventors have obtained an excellent organic acid production promoting effect by using polyphenols. I found it. Such polyphenols include, among others, quercetin, taxifolin, phloretin, epigallocatechin gallate, and procyanidins.

Here, as a technique related to the improvement of the intestinal flora using polyphenols, the composition for intestinal regulation described in Patent Document 3 can be mentioned. It is described that this intestinal regulating composition contains epigallocatechin gallate and the like, and can lower the pH in the human intestine to improve the intestinal flora and constipation.
However, the intestinal regulating composition 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 bifidobacteria and lactic acid bacteria regardless of the number. It wasn't possible.

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

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 number of bifidobacteria and lactic acid bacteria, and an organic acid production promoter. The purpose is to provide a preventive and / or ameliorating agent for inflammatory bowel disease.

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

In the organic acid production promoter of the present invention, examples of the organic acid produced by bifidobacteria and / or lactic acid bacteria include lactic acid and acetic acid. In addition, bifidobacteria and / or lactic acid bacteria produce lactic acid and acetic acid, which 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, it is preferable that the polyphenol contains any one or more of flavonoids, phloretin, and procyanidins. At this time, it is preferable to use quercetin, taxifolin, and epigallocatechin gallate as flavonoids. Moreover, it is preferable to use cacao polyphenol as the Prussian carrot.

Further, in the organic acid production promoter of the present invention, it is preferable to contain the above polyphenol and the above bifidobacteria as active ingredients, and it is also preferable to contain the above polyphenol and the above lactic acid bacteria 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, an organic acid production promoter capable of improving the organic acid production efficiency of these useful bacteria themselves, and a preventive and / or ameliorating agent for inflammatory bowel disease, regardless of the number of bifidobacteria and lactic acid bacteria. Can be provided.

It is a figure which shows the graph which showed the concentration of the organic acid produced by the bifidobacteria culture | cultivated using the culture medium containing each of various polyphenols. It is a figure which shows the graph which showed the concentration of the organic acid produced by the bifidobacteria cultured using the culture medium containing cacao polyphenol as procyanidin. It is a figure which shows the graph which showed the concentration of the organic acid produced by the lactic acid bacterium culture | cultivated using the culture medium containing each of 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 the present embodiment is an organic acid production promoter for promoting the production of organic acid by bifidobacteria or lactic acid bacteria, which contains polyphenol as an active ingredient.

Bifidobacterium is a type of gram-positive obligate anaerobic bacillus that is widely inhabited in the living body of animals. Bifidobacterium is a microorganism that decomposes sugar to produce lactic acid and acetic acid. In order to maintain human health, it is desirable that bifidobacteria exist as a dominant species in the intestine for the rest of their lives.

In the organic acid production promoter of the present embodiment, the bifidobacteria are not particularly limited, but for example, Bifidobacterium longum OLB6290 (NITE P-75), Bifidobacterium longum No. 7 (FERM BP-11242) or Bifidobacterium adolescentis JCM1275 ^T or the like is preferable.

Bifidobacterium longum OLB6290 (Bifidobacterium OLB6290) was deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center under the accession number: NITE P-75 on February 3, 2005. It is a microorganism.

Bifidobacterium longum No. Bifidobacterium No. 7 (Bifidobacterium No. 7) is a microorganism deposited on April 20, 1993 at the Patent Microorganisms Depositary Center, National Institute of Technology and Evaluation, under the accession number: FERM BP-11242.

Lactic acid bacteria are microorganisms that decompose sugar to produce lactic acid, and are classified into homolactic acid bacteria that produce only lactic acid as the final product and heterolactic acid bacteria that simultaneously produce substances other than lactic acid. Examples of lactic acid bacteria include lactic acid bacteria of the genus Lactobacillus, lactic acid bacteria of the genus Bifidobacterium, lactic acid bacteria of the genus Enterococcus, lactic acid bacteria of the genus Lactococcus, lactic acid bacteria of the genus Pediococcus, and lactic acid bacteria of the genus Leuconostock. As used herein, 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 bacillus, 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 OLL1181) has been assigned to the Patent Microorganisms Depositary Center, Product Evaluation Technology Infrastructure Organization, as a consignment number: FERM BP-11269, July 16, 2010. It is a microorganism deposited on a daily basis.

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. Further, 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 the present 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 intestine, improve the intestinal flora, and suppress or prevent constipation and diarrhea. Is possible. Butyric acid is also known to have an excellent preventive and / or ameliorating effect on inflammatory bowel disease.

According to the organic acid production promoter of the present embodiment, it is possible to increase the amount of lactic acid and acetic acid produced by bifidobacteria and lactic acid bacteria without increasing the number (number of bacteria) of bifidobacteria and lactic acid bacteria. Is. Therefore, even when a person who has difficulty in growing bifidobacteria or lactic acid bacteria in the intestine ingests the organic acid production promoter of the present embodiment, the amount of lactic acid and acetic acid should be increased in the intestine. Becomes possible. Then, by increasing the amount of lactic acid and acetic acid in the intestine, the intestinal flora is improved, and it is possible to obtain a preventive effect and / or an improving effect on inflammatory bowel disease.

In the organic acid production promoter of the present 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 the present embodiment, the content of taxifolin is preferably 0.001 to 5.0% by weight, more preferably 0.01 to 4.0% by weight, still more preferably 0.1 to 1% by weight. It is 3.0% by weight.

Further, in the organic acid production promoter of the present embodiment, the effective amount (intake, 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.
The effective amount per day for humans may be ingested (administered) once, or may be ingested two or more times. The same applies to the following embodiments.

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

Further, 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. Is preferably included.
In the organic acid production promoter of the present embodiment, the content of quercetin is preferably 0.0001 to 50% by weight, more preferably 0.001 to 30% by weight, still more preferably 0.01 to 15% by weight. is there. Further, in the organic acid production promoter of the present embodiment, the content of epigallocatechin gallate is preferably 0.0001 to 50% by weight, more preferably 0.001 to 30% by weight, still more preferably 0.01. ~ 15% by weight. Further, in the organic acid production promoter of the present embodiment, the content of cacao polyphenol is preferably 0.0001 to 50% by weight, more preferably 0.001 to 30% by weight, still more preferably 0.01 to 15% by weight. By weight%.

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

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

In the organic acid production promoter of the present embodiment, the method of ingesting (administering) the organic acid is not particularly limited, and known ingestion (oral, tube, intestinal, vascular injection, ointment, suppository, etc.) It is possible to apply all of the forms (administration), and it is preferable to apply oral, tube, and intestinal, and it is particularly preferable to apply oral. In addition, in the organic acid production promoter of the present embodiment, other ingestible components, various additives, raw materials for pharmaceutical products, and the like may be contained as components other than polyphenols. 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 promoting the production of an organic acid by bifidobacteria, and is the first in that it contains not only polyphenols but also bifidobacteria as active ingredients. It is different from one embodiment. Other points can be the same as in the first embodiment.
If the organic acid production promoter is configured as in the second embodiment, it is possible to simultaneously ingest the bifidobacteria whose organic acid production has been promoted by the polyphenols, so that the bifidobacteria can be more reliably taken. Can produce organic acids.

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

Further, in production promoter of the organic acids of the present embodiment, if the per 1g of the culture of Bifidobacterium is contained bacterial count of bifidobacteria in ¹⁰⁷ or more, effective culture per day bifidobacteria to human The amount (intake amount, dose) is preferably 5 to 1000 g, more preferably 50 to 500 g, and even 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, be contained in such 10 ⁹ or more well, 10 ^7, 10 ^8, may be contained in such 10 ^9. In the organic acid production promoter of the present embodiment, if the number of bifidobacteria per gram of the culture of bifidobacteria is increased, the culture of bifidobacteria is contained in an effective amount while containing the number of bifidobacteria in an effective amount. It is possible to reduce the effective amount of bifidobacteria, and by ingesting a smaller amount of the culture of bifidobacteria, 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. This also applies to the lactic acid bacterium in the third embodiment.

In the organic acid production promoter of the present embodiment, the culture of bifidobacteria can be obtained by culturing (proliferating) bifidobacteria with known medium components. Then, the number of bifidobacteria per unit weight of the culture solution can be increased by centrifuging the obtained culture solution of bifidobacteria. In the organic acid production promoter of the present embodiment, the bifidobacteria may be, for example, in a state of being cultured (proliferated), in a state of being mixed with a cryoprotectant and frozen, or in a state of being freeze-dried. It may be. This also applies to the lactic acid bacterium in the third embodiment.

Further, in the organic acid production promoter of the present embodiment, the temperature at which it is ingested is preferably -30 to 50 ° C, more preferably -20 to 45 ° C, still more preferably 0 to 45 ° C, and further. The temperature is preferably 0 to 30 ° C, particularly preferably 0 to 20 ° C, and most preferably 0 to 10 ° C. This also applies to the lactic acid bacterium in the third embodiment.

[Third Embodiment]
The organic acid production promoter according to the third embodiment of the present invention is for promoting the production of organic acid by lactic acid bacteria, and is the 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 in the second embodiment.
If the organic acid production promoter is configured as in the third embodiment, it is possible to simultaneously ingest the lactic acid bacterium whose organic acid production is promoted by the polyphenol together with the polyphenol, so that the lactic acid bacterium is more reliably organic. It is possible to produce acid.

In the organic acid production promoter of the present embodiment, the effective amount (intake, 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 pieces, more preferably 5 × 10 ⁸ to 2 × 10 ¹⁰ pieces.

Further, in production promoter of the organic acids of the present embodiment, if the inclusion of number of bacteria in lactic acid bacteria ¹⁰⁷ or more per 1g of the culture of lactic acid bacteria, the effective amount of the culture per day of lactic acid bacteria to humans (ingestion The amount (amount, dose) is preferably 5 to 1000 g, more preferably 50 to 500 g, and even 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 according to any one of the first to third embodiments. ..
By ingesting 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. Therefore, the actually produced organic acid suppresses the growth of harmful bacteria in the intestine, and through the effect of improving the intestinal flora, an excellent preventive effect and / or improving effect on inflammatory bowel disease is obtained. 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 of the present 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. The method. In addition, the method for producing lactic acid of the present embodiment uses a composition that contains polyphenol and bifidobacteria and / or lactic acid bacteria as active ingredients and promotes the production of organic acids by bifidobacteria and / or lactic acid bacteria. It is also preferable to use a method for producing lactic acid by bacteria or lactic acid bacteria.
In the method for producing lactic acid 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 (in absolute value), more preferably 0.1 pM / cfu. Above, more preferably 1 pM / cfu or more, particularly preferably 0.01 nM / cfu or more. Further, when compared with the method of producing lactic acid by bifidobacteria or lactic acid bacteria without adding polyphenol, in the method of producing lactic acid of the present embodiment, the concentration of lactic acid produced per the number of bifidobacteria or lactic acid bacteria Is increased (promoted) by (relatively) preferably 1% or more, more preferably 2% or more, still more preferably 5% or more, and particularly preferably 10% or more.

In the method for producing lactic acid according to the present embodiment, the concentration of lactic acid produced in 10 ⁸ per bifidobacteria and lactic acid bacteria, (in absolute value) preferably 1 nM / 10 8 ^cfu or more, and more preferably 0. 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. Further, when compared with the method of producing lactic acid by bifidobacteria or lactic acid bacteria without adding polyphenol, in the method of producing lactic acid of the present embodiment, the concentration of lactic acid produced per the number of bifidobacteria or lactic acid bacteria Is increased (promoted) by (relatively) preferably 1% or more, more preferably 2% or more, still more preferably 5% or more, and particularly preferably 10% or more.
In this embodiment, it may be expressed as the use of polyphenols for producing lactic acid by bifidobacteria and / or lactic acid bacteria, the use of polyphenols for promoting lactic acid production 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 of 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. The method. In addition, the method for producing acetic acid of the present embodiment uses a composition that contains polyphenol and bifidobacteria and / or lactic acid bacteria as active ingredients and promotes the production of organic acids by bifidobacteria and / or lactic acid bacteria. It is also preferable to use a method for producing acetic acid using bacteria or lactic acid bacteria.
In the method for producing acetic acid of the present embodiment, the concentration of acetic acid produced per number of bifidobacteria and lactic acid bacteria is preferably 1 fM / cfu or more (in absolute value), 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. Further, when compared with the method of producing acetic acid by bifidobacteria or lactic acid bacteria without adding polyphenol, in the method of producing acetic acid of the present embodiment, the concentration of acetic acid produced per the number of bifidobacteria or lactic acid bacteria Is increased (promoted) by (relatively) preferably 1% or more, more preferably 2% or more, still more preferably 5% or more, and particularly preferably 10% or more.

In the method for producing acetic acid of the present embodiment, the concentration of acetic acid produced in 10 ⁸ per bifidobacteria and lactic acid bacteria, (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. Further, when compared with the method of producing acetic acid by bifidobacteria or lactic acid bacteria without adding polyphenol, in the method of producing acetic acid of the present embodiment, the concentration of acetic acid produced per the number of bifidobacteria or lactic acid bacteria Is increased (promoted) by (relatively) preferably 1% or more, more preferably 2% or more, still more preferably 5% or more, and particularly preferably 10% or more.
In addition, as this embodiment, it may be expressed as the use of polyphenol for producing acetic acid by bifidobacteria and / or lactic acid bacteria, the use of polyphenol for promoting the production of acetic acid by bifidobacteria and / or lactic acid bacteria, and the like. it can.

Hereinafter, the test carried out for confirming 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 configurations.

<Test 1>
(Test method)
Four types of polyphenols were prepared: quercetin, taxifolin, phloretin, and epigallocatechin gallate.
Specifically, quercetin (Cayman Chemical Company, Inc.) was dissolved in DMSO (Dimethyl sulfoxide) to prepare 20 mM. In addition, taxifolin ((+)-Taxifolin 1036 Extrasynthese, Funakoshi Co., Ltd.) was dissolved in DMSO to prepare 40 mM. In addition, phloretin (Phloretin, Sigma-Aldrich Japan) was dissolved in DMSO to prepare 40 mM. In addition, Epigallocatechin gallate (Cayman Chemical Company, Inc.) was dissolved in DMSO to prepare 20 mM. Then, each DMSO solution of these prepared quercetin, taxifolin, floretin, 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 DMEM medium at 0.01%.

Each DMEM medium prepared above, as bifidobacteria, inoculated Bifidobacterium address Sen infantis the (Bifidobacterium adolescentis) JCM1275 ^T at ^{10 8 cfu / ml, 37 ℃} , and anaerobically cultured for 3 hours. Then, after labeling the lactic acid and acetic acid in the culture supernatant with a labeling reagent (YMC Co., Ltd.) for long-chain and short-chain fatty acid analysis, high performance liquid chromatography (HPLC) is used. , The concentrations of lactic acid and acetic acid in each medium after culturing were measured. As the HPLC conditions, an Ascentis RP-amide column (100 × 30 mm, 3 μm, Sigma-Aldrich Japan) was used, and the mobile phase was phase A (0.01 M hydrochloric acid, pH 4.5): phase B (acetonitrile: methanol). = 2: 1) was 13: 7, and 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 culturing was calculated from the turbidity (absorbance of 650 nm) of each medium before and after culturing and the calibration curve prepared in advance.
As a specific method for preparing a calibration curve, after measuring the turbidity (absorbance at 650 nm) of the number of bifidobacteria in a solution, the bifidobacteria are anaerobically cultured on a BCP-added plate count agar medium at 37 ° C. for 72 hours. Then, the number of colonies was measured. Then, a calibration curve of the number of bifidobacteria (cfu / ml) and turbidity was prepared.

(Test results)
The concentration of organic acid in the medium (DMSO) to which no polyphenol was added was 3.46 mM for lactic acid and 4.86 mM for acetic acid. The concentration of organic acid in the medium (QUE) to which quercetin was added was 3.88 mM for lactic acid and 5.41 mM for acetic acid. The concentration of organic acid in the taxifolin-added medium (TAX) was 5.88 mM for lactic acid and 7.44 mM for acetic acid. The concentration of organic acid in the medium (PHL) to which phloretin was added was 4.91 mM for lactic acid and 6.26 mM for acetic acid. The concentration of organic acid in the medium (EGCG) to which epigallocatechin gallate was added was 5.25 mM for lactic acid and 7.81 mM for acetic acid.

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

As a result of the calculation of the number of bacteria of bifidobacteria in each medium before and after culture, cell count of bifidobacteria of each medium was approximately 10 8 ^cfu / ml. From this, it was confirmed that the number of bifidobacteria in the medium before and after the culture did not change.
Therefore, from the results of Test 1, it was clarified that quercetin, taxifolin, phloretin, and epigallocatechin gallate can improve the production efficiency of lactic acid and acetic acid by bifidobacteria without changing the number of bifidobacteria. It was.

<Test 2>
(Test method)
Cacao polyphenols were prepared as procyanidins.
Specifically, the 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 DMEM medium at 0.01%.

Cacao polyphenols were produced as follows.
First, 150 kg of cacao beans were heated at 80 ° C. for 1 hour. The heated cocoa beans were then degreased using an expeller to give 80 kg of cocoa powder. 800 kg of ethanol (50%) was added to the degreased cocoa powder and then heated. Then, after the temperature was reached to 50 ° C., the mixture was stirred and extracted for 1 hour, and then the extract was left at 4 ° C. overnight. The extract was filtered using a screw decanter and a filter press, the filtrate was concentrated under reduced pressure to 27 kg, and then 13 kg of ethanol (95%) was added. Then, this concentrated solution was stirred at 40 ° C. for 1 hour and then refrigerated overnight. The concentrate was centrifuged (25 ° C., 12000 rpm) using a Sharpless centrifuge. Then, 35 kg of acid clay (Mizuka Ace # 20) was added to the supernatant of this centrifugation, and the mixture was stirred for 1 hour. This was filtered using a Nutche, the filtrate was concentrated under reduced pressure and then lyophilized to give 6.3 kg of powder. Then, 25 L of resin and 50 L of water were added to 0.5 kg of this powder, and the mixture was stirred for 30 minutes or more. After recovering this resin, 75 L of ethanol (80%) was added and stirred for 20 to 30 minutes. This was filtered using Nutche, and the filtrate was concentrated under reduced pressure and then lyophilized to obtain cocoa polyphenols.

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

(Test results)
The concentration of organic acid in the medium (DMSO) to which no polyphenol was added was 5.70 mM for lactic acid and 11.80 mM for acetic acid. The concentration of organic acid in the medium (CBP) to which cacao polyphenol was added was 6.34 mM for lactic acid and 13.18 mM for acetic acid.

That is, the addition of cocoa polyphenols increased lactic acid by 11% and acetic acid by 12%.

As a result of the calculation of the number of bacteria of bifidobacteria in each medium before and after culture, cell count of bifidobacteria of each medium was approximately 10 8 ^cfu / ml. From this, it was confirmed that the number of bifidobacteria in the medium before and after the culture did not change.
Therefore, from the results of Test 2, it was clarified that cacao polyphenols can improve the production efficiency of lactic acid and acetic acid by bifidobacteria without changing the number of bifidobacteria.

<Test 3>
(Test method)
Four types of polyphenols were prepared: quercetin, taxifolin, phloretin, and epigallocatechin gallate.
Specifically, quercetin (Cayman Chemical Company, Inc.) was dissolved in DMSO (Dimethyl sulfoxide) to prepare 200 mM. In addition, taxifolin ((+)-Taxifolin 1036 Extrasynthese, Funakoshi Co., Ltd.) was dissolved in DMSO to prepare 400 mM. In addition, phloretin (Sigma-Aldrich Japan) was dissolved in DMSO to prepare 400 mM. In addition, Epigallocatechin gallate (Cayman Chemical Company, Inc.) was dissolved in DMSO to prepare 200 mM. Then, each DMSO solution of these prepared quercetin, taxifolin, floretin, and epigallocatechin gallate was added to DMEM medium (DMEM, Sigma-Aldrich Japan) at 0.01%. As a control, DMSO to which polyphenol was not added was added to DMEM medium at 0.01%.

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

(Test results)
The concentration of organic acid in the medium (DMSO) to which no polyphenol was added was 0.63 mM for acetic acid. The concentration of organic acid in the quercetin-added medium (QUE) was 0.61 mM for acetic acid. The concentration of the organic acid in the taxifolin-added medium (TAX) was 0.97 mM for acetic acid. The concentration of organic acid in the medium (PHL) to which phloretin was added was 0.72 mM for acetic acid. The concentration of organic acid in the medium (EGCG) to which epigallocatechin gallate was added was 0.90 mM for acetic acid.

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

As a result of calculating the number of lactic acid bacteria in each medium before and after culturing, the number of lactic acid bacteria in each medium was about 108 cfu / ml. From this, it was confirmed that the number of lactic acid bacteria in the medium before and after the culture did not change.
Therefore, from the results of Test 3, data Kishiforin, phloretin, and epigallocatechin gallate, without changing the number of bacteria of lactic acid bacteria, it has been found that can improve the production efficiency of acetic acid by lactic acid bacteria.

As described above, according to the organic acid production promoter of the present 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. Is. According to the preventive and / or ameliorating agent for inflammatory bowel disease of the present embodiment containing such an organic acid production promoter, the growth of harmful bacteria is suppressed in the intestine, and the intestinal flora is affected. Through the ameliorating effect, it is possible to obtain an excellent preventive effect and / or ameliorating effect on 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, in the above-mentioned embodiments and examples, quercetin, taxifolin, phloretin, epigallocatechin gallate, and procyanidin are used as polyphenols, but other polyphenols having the same effects as these are also used. Further, of course, as the bifidobacteria and lactic acid bacteria, microorganisms other than the strains shown in the embodiments may be used. Further, the second embodiment and the third embodiment may be combined to contain polyphenol, bifidobacteria and lactic acid bacteria as active ingredients.

The present invention can be suitably used for preventing and / or ameliorating diarrhea, constipation, inflammatory bowel disease and the like.

Claims (12)

To promote the production of lactic acid and / or acetic acid by Bifidobacterium adolescentis, which contains any one or more of quercetin, taxifolin, floretin and epigallocatechin gallate as an active ingredient. A production promoter for lactic acid and / or acetic acid
When the quercetin is contained, the content of the quercetin is 0.0001 to 50% by weight.
When the taxifolin is contained, the content of the taxifolin is 0.001 to 5.0% by weight.
When the phloretin is contained, the content of the phloretin is 0.0001 to 50% by weight.
When the epigallocatechin gallate is contained, the production promoter having an epigallocatechin gallate content of 0.0001 to 50% by weight. Containing Takishifori in as an active ingredient, a production promoter of lactic acid and / or acetic acid to promote the production of lactic acid and / or acetic acid by Bifidobacterium address Sen infantis (Bifidobacterium adolescentis),
The dosage per day for human pre Symbol taxifolin is a 0.1-100 mg, the production accelerator. The lactic acid and / or acetic acid production promoter according to claim 1 or 2, further comprising Bifidobacterium adolescentis. The production of lactic acid and / or acetic acid according to claim 3 , wherein the daily dose of Bifidobacterium adolescentis to humans is 2 × 10 ^{7 to} 5 × 10 ^10. Accelerator. Bifidobacterium ¹⁰⁷ or more per 1g of the culture of the Bifidobacterium address Sen infantis contained in production promoter of lactic acid and / or acetic acid according to claim 3 or 4 (Bifidobacterium adolescentis) - The lactic acid according to claim 3 or 4 , wherein when Bifidobacterium adolescentis is included, the daily dose of the culture of Bifidobacterium adolescentis to humans is 5 to 1000 g. And / or an acetic acid production promoter. The Bifidobacterium adolescentis is replaced by Bifidobacterium adolescentis JCM1275. ^T The lactic acid and / or acetic acid production promoter according to any one of claims 1 to 5. Production of acetic acid by Lactobacillus delbrueckii subsp. Bulgaricus, which contains one or more of taxifolin, floretin and epigallocatechin gallate as an active ingredient, to promote the production of acetic acid. It ’s an accelerator,
When the taxifolin is contained, the content of the taxifolin is 0.001 to 5.0% by weight.
When the phloretin is contained, the content of the phloretin is 0.0001 to 50% by weight.
When the epigallocatechin gallate is contained, the production promoter having an epigallocatechin gallate content of 0.0001 to 50% by weight. Containing Takishifori in as an active ingredient, a production promoter of acetic acid to promote the production of acetic acid by Lactobacillus delbrueckii bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus),
The dosage per day for human pre Symbol taxifolin is a 0.1-100 mg, the production accelerator. The acetic acid production promoter according to claim 7 or 8, further comprising Lactobacillus delbrueckii subsp. Bulgaricus. The promotion of acetic acid production according to claim 9 , wherein the daily dose of Lactobacillus delbrueckii subsp. Bulgaricus to humans is 2 × 10 ^{7 to} 5 × 10 ^10. Agent. The Lactobacillus delbrueckii bulgaricus contained in production promoter of acetic acid according to claim 9 or 10 (Lactobacillus delbrueckii subsp. Bulgaricus) 10 7 or more of Lactobacillus delbrueckii bulgaricus per 1g of a culture of When cas (Lactobacillus delbrueckii subsp. Bulgaricus) is included, the daily dose of the culture of Lactobacillus delbrueckii subsp. Bulgaricus to humans is 5 to 1000 g , claim 9 or 10. The acetic acid production promoter according to 10 . The Lactobacillus delbrueckii subsp. Bulgaricus is Lactobacillus delbrueckii subsp. Bulgaricus OLL1181 (FERM BP-11269), claim 7-11. The acetic acid production promoter described.

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