JP7587971B2 - Promotes the production of short-chain fatty acids in the intestines - Google Patents

Description

Article 30, paragraph 2 of the Patent Act applies. 1. Website address: http://nishinihon.jsbba.or.jp/presentation/2020%E5%B9%B4%E5%BA%A6%E6%94%AF%E9%83%A8%E9%96%A2%E9%80%A3%E8%A1%8C%E4%BA%8B/332-2.html http://nishinihon.jsbba.or. jp/presentation/2020%e5%b9%b4%e5%ba%a6%e6%94%af%e9%83%a8%e9%96%a2%e9%80%a3%e8%a1%8c%e4%ba%8b/abstract.html Website publication date: November 24, 2020

The present invention relates to a short-chain fatty acid production promoter that effectively promotes the production of short-chain fatty acids in the intestine.

When intestinal bacteria assimilate indigestible foods and polysaccharides derived from the diet, short-chain fatty acids such as propionic acid, butyric acid, and succinic acid are produced as metabolic products. Conventionally, it has been thought that short-chain fatty acids produced in the intestine are used as an energy source for the host, but in recent years, it has been revealed that they also contribute to preventing infection by pathogenic bacteria and improving allergic inflammation by enhancing the intestinal barrier function. Furthermore, it has recently been elucidated that short-chain fatty acids produced in the intestine also act as signal molecules mediated by G protein-coupled receptors (GPCRs), and GPR41, GPR43, GPR91, GPR109a, and Olfr78 have been identified as short-chain fatty acid receptors (see Non-Patent Documents 1 to 3).

The biological regulation functions of short-chain fatty acid receptors have also been elucidated. Among the short-chain fatty acid receptors, GPR41 and GPR43 are known to have a very high affinity for short-chain fatty acids and to express signal transduction effects even at low concentrations, and have been reported to be involved in promoting the secretion of PYY (peptide YY) and GLP-1 (glucagon-like peptide-1) in the intestine and controlling insulin secretion in the pancreas. It has also been reported that GPR43, which is expressed in sympathetic ganglia by short-chain fatty acids, activates the sympathetic nerves and promotes the secretion of noradrenaline (Non-Patent Document 4).

In this way, short-chain fatty acid receptors play a variety of biological regulatory roles, and enhancing signal transduction via short-chain fatty acid receptors is effective not only for maintaining health, but also for treating symptoms and diseases that can be prevented, treated, or improved by enhancing signal transduction via short-chain fatty acid receptors.

I Kimura et al., The gut microbiota suppresses insulin-mediated fat accumulation via the short-chain fatty acid receptor GPR43. Nature Communications. 4, 1829, 2013. Ikuo Kimura, Functional analysis of fatty acid receptors for drug discovery applications: Pharmacia. 50, 867-868, 2014. Jun Miyamoto et al., Fatty acid metabolism and intestinal environment: Anti-aging Medicine 14:030-035,2018 Miyamoto J et al., Nutritional Signaling via Free Acid Receptors. Int J Mol Sci. 17, 450, 2016.

Increasing the production of short-chain fatty acids in the intestine not only enhances signal transduction via short-chain fatty acid receptors in the intestine, but also increases the amount of short-chain fatty acids transferred into the blood, which can ultimately enhance signal transduction via short-chain fatty acid receptors throughout the body. Therefore, the present invention aims to provide a short-chain fatty acid production promoter that effectively promotes the production of short-chain fatty acids in the intestine.

The inventors conducted extensive research to solve the above problems and discovered that red koji or processed products thereof have the effect of effectively promoting the production of short-chain fatty acids in the intestines. The present invention was completed through further research based on this knowledge.

That is, the present invention provides the following aspects.
Item 1. A promoter for promoting the production of short-chain fatty acids in the intestines, comprising red koji or a processed product thereof.
Item 2. The short-chain fatty acid production promoter according to Item 1, wherein the red koji or a processed product thereof is produced using Monascus pilosus.
Item 3. The short-chain fatty acid production promoter according to Item 1 or 2, which is used to promote production in the intestine of at least one short-chain fatty acid selected from the group consisting of lactic acid, succinic acid, and butyric acid.
Item 4. The short-chain fatty acid production promoter according to any one of Items 1 to 3, which is used by being added to a food or drink, an internal medicine, an animal feed, or a pet food.
Item 5. A method for promoting intestinal production of short-chain fatty acids, comprising a step of having a person in need of promoting intestinal production of short-chain fatty acids ingest red koji or a processed product thereof.
Item 6. Use of red koji or a processed product thereof for the manufacture of an agent for promoting the production of short-chain fatty acids in the intestine.
Item 7. Red koji or a processed product thereof used to promote the production of short-chain fatty acids in the intestine.
Item 8. Non-therapeutic use of red yeast rice or a processed product thereof for promoting the production of short-chain fatty acids in the intestine.

The short-chain fatty acid production promoter of the present invention can promote the production of short-chain fatty acids, which are metabolic products of intestinal bacteria, in the intestine. The short-chain fatty acid production promoter of the present invention is expected to have effects such as preventing pathogenic infections and improving allergic inflammation by increasing the amount of short-chain fatty acids produced in the intestine and enhancing the intestinal barrier function. In addition, the short-chain fatty acid production promoter of the present invention can enhance signal transduction via short-chain fatty acid receptors by increasing the amount of short-chain fatty acids produced in the intestine, and is therefore expected to have effects such as improving homeostasis function, promoting secretion of PYY and GLP-1, regulating insulin secretion, activating sympathetic nerves, and preventing, treating, or improving symptoms and diseases in which signal transduction via short-chain fatty acid receptors is impaired.

The short-chain fatty acid production promoter of the present invention is used to promote the production of short-chain fatty acids in the intestine, and is characterized by containing red koji or a processed product thereof. The short-chain fatty acid production promoter of the present invention is described in detail below.

[Definition]
As used herein, the term "short-chain fatty acid production promoter" refers to an agent used to promote the production of short-chain fatty acids in the intestine.

[Red yeast rice or its processed products]
The short-chain fatty acid production promoter of the present invention contains red koji or a processed product thereof as an active ingredient. Red koji is koji made by fermenting cereals with red koji mold.

The red koji mold used in the production of the red koji used in the present invention may be any filamentous fungus belonging to the Monascaceae family. Examples of filamentous fungi belonging to the Monascaceae family include filamentous fungi belonging to the genera Monascus and Basipetospora, and preferably the genus Monascus.

Examples of filamentous fungi belonging to the genus Monascus include Monascus pilosus, Monascus purpureus, Monascus pubigerus, Monascus anka, Monascus serorubescens, Monascus rubellus, Monascus kaoliang, Monascus rubiginosus, Monascus major, Monascus ruber, Monascus paxii, Monascus fuliginosus, Monascus vitreus, Monascus barkeri, Monascus sp. sp.), and their variants or mutants. The red koji used in the present invention may be produced using one type of red koji mold, or may be produced by combining two or more types of red koji mold. Among these red koji molds, Monascus pilosus is preferred from the viewpoint of exerting a more excellent effect of promoting the production of short-chain fatty acids.

The type of grain used as the raw material for the red koji used in the present invention is not particularly limited, so long as it is edible and can be fermented by Red Koji mold, and examples of such grains include rice such as white rice and brown rice; wheat, barley, rye, oats, and other grains; soybeans (white soybeans, black soybeans, green soybeans, etc.), defatted soybeans, and legumes such as hypocotyls thereof. The red koji used in the present invention may be produced using one type of grain, or may be produced by combining two or more types of grains. Among these grains, rice is preferable, and white rice is more preferable.

Red koji can be produced by a conventional method using Red koji mold and grains. Specifically, Red koji can be produced by inoculating Red koji mold into grains that have been steamed (cooked or steamed) and allowing them to stand under aerobic conditions at about 20 to 40°C. Furthermore, the grains used as raw materials may be chopped or crushed as necessary before or after steaming.

In the present invention, the fermented red koji may be used as is, or a processed product of the fermented red koji may be used, which has been subjected to one or more of the following treatments: heating, drying, crushing, pulverization, grinding, extraction, etc.

Specific examples of processed red koji include dried products such as dried red koji, dried red koji powder, dried red koji fine granules, and red koji extract powder; red koji paste; and red koji extract (including concentrated extract). Among these processed red koji products, preferred are dried products, more preferred are dried red koji powder, dried red koji fine granules, and even more preferred are dried red koji powder. In addition, the red koji fungus and enzymes contained in the processed red koji product may be live or may maintain their activity, or may be inactivated by heat treatment or the like.

Commercially available processed red koji products can be used. For example, powdered dried rice red koji is sold under the product names "V. Red koji 3P-D" and "V. Red koji 3P-D20" (both manufactured by Kobayashi Value Support Co., Ltd.), and these commercially available products can be used in the present invention.

[Dosage form/form of use, etc.]
The short-chain fatty acid production promoter of the present invention can be provided in a desired dosage form by using red koji or a processed product thereof as an active ingredient, either as is or in combination with other edible ingredients. The dosage form of the fatty acid production promoter is not particularly limited, and may be any of solid, semi-solid, and liquid.

The short-chain fatty acid production promoter of the present invention is used for the purpose of promoting the production of short-chain fatty acids in the intestine, so it may be in any form that allows it to be transported into the intestine, and is preferably in a form that is taken orally orally administered, but may also be in a form that is administered enterally.

Specific examples of the forms of use of the short-chain fatty acid production promoter of the present invention include food and beverages, oral medicines (including oral quasi-drugs), feed, pet food, etc. Among these, food and beverages and oral medicines are preferred, and food and beverages are more preferred.

When the short-chain fatty acid production promoter of the present invention is used in food and beverages (i.e., when it is provided as a food and beverage for promoting the production of short-chain fatty acids), red koji or a processed product thereof may be prepared into a desired form, either as is or in combination with other food and beverage ingredients or additives. Examples of such food and beverages include general food and beverages, as well as foods with health claims (including foods for specified health uses, foods with nutrient functions, supplements, etc.) and foods for the sick. The form of these food and beverages is not particularly limited, but specific examples include supplements such as capsules (soft capsules, hard capsules), tablets, granules, powders, and jellies; beverages such as tea drinks, nutritional drinks, fruit juice drinks, carbonated drinks, and lactic acid drinks; and luxury items such as gummies, candies, and jellies. Among these food and beverages, supplements are preferred, and capsules, tablets, granules, and powders are more preferred.

When the short-chain fatty acid production promoter of the present invention is used in an oral medicine (including oral quasi-drugs), red koji or a processed product thereof may be prepared into a desired form either as is or in combination with other additives. Specific examples of such oral medicines include tablets, pills, powders, fine granules, granules, capsules (including hard capsules and soft capsules), lozenges, chewable tablets, drinks, extracts (including soft extracts, dry extracts, etc.), jellies, syrups, spirits, elixirs, liposome preparations, etc.

When the short-chain fatty acid production promoter of the present invention is used in feed or pet food, red koji or a processed product thereof may be prepared into a desired form either as is or in combination with other materials, additives, etc. Specific examples of feed include feeds fed to livestock such as cows, pigs, and sheep; and poultry such as chickens, quails, and ostriches. Specific examples of pet food include pet foods fed to dogs, cats, rabbits, hamsters, etc.

The content of red koji or a processed product thereof in the short-chain fatty acid production promoter of the present invention may be appropriately set based on the daily intake/administration amount of red koji or a processed product thereof, taking into consideration the physique, age, and symptoms of the subject, the level of short-chain fatty acid production in the intestine, the form of the short-chain fatty acid production promoter, etc., and may be, for example, 1 to 100% by weight.

[Use and dosage]
The short-chain fatty acid production promoter of the present invention is used for promoting the production of short-chain fatty acids in the intestine. In the present invention, the short-chain fatty acid is a short-chain fatty acid generated as a metabolic product of intestinal bacteria, and specifically includes short-chain fatty acids having 1 to 6 carbon atoms, such as lactic acid, succinic acid, butyric acid, isobutyric acid, acetic acid, propionic acid, formic acid, isovaleric acid, valeric acid, isocaproic acid, caproic acid, and pyruvic acid. The short-chain fatty acid production promoter of the present invention preferably includes short-chain fatty acids having 1 to 5 carbon atoms, more preferably short-chain fatty acids having 1 to 4 carbon atoms, and even more preferably, particularly preferably short-chain fatty acids having 2 to 4 carbon atoms. Among these short-chain fatty acids, the short-chain fatty acid production promoter of the present invention is particularly excellent in the effect of promoting the production of lactic acid, succinic acid, and butyric acid, and is therefore preferably used for promoting the production of at least one of lactic acid, succinic acid, and butyric acid.

In addition, an increase in the production of short-chain fatty acids in the intestine enhances the intestinal barrier function and is effective in preventing infection by pathogenic bacteria and improving allergic inflammation in the intestine, so the short-chain fatty acid production promoter of the present invention can be used for enhancing the intestinal barrier function; preventing infection by pathogenic bacteria via the intestine; improving allergic inflammation in the intestine, etc.

In addition, since a high-fat diet leads to a decrease in the amount of short-chain fatty acids produced in the intestine, the short-chain fatty acid production promoter of the present invention can also be used to suppress the decrease in the amount of short-chain fatty acid produced due to the intake of a high-fat diet. Here, a high-fat diet is a food in which the proportion of fat in the total intake energy (fat weight ratio) is 30% or more.

In addition, an increase in the production of short-chain fatty acids in the intestine not only enhances signal transduction via short-chain fatty acid receptors in the intestine, but also increases the amount of short-chain fatty acids transferred into the blood, which can result in enhanced signal transduction via short-chain fatty acid receptors throughout the body. Therefore, the short-chain fatty acid production promoter of the present invention can provide effects based on enhanced signal transduction via short-chain fatty acid receptors not only in the intestine but throughout the entire body.

For example, since enhancement of signal transduction via short-chain fatty acid receptors (GPR41, GPR43, GPR91, GPR109a, and Olfr78) can result in enhancement of homeostatic functions, the short-chain fatty acid production promoter of the present invention can be used for enhancing homeostatic functions; for preventing, treating, or improving symptoms or diseases in which signal transduction via short-chain fatty acid receptors is impaired, etc.

Since the enhancement of signal transduction via GPR41 and GPR43 promotes the secretion of PYY and GLP-1 in the intestine, the short-chain fatty acid production promoter of the present invention can also be used for promoting PYY secretion in the intestine; for preventing, treating or improving symptoms or diseases caused by reduced PYY secretion; for promoting GLP-1 secretion in the intestine; and for preventing, treating or improving symptoms or diseases caused by reduced GLP-1 secretion.

In addition, since signal transduction via GPR41 and GPR43 is also involved in regulating insulin secretion in the pancreas, the short-chain fatty acid production promoter of the present invention can also be used for regulating insulin secretion; for preventing, treating, or improving type 2 diabetes, etc.

Furthermore, since enhancement of signal transduction via GPR43 expressed in sympathetic ganglia activates sympathetic nerves and promotes the secretion of noradrenaline, the short-chain fatty acid production promoter of the present invention can also be used for activating sympathetic nerves; promoting the secretion of noradrenaline, etc.

In the short-chain fatty acid production promoter of the present invention, the daily intake/administration amount of red koji or a processed product thereof may be set according to the subject's physique, age, symptoms, the level of short-chain fatty acid production in the intestine, the purpose of use of the short-chain fatty acid production promoter, etc., but may be set to, for example, about 100 to 60,000 mg, preferably about 200 to 30,000 mg, calculated as the dry weight of red koji. Here, "red koji calculated as the dry weight" refers to the dry weight of unprocessed red koji when used, to the dry weight of red koji contained in the processed product when used as a processed product other than red koji extract and red koji extract powder, and to the dry weight of red koji used as the extraction raw material for red koji extract or red koji extract powder when used as the red koji extract or red koji extract powder.

The short-chain fatty acid production promoter of the present invention may be ingested or administered once a day or in multiple divided doses per day, but it is preferable to ingest or administer it once or in two or three divided doses per day.

The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these.

Test Example
1. Test materials and methods
1-1. Processed red koji As a processed red koji, a powder of dried red koji rice (product name "V. Red koji 3P-D20", manufactured by Kobayashi Value Support Co., Ltd.) was used. The powder of dried red koji rice is a dried powder of red koji made by fermenting white rice with Monascus pilosus, and the red koji fungus and enzymes are inactivated by heat treatment.

1-2. Measurement of the amount of succinic acid and lactic acid produced by enterobacteria Feces from 10-week-old male mice (C57BL/6) were inoculated onto a GAM agar medium using modified GAM bouillon (Nissui Pharmaceutical Co., Ltd.) to culture the enterobacteria. Next, one platinum loop of the enterobacteria cultured on the agar medium was inoculated into a test tube containing 3 ml of GAM liquid medium, and 1 ml of liquid paraffin was added to the upper layer of the medium, followed by anaerobic culture at 37°C for 24 hours to prepare an enterobacteria liquid.

30 μl of a 1.5 wt % aqueous solution of sodium cholate and the intestinal bacteria liquid prepared above (amount equivalent to an OD ₆₀₀ value of 0.1; i.e., 1.0×10 ⁷ cfu of intestinal bacteria) were added to a test tube, and GAM liquid medium was added so that the total volume in the test tube was 3 ml. Then, 40 mg of dried red rice koji powder (Example 1) or 40 mg of polished rice powder (Comparative Example 1) was added. Furthermore, 1 ml of liquid paraffin was added to the upper layer in the test tube, and anaerobic cultivation was performed at 37° C. for 48 hours. For comparison, cultivation was performed under the same conditions as above except that the dried red rice koji powder and polished rice powder were not added (Comparative Example 2).

After the culture was completed, the upper layer of liquid paraffin was discarded, and 500 μl of hexane was added to the test tube to remove any remaining liquid paraffin. Next, 1.5 ml of the contents of the test tube were collected and subjected to freeze-drying. 0.9 ml of distilled water and 0.5 ml of chloroform were added to the obtained freeze-dried product and stirred, and then 0.5 ml of chloroform was added and stirred. Next, centrifugation (2300 rpm, 15 minutes) was performed to collect the upper layer. 200 μl of the obtained upper layer, 60 μl of an aqueous solution containing 0.02 wt% ribitol (external standard), and 800 μl of distilled water were added to a microtube and freeze-dried. 300 μl of methoxyamine was added to the obtained freeze-dried product and incubated for 90 minutes while stirring at 1500 rpm to obtain a sample for gas chromatography mass spectrometry (GC/MS). The obtained sample was subjected to gas chromatography mass spectrometry to measure the succinic acid and lactic acid contents. For comparison, the amounts of succinic acid and lactic acid at the start of cultivation were also measured in the same way when dried rice red koji powder was added.

1-3. Measurement of butyric acid production by enterobacteria Feces from 10-week-old male mice (C57BL/6) were inoculated onto GAM agar medium to culture enterobacteria. Next, one platinum loop of the enterobacteria cultured on the agar medium was inoculated into a test tube containing 3 ml of YCFA liquid medium, and 1 ml of liquid paraffin was added to the upper layer of the medium, followed by anaerobic culture at 37°C for 48 hours (preculture).

30 mg (Example 2) or 100 mg (Example 3) of dried red koji powder and 5 ml of YCFA liquid medium were added to a vial, and then nitrogen was injected into the vial, the lid was closed, and the vial was autoclaved. Next, 50 μl of an aqueous vitamin solution (containing 0.2 wt% biotin, 0.2 wt% folic acid, 1 wt% pyridoxine hydrochloride, 0.5 wt% thiamine hydrochloride hydrate, 0.5 wt% riboflavin, 0.5 wt% nicotinic acid, 0.5 wt% calcium D-pantothenate, 0.01 wt% vitamin B12, 0.5 wt% p-aminobenzoic acid, and 0.5 wt% DL-α-lipoic acid) was added to the vial. Separately, the pre-cultured enterobacteria culture solution (amount equivalent to an _OD600 value of 0.1; i.e., 1.0 x ¹⁰⁷ cfu of enterobacteria) was centrifuged to remove the supernatant, and 50 to 100 μl of YCFA liquid medium was added to the resulting bacterial cells to prepare an enterobacteria solution. The entire amount of the resulting enterobacteria solution was added to the vial and cultured anaerobically at 37°C for 48 hours. For comparison, culture was performed under the same conditions as above, except that no powder of dried red rice koji was added (Comparative Example 3).

After the cultivation was completed, 1.5 ml of the contents of the vial was collected and freeze-dried. Using the obtained freeze-dried material, samples for GC/MS were prepared in the same manner as above, and subjected to gas chromatography mass spectrometry to measure the butyric acid content. For comparison, the amount of butyric acid at the start of cultivation was also measured in the same manner under conditions in which dried rice red koji powder was added.

2. Test results The results are shown in Tables 1 to 3. Tables 1 to 3 show the amount of each short-chain fatty acid produced, calculated as the ratio of the peak area of each short-chain fatty acid to the peak area of ribitol (external standard) measured by GC/MS. As a result, it was confirmed that the amounts of succinic acid, lactic acid, and butyric acid produced by intestinal bacteria increased in the presence of dried red rice koji powder, making it clear that rice koji and its processed products have the effect of promoting the production of short-chain fatty acids in the intestines.

Claims (4)

An agent that promotes the production of short-chain fatty acids in the intestines, containing red koji or processed products thereof. The short-chain fatty acid production promoter according to claim 1, wherein the red koji or a processed product thereof is red koji or a processed product thereof produced using Monascus pilosus. The short-chain fatty acid production promoter according to claim 1 or 2, which is used to promote the production in the intestine of at least one short-chain fatty acid selected from the group consisting of lactic acid, succinic acid, and butyric acid. The short-chain fatty acid production promoter according to any one of claims 1 to 3, which is used by being added to food and drink, oral medicine, feed, or pet food.