JP6675752B2 - Intestinal Blautia coccoides growth stimulant - Google Patents

Description

  The present invention relates to an intestinal Blautia coccoides growth stimulant containing glycosphingolipids.

  Many foods that are ingested orally are known to be directly digested and absorbed as nutrients such as carbohydrates, lipids, proteins, vitamins, and minerals, and as components that have pharmacological effects and functions. I have. Although involved in this digestion and absorption, there are intestinal bacteria resident in the human intestine, and these intestinal bacteria are known to have an important relationship with human health.

  Intestinal bacteria include a wide variety of bacteria, which are generally referred to as intestinal flora and their balance is considered to be important. It is known that this balance greatly varies depending on factors such as an individual, age, physical condition, nutritional status, and stress. Generally, useful microorganisms in the human digestive tract are also called good bacteria, and bifidobacteria, lactic acid bacteria, and the like are known as good bacteria.

  The regulation of intestinal flora, especially the growth of good bacteria, is affected by foods, and it is known that yogurt and other foods containing lactic acid bacteria, which are also good bacteria in the first place, have a function of regulating intestinal flora. Have been. However, even Japanese foods and the like, which are widely said to be good for health, have not yet been sufficiently studied on the effects of such diets on intestinal microflora.

  In recent years, in addition to bacteria generally known as good bacteria, the influence of Clostridium, which is confirmed to be present as intestinal bacteria, has begun to attract attention. For example, it is disclosed that direct ingestion of this bacterium has an effect of improving anxiety and hyperactivity (Patent Document 1). In addition, a technique relating to the proliferation of regulatory T cells by a bacterial composition containing a bacterium belonging to the genus Clostridium has been disclosed, and thereby a technique for treating or preventing an autoimmune disease, an inflammatory disease, an infectious disease, and the like has been disclosed. (Patent Document 2).

  On the other hand, glycosylceramide, which is a kind of glycosphingolipid, is extracted from plants, mushrooms and the like, is widely used as cosmetics for dry skin, and is reported to be effective in improving skin functions ( Patent Document 3). Furthermore, there is disclosed a method for improving the composition of intestinal microflora by administering a food containing an excess of sphingolipid to a bird or mammal (Patent Document 4).

JP 2013-119546 A JP-T-2015-500792 JP 06-145039 A JP 2006-516278 A

  Traditionally, Japanese foods are said to be good for health, but how they change the intestinal flora has not been thoroughly studied. On the other hand, Patent Document 4 discloses a method of improving intestinal microflora by ingesting sphingolipids, but here, many types of sphingolipids are not desired in the intestine. It is based on the finding that bacteria can be selectively killed while other bacteria remain virtually undisturbed.

  In other words, sphingolipids are considered to have only their growth inhibitory effect on specific bacteria, and by eliminating only bacteria corresponding to so-called bad bacteria, by maintaining other good bacteria, etc., relatively. It seeks to increase the composition and improve the intestinal flora. It is said to be used as a substitute for antibiotics and to suppress various symptoms (diarrhea, infection of the gastrointestinal tract, liver damage, intestinal cancer) due to harmful bacteria.

  Here, Blautia coccoides, which is a kind of intestinal bacteria, is expected to obtain various functions by directly ingesting, as disclosed in Patent Documents 1 and 2. However, there is no known technique for selectively growing this bacterium despite its presence in the intestinal flora.

  Under such circumstances, an object of the present invention is to provide a new regulator of intestinal bacterial flora. Here, it is an object of the present invention to provide a growth stimulating agent for intestinal Blautia coccoides, whose importance has begun to be particularly noticed. In addition, by stimulating the growth of intestinal Blautia coccoides, effects of stabilizing the mind and suppressing enteritis can be expected.

  The present inventors have selectively concentrated glycosphingolipids, particularly glycosylceramides contained in koji and the like, and orally ingested them to reduce intestinal bacterial flora, which was previously unknown, in intestinal bacterial flora. It was found that they proliferate selectively.

  The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that the following inventions meet the above objects, and have accomplished the present invention.

That is, the present invention relates to the following inventions.
<1> An intestinal Blautia coccoides growth stimulant containing glycosphingolipid.
<2> An anxiety reducing agent containing glycosphingolipid.
<3> A therapeutic agent for enteritis containing glycosphingolipid.
<4> The intestinal Blautia coccoides growth stimulating agent according to <1>, wherein the glycosphingolipid is glycosylceramide.
<5> The anxiety-reducing agent according to <2>, wherein the glycosphingolipid is glycosylceramide.
<6> The therapeutic agent for enteritis according to <3>, wherein the glycosphingolipid is glycosylceramide.

  According to the present invention, there is provided a growth stimulating agent for stimulating the growth of intestinal Blautia coccoides. In addition, anxiety-reducing agents and enteritis-suppressing agents are provided by growing intestinal Blautia coccoides.

It is a figure which shows the abundance ratio of Blautia coccoides in the gut microbiota of the mouse which fed the Blautia coccoides growth stimulant which concerns on this invention. 1 is a graph showing the effect of stimulating the growth of Blautia coccoides by a Blautia coccoides growth stimulant according to the present invention.

  Hereinafter, embodiments of the present invention will be described in detail. However, the description of constituent elements described below is an example (representative example) of an embodiment of the present invention, and the present invention is described below unless the gist is changed. It is not limited to the content of.

  The present invention relates to a composition having various functions containing glycosphingolipid. Here, the present invention relates to a growth stimulant for Blautia coccoides, an anxiety ameliorating agent, and a therapeutic agent for enteritis. Since all of the present invention relates to a composition containing glycosphingolipid, these are collectively referred to below as “the present invention. Glycosphingolipid-containing composition ".

[Glycosphingolipid]
The glycosphingolipid-containing composition of the present invention contains glycosphingolipid as its active ingredient. Glycosphingolipids are lipids having sphingoid bases, which are long-chain amino alcohols. As glycosphingolipids, galactosylceramide in which ceramide is bonded to galactose, glucosylceramide in which ceramide is bonded to glucose, and the like are known, and those generally called glycosylceramide can be used.

  As described above, the glycosphingolipid-containing composition of the present invention contains the glycosphingolipid. As this glycosphingolipid, besides those derived from Aspergillus, those derived from animals such as bovine brain, plants and microorganisms can be used. These glycosphingolipids of a plurality of origins may be used as a mixture, or may be used alone or in combination with other components as necessary. Examples of plant-derived glycosphingolipids include rice, corn, konjac, maitake, stalk mushroom, soybean, beet, and wheat-derived glycosphingolipids.

[Lipids derived from koji mold]
The glycosphingolipid-containing composition of the present invention can contain a glycosphingolipid derived from Aspergillus oryzae as the origin of the glycosphingolipid. Lipids derived from Aspergillus are mainly lipid components obtained by extracting lipid components from Aspergillus or removing components other than lipid such as water from Aspergillus. Specifically, the lipid component of the koji mold is extracted into the organic solvent by mixing the koji mold with an organic solvent such as alcohol, or the components are separated by crushing the cell membrane of the koji mold with a homogenizer or the like and centrifuging the mixture. The lipid component of Aspergillus is obtained by drying to remove water or by combining these steps. The lipid derived from Aspergillus contains glycosphingolipids such as glycosylceramide and the like. This koji-derived lipid is a lipid and, unlike koji mold and koji itself, does not need to be maintained alive or by freeze-drying or the like, and is hardly denatured even in a normal environment such as normal temperature or cold temperature, and has good storage stability. Are better. In addition, the lipid derived from Aspergillus can be obtained from a culture of Aspergillus, and the production efficiency is high.

[Koji mold]
Aspergillus is a bacterium used for koji. The koji is obtained by breeding bacteria such as rice, wheat, soybeans, etc. effective for food fermentation such as Aspergillus aspergillus described later. This koji is used when producing fermented foods such as sake, miso, vinegar, pickles, soy sauce, shochu, and awamori. Here, the koji mold of the present invention is not particularly limited as long as it has glycosphingolipid as an active ingredient. For example, koji mold belonging to the genus Aspergillus can be used. In the present invention, shochu lees and the like that have been treated as industrial waste containing these koji molds can be used, and by using as a raw material of the glycosphingolipid-containing composition of the present invention, the industrial waste containing this koji mold It is also useful as a processing method.

  More specifically, examples of Aspergillus include Aspergillus oryzae, Aspergillus oryzae, Aspergillus sojae, Aspergillus kawachii, Aspergillus sp., And Aspergillus sp. (Aspergillus awamori), Aspergillus nakazawai, Aspergillus usamii, Aspergillus luchensis, Aspergillus niger and the like.

  Further, the koji mold which can be used in the present invention is not limited to the koji mold belonging to the genus Aspergillus, and also includes other genera such as a bacterium belonging to the genus Rhizopus, a bacterium belonging to the genus Monuscus and a bacterium belonging to the genus Penicillium. be able to. Fungi belonging to these genera are exemplified below.

  Bacteria belonging to the genus Rhizopus: Rhizopus oryzae, Rhizopus oligosporus, Rhizopusniveus, Rhizopus microspores, Rhizopus Ronus, etc. , Fungi belonging to the genus Monascus: Monuscus anka, Monuscus purpureus, Monascus louver, Monuscus pilosus, Monuscus aurantiacus Fungi belonging to the genus Penicillium, such as Monascus kaoliang, Monicus kaoliang: Penicillium camemberti, Penicillium roqueforti, Penicillium roqueforti (Penicillium glaucum), such as Penicillium casei column (Penicillium caseicolum), in the present invention may be used in combination of Aspergillus singly or in combination. Among them, it is preferable to contain Aspergillus kawachi, which is widely used for fermentation of alcoholic beverages such as shochu.

[Blautia coccoides growth improving agent]
Blautia coccoides is a kind of bacteria present in the intestinal flora. By using the glycosphingolipid-containing composition of the present invention, intestinal Blautia coccoides can be prominently grown. Therefore, the intestinal flora can be prepared focusing on this bacterium.

[Anxiety improver]
The glycosphingolipid-containing composition of the present invention can be used as an anxiety-relieving agent to achieve the effects of improving anxiety and improving hyperactivity. The effect of this anxiety relieving agent is due to the proliferation of Blautia coccoides in the intestine. With respect to anxiety improvement and hyperactivity improvement, a developmental disorder called attention deficit / hyperactivity disorder is known. More specifically, there are symptoms such as maintenance of attention, abnormal sense of time, decreased information processing, and symptoms that may interfere with daily life due to overactivity and impulsivity. . Particularly in children, these symptoms are often seen, but some symptoms may be seen in adults. Heretofore, no specific treatment has been established for this symptom, but according to the present invention, an anxiolytic agent which suppresses these developmental disorders by ingesting the glycosphingolipid-containing composition of the present invention. Is provided.

[Enteritis treatment agent]
The glycosphingolipid-containing composition of the present invention can exhibit a therapeutic effect on enteritis when used as a therapeutic agent for enteritis. This therapeutic effect on enteritis is due to the proliferation of Blautia coccoides in the intestine. Regulatory T cells (Treg cells) are a kind of CD4-positive helper T cells and are said to have an immunosuppressive function and the like. Proliferating and accumulating Treg cells suppresses the immune response to self and secretes anti-inflammatory cytokines such as IL-10 and TGFβ to suppress excessive inflammation. Treg cells can be grown and accumulated by ingesting Blautia coccoides. Accumulation can be achieved in the intestine. Thereby, inflammation in digestive organs such as the intestine can be improved and suppressed.

  Therefore, the present invention can also be achieved as a use of a glycosphingolipid (preferably, glycosylceramide) for the production of a Blautia coccoides growth stimulating agent and any of anxiety ameliorating agents and therapeutic agents for enteritis.

  The glycosphingolipid-containing composition of the present invention preferably consumes 70 to 5,000 mg of a glycosphingolipid as an active ingredient per day for a human weighing 60 kg. In addition, humans can consume 1.2 to 80 mg / kg body weight per day. If the intake is lower than this, effects such as stimulation of intestinal growth of Blautia coccoides, improvement of anxiety, and treatment of enteritis may not be sufficiently exhibited. On the other hand, the glycosphingolipid of the present invention is low in toxicity and has little adverse effect due to excessive intake, but its effect is saturated even if it is excessively taken, so that the necessity of taking the upper limit amount or more is low. The daily intake of a human (adult) weighing 60 kg is more preferably 200 mg or more, and even more preferably 600 mg or more.

  The glycosphingolipid content in the composition of the glycosphingolipid-containing composition of the present invention is appropriately changed depending on the dosage form and the like, and is not particularly limited, but the content assumed as a dosage form for oral ingestion Is about 0.1 to 50% by weight.

  The glycosphingolipid-containing composition of the present invention may contain a glycosphingolipid to form a pharmaceutically acceptable solid capsule such as a soft capsule, a hard capsule, or a capsule-type tablet such as a compressed tablet, or a jelly. It can be suitably formulated into the form or a liquid form suitable for oral administration such as liquid. And it is orally administered as a formulation, and the oral dose can be controlled. Therefore, this intake amount is an amount to be ingested from a composition prepared as a composition, and can be set as an amount to be ingested separately from a normal meal, for example, as a tablet or a capsule. In addition, although glycosphingolipids are also contained in general foods, the amount ingested by a general diet may not be sufficient as an amount for achieving the effects of the present invention, and the amount of ingestion may be regarded as the concentration of the active ingredient. It is thought that there is a certain number of layers that are inadequate for normal food intake from these foods in management. Therefore, it is preferable to manage the intake amount as a sufficient amount using the glycosphingolipid-containing composition of the present invention.

  The glycosphingolipid-containing composition of the present invention can also be achieved as a functional food for stimulating intestinal Blautia coccoides growth, improving anxiety, and treating enteritis. The glycosphingolipid content in the composition of the glycosphingolipid-containing composition as this food is appropriately changed depending on the food, but can be about 0.1 to 50% by weight in each food. Foods may be of any kind, for example, bread, noodles and other flour and starch, chocolate and jelly, gummy and other confectionery, yogurt and cheese and other dairy products, fermented foods, Drinks, seasonings and the like can be mentioned. As a method of blending glycosphingolipid, it may be blended at the stage of the production process as appropriate according to various foods.

  Glycosphingolipids, which are active ingredients of the present invention, also include foods that have been eaten conventionally, and have low toxicity. Also, in the case of food forms containing glycosphingolipids, it is preferable that the concentration is adjusted to be higher than those generally contained so that an effective amount can be ingested efficiently. In addition, since glycosphingolipids have excellent palatability, they can be easily used as various foods suitable for continuous ingestion.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples unless the gist is changed.

[Example 1] Existence ratio of Blautia coccoides in intestinal flora By ingesting a glycosphingolipid-containing composition orally, a change in the abundance ratio of Blautia coccoides in the intestinal flora was evaluated.

[Evaluation item]
[Intestinal flora analysis]
The intestinal bacterial flora of the mouse was analyzed using a next-generation sequencer NGS “Miseq” (manufactured by Illumina).

[Sphingolipid (glycosylceramide)]
<Extraction of koji lipid components>
(1) A lipid component is extracted from dried koji (Aspergillus oryzae breeding, rice polishing rate 70%) with chloroform / methanol (1: 1), crudely purified by Bligh & Dyer method, dried and dried to form a koji lipid component I got When the koji lipid component was analyzed, at least “N-2′-hydroxy-3′-octadecanoyl-1-O-β-D-, which is glycosylceramide (a kind of glycosphingolipid) specific to koji mold, was analyzed. It was confirmed that glycosylceramides such as "glucopyranosyl-9-methyl-4,8-sphingadanin" were included. This koji lipid component is a lipid containing about 10% glycosylceramide.

[Glycosphingolipid-containing composition]
The koji lipid component (containing 10% of glycosylceramide) was prepared on the basis of a mouse feed "AIN-76" (a standard purified feed for nutrition research using mice and rats by the US National Nutrition Research Institute (AIN)). , And sucrose to prepare a glycosphingoid-containing composition having a yellow koji lipid component concentration of 1 wt%. This bait is hereinafter referred to as “GlcCer bait” (Kgc). Further, the yellow koji lipid component of the GlcCer bait was replaced with sucrose, and a bait containing no yellow koji lipid component was used as a “standard bait” (Nor) for comparison. Table 1 shows the more specific component ratios (% by weight).

^* Prepared based on AIN-76 composition.

[Glycosylceramide donation to mice]
"Experimental"
The experimental mice (C57BL / 6N) were divided into two groups of three each, and a group fed with the “GlcCer diet (Kgc)” (“Kgc group”) and a general mouse diet (“Standard diet” in Table 1) (Nor) ”) as a feeding group (“ Nor group ”). Each group was fed with each diet for one week, the genome was extracted from the feces, the 16S rRNA gene V1-V2 variable region of all bacteria was amplified by PCR, and the above-described next-generation genome analyzer (" Sequence analysis was performed using “Miseq” (manufactured by Illumina), and microflora data was obtained using a MiSeq Reporter 16S metagenomics system attached to the apparatus. For the cluster analysis, intestinal bacteria analysis software in the R environment of EMBL was used (see “http://enterotype.embl.de/enterotypes.html”).

  FIG. 1 shows the frequency of occurrence (relative value) of the abundance ratio of Blautia coccoides in feces of mice fed with the Blautia coccoides growth stimulant of the present invention (* p <0.05 (Student's t-test)).

  As a result of the analysis of the intestinal flora, it was confirmed that the gram-positive bacterial phylum was most present. Then, as shown in FIG. 1, the median value in the Kgc group was about 4.2%, and a significant increase in Blautia coccoides was found with respect to the median value in the Nor group of about 2.1%.

Example 2 Growth of Blautia coccoides in Koji Lipid Component-Containing Medium The effect of the presence of koji lipid components on the growth of Blautia coccoides was evaluated.

[Evaluation item]
[Turbidity (OD600)]
By measuring the turbidity of the fermentation test solution using a turbidity meter (“UV-1800” manufactured by Shimadzu Corporation: test wavelength 600 nm, use of quartz cell, 1 cm optical path length), the degree of bacterial propagation was measured. It was an index to be evaluated. Note that distilled water was used as a blank having a turbidity (OD600) of 0.

[Basic medium]
A mixture of 1.5 mL of YPD medium and 1.5 μL of sodium cholate (1.5% w / v) was used as a basic medium.

[Medium (A): Glycosylceramide-containing medium]
Further, 7.5 μL of glycosylceramide-containing ethanol (glycosylceramide concentration 4 μg / μL) was mixed with the above-mentioned basic medium to obtain a medium (A) (glycosylceramide-containing medium). As the glycosylceramide, the dried koji lipid component described in Example 1 was used.

[Medium (B): Control medium]
Further, in the medium for the control test, the same amount of ethanol was mixed with the basic medium instead of the glycosylceramide-containing ethanol in the medium (A) to obtain a medium (B).

[Culture test]
Blautia coccoides was ingested into the medium (A) or the medium (B) so that OD600 = 0.1, and anaerobically cultured at 37 ° C. under anaerobic conditions (Anelopack) for 24 hours. The results of the culture are shown in FIG. 2 (stimulation of growth of Blautia coccoides by addition of glycosylceramide).

  As shown in FIG. 2, in the group cultured in the medium (A) containing glycosylceramide, Blautia coccoides grew because OD600 increased.

  By using the glycosphingolipid-containing composition according to the present invention, a significant increase in Blautia coccoides in the intestinal microflora was confirmed, so that a more stable effect by Blautia coccoides than by direct oral ingestion of Blautia cocides can be obtained. It can be expected to improve anxiety and treat enteritis.

  According to the present invention, intestinal flora can be prepared. Specifically, it is used as an intestinal Blautia coccoides growth stimulating agent for selectively growing intestinal Blautia coccoides. In addition, by selectively growing intestinal Blautia coccoides, anxiety can be alleviated and enteritis can be treated.

Claims (1)

An intestinal Blautia coccoides growth stimulant comprising glycosylceramide which is a glycosphingolipid.