Intestinal flora improvement components

Abstract

The present invention aims to provide a food composition containing Lactobacillus mucosae as an active ingredient, which has the function of improving the intestinal microbiota, namely the function of increasing short-chain fatty acids and / or increasing diversity. Furthermore, it aims to construct a novel intestinal microbiota model that possesses the characteristics of the intestinal microbiota of Japanese people. [Solution] The present invention provides a composition for improving the intestinal microbiota, comprising Lactobacillus mucosae cells or bacterial cultures as an active ingredient. The present invention also provides a human intestinal microbiota model for evaluating the effect of a target food composition on increasing short-chain fatty acids and / or increasing diversity in the human intestine, the human intestinal microbiota model comprising an evaluation bacterial culture containing at least nine or more bacterial species, which does not include bacteria belonging to the phylum Proteobacteria, genus Lactobacillus, but includes bacteria belonging to the genus Bifidobacterium.

Description

Technical Field

[0001] The present invention relates to a food composition for improving intestinal bacteria, containing Lactobacillus mucosae as an active ingredient. Furthermore, the present invention relates to a method for evaluating a food composition suitable for improving the intestinal flora.

Background Art

[0002] There are 1,000 species and 40 trillion bacteria inhabiting the human intestine, forming a complex symbiotic relationship (intestinal flora). With the progress of omics analysis, in addition to the fact that health status, aging, diet, and lifestyle habits affect the intestinal flora, it has become clear that the disruption of the intestinal flora balance (the diversity of intestinal bacteria) leads to the onset and exacerbation of diseases. It has also been demonstrated that the diversity of intestinal bacteria decreases in obese and diabetic patients, and that the intake of dietary fibers increases the diversity of intestinal bacteria and improves related clinical symptoms.

[0003] In addition, various health functional effects have been reported for short-chain fatty acids, which are the main metabolites of intestinal bacteria, and their relationship with the diversity of intestinal bacteria has also been suggested. Short-chain fatty acids refer to fatty acids having 6 or less carbon atoms, represented by acetic acid, propionic acid, and butyric acid. These not only suppress the growth of pathogenic microorganisms by making the pH in the intestine acidic, but also activate intestinal epithelial cells and are intricately involved in the host's biological defense and immune system control. It has also been reported that they are utilized by intestinal bacteria that produce metabolites that promote the peristaltic movement of the intestinal tract and intestinal bacteria related to maintaining the homeostasis of the intestinal mucosa. As methods for increasing the diversity of intestinal bacteria that make up the intestinal flora and short-chain fatty acids in the intestinal flora, the main methods include ingesting a food composition (prebiotics) that promotes the growth and metabolite production of intestinal bacteria, and directly ingesting a food composition (probiotics) containing lactic acid bacteria and bifidobacteria.

[0004] The human gut microbiota, composed of a wide variety of intestinal bacteria, differs significantly from that of model animals such as mice, where Lactobacillus is the dominant bacterium in the gut. Furthermore, it is known that the gut microbiota varies greatly among individuals, and that the gut microbiota of Japanese people is more distinctive than that of people from other nationalities (Non-Patent Literature 1). Given the current lack of a model evaluation system to standardize the variability of gut microbiota among individuals and races, selecting food compositions that increase the diversity of intestinal bacteria constituting the gut microbiota and the amount of short-chain fatty acids in the gut microbiota is extremely difficult. While several in vitro evaluation systems have been reported that aim for standardization by artificially reconstructing the gut microbiota by extracting multiple intestinal bacteria from the gut microbiota (Non-Patent Literature 2, Non-Patent Literature 3), no systems that artificially reconstruct the gut microbiota of Japanese people have been reported. Furthermore, there are no evaluation systems that have been constructed with the aim of selecting food compositions that increase the diversity of intestinal bacteria constituting the gut microbiota or that increase short-chain fatty acids in the gut microbiota.

[0005] Non-Patent Literature 1 describes the characteristics of the Japanese gut microbiota through metagenomic analysis, reporting 50 dominant gut bacteria species and indicating that the Japanese gut microbiota is most abundant in the Japanese gut compared to the gut microbiota of foreign subjects from 11 countries. It also states that the Japanese gut microbiota is most rich in the genera Blautia, Bifidobacterium, Cholincera, and Streptococcus. Furthermore, the above 50 species do not include the phyla Proteobacteria or Lactobacillus, indicating that these gut bacteria are not dominant in the Japanese gut. In addition, metagenomic data from 106 individuals collected in Non-Patent Literature 1 revealed that the Japanese gut microbiota contains approximately 57%, 24%, and 19% of gut bacteria belonging to the phyla Firmicutes, Actinobacteria, and Bacteroidetes, respectively. However, cultivation of these dominant gut bacteria in the Japanese gut microbiota has not been carried out, and cultivable combinations and cultivation methods have not been investigated.

[0006] Regarding the number of bacterial species to extract, Non-Patent Literature 2 reports a combination of eight intestinal bacteria called ASF (Altered Schaedler Flora) as the smallest unit model of the gut microbiota. Non-Patent Literature 3 also reports a combination of 15 intestinal bacteria as a model that has characteristics of the gut microbiota of Europeans. However, this model includes intestinal bacteria that are not dominant in the Japanese gut microbiota, and does not include the Bifidobacterium genus, which is characteristic of the Japanese gut microbiota.

[0007] Intestinal bacteria have a high anaerobic requirement compared to lactic acid bacteria and bifidobacteria, making them extremely difficult to culture. Therefore, it is common practice to culture them in a medium tailored to the characteristics of each type of intestinal bacteria. Non-patent document 4 reports that it is possible to culture several types of intestinal bacteria, which have been reported to be dominant in the gut microbiota of Europeans, in a single medium (GAM broth medium, Nissui). However, as mentioned above, the dominant bacteria in the gut differ significantly between Europeans and Japanese, and it cannot be expected that this medium will stably cultivate the dominant gut bacteria of Japanese people as a single medium. Furthermore, while the short-chain fatty acids produced by each bacterial species are compared, only the production of a single type of intestinal bacterium is measured, and the total amount of short-chain fatty acids produced by the entire bacterial community remains unknown.

[0008] Patent Document 1 reports a method and apparatus for simulating the cultivation of gut microbiota, which can cultivate the gut microbiota while maintaining almost the compositional balance of the gut microbiota. Non-Patent Document 5 evaluates in vitro that dietary fiber increases short-chain fatty acids in the gut microbiota using a similar method and apparatus. Both Patent Document 1 and Non-Patent Document 5 are characterized by the use of feces as the culture material for evaluation, but the human gut microbiota varies greatly from person to person, and when using human-derived feces, it is affected by the differences in the gut microbiota of the individuals providing the feces. Therefore, the selection of food compositions that increase the diversity of gut bacteria constituting the gut microbiota and short-chain fatty acids in the gut microbiota by using feces is limited, and there has been a need to construct an evaluation system that does not use feces. Furthermore, there has been a need for gut microbiota improving compositions that promote the diversity of the gut microbiota and the increase in short-chain fatty acid production, which can be selected using such an evaluation system. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Patent No. 6482135 [Non-patent literature]

[0010] [Non-Patent Document 1] DNA Res. 2016 Apr;23(2):125-33. [Non-Patent Document 2] Appl Environ Microbiol. 1999 Aug;65(8):3287-92. [Non-Patent Document 3] Sci Transl Med. 2011 Oct 26;3(106):106ra106. [Non-Patent Document 4] Biosci Biotechnol Biochem. 2017 Oct;81(10):2009-2017. [Non-Patent Document 5] Sci Rep. 2018 Jan 11;8(1):435. [Overview of the project] [Problems that the invention aims to solve]

[0011] The present invention aims to provide a food composition and food / beverage containing Lactobacillus mucosae as an active ingredient, which has a function of improving the intestinal flora, namely a function of increasing short-chain fatty acids and / or increasing diversity. Furthermore, the objectives are to construct a novel gut microbiota model that possesses the characteristics of the Japanese gut microbiota, to provide a new model evaluation system that can assess the gut microbiota improvement function using this model, namely the function of increasing short-chain fatty acids and / or increasing diversity, and to provide a screening method for food compositions using this system. Furthermore, another objective is to provide food and beverages that have a function to improve the gut microbiota, namely a function to increase short-chain fatty acids and / or increase diversity, using food compositions obtained by the aforementioned new evaluation and screening methods. [Means for solving the problem]

[0012] The present invention aims to solve the above-mentioned problems by extracting multiple intestinal bacteria that are dominant in the intestinal microbiota of Japanese people and artificially reconstructing the Japanese intestinal microbiota. In this process, the intestinal microbiota was extracted while maintaining the taxonomic proportions in the intestines and including intestinal bacteria that show a high abundance compared to foreigners, which were considered characteristics of the Japanese intestinal microbiota. Then, the mixture of extracted intestinal bacteria was cultured together with indigestible dietary fiber, which has already been reported to increase short-chain fatty acids in the human intestine when ingested, and a reproducible increase in short-chain fatty acids was confirmed. From this, it was demonstrated that the present invention is effective as a model evaluation system for the Japanese intestinal microbiota to standardize the variability of intestinal microbiota among individuals, and it was also found that it can be used to evaluate the increase in short-chain fatty acids in the Japanese intestinal microbiota. Similarly, it was found that this model evaluation system can also be used to evaluate the increase in the diversity of intestinal bacteria that constitute the intestinal microbiota. Furthermore, using the aforementioned model evaluation system for the Japanese gut microbiota, the present invention selected various lactic acid bacteria and bifidobacteria as food compositions having functions to increase short-chain fatty acids and increase diversity. As a result, it was confirmed that Lactobacillus mucosae possesses these functions. The short-chain fatty acid increase-promoting effect and diversity-promoting effect of Lactobacillus mucosae were previously unknown, and it has become clear that it can be used for promoting the increase of short-chain fatty acids and increasing the diversity of the Japanese gut microbiota.

[0013] In other words, the present invention has the following configuration. <1> A composition for improving the intestinal microbiota, comprising Lactobacillus mucosae cells or cultures of Lactobacillus mucosae as an active ingredient. <2> Food and beverages for improving the intestinal flora, containing Lactobacillus mucosae cells or cultures as the active ingredient. <3> A composition for promoting the increase of short-chain fatty acids, comprising Lactobacillus mucosae cells or cell cultures as an active ingredient. <4> Food and beverages for promoting the increase of short-chain fatty acids, containing Lactobacillus mucosae cells or cultures as the active ingredient. <5> A composition for promoting increased diversity of intestinal bacteria, comprising Lactobacillus mucosae cells or cultures of Lactobacillus mucosae as an active ingredient. <6> Food and beverages for promoting increased diversity of intestinal bacteria, containing Lactobacillus mucosae cells or cultures as the active ingredient. <7> A human gut microbiota model for evaluating the effect of a target food composition on increasing and / or increasing the diversity of short-chain fatty acids in the human gut, The human gut microbiota model is comprised of an evaluation culture containing at least nine bacterial species, excluding bacteria belonging to the phylum Proteobacteria and genus Lactobacillus, but including bacteria belonging to the genus Bifidobacterium. <8> The nine or more bacterial species mentioned above are bacteria belonging to the phylum Firmicutes, phylum Actinobacteria, or phylum Bacteroidetes, and include bacteria belonging to the genera Brautia, Cholincera, or Streptococcus. <7> A human gut microbiota model described below. <9> Bacteria belonging to the phylum Firmicutes account for 56-59% of the species, those belonging to the phylum Actinobacteria account for 21-24%, and those belonging to the phylum Bacteroidetes account for 17-22%. <8> A human gut microbiota model described below. <10> At least 9 bacterial species were found, including Blautia wexlerae, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Eubacterium rectore, Bifidobacterium adolescentis, Cholincera aerofasciens, Bacteroides uniformis, Dorea longicatena, Bacteroides bulgatus, Ruminococcus gunavas, Faecalibacterium prausnitzi, Parabacteroides distasonis, Dorea formisigenerans, Ruminococcus obeum, Ruminococcus troques, Bacteroides dreyi, Flavonifracter prauti, Parabacteroides meldae, Rosebria inulinivorans, Clostridium nexile, Streptococcus salivarius, Egasella lenta, and Clostridium. Selected from the group consisting of Volteae, Rosebria intestinalis, Coprococcus comes, Bacteroides obatus, Eubacterium harii, Rosebria hominis, and Bacteroides tetaiotaomicron. <7> ~ <9> A human gut microbiota model described in any of the following. <11> At least nine bacterial species are identified as Brautia wexlerae, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Eubacterium rectore, Bifidobacterium adolescentis, Chorinthera aerofasiens, Bacteroides uniformis, Drea longicatena, Bacteroides bulgatus, Ruminococcus gunavas, Faecalibacterium prausnitzii, Brautia obeum, Bacteroides dreyi, Flavonifracter prauti, Streptococcus salivarius, Clostridium volteae, and Coprococcus comes. <7> ~ <10> A human gut microbiota model described in any of the following. <12>A method for culturing a human gut microbiota model for evaluating the promoting effect of increasing short-chain fatty acids and / or the promoting effect of increasing diversity in the human gut of a target food composition, the culturing method comprising the following steps. (1) A step of adding at least 9 bacterial species including bacteria belonging to the genus Bifidobacterium but not including bacteria belonging to the phylum Proteobacteria and the genus Lactobacillus to a modified GAM broth medium (2) A step of culturing under anaerobic conditions <13>A method for evaluating the promoting effect of increasing short-chain fatty acids and / or the promoting effect of increasing diversity in the human gut of a target food composition, the evaluation method comprising the following steps. (1) A step of adding a food composition to be evaluated to the human gut microbiota model according to any one of <7> to <11> and culturing (2) A step of measuring the amount of short-chain fatty acid production and / or calculating a diversity index of the culture obtained in (1) (3) A step of evaluating that there is a promoting effect of increasing short-chain fatty acids and / or a promoting effect of increasing diversity when the measured value and / or calculated value in (2) is 1.0 times or more the relative value compared to the case where the food composition is not added <14>A method for producing a food or drink for improving gut microbiota, the method comprising producing a food using a food composition evaluated to have a promoting effect of increasing short-chain fatty acids and / or a promoting effect of increasing diversity in the evaluation method according to <13>. <15>A strain belonging to Lactobacillus mucosae, selected from the group consisting of strain SBT10028 (NITE BP-03275), strain SBT10217 (NITE P-03276), strain SBT10027 (NITE P-03274), strain SBT10038 (NITE P-03283), strain SBT2261 (NITE P-03272), strain SBT2027 (NITE P-03271) and strain SBT2271 (NITE P-03273) selected strain.

Advantages of the Invention

[0014] The Japanese gut microbiota model evaluation system of the present invention is characterized by being composed of gut bacteria that are predominantly present in the intestines of Japanese people, maintaining the taxonomic proportions of gut bacteria that constitute the Japanese gut microbiota, and including gut bacteria that show a higher abundance compared to foreigners. Therefore, by using this model evaluation system, the gut environment improvement function of various food compositions can be evaluated. Furthermore, the present invention makes it possible to select food compositions that improve the intestinal microbiota without using humans or animals, and without using human feces, which are biological samples. Furthermore, by ingesting the intestinal microbiota improvement composition containing Lactobacillus mucosae, discovered by the present invention, it is expected that the diversity of intestinal bacteria constituting the intestinal microbiota and short-chain fatty acids in the intestinal microbiota will increase, and in addition, improvements in associated metabolic diseases and obesity will be expected. [Modes for carrying out the invention]

[0015] (Lactobacillus mucosae) In the present invention, Lactobacillus mucosae refers to bacteria belonging to the Lactobacillus mucosae phylum. Specifically, it refers to a bacterial strain whose 16S ribosomal RNA gene sequence homology to the Lactobacillus mucosae reference strain JCM12515 is 97% or higher, more preferably 98% or higher, and even more preferably 99% or higher. The isolation source may be any, but is more preferably human-derived. The Lactobacillus mucosae strain used as the active ingredient in the present invention can be any strain that increases the diversity of intestinal bacteria constituting the intestinal flora and short-chain fatty acids in the intestinal flora. Examples include SBT10028, SBT10043, SBT10217, SBT10027, SBT10038, SBT2261, SBT2027, and SBT2271, which have shown activity in the examples described later.

[0016] The compositions for improving the gut microbiota, promoting the increase of short-chain fatty acids, or promoting the increase of diversity in the human gut microbiota as described herein, as well as the active ingredients of the foods and beverages for improving the gut microbiota, foods and beverages for promoting the increase of short-chain fatty acids, and foods and beverages for promoting the increase of diversity, Lactobacillus mucosae, may be in any state that has short-chain fatty acid increase-promoting activity or diversity-promoting activity in the human gut, and are not limited to purely isolated bacterial cells, but may also be bacterial cell cultures, bacterial cell suspensions, or other bacterial cell-containing materials. The bacterial cells may be live or dead, of which live cells are preferred. Examples of live bacterial cells include bacterial cell concentrates obtained by culturing and collecting bacterial cells, dried bacterial cells, and freeze-dried bacterial cells. Examples of bacterial cell cultures include concentrated, dried, and freeze-dried bacterial cell culture solutions, as well as fermented dairy products containing Lactobacillus mucosae. Examples of fermented dairy products include cheese, fermented milk, dairy products, and lactic acid bacteria beverages, but are not particularly limited. Lactobacillus mucosae can be cultured according to conventional methods. Various culture media can be used, including milk media, media containing milk components, and semi-synthetic media that do not contain milk components. Examples of such media include reduced skim milk media.

[0017] (Fermented dairy products) In this specification, fermented dairy products refer to a liquid or paste containing milk or an equivalent or greater amount of nonfat milk solids in which Lactobacillus mucosae has been cultured. The milk can be raw milk, cow's milk, raw goat's milk, raw sheep's milk, etc., and skim milk powder may also be used after being reconstituted with water. When using skim milk powder, the concentration is not limited as long as the nonfat solids are equivalent to or greater than those of milk and the concentration is sufficient to culture Lactobacillus mucosae, but it is desirable that it be 8% (w / w) or more, preferably 9% (w / w) or more, and more preferably 10% (w / w) or more.

[0018] (Ingredients other than skim milk powder) The fermented milk product may contain nutrients other than skim milk powder, and if yeast extract is added, it is desirable that the amount be 0.1% (w / w) or more, preferably 0.2% (w / w) or more, and more preferably 0.5% (w / w) or more. Other nutrients include sugars, amino acids, minerals, and vitamins. The fermented milk product of the present invention may contain bacteria other than Lactobacillus mucosae. For example, as a fermentation starter for fermented milk, commonly used lactic acid bacteria, such as Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus, may also be included.

[0019] (Number of viable Lactobacillus mucosae bacteria in fermented dairy products) Regarding Lactobacillus mucosae in fermented dairy products, it is desirable that they be included as live bacteria. In this specification, "live bacteria" refers to the formation of colonies on a solid culture medium containing nutrients on a support such as agar (colony forming), and the number of live bacteria refers to the colony-forming units (cfu). c olony f orming u It is expressed as nit. Examples of solid media for measuring the number of viable Lactobacillus mucosae include MRS agar medium, which is a medium for lactic acid bacteria, and LBS agar medium, which is a medium for lactobacilli. The number of viable Lactobacillus mucosae in fermented dairy products is preferably 1.0E+06 cfu / mL or more, more preferably 1.0E+07 cfu / mL or more, and most preferably 1.0E+08 cfu / mL or more.

[0020] (Effect of promoting the increase of short-chain fatty acids) In this specification, "promotion of short-chain fatty acid increase" refers to promoting the increase of short-chain fatty acids in the human gut. Short-chain fatty acid increase-promoting activity is defined as the activity occurring when, in a gut microbiota model having characteristics of the Japanese gut microbiota, the increase in short-chain fatty acids is greater when the food composition is added compared to when it is not. For example, in the gut microbiota model described later, if the concentration of short-chain fatty acids obtained when cultured without the addition of bacteria such as lactic acid bacteria and bifidobacteria, and indigestible dietary fiber, is set to 1.0 times, then this activity is defined as the activity occurring when the concentration of short-chain fatty acids obtained when bacteria such as lactic acid bacteria and bifidobacteria, and indigestible dietary fiber are added is greater than 1.0 times. In this invention, a food composition having this activity is referred to as a short-chain fatty acid increase-promoting composition. The Lactobacillus mucosae strain used as the active ingredient in the short-chain fatty acid increase promoting composition of the present invention is preferably a strain that, when the short-chain fatty acid concentration obtained when Lactobacillus mucosae is added is set to 1.0 times, the short-chain fatty acid concentration obtained when Lactobacillus mucosae is added is 1.1 times or more, more preferably 1.5 times or more, and even more preferably 2.0 times or more. In the examples described later, it has been confirmed that both butyric acid and total short-chain fatty acids are greater than 1.0 times when Lactobacillus mucosae is added compared to when it is not added.

[0021] Here, short-chain fatty acids include acetic acid, butyric acid, propionic acid, or total short-chain fatty acids (the sum of these), and among these, it is preferable to evaluate butyric acid, which is said to succinctly indicate changes in the intestinal environment. Butyric acid is known to have higher proliferative activity of intestinal epithelial cells compared to acetic acid and propionic acid, and a decrease in the gut microbiota and a decrease in butyric acid-producing gut bacteria have been reported in various diseases. In patients with inflammatory bowel diseases such as ulcerative colitis, it has been reported that *Faecalibacterium prausnitzii*, a representative butyric acid-producing gut bacterium, decreases and increases with the exacerbation and remission of symptoms.

[0022] (Promotes increased diversity) In this specification, "promoting increased diversity" refers to promoting increased diversity in the human gut microbiota. Indices commonly used to represent the diversity of gut bacteria constituting the gut microbiota include the Shannon index, which indicates the degree of species equality, and the Phylogenetic diversity index, which takes phylogenetic information into account.

[0023] The activity that promotes increased diversity is described as occurring when, in a gut microbiota model with characteristics of the Japanese gut microbiota, the index representing the diversity of the gut microbiota is higher when the food composition is added compared to when it is not. For example, in the gut microbiota model described later, if the index representing diversity obtained when cultured without adding bacteria such as lactic acid bacteria and bifidobacteria, and indigestible dietary fiber, is set to 1.0, then the activity is described as occurring when the index representing diversity obtained when bacteria such as lactic acid bacteria and bifidobacteria, and indigestible dietary fiber are added is greater than 1.0. In the present invention, a composition having this activity is referred to as a composition that promotes increased diversity. The Lactobacillus mucosae used as the active ingredient in the diversity-enhancing composition of the present invention is preferably a strain that, when the diversity index obtained when Lactobacillus mucosae is added is set to 1.0, the diversity index obtained when Lactobacillus mucosae is added is 1.1 times or more, more preferably 1.2 times or more, and even more preferably 1.3 times or more. In the present invention, if a composition possesses either the activity of promoting the increase of short-chain fatty acids or the activity of promoting the increase of diversity, or both of these activities, it is said to have activity that improves the intestinal microbiota. Therefore, compositions for promoting the increase of short-chain fatty acids and compositions for promoting the increase of diversity are also compositions for improving the intestinal microbiota. Similarly, foods and beverages for promoting the increase of short-chain fatty acids and foods and beverages for promoting the increase of diversity are also foods and beverages for improving the intestinal microbiota.

[0024] (Compositions for improving the gut microbiota in the human gut, compositions for promoting the increase of short-chain fatty acids, and compositions for promoting the increase of diversity) As described above, the active ingredients of the compositions for improving the intestinal microbiota, promoting the increase of short-chain fatty acids, and promoting the increase of diversity in the human intestinal microbiota according to the present invention can be the fermented culture of Lactobacillus mucosae itself or the bacterial cells of Lactobacillus mucosae themselves. The fermented culture is preferably the fermented dairy product mentioned above. The compositions for improving the gut microbiota, promoting the increase of short-chain fatty acids, and promoting the increase of diversity can also be further formulated and used. When formulating, excipients, stabilizers, flavoring agents, etc., approved for pharmaceutical use can be appropriately mixed in. Possible dosage forms include tablets, capsules, granules, powders, syrups, etc. These include pharmaceutical preparations and supplements. Oral intake of these is expected to improve the gut microbiota through its effect of promoting the increase of short-chain fatty acids and / or promoting the increase of diversity in the human gut microbiota.

[0025] (Foods and beverages for improving the gut microbiota in the human gut, foods and beverages for promoting the increase of short-chain fatty acids, and foods and beverages for promoting the increase of diversity) The active ingredients in the food and beverages for improving the human gut microbiota, promoting the increase of short-chain fatty acids, and promoting the increase of diversity in the human gut microbiota according to the present invention can be the fermented culture of Lactobacillus mucosae itself or the bacterial cells of Lactobacillus mucosae themselves. Examples of fermented cultures used as food products include the fermented dairy products mentioned above. Furthermore, the foods and beverages for improving the gut microbiota, promoting the increase of short-chain fatty acids, and promoting diversity can also be obtained by incorporating the gut microbiota-improving composition, the short-chain fatty acid-promoting composition, and the diversity-promoting composition into appropriate foods and beverages. These formulations may be added to the raw materials during the manufacturing process of the foods and beverages, incorporated into the ingredients, or incorporated into the final food and beverage product. Examples of food and beverages include, but are not limited to, cheese, fermented milk, dairy products, lactic acid bacteria beverages, lactic acid bacteria beverages, dairy products such as butter and margarine, milk beverages, fruit juices and soft drinks, egg products such as jelly, candy, pudding and mayonnaise, sweets and bread such as butter cakes, and various types of powdered milk, as well as infant foods and nutritional compositions. Furthermore, the food and beverages for improving the intestinal flora, the food and beverages for promoting the increase of short-chain fatty acids, and the food and beverages for promoting the increase of diversity of the present invention can also be used as functional foods, foods for specified health uses, nutritional functional foods, and beauty foods. By consuming these foods and beverages, improvement of the gut microbiota is expected through the promotion of short-chain fatty acid increase and / or increased diversity in the human gut microbiota.

[0026] When manufacturing compositions for improving the intestinal flora in the human gut microbiota, compositions for promoting the increase of short-chain fatty acids, and compositions for promoting the increase of diversity, as well as foods and beverages for improving the intestinal flora in the human gut microbiota, foods and beverages for promoting the increase of short-chain fatty acids, and foods and beverages for promoting the increase of diversity, by incorporating Lactobacillus mucosae cells and / or cultures, the mixing ratio is not particularly limited and should be adjusted as appropriate according to the ease of manufacture and the preferred daily dose. The amount should be determined individually considering the symptoms and age of the recipient, but for adults, it is usually sufficient to adjust the amount so that 10 to 200 g of Lactobacillus mucosae cell culture or 0.1 to 100 mg of the cells themselves can be ingested.

[0027] (Gut microbiota model) The gut microbiota model of the present invention is a mixed bacterial culture that models the average gut microbiota of Japanese people, and is characterized by containing at least nine bacterial species, including bacteria belonging to the Bifidobacterium genus, but not bacteria belonging to the Proteobacteria phylum or Lactobacillus genus. Furthermore, as bacteria included in the average gut microbiota of Japanese people, it is composed of bacteria belonging to the Firmicutes phylum, Actinobacteria phylum, and Bacteroidetes phylum, and it is desirable that it also contains bacteria belonging to the Brautia, Cholinella, and Streptococcus genera. It is desirable that the gut bacteria belonging to the Firmicutes phylum, Actinobacteria phylum, and Bacteroidetes phylum constitute 56-59%, 21-24%, and 17-22% of the bacterial species, respectively. More preferably, they constitute approximately 57%, 24%, and 19%, respectively. Furthermore, it is desirable that the combination of Enterobacteria belonging to the phyla Firmicutes, phyla Actinobacteria, and phyla Bacteroidetes make up 56-59%, 21-24%, and 17-22%, respectively, and that it also includes the genera Brautia, Cholincera, and Streptococcus. In this specification, "bacterial species" refers to the "species" of bacteria. That is, the systematic classification of bacteria is called "phylum," "genus," "species," and "strain" in descending order of size, and "species" refers to the "species."

[0028] The aforementioned at least nine bacterial species include: Blautia wexlerae, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Eubacterium rectore, Bifidobacterium adolescentis, Cholincera aerofasciens, Bacteroides uniformis, Dorea longicatena, Bacteroides bulgatus, Ruminococcus gunavas, Faecalibacterium prausnitzi, Parabacteroides distasonis, Dorea formisigenerans, Ruminococcus obeum, Ruminococcus troques, Bacteroides dreyi, Flavonifracter prauti, Parabacteroides meldae, Rosebria inulinivorans, Clostridium nexile, Streptococcus salivarius, Egasella lenta, and Clostridium. It is sufficient to select at least 9 species from the following 29 species: Volteae, Rosebria intestinalis, Coprococcus comes, Bacteroides obatus, Eubacterium harii, Rosebria hominis, and Bacteroides tetaiotaomicron. More preferably, Brautia wexlerae, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Eubacterium rectore, Bifidobacterium adolescentis, Chorinthera aerofasiens, Bacteroides uniformis, Drea longicatena, Bacteroides bulgatus, Ruminococcus gunavas, Faecalibacterium prausnitzi, Brautia obeum, Bacteroides dreyi, Flavonifracter prauti, and Streptococcus. It is sufficient to have at least nine species selected from Salivarius, Clostridium volteae, and Coprococcus comes (17 species in total), more preferably a mixed culture containing these 17 species, and most preferably a mixed culture consisting of these 17 species. The bacteria were selected from 29 strains available from JCM (RIKEN BioResource Research Center), a domestic distribution agency, out of 50 dominant gut bacteria species in the Japanese gut microbiota. These strains were cultured and confirmed to grow, and then multiple gut bacteria were extracted to create a combination that reflects the characteristics of the Japanese gut microbiota.

[0029] (Culture method) The culture of a bacterial cell mixture, which is a model of the gut microbiota, can be performed under any conditions that allow multiple intestinal bacteria to grow under a single condition. Examples of culture media include YCFA medium (JCM medium no. 1130) and EG medium (JCM medium no. 14), but GAM broth and modified GAM broth are preferred due to their simpler preparation. Of these, modified GAM broth medium is preferred. The culture temperature is around human body temperature, such as 36-38°C, with 37°C being preferred. The large intestine is known to have different pH levels depending on the region (transverse colon, distal colon, rectum), and the pH changes due to the production of short-chain fatty acids or their absorption by the host. Accordingly, the pH of the culture medium before culturing is preferably 6 or higher, preferably 7 or higher, and even more preferably around 7.2. The large intestine is an anaerobic environment, and the intestinal bacteria that grow there are obligate anaerobes. Therefore, it is necessary to maintain an anaerobic state in the culture medium and the culture medium gas layer using nitrogen gas. Furthermore, since carbon dioxide and hydrogen gas produced by intestinal bacteria are also present in the intestines, it is preferable to include these as well. A suitable ratio is 80-90% nitrogen, 5-10% carbon dioxide, and 5-10% hydrogen gas. The incubation time should preferably be continued until the pH of the culture medium becomes constant, with 16-32 hours being preferable, and 16 hours being even more preferable. The various enterobacteria constituting the bacterial cell mixture are added to 100 mL of culture medium at a concentration of approximately 1.0E+07 to 1.0E+08 cfu / mL. The proportion of each enterobacterial bacterium in the overall bacterial cell mixture can be calculated by ensuring that the number of bacteria added is equal for all species.

[0030] (Method for evaluating the effect of promoting the increase of short-chain fatty acids) The method for evaluating the short-chain fatty acid increase-promoting effect of the present invention involves adding the substance to be evaluated to a bacterial cell mixture as a model of the intestinal microbiota, culturing it using the aforementioned culture method, measuring the amount of short-chain fatty acids after a certain period of time, and comparing it with the case without the substance. The measurement can be performed, for example, by subjecting the culture supernatant to HPLC.

[0031] (Method for evaluating the effect of promoting increased diversity) The method for evaluating the diversity-enhancing effect of the present invention involves adding the substance to be evaluated to a bacterial mixture as a gut microbiota model, culturing it using the aforementioned culture method, measuring an index representing diversity after a certain period of time, and comparing it with the case without the substance. The measurement can be performed by calculating an index representing diversity (quantification of diversity) from genomic DNA extracted from the supernatant of the culture using a next-generation sequencer and analysis pipeline.

[0032] (Screening method) The screening method of the present invention involves adding a target substance to a bacterial cell mixture used as a gut microbiota model, culturing it using the aforementioned culture method, and selecting substances that have an effect of promoting the increase of short-chain fatty acids or an effect of promoting the increase of diversity after a certain period of time has elapsed. One way to increase the diversity of gut bacteria and short-chain fatty acids in the gut microbiota is to ingest lactic acid bacteria and bifidobacteria that act on the gut bacteria and increase short-chain fatty acids and diversity. For example, this can be done by consuming fermented dairy products containing them. Representative types of lactic acid bacteria used in fermented dairy products include those belonging to the Lactobacillus genus. Commonly used species include Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus reuteri, and Lactobacillus helveticus. On the other hand, Lactobacillus mucosae is a species that is not widely utilized. It is listed as a species with no safety concerns in the EFSA (European Food Safety Authority) QPS (Qualified Presumption of Safety) and is attracting attention as a probiotic strain that has the effect of suppressing the colonization of pathogenic bacteria and lowering cholesterol. Using the screening method of the present invention, we screened lactic acid bacteria of various Lactobacillus species and discovered for the first time that Lactobacillus mucosae has the effect of promoting the increase of short-chain fatty acids and the increase of diversity. The substances to be screened in this method include not only bacteria such as lactic acid bacteria and bifidobacteria, but also known food compositions. In the examples described later, when the indigestible dietary fiber α-CD (alpha-cyclodextrin), which is already known to increase short-chain fatty acids, was evaluated (screened) using this evaluation method, it was confirmed that it increases both short-chain fatty acids and their diversity.

[0033] (Method for manufacturing food and beverages for improving the gut microbiota) The present invention relates to a method for producing food and beverages for improving the intestinal flora, which involves producing food using a food composition that has been evaluated as having an effect of promoting the increase of short-chain fatty acids and / or promoting the increase of diversity in the evaluation method or screening method described above. Food and beverages can be produced by incorporating the food composition of the present invention into food and beverages. These formulations may be added to raw materials during the food and beverage manufacturing process, incorporated into ingredients, or incorporated into the final food and beverage product. Furthermore, if the food composition of the present invention is lactic acid bacteria or bifidobacteria, it can be produced by fermenting these to obtain a culture. The following describes in detail embodiments of the present invention, but the present invention is not limited to these embodiments. [Examples]

[0034] [Test Example 1] Preparation of cultures for evaluation system 1. Extraction of intestinal bacteria that have characteristics of the Japanese gut microbiota. (1) Obtaining and selecting intestinal bacteria We obtained available species of intestinal bacteria that are dominant in the gut microbiota of Japanese people from the distribution agency JCM (RIKEN BioResource Research Center) and cultured them in modified GAM broth medium (product code 05433, Nissui). From the 29 types of intestinal bacteria whose growth was confirmed by this culture, we extracted intestinal bacteria in combinations that maintain the taxonomic proportions of the Japanese gut. Specifically, we selected combinations containing approximately 57%, 24%, and 19% of intestinal bacteria belonging to the phyla Firmicutes, Actinobacteria, and Bacteroidetes, respectively, in terms of the number of bacterial species. Furthermore, we extracted intestinal bacteria in combinations that maintain these proportions and also include genera that show a high abundance in Japanese people compared to non-Japanese people, such as Blautia, Bifidobacterium, Cholincera, and Streptococcus.

[0035] (2) Preparation of various concentrated bacterial cells Modified GAM broth medium was prepared and sterilized by heat treatment at 121°C for 15 minutes. The 29 species of Enterobacteria were inoculated into each sterile medium and cultured anaerobically at 37°C using an anaerobic workstation (Concept 400, Central Scientific Trading). Each culture obtained was concentrated by centrifugation, and glycerol was added to a concentration of 10% (v / v) to obtain various concentrated bacterial cells. These concentrated bacterial cells were frozen at -80°C, then thawed, and serially diluted. The cells were then spread onto modified GAM broth agar medium, and the number of viable cells was measured. It was confirmed that each strain contained a viable cell count of 1.0E+07 cfu / mL or higher. The composition of the modified GAM broth medium used is shown in Table 1 below.

[0036] [Table 1]

[0037] 2. Test Results In accordance with the approach described in 1(1) above, intestinal bacteria belonging to the phyla Firmicutes, Actinobacteria, and Bacteroidetes were extracted in such combinations as 5 species, 2 species, and 2 species respectively (total of 9 species); 7 species, 3 species, and 2 species (total of 12 species); 8 species, 3 species, and 3 species (total of 14 species); and 10 species, 4 species, and 3 species (total of 17 species). Table 2 shows the 17 combinations of bacteria that include all 4 species belonging to the Actinobacteria phylum among the 29 species obtained.

[0038] [Table 2]

[0039] 3. Culture method for the model evaluation system In a small-volume culture device capable of monitoring temperature and pH over time, sterile modified GAM broth medium was added, and a mixture of nitrogen, carbon dioxide, and hydrogen gases was aerated for a certain period of time before culturing to remove as much dissolved oxygen as possible from the medium. Specifically, a small-volume multi-incubator (Bio Jr. 8, Able) was used, in which silicone packing materials were replaced with nitrile (NBR) materials and tubing materials with Pharmed tubing. The aerated gas mixture consisted of 80% nitrogen, 10% carbon dioxide, and 10% hydrogen. A mixed culture of 17 bacterial species shown in the reconstructed Table 2 was inoculated, and the culture was incubated with the above mixed gas aerated through and stirred until the pH of the medium became constant. In the model evaluation system, the mixed culture contained approximately 1.0E+07 to 1.0E+08 cfu / mL of each enterobacteria per 100 mL of medium, stirring was performed at 100 rpm or less, the pH at the start of incubation was 7.2, and the incubation time was 16 hours. In the following test example, candidate food compositions are added to a mixed culture in a model evaluation system, and the cultures are collected after culturing using the above cultivation method. The amount of short-chain fatty acids produced is measured, and an index representing diversity is calculated and evaluated.

[0040] [Test Example 2] Establishment of an evaluation method for promoting the increase of short-chain fatty acids and their diversity. While it is widely known that the intake of indigestible dietary fiber increases short-chain fatty acids in the gut microbiota in humans, no food composition that stably increases diversity has been reported to date. However, in patients with obesity, diabetes, and dyslipidemia, not only has a decrease in short-chain fatty acids but also a decrease in diversity has been reported, suggesting that there is a certain degree of relationship between short-chain fatty acids and diversity. Because this evaluation system utilizes a standardized gut microbiota model, it can accurately confirm the effect of promoting diversity, which was difficult to compare due to differences in gut microbiota among individuals.

[0041] (1) Test method As the test subject, α-CD (alpha-cyclodextrin, CAVAMAX WG Food, Cyclochem), a non-digestible dietary fiber that has been confirmed to increase the aforementioned short-chain fatty acids, was added to this evaluation system to confirm whether short-chain fatty acids increased, and its diversity was also evaluated. The amount of non-digestible dietary fiber added was 0.2%, which is the amount that can be consumed by humans (assuming the addition of 6g per 3000g of food).

[0042] (2) Method for evaluating the effect of promoting the increase in short-chain fatty acid production Butyrate and total short-chain fatty acid production (sum of acetate / propionic acid / butyrate) were calculated and evaluated from the supernatant of the culture using HPLC (ICS2100, Diatec). The three main short-chain fatty acids produced by intestinal bacteria in the human large intestine are acetate, propionic acid, and butyrate.

[0043] (3) Method for evaluating the effect of promoting increased diversity We performed a diversity index calculation (quantification of diversity) using a next-generation sequencer (Ion PGM, Thermo Fisher Scientific) and an analysis pipeline (QIIME2) on genomic DNA extracted from the supernatant of the culture, and then evaluated the results. We selected two indices to represent the diversity of the gut microbiota: the Shannon index and the Phylogenetic diversity index.

[0044] (4) Test results Compared to the control group without α-CD (set as 1.0), the increase in short-chain fatty acid production was 1.57 times for butyric acid, and total short-chain fatty acid production (sum of acetic acid / propionic acid / butyric acid) also increased 1.13 times. Furthermore, compared to the no-additive sample (set as 1.0), the Shannon index increased 1.25 times, and the Phylogenetic diversity index also increased 1.05 times. Based on the above, the addition of α-CD in this evaluation method reproducibly confirmed an increase in short-chain fatty acids. Furthermore, an increase in diversity was also reproducibly confirmed. Therefore, this evaluation method has been demonstrated to be a highly accurate evaluation method as a model evaluation system.

[0045] [Test Example 3] Screening for lactic acid bacteria that increase short-chain fatty acids and / or gut microbiota diversity (1) Test method In this study, we used the evaluation method demonstrated in Test Example 2 to confirm whether lactic acid bacteria belonging to Lactobacillus mucosae increased short-chain fatty acids, and also evaluated the increase in diversity. Specifically, each lactic acid bacterium belonging to Lactobacillus mucosae isolated from human samples owned by the applicant (Table 2) was added to 100 mL of culture medium at approximately 1.0E+07 cfu / mL.

[0046] (2) Method for evaluating the effect of promoting the increase in short-chain fatty acid production The procedure was carried out in the same manner as in Test Example 2.

[0047] (3) Method for evaluating the effect of promoting increased diversity The procedure was carried out in the same manner as in Test Example 2.

[0048] (4) Test results In a model evaluation system, we searched for various lactic acid bacteria and bifidobacteria that increase short-chain fatty acids upon addition. We found that the increase in short-chain fatty acids was reproducibly confirmed with the addition of Lactobacillus mucosae culture. Furthermore, an increase in diversity was also reproducibly confirmed. For example, when Lactobacillus rhamnosus and Lactobacillus delbrueckii subspecies bulgaricus, isolated from human sources owned by the applicant, were added, the increase in short-chain fatty acid production was 0.66 times and 0.85 times for butyric acid, respectively, and the total short-chain fatty acid production (sum of acetic acid / propionic acid / butyric acid) also decreased to 0.94 times and 0.95 times, respectively. In addition, the increase in diversity indices was 0.97 times and 0.85 times for the Shannon index, and the Phylogenetic diversity index also decreased to 0.95 times and 0.95 times, respectively. Table 3 shows the rate of increase in short-chain fatty acid production, and Table 4 shows the rate of increase in the diversity index, compared to the control group without Lactobacillus mucosae, which was set to 1.0.

[0049] [Table 3]

[0050] [Table 4]

[0051] (5) Discussion When α-CD and Lactobacillus mucosae, food compositions that promote the growth of intestinal bacteria, were added to a gut microbiota model, which is a mixed culture reconstructed using the dominant gut bacteria of Japanese people, and then cultured together, it was confirmed that an increase in short-chain fatty acid production and / or diversity was promoted compared to the case without these additives. [Industrial applicability]

[0052] According to the present invention, by using a model evaluation system that artificially reconstructs the gut microbiota of Japanese people, it is possible to search for food compositions that increase the diversity of gut bacteria constituting the gut microbiota and short-chain fatty acids in the gut microbiota, without using humans or animals, and without using human feces, which are biological samples. Furthermore, by ingesting foods and beverages containing bacteria belonging to Lactobacillus mucosae, which have been screened using the above model evaluation system, it is expected that these products will act on the gut bacteria constituting the gut microbiota and improve the gut microbiota. [Accession Number]

[0053] [Reference to deposited biological materials] (1) SBT10028 (i) The name and address of the depositary institution that deposited the biological material. National Institute of Technology and Evaluation (NITE), Patent Microorganism Depositary Center (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture (Postal Code 292-0818)) Date of deposit of biological material with the depositary in Roy September 15, 2020 The depositary number assigned by the depositary in Hai to the deposit NITE BP-03275 (2) SBT10217 (i) The name and address of the depositary institution that deposited the biological material. Same as (1) above Date of deposit of biological material with the depositary in Roy September 15, 2020 The depositary number assigned by the depositary in Hai to the deposit NITE P-03276 (3) SBT10027 (i) The name and address of the depositary institution that deposited the biological material. Same as (1) above Date of deposit of biological material with the depositary in Roy September 15, 2020 The depositary number assigned by the depositary in Hai to the deposit NITE P-03274 (4) SBT10038 (i) The name and address of the depositary institution that deposited the biological material. Same as (1) above Date of deposit of biological material with the depositary in Roy September 15, 2020 The depositary number assigned by the depositary in Hai to the deposit NITE P-03283 (5) SBT2261 (i) The name and address of the depositary institution that deposited the biological material. Same as (1) above Date of deposit of biological material with the depositary in Roy September 15, 2020 The depositary number assigned by the depositary in Hai to the deposit NITE P-03272 (6) SBT2027 (i) The name and address of the depositary institution that deposited the biological material. Same as (1) above Date of deposit of biological material with the depositary in Roy September 15, 2020 The depositary number assigned by the depositary in Hai to the deposit NITE P-03271 (7) SBT2271 (i) The name and address of the depositary institution that deposited the biological material. Same as (1) above Date of deposit of biological material with the depositary in Roy September 15, 2020 The depositary number assigned by the depositary in Hai to the deposit NITE P-03273 (8) SBT10043 (i) The name and address of the depositary institution that deposited the biological material. Same as (1) above Date of deposit of biological material with the depositary in Roy March 27, 2020 The depositary number assigned by the depositary in Hai to the deposit NITE BP-03187

Claims

1. A human gut microbiota model for evaluating the effect of a target food composition on increasing and / or increasing the diversity of short-chain fatty acids in the human gut, The human gut microbiota model is comprised of an evaluation culture containing at least nine bacterial species, excluding bacteria belonging to the phylum Proteobacteria and genus Lactobacillus, but including bacteria belonging to the genus Bifidobacterium.

2. The human gut microbiota model according to claim 1, wherein the nine or more bacterial species are bacteria belonging to the phylum Firmicutes, phylum Actinobacteria, or phylum Bacteroidetes, and include bacteria belonging to the genera Brautia, Cholincera, or Streptococcus.

3. The human gut microbiota model according to claim 2, comprising 56-59% bacteria belonging to the phylum Firmicutes, 21-24% bacteria belonging to the phylum Actinobacteria, and 17-22% bacteria belonging to the phylum Bacteroidetes.

4. At least nine bacterial species were found, including Blautia wexlerae, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Eubacterium rectore, Bifidobacterium adolescentis, Cholincera aerofasciens, Bacteroides uniformis, Dorea longicatena, Bacteroides bulgatus, Ruminococcus gunavas, Faecalibacterium prausnitzi, Parabacteroides distasonis, Dorea formisigenerans, Ruminococcus obeum, Ruminococcus troques, Bacteroides dreyi, Flavonifracter prauti, Parabacteroides meldae, Rosebria inulinivorans, Clostridium nexile, Streptococcus salivarius, Egasella lenta, and Clostridium. A human gut microbiota model according to any one of claims 1 to 3, selected from the group consisting of Volteae, Rosebria intestinalis, Coprococcus comes, Bacteroides obatus, Eubacterium hali'i, Rosebria hominis, and Bacteroides tethaiotaomicron.

5. A human gut microbiota model according to any one of claims 1 to 4, wherein at least nine or more bacterial species are Brautia wexlerae, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Eubacterium rectore, Bifidobacterium adolescentis, Cholincera aerofasciens, Bacteroides uniformis, Drea longicatena, Bacteroides bulgatus, Ruminococcus gunavas, Faecalibacterium prausnitzi, Brautia obeum, Bacteroides dreyi, Flavonifractor prauti, Streptococcus salivarius, Clostridium volteae, and Coprococcus comes.

6. A method for culturing a human gut microbiota model to evaluate the effect of a target food composition on increasing and / or increasing the diversity of short-chain fatty acids in the human gut, comprising the following steps. (1) A step of adding at least nine species of bacteria, including bacteria belonging to the genus Bifidobacterium, but not bacteria belonging to the phylum Proteobacteria and genus Lactobacillus, to a modified GAM broth medium. (2) Step of culturing under anaerobic conditions

7. A method for evaluating the effect of a target food composition on increasing and / or increasing the diversity of short-chain fatty acids in the human gut, the evaluation method comprising the following steps. (1) A step of adding the food composition to be evaluated to the human gut microbiota model according to any one of claims 1 to 5 and culturing it. (2) A step of measuring the amount of short-chain fatty acids produced by the culture obtained in (1) and / or calculating the diversity index. (3) A step in which the measured values ​​and / or calculated values ​​in (2) are evaluated as having a short-chain fatty acid increase-promoting effect and / or diversity increase-promoting effect when the relative value is 1.0 times or more compared to the case where the food composition is not added.

8. A method for producing food and beverages for improving the intestinal flora, wherein the food is produced using a food composition that has been evaluated to have an effect of promoting the increase of short-chain fatty acids and / or an effect of promoting the increase of diversity in the evaluation method described in claim 7.

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