Intestinal-environment-improving agent

Abstract

This intestinal-environment-improving agent contains a yeast deposited to the National Institute of Technology and Evaluation under international deposition number NITE BP-04012. The intestinal-environment-improving agent of the present invention can be used as a useful intestinal-flora-improving agent, a short-chain fatty-acid-producing bacteria increase or decrease inhibitor, a short-chain fatty-acid-producing agent, a putrefaction product production inhibitor, an agent used for increasing or decreasing a specific bacterial genus or group, a bowel-movement-improving agent, a secondary bile acid production inhibitor, a valeric acid production inhibitor, and the like. The putrefaction product is preferably one or more selected from indole, phenol, p-cresol, 4-ethylphenol, and skatole.

Description

Intestinal environment improver

[0001] The present invention relates to an agent for improving the intestinal environment.

[0002] Intake of foods containing probiotics such as oligosaccharides and bifidobacteria has been recommended for improving the intestinal environment (Patent Document 1, Patent Document 2).

[0003] Furthermore, a method has been disclosed in which a composition having an intestinal environment-improving effect is ingested together with dietary fiber such as agar in order to prevent the composition from being inactivated by gastric juice (Patent Document 3).

[0004] In recent years, attention has been focused on intestinal environment improvers that can effectively improve the intestinal microflora. For example, butyrate produced by intestinal butyric acid bacteria is known to be a very important nutrient that provides energy to colonic mucosal epithelial cells. It is known to have effects such as immunomodulation, oxidative stress reduction, glucose tolerance improvement, obesity suppression, inflammation suppression, cancer suppression, and bowel movement control, and is known to be a cellular mediator that regulates various functions not only in the intestine but also in the gut. In addition, butyric acid is thought to promote the metabolism of intestinal mucosal epithelial cells, consuming oxygen, and maintaining an anaerobic state in the large intestine, thereby suppressing the growth of pathogenic bacteria and also functioning as an intestinal barrier. For these reasons, short-chain fatty acid-producing bacteria such as butyric acid bacteria have attracted attention (e.g., Patent Document 4). In addition to short-chain fatty acid-producing bacteria, various functions and properties of intestinal bacteria in the intestinal microflora have been discovered, such as beneficial bacteria that suppress intestinal inflammation and harmful bacteria that reduce glucose tolerance (e.g., Non-Patent Documents 1 and 2). Therefore, intestinal environment improvers that can increase / reduce these functions are anticipated. Furthermore, decay products have a high carcinogenic effect, and attention is being paid to agents that can inhibit their production (Patent Document 5).

[0005] JP 2024-005551 A JP 2023-140651 A JP 2004-215561 A International Publication No. 2022 / 036225 JP 2023-043511 A

[0006] Inflamm Bowel Dis Volume 22, Number 12, December 2016, p2802-2810Cell 175, 947-961, November 1, 2018, p947-961

[0007] An object of the present invention is to provide an intestinal environment improving agent that has an excellent effect of improving the intestinal environment.

[0008] As a result of extensive research, the present inventors have found that a particular yeast strain is particularly excellent in functions as an intestinal environment improver, such as improving the intestinal flora, producing short-chain fatty acids, inhibiting the production of putrefactive products, and improving bowel movements, and have thus completed the present invention. That is, the present invention provides an intestinal environment improver comprising a yeast deposited with the National Institute of Technology and Evaluation under International Deposit Number NITE BP-04012.

[0009] According to the present invention, an intestinal environment improving agent having excellent effects of improving the intestinal environment can be provided. The intestinal environment improving agent of the present invention can provide excellent effects of improving the intestinal flora, producing short-chain fatty acids, improving bowel movements, reducing putrefactive products, etc.

[0010] 1 is a table showing the results of analysis of the ASV (Amplicon Sequence Variant) composition of the stool culture fluid of subjects No. 1 to 3. 2 is a table showing the results of analysis of the ASV composition of the stool culture fluid of subjects No. 4 to 6. 3 is a graph showing the amounts of acetic acid and propionic acid in the stool culture fluid. 4 is a graph showing the amount of lithocholic acid in the stool culture fluid. 5 is a graph showing the results of analysis of changes in the genus composition of the intestinal bacterial flora in humans due to the ingestion of a specific yeast. 6 is a graph showing the results of analysis of changes in the genus composition of the intestinal bacterial flora in humans due to the ingestion of a specific yeast. 7 is a graph showing the results of analysis of changes in the species composition of the intestinal bacterial flora in humans due to the ingestion of a specific yeast. 8 is a graph showing the results of analysis of changes in the amount of short-chain fatty acids in feces in humans due to the ingestion of a specific yeast. 9 is a graph showing the results of analysis of changes in the amount of putrefactive products in feces in humans due to the ingestion of a specific yeast. 1 is a graph showing the results of an analysis of changes in fecal pH caused by the ingestion of a specific yeast in humans.

[0011] The present invention will be described below based on preferred embodiments thereof, but the present invention is not limited thereto.

[0012] The present invention relates to an intestinal environment improving agent containing a yeast (hereinafter also referred to as "specific yeast") deposited with the National Institute of Technology and Evaluation under international deposit number NITE BP-04012. This specific yeast was deposited with the National Institute of Technology and Evaluation under international deposit number NITE P-04012 on November 14, 2023, and transferred to international deposit on September 11, 2024. The yeast deposited with the National Institute of Technology and Evaluation under international deposit number NITE BP-04012 is known by the scientific name Saccharomyces cerevisiae. Its scientific properties are as follows: 1. Scientific properties: It forms white to pale yellow colonies in a nutrient medium containing a carbon source and a nitrogen source. Under an optical microscope, growth by budding is observed as a division morphology. It is possible to distinguish between mother and daughter cells.

[0013] 2. Taxonomic position... Yeast: Edible yeast (genus Saccharomyces) 3. Culture conditions (1) Name of medium... YPD medium (2) Medium composition... 10g of yeast extract, 20g of peptone, and 20g of glucose per 1000ml of medium (3) Medium pH... 4-7 (optimum pH 5-6) (4) Medium sterilization conditions... 121°C for 10 minutes (5) Culture temperature... 30°C (6) Culture period... 2-3 days (7) Oxygen requirement... Facultative anaerobic 4. Storage conditions Can be stored by freezing. (1) Freezing conditions... -80°C (2) Protecting agent... 10-20% glycerin aqueous solution (optimum 15%) (3) Recovery rate after freezing... 40-80% in 2 years 5. Conditions for survival test (1) Restoration of microorganisms...30°C (2) Inoculation, cultivation and confirmation method...same conditions as cultivation conditions.

[0014] As described above, the specific yeast of the present invention is preferably cultured in a YPD medium at a pH of 4 to 7, preferably at a pH of 5 to 6. The culture may be carried out under aerobic conditions, such as in the air.

[0015] The specific yeast used in the present invention may be in the form of a yeast cell wall, a yeast content, or a yeast cell body containing both the yeast cell wall and the yeast content. The yeast cell wall is a fraction insoluble in aqueous liquid media such as water, and is obtained by removing content soluble in aqueous liquid media, such as proteins, carbohydrates, amino acids, nucleic acids, and organic acids, from yeast cells. The yeast cell wall may be prepared by any method, including crushing or disrupting yeast cells to allow the water-soluble content to be eluted, followed by solid-liquid separation to obtain the water-insoluble fraction, which is then dried. Examples of methods for crushing or disrupting yeast cells include physical disruption methods such as ultrasonication, grinding using a bead mill, and pressurized liquid shearing using a French press, as well as chemical disruption methods using surfactants or lytic enzymes such as cell wall-degrading enzymes. Any drying method, such as freeze-drying or spray-drying, can be used. Physical disruption is preferred as a method for allowing the content of yeast cells to be eluted into an aqueous liquid medium. Examples of aqueous liquid media include water and ethanol, with water being preferred. When the aqueous liquid medium is water, the water may contain components other than water, such as a culture medium, and may have a water content of more than 50% by mass, preferably 60% by mass or more.

[0016] Furthermore, the contents of a specific yeast can be obtained by disrupting yeast cells to make the contents elutable in an aqueous liquid medium, and then extracting the resulting disrupted material with an aqueous liquid medium. Instead of disrupting yeast cells, the yeast cell walls may be dissolved with a cell wall-degrading enzyme to make the contents elutable in an aqueous liquid medium. The contents of a yeast are also called yeast extract. The yeast extract may be a solid that has been dried by any method.

[0017] To achieve the intestinal environment-improving effects of the present invention (e.g., intestinal flora improvement, short-chain fatty acid production, bowel movement improvement, putrefactive product production inhibition, secondary bile acid production inhibition, and valeric acid production inhibition), all or part of a specific yeast can be used. For example, when a portion is used, it may be any of the yeast cell wall, the yeast contents, and a portion of a yeast cell fraction containing the yeast cell wall and the yeast contents. Examples of yeast cell fractions containing the yeast cell wall and the yeast contents include mixtures obtained by crushing or disrupting yeast cells, in which the yeast cell wall and the yeast contents are mixed. By contacting such a mixture with an aqueous liquid and subjecting it to solid-liquid separation, the yeast extract can be separated from the cell wall. The proportion of yeast extract in the mixture is preferably 1% by mass or more, more preferably 2% by mass or more, even more preferably 3% by mass or more, even more preferably 4% by mass or more, and particularly preferably 5% by mass or more. The proportion of yeast extract in the mixture may be 90% by mass or less, or even 100% by mass or less. The proportion of the cell walls in the mixture may be, for example, 5% by mass or more, or 10% by mass or more, and the proportion of the cell walls in the mixture may be, for example, 90% by mass or less, or 100% by mass or less.

[0018] In this specification, examples of "improvement of the intestinal environment" include improvement of the intestinal flora, production of short-chain fatty acids, inhibition of an increase or decrease in short-chain fatty acid-producing bacteria, improvement of bowel movements, inhibition of production of putrefactive products, inhibition of production of secondary bile acids, inhibition of production of valeric acid, and reduction of stool pH.

[0019] For example, as shown in the examples described below, in one embodiment of the present invention, ingesting the intestinal environment improving agent of the present invention can provide a bowel movement improving effect. Examples of bowel movement improvement include normalizing bowel movement frequency and improving stool properties. Examples of normalizing bowel movement frequency include increasing bowel movement frequency. The bowel movement improving effect can be measured, for example, with reference to the index of the constipation assessment scale CAS-MT (Constipation Assessment Scale). In this specification, when simply referring to "stool," it means feces unless otherwise specified.

[0020] In another embodiment of the present invention, ingestion of the intestinal environment improver of the present invention reduces intestinal putrefactive products. Intestinal putrefactive products refer to malodorous substances in feces. However, short-chain fatty acids with four or fewer carbon atoms, such as butyric acid and isobutyric acid, are not included in the putrefactive products. Examples of putrefactive products include ammonia, sulfides, indole, phenol, p-cresol, skatole, and ethylphenol. The putrefactive products produced by the intestinal environment improver of the present invention are preferably at least one selected from indole, phenol, p-cresol, and skatole, and more preferably at least one selected from indole, phenol, and p-cresol. These putrefactive products tend to accumulate deep in the large intestine, which tends to become alkaline, causing inflammation and leading to the development of colon cancer and ulcerative colitis. Therefore, by suppressing the production of putrefactive products using the intestinal environment improver of the present invention, it is expected that the intestinal inflammation and the resulting development of colon cancer and ulcerative colitis will be suppressed.

[0021] Furthermore, in another embodiment, the intestinal environment-improving agent of the present invention has the effect of improving the intestinal bacterial flora. For example, as shown in the examples described below, the intestinal environment-improving agent of the present invention has the effect of promoting the increase of bacteria that produce short-chain fatty acids and organic acids, such as butyric acid bacteria of the genus Anaerobutyricum, Ruminococcus torques, and Eubacterium ventriosum, Parabacteroides genus such as Parabacteroides distasonis that produce succinic acid and short-chain fatty acids, propionic acid-producing bacteria of the genus Dialister, acetic acid bacteria of the genus Muricomes such as Muricomes intestini, Blautia genus such as Blautia faecis that produce lactic acid and acetic acid, and Dialister genus. The intestinal environment-improving agent of the present invention can be used as an agent for increasing or suppressing the decrease of these short-chain fatty acid-producing bacteria. Furthermore, as shown in the results of fecal culture tests and human tests described below, the intestinal environment-improving agent of the present invention can promote the production of short-chain fatty acids such as acetic acid, butyric acid, and propionic acid.

[0022] The roles of short-chain fatty acids produced in the intestine are briefly summarized below. (Immunomodulatory function, barrier function, inflammation suppression) Acetic acid is said to enhance the barrier function of the large intestine. Furthermore, butyric acid is an important energy source for the large intestine and is said to promote the secretion of mucin, a mucosal substance, and protect the large intestine. The barrier function of the large intestine is known to be closely related to intestinal immunity. Approximately 60% of the body's immune cells are concentrated in the intestine, and it is said to have an extremely strong influence on intestinal immunity throughout the body. Butyric acid has the effect of increasing Treg cells, immune cells that suppress excessive immune responses. Short-chain fatty acids such as butyric acid, acetic acid, and propionic acid are also said to be useful in suppressing inflammation in inflammatory bowel disease and other conditions. Acetic acid is known to be effective in preventing infection by O157.

[0023] (Cancer suppression) Butyric acid is said to suppress the onset of colon cancer by suppressing the abnormal proliferation of colon cells, promoting apoptosis, and suppressing lesions in colon cells. Research has shown that propionic acid acts on short-chain fatty acid receptors in liver cancer cells, suppressing the proliferation of liver cancer cells.

[0024] (Suppression of obesity, improvement of glucose tolerance) Short-chain fatty acids such as acetic acid, butyric acid, and propionic acid act on short-chain fatty acid receptors in fat cells to suppress the uptake of energy into fat cells and prevent fat cell hypertrophy. They also act on short-chain fatty acid receptors in nerve cells, promoting energy consumption via the sympathetic nervous system and thus regulating energy balance. Butyric acid and propionic acid also act on L cells in the intestinal tract to promote the secretion of GLP-1. GLP-1 has the effect of preventing and improving diabetes, suppressing the decrease in the number of pancreatic beta cells that secrete insulin, and promoting insulin secretion.

[0025] (Benefits of bowel movements) It is thought that bowel movements improve by promoting intestinal peristalsis (utilizing it as energy) through the production of butyrate as a result of an increase in butyrate-producing bacteria.

[0026] From the above, the intestinal environment improving agent of the present invention, which has the effect of increasing the short-chain fatty acid-producing bacteria and promoting the production of short-chain fatty acids, can be expected to have the above-mentioned effects.

[0027] Furthermore, the genus Fusicatenibacter has an anti-inflammatory effect on the intestines, and an increase in the number of Enterocloster species, such as Bacteroides and Enterocloster citroniae, is generally believed to have anti-inflammatory and immune-improving effects. The intestinal environment-improving agent of the present invention can promote the increase of these bacteria. Therefore, the intestinal environment-improving agent of the present invention can be used as an agent for inhibiting the increase or decrease of these bacteria.

[0028] On the other hand, bacteria of the genera Eggerthella and Bilophila are harmful bacteria that are thought to cause impaired glucose tolerance and diabetes. The genera Tyzzerella and Flavonifractor are generally thought to increase during inflammation. Furthermore, Clostridium scindens metabolizes the primary bile acids cholic acid and chenodeoxycholic acid, dehydrogenating the 7-hydroxyl group (7α-dehydrogenation) to convert them into secondary bile acids deoxycholic acid and lithocholic acid. These substances have been reported to be involved in the progression and contribution of colorectal cancer. The intestinal environment improving agent of the present invention can reduce or inhibit the growth of these bacteria. Therefore, the intestinal environment improving agent of the present invention can be used to reduce or inhibit the growth of these bacteria, thereby suppressing inflammation, improving immunity, inhibiting impaired glucose tolerance and diabetes, and inhibiting the progression of colorectal cancer. Furthermore, as shown in the examples below, the intestinal environment improving agent of the present invention is effective in improving the intestinal flora in women who tend to suffer from constipation.

[0029] Furthermore, as will be described later, the intestinal environment-improving agent of the present invention has an inhibitory effect on the production of lithocholic acid (LCA), a secondary bile acid that has been reported to promote colon cancer, and can be used as an inhibitor of secondary bile acid production. Therefore, the intestinal environment-improving agent of the present invention is expected to have an inhibitory effect on colon cancer.

[0030] Furthermore, as will be shown in the examples below, the intestinal environment-improving agent of the present invention has the effect of inhibiting the production of n-valeric acid, which causes bad odor in stool, and can be used as an agent for inhibiting the production of valeric acid.

[0031] Furthermore, as shown in the examples below, the intestinal environment improving agent of the present invention can lower the pH of feces by orally ingesting it. It has been reported that making the intestinal pH slightly acidic can suppress the growth of harmful bacteria that prefer an alkaline environment, activate the intestinal immune function, and play an important role in maintaining human health.

[0032] There are no strict limitations on the dosage of the specific yeast, which is the active ingredient of the intestinal environment improving agent of the present invention. Because the effects obtained vary depending on various usage modes, such as the subject and the applicable disease, it is desirable to set the dosage appropriately. A suitable dosage for the specific yeast is 10 mg to 100 g, more preferably 160 mg to 4 g, of cell weight per day for an adult. The effective dosage of the intestinal environment improving agent when ingested (including for humans) can be appropriately adjusted depending on the species, symptoms, age, sex, etc., of the individual to whom the agent is administered. The agent is not limited to humans; it can also be used for animals such as dogs and cats. The daily dosage may be administered in a single dose or in several divided doses.

[0033] The agent of the present invention can be taken continuously, and can be taken continuously for one week or more, two weeks or more, or four weeks or more.

[0034] The specific yeast of the present invention can be ingested by oral ingestion, transdermal ingestion, transmucosal ingestion, enteral ingestion, etc., with oral ingestion being particularly preferred.

[0035] The intestinal environment improving agent of the present invention can be used as a pharmaceutical, quasi-drug, or food or beverage for animals, including humans, or for producing them. The intestinal environment improving agent of the present invention can be directly administered to or ingested by animals, including humans, as a pharmaceutical, quasi-drug, or food or beverage, or can be added or blended into food, beverage, or animal feed such as pet food to be used as a food, beverage, or animal feed for improving the intestinal environment. In the latter case, the method of adding or blending the specific yeast into the food, beverage, or animal feed is not particularly limited. For example, the specific yeast can be directly blended into the raw materials or ingredients before the production of the food, beverage, or animal feed, or it can be added during the production process of the food, beverage, or animal feed, or it can be added to the produced food, beverage, or animal feed.

[0036] The term "food and drink" refers to substances that can be ingested by humans, including general foods and drink including so-called health foods, as well as health functional foods such as foods for specified health uses and foods with nutrient functions as defined by the Ministry of Health, Labor and Welfare's Health Functional Food System, and supplements. The term "animal feed" refers to substances given as feed to non-human animals (animals kept by humans), such as livestock, poultry, and farmed fish, including livestock feed and pet food. When the intestinal environment improving agent of the present invention is used as a drug or quasi-drug, it may contain the specific yeast as an active ingredient alone, or may further contain a pharmaceutically acceptable carrier, or may further contain other active ingredients or pharmacological ingredients to the extent that the intestinal environment improving effect of the specific yeast is not impaired. Examples of such carriers include excipients, coating agents, binders, fillers, disintegrants, surfactants, lubricants, diluents, dispersants, buffers, osmotic pressure adjusters, pH adjusters, emulsifiers, preservatives, stabilizers, antioxidants, colorants, ultraviolet absorbers, moisturizers, thickeners, activity enhancers, disinfectants, flavorings, and odor enhancers.

[0037] When the agent of the present invention is used as a pharmaceutical or quasi-drug, it can be administered in any dosage form.The dosage form may be oral or parenteral.For example, oral dosage forms include solid dosage forms such as tablets, coated tablets, granules, powders, and capsules, and liquid dosage forms such as elixirs, syrups, and suspensions.Parenteral dosage forms include injections, infusions, transdermal, transmucosal, nasal, enteral, inhalation, suppositories, boluses, patches, etc.Among these, oral dosage forms are preferred.

[0038] In the agent of the present invention, the amount of specific yeast can be any amount as long as it can be the amount that can be an active ingredient, and in the solid content of the agent, specific yeast can be 5% by mass or more, or 10% by mass or more, or 20% by mass or more, or 30% by mass or more, or 40% by mass or more, or 50% by mass or more, or 70% by mass or more.It should be noted that, here, solid content refers to the total amount excluding solvent, and solvent can be water, ethanol or organic solvent that is generally used as solvent.It should be noted that, in the case of capsule, the content of capsule is used as denominator.

[0039] When the agent of the present invention is used as a food or beverage, it may contain the specific yeast as an active ingredient alone, or may further contain various additives used in the production of foods and beverages, as long as the intestinal environment-improving effect of the specific yeast is not impaired. Such additives include, for example, various oils and fats, herbal medicines, amino acids, polyhydric alcohols, natural polymers, vitamins, dietary fiber, surfactants, purified water, excipients, stabilizers, pH adjusters, antioxidants, sweeteners, taste components, acidulants such as organic acids, stabilizers, flavors, colorants, fragrances, etc.

[0040] Examples of foods and beverages include oral preparations (gum, candy, etc.), processed seafood paste products such as kamaboko (fish cake) and chikuwa (fish cake), livestock products such as sausages and ham, bread, Western confectionery, Japanese confectionery, noodles such as fresh noodles, Chinese noodles, boiled noodles, and buckwheat, seasonings such as sauces, soy sauce, dressings, sugar, honey, powdered sugar, and starch syrup, spices such as curry powder, mustard powder, and pepper powder, jam, marmalade, chocolate spread, pickles, greens, furikake (rice seasoning), and processed vegetables and fruits such as various canned and bottled vegetables and fruits, dairy products such as cheese, butter, and yogurt, beverages such as miso soup, soup, fruit juice, vegetable juice, whey drinks, soft drinks, and alcoholic beverages, as well as other general foods and beverages such as health foods.

[0041] The present invention encompasses a package containing a package that describes its use for improving the intestinal environment and a food, drink, or animal feed containing a specific yeast contained in the package. The package is not particularly limited in shape or material, as long as it can contain the agent, food, drink, or animal feed of the present invention and can print ingredient information, etc. Examples of the shape of the package include a box and a bag. Examples of the material of the package include paper, plastic, woven fabric, metal, etc.

[0042] The agent of the present invention can be used for various purposes, such as improving the intestinal flora, inhibiting the increase or decrease of short-chain fatty acid-producing bacteria, production of short-chain fatty acids (including "promoting production" and "inhibiting decrease"), inhibitor of the production of putrefactive products (including "reducing", "promoting decrease", "inhibiting increase", etc.), inhibiting the increase or decrease, decrease or increase inhibition of the above-mentioned specific bacterial genera and groups, agent for improving laxation, inhibitor of secondary bile acid production (including "reducing", "promoting decrease", "inhibiting increase", etc.), inhibiting the production of valeric acid (including "reducing", "promoting decrease", "inhibiting increase", etc.), and can also be used for purposes caused by the increase or decrease, etc. of each bacterial genera described in this specification.

[0043] The package clearly indicates various information such as the content of the specific yeast in the agent of the present invention, food, drink, or animal feed contained in the package. The method of presenting the information on such a package is not particularly limited, and for example, 1) the information may be printed on the outer or inner surface of the package, 2) any of the above-mentioned uses, including the use for improving the intestinal environment, may be printed on a printing medium such as printing paper contained inside the package together with the food, drink, or animal feed, or 3) the package or the printing medium contained therein may be printed with a QR code (registered trademark) containing an internet URL or information that allows access to a site introducing the agent of the present invention, food, drink, animal feed, etc., and the information may be displayed by accessing the URL.

[0044] As described above, the agent of the present invention can be used to inhibit the increase or decrease of at least one species selected from the genera Anaerococcus, Anaerobutyricum, Bacteroides, Blautia, Dialister, Enterocloster, Haemophilus, Fusicatenibacter, Muricomes, and Parabacteroides, and may be used to inhibit the increase or decrease of any of these bacteria.

[0045] As described above, the agent of the present invention can be used to reduce or inhibit the increase or decrease of at least one species selected from the genera Bilophila, Butyricimonas, Eggerthella, Flavonifractor, Intestinimonas, Lachnoclostridium, Massiliimalia, Phocaeicola, Streptococcus, and Tyzzerella, and may be used to inhibit the increase or decrease of any of these bacteria.

[0046] Furthermore, the agent of the present invention can be used to reduce or inhibit the growth of at least one selected from Anaerococcus vaginalis, Blautia faecis, Enterocloster citroniae, Eubacterium ventriosum, Haemophilus parainfluenza, Muricomes intestini, Parabacteroides distasonis, and Ruminococcus torques, and may be used to reduce or inhibit the growth of any of these bacteria.

[0047] The agent of the present invention can be used to reduce or inhibit the growth of at least one selected from Anaerotignum aminivorans, Bacteroides stercoris, Butyricimonas paravirosa, Clostridium scindens, Intestinimonas massiliensis, Enterocloster lavalensis, Lachnochrostridium Phocaeicola, Massiliimalia timonensis, and Streptococcus vestibularis, and may be used to reduce or inhibit the growth of any of these bacteria.

[0048] The present invention provides the following: [1] An intestinal environment improving agent comprising a yeast deposited at the National Institute of Technology and Evaluation under International Deposit Number NITE BP-04012. [2] The intestinal environment improving agent according to [1], which is an intestinal microflora improving agent. [3] The intestinal environment improving agent according to [1] or [2], which is an agent for suppressing an increase or decrease of short-chain fatty acid-producing bacteria or an agent for producing short-chain fatty acids. [4] The intestinal environment improving agent according to any one of [1] to [3], which is an agent for suppressing the production of putrefactive products. [5] The intestinal environment improving agent according to [4], wherein the putrefactive products are one or more selected from indole, phenol, p-cresol, 4-ethylphenol, and skatole. [6] The intestinal environment-improving agent according to any one of [1] to [5], which is used to increase or suppress the decrease of at least one species selected from the genera Anaerococcus, Anaerobutyricum, Bacteroides, Blautia, Dialister, Enterocloster, Haemophilus, Fusicatenibacter, Muricomes, and Parabacteroides, or to decrease or suppress the increase of at least one species selected from the genera Bilophila, Butyricimonas, Eggerthella, Flavonifractor, Intestinimonas, Lachnoclostridium, Massiliimalia, Phocaeicola, Streptococcus, and Tyzzerella.[7] The intestinal environment-improving agent according to any one of [1] to [6], which is used to increase or suppress the decrease of at least one species selected from Anaerococcus vaginalis, Blautia faecis, Enterocloster citroniae, Eubacterium ventriosum, Haemophilus parainfluenzae, Muricomes intestini, Parabacteroides distasonis, and Ruminococcus torques, or to decrease or suppress the increase of at least one species selected from Anaerococcus aminivorans, Bacteroides stercoris, Butyricimonas paravirosa, Clostridium scindens, Intestinimonas massiliensis, Enterocloster lavalensis, Lachnochrostridium Phocaeicola, Massiliimalia timonensis, and Streptococcus vestibularis. [8] The intestinal environment improving agent according to any one of [1] to [7], which is a laxative. [9] The intestinal environment improving agent according to any one of [1] to [8], which is a secondary bile acid production inhibitor.

[10] The intestinal environment improving agent according to any one of [1] to [9], which is a valeric acid production inhibitor.

[11] The intestinal environment improving agent according to any one of [1] to

[10] , which is an intestinal pH lowering agent.

[12] Use of a yeast deposited with the National Institute of Technology and Evaluation under International Deposit Number NITE BP-04012, for the production of an intestinal environment improving agent.

[0049] 1. <Cultivation and drying of yeast> A yeast strain (international deposit number NITE BP-04012) was cultured by the following method. First, an inoculum stored under the above conditions was collected, and the inoculum was inoculated onto a YPD agar medium by streaking. The agar medium was cultured in air at 30°C for 3 days, and then multiple colonies that appeared on the agar medium were cultured under the above conditions. After culturing, the yeast was dried by spray drying.

[0050] <Preparation of yeast sample> 150 mg of the dried powder obtained in the above <Cultivation and drying of yeast> was suspended in 1.5 mL of sterilized water and placed in a tube. The cell walls were disrupted by grinding using a bead cell disrupter (Tomy Medico, Micro Smash™ MS-100R), and then freeze-dried to obtain a disrupted product with the contents eluted. 90 mg of the obtained yeast powder was dissolved in 1 mL of pure water at pH 4.5. The yeast sample was transferred to a tube whose gas phase had been replaced in an anaerobic chamber beforehand, and a yeast sample was obtained. The obtained yeast sample contained both the cell wall and the extract. The obtained yeast sample contained both the yeast cell wall and the yeast extract, with the proportion of yeast extract being approximately 5% by mass.

[0051] 1. Human Study (1) Preparation and Culture of Fecal Suspension Six subjects (healthy adults) submitted feces. Fresh feces were used to prepare the fecal suspension. The feces were quickly transferred to an anaerobic chamber, homogenized, and then added to anaerobic culture medium (YCFA medium, composition see International Publication No. 2022 / 050374) to create the same final concentration (0.1 mass volume percent concentration [w / v]). A yeast sample was dissolved in ultrapure water from which oxygen had been removed by bubbling carbon dioxide. The resulting yeast lysate was added to the fecal suspension to create the same final yeast concentration (0.3 mass volume percent concentration [w / v]). The yeast-added liquid was mixed to create a fecal culture medium, which was then dispensed in 300 μl aliquots into a 96-well plate and cultured for 16 hours at 37°C under anaerobic conditions (mainly containing nitrogen) (yeast-added group). Separately, an equal volume of ultrapure water was added to the fecal suspension, mixed, and similarly dispensed and cultured (control). After culturing, the fecal culture was centrifuged to precipitate the bacterial cells. The resulting pellet was used for nucleic acid extraction for the microbiome analysis described below in (2), and the supernatant was used for metabolite extraction for the metabolome analysis described below in (3).

[0052] (2) Nucleic Acid Extraction and Analysis To clarify the bacterial phylogenetic composition, metagenomic analysis using 16S rRNA gene sequences was performed on each stool culture obtained above. The metagenomic analysis was performed according to the method described by Murakami et al. (Evidence-Based Complementary and Alternative Medicine, Volume 2015, Article ID 824395). First, DNA was extracted from the centrifugal pellet of the stool culture. Using the extracted DNA as a template, DNA fragments from the V1-V2 region of the 16S rRNA gene were amplified by PCR. The PCR products were then sequenced using the paired-end method with an Illumina MiSeq. The bacterial phylogenetic composition was calculated from the resulting sequences according to the QIIME2 workflow. First, primer sequences were deleted using cutadapt, followed by 3'-terminal deletion, removal of PhiX-derived sequences, and quality filtering to obtain high-quality sequences. Afterwards, noise removal (correction of sequencing errors), paired-end sequence merging, and chimeric sequence removal were performed to obtain representative ASV (Amplicon Sequence Variant) sequences. The bacterial genus of these representative sequences was then identified using the Naive Bayes classifier in QIIME2. In this analysis, the representative sequences obtained by clustering the SILVA SSU Ref provided by Silva (https: / / www.arb-silva.de) at a 99% threshold were used as training data. Genus compositions were created from the ASV compositions obtained using the above method. The results are shown in Figures 1 and 2. In Figures 1 and 2, "Unclassified" and "Uncultured" refer to bacterial genera not listed in the database.

[0053] Furthermore, for each test product added to the medium, a comparison was made between the control (no test product added) and the yeast-added group, and the presence or absence of bacteria that showed a significant increase or decrease in relative occupancy (p<0.05) was confirmed using a Wilcoxon signed rank test. The results are shown in Table 1.

[0054]

[0055] As shown in Table 1, the addition of yeast significantly increased short-chain fatty acid-producing bacteria, such as Eubacterium ventriosum and Ruminococcus torques, as well as beneficial bacteria such as Parabacteroides, Bacteroides, and Fusicatenibacter. Furthermore, the harmful bacteria Tyzzerella and Eggerthella were significantly reduced.

[0056] (3) Metabolomic Analysis Pipeline To evaluate the effect of yeast addition on metabolic reactions derived from the gut microbiota, we quantitatively evaluated the metabolites contained in the supernatant of fecal culture broth. First, as described above in (1), the supernatant obtained by centrifuging the fecal culture broth was supplemented with an internal standard for concentration and elution time correction, and then filtered through a tube with a filter unit to separate the samples for organic acid / short-chain fatty acid measurement and bile acid measurement. The sample for organic acid / short-chain fatty acid measurement was derivatized according to the method described in WO 2022 / 050374, while the sample for bile acid measurement was used directly. Metabolites were measured using a liquid chromatograph time-of-flight mass spectrometer (LCTOF / MS), and the column retention time, mass-to-charge ratio (m / z), and peak area of ​​the detected peaks were obtained. The metabolites corresponding to each peak were identified by comparing this information with the measurement results of a standard sample. These peaks were corrected so that the area ratio with the internal standard was constant for each sample, and converted into values ​​(relative area) that allow relative quantification between stool cultures. Absolute quantification was performed by comparing with a calibration curve prepared using standard samples of known concentrations using the above method. The quantitative results for acetic acid and propionic acid are shown in Figure 3, and the quantitative results for the secondary bile acid lithocholic acid (LCA) are shown in Figure 4.

[0057] As shown in Figure 3, the addition of specific yeast to the fecal suspension significantly increased acetic acid and propionic acid. Furthermore, the secondary bile acid lithocholic acid (LCA) in the fecal suspension significantly decreased. As described above, acetic acid produced by intestinal bacteria is known to have anti-obesity and anti-inflammatory effects, as well as to prevent infection by O157. Furthermore, propionic acid produced by intestinal bacteria is known to improve glucose tolerance, suppress obesity, suppress inflammation, and inhibit cancer. It has also been reported that excessive secretion of lithocholic acid (LCA) in the intestine promotes colon cancer. As described above, the ingestion of specific yeast is expected to improve the production of acetic acid and propionic acid, thereby improving the effects of anti-obesity, anti-inflammatory, infection prevention, improved glucose tolerance, and cancer prevention or suppression in the intestine. Furthermore, the reduction of lithocholic acid, a carcinogen, is expected to inhibit or prevent colon cancer. These findings demonstrate that specific yeast is effective as an intestinal environment improver.

[0058] 2. Human Test A double-blind crossover comparative study was conducted. The test food was a food containing a specific yeast powder, and the specific yeast powder was the yeast sample produced by the above-mentioned <Preparation of Yeast Sample>.

[0059] The test food was a hard capsule (10 capsules / day, 230 mg of each capsule) containing 83% by mass of specific yeast powder. The other ingredients in the food, other than the specific yeast, were 16% by mass of dextrin and 1% by mass of calcium stearate. The control food was the same as the test food, except that it did not contain the specific yeast powder but used dextrin instead.

[0060] Sixteen people who met the conditions shown in Table 2 were selected as subjects. These 16 people were divided into two groups, with eight people in the first group (three men and five women) and eight people in the second group (three men and five women) equalized in terms of age, sex, BMI, and defecation frequency, and they ingested the test diet / control diet according to the following schedule: Group 1: 2 weeks of control diet intake → 2 weeks of no intake → 2 weeks of test diet intake Group 2: 2 weeks of test diet intake → 2 weeks of no intake → 2 weeks of control diet intake The intestinal flora, defecation frequency, and amount of putrefactive products in the intestines of each of the 16 people were investigated using the following methods at the start of each 2-week intake period (0W) and at the end of the period (2W).

[0061]

[0062] <Intestinal bacterial flora> Each fecal sample was placed in a stool collection container and stored frozen until DNA extraction.

[0063] The DNA extracted from the feces obtained above was subjected to metagenomic analysis using 16S rRNA gene sequencing. The method for fecal metagenomic analysis was as follows: Takahashi S, Tomita J, Nishioka K, Hisada T, Nishijima M. Development of a prokaryotic universal primer for simultaneous analysis of Bacteria and Archaea using next-generation sequencing. PLoS One 2014;9:e105592. Using the extracted DNA as a template, a DNA fragment from the V1-V2 region of the 16S rRNA gene was amplified by PCR. Primers used were Pro341FPro805R (bacteria and archaea 16S rDNA approximately 430 bp*). The PCR product sequences were then subjected to application sequencing analysis using an Illumina MiSeq. The ASV composition obtained above was analyzed. First, only bacterial genera detected in three or more individuals in both groups in the 0W and 2W test results were analyzed. Among these, t-tests were used to extract enterobacteria genera whose relative abundance changed significantly between the test food intake group and the control food intake group (p<0.05). Note that for comparisons between groups, a "paired t-test" was used as the basis, and for items that did not correspond between groups, an "unpaired t-test" was used.

[0064]

[0065] The analysis results are shown in Table 3. As shown in Table 3, the butyric acid-producing bacteria Anaerobutyricum increased compared to the control food intake group. The Eggerthella genus, which is thought to reduce glucose tolerance, decreased. This shows that the specific yeast improves the intestinal flora and the intestinal environment.

[0066] Table 4 shows the results of the above-mentioned intestinal microbiota analysis, limited to women. Table 4 also shows some bacterial genera that showed an increase in occupancy over time since the start of yeast feeding, although there was no significant difference from the control.

[0067] In women, as in the analysis of all subjects, the genus Anaerobutyricum increased compared to the control food group, while the genus Eggerthella, which is known to impair glucose tolerance, decreased. Furthermore, the genus Fusicatenibacter, which has the effect of suppressing intestinal immune responses, significantly increased compared to the control food group, and the genus Flavonifractor, which tends to increase during inflammation, significantly decreased. Furthermore, although there was no increase compared to the control food group, the genus Actinomyces, which may prevent cardiovascular disease and high blood pressure, increased with the addition of yeast.

[0068] (Measurement of putrefaction products) Fecal samples were accurately weighed into centrifuge tubes, and phosphate buffer containing an internal standard (4-isopropylphenol) was added and mixed, followed by heat treatment at 85°C for 15 minutes. After cooling, solvent extraction was performed using acetonitrile as the extraction solvent, and the mixture was purified using a solid-phase cartridge. The resulting extract was then analyzed by gas chromatography-mass spectrometry with selected ion monitoring (GC-MS-SIM). The results are shown in Table 5.

[0069] (Analysis conditions) System: GC-MS (5977A, Agilent Technologies, USA) Column: DB-WAX UI+G (60 m × 0.25 mm id, 0.25 μm film thickness, Agilent Technologies, USA) Column flow rate: 1.7 mL / min Column temperature: 70°C (hold 2 min) → 25°C / min → 190°C (hold 2 min) → 2°C / min → 200°C (hold 1.5 min) → 25°C / min → 240°C (hold 8 min) Carrier gas: Helium Injection mode: Splitless Injection port: 70°C → 200°C / min → 240°C Injection volume: 0.8 μL Ion source temperature: 230°C Interface temperature: 240°C

[0070]

[0071] As described above, the specific yeast of the present invention showed a tendency to inhibit the production of putrefactive products such as indole, phenol, and p-cresol.

[0072] (Measurement of valeric acid) Valeric acid in feces was measured using the following method. The results are shown in Table 6. A fixed amount of fecal specimen was precisely weighed into a bead tube, suspended in an extraction solution (ultrapure water), and then heat-treated (85°C, 15 minutes). After crushing with beads, the specimen was centrifuged (18,400 × g, 10 minutes). The supernatant was filtered through a membrane filter with a pore size of 0.20 μm to obtain a sample solution, which was then measured by high-performance liquid chromatography under the following conditions. System: Shimadzu Organic Acid Analysis System (Shimadzu, Japan) Column: Shim-pack Fast-OA, 100 mm × 7.8 mm ID, three columns in series. Guard column: Shim-pack Fast-OA, 10 mm × 4.0 mm ID. Eluent: 5 mmol / L p-toluenesulfonic acid. Reaction solution: 5 mmol / L p-toluenesulfonic acid, 100 μmol / L EDTA, 20 mmol / L Bis-Tris. Flow rate: 0.8 mL / min. Oven temperature: 50°C. Detector: Electrical conductivity detector CDD-10Avp.

[0073]

[0074] As shown in Table 6, the specific yeast of the present invention exhibited an effect of inhibiting the production of n-valeric acid.

[0075] (Number of days with defecation and number of defecations) The subjects were asked to report the number of days with defecation and the number of defecations for one week before the start of test food intake, the first week of the test food intake period, and the last week of the test food intake period on days 0 (0W), 7 (1W), and 14 (2W) of test food intake. The results are shown in Table 7.

[0076]

[0077] The subjects were also asked to indicate whether their bowel movements were frequent or infrequent on day 0 (0W) and day 14 (2W) of test diet intake, and to indicate which of the following items in Table 8 they corresponded to. The results are shown in Table 8.

[0078]

[0079] As described above, the intestinal environment improving agent of the present invention can effectively improve bowel movements.

[0080] 3. Human Test A double-blind crossover comparative study was conducted. The test food was a food containing a specific yeast powder, and the specific yeast powder was the yeast sample produced by the above-mentioned <Preparation of Yeast Sample>.

[0081] The test food was a hard capsule (10 capsules / day, 233 mg of each capsule) containing 83% by weight of specific yeast powder. The other ingredients in the food, other than the specific yeast, were 16% by weight of dextrin and 1% by weight of calcium stearate. The control food was the same as the test food, except that it did not contain the specific yeast powder and instead contained 99% by weight of dextrin.

[0082] Thirty-four Japanese men and women aged 20 to 65 years, who met the criteria listed in Table 2 and had a bowel movement frequency of 3 to 5 times per week, were enrolled. These 34 individuals were divided into two groups: Group 1 (17 men, 5 men, 12 women) and Group 2 (17 men, 5 men, 12 women), with equal distribution of age, sex, BMI, and bowel movement frequency. They were administered the test and control diets according to the following schedule: Group 1: 4 weeks of control diet followed by 4 weeks of no diet followed by 4 weeks of test diet; Group 2: 4 weeks of test diet followed by 4 weeks of no diet followed by 4 weeks of control diet. Feces were collected from each of the 34 individuals before the start of test food intake and on days 25 to 28 after the start of test food intake (33 valid subjects for analysis). The collected feces were analyzed for genus and species of intestinal microbiota using the same method as described above. The short-chain fatty acid content of the collected feces was also measured using the following method, and putrefactive products and other analyses were also performed using the same method as described above. Furthermore, the pH of the feces was measured using the following method. The results are shown below.

[0083] (Genus analysis of intestinal microbiota)

[0084] The main genera that showed changes in the between-group comparison in the genus analysis are shown in Table 9, Figures 5, and 6. As shown in Table 9 and Figure 5, the genus Phocaeicola, which is a pathogenic infectious bacterium with a relatively high abundance in feces, was significantly reduced by ingestion of the specific yeast. Furthermore, as shown in Table 9 and Figure 5, the genus Dialister is a propionibacterium-producing bacterium, and the genus Parabacteroides is a short-chain fatty acid-producing bacterium, both of which were relatively abundant, and were significantly increased by ingestion of the specific yeast. Similarly, the genus Muricomes, a short-chain fatty acid-producing bacterium (acetic acid bacterium), was significantly increased (Figure 6). Furthermore, Lachnoclostridium, a bacterium that is increased in patients with heart failure and atrial fibrillation, was significantly reduced by ingestion of the specific yeast (Figure 5).

[0085] (Species analysis of gut microbiota)

[0086] Table 10 and Figures 7 and 8 show the bacterial species that showed significant differences between groups after four weeks of intake. As can be seen from Table 10 and Figure 7, the abundance of Blautia faecis, a bacterium that produces lactic acid and acetic acid through glucose fermentation, and Parabacteroides distasonis, a bacterium that produces short-chain fatty acids, was relatively high. Furthermore, the abundance of Muricomes intestini, an acetic acid bacterium, also increased significantly (Figure 8). Meanwhile, the abundance of Clostridium scindens, which converts primary bile acids into secondary bile acids involved in the progression and contribution of colorectal cancer, decreased in the test food group compared to the control food group.

[0087] (Measurement of short-chain fatty acids) The amounts of short-chain fatty acids (succinic acid, lactic acid, acetic acid, propionic acid, iso-butyric acid, n-butyric acid, and iso-valeric acid) in feces were measured using the following method. The results are shown in Table 11 and Figure 9. A certain amount of fecal sample was precisely weighed into a bead tube, suspended in extraction solution (ultrapure water), and then heat-treated (85°C, 15 minutes). After disruption with beads, the sample was centrifuged (18,400 × g, 10 minutes). The supernatant was filtered through a membrane filter with a pore size of 0.20 μm to obtain a sample solution, which was then measured by high-performance liquid chromatography under the following conditions. System: Shimadzu Organic Acid Analysis System (Shimadzu, Japan) Column: Shim-pack Fast-OA, 100 mm × 7.8 mm ID, three columns in series. Guard column: Shim-pack Fast-OA, 10 mm × 4.0 mm ID. Eluent: 5 mmol / L p-toluenesulfonic acid. Reaction solution: 5 mmol / L p-toluenesulfonic acid, 100 μmol / L EDTA, 20 mmol / L Bis-Tris. Flow rate: 0.8 mL / min. Oven temperature: 50°C. Detector: Electrical conductivity detector CDD-10Avp.

[0088]

[0089] As shown in Table 11 and Figure 9, the amount of acetic acid, propionic acid, n-butyric acid, etc. in the feces increased by ingestion of the test food. The total amount of the measured values ​​of acetic acid, propionic acid, and n-butyric acid in the feces is shown as the amount of short-chain fatty acids in Table 12. As shown in Table 12, the amount of short-chain fatty acids in the feces of the test food intake group was 16% higher than that of the control food intake group four weeks after the start of the test.

[0090]

[0091] The results of measuring the amount of putrefactive products in the feces are shown in Table 13 and Figure 10. As shown in Table 13 and Figure 10, the amounts of phenol, p-cresol, 4-ethylphenol, indole, and skatole all decreased when the test food was consumed compared to when the control food was consumed. In particular, significant decreases were observed in p-cresol and skatole in the test food group after 4 weeks compared to the control food group.

[0092] (Fecal pH) 1 g of collected feces was suspended in 10 ml of water, and the pH of the suspension was measured at 25° C. The results are shown in Figure 11 and Table 14. As shown in Figure 11 and Table 14, a significant decrease in pH was confirmed in the test food group after 4 weeks compared to the control food group.

[0093]

Claims

1. An agent for improving the intestinal environment, which contains yeast deposited at the National Institute of Technology and Evaluation under international deposit number NITE BP-04012.

2. The intestinal environment improving agent according to claim 1, which is an agent for improving the intestinal flora.

3. The intestinal environment improving agent according to claim 1, which is an agent for suppressing the increase or decrease of short-chain fatty acid-producing bacteria or an agent for producing short-chain fatty acids.

4. The intestinal environment improving agent according to claim 1, which is an inhibitor of putrefaction product production.

5. The intestinal environment improving agent according to claim 4, wherein the putrefactive product is one or more selected from indole, phenol, p-cresol, 4-ethylphenol, and skatole.

6. The intestinal environment improving agent according to claim 1, which is used to increase or suppress the decrease of at least one species selected from the genera Anaerococcus, Anaerobutyricum, Bacteroides, Blautia, Dialister, Enterocloster, Haemophilus, Fusicatenibacter, Muricomes, and Parabacteroides, or to decrease or suppress the increase of at least one species selected from the genera Bilophila, Butyricimonas, Eggerthella, Flavonifractor, Intestinimonas, Lachnoclostridium, Massiliimalia, Phocaeicola, Streptococcus, and Tyzzerella.

7. The intestinal environment improving agent according to claim 1, which is used to suppress the increase or decrease of at least one species selected from Anaerococcus vaginalis, Blautia faecis, Enterocloster citroniae, Eubacterium ventriosum, Haemophilus parainfluenzae, Muricomes intestini, Parabacteroides distasonis, and Ruminococcus torques, or to reduce or suppress the increase of at least one species selected from Anaerococcus aminivorans, Bacteroides stercoris, Butyricimonas paravirosa, Clostridium scindens, Intestinimonas massiliensis, Enterocloster lavalensis, Lachnochrostridium Phocaeicola, Massiliimalia timonensis, and Streptococcus vestibularis.

8. The intestinal environment improving agent according to claim 1, which is a laxative.

9. The intestinal environment improving agent according to claim 1, which is a secondary bile acid production inhibitor.

10. The intestinal environment improving agent according to claim 1, which is an inhibitor of valeric acid production.

11. The intestinal environment improving agent according to claim 1, which is an agent for lowering intestinal pH.

12. Use of yeast deposited with the National Institute of Technology and Evaluation under international deposit number NITE BP-04012 for the production of an agent for improving the intestinal environment.

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