Intestinal microbiota improving agent

Abstract

To provide an intestinal bacterial flora improver.SOLUTION: An intestinal bacterial flora improver contains royal jelly.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an intestinal microbiota improving agent.

Background Art

[0002] The intestinal environment, which is determined by the composition and the like of the intestinal microbiota, is known to affect various health conditions, and it is important to maintain the intestinal environment in a good state. Therefore, materials for improving the intestinal environment have been demanded and investigated.

[0003] For example, Patent Document 1 discloses that by orally ingesting a composition containing a black soybean seed coat extract, the intestinal microbiota can be improved, the abundance ratio of short-chain fatty acid-producing bacteria in intestinal bacteria can be increased, the abundance ratio of equol-producing bacteria in intestinal bacteria can be increased, and the abundance ratio of pathogenic bacteria in intestinal bacteria can be reduced.

[0004] In addition, Patent Document 2 reports that water-soluble dietary fibers, particularly indigestible dextrin, increase bacteria belonging to the genus Faecalibacterium in the human intestine.

[0005] On the other hand, today, apicultural products are widely used in health foods, pharmaceuticals, cosmetics and the like, and various pharmacological actions such as vasodilation, hypotensive action and antibacterial action, as well as nutritional physiological actions, have been reported.

[0006] For example, propolis, which is one type of apicultural product, has been used in people's lives since ancient times, and in Europe, it is still used for people's health together with other apicultural products such as royal jelly, honey, and bee pollen, as apitherapy (therapy using apicultural products). Propolis is composed of a wide variety of natural components such as flavonoids, terpenoids, organic acids, amino acids, polysaccharides, and minerals.

[0007] Patent Document 3 discloses an agent containing propolis as an active ingredient for adjusting the number of bacteria in the intestines of at least one family or genus selected from the group consisting of Ruminococcusceae, Catenibacterium, Subdrigranulum, Family XIII AD3011 group, and Anaeroturuncus.

[0008] Royal jelly, also a type of beekeeping product, is a milky substance synthesized in the salivary glands of honeybees and secreted from the Mandebler's gland in their heads. Worker bees feed it to larvae placed in cells for queen bee larvae in order to create queen bees. Currently, royal jelly is widely used worldwide in health foods, pharmaceuticals, and cosmetics, and its reported effects include increased blood flow, lowering blood pressure, growth promotion, sex hormone-like effects, antibacterial effects, antitumor effects, wound healing promotion, treatment of autonomic nervous system disorders, anti-stress effects, calcium absorption promotion, blood sugar lowering effects, and antioxidant effects. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2021-155375 [Patent Document 2] Japanese Patent Publication No. 2020-083830 [Patent Document 3] Japanese Patent Publication No. 2021-195334 [Overview of the project] [Problems that the invention aims to solve]

[0010] The present invention aims to provide an agent for improving the intestinal microbiota. [Means for solving the problem]

[0011] The inventors conducted extensive research to achieve the above objectives and, as a result, prepared 10 types of stool samples categorized by the type of intestinal bacterial composition. They cultured these samples with and without royal jelly and analyzed the bacterial flora. The results showed that the addition of royal jelly increased three types of beneficial bacteria (Bifidobacterium, Blautia, and Fusicatenibacter) and decreased three types of harmful bacteria (Bilophila, Eggerthella, and Fusobacterium). Thus, it was found that royal jelly has the potential to improve the intestinal environment.

[0012] Based on these findings and further research, this invention was completed and provides the following intestinal microbiota improving agent.

[0013] Item 1. An intestinal microbiota improving agent containing royal jelly. Item 2. The agent described in Item 1, used to regulate the number in the intestines of at least one species selected from the group consisting of Bifidobacterium, Blautia, Fusicatenibacter, Virophila, Egasella, and Fusobacterium in the intestinal microbiota. Item 3. Used to increase the proportion of at least one species selected from the group consisting of Bifidobacterium, Blautia, and Fusicatenibacter in the intestinal flora, and / or Used to reduce the proportion of at least one species selected from the group consisting of *Virophila*, *Egasella*, and *Fusobacterium* in the gut microbiota. The agent described in item 1. Item 4. Used to increase the proportion of Bifidobacterium, Blautia, and Fusicatenibacter species in the intestinal flora, and to decrease the proportion of Virophila, Egasella, and Fusobacterium species. The agent described in item 1. Item 5. A food or beverage, pharmaceutical or quasi-drug, as described in any one of items 1 to 4. [Effects of the Invention]

[0014] Royal jelly is useful as an active ingredient of an intestinal microbiota improving agent. The intestinal microbiota improving agent of the present invention can increase the proportion of beneficial bacteria and decrease the proportion of harmful bacteria in the intestinal microbiota, and in particular, can increase the proportion of beneficial bacteria belonging to the genera Bifidobacterium, Blautia, and Fusicatenibacter in the intestinal microbiota, and decrease the proportion of harmful bacteria belonging to the genera Bilophila, Eggerella, and Fusobacterium.

[0015] In addition, royal jelly has been conventionally used as a food material, and thus has high safety. [Brief Description of the Drawings]

[0016] [Figure 1] This is a graph showing the results of fluctuation analysis of intestinal bacteria (genera Bifidobacterium, Blautia, Fusicatenibacter, Bilophila, Eggerella, and Fusobacterium) with and without the addition of royal jelly. The vertical axis represents the relative abundance ratio, left: no royal jelly added, right: royal jelly added [Mode for Carrying Out the Invention]

[0017] Hereinafter, the modes for carrying out the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0018] The intestinal microbiota improving agent of the present invention is characterized by containing royal jelly as an active ingredient (hereinafter, may also be referred to as "the agent of the present invention").

[0019] One embodiment of the agent of the present invention relates to an agent for regulating the intestinal population of enteric bacteria belonging to a specific genus. Specifically, one embodiment of the agent of the present invention relates to an agent used to regulate the intestinal population of at least one enteric bacterium selected from the group consisting of bacteria of the genus Bifidobacterium, bacteria of the genus Blautia, bacteria of the genus Fusicatenibacter, bacteria of the genus Bilophila, bacteria of the genus Eggerthella, and bacteria of the genus Fusobacterium in the intestinal microbiota. Regulating the intestinal population of the above bacteria includes increasing or decreasing the intestinal population of these bacteria. In addition, regulating the intestinal population of the above bacteria also includes increasing or decreasing the abundance ratio of these bacteria in the intestinal microbiota.

[0020] As used herein, the description of "bacteria of the genus ~" such as bacteria of the genus Bifidobacterium shall mean "bacteria belonging to the genus ~" as in bacteria belonging to the genus Bifidobacterium.

[0021] More specifically, one embodiment of the agent of the present invention is used to increase the proportion of at least one species selected from the group consisting of Bifidobacterium bacteria, Blautia bacteria, and Fusicatenibacter bacteria in the intestinal microbiota, and / or to reduce the proportion of at least one species selected from the group consisting of Bilophila bacteria, Eggerthella bacteria, and Fusobacterium bacteria in the intestinal microbiota.

[0022] Even more specifically, one embodiment of the agent of the present invention relates to an agent used for increasing the proportions of Bifidobacterium bacteria, Blautia bacteria, and Fusicatenibacter bacteria in the intestinal microbiota, and reducing the proportions of Bilophila bacteria, Eggerthella bacteria, and Fusobacterium bacteria.

[0023] The species of Bifidobacterium, Blautia, Fusicatenibacter, Virophila, Egasella, and Fusobacterium are not particularly limited. Examples of these bacteria include species that exist as commensal bacteria in the gut.

[0024] Of these, Bifidobacterium, Blautia, and Fusicatenibacter are considered beneficial to humans, while Virophila, Egasella, and Fusobacterium are considered harmful to humans. Bifidobacterium bacteria prevent O157:H7 infection and improve bowel movements through acetic acid production (Fukuda et al., 2011, Nature, 469(7331):543-547; Ogata et al., 1997, Biosci Microflora, 16(2):53-58); Blautia bacteria decrease in the intestines of patients with multiple diseases and show a negative correlation with visceral fat area (Larsen et al., 2010, PLoS One, 5(2):e9085; Kakiyama et al., 2013, J Hepatol, 58(5):949-955; Ozato et al., 2019, NPJ Biofilms Microbiomes, 5(1):28); and Fusicatenibacter bacteria have an anti-inflammatory effect in the intestines (Takeshita et al., 2016, Inflamm Bowel). Dis, 22(12):2802-2810., *Virophila* species are associated with deoxycholic acid and impaired glucose tolerance (Yachida et al., 2019, Nat Med, 25(6):968-976.; Natividad et al., 2018, Nat Commun, 9(1):2802.), and *Egasella* species are associated with inactivation of cardiac stimulants, type 2 diabetes, and induction of inflammation through Th17 activation (Haiser et al., 2013, Science, 341(6143):295-298.; Koh et al., 2018, Cell, 175(4):947-961.e17.; Alexander et al., 2022, Cell Host). Microbe, 30(1):17-30.e9.) Fusobacterium species are thought to be correlated with increased levels in the intestines of colorectal cancer patients and with the progression of colorectal cancer (Castellarin et al., 2012, Genome Res, 22(2):299-306.; Kostic et al., 2013, Cell Host Microbe, 14(2):207-215.).

[0025] From the above perspective, the intestinal microbiota improving agent according to this embodiment can improve the intestinal environment and prevent or improve various diseases. That is, one embodiment of the present invention can be said to provide an agent for suitably adjusting the intestinal environment by adjusting the number of the above-mentioned intestinal bacteria in the intestines. This intestinal microbiota improving agent can be used for purposes such as preventing, improving, or treating the above-mentioned diseases or symptoms by increasing or decreasing the number of the above-mentioned bacteria in the intestines. That is, one embodiment of the present invention can also be said to provide an agent for use in these purposes.

[0026] Royal jelly is a milky white, jelly-like substance produced by mixing secretions from the hypopharyngeal gland and mandibular gland of worker bees aged 3 to 12 days. The main physiologically active components in royal jelly include, for example, organic acids such as 10-hydroxy-2-decenoic acid (hereinafter referred to as "decenoic acid"), which is unique to royal jelly, as well as proteins, lipids, sugars, B vitamins, vitamins such as folic acid, nicotinic acid, and pantothenic acid, and various minerals. The royal jelly used in this invention includes raw royal jelly, concentrated or diluted royal jelly, dried royal jelly, dried royal jelly powder, enzyme-treated royal jelly, and royal jelly extract. things It contains [various ingredients]. Furthermore, the royal jelly may be sourced from any of the following countries: European countries, Oceania countries, the United States, Brazil, Japan, China, or other Asian countries.

[0027] Royal jelly concentrate can be obtained, for example, by removing water from raw royal jelly. Royal jelly dilution can be obtained, for example, by adding water to raw royal jelly.

[0028] Dried royal jelly powder is obtained by drying and pulverizing fresh royal jelly. Any known drying method used in general food processing can be used, such as natural drying (air drying or sun drying), forced drying (heating by electricity, etc.), or freeze-drying. Freeze-drying is preferred. The drying time is not particularly limited; for natural drying (air drying or sun drying), it takes about 3 days, and for forced drying (heating by electricity, etc.), it takes about 1 to 3 days at around 50°C. It is generally preferable to dry the royal jelly so that the moisture content is 10% by mass or less, preferably 5% by mass or less. If it is difficult to reduce the moisture content to 10% by mass or less, as in the case of natural drying (air drying or sun drying), the royal jelly may be further reduced by freeze-drying. Alternatively, royal jelly powder may be obtained by grinding the royal jelly in a pulverizer (e.g., pin mill, hammer mill, ball mill, jet mill) after freeze-drying or spray-drying.

[0029] Enzyme-treated royal jelly is royal jelly treated with a protease (protein-degrading enzyme). Preferably, it is a low-allergen enzyme-treated royal jelly in which allergic reactions caused by proteins contained in royal jelly are suppressed by the protease treatment. Therefore, the components contained in enzyme-treated royal jelly include not only the protease hydrolysates of proteins contained in royal jelly, but also organic acids such as 10-hydroxy-2-decenoic acid, lipids, sugars, vitamins, and various minerals.

[0030] The royal jelly used in the production of enzyme-treated royal jelly is not particularly limited and may include, for example, raw royal jelly, royal jelly powder obtained by drying and pulverizing raw royal jelly, or raw royal jelly extracted with water or aqueous ethanol.

[0031] Enzyme-treated royal jelly can be produced by treating royal jelly raw material with an enzyme having at least endopeptidase activity, an enzyme having at least exopeptidase activity, and / or an enzyme having both endopeptidase and exopeptidase activity.

[0032] Examples of proteolytic enzymes that possess at least endopeptidase activity include endopeptidases derived from animals (e.g., trypsin, chymotrypsin, etc.), plants (e.g., papain, etc.), and microorganisms (e.g., lactic acid bacteria, yeast, mold, Bacillus subtilis, actinomycetes, etc.).

[0033] Examples of proteolytic enzymes that possess at least exopeptidase activity include carboxypeptidases, aminopeptidases, exopeptidases derived from microorganisms (e.g., lactic acid bacteria, Aspergillus species, Rhizopus species, etc.), and pancreatin and pepsin, which also possess endopeptidase activity.

[0034] Among these various enzymes, preferred examples of enzymes possessing both exopeptidase activity and endopeptidase activity include Streptomyces griseus-produced peptidase (product name: Actinase AS), Aspergillus oryzae-produced peptidase (product names: Protease A, Flavorzyme, Proteax), and Aspergillus melleus-produced peptidase (product name: Protease P). Preferred examples of enzymes possessing exopeptidase activity include Aspergillus oryzae-produced peptidase (product names: Umamizyme G, Promod 192P, Promod 194P, Sumizyme FLAP), and Aspergillus soe (Aspergillus Examples include peptidases produced by *Aspergillus sojae* (product name: Sternzyme B15024), peptidases produced by *Aspergillus* species (product name: Cochlase P), and peptidases produced by *Rhizopus oryzae* (product name: Peptidase R). Furthermore, preferred examples of enzymes possessing endopeptidase activity include peptidase produced by Bacillus subtilis (trade names: Orientase 22BF, Nucleicin), peptidase produced by Bacillus licheniformis (trade name: Alcalase), peptidase produced by Bacillus stearothermophilus (trade name: Protease S), peptidase produced by Bacillus amyloliquefaciens (trade name: Neutralase), and peptidase produced by Bacillus species (trade name: Protamex).

[0035] Enzymatic treatment to reduce the allergenicity of royal jelly can be carried out, for example, in accordance with the descriptions in Japanese Patent Publication No. 2007-295919 and Japanese Patent Publication No. 2007-295920.

[0036] Royal jelly extract is obtained by extracting royal jelly (including raw, dried, and pulverized materials) with water or aqueous ethanol. The extraction time can be appropriately set depending on the form of royal jelly used as a raw material, the type and amount of solvent, the temperature and stirring conditions during extraction, etc. After extraction, solid components may be removed by filtration, centrifugation, etc. The extracted solution may be used as is, or the solvent may be removed from the solution and used as a concentrated liquid or powder. Royal jelly ethanol extract is preferred as the royal jelly extract.

[0038] Other known components may be appropriately incorporated into the preparation of the present invention, as long as they do not interfere with the effects of the present invention.

[0039] The royal jelly content in the preparation of the present invention is not particularly limited as long as the effects of the present invention are obtained, and can be appropriately adjusted depending on the final form, etc., relative to the total amount of solids in the preparation, for example, 0.1% by mass or more, 0.2% by mass or more, 0.5% by mass or more, 1% by mass or more, 3% by mass or more, 5% by mass or more, 7% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, 95% by mass or more. % by mass or more, 98% by mass or more, 99% by mass or more, 99.5% by mass or more, 100% by mass or less, 99.5% by mass or less, 99% by mass or less, 98% by mass or less, 95% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass The amount may be % or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, 7% by mass or less, 5% by mass or less, 3% by mass or less, 1% by mass or less, 0.5% by mass or less, 0.2% by mass or less, 0.1% by mass or less.

[0040] Furthermore, royal jelly is preferably applied regularly to obtain the effects of the present invention, and is particularly preferably applied once a day or in several divided doses. While not particularly limited, in terms of the amount of active ingredient as raw royal jelly, for example, per day for an adult weighing 60 kg, the amounts are: 10 mg or more, 100 mg or more, 150 mg or more, 200 mg or more, 300 mg or more, 500 mg or more, 800 mg or more, 1000 mg or more, 1200 mg or more, 1600 mg or more, 2000 mg or more, 2400 mg or more, 2800 mg or more, 3200 mg or more, 3600 mg or more, 4000 mg or more, 4800 mg or more, 540 mg The dose may be 0 mg or more, 6000 mg or more, 6600 mg or more, or 7200 mg or more, and may be 15000 mg or less, 10000 mg or less, 8000 mg or less, 7200 mg or less, 6600 mg or less, 6000 mg or less, 5400 mg or less, 4800 mg or less, 4000 mg or less, 36000 mg or less, 3000 mg or less, 2800 mg or less, 2400 mg or less, 2000 mg or less, 1000 mg or less, 800 mg or less, or 500 mg or less. The dose can be appropriately set within the above range depending on factors such as the health condition of the person being administered the drug, the method of administration, and combinations with other drugs.

[0041] The agent of the present invention can be used in food and beverages (especially food and beverages intended for health promotion, health maintenance, etc. (e.g., health foods, functional foods, nutritional compositions, nutritional supplements, supplements, health foods, foods for specified health uses, nutrient function foods, or foods with functional claims)), quasi-drugs, pharmaceuticals, etc. Furthermore, the agent of the present invention also encompasses the meaning of an additive that imparts an effect of improving the intestinal microbiota.

[0042] The above-mentioned food and beverages may use the royal jelly as is, or, if necessary, may be combined with minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, flavorings, stabilizers, gelling agents, preservatives, sustained-release regulators, surfactants, solvents, humectants, etc.

[0043] Food and beverages include all foods and beverages that can be consumed by animals (including humans). The types of food and beverages are not particularly limited and include, for example, dairy products; fermented foods (yogurt, etc.); beverages (soft drinks such as coffee, juice, and tea, milk drinks, lactic acid bacteria drinks, lactic acid bacteria drinks, yogurt drinks, carbonated drinks, sake, Western liquors, fruit wines, etc.); spreads (custard cream, etc.); pastes (fruit paste, etc.); Western sweets (chocolate, donuts, pies, cream puffs, gum, gummies, jelly, candy, cookies, cakes, puddings, etc.); Japanese sweets (daifuku, mochi, manju, castella, anmitsu, yokan, etc.); frozen desserts (ice cream, ice pops, sherbet, etc.); food products (curry, gyudon, zosui, miso soup, soup, meat sauce, pasta, pickles, jam, etc.); and seasonings (dressings, furikake, umami seasonings, soup bases, etc.).

[0044] The manufacturing method for food and beverages is not particularly limited and may be carried out according to publicly known methods as appropriate.

[0045] There are no particular restrictions on the dosage unit form when used as a supplement; it can be selected as appropriate. Examples include chewable tablets, lozenges, capsules, granules, liquids, powders, syrups, pastes, drinks, gummies, etc.

[0046] The above-mentioned pharmaceuticals may use only the royal jelly, or they may be used in combination with other medicinal ingredients listed in the Japanese Pharmacopoeia, such as vitamins and herbal medicines.

[0047] When preparing the agent of the present invention as a pharmaceutical product, the royal jelly can be prepared together with pharmaceutical-permissible components in the form of tablets (including uncoated tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, lozenges, etc.), capsules, pills, powders, granules, liquids, suspensions, emulsions, syrups, pastes, suppositories, and injections (including cases where the agent is mixed with distilled water or an intravenous solution such as an amino acid solution or an electrolyte solution at the time of use to prepare a liquid product) to create a pharmaceutical preparation.

[0048] The administration of the drug may be local or systemic. There are no particular restrictions on the method of administration; it may be administered orally or parenterally. Parenteral administration routes include intravenous or intra-arterial administration, and rectal administration.

[0049] In addition to royal jelly, the pharmaceutical product of the present invention may contain, as needed, pharmaceutically acceptable ingredients such as excipients, binders, disintegrants, lubricants, colorants, suspending agents, thickeners, antioxidants, absorption enhancers, pH adjusters, preservatives, stabilizers, surfactants, sweeteners, flavoring agents, and fragrances.

[0050] Furthermore, the pharmaceuticals of this invention also include quasi-drugs.

[0051] The agent of the present invention described above is applicable to mammals, including humans (preferably humans). The agent of the present invention may also be used in healthy individuals who are not suffering from any disease.

[0052] The agent of the present invention can increase the proportion of beneficial bacteria and decrease the proportion of harmful bacteria in the intestinal flora based on the action of royal jelly. In particular, it can increase the proportion of beneficial bacteria such as Bifidobacterium, Blautia, and Fusicatenibacter in the intestinal flora, while decreasing the proportion of harmful bacteria such as Virophila, Egasella, and Fusobacterium. Therefore, royal jelly is useful as an active ingredient in intestinal flora improving agents.

[0053] Furthermore, since royal jelly has been used as a food ingredient for a long time, it is considered highly safe.

[0054] In this specification, "comprise" encompasses both the meanings of "essentially consist of" and "consist of." [Examples]

[0055] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0056] <Method> A. Preparation of royal jelly 90 mg of royal jelly was weighed out in a fume hood. A negative control sample without royal jelly was also prepared. The weighed royal jelly was dissolved in 1 mL of pH 4.5 PureWater in an anaerobic chamber. The solution was then transferred through a 0.22 μm filter into a tube that had been pre-purged of gas in the anaerobic chamber.

[0057] B. Preparation of culture medium The composition of the YCFA medium was as follows (per 1L): Triptycase (2.5g), Peptone (2.5g), Yeast Extract (5g), K2HPO4 (0.9g), KH2PO4 (0.9g), NaCl (1.8g), NH4SO4 (1.8g), CaCl2·2H2O (0.24g), MgSO4·7H2O (0.375g), Potassium Acetate (1g), Sodium Propionate (0.5g), Sodium n-Butyrate (0.3g), Valeric Acid (0.1g), Isobutyrate (0.1g), Isovaleric Acid (0.1g), 2-Methyl Butyrate (0.1g), Citrate NH4Fe(III) (0.01g), MnCl2 (0.006g), CoCl2 (0.002g), NiCl2 (0.0004g), (NH4)6(Mo7O 24 )·4H2O (0.0004g), CuSO4 (0.00015g), AlK(SO4)2 (0.0003g), Na2B4O7 (0.0003g), ZnSO4 (0.002g), Na2SeO3 (0.0003g), Pyridoxal phosphate (0.002g), p-aminobenzoate (0.0005g), Biotin (0.0002g), Phenylpropionate (0.002g), l-Arginine (0.002g), Panvitan (0.1g), Cysteine ​​hydrochloride (1g), Resazurin (7-hydroxy-3H-phenoxazine-3-one-10-oxide) (0.005g), and Vitamin K (0.001g)

[0058] C. Preparation of fecal juice In an anaerobic chamber, 12 mg of stool was weighed into an Eppendorf tube that had been pre-purged of gas. 500 μL of YCFA medium prepared in B was added, homogenized, and then more YCFA medium was added to prepare stool juice. The stool concentration in the stool juice was 0.1% (w / v). In this example, stool samples were collected from 10 different healthy men and women.

[0059] D. Culture 10 μL of royal jelly prepared in A was added to each well of a 96-well plate. Next, 290 μL of fecal juice prepared in C was added to each well. The final concentration of royal jelly was 0.3% (w / v). The samples were then incubated in a 37°C incubator in an anaerobic chamber for 16 hours.

[0060] E. Variation Analysis Metagenomic analysis was performed using 16S rRNA gene sequences in stool cultures with and without royal jelly. The metagenomic analysis followed the method of Murakami et al. (Evid.Based.Complement Alternat.Med., 2015:824395). First, DNA extracted from centrifuged pellets of stool and stool-test compound mixtures was used as a template to amplify DNA fragments of the V1-V2 region of the 16S rRNA gene by PCR. Subsequently, the sequences of the PCR products were analyzed using the paired-end method with Illumina MiSeq. From the obtained nucleotide sequences, bacterial lineage composition was calculated according to the QIIME2 (version 2019.10) workflow. First, primer sequences were removed using cutadapt, then 3' end base deletion, PhiX-derived nucleotide sequence removal, and quality filtering were performed using DADA2 to obtain high-quality nucleotide sequences. Subsequently, noise reduction (correction of sequence errors), paired-end sequence merging, and chimeric sequence removal were performed using DADA2 to obtain representative ASV (Amplicon Sequence Variant) sequences. Next, the bacterial genera of these representative sequences were identified using the Naive Bayes classifier in QIIME2. In this analysis, sequences extracted from the V1-V2 region of representative sequences obtained by clustering the SILVA 132 SSU Ref provided by Silva with a 99% threshold were used as training data.

[0061] A comparative study was conducted between two groups—one without royal jelly supplementation and the other with royal jelly—using the Wilcoxon signed-rank test for each group of intestinal bacteria. The significance level was set at less than 5%.

[0062] <Result> Ten subjects with different gut microbiota were selected. While the gut microbiota of Japanese individuals can be categorized into three patterns, the ten subjects represented all of these patterns. Stool samples from the ten subjects were cultured with and without royal jelly added, and the gut microbiota was analyzed.

[0063] Profile analysis of the gut microbiota revealed that some individuals experienced changes in their gut microbiota depending on whether or not they added royal jelly, while others did not.

[0064] We conducted a change analysis for bacteria known from the literature to be beneficial or harmful to humans, comparing the levels with and without royal jelly. The results for bacteria that showed a statistically significant difference are shown in Figure 1. As shown in Figure 1, bacteria belonging to the genera Bifidobacterium, Blautia, and Fusicatenibacter, which are beneficial to humans, significantly increased with the addition of royal jelly, while bacteria belonging to the genera Virophila, Egasella, and Fusobacterium, which are harmful to humans, significantly decreased with the addition of royal jelly.

Claims

1. An intestinal microbiota improving agent containing royal jelly, Used to increase the proportion of at least one species selected from the group consisting of Blautia and Fusicatenibacter species in the intestinal flora, and / or An agent used to reduce the proportion of at least one species selected from the group consisting of *Virophila* and *Egasella* species in the intestinal flora.

2. Used to increase the proportion of Bifidobacterium, Blautia, and Fusicatenibacter species in the gut microbiota, and to decrease the proportion of Virophila, Egasella, and Fusobacterium species. The agent according to claim 1.

3. The agent according to claim 1 or 2, which is a food or beverage, a pharmaceutical product, or a quasi-drug.

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