Composition for regulating gut microbiota

Abstract

[Problem] To provide a novel composition having gut microbiota regulating effect. [Solution] A composition for regulating gut microbiota, the composition being characterized by containing a sulfated polysaccharide that induces gut bacteria interactions in a host and alters the profile of the microbiota so that bacteria contributing to improving the host's health proliferate and pathogens are suppressed. The sulfated polysaccharide is preferably rhamnan sulfate. It is also preferable that the gut microbiota regulation (1) augments at least one selected from the group consisting of Prevotellaceae UCG-001, the Clostridia vadinBB60 group, and Mucispirillum schaedleri and (2) decreases at least one selected from the group consisting of the Rikenellaceae RC9 gut group, ASF356, and Staphylococcus.

Description

Composition for regulating gut microbiota

[0001] The present invention relates to a composition for regulating gut microbiota, and particularly to those that induce interactions between gut bacteria of a host and change the profile of the microbiota so as to promote the growth of bacteria contributing to the improvement of the host's health and suppress pathogens.

[0002] The gut microbiota plays an important biological role in the physiological and pathological functions of the host throughout its life. There is a growing view that a healthier life can be achieved by making use of the gut microbiota. Since diet is important for the formation of the gut microbiota, attempts have been made to search for and develop substances that may be able to beneficially regulate the gut microbiota (Non-Patent Document 1, Patent Documents 1, 2). Ramnan sulfate (RS) is a substance composed of linear and branched chains of rhamnose, with approximately 25% of rhamnose being sulfated. Ramnan sulfate is mainly isolated from the cell wall of Monostroma nitidum, which is commercially cultivated in Japan. Ramnan sulfate exhibits numerous physiological activities such as anti-tumor, anti-obesity, anti-thrombotic, anti-inflammatory, anti-hyperglycemic, and anti-hypercholesterolemic properties, and thus has the potential to be effectively used for the treatment of diseases. Many bacterial species that make up the gut microbiota can break down polysaccharides containing ramnan sulfate. According to recent research, the gut microbiota is known to be involved in cancer, obesity, thromboembolism, immune regulation, diabetes, and lipid metabolism. The biological function of ramnan sulfate is presumed to be related to its regulatory effect on the gut microbiota. To support this presumption, there is a report that administration of ramnan sulfate to human subjects induced a significant change in the gut microbiota profile (Non-Patent Document 2).

[0003] JP-A No. 2024-114358, JP-A No. 2024-18368

[0004] Zang, L., Baharlooeian, M., Terasawa, M., Shimada, Y. &Nishimura, N. 2023. Beneficial effects of seaweed-derived components on metabolic syndrome via gut microbiota modulation. Front Nutr, 10, 1173225.Shimada, Y., Terasawa, M., Okazaki, F., Nakayama, H., Zang, L., Nishiura, K., Matsuda, K. & Nishimura, N. 2021. Rhamnan sulphate from greenalgae Monostroma nitidum improves constipation with gut microbiome alterationin double-blind placebo-controlled trial. Sci Rep, 11, 13384.

[0005] However, there is little data on the effects of rhamnan sulfate on the gut microbiota, and much research remains to be done. This invention was made in view of the above circumstances, and its purpose is to provide a novel composition that induces interactions between gut bacteria in the host, alters the profile of the bacterial flora to promote the growth of bacteria that contribute to the improvement of host health, and suppresses pathogens.

[0006] The inventors of the present invention basically completed the present invention by administering rhamnan sulfate to mice and analyzing the changes that occurred in the gut microbiota, finding that it caused significant changes in the bacterial profile, including specific bacteria. These bacteria are also known to exhibit anti-inflammatory effects. Thus, the gut microbiota regulating composition according to the present invention is characterized by containing a sulfated polysaccharide. In this case, it is preferable that the sulfated polysaccharide is rhamnan sulfate. It is also preferable that the composition is for oral administration. Furthermore, it is preferable that the regulation of the gut microbiota is (1) an increase in at least one selected from the group consisting of Prevotellaceae UCG-001, Clostridia vadin BB60 group, and Mucispirillum schaedleri, and (2) a decrease in at least one selected from the group consisting of Rikenellaceae RC9 gut group, ASF356, and Staphylococcus. The oral dosage of rhamnan sulfate is preferably 10 mg to 1000 mg per day, more preferably 30 mg to 500 mg, and even more preferably 50 mg to 300 mg. Furthermore, the composition of the present invention can be provided as a food or beverage for regulating the intestinal flora.

[0007] Compositions for regulating the intestinal flora are provided by combining an effective amount of rhamnan sulfate for regulating the intestinal flora with pharmaceutically acceptable carriers and additives. This composition is provided as a pharmaceutical or quasi-drug. The pharmaceutical composition is used internally or externally. This pharmaceutical composition can be used in formulations such as oral preparations, intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections and / or intraperitoneal injections, transmucosal applications, and transdermal applications. In particular, rhamnan sulfate is effective even when administered orally or transdermally, so it is preferably used as an oral preparation, transmucosal application, or transdermal application. The dosage form of the pharmaceutical composition can be appropriately determined, but examples include solid preparations such as tablets, granules, capsules, powders, and powders; liquid preparations such as solutions and suspensions; and semi-solid preparations such as ointments or gels.

[0008] In the food and beverage sector, rhamnan sulfate can be provided as a food ingredient by incorporating it into various foods. In this case, food and beverages can be provided that are labeled as such for regulating the intestinal flora. Examples of food and beverages include general foods, as well as foods for specified health uses, foods with nutritional function claims, foods with functional claims, foods for hospital patients, supplements, etc. Furthermore, the food and beverages of the present invention can also be used as food additives. Examples of food compositions include beverages (soft drinks, alcoholic beverages, carbonated drinks, milk beverages, fruit juices, tea, coffee, nutritional drinks, concentrated beverages, etc.), powdered beverages (powdered juices, powdered soups, etc.), confectionery (candies (throat lozenges), cookies, biscuits, gum, gummies, chocolate, etc.), bread, cereals, seasonings, etc.

[0009] In the case of Foods for Specified Health Uses, Foods with Nutrient Function Claims, and Foods with Function Claims, they can also be provided in the form of capsules, lozenges, syrups, granules, powders, etc. Foods for Specified Health Uses are foods that contain health-promoting functional ingredients that affect physiological functions, etc., and can be labeled as suitable for specific health uses with the permission of the Commissioner of the Consumer Affairs Agency. In the present invention, the food will be sold with a label indicating a specific use, such as the effect of regulating the intestinal flora. Foods with Nutrient Function Claims are foods used to supplement nutrients (vitamins, minerals), and display the function of the nutrients. In order to be sold as a Food with Nutrient Function Claims, the amount of nutrients contained in the recommended daily intake must be within the range of the specified upper and lower limits, and not only nutritional function claims but also cautionary statements, etc., must be made.

[0010] Foods with functional claims are foods that, under the responsibility of the business operator, display functional claims based on scientific evidence. Prior to sale, information regarding the safety and evidence of functionality is submitted to the Commissioner of the Consumer Affairs Agency. This invention contains rhamnan sulfate as an active ingredient and is used as a Food for Specified Health Uses, a Food with Nutrient Function Claims, and a Food with Functional Claims for the regulation of the intestinal microbiota. This invention contains rhamnan sulfate as an active ingredient and is used as a Food with Functional Claims for the regulation of the intestinal microbiota in humans.

[0011] According to the present invention, it is possible to provide a novel gut microbiota regulating composition that induces interactions between gut bacteria in the host, alters the profile of the bacterial flora to promote the growth of bacteria that contribute to the improvement of the host's health, and suppresses pathogens.

[0012] This is a box plot showing the gut microbiota profile before RS administration (Day 0) and 7 days after administration (Day 7). (A) Estimated bacterial cell count in fecal samples on Day 0 and Day 7, and (B) Changes in alpha diversity of the gut microbiota as measured by Shannon's index, Simpson's index, and Pielou's evenness index. The p-value was calculated using the Kruskal-Wallis test (pairwise). Alpha diversity of the gut microbiota decreased significantly after RS ​​administration. The number of data points was n=8 for Day 0 and Day 7 (the same for Figures 2 and 3). This is a graph showing the gut microbiota profile before (Day 0) and after (Day 7) RS administration. (C) Principal Coordinate Analysis (PCoA) plot based on weighted UniFrac distance (PERMANOVA p-value = 0.001 (999 permutations)), (D) Volcano plot showing bacterial species variation (decrease or increase) after RS ​​administration. Principal coordinate analysis revealed that the bacterial flora profile differed significantly before and after RS ​​administration. Furthermore, the Volcano plot indicated that the bacteria affected by RS were limited. Box plots showing the gut microbiota profile before (Day 0) and after (Day 7) RS administration. (E) Graph showing the relative abundance of Prevotellaceae UCG-001, Clostridia vadin BB60 group, and Mucispirillum before and after RS ​​administration. The relative abundance of these genera significantly increased after RS ​​administration. (F) Graph showing the relative abundance of Rikenellaceae RC9 intestinal group, ASF356, and Staphylococcus before and after RS ​​administration. The relative abundance of these genera decreased significantly after RS ​​administration.

[0013] This figure shows the functional changes in the gut microbiota after RS ​​administration, as revealed by PICRUSt2 analysis of 16s rRNA sequencing data. While 34 defined pathways showed statistically significant differences (adjusted p-value < 0.05) before and after RS ​​administration (Supplementary Data 2), the 20 pathways with the most significant changes (p < 0.02) were shown. Analysis revealed that the function of gut bacteria associated with human diseases such as small cell lung cancer, colorectal cancer, viral myocarditis, Staphylococcus aureus infection, toxoplasmosis, and Parkinson's disease was significantly reduced after RS ​​administration. In contrast, functions related to biofilm formation and carbohydrate digestion and absorption were significantly increased after RS ​​administration. The box plots show the changes in bile acids in feces before (Day 0) and after (Day 7) RS administration. (A) Cholic acid, (B) α-Mulicolic acid, (C) β-Mulicolic acid, and (D) Deoxycholic acid are shown, respectively. A decreasing trend was observed for each bile acid. The number of data points was n=8 for Day 0 and Day 7, and the p-value was calculated using an unpaired t-test (the same method was used in Figure 6). These are box plots showing the changes in short-chain fatty acids in feces before (Day 0) and after (Day 7) RS administration. (E) Acetic acid, (F) Propionic acid, (G) n-butyric acid, (H) Isobutyric acid, and (I) n-valeric acid are shown, respectively.

[0014] This is a schematic diagram illustrating the potential association between RS and anti-inflammatory effects. RS is metabolized by specific bacterial species in the gut microbiota (Prevotellaceae UCG-001 and / or Clostridia vadin BB60 group), and their numbers increased after RS ​​administration. This process creates opportunities for nutrient utilization and a niche environment for M. schaedleri. Since M. schaedleri possesses genomic functions that allow it to remove oxygen and reactive oxygen species during inflammation, the anti-inflammatory effects of M. schaedleri may contribute to the overall anti-inflammatory effect of RS on the host. The results show changes in body weight and gut microbiota profile before (Day 0) and after (Day 7) RS administration. (A) Graph showing changes in body weight before and after RS ​​administration, (B1) Chao 1 index, (B2) Faith phylogenetic diversity, and (B3) Box plots showing changes in alpha diversity of the gut microbiota as measured by Simpson homogeneity index. p-values ​​were calculated using the Kruskal-Wallis test (pairwise). Alpha diversity in the gut microbiota showed a decreasing trend after RS ​​administration.

[0015] This graph shows the gut microbiota profile before (Day 0) and after (Day 7) RS administration. (C) Principal coordinate analysis (PCoA) plot based on Bray-Curtis distance (PERMANOVA p-value = 0.001 (999 permutations). The bacterial microbiota profile differed significantly before and after RS ​​administration), (D) Volcano plot showing bacterial species variation (decrease or increase) after RS ​​administration. Only a limited number of bacteria were affected by RS administration. This is a box plot showing the gut microbiota profile before (Day 0) and after (Day 7) RS administration. (E) A graph showing the relative abundance of Clostridia vadin BB60 group s_uncultured bacteria, Prevotellaceae UCG-001; and Mucispirillum schaedleri (the relative viability of these bacteria increased after RS ​​administration). (F) A graph comparing the relative abundance of Rikenellaceae RC9 intestinal group s_unclutured bacteria and ASF356 s_unclutured bacteria (the relative viability of these bacteria decreased significantly after RS ​​administration). The number of data points was n=8 on Day 0 and Day 7 (the same in Figure 11). This graph shows the changes in bacterial composition at the phylum level after RS ​​administration. (A) A bar graph showing the proportion of each bacterium (showing the relative abundance of each bacterial phylum in each mouse and the average before (Day 0) and after (Day 7) RS administration). (B) A box plot showing the Firmicutes / Bacterioidota ratio before (Day 0) and after (Day 7) RS administration.

[0016] Next, embodiments of the present invention will be described with reference to the figures and tables. The technical scope of the present invention is not limited by these embodiments, and it can be carried out in various forms without changing the gist of the invention. <Test Method> 1. Ethical Conditions The tests described herein were conducted in accordance with the prescribed ethical guidelines. All animal experiments were conducted in accordance with the ARRIVE guidelines <https: / / arriveguidelines.org / arrive-guidelines> and the National Institutes of Health guidelines on the rearing and use of laboratory animals. Ethical approval for animal manipulation was granted by the Animal Research Ethics Committee of Suzuka University of Medical Science (Approval No. 63). 2. Test Animals and Sample Collection Ten-week-old male SPF BALB / c mice were purchased from SLC Japan. The mice were housed in groups of 3 to 5 mice per cage in an air-conditioned room maintained at 24°C, 50% relative humidity, and a 12-hour / 12-hour light-dark cycle. The mice were given Rodent Diet EQ 5L37 (manufactured by SLC Japan) and allowed to spontaneously consume drinking water. Mice were orally administered either water or 1 mg of rhamnan sulfate once daily from before the start of the study (Day 0) to day 7 (Day 7). Oral administration consisted of 100 μL of 10 mg / mL rhamnan sulfate (1 mg as RS). The body weight of male mice was 20.5 g to 24.8 g before rhamnan sulfate administration (Figure 8(A)). Fresh fecal samples were collected in clean tubes before and after the rhamnan sulfate administration period and subsequently analyzed.

[0017] 3. Preparation of Rhamnan Sulfate The rhamnan sulfate used was manufactured by Kounan Chemical Co., Ltd. The specific manufacturing method is as follows: Any naturally derived rhamnan sulfate can be used. In this embodiment, we used one obtained by hot water extraction from Monostroma nitidum. Dried seaweed was washed with water, extracted with hot water, and the resulting hot water extract was filtered to obtain an extract with rhamnan sulfate as the main component (60% to 97%). In this test, the purity was 94% and the average molecular weight was 5 × 10⁶. 5The Da variety was used. In addition, rhamnan sulfate usable in this invention can be prepared by various methods other than those described above. The raw materials are not limited to *Monostroma*; rhamnan sulfate can also be prepared using *Ulva*, *Ulva*, and others.

[0018] 4. Analysis of the Gut Microbiota 16S rRNA metagenomic sequencing and bacterial quantification were commissioned to the Kyoto Institute of Nutritional Pathology. Bacterial DNA extraction was performed according to known methods. Preparation of the 16S rRNA gene metagenomic sequencing library and sequencing using Miseq (Illumina) were performed. The variable regions V3 and V4 of the rRNA gene were amplified using the forward primer 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3' (SEQ ID NO: 1) and the reverse primer 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3' (SEQ ID NO: 2) for 16S amplicon PCR, and then performed according to the method outlined in the guide provided by Illumina (https: / / jp.support.illumina.com / content / dam / illumina-support / documents / documentation / chemistry_documentation / 16s / 16s-metagenomic-library-prep-guide-15044223-b-jpn.pdf). For bacterial quantification, quantitative PCR amplification was performed using a Roter-gene6200 (Qiagen) with Uni331F primer 5'-TCCTACGGGAGGCAGT-3' (SEQ ID NO: 3) and Uni797R primer 5'-GGACTACCAGGGTATCTATCCTGTT-3' (SEQ ID NO: 4). SYBR premix Ex Taq (Takara Bio Inc.) was used for amplification in a 10 μL reaction volume containing 1 μL of DNA extract and 0.2 μmol / L of each primer. The PCR amplification protocol consisted of initial denaturation at 95°C for 1 minute, followed by denaturation at 95°C for 20 seconds, annealing at 63°C for 30 seconds, and extension at 72°C for 45 seconds, with 30 cycles. The total number of bacterial cells was estimated based on the number of Bifidobacterium longum JCM1217T cells.

[0019] Sequencing data was analyzed using the official QIIME2 (version 2023.2) plugin. Sequence quality control was performed using the "dada2" plugin. The weighted Silva 138 99% OTU full-length sequence (MD5: 48965bb0a9e63c411452a460d92cfc04) was used as a classifier for classification in the "feature-classifier" plugin. Differential abundance analysis of microbial composition data was performed using the "Bias-Corrected Microbial Composition Analysis (ANCOMBC) method" included in the "composition" QIIME2 plugin. Prediction of microbial genome function was performed using PICRUSt2 as the "picrust2" plugin on the QIIME2 platform. The results of the PICRUSt2 analysis were further analyzed and visualized using the "ggpicrust2" package in "R". Since specific information about this pathway could not be obtained from the PICRUSt2 analysis, pathway ko00281 was excluded from further analysis.

[0020] 5. Analysis of Short-Chain Fatty Acids and Bile Acids in Feces The analysis of short-chain fatty acids (SCFAs) and bile acids (BAs) in feces was outsourced to the Kyoto Institute of Nutritional Pathology. Fecal samples were diluted 10-fold (wt / vol) with water, and the diluted samples were centrifuged at 15,000 × g at 4°C for 10 minutes, with the supernatant preserved. For SCFA analysis, 10 μL of diluted fecal sample was mixed with 440 mL of water and 10 μL of internal standard solution. As the internal standard solution, 10 μL of a mixture containing 5.0 mmol / L each of sodium acetate (D3), propionic acid (D3), n-butyric acid (D7), isobutyric acid (D1), and n-valeric acid (D1) was added, and the mixture was vortexed for 10 seconds. Next, 10 μL of hydrochloric acid (35%) was added, followed by 3 mL of diethyl ether and stirring for 30 minutes. The mixture was then centrifuged (1,200 × g, 10 min). The ether layer was collected, 8 μL of tert-butyldimethylchlorosilane was added, and the mixture was heated (60°C, 30 min) to derivatize it, which was then used as a sample for GC-MS analysis. Bile acids (BA) were extracted from the fecal supernatant diluted 10-fold and analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS (Waters)) according to known methods.

[0021] 6. Statistical Analysis Measured values ​​are shown as box plots unless otherwise specified. Statistical analysis was performed using GraphPad Prism version 10.3.1 (GraphPad Software). For comparisons between two groups, independent two-tailed t-tests were performed according to the instructions in GraphPad Prism unless otherwise specified. Pearson correlation coefficients were calculated using GraphPad Prism. To compare alpha diversity between two groups, the Kruskal-Wallis test (pairwise) was performed using the "Diversity" plugin in QIIME2. Beta diversity between two microbial groups was evaluated using PERMANOVA with 999 permutations. This analysis was also performed using the "Diversity" plugin in QIIME2. Differences in the abundance of specific bacterial groups between two groups were evaluated using the adjusted p-value (q-value) for ANCOMBC included in the "Composition" plugin in QIIME2. A p-value less than 0.05 (p < 0.05) was considered statistically significant.

[0022] <Test Results> As shown in Figure 1(A), administration of rhamnan sulfate for 7 days did not significantly increase the number of bacteria in the feces. However, as shown in Figure 1(B), 16S rRNA gene analysis revealed a significant decrease in alpha diversity of the gut microbiota, as indicated by multiple indices including Shannon, Simpson, and Pierreux homogeneity. Other indices, such as Chao1, Faith phylogenetic diversity, and Simpson homogeneity index, also showed a decreasing trend in alpha diversity with rhamnan sulfate administration (Figure 8(B)). Principal coordinate analysis based on weighted UniFrac distance (Figure 2(C)) and Bray-Curts distance (Figure 9(C)) revealed clear differences in the gut microbiota profile before and after rhamnan sulfate administration. These differences were statistically significant by PERMANOVA analysis (weighted UniFrac distance, q=0.001; Bray-Curts distance, q=0.001). Furthermore, as shown in Figure 2(D), differential abundance analysis revealed changes in specific genera after 7 days of rhamnan sulfate administration. Specifically, administration of rhamnan sulfate increased the relative abundance of Mucispirillum, Clostridia vadin BB60 group, and Prevotellaceae UCG-001 (Figures 2(D) and 3(E)). In contrast, the abundance of Rikenellaceae RC9 intestinal group, Staphylococcus, and ASF356 decreased significantly (Figures 2(D) and 3(F)). At the species level, five species, including Mucispirillum schaedleri, changed after rhamnan sulfate administration (Figures 10(E) and 10(F)). Specifically, Mucispirillum schaedleri, the only species in the genus Mucispirillum, increased significantly after rhamnan sulfate administration (Figures 9(D) and 10(E)). In contrast to previous reports (Non-Patent Literature 2) showing that administration of rhamnan sulfate reduces the Firmicutes / Bacteroidota ratio, a marker often associated with obesity in humans and animals, no significant changes were observed in the abundance at the phylum level or the Firmicutes / Bacteroidota ratio (Figure 11(A), (B)).

[0023] Next, PICRUSt2 analysis was performed on 16s rRNA sequencing data to investigate changes in the gut microbiota after rhamnan sulfate administration. The analysis identified 34 defined pathways that showed statistically significant differences (adjusted p-value < 0.05) after rhamnan sulfate administration. Of these, the 20 pathways with the most significant changes (p < 0.02) are shown in Figure 4. After rhamnan sulfate administration, several gut bacteria associated with human diseases such as small cell lung cancer, colorectal cancer, viral myocarditis, Staphylococcus aureus infection, toxoplasmosis, and Parkinson's disease decreased significantly. Furthermore, gut bacteria associated with carbohydrate digestion and absorption significantly increased after rhamnan sulfate administration. Mucispirillum schaedleri increased in abundance after rhamnan sulfate administration (Figure 9(D), Figure 10(E)). Since this microorganism is known to form biofilms, it is reasonable to assume that microbial genomic functions related to biofilm formation also increased after rhamnan sulfate administration.

[0024] PICRUSt2 analysis revealed that microbial genomic functions related to the biosynthesis of primary and secondary bile acids were reduced in bacterial communities exposed to rhamnan sulfate. To verify this prediction, fecal concentrations of primary and secondary bile acids were measured. In mice administered rhamnan sulfate, the concentrations of primary bile acids such as cholic acid, α-mulicolic acid, and β-mulicolic acid tended to decrease, as shown in Figures 5(A) to 5(C). Furthermore, as shown in Figure 5(D), a decreasing trend was observed in deoxycholic acid, a secondary bile acid. Considering that the gut microbiota metabolizes primary bile acids, the changes in secondary bile acids were thought to be due to changes in the composition of the gut microbiota. Correlation analysis showed that deoxycholic acid concentrations correlated with 23 genera of bacteria, including ASF356 (r=0.52449, p=0.03668) and Staphylococcus aureus (r=0.5214, p=0.0383, see Table 2). The abundance of these microorganisms was significantly reduced by the administration of rhamnan sulfate, as shown in Figure 3(F).

[0025] Next, we investigated the effect of rhamnan sulfate administration on the fecal concentration of SCFAs, major metabolites of the gut microbiota. As shown in Figure 6(E), rhamnan sulfate significantly increased the fecal concentration of acetic acid. As shown in Figure 6(F), propionic acid also showed an increasing trend, but there was no significant change in the concentration of n-butyric acid (Figure 6(G)). Conversely, the concentrations of isobutyric acid and n-valeric acid tended to decrease after rhamnan sulfate administration (Figures 6(H), (I)). Further correlation analysis revealed a positive correlation between acetic acid concentration and Negativibacillus (r = 0.5403, p = 0.0307). On the other hand, negative correlations were found with Lactobacillus (r=-0.6709, p=0.0044), the Eubacterium coprostanoligenes group (r=-0.6602, p=0.0054), Romboutsia (r=-0.6386, p=0.0078), and UCG-010 (r=-0.5336, p=0.0333, Tables 1 and 2). These results indicate that changes in the gut microbiota induced by rhamnan sulfate lead to modifications of microbial metabolism and ultimately affect the interaction between the gut microbiota and the host.

[0026]

[0027]

[0028] <Discussion> The inventors analyzed changes in the fecal microbiota profile induced by the administration of rhamnan sulfate. Rhamnan sulfate significantly altered the composition of specific bacterial groups in the gut microbiota, increasing the abundance of Prevotellaceae UCG-001, Clostridia vadin BB60 group, and Mucispirillum, while decreasing the abundance of Rikenellaceae RC9 gut group, ASF356, and Staphylococcus. Metagenomic functional analysis revealed that rhamnan sulfate administration shifted the gut microbiota to a more anti-inflammatory profile. These findings suggest that rhamnan sulfate plays a beneficial role in host health, possibly in part through its effects on the gut microbiota.

[0029] Administration of rhamnan sulfate induced significant changes in the gut microbiota, particularly in the bacterial community including Mucispirillum schaedleri. M. schaedleri, the only species in the genus Mucispirillum, is an anaerobic, Gram-negative, non-spore-forming, spiral-shaped rod with bipolar flagella, known to form cord-like biofilms. This gut bacterium colonizes the mucus layer of the digestive tract from the stomach to the colon. Detailed genomic analysis indicates that, despite its preference for the mucus layer, M. schaedleri has a limited ability to degrade complex polysaccharides. Instead, genomic analysis predicts that M. schaedleri can utilize monosaccharides, oligopeptides, amino acids, glycerol, and SCFAs as energy substrates.

[0030] This indicates that M. schaedleri does not directly consume administered rhamnan sulfate. That is, other bacteria with polysaccharide utilization capabilities digest the rhamnan sulfate, providing M. schaedleri with opportunities for reciprocal feeding and creating a niche environment within the gut microbiota. M. schaedleri also plays a unique role in bacterial interactions within the gut ecosystem. M. schaedleri possesses catabolic genes that reduce nitrates to ammonia. Furthermore, it is known to suppress infection by Salmonella enterica serotype typhi (S.Tm) by inhibiting the expression of toxic factors. In addition, it is known to inhibit the expression of S.Tm invasive genes by competing for nitrates in the gut.

[0031] Furthermore, M. schaedleri is associated with inflammation in host tissues. The relative abundance of M. schaedleri increases in inflammatory states, such as in the gut of a mouse model of dextran sulfate sodium-induced colitis. Genomic analysis has revealed that M. schaedleri possesses a system to remove oxygen and reactive oxygen species during inflammation. This indicates that M. schaedleri can utilize inflammation to proliferate even in inflammatory states. Previous studies have shown that rhamnan sulfate has significant anti-inflammatory effects. For example, rhamnan sulfate reduced the inflammatory response in human umbilical vein endothelial cells treated with either lipopolysaccharides, tumor necrosis factor-α, or thrombin. Oral administration of rhamnan sulfate to mice is known to protect vascular endothelium from LPS-induced inflammation. Although the detailed mechanism of rhamnan sulfate's anti-inflammatory effect is unknown, these test results suggest a potential link between rhamnan sulfate and its anti-inflammatory effects (Figure 7). Specifically, rhamnan sulfate is digested by certain bacterial species in the gut microbiota.

[0032] Following administration of rhamnan sulfate, the number of Prevotellaceae UCG-001 and / or Clostridia vadinBB60 groups increased, as shown in Figure 3(E). This creates a new niche relationship with M. shcaedleri regarding its nutrient utilization opportunities. Since M. shcaedleri can cope with and utilize inflammation, it is hypothesized that the anti-inflammatory effect of M. shcaedleri contributes to the anti-inflammatory effect of rhamnan sulfate on host tissues. However, this hypothesis needs further investigation in future studies.

[0033] Thus, this study revealed that administration of rhamnan sulfate significantly altered the composition of specific bacterial groups within the gut microbiota, resulting in a remarkable change in the dynamics of the microbial community. Among these changes, the increase in M. schaedleri is a key result of the interaction of rhamnan sulfate-induced bacteria. The anti-inflammatory properties of M. schaedleri may contribute to the overall anti-inflammatory response of rhamnan sulfate, suggesting a potential link between rhamnan sulfate intake and the health benefits to the host. In this way, this embodiment provides a novel and safe composition for regulating the gut microbiota.

Claims

1. A composition for regulating the gut microbiota, characterized by containing sulfated polysaccharides that induce interbacterial interactions in the host's gut, thereby altering the profile of the bacterial flora to promote the growth of bacteria that contribute to the health of the host and suppress pathogens.

2. The intestinal microbiota regulating composition according to claim 1, wherein the sulfated polysaccharide is rhamnan sulfate.

3. The intestinal microbiota regulating composition according to claim 2, wherein the composition is for oral administration.

4. The intestinal microbiota regulating composition according to any one of claims 1 to 3, wherein the regulation of the intestinal microbiota is (1) increasing at least one selected from the group consisting of Prevotellaceae UCG-001, Clostridia vadin BB60 group, and Mucispirillum schaedleri, and (2) decreasing at least one selected from the group consisting of Rikenellaceae RC9 intestinal group, ASF356, and Staphylococcus.

5. A food or beverage for regulating the intestinal flora containing the intestinal flora regulating composition according to any one of claims 4.

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