Composition for promoting proliferation of bacterium belonging to genus phascolarctobacterium

Abstract

Disclosed is a composition for promoting the proliferation of a bacterium belonging to the genus Phascolarctobacterium, the composition containing sialyllactose or a pharmaceutically acceptable salt thereof.

Description

Composition for promoting the growth of bacteria of the genus Phascolactomyces

[0001] The present invention relates to a composition for promoting the growth of bacteria of the genus Phascolactomyces.

[0002] It has been reported that useful metabolites such as short-chain fatty acids produced by intestinal bacteria have various functions, such as the alleviation of allergic symptoms and anti-obesity effects. Among intestinal bacteria, the genus Phascolactomyces is a type of intestinal bacterium that is widely present in the human intestine. Phascolactomyces has attracted attention as one of the useful intestinal bacteria, and it has been reported that this bacterium produces propionic acid, a useful metabolite of short-chain fatty acids, and that in observational studies of allergies, this bacterium is more abundant in the intestines of healthy subjects than in patients (Non-Patent Documents 1 and 2). Therefore, it is believed that growing Phascolactomyces bacteria in the intestine is important for human health.

[0003] Phascolactomy bacteria are known to have distinctive carbohydrate metabolic activity and are difficult to effectively grow. For example, single-cell culture tests using Phascolactomy bacteria, a representative species of the genus, Phascolactomy bacteria, have revealed that the bacteria cannot decompose common sugars such as glucose and sucrose, but grow using succinic acid as the sole carbon source. Methods for growing Phascolactomy bacteria have been confirmed under limited conditions, such as in diabetic model mice (Non-Patent Documents 3 and 4). Furthermore, rhamnogalacturonan, a type of polysaccharide, is known to have a growth effect on these bacteria (Non-Patent Document 5), but a method for effectively growing Phascolactomy bacteria in the intestines using food has not yet been developed.

[0004] Human milk oligosaccharides (HMOs), the third most abundant solid component in human breast milk, have been reported to have diverse physiological activities, including promoting the proliferation of bifidobacteria and the development of immune function in infants. Orally ingested HMOs are barely absorbed or degraded by humans, but are delivered intact to the large intestine and utilized by intestinal bacteria. Therefore, functional research into HMOs as a new prebiotic material is being conducted not only for infants but also for adults. Furthermore, sialyllactose (hereinafter also referred to as "SL"), a type of HMO, is a sialic acid compound formed by the bond between lactose and N-acetylneuraminic acid. Among these, 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) are known to promote the survival and maintenance of neuronal function (Patent Document 1) and to have inhibitory effects on hyperlipidemia and other conditions (Patent Document 2). However, the proliferation-promoting effect of HMOs on the genus Phascolactomabacterium is completely unknown.

[0005] JP-A-7-258093 Publication Special Publication No. 2011-529877

[0006] Louis and Flint, "Formation of propionate and butyrate by the human colonic microbiota", Environ Microbiol. 2017 Jan; 19(1): 29-41.Bao et al., "Fecal microbiome and metabolome differ in healthy and food-allergic twins", J Clin Invest. 2021 Jan 19; 131(2): e141935.Song et al., "Feruloylated oligosaccharides and ferulic acid alter gut microbiome to alleviate diabetic syndrome", Food Res Int. 2020 Nov; 137: 109410.Liu et al., "Pumpkin polysaccharide modifies the gut microbiota during alleviation of type 2 diabetes in rats", Int J Biol Macromol. 2018 Aug; 115: 711-717.Van den Abbeele et al., "Consistent Prebiotic Effects of Carrot RG-I on the Gut Microbiota of Four Human Adult Donors in the SHIME(R) Model despite Baseline Individual Variability", Microorganisms. 2021 Oct 13; 9(10): 2142.

[0007] An object of the present invention is to provide a composition for promoting the growth of bacteria of the genus Phascolactomabacterium.

[0008] The present inventors have discovered that sialyllactose, a type of HMO, has an excellent growth-promoting effect on bacteria of the genus Phascolactomyces, and have completed the present invention. Specifically, the present invention relates to the following [1] to [9]. [1] A composition for promoting the growth of bacteria of the genus Phascolactomyces, comprising sialyllactose or a pharmaceutically acceptable salt thereof. [2] The composition according to [1], which is a food or drink. [3] The composition according to [1], which is a drug or quasi-drug. [4] A method for promoting the growth of bacteria of the genus Phascolactomyces, comprising administering sialyllactose or a pharmaceutically acceptable salt thereof to a subject in need thereof. [5] Sialyllactose or a pharmaceutically acceptable salt thereof for use in a therapeutic method for promoting the growth of bacteria of the genus Phascolactomyces. [6] Use of sialyllactose or a pharmaceutically acceptable salt thereof in a non-therapeutic method for promoting the growth of bacteria of the genus Phascolactomyces. [7] Use of sialyllactose or a pharmaceutically acceptable salt thereof for producing a composition for promoting the growth of bacteria of the genus Phascolactomyces. [8] The composition, method, compound for use or a pharmaceutically acceptable salt thereof, or use according to [1] to [7], wherein sialyllactose is 3'-sialyllactose or 6'-sialyllactose. [9] The composition, method, compound for use or a pharmaceutically acceptable salt thereof, or use according to [1] to [7], wherein sialyllactose is 6'-sialyllactose.

[0009] According to the present invention, a composition for promoting the growth of bacteria of the genus Phascolactomabacterium can be provided.

[0010] FIG. 1 is a diagram showing the configuration of the human intestinal microbial ecosystem simulator SHIME (registered trademark). PJ represents pancreatic enzyme / bile solution, FEED represents basal medium, ST / SI represents reactor for stomach and small intestine, and LI represents reactor for large intestine. NC represents negative control, 3'-SL represents 3'-sialyllactose addition group, and 6'-SL represents 6'-sialyllactose addition group. FIG. 2 is a graph showing the change in the relative abundance ratio of bacteria of the genus Phascolactomyces before and after treatment with each sample. FIG. 3 is a graph showing the rate of change in propionic acid concentration before and after treatment with each sample.

[0011] In one embodiment, a composition for promoting the growth of bacteria of the genus Phascolactomyces contains sialyllactose or a pharmaceutically acceptable salt thereof. Sialyllactose is a sialic acid compound in which lactose and N-acetylneuraminic acid are bonded, and the bonding position of the N-acetylneuraminic acid is not particularly limited. Examples of sialyllactose include 3'-sialyllactose (O-(N-acetyl-α-neuraminosyl)-(2→3)-O-β-D-galactopyranosyl-(1→4)-D-glucose; 3'-SL) and 6'-sialyllactose (O-(N-acetyl-α-neuraminosyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose; 6'-SL).

[0012] Pharmaceutically acceptable salts of sialyllactose include, for example, salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; salts with organic acids such as acetic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, lactic acid, stearic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, etc.; salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; salts with amino acids such as arginine, etc. The preferred salt is the sodium salt.

[0013] Sialyl lactose or its pharmaceutically acceptable salt may have crystalline polymorphism, but is not limited to any one, and may be any single crystalline form or a mixture.Sialyl lactose or its pharmaceutically acceptable salt also includes amorphous form.Sialyl lactose or its pharmaceutically acceptable salt also includes anhydrous form and solvate (particularly hydrate).

[0014] 3'-SL or a pharmaceutically acceptable salt thereof can be produced by known methods, for example, crystals of the sodium salt of 3'-SL can be produced by the method described in WO 2017 / 195743. 6'-SL or a pharmaceutically acceptable salt thereof can be produced by known methods, for example, crystals of the sodium salt of 6'-SL can be produced by the method described in WO 2017 / 086443.

[0015] In one embodiment, a composition for promoting the growth of bacteria of the genus Phascolactomyces contains sialyllactose or a pharmaceutically acceptable salt thereof, thereby promoting the growth of bacteria of the genus Phascolactomyces in the intestine. Here, the intestine refers to the small intestine and / or the large intestine, and in one embodiment, the large intestine.

[0016] Species belonging to the genus Phascolactomacterium include P. faecium, P. succinatutens, P. wakonense, and the like.

[0017] The proliferation of bacteria of the genus Phascolactomyces may refer to an increase in the absolute number of the bacteria in the intestinal flora, or an increase in the relative abundance ratio of the bacteria in the intestinal flora. The method for determining the relative abundance ratio of bacteria of the genus Phascolactomyces in the intestinal flora is not particularly limited, and can be calculated, for example, by amplicon sequence analysis targeting the V3-V4 region of the 16S rRNA gene. The degree of increase in the relative abundance ratio is not particularly limited, and refers to, for example, an increase of 5% or more compared to the relative abundance ratio before application of the composition of this embodiment.

[0018] Promoting the growth of Phascolactomyces bacteria in the intestines increases the production of useful metabolites such as propionic acid, which may have the effect of maintaining the barrier function of intestinal epithelial cells, alleviating allergic symptoms, anti-obesity effects, and cardiovascular protection.

[0019] The composition for promoting the growth of bacteria of the genus Phascolactomyces according to one embodiment can be applied to humans and mammals other than humans, with humans being preferred. Examples of mammals other than humans include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, and monkeys, and the composition according to the present disclosure can be used as feed, which is a form of food. Human subjects may be adults, elderly people, children, infants, or newborns.

[0020] In one embodiment, the composition for promoting the growth of Phascolactomyces bacteria can be administered or ingested orally or parenterally, but is preferably administered or ingested orally.

[0021] The content of sialyllactose or a pharmaceutically acceptable salt thereof in a composition for promoting the growth of Phascolactomyces bacteria in one embodiment is not particularly limited, based on the total amount of the composition, but can be, for example, preferably 0.01% by mass to 90% by mass, more preferably 0.1% by mass to 80% by mass.

[0022] The dosage or intake of a composition for promoting the growth of Phascolactomyces bacteria in one embodiment is not particularly limited, but for example, for a subject weighing 60 kg, the dosage or intake of sialyllactose or a pharmaceutically acceptable salt thereof per day can be preferably 50 mg to 30 g, more preferably 100 mg to 10 g.

[0023] The number of times that the composition for promoting the growth of bacteria of the genus Phascolactomyces in one embodiment is administered (ingested) can be one to three times per day.

[0024] In one embodiment, the composition for promoting the growth of bacteria of the genus Phascolactomyces may be a food or drink, a drug, or a quasi-drug. The food or drink, drug, or quasi-drug can each be produced according to a conventional method. The content of sialyllactose or a pharmaceutically acceptable salt thereof in the food or drink, drug, or quasi-drug is not particularly limited and can be freely set according to the purpose.

[0025] When the composition for promoting the growth of bacteria of the genus Phascolactomyces according to one embodiment is a food or beverage, a drug, or a quasi-drug, the food or beverage, drug, or quasi-drug may contain, in addition to sialyllactose or a pharmaceutically acceptable salt thereof, ingredients typically used in food or beverage, drugs, or quasi-drugs. The food or beverage, drug, or quasi-drug according to one embodiment may contain bases, carriers, additives, etc. typically used in food or beverage, drugs, or quasi-drugs. Examples of additives include excipients, oils, powders, buffers, solubilizers, antioxidants, surfactants, thickeners, preservatives, pH adjusters, chelating agents, stabilizers, irritation reducers, antiseptics, pigments, colorants, fragrances, gloss-imparting agents, gelling agents, alcohols, water-soluble polymers, film-forming agents, resins, etc. The bases, carriers, and various additives described above may be used singly or in combination as needed.

[0026] When the composition for promoting the growth of bacteria of the genus Phascolactomyces according to one embodiment is a food or drink, the food or drink may be a general food or drink, a food for specified health uses, a food with nutrient function claims, a food with functional claims, a food for hospital patients, a supplement, etc. Furthermore, the composition for promoting the growth of bacteria of the genus Phascolactomyces according to one embodiment can also be used as a food additive.

[0027] Examples of such foods and drinks include seasonings, processed meat products, processed agricultural products, beverages (lactic acid bacteria drinks, soft drinks, alcoholic drinks, carbonated drinks, dairy drinks, fruit juice drinks, tea, coffee, energy drinks, etc.), powdered drinks (powdered juice, powdered soup, powdered milk, etc.), concentrated drinks, confectioneries (candy (throat lozenges), cookies, biscuits, gum, gummy candies, chewable tablets, tablets, chocolate, etc.), bread, cereal, etc. In addition, in the case of foods for specified health uses, foods with nutrient functions, foods with functional claims, etc., they may be in the form of capsules, tablets, granules, powders, syrups, lozenges, etc.

[0028] Here, a food for specified health uses is a food containing functional health 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 of Japan. When a composition for promoting the growth of bacteria of the genus Phascolactomyces is a food for specified health uses, it may be a food that is sold with a label indicating a specific health use related to promoting the growth of bacteria of the genus Phascolactomyces.

[0029] A food with nutrient functions is a food used to supplement nutrients (vitamins, minerals), and displays the function of the nutrients. To be sold as a food with nutrient functions, the amount of nutrients contained in the recommended daily intake must be within the specified upper and lower limits, and in addition to displaying the nutritional function, warning labels must also be displayed.

[0030] Foods with functional claims are foods that display scientifically based functionality at the responsibility of the business operator, and information on the basis of safety and functionality is submitted to the Commissioner of the Consumer Affairs Agency before sale.

[0031] When the composition for promoting the growth of bacteria of the genus Phascolactomyces according to one embodiment is a pharmaceutical or quasi-drug, the dosage form of the pharmaceutical or quasi-drug may be, for example, a liquid, suspension, capsule, granule, pill, powder, tablet, syrup, lozenge, etc. Possible indications for the pharmaceutical include allergies, obesity, heart failure, etc.

[0032] The effect of 6'-SL on the proliferation of Phascolactomyces bacteria in the human intestine was investigated using the following method. The intestinal bacterial culture environment in the human intestine was simulated using the human intestinal microbial ecosystem simulator "SHIME (registered trademark)" (ProDigest - Ghent University, Belgium). The configuration of SHIME (registered trademark) is discussed in more detail in Molly et al., "Development of a 5-step multi-chamber reactor as a simulation of the human intestinal microbial ecosystem," Appl. Microbiol. Biotechnol., 39(2): 254-258 (1993). Use of SHIME (registered trademark) allows the effectiveness of test materials to be verified in an environment more similar to the human intestine than a simple in vitro system.

[0033] As shown in Figure 1, the configuration of the SHIME® modified for this example includes three reactors "ST / SI" for the stomach and small intestine, and nine reactors "LI" for the large intestine. The configuration of each reactor is as follows: To mimic the human body, all reactors were maintained at 37°C. The ST / SI reactors mimicked the stomach in terms of temperature, pH, and anaerobic conditions, and the LI reactors mimicked the distal colon in terms of temperature, human bacterial flora, pH, and anaerobic conditions.

[0034] Each ST / SI reactor was connected to three LI reactors to represent the human digestive process (ST / SI1-LI1 or LI2 or LI3, ST / SI2-LI4 or LI5 or LI6, and ST / SI3-LI7 or LI8 or LI9). ２The ST / SI and LI reactors were maintained anaerobic by flowing and continuously stirring the reactors. Each reactor in the series was connected via a tube connected to a pump. The flow rate of the reactor contents from start to finish was intended to mimic human digestion by using the pumps connected to the reactors. The pH in the reactors was also adjusted to match each segment of the digestive tract. The pH of the SHIME® medium fed to the ST / SI reactors was adjusted to 2.0, and the pH of the LI reactors was about 6.6 to about 6.9. The pH in the LI reactors was controlled by adding an appropriate amount of acid or base.

[0035] Stabilization period (2 weeks): Fecal samples obtained from three healthy adults (donors A, B, and C) who had not taken antibiotics within the past 3 months were inoculated into nine LI reactors (LI1, LI4, and LI7 for donor A; LI2, LI5, and LI8 for donor B; and LI3, LI6, and LI9 for donor C) filled with 250 mL of SHIME® medium. After 24 hours of incubation, the pump was turned on and 300 mL of SHIME® medium was administered to the three ST / SI reactors three times a day to simulate breakfast, lunch, and dinner. The SHIME® medium administered to the ST / SI reactors was divided into three equal portions and administered to each LI reactor. The liquid volume in each LI reactor was maintained at 250 mL by automatically pumping out excess liquid. A two week stabilization period allowed the microbial population to stabilize within the reactor prior to the start of test material treatment.

[0036] Treatment period (2 weeks): After a 2-week stabilization period, the SHIME® medium administered to each reactor was changed and operated for another 2 weeks to verify the effectiveness of the test material. The same SHIME® medium as used during the stabilization period was added to ST / SI1, which served as a control (NC group). 3'-sialyllactose (3'-SL) was added to the SHIME® medium added to ST / SI2, and 6'-sialyllactose (6'-SL) was added to the SHIME® medium added to ST / SI3, both at 4.2 g / L.

[0037] SHIME® medium was prepared by mixing basal medium (FEED) with pancreatic enzyme / bile solution (PJ) in a 7:3 ratio. FEED consisted of arabinogalactan (1.2 g / L), pectin (2 g / L), xylan (0.5 g / L), glucose (0.4 g / L), yeast extract (3 g / L), peptone (1 g / L), mucin (2 g / L), L-cysteine-HCl (0.5 g / L), and starch (4 g / L). PJ consisted of Oxgall (6.5 g / L), pancreatin (0.9 g / L), and NaHCO . ３ The solution consisted of 12.5 g / L of mucin. 210 mL of feed was administered to each ST / SI reactor, followed by 90 mL of protease inhibitor (PJ). This brought the total volume of the ST / SI reactor to 300 mL. Then, 100 mL of the contents of each ST / SI reactor were administered to the corresponding LI reactor. Each LI reactor contained 18 net-wrapped mucin beads, which were replaced with half of the beads every 2 to 3 days to mimic mucin secretion by colonic cells. The mucin beads were prepared by coating plastic beads with a mucin solution consisting of mucin (50 g / L), agar (10 g / L), and 10 N NaOH (3.5 mL / L).

[0038] On the final day of the stabilization period and the final day of the treatment period, 10 mL of culture medium was harvested from each LI reactor. The harvested culture medium was centrifuged at 15,000 rpm for 5 minutes to separate the culture supernatant and bacterial pellet, which were then frozen at -20°C for subsequent analysis. The bacterial flora in the bacterial pellet was analyzed by amplicon sequencing targeting the V3-V4 region of the 16S rRNA gene to calculate the relative abundance of each bacterial genus.

[0039] Table 1 shows the relative abundance of Fascolactomyces bacteria in the bacterial pellets recovered from the culture medium of the control (NC group), 3'-SL-added group, and 6'-SL-added group. The abundance of Fascolactomyces bacteria in the NC group decreased in all donors on the final day of the treatment period compared to the final day of the stabilization period, while the abundance increased in two of the three donors in the 3'-SL-added group. Furthermore, the abundance of Fascolactomyces bacteria in the 6'-SL-added group increased in all donors, confirming a greater growth-promoting effect. Figure 2 shows the change in the relative abundance of Fascolactomyces bacteria before and after treatment with each sample.

[0040]

[0041] In addition, the culture supernatant obtained by centrifugation of the culture medium was analyzed for short-chain fatty acid content using the YMC-Pack FA long-chain / short-chain fatty acid analysis kit (YMC Co., Ltd.). As a result, it was confirmed that propionic acid, a useful metabolite of Phascolactomyces bacteria, increased in all subjects in the 3'-SL-added group and the 6'-SL-added group. Table 2 shows the propionic acid concentration (mM) in the culture medium of the NC group, the 3'-SL-added group, and the 6'-SL-added group. Figure 3 also shows the rate of change in propionic acid concentration before and after treatment with each sample.

[0042]

Claims

1. A composition for promoting the growth of bacteria of the genus Fascolactomyces, comprising sialyllactose or a pharma- ceutical acceptable salt thereof.

2. The composition according to claim 1, wherein the sialyllactose is 3'-sialyllactose or 6'-sialyllactose.

3. The composition according to claim 1 or 2, which is a food or beverage.

4. The composition according to claim 1 or 2, which is a drug or quasi-drug.

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