Secondary bile acid increasing agent, and agent for increasing intestinal bacteria with secondary bile acid metabolism ability.

Abstract

To provide a composition (secondary bile acid increasing agent) used to increase the amount of secondary bile acids in the gut microbiota, and to provide a composition (intestinal bacteria increasing agent) used to increase bacteria (intestinal bacteria) that have secondary bile acid metabolic ability and are involved in the metabolic production of secondary bile acids in the gut microbiota. [Solution] A secondary bile acid increasing agent or intestinal bacteria increasing agent comprising a composition containing low molecular weight alginic acid or a salt thereof having a weight-average molecular weight of 50,000 to 1,000,000, or at least one thereof, as an active ingredient.

Description

[Technical Field]

[0001] The present invention relates to a composition (secondary bile acid increasing agent) used to increase the amount of secondary bile acids in the body, particularly in the intestines. The present invention also relates to a composition (intestinal bacteria increasing agent) used to increase bacteria (intestinal bacteria) that have secondary bile acid metabolic ability and are involved in the metabolic production of secondary bile acids in the intestinal microbiota. [Background technology]

[0002] In recent years, bile acids have attracted attention as regulatory factors of the gut microbiota, and it has been suggested that bile acids, which increase with high-fat diet intake, influence changes in the gut microbiota (Non-Patent Literature 1). Bile acids are involved in lipid absorption and are synthesized in the liver from cholesterol as conjugated bile acids (primary bile acids), then secreted into the intestines. After their function is complete, the conjugated bile acids are reabsorbed in the small intestine and returned to the liver (enterohepatic circulation). However, some conjugated bile acids reach the large intestine and are metabolized by intestinal bacteria into deconjugated bile acids (primary bile acids) and then secondary bile acids. When converted to secondary bile acids, the hydrophobicity of the molecules increases and they exhibit strong bactericidal activity. For example, it is known that the secondary bile acid deoxycholic acid (DCA) exhibits 10 times the bactericidal activity of the primary bile acid cholic acid (CA) (Non-Patent Literature 2). Based on these findings, the bile acid hypothesis has proposed that a high-fat diet leads to increased concentration of secondary bile acids such as DCA, which are secreted in excess and produced by intestinal bacteria in the large intestine. This increased concentration acts as a selective pressure, causing changes in the bacterial flora (Non-Patent Literature 1). Thus, secondary bile acids have generally been considered to be components that have adverse effects on the host.

[0003] However, a 2021 Nature paper (Non-Patent Literature 3) reported that the stool of centenarians (average age 107) of Japanese people over 100 years old contains particularly high levels of secondary bile acids such as isoallo-LCA, 3-oxolitocholic acid, and isolitocholic acid. In this paper, it is suggested that Isoallo-LCA, in particular, exhibits strong bactericidal activity against various Gram-positive pathogenic bacteria and contributes to maintaining the intestinal environment. Recently, it has become clear that IsoalloLCA, 3-Oxo-LCA, and IsoLCA are also involved in regulating the immune response. It has been reported that IsoalloLCA promotes the differentiation of regulatory T cells (Treg), while 3-Oxo-LCA and IsoLCA suppress the differentiation of Th17 cells (Non-Patent Literature 4 and 5). Therefore, IsoalloLCA, 3-Oxo-LCA, and IsoLCA are attracting attention as "anti-aging hormones" that act on inflammation and immunity in the lumen of the large intestine (Non-Patent Literature 6).

[0004] Furthermore, the authors of the aforementioned Nature article (Non-Patent Literature 3) analyzed the gut microbiota and metabolites of stool samples from centenarians to investigate the secrets of longevity. As a result, they discovered that bacteria belonging to the families Parabacteroides merdae, Odoribacter laneus, and Odoribacteraceae effectively metabolize and produce IsoalloLCA. It has also been revealed that these bacterial strains commonly possess an enzyme homologous to 3-oxo-5α-steroid 4-dehydrogenase (5α-reductase, 5AR), as well as a gene for 3β-hydroxysteroid dehydrogenase (3β-HSDH), which is thought to be involved in bile acid metabolism. Furthermore, it has been confirmed that deleting the 5AR and 3β-HSDH genes in Odoribacteraceae sp. strains isolated from centenarians eliminates IsoalloLCA biosynthesis, and that administering Odoribacteraceae sp. to germ-free mice enables them to synthesize IsoalloLCA in vivo (see Non-Patent Document 7).

[0005] Furthermore, Non-Patent Literature 7 states that IsoalloLCA has the effect of suppressing the growth of Gram-positive pathogenic bacteria at extremely low concentrations, and that when Odoribacteraceae sp. producing IsoalloLCA is orally administered to mice infected with Clostridioides difficile, which is a problem in hospital-acquired infections, Clostridioides difficile is eliminated as IsoalloLCA increases due to the Odoribacteraceae sp. that have colonized the intestinal tract. From this, it is considered that there are many bacteria that metabolize and produce IsoalloLCA in the intestines of centenarians, and as a result the abundance of IsoalloLCA, the elimination of Gram-positive pathogenic bacteria is promoted and a healthy intestinal environment is maintained. Non-Patent Literature 7 also states that intestinal bacteria that metabolize and produce IsoalloLCA may have the potential to be applied to new prevention and treatment of intractable infections. [Prior art documents] [Patent Documents]

[0006] [License 1] Special Announcement No. 2-303468 [License 2] Special Announcement No. 3-273002 [License 3] Special Announcement No. 6-7093 [License 4] Special Announcement No. 2009-60890 [Non-licensed literature]

[0007] [Non-licensed Document 1] Yokota et al., Is bile acid a determinant of the gut microbiota on a high-fat diet? Gut Microbes, 3(5), 455-459 (2012) [Non-licensed Document 2] Kurdi et al., Mechanism of growth inhibition by free bile acids in lactobacilli and bifidobacteria. J Bacteriol, 188(5), 1979-1986 (2006) [Non-licensed Document 3] Sato et al., Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians. Nature, 599(7885): 458-464 (2021) [Non-licensed Document 4] Paik et al., Human gut bacteria produce ΤH17-modulating bile acid metabolites. Nature, 603(7903), 907-912 (2022) [Non-licensed Document 5] Hang et al., Bile acid metabolites control TH17 and Treg cell differentiation. Nature, 576(7785): 143-148 (2019) [Non-Patent Document 6] Yuji Naito, "Amazing Guts and a Disappointing Brain," Sogo Horei Publishing, pp. 123-125 (2023) [Non-Patent Document 7] National Research and Development Agency, Japan Agency for Medical Research and Development / Results Information / Publication Date: August 10, 2021 (https: / / www.amed.go.jp / news / seika / kenkyu / 20210810-02.html), Bile Acids Involved in Healthy Longevity Produced by Intestinal Bacteria - A Novel Bile Acid Biosynthesis Pathway Increased in the Microbiome of Centenarians - [Non-Patent Document 8] Atsushi Shimonaka et al., "Consumption of high-cacao chocolate increases the proportion of Faecalibacterium species in the gut microbiota," 21st Annual Meeting of the Japanese Society for Intestinal Microbiota (2017). [Non-Patent Document 9] Sasaki et al., Low amounts of dietary fiber increase in vitro production of short-chain fatty acids without changing human colonic microbiota structure. Sci Rep, 8(1): 435 (2018) [Non-Patent Document 10] Nagata et al., In vitro colonic fermentation characteristics of barley-koji differ from those of barley. Biosci Biotechnol Biochem, 86(9): 1286-1294 (2022). [Non-Patent Document 11] Hokkaido Prefectural Industrial Research Institute Report No. 305 (published in 2006) [Non-Patent Document 12] Journal of the Japanese Society for Nutrition and Food Science, Vol. 36, No. 1, p21-24, 1983

Non-Patent Document 13

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a composition (secondary bile acid increasing agent) used for increasing the amount of specific secondary bile acids such as isoalloLCA in the human intestine. Another object of the present invention is to provide a composition (intestinal bacterium increasing agent) having the ability to metabolize secondary bile acids and used for increasing intestinal bacteria involved in the metabolic production of the secondary bile acids in the human intestine. In particular, an object of the present invention is to provide a composition useful as a prebiotic that can increase intestinal bacteria that metabolically produce useful secondary bile acids such as IsoalloLCA in the human intestine.

Means for Solving the Problems

[0009] The inventors of the present invention have conducted intensive studies to solve the above problems. As a result, they have found that by orally ingesting alginic acid having a weight average molecular weight of 50,000 to 1,000,000 (hereinafter also referred to as "low molecular weight alginic acid"), its salt, or a composition containing at least one of them (low molecular weight alginic acid-containing composition), the amount of secondary bile acids in feces excreted from the human body increases, and specific intestinal bacteria involved in the metabolic production of secondary bile acids also increase. Moreover, it was confirmed that such a phenomenon occurs by orally ingesting the low molecular weight alginic acid-containing composition for a short period of 1 to 2 weeks.

[0010] Based on these findings, the present inventors are convinced that a composition containing the low molecular weight alginic acid or a salt thereof, or at least one thereof (hereinafter also referred to as "low molecular weight alginic acid-containing composition") can serve as a prebiotic that can create an environment in the intestinal flora of mammals, including humans, that is suitable for the growth and proliferation of beneficial bacteria involved in the metabolic production of secondary bile acids such as IsoalloLCA, and have thus completed the present invention.

[0011] The present invention has the following embodiments. (I) Secondary bile acid increasing agents (I-1) A secondary bile acid increasing agent comprising an alginic acid or a salt thereof having a weight-average molecular weight of 50,000 to 1,000,000, or a composition containing at least one of the same (low molecular weight alginic acid-containing composition) as an active ingredient, A secondary bile acid increasing agent wherein the secondary bile acid is at least one selected from the group consisting of IsoalloLCA, AlloLCA, 3-OxoalloLCA, IsoLCA, and 3-OxoLCA. (I-2) The secondary bile acid increasing agent described in (I-1), wherein the composition is a low molecular weight alginic acid-containing composition prepared from kelp. (I-3) The secondary bile acid increasing agent described in (I-2), wherein the low molecular weight alginic acid-containing composition is prepared by a method comprising the step of treating alginic acid or a salt thereof contained in kelp to reduce its molecular weight so that its weight-average molecular weight is in the range of 50,000 to 1,000,000. (I-4) The secondary bile acid increasing agent described in (I-3), wherein the molecular weight reduction treatment includes a step of heating kelp at a temperature of 100°C or higher. (I-5) The kelp is obtained by iodine elution treatment by soaking or boiling the kelp in water, as described in any of (I-2) to (I-4).

[0012] (II) Agents that increase intestinal bacteria (II-1) An agent for increasing intestinal bacteria having secondary bile acid metabolism ability, comprising as an active ingredient a composition containing alginic acid or a salt thereof having a weight-average molecular weight of 50,000 to 1,000,000, or at least one thereof (low molecular weight alginic acid-containing composition), The secondary bile acid is at least one selected from the group consisting of IsoalloLCA, AlloLCA, 3-OxoalloLCA, IsoLCA, and 3-OxoLCA. An intestinal bacteria increasing agent wherein the aforementioned intestinal bacteria are at least one selected from the group consisting of Parabacteroides merdae, bacteria of the family Odoribacteraceae, bacteria of the genus Odoribacter, Parabacteroides distasonis, Bacteroides uniformis, Bacteroides thetaiotaomicron, Alistipes onderdonkii, Alistipes finegoldii, Clostridium innocuum, Eggerthella lenta strain, Gordonibacter pamelaeae, and Hungatella hathewayi. (II-2) The intestinal bacteria increasing agent described in (II-1), wherein the composition is a low molecular weight alginic acid-containing composition prepared from kelp. (II-3) The intestinal bacteria increasing agent described in (II-2), wherein the low molecular weight alginic acid-containing composition is prepared by a method comprising the step of treating alginic acid or a salt thereof contained in kelp to reduce its molecular weight so that its weight-average molecular weight is in the range of 50,000 to 1,000,000. (II-4) The intestinal bacteria increasing agent described in (II-3), wherein the molecular weight reduction treatment includes a step of heating kelp at a temperature of 100°C or higher. (II-5) An intestinal bacteria increasing agent according to any of (II-2) to (II-4), wherein the kelp is subjected to iodine elution treatment by soaking or boiling the kelp in water.

[0013] (III) Method for producing processed kelp products (III-1) A method for producing kelp products used to increase intestinal bacteria that have secondary bile acid metabolism ability, The method is characterized by having a step of reducing the molecular weight of alginic acid or its salt in kelp so that its weight-average molecular weight is in the range of 50,000 to 1,000,000. The secondary bile acid is at least one selected from the group consisting of IsoalloLCA, AlloLCA, 3-OxoalloLCA, IsoLCA, and 3-OxoLCA. The aforementioned intestinal bacteria are selected from at least one of the following groups: Parabacteroides merdae, bacteria of the family Odoribacteraceae, bacteria of the genus Odoribacter, Parabacteroides distasonis, Bacteroides uniformis, Bacteroides thetaiotaomicron, Alistipes onderdonkii, Alistipes finegoldii, Clostridium innocuum, Eggerthella lenta strain, Gordonibacter pamelaeae, and Hungatella hathewayi. The aforementioned manufacturing method. (III-2) The manufacturing method according to (III-1), wherein the molecular weight reduction treatment includes a step of heating the kelp at a temperature of 100°C or higher. (III-3) The manufacturing method according to (III-2), further comprising a step of iodine elution treatment by soaking or boiling the kelp in water before the low molecular weight treatment step.

[0014] (IV) A method for imparting to processed kelp the function of increasing intestinal bacteria capable of producing secondary bile acids. (IV-1) A method for imparting to a processed kelp product the function of increasing intestinal bacteria that have the ability to produce secondary bile acids, The method is characterized by having a step of reducing the molecular weight of alginic acid or its salt in kelp so that its weight-average molecular weight is in the range of 50,000 to 1,000,000. The secondary bile acid is at least one selected from the group consisting of IsoalloLCA, AlloLCA, 3-OxoalloLCA, IsoLCA, and 3-OxoLCA. The aforementioned intestinal bacteria are selected from at least one of the following groups: Parabacteroides merdae, bacteria of the family Odoribacteraceae, bacteria of the genus Odoribacter, Parabacteroides distasonis, Bacteroides uniformis, Bacteroides thetaiotaomicron, Alistipes onderdonkii, Alistipes finegoldii, Clostridium innocuum, Eggerthella lenta strain, Gordonibacter pamelaeae, and Hungatella hathewayi. The aforementioned method. (IV-2) The method according to (IV-1), wherein the molecular weight reduction treatment includes a step of heating the kelp at a temperature of 100°C or higher. (IV-3) The method according to (IV-2), further comprising a step of iodine elution treatment by soaking or boiling the kelp in water before the low molecular weight treatment step. [Effects of the Invention]

[0015] According to the intestinal bacteria increasing agent of the present invention, oral ingestion can increase the abundance of bacteria in the coliform microbiota that have the ability to metabolize and produce specific secondary bile acids (IsoalloLCA, AlloLCA, 3-OxoalloLCA, IsoLCA, and 3-OxoLCA), including IsoalloLCA. Furthermore, by doing so, the amount of the aforementioned secondary bile acids in the intestines can be increased. Furthermore, according to the secondary bile acid increasing agent of the present invention, oral intake thereof can increase the amount of the aforementioned specific secondary bile acids, including IsoalloLCA, present in the intestines. The intestinal bacteria-increasing agent and secondary bile acid-increasing agent of the present invention are useful as prebiotic compositions that can create an environment in the intestinal microbiota of mammals, including humans, that is suitable for the growth and proliferation of beneficial bacteria involved in the metabolic production of secondary bile acids, such as IsoalloLCA. [Brief explanation of the drawing]

[0016] [Figure 1] This shows the biosynthetic pathways of bile acids (primary bile acids and secondary bile acids). It references extended data figure 6a from Non-Patent Document 3. [Figure 2] This diagram shows the bile acid biosynthesis pathway and the intestinal bacteria involved in the synthesis of various bile acids. It is a modified version of Figure 8a from Non-Patent Document 3. [Figure 3] This table summarizes the substrates and resulting secondary bile acids in the biosynthesis pathway of secondary bile acids, as well as the intestinal bacteria involved in their production, based on the information in Non-Patent Document 3. [Figure 4-1] The results of Experimental Example 1 are shown. Specifically, the results compare the amount of bacteria in the stool of subjects who orally ingested the test sample (heat-treated kelp powder), a low-molecular-weight alginate composition, daily, before ingestion (0w), one week after ingestion (1w), and two weeks after ingestion (2w). (a) Parabacteroides merdae, (b) bacteria of the family Odoribacteraceae, (c) bacteria of the genus Odoribacter. The vertical axis (relative abundance (%)) in each figure represents the relative abundance (occupancy rate) of each intestinal bacterium relative to the total amount of intestinal bacteria present in the stool (the same applies to Figures 4-2 to 4-5). [Figure 4-2] The results of Experimental Example 1 are shown. (d) Parabacteroides distasonis, (e) Bacteroides uniformis, (f) Bacteroides thetaiotaomicron. [Figure 4-3] The results of Experimental Example 1 are shown. (g) Alistipes onderdonkii, (h) Alistipes finegoldii. [Figure 4-4]The results of Experimental Example 1 are shown. (i) Clostridium innocuum, (j) Eggerthella lenta strain. [Figure 4-5] The results of Experimental Example 1 are shown. (k) Gordonibacter pamelaeae, (l) Hungatella hathewayi. [Figure 5] The results of Experimental Example 2 are shown. Specifically, the amount of secondary bile acid (μmol / g stool) in the stool of subjects who orally ingested the test sample (heat-treated kelp powder), a low molecular weight alginate composition, daily, is compared before ingestion (0w) and one week after ingestion (1w). (a) IsoalloLCA, (b) 3-OxoLCA, and (c) IsoLCA. [Figure 6-1] The results of Experimental Example 3, using heat-treated kelp powder (a low-molecular-weight alginic acid-containing composition) (weight-average molecular weight of low-molecular-weight alginic acid: approximately 500,000) as the test sample, are shown. Specifically, in an intestinal tract model test, 3-Oxo-Δ4-LCA was added as the substrate and heat-treated kelp powder as the test sample, and the production amounts of the substrate (3-Oxo-Δ4-LCA) and various secondary bile acids (3-Oxo-alloLCA, AlloLCA) were measured over time. (a) 3-Oxo-Δ4-LCA, (b) 3-Oxo-alloLCA, (c) AlloLCA. [Figure 6-2] The results from Experimental Example 3, using heat-treated kelp powder (a composition containing low molecular weight alginic acid) as the test sample, are shown below. (d) IsoalloLCA, (e) 3-OxoLCA, (f) IsoLCA. [Figure 7-1] The results of Experimental Example 3, using sodium alginate IL-6M (weight-average molecular weight of sodium alginate: approximately 500,000) as the test sample, are shown. Specifically, in an intestinal tract model test, 3-Oxo-Δ4-LCA was added as the substrate and sodium alginate as the test sample, and the production amounts of the substrate (3-Oxo-Δ4-LCA) and various secondary bile acids (3-Oxo-alloLCA, AlloLCA) were measured over time. (a) 3-Oxo-Δ4-LCA, (b) 3-Oxo-alloLCA, (c) AlloLCA. [Figure 7-2] The results from Experimental Example 3, using sodium alginate IL-6M as the test sample, are shown below: (d) IsoalloLCA, (e) 3-Oxo-LCA, (f) IsoLCA. [Figure 8-1] The results from Experimental Example 3, using heat-treated kelp powder (composition containing low molecular weight alginic acid) (weight-average molecular weight of low molecular weight alginic acid: approximately 80,000) as the test sample, are shown below. (a) 3-Oxo-Δ4-LCA, (b) AlloLCA. [Figure 8-2] The results from Experiment Example 3, using heat-treated kelp powder as the test sample, are shown below. (c) IsoalloLCA, (d) IsoLCA. [Figure 9] The results from Experiment Example 3, using sodium alginate ULV-1 (weight-average molecular weight of sodium alginate: approximately 80,000) as the test sample, are shown below. (a) AlloLCA, (b) IsoalloLCA, (c) IsoLCA. [Modes for carrying out the invention]

[0017] Abbreviations may be used throughout this specification and in the drawings, but their full names are as follows: 3-Oxo-Δ 4 -LCA:3-Oxo-Δ 4 - Lithocholic acid 3-Oxo-alloLCA: 3-oxoallolic acid AlloLCA: Alolitocholic acid IsoalloLCA: Isoallocholic acid 3-Oxo-LCA:3-Oxolitocholic acid LCA: Lithocholic acid IsoLCA: Isolithocholic acid 5AR:3-oxo-5α-steroid 4-dehydrogenase 3α-HSDH: 3α-hydroxysteroid dehydrogenase 3β-HSDH: 3β-hydroxysteroid dehydrogenase 5BR: Flavin oxidoreductase

[0018] (I) Low molecular weight alginates and compositions containing low molecular weight alginates The secondary bile acid increasing agent and the intestinal bacteria increasing agent targeted by the present invention are both characterized by containing a composition as an active ingredient that includes alginic acid or a salt thereof having a weight-average molecular weight of 50,000 to 1,000,000, or at least one of the above.

[0019] In this specification, alginic acid with a weight-average molecular weight in the range of 50,000 to 1,000,000 is collectively referred to as "low molecular weight alginic acid." Low molecular weight alginic acid and its salts are collectively referred to as "low molecular weight alginic acids," and compositions containing at least one of low molecular weight alginic acid and its salts are collectively referred to as "low molecular weight alginic acid-containing compositions." The weight-average molecular weight range of 50,000 to 1,000,000 may also be abbreviated as the "specific range." Furthermore, in this specification, numerical ranges described as "〇 to 〇〇" (where 〇 and 〇〇 are both numbers, but 〇 < ○○) mean "〇 or more and 〇〇 or less," and both the lower and upper limits are included within that range.

[0020] The salts of low molecular weight alginic acid are not particularly limited as long as they can be taken orally, but specifically, examples include alkali salts such as sodium salts and potassium salts; ammonium salts; and alkaline earth salts such as calcium salts. Preferably, alkali metal salts and ammonium salts are used, and more preferably, sodium salts.

[0021] The weight-average molecular weight of low-molecular-weight alginates can be calculated using the method described in Experimental Methods 1-4 of the Examples below, that is, from the molecular weight distribution measured by gel filtration chromatography, using a calibration curve with pullulan as the standard substance. The weight-average molecular weight of low-molecular-weight alginates is not particularly limited as long as it has the effects of the present invention (increased secondary bile acid content, increased intestinal bacteria content), but preferably, the lower limit of the weight-average molecular weight can be exemplified by 50,000, 70,000, 80,000, and 100,000. Similarly, the upper limit of the weight-average molecular weight can be exemplified by 1,000,000, 900,000, 800,000, 750,000, 700,000, 650,000, 600,000, and 500,000. These lower and upper limits can be arbitrarily selected to set a range. For example, ranges of 50,000 to 1,000,000, 70,000 to 900,000, 80,000 to 800,000, 80,000 to 700,000, 80,000 to 600,000, and 80,000 to 500,000 can be given. Although not limited, preferably, the range of 80,000 to 500,000 can be exemplified.

[0022] Low molecular weight alginates can be prepared by using high molecular weight alginates found in brown algae such as kelp and wakame, or commercially available high molecular weight alginates or salts thereof with a weight-average molecular weight exceeding 1 million (hereinafter also referred to as "high molecular weight alginates") as raw materials, and performing a molecular weight reduction treatment to bring them within a specific range. Alternatively, commercially available low molecular weight alginates with a weight-average molecular weight within the aforementioned specific range can be used for convenience. While not limited, such low molecular weight alginates can be obtained from companies such as Kimika Co., Ltd.

[0023] The method for reducing the molecular weight of high-molecular-weight alginates is not particularly limited, as long as it reduces the molecular weight of the high-molecular-weight alginates so that the weight-average molecular weight falls within a specific range. Conventionally known methods can be used. For example, methods include reducing the molecular weight of high-molecular-weight alginates using alginases such as lyase (see Patent Documents 1 and 2); reducing the molecular weight of high-molecular-weight alginates by hydrolysis using chemical substances such as acids or hydrogen peroxide; reducing the molecular weight of high-molecular-weight alginates using supercritical or subcritical water (see Non-Patent Document 11); and reducing the molecular weight of high-molecular-weight alginates by heat treatment (see Patent Documents 3 and 4). These molecular weight reduction methods can be applied not only to purified high-molecular-weight alginates but also to high-molecular-weight alginates in a crude state, brown algae containing high-molecular-weight alginates, and their processed products (hereinafter collectively referred to as "high-molecular-weight alginate-containing products"). In terms of post-treatment and flavor, heat treatment is preferred as a molecular weight reduction method.

[0024] A heat treatment method suitably used in the present invention as a low-molecular-weight treatment method is a method of heat treatment of high-molecular-weight alginates or high-molecular-weight alginate-containing materials at a temperature of 100°C or higher. Preferably, the heat treatment is performed at a temperature of 100°C to 180°C. If the heating temperature is less than 100°C, it is difficult to reduce the molecular weight of high-molecular-weight alginates to a specific range within a reasonable time for manufacturing (for example, within 90 minutes, preferably within 60 minutes). Also, if the heating temperature significantly exceeds 180°C, it is undesirable because the high-molecular-weight alginates may be reduced to too much molecular weight in a relatively short time, and bitterness and burnt odor may occur (see Experimental Example 4).

[0025] The heat treatment can be carried out under any of the following conditions: pressurized, atmospheric, or reduced pressure. Preferably, the heat treatment is carried out under conditions of atmospheric pressure or higher (at atmospheric pressure and under pressurized). Here, atmospheric pressure refers to a state where the gauge pressure (atmospheric pressure reference pressure) is 0 MPa. The heat treatment can be carried out in or without the presence of water vapor. Unsaturated water vapor, saturated water vapor, and superheated water vapor can all be used. The heat treatment apparatus can be any apparatus capable of heat-treating polymeric alginates or polymeric alginate-containing materials at a temperature of 100°C or higher, preferably 100°C to 180°C, and can be appropriately selected depending on the presence or absence of pressurization and water vapor. Although not limited, high-pressure steam sterilizers (autoclaves), ovens, vacuum kettles, steamers, etc. can be used as desired. Furthermore, it may be an open system or a closed system.

[0026] For example, when heat-treating high-molecular-weight alginates or materials containing high-molecular-weight alginates using a high-pressure steam sterilizer (pressurized moist heat treatment), the treatment conditions are not limited, but examples include saturated steam, a pressure of 0.1 to 0.3 MPa (preferably 0.1 to 0.25 MPa), and a temperature of 100°C to 130°C (preferably 100°C to 125°C). The heat treatment time can be appropriately selected in relation to the heating temperature, pressurization conditions, etc., under conditions that result in the weight-average molecular weight of the alginates being within a specific range. For example, when using a high-pressure steam sterilizer with a pressure setting of 0.1 MPa, high-molecular-weight alginates or materials containing high-molecular-weight alginates can be reduced to a specific molecular weight range by heating and moist-heat treatment at 100°C for 3 to 60 minutes, 111°C for 15 to 60 minutes, 121°C for 15 to 30 minutes, or 130°C for 15 minutes (see Experimental Example 4). However, the treatment is not limited to these conditions; any pressure-induced moist-heat treatment conditions that yield a thermal history equivalent to that obtained by pressure-induced moist-heat treatment at 100°C for 3 minutes to 111°C for 60 minutes can be adopted.

[0027] Furthermore, for example, when heat-treating high-molecular-weight alginates or materials containing high-molecular-weight alginates using an oven under atmospheric pressure (atmospheric pressure dry heat treatment), the treatment conditions are not limited, but the temperature conditions are preferably above 100°C, for example, 130°C to 180°C (preferably 130°C to 150°C) can be exemplified. The heat treatment time can be appropriately selected in relation to the heating temperature, etc., under conditions where the weight-average molecular weight of the alginates falls within a specific range. For example, when using an oven, high molecular weight alginates or materials containing high molecular weight alginates can be reduced to a specific molecular weight range by dry heat treatment at atmospheric pressure at 130°C for 15-90 minutes, 150°C for 30-90 minutes, and 180°C for 15 minutes (see Experimental Example 4). However, the method is not limited to these, and atmospheric pressure dry heat treatment conditions that yield a thermal history equivalent to that obtained by dry heat treatment at atmospheric pressure at 130°C for 15 minutes to 150°C for 90 minutes can be adopted.

[0028] In the present invention, kelp or processed products thereof can preferably be used as the high-molecular-weight alginic acid-containing material. The kelp used can be any type, such as Makonbu, Rishiri kelp, Hidaka kelp, Naga kelp, Nekoashi kelp, or Rausu kelp; the type is not particularly limited. The part of the kelp used can be either the leaf kelp or the root kelp; there is no particular limitation on the part used. The origin of the kelp can also be any type, such as domestic kelp from Hokkaido or Aomori prefecture, or kelp from China or Russia; there is no particular limitation on the origin. Furthermore, the kelp may be used alone or in combination of two or more types.

[0029] The kelp used can be dried, fresh, frozen, or salted. While the kelp can be used in its raw state, it may also be crushed, shredded, or pulverized beforehand. Alternatively, kelp that has been soaked in water or boiled may be used. It is known that more than 90% of the iodine contained in kelp can be leached out by soaking it in cold or hot water for about 20 minutes (see Non-Patent Literature 12). Because iodine can be removed from kelp through this soaking or boiling process, the risk of developing hypothyroidism, goiter, or thyrotoxicosis due to excessive iodine intake can be reduced. The temperature and time of the soaking or boiling process can be adjusted as needed to ensure iodine leaching. However, as shown in Experimental Example 4, heating kelp under pressure at 100°C or higher reduces high molecular weight alginic acid, so it is preferable to use water at a temperature of 95°C or lower. Furthermore, when using kelp that has been used to extract kelp broth as a high-molecular-weight alginic acid-containing material, the iodine has already been removed, so further boiling is unnecessary.

[0030] (II) Secondary bile acid increasing agents The secondary bile acid increasing agent of the present invention contains the low molecular weight alginic acids or low molecular weight alginic acid-containing compositions described above as active ingredients. Preferably, it is a low molecular weight alginic acid-containing composition, and more preferably, it is a low molecular weight alginic acid-containing composition prepared from kelp, which is a high molecular weight alginic acid-containing material, by the method described above. As described above, the kelp may be in its raw state, but it may also be pre-processed by crushing, shredding or grinding, and / or by soaking in water or boiling. Preferably, it is a low molecular weight alginic acid-containing composition prepared from kelp that has been pre-treated with iodine elution by soaking in water or boiling.

[0031] Low molecular weight alginic acid-containing compositions prepared from kelp (hereinafter also referred to as "kelp-derived low molecular weight alginic acid-containing compositions") contain many dietary fibers in addition to low molecular weight alginic acids, such as laminaran, fucoidan, and cellulose. Although not limited, the total dietary fiber content of kelp-derived low molecular weight alginic acid-containing compositions can be exemplified as 60-90% by mass, preferably 70-90% by mass, and more preferably 70-80% by mass. The total dietary fiber content can be determined based on the modified Prosky method (AOAC official method 991.43).

[0032] Furthermore, while there are no limitations on the proportion of low molecular weight alginic acids contained in the kelp-derived low molecular weight alginic acid-containing composition, lower limits can be 30% by mass or more, 40% by mass or more, 50% by mass or more, 55% by mass or more, and 60% by mass or more, and upper limits can be 90% by mass or less, 85% by mass or less, 80% by mass or less, 75% by mass or less, and 70% by mass or less. Preferably, it is 40 to 80% by mass, more preferably 50 to 70% by mass.

[0033] The amount of low molecular weight alginates or low molecular weight alginate-containing compositions in the secondary bile acid increasing agent of the present invention may be any amount that exerts a secondary bile acid increasing effect, and can be appropriately adjusted so that low molecular weight alginates are present in a range of 1 to 100% by mass.

[0034] The secondary bile acids targeted in this invention are those that, in the human intestine, are produced by the action of 5AR, 3α-HSDH, and 3β-HSDH, resulting in 3-Oxo-Δ 4 3-Oxo-alloLCA, AlloLCA, and IsoalloLCA are generated using -LCA as a substrate; and 3-Oxo-Δ is produced by the action of 5BR, 3α-HSDH, and 3β-HSDH. 4Examples of secondary bile acids that can be produced using -LCA as a substrate include 3-OxoLCA and IsoLCA (see Figure 1). Although not limited, preferred secondary bile acids include IsoalloLCA, 3-OxoLCA, and IsoLCA, which are known as anti-aging hormones. More preferably as a secondary bile acid is IsoalloLCA, which is abundant in the intestines of centenarians and is thought to exhibit strong bactericidal activity against Gram-positive pathogenic bacteria, contributing to the maintenance of the intestinal environment.

[0035] The secondary bile acid increasing agent targeted by the present invention has the effect of increasing the amount of the aforementioned secondary bile acids present in the intestines and is used for that purpose. It may also have the effect of increasing the amount of at least one of the aforementioned secondary bile acids present in the intestines, but for the reasons stated above, it is preferable that it has the effect of increasing the amount of at least IsoalloLCA present in the intestines and is used for that purpose (use). More preferably, it has the effect of increasing the amount of at least one of the aforementioned secondary bile acids (IsoalloLCA, 3-OxoLCA, IsoLCA) known as anti-aging hormones present in the intestines and is used for that purpose (use).

[0036] The amount of secondary bile acids present in the intestines can be evaluated by analyzing the amount of secondary bile acids in human feces, as will be explained in Experimental Example 2 below. For example, if a candidate substance for a secondary bile acid increasing agent (in this invention, low molecular weight alginates or low molecular weight alginate-containing compositions) is continuously orally administered to humans (for example, once a day for about one to two weeks), and the amount of secondary bile acids in the excreted stool is analyzed over time, and an increase in the amount of secondary bile acids is observed compared to before ingestion, then it can be determined that the candidate substance has the effect of increasing the amount of secondary bile acids in the human intestine and can be used as a secondary bile acid increasing agent or its active ingredient.

[0037] Furthermore, this evaluation can also be performed using an in vitro test with a cultured human gut microbiota model (human intestinal tract model) that mimics the human gut microbiota, instead of an in vivo test using humans (Non-Patent Documents 9 and 10). As shown in Experimental Example 3 described later, this in vitro test yields 3-Oxo-Δ 4 -When an increase in the amount of secondary bile acids is observed by anaerobic culturing a fecal dilution (test medium) to which LCA (substrate) and a candidate substance for a secondary bile acid increasing agent (in this invention, low molecular weight alginates or low molecular weight alginate-containing compositions) have been added as a test sample, it can be determined that the candidate substance has the effect of increasing the amount of secondary bile acids in the human intestine and can be used as a secondary bile acid increasing agent or its active ingredient.

[0038] Secondary bile acid increasing agents are used to alter (improve) the intestinal environment so as to increase the amount of the aforementioned secondary bile acids in the human intestine. By improving the intestinal environment in this way, it becomes possible to maintain or promote health. Maintaining or promoting health includes maintaining or promoting health by preventing and / or improving diseases or pathological conditions. The target diseases or pathological conditions are not limited to those that can be prevented or improved by increasing the amount of the aforementioned secondary bile acids in the human intestine, but include Clostridium difficile infection, for example (see Non-Patent Document 13). Furthermore, secondary bile acids are known to have bactericidal effects against pathogenic bacteria such as vancomycin-resistant enterococci (VRE) (bacteria known to cause infections such as peritonitis, surgical wound infections, pneumonia, and sepsis in postoperative patients and patients with impaired infection defense function), methicillin-resistant Staphylococcus aureus (bacteria known to cause MRSA infections), group C and G hemolytic streptococci (bacteria known to cause suppurative diseases such as sepsis, cellulitis, and suppurative arthritis), group A hemolytic streptococci (Streptococcus pyogenes) (bacteria known to cause upper respiratory tract infections and suppurative skin infections), Streptococcus sanguinis (oral streptococci) (bacteria known to cause biofilm formation and infectious endocarditis), Clostridium perfringens (bacteria known to cause Clostridium perfringens infections), and Bacillus cereus (bacteria known to cause Bacillus cereus infections). Therefore, secondary bile acid increasing agents are useful for preventing or improving diseases and conditions caused by these pathogens.

[0039] (III) Intestinal bacteria increasing agents The intestinal bacteria increasing agent of the present invention contains the low molecular weight alginic acid compounds or low molecular weight alginic acid-containing compositions described above as active ingredients. Preferably, it is a low molecular weight alginic acid-containing composition, and more preferably, it is a low molecular weight alginic acid-containing composition prepared from kelp, which is a high molecular weight alginic acid-containing material, by the method described above. As described above, the kelp may be in its raw state, but it may also be pre-processed by crushing, shredding or grinding, and / or by soaking in water or boiling. Preferably, it is a low molecular weight alginic acid-containing composition (kelp-derived low molecular weight alginic acid-containing composition) prepared from kelp that has been pre-treated with iodine elution by soaking in water or boiling. The total amount of dietary fiber and the proportion of low molecular weight alginic acid compounds contained in the kelp-derived low molecular weight alginic acid-containing composition can be exemplified by the range described in (II) above.

[0040] The amount of low molecular weight alginic acid or low molecular weight alginic acid-containing composition in the intestinal bacteria-increasing agent of the present invention may be any amount that exerts an intestinal bacteria-increasing effect, and can be appropriately adjusted so that low molecular weight alginic acid is contained in a range of 1 to 100% by mass.

[0041] The intestinal bacteria targeted by the present invention are bacteria present in the intestinal microbiota (preferably the human intestinal microbiota) that have the ability to metabolize and produce the secondary bile acids described above (intestinal bacteria with secondary bile acid metabolic production ability). The intestinal bacteria only need to have the effect of increasing the amount of at least one of the five secondary bile acids described above in the intestines by exerting their secondary bile acid metabolic production ability. For the reasons described above, it is preferable that the intestinal bacteria have the effect of metabolizing and producing at least IsoalloLCA among the secondary bile acids described above, have the effect of increasing the amount of IsoalloLCA in the intestines, and are used for that purpose (use). More preferably, the intestinal bacteria have the effect of metabolizing and producing secondary bile acids known as anti-aging hormones (one or more selected from the group consisting of IsoalloLCA, 3-OxoLCA, and IsoLCA), have the effect of increasing their amount in the intestines, and are used for that purpose (use).

[0042] The intestinal bacteria present in the human gut microbiota that possess the ability to produce secondary bile acids as described above (hereinafter also referred to as "these intestinal bacteria") include: (a) Parabacteroides merdae (hereinafter, P. merdae), (b) bacteria of the family Odoribacteraceae, (c) bacteria of the genus Odoribacter, (d) Parabacteroides distasonis (hereinafter, P. distasonis), (e) Bacteroides uniformis (hereinafter, B. uniformis), (f) Bacteroides thetaiotaomicron (hereinafter, B. thetaiotaomicron), (g) Alistipes onderdonkii (hereinafter, A. onderdonkii), (h) Alistipes finegoldii (hereinafter, A. finegoldii), (i) Clostridium innocuum (hereinafter, C. innocuum), and (j) Eggerthella lenta strain (hereinafter, E. lenta) This includes (k) Gordonibacter pamelaeae (hereinafter, G. pamelaeae) and (l) Hungatella hathewayi (hereinafter, H. hathewayi). Of these intestinal bacteria, (e) B. uniformis and (f) B. thetaiotaomicron, as well as (d) P. distasonis, have all been reported to decompose alginate to produce short-chain fatty acids (see Non-Patent Literature 8). As shown in Figures 2 and 3, the aforementioned Bacteroides intestinal bacteria produce 3-Oxo-Δ 4 -It produces 3-OxoalloLCA using LCA as a substrate. In addition, (d)P. distasonis produces LCA and 3-Oxo-Δ 4 In addition to producing IsoLCA using -LCA as a substrate, they also produce AlloLCA using 3-OxoalloLCA as a substrate. Furthermore, among the aforementioned intestinal bacteria, (a) P. merdae, (b) bacteria of the family Odoribacteraceae, and (c) bacteria of the genus Odoribacter all produce 3-Oxo-Δ 4-LCA as a substrate to produce 3-OxoalloLCA and IsoalloLCA. Furthermore, (g) A. onderdonkii, and (h) A. finegoldii are 3-Oxo-Δ 4 -LCA as a substrate to produce 3-OxoalloLCA: (i) C. innocuum is 3-Oxo-Δ 4 -LCA as a substrate to produce IsoLCA: (j) E. lenta strain and (k) G. pamelaeae are LCA as a substrate to produce 3-OxoLCA and IsoLCA: (l) H. hathewayi is 3-Oxo-Δ 4 -LCA as a substrate to produce 3-OxoLCA, respectively contributing thereto. Thus, the above-mentioned (a) to (l) indigenous intestinal bacteria are intestinal bacteria involved in the biosynthesis of the secondary bile acids described above in the human intestinal flora.

[0043] The abundance of the indigenous intestinal bacteria in the intestinal flora can be evaluated by analyzing the composition of the bacterial flora contained in human excreted feces, as described in Experimental Example 1 below. For the analysis of the composition of bacteria in the bacterial flora (flora analysis), generally, shotgun metagenomic analysis targeting the whole genome of the bacterial flora and 16S rRNA metagenomic analysis targeting the 16S rRNA gene are used. In terms of being relatively easy to perform, the latter 16S rRNA metagenomic analysis is preferably used.

[0044] This 16S rRNA metagenomic analysis can be performed by amplicon sequencing, which involves extracting DNA from a sample containing various bacteria, amplifying a specific region (the third to fourth variable region) of the bacterial 16S rRNA gene by PCR, and then analyzing the resulting PCR amplification product (amplicon). This amplicon sequencing can be performed using a next-generation sequencer. Subsequently, the vast amount of base sequence data obtained from the sequencing is analyzed by computer. This bacterial community composition analysis (16S amplicon analysis) can be performed using amplicon sequencing data analysis software (e.g., QIIME2) according to its manual. QIIME2 is an open-source analysis pipeline that bundles the software necessary for amplicon sequencing analysis (https: / / qiime2.org / ).

[0045] By analyzing the composition of the bacterial flora in this way, it is possible to calculate the total number of bacteria in the gut microbiota and the number of the target gut bacteria, and from there, the proportion of the target gut bacteria to the total number of bacteria in the gut microbiota can be determined. For example, if a candidate substance for increasing intestinal bacteria (in this invention, low molecular weight alginates or low molecular weight alginate-containing compositions) is continuously orally administered to a human (for example, once a day for about one to two weeks), and the composition of the bacterial flora in the human's stool is analyzed, and an increase in the proportion of any of the intestinal bacteria is observed compared to the composition before ingestion, then it can be determined that the candidate substance has the effect of increasing the proportion of the intestinal bacteria in the human intestinal flora and can be used as an active ingredient in the intestinal bacteria increasing agent. This evaluation can also be performed by an in vitro test using the human intestinal tract model described above, instead of an in vivo test using a human. In the in vitro test, if an increase in the proportion of the intestinal bacteria is observed when a fecal dilution (test medium) to which the candidate substance for increasing secondary bile acids (in this invention, low molecular weight alginates or low molecular weight alginate-containing compositions) has been added is cultured anaerobically, then it can be determined that the candidate substance has the effect of increasing the proportion of the intestinal bacteria in the human intestinal flora and can be used as an active ingredient in the intestinal bacteria increasing agent.

[0046] In the above, "proportion of this intestinal bacterium" refers to the ratio to the total number of bacteria detected in the gut microbiota, and can also be rephrased as "relative abundance" or "occupancy rate." An increase in this intestinal bacterium in the gut microbiota means an increase in its occupancy rate, and to that extent, an increase or decrease in bacteria other than this intestinal bacterium in the gut microbiota may occur simultaneously. In other words, this includes not only cases where the occupancy rate of this intestinal bacterium increases because its growth in the gut microbiota is faster than that of other bacteria, but also cases where the amount of this intestinal bacterium relatively increases due to a decrease in other bacteria, and thus the occupancy rate of this intestinal bacterium increases.

[0047] An increase in the proportion of these intestinal bacteria within the gut microbiota means a relative increase compared to the proportion before the application of the intestinal bacteria-increasing agent. The degree of increase is not particularly limited as long as an increase is observed, but for example, if the proportion before the application of the intestinal bacteria-increasing agent is set to 100, the value may be greater than 100, preferably 110 or more, more preferably 120 or more, and even more preferably 130 or more.

[0048] This intestinal bacteria increasing agent is used to improve the composition of the intestinal microbiota so that the proportion (occupancy rate) of this intestinal bacteria in the intestinal microbiota increases. By improving the intestinal microbiota in this way, it becomes possible to maintain or improve health in response to the increase in this intestinal bacteria. This maintenance or improvement of health includes maintaining or improving health by preventing and / or improving diseases or pathological conditions. The target diseases or pathological conditions are not limited to those that can be prevented or improved by the proliferation of this intestinal bacteria in the intestinal microbiota, but include the diseases or pathological conditions described in (II).

[0049] (IV) Form and usage of secondary bile acid increasing agents and intestinal bacteria increasing agents, etc. The target population (administered or ingested persons) of the secondary bile acid increasing agent and intestinal bacteria increasing agent of the present invention (hereinafter collectively referred to as "the agent") is mammals, preferably humans. Furthermore, adults, children, infants, neonates (including low-birth-weight infants), etc., are also included. Sex is not particularly limited. The dosage (ingestion amount) can be appropriately set according to the type, age, sex, and condition of the target mammal. While not limited, the dosage (ingestion amount) can be appropriately set and adjusted within the range of 0.5 to 15 g / day when converted to the dosage (ingestion amount) of low-molecular-weight alginates.

[0050] The preferred route of administration (ingestion) of this drug is orally. Alternatively, parenteral administration can be performed via enteral injection.

[0051] When this drug is prepared as an oral composition, it is preferable to prepare it in the form of food or beverage, quasi-drug, or pharmaceutical product. Food or beverage products may contain a nutritional composition. This nutritional composition may include an enteral nutritional supplement that is administered into the intestines through a tube, such as a gastrostomy tube. The form of these oral compositions is not particularly limited, and can be arbitrarily selected from conventional forms such as solid, powder, granules, cream, gel, sol, liquid, and suspension, depending on the form of administration and purpose.

[0052] In addition to the low molecular weight alginic acids or low molecular weight alginic acid-containing compositions mentioned above, food and beverages may also contain, to the extent that they do not impair the effects of the alginic acids, animal and plant proteins; oils and fats derived from animals and plants; carbohydrates such as sugars, starches, and dietary fiber; vitamins; minerals; and other seasonings such as sweeteners, acidulants, and spices, stabilizers, and colorants that are commonly used in food and beverages.

[0053] Among the agents in this invention, the intestinal bacteria increasing agent can be described as "a food component that enhances the growth of the intestinal bacteria in the intestinal flora, thereby having a beneficial effect on the host and improving the host's health," and in this sense, it can also be positioned as a prebiotic. For this reason, foods and beverages containing the intestinal bacteria increasing agent can be called foods and beverages containing prebiotics (prebiotic foods and beverages). Foods and beverages targeted by this invention include, in addition to such labeling, food and beverage compositions (for example, foods with functional claims, foods for specified health uses, etc.) that are permitted to display descriptions regarding the increase of secondary bile acids and / or the intestinal bacteria mentioned above, as well as the effects and uses associated with such increase (uses related to health promotion).

[0054] Furthermore, food and beverages include, as one form, feed compositions which are food and beverages for mammals other than humans, such as pets (dogs, cats, rabbits, etc.), test animals (mice, rats, rabbits, monkeys, etc.), and livestock (cattle, horses, etc.).

[0055] When this drug is prepared as a quasi-drug or pharmaceutical product, for oral administration, it can be formulated as a solid preparation such as a powder, granules, tablet, or capsule; or as a liquid preparation such as a solution, syrup, suspension, or emulsion. For parenteral administration, it can be formulated as a tube-administered preparation such as a gastrostomy tube.

[0056] In formulation, in addition to the low molecular weight alginates or low molecular weight alginate-containing compositions described above, ingredients such as excipients, pH adjusters, colorants, and flavoring agents commonly used in formulation can be used. Furthermore, other pharmacoactive ingredients can be used in combination. In addition, formulation can be carried out using known methods as appropriate, depending on the dosage form.

[0057] (V) Method for manufacturing kelp products The present invention provides a method for producing kelp products used to increase intestinal bacteria (intestinal bacteria) that have the ability to produce secondary bile acids. The method includes a step of reducing the molecular weight of alginic acid compounds in kelp so that their weight-average molecular weight is in the range of 50,000 to 1,000,000. The kelp used for the molecular weight reduction treatment may also be kelp that has been treated with iodine elution by soaking in water or boiling in water beforehand.

[0058] The "secondary bile acids" and "intestinal bacteria" discussed here are as described above. Furthermore, "increase in intestinal bacteria" refers to an increase in the proportion (occupancy rate) of intestinal bacteria in the human gut microbiota. The meaning of increased occupancy rate and its evaluation method are also as described above. The "kelp," "low molecular weight processing method," and "iodine elution treatment" are also as described above and can all be applied here.

[0059] (VI) A method for imparting to processed kelp the function of increasing intestinal bacteria capable of producing secondary bile acids. The present invention provides a method for imparting to processed kelp the function of increasing intestinal bacteria (proto-intestinal bacteria) that have the ability to produce secondary bile acids. The method includes a step of preparing a kelp product containing low-molecular-weight alginic acid by performing a molecular weight reduction treatment on the alginic acid in the kelp so that the weight-average molecular weight of the alginic acid is in the range of 50,000 to 1,000,000. The kelp subjected to the molecular weight reduction treatment may be kelp that has been treated with iodine elution treatment in advance by soaking in water or boiling in water.

[0060] The "secondary bile acids" and "intestinal bacteria" discussed here are as described above. Furthermore, "increase in intestinal bacteria" refers to an increase in the proportion (occupancy rate) of intestinal bacteria in the human gut microbiota. The meaning of increased occupancy rate and its evaluation method are also as described above. The "kelp," "low molecular weight processing method," and "iodine elution treatment" are also as described above and can all be applied here.

[0061] In this specification, the terms “contains” and “includes” include the meanings of “consisting of” and “substantially consisting of.” [Examples]

[0062] The present invention will be described below using experimental examples to aid in understanding its structure and effects. However, the present invention is not limited in any way by these experimental examples. Unless otherwise specified, the following experiments were conducted at room temperature (25±5℃) and under atmospheric pressure conditions. Unless otherwise specified, "%" below means "mass%" and "parts" means "parts by mass".

[0063] 1. Materials and experimental methods 1-1. Preparation of the test sample (heat-treated kelp powder) 200-300g of dried kelp (raw seaweed) (second-grade Makonbu from Yasuura) was cut into 2cm x 5cm pieces, placed in 20 times its volume of water, and heated at 95°C for 60 minutes to obtain boiled kelp. After removing excess water by placing it in a colander, it was placed in a beaker and autoclaved (pressurized moist heat treatment) under the conditions described in Table 1 (heat-treated kelp 1 and 2).

[0064] [Table 1]

[0065] Furthermore, approximately 300g of dried kelp (raw seaweed) (second-grade Makonbu from Yasuura) was cut into 2cm x 5cm pieces, placed in 20 times its volume of water, stirred at room temperature for 30 minutes, and then drained in a sieve. It was then placed again in 20 times its volume of water, stirred at room temperature for 30 minutes, drained in a sieve, and the water was removed to obtain water-soaked kelp.

[0066] The heat-treated kelp 1 and 2, and the water-soaked kelp prepared as described above, were spread on stainless steel rectangular trays and dried overnight at 50°C, after which they were pulverized in a mixer. The resulting pulverized material was passed through a sieve with a pore size of 500 μm, and the fraction smaller than 500 μm that passed through the sieve was collected. The pulverized material remaining on the sieve was again put through the mixer and pulverized, and the fraction smaller than 500 μm was collected again and combined with the previous fraction. This process was repeated until powders with a particle size of 500 μm or less (Test sample 1: heat-treated kelp 1 powder, Test sample 2: heat-treated kelp 2 powder, Test sample 3: water-soaked kelp powder) (hereinafter, these may be collectively referred to as "test samples") were obtained in the proportions shown in Table 2.

[0067] [Table 2]

[0068] 1-2. Measurement of dietary fiber content The dietary fiber content in the test samples prepared using the method described above was measured using the Total Dietary Fiber Assay Kit (Megazyme), based on the modified Prosky method. Specifically, 0.5 g of the test sample was suspended in 40 mL of 50 mM MES-TRIS buffer (pH 8.2) and sequentially treated with thermostable α-amylase (100°C, 30 min), protease (60°C, 30 min), and amyloglucosidase (60°C, 30 min). This series of enzymatic treatments decomposed and removed digestible starch and protein from the test sample. The MES-TRIS buffer was prepared by dissolving 9.8 g of MES(2-(N-morpholino)ethanesulfonic acid) (Nacalai Tesque) and 6.1 g of Tris(2-amino-2-hydroxymethyl-1,3-propanediol) (Fujifilm Wako Pure Chemical Industries, Ltd.) in distilled water, adjusting the pH to 8.2 with sodium hydroxide aqueous solution, and then making up to 1 L with distilled water. The enzyme reaction solution was filtered using a glass filter (pore size 40-50 μm). The insoluble residue on the glass filter was used to measure the insoluble dietary fiber content. Specifically, the insoluble residue was sequentially washed with ethanol and acetone, dried overnight at 105°C, and then its dry weight was measured. Furthermore, the protein and ash content contained in the residue were separately quantified using the method described below, and the insoluble dietary fiber content in the test sample was calculated by subtracting these from the dry weight.

[0069] The filtrate that passed through the glass filter was used to measure the water-soluble dietary fiber content. Specifically, four times the volume of ethanol was added to the filtrate and allowed to stand at room temperature for one hour. The resulting precipitate was then filtered using a glass filter (pore size 40-50 μm). The residue on the glass filter was washed with ethanol and acetone in the same manner as described above, dried overnight, and the dry weight was measured. Furthermore, the amount of protein and ash contained in the residue was separately quantified using the method described below, and the water-soluble dietary fiber content in the test sample was calculated by subtracting these from the dry weight. The dietary fiber content in the test sample was calculated by adding the calculated insoluble dietary fiber content and water-soluble dietary fiber content.

[0070] [Method for quantifying proteins] The amount of protein in the residue, whose dry weight was measured in the above process, was determined using the Kjeldahl method. The entire residue was transferred to weighing paper and then to a decomposition bottle containing boiling chips and 2 g of a decomposition accelerator (a mixture of 10 g of copper sulfate and 90 g of potassium sulfate, ground in a mortar). 20 mL of concentrated sulfuric acid was added to the decomposition bottle and heated in a Kjeldahl decomposition apparatus until it turned emerald blue. After heating, it was allowed to cool at room temperature and the volume was increased to 100 mL by adding distilled water. Neutralization titration was performed using a Kjeldahl distillation apparatus (K-350, BUCHI), and the amount of protein was calculated from the titration value.

[0071] [Method for determining ash content] The amount of ash in the residue, whose dry weight was measured in the aforementioned process, was determined using the direct ashing method. The residue was calcined in a muffle furnace at 550°C for 5 hours, cooled in a constant temperature oven at 105°C, and then further cooled at room temperature. The amount of ash was calculated by measuring the weight of the cooled residue.

[0072] 1-3. Measurement of alginate content The residue obtained from the above treatment was suspended in a 1% (v / v) HCl aqueous solution, then centrifuged (2,000 rpm, 5 min, 20°C), and the supernatant was collected. This was filtered using a glass filter (pore size 40-50 μm), and the residue on the glass filter was suspended in 100 mL of a 1% sodium hydroxide aqueous solution and allowed to stand overnight to extract alginic acid. After removing the residue by filtration using filter paper (qualitative filter paper No. 2, ADVANTEC), the collected filtrate was diluted to 200 mL with distilled water. The amount of alginic acid in this filtrate (alginic acid-containing sample) was determined by subjecting it to the carbazole sulfuric acid method.

[0073] [Carbazole sulfuric acid method] 5 ml of borax-sulfuric acid solution (1.9 g of borax dissolved in 200 mL of concentrated sulfuric acid) was added to 1 ml of the alginic acid-containing sample described above, and the mixture was heated at 100°C for 10 minutes. After cooling with ice, 0.2 ml of carbazole reagent (0.125 g of carbazole dissolved in 100 mL of ethanol) was added, and the mixture was heated at 100°C for 15 minutes. After cooling with ice, the absorbance at 530 nm was determined using a spectrophotometer.

[0074] Calibration curves were prepared using standard sodium alginate (IL-6M, Kimika). Specifically, standard sodium alginate was dissolved in distilled water to prepare samples at various concentration series (0, 16, 31, 63, 125, 250 μg / mL), and the absorbance (530 nm) was measured to create the calibration curve. The linearity of the calibration curve was confirmed to be good from 0 to approximately 250 μg / mL (mg / L).

[0075] The alginic acid content in each filtrate (alginic acid-containing sample) prepared as described above was determined from the calibration curve, and the alginic acid content (on a dry weight basis) in test samples 1 and 2 was calculated from there. The alginic acid content and dietary fiber content of test samples 1 and 2 are shown in Table 3.

[0076] [Table 3]

[0077] 1-4. Measurement of the average molecular weight of alginate 0.1 g of the test sample prepared by the above method was placed in 50 mL of 1% (W / V) sodium carbonate aqueous solution and stirred overnight (16 hours) at room temperature (extraction treatment). Next, this was filtered using a glass filter (pore size 40-50 μm), and the recovered filtrate was adjusted to pH 7 with 3 M HCl (neutralization). This was placed in a dialysis tube (regenerated cellulose membrane, molecular weight cutoff 3500, product name SERVAPOR® 3 dialysis tubing, manufactured by SERVA Electrophoresis GmbH) and dialyzed against 2000 mL of distilled water. After overnight dialyzing, 0.85 g of sodium nitrate was added to the dialysate and stirred, then the volume was made up to 100 mL with pure water. After filtering this solution through a membrane filter (0.45 μm, product name Filtrex, manufactured by LaboLabo Company), the recovered filtrate was subjected to gel filtration chromatography (GPC) under the following conditions to measure the molecular weight distribution of alginic acid contained in the filtrate.

[0078] [GPC conditions] Column: Shodex OHpak SB-806M HQ (8.0mm I.D. x 300mm) Detector: Differential refractive index detector (RI) Mobile phase: 0.1 M sodium nitrate aqueous solution Flow rate: 0.7 ml / min Column temperature: 50℃ Injection volume: 100 μl

[0079] From the GPC results (retention time) and the calibration curve using pullulan as a standard reagent, the weight-average molecular weight (Mw), number-average molecular weight (Mn), peak-top molecular weight (Mp), and degree of dispersion (Mw / Mn) of alginate were determined. The results are shown in Table 4.

[0080] [Table 4]

[0081] 1-5. Subjects and Administration Methods Three male subjects (in their 20s to 40s) were informed of the study's procedures in advance, and their consent was obtained before the study was conducted. Each subject was given 5g of the test sample 1 prepared in 1-1 above, once a day, either with or after a meal, for two weeks. Stool samples were collected before the start of intake and at 1 and 2 weeks after intake.

[0082] 1-6. Collection of stool sample Stool samples were collected in two types of stool collection containers (FS-0003: no preservative solution, spoon-shaped; FS-0013: with preservative solution, long brush-shaped; both manufactured by Techno Suruga Lab). The stool collected in the FS-0003 container was stored at -80°C until use for bile acid measurement. The stool collected in the FS-0013 container was stored at 4°C for intestinal microbiota analysis.

[0083] 1-7. Method for extracting bile acids 100 mg of stool sample, stored at -80°C, was weighed into a 2 mL lidded microtube and then freeze-dried. 1 mL of ethanol and 10 μL of 100 μM nordeoxycholic acid (NDCA, internal standard) were added to the dried stool sample. Then, 2.8 mm ceramic beads (2BT1B-03961CK28) were added, and the sample was treated in a bead-type lyserator (MagNA Lyser, Roche) at 6000 rpm for 20 seconds twice (with a 20-second interval) to disrupt the stool sample and the bacteria it contained. Next, the sample was heated in a constant temperature bath at 60°C for 30 minutes, then stirred and heat-treated at 85°C for 3 minutes.

[0084] Next, after returning to room temperature and stirring, the mixture was centrifuged (10,000 rpm, 3 minutes, RT), the supernatant was collected, and concentrated using a centrifugal concentrator (supernatant 1). Meanwhile, 1 mL of ethanol was added to a microtube containing the residue (pellet), stirred, and then centrifuged again (10,000 rpm, 3 minutes, RT). The supernatant was collected, placed in the microtube containing supernatant 1, and concentrated using a centrifugal concentrator (supernatant 2). Furthermore, 1 mL of ethanol was added to the microtube containing the residue (pellet) once more, and the same procedure (centrifugation and supernatant collection) was performed. The collected supernatant was placed in the microtube containing supernatant 2 and concentrated again using a centrifugal concentrator (supernatant 3) (total of 3 centrifugal concentration processes). 1 mL of methanol was added to the microtube containing the centrifuged supernatant 3 and stirred, and then further mixed by pipetting. This was centrifuged (10,000 rpm, 1 minute, RT), and the supernatant was collected and used as the test sample for bile acid analysis.

[0085] A solid-phase extraction column (Oasis HLB 1 cc Vac Cartridge, Waters) was washed with methanol and 10 mM ammonium acetate aqueous solution. A mixture of the bile acid analysis sample prepared by the above method and the 10 mM ammonium acetate aqueous solution was added to the washed solid-phase extraction column. Next, the column was washed with 10 mM ammonium acetate aqueous solution, and then ethanol was passed through it (extraction), and the resulting liquid (extract) was collected. The collected liquid (extract) was concentrated using a centrifugal concentrator, then dried (bile acid measurement sample), and stored at -30°C until used for bile acid analysis. When analyzing bile acids, methanol was added to the frozen bile acid sample before the bile acid analysis was performed.

[0086] 1-8. Bile acid analysis Bile acid analysis was performed using a UPLC-MS system consisting of a Nexera X2 LC system (pump: LC-30AD, autosampler: SIL-30AC, system controller: CBM-20A, Shimadzu) connected to a triple quadrupole mass spectrometer (LCMS-8040, Shimadzu) using an ESI probe as the ion source. The substances to be measured were ionized in negative mode and analyzed in SIM (Single ion monitoring) mode.

[0087] [Analysis conditions] Column: InertSustain® C18 column (2 μm, 2.1 x 150 mm, manufactured by GL Sciences Co., Ltd.) Column temperature: 40℃ Mobile phase: Solution A (10 mM ammonium acetate + 0.01% formic acid + 20% acetonitrile), Solution B (30% acetonitrile, 70% methanol) Gradient conditions: The following "〇:〇" (where 〇 is a numerical value) indicates the capacity ratio (A:B, v / v) between mobile phase A and mobile phase B; 80:20 (0~0.1 min) → 48:52 (0.1~1 min) → 30:70 (1~27 min) → 0:100 (27~27.1 min) → 0:100 (27.1~33 min) → 80:20 (33~33.1 min) → 80:20 (33.1~38 min). Flow rate: 0.2 mL / min Injection volume: 5μL Analysis time: 38 minutes.

[0088] Figure 1 shows the biosynthetic pathway of bile acids (primary and secondary bile acids). Primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), synthesized in the liver, are converted in the intestines by intestinal bacteria, mainly producing secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA). However, it has become clear that various other secondary bile acids are also produced from primary bile acids by intestinal bacteria. In addition to LCA, ursodeoxycholic acid (UDCA), 3-oxolitocholic acid (3-OxoLCA), isolitocholic acid (IsoLCA), and isoallocholic acid (IsoalloLCA) are produced from CDCA. In particular, IsoLCA has an intermediate, 3-Oxo-Δ 4 -LCA is produced via the conversion reaction of flavin oxidoreductase (5BR) and 3β-hydroxysteroid dehydrogenase (3β-HSDH), and IsoalloLCA is 3-Oxo-Δ 4 -It is produced via a pathway involving the conversion reaction of LCA to 3-oxo-5α-steroid 4-dehydrogenase (5AR) and 3β-HSDH (Non-Patent Literature 3).

[0089] In this study, among the bile acids shown in Figure 1, 3-Oxo-Δ 4 -LCA (m / z value: 371.2), IsoalloLCA (m / z value: 375.2), AlloLCA (m / z value: 375.2), 3-Oxo-alloLCA (m / z value: 374.2), 3-Oxo-LCA (m / z value: 373.05), and IsoLCA (m / z value: 375.2) were included in the analysis. The analysis software used was LabSolutions (manufactured by Shimadzu).

[0090] 1-9.DNA extraction method Using the ISOSPIN Fecal DNA kit (manufactured by Nippon Gene Co., Ltd.), genomic DNA was extracted and purified from stool samples stored at 4°C, following the standard protocol (Beads Beat method) recommended for the kit. Cell disruption was performed by adding FE1 Buffer and RNase A (both included with the kit) to a Beads Tube (included with the kit) containing a stool sample (0.2g), and then using a bead-type lyserator (MagNA Lyser, manufactured by Roche) at 5000 rpm for 1 minute x 3 or 5 cycles (with a 90-second interval). The DNA solution prepared using the aforementioned kit product was subjected to a micro-UV-Vis spectrophotometer (NanoDrop, Thermo Fisher Scientific) to quantify the amount of DNA. Based on this quantification, the DNA solution was subjected to electrophoresis on a 1% agarose gel, and the presence of bands confirmed that purified, high-purity DNA had been obtained.

[0091] 1-10. Next-generation sequencing analysis The DNA extracted and purified using the aforementioned method was subjected to next-generation sequencing analysis by Biotechnology Research Institute Co., Ltd. The DNA solution was sent to Biotechnology Research Institute Co., Ltd., where 1st and 2nd PCR amplification and analysis were performed using the 16S rRNA V3 / V4 region as the target gene. QIIME2 was used for OTU classification, and Greengene was used as the phylogenetic name assignment database.

[0092] Figure 2 shows the bile acid biosynthesis pathway and the intestinal bacteria involved in the synthesis of various bile acids. Figure 3 shows a table summarizing the substrates and bile acids produced in the bile acid biosynthesis pathway, as well as the intestinal bacteria involved in their production.

[0093] 2. Experiment and Results Experimental Example 1: Analysis of the bacterial flora of stool samples and its results Using the method described above, genomic DNA extracted and purified from human stool samples was subjected to next-generation sequencing analysis to confirm how the composition of the bacterial flora contained in the stool samples changed over time after oral ingestion (once a day) of test sample 1 (heat-treated kelp powder 1). The results are shown in Figures 4-1 to 4-5 (collectively referred to as "Figure 4").

[0094] As a result, as shown in Figure 4, oral ingestion of test sample 1 (once a day) resulted in the removal of the following bacteria in a short period of 1 to 2 weeks: (a) Parabacteroides merdae (P. merdae), (b) bacteria of the family Odoribacteraceae, (c) bacteria of the genus Odoribacter (see Figure 4-1), (d) Parabacteroides distasonis (P. distasonis), (e) Bacteroides uniformis (B. uniformis), (f) Bacteroides thetaiotaomicron (B. thetaiotaomicron) (see Figure 4-2), (g) Alistipes onderdonkii (A. onderdonkii), (h) Alistipes finegoldii (A. finegoldii) (see Figure 4-3), (i) Clostridium innocuum (C. innocuum), and (j) Eggerthella lenta An increasing trend in the abundance of strain (E. lenta strain) (see Figure 4-4), (k) Gordonibacter pamelaeae (G. pamelaeae), and (l) Hungatella hathewayi (H. hathewayi) (see Figure 4-5) was observed in the feces.

[0095] Of these bacteria, (e) B. uniformis and (f) B. thetaiotaomicron, which belong to the genus Bacteroides, and (d) P. distasonis are all bacteria that have been reported to decompose alginate to produce short-chain fatty acids (see Non-Patent Literature 8). As shown in Figures 2 and 3, the Bacteroides bacteria have 3-Oxo-Δ 4-It is an intestinal bacterium that produces 3-OxoalloLCA using LCA as a substrate. Also, (d)P. distasonis produces LCA and 3-Oxo-Δ 4 In addition to producing IsoLCA with -LCA as a substrate, they also produce AlloLCA with 3-OxoalloLCA as a substrate. Furthermore, among the aforementioned bacteria, (a) P. merdae, (b) bacteria of the family Odoribacteraceae, and (c) bacteria of the genus Odoribacter all produce 3-Oxo-Δ 4 -LCA is used as a substrate to produce 3-OxoalloLCA and IsoalloLCA. Furthermore, (g) A. onderdonkii and (h) A. finegoldii produce 3-Oxo-Δ 4 -Produces 3-OxoalloLCA using LCA as a substrate: (i) C. innocuum produces 3-Oxo-Δ 4 -IsoLCA is produced using LCA as a substrate: (j) E. lenta strain and (k) G. pamelaeae produce IsoLCA using LCA as a substrate: (l) H. hathewayi produces 3-Oxo-Δ 4 These are bacteria that contribute to the production of 3-OxoLCA using -LCA as a substrate. Thus, oral ingestion of the test sample prepared by experimental method 1-1 was confirmed to increase several intestinal bacteria involved in the biosynthesis of secondary bile acids, including IsoalloLCA, in the colonic microbiota.

[0096] Experimental Example 2: Analysis of bile acids in stool samples and results Using the methods described in Experimental Methods 1-7 and 1-8, bile acids were extracted from human stool samples of subjects in Experimental Example 1 in which the presence of (b) bacteria of the family Odoribacteraceae or (c) bacteria of the genus Odoribacter was confirmed. These samples were then subjected to bile acid analysis to determine how the composition of bile acids in the stool samples changed over time after oral ingestion (once a day) of test sample 1.

[0097] As a result, as shown in Figure 5, it was confirmed that oral intake of test sample 1 once a day for one week increased the content of secondary bile acids ((a) IsoalloLCA, (b) 3-OxoLCA, (c) IsoLCA) in the stool. As mentioned above, oral intake of test sample 1 has been confirmed to increase the abundance of IsoalloLCA-producing bacteria ((a) P. merdae, (b) bacteria of the family Odoribacteraceae, and (c) bacteria of the genus Odoribacter), 3-OxoLCA-producing bacteria ((d) P. distasonis, (j) E. lenta strain, (k) G. pamelaeae), and IsoLCA-producing bacteria ((d) P. distasonis, (j) E. lenta strain, (k) G. pamelaeae, (i) C. innocuum) in the stool (in the colonic flora). Therefore, the increase in the content of secondary bile acids such as IsoalloLCA, 3-OxoLCA, and IsoLCA in stool is thought to be due to a change in the colonic microbiota caused by oral ingestion of the test sample, resulting in an increase in the amount of intestinal bacteria capable of producing these secondary bile acids.

[0098] Although 3-OxoalloLCA was not included in the analysis in this study, the production of its metabolite, IsoalloLCA, increased in the aforementioned study. Furthermore, in Experimental Example 1, oral ingestion of test sample 1 increased the amount of 3-OxoalloLCA-producing bacteria ((a) P. merdae, (b) bacteria of the family Odoribacteraceae, (c) bacteria of the genus Odoribacter, (e) B. uniformis, (f) B. ovatus, (g) A. onderdonkii, (h) A. finegoldii) in the coliform microbiota. Therefore, it is considered that oral ingestion of test sample 1 also increases the amount of 3-OxoalloLCA.

[0099] As shown in Figure 5, ingestion of test sample 1 resulted in an increase in various secondary bile acids, with a particularly significant increase in IsoalloLCA. IsoalloLCA is produced from the primary bile acid CDCA through conversion by various intestinal bacteria, with the intermediate being 3-Oxo-Δ4 -LCA is produced via the conversion of 3-oxo-5α-steroid 4-dehydrogenase (5AR) and 3β-hydroxysteroid dehydrogenase (3β-HSDH). On the other hand, 3-Oxo-LCA and IsoLCA are produced via 3-Oxo-Δ 4 -LCA is produced via the conversion reaction of flavin oxidoreductase (5BR) and 3β-HSDH. In this study, it is possible that the amount of IsoalloLCA significantly increases by strengthening the 5AR and 3β-HSDH pathways upon ingestion of test sample 1. In fact, the Odoribacteraceae bacteria B. uniformis and P. merdae, which possess the genes for 5AR and 3β-HSDH, tend to increase in the gut microbiota after oral ingestion of the test sample. In particular, since B. uniformis is also an alginate-degrading bacterium, it is suggested that oral ingestion of the test sample containing low molecular weight alginate may further increase B. uniformis, and consequently increase IsoalloLCA production.

[0100] Experimental Example 3 (Human Intestinal Model Test): Analysis of Bile Acids by Adding Heat-Treated Kelp Powder or Sodium Alginate and Results To evaluate the effects of heat-treated kelp powder and sodium alginate on the human gut microbiota in vitro, the following tests were conducted using a cultured human gut tract model that mimics the human gut microbiota. The cultured human gut tract model was modified from those described in Non-Patent Documents 9 and 10.

[0101] A 5g fresh stool sample was collected from a healthy Japanese male (27 years old) and suspended in 20ml of sterile saline. After filtering the fecal suspension, it was diluted to 25ml with sterile saline and transferred to an anero pouch, where it was stored at -80°C until use. 22ml of fecal filtrate, 63ml of sterile saline, and 25ml of sterile basal nutrition medium (Nutrition Broth; Biokar Diagnostics) were added to a pH-controlled small culture tank (BME-25NC-M; Able Co., Ltd.), and pre-cultured under anaerobic conditions (nitrogen gas; flow rate 40mL / min) at 37°C for 17 hours with stirring (180rpm). 50μM 3-Oxo-Δ was added to the culture tank as a substrate. 4-LCA and the test sample (see Table 5) were added and incubated at 37°C for 48 hours under anaerobic conditions (nitrogen gas: flow rate 40 mL / min) with stirring (180 rpm). 2M sodium hydroxide aqueous solution was automatically added to maintain the lower limit of the culture vessel's pH at 5.5. 1 ml of the culture solution was collected at various points in time and stored at -80°C until use for bile acid measurement.

[0102] Table 5 shows the test samples used in the experiment. The weight-average molecular weight (Mw), number-average molecular weight (Mn), peak-top molecular weight (Mp), and dispersion (Mw / Mn) of each test sample were determined by the GPC method described in Experimental Method 1-4.

[0103] [Table 5]

[0104] Method for extracting bile acids Fecal cultures stored at -80°C were centrifuged (10,000 rpm, 10 min, 4°C), and the supernatant was collected. Five times the volume of ethyl acetate was added to the culture supernatant, and the mixture was concentrated using a centrifugal concentrator to obtain the fecal sample. The fecal sample was purified using a solid-phase extraction column according to the method described in Experimental Method 1-7, and then subjected to bile acid analysis as described in Experimental Method 1-8.

[0105] The results of bile acid analysis using test sample 1 (heat-treated kelp powder: weight-average molecular weight approximately 500,000) and test sample 4 (sodium alginate: weight-average molecular weight approximately 500,000) are shown in Figures 6-1 to 6-2 (collectively referred to as Figure 6) and Figures 7-1 to 7-2 (collectively referred to as Figure 7), respectively. As shown in Figure 6, by using heat-treated kelp powder as the test sample, the 3-Oxo-Δ used as the substrate was used. 4-LCA decreased over time (Figure 6-1(a)), while the content of 3-Oxo-alloLCA, AlloLCA, IsoalloLCA, 3-Oxo-LCA, and IsoLCA increased (Figures 6-1(b)-(c), Figures 6-2(d)-(f)). As confirmed in Experimental Example 2, a significant increase in IsoalloLCA was also observed in this experiment. Furthermore, as shown in Figure 7, by using sodium alginate as the test sample, the 3-Oxo-Δ used as the substrate was used. 4 -LCA decreased over time (Figure 7-1(a)), while the content of 3-Oxo-alloLCA, AlloLCA, IsoalloLCA, 3-Oxo-LCA, and IsoLCA increased (Figures 7-1(b)-(c), 7-2(d)-(f)).

[0106] The results of bile acid analysis when using test sample 2 (heat-treated kelp powder: weight-average molecular weight approximately 80,000) as the test sample are shown in Figures 8-1 to 8-2 (collectively referred to as Figure 8). As shown in Figure 8, by using heat-treated kelp powder, similar to test sample 1, the 3-Oxo-Δ used as the substrate was analyzed. 4 -LCA decreased over time (Figure 8-1(a)), while the content of AlloLCA, IsoalloLCA, and IsoLCA increased (Figure 8-1(b), Figures 8-2(c)-(d)). As confirmed in Experimental Example 2, a significant increase in IsoalloLCA was also observed in this experiment.

[0107] Figure 9 shows the results of bile acid analysis using test sample 6 (sodium alginate: weight-average molecular weight approximately 80,000) as the test sample. As shown in Figure 9, similar to test sample 4, it was confirmed that the content of AlloLCA, IsoalloLCA, and IsoLCA increased when sodium alginate was used (Figures 9(a)-(c)). As confirmed in Experimental Example 2, a significant increase in IsoalloLCA was also observed in this experiment.

[0108] As shown in Experimental Example 3, heat-treated kelp powder containing low molecular weight alginic acid with a weight-average molecular weight in the range of 50,000 to 1,000,000, preferably 80,000 to 500,000, and low molecular weight alginic acid (or its salt) with the same weight-average molecular weight, both exhibit the following effects in the gut microbiota: 3-Oxo-Δ 4 It was confirmed that the heat-treated kelp powder has an effect of promoting the production of secondary bile acids (3-Oxo-alloLCA, AlloLCA, IsoalloLCA, 3-Oxo-LCA, and IsoLCA) using LCA as a substrate (secondary bile acid increasing effect). From this, it is suggested that the secondary bile acid increasing effect of the heat-treated kelp powder shown in Experimental Example 2 is partly due to the low molecular weight alginic acid or its salt contained in the heat-treated kelp powder. Furthermore, it is suggested that the intestinal bacteria increasing effect of the heat-treated kelp powder shown in Experimental Example 1 is also partly due to the low molecular weight alginic acid or its salt contained in the heat-treated kelp powder.

[0109] Experimental Example 4 The following studies were conducted on methods for heat-treating kelp. [Preparation of kelp powder] Dried kelp (raw seaweed) (Grade 2 Makonbu from Ofuna) was cut into approximately 5 cm squares and boiled in water at 95°C for 60 minutes to obtain boiled kelp. Next, the boiled kelp was subjected to either pressurized moist heat treatment or atmospheric pressure dry heat treatment to prepare heat-treated kelp. Pressurized moist heat treatment was carried out using an autoclave (pressure condition: 0.1 MPa) under the heating conditions described in Table 8 below. Atmospheric pressure dry heat treatment was carried out using an oven under the heating conditions described in Table 9 below. Next, the heat-treated kelp was dried at 60°C to a moisture content of 6%, then crushed using a pulverizer and sieved (mesh size (#) = 500 μm) to obtain various kelp powders with a particle size of 500 μm or less. Dried kelp (raw seaweed) and boiled kelp were dried in the same manner as above (moisture content 6%) and then crushed to obtain kelp powders with a particle size of 500 μm or less.

[0110] [Analysis of kelp powder] 1. Analysis of dietary fiber content The amount of dietary fiber in each was measured using the method described below. (1) Total dietary fiber: The results were obtained using the modified Prosky method, following the method described in Experimental Method 1-2. (2) Amount of alginate: The amount of alginic acid in the kelp powder was measured according to the method described in Experimental Method 1-3. (3) Amount of fucoidan: The kelp powder was extracted with dilute hydrochloric acid, then fractionated using cecilpyridinium chloride, and the weight of the precipitate formed by adding ethanol was used to determine the composition. (4) Amount of laminarane: The results were obtained by the glucose oxidase method after hydrolysis of kelp powder with 2N sulfuric acid. (5) Cellulose content: After removing the dilute acid hydrolysis extract according to the Southgate method, the cellulose fraction obtained by hydrolysis with a strong acid was measured by the phenol-sulfuric acid method.

[0111] Table 6 shows the results of analysis using the above method on kelp powder prepared from dried kelp (raw seaweed) and boiled kelp, respectively, as well as on kelp powder prepared from kelp treated with pressurized moist heat (121°C for 15 minutes, 130°C for 60 minutes).

[0112] [Table 6]

[0113] As shown in Table 6, it was confirmed that boiling and pressurized moist heat treatment of kelp increased the total dietary fiber and alginic acid content. However, there was no significant difference in total dietary fiber and alginic acid content between boiled kelp and pressurized moist heat treated kelp (121°C for 15 minutes, 130°C for 60 minutes).

[0114] 2. Analysis of iodine Various types of kelp powder were ashed in a 50% sodium hydroxide solution (W / V), and the filtrate was diluted to a fixed volume with distilled water. This was then adjusted (neutralized) to pH 7 with 3M HCl, boiled with 1M sodium hypochlorite solution, and then potassium iodide and 3M sulfuric acid were added to liberate iodine. The amount of iodine was calculated by titration with a 0.01M sodium thiosulfate standard solution using starch as an indicator. Table 7 shows the results of analysis using the above method on kelp powder prepared from dried kelp (raw seaweed) and boiled kelp, respectively, as well as kelp powder prepared from kelp treated with pressurized moist heat (121°C - 15 minutes).

[0115] [Table 7]

[0116] As shown in Table 7, the amount of iodine contained in 100g of kelp powder was negligible for both boiled kelp (1.7mg) and kelp treated with humid heat and pressure at 121°C for 15 minutes (2.3mg), compared to dried kelp (57.7mg). From this, it was found that the iodine contained in kelp can be almost completely removed by boiling it.

[0117] 3. Measurement of the average molecular weight of alginate The weight-average molecular weight of alginic acid contained in the kelp powder prepared by the above method was analyzed according to the method described in Experimental Method 1-4. Table 8 shows the results of the analysis of kelp powder treated with pressurized moist heat, and Table 9 shows the results of the analysis of kelp powder treated with atmospheric pressure dry heat, along with the analysis results of dried kelp (raw seaweed) and boiled kelp, respectively.

[0118] [Table 8]

[0119] [Table 9]

[0120] As shown in Tables 8 and 9, it was found that the weight-average molecular weight of alginic acid in kelp powder can be adjusted to a range of 50,000 to 1,000,000 by pressurized moist heat treatment of boiled kelp under conditions of 100°C for 3 minutes to 130°C for 15 minutes, or by atmospheric pressure dry heat treatment under conditions of 130°C for 15 minutes to 180°C for 15 minutes. In this way, kelp processed products obtained by reducing the molecular weight of alginic acid contained in kelp to a weight-average molecular weight in the range of 50,000 to 1,000,000 can be effectively used as the aforementioned low-molecular-weight alginic acid-containing composition as a raw material for a secondary bile acid increasing agent or an intestinal bacteria increasing agent having the ability to metabolize and produce secondary bile acids.

Claims

1. A secondary bile acid increasing agent comprising a composition containing alginic acid or a salt thereof having a weight-average molecular weight of 50,000 to 1,000,000, or at least one of these, as an active ingredient, A secondary bile acid increasing agent wherein the secondary bile acid is at least one selected from the group consisting of isoalolitocholic acid, allolitocholic acid, 3-oxoalolitocholic acid, isolitocholic acid, and 3-oxolitocholic acid.

2. The secondary bile acid increasing agent according to claim 1, wherein the composition is an alginic acid-containing composition prepared from kelp.

3. The secondary bile acid increasing agent according to claim 2, wherein the alginic acid-containing composition is prepared by a process of reducing the molecular weight of alginic acid or a salt thereof contained in kelp so that its weight-average molecular weight is in the range of 50,000 to 1,000,000.

4. The secondary bile acid increasing agent according to claim 3, wherein the demolecular-weight treatment includes a step of heating kelp at a temperature of 100°C or higher.

5. An agent for increasing intestinal bacteria having secondary bile acid metabolism ability, comprising a composition containing alginic acid or a salt thereof having a weight-average molecular weight of 50,000 to 1,000,000, or at least one of the above, as an active ingredient, The secondary bile acid is at least one selected from the group consisting of isoalolitocholic acid, allolitocholic acid, 3-oxoalolitocholic acid, isolitocholic acid, and 3-oxolitocholic acid. An intestinal bacteria increasing agent wherein the aforementioned intestinal bacteria are at least one selected from the group consisting of Parabacteroides merdae, bacteria of the family Odoribacteraceae, bacteria of the genus Odoribacter, Parabacteroides distasonis, Bacteroides uniformis, Bacteroides thetaiotaomicron, Alistipesonde rdonkii, Alistipes finegoldii, Clostridium innocuum, Eggerthella lenta strain, Gordonibacter pamelaeae, and Hungatella hathewayi.

6. The intestinal bacteria increasing agent according to claim 5, wherein the composition is prepared by a method comprising the step of demolecularizing alginic acid or a salt thereof contained in kelp so that its weight-average molecular weight is in the range of 50,000 to 1,000,000.

7. The intestinal bacteria increasing agent according to claim 6, wherein the low molecular weight treatment includes a step of heating kelp at a temperature of 100°C or higher.

8. A method for producing processed kelp products used to increase intestinal bacteria capable of producing secondary bile acids, The method is characterized by having a step of reducing the molecular weight of alginic acid or its salt in kelp so that its weight-average molecular weight is in the range of 50,000 to 1,000,000. The secondary bile acid is at least one selected from the group consisting of isoalolitocholic acid, allolitocholic acid, 3-oxoalolitocholic acid, isolitocholic acid, and 3-oxolitocholic acid. The aforementioned intestinal bacteria are selected from at least one of the following groups: Parabacteroides merdae, bacteria of the family Odoribacteraceae, bacteria of the genus Odoribacter, Parabacteroides distasonis, Bacteroides uniformis, Bacteroides thetaiotaomicron, Alistipes onderdonkii, Alistipes finegoldii, Clostridium innocuum, Eggerthella lenta strain, Gordonibacter pamelaeae, and Hungatella hathewayi. The aforementioned manufacturing method.

9. The manufacturing method according to claim 8, wherein the demolecular-weight treatment includes a step of heating the kelp at a temperature of 100°C or higher.

10. A method for imparting to a processed kelp product the function of increasing intestinal bacteria capable of producing secondary bile acids, The method is characterized by having a step of reducing the molecular weight of alginic acid or its salt in kelp so that its weight-average molecular weight is in the range of 50,000 to 1,000,000. The secondary bile acid is at least one selected from the group consisting of isoalolitocholic acid, allolitocholic acid, 3-oxoalolitocholic acid, isolitocholic acid, and 3-oxolitocholic acid. The aforementioned intestinal bacteria are selected from at least one of the following groups: Parabacteroides merdae, bacteria of the family Odoribacteraceae, bacteria of the genus Odoribacter, Parabacteroides distasonis, Bacteroides uniformis, Bacteroides thetaiotaomicron, Alistipes onderdonkii, Alistipes finegoldii, Clostridium innocuum, Eggerthella lenta strain, Gordonibacter pamelaeae, and Hungatella hathewayi. The aforementioned method.

11. The method according to claim 10, wherein the molecular weight reduction treatment includes a step of heating the kelp at a temperature of 100°C or higher.

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