Novel microorganisms derived from pigs

Abstract

Provided is a microorganism characterized in that: it can be used as probiotic for animals such as pigs, it belongs to the genus clostridium, and in that it has the ability to produce isoallolithocholic acid in the presence of bile acid.

Description

[Technical Field]

[0001] The present invention relates to a novel microorganism derived from pigs, a probiotic for animals, a growth promoter for useful intestinal microorganisms, and a method for screening a substance effective in promoting animal growth. The microorganism of the present invention has effects such as promoting growth and improving disease resistance in animals such as pigs. [Background technology]

[0002] Intestinal bacteria inhabit the bodies of animals, including humans, and are often referred to as the last organ due to their profound connection to health. Therefore, research into intestinal bacteria is being conducted to isolate strains that affect the host and analyze their beneficial metabolites. Antibiotics are used in livestock, but due to the emergence of resistant bacteria, there is a trend toward banning antibiotics, including in the EU. Microorganisms have therefore attracted attention as alternatives to antibiotics, including lactic acid bacteria that produce antibacterial substances and activate the immune system. Furthermore, because pigs are similar to humans, they also serve as model animals for humans, with human organs and feces transplanted into pigs. Research on pigs has been applied to human research, and porcine intestinal bacteria research is actively conducted, primarily in Europe and China.

[0003] To date, probiotics developed for pigs have primarily consisted of lactic acid bacteria, butyric acid bacteria, bifidobacteria, etc. (Patent Document 1, Non-Patent Document 1), but no probiotics using intestinal bacteria have been established. One reason for this is the difficulty of isolating and culturing intestinal bacteria. Large-scale isolation of porcine intestinal bacteria has been conducted worldwide (Non-Patent Document 2), and the relationship between the entire intestinal microflora and the host has also been studied (Non-Patent Document 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 2799273 [Non-patent literature]

[0005] [Non-Patent Document 1] Muneta, Journal of the Japanese Society for Swine Disease Research (76), 15-23, 2020-08 [Non-patent document 2] Wylensek et al., 2020. Nat Commun.;11(1):6389. doi: 10.1038 / s41467-020-19929-w. [Non-patent document 3] Yang et al., 2022. Nature;606(7913):358-367.doi: 10.1038 / s41586-022-04769-z Summary of the Invention [Problem to be solved by the invention]

[0006] Because the main analytical techniques used are bacterial flora analysis and metabolite analysis using genes extracted from intestinal contents, the bacterial species or strains that actually play important roles have not yet been identified or elucidated, and no knowledge has been obtained that can be directly applied to improving productivity.

[0007] The present invention has been made under the above circumstances, and aims to provide a novel microorganism that can be used as a probiotic for animals such as pigs. [Means for solving the problem]

[0008] As a result of extensive research to solve the above problems, the inventors discovered that microorganisms isolated from the intestines of fast-growing pigs produce the antioxidant ferulic acid, as well as formic acid and isoallorithocholic acid, which have antibacterial properties, and thus completed the present invention. That is, the present invention provides the following [1] to

[22] .

[0009] [1] A microorganism belonging to the genus Clostridium, characterized by having the ability to produce isoallorithocholic acid in the presence of bile acids.

[0010] [2] The microorganism described in [1], characterized in that it has the ability to produce isoallothocholic acid in the presence of bile acids, and the ability to produce ferulic acid and formic acid in the presence or absence of bile acids.

[0011] [3] The microorganism described in [1], characterized in that it has a 16S rRNA gene consisting of the base sequence set forth in SEQ ID NO: 1.

[0012] [4] The microorganism according to [1], characterized in that it is a strain identified by accession number NITE BP-04072.

[0013] [5] A probiotic for animals, characterized by containing a microorganism belonging to the genus Clostridium and capable of producing isoallorithocholic acid in the presence of bile acid.

[0014] [6] The probiotic agent for animals according to [5], characterized in that the microorganism is a microorganism capable of producing isoallothocholic acid in the presence of bile acids, and capable of producing ferulic acid and formic acid in the presence or absence of bile acids.

[0015] [7] The probiotic agent for animals according to [5], characterized in that the microorganism is a microorganism having a 16S rRNA gene consisting of the base sequence set forth in SEQ ID NO: 1.

[0016] [8] The probiotic agent for animals according to [5], characterized in that the microorganism is a strain identified by accession number NITE BP-04072.

[0017] [9] The probiotic agent for animals according to [5], characterized in that the animal is a pig.

[0018]

[10] The probiotic agent for animals according to [5], which is used to promote the growth of animals.

[0019]

[11] The probiotic agent for animals according to [5], which is used to improve the disease resistance of animals.

[0020]

[12] An agent for promoting the proliferation of useful intestinal microorganisms, characterized by containing raffinose and / or stachyose as active ingredients.

[0021]

[13] The agent for promoting the proliferation of useful intestinal microorganisms according to

[12] , further comprising bile acid and / or manganese as active ingredients.

[0022]

[14] The growth agent for useful intestinal microorganisms according to

[12] , which does not contain any sugars other than raffinose and stachyose.

[0023]

[15] The agent for proliferation of useful intestinal microorganisms according to

[12] , characterized in that the useful intestinal microorganisms belong to the genus Clostridium and are capable of producing isoallorithocholic acid in the presence of bile acids.

[0024]

[16] The agent for proliferation of useful intestinal microorganisms according to

[12] , characterized in that the useful intestinal microorganisms belong to the genus Clostridium, have the ability to produce isoallothocholic acid in the presence of bile acids, and have the ability to produce ferulic acid and formic acid in the presence or absence of bile acids.

[0025]

[17] The proliferation agent for useful intestinal microorganisms according to

[12] , wherein the useful intestinal microorganisms are microorganisms having a 16S rRNA gene consisting of the base sequence set forth in SEQ ID NO: 1.

[0026]

[18] The agent for proliferation of useful intestinal microorganisms according to

[12] , wherein the useful intestinal microorganisms are strains identified by accession number NITE BP-04072.

[0027]

[19] The agent for proliferation of useful intestinal microorganisms according to

[12] , which is administered to pigs.

[0028]

[20] The agent for proliferation of useful intestinal microorganisms according to

[12] , further comprising the microorganism according to any one of [1] to [4].

[0029]

[21] A method for screening a substance effective in promoting animal growth, comprising the following steps (1) and (2): (1) culturing the microorganism according to any one of [1] to [4] in the presence of a test substance; (2) A step of selecting test substances that cause the growth of the microorganisms as substances that are effective in promoting the growth of animals.

[0030]

[22] The screening method according to

[21] , wherein the animal is a pig.

[0031] The present invention also provides the following

[23] to

[25] .

[0032]

[23] A method for raising animals, comprising the step of administering to an animal a probiotic agent for animals according to any one of [5] to [9] and / or an agent for proliferation of useful intestinal microorganisms according to any one of

[12] to

[19] .

[0033]

[24] A method for promoting the growth of animals, comprising the step of administering to an animal a probiotic agent for animals according to any one of [5] to [9] and / or a growth agent for useful intestinal microorganisms according to any one of

[12] to

[19] .

[0034]

[25] A method for improving the disease resistance of an animal, comprising the step of administering to the animal a probiotic agent for animals according to any one of [5] to [9] and / or a growth agent for useful intestinal microorganisms according to any one of

[12] to

[19] .

[0035] This specification includes part or all of the contents as disclosed in the specification and / or drawings of Japanese Patent Application No. 2024-027311, which is a priority document of the present application. [Effects of the Invention]

[0036] The present invention provides a novel microorganism belonging to the genus Clostridium. This microorganism was found to be dominant in pigs with good growth potential and has disease resistance, so it can be used to promote growth and improve disease resistance in pigs, contributing to a system that provides safe food without the use of antibiotics. [Brief explanation of the drawings]

[0037] [Figure 1] FIG. 1 shows the growth of the NP-1 strain when bile acid or manganese is added. [Figure 2] FIG. 1 shows the growth of the NP-1 strain when sugars were added. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention will be described in detail below. (A) Microorganisms The microorganism of the present invention belongs to the genus Clostridium and is characterized by having the ability to produce isoallorithocholic acid in the presence of bile acids.

[0039] The microorganism of the present invention can be obtained, for example, by isolating intestinal bacteria from pigs, culturing the isolated microorganisms in a bile acid-added medium, selecting microorganisms that produce isoallorithocholic acid from the cultured microorganisms, and then selecting microorganisms belonging to the genus Clostridium from among them. The selection of microorganisms belonging to the genus Clostridium can be carried out, for example, by examining the sequence of the 16S rRNA gene.

[0040] The method for culturing the microorganism of the present invention is not particularly limited, and the microorganism can be cultured by a method generally applied to microorganisms belonging to the genus Clostridium. For example, YCFA medium can be used as the culture medium. The culture temperature can be around 37°C (e.g., 35 to 40°C). The culture is carried out under anaerobic conditions.

[0041] The microorganism of the present invention may be any microorganism capable of producing isoallothocholic acid in the presence of bile acids, but preferably also capable of producing ferulic acid and formic acid in the presence or absence of bile acids. Furthermore, the microorganism of the present invention preferably has a 16S rRNA gene consisting of the following nucleotide sequence (SEQ ID NO: 1):

[0042] tattgagagt ttgatcctgg ctcaggacga acgctggcgg cgtgcctaac acatgcaagt 60 cgagcgaatg aagttccttc gggaacggat ttagcggcgg acgggtgagt aacacgtggg 120 caacctacct catagagggg aatagccttc cgaaagggag attaataccg cataagattg 180 tagtaccgca tggtacagca attaaaggag caatccacta tgagatgggc ccgcggcgca 240 ttagctagtt ggtgaggtaa cggctcacca aggcgacgat gcgtagccga cctgagaggg 300 tgatcggcca cattgggact gagacacggc ccagactcct acgggaggca gcagtgggga 360 atattgcaca atgggggaaa ccctgatgca gcaacgccgc gtgagtgatg acggccttcg 420 ggttgtaaag ctctgtcttc agggacgata atgacggtac ctgaggagga agccacggct 480 aactacgtgc cagcagccgc ggtaatacgt aggtggcgag cgttgtccgg atttactggg 540 cgtaaaggga gcgtaggcgg atttttaagt gagatgtgaa atactcgggc ttaacctgag 600 tgctgcattt caaactggaa gtctagagtg caggagagga gaagggaatt cctagtgtag 660 cggtgaaatg cgtagagatt aggaagaaca ccagtggcga aggcgcttct ctggactgta 720 actgacgctg aggctcgaaa gcgtggggag caaacaggat tagataccct ggtagtccac 780 gccgtaaacg atgaatacta ggtgtagggg ttgtcatgac ctctgtgccg ccgctaacgc 840 attaagtatt ccgcctgggg agtacggtcg caagattaaa actcaaagga attgacgggg 900 gcccgcacaa gcagcggagc atgtggttta attcgaagca acgcgaagaa ccttacctag 960 acttgacatc tcctgcatta ctcttaatcg aggaagtcct ttcggggaca ggatgacagg 1020 tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080 caacccttat tgttagttgc catcattaag ttgggcactc tagcgagact gcccgggtta 1140 accgggagga aggtggggat gacgtcaaat catcatgccc cttatgtcta gggctacaca 1200 cgtgctacaa tggtcggtac aataagacgc aagcccgcga gggggagcaa aactggaaaa 1260 ccgatctcag ttcggattgt aggctgaaac tcgcctacat gaagctggag ttgctagtaa 1320 tcgcgaatca gcatgtcgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1380 ccatgagagt tggcaatacc caaagtacgt gatctaaccc gcaagggagg aagcgtccta 1440 aggtagggtc agcgattggg gtgaagtcgt aacaaggtag ccgtaggaga acctgcggct 1500 ggatcacctc cttt 1514

[0043] The strain included in the microorganism of the present invention can be NP-1 strain. The NP-1 strain has been internationally deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation. Detailed information regarding the international deposit is as follows.

[0044] 1) Name and address of the international depository institution Name: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center Address: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan Postal code: 292-0818 2) Domestic deposit date: January 30, 2024 3) Domestic accession number: NITE P-04072 4) Transfer date to international deposit: November 22, 2024 5) International accession number: NITE BP-04072 6) Identification mark: NP-1

[0045] (B) Live bacterial agents for animals The probiotic agent for animals of the present invention is characterized by containing the above-mentioned microorganism of the present invention.

[0046] The target animal is usually a pig, but may be an animal other than a pig. The animal may include or exclude humans. The probiotic agent for animals of the present invention is used, for example, to promote growth or improve disease resistance of the target animal.

[0047] The probiotic agent for animals of the present invention may be prepared by directly using the microbial cells of the present invention, or by adding an excipient to the microbial cells to form a formulation. Examples of excipients include glucose, sucrose, lactose, glutamic acid, ascorbic acid, skim milk, egg albumin, starch, casein, and sucrose. Furthermore, the microbial cells may be freeze-dried or sporulated to enhance their shelf life. The method for preparing the microbial cells of the present invention is not particularly limited and can be performed according to conventional methods. For example, the microbial cells of the present invention may be cultured for a certain period in a medium in which the microbial cells of the present invention can grow, and then the cells may be recovered by centrifugation or other means. However, the culture solution may also be used as is. The microbial cells of the present invention are obligate anaerobes and are highly susceptible to oxygen. Therefore, it is preferable to sporulate the microbial cells of the present invention or encapsulate them to prevent exposure to oxygen.

[0048] The dosage of the probiotic agent for animals of the present invention is not particularly limited as long as it is an amount that can achieve the desired effect (for example, growth promotion or improvement of disease resistance of the target animal). For example, the dosage of 1000 mg of microbial cells per day for one pig can be 10 ~10 12 cfu, preferably 10 10 ~10 11 It can be expressed as cfu.

[0049] The probiotic agent for animals of the present invention may be administered orally alone, or may be mixed with feed ingredients containing carbohydrates, proteins, lipids, fiber, vitamins, minerals, etc. to form powdered or pelleted feed. The feed ingredients used in this case may be any that can be used in animal feed, including animal feed ingredients (skim milk powder, whey, fish meal, fish, etc.), grains (wheat, corn, rice, etc.), meal (soybean meal, soybean oil cake, etc.), bran (bran, etc.), and leaf meal (alfalfa meal, etc.). It can also be produced by adding bile acids to a basal feed, adding the above-mentioned probiotic agent for animals, and fermenting the mixture. It is also possible to use bile acids other than isoallothocholic acid without adding any other bile acids. The type of basal feed is not particularly limited, and examples thereof include green grass, silage, grains, vegetables, root vegetables, dairy products (such as skim milk), unused or underutilized food processing by-products (such as starch cake, soy lees, and pumpkin pulp), and agricultural processing by-products (such as apple juice pomace, potato pulp, and brewer's yeast). The use of food processing by-products or agricultural processing by-products as basal feed can improve feed self-sufficiency. Furthermore, for feed given to young animals, known milk replacers, in which the crude protein, crude fat, crude fiber, crude ash, minerals, etc. are adjusted according to the individual animal, can be used as the basal feed.

[0050] (C) Proliferation agent for beneficial intestinal microorganisms The agent for promoting the proliferation of useful intestinal microorganisms of the present invention is characterized by containing raffinose and / or stachyose as active ingredients.

[0051] The useful intestinal microorganisms to be propagated are, for example, the microorganisms of the present invention described above, preferably the NP-1 strain.

[0052] The growth agent for useful intestinal microorganisms of the present invention contains raffinose or stachyose, or both, but may also contain other components. For example, it may contain bile acid or manganese, or both. Furthermore, the growth agent for useful intestinal microorganisms of the present invention may or may not contain sugars (monosaccharides, disaccharides, oligosaccharides, polysaccharides) other than raffinose. Furthermore, since the growth agent for useful intestinal microorganisms of the present invention grows the microorganisms of the present invention, it may also contain the microorganisms of the present invention themselves that are the target of growth.

[0053] The target animal for administration is usually a pig, but may be an animal other than a pig. The target animal may include or exclude humans. The agent for proliferation of useful intestinal microorganisms of the present invention promotes the proliferation of microorganisms that have the effect of promoting animal growth or improving disease resistance, and therefore can be used to promote growth or improve disease resistance in the target animal.

[0054] The agent for promoting the proliferation of useful intestinal microorganisms of the present invention may consist solely of the active ingredient such as raffinose, or may be formulated by adding an excipient, which may be the same as the excipient used in the probiotic agent for animals of the present invention.

[0055] The dosage of the agent for proliferation of useful intestinal microorganisms of the present invention is not particularly limited as long as it is an amount that can proliferate useful intestinal microorganisms. For example, the daily dosage of raffinose per pig can be 0.01 to 99% of the feed weight, preferably 0.1% to 10%, and the daily dosage of stachyose per pig can be 0.01 to 99% of the feed weight, preferably 0.1% to 10%. Furthermore, when bile acids are contained, the daily dosage of bile acids per pig can be 0.01 to 10% of the feed weight, preferably 0.1 to 1%. When manganese is contained, the daily dosage of manganese per pig can be 0.001 to 50% of the feed weight, preferably 0.01 to 0.5%.

[0056] The agent for growing useful intestinal microorganisms of the present invention may be orally administered alone, or may be mixed with feed ingredients to prepare feed. The feed can be prepared in the same manner as the above-mentioned probiotic agent for animals of the present invention.

[0057] (D) Screening method The screening method of the present invention is a method for screening for a substance that is effective in promoting the growth of an animal, and is characterized by comprising the following steps (1) and (2):

[0058] In step (1), the microorganism of the present invention is cultured in the presence of a test substance. The type of test substance is not particularly limited and may be either an organic compound or an inorganic compound. Examples of organic compounds that can be used as test substances include sugars, amino acids, peptides, lipids, and nucleic acids. As described above, the microorganism of the present invention can be cultured by a method generally applied to microorganisms belonging to the genus Clostridium.

[0059] In step (2), a test substance that causes the growth of the microorganism of the present invention is selected as a substance effective in promoting animal growth. Since the NP-1 strain contained in the microorganism of the present invention was abundant in animals with good growth potential (pigs), substances that cause the growth of the NP-1 strain or similar microorganisms are expected to be "substances effective in promoting animal growth." Whether or not the test substance caused the microorganism to grow can be determined by performing a similar culture in the absence of the test substance and comparing the bacterial cell mass after culture. The type of animal is usually pigs, but animals other than pigs may also be used. [Example]

[0060] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0061] [Example 1] Isolation of the novel strain Based on the widely conducted intestinal microbiota analysis of pigs with good and poor growth performance (see Microbiome. 2020;8(1):110, Front Microbiol. 2023;14:1239847), we focused on Clostridium_sensu_scrict1, which is characteristic of pigs with good growth performance and feed efficiency, and isolated Clostridium spp. Isolation was performed using a modified YCFA medium (https: / / www.jcm.riken.jp / cgi-bin / jcm / jcm_grmd?GRMD=1130) (with bile acid addition, excluding VFA mix) based on a previous report (Nature, 2016. 533, 543-546). As a result, strain NP-1 was isolated.

[0062] [Example 2] Organic acid and metabolome analysis of the novel strain Metabolomic analysis The culture medium used was 10 mL of YCFA medium. The serum bottle containing the medium was sealed with a butyl stopper and an aluminum cap seal, and the interior was filled with anaerobic gas (75% nitrogen, 20% carbon dioxide, 5% hydrogen) using a gas exchanger. If necessary, 100 μL of 5% porcine bile powder (Fujifilm Wako Pure Chemical Industries) solution was added to the medium. After inoculation with the NP-1 strain, the medium was cultured at 37°C for 48 hours. The culture medium was centrifuged and filtered through a 0.2 μm filter, and then metabolomic analysis of the culture medium was performed. Using LC-MS with the HM400 Metabolomics (https: / / www.nature.com / articles / nm.4358), metabolomic analysis was performed on the culture medium cultured in bile acid-free medium (YC-1, 2, 3), the culture medium cultured in bile acid-supplemented medium (Yb-1, 2, 3), and the medium before culture in bile acid-supplemented medium (Y1, 2, 3).

[0063] organic acid analysis The culture medium was diluted appropriately and analyzed using an HPLC system (LC-UV; Shimadzu) equipped with a Shim-pack SCR-102H column (8 × 300 mm; Shimadzu, equipped with an SCR-102H guard column and a CDD-10Avp conductivity detector). The mobile phase was 2.5 mM p-toluenesulfonic acid, and the post-column buffer was 2.5 mM p-toluenesulfonic acid, 50 μM EDTA, and 10 mM Bis-Tris.

[0064] Table 1 shows representative metabolites produced by the NP-1 strain in bile acid-supplemented medium. [Table 1]

[0065] As shown in Table 1, the NP-1 strain was found to produce the antioxidant ferulic acid, as well as formic acid and isoallothocholic acid, which have antibacterial properties. Isoallothocholic acid is a metabolic product that was not predicted from the strain's genome information. Furthermore, a group at Keio University has revealed that isoallothocholic acid is abundant in long-lived humans (over 100 years old) (Nature, 2021. 599,458-464. https: / / doi.org / 10.1038 / s41586-021-03832-5). The fact that this substance is produced by a strain that was dominant in pigs with good growth potential suggests that it may affect pig growth.

[0066] [Example 3] Comparison of 16S ribosomal RNA between the novel strain and the same species strain isolated from humans Porcine cecal contents (approximately 1 g / 10 mL) were suspended in 70% ethanol at a 1:1 ratio and left at room temperature for 4 hours. The supernatant was then removed by centrifugation and suspended in PBS buffer. This suspension was spread on YCFA agar medium (supplemented with: 5 g / L meat extract, 2 g / L starch, 500 μL vitamin K (0.2 g / 20 mL ethanol)) and anaerobically cultured at 37°C for 72 hours. All procedures were performed in an anaerobic chamber. After 72 hours, colonies were picked and subjected to 16S rRNA gene analysis.

[0067] The primers used were universal primers 27F (5-AGAGTTTGATCCTGGCTCAG-3, SEQ ID NO: 2) and 1525R (5-AAAGGAGGTGATCCAGCC-3, SEQ ID NO: 3), designed using a highly conserved region in the 16S rRNA gene of eubacteria. The DNA polymerase used was Takara Ex Taq Hot Start version.

[0068] The PCR product (20 μl) was subjected to agarose gel electrophoresis (1% Wako Pure Chemical Industries Agarose S, 100 V). The buffer used was 1x TAE (40 mM Tris-acetate, 1 mM EDTA, pH 7.2). After agarose gel electrophoresis, DNA was extracted from the gel using a Gel Extraction Kit (QIAGEN) and used as a template for sequencing. The BigDye Terminator v.3.1 kit was used for sequencing. The reaction solution was precipitated with ethanol, suspended in formamide, and sequenced using a GENETIC Analyzer 3500xL DNA sequencer. The sequence was then searched using BLAST to select the bacterial species.

[0069] The nucleotide sequence of the 16S rRNA gene of the NP-1 strain is shown in SEQ ID NO: 1. The 16S rRNA gene of the NP-1 strain showed 99.7% homology with the 16S rRNA gene of a Clostridium strain isolated from a human. This suggests that the NP-1 strain belongs to the Clostridium genus. However, the NP-1 strain is considered to be a different strain from the human isolate because the 16S rRNA gene sequence does not completely match and the isolation source is significantly different. [Example 4] Examination of substances that promote the growth of this novel strain The NP-1 strain was cultured in bacterial medium supplemented with bile acids or manganese to examine the effects of these substances on the growth of the NP-1 strain. YCFA medium was used as the bacterial medium. For bile acids, 100 μL of a 5% porcine bile powder (Fujifilm Wako Pure Chemical Industries) solution was added per 10 mL of medium. For manganese, 100 μL of manganese sulfide pentahydrate (Nacalai Tesque, Inc.: 5 mg / 100 mL distilled water) was added per 10 mL of medium. The turbidity (OD600) of the medium after 18 hours of culture at 37°C was measured to evaluate the growth of the NP-1 strain.

[0070] The turbidity when neither bile acid nor manganese was added (Control), when only bile acid was added (bile), when only manganese was added (Mn), and when bile acid and manganese were added (bile+Mn) is shown in Figure 1. As shown in the figure, it was revealed that bile acid and manganese improved the growth of the NP-1 strain.

[0071] [Example 5] Examination of sugars that promote the growth of the novel strain The NP-1 strain was cultured in a bacterial medium supplemented with glucose, xylobiose, lactose, rhamnose, raffinose, arabinose, xylane, mannose, D-pinitol, or stachyose as a single sugar source. Each sugar was added to the medium to achieve a concentration of 0.5%. Glucose was used as a control, as it can generally be assimilated by the entire bacterium. The culture and growth of the NP-1 strain were evaluated as in Example 4.

[0072] Figure 2 shows the turbidity when each sugar was added. As shown in the figure, it was revealed that the NP-1 strain can assimilate raffinose and stachyose. Growth was also confirmed, albeit to a small extent, on xylobiose, lactose, and xylan. It was confirmed that there was almost no growth on other sugars. From these results, it is believed that raffinose and stachyose are promising prebiotics for the NP-1 strain. Furthermore, the NP-1 strain is abundant in pigs with good growth potential. This means that substances that improve the growth of the NP-1 strain are expected to be beneficial for pig growth. Therefore, the NP-1 strain can be used to screen for substances that are effective in improving pig growth.

[0073] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. [Industrial Applicability]

[0074] The present invention can be used in industrial fields such as the livestock industry.

Claims

1. A microorganism belonging to the genus Clostridium, characterized in that it has the ability to produce isoallothocholic acid in the presence of bile acids, and characterized in that it has a 16S rRNA gene consisting of the base sequence set forth in SEQ ID NO:

1.

2. A microorganism belonging to the genus Clostridium, characterized in that it has the ability to produce isoallothocholic acid in the presence of bile acids, and characterized in that it is a strain identified by accession number NITE BP-04072.

3. A live bacterial agent for animals, characterized in that it contains a microorganism belonging to the genus Clostridium, which has the ability to produce isoallothocholic acid in the presence of bile acids, and which has a 16S rRNA gene consisting of the base sequence set forth in SEQ ID NO:

1.

4. A live bacterial agent for animals, characterized by containing a microorganism belonging to the genus Clostridium, characterized by having the ability to produce isoallorithocholic acid in the presence of bile acids, and which is a strain identified by accession number NITE BP-04072.

5. 5. The probiotic agent for animals according to claim 3, wherein the animal is a pig.

6. A growth agent for useful intestinal microorganisms, characterized in that it contains raffinose and / or stachyose as active ingredients, wherein the useful intestinal microorganisms are microorganisms having a 16S rRNA gene consisting of the base sequence set forth in SEQ ID NO:

1.

7. An agent for proliferation of useful intestinal microorganisms, characterized in that it contains raffinose and / or stachyose as active ingredients, wherein the useful intestinal microorganisms are strains identified by accession number NITE BP-04072.

8. A proliferation agent for useful intestinal microorganisms as described in claim 6 or 7, characterized in that it further contains the microorganisms as described in claim 1 or 2.

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