Carbohydrate preference inhibitor and carbohydrate preference inhibiting method

Abstract

To provide a novel carbohydrate preference inhibitor using a compound derived from A. muciniphila.SOLUTION: A carbohydrate preference inhibitor contains Amuc_1100. The Amuc_1100 may be a protein comprising the amino acid sequence shown in SEQ ID NO:1; may be a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NO:1 and having inhibitory activity on carbohydrate preference; or may be a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1 and having inhibitory activity on carbohydrate preference.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] The present invention relates to an inhibitor of carbohydrate appetite and a method for inhibiting carbohydrate appetite. [Background technology]

[0002] Patent Document 1 describes that administration of A. muciniphila (Akkermansia muciniphila), a gram-negative obligate anaerobic bacterium, enhances the barrier function associated with obesity and related disorders.

[0003] Meanwhile, the present inventors conducted research into the relationship between a host's food preferences and the host's intestinal bacterial flora, focusing on A. muciniphila.

[0004] The inventors have previously found that administering A. muciniphila to mice suppresses their carbohydrate preference ("carbohydrate preference" means a preference for food that contains a high proportion of carbohydrates) (journal article to be submitted).

[0005] Because A. muciniphila is digested in the mouse body, it is highly likely that the A. muciniphila cells contain some compound that inhibits carbohydrate preference, and that this compound exerts its inhibitory effect on carbohydrate preference after digestion. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2019-500004 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the compounds contained in A. muciniphila that suppress carbohydrate preference have not been identified.

[0008] The present invention has been made in consideration of the above circumstances, and provides a novel carbohydrate preference inhibitor and method for inhibiting carbohydrate preference using a compound derived from A. muciniphila. [Means for solving the problem]

[0009] As a result of extensive research aimed at achieving the above-mentioned object, the inventors discovered that Amuc_1100, a membrane protein present in a species-specific manner in A. muciniphila, has the effect of suppressing carbohydrate preference, and thus completed the present invention.

[0010] That is, one aspect of the present invention includes the following aspects.

[0011] [1] A carbohydrate preference suppressant that contains Amuc_1100.

[0012] [2] The carbohydrate preference inhibitor according to [1], wherein the Amuc_1100 is any one of the following proteins (a) to (c): (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 1; (b) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1, and having the ability to inhibit carbohydrate preference; or (c) a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and having the ability to inhibit carbohydrate preference.

[0013] [3] A carbohydrate preference suppressant described in [1] or [2], for oral administration.

[0014] [4] A method for suppressing carbohydrate preference, comprising the step of administering Amuc_1100 to a mammal.

[0015] [5] The method for suppressing carbohydrate preference described in [4], wherein the mammal is a mammal other than a human.

[0016] [6] The method for suppressing carbohydrate preference according to [4] or [5], wherein the Amuc_1100 is any one of the following proteins (a) to (c): (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 1; (b) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1, and having the ability to suppress carbohydrate preference; or (c) a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and having the ability to suppress carbohydrate preference.

[0017] [7] The method for suppressing carbohydrate preference described in any one of [4] to [6], wherein the administration is oral administration. Effect of the Invention

[0018] According to the present invention, it is possible to provide a novel carbohydrate preference inhibitor and a novel method for suppressing carbohydrate preference using a compound derived from A. muciniphila. [Brief description of the drawings]

[0019] [Figure 1] 13 is an image showing the environment in the breeding cage during the breeding period with high carbohydrate and high lipid diets. [Diagram 2] 1 is an image showing high carbohydrate food contained within a food box. [Diagram 3] 1 is an image showing high lipid food contained within a feeding box. [Figure 4] 1 is a graph showing the intake of high carbohydrate feed, the intake of high lipid feed, and the percentage of high carbohydrate feed intake to total intake in the comparative group. [Diagram 5] FIG. 5 is a graph showing the intake of high-carbohydrate feed, the intake of high-fat feed, and the proportion of high-carbohydrate feed intake in total intake in the example group. [Figure 6]FIG. 6 is a graph showing the ratio of high-carbohydrate feed intake to total intake shown in FIGS. 4 and 5 superimposed thereon. [Figure 7] 1 is a graph showing the total intake during feeding periods with high-carbohydrate and high-fat diets. [Figure 8] 1 is a graph showing the transition of the average body weight of mice in the example group and the comparative example group. [Figure 9] 1 is a graph showing the length of the intestine and the weight of white fat measured at the time of dissection of mice in the comparative example group and the example group. [Figure 10] 1 is a graph showing the expression levels of GLP-1, TLR2, and TGF-β in the intestinal tissues of mice in the comparative example group and the example group. [Figure 11] 1 is a graph showing the expression levels of NPY and FGF21 in the hypothalamic tissue of mice in the Comparative Example group and the Example group. [Figure 12] 1 is a graph showing the results of an analysis of the amount of A. muciniphila in the intestines of mice in the Comparative Example group and the Example group. [Figure 13] 1 is a graph showing rectal temperatures of mice in a comparative example group and an example group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Preferred embodiments of the present invention will now be described in detail.

[0021] <Carbohydrate appetite suppressant> The carbohydrate preference suppressant of this embodiment contains Amuc_1100.

[0022] Amuc_1100 is a species-specific membrane protein found in A. muciniphila, and is any one of the following proteins (a) to (c):

[0023] (a) a protein comprising the amino acid sequence set forth in SEQ ID NO:1; (b) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO:1 and having the ability to suppress carbohydrate preference; or (c) a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1 and having the ability to suppress carbohydrate preference. SEQ ID NO: 1: MGSSHHHHHSSGLVPRGSHSDEVDAHMIVNSKRSELDKKISIAAKEIKSANAAEITPSRSSNEELEKELNRYAKAVGSLETAYKPFLASSALVPTTPTAFQNELKTFRDSLISSCKKKNILTDTSSWLGFQVYSTQAPSVQAASTLGFELKAINSLVNKLAECGLSKFIKVYRPQLPIETPANNPEESDEADQAPWTPMPLEIAFQGDRESVLKAMNAITGMQDYLFTVNSIRIRNERMMPPPIANPAAAKPAAAQPATGAASLTPADEAAAPAAPAIQQVIKPYMGKEQVFVQVSLNLVHFNQPKAQEPSED

[0024] By ingesting or administering a carbohydrate preference inhibitor containing the above-mentioned Amuc_1100, the carbohydrate preference inhibitor effect can be obtained, as will be described in detail in the Examples below, and carbohydrate restriction can be easily achieved.

[0025] In the amino acid sequence of (b), the number of deleted, inserted, substituted or added amino acids is preferably 1 to 50, more preferably 1 to 20, even more preferably 1 to 10, particularly preferably 1 to 5, and most preferably 1 to 2.

[0026] The sequence identity in the amino acid sequence of (c) above is preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more.

[0027] In this specification, the sequence identity of a subject amino acid sequence to the reference amino acid sequence shown in SEQ ID NO: 1 can be determined, for example, as follows. First, the reference amino acid sequence and the subject amino acid sequence are aligned. Each amino acid sequence may contain gaps to maximize sequence identity. Next, the number of matching amino acids (residues) in the reference amino acid sequence and the subject amino acid sequence is calculated, and the sequence identity can be calculated according to the following formula (1). Sequence identity (%) = number of matching amino acids (residues) / total number of amino acids (residues) in the target amino acid sequence × 100…(1)

[0028] The number of amino acid residues in Amuc_1100 is not particularly limited, but may be, for example, 250 to 350.

[0029] The proportion of Amuc_1100 in the carbohydrate preference inhibitor is not particularly limited, but is preferably 30 to 100 mass%, more preferably 50 to 100 mass%, and most preferably 80 to 100 mass%, calculated on a dry matter basis.

[0030] The carbohydrate preference suppressant may contain optional ingredients in addition to the above-mentioned Amuc_1100. Examples of optional ingredients include carriers, additives, etc. that are acceptable for food or pharmacopoeia. A "carrier, additive that is acceptable for food or pharmacopoeia" is a carrier, additive that is not substantially toxic to an animal that ingests the ingredient or an animal (subject) that is administered the ingredient. "Not substantially toxic" means that the ingredient is not toxic to an animal that ingests the ingredient or an animal (subject) that is administered the ingredient at a dosage or intake amount that is normally used.

[0031] The type of edible or pharma- ceutically acceptable carrier is not particularly limited, but examples include binders such as gelatin, corn starch, gum tragacanth, and gum arabic; excipients such as starch and crystalline cellulose; and leavening agents such as alginic acid.

[0032] The types of food- or pharma- ceutically acceptable additives are not particularly limited, but examples include lubricants such as magnesium stearate and calcium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; acidulants; flavorings such as peppermint and red bean oil; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antifoaming agents; antioxidants; and preservatives.

[0033] The form of the carbohydrate preference suppressant is not particularly limited, but examples include tablets, powders, granules, solutions, sheets, liquids, and the like.

[0034] (Use of carbohydrate appetite suppressant) The uses of the carbohydrate preference suppressant are not particularly limited, but examples include incorporating it into foods, medicines, quasi-drugs, etc. as an active ingredient for suppressing carbohydrate preference.

[0035] Foods, medicines, or quasi-drugs containing a carbohydrate preference suppressant are considered to be effective for carbohydrate restriction, obesity treatment, diabetes treatment, etc. Specific examples of foods will be described in detail in the embodiment related to foods to be explained later.

[0036] (Animal to be ingested, administration target, administration method) The animals (including humans) that take the carbohydrate preference suppressant or the animals to which the agent is administered are not particularly limited, but are preferably mammals.

[0037] Examples of mammals include humans, mice, rats, rabbits, guinea pigs, hamsters, hedgehogs, dogs, cats, monkeys, horses, cows, pigs, sheep, etc. Among these, humans are particularly preferred.

[0038] The method of administration of the carbohydrate preference suppressant may be intra-arterial injection, intravenous injection, subcutaneous injection, intramuscular injection, or oral administration, with oral administration being particularly preferred, i.e., the carbohydrate preference suppressant may be for oral administration.

[0039] (Amount of intake, dosage) The intake or administration amount of the carbohydrate preference suppressant varies depending on the weight and age of the animal taking the carbohydrate preference suppressant or the animal to which the carbohydrate preference suppressant is administered, as well as the condition of the animal, such as symptoms, but a person skilled in the art can appropriately select an appropriate intake or administration amount. The same applies to the number of intakes or administrations and the frequency of intake or administration of the carbohydrate preference suppressant. The frequency of intake or administration is not particularly limited, and may be, for example, once or more per day, once every two days, or once per week.

[0040] The intake or administration amount of the carbohydrate preference suppressant may be, for example, an amount containing 50 μg or more, 100 μg or more, 500 μg or more, or 1 mg or more of Amuc_1100 per kg of body weight.

[0041] The intake or administration amount of the carbohydrate appetite suppressant may be, for example, 10 mg or less, 5 mg or less, 1 mg or less, 700 μg or less, 300 μg or less, or 100 μg or less per kg of body weight.

[0042] The upper and lower limits of the above intake or administration amount can be combined in any desired manner.

[0043] The intake period or administration period is preferably one week or longer, more preferably two weeks or longer, even more preferably three weeks or longer, and most preferably one month or longer.

[0044] <How to curb your craving for carbohydrates> The method for suppressing carbohydrate preference of this embodiment includes a step of administering Amuc_1100 to a mammal. By administering Amuc_1100, carbohydrate preference can be suppressed.

[0045] Amuc_1100 is similar to that detailed in the above embodiment of the inhibitor.

[0046] Like the above-mentioned inhibitors, Amuc_1100 may be administered to a mammal together with optional ingredients such as food- or pharma- ceutical acceptable carriers, additives, and the like.

[0047] The mammal is similar to the mammals described above and may be a human, but may also be a mammal other than a human.

[0048] The method of administration to mammals may be intraarterial injection, intravenous injection, subcutaneous injection, intramuscular injection, or oral administration, as in the case of the above-mentioned inhibitors, but oral administration is particularly preferred. The number of administrations, frequency of administration, duration of administration, etc. are the same as those described above.

[0049] [Other embodiments] In one embodiment, the present invention provides a food product containing the carbohydrate appetite suppressant described above.

[0050] Examples of foods containing carbohydrate preference inhibitors include, but are not limited to, breads, fermented foods, dried foods, paste products, frozen foods, retort foods, instant foods (instant noodles, dry foods), processed foods (processed fish products, processed livestock products), sweets and other luxury foods, health foods (functional foods) such as supplements, foods for special purposes (foods for the sick, foods for children, foods for the elderly), functional foods, foods for specified health uses, water, coffee, soft drinks, alcoholic beverages, tea, etc.

[0051] In one embodiment, the present invention provides a pharmaceutical product containing the carbohydrate appetite suppressant described above.

[0052] In one embodiment, the present invention provides a quasi-drug containing the carbohydrate preference suppressant described above.

[0053] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these modifications are also included in the scope of the present invention. EXAMPLES

[0054] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples. Table 1 below shows the base sequences of each primer used in the examples.

[0055] [Table 1]

[0056] <Experimental Overview> In this example, a carbohydrate preference inhibitor containing Amuc_1100 was administered to 6-week-old male mice of the C57BL6 / JJcl strain, and the effect on carbohydrate preference was evaluated.

[0057] <Preliminary breeding period> The mice were kept at 20°C and fed normal food, neither high-carbohydrate nor high-fat, such as MF feed (manufactured by Oriental Yeast Co., Ltd.) powdered in a mini speed mill, and pre-raised (acclimatised) at 20°C for one week.

[0058] <Feeding period with high carbohydrate and high fat diets> Figure 1 shows images of the environment inside the cage during the feeding period with high-carbohydrate and high-fat diets.

[0059] 2 is an image showing high-carbohydrate feed contained in the feed box 2, and FIG. 3 is an image showing high-fat feed contained in the feed box 2. As shown in FIG.

[0060] After the above preliminary rearing, mouse 1 was reared for 5 weeks in an environment in which a high-carbohydrate diet (HCD) rich in carbohydrates and a high-fat diet (HFD) rich in lipids were housed in separate feeding boxes 2 of the same shape, and mouse 1 was allowed to freely consume the HCD and HFD in the rearing case.

[0061] Each feeding box 2 was formed with an opening 2a, and the mouse 1 was free to choose between the high-carbohydrate food and the high-fat food in the feeding box 2 and eat them through the opening 2a.

[0062] During the five weeks, the intake of high-carbohydrate and high-lipid feed was measured once every two days based on the amount of feed remaining in feed box 2 containing the high-carbohydrate feed and the amount of feed remaining in feed box 2 containing the high-lipid feed.

[0063] During the first two weeks of the five-week study, antibiotics were administered to Mouse 1 in its drinking water to reduce gut bacteria, including A. muciniphila.

[0064] For the next three weeks, PBS containing 0.04 μg of Amuc_1100 (an example of an inhibitor of carbohydrate preference) or control PBS was orally administered to Mouse 1 using a probe once every two days. The amount of PBS containing Amuc_1100 or control PBS administered per dose was 0.2 mL.

[0065] Hereinafter, the group administered with PBS containing Amuc_1100 will be referred to as the "Example group" ("Amuc_1100 group" in the drawings), and the group administered with simple PBS will be referred to as the "Comparative Example group" ("PBS group" in the drawings).

[0066] Amuc_1100 was synthesized by an external company and dissolved in water. This Amuc_1100 was a protein having the amino acid sequence shown in SEQ ID NO:1.

[0067] (Feed preparation for high carbohydrate and high fat diets) Table 2 below shows the composition of high-carbohydrate and high-fat feeds and the calorie content of each ingredient.

[0068] [Table 2]

[0069] To prepare the high-carbohydrate and high-lipid feeds, powdered reagents (α-cornstarch, β-cornstarch, dextrin, casein, cellulose, mineral mix, vitamin mix (manufactured by Oriental Yeast Co., Ltd.), L-cystine, calcium carbonate, and choline bitartrate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)) were first mixed together.

[0070] Next, soybean oil and lard (manufactured by Nacalai Tesque and Oriental Yeast Co., Ltd.) were added and mixed.

[0071] Then, sucrose (manufactured by Oriental Yeast Co., Ltd.) dissolved in distilled water was added and mixed evenly.

[0072] The mixture was then frozen in a -80°C freezer for 24 hours or more, freeze-dried in a freeze dryer (Tokyo Rikakikaisha) for 24 hours or more, and then pulverized into powder form to be used as food for Mouse 1.

[0073] As shown in Table 2, the high carbohydrate diet contained α-cornstarch and β-cornstarch, and the high lipid diet contained more lard than the high carbohydrate diet.

[0074] Table 3 below shows the calorie ratio of the three major nutrients (carbohydrates, lipids, and proteins) in high-carbohydrate and high-lipid diets, and the amount of energy per gram of diet.

[0075] [Table 3]

[0076] As shown in Table 3, high-fat diets provide more energy per gram than high-carbohydrate diets due to the higher amount of fat.

[0077] (Amount of antibiotic) Table 4 below shows the amount of antibiotics contained in 1 litre of drinking water.

[0078] [Table 4]

[0079] <Analysis of the abundance of A. muciniphila in the intestine> At the start of the preliminary feeding period, and at the start and end of feeding with the high-carbohydrate and high-lipid diets, feces were collected from mouse 1, and the amount of A. muciniphila in the feces was measured by real-time PCR.

[0080] Specifically, first, feces coming out of the anus of Mouse 1 was collected using tweezers, and approximately 180 mg was immediately stored at -80°C.

[0081] Next, DNA was extracted from the feces using a Simple DNA Extraction Kit Version 2 (Kaneka Corporation).

[0082] The concentration of the extracted DNA solution was then measured using a spectrophotometer (Shimadzu Corporation), adjusted to 0.2 mg / 20 μL, and analyzed by real-time PCR. The subject of analysis was A. muciniphila, and a primer for 16S rDNA encoding the 16S rRNA of A. muciniphila was used (see Table 1).

[0083] On the other hand, as an internal standard for enterobacteria, a universal primer (see Table 1), which is a primer for eubacterial 16S rRNA, was used.

[0084] For real-time PCR, first, 5 μL of Power SYBR Green PCR Master Mix (Applied Biosystems (registered trademark)), 2 μL of a solution obtained by adding 1 μL of 50 μM (μmol / L) forward primer and 1 μL of 50 μM reverse primer to 13 μL of sterile water, and 8 μL of sterile water were each injected into a 96-well plate for the number of samples.

[0085] Thereafter, 5 μL of the DNA-extracted sample was added to the above-mentioned 96-well plate and subjected to a QuantStudio (registered trademark) 5 real time PCR system (manufactured by Thermo Fisher Scientific) to carry out a PCR reaction.

[0086] The PCR reaction conditions are shown in Table 5 below.

[0087] [Table 5]

[0088] After the PCR reaction, the amount of A. muciniphila DNA was normalized using the results of amplification using the internal standard primers described above, and the amount of A. muciniphila present was analyzed using the ΔΔCt method.

[0089] <Measurement of rectal temperature> When collecting the feces as described above, about 20 mm of the probe tip of an electronic thermometer (manufactured by Physitemp) was inserted into the anus of Mouse 1 and held there for about 30 seconds. When the temperature became constant, it was recorded as the rectal temperature.

[0090] <Analysis of gene expression levels> After a 5-week feeding period on high-carbohydrate and high-lipid diets, samples of intestinal (small intestinal) tissue and hypothalamic tissue collected from Mouse 1 were placed in 500 μL of Sepasol (registered trademark) I Super G (Nacalai Tesque) and crushed using a pestle.

[0091] Next, 100 μL of chloroform (Kokusan Kagaku Co., Ltd.) was added to the ground sample, which was then shaken and stirred using a vortex mixer, allowed to stand for 5 minutes, and then centrifuged at 4° C. and 21,200 g for 15 minutes.

[0092] Thereafter, 1 μL of Etachinmate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 3.3 μL of 3M Sodium acetate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a new 1.5 mL tube, and 300 μL of the supernatant after the above centrifugation was added and mixed by inversion.

[0093] Then, 300 μL of 2-Propanol (Kokusan Chemical Co., Ltd.) was added, and the mixture was centrifuged at 4° C. and 21,200 g for 10 minutes.

[0094] Thereafter, the supernatant after centrifugation was discarded, and 250 μL of 75% ethanol was added to each well and mixed by inversion, followed by centrifugation at 4° C. and 21,200 g for 10 minutes. The supernatant was discarded and the mixture was air-dried.

[0095] Next, 15 μL of DPEC-treated water (manufactured by Nippon Gene Co., Ltd.) was added and pipetted to dissolve the pellet, and the pellet was used as a sample.

[0096] To determine the RNA concentration of the sample from the absorbance, 48 μL of RNA sterile water and 2 μL of the sample were added to a 0.6 mL tube and shaken with a vortex mixer to prepare a sample for concentration measurement. To measure the absorbance, the RNA absorbance at 260 nm was measured using a NanoDrop Spectrophotometer (Thermo Fisher Scientific), and the cDNA concentration after reverse transcription was diluted with sterile water to 100 ng / μL.

[0097] Using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), 2 μL of 10× Reverse Transcription Buffer, 0.8 μL of 25× dNTPs, 2 μL of 10× Random Primer, 1 μL of MultiScribe Reverse Transcriptase, 1 μL of RNase Inhibitor, and 3.2 μL of sterile water were added to each of 8 PCR tubes, and 10 μL of sample diluted with sterile water was then added. Reverse transcription reaction was performed using a thermal cycler GeneAtlasG02 (Astec).

[0098] Table 6 below shows the conditions for reverse transcription reaction (RT-PCR).

[0099] [Table 6]

[0100] After the reverse transcription reaction, the concentration of each sample was adjusted to 0.2 μg / 20 μL. The reaction conditions for the subsequent Real-Time PCR are shown in Table 7 below. The expression levels of GLP-1, TLR2, and TGF-β were measured for the intestinal tissue samples, and the expression levels of NPY and FGF21 were measured for the hypothalamic tissue samples (see Table 1 for primer sequences).

[0101] [Table 7]

[0102] <Measurement results of high carbohydrate and high fat diet intake> Figure 4 is a graph showing the intake of high-carbohydrate feed, the intake of high-fat feed, and the ratio of high-carbohydrate feed intake to total intake in the comparative group. Figure 5 is a graph showing the intake of high-carbohydrate feed, the intake of high-fat feed, and the ratio of high-carbohydrate feed intake to total intake in the example group. Figure 6 is a graph showing the ratio of high-carbohydrate feed intake to total intake shown in Figures 4 and 5 superimposed.

[0103] In Figures 4 to 6, the results are shown as mean ± SD, n = 3 to 4, and comparisons of each data were performed using Student's t-test. "**" indicates a significant difference at p < 0.01, "*" indicates a significant difference at p < 0.05, and "†" indicates p < 0.1.

[0104] Figure 7 is a graph showing the total intake during the feeding period of high-carbohydrate and high-fat diets. In Figure 7, the results are shown as mean ± SD, n = 3 to 4, and no significant difference was observed between the two groups by Student's t-test.

[0105] As shown in Figures 4 and 6, in the comparative example group, the ratio of high-carbohydrate feed intake to the total intake, which is the combined intake of high-carbohydrate feed and high-fat feed, i.e., carbohydrate preference, was increased even after 15 days from the start of oral administration of PBS.

[0106] In contrast, as shown in Figures 5 and 6, in the Example group, it was observed that the preference for carbohydrates gradually decreased from the 15th day after the start of oral administration of PBS containing Amuc_1100.

[0107] These results demonstrated that administration of a carbohydrate preference inhibitor containing Amuc_1100 to animals can actually reduce carbohydrate preference.

[0108] Furthermore, as shown in FIG. 7, there was almost no difference in the total amount of food intake between the comparative example group and the example group, and as shown in FIG. 5, the intake of high-carbohydrate feed gradually decreased, revealing that the intake of high-fat feed tended to increase relatively.

[0109] Figure 8 is a graph showing the transition of the average body weight of Mouse 1 in the Example group and Mouse 1 in the Comparative Example group. In Figure 8, the results are shown as mean ± SD, n = 3 to 4, and data comparison was performed using Student's t-test. "†" indicates p < 0.1.

[0110] As shown in FIG. 8, Mouse 1 in the Example group tended to have a lower body weight than Mouse 1 in the Comparative Example group, but no significant difference was observed between the body weights of the two groups.

[0111] As described above, in the Example group, carbohydrate preference gradually decreased and the intake of high-fat feed increased, which suggests that the total amount of energy ingested had increased. However, since there was no significant increase in body weight between the Example group and Mouse 1 in the Comparative Example group, it is suggested that the administration of an inhibitor of carbohydrate preference containing Amuc_1100 increased the metabolism of Mouse 1.

[0112] <Measurement results of intestinal length and white fat weight> Fig. 9 is a graph showing the length of the intestine and the weight of white fat measured at the time of dissection of Mouse 1 in the Comparative Example group and the Example group. In Fig. 9, the results are shown as mean ± SD, n = 3 to 4, and data comparison was performed using Student's t-test. "*" indicates a significant difference at p < 0.05.

[0113] As shown in Figure 9, the length of the intestine was significantly shorter in Mouse 1 of the Example Group. It has been reported that in a low-temperature environment with little A.muciniphila, the host's intestine elongates, and that administration of A.muciniphila suppresses changes in intestinal length (Claire Chevalier. et al., Gut Microbiota Orchestrates Energy Homeostasis during Cold. Cell, 163, 1360-1374, 2015).

[0114] In this experiment, it is believed that administration of antibiotics to Mouse 1 for two weeks caused the intestinal flora to become in a state similar to that in a low-temperature environment, which resulted in the elongation of the intestinal tract in the comparison group.

[0115] In contrast, in the Example group, it is considered that the administration of Amuc_1100 suppressed the change in the length of the intestinal tract.

[0116] On the other hand, as shown in Fig. 9, the weight of white fat was significantly lower in the Example group than in the Comparative Example group. This result suggests that the administration of Amuc_1100 increased metabolism, thereby decreasing the weight of white fat.

[0117] <Analysis of gene expression levels in intestinal tissue> Fig. 10 is a graph showing the expression levels of GLP-1, TLR2, and TGF-β in the intestinal tissue of Mouse 1 in the Comparative Example group and the Example group. In Fig. 10, the results are shown as mean ± SD, n = 3 to 4, and data comparison was performed using Student's t-test. "*" indicates a significant difference at p < 0.05, and "†" indicates p < 0.1.

[0118] FIG. 10 shows the relative expression level of each gene in the group of the Example when the expression level of each gene in the group of the Comparative Example is set to 1.

[0119] TLR2 is a receptor for Amuc_1100. When Amuc_1100 binds to TLR2, it is expected that the expression of TGF-β will increase. However, as shown in FIG. 10, the expression of TGF-β was significantly lower in the group of the embodiment than in the group of the comparative example.

[0120] These results suggested that the suppression of carbohydrate preference by Amuc_1100 was not due to regulation of TGF-β expression.

[0121] Furthermore, since no significant difference was observed in the expression levels of GLP-1 between the group of the example and the group of the comparison example, it was suggested that the effect of Amuc_1100 in suppressing carbohydrate preference was not mediated by GLP-1.

[0122] <Analysis of gene expression levels in hypothalamic tissue> Fig. 11 is a graph showing the expression levels of NPY and FGF21 in the hypothalamic tissue of Mouse 1 in the Comparative Example group and the Example group. In Fig. 11, the results are shown as mean ± SD, n = 3 to 4, and data comparison was performed using Student's t-test. "†" indicates p < 0.11.

[0123] FIG. 11 shows the relative expression level of each gene in the group of the Example when the expression level of each gene in the group of the Comparative Example is set to 1.

[0124] As shown in FIG. 11, no significant difference was observed in the expression levels of either NPY, which enhances carbohydrate preference, or FGF21, which suppresses carbohydrate preference, between the Example group and the Comparative Example group.

[0125] These results suggested that the suppression of carbohydrate preference by Amuc_1100 was not mediated by NPY or FGF21.

[0126] Furthermore, although no significant difference was observed in the expression level of FGF21, the carbohydrate preference-suppressing effect of Amuc_1100 was confirmed, suggesting that the carbohydrate preference-suppressing effect of Amuc_1100 is stronger than that of FGF21.

[0127] <Analysis of the abundance of A. muciniphila in the intestine> Fig. 12 is a graph showing the results of analysis of the amount of A. muciniphila in the intestine of Mouse 1 in the Comparative Example group and the Example group. In Fig. 12, the results are shown as mean ± SD, n = 3 to 4, and data comparison was performed using Student's t-test. "†" indicates p < 0.1.

[0128] FIG. 12 shows the relative amount of A. muciniphila in the intestines of the group of the Example when the amount of A. muciniphila in the intestines of the group of the Comparative Example was set to 1.

[0129] As shown in FIG. 12, the abundance of A. muciniphila in the intestines tended to increase in the group of the Example compared to the group of the Comparative Example.

[0130] Previous studies by the present inventors have demonstrated that administration of live or pasteurized A. muciniphila suppresses carbohydrate preference. This suggests that the carbohydrate preference suppression effect may include the effect of A. muciniphila, which increases in the intestine due to administration of Amuc_1100, in addition to the effect of Amuc_1100 itself.

[0131] <Rectal temperature measurement results> Fig. 13 is a graph showing the rectal temperatures of Mouse 1 in the Comparative Example group and the Example group. In Fig. 13, the results are shown as mean ± SD, n = 3 to 4, and no significant difference was observed between the two groups by Student's t-test.

[0132] This suggests that changes in food preference due to Amuc_1100 administration are not related to changes in body temperature. [Industrial Applicability]

[0133] According to the present invention, carbohydrate preference can be suppressed by administration of Amuc_1100, and therefore, the present invention is industrially applicable. [Explanation of symbols]

[0134] 1: Mouse 2: Feeding box 2a: Opening

Claims

1. A carbohydrate preference suppressant containing Amuc_1100.

2. The carbohydrate preference inhibitor according to claim 1, wherein the Amuc_1100 is any one of the following proteins (a) to (c): (a) a protein comprising the amino acid sequence set forth in SEQ ID NO:1; (b) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1 and having the ability to suppress carbohydrate preference; or (c) a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1 and having the ability to suppress carbohydrate preference.

3. The carbohydrate preference suppressant according to claim 1 or 2, which is for oral administration.

4. A method for suppressing carbohydrate preference comprising administering Amuc_1100 to a mammal.

5. 5. The method for suppressing carbohydrate preference according to claim 4, wherein the mammal is a mammal other than a human.

6. The method for suppressing carbohydrate preference according to claim 4 or 5, wherein the Amuc_1100 is any one of the following proteins (a) to (c): (a) a protein comprising the amino acid sequence set forth in SEQ ID NO:1; (b) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 1 and having the ability to suppress carbohydrate preference; or (c) a protein comprising an amino acid sequence having 60% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1 and having the ability to suppress carbohydrate preference.

7. The method for suppressing carbohydrate preference according to claim 4 or 5, wherein the administration is oral administration.

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