Composition and method for increasing intestinal butyric acid, composition and method for increasing muscle mass, composition and method for preventing or ameliorating obesity, composition and method for increasing number of bacteria of genus alistipes, composition and method for reducing number of bacteria of genus romboutsia, and use of silkworm powder for production of these compositions

Abstract

[Problem] To provide a novel use of silkworm on the basis on novel knowledge relating to the functionality of the silkworm. According to the present invention, butyric acid can be increased in the intestine. As a result, known useful effects of butyric acid (e.g., prevention and amelioration of obesity, muscle atrophy, intestinal inflammations, rheumatoid arthritis, graft versus host diseases, fatty liver, colorectal cancer, etc.) can be expected. Further, according to the present invention, muscle mass can be increased. As a result, effects such as the prevention of sarcopenia and muscle atrophy, the enhancement of physical strength, the improvement of activity and the like can be expected. [Solution] A composition for increasing intestinal butyric acid, the composition containing a silkworm powder as an active ingredient.

Description

Composition and method for increasing intestinal butyric acid, composition and method for increasing muscle mass, composition and method for preventing or ameliorating obesity, composition and method for increasing the number of Alistipes bacteria, composition and method for suppressing the number of Rhombosia bacteria, and use of silkworm powder for producing these compositions

[0001] The present invention relates to compositions and methods using silkworm powder as an active ingredient, including compositions and methods for increasing intestinal butyric acid, increasing muscle mass, preventing or ameliorating obesity, increasing the number of bacteria in the genus Alistipes, and suppressing the number of bacteria in the genus Rhombosia, as well as the use of silkworm powder to produce these compositions.

[0002] Silkworms are insects belonging to the family Bombycidae in the order Lepidoptera. They were domesticated through repeated breeding of wild silkworms (Bombyx mandarina) for use in the silk industry approximately 5,000 to 10,000 years ago, and their survival is now dependent on humans.

[0003] As shown in Table 1 (taken from "Authors: Kaneko Yu, Shiomi Kunihiro, Tenjiku Katsura Hiroko, Togawa Toru, Yokoyama Gaku; Supervisor: The Japanese Society of Sericulture; Publisher: NTS; Biological Science Genetics Special Issue No. 23: Silkworm Experiments: Understanding Life Science Through Silkworms!; Release Date: March 22, 2019"), the silkworm completes its life cycle of egg, larva, pupa, and adult in approximately two months. After hatching from the egg, the larvae molt four times and then form a cocoon to protect themselves from predators and pathogenic microorganisms as they transform from larvae into pupae. The cocoon has a structure with multiple layers of silk fibers to protect the pupa, and silk thread is extracted from these cocoons for industrial use. Silk fibers are primarily composed of the proteins fibroin and sericin, of which the water-soluble protein sericin is usually removed (degummed) during the silk thread manufacturing process, so silk thread is composed almost entirely of fibroin protein. Fibroin and sericin have been reported to have various physiological functions, but the only reported effect of silkworms themselves is that they have a blood sugar lowering effect (Patent Document 1).

[0004] Patent No. 2757937

[0005] National Agriculture and Food Research Organization, Home > Press Releases and Public Relations > NARO "Seasonal Topics" > The Secret of Silkworms, Information Release Date: August 27, 2020 [Retrieved July 7, 2023], Internet<URL:https: / / www.naro.go.jp / publicity_report / season / 134952.html>

[0006] As mentioned above, little is known about the functionality of silkworms themselves, and they are not being fully utilized. The present invention has been made to solve this problem, and aims to provide new uses for silkworms based on new findings regarding their functionality.

[0007] As a result of extensive research, the present inventors have found that oral ingestion of silkworm powder can increase intestinal butyric acid, muscle mass, the number of Alistipes bacteria, and the number of Romboutsia bacteria. Based on these findings, the present inventors have completed the following inventions.

[0008] (1) The composition for increasing intestinal butyric acid according to the present invention contains silkworm powder as an active ingredient.

[0009] (2) The composition for increasing muscle mass according to the present invention contains silkworm powder as an active ingredient.

[0010] (3) The composition for preventing or ameliorating obesity according to the present invention contains silkworm powder as an active ingredient.

[0011] (4) The composition for increasing the number of Alistipes bacteria according to the present invention contains silkworm powder as an active ingredient.

[0012] (5) The composition for inhibiting the number of Rhombosia fungi according to the present invention contains silkworm powder as an active ingredient.

[0013] (6) In the present invention, the silkworm powder has a wave number of (1635±9) cm in the infrared absorption spectrum of FT-IR. －１ Region and wavenumber (1537±20) cm －１The compound may have at least one absorption peak in each of the above regions.

[0014] (7) In the present invention, the silkworm powder may have trehalase activity.

[0015] (8) In the present invention, the silkworm powder may not have been subjected to heat treatment at 90°C or higher.

[0016] (9) The composition for increasing intestinal butyric acid according to the present invention may be used to prevent and / or improve a disease or unhealthy condition selected from obesity, muscle atrophy, intestinal inflammation, rheumatoid arthritis, graft-versus-host disease, fatty liver, and colorectal cancer.

[0017] (10) The composition for increasing the number of Alistipes bacteria of the present invention may be used to prevent and / or improve a disease selected from enteritis, liver cirrhosis, hepatic encephalopathy, non-alcoholic steatohepatitis, hepatocellular carcinoma, atrial fibrillation, heart failure, atherosclerotic cardiovascular disease, and autism spectrum disorder.

[0018] (11) The method for increasing intestinal butyric acid according to the present invention includes a step of increasing intestinal butyric acid in a human or animal by administering silkworm powder to the human or animal.

[0019] (12) The method for increasing muscle mass according to the present invention comprises the step of administering silkworm powder to a human or an animal to increase the muscle mass of the human or animal.

[0020] (13) The method for increasing the number of Alistipes bacteria of the present invention includes a step of increasing the number of Alistipes bacteria in the body of a human or an animal by administering silkworm powder to the human or animal.

[0021] (14) The method for suppressing the number of Romboutsia bacteria according to the present invention includes a step of suppressing the number of Romboutsia bacteria in the living body of a human or an animal by administering silkworm powder to the human or animal.

[0022] (15) The method according to (11), (13) or (14) above may be a method for preventing or ameliorating obesity.

[0023] (16) The method described in (11) above may be a method for preventing or ameliorating a disease or unhealthy condition selected from obesity, muscle atrophy, intestinal inflammation, rheumatoid arthritis, graft-versus-host disease, fatty liver, and colorectal cancer.

[0024] (17) The method described in (13) above may be a method for preventing or ameliorating a disease selected from enteritis, liver cirrhosis, hepatic encephalopathy, non-alcoholic steatohepatitis, hepatocellular carcinoma, atrial fibrillation, heart failure, atherosclerotic cardiovascular disease, and autism spectrum disorder.

[0025] (18) The use according to the present invention is the use of silkworm powder for producing the composition according to the present invention.

[0026] The method of the present invention may exclude medical intervention.

[0027] According to the present invention, butyric acid can be increased in the intestine, and useful effects known to be associated with butyric acid (e.g., prevention and improvement of obesity, muscle atrophy, intestinal inflammation, rheumatoid arthritis, graft-versus-host disease, fatty liver, colon cancer, etc.) can be expected.

[0028] According to the present invention, muscle mass can be increased, and therefore, effects such as suppression of sarcopenia and muscle atrophy, improvement of physical strength, and improvement of activity can be expected.

[0029] According to the present invention, obesity can be prevented or ameliorated.

[0030] According to the present invention, the Alistipes genus can be increased in the intestine, and therefore useful effects known to be associated with the Alistipes genus (e.g., prevention or amelioration of enteritis, liver cirrhosis, hepatic encephalopathy, non-alcoholic steatohepatitis, hepatocellular carcinoma, atrial fibrillation, heart failure, atherosclerotic cardiovascular disease, or autism spectrum disorder) can be expected.

[0031] According to the present invention, the number of Rhombussia bacteria in the intestines can be suppressed, and therefore, it is expected that the present invention will be effective in preventing and improving unhealthy conditions (e.g., obesity) that are known to be correlated with Rhombussia bacteria.

[0032] This is a bar graph showing the absorbance (wavelength 465 nm) of the free glucose produced when a trehalose decomposition reaction was carried out using a solution containing powder of silkworm larvae (silkworm powders A to C) as an enzyme solution, as measured by the glucose oxidase / peroxidase method. "Background" is a sample in which a solution containing no silkworm powder was used as the enzyme solution. This is an absorption spectrum (FT-IR spectrum) obtained by measuring silkworm powders A to C and silk thread using a Fourier transform infrared spectrophotometer. This is a diagram in which the FT-IR spectra of silkworm powders A to C and silk thread are superimposed. The downward arrows indicate peaks where there was a clear difference between silkworm powders A to C and silk thread. In the FT-IR spectra of silkworm powders A to C and silk thread, the peaks at wavenumbers of 1800 to 1400 cm －１ Silkworm powders A to C and silk threads with wave numbers of 1800 to 1400 cm －１FT-IR spectra of silkworm powders A to C and silk thread are overlaid. Downward arrows indicate peaks (i, iv) that showed clear differences between silkworm powders A, B, and C and silk thread, or peaks (ii, iii) that showed clear differences between silkworm powders A, B, and C and silk thread. Bar graphs showing the percentage of left paw weight in body weight for mice that orally ingested silkworm powders A to C (groups A, B, and C) and mice that did not ingest silkworm powder (control group) (★: p<0.05). Bar graphs showing the concentration of n-butyric acid in the cecal contents of mice that orally ingested silkworm powders A to C (groups A, B, and C) and mice that did not ingest silkworm powder (control group) (★: p<0.05). 1 is a bar graph showing the abundance ratio of the genus Alistipes in the cecal content of mice that orally ingested silkworm powders A to C (groups A, B, and C) and mice that were not given silkworm powder (control group) (★★★: p<0.001). 2 is a bar graph showing the abundance ratio of the genus Rhombosia in the cecal content of mice that orally ingested silkworm powders A to C (groups A, B, and C) and mice that were not given silkworm powder (control group) (★: p<0.05, ★★: p<0.01). 3 is a bar graph showing the abundance ratio of the genus Hydrogenoanaerobacterium in the cecal content of mice that orally ingested silkworm powders A to C (groups A, B, and C) and mice that were not given silkworm powder (control group) (★★★: p<0.001).

[0033] The present invention provides a composition and method for increasing intestinal butyric acid, a composition and method for increasing muscle mass, a composition and method for preventing or ameliorating obesity, a composition and method for increasing the number of bacteria of the genus Alistipes, a composition and method for suppressing the number of bacteria of the genus Rhombosia, and the use of silkworm powder for producing these compositions.

[0034] In the present invention, silkworm powder refers to powdered silkworm moths, which are insects belonging to the genus Bombyx in the family Bombycidae of the order Lepidoptera.

[0035] Silkworm powder can be produced by processing silkworm moths into powder using conventional methods. Silkworm moths can be powdered at any stage, including eggs, larvae, pupae, and adults. However, larvae are preferred in terms of the effort required for processing (productivity and yield). Silkworm powder can be prepared at home or commercially available. Examples of commercially available products made from silkworm larvae include "Silkworm Powder" (Ebiya Kusakian Co., Ltd.). Examples of commercially available products made from silkworm pupae include "Silkworm Powder" (Art Studio Wao Co., Ltd.), "Silkworm Pupae Powder" (R.O.N. Co., Ltd.), "Powdered Silkworm" (TAKEO Co., Ltd.), and "Silk Food" (Ellie Co., Ltd.).

[0036] As will be shown in the examples described below, silkworm powder that has been processed into a powder without being heat-treated at 90°C or higher has unique physical properties, such as having trehalase activity or having fibroin protein with a significantly loose higher-order structure, and exhibits high physiological effects in terms of increasing intestinal butyric acid, increasing muscle mass, and increasing the number of Alistipes bacteria or inhibiting the number of Rhombousia bacteria. For this reason, silkworm powder may not have been heat-treated at 90°C or higher.

[0037] In other words, as shown in the examples described below, silkworm powder having trehalase activity exhibits high physiological effects in terms of increasing intestinal butyric acid, increasing muscle mass, increasing the number of Alistipes bacteria, and suppressing the number of Rhombousia bacteria. For this reason, silkworm powder may have trehalase activity. Here, trehalase activity refers to the activity of an enzyme that catalyzes the decomposition of trehalose into two glucose molecules. The presence or absence of trehalase activity can be confirmed by standard methods, such as a method in which an enzymatic reaction is carried out using trehalose as a substrate, and then the amount of glucose produced is measured by the glucose oxidase / peroxidase method (see, for example, Example 2).

[0038] In other words, silkworm powder having a fibroin protein with a loose higher-order structure exhibits high physiological effects in terms of increasing intestinal butyric acid, increasing muscle mass, increasing the number of Alistipes bacteria, and suppressing the number of Rhombousia bacteria, as shown in the examples described below. For this reason, silkworm powder may have a fibroin protein with a loose higher-order structure. A fibroin protein with a loose higher-order structure can be identified, for example, as one that has an absorption peak in one or more regions selected from the following (i) to (iv) in the infrared absorption spectrum of FT-IR: (i) wavenumber (1740±39) cm －１ , (1740±35)cm －１ , (1740±30)cm －１ , (1740±25)cm －１ or (1740±20) cm －１ (ii) Wavenumber (1635±16) cm －１ , (1635±15)cm －１ , (1635±14)cm －１ , (1635±13)cm －１ , (1635±12)cm －１ or (1635±11) cm －１ (iii) Wavenumber (1537±25) cm －１ , (1537±24)cm －１ , (1537±23)cm －１ or (1537±22) cm －１ (iv) Wavenumber (1455±9) cm －１ , (1455±8)cm －１ , (1455±7)cm －１ , (1455±6)cm －１ or (1455±5) cm －１ area.

[0039] Furthermore, fibroin proteins with a significantly looser higher-order structure can be identified, for example, as those having at least one absorption peak in each of the following regions (ii-a) and (iii-a) in the infrared absorption spectrum of FT-IR: (ii-a) wavenumber (1635±9) cm －１, (1635±8)cm －１ , (1635±7)cm －１ , (1635±6)cm －１ or (1635±5) cm －１ (iii-a) Wave number (1537±20) cm －１ , (1537±19) cm －１ , (1537±18) cm －１ , (1537±17) cm －１ , (1537±16)cm －１ or (1537±15) cm －１ In other words, silkworm powder has a wave number of (1635±9) cm in the infrared absorption spectrum of FT-IR. －１ Region and wavenumber (1537±20) cm －１ The compound may have at least one absorption peak in each of the above regions.

[0040] Next, a method for using the silkworm powder according to the present invention will be described. As shown in the examples below, oral ingestion of silkworm powder increases the concentration of butyric acid in the intestine, increases muscle mass, increases the number of Alistipes bacteria in the intestine, or suppresses the number of Rhombousia bacteria in the intestine. That is, silkworm powder exerts its functions in the human or animal body (e.g., in areas containing bacterial flora, such as the intestine, oral cavity, vagina, and skin). Therefore, a method for using silkworm powder can include administering the silkworm powder, which is an active ingredient, to a human or animal. Specific examples of such administration methods include oral administration, transdermal administration, enteral administration, vaginal administration, and mucosal administration, such as sublingual administration and nasal administration. Oral administration can include orally ingesting silkworm powder directly or in the form of a food, drink, or pharmaceutical product. Enteral administration can include administering silkworm powder directly via the anus or via an inserted tube, or adding silkworm powder to an enteral nutrient solution and administering the resulting solution via a tube inserted into the digestive tract, such as the stomach or small intestine.

[0041] Silkworm powder can be used in the form of conventional foods and beverages, such as health foods such as pharmaceuticals, food additives, feed additives, and supplements, functional foods, foods for specified health uses, nutritional supplements, foods for special dietary uses (such as foods for formula-fed infants or for the sick), infant foods, confectioneries, beverages, and processed foods, as well as feed. When used in the form of pharmaceuticals, food additives, feed additives, or supplements, the dosage form can be, for example, a solid or liquid dosage form such as powders, tablets, sugar-coated tablets, capsules, granules, dry syrups, liquids, syrups, drops, drinks, creams, and ointments. When used in the form of foods and beverages, the active ingredient can be added during the normal manufacturing process.

[0042] When silkworm powder is used for humans or animals, the intake amount (administration amount) of the active ingredient can be, for example, 1 to 2 g, 1 to 10 g, 1 to 20 g, 1 to 30 g, 1 to 40 g, 1 to 50 g, 1 to 60 g, 1 to 70 g, 1 to 80 g, 1 to 90 g, or 1 to 100 g of silkworm powder per day. The intake amount is not limited to a single dose per day, and may be divided into multiple doses. Because silkworm powder has traditionally been used as a food product and there is no particular concern about side effects from taking large amounts, there are no particular upper or lower limits for the intake amount.

[0043] Silkworm powder has the effects of increasing the concentration of butyric acid in the intestines, increasing muscle mass, increasing the number of Alistipes bacteria in the intestines, or suppressing the number of Rhombosia bacteria in the intestines, and therefore can be used to produce a composition for increasing intestinal butyric acid, a composition for increasing muscle mass, a composition for increasing the number of Alistipes bacteria, or a composition for suppressing the number of Rhombosia bacteria.

[0044] Butyric acid, also known as butanoic acid or n-butyric acid, is a straight-chain carboxylic acid with the molecular formula C ４ H ８ O ２ , rational formula is CH ３ (CH ２ ) ２Butyric acid is a type of short-chain fatty acid, and is known to be produced by butyric acid-producing bacteria that are normally present in the intestines of humans and animals.

[0045] Butyric acid is known to have various beneficial effects in the body, and increasing its concentration in the intestine is expected to have favorable effects. For example, it has been reported that administration of butyric acid to mice fed a high-fat diet increases insulin sensitivity and prevents the development of obesity (Chen, LH et al., (2022). J Cachexia Sarcopenia Muscle 13, 515-531. 10.1002 / jcsm.12849.). It has also been shown that mice fed a high-fat diet containing 5% sodium butyrate by weight experienced reduced increases in body fat percentage and weight gain (Hua V. Lin et al., PLoS ONE, Vol. 7, Issue 4, e35240, April 2012; Fig. 1A, etc.). Furthermore, it has been shown that oral administration of butyric acid at a dose of 5 g / kg body weight (dosage of sodium butyrate) per day reduces body weight and body fat percentage in mice in which obesity has been induced by a high-fat diet, and suppresses weight gain and body fat percentage increases caused by the ingestion of a high-fat diet in non-obese mice (Zhanguo Gao et al., DIABETES, VOL. 58, July 2009, pp. 1509-1517; FIG. 1, E and F, and FIG. 7, A and B, etc.). Therefore, silkworm powder can be used to prevent or improve obesity by increasing butyric acid in the intestines of humans and animals.

[0046] In addition, it has been reported that the inflammatory reaction caused by the decrease in butyric acid is involved in muscle atrophy due to aging <General Incorporated Association, National Fermented Milk and Lactic Acid Bacteria Beverage Association, Top> Papers on lactic acid bacteria> January 2022, Yuji Naito, Gut microbiota and Well-being? Prevention of frailty and sarcopenia focuses on the gut environment?, [Searched on July 10, 2023], Internet <URL:https: / / www.nyusankin.or.jp / wp / wp-content / uploads / 2022 / 01 / NSKnewsNo515cc_7-12.pdf>, Butyric acid acts as a histone deacetylase inhibitor and increases the cross-sectional area of muscle fibers <Walsh, M.E.et al., (2015). Aging Cell 14, 957-970. 10.1111 / acel.12387.>. Therefore, silkworm powder can be used to prevent and improve muscle atrophy by increasing butyric acid in the intestines of humans and animals.

[0047] In addition, it has been reported that when the concentration of butyric acid is increased in the large intestine of mice, regulatory T cells (Treg) are induced, alleviating the symptoms of experimental colitis (Furusawa, Y. et al., Nature. 2013; 504: 446-450), suggesting that it may contribute to the suppression of intestinal inflammation (Koji Hasegawa, Immunoregulation by gut bacteria, Modern Media, Vol. 63, No. 2, 2017, pp. 16-21). Furthermore, in a rat model of ulcerative colitis induced by dextran sulfate sodium (DSS), a significant anti-inflammatory effect has been confirmed by injecting butyric acid into the intestine. In the same model rats, oral administration of the spore preparation of Clostridium butyricum M588, a butyric acid-producing bacterium, has also been confirmed to have a significant anti-inflammatory effect. In addition, butyric acid injection therapy has been performed for human ulcerative colitis (Journal of Enterobacteriology, Vol. 19, No. 1, pp. 1-8, 2005; right column on page 2, Fig4-1 and 4-2, etc.). Therefore, silkworm powder can be used to prevent and improve intestinal inflammation by increasing butyric acid in the intestines of humans and animals.

[0048] Here, in the present invention, "intestinal inflammation" refers to a state in which inflammation occurs in the intestinal tract, and includes a state in which it has led to a disease. An example of a disease that can result from intestinal inflammation is inflammatory bowel disease. Inflammatory bowel disease can be interpreted in a narrow sense as referring to ulcerative colitis and Crohn's disease, or in a broad sense as referring to any inflammatory disease of the intestinal tract (Latest Internal Medicine Series <Progress 8>, Digestive Diseases, pp. 320, 1997, Nakayama Shoten). In the present invention, inflammatory bowel disease refers to the latter, any inflammatory disease of the intestinal tract.

[0049] Furthermore, it has been reported that increasing the concentration of butyric acid in the intestine suppresses the onset of arthritis in a mouse model of rheumatoid arthritis (Hase Koji, "Immune Regulation by Intestinal Bacteria," Modern Media, Vol. 63, No. 2, 2017, pp. 16-21). Therefore, silkworm powder can be used to prevent and improve rheumatoid arthritis by increasing butyric acid in the intestines of humans and animals.

[0050] Furthermore, it has been reported that oral administration of butyric acid to mice enhances histone H4 acetylation in intestinal epithelial cells, thereby increasing the expression of epithelial barrier-related genes and alleviating graft-versus-host disease (GVHD) caused by allogeneic bone marrow transplantation (Kang JY., Dig Dis Sci 1993; 38: 257-268) (Hase Koji, Immune Regulation by Intestinal Bacteria, Modern Media, Vol. 63, No. 2, 2017, pp. 16-21). Therefore, silkworm powder can be used to prevent or ameliorate graft-versus-host disease by increasing butyric acid in the intestines of humans and animals.

[0051] In addition, oral administration of butyric acid at a dose of 0.02 g / kg body weight per day in rats has been shown to suppress hepatic steatosis and inflammation caused by high-fat diet intake, and normalize blood AST (aspartate aminotransferase), ALT (alanine aminotransferase), cholesterol, LDL (low-density lipoprotein), triglycerides, and fasting blood glucose levels, as well as the insulin resistance index (HOMA-IR) (G. Mattace Raso et al., PLoS ONE, vol. 8, no. 7, e68626, 2013; Abstract, Results, etc.). Therefore, silkworm powder can be used to prevent and improve fatty liver by increasing butyric acid in the intestines of humans and animals.

[0052] It has also been shown that adding butyric acid to the culture medium of human colon cancer cells LIM1215 at a final concentration of 1 mmol / L results in an extension of the growth time and a decrease in the growth rate (Whitehead RH et al., Gut 27, pp. 1457-1463, 1986; SUMMARY, etc.). Furthermore, it has been reported that butyric acid inhibits the abnormal proliferation of colonic mucosal epithelial cells, promotes cell death and differentiation of mutant cells, suppresses the transcription of abnormal proteins, and inhibits the development of aberrant multiple crypts (ACF), an early lesion of colon cancer, thereby suppressing the onset of colon cancer (Journal of Intestinal Microbiology, Vol. 16, No. 1, pp. 35-42, 2002; p. 40, left column and Table 5, etc.). Therefore, silkworm powder can be used to prevent or improve colon cancer by increasing butyric acid in the intestines of humans and animals.

[0053] In the present invention, "increasing the number of bacteria" of a specific bacterium means increasing the number of bacteria in a living body compared to when the active ingredient is not ingested, and is synonymous with "growing" or "promoting growth." Furthermore, "suppressing the number of bacteria" of a specific bacterium means decreasing the number of bacteria in a living body compared to when the active ingredient is not ingested, and is synonymous with "suppressing growth."

[0054] Alistipes is a bacterium belonging to the genus Alistipes in the family Rikenellaceae, and is a gram-negative, anaerobic bacillus. Alistipes are indigenous intestinal bacteria found in the intestines of healthy humans. As of April 2020, the National Center for Biotechnology Information (NCBI) database lists 13 known species: Alistipes finegoldii, Alistipes putredinis, Alistipes onderdonkii, Alistipes shahii, Alistipes indistinctus, Alistipes senegalensis, Alistipes timonensis, Alistipes obesi, Alistipes ihumii, Alistipes inops, Alistipes megaguti, Alistipes provencensis, and Alistipes massiliensis. (Parker BJ et al., (2020) Front. Immunol. 11:906. doi: 10.3389 / fimmu.2020.00906)

[0055] The Alistipes genus is known to have various beneficial effects on the body, so increasing its population in the intestine (promoting its proliferation) is expected to have beneficial effects. For example, it has been reported that the Alistipes genus is negatively correlated with obesity indicators such as total cholesterol and triglyceride serum concentrations (Dai, K. et al., (2022). Food Res Int 157, 111255. 10.1016 / j.foodres.2022.111255.). Additionally, the Alistipes genus has been reported to have protective and preventive effects in enteritis, liver cirrhosis, hepatic encephalopathy, non-alcoholic steatohepatitis, hepatocellular carcinoma, atrial fibrillation, heart failure, atherosclerotic cardiovascular disease, and autism spectrum disorder (Parker BJ et al., (2020) Front. Immunol. 11:906. doi: 10.3389 / fimmu.2020.00906, TABLE 3, etc.). Therefore, by increasing the number of Alistipes bacteria in the intestines of humans and animals, silkworm powder can be used to prevent or improve diseases selected from enteritis, liver cirrhosis, hepatic encephalopathy, non-alcoholic steatohepatitis, hepatocellular carcinoma, atrial fibrillation, heart failure, atherosclerotic cardiovascular disease, and autism spectrum disorder.

[0056] Romboutsia is a group of enterobacteria belonging to the Peptostreptococcaceae family. In 2018, the Gram-positive, anaerobic, motile Romboutsia hominis DSM 28814 was proposed as the type strain (Gerritsen et al., Int J Syst Evol Microbiol 2018;68:3479-3486, DOI 10.1099 / ijsem.0.003012). According to the same publication, in addition to R. hominis, Romboutsia lituseburensis DSM 797 (M59107), Romboutsia sedimentorum JCM 19607 (KF443808), and Romboutsia ilealis DSM 25109 (JN381505) have also been reported as Romboutsia species.

[0057] Rhombussia species have been reported to increase in various diseases and ill health conditions, so suppressing their intestinal population may have beneficial effects. For example, Rhombussia species are increased in the intestinal microbiota of obese patients (Zhou, Z. et al., (2021). Front Cell Dev Biol 9, 732204. 10.3389 / fcell.2021.732204.) and have been shown to be an indicator for predicting metabolic disorders in obese patients (Zeng, Q. et al., (2019). Sci Rep 9, 13424. 10.1038 / s41598-019-49462-w.). Furthermore, fecal Rhombussia species were significantly higher in patients with esophageal cancer compared with healthy controls (YaLi Deng et al., Microbial Pathogenesis, Volume 150, January 2021, 104709). It has also been reported that fecal counts of Rhombosia bacteria were higher in patients with Hashimoto's thyroiditis than in healthy controls (Fuya Zhao et al., Thyroid Vol. 28, No. 2, Published Online:1 Feb 2018 https: / / doi.org / 10.1089 / thy.2017.0395). Furthermore, it has been reported that Rhombosia bacteria induced bone marrow necrosis in patients with acute myeloid leukemia (Dale Seviar et al., British Journal of Haematology, 2022, 196, 262, First published online 28 September 2021, doi: 10.1111 / bjh.17812).

[0058] As shown in the examples below, silkworm powder increases the intestinal concentration of butyric acid, which has been reported to have an obesity-preventing effect, increases the number of Alistipes bacteria, which have a negative correlation with obesity, and suppresses the number of Rhombosia bacteria, which have a positive correlation with obesity. In other words, silkworm powder increases the amount of substances with an obesity-preventing effect in the body and improves indicators of obesity, so it can be used as a composition for preventing or ameliorating obesity. Silkworm powder can also be used to produce a composition for preventing or ameliorating obesity.

[0059] The present invention will be described below based on examples, but the technical scope of the present invention is not limited to the features shown in these examples.

[0060] Example 1 Preparation of Silkworm Powder (1) Preparation of Silkworm Powder A Silkworm moth (Bombyx mori) larvae were collected from Silkworm Room A in Nagano Prefecture and frozen in a freezer at -27°C or below. Subsequently, 150 g of frozen larvae per layer were placed in the dry chamber of a freeze dryer (EYELA Tokyo Rikakikai) and dried at -86°C and 4 Pa ​​for 24 hours. The dried larvae were loaded into 50 mL freeze-crushing tubes at approximately 3-5 g per tube, capped with a large metal cone, and crushed in a second-speed crusher (Multi-Beads Shocker, Yasui Kikai) at 3,000 rpm for 20-30 seconds. The resulting powder was designated "Silkworm Powder A."

[0061] (2) Preparation of Silkworm Powder B Silkworm larvae were collected from Silkworm Room B in Nagano Prefecture and frozen in a freezer. The frozen larvae were then crushed and sterilized by heating at 90°C or higher. After cooling, they were freeze-dried. The freeze-dried product was crushed, and the resulting powder was designated "Silkworm Powder B."

[0062] (3) Preparation of Silkworm Powder C Silkworm larvae were collected from silkworm room C in Yamanashi Prefecture and treated in the same manner as in Example 1(2). The obtained powder was designated "Silkworm Powder C."

[0063] Example 2 Evaluation of the Physical Properties of Silkworm Powder (1) Contained Components Silkworm powders A to C prepared in Example 1 were subjected to component analysis. The results are shown in Table 2. As shown in Table 2, there was no significant difference between silkworm powders A to C in terms of the content of moisture, protein, lipid, ash, and carbohydrates.

[0064] (2) Trehalase Activity Silkworm powders A to C prepared in Example 1 were dispersed in a 10-fold volume of tissue grinding buffer (composition: 0.3 M NaCl, 5 mM EDTA, 10 mM HEPES-NaOH, pH 7.5) and shaken in a vortex mixer for 5 minutes. The mixture was then centrifuged at 20,000 × g at 4°C for 30 minutes, and the supernatant was collected and used as an enzyme solution. 20 μL of the enzyme solution was aliquoted, and 230 μL of substrate solution (trehalose dissolved to 100 mM in 0.1 M phosphate buffer (pH 6.0)) was added. The enzyme reaction was carried out in a 37°C incubator for 20 or 40 minutes. The enzyme protein was denatured by placing the mixture in a 75°C heat block for 10 minutes, and the enzyme reaction was terminated. The mixture was centrifuged at 20,000 × g at 4°C for 5 minutes, and the supernatant was collected and used as an enzyme reaction solution.

[0065] Free glucose in the enzyme reaction solution was measured by the glucose oxidase / peroxidase method (GOD-POD method). The measurement principle is that glucose oxidase acts on β-D-glucose to produce D-gluconic acid and hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide oxidizes 3,3'-diaminobenzidine (DAB) with the oxygen generated from the hydrogen peroxide, producing a dark brown precipitate of oxidized DAB. This pigment is measured at a wavelength of 465 nm. The greater the absorbance, the greater the amount of pigment produced, and the amount of pigment produced is proportional to the amount of glucose present. This means that the greater the absorbance, the greater the trehalase activity.

[0066] Specifically, 100 μL of the enzyme reaction mixture was placed in a 1.5 mL tube, and 1.0 mL of GO / PO reagent (final concentrations: 30 μL of glucose oxidase (10,000 units / mL), 500 μL of peroxidase (1.0 mg / mL), 1.0 mL of 20% Triton X-100, and 500 μL of DAB hydrochloride (1.0 mg / mL) dissolved in 100 mL of 0.5 mM Tris-Cl buffer (pH 7.0)) was added. The mixture was incubated in a 37°C incubator for 50 minutes and then placed on ice. The reaction mixture was subjected to spectrophotometric measurement of absorbance at a wavelength of 465 nm. Four trials (N=4) were performed, and the average value was calculated. For comparison, a sample without added silkworm powder was also treated in the same manner as the "background" and its absorbance was measured. The results are shown in Figure 1.

[0067] As shown in Figure 1, Silkworm powder A had an absorbance significantly higher than the background value for both 20 and 40 minutes of enzyme reaction. Furthermore, the longer the enzyme reaction time, the higher the absorbance value. This demonstrated that Silkworm powder A had high trehalase activity. On the other hand, Silkworm powder B and Silkworm powder C both had absorbances lower than the background value, regardless of the length of the enzyme reaction time. This demonstrated that Silkworm powder B and Silkworm powder C did not have trehalase activity (trehalase was inactivated).

[0068] (3) FT-IR Spectrum The silkworm powders A to C prepared in Example 1 were subjected to measurement using a Fourier transform infrared spectrophotometer (IR Prestige-21; Shimadzu Corporation) by the attenuated total reflection (ATR) method to obtain an absorption spectrum. The measurement conditions were: crystal: diamond, number of integrations: 30, resolution: 2 cm －１ ~1cm －１ And wave numbers 600 to 4000 cm －１ As a control, refined silk thread (which can be said to be composed almost entirely of fibroin protein) was measured in the same manner, and the absorption spectrum was obtained. －１The spectra of each sample are shown in FIG. 2, and the spectra of each sample are superimposed in FIG.

[0069] As shown in Figures 2 and 3, －１ In the vicinity of 2922 cm, the peak for silk thread was large, and the peaks for silkworm powders A to C were smaller than that. －１ Near 2852 cm －１ Near 1395-1389 cm －１ and 1069-1030 cm －１ In the FT-IR spectra, prominent peaks were observed in silkworm powders A to C, whereas the peaks were small or not observed in silk thread. Since the reduction or absence of peaks in the FT-IR spectrum is thought to correlate with the amount or presence of contained components, these results revealed that the contained components of silk thread and silkworm powder are clearly different.

[0070] In addition, both silk thread and silkworm larvae are thought to contain fibroin protein, and the higher-order structure of fibroin protein is expressed by a wave number of 1800 to 1400 cm －１ (Xiao Hu, David Kaplan, Peggy Cebe, "Determining beta-sheet crystallinity in fibrous proteins by thermal analysis and infrared spectroscopy." Macromolecules, 2006, 39, 6161-6170, doi: 10.1021 / ma0610109). The same paper also reports that as fibroin protein crystallizes through high-temperature heating or methanol treatment, its peak shifts to longer wavelengths, indicating an increase in regular, rigid structures such as beta-sheets. Conversely, peaks at shorter wavelengths indicate an increase in irregular structures such as random coils. Table 3 below shows excerpts from Table 1 and Figure 1 of the paper.

[0071] Therefore, based on this, the wave number 1800 to 1400 cm －１The spectra of the wavenumber region were compared. Enlarged views of the relevant wavenumber region are shown in Figure 4 (spectrum of each sample) and Figure 5 (overlapped spectrum of each sample).

[0072] As shown in (i) to (iv) of Figures 4 and 5, different peaks were observed between the samples in at least four regions. In (i), the peaks of silkworm powders A to C all had peaks at about 1740 cm －１ whereas the peak of silk thread was 1700 cm －１ In (iv), the peaks of silkworm powders A to C were all at about 1455 cm －１ whereas the peak of silk thread was 1445 cm －１ That is, in both regions (i) and (iv), the peaks of the silk thread were shifted to the longer wavelength side compared with the silkworm powders A to C.

[0073] In addition, in (ii), the peak of silkworm powder A is 1635 cm －１ , Silkworm powder B and Silkworm powder C have a peak at 1625 cm －１ , the peak of the silk thread is 1618 cm －１ In (iii), the peak of silkworm powder A was 1537 cm －１ , Silkworm powder B and Silkworm powder C have a peak of 1516 cm －１ , the peak of the silk thread is 1512 cm －１ That is, in both regions (ii) and (iii), the peaks were arranged in the order of silkworm powder A, silkworm powder B, silkworm powder C, and silk thread, from the short wavelength side to the long wavelength side.

[0074] That is, silkworm powders A to C had peaks at shorter wavelengths than silk fiber in regions (i) and (iv), while silkworm powder A had peaks at shorter wavelengths than silk fiber in regions (ii) and (iii). These results revealed that silk fiber contains fibroin protein with a regular and hard structure, while silkworm powder contains fibroin protein with a structure that is less regular or less crystalline (loose) than silk fiber. Furthermore, it was revealed that, among silkworm powders, silkworm powder A contains fibroin protein with the least regular or less crystalline (loose) structure.

[0075] From the results of this Example 2, it became clear that silkworm powder differs from silk thread at least in that it contains different components and has a loose, higher-order structure of fibroin protein, and that it has unique physical properties.

[0076] Furthermore, it was revealed that Silkworm Powder A has unique physical properties, differing from Silkworm Powder B and Silkworm Powder C in that it has at least trehalase activity and a fibroin protein with a significantly looser higher-order structure. Considering the processing procedure for silkworm larvae shown in Example 1, it was thought that such unique physical properties could be obtained by performing powder processing without heat treatment at 90°C or higher.

[0077] Example 3: Evaluation of the Functionality of Silkworm Powder (1) Raising Mice Fed Silkworm Powder The following two types of mouse feed were used. [Laboratory Animal Feed "MF" (Oriental Yeast Co., Ltd.)] Ingredients (per 100 g): moisture 8.1 g, crude protein 23.2 g, crude fat 4.9 g, crude ash 5.9 g, crude fiber 3.3 g, nitrogen-free soluble matter 54.7 g, 355.7 kcal. [Standard Feed] Composition (units: mass %): cornstarch 39.7486, milk casein 20, pregelatinized cornstarch 13.2, sucrose 10, refined soybean oil 7, crystalline cellulose 5, mineral mix 3.5, vitamin mix 1, L-cystine 0.3, choline bitartrate 0.25, tert-butylhydroquinone 0.0014.

[0078] Five-week-old female C57BL / 6 mice (Japan SLC) were acclimatized for one week with free access to laboratory animal diet "MF" and sterile distilled water. Subsequently, the mice were randomly divided into four groups of 13 mice each: Groups A, B, and C, and a control group. Groups A, B, and C were fed a diet containing 95 parts by weight of standard diet supplemented with 5 parts by weight of Silkworm Powder A, Silkworm Powder B, or Silkworm Powder C prepared in Example 1. The control group was fed a standard diet. The mice were also allowed free access to sterile distilled water. The rearing conditions were a temperature of 24±1°C with a 12-hour light / 12-hour dark cycle throughout the entire period.

[0079] During the rearing period, body weight was measured once a week, and water intake and food intake were measured twice a week. Based on these measurement records, the intake of silkworm powder A to C was calculated. The results were 0 g / kg body weight / day in the control group, 7.595 g / kg body weight / day in Group A, 7.896 g / kg body weight / day in Group B, and 7.711 g / kg body weight / day in Group C, with no significant differences between the groups. In all groups, body weight increased by approximately 120% by the end of the study, with 100% weight at the start of the study. However, the weight at the start of the study was used to calculate silkworm powder intake. No significant differences were observed between the groups in body weight, weight change rate, water intake, or food intake during the study period.

[0080] It has also been reported that the drug intake in mice can be converted to the intake in adult humans using the following formula 1 (paragraph

[0065] of JP 2014-526521 A; Shannon Reagan-Shaw et al., The FASEB Journal, Vol. 22, March 2007, pp. 659-661): {Equation 1} Daily intake of silkworm powders A to C in an adult human (g / kg body weight) = Daily intake of silkworm powders A to C in a mouse (g / kg body weight) × 3 / 37 Thus, the daily intake of silkworm powders A to C in a mouse was converted to that in an adult human using formula 1. As a result, the intake amount of silkworm powder (equivalent value) in human adults was 0 g / kg body weight / day in the control group, 0.62 g / kg body weight / day in Group A, 0.64 g / kg body weight / day in Group B, and 0.63 g / kg body weight / day in Group C.

[0081] (2) Left Paw Weight Mice from each group in Example 3(1) were euthanized by cervical dislocation, weighed, and then dissected to collect the paws, including the quadriceps femoris, the largest muscle in the body. Paw weights were measured to the first decimal place (mg) using an electronic balance (SEFI IBA-200 Calibration, MICRO PRECISION CALIBRATION INC., CA, USA). The weight of the left paw was divided by the body weight measured immediately before dissection to calculate the percentage (%) of the left paw weight (average value for N = 13). For Group B, the average value for N = 12 was used, excluding outliers. The results are shown in Figure 6.

[0082] As shown in Figure 6, the ratio of left leg weight to body weight was higher in Groups A, B, and C compared to the control group. In particular, it was significantly (p<0.05) higher in Group A compared to the control group. These results demonstrate that silkworm powder increases muscle mass.

[0083] (3) Intestinal Butyric Acid Amount Mice from each group in Example 3(1) were euthanized by cervical dislocation, dissected, and the cecum was removed. The cecal contents were collected and weighed. Next, the butyric acid concentration in the cecal contents was measured using high-performance liquid chromatography (HPLC) under the following conditions. Subsequently, the average butyric acid concentration (μmol / g) was calculated for each group and represented in a bar graph. The results are shown in Figure 7. <HPLC Measurement Conditions> Solvent: acetonitrile / methanol / water = 24 / 12 / 64, pH 4-5 Flow rate: 0.25 mL / min Column temperature: 30°C Column: Waters ACQUITY UPLC BEH C18 (2.1 mm x 150 mm) Injection volume: 0.8 μL

[0084] As shown in Figure 7, the butyric acid concentration in the cecal contents was higher in Groups A, B, and C compared to the control group. In particular, it was significantly (p<0.05) higher in Group A compared to the control group. These results demonstrate that silkworm powder increases butyric acid in the intestine.

[0085] (4) Intestinal Bacteria Abundance Ratio Mice from each group in Example 3(1) were dissected, their cecums were removed, and the cecal contents were collected and frozen. Total DNA was extracted from the cecal contents according to the method described in Shunsuke Takahashi et al., PLosONE, Vol. 9, No. 8, e105592, August 2014: Reference 1. Specifically, 100 mg of cecal contents thawed on ice were suspended in an aqueous solution containing 4 M guanidine thiocyanate, 100 mM Tris-HCl (pH 9.0), and 40 mM EDTA. The suspension was then crushed with zirconia beads using a FastPrep FP100A (MP Biomedicals). DNA was extracted from this suspension using a Magtration System 12GC (Precision System Science) and a GC series MagDEA DNA 200 (Precision System Science). The DNA concentration was measured using a spectrophotometer ND-1000 (NanDrop Technologies) and adjusted to 10 ng / μL, which was used as the total DNA of the cecal contents.

[0086] A comprehensive analysis of the types and abundance ratios of bacteria contained in cecal contents was performed according to the method described in Reference 1 above. Specifically, first, using the total DNA of cecal contents as a template, polymerase chain reaction (PCR) was performed using the universal primers of SEQ ID NOs: 1 and 2 below by the Dual-index method (Hisada Takayoshi et al., Arch Microbiol, Vol. 197, No. 7, pp. 919-34, June 2015) to amplify the V3-V4 region of bacterial 16S rDNA. Forward primer (341f): CCTACGGGAGGCAGCAG (SEQ ID NO: 1) Reverse primer (R806): GGACTACHVGGGTWTCTAAT (SEQ ID NO: 2)

[0087] The PCR amplification products were then sequenced using a next-generation sequencer, MiSeq (Illumina), using the paired-end method. The sequence was analyzed using the bacterial flora analysis software Qiime2 to identify the classification at the genus level and determine the abundance ratio. The abundance ratio was calculated as the percentage of the number of reads for each bacterial species (genus) out of the total number of reads, and the average value was calculated for each group.

[0088] The abundance ratio of the genus Alistipes in this comprehensive analysis is shown in Figure 8. Of the sequences identified as the genus Alistipes (partial sequences of 16S rDNA), three species, Alistipes senegalensis, Alistipes putredinis, and Alistipes finegoldii, are shown in SEQ ID NOs: 3 to 5, respectively. [SEQ ID NO: 3] Partial sequence of 16S rDNA of Alistipes senegalensis TCAATGGACGGAAGTCTGAACCAGCCATGCCGTGCAGGAAGACGGCTCTATGAGTTGTAAACTGCTTTTGTACAAGGGTAAACCTGAATACGTGTATTCAGCTGAAAGTACTGTACGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATCCAAGCGTTATCCGGATTTATTGGGTTTAAAG GGTGCGTAGGCGGATTGATAAGTTAGGGTGAAATGTCCGAGCTTAACTCGGGAACTGCCTCTAATACTGTTGATCTAGAGAGTAGATGCGGTAGGCGGAATGTATGGTGTAGCGGTGAAATGCTTAGAGATCATACAGAACACCGATTGCGAAGGCAGCTTACCAATCTATATCTGACGTTGAGGCACGAAAGCGTGGG

[0089] [SEQ ID NO: 4] Partial sequence of 16S rDNA of Alistipes putredinis TCAATGGACGCAAGTCTGAACCAGCCATGCCGCGTGCAGGAAGACGGCTCTATGAGTTGTAAACTGCTTTTGTACGAGGGTAAACGCAGATACGTGTATCTGTCTGAAAGTATCGTACGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATTCAAGCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGCGGTTGGATAAGTTAGAGGTGAAATCCCGGGGCTCAACTCCGGAATTGCCTCTGATACTGTTCGGCTAGAGATCAGTTGCGGTAGGCGGAATGTATGGTGTAGCGGTGAAATGCTTAGAGATCATACAGAACACCGATTGCGAAGGCAGCTTACCAAGCTGAATCTGACGTTGAGGCACGAAAGCGTGGG

[0090] [SEQ ID NO: 5] Partial sequence of 16S rDNA of Alistipes finegoldii TCAATGGACGGAAGTCTGAACCAGCCATGCCGCGTGCAGGAAGACGGCTCTATGAGTTGTAAACTGCTTTTGTACAAGGGTAAACCTGAATACGTGTATTCAGCTGAAAGTACTGTACGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATCCAAGCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGCGGATTGATAAGTTAGAGGTGAAATGTCCGAGCTCAACTCGGGAACTGCCTCTAATACTGTTGATCTAGAGAGTAGATGCGGTAGGCGGAATGTATGGTGTAGCGGTGAAATGCTTAGAGATCATACAGAACACCGATTGCGAAGGCAGCTTACCAATCTATATCTGACGTTGAGGCACGAAAGCGTGGG

[0091] As shown in Figure 8, the abundance ratio of the genus Alistipes was higher in Groups A, B, and C compared to the control group. In particular, it was significantly (p<0.001) higher in Group A compared to the control group. This result demonstrated that silkworm powder can effectively increase the number of bacteria of the genus Alistipes in the intestines.

[0092] Next, the abundance ratio of the genus Romboutsia in this comprehensive analysis (median value in each group (N = 7)) is shown in Figure 9. In addition, among the sequences identified as the genus Romboutsia (partial sequences of 16S rDNA), those of Romboutsia timonensis are shown in SEQ ID NOs: 6 to 7. [SEQ ID NO: 6] Partial sequence of 16S rDNA of Romboutsia timonensis ACAATGGGCGAAAGCCTGATGCAGCAACGCCGCGTGAGCGATGAAGGCCTTCGGGTCGTAAAGCTCTGTCCTCAAGGAAGATAATGACGGTACTTGAGGAGGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCTAGCGTTATCCGGAATTACTGGGCGTAAAGGGTGCGTAG GTGGTTTCTTAAGTCAGAGGTGAAAGGCTACGGCTCAACCGTAGTAAGCCTTTGAAACTGGGAAACTTGAGTGCAGGAGAGGAGAGTGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTTGCGAAGGCGGCTCTCTGGACTGTAACTGACACTGAGGCACGAAAGCGTGGG

[0093] [SEQ ID NO: 7] Partial sequence of 16S rDNA of Romboutsia timonensis ACAATGGGCGAAAGCCTGATGCAGCAACGCCGCGTGAGCGATGAAGGCCTTCGGGTCGTAAAGCTCTGTCCTCAAGGAAGATAATGACGGTACTTGAGGAGGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCTAGCGTTATCCGGAATTACTGGGCGTAAAGGGTGCGTAG GTGGTTTCTTAAGTCAGAGGTGAAAGGCTACGGCTCAACCGTAGTAAGCCTTTGAAACTGGGGAACTTGAGTGCAGGAGAGGAGAGTGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTTGCGAAGGCGGCTCTCTGGACTGTAACTGACACTGAGGCACGAAAGCGTGGG

[0094] As shown in Figure 9, the abundance ratio of Rhombosia was significantly lower in Groups A, B, and C compared to the control group. In particular, it was significantly (p<0.01) lower in Group A compared to the control group. This result demonstrates that silkworm powder can effectively suppress the number of Rhombosia bacteria in the intestines.

[0095] Finally, the abundance ratio of the genus Hydrogenoanaerobacterium in this comprehensive analysis is shown in Figure 10. Furthermore, among the sequences identified as the genus Hydrogenoanaerobacterium (partial sequences of 16S rDNA), that of Hydrogenoanaerobacterium saccharovorans is shown in SEQ ID NO:8. [SEQ ID NO: 8] Partial sequence of 16S rDNA of Hydrogenoanaerobacterium saccharovorans ACAATGGGCGGAAGCCTGATGCAGCGACGCCGCGTGAGGGAAGACGGTCTTCGGATTGTAAACCTCTGTCATCGGGGACGATAATGACGGTACCCGAGAAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCGAGCGTTGTCCGGAATTACTGGGTGTAAAGGGAGCGTAG GCGGGAATGCAAGTTGGATGTCAAATCTATCGGCTTAACCGGTAATCGCATTCAAAACTGCATTTCTTGAGTGAAGTAGAGGTTGGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCCAACTGGGCTTTTACTGACGCTGAGGCTCGAAAGCGTGGG

[0096] 10, the abundance ratio of the genus Hydrogenoanaerobacterium was significantly (p<0.001) higher only in Group A compared to the control group. This result demonstrated that Silkworm Powder A can effectively increase the number of bacteria of the genus Hydrogenoanaerobacterium in the intestines.

[0097] It has been reported that when mice given antibiotics ingested a suspension of Hydrogenoanaerobacterium saccharovorans, the weight of the tibialis anterior muscle located in the front of the calf significantly increased (Xie, C. et al., (2022). Anim Nutr 9, 84-99. 10.1016 / j.aninu.2021.10.010.). This suggests that the Hydrogenoanaerobacterium genus may contribute to increasing muscle mass. Therefore, considering the results of Example 3(2) and Example 3(4), it has been revealed that Silkworm Powder A can be expected to have a high effect in increasing muscle mass.

Claims

1. A composition for increasing intestinal butyric acid, containing silkworm powder as an active ingredient.

2. A composition for increasing muscle mass, containing silkworm powder as an active ingredient.

3. A composition for preventing or improving obesity, containing silkworm powder as an active ingredient.

4. A composition for increasing the number of Alistipes bacteria, containing silkworm powder as an active ingredient.

5. A composition for suppressing the number of Romboutsia bacteria, containing silkworm powder as an active ingredient.

6. The silkworm powder has a wave number of (1635±9) cm in the infrared absorption spectrum of FT-IR. －１ Region and wave number (1537±20) cm －１ The composition according to any one of claims 1 to 5, which has at least one absorption peak in each of the regions.

7. The composition according to any one of claims 1 to 5, wherein the silkworm powder has trehalase activity.

8. The composition according to any one of claims 1 to 5, wherein the silkworm powder has not been subjected to heat treatment at 90°C or higher.

9. The composition according to claim 1, which is used to prevent and / or ameliorate a disease or unhealthy condition selected from obesity, muscle atrophy, intestinal inflammation, rheumatoid arthritis, graft-versus-host disease, fatty liver and colon cancer.

10. The composition according to claim 4, which is used to prevent and / or improve a disease selected from enteritis, liver cirrhosis, hepatic encephalopathy, non-alcoholic steatohepatitis, hepatocellular carcinoma, atrial fibrillation, heart failure, atherosclerotic cardiovascular disease and autism spectrum disorder.

11. A method for increasing intestinal butyric acid, comprising the step of increasing intestinal butyric acid in a human or animal by administering silkworm powder to the human or animal.

12. A method for increasing muscle mass, comprising the step of increasing the muscle mass of a human or animal by administering silkworm powder to the human or animal.

13. A method for increasing the number of Alistipes bacteria in the human or animal body, comprising the step of increasing the number of Alistipes bacteria in the human or animal body by administering silkworm powder to the human or animal.

14. A method for suppressing the number of Romboutsia bacteria in the human or animal body, comprising the step of suppressing the number of Romboutsia bacteria in the human or animal body by administering silkworm powder to the human or animal.

15. The method according to claim 11, claim 13 or claim 14, which is a method for preventing or ameliorating obesity.

16. The method according to claim 11, which is a method for preventing or ameliorating a disease or unhealthy condition selected from obesity, muscle atrophy, intestinal inflammation, rheumatoid arthritis, graft-versus-host disease, fatty liver, and colon cancer.

17. The method according to claim 13, which is a method for preventing or ameliorating a disease selected from enteritis, liver cirrhosis, hepatic encephalopathy, non-alcoholic steatohepatitis, hepatocellular carcinoma, atrial fibrillation, heart failure, atherosclerotic cardiovascular disease and autism spectrum disorder.

18. Use of silkworm powder for producing a composition according to any one of claims 1 to 10.

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