Bioactive substances, epidermal metabolism stimulants, lipid accumulation inhibitors, limonocyte inhibitors, adenopectin production stimulants, functional foods, cosmetic products, and methods for the production of bioactive substances.

Abstract

The biological function regulating agent consists of: Includes fermented collagen peptides and fermented collagen peptides with added extracts. At least one effect selected from a group consisting of metabolic stimulating agents. Actions affecting the epidermis, inhibiting fat accumulation, and stimulating fat breakdown. And its mechanism of action in biologically regulating the amount of adipoxytokines in the body;

Description

Biofunction regulator, epidermal metabolism promoter, fat accumulation inhibitor, lipolysis promoter, adiponectin production promoter, functional food, cosmetic, and method for producing biofunction regulator

[0001] The present invention relates to a biological function regulator, an epidermal metabolism promoter, a fat accumulation inhibitor, a lipolysis promoter, an adiponectin production promoter, a functional food, a cosmetic, and a method for producing a biological function regulator.

[0002] International Publication No. 2017 / 014149 (Patent Document 1), a paper by Woo et al. (Non-Patent Document 1), and a publication by Kobayashi et al. (Non-Patent Document 2) report that collagen peptides have anti-obesity effects. JP 2018-023326 A (Patent Document 2) discloses that collagen peptides were obtained by fermenting collagen with lactic acid bacteria.

[0003] International Publication No. 2017 / 014149 Japanese Patent Application Laid-Open No. 2018-023326

[0004] M Woo et al., "Anti-Obesity Effects of Collagen Peptide Derived from Skate (Raja kenojei) Skin Through Regulation of Lipid Metabolism", Marine Drugs, 2018, Vol.16(9), 306; Kobayashi et al., "Changes in Liver Gene Expression and Anti-Obesity Effects of Oral Intake of Salmon Skin Collagen Peptide", Proceedings of the 2012 Annual Meeting of the Japanese Society for Food Science and Technology, August 29, 2012, Vol. 59th Page. 97

[0005] The above Patent Document 2 does not disclose obtaining collagen peptides by fermenting any of collagen, gelatin, and gelatin hydrolysates with koji. Furthermore, the collagen peptides reported to have anti-obesity effects in the above Patent Document 1, Non-Patent Document 1, and Non-Patent Document 2 are not collagen peptides obtained by fermenting any of collagen, gelatin, and gelatin hydrolysates with koji (hereinafter also referred to as "fermented collagen peptides"). In other words, the effects of fermented collagen peptides on the body have not yet been elucidated.

[0006] In view of the above circumstances, the present invention aims to provide a biological function regulator, an epidermal metabolism promoter, a fat accumulation inhibitor, a fat decomposition promoter, an adiponectin production promoter, a functional food, a cosmetic, and a method for producing a biological function regulator, which contain a fermented collagen peptide having at least one effect selected from the group consisting of an epidermal metabolism promoting effect, a fat accumulation inhibiting effect, a fat decomposition promoting effect, and an effect of regulating the amount of adipocytokines in the body.

[0007] The present inventors have discovered that fermented collagen peptides obtained by fermenting collagen-containing raw materials such as the hide, skin, bones, cartilage, and tendons of tetrapods, and the bones, skin, and scales of fish, or collagen, gelatin, or gelatin hydrolysates with koji, have at least one effect selected from the group consisting of the effect of promoting epidermal metabolism, the effect of inhibiting fat accumulation, the effect of promoting lipolysis, and the effect of regulating the amount of adipocytokines in the body, and have completed the present invention.

[0008] That is, the present invention has the following characteristics. [1] The biofunction regulator according to the present invention comprises a fermented collagen peptide, and the fermented collagen peptide has at least one effect selected from the group consisting of an epidermal metabolism promoting effect, an adipose accumulation inhibiting effect, an adipocytokine degradation promoting effect, and an adipocytokine level regulating effect in the body. [2] The fermented collagen peptide preferably comprises a collagen peptide and at least one first compound selected from the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methional. [3] The fermented collagen peptide preferably comprises a collagen peptide and at least three first compounds selected from the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methional. [4] The epidermal metabolism enhancer according to the present invention comprises the biofunction regulator described above. [5] The fat accumulation inhibitor according to the present invention comprises the biofunction regulator described above. [6] The adiponectin production enhancer according to the present invention comprises the biofunction regulator described above. [7] The lipolysis promoter according to the present invention comprises the biofunction regulator described above. [8] The functional food according to the present invention comprises the biofunction regulator described above. [9] The cosmetic according to the present invention comprises the biofunction regulator described above.

[10] The method for producing a biofunction regulator according to the present invention is a method for producing a biofunction regulator containing a fermented collagen peptide, comprising the steps of preparing koji containing koji mold and a collagen raw material, and fermenting the collagen raw material with the koji to obtain the biofunction regulator containing the fermented collagen peptide, wherein the koji mold is a fungus species belonging to the genus Aspergillus, and the collagen raw material is at least one species selected from the group consisting of the following groups 1 to 6, collagen extracted from at least one species selected from the above groups, gelatin obtained by processing the collagen, and a gelatin degradation product obtained by hydrolyzing the gelatin.Group 1: A group consisting of cow hide, skin, bones, cartilage and tendons Group 2: A group consisting of pig hide, skin, bones, cartilage and tendons Group 3: A group consisting of sheep hide, skin, bones, cartilage and tendons Group 4: A group consisting of chicken hide, skin, bones, cartilage and tendons Group 5: A group consisting of ostrich hide, skin, bones, cartilage and tendons Group 6: A group consisting of fish bones, skin and scales

[11] The biological function regulator according to the present invention contains fermented collagen peptides produced by fermenting a collagen raw material with koji.

[0009] According to the above, it is possible to provide a biological function regulator, an epidermal metabolism promoter, a fat accumulation inhibitor, a lipolysis promoter, an adiponectin production promoter, a functional food, a cosmetic, and a method for producing a biological function regulator, which contain a fermented collagen peptide having at least one effect selected from the group consisting of an epidermal metabolism promoting effect, a fat accumulation inhibiting effect, a fat decomposition promoting effect, and an effect of regulating the amount of adipocytokine in the body.

[0010] FIG. 1 is a graph showing the change in body weight of mice in each group in the first study. FIG. 2 is a graph showing the mesenteric fat mass of mice in each group in the first study. FIG. 3 is a graph showing the perirenal fat mass of mice in each group in the first study. FIG. 4 is a graph showing the peritellar fat mass of mice in each group in the first study. FIG. 5 is a graph showing the total visceral fat mass of mice in each group in the first study. FIG. 6 is a graph showing the leptin concentration in the serum of mice in each group in the first study. FIG. 7 is a graph showing the adiponectin concentration in the serum of mice in each group in the first study. FIG. 8 is a graph showing the intensity of FAS activity in the liver of mice in each group in the first study. FIG. 9 is a graph showing the intensity of CPT activity in the liver of mice in each group in the first study. FIG. 10 is a graph showing the leptin concentration in the serum of mice in each group in the second study. FIG. 11 is a graph showing the adiponectin concentration in the serum of mice in each group in Study 2. FIG. 12 is a graph showing the change in body weight of mice in each group in Study 7. FIG. 13 is a graph showing the body weight of mice in each group in Study 8. FIG. 14 is a graph showing the mesenteric fat mass of mice in each group in Study 8. FIG. 15 is a graph showing the perirenal fat mass of mice in each group in Study 8. FIG. 16 is a graph showing the peritellar fat mass of mice in each group in Study 8. FIG. 17 is a graph showing the total visceral fat mass of mice in each group in Study 8. FIG. 18 is a graph showing the blood glucose levels in the serum of mice in each group in Study 8. FIG. 19 is a graph showing the insulin levels in the serum of mice in each group in Study 8. FIG. 20 is a graph showing the leptin concentration in the serum of mice in each group in Study 8. Figure 21 is a graph showing the intensity of FAS activity in the liver of mice in each group in Test 8. Figure 22 is a graph showing the intensity of CPT activity in the liver of mice in each group in Test 8.

[0011] Hereinafter, an embodiment of the present invention (hereinafter also referred to as "the present embodiment") will be described in further detail. Herein, in this specification, an expression in the form of "A to B" means the upper and lower limits of a range (i.e., A or more and B or less), and when no unit is specified for A and a unit is specified only for B, the unit of A and the unit of B are the same.

[0012] As used herein, "biomodulators," "epidermal metabolism promoters," "fat accumulation inhibitors," "lipolysis promoters," "adiponectin production promoters," and "fermented collagen peptides" may be in a solid form such as a powder, or in a liquid form such as an aqueous solution dissolved in water. Furthermore, as used herein, "fermented collagen peptide" refers to a peptide mixture obtained by fermenting a collagen raw material, described below, with koji. As used herein, "fermentation" refers to the overall process by which useful organic matter is produced from raw materials through the activity of the koji mold contained in koji, and is distinguished from "putrefaction," in which unhelpful organic matter is produced from raw materials through the activity of microorganisms.

[0013] In this specification, the term "gelatin" may be used to refer to the substance name, gelatin gel, and gelatin solution, respectively. Similarly to the above-mentioned gelatin, the term "collagen peptide" may be used to refer to the substance name and collagen peptide solution, respectively.

[0014] In this specification, the term "collagen raw material" may refer collectively to at least one type selected from the following Groups 1 to 6: "collagen itself," "collagen" extracted from at least one type selected from the following Groups 1 to 6, "gelatin" obtained by treating the above collagen using known methods such as hot water extraction, and "gelatin hydrolyzate" obtained by hydrolyzing the above gelatin. Furthermore, the "hydrolysis" of gelatin includes hydrolysis using an acid, a base, an enzyme, and heat. Group 1: A group consisting of cowhide, skin, bones, cartilage, and tendons; Group 2: A group consisting of pig skin, skin, bones, cartilage, and tendons; Group 3: A group consisting of sheep skin, skin, bones, cartilage, and tendons; Group 4: A group consisting of chicken skin, skin, bones, cartilage, and tendons; Group 5: A group consisting of ostrich skin, skin, bones, cartilage, and tendons; and Group 6: A group consisting of fish bones, skin, and scales.

[0015] [Biological function regulator] The biological function regulator according to this embodiment contains a fermented collagen peptide. The fermented collagen peptide has at least one effect selected from the group consisting of an epidermal metabolism promoting effect, a fat accumulation inhibiting effect, a lipolysis promoting effect, and an effect of regulating the amount of adipocytokines in the body. A biological function regulator having such characteristics can exert at least one effect on the body selected from the group consisting of an epidermal metabolism promoting effect, a fat accumulation inhibiting effect, a lipolysis promoting effect, and an effect of regulating the amount of adipocytokines in the body (hereinafter also referred to as a "biological function regulating effect").

[0016] <Fermented Collagen Peptide> The biofunction regulator of this embodiment contains a fermented collagen peptide, as described above. The fermented collagen peptide preferably contains a collagen peptide and at least one first compound selected from the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methional. More preferably, the fermented collagen peptide contains a collagen peptide and at least three first compounds selected from the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methional. This allows the biofunction regulator to more fully exert at least one biofunction regulator selected from the group consisting of epidermal metabolism promotion, fat accumulation inhibition, lipolysis promotion, and regulation of adipocytokine levels in the body. Furthermore, as described below, the biofunction regulator of this embodiment is produced by fermenting a collagen raw material with koji. That is, the biofunction regulator of this embodiment is a biofunction regulator containing a fermented collagen peptide.

[0017] The fermented collagen peptide has an odor (so-called collagen odor) that is characteristic of conventional collagen peptides, which is suppressed by the first compound. Therefore, the biological function regulator can be used in, for example, epidermal metabolism promoters, fat accumulation inhibitors, lipolysis promoters, adiponectin production promoters, functional foods, and cosmetics, as described below, with simplified or no deodorizing treatment.

[0018] (Collagen Peptide) The fermented collagen peptide preferably contains a collagen peptide as described above. This collagen peptide appears to be the same as a conventionally known collagen peptide. That is, the collagen peptide contained in the fermented collagen peptide appears to be a peptide mixture and may contain various peptides such as dipeptides, tripeptides, oligopeptides, and polypeptides obtained by subjecting collagen or gelatin to conventionally known treatments. However, as described above, the fermented collagen peptide is obtained by fermenting a collagen raw material with koji. Therefore, the collagen peptide contained in the fermented collagen peptide is obtained by fermenting a collagen raw material with koji and is produced together with the first compound described below.

[0019] <Weight-average molecular weight> The collagen peptide contained in the fermented collagen peptide preferably has a weight-average molecular weight of 20,000 or less. When the weight-average molecular weight of the collagen peptide is 20,000 or less, the biofunction regulator can be easily applied to applications such as epidermal metabolism promoters, fat accumulation inhibitors, lipolysis promoters, adiponectin production promoters, functional foods, and cosmetics without additional processing. The weight-average molecular weight of the collagen peptide is more preferably 10,000 or less, and even more preferably 6,000 or less. The lower limit of the weight-average molecular weight of the collagen peptide is 76. When the weight-average molecular weight of the collagen peptide is within the above-mentioned range, the biofunction regulator can fully exert the above-mentioned effects on the living body when used as an epidermal metabolism promoter, fat accumulation inhibitor, lipolysis promoter, adiponectin production promoter, functional food, or cosmetic.

[0020] The weight-average molecular weight of the collagen peptide contained in the biofunction regulating agent can be determined by performing size exclusion chromatography (SEC) under the following measurement conditions. The inventors have confirmed that this measurement method is also valid for measuring molecular weights exceeding 12,000. Equipment: High-performance liquid chromatography (HPLC) (manufactured by Tosoh Corporation) Column: TSKGel (registered trademark) G2000SW XL Column temperature: 40°C Eluent: 45% by mass acetonitrile (containing 0.1% by mass TFA) Flow rate: 1.0 mL / min Injection volume: 10 μL Detection: UV 214 nm Molecular weight markers: The following five types were used: Cytochrom C Mw: 12,000 Aprotinin Mw: 6,500 Bacitracin Mw: 1,450 Gly-Gly-Tyr-Arg Mw: 451 Gly-Gly-Gly Mw: 189.

[0021] (First Compound) As described above, the fermented collagen peptide preferably contains at least one first compound selected from the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methional, and more preferably contains at least three of the first compounds. In the fermented collagen peptide, the first compound is thought to be produced together with the collagen peptide by fermenting the collagen raw material with koji. The first compound can function as a marker indicating that the fermented collagen peptide was obtained by fermenting the collagen raw material with koji.

[0022] The fermented collagen peptide may include as the first compound any one of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methional. The fermented collagen peptide may include as the first compound isovaleraldehyde and 1-octen-3-ol, may include isovaleraldehyde and phenylacetaldehyde, may include isovaleraldehyde and methional, may include 1-octen-3-ol and phenylacetaldehyde, may include 1-octen-3-ol and methional, or may include phenylacetaldehyde and methional.

[0023] The fermented collagen peptide may contain isovaleraldehyde, 1-octen-3-ol, and phenylacetaldehyde as the first compound; it may contain isovaleraldehyde, 1-octen-3-ol, and methional; it may contain isovaleraldehyde, phenylacetaldehyde, and methional; or it may contain 1-octen-3-ol, phenylacetaldehyde, and methional. The fermented collagen peptide may contain four types of first compounds (isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methional). In these cases, the biological function regulator can more fully exert at least one effect selected from the group consisting of an epidermal metabolism promoting effect, an adipose accumulation inhibiting effect, an lipolysis promoting effect, and an effect of regulating the amount of adipocytokines in the body.

[0024] (Isovaleraldehyde) Isovaleraldehyde is a compound that is also called isovaleric aldehyde, 3-methylbutanal, or 3-methylbutyraldehyde, and is a compound that has conventionally been used as a flavoring agent (food additive) and the like.

[0025] (1-Octen-3-ol) 1-Octen-3-ol is a type of unsaturated alcohol, and is a compound that has been known to be a component that contributes to the aroma of matsutake mushrooms.

[0026] (Phenylacetaldehyde) Phenylacetaldehyde is a type of aromatic aldehyde, and is a compound that has been conventionally used as a raw material for blending fragrances and flavors.

[0027] (Methional) Methional is a type of organic sulfur compound, also known as 3-methylthio-1-propanol. Methional has long been known to be contained in soy sauce. Furthermore, methional is known to have the effect of reducing the fishy odor of meat and fish.

[0028] (Content) The first compound is preferably contained in the biofunction regulator in a total amount (total of at least one or more compounds) of 0.05 ppm or more. That is, the biofunction regulator preferably contains the first compound in an amount of 0.05 ppm or more. Furthermore, the first compound is more preferably contained in the biofunction regulator in a total amount of 0.4 ppm or more. That is, the biofunction regulator more preferably contains the first compound in an amount of 0.4 ppm or more. The lower limit of the content of the first compound in the biofunction regulator is not particularly limited, but it is preferable that the total amount of the first compound is 0.01 ppm or more. The upper limit of the content of the first compound is also not particularly limited, but it is preferable that the total amount of the first compound is 5 ppm or less.

[0029] The quality and quantity of the first compound contained in the biofunction regulator can be determined by the following procedure. First, a dry powder of the biofunction regulator is obtained using the manufacturing method described below. A measurement sample is then obtained by dissolving 0.5 g of the dry powder in 4.5 mL of RO water. The measurement sample is then loaded into a gas chromatograph mass spectrometer (trade name: "7890A GC System," manufactured by Agilent Technologies, Inc., and trade name: "JMS-Q1050GC," manufactured by JEOL Ltd.), vaporized, and then transferred to a column equipped with the analyzer using ultra-high purity helium as a carrier gas, thereby separating the components contained in the measurement sample into individual compounds. The compounds are then detected with a detector equipped with the analyzer, and the data (spectral data) obtained from the detector is compared with standard data to qualitatively determine the first compound. Additionally, the first compound can be quantified based on the spectral data (peak area) obtained from the detector.

[0030] [Epidermal metabolism promoter] The epidermal metabolism promoter according to this embodiment includes the biofunction regulator. The epidermal metabolism promoter, due to its epidermal metabolism-promoting effect, can promote the excretion of melanin granules from the skin surface (in the epidermis) without stagnation as age spots, freckles, etc. Specifically, the epidermal metabolism promoter can enhance the expression level of at least one gene selected from the group consisting of transglutaminase 1 (TGM1), involucrin (Ivl), and keratin 10 (KRT10). These genes are known to contribute to the maturation and differentiation of each layer constituting the epidermis (stratum corneum, stratum granulosum, stratum spinosum, and stratum basale). Therefore, enhanced expression of the aforementioned genes is thought to promote metabolism in the epidermis (so-called turnover). Furthermore, enhanced metabolism in the epidermis may provide skin moisturizing effects, wrinkle prevention and / or wrinkle reduction effects, etc.

[0031] The concentration of the biofunction regulator in the epidermal metabolism enhancer may be 0.01 to 100% by mass. Since the content of the first compound is very small, the concentration of the biofunction regulator in the epidermal metabolism enhancer refers to the concentration of collagen peptide in the biofunction regulator. Therefore, the concentration of the biofunction regulator in the epidermal metabolism enhancer can be determined by a conventionally known method for measuring collagen peptide concentration. For example, the concentration of the biofunction regulator in the epidermal metabolism enhancer can be determined by measuring the mass percentage of hydroxyproline in the collagen peptide using the chloramine T method. Furthermore, it can also be determined by measuring the mass percentage of hydroxyproline in the collagen peptide using an amino acid analyzer.

[0032] [Fat Accumulation Inhibitor] The fat accumulation inhibitor according to this embodiment includes the biofunction regulator. The fat accumulation inhibitor can achieve an effect of inhibiting the accumulation of fat (so-called visceral fat) in the liver, intestinal tract, kidneys, testes, etc., due to the fat accumulation inhibitory effect of the biofunction regulator. Furthermore, the fat accumulation inhibitor can achieve an effect of inhibiting fat accumulation through the effect of reducing the blood concentration of leptin, known as an appetite hormone, and the effect of increasing the blood concentration of adiponectin, which is known to be inversely correlated with the amount of visceral fat in the body, based on the effect of the biofunction regulator to regulate the amount of adipocytokines in the body. The leptin and adiponectin are known as proteins classified as adipocytokines secreted from adipocytes. That is, the adiponectin production promoter according to this embodiment includes the biofunction regulator.

[0033] The concentration of the biofunction regulator in the fat accumulation inhibitor may be 0.01 to 100% by mass. The concentration of the biofunction regulator in the adiponectin production enhancer may also be 0.01 to 100% by mass. The concentration of the biofunction regulator in the fat accumulation inhibitor and adiponectin production enhancer refers to the concentration of collagen peptide in the biofunction regulator, since the content of the first compound is very small. Therefore, the concentration of the biofunction regulator in the fat accumulation inhibitor and adiponectin production enhancer can be measured using the same method as the concentration of the biofunction regulator in the epidermal metabolism enhancer described above.

[0034] [Lipolysis Promoter] The lipolysis promoter according to this embodiment includes the biofunction regulator. The lipolysis promoter can achieve the effect of promoting the decomposition of fat (so-called visceral fat) in the liver, intestinal tract, kidneys, testes, etc., due to the lipolysis-promoting activity of the biofunction regulator. The concentration of the biofunction regulator in the lipolysis promoter may be 0.01 to 100% by mass. Since the content of the first compound is negligible, the concentration of the biofunction regulator in the lipolysis promoter refers to the concentration of collagen peptide in the biofunction regulator. Therefore, the concentration of the biofunction regulator in the lipolysis promoter can be measured using the same method as the concentration of the biofunction regulator in the epidermal metabolism promoter described above.

[0035] <Method of Use and Dosage> The epidermal metabolism promoter, fat accumulation inhibitor, lipolysis promoter, and adiponectin production promoter described above can be administered orally or parenterally in various forms as supplements, pharmaceuticals, or quasi-drugs. When administered orally, the forms can be, for example, tablets, granules, capsules, powders, liquids, suspensions, emulsions, pastes, and the like. Furthermore, the above-mentioned dosage forms can also be mixed with functional foods, which will be described later.

[0036] When administered parenterally, the above-mentioned epidermal metabolism enhancers, fat accumulation inhibitors, lipolysis enhancers, and adiponectin production enhancers can be in the form of, for example, an infusion, an injection, a transdermal preparation (an application preparation, a patch, and an aerosol), a suppository, a nasal drop, an inhalant, etc. Preferred dosage forms of the above-mentioned epidermal metabolism enhancers, fat accumulation inhibitors, lipolysis enhancers, and adiponectin production enhancers include tablets, granules, capsules, powders, liquids, and transdermal preparations.

[0037] The dosages of the epidermal metabolism enhancer, fat accumulation inhibitor, lipolysis enhancer, and adiponectin production enhancer vary depending on the subject's age, sex, weight, sensitivity, administration method, administration interval, type of formulation, etc. When the epidermal metabolism enhancer, fat accumulation inhibitor, lipolysis enhancer, and adiponectin production enhancer are orally administered, the respective dosages are preferably 0.0001 to 2500 mg / kg, and more preferably 0.0001 to 500 mg / kg, per day for an adult. When the epidermal metabolism enhancer, fat accumulation inhibitor, lipolysis enhancer, and adiponectin production enhancer are in the dosage form of, for example, a tablet, the tablet contains 0.001 to 80% by mass of the epidermal metabolism enhancer, fat accumulation inhibitor, lipolysis enhancer, or adiponectin production enhancer per tablet. When the epidermal metabolism enhancer, fat accumulation inhibitor, lipolysis enhancer, or adiponectin production enhancer is in the dosage form of, for example, a powder, the powder contains 0.001 to 100% by mass of the epidermal metabolism enhancer, fat accumulation inhibitor, lipolysis enhancer, or adiponectin production enhancer. The dosage of the epidermal metabolism enhancer, fat accumulation inhibitor, lipolysis enhancer, and adiponectin production enhancer can be determined appropriately when administered parenterally or orally, taking into account the dosage when administered orally. The epidermal metabolism enhancer, fat accumulation inhibitor, lipolysis enhancer, and adiponectin production enhancer can be administered once or several times a day, or can be administered once every one to several days.

[0038] The above-mentioned epidermal metabolism promoter, fat accumulation inhibitor, lipolysis promoter and adiponectin production promoter may contain other active ingredients, carriers for formulations, etc. as appropriate, within the scope that does not adversely affect the effects of the present invention. Other active ingredients include, for example, (±)-α-tocopherol phosphate disodium salt, heparinoids, allantoin, glycyrrhizic acid, glycyrrhetin, D-amino acids, aminosilane compounds, tiliroside, yuzu extract, α-glucosyl hesperidin, mulberry leaf extract, mangosteen peel extract, α-mangosteen, γ-mangosteen, cacao seed extract, cacao husk extract, ginseng extract, lychee polyphenols, winged bean (Psophocarpus) extract, Peucedanum japonicum (Choumeisou) extract, lactic acid bacteria, glabridin, eyebright extract, dried kudzu flower extract, water shield extract, giant chanterelle extract, crocetin extract, N-acetylglucosamine, anserine, and raspberry ketone. Further, pharmaceutically acceptable carriers used in formulating pharmaceutical preparations include diluents, binders (syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone), excipients (lactose, sucrose, corn starch, potassium phosphate, sorbitol, glycine), lubricants (magnesium stearate, talc, polyethylene glycol, silica), disintegrants (potato starch), and wetting agents (sodium lauryl sulfate).

[0039] [Functional Food] The functional food according to this embodiment includes the biofunction regulator. Examples of such functional foods include foods for specified health uses and foods with functional claims. The functional food, for example, as a food for specified health uses or a food with functional claims, can exhibit at least one effect selected from the group consisting of the epidermal metabolism promoting effect, the fat accumulation inhibiting effect, the lipolysis promoting effect, and the regulating effect of the biofunction regulator in the body. The concentration of the biofunction regulator in the food for specified health uses or a food with functional claims can be, for example, 0.01 to 100% by mass. Since the content of the first compound is negligible, the concentration of the biofunction regulator in the functional food refers to the concentration of collagen peptide in the biofunction regulator. Therefore, the concentration of the biofunction regulator in the functional food can be measured using the same method as that used for measuring the concentration of the biofunction regulator in the epidermal metabolism promoter described above.

[0040] [Cosmetics] The cosmetic according to this embodiment contains the biofunction regulator. The cosmetic can be provided as a cosmetic having, for example, a whitening effect, a moisturizing effect, a wrinkle prevention effect, and / or a wrinkle reduction effect, based on the epidermal metabolism-promoting activity of the biofunction regulator. The concentration of the biofunction regulator in the cosmetic may be 0.01 to 100% by mass. Since the content of the first compound is very small, the concentration of the biofunction regulator in the cosmetic refers to the concentration of collagen peptide in the biofunction regulator. Therefore, the concentration of the biofunction regulator in the cosmetic can be measured using the same method as that used for measuring the concentration of the biofunction regulator in the epidermal metabolism promoter described above.

[0041] [Method for producing a biological function regulator] The method for producing a biological function regulator according to this embodiment is a method for producing a biological function regulator containing a fermented collagen peptide. The method for producing the biological function regulator includes a first step of preparing koji containing koji mold and a collagen raw material, and a second step of fermenting the collagen raw material with the koji to obtain the biological function regulator containing the fermented collagen peptide.

[0042] In the method for producing a biological function regulating agent, the koji mold is a fungus species belonging to the genus Aspergillus. The collagen raw material is at least one selected from the group consisting of the following groups 1 to 6, collagen extracted from at least one selected from the above groups, gelatin obtained by processing the collagen, and a gelatin degradation product obtained by hydrolyzing the gelatin. Group 1: A group consisting of cowhide, skin, bones, cartilage, and tendons. Group 2: A group consisting of pig skin, skin, bones, cartilage, and tendons. Group 3: A group consisting of sheep skin, skin, bones, cartilage, and tendons. Group 4: A group consisting of chicken skin, skin, bones, cartilage, and tendons. Group 5: A group consisting of ostrich skin, skin, bones, cartilage, and tendons. Group 6: A group consisting of fish bones, skin, and scales.

[0043] The method for producing a biological function regulator having such characteristics can produce a biological function regulator containing fermented collagen peptides that have at least one effect selected from the group consisting of the effect of promoting epidermal metabolism, the effect of inhibiting fat accumulation, the effect of promoting lipolysis, and the effect of regulating the amount of adipocytokines in the body.

[0044] The reason why the biological function regulator produced by the above production method has at least one effect selected from the group consisting of the effect of promoting epidermal metabolism, the effect of inhibiting fat accumulation, the effect of promoting lipolysis, and the effect of regulating the amount of adipocytokines in the body is not clear in detail, but is thought to be due to the following mechanism. That is, the above production method includes a step (step 2) of fermenting a collagen raw material with koji to obtain a biological function regulator containing fermented collagen peptides. The koji is known to contain a wide variety of enzymes produced by the proliferation of koji mold. Therefore, in step 2, the action of these wide variety of enzymes may decompose or oxidize or reduce polypeptides in the collagen raw material and carbohydrates in the koji.

[0045] Thus, in the second step, when the fermented collagen peptide is produced by the action of the various enzymes described above, it is presumed that the fermented collagen peptide contains collagen peptides containing dipeptides, tripeptides, oligopeptides, or polypeptides that have at least one of the physiological activities of promoting epidermal metabolism, inhibiting fat accumulation, promoting lipolysis, and regulating the amount of adipocytokines in the body. It is also presumed that when the fermented collagen peptide is produced by the action of the various enzymes described above, the fermented collagen peptide contains compounds (non-peptides) that have at least one of the physiological activities described above. Therefore, it is believed that the above production method can produce a biofunction regulator containing a fermented collagen peptide that has at least one activity selected from the group consisting of promoting epidermal metabolism, inhibiting fat accumulation, promoting lipolysis, and regulating the amount of adipocytokines in the body. Below, each step in the production method of a biofunction regulator according to this embodiment is described.

[0046] <First Step> The first step is a step of preparing koji containing koji mold and a collagen raw material. The first step is carried out for the purpose of preparing the materials required to produce the biological function regulating agent (koji containing koji mold and a collagen raw material).

[0047] (Collagen Raw Material) As described above, the collagen raw material may be at least one of the following: "collagen itself" selected from the group consisting of Groups 1 to 6 below; "collagen" extracted from at least one selected from the group consisting of Groups 1 to 6 below; "gelatin" obtained by treating the above collagen using a known method such as hot water extraction; and "gelatin hydrolyzate" obtained by hydrolyzing the above gelatin. Group 1: A group consisting of cow hide, skin, bone, cartilage, and tendon; Group 2: A group consisting of pig hide, skin, bone, cartilage, and tendon; Group 3: A group consisting of sheep hide, skin, bone, cartilage, and tendon; Group 4: A group consisting of chicken skin, skin, bone, cartilage, and tendon; Group 5: A group consisting of ostrich hide, skin, bone, cartilage, and tendon; and Group 6: A group consisting of fish bone, skin, and scale.

[0048] That is, in the first step, it is preferable that at least one type selected from the group consisting of the above Groups 1 to 6, and at least one type selected from the group consisting of the above collagen, the above gelatin, and the above gelatin degradation products be prepared as the collagen raw material. In the first step, one type of collagen raw material selected from these may be prepared, or two or more types of collagen raw materials may be prepared in combination. The group consisting of the above Groups 1 to 6, the above collagen, the above gelatin, and the above gelatin degradation products may all be prepared by conventionally known methods.

[0049] Here, it is more preferable that the gelatin be obtained by subjecting collagen extracted from at least one type selected from the group consisting of Groups 1 to 6 to a pretreatment using acid or alkali treatment, followed by hot water extraction, purification, and sterilization, in that order. This allows for the preparation of gelatin that is highly safe for the human body, etc., and therefore the biofunction regulator to be produced in this embodiment can be used for the aforementioned epidermal metabolism promoter, fat accumulation inhibitor, lipolysis promoter, adiponectin production promoter, functional food, and cosmetic products. Furthermore, such gelatin is also economical. The pretreatment using acid or alkali treatment, hot water extraction, purification, and sterilization can all be performed by conventionally known methods.

[0050] The gelatin degradation product can be obtained by subjecting the gelatin to any of the conventionally known methods of hydrolysis using an acid, a base, an enzyme, and heat. The weight-average molecular weight of the gelatin degradation product is not particularly limited, but is preferably 20,000 or less, and more preferably 10,000 or less. The lower limit of the weight-average molecular weight of the gelatin degradation product is 76. The weight-average molecular weight of the gelatin degradation product can be determined by the same measurement method as the weight-average molecular weight of the collagen peptide described above.

[0051] (Koji containing Aspergillus oryzae) As long as koji containing Aspergillus oryzae is selected that can achieve the effects of this embodiment by performing the second step described below, it can be prepared by a conventionally known method. That is, koji that will become seed koji is inoculated into grains such as rice, barley, wheat, or soybeans, and then allowed to grow in the rice, barley, wheat, or grain. The Aspergillus oryzae is preferably inoculated in an amount that corresponds to 0.01 to 1% by mass of the rice, barley, wheat, or grain. In this specification, "grains" includes not only soybeans, but also rice bran, bran, soybean lees, and defatted soybeans. When preparing koji containing Aspergillus oryzae, it is preferable to provide a koji room to prevent contamination by other bacteria, thereby creating an environment that is conducive to the growth of Aspergillus oryzae, and to carry out the necessary operations in the koji room.

[0052] The koji mold species is preferably a species belonging to the genus Aspergillus. It is even more preferable that the koji mold species is at least one species selected from the group consisting of Aspergillus sojae, Aspergillus oryzae, and Aspergillus luciensis. These species have been confirmed to be safe for the human body, and therefore the biofunction regulator produced in this embodiment can be easily applied to the aforementioned applications of epidermal metabolism promoters, fat accumulation inhibitors, lipolysis promoters, adiponectin production promoters, functional foods, and cosmetics. In the first step, koji containing one species selected from this group of fungal species may be prepared, or koji containing two or more species selected from the group of fungal species may be prepared.

[0053] <Second Step> The second step is a step of fermenting the collagen raw material with the koji to obtain a biofunction regulator containing the fermented collagen peptide. The second step is carried out for the purpose of obtaining the fermented collagen peptide contained in the biofunction regulator. In the second step, for example, the collagen raw material and the koji are placed in warm water and cultured in the warm water for a predetermined period of time to ferment the collagen raw material with the koji, thereby obtaining a biofunction regulator containing the fermented collagen peptide. The pH during culture is preferably 2 to 10, and more preferably 5 to 8. If the pH during culture is less than 2 or more than 10, there is a risk that the collagen raw material will be insufficiently reduced in molecular weight and the collagen odor will be insufficiently reduced.

[0054] Specifically, it is preferable to prepare a dispersion of 0.1 to 75% by mass of the collagen raw material, 0.1 to 20% by mass of the koji in terms of dry mass (dry weight), and 5 to 99.8% by mass of water, totaling 100% by mass, and then adjust the pH of the dispersion to 2 to 10, followed by culturing the dispersion for 1 to 24 hours while maintaining the temperature at 10 to 65° C. This allows for the first production of a fermented product containing fermented collagen peptides.

[0055] The fermented product can also be preferably obtained by the following method. Specifically, a dispersion is first prepared from 0.1 to 40% by dry mass (dry weight) of the koji and 60 to 99.9% by mass of water, totaling 100% by mass. The dispersion is then cultured for 1 to 24 hours while maintaining a temperature of 10 to 65°C, and then roughly filtered through a nylon mesh and filtered through diatomaceous earth and cellulose to obtain a koji extract. Next, a dispersion is prepared from 0.1 to 75% by mass of the collagen raw material and 0.1 to 99.9% by mass of the koji extract, totaling 100% by mass. The pH of the dispersion is then adjusted to 2 to 10, and the dispersion is then cultured for 1 to 24 hours while maintaining a temperature of 10 to 65°C. This method also allows for the production of a fermented product containing fermented collagen peptides. When a fermented product is obtained by this method, a biofunction regulator containing fermented collagen peptides can be obtained without subjecting the fermented product to the separation process described below. However, this does not exclude the possibility of subjecting the fermented product to at least one of a purification step and a deodorization step, which will be described later.

[0056] Here, the temperature of the hot water during cultivation is preferably 15 to 60° C., and more preferably 20 to 50° C. If the temperature of the hot water during cultivation is below 10° C. or above 65° C., the efficiency of fermentation by koji may decrease, and sufficient fermented collagen peptide may not be obtained.

[0057] Furthermore, the culture time is preferably 2 to 18 hours, and more preferably 4 to 8 hours. Culture times longer than 24 hours may be economically inefficient. Culture times shorter than 1 hour may result in insufficient fermentation by koji.

[0058] The content of the collagen raw material in the warm water during cultivation is preferably 10 to 45% by mass, more preferably 20 to 40% by mass. If the content of the collagen raw material in the warm water during cultivation is less than 0.1% by mass, economic efficiency may be low. If the content of the collagen raw material in the warm water during cultivation exceeds 75% by mass, the process may become inefficient.

[0059] The koji content in the dispersion liquid consisting of the koji, the collagen raw material, and water is preferably 1 to 15% by mass, more preferably 5 to 10% by mass, in terms of dry mass (dry weight). If the koji content in the warm water during cultivation is less than 0.1% by mass, in terms of dry mass (dry weight), fermentation by the koji may be insufficient. If the koji content in the warm water during cultivation exceeds 20% by mass, in terms of dry mass (dry weight), economic inefficiency may result. The koji content in the koji extract liquid is preferably 2 to 25% by mass, more preferably 8 to 16% by mass. If the koji extract liquid has a dry mass (dry weight) of less than 0.1% by mass, fermentation by the koji may be insufficient. If the koji content in the koji extract liquid has a dry mass (dry weight) of more than 40% by mass, economic inefficiency may result.

[0060] In the second step, after obtaining the fermented product through the above steps, the temperature can be raised to 75°C or higher depending on the purpose to inactivate the action (activity) of the koji mold, thereby halting the fermentation of the collagen raw material by the koji. Specifically, the weight-average molecular weight of the collagen peptide in the fermented product can be measured to confirm that it has a lower molecular weight than the collagen raw material, or after a predetermined culture time, for example 24 hours, the temperature of the fermented product can be raised to 75°C or higher to halt the fermentation of the collagen raw material by the koji. The weight-average molecular weight of the collagen peptide in the fermented product can be measured, for example, using the same method as that for measuring the weight-average molecular weight of collagen peptide described above.

[0061] (Other Steps) The second step preferably includes a separation step for separating and obtaining a biofunction regulator containing fermented collagen peptide from the fermented product. Conventional separation processes can be used for this separation step. For example, the fermented collagen peptide can be separated from the fermented product by separation processes such as coarse filtration using a nylon mesh, centrifugation, or filter filtration using commercially available filter paper. This preferably results in a biofunction regulator containing fermented collagen peptide, which contains collagen peptide and at least one, more preferably three, first compounds selected from the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methional. Since the fermented product contains fermented collagen peptide, the fermented product itself can also be considered a biofunction regulator.

[0062] Furthermore, the second step preferably includes a step (purification step) of purifying the biological function regulating agent or the fermented product obtained by applying the above-mentioned separation step in order to increase its transparency, etc. In this purification step, conventionally known purification treatments can be applied, such as a purification treatment using diatomaceous earth or a purification treatment by microfiltration. Furthermore, a deodorization treatment (deodorization step) can also be performed using activated carbon, etc.

[0063] The biofunction regulator obtained as described above can be stored as a solution. Furthermore, the biofunction regulator in solution form can be dried into a powder by conventional methods such as spraying or drum drying, and stored in that state. Furthermore, the dried powder of the biofunction regulator can be formulated into various dosage forms, as described above, by conventional formulation techniques.

[0064] <Action and Effect> From the above, the method for producing a biological function regulating agent according to this embodiment can obtain a biological function regulating agent that contains fermented collagen peptide and has at least one biological function regulating action selected from the group consisting of an epidermal metabolism promoting action, an fat accumulation inhibiting action, a lipolysis promoting action, and an action of regulating the amount of adipocytokines in the body, based on the fermented collagen peptide.

[0065] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these. In the following description, Samples 1 to 5 and Samples 41 to 49 are biological function regulators of the examples, and Samples 101 to 104 are collagen peptides or gelatins of comparative examples.

[0066] [Preparation of Samples] <Sample 1> (First Step) Koji containing koji mold and a collagen raw material were prepared according to the following procedure.

[0067] <Preparation of Koji Containing Koji Mold> Barley bran koji (manufactured by Higuchi Matsunosuke Shoten Co., Ltd.) inoculated with Aspergillus sojae was prepared as koji containing Koji mold.

[0068] <Preparation of Collagen Raw Material> Gelatin derived from pigskin (trade name: "BCN-HL", manufactured by Nitta Gelatin Co., Ltd., weight average molecular weight: approximately 65,000) was prepared as a collagen raw material.

[0069] (Step 2) The collagen raw material was fermented with the koji to obtain a fermented product containing fermented collagen peptides according to the following procedure. First, a dispersion consisting of 5 g of the collagen raw material, 1 g (dry weight) of the barley bran koji, and 50 mL of RO water was prepared, and the dispersion was cultured for 5 hours while maintaining the temperature at 40°C. The temperature of the dispersion was then increased to 75°C, and the dispersion was maintained at a temperature around 75°C for 10 minutes to inactivate the koji mold in the barley bran koji, thereby obtaining a fermented product containing fermented collagen peptides.

[0070] Next, the fermented product was filtered using ADVANTEC FILTER PAPER No. 2 (manufactured by Toyo Roshi Kaisha, Ltd.) to obtain Sample 1, a biological function regulating agent.

[0071] The biofunction regulator of Sample 1 was an aqueous solution, and measurement of its weight-average molecular weight confirmed that it was lower than the weight-average molecular weight of the collagen raw material. Furthermore, analysis using the gas chromatograph mass spectrometer confirmed that the biofunction regulator of Sample 1 contained isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methional as first compounds.

[0072] <Sample 2> In the second step, a dispersion consisting of 1 kg of the collagen raw material, 200 g (dry weight) of the barley bran koji, and 1500 mL of RO water was prepared, and the dispersion was cultured for 6 hours while maintaining the temperature at 40°C. The temperature of the dispersion was then increased to 70°C, and the dispersion was maintained at a temperature of around 70°C for 1 hour to inactivate the koji mold in the barley bran koji, thereby obtaining a fermented product containing fermented collagen peptides. The fermented product was then filtered using ADVANTEC FILTER PAPER No. 5 (manufactured by Toyo Roshi Kaisha, Ltd.) and further purified by diatomaceous earth filtration. The biofunction regulator of Sample 2 was obtained using the same method as for obtaining Sample 1, except for the above.

[0073] The biological function regulator of Sample 2 was dried into powder using a spray dryer (manufactured by Okawara Manufacturing Co., Ltd.).

[0074] The biofunction regulator of Sample 2 was a dry powder, and its weight-average molecular weight was measured and confirmed to be lower than that of the collagen raw material. Furthermore, analysis using the gas chromatograph mass spectrometer confirmed that the biofunction regulator of Sample 2 contained isovaleraldehyde, phenylacetaldehyde, and methional as the first compounds.

[0075] <Sample 3> (First Step) Koji containing koji mold and a collagen raw material were prepared according to the following procedure.

[0076] <Preparation of Koji Containing Koji Mold> Barley bran koji (manufactured by Higuchi Matsunosuke Shoten Co., Ltd.) inoculated with Aspergillus sojae was prepared as koji containing Koji mold.

[0077] <Preparation of Collagen Raw Material> Gelatin derived from pigskin (trade name: "BCN-HL", manufactured by Nitta Gelatin Co., Ltd., weight average molecular weight: approximately 65,000) was prepared as a collagen raw material.

[0078] (Step 2) The collagen raw material was fermented with the koji to obtain a fermented product containing fermented collagen peptides according to the following procedure. First, a dispersion consisting of 13% by mass (dry weight) of the barley bran koji and 87% by mass of RO water was prepared, and the dispersion was stirred for 1 hour while maintaining the temperature at 40°C. Subsequently, a koji extract was obtained by coarse filtration through a nylon mesh and filtration through diatomaceous earth and cellulose. Next, a dispersion consisting of 40% by mass of the collagen raw material and 60% by mass of the koji extract was prepared, and the dispersion was cultured for 6 hours while maintaining the temperature at 40°C. The temperature of the dispersion was then raised to 80°C, and the dispersion was maintained at a temperature around 80°C for 60 minutes to perform low-temperature sterilization. The dispersion was then dried and powdered using a spray dryer (manufactured by Okawara Manufacturing Co., Ltd.), thereby obtaining Sample 3, a biofunction regulator.

[0079] The biological function regulator of Sample 3 was a dry powder, and measurement of its weight-average molecular weight confirmed that it was lower than the weight-average molecular weight of the collagen raw material. Furthermore, analysis using the gas chromatograph mass spectrometer described above confirmed that the biological function regulator of Sample 3 contained isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methional as first compounds.

[0080] <Sample 4> A dispersion liquid consisting of 40% by mass of the collagen raw material and 60% by mass of the koji extract liquid was prepared, and after culturing for 6 hours while maintaining the temperature of the dispersion liquid at 40°C, the temperature of the dispersion liquid was increased to 60°C and the dispersion liquid was maintained at a temperature of around 60°C for 60 minutes for low-temperature sterilization, and the biological function regulator of Sample 4 was obtained in the same manner as Sample 3.

[0081] The biofunction regulator of Sample 4 was a dry powder, and measurement of its weight-average molecular weight confirmed that it was lower than the weight-average molecular weight of the collagen raw material. Furthermore, analysis using the gas chromatograph mass spectrometer described above confirmed that the biofunction regulator of Sample 4 contained isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methional as first compounds.

[0082] <Sample 5> (First Step) Koji containing koji mold and a collagen raw material were prepared according to the following procedure.

[0083] <Preparation of Koji Containing Koji Mold> Barley bran koji (manufactured by Higuchi Matsunosuke Shoten Co., Ltd.) inoculated with Aspergillus sojae was prepared as koji containing Koji mold.

[0084] <Preparation of Collagen Raw Material> Gelatin derived from tilapia scales (manufactured by Nitta Gelatin Co., Ltd., weight average molecular weight: approximately 150,000) was prepared as a collagen raw material.

[0085] (Step 2) The collagen raw material was fermented with the koji to obtain a fermented product containing fermented collagen peptides according to the following procedure. First, a dispersion consisting of 10% by mass of the collagen raw material, 2% by mass (dry weight) of the barley bran koji, and 88% by mass of RO water was prepared, and the dispersion was cultured for 6 hours while maintaining the temperature at 40°C. The temperature of the dispersion was then increased to 75°C, and the dispersion was maintained at a temperature around 75°C for 60 minutes to inactivate the koji mold in the barley bran koji, thereby obtaining a fermented product containing fermented collagen peptides.

[0086] Next, the fermented product was centrifuged at a centrifugal acceleration of 1610 G for 30 minutes, and the supernatant was collected to obtain Sample 5, a biological function regulating agent.

[0087] The biological function regulator of Sample 5 was an aqueous solution, and measurement of its weight-average molecular weight confirmed that it was lower than the weight-average molecular weight of the collagen raw material. Furthermore, analysis using the gas chromatograph mass spectrometer confirmed that the biological function regulator of Sample 5 contained isovaleraldehyde, 1-octen-3-ol, and phenylacetaldehyde as first compounds.

[0088] <Sample 101> A dry powder of collagen peptide (product name: "Colapep PU", manufactured by Nitta Gelatin Co., Ltd., weight-average molecular weight: 630) was prepared as Sample 101. Analysis using the gas chromatograph mass spectrometer described above confirmed that Sample 101 did not contain the first compound.

[0089] <Sample 102> A dry powder of gelatin derived from pigskin (product name: "BCN-HL", manufactured by Nitta Gelatin Co., Ltd., weight average molecular weight: approximately 65,000) was prepared as Sample 102. It was confirmed that Sample 102 did not contain collagen peptide, and that it did not contain the first compound through analysis using the gas chromatograph mass spectrometer described above.

[0090] <Sample 103> A dry powder of collagen peptide (product name: "CP Prototype", manufactured by Nitta Gelatin Co., Ltd., weight average molecular weight: 500 to 1000) was prepared as Sample 103. Analysis using the gas chromatograph mass spectrometer described above confirmed that Sample 103 did not contain the first compound.

[0091] <Sample 104> A dry powder of collagen peptide (product name: "SCP-5200", manufactured by Nitta Gelatin Co., Ltd., weight average molecular weight: 3000 to 6000) was prepared as Sample 104. Analysis using the gas chromatograph mass spectrometer described above confirmed that Sample 104 did not contain the first compound.

[0092] [First Test] The biological function regulating agent of Sample 2 was administered to mice to test whether the biological function regulating agent has an action of inhibiting fat accumulation. Specifically, the first test was carried out according to the following procedure.

[0093] <Test Method> Thirty 5-week-old male C57BL / 6J mice were purchased from CLEA Japan, Inc. The mice were divided into three groups (n = 10): a low-fat diet group (hereinafter also referred to as "Group L"), a high-fat diet group (hereinafter also referred to as "Group H"), and a high-fat diet and Sample 2 (5% by mass) group (hereinafter also referred to as "Group FCP"). Each group was fed the corresponding diet for 30 days (pair-feeding). During feeding, the mice's food intake and body weight were measured daily at predetermined times. The composition of the diet (unit: % by mass) given to the mice in each group is shown in Table 1. Table 1 also shows the composition of the diet used in the second test described below.

[0094]

[0095] Next, mice from each group were anesthetized with isoflurane, decapitated, and dissected to obtain visceral fat, serum, and liver. The visceral fat samples included mesenteric fat, perirenal fat, and peritestrical fat. The fat mass (g) in each organ was measured and summed to determine total visceral fat mass. Furthermore, serum leptin and adiponectin concentrations were quantified using ELISA. Leptin concentrations were determined according to the protocol of the Mouse Leptin Measurement Kit (catalog number: MS333, Morinaga Biological Science Institute, Inc.). Adiponectin concentrations were determined according to the protocol of the Lebis High Molecular Weight Adiponectin-Mouse / Rat (catalog number: 634-13071, Fujifilm Wako Pure Chemical Corporation).

[0096] The liver was subjected to the following treatment. First, 0.5 g of the liver was homogenized with 2.5 mL of 1.15% by mass potassium chloride (KCl) solution and placed in an ice-cooled test tube. The test tube was then centrifuged (gravitational acceleration 9830 G, 10 minutes, 4 ° C), and the supernatant was used as a crude enzyme solution. Next, the crude enzyme solution was analyzed based on the method described by Nepokroeff et al. (Methods Enzymol, 35: 37-44, 1975) and Kelley et al. (Biochem. J, 235: 87-90, 1986). The enzyme activity was measured (wavelength 340 nm) from the rate of decrease of NAPDH in the presence of 100 μM malonyl-CoA and 25 μM acetyl-CoA, and the strength of fatty acid synthase (FAS) activity was determined. Furthermore, the crude enzyme solution was analyzed for the activity of fatty acid decomposition enzyme (carnitine palmitoyltransferase: CPT) by measuring the enzyme activity (wavelength 412 nm) from the reaction rate of dithionitrobenzoic acid (DTNB) in the presence of 2 mM palmitoyl-CoA and 125 mM L-carnitine, based on the method described by Markwell et al. (J. Biol. Chem., 248: 3426-3432, 1973). The results are shown in Figures 1 to 9. Regarding the significant differences between the groups (L group, H group, and FCP group) in Figures 1 to 9, a Bonferrioni multiple comparison test was performed, and P<0.05 was determined to be statistically significant.

[0097] <Discussion> Figure 1 is a graph showing the changes in body weight of mice in each group in Test 1. From Figure 1, it can be seen that weight gain in the FCP group was significantly suppressed compared to the H group.

[0098] Figure 2 is a graph showing the mesenteric fat mass of mice in each group in the first study. Figure 3 is a graph showing the perirenal fat mass of mice in each group in the first study. Figure 4 is a graph showing the peritestrical fat mass of mice in each group in the first study. Figure 5 is a graph showing the total visceral fat mass of mice in each group in the first study. Figures 2 to 5 indicate that fat accumulation in each organ in the FCP group is significantly suppressed compared to that in Group H. This suggests that a fat accumulation suppression effect is achieved in the FCP group.

[0099] Figure 6 is a graph showing the leptin concentration in the serum of mice in each group in Test 1. Figure 7 is a graph showing the adiponectin concentration in the serum of mice in each group in Test 1. From Figures 6 and 7, it can be assessed that the blood leptin concentration in the FCP group was significantly decreased compared to that in Group H, and the blood adiponectin concentration in the FCP group was significantly increased compared to that in Group H. This suggests that the FCP group may have an effect of suppressing fat accumulation through the regulation of adipocytokine levels in the body.

[0100] Figure 8 is a graph showing the intensity of FAS activity in the livers of mice in each group in the first test. Figure 9 is a graph showing the intensity of CPT activity in the livers of mice in each group in the first test. According to Figures 8 and 9, it can be evaluated that fatty acid synthesis in the FCP group is significantly suppressed compared to that in the H group, and fatty acid breakdown in the FCP group is significantly promoted compared to that in the H group. This suggests that a fat accumulation inhibitory effect is obtained in the FCP group.

[0101] [Second Test] The biological function regulating agent of Sample 2 was administered to mice to test whether the biological function regulating agent has an action of inhibiting fat accumulation. Specifically, the second test was carried out according to the following procedure.

[0102] <Test Method> Forty 5-week-old male C57BL / 6J mice were purchased from CLEA Japan, Inc. The mice were divided into five groups (n = 8): L group, H group, FCP group, high-fat diet and Sample 101 (5% by mass) intake group (hereinafter also referred to as the "CP group"), and high-fat diet and Sample 102 (5% by mass) intake group (hereinafter also referred to as the "GL group"), and were fed the corresponding diet for 30 days (pair-fed feeding). During feeding, the mice's food intake and body weight were measured daily at predetermined times. The composition of the diets fed to the mice in each group is shown in Table 1 above.

[0103] Next, the mice in each group were anesthetized with isoflurane, decapitated, and dissected to obtain visceral fat and serum. The fat mass in each organ and total visceral fat mass were then determined from the visceral fat using the same method as in the first study described above. Furthermore, the concentrations of leptin and adiponectin in the serum were quantified using the same method as in the first study described above. The results for fat mass in each organ and total visceral fat mass (units: g) are shown in Table 2. The results for serum leptin and adiponectin concentrations are shown in Figures 10 and 11. Regarding significant differences between groups (L group, H group, GL group, CP group, and FCP group) in Figures 10 and 11, a Bonferrioni multiple comparison test was performed, and P<0.05 was determined to be statistically significant.

[0104]

[0105] <Discussion> According to Table 2, fat accumulation in each organ of the FCP group is evaluated as being suppressed compared to that of the H group. Furthermore, fat accumulation in each organ of the FCP group is evaluated as being suppressed compared to that of the CP group and the GL group. This suggests that the FCP group has a fat accumulation suppression effect.

[0106] Figure 10 is a graph showing the leptin concentration in the serum of mice in each group in the second study. Figure 11 is a graph showing the adiponectin concentration in the serum of mice in each group in the second study. According to Figures 10 and 11, the blood leptin concentration in the FCP group is assessed to be decreased relative to that in the H group, and the blood adiponectin concentration in the FCP group is assessed to be increased relative to that in the H group. Furthermore, the blood leptin concentration in the FCP group is assessed to be decreased relative to those in the CP and GL groups, and the blood adiponectin concentration in the FCP group is assessed to be increased relative to those in the CP and GL groups. This suggests that the FCP group exhibits a fat accumulation inhibitory effect based on its regulating effect on the amount of adipocytokines in the body.

[0107] [Third Test] The biological function regulating agents of Samples 1 and 2, as well as Samples 101 and 103, were added to adipocytes differentiated from mouse-derived preadipocytes (3T3-L1, passage number: 5 passages, 9 PDL) to test whether the biological function regulating agents of Samples 1 and 2 have an effect of inhibiting fat accumulation. Specifically, the third test was carried out according to the following procedure.

[0108] <Test Method> The mouse-derived preadipocytes (manufactured by Research Resources Bank, resource number JCRB9014, Lot. No. 01282009) were pre-cultured in a culture medium (DMEM / F12 medium (passage medium, catalog number: "11330-032", manufactured by Gibco, containing 10% by mass FBS, penicillin, and streptomycin). Then, from the pre-culture medium and the cells, 3 × 10 cells were cultured. 4 A cell suspension was prepared to give a cell concentration of 1.5 × 10 cells / mL. Five mL of the cell suspension was then seeded into each of 60 mm dishes (Corning, Cat. No. 430166). 5The cells were cultured at 37°C (5% CO2) for two days. The medium in each dish was then replaced with differentiation-inducing medium supplemented with isobutylmethylxanthine (IBMX) and dexamethasone (final concentrations of 0.5 mM and 1 μM, respectively) included in the Adipogenesis Assay Kit (catalog number: ECM950, Millipore). The cells were then cultured for another two days at 37°C (5% CO2). The differentiation-inducing medium was then replaced with differentiation medium supplemented with insulin (final concentration of 10 μg / mL) included in the Adipogenesis Assay Kit (catalog number: ECM950, Millipore). The cells were then cultured for two days at 37°C (5% CO2). After confirming that the cells in each dish had differentiated into adipocytes, the differentiation medium was replaced with the passage medium. To the adipocytes in this passage medium, the biological function regulators (Samples 1 and 2), as well as Samples 101 and 103, were added to a final concentration of 0.1% by mass. Berberine chloride solution (catalog number: "027-11781," manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 2 μg / mL. RO water was added to the remaining adipocytes in the passage medium, and the adipocytes were cultured for 2 days at 37°C (5% CO2 by volume). Samples 1, 2, 101, 103, berberine chloride solution, and RO water were added to three dishes each. The passage medium was then replaced with fresh medium, and Samples 1, 2, 101, and 103 were added again to a final concentration of 0.1% by mass. Berberine chloride solution was added again to a final concentration of 2 μg / mL. RO water was also added to the corresponding dishes.

[0109] Next, according to the protocol of the Adipogenesis Assay Kit, the Oil Red O solution included with the kit was added to each dish at room temperature. Specifically, adipocytes in each medium containing Sample 1, Sample 2, Sample 101, Sample 103, berberine chloride solution, and RO water were washed twice with phosphate-buffered saline (PBS), and then 1.25 mL of the 36% by weight Oil Red O solution was added to each dish. The dish was then washed twice with 2.5 mL of 60% by weight isopropanol, and 625 μL of 99% by weight isopropanol was added to obtain an extract containing adipocytes and Oil Red O from each dish. 200 μL of the extract was transferred to a 96-well plate, and the absorbance at OD520 nm was measured using an absorption spectrophotometer (trade name: "Synergy HTX," manufactured by Biotech Japan). The higher the absorbance at OD520nm, the more fat is accumulated in the adipocytes. The extract obtained from the dish containing RO water served as a blank, while the extract obtained from the dish containing berberine chloride solution served as a positive control.

[0110] The fat accumulation rate in adipocytes to which each sample, berberine chloride solution, or RO water had been added was calculated using the following formula: Fat accumulation rate (%) = [(measured OD520nm value of each sample) / (measured OD520nm value of blank) x 100].

[0111] The results are shown in Table 3. Table 3 shows the average (Ave) and standard deviation of the fat accumulation rate of three specimens for each sample. Furthermore, in Table 3, statistical processing was performed on each measurement value to determine the significance of whether fat accumulation was suppressed. For the statistical processing, analytical processing software (trade name: "STAT Mate V", manufactured by Atoms Corporation) was used. The significance of the above was determined by one-way analysis of variance (One-way Anova) with a confidence interval of 95% and Tukey's test as a post-hoc test. In Table 3, p<0.001 is represented by ***, p<0.01 is represented by **, and p<0.05 is represented by *.

[0112]

[0113] <Discussion> According to Table 3, the biological function regulating agents of Samples 1 and 2 are evaluated as having a greater suppression of fat accumulation in adipocytes than Samples 101 and 103.

[0114] [Fourth Test] The biofunction regulating agents of Samples 41 to 49 described below were added to adipocytes differentiated from mouse-derived preadipocytes (3T3-L1, passage number: 7 passages, 13 PDL) to test whether the biofunction regulating agents of Samples 41 to 49 have an effect of inhibiting fat accumulation. Specifically, the fourth test was carried out according to the following procedure.

[0115] <Test Method> (Preparation of Samples 41 to 49) The biological function regulators of Samples 41 to 49 were prepared by the same method as for obtaining the biological function regulator of Sample 2, except that the conditions for fermenting the collagen raw material with the koji in the second step were as shown in Table 4. For Sample 43, the conditions for fermenting the collagen raw material with the koji were the same as for Sample 2. Table 4 also shows the weight-average molecular weight of the collagen peptide contained in each sample.

[0116]

[0117] (Test Method) Test 4 was carried out in the same manner as Test 3, except that the passage number of mouse-derived preadipocytes (3T3-L1) was set to "7 passages, 13 PDL" and the final concentration of Samples 41 to 49 added to the adipocytes in the dish was set to 0.2% by mass. The results are shown in Table 5.

[0118]

[0119] <Discussion> According to Table 5, the biological function regulating agents of Samples 41 to 49 are all evaluated as suppressing fat accumulation in adipocytes compared to the blank. Note that in the fourth test, the presence or absence of a significant difference in the suppression of fat accumulation was not determined.

[0120] [Fifth Test] The biological function regulators of Samples 1 and 2, and Sample 104 were added to human normal epidermal keratinocytes NHEK (NB) (catalog number: "KK4009", Lot. 05298, manufactured by Kurabo Industries, Ltd.) to test whether the biological function regulators of Samples 1 and 2 have an epidermal metabolism-promoting effect. Specifically, the fifth test was carried out according to the following procedure.

[0121] <Test Method> Human normal epidermal keratinocytes NHEK (NB) (manufactured by Kurabo Industries, Ltd.) were pre-cultured in a culture medium (trade name: "HuMedia KG2", manufactured by Kurabo Industries, Ltd.). Then, 1.5 × 10 cells were cultured from the pre-culture medium and the cells. 4 A cell suspension was prepared at 3 × 10 cells / mL, and the cell suspension was placed in each well of a 6-well plate (catalog number: 353046, manufactured by Falcon). 4 The cells were seeded at 1000 x 1000 cells per well and cultured for 4 days. Next, after confirming that the cells were subconfluent at 90% area in the plate, the medium in the dish was replaced with test medium (trade name: "HuMedia KB2", manufactured by Kurabo Industries, Ltd.). Furthermore, the biofunction regulators of Samples 1 and 2, Sample 104, and RO water were added to each well of each plate to a final concentration of 0.1% by mass, and the cells were cultured at 37°C (5% CO2 by volume). The cells in the plate used to measure the gene expression level of keratin 10 (KRT10), described below, were cultured for 24 hours, while the cells in the plate used to measure the gene expression levels of involucrin (Ivl) and transglutaminase (TGM1), described below, were cultured for 48 hours.

[0122] Each well was then washed twice with PBS, and 1 mL of TRIzol reagent (catalog number: 15506-026, Thermo Fisher Scientific) was added to each well. After 1 minute, all cells in each well were collected into a 1.5 mL centrifuge tube using a scraper. Total RNA was then extracted from the cells according to the TRIzol protocol, and adjusted to a concentration of 1 μg / mL by measuring absorbance at 260 nm. Total RNA purity was determined by calculating the A260 / A280 ratio of 1.8 or higher. cDNA was obtained by reverse transcription of the total RNA using the High Capacity RNA to cDNA Kit (catalog number: 4387406, Life Technologies). The cDNA was then subjected to real-time RT-PCR.

[0123] In the real-time RT-PCR, the mRNA levels of the target genes KRT10 (primer: Hs01043114_gl, Thermo Fisher Scientific), TGM1 (primer: Hs01070310_ml, Thermo Fisher Scientific), and Ivl (primer: Hs00846307_sl, Thermo Fisher Scientific) were measured for each sample. GAPDH (catalog number: 4352934E, Thermo Fisher Scientific) was used as an internal standard (correction gene). Calculations were performed using the calibration curve method. FAM dye was used as the primer and probe. Real-time RT-PCR was performed using an instrument (trade name: "Step One Plus," manufactured by Applied Biosystems) and a reagent kit (trade name: "TaqMan® fast advanced master mix," catalog number: "4444556," manufactured by Applied Biosystems). The PCR conditions consisted of 40 cycles of initial denaturation (95°C, 20 seconds, 1 cycle), annealing (95°C, 1 second), and polymerization (60°C, 20 seconds). The total RNA (cDNA) obtained from the well to which RO water was added served as blank. The results are shown in Tables 6 to 8.

[0124] Table 6 shows the relative values ​​of the gene expression levels of KRT10 in Sample 1, Sample 2, and Sample 104 relative to Blank (average values ​​(Ave) and standard deviations for each of three samples). Table 7 shows the relative values ​​of the gene expression levels of TGM1 in Sample 2 and Sample 104 relative to Blank (average values ​​(Ave) and standard deviations for each of three samples). Table 8 shows the relative values ​​of the gene expression levels of Ivl in Sample 1, Sample 2, and Sample 104 relative to Blank (average values ​​(Ave) and standard deviations for each of three samples). Furthermore, Tables 6 to 8 also show the significant difference in whether or not the expression of each gene was enhanced, determined by performing statistical processing on each measurement value. For the above statistical processing, analysis processing software (trade name: "Excel", manufactured by Microsoft) was used. The above significant differences were determined by a paired T-test, with p<0.001 represented by ***, p<0.01 represented by **, and p<0.05 represented by *.

[0125]

[0126]

[0127]

[0128] <Discussion> According to Tables 6 to 8, the biological function regulating agents of Samples 1 and 2 can increase the expression levels of KRT10, TGM1, and Ivl genes in human epidermal cells, suggesting that increased expression of the above-mentioned genes promotes metabolism in the epidermis.

[0129] [Sixth Test] The fifth test described above was carried out using the biofunction regulating agents of Samples 41 to 49 to determine whether or not the biofunction regulating agents of Samples 41 to 49 have an epidermal metabolism promoting effect. The results are shown in Tables 9 to 11.

[0130] Table 9 shows the relative values ​​of the gene expression levels of KRT10 in Samples 41 to 49 relative to Blank (average values ​​(Ave) and standard deviations for each of three samples). Table 10 shows the relative values ​​of the gene expression levels of TGM1 in Samples 41 to 49 relative to Blank (average values ​​(Ave) and standard deviations for each of three samples). Table 11 shows the relative values ​​of the gene expression levels of Ivl in Samples 41 to 49 relative to Blank (average values ​​(Ave) and standard deviations for each of three samples). In the sixth test, the presence or absence of significant differences in the epidermal metabolism-promoting effect (enhanced expression of the above-mentioned genes) was not determined.

[0131]

[0132]

[0133]

[0134] <Discussion> According to Tables 9 to 11, the biological function regulating agents of Samples 41 to 49 can increase the expression levels of the KRT10, TGM1, and Ivl genes in human epidermal cells, suggesting that increased expression of the above-mentioned genes promotes metabolism in the epidermis.

[0135] [Seventh Test] The biological function regulator of Sample 2 was administered to mice in the same manner as in the first test, and a test was conducted to determine whether the biological function regulator had an effect of inhibiting fat accumulation, using changes in mouse body weight as an indicator. However, for the FCP group, the concentration of Sample 2 ingested was half that of the first test (2.5% by mass in the feed).

[0136] <Discussion> Figure 12 is a graph showing changes in body weight of mice in each group in Test 7. From Figure 12, it can be seen that weight gain in the FCP group was significantly suppressed compared to the H group.

[0137] [Test 8] The biological function regulating agents of Samples 3 and 4 were administered to mice induced to be obese by glucose loading to test whether the biological function regulating agents have an effect of inhibiting fat accumulation. Specifically, Test 8 was carried out according to the following procedure.

[0138] <Test Method> Forty-eight 5-week-old male C57BL / 6J mice were purchased from CLEA Japan, Inc. The mice were fed a normal diet (AIN-93G purified feed) and water containing 15% fructose (Fujifilm Wako Pure Chemical Industries, Ltd.) (hereinafter also referred to as "15% fructose water") for 42 days to intentionally increase their body weight. The 48 mice whose weight had increased were then divided into four groups, with the weights maintained constant across the groups: a normal group (Group A) fed the normal diet and water; a negative control group (Group B) fed the normal diet and 15% fructose water; an FCP-3 group (Group C) fed a diet in which 6% of the 20% casein in the normal diet was replaced with the biofunction regulator Sample 3 and 15% fructose water; and a control group (Group D) fed a diet in which 6% of the 20% casein in the normal diet was replaced with the biofunction regulator Sample 3 and 15% fructose water. The subjects were divided into six groups (n=8): the FCP-4 group (Group D) which was given a diet in which the casein in the 20% by mass of the normal diet had been replaced with Sample 101 and 15% by mass fructose water; the ColapepPU group (Group E) which was given a diet in which 6% by mass of the 20% by mass of the casein in the normal diet had been replaced with Sample 101 and 15% by mass fructose water; and the SCP-5200 group (Group F) which was given a diet in which 6% by mass of the 20% by mass of the casein in the normal diet had been replaced with Sample 104 and 15% by mass fructose water.

[0139] Mice in each group were then pair-fed for 35 days, providing the corresponding diet and water. During this time, the food intake and body weight of each mouse were measured daily at designated times. The diets (units: % by mass) and water content provided to the mice in each group are listed in Table 12. In Table 12, "W" in the "Water / 15% Fructose Water" column indicates that water was provided, and "F" indicates that fructose water was provided. The normal diet (AIN-93G purified diet) consisted of 20% casein by mass, 26.75% cornstarch by mass, 10% sucrose by mass, 20% corn oil by mass, 13.2% α-cornstarch by mass, 5% cellulose by mass, 0.25% choline bitartrate by mass, 3.5% mineral mix (AIN-93), 1% vitamin mix (AIN-93G), and 0.3% L-cystine by mass.

[0140]

[0141] Next, mice from each group were anesthetized with isoflurane, decapitated, and dissected to obtain visceral fat, serum, and liver. The visceral fat samples included mesenteric fat, perirenal fat, and peritestrical fat. The fat mass (g) in each organ was measured and added together to determine total visceral fat mass. Furthermore, serum glucose, insulin, and leptin concentrations were quantified. Blood glucose levels were measured using a Glutest Sensor (Sanwa Chemical Research Institute, Inc.). Insulin levels were measured using a Lebis Insulin-Mouse T (Fujifilm Wako Pure Chemical Industries, Ltd.). Leptin concentrations were determined according to the protocol of the Morinaga Mouse / Rat Leptin Assay Kit (Morinaga Biological Science Institute, Inc.).

[0142] The liver was subjected to the following treatment. First, 0.5 g of the liver was homogenized with 2.5 mL of 1.15% by mass potassium chloride (KCl) solution and placed in an ice-cooled test tube. The test tube was then centrifuged (gravitational acceleration 9830 G, 10 minutes, 4 ° C), and the supernatant was used as a crude enzyme solution. Next, the crude enzyme solution was analyzed based on the method described by Nepokroeff et al. (Methods Enzymol, 35: 37-44, 1975) and Kelley et al. (Biochem. J, 235: 87-90, 1986). The enzyme activity was measured (wavelength 340 nm) from the rate of decrease of NAPDH in the presence of 100 μM malonyl-CoA and 25 μM acetyl-CoA, and the strength of fatty acid synthase (FAS) activity was determined. Furthermore, the crude enzyme solution was analyzed for the activity of fatty acid decomposition enzyme (carnitine palmitoyltransferase: CPT) by measuring the enzyme activity (wavelength 412 nm) from the reaction rate of dithionitrobenzoic acid (DTNB) in the presence of 2 mM palmitoyl-CoA and 125 mM L-carnitine, based on the method described by Markwell et al. (J. Biol. Chem., 248: 3426-3432, 1973). The results are shown in Figures 13 to 22. Regarding the significant differences between groups (Groups A to F) in Figures 13 to 22, a Tukey HSD multiple comparison test was performed, and P<0.05 was determined to be statistically significant.

[0143] <Discussion> Figure 13 is a graph showing the body weight of mice in each group in Test 8. From Figure 13, it can be seen that weight gain was not suppressed in groups B, E, and F compared to group A, but weight gain was significantly suppressed in groups C and D compared to group A.

[0144] Figure 14 is a graph showing the mesenteric fat mass of mice in each group in Test 8. Figure 15 is a graph showing the perirenal fat mass of mice in each group in Test 8. Figure 16 is a graph showing the peritestrical fat mass of mice in each group in Test 8. Figure 17 is a graph showing the total visceral fat mass of mice in each group in Test 8. According to Figures 14 to 17, fat accumulation in each organ of groups C and D is evaluated as being significantly suppressed or tending to be suppressed compared to groups B, E, and F. This suggests that groups C and D exhibit the effect of suppressing visceral fat accumulation when administered fructose.

[0145] Figure 18 is a graph showing the blood glucose levels in the serum of mice in each group in Test 8. Figure 19 is a graph showing the amount of insulin in the serum of mice in each group in Test 8. Figure 18 suggests that blood glucose levels are significantly lower in groups C and D compared to group B. Furthermore, Figure 19 suggests that insulin levels are significantly lower in groups C and D compared to groups B, E, and F.

[0146] Figure 20 is a graph showing the serum leptin concentration of mice in each group in Test 8. Figure 20 suggests that the amount of leptin produced in the body is reduced in Groups C and D, and therefore, fructose loading may be effective in suppressing the accumulation of visceral fat.

[0147] Figure 21 is a graph showing the intensity of FAS activity in the livers of mice in each group in Test 8. According to Figure 21, it is evaluated that fatty acid synthesis in groups C and D is significantly suppressed compared to groups B, E, and F. This suggests that groups C and D have the effect of suppressing visceral fat accumulation during fructose loading due to the suppression of fatty acid synthesis. Furthermore, Figure 22 is a graph showing the intensity of CPT activity in the livers of mice in each group in Test 8. According to Figure 22, it is evaluated that fatty acid breakdown in groups C and D is significantly promoted compared to groups B, E, and F. This suggests that groups C and D have the effect of suppressing visceral fat accumulation during fructose loading due to the promotion of fatty acid breakdown.

[0148] [Ninth Test] The biofunction regulating agent of Sample 3 was administered to Hos:HR-1 mice, which are suitable for observing changes in the skin, to test whether the biofunction regulating agent has a skin metabolism-promoting effect. Specifically, the ninth test was carried out according to the following procedure.

[0149] <Test Method> Fifteen 7-week-old female Hos:HR-1 mice were purchased from Hoshino Laboratory Animal Breeding Co., Ltd. and bred until they reached 8 weeks of age. Subsequently, five mice were assigned to three groups (n = 5): Group X, which consumed a normal diet of Labo MR Stock Feed (manufactured by Nippon Nosan Kogyo Co., Ltd.) and water; Group Y, which consumed HR-AD purified feed and water; and Group Z, which consumed HR-AD purified feed supplemented with 5% by mass of the biofunction regulator (Sample 3) and water. Each group was then bred for 8 weeks (56 days) by providing the appropriate feed and water. During breeding, the feed intake and body weight of each mouse were measured and photographed once a week. Furthermore, mice were selected from each group to be euthanized before and after breeding, and skin samples were collected from the back and abdomen (lumbar region) of the euthanized mice. Furthermore, the skin samples were analyzed for H.E. Stained specimens were prepared and subjected to histopathological observation, including measurement of the thickness of the stratum corneum of the skin. The thickness of the stratum corneum measured in each area of ​​the back (neck and central back) and abdomen (lumbar region) of the mice is shown in Table 13. HR-AD purified diet is a diet that can be used to evaluate atopic dermatitis (AD), as atopic dermatitis (AD)-like symptoms are caused by a deficiency of polyunsaturated fatty acids (n-6 PUFAs).Its composition includes the following amino acids by mass: arginine 0.75%, histidine 0.60%, isoleucine 1.03%, leucine 2.02%, lysine 1.69%, methionine 0.69%, tyrosine 1.17%, alanine 0.62%, proline 2.32%, phenylalanine 1.03%, tryptophan 0.07%, valine 1.27%, cystine 0.08%, glycine 0.39%, threonine 0.87%, serine 1.18%, aspartic acid 1.47%, and glutamic acid 4.74%. The vitamins contained per kg are: vitamin A 32,157 IU, vitamin D3 4,799 IU, vitamin E 160 mg, vitamin K 5 mg, choline 868.2 mg, and folic acid. It contains 0.09 mg of niacin, 320 mg of biotin, 0.8 mg of vitamin B1, 13 mg of vitamin B2, 16.3 mg of vitamin B6, 52.7 mg of vitamin B12, 0.08 mg of vitamin C, 129.6 mg of vitamin C, and 29.7 mg of pantothenic acid, and the minerals are calcium 0.9 mass%, chlorine 0.33 mass%, magnesium 0.02 mass%, phosphorus 0.77 mass%, potassium 0.42 mass%, sodium 0.2 mass%, selenium 0.00 mass%, iodine 0.22 mg / kg, iron 276.78 mg / kg, cobalt 0.002 mass%, manganese 79.28 mg / kg, zinc 122.52 mg / kg, and copper 21.50 mg / kg. In Table 13, "Mean" means median, and "S.D." means standard deviation.

[0150]

[0151] <Discussion> First, in Group Y, desquamation was observed in the skin of the back (cervical and central back) and abdomen (lumbar), and a significant increase in the thickness of the stratum corneum was observed. This confirmed that the mice in Group Y were established as an atopic dermatitis-like model accompanied by dry skin and epidermal thickening. On the other hand, in Group Z, although there was no change in the dryness of the skin compared to Group Y, as shown in Table 13, the thickness of the stratum corneum in the cervical and dorsal regions was significantly lower, suggesting that the skin metabolism-promoting effect of the biofunction regulator was responsible for the suppression of epidermal thickening.

[0152] [Tenth Test] A test was conducted to determine whether the biofunction regulating agent of Sample 4 has an effect of inhibiting fat accumulation in humans by having healthy men and women aged 20 to 70 ingest the biofunction regulating agent of Sample 4. Specifically, the tenth test was carried out according to the following procedure.

[0153] <Test Method> This test was conducted in accordance with the "Declaration of Helsinki (2004 Tokyo Supplementary Edition)" and the "Ethical Guidelines for Epidemiological Research (2004 Ministry of Education, Culture, Sports, Science and Technology and Ministry of Health, Labour and Welfare Notification No. 1)" and in compliance with the test protocol. The test protocol was approved by the Institutional Review Board (approval number: RCB2020-001-02) and registered with UMIN (UMIN 000040736). After obtaining written consent from the subjects, a randomized, double-blind, placebo-controlled, parallel-group study was conducted. Three measurements were performed: at screening assignment, before ingestion (time 0), and after ingesting the test food at 5.0 g / day (bone-dry weight) for three months from the pre-ingestion time point. Each measurement was followed by a one-week adjustment period. The specific test contents are described below.

[0154] 1) Subject selection criteria According to the above protocol, the inclusion criteria were i) men and women aged 20 to 70 years, ii) healthy individuals with no abnormalities found in a health checkup within one year prior to the start of the study, and iii) individuals with an initial visceral fat area of ​​80 cm 2 and iv) those who can maintain an intake rate of 80% or more for the next six months.In addition, the exclusion criteria were as follows: A) those with a history of serious illness, B) those who showed abnormalities in liver function and kidney function tests in a health check, C) those who showed impaired cardiopulmonary function, D) those with food or drug allergies, E) those who had undergone gastrointestinal surgery, F) those who a doctor determined to have developed a chronic or acute infection, G) those who were participating in other clinical research at the start of this study, H) those who play strenuous sports or are on a diet, I) pregnant women, and J) those who the study director or person in charge deemed inappropriate for participation.

[0155] 2) Subject Allocation Of the 109 people who agreed to participate in this study, 55 were enrolled in the study according to the selection criteria. The principal investigator created and sealed a stratified random allocation table using random numbers, taking care to avoid imbalance in visceral fat area between groups at screening. The allocation table was provided only to the person in charge of test food management. The test food described below was distributed to the 55 study participants. The organizers, principal investigator, study participants, all staff at the study institution, including the person in charge of test food management, and members of the ethical review committee were blinded. The allocation table was opened by the person in charge of statistical analysis after the study was completed.

[0156] 3) Test Foods Sample 101 (CP group) and Sample 4 (FCP group) were used as test foods. Maltodextrin (trade name: "Pinex #2", manufactured by Matsutani Chemical Industry Co., Ltd.) was used as a control food (PL group (placebo group)).

[0157] 4) Intake method: The test and control foods were placed in aluminum pouches and distributed without revealing the contents. The test participants (subjects) were instructed to take each food (5.0 g / day) at their preferred time for three months. If they forgot to take the food, they were instructed to take it on the same day, up to one packet (5.0 g) per day.

[0158] 5) Evaluation Items 5-1) Test Schedule Various measurements were taken before intake (time 0) and 3 months after intake (3 months), and the actual measured values ​​at each time point and the change from time 0 were used as outcomes.

[0159] 5-2) Measurement of visceral fat area Visceral fat area was measured using a Dualscan HDS-2000 (Omron Corporation, Medical Device Approval Number: 22300BZX00104000). The Dualscan HDS-2000 is a visceral fat measuring device that can calculate visceral fat area simply and safely using the dual impedance method. It distinguishes between visceral fat and abdominal subcutaneous fat by passing current through two paths, and can measure visceral fat area non-invasively without the risk of exposure.

[0160] 5-3) Skin quality measurement Skin quality measurements were conducted only on subjects who agreed to have their facial photographs taken. Male subjects only washed their faces, while female subjects cleansed and washed their faces, and then allowed to acclimate to room temperature for at least 20 minutes before measurement. The above measurements were conducted using a Robo Skin Analyzer (manufactured by Shibuya Kogyo Co., Ltd.). Specifically, photographs of the face were taken from the front, facing right, and facing left, and pigmentation was evaluated using the Robo Skin Analyzer's algorithm.

[0161] 6) Statistical Analysis Statistical analysis was performed using STATMATEV for Windows (manufactured by Atoms Corporation). All statistical analyses were performed using two-sided tests, with a 95% confidence interval and a significance level of 5%. A Wilcoxon Signed Ranks Test was performed for intra-group comparisons before and after intake. A Mann-Whitney U Test was performed for two-group comparisons between the FCP group and the PL group, and between the CP group and the PL group. A one-way Anova test was performed for three-group comparisons between the FCP group, the PL group, and the CP group, and a two-sided Tukey test was also performed as a post-hoc test to calculate significant differences. Changes in visceral fat area are shown in Table 14, and the results of skin quality (evaluation of increase or decrease in pigmentation) are shown in Table 15.

[0162]

[0163]

[0164] <Discussion> According to Table 14, no significant difference was confirmed between the CP group and the PL group or the 0 time group in terms of visceral fat area. However, a significant difference was confirmed between the FCP group and the 0 time group. Furthermore, the 110 cm 2 In the above groups, the FCP group had a significantly reduced visceral fat area compared to the PL group. In terms of skin quality (evaluation of increase or decrease in pigmentation), the FCP group also had a significantly reduced area and number compared to the PL group. At the same time, Δ (3 months - 0 time) also significantly decreased compared to the PL group. Furthermore, according to Table 15, in terms of skin quality (evaluation of increase or decrease in pigmentation), the CP group had a significantly reduced Δ (3 months - 0 time) compared to the PL group.

[0165] Seven of the subjects were unable to attend the clinic for unavoidable reasons and dropped out at the third month of measurement. Other than the dropouts, no adverse events occurred in the subjects due to the intake of the test food. From the above, it was concluded that the intake of 5.0 g / day of the biofunction regulating agent of Sample 4 for three months is safe, and that the intake of the biofunction regulating agent is effective in reducing visceral fat area by 110 cm. 2 These results suggest that visceral fat area can be reduced in healthy male subjects with a visceral fat area of ​​110 cm or more. 2 The significant difference among the above subjects was observed at 110cm 2 Because the amount of visceral fat was higher than that of people under 110 cm, it is assumed that taking the biofunction regulators mentioned above may have made it easier to burn visceral fat, and that this may have made it easier to see a difference within three months. 2 It is thought that even people under 18 years of age may be able to achieve similar effects by continuing to take the above-mentioned biofunction regulator. Furthermore, it was suggested that taking the above-mentioned biofunction regulator not only suppresses visceral fat area but also skin pigmentation.

[0166] [11th Test] The biofunction regulating agent of Sample 5 was added to adipocytes differentiated from mouse-derived preadipocytes (3T3-L1, passage number: 7 passages, 13 PDL) to test whether the biofunction regulating agent of Sample 5 has an effect of inhibiting fat accumulation. Specifically, the 11th test was carried out according to the following procedure.

[0167] (Test Method) Test No. 11 was carried out in the same manner as Test No. 3, except that the sample added to the adipocytes was changed to Sample 5. The results are shown in Table 16.

[0168]

[0169] <Discussion> According to the results of this test and the third test, Sample 5 was confirmed to have the same fat accumulation inhibitory effect as Sample 1 and Sample 2. This confirmed that similar effects can be obtained even when different raw materials are used.

[0170] [Summary] From the above, it is understood that the biological function regulating agents of Samples 1 to 5 and Samples 41 to 49 have the effect of inhibiting fat accumulation and promoting epidermal metabolism. Furthermore, it is suggested that Samples 1 to 5 and Samples 41 to 49 also have the effect of regulating the amount of adipocytokines in the body.

[0171] The embodiments and examples disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims, not by the above description, and is intended to include all modifications within the meaning and scope of the claims.

Claims

1. A biological function-regulating agent comprising fermented collagen peptides exhibiting at least one selected activity from a group including epidermal metabolism-stimulating activity, lipid accumulation inhibition activity, lipolysis-stimulating activity, and biological activity in regulating adipose-cytokines.

2. A biological function-regulating agent under claim 1 in which fermented collagen peptides comprise at least one selected collagen peptide and compound I from a group including isovaleric aldehyde, 1-octane-3-ol, phenylacetaldehyde, and methional.

3. A biological function-regulating agent under claim 1 in which fermented collagen peptides comprise at least three selected collagen peptides and compound I from a group including isovaleric aldehyde, 1-octane-3-ol, phenylacetaldehyde, and methional. 4.

5. An epidermal metabolism stimulant incorporating any one of the bioregulatory agents under claims 1-3.

6. An inhibitor of lipid accumulation incorporating any one of the bioregulatory agents under claims 1-3.

7. An adenopectin production stimulant incorporating any one of the bioregulatory agents under claims 1-3.

8. A lipid breakdown stimulant incorporating any one of the bioregulatory agents under claims 1-3.

9. A functional food incorporating any one of the bioregulatory agents under claims 1-3.

10. A cosmetic product incorporating any one of the bioregulatory agents under claims 1-3.

11. A method for the production of a bioregulatory agent incorporating fermented collagen peptides; the method incorporating: provision of koji containing koji and collagen raw materials; and 1.A biological function-regulating agent comprising fermented collagen peptides exhibiting at least one selected activity from a group including epidermal metabolism-stimulating activity, lipid accumulation inhibition activity, lipolysis-stimulating activity, and biological activity in regulating the amount of adipoxytokines in the body.

2. A biological function-regulating agent under claim 1 in which fermented collagen peptides are comprising at least one collagen peptide and compound I selected from a group including isovaleric aldehyde, 1-octane-3-ol, phenylacetaldehyde, and methional.

3. A biological function-regulating agent under claim 1 in which fermented collagen peptides are comprising at least three collagen peptides and compound I selected from a group including isovaleric aldehyde, 1-octane-3-ol, phenylacetaldehyde, and methional. 4.

5. An epidermal metabolism stimulant incorporating any one of the bioregulatory agents under claims 1-3.

6. An inhibitor of lipid accumulation incorporating any one of the bioregulatory agents under claims 1-3.

7. An adenopectin production stimulant incorporating any one of the bioregulatory agents under claims 1-3.

8. A limonogenic stimulant incorporating any one of the bioregulatory agents under claims 1-3.

9. A functional food incorporating any one of the bioregulatory agents under claims 1-3.

10. A cosmetic product incorporating any one of the bioregulatory agents under claims 1-3.

11. A method for the production of a bioregulatory agent incorporating fermented collagen peptides; a method incorporating: provision of koji containing koji and collagen raw materials; and;

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