Fermented collagen peptides and their applications

Abstract

A biological function modifier, comprising fermented collagen peptides, said fermented collagen peptides having at least one effect selected from the group consisting of promoting epidermal metabolism, inhibiting fat accumulation, promoting fat breakdown, and regulating the amount of adipokines in the body.

Description

Technical Field

[0001] This invention relates to a biological function modifier, an epidermal metabolism promoter, a fat accumulation inhibitor, a fat breakdown promoter, an adiponectin production promoter, a cosmetic, and a method for manufacturing the biological function modifier. Background Technology

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

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: International Publication No. 2017 / 014149

[0006] Patent Document 2: Japanese Patent Application Publication No. 2018-023326.

[0007] Non-patent literature

[0008] Non-patent literature 1: M Woo et al., "Anti-Obesity Effects of Collagen PeptideDerived from Skate(Raja kenojei)Skin Through Regulation of Lipid Metabolism", Marine Drugs, 2018, Vol.16(9),306

[0009] Non-patent literature 2: Kobayashi et al., “Changes in liver gene expression and anti-obesity effect of salmon skin collagen peptides after oral ingestion”, Proceedings of the 2012 Japan Society of Food Science and Engineering Conference, August 29, 2012, Vol. 59th, Page 97. Summary of the Invention

[0010] The problem that the invention aims to solve

[0011] Patent Document 2 does not disclose obtaining collagen peptides by fermenting any one of collagen, gelatin, and gelatin decomposition products with koji. Furthermore, the collagen peptides with anti-obesity effects reported in Patent Document 1, Non-Patent Document 1, and Non-Patent Document 2 are not obtained by fermenting any one of collagen, gelatin, and gelatin decomposition products with koji (hereinafter referred to as "fermented collagen peptides"). That is, the effects of fermented collagen peptides on organisms have not been elucidated.

[0012] In view of the above-mentioned circumstances, the object of the present invention is to provide a biological function modifier, epidermal metabolism promoter, fat accumulation inhibitor, fat breakdown promoter, adiponectin production promoter, cosmetic, and a method for manufacturing a fermented collagen peptide comprising at least one of the group consisting of an epidermal metabolism promoting effect, a fat accumulation inhibitor, a fat breakdown promoting effect, and an adiponectin production regulating effect.

[0013] Solution for solving the problem

[0014] The inventors have discovered that fermented collagen peptides obtained by fermenting any of the following raw materials containing collagen, such as the skin, bones, cartilage, and tendons of tetrapods, as well as the bones, skin, and scales of fish, along with collagen, gelatin, and gelatin decomposition products, have at least one effect selected from the group consisting of promoting epidermal metabolism, inhibiting fat accumulation, promoting fat decomposition, and adjusting the amount of adipocyte cytokines in the body.

[0015] That is, the present invention has the following features.

[0016] [1] The biological function modifier of the present invention comprises fermented collagen peptides, wherein the fermented collagen peptides have at least one effect selected from the group consisting of promoting epidermal metabolism, inhibiting fat accumulation, promoting fat decomposition, and adjusting the amount of adipocyte kinases in the body.

[0017] [2] Preferably, the fermented collagen peptide comprises collagen peptide and a first compound selected from the group consisting of at least one of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde and methylthiopropionaldehyde.

[0018] [3] Preferably, the fermented collagen peptide comprises collagen peptide and a first compound selected from at least three of the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde and methylthiopropionaldehyde.

[0019] [4] The epidermal metabolism promoter of the present invention includes the above-mentioned biological function modulator.

[0020] [5] The fat accumulation inhibitor of the present invention comprises the above-mentioned biological function modulator.

[0021] [6] The adiponectin production promoter of the present invention comprises the above-mentioned biological function modulator.

[0022] [7] The fat decomposition promoter of the present invention includes the above-mentioned biological function modifier.

[0023] [8] The cosmetics of the present invention contain the above-mentioned biological function modifier.

[0024] [9] The method for manufacturing the bio-function modifier of the present invention is a method for manufacturing a bio-function modifier comprising fermented collagen peptides, the method comprising: a step of preparing koji containing Aspergillus and collagen raw material; and a step of obtaining a bio-function modifier comprising fermented collagen peptides by fermenting the collagen raw material with the koji, wherein the Aspergillus species is a species belonging to the Aspergillus genus, and the collagen raw material is at least any one of the following raw materials: at least one selected from the group consisting of the first to sixth groups below; collagen extracted from at least one selected from the group above; gelatin obtained by processing the collagen; and gelatin decomposition product obtained by hydrolyzing the gelatin.

[0025] Group 1: Composed of the hide, skin, bones, cartilage, and tendons of cattle.

[0026] Group 2: Composed of pig skin, bones, cartilage, and tendons.

[0027] Group 3: Composed of sheep skin, bones, cartilage, and tendons.

[0028] Group 4: The group consisting of chicken skin, bones, cartilage, and tendons.

[0029] Group 5: The group consisting of ostrich skin, bones, cartilage, and tendons.

[0030] Group 6: The group consisting of fish bones, skin, and scales.

[0031]

[11] The biological function modifier of the present invention comprises fermented collagen peptides produced by fermenting collagen raw materials with koji.

[0032] Invention Effects

[0033] Based on the above, a method for manufacturing a bio-function modifier, epidermal metabolism promoter, fat accumulation inhibitor, fat breakdown promoter, adiponectin production promoter, cosmetic, and bio-function modifier can be provided, comprising fermented collagen peptides having at least one of the effects selected from the group consisting of epidermal metabolism promotion, fat accumulation inhibition, fat breakdown promotion, and adjustment of the amount of adipokines in the body. Attached Figure Description

[0034] Figure 1 This is a graph showing the changes in body weight of mice in each group during the first experiment.

[0035] Figure 2 This is a chart showing the amount of fat in the intestinal membrane of mice in each group in the first experiment.

[0036] Figure 3 This is a chart showing the amount of fat around the kidneys in each group of mice in the first experiment.

[0037] Figure 4 This is a chart showing the amount of fat around the testes in each group of mice in the first experiment.

[0038] Figure 5 This is a chart showing the total visceral fat content of mice in each group during the first experiment.

[0039] Figure 6 This is a graph showing the serum leptin concentrations of mice in each group during the first experiment.

[0040] Figure 7 This is a graph showing the concentration of adiponectin in the serum of mice in each group during the first experiment.

[0041] Figure 8 This is a graph showing the intensity of FAS activity in the livers of mice in each group during the first experiment.

[0042] Figure 9 This is a graph showing the intensity of CPT activity in the livers of mice in each group during the first experiment.

[0043] Figure 10 This is a graph showing the serum leptin concentrations of mice in each group during the second experiment.

[0044] Figure 11 This is a graph showing the concentration of adiponectin in the serum of mice in each group during the second experiment.

[0045] Figure 12 This is a graph showing the changes in body weight of mice in each group during the seventh experiment.

[0046] Figure 13This is a chart showing the body weight of mice in each group during the eighth experiment.

[0047] Figure 14 This is a chart showing the amount of fat in the intestinal membrane of mice in each group during the eighth experiment.

[0048] Figure 15 This is a chart showing the amount of fat around the kidneys in each group of mice in the eighth experiment.

[0049] Figure 16 This is a chart showing the amount of fat around the testes of mice in each group during the eighth experiment.

[0050] Figure 17 This is a chart showing the total visceral fat content of mice in each group during the eighth experiment.

[0051] Figure 18 This is a chart showing the blood glucose levels in the serum of mice in each group during the eighth experiment.

[0052] Figure 19 This is a chart showing the amount of insulin in the serum of mice in each group during the eighth experiment.

[0053] Figure 20 This is a chart showing the serum leptin concentrations of mice in each group during the eighth experiment.

[0054] Figure 21 This is a graph showing the intensity of FAS activity in the livers of mice in each group during the eighth experiment.

[0055] Figure 22 This is a graph showing the intensity of CPT activity in the livers of mice in each group during the eighth experiment. Detailed Implementation

[0056] Hereinafter, embodiments of the present invention will be described in further detail (hereinafter also referred to as "this embodiment"). In this specification, descriptions in the form of "A to B" refer to the upper and lower limits of the range (i.e., above A and below B), and when only the unit of B is described without specifying the unit of A, the unit of A is the same as the unit of B.

[0057] Regarding the terms "biofunction regulator," "epidermal metabolism promoter," "fat accumulation inhibitor," "fat breakdown promoter," "adiponectin production promoter," and "fermented collagen peptides" in this specification, their states are sometimes solids such as powders, and sometimes liquids such as aqueous solutions dissolved in water. Furthermore, "fermented collagen peptides" in this specification refers to a mixture of peptides obtained by fermenting the collagen raw materials described later using koji. "Fermentation" in this specification refers to the entire process of generating beneficial organic matter from raw materials using the activity of Aspergillus contained in the koji, distinguished from "putrefaction," which generates non-beneficial organic matter from raw materials using the activity of microorganisms.

[0058] The term "gelatin" in this specification is sometimes used to refer to the name of the substance, gelatin gel, and gelatin solution. Similarly, the term "collagen peptide" is sometimes used to refer to the name of the substance and collagen peptide solution.

[0059] In this specification, "collagen raw material" is sometimes used collectively to refer to the following: at least one of the substances selected from the first to sixth groups below; "collagen" extracted from at least one of the first to sixth groups below; "gelatin" obtained by processing the collagen using known methods such as hot water extraction; and "gelatin decomposition products" obtained by hydrolyzing the gelatin. Furthermore, the "hydrolysis" of the gelatin includes hydrolysis using acid, hydrolysis using alkali, hydrolysis using enzymes, and hydrolysis using heat.

[0060] Group 1: Composed of the hide, skin, bones, cartilage, and tendons of cattle.

[0061] Group 2: Composed of pig skin, bones, cartilage, and tendons.

[0062] Group 3: Composed of sheep skin, bones, cartilage, and tendons.

[0063] Group 4: The group consisting of chicken skin, bones, cartilage, and tendons.

[0064] Group 5: The group consisting of ostrich skin, bones, cartilage, and tendons.

[0065] Group 6: The group consisting of fish bones, skin, and scales.

[0066] [Bio-functional modulators]

[0067] The bio-function modifier of this embodiment comprises fermented collagen peptides. These fermented collagen peptides have at least one effect selected from the group consisting of promoting epidermal metabolism, inhibiting fat accumulation, promoting lipolysis, and adjusting the amount of adipokines in the body. A bio-function modifier possessing such characteristics can exert at least one effect on an organism selected from the group consisting of promoting epidermal metabolism, inhibiting fat accumulation, promoting lipolysis, and adjusting the amount of adipokines in the body (hereinafter also referred to as "bio-function modulatory effect").

[0068] <Fermented Collagen Peptides>

[0069] As described above, the bio-function modifier of this embodiment comprises fermented collagen peptides. Preferably, the fermented collagen peptide comprises a first compound containing collagen peptides and at least one selected from the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methylthiopropionaldehyde. More preferably, the fermented collagen peptide comprises collagen peptides and at least three selected from the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methylthiopropionaldehyde. This allows for a more substantial bio-function modulating effect on the organism, including at least one of the group consisting of promoting epidermal metabolism, inhibiting fat accumulation, promoting lipolysis, and adjusting the amount of adipocyte cytokines within the organism. Furthermore, as described later, the bio-function modifier of this embodiment can be manufactured by fermenting collagen raw materials using koji. That is, the bio-function modifier of this embodiment is a bio-function modifier comprising fermented collagen peptides.

[0070] Here, regarding the aforementioned fermented collagen peptides, the odor (so-called collagen odor) characteristic of conventional collagen peptides is suppressed by the first compound. Therefore, the aforementioned biofunctional modifier can simplify or eliminate the need for deodorization treatment, and can be used, for example, in epidermal metabolism promoters, fat accumulation inhibitors, fat breakdown promoters, adiponectin production promoters, and cosmetics described later.

[0071] (Collagen peptides)

[0072] As described above, the fermented collagen peptide preferably contains collagen peptides. These collagen peptides appear identical to conventionally known collagen peptides. That is, the collagen peptides contained in the fermented collagen peptide, as a peptide mixture, may contain various peptides such as dipeptides, tripeptides, oligopeptides, and polypeptides obtained by conventionally known treatments of collagen or gelatin. As described above, the fermented collagen peptide can be obtained by fermenting collagen raw materials with koji. Therefore, the collagen peptides contained in the above-mentioned fermented collagen peptide can be obtained by fermenting collagen raw materials with koji and produced together with the first compound described later.

[0073] <Weight-average molecular weight>

[0074] The weight-average molecular weight of the collagen peptides contained in the fermented collagen peptides is preferably 20,000 or less. When the weight-average molecular weight of the collagen peptides is 20,000 or less, the biofunctional modifier can be easily applied without additional treatment to various uses as an epidermal metabolism promoter, fat accumulation inhibitor, lipolysis promoter, adiponectin production promoter, and cosmetic. More preferably, the weight-average molecular weight of the collagen peptides is 10,000 or less, and even more preferably 6,000 or less. The lower limit value of the weight-average molecular weight of the collagen peptides is 76. When the weight-average molecular weight of the collagen peptides is within the above range, the biofunctional modifier can fully exert the aforementioned effects on the organism in various uses such as an epidermal metabolism promoter, fat accumulation inhibitor, lipolysis promoter, adiponectin production promoter, or cosmetic.

[0075] Here, the weight-average molecular weight of the collagen peptides contained in the aforementioned biofunctional modifier can be determined by performing size exclusion chromatography (SEC) under the following determination conditions. It should be noted that the inventors have confirmed that this determination method is robust for molecular weights exceeding 12,000.

[0076] Equipment: High performance liquid chromatography (HPLC) (manufactured by Tosoh Corporation).

[0077] Chromatographic column: TSKGel (registered trademark) G2000SW XL .

[0078] Column temperature: 40℃.

[0079] Eluent: 45% acetonitrile (containing 0.1% TFA).

[0080] Flow rate: 1.0 mL / min.

[0081] Injection volume: 10 μL.

[0082] Detection: UV214nm.

[0083] Molecular weight markers: The following five are used.

[0084] Cytochrom C Mw: 12000.

[0085] Aprotinin Mw: 6500.

[0086] Bacitracin Mw: 1450.

[0087] Glycine-Glycine-Tyrine-Arg (Gly-Gly-Tyr-Arg) Mw: 451.

[0088] Glycine-glycine-glycine (Gly-Gly-Gly) Mw: 189.

[0089] (First compound)

[0090] As described above, the fermented collagen peptide preferably comprises a first compound selected from the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methylthiopropionaldehyde, and more preferably comprises at least three of the above-mentioned first compounds. In the above-mentioned fermented collagen peptide, the first compound can be considered to be produced together with the collagen peptide by fermenting collagen raw materials with koji. The first compound can function as a marker indicating that the above-mentioned fermented collagen peptide is a substance obtained by fermenting collagen raw materials with koji.

[0091] Fermented collagen peptides, as the first compound, sometimes contain any one of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methylthiopropionaldehyde. Fermented collagen peptides, as the first compound, sometimes contain isovaleraldehyde and 1-octen-3-ol, sometimes contain isovaleraldehyde and phenylacetaldehyde, sometimes contain isovaleraldehyde and methylthiopropionaldehyde, sometimes contain 1-octen-3-ol and phenylacetaldehyde, sometimes contain 1-octen-3-ol and methylthiopropionaldehyde, and sometimes contain phenylacetaldehyde and methylthiopropionaldehyde.

[0092] Fermented collagen peptides, as the first compound, sometimes contain isovaleraldehyde, 1-octen-3-ol, and phenylacetaldehyde; sometimes they contain isovaleraldehyde, 1-octen-3-ol, and methylthiopropionaldehyde; sometimes they contain isovaleraldehyde, phenylacetaldehyde, and methylthiopropionaldehyde; and sometimes they contain 1-octen-3-ol, phenylacetaldehyde, and methylthiopropionaldehyde. Fermented collagen peptides may also contain four (isovalleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methylthiopropionaldehyde) first compounds. In these cases, the bio-function modulator can more fully exert at least one of the following effects on the organism: promoting epidermal metabolism, inhibiting fat accumulation, promoting lipolysis, and regulating the amount of adipocyte cytokines in the organism.

[0093] (Isovaleraldehyde)

[0094] Isovaleraldehyde, also known as isovaleraldehyde, 3-methylbutanal, or 3-methylbutyraldehyde, is a compound that has been used in the past as a flavoring agent (food additive).

[0095] (1-Octen-3-ol)

[0096] 1-Octen-3-ol is an unsaturated alcohol and a compound known from the past to contribute to the aroma of matsutake mushrooms.

[0097] (phenylacetaldehyde)

[0098] Phenylacetaldehyde is an aromatic aldehyde that has been used since ancient times as a blending ingredient in perfumes and flavorings.

[0099] (Methylthiopropionaldehyde)

[0100] Methional is an organosulfur compound, also known as 3-methylthio-1-propanal. It is a compound previously known to be found in soy sauce. Furthermore, methional has the effect of reducing the fishy or gamey smell of meat and fish.

[0101] (content)

[0102] The first compound is preferably contained in the above-mentioned bio-function modifier at a total amount of 0.05 ppm or more. That is, the above-mentioned bio-function modifier preferably contains 0.05 ppm or more of the first compound. Moreover, the first compound is more preferably contained in the above-mentioned bio-function modifier at a total amount of 0.4 ppm or more. That is, the above-mentioned bio-function modifier more preferably contains 0.4 ppm or more of the first compound. The lower limit of the content of the first compound in the above-mentioned bio-function modifier is not particularly limited, but it is preferred 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 not particularly limited, but it is preferred that the total amount of the first compound is 5 ppm or less.

[0103] The qualitative and quantitative determination of the first compound contained in the aforementioned bio-functional regulator can be performed as follows: First, a dried powder of the bio-functional regulator is obtained by the manufacturing method described later. Then, 0.5 g of the dried powder is dissolved in 4.5 mL of RO water to obtain a sample for analysis. Next, the sample for analysis is introduced into a gas chromatograph-mass analyzer (trade name: "7890AGC system", manufactured by Agilent Technologies, and trade name: "JMS-Q1050GC", manufactured by Nippon Electronics Corporation), where it is vaporized. Using ultra-high purity helium as a carrier gas, it is moved to the chromatographic column of the analyzer, thereby separating the components contained in the sample for analysis one by one. Then, the compound is detected using a detector of the analyzer, and the first compound can be qualitatively determined by comparing the data obtained from the detector (spectral data) with data set as a standard. Simultaneously, the first compound can be quantified based on the spectral data (peak area) obtained from the detector.

[0104] [Epidermal metabolism promoters]

[0105] The epidermal metabolism promoter of this embodiment includes the aforementioned biological function modifier. Through the epidermal metabolism-promoting effect of the aforementioned biological function modifier, the epidermal metabolism promoter can promote the excretion of melanin granules from the skin surface (epidermis) instead of leaving them as spots, freckles, etc. Specifically, the epidermal metabolism promoter can increase the expression of at least one gene selected from the group consisting of glutaminase 1 (TGM1), inner laminarin (IV1), and keratin 10 (KRT10). These genes are known to contribute to the maturation and differentiation of the various layers constituting the epidermis (stratum corneum, stratum granulosum, stratum spinosum, and stratum basale). Therefore, it can be considered that by increasing the expression of the aforementioned genes, metabolism (so-called turnover) in the epidermis is promoted. Moreover, based on the promotion of metabolism in the epidermis, it is possible to obtain skin moisturizing effects, wrinkle prevention and / or wrinkle improvement effects, etc.

[0106] The concentration of the bio-functional modifier in the aforementioned epidermal metabolism promoter is sometimes 0.01 to 100% by mass. The concentration of the bio-functional modifier in the aforementioned epidermal metabolism promoter is set as a value representing the concentration of collagen peptides in the aforementioned bio-functional modifier because the content of the first compound is extremely small. Therefore, the concentration of the bio-functional modifier in the aforementioned epidermal metabolism promoter can be determined using conventionally known methods for determining the concentration of collagen peptides. For example, the concentration of the bio-functional modifier in the aforementioned epidermal metabolism promoter can be determined by measuring the mass percentage of hydroxyproline in collagen peptides using the chloramine-T method. Furthermore, it can also be determined by measuring the mass percentage of hydroxyproline in collagen peptides using an amino acid analyzer.

[0107] [Lipid accumulation inhibitor]

[0108] The fat accumulation inhibitor of this embodiment includes the aforementioned biological function modifier. Through the fat accumulation inhibitory effect of the aforementioned biological function modifier, the fat accumulation inhibitor can achieve an inhibitory effect on the accumulation of fat (so-called visceral fat) in the liver, intestines, kidneys, testes, etc. Furthermore, based on the regulatory effect of the aforementioned biological function modifier on the amount of adipokines in the body, the fat accumulation inhibitor achieves an inhibitory effect on fat accumulation by reducing the blood concentration of leptin, a known appetite-suppressing hormone, and increasing the blood concentration of adiponectin, which is known to be inversely correlated with the amount of visceral fat in the body. Leptin and adiponectin are known to be proteins classified as adipokines secreted by adipocytes. That is, the adiponectin production promoter of this embodiment includes the aforementioned biological function modifier.

[0109] The concentration of the bio-regulatory agent in the aforementioned fat accumulation inhibitor is sometimes 0.01–100% by mass. The concentration of the bio-regulatory agent in the aforementioned adiponectin production promoter can also be set to 0.01–100% by mass. The concentrations of the bio-regulatory agents in the aforementioned fat accumulation inhibitor and adiponectin production promoter are set to values ​​that represent the concentration of collagen peptides in the aforementioned bio-regulatory agents because the content of the first compound is extremely small. Therefore, the concentrations of the bio-regulatory agents in the aforementioned fat accumulation inhibitor and adiponectin production promoter can be determined using the same method as the method used to determine the concentration of the bio-regulatory agent in the aforementioned epidermal metabolism promoter.

[0110] [Fat breakdown promoter]

[0111] The lipolysis promoter of this embodiment includes the aforementioned bio-functional modifier. Through the lipolysis-promoting effect of the aforementioned bio-functional modifier, the lipolysis promoter can achieve a promoting effect on the breakdown of fat (so-called visceral fat) in the liver, intestines, kidneys, testes, etc. The concentration of the bio-functional modifier in the lipolysis promoter is sometimes 0.01 to 100% by mass. The concentration of the bio-functional modifier in the lipolysis promoter is set to a value that represents the concentration of collagen peptides in the aforementioned bio-functional modifier because the content of the first compound is extremely small. Therefore, the concentration of the bio-functional modifier in the lipolysis promoter can be determined using the same method as the method for determining the concentration of the bio-functional modifier in the aforementioned epidermal metabolism promoter.

[0112] <Usage and dosage, etc.>

[0113] Here, the aforementioned epidermal metabolism promoters, fat accumulation inhibitors, lipolysis promoters, and adiponectin production promoters can be administered in various forms, including as additives, pharmaceuticals, or quasi-drugs, via oral or non-oral routes. In the case of oral administration, these can be dosage forms such as tablets, granules, capsules, powders, liquids, suspensions, emulsions, and ointments.

[0114] When administered non-orally, the aforementioned epidermal metabolism promoters, fat accumulation inhibitors, lipolysis promoters, and adiponectin production promoters can be formulated as injections, transdermal preparations (topics, patches, and aerosols), sachets, nasal drops, and inhalers. Preferred dosage forms for these epidermal metabolism promoters, fat accumulation inhibitors, lipolysis promoters, and adiponectin production promoters include tablets, granules, capsules, powders, liquids, and transdermal preparations.

[0115] The dosage of the aforementioned epidermal metabolism promoters, fat accumulation inhibitors, lipolysis promoters, and adiponectin production promoters varies depending on the subject's age, sex, weight, susceptibility differences, administration method, dosing interval, and type of formulation. When administering the aforementioned epidermal metabolism promoters, fat accumulation inhibitors, lipolysis promoters, and adiponectin production promoters orally, the respective dosage, for example, for adults, is preferably 0.0001–2500 mg / kg per day, more preferably 0.0001–500 mg / kg. Regarding the aforementioned epidermal metabolism promoters, fat accumulation inhibitors, lipolysis promoters, and adiponectin production promoters, their dosage forms, for example, in the case of tablets, can be formulated as follows: the tablets contain 0.001 to 80% by mass of the epidermal metabolism promoter, fat accumulation inhibitor, lipolysis promoter, or adiponectin production promoter; or, in the case of powders, the powders contain 0.001 to 100% by mass of the epidermal metabolism promoter, fat accumulation inhibitor, lipolysis promoter, or adiponectin production promoter. The dosage of the aforementioned epidermal metabolism promoters, fat accumulation inhibitors, lipolysis promoters, and adiponectin production promoters can be appropriately determined with reference to the dosage for non-oral administration or oral administration. The aforementioned epidermal metabolism promoters, fat accumulation inhibitors, lipolysis promoters, and adiponectin production promoters can be administered once or several times a day, or once a day or every few days.

[0116] The aforementioned epidermal metabolism promoters, fat accumulation inhibitors, fat decomposition promoters, and adiponectin production promoters may contain other effective ingredients, formulation carriers, etc., within the range that does not adversely affect the effects of the present invention. Other active ingredients include, for example, (±)-α-tocopherol phosphate disodium salt, heparin analogues, allantoin, glycyrrhetinic acid, glycyrrhetinic acid, D-type amino acids, aminosilane compounds, tiliroside, grapefruit extract, α-glucosylhesperidin, mulberry leaf extract, mangosteen peel extract, α-mangosteen, γ-mangosteen, cocoa seed extract, cocoa peel extract, Korean ginseng extract, litchi polyphenols, Psophocarpus extract, Peucedanum praeruptorum extract, lactic acid bacteria, glycyrrhizin, eyebright extract, dried kudzu flower extract, water shield extract, *Vallisneria natans* extract, Tibetan flower extract, N-acetylglucosamine, anserine, and raspberry ketone. Furthermore, pharmaceutically acceptable carriers used in the formulation of pharmaceutical preparations include diluents, binders (syrups, gum arabic, gelatin, sorbitol, tragacanth gum, polyvinylpyrrolidone), excipients (lactose, sucrose, corn starch, potassium phosphate, sorbitol, glycine), lubricants (magnesium stearate, talc, polyethylene glycol, silicon dioxide), disintegrants (potato starch), and wetting agents (sodium lauryl sulfate).

[0117] [Cosmetic Materials]

[0118] The cosmetic material of this embodiment includes the aforementioned bio-functional modifier. Based on, for example, the epidermal metabolism-promoting effect of the aforementioned bio-functional modifier, the cosmetic material can provide whitening, moisturizing, wrinkle prevention, and / or wrinkle improvement effects. The concentration of the bio-functional modifier in the aforementioned cosmetic material is sometimes 0.01 to 100% by mass. The concentration of the bio-functional modifier in the aforementioned cosmetic material is set to a value that represents the concentration of collagen peptides in the aforementioned bio-functional modifier because the content of the first compound is extremely small. Therefore, the concentration of the bio-functional modifier in the aforementioned cosmetic material can be determined using the same method as the method for determining the concentration of the bio-functional modifier in the aforementioned epidermal metabolism promoter.

[0119] [Manufacturing method of biological function modifier]

[0120] The method for manufacturing the bio-functional modifier of this embodiment is a method for manufacturing a bio-functional modifier containing fermented collagen peptides. The method for manufacturing the bio-functional modifier includes: a step of preparing koji containing Aspergillus and collagen raw materials (first step); and a step of obtaining a bio-functional modifier containing fermented collagen peptides by fermenting the collagen raw materials with the koji (second step).

[0121] In the manufacturing method of the above-mentioned biological function regulator, the strain of Aspergillus is a strain belonging to the genus Aspergillus. The above-mentioned collagen raw material is at least any one of the following raw materials: at least one selected from the group consisting of the first to the sixth groups below; collagen extracted from at least one selected from the group above; gelatin obtained by processing the above-mentioned collagen; and gelatin decomposition products obtained by hydrolyzing the above-mentioned gelatin.

[0122] Group 1: Composed of the hide, skin, bones, cartilage, and tendons of cattle.

[0123] Group 2: Composed of pig skin, bones, cartilage, and tendons.

[0124] Group 3: Composed of sheep skin, bones, cartilage, and tendons.

[0125] Group 4: The group consisting of chicken skin, bones, cartilage, and tendons.

[0126] Group 5: The group consisting of ostrich skin, bones, cartilage, and tendons.

[0127] Group 6: The group consisting of fish bones, skin, and scales.

[0128] The method for manufacturing a bio-function modifier with such characteristics can produce a bio-function modifier comprising fermented collagen peptides having at least one of the following effects: promoting epidermal metabolism, inhibiting fat accumulation, promoting fat breakdown, and regulating the amount of adipokines in the body.

[0129] The reason why the bio-functional regulator manufactured by the above-described manufacturing method can have at least one of the following effects: promoting epidermal metabolism, inhibiting fat accumulation, promoting fat breakdown, and adjusting the amount of adipocyte cytokines in the body, although the details are not yet clear, can be considered to be based on the following mechanism. That is, the above-described manufacturing method includes a step (second step) of obtaining a bio-functional regulator containing fermented collagen peptides by fermenting collagen raw materials with koji. It is known that the koji contains a variety of enzymes produced by the propagation of Aspergillus. Therefore, in the second step, it is possible that through the action of these various enzymes, polypeptides in the collagen raw materials and sugars in the koji are decomposed or oxidized and reduced.

[0130] Therefore, in the second step, when fermented collagen peptides are produced by the action of the aforementioned various enzymes, it is presumed that the fermented collagen peptides contain dipeptides, tripeptides, oligopeptides, or polypeptides that have at least any one of the physiological activities described above, including promoting epidermal metabolism, inhibiting fat accumulation, promoting lipolysis, and adjusting the amount of adipocyte cytokines in the body. Furthermore, when fermented collagen peptides are produced by the action of the aforementioned various enzymes, it is also presumed that the fermented collagen peptides contain compounds (non-peptides) that have at least any one of the aforementioned physiological activities. Therefore, it can be considered that, through the above manufacturing method, a bio-function modifier containing fermented collagen peptides having at least one of the activities selected from the group consisting of promoting epidermal metabolism, inhibiting fat accumulation, promoting lipolysis, and adjusting the amount of adipocyte cytokines in the body can be obtained. Hereinafter, each step in the manufacturing method of the bio-function modifier of this embodiment will be described.

[0131] <First Process>

[0132] The first step is to prepare koji containing Aspergillus and collagen raw materials. The first step is performed with the aim of preparing the necessary materials (including koji containing Aspergillus and collagen raw materials) for the purpose of manufacturing the above-mentioned biological function regulator.

[0133] (Collagen raw materials)

[0134] As described above, the collagen raw material is at least any one of the following: at least one of the following groups (first to sixth groups) "the substance itself"; "collagen" extracted from at least one of the following groups (first to sixth groups); "gelatin" obtained by processing the collagen using known methods such as hot water extraction; and "gelatin decomposition products" obtained by hydrolyzing the gelatin.

[0135] Group 1: Composed of the hide, skin, bones, cartilage, and tendons of cattle.

[0136] Group 2: Composed of pig skin, bones, cartilage, and tendons.

[0137] Group 3: Composed of sheep skin, bones, cartilage, and tendons.

[0138] Group 4: The group consisting of chicken skin, bones, cartilage, and tendons.

[0139] Group 5: The group consisting of ostrich skin, bones, cartilage, and tendons.

[0140] Group 6: The group consisting of fish bones, skin, and scales.

[0141] Preferably, in the first step, at least one of the following components is selected as the collagen raw material: at least one of the components selected from the first to sixth groups; the collagen; the gelatin; and the gelatin decomposition product. In the first step, a collagen raw material selected from one of these components may be prepared, or two or more collagen raw materials may be prepared in combination. The components of the first to sixth groups, the collagen, the gelatin, and the gelatin decomposition product can all be prepared using conventionally known methods.

[0142] Here, the gelatin described above is more preferably obtained by the following method: collagen extracted from at least one of the groups selected from the first to sixth groups is subjected to a pretreatment process performed by acid or alkali treatment, followed by hot water extraction, purification, and sterilization. This allows for the preparation of gelatin with high safety for the human body, enabling the application of the bio-functional modifier desired to be manufactured in this embodiment to various uses such as epidermal metabolism promoters, fat accumulation inhibitors, lipolysis promoters, adiponectin production promoters, and cosmetics. Furthermore, such gelatin is economically advantageous. The pretreatment process performed by acid or alkali treatment, hot water extraction, purification, and sterilization can all be performed using methods known in the art.

[0143] The aforementioned gelatin hydrolysate can be obtained by hydrolyzing the gelatin using conventionally known methods such as acid hydrolysis, alkaline hydrolysis, enzymatic hydrolysis, and thermal hydrolysis. The weight-average molecular weight of the gelatin hydrolysate is not particularly limited, but is preferably 20,000 or less, more preferably 10,000 or less. The lower limit of the weight-average molecular weight of the aforementioned gelatin hydrolysate is 76. The weight-average molecular weight of the aforementioned gelatin hydrolysate can be determined using the same method as that used for determining the weight-average molecular weight of the aforementioned collagen peptides.

[0144] (Aspergillus-containing mold)

[0145] Koji containing Aspergillus can be prepared using conventionally known methods, provided that the desired effect of this embodiment can be achieved by performing the second step described later. Specifically, Aspergillus, which will serve as the Aspergillus strain, is inoculated into grains such as rice, barley, wheat, or soybeans, and then propagated in those grains. Preferably, the Aspergillus is inoculated at an amount of 0.01 to 1% by mass relative to the rice, barley, wheat, or grains. In this specification, "grains" includes not only soybeans but also bran, wheat bran, soybean residue, and defatted soybeans. Preferably, in the preparation of koji containing Aspergillus, the necessary operations are performed in an Aspergillus inoculation chamber to prevent the contamination of other bacteria and to adjust the environment conducive to Aspergillus propagation.

[0146] The strain of Aspergillus mentioned above is preferably a species belonging to the genus Aspergillus. More preferably, the strain is selected from at least one of the group consisting of Aspergillus sojae, Aspergillus oryzae, and Aspergillus luchuensis. These strains have been confirmed to be safe for humans, etc., therefore the bio-functional modifier manufactured in this embodiment can be readily applied to various uses such as epidermal metabolism promoters, fat accumulation inhibitors, fat breakdown promoters, adiponectin production promoters, and cosmetics. In the first step, a koji containing one strain selected from this group of strains can be prepared, or a koji containing two or more strains selected from the aforementioned group of strains can be prepared.

[0147] <Second Process>

[0148] The second step involves fermenting the collagen raw material using the aforementioned koji (fermentation starter) to obtain a bio-functional regulator containing the fermented collagen peptides. This second step is performed with the aim of obtaining the fermented collagen peptides contained in the bio-functional regulator. In this second step, for example, the collagen raw material and the aforementioned koji are placed in warm water and cultured in warm water for a predetermined time, thereby fermenting the collagen raw material using the aforementioned koji, thus obtaining a bio-functional regulator containing the aforementioned fermented collagen peptides. The pH value during culture is preferably 2 to 10, more preferably 5 to 8. If the pH value during culture is less than 2 or greater than 10, the reduction of the collagen raw material's molecular weight and the decrease in collagen odor may become insufficient.

[0149] Specifically, it is preferable to prepare a dispersion with a total mass of 100% by weight from 0.1 to 75% by mass of the aforementioned collagen raw material, 0.1 to 20% by mass of the aforementioned koji (dried weight), and 5 to 99.8% by mass of water. The dispersion is then adjusted to a pH of 2 to 10, and the mixture is cultured at a temperature of 10 to 65°C for 1 to 24 hours. This yields a ferment containing fermented collagen peptides.

[0150] Furthermore, the aforementioned fermented product is preferably obtained by the following method: First, a dispersion of 0.1–40% by weight of the aforementioned koji and 60–99.9% by weight of water, totaling 100% by weight, is prepared. The dispersion is then incubated at 10–65°C for 1–24 hours, followed by coarse filtration using a nylon mesh and filtration with diatomaceous earth and cellulose to obtain a koji extract. Next, a dispersion of 0.1–75% by weight of the aforementioned collagen raw material and 0.1–99.9% by weight of the aforementioned koji extract, totaling 100% by weight, is prepared. The pH of the dispersion is then adjusted to 2–10, and the dispersion is incubated at 10–65°C for 1–24 hours. This method also yields a fermented product containing fermented collagen peptides. When the fermented product is obtained by this method, a bio-functional modulator containing fermented collagen peptides can be obtained without performing the separation and processing steps described later on the fermented product. It is not excluded that at least any one of the refining and deodorizing processes described later may be performed on the fermented product.

[0151] Here, the temperature of the warm water used for cultivation is preferably 15–60°C, more preferably 20–50°C. If the temperature of the warm water used for cultivation is below 10°C or above 65°C, the fermentation efficiency carried out by the koji process may be reduced, and fermented collagen peptides may not be fully obtained.

[0152] Furthermore, the optimal incubation time is 2–18 hours, and more preferably 4–8 hours. Incubation times exceeding 24 hours may become economically inefficient. Incubation times below 1 hour may result in insufficient fermentation via koji.

[0153] The collagen content in the warm water used for culturing is preferably 10–45% by mass, more preferably 20–40% by mass. If the collagen content in the warm water used for culturing is less than 0.1% by mass, it may become economically inefficient. If the collagen content in the warm water used for culturing exceeds 75% by mass, the operation may become inefficient.

[0154] The koji content in the dispersion composed of the above-mentioned koji, the above-mentioned collagen raw material, and water is preferably 1-15% by mass, more preferably 5-10% by mass, based on dry weight. If the koji content in the warm water used for cultivation is less than 0.1% by mass, fermentation by koji may become incomplete. If the koji content in the warm water used for cultivation exceeds 20% by mass, it may become economically inefficient. The koji content in the above-mentioned koji extract is preferably 2-25% by mass, more preferably 8-16% by mass. If the koji content in the koji extract is less than 0.1% by mass, fermentation by koji may become incomplete. If the koji content in the koji extract exceeds 40% by mass, it may become economically inefficient.

[0155] In the second step, after obtaining the fermented product through the above steps, the temperature is set to 75°C or higher according to the purpose, thereby inactivating the Aspergillus fungus and stopping the fermentation of collagen raw materials by koji. Specifically, the weight-average molecular weight of collagen peptides in the fermented product is measured. When it is confirmed that the peptides are lower in molecular weight than collagen raw materials, or when the culture time has exceeded a specified time, such as 24 hours, the temperature of the fermented product is set to 75°C or higher, thereby stopping the fermentation of collagen raw materials by koji. The weight-average molecular weight of collagen peptides in the fermented product can be measured using, for example, the same method as the method for measuring the weight-average molecular weight of collagen peptides described above.

[0156] (Other processes)

[0157] To isolate a bio-functional modifier containing fermented collagen peptides from the above-mentioned fermentation product, the second step preferably includes a separation process. This separation process can employ conventionally known separation methods. For example, fermented collagen peptides can be separated from the fermentation product through coarse filtration using a nylon mesh, centrifugation, or filtration using commercially available filter paper. This yields a bio-functional modifier containing fermented collagen peptides, preferably comprising collagen peptides and a first compound selected from at least one of the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methylthiopropionaldehyde, more preferably three of the group consisting of. It should be noted that since the above-mentioned fermentation product contains fermented collagen peptides, the fermentation product itself can also be considered a bio-functional modifier.

[0158] Furthermore, the second step preferably includes a refining process (refining step) of the bio-functional modifier or the fermentation product obtained by applying the above-described separation process, with the aim of improving its transparency, etc. In this refining process, conventionally known refining methods can be applied, such as refining using diatomaceous earth or refining using microfiltration. Additionally, a deodorization process (deodorization step) using activated carbon or the like can also be performed.

[0159] The biomodulators obtained in the above manner can be stored directly in solution. Furthermore, the biomodulators in solution can also be dried into powder using conventionally known methods such as spray drying or drum drying, and stored in this state. Moreover, by using conventionally known formulation techniques on the dried powder of the aforementioned biomodulators, various dosage forms as described above can be formed.

[0160] <Effects>

[0161] In summary, the method for manufacturing the biological function modifier of this embodiment can produce a biological function modifier that includes fermented collagen peptides and, based on the fermented collagen peptides, has at least one biological function modulating effect selected from the group consisting of epidermal metabolism promotion, fat accumulation inhibition, fat decomposition promotion, and adjustment of the amount of adipocyte cytokines in the body.

[0162] Example

[0163] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. In the following description, samples 1 to 5 and samples 41 to 49 are biological function modifiers of the examples, and samples 101 to 104 are collagen peptides or gelatin of comparative examples.

[0164] [Sample Preparation]

[0165] <Sample 1>

[0166] (First process)

[0167] Prepare koji containing Aspergillus and collagen raw materials according to the following method.

[0168] <Preparation of koji containing Aspergillus>

[0169] As a koji containing Aspergillus, barley bran koji (manufactured by Matsunosuke Higuchi Co., Ltd.) was prepared by inoculating Aspergillus oryzae in soy sauce.

[0170] <Preparation of Collagen Raw Materials>

[0171] As a collagen raw material, gelatin derived from pig skin (trade name: "BCN-HL", manufactured by Nitta Gelatin Co., Ltd., weight average molecular weight: approximately 65,000) is prepared.

[0172] (Second process)

[0173] A fermented product containing fermented collagen peptides was obtained by fermenting the collagen raw material with the aforementioned koji. First, a dispersion consisting of 5g of the collagen raw material, 1g of the aforementioned barley bran koji (dry weight), and 50mL of RO water was prepared and cultured at 40°C for 5 hours. Then, the temperature of the dispersion was set to 75°C and maintained at approximately 75°C for 10 minutes to inactivate the Aspergillus in the barley bran koji, thereby obtaining a fermented product containing fermented collagen peptides.

[0174] Next, the above-mentioned fermentation product was filtered using ADVANTEC filter paper No.2 (manufactured by Toyo Filter Paper Co., Ltd.) to obtain the biological function modifier of sample 1.

[0175] The biofunctional modifier of Sample 1 is an aqueous solution. When its weight-average molecular weight was determined, it was confirmed that the biofunctional modifier of Sample 1 was of lower molecular weight compared to the weight-average molecular weight of the aforementioned collagen raw material. Furthermore, analysis using the aforementioned gas chromatographic mass analyzer confirmed that the biofunctional modifier of Sample 1 contains isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methylthiopropionaldehyde as the first compound.

[0176] <Sample 2>

[0177] In the second step, a dispersion consisting of 1 kg of the aforementioned collagen raw material, 200 g (dry weight) of the aforementioned barley bran koji, and 1500 mL of RO water was prepared and cultured at 40°C for 6 hours. Then, the temperature of the dispersion was set to 70°C and maintained at approximately 70°C for 1 hour to inactivate the Aspergillus in the barley bran koji, thereby obtaining a ferment containing fermented collagen peptides. Furthermore, the ferment was filtered using ADVANTEC filter paper No. 5 (manufactured by Toyo Filter Paper Co., Ltd.) and purified by diatomaceous earth filtration. In addition, the bio-functional modifier of Sample 2 was obtained using the same method as that used to obtain Sample 1.

[0178] It should be noted that the biofunctional modifier of sample 2 was made into a dry powder using a spray dryer (manufactured by Okawahara Corporation).

[0179] The biofunctional modifier of Sample 2 is a dry powder. When its weight-average molecular weight was determined, it was confirmed that the biofunctional modifier of Sample 2 was of lower molecular weight compared to the weight-average molecular weight of the aforementioned collagen raw material. Furthermore, analysis using the aforementioned gas chromatographic mass analyzer confirmed that the biofunctional modifier of Sample 2 contains isovaleraldehyde, phenylacetaldehyde, and methylthiopropionaldehyde as the first compound.

[0180] <Sample 3>

[0181] (First process)

[0182] Prepare koji containing Aspergillus and collagen raw materials according to the following method.

[0183] <Preparation of koji containing Aspergillus>

[0184] As a koji containing Aspergillus, barley bran koji (manufactured by Matsunosuke Higuchi Co., Ltd.) was prepared by inoculating Aspergillus oryzae in soy sauce.

[0185] <Preparation of Collagen Raw Materials>

[0186] As a collagen raw material, gelatin derived from pig skin (trade name: "BCN-HL", manufactured by Nitta Gelatin Co., Ltd., weight average molecular weight: approximately 65,000) is prepared.

[0187] (Second process)

[0188] A ferment containing fermented collagen peptides was obtained by fermenting the collagen raw material with the aforementioned koji as follows: First, a dispersion consisting of 13% by mass (dry weight) of the aforementioned barley bran koji and 87% by mass of RO water was prepared and stirred for 1 hour while maintaining the temperature of the dispersion at 40°C. Then, the dispersion was coarsely filtered using a nylon mesh and filtered using diatomaceous earth and cellulose to obtain a koji extract. Next, a dispersion consisting of 40% by mass of the aforementioned collagen raw material and 60% by mass of the aforementioned koji extract was prepared and cultured for 6 hours while maintaining the temperature of the dispersion at 40°C. Then, the temperature of the dispersion was set to 80°C, and the dispersion was sterilized by maintaining the temperature at approximately 80°C for 60 minutes. The resulting powder was then produced using a spray dryer (manufactured by Okawahara Manufacturing Co., Ltd.) to obtain the bio-functional modifier of Sample 3.

[0189] The biofunctional modifier of sample 3 is a dry powder. When its weight-average molecular weight was determined, it was confirmed that the biofunctional modifier of sample 3 was of lower molecular weight compared to the weight-average molecular weight of the aforementioned collagen raw material. Furthermore, analysis using the aforementioned gas chromatographic mass analyzer confirmed that the biofunctional modifier of sample 3 contains isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methylthiopropionaldehyde as the first compound.

[0190] <Sample 4>

[0191] A dispersion consisting of 40% by mass of the above-mentioned collagen raw material and 60% by mass of the above-mentioned koji extract was prepared. After culturing the dispersion at 40°C for 6 hours, the temperature of the dispersion was set to 60°C. The dispersion was sterilized at low temperature by maintaining it at a temperature of around 60°C for 60 minutes. Otherwise, the biological function modifier of sample 4 was obtained by the same method as that used to obtain sample 3.

[0192] The biofunctional modifier of sample 4 is a dry powder. When its weight-average molecular weight was determined, it was confirmed that the biofunctional modifier of sample 4 was of lower molecular weight compared to the weight-average molecular weight of the aforementioned collagen raw material. Furthermore, analysis using the aforementioned gas chromatographic mass analyzer confirmed that the biofunctional modifier of sample 4 contains isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methylthiopropionaldehyde as the first compound.

[0193] <Sample 5>

[0194] (First process)

[0195] Prepare koji containing Aspergillus and collagen raw materials according to the following method.

[0196] <Preparation of koji containing Aspergillus>

[0197] As a koji containing Aspergillus, barley bran koji (manufactured by Matsunosuke Higuchi Co., Ltd.) was prepared by inoculating Aspergillus oryzae in soy sauce.

[0198] <Preparation of Collagen Raw Materials>

[0199] As a collagen raw material, gelatin derived from tilapia scales is prepared (manufactured by Nitta Gelatin Co., Ltd., weight average molecular weight: approximately 150,000).

[0200] (Second process)

[0201] A fermented product containing fermented collagen peptides was obtained by fermenting the collagen raw material with the aforementioned koji. First, a dispersion consisting of 10% by mass of the collagen raw material, 2% by mass (dry weight) of the aforementioned barley bran koji, and 88% by mass of RO water was prepared and cultured at 40°C for 6 hours. Then, the temperature of the dispersion was set to 75°C and maintained at approximately 75°C for 60 minutes to inactivate the Aspergillus in the barley bran koji, thereby obtaining a fermented product containing fermented collagen peptides.

[0202] Next, the above fermentation product was centrifuged at a centrifugation acceleration of 1610G for 30 minutes to obtain its supernatant, thereby obtaining the biological function modifier of sample 5.

[0203] The biofunctional modifier of sample 5 is an aqueous solution. When its weight-average molecular weight was determined, it was confirmed that the biofunctional modifier of sample 5 was of lower molecular weight compared to the weight-average molecular weight of the aforementioned collagen raw material. Furthermore, analysis using the aforementioned gas chromatographic mass analyzer confirmed that the biofunctional modifier of sample 5 contains isovaleraldehyde, 1-octen-3-ol, and phenylacetaldehyde as the first compound.

[0204] <Sample 101>

[0205] A dried powder of collagen peptides (trade name: "Collapep PU", manufactured by Nitta Gelatin Co., Ltd., weight average molecular weight: 630) was prepared as sample 101. Analysis using the aforementioned gas chromatograph-mass analyzer confirmed that sample 101 did not contain the first compound.

[0206] <Sample 102>

[0207] A dried powder of gelatin derived from pigskin (trade name: "BCN-HL", manufactured by Nitta Gelatin Co., Ltd., weight average molecular weight: approximately 65,000) was prepared as sample 102. Sample 102 did not contain collagen peptides and was confirmed by analysis using the gas chromatograph-mass analyzer described above to not contain the first compound.

[0208] <Sample 103>

[0209] A dried powder of collagen peptides (trade name: "CP Prototype", manufactured by Nitta Gelatin Co., Ltd., weight average molecular weight: 500-1000) was prepared as sample 103. Analysis using the aforementioned gas chromatograph-mass analyzer confirmed that sample 103 did not contain the first compound.

[0210] <Sample 104>

[0211] A dried powder of collagen peptides (trade name: "SCP-5200", manufactured by Nitta Gelatin Co., Ltd., weight average molecular weight: 3000-6000) was prepared as sample 104. Analysis using the aforementioned gas chromatograph-mass analyzer confirmed that sample 104 did not contain the first compound.

[0212] [First Test]

[0213] The effect of the aforementioned biomodulatory agent on lipid accumulation was tested by administering the biomodulatory agent of sample 2 to mice. Specifically, the first test was performed as follows.

[0214] <Experimental Methods>

[0215] Thirty 5-week-old male C57BL / 6J mice were purchased from CREA Corporation, Japan. These mice were divided into three groups (n=10): a low-fat diet group (hereinafter referred to as "L group"), a high-fat diet group (hereinafter referred to as "H group"), and a high-fat diet plus sample 2 (5% by mass) diet group (hereinafter referred to as "FCP group"). Each group was fed the corresponding diet for 30 days (paired feeding). During feeding, the mice's feed intake and body weight were measured daily at specified times. The composition of the diets (in % by mass) for each group is shown in Table 1. It should be noted that Table 1 also clearly shows the composition of the diets used in the second experiment described later.

[0216] [Table 1]

[0217]

[0218] Next, after anesthetizing the mice in each group with isoflurane, decapitation and dissection were performed to obtain visceral fat, serum, and liver. As visceral fat, intestinal membrane fat, perirenal fat, and peritesticular fat were removed. The amount of fat (mass (g)) in each organ was measured, and the total visceral fat was calculated by summing them. Furthermore, the concentrations of leptin and adiponectin in the serum were quantified using the serum. ELISA was used as the quantification method. For leptin concentration, the protocol of the mouse leptin assay kit (catalog number: "MS333", manufactured by Morinaga Science & Technology Co., Ltd.) was followed. For adiponectin concentration, the protocol of the LBIS high molecular weight adiponectin-mouse / rat kit (catalog number: "634-13071", manufactured by Fujifilm and Hikari Pure Chemicals Co., Ltd.) was followed.

[0219] For the liver, the following treatment was performed. First, 0.5 g of the liver was homogenized with 2.5 mL of 1.15% potassium chloride (KCl) solution and placed in an ice-cold test tube. Then, the test tube was centrifuged (9830 G, 10 min, 4 °C), and the supernatant was used to prepare a crude enzyme solution. Next, based on the methods 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) according to the rate of NAPDH reduction in the presence of 100 μM malonyl-CoA and 25 μM acetyl-CoA, thereby determining the intensity of fatty acid synthase (FAS) activity. Furthermore, for the aforementioned crude enzyme solution, based on the method described by Markwell et al. (J Biol. Chem, 248: 3426-3432, 1973), the enzyme activity was determined (wavelength 412 nm) according to the reaction rate of dithionitrobenzoic acid (DTNB) in the presence of 2 mM palmitoyl-CoA and 125 mM L-carnitine, thereby calculating the intensity of the fatty acid-degrading enzyme (carnitine palmitoyltransferase: CPT) activity. The results are shown in... Figures 1-9 .for Figures 1-9 To determine the significant differences among the groups (L group, H group, and FCP group), a Bonferrioni multiple comparison test was performed, and P < 0.05 was considered statistically significant.

[0220] <Inspection>

[0221] Figure 1 This is a graph showing the changes in body weight of mice in each group during the first experiment. According to... Figure 1 This can be understood as the FCP group experiencing significantly suppressed weight gain compared to the H group.

[0222] Figure 2 This is a chart showing the amount of fat in the intestinal membrane of mice in each group in the first experiment. Figure 3 This is a chart showing the amount of fat around the kidneys in each group of mice in the first experiment. Figure 4 This is a chart showing the amount of fat around the testes in each group of mice in the first experiment. Figure 5 This is a chart representing the total visceral fat content of mice in each group during the first experiment. According to... Figures 2-5 Compared to the H group, the FCP group showed significantly inhibited fat accumulation in all organs. This indicates that the FCP group can achieve an inhibitory effect on fat accumulation.

[0223] Figure 6 This is a chart showing the serum leptin concentrations of mice in each group during the first experiment. Figure 7This is a graph showing the concentration of adiponectin in the serum of mice in each group during the first experiment. According to... Figures 6-7 Compared to group H, the FCP group showed a significant decrease in serum leptin concentration and a significant increase in serum adiponectin concentration. This indicates that the FCP group can inhibit fat accumulation by adjusting the levels of adipokines in the body.

[0224] Figure 8 This is a graph showing the intensity of FAS activity in the livers of mice in each group during the first experiment. Figure 9 This is a graph representing the intensity of CPT activity in the livers of mice in each group during the first experiment. According to... Figures 8-9 The results showed that, compared to the H group, fatty acid synthesis was significantly inhibited in the FCP group, and fatty acid breakdown was significantly promoted in the FCP group compared to the H group. This indicates that the FCP group can achieve an inhibitory effect on fat accumulation.

[0225] [Second Test]

[0226] The efficacy of the biomodulator of sample 2 in inhibiting fat accumulation was investigated by administering the biomodulator to mice. Specifically, the second test was performed as follows.

[0227] <Experimental Methods>

[0228] Forty 5-week-old male C57BL / 6J mice were purchased from CREA Corporation, Japan. These 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 "CP group"), and high-fat diet and sample 102 (5% by mass) intake group (hereinafter also referred to as "GL group"). Each group was fed the corresponding diet for 30 days (paired rearing). During rearing, the mice's feed intake and body weight were measured daily at specified times. The composition of the diets given to each group of mice is shown in Table 1 above.

[0229] Next, after anesthetizing the mice in each group with isoflurane, visceral fat and serum were obtained through decapitation and dissection. Then, using the same method as in the first experiment, the amount of fat in each organ and the total amount of visceral fat were determined from the visceral fat. Furthermore, using the same method as in the first experiment, the concentrations of leptin and adiponectin in the serum were quantified from the serum. The results of the amount of fat in each organ and the total amount of visceral fat (in g) are shown in Table 2. The results of the serum leptin and adiponectin concentrations are shown in... Figures 10-11 .for Figures 10-11To determine the significant differences among the groups (L, H, GL, CP, and FCP), a Bonferrioni multiple comparison test was performed, with P < 0.05 considered statistically significant.

[0230] [Table 2]

[0231] Group L Group H GL group CP Group FCP Group Fat content in intestinal membrane 0.311±0.024 0.500±0.034 0.553±0.055 0.453±0.029 0.339±0.012 fat around the kidneys 0.089±0.003 0.388±0.037 0.375±0.044 0.344±0.025 0.225±0.019 fat around the testes 0.398±0.016 1.09±0.05 1.08±0.12 0.945±0.057 0.728±0.027 Total visceral fat 0.798±0.030 1.98±0.11 2.01±0.21 1.74±0.10 1.29±0.05

[0232] <Inspection>

[0233] According to Table 2, compared to group H, fat accumulation in all organs of the FCP group was inhibited. Furthermore, compared to groups CP and GL, fat accumulation in all organs of the FCP group was also inhibited. This indicates that fat accumulation inhibition can be achieved in the FCP group.

[0234] Figure 10 This is a graph showing the serum leptin concentrations of mice in each group during the second experiment. Figure 11 This is a graph showing the concentration of adiponectin in the serum of mice in each group during the second experiment. According to... Figures 10-11 Compared to the H group, the FCP group showed a decrease in serum leptin concentration and an increase in serum adiponectin concentration. Furthermore, compared to the CP and GL groups, the FCP group showed a decrease in serum leptin concentration and an increase in serum adiponectin concentration. This indicates that the FCP group, based on the adjustment of adipokine levels in the body, can achieve an inhibitory effect on fat accumulation.

[0235] [Third Test]

[0236] The effects of the biomodulators of Samples 1 and 2 on lipid accumulation were investigated by adding the biomodulators of Samples 1 and 2, as well as Samples 101 and 103, to adipocytes differentiated from mouse preadipocytes (3T3-L1, passage number: 5, 9PDL). Specifically, the third experiment was performed as follows.

[0237] <Experimental Methods>

[0238] The aforementioned mouse preadipocytes (from the Research Resource Bank, Lot No. JCRB9014, 01282009) were precultured in DMEM / F12 medium (passaged medium, catalog number: "11330-032", manufactured by Gibco, containing 10% FBS, penicillin, and streptomycin). Subsequently, 3 × 10⁶ cells were prepared from the precultured medium and the aforementioned cells.4 Cell suspensions of cells / mL were prepared. Then, 5 mL of this cell suspension was seeded into each 60 mm culture dish (catalog number: Corning, Cat. No. 430166) and cultured at 37°C (5% CO2) for two days. Next, the culture medium in each dish was replaced with differentiation induction medium, to which isobutylmethylxanthine (IBMX) and dexamethasone (as per the lipogenesis assay kit, catalog number: "ECM950", manufactured by Millipore) were added at final concentrations of 0.5 mM and 1 μM, respectively, and cultured for another two days at 37°C (5% CO2). Then, the differentiation induction medium was replaced with differentiation medium, to which insulin (as per the lipogenesis assay kit, catalog number: "ECM950", manufactured by Millipore) was added at a final concentration of 10 μg / mL, and cultured for two days at 37°C (5% CO2). After confirming that the cells in each culture dish had differentiated into adipocytes, the differentiation medium was replaced with the passage medium. For the adipocytes in this passage medium, the biomodulators for Sample 1 and Sample 2, as well as Sample 101 and Sample 103, were added at a final concentration of 0.1% by mass. Berberine chloride solution (catalog number: "027-11781", manufactured by Fujifilm and Hikari Pure Chemical Industries Co., Ltd.) was added at a final concentration of 2 μg / mL. RO water was added to the remaining adipocytes in the passage medium, and the cells were cultured at 37°C (5% CO2) for two days. It should be noted that Sample 1, Sample 2, Sample 101, Sample 103, berberine chloride solution, and RO water were added to three separate culture dishes. Then, the above-mentioned subculture medium was replaced with a new subculture medium, and samples 1, 2, 101 and 103 were added again at a final concentration of 0.1% by mass, and berberine chloride solution was added again at a final concentration of 2 μg / mL. RO water was then added back to the corresponding culture dishes.

[0239] Next, following the protocol of the aforementioned adipogenesis assay kit, Oil Red O solution, provided with the kit, was added to each culture dish at room temperature. Specifically, adipocytes in each culture medium containing Sample 1, Sample 2, Sample 101, Sample 103, berberine chloride solution, and RO water were washed twice with PBS (phosphate-buffered saline), followed by the addition of 1.25 mL of the 36% (w / w) Oil Red O solution to each culture dish. Then, the dishes were washed twice with 2.5 mL of 60% (w / w) isopropanol, and an extract containing adipocytes and Oil Red O was obtained by adding 625 μL of 99% (w / w) isopropanol. 200 μL of the extract was transferred to a 96-well plate, and the absorbance at OD520 nm was measured using an absorbance meter (trade name: "Synergy HTX", manufactured by Biotech Japan). A higher absorbance at OD520 nm was considered an increase in adipocyte fat accumulation. Here, the extract obtained from a petri dish containing RO water served as a blank test. Additionally, the extract obtained from a petri dish containing berberine chloride solution served as a positive control.

[0240] The fat accumulation rate in adipocytes after adding each sample, berberine chloride solution, or RO water is calculated based on the following formula.

[0241] Fat accumulation rate (%) = [(OD520nm measured value of each sample) / (OD520nm measured value of blank test) × 100].

[0242] The results are shown in Table 3. Table 3 shows the mean and standard deviation of the fat accumulation rate of the three specimens in each sample. Furthermore, Table 3 also identifies significant differences related to whether fat accumulation was inhibited through statistical processing of each measurement. The statistical processing was performed using analytical processing software (trade name: "STAT Mate V", manufactured by ATOMS Co., Ltd.). Significant differences were determined using one-way ANOVA based on 95% confidence intervals, and a post-hoc test was performed using the Tugi test. In Table 3, ** indicates p < 0.001, ** indicates p < 0.01, and * indicates p < 0.05.

[0243] [Table 3]

[0244]

[0245] <Inspection>

[0246] According to Table 3, compared with Sample 101 and Sample 103, the biofunction modulators of Sample 1 and Sample 2 inhibited lipid accumulation in adipocytes.

[0247] [Fourth Test]

[0248] The effects of the biomodulators of samples 41 to 49 (described later) on the inhibition of fat accumulation were investigated by adding them to adipocytes differentiated from mouse preadipocytes (3T3-L1, passage number: 7, 13PDL). Specifically, the fourth experiment was performed as follows.

[0249] <Experimental Methods>

[0250] (Preparation of Samples 41 to 49)

[0251] The conditions for fermenting the collagen raw materials with the aforementioned koji in the second step were set as shown in Table 4. Except for this, the biofunctional regulators of samples 41 to 49 were prepared using the same method as for obtaining sample 2. It should be noted that the fermentation conditions for the collagen raw materials of sample 43 using the aforementioned koji were the same as for sample 2. Table 4 also shows the weight-average molecular weight of the collagen peptides contained in each sample.

[0252] [Table 4]

[0253] sample Reaction temperature (°C) Reaction time (hours) reaction pH Weight-average molecular weight (Mw) Sample 41 40 2 5.57 2995 Sample 42 40 4 5.57 1949 Sample 43 40 6 5.57 1747 Sample 44 60 2 5.75 2536 Sample 45 60 4 5.75 2286 Sample 46 60 6 5.75 2182 Sample 47 40 2 6.47 2727 Sample 48 40 4 6.47 1762 Sample 49 40 6 6.47 1578

[0254] (Experimental Methods)

[0255] The passage number of mouse preadipocytes (3T3-L1) was set to "7 passages, 13PDL", and the final concentration of samples 41 to 49, which were added to the adipocytes in the above culture dish, was set to 0.2% by mass. Otherwise, the fourth experiment was performed in the same manner as the third experiment described above. The results are shown in Table 5.

[0256] [Table 5]

[0257]

[0258] <Inspection>

[0259] According to Table 5, the biomodulators in samples 41–49 were evaluated as inhibiting lipid accumulation in adipocytes compared to the blank test. It should be noted that the presence or absence of significant differences related to the inhibition of lipid accumulation was not determined in the fourth test.

[0260] [Fifth Test]

[0261] The effects of the biomodulators of Sample 1 and Sample 2 on epidermal metabolism were investigated by adding the biomodulators of Sample 1 and Sample 2, as well as Sample 104, to normal human epidermal keratinocytes NHEK(NB) (catalog number: "KK4009", Lot. 05298, manufactured by Kurashiki Spinning Co., Ltd.). Specifically, the fifth test was performed as follows.

[0262] <Experimental Methods>

[0263] Human normal epidermal keratinocytes (NHEK(NB)) manufactured by Kurashiki Spinning Co., Ltd. were pre-cultured using a culture medium (trade name: "HuMedia KG2", manufactured by Kurashiki Spinning Co., Ltd.). Then, cells of 1.5 × 10⁻⁶ were prepared using this pre-cultured culture medium and the aforementioned cells. 4 A cell suspension of 3 × 10⁻⁶ cells / mL was prepared. 4 Cells were seeded one by one into each well of a 6-well plate (catalog number: "353046", manufactured by Falcon) and cultured for four days. Then, after confirming that the cells had reached 90% sub-confluent concentration within the plate, the culture medium in the culture dish was replaced with experimental medium (trade name: "HuMedia KB2", manufactured by Kurashiki Spinning Co., Ltd.). Next, the bio-modulators of Sample 1 and Sample 2, Sample 104, and RO water were added to each well of each plate at a final concentration of 0.1% by mass, and cultured at 37°C (5% by volume CO2). Cells used to determine the gene expression level of keratin 10 (KRT10) (described later) were cultured for 24 hours, and cells used to determine the gene expression levels of inner vesicle protein (IVL) and glutamine transferase 1 (TGM1) (described later) were cultured for 48 hours.

[0264] Next, each well was washed twice with PBS, and 1 mL of TRIzol reagent (catalog number: "15506-026", manufactured by 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. Then, total RNA was extracted from the cells according to the TRIzol protocol, and the absorbance at 260 nm was measured to prepare a total RNA concentration of 1 μg / mL. Total RNA with a purity of 1.8 or higher when calculated using A260 / A280 was used. All the RNA was reverse transcribed using the High Capacity RNAtocDNA Kit (catalog number: "4387406", manufactured by Life Technologies) to obtain cDNA, which was then used for real-time RT-PCR.

[0265] In the above real-time RT-PCR, the mRNA levels of 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 as the target gene. GAPDH (catalog number: "4352934E", Thermo Fisher Scientific) was used as an internal standard (calibration gene). The calibration curve method was used for calculations. FAM dye was used as primers and probes. Real-time RT-PCR was performed using a device (trade name: "Step One Plus", Applied Biosystems) and a kit (trade name: "TaqMan (registered trademark) fast advanced master mix", catalog number: "4444556", Applied Biosystems). PCR conditions were as follows: initial denaturation (95°C, 20 seconds, 1 cycle), annealing (95°C, 1 second), and extension (60°C, 20 seconds) for 40 cycles. Total RNA (cDNA) obtained from wells containing RO-treated water served as a blank assay. Results are shown in Tables 6–8.

[0266] Table 6 shows the relative values ​​of KRT10 gene expression levels in Sample 1, Sample 2, and Sample 104 compared to the blank test (mean and standard deviation of the three samples). Table 7 shows the relative values ​​of TGM1 gene expression levels in Sample 2 and Sample 104 compared to the blank test (mean and standard deviation of the three samples). Table 8 shows the relative values ​​of Ivl gene expression levels in Sample 1, Sample 2, and Sample 104 compared to the blank test (mean and standard deviation of the three samples). Tables 6-8 also show significant differences related to whether gene expression was hyperactive, determined by statistical analysis of the measured values. The statistical analysis was performed using Excel software (Microsoft Corporation). Significant differences were determined using paired t-tests, with ** indicating p < 0.001, ** indicating p < 0.01, and * indicating p < 0.05.

[0267] [Table 6]

[0268]

[0269] [Table 7]

[0270]

[0271] [Table 8]

[0272]

[0273] <Inspection>

[0274] According to Tables 6-8, the biomodulators in Samples 1 and 2 can increase the expression levels of the genes KRT10, TGM1, and Iv1 in human epidermal cells. This indicates that the increased expression of these genes promotes metabolism in the epidermis.

[0275] [Sixth Test]

[0276] The fifth experiment described above was conducted using the bio-function modifiers of samples 41 to 49 to test whether the bio-function modifiers of samples 41 to 49 had an epidermal metabolism-promoting effect. The results are shown in Tables 9 to 11.

[0277] Table 9 shows the relative values ​​of KRT10 gene expression levels in samples 41–49 compared to the blank test (mean and standard deviation of each of the three samples). Table 10 shows the relative values ​​of TGM1 gene expression levels in samples 41–49 compared to the blank test (mean and standard deviation of each of the three samples). Table 11 shows the relative values ​​of Ivl gene expression levels in samples 41–49 compared to the blank test (mean and standard deviation of each of the three samples). It should be noted that in the sixth test, the presence or absence of significant differences related to the promoting effect on epidermal metabolism (the increased expression of the aforementioned genes) was not determined.

[0278] [Table 9]

[0279]

[0280] [Table 10]

[0281]

[0282] [Table 11]

[0283]

[0284] <Inspection>

[0285] According to Tables 9-11, the bio-function modulators in samples 41-49 can increase the expression levels of the genes KRT10, TGM1, and IV1 in human epidermal cells. This indicates that the increased expression of these genes promotes metabolism in the epidermis.

[0286] [Seventh Test]

[0287] Following the same procedure as the first experiment described above, mice were administered the biomodulator of sample 2, and the change in mouse body weight was used as an indicator to test whether the biomodulator had an inhibitory effect on fat accumulation. For the FCP group, the concentration of sample 2 ingested was set to half that in the first experiment (feed containing 2.5% FCP by mass).

[0288] <Inspection>

[0289] Figure 12 This is a graph showing the changes in body weight of mice in each group during the seventh experiment. According to... Figure 12 This can be understood as the FCP group experiencing significantly suppressed weight gain compared to the H group.

[0290] [Eighth Test]

[0291] The efficacy of the biomodulators of samples 3 and 4 in inhibiting fat accumulation was investigated by administering them to mice obese due to glycemic load. Specifically, the eighth test was conducted as follows.

[0292] <Experimental Methods>

[0293] Forty-eight 5-week-old male C57BL / 6J mice were purchased from CREA Corporation of Japan for preparation. These mice were intentionally fed a standard diet (AIN-93G refined feed) and water containing 15% fructose (manufactured by Fujifilm and Koh Geny Pharmaceutical Co., Ltd.) for 42 days (hereinafter also referred to as "15% fructose water"). Next, the 48 mice that had gained weight were divided into six groups (n=8) to maintain constant weight between groups: Standard group (Group A), fed the standard diet and water; Negative control group (Group B), fed the standard diet and 15% fructose water; FCP-3 group (Group C), fed a diet in which 6% of the 20% casein in the standard diet was replaced with the bio-modulator of sample 3, and 15% fructose water; FCP-4 group (Group D), fed a diet in which 6% of the 20% casein in the standard diet was replaced with the bio-modulator of sample 4, and 15% fructose water; Collapep The PU group (Group E) consumed a diet made by replacing 6% of the 20% casein in the standard diet with sample 101, and 15% fructose water; and the SCP-5200 group (Group F) consumed a diet made by replacing 6% of the 20% casein in the standard diet with sample 104, and 15% fructose water.

[0294] Then, the mice in each group were fed the appropriate diet and water for 35 days (paired rearing). During rearing, the feed intake and body weight of each mouse were measured at specified times every day. A summary of the feed (in % by mass) and water given to the mice in each group is shown in Table 12. It should be noted that in the item "Water / 15% fructose water" in Table 12, "W" indicates that water was given and "F" indicates that fructose water was given. Here, the standard diet (AIN-93G refined feed) consists of 20% by mass casein, 26.75% by mass corn starch, 10% by mass sucrose, 20% by mass corn oil, 13.2% by mass α corn starch, 5% by mass cellulose, 0.25% by mass choline bitartrate, 3.5% by mass mineral mixture (AIN-93), 1% by mass vitamin mixture (AIN-93G), and 0.3% by mass L-cysteine.

[0295] [Table 12]

[0296]

[0297] Next, after anesthetizing the mice in each group with isoflurane, visceral fat, serum, and liver were obtained through decapitation, blood collection, and dissection. As visceral fat, intestinal membrane fat, perirenal fat, and peritesticular fat were removed. The total visceral fat mass was calculated by summing the fat content (mass (g)) of each organ. Furthermore, serum glucose levels, insulin levels, and leptin concentrations were quantified using the serum. Glucose levels were measured using a glucose sensor (manufactured by Sanwa Chemical Research Institute Co., Ltd.). Insulin levels were measured using LBIS Insulin-Mouse T (manufactured by Fujifilm and Kazumitsu Pharmaceutical Co., Ltd.). Leptin concentrations were determined according to the protocol of the Morinaga Mouse / Rat Leptin Assay Kit (manufactured by Morinaga Science Research Institute Co., Ltd.).

[0298] For the liver, the following treatment was performed. First, 0.5 g of the liver was homogenized with 2.5 mL of 1.15% potassium chloride (KCl) solution and placed in an ice-cold test tube. Then, the test tube was centrifuged (9830 G, 10 min, 4 °C), and the supernatant was used to prepare a crude enzyme solution. Next, based on the methods 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) according to the rate of NAPDH reduction in the presence of 100 μM malonyl-CoA and 25 μM acetyl-CoA, thereby determining the intensity of fatty acid synthase (FAS) activity. Furthermore, for the aforementioned crude enzyme solution, based on the method described by Markwell et al. (J Biol. Chem, 248: 3426-3432, 1973), the enzyme activity was determined (wavelength 412 nm) according to the reaction rate of dithionitrobenzoic acid (DTNB) in the presence of 2 mM palmitoyl-CoA and 125 mM L-carnitine, thereby calculating the intensity of the fatty acid-degrading enzyme (carnitine palmitoyltransferase: CPT) activity. The results are shown in... Figures 13-22 .for Figures 13-22 To determine the significant differences among the groups (Group A to Group F), a Tuki HSD multiple comparison test was performed, and P < 0.05 was considered statistically significant.

[0299] <Inspection>

[0300] Figure 13 This is a chart showing the body weights of the mice in each group during the eighth experiment. According to... Figure 13This can be understood as meaning that, compared to group A, the weight gain of groups B, E, and F was not suppressed, but compared to group A, the weight gain of groups C and D was significantly suppressed.

[0301] Figure 14 This is a chart showing the amount of fat in the intestinal membrane of mice in each group during the eighth experiment. Figure 15 This is a chart showing the amount of fat around the kidneys in each group of mice in the eighth experiment. Figure 16 This is a chart showing the amount of fat around the testes of mice in each group during the eighth experiment. Figure 17 This is a chart showing the total visceral fat content of mice in each group during the eighth experiment. According to... Figures 14-17 Compared to groups B, E, and F, fat accumulation in all organs of groups C and D was significantly inhibited or showed a tendency to be inhibited. This indicates that visceral fat accumulation can be inhibited in groups C and D under fructose loading.

[0302] Figure 18 This is a chart showing the blood glucose levels in the serum of mice in each group during the eighth experiment. Figure 19 This is a chart showing the serum insulin levels in each group of mice during the eighth experiment. According to... Figure 18 This indicates that, compared to group B, the blood glucose levels in groups C and D were significantly lower. Furthermore, according to... Figure 19 This indicates that, compared with groups B, E, and F, the insulin levels in groups C and D were significantly lower.

[0303] Figure 20 This is a graph showing the serum leptin concentrations of mice in each group during the eighth experiment. According to... Figure 20 This indicates that in groups C and D, the reduction in leptin production in the body resulted in an inhibitory effect on visceral fat accumulation, even under fructose load.

[0304] Figure 21 This is a graph showing the intensity of FAS activity in the livers of mice in each group during the eighth experiment. According to... Figure 21 Compared to groups B, E, and F, fatty acid synthesis was significantly inhibited in groups C and D. This indicates that, in groups C and D, the inhibition of fatty acid synthesis leads to an inhibitory effect on visceral fat accumulation under fructose loading. Furthermore, Figure 22 This is a graph showing the intensity of CPT activity in the livers of mice in each group during the eighth experiment. According to... Figure 22 Compared with groups B, E, and F, the breakdown of fatty acids was significantly promoted in groups C and D. This indicates that, in groups C and D, the promotion of fatty acid breakdown can inhibit the accumulation of visceral fat under fructose load.

[0305] [Ninth Test]

[0306] The efficacy of the aforementioned biomodulatory agent in promoting skin metabolism was investigated by administering sample 3 to Hos:HR-1 mice, which were suitable for observing changes in the skin. Specifically, the ninth test was performed as follows.

[0307] <Experimental Methods>

[0308] Fifteen 7-week-old female Hos:HR-1 mice were purchased from Hoshino Laboratory Animal Science, Ltd., and raised until they were 8 weeks old. The mice were then divided into three groups of five (n=5): Group X received a standard Labo MR Stock diet (manufactured by Nippon Agricultural Industries, Ltd.) and water; Group Y received a refined HR-AD diet and water; and Group Z received a refined HR-AD diet supplemented with 5% by mass of the bio-function modifier Specimen 3 and water. Each group of mice was fed the appropriate diet and water for 8 weeks (56 days). During this period, feed intake and body weight were measured and photographed weekly. Furthermore, mice euthanized before and at the end of the rearing period were selected from each group, and skin samples were collected from their backs and abdomens (waists). Furthermore, HE-stained specimens related to the aforementioned skin were prepared for histopathological observation, including the measurement of the thickness of the stratum corneum. The thickness of the stratum corneum measured in various parts of the back (neck and central back) and abdomen (waist) of the mice is shown in Table 13. HR-AD refined feed refers to a feed that can evaluate atopic dermatitis because atopic dermatitis (AD)-like symptoms are caused by a deficiency of polyunsaturated fatty acids (n-6 PUFAs).Its composition includes: as amino acids, 0.75% arginine, 0.60% histidine, 1.03% isoleucine, 2.02% leucine, 1.69% lysine, 0.69% methionine, 1.17% tyrosine, 0.62% alanine, 2.32% proline, 1.03% phenylalanine, 0.07% tryptophan, 1.27% valine, 0.08% cysteine, 0.39% glycine, 0.87% threonine, 1.18% serine, 1.47% aspartic acid, and 4.74% glutamic acid; as vitamins, it contains 32,157 IU of vitamin A, 4,799 IU of vitamin D3, and 160 mg of vitamin D1 per kg. It contains vitamin E, 5 mg of vitamin K, 868.2 mg of choline, 0.09 mg of folic acid, 320 mg of niacin, 0.8 mg of biotin, 13 mg of vitamin B1, 16.3 mg of vitamin B2, 52.7 mg of vitamin B6, 0.08 mg of vitamin B12, 129.6 mg of vitamin C, and 29.7 mg of pantothenic acid; as minerals, it contains 0.9% by weight of calcium, 0.33% by weight of chlorine, 0.02% by weight of magnesium, 0.77% by weight of phosphorus, 0.42% by weight of potassium, 0.2% by weight of sodium, 0.00% by weight of selenium, 0.22 mg / kg of iodine, 276.78 mg / kg of iron, 0.002% by weight of cobalt, 79.28 mg / kg of manganese, 122.52 mg / kg of zinc, and 21.50 mg / kg of copper. In Table 13, “Mean” refers to the median value and “SD” refers to the standard deviation.

[0309] [Table 13]

[0310]

[0311] **:p<0.01 Student t-test compared to standard diet group

[0312] $:p<0.05$$:p<0.01 Welch-t test compared to the standard diet group

[0313] + :p<0.05++:p<0.01 Dunnett-t test compared to the HR-AD diet group

[0314] <Inspection>

[0315] First, in group Y, desquamation was observed on the skin of the back (dorsum of the neck and central back) and abdomen (waist), and a significant increase in the thickness of the stratum corneum was confirmed. This confirms that group Y mice are a valid atopic dermatitis-like model with dry skin and thickened epidermis. On the other hand, compared with group Y, the dryness of the skin did not change in group Z, but as shown in Table 13, the thickness of the stratum corneum on the back of the neck was significantly lower, which can be attributed to the skin metabolism-promoting effect of the aforementioned biological function modulators, thereby inhibiting epidermal thickening.

[0316] [Tenth Test]

[0317] The biomodulatory agent (sample 4) was administered to healthy men and women aged 20–70 years to investigate whether it had an inhibitory effect on fat accumulation in humans. Specifically, the tenth test was conducted as follows.

[0318] <Experimental Methods>

[0319] This trial was conducted in accordance with the Declaration of Helsinki (2004 Tokyo Annotated Edition) and the Ethical Guidelines for Epidemiological Research (Ministry of Education, Culture, Sports, Science and Technology / Ministry of Health, Labour and Welfare Announcement No. 1, 2004), and adhered to the trial implementation plan. The trial protocol was approved by the Ethics Review Committee (Approval No.: RCB2020-001-02) and registered with UMIN (UMIN000040736). Furthermore, this trial was a randomized, double-blind, placebo-controlled, parallel intergroup trial conducted with the written consent of the participants. A total of three measurements were performed: at the screening allocation time point, at the pre-ingestion (0-time) time point, and at the time point three months after consuming the test food at an absolute dry weight of 5.0 g / day from the aforementioned pre-ingestion time point. Each measurement included a one-week adjustment period. The specific trial content is described below.

[0320] 1) Selection criteria for subjects

[0321] According to the above plan, the selection criteria are set as follows: i) men and women aged 20 to 70; ii) healthy individuals who have not had any abnormalities detected in a physical examination within one year prior to the start of the trial; iii) visceral fat area of ​​80 cm² at the beginning of the trial. 2The following individuals are eligible for inclusion: (i) those who meet the above criteria; and (ii) those who can maintain an intake rate of 80% or higher over the next six months. Exclusion criteria are as follows: A) those with a history of serious illness; B) those with abnormal liver and kidney function test results during a physical examination; C) those exhibiting cardiopulmonary dysfunction; D) those with food or drug allergies; E) those who have undergone surgery on the digestive tract; F) those diagnosed by a physician with a chronic or acute infectious disease; G) those participating in other clinical trials at the start of this trial; H) those engaged in strenuous exercise or those trying to lose weight; I) pregnant women; and J) others deemed unsuitable by the trial administrator or trial leader.

[0322] 2) Subject allocation

[0323] Of the 109 participants who agreed to participate in this trial, 55 were selected based on the selection criteria. A stratified randomization table was created using random numbers and sealed to ensure that the screening process would not result in an uneven distribution of visceral fat area between groups. This allocation table was provided only to the person in charge of the experimental food administration. The experimental food, described below, was distributed to the aforementioned 55 participants. Blinding was performed on all staff of the trial implementing agency, including the organizers, the responsible physician, the trial practitioners, and the person in charge of the experimental food administration, as well as members of the ethics review committee. The allocation table was opened by the person in charge of statistical analysis after the trial concluded.

[0324] 3) Experimental foods

[0325] As test foods, Sample 101 (CP group) and Sample 4 (FCP group) were used. As control foods (PL group (placebo group)), maltodextrin (trade name: "Pinedex#2", manufactured by Matsutani Chemical Industry Co., Ltd.) was used.

[0326] 4) Ingestion method

[0327] The test and control foods were placed in separate aluminum bags and distributed in a manner where their contents were unknown. Participants (subjects) were instructed to consume each food (5.0g / day) at their preferred times for three months. If a participant forgot to consume the food, they were instructed to consume one packet (5.0g) per day on that day.

[0328] 5) Evaluation Items

[0329] 5-1) Experiment Schedule

[0330] Various measurements were performed before intake (0 time) and three months after intake (3 months), and the measured values ​​at each time point and the changes from the pre-intake level were used as the results.

[0331] 5-2) Measurement of visceral fat area

[0332] The visceral fat area was measured using a Dualscan HDS-2000 (manufactured by OMRON Corporation, Medical Device Approval Number: 22300BZX00104000). The Dualscan HDS-2000 is a visceral fat measurement device that can easily and safely calculate visceral fat area using a dual impedance method. It identifies visceral fat and subcutaneous abdominal fat by flowing current through two paths, enabling non-invasive measurement of visceral fat area without the risk of radiation exposure.

[0333] 5-3) Skin texture determination

[0334] Skin texture measurements were performed only on subjects who consented to have their faces photographed. Male subjects washed their faces only, while female subjects removed makeup and washed their faces. All subjects were allowed at room temperature for at least 20 minutes before the measurements were taken. The measurements were performed using a Roboskin analyzer (manufactured by Shibuya Kogyo Co., Ltd.). Specifically, photographs of the face were taken from the front, right, and left sides, and pigmentation was evaluated using the Roboskin analysis algorithm.

[0335] 6) Statistical analysis

[0336] For statistical analysis, STATMATEV (manufactured by Atoms Corporation) for Windows (registered trademark) was used. All statistical analyses were performed using two-tailed tests, with a significance level set at 5% within the 95% confidence interval. Wilcoxon's signed-rank test was used for within-group comparisons before and after intake. The Mann-Whitney U test was used for comparisons between the FCP and PL groups, and between the CP and PL groups. One-way ANOVA was used for comparisons among the FCP, PL, and CP groups, followed by a two-tailed test in the Tugi test as a post-hoc test to calculate significance. Changes in visceral fat area are shown in Table 14, and results for skin texture (evaluation of increases and decreases in pigmentation) are shown in Table 15.

[0337] [Table 14]

[0338]

[0339] Mean ± Standard Deviation

[0340] Within-group control: Mann-Whitney U test, p<0.05, p<0.01 (compared to PL group)

[0341] Within-group control: Wilcoxon signed-rank test, #p<0.05, ##p<0.01 (compared to before intake).

[0342] [Table 15]

[0343]

[0344] Mean ± Standard Deviation

[0345] Within-group control: Mann-Whitney U test, p<0.05, p<0.01 (compared to PL group)

[0346] Within-group control: Wilcoxon signed-rank test, #p<0.05, ##p<0.01 (compared to before intake).

[0347] Within-group control: Based on ANOVA, graphical test, *p<0.05, **p<0.01 (compared to PL group).

[0348] <Inspection>

[0349] According to Table 14, regarding visceral fat area, no significant difference was found between the CP group and the PL group or before intake. However, a significant difference was found between the FCP group and before intake. Furthermore, the 110cm area of ​​males from different social classes... 2 Among the groups listed above, the FCP group showed a significant reduction in visceral fat area compared to the PL group. In skin texture (evaluation of pigmentation changes), both the area and amount of visceral fat were significantly reduced in the FCP group compared to the PL group. Furthermore, the Δ(three months post-intake – before intake) was also significantly reduced compared to the PL group. Additionally, according to Table 15, in skin texture (evaluation of pigmentation changes), the Δ(three months post-intake – before intake) was significantly reduced in the CP group compared to the PL group.

[0350] Seven participants were excluded from the third month of testing due to unavoidable reasons. No adverse events occurred among the remaining participants due to the intake of the test food. In conclusion, the three-month intake of 5.0 g / day of the bio-modulator from Sample 4 is safe, and the intake of the aforementioned bio-modulator resulted in a reduction of visceral fat area to 110 cm². 2 The visceral fat area was reduced in the above-mentioned healthy men. The visceral fat area was 110 cm². 2 The reason for the significant differences observed among the above subjects is speculated to be: compared to those less than 110cm 2 Compared to individuals with a higher visceral fat content, they are more likely to burn visceral fat through the intake of the aforementioned biological function modifiers, and the difference is more likely to be noticeable within three months. Therefore, it can be considered that even if the visceral fat area is less than 110 cm², the difference is negligible. 2 Individuals who continue to take the aforementioned bio-regulatory agents may also achieve the same effects. Furthermore, it has been shown that the intake of these bio-regulatory agents not only inhibits visceral fat area but also simultaneously inhibits skin pigmentation.

[0351] [Eleventh Test]

[0352] The effect of the biomodulator of Sample 5 on inhibiting lipid accumulation was investigated by adding the biomodulator of Sample 5 to adipocytes differentiated from mouse preadipocytes (3T3-L1, passage number: 7, 13PDL). Specifically, Experiment 11 was performed as follows.

[0353] (Experimental Methods)

[0354] The sample added to the aforementioned fat cells was replaced with sample 5. Otherwise, the eleventh test was performed in the same manner as the third test described above. The results are shown in Table 16.

[0355] [Table 16]

[0356]

[0357] <Inspection>

[0358] Based on the results of this experiment and the third experiment, the same fat accumulation inhibition effect was confirmed for sample 5 as for samples 1 and 2. This confirms that the same effect can be obtained even with different raw materials.

[0359] 〔Summarize〕

[0360] In summary, it can be understood that the bio-function modulators of samples 1-5 and 41-49 have an inhibitory effect on fat accumulation and a promoting effect on epidermal metabolism. Furthermore, it is shown that samples 1-5 and 41-49 have a regulatory effect on the amount of adipokines in the body.

[0361] The embodiments and examples disclosed herein should be considered illustrative and not limiting in any way. The scope of the invention is given by the appended claims rather than the foregoing description, and all modifications falling within the scope of the appended claims and their equivalents are included therein.

Claims

1. A biological function adjusting agent, wherein, The biological function adjusting agent contains a fermented collagen peptide produced by fermenting a collagen raw material with koji, The fermented collagen peptide has at least one action selected from the group consisting of an epidermis metabolism promoting action, a fat accumulation inhibiting action, a fat decomposition promoting action, and an adjusting action of the amount of a fat cell factor in a living body, 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 methylthiopropanal, The collagen raw material is at least one selected from the group consisting of at least one selected from the first to sixth groups below; collagen extracted from at least one selected from the group; gelatin obtained by processing the collagen; and gelatin decomposition product obtained by hydrolyzing the gelatin, The first group: a group consisting of a hide, skin, bone, cartilage, and tendon of a cow, The second group: a group consisting of a hide, skin, bone, cartilage, and tendon of a pig, The third group: a group consisting of a hide, skin, bone, cartilage, and tendon of a sheep, The fourth group: a group consisting of a hide, skin, bone, cartilage, and tendon of a chicken, The fifth group: a group consisting of a hide, skin, bone, cartilage, and tendon of an ostrich, The sixth group: a group consisting of a fish bone and scale.

2. The biological function adjusting agent according to claim 1, wherein The method for producing the biological function adjusting agent includes: a step of preparing koji containing Aspergillus and a collagen raw material; and a step of obtaining a biological function adjusting agent containing the fermented collagen peptide by fermenting the collagen raw material with the koji, The species of the Aspergillus is a species belonging to the genus Aspergillus. The epidermis metabolism promoter contains a fermented collagen peptide produced by fermenting a collagen raw material with koji, 3. An epidermal metabolism promoting agent, wherein, The fermented collagen peptide has at least one action selected from the group consisting of an epidermis metabolism promoting action, a fat accumulation inhibiting action, a fat decomposition promoting action, and an adjusting action of the amount of a fat cell factor in a living body, the epidermis metabolism promoting action being an action of enhancing the expression amount of at least one gene selected from the group consisting of glutamine synthetase 1, filaggrin, and keratin 10, 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 methylthiopropanal, The collagen raw material is at least one selected from the group consisting of at least one selected from the first to sixth groups below; collagen extracted from at least one selected from the group; gelatin obtained by processing the collagen; and gelatin decomposition product obtained by hydrolyzing the gelatin, The first group: a group consisting of a hide, skin, bone, cartilage, and tendon of a cow, The second group: a group consisting of a hide, skin, bone, cartilage, and tendon of a pig, The third group: a group consisting of a hide, skin, bone, cartilage, and tendon of a sheep, The fourth group: a group consisting of a hide, skin, bone, cartilage, and tendon of a chicken, The fifth group: a group consisting of a hide, skin, bone, cartilage, and tendon of an ostrich, The sixth group: a group consisting of a fish bone and scale. ​ Sixth group: a group consisting of bones and scales of fish.

4. A fat accumulation inhibitor, wherein, The fat accumulation inhibitor includes a fermented collagen peptide produced by fermenting a collagen raw material with a koji, The fermented collagen peptide has at least one action selected from the group consisting of an epidermis metabolism promoting action, a fat accumulation inhibiting action, a fat decomposition promoting action, and an adjustment action of an amount of adipokine in a living body, The fermented collagen peptide includes: a collagen peptide; and at least three kinds of a first compound selected from the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methylthiopropanal, The collagen raw material is at least one of: at least one selected from the group consisting of the following first to sixth groups; collagen extracted from at least one selected from the group; gelatin obtained by processing the collagen; and gelatin decomposition product obtained by hydrolyzing the gelatin, First group: a group consisting of a hide, skin, bone, cartilage, and tendon of a cow, Second group: a group consisting of a hide, skin, bone, cartilage, and tendon of a pig, Third group: a group consisting of a hide, skin, bone, cartilage, and tendon of a sheep, Fourth group: a group consisting of a hide, skin, bone, cartilage, and tendon of a chicken, Fifth group: a group consisting of a hide, skin, bone, cartilage, and tendon of an ostrich, Sixth group: a group consisting of bones and scales of fish.

5. An adiponectin production promoter, wherein, The fat accumulation inhibitor includes a fermented collagen peptide produced by fermenting a collagen raw material with a koji, The fermented collagen peptide has at least one action selected from the group consisting of an epidermis metabolism promoting action, a fat accumulation inhibiting action, a fat decomposition promoting action, and an adjustment action of an amount of adipokine in a living body, The fermented collagen peptide includes: a collagen peptide; and at least three kinds of a first compound selected from the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methylthiopropanal, The collagen raw material is at least one of: at least one selected from the group consisting of the following first to sixth groups; collagen extracted from at least one selected from the group; gelatin obtained by processing the collagen; and gelatin decomposition product obtained by hydrolyzing the gelatin, First group: a group consisting of a hide, skin, bone, cartilage, and tendon of a cow, Second group: a group consisting of a hide, skin, bone, cartilage, and tendon of a pig, Third group: a group consisting of a hide, skin, bone, cartilage, and tendon of a sheep, Fourth group: a group consisting of a hide, skin, bone, cartilage, and tendon of a chicken, Fifth group: a group consisting of a hide, skin, bone, cartilage, and tendon of an ostrich, Sixth group: a group consisting of bones and scales of fish.

6. A fat decomposition accelerator, wherein, The fat accumulation inhibitor includes a fermented collagen peptide produced by fermenting a collagen raw material with a koji, The fermented collagen peptide has at least one action selected from the group consisting of an epidermis metabolism promoting action, a fat accumulation inhibiting action, a fat decomposition promoting action, and an adjustment action of an amount of adipokine in a living body, The fermented collagen peptide includes: a collagen peptide; and at least three kinds of a first compound selected from the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methylthiopropanal, at least three kinds selected from the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methylthiopropanal, the collagen raw material is at least one of: at least one selected from the group consisting of a first group to a sixth group below; collagen extracted from at least one selected from the group; gelatin obtained by treating the collagen; and gelatin decomposition product obtained by hydrolyzing the gelatin, the first group: a group consisting of a hide, skin, bone, cartilage, and tendon of a cow, the second group: a group consisting of a hide, skin, bone, cartilage, and tendon of a pig, the third group: a group consisting of a hide, skin, bone, cartilage, and tendon of a sheep, the fourth group: a group consisting of a hide, skin, bone, cartilage, and tendon of a chicken, the fifth group: a group consisting of a hide, skin, bone, cartilage, and tendon of an ostrich, the sixth group: a group consisting of a bone and scale of a fish.

7. A cosmetic material, wherein, the cosmetic material contains fermented collagen peptide produced by fermenting a collagen raw material with koji, the fermented collagen peptide has at least one action selected from the group consisting of an epidermis metabolism promoting action, a fat accumulation inhibiting action, a fat decomposition promoting action, and an adjustment action of an amount of adipokine in a living body, the fermented collagen peptide contains: a collagen peptide; and at least three kinds selected from the group consisting of isovaleraldehyde, 1-octen-3-ol, phenylacetaldehyde, and methylthiopropanal, the collagen raw material is at least one of: at least one selected from the group consisting of a first group to a sixth group below; collagen extracted from at least one selected from the group; gelatin obtained by treating the collagen; and gelatin decomposition product obtained by hydrolyzing the gelatin, the first group: a group consisting of a hide, skin, bone, cartilage, and tendon of a cow, the second group: a group consisting of a hide, skin, bone, cartilage, and tendon of a pig, the third group: a group consisting of a hide, skin, bone, cartilage, and tendon of a sheep, the fourth group: a group consisting of a hide, skin, bone, cartilage, and tendon of a chicken, the fifth group: a group consisting of a hide, skin, bone, cartilage, and tendon of an ostrich, the sixth group: a group consisting of a bone and scale of a fish.

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