KR20130048164A - Jellyfish collagen peptide mixture - Google Patents

Abstract

PURPOSE: A jellyfish collagen peptide mixture is provided to repair a barrier function of a dead skin cell. CONSTITUTION: A jellyfish collagen peptide mixture contains an amino acid sequence among: Gly-Pro-Ala-Gly(sequence number 1); Pro-Gly-Val-Lys-Gly-Glu-Val-Gly-Glu-Ala-Gly-Lys-Arg-Gly(sequence number 2); Val-Gly-Pro-Val-Thr-Glu-Glu-Val-Ser-Pro-Ala-Lys(sequence number 3); Ser-Ala-Gly-Gln-Asn-Pro-Ala-Asp-Asp-Val(sequence number 4); Gly-Thr-Pro(OH)-Gly-Ala-Gly-Gly-Ser-Arg(sequence number 5); and Gln-Gly-Pro-Gln-Gly-Glu-Leu(sequence number 6). In the sequences, Pro(OH) indicates hydroxy proline. The jellyfish collagen peptide mixture is prepared by hydrolysis of jellyfish collagen.

Description

Jellyfish collagen peptide mixture {JELLYFISH COLLAGEN PEPTIDE MIXTURE}

The present invention relates to a jellyfish collagen peptide mixture comprising a specific amino acid sequence. The present invention also relates to an external preparation containing the jellyfish collagen peptide, and more particularly, to an external preparation as a keratinocyte activator, an involuclin production promoter, a transglutaminase production promoter and / or an anti-inflammatory agent.

In recent years, the occurrence of large numbers of jellyfish in the waters of Japan has become a social problem, and the large number of Nomura inhabited jellyfish have caused serious damage to fishing, such as damaging fishing nets and lowering catches. In addition, damaging jellyfish and Nomura inhabited jellyfish are blocked in intakes of factories, thermal power plants, and nuclear power plants. Although large-scale jellyfish are mainly captured and rescued, the disposal of captured jellyfish requires a cost, and therefore, effective solutions to replace them are desired.

As one of the countermeasures, it has been proposed to use jellyfish as a collagen raw material.

Collagen, which is an important item of basic cosmetics, is mainly derived from mammals such as cows and pigs, and in mammals, 1/3 of protein is known to be collagen. In contrast, jellyfish is composed of 95 to 98% by mass of water and 2 to 5% by mass of solids, and 20 to 30% by mass of the solids is protein, and 80% by mass is known to be collagen.

In general, while collagen derived from mammals is an extract from hard tissues such as skin or bones of animals, jellyfish collagen composed of soft tissues containing a large amount of water has a function and activity that other animal collagens do not have. It is expected to be there.

As a study on the jellyfish extract, it has been reported that by using a liquid substance obtained from a mule jellyfish or a Nomura fish jelly in combination with an external preparation for skin, the moisture retention ability of the skin is renewed and the turnover of each layer can be normalized. (Patent Document 1). However, the effect of epidermal cell activation by liquids obtained from jellyfish, that is, protein containing jellyfish collagen, was not sufficiently satisfactory.

The skin protects the cells inside from physical stimuli such as drying and ultraviolet rays and prevents the loss of components in the body. This function is called the barrier function of the skin or the barrier function of the keratin, and each layer plays an important role of the function.

The stratum corneum consists of keratin layers formed by the differentiation of epidermal keratinocytes and intercellular lipids that fill the cells. The stratum corneum consists of keratin fibers and confined envelope (CE). It is formed by crosslinking with enzymes such as transglutaminase 1. It is known that the barrier function of keratin is formed by covalent bond between a part of CE and ceramide, an intercellular lipid.To improve the barrier function of keratin, the growth of epidermal keratinocytes, the increase of involukrin and transglutaminase Is considered important.

About the involukrin production | generation promotion effect and the transglutaminase production | generation promotion effect, the effect by natural substances, such as a mulberry, an iron tree, a birch, a royal jelly, and its extract is reported (patent document 2, 3, 4) ). However, the activating effect of the jellyfish collagen peptides on the epidermal cells, and the involukrin and transglutaminase production promoting effects have not been reported yet.

Japanese Unexamined Patent Publication No. 2011-116672 Japanese Laid-Open Patent Publication 2005-213187 Japanese Unexamined Patent Publication No. 2007-217326 Japanese Unexamined Patent Publication No. 2009-184955

An object of the present invention is to provide a jellyfish collagen peptide mixture having a repair improvement effect of the keratin barrier function.

MEANS TO SOLVE THE PROBLEM As a result of earnestly examining in order to solve the said subject, in the mixture of the jellyfish collagen peptide containing a specific amino acid sequence, the outstanding epidermal keratinocyte reactivation effect, the involukrin production promotion effect, and the transglutaminase production promotion effect The anti-inflammatory effect was discovered and the present invention was completed based on this knowledge. These effects make it possible to repair and improve the barrier function of the keratin.

That is, this invention relates to following (1)-(13).

(1) A jellyfish collagen peptide mixture containing a jellyfish collagen peptide comprising any of the following amino acid sequences.

Pro (OH) stands for hydroxyproline.

(2) The jellyfish collagen peptide mixture according to item 1, obtained by hydrolyzing jellyfish collagen.

(3) The jellyfish collagen peptide mixture according to item 2, wherein hydrolysis is performed using at least one peptide degrading enzyme selected from the group consisting of trypsin, pepsin and papain.

(4) The jellyfish collagen peptide mixture according to item 2, wherein hydrolysis is performed using a solid acid catalyst.

(5) The jellyfish collagen peptide mixture according to item 4, wherein the solid acid catalyst is at least one solid acid catalyst selected from the group consisting of cation exchangers, zeolites and diatomaceous earth.

(6) The jellyfish collagen peptide mixture according to any one of items 1 to 5, wherein the jellyfish collagen is a mule jelly collagen or a nomura intake jellyfish collagen.

(7) A jellyfish collagen peptide mixture obtained by hydrolyzing jellyfish collagen using a peptide degrading enzyme or a solid acid catalyst.

(8) A peptide comprising any of the following amino acid sequences.

Pro (OH) stands for hydroxyproline.

(9) An external preparation containing the jellyfish collagen peptide mixture according to any one of items 1 to 7 and / or the peptide according to item 8.

(10) A keratinocyte activator comprising the jellyfish collagen peptide mixture according to any one of items 1 to 7 and / or the peptide according to item 8.

(11) An involukrin production promoter containing the jellyfish collagen peptide mixture according to any one of items 1 to 7 and / or the peptide according to item 8.

(12) A transglutaminase production promoter containing the jellyfish collagen peptide mixture according to any one of items 1 to 7 and / or the peptide according to item 8.

(13) An anti-inflammatory agent containing the jellyfish collagen peptide mixture according to any one of items 1 to 7, and / or the peptide according to item 8.

According to the present invention, the jellyfish which is an unused resource can be effectively utilized, and a mixture of jellyfish collagen peptides can be provided relatively inexpensively. In addition, by the transdermal administration of the jellyfish collagen peptide excellent in the keratinocyte activating effect, the involuclin production promoting effect, the transglutaminase production promoting effect, and the anti-inflammatory effect of the present invention as an external preparation, the formation of each layer is promoted, and The barrier function can be repaired and improved.

Figure 1 shows the mass spectrometry results of the jellyfish collagen peptide mixture prepared in Example 1.
Figure 2 shows the mass spectrometry results of the jellyfish collagen peptide mixture prepared in Example 9.
Figure 3 shows the molecular weight distribution of the jellyfish collagen peptide mixture prepared in Examples 12-14. In the figure, 1-3 show the molecular weight distribution of the jellyfish collagen peptide mixture produced in Examples 12-14, respectively, 4 shows the molecular weight of the molecular weight marker (the numerical value of the lower table shows molecular weight (kDa)). .

The term "jellyfish collagen peptide" used in the present specification refers to a peptide containing an amino acid sequence derived from jellyfish collagen. In this specification, the mixture (jellyfish collagen peptide mixture) of a jellyfish collagen peptide may be called jellyfish collagen peptide, and the peptide obtained by synthesis | combination based on the amino acid sequence information derived from jellyfish collagen may be called jellyfish collagen peptide. .

The mixture of jellyfish collagen peptides of the present invention can be obtained by hydrolysis of jellyfish collagen. In one embodiment, the mixture of jellyfish collagen peptides is one containing jellyfish collagen peptides comprising any of the following amino acid sequences. In addition, a peptide containing the following amino acid sequence obtained by synthesis may be used, or a mixture of a peptide comprising the following amino acid sequence obtained by synthesis and a jellyfish collagen peptide obtained by hydrolyzing jellyfish collagen may be used.

Peptidase or a solid acid catalyst can be used for the hydrolysis of the jellyfish collagen of the present invention, and jellyfish collagen can be obtained from the protein of the jellyfish.

There is no restriction | limiting in particular in the kind of jellyfish used by this invention, For example, the jellyfish which belongs to the jellyfish river (Scyphozoa), the box jellyfish river (Cubozoa), the cross jellyfish river (Staurozoa), the Hydrazoa river, etc. can be used, Specifically, mussel jelly, red jellyfish, forest root jellyfish, Nomura wing jellyfish, coffin polyp jellyfish, atoll Baird, peripila peripila, sanderia jellyfish, nettle jellyfish, ghost jellyfish, parumbrosa polylobata, Pacelophora Ambiga, Netostoma Setouchiana, Cefea Sepea, Cassiopeia Ornata, Purple Jellyfish, Aurelia Limbata, etc. can be used. Among them, from the viewpoint of resource abundance, mule jellyfish and nomura puffer jellyfish can be preferably used, and mule jellyfish can be particularly preferably used. For example, jellyfish collagen containing any of amino acid sequence <1>-<6> can be used preferably in this invention.

First, the method of extracting a protein from a jellyfish is demonstrated.

When extracting a protein from a jellyfish, what was crushed using a cutting machine, a meat chopper, a cutter, etc. can be used, and it can also be used without crushing a jellyfish. The protein is immersed in a jellyfish or jellyfish lysate at a low temperature of about 4 ° C. for 1 to 36 hours, or immersed in a phosphate buffer or a 0.2% sodium chloride solution at about 4 ° C., or in water for 1 to 36 hours. The aqueous solution containing is obtained.

The aqueous solution containing the protein can be filtered, and further, salted out using inorganic salts such as sodium chloride or ammonium sulfate, or precipitated from a lower alcohol such as ethanol to remove impurities.

There is no restriction | limiting in particular in the method of obtaining jellyfish collagen from the crude protein obtained in this way, For example, the solid obtained by centrifuging the aqueous solution containing a protein is disperse | distributed to lower alcohols, such as ethanol, and also the method of centrifuging further is mentioned. By this operation, collagen of jellyfish can be obtained as a solid. The jellyfish collagen is dispersed in water, and desalted and purified with a dialysis membrane.

After dispersing by adding water to the solid obtained by centrifugation, it is further centrifuged to separate jellyfish collagen and precipitated jellyfish collagen from the supernatant. In the present specification, the precipitated jellyfish collagen is referred to as "acidic soluble collagen" because it is transparently dissolved in an acidic solution of pH 1-4. In addition, this acid-soluble collagen is insoluble in water in the range of pH 4-10, but can take a dispersed state in the alkaline region of pH 10 or more. In addition, in this specification, jellyfish collagen in supernatant is called "neutral soluble collagen" with respect to acidic soluble collagen. In addition, this neutral soluble collagen is collagen soluble in water regardless of the pH range.

In the preparation of the jellyfish collagen peptide, neutral soluble collagen can be used more preferably than acid soluble collagen. In addition, the ratio of acidic soluble collagen can be reduced by removing a metal component previously with a chelating agent.

(Hydrolysis by Peptidase of Jellyfish Collagen)

The term "peptide degrading enzyme" used in the present specification refers to an enzyme that decomposes a protein to produce a peptide. It is preferable that it is 150-35,000 Da, and, as for the molecular weight of the peptide obtained by decomposition, it is more preferable that it is 250-30,000 Da.

Although there is no restriction | limiting in particular in the peptidase used for hydrolysis of jellyfish collagen, It is preferable that it is at least 1 sort (s) chosen from the group which consists of trypsin, pepsin, and papain.

Among these, trypsin is a protease with a molecular weight of about 23000, which is secreted from the pancreas as trypsinogen of an inactive precursor, and is changed into an active form by self-limiting degradation. In biological experiments, the hydrolysis of the C-terminal side of arginine and lysine is used for structural analysis of proteins, and is used for passage of adherent cells. It is preferable that it is 0.0001-5 mass% of jellyfish collagen used as a substrate, as for the usage-amount of trypsin, it is more preferable that it is 0.001-2 mass%, and it is still more preferable that it is 0.01-1 mass%. Although there is no restriction | limiting in particular in the processing conditions by trypsin, It is preferable that it is pH 7-9, 30-40 degreeC.

Pepsin is a kind of protease with a molecular weight of about 35000, in which precursor pepsinogen secreted in gastric juice of vertebrates is converted into pepsin by the action of hydrochloric acid or conventional pepsin. It is preferable that the usage-amount of pepsin is 0.0001-5 mass% of the jellyfish collagen used as a substrate, It is more preferable that it is 0.001-2 mass%, It is further more preferable that it is 0.01-1 mass%. Although there is no restriction | limiting in particular in the treatment conditions with pepsin, It is preferable that it is pH 1-4, 30-40 degreeC.

Papain is a protease with a molecular weight of about 23000 contained in the papaya flesh, and is an enzyme used for digestive agents, beer clarifiers, and the like. It is preferable that the usage-amount of papain is 0.0001-5 mass% of jellyfish collagen used as a substrate, It is more preferable that it is 0.001-2 mass%, It is further more preferable that it is 0.01-1 mass%. Although there is no restriction | limiting in particular in the treatment conditions by papain, It is preferable that it is pH 4-10, 20-80 degreeC.

One kind of these peptide degrading enzymes (trypsin, pepsin, papain) can be used, and two or more kinds thereof can be used in combination, but it is preferable to use two or more kinds of peptide degrading enzymes.

In the case of hydrolyzing jellyfish collagen with two or more peptidases, when jellyfish collagen is acid soluble collagen or a mixture of acid soluble collagen and neutral soluble collagen, it is preferable to first hydrolyze with pepsin. By hydrolyzing with pepsin with degradation activity under acidic conditions, a soluble collagen peptide can be obtained even in the neutral pH range, and further hydrolyzed with trypsin and / or papain. After hydrolysis by pepsin, further hydrolysis with trypsin and / or papain can also be used for hydrolysis of neutral soluble collagen. For example, a combination of pepsin and trypsin and pepsin, papain and trypsin can be preferably used in the present invention.

(Hydrolysis by Jellyfish Collagen Solid Acid Catalyst)

The mixture of jellyfish collagen peptides can also be obtained by hydrolyzing jellyfish collagen with a solid acid catalyst. Hydrolysis by a solid acid catalyst is a method of hydrolyzing jellyfish collagen by bringing a solid acid catalyst into contact with jellyfish collagen in the presence of water and under heating. In view of hydrolysis efficiency, the jellyfish collagen is preferably neutral soluble collagen.

In the present invention, a solid catalyst capable of functioning as an acid can be used as a solid acid catalyst without particular limitation, but from the viewpoint of hydrolysis efficiency, it is preferably at least one selected from the group consisting of cation exchangers, zeolites and diatomaceous earth. And it is more preferable that it is a cation exchanger. Only 1 type may be used for these solid acid catalysts, and they may be used in combination of 2 or more type.

It is preferable that a cation exchanger is resin which has at least 1 sort (s) of a sulfone group and a carboxyl group, A sulfone group and a carboxyl group may respectively be a sulfopropyl group and a carboxymethyl group. When the counter ion of a cation exchanger is other than a proton, it is preferable to use, after substituting for a proton type. Particularly preferred cation exchangers are sulfonic acid type cation exchangers having strongly acidic cation exchange groups such as sulfopropyl groups, and cation exchange resins and cation exchange membranes can be cited as preferred embodiments. Specific examples of the sulfonic acid type cation exchange resin include those containing a hydrophilic vinyl polymer as a substrate such as TOYOPEARL SP-650C, SP-550C, etc. manufactured by Tosoh Corporation, or a perfluorosulfonic acid such as Nafion (registered trademark). Polytetrafluoroethylene copolymer is mentioned.

As a zeolite, the zeolite generally used as a zeolite catalyst can be used, Especially, the zeolite (registered trademark) by Toso Corporation can be used preferably.

As diatomaceous earth, the diatomaceous earth (granular form) made from Wako Junyaku Industrial Co., Ltd. is mentioned, for example.

There is no restriction | limiting in the shape of the said solid acid catalyst, It can take arbitrary forms, such as a granular form and a powder form. If it is granular or powdery, it can also be used for the batch method of mixing a solid acid catalyst in the aqueous solution containing jellyfish collagen to hydrolyze, and can also be used for the continuous method of continuously sending a jellyfish collagen aqueous solution to the column filled with the solid acid catalyst. In addition, the jellyfish collagen aqueous solution can also be passed to the film | membrane which consists of a solid acid catalyst, and the film | membrane which fixed the granular or powdery solid acid catalyst.

As the solid acid catalyst, for example, those having an average particle diameter of about 2 μm to 2 mm and an ion exchange capacity of about 0.01 to 1 eq / L can be used. The hydrolysis of the jellyfish collagen in the present invention is considered to proceed by the action of the acid point on the solid acid catalyst. Therefore, it is preferable to use the solid acid catalyst with a high jellyfish collagen adsorption capacity, and it is preferable to use the solid acid catalyst whose jellyfish collagen adsorption amount is 5-150 g / L. For example, the above-mentioned jellyfish collagen adsorption amount of TOYOPEARL SP-650C by Tosoh Corporation is about 35-55 g / L, and the jellyfish collagen adsorption amount of TOYOPEARL SP-550C is about 80-120 g / L.

As said solid acid catalyst, it is more preferable that it is a porous body. It is preferable that the porous body has a small pore size from the viewpoint of adsorbability as the specific surface area increases, but since hydrolysis in pores of jellyfish collagen larger than the pore size becomes difficult, a size suitable for hydrolysis of jellyfish collagen is obtained. It is preferable to select a solid acid catalyst having pores. The diameter of the preferable pore of the porous body used as a solid acid catalyst is 0.01-0.75 micrometers, More preferably, it is 0.05-0.6 micrometer.

Hydrolysis of jellyfish collagen by solid acid catalyst is carried out by contacting the solid acid catalyst and jellyfish collagen in the presence of water and under heating. It is difficult to advance the hydrolysis reaction well without heating. As heating temperature, it is preferable that it is about 80-160 degreeC, and processing time is about 5 minutes-about 40 hours. It is important to stop the decomposition of jellyfish collagen from the decomposition to jellyfish collagen peptide. When treating at high temperature for a long time, jellyfish collagen may be hydrolyzed to amino acid. Therefore, the higher the treatment temperature, the shorter the treatment time. It is preferable. More preferable treatment conditions are 5 minutes-20 hours at 80-120 degreeC, More preferably, they are 5 minutes-5 hours at 80-120 degreeC, More preferably, they are 5 minutes-2 hours at 90-110 degreeC (solid For acid catalysts see also eg International Publication No. 2010/058590).

These jellyfish collagen peptide mixtures obtained by hydrolysis using peptide degrading enzymes or solid acid catalysts have increased keratinocyte reactivation activity, involukrin production promoting activity, and transglutaminase production promotion compared to jellyfish collagen which is not hydrolyzed. Active and / or anti-inflammatory activity.

In this invention, the peptide containing the amino acid sequence of any of amino acid sequence <1>-<6> can also be synthesize | combined. There is no restriction | limiting in particular in the synthesis | combining method of the peptide containing the said amino acid sequence, For example, C-terminal amino acid is fixed to the solid phase which consists of polystyrene, and an amino group can be extended | stretched by binding a protective amino acid to this, For amino acid protection, Fmoc (Fluorenyl-MethOxy-Carbonyl) groups or Boc (tert-ButOxy-Carbonyl) groups can be used. It is preferable to wash | clean a solid phase and remove unreacted protective amino acid after completion | finish of reaction of a solid amino group and a protective amino acid. Then, the amino group which becomes the next reaction point is obtained by removing (deprotecting) the protecting group of the terminal of the amino acid couple | bonded with the solid phase. By sequentially changing the protective amino acid used, a peptide containing the desired amino acid sequence can be obtained. The peptide of the present invention is a keratinocyte activating activity, involu, which is increased 1.2 times, preferably 1.5 times, more preferably 1.8 times, and even more preferably 2 times, compared to jellyfish collagen that is not hydrolyzed. It exhibits clean production promoting activity, transglutaminase production promoting activity and / or anti-inflammatory activity. The peptide containing any of amino acid sequence <1>-<6> may be a peptide which consists of any of amino acid sequence <1>-<6>.

Jellyfish collagen peptides excellent in cell activating effect, involuclin production promoting effect, transglutaminase production promoting effect, and anti-inflammatory effect of the present invention can be transdermally administered in the form of external preparations such as external preparations for skin, cosmetics, and bathing agents. . From the viewpoint of transdermal administration, the molecular weight of the jellyfish collagen peptide is preferably from 150 to 35,000 Da, more preferably from 250 to 30,000 Da.

There is no restriction | limiting in particular in the formulation at the time of formulating the mixture of the jellyfish collagen peptide of this invention as an external preparation for skin external preparations or cosmetics, It can be set as a paste, cream, a gel, an ointment, a lotion, an emulsion, a pack, a powder, a pape agent It can also be used by impregnating paper products such as wet tissues. Although there is no restriction | limiting in particular in the quantity of the jellyfish collagen peptide mix | blended with a skin external preparation and cosmetics, It is preferable that it is 0.0005-50 mass% in cosmetics among skin external preparations. Although there is no restriction | limiting in particular also in the usage-amount of the external preparation for skin and cosmetics which mix | blended the jellyfish collagen peptide of this invention, It is preferable to divide the quantity which becomes 0.1-1000 mg per adult into 1 to several times by the quantity of jellyfish collagen peptide.

The formulation of the jellyfish collagen peptide mixture of the present invention in the form of a bath solvent as an external preparation is not particularly limited, and may be used as a solid preparation such as powder, granules, liquid preparations such as emulsion, paste, or the like. Although there is no restriction | limiting in particular in the quantity of the jellyfish collagen peptide which can be mix | blended with a bath solvent, It is preferable that it is 0.0005-50 mass% in a bath solvent.

In external preparations such as the external preparation for skin, cosmetics and bathing agents of the present invention, in addition to the jellyfish collagen peptide of the present invention, known functional ingredients used in external preparations for skin, cosmetics or bathing agents, for example, glycerin, butylene glycol, urea, Moisturizing agents such as amino acids; Emollients, such as squalane, macadamia nut oil, and jojoba oil; Blood circulation promoters such as vitamin E and pepper tincture; Antioxidants such as dibutylhydroxytoluene (BHT), dibutylhydroxyanisole (BHA), tocopherol acetate, ascorbic acid; Anti-inflammatory agents such as glycyrrhizin and allantoin; Antibacterial agents such as hinokithiol, benzalkonium chloride, chlorhexidine salt and parahydroxybenzoic acid ester; Whitening agents such as ascorbic acid and arbutin; Various known substances, such as peroxide inhibitors, such as a superoxide dismutase (SOD), can be mix | blended. In addition, various extracts derived from plants, animals, and microorganisms such as ginkgo biloba extract, placental extract, and lactic acid bacteria culture extract can be added.

In addition, to the external preparations such as the external preparation for skin, cosmetics, and bathing agents, base ingredients such as surfactants and fats and oils can be blended for the formulation thereof, and thickeners, preservatives, tonicity agents, antioxidants, ultraviolet absorbers, Various additives, such as a chelating agent, a fragrance | flavor, and a coloring agent, can also be used together.

There is no restriction | limiting in the surfactant which can be used, Alkylene oxide adduct of a higher alkylamine, Alkylene oxide adduct of a higher fatty acid amide, Fatty acid ester of a polyhydric alcohol, Alkylene oxide adduct of hardened castor oil, Polyethylene glycol sorbitan alkyl ester Nonionic surfactants such as alkylene oxide adducts such as sterols, anionic surfactants such as sodium alkyl sulfates, sodium alkyloylmethyltaurine, sodium α-olefin sulfonates, sodium polyoxyalkylene alkyl ether sulfates, alkylpyridinium chlorides, Zwitterionic surfactants, such as cationic surfactant, such as distearyl dimethylammonium chloride, sodium alkylaminopropionate, and alkyl polyamino ethylglycine, can be used. These surfactant can use 1 type and can also use 2 or more types simultaneously.

There is no restriction | limiting in particular in the component used as said base material, Fatty oils, such as olive oil, camellia oil, jojoba oil, avocado oil, macadamia nut oil, almond oil, squalane, squalene, horse oil, can be used.

Although there is no restriction | limiting in particular in the component used as said thickener, Water-soluble polymer is preferable, For example, polyvinyl alcohol, polyacrylamide, polyethyleneglycol, and derivatives thereof; Celluloses such as hydroxyalkyl cellulose and derivatives thereof; Gums such as dextran, gelatin, gum arabic and tragacanth gum; Carboxyvinyl polymer and the like can be used.

There is no restriction | limiting in particular in the component used as said preservative, For example, parahydroxy benzoic acid ester, alkylpyridinium chloride, benzalkonium chloride, chlorhexidine salt, a hinokthiol, etc. can be used.

There is no restriction | limiting in particular in the component used as said isotonic agent, For example, inorganic salts, such as sodium chloride, potassium chloride, a calcium chloride, can be used.

There is no restriction | limiting in particular in the component used as said ultraviolet absorber, For example, a paraamino benzoic acid, a benzophenone type ultraviolet absorber, a benzotriazole type ultraviolet absorber can be used.

There is no restriction | limiting in particular in the chelating agent which can be used, For example, ethylenediaminetetraacetic acid, phytic acid, a citric acid, these water-soluble salts, etc. are mentioned.

Example

Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited.

1. Preparation of Jellyfish Collagen

A total of 1 kg of mussel jellyfish (picked from Tokyo Bay) washed with distilled water was cut into 5 mm-1 cm square pieces. This was stirred for 24 hours at 4 degreeC, and the solution containing a protein was obtained. This was filtered with gauze, and the same amount of Japanese pharmacy room ethanol (A) was added to the filtrate, followed by stirring to obtain a precipitate. To this precipitate, Japanese pharmacy room ethanol (A) and the same amount of Japanese pharmacy room ethanol were added again, and centrifugation (10000 * G) was performed. The obtained solid was dispersed in distilled water, and desalting treatment was performed using a dialysis membrane having a fractional molecular weight (MWCO) of 14000. This was centrifuged (10000 * G), and it divided into the supernatant layer and the precipitation layer, and obtained neutral soluble collagen (supernatant) and acidic soluble collagen (precipitate).

2. Amino Acid Analysis of Extracted Neutral Soluble Collagen

In the analysis of amino acids, 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) was used as the fluorescent derivatization reagent.

10 μg of lyophilized neutral soluble collagen was dissolved in 10 μl of water and dried in a glass tube (6 × 32 mm, CHROMACOL 03-CVG).

Into a glass vial (WHEATON 225289), 200 µl of 6 N hydrochloric acid to which one phenol crystal was added and the glass tube were placed, and the glass vial was sealed after depressurizing with a rotary vacuum pump. This glass vial was set in an aluminum block heater and heated at 110 ° C. for 20 hours to hydrolyze the neutral soluble collagen to amino acid.

10 µl of 20 mM hydrochloric acid, 30 µl of 0.2 M boric acid buffer (pH 8.8), 10 µl of AQC solution (AQC 3 mg / ml acetonitrile solution) were added to the hydrolyzate (amino acid) of the neutral soluble collagen, and 55 ° C. After heating for 10 minutes at, the hydrolyzate (amino acid) of collagen was used as an amino acid fluorescent derivative and separated by high performance liquid chromatography (HPLC).

a) HPLC apparatus: 1200 series by Agilent (including vacuum degasser G1379B, binary pump G1312B, autosampler G1329B, column thermostat G1316B, diode array detector G1315C, fluorescence detector G1321A)

b) Column: Inertsustain C18HP (3.0 × 250 mm, 3 μm), manufactured by GL Science

c) mobile phase

A solution: 95% 5 mM tetrabutylammonium bromide 30 mM phosphate buffer (pH 7.3) / 5% acetonitrile

B solution: 50% 30 mM phosphate buffer (pH 7.3) / 50% acetonitrile

Starting from 98% of A liquid and 2% of B liquid, concentration gradient was formed so that B liquid after 3.75 minutes might be 7.3%, and B liquid after 50 minutes might be 72.3%.

d) flow rate: 0.4 ml / min

e) Temperature: 50 ℃

f) Fluorescence excitation wavelength (Ex): 250 nm

g) Monitor wavelength (Em): 395 nm

Amino acids were quantified using a 10 mmol / L solution of Amino Acid Standard H (2.5 μmol / mL) manufactured by PIA's as a standard amino acid solution. The results are shown in the following table.

Jellyfish collagen contained 30% or more of Gly.

* Lys (OH), 3Pro (OH), 4Pro (OH) represents a hydroxyl group containing amino acid, respectively.

3. Determination of Metal Powders from Jellyfish Collagen Extracted

0.1 g of the freeze-dried neutral soluble collagen was precisely weighed, 10 ml of nitric acid was added, and wet decomposition was performed by microwave (manufactured by MARS5 CEM). The decomposition solution was made up to 50 ml, and the metal powder was measured by a luminescence spectrometer (Optim 5300 DV manufactured by PerkinElmer). The same operation was performed also about acid soluble collagen.

4. Preparation of Jellyfish Collagen Peptides and Interpretation of Molecular Weight Distribution, Mass Spectrometry and Amino Acid Sequence

The molecular weight distribution of the jellyfish collagen peptide mixture in the following example was measured using the gel filtration chromatography (column: TSKgel Super SW2000, Toso).

Solvent: 6 M Guanidine Hydrochloric Acid / 10 mM Phosphate Buffer (pH 6)

Flow rate: 0.15 ml / min

Measurement wavelength: 215 nm

In addition, the obtained jellyfish collagen peptide mixture was separated by reverse phase chromatography, and subjected to mass spectrometry (Ultraflex TOF / TOF, BRUKER DALTONICS) and amino acid sequence analysis (PROCISE 494cLC protein sequencer, Applied Biosystems).

Separation Conditions by Reversed Phase Chromatography

Column: Inertsil ODS-3 (manufactured by GL Science)

Measurement wavelength: 215 nm

Flow rate: 20 μl / min

Mobile phase

  A solution: 0.085% trifluoroacetic acid aqueous solution

  B liquid: 0.075% trifluoroacetic acid / 80% acetonitrile solution

Starting from 100% of A liquid, the concentration gradient was formed so that B liquid might become 12.5% in 10 minutes, 50% in 70 minutes, and 100% in 75 minutes.

About specific peaks, such as the peak of the molecular weight 1112.498 of Example 1, it started from 100% of A liquid and formed the density | concentration gradient like B liquid turned into 100% in 20 minutes.

4-1) Preparation of Jellyfish Collagen Peptides by Peptidase

Example 1

0.01 g of lyophilized neutral soluble collagen was dissolved in 10 ml of water and adjusted to pH 2 with 1 N HCl. 0.5 mass% pepsin (a reagent, Nakarai, Pepsin (1: 10,000)) was added with respect to collagen, and it reacted at 37 degreeC for 3 hours. Then, pepsin was inactivated by adjusting to pH 7 with 1N NaOH. Next, 0.5 mass% papain (a reagent from Wako Junyaku) was added to the collagen, reacted at 60 ° C. for 3 hours, and 0.5 mass% trypsin (a reagent from Wako Junyaku) was added to the collagen. Was added and reacted at 37 degreeC for 3 hours. Thereafter, the enzyme was deactivated by holding at 95 ° C for 30 minutes. This was lyophilized to obtain a mixture of collagen peptides having a molecular weight of 500 to 6000 Da.

In addition, as a result of mass spectrometry and amino acid sequence analysis, the collagen peptide of Example 1 contained a collagen peptide having a molecular weight of 1112.498, and this collagen peptide

Amino Acid Sequence <1> Gly-Pro-Ala-Gly (SEQ ID NO: 1)

It was confirmed to include.

The mass spectrometry result of Example 1 is shown in FIG. 1, and the relationship between the amino acid sequence of molecular weight 1112.498, and the estimated fragment mass of a tandem mass spectrometer is shown in Table 3 below.

Example 2

Except having made neutral soluble collagen of Example 1 into acidic soluble collagen, operation similar to Example 1 was performed and the mixture of the collagen peptide of molecular weight 500-6000 Da was obtained.

Example 3

0.01 g of lyophilized neutral soluble collagen was dissolved in 10 ml of water and adjusted to pH 2 with 1 N HCl. 0.5 mass% pepsin (Reagent, Nakarai, Pepsin (1: 10,000)) was added with respect to collagen, and it reacted at 37 degreeC for 3 hours. Then, pepsin was inactivated by adjusting to pH 8 with 1N NaOH. This was lyophilized to obtain a mixture of collagen peptides having a molecular weight of 2 kDa to 30 kDa.

Example 4)

Except having made neutral soluble collagen of Example 3 into acidic soluble collagen, operation similar to Example 3 was performed and the mixture of the collagen peptide of molecular weight 2kDa-30kDa was obtained.

Example 5

In the operation of Example 3, after deactivation of pepsin, 0.5% by mass of trypsin (a reagent manufactured by Wako Junyaku) was added to collagen, and the mixture was reacted at 37 ° C for 3 hours. Thereafter, the mixture was treated at 95 ° C. for 30 minutes to inactivate trypsin. This was lyophilized to obtain a mixture of collagen peptides having a molecular weight of 500 to 6000 Da.

Example 6

Except having made neutral soluble collagen of Example 5 into acidic soluble collagen, operation similar to Example 5 was performed and the mixture of the collagen peptide of molecular weight 500-6000 Da was obtained.

Example 7

In the operation of Example 3, after reacting for 3 hours at 37 ° C., after adjusting the pH to 1 with 1 N NaOH to inactivate pepsin, 0.5% by mass of papain (a reagent manufactured by Wako Junyaku) was added to collagen. And it was made to react at 60 degreeC for 3 hours. Thereafter, papain was inactivated under the condition of 95 ° C and 30 minutes, and a mixture of collagen peptides having a molecular weight of 1 kDa to 20 kDa was obtained by freeze drying.

Example 8

Except having made neutral soluble collagen of Example 7 into acidic soluble collagen, operation similar to Example 7 was performed and the mixture of the collagen peptide of molecular weight 1 kDa-20 kDa was obtained.

Preparation of Jellyfish Collagen Peptides by Solid Acid Catalyst

Example 9

0.5 g of cation exchange resin (TOYOPEARL SP-550C) and 0.5 ml of a 1% by mass aqueous solution of neutral soluble collagen were mixed, the neutral soluble collagen was adsorbed to the cation exchange resin, and then heated at 90 ° C. for 30 minutes to give neutral soluble collagen. Was hydrolyzed. After cooling, the decomposed product was extracted from the resin with a 5% triethylamine / 10% acetonitrile solution. This was lyophilized to obtain a mixture of collagen peptides having a molecular weight of 2 kDa to 20 kDa.

In addition, as a result of mass spectrometry and amino acid sequence analysis of the collagen peptide of Example 9, the collagen peptide having a molecular weight of 1950.915, and the collagen peptide having a molecular weight of 1950.915,

Amino Acid Sequence <2> Pro-Gly-Val-Lys-Gly-Glu-Val-Gly-Glu-Ala-Gly-Lys-Arg-Gly (SEQ ID NO: 2)

It was confirmed to include.

The result of the mass spectrometry of Example 9 is shown in FIG. 2, and the relationship between the amino acid sequence of molecular weight 1950.915 and the expected fragment mass of a tandem mass spectrometer is shown in following Table 4.

Example 10)

Except having made heating time of Example 9 into 15 minutes, operation similar to Example 9 was performed and the mixture of the collagen peptide of 6 kDa-100 kDa of molecular weight was obtained.

Example 11

Except having made heating time of Example 9 into 60 minutes, operation similar to Example 9 was performed and the mixture of the collagen peptide of molecular weight 1 kDa-10 kDa was obtained.

Example 12

The same operation as in Example 9 was carried out except that the heating temperature of Example 9 was 110 ° C and the heating time was 5 minutes, to obtain a mixture of collagen peptides having a molecular weight of 1 kDa to 10 kDa.

Example 13

Except having made heating time of Example 12 into 30 minutes, operation similar to Example 12 was performed and the mixture of the collagen peptide of molecular weight 1 kDa-5 kDa was obtained.

Example 14

Except having made heating time of Example 12 into 60 minutes, operation similar to Example 12 was performed and the mixture of the collagen peptide of 200-2000 Da of molecular weight was obtained.

Example 15)

Except having made heating time of Example 12 into 2 hours, operation similar to Example 12 was performed and the mixture of the collagen peptide of 200-1000 Da of molecular weight was obtained.

Example 16

Except having made heating time of Example 12 into 4 hours, operation similar to Example 12 was performed and the mixture of the collagen peptide of 200-1000 Da of molecular weight was obtained.

The molecular weight distribution of Examples 12-14 was shown in FIG.

5. Interpretation of Cyanide Degraded Collagen Peptides

10 mass% cyanide bromide was added to the freeze-dried neutral soluble collagen with respect to neutral soluble collagen, and it reacted at 37 degreeC for 20 hours, and the hydrolyzate of neutral soluble collagen was obtained. Hydrolysis by cyanide bromide cleaved at the C-terminal side of methionine has a high specificity and is a method generally used for the analysis of amino acid sequence. The hydrolyzate of neutral soluble collagen was concentrated and centrifuged to evaporate cyanide bromide, and then redissolved in water and subjected to SDS-polyacrylamide gel electrolysis using acrylamide gel (manufactured by Biocraft) having a gel concentration of 5 to 20% by mass. Separation was carried out by electrophoresis. The obtained gel information was blotted for 4 hours to a PVDF membrane at a voltage of 60 V, and the transferred band was analyzed by a protein sequencer (PROCISE 494HT, Applied Biosystems) to obtain the following amino acid sequence information.

Pro (OH) stands for hydroxyproline.

The amino acid sequence <1> (decomposition product by peptide degrading enzyme), amino acid sequence <2> (decomposition product by solid acid catalyst), and said amino acid sequence <3>-<6> are all of the amino acids contained in jellyfish collagen. It is an array and jellyfish collagen before hydrolysis has all of the amino acid sequence <1>-<6>. Therefore, the mixture of the jellyfish collagen peptide obtained by hydrolyzing this jellyfish collagen will contain the collagen peptide containing amino acid sequence <1>-<6>, regardless of a means of degradation.

Of these six amino acid sequences, a synthetic product (consigned to Hokkaido Systems Science Co., Ltd.) consisting of the amino acid sequences of amino acid sequences <3> to <6> was prepared, which were referred to as Examples 17 to 20.

6. Evaluation of the Effect of Jellyfish Collagen Peptides

Evaluation Test 1 (Resurrection Test Using Keratinocytes)

The cell activating effect was evaluated by the cell proliferation effect using the activated culture cells and the cell proliferation reagent WST-1 (Roche).

Human skin-derived immortalized keratinocytes PHK16-0b received from the Human Science Promotion Foundation were seeded in 2 x 10 ⁴ cells in 48 well multiplates, and cultured for 2 days with addition of 300 µl KGM medium. Thereafter, the mixture was replaced with 300 µl of KGM medium having a jellyfish collagen concentration of 10 µg / ml, and cultured in an incubator at 37 ° C for 7 days. It was again exchanged with 270 µl of fresh KGM medium, 30 µl of cell proliferation reagent WST-1 was added and reacted in an incubator at 37 ° C for 3 hours, and then the absorbance at 440 nm of the medium was measured. The cell proliferation rate of the collagen peptide was evaluated (n = 3) by making the cell viability of the control cultured in the same conditions without adding collagen peptide 100%. The results are shown below.

The cell proliferation effect was also confirmed in the jellyfish collagen peptide obtained under any hydrolysis conditions, and the peptide degradation enzyme was especially excellent in the collagen peptide obtained by hydrolyzing with pepsin and further hydrolyzing with papain and trypsin.

The cell proliferation effect of the jellyfish collagen peptide obtained by the solid acid catalyst was also favorable, and the result was favorable for the jellyfish collagen peptide within 30 minutes of treatment time in 110 degreeC treatment. The cell proliferation effect of the peptide prepared based on the amino acid sequence information of jellyfish collagen was also favorable, and the outstanding effect was confirmed especially in Example 17. On the other hand, the jellyfish collagen itself could not confirm the cell proliferation effect of neutral soluble collagen (Comparative Example 1) and acid soluble collagen (Comparative Example 2).

Evaluation test 2 (Involukrin expression test using keratinocytes)

Adult human skin-derived normal keratinocytes (manufactured by Kozine Bio) were seeded in 48 well multiplates at 1 × 10 ⁵ , and 300 μl of KGM medium was added thereto, followed by incubation at 37 ° C. for 24 hours. The jellyfish collagen peptide was replaced with 300 µl of KGM medium having a concentration of 10 µg / ml and incubated at 37 ° C for 48 hours. The cells were collected and the amount of involuclin produced was measured by Western blotting. In addition, anti-human involukrin (Cosmobio, sc-28557) was used as a primary antibody, and anti-rabbit IgG-HRP (Cosmobio, sc-2004) was used as a secondary antibody. The results were interpreted by ImageJ to evaluate the involukrin expression rate for the no addition group (control). The results are shown below.

It was confirmed that a good involukrin expression effect on the mixture of jellyfish collagen peptide obtained by hydrolyzing jellyfish collagen with a peptide degrading enzyme and a solid acid catalyst. The involukrin production effect was recognized also in the peptide synthesize | combined based on amino acid sequence information.

Evaluation test 3 (transglutaminase 1 expression test using keratinocytes)

20 × 10 ⁴ human skin-derived immortalized keratinocytes PHK16-0b were seeded in 48 well multiplates, and 300 µl of KGM medium was added thereto, followed by incubation at 37 ° C for 24 hours. The jellyfish collagen peptide was replaced with 300 µl of KGM medium having a concentration of 10 µg / ml, cultured at 37 ° C for 48 hours, and the cells were recovered, and the amount of transglutaminase 1 produced by Western blotting was measured. In addition, antitransglutaminase 1 (Cosmobio, PAB15545) was used as a primary antibody, and anti-rabbit IgG-HRP (Cosmobio, sc-2004) was used as a secondary antibody. The results were interpreted by ImageJ to evaluate the transglutaminase 1 expression rate for the no addition group (control). The results are shown below.

A favorable effect of transglutaminase 1 expression on the mixture of jellyfish collagen peptides obtained by hydrolyzing jellyfish collagen with a peptidase was confirmed. The effect of transglutaminase 1 production was also confirmed in the peptide synthesized based on the jellyfish collagen peptide mixture obtained by hydrolysis with a solid acid catalyst and the amino acid sequence information.

Evaluation test 4 (COX-2 inhibitory effect test using keratinocytes)

The anti-inflammatory effect was evaluated by the amount of PGE ₂ produced by COX-2 of keratinocytes subjected to ultraviolet irradiation. The lower the PGE ₂ production, the more jellyfish collagen peptide is anti-inflammatory.

20 × 10 ⁴ human skin-derived immortalized keratinocytes PHK16-0b were seeded in 48 well multiplates, 300 μl of KGM medium was added, and cultured overnight. 300 µL of KGM medium having an aspirin concentration of 500 µM was exchanged and cultured for 4 hours to inactivate COX-2 already expressed. Washing with PBS (-) was repeated three times, exchanged with DMEM medium having a jellyfish collagen peptide concentration of 10 µg / ml, and irradiated with 50 mJ of UVB, followed by incubation for 48 hours. Thereafter, the amount of PGE ₂ in the medium supernatant was measured by a prostaglandin E ₂ EIA kit (manufactured by Cayman Chemical) (n = 3).

A good anti-inflammatory effect can be confirmed in the jellyfish collagen peptide mixture, and in particular, the excellent anti-inflammatory effect was confirmed in the mixture of jellyfish collagen peptides obtained by digesting jellyfish collagen with pepsin and further digesting with trypsin.

7. Formulation Example for Cosmetics

The component A and component B were each heated to 80 ° C. to dissolve, and the component B was gradually added to component A while emulsifying while stirring. Then, it stirred, stirring, and added C component adjusted to the 0.2 mass% aqueous solution at 40 degreeC, and adjusted the lotion for whole body.

(A) component

    Grape Seed Oil 0.1 (g)

    Polyoxyethylene (60 Mole) Cured Castor Oil 0.7

    Diglyceryl Monoisostearate 0.3

Component (B)

    Citric Acid 0.08

    Citrate Na 0.3

    1,3-butanediol 5

    Glycerin 3

    Ethanol 5

    Xanthan Gum 0.1

    Methylparaben 0.2

    Purified water level

(C) Component

    Jellyfish Collagen Peptides (0.2% by mass) 0.5

  100 total

evaluation

The whole body lotion of the said composition is used twice a day (morning, night), a face, and a lower arm suitably, and the cosmetic or acidic formulation which mix | blended the jellyfish collagen peptide of Examples 1-20 on one side of a face and a lower arm The cosmetics which mix | blended soluble collagen (comparative example 2) and the cosmetics which mix | blended neutral soluble collagen (comparative example 1) were used for the other one. This was continued for 1 month and it judged by the following evaluation criteria. The results are shown in Table 9.

In addition, no one complained of abnormal skin during this period of use.

Score value

+3: I felt the moisture stronger than the cosmetics which combined the comparative example 1

+2: I could feel quite gloss than the cosmetics which contained the comparative example 1

+1: It could feel a little more moisture than the cosmetics which contained the comparative example 1

 0: no difference

-1: The cosmetics which contained the comparative example 1 could feel a little moisture

-2: The cosmetics containing the comparative example 1 could feel quite gloss

-3: The cosmetics containing the comparative example 1 felt moisture strongly

Also in any mixture of jellyfish collagen peptides, the skin shine effect exceeding the cosmetics which contained neutral soluble collagen was obtained.

As the hydrolyzate by the peptidase, a particularly good moistening effect was obtained in the jellyfish collagen peptide mixture obtained by hydrolyzing neutral or acidic soluble collagen with pepsin and then further hydrolyzing with papain and trypsin.

In the hydrolyzate by a solid acid catalyst, favorable moisture effect was recognized by the jellyfish collagen peptide mixture obtained by the process of 90 degreeC of treatment temperature, and processing time for 30 minutes.

Also in the cosmetics which mix | blended the peptide synthesize | combined based on the amino acid sequence information of jellyfish collagen, the skin moisture effect was favorable.

Industrial availability

By using collagen peptides derived from jellyfish collagen, keratinocyte activating effect, involuclin production promoting effect, transglutaminase production promoting effect, and anti-inflammatory effect can be obtained. By repairing and improving, it becomes possible to normalize the function of epidermal keratinocytes.

                SEQUENCE LISTING <110> NICCA CHEMICAL CO., LTD   <120> Jellyfish collagen peptide mixture <130> FP1177-KR <160> 6 <170> PatentIn version 3.3 <210> 1 <211> 4 <212> PRT <213> Aurelia aurita <400> 1 Gly Pro Ala Gly One <210> 2 <211> 14 <212> PRT <213> Aurelia aurita <400> 2 Pro Gly Val Lys Gly Glu Val Gly Glu Ala Gly Lys Arg Gly 1 5 10 <210> 3 <211> 12 <212> PRT <213> Aurelia aurita <400> 3 Val Gly Pro Val Thr Glu Glu Val Ser Pro Ala Lys 1 5 10 <210> 4 <211> 10 <212> PRT <213> Aurelia aurita <400> 4 Ser Ala Gly Gln Asn Pro Ala Asp Asp Val 1 5 10 <210> 5 <211> 9 <212> PRT <213> Aurelia aurita <220> <221> MOD_RES (222) (3) .. (3) <223> Hyp <400> 5 Gly Thr Pro Gly Ala Gly Gly Ser Arg 1 5 <210> 6 <211> 7 <212> PRT <213> Aurelia aurita <400> 6 Gln Gly Pro Gln Gly Glu Leu 1 5

Claims (13)

Jellyfish collagen peptide mixture containing a jellyfish collagen peptide comprising any of the following amino acid sequences.

Pro (OH) stands for hydroxyproline. The method of claim 1,
Jellyfish collagen peptide mixture obtained by hydrolyzing jellyfish collagen. The method of claim 2,
A jellyfish collagen peptide mixture, characterized in that hydrolysis is performed using at least one peptide degrading enzyme selected from the group consisting of trypsin, pepsin, and papain. The method of claim 2,
Jellyfish collagen peptide mixture characterized in that the hydrolysis is carried out using a solid acid catalyst. The method of claim 4, wherein
Jellyfish collagen peptide mixture, characterized in that the solid acid catalyst is at least one solid acid catalyst selected from the group consisting of cation exchanger, zeolite and diatomaceous earth. 6. The method according to any one of claims 1 to 5,
Jellyfish collagen peptide mixture wherein jellyfish collagen is mule jelly collagen or nomura nugget jelly collagen. Jellyfish collagen peptide mixture obtained by hydrolyzing jellyfish collagen using a peptide decomposing enzyme or a solid acid catalyst. Peptides comprising any of the following amino acid sequences.

Pro (OH) stands for hydroxyproline. The external preparation containing the jellyfish collagen peptide mixture of any one of Claims 1-7, and / or the peptide of Claim 8. The keratinocyte activator containing the jellyfish collagen peptide mixture of any one of Claims 1-7, and / or the peptide of Claim 8. An involukrin production promoter comprising the jellyfish collagen peptide mixture according to any one of claims 1 to 7, and / or the peptide according to claim 8. The transglutaminase production promoter containing the jellyfish collagen peptide mixture of any one of Claims 1-7, and / or the peptide of Claim 8. The anti-inflammatory agent containing the jellyfish collagen peptide mixture of any one of Claims 1-7, and / or the peptide of Claim 8.

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