KR102831123B1 - Cosmetic composition containing alpha-1,4-1,6-glucan polymer and method for preparing thereof - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising an alpha-1,4-1,6-glucan polymer obtained by reacting sucrose with amylosecrase and glycogen branching enzyme, and a method for producing the same. The cosmetic composition of the present invention is environmentally friendly, has excellent UV blocking ability, and has excellent skin whitening ability.

Description

{COSMETIC COMPOSITION CONTAINING ALPHA-1,4-1,6-GLUCAN POLYMER AND METHOD FOR PREPARING THEREOF}

The present invention relates to a cosmetic composition comprising alpha-1,4-1,6-glucan polymer.

Polysaccharides are among the most abundant macromolecules in living systems, and are components of cell walls and have diverse functions, including transport, adhesion, immune responses, and energy storage. Glycogen is a hyperbranched homopolysaccharide that is the glucose storage site in bacteria, fungi, animals, and plants. Glycogen isolated from natural sources has a molecular weight of about 10 ⁷ -10 ⁹ g mol ^-1 and consists of a chain backbone of D-glucose residues linked by α-1,4-1,6-glycosidic bonds.

The composition of glycogen has the advantages of very high biocompatibility, biodegradability and water solubility compared to other hydrocolloids, but the glycogen content of animal tissues is generally very low and it is uneconomical to extract glycogen from animal tissues. Therefore, there are limits to producing animal-derived glycogen, so various synthetic methods have been studied, and particles similar to glycogen having the aforementioned α-1,4-1,6-glycosidic bonds can be produced using amylosecrase and glycogen branching enzyme.

Ultraviolet (UV) rays are one of the environmental stress factors that can damage human skin. UV rays (100-400 nm) are classified into UVA, UVB, and UVC depending on their wavelength. In particular, UVB (280-320 nm) is a major factor that is absorbed into the epidermis and damages the skin (Piao, M. J., Hyun, Y. J., Cho, S. J., Kang, H. K., Yoo, E. S., Koh, Y. S., Hyun, J. W. (2012). An ethanol extract derived from Bonnemaisonia hamifera scavenges ultraviolet B (UVB) radiation-induced reactive oxygen species and attenuates UVB-induced cell damage in human keratinocytes. Marine drugs, 10(12), 2826-2845.). UVA (320-400 nm) penetrates deep into the skin and causes wrinkle formation and skin aging. Therefore, in order to prevent health problems caused by ultraviolet rays, it is important to block ultraviolet rays with sunscreen. However, oxybenzone (2-hydroxy-4-methoxybenzophenone), which is currently widely used as an ingredient in sunscreen, has become a problem because many studies have shown that chemicals such as oxybenzone and octinoxate can be harmful to health and the environment. According to the American environmental group EWG, it warns that there is a risk of contact dermatitis or respiratory disorders, and it can cause hormonal imbalance in adult males or infertility in women by decreasing the male hormone testosterone. Recently, a law was passed in the state of Hawaii to ban the sale of sunscreens containing oxybenzone and octinoxate. Therefore, there is a need for a substance that can protect both the environment and health and has ultraviolet ray blocking properties.

In addition, melanin is synthesized in organelles called melanosomes of melanocytes that exist in the basal layer of the epidermis. Melanin is related to the pigmentation of human skin produced by epidermal melanocytes, and the production of melanin is one of the important skin defense mechanisms that protects the skin from harmful ultraviolet rays in the external environment. However, overexpressed melanin can cause skin diseases such as freckles, melasma, and cancer. In the process of melanin synthesis, tyrosinase is an enzyme that promotes melanin synthesis, and the biosynthesis of melanin pigment is regulated by various enzymes including tyrosinase enzyme. Among them, tyrosinase is involved in the initial biosynthetic process of oxidizing tyrosine to convert it into L-dopaquinone, and then oxidizes dihydroxyindole to participate in the synthesis of melanin (Decker & Tuczek, 2000; Iozumi, Hoganson, Pennella, Everett, & Fuller, 1993; Jimenez-Atienzar, Escribano, Cabanes, Gand a-Herrero, & Garcia-Carmona, 2005). Therefore, many studies are being conducted to control melanin synthesis by inhibiting tyrosinase in the development of whitening cosmetics.

Korean Patent No. 10-1561231

The present invention aims to provide an environmentally friendly cosmetic composition having excellent UV protection efficacy, comprising an alpha-1,4-1,6-glucan polymer, and a method for producing the same.

The present invention aims to provide an environmentally friendly cosmetic composition having excellent skin whitening efficacy, comprising an alpha-1,4-1,6-glucan polymer, and a method for producing the same.

1. A method for producing a cosmetic composition comprising the step of reacting sucrose as a substrate with amylosecrase and glycogen branching enzyme to obtain an alpha-1,4-1,6-glucan polymer.

2. A method for producing a cosmetic composition according to the above 1, wherein the amylosucrase is composed of any one of the amino acid sequences of sequence numbers 1 to 3.

3. A method for producing a cosmetic composition according to the above 1, wherein the glycogen branching enzyme is composed of any one of the amino acid sequences of sequence numbers 4 to 6.

4. A method for manufacturing a cosmetic composition in the above 1, wherein the activity ratio of the amylosucrase and the glycogen branching enzyme is 1:0.8 to 5.2.

5. A method for manufacturing a cosmetic composition for skin whitening or ultraviolet ray blocking in the above 1.

6. A cosmetic composition manufactured by any one of the methods 1 to 4 above.

7. A cosmetic composition for skin whitening or ultraviolet ray blocking according to 6 above.

The cosmetic composition manufactured by the manufacturing method of the present invention is non-toxic, safe for the skin, and has an excellent UV protection effect.

The cosmetic composition manufactured by the manufacturing method of the present invention has an excellent skin whitening effect by inhibiting melanin synthesis in skin cells.

Figure 1 shows a portion of an alpha-1,4-1,6-glucan polymer in which glucose units form alpha-1,4 glycosidic linkages and alpha-1,6 glycosidic linkages.
Figure 2 is a schematic diagram illustrating a method for producing glucan polymer (GLP) by simultaneously applying amylosucrase and glycogen branching enzyme.
Figures 3 and 4 show the distribution of branch chain lengths of Np/Vv GLP. Figure 3 (A) shows the distribution of branch chain lengths of Np/Vv GLP of r1; (B) shows the distribution of branch chain lengths of Np/Vv GLP of r2; (C) shows the distribution of branch chain lengths of Np/Vv GLP of r3; and (D) shows the distribution of branch chain lengths of Np/Vv GLP of r4. Figure 4 (E) shows the distribution of branch chain lengths of Np/Vv GLP of r5; (F) shows the distribution of Np/Vv GLP of r6; (G) shows the distribution of Np/Vv GLP of r7; (H) shows the distribution of Np/Vv GLP of r8; (I) shows the distribution of Np/Vv GLP of r9; (J) shows the distribution of Np/Vv GLP of r10; and (K) shows the distribution of branch chain lengths of oyster glycogen.
Figures 5 to 8 show the UV absorbance of alpha-1,4-1,6-glucan polymers (GLP) according to the activity ratios of various types of amylosucrase (As) and glycogen branching enzyme (GBE).
Figure 5 shows the UV absorbance of Np/Vv GLP in terms of the activity ratio of As derived from Np and GBE derived from Vv. ● indicates oxybenzone, △ indicates glycogen, ○ indicates Np/Vv GLP r1, ▼ indicates Np/Vv GLP r2, and ■ indicates Np/Vv GLP r5 conditions.
Figure 6 shows the UV absorption rate of Np/Vv GLP according to the activity ratio of Bt-derived As and Bt-derived GBE. ○ indicates oxybenzone, ● indicates glycogen, ▼ indicates Np/Vv GLP r1, △ indicates Np/Vv GLP r3, and ■ indicates Np/Vv GLP r5 conditions.
Figure 7 shows the UV absorption rate of Bt/Ro GLP according to the activity ratio of Bt-derived As and Ro-derived GBE. ■ indicates oxybenzone, ● indicates glycogen, ○ indicates Bt/Ro GLP r1, ▼ indicates Bt/Ro GLP r3, and △ indicates Bt/Ro GLP r5 conditions.
Figure 8 shows the UV absorption rates of Ns/Bt GLP and Ns/Ro GLP according to the ratio of As derived from Ns and GBE activity derived from Bt or Ro. ○ indicates oxybenzone, ● indicates glycogen, ▼ indicates Ns/Bt GLP r1, △ indicates Ns/Bt GLP r3, ■ indicates Ns/Bt GLP r5, and □ indicates Ns/Ro GLP r5 conditions.
Figure 9 shows the cytotoxicity of Np/Vv GLP prepared by varying the activity ratio of amylosucrase and glycogen branching enzyme at different concentrations. Control is the untreated control group.
Figures 10 and 11 show the concentration-dependent tyrosinase activity inhibition rates of alpha-1,4-1,6-glucan polymers prepared by varying the activity ratios of various types of amylosucrase (As) and glycogen branching enzyme (GBE).
Figure 10 shows the activity ratio and the tyrosinase activity inhibition rate according to the concentration of Np/Vv GLP. Control is the control group treated only with tyrosinase enzyme and distilled water.
Figure 11 shows the activity ratio and the concentration-dependent tyrosinase activity inhibition rate of Bt/Ro GLP, Ns/Ro GLP, Bt/Bt GLP, Ns/Bt GLP, and Np/Vv GLP. NC is a negative control group treated only with tyrosinase enzyme and distilled water, and PC is a positive control group treated with tyrosinase enzyme, L-DOPA, and DW.
Figures 12 and 13 show the inhibition rate of melanin production in B16F10 melanoma cells according to the treatment concentration of alpha-1,4-1,6-glucan prepared by varying the activity ratios of amylosucrase and glycogen branching enzyme. B16F10 melanoma cells were treated with 100 nM α-MSH and the alpha-1,4-1,6-glucan polymer (GLP) of the present invention and cultured in a CO ₂ incubator for 72 hours, and the amount of melanin synthesized by the cells was measured. When B16F10 melanoma cells were treated with only a-MSH, the melanin production amount was set as 100%. Control is a group in which melanoma cells were not treated with anything.
Figure 12 shows the results of measuring the amount of melanin synthesized by cells when treated with Np/Vv GLP at different concentrations (0.625% (w/v), 0.125% (w/v), 0.25% (w/v)).
Figure 13 shows the results of measuring the amount of melanin synthesized by cells when treated with Ns/Bt GLP, Ns/Ro GLP, Bt/Bt GLP, or Bt/Ro GLP at a concentration of 0.125% (w/v).

The present invention relates to a method for producing a cosmetic composition, comprising a step of reacting sucrose as a substrate with amylosesucrase (AS) and glycogen branching enzyme (GBE) to obtain an alpha-1,4-1,6-glucan polymer.

The above alpha-1,4-1,6-glucan polymer is a polymer composed of alpha-1,4 and alpha-1,6 glycosidic bonds of glucose units, and can be obtained by reacting sucrose with amylosecrase and glycogen branching enzyme. The alpha-1,4 glycosidic bond allows a linear glucan polymer to be formed from glucose. The alpha-1,6 glycosidic bond is a bond between an internal glucose unit of a linear glucan and a glucose unit of another linear glucan, and allows a branch to be formed in the linear glucan. In addition, another linear glucan can be bonded to the branch formed in the linear glucan, and thus an alpha-1,4-1,6 glucan polymer having a plurality of side branches as illustrated in FIG. 2 can be formed.

The above amylosucrase is an enzyme that uses sucrose as a substrate, hydrolyzes sucrose into glucose and fructose, and then transfers glucose to an acceptor substance having a different alpha 1,4 glycosidic bond, thereby producing an insoluble or water-soluble polysaccharide. In this specification, the term "AS" means amylosucrase unless otherwise indicated.

The above amylosucrase may be derived from, for example, Neisseria Polysaccharea (Np), Bifidobacterium thermophilum (Bt) or Neisseria subflava (Ns).

The above amylosucrase (As) may be composed of, for example, any one of the amino acid sequences of SEQ ID NOS: 1 to 3. SEQ ID NOS: 1 to 3 are, in order, the amino acid sequences of amylosucrase (As) derived from Neisseria Polysaccharea (Np), Bifidobacterium thermophilum (Bt), and Neisseria subflava (Ns).

The above glycogen branching enzyme (GBE) is an enzyme that hydrolyzes straight-chain glucans having alpha 1,4 glycosidic bonds and transfers them to an alpha 1,6 glycosidic bond in an acceptor substance.

The above glycogen branching enzyme may be derived from, for example, Vibrio vulnificus (Vv), Rhodothermus obamensis (Ro), Bifidobacterium thermophilum (Bt), Deinococcus geothermalis (Dg), or Synechocytis.

The above glycogen branching enzyme may be composed of, for example, any one of the amino acid sequences of SEQ ID NOs: 4 to 6. SEQ ID NOs: 4 to 6 are, in order, amino acid sequences of glycogen branching enzymes derived from vulnificus (Vv), Rhodothermus obamensis (Ro), and Bifidobacterium thermophilum (Bt), respectively.

The above amylosucrase and glycogen branching enzyme can be simultaneously treated with sucrose as a substrate to produce alpha-1,4-1,6-glucan polymer in a single process (one-pot synthesis).

The above alpha-1,4-1,6-glucan polymer can control the branch length of the alpha-1,4-1,6-glucan polymer produced by controlling the ratio of the enzyme activities of amylosucrase and glycogen branching enzyme. The greater the enzyme activity of glycogen branching enzyme compared to amylosucrase, the more short-length branches are formed.

In the present invention, the amylosucrase enzyme activity was measured using the amount of reducing sugar produced by reacting with sucrose, a substrate. The reaction was performed for 10 minutes in 50 mM Tris-HCl buffer (pH 7.0) containing 0.1 M sucrose and 0.1% (w/v) waxy corn starch at 35°C, and then 500 μL DNS solution was added to terminate the reaction. The reaction mixture was then colored by heating in boiling water for 5 minutes. The mixture was cooled on ice to stabilize the color. The absorbance change at 575 nm (Dabs) was measured using a spectrophotometer (Beckman DU 730; CA). 1 unit (1 U) of enzyme activity was defined as the amount of enzyme that causes 1 unit of absorbance change (Dabs) per minute.

In the present invention, the enzyme activity of glycogen branching enzyme was measured using potato amylose type 3 (Sigma-Aldrich chemical Co., St. Louis, MO). The enzyme reaction was performed for 20 minutes using 0.05% (w/v) potato amylose type 3 (Sigma-Aldrich chemical Co., St. Louis, MO) in 50 mM Tris-HCl (pH 7.0) at 30°C. Thereafter, the reacted mixture was mixed with 0.02% Lugol's solution, and the absorbance change (Dabs) of the solution was measured at 620 nm. One unit (1 U) of enzyme activity was defined as the amount of enzyme that causes an absorbance change of 1 unit per minute.

The enzyme activity ratio of the above amylosucrase and glycogen branching enzyme may be 1:0.8 to 5.2. For example, it may be 1:0.8 to 3.2, and preferably, it may be 1:0.8 to 1.2.

In one embodiment of the present invention, it was confirmed that the ultraviolet absorbing ability of alpha-1,4-1,6-glucan produced at an enzyme activity ratio of amylosucrase and glycogen branching enzyme in the range of 1:1 to 3 was excellent, and among them, the ultraviolet absorbing ability was the best at 1:1 (Figs. 5 to 8).

In one embodiment of the present invention, it was confirmed that the melanin synthesis inhibition effect was excellent when the enzyme activity ratio of amylosucrase and glycogen branching enzyme was in the range of 1:1 to 5, and among them, the melanin synthesis inhibition effect was the best at 1:1 (Figs. 12 and 13).

The time, temperature, pH conditions, etc. for reacting the above substrates, sucrose, amylosucrase, and glycogen branching enzyme, can be appropriately controlled by those skilled in the art.

The above reaction temperature may be, but is not limited to, 25 to 50°C, 30 to 50°C, 40 to 55°C or 30 to 45°C.

The reaction time of the above substrates, sucrose, amylosucrase and glycogen branching enzyme, may be 10 hours, 20 hours, 30 hours, 40 hours or more, 50 hours or less, or 60 hours or less, but is not limited thereto.

The above-mentioned manufactured alpha-1,4-1,6-glucan polymer may have an average molecular weight of 1 x 10 ⁵ to 1 x 10 ⁷ g/mol, 1 x 10 ⁵ to 1 x 10 ⁶ g/mol or 1 x 10 ⁵ to 5 x 10 ⁵ g/mol, but is not limited thereto.

Since the alpha-1,4-1,6-glucan polymer manufactured by the above method has excellent ultraviolet ray absorption rate, a cosmetic composition containing it can protect the skin from ultraviolet ray.

Since the alpha-1,4-1,6-glucan polymer manufactured by the above method has an excellent ability to inhibit melanin synthesis in skin cells (Figs. 12 and 13), a cosmetic composition containing it has a skin whitening effect.

The present invention provides a cosmetic composition for skin whitening or UV protection, manufactured by the method described above.

The cosmetic composition manufactured by the method described above has an excellent skin whitening effect through inhibition of melanin production.

The cosmetic composition manufactured by the method described above has excellent UV protection effect.

A cosmetic composition comprising an alpha-1,4-1,6-glucan polymer manufactured according to the present invention may include conventional auxiliary agents and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and may be manufactured in any formulation conventionally manufactured in the art, and may be formulated as, for example, a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleansing, an oil, a powder foundation, an emulsion foundation, a wax foundation and a spray, but is not limited thereto. More specifically, the cosmetic composition may be manufactured in the form of a flexible toner, a nourishing toner, a nourishing cream, a massage cream, an essence, an eye cream, a hair tonic, a shampoo, a rinse, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane/butane or dimethyl ether may be additionally included.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizer or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as carrier components.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkyl amidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used as a carrier component.

If the formulation of the present invention is a lotion, it may contain natural essential oils and a moisturizer.

As a natural essential oil, it is preferable to use at least one selected from the group consisting of vegetable oils including lavender oil, olive oil, jojoba oil, and macadamia oil, but is not limited thereto.

Examples of moisturizers include water-soluble low-molecular-weight moisturizers, fat-soluble molecular-weight moisturizers, water-soluble polymers, and fat-soluble polymers.

Examples of water-soluble low-molecular-weight moisturizers include serine, glutamine, sorbitol, mannitol, sodium pyrrolidone-carboxylate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B (polymerization degree n = 2 or higher), polypropylene glycol (polymerization degree n = 2 or higher), polyglycerin B (polymerization degree n = 2 or higher), lactic acid, and lactate salts.

Examples of fat-soluble low-molecular-weight moisturizers include cholesterol and cholesterol esters.

Examples of water-soluble polymers include carboxyvinyl polymer, polyaspartate, tragacanth, xanthan gum, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, water-soluble chitin, chitosan, dextrin, etc.

Examples of lipid-soluble polymers include polyvinylpyrrolidone-eicosene copolymer, polyvinylpyrrolidone-hexadecene copolymer, nitrocellulose, dextrin fatty acid ester, and polymer silicone.

The term “GLP” in this specification is an abbreviation for Glycogen-like particles and refers to alpha-1,4-1,6-glucan polymer. In addition, the term “A/B GLP” refers to alpha-1,4-1,6-glucan polymer obtained by reacting sucrose with amylosecrase (As) from organism A and glycogen branching enzyme (GBE) from organism B. For example, “Np/Vv GLP” is an alpha-1,4-1,6-glucan polymer obtained by reacting sucrose with NpAS and VvGBE.

The term “rX (X is any number)” herein means that the enzyme activity ratio of amylosucrase and glycogen branching enzyme is 1: X. For example, “Np/Vv GLP rX” is an alpha-1,4-1,6-glucan polymer obtained by adjusting the activity ratio of NpAs and VvGBE to 1:X. That is, Np/Vv GLP r1 is an alpha-1,4-1,6-glucan polymer obtained by adjusting the enzyme activity ratio of NpAs and VvGBE to 1:1, and Np/Vv GLP r5 is an alpha-1,4-1,6-glucan polymer obtained by adjusting the enzyme activity ratio of NpAs and VvGBE to 1:5.

Hereinafter, the present invention will be described in more detail through examples.

Experimental method

1. Materials

Sucrose, potato amylose, and oyster glycogen were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Waxy corn starch was provided by Daesang (Seoul, Korea). Pullulan standard P-20 was obtained from Showa Denko KK (Tokyo, Japan).

2. Expression and purification of enzymes

Amylosucrase genes from Neisseria polysaccharea (Np, ATCC 43768), Bifidobacterium thermophilum (Bt, ATCC 25525), and Neisseria subflava (Ns, ATCC 49275) and GBE genes from Rhodothermus obamensis (Ro), Bifidobacterium thermophilum (Bt, JCM 1207), and Vibrio vulnificus (Vv, MO6-24/O) were cloned and expressed according to methods reported in previous studies (Wang et al., 2012; Palomo et al., 2009; Jo et al., 2015; Lee et al., 2008). That is, recombinant As and GBE plasmids were transformed into E. coli BL21 (DE3) host cells for cloning and expression.

GBE isolated from Rhodothermus obamensis was obtained using a commercial product (Branchzyme®) from Novozymes (Bagsvaerd, Denmark).

E. coli BL21 cells harboring the As and GBE genes were grown at 37°C in LB medium supplemented with 50 μg mL ^-1 ampicillin. When Abs at 600 nm of the cell culture reached 0.6-0.8, 0.2 M IPTG was added and the cells were cultured for an additional 18 h at 18°C to induce the expression of the enzyme protein.

E. coli BL21 cells were grown at 30°C in LB broth supplemented with antibiotics (50 μg mL ^-1 ampicillin or 50 μg mL ^-1 of kanamycin), and proteins were expressed by the same IPTG induction. The cell pellet was harvested by centrifugation (5,000 × g for 10 min at 4°C), resuspended in 50 mM Tris-HCl buffer (pH 7.0), and disrupted using a sonicator (VibraTM Cell VC 750 disruptor, Sonics Materials, Inc, Newtown, CT) on an ice bath. The disrupted cells were centrifuged (10,000 × g for 20 min at 4°C) and filtered through a 0.45-μm syringe filter. Enzymes extracted from cells were purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography (Qiagen, Hilden, Germany).

The purified enzyme solution was concentrated by centrifugation (5,000 rpm for 20 min at 25°C) using an Amicon ultra-15 centrifugal filter (MWCO=30 kDa; Merck Millipore, Carrigtwohill, Ireland) and a centrifuge.

3. Enzyme activity test

Amylosucrase (ASase) activity of NpAS was determined by measuring the amount of reducing sugar produced during the reaction on the sucrose substrate. The enzymatic reaction was performed for 10 min in 50 mM Tris-HCl buffer (pH 7.0) containing 0.1 M sucrose and 0.1% (w/v) waxy corn starch at 35°C, and then the reaction was terminated by the addition of 500 μL DNS solution. The reaction mixture was then colored by heating in boiling water for 5 min. The mixture was cooled on ice for color stabilization. The change in absorbance at 575 nm (Δabs) was measured using a spectrophotometer (Beckman DU 730; CA). To measure the enzyme activity of VvGBE, the enzyme reaction was performed in 50 mM Tris-HCL (pH 7.0) supplemented with 0.05% (w/v) potato amylose type 3 (Sigma-Aldrich chemical Co., St. Louis, MO) for 20 min. The reaction mixture was then mixed with 0.02% Lugol's solution, and the Dabs of the solution were measured at 620 nm. In both NpAS and VvGBE, 1 unit of enzyme activity (1 U) means the amount of enzyme that induces 1 unit of absorbance change (Δabs) per min.

4. Production of Glycogen-Like Particles (GLP) Using Amylosucrase (AS) and Glycogen Branching Enzyme (GBE)

To produce glycogen-like particles (GLPs), a one-pot enzymatic reaction of AS and GBE was performed in 50 mM HCl-Tris buffer (pH 7.0) at 30 °C for 48 h using 0.7 M sucrose as a substrate. The AS activity level was fixed at 6 U/mL, and 10 different activity levels of GBE were applied in increments of 0.001 U from 0.001 U to 0.01 U to evaluate the effect of changing the GBE/ASase activity ratio. After the enzymatic reaction, the sample mixture was boiled for 10 min to inactivate the enzyme. To remove by-products such as fructose, monosaccharides, disaccharides, and oligosaccharides, the reaction mixture was filtered through an Amicon μLtra-15 centrifugal filter tube (100 kDa MWCO, Merck Millipore Ltd, Burlington, MA). The residue was transferred to a regular centrifuge tube and mixed with 75% (v/v) ethanol before centrifugation (10,000 ×g for 10 min at 25 °C) to completely recover and isolate GLP from the residual byproduct. The yield of GLP after lyophilization was calculated based on the ratio of the product weight to the initial sucrose weight used as the substrate.

5. Chemical structure analysis of GLP

5.1. Preparation of GLP solutions for HPAEC and HPSEC analysis

The preparation of GLP dispersions was performed according to the starch preparation method with slight modifications. Fifty milligrams of powdered GLP were dissolved in 90% DMSO at a concentration of 1% (w/v). The dissolved sample was boiled for 1 h and stirred overnight. An aliquot of 2 mL was mixed with 6 volumes of absolute ethanol (12 mL). The precipitated GLP sample was centrifuged (5,000 g , 25 °C, 20 min), and the supernatant was removed. The sample paste was resuspended in an equal volume of ethanol, and the ethanol precipitation step was repeated twice.

5.2. Determination of GLP branch pattern by HPAEC-PAD

The GLP pellets prepared by the ethanol precipitation step were resuspended in preheated 40 mM sodium acetate buffer (pH 3.5) to a concentration of 0.5% (w/v) and boiled with stirring for 1 h. To confirm the branching reaction pattern when forming branched α-glucan, isoamylase (EC 3.2.1.68; 500 unit/240 mg; Megazyme International Ireland Ltd, Wicklow, Ireland) was added to the branched α-glucan product. The debranching reaction was performed in 40 mM sodium acetate buffer (pH 3.5) at 40°C for 24 h. After the enzyme reaction, the sample mixture was boiled for 10 min to inactivate the enzyme. Thereafter, activated resins IRA-200C and IRA-67 were mixed in a 1:1 ratio to remove the salts in the sample. After removing the water, 0.2 g per mL of sample was added and shaken gently for 2 minutes. The sample solution was filtered through a 0.45 μm syringe filter (Whatman, Maidstone, UK) and 25 μL of the sample was injected into a high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD, Dionex Dx-300, Sunnyvale, CA). The column used was a CarboPac PA200 analytical column (3 × 250 mm, Dionex, Sunnyvale, CA) maintained at 30 °C. The eluent A was 150 mM sodium hydroxide, and the eluent B was 150 mM sodium hydroxide containing 500 mM sodium acetate, and the flow rate for the change of sodium acetate (0 to 500 mM in 135 min) was 0.5 mL·min-1.

5.3. Molecular weight and size distribution measurement by HPSEC-RI-MALS

The GLP pellets prepared by the ethanol precipitation step were resuspended in preheated water to a concentration of 0.5% (w/v) and boiled with stirring for 1 h. The resuspended GLP solution was filtered through a 5 μm syringe filter (Whatman, Maidstone, UK) and injected into a high-performance size exclusion chromatography equipped with a multi-laser light scattering system (HPSEC-RI-MALS). Shodex OH pak SB-806 HQ and SB-804 HQ columns (8.0 mm ID x 300 mm, Showa, Denko, Japan) were used as analytical columns in an oven set at 50 °C. A Dawn Heleos MALLS system equipped with a K5 flow cell and a GaAs laser (λ = 658 nm) from Wyatt Technology Corporation (Santa Barbara, CA) and a RID-10A refractometer from Shimadzu (Kyoto, Japan) were used for detection. Molecular weight and size distribution were calculated by Astra software (version 5.3.4.20 for Windows, Wyatt Technology Corporation, Santa Barbara, CA). The refractive index increment (dn/dc) value was 0.145 mL·g ^-1 , and low molecular weight pullulan was used as a standard (P20 was used to achieve normalization of the photodiode).

6. GLP Cosmetic Composition

6.1. Cell culture

HaCaT cells and B16F10 melanoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin streptomycin at 37 °C in a humidified 5% CO ₂ incubator.

6.2. GLP cytotoxicity assessment

Cell viability was assessed by the Cell Titer 96 AQueous one solution cell proliferation assay (Promega). HaCaT cells were seeded at 2 × 10 ⁵ per well in 96-well cell culture plates and cultured for 2 days. The cells were exposed to glycogen-like particles (GLP) at concentrations of 1.25 and 2.5 mg/mL (0.125%, 0.25%) for 24 h at 37 °C in a 5% CO ₂ incubator. After 20 μL MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] solution was added to each well, the cells were cultured for 2 h at 37 °C in a humidified 5% CO ₂ incubator. Cell viability was measured at 490 nm using a cell imaging multimode reader (Biotek, Cytation 1).

6.3. Evaluation of GLP’s ability to inhibit mushroom tyrosinase activity in vitro

40 μL of each sample was pre-incubated with 20 μL of mushroom tyrosinase (0.02 mg/mL) at room temperature for 10 min. 1 mM L-DOPA (L-3,4-dihydroxyphenylalanine) was added and incubated for 30 min at room temperature. Dopachrome formation was detected by measuring the absorbance at 475 nm using a cell imaging multi-mode reader (Biotek, Cytation 1).

6.4. GLP and Melanin Content Evaluation

B16F10 melanoma cells were seeded at 2 × 10 ⁵ per well in 24-well cell culture plates and cultured at 37 °C for 24 h in a humidified 5% CO ₂ incubator. The cells were treated with samples and incubated for 1 h. Then, 100 nmol of α-MSH (α-melanocyte-stimulating hormone) was added to produce melanin and cultured for 3 days. The culture medium was removed and the cells were collected. The collected cells were centrifuged twice (12,000 rpm for 1 min) and the supernatant was removed. 200 μL of 1 N NaOH was added to the remaining pellet and treated on a heat block at 90 °C for 20 min to dissolve the pellet. The melanin content was measured at 490 nm using a cell imaging multimode reader (Biotek, Cytation 1).

6.5. UV protection effect of GLP

To determine the UV-blocking effect, the absorbance of GLP solution and oxybenzone in the wavelength range of 200–400 nm was measured using a spectrophotometer (Beckman DU 730; CA, U.S.A.).

7. Statistical Analysis

All statistical analyses were performed using SPSS software. Means were evaluated using analysis of variance. Means were compared using Tukey's honestly significant difference procedure at a significance level of 5%.

result

1. Production yield and chemical structure of Np/Vv GLP

In this study, biosynthetic glycogen-like particle (GLP) was synthesized at pH 7.0 and 30 °C considering the optimal reaction conditions of these two enzymes, which are slightly different (pH 7.0 and 35 °C for NpAS and pH 7.0-8.0 and 30 °C for VvGBE). Different chemical structures of GLP were expected by controlling the ratio of VvGBE/NpAS (ref), and the activity ratio of VvGBE/NpAS was adjusted from 1 (r1) to 10 (r10). Considering these reaction parameters, GLP was synthesized by the co-reaction of NpAS and VvGBE in simultaneous mode. As a result, the yield of GLP was in the range of r1 (27.54%) to r10 (38.40%) depending on the VvGBE/NpAS ratio.

The increase in GBE activity level generated a greater number of branched chains with non-reducing termini within the same reaction time, providing more reactive sites for NpAS. This in turn accelerated the growth rate of linear glucans long enough to react again with GBE, resulting in a greater increase in GLP mass.

Np/Vv GLPs produced with two different activity levels of VvGBE previously showed relatively short chain length distribution patterns compared with GLPs synthesized from other bacterial GBE sources. This result can be explained by the unique property of VvGBE to deliver more very short chains with a degree of polymerization (DP) of 3–5. As the activity ratio of VvGBE/NpAS increased from r1 to r10, the DP of the maximum peak decreased from 8 to 6 and the relative ratio increased. In addition, the maximum detectable DP gradually decreased from 60 to 26, from 13.37 to 8.01, and from 9.17 to 7.01 with VvGBE activity, respectively (Table 1).

Branch length distribution of Np/Vv GLP according to VvGBE/NpAS enzyme activity ratio

division Degree of polymerization (DP) (%) Detectable DP DP≤5 DP 6-12 DP 13-24 DP 25-36 DP ≥ 37 DP _w ¹⁾ DP _n ²⁾ Glycogen oyster 16.70±0.22 ^d 39.49±0.06 ^d 35.20±0.04 ^a 8.02±0.13 ^a 0.59±0.01 ^b 12.76 7.86 41±0 ^b r 1 9.43±0.07 ^g 58.23±0.77 ^c 18.99±0.30 ^b 8.05±0.20 ^a 5.30±0.35 ^a 13.37 9.17 60±0 ^a r 2 13.37±0.04 ^f 71.84±0.18 ^a 13.29±0.23 ^c 1.38±0.01 ^b 0.11±0.01 ^b 9.13 7.68 38±0 ^c r 3 15.03±0.13 ^e 72.74±0.26 ^ab 11.41±0.11 ^d 0.84±0.01 ^c - 8.71 7.43 35±0 ^d r 4 16.31±0.02 ^d 72.94±0.21 ^ab 10.16±0.16 ^e 0.62±0 ^cd - 8.48 7.18 33±0 ^e r 5 16.80±0.09 ^d 73.95±0.01 ^a 9.06±0.10 ^f 0.19±0.01 ^e - 8.28 7.18 28±0 ^f r 6 16.99±0.11 ^cd 73.90±0.06 ^a 8.87±0.06 ^fg 0.24±0.01 ^de - 8.25 7.21 28±0 ^f r 7 17.54±0 ^bc 73.85±0.04 ^a 8.45±0.05 ^fgh 0.17±0.01 ^e - 8.17 7.16 27±0 ^g r 8 18.19±0.19 ^ab 73.54±0.08 ^ab 8.10±0.11 ^ghi 0.17±0.01 ^e - 8.10 7.05 27±0 ^g r 9 18.58±0.03 ^a 73.48±0.02 ^ab 7.77±0.04 ^hi 0.15±0.01 ^e - 8.05 7.03 27±0 ^g r 10 18.60±0.12 ^a 73.76±023 ^a 7.53±0.06 ⁱ 0.10±0.01 ^e - 8.01 7.01 26±0 ^h

More specifically, when the VvGBE/NpAS ratio reached r5, the ratios of individual chain lengths, average DP, and detectable DP were found to level off. Further increasing the GBE ratio resulted in no further mass accumulation and no further increase in the size of the GLPs. In fact, as mass accumulation increased within the tested activity ratios up to r5, the GLP size decreased, indicating that the apparent density of the GLP structure increased accordingly. After passing this ratio point of VvGBE/NpAS activity, no significant mass accumulation was observed due to the very dense GLP structure, which prevented the enzyme from accessing the physically non-reducing terminal reaction site. In general, as GBE activity increased, the amount of chain lengths DP≥13 decreased, while the relative amount of chain lengths DP≤12 tended to increase. In actively growing GLPs, freely accessible DP≥13 chains were considered to be efficiently glucosylated, resulting in an increase in the relative amount of DP≤12 (Figure 2 ). These results indicate that the branching pattern of Np/Vv GLP can be precisely managed by controlling the VvGBE/NpAS ratio.

The molecular weights of all synthesized Np/Vv GLPs were lower than that of native glycogen from oysters. The HPSEC-RI peak widths of Np/Vv GLP products narrowed with increasing ratios of GBE/ASase (Fig. 3), indicating that the polydispersity of GLPs decreased and their molecular structures and sizes might become more homogeneous. Under these biosynthetic conditions, the molecular weights of GLPs did not reach those of native glycogens from animals, and among them, their Rz's were consistently larger, which resulted in significantly lower apparent densities (αapp). These structural parameters of GLPs suggested that this in vitro enzymatic synthesis produced branched structures that were quite different from those of native glycogen. As the activity of VvGBE increased, the molecular weight of GLPs seemed to increase due to the shorter chain length transfer that fit into a smaller space, resulting in an increase in packing mass in the same volume of space, thus resulting in an increase in density and a more compact structure (Table 2). However, the molecular weight did not increase significantly beyond a certain level, indicating that the enzyme could no longer utilize the self-enzymatically formed polymer particles as substrates. Table 2 shows the average molecular weight, radius of gyration, and apparent density of Np/Vv GLP.

division Molecular weight Turning radius
(Gyration radius) Apparent density
(Apparent density) (M _w , g mol ^-1 ) (R _z , nm) (ρ _app , g mol ^-1 nm ^-3 ) Oyster glycogen 2.64±0.03 × 10 ⁷ 28.60±0.80 270±0.21 Np/Vv GLP r1 2.30±0.04 × 10 ⁵ 39.75±0.45 0.88±0.02 r2 3.06±0.00 × 10 ⁵ 36.95±0.25 1.45±0.03 r3 3.25±0.00 × 10 ⁵ 35.65±0.05 1.72±0.00 r4 3.27±0.00 × 10 ⁵ 35.00±0.40 1.82±0.05 r5 3.70±0.08 × 10 ⁵ 35.60±0.40 1.96±0.02 r6 3.70±0.08 × 10 ⁵ 35.80±0.35 1.91±0.02 r7 3.70±0.04 × 10 ⁵ 35.35±0.65 2.00±0.08 r8 3.70±0.00 × 10 ⁵ 35.55±0.25 1.97±0.04 r9 3.82±0.02 × 10 ⁵ 35.45±0.05 2.05±0.00 r10 3.82±0.01 × 10 ⁵ 33.15±0.05 2.51±0.01

2. GLP’s UV protection effect

To investigate the UV blocking effect of GLP synthesized using sucrose and enzymes without chemical treatment, the absorbance was measured at a wavelength of 200 to 400 nm (Figs. 5 to 8). Figs. 5 to 8 compare the UV blocking effects between oxybenzone and GLP. It was confirmed that GLP manufactured by combinations of various amylosucrases and glycogen branching enzymes had excellent UV blocking ability when the enzyme activity ratio was r1 to r3, and that the UV blocking ability was the best when r1. In particular, Np/Vv GLP r1 showed a UV blocking effect similar to oxybenzone, a commonly used UV blocking material. Through this, it was confirmed that GLP can be utilized as a substitute for oxybenzone, which can potentially induce allergies and cause hormonal imbalance.

3. GLP’s skin whitening effect

(1) Cytotoxicity evaluation

Before investigating the melanin production inhibitory effect of Np/Vv GLP, cytotoxicity was measured using human keratinocytes (HaCaT cells) to determine whether GLP is toxic to human skin (Fig. 9). When HaCaT cells were treated with 1.25 and 2.5 mg/mL (0.125%, 0.25%) of Np/Vv GLP, no cytotoxicity was observed.

(2) Evaluation of tyrosinase activity inhibition ability

To investigate whether Np/Vv GLP could regulate melanin synthesis by inhibiting tyrosinase activity, mushroom tyrosinase activity was measured (Figs. 10 and 11). When 1.25 and 2.5 mg/mL (0.125, 0.25% (w/v)) of each sample were added to HaCaT cells, tyrosinase activity was inhibited in a concentration-dependent manner, except for glycogen (native). In particular, at a concentration of 2.5 mg/mL (0.25% (w/v)), r1, r2, and r5 showed tyrosinase activity inhibition rates of 22.2, 20.1, and 14.4%, respectively.

In addition, in order to confirm the inhibition ability of tyrosinase activity when other types of amylosucrase and glycogen branching enzyme were used, the activity ratios (r1, r3, r5) and tyrosinase activity inhibition rates by concentration (1.25 and 2.5 mg/mL) of Bt/Ro GLP, Ns/Ro GLP, Bt/Bt GLP, and Ns/Bt GLP were measured. As a result, when other types of amylosucrase and glycogen branching enzyme were used, no common characteristics, such as tyrosinase activity being inhibited in a concentration-dependent manner or tyrosinase activity being inhibited as the glycogen branching enzyme activity ratio increased, could be confirmed (Fig. 11).

(3) Evaluation of melanin production inhibition ability

The effect of GLP on melanin production induced by α-MSH in B16F10 melanoma cells was evaluated (Figs. 12 and 13). The cells were treated with 0, 0.625, 1.25, and 2.5 mg/mL (0, 0.0625, 0.125, and 0.25% (w/v)) of Np/Vv GLP together with 100 nM α-MSH and cultured in a CO ₂ incubator for 72 h, after which the amount of melanin was measured (Fig. 12). Fig. 12 showed that melanin synthesis was inhibited in a concentration-dependent manner by Np/Vv GLP treatment. Melanin synthesis was inhibited by 45%, 33%, and 32% at a concentration of 2.5 mg/mL (0.25% (w/v)) of r1, r2, and r5, respectively. That is, Np/Vv GLP exhibited an inhibitory effect on melanin synthesis induced by α-MSH, and in particular, Np/Vv GLP r1 showed that it could be used as an effective substance for skin whitening due to its strong melanin synthesis inhibitory activity. These results implied that the lower the ratio of VvGBE to NpAS and the higher the long-chain ratio, the higher the melanin synthesis inhibitory effect.

Additionally, GLPs prepared with various amylosucrase/glycogen branching enzyme combinations (Ns/Bt GLP, Ns/Ro GLP, Bt/Bt GLP, Bt/Ro GLP) together with 100 nM α-MSH were treated in B16F10 melanoma cells at a concentration of 1.25 mg/ml (0.125% (w/v)), and the amount of melanin secretion was measured.

As a result, it was confirmed that even in the case of GLP that does not have an effect of inhibiting tyrosinase activity, as confirmed in the “tyrosinase inhibition ability evaluation,” the effect of inhibiting melanin synthesis was excellent. In light of the fact that melanin synthesis in skin cells can be inhibited when i) the expression amount of tyrosinase itself is low, or ii) the activity is inhibited even if tyrosinase is expressed, it was found that the GLP of the present invention inhibited melanin synthesis by inhibiting the expression amount of tyrosinase itself rather than inhibiting tyrosinase activity.

Attach electronic file of sequence list

Claims (7)

A method for producing a skin whitening or UV-blocking cosmetic composition, comprising a step of reacting sucrose as a substrate with amylosecrase and glycogen branching enzyme to obtain an alpha-1,4-1,6-glucan polymer.
A method for producing a cosmetic composition for skin whitening or ultraviolet ray blocking according to claim 1, wherein the amylosucrase is composed of any one of the amino acid sequences of sequence numbers 1 to 3.
A method for producing a cosmetic composition for skin whitening or ultraviolet ray blocking according to claim 1, wherein the glycogen branching enzyme is composed of any one of the amino acid sequences of sequence numbers 4 to 6.
A method for producing a cosmetic composition for skin whitening or ultraviolet ray blocking according to claim 1, wherein the activity ratio of the amylosucrase and the glycogen branching enzyme is 1:0.8 to 5.2.
delete A cosmetic composition for skin whitening or UV protection prepared by the method of any one of claims 1 to 4. delete