KR20230101357A - Manufacturing method of high-speed fermented sesame with enhanced content of episesaminone diglucoside and sesaminol diglucoside - Google Patents

Abstract

The present invention relates to a method for producing a fast-fermented sesame product with increased content of episesaminon diglucoside and sesaminol diglucoside, and skin whitening containing the fast-fermented sesame product prepared by the above method or a compound isolated therefrom as an active ingredient. And a composition for improving wrinkles, wherein the high-speed fermented sesame seeds prepared by the manufacturing method of the present invention are significantly superior in whitening and wrinkle-improving effects compared to the sesame hot water extract, and after separating the compound produced by high-speed fermentation of sesame seeds, whitening And as a result of confirming the anti-wrinkle effect, episesaminon diglucoside and sesaminol diglucoside have excellent whitening and anti-wrinkle effects, so the high-speed fermented sesame seeds prepared by the production method of the present invention and the compounds isolated therefrom are effective for skin It can be usefully used as a material for cosmetics for whitening and wrinkle improvement or health functional food.

Description

Manufacturing method of high-speed fermented sesame with enhanced content of episesaminone diglucoside and sesaminol diglucoside}

The present invention relates to a method for producing a fast-fermented sesame product with increased content of episesaminon diglucoside and sesaminol diglucoside, and skin whitening containing the fast-fermented sesame product prepared by the above method or a compound isolated therefrom as an active ingredient. And it relates to a composition for improving wrinkles.

Sesame ( Sesamum indicum L. ) is an annual herb belonging to the Sesame family Sesame genus and is a world-class oil crop cultivated in the tropics to cool and temperate zones.

Looking at the nutritional components of sesame seeds, they contain 51% lipid, 20% protein, and 15% sugar, and are rich in calcium, phosphorus, zinc, iron, vitamins B1, B2, and unsaturated fatty acids compared to rice and wheat. In addition, sesame seeds contain 0.1 to 1.0% of sesamin and 0.3 to 0.7% of sesaminol, which have antioxidant effects.

On the other hand, the skin is the foremost defense organ of the human body that protects our body from external stimuli, and interest in it is gradually increasing from an aesthetic point of view. Accordingly, research on various functional cosmetics is active, and among them, various functional cosmetics for wrinkle reduction and whitening functions are being released.

Various causes of wrinkles have been reported. First of all, when the skin is exposed to excessive ultraviolet rays, the decomposition of collagen, which is a skin component, is promoted, causing the skin to lose elasticity and wrinkles to be generated. In addition, when the skin becomes excessively dry due to excessive temperature changes, low humidity, wind, etc., the function of the skin as a barrier against the outside deteriorates, resulting in wrinkles. In addition, when the skin is exposed to reactive oxygen species or free radicals, lipid peroxide is produced by oxidation, and as a result, skin constituent proteins such as collagen are modified to form wrinkles. In order to suppress the occurrence of wrinkles caused by these external and internal factors, various cosmetic compositions are being studied.

When the skin is irradiated with ultraviolet rays, melanocytes in the skin are activated, and melanin production is promoted by the action of tyrosinase, TRP1 (tyrosinase related protein 1) and TRP2 (tyrosinase related protein 2). The produced melanin is deposited on the skin and develops into age spots or freckles. In order to prevent this, whitening cosmetics containing various compositions are used. In addition, ultraviolet rays have been found to cause various skin disorders by promoting oxidation of sebum and cell membranes, and in particular, with the recent increase in ultraviolet rays due to the destruction of the ozone layer, more effective measures to prevent skin oxidation are required.

Conventionally known components exhibiting a whitening effect include ascorbic acid, phosphate ester salts, hydroquinone derivatives, placenta extracts, kojic acid, ellagic acid, and the like, and cosmetic compositions containing these components are common. Recently, polyphenols contained in plants have been reported to have a high whitening effect, and cosmetic compositions using them have been proposed. In addition, the whitening effect of flavanones and hydroxyflavones has already been disclosed. However, these whitening ingredients are somewhat insignificant in their effects and have problems during storage, and do not sufficiently exhibit wrinkle alleviating effects and anti-aging effects in addition to the whitening effects. In particular, in the case of functional cosmetics containing chemical components, there is a problem that causes skin irritation and is not suitable for sensitive skin, so attention is focused on functional cosmetics derived from natural plants that are less irritating to the skin and are eco-friendly.

On the other hand, Korean Patent Registration No. 0239206 discloses a method for separating and purifying sesaminol components from sesame oil and sesame meal, and Korean Patent Publication No. 2018-0064848 discloses skin containing physiologically active substances derived from Baeksuo extract. A composition for whitening and improving wrinkles is disclosed, and Korean Patent Publication No. 2021-0044952 discloses a composition for skin whitening and wrinkle improvement containing fermented jujube seeds as an active ingredient. A method for producing a fast-fermented sesame seed with increased content of seeds and sesaminol diglucoside, a composition for skin whitening and wrinkle improvement containing the fast-fermented sesame seed produced by the above manufacturing method or a compound isolated therefrom as an active ingredient is not known at all. has not been disclosed

The present invention has been derived from the above needs, and provides a method for producing a high-speed fermented sesame product in which an enzyme source derived from mushrooms is added to a hot-water extract of sesame seeds and then fermented at room temperature at high speed, and the high-speed fermentation of sesame produced by the above method is provided. The present invention was completed by confirming the whitening and wrinkle-improving effects of water and a compound separated therefrom.

In order to solve the above problems, the present invention is a step of fermenting for 20 to 180 minutes after mixing the hot water extract of sesame seeds concentrated under reduced pressure and the enzyme source derived from button mushrooms; episesaminon diglucoside and sesaminol diglucoside, including Provided is a method for producing a high-speed fermented sesame seed content.

In addition, the present invention provides a high-speed fermented sesame seed with increased content of episesaminon diglucoside and sesaminol diglucoside prepared by the above production method.

In addition, the present invention provides a cosmetic composition for skin whitening and wrinkle improvement containing, as an active ingredient, a fast-fermented sesame seed with increased content of episesaminon diglucoside and sesaminol diglucoside.

In addition, the present invention provides a health functional food composition for skin whitening and wrinkle improvement containing, as an active ingredient, a fast-fermented sesame seed with increased content of episesaminon diglucoside and sesaminol diglucoside.

In addition, the present invention provides a cosmetic composition for skin whitening and wrinkle improvement containing episesaminon diglucoside or an acceptable salt thereof isolated from fast-fermented sesame seeds as an active ingredient.

In addition, the present invention provides a health functional food composition for skin whitening and wrinkle improvement containing episesaminon diglucoside or an acceptable salt thereof isolated from the fast fermented sesame seeds as an active ingredient.

In addition, the present invention provides a cosmetic composition for skin whitening containing sesaminol diglucoside or an acceptable salt thereof separated from the fast fermented sesame seeds as an active ingredient.

In addition, the present invention provides a health functional food composition for skin whitening containing sesaminol diglucoside or an acceptable salt thereof isolated from the fast fermented sesame seeds as an active ingredient.

The present invention relates to a method for producing a fast-fermented sesame product with increased content of episesaminon diglucoside and sesaminol diglucoside, and skin whitening containing the fast-fermented sesame product prepared by the above method or a compound isolated therefrom as an active ingredient. And a composition for improving wrinkles, wherein the high-speed fermented sesame seeds prepared by the manufacturing method of the present invention are significantly superior in whitening and wrinkle-improving effects compared to the sesame hot water extract, and after separating the compound produced by high-speed fermentation of sesame seeds, whitening And as a result of confirming the anti-wrinkle effect, it was confirmed that the whitening and anti-wrinkle effects of episesaminon diglucoside and sesaminol diglucoside were excellent. In addition, the manufacturing method of high-speed fermented sesame seeds was optimized to increase the content of sesaminol diglucoside, which has whitening and wrinkle-improving effects.

Figure 1 is a flow chart of the separation process of the compound produced from the high-speed fermented sesame seeds of the present invention.
Figure 2 is an HPLC chromatogram result of a hot water extract of sesame (A), fast fermented sesame (B), an EtOAc fraction of fast fermented sesame (C), and single substances (C, D) isolated from fast fermented sesame.
3 is a result of identifying the structures of single substances SOP-1 (A) and SOP-2 (B) isolated from high-speed fermented sesame seeds.
Figure 4 confirms the whitening effect of the high-speed fermented sesame seeds of the present invention by enzyme-derived tyrosinase inhibitory activity (A), tyrosinase inhibitory activity in cells (B) and melanin content analysis in cells (C) This is the result. α-MSH (α-melanocyte-stimulating hormone) is a substance that induces tyrosinase activity and melanin production. Different letters a to d in the figure mean that there is a statistically significant difference, p<0.05.
Figure 5 shows the whitening effect of episesaminon diglucoside (SOP-1) and sesaminol diglucoside (SOP-2) isolated from the high-speed fermented sesame seeds of the present invention by enzyme-derived tyrosinase inhibitory activity ( This is the result confirmed by A), tyrosinase inhibitory activity in cells (B) and melanin content analysis in cells (C). α-MSH (α-melanocyte-stimulating hormone) is a substance that induces tyrosinase activity and melanin production. STG is (+)-sesaminol triglucoside, a major component of sesame seeds, and vitamin C and arbutin were used as positive controls.
Figure 6 is the result of confirming the anti-wrinkle effect of the high-speed fermented sesame seeds of the present invention by collagenase inhibitory activity (A) and the degree of procollagen synthesis (B). UVB was irradiated to inhibit procollagen synthesis. Different letters a to d in the figure mean that there is a statistically significant difference, p<0.05.
Figure 7 shows the collagenase inhibitory activity (A), MMP of the anti-wrinkle effect of episesaminon diglucoside (SOP-1) and sesaminol diglucoside (SOP-2) isolated from the fast-fermented sesame seeds of the present invention. -1 This is the result confirmed by the content change (B) and the degree of procollagen synthesis (C). UVB irradiation promoted the expression of MMP-1 and inhibited procollagen synthesis. STG is (+)-sesaminol triglucoside, a major component of sesame seeds, and EGCG (Epigallocatechin gallate) was used as a positive control.
Figure 8 shows the cytotoxicity of episesaminon diglucoside (SOP-1) and sesaminol diglucoside (SOP-2) isolated from sesame hot water extract, sesame fast-fermented product and sesame fast-fermented product in B16F10 cells (A, These are the results confirmed in B) and CCD-986sk (C, D) cells. STG is (+)-sesaminol triglucoside, a major component of sesame seeds, and EGCG is epigallocatechin gallate used as a positive control.
Figure 9 shows the optimal manufacturing method of high-speed fermentation of sesame seeds significantly increasing the content of SOP-2 by high-speed fermentation time (A), enzyme concentration (B), substrate concentration (C), pH (D) and fermentation temperature (E). This is the result of dividing it.

In order to achieve the object of the present invention, the present invention is a step of fermenting for 20 to 180 minutes after mixing the hot water extract of sesame seeds concentrated under reduced pressure and the enzyme source derived from button mushrooms; episesaminon diglucoside and sesaminol, including Provided is a method for producing high-speed fermented sesame seeds with increased diglucoside content.

The hot-water extract of sesame seeds concentrated under low temperature and reduced pressure may be obtained by adding 1 to 10 L of water to 1 kg of dried sesame seeds, followed by hot water extraction at 60 to 80 ° C. for 80 to 100 minutes, and then concentrated under reduced pressure at low temperature. It may be obtained by adding 4 to 5 L of water to 1 kg of dried sesame seeds, performing hot water extraction at 65 to 75 ° C. for 85 to 95 minutes, and then concentrating under reduced pressure at low temperature. More preferably, 5 L of water is added to 1 kg of dried sesame seeds. After addition, it was obtained by hot water extraction at 70° C. for 90 minutes and concentration under reduced pressure at low temperature, but is not limited thereto.

The enzyme source derived from button mushrooms may be a filtrate obtained by adding 3 to 5 L of water to 1 kg of mushrooms, pulverizing, and then filtering. It may be a filtrate, more preferably a filtrate obtained by adding 4 L of water to 1 kg of button mushrooms, pulverizing, and then filtering, but is not limited thereto.

In one embodiment of the present invention, the concentration of the button mushroom-derived enzyme source is to have a concentration of 250 g / L when 4 L of water is added to 1 kg of button mushrooms.

In one embodiment of the present invention, the method for producing a high-speed fermented sesame seed with increased content of episesaminon diglucoside and sesaminol diglucoside is specifically

1) adding 1 to 10 L of water to 1 kg of dried sesame seeds, extracting with hot water at 60 to 80 ° C for 80 to 100 minutes, and then concentrating under reduced pressure at low temperature;

2) adding 3 to 5 L of water to 1 kg of button mushrooms, pulverizing and filtering to obtain an enzyme source derived from button mushrooms at a concentration of 20 to 340 g/L as a filtrate; and

3) After mixing the hot water extract of sesame seeds concentrated under low temperature and reduced pressure in step 1) and the enzyme source derived from button mushrooms in step 2), pH was adjusted to 1.5 to 8.5, and then stirred at 0 to 75 ° C for 20 to 180 minutes and fermenting; may include,

more specifically

1) After adding 4 to 5 L of water to 1 kg of dried sesame seeds, hot water extraction at 65 to 75 ° C. for 85 to 95 minutes, followed by low-temperature vacuum concentration;

2) adding 3.5 to 4.5 L of water to 1 kg of button mushrooms, grinding them, and then filtering to obtain a filtrate, a button-derived enzyme source, at a concentration of 222 to 285 g/L; and

3) After mixing the hot water extract of sesame seeds concentrated under low temperature and reduced pressure in step 1) and the enzyme source derived from button mushrooms in step 2), the pH was adjusted to 4.5 to 6.5, and then stirred at 15 to 60 ° C for 80 to 140 minutes and fermenting; may include,

more specifically

1) After adding 5 L of water to 1 kg of dried sesame seeds, extracting with hot water at 70 ° C. for 90 minutes, and then concentrating under reduced pressure at low temperature;

2) adding 4 L of water to 1 kg of button mushrooms, pulverizing and filtering to obtain an enzyme source derived from button mushrooms at a concentration of 250 g/L as a filtrate; and

3) After mixing the hot water extract of sesame seeds concentrated under low temperature and reduced pressure in step 1) and the enzyme source derived from button mushrooms in step 2), pH was adjusted to 5-6, and then stirred at 25-35 ° C for 100-120 minutes and fermenting; may include, but is not limited thereto.

The button-derived enzyme source in step 2) has an absorbance of 0.7 to 0.85 when the amount of benzotropolone produced after the oxidation reaction using pyrogallol as a substrate is measured at 360 nm using a UV spectrometer. It can be used after confirming that it represents, but is not limited thereto.

In addition, the present invention provides a high-speed fermented sesame seed with increased content of episesaminon diglucoside and sesaminol diglucoside prepared by the above production method.

The high-speed fermented sesame seeds significantly increased the contents of episesaminon diglucoside and sesaminol diglucoside compared to the hot water extract of sesame seeds.

In addition, the present invention provides a cosmetic composition for skin whitening and wrinkle improvement containing, as an active ingredient, a fast-fermented sesame seed with increased content of episesaminon diglucoside and sesaminol diglucoside.

The cosmetic composition of the present invention is used for skin, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, eye cream, moisture cream, hand cream, essence, nutrient essence, pack, It may be prepared in any one formulation selected from the group consisting of cleansing foam, cleansing water, cleansing lotion, cleansing cream, body lotion, body cleanser, soap and powder, but is not limited thereto. The composition of each of these formulations may contain various bases and additives necessary and appropriate for the formulation of the formulation, and the types and amounts of these components can be easily selected by those skilled in the art.

The cosmetic composition of the present invention may further contain one or more components for skin whitening or wrinkle improvement that exhibit the same or similar functions in addition to the above active ingredients. Skin whitening Skin whitening or wrinkle improvement components include kojic acid and its derivatives, arbutin, ascorbic acid and its derivatives, hydroquinone and its derivatives, resorcinol, cycloalkanone, methylenedioxyphenyl alkanol, 2,7-di Nitroindazole or plant extracts such as tangerine extract, rice extract, licorice extract, etc., but are not limited thereto.

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

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 in particular, in the case of a spray, additionally chlorofluorohydrocarbon, propellants such as propane/butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butyl glycol oil, fatty acid esters of glycerol, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline Cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like may be used.

When the formulation of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The cosmetic composition of the present invention may further contain excipients including fungicides, hydrotropes, humectants, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, and the like.

In addition, the present invention provides a health functional food composition for skin whitening and wrinkle improvement containing, as an active ingredient, a fast-fermented sesame seed with increased content of episesaminon diglucoside and sesaminol diglucoside.

The health functional food composition for skin whitening or wrinkle improvement of the present invention may be prepared in one formulation selected from the group consisting of powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto. Preferably, the health functional food for skin whitening or wrinkle improvement has the advantage of having a more excellent effect by ingesting it in the form of inner beauty food. The inner beauty food is a food referred to as 'eating cosmetics or beauty food', and refers to food that changes the skin constitution to be healthy by absorbing various ingredients that are good for the skin into the body. You can select and consume inner beauty foods that are suitable for each individual by considering skin condition and lifestyle. More preferably, when cosmetics containing the high-speed fermented sesame seeds and inner beauty foods containing the fermented product are mixed, tyrosinase activity and melanin synthesis are inhibited, collagenase activity and The effect of suppressing the production of MMP-1 is significantly increased, so it has the advantage of more effective skin whitening and wrinkle improvement.

In addition, there is no particular limitation on the type of food. Examples of foods to which the health functional food composition can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea There are drinks, alcoholic beverages, and vitamin complexes, and includes all health foods in a conventional sense. The beverage includes carbonated beverages, functional ionic beverages, juice (eg, apple, pear, grape, aloe, tangerine, peach, carrot, tomato juice, etc.), sikhye, and the like.

When the fast fermented sesame seeds of the present invention is used as a food additive, the fermented product may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. Active ingredients can be appropriately used depending on their purpose of use (prevention or improvement). In general, when producing food or beverage, the fermented product of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health control, it can be used in an amount within a safe range.

The functional food of the present invention includes components commonly added during food preparation, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared as a drink, natural carbohydrates or flavors may be included as additional ingredients in addition to active ingredients. The natural carbohydrates are monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrins, cyclodextrins, etc.) or sugar alcohols (eg, maltose, sucrose, etc.) , xylitol, sorbitol, erythritol, etc.) are preferred. As the flavoring agent, natural flavoring agents (eg, thaumatin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used. In addition to the health functional food composition, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonated beverages It may further contain a carbonation agent used for

In addition to the health functional food composition, various nutrients, vitamins, inhibitors, flavoring agents, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, It may further contain a carbonating agent used in carbonated beverages. The ratio of the components to be added is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.

In addition, the present invention provides a cosmetic composition for skin whitening and wrinkle improvement containing episesaminon diglucoside or an acceptable salt thereof isolated from fast-fermented sesame seeds as an active ingredient.

The active ingredient is characterized by showing significant whitening and wrinkle improvement effects compared to STG [(+)-sesaminol triglucoside], which is the main component of sesame seeds.

In addition, the present invention provides a health functional food composition for skin whitening and wrinkle improvement containing episesaminon diglucoside or an acceptable salt thereof isolated from the fast fermented sesame seeds as an active ingredient.

The active ingredient is characterized by showing significant whitening and wrinkle improvement effects compared to STG [(+)-sesaminol triglucoside], which is the main component of sesame seeds.

In addition, the present invention provides a cosmetic composition for skin whitening containing sesaminol diglucoside or an acceptable salt thereof separated from the fast fermented sesame seeds as an active ingredient.

The active ingredient is characterized by showing a remarkable whitening effect compared to STG [(+)-sesaminol triglucoside], which is the main component of sesame seeds.

In addition, the present invention provides a health functional food composition for skin whitening containing sesaminol diglucoside or an acceptable salt thereof isolated from the fast fermented sesame seeds as an active ingredient.

The active ingredient is characterized by showing a remarkable whitening effect compared to STG [(+)-sesaminol triglucoside], which is the main component of sesame seeds.

Hereinafter, the present invention will be described in more detail using preparation examples and examples. These Preparation Examples and Examples are only for explaining the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

Preparation Example 1. Preparation of high-speed fermented sesame seeds

Sesame ( Sesamum indicum L.) used in the present invention was harvested and dried in October 2017 in Yecheon-gun, Gyeongsangbuk-do.

After adding 5 L of water to 1 kg of dried sesame seeds, hot water extraction was performed at 70 ° C. for 90 minutes, and then concentrated under reduced pressure at low temperature to prepare a sesame hot water extract (used as a comparative example).

The enzyme source derived from button mushrooms was prepared by adding 4 L of water to 1 kg of button mushrooms, grinding them, and then filtering to obtain a filtrate having a concentration of 250 g/L. The polyphenol oxidase activity of the mushroom-derived enzyme source was measured at 360 nm using a UV spectrometer to measure the amount of benzotropolone produced after the oxidation reaction using pyrogallol as a substrate. Confirmed. The specifically prepared button mushroom-derived enzyme source (1.5mL) was put into a quartz cell together with 1.5mL of 200mM pyrogallol diluted in 50mM sodium phosphate buffer solution (pH 7.2), oxidized for 5 minutes, and then absorbance was measured. , The absorbance was measured by adding 1.5 mL of substrate solution and 1.5 mL of distilled water to the control group. The polyphenol oxidase activity of the button mushroom-derived enzyme source had an O.D value of 0.78 to 0.79, and then the sesame extract was fermented.

5 g of the hot water extract of sesame seeds concentrated under reduced pressure and 240 g of the enzyme source derived from button mushrooms were mixed with respect to a total of 1 L, the remaining amount of water was added, and the pH was adjusted to 5-6. Thereafter, by stirring and fermenting at room temperature (25 ± 5 ° C.) for 100 minutes, a high-speed fermented sesame seed was obtained.

Example 1. Isolation and Identification of Newly Produced Compounds from Fast Fermented Sesame Seeds

1) Separation of newly produced compounds from high-speed fermentation of sesame seeds

In order to confirm the component changes of the sesame fast fermented product and sesame hot water extract prepared in Preparation Example 1, HPLC was performed. HPLC analysis conditions are as disclosed in Table 1 below. 0.1% (v/v) formic acid (HCOOH) was used as the mobile phase solvent A, acetonitrile (MeCN) was used as the solvent B, and gradient elution was performed with 100% solvent A. Starting with the final 100% solvent B for 30 minutes, the compound was detected at 280 nm while maintaining a flow rate of 1.0 mL/min of the mobile phase.

parameter condition device Shimadzu LC20AD detector Shimadzu SPD-M20A photodiode-array detector (PDA) wavelength 280 nm column YMC-Pack ODS A-302 Column (4.6mm i.d.x150mm) oven temperature 40℃ flow rate 1.0mL/min injection volume 5 μl mobile phase time (minutes) Solvent A: 0.1% HCOOH Solvent B: MeCN 0 100 0 10 80 20 15 50 50 20 30 70 25 0 100 30 0 100 - End

Specifically, as shown in Figure 1, after diluting by adding 2L of 10% (v / v) MeOH to 40 g of the sesame fast fermented product prepared in Preparation Example 1, n -hexane, EtOAc and n -BuOH The fractions were sequentially fractionated, and each fraction was concentrated under reduced pressure and dried to obtain n -hexane fraction (658.6 mg), EtOAc fraction (3.6 g), n -BuOH (17.1 g), and water fraction (17.2 g).

As a result of confirming the compounds in the obtained fraction through HPLC analysis, it was confirmed that the compounds produced in the fast fermented sesame seeds were all included in the EtOAc fraction, as shown in FIG. cm id × 42 cm) and ODS AQ 120-50S (1.0 cm, id × 45 cm) as a filler, and SOP-1 (25.6 mg, t _R 17.2 minutes) from 60% (v / v) MeOH eluate ) and SOP-2 (163.7 mg, t _R 17.5 min). The separated single material was measured for NMR and ESIMS spectra to determine the structure of the compound.

2) Identification of newly produced compounds from high-speed fermentation of sesame seeds

(1) Identification of SOP-1 structure

The newly produced compound SOP-1 from the fast fermented sesame seeds was obtained in the form of a yellow gum and was measured as [α] _D +35.1 ( c 0.1, MeOH). The molecular weight of the compound was confirmed by confirming the base peak of m/z 717.1996[M+Na] ⁺ as a result of HRESIMS spectrum measurement. As a result of ¹ H NMR spectrum measurement, δ _H 7.79 (1H, d, J =7.8Hz, H-5) and 7.71 (1H, dd, J =8.4, 1.8Hz, H-6) of two ABX-type aromatic signals '), 7.52 (1H, d, J =1.8Hz, H-2'), 6.98 (1H, d, J =8.4Hz, H-5'), 6.95 (1H, dd, J =7.8, 1.8Hz, H-6) and 6.79 (1H, d, J = 7.8 Hz, H-5), and the proton signals corresponding to two methylenedioxy were measured as δH _6.19 (2H, s, H-10') and 5.97 (2H , s, H-10). In addition, the signals corresponding to the tetrahedron skeleton are δ _H 4.98 (1H, d, J =8.4 Hz, H-7), 4.31 (1H, m, H-8'), 4.26 (1H, t, J =8.4 Hz, H-9'), 4.12 (1H, dd, J =8.4, 4.8Hz, H-9'), 2.72 (1H, m, H-8), and one hydroxymethyl proton signal was measured at δ _H 4.08 (1H , dd, J = 10.2, 4.8 Hz, H-9) and 3.56 (1H, dd, J = 10.2, 6.0 Hz, H-9). δ _H 4.55 (1H, d, J = 7.8 Hz, H-1'') and 4.41 (1H, d, J = 7.8 Hz, H-1'') with two anomeric protons. Metain protons were measured, and the coupling constant of anomeric hydrogen was confirmed to be 7.8 Hz, suggesting the presence of two β-glucoses. δ _C 198.2 (C-9') was measured by ¹³ C NMR and HSQC spectral analysis, confirming the presence of a ketone group. Additional 2D NMR spectral analysis of HMBC, COSY and NOESY determined the structure of SOP-1 as a previously reported (+)-episesaminone diglucoside (Fig. 3A).

In a previous study, (+)-epicesaminol diglucoside was reported as a compound produced by an oxidation reaction from a (+)-sesaminol derivative. It was confirmed that (+)-epicesaminon diglucoside was produced from (+)-sesaminol triglucoside, which is a major component.

(2) Identification of SOP-2 structure

The newly produced compound SOP-2 from the fast fermented sesame seeds was obtained as a yellow amorphous powder, and as a result of HRESIMS spectrum measurement, the molecular weight was estimated by confirming the base peak of m/z 717.2001[M+Na] ⁺ . As a result of ¹ H NMR spectrum measurement, 1,2,4,5-tetrasubstituted aromatic proton signals were measured as δ _H 6.87 (1H, s, H-6') and 6.83 (1H, s, H-3'), and ABX -type aromatic signals δ _H 6.88 (1H, d, J =2.4Hz, H-2''), 6.85 (1H, dd, J =8.4, 2.4Hz, H-6'') and 6.79 (1H, d, J = 8.4 Hz, measured at H-5″). Proton signals corresponding to the two methylenedioxy were measured at δ _H 5.94 (2H, s, H-7'') and 5.91 (2H, s, H-7'), and at δ _H 5.02 (1H, d, J = 7.8 Twelve oxidized metane protons were measured, along with two anomeric protons at Hz, H-1''') and 4.92 (1H, d, J = 7.8 Hz, H-1''''). The hydrogen coupling constant was confirmed to be 7.8 Hz, suggesting the presence of two β-glucoses. In addition, the signals corresponding to the furfuran backbone were δ _H 5.26 (1H, d, J =5.4Hz, H-2), 4.74 (1H, d, J =5.4Hz, H-6), 4.30 (1H-6), respectively. , dd, J =9.0, 6.6Hz, H-4), 4.22 (1H, dd, J =9.0, 6.6Hz, H-8), 3.92 (1H, dd, J =9.0, 7.2Hz, H-8) , 3.84 (1H, dd, J = 9.0, 7.2 Hz, H-4), 3.07 (1H, m, H-5) and 2.94 (1H, m, H-1). In ¹³ C NMR and HSQC spectral analysis, sesamol, furfuran, and two glucose signals were also observed. 2D NMR spectrum analysis identified the structure of SOP-2 as (+)-sesaminol diglucoside (FIG. 3B).

Through the above results, it was confirmed that in the high-speed fermentation of sesame seeds, the main component, (+)-sesaminol triglucoside, is hydrolyzed into diglucoside. It was confirmed that the reaction of hydrolyzing (+)-sesaminol triglucoside, which is a major component of sesame seeds, into diglucoside mainly occurs.

Example 2. Whitening effect of sesame high-speed fermented product

1) Evaluation of enzyme-derived tyrosinase inhibitory activity

The tyrosinase inhibitory activity related to the whitening activity of the fast-fermented sesame seeds and hot-water extract of sesame seeds prepared in Preparation Example 1 of the present invention was evaluated.

Specifically, 80 μl of mushroom tyrosinase (110 U/mL) in a mixture of 50 μl of 67 mM sodium phosphate buffer (pH 6.8), 80 μl of 10 mM L-DOPA, and 40 μl of the sample of Preparation Example 1 was added and then reacted at 25° C. for 2 minutes, and DOPA chrome generated in the reaction solution was measured at 475 nm. As shown in Equation 1 below, the tyrosinase inhibitory activity was expressed as a decrease in absorbance of the sample solution added and unadded.

[Equation 1]

Tyrosinase inhibitory activity (%) = (1-absorbance of sample addition group/absorbance of no addition group) × 100

As a result, as shown in FIG. 4A, it was confirmed that the tyrosinase inhibitory activity of the high-speed fermented sesame prepared by the method of the present invention was remarkably enhanced compared to the hot water extract of sesame.

2) Analysis of tyrosinase inhibitory activity and melanin content in cells

The whitening effect of the sesame hot water extract and the high-speed fermented sesame seeds was confirmed by analyzing the tyrosinase inhibitory activity and melanin content in the cells using B16F10 melanoma cells.

Specifically, B16F10 (melanoma cell) cells were cultured in DMEM (Dulbeco's Using modified eagle'medium medium, 37 ℃, 5% CO ₂ It was subcultured while adapting to an incubator (incubator). Stabilized B16F10 cells were inoculated with 1 × 10 ⁵ cells per well in a 12-well plate, and each well was pretreated with the sample or positive control of Preparation Example 1 for 1 hour, and then treated with α-MSH (10 nM / mL). , and cultured for 48 hours. After 48 hours, 250 μl of 10 mM phosphate buffer (pH 6.8) containing 1% (w/v) Triton X-100 was added and stirred for 5 minutes. Transferred to an e-tube and centrifuged at 10,000 rpm for 5 minutes to obtain a supernatant, and then the obtained supernatant was used for tyrosinase activity analysis, and the cell pellet was used for melanin content analysis.

40 μl of the supernatant obtained after the centrifugation was dispensed into a 96-well plate, and 200 μl of 2 mg/mL L-DOPA dissolved in 0.1 M sodium phosphate buffer (pH 7.2) was added thereto, followed by incubation at 37° C. for 30 minutes. DOPA chromium produced by tyrosinase was confirmed by measuring absorbance at 475 nm.

100 μl of 1N NaOH and 200 μl of distilled water were added to the cell pellet, left at 60° C. for 1 hour to completely dissolve melanin, and then 200 μl was transferred to a 96-well plate and absorbance was measured at 405 nm, The amount of melanin produced in each well was calculated using a standard calibration curve obtained with a melanin standard.

As a result, as shown in FIG. 4B, the high-speed fermented sesame produced by the production method of the present invention was superior to the hot-water sesame extract in inhibiting cellular tyrosinase activity, and as shown in FIG. 4C, the melanin content was reduced. The inhibitory effect was also remarkably excellent.

Example 3. Whitening effect of the compound isolated from sesame fast fermentation

1) Evaluation of enzyme-derived tyrosinase inhibitory activity

The enzyme-derived tyrosinase inhibitory activity of SOP-1 and SOP-2, which are compounds isolated from fast-fermented sesame seeds, was evaluated in the same manner as in Example 2 above. Vitamin C was used as a positive control group, and STG [(+)-sesaminol triglucoside], a major component of sesame seeds, was used as a control group.

As a result, as shown in FIG. 5A, it was confirmed that the tyrosinase inhibitory activity of SOP-1 and SOP-2, which are compounds isolated from fast-fermented sesame seeds, was similar to or superior to that of STG, a major component of sesame seeds.

2) Analysis of tyrosinase inhibitory activity and melanin content in cells

Tyrosinase inhibitory activity and melanin content in cells of SOP-1 and SOP-2, which are compounds isolated from fast-fermented sesame seeds, were evaluated in the same manner as in Example 2. Arbutin (100 μg/mL) was used as a positive control.

As a result, as shown in FIG. 5B, the cell tyrosinase inhibitory activity of SOP-1 and SOP-2, which are compounds isolated from fast-fermented sesame seeds, was significantly superior to that of STG, a major component of sesame seeds.

In addition, as shown in FIG. 5C, it was confirmed that the melanin content reduction effect of SOP-1 and SOP-2, which are compounds isolated from fast-fermented sesame seeds, was similar or superior to that of STG, the main component.

Example 4. Wrinkle improvement effect of sesame high-speed fermented product

1) Evaluation of intracellular collagenase inhibitory activity of high-speed fermented sesame seeds

The anti-wrinkle effect of the high-speed fermented sesame seeds was confirmed by evaluating the intracellular collagenase inhibitory activity by the amount of collagenase biosynthesis using CCD-986sk cells (Human skin fibroblast cells).

Specifically, CCD-986sk cells were prepared in DMEM (Dulbeco's modified eagle) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin (100 U/mL). Using 'medium) medium, 37 ℃, 5% CO ₂ It was subcultured while adapting to an incubator (incubator). Stabilized CCD-986sk cells were dispensed in 1×10 ⁵ per well in a 12-well plate, and then, after adding the experimental sample or positive control (EGCG) to each well, the cells were cultured in a CO ₂ incubator for 3 days. After incubation, trypsin was treated, dissolved in 100 mM Tris HCl (0.5 mM MgCl ₂ , 0.1% Triton X-100) buffer, and 50 μl of the supernatant was put into a 96-well plate, reacted at 37 ° C for 16 hours, and then dried. oven) for 24 hours. After the reaction, it was washed three times with 200 μl of distilled water, and a saturated picoric acid solution containing 0.1% Sirius Red F3BA was prepared, and then 100 μl was added to each well and incubated at 37 ° C. reacted for 1 hour. Thereafter, 200 μl of 10 mM HCl solution was added to each well and washed 5 times. 200 μl of 0.1N NaOH was added to each well, reacted for 5 minutes, transferred to a new plate, and absorbance was measured at 540 nm.

As a result, as shown in Figure 6A, it was confirmed that the collagenase inhibitory activity of the sesame fast-fermented product was significantly superior to that of the sesame hot water extract.

2) Measurement of type 1 procollagen synthesis in cells of fast-fermented sesame seeds

In CCD-986sk cells irradiated with ultraviolet rays, the ability to synthesize type 1 propolagen of the high-speed fermented sesame seeds prepared by the production method of the present invention was measured. After inoculating 5×10 ⁴ cells per well in a 24-well plate, they were cultured for 24 hours. After culturing, the cells were washed twice with PBS (phosphate buffer saline) and cultured for 24 hours by adding serum-free culture medium. Thereafter, 20mJ/cm 2 of ultraviolet (UVB) was pretreated, and the sample of Preparation Example 1 was added and cultured for 48 hours, and then the culture medium was collected and used to measure the amount of synthesized type 1 procollagen. The degree of procollagen synthesis in the cell culture medium was measured using a type 1 procollagen C-peptide assay kit (Takara Bio Inc., Japan) according to the manufacturer's protocol.

As a result, as shown in FIG. 6B, the amount of type 1 procollagen synthesis reduced by ultraviolet irradiation was significantly increased by the treatment of the sesame fast fermented product of the present invention, which was confirmed to be an excellent result compared to the sesame hot water extract.

Example 5. Anti-wrinkle effect of compound isolated from sesame fast fermented product

1) Evaluation of collagenase inhibitory activity of compounds isolated from high-speed fermentation of sesame seeds

The collagenase inhibitory activity of the compound isolated from the fast fermented sesame seeds was evaluated by measuring the amount of collagenase biosynthesis as described in Example 4 above.

As a result, as shown in Figure 7A, it was confirmed that SOP-1 and SOP-2, which are compounds isolated from sesame fast fermentation, have excellent collagenase inhibitory activity, and collagenase inhibition even compared to STG, a major component of sesame seeds. It was confirmed that the activity was significant.

2) Measurement of intracellular MMP-1 inhibitory effect of compounds isolated from high-speed fermentation of sesame seeds

The intracellular MMP-1 inhibitory effect of the compound isolated from the fast fermented sesame seeds was measured.

CCD-986sk cells were prepared in DMEM (Dulbeco's modified eagle'medium) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin (100 U/mL). Using the medium, subculture was performed while adapting to a 37°C, 5% CO ₂ incubator. Stabilized CCD-986sk cells were dispensed in 1 × 10 ⁵ per well in a 12-well plate and cultured for 24 hours. After washing the cells twice with PBS, serum-free culture medium was added, cultured for 24 hours, and 20mJ / cm 2 of ultraviolet rays (UVB) were pretreated, and samples of the experiment were added at various concentrations, followed by incubation in a CO ₂ incubator for 3 days. After culturing, the culture medium was collected and used to measure the MMP-1 protein content. The MMP-1 standard solution of the MMP-1 ELISA kit (R&D Systems, Minneapolis, MN, USA) was diluted 1/2 to 6.25-100 ng/mL, and 100 μl of the standard solution and 100 μl of the sample were mixed into the MMP-1 ELISA kit. After dispensing on the plate, 100 μl of HRP conjugate (horseradish peroxidase conjugate) was added, followed by reaction at 37° C. for 2 hours. Thereafter, each well was repeatedly washed 5 times with a buffer solution to remove unbound antigen, and 100 μl of chromogenic tetramethyl benzidine was added thereto and reacted for 30 minutes. Thereafter, 100 μl of stop reagent was added, and after 15 minutes, absorbance was measured at 450 nm using an ELISA plate reader (Tecan Austria GmBH).

As a result, as shown in FIG. 7B, it was confirmed that the MMP-1 content increased by ultraviolet irradiation was reduced by treatment with SOP-1 and SOP-2, which are compounds isolated from sesame fast fermented material, and STG, a major component of sesame seeds. Even compared to SOP-1 and SOP-2 treatment, the MMP-1 content reduction effect was excellent.

3) Measurement of type 1 procollagen synthesis in cells of compounds isolated from sesame high-speed fermentation

In the same manner as described in Example 4, the amount of procollagen type 1 synthesized in cells of the compound isolated from the fast fermented sesame seeds was measured.

As a result, as shown in FIG. 7C, the amount of type 1 procollagen synthesis reduced by ultraviolet irradiation was increased by treatment with SOP-1 and SOP-2, which are compounds isolated from fast fermented sesame seeds.

Example 6. Cytotoxicity evaluation of fast fermented sesame seeds and compounds isolated therefrom

Cytotoxicity was confirmed by MTT assay. In the MTT assay, MTT tetrazolium, a yellow water-soluble substrate, is converted into blue-purple water-insoluble MTT formazan [formazan, (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl- tetrazolium bromide], the concentration of MTT formazan was measured using an ELISA plate reader (Tecan Austria GmBH, Grodig, Austria) at 540 nm.

B16F10 or CCD-986sk was dispensed to 5×10 ⁴ per well in a 96-well plate, samples were prepared by concentration, 0.02 mL was added, and incubated at 37° C., 5% CO ₂ incubator for 24 hours. The control group was cultured under the same conditions by adding the same amount of distilled water as the sample. 0.02 mL of MTT solution prepared at a concentration of 5 mg/mL was added thereto and incubated for 4 hours, then the culture medium was removed, 0.15 mL of DMSO was added to each well, reacted at room temperature for 30 minutes, and absorbance was measured at 540 nm with an ELISA reader. The cell viability was measured by the decrease in absorbance of the sample solution added group and non-added group.

As a result, as shown in FIG. 8, the high-speed fermented sesame seeds, hot-water sesame extract, STG, SOP-1, SOP-2, arbutin, and EGCG of the present invention are safe substances without cytotoxicity at concentrations showing whitening and wrinkle-improving effects. It was confirmed that

Example 7. Establishment of Optimal Conditions for Manufacturing High-speed Fermented Sesame Seeds with Increased Episesaminon Diglucoside and Sesaminol Diglucoside Contents

Through the above examples, the increased whitening and wrinkle improvement effects of the sesame high-speed fermented sesame seeds prepared by the manufacturing method of the present invention compared to the sesame hot water extract were determined to be due to SOP-1 or SOP-2 generated in the high-speed fermentation process, and these contents The optimal manufacturing method for significantly increasing the was confirmed through SOP-2 content analysis.

In order to confirm the optimal conditions, as shown in FIG. 9, the content of SOP-2 was adjusted by adjusting the high-speed fermentation time, enzyme (mushroom-derived enzyme source) concentration, substrate (sesame hot water extract concentrated under low temperature and reduced pressure) concentration, pH, and fermentation temperature. confirmed.

As a result, after mixing the substrate (hot water extract of sesame seeds concentrated under low temperature and reduced pressure) and the enzyme (an enzyme source derived from button mushrooms), adjusting the pH to 5.5 and then fermenting with stirring at 30 ° C for 120 minutes, the content of SOP-2 was found to increase the most.

Claims (12)

Preparation of high-speed fermented sesame seeds with enhanced content of episesaminon diglucoside and sesaminol diglucoside, including mixing a hot water extract of sesame seeds concentrated under reduced pressure and an enzyme source derived from button mushrooms, followed by fermentation for 20 to 180 minutes method. The method of claim 1, wherein the hot water extract of sesame seeds concentrated under low temperature and reduced pressure is obtained by adding 1 to 10 L of water to 1 kg of dried sesame seeds, followed by hot water extraction at 60 to 80 ° C. for 80 to 100 minutes, and then concentrated at low temperature and reduced pressure. A method for producing a high-speed fermented sesame seed product with increased content of episesaminon diglucoside and sesaminol diglucoside. The method of claim 2, wherein the enzyme source derived from button mushrooms is a filtrate obtained by adding 3 to 5 liters of water to 1 kg of button mushrooms, pulverizing, and then filtering. Method for producing high-speed fermented sesame seeds. The method of claim 1, wherein the manufacturing method
1) After adding 4 to 5 L of water to 1 kg of dried sesame seeds, hot water extraction at 65 to 75 ° C. for 85 to 95 minutes, followed by low-temperature vacuum concentration;
2) adding 3.5 to 4.5 L of water to 1 kg of button mushrooms, grinding them, and then filtering to obtain a filtrate, a button-derived enzyme source, at a concentration of 222 to 285 g/L; and
3) After mixing the hot water extract of sesame seeds concentrated under low temperature and reduced pressure in step 1) and the enzyme source derived from button mushrooms in step 2), the pH was adjusted to 4.5 to 6.5, and then stirred at 15 to 60 ° C for 80 to 140 minutes A method for producing a high-speed fermented sesame product with increased content of episesaminon diglucoside and sesaminol diglucoside, comprising: The method of claim 4, wherein the button-derived enzyme source in step 2) measured the amount of benzotropolone produced after oxidation using pyrogallol as a substrate at 360 nm using a UV spectrometer. A method for producing a high-speed fermented sesame seed product with increased content of episesaminon diglucoside and sesaminol diglucoside, characterized in that the absorbance is 0.7 to 0.85. A high-speed fermented sesame seed with increased content of episesaminon diglucoside and sesaminol diglucoside, prepared by the method of any one of claims 1 to 5. A cosmetic composition for skin whitening and wrinkle improvement containing, as an active ingredient, the fast-fermented sesame seed with increased content of episesaminon diglucoside and sesaminol diglucoside according to claim 6. A health functional food composition for skin whitening and wrinkle improvement containing, as an active ingredient, the fast-fermented sesame seed with increased content of episesaminon diglucoside and sesaminol diglucoside according to claim 6. A cosmetic composition for skin whitening and wrinkle improvement containing episesaminon diglucoside or an acceptable salt thereof as an active ingredient. A health functional food composition for skin whitening and wrinkle improvement containing episesaminon diglucoside or an acceptable salt thereof as an active ingredient. A cosmetic composition for skin whitening containing sesaminol diglucoside or an acceptable salt thereof as an active ingredient. A health functional food composition for skin whitening containing sesaminol diglucoside or an acceptable salt thereof as an active ingredient.

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