KR102249711B1 - Composition for enhancing skin elasticity or improving skin wrinkels comprising enzyme treated extract of leguminous plants cultured root - Google Patents

Abstract

The present invention is a composition for improving skin elasticity or skin wrinkles containing an enzyme-treated extract of a legume cultured root as an active ingredient, wherein the enzyme-treated extract is added to the extract of the legume cultured root by adding an enzyme or a microorganism producing the same One, it discloses a composition for improving skin elasticity or for improving skin wrinkles. Specifically, the composition according to the present invention contains, as an active ingredient, an enzyme-treated extract of cultured legumes containing a high content of coumestrol obtained by treating the extract of cultured legumes with enzymes to enhance skin elasticity or It can show the effect of improving skin wrinkles.

Description

A composition for enhancing skin elasticity or improving skin wrinkles containing enzyme-treated extracts of cultured legumes of legumes {COMPOSITION FOR ENHANCING SKIN ELASTICITY OR IMPROVING SKIN WRINKELS COMPRISING ENZYME TREATED EXTRACT OF LEGUMINOUS PLANTS CULTURED ROOT}

The present invention relates to a composition for enhancing skin elasticity or improving skin wrinkles containing an enzyme-treated extract of legume cultured root, and more particularly, to a composition for enhancing skin elasticity or improving skin wrinkles, and more specifically, the content of cumestrol is large by enzymatic treatment of the extract of the legume cultured root. It relates to a composition that is increased to exhibit excellent skin elasticity enhancement or skin wrinkle improvement effect.

Among the changes in human skin, the most representative changes are the decrease in skin function and visual beauty caused by aging. The final appearance of the skin as a result of skin aging includes the formation of wrinkles, loss of skin elasticity, and formation of senile spots, and wrinkle formation and loss of elasticity are considered the most representative phenomena.

Recently, many physiological studies have been conducted on the causes of skin wrinkles and elasticity reduction, and to prevent this, methods such as maintaining moisture in the stratum corneum by using a moisturizer, protecting the skin from ultraviolet rays or the environment, and promoting new cell activity have been applied. .

In general, enzymes, fat-soluble and water-soluble antioxidants, radical scavengers, etc., which are used for the purpose of inhibiting skin aging, have a preventive effect of suppressing wrinkle formation and loss of elasticity due to skin aging, and vitamin A, which is recently in the spotlight as an anti-aging substance, Alpha hydroxy acids and the like are known to have a healing effect that relieves wrinkles and improves elasticity generated by promoting collagen synthesis in the dermis. However, these antioxidants, radical scavengers, vitamin A, alpha hydroxy acids, and the like have limitations in that stability and skin stability are not good when formulated in cosmetics.

Accordingly, in the cosmetic field, the manufacture of cosmetics using natural substances is in the spotlight in order to solve this problem.

On the other hand, coumestrol is a substance known to be the most powerful among plant estrogens so far. It is mainly found in seeds, roots and leaves of leguminosae and compositae plants. In general, it is classified as a coumestan-based compound. Coumestrol is a substance that received attention as it was known to play a role in preventing infection by acting as antibacterial, antifungal and antiviral through antioxidant, anti-inflammatory and anti-toxin effects as it is secreted in a dark concentration at the wound site when plants are injured. . This phenomenon is because infections with various bacteria, fungi, and viruses induce the synthesis of various aromatic compounds, including coumestrol. The source of this antibiotic action is known to be that it has a phenolic structure, which is a basic chemical skeleton as an antioxidant, and inhibits the influx of free radicals oxidizing agents to prevent the formation of peroxidants in the body. . In addition, it is known that only cumestrol has an estrogen effect among various natural coumestane derivatives.

However, because the coumestrol is present in a very small amount in some plants, the currently commercially available natural coumestrol is on the market at a high price. For this reason, studies to obtain cumestrol through synthesis have been intermittently attempted, but due to the rather complex chemical structure in which several aromatic rings are fused, no approach to the development of an easy synthesis method has been introduced, although several synthetic methods have been reported. However, since each synthesis method has various problems, there are many difficulties in commercialization. In addition, since natural ingredients have a good image in terms of safety than ingredients produced by chemical synthesis in recent years, there is a need for research on a method that can be mass-produced from natural substances.

Korean Registered Patent Publication No. 10-1502687 Korean Patent Publication No. 10-0706279

An aspect of the present invention is to provide an enzyme-treated extract of cultured legumes with an increased content of coumestrol.

An aspect of the present invention is to provide a composition for enhancing skin elasticity or improving skin wrinkles, comprising an enzyme-treated extract of a legume cultured root containing a high content of coumestrol as an active ingredient.

In order to achieve the above object, an embodiment of the present invention is a composition for improving skin elasticity or skin wrinkles containing an enzyme-treated extract of cultured legumes as an active ingredient, wherein the enzyme-treated extract is the legume It provides a composition for enhancing skin elasticity or improving skin wrinkles, by adding an enzyme or a microorganism that produces the same to the extract of cultured root.

The composition according to the present invention contains, as an active ingredient, an enzyme-treated extract of cultured legumes containing a high content of coumestrol obtained by treating the extract of cultured legumes with enzymes as an active ingredient to enhance skin elasticity or improve skin wrinkles. Can represent.

Figure 1 shows the results of confirming the results of the HPLC chromatogram of cumestrin and cumestrol in the soybean culture root extract before and after the enzymatic reaction.
Figure 2 shows the results of measuring the amount of collagen produced in the soybean culture root extract before and after the enzyme reaction.

In the present specification, the term “explantation” is also referred to as explantation or in vitro culture, and is a technique of mainly separating a part of an animal or plant individual and culturing it in vitro. Mainly, it survives in vitro over a period of time and involves culturing over a period of time so that certain developmental changes are observed. General tissue culture (組織培養) is also a method of in vitro culture. In addition, in-flight culture according to an aspect of the present invention is also included in in vitro culture, and may also be referred to as aseptic culture or plant stem cell culture.

In the present specification, the explantation technology is a culture medium containing nutrients in-vitro by extracting at least one selected from the group including protoplasts, cells, tissues, organs, embryos, seeds, cultured roots, and parts of plants. It includes regenerating a callus or a single cell population into an organic or fully functional plant by culturing aseptically using an artificial culture device such as an artificial culture device.

In the present specification, the term "enzyme preparation" is a preparation containing a single or several types of active enzymes, excipients, and additives, and may be commercially available as well as arbitrarily prepared in a laboratory. An example of an enzyme preparation in the present specification is an enzyme preparation containing polygalacturonase, a kind of pectinase derived from Aspergillus aculeatus, preferably pecti It may be Nex Ultra SP-L (Pectinex Ultra SP-L, Novozyme, Denmark).

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention is a composition for enhancing skin elasticity or improving skin wrinkles containing an enzyme-treated extract of a legume cultured root as an active ingredient, wherein the enzyme-treated extract is an enzyme or a production thereof in the extract of the legume cultured root It may be a composition for improving skin elasticity or for improving skin wrinkles, to which microorganisms are added.

As an embodiment, the legume may be beans. As another embodiment, the beans may be one or more selected from soybeans, flat beans, seoritae, seomoktae, black tae, cheongtae, yellow tae, hedge beans, kidney beans, green beans, red beans, reaps, new beans and bean sprouts, preferably May be at least one selected from soybeans, flat beans, seoritae, seomoktae, black tae, cheongtae, yellow tae, hedge beans, red beans, reaps and shinhwa beans. The cultivar of the beans is not limited, but in one aspect, it may be a cultivar for soybean paste and tofu, for namul, for rice, or for green beans. Varieties for soybean and tofu include Daepung (application for variety protection application-2003-152), Hojang (application for variety protection application-2003-155), Jangwon (application for variety protection application-2001-34), Rhubarb (applied for variety protection-2000-19), Sodam (applied for variety protection-1999-19), Songhak (applied for variety protection-1999-22), Daewon (Daewonkong, variety name registration number 01-0003) -38), Genuine (Jinpumkong, application for variety protection -1998-204), Protein (Danbaegkong, application for variety protection -1998-201), Soymilk (Duyoukong, application for variety protection -1998-151), Shinpaldal ( Shinpaldalkong, Variety Protection Application-1998-199), Taekwangkong, Variety Protection Application-1998-198), Manli (Mallikong, Variety Name Registration No. 01-0003-11), Jangsukong, Variety Name Registration No. 01-0003-9), Wuhan (Muhankong, variety name registration No. 01-0003-7), Baegunkong (applied for variety protection application-1998-193), Saealkong, applied for variety protection application- 1998-143), gold (kind name registration number 01-0003-1) and Jangyeop (variety name registration number 01-0003-41). Varieties for namul include Shinhwa (kind name registration no. 01-0003-134), Sowon (applied for variety protection application-2000-16), and Anpyeong (applied for variety protection application-2003-151). , Sunam (applied for variety protection-2003-153), Dachae (applied for variety protection-2003-148), Sorok (applied for variety protection-2002-116), Sohokong (applied for variety protection- 2001-36), Somyeongkong, Variety Protection Application-1998-18), Dawon (Variety Protection Application-1998-209), Pungsannamul, Variety Protection Application-1998-140), Iksannamul , Variety protection application application-1998-139), Sobaek namul (variety name registration number 01-0003-31), Gwangan (Kwangankong, variety protection application application-1998-202), Single leaf and galaxy (Eunhakong, variety name registration) No. 01-0003-6). Varieties for rice include Cheongjakong (applied for variety protection application-2001-38), Heugcheong (applied for variety protection application-2000-27), Galmi (applied for variety protection application-2000-25), and Seonheukkong, Variety protection application application-1999-20), black beans and excellent black beans (variety protection application application-1998-158). In addition, the varieties for green beans are Daol (Daol, application for variety protection application-2003-149), Shinrok (application for variety protection application-2001-35), Saeul, Gyeul, Seokryang green beans, Hwaom green beans (variety name registration number) 01-0003-21) and Big Autumn. In another aspect of the present invention, it is preferable that the bean is a cultivar that is capable of germination and is resistant to pests and diseases. Such beans include, for example, Shinhwa, Sowon, Anpyeong, Seonam, Dachae, Sorok, Soho, Somyeong, Dawon, Poongsan Green, Iksan Green, Sobaek Namul, Gwangan, Danyeop and Galaxy. However, the bean according to an aspect of the present invention is not limited to the bean variety.

The flat bean according to an aspect of the present invention is variously called as lead bean, lead bean, lead bean, lead bean, and the like.

In one embodiment, the enzyme may be pectinase or polygalacturonase.

As another embodiment, the enzyme may be added in the form of an enzyme preparation containing the enzyme, and the amount of the enzyme in the enzyme preparation may be 0.5 to 10% by weight based on the total weight of the enzyme preparation.

In addition, as an embodiment, the microorganism may be Aspergillus aculeatus. Since the enzyme or enzyme preparation is derived from Aspergillus aculeatus, the same effect as using the enzyme or enzyme preparation can be obtained through treatment or fermentation using the microorganism.

As an embodiment, the amount of the enzyme added may be 0.8 to 8 parts by weight based on 100 parts by weight of the extract of the legume culture root. For example, the amount of the enzyme added is 0.8 parts by weight or more, 0.9 parts by weight or more, 1 part by weight or more, 1.2 parts by weight or more, 1.5 parts by weight or more, 2 parts by weight or more, 2.5 parts by weight or more, 3 parts by weight or more, 3.5 parts by weight or more, 4 parts by weight or more, 4.5 parts by weight or more, 5 parts by weight or more, 5.5 parts by weight or more, 6 parts by weight or more, 6.5 parts by weight or more, 7 parts by weight or more, or May be 7.5 parts by weight or more, 8 parts by weight or less, 7.5 parts by weight or less, 7 parts by weight or less, 6.5 parts by weight or less, 6 parts by weight or less, 5.5 parts by weight or less, 5 parts by weight or less, 4.5 parts by weight or less, 4 parts by weight Hereinafter, it may be 3.5 parts by weight or less, 3 parts by weight or less, 2.5 parts by weight or less, 2 parts by weight or less, 1.8 parts by weight or less, 1.5 parts by weight or less, 1.2 parts by weight or less, or 1 part by weight or less.

In another embodiment, the amount of the enzyme preparation added is 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, 90% by weight or more, 100% by weight or more, based on the total weight of the extract of the legume culture root. Alternatively, it may be 110% by weight or more, and may be 120% by weight or less, 110% by weight or less, 100% by weight or less, 90% by weight or less, 80% by weight or less, 70% by weight or less, or 60% by weight or less. Preferably, it may be 90 to 110% by weight based on the total weight of the extract of the legume culture root.

As an embodiment, the enzyme of the enzyme preparation may be an enzyme that degrades pectin in plant tissues. Specifically, the enzyme preparation may include polygalacturonase, a kind of pectinase derived from Aspergillus aculeatus, as an enzyme, and as another embodiment, The enzyme preparation may further include pectintranseliminase, pectinesterase, hemicellulase, and cellulases as enzymes. In another embodiment, the enzyme preparation may be pectinex ultra SPL (Novozyme, Denmark).

As an embodiment, the amount of polygalacturonase contained in the enzyme preparation is 0.1% by weight or more, 0.5% by weight or more, 0.8% by weight or more, 1% by weight or more, 1.2% by weight or more based on the total weight of the enzyme preparation. , 1.4% or more, 1.6% or more, 1.8% or more, 2% or more, 2.2% or more, 2.6% or more, 2.8% or more, 3% or more, 3.2% or more, 3.4% or more , 3.6% or more, 3.8% or more, 4% or more, 4.2% or more, 4.4% or more, 4.6% or more, 4.8% or more, 5% or more, 5.2% or more, 5.4% or more , 5.6 wt% or more, 6 wt% or more, 6.5 wt% or more, or 7 wt% or more, and 8 wt% or less, 7.5 wt% or less, 7 wt% or less, 6.5 wt% or less, 6 wt% or less, 5.8 wt. % Or less, 5.6 wt% or less, 5.4 wt% or less, 5.2 wt% or less, 5 wt% or less, 4.8 wt% or less, 4.6 wt% or less, 4.4 wt% or less, 4.2 wt% or less, 4 wt% or less, 3.8 wt. % Or less, 3.6% or less, 3.4% or less, 3.2% or less, 3% or less, 2.8% or less, 2.6% or less, 2.4% or less, 2.2% or less, 2% or less, 1.8% % Or less, 1.6 wt% or less, 1.4 wt% or less, 1.2 wt% or less, 1 wt% or less, 0.8 wt% or less, 0.6 wt% or less, 0.4 wt% or less, or 0.2 wt% or less. The preferred amount of polygalacturonase in the enzyme preparation may be 1 to 5% by weight.

As an embodiment, the amount of polygalacturonase contained in the enzyme preparation is 2000 units/mL or more, 2200 units/mL or more, 2400 units/mL or more, 2600 units/mL or more, 2800 units/mL or more based on enzymatic activity. , 3000units/mL or more, 3200units/mL or more, 3400units/mL or more, 3600units/mL or more, 3800 units/mL or more, 4000units/mL or more, 4200units/mL or more, 4400units/mL or more, 4600units/mL or more, or 4800units/mL It can be more than that In addition, the above amount is 5000units/mL or less, 4800units/mL or less, 4600units/mL or less, 4400units/mL or less, 4200units/mL or less, 4000units/mL or less, 3800units/mL or less, 3600units/mL or less, 3400units/mL or less, 3200units or less /mL or less, 3000 units/mL or less, 2500 units/mL or less, or 2400 units/mL or less. Preferably, the amount of polygalacturonase contained in the enzyme preparation may be 3800 units/mL or more.

As an embodiment, the amount of the enzyme added may be 2000 to 5000 units compared to 1 g of the extract of the leguminous plant culture root based on the enzyme activity. For example, the amount of the enzyme added is 2000 units or more, 2200 units or more, 2400 units or more, 2600 units or more, 2800 units or more, 3000 units or more, 3200 units or more, 3400 units or more, 3600 units or more, May be 3800 units or more, 4000 units or more, 4200 units or more, 4400 units or more, 4600 units or more, or 4800 units or more, and 5000 units or less, 4800 units or less, 4600 units or less, 4400 units or less, 4200 units or less, 4000 units or less, 3800 units or less, 3600 units or less, 3400 units or less, 3200 units or less , 3000units or less, 2500units or less, or 2400units or less. Preferably, the addition amount may be 3500 to 4200 units compared to 1 g of the extract of the leguminous plant culture root based on the enzyme activity.

In one aspect, the function and role of the enzyme or the enzyme preparation is to remove the sugar from the coumestrin in the extract of the legume culture root so that the coumestrin can be converted to coumestrol. By combining the technical characteristics of this enzyme preparation with an xplantation method that maintains uniform quality and mass-produces, the yield of coumestrol can be remarkably improved while maintaining uniformity.

In one embodiment, the enzyme-treated extract is centrifuged after maintaining the reaction product obtained by adding an enzyme or a microorganism producing the same to the extract of the legume culture root for 38 to 58 hours at 40 to 50°C and 60 to 100 rpm for 38 to 58 hours. It may be a sediment recovered by separating. In one aspect, the temperature may be 40°C or more, 42°C or more, 44°C or more, 46°C or more, or 48°C or more, and may be 50°C or less, 48°C or less, 46°C or less, 44°C or less, or 42°C or less. . In one aspect, the rpm may be 60 or more, 70 or more, 80 or more, 90 or more, or 95 or more, and may be 100 or less, 95 or less, 90 or less, 85 or less, 80 or less, 75 or less, or 65 or less. In one aspect, the time may be 38 hours or more, 40 hours or more, 42 hours or more, 44 hours or more, 46 hours or more, 48 hours or more, 50 hours or more, 52 hours or more, 54 hours or more, or 56 hours or more, and 58 hours It may be less than or equal to 56 hours, less than 54 hours, less than 52 hours, less than 50 hours, less than 48 hours, less than 46 hours, less than 44 hours, less than 42 hours, or less than 40 hours.

As an embodiment, the reactant may be obtained by adding distilled water to the result of adding enzymes or microorganisms to the extract of the legume culture root, and the concentration thereof is 0.5% by weight or more, 1% by weight or more, 1.5% by weight or more, 2% by weight or more, 2.5% by weight or more, 3% by weight or more, 5% by weight or more, or 8% by weight or more, and may be 10% by weight or less, 8% by weight or less, 5% by weight or less, 3% by weight or less, 2.5% by weight It may be 2% by weight or less, 1.5% by weight or less, 1% by weight or less, or 0.8% by weight or less.

As an exemplary embodiment, the extract of the legume culture root may be obtained by extracting the legume culture root using water, C1 to C6 lower alcohol, or a mixture thereof as a solvent.

As an embodiment, the alcohol concentration of the mixture may be 60 to 100% (w/v). For example, the alcohol concentration is 60% (w/v) or more, 70% (w/v) or more, 75% (w/v) or more, 80% (w/v) or more, 85% (w/v) ) Or more, 90% (w/v) or more, or 95% (w/v) or more, and 100% (w/v) or less, 95% (w/v) or less, 90% (w/v) or less , 85% (w/v) or less, 80% (w/v) or less, 75% (w/v) or less, 70% (w/v) or less, or 65% (w/v) or less.

As another embodiment, the lower alcohol may be ethanol.

As another embodiment, the extract of the leguminous plant culture root may be obtained by drying the legume culture root and then extracting the dried culture root using the solvent.

More specifically, the extract of the leguminous plant culture root may be extracted by the weight ratio of the culture root dried the culture root: the extraction solvent of 1:10 to 1:70. For example, the weight ratio is 1:10 or more, 1:15 or more, 1:20 or more, 1:25 or more, 1:30 or more, 1:35 or more, 1:40 or more, 1:45 or more, 1: It may be 50 or more, 1:55 or more, 1:60 or more, or 1:65 or more. In addition, the weight ratio is 1:70 or less, 1:65 or less, 1:60 or less, 1:58 or less, 1:55 or less, 1:52 or less, 1:50 or less, 1:48 or less, 1:45 or less, It may be 1:42 or less, 1:40 or less, 1:38 or less, 1:36 or less, 1:35 or less, 1:30 or less, 1:25 or less, 1:20 or less, or 1:15 or less.

As an embodiment, the extraction time may be 20 to 28 hours. Specifically, the extraction time may be 20 hours or more, 22 hours or more, 24 hours or more, 25 hours or more, or 26 hours or more, and 28 hours or less, 26 hours or less, 25 hours or less, 24 hours or less, 22 hours or less, Or 21 hours or less.

As an embodiment, the extract of the leguminous plant culture root may be an extract of the cultured root grown while maintaining a constant air supply amount in the bioreactor by placing the legume culture root in a medium in a bioreactor.

As an exemplary embodiment, the bioreactor is a Stirred Tank Reactor, a Bubble Column Reactor, an Air Lift Reactor, a Fludized Bed Reactor, and a Pick / May be a fixed/packed bed reactor, or a tower fermenter, preferably a bulb type bubble bioreactor.

As an embodiment, the _{concentration of ammonium nitrate (NH 4} NO ₃ ) in the medium is 650 mg/L or more, 660 mg/L or more, 700 mg/L or more, 740 mg/L or more, 760 mg/L or more, 800 mg/L or more , 825mg/L or more, 850mg/L or more, 900mg/L or more, 1000mg/L or more, 1200mg/L or more, or 1400mg/L or more, 1500mg/L or less, 1400mg/L or less, 1200mg/L or less, 1000mg/ L or less, 990mg/L or less, 900mg/L or less, 850mg/L or less, 825mg/L or less, 800mg/L or less, 760mg/L or less, 740mg/L or less, 700mg/L or less, or 660mg/L or less.

As an embodiment, the _{concentration of calcium chloride (CaCl 2} · 2H ₂ O) in the medium is 175 mg/L or more, 176 mg/L or more, 190 mg/L or more, 200 mg/L or more, 220 mg/L or more, 240 mg/L or more, May be 260mg/L or more, 264mg/L or more, 270mg/L or more, 280mg/L or more, 300mg/L or more, 350mg/L or more or 380mg/L or more, 400mg/L or less, 380mg/L or less, 350mg/L Or less, 300mg/L or less, 280mg/L or less, 270mg/L or less, 264mg/L or less, 260mg/L or less, 240mg/L or less, 220mg/L or less, 200mg/L or less, 190mg/L or less, or 176mg/L It can be below.

As an embodiment, the concentration of magnesium sulfate (MgSO ₄ 7H ₂ O) in the medium is 145 mg/L or more, 148 mg/L or more, 150 mg/L or more, 160 mg/L or more, 180 mg/L or more, 185 mg/L or more , 190mg/L or more, 200mg/L or more, 215mg/L or more, 222mg/L or more, 240mg/L or more, 280mg/L or more, or 300mg/L or more, and 320mg/L or less, 300mg/L or less, 280mg/ L or less, 240 mg/L or less, 222 mg/L or less, 215 mg/L or less, 200 mg/L or less, 190 mg/L or less, 185 mg/L or less, 180 mg/L or less, 160 mg/L or less, 150 mg/L or less, or 148 mg/ It may be less than or equal to L.

In one embodiment, the _{concentration of potassium phosphate (KH 2} PO ₄ ) in the medium is 65 mg/L or more, 68 mg/L or more, 70 mg/L or more, 75 mg/L or more, 80 mg/L or more, 85 mg/L or more, It can be 90mg/L or more, 95mg/L or more, 100mg/L or more, 102mg/L or more, 120mg/L or more, or 140mg/L or more, and 150mg/L or less, 140mg/L or less, 120mg/L or less, 110mg/ L or less, 102mg/L or less, 100mg/L or less, 95mg/L or less, 90mg/L or less, 85mg/L or less, 80mg/L or less, 75mg/L or less, 70mg/L or less, or 68mg/L or less.

As an embodiment, the _{concentration of potassium nitrate (KNO 3} ) in the medium is 750mg/L or more, 760mg/L or more, 800mg/L or more, 850mg/L or more, 900mg/L or more, 950mg/L or more, 1000mg/L Or more, 1140mg/L or more, 120mg/L or more, or 1400mg/L or more, and 1500mg/L or less, 1400mg/L or less, 1300mg/L or less, 1140mg/L or less, 1000mg/L or less, 980mg/L or less, 950mg /L or less, 900mg/L or less, 850mg/L or less, 800mg/L or less, or 760mg/L or less.

As another embodiment, the medium may be one to which IBA (indole-3-butyric acid) and a carbon source are added. At this time, the carbon source is one selected from the group consisting of glucose, fructose, mannose, ribose, arabinose, xylose, galactose, sucrose, cellobiose, trehalose, lactose, raffinose, amylose, starch, sorbitol, mannitol, and glycerol. It can be more than that

As an embodiment, the medium may be MS medium (Murashige and Skoog medium). In one aspect, the type of the component in the medium is the same as the type of the component in the MS medium, and the concentration of the component is 40% or more, 45% or more, 50% or more, 55% or more, or 58 of the component concentration in the MS medium. % Or more, and may be 60% or less, 55% or less, 50% or less, or 45% or less.

As an embodiment, when the culture root is placed in the medium, the pH of the medium may be 4.8 to 6.8. Specifically, the pH may be 4.8 or more, 5 or more, 5.2 or more, 5.4 or more, 5.6 or more, 5.8 or more, 6.0 or more, 6.2 or more, 6.4 or more, or 6.6 or more. In addition, the pH may be 6.8 or less, 6.6 or less, 6.4 or less, 6.2 or less, 6.0 or less, 5.8 or less, 5.6 or less, 5.4 or less, 5.2 or less, or 5.0 or less.

As an embodiment, when placing the cultured muscle in a medium, the inoculation density of the cultured muscle may be 2 to 6 g/L. Specifically, the density may be 2g/L or more, 3g/L or more, 4g/L or more, or 5g/L or more. In addition, the density may be 6g/L or less, 5g/L or less, 4g/L or less, or 3g/L or less.

As an embodiment, the proliferation may be performed for 3 to 5 weeks in a dark condition of 19 to 25°C. Specifically, the temperature may be 19°C or higher, 20°C or higher, 21°C or higher, 22°C or higher, 23°C or higher, or 24°C or higher. In addition, the temperature may be 25°C or less, 24°C or less, 23°C or less, 22°C or less, 21°C or less, or 20°C or less. In addition, the proliferation period may be performed for 3 weeks or more or 4 weeks or more, and may be performed for 5 weeks or less or 4 weeks or less.

As an embodiment, the air supply amount may be 0.05 to 0.4 vvm (air volume/culture volume per min). Specifically, the amount of air supplied constant in the bioreactor is 0.05vvm or more, 0.08vvm or more, 0.1vvm or more, 0.12vvm or more, 0.14vvm or more, 0.16vvm or more, 0.18vvm or more, 0.2vvm or more, 0.22vvm or more. , 0.24vvm or more, 0.26vvm or more, 0.28vvm or more, 0.3vvm or more, 0.32vvm or more, 0.34vvm or more, 0.36vvm or more, or 0.38vvm or more. In addition, the air supply amount is 0.4vvm or less, 0.38vvm or less, 0.36vvm or less, 0.34vvm or less, 0.32vvm or less, 0.3vvm or less, 0.28vvm or less, 0.26vvm or less, 0.24vvm or less, 0.22vvm or less, 0.2vvm or less, It may be 0.18vvm or less, 0.16vvm or less, 0.14vvm or less, 0.12vvm or less, 0.1vvm or less, 0.08vvm or less, or 0.06vvm or less.

As described above, the enzyme-treated extract of the legume culture root of the present invention can exert an eco-friendly and uniform effect by using an eco-friendly and uniformly obtained legume culture tissue in large quantities without the use of growth promoters, fertilizers, pesticides, etc. have.

As an embodiment, the content of coumestrol in the enzyme-treated extract may be 20 to 200 mg compared to 1 g of the enzyme-treated extract. For example, the content is 20mg or more, 40mg or more, 60mg or more, 80mg or more, 100mg or more, 110mg or more, 120mg or more, 130mg or more, 140mg or more, 142mg or more, 143mg or more, 144mg or more, 144.27mg or more compared to 1g of the enzyme-treated extract. mg or more, 145 mg or more, 145.5 mg or more, 146 mg or more, 146.4 mg or more, 147 mg or more, 147.5 mg or more, 148 mg or more, 148.5 mg or more, 148.53 mg or more, 149 mg or more, 150 mg or more, 155 mg or more, 160 mg or more, 170 mg or more, It may be 180mg or more or 190mg or more, and it may be 200mg or less, 190mg or less, 180mg or less, 170mg or less, 160mg or less, 155mg or less, 150mg or less, 149mg or less, 148.6mg or less, 148.53mg or less, 148.5mg or less, 148mg or less, 147.5mg Or less, 147mg or less, 146.4mg or less, 146mg or less, 145.5mg or less, 145mg or less, 144.5mg or less, 144.27mg or less, 144mg or less, 143mg or less, 142mg or less, 140mg or less, 130mg or less, 120mg or less, 110mg or less, 100mg or less , 80mg or less, 60mg or less, or may be 40mg or less. Preferably, the content of the cumestrol may be 140 to 150 mg compared to 1 g of the enzyme-treated extract. The content is significantly increased to about 100 times as compared to the case where the general soybean root extract grown in the field is not treated with an enzyme preparation (1.7 to 1.9 mg or less per 1 g of the extract). In addition, the content is about three times higher than that of using a conventional MS medium (Murashige and Skoog medium).

As an embodiment, the enzyme-treated extract of the legume culture root may be contained in an amount of 0.1 to 100 μg/ml based on the total weight of the composition. Specifically, the enzyme-treated extract of the legume culture root is 0.1 μg/ml or more, 0.5 μg/ml or more, 1 μg/ml or more, 5 μg/ml or more, 10 μg/ml or more, 15 μg/ml or more, 20 μg/ml or more, 25 μg/ml or more, 30 μg/ml or more, 35 μg/ml or more, 40 μg/ml or more, 45 μg/ml or more, 50 μg/ml or more, 55 μg/ml or more, 60 μg/ml or more, 65 μg/ml or more, 70μg/ml or more, 75μg/ml or more, 80μg/ml or more, 85μg/ml or more, 90μg/ml or more, or 95μg/ml or more, and also 100μg/ml or less, 95μg/ml or less, 90μg/ml or less, 85μg /ml or less, 80μg/ml or less, 75μg/ml or less, 70μg/ml or less, 65μg/ml or less, 60μg/ml or less, 55μg/ml or less, 50μg/ml or less, 45μg/ml or less, 40μg/ml or less, 35μg /ml or less, 30 μg/ml or less, 25 μg/ml or less, 20 μg/ml or less, 15 μg/ml or less, 10 μg/ml or less, 5 μg/ml or less, 1 μg/ml or less, or 0.5 μg/ml or less. Preferably, it may be contained in an amount of 1 to 10 μg/ml.

As an embodiment, the composition may be a cosmetic composition, and the formulation of the cosmetic composition is not particularly limited, and may be appropriately selected depending on the purpose. For example, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, foundation, essence, nutrition essence, pack, soap, cleansing Foam, cleansing lotion, cleansing cream, body lotion, and may be prepared in one or more formulations selected from the group consisting of a body cleanser, but is not limited thereto.

Depending on the formulation of the cosmetic composition according to the present invention, a generally used carrier component may be additionally included, and in addition to the enzyme-treated extract of the legume cultured root, functional additives and components included in the general cosmetic composition may be additionally included. The functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingo lipids, and seaweed extract.

In the cosmetic composition according to the present invention, in addition to the functional additives, components included in general cosmetic compositions may be blended as needed. Other ingredients included include oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation Examples include accelerators, cooling agents, restrictors, and purified water.

The blending amount of the ingredients is not particularly limited, but can be easily selected by a person skilled in the art within a range that does not impair the object and effect of the present invention.

In one embodiment, the composition may be a food composition, and the formulation of the food composition is not particularly limited, but, for example, tablets, granules, pills, powders, liquids such as drinks, caramels, gels, bars, tea bags And the like. In the food composition of each formulation, in addition to the active ingredients, ingredients commonly used in the field can be appropriately selected and blended without difficulty by a person skilled in the art according to the formulation or purpose of use, and synergistic effect when applied simultaneously with other ingredients Can happen. In addition, the food may be a health functional food.

The composition may be administered by a variety of methods such as simple ingestion, drinking, injection administration, spray administration, or squeeze administration.

In the food composition according to an aspect of the present invention, the determination of the dosage of the active ingredient is within the level of a person skilled in the art, and may vary depending on various factors such as age, health condition, and complications of the subject to be administered. .

Food compositions according to one aspect of the present invention include, for example, various foods such as chewing gum, caramel products, candies, frozen desserts, and confectionery, beverage products such as soft drinks, mineral water, alcoholic beverages, and health including vitamins or minerals. It may be a nutraceutical product.

In addition to the above, the food composition according to an aspect of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and its Salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. In addition, food compositions according to an aspect of the present invention may include flesh for the manufacture of natural fruit juice and fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not so important, but is generally included in the range of 0 to about 60 parts by weight per 100 parts by weight of the composition according to an aspect of the present invention.

In another aspect, the present invention includes the steps of: (1) placing the legume culture root in a medium in a bioreactor to maintain a constant air supply amount in the bioreactor and proliferate it; (2) obtaining an extract from the cultured root passed through the step (1); And (3) adding an enzyme preparation, an enzyme, or a microorganism producing the same to the extract. It may relate to a method for preparing an enzyme-treated extract of cultured legumes for improving skin elasticity or for improving skin wrinkles.

As an embodiment, the method may include germinating a legume plant in a medium prior to step (1). As another embodiment, the method may include inducing a legume culture root from a legume plant before step (1).

On the other hand, the content of the main composition of steps (1) to (3) of the above method is for the composition for enhancing skin elasticity or improving skin wrinkles containing the enzyme-treated extract of the cultured root of the legume of the present invention as an active ingredient. Since it is the same as described in detail, it will be omitted below.

Hereinafter, the content of the present invention will be described in more detail through Examples and Test Examples. However, these examples and test examples are only presented to understand the content of the present invention, and the scope of the present invention is not limited to these examples and test examples, and modifications and substitutions commonly known in the art And insertion and the like can be performed, and this is also included in the scope of the present invention.

[Example 1] Soybean seed germination and induction of in-flight plants

Shinhwa bean seeds were sterilized for 20 minutes with a 2% by weight aqueous sodium hypochlorite solution, and then washed three times with sterile water. Then, using 0.5 MS medium (Murashige and Skoog Medium, Haarlem, The Netherlands) to which 30g/L of sucrose was added, the plants were induced for 2 weeks under bright conditions maintaining 25±1℃ in the cabin.

[Example 2] Soybean cultured root induction and proliferation

IBA (indole-3-butyric acid, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and NAA (Naphthalene Acetic Acid; Duchefa, Netherlands) each 4mg/L and sucrose 30g/L Cultured roots were induced in the added 1MS medium, and other culture conditions were maintained for 2 weeks under dark conditions maintaining 22±1°C. Then, IBA (indole-3-butyric acid, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) 4 mg/L in a bulb type bioreactor (commercially available) with an air volume of 3 L, Cultured roots were proliferated for 4 weeks using 2L of 0.5 MS medium (Murashige and Skoog medium, Duchefa, Netherlands) to which 30g/L of sucrose was added. The 0.5MS medium refers to a medium prepared by making the concentration of raw materials such as minerals used in the medium 1/2 of that of a normal MS medium. At this time, the medium was adjusted to pH 5.8 using 1N NaoH and sterilized for 35 minutes at 121°C and 1.2 atm. In the culture, the cultured muscle was cut into 1-1.5cm to obtain a 4g/L inoculation density based on the fresh weight. After inoculation in the medium, it was carried out in dark conditions maintained at 22±1°C. The amount of air supplied was constantly adjusted to 0.1vvm (air volume/culture volume per min) using air flow meters (RMA series; Dwyer Instruments, Inc., USA) during the entire culture period. The supplied air is sequentially passed through an air condenser that can condense compressed air, a filter that can remove impurities, and an air dryer, and then goes into the bioreactor using an air compressor that does not use oil. Supplied.

[Example 3] Extract preparation and enzyme treatment

1. Extract preparation

The soybean culture root obtained and dried according to Examples 1 and 2 was immersed in 80% (w/v) ethanol aqueous solution so that the weight ratio of the culture root: ethanol aqueous solution was 1:30, and extracted at room temperature for 24 hours. The extract was filtered using filter paper, and the solvent was evaporated to dryness to obtain a powdery extract.

2. Enzyme treatment

After treating the extract powder with a liquid pectinase enzyme preparation (pectinex ultra SP-L, Novozyme, Denmark) having the same weight as the soybean culture root extract powder obtained in 1. of Example 3, distilled water A reaction product was prepared at a concentration of 2% by weight (2% by weight of a mixture of an enzyme preparation and an extract powder and 98% of distilled water) (the enzyme preparation contains 1 to 5% by weight of polygalacturonase as a pectinase. , It contains 3800 units/mL or more of polygalacturonase on the basis of enzyme activity). The reactant was maintained at 45° C. and 80 rpm for 48 hours to react, and then the precipitate was recovered using a centrifugal separator. The recovered precipitate, that is, the enzyme-treated extract of the soybean culture root was freeze-dried and powdered.

[Experimental Example 1] Coumestrol assay test

The content of coumestrol in the soybean culture root extract was determined by filtering the extract with a 0.45μm filter before and after (the extract of Example 3) and after the enzyme treatment (the precipitate of Example 3) with a UV detector. Analysis was performed by injecting 10 μL into High Performance Liquid Chromatography (HPLC). The column was Mightysil RP-18 GP 250-4.6 (5 μm, KANTO CHEMICALS, JAPAN), and the content of cumestrol in the extract was measured at a wavelength of 342 nm. The measurement results are shown in Table 1 and FIG. 1 below.

Table 1 below is a comparison of the content of cumestrol in the soybean culture root extract before and after the enzyme treatment. In Table 1, the extraction yield refers to the weight of the extract according to Example 3, 1, and the weight of the sediment according to Example 3, 2, compared to the input weight of the soybean culture root, and the weight of the precipitate according to Example 3, respectively, as a percentage (%). It is represented by

Extraction yield (%) Cumestrol content in extract (mg/g) Before enzyme treatment 33.7±0.04 1.8±0.10 After enzyme treatment 5.6±0.20 146.4±2.13

In addition, Figure 1 shows the results confirmed by HPLC chromatogram for coumestrin and cumestrol in the soybean culture root extract before and after the enzymatic reaction.

From the results of Table 1 and Fig. 1, it can be seen that the sugar of coumestrin in the extract is removed by enzyme treatment and converted to coumestrole, and the amount converted to coumesrol is more remarkable than the amount by the conventional method. It could be confirmed that it increased significantly. In addition, when using a bioreactor, unlike conventional soybean (seed, plant-derived) extracts cultivated in the open field or outdoors, soybean culture root extracts of uniform quality throughout the year can be produced. If so, it can be seen that the soybean culture root extract containing such a high content of cumestrol can be uniformly and continuously produced.

[Experimental Example 2] Collagen production amount measurement test

In order to measure the amount of collagen-I produced in the soybean culture root extract before and after the enzyme treatment, the amount of collagen produced in each sample was measured using a Procollagen Type I C-peptide (PIP) ELISA kit.

Specifically, normal human dermal fibroblast (HDF) (purchased from Korea Cell Line Bank) ^{was dispensed into a 40mm Petri plate at a concentration of 1×10 4} cells/well, and then cultured at 37°C for 24 hours. Then, each of the cells was daidzein, coumestrol, 1 μg/ml and 5 μg/ml of soybean cultured root extract before enzyme treatment (extract of 1. in Example 3), respectively, and soybean cultured root extract after enzyme treatment. (Sediment 2 of Example 3) Treated with 1 μg/ml and 5 μg/ml, respectively, and incubated at 37° C. for 72 hours, and then collected the supernatant, respectively, and a Procollagen Type I C-peptide (PIP) ELISA kit (Takara) Was used to measure the amount of collagen-1 produced. The results are shown in FIG. 2.

From the results of Figure 2, the amount of collagen production (2.5 ng/ml and 3.1 ng/ml at 1 μg/ml and 5 μg/ml, respectively) of the soybean culture root extract before enzyme treatment was untreated control (2.3 ng/ml), a kind of isoflavone It was found to be higher or equivalent to daidzein (2.5 ng/ml) and cumestrol (2.3 ng/ml). On the other hand, in the case of the soybean culture root extract (2.7 ng/ml and 3.9 ng/ml at 1 μg/ml and 5 μg/ml, respectively) after the enzyme treatment according to the present invention, the control (untreated), daidzein and coo, which are a kind of isoflavones It was confirmed that the amount of collagen production was measured higher than not only mestrol, but also compared to the soybean cultured root extract before enzyme treatment.

That is, in the case of the sediment (enzyme-treated extract of soybean cultured root) of Example 3, which is the active ingredient of the composition according to the present invention, it contains a high content of coumestrol to show excellent collagen production effect, and thus skin elasticity. It has the effect of promoting skin wrinkles and preventing or improving skin wrinkles.

Claims (22)

A composition for enhancing skin elasticity or improving skin wrinkles, comprising an enzyme-treated extract of cultured legumes as an active ingredient,
The enzyme-treated extract is obtained by adding an enzyme or a microorganism producing the same to the extract of the legume culture root,
The content of cumestrol in the enzyme-treated extract is 20 to 200mg compared to 1g of the enzyme-treated extract, a composition for enhancing skin elasticity or improving skin wrinkles. The method of claim 1,
The enzyme is pectinase or polygalacturonase, a composition for enhancing skin elasticity or improving skin wrinkles. The method of claim 1,
The microorganism is Aspergillus aculeatus (Aspergillus aculeatus), a composition for enhancing skin elasticity or improving skin wrinkles. The method of claim 1,
The amount of the enzyme added is 0.8 to 8 parts by weight based on 100 parts by weight of the extract of the legume culture root, for enhancing skin elasticity or improving skin wrinkles. The method of claim 1,
The amount of the enzyme added is 2000 to 5000 units compared to 1 g of the extract of the leguminous plant culture root based on the enzyme activity, for enhancing skin elasticity or improving skin wrinkles. The method of claim 1,
The enzyme-treated extract is a precipitate recovered by centrifugation after maintaining the reaction product obtained by adding an enzyme or a microorganism producing the same to the extract of the legume culture root for 38 to 58 hours at a condition of 40 to 50°C and 60 to 100 rpm Phosphorus, a composition for enhancing skin elasticity or improving skin wrinkles. delete The method of claim 1,
The legumes are soybeans, flat beans, seoritae, seomoktae, black tae, cheongtae, yellow tae, hedge beans, red beans, reaps, or any one selected from Shinhwa beans, skin elasticity enhancement or skin wrinkle improvement composition. The method of claim 1, wherein the extract of the legume culture root,
The legume culture root is extracted with water, a lower alcohol of C1 to C6, or a mixture thereof as a solvent, a composition for enhancing skin elasticity or improving skin wrinkles. The method of claim 9,
The alcohol concentration of the mixture is 60 to 100% (w/v), a composition for improving skin elasticity or improving skin wrinkles. The method of claim 10,
The lower alcohol is ethanol, a composition for enhancing skin elasticity or improving skin wrinkles. The method of claim 9,
The extraction time is 20 to 28 hours, a composition for enhancing skin elasticity or improving skin wrinkles. The method of claim 1, wherein the extract of the legume culture root,
A composition for enhancing skin elasticity or improving skin wrinkles, which is an extract of cultured root grown while maintaining a constant air supply amount in the bioreactor by placing the legume cultured root in a medium in a bioreactor. The method of claim 13,
The bioreactor is a bulb type bubble bioreactor, a composition for enhancing skin elasticity or improving skin wrinkles. The method of claim 13,
The concentration of ammonium nitrate (NH ₄ NO ₃ ) in the medium is 650 to 1500 mg/L, the concentration of calcium chloride (CaCl ₂ ·2H ₂ O) is 175 to 400 mg/L, and magnesium sulfate (MgSO ₄ ·7H ₂ The concentration of O) is 145 to 320 mg/L, the concentration of potassium phosphate (KH- ₂ PO ₄ ) is 65 to 150 mg/L, and _{the concentration of potassium nitrate (KNO 3} ) is 750 to 1500 mg/L, A composition for enhancing skin elasticity or improving skin wrinkles. The method of claim 13,
The medium is IBA (indole-3-butyric acid) and a carbon source is added, the composition for improving skin elasticity or skin wrinkles. The method of claim 13,
The medium is MS medium (Murashige and Skoog medium), a composition for enhancing skin elasticity or improving skin wrinkles. The method of claim 13,
When the cultured muscle is placed in the medium, the medium pH is 4.8 to 6.8, and the inoculation density of the cultured muscle is 2 to 6g/L, a composition for enhancing skin elasticity or improving skin wrinkles. The method of claim 13,
The proliferation is to be carried out for 3 to 5 weeks in the dark conditions of 19 to 25 ℃, the composition for improving skin elasticity or skin wrinkles. The method of claim 13,
The air supply amount is 0.05 to 0.4vvm (air volume/culture volume per min), a composition for enhancing skin elasticity or improving skin wrinkles. The method of claim 1,
The enzyme-treated extract of the legume culture root is contained in an amount of 0.1 to 100 μg/ml based on the total weight of the composition, a composition for enhancing skin elasticity or improving skin wrinkles. The method of claim 1,
The composition is a cosmetic composition or a food composition, a composition for enhancing skin elasticity or improving skin wrinkles.

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