KR20250113023A - Method for producing whitening functional peptide derived from black bean fermentation filtrate and cosmetic composition using the same - Google Patents

Abstract

Disclosed are a fermentation and separation and purification method for increasing the content of highly functional substances in a fermented product through an effective fermentation process and separation and purification process of a black bean extract, and a method for utilizing the same as a cosmetic composition having excellent skin whitening effects. The present invention provides a method for producing a peptide having a skin whitening function in a black bean fermented product, comprising the steps of inoculating and fermenting a black bean ( Glycine max Merr. ) extract with bacteria; filtering the fermented product to remove microorganisms; and separating and purifying a peptide.

Description

Method for producing whitening functional peptide derived from black bean fermentation filtrate and cosmetic composition using the same

The present invention relates to a fermentation process and separation and purification using microorganisms, and more specifically, to a fermentation process and separation and purification using microorganisms using black beans.

Fermentation is the process in which enzymes contained in microorganisms change raw materials to create beneficial ingredients. During the fermentation process, effective ingredients are extracted and the content of functional substances increases.

And fermentation technology overlaps in meaning with terms such as bioconversion, biosynthesis, and biocatalysis, and refers to the technology of manufacturing a desired product from a precursor by utilizing the enzymatic function of microorganisms. Therefore, fermentation technology that utilizes biological functions at the cellular or enzymatic level can be said to be a major process that constitutes the current bioindustry with the main purpose of producing useful substances. If fermented foods utilized a one-dimensional fermentation technology that utilizes naturally occurring microorganisms, fermented cosmetics require more advanced technology, and if the first-generation fermentation simply utilized traditional fermentation methods, the second-generation fermentation was the development of practical use technology for effective substances, and the bioconversion fermentation technology that laid the foundation for the cosmetics functional materials industry through an advanced fermentation production technology that can mass-produce only useful physiologically active substances is established as the third-generation fermentation technology.

Koreans are very familiar with the concept of fermentation because they use a lot of fermented foods such as kimchi and doenjang, and they also use cosmetics using fermented ingredients without any resistance. In addition, it is known that when new materials are developed using fermentation technology, they show completely new effects compared to existing materials, or many harmful ingredients are changed into safe ingredients, so fermented cosmetics are preferred, and the level of technology development according to this is not far behind that of other advanced countries. However, it is known that many fermented materials currently applied to cosmetics use natural fermentation or fermentation methods close to natural maturation.

That is, the recent research trend in domestic fermentation technology is known to use a fermentation method close to natural fermentation or natural maturation. Natural fermentation is a method that naturally produces ingredients useful for the skin by using the power of nature such as water, wind, earth, and seasons by leaving various unprocessed raw materials as they are. Although it takes a long time, its advantage is that various substances can be obtained, so it can achieve effects such as moisturizing, anti-oxidation, wrinkle improvement, cell activation, and skin texture improvement. However, natural fermentation or natural maturation method fermentation has the disadvantage that it is difficult to obtain fermented products of consistent quality for each production batch, and quality management is also difficult.

The fermented cosmetics market in Korea has been growing at a rate of 40% per year for the past 3-4 years, and is expected to reach a size of approximately KRW 450 billion in 2012, KRW 650 billion in 2013, and KRW 1 trillion (estimated) in 2014.

Fermented products can be broadly classified into ferment filtrate and ferment lysate. Fermented filtrate utilizes the supernatant after fermentation is completed by removing microorganisms through a filter, and fermented lysate utilizes the method of killing microorganisms using heat, pH, enzymes (lysozyme), high pressure, etc. after fermentation is completed.

Meanwhile, black beans ( Glycine max Merr. ) do not refer to a single type of bean, but rather a general term for black beans such as heuktae, seoritae, and seomoktae. Black beans are known to contain healthy ingredients in their black seed coats, unlike regular beans. Isoflavones, a plant-based female hormone found in large quantities in black beans, have a similar effect to estrogen, the female hormone in our body, and are good for the health of menopausal women. In particular, black beans are a grain with a very high protein content, and these proteins exist in concentrations close to 40% of the total mass, so they have been utilized at a level where they can be used as a substitute for meat. Some of these proteins contain useful peptides that can be used as raw materials for cosmetics, and research on these is also actively being conducted.

The present invention proposes a fermentation and separation and purification method for increasing the content of highly functional substances in a fermented product through an effective fermentation process and separation and purification process of a black bean extract, and a method for utilizing the same as a cosmetic composition having an excellent skin whitening effect.

In order to solve the above problem, the present invention provides a method for producing a peptide having a whitening function in a fermented black bean ( Glycine max Merr. ) extract, comprising the steps of inoculating and fermenting a fungus into a black bean (Glycine max Merr.) extract; filtering the fermented product to remove microorganisms; and separating and purifying a peptide and a peptide derivative.

In addition, the present invention provides a method for producing a peptide, characterized in that the fermentation is performed at a temperature of 30 to 50°C for 2 to 4 days.

In addition, a method for producing a peptide is provided, characterized in that the fungus is a fungus of the genus Bacillus ( Bacillus sp.).

In addition, the present invention provides a method for producing a peptide, characterized in that the separation and purification are performed through solvent fractionation and size exclusion chromatography.

In addition, the present invention provides a method for producing a peptide characterized in that the peptide has an ability to inhibit inducible nitric oxide synthase (iNOS) production and an ability to inhibit nitric oxide (NO) production in macrophages.

In addition, the present invention provides a method for producing a peptide characterized in that the peptide has skin cell proliferation ability and skin cell regeneration inducing ability.

In addition, the present invention provides a method for producing a peptide, characterized in that the peptide is obtained from a butanol fraction layer among the solvent fractions.

In order to solve the above another problem, the present invention provides a cosmetic composition containing a peptide manufactured by the above method.

According to the present invention, it was confirmed that the concentration of peptides and peptide derivatives in black beans increased through fermentation of black beans, and it was also confirmed that as the content of peptides increased through separation and purification, a higher skin whitening activity was exhibited.

In particular, a peptide showing excellent skin whitening efficacy was finally separated, and the toxicity, efficacy, and physical properties of a single substance were confirmed. From this, a fermentation and separation and purification method for increasing the content of a highly functional substance in a fermented product can be provided, and through this, it can be utilized as a cosmetic composition with excellent skin whitening efficacy.

Figure 1 is a photograph showing the TLC results of a black bean fermentation product purified through solvent fractionation and open column in an embodiment of the present invention.
Figure 2 is a graph showing the HPLC results of the black soybean fermentation fraction after Prep LC purification.
Figure 3 is a schematic diagram showing a separation example in the present invention.
Figure 4 is a graph showing the TLC results of fractions separated in a purification example of the present invention.
Figure 5 is a graph showing the HPLC analysis results of the butanol layer separated in the purification example of the present invention.
Figure 6 is a graph confirming the melanin expression inhibition effect of each peptide separated in a separation example according to the present invention.
Figure 7 is a graph showing the HPLC analysis results of the peptide separated in the present invention.
Figures 8 and 9 are graphs showing the results of measurements of light stability and thermal stability of Experimental Example 10 of the present invention.

Hereinafter, the present invention will be described in detail through preferred embodiments. Prior to this, the terms or words used in this specification and claims should not be interpreted as limited to their usual or dictionary meanings, and should be interpreted as meanings and concepts that conform to the technical idea of the present invention based on the principle that the inventor can appropriately define the concept of the term in order to explain his or her own invention in the best way. Therefore, the configuration of the embodiments described in this specification is only the most preferred embodiment of the present invention and does not represent all of the technical idea of the present invention, so it should be understood that there may be various equivalents and modified examples that can replace them at the time of filing this application. In addition, throughout the specification, when a part is said to "include" a certain component, this does not mean that other components are excluded, but that other components can be further included, unless specifically stated otherwise.

The present inventors have conducted repeated studies to develop a fermentation and separation and purification method for increasing the content of a highly functional substance derived from black beans that can be used as a cosmetic composition that effectively prevents or improves skin inflammation reactions and is effective in improving skin barriers, and as a result, they have confirmed that when a black bean extract is fermented or a peptide derived from a fermented black bean is used, the effect of inhibiting melanin production in melanocytes is increased, resulting in a skin whitening effect that is significantly improved compared to the existing one, leading to the present invention.

Accordingly, the present invention discloses a method for producing a peptide having a skin whitening function in a fermented black bean ( Glycine max Merr.) extract, comprising the steps of inoculating and fermenting a fungus into a black bean (Glycine max Merr. ) extract; filtering the fermented product to remove microorganisms; and isolating and purifying the peptide.

In the present invention, the black bean is an annual plant belonging to the legume order and legume family, and is widely distributed in Asia, Africa, Australia, etc. Its origin is estimated to be the area from the northern part of the Korean peninsula to the northern part of China, from the Tumen River basin to the northern part of China, to the northern part of China. In particular, it is widely cultivated as an edible crop, and various pharmacological actions of black beans have been discovered, and it is being developed into a cosmetic, and is being used for skin care and anti-aging.

In the present invention, the black bean fermentation refers to a fermentation product obtained by drying and extracting black beans and then fermenting them. Black bean extract has been used as a medicinal herb effective for various diseases, and is a medicinal herb that is rich in various efficacies such as lecithin and isoflavones, and has been confirmed to have anticancer, antioxidant, anticancer, etc. efficacies.

In the present invention, the black bean fermentation product or the black bean fermentation purified product can be used as a raw material that acts on the skin and helps improve skin whitening, and by purifying a specific ingredient with efficacy, the effect is further enhanced compared to before purification. At this time, the black bean fermentation product and the black bean fermentation purified product can be used together.

In these black bean fermented products or black bean fermented purified products, the highly functional effective ingredients that enable them to exhibit efficacy on the skin are peptides or peptide derivatives, and in the present invention, the method for increasing the content of the highly functional substances is performed through the steps of inoculating microorganisms into black bean fruit extracts and fermenting them to produce a fermented product, and the step of removing the microorganisms from the fermented product.

In the present invention, the step of inoculating microorganisms into the black bean fruit extract and fermenting it to produce a fermented product is a step for increasing the content of functional substances in the black bean fruit.

The above black bean fruit extract can be prepared using a conventional extraction method known in the art, for example, a solvent extraction method. The extraction solvent used in the preparation of the mixed extract using a solvent extraction method can be selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms (for example, methanol, ethanol, propanol, and butanol) or hydrous lower alcohols which are mixtures thereof, propylene glycol, 1,3-butylene glycol, glycerin, acetone, diethyl ether, ethyl acetate, butylacetate, dichloromethane, dichloromethane, hexane, and mixtures thereof, and among these, it is preferable to select from water, alcohol, or mixtures thereof, and when alcohol is used as the extraction solvent, it is preferable that the alcohol is a lower alcohol having 1 to 4 carbon atoms, and it is more preferable that the lower alcohol is selected from methanol or ethanol, and even more preferably ethanol.

Here, when ethanol is used as an extraction solvent for the black bean fruit, for example, 99.5% (v/v) ethanol can be added in an amount 5 to 20 times the weight of the black bean, and extraction can be performed at room temperature for 18 to 30 hours. At this time, the sugar content of the black bean extract can be 4 to 10 brix, preferably 6 to 8 brix.

In addition, when extracting the black bean fruit by hot water extraction, for example, purified water may be added in an amount 5 to 20 times the weight of the black bean fruit, and extraction may be performed at 100 to 140°C for 4 to 8 hours. At this time, the sugar content of the black bean fruit extract may be 4 to 10 brix, preferably 6 to 8 brix.

In the present invention, the microorganism used for fermentation may be a Bacillus sp. strain, a Lactobacillus sp. strain, or a mixed strain thereof. The Bacillus sp. strain may be Bacillus methylotrophicus , Bacillus subtilis , or Bacillus licheniformis , and the Lactobacillus sp. strain may be Lactobacillus paracasei. In the present invention, in terms of maximizing the content of highly functional effective ingredients, a mixed strain of Bacillus subtilis, Bacillus methylotrophicus, and Bacillus licheniformis may be preferably used in a weight ratio of 1:0.8 to 1.2:0.8 to 1.2.

In the present invention, fermentation refers to a process in which enzymes possessed by microorganisms change raw materials to produce beneficial ingredients, and during the fermentation process, effective ingredients are extracted and the content of functional substances increases. In addition, in the present invention, the fermentation may be performed by adjusting the culture solution of the microbial strain to 10 ⁷ to 10 ¹⁰ cfu/㎖, inoculating 0.1 to 10 wt%, preferably 0.5 to 5 wt%, of the black bean fruit extract, and for 1 to 5 days, preferably 2 to 4 days, at 30 to 50°C, preferably 35 to 45°C.

In addition, the medium composition during the microbial culture and fermentation may include 0.5 to 2 wt% of beef extract, 1 to 3 wt% of enzyme extract, 3 to 7 wt% of peptone, 3 to 7 wt% of sodium chloride, and distilled water (balance) in terms of maximizing the content of highly functional effective ingredients.

The step of manufacturing a black bean fermented product by removing microorganisms from the above fermented product is a step of removing microorganisms to prevent further fermentation of the black bean fermented product. The microorganisms can be removed through centrifugation and filtration. For example, the microorganisms can be removed by separating the microorganisms from the fermented product through a centrifuge and then filtering the fermented product through a 0.1 to 0.3 ㎛ filter.

In the present invention, the separation and purification of the black bean fermentation product can be performed through solvent fractionation and chromatography. The step of solvent fractionating the black bean fermentation product is a step of purifying the black bean fermentation product to extract the target component, and the solvent fractionation can be performed sequentially through hexane, dichloromethane, ethyl acetate, butanol, and water. In addition, purification can be performed through a column after the solvent fractionation.

In addition, the whitening efficacy component confirmed through the black bean fermentation purification in the present invention may be a peptide derived from black bean fruit, and the molecular weight of the peptide may be about 390.

In the present invention, the cosmetic composition may contain 1 to 90 wt% of the black bean fermentation product or the black bean fermentation purified product, and preferably 1 to 20 wt%. In addition, the cosmetic composition may contain both the black bean fermentation product and the black bean fermentation purified product, and may contain them in an amount of 1 to 20 wt% each.

In another aspect, the present invention provides a cosmetic composition containing a peptide manufactured by the above method.

It was confirmed that the cosmetic composition according to the present invention has an excellent effect of inhibiting melanin production in melanocytes by including a fermented black bean containing a peptide.

The cosmetic composition according to the present invention can be applied as a gel type, skin type, cream type, ointment type, etc., but is not limited thereto. The composition can be appropriately prepared by a known method by adding an appropriate conventional softener, emulsifier, thickener, or other materials known in the art according to its type.

The above gel type composition can be manufactured by adding a softener such as trimethylolpropane, polyethylene glycol or glycerin, a solvent such as propylene glycol, ethanol or isocetyl alcohol, purified water, etc.

The above skin type composition can be prepared by adding fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, and isocetyl alcohol; butylene glycol, glycerin, allantoin, methyl paraben, sodium EDTA-2, xanthan gum, dimethicone, polyethylene glycol-60 hydrogenated castor oil, polysorbate 60, and purified water.

The above cream type composition can be manufactured by adding fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, and isocetyl alcohol; lipids such as lecithin, phosphatidylcholine, phosphatidylethanolamine, sophatidylserine, and sophatidyl inositol; derivatives thereof; emulsifiers such as glyceryl stearate, sorbitan palmitate, and sophatidyl stearate; natural fats or oils such as avocado oil, apricot oil, babassu oil, gromwell oil, and camellia oil; solvents such as propylene glycol; and purified water.

The above ointment type composition can be manufactured by adding a softener, an emulsifier, and a wax such as microcrystalline wax, paraffin, ceresin, beeswax, spermaceti, and vaseline.

The cosmetic composition of the present invention may additionally contain one or more anti-inflammatory ingredients having the same or similar function in addition to the effective ingredient. The anti-inflammatory ingredients may be at least one selected from Machihyeon extract, Byungpung extract, Dipotassium Glycyrrhizate, Ammonium glycyrrhizate, and anti-inflammatory functional peptides, but are not limited thereto. In addition, the cosmetic composition of the present invention may additionally contain excipients including fluorescent substances, fungicides, hydrotropism inducers, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunblocks, vitamins, plant extracts, etc.

In the present invention, the cosmetic composition may be formulated as a softening toner, an astringent toner, a nourishing toner, an eye cream, a serum, a nourishing cream, a massage cream, a cleansing cream, a cleansing lotion, a cleansing foam, a cleansing water, a powder, an essence, a pack, a hair tonic, a hair treatment, a shampoo or a rinse.

The above cosmetic composition can be formulated by a conventional method. For the formulation of a skin external preparation, reference can be made to the contents disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, and for the formulation of a cosmetic composition, reference can be made to the contents disclosed in International cosmetic ingredient dictionary, 6th ed (The cosmetic, Toiletry and Fragrance Association, Inc, Washington, 1995).

Specifically, the cosmetic composition can be prepared as a general emulsified formulation and a solubilized formulation. For example, the cosmetic composition can be formulated as a toner such as a flexible toner or a nourishing toner; an emulsion such as a facial lotion or a body lotion; a cream such as a nourishing cream, a moisture cream, or an eye cream; an essence; a cosmetic ointment; a spray; a gel; a pack; a sunscreen; a makeup base; a foundation such as a liquid type, a solid type, or a spray type; a powder; a makeup remover such as a cleansing cream, a cleansing lotion, or a cleansing oil; or a cleanser such as a cleansing foam, a soap, or a body wash, but is not limited thereto. In addition, the skin external preparation can be formulated as an ointment, a patch, a gel, a cream, or a spray, but is not limited thereto.

In addition to the essential ingredients, other ingredients may be appropriately blended into each formulation of the above cosmetic composition, within a range that does not impede the purpose of the present invention, depending on the type of formulation or intended use.

The above cosmetic composition may typically include an acceptable carrier, such as oil, water, a surfactant, a moisturizer, a lower alcohol, a thickener, a chelating agent, a pigment, a preservative, a fragrance, etc., but is not limited thereto.

The above acceptable carriers may vary depending on the formulation. For example, when formulated as an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or mixtures thereof may be used as carrier components.

When the above cosmetic composition is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or a mixture thereof may be used as a carrier component, and in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane, butane or dimethyl ether may be further included.

When the above cosmetic composition is formulated as a solution or emulsion, a solvent, a solubilizer, or an emulsifier can be used as a carrier component, and for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil can be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan can be used.

When the above cosmetic composition is formulated as a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as carrier components.

The above cosmetic composition may additionally contain adjuvants commonly used in the field of cosmetology or dermatology, such as fatty substances, organic solvents, solubilizers, thickeners, gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestering agents, chelating agents, preservatives, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, and any other ingredients commonly used in cosmetics, depending on the quality or function of the final product. However, the adjuvants and the mixing ratio thereof may be appropriately selected so as not to affect the desirable properties of the cosmetic composition according to the present invention.

A cosmetic composition comprising a fermented black bean product or a fermented black bean purified product manufactured according to the present invention as an active ingredient has a skin whitening effect by inhibiting the production of melanin in melanocytes by including the fermented black bean product or the fermented black bean purified product.

Hereinafter, specific examples according to the present invention will be described.

Manufacturing example 1

Dried black beans were rinsed with purified water and soaked for a day. Then, 1 kg of purified water was added to 1 kg of black beans and hot water was extracted at 121°C for 30 minutes. After extraction, a black bean extract with a sugar content of 6 to 8 birx was produced. After extract preparation, a mixed strain of Bacillus licheniformis (purchased from the Korean Cell Line Bank), Bacillus methylotrophicus (Apec microrganism S001, purchased from the Korean Cell Line Bank), and Bacillus subtilis (Apec microrganism K007, purchased from the Korean Cell Line Bank) in a weight ratio of 1:1:1 was cultured, and 1 wt% of the culture solution, whose concentration was measured by a spectrophotometer as 1.0× ¹⁰⁹ cfu/ml, was inoculated into the black soybean extract, and fermented at 40°C for 3 days. The medium used for culture consisted of 1 wt% of beef extract, 2 wt% of enzyme extract, 5 wt% of peptone, 5 wt% of sodium chloride, and distilled water (balance). After fermentation, the fermented solution was centrifuged at 4,000 rpm for 10 minutes to separate the bacteria and the culture solution, and then the culture solution was filtered through a 0.2 ㎛ filter to produce a fermented black bean product.

Manufacturing example 2

2 kg of 80% (v/v) ethanol was added to 200 g of dried black beans, and extraction was performed at 25°C for 24 hours, producing a black bean extract with a sugar content of 6 to 8 birx after extraction. After preparing the extract, a mixed strain of Bacillus licheniformis (purchased from the Korean Cell Line Bank), Bacillus methylotrophicus (Apec microrganism S001, purchased from the Korean Cell Line Bank), and Bacillus subtilis (Apec microrganism K007, purchased from the Korean Cell Line Bank) in a weight ratio of 1:1:1 was cultured, and 1 wt% of the culture solution, whose concentration was measured by a spectrophotometer as 1.0× ¹⁰⁹ cfu/ml, was inoculated into the black soybean extract, and fermented at 37°C for 3 days. The medium used for culture consisted of 1 wt% of beef extract, 2 wt% of enzyme extract, 5 wt% of peptone, 5 wt% of sodium chloride, and distilled water (balance). After fermentation, the fermented solution was centrifuged at 4,000 rpm for 10 minutes to separate the bacteria and the culture solution, and then the culture solution was filtered through a 0.2 ㎛ filter to produce a fermented black bean product.

Manufacturing example 3

2 kg of purified water was added to 200 g of dried black beans, and hot water extraction was performed at 120°C for 6 hours to produce a black bean extract with a sugar content of 6 to 8 birx.

Manufacturing example 4

A black bean extract with a sugar content of 6 to 8 birx was prepared by adding 2 kg of 80% (v/v) ethanol to 200 g of dried black beans and extracting the extract at 25°C for 24 hours.

Refined example

(1) n-Hexane, dichloromethane, ethyl acetate and butanol were sequentially added to the black bean fermentation product manufactured in the above Manufacturing Example 2 to separate it into an n-hexane fraction, a dichloromethane fraction, an ethyl acetate fraction, a butanol fraction and a water fraction, thereby manufacturing fractions.

(2) After concentrating the separated butanol fraction under reduced pressure, silica column chromatography was performed. At this time, a silica column was used as the column, and the mobile phase solvent was distilled water CH ₂ Cl ₂ : MeOH = 4:1, 3:1, 2:1 in that order, changing 1 L each time, and separated into a total of 10 fractions: 1) No. 3 2) No. 4~5 3) No. 6~7 4) No. 8~11 5) No. 12~15 6) No. 16~19 7) No. 20~22 8) No. 23~30 9) No. 31~35 10) No. 36~41, and the results are shown in Fig. 1.

(3) The separated fr.1 was purified by Prep. HPLC (C18 ODS column, 3 ml/min). The solvent was solvent A: water in 0.1% TFA, solvent B: acetonitrile (ACN) in 0.1% TFA, 85:15, and Compound 1 was purified from the black bean fermentation purified product. The HPLC analysis results after purification are shown in Fig. 2. In addition, the overall separation process through the aforementioned fractionation and purification is schematically shown in Fig. 3.

Experimental Example 1

In order to confirm the whitening effect of the black bean fermentation product or black bean extract manufactured in the above Manufacturing Examples 1 to 4, a melanin production inhibition experiment was performed according to the following method, and the results are shown in Table 1 below.

[Experimental Method]

Mouse melanocyte B16F1 cells were cultured in DMEM (Dulbecco's modified Eagle's media, Sigma) containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Cells were cultured at a concentration of 1 × 105 cells/well in a 24-well plate, and cell attachment was confirmed. The control group was treated with only the solvent, the positive control group was treated with 200 ng/ml of a-MSH, and the remaining dishes were treated with 200 ng/ml of α-MSH and 10, 50, and 100 μg/ml of arbutin as experimental and control groups. Test substances were added to each dish and cultured for 3 days. After 3 days, the culture medium was removed by centrifugation, and the cells were lysed to measure the amount of intracellular melanin production, and the intracellular melanin was collected and observed at 490 nm.

Each experimental group was treated with concentration and the melanin production inhibition effect was measured. It was confirmed that the amount of melanin decreased in most experimental groups compared to the control group, arbutin, and the experimental group was confirmed to be effective. From these results, it is thought that when a factor that induces melanin production in the skin is applied, it is more useful to use a mixture of various combinations than when using a single raw material, and it is thought that it can suppress melanin expression activity and brighten the skin tone.

Example of manufacturing a cosmetic composition

A cream type composition was prepared by the following method. The cream type composition contained the above-mentioned fermented product at a weight ratio of 1% and formed a formulation as shown in Table 1 below.

(1) [Award B] Manufacturing

Heat the water to 60 to 70℃ and stir while adding powdered raw materials one by one, and stir with a disperser mixer until thickened to evenly dissolve, then cool. Prepare in advance for ease of process.

(2) [Award A] Manufacturing

After sufficiently impregnating the powdered raw material with glycerin, add water, heat to 50 to 60℃, and dissolve evenly using a disperser mixer.

(3) [Award A] + [Award B] Mixed Stirring Manufacturing

Add previously prepared solution B to solution A and heat while mixing evenly. Prepare so that the temperature can be maintained at 65 to 75℃.

(4) [Paid] Mixed and stirred manufacturing

Weigh each raw material in an individual beaker and melt it by heating and stirring. (Above 80℃) Prepare the melted phase so that it can be maintained at a temperature above 75℃.

(5) Whole cream production

The oil phase is gradually added to the prepared water mixture and the emulsification process is carried out by vigorously stirring (6,000 rpm) for about 10 minutes. During this time, the temperature should be maintained at 65 to 75°C.

After completion, prepare additive A and proceed with the pH adjustment and neutralization process. At this time, the viscosity of the contents increases, so stir vigorously (6,000 rpm) and evenly for more than 5 minutes.

When the contents cool to below 50℃, add additive B and mix slowly for about 2 minutes (3,000~4,000rpm). When it becomes below 40℃, add additive C and additive D sequentially and mix slowly for about 2 minutes (3,000~4,000rpm). Then, collect the contents through a degassing/filtration process.

After completion, proceed with the pH adjustment and neutralization process. At this time, the viscosity of the contents increases, so stir vigorously (6,000 rpm) and evenly for more than 5 minutes.

When the contents cool to below 50℃, add the preservative and mix slowly for about 2 minutes (3,000~4,000rpm). When it becomes below 40℃, add the nignan analogue (compound 1) and fragrance, stir slowly for about 2 minutes (3,000~4,000rpm), and then collect the contents through a degassing/filtration process.

The manufactured cream was stored at low temperature (4℃), high temperature (50℃), room temperature (25℃), and under sunlight conditions to check the stability of the cream. No phenomena such as discoloration, odor change, phase change, or pH change were observed in the cream for 4 weeks under all conditions, confirming that the stability of the cream was maintained. In particular, the stability of the black bean-derived peptide (compound 1) was maintained, and the HPLC analysis results confirmed that more than 90% of the content was maintained for 4 weeks under all conditions. Table 2 below shows the change in the content (%) of black bean peptide (compound 1) for 4 weeks by storage condition.

division Low temperature (4℃) Room temperature (25℃) High temperature (50℃) daylight 1 week later 99 98 97 97 2 weeks later 98 95 95 96 3 weeks later 97 95 92 93 4 weeks later 97 93 90 91

Through the development of the technology of this patent, it was confirmed that the protein and peptide patterns in black beans were changed through fermentation of black beans, and it was also confirmed that the skin whitening activity was higher as the peptide content increased through separation and purification. In particular, the peptide showing the best efficacy was finally separated, and the toxicity, efficacy, and physical properties of the single peptide were confirmed.

It is expected that the functional peptide derived from black beans developed through the present invention will have various uses as a cosmetic composition.

The preferred embodiments of the present invention have been described in detail above. The description of the present invention is for illustrative purposes, and those skilled in the art to which the present invention pertains will understand that other specific forms can be easily modified without changing the technical idea or essential features of the present invention.

Accordingly, the scope of the present invention is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning, scope, and equivalent concepts of the claims should be interpreted as being included in the scope of the present invention.

Claims (7)

A step of inoculating and fermenting a black bean ( Glycine max Merr. ) extract with a fungus;
A step of filtering the fermented product to remove microorganisms; and
Step of isolating and purifying the peptide;
A method for producing a peptide having a skin whitening function in a fermented black bean containing the same. In the first paragraph,
A method for producing a peptide, characterized in that the above fermentation is performed at a temperature of 30 to 50°C for 2 to 4 days. In the first paragraph,
A method for producing a peptide, characterized in that the above-mentioned fungus is a fungus of the genus Bacillus ( Bacillus sp.). In the first paragraph,
A method for producing a peptide, characterized in that the above separation and purification are performed through solvent fractionation and size exclusion chromatography. In the first paragraph,
A method for producing a peptide, characterized in that the above peptide has the ability to inhibit melanin production in melanocytes. In the first paragraph,
A method for producing a peptide, characterized in that the above peptide has a molecular weight of 390 g/mol. A cosmetic composition containing a peptide manufactured by the method of any one of claims 1 to 6.