KR102362960B1 - Functional composition for anti-inflammatory and skin improvement comprising caviar enzyme-treated powder extract as an active ingredient. - Google Patents

Abstract

The present invention relates to a functional composition for anti-inflammatory and skin condition improvement, comprising a caviar enzyme-treated powder extract as an active ingredient. More specifically, the present invention provides a method for manufacturing a functional composition for anti-inflammatory and skin condition improvement, and a functional food and health functional food composition for anti-inflammatory and skin condition improvement that can be safely used by using Korean natural products by providing extracts, wherein the method comprises the following steps: freeze-drying raw caviar; supercritical-extracting caviar freeze-dried powder to obtain caviar skim powder; after adding 10 times distilled water to the caviar skim powder, and performing an enzyme treatment; and concentrating a filtrate to 15 brix after filtering a caviar enzyme treatment solution, and freeze-drying the same.

Description

Functional composition for anti-inflammatory and skin improvement comprising caviar enzyme-treated powder extract as an active ingredient.

The present invention relates to a functional composition for anti-inflammatory and skin improvement comprising a caviar enzyme-treated powder extract as an active ingredient. More specifically, it provides an anti-inflammatory and skin-improving functional food and health functional food composition that can be safely used by providing a powdered extract by enzymatic treatment of caviar.

Immunity is a phenomenon in which the internal environment of a living body defends against antigens such as pollen, food, drugs, microorganisms, and viruses. Innate immunity responds non-specifically to antigens, and skin, mucous tissue, gastric acid, and macrophages are responsible for it. Acquired immunity responds specifically to antigens and is divided into humoral immunity and cellular immunity. A typical result of an immune response is an inflammatory response. Inflammation is trauma, tissue damage such as burns, bacterial infection, or a reaction to a specific antigen, and is a symptom that causes swelling, fever, and pain.

Such inflammation ends with acute inflammation when the tissue damage or infection site is small or temporary, whereas chronic inflammation progresses when the chronic infection state or inflammation site is large. According to the Ministry of Food and Drug Safety's <2018 Food Production Performance Statistics>, the immune function health functional food accounted for the highest proportion in sales of 1.25 trillion won in 2018, and various diseases that can be caused by inflammation and The demand for related functional foods is constantly increasing.

Also, inner beauty refers to eating cosmetics, and it refers to improving the health of the skin and improving the skin itself by changing eating habits and lifestyle. According to the Industry Analyst Report, the global market for 'Nutricosmetics', which refers to cosmetics or beauty foods to eat, was $4.1 billion in 2013, but is expected to grow to $7.4 billion in 2020, so its value is limitless. In particular, the domestic inner beauty market has grown rapidly at an average annual rate of 68.2% for five years from 2011, and is establishing itself as a major market in the beauty industry. However, the main raw materials for production products are limited raw materials linked to existing cosmetics such as collagen, hyaluronic acid, and vitamins.

On the other hand, the sturgeon ( Acipenser sinensis ) is a kind of fish belonging to the sturgeon family, and it is a large fish species with a streamlined body without scales and a body length of 2 to 5 m. Sturgeon eggs are called caviar and are classified according to the type of sturgeon, such as beluga, osetra, and sevruga.

Caviar is low in fat and high in vitamins and protein, so it is used as an ingredient in high-end cuisine. After surgery, patients only drank oil as a recovery meal, and they acted as a substitute for vitamin intake, which was lacking in cold regions where vegetables were not consumed. In addition, it is very effective in anti-aging, so many cosmetics using caviar have appeared.

The present invention intends to utilize caviar used as a food or cosmetic raw material as a functional composition showing anti-inflammatory and skin improvement effects by reducing fish odor through supercritical extraction and enzyme treatment and using caviar material from which lipids have been removed.

Korean Patent Publication No. 10-2020-0108616 discloses a first step of preparing a caviar extract; A second step of preparing purified water, a first component containing adenosine, a second component containing oil and an emulsifier, a third component containing a lubricant, and a pH adjusting agent as cosmetic raw materials; A third step of heating the purified water to 70 ~ 80 ℃; a fourth step of adding the collagen peptide and the first component to the heated purified water and stirring at 60-70°C; a fifth step of mixing and stirring the second component and the third component at 60 to 70°C; a sixth step of mixing and stirring the agitated material of the fourth step and the agitated material of the fifth step at 55 to 65°C; a seventh step of cooling the agitated material of the sixth step to 40-50°C; And, a cosmetic composition containing caviar comprising the eighth step of adding caviar extract to the cooling water of the seventh step, stirring at 40-50° C., and adjusting the pH by adding a pH adjuster, and a cosmetic manufacturing method using the same start Korean Patent Publication No. 10-2020-0108616 discloses a step of mixing raw caviar and water heated to 80, followed by stirring; Natural cooling to room temperature; Fermenting by inoculating Bacillus subtilis and fermenting microorganisms in the step; Aging the fermented product; Disclosed is a fermented product prepared by the method and a cosmetic composition for moisturizing, elasticity, anti-inflammatory, antioxidant, anti-atopic, whitening, and skin wrinkle improvement containing the same as an active ingredient as a fermentation method comprising: Korean Patent Publication No. 10-2013-0088997 discloses a fermented product produced using Bacillus subtilis, lactic acid bacteria and yeast in raw caviar extract and raw caviar, and hair growth and hair growth effects or hair loss prevention and scalp improvement effects containing its active ingredients Disclosed is a pharmaceutical composition and cosmetic composition for external use on the skin having a. Korean Patent Application Laid-Open No. 10-2019-0090703 discloses (a) mixing lecithin and ethanol; (b) mixing the lecithin-ethanol mixed solution with caviar and purified water; (c) homogenizing the caviar-purified water-lecithin-ethanol mixed solution by stirring at room temperature; and (d) filtering the homogeneous solution.

An object of the present invention is to provide a composition showing anti-inflammatory and skin improvement effects by reducing fish odor through supercritical extraction of caviar used as a food or cosmetic raw material and enzymatically treating caviar protein from which lipids have been removed.

In order to achieve the above object, the present invention provides a functional composition for anti-inflammatory and skin improvement comprising a caviar enzyme-treated powder extract as an active ingredient.

The caviar is an extract available from the supercritical extraction method, and the supercritical extraction pressure is 400 bar, a temperature of 40° C., a flow rate of 60 ml/min, and an extraction time of 300 minutes.

The caviar enzyme-treated powder extract is anti-inflammatory and skin obtained by adding distilled water 10 times that of caviar skim powder, and simultaneously treating enzymes Prozyme 1000L and Protamex, or primary treatment with enzyme Prozyme 1000L and secondary treatment with enzyme Protamex Provided is a functional composition for improvement.

In the composition, the caviar enzyme-treated powder extract may be prepared in an amount of 0.01 to 10 g/day, or the caviar enzyme-treated powder extract in an amount of 0.01 to 100 wt% Anti-inflammatory and skin improvement functional composition provides

The anti-inflammatory and skin improvement composition comprising the caviar enzyme-treated powder extract of the present invention is supercritically extracted with carbon dioxide, and it is safe by decomposing it with an edible enzyme in caviar used as food, easy to take, and can be stored for a long time. , has excellent anti-inflammatory and skin improvement effects.

1 shows a schematic diagram of a caviar enzyme-treated powder manufacturing method.
2 is a cytotoxicity result of caviar enzyme-treated powder and hot water extract powder in RAW264.7 cells.
3 shows the IL-6 reduction effect of hot water extract powder and enzyme-treated powder for each condition in RAW264.7 cells.
4 shows the TNF-α reduction effect of hot water extract powder and enzyme-treated powder for each condition in RAW264.7 cells.
5 shows the effect of reducing MMP-1 expression of hot water extract powder and enzyme-treated powder for each condition in Hs68 cells.
6 shows the effect of increasing COL1A of hot water extract powder and enzyme-treated powder for each condition in Hs68 cells.
7 shows the effect of increasing the fibrillin expression of hot water extract powder and enzyme-treated powder for each condition in Hs68 cells.
8 shows the caviar enzyme-treated powder composition completed from the present invention.

The present invention relates to a composition for anti-inflammatory and skin improvement comprising a caviar enzyme-treated powder extract as an active ingredient. Hereinafter, it will be described in detail with reference to specific examples.

I. Preparation of caviar enzyme-treated powder and comparison of yields by condition

1. Preparation of caviar enzyme-treated powder

1) Freeze drying

In the caviar enzyme-treated powder manufacturing method of the present invention, 3,000 g of raw caviar is lyophilized and powdered. When freeze-dried powder is evaporated, the yield is 40% compared to the raw material at 1,200 g.

2) Supercritical extraction

1,200 g of freeze-dried caviar powder is supercritically extracted to obtain caviar skim powder. Supercritical extraction is performed under the conditions of a pressure of 400 bar, a temperature of 40 °C, a flow rate of 60 ml/min, and an extraction time of 300 minutes. Through supercritical extraction, 960 g can be obtained with a yield of 80% compared to the freeze-dried powder.

The supercritical fluid extraction (SFE) used in the present invention is an extraction method using a supercritical fluid, which is a fluid in a state exceeding the critical temperature and boundary pressure, and generally uses inert, tasteless, odorless, harmless carbon dioxide. In addition, since the fluid vaporizes in the vicinity of atmospheric pressure, it has the advantage of obtaining only an extractable component, so it is used in pharmaceuticals and food. Examples of applications of supercritical extraction include decaffeination of coffee, EPA extraction from fish, and low-fat and low-cholesterolization treatment of food.

3) Enzyme treatment

After adding distilled water 10 times the amount of skim caviar powder, enzyme treatment is performed. For enzymes, Prozyme 1000L and Protamex were treated with 1%, 2.5%, and 5% of the raw material weight, respectively. Distilled water and enzyme-treated caviar skim powder lose enzyme activity at 90°C for 30 minutes after heating at 50°C for 4 hours.

As the enzyme used in the present invention, Prozyme 1000L is an endo-type protease obtained from cultures of Bacillus licheniformis and Bacillus amyloliquefaciens . Since it rapidly decomposes proteins to produce water-soluble peptides, it is suitable for the primary decomposition of proteins. Also, Protamex is an exo-type protease obtained from a culture of the genus Bacillus . Proteins firstly degraded by endo-type proteases are secondarily degraded by exo-type proteases, Protamex.

4) Filtration concentration and freeze-drying

After filtering the caviar enzyme-treated solution, the filtrate was concentrated to 15brix, and the concentrated 15brix concentrated solution was freeze-dried to prepare a caviar enzyme-treated powder functional composition.

2. Comparison of Yield of Caviar Enzyme-treated Powder Composition by Enzyme Treatment Conditions

Table 1 shows the caviar extraction yield for each composition preparation condition. In the description of the table, the primary enzyme treatment shows the results of enzyme treatment as it is, and the secondary enzyme treatment shows the results of enzyme treatment after pulverization of the skim powder (140mesh). The tertiary enzyme treatment shows the result of enzyme treatment by increasing the amount of enzyme input from 1% to 5% or 10% as it is with skim powder.

In addition, in the sample name, enzyme treatment x indicates the result of heating under the same conditions as in the enzyme treatment group without adding enzyme, and swelling + enzyme treatment indicates the result of swelling in distilled water for 1 hour to soften the tissue, and then enzyme treatment .

Looking at the results, the yield increased during pulverization of skim powder (secondary enzyme treatment), but it is judged that it is not suitable due to loss in the actual pulverization process in consideration of the unit cost of caviar raw material. (No. 1 to 5) Except for pulverization process, enzyme As a result of increasing the input amount (tertiary enzyme treatment), the yield was significantly increased (No. 6 to 11), and it was found that the yield was high when mixed enzymes were used rather than single enzymes (No. 8-11).

Caviar extraction yield by condition Caviar extraction yield by condition number sample name 3rd amount of skim powder input (g) 1st experiment 2nd experiment 3rd experiment Yield (%) compared to skim powder One hot water (pH6) 25 2 g (5%) 1 g (4%) - 1.3 2 Enzyme treatment X 25 2.5 g (10%) 2 g (8%) - 2.6 3 Prozyme 1% 25 6.16 g (24%) 7.89 g (31.92%) - 10.2 4 Protamex 1% 25 3.25 g (13%) 3.89 g (15.56%) - 5.0 5 Prozyme + Protamex 1% 25 4.19 g (16.8%) 6.9 g (27.6%) - 8.8 6 Prozyme 5% 40.07 - - 19.15 g (47.88%) 14.3 7 Protamex 5% 40.03 - - 17.23 g (43.08%) 12.9 8 Prozyme + Protamex 5% 48.33 - - 31.63 g (65.89%) 19.6 9 Prozyme + Protamex 10% 40.06 - - 28.05 g (70.13%) 21.0 10 Swelling + Prozyme + Protamex 5% 40.12 - - 20.19 g (50.32%) 15.1 11 Swelling + Prozyme + Protamex 10% 40.03 - - 25.29 g (63.23%) 19.0

In addition, it can be seen that, after swelling for the purpose of tissue softening, the yield of the enzyme treatment was not higher than that before the swelling treatment. Although it was the highest, it was judged that condition 8 was most suitable for mass production as it took a long time for filtration due to poor filtration during the filtration process, and there were many problems with the use of filters. That is, for mass production, the amount of skim powder input was increased to 500 g under condition No. 8, and the yield was 18.19% compared to skim powder even when a large amount of enzyme treatment was performed, completing the caviar enzyme-treated powder extraction method under condition No. 8.

II. Anti-inflammatory and skin test functional evaluation

In the present invention, mouse-derived macrophages, RAW264.7, were used, and as an antigen, LPS, a cell wall material of Gram-negative bacteria, was used for stimulation. In the case of the caviar enzyme-treated extract, the LPS-stimulated macrophages were treated for each concentration, and as a result, the inflammatory cytokines IL-6 and TNF-a were inhibited, thereby showing an anti-inflammatory effect. In addition, as a result of UV irradiation on Hs68, a human-derived fibroblast, it was found that it was effective in improving skin wrinkles and elasticity by decreasing MMP-1, a collagen degrading enzyme, and increasing elastic fibers.

1. Anti-inflammatory experiment

1.1 Cytotoxicity Results

2 shows the cytotoxicity results of caviar enzyme-treated powder and hot-water extract powder in RAW264.7 cells. Caviar enzyme-treated powder and hot water extract powder were treated with RAW264.7 (10, 50, 100, 250, 500 ppm) and cultured for 24 hours, and then MTT solution was added at a concentration of 5 mg/ml. After dissolution with DMSO, cytotoxicity was measured at a wavelength of 450 nm. RAW264.7 was cultured in a medium composition of 10% FBS and 1% P/S at 37° C., 5% CO ₂ conditions.

Looking at the results of Figure 1, it was found that there was no cytotoxicity in all conditions and concentrations except for 250 and 500 ppm of the No enzyme group. That is, if it shows a functional effect within a concentration without cytotoxicity, it can be confirmed that it is due to the raw material effect rather than the decrease or increase of the indicator due to apoptosis.

1.2. Cytokine IL-6 changes

3 shows the IL-6 reduction effect of hot water extract powder and enzyme-treated powder for each condition in RAW264.7 cells. RAW264.7 was treated with caviar enzyme-treated powder and caviar hot water extract powder (10, 100 ppm) for 24 hours, and then treated with LPS at a final concentration of 500 ng/ml and cultured again for 24 hours. After taking the supernatant, the inflammatory cytokine IL-6 was quantified with an ELISA kit. RAW264.7 was cultured in a medium composition of 10% FBS and 1% P/S at 37° C., 5% CO ₂ conditions.

Looking at the results of FIG. 3, IL-6 significantly decreased at 100 ppm of the single and complex enzyme treatment groups. The caviar hot water extraction and enzyme untreated group did not show any change in IL-6, an inflammatory cytokine, compared to the control group (LPS treated group), but showed a significant decrease in IL-6 in the three enzyme treatment groups. Also, there was no difference between each enzyme type. From this, it was found that the enzyme treatment of caviar skim powder showed an anti-inflammatory effect.

1.3. TNF-α reducing effect

4 shows the TNF-α reduction effect of hot water extract powder and enzyme-treated powder for each condition in RAW264.7 cells. RAW264.7 was treated with caviar enzyme-treated powder and caviar hot water extract powder (10, 100 ppm) for 24 hours, and then treated with LPS at a final concentration of 500 ng/ml and cultured again for 24 hours. After taking the supernatant, TNF-α, an inflammatory cytokine, was quantified with an ELISA kit. RAW264.7 was cultured in a medium composition of 10% FBS and 1% P/S at 37° C., 5% CO ₂ conditions.

Looking at the results in FIG. 4, TNF-a significantly decreased in 100 ppm of the single and complex enzyme treatment groups. There was no change in TNF-α, an inflammatory cytokine, in the caviar hot water extract and enzyme-untreated groups compared to the control group (LPS-treated group), but the enzyme-treated group showed a significant decrease in TNF-α in 3 groups. There was no difference between each enzyme type. From this, it was found that the enzyme treatment of caviar skim powder showed an anti-inflammatory effect.

2. Results of skin functional improvement test

2.1 Effect of reducing MMP-1 expression

5 shows the effect of reducing MMP-1 expression of hot water extract powder and enzyme-treated powder for each condition in Hs68 cells. After culturing human skin fibroblasts for 24 hours, they were irradiated with UV of 20 mJ/cm ² _, and 24 hours later, the caviar enzyme-treated powder was treated with a final concentration of 10 ppm. , and cultured in a composition of 1% P/S, and cultured in a composition excluding FBS when processing samples. RNA was isolated from Ps68 cells cultured with the experimental group using the RNeasy kit, and the change in MMP-1 mRNA expression was confirmed using real-time RT-PCR.

Looking at the results of Figure 5, the collagen-degrading enzyme MMP-1 was decreased in the Prozyme and mixed enzyme (Prozyme + Protamex) treatment group compared to the UV treatment group, and there was no significant difference compared to the Control group not treated with UV. decreased. This indicates that the collagen degrading enzyme expression can be reduced to the level before UV treatment in the Prozyme group and the mixed enzyme treatment group. In addition, it showed a superior collagen degrading enzyme inhibitory effect than the hot water extract and the enzyme-untreated group.

2.2 Effect of increasing COL1A

6 shows the effect of increasing COL1A of hot water extract powder and enzyme-treated powder for each condition in Hs68 cells. After culturing human skin fibroblasts for 24 hours, the caviar enzyme-treated powder was treated with a final concentration of 10 ppm and further cultured for 24 hours. Hs68 cells were cultured at 37°C, 5% CO _2, 10% FBS, and 1% P/S. RNA was isolated from Hs68 cells cultured with the experimental group using the RNeasy kit, and the change in COL1A mRNA expression was confirmed using real-time RT-PCR.

Looking at the results of FIG. 6 , COL1A expression was significantly increased in the Protamex and mixed enzyme treatment groups compared to the control group, although not as much as the EGCG group level. There was no significant difference between the enzyme-treated group and the enzyme-untreated group. In the case of enzyme treatment, there was an effect of increasing collagen expression which was superior to that of untreated and hot water extracts.

2.3 Effect of Increasing Fibrillin Expression

7 shows the effect of increasing the fibrillin expression of hot water extract powder and enzyme-treated powder for each condition in Hs68 cells. After culturing human skin fibroblasts (Ps68) for 24 hours, the caviar enzyme-treated powder was treated with a final concentration of 10 ppm and further cultured for 24 hours. Hs68 cells were cultured at 37°C, 5% CO _2, 10% FBS, and 1% P/S. RNA was isolated from Hs68 cells cultured with the experimental group using the RNeasy kit, and the change in the mRNA expression of Fibrillin was confirmed using Real-time RT-PCR.

Looking at the results of FIG. 7 , the expression level of Fibrillin was significantly increased in the enzyme-untreated group and the mixed enzyme-treated group compared to the CTRL group, and increased to a value similar to that of EGCG. Among the enzyme-treated groups, the single enzyme treatment did not show a significant difference, and only the mixed enzyme treatment group showed a significant difference. It has an effect of increasing fibrillin expression that is superior to that of the hot water extract and enzyme-only treatment group, so skin elasticity is increased and wrinkles are improved.

Through the above experiments, it was confirmed that the caviar enzyme-treated powder is a raw material that can help anti-inflammatory and skin improvement. In addition, it was confirmed that the caviar enzyme-treated powder had superior activity than the unenzymatically-treated extract.

8 shows the caviar enzyme-treated powder composition completed from the present invention. By using the caviar enzyme-treated powder extract prepared from the present invention, it can be prepared as a health functional food for anti-inflammatory and skin improvement. Health functional food is prepared by formulating in any one form selected from powders, granules, tablets, capsules, soft capsules, granules, and liquids so that the caviar enzyme-treated powder extract is contained in an amount of 0.01 to 99.9% by weight.

The composition for anti-inflammatory and skin improvement comprising the caviar enzyme-treated powder extract of the present invention as an active ingredient has industrial applicability because it can be used as an anti-inflammatory and skin-improving functional food that can be safely used without toxicity and side effects. .

Claims (4)

delete It contains a caviar enzyme-treated powder extract as an active ingredient, and the caviar enzyme-treated powder extract is an extract available from the supercritical extraction method, and the supercritical extraction pressure is 400 bar, temperature 40° C., flow rate 60 ml/min, extraction time 300 minutes. Functional composition for anti-inflammatory and skin improvement, characterized in that it consists of
A functional composition for anti-inflammatory and skin improvement comprising caviar enzyme-treated powder extract as an active ingredient, wherein the caviar enzyme-treated powder extract is obtained by primary enzyme treatment with Prozyme 1000L and secondary enzyme treatment with Protamex
Freeze-drying the raw caviar; supercritically extracting caviar freeze-dried powder to obtain caviar skim powder;
After adding 10 times distilled water to the caviar skim powder, performing an enzyme treatment;
Method for producing a functional composition for anti-inflammatory and skin improvement, characterized in that it is prepared by filtration of the caviar enzyme treatment solution and then freeze-drying the filtrate by concentrating the filtrate to 15brix

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