KR102453633B1 - Fermented pomegranate composition containing high content of ellagic acid and gallic acid with increased absorption into the body and its use having skin whitening, wrinkle improvement or antioxidant function, and method for manufacturing the same by using EFC method - Google Patents

Abstract

The present invention relates to a fermented pomegranate composition containing a high content of ellagic acid and gallic acid with increased absorption into body, uses having skin whitening, wrinkle alleviation or antioxidant functions, and a method for manufacturing thereof by using an EFC method and, more specifically, to a fermented pomegranate composition with significantly increased active ingredients such as gallic acid, ellagic acid and the like by fermenting the pomegranate with specific enzymes and specific strains by using the EFC method, a manufacturing method thereof, and uses of the composition for antioxidant activity, skin whitening or wrinkle alleviation.

Description

Fermented pomegranate composition containing a high content of ellagic acid and gallic acid with increased body absorption, and a use for skin whitening, wrinkle improvement or antioxidant function, and a method for manufacturing the same using EFC method with increased absorption into the body and its use having skin whitening, wrinkle improvement or antioxidant function, and method for manufacturing the same by using EFC method}

The present invention relates to a fermented pomegranate composition containing a high content of ellagic acid and gallic acid with increased body absorption, a use for skin whitening, wrinkle improvement or antioxidant function, and a method for producing the same using the EFC method, more particularly, the EFC method to a fermented pomegranate composition in which active ingredients such as gallic acid and ellagic acid are significantly increased by fermenting pomegranate with specific enzymes and strains using

Pomegranate is a deciduous broad-leaved small arboreous tree belonging to the Pomegranate family, Pomegranate family, Pomegranate family, Pomegranate family, and Pomegranate species, and there are about 500 species. Pomegranate is native to Iran and is mainly produced in South Asia such as India and Pakistan, and was introduced to Korea during the Joseon Dynasty.

The fruit is divided into six small chambers with thin partitions, which contain numerous seeds. Pomegranate is regarded as one of the most popular functional foods in the world because of its sweet and sour taste and excellent various health benefits, and its consumption is rapidly expanding.

Pomegranate contains abundant nutrients such as carbohydrates, proteins, fats, B vitamins, C, E, K, and minerals. In particular, it contains various kinds of phytochemicals such as phenolic compounds with strong physiological activities such as antioxidant, anticancer, antibacterial, and anti-inflammatory, as well as plant estrogens.

The representative efficacy of pomegranate is improvement of menopausal disorders and skin protection, and it is already registered as a health functional food ingredient in the Ministry of Food and Drug Safety of Korea. This effect is due to the strong antioxidants from estrogenic substances such as urolithin and phenolic compounds such as gallic acid produced from ellagic acid contained in pomegranate.

However, these two representative physiologically active substances are actually contained in pomegranate juice, but contain very small amounts, and most of them exist as hydrolyable tannins such as precursors ellagtannin and gallotannin.

Therefore, in order to improve the activity of active ingredients in pomegranate juice, it is necessary to develop a technology for producing functional raw materials that can promote absorption in the body by increasing ellagic acid and gallic acid.

As a method of increasing useful physiologically active ingredients in pomegranates, fermented pomegranate for improving menopausal symptoms in Korea Patent Publication No. 10-2020-0064501, and pomegranate for improving menopause containing high content of ellagic acid in Korean Patent No. 10-1272602 Extract, pomegranate concentrate for skin improvement in Korean Patent No. 10-1933152, lactic acid bacteria fermented product of pomegranate having anti-obesity and anti-inflammatory activity in Korean Patent Registration No. 10-2214532, and Korean Patent Publication No. 10-2020- No. 0064249 discloses a method for extracting ellagic acid from pomegranate peel powder.

However, in this prior art, pomegranate skins of high-content varieties or pomegranate peels, which were originally prohibited for use as food, were used, or hydrolytic enzymes such as pectinase or celluase that decompose plant cell membranes were used, and some were treated with lactic acid bacteria, acetic acid bacteria, etc. Thus, examples of slightly improved flavonoid compounds and antioxidant activity can be seen, but there is a limit to increasing the content of ellagic acid and gallic acid, which are key physiologically active ingredients, and the activity enhancing effect did not show the expected performance.

Korean Patent Publication No. 10-2020-0064501 Korean Patent Registration No. 10-1272602 Korean Patent No. 10-1933152 Korean Patent Registration No. 10-2214532 Korean Patent Publication No. 10-2020-0064249

In order to solve the above problems, the present invention provides a processed pomegranate product containing a high content of active ingredients that can significantly increase the content of active ingredients such as ellagic acid and gallic acid contained in pomegranates and improve the absorption rate in the body. do it with

Accordingly, an object of the present invention is to provide a processed pomegranate product containing a high content of the active ingredient by using an enzyme and a strain that act directly on the precursor of the active ingredient contained in the pomegranate.

Another object of the present invention is to provide a fermented pomegranate composition in which the contents of ellagic acid and gallic acid, which are core phytoestrogens contained in pomegranate, are remarkably increased by fermenting the pomegranate concentrate with specific enzymes and specific lactic acid bacteria.

In addition, another object of the present invention is to provide a fermented pomegranate composition having skin whitening, wrinkle improvement or antioxidant functions including high content of ellagic acid and gallic acid with increased body absorption.

In addition, another object of the present invention is to provide a method that can dramatically increase active ingredients such as ellagic acid and gallic acid in a short period of time by fermenting pomegranate using the EFC method (enzyme fermentation method).

In order to solve the problems of the present invention as described above, the present invention provides a fermented pomegranate composition comprising a fermented product obtained by enzymatic fermentation of pomegranate with tannaise and Lactobacillus vespulae DCY75 as an active ingredient for skin protection.

According to a preferred embodiment of the present invention, the fermented product may include a fermented product obtained by fermenting at least one of pomegranate juice, pomegranate juice, or pomegranate concentrate.

According to a preferred embodiment of the present invention, 0.01 to 1% by weight of the tannaise is used, and Lactobacillus vespulae DCY75 may include a fermented product at 0.5x10 ⁷ cfu/ml to 3x10 ⁷ cfu/ml.

According to a preferred embodiment of the present invention, the present invention includes a composition for antioxidant, skin wrinkle improvement or skin whitening using the fermented product as an active ingredient.

According to a preferred embodiment of the present invention, the present invention provides a cosmetic composition containing the antioxidant, skin wrinkle improvement or skin whitening composition.

According to a preferred embodiment of the present invention, the present invention provides a health food containing the antioxidant, skin wrinkle improvement or skin whitening composition.

Also, the present invention

preparing pomegranate juice, pomegranate juice, or pomegranate concentrate from the pomegranate pulp;

The pomegranate stock solution is diluted with 5-10brix, treated with 0.01 to 1% by weight of tannaise and 0.5x10 ⁷ cfu/ml to 3x10 ⁷ cfu/ml of Lactobacillus vespulae DCY75 lactic acid bacteria, and cultured with shaking at 30-40 ℃ for 2 to 48 hours. step; and

Inactivating the enzyme at 70-80 ° C. in the enzyme reaction solution obtained by the shaking culture

It provides a method for producing a pomegranate fermented composition comprising a.

According to a preferred embodiment of the present invention, the shaking culture may be performed under agitation at 80 to 200 rpm.

According to a preferred embodiment of the present invention, the step of inactivating the enzyme may be performed for 5 to 30 minutes.

The present invention has the effect of selectively producing large amounts of active ingredients such as ellagic acid, gallic acid, and phenolic compounds in a short time by fermenting hydrolyzable tannins present in the pomegranate extract by the EFC method.

Since the present invention contains a high content of the active ingredients as described above, the antioxidant effect is excellent due to the strengthening of these active ingredients, and the effects such as skin wrinkle improvement and skin whitening are remarkably increased.

In addition, through the reaction, tannin in pomegranate juice, pomegranate juice or pomegranate concentrate is reduced, thereby reducing the astringent taste, thereby significantly improving the quality.

1 is a diagram showing the increase in gallic acid and ellagic acid in a pomegranate fermented composition fermented by the EFC method.
2 is a diagram showing the effect on intracellular tyrosinase activity in B16F10 cells of a fermented pomegranate composition fermented by the EFC method.
3 is a diagram showing the effect on extracellular melanin production in B16F10 cells of a pomegranate fermented composition fermented by the EFC method.
4 is a diagram showing the effect on intracellular melanin production in B16F10 cells of a pomegranate fermented composition fermented by the EFC method.
5 is a diagram showing the effect on the activity of Elastase in HaCaT cells of the fermented pomegranate composition fermented by the EFC method.
6 is a diagram showing the effect on Elastase activity in HDF cells of the fermented pomegranate composition fermented by the EFC method.

Hereinafter, the present invention will be described in more detail as one embodiment as follows.

The present invention is characterized in that the pomegranate is prepared as a fermented pomegranate composition using the so-called EFC (enzyme fermentation method).

According to a preferred embodiment of the present invention, by enzymatic fermentation of pomegranate, hydrolysable tannins present in pomegranate juice, pomegranate juice or pomegranate concentrate (this may be collectively referred to as 'pomegranate juice' or 'pomegranate juice') ) was fermented with tannaise and new lactic acid bacteria ( Lactobacillus vespulae DCY75) to dramatically increase the content of ellagic acid and gallic acid.

According to a preferred embodiment of the present invention, the fermented pomegranate composition of the present invention can be provided as a pomegranate fermented liquid with significantly increased contents of ellagic acid and gallic acid, which are excellent in antioxidant and skin protection effects. As described above, the composition obtained by fermenting pomegranate with tannaise and lactic acid bacteria by the method of the present invention increases the physiologically active ingredients in the pomegranate juice, thereby exhibiting remarkably excellent physiological activity.

According to a preferred embodiment of the present invention, pomegranate can be fermented using an enzyme fermentation method. Here, the enzyme fermentation method (EFC; Enzyme Fermentation Control System) is a method of fermentation in the presence of enzymes. If the structure of a physiologically active substance is converted using this method, absorption into the body is remarkably good, and effective It can be said that it is a specialized method that enables mass production of ingredients.

According to a preferred embodiment of the present invention, in the present invention, ellagic acid and gallic acid, which are key phytoestrogens in the pomegranate extract, are used directly with enzymes and special strains acting on their precursors. It can dramatically increase active ingredients such as gallic acid.

According to a preferred embodiment of the present invention, the fermented product may include a fermented product obtained by fermenting at least one of pomegranate juice, pomegranate juice, or pomegranate concentrate, which is a pomegranate extract that is the fermented product.

According to a preferred embodiment of the present invention, the tannaise is characterized in that 0.01 to 1% by weight and Lactobacillus vespulae DCY75 0.5x10 ⁷ cfu/ml to 3x10 ⁷ cfu/ml are simultaneously applied and fermented, where only the enzyme is applied or When only lactic acid bacteria are applied, it is difficult to obtain a remarkable physiologically active effect according to the present invention.

In particular, according to the present invention, tannaise can be preferably used as the enzyme, and Lactobacillus vespulae DCY75 is selectively and preferably used as the lactic acid bacterium.

As such, according to a preferred embodiment of the present invention, it is difficult to achieve the object of the present invention when the present invention is fermented using tannaise alone or Lactobacillus vespulae DCY75 alone. In addition, it is difficult to achieve the object of the present invention even when using Lactobacillus vespulae DCY75 after using tannaise or using tannaise after using Lactobacillus vespulae DCY75 lactic acid bacteria.

Therefore, the present invention has technical significance in obtaining a synergistic effect by fermenting using tannaise and Lactobacillus vespulae DCY75 at the same time. When these two components are used at the same time, excellent effects of the present invention can be obtained. Therefore, when tannaise and Lactobacillus vespulae DCY75 are used at the same time, bioactive ingredients such as gallic acid or ellagic acid, which are active ingredients selectively, can show remarkably superior results with a synergistic effect compared to other cases.

According to a preferred embodiment of the present invention, in order to apply the enzyme fermentation method as described above, in the present invention, a pomegranate extract can be prepared from the pomegranate flesh by a pressing method. Such a pomegranate extract may be prepared, for example, as pomegranate juice, pomegranate juice, or pomegranate concentrate.

The pomegranate extract thus prepared is fermented with specific enzymes and specific lactic acid bacteria, for example, by diluting the pomegranate extract with 5-10 brix to obtain 0.01 to 1% by weight of tannaise. More preferably, 0.05-0.5 wt% and Lactobacillus vespulae DCY75 of 0.5x10 ⁷ cfu/ml to 3x10 ⁷ cfu/ml. More preferably, it may be subjected to a step of fermenting by treatment with 0.8x10 ⁷ cfu/ml to 1.5x10 ⁷ cfu/ml and culturing with shaking at 30 to 40° C. for 2 to 48 hours.

According to a preferred embodiment of the present invention, the desired fermented pomegranate composition can be prepared by subjecting the enzyme reaction solution obtained by fermenting and culturing with shaking as described above to inactivating the enzyme at 70 to 80°C.

According to a preferred embodiment of the present invention, the shaking culture may be performed under agitation at 80 to 200 rpm, more preferably at 100 to 150 rpm. In addition, the step of inactivating the enzyme after the fermentation culture may be performed for 5 to 30 minutes.

According to a preferred embodiment of the present invention, the fermented pomegranate composition prepared as described above can be obtained as a result of remarkably improved physiologically active ingredients such as ellagic acid or gallic acid, and such a fermented pomegranate composition can be used for antioxidant, skin wrinkle improvement or skin care. It has a very good effect on whitening.

Accordingly, the present invention includes a composition for antioxidant, skin wrinkle improvement, or skin whitening using the fermented pomegranate composition as described above as an active ingredient.

According to a preferred embodiment of the present invention, such antioxidant, skin wrinkle improvement or skin whitening composition is very useful as a cosmetic composition or health food. In particular, when the composition of the present invention is used for the above purpose, the bioabsorption rate is excellent, and thus an excellent use effect can be exhibited.

According to a preferred embodiment of the present invention, the cosmetic composition can be formulated and used in various forms such as skin, cream, lotion, essence, celeb, ointment, and the like.

In addition, when the composition of the present invention is used as a health food, it can be prepared and used in various forms such as tablets, capsules, granules, granules, pills, gels, powders, jellies, liquids, drinks, carbonated drinks, and soft drinks.

As described above, the fermented pomegranate composition according to the present invention can selectively produce large amounts of ellagic acid, gallic acid and phenolic compounds in a short time by fermenting hydrolyzable tannins present in the pomegranate extract by the EFC method. Therefore, the present invention has an effect that can dramatically increase active ingredients such as ellagic acid and gallic acid in a short period of time.

The fermented pomegranate composition of the present invention has the effect of remarkably increasing the efficacy of wrinkle improvement and skin whitening due to the strengthening of the active ingredient with excellent physiological activity.

In addition, according to the present invention, the tannin in the pomegranate juice, pomegranate juice or pomegranate concentrate is reduced through an enzyme fermentation reaction with respect to the pomegranate stock solution of at least one of pomegranate juice, pomegranate juice or pomegranate concentrate, so that the astringent taste can be greatly reduced and the taste can be improved. , there is an effect of improving the quality of pomegranate extract or health food using the same.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited by the examples.

Example: Preparation of fermented pomegranate composition

(1) A step of preparing a pomegranate juice undiluted solution (pomegranate juice) by a pressing method from the pomegranate flesh is performed.

(2) The pomegranate juice stock solution prepared in step (1) was diluted with 8brix, treated with 0.2 wt% of tannaise and Lactobacillus vespulae DCY75 (1x10 ⁷ cfu/ml), fermented at 34° C. for 24 hours, and stirred at 120 rpm. An enzyme reaction solution is obtained through a shaking incubation step.

(3) The enzyme reaction solution obtained in step (2) is treated at 70-80° C. for 10 minutes to inactivate the enzyme.

(4) Prepare a pomegranate fermented composition by preparing the enzyme reaction solution deactivated in the above step as a pomegranate concentrate.

Comparative Example 1

A pomegranate fermented composition was prepared by performing fermentation under the same comparative conditions as in the above example, using only tannin acylhydrolase (TAH) as an enzyme, but not using lactic acid bacteria, and then fermenting and culturing with shaking.

Comparative Example 2

A pomegranate fermented composition was prepared by fermenting with Lactobacillus vespulae DCY75 as a lactic acid bacterium after using tannaise (TAH) as an enzyme and culturing with shaking under the same comparative conditions as in the above example.

Comparative Example 3

A pomegranate fermented composition was prepared by fermenting using Lactobacillus vespulae DCY75 as a lactic acid bacterium and then culturing with shaking after using tannaise (TAH) as an enzyme under the same comparative conditions as in the above example.

Comparative Example 4

A pomegranate fermented composition was prepared by fermenting using Lactobacillus vespulae DCY75 as a lactic acid bacterium and culturing with shaking under the same comparative conditions as in the above Example.

Experimental Example 1: Comparison of fermentation effect for lactic acid bacteria selection

In order to compare the fermentation effect of lactic acid bacteria, fermentation was performed using the same raw materials as in the above example, but without using enzymes, Lactobacillus brevis, Lactobacillus vespulae DCY75, Lactobacillus panaxibrevis DCY65, Bacillus amyloliquefaciens, Saccharomyces cerevisiae were used individually. It was fermented to prepare a fermented pomegranate composition.

The gallic acid production amount was measured for each of the lactic acid bacteria-using fermented compositions compared with the control group. As a result, Table 1 below is the result shown based on the gallic acid production amount for strain selection for fermentation.

Referring to Table 1 above, Lactobacillus vespulae DCY75 in gallic acid production showed an excellent effect compared to other strains. Therefore, it shows that the use of Lactobacillus vespulae DCY75 as a lactic acid bacterium has an excellent selective effect on the increase of effective bioactive substances such as gallic acid.

Experiment 2: Effect comparison experiment through comparison of gallic acid production

For each of the above Examples and Comparative Examples 1 to 4, the amount of gallic acid production was measured and compared with the pomegranate fermented composition finally produced after fermentation for each sample.

The results are shown in the table below. Table 2 below compares the results of gallic acid production under the same conditions according to tannaise (TAH) and Lactobacillus vespulae DCY75 alone, sequential use (indicated by arrows), and simultaneous mixed use (indicated by +), respectively. .

Referring to Table 2, the case of using tannaise (TAH) and Lactobacillus vespulae DCY75 at the same time showed a significantly superior increase in gallic acid production compared to other comparative groups. From this result, it can be confirmed that tannaise (TAH) and Lactobacillus vespulae DCY75 each have a remarkably excellent synergistic effect compared to the case of using them alone or sequentially.

Experimental Example 3: Antioxidant effect experiment

For the samples according to Examples and Comparative Example 1, free radical scavenging ability was measured under the same conditions in a conventional manner and reducing power was measured and compared.

In addition, the total polyphenolic compounds and total flavonoid contents, which are antioxidants, were measured and compared for each sample.

In the free radical scavenging activity test, the DPPH (Sigma-Aldrich Co., St. Louis, MO, USA) radical scavenging activity of each sample was measured according to the method of Blois (21). 0.1 mL of each sample by concentration (7.8, 15.6, 31.2, 62.5, 125, and 250 μg/mL) dissolved in ethanol was mixed with the same amount of 0.2 mM DPPH solution and reacted at 37°C for 15 minutes, followed by a spectrophotometer (UV- 1600 PC, Shimadzu, Tokyo, Japan) was used to measure the absorbance in the region of 517 nm.

The comparison with the control group (before fermentation or enzyme treatment) is shown in Table 3 below.

Table 3 shows the changes in antioxidant activity and content of antioxidants in the fermented pomegranate composition fermented by the EFC method. From this experimental result, it is shown that the Example in which tannaise (TAH) and Lactobacillus vespulae DCY75 are used simultaneously has a superior antioxidant activity effect compared to other comparative groups. From this result, it can be confirmed that when tannaise (TAH) and Lactobacillus vespulae DCY75 are mixed, there is a superior synergistic effect compared to tannaise alone.

Experimental Example 4: Hydrolyzable tannin content test

For the samples according to Example and Comparative Example 1, the content of hydrolyzable tannins was measured and compared by a conventional method under the same conditions.

The comparison with the control group (before fermentation or enzyme treatment) is shown in Table 4 below.

Table 4 shows a comparison of the hydrolyzable tannin content of the pomegranate fermented composition fermented by the EFC method. From this experimental result, it is shown that the case of using tannaise (TAH) and Lactobacillus vespulae DCY75 at the same time has a significantly superior tannin content reduction effect compared to other comparative groups. From this result, it can be confirmed that when a mixture of tannaise (TAH) and Lactobacillus vespulae DCY75 is used, there is a superior synergistic effect on tannin reduction compared to the use of tannae alone.

Experimental Example 5: Sensory evaluation

For the samples according to the Example and Comparative Example 1, a sensory evaluation was performed to compare the taste under the same conditions.

The sensory evaluation was a 5-point evaluation method, and 60 students including college students (36 males, 13 females) and housewives (11 people) were evaluated by comparing astringency and sweetness.

The comparison with the control group (before fermentation or enzyme treatment) is shown in Table 5 below.

Table 5 shows a comparison of the sensory astringency and sweetness of the fermented pomegranate composition fermented by the EFC method. From this experimental result, it is shown that the case of using tannaise (TAH) and Lactobacillus vespulae DCY75 simultaneously has an excellent taste improvement effect compared to other comparative groups. From this result, it can be confirmed that when tannaise (TAH) and Lactobacillus vespulae DCY75 are mixed and used, there is an excellent synergistic effect in taste compared to tannaide alone.

Experimental Example 6: Comparative experiment of increasing gallic acid and ellagic acid

The analysis of ellagic acid and gallic acid for the sample of the above example was analyzed by the HPLC system. The HPLC system was an Agilent 1260 Infinity Quaternary pump (G1311B), an Agilent 1260 Infinity, a standard autosampler (G1329B), an Agilent 1260 Infinity column thermostat compartment (G1316A), and an Agilent 1260 Infinity Variable Wavelength Detector (G1314F) as instruments. was used

As the column used, a ZORBAX Eclipse Plus C18 column (250 mm × 4.6 mm, 5 μm particle size) (Mil ford, MA, USA) was selected as a stationary phase.

The mobile phase is a concentration gradient, and the eluent composition is as follows.

(0-16 min, 15-30% B, 16-34 min, 30-100% B, 34-40 min, 100-100% B, 40-41 min, 100-15% B; 41-42 min 15 -15% B)

The experimental results are shown in comparison with FIG. 1 . 1 is a comparative graph showing the increase in gallic acid and ellagic acid in pomegranate juice fermented by the EFC method. From this result, it was confirmed that in the case of the fermented pomegranate composition prepared according to the method of Example (TAH+DCY75), the production of gallic acid and ellagic acid was significantly increased compared to the control. In particular, the increase in gallic acid shows a more remarkable difference. From this, it is shown that the sample of Example has a remarkable effect on the increase of gallic acid and ellagic acid, which are physiologically active ingredients.

Experimental Example 7: Effect on intracellular tyrosinase activity in B16F10 cells

Intracellular tyrosinase inhibitory activity was evaluated by measuring the degree of L-DOPA oxidation. That is, B16F10 cells were seeded to become 2x104 cells in a 6 well plate and cultured for 1 day (overnight). Then, samples such as the composition of the above examples were treated from 125 to 500 ug/ml, treated with 100 uM IBMX together with a control without a sample and cultured for 72 hours, and 125 uM Kojic acid was used as a positive control,

After the reaction was completed, the cells were washed with PBS buffer, lysed with 1% TritonX-100, and left in a -80°C deep freezer for 1 hour. After thawing the cells, 80ul of the supernatant and 25ul of L-DOPA (2mg/ml) were taken in a 96-well plate, incubated for 1 hour, and absorbance was measured at 475nm.

The measurement result is shown in FIG. 2 . 2 is a diagram showing the effect on intracellular tyrosinase activity in B16F10 cells of the pomegranate fermented composition fermented by the EFC method of the above example.

From this result, it is confirmed that the sample has a significant concentration-dependent effect on tyrosinase activity.

In the drawings, PG-C is a control, PG-T is a case in which only tannaise is used (Comparative Example 1), and PG-TL is a case in which tannaise and Lactobacillus vespulae DCY75 are used simultaneously (Example).

Experimental Example 8: Effect on extracellular melanin production

As part of the skin whitening efficacy evaluation of the sample of the above example, the inhibitory effect on tyrosinase isolated and purified from mushrooms that catalyze melanin formation was investigated. That is, Tyrosinase activity inhibition was measured as follows. For the experiment, 40ul of 0.1M PBS buffer, 40ul of 2mM L-tyrosine, 40ul of mushroom tyrosinase (125units/ml), and an appropriate amount of sample 40ul were added to a 96 well plate to adjust the total amount to 120ul and stirred at 37℃ for 10 minutes. After the reaction, absorbance was measured at 475 nm to calculate the degree of inhibition of tyrosinase activity.

The results are shown in FIG. 3 . 3 is a diagram showing the effect on the extracellular melanin production of pomegranate juice fermented by the EFC method.

From this result, it was confirmed that the sample of Example exhibited an excellent effect on whitening activity.

Experimental Example 9: Effect on Elastase activity in B16F10 cells and HaCaT cells

Elastase activity was measured using several modifications using N-succinyl-tri-alanyl-p-nitroanilide (STANA, elastase substrate).

Since dermal fibroblasts (HDFs) are primary cells that secrete elastase, HDF cells, each cell group (UVB non-irradiated cells, UVB-irradiated cells, UVB-irradiated cells) After exposure to mJ/cm2 UVB irradiation, incubation was carried out for 24 hours In HaCat cells, elastase (0.3 units/mL) was treated with each sample and incubated together, namely, PG, PG-F (50, 100, 200). μg/mL) or ursolic acid (500 μM) in a 5% CO2 incubator for 30 minutes, then the substrate STANA (400 μM) is added and the cells are harvested after incubation for 15 minutes in a 5% CO2 incubator for reaction with 0.2M Tris After dissolving in 0.1% Triton X-110 in -HCl buffer (pH 8.0), freezing and thawing were repeated three times.

Cell debris was removed through microcentrifugation (3000 rpm, 20 minutes, 4° C.), and the remaining sample containing the protein was used. These samples were placed in 96-well plates of each sample group, and the volume of each well was adjusted to 98 μL with 0.2M Tris-HCl. 2 microliters of 400 µM STANA solution were aliquoted into each well and then the mixture was incubated at 37 °C for 90 min. Elastase activity was determined by measuring absorbance at 410 nm using a microplate reader.

Experimental results are shown in Figs. 4a and 4b. 4a and 4b are graphs showing the effect on Elastase activity in B16F10 cells and HaCaT cells of Example samples fermented by the EFC method, respectively.

From the results of the above experiments, the fermented pomegranate composition fermented by the EFC method of Examples according to the present invention has a technical significance of a synergistic effect due to the selection and mixed use of the specific enzyme and specific lactic acid bacteria, ellagic acid having an increased rate of absorption into the body, It was confirmed that the effect of remarkably improved skin whitening, wrinkle improvement, and antioxidant functions containing high gallic acid content. In addition, it was confirmed that the composition has a very high absorption rate in the body and has a distinct improvement effect in taste, such as astringency and sweetness.

Claims (9)

A fermented pomegranate composition containing the fermented product obtained by simultaneous enzymatic fermentation of pomegranate with tannaise and Lactobacillus vespulae DCY75 as an active ingredient for skin protection. The fermented pomegranate composition according to claim 1, wherein the fermented product comprises a fermented product obtained by fermenting at least one of pomegranate juice, pomegranate juice, and pomegranate concentrate. The fermented pomegranate composition according to claim 1, wherein the tannaise is used in an amount of 0.01 to 1% by weight and Lactobacillus vespulae DCY75 is fermented using 0.5x10 ⁷ cfu/ml to 3x10 ⁷ cfu/ml. A composition for antioxidant, skin wrinkle improvement, or skin whitening, containing, as an active ingredient, a fermented product obtained by simultaneous enzymatic fermentation of pomegranate with tannaise and Lactobacillus vespulae DCY75. A cosmetic composition for antioxidant, skin wrinkle improvement, or skin whitening containing, as an active ingredient, a fermented product obtained by simultaneous enzymatic fermentation of pomegranate with tannaise and Lactobacillus vespulae DCY75. A health food for antioxidant, skin wrinkle improvement or skin whitening containing fermented products obtained by simultaneously enzymatic fermentation of pomegranate with tannaise and Lactobacillus vespulae DCY75. preparing pomegranate juice, pomegranate juice, or pomegranate concentrate from the pomegranate pulp;
The pomegranate stock solution is diluted with 5-10brix, treated with 0.01 to 1% by weight of tannaise and 0.5x10 ⁷ cfu/ml to 3x10 ⁷ cfu/ml of Lactobacillus vespulae DCY75 lactic acid bacteria, and cultured with shaking at 30-40 ℃ for 2 to 48 hours. step; and
Inactivating the enzyme at 70-80 ° C. in the enzyme reaction solution obtained by the shaking culture
A method for producing a fermented pomegranate composition comprising a. The method according to claim 7, wherein the shaking culture is performed under stirring at 80 to 200 rpm. The method according to claim 7, wherein the step of inactivating the enzyme is performed for 5 to 30 minutes.

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