KR102711955B1 - Composition for anti-inflammatory, antioxidation and skin whitening containing fermented Lindera obtusiloba flower extract - Google Patents

Abstract

The present invention relates to a functional composition containing a fermented ginger flower extract as an effective ingredient, which can be utilized as a cosmetic composition, a food composition, or a health functional food composition having excellent anti-inflammation, anti-oxidation, and skin whitening effects.

Description

{Composition for anti-inflammatory, antioxidation and skin whitening containing fermented Lindera obtusiloba flower extract}

The present invention relates to an anti-inflammatory, anti-oxidant and skin whitening composition, and more specifically, to an anti-inflammatory, anti-oxidant and skin whitening composition containing a fermented ginger flower extract as an effective ingredient.

The functional cosmetics market that can satisfy both skin health and aesthetic satisfaction is showing a growth trend every year. Among them, the most active research fields are whitening and anti-aging cosmetics. In particular, many people are suffering from aesthetically negative effects such as skin darkening and pigmentation due to the increase in ultraviolet rays caused by environmental pollution. These negative effects are mainly caused by melanin increased by ultraviolet rays, and although it generally plays a role in protecting the skin from ultraviolet rays, if melanin is produced excessively, it can cause skin spots, freckles, and spots.

Melanin is produced in the melanocytes of the skin's epidermis, and is divided into dark brown eumelanin and reddish-yellow pheomelanin depending on the color of the melanin, but they are commonly produced based on the oxidation of tyrosine. The oxidation process is mainly promoted by tyrosinase and tyrosinase-related proteins, and whitening cosmetics whiten the skin by inhibiting the elements necessary for this oxidation process, such as inhibition of tyrosinase activity, inhibition of tyrosinase production, removal of melanin precursors, inhibition of melanosome movement, direct destruction of melanin, and direct destruction of melanocytes. Among these, the whitening mechanism mainly used in whitening cosmetics is inhibition of tyrosinase activity.

Inhibition of tyrosinase activity is a method for achieving a whitening effect without affecting cells or tissues, and is preferred as a mechanism for whitening cosmetics. The activity of tyrosinase can be reduced by substances that can inhibit the oxidation of tyrosinase, that is, antioxidant substances. For this reason, antioxidant activity is one of the factors that can be evaluated before examining the whitening effect.

Antioxidant activity refers to the ability to inhibit oxidation or eliminate active oxygen. Active oxygen is a general term for oxygen that is not electronically stable and easily reacts with other substances to oxidize them. When active oxygen exists in an appropriate amount, it can perform beneficial functions such as synthesizing cell components and immune function, but when it is produced excessively, it oxidizes and denatures DNA, RNA, proteins, lipids, etc., causing cell aging. Accordingly, many studies have been conducted to remove active oxygen, and interest in antioxidant substances has increased accordingly.

Ginger ( Lindra obtusiloba ) is a plant in the Lauraceae family that grows in the shade of forests and mountain valleys in Korea, Japan, and China. It is called ginger because its branches emit a ginger-like smell when broken. The bark, leaves, and branches of the ginger tree contain capric acid, lauric acid, myristic acid, linderic acid, oleic acid, and linoleic acid, which are known to have the effects of promoting blood circulation, dispersing blood stasis, reducing swelling, and relieving muscle pain, and are known to treat bruises, blood stasis, and swelling.

Research on ginger flowers is being conducted on their antioxidant, anti-inflammatory, whitening, and antibacterial properties, and ginger flowers in particular can be used as a raw material for tea.

Accordingly, the inventors of the present invention developed a technology capable of increasing the anti-inflammation, anti-oxidation, and whitening effects of ginger flower through lactic acid bacteria fermentation, and completed the present invention.

The matters described as background technology above are only intended to enhance understanding of the background of the present invention, and should not be taken as an acknowledgment that they correspond to prior art already known to those skilled in the art.

[National Research and Development Project]

① Assignment number: 2022-014

② Ministry name: Ministry of Education

③ Project Management (Professional) Organization Name: National Research Foundation of Korea

④ Research Project Name: 3-Stage Industry-Academia-Research Cooperation Leading Specialized University Development Project (LINC3.0)

⑤ Research Project Name: Development of Cosmetic Raw Material through Lactic Acid Bacteria Fermentation of Ginger Flower Extract

⑥ Contribution rate: 1

⑦ Project implementation organization name: Yonsei University Industry-Academic Cooperation Foundation, Elfounder Co., Ltd.

⑧ Research period: 2022.11.01. ~ 2023.10.31.

Patent Document 1: KR 10-2022-0125190 A (2022.09.14) "Health functional food and pharmaceutical composition for improving inflammatory diseases or memory containing ginger extract as an effective ingredient" Patent Document 2: KR 10-2022-0085987 A (2022.06.23) "Antibacterial composition containing a mixed extract of ginger and sagebrush as an effective ingredient"

In order to solve the problems of the above-mentioned prior art, the present invention aims to provide a method for producing a fermented ginger flower extract.

In addition, the present invention aims to provide an anti-inflammatory, anti-oxidation and whitening cosmetic composition containing a fermented ginger flower extract as an effective ingredient.

In addition, the present invention aims to provide an anti-inflammatory, anti-oxidant and whitening food or health functional food composition containing a fermented ginger flower extract as an effective ingredient.

Other objects and advantages of the present invention will become more apparent from the detailed description of the invention, the claims and the drawings which follow.

In order to solve the above technical problems, the present invention provides a method for producing a fermented ginger flower extract exhibiting anti-inflammatory, antioxidant and skin whitening effects, comprising the steps of precipitating dried ginger flowers in a 10-fold diluted solution of MRS broth; and inoculating a fermentation strain into the solution of the precipitation step to ferment the ginger flowers.

In the present invention, in the precipitation step, the MRS broth may be composed of 10 g/L peptone, 10 g/L beef extract, 5 g/L yeast extract, 20 g/L glucose, 5 g/L sodium acetate, 2 g/L ammonium citrate, 2 g/L potassium phosphate, 0.1 g/L magnesium sulfate, and 0.05 g/L manganesum sulfate.

Here, MRS broth can be used by diluting it 10 times its volume, and it is preferable to extract dried ginger flowers by adding 1% of the solvent volume.

In one embodiment of the present invention, 1 g of dried ginger flower can be precipitated in 100 mL of a 10-fold diluted solution of MRS broth (peptone (10 g/L), beef extract (10 g/L), yeast extract (5 g/L), glucose (20 g/L), sodium acetate (5 g/L), ammonium citrate (2 g/L), potassium phosphate (2 g/L), magnesium sulfate (0.1 g/L), manganesum sulfate (0.05 g/L)).

In another embodiment of the present invention, the composition of 100 mL of a 10-fold diluted solution of the MRS broth may include 0 to 2 g of glucose, 0 to 2 g of peptone, 0 to 5 g of yeast extract, 1 g of beef extract, 0.5 g of sodium acetate, 0.2 g of ammonium citrate, 0.2 g of potassium phosphate, 0.01 g of magnesium sulfate, and 0.005 g of manganese sulfate, and preferably, the composition may be 0 g of glucose, 0.4646 g of peptone, and 0 g of yeast extract, and may exhibit the best antioxidant effect among the fermented ginger flower extract manufactured through the fermentation step. At this time, 0.5 to 5 g of ginger flower can be added and precipitated.

In the present invention, the strain pre-cultured in the fermentation step can be inoculated at 1% of the volume of the solvent based on an OD of 0.7, and then cultured at 37°C for 24 hours.

In the present invention, in the fermentation step, Lactococcus lactis KCTC 3115 can be used as the fermentation strain.

According to one embodiment of the present invention, the Lindera obtusiloba flower fermented extract is manufactured by precipitating the Lindera flower in MRS broth, inoculating and culturing a fermentation strain, and then fermenting.

In the present invention, the ginger flower fermentation extract produced in the fermentation step can be filtered, concentrated, or dried to remove the solvent, and filtration, concentration, and drying can all be performed. For example, filtration can be performed using a filter paper or a vacuum filter, concentration can be performed using a vacuum concentrator, and drying can be performed using a freeze-drying method, but is not limited thereto.

The present invention provides an anti-inflammatory, anti-oxidant and skin whitening cosmetic composition containing a fermented ginger flower extract produced by the above-mentioned production method as an effective ingredient.

The present invention provides an anti-inflammatory, anti-oxidant and whitening food or health functional food composition containing a fermented ginger flower extract manufactured by the above manufacturing method as an effective ingredient.

The cosmetic composition of the present invention may have any one formulation selected from the group consisting of cream, emollient toner, nourishing toner, pack, essence, hair tonic, shampoo, rinse, hair conditioner, hair treatment, gel, skin lotion, skin softener, skin toner, astringent, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, nourishing essence, sunscreen, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and body cleanser, but is not limited thereto. The cosmetic composition composed of each of these formulations may contain various bases and additives necessary and appropriate for formulating the formulation, and the types and amounts of these ingredients can be easily selected by those skilled in the art.

The food composition of the present invention may be in the form of a pill, powder, granule, infusion, tablet, capsule or liquid, and there is no particular limitation on the type of food to which the ginger flower fermented extract or the fraction thereof of the present invention can be added, and examples thereof include various beverages, gum, tea, vitamin complexes, health supplements, health functional foods, etc. In addition to the ginger flower fermented extract or the fraction thereof, other ingredients may be added to the food composition, and the types thereof are not particularly limited. For example, like conventional foods, various herbal extracts, food-wise acceptable food additives or natural carbohydrates may be included as additional ingredients, but are not limited thereto.

The above "food supplement additives" are components that can be added to foods as auxiliary components, can be added when manufacturing health functional foods of each formulation, and can be appropriately selected and used by those skilled in the art. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but the types of food supplement additives of the present invention are not limited by the above examples.

Examples of the above "natural carbohydrates" include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. In addition to the above, natural flavoring agents (thaumatin, etc.), stevia extracts (rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used as flavoring agents.

The present invention relates to an anti-inflammatory, anti-oxidant and skin-whitening composition that is differentiated from existing compositions, and can be utilized as a cosmetic composition, a food composition, a health functional food composition, etc., having improved anti-inflammatory, anti-oxidant and skin-whitening effects by containing a fermented ginger flower extract as an effective ingredient.

FIG. 1 is a graph schematically illustrating a process for manufacturing a fermented ginger flower extract according to one embodiment of the present invention (A), and showing the results of measuring (B) the proliferation of lactic acid bacteria and (C) the pH level.
Figure 2 is a graph showing the DPPH measurement results of a fermented ginger flower extract according to one embodiment of the present invention.
Figure 3 is a graph showing the ABTS measurement results of a fermented ginger flower extract according to one embodiment of the present invention.
Figure 4 is a graph showing the FRAP measurement results of a fermented ginger flower extract according to one embodiment of the present invention.
Figure 5 is a graph showing the TPC measurement results of a fermented ginger flower extract according to one embodiment of the present invention.
Figure 6 is a graph showing the results of TFC measurement of a fermented ginger flower extract according to one embodiment of the present invention.
Figure 7 shows the DPPH optimization measurement results (DPPH scavenging rate (%)) in ginger flower fermentation according to one embodiment of the present invention.
A: Glucose (g); B: Pepton (g); C: Yeast extract (g); D: L. obtusiloba powder (g)
Figure 8 shows the DPPH optimization measurement results (DPPH scavenging rate yield (%/g)) compared to raw materials in ginger flower fermentation according to one embodiment of the present invention.
A: Glucose (g); B: Pepton (g); C: Yeast extract (g); D: L. obtusiloba powder (g)
Figure 9 shows the results of β-glucosidase optimization measurement (β-glucosidase activity (unit)) in ginger flower fermentation according to one embodiment of the present invention.
A: Glucose (g); B: Pepton (g); C: Yeast extract (g); D: L. obtusiloba powder (g)
Figure 10 is a graph showing the results of a cytotoxicity analysis of a fermented ginger flower extract according to an embodiment of the present invention against B16F10 cells.
Figure 11 is a graph showing the results of confirming the inhibition of melanin production by a fermented ginger flower extract according to one embodiment of the present invention.
Figure 12 is a graph showing the results of measuring the inhibition of melanin production enzyme of a fermented ginger flower extract according to one embodiment of the present invention.
Figure 13 is a graph showing the cytotoxicity and NO measurement results for RAW 264.7 cells of a fermented ginger flower extract according to one embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to the attached drawings. These embodiments are only intended to explain the present invention more specifically, and it will be apparent to those skilled in the art to which the present invention pertains that the scope of the present invention is not limited by these embodiments according to the gist of the present invention.

The features of the present invention can be more clearly explained through the following examples.

[Example 1] Method for producing fermented ginger flower extract

The fermentation strains used in the present invention were Lactiplantibacillus paracasei KCTC 3074, Lactiplantibacillus casei KCTC 3109, Lactococcus lactis KCTC 3115, Lactiplantibacillus rhamnosus KCTC 3237, and Lactiplantibacillus plantarum KCTC 3107 distributed by KCTC (Korea). Washed and dried ginger flowers were purchased from Kkotchasidae (Korea) and used. Ginger flower extract (NF) was obtained by precipitating 1 g of dried Ginger flower in 100 mL of a 10-fold diluted solution of MRS broth (peptone (10 g/L), beef extract (10 g/L), yeast extract (5 g/L), glucose (20 g/L), sodium acetate (5 g/L), ammonium citrate (2 g/L), potassium phosphate (2 g/L), magnesium sulfate (0.1 g/L), manganesum sulfate (0.05 g/L)), inoculating 1 mL of the pre-cultured strain based on OD 0.7, culturing for 24 h at 37°C, and then filtering through No. 2 filter paper (whatman, UK).

Hereinafter, the fermented ginger flower extracts manufactured by fermenting each of the five types of lactic acid bacteria in the present invention are indicated by the KCTC numbers of the lactic acid bacteria.

In the present invention, the fermented ginger flower extract was prepared as shown in Fig. 1A.

As shown in Fig. 1B and Fig. 1C, the growth of lactic acid bacteria and the pH level were checked for a total of 24 hours at 3-hour intervals. Based on OD600 nm, the growth rate of bacteria was the highest in group 3107, followed by group 3237, and the remaining groups also showed growth. Except for group NF, the pH level was close to 4 in all groups, confirming that fermentation was properly performed.

[Experimental Example 1] Measurement of antioxidant activity of fermented ginger flower extract

DPPH assay measurement

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay for measuring antioxidant capacity was performed by modifying the method of a previous study to a level suitable for the experimental conditions of this study. DPPH (Sigma, USA) was dissolved in 95% ethanol and used as a DPPH solution with a final absorbance of 0.5 at 540 nm. 0.18 mL of DPPH solution and 0.02 mL of fermented ginger flower extract were dispensed into a 96-well plate, reacted for 30 minutes, and then the absorbance at 540 nm was measured using a microplate reader (synergy HT, Biotek, USA).

The results of measuring the antioxidant activity of NF and fermented extracts against DPPH are shown in Fig. 2. In the case of the NF group, it can be seen that the antioxidant activity increases as the extraction time increases. On the other hand, the 3107 group showed a higher antioxidant activity than the NF group up to 3 hours of fermentation, but showed a gradual decreasing trend thereafter. At 3 hours of fermentation, the 3115 and 3109 groups showed higher antioxidant activity than the NF group, but at 24 hours of fermentation, only the 3115 group showed a significantly higher ( p < 0.001) antioxidant activity.

ABTS assay measurement

2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay was performed by modifying the method of a previous study to a level suitable for the conditions of this experiment. ABTS (Sigma, USA) was dissolved in distilled water as a solvent at a concentration of 7.4 mM, and ammonium persulfate was added to make 2.45 mM and the color was developed for 8 hours. It was then diluted to a final absorbance of 1.0 at 740 nm before use. 0.19 mL of the pre-diluted ABTS solution and 0.01 mL of the fermented ginger flower extract were dispensed into a 96-well plate, reacted for 30 minutes, and the absorbance at a wavelength of 740 nm was measured using a microplate reader.

The results of measuring the antioxidant activity of NF and fermented extracts for ABTS in the present invention are as shown in Fig. 3. In the case of the NF group, it can be seen that the antioxidant activity increases as the extraction time increases. Meanwhile, the 3107 group showed a higher antioxidant activity than the NF group up to 6 hours of fermentation, but showed a gradual decreasing trend thereafter. At 3 hours of fermentation, the 3074, 3107, 3109, 3115, and 3237 groups showed higher antioxidant activity than the NF group, but at 24 hours of fermentation, the 3074 and 3115 groups showed significantly higher ( p < 0.01, p < 0.001) antioxidant activities than the NF group.

Both DPPH and ABTS showed an increase in antioxidant activity in groups 3115 and 3107 at 3 hours of fermentation, but only group 3115 showed an increase at 24 hours. In general, enzymes such as β-glucosidase, cellulase, pectinase, and lignin-degrading enzyme produced during the fermentation process help to decompose the cell walls of plants, thereby facilitating the release of various effective ingredients. Among them, β-glucosidase increases activity by decomposing the bond with sugar in substances such as quercetin glycoside. However, enzymes that decompose antioxidant substances are produced, and a decrease in antioxidant activity may occur due to active oxygen generated during the metabolic process. In light of this, the reason why the antioxidant activity of group 3107 began to decrease at 6 hours of fermentation when the strains had sufficiently grown is thought to be due to a decrease in antioxidant activity caused by decomposing enzymes and active oxygen produced in the strain.

FRAP assay measurement

Ferric ion reducing antioxidant power (FRAP) assay was performed based on previous studies. Sodium acetate and acetate buffer were mixed to prepare sodium acetate buffer (30 mM, pH 3.6), and 40 mM HCl and TPTZ (2,4,6-tripyridyl-s-triazine) were mixed to prepare a 10 mM TPTZ solution. The reaction solution was prepared by mixing sodium acetate buffer, 10 mM TPTZ solution, and FeCl ₃ (20 mM in DW) in a volume ratio of 10:1:1, and incubating and preheating at 37°C for 15 minutes to prepare the FRAP solution. 10 μL of the sample and 190 μL of the FRAP solution were dispensed into a 96-well plate, reacted at 37°C for 15 minutes, and the absorbance was measured at 560 nm. At this time, FRAP activity was measured using ascorbic acid.

FARP is known as an antioxidant power that can be measured through absorbance of a colored ferrous tripyridyl triazine complex obtained by reducing ferric ion to ferrous by an antioxidant present in the sample.

As shown in Figure 4, it was confirmed that the value gradually increased as the fermentation time passed, similar to other antioxidant values, and in particular, the value of 3237 was significantly higher than other groups in proportion to the fermentation time, followed by 3109 and 3115, which were significantly higher ( p < 0.01, p < 0.001).

Total phenolic contents (TPC) and total flavonoid contents (TFC) assay measurement

The TPC assay was performed based on a previous study method. 2N Folin-ciocalteu reagent (phenol reagent, Junsei, Japan) and 10% Na ₂ CO ₃ were used in the experiment. 0.02 mL of Folin-ciocalteu reagent and 0.16 mL of diluted sample were mixed, and the reaction was waited for 15 minutes. Then, 0.02 mL of 10% Na ₂ CO ₃ was additionally mixed to develop the color. 200 μL of the color-developed sample was transferred to a 96-well plate, and the absorbance at a wavelength of 740 nm was measured using a microplate reader, and the value was converted based on gallic acid.

The TFC assay was performed based on the method of a previous study. 0.15 mL of 10% AlCl ₃ and 0.05 mL of diluted sample were mixed and reacted for 15 minutes. 200 μL of the sample after reaction was transferred to a 96-well plate, and the flavonoid concentration of the sample was measured by measuring the absorbance at a wavelength of 450 nm using a microplate reader. The results were converted based on quercetin.

Phenolic substances are substances found in various organisms and scavenge radicals such as active oxygen. On the other hand, flavonoids belong to phenolic substances, are found in plants, and are known to play an important role in the antioxidant capacity of plants. The results of measuring phenolic substances and flavonoids in NF and fermented extracts are as shown in Figures 5 and 6.

In the measurement of phenolic substances, it was found that phenolic substances increased in the NF group as the extraction time increased. All fermented groups showed an overall increase and showed higher concentrations than the control group. At 3 hours of fermentation, all showed higher antioxidant activity than the NF group, and at 24 hours of fermentation, all lactic acid bacteria fermented groups showed significantly higher ( p < 0.001) concentrations. In the measurement of flavonoids, it was found that phenolic substances increased in the NF group as the extraction time increased. On the other hand, the 3107 group showed a higher flavonoid concentration than the flower extract until 3 hours of fermentation, but thereafter, there was no significant difference from the NF group. At 3 hours of fermentation, groups 3107, 3109, 3115, and 3237 showed significantly higher ( p < 0.01, p < 0.001) antioxidant activity than the NF group, and at 24 hours of fermentation, groups 3074, 3109, 3115, and 3237 showed significantly higher ( p < 0.001) flavonoid concentrations.

In summary, the lactic acid bacteria L. lactis KCTC 3115 further enhanced the antioxidant activity of ginger flower through the fermentation process, and surprisingly, the plant-derived strain L. plantarum KCTC 3107 showed a lower antioxidant activity level than the other strains through the fermentation process of ginger flower. Antioxidant activity is known to have a cell protective effect and is related to anti-inflammation, aging, and skin pigmentation inhibition by suppressing oxidative stress. Based on the results to date, L. lactis KCTC 3115 is likely to be the lactic acid bacteria that can maximize the whitening effect. Therefore, based on the antioxidant activity level, we decided to verify the ginger flower fermentation conditions and confirm the anti-inflammation and whitening effects through 3115.

[Experimental Example 3] Optimal fermentation conditions for ginger flowers

Optimization of Ginger Flower Fermentation

Minitab (Minitab, USA) was used for response surface analysis. Experimental design was performed using Box-Behnken, and changes in DPPH scavenging activity, antioxidant yield, and β-glucosidase activity according to the concentration of glucose, peptone, yeast extract, and ginger flower were measured. Based on this, the function between variables and responses was estimated and optimized.

The results of optimizing ginger flower fermentation using response surface analysis are shown in Table 1 and Figs. 7 to 9. The culture solution was prepared according to the conditions of [Table 1], and 100 mL of distilled water was added with A-D corresponding to each row, and the pre-cultured strain was inoculated with 1% lactic acid bacteria pre-culture solution based on O.D 0.7, and cultured for 24 hours in an environment of 37℃, and then DPPH scavenging activity (A) and β-glucosidase (C) activity were measured. The yield was determined by adding 100 mL of distilled water with A-D corresponding to each row, inoculating 1 mL of the pre-cultured strain based on O.D 0.7, and cultured for 24 hours in an environment of 37℃, measuring DPPH, and then measuring the yield (B) based on the raw material ginger flower. The optimization results were as follows.

Result (A) = 35.6 - 4.4 A - 6.1 B - 7.9 C + 19.08 D + 2.16 A ² - 0.3 B ² - 4.8 C ² - 1.873 D ² + 2.05 A×B - 1.0 A×C - 3.42 A×D + 5.7 B×C - 0.27 B×D + 6.24 C×D

Result (B) = 102.7 - 14.5 A + 3.0 B - 12.6 C - 38.42 D + 4.09 A ² - 3.38 B ² - 2.2 C ² + 4.141 D ² + 0.75 A×B -0.4 A×C + 0.86 A×D + 2.1 B×C + 0.32 B×D + 4.81 C×D

Result (C) = 102.67 - 14.5 A + 3.0 B - 12.6 C - 38.42 D + 4.09 A ² - 3.38 B ² - 2.2 C ² + 4.141 D ² + 0.75 A×B - 0.4 A×C + 0.86 A×D + 2.1 B×C + 0.32 B×D + 4.81 C×D

The model showed p value = 0.000, R2 = 0.9509. The optimal conditions for A were glucose 0 g, peptone 0 g, yeast extract 0.5 g, and ginger flower 5 g, the optimal conditions for B were glucose 0 g, peptone 0.4646 g, yeast extract 0 g, and ginger flower 0.5 g, and the optimal conditions for C were glucose 0 g, peptone 2 g, yeast extract 0.1010 g, and ginger flower 5 g. The addition of glucose and yeast extract did not help the fermentation of ginger flower. However, the antioxidant value changed depending on the amount of peptone added, and the optimal DPPH activity compared to the amount of raw material was shown under the condition where 0.4646 g of peptone was added. Based on this, a re-experiment was conducted under the corresponding conditions, and the optimal yield (B) compared to DPPH scavenging activity was 85.57±0.377%/g, which did not show a significant difference with p > 0.05. In addition, the re-experiment results of A and C were 94.04±0.696% and 0.2441±0.377 units, respectively, and did not show a significant difference with p > 0.05.

[Experimental Example 4] Measurement of the whitening effect of fermented ginger flower extract

B16F10 MTT assay

Before examining the whitening effect of the fermented ginger flower extract, the cytotoxicity of the fermented ginger flower extract on B16F10 (KCLB, Korea) cells was confirmed to set the experimental concentration. The fermented extract used here was 3115. First, to confirm the cytotoxicity on B16F10 cells, FBS (fetal bovine serum, Sigma, USA) was added to DMEM broth (Dulbecco's Modified Eagle Medium, GE healthcare, USA) so that the ratio of FBS was 5%. Penicillin-Streptomycin (100×) (Sigma, USA) was added as an antibiotic and used as a culture medium. The fermented ginger flower extract was diluted in DMEM broth and then sterilized through a 0.22 μm syringe filter (whatman, UK) and processed. B16F10 was pre-cultured using the above culture medium, detached from the dish using trypsin, and then 5.0×10 ³ cells were injected per well into a 96-well plate and cultured for 24 hours. The supernatant was removed, and DMEM containing various concentrations of ginger flower fermentation extract based on 100 μg/mL was dispensed, and cultured for 48 hours. After culture, the supernatant was removed, 100 μL of MTT solution (5 mg/mL) was added, and MTT was crystallized for 3 hours in an environment of 37℃ and _CO2 concentration 5%. After that, the supernatant was removed to prevent the crystals formed in each well from being removed, and the crystals were dissolved in DMSO and the absorbance at a wavelength of 540 nm was measured to confirm the cell viability.

B16F10 cell cytotoxicity

The results of measuring cell viability after treating B16F10 cells with the NF group and the 3115 group in this experiment are as shown in Fig. 10. As a result of the experiment, the NF group showed 85.3% ± 1.8% at 100 μg/mL, 91.3% ± 0.6% at 50 μg/mL, 95.6% ± 1.0% at 25 μg/mL, and 100.1% ± 0.7% at 12.5 μg/mL. The 3115 group showed 82% ± 1.6% at 100 μg/mL, 87.5% ± 1.1% at 50 μg/mL, 93.4% ± 0.4% at 25 μg/mL, and 96.8% ± 0.6% at 12.5 μg/mL. In this experiment, all concentrations showed less than 20% cytotoxicity according to the ISO 10993-5:2009 standard, so it can be judged as non-cytotoxic. Subsequent experiments were conducted at concentrations of 12.5-100 μg/mL, which were confirmed to be non-cytotoxic.

Extracellular melanin measurement

In order to investigate the whitening effect of the fermented ginger flower extract, an experiment was conducted using B16F10 cells. The cell culture medium was the same as that used in the MTT assay, and α-MSH was diluted in DMEM broth after sterilization through a 0.22 μm syringe filter (whatman, UK). B16F10 cells were pre-cultured using the above culture medium, detached from the dish using trypsin, and 5.0 × 10 ³ cells were injected per well into a 96-well plate and cultured for 24 hours. After removing the supernatant, DMEM containing α-MSH at a concentration of 100 nM and various concentrations of the fermented ginger flower extract based on 100 μg/mL was dispensed, and cultured for 48 hours. The culture supernatant was transferred to a new 96-well plate at 100 μL each, and the absorbance was measured at 475 nm. This was used as extracellular melanin and compared between the control and experimental groups.

The results of measuring the inhibition rate of melanin production by NF and 3115 are shown in Figs. 11A and 11B. As confirmed with the naked eye in Fig. 11A, the difference in melanin production in the 3115 group could be confirmed compared to the MSH group in which melanization was induced by treating B16F10 cells with α-MSH. As a result of the experiment, the NF group showed 74.9% ± 1.2% at 100 μg/mL, 79.0% ± 1.0% at 50 μg/mL, 89.7% ± 1.8% at 25 μg/mL, and 96.2% ± 1.4% at 12.5 μg/mL. For group 3115, the results were 75% ± 1.4% at 100 μg/mL, 68.8% ± 1.7% at 50 μg/mL, 77.6% ± 0.9% at 25 μg/mL, and 90.7% ± 0.8% at 12.5 μg/mL. In this experiment, all groups except the NF group treated with a concentration of 12.5 μg/mL were compared to the MSH group, and showed significant differences ( p < 0.001). The melanin production inhibition rate decreased in proportion to the concentration. Based on this, it was confirmed that the NF and 3115 groups had a whitening effect, and at the same time, it was confirmed that the whitening effect statistically significantly increased through fermentation ( p < 0.001).

Tyrosinase measurement: Measurement of melanin-producing enzyme

The cells were cultured for 48 hours in the same manner as for measuring extracellular melanin. After the culture was completed, 100 μL of RIPA buffer was treated to each cultured cell to prepare a cell lysate, which was used for measurement. The concentration of tyrosinase was measured using an ELISA kit (MyBioSource, USA).

The results of measuring the inhibition rate of tyrosinase production, a melanin-producing enzyme, by ginger flower extract and fermented extract are as shown in Fig. 12. As a result of the experiment, the inhibition rate of tyrosinase production in the NF group was 92.5% ± 1.5% at 100 μg/mL, 96.1% ± 1.6% at 50 μg/mL, 99.3% ± 1.2% at 25 μg/mL, and 100.1% ± 0.6% at 12.5 μg/mL. In the case of the 3115 group, the inhibition rates were 74.5% ± 1.4% at 100 μg/mL, 85.7% ± 2.2% at 50 μg/mL, 95.6% ± 1.7% at 25 μg/mL, and 99.3% ± 1.6% at 12.5 μg/mL. There was a significant difference between the NF group at a concentration of 50 μg/mL and the 3115 group at concentrations of 50 μg/mL and 100 μg/mL ( p < 0.001), and tyrosinase was shown to decrease in proportion to the concentration.

Tyrosinase is an enzyme that is directly involved in inducing melanin by producing melanocytes in the skin epidermis, and melaninization progresses when tyrosinase is produced. Overall, 3115 effectively inhibited the production of tyrosinase compared to NF in proportion to its concentration. In addition, these results confirmed that the inhibition of tyrosinase production is one of the mechanisms of the whitening effect of NF and 3115, and at the same time, it was confirmed that the whitening effect was statistically significantly increased ( p < 0.001) through fermentation.

[Experimental Example 5] Measurement of the anti-inflammatory effect of fermented ginger flower extract

To investigate the anti-inflammatory response of NF and 3115, cytotoxicity and NO production rate on RAW 264.7 cells were measured.

RAW 264.7 MTT assay

To check cytotoxicity on RAW 264.7 (KCLB, Korea) cells, DMEM broth was added with 11% FBS and 1% antibiotics. When the pre-culture of the cells was completed, the cells were harvested and seeded into a 96-well plate at 1 × 10 ⁴ cells/well and cultured for 24 hours to allow the cells to attach to the plate. After confirming cell attachment, the main culture was treated with 10 μg/mL of LPS (lipolysaccharide) and each concentration of ginger flower extract, and cultured for 24 hours. After the incubation was completed, the supernatant was removed and 20 μL of MTT solution (5 mg/mL) was dispensed and left in a dark environment at 37℃ and _CO2 concentration of 5% for 2 hours to induce crystallization of MTT. After that, the supernatant of the MTT solution was removed, redissolved in DMSO, and the absorbance was measured at a wavelength of 560 nm.

RAW 264.7 cell cytotoxicity measurement

The cell viability for RAW 264.7 cells is as shown in Fig. 13A. The NF group showed 88.2% ± 0.3% at 100 μg/mL, 91.1% ± 0.8% at 50 μg/mL, 95.7% ± 0.5% at 25 μg/mL, and 99.0% ± 0.7% at 12.5 μg/mL. In the case of the 3115 group, the results were 85.2% ± 0.5% at 100 μg/mL, 89.0% ± 0.6% at 50 μg/mL, 92.6% ± 0.6% at 25 μg/mL, and 95.0% ± 1.2% at 12.5 μg/mL. The above experimental results showed that there was no cytotoxicity for all concentrations of NF and 3115.

Measurement of nitric oxide (NO) production rate

The culture supernatant was transferred to a new 96-well plate at a 100 μL volume, reacted with Griess reagent (0.2% N-(1-naphthyl) ethyl-enediamine dihydrochlorided and 2% sulfanilamide in 5% phosphoric acid, mixed at a 1:1 ratio), and the absorbance was measured at 540 nm. This was set as nitric oxide (NO), and the control and experimental groups were compared.

As shown in Fig. 13B, the NO production rates were 69.4±1.0% at 100 μg/mL, 76.6±0.7% at 50 μg/mL, 84.3±2.5% at 25 μg/mL, and 91.3±1.2% at 12.5 μg/mL in the NF group. In the 3115 group, the rates were 65.8±1.3% at 100 μg/mL, 73.1±1.3% at 50 μg/mL, 83.3±1.4% at 25 μg/mL, and 87.4±3.0% at 12.5 μg/mL. NO is produced in large quantities when macrophages are stimulated with LPS or IFN-γ (interferon-γ) by the expression of iNOS (inducible NO synthase). NO produced in such large quantities acts as a mediator of inflammatory response. In the above experiment, the NF group and the 3115 group suppressed NO production proportionally to the concentration, and in particular, compared to the LPS group that induced NO production, the NF group and the 3115 group significantly ( p < 0.01, p < 0.001) suppressed it at all concentrations.

While the specific parts of the present invention have been described in detail above, it should be clear that these specific descriptions are merely preferred implementation examples for those skilled in the art, and that the scope of the present invention is not limited thereto. The terms or words used in this specification and claims should not be interpreted as being 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 own invention in the best way. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (5)

A step of precipitating dried ginger flowers in a 10-fold diluted solution of MRS broth consisting of glucose, peptone, yeast extract, beef extract, sodium acetate, ammonium citrate, potassium phosphate, magnesium sulfate and manganese sulfate; and
A method for producing a fermented ginger flower extract exhibiting anti-inflammatory, antioxidant and skin whitening effects, characterized by producing the extract by inoculating Lactococcus lactis KCTC 3115 as a fermentation strain into the solution of the above precipitation step and fermenting ginger flower. In paragraph 1,
A method for producing a fermented ginger flower extract exhibiting anti-inflammatory, antioxidant, and skin whitening effects, characterized by inoculating the pre-cultured strain in the above fermentation step at a volume ratio of 1% based on an OD of 0.7 and then culturing at 37°C for 24 hours to ferment the ginger flower. A cosmetic composition exhibiting anti-inflammatory, anti-oxidant and skin whitening effects, containing a fermented ginger flower extract as an active ingredient, characterized in that it is manufactured by the manufacturing method of any one of claims 1 or 2. A food composition exhibiting anti-inflammatory, anti-oxidant and skin whitening effects, containing a fermented ginger flower extract as an active ingredient, characterized in that it is manufactured by the manufacturing method of any one of claims 1 or 2. A health functional food composition exhibiting anti-inflammatory, anti-oxidant and skin whitening effects, containing a fermented ginger flower extract as an active ingredient, characterized in that it is manufactured by the manufacturing method of any one of claims 1 or 2.