KR101591816B1 - Manufacturing method of gastrodia elata blume fermentation extracts and functional materials thereof - Google Patents

Abstract

The present invention relates to a method for producing a fermented extract of Gastrodia elata and a functional material using the same. To this end, the production method comprises the following steps: producing an extract of Gastrodia elata by solvent-extracting Gastrodia elata; and fermenting the extract of Gastrodia elata by using a strain containing at least one or two strains selected from an effective microorganism group or a lactobacillus. Thus, the content of effective components such as flavonoid and polyphenol increases, and the method also offers excellent advantages in terms of 2,2-diphenylpicrylhydrazyl (DPPH) radical scavenging ability, reactive oxygen species inhibition, and cell viability.

Description

Technical Field [0001] The present invention relates to a fermented milk extract,

The present invention relates to a method for producing a fermented Chunmei extract and a functional material using the same.

Gastrodia elata blume is a perennial herb that grows in the deep mountains of our country. Growing environment grows in rotten place where oak tree collapses in moisturey stone gap, shaded area or semi-shade. Most of the components of the horse chestnut include phenolic compounds such as Gastrodin and non-phenolic compounds such as p-hydroxybenzylalcohol, pasishin, p-hydroxybenzyloxymethyl, phnol glucose, vanillyl alcohol, triterpene and circium aldehyde. Fresh chunmeans have a disadvantage of having a very high flavor, a heterogeneous smell and a short shelf-life, and most of the studies have been used to increase the amount of raw materials, and to produce dried, extracted and powdered products. Studies on various parts such as extractive optimization method of effective ingredients of chunmein, analysis and change of composition of chunmein according to drying difference, antioxidant activity and compositional change of chunma extract and fermented product of chunma, antioxidant and anticancer effect of extract , And it is widely used in food fields such as Chunmahwan using Gunma, sponge cake added with Gunma powder, and Chunma beverage.

Fermentation refers to the process of decomposing organic matter using enzymes of microorganisms. The fermentation process, which is the oldest technology in history, has been widely used in various fields such as food, medicine, and cosmetics. Among them, food fermentation is most widely used not only in Korea but also in the whole world. In the case of food fermentation, processing is the traditional way of fermenting food's enzymes through the action of microorganisms. zest. It is known that it has the effect of improving the texture, destroying the toxic substance through the fermentation process, and promoting the physiologically active substance.

Korean Patent Publication No. KR2013-0026123A discloses a fermented chestnut fermented extract produced by fermenting a horse mackerel with a probiotic strain and materials of various chewing gum functional products using the same.

Korean Patent Registration No. KR1494592B1 discloses a fermented product of Chumma and a fermentation method thereof in which the fermentation of chimma is carried out in a solid form to increase the extraction yield and physiologically active ingredient.

These techniques are intended to maximize the effective ingredients of chimaese and to maximize the effective ingredients or the effective effects generated by them through fermentation. However, the efficiency of extracting and activating the intrinsic effective ingredients of chimaera itself is still low .

Therefore, there is a need for a manufacturing method capable of extracting intrinsic effective ingredients of the TMM itself, activating and maximizing it, and a method applicable to various fields as functional materials without deteriorating the above components.

Korean Patent Publication No. KR2013-0026123A Korean registered patent KR1494592B1

It is an object of the present invention to provide a method for producing fermented Chamae plum extract capable of activating and maximizing intrinsic effective ingredients of Chromosome itself at high concentration and a functional material which can be applied to various fields while maintaining the above components intact.

The present invention relates to a method for fermenting a chamois extract comprising the steps of: a) preparing a chewing gum extract by solvent extraction of a chewing gum, and b) fermenting the chewing gum extract with a strain comprising any one or more selected from the group consisting of useful microorganisms or lactic acid bacteria And a method for producing the fermented extract.

In one embodiment of the present invention, the method may further include a step of filtering before step b), a step of concentrating under reduced pressure, or a step of concentrating under reduced pressure after filtration.

In one embodiment of the present invention, the method may further include lyophilizing.

The fermented Chumma extract prepared by the production method according to the present invention can be used as an external preparation for skin.

The fermented Chumma extract prepared by the method of the present invention can be used as a functional material.

The fermented Chumma extract prepared by the fermentation method of the present invention has an advantage of being able to contain active ingredients such as flavonoids and polyphenols at a high concentration and to activate and maximize them.

In addition, the fermented Chumma extract prepared by the method of the present invention for fermentation of the fermented Chumma extract has the advantages of increasing active ingredients such as flavonoids and polyphenols, and also having excellent properties such as DPPH radical scavenging ability, active oxygen species suppression, and survival rate.

The fermented Chumma extract prepared by the method of the present invention can be applied as functional materials in various fields without deteriorating the characteristics.

1 is a graph showing the results of measurement of the DPPH radical scavenging ability according to Examples 1 to 6 by modifying the method of Biois et al.
2 is a graph showing the results of measurement of the DPPH radical scavenging ability according to Examples 1 to 6 by modifying the method of Biois et al.
FIG. 3 is a graph showing the results of measurement of cell viability in RAW 264.7 cells (Mosmann) according to Example 1, Examples 3 to 5, Comparative Examples 1 and 3 on the basis of normal cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be described in detail with reference to the accompanying drawings.

Where a drawing is described, it is provided as an example to enable those skilled in the art to fully understand the spirit of the invention. Therefore, the present invention is not limited to the illustrated drawings, but may be embodied in other forms, and the drawings may be exaggerated in order to clarify the spirit of the present invention.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

Also, units of% used unclearly herein, unless otherwise specified, means weight.

According to the present invention,

a) a step of preparing chondroma extract by solvent extraction of a horse mackerel, and

b) fermenting the chimpanzee extract with a strain comprising any one or two or more selected from the group consisting of useful microorganisms or lactic acid bacteria

And a method for producing the fermented milk fermentation extract.

In one embodiment of the present invention, the method for preparing a fermented milk fermentation extract may further include drying the chymase before the step a). As a specific but nonlimiting example, it is possible to exemplify the step of drying at 40 to 70 DEG C for 10 to 60 minutes and then the second drying at 10 to 35 DEG C for 2 to 10 hours. Drying at a high temperature may be efficient in terms of time, but if exposed to a high temperature for a long time, physical / chemical denaturation may occur, and drying at a lower temperature in a multistage manner may prevent degradation or denaturation of active ingredients such as polyphenols And may be preferable.

In one embodiment of the present invention, the solvent is not limited as long as the object of the present invention is achieved. However, the solvent may include any one or two or more selected from water, ethanol, and the like. When the above range is satisfied, the content of active ingredient such as flavonoid, polyphenol and the like can be further increased.

In one example of the present invention, the solvent is not limited to achieve the object of the present invention, but may include, for example, from 180 to 280 parts by weight of ethanol relative to 100 parts by weight of water. When the above range is satisfied, the content of the active ingredient such as flavonoid can be remarkably increased.

In one embodiment of the present invention, the solvent extraction method in step a) is not limited to achieve the object of the present invention, but may be, for example, a reflux cooling method. The reflux cooling method is an extraction method using a structure in which a vapor such as a solvent is cooled in a vertical or inclined position and the condensate is returned to the distillation flask. As a specific but non-limiting example, the sample is placed in a filter paper and inserted into an intermediate tube, then the solvent is added to the bottom flask and heated to vaporize the solvent. The vapors rise up, are condensed by a reflux condenser, and the condensed solvent again falls on the filter paper to produce an extract containing the sample component. By such a reflux cooling method, it is possible to prepare a chewing gum extract which can extract chymus and increase contents of effective components such as flavonoids and polyphenols.

In one embodiment of the present invention, in the step a), when the reflux cooling method is used, water and ethanol may be independently arranged and injected into the sample at the same time. The aqueous ethanol solution containing water and ethanol can be used as it is. However, a specific but non-limiting example is a system in which water and ethanol are separately supplied to the sample (chimma) Is added to the intermediate tube in which the sample is placed at the same time, it is possible to produce a high concentration of the Ganoderma extract having an increased content of effective components such as flavonoids and polyphenols. As a specific, but non-limiting example, the input flow rate ratio of steam to water and ethanol, respectively, is not limited to achieve the object of the present invention, but may be, for example, 1: 1.8 to 2.8.

In an example of the present invention, step (a) is not limited as far as the object of the present invention is achieved. For example, 0.01 to 1,000 parts by weight, preferably 0.1 to 500 parts by weight, And 1 to 100 parts by weight of a solvent.

In one embodiment of the present invention, the step a) is not limited as far as the object of the present invention is achieved. For example, the step a) may include a step of solvent extraction at 1 to 50 times based on the horse root and the solvent.

In one embodiment of the present invention, the step a) is not limited to achieve the object of the present invention, but may include, for example, extraction at 30 to 90 ° C for 4 to 12 hours.

In one embodiment of the present invention, the step b) is not limited to achieve the object of the present invention, but may include, for example, fermentation at 20 to 45 캜 for 1 to 12 days.

In one example of the present invention, the step b) is not limited to achieve the object of the present invention, but may include, for example, fermentation at a relative humidity of 50 to 90%.

In one embodiment of the present invention, when the fermentation is carried out using the lactic acid bacterium, the step b) is not limited to attain the object of the present invention. For example, the fermentation may be performed by inoculating the strain on MRS (de Man, Rogosa and Sharpe) And fermenting the chymus extract with the cultured lactic acid bacteria. As a specific but non-limiting example, the content of the cultured lactic acid bacteria may be 0.001 to 10% by weight based on the total weight. However, this is a preferable example, but the present invention is not limited thereto.

In one embodiment of the present invention, the fermentation using the cultured lactic acid bacterium may include, for example, fermentation for 0.5 to 5 days, although the present invention is not limited thereto.

In one embodiment of the present invention, the strain of step b) is not limited to achieve the object of the present invention, but may be more preferably an effective microorganisms (EM). The useful microorganism group is a combination of several microorganisms beneficial to humans among many microorganisms existing in nature, and / or cultivated. The useful microorganism group is made by cultivating 80 kinds of microorganisms beneficial to human and environment. As a specific but non-limiting example, a stock solution of beneficial microorganism group (seed bacterium) can be used, and EM activated solution containing useful microorganism group can be used.

In one embodiment of the present invention, the step b) is not limited to achieve the object of the present invention, but may include, for example, 0.1 to 100 parts by weight of the EM activated solution per 100 parts by weight of the extract of Chimpanzee extract. And the like. As a specific but non-limiting example, it may be preferable to control the amount of the EM activated solution and the amount of the sugar to be initially introduced, as the fermentation time progresses, since the fermentation can proceed efficiently. However, this is a preferable example, but the present invention is not limited thereto.

In one embodiment of the present invention, the method for preparing a fermented milk fermentation extract may further comprise drying the chymus before the step a), as described above, wherein the step of dewatering and drying with a certain amount of sugar is performed . As a specific but non-limiting example, 10 to 200 parts by weight of the powdery sugar per 100 parts by weight of the horse chestnut is mixed with the chewing gum, followed by dehydration and drying.

In one embodiment of the present invention, the sugar is not limited in the step of dewatering and drying, but it may be preferable to be in powder form. For example, in the case of a liquid sugar, since the intended drying itself may be difficult, the sugar contained in the powdered sugar may be discharged to the outside and sugar content may be contained therein.

In one example of the present invention, the dehydrating and drying step is not limited to achieve the object of the present invention, but it may be preferable to dry it in a vibration or flow state, for example. Since the powdery sugar containing water in the process of dehydration may not be dried properly by a general method, a physical force is applied by a gas such as air using a vibrating or flowing state such as a fluid bed dryer By drying and widening the space between the sugar particles and the particles, it is possible to minimize drying of the active ingredient within a shorter period of time.

As described above, when the fermentation is carried out into the useful microorganism group using the above-mentioned Chunmei extract which has been subjected to the dehydration and drying step using the powdery sugar, a fermentation step is carried out using a lesser amount of sugar, And fermentation can proceed more efficiently. Therefore, it is possible to produce a fermented Chamai extract having enhanced potency of flavonoids, polyphenols and the like.

In one example of the present invention, the sugar is not limited, and various components other than the sugar can be used as long as the useful microorganism group is available for metabolism.

The useful microorganism group is a useful microorganism having excellent antioxidant ability, revitalization power, and purification ability. Especially, when used for fermentation of Chumoma extract, the resulting fermented product contains various organic products having antioxidative function, And metabolic activities, thereby increasing the content of active ingredients such as flavonoids and polyphenols, as well as improving the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging ability. That is, free radicals in the body react with lipids, proteins, and the like to accelerate aging of a living body, and it is possible to produce fermented Chumma extract having excellent radical scavenging properties.

In one embodiment of the present invention, the step (a) may include, for example, fermentation for 3 to 10 days, when the fermentation is carried out using the useful microorganism group, but the present invention is not limited thereto.

In one embodiment of the present invention, when the fermentation is carried out using the group of useful microorganisms, the step a) is not limited to the achievement of the object of the present invention, but includes a step of operating in a low vacuum state of 0.2 to 0.6 atm . When the fermentation environment is not appropriate, the probability of contamination from germs and the like increases due to factors such as the external environment, The fermentation may not proceed smoothly or aging. Therefore, fermentation or aging in an appropriate vacuum state can minimize the contamination of germs and the like caused by excessive oxygen inflow, and can minimize the problem caused by poor metabolism of aerobic microbes due to hypoxic environment.

In one embodiment of the present invention, the method for preparing a fermented milk fermented extract may further include one or more selected from the steps of grinding the chitin or sterilizing the chitin before the step a).

In one embodiment of the present invention, the method for preparing a fermented milk fermentation extract may further include a step of filtering before step b), a step of concentrating under reduced pressure, or a step of concentrating under reduced pressure after filtration.

In one embodiment of the present invention, the method for preparing a fermented milk fermentation extract may further include any one or two or more selected from aging in step b) or lyophilizing.

In one embodiment of the present invention, the aging step is not limited to achieve the object of the present invention, but may include, for example, a low temperature aging step. As a specific but non-limiting example, it may be a step of aging at a low temperature of 0 to 15 캜, preferably 2 to 10 캜. When the fermentation is carried out at a low temperature under the above range, inhibition of harmful microorganisms can be minimized, so that normal fermentation can be induced, which is preferable.

In one embodiment of the present invention, the concentration of flavonoid in the fermented Chunmajara extract prepared by the fermented fermented milk production method is not less than 15 mg / g, May be 17 to 45 mg / g, more preferably 20 to 40 mg / g.

In one example of the present invention, the concentration of polyphenol in the fermented Chumma extract prepared by the fermented fermented milk production method is not less than 80 mg / g, G may be 85 to 140 mg / g, more preferably 89 to 130 mg / g.

In one embodiment of the present invention, the DPPH radical scavenging ability of the fermented Chumma extract prepared by the fermentation method of the present invention is not less than 25%, but the upper limit thereof is not limited, but is preferably 25 to 60%, more preferably 25 to 60% 50%. ≪ / RTI > The DPPH radical scavenging activity is the difference in absorbance at a wavelength of 517 nm between the sample addition group and the no addition group after inducing the reaction for 30 minutes at room temperature and light shielding condition at 20 to 30 DEG C as a percentage.

In one example of the present invention, the cell viability of RAW 264.7 cells (Mosmann) of the fermented Chunma extract prepared by the fermentation method of Chunma fermented extract was measured by using the normal cell as a reference and the difference in absorbance between the sample addition group and the no addition group as a percentage , Which may be 85% or more. Therefore, it is suitable for use as a bio-contact functional material such as a skin external preparation for cosmetics and the like.

In one example of the present invention, the fermented milk extract prepared according to the method of manufacturing a fermented milk fermentation extract is not limited to attain the object of the present invention, but can be applied to, for example, external preparation for skin. As specific and non-limiting examples, when the fermented milk extract is applied to an external preparation for skin, a melanin synthesis inhibitor, an anti-aging agent, a preservative, an isotonic agent, a stabilizer, a disinfectant, a wound healing agent, An antiseptic agent, an antimicrobial agent, a chelating agent, a surfactant, a foaming agent, a stabilizer, a penetrating agent, an antioxidant, an antioxidant, an antioxidant, an ultraviolet absorber, It may be applied together with any one or two or more selected from ingredients such as an emulsifier, an emulsifier, an emulsifier, a pressure-sensitive adhesive, an adhesive, a perfume and a pigment. In addition, the above components may be appropriately selected by a person having ordinary skill in the art to which the present invention belongs. In addition, various components for improving the physical properties of the external preparation for skin may be further applied.

In one example of the present invention, examples of the external preparation for skin include softening lotion, convergent lotion, nutritional lotion, baboon cream, eye cream, nutritional cream, massage cream, cleansing cream, ultraviolet screening cream, cleansing foam, And may be prepared and used in various formulations such as liquid, solid, powder, hydrogel, essence, spray, patch, ointment, lotion or pack. In addition, each of these formulations may contain various kinds of bases and additives necessary for the formulation of the formulation, and the kind and amount of these ingredients can be easily adjusted. Do not. Also, the method of manufacturing the above-described formulation is well known, and a known method may be used.

In one example of the present invention, the fermented Chumam extract prepared according to the method of manufacturing a fermented milk fermented extract can be applied not only to external preparations for skin but also to various functional materials such as food, herbal medicines, medicines, and life products (shampoo, soap, etc.).

Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples. However, the present invention is described in more detail with reference to the following Examples, but the scope of the present invention is not limited by the following Examples.

10 g of dried gum and 100 g of water as a solvent were put into a reflux condenser and then extracted 10 times at 60 DEG C for 8 hours. Impurities were removed from the obtained extract solution using a filter paper No. 2 (Advantec, TOYO), and then concentrated under reduced pressure using a rotary evaporator (N-1000SW L, Eyela). The resulting concentrate was stored in a cryogenic freezer (Deep freezer DE8514, Ilshin Lab co. Ltd., Korea) for one day, and then chitosan extract was prepared using a freeze dryer (Lyophilizer FD5508, Ilshin Lab co.

The mixture was adjusted to maintain a constant EM active solution (EM solution, EM bio) of 6% and sugar of 5% based on the total weight of the chymus extract, and incubated at 37 ° C for 7 days using an incubator (Wonil tech. Fermented EM fermented extract was prepared.

The procedure of Example 1 was repeated except that ethanol was used instead of water.

Except that a 70% by weight aqueous ethanol solution was used in place of the water of Example 1.

Lactobacillus casei KCTC 2180, Sahmyook University, Seoul, Korea) was inoculated in 10 ml of sterilized MRS medium and cultured at 37 ° C for 1 day. 0.1 ml of the cultured lactic acid bacterium and 10 ml of distilled water were mixed with 0.1 g of the chewing gum extract prepared in Example 1 and cultured at 37 ° C for 2 days using an incubator (Wonil tech. Korea) Respectively.

The procedure of Example 4 was repeated except that ethanol was used instead of water.

Except that 70% by weight of an aqueous ethanol solution was used in place of the water of Example 4.

[Comparative Example 1]

Only the step of preparing the chewing gum extract of Example 1 was performed.

[Comparative Example 2]

Only the step of preparing the chewing gum extract of Example 2 was performed.

[Comparative Example 3]

Only the step of preparing the chewing gum extract of Example 3 was performed.

1. Determination of flavonoid content

The total flavonoid content per gram of the final manufactured samples in Examples 1 to 6 and Comparative Examples 1 to 3 was measured using a diethylene glycol colorimetric method. Specifically, 100 μl of the above sample, 100 μl of 1 N NaOH and 1 ml of diethylene glycol were mixed in a tube using a vortex mixer, and the mixture was reacted with water at 30 ° C. for 1 hour. Then, the content of flavonoid was measured at a wavelength of 420 nm using an ultraviolet-visible spectrophotometer (Mecasys co. Korea).

The content of flavonoid in the fermented Angelica keisjosa fermented extract prepared in Examples 1 to 6 was measured and it was confirmed that ethanol or 70% by weight ethanol aqueous solution as the solvent was about 4 to 6 times higher than that in the case of using only water Respectively.

It was also confirmed that the use of 70 wt% ethanol aqueous solution as a solvent was about 69% higher than that of ethanol alone.

2. Polyphenol content measurement

The total polyphenol content per gram of the final manufactured samples in Examples 1 to 6 and Comparative Examples 1 to 3 was measured using the Folin-Danis method. Specifically, 100 μl of the above sample and 1 ml of 2% Na ₂ CO ₃ were mixed in a tube and reacted at room temperature for 2 minutes, respectively. Then, 100 μl of 50% Folin-ciocalteu's phenol regent was added, mixed using a vortex mixer, and reacted at room temperature for 30 minutes. Then, polyphenol contents were measured at 750 nm wavelength using a spectrophotometer (UV-VIS Spectrophotometer, Mecasys co. Korea).

The content of polyphenols in the fermented Chumma extract prepared in Examples 1 to 6 was measured to be about 5 to 6 times higher than that in the case of using ethanol or 70% by weight ethanol aqueous solution as a solvent Respectively. It was also confirmed that the solvent extraction of water and ethanol at the same time independently increased the water content by 1.5 times as compared with the aqueous solution of 70 wt% ethanol.

3. Measurement of DPPH radical scavenging ability

DPPH (1,1-diphenyl-2-picrylhydrazyl) radicar assay was used to modify each of the 2.5 mg / ml samples prepared in Examples 1 to 6 and Comparative Examples 1 to 3 by modifying the method of Biois et al. The DPPH radical scavenging activity of the 5 mg / ml sample was measured. Specifically, 0.1 M Triz buffer (Trizma base-HCl buffer) of pH 7.4 was prepared, and then 500 mM DPPH was prepared using methanol. 100 μl of the above sample, 400 μl of 0.1 M Triz buffer and 500 μl of 500 mM DPPH were mixed in a vortex mixer and reacted at room temperature for 30 minutes in a shade state. The absorbance at 517 nm was measured using a spectrophotometer (UV-VIS Spectrophotometer) (Mecasys co. Korea). The DPPH radical scavenging activity was calculated by converting the absorbance difference between the sample addition group and the non- 1 and 2 (Water: Chimpanzee extract prepared in Comparative Example 1, Water-LB: Lactobacillus fermentation extract obtained from Example 4, Water-EM: Examples 1 and 2) 70% EtOH-EM: Ethanol-fermented extract prepared in Example 6, 70% EtOH-EM: Ethanol-fermented extract prepared in Example 6, 70% EtOH: EtOH-EM: fermented EM fermented extract prepared in Example 2), EtOH-fermented extract prepared in Comparative Example 2, EtOH-LB: lactic acid fermented Ganoderma lucidum fermented extract prepared in Example 5,

FIGS. 1 and 2 are graphs showing the results of measurement of the DPPH radical scavenging ability according to Examples 1 to 6 and Comparative Examples 1 to 3 by modifying the method of Biois et al. As the concentration (2.5, 5 mg / ml) increases, the DPPH radical scavenging ability increases. It can be seen that ethanol or 70 wt% ethanol aqueous solution> water increases in the order of extraction solvent. By fermentation, it can be confirmed that the number of useful microorganisms> lactobacillus> non fermentation increases.

4. Measurement of cell viability (MTT assay)

The MTT assay is a standard colorimetric assay as a test method for measuring the cell survival rate. The samples of Examples 1, 3 to 5, Comparative Examples 1 and 3 Respectively. Specifically, according to the method of Mosmann, RAW 264.7 cells were seeded at a density of 3 × 10 5 cells / well in a 96-well plate, and then cultured in a 5% CO ₂ interviewer at 37 ° C. Then, Respectively. After incubation, 100 μl of MTT [3- (4,5-dimethylthiazol-2yl) -2,5-diphenyltetrazoliumbromide] solution was added to each well and incubated for 4 hours. Then, the formazan formed by removing the supernatant was dissolved in 100 μl of DMSO (dimethylsulfoxide), and the absorbance was measured at a wavelength of 570 nm using a microplate reader. 3 shows the difference in absorbance between the sample added group and the no-added group as a percentage. (In FIG. 3, Water: the Chunma extract prepared in Comparative Example 1, Water-LB: the lactic acid fermented milk fermented in Example 4 Water-EM: EM Chunmagyung's fermented extract prepared in Example 1, 70% EtOH: Chimma extract prepared in Comparative Example 3, 70% EtOH-LB: Fermented lactic acid bacteria prepared in Example 6, 70% EtOH -EM: EM chestnut fermentation extract prepared in Example 3)

FIG. 3 is a graph showing the results of measurement of cell viability in RAW 264.7 cells (Mosmann) according to Example 1, Examples 3 to 5, Comparative Examples 1 and 3 on the basis of normal cells, 1 and Examples 3 to 5 exhibited cell viability of 90% or more, which indicates that they are suitable for use as cosmetics and functional depressants.

5. Measurement of reactive oxygen species

To measure the production of reactive oxygen species (ROS), the fluorescence was measured using Dichlorofluorescein (DCF) and Dichlorodihydrofluorescein diacetate (DCFH-DA). Dichlorodihydrofluorescein diacetate (DCFH-DA) is a non-mineral substance that diffuses through the cell membrane and enters the cell. When it enters the cell, it is hydrolyzed in the cell. When active oxygen species are present, it is converted to dichlorofluorescein and oxidized as a high-performance fluorescent substance . Specifically, RAW 264.7 cells were seeded at a density of 3 × 10 5 cells / well on a 96-well plate, and the cells were stably cultured with the above culture medium. (Samples of Examples 1 to 6 and Comparative Examples 1 to 3 25, 50 and 100 μl) (n = 3 per supporter). As a negative control, only RAW 264.7 cells cells were cultured in the culture medium. RAW 264.7 cells cells and Lipopolysaccharide , LPS) was added and cultured for 12 hours. Then, 5 μM dichlorofluorescein diacetate was added, and the cells were incubated at 37 ° C. for 20 minutes. Cells were obtained by trypsinization and phosphate-buffered saline (PBS), and analyzed by using a flow cytometer (FACS Calibur ) (BENTON-Dickson).

As a result of the measurement, active oxygen species increased in the order of the useful microorganism group> non-fermentation> lactic acid bacteria. Thus, it can be confirmed that lactic acid fermentation is not suitable because it produces more active species than non-fermentation .

As described above, it can be seen that the fermentation with the useful microorganism group proceeds to produce the fermented Chumma extract, showing an excellent antioxidative effect. In addition, it can be seen that the whitening material can be used as a better material as in the case of lactic acid bacteria.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims and their equivalents.

Claims (11)

Mixing 10 to 200 parts by weight of the powdery sugar per 100 parts by weight of gum with gum, and dewatering to prepare a dehydrated gel;
Preparing a chymus extract by solvent extraction of the dehydrated chymase; and
Fermenting the chondroitin extract with an EM activating solution,
Wherein the solvent comprises ethanol or water and ethanol, and the solvent comprises 180 to 280 parts by weight of ethanol relative to 100 parts by weight of water when the solvent comprises water and ethanol. delete delete The method according to claim 1,
Wherein the step of preparing the chewing gum extract comprises extracting at 30 to 90 ° C for 4 to 12 hours. delete The method according to claim 1,
Wherein the fermenting step comprises fermenting at 20 to 45 DEG C for 1 to 12 days. The method according to claim 1,
Further comprising the step of filtrating before the step of fermentation, the step of concentrating under reduced pressure, or the step of filtration and concentration under reduced pressure. 8. The method of claim 7,
And further comprising a step of freeze-drying. delete delete delete

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