WO2020091192A9 - Matured mixed fermentation product of wild-cultivated ginseng in which ginsenoside absorbable in human body and omega-6 fatty acid are increased, and preparation method therefor - Google Patents

Abstract

Disclosed are a matured mixed fermentation product of wild-cultivated ginseng in which the amounts of ginsenosides Rd2, Rg3, F2, and compound K that are easily absorbed by the human body, omega-6 fatty acids (ω-6 fatty acids: linoleic acid and arachidonic acid), GABA, phenolics, and flavonoids are significantly increased, and a preparation method therefor. The matured mixed fermentation product of wild-cultivated ginseng according to the present invention has significantly increased amounts of ginsenosides Rd2, Rg3, F2, and compound K, omega-6 fatty acids, GABA, phenolics, and flavonoids, and has excellent antioxidant effects, and thus can be used as a material for functional foods and pharmaceuticals. Functional foods and cosmetics according to the present invention have excellent antioxidant activity, thus being useful for improving lowered immunity due to oxidative stress caused by radicals or the like, improving cognitive ability, overcoming fatigue, and for use in oral cosmetics and skin care.

Description

Fermented wild ginseng matured complex fermented product with enhanced human absorption-type ginsenoside and omega-6 fatty acid and its manufacturing method

The present invention relates to a complex fermentation of wild ginseng matured with enhanced human absorption ginsenoside and omega-6 fatty acids and a method for manufacturing the same, and more specifically, steaming/sealed aging and steaming/open aging of wild ginseng four times alternately After performing, it is prepared by complex fermentation with Lactobacillus brevis BMK484 strain and Lactobacillus plantarium P1201 strain, and is easily absorbed by the human body, ginsenosides Rd2, Rg3, F2 and compound K, omega-6 fatty acids (ω-6 fatty acids; Linoleic acid and arachidonic acid), gaba, phenolics, and the content of the flavonoids is significantly improved fermented sanyangsams fermented complex and a method for producing the same.

Ginseng is classified into ginseng (人蔘), wild ginseng (山蔘) or wild ginseng (山羊蔘) according to the difference between artificial growth and natural growth according to the cultivation environment and morphological differences. In particular, wild ginseng belongs to the family Ogapidae and refers to the artificially grown wild ginseng seeds or yusams grown in the mountains by Panax ginseng , a perennial plant, in the wild under the forest in the mountains, and these are called wild ginseng. In addition, wild ginseng grown in the wild from ancient times has been hit as the best wild ginseng and is also called camphor ginseng. Most of ginseng has 3-7 outcrops, but wild ginseng is more than that depending on age. The roots of ginseng are thick and short, but wild ginseng is long and thin. Wild ginseng is known to be more effective than cultivated ginseng in terms of various efficacy, but there is not much scientific evidence to support it than ginseng.

Ginseng is reported to have various pharmacological effects on body function control by affecting the central nervous system, endocrine system, immune system, and metabolic system, and skin health effects such as whitening, wrinkle improvement, moisturizing and anti-inflammatory effects have also been reported. Such pharmacological effects are known to be due to ginsenosides, which are easily absorbed by the human intestine or skin cells, that is, human absorption.

Examples of ginsenosides absorbed by the human body include ginsenosides Rg1, Rg3, Rd, Rf, F2, and compound K. Ginsenoside Rg1 improves learning function, anti-fatigue, ginsenoside Rf has analgesic action, lipid and oxidation inhibitory action, ginsenoside Rg3 inhibits cancer cell metastasis, hepatoprotective action, inhibits anticancer drug resistance, ginsenoside Rd series has been shown to promote the secretion of corticosteroids, and ginsenoside compound K has anti-cancer, hepatoprotective, skin-protective effects, tumor growth inhibition, antioxidant, and anti-allergic effects. Ginsenoside F2 has been recently known to have an effect of reducing inflammation of atopic skin and suppressing itching. Ginsenoside Compound K (20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol) is a metabolite of ginsenosides. It is known to have excellent absorption in the body and excellent pharmacological activity. Recently, it is known to treat macular degeneration disease. , Neuropathic pain treatment, whitening and wrinkle improvement, etc. effects have also been reported (Registration Patent 10-1539573, Registration Patent 10-1300775, Patent Publication 10-2014-0003198).

However, since these human-absorbable ginsenosides are hardly contained in ginseng itself, several technologies have been developed to convert ginsenosides contained in ginseng into human-absorbable types using enzymes to solve this problem. Since this high or low enhancement rate of human-absorbable ginsenoside is implicated, there is still a demand for the development of processed sanyang ginseng products with enhanced human-absorbable ginsenoside.

Fatty acids have a carboxyl group (-COOH) at one end and a methyl group (-CH ₃ ) at the other end, and omega-3 (ω-) depends on the number of carbons from the carbon to which the methyl group is attached (ω carbon). 3) or omega-6 (ω-6) fatty acids. Omega-6 fatty acid refers to an unsaturated fatty acid having a double bond on the 6th carbon from the ω carbon, and there are linoleic acid and arachidonic acid. Omega-6 fatty acids are not synthesized in animals, so they are called essential fatty acids, and vegetable oils such as corn oil and peanut oil are known as source foods. Omega-6 fatty acids are effective in lowering blood cholesterol levels.

Gabaro (Gamma-aminobutyric acid, GABA) is a non-proteinaceous amino acid composed of four carbons. By glutamate decarboxylase (GADase) from glutamic acid (GA), carbon dioxide is released along with decarboxylation reactions and carbon dioxide is released. Is converted. Gaba not only plays a role as a neurotransmitter in the brain, but also has a wide variety of physiological functions such as promoting brain function, mental stability, lowering blood pressure, diuretic, improving liver function, preventing obesity, promoting alcohol metabolism, and deodorizing. It is also registered as a medicine and is used for the treatment of headache, tinnitus, and decreased motivation due to stroke or cerebral artery sequelae. Normally, the human body produces gaba, but as aging progresses due to excessive intake of inappropriate foods or additives, and insufficient intake of vitamins or minerals, the production of gaba in the body is limited. Accordingly, it is necessary to consume foods containing gava.

Regarding processed wild ginseng products, processed wild wild ginseng products in which the contents of human absorbable ginsenosides, omega-6 fatty acids, gaba, phenolics, and flavonoids are all significantly improved have not been developed yet.

Accordingly, as a result of continuing research to meet the requirements of the prior art, the present inventors performed four times alternately steaming and sealed ripening, steaming and open ripening of wild ginseng, and then Lactobacillus brevis BMK484 strain and Lactobacillus plantarium P1201. In the case of complex fermentation and processing as a strain, it was confirmed that the contents of human absorbable ginsenosides Rd2, Rg3, F2 and compound K, omega-6 fatty acids (linoleic acid and arachidonic acid), gaba, phenolics, and flavonoids were significantly improved. The present invention has been completed.

Accordingly, it is an object of the present invention to provide a complex fermented fermented sanyangsam with improved ginsenosides Rd2, Rg3, F2 and compound K, omega-6 fatty acids, gaba, phenolics, and flavonoids content.

Another object of the present invention is to provide a method for producing a complex fermented fermented mountain ginseng with improved ginsenosides Rd2, Rg3, F2 and compound K, omega-6 fatty acids, gaba, phenolics, and flavonoids content.

Another object of the present invention is to provide a functional food for improving immunity, improving cognitive ability, recovering from fatigue, and eating cosmetics (inner beauty), including the fermented fermented fermented fermented fermented wild ginseng of the present invention.

Another object of the present invention is to provide a functional cosmetic for whitening and wrinkle improvement comprising the fermented fermented fermented mountain ginseng of the present invention.

In order to achieve the above object, the present invention is a complex fermentation of ginsenosides Rd2, Rg3, F2 and compound K, omega-6 fatty acids, gaba, phenolics, and enhanced content of flavonoids including the following steps. Provides a method of making water:

i) performing steaming and sealed ripening of wild ginseng and performing steaming and open ripening alternately four times; And

ii) Fermenting with a complex seed of Lactobacillus brevis BMK484 strain and Lactobacillus plantarium P1201 strain.

The manufacturing method of the present invention may further include the step of drying and powdering the fermented wild wild ginseng from step ii).

Step i): Sanyangsam Steaming And ripening

The wild ginseng is steamed, sealed, and steamed and opened four times in turn.

In the present invention, "sanyangsam" means sanyangsam or sanyang sprout ginseng.

In the present invention,'goat sprout ginseng' refers to wild ginseng outposts including the entire root, leaves and stems of wild ginseng grown in a mountainous area of 500 m or more above sea level between May and July, or seedlings of wild ginseng of 2 years or more in an artificial environment for 90 days. It means that it has been cultivated in hydroponic or soil culture. On the other hand, goat sprout ginseng is an eco-friendly agricultural product that does not use any pesticides during cultivation, and has an advantage over mountain ginseng because it forms a lower price than conventional mountain ginseng due to hydroponics or soil cultivation, year-round production, and shortening the cultivation period.

In the present invention, it is preferable to use the goat sprout ginseng harvested in May cultivated in a natural environment or the goat sprout ginseng grown for 30 days in an artificial environment, and more preferably, the goat sprouts cultured for 30 days in an artificial environment. Use hemp.

Wild ginseng and wild sprout ginseng can be used as outposts including roots, leaves and whole stems.

Wash wild wild ginseng or wild wild sprout ginseng 3 times with running water, remove moisture, and steam.

Steaming can be performed using a steamer such as a conventional steamer, and is performed at 100°C for 30 to 120 minutes. If the steaming time is less than 30 minutes, sufficient steaming does not proceed, so subsequent steps such as ripening may not be smooth and contamination of various bacteria may occur. If it is steamed for more than 120 minutes, the nutrients of wild wild ginseng or wild wild sprout ginseng may be destroyed by long heat treatment.

During steaming, if necessary, moisture may be added to the wild ginseng or the wild sprout ginseng so that the moisture content is 40-50%.

Sealed aging is carried out for 2-3 days at 70-80℃ by placing steamed wild wild ginseng or wild wild sprout ginseng in a sealed aging machine (container).

Open maturation is carried out for 2-3 days at 60 ~ 70 ℃ after putting the steamed wild wild ginseng or wild mountain sprout ginseng in the open maturation period.

Sealed aging and open aging are performed alternately (alternately), and the order of sealed aging and open aging is irrelevant.

As an example, as exemplified in the following Examples (Preparation Example), wild ginseng is first steamed and first sealed-aged, second steamed and second opened-aged, third steamed, third-sealed, and fourth steamed And it can be carried out by 4th open ripening.

The sanyangsam or sanyang sprout ginseng steamed and aged as described above is sufficiently matured and decomposition of the produced physiologically active substances is minimized.

Step ii) Complex fermentation

The steamed and matured wild ginseng or wild sprout ginseng from step i) is fermented as a complex seed of Lactobacillus brevis BMK484 strain and Lactobacillus plantarium P1201 strain.

Lactobacillus brevis BMK484 strain, which is one strain constituting the complex seed in the present invention, was isolated/identified by the present inventors from kimchi and deposited with the National Academy of Agricultural Sciences Agricultural Genetic Resource Center (KACC) on December 12, 2016 (trusted No. KACC92157P), the productivity of GABA and the productivity of ginsenoside compound K are excellent (patent application 10-2017-0152033).

Lactobacillus plantarium P1201 strain, another strain constituting the complex spawn in the present invention, was separated/identified from fermented food by the present inventors and was sent to the National Academy of Agricultural Sciences Agricultural Genetic Resource Center (KACC) on July 19, 2013. As the deposited strain (Accession No. KACC91848P), it has excellent properties of probiotic preparation and productivity of physiologically active substances (Registration Patent No. 10-154418).

The Lactobacillus brevis BMK484 strain and Lactobacillus plantarium P1201 strain constituting the composite seed of the present invention exhibit a synergistic effect by complementing the fermentation properties. Lactobacillus brevis BMK484 strain is a hetero-lactic acid fermentation lactic acid bacterium, which does not produce enough lactic acid when fermented alone, and Lactobacillus plantarium P1201 strain is fermented with homo-lactic acid fermentation lactic acid bacteria alone. When lactic acid is generated excessively, surprisingly, the fermentation characteristics of both strains are complemented by the use of these two seed germs together, surprisingly, and human absorption-type ginsenosides, omega-6 fatty acids, gaba, The content of phenolics and flavonoids was remarkably improved (Test Examples 1 to 4).

In the present invention, the complex spawn may be added as a separate culture solution for each of the Lactobacillus brevis BMK484 strain and the Lactobacillus plantarium P1201 strain, or may be added as a mixed culture solution mixed culture.

In the complex seed, the mixing ratio of the two strains may be 3: 1 to 1: 3 (v/v).

Fermentation may be performed by inoculating the culture broth of the complex seed into steamed and matured wild ginseng or wild wild ginseng at a concentration of 2 to 10% (v/w) and fermenting for 2 to 7 days at 25 to 40°C.

If the inoculation amount of the complex seed is less than 2% (v/w), the fermentation rate may be delayed. If it exceeds 10% (v/w), the cell growth rate is high, so the conversion rate of active ginsenosides is low, and the sour taste is excessive. If the fermentation temperature is less than 25℃, the fermentation period is prolonged, causing contamination of various germs. If the fermentation temperature exceeds 40℃, the growth of the strain may be stopped. If the fermentation period is less than 2 days, fermentation is insufficient and menstruation The production of active substances, etc. may be poor, and if it exceeds 7 days, physiologically active substances may be decomposed by overfermentation.

In addition, during fermentation, if necessary, water is added to steamed and aged wild ginseng or wild sprout ginseng to adjust and/or sterilize the moisture to 40-50% (v/w), and then inoculate complex seed to ferment. I can. Fermentation efficiency is highest when the moisture content is within the above range. If the moisture content is less than 40%, smooth fermentation does not proceed due to insufficient moisture for microorganisms to survive or grow, and if it exceeds 50%, drying time and cost may be high after fermentation is terminated, and if sterilization is not performed Smooth fermentation may not proceed due to contamination of various germs.

The fermented fermented wild mountain ginseng produced by the above steps of the present invention has 1.5 times more ginsenoside Rd2, 8.1 times more ginsenoside Rg3, 11.6 times more ginsenoside F2 than before processing, The content of senoside compound K was increased by more than 12.4 times, the contents of phenolis and flavonoids were also improved by more than 3.6 and 1.4 times, respectively, and the omega-6 fatty acids linoleic acid and arikidonic acid were improved by more than 3.1 and 3.7 times, respectively. It was enhanced, and Gaba was improved by more than 3.1 times (Table 2, Figure 2, Figure 3a, Figure 3b, Table 3).

Another object of the present invention is to prepare a complex fermented fermented mountain ginseng with improved ginsenosides Rd2, Rg3, F2 and compound K, omega-6 fatty acids, gaba, phenolics, and flavonoids contents prepared by the above manufacturing method. to provide.

The complex fermented fermented fermented sanyangsam according to the present invention has ginsenoside Rd2 of 2.75 mg/g or more, ginsenoside Rg3 of 2.90 mg/g or more, ginsenoside F2 of 3.61 mg/g or more, and ginsenoside compound K of 6.20 mg/g or more, phenolics 8.48 mg/g or more, flavonoids 2.22 mg/g or more, and omega-6 fatty acids linoleic acid and arikidonic acid, respectively, containing 282.6 mg/100 g or more and 6.6 mg/100 g or more, Gaba contains 100.33 mg/100g or more (Table 2, Figure 2, Figure 3a, Figure 3b, Table 3).

The complex fermented fermented mountain ginseng according to the present invention has a remarkably enhanced antioxidant activity (Test Example 4).

According to another object of the present invention, the present invention is prepared by the above production method, and the contents of ginsenosides Rd2, Rg3, F2 and compound K, omega-6 fatty acids, gaba, phenolics, and flavonoids are enhanced. Provides functional foods with antioxidant activity, including aged complex fermented products as an active ingredient.

In the present invention, the functional food is dried and powdered of the fermented wild ginseng of the present invention as it is, or extracted and used as a food additive, or powder or extract in the form of granules, pills, beverages, jelly, etc. It can be manufactured as a product of

The food according to the present invention is useful for improving immunity, improving cognitive ability, recovering from fatigue and eating cosmetics (inner beauty).

According to another object of the present invention, the present invention is prepared by the above production method, and the contents of ginsenosides Rd2, Rg3, F2 and compound K, omega-6 fatty acids, gaba, phenolics and flavonoids are enhanced. Provides cosmetics with antioxidant activity containing complex fermented products as an active ingredient.

Cosmetics according to the present invention are useful for whitening and wrinkle improvement.

The fermented wild wild ginseng composite presentation according to the present invention is dried and powdered as it is or extracted and used as an additive in cosmetics for whitening, wrinkle improvement or moisturizing, or manufactured as a product in the form of a mask pack, skin, cream, lotion, etc. Can be.

The production method of the present invention produces a processed wild ginseng product with significantly improved ginsenosides Rd2, Rg3, F2 and compound K, omega-6 fatty acids, gaba, phenolics, and flavonoids content and antioxidant activity compared to conventional methods. Make it.

The complex fermented fermented sanyangsam according to the present invention has significantly improved ginsenosides Rd2, Rg3, F2 and compound K, omega-6 fatty acids, gaba, phenolics, and flavonoids content, and also has excellent antioxidant effects. , It can be used as a material for cosmetics and pharmaceuticals.

Functional foods according to the present invention have excellent antioxidant activity, and are useful for improving immunity due to oxidative stress, improving cognitive ability, recovering from fatigue, eating cosmetics and skin care.

1 is an example of a manufacturing process of a composite fermented fermented sanyangsam according to the present invention.

Figure 2 shows the ginsenoside compound HPLC chromatogram of the complex fermentation of sanyangsam according to the present invention. 2A is an HPLC chromatogram of 21 types of ginsenoside standards, FIG. 2B is an HPLC chromatogram of a ginsenoside compound of Comparative Example 1, and FIG. 2C is an HPLC chromatogram of a ginsenoside compound of Comparative Example 2, and FIG. 2D Is a ginsenoside compound HPLC chromatogram of Example 1, and 2e is a ginsenoside compound HPLC chromatogram of Example 2.

Figure 3 is a graph showing the content of phenolics and flavonoids of fermented wild wild ginseng aging complex according to the present invention. Figure 3a shows the total phenolics content, Figure 3b shows the total flavonoids content.

Figure 4 is a graph showing the antioxidant activity of the fermentation complex fermented sanyangsam according to the present invention. Figure 4a shows the DPPH radical scavenging activity, Figure 4b shows the ABTS radical scavenging activity, Figure 4c shows the hydroxyl radical scavenging activity, Figure 4d shows the FRAP reducing power.

The present invention is explained in more detail by the following examples. These examples are intended to illustrate the invention, and the scope of the invention should not be limited by them.

Manufacturing example One. Sanyangsam ferment Compound fermentation Produce

After washing 10 kg of wild ginseng (outpost) over 3 years old in running water 3 times and completely draining it, 5 kg was put in a steamer and then steamed for 60 minutes at 100°C. The first steamed wild ginseng was put in 2 kg each in a closed maturation machine, and the first sealed maturation was carried out at 75°C for 3 days while moisture was evaporated. Moisture (about 40% moisture content) was added to the first aged wild ginseng, put in a steamer, and then steamed for a second time at 100°C for 60 minutes. The second steamed wild ginseng was transferred to an open aging machine, and then the second open aging was performed at 65°C for 3 days. Water (about 40% moisture content) was added to the second-aged wild ginseng, and after being placed in a steamer, the third steamed for 60 minutes was performed at 100°C, and the third steamed wild ginseng was again placed in a sealed maturity for 3 days at 75°C. Sealed aging was performed. Finally, add moisture (about 40% of moisture content) to the third-aged wild ginseng, put it in a steamer, and cultivate for 4 times at 100°C for 60 minutes, and transfer the fourth steamed wild ginseng to an open-type maturation unit and place 3 at 65°C. Steamed and aged wild ginseng was prepared by performing 4 open aging for a day (Fig. 1).

Put 500 g of the prepared steamed and aged wild ginseng into a fermentation container, add twice as much clean water (1 L), and hydrate for 1 hour to adjust the moisture content to about 50%, then sterilize at 120°C for 30 minutes, Lactobacillus plantarium P1201 strain and Lactobacillus brevis BMK484 strain were each cultured as a seed in malt extract liquid medium, and a complex seed mixed at a ratio of 1:1 was inoculated at 5% (v/w) and fermented at 30°C for 7 days. To prepare a composite fermented fermented wild ginseng (Example 1).

On the other hand, 10 kg of goat sprout ginseng (outpost) grown on the soil for 30 days or longer of 2 years old wild ginseng was prepared by steaming, aging and complex fermentation of wild ginseng (goat sprout ginseng) aged fermentation composition (Example 2 ).

For comparison, the raw material wild ginseng was prepared as Comparative Example 1, and as Comparative Example 2, 5 kg of wild ginseng was steamed at 100°C for 1 hour, placed in a dry tray (open container), and dried at 65°C for 3 days, and the process was repeated 4 times. Then, after aging for 12 days, a fermented sanyangsam fermentation composition fermented in the same manner as above was prepared.

<Physical and chemical properties>

Physicochemical properties were measured for Example 1, Example 2, and Comparative Examples 1 and 2. The pH was measured using a pH meter, and the total acidity was calculated in terms of lactic acid by performing the neutralization titration method, and the reducing sugar was performed through the DNS method (Mille, 1953) and converted into glucose. .

Analysis item	Comparative Example 1	Comparative Example 2	Example 1	Example 2
pH	5.05	4.21	4.31	4.26
Acidity (%, based on lactic acid)	1.81	3.02	2.91	3.12
Reducing sugar (mg/g)	5.02	6.24	5.85	5.55
*All experiments are repeated three times and expressed as an average value.

It is judged that the fermented sanyangsam prepared in accordance with the present invention (Examples 1 and 2) had a lower pH and increased total acidity than Comparative Example 1 (raw sanyangsam), thereby improving preservation and palatability. There was no significant difference in reducing sugar (Table 1).

Test example 1. Analysis of ginsenoside content

The fermented fermented fermented fermented mountain ginseng of Example 1, the fermented fermented fermented fermented fermented mountain ginseng of Example 2, and the fermented fermentation composition of the raw material mountain ginseng of Comparative Example 1 and the fermented mountain ginseng of Comparative Example 2 were dried at 50 to 55°C for 2 days, respectively. After that, it was pulverized to 100 mesh or less with a grinder and powdered.

<Preparation of sample for analysis>

The powder prepared above was extracted from samginsenoside according to the health functional food analysis method. Specifically, 1 g of each dry powder was accurately taken in a 250 ml Erlenmeyer flask, 20 ml of 70% methanol was added, allowed to stand in a constant temperature water bath at 80° C. for 1 hour, and then cooled. This was centrifuged to take only the supernatant, and this was repeated twice. The supernatant was concentrated under reduced pressure at 60°C, the residue was dissolved in 2 ml of distilled water, filtered through a 0.45 μm membrane filter, and used as an analysis sample.

<Ginsenoside compound analysis>

Ginsenoside compound analysis was performed by high press liquid chromatography (HPLC) by modifying the method described in the functional food analysis method. TSKgel ODS-100Z was used as the analysis column, and the sample injection amount was 10 μl, the temperature was 30°C, the measurement wavelength was 203 nm, and the flow rate was 1.0 ml/min. As the mobile phase, HPLC water was used for solution A and acetonitrile was used for solution B. . As for the HPLC analysis conditions, when the mobile phase solution was 0 minutes, flow A solution 81%: B solution 19%, 15 minutes, A solution 80%: B solution 20%, 40 minutes, A solution 77%: B solution 23%, Solution A 70%: Solution B 30% at 42 minutes, Solution A 65%: Solution B 35% at 75 minutes, Solution A 30% at 80 minutes: Solution B 70%, Solution A 10% at 90 minutes: Solution B 9 0 The mobile phase was shed in %. The analyzed ginsenoside content of Comparative Example 1, Comparative Example 2, Example 1, and Example 2 are shown in Table 2 and FIG. 2.

Content (mg/g d.w.)	Comparative Example 1	Comparative Example 2	Example 1	Example 2
Ginsenoside Rd2	1.81	2.77	2.75	4.63
Ginsenoside F2	0.31	2.27	3.61	4.09
Ginsenoside Rg3	0.36	1.93	3.03	2.90
Ginsenoside C.K	0.50	4.13	6.20	6.30
*All experiments are repeated three times and expressed as an average value.

As shown in Table 2 and FIG. 2, in the case of the complex fermented fermented mountain ginseng of Example 1, compared to Comparative Example 1 (raw material mountain ginseng, FIG. 2b), ginsenoside Rd2 was about 1.5 times (2.75 mg/g) or more, Ginsenoside Rg3 is about 8.4 times (3.03 mg/g) or more, ginsenoside F2 is about 11.6 times (3.61 mg/g) or more, and ginsenoside compound K is about 12.4 times (6.20 mg/g) or more. Was enhanced (Fig. 2d). In the case of the complex fermentation of the fermented goat sprout ginseng of Example 2, ginsenoside Rd2 was increased by about 2.6 times (4.63 mg/g) or more, and ginsenoside Rg3 was improved by about 8.1 times (2.90 mg/g) or more, especially ginsenoside. Side F2 was significantly improved by about 13.2 times (4.09 mg/g) or more and ginsenoside compound K by about 12.6 times (6.30 mg/g) or more (Fig. 2e).

In addition, ginsenoside Rg3, ginsenoside F2, and compound K were much more improved compared to the fermented wild ginseng composition of Comparative Example 2.

Test example 2. Phenolics And Flavonoids Content analysis

The contents of phenolics and flavonoids, which are bioactive ingredients, were analyzed.

<Preparation of sample for analysis>

10 ml of 50% fermented alcohol was added to 10 g of each powder prepared as in Test Example 1, extracted for 24 hours at room temperature (20±5° C.) and centrifuged at a speed of 3,000 rpm to prepare an extract by taking only the supernatant. Using this extract as a sample, total phenolics and total flavonoids were analyzed.

<Total phenolics content>

Total phenolics content was measured by the Folin Denis method (1912).

Specifically, 0.5 ml of each analysis sample prepared above was dispensed into a test tube, 0.5 ml of a 25% Na ₂ CO ₃ solution was added thereto, and allowed to stand for 3 minutes. Again, 0.25 ml of 2N-Folin-Ciocalteu phenol reagent was added, mixed, and allowed to stand at 30° C. for 1 hour, and the absorbance was measured at 750 nm. At this time, the total phenolics content was calculated as an amount equivalent to gallic acid by obtaining the content from a standard curve prepared using gallic acid, and the results are shown in FIG. 3A.

As shown in Figure 3a, the total phenolics content was compared to Comparative Example 1 (wild ginseng raw material) and Comparative Example 2 (open-aged wild ginseng fermentation composition), the fermented wild wild ginseng complex fermentation according to the present invention (Example 1), respectively It was improved about 3.6 times and 1.5 times, and the complex fermentation of goat sprout ginseng (Example 2) was enhanced by about 3.7 times and 1.4 times, respectively.

<Total flavonoid content>

The total flavonoids content was determined by the Davis variant.

Specifically, 0.01 ml of 1 N NaOH was added to 0.1 ml of the sample prepared above, 1.0 ml of diethylene glycol was added, and the mixture was allowed to stand at 37° C. for 1 hour, and the absorbance at 420 nm was measured. At this time, the total flavonoid content was obtained from a standard curve prepared by preparing the final concentration of rutin at 0, 0.25, 0.5, and 1.0 mg/ml, and the result is shown in FIG. 3B.

As shown in Figure 3b, the total flavonoids content, compared to Comparative Example 1 (wild ginseng raw material) and Comparative Example 2 (open ripened wild ginseng fermentation composition), the wild ginseng aged complex fermented product (Example) according to the present invention, respectively It was improved about 13.1 times and 2.5 times, and the complex fermentation of goat sprout ginseng maturation (Example 2) was improved about 13.2 times and 2.6 times, respectively.

Test example 3. Omega 6 fatty acids (linoleic acid and arachidonic acid) and Gaba Content analysis

The content of omega 6 fatty acids was analyzed by gas chromatography (GC), and the content of Gaba was analyzed by an automatic amino acid analyzer.

<Omega 6 fatty acid content analysis>

It was carried out according to the fatty acid analysis method of the Food Code.

Specifically, 1 g of each powder prepared as in Test Example 1 was taken, 3 ml of methanolic 0.5 N NaOH was added, and heat treatment was performed at 100°C for 10 minutes. Then, 2 ml of BF ₃ was added and stirred, followed by methylation of fatty acids for 30 minutes. After the reaction was completed, 1 ml of isooctane was added and mixed, and only isooctane was recovered, dehydrated with sodium sulfite anhydrous, and filtered through a 0.45 μm membrane filter (Dismic-25CS) to be used as an analysis sample.

GC was used for analysis, and after mounting SP-2560 capillary column (100 m×0.25 mm, 0.20 µm, Sigma-Aldrich Co.), nitrogen gas was used as the mobile phase. 20 µl of the analysis sample was injected to maintain a mobile phase speed of 1 ml/min. At this time, the temperature of the oven was raised to 200°C and then maintained at 230°C for 30 minutes and detected using a fluorescence detector, and the content is shown in Table 3.

<Gabar content analysis>

4 ml of distilled water was added to 1 g of each of the wild ginseng powder prepared as in Test Example 1, hydrolyzed at 60°C for 1 hour, and then 1 ml of 10% 5-sulfosalicylic acid was added and left at 4°C for 2 hours. Then, the supernatant obtained by centrifugation was filtered through a 0.45 μm membrane filter and concentrated under reduced pressure at 60°C. After concentration, 2 ml of a pH 2.2 lithium buffer was added to the dried product, dissolved and filtered through a 0.45 μm membrane filter, and analyzed by an automatic amino acid analyzer, and the contents are shown in Table 3.

Content (mg/100 g d.w.)	Comparative Example 1	Comparative Example 2	Example 1	Example 2
Linoleic acid (C18:2c)	261.6	300.5	329.1	282.6
Arachidonic acid (C20:4n6)	1.8	4.3	7.4	6.6
GABA	75.12	83.17	100.61	100.33
*All experiments are repeated three times and expressed as an average value.

As shown in Table 3, compared to Comparative Example 1 (wild ginseng raw material), the fermented wild wild ginseng fermented product (Example 1) according to the present invention was about 3.1 times the amount of linoleic acid and gava, respectively (329.1 mg/100 g: linoleic acid and 100.61). mg/100 g: Gaba) was enhanced, and in particular, arikidonic acid was significantly improved to about 4.1 times (7.4 mg/100 g), and the complex fermented product (Example 2) aged goat sprout ginseng had linoleic acid content (282.6 mg/100 g) ) Was enhanced, and gaba and arikidonic acid were enhanced by about 3.1 and 3.7 times, respectively. In addition, compared to Comparative Example 2 (fermented composition of openly aged wild ginseng), the complex fermented product of fermented wild wild ginseng (Examples 1 and 2) according to the present invention showed much improved arachidonic acid and gaba.

Test example 4. Antioxidant activity assay

Antioxidant activity was analyzed by measuring DPPH radical scavenging activity, ABTS radical scavenging activity, hydroxyl radical scavenging activity, and FRAP reducing power.

<Sample preparation>

10 ml of 50% fermented alcohol was added to 10 g of each powder prepared as in Test Example 1, extracted for 24 hours at room temperature (20±5° C.) and centrifuged at a speed of 3,000 rpm to prepare an extract by taking only the supernatant. , Each extract was concentrated under reduced pressure at 60° C. and freeze-dried, followed by analysis of antioxidant activity on samples prepared at concentrations of 0.25, 0.5, and 1.0 mg/ml.

<DPPH radical scavenging activity>

DPPH radical scavenging activity was obtained by adding 0.8 ml of DPPH solution (1.5×10 ^-4 M) to 0.2 ml of the sample (0.25, 0.5, 1 mg/ml concentration) prepared above, uniformly mixing, and allowing to stand for 30 minutes and then 525 nm. It was carried out by measuring the absorbance at. The negative control of DPPH radical scavenging activity was performed in the same manner using distilled water instead of the sample, and the difference in absorbance was calculated as a percentage (%) by the following equation, and the result is shown in FIG. 4A.

Radical scavenging activity (%) = [1-(absorbance of negative control ÷ absorbance of experimental sphere)]×100

<ABTS radical scavenging activity>

ABTS radical scavenging activity was obtained by mixing 5 ml of 7 mM ABTS reagent and 5 ml of 140 mM K ₂ S ₂ O ₈ (FW 270.3, Sigma 9392) and standing in a dark place for 16 hours to generate cationic radicals, which were then mixed with methanol to 732 nm. In the ABTS solution adjusted so that the absorbance value of the control was 0.7±0.02 was used.

0.1 ml of the sample prepared above (0.25, 0.5, 1 mg/ml concentration) and 0.9 ml of ABTS solution were mixed, reacted for 3 minutes, and absorbance was measured at 732 nm. The ABTS radical scavenging activity was also negative control was performed in the same manner using distilled water instead of the sample, and the difference in absorbance was calculated as a percentage (%) by the above equation, and the result is shown in FIG. 4B.

<Hydroxy radical scavenging activity>

The hydroxyl (·OH) radical scavenging ability is FeSO _{4 .} 7H ₂ 0-EDTA (10 mM) 2 ml and 2-deoxyribose (10 mM) 0.2 ml, H ₂ O ₂ (10 mM) 0.2 ml and 1.4 ml of each composition of Preparation Example were mixed and reacted at 37° C. for 4 hours, and 1% thiobarbituric acid (TBA) and 2.8% trichloroaceric acid (TCA) were added to the mixture. ) Was added to each of 1 ml, color was developed at 100°C for 20 minutes, cooled, and absorbance was measured at 520 nm. The negative control for hydroxyl radical scavenging activity was performed in the same manner using distilled water instead of the sample, and the difference in absorbance was calculated as a percentage (%) by the above equation, and the result is shown in FIG. 4C.

<FRAP reducing power analysis>

FRAP (Ferric reducing antioxidant power) The reducing power analysis is a method of measuring the reducing power of a compound, and is a method of measuring the power to reduce Fe ^{3 +} to Fe ^{2 +} . Specifically, when FeIII-TPTZ (ferric tripyridyl triazine) is reduced to blue FeII-TPTZ (ferrous tripyridyl triazine) by the reducing power of the sample, the absorbance is measured to determine the antioxidant activity.

In the FRAP reducing power analysis, the reaction solution was 30 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-s-triazine dissolved in 40 mM hydrochloric acid (TPTZ, T1253, C ₁₈ H ₁₂ N ₆ , MW312.33) and 20 mM FeCl ₃ (F7134, MW 162.20, in DW) were prepared, and acetate buffer, TPTZ solution and FeCl ₃ solution were mixed in 10:1:1 (v/v/v) at 37°C. It was allowed to pre-react for 15 minutes.

50 µl of each of the assay samples (0.25, 0.5, 1 mg/ml concentration) prepared above and 950 µl of FRAP reagent were dispensed into a test tube, reacted for about 15 minutes, and 593 using a microplate reader (Biorad 3055, Sweden) The absorbance was measured at nm and the results are shown in FIG. 4D.

As shown in Figures 4a to 4d, compared to Comparative Example 1 (wild ginseng raw material) and Comparative Example 2 (open ripened wild ginseng fermentation composition), the fermented wild wild ginseng complex fermentation according to the present invention (Example 1) and goat sprouts The three-aged fermentation complex (Example 2) significantly improved DPPH radical scavenging activity, ABTS radical scavenging activity, hydroxyl radical scavenging activity, and FRAP reducing power. In particular, when treated with 1 mg/ml sample, the examples 1 and 2 were The DPPH radical scavenging activity was 90.66% and 89.92%, respectively, the ABTS radical scavenging activity was 89.77% and 94.37%, respectively, the hydroxyl radical scavenging activity was 64.57% and 67.72%, respectively, and the FRAP reducing power was very high at 1.752 and 1.859, respectively.

From the above results, it can be seen that the fermented fermented fermented sanyangsam or sanyang sprout ginseng according to the present invention remarkably enhances its antioxidant activity and thus is useful as a material for functional foods or cosmetics.

[Amendment 26.09.2019 under Rule 91]

Claims (8)

1. A method for producing a complex fermented fermented fermented mountain ginseng with improved ginsenosides Rd2, Rg3, F2 and compound K, omega-6 fatty acids, gaba, phenolics, and flavonoids,

   The above method is

   i) performing steaming and sealed ripening and steaming and open ripening of wild wild ginseng or wild mountain sprout ginseng alternately 4 times; And

   ii) fermenting with a complex seed of the Lactobacillus brevis BMK484 strain deposited with the accession number KACC 92157P and the Lactobacillus plantarium P1201 strain deposited with the accession number KACC91848P,

   The steaming is performed at 100° C. for 30 to 120 minutes,

   The sealed aging is performed for 2-3 days in a sealed aging machine at 70 to 80°C,

   The open aging process is to perform aging for 2 to 3 days in an open maturation period of 60 to 70 ℃.
2. It is prepared by the method according to claim 1, and ginsenosides Rd2, Rg3, F2 and compound K, omega-6 fatty acids, gaba, phenolics, and a fermented wild wild ginseng with enhanced content of flavonoids.
3. The method of claim 2, wherein the fermented fermented wild ginseng is 2.75 mg/g or more of ginsenoside Rd2, 2.90 mg/g or more of ginsenoside Rg3, 3.61 mg/g or more of ginsenoside F2, ginsenoside compound K contains at least 6.20 mg/g, phenolics at least 8.48 mg/g, flavonoids at at least 2.22 mg/g, and omega-6 fatty acids linoleic acid and arikidonic acid at least 282.6 mg/100 g and 6.6 mg/100 g, respectively Ginsenosides Rd2, Rg3, F2 and compound K, omega-6 fatty acids, gaba, phenolics, and flavonoids, characterized by containing more than 100.33 mg/100g of gaba water.
4. The content of ginsenosides Rd2, Rg3, F2, and compound K, omega-6 fatty acids, gaba, phenolics, and flavonoids according to claim 2, wherein the fermented wild ginseng aged complex has enhanced antioxidant activity. Enhanced fermented wild ginseng aged complex.
5. A food comprising the fermented wild ginseng aged complex according to any one of claims 2 to 4.
6. Cosmetics comprising the fermented wild ginseng aged complex according to any one of claims 2 to 4.
7. The food according to claim 5, wherein the food is for improving immunity, improving cognitive ability, recovering from fatigue and eating cosmetics (inner beauty).
8. The cosmetic according to claim 6, wherein the cosmetic is for whitening and wrinkle improvement.

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