KR20150050726A - fermented red ginseng concentrate having hepatoprotective activity and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a method for producing a fermented red ginseng concentrate having an increased content of ginsenoside Rg3, wherein the method comprises the steps of: a) preparing an extract from a red ginseng; b) inoculating and fermenting Lactobacillus plantarum in the red ginseng extract; c) heating the fermented liquid at a high temperature; and d) filtering and concentrating the same. According to the present invention, a red ginseng fermented concentrate produced by inoculating and fermenting Lactobacillus plantarum in a red ginseng extract, heating the fermented liquid at a high temperature, and filtering and concentrating the same has an increased content of ginsenoside Rg3 and may exhibit a hepatoprotective activity.

Description

TECHNICAL FIELD The present invention relates to a fermented red ginseng concentrate composition having liver protecting activity and a fermented red ginseng concentrate having a hepatoprotective activity and a manufacturing method thereof,

The present invention relates to a fermented red ginseng concentrate composition having increased content of ginsenoside Rg3 having liver protective activity and a preparation method thereof, more specifically, a) preparing an extract from red ginseng; b) To the above red ginseng extract was added Lactobacillus plantarum ) and fermenting the same; c) heat treating the fermentation broth at a high temperature; And d) filtering and concentrating the fermented red ginseng concentrate, wherein the content of ginsenoside Rg3 is increased.

The liver is one of the most important organs in the human body and plays an important role in detoxifying toxins from the outside. Chronic intake of alcohol, which is a social problem, causes oxidative stress toxicity in hepatocytes. The cytochrome P450 2E1 (CYP2E1), which is induced during the metabolism of alcohol in the liver, produces active oxygen, causing oxidative stress, lipid peroxidation, mitochondrial dysfunction and DNA damage. These liver diseases are the most frequently occurring in Koreans with cancer and diabetes.

Ginseng is a perennial herbaceous perennial plant belonging to ginseng belonging to the genus Arangaiaceae. It has been widely used for health promotion in Korea, China and Japan for thousands of years. Recently, it has been known that red ginseng, which contains a small amount of ginsenoside useful for the human body, is superior in the efficacy of steam ginseng steamed several times. Saponine, a representative active ingredient of ginseng, is a kind of compound called ginsenoside. Saponin, which is the main effect of ginseng extract extracted with water and ethanol, consists of more than 30 kinds of ginsenosides. Ginsenoside is a major component of red ginseng extract and has been reported to have immunity, anti-inflammatory action, anti-allergic action, anti-cancer effect, hypotensive action, anti-cholesterol action, antithrombotic action, have.

Saponins ingested in the body are degraded by microorganisms in the intestine and absorbed into the body [Panta Med. 62, pp 453-457, 1996]. In the case of ginsenoside Rg3, it is sequentially converted to ginsenosides Rb1, Rd, and Rg3 by intestinal microorganisms. Intestinal microorganisms that decompose saponin of ginseng differ in the presence or absence and presence of the microorganisms depending on the constitution of humans according to the eating habits, and there is a difference in conversion to metabolites. Therefore, there is a difference in the efficacy after taking ginseng . In addition, since intestinal microflora vary from person to person, the conversion rate and bioavailability of ginsenoside are different [J. Pharm. Pharmacol., 50, pp1155-1160, 1998]. Therefore, in order to eliminate the difference between the efficacy and the absorption rate due to differences in intestinal microorganisms, the use of lactic acid bacteria fermented product of ginseng extract using microorganisms has an effect of overcoming individual differences and increasing the body absorption rate. Numerous researchers have been conducting research to increase conversion rates.

Accordingly, the present inventors have made extensive efforts to overcome the problems of the prior art. As a result, they have found that fermentation of Lactobacillus plantarum is performed by inoculation of red ginseng extract, followed by heat treatment of the fermentation broth at a high temperature, followed by filtration and concentration It was confirmed that the content of ginsenoside Rg3 was increased in the case of the fermented concentrate of red ginseng to be produced and that it exhibited liver protective activity, thus completing the present invention.

Accordingly, it is a main object of the present invention to provide a method for producing a fermented red ginseng concentrate having an increased content of ginsenoside Rg3 and having liver protecting activity.

Another object of the present invention is to provide a liver protecting active composition comprising the fermented red ginseng concentrate prepared by the above production method.

It is still another object of the present invention to provide a health functional food comprising the fermented red ginseng concentrate prepared by the above production method.

According to one aspect of the present invention, the present invention provides a method for preparing fermented red ginseng concentrate having an increased content of ginsenoside Rg3 comprising the steps of:

a) preparing an extract from red ginseng;

b) inoculating the red ginseng extract with Lactobacillus plantarum and fermenting the same;

c) heat treating the fermentation broth at a high temperature; And

d) filtering and concentrating

In the present invention, the term " fermented red ginseng concentrate " refers to a product obtained by inoculating and culturing microorganisms, particularly Lactobacillus plantarum , in the red ginseng extract. In addition, "the content of ginsenoside Rg3 increased" means that the content of ginsenoside Rg3 is increased compared to that of the red ginseng without fermentation and heat treatment.

Saponin, which represents the major efficacy of ginseng extract, is composed of more than 30 kinds of ginsenosides. Ginsenoside is a major component of red ginseng extract. Saponin ingested into the body is degraded by the microorganisms in the intestine and absorbed into the body. In the case of ginsenoside Rg3, intestinal microorganisms convert ginsenosides Rb1, Rd, and Rg3 sequentially. The intestinal microorganisms that degrade saponin may have different efficacy after administration of ginseng because the presence or absence of the microorganism differs depending on the human body's eating habits and dietary habits.

Therefore, the inventors of the present invention prepared fermented red ginseng concentrate in order to eliminate the difference between the efficacy and the absorption rate due to differences in intestinal microorganisms, and found that the ginsenoside Rg3 content was higher than that of the non-fermented red ginseng extract and was effective for liver protecting activity And completed the present invention.

In the step a) of the present invention, the step of extracting red ginseng with 60 to 80% alcohol concentrate, diluting the extract to 10 to 30%, sterilizing the extract at 90 to 120 ° C, and cooling to 35 to 40 ° C And a control unit.

According to the embodiment of the present invention, when the fermented red ginseng concentrate is prepared, 60% of the red ginseng extract is diluted to 20%, since the lactic acid bacteria do not grow when the red ginseng extract is thick, so the red ginseng extract is diluted to 10 ~ 30% do.

In the present invention, the step (b) may include adding the red ginseng extract to a Lactobacillus plantarum ) and then cultured at 35 to 40 ° C for 24 to 48 hours.

If the temperature of the lactic acid bacteria deviates from the culture temperature, it is difficult to proliferate the bacteria. In addition, if the bacteria are cultured for more than 48 hours, the bacteria will self-divide and die.

In the present invention, the Lactobacillus plant plantarum is inoculated into an MRS medium and then added to an extract of red ginseng after culturing in a shaking incubator.

In the present invention, the above-mentioned Lactobacillus plantarum ) is added in an amount of 2 to 10% (v / v) relative to the extract of red ginseng.

If the amount of the inoculum of the microorganism is small, the microorganism can not grow and die and can not ferment. If the microorganism is too much, the time point of reaching the apex of the growth curve comes soon. Ginsenoside is converted by microorganism metabolism. It is difficult to determine the end point of culture if the number of germs is large because of the large number of initial bacteria. Metabolic process is insufficient.

In the present invention, Lactobacillus < RTI ID = 0.0 > plantarum ), the agitation is performed at 20 to 100 rpm.

In the present invention, the step (c) is characterized in that the heat treatment is performed at 100 to 150 ° C for 1 to 5 hours.

When the heat treatment is performed at such a high temperature as described above, the content of ginsenosides Rb1, Rb2 and Rd, which are precursors of ginsenoside Rg3, falls to 0.1 mg / g or less and the ginsenoside Rg3 is converted to 10 mg / g or more. According to the experimental example of the present invention, as shown in FIG. 4, in the case of the non-fermented red ginseng extract (Comparative Example 1), the content of ginsenoside Rg3 was 0.5 mg / g, but the fermented red ginseng concentrate In the case of Example 1), it can be confirmed that the content increases as ginsenoside Rb1 is converted to ginsenoside Rg3. Further, in the case of Example 2 in which the heat treatment was performed at a different temperature and time than in Example 1, the content of ginsenoside Rg3 was increased (Comparative Example 1), but it was lower than that of Example 1 Can be confirmed. Therefore, it can be seen that the fermented red ginseng concentrate prepared according to the preparation method of the present invention contains a higher content of ginsenoside Rg3 than the red ginseng extract (Test Example 1).

In the present invention, the fermented red ginseng concentrate is characterized in that the crude noside Rg3 content is 10 mg / g or more, preferably 10 to 30 mg / g.

According to the embodiment of the present invention, it can be seen that the fermented red ginseng concentrate (Experimental Example 1) contains a higher content of ginsenoside Rg3 than the red ginseng extract (Comparative Example 1), and as a result, the red ginseng fermented concentrate Can have greater efficacy.

According to another aspect of the present invention, there is provided a liver protective active composition comprising a fermented red ginseng concentrate having an increased content of ginsenoside Rg3 produced by the method of the first aspect.

In the present invention, the fermented red ginseng concentrate is characterized by exhibiting liver protective activity against ethanol, liver protective activity against acetaldehyde, and liver protective activity against nonalcoholic fatty liver.

Acetaldehyde is an alcohol metabolite. It is highly reactive and has a strong ability to bind to proteins and DNA. It is a substance that deteriorates cell function. Fatty acid is a component of alcoholic fatty liver Causing toxicity to the liver.

According to the experimental example of the present invention, hepatoprotective activity against alcoholic oxidative stress in HepG2 cells (cells derived from liver cancer) was examined. As a result, in case of the red ginseng extract (Comparative Example 1) But the cell viability of fermented red ginseng concentrate (Example 1) was 100% or more at all concentrations. From these results, it can be seen that the fermented red ginseng concentrate of the present invention exhibits liver protective activity against alcoholic oxidative stress (Test Example 2 and Fig. 1).

In addition, the hepatoprotective effect of acetaldehyde on the cytotoxicity of HepG2 cells (hepatocarcinoma cells) was examined. As a result, the cell survival rate of red ginseng extract (Comparative Example 1) was similar to that of acetaldehyde treated group However, in the case of the fermented red ginseng concentrate of the present invention (Example 1), the cell viability was 30 to 50% at all concentrations, compared with the acetaldehyde-treated group. From these results, it can be seen that the fermented red ginseng concentrate of the present invention exhibits liver protection against the cytotoxicity of acetaldehyde (Test Example 3 and Fig. 2).

Furthermore, as a result of confirming the hepatoprotective activity against nonalcoholic fatty liver in HepG2 cell (hepatocarcinoma cell) cells, the effect of inhibiting fat accumulation in the case of red ginseng extract (Comparative Example 1) was not confirmed, but the fermented red ginseng concentrate of the present invention In case of Example 1), fat accumulation was inhibited in a concentration-dependent manner. From these results, it can be seen that the fermented red ginseng concentrate of the present invention exhibits liver protective activity against nonalcoholic fatty liver (Test Example 4 and Fig. 3 ).

The fermented concentrate of red ginseng according to the present invention may be prepared by pulverization, granulation, tableting or liquefying according to the purpose and use of the present invention, but is not limited thereto. In addition, the amount of the fermented red ginseng concentrate composition may be suitably used depending on the purpose of use, the application form and the article to be applied, and may include, for example, 0.01 to 50 parts by weight based on the total composition.

According to another aspect of the present invention, there is provided a health functional food comprising fermented red ginseng extract having an increased content of ginsenoside Rg3 produced by the production method of claim 1.

In the present invention, the health functional food may be formulated into any one form selected from the group consisting of podwer, pill, granules, capsule, tablet and drink. .

According to the present invention, when the extract of the present invention is used as a food additive, it can be used as it is or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The ingredients other than the extract may be appropriately selected and blended by those skilled in the art according to the formulations, and the active ingredients may be used alone or in combination of two or more. The amount of mixing can be suitably determined according to the intended use (prevention, health or therapeutic treatment).

In the case of the fermented red ginseng concentrate prepared by the production method of the present invention, the content of ginsenoside Rg3 is increased and it exhibits liver protecting activity against ethanol, acetaldehyde and fatty acid, and thus it can be used variously in health functional food or medicine field .

Figure 1 is a plot of liver protective activity against alcoholic oxidative stress.
Figure 2 is a plot of liver protective activity against the cytotoxicity of acetaldehyde.
Figure 3 is a plot of non-alcoholic fatty liver inhibitory activity.
4 is a ginsenoside analysis chart.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

The fermented red ginseng concentrate according to the present invention means that the ginsenosides Rb1, Rb3 and Rd are converted stepwise by fermentation of the red ginseng extract to increase the content of ginsenoside Rg3. More specifically, the Rg3 content of ginsenosides may be 10 mg / g or more. In addition, the test for liver protective activity was tested in Example 1 in which the content of ginsenoside Rg3 was the highest.

Hereinafter, the present invention will be described in more detail with reference to the following examples.

Comparative Example  1: Production of red ginseng extract

To 1 kg of red ginseng purchased from Punggi ginseng cooperative, 10 L of 70% alcohol was added and the mixture was refluxed at 80 ° C for 8 hours. The extract was filtered and concentrated to a solid content of 60% to yield 427 g.

Example  One : Of fermented red ginseng concentrate  Produce

400 g of constant water was added to 200 g of red ginseng extract prepared in the same manner as in Comparative Example 1, diluted to a solid content of 20%, and sterilized at 100 DEG C for 30 minutes. After cooling to 37 < 0 > C, MRS medium was supplemented with lactobacillus plantarum , KCCM11019P), cultured at 37 ° C for 24 hours, and cultured at 50 rpm for 36 hours. After the incubation, the mixture was heat-treated at 121 ° C for 2 hours, cooled to 50 ° C and filtered. And concentrated to a solid content of 60% to prepare 171 g of fermented red ginseng concentrate.

Example  2 : Of fermented red ginseng concentrate  Produce

400 g of constant water was added to 200 g of red ginseng extract prepared in the same manner as in Comparative Example 1, diluted to a solid content of 20%, and sterilized at 100 DEG C for 30 minutes. After cooling to 37 < 0 > C, MRS medium was supplemented with lactobacillus plantarum , KCCM11019P), cultured at 37 ° C for 24 hours, and cultured at 50 rpm for 36 hours. After the incubation, the mixture was heat-treated at 100 ° C for 30 minutes, cooled to 50 ° C and filtered. And concentrated to a solid content of 60% to prepare 175 g of fermented red ginseng concentrate.

Test Example  One : High performance liquid chromatography  Of the extract used Gin Senocide  analysis

High Performance Liquid Chromatography (UPLC, Waters) was used to measure the content of the ginsenoside components contained in the red ginseng extract and fermented red ginseng concentrate prepared in Comparative Example 1 and Examples 1 and 2 . The experimental procedure for measuring the content of ginsenosides is described in detail below.

First, 1 g of the red ginseng extract of Comparative Example 1 and the fermented red ginseng concentrate of each of Examples 1 and 2 were weighed into a 50 mL mass flask, and then dissolved in ionized water. The solution was passed through a C ₁₈ cartridge (Sep-Pak Plus, WAT020515, Waters, USA) in which the solution was activated, washed with water and 30% (v / v) methanol, eluted with 20 mL of methanol, Concentrated under reduced pressure, dissolved in 2 mL of methanol and used for analysis. As a standard, ginsenosides Rb1, Rg3-S and Rg3-R were dissolved in methanol at a concentration of 1 mg / ml to prepare stock solutions, and 1.0, 0.1 and 0.01 mg / ml of a sample solution was prepared. To prepare a calibration curve. The measurement results of each extract are shown in Table 1.

ginsenoside Rb1 (mg / g) ginsenoside Rg3 (mg / g) Comparative Example 1 28.9 0.5 Example 1 0.05 12.5 Example 2 19.2 3.4

As a result, as shown in Table 1 and Fig. 4, the content of ginsenoside Rg3 in Example 2 was increased about 6.8 times, and the content of ginsenoside Rb1, which is a precursor of ginsenoside Rg3 in Example 1, %, The content of ginsenoside Rg3 increased by 25 times. These results indicate that the fermentation of red ginseng concentrate is accompanied by the conversion of fermentation and the increase of ginsenoside Rg3 content after heat treatment at high temperature after fermentation.

Test Example  2: Alcoholic Oxidative  Liver protective activity against stress

HepG2 cells were seeded on a 24-well plate at a density of 5 × 10 ⁴ cells / well and cultured for 24 hours. Then, the existing medium was removed and 900 μl of a medium containing 5% FBS and 0.015 μg / ml PMA, (Comparative Example 1 and Example 1) were diluted to 0.1, 0.5, and 1.0 mg / ml, respectively, and 100 쨉 l of each concentration was added thereto. After culturing for 2 hours, the cells were treated with 300 mM of ethanol and cultured for 24 hours. The medium was washed twice with PBS, replaced with fresh medium, and then incubated with 50 μL of MTT solution for 2 hours. After removing the medium, the formazan crystals formed in the cells were dissolved by adding 500 ul of DMSO and the absorbance was measured at 540 nm using a microplate reader (BIORAD 680, BIORAD, USA).

As a result, as shown in FIG. 1, the cell survival rate was 54.4% in the 300 mM ethanol-treated group, and improved to 70.7% in the 0.1 mg / ml in the Comparative Example 1, and decreased to 37.6% in the 1 mg / mL . In other words, it showed an alcoholic liver protective activity at low concentration and some cytotoxicity at high concentration. On the other hand, in the case of Example 1 which is a fermented concentrate of the present invention, the cell survival rate was 100% or more at all concentrations. As described above, in Example 1, no toxicity was observed at a high concentration.

Test Example  3: Acetaldehyde Protective Effect on Cytotoxicity

HepG2 cells were seeded on a 24-well plate at 5 × 10 ⁴ cells / well and incubated for 24 hours. Each sample was diluted to 0.1, 0.5, and 1.0 mg / ml, diluted 100 μl per concentration, cultured for 24 hours, and then cultured for 24 hours with 4 mM acetaldehyde. The medium was washed twice with PBS, replaced with fresh medium, and then added with 50 μl of MTT solution and cultured for 2 hours. The medium was removed and the formazan crystals formed in the cells were dissolved by adding 500 ul of DMSO and the absorbance was measured at 540 nm using a microplate reader.

As a result, as shown in FIG. 2, the hepatoprotective effect of acetaldehyde on the cytotoxicity was measured, and the cell survival rate was reduced to 55.5% by 10 mM acetaldehyde. In Comparative Example 1, it was difficult to confirm the improvement effect. On the other hand, in Example 1, 30 to 50% of the liver protective activity was confirmed depending on the concentration.

Test Example  4: Non-alcoholic fatty liver inhibitory activity

HepG2 cells were seeded on a 24-well plate at 2 × 10 ⁴ cells / well and cultured for 24 hours. Each sample was diluted to a constant of 0.1, 0.5, and 1.0 mg / ml, treated with 100 μl of each concentration, cultured for 24 hours, and then the medium was removed. After washing twice with PBS, 100 μl of 3 mM oleic acid dissolved in 900 μl of 1% BSA solution was added and incubated for 24 hours. Oil Red O (ORO) staining was performed as follows. After removing the medium from each well and washing twice with ice PBS, 200 μl of 10% formalin was treated for 1 hour, washed twice with distilled water, treated with 200 μl of ORO working solution, and reacted at room temperature for 2 hours . Remove ORO solution and remove unlabeled ORO by washing twice with distilled water. Add 300 μl of isopropanol to the stained ORO, and transfer 100 μl of the solution to a 96-well plate. Add absorbance at 490 nm using a microreader Respectively. Results were expressed as a percentage of the control group. As a positive control, oltipraz was treated at 5 ug / mL.

As a result, as shown in Fig. 3, the positive control oltipraz inhibited about 20% fat accumulation at 5 ug / mL. In Comparative Example 1, almost no inhibitory effect was confirmed, but in Example 1, the activity was confirmed in a concentration-dependent manner. At 1 mg / mL of Example 1, about 15% of the fat accumulation was inhibited.

Korea Microorganism Conservation Center (overseas) KCCM11019P 20090721

Claims (12)

A method for preparing a fermented red ginseng concentrate having an increased content of ginsenoside Rg3 comprising the steps of:
a) preparing an extract from red ginseng;
b) inoculating the red ginseng extract with Lactobacillus plantarum and fermenting the same;
c) heat treating the fermentation broth at a high temperature; And
d) filtering and concentrating.
The method according to claim 1, wherein the step a) comprises: extracting red ginseng with 60 to 80% alcohol concentrate; diluting the extract to 10 to 30% by weight; sterilizing the extract at 90 to 120 ° C; Wherein the concentration of ginsenoside Rg3 is increased. ≪ RTI ID = 0.0 > 11. < / RTI >
The method of claim 1, wherein step b) comprises adding the red ginseng extract to a Lactobacillus plantarum ), and culturing the cells at 35 to 40 ° C for 24 to 48 hours. The method for producing the fermented red ginseng concentrate according to claim 1, wherein the ginsenoside Rg3 content is increased.
4. The method according to claim 3, wherein the Lactobacillus plantarum ) is inoculated into an MRS medium and then added to an extract of red ginseng after culturing in a shaking incubator to produce a fermented red ginseng concentrate having an increased content of ginsenoside Rg3.
4. The method according to claim 3, wherein the Lactobacillus plantarum ) is added in an amount of 2 to 10% (v / v) relative to the extract of red ginseng. The method for producing fermented red ginseng concentrate having increased ginsenoside Rg3 content.
4. The method according to claim 3, wherein the Lactobacillus plantarum ), wherein the agitation speed is 20 to 100 rpm. The method for producing fermented red ginseng concentrate according to claim 1, wherein the ginsenoside Rg3 content is increased.
The method for preparing fermented red ginseng concentrate according to claim 1, wherein the step c) is heat-treated at 100 to 150 ° C for 1 to 5 hours.
The fermented red ginseng concentrate according to claim 1, wherein the fermented red ginseng concentrate contains 10 to 30 mg / g of crude tertiary Rg3.
A liver protecting active composition comprising a fermented red ginseng concentrate having an increased content of ginsenoside Rg3 produced by the method of claim 1.
[10] The composition of claim 9, wherein the fermented red ginseng concentrate exhibits liver protective activity against ethanol, liver protective activity against acetaldehyde, and liver protective activity against nonalcoholic fatty liver.
A health functional food comprising fermented red ginseng extract having increased content of ginsenoside Rg3 produced by the production method of claim 1.
12. The method of claim 11, wherein the health functional food is formulated into any one form selected from the group consisting of a podwer, a pill, granules, a capsule, a tablet, Wherein the functional food is selected from the group consisting of:

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