KR101203096B1 - Producing method for fermented wild grass extracts with biological activity - Google Patents

Abstract

PURPOSE: A method for preparing fermented Artemisia princeps and pine needle extract is provided to reduce fat accumulation and to suppress ROS generation. CONSTITUTION: A method for preparing fermented medicinal herb extract comprises a step of inoculating lactic acid bacteria to Artemisia princeps and pine needle extract with the lactic acid bacteria and fermenting. The lactic acid bacteria are Leuconostoc mesenteroides 1076. The extract is prepared by pulverizing Artemisia princeps and pine needles, adding distilled water, and extracting at 24-26 Deg. C. for 23-25 hours. The method comprises: a step of pulverizing one of pine needles or Artemisia princeps; a step of adding water and extracting at 25 Deg. C. for 24 hours; a step of placing the extract at 0-4 Deg. C.; a step of inoculating Leuconostoc mesenteroides 1076 isolated from Kimchi; a step of fermenting at 30 Deg. C. and 200 rpm for 36 hours and centrifuging; a step of freeze-drying at 40 Deg. C. [Reference numerals] (AA) Selecting Artemisia princeps and pine needles; (BB) Washing and drying; (CC) Pulverizing (20-30 mesh); (DD) Incubating (0-4°C,24hr); (EE) Low temperature extraction (25°C,24hr); (FF) Adding 10 times of D.W; (GG) Inoculating strain into medicinal herb extract; (HH) Preculture of Leuconostoc mesenteroides 1076; (II) Selecting excellent strains with mannitol high productivity; (JJ) Fermentation process(30°C, 200rpm, 36 hr); (KK) Centrifuging(13,200 rpm); (LL) concentration at reduced pressure(40°C); (MM) Pulverizing; (NN) Freeze-drying

Description

Producing method for fermented wild grass extracts with biological activity

The present invention relates to a method for producing fermented mugwort and fermented pine needle extract, and relates to a method for producing fermented mugwort and fermented pine needle extract enhanced by physiological activity by fermentation using lactic acid bacteria.

As the national income level improves, consumers' interest in health increases and the well-being culture spreads, and the demand for medicinal crops increases. This change, coupled with the high quality and branding of agricultural products and the trend of expansion of the agricultural industry by means of linkage with various secondary and tertiary industries, is increasing the demand for medicinal crops (Source: Korea Rural Economic Institute, Medicinal Crop Region). Strategic Industry Development Plan, 2008).

In Korea, about 60 kinds of medicinal crops such as Dangui and Cheongung are cultivated. The annual output of medicinal crops in Korea, excluding ginseng, is 563 billion won, accounting for 0.6% of total agricultural production. As of 2007, the area of cultivation is 11,433 ha and the output is 59,764 tons. Currently, many farming farmers who face the deteriorating market environment are turning crops to cultivate medicinal crops, and productivity is improved by improving cultivation technology (Source: Korea Rural Economic Institute, Fostering Regional Strategic Industries for Medicinal Crops) , 2008).

Most of these herbs are sold through a fermentation process using sugar, but the existing high sugar fermentation process has a disadvantage in that it cannot be used for diabetic patients due to the high concentration of sugar added during fermentation. However, according to a survey of consumers' selection criteria, consumers replied that the emphasis was on quality and taste as much as the functionality of herbs. Therefore, there is a need to use sweeteners to replace high sugar content while maintaining product quality and shelf life.

In addition, the existing high sugar fermentation process has a long fermentation period using the natural fermentation method, it is difficult to optimize the extraction process of the active ingredient and there is also a problem that it is difficult to expect the increase in the physiological activity (antioxidant, anti-obesity effect, etc.). Therefore, there is a need to optimize the fermentation process that can improve the functionality of the product.

On the other hand, Kimchi, a traditional Korean fermented food, produces acetic acid, ethanol, carbon dioxide, and mannitol other than lactic acid through heterogeneous fermentation of lactic acid bacteria, among which mannitol prevents aging of kimchi and prevents it from being soaked. It is known to give.

Mannitol is a kind of 6-carbon sugar alcohol, which has a sweetness of about 30-40% of sugar, and is used as a substitute sweetener in food production where the use of sugar is limited.It has properties such as low calorie, cold taste, and low hygroscopicity. It is widely used as an additive in confectionery such as candy and candy (Source: Korea Biotechnology Association, Leuconostoc Mannitol production using mesenteroides NRRL B-1149, 2002).

Accordingly, the inventors of the present invention intend to ferment herbs using lactic acid bacteria isolated from kimchi which has been traditionally used for food fermentation, thereby improving the functionality and preservation by mannitol production and greatly improving the functionality.

Korean Registered Patent No. 10-0353481 (pine needle fermented beverage and its manufacturing method) is a fine pine needles, pine needles or pine needles and the sweetener and water selected from white sugar, yellow sugar, brown sugar, honey and starch syrup to the weight of pine needles, pine needles or pine needles Disclosed is a method for preparing a fermented beverage, comprising the step of pouring a boiled and cooled sweetener solution by mixing in a weight ratio of 1 to 1.2: 19 to 21 and naturally fermenting at 20 ± 1 ° C. for 5 days, followed by filtration and can packaging for sterilization. This is related to the natural fermentation of pine needles is different from the present invention. Korean Patent No. 1000412425 (Multi-stage fermentation extract of pine needles having a hangover effect) includes a first fermentation step of naturally fermenting pine needles with water and sugar; Separating the fermentation broth from the fermentation broth obtained from the first fermentation step and pine needles; Mixing the fermentation residues of the pine needles with pine needles and extracting them by heat, then concentrating under reduced pressure to obtain a concentrated extract; A second fermentation step of mixing and fermenting the fermentation broth and the concentrated extract solution; A functional food and beverage for hangover elimination comprising a multi-stage fermentation extract of pine needles prepared by the method comprising the step of aging the mixed solution fermented in the second fermentation step in an anaerobic environment as an active ingredient, which is a multi-stage fermentation The present invention is different from the present invention.

The present invention is to provide a method for producing a fermented herbal extract that can maintain a high degree of preference even without using sugar when fermenting herbs in order to solve the problems of the prior art.

In addition, the present invention is to provide a method for producing a fermented herbal extract with a large amount of phenolic compound is significantly enhanced antioxidant capacity.

In addition, the present invention is to provide a method for producing a fermented herbal extract excellent in anti-obesity effect by excellently reducing fat accumulation and inhibiting ROS production.

In order to achieve the above object, the present invention provides a method for producing a fermented herbal extract, characterized in that the fermentation by inoculating lactic acid bacteria in one or more extracts of mugwort or pine needles.

The present invention also provides a method for producing a fermented herbal extract, characterized in that the lactic acid bacteria is Leuconostoc mesenteroides 1076.

In addition, the present invention after the one or more extracts of the mugwort or pine needles pulverized one or more of the mugwort or pine needles, the method of producing a fermented herbal extract characterized in that the extract for 23 to 25 hours at 24 ~ 26 ℃ by adding distilled water to provide.

In addition, the present invention provides a method for producing a fermented herbal extract, characterized in that the fermentation is carried out for 35 to 37 hours at 29 ~ 31 ℃ at 190 ~ 210 rpm.

The present invention relates to a method for producing a fermented herbal extract, characterized in that the fermentation by inoculating lactic acid bacteria to one or more extracts of mugwort or pine needles. As such, the present invention has a process advantage by significantly shortening the fermentation time by fermenting one or more of the mugwort or pine needles using lactic acid bacteria rather than the conventional natural fermentation method. Many physiological activities such as are greatly enhanced.

Here, the lactic acid bacteria is preferably lactic acid bacteria derived from kimchi, most preferably Leuconostoc ( Leuconostoc) mesenteroides ) 1076. Leuconostoc mesenteroides ) 1076 is very excellent in mannitol productivity, when using the fermentation of one or more extracts of mugwort or pine needles can be prepared fermentation extract having a very good sweet taste even after fermentation without adding sugar is very good.

At this time, the fermentation is preferably carried out for 35 to 37 hours at 29 ~ 31 ℃ at 190 ~ 210 rpm. If the above conditions are satisfied, a large amount of mannitol is produced, and the taste is very good, and a large amount of phenolic compounds are also produced, and thus, fermentation extracts having excellent physiological activities such as antioxidant and anti-obesity can be prepared.

In addition, the extract of one or more of the mugwort or pine needles is preferably pulverized one or more of the mugwort or pine needles, and then extracted for 23 to 25 hours at 24 ~ 26 ℃ by adding distilled water, so as to extract various physiological activity There is an advantage that the components are not destroyed.

Preferably, the present invention is coarsely pulverized at least one of pine needles or wormwood, and then extracted with low temperature (25 ℃, 24 hours) by adding 10 times of water and left at 0 ~ 4 ℃, mannitol production ability separated from kimchi Inoculated with this excellent Leuconostoc mesenteroides 1076 and fermented at 200 rpm for 30 hours at 30 ℃, centrifugation (13,200 rpm) and concentrated under reduced pressure (40 ℃) by lyophilization, greatly physiological activity Enhanced fermentation extracts can be obtained.

Thus obtained fermented extract of the present invention increased the content of the phenolic compound by approximately 11.5 times (fermented pine needle extract), 27.5 times (fermented mugwort extract) compared to the conventional herbal fermentation broth with sugar, accordingly Antioxidant and anti-obesity activities have also been enhanced compared to conventional herbal fermentation broths.

Obesity is an abnormal accumulation of fat tissue in the body due to abnormal increase in the size and number of adipocytes, the increase in the number of adipocytes is the active proliferation of preadipocytes and their activated fat It is caused by the process of adipogensis. Currently, gene-level studies on adipogenesis have been studied using several cell lines, and murine C3H10T1 / 2 embryonic cells, 3T3-F442A and 3T3-L1, are used as representative in vitro models for the study of differentiation and function of adipocytes.

In the case of 3T3-L1 adipocytes, fat accumulation and reactive oxygen species (ROS) production increase rapidly during the process of preadipocyte differentiation into adipocytes. The resulting reactive oxygen species has been found to be the cause of insulin resistance and several metabolic syndromes.

However, the fermented extract of the present invention is very excellent in inhibiting fat accumulation and reducing ROS production, and thus may be effectively used as a functional material related to metabolic syndrome. Preferably, the fermented extract may be used as an antioxidant or anti-obesity functional food composition. .

Furthermore, the fermentation extract of the present invention is fermented using Leuconostoc mesenteroides 1076, which is an excellent strain of mannitol production, so that even when fermented without adding sugar, fermentation odor, bitter taste, The astringent taste is greatly reduced, and consumer preference is very high.

According to the present invention it is possible to provide a fermented medicinal herb extract with high preference even if sugar is not added.

In addition, according to the present invention it is possible to provide a fermented medicinal herb extract having a very high content of a phenolic compound and having excellent antioxidant and anti-obesity effects.

In addition, the present invention can provide a fermented herbal extract with a very high anti-oxidation and anti-obesity effect even with short-term fermentation.

1,Lactobacillus The production amount of mannitol of the genus Lactic acid bacteria (19 species) is shown.
2 isLeuconostoc The production amount of mannitol of the genus Lactic acid bacteria (27 species) is shown.
3 isWeissella The production amount of mannitol of the genus Lactic acid bacteria (5 types) is shown.
4 isLactobacillus The yield of mannitol production of the genus Lactic acid bacteria (19 species) is shown.
5 isLeuconostoc The yield of mannitol production of the genus Lactic acid bacteria (27 species) is shown.
6 isWeissella The yield of mannitol production of the genus Lactic acid bacteria (5 species) is shown.
7 isLactobacillus Mannitol productivity of the genus Lactic acid bacteria (19 species) is shown.
8 isLeuconostoc Mannitol productivity of the genus Lactic acid bacteria (19 species) is shown.
9 isWeissella Mannitol productivity of the genus Lactic acid bacteria (5 types) is shown.
10 is leukonostock mesenteroides by Lactic acid concentration (Leuconostoc mesenteroides) Shows a specific growth rate of 1076.
Figure 11 shows the leukonostock mesenteroides for each acetic acid concentration (Leuconostoc mesenteroides) Shows a specific growth rate of 1076.
Figure 12 shows the total phenolic content of wild vinegar fermentation broth, fermented mugwort extract, fermented pine needle extract and mixed fermented extract.
Figure 13 shows the total flavonoid content of wild vinegar fermentation broth, fermented mugwort extract, fermented pine needle extract and mixed fermented extract.
Figure 14 shows the DPPH radical scavenging ability of wild vinegar fermentation broth, fermented mugwort extract, fermented pine needle extract and mixed fermented extract.
Figure 15 shows the ABTS radical scavenging ability of wild vinegar fermentation broth, fermented mugwort extract, fermented pine needle extract and mixed fermented extract.
Figure 16 shows the FRAP activity of wild vinegar fermentation broth, fermented mugwort extract, fermented pine needle extract and mixed fermented extract.
Figure 17 shows the reducing power (Reducing Power) of wild vinegar fermentation broth, fermented mugwort extract, fermented pine needle extract and mixed fermented extract.
Figure 18 shows the cytotoxicity measurement results of the sample through the XTT assay.
Figure 19 shows the result of measuring the fat accumulation inhibitory effect through ORO staining.
20 shows the results of measuring the inhibition of ROS production through NBT assay.
21 shows the effect of inhibiting fat accumulation and reducing ROS production of fermented mugwort extract and fermented pine needle extract.
Figure 22 shows the sensory characteristics of fermented mugwort and fermented pine needle extract.
Figure 23 is a production process of the fermented pine needle extract, fermented mugwort extract of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

Experimental Example  One : Mannitol  Selection of strains with high productivity

In order to select strains with high mannitol productivity, 51 kimchi-derived lactic acid bacteria (19 species of Latobacillus , 27 species of Leuconostoc , and 5 species of Weissella ) were used in the experiment. Kimchi-derived lactic acid bacteria were used by the Korea Microbial Resources Center for preservation and laboratory strains. Lactobacillus pre-cultured in 2% glucose MRS (BD, USA) medium was washed twice with sterile water, and then inoculated at 5% fructose MRS (BD, USA) medium at an initial concentration of 0.05 (OD ₆₀₀ ) of 0.05 for 24 hours. (30, 200 rpm). The supernatant was obtained by recovering 1 mL of MRS (5% fructose) inoculation and after 24 hours of incubation, and mannitol production was measured using a mannitol assay kit (Megazyme, Ireland).

The results are shown in FIGS. 1 to 9. A total of 51 lactic acid bacteria screenings were performed to compare the production, yield, and productivity of mannitol, showing the results of Leuconostoc. mesenteroides ) 1076 showed the highest mannitol productivity.

Experimental Example  2: Leukono Stock Mecenteroides ( Leuconostoc mesenteroides ) Osmosis Resistance Verification of 1076

Since lactic acid bacteria were inhibited by acetic acid and lactic acid produced during the culture, the resistance to these by-products was determined by measuring the osmotic resistance.

Leuconostoc in a medium containing lactic acid at 1, 2, 3, 4, 5, 6, 10 g / L mesenteroides ) 1076 was shown in Figure 10, it was confirmed that the specific growth rate of L. mesenteroides significantly decreased.

Leuconostoc in a medium containing 1, 2, 3, 4, 5, 6, 10 g / L of Acetic acid mesenteroides ) 1076 was shown in Figure 11, it was confirmed that the specific growth rate of L. mesenteroides significantly decreased.

Leuconostoc mesenteroides ) 1076 was able to confirm the osmotic resistance.

Manufacturing example  1: Preparation of Fermented Pine Needle Extract

10 times distilled water was added to the coarsely pulverized pine needle powder, followed by low temperature extraction with stirring at 25 ° C. for 24 hours, followed by Leuconostoc , a highly mannitol high-productive strain, Leuconostoc. mesenteroides ) 1076 was precultured in MRS (2% glucose) medium, and then cultured for 36 hours at 30 ° C. and 200 rpm in 1 L of the pine needle extract. Fermented pine needle extract was concentrated under reduced pressure and lyophilized to powder.

Manufacturing example  2: Preparation of Fermented Mugwort Extract

Mugwort extract fermented in the same manner as in Preparation Example 1 was prepared except that instead of using pine needles.

Manufacturing example  3: Preparation of Fermented Mixed Extract

A mixed extract (fermented pine needles: fermented mugwort = 1: 1, w / w) of the same amount of fermented mugwort leaf extract and fermented mugwort extract of Preparation Examples 1 and 2 was prepared.

compare Manufacturing example  : Manufacture of Sanyacho Fermentation Broth

After washing the outposts of wild vinegar (pine needles, mugwort, purslane, etc.), put them in a jar, add sugar, and ripen them at room temperature for 180 days, and then fermented wild vinegar by filtering the wild vinegar fermentation broth for more than 180 days. .

Experimental Example  3: Analysis of Useful Components of Fermented Mugwort Extract and Fermented Pine Needle Extract

<Total Phenolic Content>

Each sample was prepared at a concentration of 1 mg / mL and then 1 mL of the sample was added to a 15 mL tube. Then 10% Folin reagent and 1 mL of 10% Na ₂ CO ₃ were added. After reacting for 1 hour, 200 μl was transferred to a 96-well plate and the absorbance was measured at 750 nm.

The results are shown in Figure 12, 116.30 ± 4.58 GAE mg / g in fermented pine needle extract, 181.63 ± 3.51 GAE mg / g in fermented mugwort extract, fermented mixed extract (fermented pine needles: fermented mugwort = 1: 1, w / w ) Shows 158.30 ± 3.00 GAE mg / g, which is higher in total phenolic content than wild vinegar fermentation broth.

<Total Flavonoid Content>

The total flavonoid content was prepared at a concentration of 1 mg / mL for each sample, and 0.5 mL of the sample was added to a 15 mL tube. After ethanol 95% was added 1.5 mL, 10% aluminum nitrate 0.1 mL, 1 M potassium acetate 0.1 mL, distilled water 2.8 mL and left at room temperature for 30 minutes. The absorbance was transferred to a 96-well plate reacted for 30 minutes at room temperature 200 ㎕ measured at 415 nm.

The results are shown in Figure 13, 87.90 ± 3.68 NE mg / g in fermented pine needle extract, 145.13 ± 16.88 NE mg / g in fermented mugwort extract, fermented mixed extract (fermented pine needles: fermented mugwort = 1: 1, w / w ), 115.04 ± 15.42 NE mg / g was measured, indicating that the total flavonoid content was higher than that of wild vinegar fermentation broth.

<Contents of Individual Phenolic Compounds from Fermented Mugwort or Fermented Pine Needle Extract and Wild Vegetable Fermentation Solution>

The contents of the individual phenolic compounds contained in the fermented pine needles, fermented mugwort extracts of Preparation Examples 1 and 2, and the Sanyacho fermentation broth of Comparative Preparation Example were analyzed.

The HPLC system used a Shimadzu model (Shimadzu-Model No. SPD-M10A VP, Kyoto, Japan), separated by an Agilent C18 column (15 cm × 4.6 mm, 5 μm, Agilent Technologies, Santa Clara, CA, USA). The components were analyzed with a diode-array detector. The mobile phase consisted of 6% acetic acid (final pH 2.55, v / v, solvent A) and acetonitrile (solvent B) containing 2 mM sodium acetate, and the flow rate was 1.0 mL / min. The composition of the solvent was analyzed for a total of 80 minutes. The composition of the solvent was 0 to 15% B (45 min), 15 to 30% B (15 min), 30 to 50% B (5 min), 50 to 100% B (5 min). ) And 100 ~ 0% B (10 min) injection volume was analyzed by 10 μl. Standards used in the analysis were 4-hydroxyl benehydrazid, coumarin, garlic acid, pyrogallol, catechin, vanillic acid, chlorogenic acid, caffeic acid, p-comaric acid, p-anisic acid, coumarin, rutin, quercitrin hydrate, myricetin, qucetein , ruteorin, hesperetin, dexamethasone, alizarin, and rhein were used.

Name Retention time
(min) ug / g extract Production Example 1 Production Example 2 Comparative Production Example 4-hydroxyl benehydrazid 2.78 ^-1) 1,715.84  - coumarin 2.82  -  - 59.72 garlic acid 3.04 0.10 11.57 84.41 pyrogallol 3.46  - 2,324.89 414.01 catechin 11.22 0.97  - 204.28 vanillic acid 16.51  - 3,390.04 79.35 chlorogenic acid 16.75 6,828.22  -  - caffeic acid 17.62  - 0.45  - p-comaric acid 28.30  - 9.86  - p-anisic acid 40.97 535.16 669.72  - coumarin 42.55 753.53  -  - rutin 47.36 4,002.70 10,789.58  - quercitrin hydrate 53.98 202.25 345.80  - myricetin 54.32  - 617.76  - qucetein 61.74  - 10.05 1.07 ruteorin 62.44 102.26  -  - hesperetin 65.48 4.61 12,720.80 0.27 dexamethasone 66.15 56.77  - 32.53 alizarin 69.25 10.78 49.90 129.05 rhein 70.40 1,134.04  - 182.01 Total amounts 13,630.39 32,656.26 1,186.7

¹⁾ Not detected

The content of phenolic compounds in fermented mugwort or fermented pine needle extract and wild vinegar fermentation broth was measured using the area ratio of peaks corresponding to retention time of 20 standard materials.

As a result of the total phenolic content, the total amount of individual phenolic compounds was found in the order of fermented mugwort extract (32,656 ug / g)> fermented pine needle extract (13,631 ug / g)> wild vinegar fermentation broth (1,186 ug / g). Compared to the preparation of Sanyacho fermentation broth, the content of these phenolic compounds was about 11.5 times higher in the fermented pine needle extract, and about 27.5 times higher in the case of fermented mugwort extract.

In addition, pyrogallol, catechin, alizarin, and rhein were analyzed as major phenolic compounds in the fermented broth of the comparative preparation, whereas chlorogenic acid, rutin, rhein, p-comaric acid, p- Anisic acid was analyzed as a major phenolic compound. On the other hand, the fermented mugwort extract of Preparation Example 2, which had the highest total amount of phenolic compounds, had a high content of hesperetin, rutin, vanillic acid, pyrogallol, and 4-hydroxyl benehydrazid, which was different from fermented pine needle extract.

In summary, it was found that the fermented pine needle extract and the fermented mugwort extract of Preparation Examples 1 and 2 had a very high total phenol and total flavonoid content as compared to the wild vegetable fermentation broth of Comparative Preparation Example.

Experimental Example  4: Antioxidant Activity of Fermented Mugwort and Fermented Pine Needle Extracts

<Electron donating ability measurement (DPPH radical scavenging activity measurement)>

Each sample was prepared at a concentration of 0.1 mg / mL and 0.5 mg / mL. The sample divided by concentration was added to 0.2 mL and 0.8 mL of 0.0004 M DPPH reagent in a 15 mL tube and allowed to stand at room temperature for 10 minutes. Thereafter, 200 μl was transferred to a 96-well plate and the absorbance was measured at 517 nm.

The results are shown in Figure 14, fermented mugwort extract, fermented mixed extract (fermented pine needles: fermented mugwort = 1: 1, w / w), the fermented pine needle extract was found to have a very high antioxidant effect compared to the fermentation of wild grass.

<Determination of ABTS radical scavenging activity>

Each sample was prepared at concentrations of 0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL. A mixed solution of 7 mM ABTS and 2.45 mM Potassium persulfate in a 1: 0.5 ratio was incubated at room temperature for 16 hours. An ABTS solution containing 1 mL of this mixed solution and 88 mL of anhydrous ethanol was prepared. 1 mL of ABTS solution and 10 L of sample were added to a 1.5 mL tube and allowed to react for 6 minutes. Thereafter, 200 μl was transferred to a 96-well plate and the absorbance was measured at 734 nm.

The results are shown in Figure 15, fermented mugwort extract, mixed fermented extract (fermented pine needles: fermented mugwort = 1: 1, w / w), the fermented pine needle extract was found to have a very high antioxidant effect compared to wild vinegar fermentation broth.

<FRAP activity measurement>

Each sample was prepared at concentrations of 0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL. Prepare a FRAP solution mixed with 300 mM sodium acetate buffer, 10 mM TPTZ, and 20 mM FeCl ₃ ˜6H ₂ O in a 10: 1: 1 mixture, add 1.9 mL of FRAP solution and 100 μl of sample to a 2 mL tube. The reaction was carried out for a minute. Thereafter, 200 μl was transferred to a 96-well plate and the absorbance was measured at 590 nm.

The results are shown in Figure 16, the fermented mugwort extract, mixed fermented extract (fermented pine needles: fermented mugwort = 1: 1, w / w), the fermented pine needle extract was found to have a very high FRAP activity compared to wild vegetable fermentation broth.

<Reducing power>

Each sample was prepared at concentrations of 0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL. 0.5 mL of the sample divided by concentration, 2.5 mL of 0.2 M sodium phosphate buffer solution and 2.5 mL of 1% Potassium ferricyanide were added to a 50 mL tube, followed by incubation for 50 to 20 minutes. Thereafter, 2.5 mL of 10% trichloroacetic acid was added to the mixture and centrifuged for 10 minutes. 2.5 mL of the supernatant was taken, and 2.5 mL of tertiary distilled water and 0.5 mL of 0.1% Iron (III) Chloride were mixed, and 200 μl of each of the 96-well plates was measured at an absorbance of 655 nm.

The results are shown in Figure 17, the fermented mugwort extract, mixed fermented extract, the fermented pine needle extract was found to have a very high antioxidant effect compared to the wild vegetable fermentation.

Experimental Example  5: Evaluation of Cytotoxicity of Fermented Mugwort and Fermented Pine Needle Extracts

Cytotoxicity evaluation of the sample for 3T3-L1 was measured using the XTT assay kit.

Increase bioreduction by adding PMS (phenazine methosulfate) that acts as an election coupling agent to XTT, and then add XTT and PMS to cells to decompose the tetrazolium ring of XTT by mitochondrial dehydrogenase in living cells to form formazan crystal When the formazan crystal is dissolved in an aqueous solution, yellow color is obtained. The yellow color was measured by ELISA (VersaMax ELISA Microplate Reader, Molecular Devices, Sunnyvale, USA) and used for evaluation of cytotoxicity. The cytotoxicity was calculated by subtracting the absorbance value of 690 nm from the 450 nm absorbance value.

The results are shown in Figure 18, the concentration of 500 ㎍ / mL does not appear to be cytotoxic compared to the control group, the efficacy evaluation of the sample for the adipocyte differentiation and intracellular fat accumulation of the following was carried out at 500 ㎍ / mL concentration .

Experimental Example  6: Metabolic syndrome using cell lines (fat accumulation inhibition and free radicals Abatement Efficacy Verification

The samples used in this experiment were dissolved in DMSO (dimethyl sulfoxide) and used for 3T3-L1 cytotoxicity and adipocyte differentiation experiments.

The 3T3-L1 cell line was used from the American Type Culture Collection (ATCC, CL-173, Manassas, VA, USA) and used for insulin, Oil Red O, Dexamethasone (DEX), 3 used for 3T3-L1 cell culture and differentiation. -isobutyl-1-methylxanthine (IBMX) was purchased from Sigma (Sigma-Aldrich Co., St. Louis, MO, USA), Dulbecco's modified Eagle's medium (DMEM), bovine serum (BS), fetal bovine serum (FBS) , penicillin-streptomycin (P / S), phosphate-buffered saline (PBS) and trypsin-EDTA were purchased from Gibco (Gaithersburg, MD, USA).

3T3-L1 precursor cells were seeded at 1 × 0 ⁶ in 24-well and 96-well, respectively, according to the experimental purpose, and then concentrated glucose DMEM (89%) containing BS (10%) and P / S (1%). Incubated until 100% confluence. Two days later, all cells were adipated with DMEM containing adipocyte differentiation-inducing substance (10 μg / mL insulin, 1 μM DEX, 0.5 mM IBMX) and FBS (10%) and P / S (10%). Differentiation was induced into cells, and when adipocyte differentiation (day 0), the samples were treated with DMEM at 500 ㎍ / mL according to the results of toxicity evaluation.

The differentiation of adipocytes was differentiated for 8 days after treatment with differentiation-inducing substances, each sample was treated with medium containing 10 μg / mL insulin, 1% P / S and 10% FBS every 2 days. Fat accumulation and ROS production were observed.

<Observation of fat accumulation by Oil red O staining>

In order to determine the amount of 3T3-L1 intracellular fat accumulation according to the differentiation process, each sample was treated to remove culture medium of differentiated 3T3-L1 cells for 8 days in 24-well, and then 500 μl of 10% formalin solution was added for 5 minutes. It was left at room temperature. After 3T3-L1 cells differentiated with the same amount of formalin solution was left at room temperature for at least 1 hour, formalin was removed and washed with 500 µl of 60% isopropanol solution to completely dry the cells. Completely dried cells were sufficiently stained fat components accumulated in the cells with a pre-prepared Oil red O working solution (Oil red O: DDW = 6: 4), washed the cells 3-4 times with distilled water and again It was dried completely. Oil red O bound to the fat component accumulated in cells was eluted with 100% isopropanol, and then absorbance was measured at 490 nm using ELISA.

The results are shown in Figure 19, it was found that the effect of inhibiting fat accumulation in fermented pine needle extract and mixed fermentation extract is excellent.

<ROS measurement by NBT assay>

In order to measure the ROS production of adipocytes according to differentiation process, first remove the culture solution of 3T3-L1 cells cultured and differentiated in 24-well, and then wash twice with sterilized PBS (Phosphate buffer saline, pH 7.4), 0.2 0.2 mL of% NBT solution was added and reacted for 90 minutes in a CO ₂ incubator. ROS accumulated in adipocytes reacted with NBT solution to produce dark blue formazan, and the absorbance was measured at 570 nm by eluting all of these dark blue formazan using 100% acetic acid.

The results are shown in Figure 20, it was found that the effect of inhibiting the ROS production in fermented pine needle extract, mixed fermentation extract.

<Expression of major genes>

To examine the changes in major genes involved in differentiation in adipocytes, total RNA in adipocytes was extracted, and cDNA was prepared using reverse transcriptase. Synthesized cDNA and primers for each gene were amplified by RT-PCR and the expression levels of the genes were measured.

-Extraction of Total RNA and Synthesis of cDNA: Total RNA was extracted from adipocytes using TRIzol reagent (phenol + guanidine isothiocyanate). Adipocytes at 8 days of differentiation were washed twice with PBS and 1 mL of TRIzol was added to harvest the cells. Add 0.2 mL of chloroform per 1 mL of TRIzol reagent, shake well for 15 seconds, react for 2-3 minutes at room temperature, centrifuge at 15,000 rpm for 10 minutes, remove the supernatant, transfer to another tube, and use isopropyl alcohol (first used 0.5 mL per 1 mL of TRIzol) was added and allowed to react for 10 minutes to precipitate RNA. After washing the precipitated RNA with 70% DEPC-ethanol (1 mL per 1 mL of the first TRIzol used), centrifuge at 11,000 rpm for about 5 minutes, dry the pellet at room temperature for 5-10 minutes, and 40 mL of DEPC water. The total RNA concentration was quantified by measuring the OD value with a spectrophotometer (260 nm). 5 μg of total RNA of the same concentration was added to cDNA Premix (Maxime RT Premix (oligo dT primer), Seongnam, South Korea), and DEPC-water was added so that the total volume was 20 μl. CDNA was synthesized by treatment at 95 ° C. for 5 minutes.

-Semi-quantitative RT-PCR: After mixing primers of major genes related to cDNA and adipocyte differentiation (Table 2), RT-PCR amplification mixture was mixed to make 12 μl total volume, denaturation, annealing, The polymerization was repeated 35 times to amplify the desired DNA portion. At this time, the annealing temperature was treated at the appropriate temperature according to the primer. To confirm the RT-PCR product, 5 μl was taken and electrophoresed on a 1.5% agar (agarose) gel, followed by staining with EtBr (ethidium bromide) to confirm the amplified DNA band using a transilluminator. DNA bands were expressed by band intensity using Carestream MI SE program.

The results are shown in Figure 21, as a result of confirming the expression level of PPAR and aP2 genes related to the production of fat, the cells treated with the fermented mugwort extract, fermented pine needle extract and mixed fermented extract inhibits fat production more than conventional wild vegetable fermentation broth. , Especially in fermented pine needle extract and mixed fermented extract.

In addition, in the case of NOX4 associated with ROS production showed a similar tendency to the NBT assay results, even in the case of the antioxidant enzyme Cu / Zn SOD in the fermented mugwort extract, fermented pine needle extract and mixed fermentation extract of the present invention than conventional wild vegetable It can be seen that the expression is strong.

On the other hand, the inhibition of differentiation of 3T3-L1 adipocytes showed the highest inhibitory effect in fermented pine needle extract, unlike the results of total phenol, total flavonoid and antioxidant activity. This is a particular phenolic compound contained in the fermentation pine needle in In vitro antioxidant activity was not shown, while it was found to have a specific inhibitory effect on 3T3-L1 adipocyte differentiation.

Experimental Example  7: Sensory Evaluation of Fermented Pine Needles and Fermented Mugwort Extracts

Descriptive tests of fermented mugwort and fermented pine needles were carried out to select the sensory characteristics of fermented odor, bitter taste, astringent taste, sweetness and preference. All sensory characteristics of fermented pine needles and fermented mugwort extracts of Preparation Examples 1 and 2 were scored on a nine-point scale using 10 of the sensory test personnel trained on the proposed sensory characteristics. Compare and evaluate.

The results are shown in Figure 22, the fermented pine needles, fermented mugwort extracts of Preparation Examples 1 and 2 was found that the fermentation odor, bitter taste, astringent taste, sweet taste was reduced compared to the wild vinegar fermentation solution of Comparative Preparation Example, while the palatability increased. According to this, it was found that the fermented broth of Sanyacho of the Comparative Preparation Example was too sweet and had a bitter astringent taste, but the fermented pine needles and Fermented Wormwood Extracts of Preparation Examples 1 and 2 increased their palatability while neutralizing excessive sweetness, bitterness and astringency.

Claims (4)

Method of producing a fermented herbal extract characterized in that the fermentation by inoculating lactic acid bacteria into one or more extracts of mugwort or pine needles.
The method of claim 1,
The lactic acid bacteria is Leuconostoc Leukonostoc mesenteroides ) 1076 method for producing a fermented herb extract, characterized in that.
The method according to claim 1 or 2,
Extract of one or more of the wormwood or pine needles
After pulverizing at least one of the wormwood or pine needles, the method of producing a fermented herbal extract characterized in that the extract for 23 to 25 hours at 24 ~ 26 ℃ by adding distilled water.
The method according to claim 1 or 2,
The fermentation is a method for producing a fermented herb extract, characterized in that carried out for 35 to 37 hours at 29 ~ 31 ℃ at 190 ~ 210 rpm.

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