KR101915058B1 - NOVEL STRAIN OF Leuconostoc mesenteroides AND COMPOSITION FOR PREVENTING OR TREATING OF INFLAMMATORY DISEASE USING THE SAME - Google Patents

Abstract

The present invention relates to a novel strain of Leuconostoc mesenteroides (KCCM 12010P) having excellent β-glycosidase activity, and to a composition for preventing, treating or alleviating inflammatory diseases using the same.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel strain of Mycobacterium cepum strain, and a composition for preventing or treating an inflammatory disease using the same. ≪ Desc / Clms Page number 1 >

The present invention relates to a novel strain of Reckon Stokaceae, and to a composition for preventing or treating inflammatory diseases using the same.

In modern society, wastes such as free radicals and peroxides accumulate in the human body due to irregular and unbalanced eating habits, lack of momentum, and excessive mental stress in relation to complex society, which causes inflammation, And are becoming vulnerable to a variety of diseases.

On the other hand, human cells can be damaged by various factors. These factors include physical factors such as hypoxia, trauma or electric shock, rapid changes in temperature, chemical factors caused by various harmful substances (drugs, pesticides, alcohol, etc.), biological factors caused by bacteria, viruses, and parasites In addition, physiological factors such as nutritional imbalance and aging can be mentioned.

As a cell damage, its symptoms appear as various phenomena. In other words, reversible acute cellular damage, the type of cell death after irreversible damage (necrosis, necrosis), apoptosis by cell death due to suicide (apoptosis), sub-system changes caused by reaction to noxious stimulation, accumulation of various organic substances (Obesity), and the like.

A defense mechanism against such cell damage is present in the human body, and one of them is an inflammatory reaction. Inflammation is a series of vital reactions that minimize local damage and restore the integrity. These inflammatory reactions are mainly caused by blood vessels and blood cells. Inflammation reactions can be divided into acute and chronic. Acute cases are immediate and nonspecific, characterized by infiltration of polymorphonuclear leukocytes. Systemic symptoms include fever, fatigue, loss of appetite, weakness, localized fever, localized fever , Swelling, pus and pain. Chronic inflammation includes monocytes (macrophages, lymphocytes, plasma cells), fibroblasts and capillary proliferation, and fibroblasts formed by fibroblasts are deposited and fibrosed. At this time, tissue destruction is more severe than acute inflammation. The morphological classification of inflammation varies depending on the type and size of the stimulation, the degree of inflammation reaction, the type of damaged tissue, and mainly manifests as serous inflammation, fibrosing inflammation, pyogenic inflammation, ulcer.

Currently, there is medical chemotherapy as a method of treating inflammation, but its kind is very limited due to side effects and safety. In recent years, there is a tendency to prefer diet through natural products due to negative perception of the possibility of adverse effects in chemotherapy of consumers .

On the other hand, ginseng has been widely used as a medicinal crop in Korea as well as in China, Japan and other Asian countries. Ginseng mainly represents functionalities such as anti-cancer effect, immunity and blood pressure lowering. A typical active substance is called ginsenoside and is a kind of glycoside. Among the ginsenosides, Rg3 is known to regulate nitric oxide (NO) secretion ability and lymphocyte division ability of macrophages. In China, Rg3 has been clinically used as a new drug, proving its efficacy as a tumor suppressor agent for cancer cells such as lung cancer, liver cancer and breast cancer.

Bioconversion refers to the transformation of the structure of the active substance by using various enzymes to enhance the physiological function or to change the physical properties such as solubility or polarity. It is mainly involved in microbial cultivation, Purified. In recent years, studies have been conducted to increase the functionality of ginseng by increasing the ginsenoside content of ginseng using fermentation. Aspergillus, bacillus, and lactic acid bacteria isolated from fermented foods are mainly used as the strains used for fermentation.

Related prior arts include a method for producing fermented ginseng extract and a composition containing the same (Korean Patent Laid-Open No. 10-2011-0123311). In this technique, ginseng was extracted with hot water and cultured in Lactobacillus fermentum, Aspergillus niger, Trichoderma reesei and then subjected to high pressure emulsification to analyze ginsenoside content. However, this technology has only fermented the water soluble components of ginseng due to hot water extraction, and the process is complicated and takes a long time until the fermentation is made by high pressure treatment after the third fermentation with three strains. There was no mention of the functionality of these fermentations.

The present invention is intended to provide a pharmaceutical composition for preventing or treating inflammatory diseases, which comprises fermented ginseng extract and the fermentation is carried out by Leuconostoc mesenteroides strain (KCCM 12010P).

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, which comprises fermented ginseng extract, wherein the fermentation is by strain Leuconostoc mesenteroides (KCCM 12010P).

The fermentation may be for enhancing the content of ginsenosides Rg3, Rh2 or Rg2 in the ginseng extract.

The fermentation may be carried out for 24 to 72 hours.

Wherein the ginsenoside Rb2, Rb1, Rb3, Rd, Rg3, Rh2, and compound K in the fermented ginseng extract contains at least one selected from the group consisting of protopanaxyldiol-based ginsenosides and Rg2, The weight ratio of the oily ginsenosides may be from 5: 1 to 10: 1.

The fermented ginseng extract may have inhibitory activity against β-carotene oxidation or inhibition of peroxidation.

The inflammatory disease is selected from the group consisting of allergies, dermatitis, atopic dermatitis, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, , Osteoarthritis, rheumatoid arthritis, shoulder inflammation, tendinitis, hay fever, perianal inflammation, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases.

In one embodiment of the present invention, the fermented ginseng extract is used, and the fermentation is carried out by leuconostoc mesenteroides ( Leuconostoc mesenteroides ) strain (KCCM 12010P). The present invention also provides a health functional food for preventing or ameliorating an inflammatory disease.

When the ginseng extract is fermented by using the novel Leuconostoc mesenteroides strain (Accession No. KCCM 12010P) according to the present invention, the ginsenosides Rb2, F2 or Re in the ginseng extract are referred to as ginsenosides Rg3, Rh2 or Rg2, it has an excellent antioxidative and anti-inflammatory effect.

Figure 1 is a schematic diagram of a new leuconostoc mesenteroides ( Leuconostoc mesenteroides ) (Accession No. KCCM 12010P).
Figure 2 is a graph showing the results of a new leuconostoc mesenteroides ( Leuconostoc (Ginsenoside Rb2? Ginsenoside Rg3) using a strain of Escherichia mesenteroides (Accession No. KCCM 12010P).
FIG. 3 is a graph showing viable cell numbers and pH of fermented ginseng extract according to Example 2. FIG.
4 is a graph showing the antioxidative effect of fermented ginseng extract according to an embodiment of the present invention.
FIG. 5 is a graph showing the anti-inflammatory effect of fermented ginseng extract according to an embodiment of the present invention.

The present inventors have found that leuconostoc mesenteroides ( Leuconostoc mesenteroides) the case where among the strains, excellent β- glucosidase which is active acids Pocono stock mesen teroyi des (Leuconostoc mesenteroides) the strain (KCCM 12010P) Isolation and Identification, and by using this fermented ginseng extract, ginseng extract in ginsenoside (Rg3), Rh2 (Rg2), and Rg2. It was confirmed that the content of the side Rg3, Rh2 or Rg2 was increased, and the inhibitory effect on β-carotene oxidation and the inhibition of peroxidation were excellent.

The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, which comprises fermented ginseng extract, wherein the fermentation is by strain Leuconostoc mesenteroides (KCCM 12010P).

First, the strain (KCCM 12010P) is a new strain and can be regarded as a subspecies of the strain Leuconostoc mesenteroides . The flow diagram of the strain (KCCM 12010P) is shown in Fig.

Leuconostoc mesenteroides ( Leuconostoc mesenteroides ) is a gram-positive anaerobic strain. This strain is a typical kimchi lactic acid bacteria, especially in the early stage of fermentation of kimchi. It also enhances the preservation of kimchi by producing bacteriocin, an antimicrobial substance. This strain heterotrophic during fermentation, producing not only lactic acid but also various organic acids and ethanol, and plays a very important role in the taste of kimchi. In addition, the monosaccharide can be applied as a dietary fiber while making a polymer carbohydrate called degradable dextrin. In particular, this strain is known to secrete a carbohydrate hydrolase called? -Glucosidase and can be applied to bioconversion through fermentation of physiologically active substances present as glycosides in plants.

Among the strain (KCCM 12010P) is different flow Pocono stock mesen teroyi des (Leuconostoc mesenteroides) characterized by having a particularly excellent β- glucosidase activity. The? -Glucosidase is an enzyme capable of degrading the glucose bond and is involved in biotransformation. Therefore, the strain (KCCM 12010P) can be used for the fermentation of ginseng extract, and the fermentation can be carried out by using the ginsenoside Rb1, Rb3, Rd, Rg3, Rh2 or compound K , Or to enhance the content of protopanaxadiol-based ginsenosides such as ginsenoside Rg2 in ginseng extract. Thus, the antioxidative and anti-inflammatory effects can be maximized. Specifically, the fermentation is preferably performed to enhance the content of ginsenosides Rg3, Rh2 or Rg2 in the ginseng extract, but is not limited thereto. At this time, the ginsenosides Rg3, Rh2 or Rg2 in the ginseng extract can be regarded as a result of biodegradation of the glucose bond in ginsenoside Rb2, F2 or Re in the ginseng extract.

In addition, the strain (KCCM 12010P) may be a live cell, a dead cell, or a culture filtrate.

The fermented ginseng extract is characterized in that the ginseng extract is fermented by the strain (KCCM 12010P).

The ginseng extract may be obtained by extracting fruits, roots, stems or leaves of ginseng with water, C1 to C4 lower alcohols or a mixture thereof.

Specifically, the extraction is preferably performed by dividing the dried ginseng and adding water, alcohol or a mixture thereof in an amount of 2 to 20 times the volume of the ginseng, more preferably 3 to 10 times But is not limited thereto. The extraction temperature is preferably 30 ° C to 100 ° C, more preferably 70 ° C to 100 ° C, but is not limited thereto. The extraction time is preferably 1 hour to 20 hours, more preferably 10 hours to 15 hours, but is not limited thereto. The extraction method may be a cold extraction, an ultrasonic extraction, or a reflux cooling extraction method, but is not limited thereto. The number of times of extraction is preferably 1 to 5 times, more preferably 2 to 3 times, but is not limited thereto. In addition, the ginseng extract may be diluted, concentrated, diluted or concentrated, and then purified.

The fermentation can be carried out by increasing the content of protopanaxyl trihydrate ginsenosides such as ginsenosides Rb1, Rb3, Rd, Rg3, Rh2 or compound K in ginseng extract, or by using a ginsenoside Rg2 And may be one for enhancing the content of the diol based ginsenoside. Thus, the antioxidative and anti-inflammatory effects can be maximized. Specifically, the fermentation is preferably performed to enhance the content of ginsenosides Rg3, Rh2 or Rg2 in the ginseng extract, but is not limited thereto. At this time, the ginsenosides Rg3, Rh2 or Rg2 in the ginseng extract can be regarded as a result of biodegradation of the glucose bond in ginsenoside Rb2, F2 or Re in the ginseng extract.

In addition, the fermentation may be performed for 24 to 72 hours, more preferably 48 to 72 hours, but is not limited thereto.

Wherein the ginsenoside Rb1, Rb2, Rb3, Rd, Rg3, Rh2, and compound K in the fermented ginseng extract contains at least one selected from the group consisting of protopanaxyldiol-based ginsenosides and Rg2, The weight ratio of the omega-3-glucoside is preferably 5: 1 to 10: 1, more preferably 7: 1 to 9: 1, but is not limited thereto. At this time, by maintaining the weight ratio of protopanaxadiol based ginsenoside and protopanaxyl triol based ginsenoside within the above range, it is possible to maximize the antioxidative and anti-inflammatory effects.

The number of viable cells of the fermented ginseng extract may be 5 Log cfu / mL or more, preferably 7 Log cfu / mL or more. The pH of the fermented ginseng extract may be 5.0 or less, preferably 4.8 or less. Such a change in pH may be attributed to organic acids produced by growing the strain (KCCM 12010P).

The inflammatory disease is a disease caused by a decrease in NO production and is a disease caused by allergies, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn disease, colitis, Sjogren's syndrome, multiple sclerosis, acute and chronic < RTI ID = 0.0 > and / or < / RTI > chronic inflammatory diseases such as rheumatoid arthritis, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, Inflammatory diseases, but is not limited thereto.

The amount of the strain (KCCM 12010P) is preferably 1 μg / ml to 100 μg / ml, but is not limited thereto. When the capacity of the strain (KCCM 12010P) is less than the above range, it is difficult to exert an inflammatory preventive or therapeutic effect. When the capacity of the strain (KCCM 12010P) exceeds the above range, toxicity including cytotoxicity There may be concerns.

The pharmaceutical compositions for the prevention or treatment of inflammatory diseases according to the present invention may be in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions And may contain suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions for formulation.

The carrier or the excipient or diluent includes lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In the case of formulation, a diluent or excipient such as a commonly used filler, a weight agent, a binder, a wetting agent, a disintegrant or a surfactant may be used.

The solid preparation for oral administration can be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the above strain (KCCM 12010P). In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous agents, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, Tween 61, cacao paper, laurin, glycerol gelatin and the like can be used.

The preferred dosage of the pharmaceutical composition for the prophylactic or therapeutic treatment of inflammatory diseases according to the present invention varies depending on the condition of the patient, the body weight, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for a desired effect, the dose may be 0.0001 to 2,000 mg / kg, preferably 0.001 to 2,000 mg / kg per day. The administration may be carried out once a day or divided into several doses. However, the scope of the present invention is not limited by the dosage.

The pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases according to the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention also provides a health functional food for preventing or ameliorating an inflammatory disease, comprising fermented ginseng extract and the fermentation by Leuconostoc mesenteroides strain (KCCM 12010P).

The fermented ginseng extract and the strain (KCCM 12010P) are as described above. In addition, the strain (KCCM 12010P) may be a live cell, a dead cell, or a culture filtrate.

The inflammatory disease is a disease caused by a decrease in NO production and is a disease caused by allergies, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn disease, colitis, Inflammatory bowel disease, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder periitis, tendinitis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, Inflammatory diseases, but is not limited thereto.

In the health functional food for preventing or ameliorating an inflammatory disease according to the present invention, when the above-mentioned strain (KCCM 12010P) is used as an additive for health functional food, it may be added as it is or may be used together with other foods or food ingredients, And the like. The amount of the active ingredient to be mixed may be suitably determined according to each use purpose such as prevention, health, or treatment.

Formulations of health functional foods may be in the form of powders, granules, pills, tablets, capsules, as well as in the form of ordinary foods or beverages.

There is no particular limitation on the type of the food, and examples of the food to which the above substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, , Various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include foods in a conventional sense.

Generally, the above-mentioned strain (KCCM 12010P) may be added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, per 100 parts by weight of the raw material. However, in the case of long-term ingestion intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range.

The beverage in the health functional food according to the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the beverage according to the present invention.

In addition to the above, the health functional food for the prevention or improvement of inflammatory diseases according to the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, Stabilizers, preservatives, glycerin, alcohols, and carbonating agents used in carbonated beverages. In addition, the health functional food for preventing or ameliorating an inflammatory disease according to the present invention may contain natural fruit juice, fruit juice drink, and pulp for producing vegetable drinks. These components may be used independently or in combination. The ratio of such additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food for the prevention or amelioration of inflammatory diseases according to the present invention.

As described above, when the ginseng extract is fermented using the novel Leuconostoc mesenteroides strain (Accession No. KCCM 12010P) according to the present invention, the ginsenosides Rb2, F2 or Re in the ginseng extract are respectively Can be bioconverted to ginsenoside Rg3, Rh2 or Rg2, which has an advantage of excellent antioxidative and antiinflammatory effects.

< Example >

Example  One: Leuconostoc mesenteroides Strain KCCM  12010P) Isolation and Identification

In order to isolate new strains, various kimchi broths such as Chinese cabbage kimchi, Korean radish kimchi, and kakdugi were sampled and used as samples. Kimchi broth was sprinkled on MRS agar medium, LBS agar medium and PES agar medium by 10 times dilution method, and after streaking and culturing several times, colonies were observed and a new strain was isolated. To identify the isolates, 16S rRNA sequence analysis was requested by Bionics (Seoul, Korea). Specifically, 16S rRNA sequencing was performed, and 16S rRNA gene was subjected to PCR using 27F and 1492R primers. Based on the results of analyzing the nucleotide sequence, other standard strains ( Lactobacillus spp ., Lactococcus spp . , Leuconostoc spp . , Pediococcus spp . , And Streptococcus spp . ) Were compared with each other. The new strain was identified as Leuconostoc mesenteroides strain and the similarity was%. In addition, the homology was analyzed and a schematic diagram was obtained using the Mega 7 program (Fig. 1). At this time, the Leuconostoc mesenteroides strain was deposited with the Korean Microorganism Conservation Center on April 5, 2017, and received the accession number KCCM 12010P.

Example  2: Preparation of fermented ginseng extract

A 60% ethanol ginseng concentrate (domestic) was purchased from FINE Korea (Yeongdeungpo, Seoul) and used as a ginseng extract. The fermentation broth was prepared by adding 400 mL of distilled water to 40 g of ginseng extract and disinfection with a high pressure sterilizer at 121 ° C for 15 minutes. In the medium Leuconostoc according to Example 1, twice subcultured in a MRS medium at 37 ℃ The mesenteroides strain (KCCM 12010P) was inoculated at the initial bacterial count of 10 ⁶ CFU / mL. Fermentation was carried out in a shaking incubator at 37 ° C for 2 days. After the fermentation, it was filtered with a 0.45 μm syringe filter.

Before and after fermentation, high performance liquid chromatography (HPLC, Agilent 1100 series) was used to measure ginsenoside content in ginseng extract. The following pretreatment was performed to extract crude saponin before analysis. Before and after fermentation, 1 mL of water-saturated butanol was added to 1 mL of ginseng extract, mixed, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was recovered. The supernatant collected three times was concentrated under reduced pressure and dissolved in methanol after analysis and analyzed. The HPLC analysis conditions are shown in Table 1.

column Eclipse XDB-C18 Flow rate 1.0 min / mL Detector wavelength UV 206 nm Mobile phase A) 14% ACN B) 100% ACN
0% B at 0 min, 5% B at 3 min, 10% B at 15 min, 15% B at 20 min, 20% B at 30 min, 60% B at 50 min, 55 0% B in minutes

The content of ginsenosides in the ginseng extract before and after fermentation was measured by HPLC. The results are shown in Table 2.

Ginsenoside content (ppm) Before fermentation After fermentation The proto party Rb2 144.4 125.6 Rb1 327.3 329.7 Rb3 200.6 207.6 Rd 96.2 97.4 Rg3 119.8 144.0 Rh2 99.1 102.1 Compound K 78.2 81.3 The proto-raccoon trio ginsenoside Rg2 114.7 122.5

As shown in Table 2, after the fermentation, the content of protopanaxadiol-based ginsenosides such as Rb1, Rb3, Rd, Rg3, Rh2 or compound K was increased compared with the ginseng extract before fermentation, It was confirmed that the content of protopanaxyl triol ginsenoside was increased.

In particular, the ginseng extract after fermentation showed that the total content of Rg3, Rh2 and Rg2 was increased by 10% by weight or more, compared with the ginseng extract before fermentation. Specifically, it was confirmed that the ginseng extract after fermentation showed a decrease of about 13% by weight of Rb2 and an increase of about 20% by weight of Rg3 compared with that of the ginseng extract before fermentation. This shows that Leuconostoc mesenteroides having excellent? -Glucosidase activity The strain (KCCM 12010P) can be seen as a result of biodegradation of ginsenoside Rg3 by degrading the glucose bond present in ginsenoside Rb2 in ginseng extract. Besides, after the entry into force of ginseng extract was confirmed that there is an increase compared to the pre-fermentation ginseng extract, Rh2 is about 3%, which Leuconostoc mesenteroides The strain (KCCM 12010P) can be seen as a result of biodegradation of the ginsenoside F2 in the ginseng extract to the biosynthesis of ginsenoside Rh2. In addition, after the entry into force of ginseng extract there is sure to be compared to the pre-fermentation ginseng extract, Rg2 have increased about 6%, which Leuconostoc mesenteroides (KCCM 12010P), which is the result of biodegradation of ginsenoside Rg2 by degradation of the glucose bond present in ginsenoside Re in ginseng extract.

On the other hand, after fermentation, the ginseng extract contained protopanaxadiol-based ginsenosides containing one or more members selected from the group consisting of ginsenosides Rb1, Rb2, Rb3, Rd, Rg3, Rh2 and compound K, And the weight ratio of protopanaxadiol based ginsenoside and protopanaxyl triol based ginsenoside is found to be in the range of 8: 1 to 9: 1.

Comparative Example  One: Leuconostoc mesenteroides Strain YLB8  Ready

Leuconostoc mesenteroides The strain YLB8 was prepared (Bioconversion of puffed red ginseng extract using β-glucosidase-producing lactic acid bacteria, Food Eng. Prog. 2014 (18)). The culture conditions were the same as those of the strain according to Example 1.

Comparative Example  2: Leuconostoc mesenteroides  Strain preparation

Leuconostoc mesenteroides strains were prepared (Purification and characterization of an intracellular β-glucosidase from Lactobacillus casei ATCC 393. Appl. Biochem. Biotech. 1998 (74)). The culture conditions were the same as those of the strain according to Example 1.

Experimental Example  One: Leuconostoc mesenteroides  Strain KCCM12010P ) Of β- Glycosidase  Activity evaluation

Leuconostoc according to Example 1 mesenteroides (KCCM 12010P) and Leuconostoc according to Comparative Examples 1 and 2 mesenteroides The β-glucosidase activities of the strains were compared.

Specifically, 50 mM sodium phos- phate buffer (pH 7.0) and 5 mM pNPG solution were used as substrates to evaluate β-glucosidase activity. The reaction was stopped by adding 100 μL of the strain to 1 mL of the substrate, reacting at 50 ° C for 10 minutes, and then adding 1 mL of 0.5 M Na ₂ CO ₃ solution. The absorbance of the reaction solution was measured at 400 nm, and the β-glucosidase activity was evaluated using the equation obtained from the standard curve. The results are shown in Table 3. The standard curve was prepared using p-nitrophenol in a concentration range of 0.1-1.0 mM and one unit of enzyme was defined as the amount of enzyme liberating 1 mM p-nitrophenol per minute at given conditions.

beta -glucosidase potency
(U / L) Example 1 117.8 Comparative Example 1 78.0 Comparative Example 2 70.0

Experimental Example  2: Fermented ginseng extract Viable cell count  And pH measurement

In order to measure the viable cell count and pH of the fermented ginseng extract according to Example 2, Leuconostoc mesenteroides strain (KCCM 12010P) according to Example 1, which was subcultured twice in the MRS medium at 37 ° C in the ginseng extract medium, Log CFU / mL, incubated at 37 ℃ incubator, and the viable cell count and pH were measured according to the incubation time. To measure viable cell counts, the cells were appropriately diluted in the MRS medium, and the cells were cultured in an incubator for 24 hours to measure viable cell counts. The pH was measured using a pH meter.

FIG. 3 is a graph showing viable cell numbers and pH of fermented ginseng extract according to Example 2. FIG.

As shown in FIG. 3, the viable cell count of the fermented ginseng extract according to Example 2 reached the stopper after 12 hours and was maintained at 7 Log CFU / mL or more for 48 hours. In addition, the pH of the ginseng extract before fermentation according to Example 2 was 4.9, and the pH of the ginseng extract reached 4.4 after 48 hours of fermentation.

Experimental Example  3: Evaluation of Antioxidative Activity of Fermented Ginseng Extract

To evaluate the antioxidant activity of fermented ginseng extract according to Example 2, β-carotene assay and ferric thiocyanate (FTC) assays were used.

Through the β-carotene assay, the oxidation inhibitory ability against β-carotene corresponding to the active ingredient was confirmed. To prepare β-carotene solution, 3 mg of β-carotene, 66 μL of linoleic acid and 300 μL of Tween 80 were dissolved in 15 mL of chloroform. The solvent was removed with a vacuum concentrator and 150 mL of distilled water was added. 4.5 mL of β-carotene solution was added to 0.5 mL of the fermented ginseng extract according to Example 2, and the reaction was allowed to proceed for 6 hours at 50 ° C. The absorbance was measured at 470 nm and the inhibitory activity was calculated as follows:

? -carotene oxidation inhibition (%) = (absorbance after reaction / absorbance before reaction) × 100.

Through the FTC assay, the ability to inhibit the production of peroxides that oxidize iron (Fe) was confirmed. To prepare the FTC solution, 100 μL of fermented ginseng extract according to Example 2, 200 μL of 25 mg / mL linoleic acid, 400 μL of 40 mM potassium phosphate buffer, and 200 μL of distilled water were mixed. 100 μL of the prepared FTC s solution, 4 mL of 70% ethanol, 100 μL of 30% ammonium thiocyanate, and 100 μL of 20 mM ferric chloride were added and the absorbance was measured at 500 nm for 24 hours. Calculated as:

(%) = [1- (absorbance of fermented ginseng extract / absorbance of control group)] × 100.

4 is a graph showing the antioxidative effect of fermented ginseng extract according to an embodiment of the present invention.

As shown in Fig. 4, the inhibitory activity against β-carotene oxidation was 39.05% at day 0 of fermentation, 52.11% at day 1, and 53.46% at day 2, indicating that β-carotene oxidation inhibition increases over fermentation period . On the other hand, the inhibition of peroxidation was 23.6% at day 0 of fermentation, 28.0% at day 1, and 29.0% at day 2, indicating that fermentation increases fermentation irrespective of fermentation time.

Experimental Example  4: Evaluation of anti-inflammatory activity of fermented ginseng extract

In order to evaluate the anti-inflammatory activity of the fermented ginseng extract according to Example 2, a decrease in the production of nitrogen oxides (NO) was measured using raw 264.7 cells. 50 μL of RAW cells (2 × 10 ⁵ / well) were added to a 96-well plate and reacted at 37 ° C. for 2 hours in a CO ₂ incubator. To induce inflammation, 100 μL of lipopolysaccharide (LPS) and 50 μL of fermented ginseng extract according to Example 2 were added and incubated for 24 hours. 100 μL of the supernatant and 100 μL of Griess reagent were added and reacted at room temperature for 10 minutes The absorbance was measured at 570 nm with a plate reader and the NO content was measured by comparing with the standard curve for the nitrate content.

FIG. 5 is a graph showing the anti-inflammatory effect of fermented ginseng extract according to an embodiment of the present invention.

As shown in FIG. 5, 46.3 μM of NO was produced in inflammation-induced cells. When the fermented ginseng extract according to Example 2 was further treated, the amount of NO was decreased. When the fermented ginseng extract according to Example 2 was diluted to 0.15%, it was found that the effect was the highest on the second day of fermentation, from 15.7 μM on day 0 of fermentation, 15.9 μM on day 1, and 11.8 μM on day 2 of fermentation. The half maximal inhibitory concentration (IC50) value was calculated as 0.055.

In a previous study related to the anti-inflammatory effect of ginseng, the IC50 value inhibiting NO production was 0.15 (Korean Patent Laid-open No. 10-2016-0065245) in the fermented extract of ginseng fermented with Lactobacillus, and the ginsenoside Rg3 The IC50 value of the fermented ginseng extract according to Example 2 showed a significant difference (p < 0.05), and the IC50 value inhibiting the NO production was 0.075 (Jo Jae Yeol, J. Ginseng Res, 2008) And the anti-inflammatory effect is also confirmed.

The preparation examples of the composition containing the fermented ginseng extract of the present invention will be described below, but the present invention is not intended to be limited thereto but is specifically described.

Formulation Example 1: Preparation of powder

20 mg of fermented ginseng extract

Lactose hydrate 100 mg

Talc 10 mg

The above ingredients were mixed and filled in an airtight container to prepare powders.

Formulation Example 2: Preparation of tablets

Fermented ginseng extract 10 mg

Corn starch 100 mg

100mg of lactose hydrate

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

Formulation Example 3: Preparation of capsules

Fermented ginseng extract 10 mg

Microcrystalline cellulose 3 mg

Lactose hydrate 14.8 mg

Magnesium stearate 0.2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the usual preparation method of capsules.

Formulation Example 4: Preparation of injection

Fermented ginseng extract 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Ginsenoside monohydrate 26 mg

After the above components were mixed, they were prepared with the above-mentioned component contents per 1 ampoule (2 mL) according to the usual injection preparation method.

Formulation Example 5: Preparation of a liquid preparation

Fermented ginseng extract 10 mg

10 g per isomer

5 g mannitol

Purified water quantity

Lemon incense quantity

The components are dissolved in purified water according to the usual preparation method, and the lemon flavor is added in an appropriate amount. Then, purified water is added to adjust the total volume to 100 mL, sterilized and filled in a brown bottle to prepare a liquid preparation.

Formulation Example 6: Preparation of Health Functional Foods

Fermented ginseng extract 10 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a component suitable for a health functional food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above components may be mixed And then granules are prepared and used in the manufacture of health functional foods according to conventional methods.

Formulation Example 7: Preparation of health drinks

Fermented ginseng extract 10 mg

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3g

Purified water quantitation

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 for about 1 hour. The resulting solution was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, &Lt; / RTI &gt;

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Korea Microorganism Conservation Center (overseas) KCCM12010P 20170405

Claims (7)

Fermented ginseng extract,
The fermentation is carried out by Leuconostoc mesenteroides strain (KCCM 12010P), which is excellent in the activity of? Glucosidase,
Wherein the fermented ginseng extract has an increased content of Rg3, Rh2 and Rg2 relative to the ginseng extract before fermentation.
delete The method according to claim 1,
Wherein the fermentation is carried out for 24 to 72 hours.
The method according to claim 1,
Wherein the ginsenoside Rb2, Rb1, Rb3, Rd, Rg3, Rh2, and compound K in the fermented ginseng extract contains at least one selected from the group consisting of protopanaxyldiol-based ginsenosides and Rg2, Wherein the weight ratio of the ovine ginsenoside is 5: 1 to 10: 1.
The method according to claim 1,
The fermented ginseng extract has anti-β-carotene oxidation inhibitory activity or inhibition of peroxidation.
delete Fermented ginseng extract,
The fermentation is carried out by Leuconostoc mesenteroides strain (KCCM 12010P), which is excellent in the activity of? Glucosidase,
The fermented ginseng extract is characterized in that the content of Rg3, Rh2, and Rg2 relative to the ginseng extract before fermentation is increased.

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