KR102582772B1 - Method for Preparing Fermented Ginseng with Increased Ginsenosides - Google Patents

Abstract

The present invention discloses a method for producing fermented ginseng products with increased ginsenoside Rg1, Re, Rd, Rb2, Rc, Rb1 and/or Rg2 content by fermenting ginseng with Bacillus sp microorganisms.

Description

Method for producing fermented ginseng with increased ginsenoside content {Method for Preparing Fermented Ginseng with Increased Ginsenosides}

The present invention relates to a method for producing fermented ginseng (ginseng root, Ginseng radix) with increased ginsenoside content.

Ginseng is a perennial herb, and 6-7 species are known worldwide, currently including Korean ginseng ( Panax ginseng CA MEYER), American Guangdong ginseng ( Panax quinquefolium L.), Chinese ginseng ( Panax notoginseng ), and Japanese bamboo ginseng ( Panax japonicus ) . CA MEYER) and others are used as medicinal ingredients. The root (Ginseng radix) is divided into white ginseng (Ginseng Radix alba) and red ginseng (Ginseng Radix Rubra) depending on the processing method. White ginseng is fresh ginseng (raw ginseng) that is dried without cooking by methods such as natural drying in the sun, and red ginseng is fresh ginseng. The roots are dried by steaming with steam, and have a light yellow-brown or reddish-brown color. The unique color of red ginseng, which is different from white ginseng, is because when red ginseng is steamed, ginseng starch is gelatinized through heat treatment and the internal tissue color changes due to an amino-carbonyl reaction.

Ginseng has been widely used in East Asian countries such as China, Korea, and Japan as a medicine or health-promoting herbal medicine for about 2,000 years. Saponin (ginsenoside), the main active ingredient of ginseng, has immune-boosting, anti-inflammatory, anti-allergic, anti-cancer, erectile dysfunction, blood pressure-lowering, anti-cholesterol, anti-thrombotic, anti-diabetic, and anti-inflammatory effects. It has been reported to have preventive and therapeutic effects on aging (J. Immunol., 148:1519-25, 1992; Lee FC., Facts about ginseng, the elixir of life. Hollyn International. New Jersey, 1992; Huang KC ., The pharmacology of Chinese herbs. CRC Press. Florida, 1999; Chang HM., Pharmacology and application of Chinese material medica. Vol1. World Scientific. Singapore, 1986; Biol. Pharm. Bull. 17:635-9, 1994; Biol. Pharm. Bull. 18:1197-1202, 1995).

As such, the main component that exhibits physiological activity in ginseng is saponin. Ginseng saponin is a neutral compound of bisdesmoside that combines glucose, arabinose, xylose, rhamnose, etc. with the dammarane skeleton of the triterpenoid series. It is a glycoside. They are named Ginsenoside Rx according to the polarity order determined by column chromatography. To date, more than 40 types of ginsenosides have been isolated and identified, and these ginsenosides are divided into the PPD (Protopanaxadiol) series and the PPT (Protopanaxatriol) series, the basic chemical formula of which is shown in [Chemical Formula 1] below. As can be seen in [Table 1] below, PPD series saponins have a basic structure of PPD where R ₂ is hydrogen (H), and ginsenosides Rb1, Rb2, Rg3, Rc, Rd, F2, compound Y, compound K, etc. In the PPT series of saponins, R ₁ is hydrogen (H), PPT is the basic structure, and ginsenosides Re, Rf, Rg1, Rh1, etc. belong to this group.

[Formula 1]

The present invention discloses a method for producing fermented ginseng products with increased ginsenoside Rg1, Re, Rd, Rb2, Rc, Rb1 and/or Rg2 content.

The purpose of the present invention is to provide a method for producing fermented ginseng products with increased ginsenoside content.

Other or specific purposes of the present invention will be presented below.

As confirmed in the examples below, the present inventors have discovered that ginseng (hereinafter referred to as ginseng means ginseng root (Ginseng radix) unless otherwise specified) powder can be fermented alone with Bacillus sp. microorganisms. It was confirmed that the total ginsenoside content increased, and when sequential fermentation was performed with two strains, the ginsenoside Rg2, Rg1, Rd, and Rb1 content increased.

The present invention is provided based on these experimental results. The present invention relates to a method for producing a fermented ginseng product with increased ginsenoside content. The method for producing a fermented ginseng product with increased ginsenoside content of the present invention is (a ) It consists of preparing a medium containing ginseng powder or its extract, and (b) inoculating and culturing Bacillus sp microorganisms in this medium. Here, ginsenoside means Rg1, Re, Rd, Rb2, Rc, Rb1 and/or Rg2 when referring to the examples below.

In the present invention, the pulverized ginseng may be pulverized ginseng (fresh ginseng) that has not been dried after collection, dried ginseng (white ginseng), or pulverized ginseng (red ginseng) that has been steamed. The pulverized product may be powder with small particle size, shredded material with large particle size, or a mixture thereof.

In addition, in the present invention, ginseng extract refers to something obtained by leaching the ginseng root, which is the extraction target, with water, an organic solvent, or a mixed solvent thereof. Organic solvents include lower alcohols with 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butylacetate, N,N-dimethylformamide (DMF), dimethyl Sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, etc., preferably ethanol. As an extraction method, any method such as cold immersion, reflux, heating, ultrasonic radiation, or supercritical extraction can be applied. After extraction, it may be desirable to remove extraction residues (subsequent solids) through filtration, centrifugation, or standing for a certain period of time. In particular, in the present invention, the ginseng extract is preferably a supernatant obtained by extracting with hot water (60-90°C), removing the extraction residue, adding ethanol to the supernatant from which the extraction residue was removed, reacting for a certain period of time (20-240 minutes), and then centrifuging. do. When ethanol is reacted and centrifuged, sugar components other than ginsenoside present in the supernatant are removed, thereby increasing the ginsenoside concentration in the supernatant after centrifugation.

In the present invention, the medium containing ginseng powder or extract can be prepared by adding an appropriate amount of water (purified water or distilled water) to the ginseng powder or extract, preferably 2 to 4 times the weight of the ginseng powder or extract. .

The medium can be adjusted to an appropriate pH for smooth growth and proliferation of the inoculated Bacillus microorganisms, for example, in the range of pH 6.0 to pH 7.5.

In addition, carbon sources, nitrogen sources, and/or trace elements known in the art may be added to the medium to promote or assist the growth and proliferation of the inoculated Bacillus microorganisms.

The carbon source is preferably a sugar such as a monosaccharide, disaccharide or polysaccharide. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, starch hydrolyzate, etc. can be used. Complex compounds such as molasses or by-products of sugar refining processes can also be used. In some cases, oils such as soybean oil and sunflower oil, organic acids such as acetic acid, and glycerol may be advantageous as carbon sources. These carbon sources may be included in the medium alone or in a mixture of two or more types, and may be included in the medium alone or in a mixture of two or more types, in an amount of 0.1% (w/w) to 20% (w/w). It can be included in the badge as a range.

The nitrogen source may typically be an inorganic nitrogen compound, an organic nitrogen compound, or a complex compound containing these compounds. For example, inorganic nitrogen compounds include ammonia, ammonium salts (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.), nitrates, etc., and organic nitrogen compounds include urea, amino acids, etc. Complex compounds include yeast extract, soytone, peptone, tryptone, malt extract, meat juice, soybean powder, soybean okara powder, cheonggukjang powder, and soybean paste. Natural nitrogen sources such as powder can be mentioned. One or more of these nitrogen sources may be used in combination, and may be included in the medium in the range of 0.1% (w/w) to 8.0% (w/w). When using a natural nitrogen source, it is desirable to sterilize it for the same reason as described in relation to using a natural carbon source.

Trace elements include calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper, iron, phosphorus, sulfur, etc. These trace elements can be added to the medium in the form of salt compounds, and the medium of these trace elements To increase solubility and maintain the solution state, chelating agents such as catechol, citric acid, etc. may be added together. Examples of the salt compound include sodium hydrogen phosphate, magnesium sulfate, iron chloride, calcium salt, manganese salt, cobalt salt, molybdate salt, and chelate metal salt. These trace elements can be used in combination of one or more types, and can be included in the medium in the range of 0.001% (w/w) to 3.0% (w/w).

Additionally, the medium may optionally be prepared containing growth promoters such as biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate, and pyridoxine.

Regarding the optimization of other medium compositions, there are many references in the art, including the literature (Applied Microbiol. Physiology, A Practical Approach (Editors P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). The literature is known, and reference may be made to these known literature.

Also, in the present invention, any Bacillus sp. microorganism may be used as the Bacillus genus microorganism. Specifically, such microorganisms include Bacillus subtilis, B. lichneformis , B. megaterium , and Bacillus as microorganisms of the Barillus genus that can be used in the method of the present invention. Representative examples include B. amyloliquefaciens and B. natto , as well as B.antharcis , B.lentus , and B. permirus. pumilus ), Bacillus thuringiensis ( B.thuringiensis ), B.alvei , B.azotofixans , B.macerans , B.polymyxa , B. popilliae , B. coagulans, B. stearothermophilus , B. pasteurii , B. sphaericus , B. pass B. fastidiosus and the like can be mentioned. Preferably, it may be the Bacillus sp. S9 strain (accession number KCTC 13945BP), S10 strain (accession number KACC 92429P), or Bacillus S11 strain (accession number KCTC13946BP) used in the examples below.

It may be desirable to use these Bacillus microorganisms by inoculating them in a medium containing ginseng extract and cultivating them for 1 to 3 days to acclimatize them to ginseng fermentation. Here, the ginseng extract may be a water or hot water (50-80°C) extract, and sonication may be performed during the extraction process to extract ginsenosides at a higher yield. The ginseng extract-containing medium can be prepared by preparing ginseng extract in powder form and adding water to it, or by adding water to ginseng powder to extract it and filtering the extract to obtain the filtrate, or leaving it alone to obtain the supernatant.

Among these strains, the Bacillus S9 strain is Stenotrophomonas sp. as disclosed in Korean Patent Publication No. 2021-0060864 (publication date: 2021.05.27). It is deposited under the deposit number KCTC 13945BP as a mixture with the S7 strain. When this mixed strain is cultured on R2A agar at a temperature of 35°C, only the Bacillus sp. S9 strain can be isolated, cultured, and used.

After the Bacillus genus microorganisms are inoculated, it may be desirable to culture them with shaking at a temperature of 25 to 45°C, especially 30 to 35°C, for more than 1 day, preferably for 1 to 10 weeks, especially for 4 to 8 weeks. there is.

In the present invention, it is preferable to sterilize the medium containing ginseng powder or extract to prevent unintended fermentation by other microorganisms. Sterilization can be performed using methods known in the art, such as high temperature/high pressure sterilization and ultraviolet irradiation.

In addition, in the present invention, in order to produce fermented ginseng products with particularly high ginsenoside Rg2, Rg1, Rd and/or Rb1 content, sequential inoculation and cultivation of Bacillus S9 strain, Bacillus S10 strain or Bacillus S11 strain is preferred. can do. Here, the sequential inoculation and culture may be specifically inoculation and culture of the Bacillus S9 strain followed by inoculation and culture of the Bacillus S10 strain, or inoculation and cultivation of the Bacillus S11 strain followed by inoculation and cultivation of the Bacillus S10 strain. At this time, each sequential culture may be 1 day or more, preferably 1 week to 8 weeks, especially 2 weeks to 4 weeks. In the examples below, when the Bacillus S10 strain is sequentially cultured with the Bacillus S9 strain or the Bacillus S11 strain, ginsenoic acid is produced compared to non-fermentation, and when each strain is cultured alone or when the Bacillus S10 strain is inoculated and cultured twice. It shows that fermented ginseng products with particularly high side Rg2, Rg1, Rd and/or Rb1 content can be produced. Although not presented as a result in the examples below, in the case of simple mixed culture (inoculation and culture of Bacillus S9 strain and Bacillus S10 strain or Bacillus S11 strain and Bacillus S10 strain once, culture) rather than sequential culture, ginsenoside Rg2, Rg1, Rd and/or Rb1 content did not increase.

Meanwhile, since the fermented ginseng product with increased ginsenoside content obtained by the production method of the present invention is known to have various physiological activities, it can be commercialized into pharmaceutical compositions, health functional foods, functional cosmetics, etc. Specifically, the pharmaceutical compositions, health functional foods, and functional cosmetics contain these ginsenoside ingredients for improving immunity, improving fatigue, improving bone health, anti-diabetes, anti-hypertension, anti-hyperlipidemia, treating or preventing cardiovascular disease, and improving atopic dermatitis. , anti-obesity, rejuvenation, skin wrinkle improvement, skin whitening, etc. may have uses or functionality known in the art.

When the ginseng fermentation product with increased ginsenoside content obtained by the production method of the present invention is commercialized and used as a pharmaceutical composition, the pharmaceutical composition contains a pharmaceutically acceptable carrier in addition to the ginseng fermentation product and is known in the art. It can be prepared as an oral formulation or a parenteral formulation depending on the route of administration by a known conventional method. Here, the route of administration may be any suitable route, including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of a combination of two or more routes is a case where two or more dosage forms of drugs according to the administration route are combined, for example, when one drug is administered firstly through an intravenous route and the other drug is administered secondarily through a local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or dosage form, and for specifics, reference can be made to the pharmacopoeia of each country, including the "Korean Pharmacopoeia".

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, it can be prepared as powder, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, and wafers using a suitable carrier according to methods known in the art. It can be manufactured in a dosage form such as: Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Cellulose such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease. Serol, etc. can be mentioned. When formulating, appropriate binders, lubricants, disintegrants, colorants, diluents, etc. can be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrite, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, and wax, and as a lubricant, oleic acid. Examples include sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium and calcium salts, and polydethylene glycol. Disintegrants include starch and methyl cellulose. , agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt, etc. Additionally, diluents include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories along with a suitable carrier according to methods known in the art. When formulated as an injection, an aqueous isotonic solution or suspension can be used as a suitable carrier. Specifically, an isotonic solution such as PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or 5% dextrose can be used. . When formulated for transdermal administration, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, paste preparations, linear preparations, and aerol preparations. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide. When formulated as a suppository, the carrier is Wethepsol ( witepsol), Tween 61, polyethylene glycols, cocoa fat, laurel paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, etc. can be used.

Regarding the specific formulation of pharmaceutical compositions, it is known in the art, and references can be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). The above documents are considered a part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, gender, age, patient's severity, and administration route. It may be in the /kg range. Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the invention in any respect.

In another aspect, when the fermented ginseng product with increased ginsenoside content obtained by the production method of the present invention is commercialized and used as a health functional food composition, the food composition can be manufactured in any form, such as tea, juice, Beverages such as carbonated drinks and electrolyte drinks, processed oils such as milk and yogurt, foods such as gum, rice cakes, Korean snacks, bread, snacks, noodles, tablets, capsules, pills, granules, liquid, powder, flake, paste, syrup , can be manufactured into health functional food preparations such as gels, jellies, and bars.

In addition, the food composition of the present invention can have any product classification in terms of legal and functional classification as long as it complies with the enforcement laws and regulations at the time of manufacture and distribution. For example, it is a health functional food according to the Korean 「Act on Health Functional Foods」, or confectionery, beans, tea, and beverages according to each food type according to the food code of the Korean 「Food Sanitation Act」 (Ministry of Food and Drug Safety Notification 「Food Standards and Specifications」) , special purpose food, etc.

The food composition of the present invention may contain food additives in addition to the fermented broth. Food additives can generally be understood as substances that are added to, mixed with, or infiltrated into food when manufacturing, processing, or preserving food. Since they are consumed daily and for a long period of time with food, their safety must be guaranteed. In the Food Additives Code of Laws of each country that regulates the manufacturing and distribution of food (in Korea, it is the Food Sanitation Act), food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korea Food Additive Code (Ministry of Food and Drug Safety Notification “Food Additive Standards and Specifications”), food additives are classified into chemical synthetics, natural additives, and mixed preparations in terms of composition. These food additives are classified into sweeteners and flavors in terms of function. It is classified into preservatives, emulsifiers, acidulants, thickeners, etc.

Sweeteners are used to impart an appropriate sweetness to foods, and either natural or synthetic ones can be used in the composition of the present invention. Preferably, a natural sweetener is used, and natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents can be used to improve taste or aroma, and both natural and synthetic ones can be used. Preferably, natural products are used. When using natural products, they can serve the purpose of enhancing nutrition in addition to flavor. Natural flavoring agents may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or may be obtained from green tea leaves, coriander leaves, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. You can also use things obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo nuts. Natural flavoring agents may be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and the synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, etc.

Preservatives include calcium sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc., and emulsifiers include acacia gum, carboxymethyl cellulose, xanthan gum, Examples include pectin, and acidulants include acidic acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. In addition to improving taste, acidulants may be added to ensure that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms.

As a thickening agent, a suspending agent, settling agent, gel forming agent, bulking agent, etc. may be used.

In addition to the food additives described above, the food composition of the present invention may contain bioactive substances or minerals known in the art and whose safety is guaranteed as food additives for the purpose of supplementing and reinforcing functionality and nutrition.

Such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, etc., and minerals include calcium preparations such as calcium citrate and magnesium stearate. Magnesium preparations such as iron citrate, iron preparations such as chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, etc. are included.

The food composition of the present invention may contain the above-described food additives in an appropriate amount to achieve the purpose of addition depending on the product type.

Regarding other food additives that can be included in the food composition of the present invention, each country's food code or food additive code can be referred to.

In another aspect, when the ginseng fermentation product with increased ginsenoside content obtained by the production method of the present invention is commercialized and used as a functional cosmetic composition, the cosmetic composition contains ingredients commonly used in cosmetic compositions in addition to the fermentation broth, such as , conventional auxiliaries such as stabilizers, solubilizers, surfactants, vitamins, colorants and perfumes, and carriers.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier ingredient. You can.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the formulation is a spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. /May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent is used as a carrier component, specifically water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, etc. can be used.

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, and microcrystals. Cellulose, aluminum metahydroxide, bentonite, agar, etc. can be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide. Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester can be used.

The cosmetic composition of the present invention can be manufactured according to a cosmetic composition manufacturing method commonly used in the art, except that it contains a fermented ginseng product with increased ginsenoside content obtained by the manufacturing method of the present invention.

As described above, according to the present invention, a method for producing a fermented ginseng product with increased ginsenoside content from ginseng can be provided. This fermented ginseng product with increased ginsenoside content can be usefully used in health functional foods, medicines, etc.

Figure 1 is an HPLC chromatogram of a ginseng fermentation product by a single strain, Figure 2 is the measurement result of the total ginsenoside content of a ginseng fermentation product by a single strain, and Figure 3 is a ginsenoside content by component of a ginseng fermentation product by a single strain. This is the measurement result.
Figure 4 shows the measurement results of the content of each ginsenoside component of fermented ginseng by sequential cultivation of two strains.

Hereinafter, the present invention will be described with reference to examples. However, the scope of the present invention is not limited to these examples.

<Example> Preparation of fermented ginseng product with increased ginsenoside content

<Example 1> Preparation of fermented ginseng product with increased total ginsenoside content

1. Preparation of inoculating strains

To prepare the inoculation strain, first, on R2A agar medium (R-2A Agar, millipore 17209-500G), Bacillus S9 strain (Bacillus sp . S9 strain, KCTC13945BP), Bacillus S10 strain (Bacillus sp . ), and Bacillus S11 strains (Bacillus sp . S11, KCTC13946BP) were each inoculated and cultured for about 12 hours until colonies were clearly visible. Afterwards, each strain prepared in the R2A medium was inoculated into 1 mL of medium containing 10% (w/v) ginseng extract, and the inoculated strains were prepared by shaking culture (80 rpm) at 30°C for more than 24 hours. The medium containing 10% (w/v) ginseng extract was extracted by suspending 10 g of ginseng powder obtained through a ball mill in 90 ml of distilled water, sonicating it at 50°C for 30 minutes, leaving it overnight at 4°C, and collecting the supernatant. The supernatant was recovered by centrifugation (8000 rpm/30 min) and then sterilized (121°C, 15 min).

2. Production of fermented ginseng products with increased total ginsenoside content

Double the weight of distilled water was added to the dried powder of ginseng root (fresh ginseng) and sterilized (121°C, 15 minutes), then 0.2ml of sterilized supernatant, which was the strain culture medium, was inoculated and cultured at 30-35°C for 8 weeks.

3. Measurement of ginsenoside contentc in fermented ginseng products

The content of each ginsenoside component of the fermented ginseng product was analyzed according to the method of Kim Geum-sook et al. (Korean J. Medicinal Crop Sci. 24(1):47-54, 2016). For HPLC analysis, 300ul of the sample was mixed with 1.2ml methanol, extracted ultrasonically, centrifuged, and 1ml of the supernatant was taken. The ginsenoside was partially purified using a solid phase extractor (SPE), the Agilent 1100 series HPLC system (Agilent Technolgies, USA) was used, and the ginsenoside standard product was purchased from Embo Research Institute.

4. Ginsenoside contentc measurement result of fermented ginseng product

The HPLC chromatogram of the fermented ginseng product is shown in Figure 1, the measurement results of the total ginsenoside content are shown in Figure 2, and the content of each ginsenoside component is shown in Figure 3.

Referring to Figures 1 to 3, it can be seen that the total ginsenoside content increased compared to the non-fermentation control (ctl) by fermentation of the S9 strain, S10 strain, and S11 strain, and was especially clearly increased by the S10 strain fermentation. It can be seen that there has been an increase. In addition, ginsenosides Rg1 and Rb1 are used as indicator components in functional foods using ginseng powder, extracts, or fermented products as functional ingredients (Ministry of Food and Drug Safety Notification No. 2022-25 “Health Functional Food Standards and Specifications”) 2-1 Ginseng), the content of these indicator components all increased compared to the control group, and in particular, it increased significantly due to S10 strain fermentation. The increase in indicator components means that it is advantageous to standardize raw materials in the development of health functional foods and can reduce the amount of raw material usage.

<Example 2> Preparation of fermented ginseng product with increased ginsenoside Rg2, Rg1, Rd and Rb1 content

Fermented ginseng was prepared in the same manner as in Example 1, and the ginsenoside content was measured according to the method of Kim Geum-sook et al., except that two strains were used sequentially to produce the fermented ginseng product. Specifically, (i) after inoculation of S9 strain (inoculated with 0.2 ml of sterilized supernatant, which is a strain culture medium), cultured for 4 weeks, and additionally inoculated with strain S10 without sterilization (inoculated with 0.2 ml of sterilized supernatant, which is a strain culture medium), Cultivate for an additional 4 weeks to produce fermented ginseng (S9+S10), or (ii) inoculate the S11 strain (inoculate 0.2 ml of sterilized supernatant, which is a strain culture medium), then culture for 4 weeks and add the S10 strain to this culture without sterilization. was inoculated (inoculated with 0.2 ml of sterilized supernatant, which is a strain culture medium) and cultured for an additional 4 weeks to produce fermented ginseng (S11+S10). As a control group, the non-fermentation control group (ctl) was inoculated with the S10 strain (inoculated with 0.2 ml of sterilized supernatant, which is a strain culture medium), then cultured for 4 weeks, and the culture was again inoculated with the S10 strain without sterilization (0.2 ml of sterilized supernatant, which is a strain culture medium). The fermented ginseng product (S10+S10) obtained by inoculating) and culturing for an additional 4 weeks was used.

The results of measuring the content of each component of the fermented ginseng product are shown in Figure 4. Referring to Figure 4, in the case of fermented ginseng products sequentially cultured S9 + S10 and ginseng fermented products sequentially cultured S11 + S10, the ginsenoside Rg2 and Rd contents were clearly increased compared to the non-fermented control group (ctl) or S10 + S10. can be seen.

Claims (7)

(a) preparing a medium containing ginseng root powder or its extract, and (b) adding Bacillus sp S9 strain (accession number KCTC 13945BP) or Bacillus sp S10 strain to this medium ( A method for producing fermented ginseng with increased ginsenoside content, comprising the step of inoculating and culturing (Accession number KACC 92429P),
The strain is a strain cultured in a medium containing ginseng extract,
A method wherein the ginsenoside is Rb1.
According to paragraph 1,
The method is characterized in that the culture is carried out by shaking culture for 1 to 10 weeks at 30 to 35 ℃.
(a) preparing a medium containing ginseng root powder or its extract, and (b) inoculating this medium with the Bacillus genus S9 strain (accession number KCTC 13945BP), then culturing it, and then inoculating the Bacillus genus S10 strain (accession number KCTC 13945BP) A method for producing fermented ginseng with increased ginsenoside content, comprising the step of inoculating and culturing (No. KACC 92429P),
The strain is a strain cultured in a medium containing ginseng extract,
A method characterized in that the ginsenoside Rg2 or Rd.
According to paragraph 3,
The method is characterized in that the culture is carried out by shaking culture for 1 to 10 weeks at 30 to 35 ℃.
(a) preparing a medium containing ginseng root powder or its extract, and (b) inoculating the medium with the Bacillus genus S11 strain (accession number KCTC13946BP) and culturing it, and then Bacillus genus S10 strain (accession number A method for producing fermented ginseng with increased ginsenoside content, comprising the step of inoculating and culturing KACC 92429P),
The strain is a strain cultured in a medium containing ginseng extract,
A method characterized in that the ginsenoside Rg2 or Rd.
According to clause 5,
The method is characterized in that the culture is carried out by shaking culture for 1 to 10 weeks at 30 to 35 ℃.


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