KR20210060864A - Fermented broth comprising ginsenoside originated the leaf of ginseng and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a fermentation solution containing ginsenosides obtained by inoculating leaves of ginseng with a microorganism strain and culturing the same, and to a preparation method thereof, and more specifically to a fermentation solution containing a large quantity of ginsenosides such as compounds K, F2, F1 and Rd obtained by inoculating a ginseng leaf suspension with a microorganism strain newly isolated by the present inventors and culturing the same under specific culture conditions, and a preparation method thereof. The fermentation solution containing ginsenosides originated from ginseng leaves according to the preparation method of the present invention can increase the total content of ginsenosides with high biological activity such as F2, F1 and Rd as well as a compound K from saponin precursors present in the ginseng leaves. Specifically, it was confirmed through comparison that a content of a ginsenoside F2, a content of a ginsenoside F1, and a content of a ginsenoside Rd, contained in the fermentation solution according to the preparation method of the present invention, were 2.6 times, 2 times, and 2 times higher than that of a control group, respectively, and the total content of ginsenosides was 619 ppm that was an increase of 36% from 454 ppm which is the total content of ginsenosides in the control group. Accordingly, the fermentation solution containing ginsenosides originated from ginseng leaves can be effectively used for health functional food, pharmaceuticals, and the like.

Description

Ginsenoside-containing fermented broth derived from ginseng leaf and its manufacturing method {FERMENTED BROTH COMPRISING GINSENOSIDE ORIGINATED THE LEAF OF GINSENG AND MANUFACTURING METHOD THEREOF}

The present invention relates to a ginsenoside-containing fermentation broth obtained by inoculating and culturing a microbial strain on a leaf of ginseng and a method for producing the same, and more specifically, inoculating a microbial strain newly isolated by the present inventor into a ginseng leaf suspension And, it relates to a fermentation broth containing a large amount of ginsenosides such as compounds K, F2, F1 and Rd obtained by culturing under specific culture conditions, and a method for producing the same.

Ginseng is a semi-neutral plant, belonging to the genus Panax, and is a medicinal herb that has been well known for its efficacy and effectiveness in the East as well as in the West for a long time.

In general, ginseng is composed of 3-6% saponin content, 12-15% nitrogen compounds, 1-2% fat-soluble components, 50-60% carbohydrates and 4-6% other ginseng species. Ginsenoside, a physiologically active substance as an active ingredient, is a type of saponin and contains about 3-6%. Ginsenoside, a type of saponin, is divided into Protopanaxadiol (PPD), Protopanaxatriol (PPT), and Oleanane, and about 33 species have been reported according to its molecular structure. , Korea Ginseng and Tobacco Research Institute, 1996].

Research on the pharmacological efficacy of ginseng has focused on the investigation of the active ingredient and pharmacological efficacy of ginseng by purely separating ginsenoside components peculiar to ginseng and revealing its chemical structure, and has been revealed through numerous research reports and papers at home and abroad. The results of the research on the efficacy of ginseng include central nervous system disease, anti-anxiety, nervous system, drug addiction detoxification effect, blood vessel function, alcohol detoxification, lipid metabolism and fatigue recovery, anti-diabetes, hyperlipidemia, anti-viral, immunity enhancement, sexual function improvement, anti-cancer and Cancer prevention efficacy has been scientifically revealed. In particular, ginsenoside Rb1, a representative ginsenoside of panaxadiol (PPD) saponin, exhibits an inhibitory effect on the central nervous system, resulting in mental stability, nerve relaxation, analgesia, anticancer, anticonvulsant, It is known to exhibit a blood pressure lowering effect. In addition, it is known that it has anti-fatigue effects by acting on the central nervous system represented by ginsenoside Rg1, a panaxatriol-based (PPT) saponin [Latest Korea Ginseng Research, Korea Ginseng Association, 2007].

On the other hand, ginseng leaves are known to contain 19.58% of irradiated ponins compared to stems (2.38%) and roots (4.78%). Ginseng leaves also contain 10.84% of ginsenosides, stems (0.64%) and roots. (2.37%) is contained in a large amount (Korean J. Ginseng Sci. 11(2). 118-112, 1987). In addition, ginsenosides have various types of ingredients, and each exhibits different efficacy, making it a major target for research.

However, studies on the analysis of ginsenosides in ginseng leaves are not actively conducted compared to the roots, and in particular, there are no studies on ginseng leaf fermentation such as increasing the content of useful saponin components by inoculating and culturing ginseng leaves with specific strains.

KR 10-2010-0043725 A KR 10-2010-0043723 A

Lee S et al., Bioconversion of Ginsenosides from Red Ginseng Extract Using Candida allociferrii JNO301 Isolated from Meju, Mycobiology, 2014;42(4):368-75 Geum-sook Kim et al., Extraction and pretreatment method for analysis of ginsenosides in ginseng, J Medicinal Crop Sci 2008;16 (6):446-54 Sanada S et al., Studies on the saponins of ginseng, Structures of ginsenoside-Ro, -Rb1, -Rb2, -Rc and -Rd, Chemical and Pharmaceutical Bulletin, 1974;22(2):421-8

An object of the present invention was conceived to solve the problems of the prior art as described above, and the problem to be solved in the present invention is to remove or convert sugars of ginsenosides using microorganisms targeting ginseng leaves, as well as high biological activity. It is intended to provide a method for preparing a ginsenoside-containing fermentation broth capable of improving the content of ginsenosides and a ginsenoside-containing fermentation broth derived from ginseng leaves.

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

In order to solve the problems as described above, the present invention comprises (a) preparing a ginseng leaf suspension, (b) in Pomona switch to stacking notes represented by the ginseng leaf suspensions, Accession No. Accession No. KCTC 13945BP (Stenotrophomonas sp .) One or more microbial strains selected from the group consisting of S7 strain, Bacillus sp. S9 strain represented by the accession number KCTC 13945BP and Lactobacillus sp. S11 strain represented by the accession number KCTC13946BP. It provides a method for producing a ginsenoside-containing fermentation broth derived from ginseng leaves, comprising the steps of inoculating and culturing the ginseng leaf suspension to ferment the ginseng leaf suspension, and (c) obtaining the fermentation broth.

The ginseng leaf suspension is (a-1) drying the ginseng leaf; (a-2) preparing dried ginseng leaves into powder; (a-3) adding the powder to distilled water and heating it; And (a-4) is preferably prepared from the step of sterilizing.

The culture is preferably cultured with shaking for 1 day or more under conditions of 25° C. or higher and pH 6.5 to 8.5.

The ginsenoside is preferably selected from the group consisting of Rb2, Rd, Re, Rf, Rg1, Rg2, Rg3, Rh1, Rh2, compound K, F1, F2, and F3.

In addition, the present invention provides a ginsenoside-containing fermented broth derived from ginseng leaves obtained by the production method of the present invention.

In addition, the present invention comprises the step of preparing a supernatant from the fermentation broth, and separating a single ginsenoside component from the supernatant, wherein the ginsenoside single component is Rb2, Rd, Re, Rf, Rg1, Rg2, Rg3 , Rh1, Rh2, Compound K, F1, F2, F3 provides a method for separating a single component of ginsenoside, characterized in that selected from the group consisting of.

The fermentation broth containing ginsenosides originating from ginseng leaves according to the manufacturing method of the present invention is to increase the total content of ginsenosides having high biological activity such as compound K as well as F2, F1 and Rd from saponin precursors present in ginseng leaves. It is possible. Therefore, the ginsenoside-containing fermented product of ginseng leaf origin of the present invention is expected to be effectively used in health functional foods and pharmaceuticals.

1 is a result of selection of primary strains by the esculin agar method from foods distributed in the traditional market.
2 is a result showing the decomposition activity of ginsenosides per selected strain.
Figure 3 is Stenotrophomonas sp. S7 and Bacillus sp. This is a result showing that the S9 mixed strain can be separated and cultured depending on the temperature.
4 is a result showing the culture characteristics according to the temperature of the selected strain.
5 and 6 are results of analysis of the components of ginsenoside according to the combination of the selected strains and fermentation conditions.
7 is a result showing the degree of growth according to the combination of the selected strains and fermentation conditions.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention target ginseng leaves, which are known to have a higher saponin content than ginseng roots, and prepare them as a suspension, and by inoculating and culturing new microbial strains therein, the ginsenoside content is increased, as well as physiological A fermentation broth containing a large amount of sugar-removed or converted ginsenoside components having excellent activity was obtained.

Accordingly, the present invention relates to (a) preparing a ginseng leaf suspension, (b) in the ginseng leaf suspension, Stenotrophomonas sp. S7 strain represented by accession number KCTC 13945BP, accession number Ginseng leaves by inoculating and culturing at least one microorganism strain selected from the group consisting of Bacillus sp. S9 strain represented by KCTC 13945BP and Lactobacillus sp. S11 strain represented by accession number KCTC13946BP. It provides a method for producing a ginsenoside-containing fermented broth derived from ginseng leaves, comprising fermenting the suspension and (c) obtaining the fermented broth.

The ginseng leaf targets only the "leaf" from which the roots and stems of ginseng have been removed. The ginseng leaf suspension is prepared by drying the prepared ginseng leaves, finely powdering them, adding distilled water, heating it to the extent of foaming, leaving it at room temperature to cool it, and then using it as a microbial medium by high pressure sterilization. It can be prepared by sterilization.

Therefore, the ginseng leaf suspension of the present invention comprises the steps of (1) drying ginseng leaves; (2) preparing dried ginseng leaves into powder; (3) adding the powder to distilled water and boiling; And (4) sterilizing.

As a preferred embodiment, the ginseng leaf suspension of the present invention is prepared by completely drying the prepared ginseng leaves at 50 to 60°C, preferably 50 to 55°C, and most preferably at 50°C for 3 days or more, and preparing a fine powder. Here, 1 to 5 g, preferably 1.5 to 3 g, most preferably 2 g of fine powder per 100 ml of distilled water is suspended, heated to the extent of foaming, allowed to cool at room temperature, and then sterilized under pressure and high temperature to sterilize microorganisms. A ginseng leaf suspension medium can be prepared for inoculation.

The novel microbial strains used in the present invention were separated from the foods of mushrooms, pastes, and milk galls distributed in the traditional market (named "S7" to "S11") (Table 1), of which the most excellent strain Finally, three species (S7, S9 and S11) were selected.

Specifically, a total of 18 foods of mushrooms, pastes, and milk galls purchased from the traditional market were prepared, and colonies showing black were considered to have β-glucosidase activity using the esculin agar method (Lee et al., 2014). Thus, microorganisms having a sugar change activity of ginsenoside were first selected (Table 1 and FIG. 1).

Thereafter, three new microorganisms were finally selected through the following process in order to select a strain having conversion activity per ginsenoside for the first selected microorganism strains.

Specifically, the first selected strain was inoculated into a conical tube containing 3 ml of the prepared ginseng leaf suspension, followed by shaking culture at 30° C. for 5 days. The culture solution was centrifuged at 13,000 rpm for 15 minutes at room temperature to remove cells and polymer substances. After centrifugation, 1 ml of the supernatant was used to measure the content of each component of ginsenoside. The measurement of the content of ginsenosides using HPLC was analyzed according to the method of Geum-suk Kim, Keum-sook Kim, Dong-yoon Hyun, Young-ok Kim, Seong-woo Lee, Young-chang Kim, Seung-eun Lee, Young-deuk Son, 2008). That is, 1 ml of the supernatant was mixed with 1 ml of methanol, and then ginsenoside was partially purified using a solid phase extractor (SPE). The partially purified product was analyzed by HPLC to analyze the content of ginsenoside components by strain. At this time, the instrument used was an Agilent 1100 series HPLC system (Agilent Technolgies, USA). Ginsenoside standards were purchased from Embo Research Institute.

The results of the analysis of ginsenoside sugar conversion activity as described above are shown in FIG. 2, and most of the selected strains showed high sugar conversion activity for two types of ginsenosides [Protopanaxadiol (PPD), Protopanaxatriol (PPT)], All the selected strains also showed activity against the polysaccharide components Rb1 and Rb2. From these results, three strains were finally selected (S7, S9 and S11).

To identify the three selected strains, sequencing was performed, and the nucleotide sequence of the 16S rRNA gene of the final selected strain was sequenced, and the similarity (%) using the NCBI database and its program Named based on the result of identification by analysis. That is, " Stenotrophomonas sp. S7" (SEQ ID NO: 1) is Stenotrophomonas sp. PRGB08 16S ribosomal RNA gene, partial sequence (GenBank: EF195342.1) showed 81% similarity, " Bacillus sp. S9" (SEQ ID NO: 2) was Bacillus sp. TS1223R 16S ribosomal RNA gene, partial sequence (GenBank: FJ449657.1) and " Bacillus sp. S11" (SEQ ID NO: 3) are Lactobacillus murinus gene for 16S ribosomal RNA, partial sequence, strain: LAP1 (GenBank: LC187241.1) showed 99% similarity. These strains were deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on September 16, 2019, and Stenotrophomonas sp. S7 and Bacillus sp. For the S9 mixed strain, accession number KCTC 13945BP, Bacillus sp. For S11, it was assigned the deposit number KCTC13946BP, respectively.

The selected new strains may be inoculated into the ginseng leaf suspension alone or in combination of two or more.

The inoculated ginseng leaf suspension undergoes a cultivation process, and the cultivation is at least 1 day, preferably 5 days or more under the conditions of 25 to 45°C, preferably 25 to 40°C, pH 6.5 to 8.5, and preferably pH 8.5. It is desirable to incubate with shaking for 5 weeks.

In addition, a carbon source, a nitrogen source, and/or a trace element known in the art may be added to the phosphate leaf suspension to promote or assist the growth of the inoculated strain.

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, and the like can be used. Complex compounds such as molasses or by-products of the sugar refining process may 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, and whether included in the medium alone or as a mixture of two or more, 0.1% (w/w) to 20% (w/w) of It can be included in the medium in a range. When natural or processed natural products such as starch or starch hydrolyzate are used as carbon sources, these carbon sources can be sterilized and used to prevent unwanted fermentation by other microorganisms. Sterilization may be performed using a method known in the art such as high temperature/high pressure sterilization method, ultraviolet irradiation, or the like.

As the nitrogen source, it may be usually an inorganic nitrogen compound, an organic nitrogen compound, or a composite compound containing these compounds. Examples of inorganic nitrogen compounds include ammonia, ammonium salts (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.), nitrates, and the like, and organic nitrogen compounds include urea and amino acids. As a complex compound, yeast extract, soytone, peptone, tryptone, malt extract, meat juice, soybean powder, soybean bean curd powder, cheonggukjang powder, and soybean paste And natural nitrogen sources such as powder. One or more of these nitrogen sources may be mixed and used, 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 preferable to use it after sterilization for the same reason as described in connection with the case of 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 In order to increase the solubility and maintain the solution state, a chelating agent such as catechol, citric acid, etc. may be added together. Examples of the salt compound form include sodium hydrogen phosphate, magnesium sulfate, iron chloride, calcium salt, manganese salt, cobalt salt, molybthenate salt, and chelate metal salt. These trace elements may be used by mixing one or more, and may be included in the medium in the range of 0.001% (w/w) to 3.0% (w/w).

In addition, the medium may optionally be prepared by including a growth promoter such as biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate, and pyridoxine.

Regarding optimization of other medium composition, there are many in the art, including Applied Microbiol. Physiology, A Practical Approach (Editors PM Rhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Documents are known, and reference may be made to these known documents.

The fermentation broth prepared according to the manufacturing method of the present invention contains a large amount of various ginsenosides, in particular, the ginsenosides are Rb2, Rd, Re, Rf, Rg1, Rg2, Rg3, Rh1, Rh2, compound K, F1 , F2, F3 may be one or more selected from the group consisting of.

The fermentation broth prepared according to the manufacturing method of the present invention may be used for separating individual components of the ginsenoside.

This separation method includes obtaining a supernatant of the fermentation broth, and separating each of the individual components from the supernatant.

Methods for separating these individual components are known in the art, such as HPLC method, and any method known in the art may be used.

Meanwhile, the fermentation broth containing ginsenoside derived from ginseng leaves obtained by the above-described manufacturing method or individual ginsenoside components separated therefrom have various physiological activities, so they are commercialized into pharmaceutical compositions, health functional foods, functional cosmetics, etc. Can be. Specifically, the pharmaceutical composition, health functional food, and functional cosmetics are anti-diabetes, anti-hypertension, anti-hyperlipidemia, cardiovascular disease treatment or prevention, atopic dermatitis improvement, anti-obesity, rejuvenation, skin wrinkles of these ginsenoside components It may have uses or functionality known in the art, such as improvement, skin whitening, and the like.

When the ginsenoside-containing fermentation broth derived from ginseng leaves of the present invention is commercialized and used as a pharmaceutical composition, the pharmaceutical composition includes a pharmaceutically acceptable carrier in addition to the fermentation broth, and is administered by a conventional method known in the art. Depending on, it can be prepared in an oral or parenteral formulation. Here, the route of administration may be any suitable route including a local route, an oral route, an intravenous route, an 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 in which two or more formulations of drugs are combined according to the route of administration, for example, when one drug is first administered by an intravenous route and the second drug is administered by a local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specifically, reference may be made to the pharmacopoeia of each country, including the "Korean Pharmacopeia."

When the pharmaceutical composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to a method known in the art together with a suitable carrier It can be prepared in a formulation such as. Examples of suitable carriers at this time include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease Serol, etc. are mentioned. In the case of formulation, appropriate binders, lubricants, disintegrants, colorants, and diluents may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferryst, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, etc., and lubricants include oleic acid. Sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polydecylene glycol, etc., and as disintegrating agents starch, methyl cellulose , Agar, bentonite, xanthan gum, starch, alginic acid, or a sodium salt thereof. Moreover, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc. are mentioned as a diluent.

When the pharmaceutical composition of the present invention is prepared in a parenteral dosage form, it may be formulated in the form of an injection, a transdermal administration, a nasal inhalation and a suppository according to a method known in the art together with a suitable carrier. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, triethanol amine-containing PBS (phosphate buffered saline), sterile water for injection, an isotonic solution such as 5% dextrose, etc. may be used. . When formulated as a transdermal administration, it can be formulated in the form of an ointment, cream, lotion, gel, external solution, pasta, liniment, air roll, and the like. In the case of nasal inhalants, suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. can be used to form an aerosol spray.When formulated as a suppository, the carrier is Withepsol ( witepsol), tween 61, polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like.

The specific formulation of pharmaceutical compositions is known in the art, and for example, Remington's Pharmaceutical Sciences (19th ed., 1995) may be referred to. This document is considered as 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, sex, age, patient severity, and route of administration. May be in the /kg range. Administration can be made once a day or divided into several times. Such dosage should not be construed as limiting the scope of the invention in any aspect.

In another aspect, when the ginsenoside-containing fermented broth derived from ginseng leaves of the present invention is commercialized and used as a health functional food composition, the food composition may be prepared in any form, such as tea, juice, carbonated drinks, ionized drinks, etc. Beverages, processed oils such as milk, yogurt, gums, rice cakes, Korean sweets, bread, snacks, foods such as noodles, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jelly, bars It can be manufactured with health functional food preparations such as.

In addition, the food composition of the present invention can be classified as a product as long as it conforms to the enforcement regulations at the time of manufacture and distribution in terms of legal and functional classification. For example, it is a health functional food according to the Korean 「Health Functional Food Act」, or confectionery, bean, tea, and beverages according to each food type according to the food code of the Korean 「Food Sanitation Act」 (``Food Standards and Standards'' notified by the Ministry of Food and Drug Safety). , Special use food, etc.

In addition to the fermented liquid, food additives may be included in the food composition of the present invention. Food additives can generally be understood as substances that are added to food and mixed or infiltrated in manufacturing, processing, or preserving food. Since they are consumed daily and for a long time with food, their safety must be ensured. Food additives with guaranteed safety are limited in terms of ingredients or functions in the Food Additives Code according to the laws of each country governing the manufacture and distribution of food (the “Food Sanitation Act” in Korea). In the Korean Food Additives Code (“Food Additive Standards and Standards” notified by the Ministry of Food and Drug Safety), food additives are classified into chemical synthetic products, natural additives, and mixed preparations in terms of ingredients.These food additives are sweeteners and flavors in terms of function. It is categorized into agents, preservatives, emulsifiers, acidulants, and thickeners.

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

Flavoring agents can be used to enhance taste or aroma, and both natural and synthetic can be used. Preferably, it is the case of using a natural one. In the case of using natural ones, the purpose of nutrient enhancement can be combined in addition to flavor. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, or the like, or from green tea leaves, roundtails, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, you can use those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo. The natural flavoring agent may be a liquid concentrate or a solid extract. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, and the like.

As a preservative, sodium calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, Pectin, etc. may be mentioned, and as the acidulant, arithmetic, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like can be used. The acidulant may be added so that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms in addition to the purpose of enhancing taste.

As the thickener, a suspending agent, a settling agent, a gel-forming agent, a swelling agent, and the like may be used.

In addition to the food additives described above, the food composition of the present invention may include a physiologically active substance or minerals known in the art for the purpose of supplementing and reinforcing functionality and nutritional properties and ensuring stability as a food additive.

Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoyl thiamine, etc., and minerals include calcium preparations such as calcium citrate, magnesium stearate. Magnesium preparations such as, iron preparations such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, and zinc.

In the food composition of the present invention, the food additive described above may be included in an appropriate amount to achieve the purpose of addition according to the product type.

Regarding other food additives that may be included in the food composition of the present invention, reference may be made to the food code of each country or the food additive code.

In another aspect, when the ginsenoside-containing fermentation broth derived from ginseng leaves of the present invention is commercialized and used as a functional cosmetic composition, the cosmetic composition includes ingredients commonly used in cosmetic compositions, such as stabilizers, solubilizers, in addition to the fermentation broth. , Surfactants, conventional auxiliaries such as vitamins, pigments and perfumes, and carriers.

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

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

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an 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, and the like may be used.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, and crystallites Castle cellulose, aluminum metahydroxide, bentonite, agar, and the like can be used.

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters may be used.

The cosmetic composition of the present invention can be prepared according to a method for preparing a cosmetic composition commonly used in the art, except for including the fermentation broth.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples describe a preferred specific example of the present invention, and the scope of the present invention is not limited and interpreted by the matters described in the following examples.

[Example]

1. Selection of microorganisms with sugar-changing activity of ginsenoside

Product samples (m13, m14, m15, m21, m27) with beta-glucosidase activity from 28 products (sample numbers m1 to m28) distributed in the traditional market m28) was first selected, from which five strains of S7 to S11 were selected (Table 1, Fig. 1). The selection method used the esculin agar method (Lee S, Lee YH et al. Mycobiology. 2014;42(4):368-75). Colonies showing black complex were considered to have beta-glucosidase activity.

Please check if it is a natural mushroom harvest.

Please fill in 28 times.

Sample number Products Product or trade name manufacturer Isolation strain name 13 Mushrooms White Mushroom Natural harvest Stenotrophomonas sp. S7 14 Mushrooms Bean Bird Sample Ewingella sp. S8 15 Fermented soybean food Traditional old miso Jeongjeongwon Sunchang Bacillus sp. S9 21 fermented food Malt gold Blues Bacillus sp. S10 27 Salted fish Handsome Mokpo-Shinan-san Shrimp Salted Umda Food Bacillus sp. S11 28 Cheonggukjang Self-manufacturing Enterococcus sp. S12

2. Assay for conversion activity per ginsenoside of selected strains

In order to determine whether there is an activity such as transformation per ginsenoside for the five strains selected in this way, shaking culture (30° C., 200 rpm) was performed using a leaf suspension (2%) as a medium. Leaf suspension used as a medium is to add 2 g of fine powder of dried leaves (more than 3 days at 50°C, dry completely) in 100 ml of distilled water, break to the extent that bubbles are formed, and then stand at room temperature for 10 minutes, and pressurize at 121°C for 10 minutes. It was sterilized at high temperature and then cooled to room temperature. The selected strain was inoculated into a conical tube containing 3 ml of this leaf suspension and cultured with shaking at 30° C. for 5 days. The culture solution was centrifuged at 1,300 rpm for 15 minutes at room temperature to remove cells and polymer substances.

After centrifugation, 1 ml of the supernatant was used to measure the content of each component of ginsenoside. The measurement of the content of ginsenosides using HPLC was analyzed according to the method of Geum-sook Kim et al. Specifically, 1 ml of this supernatant was mixed with 1 ml of methanol, and then ginsenoside was partially purified using a solid phase extractor (SPE). The partially purified product was analyzed by HPLC to analyze the ginsenoside component content of each strain. At this time, the instrument used was an Agilent 1100 series HPLC system (Agilent Technolgies, USA). Ginsenoside standard products were purchased from Ambo Research Institute (Daejeon, Korea).

Fig. 2 shows the results of analyzing the content of each component of ginsenoside.

According to Figure 2, Bacillus sp. Only S11 strain showed CK (ginsenocide compound K) conversion activity, and F1 (ginsenocide compound F1) was Bacillus sp. S9 strain and Bacillus sp. All of the S11 strains showed high conversion activity.

Based on these results, the final Bacillus sp. S9 strain and Bacillus sp. S11 strain and Stenotrophomonas sp. S7 strain was selected.

3. Identification and deposit of the selected strain through 16S rRNA sequencing

The nucleotide sequence of the 16S rRNA gene of the finally selected strain was sequenced and named based on the identification result by similarity (%) analysis using the NCBI database and its program. That is, Stenotrophomonas sp. S7 (SEQ ID NO: 1) is Stenotrophomonas sp. PRGB08 16S ribosomal RNA gene, partial sequence (GenBank: EF195342.1) and 81%, and Bacillus sp. S9 (SEQ ID NO: 2) is Bacillus sp. TS1223R 16S ribosomal RNA gene, partial sequence (GenBank: KU587453.1) and 99%, Bacillus sp. S11 (SEQ ID NO: 3) was 99% similar in base sequence to Bacillus velezensis strain MHNK1 16S ribosomal RNA gene, partial sequence (GenBank: KX863524.1). These strains were deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on September 16, 2019, and Stenotrophomonas sp. S7 and Bacillus sp. For the S9 mixed strain, accession number KCTC 13945BP, Bacillus sp. For S11, it was assigned the deposit number KCTC13946BP, respectively.

Stenotrophomonas sp. S7 and Bacillus sp. When the S9 mixed strain was cultured for 15 hours in the R2A agar mixed strain, Stenotrophomonas sp. Only S7 was cultured, and Bacillus sp. Since only S9 is cultured, each strain can be separated and used if necessary. The separation of the mixed strain according to the culture temperature can be confirmed from the results of FIG. 4 below.

4. Growth characteristics according to culture temperature of the selected strain

The growth characteristics according to the culture temperature of the selected strains were observed in R2A agar medium (R2A agar), and the results are shown in FIG. 4. As in the results of Fig. 3, Bacillus sp. S9 strain was found to have an appropriate culture temperature of 35°C or higher, and Stenotrophomonas sp. S7 strain and Bacillus sp. S11 strain was found to have an appropriate culture temperature of 25 ℃ to 30 ℃.

5. Ginsenoside fermentation medium or saponin extract preparation

In order to use ginseng leaf extract as a fermentation medium, it was treated as follows. Ginseng leaves were dried, finely powdered, and then sterilized distilled water was added to a concentration of 2%. This suspension was heated to the extent of foaming, and then allowed to stand at room temperature for 10 minutes. After washing this solution with distilled water for medical gauze, it was filtered in 8 layers. The recovered solution was adjusted for acidity with 1N NaOH as necessary. The pH meter (Metter Toledo Group, Switzerland) was used to measure the acidity. This extract was sterilized at 121° C. for 10 minutes and used as a microorganism fermentation medium for bioconversion such as ginsenoside.

6. Investigation of strain combinations and fermentation conditions to maximize ginsenoside active ingredient

The selected strain culture medium grown in LB liquid medium was mixed with the ginseng leaf medium sterilized solution prepared above in the amount shown in the table below, and the contents of ginsenoside components were shaken (200 rpm) during the cultivation temperature, pH and cultivation period in the table below. Was analyzed. Analysis of the content of the ginsenoside component was performed by centrifuging the culture medium (5 minutes at 1,300 rpm), taking the supernatant, and then using HPLC and TLC.

Measurement of the ginsenoside component content using HPLC was performed according to the method of Geum-sook Kim et al. (Kum-sook Kim et al., J Medicinal Crop Sci 2008;16(6):446-54), as described above, and analysis of ginsenosides of the extract using TLC. Followed the method of Sanada et al. (Sanada S et al. Chemical and Pharmaceutical Bulletin. 1974;22(2):421-8). In the analysis using TLC, TLC Silica gel 60 RP-18 F254s (Merk, hx43662759) was used, and the developing solvent was chloroform: methanol: water=7:3:2.

The ginsenoside component analysis results were divided into PPD-based ginsenoside and PPT-based ginsenoside, and are shown in FIGS. 5 and 6, respectively. Referring to FIGS. 5 and 6, the overall component content of both PPD and PPT ginsenoside was the best in the case of pH 8.5-3. In the case of pH 8.5-3, ginsenoside F2 content increased 2.6 times, F1 2 times, and Rd 2 times higher than that of the control, and the total ginsenoside content was about 36% from 454 ppm to 619 ppm in the control content. Increased.

Meanwhile, in the case of ginsenoside compound K, Stenotrophomonas sp. S7 strain was produced when cultured under pH 6.5 conditions (see pH6.5_1 section), and Stenotrophomonas sp. S7 strain and Bacillus sp. In the case of mixed culture of S9 strain, (i) shaking culture at 30℃ for 2 weeks (200rpm) and then shaking culture at 40℃ for 2 weeks, regardless of pH conditions, were produced (pH6.5_2, pH7.5_2 and pH8). .5_2), (ii) shaking culture at 30℃ for 2 weeks (200rpm) and then shaking culture at 40℃ for 1 week, it was produced under pH 7.3 condition (refer to pH7.5_3 item).

Meanwhile, the absorbance (A600) of the strains labeled A, B, C, and D in Table 2 according to the pH was measured and shown in FIG. 7. At pH 6.5, the strain labeled A ( Stenotrophomonas sp. S7 strain) and labeled strain B ( a mixed strain of Stenotrophomonas sp. S7 strain and Bacillus sp. S9 strain) showed excellent growth.

<110> Kyongsangbuk-do Agricultural Technology Administration <120> FERMENTED BROTH COMPRISING GINSENOSIDE ORIGINATED THE LEAF OF GINSENG AND MANUFACTURING METHOD THEREOF <130> PP19-000-KBA <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 893 <212> DNA <213> Unknown <220> <223> Stenotrophomonas sp. S7 <400> 1 ccccatggtg gacacttaac gcgttagctt cgacactgag agcgtatggc accctccgtc 60 catttcccac cgttaagggc gggaacaacc agggtatcta acccggtttg ccccccaccc 120 tttcgtgcct cagtgtcatt gttggtccag gtatctgcct tccccatgga tgttcctcca 180 tatctctact catttgactg ctgcaccggg cagtccacta ccctctaact cactccatac 240 acccactatg aacgggcatt cccaggttga gcccgggact ttcacaccaa acttacaaaa 300 ccccctacgc ccgctttacc cccaatattt ccaagaaacc ctggaccccc tctaattacc 360 gggcctgcgg gccagaattt acccggggct aattctttgg gtacggtcaa aacaaccggg 420 tattagccaa tggcttttct ttcccatcaa aagggcttaa caacccaaaa gccttcttca 480 cccacgcggg ttggctggac caggcttgcc ccaatttccc aatattcccc atggctgcct 540 ccctgggaag tgtggaccgt gtctcatttc cggagtggct gatcatcctc ccaaaacatt 600 tacggatggt gacttggtgg gcctttaccc cgccaatgta gctaatccaa catcggatca 660 tctatatacg caaggcccaa aggtcccctg tttatgacgt aaggcggtat gcggttttcc 720 cgtaagtttc tgtcccttat ccccccgcgt acggggtaga ttccgatgta ttcctccagc 780 cgttccgccc ctcaccacgc aaaaaactac tctctgactg tgctcccact tgagtgtaat 840 gtgtccggcc gcaccccctc tgctcgcgtt gcctcaaaaa aaaaaaaaaa aaa 893 <210> 2 <211> 992 <212> DNA <213> Unknown <220> <223> Bacillus sp. S9 <400> 2 acgtcagtct cccaggcgga gtgcttatgc gttagctgca gcactaaggg gcggaaaccc 60 cctaacactt agcactcatc gtttacggcg tggactacca gggtatctaa tcctgttcgc 120 tccccacgct ttcgctcctc agcgtcagtt acagaccaga gagtcgcctt cgccactggt 180 gttcctccac atctctacgc atttcaccgc tacacgtgga attccactct cctcttctgc 240 actcaagttc cccagtttcc aatgaccctc cccggttgag ccgggggctt tcacatcaga 300 cttaagaaac cgcctgcgag ccctttacgc ccaataattc cggacaacgc ttgccaccta 360 cgtattaccg cggctgctgg cacgtagtta gccgtggctt tctggttagg taccgtcaag 420 gtaccgccct attcgaacgg tacttgttct tccctaacaa cagagcttta cgatccgaaa 480 accttcatca ctcacgcggc gttgctccgt cagactttcg tccattgcgg aagattccct 540 actgctgcct cccgtaggag tctgggccgt gtctcagtcc cagtgtggcc gatcaccctc 600 tcaggtcggc tacgcatcgt tgccttggtg agccgttacc tcaccaacta gctaatgcgc 660 cgcgggtcca tctgtaagtg gtagccgaag ccacctttta tgtttgaacc atgcggttca 720 aacaaccatc cggtattagc cccggtttcc cggagttatc ccagtcttac aggcaggtta 780 cccacgtgtt actcacccgt ccgccgctaa catcagggag caagctccca tctgtccgct 840 cgacttgcat gtattaggca cgccgccagc gttcgtcctg acgaggggaa aaacccctat 900 ataaaccccc cccccctctt tccccccttt ttttttttat tttaaaaatt aaagggatat 960 aacatttaac aaggttcttg ggattccact ga 992 <210> 3 <211> 981 <212> DNA <213> Unknown <220> <223> Bacillus sp. S11 <400> 3 acggagtctc ccatgcggag tgcttatgcg ttagctgcag cactaagggg cggaaacccc 60 ctaacactta gcactcatcg tttacggcgt ggactaccag ggtatctaat cctgttcgct 120 ccccacgctt tcgctcctca gcgtcagtta cagaccagag agtcgccttc gccactggtg 180 ttcctccaca tctctacgca tttcaccgct acacgtggaa ttccactctc ctcttctgca 240 ctcaagttcc ccagtttcca atgaccctcc ccggttgagc cgggggcttt cacatcagac 300 ttaagaaacc gcctgcgagc cctttacgcc caataattcc ggacaacgct tgccacctac 360 gtattaccgc ggctgctggc acgtagttag ccgtggcttt ctggttaggt accgtcaagg 420 taccgcccta ttcgaacggt acttgttctt ccctaacaac agagctttac gatccgaaaa 480 ccttcatcac tcacgcggcg ttgctccgtc agactttcgt ccattgcgga agattcccta 540 ctgctgcctc ccgtaggagt ctgggccgtg tctcagtccc agtgtggccg atcaccctct 600 caggtcggct acgcatcgtt gccttggtga gccgttacct caccaactag ctaatgcgcc 660 gcgggtccat ctgtaagtgg tagccgaagc caccttttat gtttgaacca tgcggttcaa 720 acaaccatcc ggtattagcc ccggtttccc ggagttatcc cagtcttaca ggcaggttac 780 ccacgtgtta ctcacccgtc cgccgctaac atcagggagc aagctcccat ctgtccgctc 840 gacttgcatg tattaggcac gccgccagcg ttcgtccgac gggggggaaa accatatata 900 aaaaaggggg ggtgtttggg gaaaaaaagg aatttaacga taatggcagc gaaaaaaatt 960 taaaaggggt tggagatttt t 981

Claims (8)

The present invention (a) preparing a ginseng leaf suspension,
(b) In the ginseng leaf suspension, Stenotrophomonas sp. S7 strain represented by accession number KCTC 13945BP, Bacillus sp. S9 strain represented by accession number KCTC 13945BP, and Fermenting a ginseng leaf suspension by inoculating and culturing at least one microorganism strain selected from the group consisting of Lactobacillus sp. S11 strain represented by deposit number KCTC13946BP, and
(c) a method for producing a ginsenoside-containing fermented broth derived from ginseng leaves, comprising the step of obtaining the fermented broth
The method of claim 1, wherein the ginseng leaf suspension is prepared from the following steps:
(1) drying ginseng leaves;
(2) preparing dried ginseng leaves into powder;
(3) adding the powder to distilled water and boiling; And
(4) sterilizing step.
The method according to claim 1, wherein the culture is performed with shaking for at least 1 day under conditions of 25°C or higher and pH 6.5 to 8.5.
The method of claim 1, wherein the ginsenoside is at least one selected from the group consisting of Rb2, Rd, Re, Rf, Rg1, Rg2, Rg3, Rh1, Rh2, compound K, F1, F2, F3. Manufacturing method.
A ginsenoside-containing fermentation broth derived from ginseng leaves obtained by the production method according to any one of claims 1 to 4.
Preparing a supernatant of a ginsenoside-containing fermentation broth derived from ginseng leaves obtained by the manufacturing method according to any one of claims 1 to 4, and separating a single ginsenoside component from the supernatant,
The ginsenoside single component is selected from the group consisting of Rb2, Rd, Re, Rf, Rg1, Rg2, Rg3, Rh1, Rh2, compound K, F1, F2, F3.
Method for separating a single component of ginsenoside.

A mixed strain of Stenotrophomonas sp. S7 and Bacillus sp. S9 strain with accession number of KCTC 13945BP.
Bacillus sp. S11 strain with an accession number of KCTC13946BP.

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