KR102376187B1 - Fermention product of ginseng extract complex GINOS with enhanced active saponin and uses thereof - Google Patents

Abstract

The present invention relates to a ginseng comprehensive fermentation product and a manufacturing method thereof, capable of increasing a content of minor ginsenosides by fermenting Ginos, which is a ginseng comprehensive extract manufactured by mixing a white ginseng extract, a red ginseng extract, a Taegeuk ginseng extract, a black ginseng extract, and a wild ginseng culture root extract which are representative processed ginseng extracts of five types with different compositions of ginsenosides, with a specific enzyme and lactic acid bacteria. The ginseng comprehensive fermentation product according to the present invention increases contents of minor ginsenosides Rh1, Rh2, and F2 and a compound K by 2.5-11 times, improves pre-radical elimination, increases reducing power, and maintains high efficiency for anti0inflammatory activity, immune enhancement activity, and lung cancer cell (A254) growth inhibition activity. The ginseng comprehensive fermentation product can be used as a raw material for manufacturing food, health functional food, cosmetics, and medicines by using excellent physiological activity.

Description

Fermented ginseng complex saponin with enhanced active saponin (GINOS) and uses thereof {Fermention product of ginseng extract complex GINOS with enhanced active saponin and uses thereof}

The present invention relates to a ginseng complex fermented product and a method for producing the same. Specifically, extracts of 5 types of processed ginseng, white ginseng, red ginseng, Taegeuk ginseng, black ginseng and wild ginseng cultured root, which have different compositions of ginsenosides, are mixed and mixed with specific enzymes and It relates to a ginseng complex fermented product prepared by two-step fermentation using specific lactic acid bacteria, a method for preparing the same, and a composition for antioxidant, anti-inflammatory or immune enhancement comprising the ginseng complex fermented product as an active ingredient.

Ginseng (Panax ginsneg C. A Meyer) is a perennial semi-shade succulent root plant belonging to the Araliaceae family, Panax , and about 11 species are known. In particular, Korean ginseng is native to the Korean Peninsula and has been known as a representative nourishing agent since ancient times, and various physiological activities have been revealed.

The representative pharmacological and physiological actions of ginseng revealed so far include antioxidant action, anticancer action, immunity enhancement, memory improvement, blood circulation improvement through inhibition of platelet aggregation, arteriosclerosis prevention, hypertension prevention, fat accumulation inhibition, anti-obesity, skin whitening, wrinkle improvement, Anti-diabetes, alcohol decomposition, liver function promotion, liver protection, central nervous system protection, menopausal improvement, anti-stress, anti-fatigue, gastric protection, etc. are very diverse.

It is reported that these various effects of ginseng are expressed by the special ingredients of ginseng and their harmony. Representative physiologically active ingredients include ginsenosides, polyphenols, acidic polysaccharides, polyacetylene, ginseng proetin, and amino acids. Among these, the most important bioactive substances are ginsenosides. Ginsenoside is a non-toxic saponin that only ginseng has. It has a different chemical structure from saponin found in other plants. They are called ginsenosides. In general, cultivated fresh ginseng contains 6-7 types of ginsenosides such as Rg1, Rb2, Rg1, Rc, Rd, and Re.

Ginseng saponins are transformed, lost, or biosynthesized into various types of saponins depending on the processing method, and about 35 types of ginsenosides, which were only 6-7 types in fresh ginseng, have been identified. The reason why various types of ginsenosides appear depending on processing is because sugars are separated from major ginsenosides with large molecular weights due to physical treatment such as heat, and low molecular weight trace ginsenosides are generated. Studies have shown that minor ginsenosides also have various physiological activities.

The various effects of ginseng depend on how many types of ginsenosides are produced depending on the processing method. For example, in red ginseng steamed and dried as the most representative processed ginseng, major ginsenoside is decomposed to produce minor ginsenoside components such as Rg3, Rh1, and Rh2, which are not found in fresh ginseng. Efficacy such as boosting immunity is increased. In addition, in the case of Taegeuk ginseng dried by being exposed to water at about 95°C to 100°C, it has an intermediate characteristic of containing both white ginseng and red ginseng. Black ginseng, which is made by repeating the steaming and drying process, which is receiving attention recently, greatly increases Rk1 and Rg5 by up to 20 times, and increases skin health effects such as suppression of melanin production. In particular, black ginseng contains many types of ginsenosides compared to other processed ginseng. This is because during the manufacturing process without peeling, starch particles in the ginseng tissue are converted from a sol state to a gel state, which increases the content of active ingredients. it is known

Various processing methods have been introduced to increase the efficacy of ginseng and to produce special ginsenosides. The most common methods are physical methods such as heating, pressurization, and steaming, which are mainly applied to white ginseng, red ginseng, Taegeuk ginseng, and black ginseng. Another method is to change the pH of ginseng by adding organic acids such as plum extract by a chemical method. However, the above methods have limitations in that several steps are required in the manufacturing process, or a desired component is lost in the manufacturing process, and low yield.

Recently, the most innovative method is using a biological method, which is a technique for bioconverting ginsenosides into other forms by fermenting ginseng or extracts of ginseng with enzymes or lactic acid bacteria. Since this method is specifically performed by a specific enzyme, there is an advantage in that a specific desired substance can be obtained in large quantities. For example, in recent years, fermentation techniques using various bacteria and enzymes have been introduced, and techniques for producing specific ginsenosides have been reported.

In addition, there is a cultured root production method that can obtain various ginseng saponins in a short period of time by inducing irregular roots from the roots of ginseng or wild ginseng and culturing them in an incubator. This method is especially used to produce precious wild ginseng root, and in particular, a lot of ginsenoside Re and Rh1 are produced. These components have an activity of increasing physiological activities such as hematopoiesis and vasodilation.

In fact, high molecular weight ginsenosides, which account for more than 90% of ginseng saponins, have a very low absorption rate in the body, so physicochemical or biological methods for converting them into easily absorbable low molecular weight ginsenosides are being developed. Bioconversion using microbial fermentation or microorganism-derived enzymes can selectively decompose glycosides of glycosides, and the reaction conditions are environmentally friendly.

Fermentation using microorganisms or enzymes derived from microorganisms is the same as preliminarily performing the biological transformation of ginsenosides that can occur in the human body. (Protopanaxatriol, PPT) may contribute to maintaining the balance of ginsenosides.

In addition, the bioconversion method using enzymes or microorganisms is very useful as a method to maximize the pharmacological effect while increasing the absorption rate of ginsenosides. Compound K, a trace ginsenoside newly produced by fermentation, is known to have high absorption rate, anticancer and antioxidant activity in the body.

However, even when using the enzymatic method, not all major ginsenosides have the activity to be converted into minor ginsenosides, there are differences in substrates and final products depending on the enzyme, and the activity is insignificant, so the yield is low. There is this. Also, as mentioned above, since ginseng contains different ingredients and effects depending on the processing method, it is not possible to obtain all the effective effects of ginseng with one processed ginseng.

Accordingly, a ginseng complex extract is prepared by mixing various types of processed ginseng, such as white ginseng, red ginseng, Taegeuk ginseng, black ginseng and wild ginseng cultured root, each containing various ginsenosides, and a special enzyme or lactic acid bacteria that can bioconvert it with high efficiency It is necessary to develop a technology that can maximize the various pharmacological effects of ginseng by using it and fermenting it. In addition, there is an urgent need for a highly functional ginseng complex fermented product with enhanced efficacy of ginseng produced as a result.

US 10-1253471 B US 10-1569089 B US 10-1345942 B US 10-1175955 B US 10-2017-0027367 A

Kang BH, Korean J Food Preserv., Vol. 20, No. 4, pp. 573-582 (2013) Kang SH, Kir. J. Pharmacogn., Vol. 50, No. 4, pp. 305-311 (2019)

The present invention was to solve the above needs and overcome the problems of the prior art, and to develop a highly functional ginseng complex fermented product capable of maximizing various pharmacological effects of ginseng and a method for manufacturing the same.

In the present invention, a ginseng complex extract prepared by mixing extracts of five types of representative processed ginseng with different ginsenoside compositions, white ginseng, red ginseng, Taegeuk ginseng, black ginseng and cultured wild ginseng, is fermented with specific enzymes and specific lactic acid bacteria to produce trace amounts of ginsenosides ( It aims to provide a ginseng complex fermented product with an increased content of minor ginsenosides).

In addition, an object of the present invention is to provide a method for producing a complex fermented product of ginseng, which is a mixed fermented product of white ginseng extract, red ginseng extract, Taegeuk ginseng extract, black ginseng extract and cultured wild ginseng extract.

The present invention also includes the ginseng complex fermented product according to the present invention, in which the content of trace ginsenosides Rh2, Rh1, F2 and compound K (compound K) is increased by 2.5 times or more compared to the ginseng extract mixture before fermentation An object of the present invention is to provide a composition for antioxidant, anti-inflammatory, immune enhancement or anticancer.

The present invention provides a method for producing a fermented product containing a ginseng complex extract having the effect of improving free radical scavenging ability, increasing reducing power, and inhibiting lung cancer cell (A254) growth, and a fermented product prepared by the above method. is to solve

Other objects of the present application and the technical problems to be achieved by the present invention are not limited to the above-mentioned objects, and will become clearer by the following description of the invention in conjunction with the claims and drawings described in the present specification. In addition, the technical problems to be achieved by the present invention not described in this specification include those that can be clearly understood and inferred by those of ordinary skill in the technical field of the present invention (hereinafter referred to as 'person of ordinary skill').

In order to solve the said subject, the following invention is provided.

The present invention provides a highly functional ginseng complex fermented product capable of maximizing various pharmacological effects of ginseng.

In one embodiment of the present invention, the ginseng complex fermented product is a ginseng comprehensive saponin prepared by mixing extracts of five representative processed ginsengs, white ginseng, red ginseng, Taegeuk ginseng, black ginseng and wild ginseng cultured root, which are five types of representative processed ginseng with different compositions of ginsenosides. (GINOS)" may be a ginseng complex fermented product in which the content of minor ginsenosides is increased by fermentation using a specific enzyme and specific lactic acid bacteria.

In another embodiment of the present invention, the ginseng complex fermented product is a two-step fermentation of the ginseng complex extract "GINOS" using specific enzymes and specific lactic acid bacteria to obtain minor ginsenosides Rh1, The content of Rh2, F2 and compound K (compound K) may be increased by 2 to 12 times in the ginseng complex fermented product after fermentation compared to the ginseng extract mixture before fermentation. Preferably, Rh1 is 10 to 12 times, Rh2 is 3 to 6 times, F2 is 2 to 4 times, and compound K (compound K) may be increased by 3 to 6 times. More preferably, Rh1 may be increased by 11 to 12 times, Rh2 by 4 to 5 times, F2 by 2 to 3 times, and compound K by 4 to 5 times.

Another object of the present invention is to provide a method for producing a complex fermented product of ginseng which is a mixed fermented product of white ginseng extract, red ginseng extract, Taegeuk ginseng extract, black ginseng extract and wild ginseng cultured root extract.

For example, the present invention may be a method for preparing a white ginseng extract, a red ginseng extract, a Taegeuk ginseng extract, a black ginseng extract, a wild ginseng cultured root extract, and a method for preparing a ginseng complex extract that is a mixture thereof. In addition, the present invention may be a method for producing a fermented product using a specific hydrolytic enzyme for the ginseng complex extract, or a method for preparing a fermented product using a specific lactic acid bacteria for the ginseng complex extract.

In one embodiment, the method for producing the ginseng complex fermented product is

(a) mixing white ginseng extract, red ginseng extract, Taegeuk ginseng extract, black ginseng extract, and wild ginseng cultured root extract;

(b) treating the mixture with a hydrolase; and

(c) inoculating the mixture with lactic acid bacteria and fermenting; It may be a method for producing a fermented product containing the complex extract, characterized in that it comprises a.

Also in one embodiment, the hydrolase is Plantase C150P or Sumizyme AC, and the lactic acid bacteria is Lactobacillus kimchicus DCY51 or Lactobacillus reuteri (Lactobacillus kimchicus DCY51). reuteri) can be

Another object of the present invention is to provide a method of increasing the content of trace ginsezzosides Rh2, Rh1, F2 and compound K (compound K) in the ginseng complex extract.

In one embodiment, the present invention prepares a white ginseng extract, a red ginseng extract, a Taegeuk ginseng extract, a black ginseng extract, and a mixture thereof, and a hydrolytic enzyme treatment on the mixture, followed by inoculation with lactic acid bacteria to ferment. it could be At this time, the hydrolase is Plantase C150P or Sumizyme AC, and the lactic acid bacteria is Lactobacillus kimchicus DCY51 or Lactobacillus reuteri. can

The present invention also provides a composition for antioxidant, immune enhancement, anti-inflammatory or anticancer comprising the ginseng complex fermented product according to the present invention.

In one embodiment, the composition compares the contents of trace ginsenosides Rh1, Rh2, F2 and compound K (compound K) before and after fermentation of the ginseng complex extract, and the composition of Rh1, Rh2, F2 and compound K (compound K). The content may be increased by 2 to 12 times. Preferably, Rh1 is 10 to 12 times, Rh2 is 3 to 6 times, F2 is 2 to 4 times, and compound K (compound K) may be increased by 3 to 6 times. More preferably, Rh1 may be increased by 11 to 12 times, Rh2 by 4 to 5 times, F2 by 2 to 3 times, and compound K by 4 to 5 times.

In one embodiment, the composition may have high efficacy in improving free radical scavenging ability, increasing reducing power, or inhibiting lung cancer cell (A254) growth.

In one embodiment, the composition may be a food composition or a cosmetic composition for preventing or improving inflammation. In addition, the composition may be a pharmaceutical composition for preventing or treating inflammation.

In one embodiment, the composition may be a composition for enhancing immunity.

Also in one embodiment, the composition may be a food composition for preventing or improving cancer. The composition may be a pharmaceutical composition for preventing or treating cancer.

The present invention is a ginseng complex extract "GINOS" prepared by mixing extracts of five representative processed ginsengs with different ginsenoside compositions, white ginseng, red ginseng, Taegeuk ginseng, black ginseng and wild ginseng cultured root, with a specific enzyme Provided are a ginseng complex fermented product in which the content of minor ginsenosides Rh1, Rh2, F2 and compound K is remarkably enhanced by two-step fermentation using specific lactic acid bacteria, and a method for manufacturing the same.

The ginseng complex fermented product according to the present invention increases the content of minor ginsenosides Rh1, Rh2, F2 and Compound K by 2.8 to 11 times or more, improving free radical scavenging ability, and reducing power ) increase, anti-inflammatory activity, immune-enhancing activity, lung cancer cell (A254) growth inhibitory activity has high efficacy. By using the excellent physiological activity of this ginseng complex fermented product, it can be used as a raw material for manufacturing food, health functional food, cosmetics and pharmaceuticals.

1 shows the manufacturing process of a mixed fermented ginseng extract
2 is a graph showing ginsenoside patterns before and after fermentation of white ginseng, red ginseng, Taegeuk ginseng, black ginseng, and wild ginseng cultured extract mixture.
3 is a diagram showing the free radical scavenging ability before and after fermentation of the processed ginseng extract mixture
4 is a diagram showing the reducing power before and after fermentation of the processed ginseng extract mixture.
5 is a diagram showing the effect of the processed ginseng extract mixture on macrophage growth before and after fermentation
6 is a diagram showing the effect on NO production before and after fermentation of the processed ginseng extract mixture
7 is a diagram showing lung cancer cell toxicity before and after fermentation of the processed ginseng extract mixture.

As described above, the present invention was to develop a highly functional ginseng complex fermented product capable of maximizing various pharmacological effects of ginseng and a method for preparing the same.

Accordingly, the present inventors specified a ginseng complex extract "GINOS" prepared by mixing each extract of five types of representative processed ginseng, white ginseng, red ginseng, Taegeuk ginseng, black ginseng and wild ginseng cultured ginseng with different compositions of ginsenosides. Through a two-step fermentation process using enzymes and specific lactic acid bacteria, a complex fermented ginseng product with significantly increased content of minor ginsenosides Rh1, Rh2, F2 and compound K and a method for manufacturing the same developed.

The method for producing a ginseng complex fermented product according to the present invention is a) preparing a ginseng complex saponin by mixing the preparation and extract of each processed ginseng, that is, white ginseng, red ginseng, Taegeuk ginseng, black ginseng and wild ginseng cultured root, b) mixing It includes the steps of fermenting the extract with enzymes and lactic acid bacteria, and c) measuring and confirming the content of minor ginsenosides and antioxidant and anticancer effects enhanced in the fermented ginseng saponin.

In addition, it was confirmed that the ginseng complex fermented product has high efficacy in improving free radical scavenging ability, increasing reducing power, increasing macrophage growth activity, inhibiting NO production and inhibiting lung cancer cell (A254) growth, and completed the present invention. .

Hereinafter, the present invention will be described in detail through the description of terms, specific examples and examples. It should be noted that the above specific examples and examples include some and not all embodiments of the invention. It is apparent that the content of the invention disclosed by the present specification is not limited to the specific embodiments described herein, and includes that those skilled in the art can implement variously. Accordingly, it should be understood that the content of the invention disclosed herein is not limited to the specific embodiments described herein, and modifications and other embodiments thereof are also included within the scope of the claims.

[Explanation of terms]

Fermentation

As used herein, the term "fermentation" basically refers to a process of decomposing organic matter using enzymes possessed by microorganisms such as yeast or bacteria. Therefore, the fermentation is greatly affected by the microorganisms used, that is, the fermenting bacteria, and the ingredients, taste, and aroma may vary greatly depending on the type of strain used, fermentation time, temperature, concentration, etc. conditions. When the fermentation process is carried out mainly using lactic acid bacteria or yeast, the organic matter cannot be completely decomposed and other types of organic matter are produced.

In addition, the bacteria used for fermentation have certain types of glycolytic enzymes, ie, β-glucosidase, β-xylosidse, α-arabinofluctosidase, and their activity has a great influence on the final product.

In the present invention, enzymes of microorganisms used for fermentation can be involved in various pathways for bioconverting major ginsenoside to minor ginsenoside to produce a final product. For example, various pathways are possible, such as Rd → F2 → CK, Rb1 → Rd, Rb1 → Rd → F2 → CK, Rb1 → Rd, gypenoside XVII → F2 → CK.

In addition, in the present invention, in order to ferment the ginseng complex extract, lactic acid bacteria were used in the second fermentation step. Lactobacillus can be an important choice whether to use a whole cell catalyst or a purified enzyme. The former has the ease of reducing labor, but the specificity of the product and production efficiency are inevitably reduced. However, there is a disadvantage that it is difficult to obtain Rh1, Rh2, or F2.

In order to bioconvert the major ginsenoside of ginseng into other high-functioning minor ginsenoside through fermentation, it is necessary to ferment without adding additional nutrients for culture. The influencing factors are the composition and concentration of ginsenoside in ginseng. Therefore, if the complex composed of various processed ginseng extracts is complex, performing a pre-treatment using a hydrolytic enzyme before the fermentation process using lactic acid bacteria may be a way to increase the efficiency of biotransformation.

saponin

As used herein, the term “saponin” refers to a substance in which a non-sugar part is composed of several cyclic compounds in glycosides widely present in the plant kingdom.

In particular, triterpene saponin, a saponin component included as a major physiologically active ingredient in ginseng or red ginseng, has a different chemical structure from saponin found in other plants.

Ginseng saponin or gensenoside

The term "ginseng saponin or gensenoside" used in the present invention refers to steroid saponin and triterpene saponin that are specifically present in the genus Panax. . To distinguish saponins that exist only in ginseng from other plant-based saponins, they are called ginsenosides in the meaning of ginseng glycosides. Ginseng has been used as a medicinal plant for a long time, and ginsenoside is known as a major active ingredient showing the pharmacological effect of ginseng.

Ginseng saponin or ginsenoside is a protopanaxadiol-type (PPD type) ginsenoside, protopanaxatriol-type, depending on the structure of aglycone. PPT type) can be classified into three types: ginsenosides and oleanolic acid type ginsenosides. These three groups are again classified according to the position and number of sugar moieties attached to the 3, 6 and 20 carbon positions of the ring by glycosidic bonds in the compound structure. . The basic skeleton of oleanolic acid-based ginsenosides is pentacyclic, and there is the only ginsenoside Ro, and its aglycone is oleanolic acid.

Currently, more than 180 ginsenosides have been isolated, and most of them are PPD-type ginsenosides. Ginsenosides of the PPD type include Rb1, Rb2, Rb3, Rc, Rd, Gypenoside XVII (Gypenoside XVII), Compound O (Compound O), Compound Mc1 (Compound Mc1), F2, Compound Y (Compound Y), Compound Mc (Compound Mc), Rg3, Rh2, compound K (Compound L) is included. PPT-type ginsenosides include Re, Rg1, Rf, Rg2, Rh1, and the like.

Ginsenoside, which exhibits physiological activity, has very high levels of sugar, arabinose, rhamnose galactose, and xylose, such as glucose, arabinose, rhamnose galactose, and xylose, in the non-saccharide site, and the composition ratio of major saponins Rb1, Rb2, Rc, Rd, Rf, and Rg1. It is important.

Major ginsenoside

The term "major ginsenoside" used in the present invention accounts for 90% or more of ginsenosides in dried ginseng. However, major ginsenoside has a very low absorption rate in vivo due to its large size of around 1,000 Daltons. Therefore, in order to increase the efficacy of ginsenosides, it is necessary to convert the major ginsenosides into minor ginsenosides that are relatively well absorbed and have better efficacy. That is, major ginsenosides require a conversion process in which glucose, arabinose, rhamnose, xylose, etc. constituting sugars are removed (deglycosylated) in order to effectively exhibit physiological activity in vivo.

Major ginsenosides include Rg1, Re, Rb1, Rb2, Rc and Rd.

Minor ginsenoside

The term "minor ginsenoside" used in the present invention is also called trace ginsenoside or rare ginsenoside because it exists in a trace amount in ginseng. Minor ginsenosides include F2, Rg3, Rk1, Rg5, Rh1, Rh2, Rk2, Rh3, gypenoside (Gyp) XVII, gypenoside LXXV and compound K (compound K), compound Mc ( Compound Mc), Compound Mc1 (Compound Mc1), and the like.

Minor ginsenoside has been reported to exhibit various physiological activities and is attracting attention as a functional ingredient.

GINOS

The term "GINOS" used in the present invention is a registered trademark of Oriental Medicine Bio Co., Ltd. for a food composition containing various ginsenosides produced from each processed ginseng.

The ginseng complex extract of each processed ginseng prepared according to the present invention may be called "GINOS" or "GINOS-C" in the present invention, and the present invention obtained by fermenting the ginseng complex extract The ginseng complex fermented product may be named "GINOS-F". Basically, the ginseng extract of each processed ginseng can be mixed in the same weight ratio, but this composition ratio can be variously changed to give different characteristics to the ginseng complex extract or ginseng complex fermented product.

All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention.

[Materials and Methods]

The manufacturing method of each processed ginseng according to the present invention, the manufacturing method of the ginseng complex extract, the fermentation method thereof, and the verification of ginsenosides and physiological activity before and after fermentation will be described in detail step by step as follows.

1. Manufacturing white ginseng, red ginseng, Taegeuk ginseng, black ginseng and wild ginseng cultured root

In the present invention, fresh ginseng raw material for white ginseng production is 4 to 6 years old, washed clean and dried naturally for 5 hours to remove moisture. Drying is performed first at 60±5 ℃ for 12 hours, second at 50±5℃ for 6 hours, and tertiary at 45±5℃ for 10 hours, then dry in the sun while observing the color, and the color is milky white or pale yellow. White ginseng with Representative ginsenosides of the prepared white ginseng are Rb1, Rb2, Rc, Rd, Re, Rg1, Rg2, and the like.

In the present invention, the raw materials for the production of red ginseng are the same as in white ginseng, and the steamed ginseng is steamed for 2-3 hours using steam heated at 95-100° C., and preferably steamed for 2.5 hours. After steaming and drying, the first drying is performed at 60±5℃ for 10 hours, and after 10 hours of drying at 50±5℃, observing the color development of red ginseng, and drying in sunlight to prepare red ginseng. The moisture of the prepared red ginseng is maintained at 14-15%. The representative ginsenosides of red ginseng are Rg3, Rh1, and Rh2.

The raw materials for the production of Taegeuk ginseng in the present disease are the same as in white ginseng, and cook for 20-30 minutes, preferably 25 minutes, at 96±2° C. using a double pot. After the heat treatment, the first drying is carried out at 60°C for 3 hours, and the secondary drying is performed at 50°C for 10 hours, followed by sun-drying while checking the color to produce Taegeuk ginseng. Taegeuk ginseng has intermediate characteristics between white ginseng and red ginseng in color and ginsenoside content.

To manufacture black ginseng, first select fresh ginseng, wash it clean with water, and dry it naturally for 5-6 hours. Steamed ginseng is steamed for 2-3 hours, preferably for 2.5 hours, using steam heated to 95-100°C. After steaming is completed, perform primary drying at 60±5℃ for 10 hours and cool to room temperature. Repeat the above steaming and drying 3 to 9 times, preferably 5 to 6 times, and in the last time, perform secondary sun drying, observe the color of black ginseng, and manage the final moisture to 15% or less.

Wild ginseng cultured root is tissue cultured by removing tissue from Cheonjong wild ginseng, inducing dedifferentiated callus, and culturing it in a nutrient solution to generate irregular roots. The strain (strain) of this cultured root may be HB-88, but is not limited thereto. Cultured roots are harvested after being put into a bioreactor and culturing for 2 months. The harvested cultured roots are washed 1 to 3 times with purified water and dried at 50° C. to a moisture content of about 15%. Ginsenosides from cultured wild ginseng root are more ginsenoside Re and Rh1 than ginseng and red ginseng, and there may be some that are not found in ginseng in particular, and the panaxatriol-based saponin ratio may be relatively high.

2. Preparing the processed ginseng extract and fermenting the extract mixture

2-1. Preparation and mixing of extracts of each processed ginseng

The processed ginseng prepared as described above, that is, white ginseng, red ginseng, Taegeuk ginseng, black ginseng and dried wild ginseng cultured root, is ground into particles having a size of about 80 to 100 mesh, preferably 80 mesh. The pulverized powder was extracted with 20 times the volume of purified water at 95-98° C. for 8 hours, repeated twice, and the combined extracts were concentrated to 60 brix. Although high concentration is required for long-term storage, it can be concentrated to 30-60 brix, and in case of immediate fermentation, it can be concentrated to 5 brix.

White ginseng, red ginseng, Taegeuk ginseng, black ginseng and wild ginseng cultured root concentrate (60 brix) are mixed at a certain ratio, diluted with purified water to 5 brix, and used for fermentation. In this case, the ratio of each processed ginseng extract may vary depending on the purpose of the final product.

2-2. One-step enzymatic digestion using hydrolase

The hydrolytic enzyme used for ginseng synthesis saponin, each of the above processed ginseng extract mixtures, may be pectinase, cellulase, Plantase or Sumizyme. In addition, the concentration to be added is 0.5 to 5% (v/v), the reaction temperature is 30 to 40° C., and the reaction time may be 3 to 7 days. Preferably, the same amount of 1% commercial enzyme plantase C150P and Sumizyme AC may be used, and may be reacted at 37° C. for 5 days.

2-3. Two-step fermentation using lactic acid bacteria

In the present invention, the lactic acid bacteria for the two-step fermentation of ginseng synthesis saponin, which is the processed ginseng extract mixture, may be Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus reuteri or Lactobacillus kimchicus DCY51 . The inoculation amount of bacteria for fermentation is 0.5 to 5% (1x10 ⁹ cfu/ml), and the reaction time at 30 to 60° C. may be 3 to 10 days. The selection of lactic acid bacteria can be selected according to the ginsenoside composition and the activity of related enzymes such as β-glucosidase contained in the lactic acid bacteria. Preferably, it may be a 1:1 mixture of Lactobacillus reuteri and Lactobacillus kimchicus DCY51 to ferment at 50° C. for 5 days. After fermentation of lactic acid bacteria is completed, the enzyme reaction is stopped by heating at 95°C for 30 minutes.

3. Measuring ginseosides content, antioxidant and anticancer efficacy in the fermented product

In the present invention, changes in major ginsenoside and minor ginsenoside content before and after fermentation of ginseng synthesis saponin, each processed ginseng extract mixture, are analyzed and quantified by HPLC. Subject is Rb1, Rb2, Rc, Rd, Rf, Re, Rg1, 20(S)-Rg2, 20(R)-Rg2, 20(S)-Rg3, 20(R)-Rg3, F1, F2, Rh1, Rh2, Rk1, and compound K (compound K, CK), and can be evaluated using their sum or ratio.

In the present invention, comparison of antioxidant activity before and after fermentation of ginseng synthesis saponin, each processed ginseng extract mixture, is measured by free radical scavenging ability using DPPH (2,2'-diphenyl-1-picrylhydrazyl radical) analysis method, and oxidation of Fe A method of measuring the reducing power using a reduction reaction is used, and other methods similar to the lipid peroxidation reaction may be used.

In the present invention, the anticancer activity before and after fermentation of each processed ginseng extract mixture, ginseng saponin, is measured using a cancer cell-derived cell line. More specifically, it can be evaluated by measuring the growth inhibitory activity in a cell culture system using the lung cancer-derived A549 cell line. Anti-shipping potency measurements can also be measured and evaluated in other cancer-derived cells.

[Example]

Hereinafter, the present invention will be described in more detail through examples. The examples of the present invention are only for illustrating the present invention, and the present invention may be implemented in various different forms, and it is common in the art that the scope of the present invention is not to be construed as being limited by these examples. It will be obvious to those who have the knowledge of

Example 1: Preparation of each processed ginseng and analysis of ginsenosides

1-1. Manufacture of white ginseng, red ginseng, Taegeuk ginseng, black ginseng and cultured wild ginseng root

(1) White ginseng production

In the present invention, fresh ginseng raw material was thoroughly washed and dried naturally for 5 hours to remove moisture. Thereafter, primary drying was performed at 60±5° C. for 12 hours, secondary drying was performed at 50±5° C. for 6 hours, and tertiary drying was dried at 45±5° C. for 10 hours. Thereafter, white ginseng having a milky white color was prepared.

(2) Manufacture of red ginseng

In the manufacturing process of red ginseng, fresh ginseng was washed clean with water and dried naturally for 5 hours. Steamed ginseng was placed in a certain container and steamed for 2 hours and 30 minutes using steam heated at 95-100°C. After completion of the steaming, drying was sequentially performed at 60±5° C. for 10 hours and at 50±5° C. for 10 hours, and thereafter, red ginseng was prepared by sun-drying while observing the color expression of red ginseng. The moisture of the prepared red ginseng was maintained at 14-15%.

(3) Manufacture of Taegeuk Ginseng

In the present outbreak, Taegeuk ginseng production was carried out by first selecting fresh ginseng, removing dirt and residues using purified water, and drying in the sun for 5 hours. Then, using a double pot, it was cooked at 96±2℃ for 25 minutes. After the heat treatment was finished, the first was dried at 60°C for 3 hours, and the second was dried at 50°C for 10 hours, and then the color was checked under sunlight to prepare Taegeuk ginseng.

(4) Black ginseng production

For the production of black ginseng, fresh ginseng was first selected, washed clean with water, and dried naturally for 5 hours. Steamed ginseng was steamed for 3 hours using steam heated at 95-100°C. After steaming, primary drying was carried out at 60±5℃ for 10 hours and cooled to room temperature. The above steaming and drying were repeated 6 times, and in the last time, a secondary sun-drying was performed, the color of black ginseng was observed, and the moisture was maintained to 15% or less.

(5) Production of cultured wild ginseng root

Cultured wild ginseng roots were removed from Cheonjong wild ginseng to induce dedifferentiated callus, and then cultured in a nutrient solution to generate irregular roots. The strain used in the present invention was HB-88, and was harvested after culturing for 2 months using a bioreactor. The harvested cultured roots were washed three times with purified water and dried at 50° C. to about 15% moisture.

1-2. Ginsenoside analysis of white ginseng, red ginseng, Taegeuk ginseng, black ginseng and wild ginseng cultured root

Ginsenosides contained in each processed ginseng were analyzed by HPLC. That is, 1 g of each processed ginseng powder was precisely weighed and extracted at 70° C. for 1 hour while refluxing with 30 ml of 80% methanol. The extract was filtered and extracted once more, and the filtrate was dried at 45° C. using a vacuum concentrator.

After that, the residue was dissolved in 10 ml of distilled water and, if necessary, completely dissolved by ultra-sonication, and transferred to a 50 ml cornical tube. Again, 5 ml of distilled water was added and washed completely, and the filtrates were combined. 20 ml of butanol saturated with water was added thereto, mixed well, and centrifuged at 8,000 rpm. The butanol layer was carefully collected in another round bottom flask, and 20 ml of water-saturated butanol was added to the precipitate again, mixed well, and extracted twice more in the same way. The butanol extract was completely dried at 50°C with a vacuum concentrator, dissolved in 5 ml of HPLC grade methanol, filtered through a 0.45um membrane filter, and analyzed by high performance liquid chromatography (HPLC).

High performance liquid chromatography (HPLC) analysis was performed on an Aglient infility 1260 HPLC system. The column used was Phenomenex's reverse-phase Kinetex C18 (4.6mm x 10mm, 5μm particle size). Separation of ginsenosides from each processed ginseng extract is performed using a mobile phase composed of deionized water and HPLC grade Acetonitrile (Burdick & Jackson, US) at a column temperature of 45 ° C. was performed by giving The flow rate was 0.6 mL/min and UV-visible detection was performed at 203 nm.

Table 1 shows the results of analyzing the content of each processed ginseng ginsenosides.

Ginsenosides content of each processed ginseng (mg/g) Ginsenosides white ginseng red ginseng Taegeuksam black ginseng wild ginseng cultured root Rg1 1.51 2.11 1.91 0.80 0.31 Re 1.39 0.74 1.40 0.58 2.55 Rf 0.83 0.80 1.04 0.93 0.02 20(S)-Rg2 0.83 0.20 0.36 0.25 0.10 Rh1 0 0 0 0 0 20(R)-Rg2 0 0 0.36 0.19 0 Rb1 4.44 3.88 4.19 2.41 1.14 Rc 2.08 2.54 1.94 1.29 0.37 Rb2 2.87 2.44 2.91 1.93 0.69 F1 0.19 0.07 0.09 0.10 0.17 Rd 0.44 0.29 0.61 0.62 0.24 F2 0 0 0.02 0 0.05 20(S)-Rg3 0.06 0.37 0 1.18 0.09 20(R)-Rg3 0 0 0 0.05 0.01 Rk1 0.61 0.02 0 2.51 0.04 Rg5 0 0 0 2.39 0 CK 0 0 0 0 0 Rh2 0 0.2 0 0 0.08

As shown in Table 1, the ginsenoside content analyzed in each processed ginseng well represents the ginsenoside content characteristics according to the processing method. In the present invention, since 18 kinds of ginsenosides were analyzed, it is not the total amount of ginsenosides, and there were cases in which the content was not indicated as 0 or difficult to quantify because the measurement limit was insufficient.

As a result of the analysis, major ginsenosides of ginseng such as Rg1, Re, Rb1, and Rc were detected in white ginseng, red ginseng, and Taegeuk ginseng, and the highest in white ginseng without heat treatment such as steaming. A significant amount of Rg3, a minor ginsenoside, was detected in red and black ginseng. Taegeuk ginseng showed a pattern similar to that of red ginseng and white ginseng. In the case of black ginseng, the contents of the minor ginsenosides, Rk1 and Rg5, were relatively high, and the sum of the confirmed ginsenosides was also rather high. In the case of cultured wild ginseng root, the content of 18 identified ginsenosides was relatively low, but the Re content was relatively high at about 40% of the total.

The above results show that each processed ginseng prepared in the present invention exhibits unique characteristics well.

Example 2: Processed ginseng extract preparation and fermentation of ginseng complex extract

2-1. Preparation of each processed ginseng extract and extract mixture

The processed ginseng prepared in Example 1, that is, white ginseng, red ginseng, Taegeuk ginseng, black ginseng, and dried wild ginseng root, was ground by basis weight and ground into 80 mesh particles. 20 times the volume of water was added to this powder, extracted twice for 8 hours at 95-98°C, and concentrated to 60 brix. Equal amounts of each concentrate obtained from the white ginseng, red ginseng, Taegeuk ginseng, black ginseng and wild ginseng cultured root thus prepared were mixed, and sterilized purified water was added, diluted to 5 brix, and used for enzyme treatment and fermentation.

2-2. One-step decomposition using hydrolase of processed ginseng extract

Hydrolytic enzymes Plantase C150P and Sumizyme AC were added to each of the above processed ginseng extract mixture 5 brix by 1.0%, respectively, and decomposed in one step at 37° C. for 3 days.

2-3. Two-step fermentation using specific lactic acid bacteria

The enzyme-treated extract mixture according to the present invention was then inoculated with 1.0% (1x10 ⁹ cfu/ml) of Lactobacillus reuteri and Lactobacillus kimchicus DCY51 , followed by two-step fermentation for 5 days. After the fermentation of lactic acid bacteria was completed, the enzyme reaction was stopped by heating at 95° C. for 30 minutes.

2-4. Comparison of ginsenosides content before and after fermentation

Ginsenoside content was measured in each processed ginseng complex extract (before fermentation) and fermented product (after fermentation) (FIG. 2 and Table 1). As shown in FIG. 3 , it can be seen that most major ginsenosides such as Rb1, Re, and Rc are converted and appear as PPD type or unknown peak.

Table 2 shows the quantitative values of ginsenoside.

Comparison of ginsenoside content before and after fermentation of processed ginseng mixed extract Ginsenosides Before fermentation (mg/ml 5 brix) After fermentation (mg/ml 5 brix) increase(%) Rg1 0.166 0.005 ▽97 Re 0.017 0.010 20(S)-Rg2 0.038 0.045 Rh1 0.005 0.061 △1120 20(R)-Rg2 0.039 0.048 Rb1 0.774 0.040 ▽95 Rc 0.415 0.011 ▽97 Rb2 0.628 0.006 ▽99 F1 0.021 0.005 Rd 0.158 0.081 ▽49 PPT 2.261 0.315 ▽86 F2 0.005 0.014 △283 20(S)-Rg3 0.102 0.122 20(R)-Rg3 0.005 0.006 Rk1 0.152 0.126 CK 0.152 0.700 △461 Rh2 0.002 0.009 △465 PPD 0.428 0.977 △228

As shown in Table 2, compared to before fermentation, through two-step fermentation, the minor ginsenoside, Rh1, was 11.2 times, Rh2 was 4.6 times, F2 was 2.8 times, and compound K (compound K) and Rh2 were 4.6 times, respectively. was doubled. On the other hand, major ginsenosides, such as Rb1, Rb2, Rg1, Re, and Rd, were reduced by 40-99%.

This is that, under the above fermentation conditions, major ginsenoside was very effectively converted into minor ginsenosides Rh1, Rh2, F2, Ck, and as a result, the ginseng complex fermented product according to the present invention was It can be seen that the complex extract has more excellent physiological activity.

Example 3: Confirmation of excellent physiological activity of processed ginseng mixed fermented product

3-1. Confirmation of antioxidant activity of fermented products

Antioxidant activity was measured in the ginseng complex extract and ginseng complex fermented product according to the present invention.

(1) Free radical scavenging activity measurement

The free radical scavenging activity was measured using the DPPH method modified from the method of Ouyang (2020) (Ouyang, et al. 2020). That is, after dissolving DPPH in ethanol to a concentration of 0.2 mM, 20ul of extract and 180ul of DPPH solution were added to a 96 wall plate and reacted for 30 minutes with strong shaking in a dark place, and then absorbance was measured at 517nm.

The free radical scavenging ability of the ginseng complex fermented according to the present invention prepared in Example 2 was increased in a concentration-dependent manner, and increased by about 20% or more compared to before fermentation (FIG. 3).

(2) Measurement of reducing power

One of the measures of antioxidant activity, reducing power, was measured according to the method of Nayan et al. (2013) as follows. That is, the sample and standard solution having a concentration of 10-100 μg/ml were dissolved in 1.0 ml of deionized distilled water, and then mixed with 2.5 ml of a phosphate buffer solution (pH 6.5) and 2.5 ml of a potassium ferricyanide solution. This solution was reacted at 50° C. for 20 minutes, cooled, and then 2.5 ml of 10% TCA solution was added, mixed well, and centrifuged at 3,000 rpm for 10 minutes. After taking 2.5ml of the supernatant, 2.5ml of distilled water and fresh 0.1% ferric chloride solution were added, and the change in absorbance at 700nm was measured. Reducing power was calculated by measuring ascorbic acid and garlic acid as controls.

The reducing ability of the ginseng complex fermented product according to the present invention prepared in Example 2 was also increased in a concentration-dependent manner, and increased by about 15% or more compared to before fermentation (FIG. 4).

The above results indicate that the complex fermented ginseng according to the present invention exhibits improved antioxidant activity than the complex extract of ginseng before fermentation, meaning that the complex fermented ginseng may be included as an active ingredient in a food composition having antioxidant activity.

3-2. Confirmation of immune enhancing activity and cytotoxicity of ginseng complex fermented product

(1) Effect on cell growth (Cell viability)

The cytotoxicity and immune-enhancing activity of the ginseng complex fermented product according to the present invention was measured as follows. First, the macrophage line RAW264.7 was cultured at a density of 70-80% in an RPMI 1640 medium containing 10% FBS, 5% CO ₂ , and 37° C. in an incubator. Penicillin (100 IU/ml) and streptomycin (100 μg/ml) were added to the media as antibiotics. In order to determine the effect on the cell survival of each sample in each of the cell lines, the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test method is used. and measured.

1x10 ⁶ cells were plated on a 96-wall plate, and cultured in a CO ₂ incubator at 37° C. for an incubation time corresponding to each experimental condition. Then, 10 μl MTT solution (stock concentration: 5 mg/ml) was added and further reaction was induced for 3 hours. For completion of the reaction and dissolution of formazan crystals, 100 μl MTT stopping solution (10% SDS in 0.01M HCl) was additionally added to each well. Cell viability was calculated using the OD570 value obtained by measuring the absorbance at 570 nm in the amount of MTT reduced to formazan.

As a result of measuring the effect of the ginseng complex extract and the ginseng complex fermented product according to the present invention on the growth of normal macrophages, both substances increased macrophage growth by about 20% at low concentrations (200 μg/ml or less), thereby enhancing immunity. showed activity.

On the other hand, in the case of unfermented ginseng complex extract (GINOS-C), macrophage growth was greatly inhibited and cytotoxicity was shown at high concentration (more than 200 μg/ml). However, the ginseng complex fermented product (GINOS-F) did not inhibit macrophage growth even at a high concentration of 400 to 800 ug/ml, and thus did not show cytotoxicity (FIG. 5).

This indicates that the complex fermented ginseng according to the present invention can maintain immune enhancing activity without inhibiting the growth of normal macrophages even when ingested at a high concentration, indicating that its utility is superior to that of the ginseng complex extract. In addition, it means that the ginseng complex fermented product may be included as an active ingredient in a food composition, cosmetic composition or pharmaceutical composition for enhancing immunity.

3-3. Confirmation of anti-inflammatory effect of ginseng complex fermented product

Next, as part of the verification of anti-inflammatory efficacy, NO production was induced by LPS in RAW 264.7 cells, and the NO production inhibitory effect was measured.

NO production inhibition test was performed on RAW 264.7 cells. RAW264.7 cells were adjusted to 1x10 ⁵ cells/ml using DMEM medium, inoculated in 96 well plate, treated with a new medium containing the sample and LPS (1 μ/ml), and cultured for 24 hours. Using Griess reagent, measure the amount of NO generated with 100 μl of cell culture supernatant and Griess reagent [1% (w/v) sulfanilamide, 0.1% (w/v) naphtyl-ethylenediamine in 2.5% (v/v) phosphoric acid] After mixing 100 μl and reacting for 10 minutes in 96 well plates, absorbance was measured at 540 nm using an ELISA microplate reader.

The concentration of nitrite produced was calculated using a standard curve in which sodium nitrite was dissolved in DMEM medium. Based on the difference in the amount of nitrite produced in the test group treated with LPS and the control group not treated with LPS, NO production of each sample was inhibited. Activity was confirmed.

In addition, compared to N-monomethyl-L-arginine (L-NMMA), which is a structural analogue of L-arginine, which is a currently known substance that inhibits NO production, the effect on the stage after iNOS induction was investigated.

As a result, as shown in FIG. 6 , the NO production inhibitory effect was remarkably confirmed in both the ginseng complex extract and the ginseng complex fermented product according to the present invention. As for the overall trend, as the concentration increased, it showed a somewhat more suppressive tendency after fermentation than before fermentation.

The above results indicate that the ginseng complex fermented product prepared in the present invention exhibits excellent anti-inflammatory activity, and the ginseng complex saponin complex may be included as an active ingredient in a food composition, a cosmetic composition or a pharmaceutical composition having anti-inflammatory activity. will be.

3-4. Measurement of anticancer efficacy of fermented ginseng complex fermented product

Human lung cancer-derived cells (A549) were cultured in 89% RPMI medium containing 10% FBS and 1% penicillin-steptomycin in 5% CO ₂ , 37° C. humidified chamber. In addition, before processing the sample, it was cultured for 24 hours so that the cells could adhere well. To confirm the anticancer activity before and after fermentation of the ginseng complex extract, lung cancer cells (A549) were stained with MTT solution.

That is, first, cancer cells were inoculated in a 96-well plate at a concentration of 1x10 ⁴ cell/ml per well, and after 24 hours, the cells were sufficiently settled, the initial culture medium was removed, and the samples were treated at different concentrations from 12.5ug to 800ug/ml, and for 24 hours. cultured. After that, 20 μl of MTT solution was added to each well and the cells were incubated for 4 hours. Finally, 100 μl of DMSO was added to dissolve the remaining formazan. Then, by shaking the microplate in a dark place, the absorbance of the purple color formed by dissolving formazan was measured at 570 nm using an ELISA Leader.

As a result, as shown in FIG. 7 , the cytotoxicity to lung cancer cells did not show a significant difference between the ginseng complex extract (GINOS-C) before fermentation according to the present invention and the ginseng complex fermented product (GINOS-F) after fermentation according to the present invention. . However, as the concentration increased, the proliferation inhibitory effect of lung cancer cells increased in the ginseng complex fermented product (GINOS-F) after fermentation. In particular, the growth inhibitory effect of lung cancer cells was significantly shown by treatment with 800 μg/ml of ginseng complex fermented product (GINOS-F).

This means that the ginseng complex fermented product according to the present invention can be used as an active ingredient in a food composition for the prevention or improvement of cancer or a pharmaceutical composition for the prevention or treatment of cancer.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be clear Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

delete delete (a) White ginseng, red ginseng, Taegeuk ginseng, black ginseng, and dried wild ginseng root powder were extracted with hot water repeatedly for 8 hours at 95-98° C., concentrated to 60 brix, and the same amount of each concentrate was mixed, preparing a ginseng complex extract by adding purified water and diluting it to 5 brix;
(b) treating the ginseng complex extract with hydrolytic enzymes Plantase C150P and Sumizyme AC and hydrolyzing at 37° C. for 5 days; and
(c) inoculating the hydrolyzed ginseng complex extract with lactic acid bacteria Lactobacillus kimchicus DCY51 and Lactobacillus reuteri and fermenting at 50° C. for 5 days; A method for producing a ginseng complex fermented product comprising a, enhanced ginsenoside Rh1 content.
delete delete (a) White ginseng, red ginseng, Taegeuk ginseng, black ginseng, and dried wild ginseng root powder were extracted with hot water repeatedly for 8 hours at 95-98° C., concentrated to 60 brix, and the same amount of each concentrate was mixed, preparing a ginseng complex extract by adding purified water and diluting it to 5 brix;
(b) treating the ginseng complex extract with hydrolytic enzymes plantase C150P and Sumizyme AC and hydrolyzing at 37° C. for 5 days; and
(c) inoculating the hydrolyzed ginseng complex extract with lactic acid bacteria Lactobacillus kimchicus DCY51 and Lactobacillus reuteri and fermenting at 50° C. for 5 days; A method of increasing the ginsenoside Rh1 content in the ginseng complex fermented product comprising a.
delete delete delete delete delete delete

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