KR20180039539A - Producing Method of Ginseng Saponin - Google Patents

Abstract

The present invention relates to a method for producing ginseng saponin. The method comprises: a step (S1) of extracting ginseng saponin by alcohol; a step (S2) of preparing a ginseng saponin solution; and a step (S3) of obtaining a concentrate. Active ginseng saponin which is not soluble in water but has a higher level of in vivo absorptivity to exhibit an effect well can be mass-produced.

Description

Production Method of Ginseng Saponin}

The present invention relates to a method for producing ginseng saponin, and more particularly, to a method for producing ginseng saponin, which can produce a large amount of active ginseng saponin, . ≪ / RTI >

The government of the Republic of Korea has unified ginseng, ginseng, mountain ginseng, goat ginseng, fennel ginseng, fire ginseng, and taekguk ginseng, and previously called ginseng for more than four years. In the present invention, ginseng refers to one or two or more selected from the group consisting of ginseng, wild ginseng, goat ginseng, jelly ginseng, fire ginseng, fresh ginseng, pure ginseng, and taekguk ginseng.

Saponin and non saponin substances (Panacen, polysaccharide, amino acid derivative, polyacetylene derivative, phenol compound) contained in ginseng root have excellent pharmacological activity, excellent efficacy in eliminating free radicals, Lipid-lowering, hepatotoxicity and so on. Ginseng saponin, which is well known as the main pharmacological component of ginseng, is divided into PPD (Protopanaxadiol) and PPT (Protopanaxatriol). PPD saponin has a structure in which various substituents are bonded to the basic structure PPD, and ginsenosides Rb1, Rb2, Rc, Rd, F2, Rg3, and Compound K are typical saponins. PPT saponin is a basic structure of PPT, and ginsenosides Re, Rf, Rg1, and Rh1 are representative.

In general, many glycoside compounds present in nature show a tendency to increase physiological activity when the saccharide is degraded to an unglycoside rather than itself (Med. Pharm. Soc. 1992, 9: 1-13) The ginsenosides Rg3, Rh1, Rh2, F2, CY and CK produced by the hydrolysis of a part of the ginsenosides Rb1, Rb2, Rd, (Ginseng Res. 2003, 27: 129-134). It is believed that the effect of the enzyme on the activity of the enzyme is enhanced. It is known that ginseng saponin, which has a high sugar content, is absorbed into the body in the small intestine. Experiments on the hydrolysis ability of ginsenoside Rb1 of intestinal microorganism extracted from human excreta showed that the intestinal microorganisms 21 % Showed no ability to degrade, and 70% of intestinal microorganisms with degradation ability were found to have a large difference in the ability to degrade ginseng saponin (Planta Medica 1998, 64: 696-700).

Generally, ginseng products including saponin components are composed of ginsenosides composed of components having high solubility in water. While these components in an inactive state are highly soluble in water, they are not easily absorbed in the intestines There is little known effect. However, when the above-mentioned components are activated and changed into the active component, they are not well dissolved in water, but the absorbency in the body is increased, and the effect is exerted well.

In the present invention, the term "active type" used in the active type saponin, active type goat's gum, active ingredient, etc. refers to the property of saponin or ginsenoside of the processed ginseng according to the present invention, As a result, the total content of saponin is higher than that of ordinary wild ginseng, ginseng ginseng or ginseng and is absorbed into human body and then exerted its effect. That is, in the group consisting of the ginsenosides Rg3, Rh1, Rh2, F2, CY and CK produced by hydrolysis of some saccharides other than the ginsenosides Rb1, Rb2, Rd, Means one or two or more selected ginsenosides which exhibit far superior effects in terms of absorption into the body and physiological activity.

Red ginseng is a representative example of ginseng that has been partially activated, and in the modern sense, it refers to a treatment of about 6 hours at 90 ° C. Particularly, it is known that Compound K, which is an active ingredient, is not produced in nature but is partially produced by intestinal bacteria, making it difficult to produce the compound K because of its complicated manufacturing method. In this regard, a manufacturing method of compound k is already known from Korean Patent Application No. 10-2010-0099241 and Korean Patent Application No. 10-2010-0096999 developed by the present inventors. However, an effective method for producing Compound K by using the strain itself has not yet been developed. Compound K prepared using enzymes can be used for medicines or cosmetics, but is not allowed for use in foods. Therefore, in order to use as a food, it must be produced using a strain. When a strain is used compared to an enzyme, the production yield is low and it is not economical.

Fermentation using lactic acid bacteria is usually carried out at around 37 캜 and difficult to ferment at 42 캜 or higher. This results in a change in the tertiary structure of the enzyme produced by the strain, which makes it difficult for the strain to live and to expect the action of the enzyme. Scientifically, it is known that when the temperature rises by 10 ° C, the enzyme capacity doubles, but when the temperature rises, it is difficult for the strain to live. Therefore, the present inventors tried to use a strain capable of growing at high temperature and producing various enzymes.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems of the related art, and has the following objectives.

It is an object of the present invention to provide a method for producing an active saponin which can produce a larger amount of active saponin which shows much better effects in terms of absorption into the living body and physiological activity, and which can also be used for foods.

The present invention provides a fermentation method of active saponin containing a large amount of compound K, Rd, Rg3, etc. as an active ingredient by removing sugar attached to ginseng saponin by fermentation at a high temperature of 42 ° C or higher, .

In order to achieve the above object, the present invention is implemented by the following means.

The method for producing ginseng saponin according to the present invention comprises the steps of: (S1) exposing ginseng to steam to decompose after heat treatment, and extracting ginseng saponin with alcohol; Bacillus mojavensis KJS- 3 culture medium and Bacillus polyfermenticus (S2) a ginseng saponin solution is prepared by adding the extracted ginseng saponin to a mixture of KJS - 2 culture medium and fermenting at a temperature of 42 ° C to 60 ° C; And centrifuging the prepared ginseng saponin solution to separate the supernatant, collecting the precipitate, resuspending it in ethanol, filtering the supernatant, and concentrating the filtrate to obtain a first concentrate (S3).

Further, the present invention is characterized in that the supernatant stored separately in step S3 is fermented at a temperature of 42 to 60 DEG C, collected by centrifugation, resuspended in ethanol and filtered, and then the filtrate is concentrated to a second concentration And acquiring water (S4).

Further, the present invention is characterized in that the above-mentioned primary condensate is once again treated with the complex strain Bacillus mojavensis KJS- 3 and Bacillus polyfermenticus (Active ginseng saponin) containing a high content of Compound K by re-fermenting the primary fermentation saponin at a temperature of 42 ° C to 60 ° C to a culture mixture of KJS - 2 strain.

The present invention has the following effects with the above-described configuration.

The active saponin including the compound k and the like having a high yield can be prepared by preparing the saponin active ingredient by heat treatment and extracting it and then applying the high temperature fermentation using the complex strain to the next step at two successive temperatures , And it has an effect that it can be used for food because it is produced by fermentation.

Brief Description of the Drawings Fig. 1 is a schematic view of Bacillus mojavensis KJS-3, Bacillus polyfermenticus KJS-2 This is the analysis condition of active type saponin by complex complex fermentation.
Figure 2 is a graphical representation of Bacillus mojavensis KJS-3 (strain KCCM 10961P).
Figure 3 shows that Bacillus polyfermenticus KJS-2 (seedling registration number KCCM 10769P).
FIG. 4 shows the results of fermentation of two strains of Bacillus mojavensis KJS-3 and Bacillus polyfermenticus KJS-2 at 45 ° C and 55 ° C at high temperature to produce activated saponin, And the active saponin containing a high content of Compound K is produced by fermentation at high temperature in two stages.

The Applicant hereunder explains in detail the means for solving the foregoing problem. The detailed description of known technology, which is considered to be unnecessarily obscured by the gist of the present invention, will be omitted.

Generally, it is known that the production yield by enzyme is twice as high at a temperature of 10 ° C under the condition that the secondary structure of the enzyme is maintained. Therefore, we tried to find a strain that grows well at a temperature higher than 37 ° C, which is a general optimum growth temperature. Through many studies, it has been found that the maximum growth of Bacillus capsicus jaejese-3 (Moja-3 Strain) was selected. In addition, Bacillus polyfermenticus kayserase-2 strain (Bp-2 strain) having a growth temperature of 60 ° C at maximum was selected and used. The maximum growth temperature of the Bacillus polyfermenticus kayserase-2 strain was 55 ° C at 45 ° C, and the highest temperature of 55 ° C was selected for fermentation. At this temperature, most of the airborne microorganisms can not grow, and thus it is possible to prevent the fermentation from being stopped due to pollution.

Hereinafter, the present invention will be described in detail with reference to examples and drawings.

Specifically, the active ingredient was prepared by heat treatment. Specifically, the active ingredient was treated at 110 ° C and 1.1 kg / cm 2 for 10 minutes, and repeated three times in total. The product was pulverized by a pulverizer and pulverized with 80% To extract the saponin component. This is because Panaxadiol (PPD) is extracted in 80% ethanol solution compared to Panaxatriol (PPT). The saponin component thus extracted was concentrated by evaporation, and then suspended in water to use active PPD as a raw material for production. The suspended material is injected into the fermentation solution containing the Moja-3 and Bp-2 strains by a predetermined amount using a peristaltic pump to decompose the sugar chain of the saponin to form a solution containing the active component, (Hereinafter referred to as " CKS " solution) was prepared. When the reaction material is not added to the enzyme in a certain amount, the yield is low and the reaction does not occur due to the aggregation phenomenon. Therefore, the PPD active component CKS solution containing the compound k was prepared by injecting the reaction materials into the fermentation broth of the Moja3 and Bp2 strains by using a tube-operated pump. The prepared CKS solution was centrifuged, resuspended in ethanol, filtered, and the filtrate was concentrated to collect CKS.

For the preparation of Compound K at a higher concentration, active fermented saponin was firstly mixed and fermented again.

Active type PPD  Produce

The ginsengs were placed on a porous shelf at 121 ° C for 10 minutes using an automatic temperature and humidity control system, which was filled with steam, and exposed to steam under a pressure of 1.1 kg / cm 2, Heat treated. The ventilation valve was opened to inject air and the heat-treated ginseng was cooled to 90 ° C or lower, and the same operation was repeated two more times. Ginseng saponin was extracted by thoroughly extracting heat-treated ginseng spirits by finely crushing the dried ginseng to finer particles of 1 mm or less and adding 80% ethanol. Specifically, 60 grams of the prepared heat-treated ginseng was crushed and extracted with 300 ml of 80% alcohol, which was then filtered through a filter paper having a particle size of about 500 μm to remove solid matter to obtain a solution of alcohol alcohol (hereinafter referred to as "heat-treated saponin solution" Respectively. The 'heat-treated saponin solution' was concentrated using a rotary evaporator. The 'heat-treated saponin concentrate' was dissolved in a small amount of alcohol (ethanol) and sterilized distilled water was added thereto to prepare a suspension. The 'heat-treated saponin suspension' was maintained at 50 ° C or higher in order to prevent the generation of harmful bacteria due to contamination.

At 45 ° C Moja3  Production of active saponin by fermentation

An active PPD containing Rg3 and the like was prepared and used as in Example 1 by using 24 g of solid of ginseng.

First, the Moja3 strain was cultured in Ttryptic soy broth (TSB) for 16 hours or more at 45 ° C. 100 ml of the culture solution was added to 1,000 ml of freshly prepared TSB, and cultured at 45 ° C. for 60 minutes. Then, 10 w / v% 100 ml of the solution was added at a rate of 20 ml / min using a peristaltic pump and further cultured at 45 ° C for a total of 6 hours. The prepared CKS solution was centrifuged, and the supernatant (referred to as 'A solution') was collected separately, and the precipitate was collected and re-suspended in a small amount of ethanol to elute the CKS. After filtration, the filtrate was concentrated to collect the CKS Respectively. The collected CKS was lyophilized and weighed to give 0.054 grams (first-order reaction CKS). In addition, the A solution was further cultured at 45 ° C for 6 hours, and then CKS was prepared and collected in the same manner as in the primary CKS preparation method. At this time, 0.18 gram CKS was collected (secondary reaction CKS). The sum of the primary and secondary CKSs was 0.234 grams.

At 55 ° C Bp2  Production of active saponin by fermentation

Activated PPD containing Rg3 was prepared by using 24 g of solid content of ginseng.

First, Bp2 strain was cultured in Ttryptic soy broth (TSB) for 16 hours at 45 ° C. 100 ml of this culture was added to 1,000 ml of newly prepared TSB, and cultured at 55 ° C for 60 minutes. Then, 10 w / v% active PPD 100 ml of the suspension solution was added at a rate of 20 ml / min using a peristaltic pump and further cultured at 55 ° C for a total of 6 hours. The prepared CKS solution was centrifuged, and the supernatant (referred to as 'B solution') was collected separately. The precipitate was collected and resuspended in a small amount of ethanol to elute the CKS. The solution was filtered and the filtrate was concentrated to collect the CKS Respectively. The resulting CKS was lyophilized and weighed to obtain 0.0645 grams (primary reaction CKS). The stomach B solution was further cultured at 55 ° C for 6 hours, CKS was prepared and recovered, wherein 0.0139 grams of CKS was collected secondarily (second-order reaction CKS). The sum of the primary and secondary CKSs was 0.0784 grams.

Production of active saponin by combined fermentation (at 45 ° C Moja3  Fermentation and at 55 ° C Bp2  Production of active saponin by fermentation)

Activated PPD containing Rg3 was prepared by using 24 g of solid content of ginseng.

First, the Moja3 and Bp2 strains were cultured in Ttryptic soy broth (TSB) for 16 hours at 45 ° C. 100 ml of each of these cultures was added to 1,000 ml of freshly prepared TSB and cultured at 45 ° C for 60 minutes. 10w / v% 100 ml of the active PPD suspension solution was added at a rate of 20 ml / min using a peristaltic pump and further cultured at 45 ° C for a total of 6 hours. The prepared CKS solution was centrifuged, and the supernatant (referred to as 'C solution') was collected separately. The precipitates were collected, resuspended in a small amount of ethanol, eluted with CKS, filtered and concentrated to collect CKS Respectively. The collected CKS was lyophilized and weighed to obtain 1.04 grams (primary reaction CKS). In addition, C solution was further cultured at 55 ° C for 6 hours, and then CKS was prepared and collected in the same manner as the first CKS preparation method. At this time, 1.315 grams of CKS was collected (secondary reaction CKS). The sum of the primary and secondary CKSs was 2.355 grams. From these results, it was confirmed that a large amount of active saponin can be produced by the combined action of two strains at high temperature.

Production of active saponin by combined fermentation (prolongation of reaction time, Moja3, 55 ≪ RTI ID = 0.0 > C, < / RTI >

Activated PPD containing Rg3 was prepared by using 24 g of solid content of ginseng.

First, the Moja3 and Bp2 strains were cultured in Ttryptic soy broth (TSB) for 16 hours at 45 ° C. Each 100 ml of this culture was added to 1,000 ml of freshly prepared TSB and incubated at 45 ° C. for 60 minutes. Then, 10 w / v% Type PPD suspension solution was added at a rate of 20 ml / min using a peristaltic pump and further cultured at 45 ° C for a total of 12 hours. The prepared CKS solution was centrifuged, and the supernatant (referred to as 'D solution') was collected separately. The precipitate was collected and resuspended in a small amount of ethanol to elute CKS. After filtration, the filtrate was concentrated to collect CKS Respectively. The collected CKS was lyophilized and weighed 1.02 grams (first-order reaction CKS). The D solution was further cultured at 55 ° C for 12 hours, and then CKS was prepared and collected in the same manner as the first CKS preparation method. At this time, 1.210 grams of CKS was collected (secondary reaction CKS).

The sum of the primary and secondary CKSs was 2.23 grams. It was found that the active saponin was produced in large amounts by the combined action of the two strains at high temperature. However, after the fermentation time exceeded 6 hours, it was confirmed that CKS was not produced much even when the time was extended.

Production of active saponin by combined fermentation of active saponin prepared by complex fermentation (CKS re - fermented product: Complex strain  Fermentation active saponin (secondary))

Activated PPD containing Rg3 was prepared by using 24 g of solid content of ginseng.

First, the Moja3 and Bp2 strains were cultured in Ttryptic soy broth (TSB) for 16 hours at 45 ° C. Each 100 ml of this culture was added to 1,000 ml of freshly prepared TSB and incubated at 45 ° C. for 60 minutes. Then, 10 w / v% Type PPD suspension solution was added at a rate of 20 ml / min using a peristaltic pump and further cultured at 45 ° C for a total of 12 hours. The prepared CKS solution was centrifuged, and the supernatant (referred to as 'E solution') was collected separately. The precipitate was collected and resuspended in a small amount of ethanol to elute CKS. After filtration, the filtrate was concentrated to collect CKS Respectively. The collected CKS was lyophilized and weighed 1.02 grams (first-order reaction CKS). E solution was fermented at 55 ° C for 12 hours and centrifuged to collect the precipitate. A small amount of the precipitate was eluted with ethanol, centrifuged, and the ethanol layer was collected and ethanol was volatilized to obtain 1.345 g of a solid.

In addition, the first-order reaction CKS was dissolved in ethanol and diluted in purified water to prepare a 1% solution. This solution was incubated at 45 ° C for 6 hours and at 55 ° C for an additional 6 hours and then centrifuged to produce active saponin (CKS re-fermented product).

The "CKS re-fermented product" shows that CKS produced in the first fermentation was further fermented, and a higher content of active-type saponin was produced in a larger amount. The yield of "CKS re-fermented product" was about 90%, 2,112 mg less than 2,355 mg of primary fermented product, and Compound K, a typical saponin of active type, increased about 5.85 times.

Analysis conditions of saponin

The specific analysis conditions are shown in Fig. 1 attached hereto. The column used was Kinex C18, and the flow rate was analyzed at 1.0 ml / min. The mobile phases were acetonitrile (ACN) and distilled water (D.W). The ACN 20v / v% and 80v / v% DW were mixed and flowed. For 40min to 45min, ACN 60v / v% and 40v / v% DW were mixed It was shed. From 45 to 60.1 minutes, only ACN flowed, and after that ACN 20 v / v% and 80 v / v% D.W were mixed and flowed.

Analysis of the manufactured saponin

Representative individual saponins of CKS from Examples 1 to 4 were analyzed. The HPLC-CAD method was used for the analysis. Compound K (CK), Rg3, Rd, Rb1 and Rf were analyzed with typical saponins. The content of each saponin was expressed by the absolute amount of saponin produced as a result of fermentation using 24 g of ginseng. As shown in Table 1 below, the CKS produced by the combined fermentation of Moja3 strain and Bp2 was 234 mg due to Moja3 alone fermentation and significantly higher than that of 78,4 mg due to Bp2 fermentation alone Of CKS. In addition, the amount of active saponin was 32.64 mg in the case of Rg3, which is an index substance of red ginseng, more than 2 times as much as that of Moja3 alone fermentation 8.39 mg and Bp2 alone fermentation 12.48 mg. In the case of Compound K, which is not found in nature but produced by intestinal bacteria or enzyme, 0.04 mg was produced in Moja3 single fermentation, 0.14 mg in Bp2 single fermentation, and 0.26 mg in Moj3 and Bp2 complex fermentation It was confirmed that the yield was very high.

division CKS total amount Rf Rb1 Rd Rg3 (S) Rg3 (R) CK Example 2 (Primary reaction) 54 mg 2.81 1.88 0.58 2.15 0.88 0.02 Example 2
(Secondary reaction) 180 mg 0.63 3.10 1.09 3.33 2.03 0.02 Example  2
(Primary, Secondary reaction total ) 234 3.44 4.98 1.67 5.48 2.91 0.04 Example 3
(Primary reaction) 64.5 mg 1.45 4.06 7.30 6.27 4.00 0.14 Example 3
(Secondary reaction) 13.9 mg 0.56 1.94 0.60 1.39 0.80 - Example  3
(Primary, Secondary reaction total ) 78.4 2.01 6 7.9 7.66 4.8 0.14 Example 4
(Primary reaction) 1,040 mg 1.64 9.13 2.90 17.28 5.33 0.21 Example 4
(Secondary reaction) 1,315 mg 1.47 5.20 1.45 7.65 2.38 0.05 Example  4 2,355 3.11 14.33 4.35 24.93 7.71 0.26

In Example 6 in which the first fermentation product of CKS was re-fermented, the yield of the prepared CKS fermented product was about 90%, and the produced amount of compound K was 5.85-fold and Rg3 was 1.63-fold.

division CKS total amount Rf Rb1 Rd Rg3 (S) Rg3 (R) CK Example 6 2.112 mg 0.80 7.16 2.78 41.38 11.95 1.52

Korea Microorganism Conservation Center (overseas) KCCM10961 20080822 Korea Microorganism Conservation Center (overseas) KCCM10769 20060816

Claims (5)

(S1) a step of exposing the ginseng to steam, crushing it after heat treatment, and extracting ginseng saponin with alcohol;
Bacillus mojavensis KJS- 3 culture medium and Bacillus polyfermenticus (S2) a ginseng saponin solution is prepared by adding the extracted ginseng saponin to a mixture of KJS - 2 culture medium and fermenting at a temperature of 42 ° C to 60 ° C; And
(Step S3) of centrifuging the ginseng saponin solution so as to separate the supernatant, collecting the precipitate, resuspending it in ethanol, filtering the supernatant, and concentrating the filtrate to obtain a first concentrate To produce ginseng saponin. The method according to claim 1,
The separately stored supernatant of step S3 is fermented at a temperature of 42 ° C to 60 ° C,
Further comprising a step (S4) of collecting the precipitate by centrifugation, resuspending the precipitate in ethanol, filtering and concentrating the filtrate to obtain an additional secondary concentrate (S4). The method according to claim 1,
Wherein the fermentation temperature in step S2 is 45 占 폚 or 55 占 폚. The method according to claim 1,
Wherein the fermentation temperature in step S4 is 45 占 폚 or 55 占 폚. (S1 ') a step of exposing ginseng to steam, breaking the ginseng after heat treatment, and extracting ginseng saponin with alcohol;
(S2 ') comprising adding the extracted ginseng saponin to a mixture of a Bacillus mojavensis KJS-3 culture broth and a Bacillus polyfermenticus KJS-2 culture broth and fermenting the mixture at a temperature of 42 ° C to 60 ° C to prepare a ginseng saponin solution; And
The obtained ginseng saponin solution is centrifuged to separate the supernatant, and the precipitate is collected, resuspended in ethanol, filtered and concentrated to obtain an initial concentrate (S3 ');
The initial concentrate obtained in step S3 'was dissolved in ethanol, diluted in purified water, and once again treated with Bacillus mojavensis It was added to the extracted ginseng saponin in a mixture of KJS -3 strain culture with Bacillus polyfermenticus KJS-2 strain culture solution to prepare a fermented ginseng saponin solution at a temperature of 42 ℃ ~ 60 ℃ (S4 ');
(Step S5 ') of collecting the precipitate by centrifuging the ginseng saponin solution prepared in the step S4', resuspending it in ethanol, filtering and concentrating the filtrate to obtain a final re-concentrate Ginseng saponin production method.

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