KR101769184B1 - Preparation for fermented ginseng or red ginseng extract containing increased ginsenoside Rd - Google Patents

Abstract

The present invention relates to a method for producing ginsenoside Rd-enhanced fermented ginseng or red ginseng extract by fermenting enzyme extracts of main roots and fine roots of ginseng or a mixture of main roots and fine roots of red ginseng. The fermented ginseng or red ginseng extract Is economical by shortening the fermentation time by adding an enzyme for food additives, and has an advantage of reinforcing ginsenoside Rd without addition of additives for pH and the like.

Description

Preparation of Fermented Ginseng or Red Ginseng Extract with Enhanced Ginsenoside Rd [Technical Field]

The present invention relates to a method for preparing fermented ginseng or red ginseng extract having enhanced ginsenoside Rd content, and more particularly, to a method for producing fermented ginseng or ginseng extract having ginsenoside Rd To a fermented ginseng or a red ginseng extract.

The ginseng was named after the Soviet scientist CAMeyer in 1843 as " Panax ginseng CA Meyer "is a botanical plant belonging to the genus Araliaceae (Panax), and its roots are used medicinally. Such ginseng is widely used as a natural health food, and has scientific proof of pharmacological efficacy and clinical , The recognition and trust is high, and the demand for medicine and functional food is increasing.

Ginseng has been used in the private sector for 2,000 years due to its excellent pharmacological effect in the Orient, and its effects are mostly attributed to saponin compounds (Ha, 2005). Ginseng is divided into red ginseng and white ginseng according to the treatment method of medicinal roots. Red ginseng is steamed and dried red ginseng without extracting ginseng, and white ginseng is dried ginseng as yellowish white. Ginsenoside, a representative pharmacological component of ginseng, has been found in about 22 kinds of white ginseng and about 30 kinds of red ginseng (Ryu, 2003). Especially, panaxatriol is a unique ingredient not found in white ginseng but only in red ginseng (Kitagawa, 1983). In addition, non-polar ginsenosides Rg2, Rg3, Rh1, Rh2, Rg5, Rk1 and polar ginsenoside Rb1, Rb2, which are unique components of red ginseng, which are not present in ginseng by heat generated during the manufacturing process of red ginseng, (Jeon et al., 1999), the neuronal cell protection and learning ability improving effect (Kim et al., 1999), and the anti- (Jung et al., 1998) and antioxidant activity (Bae and Kim, 1998). Therefore, it is expected that the excellent pharmacological efficacy of Korean red ginseng alone It is known to be able to.

In addition, G-Rh2, a specific saponin component of red ginseng, and polyacetylene components such as panaxydol, panaxynol, and panaxytriol, as non-saponin substances, are the main active ingredients inhibiting the proliferation of cancer cells. These components are known to have cancer cell proliferation inhibitory action. In addition, polysaccharide components have been known to have anti-diabetic effect, immune enhancing effect, anti-gastric effect.

In the manufacturing process of red ginseng, fresh ginseng is washed thoroughly with water, put in a certain container, and steamed for a certain time according to its size by using heated steam. After the first hot air drying, To about 13.5%, and it is called red ginseng in which the root roots are removed in the process of manufacturing the red ginseng.

The red ginseng is not significantly different from red ginseng in that the root roots and components of red ginseng are glycosides, ginseng flavor components (panacen), polyacetylene compounds, nitrogen nitrogen components, flavonoids, vitamins (B group) , Antioxidant substances, organic acids and amino acids. It has sedative and excitatory action on the central nervous system, acts on the circulatory system to prevent hypertension and arteriosclerosis, and has a hematopoietic effect (hematopoietic action) and blood glucose level It protects the liver and acts on the endocrine system and acts indirectly on sexual behavior and reproductive effects and has anti-inflammatory and anti-tumor effects (anti-tumor effect) , The effect of protection against radiation, the skin is known to protect and soften the action.

However, it has been found that the saponin is decomposed and converted into intestinal microorganisms and is absorbed into the body. As a result, the degree of absorption of saponin has been determined to be different according to a person. For example, the 2004 International Conference of the Korean Society of Food Science and Nutrition Ham et al. , 20.3% were able to absorb only saponins of protopanaxadiol (PPD), except for the group capable of absorbing saponin, and Protopanaxatriol (hereinafter referred to as " PPT '), and the group that can not absorb saponin at a whopping 4.7%.

Therefore, fermentation of ginseng using a microorganism useful for increasing the absorption of saponin in the body, and induction of ginsenoside component conversion by using glycosidase of microorganism, etc., (Kim et al., 2007). For example, the most suitable lactic acid strains and optimal processes for the production of fermented ginseng using lactic acid bacterium have been studied (Park et al., 2006) (Kong et al., 2008), which has been reported to investigate the compositional characteristics of general components of fermented ginseng.

In addition, studies have been made to develop fermented foods by fermenting or adding various ingredients including ginseng to improve functionality and sensory quality of foods (Kim and Han, 2005) is distributed in the intestine by woosegyun and by the fermentation of sugars as a live cell activator (probiotics) which promotes the growth of body yuikgyun the lactic acid bacteria such as bacteria of the Lactobacillus Bashile (Lactobacilli) and Bifidobacterium (Bifidobacterium) for generating a lactic reported. (Goldin, 1998).

Korean Patent No. 10-0884252 " Lactobacillus plantarum NUC-JiKCCM10582 ", and fermented red ginseng produced by the method described in " Lactobacillus plantarum NUC-J1 KCCM 10852P is used to prepare fermented red ginseng containing ginsenosides Rg3 and Rh2, Compound K. However, in order to industrialize the fermentation of the extract by these microorganisms, it is difficult to standardize the process to maintain a certain quality. The principle of microbial fermentation is that the enzymes that secrete as the microorganism grows ultimately convert the ingredients contained in the food or herbal medicine. By putting the microbes into the herbal medicines directly and fermenting them, Which simplifies the process and facilitates quality control.

KR 10-0884252 B1, 03.11.2009

Accordingly, the inventors of the present invention have conducted intensive studies to solve the above problems, and have found that when an extract prepared by mixing main roots of ginseng and main roots and fine roots of red ginseng at a certain ratio is fermented by enzyme, a large amount of ginsenoside Rd And discovered a method for producing a new fermented ginseng or red ginseng extract, thereby completing the present invention.

The object of the present invention is to prepare an extract by mixing main roots of ginseng having a high total saponin content including ginsenoside Rd and main roots and fine roots of red ginseng or red ginseng, and fermenting the extract to obtain a useful saponin, ginsenoside Rd-enhanced fermentation And to provide a method for producing ginseng or red ginseng extract.

Another object of the present invention is to provide fermented ginseng or red ginseng extract prepared by the above method.

In order to achieve the object of the present invention, the present invention provides a method for producing a ginseng extract, comprising the steps of: (1) extracting an extracting solvent by adding an extracting solvent to a mixture of a main muscle of a ginseng and a main muscle of a red ginseng or red ginseng, Fermented ginseng or ginseng extract having enhanced ginsenoside Rd, which comprises a fermentation step (step 2).

The present invention relates to a method for producing a ginseng or red ginseng mixed extract, which comprises concentrating the ginseng or red ginseng mixed extract prepared in the above step 1 and diluting the concentrated extract with water and ultrafiltering with a molecular cut-off of 2 kDa to 6 kDa to obtain a filtrate Wherein the ginsenoside Rd-enriched fermented ginseng or red ginseng extract further comprises the step of adding ginsenoside Rd.

The present invention provides ginsenoside Rd enhanced fermented ginseng or red ginseng extract prepared by the above method.

Hereinafter, the present invention will be described in detail.

The ginseng saponin of the present invention refers to ginsenoside.

The ginsenoside Rd of the present invention is a kind of PPD dyesenoside, which is a compound having a structure represented by the following general formula (1).

[Chemical Formula 1]

The ginsenoside Rd is present in a trace amount of less than 1% in ginseng or red ginseng and has not been widely used industrially. However, it is known that it has the effects of arthritis improvement, cartilage regeneration, collagen formation and the like.

 The present invention relates to a method for preparing a fermented ginseng or red ginseng extract having enhanced ginsenoside Rd, which is a useful saponin, comprising the steps of extracting a mixture of main roots and fine roots of red ginseng or red ginseng with an extraction solvent, And a fermented ginseng or ginseng extract having enhanced ginsenoside Rd, comprising the step of fermenting the enzyme. Please refer to Fig.

The preparation method may further include a step of sterilizing according to a conventional method after fermentation. In one embodiment of the invention, it may be sterilized at 80-100 ° C for 5-20 minutes.

The preparation method may further include a step of vacuum concentration, spray drying or lyophilization according to a conventional method after fermentation. In one embodiment of the present invention, it can be concentrated under reduced pressure at 50 to 70 占 폚.

In the present invention, the step of concentrating the ginseng or red ginseng mixed extract prepared in step 1 and diluting the concentrated extract with water and ultrafiltration with a molecular cut-off of 2 kDa to 6 kDa to obtain a filtrate Step < / RTI >

In the present invention, the dilution is not limited, but 3 to 20 times of water may be added to the total weight of the fermented ginseng or red ginseng extract.

In the present invention, the molecular weight cut of the filtration step is not limited but may be set to 3 kDa or 5 kDa. The ultrafiltration in the filtration step is not limited, but the ratio of the inner liquid to the outer liquid is in the range of 75 to 85:25 to 15 And the like.

In the present invention, the ginseng is ginseng ( Panax ginseng CA Meyer), ginseng, white ginseng, Panax quinquefolium , Panax at least one member selected from the group consisting of notoginseng , Panax japonicum , Panax trifolium and Panax pseudoginseng may be used, but it is not limited thereto.

The fresh ginseng refers to raw ginseng that has not been processed after being extracted from the field, and white ginseng is dried or processed without removing or removing the epidermis, mainly using 4-6 years old ginseng as a raw material.

In the present invention, red ginseng refers to pale yellow-brown or light reddish brown ginseng which has been steamed with a steam without removing the epidermis by strictly selecting 4 to 6 years old fresh ginseng.

In the present invention, ginseng roots or red ginseng root refers to main roots of ginseng or red ginseng, which is the body portion of the ginseng or red ginseng.

According to the present invention, red ginseng is a red ginseng root which is obtained by steaming steam ginseng for 4 to 6 years to produce dried ginseng root, Were used separately.

In the present invention, ginseng can be used as a name encompassing common ginseng including processed ginseng, white ginseng or red ginseng and processed ginseng thereof.

In the present invention, the main roots of ginseng and the mixture of main roots and fine roots of fine rosin or red ginseng may have a ratio of fine roots to main roots of 0% to 40% of main roots versus 60% to 100% of fine roots, It is appropriate that fine roots are 60% or more.

In the present invention, the major roots of ginseng and the mixture of main roots and fine roots of fine roots or red ginseng can be pulverized or powdered to enhance extraction efficiency prior to extraction, and can be carried out according to a conventional extraction method known in the art. For example, a water extraction method, an alcohol extraction method, an organic solvent extraction method, a supercritical extraction method and the like can be used, and water extraction or alcohol extraction is preferably used, but not limited thereto.

The extraction solvent used in the alcohol extraction method may be a lower alcohol having 1 to 6 carbon atoms such as methanol, ethanol, propanol, isopropanol, and butanol. As the extraction solvent of the organic solvent extraction method, organic solvents such as acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, hydrochloric acid, acetic acid, formic acid, citric acid, cyclohexane and petroleum ether, .

The ratio of the extraction solvent added during the extraction is not particularly limited, but the extraction solvent may be used in an amount of 2 to 20 times (based on the weight) for the mixture (dry weight) of the main roots of ginseng and the main roots and fine roots of fine roots or red ginseng. In order to increase the extraction efficiency, preferably, the mixture of the main muscle of the ginseng and the main muscle of the red ginseng or the red ginseng may be repeated several times at least twice using an extraction solvent 5 to 15 times (weight basis).

The extraction temperature is suitably 50 to 110 캜, preferably 70 to 100 캜, more preferably 75 캜. The extraction time varies depending on the extraction temperature, but is extracted for 1 hour to 48 hours, preferably 2 hours to 8 hours, more preferably 6 hours. In addition, extraction efficiency can be further increased when stirring is carried out with a shaker at the time of extraction.

 In the present invention, the main extract of the ginseng and the extract of the mixture of the main muscle and the fine muscle of the red ginseng can be used at a reduced pressure to have a solid content of 1 to 20 Brix. Preferably, ginseng or red ginseng containing 60% or more of fine roots relative to the main muscle can be extracted into water, alcohol, or alcohol, and then concentrated to about 6 Brix.

If the solid content is not adjusted to 1 to 20 Brix, the solid content of the ginseng main root and the extract of the mixture of main muscle and fine muscle of red ginseng is 1 to 20 It is preferable to control with Brix.

In the present invention, the enzyme fermentation step is performed by adding the enzyme to the weight of the main extract of ginseng and the extract of the mixture of the main muscle and the fine root or red ginseng, preferably about 10% to about 15% Deg.] C for 2 to 5 days, preferably 50 [deg.] C for about 5 days. More preferably, 10% (w / w) pectinase enzyme is added to the ginseng or red ginseng extract of 6 bricks, and the mixture is incubated at 50 ° C for 3 days with stirring, and then the enzyme reaction is stopped by heating at 95 ° C for 30 minutes Ginsenoside Rd-enhanced fermented ginseng or red ginseng extract (GS-E3D).

In the present invention, it is preferable to sterilize the enzyme fermentation broth at 80 to 100 ° C for 5 to 30 minutes, preferably at 95 ° C for 10 to 30 minutes, in order to stop the enzymatic fermentation reaction of the enzyme fermentation broth. At this time, the activity of the added enzyme can be suppressed by heating.

In the present invention, the enzyme fermentation broth is sterilized and then cooled to 30 to 40 캜, preferably 37 캜.

At this time, the enzyme can be inactivated by treatment at 90 to 95 ° C for 5 to 30 minutes, and it is preferable to treat at 95 ° C for 30 minutes.

At this time, fermentation is accelerated by the added enzyme to shorten the fermentation time, which is economical, and the ginsenoside Rd can be strengthened without separately adding an additive for controlling the pH and the like.

In the enzymatic fermentation step of the present invention, pectinase is used as an enzyme. Preferably, pectinase is selected from the group consisting of Ultrazyme AFP, Pectinex Ultra AFP, Pectinex Ultra clear, ,? -herbzym, Novozym 33095, and more preferably Pectinex Ultra clear or Novozym 33095. In addition,

More specifically, by converting the ginsenosides Rb1 and Rc into ginsenoside Rd by the enzyme fermentation step of the present invention, a large amount of ginsenoside Rd can be contained.

The present invention provides fermented ginseng or red ginseng extract prepared according to the above method.

Therefore, the fermented ginseng or red ginseng extract prepared according to the present invention is economical by shortening the fermentation time by adding an enzyme for food additives, and can enhance the ginsenoside Rd without adding an additive for controlling pH or the like. Also, by preparing a fermented enzyme extract using a main ginseng of high total ginsenoside content including ginsenoside Rd and a mixture of main muscle and fine root of red ginseng or red ginseng, a ginsenoside Rd It can make a lot of structural changes and can be differentiated from existing ginseng or red ginseng.

The method for producing fermented ginseng or red ginseng extract according to the present invention is advantageous in that it can easily and economically convert ginsenoside Rd present in minor amounts in main muscle and fine muscle of ginseng or main muscle and fine muscle of red ginseng using enzymatic fermentation have. The fermented ginseng or red ginseng extract according to the present invention is advantageous in that it is economical by shortening the fermentation time by adding an enzyme for food additives, and enhances the ginsenoside Rd without adding an additive for controlling the pH and the like.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view of a method for producing ginsenoside Rd-enhanced fermented ginseng or red ginseng extract of the present invention.
FIG. 2 is a graph showing the analysis of saponin content of crude saponin fortified with ginsenoside Rd by enzyme.
Fig. 3 is a graph showing the analysis of saponin content of ginsenoside Rd-enhanced ginseng or red ginseng concentrate by enzyme.
FIG. 4 is a graph showing the analysis of the saponin content of the ginseng or red ginseng concentrate according to the temperature and concentration after enzyme fermentation.
FIG. 5 is a graph showing an analysis of the content of ginsenoside Rd over time according to the enzyme concentration in 12 Brix of ginseng or red ginseng concentrate.
FIG. 6 is a graph showing the extraction yields according to the mixing ratio of the main roots and fine roots of red ginseng or main roots and fine roots of red ginseng.
FIG. 7 is a graph showing the analysis of the content of ginsenoside and the change of Rb1 / Rg1 according to the ultrafiltration membrane filtration rate.
Figure 8 shows HPLC chromatograms according to ultrafiltration membrane filtration rate of 5 kDa.
9 shows HPLC chromatograms according to 3 kDa ultrafiltration membrane filtration rate
FIG. 10 is a graph showing changes in the content of ginsenoside according to the blending ratio of the main muscle of the ginseng and the main muscle and fine muscle of the red ginseng or red ginseng.
11 is a graph showing an analysis of Rb1 / Rg1 and PPD / PPT series saponin ratios according to blending ratios of main roots and fine roots of red ginseng or main roots and fine roots of ginseng.
12 is a graph showing changes in the content of ginsenoside Rd according to the extract concentration.
13 is a graph showing changes in the content of ginsenoside Rd with respect to the reaction time.
FIG. 14 is a graph showing an analysis of the functional ingredient content of the main ginseng root extract and the extract (GS-C46) of the main muscle and the fine root mixture of red ginseng and the fermented ginseng or red ginseng extract (GS-E3D) according to the present invention.

The present invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

Hereinafter, in the present invention, ginseng can be used as a name encompassing common ginseng including processed ginseng, white ginseng or red ginseng and processed ginseng thereof.

1. Experimental material

GAP certified 4 year old red ginseng main roots were purchased from Ginseng Ginseng Nonghyup and red ginseng was purchased from Woosin Industry. Pectinase enzymes are widely used as food additives such as Ultrazyme AFP (E1), Pectinex Ultra AFP (E2), Pectinex Ultra clear (E3), Novozym 33095 (Novozym) 33095) (E5) were purchased from Novozymes (Denmark) and used. In addition, α-herbzym (E4) was purchased from Korean Enzyme Source (Korea).

2. Extract preparation method for selection ratio of main roots and fine roots of ginseng or red ginseng

In order to determine the content of ginsenoside according to the blending ratio of main roots and fine roots of ginseng or red ginseng and to find the optimum blending ratio for the conversion of ginsenoside Rd, the proportion of main roots and fine roots of ginseng or red ginseng was set at 0: ), Main roots 2: fine roots 8 (C-28), main roots 4: fine roots 6 (C-46), main roots 6: fine roots 4 (C-64), main roots 8: The mixture was weighed in a ratio of 0 (C-10) and adjusted to a total weight of 100 g. Five times the weight of water was added, and the mixture was extracted three times for 6 hours on a 75 ° C water bath. The filtrate was concentrated on a water bath at 60 ° C, Brix, and 12 Brix.

 3. Production of ginsenoside Rd-fortified enzyme-treated ginseng or red ginseng extract

10% (w / w) of Novozym 33095 (E5) was added to the above ginseng or red ginseng extract to prepare ginsenoside Rd from the ginsenosides Rb1, Rb2 and Rc of the PPD series, (Shaking incubator) for 3 days and 5 days, followed by treatment at 95 ° C for 30 minutes to stop the enzyme reaction.

4. Ginsenoside analysis

For the analysis of ginsenoside content, extracts of ginseng or red ginseng extracts by main blending ratio and blend ratio and ginsenoside Rd - fortified enzyme treated ginseng or red ginseng extract were respectively dried in a freeze dryer and powdered and used as a sample. Powdered samples were precisely weighed 10 mg and dissolved in 1 ml of methanol. Ultrasonic extraction was performed for 1 hour, and the supernatant was recovered by centrifugation. The supernatant was collected and filtered through a 0.45-μm membrane filter, and the filtrate was used as an analytical sample. The conditions of the analyzer are as follows.

The water content (A solution) and ACN (B solution) were measured under the solvent conditions shown in Table 1 and Table 2 using a Waters-PDA (Waters 1525, detector 2998, USA) And analyzed by HPLC (high performance liquid chromatography).

[Table 1] Solvent conditions for ginsenoside analysis

[Table 2] HPLC analysis conditions for ginsenoside analysis

[ Experimental Example  1] Experiments for selection of optimal enzymes

One. Gin Senocide Rd  Enrichment Optimal Enzyme Screening Experiment

Ginseng extract (Sungshin BST) and red ginseng (Woo Shin Industry) were purchased and used. Red ginseng was refluxed with 80% ethanol to obtain an extract. Ginseng or red ginseng extract was lyophilized.

5 mg of the sample was completely dissolved in 2 mL of distilled water, and 0.5 mL of the enzyme solution was adjusted to 1 mL (v / v) to 2.5 mL. And reacted at 120 RPM for 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours in a shaking incubator at 50 ° C. The untreated group was reacted by adding distilled water instead of the enzyme.

(E2), Pectinex Ultra AFP (E2), and Pectinex Ultra clear (E2), which are commercially available for food additives in order to select the most suitable enzyme for enhancing ginsenoside Rd. (E3) and α-herbzym (E4) were added to crude saponin, ginseng concentrate and red ginseng concentrate to 1% (v / v) , 12 hours, 24 hours, and analyzed for ginsenoside content.

For the analysis of ginsenoside content, 1: 1 of water-saturated n-butanol was added to the extract, the mixture was thoroughly mixed and centrifuged to recover the water-saturated n-butanol layer twice more. The recovered water-saturated n-butanol layer was washed with 3 mL of distilled water, centrifuged to collect only the butanol layer, and concentrated in a 70 ° C water bath to recover saponin components for analysis.

The content of ginsenoside of the crude saponin fermented product after enzyme fermentation by the four enzymes was examined and shown in Tables 3, 4 and 2 below.

[Table 3] Changes in the content of ginsenosides after crude enzyme saponin treatment with enzymes E1 and E2

[Table 4] Changes in the content of ginsenosides after crude enzyme saponin treatment with enzymes E3 and E4

The content of ginsenosides in the ginseng or red ginseng fermented product after enzyme fermentation by the four enzymes was examined and shown in Tables 5, 6 and 3 below.

[Table 5] Changes in contents of ginsenosides after treatment with enzymes E1 and E2 in ginseng or red ginseng extract

[Table 6] Changes in content of ginsenosides after treatment with enzymes E3 and E4 in ginseng or red ginseng extract

As shown in Tables 3 to 6, all of the four enzymes did not significantly affect the content of PPT saponins, but they were affected by changes in the content of PPD-based saponins and were considered to be enzymes acting on PPD saponins.

As shown in Tables 3 to 6 and FIG. 2 and FIG. 3, the enzyme that selectively increases the content of ginsenoside Rd is represented by Pectinex Ultra clear, which is E3 among four enzymes, The optimum enzyme for Rd production was determined by Pectinex Ultra clear (E3). Ultrazymes AFP (E1) and α-herbzym (E4) increase the content of ginsenoside Rd, but the absolute value of ginsenoside Rd is higher than that of Pectinex Ultra clear (E3) (Pectinex Ultra AFP) (E2), the reaction proceeded very rapidly, and the amount of F2 and Compound K produced through ginsenoside Rd at the same time was large, so that F2 and Compound K were the final products Although it was judged to be a useful enzyme, standardization of scale up process for producing ginsenoside Rd was considered to be difficult and was excluded. It was found that the crude saponin and ginseng or red ginseng extracts showed the highest ginsenoside Rd values in 1 to 6 hours, although there was a difference in the production of ginsenoside Rd.

2. Optimum concentration and temperature selection test for economic efficiency

Ginseng or red ginseng concentrate was diluted with distilled water to 3, 6, and 12 Brix. To 2 mL of extract, add 0.5 mL of enzyme solution (E2, E3) at 10% (v / v) to 2.5 mL. The temperature was set at 37 ° C and 50 ° C, respectively, and reacted at 120 RPM for 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours in a shaking incubator. The untreated group was reacted by adding distilled water instead of crude enzyme. The reaction was stopped by treating the enzyme reaction product at 95 DEG C for 10 minutes.

In order to commercialize this technology, a more economical production method should be adopted. To select the optimal temperature for the Rd enzyme conversion, the enzyme Pectinex Ultra clear (E3) , Extract concentration and temperature selection experiment were conducted by adjusting the concentration of ginseng or red ginseng extract to 3 Brix, 6 Brix, and 12 Brix. As a result, the content of ginsenoside was determined by the enzyme E3 concentration at 50 ° C. (Table 7), the content of ginsenoside by enzyme E3 at the extract concentration at 37 ° C. (Table 8) Analysis of saponin content after enzyme conversion by temperature and concentration of ginseng or red ginseng concentrate (FIG. 4) is shown.

[Table 7] Changes in the content of ginsenosides by enzyme E3 at the extract concentration at 50 < 0 > C

[Table 8] Changes in the content of ginsenoside by enzyme E3 at the extract concentration at 37 ° C

As shown in Tables 7 and 8 and FIG. 4, the temperature condition for raising the content of ginsenoside Rd by Pectinex Ultra clear (E3) is much higher than 37 ° C at 50 ° C Conversion of ginsenoside Rd rapidly occurred at low temperature at 50 ℃, and it was necessary to extend the reaction time and adjust the enzyme concentration when the extract concentration was increased. However, when the concentration of the extract is low, the time and economic costs such as concentration are increased. Therefore, it is considered that the economical production method should increase the concentration of the extract and slightly increase the reaction time. Therefore, the present study was carried out to increase the content of ginsenoside Rd The optimal enzyme temperature was 50 ℃ and the extract concentration was determined to be 12 Brix, but it was considered to be preferable to adjust the reaction time and enzyme concentration.

3. Conditions for determining enzyme concentration and time

10% or 15% (w / w) of the enzyme solution (Novozym 33095, E5) was added to the solution, and the solution was diluted with distilled water to a concentration of 50 Brix The reaction was carried out for 6 hours, 1 day, 2 days, 3 days, and 4 days at 120 ℃ with RPM at shaking incubator. The reaction was stopped by treating the enzyme reaction product at 95 DEG C for 10 minutes.

To prepare Rd - enhanced fermented ginseng or red ginseng with the minimum enzyme concentration, enzyme concentration and enzyme reaction time setting experiment were performed. (Novozym 33095) (E5) were added to ginseng or red ginseng extracts adjusted to 12 Brix at 10% and 15%, respectively, and reacted at 50 ° C for 1 day, 2 days, 3 days and 4 days. Side contents were analyzed and are shown in Table 9 and FIG. 5 below.

[Table 9] Change in content of ginsenoside over time according to the concentration of enzyme Novozym 33095 (E5)

As shown in Table 9 and FIG. 5, the content of ginsenoside Rd was increased with increasing time from the analysis of ginsenoside content. However, the content of ginsenoside Rd did not increase after 2 days, And 15%, respectively. Therefore, when 10% of Novozym 33095 (E5) was added to ginseng or red ginseng extract of 12 Brix and reacted at 50 ℃ for 3 days, the sum of Rg1 and Rb1, which are ginseng and red ginseng components, It was confirmed that an enzyme reaction product with a significantly increased side Rd level can be obtained.

[ Experimental Example  2] Main roots of ginseng or red ginseng Fine  Experiment for selection of compounding ratio

1. Main roots of ginseng or red ginseng Fine  Depending on the compounding ratio Extraction yield  compare

Ginsenoside Rd is produced by microorganisms or enzymes with one sugar removed from the PPD family of ginsenosides Rb1, Rb2, Rc, and the like. Therefore, in the present invention, the higher the content of PPD-based ginsenosides such as ginsenosides Rb1, Rb2, and Rc in ginseng or red ginseng, is advantageous to increase the ginsenoside Rd content. In order to investigate the content of ginsenoside according to the mixing ratio of main and fine roots of ginseng or red ginseng, the ratio of main roots and fine roots of ginseng or red ginseng was divided into main roots 0: fine roots 10 (C-01), main roots 2: Extracted at six ratios of main root 4: fine root 6 (C-46), main root 6: fine root 4 (C-64), main root 8: fine root 2 (C-82), main root 10: fine root 0 (C-10) Concentrated and then lyophilized to give an extraction yield and ginsenoside analysis. The extraction yield was the lowest at 29.9% when only ginseng or red ginseng root was used and the yield was increased with increasing ratio of ginseng or red ginseng root to the main ginseng. When using ginseng or red ginseng root alone, 54.0% It was confirmed that the extraction yield was about twice as high as that using only fine root (Table 10 and FIG. 6). This is thought to be due to the fact that the main muscle has a thick skin and a lot of fiber, pectin and starch which are not eluted in water.

[Table 10] Extraction yields of ginseng or red ginseng according to the proportion of main roots and fine roots

2. Main roots of ginseng or red ginseng Fine  Depending on the compounding ratio Extraction yield  compare

The ginsenosides were analyzed for seven main components of ginseng or red ginseng (Table 11, Figs. 7 and 8). The ginsenosides Rg1, Re and Rf as the PPT series ginsenosides and Rb1 as the PPD series ginsenosides , Rc, Rb2 and Rd were analyzed. The total ginsenoside contents of these seven ginsenosides were 75.06 mg / g of ginseng or red ginseng extract (C01) and 30.17 mg of ginseng or red ginseng root extract (C10) / g. The ginsenoside contents decreased with increasing ratio of ginseng or red ginseng root in ginseng or red ginseng roots. The sum of ginsenoside or red ginseng extract (C01) was 57.64 mg / g, and the sum of ginseng or red ginseng root extract (C10) was 18.08 mg / g. The content of ginsenosides in PPD was also decreased with increasing ratio of ginseng or red ginseng root in ginseng or red ginseng roots. These results suggest that it is more advantageous to use ginseng or red ginseng roots rather than ginseng or red ginseng roots as a sample for enhancing ginsenoside Rd.

[Table 11] Analysis of ginsenosides according to the mixing ratio of main roots and fine rosins of ginseng or red ginseng

3. Ultrafiltration  Preparation of component fractions

Main root: The fine roots were mixed at a ratio of 4: 6 (R4S6) to obtain a total weight of 500 g, and 5 L of water was added. The mixture was repeatedly extracted and filtered three times at 80 ° C for 6 hours, kDa and 5 kDa molecular cut-off ultrafiltration membranes were used to perform ultrafiltration by 10% (internal solution: external solution 9: 1), 20%, 40%, 60%, and 80% The inner liquid was lyophilized.

The yield of the ultrafiltration concentrate (inner solution) did not show a yield difference according to the filtration rate in the 3 kDa and 5 kDa ultrafiltration membranes (Table 12 and Fig. 7). According to the yield by the ultrafiltration membrane having 3 kDa pore size, when the ultrafiltration was performed at 20%, the yield was 89.2%, which was about 90%, the yield was 70.9% when 50% ultrafiltration was achieved, Yield was 54.9% (Table 12 and Figure 7).

[Table 12] Filtration yields of ultrafiltration membranes of 3 kDa and 5 kDa

The ratio of the inner liquid to the outer liquid was 10% (inner liquid: outer liquid 9: 1), 20% (inner liquid: outer liquid 8: 2), 30% 50% (inner liquid: outer liquid 5: 5), 60% (inner liquid: outer liquid 4: 6), 70% (inner liquid: outer liquid 3: 7) and 80%

[ Example  1] According to the present invention Gin Senocide Rd  Ginseng extract or ginseng extract GS -E3D) manufacture

Ginsenoside Rd fortified ginseng or red ginseng extract was prepared by using 6 samples C-01, C-28, C-46, C-64, C-82 and C-10 according to the blending ratio of ginseng or red ginseng Experiments were conducted to set conditions for manufacturing. Pectinase (10%, w / w) was added to ginseng or red ginseng extract and reacted for 4 days, 5 days, and 6 days in a shaking incubator at 50 ° C, followed by treatment at 95 ° C for 30 minutes to stop the enzyme reaction Rc, Rb2, Rb3, Rd, F2, and CK contents as PPD series were analyzed as the PPT series, and the content of ginsenosides Rg1, Re, Rf and PPD series were analyzed. The ginsenoside Rd having three sugar chains is formed by one enzyme from the ginsenosides Rb1, Rc, Rb2, and Rb3 having four sugar chains, and F2 having two sugar chains from the ginsenoside Rd It is converted to CK with one sugar chain. Therefore, in order to increase the content of ginsenoside Rd, it is advantageous that the content of ginsenosides Rb1, Rc, Rb2 and Rb3 of the PPD series is high in the substrate extract.

As shown in the following Tables 13 and 14, ginsenoside Rd was found to be high in C01 which is a 100% extract of fine hair in all conditions, and gradually decreased as the content of the main muscle increased, and the concentration of the extract was changed to a brix As a result, it was confirmed that the lower the concentration, the faster the reaction occurred, and the concentration of senoside Rd was increased and decreased. When the concentration was high, the reaction was observed to be slow and continuous (FIGS. 12 and 13) 13). However, for industrial application of this product, it is important to shorten the reaction time. However, since the stability and economical efficiency of the reaction are important, it is important to determine the proper concentration and reaction time since the concentration of the extract can not be too low to shorten the reaction time. Therefore, the inventor selected the most economical condition by increasing the content of ginsenoside Rd and prepared ginsenoside Rd-fortified ginseng or red ginseng extract and named it "GS-E3D". In summary, ginseng or red ginseng containing 60% or more of fine roots relative to the main muscle is extracted into a water, alcohol, or alcohol-containing solvent and concentrated to about 6 bricks (1 to 12 Bricks). 6) 10% (w / w) of pectinase enzyme was added to ginseng or red ginseng extract of Briggs, and the mixture was incubated at 50 ℃ for 3 days and then heated at 95 ℃ for 30 minutes to stop the enzyme reaction. Ginseng or red ginseng extract (GS-E3D). The manufacturing process is shown in Fig.

[Table 13] Ginsenoside Rd Enhanced Ginsenoside Content of Ginseng or Red Ginseng Extract (mg / g)

[Table 14] Ginsenoside Rd enhanced ginsenoside content of ginseng or red ginseng extract (mg / g)

[ Example  2] Gin Senocide Rd  Ginseng extract or ginseng extract GS - E3D ) Standard specification

In this embodiment, the raw material is ginseng Panax ginseng CA Meyer (Araliaceae) Roots of dried white ginseng or ginseng are steamed and steamed, and dried red ginseng is extracted with alcohol, pure water or mixed liquor. Then, enzyme-treated pectinase for food additives is added and fermented ginseng Red ginseng extract.

In this example, 1 kg of ginseng or red ginseng (containing 60% or more of fine roots relative to main muscle) was added with 10 L of water or 70% alcohol, and the mixture was first extracted at 75 ° C. for 18 hours. 9 L of 30% (60 Brix, solid content of 60% or more), and then 100 g of the ginseng or red ginseng concentrate was diluted to 1000 L with purified water. After the second extraction, the first extract and the second extract were combined and concentrated to obtain ginseng or red ginseng concentrate (6 Brix), and then 10 g of the enzyme was added thereto, followed by reaction at 50 ° C for 3 days. This specimen is reddish brown with a characteristic odor.

0.2 g of the sample was precisely weighed, dissolved in 10 mL of methyl alcohol, and filtered through a 0.45-μm membrane filter. The test solution was injected into the analyzer according to the analytical conditions shown in Tables 15 and 16 below and subjected to HPLC analysis to determine the peak (ginsenoside Rd) Retention time (RT) was confirmed.

[Table 15]

[Table 16]

The functional component of this sample is ginsenoside Rd and the ginsenoside Rd content should not be less than 15 mg / g when tested according to the Identification Test. When this test sample is tested according to Method 1 of the General Tests Method for Specific Gravity Measurement according to KP, it shall have a specific gravity of 1.000 ~ 1.040. When 3.0 g of this sample is put into 30 ml of freshly boiled and cooled water and tested according to the General Methods for Testing pH, the pH should be 5.0 to 7.0. When this test sample is tested according to the general test method Refractive Index of General Tests, the refractive index should be 1.340 to 1.380.

The purity test method for this sample is as follows (Table 17). Heavy metals are tested by operating in accordance with Method 2 of the Heavy Metals General Tests. Add 2.0 mL of lead standard solution (20 ppm or less) to the comparison solution. Arsenic is tested in accordance with Method 3 of the Arsenic Test of General Test Methods in KP. The evaporation residue shall be 1.0 to 5.0% when 1.0 g of this sample is tested and tested according to the Vapor Deposition Residue Test in the General Test Methods. Residual pesticide test shall be carried out with this sample in accordance with the standards and test methods of residual contaminants such as medicinal herb and medicinal products. When the microbial limit test is conducted according to the Microbial Limit Test Method of the General Practice of the Pharmacopoeia, the number of aerobic live cells should not be more than 100 per 1 g. Unless otherwise specified, the General Rules and General Test Methods shall apply mutatis mutandis to this standard and test methods.

[Table 17] Analysis of Purity of Ginsenoside Rd Reinforced Fermented Ginseng or Red Ginseng Extract (GS-E3D)

[ Example  3] Gin Senocide Rd  Enhanced fermented ginseng or red ginseng extract ( GS - E3D ) Component analysis

4-year-old ginseng or red ginseng was used to prepare enzyme-fermented ginseng or ginseng extract (GS-E3D) according to the present invention. To increase the content of ginsenoside Rd, the ratio of fine roots to main roots was set to 60% For 6 hours, and then concentrated to 6 Brix to prepare an extract ((GS-C46)) of the main and fine mixture. 10% of pectinase (Novozyme ^® 33095, Denmark) was added to the mixed extract (GS-C46) and reacted for 3 days in a stirred incubator at 50 ° C to obtain ginsenosides Rb1 and Rc as ginsenoside Rd Respectively. In order to stop the further reaction, the fermented ginseng or red ginseng extract was prepared by inactivation of the enzyme at 95 ° C for 30 minutes. The fermented ginseng or red ginseng extract was concentrated and made into an extract or powder to be used for analysis. The functional ingredient setting is Ginenoside Rd (range: 15 mg / g or more), and the functional ingredient content is shown in the following Table 18 and FIG.

[Table 18] Analysis of functional components of ginsenoside Rd-fortified fermented ginseng or red ginseng extract (GS-E3D)

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

Extracting the fine roots of ginseng or red ginseng with a blending ratio of fine roots relative to the main roots of 100 wt% of fine roots by extracting solvent (step 1);
Concentrating the ginseng or red ginseng mixed extract solution prepared in the above step 1 under reduced pressure to a solid content of 6 grix (step 2);
Diluting the concentrated extract with water and ultrafiltering with a molecular cut-off of 5 kDa to obtain filtrate internal solution so that the ratio of the inner filtrate to the filtrate is 8: 2 (step 3); And
Converting the ginsenosides Rb1, Rc, Rb2, and Rb3 to ginsenoside Rd (step 4) by adding 10% by weight of a pectinase enzyme to the filtration stock solution, and fermenting the enzyme to increase the ginsenoside Rd Wherein the fermented ginseng or red ginseng extract is prepared by a method comprising:
delete delete delete delete The method according to claim 1,
Wherein the enzyme fermentation step is carried out in a reactor at 50 DEG C for 3 days and sterilized at 95 DEG C for 30 minutes to inactivate the enzyme. delete

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