KR102458031B1 - Composition and method for producing ginsenoside compound k using enzyme liquid of aspergillus niger - Google Patents

Abstract

The present invention is Aspergillus niger ( Aspergillus niger ) Ginsenoside compound containing an enzyme solution cultured in an expression medium containing carboxymethyl cellulose (carboxymethyl cellulose) or ginseng powder (ginseng powder) ginsenoside compound K production composition and method is about

Description

Composition and method for preparing ginsenoside compound K using Aspergillus niger enzyme solution

The present invention relates to a composition and method for preparing a ginsenoside compound K using Aspergillus niger enzyme solution.

Ginseng has been traditionally used as a cardiac agent or a diuretic in oriental medicine, and among them, ginsenoside, which refers to saponin present in ginseng, is known as a substance with pharmacological action among various active ingredients of ginseng.

Among them, ginsenoside compound K contains sugars such as glucose and arabinose from ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc and ginsenoside Rd, which are protophanaxadiol-based saponins present in large amounts in ginseng. It is decomposed into a form that is much better absorbed in the body, and experiments on many effects such as anti-cancer, anti-inflammatory, anti-allergy, immune enhancement, UV-induced skin damage recovery, skin aging prevention, and wrinkle suppression have been reported in several papers.

Until now, as a high-efficiency production technology of ginsenoside compound K, a production method capable of producing high-efficiency ginsenoside compound K with an enzyme obtained by inserting a recombinant gene derived from Caldicellulosiruptor bescii into E. coli is known. , because it is a method that reacts with an enzyme obtained by genetic manipulation, it has limitations in its use in food. Since there is a problem of low conversion rate, a fungal enzyme that can be used in food and produces ginsenoside compound K with a higher yield than lactic acid bacteria was used.

As a method using existing fungal enzymes, protopanaxadiol (PPD)-based saponins are produced using cellulase isolated from Trichoderma or Penicillium or beta-galactosidase isolated from Aspergillus. Methods have been reported to prepare ginsenoside compound K using the saponin fraction isolated from ginseng root, which is mainly contained, and ginseng extract as a substrate, but there is a limitation in yield. There was a disadvantage in that it could be converted to compound K only after a long time of reaction in compound Y (compound Y) generated when it was used.

The present invention is for producing ginsenoside compound K with high productivity and yield, (a) Aspergillus niger ( Aspergillus niger ) As a carbon source, cultured in an expression medium containing carboxymethyl cellulose (carboxymethyl cellulose) to obtain an enzyme solution; And (b) to provide a method for producing a ginsenoside compound K, including the step of treating the obtained enzyme solution to a substrate.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention is (a) Aspergillus niger ( Aspergillus niger ) as a carbon source, culturing in an expression medium containing carboxymethyl cellulose (carboxymethyl cellulose) or ginseng powder (ginseng powder) to obtain an enzyme solution; And (b) provides a method for producing a ginsenoside compound K comprising the step of treating the obtained enzyme solution to a substrate.

The Aspergillus niger strain in step (a) may be an Aspergillus niger strain (KACC 46495).

As a carbon source in the expression medium in step (a), the concentration of carboxymethyl cellulose or ginseng powder may be 3 g/L to 20 g/L.

In step (a), the expression medium may include corn steep solid as a nitrogen source.

The expression medium in step (a) is KH ₂ PO ₄ , MgSO ₄ ^. 7H ₂ O, CaCl ₂ , FeSO ₄ ^. 7H ₂ O, ZnSO ₄ ^. 7H ₂ O, MnSO ₄ ^. H ₂ O and CoCl ₂ ^. 6H ₂ O may be further included.

The enzyme solution in step (a) may be an extracellular enzyme solution of the Aspergillus niger strain.

In step (b), the substrate is one or more protopanaxadiol-based saponins selected from the group consisting of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc and ginsenoside Rd; Or it may include a ginseng extract.

The concentration of protopanaxadiol-based saponin in the substrate in step (b) may be 0.1 mg/ml to 10 mg/ml.

The treatment in step (b) may be performed for 2 hours to 24 hours at pH 4.0-6.5 and 40-65°C conditions.

In one embodiment of the present invention, Aspergillus niger ( Aspergillus niger ) Ginseno containing an enzyme solution cultured in an expression medium containing a strain as a carbon source, carboxymethyl cellulose (carboxymethyl cellulose) or ginseng powder (ginseng powder) A composition for preparing side compound K is provided.

The expression medium may include corn steep solid as a nitrogen source.

The method for producing ginsenoside compound K according to the present invention is (a) Aspergillus niger strain as a carbon source, in an expression medium containing carboxymethyl cellulose or ginseng powder culturing to obtain an enzyme solution; and (b) treating the obtained enzyme solution on a substrate, using a low-cost carbon source, but through optimal hydrolysis of protopanaxadiol-based saponins in the substrate, ginsenoside compound K is produced with high productivity and It can be prepared in yield.

In particular, Aspergillus niger ( Aspergillus niger ) The strain is a safe mold available for food, the ginsenoside compound K prepared in the same way as above has the advantage that can be usefully utilized for food.

1 (a) to (c) show that the extracellular enzyme solution is cultured by varying the carbon source of the expression medium, and then as a substrate, (a) ginsenoside Rb1, (b) ginsenoside Rb2 and (c) ginsenoside It is a graph showing the relative activity when each side Rc was treated.
Figure 2 is a graph showing the relative activity when the extracellular enzyme solution is cultured by varying the carbon source concentration of the expression medium, and then treated with ginsenoside Rb2 as a substrate.
3 (a) and (b) are graphs showing the relative activity and stability according to changes in (a) temperature, (b) pH of the extracellular enzyme solution cultured through the optimized expression medium, in which case the relative activity is The senoside Rb1 was shown according to the degree of decrease.
4(a) to (c) show that the extracellular enzyme solution is cultured through an optimized expression medium, and then as a substrate, as a substrate, (a) ginsenoside Rb1, (b) ginsenoside Rb2 and (c) gene It is a graph showing the degree of conversion by time when each treatment is performed on the senoside Rc.
5 is a diagram showing how ginsenoside Rb1, ginsenoside Rb2 and ginsenoside Rc are converted into ginsenoside compound K through which intermediate materials.
Figure 6 (a) shows the degree of conversion by time when the extracellular enzyme solution is cultured through an optimized expression medium and then treated with a Korean ginseng root extract containing a protopanaxadiol-based saponin mixture as a substrate, as a substrate. , and FIG. 6(b) is a diagram showing the measurement results of high performance liquid chromatography (HPLC) before and after the reaction.

The present inventors were studying a method for increasing the productivity and yield thereof in preparing a ginsenoside compound K using an Aspergillus niger enzyme solution, Aspergillus niger As an enzyme solution, Culturing the Aspergillus niger strain through an expression medium optimized for carbon source, confirming the excellent activity of its extracellular enzyme solution, and then treating it with a substrate containing protopanaxadiol-based saponin to compound a ginsenoside compound By making a K, the present invention was completed.

In the present specification, "ginsenoside compound K (C-K)" refers to a compound represented by the following Chemical Formula 1, and various physiological activities have been previously reported:

[Formula 1]

Manufacturing method of ginsenoside compound K

The present invention (a) Aspergillus niger ( Aspergillus niger ) as a carbon source, carboxymethyl cellulose (carboxymethyl cellulose) or culturing in an expression medium containing ginseng powder (ginseng powder) to obtain an enzyme solution; And (b) provides a method for producing a ginsenoside compound K comprising the step of treating the obtained enzyme solution to a substrate.

First, the method for producing a ginsenoside compound K according to the present invention is cultured in an expression medium containing Aspergillus niger strain as a carbon source, carboxymethyl cellulose or ginseng powder to obtain an enzyme solution [step (a)].

The Aspergillus niger ( Aspergillus niger ) strain is a commercial mold available for food, belonging to the genus Ascomycetes yeast mold. The Aspergillus niger ( Aspergillus niger ) strain is preferably an Aspergillus niger ( Aspergillus niger ) strain (KACC 46495), but is not limited thereto. Specifically, the present inventors from the National Academy of Agricultural Sciences (KACC), Rural Development Administration, Aspergillus oryzae, which are strains available for about 50 foods, Aspergillus oryzae , Aspergillus niger ( Aspergillus niger ), Aspergillus tubingen Sis ( Aspergillus tubingensis ), Penicillium chrysogenum ( Penicillium chrysogenum ), etc. were sold. As a result of reacting the extracellular enzyme reactant obtained by culturing these fungal strains with ginsenoside Rb1, Rb2 or Rc, Aspergillus niger KACC 46495 strain appeared as the most active strain, so the above strain was selected .

In order to obtain a culture solution from the Aspergillus niger strain, plate-culturing the strain to obtain spores; activating the mycelium by culturing the spores in a growth medium; And after removing the growth medium, transferring the activated mycelium to an expression medium and culturing to obtain a culture solution.

Additionally, in order to obtain an extracellular enzyme solution from the culture solution, filtering the culture solution; recovering the filtrate, adding and mixing ammonium sulfate to obtain an enzyme precipitate; dialyzing the enzyme precipitate solution into a dialysis membrane; and further removing and concentrating the remaining salt using a centricon.

That is, the Aspergillus niger ( Aspergillus niger ) enzyme solution can be obtained by culturing the Aspergillus niger strain through an expression medium, and the present invention is characterized in that the expression medium is optimized.

The expression medium is characterized in that it contains carboxymethyl cellulose or ginseng powder as a carbon source, and preferably contains carboxymethyl cellulose, ginsenoside Rb1, Rb2 or Rc can be effectively converted into ginsenoside compound K.

At this time, carboxymethyl cellulose is a carboxymethylated hydroxyl group of cellulose. It is chemically very stable and especially harmless and non-toxic to the human body, so it is a compound suitable for use in the food industry and cosmetics industry. It is widely used.

In addition, ginseng powder is derived from Panax ginseng, and may be Panax ginseng extract powder purified with HP-20, and the ginseng powder includes ginsenosides Rb1, Rb2, Rc and Rd. Protopanaxadiol-based saponins in a total concentration of 120 mg/g to 140 mg/g, and protopanaxatriol-based saponins including ginsenoside Rg1, Rh1, Re, Rg2, Rf1 and F1 total 40 mg/g to 60 mg/g may be included. Specifically, the detailed contents and concentrations of Panax ginseng (4-year-old root) derived ginseng powder used in the present invention are shown in Table 1 below.

Ginsenoside ginseng powder Content ((w/w)%) Concentration (mg/g) Protopanaxadiol-based saponins Rb1 21.4 38.0 Rb2 18.8 33.4 Rc 17.5 31.1 Rd 14.3 25.4 Protopanaxatriol-based saponins Rg1 2.6 4.6 Rh1 0.0 0.0 Re 9.7 17.3 Rg2 1.3 2.3 Rf1 0.6 1.2 F1 13.6 24.2 gun 100.0 177.5

The concentration of carboxymethyl cellulose or ginseng powder in the expression medium may be 3 g/L to 20 g/L, preferably 10 g/L to 15 g/L, but limited thereto doesn't happen

On the other hand, when cellulose, lactose, or pectin from citrus is used as a carbon source, ginsenoside Rb1, Rb2 or Rc cannot be effectively converted into ginsenoside compound K.

In addition, the expression medium preferably includes corn steep solid as a nitrogen source, but is not limited thereto. Likewise, ginsenoside Rb1, Rb2 or Rc can be effectively converted into ginsenoside compound K. At this time, the corn steep solid is a by-product discharged from the soaking process during the process of staking corn starch, and not only has the advantage of low cost, but also contains soluble protein, lactic acid, sugars, vitamins, minerals, etc. Because of its high nutritional value, it is suitable for addition to feed or as a nitrogen source for microbial media.

The concentration of corn steep solid in the expression medium may be 5 g/L to 20 g/L, preferably 10 g/L to 15 g/L, but is not limited thereto.

In addition, the expression medium is KH ₂ PO ₄ , MgSO ₄ 7H ₂ O, CaCl ₂ , FeSO ₄ ·7H ₂ O, ZnSO ₄ ·7H ₂ O, MnSO ₄ ·H ₂ O and CoCl ₂ ·6H ₂ O In addition It may include, specifically, KH ₂ PO ₄ 1-3 g/L, MgSO ₄ 7H ₂ O 0.1-1.0 g/L, CaCl ₂ 0.1-1.0 g/L, FeSO _{4.7H 2} O 1.0-10.0 mg /L, ZnSO _{4.7H 2} O 0.5 to 5.0 mg/L, MnSO _4· H ₂ O 0.5 to 5.0 mg/L, and CoCl ₂ ·6H ₂ O 1.0 to 10.0 mg/L may be further included.

On the other hand, the Aspergillus niger ( Aspergillus niger ) As described above, the enzyme solution may be an extracellular enzyme solution of the Aspergillus niger ( Aspergillus niger ) strain.

Next, the method for producing a ginsenoside compound K according to the present invention includes the step [(b) step] of treating the obtained enzyme solution to a substrate.

The substrate preferably includes one or more protopanaxadiol-based saponins selected from the group consisting of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc and ginsenoside Rd, but is not limited thereto.

In addition, the substrate may be a Korean ginseng extract, and the Korean ginseng extract may be a solvent-extracted product of the root, fruit, stem or leaf of ginseng. In this case, the solvent extraction may be performed using water, a C1 to C2 lower alcohol or a mixture thereof as a solvent, and the lower alcohol may be ethanol or methanol.

The concentration of protopanaxadiol-based saponin in the substrate may be 0.1 mg/ml to 10 mg/ml, preferably 0.4 mg/ml to 7.0 mg/ml, but is not limited thereto.

In addition, the treatment may be carried out for 2 hours to 24 hours at pH 4.0 ~ 6.5 and 40 ~ 65 ℃ conditions. Specifically, the pH condition for the treatment is preferably pH 4.5 to 5.0, but is not limited thereto. At this time, when the pH exceeds 5.0, the stability of the extracellular enzyme solution may be significantly reduced. Therefore, in order to maintain such a pH condition, a buffer solution may be used as a reaction solvent. In addition, the temperature condition for the treatment is preferably 55 ~ 60 ℃, but is not limited thereto. At this time, when the temperature is 50 ℃, the relative activity may decrease to about 60% or less, and when the temperature is 65 ℃, the stability of the extracellular enzyme solution may be significantly reduced. That is, by maintaining these pH and temperature conditions, the activity of the extracellular enzyme solution is high, and the stability of the extracellular enzyme solution can be maintained even for a long time reaction. Therefore, it is possible to finally produce ginsenoside compound K with high productivity and yield through optimal hydrolysis of protopanaxadiol-based saponins in the substrate.

On the other hand, when through the above treatment, in addition to ginsenoside compound K (compound K), as a by-product, ginsenoside compound Y (compound Y) or ginsenoside F2 (F2) may be prepared.

Composition for preparing ginsenoside compound K

The present invention is Aspergillus niger ( Aspergillus niger ) as a carbon source, carboxymethyl cellulose (carboxymethyl cellulose) or ginseng powder (ginseng powder) ginsenoside compound K production composition comprising an enzyme solution cultured in an expression medium containing provides

The composition for preparing ginsenoside compound K according to the present invention uses an Aspergillus niger strain as a carbon source, and an enzyme solution cultured in an expression medium containing carboxymethyl cellulose or ginseng powder. Including, the expression medium may include a corn steep solid as a nitrogen source.

The Aspergillus niger ( Aspergillus niger ) strain, the expression medium and the enzyme solution have been described above, and thus a redundant description will be omitted.

The concentration of the enzyme solution in the composition is preferably 0.5 mg/ml to 10.0 mg/ml, but is not limited thereto.

In addition, the composition may be for treatment on a substrate.

The substrate preferably includes one or more protopanaxadiol-based saponins selected from the group consisting of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc and ginsenoside Rd, but is not limited thereto.

In addition, the substrate may be a Korean ginseng extract, and the Korean ginseng extract may be a solvent-extracted product of the root, fruit, stem or leaf of ginseng. In this case, the solvent extraction may be performed using water, a C1 to C2 lower alcohol or a mixture thereof as a solvent, and the lower alcohol may be ethanol or methanol.

The concentration of protopanaxadiol-based saponin in the substrate may be 0.1 mg/ml to 10 mg/ml, preferably 0.4 mg/ml to 7.0 mg/ml, but is not limited thereto.

As described above, the manufacturing method of the ginsenoside compound K according to the present invention is (a) Aspergillus niger strain as a carbon source, carboxymethyl cellulose (carboxymethyl cellulose) or ginseng powder (ginseng powder) Obtaining an enzyme solution by culturing in an expression medium containing; and (b) treating the obtained enzyme solution on a substrate, using a low-cost carbon source, but through optimal hydrolysis of protopanaxadiol-based saponins in the substrate, ginsenoside compound K is produced with high productivity and It can be prepared in yield.

In particular, Aspergillus niger ( Aspergillus niger ) The strain is a safe mold available for food, the ginsenoside compound K prepared in the same way as above has the advantage that can be usefully utilized for food.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1: Comparison of conversion to ginsenoside compound K according to the carbon source and its concentration in the expression medium

In the present invention, Aspergillus niger ( Aspergillus niger ) In preparing a ginsenoside compound K using an enzyme solution, Aspergillus niger ( Aspergillus niger ) The expression medium of the strain was optimized.

Specifically, the Aspergillus niger strain (KACC 46495) received from the National Academy of Agricultural Sciences (KACC), Rural Development Administration, was plate-cultured for 7 days at 26° C. in Potato dextrose agar medium (PDA). The spores in the plate cultured plate were collected by filtering with gauze, and 1 ml of the spore solution diluted to a spore concentration of 1.0 X 10 ⁶ spores/mL was inoculated into 4 ml of potato dextrose medium (PDB). , 26 ℃ and 150 rpm growth culture for 24 hours. After washing the mycelium in the growth medium made of the growth culture with an expression medium excluding carbon and nitrogen sources, only the mycelium was transferred to 100 ml of expression medium, and then cultured at 26° C. and 150 rpm for 6 days.

At this time, the composition of the expression medium is carbon source 20 g/L, nitrogen source 10 g/L, KH ₂ PO ₄ 2 g/L, MgSO ₄ 7H ₂ O 0.3 g/L, CaCl ₂ 0.3 g/L, FeSO _{4.7H 2} O 5.0 mg/L, ZnSO _{4.7H 2} O 1.4 mg/L, MnSO _4· H ₂ O 1.3 mg/L, and CoCl ₂ ·6H ₂ O 3.7 mg/L. As a carbon source, carboxymethyl cellulose (Sigma Aldrich Co., Ltd.), cellulose (Daejeong Chemical Co., Ltd.), Panax ginseng (4-year-old) derived ginseng powder (Aceem Biology, China), lactose A total of 6 types were used as (lactose) (Daejeong Hwageum Co., Ltd.), pectin from citrus (Daejeong Hwa Geum Co., Ltd.) and wheat bran (Sims Pet) extracted from citrus fruits. (corn steep solid) (Sigma Aldrich) was used.

Then, the culture solution was filtered using Whatman filter paper, the filtrate was recovered, ammonium sulfate was added to 30 to 80% saturation, and the mixture was mixed for 24 hours to obtain an enzyme precipitate solution. To remove ammonium sulfate in the enzyme precipitation solution, dialysis was performed with 200 mM Mcilvaine buffer solution for 24 hours using a cellulose dialysis membrane. was obtained.

Then, 1.0 mg/ml of the extracellular enzyme solution and a buffer solution containing 0.4 mg/ml of ginsenosides Rb1, Rb2, and Rc as substrates were reacted at 50° C. and pH 5.5 for 6 hours, and the activity of the extracellular enzyme solution was was measured.

As a result, as shown in Figures 1 (a) to 1 (c), as a carbon source, while using carboxymethyl cellulose or ginseng powder (particularly, carboxymethyl cellulose)) , it is confirmed that the activity of the extracellular enzyme solution is high when corn steep solid is used as the nitrogen source. However, when cellulose, lactose, pectin from citrus and wheat bran are used as carbon sources, it is confirmed that the activity of the extracellular enzyme solution is greatly reduced.

On the other hand, as a substrate, using the ginsenoside Rb2, which was not converted well among the protopanaxadiol-based saponins, as a carbon source in the expression medium, the optimal concentration of carboxymethyl cellulose was confirmed.

As a result, as shown in Figure 2, as a carbon source in the expression medium, when carboxymethyl cellulose is 10 g/L to 15 g/L (particularly, 10 g/L), the activity of the extracellular enzyme solution is found to be significantly higher.

Therefore, the composition of the optimized expression medium is a carbon source, carboxymethyl cellulose 10 g / L, corn steep solid 10 g / L, KH ₂ PO ₄ 2 g / L, MgSO ₄ 7H ₂ O 0.3 g/L, CaCl ₂ 0.3 g/L, FeSO _{4.7H 2} O 5.0 mg/L, ZnSO _{4.7H 2} O 1.4 mg/L, MnSO ₄ H ₂ O 1.3 mg/L and CoCl ₂ 6H ₂ O 3.7 mg/L.

Example 2: Confirmation of activity and stability according to temperature and pH change of extracellular enzyme solution

In the present invention, the activity and stability according to the temperature and pH change of the extracellular enzyme solution cultured through the optimized expression medium was confirmed.

(1) Confirmation of temperature effect on extracellular enzyme solution

In order to confirm the effect of temperature on the extracellular enzyme solution, a buffer solution (pH 5.5) containing 1.0 mg/ml of extracellular enzyme solution and 0.4 mg/mL ginsenoside Rb1 was reacted in the range of 40°C to 65°C for 10 minutes. did it In the case of the reaction solution, butanol and 1.0 mg/mL of digosine, an internal standard, are added together to terminate the reaction, the butanol layers are collected, the solvent is evaporated under vacuum, and methanol is added to prepare the measurement sample. prepared. In the case of the measurement sample, the absorbance was measured at 203 nm using HPLC (High performance liquid chromatography), and the degree of conversion was confirmed by a quantitative method according to the standard curve obtained by measuring each ginsenoside standard material.

On the other hand, after the extracellular enzyme solution was left in the range of 40°C to 65°C for 24 hours, the buffer solution (pH 5.5) containing the extracellular enzyme solution 1.0 mg/ml and 0.4 mg/mL ginsenoside Rb1 was added at 55°C. The reaction was carried out for 10 minutes. Then, the reaction was terminated and measured in the same manner as previously mentioned to confirm the stability (glycosidase activity) of the extracellular enzyme solution according to the temperature.

As a result, as shown in Figure 3 (a), the high temperature of the extracellular enzyme solution is confirmed to be 55 ~ 65 ℃ (in particular, 55 ℃), but when the stability of the extracellular enzyme solution exceeds 60 ℃, It is confirmed that there is a problem in that the stability of the extracellular enzyme solution is greatly reduced compared to the case of 60° C. or less. Therefore, it is confirmed that the optimum temperature is 55 to 60° C., which is a temperature that maintains the stability of the extracellular enzyme solution even after a long reaction while the activity of the extracellular enzyme solution is high.

(2) Confirmation of pH effect on extracellular enzyme solution

In order to confirm the effect of pH on the extracellular enzyme solution, a buffer solution containing 1.0 mg/ml and 0.4 mg/mL ginsenoside Rb1 of the extracellular enzyme solution was reacted at 55° C. in a pH range of 4.0 to 6.5 for 10 minutes. , the reaction was terminated, and the absorbance was measured at 203 nm using HPLC, and the activity (glycosidase activity) of the extracellular enzyme solution was confirmed.

On the other hand, after leaving the extracellular enzyme solution in a pH range of 4.0 to 6.5 for 24 hours, the pH was changed to 5.0 using a centricon, and 1.0 mg/ml and 0.4 mg/mL ginsenoside Rb1 was contained in the extracellular enzyme solution. The buffer solution (pH 5.0) was reacted at 55° C. for 10 minutes. Then, the reaction was terminated and the absorbance was measured at 203 nm using HPLC, and the stability (glycosidase activity) of the extracellular enzyme solution was confirmed.

As a result, as shown in Figure 3 (b), it is confirmed that all of the high pH of the extracellular enzyme solution activity and stability is 4.5 ~ 5.0. When the pH exceeded 5.0, the activity of the extracellular enzyme solution was greatly reduced, and the stability of the extracellular enzyme solution was also decreased in the long-time reaction.

Example 3: Preparation of ginsenoside compound K using extracellular enzyme solution

In the present invention, a ginsenoside compound K was prepared using an extracellular enzyme solution cultured through an optimized expression medium.

First, 2.5 mg/ml of the extracellular enzyme solution was used as a substrate, and a reagent containing 1.0 mg/ml of ginsenosides Rb1, Rb2 and Rc was reacted at 55° C. and pH 5.0 for 2 hours or 24 hours.

As a result, as shown in FIGS. 4(a) and (c), ginsenoside Rb1 or Rc was completely converted to ginsenoside C-K in 2 hours, and as shown in FIG. 4(b), ginsenoside Rb1 or Rc It was confirmed that side Rb2 was converted to ginsenoside C-K in a yield of about 94 mol% in 24 hours. Through this, it could be suggested that each protopanaxadiol-based saponin can be converted into a ginsenoside compound K along the conversion path as shown in FIG.

On the other hand, when 8.0 mg/ml of the extracellular enzyme solution was treated with a Korean ginseng root extract containing 6.0 mg/ml of protopanaxadiol-based saponin as a substrate, it was reacted at 55° C. and pH 5.0 for 18 hours.

As a result, as shown in FIG. 6(a), in the case of ginsenosides Rb1, Rb2, Rc and Rd, which are protophanaxadiol-based saponins that were present before the reaction, all were converted to intermediate substances at the beginning of the reaction, and after 6 hours The degree of conversion showed a decreasing pattern, and the reaction over 18 hours did not show any more conversion to the ginsenoside compound K. In particular, the conversion yield of the ginsenoside compound K produced as a result of the reaction for 9 hours was 80 mol%, the amount was 2.8 mg/mL (4.5 mM), and the productivity was 313.0 mg/L/h (502.4 μM/h) is confirmed to be

That is, as shown in Figure 6 (b), protopanaxadiol-based saponins ginsenoside Rb1, Rb2, Rc and Rd are converted into many ginsenoside compound K in high yield for a short reaction time by the extracellular enzyme solution was able to confirm

The foregoing description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (11)

(A) Aspergillus niger (Aspergillus niger) strain as a carbon source, cultured in an expression medium containing carboxymethyl cellulose at a concentration of 10 g / L to 15 g / L Aspergillus niger (Aspergillus niger) niger) obtaining an extracellular enzyme solution of the strain; and
(b) comprising the step of treating the obtained enzyme solution to a substrate,
In step (b), the substrate is one or more protopanaxadiol-based saponins selected from the group consisting of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc and ginsenoside Rd; or ginseng extract,
Method for producing ginsenoside compound K.
According to claim 1,
In the step (a) Aspergillus niger ( Aspergillus niger ) strain is Aspergillus niger ( Aspergillus niger ) strain (KACC 46495), the method of producing a ginsenoside compound K.
delete According to claim 1,
In the step (a), the expression medium is a nitrogen source, and a method for producing a ginsenoside compound K, including a corn steep solid.
According to claim 1,
The expression medium in step (a) is KH ₂ PO ₄ , MgSO ₄ ^. 7H ₂ O, CaCl ₂ , FeSO ₄ ^. 7H ₂ O, ZnSO ₄ ^. 7H ₂ O, MnSO ₄ ^. H ₂ O and CoCl ₂ ^. 6H ₂ O A method for producing a ginsenoside compound K, further comprising:
delete delete According to claim 1,
The concentration of protopanaxadiol-based saponin in the substrate in step (b) is 0.1 mg/ml to 10 mg/ml, a method for producing a ginsenoside compound K.
According to claim 1,
The method of producing a ginsenoside compound K, wherein the treatment in step (b) is carried out for 2 hours to 24 hours at pH 4.0-6.5 and 40-65°C conditions.
Aspergillus niger (Aspergillus niger) strain as a carbon source, Aspergillus niger (Aspergillus niger) strain cultured in an expression medium containing carboxymethyl cellulose (carboxymethyl cellulose) at a concentration of 10 g / L to 15 g / L of extracellular enzymes; and at least one protopanaxadiol-based saponin selected from the group consisting of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc and ginsenoside Rd; Or a composition for preparing a ginsenoside compound K comprising a ginseng extract.
11. The method of claim 10,
The expression medium is a nitrogen source, comprising a corn steep solid (corn steep solid), ginsenoside compound K composition for producing.

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