KR102440206B1 - Composition and method for producing ginsenoside compound k using enzyme liquid of aspergillus tubingensis - Google Patents

Abstract

The present invention relates to Aspergillus tubingensis ( Aspergillus tubingensis ) A composition for preparing a ginsenoside compound K containing an enzyme solution and a manufacturing method.

Description

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

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

Ginseng has been used as a traditional herbal medicine to strengthen immune function, provide nutrition, and reduce fatigue in many Asian countries since ancient times. Among them, ginsenoside of ginseng is the main component responsible for these various biological and medicinal chemical conversions, and it is known that 1-3% of total ginseng can be extracted. Ginsenoside compound K, which is produced by decomposition of sugars such as glucose and arabinose from ginsenosides Rb1, Rb2, Rc, and Rd, which are protophanaxadiol-based saponins present in large amounts in ginseng, lowers cholesterol and lowers blood pressure. , cytotoxicity and cancer suppression, memory increase, anti-stress, anti-aging, etc. have several activities. However, although ginsenoside compound K is known to be produced by intestinal bacteria, it is difficult to expect a pharmaceutical effect in those who have little or no decomposition ability. Therefore, in order to see this effect, the health food and cosmetic industry using ginsenoside compound K is developing a lot, and a method for mass production thereof is required.

To date, the technology for producing ginsenoside compound K includes methods using enzymes and methods using bacteria. The method using the enzyme showed high activity because it is a method for producing by reaction with an enzyme obtained by genetic manipulation, but it is not suitable for use in food. This is a required problem. Due to the nature of the conversion reaction using bacteria, there is a limitation in growth and a low production titer compared to the reaction using fungi. On the other hand, in the case of a method using a conventional fungal enzyme, to obtain such a fungal-derived enzyme is a method of direct isolation and purification from a strain, or a method of cloning an enzyme gene from a strain, expressing it in a recombinant expression vector, and purifying it. is commonly used, which is not suitable for industrialization because it can cause environmental pollution due to refining costs and by-products for industrial use.

Therefore, in order to solve the above problems, various patents have tried to solve the problems by using molds or enzymes isolated from molds that can be used in food.

In addition, when producing ginsenoside compound K using ginseng extract, one of the problems is that it hardly decomposes ginsenoside Rb2, which is one of the main ginsenosides. Ginsenoside Rb2 is one of the major ginsenosides present in ginseng, but it is difficult to decompose because it contains arabinose sugar, and few studies have been conducted to find an enzyme that decomposes it.

Therefore, in order to solve these problems, it is necessary to screen a fungus that produces an enzyme that effectively decomposes ginsenoside Rb2, and to increase the activity of the enzyme in a method suitable for use in food for industrial use.

The present invention is to provide a composition for producing a ginsenoside compound K including Aspergillus tubingensis ( Aspergillus tubingensis ) strain enzyme solution.

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 provides a composition for preparing a ginsenoside compound K, including an Aspergillus tubingensis strain enzyme solution.

The Aspergillus tubingensis ( Aspergillus tubingensis ) strain may be deposited as KCTC 14166BP.

The enzyme solution may be an extracellular enzyme solution of the Aspergillus tubingensis strain.

The enzyme solution is the Aspergillus tubingensis ( Aspergillus tubingensis ) strain as a carbon source, citrus pectin (citrus pectin) or sugar beet sludge (sugar beet sludge) in an expression medium comprising a concentration of 5 g / L to 25 g / L It may be obtained by culturing.

The concentration of the enzyme solution in the composition may be 1 mg/ml to 15 mg/ml.

The composition comprises, as a substrate, 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 the protopanaxadiol-based saponin in the composition may be 1 mg/ml to 20 mg/ml.

The composition may further include one or more mineral additives selected from the group consisting of magnesium sulfate (MgSO ₄ ), manganese chloride (MnCl ₂ ), iron sulfate (FeSO ₄ ), and iron chloride (FeCl ₃ ).

In one embodiment of the present invention, Aspergillus tubingensis ( Aspergillus tubingensis ) It provides a method for producing a ginsenoside compound K comprising the step of treating a strain enzyme solution to a substrate.

The treatment may be performed for 2 hours to 24 hours at 40-65° C. and pH 3.5-6.0 conditions.

In another embodiment of the present invention, aspergillus tubingensis ( Aspergillus tubingensis ) strain (KCTC 14166BP) produced by a ginsenoside compound having a glucose or arabinose hydrolysis activity in protopanaxadiol-based saponin ) is provided.

According to the present invention, Aspergillus tubingensis ( Aspergillus tubingensis ) It is characterized by using a strain enzyme solution, a ginsenoside compound through protopanaxadiol-based saponin glucose (glucose) or arabinose (arabinose) hydrolysis K can be produced with high productivity and yield. In particular, the Aspergillus tubingensis ( Aspergillus tubingensis ) As a carbon source in the expression medium for culturing the strain, citrus pectin or sugar beet sludge is included at an optimal concentration, and the substrate at the optimum temperature and pH conditions By processing to, it is possible to further increase the productivity and yield of the ginsenoside compound K.

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

1 is a new Aspergillus tubingensis ( Aspergillus tubingensis ) It is a figure showing a phylogenetic diagram of the KU-509 strain (KCTC 14166BP).
Figure 2 (a) is a graph showing the glycosidase activity when the extracellular enzyme solution is cultured by varying the carbon source of the expression medium and then treated with ginsenoside Rb2 as a substrate, respectively, and Figure 2 (b) is This is a graph showing the glycosidase activity when the extracellular enzyme solution is cultured with various carbon source concentrations in the expression medium and then treated with ginsenoside Rb2 as a substrate.
Figure 3 (a) is a graph showing the glycosidase activity and stability of the extracellular enzyme solution according to the change in temperature of the extracellular enzyme solution cultured through the optimized expression medium, Figure 3 (b) is through the optimized expression medium It is a graph showing the glycosidase activity and stability of the extracellular enzyme solution according to the change in the pH of the cultured extracellular enzyme solution.
Figure 4 (a) is a graph showing the glycosidase activity when the extracellular enzyme solution is cultured through an optimized expression medium, then treated with ginsenoside Rb2 as a substrate, and various mineral additives are added, respectively. 4(b) is a graph showing the glycosidase activity when the extracellular enzyme solution is cultured through an optimized expression medium, then treated with ginsenoside Rb2 as a substrate, and added with varying concentrations of MgSO ₄ . to be.
Figure 5 (a) is a graph showing the productivity of the reaction using an extracellular enzyme solution cultured through an optimized expression medium, but varying the concentration of protopanaxadiol-based saponin, and Figure 5 (b) is When reacted by varying the concentration of the extracellular enzyme solution cultured through the optimized expression medium, it is a graph showing the productivity, FIG. 3 (c) is the extracellular enzyme solution and protopanaxadi cultured through the optimized expression medium It is a graph showing the productivity and the conversion yield when the total concentration of the ol-based saponin is varied and reacted.
Figure 6 (a) is a graph showing the degree of conversion by time when the extracellular enzyme solution is cultured through an optimized expression medium and then reacted according to the optimized conditions, and Figure 6 (b) is a high-performance liquid before and after the reaction The figure shows the results of high performance liquid chromatography (HPLC) measurements.

The present inventors were studying a method for producing a ginsenoside compound K using an Aspergillus tubingensis enzyme solution, and by culturing the Aspergillus tubingensis strain through an expression medium optimized for a carbon source, After obtaining the extracellular enzyme solution, by reacting under optimal conditions (temperature, pH, addition at the optimum concentration of mineral additives, the concentration of the extracellular enzyme solution and the concentration of propophanaxadiol-based saponin), the productivity and It was confirmed that the yield could be further increased, and 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]

Composition for preparing ginsenoside compound K

The present invention provides a composition for preparing a ginsenoside compound K, including an Aspergillus tubingensis strain enzyme solution.

The Aspergillus tubingensis ( Aspergillus tubingensis ) strain is a safe fungus that can be used in food, and belongs to the genus Ascomycetes yeast mold. The Aspergillus tubingensis ( Aspergillus tubingensis ) strain is preferably an Aspergillus tubingensis ( Aspergillus tubingensis ) strain (KCTC 14166BP), but is not limited thereto.

Specifically, the strain (KCTC 14166BP) has a hydrolytic activity of glucose or arabinose in the protopanaxadiol-based saponin among Aspergillus tubingensis , and in particular, the protopa It can easily hydrolyze glucose in naxadiol-based saponin, and when the protopanaxadiol-based saponin is ginsenoside Rb2, it can easily hydrolyze arabinose present here. . The phylogenetic diagram of the strain (KCTC 14166BP) is as shown in FIG. 1 , and may be isolated from Meju. The strain (KCTC 14166BP) was deposited with the Korea Institute of Biotechnology and Biotechnology Biological Resources Center (KCTC: Korean Collection for Type Cultures) on April 7, 2020 and was given an accession number KCTC 14166BP.

In order to obtain a culture solution from the Aspergillus tubingensis 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 tubingensis ( Aspergillus tubingensis ) strain enzyme solution can be obtained by culturing the Aspergillus tubingensis strain through an expression medium, and the present invention is characterized in that the expression medium is optimized .

The expression medium may contain citrus pectin or sugar beet sludge as a carbon source, and preferably contains citrus pectin, and as a substrate, glucose in protopanaxadiol-based saponins. Through optimal hydrolysis of (glucose) or arabinose, ginsenoside compound K can be finally prepared with high productivity and yield.

As a carbon source in the expression medium, the concentration of cellulose may be 5 g/L to 25 g/L, preferably 5 g/L to 20 g/L, and 15 g/L to 20 g/L It is more preferable, but is not limited thereto.

On the other hand, as a carbon source, cellulose, carboxymethyl cellulose, starch, wheat bran, ginseng root extract, grapefruit pectin and apple pectin ), there is a limit in not effectively converting ginsenoside compound K through optimal hydrolysis of glucose or arabinose in protopanaxadiol-based saponin.

In addition, the expression medium preferably includes corn steep solid as a nitrogen source, but is not limited thereto. Likewise, through optimal hydrolysis of glucose or arabinose in protopanaxadiol-based saponin, ginsenoside compound K can be finally prepared with high productivity and yield.

The concentration of the nitrogen source (in particular, 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 limited thereto doesn't happen

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.

Meanwhile, the Aspergillus tubingensis ( Aspergillus tubingensis ) enzyme solution may be an extracellular enzyme solution of the Aspergillus tubingensis ( Aspergillus tubingensis ) strain, as described above.

The concentration of the protopanaxadiol-based saponin in the composition may be 1 mg/ml to 20 mg/ml, preferably 2 mg/ml to 20 mg/ml, and 7 mg/ml to 20 mg/ml is more preferable, but is not limited thereto.

On the other hand, the composition is for treating a substrate, wherein the substrate contains one or more protopanaxadiol-based saponins selected from the group consisting of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc and ginsenoside Rd. It is preferred, but not limited thereto.

In addition, the substrate may be a ginseng extract, and the ginseng extract may be a solvent extraction 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 composition may be 1 mg/ml to 20 mg/ml, preferably 8 mg/ml to 20 mg/ml, but is not limited thereto.

Meanwhile, the composition may further include one or more mineral additives selected from the group consisting of magnesium sulfate (MgSO ₄ ), manganese chloride (MnCl ₂ ), iron sulfate (FeSO ₄ ) and iron chloride (FeCl ₃ ), magnesium sulfate (MgSO ₄ ) It is preferable to further include, but is not limited thereto. The magnesium sulfate (MgSO ₄ ) is an additive widely used in food, such as when coagulating tofu, and is one of essential minerals for humans.

The concentration of the mineral additive in the composition may be 0.01 mM to 1.0 mM, preferably 0.01 mM to 0.1 mM, but is not limited thereto.

Manufacturing method of ginsenoside compound K

The present invention provides a method for producing a ginsenoside compound K, comprising the step of treating the Aspergillus tubingensis strain enzyme solution to a substrate.

Since the Aspergillus tubingensis ( Aspergillus tubingensis ) strain enzyme solution has been described above, a redundant description will be omitted.

Meanwhile, one or more mineral additives selected from the group consisting of magnesium sulfate (MgSO ₄ ), manganese chloride (MnCl ₂ ), iron sulfate (FeSO ₄ ) and iron chloride (FeCl ₃ ) may be added to the enzyme solution, and magnesium sulfate (MgSO ₄ ) is preferably added, but is not limited thereto. The magnesium sulfate (MgSO ₄ ) is an additive widely used in food, such as when coagulating tofu, and is one of essential minerals for humans.

In addition, 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 ginseng extract, and the ginseng extract may be a solvent extraction 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.

In addition, the treatment may be carried out for 2 hours to 24 hours at 40 ~ 65 ℃ and pH 3.5 ~ 6.0 conditions. Specifically, the temperature condition for the treatment is preferably 50 to 60 ℃, but is not limited thereto. At this time, if the temperature is less than 50 ℃, the glycosidase activity of the extracellular enzyme solution may be greatly reduced, and if the temperature is more than 60 ℃, the stability of the extracellular enzyme solution may be significantly reduced. In addition, the pH conditions for the treatment are preferably pH 4.0 to 5.0, but is not limited thereto. At this time, when the pH is less than 4.0 or the pH is more than 5.0, both the glycosidase activity of the extracellular enzyme solution and 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, by maintaining these pH and temperature conditions, the glycosidase 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 prepare ginsenoside compound K with high productivity and yield through optimal hydrolysis of glucose or arabinose in protopanaxadiol-based saponin.

In addition, the present invention provides a ginsenoside compound K producing Aspergillus tubingensis strain (KCTC 14166BP) having glucose or arabinose hydrolysis activity in protopanaxadiol-based saponin do.

The strain (KCTC 14166BP) has glucose or arabinose hydrolysis activity in protopanaxadiol-based saponins, and in particular, easily hydrolyzes glucose in the protopanaxadiol-based saponins. In the case where the protopanaxadiol-based saponin is ginsenoside Rb2, arabinose present therein can be easily hydrolyzed. Since other features have been described above, redundant description will be omitted.

As described above, according to the present invention, Aspergillus tubingensis ( Aspergillus tubingensis ) It is characterized in that the strain enzyme solution is used, glucose (glucose) or arabinose (arabinose) hydrolysis in protopanaxadiol-based saponins Through this, ginsenoside compound K can be manufactured with high productivity and yield. In particular, the Aspergillus tubingensis ( Aspergillus tubingensis ) As a carbon source in the expression medium for culturing the strain, citrus pectin or sugar beet sludge is included at an optimal concentration, and the substrate at the optimum temperature and pH conditions By processing to, it is possible to further increase the productivity and yield of the ginsenoside compound K.

In particular, Aspergillus tubingensis ( Aspergillus tubingensis ) The strain is a safe mold available for food, the ginsenoside compound K prepared in the same way as above has the advantage that it 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: Aspergillus tubing ensis ( Aspergillus tubingensis ) KU-509 strain isolation and identification

To isolate new strains, meju was used as a sample. Meju was spread on a potato dextrose agar medium (PDA) using a 10-fold dilution method, streaked several times, and cultured, followed by observing the colony form to isolate a new strain. For the identification of the isolated new strain, genomic DNA was obtained, and the β-tubulin gene was amplified and sequenced through polymerase chain reaction (PCR) with the following PCR primers:

Forward: 5'-GGT AAC CAA ATC GGT GCT GCT TTC-3'

Reverse: 5'-ACC CTC AGT GTA GTG ACC CTT GGC-3'

The nucleotide sequence of the β-tubulin gene obtained by sequencing is shown in SEQ ID NO: 1, and a systematic diagram thereof is shown in FIG. 1 . At this time, Aspergillus tubingensis ( Aspergillus tubingensis ) KU-509 strain was deposited at the Korea Research Institute of Biotechnology and Biotechnology Biological Resources Center (KCTC: Korean Collection for Type Cultures) on April 7, 2020 and was given an accession number KCTC 14166BP.

Example 2: Confirmation of glycosidase activity of the extracellular enzyme solution according to the carbon source and its concentration in the expression medium

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

Specifically, the amount of the enzyme protein was measured using the Bradford method based on bovine serum albumin, and the glycolytic protein was measured using a para-nitrophenol enzyme assay method. The sidase activity was measured. Specifically, in the para-nitrophenol enzyme analysis method, para-nitrophenyl glucose ( p NP-glu) 1 mM was used as a substrate to react with the enzyme, and then Na ₂ CO ₃ was added to terminate the reaction. Glycosidase activity was confirmed by measuring the absorbance value at absorbance at 450 nm to see how much para-nitrolphenol (which causes a yellow color reaction under basic conditions) was produced.

및 150 rpm으로 7일간 배양하였다. First, Aspergillus tubingensis ( Aspergillus tubingensis ) strain (KCTC 14166BP) isolated and identified in Example 1 was plate-cultured at 26 ° C. for 7 days in Potato dextrose agar medium (PDA). The spores in the plate cultured plate were collected by filtering with gauze, and the spore solution diluted to a spore concentration of 2.0 X 10 ⁶ spores/mL in 4 ml of liquid potato dextrose medium (PDB) was added to 1 ㎖ inoculated, and grown and cultured for 24 hours at 26 ℃ and 150 rpm. After washing the mycelium in the growth medium made from the growth culture with an expression medium excluding carbon and nitrogen sources, and transferring only the mycelium to 100 ml of expression medium, 26

and 150 rpm for 7 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, cellulose (Daejeong Chemical Co., Ltd.), carboxymethyl cellulose (Sigma Aldrich), starch (Biosang Co., Ltd.), sugar beet sludge (Holy Medcam), Wheat bran (Sims Pet), ginseng root extract (Ace Gem), citrus pectin (Daejeong Hwageum Co., Ltd.), grapefruit pectin (Source Naturals) and apple pectin A total of six were used as (apple pectin) (Naturals Plus), and as a nitrogen source, 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 80% saturation, and the mixture was mixed at 4° C. for 24 hours to obtain an enzyme precipitate solution. To remove ammonium sulfate in the enzyme precipitation solution, dialysis was performed at 4° C. for 24 hours with 200 mM Mcilvaine buffer using a cellulose dialysis membrane, and the remaining ammonium sulfate was further removed and concentrated using Centricon. An extracellular enzyme solution was obtained.

Then, a buffer solution containing 0.5 mg/ml of extracellular enzyme solution and 0.4 mg/ml of ginsenoside Rb2 (Ambo Institute) as a substrate was reacted at 55° C. and pH 4.0 for 10 minutes, and glycosidase of the extracellular enzyme solution Activity was measured.

As a result, as shown in Figure 2 (a), as a carbon source in the expression medium, when using citrus pectin or sugar beet sludge (in particular, citrus pectin), extracellular enzyme solution It is confirmed that the glycosidase activity of However, as a carbon source, cellulose, carboxymethyl cellulose, starch, wheat bran, ginseng root extract, grapefruit pectin and apple pectin ), it is confirmed that the glycosidase activity of the extracellular enzyme solution is relatively significantly decreased.

In addition, as shown in Figure 2(b), as a carbon source in the expression medium, when citrus pectin is 5 g/L to 20 g/L (in particular, 15 g/L to 20 g/L), It is confirmed that the glycosidase activity of the extracellular enzyme solution is greatly improved.

Therefore, the composition of the optimized expression medium as a carbon source, citrus pectin (citrus pectin) 20 g / L, corn steep solid (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 is 3.7 mg/L.

Example 3: Confirmation of glycosidase activity and stability of extracellular enzyme solution according to temperature and pH change of extracellular enzyme solution

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

(1) Confirmation of temperature effect on extracellular enzyme solution

In order to confirm the temperature effect on the extracellular enzyme solution, a buffer solution (pH 4.0) containing 0.5 mg/ml of extracellular enzyme solution and 0.4 mg/ml of ginsenoside Rb2 (Ambo Institute) as a substrate was prepared at 40°C to 65°C. The glycosidase activity of the extracellular enzyme solution was confirmed by reacting in the range for 10 minutes.

In addition, 0.5 mg/ml of the extracellular enzyme solution was left in the range of 40°C to 65°C for 12 hours, and a buffer solution (pH 4.0) containing 0.4 mg/mL of ginsenoside Rb2 (Ambo Institute) as a substrate was added thereto. The stability of the extracellular enzyme solution was confirmed by reacting for 10 minutes at each temperature.

As a result, as shown in FIG. 3(a), it was confirmed that the temperature at which the glycosidase activity of the extracellular enzyme solution is high is 50-60°C (in particular, 55°C), but the stability of the extracellular enzyme solution at a temperature of 60°C or higher It is confirmed that there is a problem that this is greatly reduced. Therefore, it is confirmed that the optimum temperature is 50 to 55° C., which is a temperature that maintains the stability of the extracellular enzyme solution even after a long reaction while having high glycosidase activity of the extracellular enzyme solution.

(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 (55° C.) containing 0.5 mg/ml of extracellular enzyme solution and 0.4 mg/ml of ginsenoside Rb2 (Ambo Institute) as a substrate was used in the pH range of 3.5 to 6.5. was reacted for 10 minutes to confirm the glycosidase activity of the extracellular enzyme solution.

In addition, after 0.5 mg/ml of the extracellular enzyme solution was left at a pH of 3.5 to 6.5 for 12 hours, a buffer solution (55° C.) containing 0.4 mg/ml of ginsenoside Rb2 (Ambo Institute) as a substrate was added thereto. By reacting for 10 minutes, the stability 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 glycosidase activity and stability of the extracellular enzyme solution is 4.0 to 5.0 (in particular, 4.0 to 4.5). When the pH exceeded 5.0, the glycosidase 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 4: Confirmation of glycosidase activity of extracellular enzyme solution according to the addition of mineral additives and the concentration thereof

In the present invention, the glycosidase activity of the extracellular enzyme solution was confirmed according to the addition and concentration of the mineral additive in the extracellular enzyme solution cultured through the optimized expression medium.

및 pH 4.0에서 10분 동안 반응시켜 세포 외 효소액의 글리코시다아제 활성을 확인하였다. First, in a buffer containing 0.5 mg/ml of extracellular enzyme solution and 0.4 mg/ml of ginsenoside Rb2 (Ambo Institute) as a substrate, 9 mineral additives (CaCl ₂ , CoCl ₂ , KCl, MgSO ₄ , MnCl ₂ , NaCl) , FeSO ₄ , FeCl ₂ and FeCl ₃ ) 1.0 mM each was added and 55

And by reacting at pH 4.0 for 10 minutes, the glycosidase activity of the extracellular enzyme solution was confirmed.

As a result, as shown in FIG. 4(a), as a mineral additive, MgSO ₄ , MnCl ₂ , FeSO ₄ and FeCl ₃ (particularly, MgSO ₄ ) When added, the glycosidase activity of the extracellular enzyme solution is high (When MgSO ₄ is added, 1.12-fold improvement). However, as a mineral additive, when CaCl ₂ , CoCl ₂ , KCl, NaCl and FeCl ₂ are added, it is confirmed that the glycosidase activity of the extracellular enzyme solution is hardly changed or rather decreased.

및 pH 4.0에서 10분 동안 반응시켜 세포 외 효소액의 글리코시다아제 활성을 확인하였다. In addition, in a buffer containing 0.5 mg/ml of extracellular enzyme solution and 0.4 mg/ml of ginsenoside Rb2 (Ambo Institute) as a substrate, 0.01~1.0 mM of MgSO ₄ was added, respectively, 55

And by reacting at pH 4.0 for 10 minutes, the glycosidase activity of the extracellular enzyme solution was confirmed.

As a result, as shown in FIG. 4(b), when 0.01 to 0.1 mM (particularly, 0.05 mM) of MgSO ₄ was added, the glycosidase activity of the extracellular enzyme solution was high (MgSO ₄ when 0.05 mM was added, 1.40) double improvement).

Example 5: Preparation of ginsenoside compound K using extracellular enzyme solution according to the concentration of protopanaxadiol-based saponin and extracellular enzyme solution in the composition

In the present invention, but using an extracellular enzyme solution cultured through an optimized expression medium, according to the concentration of protopanaxadiol-based saponin and extracellular enzyme solution in the composition, a ginsenoside compound K was prepared.

First, a buffer solution containing 7 mg/ml of extracellular enzyme solution and 1 to 11 mg/ml of protopanaxadiol-based saponin as a substrate (ginseng root extract) was reacted at 55° C. and pH 4.0 for 5 hours.

As a result, as shown in Figure 5 (a), when the concentration of the protopanaxadiol-based saponin in the composition is 8 to 11 mg/ml, it is confirmed that the productivity of the ginsenoside compound K can be increased.

및 pH 4.0에서 5시간 동안 반응시켰다.In addition, a buffer solution containing 9 mg/ml of protopanaxadiol-based saponin was added to 55 as an extracellular enzyme solution 1 to 10 mg/ml and a substrate (ginseng root extract).

and pH 4.0 for 5 hours.

As a result, as shown in Figure 5 (b), when the concentration of the extracellular enzyme solution in the composition is 2 ~ 10 mg / ml (particularly, 7 ~ 10 mg / ml), ginsenoside compound K productivity can be increased that is confirmed

및 pH 4.0에서 5시간 동안 반응시켰다.From the above results, it was confirmed that the optimal concentrations of protopanaxadiol-based saponin and extracellular enzyme solution in the composition were 9 mg/ml and 7 mg/ml, respectively, the optimal concentration of protopanaxadiol-based saponin and extracellular enzyme solution in the composition The concentration ratio was set to 1.29:1, but the total concentration was increased. That is, a buffer solution containing 1.29 to 19.35 mg/ml of protopanaxadiol-based saponin was added to 55 as an extracellular enzyme solution at 1 to 15 mg/ml and a substrate (ginseng root extract).

and pH 4.0 for 5 hours.

As a result, as shown in FIG. 5(c), when the optimal concentrations of protopanaxadiol-based saponin and extracellular enzyme solution in the composition were 9 mg/ml and 7 mg/ml, respectively, ginsenoside compound K 2.0 mg/ ml, and the conversion yield is found to be 55%. On the other hand, when the optimal concentrations of protopanaxadiol-based saponin and extracellular enzyme solution in the composition are 14.19 mg/ml and 11 mg/ml, respectively, ginsenoside compound K 3.9 mg/ml is produced, and the conversion yield is 47% is confirmed to be That is, as the total concentration of protopanaxadiol-based saponin and extracellular enzyme solution in the composition was increased, productivity was increased 1.35 times, but the conversion yield was reduced by 0.85 times.

Example 6: Preparation of ginsenoside compound K using an extracellular enzyme solution according to optimized conditions

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

11 mg/ml of extracellular enzyme solution and as a substrate (ginseng root extract), 0.05 mM MgSO ₄ was added to a buffer containing 14.19 mg/ml of protopanaxadiol-based saponin and reacted at 55° C. and pH 4.0 for 24 hours. .

As a result, as shown in FIGS. 6(a) and (b), the protophanaxadiol-based saponins (ginsenosides Rb1, Rb2, Rc and Rd) in the ginseng root extract were ginsenoside compound K after 20 hours of reaction. It was confirmed that it was completely converted to 8.34 mg/ml.

For reference, Korean Patent Application Laid-Open No. 10-2011-0113228 discloses the production of compound K using Aspergillus niger , and the enzyme obtained when using a mixture of bran and lactose as a carbon source and protopanaxadi As a result of reacting all powder, the maximum conversion to compound K was only 55%. In addition, in Korea Patent Publication No. 10-2018-0040784, the content of ginsenoside in red ginseng by liquid and solid fermentation is confirmed using Aspergillus sp. strains, and at this time, the content of ginsenoside Rb2 is changed It was confirmed that it was not.

Therefore, when producing a ginsenoside compound K using a fungus of the genus Aspergillus, there was no strain that reached 100% conversion, and the strain that converts the ginsenoside Rb2 is Aspergillus tubingensis ( Aspergillus tubingensis ) ) strain is identified as unique.

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.

Korea Institute of Bioscience and Biotechnology KCTC14166BP 20200407

<110> Konkuk University Industrial Cooperation Corp <120> COMPOSITION AND METHOD FOR PRODUCING GINSENOSIDE COMPOUND K USING ENZYME LIQUID OF ASPERGILLUS TUBINGENSIS <130> 2020-I098 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 463 <212> DNA <213> Aspergillus tubingensis <400> 1 cttagcttga gttcagatgt tatccatcgg ggatatagct acggttaaga acacgtctaa 60 caactcaaca ggcagaccat ctctggcgag cacggccttg acggctccgg tgtgtaagta 120 caactttttc acacctctca attggtcaac aatgtggaaa ggattgggtt tcctgacgcg 180 caggatagtt acaatggcac ctccgacctc cagctggagc gcatgaacgt ctacttcaac 240 gaggttagat cacaccgtcc ctgagttttt tcacgacaat atcatcaatg tcctgaccac 300 ttcagcaggc tagcggtaac aagtatgtcc cccgtgccgt cctcgtcgat ctcgagcccg 360 gtaccatgga cgccgtccgt gccggtccct tcggccagct cttccgcccc gacaacttcg 420 tcttcggcca gtccggtgct ggtaacaact gggccaaggg tca 463

Claims (11)

Aspergillus tubingensis deposited as KCTC 14166BP A composition for producing a ginsenoside compound K containing an enzyme solution of the strain Aspergillus tubingensis .
delete According to claim 1,
The enzyme solution is the extracellular enzyme solution of the Aspergillus tubingensis strain, ginsenoside compound K production composition.
According to claim 1,
The enzyme solution is the Aspergillus tubingensis ( Aspergillus tubingensis ) strain as a carbon source, citrus pectin (citrus pectin) or sugar beet sludge (sugar beet sludge) in an expression medium comprising a concentration of 5 g / L to 25 g / L A composition for preparing a ginsenoside compound K obtained by culturing.
According to claim 1,
The concentration of the enzyme solution in the composition is 1 mg / ml to 15 mg / ml, ginsenoside compound K composition for preparing.
According to claim 1,
The composition comprises, as a substrate, one or more protopanaxadiol-based saponins selected from the group consisting of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc and ginsenoside Rd; Or, a composition for preparing a ginsenoside compound K, including a ginseng extract.
7. The method of claim 6,
The concentration of the protopanaxadiol-based saponin in the composition is 1 mg/ml to 20 mg/ml, a composition for preparing a ginsenoside compound K.
According to claim 1,
The composition further comprises one or more mineral additives selected from the group consisting of magnesium sulfate (MgSO ₄ ), manganese chloride (MnCl ₂ ), iron sulfate (FeSO ₄ ) and iron chloride (FeCl ₃ ) For producing ginsenoside compound K composition.
Aspergillus tubingensis deposited as KCTC 14166BP ( Aspergillus tubingensis ) A method of producing a ginsenoside compound K, comprising the step of treating a strain enzyme solution to a substrate.
10. The method of claim 9,
The method for producing a ginsenoside compound K, wherein the treatment is carried out for 2 hours to 24 hours at 40 ~ 65 ℃ and pH 3.5 ~ 6.0 conditions.
Protopanaxadiol-based saponin in glucose (glucose) or arabinose (arabinose) ginsenoside compound K production having a hydrolytic activity Aspergillus tubingensis ( Aspergillus tubingensis ) strain (KCTC 14166BP).

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