KR102258788B1 - Novel Aspergillus niger C2-2 isolated from Nu-ruk producing ginsenoside compound K biotransformation enzyme and use thereof - Google Patents

Abstract

The present invention relates to a novel yeast Aspergillus niger C2-2 strain and its use, specifically, the Aspergillus niger C2-2 ( Aspergillus niger C2-2) strain deposited with accession number KACC 93333P, of the strain It relates to a culture, an enzyme composition obtained by purifying the culture, a method for increasing the compound K content of ginseng or processed ginseng using the same, and a composition for the method.
By using the strain, culture or enzyme composition of the present invention, it is possible to efficiently convert major ginsenosides of ginseng PPD type into compound K. Therefore, by using them, it is possible to manufacture a ginseng product containing a high concentration of compound K, which is more easily absorbed into the body and has excellent physiological activity.

Description

Novel Aspergillus niger C2-2 isolated from Nu-ruk producing ginsenoside compound K biotransformation enzyme and use thereof

The present invention relates to a novel yeast Aspergillus niger C2-2 strain producing a ginsenoside compound K-converting enzyme and its use, specifically, Aspergillus niger C2-2 deposited with accession number KACC 93333P ( Aspergillus niger C2-2 ( Aspergillus niger C2-2) niger C2-2) strain, a culture of the strain, an enzyme composition obtained by purification from the culture, a method for increasing the compound K content of ginseng or ginseng processed using them, and a composition for the method will be.

Saponin refers to a substance composed of several cyclic compounds in the glycosides that are widely present in the plant kingdom, which are not sugars. Since triterpene saponin, a saponin component included as a major physiologically active ingredient in ginseng or red ginseng, has a different chemical structure from saponins found in other plants, in order to distinguish these ginseng saponins from other plant-based saponins, ginseng ) is called ginsenoside, meaning glycoside.

Ginsenosides are protopanaxadiol-type (PPD type) ginsenosides, protopanaxatriol-type (PPT type) ginsenosides and oleanoric acid based on the structure of aglycone (oleanolic acid type) It can be classified into three types of ginsenosides. These three groups are, again, the positions of sugar moieties (aglycones, sugar moieties, aglycones) attached to carbon 3, carbon 6 and carbon 20 positions of the ring by a glycosidic bond in the compound structure and classified according to number. The basic skeleton of oleanolic acid-based ginsenosides is pentacyclic, and there is the only ginsenoside Ro, and its aglycone is oleanolic acid. Currently, more than 40 types of ginsenosides have been isolated, and most of them are PPD-type ginsenosides. PPD-type ginsenosides include Rb1, Rb2, Rb3, Rc, Rd, gypenoside XVII, compound O, compound Mc1, F2, compound Y, These include compound Mc, Rg3, Rh2, and compound K (CK). PPT-type ginsenosides include Re, Rg1, Rf, Rg2, Rh1, and the like.

In addition, major ginsenosides account for more than 90% of ginsenosides in dried ginseng, but their absorption in the body is very low due to their large size of around 1,000 daltons. Therefore, in order to increase the medicinal efficacy of ginsenosides, a process of converting major ginsenosides into minor ginsenosides with relatively good absorption and better medicinal efficacy is required. That is, major ginsenosides require a conversion process in which glucose, arabinose, rhamnose, xylose, etc. constituting sugars are removed (deglycosylated) in order to effectively exhibit physiological activity in vivo. Major ginsenosides include Rg1, Re, Rb1, Rb2, and Rc, and minor ginsenosides (rare ginsenosides) that are soluble in the living body present in trace amounts include Rd, F2, Rg3, Rh1, Rh2, and gypheno Side XVII, Zyphenoside LXXV and Compound K, Compound MC, Compound MC One, and the like are included.

Among the minor ginsenosides, compound K is a saponin contained only in ginseng, which is easily absorbed into the body and has strong pharmacological effects. It also has an anti-inflammatory function, which is very important for treatment, and additionally inhibits the formation of atheroma to prevent arteriosclerosis, and it is also excellent in improving immunity and recovering from fatigue due to liver function recovery. It has even been reported in many academic societies that it has anticancer effects. Therefore, compound K is applied to pharmaceuticals or health functional foods, and its development potential is limitless in the future. However, all the enzymes currently used by Korean compound K producers depend on imports, and there is a limitation in that they are not even edible.

Accordingly, the present inventors have researched and tried to develop a method that can easily convert ginseng PPD-type major ginsenosides (Rb1, Rb2, Rb3, Rc) into compound K, in particular, a method that is safe for the human body and can be used in food. As a result, it was confirmed that PPD-type ginsenoside derived from ginseng can be efficiently converted into compound K using the newly discovered strain of the present invention classified as a microorganism that is safe for the human body and can be used in food, and the present invention has been completed

Republic of Korea Patent No. 10-1718465

Accordingly, the main object of the present invention is to provide a novel strain that can be used to convert major ginsenosides of ginseng PPD type into compound K.

Another object of the present invention is to provide a method capable of increasing the content of compound K compared to the original ginseng or ginseng processed product using the strain.

According to one aspect of the present invention, the present invention provides an Aspergillus niger C2-2 (Aspergillus niger C2-2) strain deposited with accession number KACC 93333P.

According to another aspect of the present invention, the present invention provides a culture of the strain.

According to another aspect of the present invention, the present invention provides an enzyme composition obtained by purification from the culture of the strain.

According to another aspect of the present invention, the present invention is the strain; a culture of the strain; Or an enzyme composition obtained by purification from the culture of the strain; provides a method of increasing the compound K (compound K) content of ginseng or processed ginseng, characterized in that by adding the fermentation.

According to another aspect of the present invention, the present invention is the strain; a culture of the strain; Or an enzyme composition obtained by purification from the culture of the strain; provides a composition for increasing the compound K (compound K) content of ginseng or processed ginseng containing as an active ingredient.

By using the strain, culture or enzyme composition of the present invention, it is possible to efficiently convert major ginsenosides of ginseng PPD type into compound K. Therefore, by using them, it is possible to manufacture a ginseng product containing a high concentration of compound K, which is more easily absorbed into the body and has excellent physiological activity.

1 shows the results of investigating the homology of the 28s rRNA sequence of the strain of the present invention based on the GenBank database.
Figure 2 is a block diagram showing the process of obtaining an enzyme composition according to an embodiment of the present invention, enzymatic reaction and compound K content confirmation.
Figure 3 shows the high performance liquid chromatography analysis results of the representative ginsenoside standard product mixture. Retention time: 27.278(Rg1 or Re), 35.775(Rb1), 36.010(Rc), 36.290(Rb2 or Rb3), 36.819(Rd), 37.187(Gyp 17), 37.577(C-Mc1), 37.862(CO), 38.897(F2), 41.748(C-Mc), 42.268(CY), 45.916(Rg5 or compound-K), 48.212(Rh2).
4 shows the results of high performance liquid chromatography analysis of the PPD extract of ginseng ginseng according to an embodiment of the present invention.
5 shows the results of thin-layer chromatography analysis for each reaction time of the crude enzyme solution and the PPD extract of Hwagi ginseng according to an embodiment of the present invention. lane 1: representative ginsenoside standard mixture (PPD standard), lane 2: ginseng PPD extract, lane 3: crude enzyme solution and ginseng ginseng PPD extract (10,000ppm) reaction solution, lane 4: crude enzyme solution and ginseng ginseng PPD extract (15,000ppm) ) reaction solution.
6 shows the results of high-performance liquid chromatography analysis of the crude enzyme solution and the PPD extract reaction solution of ginseng ginseng (10,000ppm, 60 hours reaction) according to an embodiment of the present invention.
7 shows the results of high-performance liquid chromatography analysis of the crude enzyme solution and the PPD extract reaction solution of ginseng ginseng (10,000ppm, 84 hours reaction) according to an embodiment of the present invention.
8 shows the results of high-performance liquid chromatography analysis of the crude enzyme solution and the PPD extract reaction solution of ginseng ginseng (15,000 ppm, 60 hours reaction) according to an embodiment of the present invention.
9 shows the results of high-performance liquid chromatography analysis of the crude enzyme solution and the PPD extract reaction solution of ginseng ginseng (15,000 ppm, 84 hours reaction) according to an embodiment of the present invention.
Figure 10 shows the results of a comparative experiment of the compound K increase effect of the strain of the present invention and other strains. lane 1: representative ginsenoside standard product mixture (PPD standard), lane 2: ginseng PPD extract, lane 3: crude enzyme solution of the present strain and ginseng PPD extract (15,000 ppm) reaction solution, lane 4: preparation of the present strain Enzyme solution and ginseng PPD extract (10,000ppm) reaction solution, lane 5: Aspergillus niger KACC40280 crude enzyme solution and ginseng PPD extract (15,000ppm) reaction solution, lane 6: A. Reaction solution, lane 7: A. oryzae KACC40250 crude enzyme solution and PPD extract (15,000ppm) reaction solution, lane 8: A. Coenzyme solution of oryzae KACC40250 and PPD extract of Korean Ginseng Ginseng PPD (10,000ppm) reaction solution.

The strain of the present invention is a new yeast strain and has been deposited with the Agricultural Genetic Resources Center (KACC) of the National Academy of Agricultural Sciences under the accession number KACC 93333P, and is classified as Aspergillus niger of the genus Aspergillus that is safe for the human body and can be used in food. do.

The sequence of 28s rRNA of the strain of the present invention is shown in SEQ ID NO: 1.

The strain of the present invention can convert ginsenosides of ginseng PPD type into compound K. And compared to other strains of the same genus, this conversion ability is very good.

The strain of the present invention may be cultured in the medium and culture conditions used for culturing the strain of the genus Aspergillus. For example, it may be cultured at a temperature of 25 ~ 33 ℃ using a medium containing malt extract or bran, and may be cultured in a liquid phase or a solid phase.

The culture of the present invention may be obtained by culturing the strain of the present invention under the same conditions as described above, and may be in a liquid or solid state, and may be in a state in which cells are contained or in a state in which cells are removed.

The enzyme composition of the present invention can be obtained by a conventional purification method of an enzyme produced by a microorganism, preferably by a method of purifying an extracellular protein from a strain of the genus Aspergillus. For example, it can be obtained by adding a buffer to the culture of the strain of the present invention, removing the cells and solids, and then adding alcohol to precipitate the enzyme. Preferably, a culture is obtained by culturing the strain of the present invention in a solid medium containing bran, an acetic acid buffer is added to the culture, mixed, and then the cells and solids are removed through centrifugation or membrane separation, and alcohol It can be obtained by adding an enzyme to precipitate the enzyme. Preferably, an acetic acid buffer of 10 to 100 mM at a pH of 4.0 to pH 6.0 can be used. After adding the acetic acid buffer and mixing at 5 to 15° C. for 10 to 20 hours, the enzyme can be well mixed in the buffer, and then the cells And the process of removing the solid content may be performed. In addition, preferably, alcohol having an alcohol content of 80 to 98% (v/v) may be used, and a method of precipitation by adding alcohol and standing at 1 to 10° C. for 10 to 20 hours may be used.

The method of the present invention is a method capable of increasing the compound K content of ginseng or processed ginseng, and it is characterized in that it is fermented by adding the strain of the present invention, the culture or the enzyme composition.

According to the present invention, the extracellular enzyme produced by the strain of the present invention can convert major ginsenosides of ginseng PPD type into compound K. Therefore, if the strain, culture or enzyme composition of the present invention is added to ginseng or processed ginseng to allow an enzyme reaction to occur, it can be increased compared to the content of compound K originally contained.

In the method of the present invention, the ginseng processed product may be red ginseng, a ginseng extract or a red ginseng extract, preferably an extract obtained through the process of extracting PPD-type ginsenosides from ginseng PPD extract, that is, ginseng. The Hwagi ginseng PPD extract can be obtained through a conventional extraction method used to extract PPD-type ginsenosides from ginseng.

By adding the strain, culture, or enzyme composition of the present invention to ginseng or processed ginseng, and maintaining a constant temperature so that the strain can grow or the enzyme can act, the enzymatic reaction can be performed. For example, it is possible to use a method of maintaining at 20 ~ 60 ℃, when adding the enzyme composition, preferably 40 ~ 60 ℃, more preferably a method of maintaining at 45 ~ 55 ℃ can be used. In addition, when it is desired to increase the compound K content of the PPD extract using the enzyme composition, the concentration of the PPD extract in the reaction solution may be preferably 5,000 to 20,000 ppm.

The composition for increasing the compound K content of ginseng or processed ginseng of the present invention is a composition that can be used to perform the method of the present invention as described above, and contains the strain of the present invention, the culture or the enzyme composition as an active ingredient do it with

At this time, the content of the active ingredient, the strain of the present invention, the culture, or the enzyme composition is not particularly limited, but may preferably be 0.001 to 100% by weight.

In addition, the composition for increasing the compound K content of the present invention may further contain a component for stabilizing the strain or the enzyme of the present invention in addition to the strain of the present invention, the culture or the enzyme composition, and other strains, a culture solution thereof Or an enzyme thereof may be further contained.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and therefore, the scope of the present invention should not be construed as being limited by these examples.

[Example]

1. Isolation and identification of strains

1 g of the yeast sample was suspended in 9 ml of sterile distilled water ^{, serially diluted at a dilution factor of 10 2} , 10 ³ , 10 ⁴ , spread on a malt extract agar, and cultured at 30°C.

The microorganisms of the generated single colony were transferred to a new solid malt extract medium and cultured, and then the generated single colonies were added to a liquid medium, that is, malt extract broth, with 1% (w/v) PPD extract (below). See Example 4) was cultured in a medium.

After 10 days of shaking culture at 30 ° C., the conversion ability of the H. ginseng PPD extract was confirmed for each of the cultured microorganisms (see Example 4 below), and ITS sequencing was performed only on the microorganisms shown to have the conversion ability.

As a result of analyzing the 28s rRNA sequence of the C2-2 strain selected as the candidate strain and attempting to identify the strain using the homology investigation based on the GenBank database, the existing Aspergillus tubingensis and 99% It was analyzed to have more homology (see FIG. 1).

Based on this, the isolated strain was deposited with the Agricultural Genetic Resources Center (KACC) of the National Academy of Agricultural Sciences under the accession number KACC 93333P.

2. Culture of the strain

Aspergillus niger C2-2 strain was inoculated in malt extract broth (BD Difco, cat. 218630) and cultured at 30° C. for about 3 days to obtain a culture solution.

After putting 250 g of bran into a tray (80 * 100 * 320 mm) to adjust the moisture content to 20%, high pressure sterilization was performed, and 75 ml of the culture solution was added to achieve a final moisture content of 50%, followed by solid-state culture. At this time, the solid-state culture was performed for 8 days in a thermo-hygrostat at 30° C. and 90% humidity.

3. Preparation of crude enzyme solution

An acetic acid buffer solution (pH 5.0, 50 mM) of 10 times the volume of the solid culture obtained through the solid-phase culture was added and mixed for 3 hours in a shaker incubator at 10 ° C. Then, the extract was removed by centrifugation or membrane separation. Then, three times the volume of the extract was mixed with alcohol (alcohol content of 95% (v/v)), and then left at 4° C. overnight to perform enzymatic precipitation. After precipitation, the supernatant was removed by centrifugation, and an acetic acid buffer solution was added to the enzyme precipitate to prepare a 10-fold concentrated crude enzyme solution.

4. Reaction of crude enzyme solution and Hwagi ginseng PPD extract

The crude enzyme solution was reacted with the PPD extract of ginseng ginseng, and the content of compound K in the reaction solution was analyzed through thin layer chromatography and high performance liquid chromatography. The reaction solution was prepared by adding crude enzyme solution and PPD extract of ginseng ginseng to acetic acid buffer solution (pH 5.0, 50 mM).

In this case, the PPD extract of ginseng ginseng obtained by the following method was used.

○ Manufacture of Hwagi Ginseng PPD extract

1 kg of Hwagi ginseng in powder form was added to 10 liters of 50% ethanol and extracted three times at 50° C. for 24 hours each. The extract was concentrated to 1/2 volume using a vacuum concentrator (a process of reducing the ethanol content in the concentrate to 5% or less), and then the concentrate was passed through a 4L HP20 column. 40 L or more of distilled water was flowed through the column to remove impurities, and 32 L of 29% ethanol was flowed to elute the PPT series first. Then, 20ℓ of 80% ethanol was flowed to elute the PPD series, and the PPD series eluate was powdered.

The reaction solution was prepared by adding the crude enzyme solution to 1/10 of the final volume of the reaction solution and setting the concentration of the PPD extract of ginseng ginseng differently to 10,000 ppm (10 mg/ml) and 15,000 ppm (15 mg/ml), respectively, and then 50 It was reacted for 60 ~ 84 hours in a shaking incubator at ℃.

The composition of the reaction solution is shown in Table 1.

concentration of substrate
10,000ppm concentration of substrate
15,000ppm Enzyme (μl) 200 200 Substrate (μl)
(Hwagi Ginseng PPD 50,000ppm) 400 600 Acetic acid buffer (μl) 1,400 1,200 Final reaction solution (μl) 2,000 2,000

As a result, as shown in FIGS. 3 to 9 , it was found that the content of compound K in the PPD extract of Hwagi ginseng was greatly increased by the reaction with the crude enzyme solution extracted from the strain of the present invention. And this phenomenon was observed at the concentrations of 10,000 ppm and 15,000 ppm, and the reaction results of 60 hours and 84 hours.

The above results indicate that the strain of the present invention has the ability to convert PPD type ginsenoside to compound K by producing a specific enzyme (one type of enzyme or two or more types of enzymes), and this ability is very good do.

5. Comparison with other strains

In order to compare the compound K-increasing effect of the strain of the present invention as described above, the same experiment as in Examples 2 to 4 was performed on Aspergillus niger KACC40280 and Aspergillus oryzae KACC40250. As a result, the strain of the present invention was It was found that the compound K increasing effect was significantly better than that of other strains.

Agricultural Biotechnology Research Institute KACC93333P 20191022

<110> AceEMzyme co., Ltd. <120> Novel Aspergillus niger C2-2 isolated from Nu-ruk producing ginsenoside compound K biotransformation enzyme and use thereof <130> PA-D19223 <140> 10-2019-0145834 <141> 2019-11-14 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 573 <212> DNA <213> Unknown <220> <223> Aspergillus niger C2-2 <400> 1 gtcgtttacg agccattatg ccagcgtccg tgccgaagcg cgttcctcgg tccaggctgg 60 ccgcattgca cccctggcta taaggtaccc cggagggtac tacattccag gggcctttga 120 ccggccgccc aaaccgacgc tggcccgccc acggggaagt acaccggcac gaatgccggc 180 tgaaccccgc gggcgagtct ggtcgcaagc gcttcccttt caacaatttc acgtgctgtt 240 taactctctt ttcaaagtgc ttttcatctt tcgatcactc tacttgtgcg ctatcggtct 300 ccggccagta tttagcttta gatgaaattt accacccatt tagagctgca ttcccaaaca 360 actcgactcg tcgaaggagc tttacagggg cacggacacc ccgcccaaga cgggattctc 420 accctctctg acggcccgtt ccagggcact tagacggggg ccgcacccaa agcatcctct 480 gcaaattaca atgcggactc cgaaggagcc agctttcaaa tttgagctct tgccgcttca 540 ctcgccgtta ctgagggcaa tcccggttgg ttt 573

Claims (5)

Aspergillus niger C2-2 strain deposited with accession number KACC 93333P. A culture of the strain of claim 1. An enzyme composition obtained by purification from the culture of claim 1 strain. The strain of claim 1; a culture of the strain; Or an enzyme composition obtained by purification from the culture of the strain; Method of increasing the compound K content of ginseng or ginseng processed product, characterized in that by adding and fermenting. The strain of claim 1; a culture of the strain; Or an enzyme composition obtained by purification from the culture of the strain; composition for increasing the compound K content of ginseng or processed ginseng containing as an active ingredient.

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