KR20210058416A - Novel Aspergillus tubingensis C2-2 isolated from Nu-ruk producing ginsenoside compound K biotransformation enzyme and use thereof - Google Patents

Abstract

The present invention relates to a new yeast bacteria asperzillus tubingensis C2-2 strain and a use thereof, and more specifically, to an aspergillus tubingensis C2-2 strain deposited under consignment number KACC 93333P, a culture of the strain, an enzyme composition obtained by being purified from the culture, a method for increasing compound K contents of ginseng or a ginseng processed article using the same, and a composition for the method. By using the strain, the culture, or the enzyme composition of the present invention, major ginsenoside of a ginseng PPD type can be efficiently converted into compound K. Therefore, the present invention can manufacture a ginseng product containing a high concentration of compound K which is easily absorbed to a human body and includes excellent physiological activity when using the same.

Description

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

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

Saponin refers to a substance composed of several cyclic compounds in which non-sugar parts are present in glycosides that are widely present in the plant world. 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. ) It is called ginsenoside in the sense of glycoside.

Ginsenosides are protopanaxadiol-type (PPD type) ginsenoside, protopanaxatriol-type (PPT type) ginsenoside and oleanolic 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 in the compound structure by a glycosicid bond. Classified according to number. The basic skeleton of oleanoline acid-based ginsenoside is 5-cyclic, and there is only ginsenoside Ro, and the 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, zipenoside XVII (gypenoside XVII), compound O, compound Mc1, F2, compound Y, Compound Mc, Rg3, Rh2, and Compound K (CK) are included. 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 rate in vivo is very low due to their large size around 1,000 Daltons. Therefore, in order to increase the medicinal effect of ginsenoside, a process of converting major ginsenosides to minor ginsenosides that are relatively well absorbed and have more medicinal effects is necessary. In other words, major ginsenosides require a conversion process that removes glucose, arabinose, rhamnose, and xylose constituting sugars 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 living organisms present in trace amounts include Rd, F2, Rg3, Rh1, Rh2, zipeno These include Side XVII, Zipenoside LXXV and Compound K, Compound MC, and Compound MC One.

Among the minor ginsenosides, Compound K is a saponin contained only in ginseng. It is easy to absorb into the body and has a strong pharmacological effect. In particular, it has an antioxidant power of 40 times that of vitamin C, as well as preventing vascular inflammatory diseases such as diabetes. It also has an anti-inflammatory function, which is very important for treatment, and additionally inhibits the formation of atheroma to prevent arteriosclerosis, and has an excellent effect of improving immunity and recovering from fatigue due to recovery of liver function. It even has anti-cancer effects, many have been reported in the Society. Therefore, Compound K is being used by incorporating it into pharmaceuticals or health functional foods, and the potential for development in the future is endless, but there is a limitation that all enzymes currently used by Korean compound K producers depend on imports and are not even edible.

Accordingly, the present inventors have made research efforts to develop a method that can easily convert ginseng PPD-type major ginsenosides (Rb1, Rb2, Rb3, Rc) into compound K, especially a method that is safe for the human body and can be used in food. As a result, when using the newly discovered strain of the present invention, that is, Aspergillus Tubing Nsis C2-2, which is classified as a microorganism that is safe for the human body and can be used in food, PPD type ginsenoside derived from ginseng is efficiently used as a compound K. It was confirmed that it can be converted to and completed the present invention.

Korean Patent Registration No. 10-1718465

Therefore, the main object of the present invention is to provide a novel strain that can be used to convert the major ginsenoside of ginseng PPD type to 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 above strain.

According to one aspect of the present invention, the present invention provides an Aspergillus tubingensis C2-2 (Aspergillus tubingensis C2-2) strain deposited under the 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 it provides a method of increasing the compound K (compound K) content of ginseng or ginseng processed product characterized in that the fermentation by adding; 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 it provides a composition for increasing the compound K (compound K) content of ginseng or ginseng processed product containing an enzyme composition obtained by purification from the culture of the strain as an active ingredient.

When the strain, culture or enzyme composition of the present invention is used, major ginsenosides of ginseng PPD type can be efficiently converted into compound K. Therefore, by using these, it is possible to manufacture ginseng products containing high concentration of Compound K, which is more easily absorbed in the body and has excellent physiological activity.

1 shows the results of investigation of the homology of the 28s rRNA sequence of the strain of the present invention based on the GenBank database.
2 is a block diagram showing a process of obtaining an enzyme composition, an enzyme reaction, and a compound K content confirmation process according to an embodiment of the present invention.
3 shows the results of high performance liquid chromatography analysis of a representative ginsenoside standard 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).
Figure 4 shows the results of high-performance liquid chromatography analysis of the Hwagisam PPD extract according to an embodiment of the present invention.
Figure 5 shows the result of thin-layer chromatography analysis by reaction time of the crude enzyme solution and the reaction solution of Hwagisam PPD extract according to an embodiment of the present invention. lane 1: representative ginsenoside standard mixture (PPD standard), lane 2: Hwagisam PPD extract, lane 3: crude enzyme solution and Hwagisam PPD extract (10,000ppm) reaction solution, lane 4: crude enzyme solution and Hwagisam PPD extract (15,000ppm) ) Reaction solution.
6 shows the results of high performance liquid chromatography analysis of the crude enzyme solution and the Hwagisam PPD extract reaction solution (10,000 ppm, 60 hour 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 Hwagisam PPD extract reaction solution (10,000 ppm, 84 hour 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 Hwagisam PPD extract reaction solution (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 a crude enzyme solution and a reaction solution of Hwagisam PPD extract (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 mixture (PPD standard), lane 2: Hwagisam PPD extract, lane 3: crude enzyme solution of the present invention strain and Hwagisam PPD extract (15,000ppm) reaction solution, lane 4: crude of the present invention strain Enzyme solution and Hwagisam PPD extract (10,000ppm) reaction solution, lane 5: Coenzyme solution of Aspergillus niger KACC40280 and Hwagisam PPD extract (15,000ppm) reaction solution, lane 6: A. niger crude enzyme solution and Hwagisam PPD extract (10,000ppm) Reaction solution, lane 7: Coenzyme solution of A. oryzae KACC40250 and Hwagisam PPD extract (15,000ppm) reaction solution, lane 8: Coenzyme solution of A. oryzae KACC40250 and Hwagisam PPD extract (10,000ppm) reaction solution.

The strain of the present invention is a novel yeast strain, deposited with the accession number KACC 93333P at the National Academy of Agricultural Sciences Agricultural Genetic Resource Center (KACC), and is classified as Aspergillus tubingensis species, which is safe for humans and can be used in food. do.

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

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

The strain of the present invention can be cultured with a medium and culture conditions used for culturing a strain of the genus Aspergillus, preferably a strain of Tubing Nsis species. For example, it may be cultured in a temperature condition of 25 ~ 33 ℃ using a medium containing malt extract or bran, it can be cultured in a liquid phase and 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 liquid or solid, and may be in a state in which cells are contained or 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 a method of purifying extracellular protein from a strain of the genus Aspergillus, more preferably a strain of Tubing Nsis species. I can. For example, it can be obtained by adding a buffer solution to the culture of the strain of the present invention, removing cells and solids, and then adding alcohol to precipitate the enzyme. Preferably, the strain of the present invention is cultivated in a solid medium containing bran to obtain a culture, and after mixing by adding an acetic acid buffer to this culture, the cells and solids are removed through centrifugation or membrane separation, and alcohol It can be obtained by a method of precipitating the enzyme by adding. Preferably, an acetic acid buffer solution of 10 to 100 mM of pH 4.0 to pH 6.0 may be used, and an acetic acid buffer solution is added and mixed at 5 to 15°C for 10 to 20 hours so that the enzyme can be well mixed in the buffer solution. And a 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 precipitating by adding alcohol and allowing it to stand 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 is characterized by fermentation by adding the strain, the culture or the enzyme composition of the present invention.

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

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

Enzymatic reaction can be performed by adding the strain, culture or enzyme composition of the present invention to ginseng or ginseng processed product, and maintaining the strain at a constant temperature so that the strain can grow or the enzyme can act. For example, a method of maintaining at 20 to 60°C may be used, and when the enzyme composition is added, a method of maintaining at preferably 40 to 60°C, more preferably at 45 to 55°C may be used. In addition, in the case of increasing the compound K content of the PPD extract by using the enzyme composition, the concentration of the PPD extract in the reaction solution may be preferably set to 5,000 to 20,000 ppm.

The composition for increasing the compound K content of ginseng or ginseng processed product 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. It is done.

At this time, the content of the strain of the present invention, the culture or the enzyme composition as an active ingredient 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, in addition to the strain of the present invention, the culture or the enzyme composition, the strain of the present invention or a component for stabilizing the enzyme, and other strains, a culture solution thereof Or it may further contain an enzyme thereof.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.

[Example]

1. Isolation and identification of strains

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

The resulting single colony of microorganisms is transferred to a new malt extract solid medium for cultivation, and then the regenerated single colony is transferred to a liquid medium, that is, malt extract broth, with 1% (w/v) of Hwagisam PPD extract (below. (See Example 4) was cultured in a medium containing.

After shaking culture at 30° C. for 10 days, the conversion ability of the Hwagisam PPD extract was confirmed for each of the cultured microorganisms (see Example 4 below), and ITS sequencing was performed only for microorganisms shown to have 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 a homology investigation based on the GenBank database, it was found that the existing Aspergillus tubingensis and 99% It was analyzed to have the above homology (see Fig. 1).

Based on this, the isolated strain was named Aspergillus tubingensis C2-2, and deposited with the National Academy of Agricultural Sciences Agricultural Genetic Resource Center (KACC) under the accession number KACC 93333P.

2. Culture of strain

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

250 g of wheat bran was put in a tray (80*100*320mm) and the moisture content was adjusted to 20%, followed by autoclaving, and 75 ml of the culture solution was added so that the final moisture content was 50%, followed by solid-state culture. At this time, the solid-state culture was performed for 8 days under the conditions of 30°C and 90% humidity in a constant temperature and humidity.

3. Preparation of crude enzyme solution

An acetic acid buffer solution (pH5.0, 50mM) of 10 times the volume of the solid culture obtained through the solid culture was added, mixed for 3 hours in a shaking incubator at 10°C, and then the extract from which the cells were removed through centrifugation or membrane separation was prepared. The resulting extract was mixed with 3 times the volume of alcohol (alcohol content 95% (v/v)), and then allowed to stand at 4° C. overnight to perform enzyme 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 Hwagisam PPD extract

The crude enzyme solution was reacted with the Hwagisam PPD extract, 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 a crude enzyme solution and Hwagisam PPD extract to an acetic acid buffer solution (pH 5.0, 50 mM).

At this time, the Hwagisam PPD extract was used as obtained by the following method.

○ Manufacture of Hwagisam PPD extract

1kg of Hwagisam in powder form was added to 10ℓ 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 (the process of making the ethanol content of the concentrate less than 5%), and then the concentrate was passed through a 4L HP20 column. Impurities were removed by flowing more than 40ℓ of distilled water through the column, and 32ℓ of 29% ethanol was flowed to elute the PPT series first. Thereafter, 20ℓ of 80% ethanol was flowed to elute the PPD series, and the PPD series eluate was powdered.

Prepare a reaction solution by adding the crude enzyme solution to 1/10 of the final volume of the reaction solution and setting the concentrations of Hwagisam PPD extract to 10,000 ppm (10 mg/ml) and 15,000 ppm (15 mg/ml), respectively. It was reacted for 60 to 84 hours in a shaking incubator at ℃.

The reaction solution composition is shown in Table 1.

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

As a result, it was found that the content of compound K of the Hwagisam PPD extract was significantly increased by the reaction with the coenzyme solution extracted from the strain of the present invention as shown in FIGS. 3 to 9. And this phenomenon appeared in both concentrations of 10,000 ppm and 15,000 ppm, and 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 ginsenosides to compound K by producing a specific enzyme (one kind of enzyme or two or more kinds of enzymes), and this ability is very excellent. 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 shown in FIG. Compared to other strains, it was found that the effect of increasing the compound K was remarkably superior.

Institute of Agricultural Biotechnology KACC93333P 20191022

<110> AceEMzyme co., Ltd. <120> Novel Aspergillus tubingensis C2-2 isolated from Nu-ruk producing ginsenoside compound K biotransformation enzyme and use thereof <130> PA-D19223 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 573 <212> DNA <213> Unknown <220> <223> Aspergillus tubingensis 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 tttacacggg 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 tubingensis C2-2 (Aspergillus tubingensis C2-2) strain deposited with the accession number KACC 93333P. A culture of the strain of claim 1. An enzyme composition obtained by purification from the culture of the strain of claim 1. Claim 1 strain; A culture of the strain; Or an enzyme composition obtained by purification from the culture of the strain; a method for increasing the compound K content of ginseng or ginseng processed product characterized in that it is fermented by adding. Claim 1 strain; A culture of the strain; Or an enzyme composition obtained by purifying from the culture of the strain; a composition for increasing the compound K content of ginseng or ginseng processed product containing as an active ingredient.

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