METHOD FOR IMPROVING PREPARATION OF FERMENTED BLACK GINSENG CONTAINING HIGH CONTENT OF FUNCTIONAL INGREDIENTS BASED ON SAFETY BY APPLYING LED

Abstract

The present invention relates to a method for improving the preparation of fermented black color ginseng containing functional ingredients at high contents based on safety, specifically to a novel Aspergillus niger KHNT-1 strain deposited with accession number KCTC14739BP; an improved process for preparing fermented black color ginseng through treatment with the strain, steaming utilizing pottery, and drying utilizing an LED dryer; fermented black color ginseng containing functional ingredients, ginsenosides Rg5, Rg3, and Rk1, and CK at high contents; and a composition for preventing, improving, or treating coronavirus infectious disease, the composition containing the fermented black color ginseng as an active ingredient.

As the fermented black color ginseng is prepared through fermentation using the novel Aspergillus niger KHNT-1 strain according to the present invention, steaming utilizing pottery as a steamer, and drying (fermentation) utilizing a medical LED as a dryer/fermenter, the method for improving the preparation of fermented black color ginseng is economical and safe since the preparation process takes 7 to 15 days and benzopyrene is not detected in the fermented black color ginseng, and the fermented black color ginseng contains ginsenosides Rg3, Rg5, and Rk1, and CK at high contents, and can be usefully utilized as a high value-added new drug raw material that is required to be contained at a high content per unit and in the field of high-functional food development.

Claims

1. A novel Aspergillus niger KHNT-1 strain deposited under accession number KCTC14739BP.

2. The strain according to claim 1, wherein the strain is isolated from a fermented soybean powder and identified.

3. The strain according to claim 1, wherein the strain is activated in response to light.

4. A method for improving preparation of fermented black color ginseng, the method comprising: a) a pretreatment step of washing and drying a fresh ginseng raw material; b) a step of immersing the fresh ginseng in an Aspergillus niger KHNT-1 strain culture solution and fermenting the fresh ginseng; and c) a step of repeating steaming and drying nine times after the fermentation step.

5. The method for improving preparation of fermented black color ginseng according to claim 4, wherein the drying step a) is performed at 40° C. to 50° C.

6. The method for improving preparation of fermented black color ginseng according to claim 4, wherein the immersion and fermentation in a strain culture solution of b) is performed for 5 to 9 hours.

7. The method for improving preparation of fermented black color ginseng according to claim 4, wherein the steaming in c) is performed by applying heat to pottery to raise an internal temperature of the pottery and thus to raise a temperature of water contained in fresh ginseng itself and at the same time emitting far-infrared rays.

8. The method for improving preparation of fermented black color ginseng according to claim 4, wherein the steaming in c) is performed at 75° C. to 90° C. for 5 to 9 hours.

9. The method for improving preparation of fermented black color ginseng according to claim 4, further comprising a step of performing immersion in an Aspergillus niger KHNT-1 strain culture solution every time after the steaming in c).

10. The method for improving preparation of fermented black color ginseng according to claim 4, wherein the drying in c) is to perform drying and fermentation utilizing a medical LED as a dryer.

11. The method for improving preparation of fermented black color ginseng according to claim 4, wherein the drying in c) is performed at 27° C. to 38° C. for 5 to 9 hours.

12. The method for improving preparation of fermented black color ginseng according to claim 10, wherein wavelengths of solar radiation excluding ultraviolet rays are applied to the LED.

13. The method for improving preparation of fermented black color ginseng according to claim 12, wherein the wavelengths of solar radiation include white wavelengths of 3000 K and 5500 K and short wavelengths of 590 nm, 620 nm, 630 nm, and 850 nm.

14. The method for improving preparation of fermented black color ginseng according to claim 13, wherein the wavelengths activate Aspergillus niger KHNT-1 to produce ginsenosides Rg5, Rg3, and Rk1, and CK (Compound K).

15. The method for improving preparation of fermented black color ginseng according to claim 4, wherein benzopyrene is not detected in the fermented black color ginseng.

16. Fermented black color ginseng containing ginsenosides Rg5, Rg3, and Rk1, and CK (Compound K), which is prepared by the method according to claim 4.

17. A pharmaceutical composition for preventing or treating coronavirus infectious disease, the pharmaceutical composition comprising the fermented black color ginseng according to claim 16 as an active ingredient.

18. The pharmaceutical composition according to claim 17, wherein the coronavirus infectious disease is SARS-CoV-2 (COVID-19).

19. A food composition for preventing or improving coronavirus infectious disease, the food composition comprising the fermented black color ginseng according to claim 16 as an active ingredient.

20. A feed composition for preventing or improving coronavirus infectious disease, the feed composition comprising the fermented black color ginseng according to claim 16 as an active ingredient.

21. An antiviral composition comprising the fermented black color ginseng according to claim 16 as an active ingredient.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0079] FIG. 1 illustrates the phylogenetic tree analysis of a novel Aspergillus niger KHNT-1 strain;

[0080] FIG. 2 is a schematic diagram of an improved process for preparing fermented black color ginseng of the present invention;

[0081] FIG. 3 is test certificates confirming that benzopyrene is not detected in fermented black color ginseng prepared through nine times of steaming and nine times of drying (FIG. 3A) and fermented black color ginseng prepared through 14 times of steaming and 14 times of drying (FIG. 38);

[0082] FIG. 4 is test certificates confirming that there is a ginsenoside content Increasing effect in a case where treatment with an Aspergillus niger KHNT-1 strain and drying treatment utilizing an LED (FIG. 4A) are performed as compared to cases where treatment with Lactobacillus plantarum strain and drying treatment utilizing an LED (FIG. 48), treatment with drinking water instead of treatment with a strain and drying treatment utilizing an LED (FIG. 40), no treatment and drying treatment utilizing an LED (FIG. 4D), and treatment with an Aspergillus nicer KHNT-1 strain and general drying treatment (FIG. 4E) are performed, respectively.

[0083] FIG. 5 is a graph illustrating the results of cytotoxicity evaluation at various concentrations of fermented black color ginseng extract; and

[0084] FIG. 6 is a graphs illustrating the evaluation results of infection inhibitory efficacy (FIG. 6A) and replication inhibitory efficacy (FIG. 68) of fermented black color ginseng extract against SARS-CoV-2.

DETAILED DESCRIPTION OF THE INVENTION

[0085] Hereinafter, the present invention will be described in more detail with reference to Examples. These Examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited by these Examples.

Experimental Example 1. Isolation and Identification of Microorganisms

[0086] The strain used in the present invention was isolated from fermented soybean (meju) powder.

[0087] Specifically, water extract was taken from dry fermented soybeans using 10 mL water, and then 100 μL of the extract diluted by serial dilution was carefully cultured at 30° C. for 2 days using a Potato Dextrose Agar (PDA) plate. Thereafter, different colony types were selected, and strains forming a single colony were selected and further subcultured on a new PDA under the same conditions. The cultured strain was preserved in POB containing 30% glycerol and stored at −70° C. for use in experiments.

Experimental Example 2. Sequencing and Phylogenetic Tree Analysis

[0088] The phylogenetic tree analysis was performed using sequencing of the selected strain.

[0089] Specifically, the internal transcribed spacer (TS) region of 18 S rDNA was amplified using universal primers through an automated DNA sequencing system (Applied Biosystems, Foster City, Calif., USA) from Macrogen, Seoul, Korea. The obtained PCR product was initially sequenced and identified by performing homology search using the online tool BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cqi), and was designated as A. niger_MT804610.1.

[0090] Based on the similarity search with the 163 ribosomal RNA sequence of A. niger MT804610.1 identified above in the GenBank database, phylogenetic tree analysis was performed using the closest organism and a partial sequence (1693 bp) of the identified strain. The phylogenetic tree was constructed by way of the maximum likelihood method using MEGA X (Molecular Evolutionary Genetic Analysis, version 10.1).

[0091] As a result, as illustrated in Fla 1, it has been confirmed that the identified strain designated as A. niger_MT804610.1 shows a bootstrap value of 92% with A. niger_MF093522.1 and forms a duster.

[0092] This was named Aspergillus niger KHNT-1 and was used in the present invention.

Experimental Example 3. Detection of Benzopyrene

[0093] The benzopyrene content in the fermented black odor ginseng prepared was analyzed based on the Food and Drug Administration (2007) Guidelines for testing benzo(a)pyrene in health functional foods (so-called black color ginseng) (KFDA Public Health Functional Food Standards Team-5454, 2007, 12, 31).

Experimental Example 4. Preparation of Cell Line and Virus

[0094] Experiments on the inhibitory efficacy on the novel BARS-Coil-2 were performed using vero cells, and the vero cells used in the present invention were cultured in Dulbecco's modified Eagle's medium (DMEM, Welgene, Gyeongsan, Korea) containing American Type Culture Collection (ATCC, CCL-81; Manassas, Va.) 10% heat-inactivated fetal bovine serum and 1× Antibiotic-Antimycotic (GibcolThermo Fisher Scientific, Waltham, Mass.) at 37° C. and 5% carbon dioxide.

[0095] SARS-COV-2 (BetaCoV/Korea/DCDC03/2020) was provided by the National Pathogen Resource Bank of the Korea Centers for Disease Control and Prevention.

Preparation Example 1. Method for Preparing Fermented Black Color Ginseng

[0096] Fermented black color ginseng was prepared by performing steaming nine times and drying nine times after a pretreatment step of washing and drying a fresh ginseng raw material and an immersion and fermentation step using the strain identified in the Experimental Example above.

[0097] Specifically, fresh ginseng raw material was pretreated by washing and drying at 40° C. to 50° C., and the fresh ginseng was immersed and fermented in an Aspergillus niger KHNT-1 strain culture solution for 5 to 9 hours, then treated with the strain, fermented, and placed in pottery. Heat was applied to raise the internal temperature of the pottery to 75° C. to 90° C. and thus to raise the temperature of water contained in the fresh ginseng itself, and steaming was performed for 5 to 9 hours. Thereafter, a step of performing immersion in the strain culture solution for 5 to 9 hours and then drying and fermentation at 27° C. to 38° C. for 5 to 9 hours utilizing a medical LED as a dryer was repeated nine times, whereby fermented black color ginseng was prepared (FIG. 2).

[0098] It has been confirmed that benzopyrene is not detected at all not only in the fermented black color ginseng of the present invention prepared through steaming nine times and drying nine times by way of the method including a step of performing drying at a low temperature utilizing an LED as a dryer, but also in the fermented black color ginseng prepared through steaming 14 times and drying 14 times by way of the preparation method (FIGS. 3A and 3B).

Preparation Example 2. Manufacture of Solar Radiation—Like LED

[0099] As a method to replace the natural drying method, an LED containing all of the solar radiation spectrum was manufactured and utilized in the drying and fermentation step of the present invention.

[0100] Specifically, the LED was manufactured to be most similar to the solar radiation containing all wavelengths including the visible ray region of 440 nm to 780 nm as well as the near-infrared rays of 850 nm and white wavelengths (including 3000 K and 5500 K) but excluding the ultraviolet region of 400 nm or less.

Example 1 and Comparative Examples 1 to 4. Microbial Activating and Ginsenoside Content Increasing Effect by Drying Utilizing LED

[0101] The effect of drying by LED utilized in the preparation process of fermented black color ginseng of the present invention on Aspergillus niger KHNT-1 strain and ginsenoside content was investigated.

[0102] Specifically, fermented black color ginseng prepared by treating fresh ginseng with Aspergillus niger KHNT-1 and drying and fermenting the fresh ginseng utilizing an LED to which short wavelengths of 590 nm, 620 nm, 630 nm, and 850 nm and white wavelengths of 3000 K and 5500 K were applied was denoted as Example 1. Fermented black color ginseng prepared by treating fresh ginseng with Lactobacillus plantarum and drying and fermenting the fresh ginseng utilizing the LED was denoted as Comparative Example 1. Fermented black color ginseng prepared by treating fresh ginseng with drinking water instead of a strain and drying the fresh ginseng utilizing the LED was denoted as Comparative Example 2. Fermented black color ginseng prepared by drying fresh ginseng utilizing the LED without any treatment was denoted as Comparative Example 3. Fermented black color ginseng prepared by treating fresh ginseng with Aspergillus niger KHNT-1 and drying the fresh ginseng by way of a general drying method was denoted as Comparative Example 4. The ginsenoside contents therein are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Comparative Comparative Comparative Example 1 Example 1 Example 2 Example 3 Comparative Treatment Treatment with Treatment with Without Example 4 with KHNT-1 Lactobacillus water and treatment and Treatment with and drying and drying drying utilizing drying utilizing KHNT-1 and Ingredients utilizing LED utilizing LED LED LED general drying Rg3(S) 9,245.49 7,634.60 6,274.20 7,752.09 2,533.39 Rg3(R) 4,961.90 3,380.41 3,757.48 3,248.11 2,042.42 C-K 34.10 26.48 32.21 52.27 29.26 Rk1 11,764.54 6,425.42 7,151.53 8,009.52 1,999.41 Rg5 16,346.40 9,756.40 11,246.04 11,566.93 3,262.77 Total 42,352.43 27,223.31 28,461.46 30,628.92 9,867.25

[0103] As a result, as illustrated in FIG. 4 and Table 1, it has been confirmed that the total amount of ginsenosides in fermented black color ginseng prepared through treatment with Aspergillus niger KHNT-1 and drying utilizing an LED is about 42 mg/g, and this means that the ginsenoside content is increased by about 55%, about 48%, and about 38% or more, respectively, compared to those of Comparative Examples 1 to 3, and thus the effect of treatment with the strain of the present invention on the ginsenoside content has been confirmed. In addition, the effect of drying utilizing an LED on the increase in ginsenoside content in Comparative Example 4 was examined, and as a result, it has been confirmed that the ginsenoside content in Example 1 is significantly increased by about 330% or more compared to that in Comparative Example 4.

[0104] Therefore, it has been confirmed that the fermented black color ginseng prepared through treatment with Aspergillus niger KHNT-1 and drying utilizing an LED of the present invention contains functional ingredients at significantly high contents since the total amount of ginsenosides Rg3, Rk1, and Rg5, and CK in the fermented black color ginseng is about 42 mg/g.

Example 2. Evaluation of Cytotoxicity

[0105] Cytotoxicity of fermented black color ginseng extract was examined using Vero E6 cells. At this time, DMEM medium containing penicillin/streptomycin and 10% fetal bovine serum was used for the cells. The cells were inoculated into a 96-well plate at 5×10.sup.4 cells/well, and cultured for 24 hours in an incubator at 37° C. and 5% CO.sub.2. After 24 hours, the cells were treated with the fermented black color ginseng extract at a concentration of 1 μg/mL, 5 μg/mL, or 25 μg/mL and cultured again for 24 hours in an incubator at 37° C. and 5% CO.sub.2. After 24 hours, the supernatant was removed, the cells were treated with 100 μL of 0.5 mg/mL. MTT reagent and cultured for 1 hour, the supernatant was removed, the cells were treated with 100 μL of DMSO, and after 10 minutes, the absorbance was measured at 540 nm using a microplate reader.

[0106] As a result, as Illustrated in FIG. 5, it has been confirmed that the cell viability of 100% is maintained at all concentrations.

Example 3. Anti-Coronavirus Efficacy of Fermented Black Color or Ginseng Containing Ginsenosides Rg3, Rg5, and Rk1, and CK at High Contents

Example 3-1. Evaluation of Infection Inhibitory Efficacy Against SARS-CoV-2

[0107] In order to examine whether the fermented black color ginseng extract interfares with cell penetration of SARS-CoV-2, the plaque formation inhibitory efficacy of the virus was evaluated.

[0108] Specifically, the Vero E6 cell line was inoculated into a 12-well plate at 3×10.sup.5 cells/well and cultured for 24 hours in an incubator at 37° C. and 5% CO.sub.2, the supernatant was removed, and washing with PBS was performed two times. The cell line was treated with fermented black color ginseng extract at a concentration of 1 μg/mL, 5 μg/mL, or 25 μg/mL in the SARS-CoV-2 (50 pfu/well) absorption step and cultured for 1 hour in an incubator at 37° C. and 5% CO.sub.2. The supernatant was then removed, and washing with PBS was performed two times. The cells were overlaid with 0.6% agarose in DMEM containing 0.3% BSA, and the 12-well plate was cultured for 3 days in an incubator at 37° C. and 5% CO.sub.2. Thereafter, the cells were stained with 0.4% crystal violet for 10 minutes, washed with PBS three times to remove the virus and extract, and additionally overlaid with agarose, and then the plaque formation inhibitory ability of SARS-CoV-2 was examined. The plague reduction rate was compared between cells treated with SARS-CoV-2 and those not treated with SARS-CoV-2. The evaluation was repeated three times, and statistical analysis was performed using Graph Pad Prism 5 software. At this time, student's t-test method was used for the standard error.

[0109] As a result, as illustrated in FIG. 6A, the fermented black color ginseng (FBCG) extract at a concentration of 1 μg/mL has exhibited a strong inhibitory effect to be about 6 times and about 1.8 times those of the positive control groups, remdesivir (Rem) and chloroquine (CP), respectively. In particular, activity close to 100% has been confirmed in the fermented black color ginseng extract at a concentration of 5 μg/mL or more, and thus the strong antiviral effect of fermented black color ginseng extract against SARS-CoV-2 has been confirmed.

Example 3-2. Evaluation of Replication Inhibitory Efficacy Against SARS-CoV-2

[0110] In order to examine whether the fermented black color ginseng extract interfares with the intracellular replication of SARS-CoV-2, the viral genes in the cell culture solution were quantitatively analyzed through rea-time PCR.

[0111] Specifically, the Vero E6 cell line was inoculated into a 12-well plate at 3×10.sup.5 cells/well and cultured for 24 hours in an incubator at 37° C. and 5% CO.sub.2. Thereafter, the supernatant was removed, and washing with PBS was performed two times. The cells were infected with SARS-CoV-2 at 0.01 mol and cultured for 1 hour in an incubator at 5% CO.sub.2. After 1 hour of viral infection, the cells were treated with fermented black color ginseng extract at a concentration of 1 μg/mL, 5 μg/mL, or 25 μg/mL and cultured for 24 hours in an incubator at 37° C. and 5% CO.sub.2. In order to obtain an RNA sample, virus-infected cells were treated with fermented black color ginseng extract, the cells and supernatant were collected after 18 hours, RNA was extracted, and the level of viral gene expression was examined through real-time PCR.

[0112] As a result, as illustrated in FIG. 6B, it has been confirmed that the fermented black color ginseng extract inhibits the proliferation of SARS-CoV-2 in a concentration dependent manner, and a similar level of activity to that in remdesivir is exhibited in the fermented black color ginseng extract at a concentration of 25 μg/mL.

[0113] Through this, the inhibitory efficacy of fermented black color ginseng containing ginsenosides Rg3, Rg5, and Rk1, and CK at high contents, which is prepared by the preparation method of the present invention, on the infection stage of SARS-CoV-2 into host cells and the proliferation after infection into host cells has been confirmed.

[0114] Based on the above description, it will be understood by those skilled in the art that the present invention may be implemented in a different specific form without changing the technical spirit or essential characteristics thereof. Therefore, it should be understood that the above embodiment is not limitative, but illustrative in all aspects. The scope of the present invention is defined by the appended claims rather than by the description preceding them, and therefore all changes and modifications that fall within metes and bounds of the claims or equivalents of such metes and bounds are therefore intended to be embraced by the claims.