KR102356058B1 - Culture method for enhancing cordycepin content of Paecilomyces javanicus Mycelium using Light-Emitting Diode - Google Patents

Abstract

The present invention relates to a culturing method for increasing the cordycepin content of Cordyceps Cordyceps mycelium using an LED, comprising: an inoculation step of inoculating a Cordyceps Cordyceps mycelium into a medium; and culturing the inoculated medium under light conditions to obtain cordycepin from the cultured Cordyceps Cordyceps mycelium and culture solution; It was confirmed that the Cordyceps Cordyceps mycelium had the maximum content according to the medium and light conditions by a culture method to increase the cordycepin content of the Cordyceps Cordyceps mycelium containing By deriving a culture time condition and using it as a culture condition, it is possible to efficiently obtain a maximum amount of cordycepin from the mycelium of Cordyceps Cordyceps.

Description

Culture method for enhancing cordycepin content of Paecilomyces javanicus Mycelium using Light-Emitting Diode using LEDs

The present invention relates to a culturing method for increasing the cordycepin content of mycelia of Cordyceps Cordyceps using LED.

Dongchunghacho (冬蟲夏草) got its name from the meaning that it comes out as an insect in winter and a mushroom in summer. About 800 species are known, most of which are entomopathogenic fungi, and a major feature is that they are infected with host insects and mycelium grows inside to produce fruiting bodies. Cordyceps cordyceps has been used as a medicinal herb in Asia (China, Korea, Japan, etc.) since ancient times. For its efficacy, it has been used as a good medicine for strengthening immunity, preventing adult diseases, asthma, and anemia.

In addition, Cordyceps Cordyceps belongs to the group with the highest protein content (more than 28% of dry content) among crops, and contains a large amount of substances that enhance immunity. In particular, 'Cordycepin (3'-deoxy-adenosine)', which has been found to be an isomer of quinic acid, is included among the immunoactive substances of Cordyceps Cordyceps. Cordycepin is a nucleic acid substance that prevents normal cells from being converted into cancer cells by activating the reduced immune function while participating in the genetic information of cells. In a clinical trial using Cordyceps Cordyceps, it was confirmed that the content of natural killer cells (NK cells) and cytokines secreted from immune cells, which kill cancer cells, increased by 18% to 25%. It is used for treatment and is receiving a lot of attention as a health functional food.

Based on these results, Cordyceps Cordyceps containing cordycepin has been certified by the Ministry of Food and Drug Safety as a health functional food in Korea, and various functions such as antiviral and anti-inflammatory effects in addition to the immune enhancing activity and anticancer activity of cordycepin have been announced.

Due to the function of cordycepin as described above, recently, a lot of research on extracting cordycepin from Cordyceps Cordyceps is being conducted. However, since the separation method disclosed in Korean Patent Application Laid-Open No. 2001-0054264 is a purification method using HPLC, there has been a problem that is not commercially suitable because it cannot separate the active material in a small amount. Studies on the optimal conditions suitable for

Therefore, there is a need for research on an optimal method for increasing the cordycepin yield by increasing the cordycepin content of the Cordyceps Cordyceps mycelium.

1. Republic of Korea Patent Publication No. 10-2001-0054264 (2001.07.02.)

It is an object of the present invention to provide a culture method for increasing the cordycepin content of the Cordyceps Cordyceps mycelium optimal for the maximum cordycepin content of Cordyceps Cordyceps.

In addition, another object of the present invention is to provide a cordycepin composition obtained from Cordyceps Cordyceps according to the above method.

In addition, another object of the present invention is to provide a method for predicting the maximum cordycepin content of the mycelium of Cordyceps Cordyceps.

In order to achieve the above object, the present invention is an inoculation step of inoculating a medium for Cordyceps Cordyceps mycelium; and culturing the inoculated medium under light conditions to obtain cordycepin from the cultured Cordyceps Cordyceps mycelium and culture solution; It provides a culture method for increasing the cordycepin content of the mycelium of Cordyceps Cordyceps, comprising a.

In addition, the present invention provides a cordycepin composition obtained according to a culturing method for increasing the cordycepin content of the Cordyceps Cordyceps mycelium.

In addition, the present invention is a method for predicting the cordycepin content of the Cordyceps Cordyceps mycelium, (a) the amount of light (X ₁ ) in the box-Behnken design (Box-Behnken design), the glucose content of the medium (X ₂ ) , for the incubation time (X ₃ ), designing the experimental range by coding -1, 0, and 1; (b) obtaining experimental values for the amount of light, the glucose content of the medium, and the incubation time in the designed experimental range of step (a); (c) deriving a quadratic regression model expressed by Equation 1 below using the experimental values of step (b); and (d) predicting the cordycepin content of the Cordyceps Cordyceps mycelium by ANOVA on the quadratic regression model represented by Equation 1 derived in step (c); It provides a method for predicting the content of cordycepin in the mycelium of Cordyceps Cordyceps, characterized in that it comprises a.

[Equation 1]

Y ₁ =3224.64+974.20X ₁ +571.52X ₂ -307.48X ₃ +250.54X ₁ X ₂ -187.12X ₁ X ₃ -95.55X ₂ X ₃ -1651.51X ₁ ² -272.72X ₂ ² -663.51X ₃ ²

(In Equation 1, Y ₁ is the cordycepin content (mg/L) of the Cordyceps Cordyceps mycelium, X ₁ is the amount of light, X ₂ is the glucose content of the medium, and X ₃ is the culture time.)

According to the present invention, the mycelium of Cordyceps Cordyceps mycelium exhibited the maximum cordycepin content when irradiated with blue and red mixed LED light in SDB (Sabouraud dextrose broth) liquid medium and cultured, and optimal light amount and medium glucose content through response surface analysis And by deriving a culture time condition, and using it as a culture condition, there is an effect of efficiently obtaining a maximum amount of cordycepin from the mycelium of Cordyceps Cordyceps.

In addition, the Cordyceps Cordyceps mycelium and culture medium obtained according to the culturing method according to the present invention have a high content of cordycepin, which has excellent immune enhancing activity, anticancer activity, antiviral and anti-inflammatory effects, and can be usefully used as a health functional food.

1 is a snow flower Cordyceps ( Paecilomyces) javanicus ) It is a diagram showing the mycelium.
2 is a view showing the culturing process of Cordyceps Cordyceps and the measuring process of the cordycepin content.
3 is a diagram showing the cordycepin content of the mycelium according to the type of liquid medium of Cordyceps sinensis (data is the mean ± standard deviation of the results of three experiments).
4 is a diagram showing the cordycepin content of the mycelium according to the culture period of Cordyceps Cordyceps (data is the mean ± standard deviation of the results of three experiments).
5 is a view showing a shaking incubator installed with a fluorescent lamp and UV-A.
6 is a view showing a control box for adjusting the amount of fluorescent lamp and UV-A light and on/off.
7 is a view showing the change in the cordycepin content of Cordyceps Cordyceps mycelium according to irradiation with a fluorescent lamp and UV-A (data is the mean ± standard deviation of the results of three experiments).
8 is a view showing a shaking incubator installed with LED single light (red, green, blue).
9 is a view showing a control box for controlling the amount of single light and on/off of the LED.
10 is a view showing the change in the cordycepin content of the mycelium according to the LED single light (red, green, blue) irradiation of Cordyceps Cordyceps (data is the mean ± standard deviation of the results of three experiments).
11 is a diagram showing the change in the cordycepin content of Cordyceps Cordyceps mycelium according to the irradiation time of a single LED light (data is the mean ± standard deviation of the results of three experiments).
12 is a view showing the change in the cordycepin content of the mycelium according to the type of LED single light and mixed light of Cordyceps Cordyceps Cordyceps (data is the mean ± standard deviation of the results of three experiments).
13 is a view showing the change in the cordycepin content according to the mixing ratio of the mixed light red*blue in the mycelium of Cordyceps Cordyceps (data are the results of three experiments as mean±standard deviation).
14 is a view showing changes in the mycelium cordycepin content according to the irradiation time of mixed light red*blue (5:5) in the mycelium of Cordyceps Cordyceps mycelium (data shows the results of three experiments as mean ± standard deviation).
15 is a view showing the correlation between the mycelium culture conditions of Cordyceps Cordyceps mycelium and the mycelium cordycepin content.
16 is a view showing a fit model between LED control conditions and the content of Cordyceps mycelium mycelium cordycepin.
17 is a view showing the optimal LED control conditions for the maximum cordycepin content of the mycelium of Cordyceps Cordyceps.
18 is a diagram showing the fit of the optimal LED control condition response model for the maximum cordycepin content of the mycelium of Cordyceps Cordyceps.

Hereinafter, the present invention will be described in detail.

The present inventors confirmed that, when irradiated with blue and red mixed LED light in SDB (Sabouraud dextrose broth) liquid medium for 2 days at 12 hours a day and cultured, the maximum cordycepin content of the Cordyceps Cordyceps mycelium was shown, and response surface analysis The present invention was completed by deriving the optimal light amount, glucose content of the medium, and culture time conditions through this, and using these as culture conditions to efficiently obtain the maximum amount of cordycepin from the mycelium of Cordyceps Cordyceps.

The present invention is an inoculation step of inoculating the medium for Cordyceps Cordyceps mycelium; and culturing the inoculated medium under light conditions to obtain cordycepin from the cultured Cordyceps Cordyceps mycelium and culture solution; It provides a culture method for increasing the cordycepin content of the mycelium of Cordyceps Cordyceps, comprising a.

In this case, the medium may be a Sabouraud dextrose broth medium, and the culture may be performed for 1 to 4 days, preferably for 2 days.

In addition, 5 to 20 g/L of rice straw having a size of 80 to 120 mesh may be added to the medium, and preferably 15 g/L of rice straw having a size of 80 mesh may be added, but is not limited thereto.

In addition, the light conditions include culturing the mycelium by irradiating the medium inoculated with LED single light or mixed light, LED single light is blue light having a wavelength of 400 nm to 500 nm, and LED mixed light is blue having a wavelength of 400 nm to 500 nm It is characterized in that light and red light having a wavelength of 620 nm to 780 nm are mixed, preferably, blue light having a wavelength of 400 nm to 500 nm and red light having a wavelength of 620 nm to 780 nm can be used. The mixing ratio of the LED blue and red mixed light is 7: 3 to 3: 7, respectively, but it is characterized in that it is preferably 5: 5. At this time, it is possible to represent the maximum cordycepin content of the mycelium of Cordyceps Cordyceps under the above-mentioned preferred light conditions.

In addition, it is characterized in that the mycelium is cultured by irradiating the LED single light or mixed light to the inoculated medium for 5 to 15 hours a day, and preferably irradiated for 12 hours a day, but is not limited thereto. .

At this time, if the culture conditions and light conditions are deviated as described above, the cordycepin content of the Cordyceps Cordyceps mycelium according to the present invention does not increase, so that the yield of cordycepin is significantly lowered, which may cause an uneconomical problem.

In addition, the cordycepin content of the Cordyceps Cordyceps mycelium may be characterized in that it has a correlation as shown in Equation 1 below with the amount of light, the glucose content of the medium, and the culture time.

[Equation 1]

Y ₁ =3224.64+974.20X ₁ +571.52X ₂ -307.48X ₃ +250.54X ₁ X ₂ -187.12X ₁ X ₃ -95.55X ₂ X ₃ -1651.51X ₁ ² -272.72X ₂ ² -663.51X ₃ ²

(In Equation 1, Y ₁ is the cordycepin content (mg/L) of the Cordyceps Cordyceps mycelium, X ₁ is the amount of light, X ₂ is the glucose content of the medium, and X ₃ is the culture time.)

At this time, the culture conditions of the light amount, the glucose content of the medium, and the culture time to obtain the maximum cordycepin content of the mycelia of Cordyceps Cordyceps mycelium are 500 to 1500 lux, 5 to 15 g/50mL, 30 to 100 hours, preferably The light amount, the glucose content of the medium, and the culture conditions of the culture time may be 990.723 lux, 9.65274 g/50mL, and 61.2026 hours, respectively, but are not limited thereto.

In addition, the present invention provides a cordycepin composition obtained according to the culturing method for increasing the cordycepin content of the above-mentioned Cordyceps Cordyceps mycelium.

Cordyceps mycelium and culture medium cultured as described above have a high content of cordycepin, so it may be usefully used as a health functional food for immune enhancing activity and anticancer activity.

In addition, the present invention is a method for predicting the cordycepin content of the Cordyceps Cordyceps mycelium, (a) the amount of light (X ₁ ) in the box-Behnken design (Box-Behnken design), the glucose content of the medium (X ₂ ) , for the incubation time (X ₃ ), designing the experimental range by coding -1, 0, and 1; (b) obtaining experimental values for the amount of light, the glucose content of the medium, and the incubation time in the designed experimental range of step (a); (c) deriving a quadratic regression model expressed by Equation 1 below using the experimental values of step (b); and (d) predicting the cordycepin content of the Cordyceps Cordyceps mycelium by ANOVA on the quadratic regression model represented by Equation 1 derived in step (c); It provides a method for predicting the content of cordycepin in the mycelium of Cordyceps Cordyceps, characterized in that it comprises a.

[Equation 1]

Y ₁ =3224.64+974.20X ₁ +571.52X ₂ -307.48X ₃ +250.54X ₁ X ₂ -187.12X ₁ X ₃ -95.55X ₂ X ₃ -1651.51X ₁ ² -272.72X ₂ ² -663.51X ₃ ²

(In Equation 1, Y ₁ is the cordycepin content (mg/L) of the Cordyceps Cordyceps mycelium, X ₁ is the amount of light, X ₂ is the glucose content of the medium, and X ₃ is the culture time.)

At this time, the culture conditions of the light amount, the glucose content of the medium, and the culture time to obtain the maximum cordycepin content of the mycelia of Cordyceps Cordyceps mycelium are 500 to 1500 lux, 5 to 15 g/50mL, 30 to 100 hours, preferably The light amount, the glucose content of the medium, and the culture conditions of the culture time may be 990.723 lux, 9.65274 g/50mL, and 61.2026 hours, respectively, but are not limited thereto.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it is to those of ordinary skill in the art to which the present invention pertains that the scope of the present invention is not limited by these examples according to the gist of the present invention. it will be self-evident

< Example 1> Establishment of liquid culture technology of Cordyceps mycelium

1. Disclosure material

In order to evaluate the cordycepin content of the Cordyceps mycelium, Paecilomyces javanicus (KCCM 60299) was sold from the Korea Microorganism Conservation Center (KCCM) (FIG. 1).

2. Snow Cordyceps Cordyceps ( Paecilomyces javanicus ) of liquid culture and cordycepin content analysis

Snowflake Cordyceps ( Paecilomyces) javanicus ) 3 species (YMG (yeast extract, malt extract, glucose medium), PDB (potato dextrose broth), SDB (Sabouraud dextrose) having the culture composition of Table 1 below to establish the culture environment conditions for increasing the cordycepin content of the mycelium broth) liquid culture medium was used.

badge person Medium pH Medium composition (g/L) yeast extract malt extract Glucose Potato extract Peptone YMG 6.0 4 10 4 - - PDB 5.2 - - 20 4 - SDB 4.5 - - 20 - 10

Snow flower Cordyceps (Paecilomyces) received from KCCM javanicus ) mycelia were cultured for 7 days at 25°C in an incubator using PDA (Potato Dextrose Agar) plate medium. After that, the edge portion of the cultured mycelium was inoculated into a liquid medium (YMG, PDB, SDB) by 3 cores using a cork borer with a diameter of 5 mm, and cultured with shaking (100 rpm) at 25 ° C for 7 days to produce a liquid spawn, one day The mycelium cordycepin content of the liquid seed culture cultured at intervals was measured.

Each of the YMG, PDB or SDB medium was put into a 100 mL Erlenmeyer flask by 50 mL, sterilized at 121° C. for 15 minutes, and then cooled before use.

In addition, the cordycepin content of the cultured Cordyceps Cordyceps mycelium and the culture medium was determined by Wang et al . (2005) was measured by the following method by the colorimetric method, and the process of culturing the Cordyceps Cordyceps mycelium and measuring the cordycepin content is shown in FIG. 2 .

① Transfer the cultured mycelium culture to a falcon tube.

② Centrifugation (4000 rpm, 10 min, 2 times, Union 32R Plus, HANIL, Korea) was performed.

③ After separating the culture medium and mycelium, keep the culture medium refrigerated and dry the mycelium at 60 ℃ for 24 hours.

④ Add 10 mL of 50% ethanol to the mycelium dried at 60 ℃ for 24 hours.

⑤ Ultrasonication (40 kHz 250 W, JINWOO 2010, Korea) was performed at 50 °C for 1 hour.

⑥ After centrifugation at 1000 rpm for 20 minutes (Union 32R Plus, HANIL, Korea), the supernatant was used for cordycepin measurement.

⑦ Prepare by dissolving 0.2 g of Anthron in 100 mL of 90% sulfuric acid (Solvent A).

⑧ Add 5mL of Solvent A to 1mL of refrigerated culture medium and mycelium ethanol extract.

⑨ After reacting for 10 minutes in a 100 ℃ water bath, absorbance was measured at 460 nm using a UV-spectrophotometer (7205, ZENWAY, UK).

⑩ The standard reagent (cordycepin, sigma) is substituted for the calibration curve y=0.0075x+0.0378 (r ² =0.9913) to derive the content of cordycepin.

3. Snowflake Cordyceps ( Paecilomyces javanicus ) Cordycepin content according to the liquid culture medium

Cordycepin content of the mycelium and the culture medium according to the type of liquid medium of the mycelium of Cordyceps Cordyceps is shown in FIG. 3 .

As a result, when the Cordyceps Cordyceps mycelium was cultured in SDB medium, the culture medium (extra-mycelium) was 124.8 µg/mL and the mycelium (intra-mycelium) was 55.9 µg/mL, indicating the highest cordycepin content, It was confirmed that it exhibited a higher cordycepin content in the culture medium than the mycelium of Cordyceps Cordyceps.

In addition, as a result of analyzing the cordycepin content of Cordyceps Cordyceps according to the culture period, as shown in FIG. 4, it significantly increased after 2 days, indicating a decrease in the cordycepin content after 3 days. The incubation period was fixed at 2 days.

< Example 2> Evaluation of Effect of Fluorescent Light and UV-A on Cordycepin Content of Cordyceps Mycelium

1. Evaluation of Cordycepin Content of Specified Materials and Mycelium of Cordyceps Cordyceps Cordyceps

Fluorescent lamp and UV-A (356 nm) were installed in a shaking incubator as shown in FIG. 5 in order to evaluate the change in the cordycepin content of the Cordyceps Cordyceps mycelium sold in <Example 1-1> according to irradiation with fluorescent lamps and UV-A. did The light source was installed at a height of about 30 cm of the culture medium, and the light intensity was set to ^{100 pmol·m -2} ·s ^-1.

In addition, the control box for on/off of the light source was purchased and installed from Bitsol LED, an LED lighting brand for plant growth of Esrez, as shown in FIG. 6, and the culture was performed as in <Example 1-2>. Liquid spawn was produced by shaking culture (100 rpm) for 7 days in SDB medium at 25 ° C., and irradiated with fluorescent lamps and UV-A for 2 days at 12 h/day. Sepin content was measured.

As a control group, the mycelium of Cordyceps Cordyceps cultured under dark culture conditions was used.

2. Cordycepin content according to fluorescent lamp and UV-A light source of Cordyceps Cordyceps.

7 shows the cordycepin content according to fluorescent lamp and UV-A irradiation of Cordyceps Cordyceps Cordyceps. It was confirmed to exhibit about 8 times more cordycepin content than

< Example 3> LED single light Cordycepin content evaluation of Cordyceps mycelium according to control conditions (wavelength type, irradiation time)

1. Evaluation of Cordycepin Content of Specified Materials and Mycelium of Cordyceps Cordyceps Cordyceps

In order to evaluate the change in the content of Cordyceps mycelium cordycepin according to the irradiation of LED single light, LED single light was installed in a shaking incubator as shown in FIG. 8 . A total of three colors were installed for the single light, red, green, and blue, and 120 pieces were installed for each color (Table 2). The light source was installed at a height of about 30 cm of the culture medium, and the light intensity was set to 64.9-108.0 pmol·m ^-2 ·s ^-1 .

In addition, as shown in FIG. 9, the LED and control box were installed by purchasing and installing Bitsol LED, an LED lighting brand for plant growth of Esrez Co., Ltd., at 25° C. in PDB medium under the same conditions as in <Example 1-2>. Shaking culture (100 rpm) was performed for 7 days to produce liquid spawn, and the three types of single light were irradiated for 2 days at 12 h/day, and the cordycepin content of the cultured Cordyceps mycelium and the culture medium was measured by a colorimetric method.

As a control, Cordyceps mycelium cultured under dark culture, fluorescent light and UV-A conditions (Example 2) was used.

colour Wavelength (nm) LED quantity PCB quantity LED quantity per PCB Red 650 120 10 12 Green 525 120 10 12 Blue 450 120 10 12

2. LED of Snow Cordyceps Cordyceps single light Cordycepin content according to conditions

Figure 10 shows the cordycepin content of the mycelium according to LED single light irradiation of Cordyceps Cordyceps, dark culture, fluorescent light culture, UV-A culture, red light culture, green light culture, blue light culture 124.8 mg/L, 301.6, respectively Cordycepin content of mg/L, 492.2 mg/L, 1186.9 mg/L, 1132.2 mg/L, and 3437.6 mg/L of cordycepin was shown. It was confirmed that the cordycepin content was 7 times higher than that of -A.

Therefore, it is determined that the LED blue light is effective in increasing the cordycepin content of the Cordyceps Cordyceps mycelium, and FIG. 11 shows the change in the cordycepin content during the irradiation time of blue light among LED single lights, when blue light is irradiated for 12 hours a day. It was confirmed that it represents the highest cordycepin content.

< Example 4> LED mixed light control condition ( mixed light Cordycepin content evaluation of Cordyceps mycelium according to type, mixing ratio, irradiation time)

1. Evaluation of Cordycepin Content of Specified Materials and Mycelium of Cordyceps Cordyceps Cordyceps

Red*green (650+525nm), red*blue (650+450nm), green*blue (525+450nm) were used as the mixed light to evaluate the change in the cordycepin content of Cordyceps mycelium according to the irradiation of LED mixed light. , The luminous intensity of the light source was set ^{to 64.9-108.0 pmol·m -2} ·s ^{-1 .}

In addition, the culture was performed with shaking culture (100 rpm) for 7 days at 25° C. in SDB medium under the same conditions as in <Example 1-2> to produce liquid spawn, and three types of mixed light were irradiated for 2 days at 12 h/day. Cordycepin content of the cultured Cordyceps mycelium and culture solution was measured by colorimetric method.

2. LED of Snow Cordyceps Cordyceps mixed light Cordycepin content according to conditions

12 is a diagram showing the cordycepin content of mycelium according to LED mixed light irradiation of Cordyceps Cordyceps, and when irradiated with red*blue, the highest cordycepin content of 3654.8 mg/L, the lowest cordycepin content when irradiated with red*green mixed light Phosphorus showed 1397.5 mg/L, and when mixed light red*blue was applied (3654.8 mg/L) than when single light blue was applied (3437.0 mg/L), a higher cordisepin content was exhibited.

Therefore, when red*blue of the mixed light was irradiated to the mycelium of Cordyceps, it was confirmed that it was most effective in increasing the content of cordycepin of the mycelium.

3. Cordycepin content according to LED mixing ratio conditions of Cordyceps Cordyceps.

As described above, the mixing ratio of red*blue mixed light, which was most effective for increasing the cordycepin content of the mycelium of Cordyceps Cordyceps mycelium, was set to red:blue as 7:3, 5:5, 3:7, and the <Example 1-2> Cordyceps mycelium and the cordycepin content of the cultured medium were measured in the same manner as described above.

As a result, as shown in FIG. 13 , it was confirmed that the highest cordycepin content was exhibited when red:blue was 5:5.

4. Cordycepin content according to LED irradiation time conditions of Cordyceps Cordyceps.

In order to evaluate the cordycepin content according to the irradiation time of the LED mixed light red*blue (5:5), it was irradiated under the conditions of 6 h/day, 12 h/day, and 24 h/day, and the <Example 1-2> and Cordyceps mycelium and cordycepin content of the cultured medium were measured in the same way.

As a control, a cancer-cultured Cordyceps mycelium was used.

As a result, as shown in FIG. 14 , when mixed light red*blue (5:5) was irradiated for 12 hours a day, it exhibited the highest cordycepin content at 3657.5 mg/L, which is about 29 times higher than that of cancer culture. It was confirmed that the content was indicated.

<Example 5> Cordycepin content evaluation of Cordyceps mycelium according to medium containing LED mixed light and rice straw

1. Evaluation of Cordycepin Content of Specified Materials and Mycelium of Cordyceps Cordyceps Cordyceps

When rice straw was included in the SDB medium in the 12-hour irradiation with LED mixed light red*blue (5:5), to evaluate the change in the cordycepin content of the Cordyceps Cordyceps mycelium, the rice straw was crushed and then crushed to ASTM standard mesh No. 80, No. 100, No. It was classified as 120 and 5 to 20 g/L was added, and the cordycepin content of the cordyceps mycelium and the culture medium cultured in the same manner as in <Example 1-2> was measured.

As a control group, a cancer-cultured Cordyceps mycelium was used, and the LED mixed light was set to red*blue (5:5) and the irradiation time was fixed to 12 hours.

2. Cordycepin content according to medium conditions containing LED mixed light and rice straw of Cordyceps Cordyceps

Table 3 shows the change in the cordycepin content according to the LED mixed light red*blue (5:5) irradiation and the size of rice straw, and the amount of rice straw added at this time was fixed at 15 g/L.

As a result, when mixed light red*blue (5:5) was applied in SDB medium without rice straw, 3657.5 mg/L, Mesh No. When mixed light red*blue (5:5) was applied in 80 pass SDB medium containing rice straw, it showed a cordycepin content of 3813.1 mg/L.

Therefore, when the LED mixed light red*blue (5:5) was applied to the SDB medium containing rice straw in the Cordyceps mycelium, it was determined that there was a synergistic effect on increasing the cordycepin content, and the size of rice straw was No. 100 mesh and No. No. than 120 mesh. 80 mesh was confirmed to be the most effective for increasing cordycepin.

In addition, Table 4 shows the cordycepin content of Cordyceps Cordyceps Cordyceps according to the amount of rice straw added (5 ~ 20 g/L) in the medium, and the size of rice straw is mesh No. It was fixed at 80 pass.

As a result, when 15 g/L of rice straw was added to the medium, it exhibited the highest cordycepin content (3915.5 mg/L), and thus, when rice straw of 80 mesh size was included in the SDB medium at 15 g/L, the snowflake It was confirmed that the increase in the cordycepin content of Cordyceps Cordyceps was high.

Condition Cordycepin content (μg/mL) Red:Blue (5:5) 3657.5 ±0.0d Red:Blue (5:5) + media based 80mesh pass rice straw 3813.1 ±0.1a Red:Blue (5:5) + media based 100mesh pass rice straw 3696.8 ±0.0b Red:Blue (5:5) + media based 120mesh pass rice straw 3681.3 ±0.0c

Condition Cordycepin content (μg/mL) Red:Blue (5:5) 3657.5 ±0.0e Red:Blue (5:5) + media based 5g rice straw/L 3662.4 ±0.0d Red:Blue (5:5) + media based 10g rice straw/L 3768.8 ±0.0c Red:Blue (5:5) + media based 15g rice straw/L 3813.1 ±0.1a Red:Blue (5:5) + media based 20g rice straw/L 3800.5 ±0.1b

< Example 6> Derivation of optimal control conditions for LED for maximum cordycepin content of mycelium

1. LED control conditions and mycelium cordycepin content interracial Correlation Derivation

Pearson correlation coefficient (Hauke and Kossowski, 2001) and heat map (Wilf et al., 2016) were used to derive the correlation between Cordyceps mycelium culture conditions including LED control conditions and mycelium cordisepin content, and the statistical program was R program ( version 3.4.3) was used.

The factors for culturing the mycelium of Cordyceps Cordyceps mycelium were set as LED wavelength, LED light amount, LED irradiation time, glucose content of the medium, and pH of the medium.

It was evaluated that the higher the coefficient in the Pearson correlation coefficient, the higher the correlation between the culture condition factor and the cordycepin content. 3) fit model was used.

As a result, the correlation between the mycelia culture conditions of Cordyceps Cordyceps mycelium and the mycelium cordycepin content is shown in FIG. 15, and the culture condition that has the greatest effect on the Cordyceps cordyceps mycelium mycelium cordycepin content is the LED wavelength, where the LED wavelength is the mycelium cordycepin content and negative correlation (-0.50).

In addition, the fit model between the LED control condition and the cordycepin content of the cordyceps mycelium is shown in FIG. 16, and the LED wavelength with the highest correlation between the LED control condition and the mycelium cordycepin content was confirmed to be the ‘Ratkowsky’ model. The regression equation using this is shown in Table 5, and through the regression equation between the derived LED control condition and the mycelium cordycepin content, it is determined that the cordycepin content can be predicted using only the LED control conditions (wavelength, light quantity, irradiation time).

No. χ y regression a constant One wavelength Cordycepin content y=a/(1+eb ^-cχ ) a=0.98; b=3.86; c=0.03 2 amount of light Cordycepin content y=a/(1+eb ^-cχ ) a=3.79; b=162.80; c=0.12 3 investigation time Cordycepin content y=(a+bχ)/(1+cχ+dχ ² ) a=27.07; b=-0.29; c=-3.05; d=2.04

2. Establishment of optimal LED conditions to increase the content of mycelium cordycepin

The optimal LED control condition for increasing the content of Cordyceps mycelium cordycepin was used using the Box-Benkhen design (BBD) experimental design of the Response Surface Model (RSM), and the program was designed in Design-Expert Software Version 10 was used.

The fixing factor was P. javanicus mycelium, and the variables were set as the light quantity condition, the glucose content of the medium, and the incubation time. In this case, the range of variables was set through preliminary experiments, and the BBD experimental design was set to a total of 17 conditions as shown in Table 6 below.

Run Independent variables
(coded) Independent variables
(actual) Cordycepin content
content), mg/L X _One X ₂ X ₃ X _One X ₂ X ₃ Y _One One One 0 -One 1338 7.5 24 2074.18 2 0 0 0 669 7.5 72 3100.92 3 One 0 One 1338 7.5 120 1320.56 4 -One One 0 0 10 72 186.37 5 0 0 0 669 7.5 72 3165.12 6 0 -One -One 669 5 24 1772.23 7 -One 0 -One 0 7.5 24 124.43 8 One -One 0 1338 5 72 1913.36 9 -One 0 One 0 7.5 120 119.28 10 0 -One One 669 5 120 1112.81 11 One One 0 1338 10 72 3008.74 12 0 One One 669 10 120 2613.48 13 0 0 0 669 7.5 72 3031.83 14 0 0 0 669 7.5 72 3112.59 15 -One -One 0 0 5 72 93.16 16 0 One -One 669 10 24 3655.11 17 0 0 0 669 7.5 72 3712.73 Independent variables Levels -One 0 One X ₁ : Light quantity, lux 0 669 1338 X ₂ : glucose content, g/50 mL 5 7.5 10 X ₃ : incubation time, h 24 72 120

17 shows a three-dimensional response surface graph showing the interrelationship between factors as a response surface and a second-order polynomial regression model derived from it. lux, when the glucose content of the medium was 9.65274 g/50mL, and the incubation period was 61.2026 hours, it was confirmed that the cordycepin content was increased to 3779.2 mg/L.

In addition, the second-order polynomial equation regression model was analyzed for variance (ANOVA ^{) to obtain R 2} (coefficient of determination) indicating the degree of fit of the response model as shown in FIG. 18 . As a result, R ² (coefficient of determination) was 0.8910, indicating high reliability, and accordingly, the quadratic polynomial equation derived from FIG. 17 was judged to be suitable for predicting the response value of optimal culture conditions for the maximum cordycepin content of Cordyceps Cordyceps. do.

Claims (16)

An inoculation step of inoculating the Snow Cordyceps mycelium in Sabouraud dextrose broth medium; and
Culturing the inoculated medium under LED blue and red mixed light conditions having a wavelength of 450 nm and a wavelength of 650 nm to obtain cordycepin from the cultured Cordyceps mycelium and culture solution; includes,
The mixing ratio of the LED blue and red mixed light is 5: 5, respectively, and 15 g/L of 80 mesh size rice straw is added to the medium, and cultured for 2 to 3 days at 12 hours/day. A culture method to increase the content of cordycepin. delete delete delete delete delete delete delete delete The method of claim 1,
The content of cordycepin in the mycelium of Cordyceps sinensis is,
A culturing method for increasing the cordycepin content of Cordyceps Cordyceps mycelium, characterized in that it has a correlation as shown in Equation 1 below with the amount of light, the glucose content of the medium, and the culture time:
[Equation 1]
Y ₁ =3224.64+974.20X ₁ +571.52X ₂ -307.48X ₃ +250.54X ₁ X ₂ -187.12X ₁ X ₃ -95.55X ₂ X ₃ -1651.51X ₁ ² -272.72X ₂ ² -663.51X ₃ ²
(In Equation 1, Y ₁ is the cordycepin content (mg/L) of the Cordyceps Cordyceps mycelium, X ₁ is the amount of light, X ₂ is the glucose content of the medium, and X ₃ is the culture time.) 11. The method of claim 10,
A culture method for increasing the cordycepin content of the Cordyceps Cordyceps mycelium, characterized in that the culturing conditions for the amount of light, the glucose content of the medium, and the culture time are 500 to 1500 lux, 5 to 15 g/50 mL, and 30 to 100 hours, respectively. 12. The method of claim 11,
A culturing method for increasing the cordycepin content of the Cordyceps Cordyceps mycelium, characterized in that the culturing conditions of the amount of light, the glucose content of the medium and the incubation time are 990.723 lux, 9.65274 g/50mL, and 61.2026 hours, respectively. delete In the method for predicting the cordycepin content of the mycelium of Cordyceps Cordyceps according to claim 1,
(a) With respect to the amount of light (X ₁ ), the glucose content of the medium (X ₂ ), and the incubation time (X ₃ ) by the Box-Behnken design, -1, 0 and 1 were coded as experiments designing a range;
(b) obtaining experimental values for the amount of light, the glucose content of the medium, and the incubation time in the designed experimental range of step (a);
(c) deriving a quadratic regression model expressed by Equation 1 below using the experimental values of step (b); and
(d) predicting the cordycepin content of the Cordyceps Cordyceps mycelium by ANOVA on the quadratic regression model represented by Equation 1 derived in step (c); Cordycepin content prediction method of the mycelium of Cordyceps sinensis, comprising:
[Equation 1]
Y ₁ =3224.64+974.20X ₁ +571.52X ₂ -307.48X ₃ +250.54X ₁ X ₂ -187.12X ₁ X ₃ -95.55X ₂ X ₃ -1651.51X ₁ ² -272.72X ₂ ² -663.51X ₃ ²
(In Equation 1, Y ₁ is the cordycepin content (mg/L) of the Cordyceps Cordyceps mycelium, X ₁ is the amount of light, X ₂ is the glucose content of the medium, and X ₃ is the culture time.) 15. The method of claim 14,
The method for predicting the cordycepin content of the Cordyceps Cordyceps mycelium, characterized in that the culturing conditions of the amount of light, the glucose content of the medium and the culture time are 500 to 1500 lux, 5 to 15 g/50mL, and 30 to 100 hours, respectively. 16. The method of claim 15,
The method for predicting the cordycepin content of the mycelium of Cordyceps Cordyceps mycelium, characterized in that the culture conditions of the amount of light, the glucose content of the medium and the incubation time are 990.723 lux, 9.65274 g/50mL, and 61.2026 hours, respectively.

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