TW202200779A - Medium and method for improving cordycepin and pentostatin production of cordyceps militaris - Google Patents

Abstract

The present invention provides a method for improving the production of Cordycepin and Pentostatin of Cordyceps militaris, which comprises inoculating Cordyceps militaris with a grain solid medium and adjusting the ratio of grain to water, and at least one of malt extract, galactose, yeast extract and peptone is added. Different media formulations are provided to increase the production of Cordyceps militaris fruit bodies or to increase the content of Cordycepin and Pentostatin of Cordyceps militaris.

Description

Culture medium and method for improving the yield of cordycepin and pentostatin of Cordyceps militaris

The invention relates to a culture medium and a method for improving the yield of cordycepin and pentostatin of Cordyceps militaris. Specifically, it relates to a medium and a method for increasing the yield of cordycepin and pentostatin of Cordyceps militaris by means of solid-state culture.

Cordyceps militaris , also known as Cordyceps militaris, is a Cordyceps species belonging to the class of Ascomycetes. The 70% alcohol extract of its mycelium and fruiting bodies has anti-inflammatory and other pharmacological effects. Currently known physiologically active substances of Cordyceps militaris include Adenosine, Cordycepin, Cordycepic acid (or D-mannitol), polysaccharide and pentostatin. (Pentostatin). Adenosine is known to act as a cardioprotective and therapeutic drug for heart failure and to inhibit the release of neurotransmitters in the central nervous system. Cordyceps has diuretic and antioxidant properties and relieves cough and asthma symptoms. Polysaccharides have anti-inflammatory, anti-cancer and immunomodulatory functions.

式1The chemical formula of cordycepin C ₁₀ H ₁₃ N ₅ O ₃ is shown in formula 1, the molecular weight is 251.24, and the chemical name is named 9-(3-deoxy-β-D-ribofuranosyl)adenine according to IUPAC, or 3'-deoxyadenosine , whose structure is a structural analog of adenosine. Therefore, when synthesizing RNA, cordycepin replaces adenosine and is inserted into the synthesizing RNA, so that the synthesized RNA cannot be extended and ends the synthesis of RNA. Studies have pointed out that cordycepin may be intercalated and combined with the phosphate group of DNA to inhibit DNA synthesis.

Formula 1

Studies have shown that cordycepin induces apoptosis of thyroid cancer cells through the calcium ion-calpain-sulfatase-polymerase signaling pathway. Cordycepin can stimulate the production of testosterone in mouse Leyden cells by binding to adenine receptors, activating the PKA transmission pathway and regulating the expression of steroid rate-controlling proteins. And some studies speculate that cordycepin can trigger the activation of thiocysteine-9, -3 and -7 by interacting with adenine receptors, and finally make the MA-10 mouse Leyden tumor cells go to apoptosis. route of. In addition, some studies have used the water extract of Cordyceps militaris to treat human lung cancer A549 cell line. Preliminary animal experiments showed that subcutaneous administration of CMHW with a dose of 15 mg/kg of cordycepin for 5 consecutive days can effectively inhibit tumor growth. The fermented products of Cordyceps militaris after hot water extraction were cultured on canine breast cancer cell lines (DTK-SM2 and DTK-SME-like) and human breast cancer cell lines (HCC1937), respectively. The results showed that when the hot water extract contained cordycepin At 10μg/mL, canine and human breast cancer cell lines can be induced to produce apoptotic bodies, showing that the hot water extract of the solid-state fermentation product of Cordyceps militaris has antitumor activity.

式2Penstostatin (Penstostatin) chemical formula is C ₁₁ H ₁₆ N ₄ O ₄ , molecular weight = 268.2691 IUPAC named (8R)-3-[(2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)oxolan- 2-yl]-3H,6H,7H,8H-imidazo[4,5-d][1,3]diazepin-8-ol, its chemical structure is shown in formula II.

Formula 2

Penstostatin (2'-deoxycoformycin) was initially isolated from cultivated soil microorganisms (Streptomyces antibioticus), its structure is similar to purine, and it can be used as an inhibitor of adenosine deaminase. In 1991, it was approved by the FDA as a commercial drug (Nipent) for the treatment of hairy cell leukemia. Academic literature is currently known to have therapeutic efficacy against leukemia-related diseases such as chronic lymphocytic leukemia, prolymphocytic leukemia, cutaneous T-cell lymphoma, adult T-cell lymphoma-leukemia, and low-grade non-Hodgkin lymphoma tumor activity. It is also used to treat hairy cell leukemia and indolent lymphoproliferative diseases. Case reports suggest that alemtuzumab in combination with pentostatin is a complete response in the treatment of adult T-cell leukemia (ATL). Application study of pentostatin on human parasite-Trypanosoma spp. In the study, feeding the combination of cordycepin (2 mg/kg) and pentostatin (0.2 mg/kg) can effectively eliminate the infected Trypanosoma evansi in mice. Another study fed Trypanosom acruzi-infected mice alone or in combination with cordycepin and pentostatin, which was not successfully cured, but reduced damage from heart inflammation.

As mentioned above, Cordyceps militaris can produce cordycepin and pentostatin with anti-cancer ability, and other species of Cordyceps do not synthesize cordycepin and pentostatin at the same time. Therefore, an object of the present invention is to provide a medium capable of increasing the yield of cordycepin and pentostatin of Cordyceps militaris and a method for increasing the yield of cordycepin and pentostatin of Cordyceps militaris.

In view of the above objects, the present invention provides a method for improving the yield of cordycepin and pentostatin of Cordyceps militaris, comprising: activating bacterial species, comprising preparing a plate medium comprising a potato glucose substrate, and inoculating the mycelium block of each strain on a plate Culture medium, cultivate and activate at 22-25 DEG C; prepare liquid bacterial species, comprise the liquid culture medium of inserting 100 mL of plate medium inoculated with mycelial block, and cultivate 1.5-2 days at 22-25 DEG C, 130-150rpm To obtain liquid strains; prepare solid culture medium, including drying the grains, adding a predetermined proportion of RO water, and inserting liquid strains for cultivation; wherein, the culturing process comprises maintaining 22-25 ° C and culturing in a dark room for 8-10 days, and maintained at 16-19°C for 46-48 days in a lighted environment.

Preferably, the grains include Black rice, Brown rice, Buckwheat, Coix seed, Oat, Pearl barly and Wheat.

Preferably, the ratio of grain to RO water is 1:1.2~1:1.8.

Preferably, the illumination environment in the cultivation process is to cultivate for 46-48 days at 16-19 ° C under the illumination of white light of 100-1200 lux.

Preferably, the solid medium further comprises fructose, glucose, galactose, peptone, yeast extract, malt extract, adenosine monophosphate, glycine, aspartic acid and glutamine. at least one of.

According to an embodiment of the present invention, the present invention further provides a solid medium for improving the yield of cordycepin and pentostatin of Cordyceps militaris, each portion comprising: 3-6 mL of a pre-medium containing strains of Cordyceps militaris; 30 g of oat medium, oat The volume ratio of medium to water is 1.2 to 1.8; and additives.

Preferably, the pre-medium is potato dextrose medium, malt extract medium or yeast malt extract medium.

Preferably, the additive is 0.5-1.5 wt% of at least one of fructose, galactose, glucose, protein gluten, malt extract or yeast extract.

Preferably, the additive is 10-100 ppm of at least one of adenosine monophosphate, glycine, aspartic acid and glutamine.

By the above-mentioned technical features, the culture medium and method for improving cordycepin and pentostatin produced by Cordyceps militaris provided by the present invention have at least the following advantages:

(1) The yield of cordycepin and pentostatin of Cordyceps militaris seeds can be improved by the method provided by an embodiment of the present invention.

(2) The concentration of cordycepin and pentostatin in Cordyceps militaris can be increased through the medium provided by an embodiment of the present invention.

(3) Through the medium provided by another embodiment of the present invention, the yield of the fruiting bodies of Cordyceps militaris can be improved.

In the following description, for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of various exemplary embodiments or implementations of the present invention. It will be apparent, however, that various illustrative embodiments may be practiced without these specific details or with one or more equivalent arrangements. Furthermore, the various exemplary embodiments may vary, but are not necessarily exclusive. For example, the specific shapes, configurations, and characteristics of an exemplary embodiment may be used or implemented in another exemplary embodiment without departing from the inventive concept.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms defined in common dictionaries, for example, should be interpreted as having meanings consistent with their meanings in the relevant art, and should not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

Numerical ranges recited herein can include all subranges within that range with the same numerical precision. For example, the range "1.0 to 10.0" may include all subranges between a minimum value of 1.0 and a maximum value of 10.0, that is, with a minimum value equal to or greater than 1.0 and a maximum value equal to or less than 10.0, eg, 2.4 to 7.6 . Any maximum numerical limitation stated herein can include all lower numerical limitations, and all minimum numerical limitations stated in this specification can include all higher numerical limitations. Accordingly, the applicant reserves the right to amend this specification (including the claimed scope) to expressly recite any sub-ranges subsumed within the ranges expressly recited herein. All of these ranges are inherent in this specification.

The research of the present invention includes screening the most suitable pre-medium, grain medium, culture conditions and the most suitable medium supplement for improving the production of cordycepin and pentostatin of Cordyceps militaris.

In each embodiment of the present invention, the methods for the activation of strains, the preparation of liquid strains, the preparation of fruit body powder after cultivation, and the measurement of adenosine/cordycepin/pentastatin are as follows:

Strain activation

The test strains were stored in a refrigerator at 4°C. When carrying out the test, configure potato dextrose (Potato Dextrose Agar (PDA)) plate medium, the mycelium block of each bacterial strain is inoculated into plate medium, cultivate and activate at 22-25 DEG C, through the bacterial strain after activation again, can carry out follow-up test. The test strains were subcultured every 2 months to avoid the aging and contamination of the strains.

Liquid strain preparation

Test bacterial strain configuration liquid culture medium, 100 ml liquid culture medium is inserted in the triangular flask of 300 ml, at 121 DEG C, 1.5 kg/cm ²⁰ minutes of sterilization, after cooling, the mycelium block (ratio be 2 cm ² /bottle) into the above-mentioned liquid culture medium, cultivate at 22-25 ° C, 130-150 rpm for 1.5-2 days, namely liquid bacterial species.

Fruit body powder preparation

The sampled fruit bodies were placed in a 50°C oven and dried to constant weight, pulverized with a pulverizer and then sieved (40 mesh) to obtain fruit body powder.

Determination of Cordycepin and Pentostatin in Fruiting Body

The extraction method is as follows: add 50 times the volume of ddH ₂ O to the fruit body powder, extract by ultrasonic for 20 minutes, and then centrifuge at 14,000 rpm for 20 minutes, collect the supernatant, filter it through a 0.45 μm microporous membrane, and filter it with a high-efficiency liquid phase layer. The content of cordycepin and pentostatin was analyzed by high performance liquid chromatograph (HPLC).

The HPLC analysis conditions of cordycepin and pentostatin are as follows: Separation column: Hypersil GOLD column (5 μm; Thermo Fisher Scientific Inc., Waltham, MA). Injection volume: 10 μL. Detection wavelength: UV 260 nm (ultraviolet/visible light detector (UV-2075 Plus; JASCO, Co. Ltd., Tokyo, Japan)). Mobile phase: 85% KH ₂ PO ₄ + 15% methanol. Flow rate: 0.5 mL/min.

Screening of the most suitable grain medium is then carried out.

Preparation and screening of grain cultures

Referring to FIG. 1 and FIG. 2, FIG. 1 shows the growth status of Cordyceps militaris in different grains and different cultivation modes according to an embodiment of the present invention. FIG. 2 is a comparison of harvesting results of Cordyceps militaris in different grains and different cultivation modes according to an embodiment of the present invention.

Inoculate 3 mL of Cordyceps militaris liquid strain on 30g of different grains (Black rice, Brown rice, Buckwheat, Coix seed, Oat, Pearl barly) respectively. It was cultured in two temperature control modes with seven kinds of grain culture medium including wheat (Wheat), mode one was 23 °C (dark room, 9 days)-18 °C (light, 47 days), mode two was 23 °C (dark room, 9 days)-18 °C (light, 5 days)-23 °C (light, 42 days). Figure 1 is the growth state of Cordyceps militaris cultured in mode 1 (23 °C (dark room, 9 days) - 18 °C (light, 47 days)). On the 12th day, the growth was obvious. On the 33rd day, the height of the fruiting body of Cordyceps militaris grown in the oat (Oat) medium had reached the bottle mouth of the culture bottle. By the end of the 47th day, except for buckwheat, barley and black rice , Cordyceps militaris grown in the rest of the grain medium of the fruit body height has filled the culture bottle, but the growth of the fruit body density and thickness, affecting the final total yield.

In Figure 2, it can be seen that the medium with higher dry weight of fruiting bodies is oat medium (mode one, day 56, 5.88 ± 0.0.14 g dry weight fruit body/bottle; mode two, day 56, 5.74 ± 0.13 g) g dry weight fruiting body/bottle) and rye medium (mode 1, day 56, 5.68±0.19g dry weight fruiting body/bottle), and the higher content of cordycepin in the fruiting body is the barley medium (mode 2, day 56, 8.06 g dry weight fruiting body/bottle) ±0.08 mg/g dry weight fruiting body), but its fruit body yield was low (4.98±0.38 g dry weight fruiting body/bottle on the 56th day) and the pentostatin content was only 0.66±0.06 mg/g dry weight on the 42nd day. bary fruit body; followed by wheat medium (mode 2, day 56, 5.43±0.34 mg/g dry weight fruit body), but its dry weight of fruit body was also low (the highest on day 56 was 4.43±0.37 g dry weight fruit body/bottle) ). The content of pentostatin in the fruiting body was higher in oat medium (Model 1, on the 42nd day, 1.1.18±0.0.32 mg/g dry weight fruiting body), but its cordycepin content on the 56th day was 1.972.06 ±0.410.44 mg/g dry weight fruiting body; buckwheat medium was in mode 1, the pentostatin content was 1.28±0.12 mg/g dry weight fruiting body on the 42nd day, and its cordycepin content was 3.99±0.89 on the 56th day mg/g dry weight fruiting body, but its fruiting body dry weight was only 4.22±0.17g dry weight fruiting body/bottle on the 56th day. In addition, in the case of using buckwheat medium, oat medium, rye medium and wheat medium of this example, the content of cordycepin in fermented grains (that is, medium with mycelium) was higher than that in fruiting bodies of Cordyceps and the content of cordycepin showed an upward trend with the passage of time.

Next is the optimum medium test for liquid strains.

Referring to Fig. 3 and Fig. 4, Fig. 3 shows the growth conditions of different liquid culture mediums according to an embodiment of the present invention. FIG. 4 is a comparison of harvesting results of different liquid culture media according to an embodiment of the present invention.

Cordyceps militaris mycelium pieces were implanted into potato dextrose (PDB) medium, malt extract (MEB) medium, and yeast extract Malt Broth (YMB) medium, respectively, and cultured for 1.5 days. That is, the liquid strain used for the test, and then take 3 mL and inoculate it in 30g oat medium, and carry out the growth state of cultivation in mode one (23 °C (9 days, dark room)-18 °C (47 days, light)). Figure 3 shows that the original matrix of Cordyceps militaris shows obvious growth on the 12th day after the illumination, and the height of the fruiting body of Cordyceps militaris has reached the bottle mouth of the culture bottle on the 40th day. The density and thickness of the fruiting bodies were significantly different, which affected the final total yield. The dry weight, cordycepin and pentostatin content of the fruiting bodies on the 42nd, 50th and 56th days after inoculation were determined as shown in Figure 4. The experimental results showed that when the current medium used yeast malt extraction medium, higher dry weight of fruit bodies (56th day, 6.26±0.09 g dry weight fruit bodies/bottle) was obtained, but when the former medium used the control potato glucose medium, The content of cordycepin and pentostatin in the fruiting bodies was higher (1.97±0.41 mg/g dry weight fruiting body on day 56; 1.18±0.32 mg/g dry weight fruiting body on day 42). In addition, in this example, the content of cordycepin in the fermented grains also showed an increasing trend, and the content of cordycepin in the yeast malt extract medium was also higher than that in the fruiting bodies.

The next step is to determine the optimal inoculum size for liquid strains.

Referring to FIG. 5 and FIG. 6, FIG. 5 shows the growth status of different liquid bacterial inoculum amounts according to an embodiment of the present invention. FIG. 6 is a comparison of harvesting results of different liquid bacterial inoculation amounts according to an embodiment of the present invention.

Cordyceps militaris liquid strain accesses 30g oat medium, respectively 3 ml/bottle (inoculation volume 10%, v/w), 4.5 ml/bottle (inoculation volume 15%, v/w) and 6 ml/bottle (inoculation volume 15%, v/w) Amount of 20%, v/w), and then in mode one (23 °C (dark room, 9 days)-18 °C (light, 47 days)) to cultivate the growth state of Cordyceps militaris, as shown in Figure 5, by The figure shows that the primordium of Cordyceps militaris grows significantly on the 12th day after the illumination, and the height of the fruiting bodies of Cordyceps militaris has reached the bottle mouth of the culture bottle on the 40th day. When the culture is completed on the 47th day, the density of the growing fruiting bodies and the There were obvious differences in thickness, and the fruiting bodies inoculated with 6 ml/bottle (20 % inoculation volume, v/w) had higher density and finer fruiting bodies. The dry weight, cordycepin and pentostatin content of fruiting bodies on the 42nd, 50th and 56th days after inoculation were determined. The experimental results are shown in Figure 6. The experimental results showed that with the increase of inoculation amount, the yield of fruiting body also increased. When the inoculation amount was 6 ml/bottle (inoculation amount 20%, v/w), a higher dry weight of fruiting body could be obtained (56th day, 6.52 ±0.16 g dry weight fruiting bodies/bottle). However, the content of cordycepin and pentostatin in the fruit bodies of the control group (inoculation volume of 3 mL) was higher (1.97±0.41 mg/g dry weight fruit bodies on the 56th day; 1.18±0.32 mg/g dry weight fruit bodies on the 42nd day) entity).

Next is the optimum water ratio for oat medium.

Referring to Figures 7 and 8, Figure 7 shows the growth conditions of the oat medium in different water ratios according to an embodiment of the present invention. FIG. 8 is a comparison of the harvesting results of the oat medium in different water ratios according to the embodiment of the present invention.

3 mL of Cordyceps militaris liquid strains were added to oat medium with different ratios of oats and water, respectively. 30 g): 1.5 (45 ml) and 1 (30 g): 1.8 (54 ml), carry out mode 1 cultivation, Figure 7 shows the growth state of Cordyceps militaris cultured, and it can be seen that the original matrix of Cordyceps militaris is in the 12th after illumination. On the 40th day, the growth was obvious. By the 40th day, the oat and water ratios of 1:1.5 and 1:1.8 of the fruit body height of the growth of the oat medium had reached the bottle mouth of the culture bottle, and when the culture was completed on the 47th day, it was observed that 1: 1.8 oat medium had higher density and finer fruit bodies. The density of fruiting bodies grown in 1:1.2 oat medium was sparse. The dry weight, cordycepin and pentostatin contents of the fruiting bodies on the 42nd, 50th and 56th days after inoculation were determined. When the water ratio was 1:1.8, the highest yield of fruiting bodies could be obtained (6.68±0.85 g dry weight fruiting bodies/bottle on the 56th day), but when the water ratio of oats was 1:1.2, it was the fruiting bodies of Cordyceps militaris. Cordycepin and pentostatin were higher in content (2.37±1.31 mg/g dry weight fruit body on day 56; 1.47±0.47 mg/g dry weight fruit body on day 42). In addition, in this example, it can be seen in Figure 8 that when the water ratio is 1:1.2, the content of cordycepin in the fruiting body increases over time, which is significantly higher than that of the other two groups, and the fermented grains contain The content of cordycepin was also slightly higher than that of the other two groups at each time point.

The following will discuss the effects of adding different additives to the grain medium on the cultured Cordyceps militaris through the examples of the present invention.

The first is the experiment of adding different carbon sources to the oat medium.

Referring to Figures 9 and 10, Figure 9 shows the growth conditions of the oat medium supplemented with different carbon sources according to an embodiment of the present invention. Fig. 10 is a comparison of the harvesting results of adding different carbon sources to the oat medium according to the embodiment of the present invention.

3 mL of Cordyceps militaris liquid strains were respectively inserted into 30 g of oat medium containing 1% fructose, galactose, and glucose, respectively, to carry out mode one cultivation. Figure 9 shows the growth state of Cordyceps militaris cultured, and it can be seen that the original matrix of Cordyceps militaris The growth is obvious on the 12th day after the illumination, and when the culture finishes on the 47th day, it can be observed that the density of the Cordyceps militaris fruiting body of the growth of the oat medium that adds glucose is sparse, and the density of the fruiting body of the growth of the oat medium that adds galactose is higher. . Figure 10 shows the changes in dry weight, cordycepin and pentostatin content of fruiting bodies of Cordyceps militaris on days 42, 50 and 56. The experimental results showed that Cordyceps militaris was cultivated in the oat medium supplemented with carbon source, and the fruit body yield on the 42nd day after inoculation was lower than that of the control group without carbon source, but the fruit body yield on the 56th day was higher. The increase in fruit body yield (6.10 ± 0.30 g dry weight fruit bodies/bottle) was most significant. However, the content of cordycepin in the fruiting bodies of Cordyceps militaris in the control group without carbon source was higher (1.97±0.41 mg/g dry weight fruiting bodies on the 56th day), and the fruiting bodies produced by the galactose-supplemented oat medium contained pentostatin The content was higher (day 42, 1.27±0.34 mg/g dry weight fruiting bodies).

This was followed by experiments with the addition of different nitrogen sources to the oat medium.

Referring to Fig. 11 and Fig. 12, Fig. 11 shows the growth conditions of the oat medium supplemented with different nitrogen sources according to an embodiment of the present invention. FIG. 12 is a comparison of the harvesting results of adding different nitrogen sources to the oat medium according to the embodiment of the present invention.

3 mL of the liquid strain of Cordyceps militaris was added to 30 g of oat medium containing 1% malt extract, protein gluten, and yeast extract, respectively, for mode 1 cultivation. Figure 11 shows the growth state of Cordyceps militaris during the cultivation. It can be seen that Cordyceps militaris On the 12th day after the illumination, the original matrix grew obviously. On the 33rd day, the fruiting body of the control group and the oat medium with the addition of malt extract grew better, and the addition of proteus and yeast extract was in the later stage of culture. quick.

Figure 12 shows the changes in the dry weight of the fruiting bodies, the content of cordycepin and pentostatin on the 42nd, 50th and 56th days after inoculation of Cordyceps militaris. The experimental results showed that the fruiting body yield (6.10±0.30g dry weight fruiting body/bottle) of Cordyceps militaris cultured in the oat medium supplemented with protein gluten on the 56th day was higher than that of the unadded control group (5.87±0.14g dry weight fruiting body/bottle), However, the yield of fruiting bodies on the 56th day (5.26±0.22g dry weight fruiting bodies/bottle) of the oat medium supplemented with yeast extract decreased compared with the control group without addition, that is, adding 1% yeast extract could not improve the fruiting bodies of Cordyceps militaris Yield. However, the Cordyceps militaris fruiting body of the unsupplemented control group had a higher content of cordycepin (day 56, 1.97±0.41 mg/g dry weight fruiting body), while the fruiting body of the oat medium supplemented with protein gluten had the highest pentostatin content (day 42). day, 1.46±0.20 mg/g dry weight fruiting bodies). In addition, with the addition of a nitrogen source, the content of cordycepin in the fermented grains was higher than that in the fruiting bodies at each time point.

The last is the experiment of adding other supplements to the oat medium.

Referring to Fig. 13 to Fig. 15, Fig. 13 shows the growth status of the oat medium with different additives according to the embodiment of the present invention. Fig. 14 shows the growth conditions of the oat medium with different additives according to the embodiment of the present invention. Fig. 15 is a comparison of the harvest results of the oat medium with different additives according to the embodiment of the present invention.

Each bottle of 30 g oats was put into a 450 mL glass bottle, and 45 mL of different concentrations (10 ppm, 100 ppm) of adenosine monophosphate (AMP), glycine (Glycine), and aspartic acid (Aspartic acid) were added. acid) and glutamine (Glutamine), after the sterilization is completed, add 3 mL of Cordyceps militaris liquid strain for mode 1 cultivation, Figure 13 shows the growth state of Cordyceps militaris when the concentration is 10 ppm, and Figure 14 shows the concentration It is the growth state of Cordyceps militaris when it is 100 ppm. It can be seen from the above two figures that adding the above chemicals will not be disadvantageous to the formation of the original matrix of Cordyceps militaris, and the growth of fruiting bodies is also good.

Figure 15 shows the changes in the dry weight of the fruiting bodies, the content of cordycepin and pentostatin on the 42nd, 50th and 56th days after inoculation of Cordyceps militaris. The experimental results show that the fruit body yield of Cordyceps militaris cultured in the oat medium supplemented with 10-100ppm of the precursor used in the test on the 56th day (5.97-6.32 g dry weight fruiting body/bottle) relative to the unadded control group (5.5.87± 0.14g dry weight fruiting bodies/bottle) were higher, and the yield of fruiting bodies on the 42nd day did not decrease significantly, among which 100 ppm adenosine monophosphate (6.32±0.40g dry weight fruiting bodies/bottle) and (6.32±0.13g dry weight fruiting bodies) and (6.32±0.13g dry weight fruiting bodies) body/bottle) was the most significant for increasing fruit body yield. Cordycepin content of the fruiting bodies of Cordyceps militaris supplemented with 100 ppm aspartic acid (day 56, 2.42±0.32 mg/g dry weight fruiting bodies) was higher than that of the non-supplemented control group, but the pupae formed in the oat medium supplemented with the precursors The pentostatin content of the fruiting bodies of Cordyceps sinensis (0.42-1.01 mg/g dry weight fruiting bodies on the 42nd day) was lower than that of the control group without supplementation.

It can be seen from the above results that potato glucose medium was used in the pre-medium of Cordyceps militaris, and the inoculum amount was 3 mL/bottle in the oat medium. The oat-to-moisture ratio of the oat medium was 1:1.2, and the addition of yeast extract could generate relatively high content of Cordyceps The fruiting bodies of vegan and pentostatin. The pre-medium used yeast malt extract medium, the inoculum volume was 6 ml/bottle, the ratio of oat to water was 1:1.8, and galactose and protein gluten were added to the oat medium to obtain high-yield fruit bodies. When the medium according to the embodiment of the present invention is used, in addition to the fruit body, the fermented grain also has a high content of cordycepin.

In this way, when producing cordycepin and pentostatin by cultivating Cordyceps militaris, a production method that can provide high content or a production method that can provide high yield can be selected according to the constraints of hardware equipment, environment, manpower, etc. , and then achieve the effect of improving efficiency and saving production costs.

While certain illustrative embodiments and implementations have been described herein, other embodiments and modifications will be apparent from this description and, therefore, the inventive concepts are not limited to these embodiments, but rather to broader aspects of the scope of the appended claims. Wide range and various obvious modifications and equivalent arrangements that will be apparent to those of ordinary skill in the art.

none

The following description of the accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the inventive concept. FIG. 1 shows the growth status of Cordyceps militaris in different grains and different culture modes according to an embodiment of the present invention. FIG. 2 is a comparison of harvesting results of Cordyceps militaris in different grains and different cultivation modes according to an embodiment of the present invention. FIG. 3 shows the growth conditions of different liquid culture mediums according to an embodiment of the present invention. FIG. 4 is a comparison of harvesting results of different liquid culture media according to an embodiment of the present invention. FIG. 5 shows the growth conditions of different liquid bacterial inoculum amounts according to an embodiment of the present invention. FIG. 6 is a comparison of harvesting results of different liquid bacterial inoculation amounts according to an embodiment of the present invention. Fig. 7 shows the growth conditions of the oat medium at different water ratios according to an embodiment of the present invention. FIG. 8 is a comparison of the harvesting results of the oat medium in different water ratios according to the embodiment of the present invention. Fig. 9 shows the growth conditions of oat medium supplemented with different carbon sources according to an embodiment of the present invention. Fig. 10 is a comparison of the harvesting results of adding different carbon sources to the oat medium according to the embodiment of the present invention. Fig. 11 shows the growth conditions of the oat medium supplemented with different nitrogen sources according to an embodiment of the present invention. FIG. 12 is a comparison of the harvesting results of adding different nitrogen sources to the oat medium according to the embodiment of the present invention. Fig. 13 shows the growth conditions of the oat medium with different additives according to the embodiment of the present invention. Fig. 14 shows the growth conditions of the oat medium with different additives according to the embodiment of the present invention. Fig. 15 is a comparison of the harvest results of the oat medium with different additives according to the embodiment of the present invention.

Claims (9)

A method for improving the output of cordycepin and pentostatin of Cordyceps militaris, comprising: Activating bacterial species, comprising preparing a flat plate medium containing potato dextrose, inoculating the mycelial block of each bacterial strain into the plate medium, and culturing and activating at 22-25°C; Preparation of liquid bacterial species, comprising inserting the plate medium inoculated with the mycelial block into the liquid medium of 100 mL, and culturing for 1.5-2 days at 22-25 ° C, 130-150 rpm to obtain a liquid bacterial species; preparing a solid culture medium, including drying the grains, adding a predetermined proportion of RO water, and inserting the liquid strain for cultivation; Wherein, the culturing process includes maintaining 22-25 °C in a dark room for 8-10 days, and maintaining 16-19 °C in a light environment for 46-48 days. The method of claim 1, wherein the grains comprise Black rice, Brown rice, Buckwheat, Coix seed, Oat, Pearl barly ) and Wheat. The method as described in claim 1, wherein the ratio of the grain to RO water is 1:1.2~1:1.8. The method as described in item 1 of the claimed scope of application, wherein the illumination environment in the cultivation process is culturing for 46-48 days at 16-19° C. under the illumination of 100 to 1200 lux of white light. The method of claim 1, wherein the solid medium further comprises fructose, glucose, galactose, malt extract, peptone, yeast extract, adenosine monophosphate, glycine , at least one of aspartic acid and glutamine. A solid medium for improving the production of cordycepin and pentostatin of Cordyceps militaris, each serving comprising: 3~6mL of pre-medium containing Cordyceps militaris strain; 30 g of oat medium, the volume ratio of oat medium to water is 1.2 to 1.8; and an additive. The solid medium of claim 6, wherein the pre-medium is a potato dextrose medium, a malt extract medium or a yeast malt extract medium. The solid medium of claim 6, wherein the additive is at least one of fructose, glucose, galactose, malt extract, protein gluten or yeast extract in an amount of 0.5-1.5 wt%. The solid medium of claim 6, wherein the additive is 10-100 ppm of at least one of adenosine monophosphate, glycine, aspartic acid and glutamine.

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