CN112005814A

CN112005814A - Cordyceps sobolifera, solid and liquid culture method thereof and application of extract thereof

Info

Publication number: CN112005814A
Application number: CN202010414468.0A
Authority: CN
Inventors: 吴建一
Original assignee: DAYEH UNIVERSITY
Current assignee: DAYEH UNIVERSITY
Priority date: 2019-05-30
Filing date: 2020-05-15
Publication date: 2020-12-01

Abstract

The invention provides cordyceps sobolifera, a solid and liquid culture method thereof and application of extracts thereof. The solid state culture method of cordyceps sobolifera comprises the following steps: culturing the cordyceps sobolifera strain in a liquid culture medium to be used as a liquid strain, wherein the sequence of the cordyceps sobolifera strain is shown as SEQ ID NO:1 is shown in the specification; inoculating liquid strains in a solid culture medium according to a specific inoculation ratio; and culturing for a predetermined time under a specific illumination intensity with a specific light source type to obtain mycelium and fruiting body of the Cordyceps cicadae strain.

Description

Cordyceps sobolifera, solid and liquid culture method thereof and application of extract thereof

Technical Field

The invention relates to a cicada fungus, a solid and liquid culture method thereof and application of an extract thereof, in particular to a cicada fungus with a sequence shown in SEQ ID NO:1, a solid and liquid culture method thereof and application of an extract thereof.

Background

The medicinal fungi have medicinal efficacy, so the medicinal fungi are popular in global markets, and the research of the medicinal fungi is a very important subject. From ancient times to date, the production of secondary metabolites by medicinal fungi has largely been through a number of different mechanisms. In humans, these secondary metabolites may be harmful substances, but may also have biological activity that can improve health or prevent diseases.

Wherein Cordyceps cicadae (Cordyceps cicadae, C. cicadae) belongs to the family of Ceramiaceae of Cordyceps, and is parasitized in pupa Cicadae or larva of cicada, and the pupa Cicadae or larva of cicada as host is converted into Cordyceps cicadae mycelium as nutrient source, and then bud-shaped fruiting body is grown. As such, cordyceps sobolifera contains numerous secondary metabolites with biological activity. The gene sequence of the cordyceps sobolifera and the growth environment conditions thereof are different, so that the active ingredients of the cordyceps sobolifera collected or cultured in various regions are greatly influenced, and the application of the cordyceps sobolifera extract is correspondingly influenced.

At present, cordyceps sobolifera can be classified as natural collection or artificial culture according to the source. However, the natural collection method is affected by timeliness, locality, and the like, and a large amount of collection may destroy the environment.

Therefore, there is still a need for a method capable of culturing cordyceps sobolifera fruiting bodies in a solid medium; and a liquid culture method of cordyceps sobolifera capable of reducing culture time and reducing the rate of field bacterial pollution is still needed.

Disclosure of Invention

In view of the above problems in the prior art, the present invention provides a method for solid-state culture of cordyceps sobolifera, comprising: culturing Cordyceps cicadae strain (SEQ ID NO:1) in liquid culture medium as liquid strain; inoculating liquid strains in a solid culture medium according to a specific inoculation ratio; and culturing for a predetermined time under a specific illumination intensity with a specific light source type to obtain mycelium and fruiting body of the Cordyceps cicadae strain.

Preferably, the solid-state culture method, wherein the solid-state culture medium comprises one or more combinations of red beans, mung beans, soybeans, black beans, rice beans, red coix seeds, white coix seeds, brown rice, white rice, purple rice, millet, decagrain, red quinoa, peru quinoa, oats, oatmeal, wheat, buckwheat, sorghum, and corn.

Preferably, the specific light source category comprises one or more combinations of red light, blue light, white light and infrared light.

Preferably, the contents of uracil, uridine, adenine and adenosine in the mycelium and the fruiting body of the cordyceps sobolifera strain are 0-0.42 mg/mg, 0-0.09 mg/mg, 0-0.07 mg/mg and 0-1.05 mg/mg respectively.

Another object of the present invention is to provide a method for liquid culture of cordyceps sobolifera, which comprises: culturing the cordyceps sobolifera strain in a liquid culture medium, wherein the sequence of the cordyceps sobolifera strain is shown as SEQ ID NO:1 to obtain mycelium of cordyceps sobolifera strain; wherein the liquid medium comprises a basal medium.

Preferably, the basal medium comprises a carbon source, and the carbon source comprises one or more combinations of glucose, fructose, xylose, sucrose, maltose, lactose, dextrin, carboxymethyl cellulose, soluble starch, and mannitol.

Preferably, the basal medium comprises a nitrogen source, and the nitrogen source comprises one or more combinations of peptone, yeast extract, malt extract, beef extract, casein, sodium nitrate, potassium nitrate, and ammonium sulfate.

Preferably, the nitrogen source accounts for 0.1-3% of the total volume of the basic culture medium.

Preferably, the contents of uracil, uridine, adenine and adenosine in the mycelium of the cordyceps sobolifera strain are 0.5-3 mg/mg, 0.05-0.6 mg/mg, 0.02-0.9 mg/mg and 0.1-2.91 mg/mg respectively.

The other purpose of the invention is to provide a cordyceps sobolifera with a sequence shown as SEQ ID NO:1, and has at least the productivity of polysaccharides, uracil, uridine, adenine and adenosine.

Another object of the present invention is to provide a use of a cordyceps sobolifera extract for preparing an antioxidant, wherein the cordyceps sobolifera extract is prepared from a cordyceps sobolifera extract with a sequence shown as SEQ ID NO:1, and extracting the cordyceps sobolifera shown in the formula 1.

Another object of the present invention is to provide a use of a cordyceps sobolifera extract for preparing an inhibitor for inhibiting tyrosinase activity, wherein the cordyceps sobolifera extract is prepared from cordyceps sobolifera extract with a sequence shown as SEQ ID NO:1, and extracting the cordyceps sobolifera shown in the formula 1.

Another object of the present invention is to provide a use of a cordyceps sobolifera extract for preparing an anti-inflammatory agent, wherein the cordyceps sobolifera extract is prepared from a cordyceps sobolifera extract having a sequence shown as SEQ ID NO:1, and extracting the cordyceps sobolifera shown in the formula 1.

The invention has the following advantages:

(1) since the Cordyceps sobolifera of the present invention is Cordyceps cicadae Wu-BFP14(SEQ ID NO:1), a different ratio of Cordyceps sobolifera extract to that of the known Cordyceps sobolifera can be obtained.

(2) The culture method of the cordyceps sobolifera not only can culture the mycelium of the cordyceps sobolifera, but also can culture the fruiting body of the cordyceps sobolifera, thereby obtaining the contents of polysaccharide, active ingredients, total phenols and flavonoid with different proportions with the mycelium of the cordyceps sobolifera.

(3) The culture method of cordyceps sobolifera of the present invention is a culture method capable of adjusting the levels of the components obtained from the extract by adjusting the parameters of the culture time, the culture humidity, the light source type, the illumination intensity, the culture humidity, the carbon source type, the nitrogen source type, the mineral elements, the culture grain type, etc., and thus can be adjusted as required.

(4) Compared with the field culture of cordyceps sobolifera, the culture method of cordyceps sobolifera can achieve the purposes of reducing culture time, reducing the field bacterial pollution rate and improving the morphological stability of cordyceps sobolifera mycelia and sporocarp.

(5) The cordyceps sobolifera extract has the total polysaccharide content of 4-28 g/L, the biomass content of 2-9 g/L, the uracil content of 0-3 mg/mg, the uridine content of 0-0.6 mg/mg, the adenine content of 0-0.9 mg/mg, the adenosine content of 0-2.91 mg/mg, the total flavonoid content of 0.1-50 mg/mg and the total phenol content of 1.5-25 mg/mg, so the cordyceps sobolifera extract and the cordyceps sobolifera extract have the corresponding application in preparing antioxidants, inhibitors for inhibiting tyrosinase activity and anti-inflammatory agents.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1a is a schematic process diagram of the solid-state culture method of Cordyceps sobolifera according to the present invention.

FIG. 1b is a schematic diagram showing the steps of the method for culturing Cordyceps sobolifera in a liquid state according to the present invention.

FIG. 2 is an image of Cordyceps cicadae according to the present invention.

FIG. 3 is a solid-state culture diagram of Cordyceps sobolifera according to the present invention.

FIG. 4 is a liquid culture diagram of Cordyceps sobolifera according to the present invention.

FIG. 5 is a solid-state culture diagram of Cordyceps sobolifera according to the present invention.

Fig. 6 to 8 are analysis diagrams of examples 1 to 11 of the present invention.

Fig. 9 to 11 are analysis diagrams of examples 12 to 21 of the present invention.

Fig. 12 to 14 are analysis diagrams of examples 22 to 30 of the present invention.

Fig. 15 to 18 are analysis diagrams of examples 31 to 28 of the present invention.

Fig. 19 to 22 are analysis diagrams of examples 39 to 45 of the present invention.

Fig. 23 to 26 are analysis diagrams of examples 46 to 53 of the present invention.

Fig. 27 to 30, which are analysis diagrams of examples 54 to 73 of the present invention.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

In order to make the aforementioned objects, technical features, and advantages of practical implementation more comprehensible to those of ordinary skill in the art, embodiments accompanying with the drawings are described in more detail below.

The invention provides cordyceps sobolifera with a sequence shown as SEQ ID NO:1, and has at least the productivity of polysaccharides, uracil, uridine, adenine and adenosine. The culture method for culturing cordyceps sobolifera is exemplified as follows.

Referring to FIG. 1a and FIG. 1b, there are shown process diagrams of a solid state culture method and a liquid state culture method of Cordyceps sobolifera, respectively, according to the present invention.

As shown in FIG. 1a, in step S10, Cordyceps sobolifera strain is cultured in liquid medium as liquid strain. Wherein the cicada fungus strain is Cordyceps cicadae Wu-BFP14, and the sequence of the cicada fungus strain is shown as SEQ ID NO:1 is shown. In step S20, liquid strains are inoculated into the solid medium at a specific inoculation ratio. In step S30, the cordyceps sobolifera strain is cultured for a predetermined time under a specific illumination intensity with a specific light source type to obtain mycelium and fruiting body of the cordyceps sobolifera strain. As shown in FIG. 1b, in step S40, Cordyceps sobolifera strain is cultured in liquid medium. Wherein the cicada fungus strain is Cordyceps cicadae Wu-BFP14, and the sequence of the cicada fungus strain is shown as SEQ ID NO:1 is shown. The liquid medium may comprise a carbon source. In step S50, a mycelium of cordyceps sobolifera strain is obtained.

Preferably, the total polysaccharide content of the cordyceps sobolifera is 4-28 g/L.

Preferably, the biomass content of the cordyceps sobolifera is 2-9 g/L.

Preferably, the content of uracil, uridine, adenine and adenosine in the cordyceps sobolifera is 0-3 mg/mg, 0-0.6 mg/mg, 0-0.9 mg/mg and 0-2.91 mg/mg respectively.

Preferably, the contents of total flavonoids and total phenols in the cordyceps sobolifera are 0.1-50 mg/mg and 1.5-25 mg/mg respectively.

Meanwhile, the invention also provides application of the cordyceps sobolifera extract in preparing the antioxidant, wherein the cordyceps sobolifera extract is prepared from cordyceps sobolifera extract with a sequence shown as SEQ ID NO:1, and extracting the cordyceps sobolifera shown in the formula 1.

Furthermore, the invention also provides the application of the cordyceps sobolifera extract in preparing the inhibitor for inhibiting the activity of tyrosinase, wherein the cordyceps sobolifera extract is prepared from cordyceps sobolifera extract with a sequence shown as SEQ ID NO:1, and extracting the cordyceps sobolifera shown in the formula 1.

In addition, the invention also discloses application of the cordyceps sobolifera extract in preparing an anti-inflammatory agent, wherein the cordyceps sobolifera extract is prepared from a cordyceps sobolifera extract with a sequence shown as SEQ ID NO:1, and extracting the cordyceps sobolifera shown in the formula 1.

The following is a detailed description of each example.

Research method

1.1 Strain selection

The cordyceps sobolifera strain selected by the method is automatically screened in the three gorges mountainous area of Taiwan, the screened strain is cultured, the mycelium of the strain is collected, the genome DNA of the strain is extracted, and the DNA is amplified by utilizing the PCR technology. After cloning and order-resolving, the sequence is compared with other cicada fungus strains in NCBI gene bank by BLAST. The cordyceps sobolifera species to be compared with the present invention include known cordyceps sobolifera species of KF740422, KF373077, EU807996, KJ173474, FJ765285, KJ173475, FJ765283, KJ173461, FJ765282, AB916360, KP771879, AY 24627, KJ857272, EU573331, JQ283963, EU573333, AB086631, FJ765284, KJ173448, FJ765280, JX488475 and KJ 173447.

1.2 liquid culture of Cordyceps sobolifera mycelia

A strain piece with the diameter of 5mm is taken from the cordyceps sobolifera mycelium of a culture dish and is inoculated into a sterilized liquid culture medium, and the influence of parameters such as different environmental factors, types and concentrations of carbon sources, types and concentrations of nitrogen sources, trace elements and the like on the growth of the cordyceps sobolifera strain is discussed. The process comprises the following steps: the medium was inoculated from a disk using a metal tube of 5mm diameter (a inoculum size is a 5mm diameter pellet) to a 125mL flat-bottomed flask containing the medium, and shake-cultured at 28 ℃ at 130rpm, followed by filtering and drying the mycelia with a filter paper of 0.45 μm pore size. Then, the steps of subpackaging, freezing the bacteria and the like can be carried out.

1.3 Strain activation

Inoculating the frozen strain to a culture dish containing a Potato glucose culture medium (PDA) for culturing, cutting 2 PDAs with the diameter of 1cm on the culture dish with a milling cutter, inoculating the PDA to 250mL of strain liquid activated culture medium, and culturing for 7 days at 25 ℃ in a constant-temperature shaking incubator at 150 rpm. Wherein the liquid activation medium is Potato Dextrose Broth (PDB).

1.4 different carbon sources

The above formula of the activation medium was used as a substrate, and the analysis was performed with the addition of no carbon source and the addition of 2% of different carbon sources. The carbon source includes Glucose (Glu), Fructose (frutose, fruu), Xylose (Xylose, Xyl), Sucrose (sum), Maltose (Maltose, Mal), lactose (Lactos, Lac), Dextrin (Dextrin, Dex), carboxymethyl cellulose (CMC), Soluble Starch (SS), and Mannitol (Mannitol, Man). Wherein the culture time is 7-14 days; the culture temperature is 23-28 ℃; and the culture humidity was 100%.

1.5 different Nitrogen sources

Glucose was used as a carbon source for comparison, and different nitrogen sources were added in an amount of 0.5% for analysis. Among them, the nitrogen source includes organic nitrogen sources such AS Peptone (PT), Yeast Extract (YT), Malt Extract (ME), Beef Extract (Beef Extract, BE), and Casein (Casein, Cas), and inorganic nitrogen sources such AS Sodium Nitrate (SN), Potassium Nitrate (PN), Ammonium Sulfate (AS), Sodium nitrite (Sodium nitrate), and Urea (Urea). Wherein the culture time is 7-14 days; the culture temperature is 23-28 ℃; the cultivation humidity was 100%.

In addition, since mineral elements are also a main element in the growth of mycelia, Glucose (Glu) and Yeast extract (YT) were used as carbon and nitrogen sources, and magnesium sulfate (MgSO 2 mM) was added thereto₄) Calcium sulfate (CaSO)₄) Sulfuric acid sulfiteIron (FeSO)₄) Iron (Fe) sulfate₂(SO₄)₃) Manganese sulfate (MnSO)₄) Zinc sulfate (ZnSO)₄) Copper sulfate (CuSO)₄) Nickel sulfate (NiSO)₄) And 5mM magnesium sulfate. Wherein the culture time is 7-14 days; the culture temperature is 23-28 ℃; the cultivation humidity was 100%.

1.6 solid-state culture of Cordyceps sobolifera

Inoculating the liquid strain of Cordyceps sobolifera strain cultured in liquid state into sterilized different cereals such as bean cereals (such as semen Phaseoli (RB), semen Phaseoli Radiati (GB), semen glycines (YB), semen Sojae Atricolor (BB), and semen oryzae Sativae (MB), rice cereals (such as semen Coicis (RY), semen Coicis (WY), brown rice (RR), White Rice (WR), Purple Rice (PR), semen Setariae (MR), and semen Setariae (Ten), quinoa cereals (TR), Chenopodium quinoa (MUR), herba Avenae Fatuae (YM), Oatmeal (OL), semen Tritici Aestivi (MM), semen Fagopyri Esculenti (CM), and other cereals (such as jowar (H) and semen Maydis (PC)) with 10% of total volume of solid culture medium.

1.7 different illumination intensities

Taking 30g of wheat as a solid culture medium, and adding 1: sterilizing culture medium with solid-liquid ratio of 1.5, inoculating 5% liquid strain, culturing in dark room until white mycelium grows on the surface, transferring with different light intensities (0, 50, 100, 250, 500, 1000 and 1500Lux), and culturing at 25 deg.C for 42 days.

1.8 different light sources

Taking 45g of wheat as a solid culture medium, and adding 1: sterilizing culture medium with solid-liquid ratio of 2, inoculating 5% liquid strain, culturing in dark room until white mycelium grows on the surface, and culturing at 25 deg.C for 42 days with different light sources. The Light source is a Light emitting diode Light source including 5 different luminescent substances (Cw) of a micro tissue culture system (MEL, Mini E Light, Light biotechnology), 100% red Light, 70% red Light and 30% blue Light, 30% red Light and 70% blue Light, and 100% blue Light, and the Light source parameters of the Light source are shown in table 1.

TABLE 1 light source parameters

Code number	Light ratio	Luminous intensity (Lux)
			2700K	100% white light, color temperature 2700K	3410
5000K	100% white light, color temperature 5000K	2479
			9R	100% Red light	538
9B	100% blue light	69.2
			3R6B	30% red light and 70% blue light	14.09
6R3B	70% red light and 30% blue light	238
			3R3B3IR	33.33% red light, 33.33% blue light, 33.33% infrared light	0.01
9IR	100% infrared ray	0.01

The above parameters are shown in tables 2 and 3. Wherein "- -" means not added.

TABLE 2 liquid culture parameters

TABLE 3 solid State culture parameters

1.9 extraction method

1g of cordyceps sobolifera mycelium or powder obtained after solid culture and drying is added with a solvent for extraction, and then the mixture is centrifuged at 9000rpm for 10 minutes, and supernatant is taken as extraction liquid. Wherein the solvent can be deionized water, alcohol, acid solution or alkali solution.

1.10 sample treatment

Adding 1g of lyophilized mycelium into 10mL of distilled water, placing in an autoclave at 121 ℃ for hot water extraction for 20min, and filtering to obtain an extract. The supernatant of the culture broth (the supernatant described in the extraction method) and the intracellular extract (the extract from the mycelium after extraction) were filtered through a 0.45 μm microfiltration membrane and analyzed by HPLC.

2.1 analysis of polysaccharide content

0.5mL of a sample was taken, 0.5mL of Phenol (Phenol, 10%, v/w) was added, 2.5mL of Sulfuric acid (36N) was added, the mixture was uniformly mixed and reacted at room temperature for 10 minutes, the mixture was placed in water at 25 ℃ and the temperature was reduced for reaction for 15 minutes, and the absorption spectrum wavelength of the reacted sample was recorded at 490 nm.

2.2 analysis of Cordycepin and adenosine content

10mg of each cordycepin and adenosine standard substance is taken, dissolved in 10mL of Methanol (Methanol, 15%), analyzed by High Performance Liquid Chromatography (HPLC) to respectively manufacture a detection line, and the peak areas of the cordycepin and the adenosine are substituted into a linear regression equation to obtain the concentrations of the cordycepin and the adenosine.

2.3 uracil, uridine, adenine and adenosine content analysis

Uracil (Uracil), Uridine (Uridine), Adenine (Adenine) and Adenosine (Adenosine) standards were prepared separately and dissolved in 10mL deionized water and analyzed by high performance liquid chromatography to make calibration lines separately. The operating conditions for HPLC were as follows: the column is Phenomenex Luna 5 mu C18100A; the Mobile phase is 0.02M potassium dihydrogen phosphate (KH)₂PO₄): methanol (85: 15); the flow rate is 0.8 mL/min; UV detector with wavelength 254 nm; and the injection amount was 20 μ.

2.4 Total Phenols Content (TPC)

1mL of the sample solution was added to 1mL of Folin-Ciocalteu reagent, mixed and reacted for 3 minutes with shaking, and then 1mL of Sodium carbonate (Sodium carbonate, 10%, v/w) was added and reacted for 30 minutes at room temperature in the absence of light, and the absorbance spectrum wavelength was recorded at 735 nm. A calibration curve was prepared using Gallic acid (galic acid) as a standard, and the total amount of phenolic compounds in the sample was represented by the relative Gallic acid equivalent (GAE mg/g) per gram of the sample by interpolation.

2.5 Total Flavonoids Content (TFC)

250 μ L of sample solution was taken and 1.25mL of deionized water and 75 μ L of sodium nitrite (NaNO) were added₂5%, v/w), mixed and reacted for 6 minutes, 150. mu.L of aluminum chloride (AlCl) was added₃10%, v/w), mixed for 5 minutes, and then 0.5mL of 1M sodium hydroxide (NaOH) and 275. mu.L of deionized water were added, and centrifuged at 9,000rpm for 3 minutes using a high speed centrifuge, and the supernatant was recorded at an absorption spectrum wavelength of 510 nm. A calibration curve is prepared by using a Quercetin (Quercetin) standard substance, and the relative Quercetin equivalent (QE mg/g) in each gram of sample is obtained by an interpolation method to represent the total amount of flavonoid compounds in the sample.

Discussion of results

3.1 morphological characteristics of Cordyceps sobolifera

Referring to fig. 2, it is an image of cordyceps sobolifera of the present invention. Wherein part (a) represents the whole appearance of wild cordyceps sobolifera; (b) part represents a fruit body; (c) part represents the polypide; (d) part represents microscope photograph of fruiting body; (e) part represents microscope photograph of fruiting body; (f) part represents microscope photograph of fruiting body; (g) part represents microscope photograph of fruiting body; (h) part represents the mycelium micrographs; and (i) part represents the mycelium micrographs.

The cicada fungus disclosed by the invention is a Chinese Taiwan indigenous big cicada fungus of a pure strain of the cicada fungus which is separated after being collected from a bamboo forest in a Chinese Taiwan mountain area. The surface of fresh cordyceps sobolifera is coated by white or grey white hypha, the dried cordyceps sobolifera becomes light yellow to brown, except the forms of the epidermis, the compound eyes and the three pairs of chest and feet are not changed, all organs are coated by the hypha, and the prolate elliptic sclerotium with the size of about 3 centimeters and the diameter of about 1-1.1 centimeters is formed. The sclerotia grow 3-4 antler-shaped or rod-shaped branches with a length of about 4-10 cm. When the wild cordyceps sobolifera fruiting body is observed by an optical microscope, a plurality of branches are shown in the cordyceps sobolifera fruiting body part, and an oval conidium exists, while in the cordyceps sobolifera fruiting body, only a plurality of filamentous structures exist.

Referring to FIG. 3, it is a solid state culture of Cordyceps sobolifera according to the present invention. Wherein part (a) represents plate culture; (b) parts represent plating close-ups; (c) part represents the mycelium micrographs; and (d) part represents mycelium micrographs.

After the cordyceps sobolifera fruiting body is cultured in a PDA culture dish for 10 days, white to off-white felty to fluffy cordyceps sobolifera mycelium with the diameter of about 6-8 cm can grow out, the back surface of the colony presents semi-yellow to orange yellow hyphae with color-releasing furrows, the inner wall of the hyphae of the cordyceps sobolifera is smooth and transparent tubular, and a plurality of dense and irregular branches are presented, and the size of the branches is about 2.0-3.5 mu m.

Referring to FIG. 4, it is a liquid culture diagram of Cordyceps sobolifera according to the present invention. Wherein part (a) represents a liquid shake flask culture; (b) part represents plate culture; (c) part represents a liquid shake flask culture close-up; (d) part represents a mycelium micrograph; (e) part represents a mycelium micrograph; (f) part represents a mycelium micrograph; (g) part represents a mycelium micrograph; (h) part represents a mycelium micrograph; and (i) part represents a mycelium micrograph.

After cordyceps sobolifera is cultured in a liquid state, the cordyceps sobolifera is gradually converted into a culture solution from light pink to light purple from a light yellow culture medium, the mycelium of the cordyceps sobolifera liquid fermentation is observed by using an optical microscope, the mycelium presents a fine thread in a hair structure shape like jute under low multiplying power, and the liquid culture mycelium of the cordyceps sobolifera is similar to the mycelium of the cordyceps sobolifera cultured on a culture dish in shape, has a transparent tubular shape with a smooth inner wall, presents a plurality of dense and irregular branches, and the thick branches are about 1.5 mu m-3.0 mu m, are smaller than the cordyceps sobolifera culture dish and have higher density than the mycelium of the cordyceps sobolifera culture dish. After the liquid culture medium is kept still for 1 to 2 weeks, the surface of the culture medium is changed to form a thin layer of cordyceps sobolifera hypha membrane, the surface of the hypha membrane is observed by an optical microscope to find that the hypha membrane has a relatively multi-layer filamentous structure, dense hemp like transparent thin threads with hair structure shapes can be gradually found along with magnification of the microscope, and after the hypha membrane is magnified to 500 times, the hypha is similar to the hypha of a mycelium and a culture dish in shape, has a transparent tubular shape with a smooth inner wall and presents a plurality of dense and irregular branches, and the branches are about 5.0 to 10 mu m thick.

Referring to FIG. 5, it is a solid state culture of Cordyceps sobolifera according to the present invention. Wherein part (a) represents a cicada fungus solid culture; (b) part represents a close-up of the fruiting body; (c) part represents a very close-up of the substrate; (d) part represents the micro-photograph of the fruiting body; and (e) part represents a mycelium micrograph.

Inoculating Cordyceps cicadae strain into grain coarse cereals, culturing for 1-2 months to obtain artificial solid cultured fruiting body and mycelium, wherein the fruiting body is white or grey white, 4-10 cm long and 0.1-0.5 cm thick, and becomes light yellow to brown after drying, but the length and thickness of the fruiting body are obviously shrunk. Further observation with an optical microscope revealed that the artificial fruiting body had a multi-layered structure, and the distribution of many hyphae was observed around the fruiting body. On the artificial mycelium, a multi-layered filamentous structure is present with similar results to those of the wild cicada bodies.

3.2 Strain identification of Cordyceps sobolifera

The local cordyceps sobolifera is firstly judged in appearance form and is respectively cultured in liquid state and solid state. The classification of general fungi is mainly based on ribosomal DNA, and the ribosomal DNA has very high retention on fungi aiming at 18SRrna gene, 5.8Rrna gene or 28SRrna gene, and is mainly used for the research of the classification of the family genus above. An Internal Transcribed Spacer (ITS) is respectively arranged between the 5.8S Rrna gene and the 18S and 28S Rrna genes, and is divided into ITS1 and ITS2, while ITS1 and ITS2 have faster evolution rate, and the gene sequences of different species in the same genus are obviously different. Therefore, the ITS sequence of the strain of Isaria cicadae was analyzed and compared with the ITS sequences of 22 different species of Isaria cicadae (Cordyceps cicadae) on the GenBank of NCBI (NCBI GenBank), and the similarity of the sequence and the sequence of the Isaria cicadae was found to be more than 99%, confirming that the selected strain was the Isaria cicadae strain and the strain was named Cordyceps cicadae Wu-BFP14 and the gene sequence thereof was uploaded to the GenBank of GenBank with the Accession number (Accession number) KX289580, hereinafter abbreviated as BFP14(SEQ ID NO: 1).

3.3 different carbon sources

Referring to fig. 6, which is an analysis diagram of examples 1 to 11 of the present invention. Wherein the parts (a) to (d) represent pH, extracellular polysaccharide (EPS, unit: g/L), Intracellular polysaccharide (IPS, unit: g/L), and Biomass (Biomass, unit: g/L), respectively.

The pH value of the fermentation broth cultured by using different carbon sources is not greatly different, the BFP14 is maintained at about pH5-pH6, and is slightly higher than that when CMC is used as the carbon source, and is about pH 7. BFP14 was found to be available in monosaccharides (Gly, Fru and Xyl), disaccharides (Sur, Mal and Lac), polysaccharides (Dex, CMC and SS) and even polyols (Man).

The BFP14 mycelium has 2.11g/L mycelium biomass in terms of biomass and carbon source adding condition, and has a phenomenon of significant increase in mycelium biomass after adding various carbon sources, wherein Gly is the preferred culture carbon source, the mycelium biomass can reach 6.55g/L, which is more than three times higher than the condition without carbon source, and Man is the second mycelium biomass which is about 6.21g/L, and is 6.15g/L of SS. In terms of intracellular polysaccharide content, the same Gly content preferably amounts to 6.19g/L, and in the following the lactose (Lac) amount to 5.25 g/L. In the case of extracellular polysaccharides, mannitol (Man) is preferably up to 17.8g/L, followed by Glu up to 14.51g/L, which is significantly different from other carbon sources for extracellular polysaccharide production.

Referring to fig. 7, which is an analysis diagram of examples 1 to 11 of the present invention. Wherein the moieties (a) to (d) represent the content (mg/mg) of uracil, uridine, adenine and adenosine, respectively.

It was found that Xyl as a carbon source had the highest contents of uracil, uridine and adenine, respectively, of 2.91mg/g, 0.53mg/g and 0.10 mg/g; under the condition that Mal is used as a carbon source, uracil and uridine are respectively 2.49mg/g and 0.44mg/g, which are the contents of xylose which is the next carbon source; followed by Sur, 2.39mg/g and 0.48mg/g, respectively, followed by glucose and fructose. Since Mal and Sur belong to disaccharides formed by polymerization of two molecules of glucose and one molecule of fructose, Glu and Fru are presumed to promote the production of uracil and uridine. In the case of adenosine, preference can be clearly found for CMC, which has a content of about 2.91mg/g, secondly for Lac having a content of 2.65mg/g and subsequently for Glu having a content of 2.22 mg/g.

Referring to fig. 8, which is an analysis diagram of examples 1 to 11 of the present invention. Wherein, the parts (a) and (b) respectively represent the content (mg/mg) of total flavonoids and total phenols.

In terms of the total phenol content, it was found that Xyl, which is the highest carbon source, had a gallic acid content of 9.87mg/g, followed by Dex having a gallic acid content of 6.93mg/g, followed by glucose having a gallic acid content of 5.35mg/g, and none of the remaining carbon sources produced gallic acid at a content higher than 4 mg/g. In terms of flavonoid content, the same Xyl carbon source was the highest, with a quercetin content of 19.76mg/g, followed by Lac with a quercetin content of 15.5 mg/g.

In summary, Glu is a carbon source that is utilized by most bacteria and fungi, is relatively easy to obtain and inexpensive in cost, and is superior to most carbon sources in terms of active ingredients such as uracil, uridine, adenine and adenosine, and glucose is used as the carbon source for the following examples.

Reference is made to fig. 9, which is an analysis diagram of examples 12 to 21 of the present invention. Wherein the components (a) to (d) represent pH, extracellular polysaccharide content, intracellular polysaccharide content, and biomass content, respectively.

All five organic nitrogen sources are available for BFP14, whereas only SN, PN and AS can be used among inorganic nitrogen sources. Overall, culture conditions with organic nitrogen sources are superior to inorganic nitrogen sources. Among the organic nitrogen sources, BFP14 mycelium biomass is preferably YT, which can achieve 8.37g/L of mycelium biomass, approximately three times higher than the condition without nitrogen source (3.11g/L), Cas (7.32g/L) and MT is only 4.50g/L of mycelium biomass at the lowest. SN is preferred as inorganic nitrogen source, which can obtain 5.05g/L mycelium biomass, and PN (5.01g/L) is the second best, and ammonium sulfate is not suitable for BFP14 mycelium biomass growth.

In terms of polysaccharides, MT is preferred, the contents of intracellular or extracellular polysaccharides are 17.54g/L and 54.96g/L, respectively, in inorganic nitrogen source polysaccharides such as mycelium biomass, SN (11.53g/L, 50.45g/L) is preferred, and PN (10.17g/L, 46.37g/L) is preferred. In the present invention, BFP14 is superior to inorganic nitrogen sources in terms of organic nitrogen sources, and in terms of inorganic nitrogen sources, nitrate nitrogen sources are superior to ammonium salt nitrogen sources, regardless of the biomass of mycelia, intracellular polysaccharides and extracellular polysaccharides.

Referring to fig. 10, which is an analysis diagram of examples 12 to 21 of the present invention. Wherein the moieties (a) to (d) represent the content (mg/mg) of uracil, uridine, adenine and adenosine, respectively.

Among the bioactive components, YT is highest in all four bioactive components uracil, uridine, adenine and adenosine, with 1.69mg/g, 0.17mg/g, 0.91mg/g and 0.98mg/g, respectively, in organic nitrogen sources, followed by Cas, PT, BT and MT in order, wherein in MT, the presence of uridine is not detected, while in inorganic nitrogen sources, the same nitrate nitrogen source is found to be superior to the ammonium nitrogen source, with the four bioactive components having advantages in SN and PN, respectively, but in SN PN.

Reference is made to fig. 11, which is an analysis diagram of examples 12 to 21 of the present invention. Wherein, the parts (a) and (b) respectively represent the content (mg/mg) of total flavonoids and total phenols.

In terms of total phenol content, YT is a nitrogen source, which is preferable to have total phenol content, and has gallic acid content of 24.21mg/g, PT has gallic acid content of 23.6mg/g, Cas has gallic acid content of 22.36mg/g, and the total phenol content of the other two organic nitrogen sources is less than the gallic acid content of 20 mg/g. Preferably, SN is used AS the inorganic nitrogen source, which corresponds to a gallic acid content of 21.62mg/g, followed by PN (20.29mg/g), which is much higher than AS (1.7 mg/g). In terms of flavonoid content, the organic nitrogen source preferably has YE, 24.36mg/g of quercetin, 23.96mg/g of PT, 23.93mg/g of Cas and more than 20mg/g of total flavonoids, and the AS has the highest total flavonoids content in the inorganic nitrogen source, and can reach 12.83 mg/g.

Reference is made to fig. 12, which is an analysis diagram of examples 22 to 30 of the present invention. Wherein the components (a) to (d) represent pH, extracellular polysaccharide content, intracellular polysaccharide content, and biomass content, respectively.

With the addition of various mineral elements, BFP14 mycelium can grow. Under the condition of no addition of mineral elements and addition of magnesium sulfate and calcium sulfate, the fermentation liquor can show red color, and under the condition of other mineral elements, the fermentation liquor can show different color changes. The BFP14 mycelium biomass is preferably mycelium biomass without mineral elements, which can reach 6.35g/L, secondly, 2mM magnesium sulfate (6.18g/L) is added, the rest trace elements are mycelium biomass which is higher than 6g/L, nickel sulfate is most unfavorable for BFP14 mycelium growth, and only 2.73g/L mycelium biomass is added, and secondly, ferric sulfate (3.43 g/L). In terms of intracellular polysaccharides, it is clearly observed that the addition of magnesium sulfate is preferred to achieve an intracellular polysaccharide yield of 7.12g/L, followed by calcium sulfate 6.40g/L, with the remainder being greater than 6g/L, and with ferric sulfate being the lowest, the intracellular polysaccharide yield being only 3.79 g/L. The addition of different mineral elements to the extracellular polysaccharide is less significant, and nickel sulfate is preferred to obtain 55.19g/L of extracellular polysaccharide, and the other conditions are to maintain the extracellular polysaccharide yield at 48-53 g/L.

Reference is made to fig. 13, which is an analysis diagram of examples 22 to 30 of the present invention. Wherein the moieties (a) to (d) represent the content (mg/mg) of uracil, uridine, adenine and adenosine, respectively.

The content of uracil, uridine, adenine and adenosine in the BFP14 mycelium extract is preferably iron sulfate in the uracil content, and can reach a content of 2.75mg/g, but the uridine content is less preferred. Under the conditions of copper sulfate, the contents of uridine, adenine and adenosine are all the worst, and it is evident that copper sulfate may be less favorable for the production of bioactive components. Manganese sulfate is preferred at adenine to be up to 0.52mg/g followed by iron sulfate (0.49mg/mg), whereas zinc sulfate is preferred at adenosine to be up to 2.46mg/g followed by nickel sulfate (2.44 mg/g).

Referring to fig. 14, which is an analysis diagram of examples 22 to 30 of the present invention. Wherein, the parts (a) and (b) respectively represent the content (mg/mg) of total flavonoids and total phenols.

Magnesium sulfate is preferred in the total phenol content, which corresponds to a gallic acid content of 9.73mg/g, while the highest manganese sulfate, corresponding to a quercetin content of 36.98mg/g, and manganese sulfate is observed in the flavonoid content.

In the invention, different mineral elements are added, which not only affect the biological quality of mycelium and intracellular polysaccharide, but also affect the composition of different bioactive components, and the extraction liquid of different elements has different ratios of bioactive components. The biological quality of the mycelium is preferably without mineral elements, then magnesium sulfate, the intracellular polysaccharide is preferably magnesium sulfate, and the biological active components are different, wherein the flavonoid component added with manganese sulfate is preferably used, but the biological quality of the mycelium is not excellent, and the manganese sulfate is a key point which is very worthy of attention in the future if the antioxidant activity and the flavonoid content are considered.

Referring to fig. 15, which is an analysis diagram of examples 31 to 38 of the present invention, wherein parts (a) and (b) represent water content (%) and yield (Product yield), respectively. The yield was calculated as product (g)/starting material (g).

In the solid culture with oatmeal as cereal for BFP14 with different days of culture, it was found that the whole cereal matrix was covered with mycelia after 7 days of culture, while 0.3-0.5cm of primordia were grown after 14 days of culture, and 1-3cm of fruiting body was grown after 21 days of culture. The height of the fruiting body can reach 3-5 cm at 28 days of culture, and the fruiting body grows to about 5-7cm at 35 days of culture, and then cell formation and fruiting body atrophy gradually at 42 days of culture. Wherein the culture humidity is about 60-80%, and the humidity is increased with the increase of culture days. In terms of productivity, the productivity decreased with the increase of the number of days of culture.

Referring to FIG. 16, there are analysis graphs of examples 31 to 38 of the present invention, which represent polysaccharide (TSC) content analysis in units of glucose equivalent (glucose equivalent) g/L.

It can be found that the total polysaccharide content is reduced with the increase of the culture days, and the total polysaccharide content is 27.55g/L on the 7 th day of the BFP14 cereal matrix solid culture, and only 8.88g/L is left after 56 days of culture.

Referring to fig. 17, which is an analysis diagram of examples 31 to 38 of the present invention. Wherein the moieties (a) to (d) represent the content (mg/mg) of uracil, uridine, adenine and adenosine, respectively.

Four bioactive components of uracil, uridine, adenine and adenosine all increased with the number of days of culture, and had the highest contents, 0.42, 0.09, 0.07 and 0.63mg/g, on the 35 th day of culture, which is also the most mature period of the fruit body.

Referring to fig. 18, which is an analysis diagram of examples 31 to 38 of the present invention. Wherein, the parts (a) and (b) respectively represent the content (mg/mg) of total flavonoids and total phenols.

The total flavonoids and the total phenols are increased along with the number of culture days, the highest total phenol content is obtained on the 42 th day of culture, when the gallic acid content of 9mg/g is higher than the total phenol content of 7 days (2.62mg/g) of culture by more than 3 times, the flavonoid content is always increased along with the number of culture days, the quercetin content equivalent to 10.74mg/g is obtained on the 7 th day of culture, and after the quercetin content equivalent to 49.83mg/g is obtained after the quercetin content is cultured for 56 days, which is improved by more than five times.

Referring to fig. 19, which is an analytical chart of examples 39 to 45 of the present invention, wherein parts (a) and (b) represent water content and yield, respectively.

It can be observed that under the condition of 0Lux illumination intensity, a layer of white mycelium membrane grows along the edge of the culture container, no sporophore is generated, and under the condition of 50Lux illumination intensity, sporophore of 5-7cm can grow, and with the increase of illumination intensity, the change is not too large, and when the illumination intensity is 1500Lux, the phenomenon that the sporophore is gradually shrunk can be found, in the grain solid state culture of BFP14, the fruiting body can be stimulated to grow by a little illumination, and the illumination intensity is not too high, and the illumination intensity is too high, so that sporulation and the gradual shrinking of the sporophore can be caused.

In the cultivation under different illumination intensity, the cultivation humidity under the illumination without light is about 70%, the cultivation humidity under other illumination intensity is maintained at about 80%, in terms of yield, under the illumination without light, 70% yield can be obtained, 30g of grains are fermented completely, 21g of fermentation products can be obtained, under the illumination condition of 50 to 1500Lux, about 50% yield can be obtained, and about 15g of fermentation products can be obtained.

Referring to fig. 20, there are analysis diagrams of examples 39 to 45 of the present invention, which represent polysaccharide content analysis.

It was found that the highest polysaccharide content (14.86g/L) was observed in the 0Lux culture conditions, but the polysaccharide content gradually decreased as the light intensity increased.

Reference is made to fig. 21, which is an analytical chart of examples 39 to 45 of the present invention. Wherein the moieties (a) to (d) represent the content (mg/mg) of uracil, uridine, adenine and adenosine, respectively.

The illumination intensity did not have much influence on the bioactive components of uracil, uridine, adenine and adenosine, but it was observed that after the incubation with illumination, the bioactive components were all higher than without illumination, especially on the uridine content, it was clearly observed that the uridine content could not be detected under the illumination condition of 0 to 100Lux, the content increased with the increase of the light intensity, and at 500Lux, the highest uridine content (0.09mg/g) was present, and then appeared to decrease.

Reference is made to fig. 22, which is an analysis diagram of examples 39 to 45 of the present invention. Wherein, the parts (a) and (b) respectively represent the content (mg/mg) of total flavonoids and total phenols.

At the total phenol content, lighting conditions of white light at 50 to 1000Lux were observed, with no significant variability; under the illumination of no illumination and 1500Lux illumination, the content of the gallic acid is respectively equal to 9.18mg/g and 10.67mg/g, and the content of the gallic acid is larger than 12mg/g under the other conditions, and the highest content is 1000Lux, which is equal to the content of the gallic acid of 13.24 mg/g. In the aspect of flavonoid content, no obvious difference exists under the illumination of 50-1000 Lux; under the conditions of no illumination and illumination of 1500Lux, the contents of the quercetin are respectively 13.17mg/g and 13.77mg/g, and the contents of the quercetin are all larger than 15mg/g under the other conditions, and the highest quercetin content is 500Lux, which is equivalent to the quercetin content of 16.77 mg/g.

In the fruit body form, the illumination intensity is between 50 and 1000Lux, and the growth of the fruit body is not greatly influenced, and besides uracil has high content in 500Lux, other biological active ingredients also do not have great difference between 50 and 1000 Lux. This is true for the total phenolic and flavonoid contents, however, the same is less pronounced in antioxidant activity, and it has been found that the lack of illumination and high illumination intensity (1500Lux) are less advantageous, preferably 1000 Lux. From the above results, it is considered that the light intensity of 500Lux is a preferable culture condition, and the subsequent studies were conducted with the light intensity of 500 Lux. The subsequent study refers to using white light as the illumination light source with the condition of 500Lux, and other light sources are different from the illumination light source with the condition of 500 Lux.

Referring to fig. 23, which is an analytical chart of examples 46 to 53 of the present invention, wherein parts (a) and (b) represent water content and yield, respectively.

The solid culture of BFP14 cereals produced fruiting bodies under various light conditions, and it was observed that the height of fruiting body growth was high at 6R3B and 9IR, about 5cm, while the height of fruiting body growth under other light conditions was about 3-4 cm.

The culture humidity of different light sources is about 80%, and in terms of yield, the culture humidity is about 40-50% or more, and is higher than 50% or more under 5000K and 6R3B, and the culture humidity is 53% and 54%, respectively.

Referring to fig. 24, which is an analysis diagram of examples 46 to 53 of the present invention, which represents polysaccharide content analysis.

It was found that the difference in total sugar content was not large, and was maintained at a polysaccharide content of 9-11g/L, preferably 6R3B, with a polysaccharide content of 11.41 g/L.

Reference is made to fig. 25, which is an analysis diagram of examples 46 to 53 of the present invention. Wherein the moieties (a) to (d) represent the content (mg/mg) of uracil, uridine, adenine and adenosine, respectively.

In uracil, the control (500Lux white light) is preferred, with a content of 1.4mg/g, followed by a content of 1.31mg/g for 3R3B3IR and further 1.27mg/g for 9 IR. Uridine is preferably 9B, with a content of 0.19mg/g, followed by 9IR with a content of 0.15mg/g, and further followed by 3R3B3IR with a content of 0.14 mg/g. Adenine is preferably 3R3B3IR, having a content of 0.49mg/g, secondly 6R3B has a content of 0.41mg/g and thirdly 9R has a content of 0.4 mg/g. Adenosine is preferably 9B, with a content of 1.05mg/g, followed by 9IR with a content of 0.92mg/g, and further followed by 3R3B3IR with a content of 0.87 mg/g. It was found that far infrared light stimulates uracil levels, blue light stimulates uridine and adenosine levels, and red light stimulates adenine levels.

Referring to fig. 26, which is an analysis diagram of examples 46 to 53 of the present invention. Wherein, the parts (a) and (b) respectively represent the content (mg/mg) of total flavonoids and total phenols.

Under different light source conditions, the gallic acid content is nearly 10mg/g, wherein 2700K is preferred, and the gallic acid content is 12.32 mg/g. In terms of flavonoid content, it was found that 3R3B3IR is preferred, corresponding to a quercetin content of 41.36mg/g, followed by 9IR corresponding to a quercetin content of 36.61 mg/g. Represents the flavonoid content of BFP14 grain solid state culture stimulated by far infrared ray.

Referring to fig. 27, which is an analytical chart of examples 54 to 73 of the present invention, wherein parts (a) and (b) represent water content and yield, respectively.

It was found that of the 20 different cereals used, the BFP14 cereal could be grown in solid state culture. Most grain solid cultures are capable of growing fruit bodies, except for a few grains. In the case of bean-type grains, except YB and BB, no fruit body grows, the other bean-type grains can grow about 1-2 cm, in the case of rice-type grains, except RR, a thick hypha grows, the fruit bodies grow from the other six rice-type grains, WR is preferred, in the case of Li-wheat-type grains, TR and MUR grow only thick hypha, the fruit bodies grow from the other four Li-wheat-type grains, and in the case of H and PC of the other grains, the fruit bodies grow. In these cereals, the fruit body type is preferably MM, then WR, and the fruit body type of the rest cereals is at least 3-4 cm, while the fruit body type of the legume cereals is inferior.

Apart from the poor growth pattern in YB and BB, the moisture content is around 60%, while the moisture content of the other 18 cereals is between 70% and 80%. In terms of yield, it was found that 22.4g and 23.7g of fermentation products were obtained with the yield of the legume grains being higher than 50% and higher than that of other grains, particularly 74% and 79% of the legume grains YB and BB, respectively. However, the yield is low in rice-based grains, with an average yield of about 40%.

Referring to fig. 28, which is an analysis diagram of examples 54 to 73 of the present invention, which represents polysaccharide content analysis.

In terms of total saccharide content, the rice grain has a highest polysaccharide content corresponding to 18.75g/L by using MR, and 13.14g/L by using WR, the bean grain has a highest polysaccharide content corresponding to 13.59g/L by using MB, and the Li-wheat grain has a highest polysaccharide content corresponding to 11.71g/L by using MM.

Reference is made to fig. 29, which is an analysis diagram of examples 54 to 73 of the present invention. Wherein the moieties (a) to (d) represent the content (mg/mg) of uracil, uridine, adenine and adenosine, respectively.

In the legume grain, uracil is MB at the highest, and can be 0.99mg/g, and GB is 0.94mg/g, and uridine and adenine are not detected in RB and BB, uridine is YB at the highest, while adenine is GB at the highest, and 0.02mg/g, while MB is MB at the highest, and 0.47 mg/g. In rice-based grains, the content of uracil is at most Ten and 1.23mg/g can be obtained, while the content of uridine, WR, PR and MR are not detected, and the content of RR is at most 0.21mg/g, while the content of adenine is PR and 0.12mg/g, while the content of adenosine is RR and 0.97 mg/g. In the Mumai-type grains, both uracil and uridine are preferably TR and have a content of 1.13mg/g and 0.21mg/g, respectively, whereas uridine is not detected in OL, YM is preferably used in adenine and has a content of 0.08mg/g, YM is also preferably used in adenosine and has a content of 0.76 mg/g. In other types of cereals, these four bioactive ingredients are present in lesser amounts, followed by cereals of the Li Mai variety.

Reference is made to fig. 30, which is an analysis diagram of examples 54 to 73 of the present invention. Wherein, the parts (a) and (b) respectively represent the content (mg/mg) of total flavonoids and total phenols.

In the total phenol content of the bean-type grains, GB is preferred, the gallic acid content is 7.8mg/g, PR is preferred, the gallic acid content is 12.77mg/g, RR is 11.02mg/g, OL is preferred for the Li-wheat-type grains, and H and PC of other grains are 8.45 and 6.63mg/g, respectively. In terms of flavonoid content, bean grains have the highest RB content and 31.88mg/g quercetin content, rice grains have great difference, while WR has 9.9mg/g quercetin content, PR has 41.22mg/g quercetin content, which is more than four times of that of PR, Li-Mai grains are preferred for TR, have 40.9mg/g quercetin content, and H and PC of other grains have 22.77 and 9.94mg/g quercetin content respectively.

BFP14 can grow in 20 kinds of grains, and preferably has MM and WR in terms of fruit body form, but its contents of bioactive components, total phenols and flavonoids are not excellent. Each different cereal has different advantages, such as the highest uridine and adenosine content in RR, but not the good total phenolic and flavonoid content, and the better antioxidant activity than other cereals, PR has the highest total phenolic and flavonoid content, and also has quite good DPPH free radical scavenging activity, but it has no detected uridine content, and other antioxidant activity is not as expected. It is only demonstrated here that BFP14 can be grown in a variety of grains and that the choice of grains and proportions can be adjusted depending on the desired characteristics.

4. Conclusion

Cordyceps cicadae belongs to medicinal fungus of Cordyceps, is rich in bioactive components related to Cordyceps sinensis and Cordyceps militaris, such as nucleotide, cordycepin and ergosterol, and has antioxidant and anti-inflammatory effects. The strain of the Taiwan indigenous cordyceps sobolifera is screened out and identified by 18S rDNA, then is named as Cordycepscicadae Wu-BFP14(NCBI: KX289580), and the influence of a carbon source and a nitrogen source on the growth and the biological activity of the cordyceps sobolifera liquid fermentation mycelium is discussed.

Carbon source studies have shown that glucose utilization results in a preferred mycelium yield (6.55g/L) with the highest intracellular polysaccharide yield (6.19 g/L). In addition, the results also showed that xylose as a carbon source had the highest uracil, uridine and adenine contents of 2.912mg/g, 0.527mg/g and 0.100mg/g, respectively. Xylose is also preferred for the total phenolic and flavonoid content. In the nitrogen source study, the yield of BFP14 mycelium was preferably 8.37g/L from yeast extract, and also preferably 1.690mg/g, 0.172mg/g, 0.905mg/g and 0.976mg/g for uracil, uridine, adenine and adenosine, respectively. The total phenol and flavonoid contents were 24.21mg GAE/g and 23.96mg QE/g, respectively.

The optimal mycelium growth, polysaccharide and bioactive ingredient content liquid medium consists of: 50g/L glucose, 5g/L yeast extract and 5mM manganese sulfate.

In solid culture, wheat was used as a culture medium, and the influence of various culture conditions on the biological activity was examined in a solid-to-liquid ratio of 2: 1. the best yield of the biological activity is obtained after the culture for 42 days at the illumination intensity of 500 Lux. When the test is carried out by different LED light sources, the content of uracil (0.19mg/g), adenosine (1.05mg/g), total phenol (12.32mg GAE/g) and flavonoid (41.36mg QE/g) is respectively optimal in blue light, white light 2700K and red light, blue light and far infrared mixed light sources. Using 20 cereals as solid culture medium of BFP14, observing the growth morphology and biological activity of different cereals, the cereal substrate best for producing uridine and adenosine is brown rice, while purple rice is obtained with best content of phenols and flavonoids.

While the invention has been described with reference to specific embodiments, the invention is not limited thereto, and various equivalent modifications and substitutions can be easily made by those skilled in the art without departing from the scope of the invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Sequence listing

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<120> cicada fungus, solid and liquid culture method thereof and application of extract thereof

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<170> PatentIn version 3.5

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<213> Cordyceps sobolifera (Cordyceps bifusispora)

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Claims

1. A solid state culture method of cordyceps sobolifera, which is characterized by comprising the following steps:

culturing Cordyceps cicadae strain (SEQ ID NO:1) in liquid culture medium as liquid strain;

inoculating the liquid strain in a solid culture medium according to a specific inoculation ratio; and

culturing for a predetermined time under a specific light intensity with a specific light source type to obtain mycelium and fruiting body of the Cordyceps cicadae strain.

2. The method for culturing cordyceps sobolifera in a solid state according to claim 1, wherein the solid state culture medium comprises one or more combinations of red beans, mung beans, soybeans, black beans, rice beans, red coix seeds, white coix seeds, brown rice, white rice, purple rice, millet, decagrain rice, red quinoa, peru quinoa, oats, oatmeal, wheat, buckwheat, sorghum, and corns.

3. The method for solid-state culture of cordyceps sobolifera according to claim 1, wherein the specific light source species comprises one or more combinations of red light, blue light, white light and infrared light.

4. The method for solid-state culture of cordyceps sobolifera as claimed in claim 1, wherein the contents of uracil, uridine, adenine and adenosine in the mycelia and the fruiting body of the cordyceps sobolifera strain are 0 to 0.42mg/mg, 0 to 0.09mg/mg, 0 to 0.07mg/mg and 0 to 1.05mg/mg, respectively.

5. A liquid culture method of cordyceps sobolifera is characterized by comprising the following steps:

culturing the cordyceps sobolifera strain in a liquid medium, wherein the sequence of the cordyceps sobolifera strain is shown as SEQ ID NO:1 to obtain mycelium of the cordyceps sobolifera strain;

wherein the liquid medium comprises a basal medium.

6. The liquid culture method of cicada fungus according to claim 5, wherein the basic medium comprises a carbon source, and the carbon source comprises one or more combination of glucose, fructose, xylose, sucrose, maltose, lactose, dextrin, carboxymethyl cellulose, soluble starch and mannitol.

7. The liquid culture method of cicada fungus according to claim 5, wherein the basic culture medium comprises a nitrogen source, and the nitrogen source comprises one or more combination of peptone, yeast extract, malt extract, beef extract, casein, sodium nitrate, potassium nitrate and ammonium sulfate.

8. The method for liquid culturing of cicada fungus according to claim 5, wherein the nitrogen source accounts for 0.1-3% of the total volume of the basic culture medium.

9. The method for liquid culture of cordyceps sobolifera as claimed in claim 5, wherein the contents of uracil, uridine, adenine and adenosine in the mycelia of the cordyceps sobolifera strain are 0.5-3 mg/mg, 0.05-0.6 mg/mg, 0.02-0.9 mg/mg and 0.1-2.91 mg/mg, respectively.

10. A cordyceps sobolifera is characterized in that the sequence of the cordyceps sobolifera is as shown in SEQ ID NO:1, and has at least the productivity of polysaccharides, uracil, uridine, adenine and adenosine.

11. The application of the cordyceps sobolifera extract in preparing the antioxidant is characterized in that the cordyceps sobolifera extract is prepared from a cordyceps sobolifera extract with a sequence shown as SEQ ID NO:1, and extracting the cordyceps sobolifera shown in the formula 1.

12. The application of the cordyceps sobolifera extract in preparing the inhibitor for inhibiting the activity of tyrosinase is characterized in that the cordyceps sobolifera extract is prepared from cordyceps sobolifera extract with a sequence shown as SEQ ID NO:1, and extracting the cordyceps sobolifera shown in the formula 1.

13. The application of the cordyceps sobolifera extract in preparing the anti-inflammatory agent is characterized in that the cordyceps sobolifera extract is prepared from cordyceps sobolifera extract with a sequence shown as SEQ ID NO:1, and extracting the cordyceps sobolifera shown in the formula 1.