CN114208586A - New Guni iso cordyceps fruiting body and artificial cultivation production method - Google Patents

Abstract

The invention belongs to the field of biotechnology, and discloses a production method for the fruiting body and artificial cultivation of Cordyceps neogoni. The strains isolated in the field are used to produce fruiting bodies, and the fruiting bodies are cultivated through cultivation techniques such as plate or slanted culture, liquid culture, solid medium preparation, inoculation, germination, primordium induction, fruiting body culture and harvesting. entity. The used strain has strong grass-producing ability, can produce a large amount of green pigments and has low angular variability. Under artificial cultivation conditions, the liquid strain quality control technology is adopted to reduce the number of conidia in the liquid strain, improve the yield of mycelium, and realize effective artificial cultivation. 6 cm, 0.5 cm thick, the biotransformation rate can reach 40%, and it is rich in active substances. The present invention solves the problems that the current New Gunny Cordyceps is mainly collected from the wild and the quality is difficult to control, and relieves the ecological pressure.

Description

New Guni iso cordyceps fruiting body and artificial cultivation production method

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a production method of a new Gunnit cordyceps (Metacadyces neogunnii) fruiting body and artificial cultivation.

Background

The cordyceps sinensis is rich in resources and has great development and application values. According to statistics, more than 500 Cordyceps fungi (cordyces fungi) are reported all over the world, and more than 100 fungi are reported in China. The Guizhou Cordyceps sinensis resources are extremely rich, about 70 recorded Cordyceps sinensis resources account for about half of the total number reported in China, and are the most abundant regions of domestic resources, wherein the species with geographical features are not lacked, and the Guizhou Cordyceps sinensis mainly comprises new Gunney iso-Cordyceps sinensis (Metadyceps neogenii), Chinese caterpillar fungus (cordyces pruinosa), and Gao-xiong mountain Cordyceps sinensis (C. takaomontana) (Song et al, 2006; Liangpecten, 2007; Wen, et al, 2017).

Through modern research and analysis, many cordyceps fungi are rich in various amino acids, trace elements, vitamins and other nutritional ingredients, and adenosine, cordycepin, cordycepic acid, polysaccharide and other active ingredients. Modern pharmacological tests prove that cordyceps sinensis has various pharmacological functions such as tumor resistance, pathogenic microorganism resistance, oxidation resistance, immunoregulation and the like, the medicinal value of rare Chinese medicinal material cordyceps sinensis (Ophiocerdyceps sinensis) is fully determined, other species such as cordyceps militaris (C. militaris), cordyceps sobolifera (C. cicadae), New Gunie isocordyceps sinensis, Liangshan cordyceps sinensis (M. liangshanesis), Baens cordyceps sinensis (C. barnesis) and the like are also concerned, the cordyceps sinensis has similar effects to cordyceps sinensis, and the active ingredients of part of the species are even higher than that of cordyceps sinensis (Chen group and the like, 1993). The varieties are widely distributed in Guizhou areas, and part of the varieties are used as Chinese herbal medicines and have a long application history in folks. For example, the Xinguli-Niisocordyceps sinensis has the functions of nourishing lung and kidney, stopping bleeding and reducing phlegm, and is a famous Chinese herbal medicine. Therefore, the active development and utilization of cordyceps fungus resources are of great significance.

The new ancient chinese caterpillar fungus (Metardyceps neogenii) is a fungus belonging to Clavicipitaceae (Clavicipitaceae), genus Heterocordyceps (Metardycceps), which is abundantly distributed in Guizhou, Hunan, Yunnan, etc. of China, and has been regarded as ancient chinese caterpillar fungus (Cordyceps gunnii) until 2017, and has been proved to be a new species, which is formally named as new ancient chinese caterpillar fungus (Metardyceps neogenii) (Wen, et al, 2017). The important progress of taxonomic research provides more accurate basic data for the research of the new Guni iso-cordyceps. Because the resource has a long application history among China, the efficacy of the resource is determined. The Xinguli-Nissan cordyceps sinensis is rich in various active substances such as flavone, polyphenol, polysaccharide, nucleotide and the like, has various pharmacological actions and has good development value. The new Guni iso cordyceps is proved to have pharmacological actions of resisting tumor, resisting oxidation, easing pain, calming, regulating immunity and the like. The artificial culture of the Xinguli-Nissan cordyceps in China dates back to 1985, and the pure culture is obtained by the pectorac tectomy in a tissue separation mode and is used for identifying the non-sexuality of the Xinguli-Nissan cordyceps. In 1990, Liujilin et al reported that fruiting bodies of new Guni iso cordyceps sinensis could be formed under artificial conditions for the first time, in 2006, Lichun et al cultured stroma of a microspore variety of Guni cordyceps sinensis, in 2016, and in grandfather et al reported artificial cultivation of fruiting bodies of new Guni iso cordyceps sinensis using millet and the like as a culture medium. Since then, no report has been made on the artificial cultivation of the fruiting body of Cordyceps sinensis. As can be seen, the difficulty of artificial cultivation of the new Guninai cordyceps sporophores is far greater than that of the common cordyceps species.

The induction of the cordyceps primordia is a key link of artificial cultivation of sporocarp, and the limiting factors of the induction mainly comprise two aspects: one is strain and the other is culture condition. Liujilin et al, 1990, used 5-19-M strain to cover pine needles and cultivated to obtain 9 sporophores/bottle at most, but the sporophores were 4cm high, while the length of the sporophores obtained in the cultivation bottle was 7cm, but the number of sporophores was only 3. The Sunpiens adopts the variant strain of the cordyceps sinensis microspore, 3 cultivated strains S3, S21 and S29 are obtained by single spore separation and screening, the highest fresh yield of sporocarp is only 2.46 g/bottle, and the maximum sporocarp is 1.91 cm. Both of these independently conducted studies were performed at 25 ℃ for hyphal culture. None of these studies has met the production needs, too few fruit bodies, or too small and short fruit bodies.

Aiming at the problems of artificial cultivation of the new Guni cordyceps sinensis, the inventor collects a batch of wild new Guni cordyceps sinensis from Dong nationality Dong autonomous prefecture of Guizhou southeast Miao of Guizhou province, and separates and screens a strain with grass-growing capacity by taking the wild new Guni cordyceps sinensis as a material, wherein the strain number is 2020031916. The cordyceps sinensis is determined to be the new gulina cordyceps sinensis through morphological and molecular identification (ITS sequence determination and comparison). The invention successfully obtains the sporophore product with excellent cultivation properties by using the strain through a series of microorganisms such as slant culture, liquid strain culture, inoculation, spawn running, primordium induction, sporophore culture and the like and edible fungus cultivation process technologies. The components of the sporocarp are determined, and the sporocarp is proved to be rich in adenosine, polysaccharide, cordycepic acid, amino acid, crude fiber and other active and nutrient substances, and is an ideal raw material for developing functional food.

The invention provides a new Guni iso cordyceps strain with strong grass-growing capacity and a cultivation technology thereof, the strain is obtained by taking fruit bodies collected in the field as materials and carrying out tissue separation, and the bacterial colony of the strain is characterized by being capable of generating a large amount of green pigments and a large amount of conidia, but having low angular transformation rate and stable genetic characters. Under artificial culture conditions, the strain 2020031916 can form fruiting bodies with good appearance by adopting a targeted culture technology, and the culture characteristics are very stable.

The invention provides a new Guni iso cordyceps fruiting body product which is divided into a fresh product and a dry product, wherein the fruiting body is 3-6cm long and 0.4-0.5cm thick. The product is rich in various nutrient substances and special active substances of cordyceps sinensis, and solves the problems that the prior new Guni iso-cordyceps sinensis is mainly collected in a wild way and the quality is difficult to control. Meanwhile, the implementation of the invention can relieve the pressure of ecological damage caused by collecting wild sporocarp.

Disclosure of Invention

The invention aims to provide a new Guni iso cordyceps fruiting body and a production method for artificial cultivation.

The invention is realized by the following technical scheme and steps:

1. separating and screening a new guli-nig-iso-cordyceps excellent strain 2020031916: wild new Guni cordyceps sinensis (figure 1) is collected in Dong nationality autonomous region of Guizhou south Guizhou Miao nationality in Guizhou province, and is brought back to a laboratory together with soil for strain separation. Cleaning soil on the surface of the Xinguli-Nissan cordyceps, tearing off filaments on the surface of a host worm body, wiping the worm body and a fruiting body with 75% alcohol, and performing surface disinfection. Cutting off fruiting body of new Guni Cordyceps with sterilized scalpel, clamping flesh tissue with sterilized forceps, and inoculating in PPDA slant culture medium; in the same way, the flesh tissue is picked from the sclerotium of the polypide and inoculated in PPDA slant culture medium, thus completing the strain separation step. All isolated bacon tissues were incubated at 20 ℃ for 10-20 days in the dark. All separated strains are purified without other mixed bacteria pollution. And (3) separating to obtain 250 parts of strains, and primarily screening to obtain a strain with good grass growing capability, wherein the strain is numbered as follows: 2020031916. the strain is preliminarily determined to be the aneuploid of the new gulina cordyceps sinensis by microscopic observation, and the strain is finally determined to be the new gulina cordyceps sinensis (Metacadyces neogenii) by ITS sequence sequencing and comparison. The strain is propagated by a rotating tube, is preserved in China Center for Type Culture Collection (CCTCC) in 2021, 4 months and 19 days, and has a preservation number: m2021416, survival is detected as the result of detecting the survivability, and the address of the preservation unit is Wuhan, Wuhan university in China.

The ITS sequence of strain 2020031916 is as follows:

TTCCGTAGGGGGGGACCTGCGGAGGGATCATTACCGAGTTCTTACAACTCCCAAACCCCTGTGAACTTATACCTATACTGTTGCTTCGGCGGGTCATCGCCCCGGGGAAAGACAGGGAGCCGGCAACGGCCCCCTGGAAAACCCCCCCGGAACCAGGCGCTCGCCGGGGGACTTAAACTCTGTATTTCTCTTTACTGTATTGTATACCGTCTGAGTGACAAAAAACATAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTACTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAGGTACCCCCCGCTGCGTCTGTGGCGGGCGGGAGCCTGGTGTTGGGGACCGGCGGAAAACCTGCCCCAGGGCAGCCGCCGCCCCCTAAATGAATTGGCGGCCTCGTCGCGGCCCTCCTCTGCGTAGTAGCACACACCTCGCAACGGGAGCCCGGCGGCGGCCACTGCCGTAAAACGCACAATCTTCTCTTCTAGAGTGACCTCGAATCAGGTAGGAGTGCCCCCCGCT

2. the excellent strain 2020031916 is cultured by a plate colony or a slant strain: inoculating the strain blocks into PPDA culture medium (formula is potato 200g, glucose 20g, agar 20g, peptone 5g, and distilled water to 1000 mL), and dark culturing at 20 deg.C for 14-20 days. The colonies produced a large amount of dark green pigment and a large amount of conidia (FIGS. 2 and 3), but the angular transformation rate was low and the genetic trait was stable. The strain is separated by single spore to obtain 12 single spore strains, mating type gene segments can be detected, and the new Guni iso-cordyceps can be conjointly matched (figure 4).

3. Culturing liquid strains at all levels: cutting plate colony or slant strain into 0.5cm pieces with size of 0.5cm, inoculating into liquid culture medium composed of glucose, peptone, yeast extract, vitamin B1, potassium dihydrogen phosphate, and magnesium sulfate, sterilizing at 115 deg.C for 30min, and cooling to room temperature. Placing the liquid culture medium with the plate bacterial colony or the slant strain into a shaking incubator for dark culture at the rotation speed of 120-. Counting conidia in the primary liquid strain by using a blood counting plate, wherein the number of the conidia cannot be higher than 105/mL. Inoculating the primary liquid strain to a secondary liquid culture medium according to the proportion of 5 percent of inoculation amount, wherein the formula of the secondary liquid culture medium comprises glucose, peptone, yeast extract, vitamin B1, potassium dihydrogen phosphate and magnesium sulfate. The culture can be carried out by combining a culture bottle with a shaking culture box at the rotation speed of 120-. Thus finishing the culture of the second-level liquid strain. The quality requirements of the secondary liquid strain are as follows: the number of conidia cannot be higher than 105/mL, the mycelium pellet is compact in center and uniform in distribution of surrounding mycelial hyphae, and the wet weight of the mycelium is not lower than 60-100 g/L.

4. Preparing a solid culture medium: the new Guni iso cordyceps fruiting body needs a cereal solid culture medium for growth, and the preparation process comprises the following steps: proper amount of rice or millet is added into the cultivation container, the silkworm chrysalis accounts for 0.5-5% of the rice or millet, and the nutrient solution or water accounts for 1.5-1.8 times of the rice or millet. The nutrient solution formula is as follows: 20-30g of carbon source, 4-10g of nitrogen source, 1-3g of yeast extract, 10.02g of vitamin B, 1g of monopotassium phosphate, 0.5-1g of magnesium sulfate and constant volume of water to 1L, wherein the carbon source can adopt sucrose, glucose, maltose, glycerol, fructose or corn starch; the nitrogen source can be beef extract powder, potato extract powder, tryptone, peptone, yeast extract powder, soybean peptone, cotton seed protein or cotton seed hull extract powder. Mixing, sealing, sterilizing at 121 deg.C for 20-40min, and cooling to room temperature for inoculation.

5. Inoculation: diluting the primary or secondary liquid strain before inoculation, wherein the diluent can be sterile water or nutrient solution containing contents such as glucose, peptone and the like, and the dilution ratio is 1: 5-10. The diluted strain contains conidium no more than 105/mL and mycelium pellet no less than 50/mL. Inoculating the strain into a solid culture medium according to the inoculum size of 10-25%, and uniformly spraying the strain on the surface of the culture medium.

6. Spawn running: after inoculation, the culture bottle is placed in a constant temperature incubator or culture room at 20 ℃ for 6-8 days in a dark place. When the hyphae grow over the whole culture medium and the production of the dark green pigment begins, the cultivation stage needs to be started to be changed into the illumination cultivation stage.

7. Light induction primordia: after the spawn running is finished, the primordium induction stage is carried out. Illuminating the culture medium full of mycelia with light source of white, yellow, blue or combination thereof at 18-20 deg.C preferably at 200-. Primordia typically begin to appear within 10-14 days of light (FIG. 5) and increase gradually. The primordial induction phase is completed when most of the primordia grow to a height of 0.5 to 1 cm.

8. And (3) fruiting body culture: after the primordium induction phase is completed, the primordium continues to grow and develop into a fruit body, and the fruit body culture phase is entered (fig. 6). The environmental conditions in the incubator or the culture room are as follows: the illumination intensity is preferably 200 and 600lx, the temperature is 18-22 ℃, the carbon dioxide concentration is not higher than 5000ppm, and the culture is continued for 40-60 days.

9. Harvesting: culturing until the top of fruiting body is slightly enlarged (FIG. 7), and harvesting.

10. Drying: drying the collected fruiting body by freeze drying or oven drying (fig. 8).

The new Guniyiiso cordyceps fruiting body provided by the invention has the following characteristics:

the fruiting body is rod-shaped, basically without branch, slightly enlarged at the top, grey-white to light grey-green, smooth in surface and without thorn (figure 7). The fruiting body is rich in protein and crude fiber, 29.2% and 20.4% respectively, and also contains active ingredients such as polysaccharide and adenosine, and does not contain cordycepin (fig. 9-11).

Drawings

FIG. 1 wild New Guni Heterocordyceps

FIG. 2 front side of colonies of New Gunie Heterocordyceps strain 2020031916 plate

FIG. 3 the reverse side of the new Guni isocordyceps strain 2020031916 plate colony

FIG. 4 shows the results of the mating type gene amplification of New Guni Heterochayotis sinensis (M, marker, from top to bottom, 2000 bp, 1000 bp, 750 bp, 250 bp, 100 bp, respectively)

FIG. 5 New Guni Heterocordyceps primordium

FIG. 6 growth stage of fruiting body of Cordyceps sinensis

FIG. 7 maturation of fruiting bodies of Cordyceps sinensis (berk.) Sacc

FIG. 8 shows the dried novel Guni-iso-Cordyceps fruiting body

FIG. 9 New Gunini Cordyceps sinensis fruiting body detection report on page 1

FIG. 10 New Guni Heterocordyceps sinensis fruiting body detection report on page 2

FIG. 11 New Gunini Cordyceps sinensis fruiting body detection report on page 3

Detailed Description

The present invention will be further illustrated with reference to specific embodiments, but the present invention is not limited to the following examples.

Example one

1. Separating new Guni iso cordyceps excellent strains: cleaning soil on the surface of wild Xinguli-Nissan cordyceps, tearing off filaments on the surface of a host worm body, wiping the worm body and a sporocarp with 75% alcohol, and performing surface disinfection. Cutting off fruiting body of new Guni Cordyceps with sterilized scalpel blade, clamping flesh tissue with sterilized forceps, inoculating to PPDA slant culture medium, and culturing at 20 deg.C in dark for 14 days. Then, the tube transfer purification is carried out to ensure that the strains are free from other mixed bacteria pollution. The strain is determined to be the new guli-ni cordyceps sinensis through microscopic observation and ITS sequence sequencing and comparison, and can be used for culture.

2. Slant culture: the seed pieces were inoculated into PPDA medium (formulation: potato 200g, glucose 20g, agar 20g, peptone 5g, distilled water to 1000 mL) and cultured in the dark at 20 ℃ for 14 days. The bacterial colony produces dark green pigment, and the bacterial colony can be used.

3. Primary liquid strain culture: cutting slant strain into 0.5cm by 0.5cm pieces, inoculating into primary liquid culture medium containing glucose 20g, peptone 4 g, yeast extract 1g, and water to constant volume of 1L, sterilizing at 115 deg.C for 30min, and cooling to room temperature. And (3) placing the primary liquid culture medium with the slant strains into a shaking incubator for dark culture at the rotation speed of 160r/min and the temperature of 20 ℃ for 5 days, thus completing the culture of the primary liquid strains. Counting conidia in the primary liquid strain by using a blood counting plate, wherein the number of the conidia cannot be higher than 105/mL, the mycelium pellet is compact in center and uniform in distribution of surrounding hairy hyphae, and the wet weight of the mycelium is not lower than 60 g/L.

4. Preparing a solid culture medium: adding 20g of millet, 0.5g of silkworm chrysalis and 45mL of water into a cultivation bottle (7 cm multiplied by 11 cm) with a breathable film cover, uniformly mixing, sealing, sterilizing at 121 ℃ for 20min, and cooling to room temperature for inoculation.

5. Inoculation: diluting the primary liquid strain before inoculation, wherein the diluent is sterile water, and the dilution ratio is 1: 5. inoculating the strain into a solid culture medium according to the inoculum size of 10%, and uniformly spraying the strain on the surface of the culture medium.

6. Spawn running: after inoculation, the culture bottle is placed in a constant temperature incubator or culture room at 20 ℃ for 6 days in a dark place. The hyphae grow over the whole culture medium, and when the production of the dark green pigment begins, the cultivation stage begins to be switched into the illumination cultivation stage.

7. Light induction primordia: illuminating the culture medium full of mycelia with white light at a light source of 1000lx and 20 deg.C, and ventilating for 0.5 hr/time and 2 times/day to ensure that the concentration of carbon dioxide in the culture workshop or incubator is not higher than 5000 ppm. Primordia began to appear within 12 days of light and increased gradually. The primordial induction phase is completed when most of the primordia grow to a height of 0.5 to 1 cm.

8. And (3) fruiting body culture: the environmental conditions in the incubator or the culture room are as follows: the illumination intensity is preferably 400lx, the temperature is 20 ℃, the carbon dioxide concentration is not higher than 5000ppm, and the culture is continued for 40 days.

9. Harvesting: culturing until the top of the fruiting body is slightly enlarged, and harvesting. The yield of the fresh sporocarp reaches 8.22 g/bottle by measuring the yield, and the biotransformation rate is 40 percent.

10. Drying: drying the collected sporocarp by drying.

Example two

1. Separating new Guni iso cordyceps excellent strains: cleaning soil on the surface of the Xinguli-Nissan cordyceps, tearing off filaments on the surface of a host worm body, wiping the worm body and a fruiting body with 75% alcohol, and performing surface disinfection. Cutting the body of the Xinguli-Ning-isocordyceps sinensis with a sterilized scalpel blade, clamping the flesh tissue from the sclerotium, inoculating into PPDA slant culture medium, and culturing at 18 ℃ in the dark for 20 days. The strain is determined to be the new guli-ni cordyceps sinensis through microscopic observation and ITS sequence sequencing, and can be used for culture.

2. Strain plate strain of superior strain: the seed pieces were inoculated into PPDA plate medium (formulation: potato 200g, glucose 20g, agar 20g, peptone 5g, distilled water to 1000 mL) and cultured in the dark at 20 ℃ for 20 days. The colonies produced a large amount of dark green pigment and a large amount of conidia, but did not have angular changes, and were used for liquid seed culture.

3. Culturing liquid strains at all levels: cutting the flat strain into blocks of 0.5cm x 0.5cm, inoculating the blocks into a primary liquid culture medium, wherein the primary liquid culture medium comprises 25 g of glucose, 5g of peptone, 2g of yeast extract, 10.02 g of vitamin B, 1.0 g of potassium dihydrogen phosphate and 0.5g of magnesium sulfate, fixing the volume of water to 1 liter, sterilizing at 115 ℃ for 30 minutes, and cooling to room temperature for use. And (3) placing the liquid culture medium connected with the plate strains into a shaking incubator for dark culture at the rotation speed of 180r/min and the temperature of 20 ℃ for 4 days, thus finishing the culture of the primary liquid strains. Inoculating the primary liquid strain to a secondary liquid culture medium according to the proportion of 5 percent of inoculation amount, wherein the formula of the secondary liquid culture medium comprises 30g of glucose, 8g of peptone, 1g of yeast extract, 10.02 g of vitamin B, 1.0 g of monopotassium phosphate, 1g of magnesium sulfate and 1 liter of water. And (3) performing secondary liquid strain culture by adopting a fermentation tank at the stirring speed of 180r/min, the ventilation quantity of 0.8L/min and the temperature of 18 ℃ for 4 days to finish the culture of the secondary liquid strain. The quality requirements of the secondary liquid strain are as follows: the number of conidia cannot be higher than 105/mL, the mycelium pellet is compact in center and uniform in distribution of surrounding mycelial hyphae, and the wet weight of the mycelium is not lower than 100 g/L.

4. Preparing a solid culture medium: 150g of millet, 2g of silkworm chrysalis and 250mL of nutrient solution are added into a cultivation box (17 cm multiplied by 11 cm) with a breathable film cover. The nutrient solution formula is as follows: 20g of sucrose, 8g of peptone, 1g of yeast extract, 10.02g of vitamin B, 1g of monopotassium phosphate, 0.5g of magnesium sulfate and water to a constant volume of 1L, uniformly mixing, sealing, sterilizing at 121 ℃ for 30min, and cooling to room temperature for inoculation.

5. Inoculation: diluting the second-stage liquid strain before inoculation, wherein the diluent is sterile water, and the dilution ratio is 1: 6. the diluted strain contains conidium no more than 105/mL and mycelium pellet no less than 60/mL. Inoculating into solid culture medium according to 15% of inoculum size, and uniformly spraying the strain on the surface of the culture medium.

6. Spawn running: after inoculation, the culture bottle is placed in a constant temperature incubator or culture room at 20 ℃ for 8 days in a dark place. When the hyphae grow over the whole culture medium and the production of the dark green pigment begins, the cultivation stage needs to be started to be changed into the illumination cultivation stage.

7. Light induction primordia: after the spawn running is finished, the primordium induction stage is carried out. Illuminating the culture medium with mycelia, wherein the light source is blue, the illumination intensity is preferably 200, the temperature is 18 deg.C, and ventilation is performed for 0.5 hr/time and 2 times/day to ensure that the concentration of carbon dioxide in the culture workshop or incubator is not higher than 5000 ppm. Primordia typically begin to appear and increase within 14 days of illumination. The primordial induction phase is completed when most of the primordia grow to a height of 0.5 to 1 cm.

8. And (3) fruiting body culture: after the primordium induction stage is completed, the primordium continues to grow and develop into a sporocarp, and the sporocarp culture stage is carried out. The environmental conditions in the culture room were as follows: the illumination intensity is preferably 300lx, the temperature is 18 ℃, the carbon dioxide concentration is not higher than 5000ppm, and the culture is continued for 50 days.

9. Harvesting: culturing until the top of the fruiting body is slightly enlarged, and harvesting. According to the test, the yield of the fresh fruit body in the second example is 50.8 g, and the biotransformation rate is about 33%.

10. Drying: drying the collected sporocarp by adopting a freeze drying mode.

Claims (2)

1. A production method of new Guni iso cordyceps fruiting body and artificial cultivation is characterized by comprising the following steps:

(1) separating and screening a new guli-nig-iso-cordyceps excellent strain 2020031916: cleaning soil on the surface of the new guli-ni cordyceps sinensis, then carrying out surface disinfection by using 75% alcohol, cutting open fruiting bodies and polypide of the new guli-ni cordyceps sinensis by using a sterilized scalpel blade, clamping flesh tissues by using sterilized tweezers, inoculating the flesh tissues into a PPDA slant culture medium, placing all separated flesh tissues at 20 ℃ for dark culture for 10-20 days, obtaining a strain through purification, and obtaining a strain with good weed producing capacity through preliminary screening, wherein the number is as follows: 2020031916, determining the strain as new Gunningyicheng Cordyceps (Metacadyces neogenii) by microscopic observation and ITS sequence sequencing comparison;

(2) the excellent strain 2020031916 is cultured by a plate colony or a slant strain: inoculating the strain blocks into PPDA culture medium (composed of potato 200g, glucose 20g, agar 20g, peptone 5g, and distilled water to 1000 mL), and dark culturing at 20 deg.C for 14-20 days;

(3) culturing liquid strains at all levels: cutting plate bacterial colony or slant strain into blocks of 0.5cm × 0.5cm, inoculating into liquid culture medium composed of glucose, peptone, yeast extract, vitamin B1, potassium dihydrogen phosphate, and magnesium sulfate, sterilizing at 115 deg.C for 30min, and cooling to room temperature; placing the liquid culture medium connected with the plate bacterial colony or the slant strain into a shaking incubator for dark culture at the rotation speed of 120-; counting conidia in the primary liquid strain by using a blood counting plate, wherein the number of the conidia cannot be higher than 105/mL; inoculating the primary liquid strain to a secondary liquid culture medium according to the proportion of 5 percent of inoculation amount, wherein the formula of the secondary liquid culture medium comprises glucose, peptone, yeast extract, vitamin B1, potassium dihydrogen phosphate and magnesium sulfate; culturing for 4-5 days by adopting a culture bottle combined with a shaking culture box at the rotating speed of 120-; the quality requirements of the secondary liquid strain are as follows: the number of conidia cannot be higher than 105/mL, the mycelium pellet is compact in center and uniform in distribution of surrounding hairy hyphae, and the wet weight of the mycelium is not lower than 60-100 g/L;

(4) preparing a solid culture medium: adding proper amount of rice or millet into the cultivation container, wherein the amount of silkworm pupae is 0.5-5% of that of the rice or millet, and the amount of nutrient solution or water is 1.5-1.8 times of that of the rice or millet; the nutrient solution formula is as follows: 20-30g of carbon source, 4-10g of nitrogen source, 1-3g of yeast extract, 10.02 g of vitamin B, 1g of monopotassium phosphate, 0.5-1g of magnesium sulfate and constant volume of water to 1L, wherein the carbon source can adopt sucrose, glucose, maltose, glycerol, fructose or corn starch; the nitrogen source can be beef extract powder, potato extract powder, tryptone, peptone, yeast extract powder, soybean peptone, cotton seed protein or cotton seed hull extract powder, mixing, sealing, sterilizing at 121 deg.C for 20-40min, cooling to room temperature for inoculation;

(5) inoculation: diluting the primary or secondary liquid strain before inoculation, wherein the diluent can be sterile water or nutrient solution containing contents such as glucose, peptone and the like, and the dilution ratio is 1: 5-10, the diluted strain contains conidium no more than 105/mL, the mycelium pellet is no less than 50/mL, the strain is inoculated into a solid culture medium according to the inoculum size of 10-25%, and the strain needs to be uniformly sprayed on the surface of the culture medium;

(6) spawn running: placing the inoculated culture bottle in a constant-temperature incubator or culture room at 20 ℃ for 6-8 days in a dark environment, wherein hyphae grow over the whole culture medium, and when the production of the dark green pigment begins, the cultivation bottle needs to be shifted to a light culture stage;

(7) light induction primordia: illuminating the culture medium full of mycelia with light source of white, yellow, blue or combination thereof at 18-20 deg.C preferably at 200-; the primordia generally begin to appear within 10-14 days of illumination and gradually increase, and when most of the primordia grow to the height of 0.5-1 cm, the primordia induction stage is completed;

(8) and (3) fruiting body culture: the environmental conditions in the incubator or the culture room are as follows: the illumination intensity is preferably 200 and 600lx, the temperature is 18-22 ℃, the concentration of carbon dioxide is not higher than 5000ppm, and the culture is continued for 40-60 days;

(9) harvesting: culturing until the top of the fruiting body is slightly enlarged, and then harvesting;

(10) drying: drying the collected fruiting body by freeze drying or oven drying.

2. The method according to claim 1, wherein (1) the isolated material is wild Cordyceps sinensis (P.nikowii) collected in the field, (2) the strain 2020031916 produces a large amount of dark green pigment by plate or slant colony, (3) the temperature is preferably 18-20 ℃, the number of conidia in the liquid culture is not higher than 105/mL, (4) the carbon source is preferably fructose, and the nitrogen source is preferably tryptone, (5) the first-stage liquid culture is preferably used directly for inoculation, preferably 25%, in the light induction primordium, the light intensity is most preferably 800 lx white light intensity, the carbon dioxide concentration is most preferably 4000ppm, and (8) the culture temperature is most preferably 22 ℃ in the culture of the fruiting body, (10) the drying is most preferably freeze-drying.

Images