阅读说明：本技术 一种虫草真菌新种及其分离鉴定方法 (Novel cordyceps sinensis fungus species and separation and identification method thereof ) 是由 罗侠 于 2021-09-24 设计创作，主要内容包括：本发明公开了一种虫草真菌新种及其分离鉴定方法,本发明运用形态和分子相结合的方法准确分离鉴定出三株野生虫草菌株,其中HGF01、XJ46分别是柱状虫草和蝉花,CH01是新种,并在此基础上进行液体培养菌丝体中虫草酸、黄酮和多糖成分检测。三种虫草菌丝体中均含有虫草酸、黄酮和多糖成分,CH01中的虫草酸、黄酮和多糖成分含量与柱状虫草和蝉花相当,不具有显著性差异,具有与柱状虫草和蝉花相同的保健食用价值。再此基础上CH01菌丝中的部分氨基酸种类的含量显著高于柱状虫草和蝉花,且自由基的清除率在一定浓度下显著高于柱状虫草和蝉花,具有较好的氧化功效。(The invention discloses a new cordyceps fungus species and a separation and identification method thereof, the invention accurately separates and identifies three wild cordyceps fungus strains by a method of combining morphology and molecules, wherein HGF01 and XJ46 are columnar cordyceps and cicada fungus respectively, CH01 is a new species, and on the basis, the cordycepic acid, flavone and polysaccharide components in liquid culture mycelia are detected. The three cordyceps mycelia contain cordycepic acid, flavone and polysaccharide components, the content of the cordycepic acid, the flavone and the polysaccharide components in CH01 is equivalent to that of cordyceps columnar and cordyceps sobolifera, the obvious difference is not generated, and the cordyceps mycelia have the same health-care edible value as the cordyceps columnar and cordyceps sobolifera. On the basis, the content of partial amino acid species in the CH01 hypha is obviously higher than that of the cordyceps columnar and the cordyceps sobolifera, the clearance rate of free radicals is obviously higher than that of the cordyceps columnar and the cordyceps sobolifera under certain concentration, and the oxidation effect is better.)

1. A new species of Cordyceps fungus is Cordyceps sinensis (Cordyceps sp.) CH01, and the preservation unit address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, with a preservation date of: on 09/08/2021, accession number: CGMCC No. 23076.

2. A method of isolating a new species of cordyceps fungus as claimed in claim 1, characterized by the steps of:

(1) collecting cordyceps fruiting bodies and worm bodies, wrapping collected samples by using tinfoil, and placing the wrapped collected samples in a collection box; drying in a 45 ℃ oven, and placing the dried sample and the label into a plastic package bag for storage;

(2) separating the mycelium in the polypide of the collected sample by adopting a tissue separation method: washing the surface of a fresh worm body for several times by using running water to remove surface soil, and preliminarily disinfecting the surface of the worm body by using 75% alcohol; under the aseptic condition, cutting the surface of the worm body by a blade, using the tip of a pair of tweezers to pick up a little of hypha in the worm body, quickly inoculating the hypha into a PDA (personal digital assistant) solid culture medium, placing the culture medium in an incubator at 28 ℃, carrying out inverted culture, observing once every other day, and timely treating a flat plate polluted by mixed bacteria; when hyphae grow initially, transferring the pollution-free hyphae to a new PDA culture medium by using a plate-scribing method for further culture; inoculating the purified mycelium into PDA slant test tube, and storing in refrigerator at-20 deg.C.

3. A method of identifying new species of cordyceps fungi as claimed in claim 1, characterized by: the method comprises the following specific steps:

(1) morphological identification: observing and recording the macroscopic morphological characteristics of the cordyceps sinensis sample in detail, including the length of a stroma, the color, the shape, the length and the diameter of a fertile part, the color, the length and the diameter of a stipe of the cordyceps sinensis, and the type of a host, and preliminarily identifying the cordyceps sinensis sample to belong to or breed by referring to Chinese large-scale fungus resource atlas; recording the colony characteristics, including morphology, color and texture, of the isolated strains;

(2) and (3) molecular identification:

a. extracting genome DNA of the cordyceps sinensis sample;

b. PCR amplifying 2 gene segments by using genome DNA as a template;

c. the results of PCR product sequencing were spliced at SeqMan and the spliced sequences were analyzed by alignment in GenBank.

4. The method for identifying a new species of cordyceps fungus according to claim 3, wherein: the primer sequence of the PCR amplification is as follows:

5. the method for identifying a new species of cordyceps fungus according to claim 3, wherein: the specific comparison analysis method comprises the following steps: sequence alignment sequencing was performed on-line using mafftv.6, with Gblocks to identify and remove spaces, introns, and unclear sequences; the phylogenetic tree was analyzed for maximum similarity and Bayesian analysis using a total of 83 sequences, 5 from the collected samples and 77 from GenBank. Phylogenetic tree analysis adopts two sequences of Tolypocladium inflatum and T.cyclindrosporum as outer group, and maximum similarity analysis is carried out by using GTRGAMMA nuclear gene model in software in RAxML v8.2.12; bayesian analysis is completed by using MrBayes v3.1 software; phylogenetic trees were opened with the FigTree v1.4.2 software and edited with Ai.

Technical Field

The invention relates to the technical field of cordyceps separation and identification, in particular to a new cordyceps fungus strain and a separation and identification method thereof.

Background

China has reported more than 130 cordyceps, but many cordyceps are not discovered or recorded, such as cordyceps sinensis (C.sinensis), cordyceps militaris (C.militaris) and Isaria tenuipes (Isaria tenuipes), which are common cordyceps fungi [ Huangluoshu, Mayufeng, Wang Yue, etc. ] the research and development and utilization of fine cordyceps resources are advanced [ J ] edible fungi academic newspaper, 2019,26(02):141- "151. Qi zong. Scientific Press 2007:1-190 ]. Cordyceps sinensis has complex morphological characteristics due to certain differences in host species and stroma morphology. Therefore, the cordyceps can be preliminarily classified and identified according to the morphological characteristics of the cordyceps, and meanwhile, the morphological identification is more convenient and quicker than other modes when the cordyceps is identified [ Helezhong, black-like cordyceps morphology, genetic differentiation and phylum fungus diversity research [ D ]. Shanxi university, Shuoshi paper, 2019.07 ]. However, due to the complexity of morphological features of cordyceps sinensis, it is difficult to accurately identify and correctly classify cordyceps sinensis by only relying on traditional morphological methods.

At present, the method combining morphological identification and molecular biology identification is widely applied to species identification of cordyceps fungi. Phylogenetic analysis is carried out on the 6 gene sequence fragments, and species identification is more accurate. Species identification and classification status of Cordyceps fungi have important significance for related researches such as active ingredients, pharmacological action and product development (Sun GH, Hywel-Jones NL, Sun JM, et al. physiogenic classification of Cordyceps and the clavicipitaceae fungi [ J ]. Sudies in Mycol,2007,57(01):5-59. Chen Zihong-Pang Cordyceps biological research [ D ]. Yunnan university, doctor, 2013, 12-21.).

The cordyceps fungus contains various active ingredients, such as cordycepin, polysaccharide, superoxide dismutase, cordycepic acid, ergosterol, protein, flavonoid, amino acid and the like, so the cordyceps fungus has important food and medicinal values and research and development potentials. Relevant studies show that cordycepic acid (D-mannitol) has positive effects of resisting tumor, promoting urination, resisting oxidation, promoting metabolism and the like, and can prevent and treat diseases such as cerebral thrombosis, cerebral hemorrhage, myocardial infarction and the like [ Wanqin, Liu Xiang, Wan Song, Cordyceps militaris liquid fermentation and research progress of application thereof [ J ] food and fermentation technology, 2017,53(1):88-91,114 ]; flavonoids have the capacities of resisting oxidation, regulating an immune system and the like, and can achieve an anti-tumor effect in ways of eliminating free radicals, inducing apoptosis of certain cells, regulating the immune system and the like [ Wangwang Cordyceps ITS sequence molecular evolution analysis and in vitro cell effect research of compound nutrient solution [ D ]. southwest traffic university, Master thesis, 2012,19-25 ]; cordyceps sinensis polysaccharide has positive effects of resisting oxidation, resisting aging, maintaining intestinal tract shape, etc.

Because the wild cordyceps sinensis resources are limited, the artificially cultivated cordyceps sinensis has the characteristics of long time, easy degradation, difficulty in mastering the cultivation technology and the like, a large amount of bioactive substances cannot be obtained, and the current market demand is difficult to meet. Through liquid culture, the production efficiency can be improved, the yield can be improved, and various bioactive substances [ Liu, A.J., and Zhang, Y.M. structural properties of polysaccharides from cultured from animals and fermentation broth [ Liu, A.J., and Zhang, Y.M. structural properties of polysaccharides from cultured from animals and bacteria Polymers,2016,142:63-72. Geneva. Cordyceps militaris liquid fermentation and active extract thereof influence research on mouse intestinal tract and physiology [ D ] university, Shuoshi paper, 2019,3 ]. Researches find that the fermentation mycelium of some cordyceps sinensis fungi and the fungus fruiting body have similar pharmacological effects and are widely applied to various food health products.

Disclosure of Invention

The technical problems to be solved by the invention are as follows: the invention utilizes morphological and molecular biology methods (ITS and TEF-1 sequences) to perform phylogenetic analysis on three wild cordyceps fungi of cordyceps, accurately identifies species, and compares cordycepic acid, flavone, polysaccharide and amino acid contents and free radical clearance rates in mycelia cultured by three cordyceps liquid on the basis.

In order to solve the technical problems, the invention provides the following technical scheme:

a new species of Cordyceps fungus is Cordyceps sinensis (Cordyceps sp.) CH01, and the preservation unit address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, with a preservation date of: on 09/08/2021, accession number: CGMCC No. 23076.

A method for separating new strains of cordyceps fungi comprises the following steps:

(1) collecting cordyceps fruiting bodies and worm bodies, wrapping collected samples by using tinfoil, and placing the wrapped collected samples in a collection box; drying in a 45 ℃ oven, and placing the dried sample and the label into a plastic package bag for storage;

(2) separating the mycelium in the polypide of the collected sample by adopting a tissue separation method: washing the surface of a fresh worm body for several times by using running water to remove surface soil, and preliminarily disinfecting the surface of the worm body by using 75% alcohol; under the aseptic condition, cutting the surface of the worm body by a blade, using the tip of a pair of tweezers to pick up a little of hypha in the worm body, quickly inoculating the hypha into a PDA (personal digital assistant) solid culture medium, placing the culture medium in an incubator at 28 ℃, carrying out inverted culture, observing once every other day, and timely treating a flat plate polluted by mixed bacteria; when hyphae grow initially, transferring the pollution-free hyphae to a new PDA culture medium by using a plate-scribing method for further culture; inoculating the purified mycelium into PDA slant test tube, and storing in refrigerator at-20 deg.C.

The identification method of the new cordyceps fungus species is characterized by comprising the following steps: the method comprises the following specific steps:

(1) morphological identification: observing and recording the macroscopic morphological characteristics of the cordyceps sinensis sample in detail, including the length of a stroma, the color, the shape, the length and the diameter of a fertile part, the color, the length and the diameter of a stipe of the cordyceps sinensis, and the type of a host, and preliminarily identifying the cordyceps sinensis sample to belong to or breed by referring to Chinese large-scale fungus resource atlas; recording the colony characteristics, including morphology, color and texture, of the isolated strains;

(2) and (3) molecular identification:

a. extracting genome DNA of the cordyceps sinensis sample;

b. PCR amplifying ITS and TEF-1 sequences by taking genome DNA as a template;

c. the results of PCR product sequencing were spliced at SeqMan and the spliced sequences were analyzed by alignment in GenBank. The primer sequence of the PCR amplification is as follows:

primer name	Primer sequences
		ITS1-F/ITS4	5＇-CTTGGTCATTTAGAGGAAGTAA-3＇/5＇-TCCTCCGCTTATTGATATGC-3＇
TEF1-983F/TEF1-2218R	5＇-GCYCCYGGHCAYCGTGAYTTYAT-3＇/5＇-ATGACACCRACRGCRACRGTYTG-3＇

The specific comparison analysis method comprises the following steps: sequence ordering was performed online using mafftv.6, with spaces, introns, and unclear sequences identified and removed with Gblocks in Geneious v.7.0.6; the phylogenetic tree was analyzed for maximum similarity and Bayesian analysis using a total of 83 sequences, 5 from the collected samples and 77 from GenBank. Phylogenetic tree analysis adopts two sequences of Tolypocladium inflatum and T.cyclindrosporum as outer group, and maximum similarity analysis is carried out by using GTRGAMMA nuclear gene model in software in RAxML v8.2.12; bayesian analysis is completed by using MrBayes v3.1 software; phylogenetic trees were opened with the FigTree v1.4.2 software and edited with Ai.

The invention has the following beneficial effects:

the invention accurately separates and identifies three wild cordyceps strains by using a method of combining morphology and molecules, wherein HGF01 and XJ46 are cordyceps columnar and cordyceps sobolifera respectively, CH01 is a new species, and cordycepic acid, flavone and polysaccharide components in liquid culture mycelia are detected on the basis. The three cordyceps mycelia contain cordycepic acid, flavone and polysaccharide components, the content of the cordycepic acid, the flavone and the polysaccharide components in CH01 is equivalent to that of cordyceps columnar and cordyceps sobolifera, the obvious difference is not generated, and the cordyceps mycelia have the same health-care edible value as the cordyceps columnar and cordyceps sobolifera. On the basis, the content of part of amino acid species in the CH01 hypha is obviously higher than that of the cordyceps columnar and the cordyceps sobolifera, the clearance rate of free radicals is obviously higher than that of the cordyceps columnar and the cordyceps sobolifera under certain concentration, and the oxidation effect is better.

Drawings

FIG. 1 is a standard curve of mannitol standard solution.

Fig. 2 is a standard curve of rutin standard solution.

FIG. 3 is a standard curve of glucose standards.

FIG. 4 is a comparison of Chinese caterpillar fungus fruiting body; wherein, a): wild Cordyceps columnar HGF 01; b) the method comprises the following steps Wild CH 01; c) the method comprises the following steps Wild cordyceps sobolifera XJ 46; the scale bar is 1.0 cm.

FIG. 5 is a comparison of the colonies of the solid culture of Cordyceps sinensis. Wherein, d): solid culture colony of the columnar cordyceps sinensis; e) the method comprises the following steps CH01 solid culture colony; f) the method comprises the following steps Culturing solid cicada fungus colony; the scale bar is 1.0 cm.

FIG. 6 is a comparison of cultured mycelia of Cordyceps liquid. Wherein, left panel g): culturing mycelium with column-shaped Cordyceps liquid; middle panel h): liquid culture mycelium of CH 01; right panel i): culturing mycelium with Cordyceps cicadae liquid; the scale bar is 1.0 cm.

FIG. 7 shows the maximum similarity of the 6 sequences of three Cordyceps fungi and their related species and Bayesian developmental tree;

wherein the values to the left of the support rate represent values for which the maximum similarity is greater than 70%, the values to the right represent values for which the Bayesian analysis is greater than 0.85, and the scale bar represents the expected value of the nucleotide change at each site.

FIG. 8 is a statistical chart of DPPH scavenging effect of mycelia of three kinds of Cordyceps cultured in liquid.

FIG. 9 is a diagram showing the results of agarose gel electrophoresis detection of amplification reaction products.

Detailed Description

The following examples are included to provide further detailed description of the present invention and to provide those skilled in the art with a more complete, concise, and exact understanding of the principles and spirit of the invention.

Example 1:

1. obtaining a cordyceps sinensis sample:

wild fresh cordyceps samples (numbered XJ46, HGF01 and CH01) were collected from Xian Ju county, Huangshan mountain valley and Dabie mountain sparrow lawn respectively in Zhejiang province. Xian Ju county is located at Zhejiang. It occupies 1612km in hilly and mountainous regions²The average annual temperature is 18.3 ℃, the average 7-month temperature is 28.5 ℃, and the plant species are rich. The yellow mountain valley was located in yellow mountain city, Anhui province, in subtropical monsoon climate areas. Due to high mountain body altitude, deep valley, large difference between the south slope and the north slope and complex terrain, the climate characteristics of high humidity and much rainfall are formed. The average temperature was 25 ℃ during months 7-10. The harry ray lawn natural protection area is located in the west of Anhui province, the plant area system is very complex, and the forest coverage rate is high.

2. Experimental methods

2.1 sample Collection and recording

Collecting time is 7-10 months between 2018 and 2019, after Cordyceps is found in forest, taking pictures to record morphological characteristics and habitat, carefully digging out Cordyceps fruiting body and insect body, wrapping collected sample with tinfoil, placing in a collecting box, and taking back to laboratory. The day, more detailed subsites and host morphology characteristics were observed and recorded. Describing and recording the finished sample, taking a part of the sample, drying the part of the sample in allochroic silica gel (for molecular identification), drying the rest part of the sample in a 45 ℃ oven, and placing the dried sample and the label into a plastic packaging bag for storage.

2.2 hypha separation, purification and Strain preservation

And separating the mycelium in the polypide of the collected sample by adopting a tissue separation method. The surface of fresh polypide is washed several times by running water to remove surface mud, and then the surface of polypide is primarily disinfected by 75% alcohol. Under the aseptic condition, a blade is used for cutting the surface of the worm body, a little hypha in the worm body is taken by tweezers at the tip of the tweezers and is quickly inoculated in a PDA solid culture medium, the culture medium is placed in an incubator at 28 ℃ for inverted culture, observation is carried out once every other day, and the flat plate polluted by the mixed bacteria is processed in time. When the hyphae grow initially, the hyphae without pollution are transferred to a new PDA culture medium for further culture by using a plate-scribing method. Inoculating the purified mycelium into a PDA slant test tube, and storing in a refrigerator at-20 deg.C.

2.3 morphological characterisation

Observing and recording the macroscopic morphological characteristics of the cordyceps sinensis sample in detail: the length of the stroma, the color, shape, length, diameter of the fertile part, the color, length, diameter of the stipe of the fungus, the host type, and the genus or species are preliminarily identified according to the graphic and identification of Chinese large-scale fungus resources. The colony characteristics of the isolated strains, including morphology, color and texture, were recorded and the size descriptions are expressed as mean ± standard deviation (min-max).

2.4 molecular characterization

2.4.1 DNA extraction

The fruiting bodies of the samples and the strains isolated from them are preliminarily classified and identified according to morphological characteristics, and then further identified on the molecular level by ITS and TEF-1 sequences. The purified DNA of the cordyceps mycelia is extracted by using a fungal genome extraction kit. And (3) taking a proper amount of purified mycelium, putting the mycelium into a 1.5mL sterile centrifuge tube, adding liquid nitrogen, fully grinding the mycelium by using a grinding rod, and extracting DNA according to the method steps of Guo Liang and the like.

2.4.2 PCR amplification

The PCR amplification reaction system comprises Taq PCR Master Mix, upstream and downstream primers (ITS1-F/ITS4, TEF1-983F/TEF1-2218R) and Sterilized ddH₂And O, forming a template. The primer sequences are shown in the table, and the PCR amplification reaction conditions are that denaturation at 94 ℃ is carried out for 4min, then continuous 35 cycles are carried out, denaturation at 94 ℃ is carried out for 40s, annealing at 50 ℃ is carried out for 40s, and extension at 72 ℃ is carried out for 150 s. For the primer ITS1-F and ITS4 primers, denaturation at 94 ℃ for 3min followed by 35 successive cycles of denaturation at 95 ℃ for 30s, (52 ℃) annealing for 1min, extension at 72 ℃ for 1min, and extension at 72 ℃ for 10min after completion of the cycles. The amplification reaction products were detected by agarose gel electrophoresis (see FIG. 9), and the resulting sequences were sent to Chuzhou general sequencing for bidirectional sequencing.

TABLE 2-1 primer names and sequences

Primer name	Primer sequences
		ITS1-F/ITS4	5＇-CTTGGTCATTTAGAGGAAGTAA-3＇/5＇-TCCTCCGCTTATTGATATGC-3＇
TEF1-983F/TEF1-2218R	5＇-GCYCCYGGHCAYCGTGAYTTYAT-3＇/5＇-ATGACACCRACRGCRACRGTYTG-3＇
		NS1/NS4	5’-GTAGTCATATGCTTGTCTC-3’/5’-CTTCCGTCAATTCCTTTAAG-3’

2.4.3 DNA sequence alignment analysis and phylogenetic Tree construction

The results of sequencing were spliced at SeqMan (see Table 2-2), and the spliced sequences were analyzed by alignment in GenBank. Sequence ordering was performed online using mafftv.6, with spaces, introns, and unclear sequences identified and removed using Gblocks in Geneious v.7.0.6. The phylogenetic tree was analyzed for maximum similarity and Bayesian analysis using a total of 83 sequences, 5 from the sample collected in this experiment and 77 from GenBank. Phylogenetic tree analysis uses two sequences, Tolypocladium inflatum and T.cyclindrosporum, as exo-cluster, and maximum similarity analysis (ML) is performed using the GTRGAMMA nuclear gene model in the software in RAxML v8.2.12. Bayesian analysis [ quality of the girl, Yunnan myceliophthora textbook fungus molecular phylogeny research, Yunnan university, Master thesis, 2019:1-96 ] is completed by using MrBayes v3.1 software. Phylogenetic trees were opened with the FigTree v1.4.2 software and edited with Ai.

TABLE 2-2 sequence results after PCR product sequencing splicing

2.5 liquid culture

Inoculating the purified three cordyceps mycelia from the PDA solid culture medium into a liquid culture bottle (250mL) under the aseptic condition, and culturing at 23 ℃ for 7d at 150r/min, wherein each cordyceps is provided with three repeats. The mycelium obtained by culture is fully ground into powder after being cleaned, filtered and dried at 60 ℃.

2.6 active ingredient study

2.6.1 preparation of samples to be tested

Sample solution of cordycepic acid to be detected: extracting cordycepic acid with hot water. Accurately weighing 0.20g of Cordyceps sinensis dry mycelium powder, adding 16mL of distilled water, boiling in water bath for 2h, filtering to obtain filtrate, and adding distilled water to constant volume of 100 mL.

Sample solution of flavone to be tested: extracting total flavone from three Cordyceps mycelia by ultrasonic extraction method. Accurately weighing 0.20g Cordyceps sinensis mycelia powder, extracting with 12ml 85% ethanol as solvent under ultrasonic wave (30 deg.C, 30min), filtering to obtain filtrate, and diluting with 85% ethanol to 100 ml.

Sample solution of polysaccharide to be tested: the extraction method of polysaccharide is the same as that of cordycepic acid, and the extraction method is hot water extraction. Precisely weighing 0.20g of each cordyceps sinensis mycelium powder, leaching in a boiling water bath for 2h, filtering to retain filtrate, adding 4 times of ethanol by volume into the filtrate, standing in a refrigerator at 4 ℃ for 12h, drying the precipitate in an oven at 60 ℃, dissolving with distilled water (decolorizing with activated carbon), and fixing the volume to 100 ml.

2.6.2 Standard Curve preparation

Preparing a mannitol standard curve: referring to the methods of extraction and determination of cordycepic acid in the shore glume, livin, wangtao, paecilomyces pupae fermentation mycelium [ J ]. science and technology in food industry, 2012,33(01). making a standard curve, drawing the standard curve (as shown in fig. 1).

Preparing a rutin standard curve: referring to Pueraria genu, Isaria cicadae Miq composition analysis and polysaccharide antioxidant activity research [ D ] Jiangsu university, Master thesis, 2019:13-18, a method for making a standard curve is used to draw the standard curve (as shown in FIG. 2).

Glucose standard curve preparation: according to the method for identifying 1 wild cordyceps sobolifera and comparing intracellular and extracellular polysaccharide anti-liver cancer activities [ J ]. The university of agriculture and forestry in northwest (Nature science edition), 2020,48(12): 117-.

2.6.3 determination of cordycepic acid, flavone and polysaccharide

The contents of cordycepic acid, flavone and polysaccharide in the three cordyceps sinensis samples to be tested are respectively measured by adopting a spectrophotometry method, an aluminum nitrate colorimetric method and a sulfuric acid-anthrone method, and the contents of cordycepic acid, flavone and polysaccharide in the mycelia are calculated according to a standard curve.

2.6.4 statistical analysis

The determined contents of cordycepic acid, flavone and polysaccharide were subjected to basic analysis including mean and Standard Deviation (SD) using STATISTICA statistical software. After the three components are subjected to basic analysis, one-way ANVOA-Tukey HSD is used for analyzing whether the contents of cordycepic acid, flavone and polysaccharide have significant difference among the three cordyceps mycelia and whether the contents of cordycepic acid, flavone and polysaccharide in the three cordyceps mycelia have significant difference.

3.1 species identification

3.1.1 fruiting bodies and colony characteristics

HGF01 is Cordyceps fungus, has no obvious stroma characteristics, and has spore powder purple, host spider, long axis of 5.0cm and short axis of 3.86cm (figure 4-a). HGF01 solid culture colony is white fluffy hypha in the initial stage, purple powdery spore grows gradually in the middle of the late stage colony, and the back of the colony is white and flat (FIG. 5-d). The total length of CH01 fruiting body is 1.96 + -0.73 cm (1.14-2.71cm) (excluding host length), and is cylindrical and yellowish, and the host is lepidopteran insect cocoon (FIG. 4-b). The initial stage of the solid culture colony of CH01 was white hairy velvet-like hyphae, the late stage hyphae extended significantly and were orange yellow, and the back of the colony was dark yellow and relatively flat (FIG. 5-e). The XJ46 fruiting body grows from head of pupa Cicadae. The total length of fruiting body is 2.38 + -1.13 cm (1.14-3.86cm), and the host is pupa Cicadae (FIG. 4-c). XJ46 solid cultured colony was pure white villous hypha in the early stage, and faint yellow spore scattered in the later stage, and the back of the colony was not wrinkled (FIG. 5-f). The mycelia with a size of 0.5-2.8cm are grown in the culture bottle for liquid culture of three kinds of Cordyceps, and have moderate density and uniform distribution (as shown in FIG. 6).

3.1.2 molecular characterization

Table 3-1 seed name and GenBank number for phylogenetic Tree construction

The results of the alignment of the ITS and TEF-1 sequences in the NCBI database and the phylogenetic tree based on the ITS and TEF-1 sequences (as shown in FIG. 7) show that the similarity between HGF01 and the C.cylindrica LC008212 ITS sequence and C.cylindrica LC008347 TEF-1 sequence is 100% and 99%, respectively. HGF01 and c. cylindrica were grouped together, with a maximum similarity analysis support rate of 100% and a bayesian analysis of 1; XJ46 shares 100% sequence similarity with both C.cicadae KF740422 ITS and C.cicadae MK770632 TEF-1. XJ46 and c.subconteuises are grouped into one, the maximum similarity analysis support rate is 100%, and bayesian analysis is 1; the alignment of HGF01 and XJ46 sequences was consistent with developmental tree results. The similarity of CH01 to the sequences of C.takamontana MF361939 ITS and C.tendipes MH521911TEF-1 was 99%, 100%, respectively. However, CH01 was clustered into one branch alone, the maximum similarity analysis support was 90%, bayesian analysis was 0.961, and was far from the experimental sample and c. According to the morphological and molecular identification and the comprehensive result of phylogenetic trees, XJ46 is cordyceps sobolifera, HGF01 is cordyceps columnar, and CH01 is cordyceps new species.

3.2 content and difference of three Cordyceps components

TABLE 3-2 contents of cordycepic acid, flavone and polysaccharide of three Cordyceps fungi and their significant differences

Note: the ingredient contents are expressed as mean ± SD; p is less than 0.05, and the difference is significant; p <0.01, with very significant variability

Cordycepic acid, flavone and polysaccharide are respectively taken as factors, and the content of three cordyceps components and the significant difference of the three cordyceps components are analyzed. In the content of cordycepic acid, no significant difference exists among cordyceps columnar (0.52mg/g), cordyceps sobolifera (0.63mg/g) and CH01(0.82 mg/g). The content of the CH01 cordycepic acid is higher than that of cordyceps columnar and cordyceps sobolifera, but the contents of the cordycepic acid in the cordyceps columnar and cordyceps sobolifera are less than 1 mg/g. The flavone content detection data shows that no significant difference exists among the cordyceps columnar, cordyceps sobolifera and the new CH01 species, the content of the new CH01 species (0.05mg/g) is higher than that of the cordyceps columnar and cordyceps sobolifera (0.04mg/g) and 0.02mg/g, and the flavone content of the CH01 is about 2 times that of the cordyceps sobolifera. There was a significant difference in polysaccharide content between cordyceps sobolifera and CH01 (P <0.01, df ═ 6.00), whereas cordyceps columnar was not statistically different from CH01 and cordyceps sobolifera. The content of cordyceps sobolifera polysaccharide (56.09mg/g) is obviously higher than that of CH01(27.22mg/g), and the content of cordyceps sobolifera polysaccharide is about 2 times of that of CH 01. The polysaccharide content of Cordyceps pillared is 44.40mg/g (Table 3-2).

3.3 composition and content of three Cordyceps amino acids

Seventeen amino acids contained in the three cordyceps mycelia are detected by an amino acid automatic analyzer, and the fourteen amino acids of aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, phenylalanine, histidine, tryptophan and arginine in the cylindrical cordyceps sinensis, cordyceps sobolifera and CH01 mycelia have no statistical difference, while the three amino acids of cysteine, methionine and lysine have difference. On cysteine, the CH01 strain and the cordyceps cylindracea and the cordyceps sobolifera have significant difference (P is less than 0.05), and the cordyceps sobolifera and the cordyceps cylindracea have no significant difference. The cysteine content of the CH01 strain (0.583%) is obviously higher than that of cylindrical cordyceps sinensis (0.097%) and cicada fungus (0.068%), wherein the CH01 strain is about 6 times of the cylindrical cordyceps sinensis and 9 times of the cicada fungus. The methionine content (0.287%) in cylindrical cordyceps sinensis was significantly higher than that of cordyceps sobolifera (0.030%) (P <0.05), while the CH01 strain (0.121%) had no statistical difference from both cylindrical cordyceps sinensis and cordyceps sobolifera. At the content of lysine, there were very significant differences (P <0.01) between cicada fungus (55.813%) and both the CH01 strain (1.382%) and the cylindrical cordyceps sinensis (1.112%), while there were no statistical differences between the CH01 strain and the cylindrical cordyceps sinensis. The lysine content of Cordyceps cicadae was significantly higher than that of CH01 strain and cylindrical Cordyceps sinensis, and was about 55 times of those of both (tables 3-3 and 3-4).

TABLE 3-3 average content (%)% of 17 amino acids in three mycelia of Cordyceps cicadae, strain CH01 and Cordyceps cylindrica

TABLE 3-4 significant differences of 17 amino acids in three mycelia of Cordyceps cicadae Miq, strain CH01 and Cordyceps cylindrica

Note: denotes P <0.05, significant difference; denotes P <0.001, very significant difference.

3.4 DPPH scavenging ability of three Cordyceps species

According to the reference: dynamic changes of main active substances and antioxidant activity in a process of fermenting needle mushroom stems by paecilomyces puparum [ J ] food research and development, 2020,41(04):19-26. The effect of liquid culture of the three cordyceps mycelia on removing DPPH was measured, and a curve was established as shown in FIG. 8 with the concentration as abscissa and the removal rate as ordinate. As can be seen from FIG. 8, when the cordyceps sobolifera concentration is less than 0.24mg/mL, the DPPH clearance rate is decreased along with the increase of the concentration of the cordyceps mycelium powder solution; a minimum of 19.56% clearance was achieved at a concentration of 0.24 mg/mL; at concentrations greater than 0.24mg/mL, the clearance rate tends to increase with increasing concentration. The cylindrical cordyceps sinensis is in the concentration of 0.08-0.24 mg/mL, the DPPH clearance rate is reduced along with the increase of the concentration of cordyceps sinensis mycelia, and the minimum value is 18.47%; within the concentration of 0.24-0.32 mg/mL, the clearance rate is increased along with the increase of the concentration; within the concentration of 0.32-0.40 mg/mL, the clearance rate is reduced along with the increase of the concentration. The CH01 strain is 0.08-0.16 mg/mL, and the DPPH clearance rate is reduced along with the increase of the concentration of the cordyceps sinensis mycelia to reach a minimum value of 18.66%; within the concentration of 0.16-0.24 mg/mL, the clearance rate is increased along with the increase of the concentration; within the concentration of 0.24-0.32 mg/mL, the clearance rate is reduced along with the increase of the concentration; within the concentration of 0.32-0.40 mg/mL, the clearance rate is increased along with the increase of the concentration.

4.1 morphological and molecular species identification of three Cordyceps species

The invention accurately identifies the three wild cordyceps fungi based on the methods of morphology and molecular biology, and the identification result shows that the morphological identification is basically consistent with the molecular identification result, and the XJ46 and the HGF01 are accurately identified to be cordyceps sobolifera and cordyceps columnar respectively. The characteristics of both the collected sample of HGF01 and the isolated colonies were consistent with the characterization of Nomuraea atypicola, a cordyceps sinensis anamorph. The XJ46 fruiting body and colony characteristics are consistent with the characterization of the identified Cordyceps cicadae. Since the macroscopic characteristics of the collected CH01 fruiting bodies are not obvious, only cordyceps genus is identified by macroscopic morphological identification, and specific species cannot be accurately identified. Although morphological identification shows a certain limitation, the method still has a certain significance for sample collection and colony culture. The results of sequence alignment in molecular identification show that the sequences tested in this study all found identical or similar sequences in the NCBI database. Wherein, the similarity of CH01 and C.takamontana MF361939 ITS sequences reaches 99%, and the similarity of CH01 and C.tenuipes MH521911 reaches 100% on the alignment of TEF-1 alpha sequences. However, phylogenetic trees based on ITS and TEF-1 α sequences showed that CH01 clustered as one branch alone, with a maximum similarity analysis support rate of 90%, bayesian analysis of 0.961, and a greater distance from c. By combining the morphological and molecular identification results, XJ46 can be identified as cordyceps sobolifera, HGF01 as cordyceps pillared and CH01 as a new species.

4.2 active ingredients of three Cordyceps

Under the same liquid culture condition, cordycepic acid, flavone and polysaccharide components in the three cordyceps liquid culture mycelia are detected, and the results show that the three cordyceps liquid culture mycelia contain cordycepic acid, flavone and polysaccharide components. No significant difference exists among cordyceps column (0.52mg/g), cordyceps sobolifera (0.63mg/g) and CH01(0.82mg/g) in cordycepic acid content. The cordycepic acid, the CH01 and the cordyceps sobolifera have no significant difference in cordycepic acid and flavone contents, and the cordyceps sobolifera have no significant difference in polysaccharide contents. Phylogenetic trees show that CH01 is close to cicada fungus relativity, so CH01 is similar to cicada fungus, and the phylogenetic trees have good pharmacological action. The Cordyceps pillared with CH01 also has edible and medicinal value.

4.3 amino acid content of three Cordyceps

The method uses an amino acid automatic analyzer to detect that the mycelium of the cylindrical cordyceps sinensis, the CH01 strain and the cicada fungus obtained by liquid culture contains 17 amino acids. The detection result shows that the content of cysteine is higher, and the CH01 strain is obviously higher than cicada fungus and cylindrical cordyceps sinensis; the content of lysine in cordyceps sobolifera is obviously higher than that of cylindrical cordyceps sinensis and CH01 strain, other 14 kinds of amino acid have no statistical difference, and the content of the amino acid is different from that of other recorded cordyceps sinensis.

4.4 radical scavenging ability of three Cordyceps

As can be seen from FIG. 8, the concentration of the mycelium of the three Cordyceps sinensis strains is within 0.08-0.32 mg/mL, and the difference of DPPH clearance rate is small. The DPPH clearance of the CH01 strain is significantly higher than that of cylindrical Cordyceps sinensis and Cordyceps sobolifera at concentrations of 0.08mg/mL, 0.24mg/mL and 0.40 mg/mL. The concentration of the whole experiment is integrated to obtain: the DPPH clearance of the CH01 strain is the highest at the same concentration of cordyceps mycelia. In addition, the half inhibitory concentration IC50 of the CH01 strain is 1.641 which is obviously higher than that of cordyceps sobolifera and cordyceps columnar.

The above embodiments are only for illustrating the technical idea of the present invention, and the protection scope of the present invention cannot be limited thereby, and any modification made on the basis of the technical scheme according to the technical idea proposed by the present invention falls within the protection scope of the present invention; the technology not related to the invention can be realized by the prior art.