Cultivation method and application of selenium and zinc-rich bugleweed

Abstract

The application provides a cultivation method of selenium and zinc enriched Cordyceps militaris and application thereof. The application also provides a Cordyceps militaris M8 with high tolerance to selenium. The cultivation method of the selenium and zinc enriched Cordyceps militaris comprises the steps of strain activation, seed liquid preparation and cultivation of the selenium and zinc enriched Cordyceps militaris. The Cordyceps militaris fruiting body cultivated by the method is enriched with both selenium and zinc, and the original active substances are maintained, so that high-quality selenium and zinc enriched Cordyceps militaris fruiting body is obtained. In particular, sodium selenite and zinc sulfate are added in the cultivation medium, and sodium selenite and zinc disodium ethylenediaminetetraacetate are added, so that the selenium content of the corresponding Cordyceps militaris fruiting body is the highest, the zinc content is relatively high, the biomass is relatively high, the adenosine content is the highest, the cordycepic acid content is the highest, and the nutritional and medicinal values of the Cordyceps militaris are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a cultivation method of selenium and zinc-rich Cordyceps cicadae and application thereof. BACKGROUND

[0002] Cordyceps cicadae belongs to Ascomycota, Cordycipitaceae and Isaria, and is a kind of entomogenous fungus with both medicinal and edible functions. Cordyceps cicadae extract contains rich bioactive components. Cordyceps cicadae polysaccharide has good antioxidant, antitumor and blood lipid-lowering effects. Cordycepic acid has the effects of lowering blood pressure and blood lipid and replacing sugar as a sweetener. Cordycepin has certain anticancer effect. At the same time, Cordyceps cicadae has similar active components to Cordyceps sinensis. Due to the short cultivation period and large biomass, Cordyceps cicadae becomes an optimal substitute for Cordyceps sinensis. Due to the limitations of natural conditions and overexploitation by human factors, the yield of wild Cordyceps cicadae cannot meet market demand, and there are differences in quality among different varieties. Therefore, artificial cultivation can be carried out, and the conditions of the culture medium can be adjusted to obtain high content of target products.

[0003] Selenium and zinc are essential trace elements for the human body, which are mainly supplemented through food intake to maintain the health of the body. Selenium deficiency in the human body can cause local diseases such as Keshan disease and Kashin-Beck disease, and also cause high incidence of diseases such as cardiovascular disease and cancer. Appropriate selenium intake is beneficial to health, but high selenium intake also poses health risks. In the natural environment, selenium mainly exists in the form of inorganic selenium such as selenate and selenite, and organic selenium such as selenomethionine, selenocystine, selenomethylselenocysteine and selenoprotein. Organic selenium has higher biological activity and safety than inorganic selenium, and is more easily digested and absorbed by animals. The Chinese Nutrition Society determines that the appropriate range of human selenium intake is 60-400 μg / d. Zinc is also an essential trace element for the human body. Zinc can promote the normal development of the human body, improve appetite, improve the immune capacity of the body, and enhance the resistance to diseases. Zinc deficiency can cause dwarfism and poor intellectual development. The Chinese Nutrition Society recommends that the zinc intake of adult men is 12.5 mg / d, the zinc intake of adult women is 7.5 mg / d, and the zinc intake of pregnant women is 9.5 mg / d. Edible fungi can be cultivated by selenium and zinc enrichment to increase the selenium and zinc content in the fruiting bodies, which has important biological significance. However, there is no report on the use of Cordyceps cicadae to simultaneously enrich selenium and zinc and increase the content of active substances such as cordycepic acid and adenosine. SUMMARY

[0004] The present application aims to provide a cultivation method of selenium and zinc-rich Cordyceps cicadae and application thereof.

[0005] Another object of the present application is to provide a strain of Cordyceps cicadae with high selenium tolerance and organic selenium conversion capacity.

[0006] In order to achieve the object of the present application, in a first aspect, the present application provides a strain of Isaria cicadae M8 with high selenium tolerance, which is isolated from wild Isaria cicadae in Tianmushan, Zhejiang, and has been preserved in the China General Microbiological Culture Collection Center, No. 3, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology of Chinese Academy of Sciences, Postcode 100101, on January 22, 2021, with the preservation number CGMCC No. 21453. The microbiological characteristics of Isaria cicadae M8 are as follows: the colony is round, the surface is fluffy, the mycelium is white in the early stage and is closely attached to the culture medium, and is yellowish in the later stage, and a large amount of conidiospores are produced, and the color turns to gray Figure 1 ). The conidial phialides are densely branched, the base of the sporulating cell is oval or spherical, the single conidial wall is smooth and transparent, and the size is (5-14) μm x (1.4-2.9) μm, and the spores are aggregated in long chains Figure 2 ), and after verification, the above characteristics are consistent with the characteristics of Isaria cicadae in Chinese Fungi.

[0007] The molecular biology identification results are as follows: the genomic DNA of M8 strain is extracted by CTAB method, and ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2R (5'-GCTGCGTTCTTCATCGATGC-3') are used as primers, and the ITS DNA fragment of M8 strain is obtained by PCR amplification, and the PCR product is sequenced after purification to obtain the ITS sequence as shown in SEQ ID NO: 1, and the result of sequence alignment analysis in NCBI (https: / / www.ncbi.nlm.nih.gov / ) online database Blast shows that the ITS sequence of M8 strain has 100% similarity with the ITS sequence of Isaria cicadae strain, and the M8 strain is identified as Isaria cicadae in combination with the morphological characteristics.

[0008] In a second aspect, the present application provides a microbial agent or composition containing the Isaria cicadae M8.

[0009] In a third aspect, the present application provides the use of the Isaria cicadae M8 in the preparation of selenium and / or zinc-rich Isaria cicadae.

[0010] In a fourth aspect, the present application provides a cultivation method of selenium and zinc-rich Isaria cicadae, comprising the following steps:

[0011] A, strain activation;

[0012] B, preparation of seed liquid;

[0013] C, cultivation of selenium and zinc-rich Isaria cicadae.

[0014] Step C is cultivation of Cordyceps using wheat medium, and inorganic selenium salt and / or nano-selenium and inorganic zinc salt and / or organic zinc salt are added to the wheat medium.

[0015] The method for preparing the wheat medium comprises mixing wheat and nutrient solution at a ratio of 1:1-1.5 (g / mL), and sterilizing at 105-121℃ for 20-30 min.

[0016] The nutrient solution is prepared as follows: 20 g of sugar, 2 g of KH2PO4 and 1 g of MgSO4·7H2O are mixed, and water is added to make up to 1000 mL. The sugar can be selected from at least one of glucose, sucrose or maltose, etc.

[0017] The inorganic selenium salt includes selenate (such as sodium selenate) and / or selenite (such as sodium selenite); the nano-selenium includes biosynthetic nano-selenium (see CN201510047365.4, CN201610566900.1, CN201610952997.X, CN201610946230.6 or CN201610946282.3) and / or chemically synthesized nano-selenium (see ZL201410520106.4); the inorganic zinc salt is, for example, zinc sulfate, etc.; and the organic zinc salt is, for example, zinc disodium ethylenediaminetetraacetate, etc.

[0018] Further, the concentration of selenate in the wheat medium is 0.5-100 mg / kg, preferably 40 mg / kg.

[0019] Further, the concentration of selenite in the wheat medium is 0.5-100 mg / kg, preferably 40 mg / kg.

[0020] Further, the concentration of zinc sulfate in the wheat medium is 60-120 mg / kg, preferably 100 mg / kg.

[0021] Further, the concentration of zinc disodium ethylenediaminetetraacetate in the wheat medium is 60-120 mg / kg, preferably 100 mg / kg.

[0022] The aforementioned method, step A comprises inoculating Cordyceps into improved PDA medium, and culturing in a constant temperature incubator at 20-25℃ for 18-25 days until a large number of spores are produced.

[0023] The improved PDA medium is prepared as follows: 200 g of potato (dioscorea), 20 g of glucose (or sucrose or maltose), 17 g of agar, 3 g of proteose peptone, 2 g of KH2PO4, 1 g of MgSO4·7H2O and 0.5 g of VB1 are mixed, and water is added to make up to 1000 mL. B30mg mixed, add water to 1000mL, autoclaved at 105-121℃ for 20-30min.

[0024] The foregoing method, the activated Coccinia fungus cake with a diameter of about 5-9mm is inoculated into a 500mL culture bottle (conical bottle) containing 200mL modified PD medium for shake culture, and the shake culture conditions are: 21℃, rotation speed 180r / min, shake culture for 3 days to obtain a seed liquid.

[0025] The modified PD medium is prepared as follows: 200g of potato, 20g of glucose, 3g of proteose peptone, 2g of KH2PO4, 1g of MgSO4·7H2O, and 0.1g of VB1 are mixed, and water is added to 1000mL. B 30mg mixed, add water to 1000mL, autoclaved at 105-121℃ for 20-30min.

[0026] The foregoing method, the liquid seed liquid is diluted 3-7 times, 5mL of the diluted bacterial solution is added to a cultivation bottle containing wheat medium, and 20-30g of wheat medium is contained in each cultivation bottle; the cultivation bottle inoculated with the bacteria is transferred to a constant-temperature cultivation room at 21℃, the relative humidity is 80%, and the cultivation is carried out in the dark for 4 days, then light cultivation is carried out for 35 days under 12 hours of white light and 12 hours of red light alternately until the fruiting bodies are mature, and the top spores are collected. The collected Coccinia fruiting bodies are dried in an oven at 50℃, weighed, and ground for use.

[0027] The intensity of the white light can be 200-400lx, and the wavelength of the red light can be 620-760nm

[0028] Preferably, the Coccinia is Isaria cicadae M8.

[0029] In a fifth aspect, the present application provides any one of the following applications of the selenium and zinc-rich Coccinia obtained according to the method:

[0030] 1) used in the field of health care products;

[0031] 2) used in the field of food;

[0032] 3) used in the field of feed;

[0033] 4) used for preparing medicines or compositions.

[0034] The Coccinia fruiting bodies obtained by cultivation according to the method are enriched with both selenium and zinc, and the original active substances are maintained, so that high-quality selenium and zinc-rich Coccinia fruiting bodies are obtained. In particular, sodium selenite and zinc sulfate are added to the cultivation medium, and sodium selenite and zinc disodium ethylenediaminetetraacetate are added, and the corresponding Coccinia fruiting bodies have the highest selenium content, the highest zinc content, the highest biomass, the highest adenosine content, and the highest cordycepic acid content, which improves the nutritional and medicinal value of Coccinia. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The solid culture tolerance of the cicada to different forms of selenium in the preferred embodiment of the present application.

[0036] Figure 2 The microscopic features of the cicada M8 mycelium and spores in the preferred embodiment of the present application.

[0037] Figure 3 The colony diameter of the cicada under the treatment of different forms of selenium in the preferred embodiment of the present application.

[0038] Figure 4 The dose-effect curve of sodium selenite fitted by the logistic equation model in the preferred embodiment of the present application.

[0039] Figure 5 The microscopic photograph of the mycelium under the concentration of 750 mg / L selenium in the preferred embodiment of the present application.

[0040] Figure 6 The influence of different forms of selenium on the growth form of the cicada fruiting body in the preferred embodiment of the present application.

[0041] Figure 7 The dry weight of the selenium-rich cultivated fruiting body in the preferred embodiment of the present application.

[0042] Figure 8 The total selenium content of the cicada fruiting body under the treatment of different forms of selenium in the preferred embodiment of the present application.

[0043] Figure 9 The influence of sodium selenate and zinc sulfate on the cicada fruiting body in the preferred embodiment of the present application; A: fruiting body form, B: fruiting body biomass. a, b represent significant difference p<0.05.

[0044] Figure 10 The influence of sodium selenate and zinc sulfate on the adenosine and cordycepin in the cicada fruiting body in the preferred embodiment of the present application; A: adenosine, B: cordycepin. a, b represent significant difference p<0.05.

[0045] Figure 11 The influence of sodium selenate and zinc sulfate on the cordycepic acid in the cicada fruiting body in the preferred embodiment of the present application. a, b represent significant difference p<0.05.

[0046] Figure 12 The influence of sodium selenate and zinc sulfate on the trace elements in the cicada fruiting body in the preferred embodiment of the present application. a, b represent significant difference p<0.05.

[0047] Figure 13Effects of sodium selenate and zinc disodium ethylenediaminetetraacetate on the fruiting bodies of Cordyceps cicadae in the preferred embodiments of the present application; A: fruiting body morphology, B: fruiting body biomass. a, b represent significant difference p<0.05.

[0048] Figure 14 Effects of sodium selenate and zinc disodium ethylenediaminetetraacetate on the adenosine and cordycepin in the fruiting bodies of Cordyceps cicadae in the preferred embodiments of the present application; A: adenosine, B: cordycepin. a, b represent significant difference p<0.05.

[0049] Figure 15 Effects of sodium selenate and zinc disodium ethylenediaminetetraacetate on the cordycepic acid in the fruiting bodies of Cordyceps cicadae in the preferred embodiments of the present application. a, b represent significant difference p<0.05.

[0050] Figure 16 Effects of sodium selenate and zinc disodium ethylenediaminetetraacetate on the various trace elements in the fruiting bodies of Cordyceps cicadae in the preferred embodiments of the present application. a, b represent significant difference p<0.05.

[0051] Figure 17 Effects of sodium selenite and zinc sulfate on the fruiting bodies of Cordyceps cicadae in the preferred embodiments of the present application; A: fruiting body morphology, B: fruiting body biomass. a, b represent significant difference p<0.05.

[0052] Figure 18 Effects of sodium selenite and zinc sulfate on the adenosine and cordycepin in the fruiting bodies of Cordyceps cicadae in the preferred embodiments of the present application; A: adenosine, B: cordycepin. a, b represent significant difference p<0.05.

[0053] Figure 19 Effects of sodium selenite and zinc sulfate on the cordycepic acid in the fruiting bodies of Cordyceps cicadae in the preferred embodiments of the present application. a, b represent significant difference p<0.05.

[0054] Figure 20 Effects of sodium selenite and zinc sulfate on the various trace elements in the fruiting bodies of Cordyceps cicadae in the preferred embodiments of the present application. a, b represent significant difference p<0.05.

[0055] Figure 21 Effects of sodium selenite and zinc disodium ethylenediaminetetraacetate on the fruiting bodies of Cordyceps cicadae in the preferred embodiments of the present application; A: fruiting body morphology, B: fruiting body biomass. a, b represent significant difference p<0.05.

[0056] Figure 22 Effects of sodium selenite and zinc disodium ethylenediaminetetraacetate on the adenosine and cordycepin in the fruiting bodies of Cordyceps cicadae in the preferred embodiments of the present application; A: adenosine, B: cordycepin. a, b represent significant difference p<0.05.

[0057] Figure 23 Effect of sodium selenite and zinc disodium ethylenediaminetetraacetate on cordycepic acid in Cordyceps cicadae fruiting bodies in the preferred embodiments of the present application. a, b represent significant difference p<0.05.

[0058] Figure 24 Effect of sodium selenite and zinc disodium ethylenediaminetetraacetate on trace elements in Cordyceps cicadae fruiting bodies in the preferred embodiments of the present application. a, b represent significant difference p<0.05.

[0059] Figure 25 Effect of nano selenium and zinc sulfate on Cordyceps cicadae fruiting bodies in the preferred embodiments of the present application; A: fruiting body morphology, B: fruiting body biomass. a, b represent significant difference p<0.05.

[0060] Figure 26 Effect of nano selenium and zinc sulfate on adenosine and cordycepin in Cordyceps cicadae fruiting bodies in the preferred embodiments of the present application; A: adenosine, B: cordycepin. a, b represent significant difference p<0.05.

[0061] Figure 27 Effect of nano selenium and zinc sulfate on cordycepic acid in Cordyceps cicadae fruiting bodies in the preferred embodiments of the present application. a, b represent significant difference p<0.05.

[0062] Figure 28 Effect of nano selenium and zinc disodium ethylenediaminetetraacetate on trace elements in Cordyceps cicadae fruiting bodies in the preferred embodiments of the present application. a, b represent significant difference p<0.05.

[0063] Figure 29 Effect of nano selenium and zinc disodium ethylenediaminetetraacetate on Cordyceps cicadae fruiting bodies in the preferred embodiments of the present application; A: fruiting body morphology, B: fruiting body biomass. a, b represent significant difference p<0.05.

[0064] Figure 30 Effect of nano selenium and zinc disodium ethylenediaminetetraacetate on adenosine and cordycepin in Cordyceps cicadae fruiting bodies in the preferred embodiments of the present application; A: adenosine, B: cordycepin. a, b represent significant difference p<0.05.

[0065] Figure 31 Effect of nano selenium and zinc disodium ethylenediaminetetraacetate on cordycepic acid in Cordyceps cicadae fruiting bodies in the preferred embodiments of the present application. a, b represent significant difference p<0.05.

[0066] Figure 32 Effect of nano selenium and zinc disodium ethylenediaminetetraacetate on trace elements in Cordyceps cicadae fruiting bodies in the preferred embodiments of the present application. a, b represent significant difference p<0.05.

[0067] Figure 33 The chromatogram of 100 mg / L mixed standard determined by HPLC in a preferred embodiment of the present invention is shown below; 1: adenosine, 2: cordycepin.

[0068] Figure 34 The results of HPLC determination of adenosine and cordycepin in the fruiting body of Cordyceps sinensis in a preferred embodiment of the present invention are shown below; 1: adenosine, 2: cordycepin.

[0069] Figure 35 This is a standard curve of selenium speciation in a preferred embodiment of the present invention.

[0070] Figure 36 The chromatograms for the determination of selenium speciation in the fruiting bodies of cicada flowers in each treatment group are shown in the preferred embodiment of the present invention. Detailed Implementation

[0071] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0072] The culture media used in the following examples are as follows:

[0073] The modified PDA medium is prepared as follows: Combine 200g potato, 20g glucose, 17g agar, 3g peptone, 2g KH2PO4, 1g MgSO4·7H2O, and V... B Mix 30mg, add water to make up to 1000mL, and autoclave at 121℃ for 20min.

[0074] The modified PD medium is prepared as follows: 200g potato, 20g glucose, 3g peptone, 2g KH2PO4, 1g MgSO4·7H2O, and V... B Mix 30mg, add water to make up to 1000mL, and autoclave at 121℃ for 20min.

[0075] The nutrient solution for cicada flower cultivation is prepared as follows: Mix 20g of glucose, 2g of KH2PO4 and 1g of MgSO4·7H2O, and add water to make up to 1000mL.

[0076] The white light intensity used in the cultivation of cicada fruiting bodies is 200-400 lx, and the red light wavelength is 620-760 nm.

[0077] The nano selenium SeNPs used in the following examples can be referred to ZL201410520106.4, ZL201610946282.3, ZL201610566900.1, ZL201510047365.4, ZL201610952997.X, ZL201610946230.6 and ZL201711460184.X.

[0078] Example 1 Isolation, screening and identification of selenium-resistant high-yield strain

[0079] A 0.5M sodium selenite solution was prepared and sterilized by filtration, and then added to a modified PDA medium at about 50°C to make the final concentration of selenium 200mg / L, thereby preparing a selenium-containing modified PDA plate. Wild Cymbidium ensifolium was collected from Tianmushan in Zhejiang, and 3 spore stalks of Cymbidium ensifolium were cut from the Cymbidium ensifolium and placed in 20ml sterile water, shaken for 5 minutes on a vortex shaker, and 200ul of sterile water containing spores was evenly spread on the modified PDA medium plate containing 200mg / L of selenium, and cultured at 21°C for 7 days, to obtain 12 strains M1-M12.

[0080] Cymbidium ensifolium M1-M12 were inoculated into modified PDA medium and cultured in a 21°C constant temperature incubator for 20 days until a large amount of spores were produced, and then 5mm diameter activated Cymbidium ensifolium fungus cakes of each strain were inoculated into 500ml conical flasks containing 150ml modified PD medium, cultured at 21°C at a speed of 180r / min for 3 days to prepare seed liquid. Wheat and Cymbidium ensifolium cultivation nutrient solution was mixed according to the ratio of 20g:25ml (1:1.25), and then sterilized at 121°C under high temperature and high pressure. The prepared Cymbidium ensifolium seed liquid was diluted 5 times with sterile water, and 5ml of diluted bacterial liquid was added to each cultivation bottle. Each strain was cultivated in 10 bottles, and the cultivation bottles inoculated with bacteria were transferred to a constant temperature cultivation room at 21°C, with a relative humidity of 80% and light-free cultivation for 4 days, followed by 12 hours of white light and 12 hours of red light alternate light illumination for 35 days, and the fruiting bodies were matured, and the top spores were harvested. The harvested Cymbidium ensifolium fruiting bodies were dried in an oven at 50°C and weighed. The biomass of the fruiting bodies of the 12 Cymbidium ensifolium strains M1-M12 cultivated was 1.22±0.23g, 1.68±0.11g, 1.37±0.28g, 1.93±0.43g, 2.11±0.33g, 1.76±0.22g, 2.39±0.15g, 2.71g±0.17g, 1.66±0.24g, 1.99g±0.33g, 1.13±0.14g and 1.45±0.12g, respectively. Among them, the biomass of the fruiting bodies of M8 was significantly higher than that of the other 11 strains, and M8 was used as the preferred strain of the present application.

[0081] The colony is round, the surface is fluffy, the mycelium is white at the initial stage and is closely attached to the culture medium, and is yellowish at the later stage, and produces a large amount of conidiospores, and is gray Figure 1 The conidial phialide is relatively dense, the base of the sporulating cell is oval or spherical, the single conidiospore wall is smooth and transparent, is long oval or slightly curved with a waist contraction, and the size is (5-14) pm x (1.4-2.9) pm, and the spores are mostly aggregated in long chains (as shown in Figure 2 ), and after searching, the above characteristics are consistent with the characteristics of Isaria cicadae in Chinese Fungi.

[0082] The CTAB method was used to extract the genomic DNA of the M8 strain, and ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2R (5'-GCTGCGTTCTTCATCGATGC-3') were used as primers to obtain the ITS DNA fragment of the M8 strain by PCR amplification, and the ITS sequence of the PCR product after purification was as shown in SEQ ID NO: 1. The results of sequence alignment analysis in the NCBI (https: / / www.ncbi.nlm.nih.gov / ) online database showed that the ITS sequence of the M8 strain had 100% similarity with the ITS sequence of the Isaria cicadae strain, and the M8 strain was identified as Isaria cicadae in combination with the morphological characteristics.

[0083] Example 2 Selenium tolerance of Isaria cicadae

[0084] Sodium selenate Se (Ⅵ), sodium selenite Se (Ⅳ) and nano selenium SeNPs were added to the modified PDA culture medium, so that the final selenium concentration (calculated as selenium) gradient range was 0-20000 mg / L, 0-10000 mg / L and 0-1000 mg / L, respectively. After the Isaria cicadae strain M8 was cultured on the modified PDA plate for 7 days, a 5 mm diameter fungus cake was inoculated on the modified PDA plate added with different selenium concentrations, and was cultured in a constant temperature incubator at 21℃ for 11 days. The colony diameter was measured by using a vernier caliper.

[0085] Selenium tolerance culture results of Isaria cicadae on solid medium Figure 1 , Figure 3) shows that: when the concentration of selenium is 1-1000mg / L, the diameter of M8 colony has no significant difference compared with the control group; when the concentration of selenium is 5000 and 10000mg / L, the diameter of M8 colony is significantly larger than the control group; when the concentration of selenium reaches 20000mg / L, the diameter of M8 colony is significantly smaller than the control group, and the growth of M8 colony is inhibited, with an inhibition rate of 24.5%. When Se(Ⅳ) is added to the culture medium, when the concentration of selenium reaches 1-50mg / L, the diameter of M8 colony has no significant difference compared with the control group; when the concentration of selenium reaches 500 and 1000mg / L, the diameter of M8 colony is significantly smaller than the control group, and the growth of M8 colony is inhibited, with an inhibition rate of 23.44% and 66.89% respectively. When SeNPs is added to the culture medium, when the concentration of selenium is 1-50mg / L, the diameter of M8 colony has no significant difference compared with the control group; when the concentration of selenium reaches 500 and 1000mg / L, the diameter of M8 colony is significantly smaller than the control group, and the growth of M8 colony is inhibited, but the diameter of M8 colony is significantly larger than that of Se(Ⅳ) treatment with the same concentration. The IC 50 of Se(Ⅳ) is calculated by fitting with Logistic Equation, which is 645.8mg / L, and the correlation coefficient is 0.9959. Figure 4 .

[0086] Example 3 Influence of selenium on the morphology of mycelium and spores of C. sinensis

[0087] Se(Ⅵ), Se(Ⅳ) and SeNPs filtered and sterilized are added to the modified PDA culture medium to make the final selenium concentration (calculated as selenium) 750mg / L. C. sinensis strain M8 is cultured on the modified PDA plate for 7 days, and then 5mm diameter of mycelium is inoculated on the modified PDA containing different forms of selenium. The culture is incubated in a constant temperature incubator at 21℃ until the 5th day. A cover glass is inserted obliquely at the edge of the mycelium, and the culture dish is sealed and returned to the incubator for incubation until the 11th day. The morphology of mycelium and spores is observed under an optical microscope. The microscope observation result Figure 5 ) shows that: the spore-producing structure of the control group is complete, and the single cell is slender. SeNPs treatment group has no significant influence on the spore-producing structure and cell morphology of C. sinensis; in Se(Ⅳ) and Se(Ⅵ) treatment groups, the cells are short and thick, and the top of the mycelium is often forked. No spore-producing structure is observed in Se(Ⅳ) treatment group, and only a large number of top cyst structures are observed in Se(Ⅵ) treatment group. It is indicated that SeNPs has less influence on the morphology of mycelium, and Se(Ⅵ) and Se(Ⅳ) have great influence on the spore-producing structure and mycelium branching.

[0088] Example 4 Cultivation of selenium-rich C. sinensis fruiting body

[0089] 1. Activation of C. sinensis strain

[0090] Cicada flower M8 was inoculated into modified PDA medium and cultured in a constant temperature incubator at 21℃ for 20 days until a large number of spores were produced before use.

[0091] 2. Preparation of Cordyceps flower seed liquid

[0092] Take a 5mm diameter activated cicada flower cake and inoculate it into a 500mL Erlenmeyer flask containing 150mL of modified PD medium. Incubate at 21℃ and 180r / min for 3 days to prepare the seed culture.

[0093] 3. Cultivation of selenium-enriched cicada flower fruiting bodies

[0094] The wheat culture medium composition was as follows: wheat (dry weight) to nutrient solution in a ratio of 20g:25mL (1:1.25). After mixing, the wheat and nutrient solution were sterilized at 121℃ under high temperature and pressure. Filter-sterilized Se(VI), Se(Ⅳ), and SeNPs were added to the wheat culture medium to create a selenium concentration gradient (calculated as selenium) of 0-100mg / kg. The prepared *Cicada nymph* seed solution was diluted 5 times, and 5mL of the diluted bacterial solution was added to each culture bottle. After inoculation, the culture bottles were transferred to a constant temperature cultivation room at 21℃ with 80% relative humidity and cultured in the dark for 4 days. Then, they were cultured under alternating light of 12 hours of white light and 12 hours of red light for 35 days. Harvesting was possible when the fruiting bodies matured and spores were produced at the top. The harvested *Cicada nymph* fruiting bodies were dried in a 50℃ oven, weighed, ground, and then tested.

[0095] 4. Effects of selenium on the biomass of Cordyceps militaris

[0096] The results of biomass showed ( Figure 6 , Figure 7 The biomass of the fruiting bodies in the control group was 2.72 ± 0.14 g per culture bottle. Within the concentration range of 0-100 mg / kg for Se(VI) and SeNPs, there was no significant effect on the biomass of the fruiting bodies. When the concentration of Se(Ⅳ) in the culture medium was in the range of 0.5-40 mg / kg, there was no significant effect on the biomass of the fruiting bodies. When the concentration of Se(Ⅳ) reached 60 mg / kg, the biomass of the fruiting bodies was significantly reduced by 40.8%, to only 1.61 ± 0.39 g.

[0097] 5. Effect of selenium on the total selenium content in the fruiting bodies of Cordyceps militaris

[0098] Take 0.20 g of crushed fruiting body sample into a microwave digestion tube, add 8 mL of pure nitric acid, cold soak overnight, use a microwave digestion furnace to digest at 180°C for 2-3 hours, cool to room temperature after digestion is complete, dilute to 25 mL, take 1 ml of the digested and diluted sample, add 3 ml of ultrapure water and 1 ml of 6 mol / L hydrochloric acid, boil in a water bath for 2 hours, then reduce to room temperature after reduction is complete, and determine the total selenium content in the fruiting body by hydride generation-atomic fluorescence spectrometry (HG-AFS). When the sample is measured on the instrument, the carrier liquid is 10% HC1, the reducing agent is 0.5% KOH and 2% KBH4; the lamp current is 80 mA, the negative high voltage is 270 V, the carrier gas is 600 mL / min, the shielding gas is 800 mL / min, and the sample injection volume is 1 mL. The total selenium determination results are shown in Table 1. Figure 8 As shown in Table 1, the selenium concentration in the culture medium is increased, and the selenium concentration in the fruiting body is also increased. When the culture medium is added with 0 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.5 mg / kg, 5.0 mg / kg, 10.0 mg / kg, 20.0 mg / kg, 40 mg / kg, 60 mg / kg, 80 mg / kg, and 100 mg / kg of Se(Ⅵ), Se(Ⅳ), and SeNPs, respectively, the selenium content in the C. cicadae fruiting body in the Se(Ⅵ) treatment group is 0.26 mg / kg, 1.13 mg / kg, 2.06 mg / kg, 4.22 mg / kg, 7.95 mg / kg, 14.00 mg / kg, 25.26 mg / kg, 59.54 mg / kg, 100.61 mg / kg, 157.18 mg / kg, and 242.93 mg / kg, respectively; the selenium content in the C. cicadae fruiting body in the Se(Ⅳ) treatment group is 0.26 mg / kg, 1.35 mg / kg, 2.04 mg / kg, 4.06 mg / kg, 6.47 mg / kg, 14.04 mg / kg, 37.58 mg / kg, 90.59 mg / kg, 152.89 mg / kg, 252.99 mg / kg, and 369.19 mg / kg, respectively; the selenium content in the C. cicadae fruiting body in the SeNPs treatment group is 0.26 mg / kg, 1.03 mg / kg, 1.77 mg / kg, 3.92 mg / kg, 5.35 mg / kg, 9.20 mg / kg, 18.12 mg / kg, 27.96 mg / kg, 37.68 mg / kg, 43.04 mg / kg, and 46.23 mg / kg, respectively; when the selenium concentration in the culture medium is 100 mg / kg, the selenium content in the C. cicadae fruiting body in the Se(Ⅵ), Se(Ⅳ), and SeNPs treatment groups is 952 times, 1447 times, and 181 times that of the blank control group, respectively. In the range of 20-100 mg / kg, the selenium concentration in the fruiting body in the Se(Ⅳ) treatment group is significantly higher than that in the Se(Ⅵ) and SeNPs.

[0099] Example 5 Method for cultivating selenium and zinc-enriched Cordyceps militaris using sodium selenate and zinc sulfate

[0100] 1. Activation of Cordyceps militaris spores

[0101] Cordyceps militaris M8 was inoculated into modified PDA medium and cultured in a constant temperature incubator at 21°C for 20 days until a large number of spores were produced for standby use.

[0102] 2. Preparation of Cordyceps militaris liquid seed solution

[0103] A 5 mm diameter activated Cordyceps militaris cake was inoculated into a 500 mL conical flask containing 200 mL of modified PD medium for shake culture. The shake culture conditions were 21°C and a rotation speed of 180 r / min for 3 days to prepare the seed solution.

[0104] 3. Cultivation of selenium and zinc-enriched Cordyceps militaris

[0105] The composition of the wheat medium was 20 g of wheat (dry weight) to 25 mL of nutrient solution. After mixing the wheat and the nutrient solution, high-temperature and high-pressure sterilization was performed at 121°C for standby use. Filter-sterilized sodium selenate and zinc sulfate were added to the medium to achieve a selenium concentration (calculated as selenium) of 40 mg / kg and a zinc concentration (calculated as zinc) of 100 mg / mL. Sodium selenate or zinc sulfate was added alone as a control, and no sodium selenate or zinc sulfate was added as a blank control. The prepared Cordyceps militaris seed solution was diluted 5-fold, and 5 mL of the diluted solution was added to each cultivation bottle. The inoculated cultivation bottles were transferred to a constant temperature cultivation room at 21°C with a relative humidity of 80% and cultured in the dark for 4 days. Then, the cultivation was performed under light conditions of 12 hours of white light and 12 hours of red light for 35 days until the fruiting bodies matured, spores were produced at the top, and the Cordyceps militaris fruiting bodies were harvested. The harvested Cordyceps militaris fruiting bodies were dried in an oven at 50°C, weighed, and ground for testing.

[0106] 4. Effect of sodium selenate and zinc sulfate on the biomass of Cordyceps militaris fruiting bodies

[0107] The results of the biomass (Figure Figure 9 , A and B) showed that the biomass of the fruiting bodies in the blank control group was 2.31 ± 0.15 g per bottle. When sodium selenate was added alone, the biomass of the Cordyceps militaris fruiting bodies increased by 6.3% to 2.45 ± 0.14 g. When zinc sulfate was added alone, the biomass of the Cordyceps militaris fruiting bodies significantly decreased by 12.9% to 2.01 ± 0.18 g. When sodium selenate and zinc sulfate were added together to the medium, the biomass of the Cordyceps militaris fruiting bodies was not significantly different from that of the blank control, reaching 2.31 ± 0.11 g. Sodium selenate significantly reduced the toxicity of zinc sulfate on the growth of Cordyceps militaris fruiting bodies.

[0108] 5. Effect of sodium selenate and zinc sulfate on the contents of adenosine, cordycepin, and cordycepic acid in Cordyceps militaris fruiting bodies

[0109] Adenosine and cordycepin contents in Cordyceps cicadae mycelia were determined by HPLC according to NY / T 2116-2012. The main steps included: 0.20 g of Cordyceps cicadae mycelia was accurately weighed into a 50 mL centrifuge tube, and about 5 mL of ultrapure water was added. After shaking, ultrasonic extraction was performed for 40 min. After centrifugation, 5 mL of ultrapure water was added to the precipitate, and the above operation was repeated. The two extraction solutions were filtered with a 0.45 μm MCM microporous filter, and the adenosine and cordycepin contents were determined by HPLC. The results are shown in Table 1. Figure 10 , Figure 33 , Figure 34 As shown in Table 1, the adenosine content of the blank control group was 1.32 ± 0.02 mg / g. The adenosine content of the treatment group with only sodium selenate added was significantly reduced by 12.4%, reaching 1.16 ± 0.05 mg / g. The adenosine content of the treatment group with only zinc sulfate added was increased by 8.9%, reaching 1.44 ± 0.10 mg / g. When sodium selenate and zinc sulfate were added together to the culture medium, the adenosine content of Cordyceps cicadae mycelia was significantly increased by 22.9%, reaching 1.62 ± 0.08 mg / g. The results of cordycepin content showed that the cordycepin content of the blank control group was 141.64 ± 4.39 mg / kg. The cordycepin content of the treatment group with only sodium selenate added was reduced by 20.8%, reaching 112.20 ± 15.35 mg / kg. The cordycepin content of the treatment group with only zinc sulfate added was reduced by 0.5%, reaching 140.89 ± 22.84 mg / kg. When sodium selenate and zinc sulfate were added together to the culture medium, the cordycepin content of Cordyceps cicadae mycelia was reduced by 4.4%, reaching 135.37 ± 8.91 mg / kg, which was not significantly different from the blank control group and the control group.

[0110] The content of cordycepic acid in Cordyceps cicadae mycelia was determined by UV spectrophotometry. 0.20 g of Cordyceps cicadae sample was accurately weighed into a 50 mL centrifuge tube, and 5 mL of 50% ethanol was added. Ultrasonic extraction was performed for 30 min, and the supernatant was taken. The precipitate was continuously added with 50% ethanol, and the above operation was repeated three times. The extraction supernatant was combined. 0.1 mL of the above sample was taken and diluted 50 times for determination. A standard solution was prepared with 1 mg / mL of mannitol as the standard sample. 1 mL of the sample was taken, 1 mL of potassium periodate solution was added, and it was mixed well. After 10 min of reaction at room temperature, 2 mL of 0.1% L-rhamnose solution was added to remove excess potassium periodate. After shaking and mixing, 4 mL of freshly prepared Nash reagent (150 g of ammonium acetate was dissolved, 2 mL of glacial acetic acid and 2 mL of acetylacetone were added, and the volume was made to 1000 mL) was added. It was colored at 53°C water bath for 15 min, and then quickly cooled to room temperature. The absorbance value was determined by spectrophotometry at a wavelength of 412 nm, with distilled water instead of the sample solution. The same method was used as a control. The content was calculated by the standard curve. The results of cordycepic acid content are shown in Table 2. Figure 11)The content of cordycepic acid in the control group was 22.67±1.76mg / g. The content of cordycepic acid in the treatment group with sodium selenate alone increased by 4.9% and reached 23.78±2.82mg / g. The content of cordycepic acid in the treatment group with zinc sulfate alone increased by 33.9% and reached 30.36±3.12mg / g. The content of cordycepic acid in the treatment group with sodium selenate and zinc sulfate together increased by 11.7% and reached 25.33±0.98mg / g.

[0111] 6、Sodium selenate and zinc sulfate affect trace elements in the fruiting body of Cordyceps cicadae

[0112] The content of elements in the fruiting body of Cordyceps cicadae was determined by inductively coupled plasma mass spectrometry (ICP-MS). The isotopes for detecting each element were 78Se, 63Cu, 66Zn, 56Fe, 55Mn, 52Cr, 75As, 112Cd and 208Pb. The specific steps were as follows: 0.10g of the sample to be tested was accurately weighed and added into a digestion tube, 4mL of high-purity nitric acid was added overnight, and then the mixture was placed in a microwave digestion instrument for sufficient digestion (180℃, 3 hours). After the acid was removed, the mixture was diluted to 25mL and stored for testing. The element mixed standard solution was diluted with 5% high-purity nitric acid. The above series of standard solutions and samples were determined by ICP-MS. The results of the content of each element were as follows: Figure 12 )Compared with the blank control group, the content of selenium in Cordyceps cicadae reached 59.45±3.6mg / kg when sodium selenate was added alone, which was significantly increased by 8989.3% compared with the control. Meanwhile, the contents of Cr, Fe, Cu, As and Pb in the fruiting body of Cordyceps cicadae were significantly reduced by 74.9%, 15.7%, 11.5%, 20.9% and 32.5%, respectively. Mn, Zn and Cd had no significant effect. When zinc sulfate was added alone, the content of zinc in Cordyceps cicadae reached 72.36±9.9mg / kg, which was significantly increased by 71.3%. The contents of Cr, Cu, As, Cd and Pb in the fruiting body of Cordyceps cicadae were significantly reduced by 27.9%, 17.0%, 40.5%, 24.2% and 29.7%, respectively. Mn, Fe and Se had no significant effect. When sodium selenate and zinc sulfate were added together, the contents of Zn and Se in the fruiting body of Cordyceps cicadae were significantly increased, reaching 58.39±3.54mg / kg and 51.53±4.13mg / kg, respectively, which were 1.4 times and 78.8 times of the blank control group. The contents of Cr, Fe, Cu, As, Cd and Pb in the fruiting body of Cordyceps cicadae were significantly reduced by 67.1%, 22.9%, 16.6%, 32.9%, 14.3% and 55.4%, respectively. The content of Mn in the fruiting body of Cordyceps cicadae had no significant change.

[0113] 7、Sodium selenate and zinc sulfate affect the selenium speciation in the fruiting body of Cordyceps cicadae

[0114] Determination method of selenium form: HPLC-HG-AFS was used to detect selenium form in Cordyceps militaris fruiting body. 0.08 g of Cordyceps militaris fruiting body powder was added with 4 mL of 8 mg / mL protease X IV, and after ultrasonic treatment at 30°C for 60 min, it was cultured at 37°C on a 150 r / min shaker for 8 hours, and then centrifuged at 8000 rpm to collect the supernatant, which was filtered through a 0.22 μm filter for testing. Hamilton PRP-X100 was used as the separation column, the mobile phase was (NH4)2HPO4, the carrier was 10% HCl, the oxidizing agent was 0.35% KOH and 0.2% KI, the reducing agent was 0.35% KOH and 2% KBH4, and HG-AFS was used as the detector to analyze the selenium form in Cordyceps militaris fruiting body. Selenium cysteine (SeCys2), selenomethyl selenocysteine (MeSeCys), selenite (Se(IV)), selenomethionine (SeMet), and selenate (Se(VI)) were used as standard samples, and the selenium form in Cordyceps militaris fruiting body was analyzed according to the retention time, and the content of each selenium form was calculated according to the peak area. Figure 35 、 Figure 36 ) shows that the selenium form analysis of Cordyceps militaris fruiting body in the blank control group and the treatment group with zinc sulfate alone is below the detection limit, and SeCys2, Se(IV) and SeMet are detected in the treatment group with sodium selenate alone and the treatment group with sodium selenate and zinc sulfate, among which the proportions of SeCys2, Se(IV) and SeMet in the treatment with sodium selenate alone are 67.7%, 9.8% and 22.5% respectively, and the proportions of SeCys2, Se(IV) and SeMet in the treatment with sodium selenate and zinc sulfate are 69.70%, 8.78% and 21.52% respectively.

[0115] Example 6 Method for cultivating selenium and zinc enriched Cordyceps militaris using sodium selenate and zinc disodium ethylenediaminetetraacetate

[0116] 1. Activation of Cordyceps militaris spores

[0117] Cordyceps militaris M8 was inoculated into modified PDA medium and cultured in a constant temperature incubator at 21°C for 20 days, and then stored for use after a large number of spores were produced.

[0118] 2. Preparation of Cordyceps militaris liquid seed solution

[0119] A 5 mm diameter activated Cordyceps militaris cake was inoculated into a 500 mL conical flask containing 200 mL of modified PD medium for shake culture, and the shake culture conditions were 21°C and a rotation speed of 180 r / min, and the shake culture was carried out for 3 days to prepare the seed solution.

[0120] 3. Cultivation of selenium and zinc enriched Cordyceps militaris

[0121] Composition of wheat medium: the ratio of wheat (dry weight) to nutrient solution was 20g:25mL. The mixture of wheat and nutrient solution was sterilized at 121℃ for later use. In the wheat medium, filtered and sterilized sodium selenite and zinc disodium ethylenediaminetetraacetate were added to make the selenium concentration (calculated as selenium) 40mg / kg and the zinc concentration (calculated as zinc) 100mg / mL. Sodium selenite or zinc disodium ethylenediaminetetraacetate was added alone as a control, and no sodium selenite or zinc disodium ethylenediaminetetraacetate was added as a blank control. The prepared Cordyceps cicadae seed liquid was diluted 5 times, and 5mL of the diluted bacteria liquid was added to each cultivation bottle. The inoculated cultivation bottles were transferred to a constant temperature cultivation room at 21℃, with a relative humidity of 80% and light-free cultivation for 4 days, followed by 12 hours of white light and 12 hours of red light illumination for 35 days. The fruiting bodies were matured, and the top of the fruiting bodies was harvested. The harvested Cordyceps cicadae fruiting bodies were dried in an oven at 50℃, weighed, and ground for testing.

[0122] 4. Effects of sodium selenite and zinc disodium ethylenediaminetetraacetate on the biomass of Cordyceps cicadae fruiting bodies

[0123] Biomass results Figure 13 The results (A and B) show that the biomass of the fruiting bodies in the blank control group was 2.31±0.15g per bottle. The biomass of the fruiting bodies increased by 6.3% when sodium selenite was added alone, reaching 2.45±0.14g. When zinc disodium ethylenediaminetetraacetate was added alone, the biomass of the fruiting bodies decreased significantly by 17.4%, reaching only 1.90±0.30g. When sodium selenite and zinc disodium ethylenediaminetetraacetate were added together in the medium, the biomass of the fruiting bodies was not significantly different from the blank control, reaching 2.26±0.11g. Sodium selenite significantly reduced the growth toxicity of zinc disodium ethylenediaminetetraacetate on Cordyceps cicadae fruiting bodies.

[0124] 5. Effects of sodium selenite and zinc disodium ethylenediaminetetraacetate on the contents of adenosine, cordycepin, and cordycepic acid in Cordyceps cicadae fruiting bodies

[0125] The contents of adenosine and cordycepin in Cordyceps cicadae fruiting bodies were detected by high-performance liquid chromatography according to the method of NY / T 2116-2012. The main steps included: accurately weighing 0.20g of Cordyceps cicadae fruiting body powder into a 50mL centrifuge tube, adding about 5mL of ultrapure water, shaking, and ultrasonic extraction for 40min. After centrifugation, 5mL of ultrapure water was added to the precipitate, and the above operation was repeated. The two extraction solutions were filtered with a 0.45μm MCM microporous filter membrane, and the contents of adenosine and cordycepin were determined by high-performance liquid chromatography. The results are shown in Figure 14 , Figure 33 , Figure 34As shown: the adenosine content of the fruiting bodies in the blank control group was 1.32±0.02 mg / g. The treatment with sodium selenate alone significantly reduced the adenosine content by 12.4% to 1.16±0.05 mg / g; the treatment with zinc disodium EDTA alone significantly increased the adenosine content of the fruiting bodies by 11.4% to 1.47±0.03 mg / g; when sodium selenate and zinc disodium EDTA were added together to the culture medium, the adenosine content of the fruiting bodies of Cordyceps sinensis significantly increased by 21.7% to 1.60±0.01 mg / g. The results of cordycepin content analysis showed that the cordycepin content in the fruiting bodies of the blank control group was 141.64±4.39 mg / kg. Treatment with sodium selenate alone reduced the cordycepin content in the fruiting bodies by 20.8% to 112.20±15.35 mg / kg, while treatment with zinc disodium EDTA alone increased the cordycepin content by 0.7% to 142.68±43.90 mg / kg. When sodium selenate and zinc disodium EDTA were added together to the culture medium, the cordycepin content in the fruiting bodies of *Cicada nycteris* was 114.51±16.66 mg / kg, which showed no significant difference from either the blank control group or the control group.

[0126] The content of cordycepic acid in the fruiting bodies of *Cicada nymph* was determined by ultraviolet spectrophotometry. The specific method is described in Example 4. The results of the cordycepic acid content (…) Figure 15 The results showed that the cordycepic acid content in the control group was 22.67±1.76 mg / g. The treatment with sodium selenate alone increased the cordycepic acid content in the fruiting bodies by 4.9%, reaching 23.78±2.82 mg / g. The treatment with zinc disodium EDTA alone decreased the cordycepic acid content in the fruiting bodies by 1.6%, to 22.32±4.11 mg / g. When sodium selenate and zinc disodium EDTA were added together to the culture medium, the cordycepic acid content in the fruiting bodies of Cicadae periostracum increased by 8.6%, to 24.62±2.95 mg / g.

[0127] 6. Effects of sodium selenate and zinc disodium ethylenediaminetetraacetate on trace elements in the fruiting bodies of Cordyceps militaris

[0128] The elemental content in the fruiting bodies of *Cicada nymph* was determined by inductively coupled plasma mass spectrometry (ICP-MS). Detailed method is described in Example 4. Results of elemental content (…) Figure 16)showed that compared with the blank control group, the selenium content of Cordyceps cicadae reached 59.45±3.6 mg / kg by adding sodium selenite alone, and the Se content was significantly increased by 8989.3% compared with the control, while the contents of Cr, Fe, Cu, Zn, As and Pb in Cordyceps cicadae fruiting bodies were significantly reduced, by 74.9%, 15.7%, 11.5%, 12.8%, 20.9% and 32.5% respectively; Mn and Cd had no significant effect; by adding zinc disodium ethylenediaminetetraacetate alone, the zinc content of Cordyceps cicadae reached 67.44±3.77 mg / kg, and the Zn content was significantly increased by 59.7%, and the contents of Cr, Cu, As and Pb in Cordyceps cicadae fruiting bodies were significantly reduced, by 41.9%, 17.0%, 46.8% and 37.7% respectively, and Mn, Fe, Se and Cd had no significant effect; after adding sodium selenite and zinc disodium ethylenediaminetetraacetate together, the contents of Se, Zn and Cd in Cordyceps cicadae fruiting bodies were significantly increased, and the contents of Zn and Se reached 60.32±3.87 mg / kg and 54.31±1.28 mg / kg respectively, which were 1.4 times and 83.0 times of the blank control group; the contents of Cr, As and Pb in Cordyceps cicadae fruiting bodies were significantly reduced, by 55.9%, 18.2% and 50.3% respectively; the contents of Mn, Fe and Cu in Cordyceps cicadae fruiting bodies had no significant change.

[0129] 7、Selenium and zinc disodium ethylenediaminetetraacetate affect selenium speciation in Cordyceps cicadae fruiting bodies

[0130] The selenium speciation in Cordyceps cicadae fruiting bodies was detected by high performance liquid chromatography coupled with hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS), and the specific method is shown in Example 4. The selenium speciation determination results ( Figure 35 、 Figure 36 )showed that the selenium speciation analysis of Cordyceps cicadae fruiting bodies in the blank control group and the treatment group with zinc disodium ethylenediaminetetraacetate alone was below the detection limit, and SeCys2, Se(Ⅳ) and SeMet were detected in the treatment group with sodium selenite alone and the treatment group with sodium selenite and zinc sulfate, wherein the proportions of SeCys2, Se(Ⅳ) and SeMet in the treatment group with sodium selenite alone were 67.7%, 9.8% and 22.5% respectively; and the proportions of SeCys2, Se(Ⅳ) and SeMet in the treatment group with sodium selenite and zinc disodium ethylenediaminetetraacetate were 69.1%, 10.6% and 20.4% respectively.

[0131] Example 7 Method for cultivating selenium-rich and zinc-rich Cordyceps cicadae by using sodium selenite and zinc sulfate

[0132] 1、Cordyceps cicadae strain activation

[0133] Cordyceps cicadae M8 was inoculated into modified PDA medium and cultured in a 21℃ constant temperature incubator for 20 days, and then stored for use after a large amount of spores were produced.

[0134] 2. Preparation of Cordyceps militaris liquid seed

[0135] The activated Cordyceps militaris cake with a diameter of 5 mm was inoculated into a 500 mL conical flask containing 200 mL of modified PD medium for shake culture. The shake culture conditions were 21°C, a rotation speed of 180 r / min, and a culture time of 3 days.

[0136] 3. Cultivation of selenium and zinc-enriched Cordyceps militaris

[0137] The composition of the wheat medium was as follows: the ratio of wheat (dry weight) to nutrient solution was 20 g:25 mL. The wheat and the nutrient solution were mixed and sterilized at 121°C under high temperature and high pressure for use. Filtered and sterilized sodium selenite and zinc sulfate were added to the medium, so that the selenium concentration (calculated as selenium) in the medium was 40 mg / kg, and the zinc concentration (calculated as zinc) was 100 mg / mL. Sodium selenite or zinc sulfate was added alone as a control, and no sodium selenite and zinc sulfate was added as a blank control. The prepared Cordyceps militaris seed liquid was diluted 5 times, and 5 mL of the diluted bacterial liquid was added to each cultivation bottle. The inoculated cultivation bottles were transferred to a constant-temperature cultivation room at 21°C, with a relative humidity of 80% and light-free cultivation for 4 days, followed by light cultivation for 35 days under 12 hours of white light and 12 hours of red light. The fruiting bodies were matured, spores were produced at the top, and the Cordyceps militaris fruiting bodies were harvested. The harvested Cordyceps militaris fruiting bodies were dried in an oven at 50°C, weighed, and ground for testing.

[0138] 4. Effects of sodium selenite and zinc sulfate on the biomass of Cordyceps militaris fruiting bodies

[0139] The results of the biomass Figure 17 , A and B) showed that the biomass of the fruiting bodies in the blank control group was 2.31±0.15 g per bottle. When sodium selenite was added alone, the biomass of the Cordyceps militaris fruiting bodies decreased by 2.7%, reaching 2.24±0.22 g per bottle. When zinc sulfate was added alone, the biomass of the Cordyceps militaris fruiting bodies decreased significantly by 12.9%, reaching only 2.01±0.18 g per bottle. When sodium selenite and zinc sulfate were added together in the medium, the biomass of the Cordyceps militaris fruiting bodies decreased significantly by 15.0%, reaching only 1.96±0.18 g per bottle.

[0140] 5. Effects of sodium selenite and zinc sulfate on the contents of adenosine, cordycepin, and cordycepic acid in Cordyceps militaris fruiting bodies

[0141] The contents of adenosine and cordycepin in the Cordyceps militaris fruiting bodies were detected by high-performance liquid chromatography according to the method of NY / T 2116-2012. The detailed method is shown in Example 4. The results are shown in Figure 18 、 Figure 33 、 Figure 34As shown: the adenosine content of the fruiting bodies in the blank control group was 1.32±0.02 mg / g. The adenosine content decreased by 3.7% to 1.27±0.03 mg / g when sodium selenite was added alone; the adenosine content increased by 8.9% to 1.44±0.10 mg / g when zinc sulfate was added alone; and the adenosine content in the fruiting bodies of Cordyceps sinensis increased significantly by 37.9% to 1.82±0.03 mg / g when sodium selenite and zinc sulfate were added together to the culture medium. The results of cordycepin content analysis showed that the cordycepin content in the fruiting bodies of the blank control group was 141.64±4.39 mg / kg. Treatment with sodium selenite alone reduced the cordycepin content in the fruiting bodies by 5.0%, to 134.52±11.95 mg / kg. Treatment with zinc sulfate alone reduced the cordycepin content in the fruiting bodies by 0.5%, to 140.89±22.84 mg / kg. When sodium selenite and zinc sulfate were added together to the culture medium, the cordycepin content in the fruiting bodies of *Cicada nymph* decreased by 15.7%, to 119.40±4.97 mg / kg, which was not significantly different from the blank control group and the control group.

[0142] The content of cordycepic acid in the fruiting bodies of *Cicada nymph* was determined by ultraviolet spectrophotometry. Detailed method is described in Example 4. Results of cordycepic acid content ( Figure 19 The results showed that the cordycepic acid content in the control group was 22.67±1.76 mg / g. The treatment with sodium selenite alone increased the cordycepic acid content in the fruiting bodies by 18.7%, reaching 26.92±2.75 mg / g. The treatment with zinc sulfate alone significantly increased the cordycepic acid content in the fruiting bodies by 33.9%, reaching 30.36±3.12 mg / g. When sodium selenite and zinc sulfate were added together to the culture medium, the cordycepic acid content in the fruiting bodies of Cicadae periostracum significantly increased by 34.1%, reaching 30.40±1.04 mg / g.

[0143] 6. Effects of sodium selenite and zinc sulfate on trace elements in the fruiting bodies of Cordyceps militaris

[0144] The elemental content in the fruiting bodies of *Cicada nymph* was determined by inductively coupled plasma mass spectrometry (ICP-MS). Detailed method is described in Example 4. Results of elemental content (…) Figure 20)showed that compared with the blank control group, the selenium content in the Cordyceps reached 95.14±3.79 mg / kg when sodium selenite was added alone, and the Se content was significantly increased by 14445.5% compared with the control, while the contents of Cr, As, and Pb in Cordyceps fruiting bodies were significantly reduced by 54.8%, 20.0%, and 31.2%, respectively; Cd was significantly increased by 20.7%, and Mn, Fe, Cu, and Zn had no significant effect; when zinc sulfate was added alone, the zinc content in Cordyceps reached 72.36±9.9 mg / kg, and Zn was significantly increased by 71.3%, and the contents of Cr, Cu, As, Cd, and Pb in Cordyceps fruiting bodies were significantly reduced by 27.9%, 17.0%, 40.5%, 24.2%, and 29.7%, respectively, and Mn, Fe, and Se had no significant effect; after adding sodium selenite and zinc sulfate together, the contents of Zn and Se reached 58.95±3.62 mg / kg and 60.97±5.80 mg / kg, respectively, and the contents of Zn and Se in Cordyceps fruiting bodies were significantly increased by 1.4 times and 93.2 times, respectively, compared with the blank control group; the contents of Cr, Fe, Cu, As, and Pb in Cordyceps fruiting bodies were significantly reduced by 55.4%, 17.7%, 20.0%, 34.7%, and 47.3%, respectively; and the contents of Mn and Cd in Cordyceps fruiting bodies had no significant change.

[0145] 7、Sodium selenite and zinc sulfate affect selenium speciation in Cordyceps fruiting bodies

[0146] Selenium speciation in Cordyceps fruiting bodies was detected by high-performance liquid chromatography coupled with hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS), and the detailed method is described in Example 4. The results of selenium speciation determination ( Figure 35 、 Figure 36 )showed that the selenium speciation analysis of Cordyceps fruiting bodies in the blank control group and the treatment group with zinc sulfate alone was below the detection limit, and SeCys2, Se(IV), and SeMet were detected in the treatment group with sodium selenite alone and the treatment group with sodium selenite and zinc sulfate added, among which the proportions of SeCys2, Se(IV), and SeMet in the treatment group with sodium selenite added alone were 70.2%, 9.4%, and 20.4%, respectively; and the proportions of SeCys2, Se(IV), and SeMet in the treatment group with sodium selenite and zinc sulfate added were 69.3%, 9.0%, and 21.8%, respectively.

[0147] Example 8 Method for cultivating selenium and zinc-rich Cordyceps using sodium selenite and zinc disodium ethylenediaminetetraacetate

[0148] 1、Cordyceps strain activation

[0149] Cordyceps M8 was inoculated into modified PDA medium and cultured in a 21°C constant temperature incubator for 20 days until a large amount of spores were produced for standby use.

[0150] 2. Preparation of Cicada Flower Liquid Seed Solution

[0151] Take a 5mm diameter activated cicada flower cake and inoculate it into a 500mL Erlenmeyer flask containing 200mL of modified PD medium for shaking culture. The shaking conditions are 21℃ and 180r / min. Shake culture for 3 days to prepare the seed culture.

[0152] 3. Cultivation of selenium- and zinc-enriched Cordyceps militaris

[0153] The wheat culture medium consisted of wheat (dry weight) at a ratio of 20g to 25mL of nutrient solution. After mixing, the wheat and nutrient solution were sterilized at 121℃ under high pressure. Filter-sterilized sodium selenite and zinc disodium EDTA were added to the wheat culture medium to achieve a selenium concentration (calculated as selenium) of 40mg / kg and a zinc concentration (calculated as zinc) of 100mg / mL. Sodium selenite or zinc disodium EDTA alone served as controls, while treatment without sodium selenite and zinc disodium EDTA served as a blank control. The prepared *Cicada nycteris* seed solution was diluted 5 times, and 5mL of the diluted bacterial solution was added to each cultivation bottle. After inoculation, the culture bottles were transferred to a constant temperature culture room at 21°C with a relative humidity of 80%. After 4 days of light-protected culture, they were cultured under 12 hours of white light and 12 hours of red light for 35 days. When the fruiting bodies matured and spores were produced at the top, they could be harvested. The harvested cicada flower fruiting bodies were dried in an oven at 50°C, weighed, ground, and prepared for testing.

[0154] 4. Effects of sodium selenite and zinc disodium EDTA on the biomass of Cordyceps sinensis fruiting bodies

[0155] Biomass results ( Figure 21 Studies A and B show that the biomass of the fruiting bodies in the blank control group was 2.31 ± 0.15 g per bottle. When sodium selenite was added alone, the biomass of the fruiting bodies of *Cicada spp.* decreased by 2.7%, reaching 2.24 ± 0.22 g per bottle. When zinc disodium EDTA was added alone, the biomass of the fruiting bodies of *Cicada spp.* decreased significantly by 17.4%, to only 1.90 ± 0.30 g. When sodium selenite and zinc disodium EDTA were added together to the culture medium, the biomass of the fruiting bodies of *Cicada spp.* decreased by 10.7%, to only 2.17 ± 0.30 g. Sodium selenite reduced the toxicity of zinc disodium EDTA to the growth of *Cicada spp.* fruiting bodies.

[0156] 5. Effects of sodium selenite and zinc disodium EDTA on the contents of adenosine, cordycepin, and cordycepic acid in Cordyceps sinensis fruiting bodies.

[0157] The contents of adenosine and cordycepin in the fruiting bodies of Cordyceps militaris were determined by high performance liquid chromatography using the method specified in NY / T 2116-2012. Detailed method is described in Example 4. Results are as follows: Figure 22 , Figure 33 , Figure 34The adenosine content of the fruiting body of the control group was 1.32 ± 0.02 mg / g. The adenosine content of the fruiting body of the treatment with sodium selenite alone was reduced by 3.7% to 1.27 ± 0.03 mg / g. The adenosine content of the fruiting body of the treatment with zinc disodium ethylenediaminetetraacetate alone was increased by 11.4% to 1.47 ± 0.03 mg / g. The adenosine content of the fruiting body of Cordyceps cicadae treated with sodium selenite and zinc disodium ethylenediaminetetraacetate together was significantly increased by 30.2% to 1.72 ± 0.33 mg / g.

[0158] The content of cordycepic acid in the fruiting body of Cordyceps cicadae was determined by ultraviolet spectrophotometry. The detailed method is shown in Example 4. The results of the content of cordycepic acid ( Figure 23 ) show that the content of cordycepic acid in the control group was 22.67 ± 1.76 mg / g. The content of cordycepic acid in the fruiting body treated with sodium selenite alone was increased by 18.7% to 26.92 ± 2.75 mg / g. The content of cordycepic acid in the fruiting body treated with zinc disodium ethylenediaminetetraacetate alone was reduced by 1.6% to 22.32 ± 4.11 mg / g. The content of cordycepic acid in the fruiting body of Cordyceps cicadae treated with sodium selenite and zinc disodium ethylenediaminetetraacetate together was reduced by 1.1% to 22.41 ± 3.12 mg / g.

[0159] 6. Effect of sodium selenite and zinc disodium ethylenediaminetetraacetate on trace elements in the fruiting body of Cordyceps cicadae

[0160] The content of elements in the fruiting body of Cordyceps cicadae was determined by inductively coupled plasma mass spectrometry (ICP-MS). The detailed method is shown in Example 4. The results of the content of elements ( Figure 24)showed that compared with the blank control group, the selenium content in the Cordyceps reached 95.14 ± 3.79 mg / kg when sodium selenite was added alone, and the Se content was significantly increased by 14445.5% compared with the control, while the Cr, As and Pb in Cordyceps fruiting bodies were significantly reduced by 54.8%, 20.0% and 31.2% respectively; Se and Cd were significantly increased by 14445.5% and 20.7%, and Mn, Fe, Cu and Zn had no significant effect; when zinc disodium ethylenediaminetetraacetate was added alone, the zinc content in Cordyceps reached 67.44 ± 3.77 mg / kg, and Zn was significantly increased by 59.7%, and Cr, Cu, As and Pb in Cordyceps fruiting bodies were significantly reduced by 41.9%, 17.0%, 46.8% and 37.7% respectively, and Mn, Fe, Se and Cd had no significant effect; after adding sodium selenite and zinc disodium ethylenediaminetetraacetate together, the contents of Zn and Se reached 62.51 ± 3.64 mg / kg and 52.67 ± 3.95 mg / kg respectively, and the contents of Zn, Se and Cd in Cordyceps fruiting bodies were significantly increased, which were 1.5 times, 80.5 times and 1.2 times of the blank control group respectively; the contents of Cr, Mn, Fe, Cu, As and Pb in Cordyceps fruiting bodies were significantly reduced by 57.9%, 10.2%, 10.8%, 16.5%, 34.6% and 55.0% respectively.

[0161] 7、Sodium selenite and zinc disodium ethylenediaminetetraacetate affect selenium speciation in Cordyceps fruiting bodies

[0162] Selenium speciation in Cordyceps fruiting bodies was detected by high performance liquid chromatography coupled with hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS), and the detailed method is shown in Example 4. The results of selenium speciation determination ( Figure 35 、 Figure 36 )showed that the selenium speciation analysis of Cordyceps fruiting bodies in the blank control group and the treatment group with zinc disodium ethylenediaminetetraacetate alone was below the detection limit, and SeCys2, Se(Ⅳ) and SeMet were detected in the treatment group with sodium selenite alone and the treatment group with sodium selenite and zinc disodium ethylenediaminetetraacetate, among which the proportions of SeCys2, Se(Ⅳ) and SeMet in the treatment group with sodium selenite alone were 70.2%, 9.4% and 20.4% respectively; and the proportions of SeCys2, Se(Ⅳ) and SeMet in the treatment group with sodium selenite and zinc disodium ethylenediaminetetraacetate were 67.3%, 9.7% and 23.0% respectively.

[0163] Example 9 Method for cultivating selenium-rich and zinc-rich Cordyceps by using nano-selenium and zinc sulfate

[0164] 1、Activation of Cordyceps fungus

[0165] The M8 of Cordyceps cicadae was inoculated into modified PDA medium and cultured in a constant temperature incubator at 21°C for 20 days until a large number of spores were produced.

[0166] 2. Preparation of liquid seed solution of Cordyceps cicadae

[0167] The activated Cordyceps cicadae cake with a diameter of 5 mm was inoculated into a 500 mL conical flask containing 200 mL of modified PD medium for shake culture, and the shake culture conditions were 21°C, 180 r / min, and 3 days.

[0168] 3. Cultivation of selenium and zinc-rich Cordyceps cicadae

[0169] The composition of the wheat medium was as follows: the ratio of wheat (dry weight) to nutrient solution was 20 g:25 mL. After mixing the wheat and the nutrient solution, high-temperature and high-pressure sterilization was performed at 121°C for standby use. Nano-selenium and zinc sulfate filtered and sterilized were added to the medium, so that the concentration of selenium (calculated as selenium) in the medium was 40 mg / kg, and the concentration of zinc (calculated as zinc) was 100 mg / mL. Nano-selenium or zinc sulfate was added alone as a control, and no nano-selenium and zinc sulfate were added as a blank control. The prepared seed solution of Cordyceps cicadae was diluted 5 times, and 5 mL of the diluted bacterial solution was added to each cultivation bottle. The inoculated cultivation bottles were transferred to a constant temperature cultivation room at 21°C, with a relative humidity of 80% and light-free cultivation for 4 days, followed by 12 hours of white light and 12 hours of red light illumination for 35 days. The fruiting bodies were matured, and the top spores were harvested. The harvested Cordyceps cicadae fruiting bodies were dried in an oven at 50°C, weighed, and ground for testing.

[0170] 4. Effect of nano-selenium and zinc sulfate on the biomass of Cordyceps cicadae fruiting bodies

[0171] The results of biomass Figure 25 , A and B) showed that the biomass of the fruiting bodies in the blank control group was 2.31±0.15 g per bottle, the biomass of the fruiting bodies of Cordyceps cicadae decreased by 0.3% when nano-selenium was added alone, reaching 2.30±0.10 g per bottle, the biomass of the fruiting bodies of Cordyceps cicadae decreased significantly by 13.0% when zinc sulfate was added alone, reaching only 2.01±0.18 g per bottle, and the biomass of the fruiting bodies of Cordyceps cicadae decreased significantly by 20.0% when nano-selenium and zinc sulfate were added together, reaching only 1.84±0.30 g per bottle.

[0172] 5. Effect of nano-selenium and zinc sulfate on the contents of adenosine, cordycepin, and cordycepic acid in Cordyceps cicadae fruiting bodies

[0173] The contents of adenosine and cordycepin in Cordyceps cicadae fruiting bodies were detected by high-performance liquid chromatography according to the method of NY / T 2116-2012, and the detailed method is shown in Example 4. The results are shown in Figure 26 、 Figure 33 、 Figure 34As shown: the adenosine content of the fruiting bodies in the blank control group was 1.32±0.02 mg / g. The adenosine content decreased by 5.2% to 1.25±0.08 mg / g when nano-selenium was added alone; the adenosine content increased by 8.9% to 1.44±0.10 mg / g when zinc sulfate was added alone; and the adenosine content in the fruiting bodies of Cordyceps sinensis increased significantly by 21.6% to 1.60±0.14 mg / g when nano-selenium and zinc sulfate were added together to the culture medium. The results of cordycepin content analysis showed that the cordycepin content in the fruiting bodies of the blank control group was 141.64±4.39 mg / kg. Treatment with nano-selenium alone reduced the cordycepin content in the fruiting bodies by 23.5% to 108.30±23.33 mg / kg. Treatment with zinc sulfate alone reduced the cordycepin content in the fruiting bodies by 0.5% to 140.89±22.84 mg / kg. When sodium selenate and zinc sulfate were added together to the culture medium, the cordycepin content in the fruiting bodies of *Cicada nymph* increased by 1.6% to 119.40±4.97 mg / kg, which was not significantly different from the blank control group and the control group.

[0174] The content of cordycepic acid in the fruiting bodies of *Cicada nymph* was determined by ultraviolet spectrophotometry. Detailed method is described in Example 4. Results of cordycepic acid content ( Figure 27 The results showed that the cordycepic acid content in the control group was 22.67±1.76 mg / g. The treatment with nano-selenium alone increased the cordycepic acid content in the fruiting bodies by 15.0%, reaching 26.07±5.57 mg / g. The treatment with zinc sulfate alone increased the cordycepic acid content in the fruiting bodies by 33.9%, reaching 30.36±3.12 mg / g. When sodium selenate and zinc sulfate were added together to the culture medium, the cordycepic acid content in the fruiting bodies of Cicadae periostracum increased by 21.8%, reaching 27.62±1.08 mg / g.

[0175] 6. Effects of nano-selenium and zinc sulfate on trace elements in the fruiting bodies of Cordyceps militaris

[0176] The elemental content in the fruiting bodies of *Cicada nymph* was determined by inductively coupled plasma mass spectrometry (ICP-MS). Detailed method is described in Example 4. Results of elemental content (…) Figure 28)showed that compared with the blank control group, the selenium content in the treatment group with nano-selenium alone reached 29.27 ± 3.63 mg / kg, which was significantly increased by 4374.4% compared with the control group, and the contents of Cr, Cu, As, Cd and Pb in the fruiting body of Cordyceps were significantly reduced by 30.2%, 10.8%, 33.0%, 20.0% and 30.0%, respectively; Mn, Fe and Zn had no significant effect; the addition of zinc sulfate alone made the zinc content in Cordyceps reach 72.36 ± 9.9 mg / kg, and the zinc content was significantly increased by 71.3%, and the contents of Cr, Cu, As, Cd and Pb in the fruiting body of Cordyceps were significantly reduced by 27.9%, 17.0%, 40.5%, 24.2% and 29.7%, respectively, and Mn, Fe and Se had no significant effect; after adding sodium selenate and zinc sulfate together, the contents of Zn and Se reached 67.86 ± 4.04 mg / kg and 26.49 ± 2.65 mg / kg, respectively, and the contents of Zn and Se in the fruiting body of Cordyceps were significantly increased by 1.6 times and 40.5 times compared with the blank control group; the contents of Fe, Cu, As, Cd and Pb in the fruiting body of Cordyceps were significantly reduced by 20.4%, 15.4%, 33.6%, 19.7% and 50.4%, respectively; the contents of Cr and Mn in the fruiting body of Cordyceps had no significant change.

[0177] 7、Nano-selenium and zinc sulfate on the influence of selenium speciation in Cordyceps fruiting body

[0178] The selenium speciation in Cordyceps fruiting body was detected by high performance liquid chromatography coupled with hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS), and the detailed method was shown in Example 4. The selenium speciation determination results ( Figure 35 、 Figure 36 )showed that the selenium speciation analysis in the blank control group and the treatment group with zinc sulfate alone was lower than the detection limit, and SeCys2, Se(Ⅳ) and SeMet were detected in the treatment group with nano-selenium alone and the treatment group with nano-selenium and zinc sulfate, wherein the proportions of SeCys2, Se(Ⅳ) and SeMet in the treatment group with nano-selenium alone were 68.4%, 9.6% and 22.0%, respectively; and the proportions of SeCys2, Se(Ⅳ) and SeMet in the treatment group with nano-selenium and zinc sulfate were 64.5%, 7.7% and 22.5%, respectively.

[0179] Example 10 Method for cultivating selenium and zinc-rich Cordyceps by using nano-selenium and zinc disodium ethylenediaminetetraacetate

[0180] 1、Cordyceps strain activation

[0181] Cordyceps M8 was inoculated into modified PDA medium and cultured in a constant temperature incubator at 21°C for 20 days, and then a large amount of spores were produced for standby use.

[0182] 2. Preparation of Cordyceps liquid seed

[0183] The activated Cordyceps cake with a diameter of 5 mm was inoculated into a 500 mL conical flask containing 200 mL of modified PD medium for shake culture. The shake culture conditions were 21 °C, 180 r / min, and 3 days for seed liquid.

[0184] 3. Cultivation of selenium and zinc-rich Cordyceps

[0185] The composition of the wheat medium was 20 g of wheat (dry weight) to 25 mL of nutrient solution. The wheat and nutrient solution were mixed and sterilized at 121 °C under high temperature and high pressure for use. Nano-selenium and ethylenediaminetetraacetic acid disodium zinc were added to the medium, with a selenium concentration (calculated as selenium) of 40 mg / kg and a zinc concentration (calculated as zinc) of 100 mg / mL. Nano-selenium or ethylenediaminetetraacetic acid disodium zinc was added alone as a control, and no nano-selenium or ethylenediaminetetraacetic acid disodium zinc was added as a blank control. The prepared Cordyceps seed liquid was diluted 5 times, and 5 mL of diluted bacteria liquid was added to each cultivation bottle. The inoculated cultivation bottles were transferred to a constant temperature cultivation room at 21 °C, with a relative humidity of 80% and light-free cultivation for 4 days, followed by 12 hours of white light and 12 hours of red light illumination for 35 days. The fruiting bodies were matured, and the top spores were harvested. The harvested Cordyceps fruiting bodies were dried in an oven at 50 °C, weighed, and ground for testing.

[0186] 4. Effect of nano-selenium and ethylenediaminetetraacetic acid disodium zinc on the biomass of Cordyceps fruiting bodies

[0187] The results of biomass Figure 29 , A and B) showed that the biomass of the fruiting bodies in the blank control group was 2.31 ± 0.15 g per bottle. When nano-selenium was added alone, the biomass of the Cordyceps fruiting bodies decreased by 0.3%, reaching 2.30 ± 0.10 g per bottle. When ethylenediaminetetraacetic acid disodium zinc was added alone, the biomass of the Cordyceps fruiting bodies decreased significantly by 20.0%, reaching only 1.90 ± 0.30 g. When nano-selenium and ethylenediaminetetraacetic acid disodium zinc were added together in the medium, the biomass of the Cordyceps fruiting bodies decreased significantly by 20.5%, reaching only 1.83 ± 0.12 g per bottle.

[0188] 5. Effect of nano-selenium and ethylenediaminetetraacetic acid disodium zinc on the contents of adenosine, cordycepin, and cordycepic acid in Cordyceps fruiting bodies

[0189] The contents of adenosine and cordycepin in Cordyceps fruiting bodies were detected by high-performance liquid chromatography according to the method of NY / T 2116-2012, and the detailed method is shown in Example 4. The results are shown in Figure 30 , Figure 33 , Figure 34The adenosine content of the fruiting body of the control group was 1.32 ± 0.02 mg / g. The adenosine content of the fruiting body treated with nano-selenium alone was reduced by 5.2% to 1.25 ± 0.08 mg / g. The adenosine content of the fruiting body treated with zinc disodium ethylenediaminetetraacetate alone was significantly increased by 11.4% to 1.47 ± 0.03 mg / g. When nano-selenium and zinc disodium ethylenediaminetetraacetate were added to the culture medium together, the adenosine content of the fruiting body of Cordyceps cicadae was significantly increased by 13.4% to 1.50 ± 0.04 mg / g. The results of the cordycepin content showed that the cordycepin content of the fruiting body of the control group was 141.64 ± 4.39 mg / kg. The cordycepin content of the fruiting body treated with nano-selenium alone was reduced by 23.5% to 108.3 ± 23.33 mg / kg. The cordycepin content of the fruiting body treated with zinc disodium ethylenediaminetetraacetate alone was increased by 0.7% to 142.68 ± 43.90 mg / kg. When nano-selenium and zinc disodium ethylenediaminetetraacetate were added to the culture medium together, the cordycepin content of the fruiting body of Cordyceps cicadae was reduced by 25.0% to 106.20 ± 12.41 mg / kg, which was not significantly different from the control group.

[0190] The content of cordycepic acid in the fruiting body of Cordyceps cicadae was determined by ultraviolet spectrophotometry, and the detailed method is shown in Example 4. The results of the cordycepic acid content ( Figure 31 )showed that the cordycepic acid content of the control group was 22.67 ± 1.76 mg / g. The cordycepic acid content of the fruiting body treated with nano-selenium alone was increased by 15.0% to 26.07 ± 5.57 mg / g. The cordycepic acid content of the fruiting body treated with zinc disodium ethylenediaminetetraacetate alone was reduced by 1.6% to 22.32 ± 4.11 mg / g. When nano-selenium and zinc disodium ethylenediaminetetraacetate were added to the culture medium together, the cordycepic acid content of the fruiting body of Cordyceps cicadae was increased by 23.4% to 27.98 ± 1.36 mg / g.

[0191] 6. Effect of nano-selenium and zinc disodium ethylenediaminetetraacetate on trace elements in the fruiting body of Cordyceps cicadae

[0192] The element content in the fruiting body of Cordyceps cicadae was determined by inductively coupled plasma mass spectrometry (ICP-MS), and the detailed method is shown in Example 4. The results of the element content ( Figure 32)showed that compared with the control group, the selenium content of Cordyceps cicadae reached 29.27 ± 3.63 mg / kg when only nano-selenium was added, and the Se content was significantly increased by 4374.4% compared with the control, while the contents of Cr, Fe, Cu, As, Cd and Pb in Cordyceps cicadae fruiting bodies were significantly reduced by 30.2%, 7.9%, 10.8%, 33.0%, 20.0% and 30.0%, respectively; Mn was significantly increased by 12.0%, and Zn had no significant effect; when only zinc disodium ethylenediaminetetraacetate was added, the zinc content of Cordyceps cicadae reached 67.44 ± 3.77 mg / kg, and the Zn content was significantly increased by 59.7%, and the contents of Cr, Fe, Cu, As and Pb in Cordyceps cicadae fruiting bodies were significantly reduced by 41.9%, 7.2%, 17.0%, 46.8% and 37.7%, respectively, and Mn, Se and Cd had no significant effect; after adding nano-selenium and zinc disodium ethylenediaminetetraacetate together, the contents of Zn and Se in Cordyceps cicadae fruiting bodies reached 71.60 ± 4.04 mg / kg and 27.54 ± 2.68 mg / kg, respectively, and the contents of Zn and Se were significantly increased by 1.7 times and 42.1 times compared with the control group; the contents of Cr, Fe, Cu, As and Pb in Cordyceps cicadae fruiting bodies were significantly reduced by 60.9%, 12.6%, 15.2%, 39.1% and 44.7%, respectively; and the contents of Mn and Cd in Cordyceps cicadae fruiting bodies had no significant change.

[0193] 7. Effects of nano-selenium and zinc disodium ethylenediaminetetraacetate on selenium speciation in Cordyceps cicadae fruiting bodies

[0194] The selenium speciation in Cordyceps cicadae fruiting bodies was detected by high performance liquid chromatography coupled with hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS), and the detailed method is shown in Example 4. The selenium speciation determination results Figure 35 、 Figure 36 )showed that the selenium speciation analysis of Cordyceps cicadae fruiting bodies in the blank control group and the treatment group with only zinc disodium ethylenediaminetetraacetate added was below the detection limit, and SeCys2, Se(IV) and SeMet were detected in the treatment group with only sodium selenite added and the treatment group with sodium selenite and zinc disodium ethylenediaminetetraacetate added, wherein the proportions of SeCys2, Se(IV) and SeMet in the treatment group with only nano-selenium added were 68.4%, 9.6% and 22.0%, respectively; and the proportions of SeCys2, Se(IV) and SeMet in the treatment group with nano-selenium and zinc disodium ethylenediaminetetraacetate added were 63.0%, 9.3% and 27.7%, respectively.

[0195] From the biomass results (Table 1), the three selenium forms and two zinc forms had no interaction on the biomass of Cordyceps cicadae fruiting bodies, but the selenium form had a main effect. The biomass of the treatment group with added sodium selenate was significantly greater than that of the treatment group with added sodium selenite and that of the treatment group with added nano-selenium. From the adenosine content (Table 2), the three selenium forms and two zinc forms had no interaction on the adenosine content of Cordyceps cicadae fruiting bodies, and there was no main effect form. From the cordycepin content (Table 2), the three selenium forms and two zinc forms had no interaction on the cordycepin content of Cordyceps cicadae fruiting bodies, but the zinc form had a main effect. The cordycepin content of the treatment group with added zinc sulfate was significantly higher than that of the treatment group with added zinc disodium ethylenediaminetetraacetate. From the cordycepic acid content (Table 2), the three selenium forms and two zinc forms had an interaction on the cordycepic acid content of Cordyceps cicadae fruiting bodies, and the zinc form had a main effect. The cordycepin content of the treatment group with added zinc sulfate was higher than that of the treatment group with added zinc disodium ethylenediaminetetraacetate. From the trace element content (Table 3), the addition of Se significantly increased the selenium content in the fruiting bodies. The effect of adding sodium selenite was better than that of adding sodium selenate and that of adding nano-selenium. The selenium form and the zinc form had an interaction on the selenium content in the fruiting bodies, and the selenium form had a main effect. The addition of Zn significantly increased the zinc content in the fruiting bodies, but there was no significant difference in the effect of the two zinc forms on the zinc content in the fruiting bodies. The selenium form and the zinc form had no interaction on the zinc form in the Cordyceps cicadae fruiting bodies, but adding different selenium forms affected the zinc content in the fruiting bodies. The effect of the treatment group with added nano-selenium was better than that of the treatment group with added sodium selenite and that of the treatment group with added sodium selenate. For the Mn content in the fruiting bodies, the selenium form and the zinc form had no interaction, but the selenium form had a main effect. The effect of the treatment group with added nano-selenium was better than that of the treatment group with added sodium selenate and that of the treatment group with added sodium selenate. For the Fe content in the fruiting bodies, the selenium form and the zinc form had no interaction, but the zinc form had a main effect. The effect of the treatment group with added zinc disodium ethylenediaminetetraacetate was better than that of the treatment group with added zinc sulfate. For the Cu content in the fruiting bodies, the selenium form and the zinc form had an interaction, and the selenium form had a main effect. The effect of the treatment group with added sodium selenate was better than that of the treatment group with added nano-selenium and that of the treatment group with added sodium selenite. For the As content in the fruiting bodies, the selenium form and the zinc form had no interaction, but compared with the blank control group, each treatment group significantly reduced the As content in the fruiting bodies. For the Cd content in the fruiting bodies, the selenium form and the zinc form had an interaction. For reducing the Cd content in the fruiting bodies, the selenium form was best with nano-selenium, and the zinc form was best with zinc sulfate. For the Pb content in the fruiting bodies, the selenium form and the zinc form had no interaction, but compared with the blank control group, the Pb content was greatly reduced in all treatment groups.

[0196] Table 1 Effect of selenium and zinc addition on the biomass of Cordyceps cicadae fruiting bodies

[0197]

[0198] Note: The data are mean ± standard deviation; n.s. indicates no significant difference; * indicates significant p < 0.05.

[0199] The best combination recommended by the present application is the sodium selenite plus zinc sulfate treatment group, which has the highest selenium content, higher zinc content, higher biomass, the second highest cordycepic acid content, and higher adenosine content; the second is the sodium selenite plus zinc disodium ethylenediaminetetraacetate treatment group, the nano-selenium plus zinc disodium ethylenediaminetetraacetate treatment group, and the nano-selenium plus zinc sulfate treatment group, the sodium selenite plus zinc disodium ethylenediaminetetraacetate treatment group has higher selenium and zinc content, the highest adenosine content, and higher cordycepin content, the nano-selenium plus zinc disodium ethylenediaminetetraacetate treatment group has the highest zinc content, and both adenosine and cordycepin are at a relatively high level, and the nano-selenium plus zinc sulfate treatment group has the highest cordycepin and cordycepic acid content, and selenium, zinc and adenosine are at a relatively high level.

[0200] Example 10 Application of selenium and zinc-rich Cordyceps in selenium and zinc-rich functional food, health products and feed products

[0201] 1. Preparation of selenium and zinc-rich Cordyceps candy tablets

[0202] The selenium and zinc-rich Cordyceps prepared in this example contains 26.48-66.28 mg / kg selenium, 58.39-71.60 mg / kg zinc, 106.2-143.9 mg / kg cordycepin, 1.60-1.89 mg / g adenosine, and 22.41-30.40 mg / g cordycepic acid. Meanwhile, the selenium-rich Cordyceps also contains a large amount of polysaccharides, which have the ability to scavenge free radicals and antioxidant, and can improve the body's immunity. Using selenium and zinc-rich Cordyceps as raw material, the development of selenium and zinc-rich Cordyceps candy tablets has good health care effect.

[0203] The selenium and zinc-rich Cordyceps candy tablets are composed of 40-60% selenium and zinc-rich Cordyceps fruiting bodies, 5-10% corn starch, 10-20% enzyme powder, 10-20% malt dextrin, and 0.3-0.7% magnesium stearate, in terms of mass percentage.

[0204] For example, take 60% selenium and zinc-rich Cordyceps, 10% corn starch, 10% enzyme powder, 10% malt dextrin, 9.5% sucrose, and 0.5% magnesium stearate, mix thoroughly, irradiate for 1 h, and then press into tablets to obtain selenium and zinc-rich Cordyceps candy tablets (1 g / tablet).

[0205] Each piece of selenium and zinc-rich Cordyceps candy tablet contains 20-40 μg selenium, 20-42 μg zinc, 60-85 μg cordycepin, 0.6-1.1 mg adenosine, and 12-18 mg cordycepic acid. Normal people can meet their daily selenium needs by taking 2-3 tablets per day, and special groups such as the sick and the elderly can take 4-6 tablets per day. The selenium-rich Cordyceps candy tablets have multiple health care functions, can improve the body's immunity, have anti-tumor, antioxidant, anti-inflammatory effects, etc. At the same time, the candy tablets are easy to carry and have a sweet taste.

[0206] 2. Preparation of functional selenium and zinc-rich tetranychus powder

[0207] (1) Selenium and zinc-rich tetranychus corn powder

[0208] Selenium and zinc-rich instant corn powder, which mainly comprises selenium and zinc-rich tetranychus powder, corn powder and rice powder.

[0209] The selenium and zinc-rich tetranychus is dried and crushed to be fine, and 20 parts are weighed. High-quality corn kernels with no mildew and water content less than 10% are selected, peeled, crushed and weighed 100 parts. After being mixed with 4% water, the corn is puffed and then sieved through a 100-mesh sieve. Fresh and clean rice with water content less than 10% is selected, crushed, mixed with 3% water, puffed and then sieved through a 100-mesh sieve.

[0210] 40-80 parts of selenium-rich cordyceps powder, 100-300 parts of puffed corn powder, 40-100 parts of puffed rice powder and 20-60 parts of DE20 malt dextrin are fully mixed, irradiated and sterilized, and then packaged to obtain selenium-rich instant corn powder. Each package of instant corn powder contains 10-60 g, including 10-100 μg of selenium and 10-100 μg of zinc. Normal people can meet their daily selenium needs by taking 1-4 packages per day, and special groups such as the sick and the elderly can take 2-8 packages per day.

[0211] (2) Selenium and zinc-rich cordyceps millet powder

[0212] Selenium and zinc-rich instant millet powder mainly comprises selenium and zinc-rich tetranychus powder, millet powder and walnut kernel powder.

[0213] The selenium-rich cordyceps is dried and crushed to be fine, and 10-40 parts are weighed. High-quality millet is selected, washed to remove dust and rice bran, dried to control the water content to be less than 10%, and then broken, polished and crushed to obtain 100 parts. After being mixed with 4% water, the millet is puffed and then sieved through a 100-mesh sieve. High-quality walnut kernels (5-20 parts) and white sesame seeds (5-20 parts) are selected, washed, dried and fried, and then ground into powder.

[0214] 40-80 parts of selenium and zinc-rich cordyceps powder, 100-300 parts of puffed millet powder, 10-40 parts of walnut kernels and 10-40 parts of white sesame seeds are fully mixed, irradiated and sterilized, and then packaged to obtain selenium-rich instant millet powder. Each package of instant millet powder contains 10-60 g, including 10-100 μg of selenium and 10-100 μg of zinc. Normal people can meet their daily selenium needs by taking 1-4 packages per day, and special groups such as the sick and the elderly can take 2-8 packages per day.

[0215] (3) Selenium and zinc-rich tetranychus bean powder

[0216] Selenium-rich instant bean powder, the main components are: selenium-rich and zinc-rich Cordyceps cicada powder, soybean powder.

[0217] Selenium-rich and zinc-rich Cordyceps cicada is dried and crushed to fine, 40-80 parts are weighed; high-quality soybeans are selected, washed, dried, baked at 130°C for 1h, soaked in alkali, peeled, ground into pulp, and the soybean pulp is spray dried to obtain 100-300 parts of soybean powder.

[0218] After the 40-80 parts of selenium-rich Cordyceps cicada powder and the 100-300 parts of soybean powder are fully mixed and irradiated for sterilization, they are packaged to obtain the selenium-rich instant bean powder. Each package of instant bean powder contains 10-60g, including 10-100μg of selenium and 10-100μg of zinc. Normal people can meet their daily selenium needs by taking 1-4 packages per day, and special groups such as the sick and the elderly can take 2-8 packages per day.

[0219] 3. Preparation of selenium-rich and zinc-rich feed

[0220] Selenium-rich and zinc-rich feed is prepared from the cultivation medium of selenium-rich and zinc-rich Cordyceps cicada. The selenium content in the medium is 35-40mg / kg, and the zinc content is 80-100mg / kg. The selenium-rich and zinc-rich feed is prepared by mixing the selenium-rich and zinc-rich Cordyceps cicada cultivation medium with soybean meal, corn, vitamins, trace elements, salt, etc. The main formula is: 3-12kg of selenium-rich and zinc-rich Cordyceps cicada cultivation medium cake, 400-500kg of soybean meal, 300-500kg of corn, 2-3g of vitamins, 2-4g of trace elements, 0.5-1g of salt, and 1-2g of additives. The selenium content of the feed reaches 150-500μg / kg, and the zinc content increases by 5-10%. The feed is rich in active substances such as cordycepin, adenosine, and cordycepic acid. After feeding the selenium-rich and zinc-rich feed to laying hens and broiler chickens for 20-30 days, the selenium content in the eggs reaches 220-580μg / kg, and the selenium content in the chicken meat reaches 150-280μg / kg; after feeding the feed to pigs for 45 days, the selenium content in the pork reaches 140-290μg / kg.

[0221] Although the present application has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of protection claimed by the present application.

[0222] SEQUENCE LISTING <110> China Agricultural University <120> Cultivation method of selenium-rich and zinc-rich Cordyceps cicada and its application <130> KHP211120483.2 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 258 <212> DNA <213> Isaria cicadae <400> 1 aagtcgtaac aaggtctccg ttggtgaacc agcggaggga tcattaccag agttttacaa 60 ctcccaaccc ttctgtgaac ctacccatcg ttgcttcggc ggactcgccc cagcgtccgg 120 acggccctgc gccggcccgc gacctggacc caggcggccg ccggagacca cgcaaccctg 180 tatccatcag tctctctgaa tccgccgcaa ggcaacacaa atgaatcaaa actttcaaca 240 acggatctct tggttctg 258

Claims

1. Isaria cicadae M8, characterized in that, CGMCC No. 21453.

2. A microbial agent containing the Cordyceps cicadae M8 of claim 1.

3. Use of the Cordyceps cicadae M8 of claim 1 in the preparation of selenium and / or zinc enriched Cordyceps cicadae.

4. The cultivation method of the selenium and zinc-rich bugleweed, characterized by, The method comprises the following steps: A. Activation of the strain; B. Preparation of the seed liquid; C. Cultivation of the selenium and zinc enriched Cordyceps cicadae; The cultivation of the Cordyceps cicadae is carried out using a wheat medium, and inorganic selenium salt and / or nano selenium and inorganic zinc salt and / or organic zinc salt are added to the wheat medium; The preparation method of the wheat medium comprises: mixing wheat and nutrient solution at a ratio of 1:1-1.5 (g / mL), and sterilizing at 105-121 ℃ for 20-30 min under high pressure; The nutrient solution is prepared as follows: 20 g of sugar, 2 g of KH2PO4 and 1 g of MgSO4·7H2O are mixed, and water is added to make up to 1000 mL; the sugar is selected from at least one of glucose, sucrose or maltose; The inorganic selenium salt comprises selenate and / or selenite; the nano selenium comprises biosynthetic nano selenium and / or chemically synthesized nano selenium; the inorganic zinc salt comprises zinc sulfate; and the organic zinc salt comprises zinc disodium ethylenediaminetetraacetate; The concentration of selenate in the wheat medium is 0.5-100 mg / kg; and / or The concentration of selenite in the wheat medium is 0.5-100 mg / kg; and / or The concentration of zinc sulfate in the wheat medium is 100 mg / kg; and / or The concentration of zinc disodium ethylenediaminetetraacetate in the wheat medium is 100 mg / kg; The Cordyceps cicadae is the Cordyceps cicadae M8 of claim 1.

5. The method of claim 4, wherein, Step A comprises: inoculating the Cordyceps cicadae into modified PDA medium, and culturing in a constant temperature incubator at 20-25 ℃ for 18-25 days until a large number of spores are produced; The improved PDA medium is prepared as follows: 200 g of potato, 20 g of glucose, 17 g of agar, 3 g of peptone, 2 g of KH2PO4, 1 g of MgSO4.7H2O and 1 g of vitamin mixture are mixed, water is added to make up to 1000 mL, and high-pressure sterilization is performed at 105-121 °C for 20-30 min. B 30 mg are mixed, water is added to make up to 1000 mL, and high-pressure sterilization is performed at 105-121 °C for 20-30 min.

6. The method of claim 5, wherein, Step B comprises: taking activated Cordyceps cicadae fungus cakes with a diameter of 5-9 mm and inoculating them into 500 mL culture bottles containing 200 mL of modified PD medium for shake cultivation, the shake cultivation conditions being: 21 ℃, rotation speed 180 r / min, shake cultivation for 3 days, to obtain the seed liquid; The improved PD medium is prepared as follows: 200 g of potato, 20 g of glucose, 3 g of proteose peptone, 2 g of KH2PO4, 1 g of MgSO4·7H2O, and 1 g of VB6 are mixed, water is added to a constant volume of 1000 mL, and high-pressure sterilization is performed at 105-121°C for 20-30 min. B 30 mg are mixed, water is added to a constant volume of 1000 mL, and high-pressure sterilization is performed at 105-121°C for 20-30 min.

7. The method of claim 6, wherein, The liquid seed liquid is diluted by 3-7 times, 5 mL of the diluted bacterial liquid is added to the cultivation bottle containing the wheat medium, and each cultivation bottle contains 20-30 g of wheat medium; the cultivation bottle inoculated with the bacteria is transferred to a constant temperature cultivation room at 21 ℃, the relative humidity is 80-90%, and the cultivation is carried out in the dark for 4 days, followed by 12 hours of white light and 12 hours of red light alternately for 35 days, until the fruiting bodies are mature and spores are produced at the top, and then the Cordyceps cicadae can be harvested.

8. Use of the selenium and zinc enriched Cordyceps cicadae obtained by the method of any one of claims 4-7 in any one of the following applications: 1) for the preparation of health care products; 2) for the preparation of food; 3) for the preparation of feed; 4) for the preparation of medicines.

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