A bacillus licheniformis fusion strain, fermentation culture thereof and application thereof

Abstract

The present application relates to a kind of Eupenicillium fusion strain and its fermentation culture and application, belong to strain fermentation technical field.The content of active ingredients such as cordycepin, polysaccharide in the secondary metabolites of Eupenicillium F2652 of the present application is significantly improved, realizes the conversion and enrichment of polysaccharide of Eupenicillium strain while improving the content of cordycepin, the marker component of Cordyceps genus fungi.The fermentation culture obtained by liquid submerged fermentation of Eupenicillium F2652, the advantages of both parents are fused, both retain excellent composite polysaccharide synthesis ability, and also fuse good cordycepin accumulation potential, possess the development high cordycepin content Eupenicillium culture, it is conducive to enrich its as functional feed additive in different animals Practical application.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of strain fermentation, in particular to a sarocladium terricola fusion strain, fermentation culture and application thereof. BACKGROUND

[0002] More than 1500 species of ascomycetes capable of infecting and killing insects have been identified in nature, collectively known as broad-sense Cordyceps fungi, which play an important role in the natural regulation of insect populations. Some complexes of Cordyceps fungi and hosts can be directly used in traditional Chinese medicine, such as Cordyceps sinensis, Cordyceps gunnii, Cordyceps militaris and Cordyceps cicadae, which have a long history of food and medicine use and have positive significance for promoting body metabolism and improving immune function. Cordyceps militaris, as one of the model species of Cordyceps fungi, has received extensive attention in scientific research and industrial application in recent years. It contains multiple active ingredients such as cordycepin, adenosine, cordycepic acid, cordyceps polysaccharide, superoxide dismutase and ergosterol, especially its strong ability to biosynthesize and accumulate cordycepin, which has been proven to have unique pharmacological activity and health function, making Cordyceps militaris show great application potential in the fields of medicine, health products and food. With the continuous progress of biotechnology, research on Cordyceps militaris strains has also made significant progress.

[0003] Sarocladium terricola, formerly known as Acremonium terricola, is a new recorded species of Cordyceps fungi and also a kind of entomogenous fungi. It was first isolated from the microspore variety of Cordyceps gunnii. It can also synthesize multiple pharmacologically active substances such as adenosine, cordycepin and polysaccharide, among which polysaccharide is one of the most important physiological active ingredients. Tests have shown that these pharmacologically active ingredients have in vitro anti-tumor activity and the function of enhancing the immune activity of animals in vivo, and are developed for animal nutrition and health care. At present, the culture of Sarocladium terricola is a kind of functional feed additive in the directory of feed additives, and its application range includes pigs, chickens and extends to lactating cows.

[0004] The content of active ingredients such as cordycepin and polysaccharide in the secondary metabolites of the currently discovered Sarocladium terricola strains is not high, which limits the further utilization of Sarocladium terricola. SUMMARY

[0005] The present application aims to overcome the shortcomings of the prior art and provide a sarocladium terricola fusion strain, fermentation culture and application thereof, which can increase the content of cordycepin, the hallmark component of Cordyceps fungi, and realize the transformation and enrichment of polysaccharide, the main active ingredient of Sarocladium terricola strain, which has significant innovative value and economic benefits for the further development of Sarocladium terricola and its culture application.

[0006] To achieve the above object, the technical scheme adopted by the present application is:

[0007] In a first aspect, the present application provides a fusant of Acremonium, which is named as Sarocladium terricola , the fusant is named as Acremonium F2652, the preservation number is GDMCC No:64892, the preservation date is October 09, 2024, the preservation unit is Guangdong Microbial Culture Collection Center, the address of the preservation unit is No. 59, Building 5, 100, Jianli Middle Road, Guangzhou, Guangdong Province; the rDNA sequence of the Acremonium F2652 is shown as SEQ ID NO:1.

[0008] The fusant of Acremonium F2652 provided by the present application is obtained by the following method: preparing protoplasts of Acremonium and Cordyceps militaris starting strains respectively, inactivating the parent protoplasts by ultraviolet and heating, using polyethylene glycol (PEG) as a fusion agent, carrying out cell fusion and regeneration, and screening the fermentation mycelium by shake flask fermentation to obtain a new fusant of Acremonium with high biomass of fermentation mycelium, significantly improved polysaccharide content, and can effectively synthesize adenosine and cordycepin.

[0009] The Acremonium starting strain is Acremonium FA1603 (Acremonium terricola FA1603), which was preserved in the China Center for Type Culture Collection (CCTCC) on June 15, 2016, the address is Wuhan University, Wuhan, China, the postcode is 430072, and the preservation number is CCTCC NO: M2016331. Acremonium FA1603 (Acremonium terricola FA1603) CCTCC NO: M2016331 is referred to as Acremonium FA1603.

[0010] The Cordyceps militaris starting strain is Cordyceps militaris CICC14015 (Cordyceps militaris CICC14015), referred to as Cordyceps militaris CICC14015, purchased from China Industrial Microbial Culture Collection Center (CICC).

[0011] The preparation of the protoplasts first prepares spore suspensions of the Acremonium strain and the Cordyceps militaris strain respectively, then collects mycelium by liquid culture, and finally performs enzymatic hydrolysis under certain conditions to prepare the protoplasts.

[0012] The *Gastrodinium* spore suspension was obtained by solid agar culture. Glyceryl spores of the *Gastrodinium* strain FA1603 were inoculated onto 18×180 mm PDA agar slants. The slants were cultured at 24-26℃ for 5-7 days. The spores were then washed off the solid agar with sterile physiological saline to prepare a spore eluent. This eluent was then filtered through sterile filter paper to obtain the spore suspension. The spore suspension contained ≥1.0×10⁻⁶ spores. 6 The concentration of each component in the PDA liquid medium is [number of components per mL]. The composition of the comprehensive PDA agar medium includes: 1000.0 mL potato extract, 20.0 g glucose, 0.1 g yeast extract, 3.0 g KH₂PO₄, 1.5 g MgSO₄·7H₂O, 20.0 g agar powder, and pH 6.0. The potato extract is prepared as follows: 200.0 g peeled potatoes are cut into small pieces, added to 1000.0 mL of water, boiled for 30 min, the potato pieces are filtered out, and the filtrate is brought to a final volume of 1000.0 mL. All components are sterilized at 121℃ for 15-20 min. No agar powder is added to the PDA liquid medium.

[0013] The Cordyceps militaris spore suspension was obtained by solid agar culture. Glycerin-rich Cordyceps militaris strain CICC14015 was inoculated onto a comprehensive PDA agar medium. The agar was cultured at 24-28℃ for 5-7 days. The spores were then washed off the solid agar with sterile physiological saline to prepare a spore eluent. This eluent was then filtered through sterile filter paper to obtain the spore suspension. The spore suspension contained ≥1.0 × 10⁻⁶ spores. 6 The volume / mL of the comprehensive PDA agar medium consists of: 1000.0 mL potato extract, 20.0 g glucose, 0.1 g yeast extract, 3.0 g KH2PO4, 1.5 g MgSO4·7H2O, and 20.0 g agar powder, pH 6.0. The potato extract is prepared by taking 200.0 g peeled potatoes, cutting them into small pieces, adding 1000.0 mL of water, boiling for 30 min, filtering out the potato pieces, and bringing the filtrate to a final volume of 1000.0 mL. All components are sterilized at 121℃ for 15 min. No agar powder is added to the liquid medium.

[0014] The liquid culture for collecting mycelium involves inoculating the above spore suspension into PDA liquid culture medium at an inoculation rate of 10-20%, transferring 50 mL of the medium into a 250 mL Erlenmeyer flask containing 10 glass beads, and culturing it in a constant temperature shaker at 100-150 r / min for 24-36 hours to obtain the liquid culture. The culture is then centrifuged at 8000 rpm for 10 min to collect the mycelium.

[0015] The enzymatic hydrolysis under certain conditions refers to using different combinations of enzymatic hydrolysis conditions for different strains. The enzymatic hydrolysis conditions for *Aureobasidium* strain are as follows: a mixed hydrolysate of lysozyme, cellulase, and snailase, with the following concentrations: lysozyme concentration: 0.5%-2.0%, optimal concentration: 1.0%-1.5%; cellulase concentration: 0.5%-2.0%, optimal concentration: 0.5%-1.5%; snailase concentration: 0.5%-2.0%, optimal concentration: 0.5%-1.0%; hydrolysis temperature range: 24-35℃, optimal temperature: 26-30℃; hydrolysis time: 2-8 hours, optimal time: 4-5 hours; hydrolysis pH: 5.0-7.0, optimal pH: 6.5-6.8; protoplast concentration: >1.0 × 10⁻⁶. 5 per mL.

[0016] The enzymatic hydrolysis conditions for Cordyceps militaris mycelium were as follows: a mixed hydrolysate of lysozyme (purchased from Guangdong Microbial Culture Collection Center), snail enzyme, and cellulase. The concentration of the lysozyme hydrolysate was 0.5%-2.5%, with an optimal concentration of 0.5%-1.0%; the concentration of the snail enzyme hydrolysate was 0.5%-2.5%, with an optimal concentration of 0.5%-1.0%; the concentration of the cellulase hydrolysate was 0.2%-1.5%, with an optimal concentration of 0.5%-1.0%; the hydrolysis temperature range was 24-35℃, with an optimal temperature of 26-30℃; the hydrolysis time was 2-8 hours, with an optimal time of 3-5 hours; the hydrolysis pH was 5.0-7.0, with an optimal pH of 6.2-6.8; and the protoplast concentration was >1.0 × 10⁻⁶. 5 per mL.

[0017] The aforementioned UV and heat inactivation methods are primarily for facilitating subsequent complementary screening of fusion proteins. The purpose of protoplast fusion is to transform and complement the genetic material of both parents, thereby obtaining a new strain possessing the desirable traits of both. However, during the recombination process, some genetic traits may not be successfully expressed in the fused strain, resulting in a bias towards one parent. The fused strain is obtained by fusing UV-inactivated *Cordyceps militaris* protoplasts with heat-inactivated *Acer tumefaciens* protoplasts. During the protoplast fusion process, the genetic material of the two strains undergoes varying degrees of change, and some genetic material is expressed in the fused strain after gene recombination, allowing the two parental strains to achieve complementarity to a certain extent, thus achieving the purpose of fusion protein selection.

[0018] The optimal heat inactivation conditions of the A. terreus protoplast are: heat inactivation treatment at 50-60°C for 15-20 min, and the protoplast is gently shaken once every 2 min to make the heat inactivation uniform, and the inactivation rate reaches 100%. The optimal ultraviolet inactivation conditions of the C. militaris strain are: the cover is opened at a distance of 10 cm from a 30W ultraviolet lamp to avoid the interference of white light, and ultraviolet irradiation inactivation is performed for 10-15 min, and the inactivation rate reaches 100%.

[0019] The cell fusion is performed by mixing 1 mL of the A. terreus protoplast and 1 mL of the M. alpina protoplast, which are centrifuged, resuspended in 1 mL of the fusion promoter, preheated at 30°C for 5 min, and then rapidly placed in a 35°C water bath shaker at 60 r / min for 30 min.

[0020] The fusion regeneration and screening are performed by centrifuging at 2000 r / min for 10 min at 4°C after the cell fusion, discarding the supernatant, washing with 0.6 mol / L KCl three times to remove PEG, diluting the obtained protoplast to a concentration of 104-105 / mL with 0.6 mol / L KCl, taking the dilution liquid to be plated on a regeneration medium, and placing it in a 25°C constant temperature incubator for dark culture.

[0021] The fusion regeneration medium includes the following raw materials: glucose 7.5 g / L, sucrose 7.5 g / L, maltose 5.0 g / L, peptone 0.5 g / L, yeast powder 1.0 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.25 g / L, CaCl2 0.2 g / L, VB1 0.2 g / L, agar 10.00 g / L, and mannitol 109.30 g / L, which are sterilized at 121°C for 20 min.

[0022] The screening is performed by designing a special characteristic primer (AT-F / AT-R) for identification according to the existing ITS sequence of A. terreus, and screening and identifying the fusion by taking the parents as a control. The specific primer sequences are as follows:

[0023] AT-F: 5-AACATACCTACGTTTCCCTCG-R

[0024] AT-R: 5-TTAAGTTCAGCGGGTATTCC-R

[0025] Then, the fusion strain with high biomass and capable of significantly improving polysaccharide and comprehensive adenosine is obtained by selecting the PCR product fragments with electrophoretic bands of about 500 bp for shake flask fermentation screening, and the culture conditions are as follows: 24 DEG C, initial pH 6.5, 250 mL of shake flask loading amount of 50 mL, inoculation amount of 10 mL, shake flask rotation speed of 200 r / min, and fermentation time of 108 h. After fermentation, the mycelium is collected by centrifugation at 8000 r / min at room temperature, dried at 60 DEG C, weighed to calculate the biomass, and the polysaccharide content and the comprehensive adenosine content are measured.

[0026] Finally, the fusion strain with the highest polysaccharide content and comprehensive adenosine content is selected for phylogenetic analysis, and the genetic stability is detected by subculture, and the strain is preserved.

[0027] The above-mentioned shake flask fermentation medium is prepared according to the volume measurement as follows: 8% of glucose, 0.5% of proteose peptone, 1.0% of yeast powder, 2.0% of soybean powder, 5.5% of corn powder, 0.5% of wheat powder, 0.5% of ammonium sulfate, 0.75% of potassium dihydrogen phosphate, 0.25% of potassium phosphate, 0.3% of magnesium sulfate, 0.5% of amino acid nutrient mother liquor, 10 mL of trace element mother liquor, 2 mg / 100 mL of vitamin B1, 1 mg / 100 mL of vitamin B2, 1 mg / 100 mL of vitamin B6, and 1 mg / 100 mL of vitamin B12, and adding tap water to 100%, adjusting pH to 6.5-7.0, and sterilizing at 121 DEG C for 25 min by the conventional method. 12 1mg / 100ml, add tap water to 100%, adjust pH to 6.5-7.0, and sterilize at 121 DEG C for 25 min by the conventional method.

[0028] The above-mentioned amino acid nutrient mother liquor includes: 0.6 g of glycine, 0.5 g of threonine, 0.3 g of valine, 0.1 g of leucine, 0.2 g of isoleucine, 0.5 g of phenylalanine, 0.2 g of serine, 0.1 g of proline, 0.06 g of hydroxyproline, 0.04 g of cysteine, 0.05 g of tryptophan, 0.1 g of methionine, 0.2 g of lysine, and 0.1 g of glutamic acid, and the volume is made up to 1000 mL, and stored at 4 DEG C for standby.

[0029] The above-mentioned trace element mother liquor includes: 16.5 mg of ferrous sulfate, 16.5 mg of calcium chloride, 0.3 mg of copper sulfate, and 3.5 mg of zinc sulfate, and the volume is made up to 1000 mL, and stored at 4 DEG C for standby.

[0030] In the second aspect, the present application provides a fermentation culture of the fusion strain of Gliocladium, which is obtained by fermentation culture of the fusion strain of Gliocladium.

[0031] In the third aspect, the present application provides a preparation method of the fermentation culture of the fusion strain of Gliocladium, which comprises the following steps:

[0032] S1, activating culture of the fusion strain of Gliocladium to obtain an activated strain;

[0033] S2, inoculating the activated strain into millet seed reinforced medium for culture to obtain a reinforced seed culture; wherein the millet seed reinforced medium comprises the following raw materials: millet, soybean extract powder, sucrose, yeast extract, peptone, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, vitamin B1, vitamin B2, vitamin B6 and vitamin B 12 ;

[0034] S3, liquid fermentation culture of the reinforced seed culture to obtain a liquid fermentation product;

[0035] S4, inoculating the liquid fermentation product into solid medium, solid fermentation under light condition, and taking out the solid fermentation product for drying to obtain the fermentation culture of the G. fimbriatum fusion strain.

[0036] Preferably, in step S1, the step of activation culture is: slant culture activation of the G. fimbriatum fusion strain on comprehensive PDA agar medium, inoculation of glycerol bacteria into a comprehensive PDA agar medium plate, culture in a culture box at 24-28°C in the dark for 5-7 days, washing off the conidia with sterile normal saline, transferring to a slant, and culture in a culture box at 24-28°C in the dark for 3-5 days to obtain the activated strain.

[0037] Preferably, in step S2, the activated strain is eluted to a conidia concentration of (4.5-5.5) × 10 6 / mL in an eluent, and the eluent is transferred to a tomato bottle containing a millet seed reinforced medium slant, and culture in a culture box at 24-28°C in the dark for 3 days to obtain the reinforced seed culture.

[0038] Preferably, the preparation method of the millet seed reinforced medium comprises the following steps:

[0039] Weigh 15-25 parts of soybean extract powder, 5-15 parts of sucrose, 5-15 parts of yeast extract, 2-5 parts of peptone, 0.2-0.6 parts of potassium dihydrogen phosphate, 0.05-0.15 parts of magnesium sulfate, 0.05-0.15 parts of ferrous sulfate, 0.01-0.03 parts of vitamin B1, 0.01-0.03 parts of vitamin B2, 0.01-0.03 parts of vitamin B6 and 0.00002-0.00006 parts of vitamin B 12 , make up to 1000ml, weigh 100 parts of millet in the amount of 1:1 by mass volume ratio, mix evenly, steam for 40-50 minutes, after steaming, weigh 100 parts into each tomato bottle, sterilize according to the conventional method to obtain the millet seed reinforced medium.

[0040] Preferably, in the step S3, the millet seed seedling culture is inoculated into a fermentation tank for cultivation, in the initial stage of the fermentation culture, the air volume of the fermentation tank is controlled to be 10-20 L / min, the stirring speed is controlled to be 150-200 rpm, the temperature is controlled to be 24-28 DEG C, the initial reducing sugar concentration is controlled to be 80-100 g / L, the reducing sugar concentration in the fermentation process is controlled to be 0.5-1 g / 100 mL by glucose feeding, the initial pH is controlled to be 6.0-7.5, the pH in the whole fermentation process is controlled to be 6.0-7.5 by ammonia water feeding, and the cultivation is performed for 20-26 hours, and then the cultivation is performed for 2-3 days under the condition that the light intensity is 200-500 Lx, the mycelium and cordycepin are intensively cultured, the whole fermentation cycle is 110-120 h, and the liquid fermentation culture is obtained.

[0041] Preferably, in the step S4, the solid fermentation is performed under the following conditions: the temperature is 25-35 DEG C, the humidity is 65-75%, the fermentation time is 4-7 days, and the light intensity is 100-200 Lx, and the light time is 8-12 hours per day; wherein the solid culture medium comprises the following components in terms of weight parts: 35-45 parts of soybean meal powder, 25-35 parts of wheat powder and 25-35 parts of bran powder.

[0042] Preferably, in the step S4, the temperature for drying the solid fermentation product is 60-80 DEG C, the solid fermentation product is dried to have a moisture content less than 10%, and the solid fermentation product is crushed to have a particle size of 30-50 meshes, so that the fermentation culture of the Mortierella hyalina fusion strain is obtained.

[0043] In a fourth aspect, the application provides an application of the fermentation culture of the Mortierella hyalina fusion strain in preparing a functional feed additive.

[0044] Compared with the prior art, the application has the following beneficial effects:

[0045] The content of active ingredients such as cordycepin and polysaccharide in the secondary metabolites of the Mortierella hyalina F2652 is obviously increased, so that the content of cordycepin, the marker component of the Cordyceps genus fungus, is increased, and the conversion and enrichment of the main active component, polysaccharide, of the Mortierella hyalina strain are realized. The fermentation culture obtained by liquid submerged fermentation of the Mortierella hyalina F2652 has a significantly increased mycelium adenosine synthesis capacity compared with the parent FA1603, and the content of cordycepin in the solid culture product is also greatly increased, which is increased by about 53% compared with the parent Mortierella hyalina FA1603 strain, and the content of polysaccharide is basically maintained at the level of the parent, so that the advantages of the two parent strains are well combined, the excellent composite polysaccharide synthesis capacity is retained, and the good cordycepin accumulation potential is combined, so that the Mortierella hyalina culture with high cordycepin content is developed, which is beneficial to enrich the practical application of the Mortierella hyalina culture as a functional feed additive in different animals. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 Screening and identification of specific primers for different fusions; in the figure, M is 100 bp DNA leader marker, A is parent Acremonium terricola FA1603, C is parent Cordyceps militaris CICC14015, 1-7 are different fusion strains;

[0047] Figure 2 Phylogenetic tree of F2652 strain based on ITS characteristic sequence. DETAILED DESCRIPTION

[0048] The following examples facilitate a better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods, unless otherwise specified. In the following examples, the experimental materials are commercially available, unless otherwise specified. In the following examples, the quantitative tests are set up in triplicate, and the results are averaged. EXAMPLE

[0049] Screening and identification of specific primers for different fusions; in the figure, M is 100 bp DNA leader marker, A is parent Acremonium terricola FA1603, C is parent Cordyceps militaris CICC14015, 1-7 are different fusion strains;

[0050] 1. Collection of mycelium of starting parent strains

[0051] One of the starting parents, Acremonium terricola FA1603, was inoculated into a comprehensive PDA solid culture medium: 18x180mm test tube slant, cultured at 25°C for 6 days, then the spores in the solid slant were washed with sterile normal saline to prepare a spore eluate, and the eluate was filtered using sterile filter paper to obtain a spore suspension, which was inoculated into PDA liquid culture medium at an inoculation amount of 15%, the volume of 250mL of a triangular flask was 50mL, containing 10 glass beads, and cultured in a constant temperature shaker at a rotation speed of 120r / min for 24 hours to obtain a liquid culture, which was centrifuged at 8000rpm for 10min to collect the mycelium.

[0052] Another parent strain, Cordyceps militaris CICC14015, was inoculated into PDA agar medium in a 18x180mm test tube slant and cultured at 25°C for 6 days. The spores were then eluted from the slant with sterile saline and filtered with sterile filter paper to obtain a spore suspension. The suspension was then inoculated into PDA liquid medium at a 15% inoculation rate and cultured in a 250ml flask containing 50ml of liquid medium with 10 glass beads at 25°C, 100-150rpm for 30h. After the culture was completed, the mycelium was collected by centrifugation at 8000rpm for 10min, washed twice with sterile saline, and dried with sterile filter paper. Finally, the mycelium was collected.

[0053] 2. Preparation of protoplast suspension of the two parent strains

[0054] The mycelium prepared in step 1 was used as the raw material, and a 45g / L potassium chloride solution was used as the osmotic pressure stabilizer. The enzyme solution was a mixture of lywallzyme (purchased from the Guangdong Microbial Culture Collection Center), lysozyme (purchased from Shanghai Generay Biotech), snailase (purchased from Shanghai Generay Biotech), and cellulase (purchased from Shanghai Generay Biotech).

[0055] The enzyme solution for the enzymatic wall-breaking of A. terreus FA1603 was a mixture of lysozyme (purchased from Shanghai Generay Biotech), cellulase (purchased from Shanghai Generay Biotech), and snailase (purchased from Shanghai Generay Biotech). The lysozyme solution had a concentration of 1%, the cellulase solution had a concentration of 1%, and the snailase solution had a concentration of 0.7%. The enzymatic wall-breaking was performed at a temperature of 27°C for 5h, at a pH of 6.5, and the protoplast concentration reached 1.0x10 6 / mL. This was the optimal condition for preparing the protoplasts of A. terreus FA1603, and the efficiency of preparing the protoplasts was the highest. Microscopic observation showed that the mycelium was almost completely converted into protoplasts, and the regeneration rate was also good. The regeneration rate was 35%, calculated as the number of regenerated colonies divided by the total number of protoplasts, multiplied by 100%. The protoplasts were resuspended in a 45g / L potassium chloride solution to prepare the protoplast suspension of A. terreus FA1603.

[0056] The enzyme solution for the enzymatic wall-breaking of Cordyceps militaris CICC14015 was a mixture of lywallzyme (purchased from the Guangdong Microbial Culture Collection Center), snailase (purchased from Shanghai Generay Biotech), and cellulase (purchased from Shanghai Generay Biotech). The lywallzyme solution had a concentration of 1%, the snailase solution had a concentration of 0.6%, and the cellulase solution had a concentration of 0.8%. The enzymatic wall-breaking was performed at a temperature of 30°C for 4h, at a pH of 6.5, and the protoplast concentration reached 1.0x10 6The best condition for preparing the protoplast of Cordyceps militaris strain CICC14015 is 1 mL / mL, which is the highest efficiency of preparing the protoplast. Microscopic observation shows that the mycelium is basically formed into protoplast, and the regeneration rate of protoplast is also good. The regeneration rate = the number of regenerated mycelium / the total number of protoplasts x 100%, which reaches 32%. The protoplast suspension of Cordyceps militaris CICC14015 is prepared by resuspending the protoplast cells with 45 g / L potassium chloride solution.

[0057] 3. Protoplast fusion

[0058] Firstly, the best inactivation condition is determined.

[0059] The heat inactivation condition of the protoplast of the strain of Gliocladium roseum FA1603 is that 1 mL of the protoplast suspension is taken into a 5 mL centrifuge tube, and heat inactivation treatment is carried out at 55°C in a constant temperature water bath, the inactivation time is 15 min, each gradient is repeated 3 times, and a control group is set. The centrifuge tube is gently shaken every 2 min. The best heat inactivation condition is that the inactivation temperature is 50°C, the time is 15 min, and the inactivation rate reaches 100%. The inactivation condition of the protoplast of Cordyceps militaris strain CICC14015 is ultraviolet irradiation. 300 μL of protoplast is uniformly coated on the regeneration medium, the cover is opened at a distance of 5-10 cm from the 30W ultraviolet lamp, and the vertical irradiation is carried out for 10-15 min under the condition of avoiding white light interference, the best inactivation condition is that the distance from the ultraviolet lamp is 10 cm, and the irradiation time is 10 min, and the inactivation rate reaches 100%. The regeneration medium includes the following raw materials in g / L: glucose 7.5 g / L, sucrose 7.5 g / L, maltose 5.0 g / L, peptone 0.5 g / L, yeast powder 1.0 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.25 g / L, CaCl2 0.2 g / L, VB1 0.2 g / L, agar 10.00 g / L, mannitol 109.30 g / L, and the above components are sterilized at 121°C for 20 min to obtain the regeneration medium.

[0060] Then, the fusion and regeneration of the inactivated parent protoplast are carried out, and the specific steps are as follows:

[0061] The 35% polyethylene glycol (PEG-6000) solution prepared by 0.05 mol / L calcium chloride solution as a fusion agent, respectively, take different inactivated Geodin FA1603 protoplast and Cordyceps militaris CICC14015 protoplast each 1 mL mixed and centrifuged, add 1 mL fusion agent resuspended, and in 30℃ water bath preheating 5 min, then quickly placed in 35℃ water bath shaker, 60 r / min, vibration fusion 30 min. After cell fusion, 2000 r / min, 4℃ centrifugation 10 min, discard the supernatant, and then use 0.6 mol / L KCl washing three times, remove PEG. The obtained protoplast precipitate was diluted to 105 / mL concentration with 0.6 mol / L KCl as an osmotic pressure stabilizer, and its diluent was coated on the regeneration medium and placed in a 25℃ constant temperature incubator, and cultured in the dark. The regenerated fusion colonies were obtained.

[0062] 4. Screening of fusion strains

[0063] Firstly, according to the existing ITS sequence of Geodin, a specially designed characteristic primer (AT-F / AT-R) for Geodin strain identification was selected. The fusion colonies in the regeneration plate were picked, and the parent strain suspension was used as a control to directly screen and identify the fusion colonies by PCR.

[0064] AT-F: 5-AACATACCTACGTTTCCCTCG-R

[0065] AT-R: 5-TTAAGTTCAGCGGGTATTCC-R

[0066] The PCR reaction system is shown in Table 1.

[0067] Table 1 PCR reaction system

[0068]

[0069] Reaction conditions: 98℃ 1m30s, {94℃ 40s, 57℃ 45s, 72℃ 45s} 30 cycles, 72℃ 10m, 4℃ preservation.

[0070] The 22 fusion colonies picked were screened by PCR, as shown in Table 2, of which only three fusion colonies had the same size bands as Geodin FA1603, which were possible new Geodin strains. Figure 1

[0071] ​Then, the three fusion colonies were selected for shake flask fermentation screening. The culture conditions were as follows: 24°C, initial pH 6.5, 250 mL of shake flask with a liquid volume of 50 mL, inoculation amount of 10 mL, shake flask rotation speed of 200 r / min, and fermentation time of 108 h. After fermentation, the mycelium was collected by centrifugation at 8000 r / min at room temperature, dried at 60°C, weighed to calculate the biomass, and the contents of polysaccharide and adenosine were measured.

[0072] The content of polysaccharide in the fermented mycelium was detected according to the determination of crude polysaccharide content in edible fungi in NYT 1676-2008. The contents of cordycepin and adenosine in the fermented mycelium were detected by high performance liquid chromatography according to the determination of cordycepin and adenosine in cordyceps products in NYT 2116-2012. The specific results are shown in Table 2. Note: The biomass refers to the total dry weight of the solid collected after centrifugation at 5000 r / min for 30 min after fermentation and dried.

[0073] Table 2. Shake flask fermentation detection results of biomass, polysaccharide, adenosine and cordycepin of different fusion strains

[0074]

[0075] Finally, the No. 3 fusion strain with the best biomass, polysaccharide and adenosine content was selected and named as Gliocladium strain F2652. The characteristic sequence was recovered for sequencing, phylogenetic analysis was performed, and slope preservation and glycerol preservation were carried out. At the same time, microbial preservation and genetic stability detection were carried out.

[0076] As shown in Figure 2 Characteristic sequence alignment and phylogenetic analysis showed that the similarity of F2652 strain to parent FA1603 reached 99.5%, and the closest relative was Sarocladium terricola CBS 243.59 with a similarity of 100%. It was confirmed that F2652 was Gliocladium (Sarocladium terricola, formerly known as Acremonium terricola).

[0077] The Gliocladium strain (Sarocladium terricola) F2652 was preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on October 9, 2024, and the preservation number was GDMCC NO: 64892. The Gliocladium strain F2652 (Sarocladium terricola F2652) GDMCC NO: 64892 is referred to as Gliocladium strain F2652 or F2652. The rDNA sequence of the Gliocladium F2652 is shown in SEQ ID NO: 1.

[0078] The F2652 strain was subcultured to investigate its genetic stability, subcultured every 3 days, subcultured for 10 times, and the cordycepin content and polysaccharide content of the strain were determined by shake flask fermentation every other generation. The results show that the cordycepin content of the fermentation mycelium of the F2652 strain does not change significantly during the subculture process, the polysaccharide synthesis ability is basically maintained stable, and the strain has good genetic stability.

[0079] The above-mentioned shake flask fermentation medium is as follows according to the volume measurement: glucose 8%, proteose peptone 0.5%, yeast powder 1.0%, soybean powder 2.0%, corn flour 5.5%, wheat flour 0.5%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.75%, potassium phosphate 0.25%, magnesium sulfate 0.3%, amino acid nutrient mother liquor 0.5%, trace element mother liquor 10 ml, vitamin B1 2 mg / 100 ml, vitamin B2 1 mg / 100 ml, vitamin B6 1 mg / 100 ml, and tap water to 100%, pH is adjusted to 6.5-7.0, and high pressure sterilization is carried out at 121℃ for 25 min according to the conventional method. 12 1mg / 100ml, add tap water to 100%, adjust pH to 6.5-7.0, and high pressure sterilization is carried out at 121℃ for 25 min according to the conventional method.

[0080] The above-mentioned amino acid nutrient mother liquor includes: glycine 0.6 g, threonine 0.5 g, valine 0.3 g, leucine 0.1 g, isoleucine 0.2 g, phenylalanine 0.5 g, serine 0.2 g, proline 0.1 g, hydroxyproline 0.06 g, cysteine 0.04, tryptophan 0.05 g, methionine 0.1 g, lysine 0.2 g, glutamic acid 0.1 g, constant volume 1000 ml, store at 4℃, and reserve.

[0081] The above-mentioned trace element mother liquor includes: ferrous sulfate 16.5 mg, calcium chloride 16.5 mg, copper sulfate 0.3 mg, zinc sulfate 3.5 mg, constant volume 1000 ml, store at 4℃, and reserve. Example

[0082] The fermentation application of the A. terreus F2652, i.e. the preparation method of the fermentation culture of the A. terreus F2652, comprises the following steps:

[0083] I. Seed activation culture of the A. terreus F2652

[0084] 1. The A. terreus F2652 glycerol inoculum is inoculated into a comprehensive PDA agar culture medium plate, cultured in a culture box at 25℃ in the dark for 7 days, then the conidia (F1) are washed off with 5 ml of sterile normal saline, and transferred to a slope 18x180 mm test tube slope (F2), and cultured in a culture box at 27℃ in the dark for 4 days, which is the activation culture of the strain.

[0085] II. Millet seed intensive culture

[0086] The activated bacterial strain was eluted with 20 ml of sterile physiological saline until the spore concentration in the eluent was 5.0 × 10⁻⁶. 6 The elution buffer was collected at a concentration of 1.0 × 10⁶ spores / mL and transferred entirely to an eggplant-shaped flask containing millet seed-enhanced medium slant. The flask was then incubated at 25°C in the dark for 3 days to obtain an enhanced seed culture with a spore concentration of up to 1.0 × 10⁶ spores / mL. 11 1 / gram of culture.

[0087] The preparation method of the millet seed-enhanced culture medium is as follows: Weigh 20g of soybean extract powder, 10g of sucrose, 5.0g of yeast extract, 3.0g of peptone, 0.5g of potassium dihydrogen phosphate, 0.1g of magnesium sulfate, 100mg of ferrous sulfate, 20mg of vitamin B1, 10mg of vitamin B2, 10mg of vitamin B6, and vitamin B... 12 Prepare a nutrient solution by mixing 50 μg of commercial millet at pH 6 with a final volume of 1000 ml. Then, weigh out 1000 g of millet (1:1 mass-to-volume ratio) for every 1000 ml of nutrient solution. Mix thoroughly and steam over a water bath for 45 minutes. After steaming, weigh out 100 g of the mixture into each eggplant-shaped bottle and autoclave at 121°C for 20-30 minutes using standard methods. This results in a millet seed-enhanced culture medium with a large surface area, good aeration, and favorable conditions for spore germination and mycelial growth.

[0088] III. Liquid fermentation culture of *Gastrodinium glomeratum* F2652

[0089] The above-mentioned enhanced millet seed culture was directly inoculated into a fermenter. Approximately 100g of millet seed culture was inoculated into a 30L fermenter containing 15L of liquid culture medium. At the beginning of fermentation, the aeration rate was controlled at 15L / min, the stirring speed at 150rpm, the temperature at 25℃, and the initial reducing sugar concentration at 100g / L. The reducing sugar concentration was controlled to 0.7g / 100mL by adding 70% glucose. The initial pH was 7.0, and the pH was maintained at 7.0 throughout fermentation by adding ammonia. After 24 hours of cultivation, diffused light was applied for 2-3 days at a light intensity of 400Lx to enhance the culture of mycelium and cordycepin. The dissolved oxygen (DO) was controlled at 30% throughout the process, and the fermentation cycle was 5 days. Fermentation was then stopped, yielding a liquid fermentation culture. The liquid fermentation mycelium was collected by centrifugation at 3000rpm for 15min, dried at 60-80℃ until the moisture content was less than 10%, and used to determine biomass, mycelial polysaccharide, and adenosine content.

[0090] The liquid culture medium is prepared according to the following method: 8% of glucose, 1.5% of proteose peptone, 1.5% of yeast powder, 5.5% of corn powder, 2.0% of soybean powder, 0.5% of wheat powder, 0.5% of ammonium sulfate, 0.5% of potassium dihydrogen phosphate, 0.3% of magnesium sulfate, 0.5% of amino acid nutrient mother liquor, 10 ml of trace element mother liquor, 2 mg / 100 ml of vitamin B1, 1 mg / 100 ml of vitamin B2, 1 mg / 100 ml of vitamin B6, and tap water to 100%, and the pH is adjusted to 6.5-7.0. The medium is sterilized at 121°C for 25 min by the conventional method. 12 1mg / 100ml, and tap water to 100%, and the pH is adjusted to 6.5-7.0. The medium is sterilized at 121°C for 25 min by the conventional method.

[0091] Four, solid secondary fermentation of the strain of Metarhizium F2652

[0092] The liquid fermentation product is directly inoculated into the solid culture medium to perform solid fermentation, and a solid fermentation product is obtained.

[0093] The solid fermentation temperature is controlled at 30°C, the humidity is 70%, the fermentation time is 5 days, and the light intensity is 150 Lx, and the light time per day is 10 hours.

[0094] The mixing ratio of the liquid fermentation liquid to the solid culture medium is 1L of liquid fermentation product: 5 kg of solid culture medium.

[0095] The solid culture medium is prepared by mixing soybean meal powder, wheat powder, bran powder, and the like, and the solid culture medium comprises the following raw materials in parts by weight: 40 parts of soybean meal powder, 30 parts of wheat powder, and 30 parts of bran powder.

[0096] The fermentation product is collected, which means that the solid fermentation product is collected after the solid fermentation is completed, dried at 60°C until the moisture content is 7%, crushed, and passed through a 40-mesh sieve, and the obtained product is the Metarhizium culture.

[0097] Comparative Example 1

[0098] The strain Metarhizium FA1603 is used instead of the strain Metarhizium F2652, and the conditions of the test group of Example 2 are used to perform steps one, two, three, and four, and the mycelium and the solid fermentation culture of the strain Metarhizium FA1603 are collected, dried at 60°C until the moisture content is 7%, and the mycelium and the solid fermentation culture of the strain Metarhizium FA1603 of Comparative Example 1 are obtained.

[0099] Comparative Example 2

[0100] The strain Cordyceps militaris CICC14015 is used instead of the strain Metarhizium F2652, and the conditions of the test group of Example 2 are used to perform steps one, two, three, and four, and the mycelium and the solid fermentation culture of the strain Cordyceps militaris CICC14015 are collected, dried at 60°C until the moisture content is 7%, and the mycelium and the solid fermentation culture of the strain Cordyceps militaris CICC14015 of Comparative Example 2 are obtained.

[0101] Performance test

[0102] The mycelium and solid fermentation culture of the strains of Example 2 and Comparative Examples 1-2 were tested for biomass, polysaccharide content, adenosine and cordycepin content, respectively. Among them, the polysaccharide content in the fermentation mycelium and solid fermentation culture was detected according to the determination of crude polysaccharide content in edible fungi in NYT 1676-2008. The cordycepin and adenosine in the fermentation mycelium and solid fermentation culture were detected by high performance liquid chromatography according to the determination of cordycepin and adenosine in cordyceps products in NYT 2116-2012. The specific results are shown in Tables 3-4. Note: Biomass refers to the total dry weight of the collected solids after 5000r / m centrifugation for 30 min at the end of fermentation.

[0103] Table 3 Detection results of biomass, polysaccharide and adenosine content of mycelium of different strains

[0104]

[0105] Table 4 Detection results of polysaccharide, adenosine and cordycepin content of solid fermentation culture of different strains

[0106]

[0107] As can be seen from Tables 3-4, the fusion strain of Metarhizium anisopliae F2652, which has absorbed the advantages of both parent strains, has significantly improved the adenosine synthesis capacity of the mycelium under liquid deep fermentation conditions compared to the parent strain FA1603. The cordycepin content of the solid culture also increased significantly, which was about 53% higher than that of the parent strain Metarhizium anisopliae FA1603 strain. The polysaccharide content was basically maintained at the level of the parent strain. The fusion strain well combined the advantages of both parent strains, retaining excellent polysaccharide synthesis capacity and good cordycepin accumulation potential. It has the potential to develop Metarhizium anisopliae culture with high cordycepin content, which is conducive to enriching its practical application as a functional feed additive in different animals.

[0108] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A Metarhizium anisopliae fusion strain, characterized in that, The strain is named Gliocladium F2652, the preservation number is GDMCC No: 64892, the preservation date is October 09, 2024, the preservation unit is Guangdong Microbial Culture Collection Center, and the address of the preservation unit is No. 59, Building 5, 100, Jiefang Road, Guangzhou, Guangdong Province; the rDNA sequence of the Gliocladium F2652 is shown as SEQ ID NO:

1.

2. A fermentation culture of a Geodictyopon conidiogenum fusion strain, characterized in that, The fermentation culture of the Gliocladium fusaroides fusion strain of claim 1 is obtained by fermentation culture.

3. The method for preparing the fermentation culture of the *Gastrodia elata* fusion strain as described in claim 2, characterized in that, The method comprises the following steps: S1, activating culture of the Gliocladium fusaroides fusion strain to obtain an activated strain; S2, inoculating the activated strain into millet seed reinforced culture medium for culture to obtain a reinforced seed culture; wherein the millet seed reinforced culture medium comprises the following raw materials: millet, soybean extract powder, sucrose, yeast extract, peptone, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, vitamin B1, vitamin B2, vitamin B6 and vitamin B 12 ; S3, liquid fermentation culture of the strengthened seed culture to obtain a liquid fermentation product; S4, inoculating the liquid fermentation product into a solid culture medium, and performing solid fermentation under light conditions to obtain a solid fermentation product, and drying the solid fermentation product to obtain the fermentation culture of the Gliocladium fusaroides fusion strain.

4. The method for preparing the fermentation culture of the *Gastrodia elata* fusion strain as described in claim 3, characterized in that, In step S1, the step of activating culture is: using a comprehensive PDA agar culture medium to perform slant culture activation of the Gliocladium fusaroides fusion strain, inoculating glycerol bacteria into a comprehensive PDA agar culture medium plate, and culturing in a culture box at 24-28 DEG C in the dark for 5-7 days, then washing off conidia with sterile normal saline, and transferring to a slant and culturing in a culture box at 24-28 DEG C in the dark for 3-5 days to obtain the activated strain.

5. The method for preparing the fermentation culture of the *Gastrodia elata* fusion strain as described in claim 3, characterized in that, In the step S2, the activated strain is eluted to a concentration of (4.5-5.5)×10 6 The eluent is then transferred to a potato bottle containing millet seed reinforced medium slant, and cultured in a dark incubator at 24-28°C for 3 days to obtain the reinforced seed culture.

6. The method of preparing a fermentation culture of a Geotrichum candidum fusion strain according to claim 3 or 5, characterized in that, The preparation method of the millet seed strengthening culture medium comprises the following steps: The soybean extraction powder, sucrose, yeast paste, peptone, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, vitamin B1, vitamin B2, vitamin B6 and vitamin B are weighed as 15-25 parts, 5-15 parts, 5-15 parts, 2-5 parts, 0.2-0.6 parts, 0.05-0.15 parts, 0.05-0.15 parts, 0.01-0.03 parts, 0.01-0.03 parts, 0.01-0.03 parts and 0.00002-0.00006 parts respectively, and then mixed with 0.00002-0.00006 parts of vitamin B 12 , and then 1000ml of nutrient solution is prepared. Millet is weighed in an amount of 1:1 by mass and volume, mixed uniformly, and then steamed for 40-50min. After steaming, 100 parts of the millet is weighed into each eggplant bottle, sterilized according to a conventional method, and then a millet seed-strengthened culture medium is obtained.

7. The method for preparing the fermentation culture of the *Gastrodia elata* fusion strain as described in claim 3, characterized in that, In step S3, the millet seed strengthening culture is inoculated into a fermentation tank for culture, in the initial stage of fermentation culture, the ventilation of the fermentation tank is controlled at 10-20 L / min, the stirring speed is controlled at 150-200 rpm, the temperature is controlled at 24-28 DEG C, the initial reducing sugar concentration is controlled at 80-100 g / L, the reducing sugar concentration in the fermentation process is controlled at 0.5-1 g / 100 mL by adding glucose, the initial pH is controlled at 6.0-7.5, and the pH in the whole fermentation process is controlled at 6.0-7.5 by adding ammonia water, and the culture is performed for 20-26 hours, then the culture is performed for 2-3 days under the condition of light intensity of 200-500 Lx, the mycelium and cordycepin are strengthened, and the whole fermentation period is 110-120 h to obtain the liquid fermentation culture.

8. The method for preparing the fermentation culture of the *Gastrodia elata* fusion strain as described in claim 3, characterized in that, In step S4, the solid fermentation conditions are: temperature of 25-35 DEG C, humidity of 65-75%, fermentation time of 4-7 days, light intensity of 100-200 Lx, and light time of 8-12 hours per day. The solid culture medium comprises the following components in parts by weight: 35-45 parts of soybean meal powder, 25-35 parts of wheat flour, and 25-35 parts of bran powder.

9. The method for preparing the fermentation culture of the *Gastrodia elata* fusion strain as described in claim 3, characterized in that, In step S4, the temperature for drying the solid fermentation product is 60-80 DEG C; the moisture content is less than 10% after drying, and the solid fermentation product is crushed to 30-50 meshes to obtain the fermentation culture of the Gliocladium fusaroides fusion strain.

10. The application of the fermentation culture of the Gliocladium fusaroides fusion strain of claim 2 in preparing a functional feed additive.

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