Application of Acremonium truncatum F2652 in the preparation of preparations for improving production performance, immunity or antioxidant of weaned piglets

Abstract

The present invention relates to the application of Acremonium terrestris F2652 in the preparation of preparations for improving the production performance, immunity or antioxidant of weaned piglets, and belongs to the field of strain fermentation technology. The contents of active ingredients such as cordycepin and polysaccharides in the secondary metabolites of Acremonium terrestris F2652 of the present invention are significantly improved, thereby achieving the goal of increasing the content of cordycepin, a characteristic component of Cordyceps fungi, while also achieving the conversion and enrichment of polysaccharides of the Acremonium terrestris strain. The fermentation culture obtained by liquid submerged fermentation of Acremonium terrestris F2652 combines the advantages of both parents, retains the ability to synthesize excellent complex polysaccharides, and integrates good cordycepin accumulation potential, and has the ability to develop Acremonium terrestris cultures with high cordycepin content, which is conducive to enriching its practical application as a functional feed additive in different animals.

Description

Technical Field

[0001] The invention relates to the technical field of strain fermentation, and in particular to application of Acremonium terrestris F2652 in preparing a preparation for improving the production performance, immunity or antioxidant properties of weaned piglets. Background Art

[0002] Over 1,500 species of ascomycetes capable of infecting and killing insects have been identified in nature, collectively known as Cordyceps sensu lato. These fungi play a vital role in the natural regulation of insect populations. Some Cordyceps-host complexes are used directly in traditional Chinese medicine. For example, Cordyceps sinensis, Cordyceps kunini, Cordyceps militaris, and Cicadae fungi have a long history of medicinal and edible uses, demonstrating their beneficial effects in promoting metabolism and enhancing immune function. Cordyceps militaris, as a model species of the Cordyceps genus, has garnered significant attention in recent years in both scientific research and industrial applications. It contains numerous active ingredients, including cordycepin, adenosine, cordycepic acid, cordyceps polysaccharides, superoxide dismutase, and ergosterol. Its robust ability to biosynthesize and accumulate cordycepin has been shown to possess unique pharmacological activities and health benefits, demonstrating its enormous potential for application in medicine, health supplements, and food. With the continuous advancement of biotechnology, research on Cordyceps militaris strains has also made significant progress.

[0003] Sarocladium terricola (formerly known as: Acremonium terricola) is a newly recorded species of Cordyceps fungi and is also an entomogenous fungus. It was first isolated from the microspore variety of Cordyceps gunnii. It can also synthesize a variety of pharmacologically active substances such as adenosine, cordycepin, polysaccharides, among which polysaccharides are one of its most important physiologically active ingredients. After testing, these pharmacologically active ingredients have in vitro anti-tumor activity and the function of enhancing immune activity in animals, and are developed for animal nutrition and health care. At present, Sarocladium terricola culture is a type of functional feed additive in the feed additive catalog. Its application range includes pigs and chickens, and has been expanded to lactating dairy cows.

[0004] The contents of active ingredients such as cordycepin and polysaccharides in the secondary metabolites of the Acremonium terrestris strains discovered so far are not high, which limits the further utilization of Acremonium terrestris. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a fusion strain of Acremonium terrestris and its fermentation culture and application, which can increase the content of cordycepin, a characteristic component of Cordyceps fungi, and realize the conversion and enrichment of polysaccharides, the main active ingredient of the Acremonium terrestris strain, and has significant innovative value and economic benefits in further developing the application of Acremonium terrestris and its culture.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In the first aspect, the present invention provides the use of Acremonium truncatum F2652 in the preparation of a preparation for improving the production performance, immunity or antioxidant of weaned piglets. The Acremonium truncatum F2652 has a preservation number of GDMCC No: 64892, a preservation date of October 9, 2024, and a preservation unit of Guangdong Provincial Microbial Culture Collection Center. The preservation unit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province; the rDNA sequence of the Acremonium truncatum F2652 is shown in SEQ ID NO: 1.

[0008] Preferably, the Acremonium terrestris F2652 comprises a fermentation culture of a fusion strain of Acremonium terrestris, and the fusion strain of Acremonium terrestris is Acremonium terrestris F2652.

[0009] The Acremonium fusion strain F2652 provided by the present invention is obtained by the following method: protoplasts are prepared from Acremonium fusium and a starting strain of Cordyceps militaris respectively, the protoplasts are inactivated by ultraviolet and heating, polyethylene glycol (PEG) is used as a fusogen, cell fusion and regeneration are performed, and a novel fusion Acremonium fusium strain with high fermentation mycelium biomass and significantly improved polysaccharide content is obtained by shake flask fermentation screening, which can effectively synthesize adenosine and cordycepin.

[0010] The starting strain of Acremonium terricola is Acremonium terricola FA1603, which was deposited with the China Center for Type Culture Collection (CCTCC; Address: Wuhan University, Wuhan, China; Postal Code: 430072) on June 15, 2016, with a deposit number of CCTCC NO: M2016331. Acremonium terricola FA1603 (CCTCC NO: M2016331) is referred to as Acremonium terricola FA1603.

[0011] The Cordyceps militaris starting strain is Cordyceps militaris CICC14015, referred to as Cordyceps militaris CICC14015, which was purchased from China Industrial Microorganism Culture Collection Center (CICC).

[0012] The preparation of the protoplasts comprises the following steps: firstly preparing spore suspensions of Acremonium truncatum and Cordyceps militaris strains respectively, then carrying out liquid culture to collect mycelia, and finally carrying out enzymatic cell wall breaking under certain conditions to prepare the protoplasts.

[0013] The spore suspension of the Acremonium truncatum strain is obtained by solid slant culture. The glycerol bacteria of the Acremonium truncatum starting strain FA1603 are inoculated into a comprehensive PDA agar medium: an 18×180 mm test tube slant. The slant bacteria are cultured at 24-26° C. for 5-7 days. The spores in the solid slant are then washed with sterile physiological saline to prepare a spore eluate. The eluate is filtered using sterile filter paper to obtain a spore suspension. The spore amount in the spore suspension is ≥1.0×10 6 The comprehensive PDA agar medium consists of: 1000.0 mL of potato extract, 20.0 g of glucose, 0.1 g of yeast extract powder, 3.0 g of KH2PO4, 1.5 g of MgSO4·7H2O, 20.0 g of agar powder, pH 6.0. The potato extract is prepared by taking 200.0 g of peeled potatoes, cutting them into small pieces, adding 1000.0 mL of water, and boiling for 30 minutes. The potato pieces are filtered out, and the filtrate is brought to a volume of 1000.0 mL. The above ingredients are sterilized at 121°C for 15-20 minutes. Agar powder is not added to the PDA liquid medium.

[0014] The Cordyceps militaris spore suspension is obtained by solid slant culture. The glycerol bacteria of the Cordyceps militaris starting strain CICC14015 are inoculated into a comprehensive PDA agar medium. The slant culture is cultured at 24-28°C for 5-7 days. The spores in the solid slant are then washed with sterile physiological saline to prepare a spore eluate. The eluate is filtered using sterile filter paper to obtain a spore suspension. The spore amount in the spore suspension is ≥1.0×10 6 The comprehensive PDA agar medium consists of: 1000.0 mL of potato extract, 20.0 g of glucose, 0.1 g of yeast extract powder, 3.0 g of KH2PO4, 1.5 g of MgSO4·7H2O, 20.0 g of agar powder, pH

[0015] 6.0; Potato extract: Take 200.0g of peeled potatoes, cut into small pieces, add 1000.0mL of water, and boil for 30 minutes. Filter out the potato pieces and make up the volume to 1000.0mL. Sterilize all ingredients at 121°C for 15 minutes. Do not add agar powder to the liquid medium.

[0016] The liquid culture and mycelium collection method comprises inoculating the above spore suspension into PDA liquid culture medium at an inoculum volume of 10-20%, transferring 50 mL of liquid into a 250 mL Erlenmeyer flask containing 10 glass beads, culturing in a constant temperature shaker at a speed of 100-150 rpm for 24-36 hours to obtain a liquid culture, and centrifuging at 8000 rpm for 10 minutes to collect the mycelium.

[0017] The enzymatic cell wall breaking under certain conditions refers to the use of different enzymatic cell wall breaking conditions according to different strains. The enzymatic cell wall breaking conditions of the Acremonium truncatum strain are: an enzymatic cellulase mixed with lysozyme, cellulase and snailase, wherein the concentration of the lysozyme enzymatic cellulase is 0.5%-2.0%, the optimal enzymatic cellulase concentration is 1.0%-1.5%, the concentration of the cellulase is 0.5%-2.0%, the optimal concentration is 0.5-1.5%, the concentration of the snailase is 0.5%-2.0%, the optimal concentration is 0.5-1.0%, the enzymatic cellulase temperature range is 24-35°C, the optimal enzymatic cellulase temperature is 26-30°C, the enzymatic cellulase time is 2-8h, the optimal enzymatic cellulase time is 4-5h, the enzymatic cellulase pH is 5.0-7.0, the optimal enzymatic cellulase pH is 6.5-6.8, and the protoplast concentration is greater than 1.0×10 5 pieces / mL.

[0018] The enzymolysis wall-breaking condition of the mycelium of the Cordyceps militaris strain is: an enzymolysis solution of a mixture of lytic enzyme (purchased from Guangdong Microbial Culture Collection Center), snail enzyme and cellulase, wherein the lytic enzyme enzymolysis solution is used at a concentration of 0.5%-2.5%, and the optimal enzymolysis concentration is 0.5%-1.0%. The concentration of snail enzyme enzymolysis solution is 0.5%-2.5%, and the optimal enzymolysis concentration is 0.5%-1.0%. The working concentration of cellulase is 0.2%-1.5%, and the optimal concentration is 0.5%-1.0%. The enzymolysis temperature range is 24-35°C, the optimal enzymolysis temperature is 26-30°C, the enzymolysis time is 2-8h, the optimal enzymolysis time is 3-5h, the enzymolysis pH is 5.0-7.0, the optimal enzymolysis pH is 6.2-6.8, and the protoplast concentration is >1.0×10 5 pieces / mL.

[0019] Described ultraviolet and heating mode inactivation, is mainly for the complementary screening that is convenient to follow-up fusion.Protoplast fusion purpose is that the genetic material of parents is transformed and complementary, thereby obtains the new bacterial strain with the excellent traits of parents concurrently.Yet in the process that genetic material is reorganized, some hereditary traits can not successfully express in fusion strain, thereby produces the phenomenon that fusion strain is biased towards a certain parent.Fusion strain is the Cordyceps sinensis protoplast through ultraviolet inactivation, merges with the Acremonium protoplast through heat inactivation and obtains, and in the parents' protoplast fusion process, the genetic material of two bacterial strains has changed to varying degrees, some genetic material is expressed in fusion strain after genetic recombination, makes two parental bacterial strains can realize complementation to a certain extent, reaches the purpose of fusion screening.

[0020] The optimal heat inactivation conditions for Acremonium terrestris protoplasts are: 50°C-60°C for 15-20 minutes, with gentle shaking every 2 minutes to ensure even heating of the protoplasts, achieving a 100% inactivation rate. The optimal UV inactivation conditions for Cordyceps militaris strains are: irradiation with UV light vertically for 10-15 minutes, with the lid open and 10 cm away from a 30W UV lamp, avoiding interference from white light.

[0021] The cell fusion is carried out by using a 35% polyethylene glycol (PEG-6000) solution prepared with a 0.05 mol / L calcium chloride solution as a fusion promoter, taking 1 mL of protoplasts of different inactivated Cordyceps militaris strains and 1 mL of protoplasts of Morchella oleracea strains, mixing and centrifuging, adding 1 mL of the fusion promoter to resuspend, and preheating in a 30°C water bath for 5 minutes, and then quickly placing in a 35°C water bath shaker, shaking at 60 rpm, and fusing for 30 minutes.

[0022] The fusion regeneration and screening process involves centrifuging the cells at 2000 rpm at 4°C for 10 minutes after fusion, discarding the supernatant, and then washing the cells three times with 0.6 mol / L KCl osmotic stabilizer to remove PEG. The resulting protoplast pellet is diluted with 0.6 mol / L KCl osmotic stabilizer to a concentration of 104-105 cells / mL. The diluted solution is spread on a regeneration medium and placed in a 25°C constant temperature incubator in the dark to grow regenerated fusion colonies.

[0023] The fusion product 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, mannitol 109.30 g / L, and is sterilized by high pressure at 121°C for 20 min.

[0024] The screening method firstly designs special identification primers (AT-F / AT-R) based on the existing ITS sequence of Acremonium truncatum, and uses the two parents as controls to screen and identify the fusion products. The specific primer sequences are as follows:

[0025] AT-F: 5-AACATACCTACGTTTCCCTCG-R (sequence shown in SEQ ID NO: 2)

[0026] AT-R: 5-TTAAGTTCAGCGGGTATTCC-R (sequence shown in SEQ ID NO: 3)

[0027] Next, fusion colonies showing an electrophoretic band around 500 bp of the PCR product were selected for shake flask fermentation screening. Culture conditions were: 24°C, initial pH 6.5, 50 mL of liquid in a 250 mL shake flask, 10 mL of inoculum, 200 rpm, and a fermentation time of 108 hours. At the end of fermentation, mycelium was collected by centrifugation at 8000 rpm at room temperature, dried at 60°C, and weighed to calculate biomass. Polysaccharide and adenosine content were also measured to identify novel fusion strains with high biomass and significantly increased polysaccharide and adenosine content.

[0028] Finally, the fusion strains with the highest polysaccharide content and comprehensive adenosine content were selected for phylogenetic analysis, and their genetic stability was tested through passage, and the strains were preserved.

[0029] The shake flask fermentation medium is calculated by weighing volume as follows: glucose 8%, 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%, dipotassium hydrogen phosphate sulfur 0.25%, magnesium sulfate 0.3%, amino acid nutrient mother solution 0.5%, trace element mother solution 10ml, vitamin B12mg / 100ml, vitamin B21mg / 100ml, vitamin B 12 1 mg / 100 ml, add tap water to 100%, adjust the pH to 6.5-7.0, and sterilize by high pressure at 121℃ for 25 min according to conventional methods.

[0030] The above-mentioned amino acid nutrient mother solution includes: glycine 0.6g, threonine 0.5g, valine 0.3g, leucine 0.1g, isoleucine 0.2g, phenylalanine 0.5g, serine 0.2g, proline 0.1g, hydroxyproline 0.06g, cysteine 0.04g, tryptophan 0.05g, methionine 0.1g, lysine 0.2g, glutamic acid 0.1g, fixed volume 1000ml, 4℃ storage for standby use.

[0031] The above-mentioned trace element mother solution 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. The volume is fixed to 1000 ml and stored at 4° C. for future use.

[0032] In a second aspect, the present invention provides a fermentation culture of a fusion strain of Acremonium truncatum, which is obtained by fermenting and culturing the fusion strain of Acremonium truncatum.

[0033] In a third aspect, the present invention provides a method for preparing a fermentation culture of the Acremonium fusion strain, comprising the following steps:

[0034] S1. activating and culturing the Acremonium fusion strain to obtain an activated strain;

[0035] S2. Inoculate the activated strain into a millet seed culture medium for cultivation to obtain a fortified seed culture; wherein the millet seed 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 ;

[0036] S3. performing liquid fermentation on the fortified seed culture to obtain a liquid fermentation product;

[0037] S4. Inoculating the liquid fermentation product into a solid culture medium, performing solid fermentation under light conditions, taking out the solid fermentation product and drying it to obtain the fermentation culture of the Acremonium fusion strain.

[0038] Preferably, in step S1, the activation culture step is: using a comprehensive PDA agar medium to activate the Acremonium fusion strain on a slant culture, inoculating glycerol bacteria onto a comprehensive PDA agar medium plate, culturing in an incubator at 24-28° C. in the dark for 5-7 days, then washing the conidia with sterile physiological saline, transferring them to a slant, and culturing in an incubator at 24-28° C. in the dark for 3-5 days to obtain the activated strain.

[0039] Preferably, in step S2, the activated strain is eluted to a spore concentration of (4.5-5.5)×10 6 The eluate was transferred to an eggplant bottle containing a slant of millet seed fortified culture medium, and cultured in an incubator at 24-28° C. in the dark for 3 days to obtain the fortified seed culture.

[0040] Preferably, the method for preparing the millet seed fortified culture medium comprises the following steps:

[0041] 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 , adjust the volume to 1000 ml to prepare a nutrient solution, then weigh millet in a mass-to-volume ratio of 1:1, mix evenly, steam in water for 40-50 minutes, weigh 100 portions after steaming and put them into each eggplant bottle, sterilize according to a conventional method to obtain the millet seed fortified culture medium.

[0042] Preferably, in step S3, the millet seed enhanced culture is inoculated into a fermenter for culture. At the initial stage of fermentation culture, the fermenter ventilation volume is controlled at 10-20 L / min, the stirring speed is 150-200 rpm, the temperature is controlled at 24-28 ° C, the initial reducing sugar concentration is controlled at 80-100 g / L, and the reducing sugar concentration during the fermentation process is controlled at 0.5-1 g / 100 mL by feeding glucose, the initial pH is 6.0-7.5, and the pH is controlled at 6.0-7.5 during the entire fermentation process by feeding ammonia water. The culture is carried out for 20-26 hours, and then cultured under a light intensity of 200-500 Lx for 2-3 days to carry out enhanced culture of mycelium and cordycepin. The full fermentation cycle is 110-120 hours to obtain the liquid fermentation culture.

[0043] Preferably, in step S4, the conditions for solid fermentation are: temperature of 25-35°C, humidity of 65-75%, fermentation time of 4-7 days, light intensity of 100-200Lx, and light exposure time of 8-12 hours per day; wherein 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.

[0044] Preferably, in step S4, the temperature for drying the solid fermentation product is 60-80° C.; the solid fermentation product is dried to a moisture content of less than 10%, and crushed to 30-50 mesh to obtain the fermentation culture of the Acremonium fusion strain.

[0045] In a fourth aspect, the present invention provides a use of a fermentation culture of the Acremonium fusion strain in the preparation of a functional feed additive.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] The contents of active ingredients such as cordycepin and polysaccharides in the secondary metabolites of Acremonium truncatum F2652 of the present invention are significantly increased, achieving both an increase in the content of cordycepin, a hallmark component of the Cordyceps fungus, and the conversion and enrichment of polysaccharides, the main active ingredient of the Acremonium strain. The fermentation culture obtained by submerging Acremonium truncatum F2652 in liquid culture has a significantly improved mycelial adenosine synthesis capacity compared to its parent FA1603. The cordycepin content of the solid-cultured strain is also significantly increased, increasing by approximately 53% compared to the parent Acremonium truncatum FA1603 strain, while the polysaccharide content remains essentially at the parent level. This effectively combines the advantages of both parents, retaining excellent complex polysaccharide synthesis capacity while incorporating good cordycepin accumulation potential. This allows for the development of Acremonium truncatum cultures with high cordycepin content, which is beneficial for enriching its practical application as a functional feed additive for various animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Screening and identification of different fusion products by using specific primers for PCR; in the figure, M is a 100 bp DNA leader marker, A is the parent Acremonium FA1603, C is the parent Cordyceps militaris CICC14015, and 1-7 are different fusion strains;

[0049] Figure 2 This is the phylogenetic tree of the F2652 strain based on the ITS signature sequence. DETAILED DESCRIPTION

[0050] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0051] Example 1

[0052] Breeding of Acremonium fusogenum strain F2652

[0053] 1. Collection of mycelium of parental strains

[0054] One of the starting parents, Acremonium terricola FA1603, was inoculated with a glycerol strain of a comprehensive PDA solid medium: an 18×180 mm test tube slant and cultured at 25° C. for 6 days. The spores in the solid slant were then washed with sterile physiological saline to prepare a spore eluate. The eluate was filtered through sterile filter paper to obtain a spore suspension, which was inoculated into PDA liquid medium at a 15% inoculum size. The liquid volume was transferred to a 250 mL Erlenmeyer flask containing 50 mL of 10 glass beads and cultured in a constant temperature shaker at 120 rpm for 24 hours to obtain a liquid culture. The culture was centrifuged at 8000 rpm for 10 minutes to collect the mycelium.

[0055] The glycerol strain of another parent strain, Cordyceps militaris CICC14015, was inoculated into a comprehensive PDA agar medium: an 18×180 mm test tube slant, cultured at 25° C. for 6 days, eluted the slant spores with sterile physiological saline, and filtered the eluate through sterile filter paper to obtain a spore suspension, which was transferred to a comprehensive PDA liquid medium at a 15% inoculum volume for further culture. The transfer volume was 50 mL in a 250 mL Erlenmeyer flask containing 10 glass beads, and cultured in a thermostatic shaker at 25° C., 100-150 rpm, for 30 hours. After the culture was completed, a liquid culture was obtained, and mycelia was collected by centrifugation at 8000 rpm for 10 minutes, washed twice with sterile physiological saline, centrifuged, and dried with sterile filter paper. The mycelia were finally collected.

[0056] 2. Preparation of protoplast suspension of parental starting strains

[0057] The mycelium prepared in step 1 was used as the raw material, a potassium chloride solution with a concentration of 45 g / L was used as the osmotic pressure stabilizer, and an enzymatic solution of a mixture of lytic enzyme (purchased from Guangdong Microbial Culture Collection Center), lysozyme (purchased from Shanghai Shengong), snail enzyme (purchased from Shanghai Shengong) and cellulase (purchased from Shanghai Shengong) was used.

[0058] The enzymatic hydrolysis conditions for the cell wall breaking of Acremonium FA1603 were as follows: a mixed enzymatic solution of lysozyme (purchased from Shanghai Shenggong), cellulase (purchased from Shanghai Shenggong) and snailase (purchased from Shanghai Shenggong), wherein the concentration of lysozyme was 1%, the concentration of cellulase was 1%, the concentration of snailase was 0.7%, the enzymatic hydrolysis temperature was 27 ° C, the enzymatic hydrolysis time was 5 h, the enzymatic hydrolysis pH was 6.5, and the protoplast concentration reached 1.0 × 10 6 100 cells / mL. These are the optimal conditions for preparing Acremonium FA1603 protoplasts, resulting in the highest protoplast preparation efficiency. Microscopic observation showed that nearly all mycelia had formed protoplasts, and the protoplast regeneration rate was also good. Regeneration rate = number of regenerated colonies / total number of protoplasts × 100%, reaching 35%. Protoplasts were resuspended in 45 g / L potassium chloride solution to prepare a Acremonium FA1603 protoplast suspension.

[0059] The enzymatic hydrolysis conditions for the mycelial wall breaking of Cordyceps militaris CICC14015 were as follows: a mixed enzymatic solution of lytic enzyme (purchased from Guangdong Microbial Culture Collection Center), snail enzyme (purchased from Shanghai Shenggong) and cellulase (purchased from Shanghai Shenggong), wherein the concentration of lytic enzyme was 1%, the concentration of snail enzyme was 0.6%, the concentration of cellulase was 0.8%, the enzymatic hydrolysis temperature was 30 ° C, the enzymatic hydrolysis time was 4 h, the enzymatic hydrolysis pH was 6.5, and the protoplast concentration reached 1.0 × 10 6100 cells / mL. These are the optimal conditions for preparing protoplasts from the Cordyceps militaris strain CICC14015, achieving the highest protoplast production efficiency. Microscopic observation showed that nearly all mycelia had formed protoplasts, and the protoplast regeneration rate was also good. Regeneration rate = number of regenerated colonies / total number of protoplasts × 100%, reaching 32%. Protoplasts were resuspended in 45 g / L potassium chloride solution to prepare a Cordyceps militaris CICC14015 protoplast suspension.

[0060] 3. Protoplast fusion

[0061] First, the optimal inactivation conditions were determined.

[0062] The heat inactivation conditions for protoplasts of the Acremonium FA1603 strain are as follows: 1 mL of protoplast suspension is placed in a 5 mL centrifuge tube and heat-inactivated at 55°C in a constant temperature water bath for 15 minutes at each gradient, repeated three times, with a control group. The centrifuge tube is gently shaken every 2 minutes. The optimal heat inactivation conditions are 50°C for 15 minutes, achieving a 100% inactivation rate. The inactivation conditions for protoplasts of the Cordyceps militaris strain CICC14015 are ultraviolet irradiation. 300 μL of protoplasts are evenly spread on regeneration medium. The tube is then placed under a 30W UV lamp, opened at a distance of 5-10 cm, and vertically irradiated for 10-15 minutes, avoiding interference from white light. The optimal inactivation conditions are 10 minutes at a distance of 10 cm, achieving a 100% inactivation rate. 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, and mannitol 109.30 g / L. The above ingredients are sterilized by high pressure at 121°C for 20 min to obtain the regeneration medium.

[0063] Then, the fusion and regeneration of the inactivated parental protoplasts are carried out, and the specific steps are as follows:

[0064] 35% polyethylene glycol (PEG-6000) solution prepared with 0.05mol / L calcium chloride solution is a fusogen. Each 1mL of the protoplasts of Acremonium FA1603 and Cordyceps militaris CICC14015 of different inactivations is mixed and centrifuged. After adding 1mL of the fusogen, the cells are preheated in a 30℃ water bath for 5min and then rapidly placed in a 35℃ shaking water bath, 60 revs / min, and fused for 30min. After cell fusion ends, the cells are centrifuged at 2000r / min for 4℃ for 10min, and the supernatant is discarded. The cells are then washed three times with 0.6mol / L KCl to remove PEG. The protoplast precipitation obtained is diluted to 105 cells / mL concentration with 0.6mol / L KCl as an osmotic pressure stabilizer. The dilution is then applied to the regeneration medium and placed in a 25℃ constant temperature incubator. Culture in lucifuge to grow the regeneration fusion bacterium colonies.

[0065] 4. Screening of fusion strains

[0066] First, based on the existing ITS sequence of Acremonium truncatum, characteristic primers (AT-F / AT-R) were specially designed for the identification of Acremonium truncatum strains. The fusion colonies in the regeneration plate were picked. At the same time, the fusion colonies were directly screened and identified by PCR using the suspension of the two parental strains as a control.

[0067] AT-F: 5-AACATACCTACGTTTCCCCTCG-R

[0068] AT-R: 5-TTAAGTTCAGCGGGTATTCC-R

[0069] The PCR reaction system is shown in Table 1.

[0070] Table 1 PCR reaction system

[0071] sample Volume (μL) PfuPCRMasterMix 25 AT-F 2 AT-R 2 Colony suspension 0.5 Sterile high-purity water 20.5

[0072] Reaction conditions: 98°C for 1m30s, 30 cycles of {94°C for 40s, 57°C for 45s, 72°C for 45s}, 72°C for 10m, and storage at 4°C.

[0073] The 22 fusion colonies were selected for PCR screening, as shown in Figure 1 As shown, only three fusion colonies had bands of the same size as one of the parent strains, Acremonium FA1603, and were possible new Acremonium strains.

[0074] Next, these three fused colonies were selected for shake flask fermentation screening. Culture conditions were: 24°C, initial pH 6.5, 50 mL of liquid in a 250 mL shake flask, 10 mL of inoculum, 200 rpm, and fermentation time of 108 hours. At the end of fermentation, mycelium was collected by centrifugation at 8000 rpm at room temperature, dried at 60°C, and weighed to calculate biomass. Polysaccharide and adenosine content were also measured.

[0075] The polysaccharide content of fermented mycelium was determined according to NYT 1676-2008, Determination of Crude Polysaccharide Content in Edible Fungi. Cordycepin and adenosine in fermented mycelium were determined according to NYT 2116-2012, Determination of Cordycepin and Adenosine in Cordyceps Products by High-Performance Liquid Chromatography. Detailed results are shown in Table 2. Note: Biomass refers to the total dry weight of the solids collected after centrifugation at 5000 rpm for 30 minutes at the end of fermentation, after drying.

[0076] Table 2 Results of shake flask fermentation of biomass, polysaccharides, adenosine and cordycepin of different fusion strains

[0077] Colony number Biomass (%) Polysaccharide (%) Adenosine (μg / g) FA1603 11.6 13.5 4650 CICC14015 12.6 8.6 6500 No. 1 10.5 8.5 3800 No. 3 11.8 11.0 5233 No. 7 10.9 9.0 2980

[0078] Finally, strain 3, which showed the highest biomass, polysaccharide, and adenosine content, was selected and named Acremonium strain F2652. Its characteristic sequence was recovered and sent for sequencing, phylogenetic analysis, and the strain was stored on slant plates and in glycerol stock. Microbial preservation and genetic stability testing were also performed.

[0079] like Figure 2 As shown, characteristic sequence alignment and phylogenetic analysis showed that the F2652 strain shared 99.5% similarity with its parent FA1603, and its closest relative was Sarocladium terricola CBS243.59, with a 100% similarity. This confirmed that F2652 was a strain of Sarocladium terricola (formerly known as Acremonium terricola).

[0080] The Sarocladium terricola strain F2652 was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on October 9, 2024, with a deposit number of GDMCC NO: 64892. The Sarocladium terricola strain F2652 (Sarocladium terricola F2652) GDMCC NO: 64892 is referred to as the Sarocladium terricola strain F2652 or F2652. The rDNA sequence of the Sarocladium terricola F2652 is shown in SEQ ID NO: 1.

[0081] The F2652 strain was subcultured to examine its genetic stability. The strain was subcultured every three days for 10 times. Shake flask fermentation was performed every other generation to determine the cordycepin content and polysaccharide content of the strain. The results showed that the cordycepin content of the fermented mycelium did not change significantly during the subculture process of the F2652 strain, and the polysaccharide synthesis ability remained basically stable, indicating good genetic stability.

[0082] The shake flask fermentation medium is calculated by weighing volume as follows: glucose 8%, 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%, dipotassium hydrogen phosphate sulfur 0.25%, magnesium sulfate 0.3%, amino acid nutrient mother solution 0.5%, trace element mother solution 10ml, vitamin B12mg / 100ml, vitamin B21mg / 100ml, vitamin B 12 1 mg / 100 ml, add tap water to 100%, adjust the pH to 6.5-7.0, and sterilize by high pressure at 121℃ for 25 min according to conventional methods.

[0083] The above-mentioned amino acid nutrient mother solution includes: glycine 0.6g, threonine 0.5g, valine 0.3g, leucine 0.1g, isoleucine 0.2g, phenylalanine 0.5g, serine 0.2g, proline 0.1g, hydroxyproline 0.06g, cysteine 0.04g, tryptophan 0.05g, methionine 0.1g, lysine 0.2g, glutamic acid 0.1g, fixed volume 1000ml, 4℃ storage for standby use.

[0084] The above-mentioned trace element mother solution 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. The volume is fixed to 1000 ml and stored at 4° C. for future use.

[0085] Example 2

[0086] The fermentation application of Acremonium truncatum F2652, i.e., the preparation method of Acremonium truncatum F2652 fermentation culture, comprises the following steps:

[0087] 1. Seed activation culture of Acremonium truncatum F2652

[0088] 1. Inoculate the glycerol strain of Acremonium truncatum F2652 onto a comprehensive PDA agar medium plate and culture it in an incubator at 25°C in the dark for 7 days. Then wash the conidia (F1) with 5 ml of sterile saline and transfer it to the slant of an 18×180 mm test tube (F2). Culture it in an incubator at 27°C in the dark for 4 days. This is the activation culture of the strain.

[0089] 2. Intensive cultivation of millet seeds

[0090] The activated strain was eluted with 20 ml of sterile saline until the spore concentration of the eluate was 5.0 × 10 6 The eluate was transferred to an eggplant bottle containing a slant of millet seed fortified culture medium and cultured in an incubator at 25°C in the dark for 3 days to obtain a fortified seed culture, so that the spore concentration of the culture was as high as 1.0×10 11 / g culture.

[0091] The preparation method of millet seed fortified culture medium is as follows: weigh 20g soybean extract powder, 10g sucrose, 5.0g yeast extract, 3.0g peptone, 0.5g potassium dihydrogen phosphate, 0.1g magnesium sulfate, 100mg ferrous sulfate, 20mg vitamin B1, 10mg vitamin B2, 10mg vitamin B6, and 10mg vitamin B6. 12 Prepare a nutrient solution with 50 μg of glutathione (glutamine) at a pH of 6, and a constant volume of 1000 ml. Commercial millet is then weighed in a 1:1 mass-to-volume ratio (e.g., 1000 g of millet per 1000 ml of nutrient solution). Mix thoroughly and steam in a double boiler for 45 minutes. After steaming, weigh 100 g into each eggplant bottle and sterilize by conventional high-temperature and high-pressure sterilization at 121°C for 20-30 minutes. This creates a millet seed-fortified medium with a large surface area, good aeration, and favorable spore germination and mycelial growth.

[0092] 3. Liquid fermentation culture of Acremonium truncatum F2652

[0093] The millet seed fortified culture was directly inoculated into a fermenter, with approximately 100 g of the millet seed culture inoculated into a 30 L fermenter containing 15 L of liquid culture medium. During the initial fermentation phase, the fermenter was aerated at 15 L / min, stirred at 150 rpm, and maintained at a temperature of 25°C. The initial reducing sugar concentration was controlled at 100 g / L, and 70% glucose was fed to maintain the reducing sugar concentration at 0.7 g / 100 mL. The initial pH was maintained at 7.0, and ammonia was fed to maintain the pH at 7.0 throughout the fermentation. After 24 hours of incubation, the culture was incubated under scattered light at 400 Lx for 2-3 days to enhance mycelium and cordycepin production. The dissolved oxygen (DO) was maintained at 30% throughout the fermentation process, and the fermentation cycle lasted 5 days. Fermentation was then terminated to yield the liquid fermentation culture. The liquid fermentation mycelium was collected by centrifugation at 3000 rpm for 15 minutes and dried at 60-80°C until the moisture content was less than 10%. This was then used to measure biomass, mycelial polysaccharide, and adenosine content.

[0094] The above liquid culture medium is calculated by weighing volume as follows: glucose 8%, peptone 1.5%, yeast powder 1.5%, corn flour 5.5%, soybean powder 2.0%, wheat flour 0.5%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.3%, amino acid nutrient solution 0.5%, trace element solution 10ml, vitamin B1 2mg / 100ml, vitamin B2 1mg / 100ml, vitamin B 12 1 mg / 100 ml, add tap water to 100%, adjust the pH to 6.5-7.0, and sterilize by high pressure at 121℃ for 25 min according to conventional methods.

[0095] 4. Solid Secondary Fermentation of Acremonium Strain F2652

[0096] The liquid fermentation product is directly inoculated into a solid culture medium to carry out solid fermentation to obtain a solid fermentation product.

[0097] The solid fermentation temperature was controlled at 30°C, the humidity was 70%, the fermentation time was 5 days, the light intensity was 150Lx, and the light time was 10 hours per day.

[0098] The mixing ratio of the above-mentioned liquid fermentation liquid and solid culture medium is 1L liquid fermentation product: 5kg solid culture medium;

[0099] The solid culture medium is prepared as follows: soybean meal powder, wheat flour, bran powder, etc. are mixed to obtain a solid culture medium, which includes the following raw materials in parts by weight: 40 parts of soybean meal powder, 30 parts of wheat flour and 30 parts of bran powder.

[0100] The collected fermentation product refers to collecting the solid fermentation product after the solid fermentation is completed, directly drying it at 60° C. to a moisture content of 7%, crushing it and passing it through a 40-mesh sieve to obtain the Acremonium culture.

[0101] Comparative Example 1

[0102] The mycelium and solid fermentation culture were collected and dried at 60° C. to a moisture content of 7%, thereby obtaining the mycelium and solid fermentation culture of the strain of Comparative Example 1.

[0103] Comparative Example 2

[0104] The Cordyceps militaris strain CICC14015 was used instead of Acremonium F2652 and the conditions of the experimental group of Example 2 were used according to steps 1, 2, 3, and 4 to collect the mycelium and solid fermentation culture of the Cordyceps militaris strain CICC14015 and dry them at 60° C. to a moisture content of 7% to obtain the mycelium and solid fermentation culture of the strain of Comparative Example 2.

[0105] Performance Testing

[0106] The mycelia and solid fermentation cultures of the strains from Example 2 and Comparative Examples 1-2 were tested for biomass, polysaccharide content, adenosine, and cordycepin content. Polysaccharide content in the fermentation mycelia and solid fermentation cultures was determined using the method described in NYT 1676-2008, "Determination of Crude Polysaccharide Content in Edible Fungi." Cordycepin and adenosine in the fermentation mycelia and solid fermentation cultures were determined using high-performance liquid chromatography (HPLC) methods described in NYT 2116-2012, "Determination of Cordycepin and Adenosine in Cordyceps Products." Specific results are shown in Tables 3-4. Note: Biomass refers to the total dry weight of the solids collected after centrifugation at 5000 rpm for 30 minutes after fermentation, after drying.

[0107] Table 3 Test results of mycelial biomass, polysaccharide and adenosine content of different strains

[0108]

[0109] Table 4 Detection results of polysaccharide, adenosine and cordycepin contents in solid fermentation cultures of different strains

[0110]

[0111] As can be seen from Tables 3-4, the fusion Acremonium strain F2652 has absorbed the advantages of both parents. Under liquid deep fermentation conditions, the mycelial adenosine synthesis ability is significantly improved compared with the parent FA1603. The cordycepin content of the solid culture species is also greatly increased, about 53% higher than that of the parent Acremonium FA1603 strain, while the polysaccharide content is basically maintained at the level of the parent. It has well integrated the advantages of both parents, retaining the ability to synthesize excellent complex polysaccharides and integrating good cordycepin accumulation potential. It has the ability to develop Acremonium cultures with high cordycepin content, which is conducive to enriching its practical application as a functional feed additive in different animals.

[0112] Example 3

[0113] Effects of Acremonium culture on production performance, immunity and antioxidant capacity of weaned piglets

[0114] Piglets' tissues and organs are not fully developed, and their immune, digestive, and absorptive functions are immature, making them prone to diarrhea, growth stagnation, disease, and even death. Acremonium culture has immune-regulating, antiviral, and growth-promoting properties, effectively reducing animal morbidity, improving animal production performance, and improving animal reproductive performance. It is a green, safe, and highly effective functional feed additive. This experiment aimed to add different levels of Acremonium culture (cordycepin ≥ 1500 μg / g) to piglet diets to study the effects of Acremonium culture on piglet production performance, immunity, and antioxidant properties, providing a scientific basis for the application of Acremonium culture in piglets.

[0115] 1. Experimental Plan

[0116] 250g / t, 500g / t and 1000g / t of Acremonium culture (cordycepin ≥1500μg / g) were added to the piglet diet respectively to study its effects on piglet growth performance, organ index, diarrhea rate, mortality rate, antioxidant index, immune performance and intestinal immune barrier.

[0117] 2. Experimental Animals

[0118] A total of 240 healthy DLY weaned piglets of similar weight and age at 35 days were randomly divided into four treatments, with 6 replicates per treatment and 10 piglets per replicate. The experimental period was 33 days (pre-test period: 5 days, main test period: 28 days).

[0119] (See Table 5 below for details)

[0120] Table 5 Experimental design

[0121]

[0122] 3. Measurement indicators

[0123] 3.1 Growth performance

[0124] After the start of the experiment, the daily feeding amount and residual feed amount were accurately recorded in replicates (pens). Feed was stopped 12 h in advance on the 1st and 28th days of the formal experiment. The pigs were weighed on an empty stomach in the morning to calculate the average daily feed intake (ADFI), average daily weight gain (ADG) and feed-to-gain ratio (F / G).

[0125] Average daily weight gain (g / d) = (weight at the end of the test - weight at the beginning of the test) / number of days of the test;

[0126] Average daily feed intake (g / d) = total feed intake per head / number of experimental days;

[0127] Feed-to-weight ratio = average daily feed intake / average daily weight gain.

[0128] 3.2 Determination of relevant indicators in blood

[0129] On the 28th day of the experiment, one piglet was selected for each replicate, 10 mL of blood was collected from the jugular vein, injected into a 10 mL centrifuge tube, tilted for 15 minutes, and then centrifuged (3500 rpm) for 10 minutes. The upper layer of serum in the centrifuge tube was collected and stored in a -80°C refrigerator for testing.

[0130] 1) Serum antioxidant index

[0131] Serum total antioxidant capacity (T-AOC) and malondialdehyde (MDA) levels, as well as glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), Cu / Zn-SOD, and catalase (CAT) activities, were measured using commercial kits from the Nanjing Jiancheng Bioengineering Institute. The microplate reader used in this study was a Thermo Multiskan FC. Specific procedures were performed according to the kit instructions.

[0132] 2) Serum immune-related indicators

[0133] Serum immunoglobulin A (IgA), IgM, and IgG were determined by enzyme-linked immunosorbent assay (ELISA), and the kits were provided by Jiangsu ELISA Industrial Co., Ltd.

[0134] 4. Conclusion and Analysis

[0135] 4.1 Growth performance

[0136] Table 6 Effects of Acremonium culture on growth performance of weaned piglets

[0137]

[0138] Data in the same industry are significantly different between groups with different letters (P<0.05); data without letters or with the same letters are not significantly different, the same below.

[0139] The results are shown in Table 6: Compared with the control group, the groups supplemented with different concentrations of Acremonium culture can significantly increase the average daily weight gain and significantly reduce the feed-to-gain ratio of weaned piglets (P < 0.05). Compared with the control group, there is no significant difference in the average daily feed intake of each experimental group from 1 to 28 days (P > 0.05).

[0140] 4.2 Immune indicators

[0141] Table 7 Effects of Acremonium culture on immune parameters of weaned piglets

[0142]

[0143] Compared with the control group, adding 500g / t of Acremonium culture to the feed can significantly increase the serum IgM index of weaned piglets (P<0.05), and adding 500g / t and 1000g / t of Acremonium culture to the feed can significantly increase the serum IgG content of weaned piglets (P<0.05).

[0144] 4.4 Antioxidant index

[0145] Table 8 Effects of Acremonium culture on serum antioxidant indices in weaned piglets

[0146]

[0147] The results showed that compared with the control group, adding 500g / t of Acremonium culture to the diet could significantly increase the serum CAT of weaned piglets (P<0.05).

[0148] 5. Results Analysis

[0149] 5.1 Effects of Acremonium culture on growth performance of weaned piglets

[0150] Average daily feed intake, average daily gain, and feed-to-gain ratio are key indicators for evaluating animal growth performance. Feed-to-gain ratio is a representative indicator of growth performance; generally, a lower feed-to-gain ratio indicates better growth performance. This study found that supplementing the diet with 250g / t, 500g / t, and 1000g / t of Acremonium culture significantly increased average daily gain and feed-to-gain ratio in weaned piglets (P < 0.05), with the most pronounced effect achieved when 500g / t of Acremonium culture was added. There were no significant differences between the groups with different Acremonium culture levels (P > 0.05).

[0151] 5.2 Effects of Acremonium culture on immune function in weaned piglets

[0152] Enhancing the immune function of piglets is crucial in actual production. In previous studies, Cordyceps polysaccharides increased the IgM in the blood of dairy calves. A report in 2022 mentioned that Cordyceps polysaccharides can improve the humoral immunity of mice and increase the level of IgG in the serum. In this study, weaned piglets eating a diet supplemented with Acremonium culture can more effectively cope with immune stress levels. The experiment found that adding 500g / t of Acremonium culture to the feed can significantly increase the serum IgM index of weaned piglets (P < 0.05), and adding 500g / t and 1000g / t of Acremonium culture to the feed can significantly increase the serum IgG content of weaned piglets (P < 0.05). This shows that Acremonium culture can significantly improve the disease resistance and stress resistance of weaned piglets.

[0153] 5.3 Effects of Acremonium culture on antioxidant function in weaned piglets

[0154] Adding 500g / t of Acremonium truncatum culture to the experimental diet effectively increased various antioxidant indicators: superoxide dismutase (SOD), catalase (CAT), copper-zinc superoxide dismutase (CuZn-SOD), and glutathione peroxidase (GSH-Px), with CAT showing a significant increase (P < 0.05). Furthermore, various studies have reported that antioxidant compounds extracted from Cordyceps can enhance antioxidant capacity by increasing the expression of antioxidant enzymes including SOD, GSH-Px, and CAT.

[0155] 6. Summary

[0156] (1) Adding 250g / t, 500g / t, and 1000g / t of Acremonium culture (cordycepin ≥ 1500μg / g) to the diet can significantly increase the average daily gain (ADG) of weaned piglets, reduce the feed-to-weight ratio, and have no significant effect on the diarrhea rate. The 500g / t Acremonium culture group had the best effect.

[0157] (2) Adding Acremonium culture to the diet can significantly improve the immune level and antioxidant capacity of weaned piglets, and has a significant improvement effect on the IgM, IgG, and CAT indicators of weaned piglets. The 500g / t Acremonium culture group has the best effect.

[0158] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of Acremonium terrestris F2652 in the preparation of a preparation for improving the production performance, immunity or antioxidant of weaned piglets, characterized in that: The Acremonium truncatum F2652 has a deposit number of GDMCC No: 64892, a deposit date of October 9, 2024, and a depository of Guangdong Provincial Microbial Culture Collection Center. The depository address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province; the rDNA sequence of the Acremonium truncatum F2652 is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The preparation includes a fermentation culture of a fusion strain of Acremonium truncatum, wherein the fusion strain of Acremonium truncatum is Acremonium truncatum F2652.

3. The use according to claim 2, characterized in that The fermentation culture is prepared by a method comprising the following steps: S1. activating and culturing the Acremonium fusion strain to obtain an activated strain; S2. Inoculate the activated strain into a millet seed culture medium for cultivation to obtain a fortified seed culture; wherein the millet seed 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. performing liquid fermentation on the fortified seed culture to obtain a liquid fermentation product; S4. Inoculating the liquid fermentation product into a solid culture medium, performing solid fermentation under light conditions, taking out the solid fermentation product and drying it to obtain the fermentation culture of the Acremonium fusion strain.

4. The use according to claim 3, characterized in that In step S1, the activation culture step is as follows: using a comprehensive PDA agar medium to activate the Acremonium fusion strain on a slant culture, inoculating glycerol bacteria onto a comprehensive PDA agar medium plate, culturing in an incubator at 24-28° C. in the dark for 5-7 days, then washing the conidia with sterile physiological saline, transferring them to a slant, and culturing in an incubator at 24-28° C. in the dark for 3-5 days to obtain the activated strain.

5. The use according to claim 3, characterized in that In step S2, the activated strain is eluted until the spore concentration of the eluate is (4.5-5.5)×10 6 The eluate was transferred to an eggplant bottle containing a slant of millet seed fortified culture medium, and cultured in an incubator at 24-28° C. in the dark for 3 days to obtain the fortified seed culture.

6. The use according to claim 3 or 5, characterized in that The method for preparing the millet seed fortified culture medium comprises the following steps: 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 , adjust the volume to 1000 ml to prepare a nutrient solution, then weigh millet in a mass-to-volume ratio of 1:1, mix evenly, steam in water for 40-50 minutes, weigh 100 portions after steaming and put them into each eggplant bottle, sterilize according to a conventional method to obtain the millet seed fortified culture medium.

7. The use according to claim 3, characterized in that In the step S3, the millet seed fortified culture is inoculated into a fermentation tank for cultivation. In the initial stage of fermentation and cultivation, the ventilation volume of the fermentation tank is controlled to 10-20 L / min, the stirring speed is 150-200 rpm, the temperature is controlled to 24-28° C., the initial reducing sugar concentration is controlled to 80-100 g / L, and the reducing sugar concentration during the fermentation process is controlled to 0.5-1 g / 100 mL by feeding glucose, and the initial pH is 6.0-7.

5. The pH during the entire fermentation process is controlled to 6.0-7.5 by feeding ammonia water, and the culture is carried out for 20-26 hours. Then, the culture is carried out under the condition of a light intensity of 200-500 Lx for 2-3 days to carry out fortified cultivation of mycelium and cordycepin. The full fermentation cycle is 110-120 hours to obtain the liquid fermentation culture.

8. The use according to claim 3, characterized in that In step S4, the solid fermentation conditions are: temperature of 25-35°C, humidity of 65-75%, fermentation time of 4-7 days, light intensity of 100-200Lx, and light exposure 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 use according to claim 3, characterized in that In the step S4, the temperature of drying the solid fermentation product is 60-80° C.; drying to a moisture content of less than 10%, and crushing to 30-50 mesh to obtain the fermentation culture of the Acremonium fusion strain.

10. The use according to claim 9, characterized in that The fermentation culture is added to the feed to a concentration of 250 g / t to 1000 g / t for piglet feeding.

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