Application of acremonium terricola F2652 in preparation of preparation for improving production performance, immunity or antioxidation of weaned piglets

Abstract

The invention relates to application of acremonium terricola F2652 in preparation of a preparation for improving the production performance, immunity or antioxidation of weaned piglets, and belongs to the technical field of strain fermentation. The contents of active components such as cordycepin, polysaccharide and the like in secondary metabolites of the acremonium terricola F2652 are obviously increased, so that the conversion and enrichment of the polysaccharide of the acremonium terricola strain can be realized while the content of a symbolic component cordycepin of cordyceps fungi is increased. The acremonium terricola F2652 is subjected to liquid submerged fermentation to obtain a fermentation culture, the advantages of amphiphilic parents are fused, the excellent compound polysaccharide synthesis capacity is reserved, the good cordycepin accumulation potential is also fused, and the acremonium terricola culture with the high cordycepin content can be developed; the practical application of the feed additive as a functional feed additive in different animals can be enriched.

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 production performance, immunity or antioxidant of weaned piglets. Background Art

[0002] More than 1,500 species of ascomycetes that can infect and kill insects have been identified in nature, collectively known as broad cordyceps fungi. These fungi play an important role in the natural regulation of insect populations. Some complexes of cordyceps fungi and hosts can be used directly as medicine in traditional Chinese medicine. For example, Cordyceps sinensis, Cordyceps gunnii, Cordyceps militaris and Cicadae fungi have a long history of food and medicine, and have positive significance in promoting metabolism and improving immune function. As one of the model species of Cordyceps fungi, Cordyceps militaris has received extensive attention in scientific research and industrial applications in recent years. It contains many types of active ingredients, such as cordycepin, adenosine, cordycepic acid, cordyceps polysaccharides, superoxide dismutase, ergosterol, etc., especially its strong ability to biosynthesize and accumulate cordycepin, which has been proven to have unique pharmacological activity and health functions, making Cordyceps militaris show great application potential in the fields of medicine, health products 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 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, etc., 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 the immune activity of 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, and 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 prior art and provide a fusion strain of Acremonium terrestris and its fermentation culture and application, which can improve 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 for 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 preparations for improving the production performance, immunity or antioxidant preparations 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 Microbiological Culture Collection Center. The address of the preservation unit is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province; the rDNA sequence of Acremonium truncatum F2652 is shown in SEQ ID NO: 1.

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

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

[0010] The starting strain of Acremonium terricola is Acremonium terricola FA1603, which was deposited in China Center for Type Culture Collection (CCTCC; address: Wuhan University, Wuhan, China; postal code: 430072) on June 15, 2016, with a deposit number of CCTCCNO: M2016331. Acremonium terricola FA1603 (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 is purchased from China Industrial Microbiological Culture Collection Center (referred to as CICC).

[0012] The preparation of the protoplasts comprises the following steps: firstly preparing spore suspensions of Acremonium truncatum strain and Cordyceps militaris strain respectively, then carrying out liquid culture to collect mycelium, 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 strain is cultured at 24-26° C. for 5-7 days. Then, the spores in the solid slant are washed with sterile physiological saline to prepare a spore eluate. The eluate is filtered with sterile filter paper to obtain a spore suspension. The spore amount in the spore suspension is ≥1.0×10 6 / mL. Among them, the comprehensive PDA agar medium composition includes: potato extract 1000.0mL, glucose 20.0g, yeast extract powder 0.1g, KH2PO43.0g, MgSO4·7H2O1.5g, agar powder 20.0g, pH6.0. The preparation method of potato extract is: take 200.0g peeled potatoes, cut into small pieces, add 1000.0mL water and boil for 30min, filter out the potato pieces, and make up the filtrate to 1000.0mL. The above ingredients are sterilized at 121℃ for 15-20min. No agar powder is added to the PDA liquid culture medium.

[0014] The spore suspension of the Cordyceps militaris strain 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 strain is cultured at 24-28°C for 5-7 days. Then, the spores in the solid slant are washed with sterile physiological saline to prepare a spore eluate. The eluate is filtered with 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 peeled potatoes, cut into small pieces, add 1000.0mL water and boil for 30min, filter out the potato pieces, and make up the filtrate to 1000.0mL. Sterilize the above ingredients at 121℃ for 15min. No agar powder is added to the liquid culture medium.

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

[0017] The enzymatic cell wall breaking under certain conditions refers to using different enzymatic cell wall breaking conditions according to different strains. The enzymatic cell wall breaking conditions of the Acremonium terrestris strain are: a mixed enzymatic cellulase, a lysozyme, a cellulase and a snail enzyme, wherein the lysozyme enzymatic cellulase is used at a concentration of 0.5%-2.0%, the optimal enzymatic cellulase concentration is 1.0%-1.5%, the cellulase is used at a concentration of 0.5%-2.0%, the optimal concentration is 0.5-1.5%, the snail enzyme is used at a concentration of 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, the protoplast concentration is greater than 1.0×10 5 Pieces / mL.

[0018] The enzymatic hydrolysis wall-breaking conditions of the mycelium of the Cordyceps militaris strain are: a mixed enzymatic hydrolysis solution of lysozyme (purchased from Guangdong Microbial Culture Collection Center), snail enzyme and cellulase, wherein the use concentration of the lysozyme enzymatic hydrolysis solution is 0.5%-2.5%, the optimal enzymatic hydrolysis concentration is 0.5%-1.0%, the concentration of the snail enzyme enzymatic hydrolysis solution is 0.5%-2.5%, the optimal enzymatic hydrolysis concentration is 0.5%-1.0%, the use concentration of the cellulase is 0.2%-1.5%, the optimal concentration is 0.5%-1.0%, the enzymatic hydrolysis temperature range is 24-35°C, the optimal enzymatic hydrolysis temperature is 26-30°C, the enzymatic hydrolysis time is 2-8h, the optimal enzymatic hydrolysis time is 3-5h, the enzymatic hydrolysis pH is 5.0-7.0, the optimal enzymatic hydrolysis pH is 6.2-6.8, the protoplast concentration is>1.0×10 5 Pieces / mL.

[0019] The ultraviolet and heating inactivation are mainly for the convenience of complementary screening of subsequent fusion. The purpose of protoplast fusion is to transform and complement the genetic material of the parents, so as to obtain a new strain with both parents' excellent traits. However, in the process of genetic material recombination, some genetic traits can not be successfully expressed in the fusion strain, so as to produce the phenomenon that the fusion strain is biased towards a certain parent. The fusion strain is the fusion of the Cordyceps sinensis protoplasts inactivated by ultraviolet, and the fusion of the Acremonium protoplasts inactivated by heat, and in the process of protoplast fusion of the parents, the genetic material of the two strains has changed to varying degrees, and some genetic material is expressed in the fusion strain after genetic recombination, so that the two parent strains can be complementary to a certain extent, and the purpose of fusion screening is achieved.

[0020] Among them, the best heat inactivation conditions for the protoplasts of Acremonium terrestris are: heat inactivation treatment at 50℃-60℃ for 15-20 minutes, gently shaking once every 2 minutes to make the protoplasts evenly heated, and the inactivation rate reaches 100%. The best ultraviolet inactivation conditions for Cordyceps militaris strains are to open the lid 10cm away from a 30W ultraviolet lamp, avoid ultraviolet irradiation inactivation under the interference of white light, and vertically irradiate for 10-15 minutes, and the inactivation rate reaches 100%.

[0021] The cell fusion is prepared by using 35% polyethylene glycol (PEG-6000) solution prepared by 0.05 mol / L calcium chloride solution as a fusion promoter, taking 1 mL of protoplasts of different inactivated Cordyceps militaris strains and Morchella strains respectively, 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 and fusing at 60 revolutions per minute for 30 minutes.

[0022] The fusion regeneration and screening is that after the cell fusion is completed, the mixture is centrifuged at 2000 rpm and 4°C for 10 minutes, the supernatant is discarded, and then washed three times with 0.6 mol / L KCl osmotic stabilizer to remove PEG. The obtained protoplast precipitate is diluted with 0.6 mol / L KCl osmotic stabilizer to a concentration of 104-105 cells / mL, and the dilution is spread on the regeneration culture medium, placed in a 25°C constant temperature incubator, cultured in the dark, and regenerated fusion colonies are grown.

[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 at 121°C under high pressure for 20 min.

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

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

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

[0027] Next, the fusion colonies with electrophoresis bands of PCR product fragments around 500bp were selected for shake flask fermentation screening. The culture conditions were: 24°C, initial pH 6.5, 250mL shake flask liquid volume of 50mL, inoculation volume 10mL, shake flask speed 200r / min, and fermentation time 108h. After the fermentation, the mycelium was collected by centrifugation at 8000r / min at room temperature, dried at 60°C, weighed to calculate the biomass, and the polysaccharide content and adenosine comprehensive content were measured to obtain a new fusion strain with high biomass and significantly increased polysaccharide and comprehensive adenosine.

[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 by 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 powder 5.5%, wheat flour 0.5%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.75%, dipotassium hydrogen phosphate 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 1mg / 100ml, add tap water to 100%, adjust pH to 6.5-7.0, and sterilize at 121℃ for 25min according to conventional methods.

[0030] The above-mentioned amino acid nutrient mother solution includes: 0.6g of glycine, 0.5g of threonine, 0.3g of valine, 0.1g of leucine, 0.2g of isoleucine, 0.5g of phenylalanine, 0.2g of serine, 0.1g of proline, 0.06g of hydroxyproline, 0.04g of cysteine, 0.05g of tryptophan, 0.1g of methionine, 0.2g of lysine, and 0.1g of glutamic acid. The volume is fixed to 1000ml and stored at 4°C 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, 3.5 mg of zinc sulfate, fixed volume 1000 ml, stored at 4° C. for standby use.

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

[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, inoculating the activated strain into a millet seed culture medium for culturing 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, subjecting the fortified seed culture to liquid fermentation 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, thereby obtaining the fermentation culture of the Acremonium fusion strain.

[0038] Preferably, in step S1, the activation culture step is: using comprehensive PDA agar medium to perform slant culture activation on the Acremonium fusion strain, inoculating glycerol bacteria to 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 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 / mL, and then transfer the eluate to the eggplant bottle containing the millet seed fortified culture medium slant, and culture 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 , make a fixed volume of 1000 ml to prepare a nutrient solution, then weigh millet in an amount with a mass volume ratio of 1:1, mix evenly, steam in water for 40-50 minutes, weigh 100 portions after steaming and put them in each eggplant bottle, sterilize according to a conventional method, and obtain the millet seed fortified culture medium.

[0042] Preferably, in step S3, the millet seed fortified culture is inoculated into a fermenter for culture. At the initial stage of fermentation culture, the ventilation volume of the fermenter is controlled to be 10-20 L / min, the stirring speed is 150-200 rpm, the temperature is controlled to be 24-28° C., the initial reducing sugar concentration is controlled to be 80-100 g / L, and the reducing sugar concentration in the fermentation process is controlled to be 0.5-1 g / 100 mL by adding glucose, and the initial pH is 6.0-7.5. The pH in the whole fermentation process is controlled to be 6.0-7.5 by adding ammonia water, and the culture is carried out for 20-26 hours, and then the culture is carried out under the condition of a light intensity of 200-500 Lx for 2-3 days to carry out the fortified culture of mycelium and cordycepin. The whole fermentation cycle is 110-120 h 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 daily illumination time of 8-12 hours; 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 meshes to obtain the fermentation culture of the Acremonium fusion strain.

[0045] In a fourth aspect, the present invention provides the 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 polysaccharide in the secondary metabolites of Acremonium truncatum F2652 of the present invention are significantly increased, and the content of cordycepin, a characteristic ingredient of Cordyceps fungi, is increased while the conversion and enrichment of polysaccharide, a main active ingredient of Acremonium truncatum strain, can be achieved. The fermentation culture obtained by liquid deep fermentation of Acremonium truncatum F2652 has a significantly improved mycelial adenosine synthesis ability compared with the parent FA1603, and the cordycepin content of the solid culture species is also greatly increased, which is increased by about 53% compared with the parent Acremonium truncatum FA1603 strain, while the polysaccharide content is basically maintained at the level of the parent, which well integrates the advantages of both parents, retains the excellent synthesis ability of complex polysaccharides, and integrates good cordycepin accumulation potential, and has the ability to develop Acremonium truncatum culture with high cordycepin content, which is conducive to enriching its practical application as a functional feed additive in different animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The specific primer PCR screening and identification of different fusions; 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 for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0051] Example 1

[0052] Breeding of Acremonium fusogenum strain F2652

[0053] 1. Collection of mycelium of the starting parent 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, cultured at 25° C. for 6 days, and then the spores in the solid slant were washed with sterile physiological saline to prepare a spore eluate, and the eluate was filtered using sterile filter paper to obtain a spore suspension, which was inoculated into a PDA liquid medium at an inoculum rate of 15%, and the transfer volume in a 250 mL triangular flask was 50 mL, containing 10 glass beads, and cultured in a constant temperature oscillator at a speed of 120 r / min for 24 hours to obtain a liquid culture, which was centrifuged at 8000 rpm for 10 min to collect mycelium.

[0055] The glycerol bacteria of another parent strain, strain Cordyceps militaris CICC14015 (Cordyceps militaris CICC14015), was inoculated into a comprehensive PDA agar medium: an 18×180 mm test tube slant, and after culturing at 25° C. for 6 days, the slant spores were eluted with sterile physiological saline, and the eluate was filtered using sterile filter paper to obtain a spore suspension, and the slurry was transferred to a comprehensive PDA liquid medium at a 15% inoculation amount for continued cultivation, and the transfer volume in a 250 mL triangular flask was 50 mL, containing 10 glass beads, and cultured for 30 hours in a constant temperature oscillator at 25° C., 100-150 rpm. After the cultivation was completed, a liquid culture was obtained, and mycelium was collected by centrifugation at 8000 rpm for 10 minutes, and washed twice with sterile physiological saline, centrifuged, and dried with sterile filter paper, and finally the mycelium was collected.

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

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

[0058] The enzymatic cell wall breaking conditions 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 enzymatic solution was 1%, the concentration of cellulase was 1%, the concentration of snailase was 0.7%, the enzymatic temperature range was 27 ° C, the enzymatic time was 5 h, the enzymatic pH was 6.5, and the protoplast concentration reached 1.0 × 10 6 / mL. This is the optimal condition for the preparation of the protoplasts of Acremonium FA1603, and the efficiency of preparing the protoplasts is the highest. Microscopic observation shows that the mycelium is basically formed into protoplasts, and the regeneration rate of the protoplasts is also good. Regeneration rate = number of regenerated colonies / total number of protoplasts × 100%, reaching 35%. Use 45g / L potassium chloride solution to resuspend the protoplast cells to prepare the Acremonium FA1603 protoplast suspension.

[0059] The conditions for enzymatic hydrolysis of mycelium of Cordyceps militaris CICC14015 were as follows: a mixture 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 6 / mL. This is the best condition for preparing the protoplasts of Cordyceps militaris strain CICC14015, and the efficiency of preparing protoplasts is the highest. Microscopic observation shows that the mycelium is basically formed into protoplasts, and the regeneration rate of protoplasts is also good. Among them, the regeneration rate = the number of regenerated colonies / the total number of protoplasts × 100%, reaching 32%. Use 45g / L potassium chloride solution to resuspend the protoplast cells to prepare the Cordyceps militaris CICC14015 protoplast suspension.

[0060] 3. Protoplast fusion

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

[0062] The heat inactivation conditions of the protoplasts of the FA1603 strain of Acremonium terrestris are as follows: take 1 mL of the protoplast suspension and put it into a 5 mL centrifuge tube, and perform heat inactivation at 55 ° C in a constant temperature water bath. The inactivation time is 15 min, and each gradient is repeated 3 times, and a control group is set. Gently shake the centrifuge tube once every 2 minutes. The best heat inactivation conditions are: inactivation temperature 50 ° C, time 15 min, and the inactivation rate reaches 100%. The inactivation conditions of the protoplasts of the Cordyceps militaris strain CICC14015 are ultraviolet irradiation. Take 300 μL of protoplasts, evenly spread on the regeneration medium, open the lid 5-10 cm away from the 30W ultraviolet lamp, and vertically irradiate for 10-15 minutes to avoid white light interference. The best inactivation conditions are: 10 cm away from the ultraviolet lamp, irradiate for 10 minutes, 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, and mannitol 109.30 g / L. The above ingredients are sterilized 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 fusion promoter, and 1mL of different inactivated Acremonium FA1603 protoplasts and Cordyceps militaris CICC14015 protoplasts are mixed and centrifuged respectively, and 1mL of fusion promoter is added to resuspend, and after preheating in a 30℃ water bath for 5min, it is quickly placed in a 35℃ water bath shaker, 60 revs / min, and fusion is shaken for 30min. After the cell fusion ends, 2000r / min, 4℃ centrifugal 10min, supernatant is abandoned, and then washed three times with 0.6mol / L KCl to remove PEG. The protoplast precipitation obtained is diluted to 105 / mL concentration with 0.6mol / L KCl as an osmotic pressure stabilizer, and its diluent is coated on the regeneration culture medium, placed in a 25℃ constant temperature incubator, and cultured in dark, and the regeneration fusion bacterium colony is grown.

[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, and the fused colonies in the regeneration plate were picked. At the same time, the suspension of the parental strains was used as a control, and the fused colonies were directly screened and identified by PCR.

[0067] AT-F: 5-AACATACCTACGTTTCCCTCG-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 1m and 30s, 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 selected fusion colonies were screened by PCR. 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, and the culture conditions were: 24°C, initial pH 6.5, 250mL shake flask liquid volume of 50mL, inoculation volume 10mL, shake flask speed 200r / min, fermentation time 108h. After the fermentation, the mycelium was collected by centrifugation at 8000r / min at room temperature, dried at 60°C, weighed to calculate the biomass, and the polysaccharide and adenosine contents were measured.

[0075] The polysaccharide content of fermented mycelium was tested with reference to NYT 1676-2008 Determination of crude polysaccharide content in edible fungi. Cordycepin and adenosine in fermented mycelium were tested with reference to NYT 2116-2012 Determination of cordycepin and adenosine in cordyceps products by high performance liquid chromatography. The specific results are shown in Table 2. Note: Biomass refers to the total dry weight of the solids collected after centrifugation at 5000r / m for 30min after fermentation and 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, the No. 3 fusion strain with the best biomass, polysaccharide and adenosine content was selected and named as Acremonium strain F2652. Its characteristic sequence was recovered and sent for sequencing, phylogenetic analysis was performed, and slant storage and glycerol seed storage were performed. At the same time, microbial preservation and genetic stability testing were performed.

[0079] like Figure 2 As shown, characteristic sequence alignment and phylogenetic analysis showed that the similarity between strain F2652 and its parent FA1603 reached 99.5%, and its closest relative was Sarocladium terricola CBS243.59, with a similarity of 100%. It was confirmed that F2652 was 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, 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 once every 3 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 ability to synthesize polysaccharides remained basically stable, indicating that the strain had 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 powder 5.5%, wheat flour 0.5%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.75%, dipotassium hydrogen phosphate 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 1mg / 100ml, add tap water to 100%, adjust pH to 6.5-7.0, and sterilize at 121℃ for 25min according to conventional methods.

[0083] The above-mentioned amino acid nutrient mother solution includes: 0.6g of glycine, 0.5g of threonine, 0.3g of valine, 0.1g of leucine, 0.2g of isoleucine, 0.5g of phenylalanine, 0.2g of serine, 0.1g of proline, 0.06g of hydroxyproline, 0.04g of cysteine, 0.05g of tryptophan, 0.1g of methionine, 0.2g of lysine, and 0.1g of glutamic acid. The volume is fixed to 1000ml and stored at 4°C 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, 3.5 mg of zinc sulfate, fixed volume 1000 ml, stored at 4° C. for standby 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 Acremonium truncatum F2652 glycerol strain into a comprehensive PDA agar medium plate, 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, transfer it to a slant 18×180 mm test tube slant (F2), and 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 the eggplant bottle containing the millet seed fortified medium slant and cultured in an incubator at 25°C in the dark for 3 days to obtain the fortified seed culture, so that the culture spore concentration 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, 10mg vitamin B 12 50μg, pH 6, constant volume 1000ml, make nutrient solution, then weigh commercial millet in a mass volume ratio of 1:1, that is, 1000ml nutrient solution, weigh 1000g millet, mix evenly, steam in water for 45min, weigh 100g in each eggplant bottle after steaming, and sterilize at high temperature and high pressure according to conventional methods, 121℃, 20-30min. This is the prepared millet seed fortified medium with large surface area, good ventilation, and easy spore germination and mycelium growth.

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

[0093] The above millet seed enhanced culture was directly inoculated into a fermenter, and about 100g of millet seed culture was inoculated into a 30L fermenter containing 15L liquid culture medium for cultivation. In the initial stage of fermentation and cultivation, the fermenter ventilation was controlled to be 15L / min, the stirring speed was 150rpm, the temperature was controlled at 25°C, the initial reducing sugar concentration was controlled at 100g / L, and the reducing sugar concentration of the fermentation process was controlled to be 0.7g / 100mL by adding 70% glucose, the initial pH was 7.0, and the pH of the whole fermentation process was controlled to be 7.0 by adding ammonia water. After 24 hours of cultivation, the scattered light was turned on for 2-3 days, wherein the light intensity was 400Lx, and the mycelium and cordycepin were enhanced. The dissolved oxygen (DO) was controlled to be 30% throughout the process, and the fermentation cycle was 5 days. The fermentation was stopped to obtain a liquid fermentation culture. The liquid fermentation mycelium can be collected by centrifugation at 3000rpm for 15min, and dried at 60-80°C until the moisture is less than 10%, which is used to detect the biomass, mycelium polysaccharide and adenosine content.

[0094] The 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 mother solution 0.5%, trace element mother solution 10ml, vitamin B1 2mg / 100ml, vitamin B2 1mg / 100ml, vitamin B 12 1mg / 100ml, add tap water to 100%, adjust pH to 6.5-7.0, and sterilize at 121℃ for 25min according to conventional methods.

[0095] 4. Solid secondary fermentation of Acremonium truncatum 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 is controlled at 30°C, the humidity is 70%, the fermentation time is 5 days, the light intensity is 150Lx, and the light time is 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, and the solid culture medium 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 conditions of the test group of Example 2 were used to replace Acremonium FA1603 with Acremonium F2652, and 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 test group of Example 2 were used to operate according to steps 1, 2, 3 and 4, the mycelium and solid fermentation culture of the Cordyceps militaris strain CICC14015 were collected, and dried 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 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. Among them, the polysaccharide content in the fermentation mycelium and solid fermentation culture was detected with reference to NYT 1676-2008 Determination of crude polysaccharide content in edible fungi. Cordycepin and adenosine in the fermentation mycelium and solid fermentation culture were detected with reference to NYT 2116-2012 Determination of cordycepin and adenosine in cordyceps products by high performance liquid chromatography. Specific results are shown in Table 3-4. Note: Biomass refers to the total dry weight of the solids collected after centrifugation at 5000r / m for 30 minutes after the fermentation is completed and dried.

[0107] Table 3 Detection results of mycelium 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 the conditions of deep liquid fermentation, the mycelium 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] Embodiment 3,

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

[0114] The tissues and organs of piglets are not fully developed, and their immune, digestive and absorptive functions are immature. They are prone to diarrhea, growth stagnation, infection and even death. Acremonium culture has the functions of regulating immunity, antiviral and growth promotion. It can effectively reduce the incidence of animals, improve animal production performance and reproductive performance. It is a green, safe and efficient functional feed additive. This experiment aims to add different levels of Acremonium culture (cordycepin ≥ 1500μg / g) to the diet of piglets, aiming to study the effects of Acremonium culture on piglet production performance, immunity and antioxidant properties, and provide 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 feed respectively to study its effects on piglet growth performance, organ index, diarrhea rate, mortality rate, antioxidant indexes, immune performance and intestinal immune barrier.

[0117] 2. Experimental Animals

[0118] 240 healthy DLY weaned piglets of 35 days old and similar weight were selected and randomly divided into 4 treatments, with 6 replicates in each treatment and 10 pigs in each 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. Determination of 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 units of replicates (pens). Feed was stopped 12 h in advance on the 1st and 28th days of the formal experiment, and the animals 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-weight 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 and injected into a 10 mL centrifuge tube. After tilting for 15 minutes, the tube was centrifuged (3500 r / min) for 10 minutes, and the upper serum of the centrifuge tube was collected and stored in a -80°C refrigerator for testing.

[0130] 1) Serum antioxidant index

[0131] The total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content in serum as well as the activities of glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), Cu / Zn-SOD and catalase (CAT) were determined. The serum antioxidant indexes were determined using commercial kits from Nanjing Jiancheng Bioengineering Institute. The microplate reader used in this test was ThermoMultiskan FC. The specific operation method was in accordance with the kit instructions.

[0132] 2) Serum immune-related indicators

[0133] Serum immunoglobulin A (IgA), IgM, and IgG were measured 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 have significant differences between groups with different letters on their shoulders (P<0.05); no letters or the same letters indicate no significant differences, 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-weight 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 indexes of 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 weight gain and feed-to-weight ratio are the main indicators for evaluating animal growth performance, among which feed-to-weight ratio is a representative indicator for evaluating growth performance. Generally speaking, the lower the feed-to-weight ratio, the better the growth performance. This experiment found that adding 250g / t, 500g / t, and 1000g / t of Acremonium culture to the diet can significantly increase the average daily weight gain and feed-to-weight ratio of weaned piglets (P < 0.05), among which the addition of 500g / t of Acremonium culture has the most obvious effect. There was no significant difference between the groups with different levels of Acremonium culture (P > 0.05).

[0151] 5.2 Effects of Acremonium culture on immune function of 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 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). It 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 culture to the experimental diet can effectively improve various antioxidant indicators: superoxide dismutase (SOD), catalase (CAT), copper-zinc superoxide dismutase (CuZn-SOD), glutathione peroxidase (GSH-Px), among which CAT has a significant increase (P < 0.05). In addition, various research reports show that antioxidant compounds extracted from Cordyceps can also improve antioxidant capacity by increasing the expression of antioxidant enzymes including SOD, GSH-Px and CAT.

[0155] 6. Summary

[0156] (1) Adding 250 g / t, 500 g / t, and 1000 g / t of Acremonium culture (cordycepin ≥ 1500 μg / 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 500 g / 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 solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. The 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 preservation number of GDMCC No: 64892, a preservation date of October 9, 2024, a preservation unit of Guangdong Microbial Culture Collection Center, and an address of the preservation unit of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province; the rDNA sequence of Acremonium truncatum F2652 is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The Acremonium terrestris F2652 comprises a fermentation culture of an Acremonium terrestris fusion strain, and the Acremonium terrestris fusion strain is Acremonium terrestris 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, inoculating the activated strain into a millet seed culture medium for culturing 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, subjecting the fortified seed culture to liquid fermentation 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, thereby obtaining the fermentation culture of the Acremonium fusion strain.

4. The use according to claim 3, characterized in that In the step S1, the activation culture step is: using a comprehensive PDA agar medium to perform slant culture activation on the Acremonium fusion strain, inoculating glycerol bacteria to 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 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 / mL, and then transfer the eluate to the eggplant bottle containing the millet seed fortified culture medium slant, and culture 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 , make a fixed volume of 1000 ml to prepare a nutrient solution, then weigh millet in an amount with a mass volume ratio of 1:1, mix evenly, steam in water for 40-50 minutes, weigh 100 portions after steaming and put them in each eggplant bottle, sterilize according to a conventional method, and obtain the millet seed fortified culture medium.

7. The use according to claim 3, characterized in that In the 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 to be 10-20 L / min, the stirring speed is 150-200 rpm, the temperature is controlled to be 24-28° C., the initial reducing sugar concentration is controlled to be 80-100 g / L, and the reducing sugar concentration in the fermentation process is controlled to be 0.5-1 g / 100 mL by adding glucose, the initial pH is 6.0-7.5, and the pH in the whole fermentation process is controlled to be 6.0-7.5 by adding ammonia water, and the culture is carried out for 20-26 hours, and then the culture is carried out under the condition of a light intensity of 200-500 Lx for 2-3 days to carry out enhanced culture of mycelium and cordycepin, and the whole 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 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 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.; the solid fermentation product is dried until the moisture content is less than 10%, and crushed to 30-50 meshes 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 of the fermentation culture in the feed for piglet feeding.

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