Cordyceps militaris strain with high yield of ergothioneine and application thereof

Abstract

The application discloses a cordyceps militaris strain with high ergothioneine yield and application thereof, and belongs to the technical field of microorganisms and fermentation engineering. On the basis of the cordyceps militaris strain with high ergothioneine yield potential screened, the cordyceps militaris strain is subjected to ultraviolet mutagenesis treatment, and a cordyceps militaris mutant strain with stable heredity and high ergothioneine yield is screened. The application improves the yield of ergothioneine, cordycepin and adenosine in the cordyceps militaris strain through optimization and screening of precursor substances and based on precursor directed metabolic remodeling. The extract of the cordyceps militaris strain of the application can improve the survival rate of lactobacillus reuteri at high temperature of 50 DEG C, has a protective effect on cells, and active ingredients thereof can provide antioxidant protection to improve the survival ability of cells in extreme environments.

Description

Technical Field

[0001] This invention relates to the fields of microbial and fermentation engineering technology, specifically to a high-yield ergothioneine-producing Cordyceps militaris strain and its applications. Background Technology

[0002] Cordyceps militaris is a fungus used both as food and medicine, possessing various pharmacological effects such as immunomodulation, anti-tumor activity, and lipid regulation. It contains multiple active ingredients including ergothioneine (EGT), cordycepin, and adenosine. Ergothioneine, a rare natural sulfur-containing amino acid derivative, possesses powerful antioxidant, anti-inflammatory, and cell-protective functions, earning it the title of "longevity vitamin." Cordycepin exhibits anti-inflammatory and immunomodulatory functions, while adenosine enhances cellular stress tolerance by regulating energy metabolism and signaling pathways.

[0003] However, the content of ergothioneine in natural Cordyceps militaris is extremely low (usually <1 mg / g dry weight), and its synthesis efficiency is limited by the genetic characteristics of the strain, metabolic flux allocation, and defects in the culture process. In recent years, although the development of genetically engineered strains has made it possible to increase EGT production (such as overexpression of the EGT1 gene), adenosine is a direct precursor of cordycepin and cordycepic acid, and the synthesis of adenosine depends on the purine metabolic pathway; while the synthesis of ergothioneine requires a histidine-derived imidazole ring and cysteine ​​as precursors, which partially overlap with purine metabolism. Therefore, when microorganisms synthesize ergothioneine, cordycepin, and cordycepic acid simultaneously, there may be a certain degree of antagonism, leading to an imbalance in resource allocation and making it difficult to simultaneously increase the content of the three active ingredients. Moreover, such methods face regulatory restrictions and strict market demands for "naturally derived" products. Therefore, how to achieve efficient synthesis of ergothioneine through non-genetic engineering methods (such as mutagenesis screening combined with metabolic regulation) while maintaining synergistic high yield of cordycepin and cordycepin adenosine has become a core technical challenge that the industry urgently needs to overcome. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to provide a high-yield Cordyceps militaris strain of ergothioneine and its applications.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a Cordyceps militaris strain that produces high levels of ergothioneine. This strain was deposited on May 28, 2025, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) with accession number CCTCC NO:M 20251217.

[0007] The Cordyceps militaris strain of this invention was obtained by ultraviolet mutagenesis of a wild-type Cordyceps militaris strain and named Cordyceps militaris FHYHC. Compared with the wild-type Cordyceps militaris strain, its ergothioneine production is greatly increased, and it is a genetically stable Cordyceps militaris mutant strain with high ergothioneine production.

[0008] In a second aspect, the present invention provides mycelium obtained by culturing the above-mentioned Cordyceps militaris strain.

[0009] In a third aspect, the present invention provides fruiting bodies obtained by cultivating the above-mentioned Cordyceps militaris strain.

[0010] The mycelium and fruiting body of Cordyceps militaris represent two distinct developmental stages in its life cycle, in which:

[0011] The mycelium is a white or light-colored filamentous body composed of branched hyphae; it is obtained through liquid submerged fermentation or solid culture, with a short culture cycle (3-7 days), making it suitable for large-scale industrial production.

[0012] The fruiting body is a mature reproductive structure, orange-red or yellow in color, with a rod-shaped or coral-shaped form. Under natural conditions, it grows from the insect pupa. Artificial cultivation requires simulating the natural environment, has a long cultivation cycle (1-3 months), and is technically challenging.

[0013] The content of active ingredients in the fruiting body is usually higher than that in the mycelium, but the cultivation of the fruiting body is technically difficult and costly; depending on actual needs, either the mycelium or the fruiting body can be selected as the application form of the Cordyceps militaris strain of this invention.

[0014] In a fourth aspect, the present invention provides a microbial agent containing the aforementioned Cordyceps militaris strain that produces high levels of ergothioneine.

[0015] Preferably, the Cordyceps militaris strain in the microbial agent exists in the form of cultured live bacteria, bacterial suspension, or fermentation broth.

[0016] A fifth aspect of the present invention provides a method for synergistically increasing the yields of ergothioneine, cordycepin, and cordycepin in Cordyceps militaris strains based on precursor-directed metabolic remodeling, comprising the following steps:

[0017] The seed culture of the above-mentioned Cordyceps militaris strain was inoculated into a liquid fermentation medium containing precursor substances for fermentation culture; the precursor substances consisted of methionine, cysteine ​​and adenosine.

[0018] Preferably, the liquid fermentation medium is composed of: 40 g / L sucrose, 20 g / L peptone, 2 g / L magnesium sulfate, 1.5 g / L potassium dihydrogen phosphate, and pH 7; the final concentrations of methionine, cysteine, and adenosine after addition are all 2 mM.

[0019] The preferred fermentation conditions are: temperature 20-30℃, rotation speed 120-180r / min, and culture in the dark for 5-7 days.

[0020] In a sixth aspect, the present invention provides the use of the extract of the above-mentioned Cordyceps militaris strain in the preparation of products that enhance the resistance of Lactobacillus reuteri to high temperature stress.

[0021] In the above applications, the extract of the Cordyceps militaris strain is prepared by the following method:

[0022] The seed culture of the above-mentioned Cordyceps militaris strain was inoculated into a liquid fermentation medium containing precursor substances for fermentation culture, and the mycelium was collected by centrifugation after fermentation.

[0023] The mycelium was dissolved in an aqueous methanol solution, extracted by ultrasonication, centrifuged, and the supernatant was collected to prepare the extract of Cordyceps militaris strain.

[0024] Preferably, the raw material composition of the liquid fermentation medium containing the precursor substance is as follows:

[0025] Sucrose 40g / L, peptone 20g / L, magnesium sulfate 2g / L, potassium dihydrogen phosphate 1.5g / L, methionine 2mM, cysteine ​​2mM, adenosine 2mM.

[0026] Preferably, the conditions for ultrasonic extraction are: 40℃, 30min, and 300W power.

[0027] The beneficial effects of this invention are:

[0028] (1) Based on the Cordyceps militaris strain with high ergothioneine production potential obtained by screening, the present invention performs ultraviolet mutagenesis on it to obtain a Cordyceps militaris mutant strain that can stably inherit and produce high ergothioneine. The ergothioneine production in its mycelium is 8.11 times that of the wild-type Cordyceps militaris strain.

[0029] (2) This invention improves the yield of ergothionein, cordycepin and cordycepin in Cordyceps militaris mutant strains by optimizing the screening of precursor substances and synergistically improving the yield of ergothionein, cordycepin and cordycepin adenosine based on precursor-directed metabolic remodeling.

[0030] (3) The extract of the Cordyceps militaris strain of the present invention can improve the survival rate of Lactobacillus reuteri at 50°C and has a protective effect on cells. Its active ingredients can provide antioxidant protection and improve the cell’s ability to survive in extreme environments. Attached Figure Description

[0031] Figure 1 Six activated slant cultures of Cordyceps militaris strains preserved in the laboratory.

[0032] Figure 2Freeze-dried mycelium powder from six Cordyceps militaris strains preserved in the laboratory after fermentation.

[0033] Figure 3 The lethality curves of mycelia of Cordyceps militaris strains 2-1 and 2-6 induced by ultraviolet mutagenesis.

[0034] Figure 4 Liquid chromatography results of ergothionein production after fermentation of Cordyceps militaris strains 2-1 (wild type), 2-1-6 (mutant), and 2-1-6 (directed remodeling).

[0035] Figure 5 Liquid chromatography results of cordycepin and cordycepin yields after fermentation of Cordyceps militaris strains 2-1 (wild type), 2-1-6 (mutant), and 2-1-6 (directed remodeling). Detailed implementation method:

[0036] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0037] As mentioned earlier, ergothioneine, cordycepin, and cordycepin are three active ingredients in Cordyceps militaris, with broad market application prospects. However, the content of ergothioneine in natural Cordyceps militaris is extremely low. To increase the yield of ergothioneine, existing technologies include constructing genetically engineered strains that produce high yields of ergothioneine through genetic engineering. However, these genetically engineered strains face regulatory restrictions and strict market demands for "naturally derived" products. Moreover, the synthesis and metabolism of these three active ingredients may partially overlap, potentially leading to antagonism when Cordyceps militaris simultaneously synthesizes these three substances, making it difficult to simultaneously increase the content of all three active ingredients.

[0038] In view of this, the present invention first conducted a preliminary screening of the ability of Cordyceps militaris in the strain library to produce ergothioneine. A Cordyceps militaris strain with a high ergothioneine production was selected as the starting strain. The starting strain was subjected to ultraviolet mutagenesis treatment. From many mutant strains, a Cordyceps militaris mutant strain 2-1-6 with a significantly increased ergothioneine production was selected.

[0039] Furthermore, in order to achieve a synergistic increase in the content of the three active ingredients, ergothioneine, cordycepin, and cordycepin, this invention uses precursor-directed metabolic remodeling technology to add low-cost precursor substances methionine, cysteine, and adenosine to the liquid fermentation medium, thereby simultaneously increasing the content of the three active ingredients, ergothioneine, cordycepin, and cordycepin, in the mycelium of the Cordyceps militaris mutant strain 2-1-6.

[0040] Furthermore, the mycelium of the Cordyceps militaris mutant strain 2-1-6, cultured based on precursor-directed metabolic remodeling technology, was subjected to ultrasonic extraction to obtain an extract that increased the survival rate of Lactobacillus reuteri at 50°C from 22.6% to 62.1%, providing an innovative solution for the industrial production of natural antioxidants.

[0041] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions of this application will be described in detail below with reference to specific embodiments. Unless otherwise specified, experimental conditions not detailed in the embodiments are generally based on conventional conditions or conditions recommended by the reagent company; reagents, consumables, etc., used in the following embodiments can be obtained commercially unless otherwise specified.

[0042] Example 1: Obtaining and preserving a Cordyceps militaris strain that produces high levels of ergothioneine.

[0043] 1. Materials and Methods

[0044] 1.1 Strains

[0045] The Cordyceps militaris strains preserved in the strain bank of the Science and Technology Innovation Center of Shandong Phoenix Biotechnology Co., Ltd. were numbered as follows: Cordyceps militaris 2-1, Cordyceps militaris 2-2, Cordyceps militaris 2-3, Cordyceps militaris 2-4, Cordyceps militaris 2-5 and Cordyceps militaris 2-6.

[0046] 1.2 Culture medium

[0047] PDA medium (modified): potato starch 20g / L, glucose 20g / L, agar 15g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate 1.5g / L, vitamin B2 0.01g / L, natural pH.

[0048] Liquid seed culture medium: glucose 30 g / L, peptone 5 g / L, KH2PO4 1 g / L, MgSO4 0.5 g / L, pH 7.

[0049] Liquid fermentation medium: sucrose 40 g / L, peptone 20 g / L, magnesium sulfate 2 g / L, potassium dihydrogen phosphate 1.5 g / L, pH 7.

[0050] 1.3 Experimental Methods

[0051] 1.3.1 Cordyceps militaris mycelium culture

[0052] Activation of Cordyceps militaris strains: Under aseptic conditions, open the slant agar of strains numbered Cordyceps militaris 2-1, 2-2, 2-3, 2-4, 2-5, and 2-6. Pick a small amount of bacterial cells (with a small amount of culture medium) near a flame and quickly streak them onto new activated PDA medium (modified) slant agar using the serpentine streak method. Invert the inoculated slant agar and incubate at 25°C in the dark for 7 days until white, fluffy colonies completely cover the slant.

[0053] Preparation of seed culture: Cut 3 mycelial blocks (approximately 1 cm each) from the activated PDA medium (modified) slant. 2 The mycelium was inoculated into Erlenmeyer flasks containing 200 mL of liquid seed culture medium. The flasks were then placed in a shaker and incubated at 25°C and 150 rpm in the dark for 7 days until mycelial balls of approximately 3 mm in diameter were formed, thus preparing the seed culture.

[0054] Fermentation culture: The prepared seed culture was inoculated into Erlenmeyer flasks containing 1.5 L of liquid fermentation medium at a 5% inoculation rate (volume ratio). Each flask was placed in a shaker at 25°C and 150 rpm for 7 days in the dark, until the mycelial ball concentration reached a level that visually indicated uniform turbidity and filled the liquid fermentation medium. After fermentation, the flasks were centrifuged at 5000 rpm for 10 min to separate the mycelium and supernatant. The mycelium was collected. The mycelium was washed three times with sterile water and then freeze-dried. After freeze-drying, the mycelium was pulverized and passed through a 60-mesh sieve to obtain Cordyceps militaris mycelium freeze-dried powder.

[0055] 1.3.2 Detection of Ergothionein Content in Cordyceps militaris Mycelium

[0056] The ergothioneine content in the freeze-dried Cordyceps militaris mycelium powder obtained in section 1.3.1 above was determined as follows:

[0057] Extraction method: To ensure the accuracy of the results, three replicates were set up for each strain. 0.2 g of the above-mentioned lyophilized Cordyceps militaris mycelium powder was weighed and added to 25 mL of 1% methanol-water mixture. Ultrasonic-assisted extraction was used under the following conditions: 40℃ for 30 minutes at 300W. After extraction, the mixture was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected to obtain a crude ergothioneine extract. This crude extract was filtered through a 0.22 μm microporous membrane, and the ergothioneine content was determined by high-performance liquid chromatography (HPLC).

[0058] Standard stock solution: Accurately weigh 10 mg of ergothioneine standard (purchased from Yuanye Biotechnology Co., Ltd.), and dilute to 100 mL with 1% methanol and water in a brown volumetric flask to prepare a 100 μg / mL standard stock solution. Store at 4°C protected from light.

[0059] Standard working solutions: The standard stock solution was serially diluted to prepare standard working solutions with concentration gradients of 5, 10, 20, 50, and 100 μg / mL for establishing standard curves.

[0060] 1% Methanol-Water: Add 1 mL of anhydrous methanol to 99 mL of deionized water and mix well.

[0061] The method for determining ergothioneine content by HPLC is as follows: A C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase was: Phase A: methanol (100%), Phase B: 1% methanol-water; flow rate: 1.0 mL / min; column temperature: 30℃; detection wavelength: 257 nm; injection volume: 10 μL. The gradient program is as follows:

[0062]

[0063]

[0064] 1.3.3 Ultraviolet Mutagenesis Breeding

[0065] Two Cordyceps militaris strains with high ergothioneine yield were selected and subjected to ultraviolet mutagenesis treatment to breed mutant strains with high ergothioneine yield. Details are as follows:

[0066] Preparation of mycelial culture: Cordyceps militaris strains were inoculated onto modified PDA medium, and then the medium was incubated in a 25℃ incubator in the dark for 7 days. Three mycelial blocks (each approximately 1 cm) were then collected. 2 Place the bacterial culture in 10.0 mL of sterile water (containing sterile glass beads with a diameter of approximately 4–5 mm), and shake on a shaker at 150 rpm for 1 hour to scrape off the mycelia from the surface of the agar block. Centrifuge to remove the supernatant and collect the mycelia, finally obtaining 5 mL of bacterial culture, which is temporarily stored at 4°C for later use.

[0067] UV mutagenesis: Turn on the UV lamp in the clean bench and preheat for at least 30 minutes to stabilize the UV light wave (253.7nm). Dilute the Cordyceps militaris bacterial solution to 10. -3 Each gradient was set up with 3 replicates. 100 μL of the diluted solution was used for UV irradiation at time gradients of 0, 3, 6, 9, 12, 15, 18, and 21 min. After irradiation, the bacterial suspension was poured into modified PDA medium using the pouring method under dark conditions and incubated at 25°C for 7 days until single colonies appeared. The bacterial suspension without UV treatment was used as a control. The mutagenic lethality rate of the strains at different mutagenic time points was calculated.

[0068] Lethality = (1 - number of viable bacteria in irradiated bacterial solution / number of viable bacteria in unirradiated bacterial solution) × 100%.

[0069] Based on the lethality curve of UV-induced Cordyceps militaris, the irradiation duration with a UV lethality of approximately 80% was selected as the optimal irradiation time. Strains treated with the optimal irradiation time were selected, and Cordyceps militaris mycelium was cultured according to method 1.3.1. Ergothionein content was then determined according to method 1.3.2.

[0070] 2 Results

[0071] 2.1 Screening of high-yield Cordyceps militaris strains yielding ergothioneine

[0072] This invention involved slant activation of six Cordyceps militaris strains (2-1, 2-2, 2-3, 2-4, 2-5, and 2-6) preserved in the laboratory. After activation, the Cordyceps militaris colonies were white, with fluffy colonies completely covering the slant. The results are as follows: Figure 1 As shown, the six Cordyceps militaris strains exhibited good activity. Subsequently, after liquid seed culture preparation (200 mL), liquid fermentation culture (1.5 L), centrifugation, freeze-drying, and pulverization through a 60-mesh sieve, Cordyceps militaris mycelium freeze-dried powder was obtained. The results are as follows... Figure 2 As shown.

[0073] The yields of ergothioneine from each Cordyceps militaris strain were determined and are shown in Table 1.

[0074] Table 1: Ergothioneine production by different Cordyceps militaris strains

[0075]

[0076]

[0077] Table 1 shows that the mycelial ergothioneine yield of Cordyceps militaris strain 2-1 was 0.240 mg / g, and that of Cordyceps militaris strain 2-6 was 0.130 mg / g. Cordyceps militaris strains 2-1 and 2-6 were the two strains with relatively high ergothioneine yields, therefore these two strains were selected for subsequent experiments.

[0078] 2.2 Ultraviolet Selection of Cordyceps militaris Strains

[0079] The lethality curves of UV-mutated Cordyceps militaris strains 2-1 and 2-6 are shown below. Figure 3As shown, the lethality of Cordyceps militaris increased with increasing irradiation time, and the lethality curve tended to flatten after 15 minutes of irradiation. Studies have shown that a lethality rate that is too low is not conducive to screening for positive mutant strains, while a lethal dose that is too high leads to an increased negative mutation rate. Therefore, based on the lethality curve of UV-induced Cordyceps militaris, this study selected an irradiation time with a UV lethality of approximately 80% as the optimal irradiation time, i.e., 15 minutes of UV mutagenesis. The lethality rate of strain 2-1 was 78.95%, and that of strain 2-6 was 77.12%. After 15 minutes of UV irradiation, 20 mutant strains were randomly selected for liquid fermentation, and the ergothioneine content in their mycelium was measured.

[0080] The ergothionein yield of the mutant strain of Cordyceps militaris 2-1 after UV mutagenesis is shown in Table 2.

[0081] Table 2: Ergothionein yield determination of Cordyceps militaris 2-1 and mutant strains

[0082]

[0083] The ergothionein yield of the mutant strains of Cordyceps militaris 2-6 after UV mutagenesis treatment is shown in Table 3.

[0084] Table 3: Ergothionein yield determination of Cordyceps militaris strains 2-6 and mutant strains

[0085]

[0086] The results showed that the mutant strain with the highest ergothioneine yield after UV mutagenesis of Cordyceps militaris strain 2-1 was Cordyceps militaris strain 2-1-6 (1.947 mg / g, an increase of 711.25% compared with Cordyceps militaris strain 2-1), and the mutant strain with the highest ergothioneine yield after UV mutagenesis of Cordyceps militaris strain 2-6 was Cordyceps militaris strain 2-6-3 (0.647 mg / g, an increase of 397.69% compared with Cordyceps militaris strain 2-6).

[0087] Based on the above, the mutant strain Cordyceps militaris 2-1-6, which underwent UV mutagenesis, was selected as the preservation strain. Its genetic stability was investigated. One subculture was counted as one generation, and a total of 10 generations were conducted. The same fermentation and culture conditions, sample treatment methods, and ergothioneine content determination methods were used for each generation. There was no significant difference in ergothioneine yield among the generations, demonstrating that the mutant strain Cordyceps militaris 2-1-6 has genetic stability.

[0088] The UV-induced mutant strain Cordyceps militaris 2-1-6 was named Cordyceps militaris FHYHC and deposited at the China Center for Type Culture Collection. The deposit information is as follows:

[0089] Culture name: Cordyceps militaris FHYHC;

[0090] Accession number: CCTCC NO:M 20251217;

[0091] Preservation date: May 28, 2025.

[0092] Example 2: Synergistic enhancement of ergothionein, cordycepin, and cordycepin yield in Cordyceps militaris strains based on precursor-directed metabolic remodeling

[0093] 1. Materials and Methods

[0094] 1.1 Strains

[0095] Wild Cordyceps militaris strain 2-1, mutant Cordyceps militaris strain 2-1-6;

[0096] 1.2 Experimental Materials

[0097] Methionine, cysteine, adenosine, arginine, aspartic acid, glutamic acid, cordycepin (chromatographic grade), and cordycepin (chromatographic grade) were purchased from Yuanye Biotechnology Co., Ltd.

[0098] Liquid seed culture medium: glucose 30 g / L, peptone 5 g / L, KH2PO4 1 g / L, MgSO4 0.5 g / L, pH 7.

[0099] Liquid fermentation medium: sucrose 40 g / L, peptone 20 g / L, magnesium sulfate 2 g / L, potassium dihydrogen phosphate 1.5 g / L, pH 7.

[0100] 1.3 Experimental Methods

[0101] 1.3.1 Effects of precursor-directed metabolic remodeling on ergothionein production

[0102] Precursor addition schemes were designed: Methionine, cysteine, adenosine, arginine, aspartic acid and glutamic acid were selected as precursors in this experiment, and different precursor addition schemes were designed (see Table 4) and added to the liquid fermentation medium.

[0103] Table 4: Precursor Addition Scheme

[0104]

[0105] Note: The CK group is a liquid fermentation medium without the addition of precursor substances.

[0106] Mycelium preparation: The *Cordyceps militaris* 2-1-6 obtained in Example 1 after UV mutagenesis for 15 min was inoculated into a 500 mL Erlenmeyer flask (containing 200 mL of liquid seed culture medium) via a slant culture tube for activation, preparing a seed culture. The prepared seed culture was inoculated at a 5% inoculation rate (volume ratio) into 1.5 L of liquid fermentation medium containing different precursor addition schemes for fermentation at 25℃, a rotation speed of 150 r / min, and incubated in the dark for 7 days, observing the formation of mycelial balls. After fermentation, the mycelium was collected by centrifugation, washed three times with sterile water, and then freeze-dried. After freeze-drying, the mycelium was pulverized and passed through a 60-mesh sieve. The ergothioneine yield of the obtained *Cordyceps militaris* mycelium freeze-dried powder was determined according to the method in 1.3.2 of Example 1.

[0107] 1.3.2 Effects of precursor-directed metabolic remodeling on the production of cordycepin and cordycepin adenosine

[0108] Cordycepin and cordycepin are recognized as the two core active products of Cordyceps militaris, both possessing clear biological activity and medicinal value. This invention further investigated the effects of precursor culture protocols on the cordycepin and cordycepin content produced by wild-type Cordyceps militaris strain 2-1 and mutant Cordyceps militaris strain 2-1-6.

[0109] Extraction Method: To ensure the accuracy of the results, three replicates were set up for each group of mycelia to be tested. 0.1 g of lyophilized mycelium powder obtained from the fermentation of wild-type Cordyceps militaris strain 2-1, lyophilized mycelium powder obtained from the fermentation of mutant strain Cordyceps militaris strain 2-1-6, and lyophilized mycelium powder obtained from the fermentation of mutant strain 2-1-6 after precursor-directed metabolic remodeling (Scheme 2 in Table 4) were accurately weighed and added to 10 mL of 50% methanol aqueous solution. Ultrasonic extraction was performed under the following conditions: 40℃ for 30 minutes, power 250W. After extraction, the mixture was centrifuged at 12,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter membrane, and the contents of cordycepin and cordycepin adenosine were determined.

[0110] The contents of cordycepin and cordycepin adenosine in the lyophilized Cordyceps militaris mycelium powder of each group were determined as follows: A C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used. Mobile phase: Phase A: 0.1% formic acid aqueous solution, Phase B: acetonitrile (100%); Flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 260 nm (maximum absorption peaks of cordycepin and adenosine). The gradient program is as follows:

[0111]

[0112] 2 Results

[0113] 2.1 Effects of precursor-directed metabolic remodeling on the yield of ergothionein from Cordyceps militaris

[0114] After culturing at 25℃, 150rpm, and in the dark for 7 days, the effects of different precursor addition schemes on the ergothionein yield of Cordyceps militaris 2-1-6 are shown in Table 5. The percentage increase is calculated as (scheme content - CK content) / CK content × 100%.

[0115] Table 5: Effects of different precursor addition schemes on the yield of Cordyceps militaris 2-1-6 ergothionein

[0116]

[0117] The above results indicate that the ergothioneine content in the freeze-dried mycelium powder of the natural Cordyceps militaris strain 2-1 was 0.240 mg / g, and the ergothioneine content in the freeze-dried mycelium powder of Cordyceps militaris strain 2-1-6 was 1.947 mg / g. The highest ergothioneine content (3.833 mg / g) was found in the freeze-dried mycelium powder of Cordyceps militaris strain 2-1-6 after precursor-directed metabolic remodeling fermentation according to scheme 2. The liquid chromatography determination results for ergothioneine are as follows: Figure 4 As shown.

[0118] 2.2 Effects of precursor-directed metabolic remodeling on the yields of 2-1-6 cordycepin and cordycepin in Cordyceps militaris

[0119] The results are as follows Figure 5 As shown, the results indicate that the contents of cordycepin and cordycepin in the freeze-dried mycelium powder obtained after fermentation of the natural Cordyceps militaris strain 2-1 were 1.5 mg / g and 8.2 mg / g, respectively. The contents of cordycepin and cordycepin in the freeze-dried mycelium powder obtained after fermentation of the Cordyceps militaris mutant strain 2-1-6 were 4.5 mg / g and 12.2 mg / g, respectively. After fermentation of the precursor-directed metabolic remodeling strain 2-1-6, the contents of cordycepin and cordycepin in the freeze-dried mycelium powder were 6.1 mg / g and 16.5 mg / g, respectively.

[0120] In summary, the *Cordyceps militaris* strain 2-1-6, after UV mutagenesis, may carry a highly active ergothioneine synthase gene, enabling it to effectively utilize exogenous precursors for ergothioneine synthesis. This overcomes the precursor utilization bottleneck of conventional strains. Methionine and cysteine ​​provide sulfur sources for ergothioneine synthesis, while adenosine, as a purine metabolism precursor, directly participates in the synthesis of cordycepin and cordycepin adenosine. The synergistic supplementation of these three substances can alleviate metabolic pathway competition, breaking through the traditional bottleneck of yield enhancement from a single precursor and achieving efficient and targeted EGT synthesis. After UV mutagenesis and targeted metabolic remodeling of precursor substances, *Cordyceps militaris* strain 2-1-6 can synergistically increase the yield of ergothioneine, cordycepin, and cordycepin adenosine, demonstrating potential for industrial-scale production.

[0121] Example 3: Effect of Cordyceps militaris strain extract on the survival rate of Lactobacillus reuteri under high temperature stress

[0122] 1. Materials and Methods

[0123] 1.1 Strains

[0124] The Cordyceps militaris strain 2-1-6 obtained in Example 1 of this invention and Lactobacillus reuteri LY0032 are patent strains preserved in the laboratory with accession number CCTCC NO: M 2015341, and are recorded in patent CN 106420847 A.

[0125] 1.2 Culture medium

[0126] MRS medium (modified): by weight percentage, peptone 1%, beef extract 1%, yeast extract 0.5%, glucose 2%, dipotassium hydrogen phosphate 0.2%, sodium acetate 0.5%, ammonium citrate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.05%, Tween-80 0.1%, pH 6.5.

[0127] 1.3 Methods

[0128] 1.3.1 Preparation of Cordyceps militaris extract

[0129] The lyophilized powder of Cordyceps militaris 2-1-6 mycelium was prepared according to the method in 1.3.1 of Example 1 of this invention. 0.2 g of the lyophilized powder of Cordyceps militaris 2-1-6 mycelium was accurately weighed and dissolved in 25 mL of 1% methanol-water. An ultrasonic-assisted extraction method was used to prepare the Cordyceps militaris extract. The ultrasonic extraction conditions were: 40℃ for 30 min, power 300 W. After extraction, the extract was centrifuged at 12,000 rpm for 10 min, and the supernatant was collected to obtain a crude ergothioneine extract. This crude extract was filtered through a 0.22 μm microporous membrane for later use.

[0130] 1.3.2 Culture and treatment of Lactobacillus reuteri

[0131] Bacterial activation: Lactobacillus reuteri LY0032 (CCTCC NO: M 2015341) was inoculated into modified MRS medium and cultured at 37°C for 18 hours under static conditions. The bacterial culture was then transferred to sterile centrifuge tubes and centrifuged at 4000 rpm for 5 minutes. The bacterial cells were collected, mixed with sterile physiological saline, and adjusted to a viable cell count concentration of 1.0 × 10⁻⁶. 9 CFU / mL was used to obtain Lactobacillus reuteri bacterial culture.

[0132] High-temperature stress experiment: This experiment was divided into 5 groups:

[0133] Control group: Take 0.5 mL of Lactobacillus reuteri bacterial culture and add 4.5 mL of 1% methanol-water mixture and shake to mix well;

[0134] Experimental group 1 (wild group): Take 0.5 mL of Lactobacillus reuteri bacterial solution and add 4.5 mL of Cordyceps militaris natural strain 2-1 mycelial extract, shake and mix well;

[0135] Experimental group 2 (mutant group): Take 0.5 mL of Lactobacillus reuteri bacterial solution and add 4.5 mL of Cordyceps militaris mutant strain 2-1-6 mycelial extract, shake and mix well;

[0136] In experimental group 3 (precursor-directed metabolic remodeling group), 0.5 mL of Lactobacillus reuteri bacterial solution was added to 4.5 mL of mycelial extract from the precursor-directed metabolic remodeling fermentation of Cordyceps militaris mutant strain 2-1-6, and the mixture was shaken and mixed.

[0137] Positive control: Add 4.5 mL of known protectant (10% skim milk) to 0.5 mL of Lactobacillus reuteri bacterial culture and vortex to mix.

[0138] The mixtures were placed in a constant temperature water bath at 50°C for 1 hour, and then cooled to room temperature for viable count.

[0139] 1.3.3 Survival rate detection

[0140] Plate count method: The above bacterial cultures after high-temperature treatment were serially diluted (10⁻⁶ ppm). -6 -10 -8 Each group was set up with 3 replicates, spread on MRS agar plates, anaerobic incubated at 37℃ for 48h, colonies were counted (CFU / mL), and the survival rate range was calculated.

[0141] 2. Results

[0142] The survival rates of Lactobacillus reuteri in each group are shown in Table 6.

[0143] Table 6: Survival rate of Lactobacillus reuteri under high temperature stress

[0144]

[0145]

[0146] The results showed that compared with the control group, the survival rate of *Lactobacillus reuteri* in experimental groups 1-3 was improved under high-temperature stress. The effects were more significant in experimental groups 2 and 3, presumably because the extracts from groups 2 and 3 contained higher levels of active ingredients such as ergothioneine, cordycepin, and cordycepin, thus enhancing the *Lactobacillus reuteri*'s resistance to high-temperature stress. In particular, the effect of experimental group 3 was close to that of the positive control group, providing an innovative solution for the industrial production of natural antioxidants.

[0147] In summary, this invention significantly increases the ergothioneine content (3.833 mg / g) in Cordyceps militaris using a non-genetic engineering approach through UV mutagenesis combined with precursor-directed metabolic remodeling technology, representing a major breakthrough in non-genetic engineering methods. Simultaneously, the yields of cordycepin (6.1 mg / g) and cordycepin adenosine (16.5 mg / g) are also increased, achieving simultaneous yield increases for multiple components for the first time. Furthermore, the low-cost precursor feedstock (methionine, cysteine, and adenosine) and conventional fermentation process demonstrate potential for large-scale scalability.

[0148] The extract of the Cordyceps militaris strain of this invention is rich in active ingredients such as ergothioneine, cordycepin, and cordycepin, making it suitable for unique applications (e.g., natural health foods). The synergistic effect of these three components increases the survival rate of Lactobacillus reuteri at 50°C from 22.6% to 62.1%, providing an innovative solution for the industrial production of natural antioxidants.

[0149] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A Cordyceps militaris strain with high ergothioneine yield, which is preserved under the accession number CCTCC NO: M 20251217.

2. Mycelium obtained by culturing the Cordyceps militaris strain of claim 1.

3. Fruiting body obtained by cultivating the Cordyceps militaris strain of claim 1.

4. An inoculant characterized in that, The bacterial agent contains the Cordyceps militaris strain of claim 1.

5. The bacterial agent of claim 4, wherein In the bacterial agent, the Cordyceps militaris strain exists in the form of cultured live bacteria, bacterial suspension or fermentation broth.

6. A method for synergistically improving the production of cordycepin, cordycepin and adenosine in Cordyceps militaris strains based on precursor-directed metabolic remodeling, characterized by, The method comprises the following steps: Seed liquid of the Cordyceps militaris strain of claim 1 is inoculated into a liquid fermentation medium added with precursor substances for fermentation culture; the precursor substances consist of methionine, cysteine and adenosine.

7. The method of claim 6, wherein, The liquid fermentation medium comprises 40 g / L sucrose, 20 g / L peptone, 2 g / L magnesium sulfate, 1.5 g / L potassium dihydrogen phosphate, and has a pH value of 7; the final concentration of methionine, cysteine and adenosine after addition is 2 mM.

8. The method of claim 6, wherein, The fermentation culture conditions are as follows: temperature 20-30℃, rotation speed 120-180 r / min, and light-avoiding culture for 5-7 days.

9. Use of an extract of the Cordyceps militaris strain of claim 1 for the preparation of a product for increasing the resistance of Lactobacillus reuteri to high temperature stress, characterized in that, The Lactobacillus reuteri is Lactobacillus reuteri LY0032 preserved under the accession number CCTCC NO: M 2015341.

10. Use according to claim 9, characterized in that, The extract of the Cordyceps militaris strain is prepared by the following method: Seed liquid of the Cordyceps militaris strain of claim 1 is inoculated into a liquid fermentation medium added with precursor substances for fermentation culture, and mycelium is collected by centrifugation after fermentation; The mycelium is dissolved in a methanol aqueous solution, ultrasonic extraction is performed, centrifugation is performed, and the supernatant is collected to prepare the extract of the Cordyceps militaris strain; The raw material composition of the liquid fermentation medium added with precursor substances is as follows: 40 g / L sucrose, 20 g / L peptone, 2 g / L magnesium sulfate, 1.5 g / L potassium dihydrogen phosphate, 2 mM methionine, 2 mM cysteine and 2 mM adenosine.

11. Use according to claim 10, characterized in that, The ultrasonic extraction conditions are as follows: 40℃, 30 min, and power 300 W.

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