Cordyceps militaris strain with high yield of ergothioneine and application thereof

Abstract

The invention discloses a cordyceps militaris strain with high yield of ergothioneine and application of the cordyceps militaris strain, and belongs to the technical field of microorganisms and fermentation engineering. On the basis of the screened cordyceps militaris strain with high-yield ergothioneine potential, the cordyceps militaris strain is subjected to ultraviolet mutagenesis treatment, and a cordyceps militaris mutant strain which can be stably inherited and has high-yield ergothioneine is obtained through screening. According to the method, the yield of ergothioneine, cordycepin and cordyceps adenosine in the cordyceps militaris strain is synergistically improved on the basis of precursor directional metabolism remodeling by optimizing and screening precursor substances. The extracting solution of the cordyceps militaris strain can improve the survival rate of lactobacillus reuteri at the high temperature of 50 DEG C and has a protection effect on cells, and the active ingredients of the cordyceps militaris strain can provide an antioxidant protection effect to improve the viability of the cells in an extreme environment.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms and fermentation engineering, and in particular to a Cordyceps militaris strain with high ergothioneine production and application thereof. Background Art

[0002] Cordyceps militaris is a medicinal and edible fungus with multiple pharmacological effects, including immunomodulatory, anti-tumor, and lipid-regulating properties. It contains several active ingredients, including ergothioneine (EGT), cordycepin, and adenosine. Ergothioneine, a rare natural sulfur-containing amino acid derivative, is known as the "longevity vitamin" for its potent antioxidant, anti-inflammatory, and cytoprotective properties. Cordycepin exhibits anti-inflammatory and immunomodulatory properties. 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 distribution and culture process defects. In recent years, although the development of genetically engineered strains has provided the possibility of increasing EGT production (such as overexpressing the EGT1 gene), adenosine is the direct precursor of cordycepin and cordycepin adenosine, and the synthesis of adenosine depends on the purine metabolic pathway; and the synthesis of ergothioneine requires histidine-derived imidazole ring and cysteine ​​as precursors, which partially overlaps with purine metabolism. Therefore, when microorganisms synthesize ergothioneine, cordycepin and cordycepin adenosine at the same time, there may be a certain degree of antagonism, which causes an imbalance in resource allocation and makes it difficult to increase the content of the three active ingredients of ergothioneine, cordycepin and cordycepin adenosine at the same time. Moreover, such methods face regulatory restrictions and strict market demand for "natural source" products. Therefore, how to achieve efficient synthesis of ergothioneine through non-genetic engineering means (such as mutagenesis screening combined with metabolic regulation) while maintaining the synergistic high yield of cordycepin and cordycepin adenosine has become a core technical problem that the industry urgently needs to break through. Summary of the Invention

[0004] In view of the problems existing in the above-mentioned prior art, the object of the present invention is to provide a Cordyceps militaris strain with high ergothioneine yield and application thereof.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A first aspect of the present invention provides a Cordyceps militaris strain having a high yield of thioneine, which has been deposited in China Center for Type Culture Collection (abbreviated as CCTCC, address: Wuhan University, Wuhan, China) on May 28, 2025, with a deposit number of CCTCC NO:M 20251217.

[0007] The cordyceps militaris strain of the invention is obtained by subjecting a wild-type cordyceps militaris strain to ultraviolet mutagenesis treatment and is named Cordyceps militaris FHYHC. Compared with the wild-type cordyceps militaris strain, the cordyceps militaris strain has greatly improved thioneine production and is a genetically stable and high-ergothioneine-producing cordyceps militaris mutant strain.

[0008] The second aspect of the present invention provides mycelia obtained by culturing the above-mentioned Cordyceps militaris strain.

[0009] The third aspect of the present invention provides a fruiting body obtained by cultivating the above-mentioned Cordyceps militaris strain.

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

[0011] The mycelium is a white or light-colored filament 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 industrial large-scale production.

[0012] The fruiting body is a mature reproductive structure that is orange-red or yellow in color and has a rod-like or coral-like shape. It grows from insect pupae under natural conditions. Artificial cultivation requires simulating the natural environment, has a long cultivation cycle (1-3 months), and is technically difficult.

[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; according to actual needs, the mycelium or the fruiting body can be selected as the application form of the Cordyceps militaris strain of the present invention.

[0014] The fourth aspect of the present invention provides a bacterial agent, wherein the bacterial agent contains the above-mentioned Cordyceps militaris strain with high ergothioneine production.

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

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

[0017] The seed liquid of the Cordyceps militaris strain is inoculated into a liquid fermentation medium added with precursor substances for fermentation culture; the precursor substances are composed of methionine, cysteine ​​and adenosine.

[0018] Preferably, 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, with a pH of 7; the final concentrations of methionine, cysteine ​​and adenosine after addition are all 2 mM.

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

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

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

[0022] inoculating the seed liquid of the Cordyceps militaris strain into a liquid fermentation medium supplemented with a precursor substance for fermentation, and collecting mycelia by centrifugation after fermentation;

[0023] The mycelium is dissolved in a methanol aqueous solution, subjected to ultrasonic extraction, centrifuged, and the supernatant is collected to prepare an extract of the Cordyceps militaris strain.

[0024] Preferably, the raw material composition of the liquid fermentation medium added with the precursor substance is:

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

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

[0027] Beneficial effects of the present invention:

[0028] (1) The present invention is based on the Cordyceps militaris strain with high thioneine yield potential obtained by screening, and is subjected to ultraviolet mutagenesis treatment, and a Cordyceps militaris mutant strain that can stably inherit and high-yield thioneine is obtained by screening, and the thioneine output in its mycelium is 8.11 times that of the wild-type Cordyceps militaris strain.

[0029] (2) The present invention optimizes and screens precursor substances and synergistically improves the production of ergothioneine, cordycepin, and cordycepin adenosine in mutant strains of Cordyceps militaris 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 a high temperature of 50°C, has a protective effect on cells, and its active ingredients can provide antioxidant protection to improve the survival ability of cells in extreme environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Activated slants of six Cordyceps militaris strains preserved in the laboratory.

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

[0033] Figure 3 : Lethality curves of mycelia of Cordyceps militaris strain 2-1 and Cordyceps militaris strain 2-6 induced by ultraviolet radiation.

[0034] Figure 4 : The results of liquid phase determination of ergothioneine production after fermentation of Cordyceps militaris strain 2-1 (wild type), Cordyceps militaris strain 2-1-6 (mutant), and Cordyceps militaris strain 2-1-6 (directed remodeling).

[0035] Figure 5 : Liquid phase determination results of cordycepin and cordycepin adenosine production after fermentation of Cordyceps militaris strain 2-1 (wild type), Cordyceps militaris strain 2-1-6 (mutant), and Cordyceps militaris strain 2-1-6 (directed remodeling). DETAILED DESCRIPTION

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

[0037] As mentioned above, thioneine, cordycepin and cordyceps adenosine are three kinds of active ingredients in Cordyceps militaris, with broad market application prospects. However, the content of thioneine in natural Cordyceps militaris is extremely low. In order to improve the yield of thioneine, there is a genetic engineering strain of high-yield thioneine constructed by means of genetic engineering in the prior art. But genetic engineering bacteria need to face regulatory restrictions and the strict demand of the market for "natural source" products. Moreover, the anabolism of the three kinds of active ingredients of thioneine, cordycepin and cordyceps adenosine may have partial overlap, causing Cordyceps militaris to have a certain degree of antagonism when synthesizing thioneine, cordycepin and cordyceps adenosine these three kinds of substances simultaneously, making the content of thioneine, cordycepin and cordyceps adenosine three kinds of active ingredients difficult to improve simultaneously.

[0038] In view of this, the present invention first carries out preliminary screening to the ability of the Cordyceps militaris producing thioneine in the strain library, using the Cordyceps militaris strain with the high thioneine production amount as the starting strain, ultraviolet mutagenesis is carried out to the starting strain, and the Cordyceps militaris mutant 2-1-6 that a strain of thioneine production is significantly improved is screened from many mutant strains.

[0039] Furthermore, in order to achieve a synergistic increase in the contents of the three active ingredients of ergothioneine, cordycepin and cordycepin adenosine, the present invention adds low-cost precursor substances methionine, cysteine ​​and adenosine to the liquid fermentation medium through precursor-directed metabolic remodeling technology, thereby increasing the contents of the three active ingredients of ergothioneine, cordycepin and cordycepin adenosine in the mycelium of the Cordyceps militaris mutant strain 2-1-6 at the same time.

[0040] Furthermore, the mycelium of the Cordyceps militaris mutant strain 2-1-6, cultured based on the precursor-directed metabolic remodeling technology, was ultrasonically extracted to obtain an extract, which was able to increase the survival rate of Lactobacillus reuteri at a high temperature of 50°C from 22.6% to 62.1%, providing an innovative solution for the industrial production of natural antioxidants.

[0041] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific examples. If the specific experimental conditions are not specified in the examples, they are usually based on conventional conditions or the conditions recommended by the reagent company; the reagents and consumables used in the following examples, unless otherwise specified, can be obtained through commercial channels.

[0042] Example 1: Acquisition and preservation of high-yield thioneine Cordyceps militaris strains

[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. are numbered as 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 20 g / L, glucose 20 g / L, agar 15 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate 1.5 g / L, VB2 0.01 g / 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 sterile conditions, open the slants of the strains numbered 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. A small amount of bacterial cells (with a small amount of culture medium) were picked near a flame and quickly streaked onto a new slants of activated PDA medium (modified) using the serpentine streak method. The inoculated slants were placed upside down in a constant temperature incubator at 25°C in the dark for 7 days until white, fuzzy colonies completely covered the slants.

[0053] Preparation of seed solution: Cut three mycelium blocks (about 1 cm each) from the slope of the activated PDA medium (modified) 2 ) were inoculated into 200 mL of liquid seed culture medium in a triangular flask. The mixture was placed in a shaker at 25°C, 150 rpm, and incubated in the dark for 7 days until mycelial balls with a diameter of about 3 mm were formed to prepare the seed solution.

[0054] Fermentation culture: The prepared seed solution was inoculated into 1.5 L of liquid fermentation medium in a conical flask at a 5% inoculum volume. The flasks were placed in a shaker at 25°C and 150 rpm in the dark for 7 days until the mycelial pellets reached a concentration that was uniformly filled with liquid fermentation medium by visual observation of the turbidity of the bacterial solution. After fermentation, the mycelium was centrifuged at 5000 rpm for 10 minutes to separate the mycelium and the supernatant, and the mycelium was collected. The mycelium was washed three times with sterile water and freeze-dried. After freeze-drying, the mycelium was pulverized and passed through a 60-mesh sieve to obtain a freeze-dried powder of Cordyceps militaris mycelium.

[0055] 1.3.2 Detection of ergothioneine content in Cordyceps militaris mycelium

[0056] The thioneine content in the freeze-dried powder of Cordyceps militaris mycelium obtained in 1.3.1 above was detected as follows:

[0057] Extraction method: to ensure the accuracy of result, every strain arranges 3 parallels, all weighs the above-mentioned Cordyceps militaris mycelium freeze-dried powder of 0.2g respectively, adds 25mL 1% methanol water.Use ultrasonic assisted extraction, extraction condition: 40 DEG C of ultrasounds 30 minutes, power 300W.After extraction is completed, 12,000rpm is centrifuged 10 minutes, collects supernatant, obtains thioneine crude extract.This crude extract, after filtering through 0.22 μm of microporous membrane, is measured the content of thioneine with high performance liquid chromatography (HPLC).

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

[0059] Standard working solution: Dilute the standard stock solution step by step to prepare standard working solutions with a concentration gradient of 5, 10, 20, 50, and 100 μg / mL for establishing a standard curve.

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

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

[0062]

[0063]

[0064] 1.3.3 UV-induced mutagenesis

[0065] Two Cordyceps militaris strains with high thioneine production were selected and subjected to ultraviolet mutagenesis treatment to breed mutant strains with high thioneine production. The details are as follows:

[0066] Preparation of bacterial solution: Inoculate the Cordyceps militaris strain into PDA medium (modified), then place the medium in a 25℃ constant temperature incubator in the dark for 7 days. Take 3 pieces of mycelium (each about 1cm 2 Place the plate in 10.0 mL of sterile water (containing sterile glass beads approximately 4-5 mm in diameter) and shake at 150 rpm for 1 hour to scrape the mycelium from the agar surface. Remove the supernatant by centrifugation and collect the mycelium to obtain 5 mL of culture solution. Store at 4°C until ready for use.

[0067] Ultraviolet mutagenesis: Turn on the ultraviolet lamp of the clean bench, preheat the ultraviolet lamp for more than 30 minutes to stabilize the ultraviolet light wave (253.7nm), dilute the Cordyceps militaris liquid to 10 -3 The dilution was 100 μL each, and three replicates were set for each group. The dilutions were irradiated for 0, 3, 6, 9, 12, 15, 18, and 21 min, respectively. The irradiated bacterial solution was poured into the modified PDA medium in a dark environment and cultured at 25°C for 7 days until single colonies appeared. The bacterial solution that had not been treated with UV was used as a control. The lethality of the strains at different mutagenesis times was calculated as follows:

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

[0069] According to the mortality rate curve of ultraviolet mutagenesis of Cordyceps militaris, the irradiation time with an ultraviolet mortality rate of about 80% was selected as the optimal irradiation time. The strain induced by the optimal irradiation time was selected, and the mycelium of Cordyceps militaris was cultured according to the method in 1.3.1, and the ergothioneine content was detected according to the method in 1.3.2.

[0070] 2 Results

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

[0072] The present invention performs slant activation on 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 are white, and the velvety colonies completely cover the slant. The results are as follows Figure 1 As shown in Figure 2, the six strains of Cordyceps militaris had good activity. Subsequently, liquid seed solution preparation (200 mL), liquid fermentation culture (1.5 L), centrifugation, freeze drying, and crushing through a 60-mesh sieve gave Cordyceps militaris mycelium freeze-dried powder. The results were as follows: Figure 2 shown.

[0073] The yield of thioneine of each Cordyceps militaris strain was determined as shown in Table 1:

[0074] Table 1: Amount of ergothioneine produced by different Cordyceps militaris strains

[0075]

[0076]

[0077] As shown in Table 1, the mycelium thioneine production of Cordyceps militaris strain 2-1 is 0.240mg / g, and the mycelium thioneine production of Cordyceps militaris strain 2-6 is 0.130mg / g. Cordyceps militaris strain 2-1, 2-6 are two strains with relatively high thioneine production, so these two strains are selected to carry out subsequent experiments.

[0078] 2.2 UV Breeding of Cordyceps militaris Strains

[0079] The mortality rate curves of UV-induced Cordyceps militaris strains 2-1 and 2-6 are shown in Figure 2. Figure 3As shown. With the increase of irradiation time, the lethality of Cordyceps militaris increases, and the lethality curve tends to be flat after 15 minutes of irradiation. Studies have shown that too low a lethality is not conducive to screening positive mutant strains, while too high a lethality leads to an increase in the negative mutation rate. Therefore, based on the lethality curve of ultraviolet-induced Cordyceps militaris, this study selected an irradiation time with an ultraviolet lethality of about 80% as the optimal irradiation time, that is, 15 minutes of ultraviolet mutagenesis was the optimal mutagenesis time. The lethality of strain 2-1 treated with this method was 78.95%, and the lethality of strain 2-6 was 77.12%. After 15 minutes of ultraviolet irradiation mutagenesis treatment, 20 mutant strains were randomly selected for liquid fermentation in this experiment, and the content of ergothioneine in their mycelium was determined.

[0080] Table 2 shows the ergothioneine production of the mutant strain of Cordyceps militaris 2-1 after UV mutagenesis.

[0081] Table 2: Determination of ergothioneine production in Cordyceps militaris 2-1 and its mutant strains

[0082]

[0083] Table 3 shows the ergothioneine production of the mutant strain of Cordyceps militaris 2-6 after UV mutagenesis.

[0084] Table 3: Determination of thioneine production in Cordyceps militaris 2-6 and mutant strains

[0085]

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

[0087] Based on the above content, the mutant Cordyceps militaris 2-1-6 after ultraviolet mutagenesis was selected as the preserved strain, and its genetic stability was investigated. The results showed that the mutant Cordyceps militaris 2-1-6 was passed down from generation to generation once, and was calculated to be passed down from generation to generation 10 times. The same fermentation culture conditions were used at each time, and the same sample processing method and thioneine content assay method were used. There was no significant difference in thioneine output between each generation. It was proved that the mutant Cordyceps militaris 2-1-6 had genetic stability.

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

[0089] Culture name: Cordyceps militaris FHYHC;

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

[0091] Preservation time: May 28, 2025.

[0092] Example 2: Synergistically increasing the production of ergothioneine, cordycepin, and cordycepin adenosine in Cordyceps militaris strains based on precursor-directed metabolic remodeling

[0093] 1 Materials and Methods

[0094] 1.1 Strains

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

[0096] 1.2 Experimental Materials

[0097] Methionine, cysteine, adenosine, arginine, aspartic acid, glutamic acid, cordycepin (chromatographic grade), and cordycepin adenosine (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 Effect of Precursor-Directed Metabolic Remodeling on Ergothioneine Production

[0102] Design of precursor addition scheme: This experiment selected methionine, cysteine, adenosine, arginine, aspartic acid, and glutamic acid as precursor substances, designed different precursor addition schemes (see Table 4), and added them to the liquid fermentation medium.

[0103] Table 4: Precursor addition scheme

[0104]

[0105] Note: CK group is liquid fermentation medium without adding prerequisite substances.

[0106] Mycelium preparation: the Cordyceps militaris 2-1-6 obtained after ultraviolet mutagenesis 15min process in embodiment 1 is inoculated into (containing 200mL liquid seed culture medium) in 500mL triangular flask by slant preservation tube and activates, prepares seed liquor.The seed liquor prepared is inoculated into the 1.5L liquid fermentation culture medium containing different precursors adding scheme by 5% inoculum size (volume ratio) and carries out fermentation culture, temperature 25 DEG C, rotating speed 150r / min, lucifuge culture 7 days, observes mycelial ball and forms.After fermentation is complete, centrifugal collection mycelium, sterile water washing mycelium three times rear freeze-drying, pulverizes 60 mesh sieves after freeze drying, and the mensuration of thioneine output is carried out by the method for 1.3.2 in embodiment 1 to the Cordyceps militaris mycelium freeze-dried powder obtained.

[0107] 1.3.2 Effects of Precursor-Directed Metabolic Remodeling on Cordycepin and Cordycepin Adenosine Production

[0108] Cordycepin and cordycepin are the two core active products recognized in Cordyceps militaris, both of which have clear biological activities and medicinal value. The present invention further studied the effects of precursor material culture protocols on the production of cordycepin and cordycepin in wild Cordyceps militaris strain 2-1 and mutant Cordyceps militaris strain 2-1-6.

[0109] Extraction method: To ensure the accuracy of the results, three parallels were set up for each group of mycelia to be tested. 0.1 g of freeze-dried mycelia powder obtained after fermentation of the wild strain Cordyceps militaris 2-1, freeze-dried mycelia powder obtained after fermentation of the mutant strain Cordyceps militaris 2-1-6, and freeze-dried mycelia powder obtained after fermentation of the mutant strain 2-1-6 with 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 used, and the ultrasonic extraction conditions were: 40°C for 30 minutes and a power of 250 W. After extraction, the mixture was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was filtered through a 0.22 μm filter membrane to determine the content of cordycepin and cordycepin adenosine.

[0110] The cordycepin and adenosine contents in each group of freeze-dried Cordyceps militaris mycelium powder were determined using a C18 reverse-phase column (250 mm × 4.6 mm, 5 μm). Mobile phases: Phase A: 0.1% formic acid in water, Phase B: 100% acetonitrile; Flow rate: 1.0 mL / min; Column temperature: 30°C; Detection wavelength: 260 nm (maximum absorption peaks for cordycepin and adenosine). The gradient program was as follows:

[0111]

[0112] 2 Results

[0113] 2.1 Effects of precursor-directed metabolic remodeling on 2-1-6-ergothioneine production in Cordyceps militaris

[0114] After culturing for 7 days at a temperature of 25° C. and a rotation speed of 150 rpm in the dark, the effects of different precursor addition schemes on the ergothioneine yield of Cordyceps militaris 2-1-6 are shown in Table 5, where the percentage increase is (scheme content-CK content) / CK content×100%.

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

[0116]

[0117] The above results show that the ergothioneine content in the freeze-dried powder of mycelium corresponding to the natural strain 2-1 of Cordyceps militaris is 0.240 mg / g, and the ergothioneine content in the freeze-dried powder of mycelium corresponding to the strain 2-1-6 of Cordyceps militaris is 1.947 mg / g. After the precursor-directed metabolic remodeling and fermentation of the strain 2-1-6 of Cordyceps militaris, the ergothioneine content in the freeze-dried powder of mycelium is the highest, which is 3.833 mg / g. The liquid phase determination results of ergothioneine are as follows Figure 4 shown.

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

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

[0120] In summary, the UV-induced mutagenesis strain 2-1-6 may carry a highly active ergothioneine synthase gene, enabling it to effectively utilize exogenous precursors for the synthesis of ergothioneine, breaking through the precursor utilization bottleneck of conventional strains. Methionine and cysteine ​​provide a sulfur source for ergothioneine synthesis, while adenosine, as a purine metabolic precursor, directly participates in the synthesis of cordycepin and cordycepin adenosine. The synergistic supplementation of these three can alleviate metabolic pathway competition, breaking through the traditional bottleneck of increasing production with a single precursor and achieving efficient, directed synthesis of EGT. After UV mutagenesis, the Cordyceps militaris strain 2-1-6, through the directed metabolic remodeling of precursor substances, can synergistically increase the production of ergothioneine, cordycepin, and cordycepin adenosine, showing the potential for industrial scale-up 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 and Lactobacillus reuteri LY0032 obtained in Example 1 of the present invention are patented strains preserved in the laboratory with a preservation number of CCTCC NO: M 2015341, which are recorded in patent CN 106420847 A.

[0125] 1.2 Culture medium

[0126] MRS medium (modified): by mass 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] Cordyceps militaris 2-1-6 mycelium freeze-dried powder is prepared by the method of 1.3.1 in Example 1 of the present invention. 0.2g of Cordyceps militaris 2-1-6 mycelium freeze-dried powder is accurately weighed and dissolved in 25mL of 1% methanol water, and Cordyceps militaris extract is prepared using the method for ultrasonic assisted extraction, and ultrasonic extraction conditions are: 40 DEG C of ultrasound 30min, power 300W. After the completion of extraction, 12,000rpm is centrifuged for 10min, and supernatant is collected to obtain thioneine crude extract. The crude extract is standby after filtering through 0.22 μm of microporous membrane.

[0130] 1.3.2 Lactobacillus reuteri culture and treatment

[0131] Bacterial activation: Lactobacillus reuteri LY0032 (CCTCC NO: M 2015341) was inoculated into MRS medium (modified) and cultured at 37°C under static conditions for 18 h. The bacterial solution was transferred to a sterile centrifuge tube and centrifuged at 4000 rpm for 5 min. The cells were collected and mixed with sterile physiological saline to adjust the viable cell count to 1.0 × 10 9 CFU / mL, and obtain Lactobacillus reuteri liquid.

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

[0133] Control group: 0.5 mL of Lactobacillus reuteri culture solution was added to 4.5 mL of 1% methanol water and shaken to mix thoroughly;

[0134] Experimental group 1 (wild group): 0.5 mL of Lactobacillus reuteri culture was added to 4.5 mL of mycelial extract of Cordyceps militaris natural strain 2-1 and the mixture was shaken and mixed.

[0135] Experimental group 2 (mutant group): 0.5 mL of Lactobacillus reuteri culture was added to 4.5 mL of mycelial extract of Cordyceps militaris mutant strain 2-1-6, and the mixture was shaken and mixed.

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

[0137] Positive control: 4.5 mL of a known protective agent (10% skim milk) was added to 0.5 mL of Lactobacillus reuteri culture and the mixture was shaken and mixed.

[0138] The above mixtures were placed in a constant temperature water bath at 50°C for 1 hour, and then the viable bacteria were counted after cooling to room temperature.

[0139] 1.3.3 Survival rate detection

[0140] Plate count method: The above bacterial solution after high temperature treatment was diluted in series (10 -6 –10 -8 ), three replicates were set up for each group, the plates were spread on MRS agar plates, and the cells were cultured anaerobically at 37°C for 48 h. The colonies (CFU / mL) were counted and the survival rate range was calculated.

[0141] 2. Results

[0142] The survival rate of Lactobacillus reuteri in each group is 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 rates of Lactobacillus reuteri in experimental groups 1-3 under heat stress were all improved, with the effects in experimental groups 2 and 3 being even more pronounced. This is presumably due to the higher levels of active ingredients such as ergothioneine, cordycepin, and cordycepin in the extracts of these groups, which enhanced Lactobacillus reuteri's ability to withstand heat stress. In particular, the effect of experimental group 3 approached that of the positive control group, providing an innovative solution for the industrial production of natural antioxidants.

[0147] In summary, the present invention combines precursor directed metabolism remodeling technology by ultraviolet mutagenesis technology, significantly improves the content of thioneine (3.833mg / g) in Cordyceps militaris with non-genetic engineering means, belongs to the major breakthrough of non-genetic engineering means;Meanwhile, the output of cordycepin (6.1mg / g) and cordyceps adenosine (16.5mg / g) is also improved synchronously, and multi-component synchronous production is realized for the first time.And low-cost precursor feed (methionine, cysteine, adenosine) and conventional fermentation process, possess scale potential.

[0148] The extract of the Cordyceps militaris strain of the present invention is rich in active ingredients such as ergothioneine, cordycepin, and cordycepin adenosine, making it suitable for unique applications (such as natural health foods). The synergistic effect of the three increases the survival rate of Lactobacillus reuteri at a high temperature of 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 the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A Cordyceps militaris strain with high ergothioneine production, whose deposit number is CCTCC NO: M 20251217.

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

3. The fruiting body obtained by cultivating the Cordyceps militaris strain according to claim 1.

4. A bacterial agent, characterized in that The bacterial agent contains the Cordyceps militaris strain according to claim 1.

5. The microbial agent according to claim 4, characterized in that In the bacterial agent, the Cordyceps militaris strain exists in the form of cultured live bacteria, bacterial suspension or fermentation liquid.

6. A method for synergistically increasing the yields of ergothioneine, cordycepin and cordycepin adenosine in Cordyceps militaris strains based on precursor-directed metabolic remodeling, characterized in that: The following steps are involved: The seed liquid of the Cordyceps militaris strain according to claim 1 is inoculated into a liquid fermentation medium supplemented with precursor substances for fermentation culture; the precursor substances are composed of methionine, cysteine ​​and adenosine.

7. The method according to claim 6, characterized in that 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, with a pH value of 7; the final concentrations of methionine, cysteine, and adenosine after addition are all 2 mM.

8. The method according to claim 6, characterized in that The fermentation culture conditions are: temperature 20-30°C, rotation speed 120-180r / min, and culture in the dark for 5-7 days.

9. Use of the extract of the Cordyceps militaris strain according to claim 1 in preparing a product for improving the resistance of Lactobacillus reuteri to high temperature stress.

10. The use according to claim 9, characterized in that The extract of the Cordyceps militaris strain is prepared by the following method: inoculating the seed liquid of the Cordyceps militaris strain according to claim 1 into a liquid fermentation medium supplemented with a precursor substance for fermentation culture, and collecting the mycelium by centrifugation after fermentation; The mycelium is dissolved in a methanol-water solution, subjected to ultrasonic extraction, centrifuged, and the supernatant is collected to prepare an extract of the Cordyceps militaris strain; Preferably, the raw material composition of the liquid fermentation medium added with the precursor substance is: Sucrose 40 g / L, peptone 20 g / L, magnesium sulfate 2 g / L, potassium dihydrogen phosphate 1.5 g / L, methionine 2 mM, cysteine ​​2 mM, adenosine 2 mM; Preferably, the conditions for ultrasonic extraction are: 40°C, 30 min, and 300W power.

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