Method for producing ergothioneine by fermenting edible mushroom boletus edulis and application

Abstract

The invention belongs to the field of agricultural food and biological medicine, discloses a method for producing ergothioneine by fermentation of edible mushroom boletus edulis and application, and comprises a method for producing ergothioneine by liquid fermentation of the edible mushroom boletus edulis and a method for separating and purifying ergothioneine from a fermentation product in a high-yield and high-purity manner. By optimizing the composition of the fermentation culture medium, the supply of precursor substances and the discharge of products are enhanced, and the ergothioneine fermentation yield of the boletus edulis is remarkably increased and is increased by 419.3% compared with that of a control group. According to the invention, a serial adsorption process combining D941 anion exchange resin with 732 cation exchange resin is provided, and a gradient elution process combining ammonia water / ethanol solution is combined, so that pigments and other impurities in the fermentation liquor are effectively removed, the high yield and high purity of the ergothioneine separation and purification process are guaranteed, the purity of a separated sample reaches 96.71%, and the total yield reaches 99.13%. The method has the characteristics of greenness, environmental protection, high safety, simple and convenient process flow and the like, and shows a huge industrial application prospect.

Description

Technical Field

[0001] The invention relates to the fields of agricultural food and biomedicine, and relates to a method and application of producing ergothioneine by fermenting an edible fungus Boletus edulis, and specifically to a method for increasing the ergothioneine content in the edible fungus Boletus edulis and increasing the efficiency of separating and purifying ergothioneine in a liquid fermentation system. Background Art

[0002] Ergothioneine (EGT) is a natural small molecule histidine thiourea derivative that exists in microbial cells and plants and animals. EGT can be synthesized by fungi, cyanobacteria, actinomycetes and other microorganisms. The human body cannot synthesize it by itself and needs to be ingested through diet. Edible fungi are one of the important dietary sources of EGT. EGT has multiple biological functions such as antioxidant, anti-inflammatory, and immunomodulatory, and has a wide range of applications in food, medicine, and cosmetics. In particular, ergothioneine has been approved by the European Union as a new food ingredient, paving the way for its application in dietary supplements. In addition, it can also be used as a color stabilizer to enhance the color retention of food, and as a preservative to extend the shelf life of food. The edible fungus Boletus edulis is a precious mountain treasure belonging to the Boletaceae family and the genus Boletus. It is common in deciduous and coniferous forests in the temperate zone of the northern hemisphere in summer and autumn. It is rich in various nutrients and has health benefits such as improving immunity and lowering blood lipids.

[0003] At present, there are many problems in the production and extraction of thioneine, in terms of production, such as using Escherichia coli fermentation to produce thioneine in CN114854660A patent, there is a low yield of thioneine in the fermentation system, and the production of endotoxin limits its application in the field of medicine and food and increases the problems of separation and purification difficulty; the high-yield strain with thiosulfate as sulfur source constructed by CN118325932A patent, except introducing endotoxin, the hydrogen sulfide generated by thiosulfate disproportionation reaction can cause adverse effects on fermentation system and environment, therefore, thioneine green biomanufacturing needs to take into account the safety of production strains and the environmental protection of process flow. In terms of product extraction, many traditional methods are often difficult to take into account the dual goals (table 1) of high yield and high purity. For example, in the extraction method of CN117924184A patent, ultrafiltration membrane can cause small molecule impurities to be incorporated and easily blocked, affecting product yield, and resin selectivity is not clear, and inorganic salts may also be introduced in pretreatment and crystallization processes; the organic solvent used in the extraction method of CN116178270A patent is volatile and pollutes the environment and recycling is not mentioned, and washing the filter cake with ethanol can reduce the yield of thioneine.

[0004] Table 1 Comparison of ergothioneine separation and purification methods

[0005]

[0006]

[0007] my country has made some progress in the field of producing ergothioneine by fermentation of edible and medicinal fungi. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, successfully screened out a natural edible fungus Pleurotus ostreatus (CN114958617B) with high ergothioneine yield, established a liquid deep fermentation process for accumulating ergothioneine, and the output of ergothioneine in the shake flask fermentation liquid reached 500mg / L. In the field of edible and medicinal fungi, there are few research reports on the production and extraction and purification technology of ergothioneine, and this field is yet to be explored and developed in depth. Summary of the invention

[0008] In view of the safety concerns caused by the current large-scale application of genetically engineered bacteria in the production of ergothioneine, the present invention aims to provide a green, safe and efficient method for producing ergothioneine. Specifically, it includes a method for producing ergothioneine by liquid fermentation of edible fungi Boletus edulis and a method for separating and purifying ergothioneine in high yield and high purity from a fermentation product.

[0009] The highlight of the present invention is that, utilizing green and safe natural edible fungus delicious boletus (Boletus edulis), it shows higher thioneine production capacity (table 2) in edible fungi, and its application in food industry has safety guarantee. The present invention, by optimizing the deep liquid fermentation system of delicious boletus, not only realizes the green production of thioneine, but also improves the production efficiency of thioneine in delicious boletus. In addition, the highlight of the present invention is also that, by optimizing the separation and purification process, anion and cation exchange resins are used in series to realize the efficient separation and purification of thioneine, not only taking into account high purity and high yield, but also can realize the effective recovery of elution solvent. This not only improves economic benefit, but also reduces environmental pollution, embodies the concept of green biomanufacturing and sustainable development.

[0010] Table 2 Comparison of ergothioneine content in different edible fungi

[0011]

[0012] The specific steps are as follows:

[0013] S1 Preparation of Boletus edulis spore suspension: inoculate the Boletus edulis stored at -80°C into PDA solid culture medium, culture at 25-30°C for 7-10 days, and after the mycelium grows to maturity, wash the spores of Boletus edulis with sterile physiological saline by shaking, filter through sterile gauze, collect the filtrate, and obtain the Boletus edulis spore suspension.

[0014] S2: Deep liquid fermentation of Boletus edulis: The spore suspension of Boletus edulis prepared in step S1 is selected and inoculated into a sterile liquid fermentation medium at an inoculation rate of 2% to 5%, and the final concentration of the spore suspension is controlled to be 2×10 8 ~6×10 8 CFU / mL, cultured at 25-30°C and 180-200 rpm for 15-25 days. During the fermentation process, Boletus edulis continuously accumulates ergothioneine using nutrients.

[0015] The fermentation medium mainly comprises: 20-80 g / L of glycerol, 7-10 g / L of yeast extract, 25-30 g / L of soybean powder, 10-15 mL / L of sunflower oil, 0.5-2 g / L of histidine, 0.5-2 g / L of cysteine, 0.5-2 g / L of methionine, 5-15 g / L of K2HPO4, 2-5 g / L of MgSO4·7H2O, 2-5 g / L of CaCO3, 10-20 mg / L of VB6, and an initial pH of 4-7.

[0016] In addition, in order to control the ball morphology of Boletus edulis to promote the excretion of intracellular ergothioneine, glass balls with a diameter of 0.2 to 1 mm were added to the fermentation medium at an addition amount of 100 to 500 per liter before inoculation of the spore suspension.

[0017] S3 Pretreatment of the fermented boletus edulis broth: The fermented boletus edulis broth containing mycelium of step S2 is collected, subjected to ultrasonic crushing treatment at a power of 200-500 W for 5-10 minutes, the treated liquid is collected, and heated in a water bath at 95-100° C. for 5-15 minutes. Further, an appropriate amount of 90-100% ethanol is added thereto, and the final ethanol concentration is controlled to reach 60-80% (v / v), and after mixing, it is allowed to stand at 0-4° C. for 30-60 minutes to precipitate macromolecular substances such as proteins. Further, the supernatant is collected by centrifugation at 8000-12000 rpm for 5-10 minutes.

[0018] S4 fermentation broth decolorization treatment: The supernatant collected in step S3 is concentrated by low-pressure rotary evaporation under the conditions of 0.05-0.1MPa and 20-35°C, so that the thioneine content is within the range of 0.5-1g / L, and the pH of the concentrated solution is adjusted to 2-4. Further, in order to improve the quality of the thioneine product, the above-mentioned concentrated solution is loaded into a D941 anion exchange resin column for depigmentation treatment, the mass ratio of the concentrated solution to the D941 anion exchange resin is 1:2-2:3, the sample flow rate is maintained at 12-20mL / min, and the filtrate is collected.

[0019] S5 adsorption and elution of thioneine: The pH of the filtrate is adjusted to 4-8 to adapt the adsorption characteristics of 732 cation exchange resins. Further, the filtrate is loaded into 732 cation exchange resin columns for adsorption. The mass ratio of the filtrate to the 732 cation exchange resin is 1:2-2:3, and the loading flow rate is maintained at 1-4 mL / min to achieve efficient adsorption of the target compound thioneine. After the adsorption is completed, a 1%-5% (v / v) ammonia / ethanol solution is used for gradient elution. The solution ratios of the gradient elution are 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 1:3, 0:10, respectively, and the elution time of each gradient is 30-40 minutes, and the elution flow rate is controlled at 1-4 mL / min.

[0020] Purification of S6 thioneine: The eluent collected in step S5 is subjected to low-pressure rotary evaporation at 35-45°C and 0.05-0.1MPa to obtain crude powder. 90-100% ethanol solution is used to dissolve the crude product at a solid-liquid ratio of 1:100-1:10, centrifuged at 0-4°C and 8000-12000rpm for 5-10 minutes, insoluble impurities are removed, and supernatant is collected. Further, low-pressure rotary evaporation is performed again at 35-45°C and 0.05-0.1MPa to obtain highly purified thioneine crystalline powder.

[0021] The content detection of ergothioneine in above-mentioned steps S4, S5 and S6 samples adopts HPLC method to analyze.Specific grammar is: adopt high performance liquid chromatograph Waters e2695, detector is Waters 2998, and ultraviolet detector wavelength is 254nm, and chromatographic column selects XCharge C18 (5 μL 100A4.6 × 250mm Column), and mobile phase is 0.1% formic acid solution, and sample size is 10 μL, gradient program: 0-20min: 0.5mL / min; 21-29min: 0.8mL / min; 30-35min: 0.5mL / min; Column temperature is 20~30 DEG C.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The invention adopts the edible fungus Boletus edulis as the production strain of ergothioneine, has the advantage of food safety, and through liquid fermentation optimization, the fermentation yield of ergothioneine is increased to 37.13 mg / L, which is increased by 419.30% compared with the non-optimized system.

[0024] In the product separation and purification link, the present invention proposes D941 anion exchange resin-732 cation exchange resin series adsorption process, optimizes the technical parameters such as adsorption / elution flow rate, feed liquid pH, upper liquid amount in the process flow, effectively removes impurities such as pigment and salt ions in the fermentation liquid, and ensures the high yield (99.13%) and high purity (96.71%) of the finished product. The thioneine separation and purification process established by the present invention is easy to operate, easy to industrial amplification, and the ethanol and ammonia used by its elution solvent can realize recycling, reduce production cost, reduce environmental pollution, and meet the concept of green chemistry and sustainable development. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a comparison chart showing the effect of adding glass balls on the production of ergothioneine by fermentation of Boletus edulis;

[0026] Figure 2 The results of the effects of carbon sources and precursors on the fermentation yield of ergothioneine in Boletus edulis are shown in Figure 1; (a) is different carbon sources; (b) is histidine; (c) is methionine; (d) is cysteine;

[0027] Figure 3 This is the result diagram of the effect of pH on the adsorption of D941 anion exchange resin;

[0028] Figure 4 This is a comparison of the fermentation broth before and after decolorization by D941 anion exchange resin;

[0029] Figure 5 This is the Zeta potential diagram of ergothioneine under different pH conditions;

[0030] Figure 6 This is the result diagram of the effect of pH on the adsorption rate of ergothioneine by 732 cation exchange resin;

[0031] Figure 7 This is the result diagram of the effect of elution method on elution rate of ergothioneine;

[0032] Figure 8 The HPLC analysis spectra of samples before and after separation and purification of ergothioneine; (a) is the initial fermentation broth sample; (b) is the reconstituted sample after purification; (c) is the ergothioneine standard. DETAILED DESCRIPTION

[0033] The present invention is described in detail below by specific examples, but the protection scope of the present invention is not limited. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0034] The present invention can use any Boletus edulis, and the following embodiments are described using Boletus edulisACCC 50559 as an example.

[0035] Example 1: Preparation of Boletus edulis spore suspension

[0036] The delicious boletus B.edulis ACCC 50559 stored at -80℃ was inoculated into solid PDA medium, and then inoculated into potato dextrose agar (PDA) solid medium under sterile conditions, and cultured in a constant temperature incubator at 25℃ for 10 days. During this period, regular observation was made to ensure that the mycelium grew healthily and without contamination. After the mycelium on the slope grew mature, the mycelium was gently washed with sterile saline, filtered through gauze, and collected into a sterile conical bottle to form a spore suspension. The concentration of the spore suspension was 2.81×10 8 CFU / mL.

[0037] Example 2: Submerged liquid fermentation of Boletus edulis

[0038] The spore suspension of Boletus edulis was inoculated into a sterile culture medium with an initial pH of 6.0 (100 glass balls with a diameter of 0.5 mm were pre-added at a concentration of 100 / L) with an inoculum volume of 5% and a final concentration of 2.81×10 8 CFU / mL, placed in liquid culture time of 20 days under 28 ℃ and rotation speed of 180rpm. The present embodiment investigates the effect of glass ball addition on the efflux of product ergothioneine. In the fermentation system without glass balls, the mycelium of Boletus edulis is easy to agglomerate and aggregate into larger bacterial agglomerates. These bacterial agglomerates are often irregular in shape, of different sizes, and adhere to each other to form a large mass of bacteria. In the fermentation process with the addition of glass balls, the morphology of the mycelium changed to form smaller and dispersed bacterial balls. These bacterial balls are relatively small, more uniform in shape, and no longer tightly adhere to each other, showing a relatively loose distribution state. This change in morphology helps to improve the fermentation efficiency and the release of ergothioneine. Figure 1 As shown, the extracellular proportion of the fermentation product ergothioneine of the glass ball group was 68.83%, which was 23.49 times that of the control group, indicating that adding an appropriate amount of glass balls during the fermentation process can significantly promote the extracellular release of ergothioneine. In addition, the present embodiment also optimizes carbon sources (glycerol, glucose, fructose, sucrose) and precursor substances (histidine His, cysteine ​​Cys, methionine Met) in the fermentation medium of delicious boletus ( Figure 2 ) and found that glycerol was more conducive to the accumulation of ergothioneine than other carbon sources ( Figure 2 a), at the same time, the addition of different concentrations of precursor substances (His, Cys and Met) was beneficial to the accumulation of ergothioneine, and the optimal concentration was 1 g / L ( Figure 2bd). After optimization, the optimal fermentation medium composition for producing ergothioneine by Boletus edulis was obtained, including: 30 g / L glycerol, 8 g / L yeast extract, 25 g / L soybean powder, 12 mL / L sunflower oil, 5 g / L K2HPO4, 3 g / L MgSO4·7H2O, 2 g / L CaCO3, 10 mg / L VB6, 1 g / L histidine, 1 g / L cysteine, and 1 g / L methionine. Based on this, deep liquid fermentation was carried out, and the fermentation yield of ergothioneine reached 37.13 mg / L, which was 419.30% higher than that of the unoptimized system.

[0039] Example 3: Pretreatment of Boletus edulis fermentation broth

[0040] Fermentation product is subjected to ultrasonic cell disruption, and power is set to 285W, ultrasonic treatment is performed for 5 minutes, and then treatment solution is placed in 100 DEG C of water bath heating for 10 minutes. Anhydrous ethanol is added to the fermentation liquid after ultrasonic treatment, so that the final concentration of ethanol reaches 70% (v / v), and it is left to stand for 30 minutes at 4 DEG C to precipitate protein and other macromolecules. The fermentation liquid after alcohol precipitation is centrifuged for 5 minutes with 8000rpm rotating speed, and supernatant is taken for next step processing. Supernatant is evaporated under low pressure under 35 DEG C, 0.05MPa conditions, and it is about 500mg / L to control thioneine final concentration in conjunction with HPLC detection result.

[0041] The specific steps of HPLC analysis are:

[0042] Preparation of standard sample: dilute 10 g / L ergothioneine standard with ultrapure water to 50 mg / L, and take 1 mL of 50 mg / L standard to pass through a 0.22 μm filter membrane for testing.

[0043] Sample preparation: Take 1 mL of sample and filter it through a 0.22 μm filter membrane for testing.

[0044] Thiothioneine detects: adopt high performance liquid chromatograph Waters e2695, detector is Waters 2998, and setting ultraviolet detector wavelength is 254nm, and chromatographic column is selected XCharge C18 (5 μL 100A4.6 × 250mm Column), and mobile phase is 0.1% formic acid, and sample size is 10 μL, flow velocity: 0-20min, 0.5mL / min; 21-29min, 0.8mL / min; 30-35min, 0.5mL / min; Column temperature: 25 ℃.

[0045] Example 4: Fermentation broth decolorization

[0046] The present embodiment investigates the adsorption of thioneine by different pH feed liquids during decolorization by D941 anion exchange resin. Figure 3As shown, at pH 4, the adsorption rate of D941 anion exchange resin to thioneine is the lowest. To avoid the loss of thioneine in the impurity removal process, the pH of the treatment solution is subsequently adjusted to 4, and the pretreated fermented liquid is loaded into a D941 anion exchange resin column with a column volume of 60 mL, and decolorization is performed. The upper liquid volume is controlled at 60 mL, and the flow rate is maintained at 15 mL / min. The filtrate is collected. Figure 4 As shown, the pigment in the fermentation broth was effectively removed under the above elution conditions.

[0047] Embodiment 5: adsorption and elution of ergothioneine

[0048] This embodiment utilizes 732 cation exchange resin to adsorb thioneine, and investigates the influence of factors such as feed liquid pH, elution scheme, etc. According to the Zeta potential information of thioneine ( Figure 5 ), because the nitrogen atom in the imidazole ring of ergothioneine can accept protons under neutral or weakly alkaline conditions to form positively charged ammonium ions. This charge state makes ergothioneine show strong positive charge at pH 7.5. Figure 6 Show, under the condition of pH 7.5, 732 cation exchange resins have the best adsorption effect on thioneine, reaching 94.2%. In order to ensure that thioneine is fully adsorbed, the pH value of the filtrate after decolouring is adjusted to 7.5. Uploaded in 732 cation exchange resin columns with a column volume of 60mL, adsorption treatment is carried out, and the upper liquid amount is controlled at 60mL, and the flow rate is reduced to 2mL / min. As shown in Figure 71%, the effect of the sodium hydroxide-ethanol and 1% ammonia-ethanol gradient elution scheme all performs well, and the elution rate reaches 98.2% and 97.7% respectively. In order to avoid the introduction of impurity salt ions in the separation process and realize the recycling of solvent at the same time, the present invention selects 1% ammonia-ethanol solution gradient elution as the elution method of thioneine. 1% ammonia: ethanol elution gradient is successively 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 1:3, 0:10. The elution time of each gradient was 35 min, and the elution flow rate was controlled at 2 mL / min.

[0049] Embodiment 6: the purification of thioneine

[0050] The collected eluate was subjected to low-pressure rotary evaporation at 40°C to obtain a crude product in the form of powder. The crude product was dissolved in 200 mL of ethanol and centrifuged at 12000 rpm for 5 minutes at -4°C, and the supernatant was taken to remove insoluble impurities. Low-pressure rotary evaporation was performed again at 35°C to obtain high-purity ergothioneine powder. Figure 4As shown, the sample is measured by high performance liquid chromatograph Waters 26952998, and compared with the chromatogram of the initial sample, the impurity peak of the purified sample is significantly reduced, which shows that the purification process has significantly improved the purity of thioneine. In the sample before purification, thioneine content is 75.23%, and the sample purity after purification is increased to 96.71%, and the final high yield reaches 99.13% ( Figure 8 ).

[0051] The above-described embodiments are only preferred embodiments of the present invention, but not all feasible embodiments of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A method for producing ergothioneine by fermenting edible fungi Boletus edulis, characterized in that, The steps include: S1. Solid activated culture of Boletus edulis to prepare spore suspension; S2: Deep liquid fermentation of Boletus edulis: The spore suspension of Boletus edulis prepared in step S1 is selected and inoculated into a sterile liquid fermentation medium at an inoculation rate of 2% to 5%, and the final spore concentration is controlled to be 2×10 8 ~6×10 8 CFU / mL, cultured for 15 to 25 days at a temperature of 25 to 30°C and a rotation speed of 180 to 200 rpm; during the fermentation process, Boletus edulis continuously accumulates ergothioneine using nutrients; glass balls with a diameter of 0.2 to 1 mm are added to the fermentation medium at an addition amount of 100 to 500 per liter; S3 Pretreatment of Boletus edulis fermentation broth; S4 fermentation broth decolorization treatment; S5 adsorption and elution of ergothioneine; S6 Purification of ergothioneine.

2. the method utilizing edible fungus Boletus edulis fermentation to produce ergothioneine according to claim 1, is characterized in that, The components of the liquid fermentation medium include: glycerol 20-80 g / L, yeast extract 7-10 g / L, soybean powder 25-30 g / L, sunflower oil 10-15 mL / L, histidine 0.5-2 g / L, cysteine ​​0.5-2 g / L, methionine 0.5-2 g / L, K2HPO4 5-15 g / L, MgSO4·7H2O 2-5 g / L, CaCO3 2-5 g / L, and VB6 10-20 mg / L.

3. The method for producing ergothioneine by fermenting edible mushroom Boletus edulis according to claim 1, wherein S3 pretreatment of the delicious boletus fermentation liquid includes: collecting the delicious boletus fermentation product containing mycelium in step S2, performing ultrasonic crushing treatment at a power of 200-500W for 5-10 minutes, collecting the treated liquid, heating it in a water bath at 95-100°C for 5-15 minutes, and collecting the supernatant by centrifugation at 8000-12000rpm for 5-10 minutes.

4. The method for producing ergothioneine by fermenting edible mushroom Boletus edulis according to claim 3, wherein After the fermentation product of Boletus edulis is subjected to ultrasonic crushing and water bath heating treatment, an appropriate amount of 90-100% ethanol is added to the treatment solution to control the final ethanol concentration to reach 60-80% (v / v). After mixing, the mixture is allowed to stand at 0-4°C for 30-60 minutes to precipitate macromolecules such as proteins.

5. The method for producing ergothioneine by fermenting edible mushroom Boletus edulis according to claim 1, wherein The decolorization treatment of the S4 fermentation liquid comprises: concentrating the supernatant collected in step S3 under low-pressure rotary evaporation at 0.05-0.1 MPa and 20-35° C. so that the ergothioneine content therein is in the range of 0.5-1 g / L, adjusting the pH of the concentrated solution to 2-4, and loading the concentrated solution into a decolorization resin column for depigmentation treatment.

6. The method for producing ergothioneine by fermenting edible fungi Boletus edulis according to claim 5, characterized in that, The decolorizing resin is D941 anion exchange resin, the mass ratio of the concentrate to the D941 anion exchange resin is 1:2-2:3, the sample loading flow rate is maintained at 12-20 mL / min, and the filtrate is collected.

7. The method for producing ergothioneine by fermenting edible fungi Boletus edulis according to claim 1, wherein S5 adsorption and elution of thioneine: the filtrate of step S4 decolorization treatment is loaded into a cation exchange resin column for adsorption, after adsorption is completed, 1% ~ 5% (v / v) ammonia / ethanol solution is used to carry out gradient elution, the solution ratio of gradient elution is 10: 0, 9: 1, 8: 2, 7: 3, 6: 4, 5: 5, 1: 3, 0: 10, and the elution time of each gradient is 30 ~ 40 minutes, and the elution flow rate is controlled at 1 ~ 4mL / min.

8. The method for producing ergothioneine by fermenting edible fungi Boletus edulis according to claim 7, wherein The cation exchange resin is 732 cation exchange resin, the pH of the filtrate is adjusted to 4-8 to adapt to the adsorption characteristics of the 732 cation exchange resin, the mass ratio of the filtrate to the 732 cation exchange resin is 1:2-2:3, and the sample flow rate is maintained at 1-4 mL / min.

9. The method for producing ergothioneine by fermenting edible fungi Boletus edulis according to claim 1, characterized in that, S6 The purification of thioneine comprises: the eluent collected in step S5 is subjected to low-pressure rotary evaporation at 35-45° C. and 0.05-0.1 MPa to obtain a crude powder; the crude product is dissolved in an ethanol solution of 90-100% at a solid-liquid ratio of 1:100-1:10, centrifuged at 0-4° C. and 8000-12000 rpm for 5-10 minutes, insoluble impurities are removed, and a supernatant is collected; low-pressure rotary evaporation is performed again at 35-45° C. and 0.05-0.1 MPa to obtain a high-purity thioneine crystalline powder.

10. The method for producing ergothioneine by fermenting edible fungi Boletus edulis according to claim 1, characterized in that: The content detection of thioneine in above-mentioned steps S4, S5 and S6 sample adopts HPLC method to analyze, and concrete grammar is: adopt high performance liquid chromatograph Waters e2695, detector is Waters 2998, and ultraviolet detector wavelength is 254nm, and chromatographic column selects XCharge C18 (5 μL 100A 4.6 × 250mm Column), and mobile phase is 0.1% formic acid solution, and sample size is 10 μL, gradient program: 0-20min: 0.5mL / min; 21-29min: 0.8mL / min; 30-35min: 0.5mL / min; Column temperature is 20~30 DEG C.

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