Method for efficiently preparing ergothioneine in pilose antler mushroom feet

Abstract

The invention discloses a method for efficiently preparing ergothioneine in pilose antler mushroom feet, which comprises the following steps: (1) pretreating the pilose antler mushroom feet to obtain mushroom foot powder; (2) carrying out mild enzymolysis on the mushroom foot powder by adopting a compound enzyme preparation to destroy a cell wall structure and release intracellular ergothioneine; (3) mixing the enzymatic hydrolysate with a specific aqueous two-phase extraction system, and performing primary extraction to selectively distribute ergothioneine to an upper phase; (4) performing acid regulation and precipitation on the upper-phase solution to obtain an ergothioneine crude product; and (5) purifying the crude product by using a green deep eutectic solvent to obtain high-purity ergothioneine. The innovation point of the method is that targeting composite enzymatic hydrolysis and an aqueous two-phase system are combined for the first time, the method is specially used for the specific raw material, namely the pilose antler mushroom feet, efficient, green and high-selectivity extraction of the ergothioneine is achieved, the extraction rate and the purity are remarkably higher than those of a traditional method, and the preparation method is simple and safe in process, low in cost and beneficial to large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for efficiently preparing ergothionein from the stem of *Deer Antler Fungus*. Background Technology

[0002] Ergothioneine (EGT) is a naturally occurring sulfur-containing amino acid derivative. Its powerful antioxidant capacity and cell protection effects have led to its widespread application in the fields of medicine, beauty and skin care, and health products, and market demand continues to grow.

[0003] However, current methods for preparing EGT have many problems. Traditional chemical synthesis methods are complex, demanding, and require sophisticated equipment. They also use large amounts of chemical reagents, resulting in numerous byproducts, significant environmental impact, and high costs, thus hindering their large-scale application. Extraction from natural raw materials, on the other hand, is also difficult to meet market demand due to the extremely low EGT content in the raw materials and the cumbersome and inefficient extraction processes.

[0004] Existing technologies, such as the invention patent with publication number CN110372597A, disclose a method for extracting ergothioneine from *Pleurotus eryngii*, which involves sequentially subjecting *Pleurotus eryngii* to vacuum freeze-drying, pulverizing and sieving, microwave extraction, and ultrasonic pulverization, finally collecting the supernatant to obtain an ergothioneine extract. Another invention patent with publication number CN109293572A discloses a method for extracting ergothioneine and polysaccharides from *Pleurotus ostreatus*, which involves sequentially pulverizing and defatting dried *Pleurotus ostreatus* fruiting bodies to obtain *Pleurotus ostreatus* powder, then subjecting the powder to water extraction, vacuum filtration, and decolorization to obtain an *Pleurotus ostreatus* extract, which contains ergothioneine. However, the ergothioneine content obtained from these two methods is limited.

[0005] Deer antler mushroom, a common edible fungus, is rich in various nutrients and bioactive substances. During the harvesting and processing of deer antler mushrooms, a large amount of mushroom stems are generated. These stems have traditionally been mostly treated as waste, resulting in resource waste and environmental burden. Research has found that deer antler mushroom stems contain a certain amount of ergothioneine (EGT). Developing an efficient extraction method to extract EGT from deer antler mushroom stems could turn waste into treasure, reducing EGT production costs, improving resource utilization, and minimizing the environmental impact of waste, thus offering significant economic and environmental benefits. Therefore, exploring an efficient method for preparing EGT from deer antler mushroom stems has important practical significance and application value. Summary of the Invention

[0006] To address the shortcomings of the aforementioned technologies, this invention provides a method for efficiently and environmentally extracting high-purity ergothioneine from the stem of *Amanita muscaria*.

[0007] The technical solution adopted by this invention to solve its technical problem is: This invention provides a method for efficiently preparing ergothionein from the stem of *Agaricus esculentus*, comprising the following steps: S1. Pretreatment: The stems of the deer antler mushroom are washed, dried and crushed to obtain mushroom stem powder; S2. Enzymatic hydrolysis: The mushroom stem powder is mixed with a buffer solution with a weakly acidic pH, and a compound enzyme preparation is added for enzymatic hydrolysis to obtain an enzymatic hydrolysate. The compound enzyme preparation contains cellulase, chitinase, and β-glucanase; S3. Initial purification: The enzymatic hydrolysate is subjected to solid-liquid separation. The supernatant is mixed with the aqueous two-phase extraction system. After standing and phase separation, the upper phase solution rich in ergothioneine is collected. S4. Precipitation: The upper phase solution is adjusted to acidic conditions and allowed to stand under refrigeration to precipitate ergothioneine, which is then separated to obtain the ergothioneine.

[0008] In step S1, the collected mushroom stems of *Pleurotus ostreatus* are rinsed with clean water to remove surface impurities, soil, and residual culture medium. The rinsed mushroom stems are then placed in an oven to dry, facilitating subsequent pulverization and storage. The dried mushroom stems are then pulverized into coarse powder using a pulverizer to increase their specific surface area and improve the dissolution efficiency of ergothioneine during subsequent extraction.

[0009] Preferably, in step S1, the moisture content of the dried mushroom stem powder is not higher than 10%, and the particle size range is 60-80 mesh.

[0010] Preferably, in step S2, the pH value of the weakly acidic buffer solution is 5.0 to 6.0; the buffer solution may be selected from, but is not limited to, a citrate-disodium hydrogen phosphate buffer solution. The ratio of mushroom stem powder to buffer solution is 1:15~30 (g / mL). The mass ratio of cellulase, chitinase, and β-glucanase is 1~4:0.5~3:1; the total amount of the compound enzyme preparation added is 1%~5% of the dry weight of the mushroom stem powder; The enzymatic hydrolysis temperature is 40~50℃, and the enzymatic hydrolysis time is 1~2 hours.

[0011] This invention uses a compound enzyme preparation to gently enzymatically hydrolyze mushroom stem powder, thereby disrupting the cell wall structure and enabling better release of intracellular ergothionein.

[0012] More preferably, after the enzymatic hydrolysis is completed, the step further includes heating the hydrolysate to 85-90°C and maintaining it for 5-10 minutes to inactivate the enzyme.

[0013] Preferably, in step S3, the aqueous two-phase extraction system is composed of a hydrophilic ionic liquid, a sugar alcohol, and an inorganic salt. The hydrophilic ionic liquid is one or more of 1-butyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium acetate, and 1-ethyl-3-methylimidazolium methyl sulfate. The sugar alcohol is one or more of sorbitol, xylitol, and maltitol; The inorganic salt is one or more of dipotassium hydrogen phosphate, potassium chloride, and sodium chloride; The mass ratio of the hydrophilic ionic liquid, sugar alcohol, and inorganic salt is 2~2.5:1.5~2:1.

[0014] This invention mixes the enzymatic hydrolysate with a specific aqueous two-phase extraction system for primary extraction, enabling ergothionein to be selectively distributed to the upper phase.

[0015] More preferably, in step S3, the mass concentration of the aqueous two-phase extraction system is 35%~50%; The volume ratio of the supernatant of the enzymatic hydrolysate to the aqueous two-phase extraction system is 1:1 to 1.5.

[0016] Preferably, in step S4, at least one of dilute hydrochloric acid, citric acid, or oxalic acid is used to adjust the pH of the upper phase solution to 3.0-4.0. The temperature for static placement under refrigeration conditions is 0~4℃, and the time is 8~12 hours.

[0017] The method of the present invention further includes step S5: dissolving the ergothione obtained in step S4 in a deep eutectic solvent, and then removing the deep eutectic solvent to obtain high-purity ergothione.

[0018] Preferably, the deep eutectic solvent is composed of choline chloride and lactic acid in a molar ratio of 1:2 to 4. The mass-to-volume ratio of ergothioneine to deep eutectic solvent is 1:50~100 (g / mL).

[0019] The method for removing the deep eutectic solvent is vacuum distillation; The vacuum distillation is carried out at a temperature of 60~80℃ and a vacuum degree of -0.08~-0.095 MPa.

[0020] Compared with the prior art, the beneficial effects of this invention are: 1. Raw material innovation: Using discarded deer antler mushroom stems as raw materials, turning waste into treasure, and reducing costs.

[0021] 2. High extraction efficiency: Through the synergistic effect of compound enzymatic hydrolysis and efficient aqueous two-phase extraction, the extraction rate of ergothionein can reach more than 90%, which is much higher than that of traditional hot water extraction (usually <60%).

[0022] 3. High product purity: After initial purification in aqueous two-phase and fine purification in deep eutectic solvent, the final product purity can reach over 98%.

[0023] 4. Green and environmentally friendly process: The entire process avoids the use of toxic organic solvents, and the enzymes, ionic liquids, sugar alcohols and deep eutectic solvents used are all environmentally friendly reagents.

[0024] 5. Simple process and easy to scale up: The process is simple and the conditions are mild, making it very suitable for large-scale industrial production. Detailed Implementation

[0025] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0026] This invention provides a method for efficiently preparing ergothionein from the stem of *Agaricus esculentus*, comprising the following steps: Step 1: Rinse the collected mushroom stems with clean water to remove surface impurities, dirt, and residual culture medium. Place the rinsed mushroom stems in an oven to dry until the moisture content is below 10%, facilitating subsequent grinding and storage. Grind the dried mushroom stems into coarse powder using a grinder, achieving a particle size of 60-80 mesh to increase their specific surface area and improve the dissolution efficiency of ergothioneine during subsequent extraction. Step 2: Mix the coarse mushroom stem powder with a citrate-disodium hydrogen phosphate buffer solution with a pH of 5.0-6.0 at a ratio of 1:15-30 (g / mL). Add a compound enzyme preparation consisting of cellulase, chitinase, and β-glucanase in a mass ratio of 1-4:0.5-3:0.5. Gently stir and enzymatically hydrolyze the mixture at 40-50℃ for 1-2 hours. The total amount of compound enzyme added is 1%-5% of the dry weight of the mushroom stem powder. After enzymatic hydrolysis, heat the mixture in a water bath at 85-90℃ for 5-10 minutes to inactivate the enzyme.

[0027] Step 3: Centrifuge the enzymatic hydrolysate, collect the supernatant, mix it with the aqueous two-phase extraction system, shake thoroughly, and allow it to stand for phase separation. The aqueous two-phase extraction system consists of a hydrophilic ionic liquid, sugar alcohol, and inorganic salt. The ionic liquid is one or more of 1-butyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium acetate, and 1-ethyl-3-methylimidazolium sulfate methyl ester. The sugar alcohol is one or more of sorbitol, xylitol, and maltitol. The inorganic salt is one or more of dipotassium hydrogen phosphate, potassium chloride, and sodium chloride. The mass ratio of the three components is 2~2.5∶1.5~2∶1, and a solution with a total concentration of 35%~50% is prepared. The enzymatic hydrolysate supernatant is mixed with the aqueous two-phase extraction system at a volume ratio of 1:1~1.5.

[0028] Step 4: Collect the upper phase solution from Step 3, adjust the pH value to 3.0-4.0 with one or more of dilute hydrochloric acid, citric acid, and oxalic acid, refrigerate at 0-4℃ for 8-12 hours, centrifuge to collect the precipitate, and obtain crude ergothioneine.

[0029] Step 5: Dissolve crude ergothioneine in a green deep eutectic solvent, and recover the deep eutectic solvent by vacuum distillation to obtain high-purity ergothioneine crystals. The deep eutectic solvent consists of choline chloride and lactic acid in a molar ratio of 1:2~4; the mass-to-volume ratio of crude ergothioneine to the deep eutectic solvent is 1:50~100 (g / mL).

[0030] Example 1 Take 1 kg of fresh deer antler mushroom stems, wash and dry them, crush them and pass them through an 80-mesh sieve to obtain about 120 g of mushroom stem powder. Mix 120 g of mushroom stem powder with 2.4 L of citrate-disodium hydrogen phosphate buffer solution with a pH of 5.5.

[0031] Add a complex enzyme consisting of 3.0 g cellulase, 2.25 g chitinase, and 0.75 g β-glucanase (total addition 5%). Incubate at 45°C and 150 rpm with shaking for 1.5 hours. After incubation, inactivate the enzyme in a 90°C water bath for 8 minutes, then rapidly cool to room temperature and centrifuge at 8000 rpm for 15 minutes. Collect the supernatant.

[0032] Preparation of the aqueous two-phase system: Weigh 140g of BF4, 140g of sorbitol, and 70g of dipotassium hydrogen phosphate, add deionized water to a total mass of 1kg, and dissolve thoroughly. Take 1L of the enzymatic hydrolysis supernatant and mix it with 1.2L of the above aqueous two-phase system solution, shake vigorously for 3 minutes, and then let it stand for 30 minutes to allow for complete phase separation.

[0033] Ergothioneine was mainly concentrated in the upper phase. Approximately 1.1 L of the upper phase solution was carefully collected, and the pH was slowly adjusted to 3.5 with 1M dilute hydrochloric acid. The solution was then placed in a 4°C refrigerator and allowed to stand for 8 hours. A white flocculent precipitate was observed to form. The precipitate was centrifuged at 8000 rpm for 10 minutes, collected, and freeze-dried to obtain approximately 0.85 g of crude ergothioneine.

[0034] 0.85 g of crude ergothioneine was dissolved in 50 mL of a deep eutectic solvent prepared from choline chloride and lactic acid (molar ratio 1:2). After complete dissolution, the solution was subjected to vacuum distillation at 70 °C and -0.085 MPa to recover the deep eutectic solvent. After the solvent was evaporated to dryness, 0.78 g of white crystalline high-purity ergothioneine product was obtained.

[0035] According to high performance liquid chromatography (HPLC) analysis, the purity of the final product in this example reached 98.5%, and the total extraction rate from raw materials to the final product was 91.2%.

[0036] Example 2 Take 5 kg of fresh deer antler mushroom stems, wash and dry them, crush them and pass them through an 80-mesh sieve to obtain about 580 g of mushroom stem powder. Mix 580 g of mushroom stem powder with 8.7 L of citrate-disodium hydrogen phosphate buffer solution with a pH of 6.0.

[0037] Add a complex enzyme consisting of 5.8g cellulase, 3.8g chitinase, and 1.9g β-glucanase (total addition 2%). Incubate at 40℃ and 150rpm with shaking for 2.0 hours. After incubation, inactivate the enzyme in an 85℃ water bath for 10 minutes, then rapidly cool to room temperature, centrifuge at 8000rpm for 15 minutes, and collect the supernatant.

[0038] Preparation of the aqueous two-phase system: Weigh 1000g of 1-ethyl-3-methylimidazolium acetate, 750g of xylitol, and 500g of sodium chloride, add deionized water to a total mass of 5kg, and dissolve thoroughly. Take 4.8L of the enzymatic hydrolysis supernatant and mix it with 7.2L of the above aqueous two-phase system solution, shake vigorously for 3 minutes, and then let it stand for 30 minutes to allow for complete phase separation.

[0039] Ergothioneine was mainly concentrated in the upper phase. Approximately 6.0 L of the upper phase solution was carefully collected, and the pH was slowly adjusted to 3.0 with 1 M oxalic acid. The solution was then placed in a 4°C refrigerator and allowed to stand for 10 hours. A white flocculent precipitate was observed to form. The precipitate was centrifuged at 8000 rpm for 10 minutes, collected, and freeze-dried to obtain approximately 4.3 g of crude ergothioneine.

[0040] 4.3 g of crude ergothioneine was dissolved in 360 mL of a deep eutectic solvent prepared from choline chloride and lactic acid (molar ratio 1:2). After complete dissolution, the solvent was recovered by vacuum distillation at 60 °C and -0.095 MPa. After the solvent was evaporated to dryness, 3.5 g of white crystalline high-purity ergothioneine product was obtained.

[0041] According to high performance liquid chromatography (HPLC) analysis, the purity of the final product in this example reached 98.2%, and the total extraction rate from raw materials to the final product was 92.7%.

[0042] Example 3 Take 10 kg of fresh deer antler mushroom stems, wash and dry them, crush them and pass them through an 80-mesh sieve to obtain about 1.15 kg of mushroom stem powder. Mix the 1.15 kg of mushroom stem powder with 30 L of citrate-disodium hydrogen phosphate buffer solution with a pH of 5.0.

[0043] Add a complex enzyme consisting of 7.5g cellulase, 5.0g chitinase, and 2.8g β-glucanase (total addition 1.3%). Incubate at 45℃ and 150rpm with shaking for 1.5 hours. After incubation, inactivate the enzyme in an 80℃ water bath for 10 minutes, then rapidly cool to room temperature, centrifuge at 8000rpm for 15 minutes, and collect the supernatant.

[0044] Preparation of the aqueous two-phase system: Weigh 7.5 kg of 1-ethyl-3-methylimidazolium sulfate methyl ester, 4.5 kg of maltitol, and 3 kg of potassium chloride, and add deionized water to a total mass of 30 kg, dissolving thoroughly. Take 28 L of the enzymatic hydrolysis supernatant and mix it with 30 L of the above aqueous two-phase system solution, shake vigorously for 3 minutes, and then let it stand for 30 minutes to allow for complete phase separation.

[0045] Ergothioneine was mainly concentrated in the upper phase. Approximately 28 L of the upper phase solution was carefully collected, and the pH was slowly adjusted to 4.0 with 1 M citric acid. The solution was then placed in a 4°C refrigerator and allowed to stand for 12 hours. A white flocculent precipitate was observed to form. The precipitate was centrifuged at 8000 rpm for 10 minutes, collected, and freeze-dried to obtain approximately 8.7 g of crude ergothioneine.

[0046] 8.7 g of crude ergothioneine was dissolved in 870 mL of a deep eutectic solvent prepared from choline chloride and lactic acid (molar ratio 1:2). After complete dissolution, the solvent was recovered by vacuum distillation at 80 °C and -0.080 MPa. After the solvent was evaporated to dryness, 8.0 g of white crystalline high-purity ergothioneine product was obtained.

[0047] According to high performance liquid chromatography (HPLC) analysis, the purity of the final product in this example reached 98.3%, and the total extraction rate from raw materials to the final product was 92.2%.

[0048] Comparative Example Based on the preliminary experiments, the results of the comparative experiments were summarized and compared with the results of the invention embodiments.

[0049] 1. Comparative Example 1: Single enzyme / combination of two enzymes, other steps are the same as in the embodiments of the invention, and the parameters and results are shown in Table 1.

[0050] Table 1

[0051] 2. Comparative Example 2: Compound enzymatic hydrolysis + conventional purification method, other steps are the same as in the invention embodiment, and the parameters and results are shown in Table 2.

[0052] Table 2

[0053] 3. Comparative Example 3: No enzymatic hydrolysis + aqueous two-phase extraction, other steps are the same as in the invention examples, and the parameters and results are shown in Table 3.

[0054] Table 3

[0055] Comparative data demonstrates that the combination of "compound enzyme combination" and "aqueous two-phase extraction" is not a simple superposition of conventional techniques, but rather produces unexpected extraction results. The comparative data visually showcases the advantages of the invented process in terms of extraction rate and purity, further illustrating the significant effectiveness of the invention.

Claims

1. A method for efficiently preparing ergothionein from the stem of *Flammulina velutipes*, characterized in that, Includes the following steps: S1. Pretreatment: The stems of the deer antler mushroom are washed, dried and crushed to obtain mushroom stem powder; S2. Enzymatic hydrolysis: The mushroom stem powder is mixed with a buffer solution with a weakly acidic pH, and a compound enzyme preparation is added for enzymatic hydrolysis to obtain an enzymatic hydrolysate. The compound enzyme preparation contains cellulase, chitinase, and β-glucanase; S3. Initial purification: The enzymatic hydrolysate is subjected to solid-liquid separation. The supernatant is mixed with the aqueous two-phase extraction system. After standing and phase separation, the upper phase solution rich in ergothioneine is collected. S4. Precipitation: The upper phase solution is adjusted to acidic conditions and allowed to stand under refrigeration to precipitate ergothioneine, which is then separated to obtain the ergothioneine.

2. The method for efficiently preparing ergothionein from the stem of *Flammulina velutipes* according to claim 1, characterized in that, In step S1, the moisture content of the dried mushroom stem powder is not higher than 10%, and the particle size range is 60-80 mesh.

3. The method for efficiently preparing ergothionein from the stem of *Flammulina velutipes* according to claim 1, characterized in that, In step S2, the pH value of the weakly acidic buffer solution is 5.0~6.0; The ratio of mushroom stem powder to buffer solution is 1:15~30; The mass ratio of cellulase, chitinase, and β-glucanase is 1~4:0.5~3:1; the total amount of the compound enzyme preparation added is 1%~5% of the dry weight of the mushroom stem powder; The enzymatic hydrolysis temperature is 40~50℃, and the enzymatic hydrolysis time is 1~2 hours.

4. The method for efficiently preparing ergothionein from the stem of *Flammulina velutipes* according to claim 1 or 3, characterized in that, After enzymatic hydrolysis, the process also includes heating the hydrolysate to 85-90°C and maintaining it for 5-10 minutes to inactivate the enzyme.

5. The method for efficiently preparing ergothionein from the stem of *Flammulina velutipes* according to claim 1, characterized in that, In step S3, the aqueous two-phase extraction system consists of a hydrophilic ionic liquid, a sugar alcohol, and an inorganic salt. The hydrophilic ionic liquid is one or more of 1-butyl-3-methylimidazolium tetrafluoroborate BF4, 1-ethyl-3-methylimidazolium acetate, and 1-ethyl-3-methylimidazolium methyl sulfate. The sugar alcohol is one or more of sorbitol, xylitol and maltitol; The inorganic salt is one or more of dipotassium hydrogen phosphate, potassium chloride, and sodium chloride; The mass ratio of the hydrophilic ionic liquid, sugar alcohol, and inorganic salt is 2~2.5:1.5~2:

1.

6. The method for efficiently preparing ergothionein from the stem of *Flammulina velutipes* according to claim 1 or 5, characterized in that, In step S3, the mass concentration of the aqueous two-phase extraction system is 35%~50%; The volume ratio of the supernatant of the enzymatic hydrolysate to the aqueous two-phase extraction system is 1:1 to 1.

5.

7. The method for efficiently preparing ergothionein from the stem of *Flammulina velutipes* according to claim 1, characterized in that, In step S4, at least one of hydrochloric acid, citric acid, or oxalic acid is used to adjust the pH of the upper phase solution to 3.0-4.0; The temperature for static placement under refrigeration conditions is 0~4℃, and the time is 8~12 hours.

8. The method for efficiently preparing ergothionein from the stem of *Flammulina velutipes* according to claim 1, characterized in that, The method further includes step S5: dissolving the ergothione obtained in step S4 in a deep eutectic solvent, and then removing the deep eutectic solvent to obtain high-purity ergothione.

9. The method for efficiently preparing ergothionein from the stem of *Flammulina velutipes* according to claim 8, characterized in that, The deep eutectic solvent is composed of choline chloride and lactic acid in a molar ratio of 1:2 to 4. The mass-to-volume ratio of ergothionein to the deep eutectic solvent is 1:50~100.

10. The method for efficiently preparing ergothionein from the stem of *Flammulina velutipes* according to claim 8, characterized in that, The method for removing the deep eutectic solvent is vacuum distillation; The vacuum distillation is carried out at a temperature of 60~80℃ and a vacuum degree of -0.08~-0.095 MPa.

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