A method for efficient purification of ergothioneine from a cordyceps mycelium

Abstract

The application discloses a high-efficiency purification method of ergothioneine in H. erinaceus mycelium, and belongs to the technical field of biochemical products and food. The method comprises the steps of drying, grinding, extracting, alcohol precipitation and impurity removal, resin purification and concentration, and the like, and a high-purity ergothioneine solution is obtained. The method has the advantages of simple production process, low cost, safety and reliability, no introduction of harmful organic solvents and other new impurities, and the like, is suitable for extracting and purifying ergothioneine from H. erinaceus mycelium, and provides a high-purity, low-cost and natural H. erinaceus ergothioneine product for food, medical, cosmetic and other industries. The purity of the final ergothioneine solution is 85% to 92%, and the recovery rate of ergothioneine is 50% to 65%, which can meet most demands of food, cosmetics, laboratories and the like.

Description

Technical Field

[0001] This invention belongs to the field of biochemical products and food technology, specifically relating to a highly efficient purification method for ergothionein from hedgehog mycelium. Background Technology

[0002] Ergothioneine (EGT), scientific name 2-mercapto-histidine-trimethyl lactone (C9H) 16 Ergothioneine (N3O2S) is a rare compound derived from histidine. Due to its high redox potential and unique thione structure, it is not easily oxidized under natural pH conditions. The pure product is a colorless crystal, highly soluble in water and ethanol, and possesses strong antioxidant properties. Since the human body cannot synthesize it and must obtain it from external sources, it has effects such as scavenging free radicals, whitening, and anti-aging. As regulations in various countries increasingly permit its use in food, it has gained greater market value. With decreasing production costs, ergothioneine is being used more and more widely in the food, pharmaceutical, and cosmetic fields.

[0003] Hericium erinaceus (Bull.) Pers., also known as hedgehog mushroom, has a long history in China. As a fungus that is both food and medicine, it is not only delicious but also contains many nutrients. Hericium erinaceus can produce ergothioneine. After extraction from the mycelium of Hericium erinaceus by heating and reflux, and after removing impurities such as protein by alcohol precipitation, the purity of ergothioneine is only about 50%, which is too low. The solution also contains many small molecule impurities, which cannot meet the application requirements.

[0004] There are various methods for purifying ergothionein from edible fungi. Patent CN 113413337 A discloses a method for preparing a fungal extract rich in ergothionein and nicotinamide, which uses chitosanase to disrupt the cell wall of the fungi to release intracellular substances. However, the enzymatic hydrolysis conditions are quite stringent, and the added enzyme acts as a new impurity, increasing the burden on subsequent purification. 113666873A discloses an industrial method for preparing high-purity ergothionein, in which the purification uses a HILIC column and multiple columns in series. The lifespan of the column will be shortened as the throughput increases, and the cost is high. The purification using multiple resin columns in series is cumbersome. There are also reports on using G-10 dextran gel to purify ergothionein from enoki mushrooms, with a high recovery rate of 93.98%, but the purity is low at only 54.83% (Mo Yuli, Zhang Yixin, Wang Yan, et al. Optimization of separation and purification process of ergothionein from enoki mushrooms [J]. Edible Fungi, 2018, 40(5):64-67,70.).

[0005] In summary, existing methods for extracting ergothionein from edible fungi mycelia cannot simultaneously achieve simplicity, low cost, extremely high purity, and industrial application; no method has been found that uses only one ion exchange resin to purify ergothionein from hedgehog mycelia with a purity of 90%. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide a highly efficient purification method for ergothioneine from *Hedgehog's tick* mycelium. The production process is simple, requires no cumbersome operations, and does not introduce harmful organic solvents or other new impurities. It is suitable for extracting and purifying ergothioneine from *Hedgehog's tick* mycelium. This provides the food, medical, and cosmetic industries with high-purity, inexpensive, and naturally occurring *Hedgehog's tick* ergothioneine products.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A highly efficient method for purifying ergothionein from hedgehog mycelium includes the following preparation steps:

[0009] (1) Drying: Drying fresh hedgehog mycelium;

[0010] (2) Grinding: Grind or crush the hedgehog mycelium obtained in step (1) into powder to obtain hedgehog mycelium powder;

[0011] (3) Extraction: The hedgehog mycelium powder obtained in step (2) is added to a certain proportion of ethanol aqueous solution according to a specific material-liquid ratio and heated and refluxed for extraction. After extraction, the extract is filtered and the filtrate is retained.

[0012] (4) Alcohol precipitation to remove impurities: Add a certain amount of anhydrous ethanol to the filtrate obtained in step (3) for alcohol precipitation, centrifuge, and retain the supernatant;

[0013] (5) Resin purification: The supernatant obtained in step (4) is used as the original solution for column loading. Macroporous weakly basic anion exchange resin is used for adsorption and impurity removal. After loading, pure water is used to remove unadsorbed impurities, and then alkaline pure water is used to elute at a certain flow rate. The eluent is collected.

[0014] (6) Concentration: The first 3 column volumes of the eluent with high purity obtained in step (5) are concentrated to obtain a high-purity ergothioneine solution.

[0015] Furthermore, the drying described in step (1) is hot air drying or vacuum freeze drying; hot air drying is carried out at 60℃~70℃ for 6~8 hours; vacuum freeze drying is carried out at -60℃ for 48 hours; the specific time is determined according to the thickness of the hedgehog mycelium.

[0016] Furthermore, the grinding or pulverizing mentioned in step (2) refers to grinding or pulverizing using liquid nitrogen or a pulverizer.

[0017] Further, in step (3), the material-to-liquid ratio is 1g:10-50mL; the ethanol content in the ethanol-water solution is 10%-90% v / v; the temperature of the electric heating mantle for reflux extraction is 100±10℃; the reflux extraction time is 10min-50min; and qualitative filter paper is used for vacuum filtration.

[0018] More preferably, in step (3), the material-to-liquid ratio is 1g:20±5mL; the ethanol content in the ethanol-water solution is 40±5% v / v; the temperature for reflux extraction is 100℃; and the reflux extraction time is 20min~30min.

[0019] Further, in step (4), the addition ratio of anhydrous ethanol is 70% to 90% v / v, the alcohol precipitation refers to a standing time of 8 to 24 hours, and the centrifugation refers to a speed of 6000 rpm to 10000 rpm and a time of 5 min to 15 min.

[0020] More preferably, in step (4), the addition ratio of anhydrous ethanol is 80±10% v / v, the alcohol precipitation refers to a standing time of 12 to 18 hours, and the centrifugation refers to a speed of 8000±1000 rpm and a time of 10 to 15 minutes.

[0021] Further, in step (5), the macroporous weakly basic anion exchange resin is preferably D318 macroporous weakly basic anion exchange resin or D941 macroporous weakly basic anion exchange resin; it has been pretreated by acid washing and alkali washing before loading, and 5 to 8 column volumes of loading stock solution are added until the resin column reaches the leakage point; the loading flow rate is 1 to 5 column volumes / h; 3 to 6 column volumes of pure water are used to elute and remove unadsorbed impurities; the pH value of alkaline pure water is 11 to 14, the elution flow rate is 1 to 9 column volumes / h, and the eluent volume is 3 to 10 column volumes.

[0022] More preferably, in step (5), the macroporous weakly basic anion exchange resin is preferably D318 macroporous weakly basic anion exchange resin; it has undergone acid washing and alkali washing pretreatment before loading, and 7 times the column volume of loading stock solution is added until the resin column reaches the leakage point; the loading flow rate is 1 to 3 times the column volume / h; 5 to 6 times the column volume of pure water is used to elute and remove unadsorbed impurities; the pH value of alkaline pure water is 12±0.5, the elution flow rate is 3 times the column volume / h, and the eluent volume is 3 to 10 times the column volume.

[0023] Further, in step (6), the concentration is achieved by heating and evaporation or vacuum concentration to obtain a high-purity ergothioneine solution with a purity of 85-92% and an ergothioneine recovery rate of 50%-65%.

[0024] The present invention has the following advantages and effects compared with the prior art:

[0025] (1) The purification method of the present invention adopts the ethanol aqueous solution heating reflux extraction process, which saves time and effort, extracts ergothionein in the mycelium of hedgehog fungus to a great extent, and does not use harmful organic solvents, so there is no need to remove artificially added impurities in the subsequent process.

[0026] (2) The purification method of the present invention is based on the amphoteric nature of ergothioneine. It uses D318 macroporous weakly basic anion exchange resin for column chromatography purification and alkaline pure water elution. The process is simple and effectively reduces impurities in the ergothioneine eluent. The final ergothioneine solution has a purity of 85% to 92%, which can meet most of the needs of food, cosmetics, laboratories and other industries.

[0027] (3) Compared with existing methods for purifying ergothionein from edible fungi, the purification method of the present invention has the advantages of simple operation, high purity of ergothionein, low cost, safety and reliability, and suitability for industrial production. Attached Figure Description

[0028] Figure 1 It represents the equilibrium adsorption capacity of 10 different resins for EGT in Hedgehog mycelium.

[0029] Figure 2 The adsorption and desorption rates of EGT in Hedgehog mycelium were measured by 10 different resins.

[0030] Figure 3 This study investigated the effect of sample loading volume on the adsorption of EGT in Hedgehog mycelium during a single-factor experiment.

[0031] Figure 4 This study investigated the effect of sample loading rate on the adsorption of EGT in Hedgehog mycelium during a single-factor experiment.

[0032] Figure 5 This study investigated the effect of different eluent types on the adsorption of EGT in Hedgehog mycelium during a single-factor experiment.

[0033] Figure 6 This study investigated the effect of different elution buffers on the purity of EGT in Hedgehog mycelium during a single-factor experiment.

[0034] Figure 7 This study investigated the effect of elution flow rate on the adsorption of EGT in Hedgehog mycelium during a single-factor experiment.

[0035] Figure 8This study investigated the effect of an elution flow rate of 1 BV / h on the purity of EGT in Hedgehog mycelium during a single-factor experiment.

[0036] Figure 9 This study investigated the effect of an elution flow rate of 3 BV / h on the purity of EGT in Hedgehog mycelium during a single-factor experiment.

[0037] Figure 10 This study investigated the effect of elution flow rates of 5 BV / h on the purity of EGT in Hedgehog mycelium during a single-factor experiment.

[0038] Figure 11 This study investigated the effect of an elution flow rate of 7 BV / h on the purity of EGT in Hedgehog mycelium during a single-factor experiment.

[0039] Figure 12 This study investigated the effect of an elution flow rate of 9 BV / h on the purity of EGT in Hedgehog mycelium during a single-factor experiment.

[0040] Figure 13 It is the loading-elution curve under optimal conditions.

[0041] Figure 14 It is a standard curve established using the enzymatic method with EGT standards.

[0042] Figure 15 This is the HPLC chromatogram of the supernatant in step (3) of Example 2;

[0043] Figure 16 This is the HPLC chromatogram of the eluent from column 1 in step (5) of Example 2;

[0044] Figure 17 This is the HPLC chromatogram of the eluent from the second column in step (5) of Example 2;

[0045] Figure 18 This is the HPLC chromatogram of the eluent from column 3 in step (5) of Example 2;

[0046] Figure 19 This is the HPLC chromatogram of the first three column eluents after mixing and concentrating in step (6) of Example 2;

[0047] Figure 20 This is an HPLC chromatogram obtained from the concentration and mixing of the eluent from the first two columns in step (6) of Example 3. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the materials and reagents used are commercially available.

[0049] In the examples, purity was calculated using the area normalization method, and recovery rate refers to the percentage of total ergothioneine in the collected eluent relative to the total ergothioneine in the original sample volume.

[0050] Example 1

[0051] 1. Screening resins using static adsorption experiments

[0052] Resin types: H103 macroporous adsorption resin, DA-201 macroporous adsorption resin, HPD100 macroporous adsorption resin, DM301 macroporous adsorption resin, D318 macroporous weakly basic anion exchange resin, D1300 macroporous adsorption resin, D941 macroporous weakly basic anion exchange resin, D101-I macroporous adsorption resin, AB-8 macroporous adsorption resin, and NKA-II macroporous adsorption resin. Among these, AB-8 macroporous adsorption resin was purchased from Shanghai Yuanye Biotechnology Co., Ltd., NKA-II macroporous adsorption resin was purchased from Shaanxi Lebo Biochemical Technology Co., Ltd., and the remaining eight resins were purchased from Zhengzhou Aino Chemical Technology Co., Ltd.

[0053] Screening steps: Weigh 5g (wet weight) of each of the 10 resins that have undergone acid and alkali washing pretreatment into an Erlenmeyer flask, add 25mL of crude EGT extract from Hedgehog mycelium (i.e., the supernatant obtained in step (3) of Example 2, with an EGT concentration of 94.50mg / L determined by the rapid EGT detection method), and place it in a shaker at 150rpm and 25℃ for adsorption; after 18h, take out the supernatant and use the rapid EGT detection method to measure the EGT content and calculate the adsorption capacity and adsorption rate; then filter the resin and wash it with ultrapure water 2-3 times, transfer it to an Erlenmeyer flask, add an equal volume of 40% v / v ethanol aqueous solution to the macroporous adsorption resin and an equal volume of 5% w / v sodium hydroxide solution to the anion exchange resin for elution, and detect the EGT content of the supernatant in a shaker at 150rpm and 25℃ for 18h to calculate the desorption rate. The calculation formula is as follows:

[0054]

[0055]

[0056] In the formula: Q e E represents the adsorption capacity of the resin when it reaches equilibrium; D represents the adsorption rate; C0 represents the desorption rate; and C represents the initial concentration of EGT in the crude extract. e The remaining EGT concentration in the solution at adsorption equilibrium; V0 is the volume of the crude extract; m is the mass of the wet resin; C d The concentration of EGT in the eluent to achieve analytical equilibrium; V d This represents the volume of the elution buffer.

[0057] Static adsorption experiment results are as follows Figure 1 and Figure 2 As shown.

[0058] By comparing the adsorption capacity, adsorption rate and desorption rate of various resins for EGT, D318 macroporous weakly basic anion exchange resin was selected.

[0059] 2. Single-factor optimization design

[0060] Single-factor experiments were conducted using four factors: sample loading volume, sample loading flow rate, type of eluent, and elution flow rate (see Table 1) to determine the effects of each factor on the adsorption amount and purity of EGT in Hedgehog mycelium.

[0061] 2.1 Effect of sample loading volume on EGT adsorption in Hedgehog mycelium

[0062] Add 8 BV of crude EGT extract (i.e., the supernatant obtained in step (3) of Example 2, with an EGT concentration of 94.50 mg / L as determined by the rapid EGT detection method) to a D318 macroporous weakly basic anion exchange resin column (1 BV, 40 mL) at a loading flow rate of 1.5 times column volume / hour.

[0063] The results are as follows Figure 3 As shown, by detecting the EGT content in the effluent of each column using a rapid EGT detection method, the optimal volume of crude EGT extract as the loading stock solution was determined to be 7 BV. Based on a loading volume of 7 BV, the adsorption capacity of 1 BV (40 mL) of D318 macroporous weakly basic anion exchange resin for EGT is approximately 26.46 mg.

[0064] 2.2 Effect of sample loading flow rate on EGT adsorption in Hedgehog mycelium

[0065] Referring to 2.1, the difference is that the volume of the stock solution is 7 BV, and the loading flow rate is shown in Table 1.

[0066] The results are as follows Figure 4 As shown, the EGT content in the effluent of each column was detected by the EGT rapid detection method at different loading flow rates, and the optimal loading flow rate was determined to be 3 BV / h.

[0067] 2.3 Effects of different eluent types on the adsorption capacity and purity of EGT in Hedgehog mycelium

[0068] Referring to 2.1, the difference is that after the sample loading is completed, the resin column is eluted with 5 column volumes of naturally pure water at pH to remove unadsorbed impurities; then, different types of elution buffers (see Table 1) are used for elution at 5 BV, and the elution flow rate is 5 column volumes / hour.

[0069] The results are as follows Figure 5As shown, the EGT content in the eluent of each column after elution was detected by the rapid EGT detection method. It can be seen that ultrapure water cannot elute EGT, while water with pH 12 as the eluent has the highest EGT content.

[0070] The results are as follows Figure 6 As shown in Table 2, the EGT purity in the concentrated eluent from the first and second columns after rotary evaporation was determined by HPLC. It was found that water at pH 12 had the highest EGT purity in the first two columns when water was used as the eluent. Therefore, water at pH 12 was determined to be the optimal eluent.

[0071] 2.4 Effect of elution flow rate on the adsorption capacity and purity of EGT in Hedgehog mycelium

[0072] Referring to 2.3, the difference is that elution is performed using 10 BV of water at pH 12, and the elution flow rate is shown in Table 1.

[0073] The results are as follows Figure 7 , Figures 8-12 As shown in Table 3, the EGT content in each column eluent after elution was detected using a rapid EGT detection method. The EGT purity in the concentrate after rotary evaporation of the first few columns of eluent was detected by HPLC. It was found that the EGT purity detected at elution flow rates of 1 BV / h, 3 BV / h, and 5 BV / h was significantly better than that at 9 BV / h. At an elution flow rate of 7 BV / h, the EGT purity showed a significant decrease in the second column volume. The sum of EGT content in the first 3 BV of eluent was ranked as follows: 3 BV / h > 7 BV / h > 1 BV / h > 5 BV / h > 9 BV / h.

[0074] Taking into account time cost, improving elution yield and purity, a flow rate of 3 BV / h was selected for elution, eluting 8 BV, and collecting the first 3 BV of eluent.

[0075] Table 1 Four Single-Factor Designs

[0076]

[0077] Table 2. Percentage of EGT peak area in different types of eluents

[0078]

[0079] Table 3. HPLC chromatograms eluted with water at different flow rates at pH 12, EGT peak percentage

[0080]

[0081] After determining the optimal conditions for each factor (sample loading volume of 7 BV, sample loading flow rate of 3 BV / h, eluent of pH 12 water, and elution flow rate of 3 BV / h), sample loading and elution were performed according to the optimal method, and a curve was plotted. The first three column volumes of eluent were collected, concentrated, and subjected to liquid chromatography purity testing. The sample loading and elution curve is shown in [Figure number missing]. Figure 13 The purity test results of the concentrated EGT eluent from the first 3 column volumes are shown in the figure. Figure 19 .

[0082] The EGT fast detection method includes the following steps:

[0083] 1) Construction of recombinant expression vector: The nucleotide sequence (SEQ ID NO: 1) of the gene encoding ergothiase (GenBank: AB699692.1) was inserted between EcoRI and XhoI of the pET-28a(+) vector to obtain the recombinant expression vector, denoted as pET28a-EGTase.

[0084] 2) Construction of recombinant engineered bacteria: pET28a-EGTase was transformed into Escherichia coli BL21(DE3) to obtain recombinant engineered bacteria;

[0085] 3) Preparation of crude ergot thiocyanate solution: Inoculate the recombinant engineered bacteria from step 2) into 10 mL of LB medium (containing 50 μg / mL Kan) and incubate at 37°C with shaking at 200 rpm for 12 hours to obtain the seed culture. Inoculate the seed culture into 50 mL of LB medium containing 50 μg / mL Kan at 1% v / v and incubate at 37°C with shaking for 2-3 hours until OD is obtained. 600 Cultivation was stopped when the value reached 0.6-0.8. The bacterial culture was cooled by gentle shaking in ice water. After cooling, 1 mM IPTG inducer was added to induce protein expression. The culture was incubated at 18°C ​​and 150 rpm for 18 hours. The expression of ergothiase was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The remaining bacterial culture was centrifuged at 8000 rpm for 10 minutes at 4°C, the supernatant was discarded, the precipitate was washed twice with 1×PBS, centrifuged again to remove the supernatant, and the precipitate was resuspended in 10 mL of 1×PBS buffer. Cells were lysed by sonication at 30% power output with a 3s on / 6s off cycle for 10 minutes (to avoid sonication heating, the bacterial culture was treated in two 5-minute intervals). The lysis buffer was centrifuged at 10000 rpm for 10 minutes at 4°C, and the supernatant was transferred to a fresh 50 mL collection tube. This was the crude ergothiase enzyme solution, which was then stored at -20°C with 15% glycerol for later use.

[0086] 4) EGT content detection: Prepare a series of ergothioneine standard solutions. First, add 100 μL of sample to the ELISA plate and detect the OD value once. The result is "OD".原液 Add 100 μL of the crude ergot thiocyanate solution prepared in step 3) and gently mix with a pipette tip. Initiate the enzymatic reaction in a microplate at room temperature (using a 96-well plate with UV light). Measure the OD value after approximately 20 minutes. The result should be "OD". 酶+原液 The reaction mixture was monitored at 311 nm using "OD". 酶+原液 -OD 原液 A standard curve is plotted with "Y" as the Y-axis and EGT concentration as the X-axis. Figure 14 Based on the standard curve, the EGT content in the sample to be tested is calculated, thus enabling rapid detection of EGT content using the enzymatic method with EGT standards.

[0087] Example 2

[0088] A highly efficient method for purifying ergothionein from hedgehog mycelium includes the following preparation steps:

[0089] (1) Take 20g of dried and pulverized hedgehog mycelium powder, add 40% v / v ethanol aqueous solution at a material-to-liquid ratio of 1g:20mL to obtain 400mL solution, and perform reflux extraction at 100℃ for 30 minutes.

[0090] (2) Filter the solution obtained in step (1) to remove the filter residue and retain the filtrate.

[0091] (3) Add an appropriate amount of anhydrous ethanol to the filtrate obtained in step (2) to obtain an 80% v / v ethanol solution. Let it stand for 12 hours, then centrifuge at 8000 rpm for 10 minutes and retain the supernatant; that is, the crude EGT extract from the mycelium of Hedgehog mycelium.

[0092] (4) The supernatant obtained after centrifugation in step (3) is used as the loading solution for the D318 macroporous weakly basic anion exchange resin. 7 BV of the loading solution is added to 1 column volume (BV, 40 mL) of the resin column, and the sample is loaded at a flow rate of 3 BV / h. After loading, the resin column is eluted with 5 column volumes of naturally purified water at pH 5 to remove unadsorbed impurities. Note: The D318 macroporous weakly basic anion exchange resin has undergone acid and alkali washing pretreatment before loading.

[0093] (5) After step (4) water elution, use 10 column volumes of pure water with pH=12 to elute ergothionein at a flow rate of 3 BV / h, collect the eluent, and test the purity and content of ergothionein in the eluent.

[0094] (6) The first 3 BV eluent collected in step (5) was concentrated by rotary evaporation. The ergothioneine in the concentrate was detected by HPLC. The purity was 92.2% and the recovery rate was 51.3%.

[0095] (7) The chromatogram of ergothionein in the supernatant of this embodiment is as follows: Figure 15 As shown, the chromatograms of the 1–3 BV eluent are as follows: Figures 16-18 As shown, the chromatogram of the concentrated ergothioneine solution from the first three column volumes is as follows. Figure 19 As shown.

[0096] The method for preparing the dried and pulverized hedgehog mycelium powder in step (1) includes the following steps:

[0097] Fresh hedgehog mycelium was dried at 60°C for 8 hours using a hot air drying method; then it was ground into powder using liquid nitrogen or a pulverizer to obtain hedgehog mycelium powder.

[0098] Example 3

[0099] A highly efficient method for purifying ergothionein from hedgehog mycelium includes the following preparation steps:

[0100] (1) Take 25g of dried and pulverized hedgehog mycelium powder, add 40% v / v ethanol aqueous solution at a material-to-liquid ratio of 1g:20mL to obtain 500mL solution, and perform reflux extraction at 100℃ for 20 minutes.

[0101] (2) Filter the solution obtained in step (1) to remove the filter residue and retain the filtrate.

[0102] (3) Add an appropriate amount of anhydrous ethanol to the filtrate obtained in step (2) to obtain an 80% v / v ethanol solution. Let it stand for 18 hours, then centrifuge at 8000 rpm for 15 minutes and retain the supernatant.

[0103] (4) The supernatant obtained after centrifugation in step (3) is used as the loading solution for the D318 macroporous weakly basic anion exchange resin. 7 BV of the loading solution is added to 1 column volume (BV) of the resin column, and the sample is loaded at a flow rate of 3 BV / h. After loading, the resin column is eluted with 6 column volumes of naturally purified water at pH 6 to remove unadsorbed impurities. Note: The D318 macroporous weakly basic anion exchange resin has undergone acid and alkali washing pretreatment before loading.

[0104] (5) After step (4) water elution, use 10 column volumes of pure water with pH=12 to elute ergothionein at a flow rate of 3 BV / h, collect the eluent, and test the purity and content of ergothionein in the eluent.

[0105] (6) The first 2 BV eluent collected in step (5) was concentrated by rotary evaporation. The ergothioneine in the concentrate was detected by HPLC. The purity was 89.8% and the recovery rate was 64.4%.

[0106] (7) In this embodiment, the first 2 BV eluent was concentrated and then ergothioneine was detected. The chromatogram is shown below. Figure 20 As shown.

[0107] The method for preparing the dried and pulverized hedgehog mycelium powder in step (1) includes the following steps:

[0108] Fresh hedgehog mycelium was dried at 70°C for 6 hours using a hot air drying method; then it was ground into powder using liquid nitrogen or a pulverizer to obtain hedgehog mycelium powder.

[0109] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A highly efficient method for purifying ergothionein from hedgehog mycelium, characterized in that, The preparation steps include the following: (1) Drying: Dry the fresh hedgehog mycelium; (2) Grinding: Grind or crush the hedgehog mycelium obtained in step (1) into powder to obtain hedgehog mycelium powder; (3) Extraction: The hedgehog mycelium powder obtained in step (2) is added to a certain proportion of ethanol aqueous solution according to a specific material-liquid ratio and heated and refluxed for extraction. After extraction, the extract is filtered and the filtrate is retained. The material-liquid ratio is 1g:20±5mL; the ethanol content in the ethanol aqueous solution is 40±5%v / v; the temperature of heating and reflux extraction is 100℃; and the heating and reflux extraction time is 20 min~30 min. (4) Alcohol precipitation to remove impurities: Add a certain amount of anhydrous ethanol to the filtrate obtained in step (3) for alcohol precipitation, centrifuge, and retain the supernatant; the ratio of anhydrous ethanol added is 80±10%v / v, alcohol precipitation refers to a standing time of 12 to 18 hours, and centrifugation refers to a speed of 8000±1000 rpm and a time of 10 to 15 minutes. (5) Resin purification: The supernatant obtained in step (4) is used as the original solution for column loading. Macroporous weakly basic anion exchange resin is used for adsorption and impurity removal. After loading, pure water is used to remove unadsorbed impurities, and then alkaline pure water is used to elute at a certain flow rate. The eluent is collected. The macroporous weakly basic anion exchange resin is D318 macroporous weakly basic anion exchange resin. (6) Concentration: The first 3 column volumes of eluent with high purity obtained in step (5) are concentrated to obtain a high-purity ergothioneine solution; In step (5), add 7 column volumes of the original loading solution; the loading flow rate is 1 to 3 column volumes / h; use 5 to 6 column volumes of pure water to elute and remove unadsorbed impurities; the pH value of the alkaline pure water is 12±0.5, the elution flow rate is 3 column volumes / h, and the eluent volume is 3 to 10 column volumes.

2. The method for efficient purification of ergothionein from hedgehog mycelium according to claim 1, characterized in that: The drying process described in step (1) is either hot air drying or vacuum freeze drying; hot air drying is carried out at 60℃~70℃ for 6~8 hours; vacuum freeze drying is carried out at -60℃ for 48 hours; the specific time depends on the thickness of the hedgehog mycelium.

3. The method for efficient purification of ergothionein from hedgehog mycelium according to any one of claims 1 to 2, characterized in that: In step (2), the grinding or pulverizing refers to grinding with liquid nitrogen or pulverizing with a pulverizer; In step (6), the concentration is either heating evaporation concentration or vacuum concentration.

4. The method for efficient purification of ergothionein from hedgehog mycelium according to any one of claims 1 to 2, characterized in that: In step (6), the purity of the high-purity ergothioneine solution is 85-92%, and the ergothioneine recovery rate is 50%-65%.

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