Process method for extracting ergothioneine from pichia pastoris fermentation liquor

Abstract

The invention relates to the technical field of ergothioneine, in particular to a process method for extracting ergothioneine from pichia pastoris fermentation liquor, which comprises the following steps: taking the pichia pastoris fermentation liquor containing ergothioneine, detecting isoelectric points of the fermentation liquor and charges carried by small molecular substances, then adding corresponding flocculants according to the charges carried, adjusting the pH value to be equal to the isoelectric points, and finally extracting the ergothioneine from the pichia pastoris fermentation liquor containing ergothioneine. (diluting the obtained first turbid solution with water, then adjusting the pH value to be equal to an isoelectric point, treating the second clear solution collected after filtration with an ultrafiltration membrane, adjusting the pH value of the collected ultrafiltration clear solution to be acidic, passing through a cationic resin column, eluting with an eluent, concentrating the collected eluent with a nanofiltration membrane, concentrating in vacuum, cooling, carrying out suction filtration, and washing to obtain the product. And drying to obtain the ergothioneine product with high yield and high purity.

Description

Technical Field

[0001] The present invention relates to the technical field of ergothioneine, and particularly relates to a process for extracting ergothioneine from Pichia pastoris fermentation broth. Background Art

[0002] Ergothioneine (EGT), also known as 2-mercapto-L-histidine trimethylammonium inner salt, with a relative molecular mass of 229.3, is a very rare natural chiral small molecule amino acid. Currently, there are mainly four preparation methods for ergothioneine: direct extraction from fungi, chemical synthesis, enzyme-catalyzed method, and microbial fermentation synthesis. The direct extraction from fungi is mainly from edible fungi. Since the content of ergothioneine in fungi is relatively low, the purity of ergothioneine extracted from edible fungi is low, it is difficult to produce in large quantities, cannot meet the market demand, and the cost is high, which is generally unacceptable to consumers. The chemical synthesis process route is relatively long, the yield is very low, and the environmental pollution is serious, so the cost has been high. The enzyme-catalyzed reaction is affected by enzyme activity, with a long catalytic cycle and low yield. Compared with the above several production methods of ergothioneine, the microbial fermentation method has a higher yield, more stable production, lower raw material cost, better benefits, and is more green and natural. Therefore, the microbial fermentation method is the mainstream for the industrial production of ergothioneine in the future.

[0003] Currently, there have been reports on using genetic engineering transformation of Escherichia coli and Saccharomyces cerevisiae to produce ergothioneine, both of which are achieved by overexpressing genes in the ergothioneine synthesis pathway. However, Escherichia coli and Saccharomyces cerevisiae are host systems that were developed earlier for heterologous gene expression, and they have limitations such as limited culture density and low secretion efficiency.

[0004] Pichia pastoris is an important industrial host bacterium, a type of yeast that can utilize methanol as the sole carbon source and energy source, and is a new generation of yeast expression system, which is widely used in the production of proteins and pharmaceutical antibodies. Chinese patent text CN116286421A discloses a Pichia pastoris strain for producing ergothioneine, which realizes the efficient synthesis of ergothioneine, and the obtained Pichia pastoris strain can achieve high-density culture and regulate the oxidation / peroxidation state, solving the problem that the oxidative damage of Pichia pastoris cells weakens the yield of ergothioneine, but there is little description of the method for extracting ergothioneine from Pichia pastoris fermentation broth. Summary of the Invention

[0005] The technical problem to be solved by the present invention is: aiming at the deficiencies of the prior art, to provide a process for extracting ergothioneine from Pichia pastoris fermentation broth, and using this process to obtain ergothioneine products with high yield and high purity.

[0006] To solve the above technical problem, the technical solution of the present invention is:

[0007] A process for extracting ergothioneine from Pichia pastoris fermentation broth, the process comprising the following steps:

[0008] (1) Take the Pichia pastoris fermentation broth containing ergothioneine, detect the isoelectric point of the Pichia pastoris fermentation broth and the charges carried by small molecules in the Pichia pastoris fermentation broth, add a flocculant according to the type of charge carried, then adjust the pH value of the Pichia pastoris fermentation broth to be equal to the isoelectric point, and after the first filtration, collect the first supernatant and the first turbid liquid respectively; Take the first turbid liquid, dilute it with water, adjust the pH value to be equal to the isoelectric point pi of the Pichia pastoris fermentation broth, and after the second filtration, collect the second supernatant for standby;

[0009] (2) Take the first supernatant and the second supernatant in step (1), after merging and treating with an ultrafiltration membrane, collect the ultrafiltration supernatant for standby;

[0010] (3) Take the ultrafiltration supernatant in step (2), adjust the pH to acidic and then pass through a cation resin column, stop injecting the sample when the concentration of ergothioneine in the effluent is greater than or equal to 50 mg / L, and perform elution treatment with an eluent, and collect the eluate for standby;

[0011] (4) Take the eluate in step (3), perform nanofiltration membrane concentration treatment, the first concentrated liquid collected is subjected to vacuum concentration treatment, and the second concentrated liquid obtained is cooled, filtered by suction, washed, and dried to obtain an ergothioneine product.

[0012] As an improved technical solution, when the small molecules in the Pichia pastoris fermentation broth in step (1) carry positive charges, the added flocculant is an anionic flocculant.

[0013] As an improved technical solution, when the small molecules in the Pichia pastoris fermentation broth in step (1) carry negative charges, the added flocculant is a cationic flocculant.

[0014] As an improved technical solution, in step (1), the first filtration is carried out using a plate and frame filter press with a filter cloth pore size of 500 meshes, and the second filtration is carried out using a plate and frame filter press with a filter cloth pore size of 300 meshes.

[0015] As an improved technical solution, in step (2), an ultrafiltration membrane with a pore size range of 5000 - 10000 Da is used for treatment.

[0016] As an improved technical solution, in step (3), the ultrafiltration filtrate is adjusted to a pH of 2-4 and enters the cation resin column at a flow rate of 2-3 BV / h. The resin in the cation resin column is macroporous weakly acidic cation exchange resin - D152, macroporous weakly acidic cation exchange resin - D113, macroporous strongly acidic cation exchange resin - D001, strong acid styrene-based cation exchange resin - 732 or macroporous weakly acidic cation exchange resin - A315.

[0017] As an improved technical solution, the eluent in step (3) is 0.4-0.8 wt% ammonia water, and the ammonia water enters the cation resin column at a flow rate of 0.5-1.5 BV / h for elution treatment.

[0018] As an improved technical solution, in step (4), the eluate is concentrated by a nanofiltration membrane with a pore size range of 100-150 Da; the first concentrated solution is subjected to vacuum concentration at a temperature of 50-70 °C and a vacuum degree of less than -0.09 MPa, and the concentration of the second concentrated solution is 200-300 g / l.

[0019] As an improved technical solution, in step (4), the second concentrated solution is cooled to 5-10 °C.

[0020] After adopting the above technical solution, the beneficial effects of the present invention are:

[0021] The present invention uses a Pichia pastoris fermentation broth with an ergothioneine content of 2-4 g / L as the treatment raw material, detects the isoelectric point of the fermentation broth and the charges carried by small molecule substances, and then adds a corresponding flocculant according to the charges carried (to promote the aggregation and sedimentation of suspended particles, thereby realizing solid-liquid separation), adjusts the pH to be equal to the isoelectric point (to balance the positive and negative charges carried by small molecule substances in the fermentation broth to form macromolecular aggregates, reducing the viscosity of the fermentation broth, and thus the filtration speed and separation effect). After filtration treatment (the first turbid liquid obtained is diluted with water, then the pH is adjusted to be equal to the isoelectric point, the second clear liquid collected after filtration is treated by an ultrafiltration membrane, the collected ultrafiltration filtrate is adjusted to an acidic pH, passed through a cation resin column, eluted with an eluent, and the collected eluate is concentrated by a nanofiltration membrane and vacuum concentrated, and then cooled, filtered, washed, and dried to obtain an ergothioneine product with high yield and high purity. Specific Embodiments

[0022] In order to make the purpose, technical solution and advantages of the present invention clearer, the following further elaborates on the present invention in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] Example 1

[0024] A process for extracting ergothioneine from Pichia pastoris fermentation broth, comprising the following steps:

[0025] (1) Take 1000 L of Pichia pastoris fermentation broth with an ergothioneine content of 2 g / L, detect the isoelectric point of the Pichia pastoris fermentation broth (which is 6.2) and the charge carried by small molecule substances (polypeptides) in the Pichia pastoris fermentation broth (positive charge), add a flocculant (polyacrylamide, with a dosage of 3 kg) according to the type of charge carried, then adjust the pH value of the Pichia pastoris fermentation broth to be equal to the isoelectric point. After the first filtration (using a plate and frame filter press with a filter cloth pore size of 500 mesh), collect 600 L of the first clear liquid and 400 L of the first turbid liquid respectively; Take the first turbid liquid and dilute it with water (dilute it according to a volume ratio of 1:2), adjust the pH value to be equal to the isoelectric point pi of the Pichia pastoris fermentation broth, and after the second filtration (using a plate and frame filter press with a filter cloth pore size of 300 mesh), collect 500 L of the second clear liquid for standby;

[0026] (2) Take 500 L of the first clear liquid and 600 L of the second clear liquid in step (1), combine them and treat them with a 5000 Da ultrafiltration membrane, and collect 1300 L of the ultrafiltration clear liquid for standby;

[0027] (3) Take 1300 L of the ultrafiltration clear liquid in step (2), adjust the pH to 2, and enter a cation resin column (macroporous weakly acidic cation exchange resin - D152) with a volume of 100 m 3 at a flow rate of 2 BV / h. Stop sampling when the concentration of ergothioneine in the effluent is greater than or equal to 50 mg / L. Use 0.4 wt% ammonia water as the eluent and enter the cation resin column at a flow rate of 0.5 BV / h for elution treatment, and collect 250 L of the eluate for standby;

[0028] (4) Take 250 L of the eluate in step (3), concentrate it through a nanofiltration membrane with a pore size range of 100 Da. The 150 L of the first concentrated liquid is subjected to vacuum concentration at a temperature of 50 °C and a vacuum degree of -0.095 MPa to obtain 80 L of the second concentrated liquid with a concentration of 200 g / l. Then, after cooling by 5 °C, filtration, washing (washing with 95 wt% ethanol), and drying, an ergothioneine product is obtained.

[0029] Example 2

[0030] A process for extracting ergothioneine from Pichia pastoris fermentation broth, comprising the following steps:

[0031] (1) Take 1000 L of Pichia pastoris fermentation broth with ergothioneine content of 2.5 g / L, detect the isoelectric point of the Pichia pastoris fermentation broth (which is 6.4) and the charge carried by small molecule substances (polypeptides) in the Pichia pastoris fermentation broth (positive charge), add a flocculant (polyacrylamide, with an addition amount of 3 kg) according to the type of charge carried, then adjust the pH value of the Pichia pastoris fermentation broth to be equal to the isoelectric point, and after the first filtration (using a plate and frame filter press with a filter cloth pore size of 500 mesh), collect 600 L of the first clear liquid and 400 L of the first turbid liquid respectively; take the first turbid liquid, dilute it with water (dilute according to a volume ratio of 1:2), adjust the pH value to be equal to the isoelectric point pi of the Pichia pastoris fermentation broth, and after the second filtration (using a plate and frame filter press with a filter cloth pore size of 300 mesh), collect 550 L of the second clear liquid for standby;

[0032] (2) Take 600 L of the first clear liquid and 550 L of the second clear liquid in step (1), combine them and treat them with a 6000 Da ultrafiltration membrane, and collect 1300 L of the ultrafiltration clear liquid for standby;

[0033] (3) Take 1300 L of the ultrafiltration clear liquid in step (2), adjust the pH to 2.5, and enter a cation resin column (macroporous weakly acidic cation exchange resin - D113) with a volume of 100 m 3 at a flow rate of 2.3 BV / h. Stop injecting the sample when the concentration of ergothioneine in the effluent is greater than or equal to 50 mg / L. Use 0.5 wt% ammonia water as the eluent and enter the cation resin column at a flow rate of 0.8 BV / h for elution treatment, and collect 280 L of the eluate for standby;

[0034] (4) Take 280 L of the eluate in step (3), concentrate it through a nanofiltration membrane with a pore size range of 120 Da, and collect 160 L of the first concentrate. Under the conditions of a temperature of 55 °C and a vacuum degree of -0.095 MPa, conduct vacuum concentration treatment to obtain 85 L of the second concentrate with a concentration of 230 g / l, and then obtain the ergothioneine product through cooling to 6.5 °C, suction filtration, washing (washing with 95 wt% ethanol), and drying.

[0035] Example 3

[0036] A process for extracting ergothioneine from Pichia pastoris fermentation broth, comprising the following steps:

[0037] (1) Take 1000 L of Pichia pastoris fermentation broth with an ergothioneine content of 3 g / L, detect the isoelectric point of the Pichia pastoris fermentation broth (which is 6.5) and the charge carried by small molecular substances (polypeptides) in the Pichia pastoris fermentation broth (positive charge), add a flocculant (sodium polyacrylate, with an addition amount of 3 kg) according to the type of charge carried, then adjust the pH value of the Pichia pastoris fermentation broth to be equal to the isoelectric point, and after the first filtration (using a plate and frame filter press with a filter cloth pore size of 500 mesh), collect 650 L of the first supernatant and 350 L of the first turbid liquid respectively; take the first turbid liquid, dilute it with water (dilute it according to a volume ratio of 1:2), adjust the pH value to be equal to the isoelectric point pi of the Pichia pastoris fermentation broth, and after the second filtration (using a plate and frame filter press with a filter cloth pore size of 300 mesh), collect 550 L of the second supernatant for standby;

[0038] (2) Take 650 L of the first supernatant and 550 L of the second supernatant in step (1), after treatment with an ultrafiltration membrane with a pore size of 8000 Da, collect 1400 L of the ultrafiltration supernatant for standby;

[0039] (3) Take 1400 L of the ultrafiltration supernatant in step (2), adjust the pH to 3, and enter a cation resin column (macroporous weakly acidic cation exchange resin - A315) with a volume of 100 m 3 at a flow rate of 2.5 BV / h. Stop injecting the sample when the concentration of ergothioneine in the effluent is greater than or equal to 50 mg / L. Use 0.8 wt% ammonia water as the eluent and enter the cation resin column at a flow rate of 1 BV / h for elution treatment, and collect 300 L of the eluate for standby;

[0040] (4) Take 300 L of the eluate in step (3), conduct nanofiltration membrane concentration treatment with a pore size range of 120 Da, collect 200 L of the first concentrate, and conduct vacuum concentration treatment under the conditions of a temperature of 60 °C and a vacuum degree of -0.095 MPa to obtain 100 L of the second concentrate with a concentration of 250 g / l. Then, after cooling by 7.5 °C, filtration, washing (washing with 95 wt% ethanol), and drying, obtain the ergothioneine product.

[0041] Example 4

[0042] A process for extracting ergothioneine from Pichia pastoris fermentation broth, comprising the following steps:

[0043] (1) Take 1000 L of Pichia pastoris fermentation broth with an ergothioneine content of 3.5 g / L, detect the isoelectric point of the Pichia pastoris fermentation broth (which is 6.7) and the charge carried by small molecule substances (polypeptides) in the Pichia pastoris fermentation broth (negative charge), and add a flocculant (polymethacryloyloxyethyl trimethyl ammonium chloride, with an addition amount of 3 kg) according to the type of charge carried. Then adjust the pH value of the Pichia pastoris fermentation broth to be equal to the isoelectric point. After the first filtration (using a plate and frame filter press with a filter cloth pore size of 500 mesh), collect 650 L of the first clear liquid and 350 L of the first turbid liquid respectively; take the first turbid liquid and dilute it with water (dilute it according to a volume ratio of 1:2), adjust the pH value to be equal to the isoelectric point pi of the Pichia pastoris fermentation broth, and after the second filtration (using a plate and frame filter press with a filter cloth pore size of 300 mesh), collect 600 L of the second clear liquid for standby;

[0044] (2) Take 650 L of the first clear liquid and 600 L of the second clear liquid in step (1), combine them and treat them with a 7000 Da ultrafiltration membrane. Collect 1400 L of the ultrafiltration clear liquid for standby;

[0045] (3) Take 1400 L of the ultrafiltration clear liquid in step (2), adjust the pH to 3.5, and enter a cation resin column (macroporous strongly acidic cation exchange resin - D001) with a volume of 100 m 3 at a flow rate of 2.8 BV / h. Stop sampling when the concentration of ergothioneine in the effluent is greater than or equal to 50 mg / L. Use 0.7 wt% ammonia water as the eluent and enter the cation resin column at a flow rate of 1.2 BV / h for elution treatment. Collect 320 L of the eluate for standby;

[0046] (4) Take 320 L of the eluate in step (3), concentrate it through a nanofiltration membrane with a pore size range of 150 Da. Collect 210 L of the first concentrate and perform vacuum concentration treatment at a temperature of 65 °C and a vacuum degree of -0.095 MPa to obtain 110 L of the second concentrate with a concentration of 280 g / l. Then cool it to 8.5 °C, filter it, wash it (wash it with 95 wt% ethanol), and dry it to obtain the ergothioneine product.

[0047] Example 5

[0048] A process for extracting ergothioneine from Pichia pastoris fermentation broth, comprising the following steps:

[0049] (1) Take 1000 L of Pichia pastoris fermentation broth with an ergothioneine content of 4 g / L, detect the isoelectric point of the Pichia pastoris fermentation broth (which is 6.6) and the charge carried by small molecule substances (polypeptides) in the Pichia pastoris fermentation broth (negative charge), add a flocculant (poly aluminum sulfate, with an addition amount of 3 kg) according to the type of charge carried, then adjust the pH value of the Pichia pastoris fermentation broth to be equal to the isoelectric point. After the first filtration (using a plate and frame filter press with a filter cloth pore size of 500 mesh), collect 700 L of the first clear liquid and 300 L of the first turbid liquid respectively; take the first turbid liquid, dilute it with water (dilute it according to a volume ratio of 1:2), adjust the pH value to be equal to the isoelectric point pi of the Pichia pastoris fermentation broth, and after the second filtration (using a plate and frame filter press with a filter cloth pore size of 300 mesh), collect 450 L of the second clear liquid for standby;

[0050] (2) Take 700 L of the first clear liquid and 450 L of the second clear liquid in step (1), combine them, and after treatment with a 10,000 Da ultrafiltration membrane, collect 1300 L of the ultrafiltration clear liquid for standby;

[0051] (3) Take 1300 L of the ultrafiltration clear liquid in step (2), adjust the pH to 4, and enter a cation resin column (strong acid type styrene-based cation exchange resin - 732) with a volume of 100 m 3 at a flow rate of 3 BV / h. Stop sampling when the concentration of ergothioneine in the effluent is greater than or equal to 50 mg / L. Use 0.6 wt% ammonia water as the eluent and enter the cation resin column at a flow rate of 1.5 BV / h for elution treatment, and collect 320 L of the eluate for standby;

[0052] (4) Take 320 L of the eluate in step (3), conduct nanofiltration membrane concentration treatment with a pore size range of 150 Da, collect 220 L of the first concentrated liquid, and under the conditions of a temperature of 70 °C and a vacuum degree of -0.095 MPa, conduct vacuum concentration treatment to obtain 120 L of the second concentrated liquid with a concentration of 300 g / l. Then, after cooling by 10 °C, filtration, washing (washing with 95 wt% ethanol), and drying, obtain the ergothioneine product.

[0053] To better prove that the process method of the present invention can improve the yield and purity of ergothioneine, taking Example 3 as a reference, the following 16 comparative examples are given. The specific yields and purities of ergothioneine in Examples 1 - 5 and the 16 comparative examples are shown in Table 1 in detail.

[0054] Comparative Example 1

[0055] Different from Example 3, in step (1) during the first filtration, the pH of the fermentation broth was adjusted to be different from the isoelectric point, and the rest of the operations were the same.

[0056] Comparative Example 2

[0057] Different from Example 3, during the second filtration in step (1), the pH of the fermentation broth was adjusted to be different from the isoelectric point, and the rest of the operations were the same.

[0058] Comparative Example 3

[0059] Different from Example 3, during the first filtration in step (1), no flocculant was added, and the rest of the operations were the same.

[0060] Comparative Example 4

[0061] Different from Example 3, in step (3), the pH of the ultrafiltrate was 1.5, and the rest of the operations were the same.

[0062] Comparative Example 5

[0063] Different from Example 3, in step (3), the pH of the ultrafiltrate was 4.5, and the rest of the operations were the same.

[0064] Comparative Example 6

[0065] Different from Example 3, in step (3), after adjusting the pH of the ultrafiltrate, it entered the cation resin column at a flow rate of 1.5 BV / h, and the rest of the operations were the same.

[0066] Comparative Example 7

[0067] Different from Example 3, in step (3), after adjusting the pH of the ultrafiltrate, it entered the cation resin column at a flow rate of 3.5 BV / h, and the rest of the operations were the same.

[0068] Comparative Example 8

[0069] Different from Example 3, in step (3), the resin in the cation resin column was macroporous weakly acidic cation exchange resin - D152, and the rest of the operations were the same.

[0070] Comparative Example 9

[0071] Different from Example 3, in step (3), the resin in the cation resin column was macroporous weakly acidic cation exchange resin - D113, and the rest of the operations were the same.

[0072] Comparative Example 10

[0073] Different from Example 3, in step (3), the resin in the cation resin column was macroporous strongly acidic cation exchange resin - D001, and the rest of the operations were the same.

[0074] Comparative Example 11

[0075] Different from Example 3, in step (3), the resin in the cation resin column was strong acid type styrene based cation exchange resin - 732, and the rest of the operations were the same.

[0076] Comparative Example 12

[0077] Different from Example 3, in step (3), ammonia water enters the cation resin column at a flow rate of 2 BV / h for elution treatment, and the rest of the operations are the same.

[0078] Comparative Example 13

[0079] Different from Example 3, in step (3), the concentration of ammonia water is 0.5 wt%, and the rest of the operations are the same.

[0080] Comparative Example 14

[0081] Different from Example 3, in step (3), the concentration of ammonia water is 1 wt%, and the rest of the operations are the same.

[0082] Comparative Example 15

[0083] Different from Example 3, in step (4), the second concentrated solution is cooled to 3°C, and the rest of the operations are the same.

[0084] Comparative Example 16

[0085] Different from Example 3, in step (4), the second concentrated solution is cooled to 12°C, and the rest of the operations are the same.

[0086] Table 1

[0087]

[0088]

[0089] It can be found from Table 1 that the yield and purity of the ergothioneine product obtained in Example 3 of the present invention are superior to those of other examples and Comparative Examples 1-16.

[0090] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. A process for extracting thioneine from a Pichia fermentation broth, characterized in that, The process comprises the following steps: (1) taking the Pichia fermented liquid containing thioneine, detecting the isoelectric point of the Pichia fermented liquid and the charge of small molecules in the Pichia fermented liquid, and adding a flocculant according to the charged type, then adjusting the pH value of the Pichia fermented liquid to be equal to the isoelectric point, and collecting the first clear liquid and the first turbid liquid respectively after filtering for the first time; taking the first turbid liquid and diluting it with water, adjusting the pH value to be equal to the isoelectric point pi of the Pichia fermented liquid, filtering for the second time, and collecting the second clear liquid for standby use; (2) taking the first clear liquid and the second clear liquid in step (1), combining them, treating them with an ultrafiltration membrane, and collecting the ultrafiltration clear liquid for later use; (3) ultrafiltration clear liquid described in step (2) is taken, pH is adjusted to acidic and then crosses cationic resin column, and injection is stopped when the concentration of thioneine in the effluent is greater than or equal to 50mg / L, and eluent is used to carry out elution treatment, and the eluent collected is standby; (4) take eluent described in step (3), through nanofiltration membrane concentration process, the first concentrated solution collected is concentrated by vacuum, and the second concentrated solution obtained is cooled, filtered, washed, and dried to obtain thioneine product.

2. a kind of processing method of extracting ergothioneine from Pichia yeast fermentation broth according to claim 1, is characterized in that, When the small molecular substances in the Pichia fermentation broth in step (1) are positively charged, the added flocculant is an anionic flocculant.

3. a kind of processing method of extracting ergothioneine from Pichia fermentation broth according to claim 1, is characterized in that, When the small molecular substances in the Pichia fermentation broth in step (1) are negatively charged, the added flocculant is a cationic flocculant.

4. a kind of processing method of extracting ergothioneine from Pichia fermentation broth according to claim 1, is characterized in that, In step (1), the first filtration is performed using a plate-and-frame filter with a filter cloth aperture of 500 mesh, and the second filtration is performed using a plate-and-frame filter with a filter cloth aperture of 300 mesh.

5. a kind of processing method of extracting ergothioneine from Pichia fermentation broth according to claim 1, is characterized in that, In step (2), an ultrafiltration membrane with a pore size range of 5000-10000Da is used for treatment.

6. a kind of processing method of extracting ergothioneine from Pichia fermentation broth according to claim 1, is characterized in that, The ultrafiltration clear liquid in step (3) is adjusted to pH 2-4 and enters a cationic resin column at a flow rate of 2-3 BV / h. The resin in the cationic resin column is a macroporous weakly acidic cation exchange resin--D152, a macroporous weakly acidic cation exchange resin--D113, a macroporous strongly acidic cation exchange resin--D001, a strongly acidic styrene-based cation exchange resin--732 or a macroporous weakly acidic cation exchange resin--A315.

7. a kind of processing method of extracting ergothioneine from Pichia fermentation broth according to claim 1, is characterized in that, In step (3), the eluent is 0.4-0.8 wt % ammonia water, and the ammonia water enters the cationic resin column at a flow rate of 0.5-1.5 BV / h for elution treatment.

8. a kind of processing method of extracting ergothioneine from Pichia yeast fermentation broth according to claim 1, is characterized in that, The eluent in step (4) is concentrated using a nanofiltration membrane with a pore size range of 100-150Da; the first concentrated solution is vacuum concentrated at a temperature of 50-70°C and a vacuum degree of less than -0.09MPa, and the concentration of the second concentrated solution is 200-300g / l.

9. a kind of processing method of extracting ergothioneine from Pichia fermentation broth according to claim 1, is characterized in that, In step (4), the second concentrated solution is cooled to 5-10°C.

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