Production system for extracting ergothioneine from pichia pastoris fermentation liquor

Abstract

The utility model relates to the technical field of ergothioneine production, in particular to a production system for extracting ergothioneine from pichia pastoris fermentation liquor, which comprises a first buffer kettle, a flocculant storage tank, a first filtering device, a first clear liquid storage tank, a second buffer kettle, a second filtering device and a second clear liquid storage tank which are communicated with a fermentation liquor conveying pipeline, a discharge port of the first clear liquid storage tank and a discharge port of the second clear liquid storage tank are communicated with a mixing tank, a discharge port of the mixing tank is communicated with a third filtering device, a third buffer kettle, a cationic resin column, first concentration equipment and second concentration equipment, a concentrated solution outlet of the second concentration equipment is communicated with a crystallization kettle, and a crystal outlet of the crystallization kettle is communicated with a washing tank. A discharge hole of the washing tank is communicated with the drying device; and a discharge hole of the drying device is communicated with the product storage tank. The production system is reasonable in design, effective extraction of the ergothioneine in the pichia pastoris fermentation liquor is achieved, and the yield and purity of the ergothioneine product are greatly improved.

Description

TECHNICAL FIELD

[0001] The utility model relates to ergothioneine production technical field especially relates to a production system for extracting ergothioneine from pichia pastoris fermentation liquor. BACKGROUND

[0002] Ergothioneine (EGT) is named 2-mercapto-histidine-trimethyl inner salt, and the relative molecular mass is 229.3, and it is a very rare natural chiral small molecule amino acid. At present, ergothioneine mainly has four preparation modes: direct extraction from fungi, chemical synthesis, enzyme catalysis and microbial fermentation synthesis. The direct extraction from fungi is extracted from edible fungi, and since the content of ergothioneine in fungi is relatively low, the purity of the ergothioneine extracted from edible fungi is low, and it is difficult to mass-produce, cannot meet the market demand, the cost is high, and general consumers are difficult to accept. The process route of chemical synthesis method is long, and the yield is also very low, and the environmental pollution is serious, so the cost is always high; enzyme catalysis is affected by enzyme activity, and the catalytic cycle is long, and the yield is low; compared with the above several ergothioneine production methods, the microbial fermentation method has higher yield, more stable production, lower raw material cost, best benefit and more green and natural. Therefore, the microbial fermentation method is the mainstream of the industrialized production of ergothioneine in the future.

[0003] Pichia pastoris is an important industrial host fungus, and it is a kind of yeast fungus that can utilize methanol as the only carbon source and energy, and it is a new generation of yeast expression system, and is widely used for producing protein and drug antibody. At present, a patent discloses a pichia pastoris strain for producing ergothioneine, realizes the efficient synthesis of ergothioneine, and the obtained pichia pastoris strain can realize high-density culture, and adjusts the oxidation / peroxidation state, solves the problem that the oxidation damage of pichia pastoris cells weakens the yield of ergothioneine, but the method for extracting ergothioneine from pichia pastoris fermentation liquor is rarely explained. In view of the above problems, it is necessary to provide a production system for extracting ergothioneine from pichia pastoris fermentation liquor. UTILITY MODEL CONTENT

[0004] The technical problem to be solved by the utility model is: in view of the defects of the prior art, a production system for extracting ergothioneine from pichia pastoris fermentation liquor is provided, and high yield and high purity ergothioneine products can be obtained from the fermentation liquor by using the production system.

[0005] To solve the above technical problems, the technical scheme of the utility model is:

[0006] The utility model provides a production system for extracting ergothioneine from pichia fermenting liquor, including the first buffer kettle who links with the fermenting liquor delivery pipeline, the flocculating agent import of first buffer kettle links with flocculating agent storage tank, the discharge port of first buffer kettle links with first filter device, the liquid outlet of first filter device links with first clear liquid storage tank, the discharge port of first filter device links with second buffer kettle, the dilution liquid import of second buffer kettle links with dilution water delivery pipeline, the discharge port of second buffer kettle links with second filter device, the liquid outlet of second filter device links with second clear liquid storage tank, the discharge port of first clear liquid storage tank and the discharge port of second clear liquid storage tank link with mixed jar respectively, the discharge port of mixed jar links with third filter device, the liquid outlet of third filter device links with third buffer kettle, the discharge port of third buffer kettle links with the feed inlet of cation resin column, the feed inlet of cation resin column also links with eluent delivery pipeline, the washing liquid outlet of cation resin column links with first concentration equipment, the concentrated liquid outlet of first concentration equipment links with second concentration equipment, the concentrated liquid outlet of second concentration equipment links with crystallization kettle, the crystal outlet of crystallization kettle links with washing tank, the discharge port of washing tank links with drying device, the discharge port of drying device links with product storage tank.

[0007] As an improved technical scheme, the first filter device is a plate-and-frame filter with a filter cloth aperture of 500 mesh.

[0008] As an improved technical scheme, the second filter device is a plate-and-frame filter with a filter cloth aperture of 300 mesh.

[0009] As an improved technical scheme, the third filter device is an ultrafiltration membrane treatment equipment with a pore size range of 5000-10000 Da.

[0010] As an improved technical scheme, the first concentration equipment is a nanofiltration membrane treatment equipment with a pore size range of 100-150 Da.

[0011] After the above technical scheme is adopted, the utility model has the beneficial effects that:

[0012] The production system for extracting ergothioneine from the fermentation liquor of Pichia pastoris comprises a first buffer tank connected with a fermentation liquor conveying pipeline, a flocculant storage tank connected with the flocculant inlet of the first buffer tank, a first filter device connected with the outlet of the first buffer tank, a first clear liquid storage tank connected with the outlet of the first filter device, a second buffer tank connected with the outlet of the first filter device, a dilution water conveying pipeline connected with the dilution liquid inlet of the second buffer tank, a second filter device connected with the outlet of the second buffer tank, and a second clear liquid storage tank connected with the outlet of the second filter device. The outlets of the first and second clear liquid storage tanks are respectively connected with a mixing tank, the outlet of the mixing tank is connected with a third filter device, the outlet of the third filter device is connected with a third buffer tank, the outlet of the third buffer tank is connected with the feed inlet of a cation resin column, the feed inlet of the cation resin column is also connected with an eluent conveying pipeline, the eluent outlet of the cation resin column is connected with a first concentration device, the concentrated liquid outlet of the first concentration device is connected with a second concentration device, the concentrated liquid outlet of the second concentration device is connected with a crystallization tank, the crystal outlet of the crystallization tank is connected with a washing tank, the outlet of the washing tank is connected with a drying device, and the outlet of the drying device is connected with a product storage tank. In actual production, the Pichia pastoris fermentation liquor containing ergothioneine is introduced into the first buffer tank through the fermentation liquor conveying pipeline and a conveying pump, then part of the fermentation liquor is taken out for detection of the charge of the amino acid, and then the flocculant in the flocculant storage tank is added according to the charge. After flocculation and sedimentation, the fermentation liquor is introduced into the first filter device, the clear liquid is stored in the first clear liquid storage tank after filtration, the turbid liquid is introduced into the second buffer tank, the dilution water is conveyed to the second buffer tank through the dilution water conveying pipeline, the turbid liquid after dilution is introduced into the second filter device, the clear liquid is stored in the second clear liquid storage tank after filtration, then the clear liquid in the first and second clear liquid storage tanks is introduced into the mixing tank, the clear liquid in the mixing tank is introduced into the third filter device, the clear liquid after filtration is collected in the third buffer tank, the second concentrated liquid is subjected to cooling treatment in the crystallization tank, the crystal is extracted after crystallization, the ergothioneine crystal is collected in the washing tank, then the crystal is treated with a washing solvent, and finally the ergothioneine product is stored in the product storage tank after drying by the drying device. The above production system is reasonable in design, realizes effective extraction of ergothioneine from the Pichia pastoris fermentation liquor, and greatly improves the yield and purity of the ergothioneine product.

[0013] The first filter device is a plate-and-frame filter with a filter cloth aperture of 500 mesh. The device can effectively remove flocculant-bound bacterial cells and other suspended particles in the fermentation liquor, and realize effective solid-liquid separation.

[0014] The second filter device is a plate and frame filter with a filter cloth aperture of 300 mesh, which can further realize recycling of the liquid containing ergothioneine.

[0015] The third filter device is an ultrafiltration membrane treatment equipment with a pore size range of 5000-10000 Da, which can effectively remove macromolecular substances in the supernatant and facilitate subsequent processing of ergothioneine.

[0016] The first concentration equipment is a nanofiltration membrane treatment equipment with a pore size range of 100-150 Da, which can effectively concentrate ergothioneine and facilitate subsequent processing. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a structure diagram of the production system for extracting ergothioneine from Pichia pastoris fermentation broth according to the present application;

[0018] 1-fermentation broth conveying pipeline, 2-first buffer kettle, 3-flocculant storage tank, 4-first filter device, 5-first supernatant storage tank, 6-second buffer kettle, 7-dilution water conveying pipeline, 8-second filter device, 9-second supernatant storage tank, 10-mixing tank, 11-third filter device, 12-third buffer kettle, 13-cation resin column, 14-eluent conveying pipeline, 15-first concentration equipment, 16-second concentration equipment, 17-crystallization kettle, 18-washing tank, 19-drying device, 20-product storage tank, and 21-suction filtration pump. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the present application clearer and more apparent, the present application will be further described in detail below with reference to the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0020] The production system for extracting ergothioneine from Pichia fermenting liquor includes a first buffer tank 2 connected with a fermenting liquor conveying pipeline 1, a flocculant inlet of the first buffer tank 2 connected with a flocculant storage tank 3, a discharge outlet of the first buffer tank 2 connected with a first filtering device 4 (a plate-and-frame filter with a filter cloth aperture of 500 mesh), a liquid outlet of the first filtering device 4 connected with a first clear liquid storage tank 5, a discharge outlet of the first filtering device 4 connected with a second buffer tank 6, a dilution liquid inlet of the second buffer tank 6 connected with a dilution water conveying pipeline 7, a discharge outlet of the second buffer tank 6 connected with a second filtering device 8 (a plate-and-frame filter with a filter cloth aperture of 300 mesh), and a liquid outlet of the second filtering device 8 connected with a second clear liquid storage tank 9; a discharge outlet of the first clear liquid storage tank 5 and a discharge outlet of the second clear liquid storage tank 9 are respectively connected with a mixing tank 10, a discharge outlet of the mixing tank 10 is connected with a third filtering device 11 (an ultrafiltration membrane treatment equipment with a pore size range of 5000-10000 Da), a liquid outlet of the third filtering device 11 is connected with a third buffer tank 12, a discharge outlet of the third buffer tank 12 is connected with a feed inlet of a cation resin column 13 (filled with macroporous weak acid cation exchange resin inside), the feed inlet of the cation resin column 13 is also connected with an eluent conveying pipeline 14, a washing liquid and liquid outlet of the cation resin column 13 is connected with a first concentration equipment 15 (a nanofiltration membrane treatment equipment with a pore size range of 100-150 Da), a concentrated liquid outlet of the first concentration equipment 15 is connected with a second concentration equipment 16 (a concentration tank concentrated by a heat medium in a jacket of the concentration tank), a concentrated liquid outlet of the second concentration equipment 16 is connected with a crystallization tank 17, a liquid outlet of the crystallization tank 17 (a filter screen is arranged on an inner wall of a tank body corresponding to the liquid outlet, and the filter screen can effectively intercept crystals) is connected with a suction filter pump 21, a crystal outlet of the crystallization tank 17 is connected with a washing tank 18 (including a tank body, the tank body is open at a top portion, a stirring shaft is horizontally arranged inside the tank body, one end of the stirring shaft is connected with a motor, a plurality of stirring plates are arranged on the stirring shaft, a washing liquid outlet is arranged at a bottom portion of the tank body, and a discharge outlet is arranged at one side of the tank body), the discharge outlet of the washing tank 18 is connected with a drying device 19 (a double-cone dryer), and a discharge outlet of the drying device 19 is connected with a product storage tank 20.

[0021] In actual production, the Pichia pastoris fermentation liquor containing ergothioneine is introduced into the interior of the first buffer tank through a fermentation liquor conveying pipeline and a conveying pump, then part of the fermentation liquor is taken to detect the charge of the amino acid, and then the flocculating agent is added according to the charge in the flocculating agent storage tank, after flocculation and sedimentation, the fermentation liquor is introduced into the interior of the first filtering device, after filtration, the flocculating agent combined with the bacterial cells and other suspended particles in the fermentation liquor are effectively removed, solid-liquid effective separation is realized, then the clear liquor is stored in the first clear liquor storage tank, the turbid material liquid enters the second buffer tank, dilution water is conveyed to the second buffer tank through a dilution water conveying pipeline, after dilution, the turbid material liquid enters the second filtering device, after filtration, the liquid containing ergothioneine components is recovered and stored in the second clear liquor storage tank, then the clear liquor in the first and second clear liquor storage tanks enters the mixing tank, the clear liquor in the mixing tank enters the interior of the third filtering device (to effectively remove the macromolecular substances contained in the clear liquor), the filtered clear liquor is collected in the third buffer tank, after pH adjustment, it enters the cation resin column (ergothioneine is adsorbed by the resin), the eluent in the eluent pipeline is used to elute the resin column, the collected eluate is treated by the first concentration equipment and the second concentration equipment, the second concentrated liquid is treated by the crystallization tank to reduce the temperature, after the crystal is separated out, the crystallization mother liquor is extracted by suction filtration, the ergothioneine crystal is collected by the washing tank, then the washing solvent is used for treatment, finally, after drying by the drying equipment, the ergothioneine product is stored in the product storage tank.

[0022] The above merely describes the preferred embodiments of the present application, and is not intended to limit the present application, and any modification, equivalent replacement and improvement within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A production system for extracting ergothioneine from Pichia pastoris fermentation broth, characterized in that, The first buffer tank is connected with the fermentation liquid conveying pipeline, the flocculant inlet of the first buffer tank is connected with the flocculant storage tank, the discharge port of the first buffer tank is connected with the first filter device, the liquid outlet of the first filter device is connected with the first clear liquid storage tank, the discharge port of the first filter device is connected with the second buffer tank, the dilution liquid inlet of the second buffer tank is connected with the dilution water conveying pipeline, the discharge port of the second buffer tank is connected with the second filter device, the liquid outlet of the second filter device is connected with the second clear liquid storage tank, the discharge ports of the first clear liquid storage tank and the second clear liquid storage tank are respectively connected with the mixing tank, the discharge port of the mixing tank is connected with the third filter device, the liquid outlet of the third filter device is connected with the third buffer tank, the discharge port of the third buffer tank is connected with the feed inlet of the cation resin column, the feed inlet of the cation resin column is also connected with the eluent conveying pipeline, the eluent discharge outlet of the cation resin column is connected with the first concentration equipment, the concentrated liquid outlet of the first concentration equipment is connected with the second concentration equipment, the concentrated liquid outlet of the second concentration equipment is connected with the crystallization tank, the crystal outlet of the crystallization tank is connected with the washing tank, the discharge port of the washing tank is connected with the drying device, the discharge port of the drying device is connected with the product storage tank.

2. The production system for extracting ergothioneine from the fermentation broth of Pichia pastoris according to claim 1, characterized in that, The first filter device is a plate-and-frame filter with a filter cloth aperture of 500 mesh.

3. The production system for extracting ergothioneine from the fermentation broth of Pichia pastoris according to claim 1, characterized in that, The second filter device is a plate-and-frame filter with a filter cloth aperture of 300 mesh.

4. The production system for extracting ergothioneine from the fermentation broth of Pichia pastoris according to claim 1, characterized in that, The third filter device is an ultrafiltration membrane treatment equipment with a pore size range of 5000-10000 Da.

5. The production system for extracting ergothioneine from the fermentation broth of Pichia pastoris according to claim 1, characterized in that, The first concentration equipment is a nanofiltration membrane treatment equipment with a pore size range of 100-150 Da.

Images