Method for separating and purifying ergothioneine from biological fermentation liquor

Abstract

The invention belongs to the field of biological fermentation, and particularly provides a method for separating and purifying ergothioneine from biological fermentation liquor, which comprises the following steps: acidifying the ergothioneine fermentation liquor, sequentially filtering the acidified solution, ultra-filtering, decolorizing, desalting, concentrating, adding a solvent to crystallize, and taking crystals, thereby obtaining the ergothioneine. The solvent is triethylamine and methanol. The ergothioneine separated and purified by the method has the advantages of high purity, high yield, short production period, low equipment requirement, low raw material cost and simple production operation, and is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation, and specifically relates to a method for separating and purifying ergothionein from bio-fermentation broth. Background Technology

[0002] Ergothioneine (EGT) is a compound discovered in 1909, initially found in a fungus called Claviceps purpurea. Its molecular structure is as follows: Ergothioneine is a derivative of histidine trimethylbetaine, containing a sulfur atom on its imidazole ring. It is not easily oxidized, exhibiting good stability and solubility, and has consistently acted as an anti-stress molecule in cells. Because ergothioneine is all-natural, has low toxicity, proven efficacy, good stability, and excellent skin compatibility, it can enter cells and mitochondria via the transport protein OCTN-1 to scavenge free radicals and protect mitochondria. Its antioxidant capacity against superoxide radicals, hydroxyl radicals, and peroxynitrite radicals is known to be higher than that of vitamin E, vitamin C, cysteine, and glutathione. Due to its unique biological functions and pharmacological effects, it is highly suitable for use in cosmetics, and ergothioneine is currently a widely accepted additive in cosmetics.

[0003] The main methods for preparing ergothioneine include natural biological extraction, bio-fermentation, and chemical synthesis. ① Natural biological extraction: Ergothioneine is extracted from natural raw materials such as mushrooms, grains, and animal tissues. Edible fungi such as Boletus, King Oyster Mushroom, and Shiitake Mushroom contain ergothioneine. This method uses natural raw materials and has high safety, but the ergothioneine content in the raw materials is low, with many impurities, and the extraction cost is high, limiting its application. ② Chemical synthesis: Ergothioneine is synthesized in aqueous solution using histidine as a starting material through multiple steps including thiolation, thiol protection, methylation, and deprotection. This method can be mass-produced, but the raw materials are expensive, prone to racemization, produce many byproducts, and have low safety, limiting its application in food and medicine. ③ Bio-fermentation: Ergothioneine is produced by fermentation using engineered bacteria or edible fungi. For example, optimizing the metabolic pathways of microbial chassis such as Escherichia coli and yeast through genome editing technology can improve the efficiency of ergothioneine synthesis. This method is low-cost, high-purity, and suitable for industrial production.

[0004] As the industrial application of ergothioneine production through bio-fermentation is gradually promoted, research on the isolation and purification of ergothioneine from bio-fermentation broth has also increased accordingly. Patent CN117924184A uses silica gel chromatography to separate and purify pretreated ergothioneine fermentation broth; patent CN104774182B uses ultrafiltration to purify fungal mycelial fermentation broth extracted with hot water; and patent CN116178270A solves the problem of difficult purification of ergothioneine produced by microbial fermentation through solid-liquid separation, extraction, Boc protection, and Boc removal. These methods for separating and purifying ergothioneine from fermentation broth either require the use of large amounts of the organic solvent acetonitrile, which is environmentally unfriendly, or involve complex separation and purification processes, becoming the main bottleneck restricting the development of ergothioneine production through bio-fermentation. Summary of the Invention

[0005] To address the problems of existing technologies, this invention provides a method for separating and purifying ergothioneine from biological fermentation broth, comprising the following steps: The fermentation broth of ergothioneine was acidified. The acidified solution was then filtered, ultrafiltered, decolorized, desalted, concentrated, and crystallized with solvent. The crystals were then collected to obtain the product. The solvent is triethylamine and methanol.

[0006] Furthermore, the acidification involves adding sulfuric acid solution to the fermentation broth until the pH value reaches 3.2-3.8.

[0007] Furthermore, sulfuric acid solution is added to the fermentation broth to bring the pH value to 3.5; the concentration of the sulfuric acid solution is 15-25%.

[0008] Furthermore, the filtration is performed using a ceramic membrane.

[0009] Furthermore, the ultrafiltration is performed using a 2540 type ultrafiltration system; the pore size of the ultrafiltration membrane in the 2540 type ultrafiltration system is 2.5-3 kDa.

[0010] Furthermore, the decolorization is performed using activated carbon; the mass-to-volume ratio of the activated carbon to the ergothioneine fermentation broth is 300-500 g: 20 L; the decolorization time is 0.8-1.2 h.

[0011] Furthermore, the desalination is performed using an electrodialysis device; the desalination is carried out until the conductivity is less than 400~600 μS / cm.

[0012] Furthermore, the concentration is vacuum concentration at a temperature of 55~58℃; concentrated to a density of 80~150g / L.

[0013] Furthermore, the volume ratio of triethylamine and methanol to ergothioneine fermentation broth is 0.3-0.7:0.6-1.5:20.

[0014] Furthermore, the crystallization temperature is 8~12℃, and the time is 3~5h.

[0015] This invention discloses a method for separating and purifying ergothioneine from bio-fermentation broth. The ergothioneine obtained through impurity removal, purification, and triethylamine-methanol crystallization has a purity of over 98% and a yield of over 85%. The method of this invention provides ergothioneine with high purity and high yield, a short production cycle, low equipment requirements, low raw material costs, and simple operation, making it suitable for industrial production.

[0016] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0017] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0018] Figure 1 Here is the liquid chromatogram of ergothioneine standard; Figure 2 This is the liquid chromatogram of ergothionein of the present invention. Detailed Implementation

[0019] The raw materials and equipment used in the specific embodiments of this invention can all be purchased commercially. The Escherichia coli fermentation broth containing ergothioneine is prepared according to the following method: Recombinant Escherichia coli expressing ergothionein was inoculated into the fermentation medium, and 30% sodium hydroxide was used as a pH adjuster to maintain the pH value at 7.0-7.2 during the fermentation process.

[0020] The fermentation medium is formulated as follows: yeast extract 5~15g / L, glycerol 10~60g / L, phosphate 3~10g / L, ammonium sulfate 1.0~5.0g / L, anhydrous magnesium sulfate 0.1~3g / L, calcium sulfate dihydrate 10~20mg / L, manganese sulfate monohydrate 10~20mg / L, calcium carbonate 10~30g / L, histidine 5~25g / L, methionine 30~50g / L, sodium thiosulfate 5~40g / L, polyether defoamer 0.1~1.2g / L, and the remainder is water.

[0021] The specific fermentation medium formulations used in Examples 1-4 and Comparative Examples 1-5 are as follows: yeast extract 10 g / L, glycerol 25 g / L, phosphate 5 g / L, ammonium sulfate 3 g / L, anhydrous magnesium sulfate 1 g / L, calcium sulfate dihydrate 15 mg / L, manganese sulfate monohydrate 15 mg / L, calcium carbonate 20 g / L, histidine 10 g / L, methionine 20 g / L, sodium thiosulfate 15 g / L, polyether defoamer 0.5 g / L, and the remainder is water.

[0022] The recombinant Escherichia coli strain used was E. coli W3110, which integrates N-methyltransferase Egt1, cysteine ​​desulfurase Egt2, cystathionine γ-lyase Cys3, and cystathionine β-synthetase Cys4, and was specifically provided by Shanghai Ruikang Biotechnology R&D Co., Ltd.

[0023] Example 1: Method for Isolation and Purification of Ergothionein 20 L of *E. coli* fermentation broth containing ergothioneine was stirred thoroughly, acidified with 20% sulfuric acid to adjust the pH to 3.5, and stirred until homogeneous. The mixture was then subjected to solid-liquid separation using a ceramic membrane to obtain the ceramic membrane filtrate. This filtrate was then subjected to ultrafiltration using a 2540 ultrafiltration system with a membrane pore size of 3 kDa to obtain the ultrafiltrate. 400 g of activated carbon was added to the ultrafiltrate for decolorization for 1 hour, followed by filtration to obtain the decolorized solution. The decolorized solution was then subjected to electrodialysis for desalination until the conductivity was less than 500 μS / cm. The desalted solution was concentrated under reduced pressure at 55 °C to 100 g / L. 0.5 L of triethylamine and 1.1 L of methanol were slowly added to the concentrate, and the mixture was cooled to 10 °C for crystallization. Crystals were grown for 4 hours, filtered, and then the ergothioneine was dried. The final product, after liquid chromatography analysis, showed a purity of 99.59% and a yield of 83%.

[0024] Example 2: Method for Isolation and Purification of Ergothionein 20 L of *E. coli* fermentation broth containing ergothioneine was stirred thoroughly, acidified with 20% sulfuric acid to adjust the pH to 3.5, and stirred until homogeneous. The mixture was then subjected to solid-liquid separation using a ceramic membrane to obtain the ceramic membrane filtrate. This filtrate was then subjected to ultrafiltration using a 2540 ultrafiltration system with a membrane pore size of 3 kDa to obtain the ultrafiltrate. 400 g of activated carbon was added to the ultrafiltrate for decolorization for 1 hour, followed by filtration to obtain the decolorized solution. The decolorized solution was then subjected to electrodialysis for desalination until the conductivity was less than 500 μS / cm. The desalted solution was concentrated at 55°C under reduced pressure to 100 g / L. 0.6 L of triethylamine and 1.3 L of methanol were slowly added to the concentrate, and the mixture was cooled to 10°C for crystallization. Crystals were grown for 4 hours, filtered, and then the ergothioneine was dried. The final product, after liquid chromatography analysis, showed a purity of 99.59% and a yield of 85%.

[0025] Example 3: Method for Isolation and Purification of Ergothionein 20 L of *E. coli* fermentation broth containing ergothioneine was stirred thoroughly, acidified with 20% sulfuric acid to adjust the pH to 3.5, and stirred until homogeneous. The mixture was then subjected to solid-liquid separation using a ceramic membrane to obtain the ceramic membrane filtrate. This filtrate was then subjected to ultrafiltration using a 2540 ultrafiltration system with a membrane pore size of 3 kDa to obtain the ultrafiltrate. 400 g of activated carbon was added to the ultrafiltrate for decolorization for 1 hour, followed by filtration to obtain the decolorized solution. The decolorized solution was then subjected to electrodialysis for desalination until the conductivity was less than 500 μS / cm. The desalted solution was concentrated under reduced pressure at 55 °C to 100 g / L. 0.3 L of triethylamine and 0.6 L of methanol were slowly added to the concentrate, and the mixture was cooled to 10 °C for crystallization. Crystals were grown for 4 hours, filtered, and then the ergothioneine was dried. The final product, after liquid chromatography analysis, showed a purity of 99.59% and a yield of 80%.

[0026] Example 4: Method for Isolation and Purification of Ergothionein 20 L of *E. coli* fermentation broth containing ergothioneine was stirred thoroughly, acidified with 20% sulfuric acid to adjust the pH to 3.5, and stirred until homogeneous. The mixture was then subjected to solid-liquid separation using a ceramic membrane to obtain the ceramic membrane filtrate. This filtrate was then subjected to ultrafiltration using a 2540 ultrafiltration system with a membrane pore size of 3 kDa to obtain the ultrafiltrate. 400 g of activated carbon was added to the ultrafiltrate for decolorization for 1 hour, followed by filtration to obtain the decolorized solution. The decolorized solution was then subjected to electrodialysis for desalination until the conductivity was less than 500 μS / cm. The desalted solution was concentrated under reduced pressure at 55 °C to 100 g / L. 0.7 L of triethylamine and 1.5 L of methanol were slowly added to the concentrate, and the mixture was cooled to 10 °C for crystallization. Crystallization was carried out for 4 hours, followed by filtration and drying of the ergothioneine. The final product, analyzed by liquid chromatography, showed a purity of 99.59% and a yield of 87%.

[0027] Comparative Example 1 20 L of *E. coli* fermentation broth containing ergothioneine was stirred thoroughly, acidified with 20% sulfuric acid to adjust the pH to 3.5, and stirred until homogeneous. The mixture was then subjected to solid-liquid separation using a ceramic membrane to obtain the ceramic membrane filtrate. This filtrate was then subjected to ultrafiltration using a 2540 ultrafiltration system with a membrane pore size of 3 kDa to obtain the ultrafiltrate. 400 g of activated carbon was added to the ultrafiltrate for decolorization for 1 hour, followed by filtration to obtain the decolorized solution. The decolorized solution was then subjected to electrodialysis for desalination until the conductivity was less than 500 μS / cm. The desalted solution was concentrated under reduced pressure at 55°C to 100 g / L. 0.7 L of triethylamine and 1.5 L of ethanol were slowly added to the concentrate, and the mixture was cooled to 10°C for crystallization. Crystals were grown for 4 hours, filtered, and then dried. The final product, after being sent for liquid chromatography, showed a purity of 99.59% and a yield of 43%.

[0028] Comparative Example 2 20 L of *E. coli* fermentation broth containing ergothioneine was stirred thoroughly, acidified with 20% sulfuric acid to adjust the pH to 3.5, and stirred until homogeneous. The mixture was then subjected to solid-liquid separation using a ceramic membrane to obtain the ceramic membrane filtrate. This filtrate was then subjected to ultrafiltration using a 2540 ultrafiltration system with a membrane pore size of 3 kDa to obtain the ultrafiltrate. 400 g of activated carbon was added to the ultrafiltrate for decolorization for 1 hour, followed by filtration to obtain the decolorized solution. The decolorized solution was then subjected to electrodialysis for desalination until the conductivity was less than 500 μS / cm. The desalted solution was concentrated under reduced pressure at 55 °C to 100 g / L. 0.7 L of ammonia and 1.5 L of methanol were slowly added to the concentrate, and the mixture was cooled to 10 °C for crystallization. Crystals were grown for 4 hours, filtered, and then the ergothioneine was dried. The final product, after liquid chromatography analysis, showed a purity of 99.59% and a yield of 32%.

[0029] Comparative Example 3 20 L of E. coli fermentation broth containing ergothioneine was stirred evenly, acidified with 20% sulfuric acid to adjust the pH to 3.5, and stirred evenly before being separated into solid and liquid phases using a ceramic membrane to obtain ceramic membrane filtrate. The ceramic membrane filtrate was then subjected to ultrafiltration using a 2540 ultrafiltration system with a membrane pore size of 3 kDa to obtain ultrafiltration filtrate. 400 g of activated carbon was added to the ultrafiltration filtrate for decolorization for 1 hour, followed by filtration to obtain a decolorized solution. The decolorized solution was then subjected to electrodialysis for desalination until the conductivity was less than 500 μS / cm. The desalted solution was concentrated under reduced pressure at 60 °C to 100 g / L. 0.7 L of sodium hydroxide and 1.5 L of methanol were slowly added to the concentrated solution, and the temperature was lowered to 10 °C. No crystals were observed.

[0030] Comparative Example 4 20 L of E. coli fermentation broth containing ergothioneine was stirred evenly, acidified with 20% sulfuric acid to adjust the pH to 3.5, and stirred evenly before being subjected to solid-liquid separation using a ceramic membrane to obtain ceramic membrane filtrate. The ceramic membrane filtrate was then subjected to ultrafiltration using a 2540 ultrafiltration system with a membrane pore size of 3 kDa to obtain ultrafiltration filtrate. 400 g of activated carbon was added to the ultrafiltration filtrate for decolorization for 1 hour, followed by filtration to obtain a decolorized solution. The decolorized solution was subjected to electrodialysis for desalination until the conductivity was less than 500 μS / cm. The desalted solution was concentrated under reduced pressure at 55 °C to 100 g / L. 0.7 L of potassium hydroxide and 1.5 L of methanol were slowly added to the concentrated solution, and the temperature was lowered to 10 °C for crystallization, but no crystals formed.

[0031] Comparative Example 5 20L of *E. coli* fermentation broth containing ergothioneine was stirred thoroughly, acidified with 20% sulfuric acid to adjust the pH to 3.5, and stirred evenly before being subjected to solid-liquid separation using a ceramic membrane to obtain ceramic membrane filtrate. The ceramic membrane filtrate was then subjected to ultrafiltration using a 2540 ultrafiltration system with a membrane pore size of 3 kDa to obtain ultrafiltration filtrate. 400g of activated carbon was added to the ultrafiltration filtrate for decolorization for 1 hour, followed by filtration to obtain a decolorized solution. The decolorized solution was then subjected to electrodialysis for desalination until the conductivity was less than 500 μS / cm. The desalted solution was concentrated under reduced pressure at 55℃ to 100 g / L. 0.7L of sodium oxide and 1.5L of methanol were slowly added to the concentrated solution, and the mixture was cooled to 10℃ for crystallization, but no crystals formed.

[0032] The following experimental examples further illustrate the beneficial effects of the present invention.

[0033] Experimental Example 1: Comparison and Validation of Ergothioneine Isolation and Purification Methods 1. Comparison of Methods and Their Effects The differences in the preparation methods of crude ergothioneine in Examples 1-4 and Comparative Examples 1-5, as well as the corresponding separation and purification effects, were compared. The results are shown in Table 1. Table 1 shows that a specific crystallization solvent combination of triethylamine and methanol is required to maximize the ergothioneine yield during the separation and purification of E. coli fermentation broth containing ergothioneine.

[0034] Table 1. Comparison of methods for preparing crude ergothioneine between the examples and the control examples. 2. Verification of the method of the present invention 2.1 Experimental Methods The product obtained in Example 4 was dissolved in water and subjected to liquid chromatography. The specific chromatographic conditions for the liquid chromatography test were as follows: column: Yuexu AQ-C18 (4.6mm*250mm, 5um); mobile phase: 5mM KH2PO4:methanol = 95:5; flow rate: 0.6ml / min; column temperature: 40℃; detection wavelength: 254nm.

[0035] 2.2 Experimental Results The results are as follows Figure 1-2 As shown, Figure 1 This is the liquid chromatogram of ergothioneine standard. Figure 2 The image shows a liquid chromatogram of ergothioneine from this invention. The chromatogram shows that the response time of ergothioneine from this invention is consistent with that of the ergothioneine standard.

[0036] In summary, this invention provides a method for preparing ergothioneine, which not only simplifies the production process, shortens the production cycle, and effectively improves production efficiency, but also improves product purity and yield, with purity reaching over 99% and yield reaching over 85%. This technology is simple to operate, low in cost, and can be industrialized.

Claims

1. A method for separating and purifying ergothioneine from biological fermentation broth, characterized in that: It includes the following steps: The fermentation broth of ergothioneine was acidified. The acidified solution was then filtered, ultrafiltered, decolorized, desalted, concentrated, and crystallized with solvent. The crystals were then collected to obtain the product. The solvent is triethylamine and methanol.

2. The method as described in claim 1, characterized in that, The acidification involves adding sulfuric acid solution to the fermentation broth until the pH value reaches 3.2-3.

8.

3. The method as described in claim 2, characterized in that, The fermentation broth is mixed with sulfuric acid solution to a pH of 3.5; the concentration of the sulfuric acid solution is 15-25%.

4. The method as described in claim 1, characterized in that, The filtration is performed using a ceramic membrane.

5. The method as described in claim 1, characterized in that, The ultrafiltration is performed using a 2540 type ultrafiltration system; the pore size of the ultrafiltration membrane in the 2540 type ultrafiltration system is 2.5-3 kDa.

6. The method as described in claim 1, characterized in that, The decolorization is performed using activated carbon; the mass-to-volume ratio of activated carbon to ergothioneine fermentation broth is 300-500g:20L; the decolorization time is 0.8-1.2h.

7. The method as described in claim 1, characterized in that, The desalination is performed using an electrodialysis device; the desalination is carried out until the conductivity is less than 400~600 μS / cm.

8. The method as described in claim 1, characterized in that, The concentration is carried out under reduced pressure at a temperature of 55-58°C until the density reaches 80-150 g / L.

9. The method as described in claim 1, characterized in that, The volume ratio of triethylamine and methanol to ergothioneine fermentation broth is 0.3-0.7:0.6-1.5:

20.

10. The method as described in claim 1, characterized in that, The crystallization temperature is 8~12℃ and the time is 3~5h.

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