Preparation method of glutathione

Abstract

The invention belongs to the field of biochemical engineering, and relates to a preparation method of glutathione, in particular to a method for synthesizing, separating and extracting glutathione by using double enzymes. Main impurities in the enzyme catalysis reaction liquid can be effectively removed through the alcohol solvent precipitation method, oxidation in the separation and extraction process can be slowed down, the alcohol solvent can be recycled, and cost can be saved. According to the method, most of macromolecular and micromolecular impurities can be effectively removed through an ultrafiltration method, and the purity of the product can be increased. The preparation method of glutathione is simple and convenient to operate, low in pollution, mild in reaction condition and suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the field of biochemical engineering and relates to a method for preparing glutathione, in particular to a method for synthesizing and separating and extracting glutathione by using double enzymes. Background Art

[0002] Glutathione (GSH) is a tripeptide thiol compound with important biological activity, which is widely used in medicine, food and cosmetics. Glutathione has two forms: reduced form (GSH) and oxidized form (GSSG). Its solid form is relatively stable, and its aqueous solution is easily oxidized in the air.

[0003] Glutathione synthesis methods include extraction, chemical synthesis, fermentation and enzyme catalysis. Currently, most of them are produced by microbial fermentation, and glutathione is further separated and extracted by copper salt method (such as patent CN113912671A). Enzyme catalysis synthesis of glutathione has become one of the most competitive methods due to its advantages of high raw material utilization and simple steps.

[0004] The extraction methods of glutathione reported so far mainly include copper salt method and ion exchange method. The copper salt method utilizes cuprous oxide to specifically bind to sulfhydryl to form glutathione copper salt, and further separates glutathione from the aqueous phase specifically through sulfide replacement. The addition of metal salts and sulfur-containing toxic substances in this separation process will cause environmental pollution and is difficult to post-process. The ion exchange method exchanges ions in ion exchange resin with ions in dilute solution to achieve the purpose of extracting or removing certain ions in the solution. Cationic resin can adsorb positively charged glutathione, but this method has low specificity and produces a large amount of wastewater. Therefore, it is urgent to develop a new and efficient glutathione separation and extraction technology and preparation process. Summary of the invention

[0005] The present invention provides a method for preparing glutathione in order to overcome the defects of low raw material utilization rate, complicated process steps, poor specificity of separation and extraction method, and easy environmental pollution in the preparation process of glutathione in the prior art.

[0006] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions: A method for preparing glutathione comprises the following steps: (S.1) Weigh the bacteria containing glutathione bifunctional enzyme and the bacteria containing polyphosphate kinase respectively, resuspend the bacteria in water, and ultrasonically disrupt them to obtain a crude enzyme solution. Add sodium L-glutamate, L-cysteine, glycine, adenosine diphosphate disodium salt, anhydrous magnesium sulfate, and sodium hexametaphosphate into the reaction kettle and adjust the pH of the reaction system to 5-9; (S.2) introducing an inert gas into the reaction system in step (S.1) and stirring the reaction, maintaining the pH of the reaction system constant with an alkaline solution during the reaction, adjusting the pH of the reaction solution to acidic after the reaction is completed, and collecting the filtrate after centrifugation; (S.3) adding an alcohol solvent to the filtrate obtained in step (S.2), allowing the mixture to settle overnight, centrifuging, collecting the supernatant and ultrafiltration, obtaining an ultrafiltration permeate and concentrating the permeate under reduced pressure, then adding activated carbon, stirring, and filtering to obtain a filtrate; (S.4) The filtrate obtained in step (S.3) is enriched and concentrated by a reverse osmosis membrane to obtain a glutathione concentrate, and an alcohol solvent is added, and the concentrate is stirred for crystallization. After the crystallization is completed, the concentrate is washed and vacuum dried to obtain a crude glutathione product, which is recrystallized, filtered, washed, and freeze-dried to obtain a pure glutathione product.

[0007] The present invention can effectively remove the main impurities in the enzyme catalytic reaction solution through the alcohol solvent precipitation method, which is helpful to slow down the oxidation in the separation and extraction process, and the alcohol solvent can be recycled and reused, which is beneficial to saving costs. The present invention can effectively remove most of the macromolecular and small molecular impurities through the ultrafiltration method, which is helpful to increase the purity of the product. The preparation method of glutathione of the present invention is simple to operate, low in pollution, mild in reaction conditions, and suitable for industrial production.

[0008] Preferably, the concentration of sodium L-glutamate added in step (S.1) is 70-160 mM; the concentration of L-cysteine ​​is 70-160 mM; the concentration of glycine is 70-160 mM; the concentration of adenosine diphosphate disodium salt is 2-10 mM; the concentration of anhydrous magnesium sulfate is 60-120 mM; and the concentration of sodium hexametaphosphate is 60-120 mM.

[0009] As a further preference, the concentration of sodium L-glutamate added in step (S.1) is 120-160 mM; the concentration of L-cysteine ​​is 120 mM; the concentration of glycine is 120 mM; the concentration of adenosine diphosphate disodium salt is 2 mM; the concentration of anhydrous magnesium sulfate is 120 mM; and the concentration of sodium hexametaphosphate is 70 mM.

[0010] Preferably, the concentration ratio of the bacteria containing glutathione bifunctional enzyme to the bacteria containing polyphosphate kinase added in step (S.1) is 2-6:1.

[0011] As a further preference, the concentration ratio of the bacteria containing glutathione bifunctional enzyme and the bacteria containing polyphosphate kinase added in step (S.1) is 4:1.

[0012] Preferably, the reaction temperature in step (S.2) is 25-45°C, and the reaction time is 6-10 hours.

[0013] As further preferred, the reaction temperature in step (S.2) is 25-30° C., and the reaction time is 8-10 h.

[0014] Preferably, in step (S.2), an alkaline solution is used to maintain the pH of the reaction system at a constant pH of 6 to 8; after the reaction is completed, the pH of the reaction solution is adjusted to 2.5 to 4.5.

[0015] As a further preference, in step (S.2), an alkaline solution is used to maintain the pH of the reaction system at a constant pH of 6.5-7.5; after the reaction is completed, the pH of the reaction solution is adjusted to 3.5-4.0.

[0016] Preferably, the alkaline solution in step (S.2) is any one or more combinations of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide and ammonia water.

[0017] Preferably, in step (S.2), the acidic solution used to adjust the pH of the reaction solution to acidic after the reaction is completed is any one or more combinations of hydrochloric acid, sulfuric acid and nitric acid.

[0018] Preferably, the alcohol solvent added in steps (S.3) to (S.4) is any one or more of methanol, ethanol, tert-butanol and butanol; the volume ratio of the alcohol solvent added in step (S.3) to the filtrate is 3 to 6:1.

[0019] As a further preference, the volume ratio of the alcohol solvent added in step (S.3) to the filtrate is 4-5:1.

[0020] Preferably, the reaction temperature for the reduced pressure concentration in step (S.3) is 45-55° C., and the rotation speed is 60-100 rpm.

[0021] Preferably, the amount of activated carbon added in step (S.3) is 5-30 g / L.

[0022] Preferably, the pore size of the ultrafiltration membrane used in the ultrafiltration process in step (S.3) is 2.5-10KDa, and the membrane flux is 50-150mL / min.

[0023] Preferably, the pore size of the reverse osmosis membrane in step (S.4) is 50-200 Da.

[0024] As further preferred, the pore size of the reverse osmosis membrane in step (S.4) is 100 Da.

[0025] Preferably, the concentration of the glutathione concentrate obtained in step (S.4) is 200-350 g / L.

[0026] Preferably, the vacuum degree of the vacuum drying process in step (S.4) is 0.07-0.09 MPa, and the drying temperature is ≤40°C.

[0027] Therefore, the present invention has the following beneficial effects: (1) The present invention can effectively remove the main impurities in the enzyme catalytic reaction solution through the alcohol solvent precipitation method, which helps to slow down the oxidation in the separation and extraction process, and the alcohol solvent can be recycled and reused, which is conducive to cost saving; (2) The present invention can effectively remove most of the macromolecular and small molecular impurities through ultrafiltration, which helps to increase the purity of the product; (3) The preparation method of glutathione of the present invention is simple to operate, has low pollution, mild reaction conditions, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figure is a comparison diagram of the reaction process of glutathione bifunctional enzyme and polyphosphate kinase coupled catalytic synthesis of glutathione in Example 1 and Example 2.

[0029] Figure 2 1 is a high performance liquid chromatogram of the reaction solution before and after alcohol precipitation in Example 1.

[0030] Figure 3 This is a process flow chart for preparing glutathione in Example 1. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0032] LB liquid culture medium composition: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, solvent is water, pH is natural.

[0033] Fermentation medium composition: peptone 15g / L, yeast powder 10g / L, sodium chloride 5g / L, ammonium sulfate 2g / L, dipotassium hydrogen phosphate 3g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate 0.5g / L, glucose 10g / L (sterilized separately).

[0034] Feed medium: glucose 600g / L, yeast powder 40g / L, peptone 75g / L, ammonium sulfate 35g / L.

[0035] Example 1 This embodiment provides a method for preparing glutathione.

[0036] A method for preparing glutathione comprises the following steps: (S.1) Preparation of crude enzyme solutions of glutathione bifunctional enzyme and polyphosphate kinase: (1) The strains containing glutathione bifunctional enzyme and polyphosphate kinase were fermented in 5L fermenters. The strains were inoculated on LB plates containing 50 μg / mL kanamycin resistance and cultured at 37°C for 12 h; single colonies were selected and inoculated into shake flasks to obtain seed solution, which was cultured at 37°C until OD 600 When the pH value is 0.6-0.8, inoculate it into a 5L fermentation tank. Use an acid-base feeding bottle to adjust the pH value during the fermentation process and control it at 6.8; set the ventilation volume to 6L / min; set the culture temperature to 37°C; set the rotation speed to 500rpm, and control the residual sugar at 2g / L through feed-batch culture. Cultivate until OD 600 Reach 30, then cool to 28°C, add inducer lactose with a final concentration of 12.5g / L to the fermenter for induction culture for 12h. After the fermentation is completed, centrifuge at 8000rpm, 4°C, 10min to collect the bacteria, and store in a refrigerator at -20°C for later use; (2) Weigh 40 g / L of bacteria containing glutathione bifunctional enzyme and 10 g / L of bacteria containing polyphosphate kinase, resuspend the bacteria in water and mix them to obtain a bacterial suspension, which is then subjected to ultrasonic disruption. The ultrasonic disruption power is 300 W, and the working time is 2 s per operation, 4 s rest, and the total working time is 40 min to obtain a crude enzyme solution.

[0037] (S.2) Synthesis of glutathione catalyzed by glutathione bifunctional enzyme coupled with polyphosphate kinase: 120mM L-glutamate sodium, 120mM glycine, 120mM L-cysteine, 120mM anhydrous MgSO4, 2mM adenosine diphosphate disodium salt, 70mM sodium hexametaphosphate were added to a 10L reactor, and then the crude enzyme solution obtained in step (S.1) was added, and the pH of the reaction system was adjusted to a constant 7.0 with 2mol / L sodium hydroxide solution. The reaction was stirred for 8h at 25°C and nitrogen was passed. Samples were taken from the reaction system every 2h, 2mol / L HCl was added to terminate the reaction, and the mixture was centrifuged at 10000rpm to remove the precipitate. The filtrate was collected and diluted 10 times, and the glutathione (GSH) content was detected by HPLC through a 0.22μm water filter membrane. The concentration of GSH in the product can be calculated through the GSH standard curve.

[0038] Chromatographic conditions for HPLC detection: C18 chromatographic column (4.6×mmol / L, 5μm), mobile phase (mixed solution prepared with 6.8g / L potassium dihydrogen phosphate and 2.02g / L sodium heptane sulfonate, pH adjusted to 2.8 with phosphoric acid, 950mL of mixed solution added with 50mL methanol, freshly prepared and used), 10μL injection volume, 1mL / min flow rate, 30°C, 20min retention time, 210nm wavelength detection. The reaction process comparison of glutathione bifunctional enzyme and polyphosphate kinase coupled catalytic synthesis of glutathione in this example and Example 2 is shown in the figure below. Figure 1 The results showed that the production of glutathione by glutathione bifunctional enzyme coupled with polyphosphate kinase could reach 26.60 g / L after 8 hours of reaction.

[0039] After the reaction was completed, 6 M hydrochloric acid was added to the prepared 10 L reaction solution to adjust the pH of the reaction solution to 4.0, and the solution was centrifuged at 8000 r / min for 10 min to remove the precipitate and collect the filtrate.

[0040] (S.3) Ethanol precipitation treatment of the dual enzyme catalytic reaction solution, ultrafiltration membrane treatment of the filtrate and decolorization by vacuum concentration: Add 4 times the volume of anhydrous ethanol to the filtrate obtained in step (S.2), place it at 4°C for 6 hours, centrifuge to remove the precipitate, and collect the supernatant. According to the method in the above step (S.2), the concentration and chromatographic purity of glutathione in the supernatant are detected by high performance liquid chromatography. The chromatographic purity of the supernatant is 83%, and the yield of glutathione is 90%. The high performance liquid chromatogram of the reaction solution before and after alcohol precipitation in this embodiment is as follows Figure 2 As shown. Figure 2 The HPLC chromatogram in Figure 2 shows that the ethanol precipitation method has a good removal effect on various major impurities in the glutathione reaction solution. After ethanol precipitation, the chromatographic purity of the supernatant reached 83.0% and the glutathione yield reached 90%.

[0041] The obtained reaction solution was centrifuged to remove the precipitate, and the supernatant was collected. The supernatant was then poured into a liquid storage tank equipped with an ultrafiltration device. The ultrafiltration membrane with a pore size of 10 kDa of PW1812 membrane tube was selected, and the operating pressure was 0.1 MPa. The ultrafiltration permeate was collected. According to the method in the above step (S.2), the concentration of the glutathione solution was determined by high performance liquid chromatography. The results showed that the yield of glutathione after ultrafiltration membrane treatment was 93.2%.

[0042] The collected ultrafiltration liquid was then concentrated under reduced pressure at 45°C and 80 rpm to recover ethanol. Activated carbon was then added to the ultrafiltration liquid after reduced pressure concentration to a final concentration of 10 g / L, and the mixture was stirred for decolorization for 60 minutes and then filtered to obtain a filtrate and a filter cake.

[0043] (S.4) Crystallization: The filtrate obtained in step (S.3) is poured into the storage tank of the ultrafiltration device, and the reverse osmosis membrane is used to circulate at 25.0Hz until there are no bubbles, and then the pressure is increased to 3.0MPa for reverse osmosis concentration and enrichment to the minimum circulation volume of 0.3-0.5L, and the pressure is turned off to collect the glutathione concentrate. 4 times the volume of anhydrous ethanol is added to the glutathione concentrate, and the mixture is stirred at 0-10°C for crystallization. After the crystallization is completed, the mixture is washed with anhydrous ethanol and vacuum dried for 6-8h to obtain crude glutathione. The crude glutathione is recrystallized with ethanol, filtered, and the filter cake is washed with ethanol. After freeze-drying, pure glutathione is obtained. According to the method in the above step (S.2), the concentration and chromatographic purity of the glutathione solution are determined by high performance liquid chromatography. The results show that the solid oxidized glutathione prepared by the method of the present invention has a low impurity level, and the yield of reduced glutathione is 83.6% and the purity is 98.2%. The glutathione preparation process flow chart in this embodiment is as follows Figure 3 shown.

[0044] Example 2 The difference between this embodiment and embodiment 1 is that: This embodiment provides a method for preparing glutathione.

[0045] A method for preparing glutathione, wherein in step (S.2), 156 mM L-sodium glutamate is added to a 10 L reactor respectively instead of 120 mM L-sodium glutamate. The rest is the same as in Example 1. According to the method in step (S.2) in Example 1, the concentration and chromatographic purity of the glutathione solution are determined by high performance liquid chromatography. The results are as follows: Figure 1 As shown, glutathione bifunctional enzyme coupled with polyphosphate kinase catalyzes the synthesis of glutathione, and the yield can reach 29.76 g / L after 8 hours of reaction. Compared with Example 1, it is found that appropriately increasing the amount of sodium L-glutamate added can effectively increase the yield of glutathione.

[0046] Example 3 The difference between this embodiment and embodiment 1 is that: This embodiment provides a method for preparing glutathione.

[0047] A method for preparing glutathione, wherein in step (S.3), 4 volumes of tert-butyl alcohol are added to the filtrate obtained in step (S.2), and the filtrate is placed at 4°C for 6 hours instead of adding 4 volumes of anhydrous ethanol to the filtrate obtained in step (S.2), and the filtrate is placed at 4°C for 6 hours. The rest is the same as in Example 1. According to the method in step (S.2) in the above Example 1, the concentration and chromatographic purity of the glutathione solution in the supernatant are determined by high performance liquid chromatography. After tert-butyl alcohol precipitation treatment, the chromatographic purity of the supernatant reaches 73.0%, and the yield of glutathione is 82%.

[0048] Example 4 The difference between this embodiment and embodiment 1 is that: This embodiment provides a method for preparing glutathione.

[0049] A method for preparing glutathione, wherein in step (S.1), 20 g / L of bacteria containing glutathione bifunctional enzyme and 10 g / L of bacteria containing polyphosphate kinase are weighed respectively; in step (S.2), 70 mM sodium glutamate, 70 mM glycine, and 70 mM L-cysteine, 60mM anhydrous MgSO4, 10mM adenosine diphosphate disodium salt, 60mM sodium hexametaphosphate, and then add the crude enzyme solution obtained in step (S.1), adjust the pH of the reaction system to a constant 6.0 with 2mol / L sodium hydroxide solution, and stir the reaction for 6h at 30℃ with helium; after the reaction is completed, add 6M hydrochloric acid to the prepared 10L reaction solution to adjust the pH of the reaction solution to 2.5; in step (S.3), add 3 times the volume of methanol to the filtrate obtained in step (S.2), and let it stand at 4℃ for 7h; select PW1812 ultrafiltration membrane with a pore size of 2.5kDa, the operating pressure is 0.2Mpa, and the ultrafiltration permeate is collected; then the collected ultrafiltration permeate is concentrated under reduced pressure at 50℃ and 60rpm to recover ethanol. Then, activated carbon is added to the ultrafiltration permeate after vacuum concentration to a final concentration of 5 g / L, and the mixture is stirred for decolorization for 60 minutes and then filtered to obtain a filtrate and a filter cake; in step (S.4), a reverse osmosis membrane is used to circulate at 25.0 Hz until there are no bubbles, and then the mixture is pressurized to 2.0 MPa for reverse osmosis concentration and enrichment to a minimum circulation volume of 0.5-0.8 L, and the pressure is turned off to collect a glutathione concentrate with a concentration of 200 g / L. The results show that the solid oxidized glutathione prepared by the method of the present invention has a low impurity level, a reduced glutathione yield of 80.5%, and a purity of 98.0%. Others are the same as in Example 1.

[0050] Example 5 The difference between this embodiment and embodiment 1 is that: This embodiment provides a method for preparing glutathione.

[0051] A method for preparing glutathione, wherein in step (S.1), 60 g / L of bacterial cells containing glutathione bifunctional enzyme and 10 g / L of bacterial cells containing polyphosphate kinase are weighed respectively; in step (S.2), 160 mM sodium glutamate, 160 mM glycine, and 100 mM L-cysteine, 90mM anhydrous MgSO4, 6mM disodium adenosine diphosphate, 120mM sodium hexametaphosphate, and then add the crude enzyme solution obtained in step (S.1), adjust the pH of the reaction system to a constant 8.0 with 2mol / L sodium hydroxide solution, and stir the reaction for 10 hours at 45°C with helium. After the reaction is completed, add 6M hydrochloric acid to the prepared 10L reaction solution to adjust the pH of the reaction solution to 4.5. In step (S.3), add 6 times the volume of butanol to the filtrate obtained in step (S.2), and let it stand at 4°C for 8 hours. Select an ultrafiltration membrane with a pore size of 6kDa of PW1812 membrane tube and an operating pressure of 0.4Mpa to collect the ultrafiltration permeate. Then, at 55°C, the collected ultrafiltration permeate is concentrated under reduced pressure at 100rpm to recover ethanol. Then, activated carbon is added to the ultrafiltration permeate after vacuum concentration to a final concentration of 30 g / L, and the mixture is stirred for decolorization for 60 minutes and then filtered to obtain a filtrate and a filter cake; in step (S.4), a reverse osmosis membrane is used to circulate at 55.0 Hz until there are no bubbles, and then the mixture is pressurized to 1.0 MPa for reverse osmosis concentration and enrichment to a minimum circulation volume of 0.4-0.6 L, and the pressure is turned off to collect a glutathione concentrate with a concentration of 350 g / L. The results show that the solid oxidized glutathione prepared by the method of the present invention has a low impurity level, a reduced glutathione yield of 81.8%, and a purity of 97.9%. Others are the same as in Example 1.

[0052] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, according to the ideas provided by the present invention, there will be changes in the specific implementation methods, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing glutathione, characterized in that: The following steps are involved: (S.1) Weigh the bacteria containing glutathione bifunctional enzyme and the bacteria containing polyphosphate kinase respectively, resuspend the bacteria in water, and ultrasonically disrupt them to obtain a crude enzyme solution. Add sodium L-glutamate, L-cysteine, glycine, adenosine diphosphate disodium salt, anhydrous magnesium sulfate, and sodium hexametaphosphate into the reaction kettle and adjust the pH of the reaction system to 5-9; (S.2) introducing an inert gas into the reaction system in step (S.1) and stirring the reaction, maintaining the pH of the reaction system constant with an alkaline solution during the reaction, adjusting the pH of the reaction solution to acidic after the reaction is completed, and collecting the filtrate after centrifugation; (S.3) adding an alcohol solvent to the filtrate obtained in step (S.2), allowing the mixture to settle overnight, centrifuging, collecting the supernatant and ultrafiltration, obtaining an ultrafiltration permeate and concentrating the permeate under reduced pressure, then adding activated carbon, stirring, and filtering to obtain a filtrate; (S.4) The filtrate obtained in step (S.3) is enriched and concentrated by a reverse osmosis membrane to obtain a glutathione concentrate, and an alcohol solvent is added, and the concentrate is stirred for crystallization. After the crystallization is completed, the concentrate is washed and vacuum dried to obtain a crude glutathione product, which is recrystallized, filtered, washed, and freeze-dried to obtain a pure glutathione product.

2. The method for preparing glutathione according to claim 1, characterized in that: The concentration of sodium L-glutamate added in step (S.1) is 70-160 mM; the concentration of L-cysteine ​​is 70-160 mM; the concentration of glycine is 70-160 mM; the concentration of adenosine diphosphate disodium salt is 2-10 mM; the concentration of anhydrous magnesium sulfate is 60-120 mM; and the concentration of sodium hexametaphosphate is 60-120 mM.

3. The method for preparing glutathione according to claim 1, characterized in that: The concentration ratio of the bacteria containing glutathione bifunctional enzyme and the bacteria containing polyphosphate kinase added in step (S.1) is 2-6:

1.

4. The method for preparing glutathione according to claim 1, characterized in that: The reaction temperature in step (S.2) is 25-45°C, and the reaction time is 6-10 hours.

5. The method for preparing glutathione according to claim 1, characterized in that: In step (S.2), an alkaline solution is used to maintain the pH of the reaction system at a constant pH of 6 to 8; after the reaction is completed, the pH of the reaction solution is adjusted to 2.5 to 4.

5.

6. The method for preparing glutathione according to claim 1, characterized in that: The alcohol solvent added in steps (S.3) to (S.4) is any one or more of methanol, ethanol, tert-butanol, and butanol; the volume ratio of the alcohol solvent added in step (S.3) to the filtrate is 3 to 6:

1.

7. The method for preparing glutathione according to claim 1, characterized in that: The reaction temperature for the reduced pressure concentration in step (S.3) is 45-55°C, and the rotation speed is 60-100 rpm.

8. The method for preparing glutathione according to claim 1, characterized in that: The amount of activated carbon added in step (S.3) is 5-30 g / L.

9. The method for preparing glutathione according to claim 1, characterized in that: The pore size of the reverse osmosis membrane in step (S.4) is 50~200Da.

10. The method for preparing glutathione according to claim 1, characterized in that: The concentration of the glutathione concentrated solution obtained in step (S.4) is 200-350 g / L.

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