CN106518962B

CN106518962B - Method for preparing reduced glutathione from yeast cells

Info

Publication number: CN106518962B
Application number: CN201610901699.8A
Authority: CN
Inventors: 邓张双; 李知洪; 杜维力; 姚鹃; 刘秀继; 李啸; 陈良立; 邓艾平; 邹坤; 龚大春
Original assignee: Angel Yeast Co Ltd; China Three Gorges University CTGU
Current assignee: Angel Yeast Co Ltd; China Three Gorges University CTGU
Priority date: 2016-10-14
Filing date: 2016-10-14
Publication date: 2021-04-02
Anticipated expiration: 2036-10-14
Also published as: CN106518962A

Abstract

A method for preparing reduced glutathione from yeast cells comprises extracting yeast cells rich in reduced glutathione with hot water to obtain glutathione solution; glutathione recognition ion Cu is added²⁺Stirring; suspending the centrifuged precipitate in water, introducing hydrogen sulfide gas, and stirring; decolorizing the centrifuged solution by active carbon, and concentrating and recrystallizing the filtrate to obtain the high-purity reduced glutathione crystal. The method has simple operation process and easily obtained raw materials, and can be industrially implemented. The reduced glutathione product produced by the method can be applied as a pharmaceutical raw material medicament, food and cosmetic additive.

Description

Method for preparing reduced glutathione from yeast cells

Technical Field

The invention relates to a purification method of high-purity reduced glutathione and a process for preparing the high-purity reduced glutathione from yeast cells. Belongs to the field of biological engineering and pharmaceutical compounds.

Background

Reduced Glutathione (GSH) is an important pharmaceutical reagent and is mainly used for the treatment and adjuvant therapy of chemotherapy, hypoxemia, liver diseases, organophosphorus, amido or nitro compound poisoning in clinic; meanwhile, the antioxidant property of the glutathione makes the glutathione to be favored as an additive in the food and cosmetic processing industry. The products on the market for sale mainly comprise: glutathione tablet (adomoplan) for protecting liver, medicine for treating toxic hepatic injury (reduced glutathione for injection), glutathione whitening emulsion, glutathione speckle removing essence, etc. In 2015, the GSH market at home and abroad has increased at a rate of 10% every year. The industrialization of commercial yeast fermentation for producing glutathione by Kyowa (Kyowa, Japan) and Ajinomoto (Ajinomoto, K.K.) has been realized in the 80 s of the 20 th century, and has become a major production and supplier of glutathione all over the world and also forms a technical barrier. The pharmaceutical chemical industry of Shandong Jincheng, China introduced the Japanese glutathione purification technology, and became the first company for producing glutathione from yeast in China.

Glutathione is derived from animals, plants, microorganisms and chemical synthesis. The yeast and wheat germ are most abundant. The glutathione purification method mainly comprises an ion exchange method, an electrodialysis method, a two-aqueous phase method and the like, and the methods are mostly limited by the structural instability of the glutathione, so that the purification multiple is low, the process flow is complicated, and the production cost is high. Therefore, the development of a low-cost industrialized reduced glutathione purification process is particularly important for the development of products related to glutathione in the food and medicine field. Meanwhile, theoretical and practical references are provided for formulating national standard compilation of 'yeast rich in nutrients'.

Disclosure of Invention

The invention aims to provide a method for preparing high-purity reduced glutathione from yeast cells, which can be applied to industrialization of producing glutathione by using the yeast cells with high efficiency and low cost and provides a high-purity reduced glutathione raw material for the food and medicine field. Meanwhile, theoretical and practical references are provided for formulating national standard compilation of 'yeast rich in nutrients'.

The purpose of the invention is realized as follows: a method for preparing high-purity reduced glutathione from yeast cells comprises the following steps:

(1) preparing the yeast cell rich in the reduced glutathione: producing reduced glutathione by yeast cell fermentation method, wherein the content of GSH is 1-5% calculated by dry weight of yeast cells, and the yeast cells rich in reduced glutathione comprise: yeast milk, yeast dry powder, yeast extract, etc.

(2) Thermal extraction of yeast cells: a certain mass of yeast cells is suspended in 5 to 15 times, preferably 10 times, volume of deionized water. Adjusting pH to 2.0, and stirring at 80-100 deg.C, preferably 85-90 deg.C for 0-30 min, preferably 5-15 min. After completion, the mixture is quickly placed in ice water to be cooled to below 20 ℃, and centrifuged at 3000-10000rpm/min, preferably 7500rpm/min for 10-30 minutes, preferably 15 minutes. The precipitate was removed and the light phase remained.

(3) Preparation of GSH-Cu complex: detecting the content of the GSH in the light phase in the step 2) by utilizing an HPLC technology. The pH of the light phase is adjusted to 5.0 to 7.0, preferably 6.0, with NaOH, with stirring at 10 to 100rpm/min, preferably 50 rpm/min. Adding CuSO according to the molar mass of GSH in the light phase and the proportion of 1:1-1:20, preferably 1:2-1:15₄. Stirring is maintained for 15-60 minutes, preferably 30 minutes. Centrifugation is carried out at 3000-10000rpm/min, preferably 7500rpm/min for 20-40 minutes, preferably 30 minutes. The heavy phase is retained.

(4) Washing: suspending a certain mass of GSH-Cu complex in purified water with the volume of 100-fold and 200-fold, preferably 150-fold, and maintaining stirring for 20-40 minutes, preferably 30 minutes. Centrifugation is carried out at 3000-10000rpm/min, preferably 7500rpm/min for 20-40 minutes, preferably 30 minutes. The heavy phase is retained.

(5) Decomplexing: a certain mass of GSH-Cu complex is suspended in 2-10 times, preferably 4 times, volume of purified water, and stirred. Introduction of H₂S gas was maintained for 5 minutes. Centrifugation is carried out at 3000-10000rpm/min, preferably 7500rpm/min for 10-30 minutes, preferably 15 minutes. The precipitate was removed and the light phase remained. Introduction of H₂The S gas comprises: pure H₂S gas, or enriched in H at high concentration₂Industrial methane waste gas of S component or H easily generated under acidic condition₂Inorganic and organic salts of S, e.g. (NH)₄)₂S，Na₂S and the like.

(6) And (3) decoloring: in the step (5), the pH value of the light phase is adjusted to 2.0 by hydrochloric acid, activated carbon is added according to the proportion of 5 per mill, and the stirring reaction is maintained for 20-60 minutes, preferably 30 minutes. After the reaction was completed, the activated carbon was removed by filtration with a 0.25 μm filter membrane, and the filtrate was retained.

(7) Concentration: and (6) concentrating the filtrate under reduced pressure to obtain a glutathione solution with the concentration of 500-1000g/L glutathione, preferably 800 g/L.

(8) And (3) recrystallization: keeping stirring, adding 50-80% (v/v) ethanol water solution, preferably 70% ethanol water solution, into the glutathione-containing solution in step (7), and adding glutathione crystal as seed crystal at-4-20 deg.C, preferably 4 deg.C. The addition amount of the seed crystal is 20 to 200mg/L, preferably 50 mg/L. After stirring is continued for 30 minutes, stirring is stopped and the temperature is maintained for 6 to 20 hours, preferably 12 hours. Obtaining crystal slurry, and filtering to obtain high-purity reduced glutathione substance with the purity of the reduced glutathione being more than 98%.

The invention uses the prepared reduced glutathione in pharmaceutical reagents, foods and cosmetic additives.

Drawings

FIG. 1 shows the results of screening GSH-metal complexes.

FIG. 2 is an aqueous phase HPLC plot of yeast cells after hot water (pH 2.0) leaching for 15 minutes at 90 ℃.

FIG. 3 is a process of treating yeast extract with Cu²⁺Light phase HPLC profile after complexation.

FIG. 4 is a light phase HPLC profile after washing with water of a GSH-Cu complex.

FIG. 5 is a light phase HPLC chromatogram after complex decomplexation.

FIG. 6 shows the composition of the complex after decomplexation and freeze-drying of the light phase¹H NMR spectrum (400MHz, D)₂O)。

FIG. 7 shows the composition of the complex after decomplexation and freeze-drying of the light phase¹³C NMR spectrum (400MHz, D)₂O)。

FIG. 8 shows the components obtained after recrystallization¹H NMR spectrum (400MHz, D)₂O)。

FIG. 9 shows the components obtained after recrystallization¹³C NMR spectrum (400MHz, D)₂O)。

Figure 10 is the reduced GSH crude and recrystallized product appearance, with the upper left corner being the recrystallized product with a purity > 98% and the right corner being the crude product with a purity > 90%.

Detailed Description

Example 1

(1) Extraction of GSH from yeast cells

Weighing 100.0g of yeast milk, adding 900mL of pure water, adjusting the pH value to 2.0 by hydrochloric acid, leaching in a water bath at 90 ℃ for 15min, stirring for 5min, and quickly cooling to room temperature in an ice bath. Centrifuging at 7500r/min for 15min, and collecting supernatant.

(2) Preparation of GSH-Cu complexes

400mL of yeast extract in the above steps is adjusted to pH 6.0 by NaOH according to GSH and CuSO₄·5H₂CuSO is added into the mixture with the molar mass ratio of O being 1:15₄·5H₂And complexing with O, maintaining stirring, and centrifuging at 7500r/min for 30min to obtain about 500mg of precipitate.

(3) Washing machine

The precipitate obtained in the above step was washed with water 3 times, suspended in 150-fold volume of purified water, stirred for 30 minutes, and then centrifuged at 7500rpm/min for 30 minutes. The heavy phase is retained.

(4) Decomplexation of

Adding 2L of water into the precipitate washed by the water in the step for suspension, and introducing H₂S gas, stirring is maintained for 5 minutes, and centrifugation is carried out at 7500rpm/min for 15 minutes. The precipitate was removed and the light phase remained. OD600 was measured to be 0.022 using a spectrophotometer. The HPLC technology is utilized to carry out qualitative and quantitative analysis on the water phase, and the purity of the reduced glutathione is 90.5 percent.

(5) Decolorization of

And (3) regulating the pH value of the light phase in the steps to be 2.0 by hydrochloric acid, adding activated carbon according to the proportion of 5 per mill, and keeping stirring and reacting for 30 minutes. After the reaction was completed, the activated carbon was removed by filtration with a 0.25 μm filter membrane, and the filtrate was retained. The OD600 value detected by a spectrophotometer is 0.002, and the decolorizing effect is obvious. The absolute content of the reduced glutathione is not obviously changed, and the purity of the reduced glutathione is 89.4 percent.

(6) Concentrating

And concentrating the water phase in the step under reduced pressure to obtain a glutathione solution with the concentration of 800 g/L.

(7) Recrystallization

While maintaining stirring, an equal volume of 70% (v/v) ethanol aqueous solution was added to the glutathione-containing solution in the above step, and glutathione crystals were added as seed crystals at 4 ℃. The amount of seed crystal added was 50 mg/L. After stirring was continued for 30 minutes, stirring was stopped and the temperature was maintained for 12 hours. Obtaining crystal slurry, and filtering to obtain the high-purity reduced glutathione substance. The purity of the reduced glutathione is 98.5 percent.

FIG. 1 shows the results of screening GSH-metal complexes. Showing Cu in the figure²⁺Form a white complex precipitate with GSH, and KMnO₄GSH is oxidized to generate brown oxidation products.

FIG. 4 is a light phase HPLC profile after washing with water of a GSH-Cu complex. The figure shows that after 2 washes most of the water soluble impurities were eluted and no reduced GSH was detected in the eluate.

FIG. 5 is a light phase HPLC profile after complex decomplexation (reduced glutathione purity > 90%, low peak line is reduced glutathione and oxidized glutathione standards cocktail).

Claims

1. A method for preparing reduced glutathione from yeast cells is characterized by comprising the following steps:

(1) extraction of glutathione from yeast cells: suspending yeast cells with glutathione content of 1-5% with deionized water of 5-15 times volume, adjusting pH value to 2.0, stirring at 80-100 deg.C for 5-30 min, rapidly cooling in ice water to below 20 deg.C, centrifuging at 3000 plus 10000rpm/min for 10-30 min, removing precipitate, and retaining light phase;

(2) preparation of GSH-Cu complex: detecting the content of the GSH in the light phase in the step (1) by using an HPLC technology, adjusting the pH value of the light phase to be 5.0-7.0 by using NaOH under the stirring state of 10-100rpm/min, and according to the mole number of the GSH in the light phase, performing the following steps of 1:1-1:20, adding CuSO₄Keeping stirring for 15-60 minutes, centrifuging for 10-40 minutes at 3000-10000rpm/min, and reserving heavy phase to obtain GSH-Cu complex;

(3) washing: suspending the GSH-Cu complex with purified water of which the volume is 200 times that of 100-;

(4) decomplexing: suspending the GSH-Cu complex washed in the step (3) with purified water with the volume of 2-10 times, maintaining stirring, and introducing H₂S gas is maintained for 5 minutes, centrifugation is carried out for 10-30 minutes at 3000-10000rpm/min, precipitates are removed, and a light phase is remained;

(5) and (3) decoloring: adjusting the pH value of the light phase in the step (4) to 2.0 by using hydrochloric acid, adding activated carbon according to the proportion of 5 per mill of mass fraction, maintaining stirring for reaction for 20-60 minutes, filtering by using a 0.25 mu m filter membrane to remove the activated carbon after the reaction is finished, and keeping the filtrate;

(6) concentration: concentrating the filtrate obtained in the step (5) under reduced pressure to obtain glutathione solution containing glutathione;

(7) and (3) recrystallization: and (3) keeping stirring, adding an equal volume of 70% (v/v) ethanol aqueous solution into the glutathione-containing solution in the step (6), adding glutathione crystals as seed crystals at 4 ℃, keeping the addition amount of the seed crystals at 50mg/L, continuing stirring for 30 minutes, stopping stirring, keeping the temperature for 12 hours to obtain crystal slurry, and filtering to obtain reduced glutathione substances, wherein the purity of the reduced glutathione is more than 98%.

2. The method of producing reduced glutathione from yeast cells according to claim 1, wherein: in the step (1), extracting glutathione from yeast cells: suspending yeast cells with glutathione content of 1-5% with 10 times volume of deionized water, adjusting pH to 2.0, stirring at 90 deg.C for 5min, rapidly cooling in ice water to below 20 deg.C, centrifuging at 7500rpm/min for 15min, removing precipitate, and retaining light phase.

3. The method of producing reduced glutathione from yeast cells according to claim 1, wherein: in the step (2), preparation of GSH-Cu complex: detecting the content of the GSH in the light phase in the step (1) by using an HPLC technology, adjusting the pH value of the light phase to be 6.0 by using NaOH under the stirring state of 50rpm/min, and according to the mole number of the GSH in the light phase, performing the following steps of 1:15, adding CuSO₄·5H₂And O, maintaining stirring for 15-60 minutes, centrifuging for 15 minutes at 7500rpm/min, and reserving heavy phase to obtain GSH-Cu complex.

4. The method of producing reduced glutathione from yeast cells according to claim 1, wherein: in the step (3), washing: the GSH-Cu complex was suspended in 150 volumes of purified water, stirred for 30 minutes, centrifuged at 7500rpm/min for 30 minutes, leaving the heavy phase.

5. The method of producing reduced glutathione from yeast cells according to claim 1, wherein: in the step (4), decomplexing: suspending the GSH-Cu complex washed in the step (3) with purified water with 4 times of volume, maintaining stirring, and introducing H₂S gas is maintained for 5 minutes, and the mixture is centrifuged at 7500rpm/min for 30 minutes to remove precipitates and leave a light phase.

6. The method of claim 1, wherein the reduced form of the valley is prepared from yeast cellsA method of cystatin, characterized by: h is introduced in the complex decomplexing process carried out in the step (4)₂The S gas comprises: pure H₂S gas, or containing H₂Industrial methane waste gas of S component or H easily generated under acidic condition₂An inorganic or organic salt of S, the inorganic salt being (NH)₄)₂S or Na₂S。