阅读说明：本技术 一种采用发酵法生产谷胱甘肽的新方法 (Novel method for producing glutathione by adopting fermentation method ) 是由 夏颖 贾伟 周雪美 王水淋 于 2019-10-30 设计创作，主要内容包括：本发明提供了一种采用发酵法生产谷胱甘肽的新方法,包括斜面菌种的制备、培养基的选择、发酵条件的优化和目标产物的提取精制等工艺步骤,本发明通过毕赤酵母菌(P.pastoris)发酵转化生产谷胱甘肽,具有生产成本低,生产原料来源简单易得,生产过程中不使用有机溶剂,发酵过程也不产生有毒有害的物质,具有工艺过程简单,生产操作安全等优点,且其发酵时间快,生产的的谷胱甘肽产量高、质量好。(The invention provides a novel method for producing glutathione by adopting a fermentation method, which comprises the process steps of preparation of slant strains, selection of a culture medium, optimization of fermentation conditions, extraction and refining of a target product and the like.)

1. A new method for producing glutathione by adopting a fermentation method is characterized in that: the production process of glutathione by fermentation of Pichia pastoris (P. pastoris) comprises the following steps:

(1) inoculating Pichia pastoris (P.pastoris) strain to a slant culture medium in a test tube to prepare slant strain;

(2) performing amplification culture on slant strains to obtain a seed solution;

(3) inoculating the seed liquid into a fermentation tank for fermentation to obtain fermentation liquid;

(4) adding reactant components such as glucose and the like;

(5) and (5) separating and purifying glutathione.

2. The method of claim 1, wherein the glutathione is produced by fermentation, which comprises the following steps: the slant culture medium in the step (1) comprises the following components in parts by weight: 5-40 parts of yeast extract, 5-40 parts of glucose, 0-40 parts of ammonia water, 3-30 parts of soybean peptone, 0-40 parts of ammonium sulfate, 0-3 parts of potassium chloride, 0-5 parts of urea, 0-10 parts of diammonium hydrogen phosphate, 0-5 parts of magnesium sulfate, 0-10 parts of glycerol, 0-5 parts of sodium glutamate, 0-5 parts of glycine, 0-5 parts of cysteine and a proper amount of water.

3. The method of claim 1 or 2, wherein the glutathione is produced by fermentation, which comprises the following steps: the preparation process conditions of the slant strain are as follows: the pH value is 4.0-7.5, the temperature is 25-40 ℃, and the culture time is 12-36 hours.

4. The method of claim 1, wherein the glutathione is produced by fermentation, which comprises the following steps: the seed liquid in the step (2) consists of the following components in parts by weight: 40-80 parts of glucose, 0-40 parts of ammonia water, 0.1-5 parts of urea, 20-40 parts of ammonium sulfate, 1-10 parts of diammonium hydrogen phosphate, 0.1-5 parts of potassium chloride, 0.1-5 parts of magnesium sulfate, 1-20 parts of yeast extract, 0.1-5 parts of sodium glutamate, 0.1-5 parts of glycine, 0.1-5 parts of cysteine and 1000 parts of water.

5. The method of claim 1, wherein the glutathione is produced by fermentation, which comprises the following steps: the fermentation process conditions in the step (3) are as follows: the inoculation amount of the seed liquid is 6-12%, the pH value is 4.0-7.5, the culture temperature is 25-40 ℃, the ventilation rate is 10-300m3/h, the stirring speed is 350-450 r/min, and the culture time is 12-36 h.

6. The method of claim 1, wherein the glutathione is produced by fermentation, which comprises the following steps: the reaction conditions in the step (4) are as follows: the mass concentration of the glucose is 100-200mmol/l, the reaction temperature is 20-40 ℃, the reaction time is 24-48 hours, and the stirring speed is 160-200 r/min.

7. The method of claim 1, wherein the glutathione is produced by fermentation, which comprises the following steps: the separation and purification of the glutathione in the step (5) comprises the following six steps:

a. breaking the cell wall of the fermentation broth after fermentation is finished;

b. b, removing impurities such as mushroom dreg protein and the like from the fermentation liquor obtained in the step a through a membrane filtration technology;

c. and c, carrying out copper salt reaction on the concentrated solution after the step b.

d. C, carrying out hydrogen sulfide reaction after the step c;

e. c, filtering, separating and purifying the reaction liquid obtained in the step d;

f. and e, crystallizing and drying the concentrated solution obtained in the step e to obtain the glutathione with the purity of more than 98 percent.

8. The method of claim 7, wherein the glutathione is produced by fermentation, which comprises the following steps: breaking the cell wall of the fermentation liquor thallus in the step a; the filtering membrane in the step b is a ceramic membrane, an organic membrane or a nanofiltration membrane; e, adopting a macroporous adsorption resin column or cation resin or a simulated moving bed chromatography for the filtration separation technology in the step e; and f, concentrating and drying the filtered solution in the step f, wherein the vacuum degree of vacuum drying is 0.05-0.5Mpa, the temperature is 65 ℃, and the purity of the dried finished product is more than 99%.

Technical Field

The invention relates to the technical field of microorganisms, in particular to a novel method for producing glutathione by adopting a fermentation method.

Background

Glutathione (GSH) is a tripeptide with important physiological functions consisting of L-glutamic acid, L-cysteine and glycine. Glutathione is widely distributed in animals, plants and microorganisms, participates in the synthesis of protein and ribonucleic acid, the transportation of oxygen and nutrient substances, maintains the activity of endogenous enzymes, the tricarboxylic acid cycle and the sugar metabolism in vivo, eliminates excessive free radicals in vivo and the like. The glutathione is clinically used for the treatment of radiation resistance, tumor resistance, antioxidant poisoning, aging resistance and endocrine coordination. Glutathione is also used as a food additive to extend the shelf life of food and fortify the amino acid content of food. Reduced glutathione is a liver disease drug which is successfully developed by Nippon synergy fermentation company and Nippon mountain company in 1995 and is first approved and produced by Nippon JFDA on the market, and is the most basic composition component for liver disease therapy because of its definite curative effect. Glutathione is used as a key intermediate of anti-hepatosis drugs, and has great economic prospect.

At present, the main production methods of glutathione include extraction methods, chemical synthesis methods, fermentation methods and enzymatic methods. The Chinese invention patent No. CN102093468A discloses a method for synthesizing reduced glutathione, but the synthesis process uses a large amount of organic solvents such as diethyl ether, benzyl alcohol, tetrahydrofuran, petroleum ether, thionyl chloride and the like, and has certain influence on environmental pollution; chinese invention patent No. CN102586369A discloses a method for producing glutathione by using recombinant Escherichia coli fermentation, but the fermentation time is a little longer, and the product is an intracellular product with low content, and secondly, the Escherichia coli is a pathogenic bacterium which is harmful to human body and has poor production safety; the preparation method of the foreign glutathione mainly catalyzes and synthesizes the glutathione by taking glycosidase as a catalyst, so the cost of the produced glutathione is high, and the yield of the glutathione prepared by the method is not high, thereby bringing great difficulty to practical popularization and application.

Disclosure of Invention

Technical problem to be solved

The invention aims to provide a novel method for producing glutathione by adopting a fermentation method, wherein glutathione is produced by adopting a microbial fermentation method, glucose is used as a substrate, and the substrate is converted into glutathione by adopting a microbial conversion method. In order to achieve the purpose, the invention adopts the following technical scheme:

(II) technical scheme

A new method for producing glutathione by adopting a fermentation method, namely, pichia pastoris (P.pastoris) is used for producing glutathione by fermentation, and the production process comprises the following process steps:

(1) inoculating Pichia pastoris (P.pastoris) strain to a slant culture medium in a test tube to prepare slant strain;

(2) performing amplification culture on slant strains to obtain a seed solution;

(3) inoculating the seed liquid into a fermentation tank for fermentation to obtain fermentation liquid;

(4) adding reactant components such as glucose and the like;

(5) and (5) separating and purifying glutathione.

Further, the slant culture medium in the step (1) comprises the following components in parts by weight: 5-40 parts of yeast extract, 5-40 parts of glucose, 0-40 parts of ammonia water, 3-30 parts of soybean peptone, 0-40 parts of ammonium sulfate, 0-3 parts of potassium chloride, 0-5 parts of urea, 0-10 parts of diammonium hydrogen phosphate, 0-5 parts of magnesium sulfate, 0-10 parts of glycerol, 0-5 parts of sodium glutamate, 0-5 parts of glycine, 0-5 parts of cysteine and a proper amount of water.

Further, the preparation process conditions of the slant strain are as follows: the pH value is 4.0-7.5, the temperature is 25-40 ℃, and the culture time is 12-36 hours.

Further, the seed liquid in the step (2) comprises the following components in parts by weight: 40-80 parts of glucose, 0-40 parts of ammonia water, 0.1-5 parts of urea, 20-40 parts of ammonium sulfate, 1-10 parts of diammonium hydrogen phosphate, 0.1-5 parts of potassium chloride, 0.1-5 parts of magnesium sulfate, 1-20 parts of yeast extract, 0.1-5 parts of sodium glutamate, 0.1-5 parts of glycine, 0.1-5 parts of cysteine and 1000 parts of water.

Further, the process conditions of the fermentation in the step (3) are as follows: the inoculation amount of the seed liquid is 6-12%, the pH value is 4.0-7.5, the culture temperature is 25-40 ℃, the ventilation rate is 10-300m3/h, the stirring speed is 350-450 r/min, and the culture time is 12-36 h.

Further, the reaction conditions in the step (4) are as follows: the mass concentration of the glucose is 100-200mmol/l, the reaction temperature is 20-40 ℃, the reaction time is 24-48 hours, and the stirring speed is 160-200 r/min.

Further, the separation and purification of glutathione in the step (5) comprises the following six steps:

a. breaking the cell wall of the fermentation broth after fermentation is finished;

b. b, removing impurities such as mushroom dreg protein and the like from the fermentation liquor obtained in the step a through a membrane filtration technology;

c. and c, carrying out copper salt reaction on the concentrated solution after the step b.

d. C, carrying out hydrogen sulfide reaction after the step c;

e. c, filtering, separating and purifying the reaction liquid obtained in the step d;

f. and e, crystallizing and drying the concentrated solution obtained in the step e to obtain the glutathione with the purity of more than 98 percent.

Further, breaking the cell wall of the fermentation liquor thalli in the step a; the filtering membrane in the step b is a ceramic membrane, an organic membrane or a nanofiltration membrane; e, adopting a macroporous adsorption resin column or cation resin or a simulated moving bed chromatography for the filtration separation technology in the step e; and f, concentrating and drying the filtered solution in the step f, wherein the vacuum degree of vacuum drying is 0.05-0.5Mpa, the temperature is 65 ℃, and the purity of the dried finished product is more than 99%.

(III) advantageous effects

Compared with the prior art, the method has obvious advantages and beneficial effects, and specifically, the method comprises the steps of selecting a proper culture medium for pichia pastoris (P.pastoris) to culture for a certain time under the optimal culture condition, adding reactant yeast extract and glucose, fermenting under the optimal reaction condition to produce glutathione, and separating and purifying the fermentation liquor through a macroporous adsorption resin column to obtain the high-purity glutathione.

The invention adopts a microbial fermentation method to produce glutathione, uses glucose as a substrate, adopts a microbial conversion method to convert the substrate into glutathione, and then adopts separation and purification processes such as membrane filtration, membrane concentration and the like to obtain the high-purity glutathione.

Detailed Description

The following detailed description of the embodiments of the present invention is intended to be illustrative, and not to be construed as limiting the invention.