CN109750073B - Method for extracting glutathione from candida utilis fermentation liquor - Google Patents
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- 108010024636 Glutathione Proteins 0.000 title claims abstract description 59
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for extracting glutathione from candida utilis fermentation liquor, which comprises the following steps: culturing and fermenting candida utilis seeds; collecting fermentation broth after fermentation culture, centrifuging, and freezing the collected cells; adding the frozen candida utilis cells into a sulfuric acid solution, standing for 15-30 minutes, performing centrifugal separation, and collecting supernatant; at this time, intracellular reduced glutathione is released from the cells and dissolved in a sulfuric acid solution, thereby completing the extraction. The invention freezes the collected cells to increase the permeability of the cell wall; the frozen cells are added into the strong acid solution, intracellular reduced glutathione is completely released in the strong acid solution, and other intracellular macromolecular substances such as protein, saccharides, nucleic acid and the like are basically left in the cells, so that the subsequent purification operation is simplified, the purification cost is reduced, and efficient and clean extraction is realized; meanwhile, the reduced glutathione is not damaged under the extraction condition of the invention.
Description
The application is a divisional application of an invention patent application with the application number of 201510720495.X and the application date of 2015, 10 months and 30 days, and the invention is named as a method for extracting glutathione from candida utilis fermentation liquor.
Technical Field
The invention relates to a preparation method of glutathione, in particular to a method for extracting glutathione from candida utilis fermentation liquor.
Background
Glutathione, gamma-L-glutamyl-L-cysteinylglycine, is a bioactive tripeptide compound with both gamma-glutamyl and sulfhydryl groups, which is formed by the condensation of L-glutamic acid, L-cysteine and glycine through peptide bonds. Glutathione, which is a non-protein sulfhydryl compound in the living body, mainly has two forms, namely, reduced form (G-SH) and oxidized form (G-S-S-G), and reduced glutathione exists in large amounts in the body and plays a major role. Reduced glutathione has unique physiological functions and is called longevity factor and anti-aging factor. Reduced glutathione as amino acid biochemical medicine may be used in treating liver disease, heavy metal poisoning and other diseases, and has also the effect of resisting AIDS virus.
The main method for producing reduced glutathione at present is a fermentation method, that is, a method for biosynthesis of reduced glutathione by using a substance in a microorganism and an energy metabolism pathway using inexpensive sugars and other raw materials as nutrients. Generally, the content of reduced glutathione in microbial cells is not high, but only 0.5 to 1.0% of dry weight, because too high a content of reduced glutathione easily destroys the in vivo balanced redox environment. Because the reduced glutathione is an intracellular product, the reduced glutathione needs to be extracted and purified after the fermentation is finished.
At present, the main methods for extracting the reduced glutathione from the cells comprise a solvent extraction method, a hot water extraction method and an ultrasonic crushing extraction method.
Wherein the solvent extraction method is to complete extraction by increasing cell wall permeability, the kind, temperature and extraction time of the solvent have great influence on the extraction rate, and the extraction rate is low when formic acid, liquid ammonia, sulfuric acid or trichloroacetic acid is used as the solvent for extraction; the extraction with ethanol can not damage cell walls, has less impurity content and low energy consumption, but needs recovery treatment after the extraction with ethanol is finished, thereby increasing the extraction cost. The main problems of ultrasonic disruption are that impurities such as intracellular protein, nucleic acid and the like are completely dissolved out after wall breaking, and are easy to be turbid after centrifugation, so that the subsequent treatment is not facilitated; the ultrasonic crushing consumes long time, needs an effective cooling system, has high energy consumption and is not beneficial to industrial production.
A hot water extraction method is a method for extracting glutathione from a fermentation broth, which is earlier applied, for example, Chinese patent document CN 100455592C (application No. 200610040606.3) discloses a method for extracting glutathione from a glutathione fermentation broth, which adopts a boiling water boiling method, wherein the glutathione fermentation broth is centrifuged to obtain yeast cell sap, the pH value of the yeast cell sap is adjusted to 1-2 by sulfuric acid, the yeast cell sap with the adjusted pH value is poured into boiling water for boiling water wall breaking, cation exchange is carried out, a concentrated solution rich in glutathione is collected, a glutathione hyperconcentrated solution is obtained by vacuum concentration, the glutathione hyperconcentrated solution is freeze-dried to obtain white powdery glutathione, and a finished product is packaged to obtain the glutathione.
Chinese patent document CN 104130310A (application No. 201410231642.2) discloses a method for separating and purifying glutathione, which comprises the steps of centrifuging yeast fermentation liquor, collecting yeast, adding hot water with the temperature of 50-90 ℃, extracting for 5-30 min, adjusting the pH value of an extracted liquid to 1.5-3, filtering or centrifuging an extracting solution, and collecting the extracting solution; removing impurities such as macromolecular protein, polysaccharide and the like from the extracting solution by adopting an ultrafiltration method, and then removing most impurities such as micromolecular amino acid, monosaccharide, inorganic salt and the like by adopting a nanofiltration membrane filtering method; taking nanofiltration concentrated solution as a raw material, adopting strong acid cation exchange resin to adsorb glutathione, and eluting after the adsorption reaches a penetration point; concentrating the eluate under reduced pressure, adding ethanol with three times of volume for precipitation, and freeze-drying the precipitate to obtain glutathione product with purity of over 95%.
The hot water extraction method is simple and has low cost, but the reduced G-SH is easily converted into G-S-S-G, and other intracellular substances such as protein, saccharide, nucleic acid and other macromolecules can be released along with the reduced G-SH, so that the subsequent purification difficulty is increased.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for extracting glutathione from candida utilis fermentation liquor, which is complete in extraction and low in cost.
The technical scheme for realizing the aim of the invention is a method for extracting glutathione from candida utilis fermentation liquor, which comprises the following steps:
culturing and fermenting candida utilis seeds.
And collecting the fermentation liquid of the candida utilis after fermentation culture, removing supernatant after centrifugation, and freezing the collected cells for 20-30 minutes at-15 to-25 ℃.
And thirdly, adding the frozen Candida utilis cells into a sulfuric acid solution, wherein the pH value of the sulfuric acid solution is 1-2, and after the Candida utilis cells are placed at the temperature of 20-35 ℃ for 15-30 minutes, the reduced glutathione in the Candida utilis cells is released from the cells and dissolved in the sulfuric acid solution, so that the glutathione is extracted from the Candida utilis fermentation liquor, and the supernatant is collected after centrifugal separation.
In the third step, the concentration of the candida utilis cells in the sulfuric acid solution is 0.08-0.12 g/mL.
Inoculating the candida utilis strain into a seed culture medium during seed culture of the candida utilis, culturing for 18-28 hours at the temperature of 28-32 ℃, stopping culturing when the rotating speed of a shaking table is 200-250 rpm and culturing to a logarithmic phase, and allowing the obtained seed solution to be subjected to fermentation culture.
Firstly, inoculating a seed solution into a fermentation minimal medium according to the inoculation amount of 8-10% to perform batch culture for 8-10 h during fermentation of candida utilis, and then performing growth rate of 0.15h according to the cell ratio-1～0.25h-1Exponential-fed culture for 11-13 h, followed by 5.0-6.0 g (h. L)-1Culturing for 23-25 h by constant flow addition; adding three amino acids into the tank, adding 5.5-6.5 mmol/L glutamic acid, 5.0-6.0 mmol/L glycine and 6.0-7.0 mmol/L cysteine into the tank, and continuously culturing for 25-35 h to complete fermentation culture of the Candida utilis.
The basic fermentation medium for batch culture comprises the following components: 15 g/L of glucose, 5 g/L of ammonium sulfate, 2 g/L of yeast extract, 1.5 g/L of monopotassium phosphate, 0.25 g/L of magnesium sulfate and 5.5 of the pH value of a fermentation minimal medium;
the exponential fed-batch culture and the constant fed-batch culture both use fed-batch culture media, and the fed-batch culture media comprise the following components: 500 g/L glucose, 36 g/L ammonium sulfate, 5 g/L potassium dihydrogen phosphate and 0.5 g/L magnesium sulfate, wherein the glucose is separately sterilized and then mixed with other components.
The invention has the positive effects that: (1) the extraction method comprises the steps of firstly culturing and fermenting Candida utilis (C.utilis), centrifugally separating the obtained fermentation liquor, removing supernatant, and freezing the collected cells at-20 to-30 ℃ to increase the permeability of the cell wall of the C.utilis; the frozen C.utilis cells are added into the strong acid solution with low pH value, intracellular reduced glutathione is completely released in the strong acid solution with low pH value, and other intracellular macromolecular substances such as protein, saccharides, nucleic acid and the like are basically left in the cells, so that the subsequent purification operation is simplified, the purification cost is reduced, and the efficient and clean extraction of G-SH in the C.utilis cells is realized; meanwhile, the reduced glutathione is not damaged under the extraction condition of the invention.
(2) When the GSH is extracted by the strong acid solution with low pH value, only a small amount of the strong acid solution is needed, and after the G-SH extraction is finished, the strong acid solution can be further recycled, so that the method is environment-friendly.
(3) The extraction method of the invention has short extraction time, and the glutathione in the cells can be completely released into the strong acid solution within about 20 minutes, so the extraction efficiency of the method is high.
(4) The extraction method disclosed by the invention is simple in process, economic and environment-friendly, meets the requirement of circular economy development, and has a good industrial application prospect.
Detailed Description
(example 1)
The method for extracting glutathione from candida utilis fermentation liquor comprises the following steps of:
culturing and fermenting candida utilis seeds. Candida utilis WSH 02-08 is used as a production strain, seed culture is firstly carried out, the culture is stopped when the culture reaches the late logarithmic phase, and the obtained seed solution is ready for fermentation culture.
The seed medium composition was as follows (g/L): 20 parts of glucose, 20 parts of peptone, 10 parts of yeast extract and water as a solvent, wherein the pH value is 5.5-6.5.
Specifically, during seed culture, a Candida utilis strain (Candida utilis WSH 02-08) is inoculated in a seed culture medium, the culture is carried out for 18-28 hours at the temperature of 28-32 ℃, the rotation speed of a shaking table is 200-250 rpm, the culture is stopped when the culture reaches the late logarithmic phase, and the obtained seed solution is ready for fermentation culture.
3L of fermentation minimal medium is filled in a 5L fermentation tank, the seed liquid is inoculated in the fermentation minimal medium according to the inoculation amount of 8-10% (v/v) for batch culture for 8-10 h (9 h in the embodiment), and the cell specific growth rate is 0.15h-1～0.25h-1Exponential-fed culture for 11-13 h (12 h in this example), followed by 5.0-6.0 g (h & L)-1After constant-number feeding culture for 23-25 h (24 h in the embodiment), the cell concentration in the fermentation tank reaches 102 g/L, at this time, three amino acids are added into the tank, and glutamic acid in the tank is added for 6 mmol/L, glycine 5.5 mmol/L, cysteine 6.5 mmol/L; and continuously culturing for 25-35 h (30 h in the embodiment) to finish the fermentation culture of the candida utilis.
The composition of the fermentation minimal medium is as follows (g/L): 15 parts of glucose, 5 parts of ammonium sulfate, 2 parts of yeast extract, 1.5 parts of monopotassium phosphate, 0.25 part of magnesium sulfate, water as a solvent and 5.5 parts of pH value.
The exponential feeding culture and the constant feeding culture both use feeding culture media, and the feeding culture media comprise the following components (g/L): 500 portions of glucose, 36 portions of ammonium sulfate, 5 portions of monopotassium phosphate, 0.5 portion of magnesium sulfate and water as a solvent; wherein the glucose is sterilized separately and then mixed with other ingredients.
And (3) centrifuging 25mL of fermentation liquor obtained after fermentation culture in the reaction tank at the rotating speed of 3000rpm for 10min, removing supernatant in a centrifugal tube, and collecting the Candida utilis cells. Adding the collected candida utilis cells into ethanol, wherein the volume ratio of the candida utilis cells to the ethanol is 0.4:1, extracting the candida utilis cells in the ethanol for 30 minutes, performing centrifugal separation, injecting supernate into a high performance liquid chromatography to measure the G-SH content, calculating the G-SH content in the candida utilis cells cultured by fermentation to be 1.81%, and further, in the fermentation liquor after the fermentation culture in a reaction tank is completed, the yield of reduced glutathione is 1847 mg/L.
In addition, intracellular glutathione is extracted according to the method of Chinese patent document CN 104130310A (application No. 201410231642.2), nanofiltration concentrated solution is injected into a high performance liquid chromatography to measure the content of GSH, and the content of GSH in the candida utilis cells cultured by fermentation in the step (i) is calculated to be 1.81%.
Collecting 5000g of fermentation broth in the fermentation tank in the step I, centrifuging the fermentation broth at 3000rpm for 10min, centrifuging to remove supernatant, and freezing the collected cells at-15 to-25 ℃ (in the embodiment, at-20 ℃) for 20 to 30min (in the embodiment, at 30 min).
And thirdly, adding 2.5g of the frozen candida utilis cells into 25mL of sulfuric acid solution, wherein the pH value of the sulfuric acid solution is 1-2 (1.2 in the embodiment), and the concentration of the candida utilis cells in the sulfuric acid solution is 0.1 g/mL.
Placing the candida utilis fermentation liquor at the room temperature of 20-35 ℃ for 15-30 minutes (20 minutes in the embodiment), releasing the reduced glutathione in the candida utilis cells from the cells and dissolving the reduced glutathione in a sulfuric acid solution, and finishing the extraction of the glutathione from the candida utilis fermentation liquor in the embodiment; then, the mixture was centrifuged at 3000rpm for 10min, and the supernatant was collected.
And step three, injecting 0.2 mu L of supernate containing the reduced glutathione into a high performance liquid chromatography to measure the GSH content, measuring that the concentration of the reduced glutathione in the supernate is 0.16wt%, then calculating to obtain that the GSH content in the Candida utilis cells cultured in the step one, and further, the yield of the reduced glutathione in the fermentation liquor after the fermentation culture in the reaction tank is completed is 1843 mg/L.
From the above data, it can be seen that the method of the present invention for extracting glutathione from Candida utilis cells is equivalent in effect to the solvent extraction method and the hot water extraction method.
Purification of glutathione from the supernatant is carried out according to the prior art, for example using cation exchange resins.
Claims (3)
1. A method for extracting glutathione from Candida utilis fermentation liquor is characterized by comprising the following steps:
culturing and fermenting candida utilis seeds; inoculating the candida utilis strain into a seed culture medium, culturing for 18-28 hours at 28-32 ℃, stopping culturing when the rotating speed of a shaking table is 200-250 rpm and the logarithmic phase is reached, and allowing the obtained seed solution to be subjected to fermentation culture;
collecting fermentation liquor of the candida utilis which is fermented and cultured in the step I, centrifuging, removing supernatant, and freezing the collected cells for 20-30 minutes at-15 to-25 ℃;
and thirdly, adding the frozen Candida utilis cells into a sulfuric acid solution, wherein the concentration of the Candida utilis cells in the sulfuric acid solution is 0.08-0.12 g/mL, the pH value of the sulfuric acid solution is 1-2, and after the Candida utilis cells are placed for 15-30 minutes at the temperature of 20-35 ℃, the reduced glutathione in the Candida utilis cells is released from cells and dissolved in the sulfuric acid solution, so that the extraction of the glutathione from the Candida utilis fermentation liquor is completed, the glutathione is centrifugally separated, and the supernatant is collected.
2. The method for extracting glutathione from candida utilis fermentation liquor according to claim 1, which is characterized in that: firstly, inoculating a seed solution into a fermentation minimal medium according to the inoculation amount of 8-10% to perform batch culture for 8-10 h during fermentation of candida utilis, and then performing growth rate of 0.15h according to the cell ratio-1～0.25h-1Exponential-fed culture for 11-13 h, followed by 5.0-6.0 g (h. L)-1Culturing for 23-25 h by constant flow addition; adding three amino acids into the tank, adding 5.5-6.5 mmol/L glutamic acid, 5.0-6.0 mmol/L glycine and 6.0-7.0 mmol/L cysteine into the tank, and continuously culturing for 25-35 h to complete fermentation culture of the Candida utilis.
3. The method for extracting glutathione from candida utilis fermentation liquor according to claim 2, wherein the glutathione extracting step comprises the following steps: the basic fermentation medium for batch culture consists of: 15 g/L of glucose, 5 g/L of ammonium sulfate, 2 g/L of yeast extract, 1.5 g/L of monopotassium phosphate, 0.25 g/L of magnesium sulfate and 5.5 of the pH value of a fermentation minimal medium;
the exponential feeding culture and the constant feeding culture both use feeding culture media, and the feeding culture media comprise the following components: 500 g/L glucose, 36 g/L ammonium sulfate, 5 g/L potassium dihydrogen phosphate and 0.5 g/L magnesium sulfate, wherein the glucose is separately sterilized and then mixed with other components.
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