CN116218934A - Method for accumulating glutathione - Google Patents
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- CN116218934A CN116218934A CN202310397845.8A CN202310397845A CN116218934A CN 116218934 A CN116218934 A CN 116218934A CN 202310397845 A CN202310397845 A CN 202310397845A CN 116218934 A CN116218934 A CN 116218934A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
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Abstract
The invention relates to a method for accumulating glutathione, which comprises the steps of fermenting and culturing activated saccharomycetes in a fermentation culture medium, adding nystatin and Sodium Dodecyl Sulfate (SDS) in the logarithmic phase or the stationary phase of the growth of the saccharomycetes, and continuing fermenting and culturing to obtain the glutathione. The invention can obviously improve the production amount of glutathione, improve the utilization rate of raw materials and reduce the production cost.
Description
The invention is a divisional application of an invention patent with the application date of 2019, 6-11, the application number of 2019105000227 and the invention name of a method for accumulating glutathione.
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for accumulating glutathione.
Background
Glutathione (GSH), a bioactive tripeptide compound consisting of L-glutamic acid, L-cysteine and glycine, is an important antioxidant and a major non-protein thiol compound (> 90%) within cells. In biological tissues, glutathione maintains the dynamic balance of oxidized and reduced forms through glutathione reductase and plays roles of cytoprotection, substance metabolism, information regulation, cell and tissue development regulation, disease resistance or induction and the like, so that the glutathione is widely applied to industries of medical health care, skin care and beauty, food addition and the like.
The current GSH production methods mainly comprise a solvent extraction method, a chemical synthesis method, a biological enzyme catalysis method and a biological fermentation method. In contrast, the biological fermentation method has the advantages of mild reaction conditions, simple reaction steps, low cost, high conversion efficiency, high production rate and the like, is a main trend of producing glutathione in the future, but most of researches remain in a laboratory stage at present, and the country realizing commercial production is mainly Japan.
The usual methods for increasing glutathione production in molecular biology, however, engineering bacteria require the addition of large amounts of antibiotics, which are not operationally permissible. Furthermore, antibiotics are generally expensive, which is not economically permissible. Most seriously, engineering bacteria are easy to degrade in the fermentation process, and are difficult to use for a long time. Therefore, it is important to find other alternatives, economical methods. The surfactant has been widely reported as a detergent for glutathione production, and the working principle is that the cell membrane permeability is changed to release intracellular glutathione, so that feedback inhibition of glutathione on the synthase can be weakened, and finally the yield is improved. The method does not involve gene manipulation, and has convenient operation and strong economic applicability. Meanwhile, the added surfactant can release the product, so that the difficulty of subsequent separation and purification is reduced.
Disclosure of Invention
The invention aims to provide a method for accumulating glutathione more efficiently.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for accumulating glutathione comprises fermenting activated yeast in a fermentation medium, adding nystatin and Sodium Dodecyl Sulfate (SDS) in logarithmic phase or stationary phase of growth of the yeast, and fermenting to obtain the glutathione.
Preferably, the formula of the fermentation medium is as follows: glucose 20-40 g/L, yeast extract 2-8 g/L, (NH) 4 ) 2 SO 4 2～8g/L，KH 2 PO 4 3～9g/L，K 2 SO 4 3～4g/L，MgSO 4 1～2g/L，FeSO 4 0.006～0.01g/L，MnSO 4 0.006～0.01g/L。
Preferably, the pH of the fermentation medium is 5.5-6.5.
Preferably, the fermentation culture is carried out at a temperature of 25 to 35 ℃.
Preferably, the addition amount of the nystatin is 0.2-0.4 mg/L.
Preferably, the addition amount of the sodium dodecyl sulfate is 5-10 mg/L.
Preferably, the activating method of the activated saccharomycetes comprises the following steps: and (3) performing activation culture on the saccharomycetes until single colonies grow out, and performing further activation culture on the single colonies for 25-35 hours to obtain the activated saccharomycetes.
Further preferably, the activation culture is performed at a temperature of 25 to 35 ℃.
Further preferably, the formulation of the medium for culturing to grow single colonies includes: 0.5 to 1.5% w/v glucose, 0.2 to 0.8% w/v peptone, 0.1 to 0.5% w/v yeast extract, and agar to make the medium solid.
Further preferably, the formulation of the medium for further activation culture comprises: 0.5-1.5% w/v glucose, 0.2-0.8% w/v peptone, 0.1-0.5% w/v yeast extract.
Preferably, the yeast is Saccharomyces cerevisiae Saccharomyces cerebisiae.
Due to the implementation of the technical scheme, compared with the prior art, the invention has the following advantages:
the invention can obviously improve the production amount of glutathione, improve the utilization rate of raw materials and reduce the production cost.
Drawings
FIG. 1 is a plot of growed yeast growth.
FIG. 2 shows the glutathione production of the added nystatin at various time points.
FIG. 3 shows the yield of SDS-added glutathione at various time points.
FIG. 4 shows the glutathione production by the addition of different concentrations of nystatin.
FIG. 5 shows the yield of glutathione with different SDS concentrations.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The following examples are examples of experimental procedures that show specific conditions, generally according to conventional conditions, such as molecular cloning: conditions described in the laboratory Manual (New York: coLd Spring Harbor Laboratory Press, 1989) or the protocol provided by the manufacturer.
In the following examples of the present invention, reagents, media, etc. used were purchased from the biological company.
In the following examples of the present invention, the strain used was Saccharomyces cerevisiae Saccharomyces cerebisiae, which was deposited in China general microbiological culture Collection center, accession number was: cgmccno.1917.
In the following examples of the invention, the shake flask fermentation medium used was formulated as follows: YPD medium included (1.0% w/v glucose, 0.5% w/v peptone, 0.3% w/v yeast extract). The formula of the fermentation medium is (g/L): glucose 30, yeast extract 5, (NH) 4 ) 2 SO 4 5,KH 2 PO 4 6,K 2 SO 4 3.6,MgSO 4 1.5,FeSO 4 0.008,MnSO 4 0.008, medium pH was adjusted to 6.0.
Example 1: fumbling of growth curves of yeasts
1.1 Strain activation and preparation of culture Medium
YPD medium was prepared in the following volume ratio, and solid medium was obtained by adding agar powder.
YPD medium formulation: 1.0% w/v glucose, 0.5% w/v peptone, 0.3% w/v yeast extract.
The deposited strain was removed from the-80℃refrigerator, streaked on an antibiotic-free solid medium and cultured in a 30℃incubator for about 24 hours. After a clearly visible spot (single colony) was grown, the single bacteria were picked up with a sterile toothpick and placed in YPD liquid medium for cultivation. After culturing under the above-mentioned culture conditions for about 30 hours, single colonies were transferred to a fermentation medium for continuous fermentation to perform subsequent experiments.
The formula of the fermentation medium is (g/L): glucose 30, yeast extract 5, (NH) 4 ) 2 SO 4 5,KH 2 PO 4 6,K 2 SO 4 3.6,MgSO 4 1.5,FeSO 4 0.008,MnSO 4 0.008, medium pH was adjusted to 6.0.
1.2 growths of Yeast growths
The above procedure was used to search the growth curve of yeast. The specific method comprises the following steps:
(1) Culturing yeast according to 1.1 method, and culturing at 30deg.C at 240 rpm;
(2) Sampling at intervals of 3 hours, and continuously sampling for more than 10 times;
(3) The culture was centrifuged, dried to constant weight, and then weighed to determine the dry cell weight.
(4) Growth curves were plotted according to the dry cell weights at various times, and the results are shown in FIG. 1, and the time points of reagent addition were determined.
Example 2: fumbly for the effect of time of nystatin addition on glutathione
The addition time is determined by a single addition metering method. Since the amount of cells in the initial stage of fermentation is relatively small, a few time points of logarithm and stationary phase are selected as undetermined addition points to add relevant reagents. The main technical flow is as follows:
(1) Culturing yeast according to method 1.1;
(2) Adding 0.2mg/L of tobramycin (obtained by previous experiments) at different periods, and culturing for 30 hours to collect bacteria;
(3) Cell dry weight of cultured cells was measured, and at the same time, intracellular and extracellular glutathione content was measured, and the test results are shown in FIG. 2.
(4) And analyzing the experimental result to determine the adding time.
Example 3: fumbling the effect of SDS addition time on glutathione
The addition time is determined by a single addition metering method. Since the amount of cells in the initial stage of fermentation is relatively small, a few time points of logarithm and stationary phase are selected as undetermined addition points to add relevant reagents. The main technical flow is as follows:
(1) Culturing yeast according to method 1.1;
(2) SDS (obtained from previous experiments) was added at 5mg/L in various periods and incubation was continued for 30 hours after addition to harvest;
(3) Cell dry weight of cultured cells was measured, and at the same time, intracellular and extracellular glutathione content was measured, and the test results are shown in FIG. 3.
(4) And analyzing the experimental result to determine the adding time.
Example 4: fumbly for influence of addition amount of nystatin on glutathione
The addition metric is determined by the method of single point in time addition. The addition metering should include the experimental metering of example 3, and the main technical flow is as follows:
(1) Culturing yeast according to method 1.1;
(2) Adding 0.1,0.2,0.4,0.8,1,2mg/L of metered nystatin into a fermentation culture medium for fermentation culture for 20 hours, and continuously culturing for 30 hours after adding for bacterial recovery;
(3) Cell dry weight of cultured cells was measured, and intracellular and extracellular glutathione contents were measured, and the test results are shown in FIG. 4.
(4) And analyzing the experimental result to determine the addition metering.
Example 5: the effect of SDS addition on glutathione was investigated
The addition metric is determined by the method of single point in time addition. The addition metering should include the experimental metering of example 2, and the main technical flow is as follows:
(1) Culturing yeast according to method 1.1;
(2) Adding 2.5,5,7.5, 10, 12.5, 15, 17.5 and 20mg/L SDS in the fermentation medium for 20h of fermentation culture, and continuing to culture for 30h for bacterial recovery after the addition;
(3) Cell dry weight of cultured cells was measured, and at the same time, intracellular and extracellular glutathione content was measured, and the test results are shown in FIG. 5.
(4) And analyzing the experimental result to determine the addition metering.
Example 6
YPD medium was prepared in the following volume ratio, and solid medium was obtained by adding agar powder.
YPD medium formulation: 1.0% w/v glucose, 0.5% w/v peptone, 0.3% w/v yeast extract.
The deposited strain was removed from the-80℃refrigerator, streaked on an antibiotic-free solid medium and cultured in a 30℃incubator for about 24 hours. After a clearly visible spot (single colony) was grown, the single bacteria were picked up with a sterile toothpick and placed in YPD liquid medium for cultivation. Culturing under the same culture conditions, transferring to a fermentation medium for continuous fermentation after single colony culturing for about 30 hours, adding 0.4mg/L of nystatin and 10mg/L of SDS after culturing for 20 hours, and continuing culturing for 30 hours after adding for bacterial recovery; the content of glutathione inside and outside the cell is 572.36mg/L.
The present invention has been described in detail with the purpose of enabling those skilled in the art to understand the contents of the present invention and to implement the same, but not to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method of accumulating glutathione comprising: fermenting and culturing the activated saccharomycetes in a fermentation culture medium, then adding the nystatin and the sodium dodecyl sulfate in the growth stabilizing period of the saccharomycetes, continuing fermenting and culturing to obtain the glutathione,
the addition amount of the nystatin is 0.4-0.8 mg/L,
the adding amount of the sodium dodecyl sulfate is 5-15 mg/L.
2. The method of accumulating glutathione according to claim 1, wherein: and adding the nystatin and the sodium dodecyl sulfate into the activated saccharomycetes for fermentation culture for 20 hours in a fermentation culture medium, and continuously culturing for 30 hours after adding for bacterial recovery.
3. The method of accumulating glutathione according to claim 1 or 2, characterized in that: the addition amount of the nystatin is 0.4mg/L, and the addition amount of the sodium dodecyl sulfate is 10mg/L.
4. The method of accumulating glutathione according to claim 1, wherein: the formula of the fermentation medium is as follows: glucose 20-40 g/L, yeast extract 2-8 g/L, (NH) 4 ) 2 SO 4 2～8g/L，KH 2 PO 4 3～9g/L，K 2 SO 4 3～4g/L，MgSO 4 1～2g/L，FeSO 4 0.006～0.01g/L，MnSO 4 0.006～0.01g/L。
5. The method of accumulating glutathione according to claim 1, wherein: the pH value of the fermentation culture medium is 5.5-6.5;
and/or the fermentation culture is carried out at a temperature of 25-35 ℃.
6. The method of accumulating glutathione according to claim 1, wherein: the activating method of the activated saccharomycetes comprises the following steps: firstly, activating and culturing the saccharomycetes until single colony grows, and then, further activating and culturing the single colony for 25-35 hours to obtain the activated saccharomycetes.
7. The method for accumulating glutathione according to claim 6, wherein: the temperature for performing the activation culture is 25-35 ℃.
8. The method for accumulating glutathione according to claim 6, wherein: the formulation of the medium for culturing until single colonies are grown includes: 0.5 to 1.5% w/v glucose, 0.2 to 0.8% w/v peptone, 0.1 to 0.5% w/v yeast extract, and agar to make the medium solid.
9. The method for accumulating glutathione according to claim 6, wherein: the formulation of the medium for further activation culture comprises: 0.5-1.5% w/v glucose, 0.2-0.8% w/v peptone, 0.1-0.5% w/v yeast extract.
10. The method of accumulating glutathione according to claim 1, wherein: the microzyme is Saccharomyces cerevisiae Saccharomyces cerebisiae.
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