A method of accumulating glutathione

Abstract

The present application relates to a method for accumulating glutathione, wherein activated yeast is fermented in a fermentation medium, nystatin and sodium dodecyl sulfate (SDS) are added in the logarithmic or stationary phase of the yeast growth, and the fermentation is continued to obtain the glutathione. The present application can significantly increase the production of glutathione, improve the utilization of raw materials, and reduce the production cost.

Description

[0001] The present application is a divisional application of the invention patent application with the application number 2019105000227, the application date of June 11, 2019, and the invention name of a method for accumulating glutathione. TECHNICAL FIELD

[0002] The present application belongs to the field of bioengineering technology, and specifically relates to a method for accumulating glutathione. BACKGROUND

[0003] Glutathione (γ-L-glutamyl-cysteinyl-glycine, GSH) is a bioactive tripeptide compound composed of L-glutamic acid, L-cysteine and glycine, and is an important antioxidant and the main non-protein sulfhydryl compound (>90%) in cells. In biological tissues, glutathione maintains the dynamic balance between oxidized and reduced forms through glutathione reductase and plays a role in cell protection, material metabolism, information regulation, regulation of cell and tissue development, resistance or induction of diseases, etc., and thus is widely used in the industries of medical care, skin care and beauty, and food additives.

[0004] Currently, the main production methods of GSH include solvent extraction, chemical synthesis, enzyme catalysis and biological fermentation. Compared with other methods, the biological fermentation method has the advantages of mild reaction conditions, simple reaction steps, low cost, high conversion efficiency and fast production rate, and is the main trend for the future production of glutathione. However, most of the current researches are still at the laboratory stage, and the countries that have realized commercial production are mainly Japan.

[0005] Molecular biology is a common method to improve the yield of glutathione, but a large amount of antibiotics needs to be added to the engineering bacteria, which is not allowed in operation. In addition, antibiotics are generally expensive, which is also not allowed in economy. The most serious problem is that the engineering bacteria are prone to strain degeneration during fermentation, and it is difficult to use them for a long time. Therefore, it is crucial to seek other alternative and economical methods. Surfactants have been widely reported as a detergent for the production of glutathione. The working principle is to change the permeability of the cell membrane to release intracellular glutathione, so that the feedback inhibition of glutathione on its synthetase can be reduced, and finally the yield can be improved. This method does not involve genetic manipulation, and is easy to operate, economical and applicable. At the same time, since the added surfactant can release the product, it also reduces the difficulty of subsequent separation and purification. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a method for more efficiently accumulating glutathione.

[0007] To solve the above technical problems, the present application adopts the following technical solutions:

[0008] The method for accumulating glutathione comprises the following steps: fermenting activated yeast in a fermentation medium, adding nystatin and sodium dodecyl sulfate (SDS) in the logarithmic phase or stationary phase of the yeast, and continuing the fermentation to obtain the glutathione.

[0009] Preferably, the fermentation medium comprises 20-40 g / L of glucose, 2-8 g / L of yeast extract, 2-8 g / L of (NH4)2SO4, 3-9 g / L of KH2PO4, 3-4 g / L of K2SO4, 1-2 g / L of MgSO4, 0.006-0.01 g / L of FeSO4, and 0.006-0.01 g / L of MnSO4.

[0010] Preferably, the pH of the fermentation medium is 5.5-6.5.

[0011] Preferably, the fermentation is carried out at a temperature of 25-35℃.

[0012] Preferably, the nystatin is added in an amount of 0.2-0.4 mg / L.

[0013] Preferably, the sodium dodecyl sulfate is added in an amount of 5-10 mg / L.

[0014] Preferably, the activation method of the activated yeast comprises the following steps: activating the yeast to grow single colonies, and further activating the single colonies for 25-35 h to obtain the activated yeast.

[0015] Further preferably, the activation is carried out at a temperature of 25-35℃.

[0016] Further preferably, the medium for growing the single colonies comprises 0.5-1.5% w / v of glucose, 0.2-0.8% w / v of peptone, 0.1-0.5% w / v of yeast extract, and agar for solidifying the medium.

[0017] Further preferably, the medium for the further activation comprises 0.5-1.5% w / v of glucose, 0.2-0.8% w / v of peptone, and 0.1-0.5% w / v of yeast extract.

[0018] Preferably, the yeast is Saccharomyces cerebisiae.

[0019] Compared with the prior art, the present application has the following advantages:

[0020] The present application can significantly increase the production of glutathione, improve the utilization rate of raw materials, and reduce the production cost.BRIEF DESCRIPTION OF DRAWINGS BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the growth curve of the yeast strain.

[0022] Figure 2 is the yield of glutathione with different time points of nystatin addition.

[0023] Figure 3 is the yield of glutathione with different time points of SDS addition.

[0024] Figure 4 is the yield of glutathione with different concentrations of nystatin addition.

[0025] Figure 5 is the yield of glutathione with different concentrations of SDS addition. DETAILED DESCRIPTION

[0026] The present application is further described in conjunction with the following examples. It should be understood that the following examples are merely illustrative of the present application and do not limit the scope of the application.

[0027] In the following examples, the experimental methods are described with specific conditions, and generally are performed according to conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the protocols provided by the manufacturers.

[0028] In the following examples of the present application, the reagents, media, etc. are purchased from Shengong Biotech Co. Ltd.

[0029] In the following examples of the present application, the strain used is Saccharomyces cerebisiae, which was deposited in China General Microbiological Culture Collection Center on January 12, 2007, and the deposit number is CGMCC No. 1917.

[0030] In the following examples of the present application, the formula of the shake flask fermentation medium used is YPD medium including (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, (NH4)2SO45, KH2PO46, K2SO43.6, MgSO41.5, FeSO40.008, MnSO40.008, and the medium pH is adjusted to 6.0.

[0031] Example 1: Exploration of the growth curve of the yeast strain

[0032] 1.1 Strain activation and medium preparation

[0033] YPD medium was prepared according to the following volume ratio, and solid medium was obtained by adding agar powder.

[0034] YPD medium formula: 1.0% w / v glucose, 0.5% w / v peptone, 0.3% w / v yeast extract.

[0035] The preserved strain was taken out from the -80°C refrigerator, streaked on the solid medium without antibiotics and placed in a 30°C incubator for about 24 hours. When clear spots (single colonies) appeared, a single colony was picked up with a sterilized toothpick and inoculated into YPD liquid medium. The culture was incubated under the same conditions as described above. After about 30 hours of single colony culture, the culture was transferred to the fermentation medium for further experiments.

[0036] The fermentation medium formula was (g / L): glucose 30, yeast extract 5, (NH4)2SO4 5, KH2PO4 6, K2SO4 3.6, MgSO4 1.5, FeSO4 0.008, MnSO4 0.008, and the medium pH was adjusted to 6.0.

[0037] 1.2 Exploration of the growth curve of yeast

[0038] The exploration of the growth curve of yeast was carried out as described above. The specific method is as follows:

[0039] (1) The yeast was cultured according to the method of 1.1, and placed at 30°C under 240 rpm culture;

[0040] (2) Sampling every 3 hours, more than 10 times continuously;

[0041] (3) The culture was centrifuged and dried to constant weight, and then weighed to determine the dry weight of the cells.

[0042] (4) The growth curve was plotted according to the dry weight of the cells at different times, and the results are shown in Figure 1 , to determine the time point of adding reagents.

[0043] Example 2: Exploration of the effect of nystatin addition time on glutathione

[0044] The addition time was determined by the method of single addition of metering. Since the amount of bacteria was small at the beginning of fermentation, several time points of logarithmic and stationary phase were selected as the addition points of related reagents. The main technical process is as follows:

[0045] (1) The yeast was cultured according to the method of 1.1;

[0046] (2) 0.2 mg / L of nystatin was added at different time points (according to the results of the previous experiment), and the culture was continued for 30 hours after the addition to collect the bacteria;

[0047] (3) Measure the dry weight of the bacteria at the end of the culture, and measure the content of glutathione in and outside the cells, and the test results are shown in Table 3. Figure 2

[0048] (4) Analyze the experimental results to determine the addition time.

[0049] Example 3: Explore the effect of SDS addition time on glutathione

[0050] The addition time is determined by the method of adding a single dose. Since the amount of bacteria is relatively small at the beginning of fermentation, several time points during the logarithmic and stationary phases are selected as the addition points for adding the relevant reagents. The main technical process is as follows:

[0051] (1) Culture the yeast according to the method of 1.1;

[0052] (2) Add 5 mg / L of SDS (according to the results of the previous experiment) at different times, and continue to culture for 30 hours after adding to collect the bacteria;

[0053] (3) Measure the dry weight of the bacteria at the end of the culture, and measure the content of glutathione in and outside the cells, and the test results are shown in Table 3. Figure 3

[0054] (4) Analyze the experimental results to determine the addition time.

[0055] Example 4: Explore the effect of nystatin addition amount on glutathione

[0056] The addition amount is determined by the method of adding at a single time point. The addition amount should include the experimental amount of Example 3, and the main technical process is as follows:

[0057] (1) Culture the yeast according to the method of 1.1;

[0058] (2) Add 0.1, 0.2, 0.4, 0.8, 1, and 2 mg / L of nystatin to the fermentation medium after 20 hours of fermentation, and continue to culture for 30 hours after adding to collect the bacteria;

[0059] (3) Measure the dry weight of the bacteria at the end of the culture, and measure the content of glutathione in and outside the cells, and the test results are shown in Table 3. Figure 4

[0060] (4) Analyze the experimental results to determine the addition amount.

[0061] Example 5: Explore the effect of SDS addition amount on glutathione

[0062] ​​​The addition amount is determined by the method of single time point addition. The addition amount should include the experimental amount of Example 2, and the main technical procedure is as follows:

[0063] (1) The yeast is cultured according to the method of 1.1;

[0064] (2) The fermentation culture is fermented for 20h, and then 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20mg / L of SDS is added, and the culture is continued for 30 hours after the addition to collect the bacteria;

[0065] (3) The cell dry weight of the bacteria at the end of the culture is determined, and the content of glutathione in and outside the cell is determined, and the test results are shown in Table 1. Figure 5

[0066] (4) The experimental results are analyzed to determine the addition amount.

[0067] Example 6

[0068] The YPD culture medium is prepared according to the following volume ratio, and the solid culture medium is obtained by adding agar powder.

[0069] The YPD culture medium formula is: 1.0% w / v glucose, 0.5% w / v peptone, and 0.3% w / v yeast extract.

[0070] The preserved strain is taken out from the -80°C refrigerator, streaked on the solid culture medium without antibiotics, and placed in a 30°C incubator for about 24 hours. When clear spots (single colonies) are grown, a single colony is picked up with a sterilized toothpick and cultured in YPD liquid culture medium. The culture is cultured under the same culture conditions as described above, and after the single colony is cultured for about 30h, it is transferred to the fermentation culture medium for further fermentation, and then 0.4mg / L of nystatin and 10mg / L of SDS are added after 20h of culture, and the culture is continued for 30 hours after the addition to collect the bacteria; the content of glutathione in and outside the cell is determined to be 572.36mg / L.

[0071] The above detailed description of the present application is intended to enable those skilled in the art to understand the content of the present application and to implement it, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made in accordance with the spirit and essence of the present application should be covered within the protection scope of the present application.​

Claims

1. A method of accumulating glutathione, characterized by: The activated Saccharomyces cerevisiae is fermented in a fermentation medium for 20 hours, and then nystatin and sodium dodecyl sulfate are added, and the fermentation is continued for 30 hours to obtain the glutathione, The nystatin is added in an amount of 0.4 mg / L, The sodium dodecyl sulfate is added in an amount of 10 mg / L, The yeast is Saccharomyces cerevisiae Saccharomyces cerevisiae , The fermentation medium comprises 20-40 g / L glucose, 2-8 g / L yeast extract, 2-8 g / L (NH4)2SO4, 3-9 g / L KH2PO4, 3-4 g / L K2SO4, 1-2 g / L MgSO4, 0.006-0.01 g / L FeSO4, and 0.006-0.01 g / L MnSO4. The pH of the fermentation medium is 5.5-6.

5.

2. The method of claim 1, wherein: The fermentation is carried out at a temperature of 25-35 °C.

3. The method of claim 1, wherein: The activation method of the activated Saccharomyces cerevisiae comprises first activating the Saccharomyces cerevisiae to grow single colonies, and then further activating the single colonies for 25-35 hours to obtain the activated Saccharomyces cerevisiae.

4. The method of claim 3, wherein: The activation is carried out at a temperature of 25-35 °C.

5. The method of claim 3, wherein: The culture medium for growing the single colonies comprises 0.5-1.5% w / v glucose, 0.2-0.8% w / v peptone, 0.1-0.5% w / v yeast extract, and agar to solidify the medium.

6. The method of claim 3, wherein: The culture medium for the further activation comprises 0.5-1.5% w / v glucose, 0.2-0.8% w / v peptone, and 0.1-0.5% w / v yeast extract.

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