YEAST STRAIN AND USE THEREOF AND PREPARATION METHOD OF ERGOTHIONEINE

Abstract

The present relates to a yeast strain and use thereof and a preparation method of ergothioneine. The present invention relates to the field of biotechnology. The yeast strain is obtained through traditional mutagenesis and screening, and its deposit number is CCTCC M 20211505. The present invention provides a preparation method of ergothioneine. The preparation method of ergothioneine comprises: mixing the aforementioned yeast strain with a fermentation medium and an optional substrate, fermenting, and then homogenizing cells and separating to obtain ergothioneine. The aforementioned yeast strain can be used for the preparation of ergothioneine, and the ergothioneine prepared by the yeast strain has the advantages of high yield, low cost and fast preparation speed. The preparation method has the advantages of low cost, environmental protection, high product quality, high yield, less impurities, less drug residues, short fermentation period and the like.

Claims

1. A yeast strain with the deposit number of CCTCC M 20211505.

2. A preparation method of ergothioneine, comprising: mixing the yeast strain according to claim 1 with a fermentation medium and an optional substrate, fermenting, and then homogenizing cells and separating to obtain ergothioneine.

3. The preparation method according to claim 2, wherein the yeast strain is first inoculated into a seed liquid culture medium for amplification culture to obtain a seed liquid, then the seed liquid is mixed with a fermentation medium and an optional substrate, fermenting, and then the cells are homogenized and separated to obtain ergothioneine.

4. The preparation method according to claim 2, wherein the yeast strain is first activated to obtain a culture medium containing the activated yeast strain, then the culture medium containing the activated yeast strain is inoculated into a seed liquid culture medium for amplification culture to obtain a seed liquid, and the seed liquid is mixed with a fermentation medium and an optional substrate, fermenting, and then the cells are homogenized and separated to obtain ergothioneine.

5. The preparation method according to claim 2, wherein the substrate comprises at least one selected from arginine, histidine, methionine or cysteine.

6. The preparation method according to claim 2, wherein the fermentation medium comprises a carbon source.

7. The preparation method according to claim 6, wherein the carbon source includes at least one selected from sucrose, fructose, xylose, ethanol, methanol, glycerol, glucose, cellulose, starch, cellobiose or other glucose-containing polymers.

8. The preparation method according to claim 4, wherein the fermentation medium comprises phosphoric acid, CaSO.sub.4, K.sub.2SO.sub.4, KOH, MgSO.sub.4.Math.7H.sub.2O, glycerol, yeast extract, peptone, defoaming oil and water.

9. The preparation method according to claim 8, wherein the fermentation medium comprises 1% vol to 7% vol phosphoric acid, 0.03 wt % to 0.3 wt % CaSO.sub.4, 1 wt % to 6 wt % K.sub.2SO.sub.4, 0.1 wt % to 2 wt % KOH, 0.5 wt % to 5 wt % MgSO.sub.4.Math.7H.sub.2O, 1 wt % to 12 wt % glycerol, 0.1 wt % to 2 wt % yeast extract, 0.1 wt % to 2 wt % peptone, 0.01% vol to 1% vol defoaming oil, and the solvent is water.

10. The preparation method according to claim 9, wherein the fermentation medium comprises 2.27% vol phosphoric acid, 0.093 wt % CaSO.sub.4, 1.82 wt % K.sub.2SO.sub.4, 0.413 wt % KOH, 1.49 wt % MgSO.sub.4.Math.7H.sub.2O, 4 wt % glycerol, 0.5 wt % yeast extract, 0.5 wt % peptone, 0.1% vol defoaming oil, and the solvent is water.

11. The preparation method according to claim 4, wherein the seed liquid culture medium comprises yeast extract, peptone and glucose.

12. The preparation method according to claim 4, wherein the seed liquid culture medium comprises yeast extract 3 g/L to 30 g/L, peptone 3 g/L to 30 g/L, and glucose 5 g/L to 50 g/L.

13. The preparation method according to claim 4, wherein the seed liquid culture medium comprises yeast extract 10 g/L, peptone 10 g/L, and glucose 20 g/L.

14. The preparation method according to claim 2, wherein the fermentation temperature is 20° C. to 40° C.; and/or the fermentation time is 16 hours to 200 hours; and/or the fermentation has an airflow rate of 0.8 L/min-8 L/min; and/or the air pressure of the fermentation is 0 to 0.6 MPa; and/or the pH of the fermentation system of the fermentation is 4.0 to 7.0; and/or the initial stirring speed of the fermentation is between 100 rpm to 900 rpm.

15. The preparation method according to claim 2, when the dissolved oxygen concentration drops to 50% during the fermentation, glycerol is supplemented, and on the basis of the initial stirring speed, the stirring speed is increased stepwise between 100 rpm to 600 rpm; glycerol is supplemented continuously and the stirring speed maintains stepwise increment until the detected wet weight biomass reaches 30 g/L to 300 g/L.

16. The preparation method according to claim 2, wherein the supplemented amount of glycerol is 20 g/L to 220 g/L.

17. The preparation method according to claim 2, comprising: inoculating the yeast strain into a seed liquid culture medium for amplification culture to obtain a seed liquid, mixing the seed liquid with a fermentation medium and an optional substrate, fermenting under the conditions of the fermentation temperature of 20° C. to 40° C., the airflow rate of 0.8 L/min to 8 L/min, the air pressure of 0 to 0.6 MPa, the pH of 4.0 to 7.0 and the stirring speed of 100 rpm to 900 rpm; supplementing glycerol and increasing the stirring speed stepwise between 100 rpm to 600 rpm on the basis of the initial stirring speed when the dissolved oxygen concentration drops to 50% during the fermentation; supplementing glycerol continuously and maintaining stepwise increment of the stirring speed until the detected wet weight biomass reaches 200 g/L; then homogenizing cells and separating to obtain ergothioneine; wherein the substrate comprises at least one selected from arginine, histidine, methionine or cysteine; the fermentation medium comprises 1% vol to 7% vol phosphoric acid, 0.03 wt % to 0.3 wt % CaSO.sub.4, 1 wt % to 6 wt % K.sub.2SO.sub.4, 0.1 wt % to 2 wt % KOH, 0.5 wt % to 5 wt % MgSO.sub.4.Math.7H.sub.2O, 1 wt % to 12 wt % glycerol, 0.1 wt % to 2 wt % yeast extract, 0.1 wt % to 2 wt % peptone, 0.01% vol to 1% vol defoaming oil, and the solvent is water.

18. The preparation method according to claim 17, wherein the yeast is first inoculated in the seed culture medium, grown at 28° C. and 250 rpm for 48 hours for activation, then the culture medium containing the activated yeast strain is inoculated in the seed solution culture medium for expanded culture to obtain the seed solution.

19. The preparation method according to claim 18, wherein the seed liquid culture medium comprises yeast extract 3 g/L to 30 g/L, peptone 3 g/L to 30 g/L, and glucose 5 g/L to 50 g/L.

Description

DESCRIPTIONS OF THE DRAWINGS

[0059] FIG. 1 is a schematic diagram of the synthesis of ergothioneine in Example 1.

[0060] FIG. 2 is a high performance liquid chromatogram of ergothioneine standard in Example 1.

[0061] FIG. 3 is a high performance liquid chromatogram of the fermentation broth of the MM1 strain in Example 1. Compared with FIG. 2, it can be proved that there is ergothioneine in FIG. 3.

[0062] FIG. 4 is a schematic diagram of the synthesis of natural ergothioneine in Example 3.

DEFINITION OF TERMS

[0063] In the present invention, regardless of whether the word “approximately”, “about” or “substantially” is used, all numbers disclosed herein are approximate values. Based on the published figures, the value of each number may have a difference of ±10% or less or a reasonable difference considered by those in the art, such as a difference of ±1%, ±2%, ±3%, ±4%, or ±5%.

[0064] The term “and/or” should be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0065] The term “option”, “optional” or “optionally” means that the subsequently described event or situation can but does not necessarily occur. For example, “mixing the yeast strain described in the first aspect with a fermentation medium and an optional substrate” means “mixing the yeast strain described in the first aspect with a fermentation medium and a substrate” or “mixing the yeast strain described in the first aspect with a fermentation medium”.

[0066] The term “inoculation amount” refers to the volume percentage of the volume of the culture medium containing the activated yeast strain to the total volume of the seed liquid culture medium and the culture medium of the activated yeast strain after the inoculation; or the volume percentage of the volume of the seed liquid to the total volume of the seed liquid and the fermentation medium after the seed liquid is inoculated into the fermentation medium.

[0067] The term “OD.sub.600” refers to the detected optical density at a wavelength of 600 nm.

[0068] The term “wt %” refers to mass percentage.

[0069] The term “% vol” refers to volume percentage.

[0070] The term “SAM” refers to S-adenosylmethionine, which is a naturally occurring intermediate metabolite in cells.

[0071] The term “SAH” refers to S-adenosylhomocysteine, which is produced by demethylation of SAM and is a naturally occurring intermediate metabolite in cells.

[0072] Reference throughout this specification to “an embodiment”, “some embodiments”, “one embodiment”, “another example”, “an example”, “a specific example” or “some examples” means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as “in some embodiments”, “in one embodiment”, “in an embodiment”, “in another example”, “in an example”, “in a specific example” or “in some examples” in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can integrate and combine different embodiments, examples or the features of them as long as they are not contradictory to one another.

EXAMPLES

[0073] In order to enable those skilled in the art to better understand the technical solutions herein, some non-limiting embodiments are further disclosed below to further describe the present invention in detail.

[0074] The reagents used herein can be purchased from the market or can be prepared by the method described in this invention.

[0075] Unless otherwise specified, the formula of each medium described in the following specific examples is as follows:

[0076] The formula of the YPD medium (yeast extract peptone dextrose medium) is: yeast extract 10 g/L, peptone 10 g/L and dextrose 20 g/L, and the solvent is water.

[0077] The formula of the YPD agar medium (yeast extract peptone dextrose agar medium) is: yeast extract 10 g/L, peptone 10 g/L, dextrose 20 g/L and agar powder 15 g/L, and the solvent is water.

[0078] The formula of the YPD agar medium containing 0.02% lithium chloride (as a mutagen) is: yeast extract 10 g/L, peptone 10 g/L, dextrose 20 g/L, agar powder 15 g/L and lithium chloride 0.2 g/L, and the solvent is water.

[0079] The combined treatments of physical and chemical mutagenic agents breeding technology described in this application is a conventional mutagenesis method well known to those skilled in the art. It should be understood that the method is only used to illustrate the application, and cannot limit the scope of protection of the application. For those skilled in the art, without departing from the spirit and essence of the application, various changes or modifications to the medium components, content, culture conditions, and mutation conditions in the method also fall within the protection scope of the application.

[0080] Preferably, in the ultraviolet (UV) mutation described in this application, the yeast strain suspension is irradiated at 25-35 cm under an ultraviolet lamp for 15-25 minutes. Preferably, in the lithium chloride mutation described in this application, a yeast strain suspension is spread to a medium containing 0.01-0.02% lithium chloride (as a mutagen), and cultured at 18-37° C. for 16-72 hours.

[0081] Specifically, the yeast strain suspension is prepared by: taking Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, Torulaspora delbrueckii or Kluyveromyces lactis broth cultured at 18-37° C. for 16-72 hours, the cells in the broth are collected by centrifugation and washed 2-3 times with sterile water, then an appropriate amount of Tween 80 is added, the mixture is resuspended and shaken to disperse the cells, the cell density is adjusted to about 1,000,000 cells/mL with sterile water.

[0082] The term “rpm” means the speed “revolutions per minute”; “° C.” means the temperature unit “degrees Celsius”; “L” means the volume unit “liter”; “mL” means the volume unit “milliliters”; “pH” means pH degree of acid or alkali; “g” means the mass unit “gram”; “Mpa” means the pressure unit “megapa”.

Example 1

Screening of Yeast Strains

[0083] Preparation of Cell Suspension

[0084] The isolated Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, Torulaspora delbrueckii or Kluyveromyces lactis was inoculated into a 50 mL flat-bottomed Erlenmeyer flask containing 10 mL of yeast extract peptone dextrose (YPD) medium, and grown at 28° C. and 250 rpm for 48 hours. The yeast cells in the broth were collected by centrifugation and washed 3 times with 10 mL of sterile water, then resuspended with 10 mL of sterile water and placed in a 50 mL flat-bottomed Erlenmeyer flask. 5 μL of Tween 80 was added with a pipette, and the resulting mixture was shaken at 28° C. and 250 rpm for 30 min to disperse the cells. The cell density of each yeast strain was obtained by detecting OD.sub.600, and an appropriate amount of sterile water was added to adjust the cell density to about 1,000,000 cells/mL. UV mutation

[0085] 10 mL of each yeast strain suspension with a cell density of about 1,000,000 cellsmL was taken and placed in a sterilized petri dish with a diameter of 90 mm. The petri dishes were placed on the stage of the magnetic stirrer, the lids of the dishes were opened, and the ultraviolet irradiation was performed 30 cm below the ultraviolet lamp, and the irradiation time was 20 min.

[0086] Lithium Chloride Mutation

[0087] 10 mL of each yeast strain suspension after UV mutation was taken and all were spread on YPD agar medium containing 0.02% lithium chloride. They were wrapped with kraft paper, and placed in a 28° C. incubator for 72 hours.

[0088] Screening of Yeast Strains

[0089] Well-growing mutant strains of each yeast strain with relatively large single colony diameters that had undergone UV mutation and lithium chloride mutation were selected and streaked on the YPD agar medium plates. They were cultured in a constant temperature incubator at 28° C. for 48 hours. About 500 mutant strains were picked for each yeast strain. Each mutant strain was inoculated into a 50 mL flat-bottomed Erlenmeyer flask containing 10 mL of YPD medium, and grown at 28° C. and 250 rpm for 48 hours to obtain a broth. The ergothioneine-containing supernatant was collected by homogenizing the cells and centrifugation, the ergothioneine standard and the supernatant were taken, and the content of ergothioneine obtained by fermentation in the supernatant was determined by high performance liquid chromatography (HPLC), as shown in FIG. 2 and FIG. 3 respectively. The solution of each yeast strain before the combined treatments of physical and chemical mutagenic agents was taken as a control.

[0090] Before the combined treatments of ultraviolet and lithium chloride, no ergothioneine was detected in each yeast strain by HPLC. After the combined treatments of ultraviolet and lithium chloride, some yeast strains were detected to have the ability to synthesize ergothioneine, and a Schizosaccharomyces pombe strain that could accumulate a certain amount of ergothioneine in cells was selected and named MM1. The yield of ergothioneine (that is, the content of ergothioneine in the supernatant) was about 62.6 mg/L, as shown in FIG. 1. The mutated Schizosaccharomyces pombe strain MM1 was stored at minus 80° C. with glycerol and was passaged 5 times, and the yield of ergothioneine was stable.

Example 2

Multiple Round of Screening of Schizosaccharomyces pombe Strain MM1 Preparation of Cell Suspension

[0091] The Schizosaccharomyces pombe strain MM1 was inoculated into a 50 mL flat-bottomed Erlenmeyer flask containing 10 mL of yeast extract peptone dextrose (YPD) medium separately, and grown at 28° C. and 250 rpm for 48 hours. The yeast cells in the broth were collected by centrifugation and washed 3 times with 10 mL of sterile water, then resuspended with 10 mL of sterile water and placed in a 50 mL flat-bottomed Erlenmeyer flask. 5 μL of Tween 80 was added with a pipette, and the resulting mixture was shaken at 28° C. and 250 rpm for 30 min to disperse the cells. The cell density of Schizosaccharomyces pombe strain MM1 was obtained by detecting OD.sub.600, and an appropriate amount of sterile water was added to adjust the cell density to about 1,000,000 cells/mL.

[0092] UV Mutation

[0093] 10 mL of Schizosaccharomyces pombe strain MM1 suspension with a cell density of about 1,000,000 cellsmL was taken separately and placed in a sterilized petri dish with a diameter of 90 mm. The petri dish was placed on the stage of the magnetic stirrer, the lid of the dish was opened, and the ultraviolet irradiation was performed 30 cm below the ultraviolet lamp, and the irradiation time was 20 min.

[0094] Lithium Chloride Mutation

[0095] 10 mL of Schizosaccharomyces pombe strain MM1 suspension after UV mutation was taken separately and all was spread on YPD agar medium containing 0.02% lithium chloride (as a mutagen). It was wrapped with kraft paper, and placed in a 28° C. incubator for 72 hours.

[0096] Screening of Schizosaccharomyces pombe

[0097] 500 well-growing mutant strains of Schizosaccharomyces pombe strain MM1 with relatively large single colony diameters that had undergone UV mutation and lithium chloride mutation were selected and streaked on the YPD agar medium plates. They were cultured in a constant temperature incubator at 28° C. for 48 hours. The mutant strains were inoculated into a 50 mL flat-bottomed Erlenmeyer flask containing 10 mL of YPD medium, and grown at 28° C. and 250 rpm for 48 hours to obtain a broth. The ergothioneine-containing supernatant was collected by homogenizing the cells and centrifugation, the ergothioneine standard and the supernatant were taken, and the content of ergothioneine in the supernatant was determined by high performance liquid chromatography (HPLC). A solution containing Schizosaccharomyces pombe strain MM1 before multiple round of screening was used as a control.

[0098] After the combined treatments of ultraviolet and lithium chloride, a Schizosaccharomyces pombe strain that could accumulate a certain amount of ergothioneine in cells was selected and named MM2. The yield of ergothioneine (that is, the content of ergothioneine in the supernatant) was about 598 mg/L. The mutated Schizosaccharomyces pombe strain MM2 was stored at minus 80° C. with glycerol and was passaged 5 times, and the yield of ergothioneine was stable.

[0099] The Schizosaccharomyces pombe strain MM2 was subjected to the combined treatments of ultraviolet and lithium chloride again. After multiple round of the combined treatments, a Schizosaccharomyces pombe strain with high yield of ergothioneine was finally selected and named OMK-79 (The deposit number of this strain is CCTCC M 20211505, and it was deposited biologically at China Center for Type Culture Collection (CCTCC, Wuhan University) on Nov. 29, 2021). The mutated Schizosaccharomyces pombe strain OMK-79 was stored at minus 80° C. with glycerol and was passaged 5 times, and the yield of ergothioneine was stable.

Example 3

Fermentation Production of Ergothioneine

[0100] The synthesis process shown in FIG. 4 specifically includes the following steps:

[0101] The Schizosaccharomyces pombe strain OMK-79 was inoculated into a 250 mL flat-bottomed Erlenmeyer flask containing 50 mL of YPD medium, and grown at 28° C. and 250 rpm for 48 hours to activate the strain. The activated OMK-79 strain was transferred to a 1 L flat-bottomed Erlenmeyer flask containing 150 mL of YPD medium at an inoculation amount of 1%, and grown at 28° C. and 250 rpm for 18 hours to obtain a seed liquid. 100 mL of Schizosaccharomyces pombe OMK-79 seed liquid was inoculated into 3 L of fermentation medium for fermentation. The fermentation was carried out in a 5 L small-scale bioreactor, and its temperature and pH were controlled at 28-30° C. and 4.0-7.0, respectively. During the whole operation period of the above fermentation, the airflow rate was maintained at 3 L/min, the air pressure was maintained at 0.08 Mpa, the pH was controlled by adding ammonia water, and the initial stirring speed was 200 rpm.

[0102] When the dissolved oxygen concentration (DO) dropped to 50% (about 24 hours after fermentation), glycerol began to be supplemented and the stirring speed was increased stepwise to 600 rpm until the detected biomass reached 200 g/L (wet weight), the total supplemented amount of glycerol was about 75 g/L. The cell density of the fermentation broth reached saturation 120 hours after supplementing glycerol, and the OD.sub.600 value could reach up to 400. During the entire fermentation period after the start of the supplementation of glycerol, the airflow rate was maintained at 4 L/min, and the air pressure was maintained at 0.10 Mpa. After the fermentation, the ergothioneine-containing supernatant was collected by homogenizing the cells and centrifugation. The content of ergothioneine in the supernatant was determined by high performance liquid chromatography (HPLC), and the content was 7.6 g/L.

Example 4

Production Optimization of Ergothioneine

[0103] The Schizosaccharomyces pombe strain OMK-79 was inoculated into a 250 mL flat-bottomed Erlenmeyer flask containing 50 mL of YPD medium, and grown at 28° C. and 250 rpm for 48 hours to activate the strain. The activated OMK-79 strain was transferred to a 1 L flat-bottomed Erlenmeyer flask containing 150 mL of YPD medium at an inoculation amount of %, and grown at 28° C. and 250 rpm for 18 hours to obtain a seed liquid. 100 mL of Schizosaccharomyces pombe OMK-79 seed liquid was inoculated into 3 L of fermentation medium for fermentation. The fermentation was carried out in a 5 L small-scale bioreactor, and its temperature and pH were controlled at 28-30° C. and 5.0-6.0, respectively. During the whole operation period of the above fermentation, the airflow rate was maintained at 3 L/min, the air pressure was maintained at 0.08 Mpa, the pH was controlled by adding ammonia water, and the initial stirring speed was 200 rpm.

[0104] When the dissolved oxygen concentration (DO) dropped to 50% (about 24 hours after fermentation), glycerol and substrate began to be supplemented. The substrate was 1 g/L arginine, 1 g/L histidine, 1 g/L methionine or 1 g/L cysteine. After starting to supplement glycerol and substrate, the stirring speed was gradually increased to 600 rpm until the detected biomass reached 200 mg/mL (wet weight). The supplemented amount of glycerol was about 75 g/L. The cell density of the fermentation broth reached saturation 120 hours after supplementing glycerol and substrate, and the OD.sub.600 value could reach up to 400. During the entire fermentation period after the start of the supplementation of glycerol and substrate, the airflow rate was maintained at 4 L/min, and the air pressure was maintained at 0.10 Mpa. After the fermentation, the ergothioneine-containing supernatant was collected by homogenizing the cells and centrifugation. The content of ergothioneine in the supernatant was determined by high performance liquid chromatography (HPLC). Through experiments, the supplementation of the substrates arginine, histidine, methionine and cysteine all promoted the production of ergothioneine, and the content of ergothioneine in the supernatant was 10.7 g/L (substrate was arginine), 9.8 g/L (substrate was histidine), 9.2 g/L (substrate was methionine) and 8.4 g/L (substrate was cysteine).

Example 5

Fermentation Optimization of Ergothioneine

[0105] The Schizosaccharomyces pombe strain OMK-79 was inoculated into a 250 mL flat-bottomed Erlenmeyer flask containing 50 mL of YPD medium, and grown at 28° C. and 250 rpm for 48 hours to activate the strain. The activated OMK-79 strain was transferred to a 1 L flat-bottomed Erlenmeyer flask containing 150 mL of YPD medium at an inoculation amount of 1%, and grown at 28° C. and 250 rpm for 18 hours to obtain a seed liquid. 100 mL of Schizosaccharomyces pombe OMK-79 seed liquid was inoculated into 3 L of fermentation medium for fermentation. The fermentation was carried out in a 5 L small-scale bioreactor, and its temperature and pH were controlled at 28-30° C. and 5.0-6.0, respectively. During the whole operation period of the above fermentation, the airflow rate was maintained at 3 L/min, the air pressure was maintained at 0.08 Mpa, the pH was controlled by adding ammonia water, and the initial stirring speed was 200 rpm.

[0106] When the dissolved oxygen concentration (DO) dropped to 50% (about 24 hours after fermentation), glycerol, 1.3 g/L arginine, 0.8 g/L histidine, 0.6 g/L methionine and 0.5 g/L cysteine began to be supplemented. After starting to supplement glycerol and substrate, the stirring speed was gradually increased to 600 rpm until the detected biomass reached 200 mg/mL (wet weight). The supplemental amount of glycerol was about 75 g/L. The cell density of the fermentation broth reached saturation 120 hours after supplementing glycerol and substrate, and the OD.sub.600 value could reach up to 400. During the entire fermentation period after the start of the supplementation of glycerol and substrate, the airflow rate was maintained at 4 L/min, and the pressure was maintained at 0.10 Mpa. After about 148 hours of fermentation, the ergothioneine-containing supernatant was collected by homogenizing the cells and centrifugation. The content of ergothioneine in the supernatant was determined by high performance liquid chromatography (HPLC), and the content was 12.5 g/L.

[0107] The method of this application has been described through the preferred embodiments. It is obvious that relevant persons can make changes or appropriate changes and combinations to the methods and applications described herein within the content, spirit and scope of this application to realize and apply the technology of this application. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application.