A culture medium for producing (-)-a-bisabolol and a method for fermenting (-)-a-bisabolol

Abstract

The present application belongs to the field of microbial fermentation, and particularly relates to a culture medium for producing (-)-alpha-bisabolol and a method for fermenting to produce (-)-alpha-bisabolol. The culture medium for producing (-)-alpha-bisabolol is composed of the following raw materials in parts by weight: glucose 8-15 parts, soybean peptone 25-35 parts, yeast powder 5-15 parts, corn syrup dry powder 15-25 parts, potassium dihydrogen phosphate 1-5 parts, magnesium sulfate heptahydrate 3-8 parts, and defoaming agent 0.01-0.02 parts. Through the culture medium with specific components and the optimized fermentation process, the yield of recombinant Saccharomyces cerevisiae for fermenting to produce (-)-alpha-bisabolol is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation, specifically relating to a culture medium for producing (-)-α-bisabolol and a method for fermenting to produce (-)-α-bisabolol. Background Technology

[0002] (-)-α-bisabolol, also known as sweet bisabolol or saposhnikovia root alcohol, is a monocyclic unsaturated sesquiterpene alcohol (molecular formula C). 15 H 26 Glycerin (O) is typically a colorless to straw-yellow viscous liquid with a density between 0.922 and 0.928, a refractive index between 1.492 and 1.498, and a slightly characteristic odor. It is readily soluble in lower alcohols such as ethanol and isopropanol, as well as fatty alcohols, glycerides, and acetic acid, but almost insoluble in glycerin and water. This ingredient is widely used in cosmetics, especially as an active ingredient for skin protection and care, suitable for sunscreens, after-sun lotions, baby care products, and after-shave products; it can also be used in oral care products such as toothpaste and mouthwash.

[0003] In natural sources, (-)-α-bisabolol is mainly found in chamomile, poplar, and some plants in the genera *Salvia* and *Salvia*. However, as a plant secondary metabolite, its extraction is constrained by the growth cycle and environment, resulting in limited yields that cannot meet market demand. Currently, industrial production mainly relies on chemical synthesis. Domestically, isopentenol and dipentene are commonly used for esterification and condensation, while BASF uses farnesol and nerolidol for acid-catalyzed cyclization followed by hydrolysis. However, due to the complex chiral structure of this compound, chemical synthesis generally faces challenges such as high difficulty, low biological activity, and insufficient purity. Therefore, utilizing microbial fermentation technology to efficiently produce high-value-added (-)-α-bisabolol with inexpensive carbon sources and culture media is considered a highly promising alternative.

[0004] Existing research has constructed various engineered strains capable of producing (-)-α-bisabolol using genetic engineering techniques. For example, in 2019, Zhen Kang's team reconstructed the biosynthetic pathway in *Saccharomyces cerevisiae*, obtaining an engineered strain with a fermentation yield of 10.26 g / L. Patent CN110982723A also discloses a method for producing this substance using *Saccharomyces cerevisiae*, with a maximum yield of only 10.26 g / L in a fermenter. CN116286576A discloses a method for producing (-)-α-bisabolol using recombinant *Escherichia coli* fermentation, with a maximum yield of only 9.88 g / L in a fermenter. However, these yield levels still need further improvement to achieve economically competitive scale-up when scaling up industrial fermenters. Summary of the Invention

[0005] To address the above problems, this invention provides a culture medium for producing (-)-α-bisabolol, which is composed of the following raw materials in parts by weight: 8-15 parts glucose, 25-35 parts soybean peptone, 5-15 parts yeast powder, 15-25 parts corn steep liquor powder, 1-5 parts potassium dihydrogen phosphate, 3-8 parts magnesium sulfate heptahydrate, and 0.01-0.02 parts defoamer.

[0006] Furthermore, it is composed of the following raw materials in parts by weight: 10 parts glucose, 30 parts soybean peptone, 10 parts yeast powder, 20 parts corn steep liquor powder, 3 parts potassium dihydrogen phosphate, 6 parts magnesium sulfate heptahydrate, and 0.01 parts defoamer.

[0007] The present invention also provides the use of the aforementioned culture medium in the fermentation production of (-)-α-bisabolol.

[0008] Finally, this invention provides a method for producing (-)-α-bisabolol by fermentation, comprising the following steps: 1) Take recombinant brewing yeast, inoculate it into a seed bottle and culture it to obtain seed liquid; 2) Take the seed liquid obtained in step 1) and inoculate it into a fermenter for fermentation for 80-100 hours; fermentation conditions include: aeration rate of 45-60 L / min; The culture medium in the fermenter is prepared by dissolving the aforementioned culture medium in water and then sterilizing it.

[0009] Furthermore, the culture medium in the seed bottle in step 1) is LB medium.

[0010] Further, the cultivation conditions in step 1) are a temperature of 27-32℃, a rotation speed of 200-600rpm, and a time of 20-30h.

[0011] Furthermore, the culture conditions are: temperature 30°C, rotation speed 200 rpm, and time 25 h.

[0012] Furthermore, the fermentation conditions described in step 2) also include: inoculum size of 10-15%, tank pressure of 0.03-0.06 MPa, stirring speed of 200-600 rpm, culture medium temperature of 27-32℃, culture medium pH of 5.0-6.0, and dissolved oxygen of 30-80%; when the sugar content in the fermentation broth is lower than 1-5 g / L, a sugar solution with a concentration of 40-80% is added until the sugar content in the fermentation broth reaches 1-5 g / L.

[0013] Furthermore, the fermentation conditions are as follows: The inoculum size is 13%, the tank pressure is 0.050 MPa, the aeration rate is 50 L / min, the stirring speed is 200-600 rpm, the culture medium temperature is 30℃, the culture medium pH is 5.25, and the dissolved oxygen is 50%. When the sugar content in the fermentation broth is lower than 1-5 g / L, add sugar solution with a concentration of 40-80% until the sugar content in the fermentation broth reaches 1-5 g / L.

[0014] Furthermore, the sugar solution is a glucose solution.

[0015] The "defoaming agent" described in this invention is a functional additive used to control and eliminate foam during microbial fermentation to meet the requirements of the fermentation process. It includes polyether-based, silicone oil-based, and natural oil-based defoaming agents. Specifically, this invention uses polyoxyethylene polyoxypropylene glycerol ether (GPE).

[0016] The present invention provides a (-)-α-bisabolol fermentation medium that, through a medium with specific components, can supplement the nutritional needs of recombinant Saccharomyces cerevisiae, thereby accelerating gene expression in recombinant Saccharomyces cerevisiae, promoting the production of mevalonase during the synthesis stage, resulting in a large production of isoprene pyrophosphate, further promoting the production of (-)-α-bisabolol, and ultimately significantly increasing the yield of fermented (-)-α-bisabolol.

[0017] Experiments have shown that the fermentation process optimized using the fermentation medium of this invention can achieve a yield of 50.0 g / L of (-)-α-bisabolol in a 50L fermenter. This represents a significant increase compared to existing fermentation methods for producing (-)-α-bisabolol, reducing the cost and environmental impact of chemical synthesis and demonstrating practical application value.

[0018] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0019] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0020] Figure 1 Brewing yeast plates; Figure 2 Saccharomyces cerevisiae fermentation mycelium. Detailed Implementation

[0021] The raw materials and equipment used in the specific embodiments of the present invention are obtained through commercial purchases. The recombinant brewer's yeast is provided by Shanghai Ruikang Biotechnology R&D Co., Ltd., and the recombinant brewer's yeast has been patented under CN202310076063.4.

[0022] Example 1: Preparation of a culture medium for producing (-)-α-bisabolol Formula: 10g glucose, 30g soy peptone, 10g yeast powder, 20g corn steep liquor powder, 3g potassium dihydrogen phosphate, 6g magnesium sulfate heptahydrate, 0.01g defoamer; Preparation method: Weigh the raw materials according to the ratio, dissolve the raw materials except glucose and defoamer in water, then add water to make up to 1000ml, and finally add glucose and defoamer in sequence to dissolve and mix well, and sterilize at 121-123℃ for 30min.

[0023] Example 2: Method for Fermentation Production of (-)-α-bisabolol 1) Take recombinant Saccharomyces cerevisiae, inoculate it into a seed bottle containing LB liquid medium, and culture it in a shaker at 30℃ and 200rpm for 25h to obtain seed liquid; 2) Take the seed culture obtained in step 1) and inoculate it into a fermenter containing the culture medium prepared according to Example 1 for fermentation for 80-100 hours; the fermentation conditions are as follows: The inoculum size is 13%, the tank pressure is 0.050 MPa, the aeration rate is 50 L / min, the stirring speed is 200-600 rpm, the culture medium temperature is 30℃, the culture medium pH is 5.25, and the dissolved oxygen is controlled at 50%. When the sugar content in the fermentation broth is lower than 1-5 g / L, add a 40-80% glucose solution until the sugar content in the fermentation broth reaches 1-5 g / L.

[0024] Example 3: Preparation of a culture medium for producing (-)-α-bisabolol Formula: 8g glucose, 20g soy peptone, 5g yeast powder, 15g corn steep liquor powder, 1g potassium dihydrogen phosphate, 3g magnesium sulfate heptahydrate, 0.01g defoamer; Preparation method: Same as in Example 1.

[0025] Example 4: Method for Fermentation Production of (-)-α-bisabolol 1) Take recombinant Saccharomyces cerevisiae, inoculate it into a seed bottle containing LB liquid medium, and culture it in a shaker at 30℃ and 200rpm for 25h to obtain seed liquid; 2) Take the seed liquid obtained in step 1) and inoculate it into a fermenter containing the culture medium prepared according to Example 3 for fermentation for 80-100 hours; the fermentation conditions are as follows: The inoculum size is 10%, the tank pressure is 0.030 MPa, the aeration rate is 45 L / min, the stirring speed is 200-600 rpm, the culture medium temperature is 27℃, the culture medium pH is 5.0, and the dissolved oxygen is controlled at 30%. When the sugar content in the fermentation broth is lower than 1-5 g / L, add a 40-80% glucose solution until the sugar content in the fermentation broth reaches 1-5 g / L.

[0026] Example 5: Preparation of a culture medium for producing (-)-α-bisabolol Formula: 15g glucose, 35g soy peptone, 15g yeast powder, 25g corn steep liquor powder, 5g potassium dihydrogen phosphate, 8g magnesium sulfate heptahydrate, 0.02g defoamer; Preparation method: Same as in Example 1.

[0027] Example 6: Method for Fermentation Production of (-)-α-bisabolol 1) Take recombinant Saccharomyces cerevisiae, inoculate it into a seed bottle containing LB liquid medium, and culture it in a shaker at 30℃ and 200rpm for 25h to obtain seed liquid; 2) Take the seed liquid obtained in step 1) and inoculate it into a fermenter containing the culture medium prepared according to Example 5 for fermentation for 80-100 hours; the fermentation conditions are as follows: The inoculum size is 15%, the tank pressure is 0.060 MPa, the aeration rate is 60 L / min, the stirring speed is 200-600 rpm, the culture medium temperature is 32℃, the culture medium pH is 6.0, and the dissolved oxygen is controlled at 80%. When the sugar content in the fermentation broth is lower than 1-5 g / L, add a 40-80% glucose solution until the sugar content in the fermentation broth reaches 1-5 g / L.

[0028] The following experimental examples illustrate the beneficial effects of the present invention.

[0029] Experimental Example 1: Study on the production of (-)-α-bisabolol by fermentation of recombinant brewer's yeast I. Methods Recombinant brewing yeast (plate colonies such as...) Figure 1 Inoculate with LB liquid culture medium The seed culture was carried out in a seed bottle at 30°C and 200 rpm for 25 h. The resulting mature seed culture was then transferred to a fermenter containing fermentation medium for fermentation. The fermentation medium and fermentation process involved in the fermentation were carried out according to the following experimental groups.

[0030] Experimental group 1 The fermentation medium formula is as follows: glucose 20g / L, yeast extract 30g / L, yeast peptone 10g / L, soybean meal powder 10g / L, corn steep liquor powder 20g / L, dipotassium hydrogen phosphate 6g / L, potassium dihydrogen phosphate 8g / L, and the remainder is water. Fermentation conditions were as follows: inoculum size 10%, tank pressure 0.030 MPa, aeration rate 50 L / min, stirring speed 200-600 rpm, temperature 33℃, pH 5.5, and dissolved oxygen controlled at 30%. When the sugar content in the fermentation broth was below 1-5 g / L, 60% glucose solution was added, with the amount calculated based on the residual sugar content of 1-5 g / L. Fermentation was carried out for 90 hours before being discharged from the tank.

[0031] Experimental group 2 The fermentation medium formula is: glucose 10g / L, soybean peptone 30g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate heptahydrate 6g / L, defoamer 0.01g / L, and the remainder is water; Fermentation conditions were as follows: inoculum size 15%, tank pressure 0.040 MPa, aeration rate 67 L / min, stirring speed 200-600 rpm, temperature 30℃, pH 5.50, and dissolved oxygen controlled at 30%. When the sugar content in the fermentation broth was below 1-5 g / L, 60% glucose solution was added, with the amount calculated based on the residual sugar content of 1-5 g / L. Fermentation was carried out for 90 hours before being discharged from the tank. Experimental group 3 The fermentation medium formula is as follows: glucose 10g / L, soybean peptone 30g / L, corn steep liquor powder 20g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate heptahydrate 6g / L, defoamer 0.01g / L, and the remainder is water; Fermentation conditions were as follows: inoculum size 13%, tank pressure 0.040 MPa, aeration rate 67 L / min, stirring speed 200-600 rpm, temperature 28℃, pH 5.5, and dissolved oxygen controlled at 40%. When the sugar content in the fermentation broth was below 1-5 g / L, 60% glucose solution was added, with the amount calculated based on the residual sugar content of 1-5 g / L. Fermentation was carried out for 90 hours before being discharged from the tank.

[0032] Experimental group 4 The fermentation medium formula is as follows: glucose 10 g / L, soybean peptone 30 g / L, yeast extract 10 g / L, corn steep liquor powder 20 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 6 g / L, defoamer 0.01 g / L, and the remainder is water. Fermentation conditions were as follows: inoculum size 13%, tank pressure 0.040 MPa, aeration rate 67 L / min, stirring speed 200-600 rpm, temperature 30℃, pH 5.25, and dissolved oxygen controlled at 50%. When the sugar content in the fermentation broth was below 1-5 g / L, 60% glucose solution was added, with the amount calculated based on the residual sugar content of 1-5 g / L. Fermentation was carried out for 90 hours before being discharged from the tank.

[0033] Experimental group 5 The fermentation medium formula is as follows: glucose 10g / L, tryptone 30g / L, yeast powder 10g / L, corn steep liquor powder 20g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate heptahydrate 6g / L, defoamer 0.01g / L, and the remainder is water; Fermentation conditions were as follows: inoculum size 13%, tank pressure 0.050 MPa, aeration rate 30 L / min, stirring speed 200-600 rpm, temperature 30℃, pH 5.25, and dissolved oxygen controlled at 50%. When the sugar content in the fermentation broth was below 1-5 g / L, 60% glucose solution was added, with the amount calculated based on the residual sugar content of 1-5 g / L. Fermentation was carried out for 90 hours before being discharged from the tank.

[0034] Experimental group 6 The fermentation medium formula is as follows: glucose 10g / L, soybean peptone 30g / L, yeast powder 10g / L, corn steep liquor powder 20g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate heptahydrate 6g / L, defoamer 0.01g / L, and the remainder is water; Fermentation conditions were as follows: inoculum size 13%, tank pressure 0.050 MPa, aeration rate 50 L / min, stirring speed 200-600 rpm, temperature 30℃, pH 5.25, and dissolved oxygen controlled at 50%. When the sugar content in the fermentation broth was below 1-5 g / L, 60% glucose solution was added, with the amount calculated based on the residual sugar content of 1-5 g / L. Fermentation was carried out for 90 hours before being discharged from the tank.

[0035] Experimental group 7 The fermentation medium formula is as follows: glucose 10g / L, soybean peptone 30g / L, yeast extract 10g / L, corn steep liquor powder 20g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate heptahydrate 6g / L, defoamer 0.01g / L, and the remainder is water; Fermentation conditions: inoculum size 13%, tank pressure 0.050 MPa, aeration rate 30 L / min, stirring 200-600 rpm, temperature 30℃, pH 5.25, dissolved oxygen controlled at 50%. When the sugar content in the fermentation broth is lower than 1-5 g / L, start adding 60% glucose solution, the amount added is calculated based on the residual sugar content of 1-5 g / L, and ferment for 90 hours before removing from the tank.

[0036] II. Results Each experimental group was repeated three times. Typical mycelial images during fermentation are shown below. Figure 2 After fermentation, the content of (-)-α-bisabolol in the fermentation broth was determined by HPLC.

[0037] The chromatographic conditions for determining the content of (-)-α-bisabolol in the fermentation broth by HPLC were as follows: Column: C18 (4.6 mm) 250 mm, 5 μm); Mobile phase A: 83.0% methanol aqueous solution; Mobile phase B: acetonitrile; Flow rate: 1 ml / min; Injection volume: 10 μl. Fermentation results are shown in Table 1 below.

[0038] Table 1. Detection values ​​of (-)-α-bisabolol produced during fermentation in experimental groups 1-5 The culture medium used in experimental group 1 in Table 1 is the culture medium formula disclosed in patent CN116286576A. When brewing with this formula, the fermentation titer of Saccharomyces cerevisiae increased significantly compared with Escherichia coli fermentation. However, the formula can still be continuously optimized and improved, so subsequent experiments were conducted to optimize it.

[0039] The culture medium used in experimental group 2 had a lower ratio of glucose and potassium dihydrogen phosphate compared to experimental group 1, and no added nutrients such as yeast extract powder and yeast peptone. The potency of (-)-α-bisabolol increased by 7.13 g / L, indicating that the fermentation process of Saccharomyces cerevisiae does not require a complex and nutrient-rich culture environment. However, it was found that the supply of nutrients was insufficient in the later stages of the process, and there is still room for optimization. It was also determined that the culture medium of experimental group 1 is not suitable for the fermentation process of Saccharomyces cerevisiae.

[0040] Experimental group 3, through further optimization—increasing the nitrogen source ratio in the culture medium, decreasing the inoculum size and culture temperature, and improving dissolved oxygen control parameters—achieved a significant increase in the (-)-α-bisabolol potency of 3.95 g / L compared to experimental group 2. This result indicates that the culture medium in experimental group 2 may lack readily available nitrogen sources that promote early cell growth. Supplementing with such substances (e.g., corn steep liquor powder) combined with lower culture temperatures and inoculum sizes can create a more suitable growth environment for the cells, thereby effectively promoting the synthesis of (-)-α-bisabolol.

[0041] Based on Experiment 3, Experiment 4 further adjusted the culture medium and fermentation process: yeast extract was added to the fermentation medium; the temperature and dissolved oxygen control parameters were improved, and the pH control value was lowered in the fermentation process. The potency of (-)-α-bisabolol significantly increased by 5.92 g / L. Yeast extract contains a large amount of amino acids in biosynthesis, which can promote the synthesis of isoprene pyrophosphate in biosynthesis. Increasing the culture temperature is beneficial to cell activity, increasing the dissolved oxygen control value is beneficial to cell metabolism, and decreasing the pH control value provides conditions for optimizing the cell metabolic environment. The increased yield of the target product resulting from these improvements further demonstrates that yeast extract and process adjustments have a promoting effect on the production of (-)-α-bisabolol.

[0042] Experimental group 5, based on experimental group 4, replaced yeast extract with yeast powder and soybean peptone with tryptone, and increased tank pressure while decreasing airflow during fermentation. The results showed that the potency of (-)-α-bisabolol increased by 3.59 g / L. Yeast powder, as a slow-release nitrogen source, can continuously provide nutrients such as amino acids to the cells, which helps promote the biosynthesis of (-)-α-bisabolol. However, replacing tryptone did not show a significant benefit, indicating that it may not be suitable for the current metabolic needs of the yeast. In terms of process, increasing tank pressure helps maintain dissolved oxygen levels during cultivation, while decreasing airflow effectively controls foam formation, thus maintaining the stability of the fermentation system.

[0043] Building upon experimental group 5, experimental group 6 replaced tryptone with soybean peptone in the fermentation medium and increased the air flow rate during fermentation. This resulted in a 4.15 g / L increase in the potency of (-)-α-bisabolol. This change indicates that soybean peptone remains a good nitrogen source for yeast fermentation metabolism, significantly enhancing its potency. Increasing the air flow rate ensures thorough mixing and oxygen contact in the fermentation broth, creating more favorable conditions for cell metabolism. Nitrogen source selection and oxygen supply regulation have a mutually reinforcing synergistic effect in increasing the yield of (-)-α-bisabolol.

[0044] While maintaining the same fermentation medium as experimental group 6, experimental group 7 adjusted the process by reducing the airflow rate, resulting in a significant decrease of 7.21 g / L in the potency of (-)-α-bisabolol. This result indicates that a lower airflow rate affects the normal growth and metabolic activity of the cells, leading to metabolic pathway obstruction and byproduct accumulation, ultimately impacting the yield of (-)-α-bisabolol. Combining the results from experimental groups 1-6, it is clear that in fermentation systems using soybean peptone as the nitrogen source, dissolved oxygen levels must be controlled within an appropriate range to fully guarantee the growth and metabolic activity of *Saccharomyces cerevisiae*, thereby maximizing the yield of (-)-α-bisabolol.

[0045] The experimental results above show that: This invention significantly improves the yield of (-)-α-bisabolol through synergistic optimization of the fermentation medium and fermentation process. Regarding the culture medium, the addition of organic nitrogen sources such as corn steep liquor powder and yeast extract effectively promotes rapid biomass accumulation in the early stages of cell growth. In terms of process control, key parameters such as inoculum size, tank pressure, air flow rate, dissolved oxygen, temperature, and pH are systematically adjusted to create a more favorable metabolic environment for the synthesis of (-)-α-bisabolol. These optimizations not only enhance cell viability but also promote the synthesis of mevalonate phosphate kinase, thereby driving the formation of isoprene pyrophosphate in the downstream metabolic pathway, ultimately efficiently driving the biosynthesis of (-)-α-bisabolol.

[0046] The method of this invention is simple and easy to implement, and it improves the production of (-)-α-bisabolol in a 50L fermenter, achieving a potency of 53.13 g / L. This has significant practical implications for industrial production.

[0047] In summary, this invention uses recombinant Saccharomyces cerevisiae as the production strain, optimizes the culture medium and fermentation process, and obtains a complete method for producing (-)-α-bisabolol. This method ensures the nutritional conditions and external environment required for the rapid growth and metabolism of the strain, has a large yield of (-)-α-bisabolol, greatly reduces production costs, and is suitable for industrial production.

Claims

1. A culture medium for producing (-)-α-bisabolol, characterized in that: It is composed of the following raw materials in parts by weight: 8-15 parts glucose, 25-35 parts soybean peptone, 5-15 parts yeast powder, 15-25 parts corn steep liquor powder, 1-5 parts potassium dihydrogen phosphate, 3-8 parts magnesium sulfate heptahydrate, and 0.01-0.02 parts defoamer.

2. The culture medium according to claim 1, characterized in that: It consists of the following raw materials in parts by weight: 10 parts glucose, 30 parts soybean peptone, 10 parts yeast powder, 20 parts corn steep liquor powder, 3 parts potassium dihydrogen phosphate, 6 parts magnesium sulfate heptahydrate, and 0.01 parts defoamer.

3. Use of the culture medium according to claim 1 or 2 in the fermentation production of (-)-α-bisabolol.

4. A method for producing (-)-α-bisabolol by fermentation, characterized in that: Includes the following steps: 1) Take recombinant brewing yeast, inoculate it into a seed bottle and culture it to obtain seed liquid; 2) Take the seed liquid obtained in step 1) and inoculate it into a fermenter for fermentation for 80-100 hours; fermentation conditions include: aeration rate of 45-60 L / min; The culture medium in the fermenter is prepared by dissolving the culture medium described in claim 1 or 2 in water and then sterilizing it.

5. The method according to claim 4, characterized in that: Step 1) The culture medium in the seed bottle is LB medium.

6. The method according to claim 4, characterized in that: The culture conditions are: temperature 27-32℃, rotation speed 200-600rpm, and time 20-30h.

7. The method according to claim 6, characterized in that: The culture conditions were 30°C, 200 rpm, and 25 h.

8. The method according to claim 4, characterized in that: Step 2) The fermentation conditions also include: inoculum size 10-15%, tank pressure 0.03-0.06 MPa, stirring speed 200-600 rpm, culture medium temperature 27-32℃, culture medium pH 5.0-6.0, dissolved oxygen 30-80%; when the sugar content in the fermentation broth is lower than 1-5 g / L, add sugar solution with a concentration of 40-80% until the sugar content in the fermentation broth reaches 1-5 g / L.

9. The method according to claim 4 or 8, characterized in that: The fermentation conditions are as follows: The inoculum size is 13%, the tank pressure is 0.050 MPa, the aeration rate is 50 L / min, the stirring speed is 200-600 rpm, the culture medium temperature is 30℃, the culture medium pH is 5.25, and the dissolved oxygen is 50%. When the sugar content in the fermentation broth is lower than 1-5 g / L, add sugar solution with a concentration of 40-80% until the sugar content in the fermentation broth reaches 1-5 g / L.

10. The method according to claim 9, characterized in that: The sugar solution is a glucose solution.

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