A method for producing single cell protein

Abstract

The present application relates to the technical field of biological fermentation, and discloses a production method of single-cell protein.The present application adopts corn syrup hydrolysate as a culture medium component, which is beneficial to promote rapid fermentation of bacteria and efficient synthesis of single-cell protein.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, specifically to a culture medium containing corn steep liquor hydrolysate and its uses, as well as a method for producing single-cell protein. Background Technology

[0002] Corn steep liquor (CSL) is a byproduct of corn starch production and can be prepared by wet milling. It contains abundant peptides, polysaccharides, proteins, and various amino acids, such as arginine, glutamic acid, histidine, phenylalanine, alanine, and β-phenylethylamine.

[0003] Corn steep liquor can be used as a water-soluble plant protein and water-soluble vitamin, a compound fertilizer additive, a fermentation nitrogen source, and a fiber feed. It is the cheapest nitrogen source, rich in nutrients, and a promoter of microbial anabolic metabolism. Currently, corn steep liquor is widely used as a nitrogen source in biopharmaceuticals, amino acid fermentation, and enzyme preparations. In microbial fermentation, to reduce fermentation costs, corn steep liquor can be used to replace or partially replace other nitrogen sources, such as soybean meal hydrolysate, yeast powder, and peptone.

[0004] However, due to the complex composition of corn steep liquor, which is entirely in polymer form, it cannot be utilized by microorganisms when used as a fermentation raw material, thus requiring processing. Summary of the Invention

[0005] The present invention aims to solve the problem of complex composition of corn steep liquor in the prior art, and provides a culture medium that uses corn steep liquor hydrolysate obtained by a specific method as a component, which can make full use of the nutrients in corn steep liquor, promote rapid fermentation of microorganisms and efficient synthesis of single-cell proteins.

[0006] In one aspect, the present invention provides a culture medium comprising corn steep liquor hydrolysate;

[0007] The method for preparing the corn steep liquor hydrolysate includes:

[0008] Corn steep liquor and sodium hydroxide are provided;

[0009] After hydrolyzing the corn steep liquor with the sodium hydroxide, the pH was adjusted to neutral to obtain the corn steep liquor hydrolysate.

[0010] In some embodiments, the final concentration of sodium hydroxide in the hydrolysis treatment is 1-4% by mass. If the final concentration of sodium hydroxide is too low, the hydrolysis effect on corn steep liquor will be poor; if the final concentration of sodium hydroxide is too high, the alkalinity will be too strong, which can easily lead to the destructive or even inactivation of proteins and peptides in the corn steep liquor hydrolysate. Specifically, the final concentration of sodium hydroxide is 1%, 2%, 3%, or 4% by mass.

[0011] In some embodiments, the pH of the hydrolysis treatment is 9-11. If the pH of the hydrolysis treatment is too low, the corn steep liquor cannot be hydrolyzed; if the pH of the hydrolysis treatment is too high, it can easily lead to the breakdown or even inactivation of proteins and peptides in the corn steep liquor hydrolysate. Specifically, the pH of the hydrolysis treatment is 9, 9.5, 10, 10.5, or 11.

[0012] In some embodiments, the hydrolysis treatment lasts for 18-24 hours at a temperature of 62-70°C. If the hydrolysis time is too short, the hydrolysis will be incomplete; if the hydrolysis time is too long, it will unnecessarily extend the production time. Appropriate temperature can promote molecular motion and improve the hydrolysis effect. If the temperature is too high, water will easily evaporate, and the sodium hydroxide concentration will be too high, leading to the destructive or even inactivation of proteins and peptides in the corn steep liquor hydrolysate. Specifically, the hydrolysis treatment time is 18, 19, 20, 21, 22, 23, or 24 hours.

[0013] In some embodiments, the culture medium further comprises at least one of anhydrous glucose, yeast extract, casein peptone, magnesium sulfate, thiamine hydrochloride, betaine, ammonium sulfate, potassium dihydrogen phosphate, and disodium hydrogen phosphate.

[0014] In some embodiments, the culture medium is a primary seed culture medium containing 50-80 g / L of the corn steep liquor hydrolysate, 1-2% by mass of anhydrous glucose, 0.6-1.5% by mass of yeast extract, and 1.5-3% by mass of casein peptone.

[0015] In some embodiments, the culture medium is a secondary seed culture medium containing 80-120 g / L of the corn steep liquor hydrolysate, 1-3% by mass of anhydrous glucose, 0.6-2% by mass of yeast extract, 1.5-3.2% by mass of casein peptone, 0.8-2.4% by mass of magnesium sulfate, and 0.2-0.8% by mass of thiamine hydrochloride.

[0016] In some embodiments, the culture medium is a fermentation medium comprising 100-200 g / L of the corn steep liquor hydrolysate, 1-3% by mass of anhydrous glucose, 0.6-4% by mass of yeast extract, 1.5-5% by mass of casein peptone, 0.8-3.2% by mass of magnesium sulfate, 0.6-1.4% by mass of thiamine hydrochloride, 0.5-1% by mass of betaine, 3.5-6.8% by mass of ammonium sulfate, 5-7% by mass of potassium dihydrogen phosphate, and 2-4% by mass of disodium hydrogen phosphate.

[0017] In a preferred embodiment of the present invention, by adding the corn steep liquor hydrolysate to the primary seed culture medium and the secondary seed culture medium, the microorganisms can adapt to the composition of the corn steep liquor hydrolysate in advance, induce the microorganisms to produce enzymes that utilize the corn steep liquor hydrolysate, and enable the microorganisms to adapt more quickly in the fermenter culture.

[0018] In another aspect, the present invention provides the use of the culture medium in fermentation production.

[0019] In some implementations, the fermentation production is the fermentation production of single-cell proteins.

[0020] In another aspect, the present invention provides a method for producing single-cell protein, comprising:

[0021] Provide the bacterial strain and the culture medium provided by the present invention;

[0022] The bacterial strain was cultured using the culture medium to obtain single-cell protein.

[0023] In some implementations, the culture includes:

[0024] The strain was inoculated into a primary seed culture medium. When the cell OD value was 4.2-6.8, it was inoculated into a secondary seed culture medium at an inoculation rate of 3-7%. When the cell OD value was 12-16, it was inoculated into a fermentation culture medium at an inoculation rate of 5-10% for cultivation.

[0025] The primary seed culture medium contains 50-80 g / L of corn steep liquor hydrolysate, 1-2% by mass of anhydrous glucose, 0.6-1.5% by mass of yeast extract, and 1.5-3% by mass of casein peptone.

[0026] The secondary seed culture medium contains 80-120 g / L of corn steep liquor hydrolysate, 1-3% by mass of anhydrous glucose, 0.6-2% by mass of yeast extract, 1.5-3.2% by mass of casein peptone, 0.8-2.4% by mass of magnesium sulfate, and 0.2-0.8% by mass of thiamine hydrochloride.

[0027] The fermentation medium comprises 100-200 g / L of corn steep liquor hydrolysate, 1-3% by mass of anhydrous glucose, 0.6-4% by mass of yeast extract, 1.5-5% by mass of casein peptone, 0.8-3.2% by mass of magnesium sulfate, 0.6-1.4% by mass of thiamine hydrochloride, 0.5-1% by mass of betaine, 3.5-6.8% by mass of ammonium sulfate, 5-7% by mass of potassium dihydrogen phosphate, and 2-4% by mass of disodium hydrogen phosphate.

[0028] In some implementations, during the culturing step in the fermentation medium, when the cell OD value is ≥80, 200 g / L of the corn steep liquor hydrolysate is added to the fermenter to provide sufficient nutrition for the cells to grow further.

[0029] In some implementations, during the culturing step in the fermentation medium, when the cell OD value is ≥200, carbon and nitrogen sources are added to the fermenter to provide sufficient carbon and nitrogen sources for cell growth.

[0030] In some preferred embodiments, the added carbon source is selected from at least one of acetic acid, formic acid, and glucose, and the added nitrogen source is selected from at least one of sterilized 30% by mass ammonium sulfate solution, 20% by mass ammonium acetate solution, and 40% by mass urea solution.

[0031] In some implementation schemes, when the bacterial cell OD value is ≥200, carbon and nitrogen sources are added to the fermenter, with a carbon-to-nitrogen ratio of (6-10):1. This carbon-to-nitrogen ratio is more suitable for nutrient utilization and absorption by the bacteria. If the carbon-to-nitrogen ratio is less than 6, there is less carbon and more nitrogen, leading to slow bacterial growth. If the carbon-to-nitrogen ratio is greater than 10, there is more carbon and less nitrogen, resulting in rapid bacterial growth but lower nitrogen utilization and lower synthesis of single-cell protein. Specifically, the carbon-to-nitrogen ratio is controlled at 6:1, 7:1, 8:1, 9:1, or 10:1.

[0032] In some implementations, during the culturing step in the fermentation medium, the carbon source concentration of the fermentation broth is controlled at 4-15 g / L, and the nitrogen source concentration is controlled at 2-7 g / L. When the carbon / nitrogen source concentration is too low, the cells exhibit slow growth; when the carbon / nitrogen source concentration is too high, cell growth is inhibited. Specifically, the carbon source concentration is controlled at 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, or 15 g / L; and the nitrogen source concentration is controlled at 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, or 7 g / L.

[0033] In some embodiments, the strain is selected from at least one of Yarrowia lipolytica, Schizochytrium, and Pseudomonas aeruginosa.

[0034] In one specific implementation scheme, the *Yarrowia lipolytica* strain was purchased from the China Industrial Microbial Culture Collection Center (CICC 32291); the *Schizochytrium* strain was purchased from the U.S. Type Culture Collection Center (ATCC 20888); and the *Pseudomonas aeruginosa* strain was purchased from the China Industrial Microbial Culture Collection Center (CICC 10351).

[0035] In one specific implementation, the method for producing the single-cell protein includes the following steps:

[0036] (1) Inoculate the strain into sterilized primary seed culture medium and culture it in a shaker at 32°C and 180 rpm for 16-18 hours. When the OD value is 4.2-6.8, primary seeds are obtained.

[0037] (2) Inoculate the primary seeds into the sterilized secondary seed culture medium at an inoculation rate of 5% and culture for 8-12 hours. When the OD value is 12-16, the secondary seeds are obtained.

[0038] (3) Add the secondary seed to the fermentation medium at an inoculation rate of 5%-10%. Take samples every hour to measure the OD, acetic acid and nitrogen source concentrations. Control the acetic acid concentration to 4-15 g / L and the nitrogen source concentration to 2-7 g / L. After 2 consecutive hours, when the OD value increases by less than 10 per hour, harvest the fermentation broth, centrifuge, wash with water and dry to obtain single-cell protein (bacterial cells).

[0039] In step (3), a feed medium, carbon source, and nitrogen source are added according to the required acetic acid concentration and nitrogen source concentration. The feed medium contains 200 g / L of the corn steep liquor hydrolysate. The added carbon source is acetic acid, and the added nitrogen source is a sterilized 30% by mass ammonium sulfate solution. The carbon-nitrogen ratio of the added carbon source and the added nitrogen source is controlled to be (6-10):1.

[0040] This invention utilizes sodium hydroxide to hydrolyze corn steep liquor, which disrupts the macromolecular structure of the liquor, breaks down large polymer bonds, and transforms it into smaller organic molecules. These smaller molecules are more readily absorbed by microorganisms through cell walls, while simultaneously increasing the content of free amino acids and soluble proteins, resulting in a nutrient-rich corn steep liquor hydrolysate. The inventors discovered that many basic amino acids in corn steep liquor with an isoelectric point (pH greater than 7) (such as lysine and arginine) are more soluble in a sodium hydroxide environment. Sodium hydroxide hydrolysis allows more organic matter, soluble proteins, and amino acids to precipitate rather than dissolve in acidic environments or enzymatic environments. Therefore, using sodium hydroxide to hydrolyze corn steep liquor is significantly superior to traditional acid or enzymatic hydrolysis methods.

[0041] The corn steep liquor hydrolysate provided by this invention is low in cost and high in organic nitrogen, making it a superior nitrogen source. When this corn steep liquor hydrolysate is used as a component of the culture medium to cultivate bacterial strains, the problem of poor stability of corn steep liquor is avoided, allowing the nutrients in the corn steep liquor to be fully utilized by the strains, promoting rapid fermentation of the cells and efficient synthesis of single-cell proteins.

[0042] The method for producing single-cell protein provided by this invention, through a specific culture medium composition, facilitates rapid fermentation of the cells and efficient synthesis of single-cell protein. Furthermore, by employing specific feeding methods and controlling the carbon-to-nitrogen ratio during fermentation, rapid fermentation of the cells can be achieved in a short time, further improving the production efficiency and yield of single-cell protein. Detailed Implementation

[0043] The present invention will be further illustrated below with specific embodiments. These embodiments are merely illustrative and do not limit the scope of the invention. Unless otherwise specified, the raw materials and ingredients used in the following embodiments are commercially available products, and the methods and conditions used are known and conventional in the art.

[0044] The term "inoculum volume" refers to the ratio of the volume of seed culture transferred to the volume of the culture system after inoculation. For example, an inoculum volume of 5-10% means that the volume fraction of the seed culture is 5-10%. In this document, when "inoculum volume" is mentioned, it refers to the volume fraction. For the sake of brevity, it will not be elaborated on in detail.

[0045] In this invention, the term "OD value" refers to the OD value of the fermentation broth, which is obtained by ultraviolet spectrophotometry. Specifically, the OD value of the fermentation broth at 600 nm is detected using an ultraviolet spectrophotometer (Alpha-1106, Shanghai Spectrum Instruments Co., Ltd.).

[0046] In the following specific embodiments of the present invention, the methods for testing dry weight, yield, and crude protein content are as follows:

[0047] (1) Dry weight of single-cell protein: in g / L

[0048] Weigh 1.00L of fermentation broth, centrifuge at 8000 rpm for 10 min to obtain bacterial cells, add 1L of water to resuspend evenly, and centrifuge again at 8000 rpm for 10 min to obtain wet bacterial cells, and weigh their mass as m1; take 5.000g from m1 and dry it at 103±2℃ according to the method of national standard GB / T6435-2014 "Determination of Moisture in Feed" to obtain dry bacterial cells, and weigh their mass as m2 (accurate to 4 decimal places).

[0049] Dry weight of bacteria (DCW) = m² / 5.000 × m¹ / 1.00

[0050] (2) Yield: Unit is g / (L·h)

[0051] Yield = Dry weight of single-cell protein ÷ High-density fermentation time (fermentation cycle).

[0052] (3) Crude protein content

[0053] According to the national standard GB / T 6432-2018 "Determination of Crude Protein in Feed - Kjeldahl Method", the crude protein content (mass fraction) was obtained by digestion, ammonia distillation, and titration.

[0054] In the following specific embodiments of the present invention, Yersinia lipolytica was purchased from the China Industrial Microbial Culture Collection Center, accession number: CICC 32291; all components of the culture medium were commercially available chemicals.

[0055] In the following specific embodiments of the present invention, the method for determining the endpoint of high-density fermentation is as follows: if the increase in OD value per hour is less than 10 for two consecutive hours, it indicates that the cell growth is very slow, and it is determined that no further growth will occur, the fermentation endpoint is reached, and the fermentation liquid is obtained by transferring the fermentation liquid to the tank.

[0056] Example 1

[0057] (1) Preparation of corn steep liquor hydrolysate

[0058] Corn steep liquor was hydrolyzed with 1% sodium hydroxide at pH 9 for 18 hours at 62-70°C. Then, hydrochloric acid was added to adjust the pH to 6 to obtain the corn steep liquor hydrolysate.

[0059] (2) Preparation of various culture media:

[0060] Prepare 200 mL of primary seed culture medium, consisting of: 50 g / L corn steep liquor hydrolysate, 1% anhydrous glucose, 0.6% yeast extract, and 1.5% casein peptone; sterilize in an autoclave at 121 °C for 20 min.

[0061] Prepare 2 L of secondary seed culture medium with the following components: 80 g / L corn steep liquor hydrolysate, 1% anhydrous glucose, 0.6% yeast extract, 1.5% casein peptone, 0.8% magnesium sulfate, and 0.2% thiamine hydrochloride. Sterilize in an autoclave at 121°C for 20 min.

[0062] A 40L fermentation medium was prepared, consisting of: 200g / L corn steep liquor hydrolysate, 3% anhydrous glucose, 0.6% yeast extract, 1.5% casein peptone, 0.8% magnesium sulfate, 0.6% thiamine hydrochloride, 0.5% betaine, 3.5% ammonium sulfate, 7% potassium dihydrogen phosphate, and 2% disodium hydrogen phosphate. The fermentation medium was added to a 100L fermenter and sterilized by steam in the fermenter.

[0063] Prepare a fed culture medium consisting of 200 g / L corn steep liquor hydrolysate.

[0064] Prepare carbon and nitrogen sources for replenishment. The carbon source is acetic acid, and the nitrogen source is a sterilized 30% ammonium sulfate solution.

[0065] Example 2

[0066] Single-cell protein production was carried out using various culture media prepared in Example 1:

[0067] (1) Add Yersinia lipophila to the primary seed culture medium, place it in a shaker, and culture it for 16 hours at a temperature of 32℃ and a rotation speed of 180rpm. The OD value is 4.2, and the primary seed is obtained.

[0068] (2) The primary seeds were added to the fermenter containing the secondary seed culture medium at an inoculation rate of 5% by fire ring inoculation and cultured for 8 hours. The OD value was 12, and the secondary seeds were obtained.

[0069] (3) Add the secondary seed at an inoculation rate of 8% to a 100L fermenter containing fermentation medium for cultivation. When the cell OD≥80, start feeding the culture medium. When the cell OD≥200, add carbon and nitrogen sources. The carbon source rate is 1.2L / h. Adjust the nitrogen source rate according to the carbon-nitrogen ratio. During the fermentation process, control the carbon-nitrogen ratio of the added carbon and nitrogen sources to 6:1.

[0070] (4) If the OD growth is less than 10 for 2 consecutive hours, it is determined that the bacterial growth is slow and the bacteria can be transferred to the tank to obtain the fermentation liquid.

[0071] (5) The fermentation broth was centrifuged, washed with water, and spray-dried to obtain single-cell protein cells. The dry weight was 160 g / L, the single-cell protein yield was 5.3 g / L / h, and the crude protein content was 56%.

[0072] Example 3

[0073] This embodiment is basically the same as Example 2, except that the components of the primary seed culture medium are: 80 g / L corn steep liquor hydrolysate, 2% anhydrous glucose, 1.5% yeast extract, and 3% casein peptone.

[0074] Example 4

[0075] This embodiment is basically the same as Example 2, except that the components of the secondary seed culture medium are: 120 g / L corn steep liquor hydrolysate, 3% anhydrous glucose, 2% yeast extract, 3.2% casein peptone, 2.4% magnesium sulfate, and 0.8% thiamine hydrochloride.

[0076] Example 5

[0077] This embodiment is basically the same as embodiment 2, except that the carbon-nitrogen ratio of the added carbon source and nitrogen source is controlled to be 2:1 during the fermentation process in step (3).

[0078] Example 6

[0079] This embodiment is basically the same as embodiment 2, except that the carbon-nitrogen ratio of the added carbon source and nitrogen source is controlled to be 16:1 during the fermentation process in step (3).

[0080] Example 7

[0081] This embodiment is basically the same as Example 2, except that the composition of the feeding culture medium is: 200g / L hydrolyzed soybean meal aqueous solution (commercially available).

[0082] Example 8

[0083] This embodiment is basically the same as Example 2, except that the composition of the feeding culture medium is: 200g / L hydrolyzed cottonseed cake solution (commercially available).

[0084] Example 9

[0085] This embodiment is basically the same as Embodiment 2, except that the inoculation amount of secondary seeds is 2%.

[0086] Example 10

[0087] This embodiment is basically the same as Embodiment 2, except that a fissuring chyme strain is used instead of Yersinia lipophila for production (ATCC 20888, purchased from the U.S. Type Culture Collection Center).

[0088] Example 11

[0089] This embodiment is basically the same as Embodiment 2, except that the corn steep liquor is treated with an acid + enzymatic hydrolysis method.

[0090] Add 2 mol / L hydrochloric acid to corn steep liquor to prepare a corn steep liquor-hydrochloric acid suspension with a solid content of 25% by mass. After reacting at 80 degrees Celsius for 16 hours, adjust the pH to 4.0 with ammonia water, and then add acidic protease (300 μg / L) and phytase (150 μg / L). After enzymatic hydrolysis at 40 degrees Celsius for 8 hours, sterilize at 115 degrees Celsius for 20 minutes to obtain the corn steep liquor hydrolysate.

[0091] The yield and crude protein content of single-cell proteins in Examples 2-11 are shown in Table 1.

[0092] Table 1

[0093] Example Single-cell protein production g / L / h Crude protein content (mass%) 2 5.3 56 3 5.4 57 4 5.5 56 5 4.8 51 6 4.6 48 7 4.2 46 8 4.3 50 9 4.1 45 10 2.1 41 11 1.9 38

[0094] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for producing single-cell protein, characterized in that, include: Provide strains and culture media; The strain was cultured using the culture medium to obtain a fermentation broth; The fermentation broth was separated to obtain single-cell protein; The culture medium contains corn steep liquor hydrolysate; the method for preparing the corn steep liquor hydrolysate includes: Corn steep liquor and sodium hydroxide are provided; After hydrolyzing the corn steep liquor with the sodium hydroxide, the pH was adjusted to neutral to obtain the corn steep liquor hydrolysate. In the hydrolysis treatment, the final concentration of sodium hydroxide is 1-4% by mass; the pH of the hydrolysis treatment is 9-11; the hydrolysis treatment time is 18-24 hours, and the temperature is 62-70℃. The cultivation includes: The strain was inoculated into a primary seed culture medium. When the cell OD value was 4.2-6.8, it was inoculated into a secondary seed culture medium at an inoculation rate of 3-7%. When the cell OD value was 12-16, it was inoculated into a fermentation culture medium at an inoculation rate of 5-10% for cultivation. The primary seed culture medium comprises 50-80 g / L corn steep liquor hydrolysate, 1-2% by mass of anhydrous glucose, 0.6-1.5% by mass of yeast extract, and 1.5-3% by mass of casein peptone. The secondary seed culture medium comprises 80-120 g / L corn steep liquor hydrolysate, 1-3% by mass of anhydrous glucose, 0.6-2% by mass of yeast extract, 1.5-3.2% by mass of casein peptone, 0.8-2.4% by mass of magnesium sulfate, and 0.2-0.8% by mass of thiamine hydrochloride. The fermentation medium comprises 100-200 g / L of the corn steep liquor hydrolysate, 1-3% by mass of anhydrous glucose, 0.6-4% by mass of yeast extract, 1.5-5% by mass of casein peptone, 0.8-3.2% by mass of magnesium sulfate, 0.6-1.4% by mass of thiamine hydrochloride, 0.5-1% by mass of betaine, 3.5-6.8% by mass of ammonium sulfate, 5-7% by mass of potassium dihydrogen phosphate, and 2-4% by mass of disodium hydrogen phosphate. The strain was selected from *Yersinia lipophila* ( Yarrowia lipolytica ), Schizochytrium ( Schizochytrium ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa At least one of the following.

2. The production method according to claim 1, characterized in that, The strain was cultured for 16-18 hours in the primary seed medium and for 8-12 hours in the secondary seed medium.

3. The production method according to claim 1, characterized in that, In the step of culturing in the fermentation medium, when the cell OD value is ≥80, 200 g / L of the corn steep liquor hydrolysate is added to the fermenter; and / or During the culturing process in the fermentation medium, when the cell OD value is ≥200, carbon and nitrogen sources are added to the fermenter.

4. The production method according to claim 3, characterized in that, The carbon source is selected from at least one of acetic acid, formic acid, and glucose; the nitrogen source is selected from at least one of sterilized 30% by mass ammonium sulfate solution, 20% by mass ammonium acetate solution, and 40% by mass urea solution.

5. The production method according to claim 3, characterized in that, When the bacterial cell OD value is ≥200, carbon and nitrogen sources are added to the fermenter, and the carbon-to-nitrogen ratio of the added carbon and nitrogen sources is (6-10):1; and / or In the culturing step in the fermentation medium, the carbon source concentration of the fermentation broth is controlled at 4-15 g / L, and the nitrogen source concentration is controlled at 2-7 g / L.