Method for producing lactone sophorolipid through fermentation

Abstract

The invention provides a method for producing lactone sophorolipid by fermentation, which comprises the following steps: inoculating candida tropicalis strain into a seed culture medium for seed culture to obtain a seed solution; inoculating the seed solution into a fermentation culture medium for fermentation, and supplementing a carbon source and a nitrogen source in the fermentation process to obtain the microbial secondary metabolite lactone type sophorolipid, carbon sources of the seed culture medium and the fermentation culture medium comprise n-hexadecane, and ricinoleic acid and oleic hydroxylase are supplemented in the fermentation process to serve as precursor substances and reaction enzymes for synthesizing sophorolipid. Through mutual compounding of specific materials and processes, the growth rate of the thalli and the synthesis rate of the lactone type sophorolipid are effectively improved, so that the method can realize fermentation growth of the thalli and efficient synthesis of the lactone type sophorolipid in a short time.

Description

Technical Field:

[0001] The present invention belongs to the field of biotechnology. More specifically, the present invention relates to a method for fermentative production of lactone-type sophorolipids. Background Art

[0002] Sophorolipids are a type of glycolipid biosurfactant produced by yeast metabolism, which have excellent surface / interface activity and stability, and are non-toxic, non-irritating, and easily biodegradable. They have good application prospects in many fields and are considered to be one of the most promising biosurfactants to replace chemical surfactants.

[0003] Sophorolipids were first discovered in 1961. Different types of sophorolipids have common structural characteristics. Each molecule consists of a hydrophilic part and a hydrophobic part. The hydrophilic part is sophorose, and the hydrophobic part is a saturated or unsaturated long-chain hydroxy fatty acid. The differences also lie in the length and saturation of the fatty acid chain, the position and degree of acetylation. According to the presence or absence of 1,4" esterification, they are also divided into lactone-type and acid-type. Lactone-type sophorolipids have better ability to reduce surface tension and stronger antibacterial activity; while acid-type sophorolipids have better foam-forming ability and solubility.

[0004] Currently, all product suppliers with definite information for industrialization adopt fermentation processes, and the strains are all naturally selected. There are no genetically engineered strains for industrial production yet. The purification process is basically physical methods, including natural sedimentation, centrifugation, membrane filtration, etc. Currently, only a few companies such as those in China and Germany can truly provide industrial products.

[0005] Although the fermentation production level of sophorolipids is higher than that of other biosurfactants, its yield and carbon source conversion rate still need to be improved. At the same time, optimizing the addition ratio of hydrophilic and hydrophobic carbon sources can further improve the product yield and substrate conversion rate. Summary of the Invention:

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for fermentative production of lactone-type sophorolipids. Through the design of jointly promoting product synthesis by carbon sources, precursor substances, and enzymes, and by using specific materials and processes in combination, the method can achieve the reproduction and growth of bacteria and the efficient synthesis of lactone-type sophorolipids in a short time, greatly improving the production efficiency and yield of lactone-type sophorolipids.

[0007] To achieve the purpose of this invention, the following technical solutions are adopted by the present invention:

[0008] A method for fermentative production of lactone-type sophorolipids, the method comprising: inoculating Candida tropicalis strain into a seed medium for seed culture to obtain a seed solution;

[0009] Inoculate the seed liquid into a fermentation medium for fermentation. During the fermentation process, a carbon source is supplemented, including any one or a combination of at least two of rapeseed oil, soybean oil, glucose, and glycerol, and rapeseed oil is further preferred; during the fermentation process, a nitrogen source is also supplemented, including any one or a combination of at least two of urea, ammonium sulfate, ammonium nitrate, and ammonia water, and urea is further preferred to obtain the fermentation broth containing lactone-type sophorolipid.

[0010] The carbon sources of the seed medium and the fermentation medium include n-hexadecane, and ricinoleic acid and oleate hydroxylase are supplemented as sophorolipid synthesis promoters during the fermentation process.

[0011] In the present invention, the Candida tropicalis is selected from Candida glabrata and Saccharomyces cerevisiae, and Candida glabrata is preferably selected.

[0012] In the present invention, the seed medium contains: calcium chloride 0.4 - 2.2%, yeast powder 0.8 - 2.4%, casein peptone 1.2 - 4.2%, magnesium sulfate 0.9 - 2.6%, ammonium sulfate 0.3 - 0.9%, riboflavin 0.029 - 0.039%, glucose 3% - 9%, n-hexadecane 0.7 - 2.2%, and the rest is water; the pH is adjusted to 2 - 4 with hydrochloric acid or ammonia water.

[0013] In the present invention, the temperature of the seed culture is 20 - 30 °C, and the time is 40 - 50 h; preferably, the seed culture is carried out under stirring conditions, and the stirring speed is 100 - 300 rpm.

[0014] In the present invention, the seed liquid is inoculated into the fermentation medium for fermentation at an inoculation amount of 3% - 6% by mass fraction.

[0015] In the present invention, the fermentation medium contains 1 - 3% of n-hexadecane; preferably, the fermentation tank medium contains the following components by mass fraction: p-aminobenzoic acid 0.003 - 0.01%, riboflavin 0.002 - 0.008%, pyridoxol 0.001 - 0.006%, yeast powder 1.5 - 3.5%, peptone 4.1 - 7.5%, dipotassium hydrogen phosphate 0.06 - 0.28%, potassium dihydrogen phosphate 0.18 - 0.24%, glucose 2% - 7%, n-hexadecane 1 - 3%, and the rest is water.

[0016] In the present invention, the temperature of the fermentation is 22 - 29 °C, preferably 24 - 27 °C; the pH of the fermentation is 2 - 4, preferably 2.5 - 3.5.

[0017] In the present invention, after 44 - 48 h of fermentation, a carbon source and a nitrogen source are supplemented. Preferably, the carbon source supplemented during the fermentation process includes any one or more of rapeseed oil, soybean oil, glucose, and glycerol, and rapeseed oil is further preferred.

[0018] In the present invention, the nitrogen source supplemented during the fermentation process includes any one or more of urea, ammonium sulfate, ammonium nitrate, and ammonia water, preferably urea; preferably, the carbon-nitrogen ratio is controlled to be 100-126:1 by using the carbon source and the nitrogen source.

[0019] In the present invention, after 55-60 h of fermentation, 0.05%-0.08% of ricinoleic acid based on the weight of the fermentation broth and 0.02%-0.04% of an aqueous solution of 0.3-2 mM oleic acid hydroxylase based on the weight of the fermentation broth are supplemented.

[0020] As a preferred method, the method includes:

[0021] 1. Inoculate the thawed Candida tropicalis into the sterilized first-stage seed medium. The composition of the first-stage seed medium is (by mass): calcium chloride 0.4-2.2%, yeast powder 0.8-2.4%, casein peptone 1.2-4.2%, magnesium sulfate 0.9-2.6%, ammonium sulfate 0.3-0.9%, riboflavin 0.029-0.039%, glucose 3%-9%, n-hexadecane 0.7-2.2%; adjust the pH to 2-4 with hydrochloric acid or ammonia water.

[0022] 2. Place the inoculated first-stage seed medium in a shaker, and the culture conditions are 20-30°C and 100-300 revolutions per minute for 40-50 hours to obtain the first-stage seed liquid.

[0023] 3. Inoculate the first-stage seed liquid into a fermenter for cultivation, and the inoculation amount is 3%-6%.

[0024] 4. The fermenter medium is (by mass ratio): p-aminobenzoic acid 0.003-0.01%, riboflavin 0.002-0.008%, pyridoxol 0.001-0.006%, yeast powder 1.5-3.5%, peptone 4.1-7.5%, dipotassium hydrogen phosphate 0.06-0.28%, potassium dihydrogen phosphate 0.18-0.24%, glucose 2%-7%, n-hexadecane 1-3%, and the rest is water;

[0025] 5. The fermenter culture conditions are: the fermentation temperature is 22-29°C and the pH is 2-4.

[0026] 6. After 44-48 h of fermentation, supplement the carbon source and the nitrogen source. The flow rate of the carbon source is 0.02%-0.04% of the volume of the fermentation broth per minute, and the carbon-nitrogen molar ratio is controlled to be (100-126:1), and the flow rate of the nitrogen source is adjusted according to the carbon-nitrogen ratio.

[0027] 7. After 55 - 60 h of fermentation, add 0.05% - 0.08% of ricinoleic acid based on the weight of the fermentation broth, and 0.02% - 0.04% of a 0.3 - 2 mM aqueous solution of oleic acid hydroxylase as a promoter for the synthesis of sophorolipid

[0028] 8. During the later stage of fermentation for 140 - 160 hours, the growth of the bacterial cells is slow, and the synthesis rate of sophorolipid no longer increases. Then the fermentation can be stopped, and a sophorolipid concentration greater than 300 g / L and a sophorolipid production rate greater than 2 g / (L·h) can be obtained. The production rate of the synthesized lactone-type sophorolipid > 1.2 g / (L·h), preferably 1.5 - 2 g / (L·h).

[0029] The present invention uses Candida tropicalis as the fermentation strain (China Center for Industrial Culture Collection of Microorganisms, accession number CICC 32626). The method designs the carbon sources in seed culture and fermentation. Adding n-hexadecane to the seed medium can promote the formation of a unique metabolic pathway in the bacterial cells; at the same time, the carbon source in the fermentation medium also includes n-hexadecane, which further induces and promotes the n-hexadecane metabolic pathway of the bacterial cells. Adding rapeseed oil and urea during the fermentation process can effectively promote the rapid growth of the bacterial cells and the synthesis of sophorolipid. Further, adding ricinoleic acid and oleic acid hydroxylase during the fermentation process plays the role of inducing precursor substances and enzymatic catalysis in the sophorolipid metabolic pathway, promoting the growth of the bacterial cells and the rapid synthesis of the product, and shortening the fermentation cycle. Specific Embodiments

[0030] The following examples will further illustrate the method provided by the present invention. However, the present invention is not limited to the listed examples, and should also include any other known changes within the scope of the claims of the present invention. Each component for preparing the culture medium is a commercially available chemical.

[0031] In the following examples and comparative examples of the present invention, the detection method for the content of sophorolipid is the sulfuric acid - anthrone method, and the specific operation method is as follows:

[0032] (1) Preparation of anthrone reagent

[0033] Accurately weigh 0.1 g of anthrone, add 100 mL of concentrated sulfuric acid, and mix well to obtain anthrone reagent.

[0034] (2) Preparation of standard glucose solution

[0035] Weigh a certain mass of anhydrous glucose, dry it to constant weight in an oven at 105 °C, weigh 0.1800 g of the dried anhydrous glucose, and make up the volume to 100 mL in a volumetric flask, and shake well. Its concentration is 10 mmol / L. Then take 0.5, 1, 2, 3, 4 mL respectively and make up the volume to 100 mL, and their concentrations are 0.5, 0.1, 0.2, 0.3, 0.4 mmol / L respectively.

[0036] (3) Drawing the glucose standard curve by the anthrone method

[0037] Measure 1 mL of anthrone reagent and standard glucose solutions of different concentrations and place them in a stoppered test tube. Quickly cool them in an ice-water bath, then heat them in a boiling water bath for 10 min, cool them in an ice bath, and measure the OD value at a wavelength of 620 nm. The experimental blank control is 1 mL of distilled water.

[0038] Use the anthrone method to measure the absorbance value and draw the standard curve.

[0039] y = ax + b

[0040] Among them, y is the glucose content and x is the absorbance. Calculate the values of a and b through the standard curve, and this formula is used to calculate the glucose content in the sample according to the absorbance in the later stage.

[0041] (4) Determination of sophorolipid content

[0042] Take the sample to be tested, dilute it 200 - 500 times with water, take 1 mL of the diluted sample and place it in a stoppered test tube, add 1 mL of anthrone reagent, quickly cool it in an ice-water bath, then heat it in a boiling water bath for 10 min, cool it in an ice bath, and measure the OD value at a wavelength of 620 nm. The experimental blank control is 1 mL of distilled water. Calculate the glucose concentration of the diluted sample according to the measured absorbance and the standard curve.

[0043] The final calculation formula for sophorolipid content is as follows:

[0044] Sophorolipid content = content of glucose in the reaction solution × dilution factor × 1.91

[0045] Use a Q-TOF high-resolution liquid chromatography-mass spectrometry instrument to determine the ratio of acid-type and lactone-type sophorolipids, and determine the ratio of lactone-type and acid-type by area normalization.

[0046] LC-MS / MS detection of lactone-type and acid-type sophorolipids:

[0047] 1) Sample: The fermentation broth is extracted with ethyl acetate, the solvent is removed, and it is dissolved with chloroform / methanol (1:3, v / v) and filtered through a 0.22 μm filter membrane for testing

[0048] 2) Instrument: Q-TOF high-resolution liquid chromatography-mass spectrometry instrument

[0049] 3) Chromatographic separation

[0050] C18 column (2.1 * 150 mm, 5 μm), flow rate 0.2 ml / min, separation temperature 35 °C, separation gradient: 0 - 3 min, 40% B - 60% B; 3 - 20 min, 60% B - 88% B; 22 min, 100% B (A: water containing 0.1% formic acid; B: acetonitrile containing 0.1% formic acid)

[0051] 4) Identification by ESI-MS: Positive ion scan, scanning range (m / z): 50–1500, scanning mode: AUTO MS / MS, nebulizer gas pressure: 15 psi, drying gas flow rate: 9 L / min, drying gas temperature: 180 °C.

[0052] In the present invention, the term "OD value" refers to the OD value of the fermentation broth, which is obtained by testing with ultraviolet spectrophotometry. Specifically: Use an ultraviolet spectrophotometer (Alpha-1106, Shanghai Puyuan Instrument Co., Ltd.) to detect the OD value of the fermentation broth at 600 nm.

[0053] Example 1:

[0054] 1. Prepare 100 ml of the first-stage seed culture medium, with the composition: calcium chloride 2.2%, yeast powder 2.4%, casein peptone 4.2%, magnesium sulfate 2.6%, ammonium sulfate hydrochloride 0.9%, riboflavin 0.039%, glucose 5%, n-hexadecane 1%; adjust the pH to 3 with hydrochloric acid;

[0055] 2. Put the prepared first-stage seed culture medium into an autoclave for high-temperature sterilization at a temperature of 121 °C for 15 minutes;

[0056] 3. Take out the first-stage seed culture medium after sterilization and wait for it to return to room temperature;

[0057] 4. Take a strain of Candida tropicalis, and add the dissolved strain into the first-stage seed culture medium in a laminar flow hood. Add 200 μl of the strain solution to every 100 ml of the culture medium,

[0058] 5. Place the inoculated first-stage seed liquid in a shaker, and the culture conditions are 25 °C, 200 revolutions per minute, and culture for 45 hours,

[0059] 6. Inoculate the first-stage seed liquid into a 5 L fermenter for culture, and the inoculation amount is 6%;

[0060] 7. The fermenter culture medium is: para-aminobenzoic acid 0.01%, riboflavin 0.008%, pyridoxol 0.006%, yeast powder 3.5%, peptone 7.5%, dipotassium hydrogen phosphate 0.28%, potassium dihydrogen phosphate 0.24%, glucose 7%, n-hexadecane 3%, adjust the pH to 3 with hydrochloric acid, and use it after sterilization;

[0061] 8. Start fermentation in the fermenter, and the fermentation temperature is 25 °C and the pH is 3

[0062] 9. After 48 h of fermentation, carbon source and nitrogen source were supplemented. The carbon source was rapeseed oil with a flow rate of 0.02% of the volume of the fermentation broth per minute, and the nitrogen source was a 30% ammonium sulfate solution after sterilization. The carbon-nitrogen ratio was controlled at 100:1, and the flow rate of the nitrogen source was adjusted according to the carbon-nitrogen ratio.

[0063] 10. After 60 h of fermentation, 0.05% aqueous ricinoleic acid solution based on the weight of the fermentation broth and 0.02% aqueous solution of 2 mM oleic acid hydroxylase based on the weight of the fermentation broth were supplemented.

[0064] 11. After 150 h of fermentation culture, the concentration of sophorolipid synthesized by the cells reached the maximum value and no longer increased. The sophorolipid concentration of 300 g / L could be obtained, and then the fermentation could be stopped. The sophorolipid production was 2 g / (L·h), and the production of lactone-type sophorolipid was 1.90 g / (L·h).

[0065] Example 2:

[0066] 1. Prepare 100 ml of the first-stage seed culture medium, and its composition is: calcium chloride 0.4%, yeast powder 0.8%, casein peptone 4.2%, magnesium sulfate 2.6%, ammonium sulfate hydrochloride 0.9%, riboflavin 0.032%, glucose 3%, n-hexadecane 0.7%; adjust the pH to 2 with hydrochloric acid.

[0067] 2. Put the prepared first-stage seed culture medium into an autoclave for high-temperature sterilization at a temperature of 121 °C for 15 minutes.

[0068] 3. Take out the first-stage seed culture medium after sterilization and wait until it returns to room temperature.

[0069] 4. Take a strain of Candida tropicalis, and add the dissolved strain into the first-stage seed culture medium in a laminar flow hood. Add 200 μl of the strain solution to every 100 ml of the culture medium.

[0070] 5. Place the inoculated first-stage seed liquid in a shaker, and the culture conditions are 20 °C and 100 revolutions per minute for 40 hours.

[0071] 6. Inoculate the first-stage seed liquid into a 5 L fermenter for culture, and the inoculation amount is 3%.

[0072] 7. The fermenter culture medium is: p-aminobenzoic acid 0.004%, riboflavin 0.008%, pyridoxol 0.004%, yeast powder 3.5%, peptone 7.5%, dipotassium hydrogen phosphate 0.26%, potassium dihydrogen phosphate 0.22%, glucose 5%, n-hexadecane 1%. Adjust the pH to 3 with hydrochloric acid and use it after sterilization.

[0073] 8. The fermenter starts to ferment, and the fermentation temperature is 22 °C and the pH is 3.

[0074] 9. After 44 h of fermentation, a carbon source and a nitrogen source are supplemented. The carbon source is soybean oil, and the flow rate is 0.03% of the volume of the fermentation broth per minute. The nitrogen source is an aqueous urea solution with a mass fraction of 30% after sterilization. The solution controls the carbon-nitrogen ratio to be 115:1, and the flow rate of the nitrogen source is adjusted according to the carbon-nitrogen ratio.

[0075] 10. After 55 h of fermentation, an aqueous ricinoleic acid solution accounting for 0.06% of the weight of the fermentation broth and an aqueous 2 mM oleic acid hydroxylase solution accounting for 0.03% of the weight of the fermentation broth are supplemented.

[0076] 11. After 151 h of fermentation culture, when the concentration of sophorolipid synthesized by the cells reaches the maximum value and no longer increases, the fermentation can be stopped. The concentration of sophorolipid can reach 302 g / L, the production of sophorolipid is 2 g / (L·h), and the production of lactone-type sophorolipid is 1.86 g / (L·h).

[0077] Example 3:

[0078] 1. Prepare 100 ml of the first-stage seed medium, and its composition is: calcium chloride 1.2%, yeast powder 1.4%, casein peptone 3.1%, magnesium sulfate 2.6%, ammonium sulfate hydrochloride 0.9%, riboflavin 0.038% g / L, glucose 9%, n-hexadecane 2.2%; adjust the pH to 4 with hydrochloric acid;

[0079] 2. Put the prepared first-stage seed medium into an autoclave for high-temperature sterilization at a temperature of 121 °C for 15 minutes;

[0080] 3. Take out the first-stage seed medium after sterilization and wait until it returns to room temperature;

[0081] 4. Take a strain of Candida tropicalis. Add the dissolved strain into the first-stage seed medium in a laminar flow hood. Add 200 μl of the strain solution to every 100 ml of the medium;

[0082] 5. Place the inoculated first-stage seed liquid in a shaker, and the culture conditions are 30 °C and 300 revolutions per minute for 50 hours;

[0083] 6. Inoculate the first-stage seed liquid into a 5 L fermenter for culture, and the inoculation amount is 4%;

[0084] 7. The medium in the fermenter is: p-aminobenzoic acid 0.007%, riboflavin 0.006%, pyridoxol 0.002%, yeast powder 3.5%, peptone 7.5%, dipotassium hydrogen phosphate 0.28%, potassium dihydrogen phosphate 0.24%, glucose 2%, n-hexadecane 2%. Adjust the pH to 3 with hydrochloric acid and use it after sterilization;

[0085] 8. The fermenter starts to ferment, and the fermentation temperature is 28 °C and the pH is 3;

[0086] 9. After 47 h of fermentation, a carbon source and a nitrogen source were supplemented. The carbon source was glycerol, and the flow rate was 0.04% of the volume of the fermentation broth per minute. The nitrogen source was an aqueous solution of ammonium nitrate with a mass fraction of 30% after sterilization. The carbon-nitrogen ratio of the solution was controlled at 126:1, and the flow rate of the nitrogen source was adjusted according to the carbon-nitrogen ratio.

[0087] 10. After 58 h of fermentation, an aqueous solution of ricinoleic acid at 0.08% of the weight of the fermentation broth and an aqueous solution of 1 mM oleic acid hydroxylase at 0.04% of the weight of the fermentation broth were supplemented.

[0088] 11. After 150 h of fermentation culture, when the concentration of sophorolipid synthesized by the bacterial cells reached the maximum value and no longer increased, the fermentation tank could be stopped. The concentration of sophorolipid obtained was 304 g / L, the yield of sophorolipid was 2.02 g / (L·h), and the yield of lactone-type sophorolipid was 1.81 g / (L·h).

[0089] Comparative Example 1

[0090] The difference between this comparative example and Example 1 was that: "1% n-hexadecane" in the first-stage seed culture medium was replaced with "1% glucose", and the other components remained unchanged; "3% n-hexadecane" in the fermentation tank culture medium was replaced with "3% glucose", and the other components remained unchanged. The other steps were the same as those in Example 1.

[0091] After 184 h of fermentation culture, when the concentration of sophorolipid synthesized by the bacterial cells reached the maximum value and no longer increased, the fermentation tank could be stopped. The concentration of sophorolipid obtained was 162 g / L, the yield of sophorolipid was 0.88 g / (L·h), and the yield of lactone-type sophorolipid was 0.46 g / (L·h).

[0092] Comparative Example 2

[0093] Steps 1-9 of this example were the same as those in Example 1.

[0094] 10. After 60 h of fermentation, an aqueous solution of 2 mM oleic acid hydroxylase at 0.02% of the weight of the fermentation broth was supplemented.

[0095] 11. After 167 h of fermentation culture, when the concentration of sophorolipid synthesized by the bacterial cells reached the maximum value and no longer increased, the fermentation tank could be stopped. The concentration of sophorolipid obtained was 145 g / L, the yield of sophorolipid was 0.87 g / (L·h), and the yield of lactone-type sophorolipid was 0.49 g / (L·h).

[0096] Comparative Example 3

[0097] Steps 1-9 of this example were the same as those in Example 1.

[0098] 10. After 60 h of fermentation, an aqueous solution of ricinoleic acid at 0.05% of the weight of the fermentation broth was supplemented.

[0099] 11. After 184 h of fermentation culture, when the concentration of sophorolipid synthesized by the cells reaches the maximum value and no longer increases, the fermentation can be stopped. The concentration of sophorolipid can reach 151 g / L, the yield of sophorolipid is 0.82 g / (L·h), and the yield of lactone-type sophorolipid is 0.47 g / (L·h).

[0100] Comparative Example 4

[0101] Steps 1-9 of this example are the same as those of Example 1.

[0102] 10. After 60 h of fermentation, a sterile aqueous solution of 0.07% of the weight of the fermentation broth is added.

[0103] 11. After 196 h of fermentation culture, when the concentration of sophorolipid synthesized by the cells reaches the maximum value and no longer increases, the fermentation can be stopped. The concentration of sophorolipid can reach 148 g / L, the yield of sophorolipid is 0.76 g / (L·h), and the yield of lactone-type sophorolipid is 0.38 g / (L·h).

[0104] The fermentation times, sophorolipid concentrations and yields in Examples 1-3 and Comparative Examples 1-4 are shown in Table 1

[0105]

Claims

1. A method for fermentatively producing lactone-type sophorolipid, characterized in that, The method includes: Inoculating Candida tropicalis strains into a seed culture medium for seed culture to obtain a seed solution; Inoculating the seed solution into a fermentation medium for fermentation, and adding a carbon source and a nitrogen source during the fermentation process to obtain the fermentation broth containing lactone-type sophorolipid; The carbon sources of the seed culture medium and the fermentation medium include n-hexadecane, and ricinoleic acid and oleate hydroxylase are added as sophorolipid synthesis promoters during the fermentation process.

2. The method according to claim 1, wherein The Candida tropicalis is selected from Candida glabrata and Saccharomyces cerevisiae, preferably Candida glabrata.

3. The method according to claim 1 or 2, characterized in that, The mass fraction of n-hexadecane in the seed culture medium is 0.7-2.2%; preferably, the seed culture medium contains, by mass fraction: calcium chloride 0.4-2.2%, yeast powder 0.8-2.4%, casein peptone 1.2-4.2%, magnesium sulfate 0.9-2.6%, ammonium sulfate 0.3-0.9%, riboflavin 0.029-0.039%, glucose 3%-9%, n-hexadecane 0.7-2.2%, and the rest is water.

4. The method according to any one of claims 1-3, characterized in that, The temperature of the seed culture is 20-30°C, and the time is 40-50h; preferably, the seed culture is carried out under stirring conditions, and the stirring speed is 100-300rpm.

5. The method according to any one of claims 1-4, characterized in that The seed solution is inoculated into the fermentation medium for fermentation at an inoculation amount of 3%-6% by mass fraction.

6. The method according to any one of claims 1-5, characterized in that, The fermentation medium contains 1-3% of n-hexadecane; preferably, the fermentation tank medium contains the following components by mass fraction: p-aminobenzoic acid 0.003-0.01%, riboflavin 0.002-0.008%, pyridoxol 0.001-0.006%, yeast powder 1.5-3.5%, peptone 4.1-7.5%, dipotassium hydrogen phosphate 0.06-0.28%, potassium dihydrogen phosphate 0.18-0.24%, glucose 2%-7%, n-hexadecane 1-3%, and the rest is water.

7. The method according to any one of claims 1 to 6, characterized in that, The temperature of the fermentation is 22-29°C, preferably 24-27°C; the pH of the fermentation is 2-4, preferably 2.5-3.

5.

8. The method according to any one of claims 1-7, characterized in that, After 44-48h of fermentation, a carbon source and a nitrogen source are added. Preferably, the carbon source added during the fermentation process includes any one or more of rapeseed oil, soybean oil, glucose, and glycerol, and rapeseed oil is further preferred.

9. The method according to any one of claims 1-8, characterized in that, The nitrogen source added during the fermentation process includes any one or more of urea, ammonium sulfate, ammonium nitrate, and ammonia water, preferably urea; preferably, the carbon-nitrogen ratio is controlled to be 100-126:1 by using the carbon source and the nitrogen source.

10. The method according to any one of claims 1-9, characterized in that, After 55-60h of fermentation, 0.05%-0.08% of ricinoleic acid by the weight of the fermentation broth and 0.02%-0.04% of an aqueous solution of 0.3-2mM oleate hydroxylase by the weight of the fermentation broth are added.

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