A method for producing acidic sophorolipids

Abstract

This invention discloses a fermentation method for producing acidic sophorolipids. This method uses palm oil as the main carbon source and achieves high yields of acidic sophorolipids through feeding at different growth stages and precise adjustment of the carbon / nitrogen source concentration and ratio. Applications of the acidic sophorolipids produced by this invention include, but are not limited to, biosurfactants, environmentally friendly cleaning agents, and the biopharmaceutical field.

Description

Technical Field

[0001] This invention relates to the field of biosurfactant technology, specifically a method for producing acidic sophorolipids. Background Technology

[0002] Surfactants have wide applications in many fields such as petroleum, environmental protection, medicine, food, and cosmetics. However, traditional chemical surfactants pose environmental pollution and ecological safety problems.

[0003] Biosurfactants such as sophorolipids are gradually becoming an important alternative to chemical surfactants due to their advantages such as low toxicity, biodegradability, ecological safety, and high surface activity. Sophorolipids are glycolipid biosurfactants produced by microorganisms through fermentation using oils and sugars as carbon sources, and are mainly divided into two categories: lactone type and acid type. Among them, acid type sophorolipids have higher application potential due to their unique open-ring structure and the presence of free carboxyl groups on their fatty acid chains.

[0004] However, the current synthesis of sophorolipids has low yield and high pollution levels, and it is difficult to accurately synthesize acidic sophorolipids. Summary of the Invention

[0005] The purpose of this invention is to provide a method for producing acidic sophorolipids.

[0006] To achieve the above-mentioned objectives and solve the technical problems, this invention provides a novel method for producing acidic sophorolipids from palm oil, mainly comprising the following steps:

[0007] (A) Primary and secondary seed culture: The strain is activated and cultured to obtain primary seeds; according to a certain inoculation amount, the activated strain seeds are inoculated into secondary seed culture medium for expansion culture to obtain secondary seeds;

[0008] (B) Fermentation culture: Inoculate the seed culture prepared in step (A) into the fermentation medium (preferably at an inoculation rate of 1-10%), control the fermentation conditions, and carry out acidic sophorolipid fermentation. The cells are cultured in stages. In the early stage, when 40 < OD of the strain < 100, adjust the carbon source flow rate and carbon-nitrogen ratio to achieve the optimal growth conditions for the cells and enable them to grow rapidly. In the later stage, when OD ≥ 100, adjust the carbon source flow rate and carbon-nitrogen ratio again, and add a promoter to increase the degree of acetylation of sophorolipids, so that the cells can synthesize acidic sophorolipids. When the concentration of acidic sophorolipids no longer increases, the fermentation can be stopped.

[0009] In a preferred embodiment, the method includes the following steps:

[0010] (1). Primary seed culture: The strains preserved at low temperature are activated and cultured.

[0011] (2). Secondary seed expansion culture: According to a certain inoculation amount, the activated strain seeds in step (1) are inoculated into the secondary seed culture medium for expansion culture.

[0012] (3). Fermentation culture: The seed culture prepared in step (2) is inoculated into the fermentation culture medium, and the appropriate fermentation conditions are controlled to produce acidic sophorolipids.

[0013] In this invention, the formulations of the primary and secondary seed culture media are as follows: yeast powder 9-20 g / L, casein peptone 14-40 g / L, magnesium sulfate 6-27 g / L, ammonium sulfate hydrochloride 3-9 g / L, and palm oil 5-15 g / L.

[0014] In one specific embodiment of the present invention, the strain described in step (1) is selected from any one or more of *Candida thymosinii* or *Candida tropicalis*, preferably *Candida tropicalis*.

[0015] The primary seed culture in step (1) can be carried out using conventional methods in the art. For example, the strain preserved at low temperature is placed in a container containing YPD medium and activated at 180-250 rpm and 27-32°C. An OD600 between 3 and 8 indicates that the strain has matured. The volume ratio of the strain to the YPD medium is preferably 3% to 8%.

[0016] In a specific implementation, preferably, the method of primary seed culture in step (1) is as follows: take 0-8 mL of glycerol tube culture (stored at -20℃) into a 500 mL Erlenmeyer flask containing 50-100 mL of YPD medium, and culture it in a reciprocating shaker at 180-250 rpm and 27-32℃ for about 8-9 hours. An OD600 between 3 and 8 indicates that the growth is mature.

[0017] In this invention, the secondary seed expansion culture in step (2) involves inoculating the activated seed liquid from step (1) into a container containing secondary seed culture medium at an inoculation rate of 3% to 8%, and culturing at a constant temperature of 27-32℃. An OD600 between 5 and 12 indicates that the seed liquid has matured.

[0018] In a specific implementation, preferably, the seed propagation process in step (2) includes: inoculating the activated seed liquid from step (1) into a 500mL Erlenmeyer flask containing 50-100mL of secondary seed culture medium at an inoculation rate of 3% to 8%, and incubating at a constant temperature of 27-32℃ for 15-20h. An OD600 between 5 and 12 indicates that the plant is mature.

[0019] In this invention, the fermentation culture described in step (3) involves inoculating the seed liquid cultured in step (2) into the fermentation medium at an inoculation rate of 1-10%, preferably 3-8%, and controlling the fermentation conditions by using segmented culture.

[0020] When the bacterial strain 40 < OD < 100, a certain amount of carbon and nitrogen sources are added to the fermentation broth, and the carbon source is controlled to be added at 0.02-0.07% of the total mass of the fermentation system per hour. The most suitable carbon-nitrogen ratio for carbon and nitrogen source regulation is 5-15:1. The flow rate of nitrogen source is adjusted according to the carbon-nitrogen ratio to promote rapid bacterial growth.

[0021] When the OD of the strain is ≥100, the carbon source is controlled at 0.07-0.20% of the total mass of the fermentation system per hour. The optimal carbon-nitrogen ratio for carbon and nitrogen sources is 15-30:1. The nitrogen source flow rate is adjusted according to the carbon-nitrogen ratio.

[0022] Preferably, when the OD of the strain is ≥100, 0.05-0.2% of chloroacetyl is added simultaneously to the fermentation system to promote the acetylation of the product, increase the degree of acetylation of sophorolipid, and thus generate acidic sophorolipid.

[0023] More preferably, the fermentation medium is formulated as follows: KH2PO4 1.8-2.4 g / L, palm oil 25-50 g / L, inositol 0.02-0.1 g / L, pantothenic acid 0.05-0.1 g / L, p-aminobenzoic acid 0.03-0.1 g / L, betaine 6.5-18 g / L, ammonium sulfate 22-65 g / L, with water as the solvent, preferably with a pH of 6.8-7.2.

[0024] More preferably, the carbon source is palm oil, and the nitrogen source is an aqueous solution of one or more of ammonium sulfate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, and ammonium chloride, with a mass percentage of 10-30%.

[0025] More preferably, the fermentation process is controlled at a temperature of 28-37℃ and a pH of 6.5-7, with the entire process being aerobic fermentation.

[0026] The concentration of acidic sophorolipid in the fermentation broth is monitored every hour during the fermentation process. The fermentation can be stopped when the concentration of acidic sophorolipid no longer increases for 2 consecutive hours.

[0027] The applications of the acidic sophorolipid produced by this invention include, but are not limited to, biosurfactants, environmentally friendly cleaning agents, and the biopharmaceutical field.

[0028] The beneficial effects of this invention are:

[0029] 1. Utilizing palm oil as the main carbon source in the fermentation process to achieve efficient production of acidic sophorolipids.

[0030] 2. By adjusting the concentration and ratio of carbon and nitrogen sources, the fermentation conditions can be optimized to improve the yield of acidic sophorolipids.

[0031] 3. During the later stages of fermentation, chloroacetyl is added as a promoter to induce acetylation of the product, increase the degree of acetylation of sophorolipids, and thus generate acidic sophorolipids.

[0032] 4. This method is scalable and suitable for industrial-scale production. Detailed Implementation

[0033] To facilitate understanding of the present invention, the following description, in conjunction with embodiments, will further illustrate the invention. It should be understood that the following embodiments are merely for a better understanding of the invention and do not imply that the invention is limited to these embodiments.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0035] 1. OD detection

[0036] Bacterial cell OD detection method: Dilute the sample to the specified range and perform detection using a spectrophotometer at a wavelength of 600 nm.

[0037] 2. LC-MS / MS detection of acidic sophorolipids

[0038] 1) Sample: The fermentation broth was extracted with ethyl acetate, desolventized, dissolved in chloroform / methanol (1:3, v / v), and filtered through a 0.22 μm filter membrane before testing.

[0039] 2) Instrument: Q-TOF high-resolution liquid chromatography-mass spectrometry system

[0040] 3) Chromatographic separation

[0041] C18 (2.1*150mm, 5µm) column, flow rate 0.2ml / min, separation temperature 35℃, separation gradient: 0-3min, 40% B-60% B, 3-20min, 60% B-88% B, 22min, 100% B (A: water, containing 0.1% formic acid; B: acetonitrile, containing 0.1% formic acid)

[0042] 4) ESI-MS identification: positive ion scan, scan range (m / z): 50–1500, scan mode: AUTO MS / MS, nebulizer gas pressure: 15psi, dryer gas flow rate: 9L / min, dryer gas temperature: 180℃.

[0043] The sophorolipid composition in the sample is quite complex. The different lengths, saturation levels, and positions and degrees of acetylation of fatty acid chains result in a variety of sophorolipid structures. During MS / MS detection, ester bonds are easily broken, and the corresponding fragment ions generated after the breakage of sophorose residues, fatty acid residues, and glycosidic bonds can be measured. By analyzing the molecular information of these fragment ions, the degree of glycosylation and the types of fatty acids can be determined, thereby determining its chemical structure.

[0044] The strains used were Wickerhamierlla domercqiae var. sophorolipid (CGMCC No. 1576) and Candida tropicalis (China Industrial Microbial Culture Collection Center, No. CICC 32626).

[0045] Example 1:

[0046] 1. Select Candida tropicalis as the strain for culture. Primary seed culture: Take 4 mL of glycerol tube of inoculum (stored at -20℃) into a 500 mL Erlenmeyer flask containing 50 mL of YPD medium, and culture at 27℃ with a reciprocating shaker at 180 rpm and an amplitude of about 8 cm until the OD600 is 8.

[0047] 2. Seed propagation: After the primary seeds mature, they are inoculated into the secondary shake flask seed culture medium at an inoculation rate of 3% and cultured at a constant temperature of 27℃ and 200rpm until the OD600 reaches 10.

[0048] The seed culture medium formula is as follows: yeast powder 15g / L, casein peptone 28g / L, magnesium sulfate 25g / L, ammonium sulfate hydrochloride 8g / L, and palm oil 10g / L.

[0049] 3. Fermentation culture: Prepare a 50L fermenter with an initial liquid volume of 25L. Inoculate the mature shake flask seed cultured in step 2 into the sterile fermentation medium at an inoculation rate of 5% for fermentation. The fermentation temperature is 29℃, the pH is controlled at 6.8 with ammonia water, and the tank pressure is 0.02~0.1Mpa.

[0050] The fermentation medium has the following formula: KH2PO4 2.2g / L, palm oil 30g / L, inositol 0.08g / L, pantothenic acid 0.07g / L, para-aminobenzoic acid 0.08g / L, betaine 10g / L, and ammonium sulfate 22g / L.

[0051] 4. Start fermentation. When the inoculum size is 40 < OD < 100, add a certain amount of carbon and nitrogen sources to the fermentation broth. The carbon source is added at a rate of 0.05% of the total mass of the fermentation system per hour, with a carbon-to-nitrogen ratio of 10:1. Adjust the nitrogen source flow rate according to the carbon-to-nitrogen ratio. The carbon source is palm oil, and the nitrogen source is 30% ammonium sulfate by mass.

[0052] When the OD of the microbial strain is ≥100, the carbon source is controlled to be added at 0.07% of the total mass of the fermentation system per hour, and the carbon-nitrogen ratio is 15:1. The flow rate of the nitrogen source is adjusted according to the carbon-nitrogen ratio.

[0053] When the bacterial strain OD≥100, 0.05% chloroacetyl is added simultaneously.

[0054] The concentration of acidic sophorolipid in the fermentation broth is monitored every hour. The fermentation can be stopped when the concentration of acidic sophorolipid no longer increases for 2 consecutive hours.

[0055] 5. After testing and calculation, the maximum concentration of acidic sophorolipid was 180 g / L, and the conversion rate of the carbon source palm oil reached 86%.

[0056] Example 2:

[0057] 1. Select *Saccharomyces cerevisiae* as the strain for culture. Primary seed culture: Take 4 mL of glycerol tube culture (stored at -20℃) and place it in a 500 mL Erlenmeyer flask containing 50 mL of YPD medium. Incubate at 27℃ with a reciprocating shaker at 180 rpm and an amplitude of about 8 cm until the OD600 reaches 4.

[0058] 2. Secondary seed propagation: After the primary seeds mature, inoculate them into the secondary shake flask seed culture medium at a 5% inoculation rate and culture at 30℃ and 200rpm until the OD600 reaches 8;

[0059] The seed culture medium formula is as follows: yeast powder 10g / L, casein peptone 36g / L, magnesium sulfate 12g / L, ammonium sulfate hydrochloride 4g / L, and palm oil 8g / L.

[0060] 3. Fermentation culture: Prepare a 50L fermenter with an initial liquid volume of 25L. Inoculate the mature shake flask seed cultured in step 2 into the sterile fermentation medium at an inoculation rate of 7% for fermentation. The fermentation temperature is 35℃, the pH is controlled at 7 with ammonia water, and the tank pressure is 0.02~0.1Mpa.

[0061] The fermentation medium has the following formula: KH2PO4 1.9g / L, palm oil 45g / L, inositol 0.05g / L, pantothenic acid 0.06g / L, para-aminobenzoic acid 0.05g / L, betaine 16g / L, and ammonium sulfate 60g / L.

[0062] 4. Start fermentation. When the inoculum size is 40 < OD < 100, add a certain amount of carbon and nitrogen sources to the fermentation broth. The carbon source is added at a rate of 0.03% of the total mass of the fermentation system per hour, with a carbon-to-nitrogen ratio of 7:1. Adjust the nitrogen source flow rate according to the carbon-to-nitrogen ratio. The carbon source is palm oil, and the nitrogen source is 10% diammonium hydrogen phosphate by mass.

[0063] When the OD of the microbial strain is ≥100, the carbon source is controlled to be added at 0.08% of the total mass of the fermentation system per hour, and the carbon-nitrogen ratio is 25:1. The nitrogen source flow rate is adjusted according to the carbon-nitrogen ratio.

[0064] When the bacterial strain OD≥100, chloroacetyl at a final mass ratio of 0.15% should be added simultaneously.

[0065] The concentration of acidic sophorolipid in the fermentation broth is monitored every hour. The fermentation can be stopped when the concentration of acidic sophorolipid no longer increases for 2 consecutive hours.

[0066] 5. After testing and calculation, the maximum concentration of acidic sophorolipid was 181 g / L, and the palm oil conversion rate reached 88%.

[0067] Example 3:

[0068] 1. Primary seed culture: Same as step 1 in Example 1.

[0069] 2. Secondary seed propagation: After the primary seeds mature, they are inoculated into the secondary shake flask seed culture medium at an inoculation rate of 8%, and cultured at a constant temperature of 32℃ and 200rpm until the OD600 reaches 10.

[0070] The seed culture medium formula is as follows: yeast extract 9 g / L, casein peptone 35 g / L, magnesium sulfate 8 g / L, ammonium sulfate hydrochloride 5 g / L, and palm oil 14 g / L.

[0071] 3. Fermentation culture: Prepare a 50L fermenter with an initial liquid volume of 25L. Inoculate the mature shake flask seed cultured in step 2 into the sterile fermentation medium at an inoculation rate of 8% for fermentation. The fermentation temperature is 37℃, the pH is controlled at 6.9 with ammonia water, and the tank pressure is 0.02~0.1Mpa.

[0072] The fermentation medium has the following formula: KH2PO4 2.4g / L, palm oil 26g / L, inositol 0.09g / L, pantothenic acid 0.06g / L, para-aminobenzoic acid 0.05g / L, betaine 16g / L, and ammonium sulfate 60g / L.

[0073] 4. Start fermentation. When the inoculum size is 40 < OD < 100, add a certain amount of carbon and nitrogen sources to the fermentation broth. Control the carbon source to be added at a rate of 0.06% of the total mass of the fermentation system per hour, with a carbon-to-nitrogen ratio of 10:1. Adjust the nitrogen source flow rate according to the carbon-to-nitrogen ratio. The carbon source is palm oil, and the nitrogen source is 20% ammonium chloride by mass.

[0074] When the OD of the microbial strain is ≥100, the carbon source is controlled to be added at 0.1% of the total mass of the fermentation system per hour, and the carbon-nitrogen ratio is 20:1. The flow rate of the nitrogen source is adjusted according to the carbon-nitrogen ratio.

[0075] When the bacterial strain OD≥100, 0.1% chloroacetyl is added simultaneously.

[0076] The concentration of acidic sophorolipid in the fermentation broth is monitored every hour. The fermentation can be stopped when the concentration of acidic sophorolipid no longer increases for 2 consecutive hours.

[0077] 5. After testing and calculation, the maximum concentration of acidic sophorolipid was 188 g / L, and the palm oil conversion rate reached 90%.

[0078] Comparative Example 1:

[0079] 1. Primary seed culture: Same as step 1 in Example 1.

[0080] 2. Secondary seed propagation: Same as step 2 in Example 1.

[0081] 3. Fermentation culture: Same as step 3 in Example 1.

[0082] 4. Start fermentation. When the inoculum size is 40 < OD < 100, add a certain amount of carbon and nitrogen sources to the fermentation broth, and control the carbon source to be added at 0.05% of the total mass of the fermentation system per hour, with a carbon-nitrogen ratio of 15:1.

[0083] The carbon source is palm oil, and the nitrogen source is 30% ammonium sulfate by mass.

[0084] When the bacterial strain OD≥100, the carbon source flow rate and carbon-nitrogen ratio remain unchanged.

[0085] When the bacterial strain OD≥100, 0.1% chloroacetyl is added simultaneously.

[0086] The concentration of acidic sophorolipid in the fermentation broth is monitored every hour. The fermentation can be stopped when the concentration of acidic sophorolipid no longer increases for 2 consecutive hours.

[0087] 5. After testing and calculation, the maximum concentration of acidic sophorolipid was 72 g / L, and the palm oil conversion rate reached 41%.

[0088] Comparative Example 2:

[0089] 1. Primary seed culture: Same as step 1 in Example 1.

[0090] 2. Secondary seed propagation: Same as step 2 in Example 1.

[0091] 3. Fermentation culture: Same as step 3 in Example 1.

[0092] 4. Start fermentation. When the inoculum size is 40 < OD < 100, add a certain amount of carbon and nitrogen source to the fermentation broth, controlling the carbon source to be added at a rate of 0.05% of the total mass of the fermentation system per hour, with a carbon-to-nitrogen ratio of 10:1.

[0093] The carbon source is palm oil, and the nitrogen source is 30% ammonium sulfate by mass.

[0094] When the OD of the microbial strain is ≥100, the carbon source is controlled at 0.07% of the total mass of the fermentation system per hour, and the carbon-nitrogen ratio is adjusted to 15:1. The flow rate of the nitrogen source is adjusted according to the carbon-nitrogen ratio.

[0095] When the bacterial culture OD≥100, add water at a final mass ratio of 0.05%.

[0096] The concentration of acidic sophorolipid in the fermentation broth is monitored every hour. The fermentation can be stopped when the concentration of acidic sophorolipid no longer increases for 2 consecutive hours.

[0097] 5. After testing and calculation, the maximum concentration of acidic sophorolipid was 68 g / L, and the palm oil conversion rate reached 44%.

[0098] It is readily understood that the above embodiments are merely illustrative examples for clear explanation and do not imply that the invention is limited thereto. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for producing acidic sophorolipids, characterized in that, The method includes the following steps: (1) Primary and secondary seed culture: The strain is activated and cultured into primary seeds; according to a certain inoculation amount, the activated strain seeds are inoculated into secondary seed culture medium and expanded to obtain secondary seeds; wherein, the strain is selected from Wicke yeast CGMCC No.1576 or Candida tropicalis CICC32626; (2) Fermentation culture: The seed liquid cultured in step (1) is inoculated into the fermentation medium, and the fermentation conditions are controlled to carry out acidic sophorolipid fermentation; The bacterial cells were cultured in stages. In the early stage, when 40 < bacterial OD < 100, a certain amount of carbon and nitrogen sources were added to the fermentation broth, and the carbon source was fed at 0.02-0.07% of the total mass of the fermentation system per hour, with a carbon-to-nitrogen ratio of 5-15:

1. The nitrogen source flow rate was adjusted according to the carbon-to-nitrogen ratio to promote bacterial growth. In the later stage, when OD ≥ 100, the carbon source was fed at 0.07-0.20% of the total mass of the fermentation system per hour, with a carbon-to-nitrogen ratio of 15-30:

1. The nitrogen source flow rate was adjusted according to the carbon-to-nitrogen ratio, and the promoter chloroacetyl was added to increase the acetylation degree of sophorolipids, enabling the cells to synthesize acidic sophorolipids. The carbon source was palm oil.

2. The method as described in claim 1, characterized in that, Step (1) The steps for primary seed culture are as follows: Take the strain preserved at low temperature and place it in a container containing YPD medium. Activate it at 180-250 rpm and 27-32℃. An OD600 between 3 and 8 indicates that the strain has matured.

3. The method as described in claim 2, characterized in that, In step (1), the volume ratio of the strain to YPD medium is 3%-8%.

4. The method according to claim 1, characterized in that, The steps for the secondary seed culture in step (1) are as follows: the activated primary seed solution from step (1) is inoculated into a container containing secondary seed culture medium at an inoculation rate of 3%-8%, and cultured at 27-32℃. An OD600 between 5 and 12 indicates that the seed culture is mature.

5. The method according to any one of claims 1-4, characterized in that, Step (1) The seed culture medium formula is: yeast powder 9-20g / L, casein peptone 14-40g / L, magnesium sulfate 6-27g / L, ammonium sulfate hydrochloride 3-9g / L, palm oil 5-15g / L.

6. The method as described in claim 1, characterized in that, In step (2), administer the vaccine at a dose of 1-10%.

7. The method according to claim 1, characterized in that, In step (2), the fermentation medium formula is: KH2PO4 1.8-2.4 g / L, palm oil 25-50 g / L, inositol 0.02-0.1 g / L, pantothenic acid 0.05-0.1 g / L, p-aminobenzoic acid 0.03-0.1 g / L, betaine 6.5-18 g / L, ammonium sulfate 22-65 g / L, and water as the solvent.

8. The method according to any one of claims 1, 6-7, characterized in that, In step (2), the fermentation temperature is 28-37℃ and the fermentation pH is 6.5-7.

0.

9. The method according to claim 1, characterized in that, In step (2), the nitrogen source is one or more of the following: ammonium sulfate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, and ammonium chloride aqueous solution, with a mass percentage of 10-30%.

10. The method according to claim 1, characterized in that, In step (2), the amount of the promoter added is 0.05-0.2% of the mass of the fermentation system.