Method for producing acid sophorolipid

Abstract

The invention discloses a fermentation production method of acid type sophorolipid, which is characterized in that palm oil is used as a main carbon source, and high yield of the acid type sophorolipid is realized by supplementing materials at different growth stages and accurately adjusting the concentration and proportion of a carbon source / nitrogen source. The application of the produced acid type sophorolipid includes but is not limited to the fields of biological surfactants, environment-friendly cleaning agents and biological medicines.

Description

Technical Field

[0001] The invention relates to the technical field of biosurfactants, in particular to a method for producing acid-type sophorolipids. Background Art

[0002] Surfactants are widely used in many fields such as petroleum, environmental protection, medicine, food, cosmetics, etc. However, traditional chemical surfactants have environmental pollution and ecological safety issues.

[0003] Biosurfactants such as sophorolipids have gradually become an important alternative to chemical surfactants due to their low toxicity, biodegradability, ecological safety and high surface activity. Sophorolipids are glycolipid biosurfactants produced by microorganisms 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 free carboxyl groups on the fatty acid chain.

[0004] However, the current synthesis of sophorolipids has a low yield and is highly polluting, and it is difficult to accurately synthesize acid-type sophorolipids. Summary of the invention

[0005] The object of the present invention is to provide a method for producing acid-type sophorolipids.

[0006] In order to achieve the above-mentioned purpose of the invention and solve the technical problem, the present invention provides a novel method for producing acid-type sophorolipids from palm oil, which mainly comprises the following steps:

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

[0008] (B) Fermentation culture: The seed liquid cultured in step (A) is inoculated (preferably at an inoculation amount of 1-10%) into the fermentation medium, and the fermentation conditions are controlled to carry out acid-type sophorolipid fermentation, wherein the bacteria are cultured in stages, and in the early stage when 40 < strain OD < 100, the carbon source flow rate and the carbon-nitrogen ratio are adjusted to achieve the optimal growth conditions of the bacteria, so that the bacteria grow at a high speed, and in the later stage when OD ≥ 100, the different carbon source flow rates and carbon-nitrogen ratios are adjusted again, and a promoter is added to increase the acetylation degree of sophorolipid, so that the bacteria synthesize acid-type sophorolipid, and the tank can be stopped when the concentration of acid-type sophorolipid no longer increases.

[0009] As a preferred embodiment, the method comprises the following steps:

[0010] (1) Primary seed culture: Activate and culture the strains stored at low temperatures.

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

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

[0013] In the present invention, the formula of the primary and secondary seed culture medium is: 9-20 g / L yeast powder, 14-40 g / L casein peptone, 6-27 g / L magnesium sulfate, 3-9 g / L ammonium sulfate hydrochloride, and 5-15 g / L palm oil.

[0014] In the present invention, in a specific embodiment, the strain in step (1) is selected from any one or more of Pseudomonas aeruginosa or Candida tropicalis, preferably Candida tropicalis;

[0015] The primary seed culture in step (1) can be carried out by conventional methods in the art, for example, taking the strain stored at low temperature into a container filled with YPD medium, and activating the culture at 180-250 rpm and 27-32° C., and an OD600 between 3 and 8 indicates that the strain has grown to maturity; wherein the volume ratio of the strain to the YPD medium is preferably 3% to 8%;

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

[0017] In the present invention, the secondary seed expansion culture in step (2) is to inoculate the seed liquid activated in step (1) into a container containing a secondary seed culture medium at an inoculum amount of 3% to 8%, and culture at a constant temperature of 27-32° C., and an OD600 between 5 and 12 indicates growth maturity;

[0018] In a specific embodiment, preferably, the seed expansion process of step (2) includes: inoculating the seed liquid activated in step (1) into a 500mL conical flask containing 50-100mL of secondary seed culture medium at an inoculation rate of 3%-8%, culturing at a constant temperature of 27-32°C for 15-20h, and OD600 between 5 and 12 indicates growth maturity.

[0019] In the present invention, the fermentation culture in step (3) is to inoculate the seed liquid cultured in step (2) into the fermentation medium at an inoculation rate of 1-10%, preferably 3-8%, to carry out fermentation culture, control the fermentation conditions, and adopt segmented culture;

[0020] Among them, when the strain is 40<OD<100, a certain carbon source and nitrogen source are added to the fermentation liquid, and the carbon source is controlled to be 0.02-0.07% of the total mass of the fermentation system per hour, and the most suitable carbon-nitrogen ratio of the carbon source and nitrogen source is 5-15:1, and the nitrogen source flow rate is adjusted according to the carbon-nitrogen ratio; so that the bacteria grow at a high speed;

[0021] When the strain OD is ≥100, the carbon source is controlled to be 0.07-0.20% of the total mass of the fermentation system per hour, and the most suitable carbon-nitrogen ratio of the carbon source and nitrogen source 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 by mass of the fermentation system is added simultaneously to promote the acetylation of the product, increase the acetylation degree of sophorolipid, and then generate acid-type sophorolipid.

[0023] More preferably, the fermentation medium formula is: KH 2 PO 4 1.8-2.4g / L, palm oil 25-50g / L, inositol 0.02-0.1g / L, pantothenic acid 0.05-0.1g / L, p-aminobenzoic acid 0.03-0.1g / L, betaine 6.5-18g / L, ammonium sulfate 22-65g / L, the solvent is water, preferably, pH 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°C and a Ph of 6.5-7, and the entire process is aerobic fermentation.

[0026] During the fermentation process, the concentration of acid-type sophorolipids in the fermentation broth was tested every hour, and the fermentation was stopped when the concentration of acid-type sophorolipids did not increase for 2 consecutive hours.

[0027] Applications of the acid-type sophorolipid produced by the present invention include but are not limited to biosurfactants, environmentally friendly cleaning agents and biomedicine.

[0028] Beneficial effects of the present invention:

[0029] 1. Use palm oil as the main carbon source in the fermentation process to achieve efficient production of acid-type sophorolipids.

[0030] 2. By adjusting the concentration and ratio of carbon source and nitrogen source, the fermentation conditions are optimized and the yield of acid-type sophorolipids is increased.

[0031] 3. In the later stage of fermentation, chloroacetyl is added as a promoter to promote the acetylation of the product, increase the acetylation degree of sophorolipids, and then generate acid-type sophorolipids.

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

[0033] In order to facilitate the understanding of the present invention, the present invention will be further described below in conjunction with examples. It should be understood that the following examples are only for a better understanding of the present invention and do not mean that the present invention is limited to the following examples.

[0034] Unless defined otherwise, 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 belongs.

[0035] 1.OD detection

[0036] Bacteria OD detection method: dilute the sample to within the range and detect it with a spectrophotometer at a wavelength of 600nm

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

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

[0039] 2) Instrument: Q-TOF high-resolution liquid-mass spectrometer

[0040] 3) Chromatographic separation

[0041] C18 (2.1*150mm, 5um) column, 0.2ml / min flow rate, separation temperature 35°C, 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 scanning, 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.

[0043] The composition of sophorolipids in the sample is relatively complex. Different lengths, saturation, positions and degrees of acetylation of fatty acid chains will produce a variety of sophorolipid structures. The ester bond is easily broken during MS / MS detection, and the corresponding fragment ions produced 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 glycosyl acetylation and the type of fatty acid can be determined, thereby determining its chemical structure.

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

[0045] Embodiment 1:

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

[0047] 2. Seed expansion: After the primary seeds mature, inoculate them into the secondary shake flask seed culture medium at a 3% inoculation rate and culture them at 27°C and 200rpm until the OD600 reaches 10;

[0048] Among them, the seed culture medium formula is: 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 fermentation tank with an initial liquid volume of 25L, inoculate the mature shake flask seeds cultured in step 2 into a sterile fermentation medium at a 5% inoculation rate for fermentation at a fermentation temperature of 29°C, ammonia water to control pH 6.8, and a tank pressure of 0.02-0.1Mpa;

[0050] Fermentation medium, formula: KH 2 PO 4 2.2g / L, palm oil 30g / L, inositol 0.08g / L, pantothenic acid 0.07g / L, p-aminobenzoic acid 0.08g / L, betaine 10g / L, ammonium sulfate 22g / L.

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

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

[0053] When the OD of the strain was ≥100, 0.05% chloroacetyl was added at the same time.

[0054] The concentration of acid-type sophorolipids in the fermentation broth was tested every hour, and the fermentation process was stopped when the concentration of acid-type sophorolipids did not increase for 2 consecutive hours.

[0055] 5. After testing and calculation, the maximum concentration of acid-type sophorolipids is 180g / L, and the conversion rate of carbon source palm oil reaches 86%.

[0056] Embodiment 2:

[0057] 1. Select Pseudomonas aeruginosa as the strain for cultivation. Primary seed culture: take 4 mL of glycerol tube strain (stored at -20°C) into a 500 mL Erlenmeyer flask containing 50 mL of YPD medium, and culture on a reciprocating shaker at 180 rpm, with an amplitude of about 8 cm, at 27°C until OD600 is 4.

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

[0059] Among them, the seed culture medium formula is: 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 fermentation tank with an initial liquid volume of 25L, inoculate the mature shake flask seeds cultured in step 2 at a 7% inoculation rate, and inoculate into a sterile fermentation medium for fermentation at a fermentation temperature of 35°C, ammonia water to control pH7, and a tank pressure of 0.02-0.1Mpa;

[0061] Fermentation medium, formula: KH 2 PO 4 1.9g / L, palm oil 45g / L, inositol 0.05g / L, pantothenic acid 0.06g / L, p-aminobenzoic acid 0.05g / L, betaine 16g / L, ammonium sulfate 60g / L.

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

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

[0064] When the OD of the strain was ≥100, 0.15% of chloroacetyl was added at the same time.

[0065] The concentration of acid-type sophorolipids in the fermentation broth was tested every hour, and the fermentation process was stopped when the concentration of acid-type sophorolipids did not increase for 2 consecutive hours.

[0066] 5. After testing and calculation, the maximum concentration of acid-type sophorolipids is 181g / L, and the palm oil conversion rate reaches 88%.

[0067] Embodiment 3:

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

[0069] 2. Secondary seed expansion: After the primary seeds mature, inoculate them into the secondary shake flask seed culture medium at an inoculum rate of 8%, and culture them at a constant temperature of 32°C and 200rpm until the OD600 reaches 10;

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

[0071] 3. Fermentation culture: prepare a 50L fermentation tank with an initial liquid volume of 25L, inoculate the mature shake flask seeds cultured in step 2 into a sterile fermentation medium at an inoculation rate of 8% for fermentation at a fermentation temperature of 37°C, ammonia water to control pH 6.9, and a tank pressure of 0.02-0.1Mpa;

[0072] Fermentation medium, formula: KH2PO4 2.4g / L, palm oil 26g / L, inositol 0.09g / L, pantothenic acid 0.06g / L, p-aminobenzoic acid 0.05g / L, betaine 16g / L, ammonium sulfate 60g / L.

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

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

[0075] When the OD of the strain was ≥100, 0.1% chloroacetyl was added at the same time.

[0076] The concentration of acid-type sophorolipids in the fermentation broth was tested every hour, and the fermentation process was stopped when the concentration of acid-type sophorolipids did not increase for 2 consecutive hours.

[0077] 5. After testing and calculation, the maximum concentration of acid-type sophorolipids is 188g / L, and the palm oil conversion rate reaches 90%.

[0078] Comparative Example 1:

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

[0080] 2. Secondary seed expansion: same as step 2 in Example 1.

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

[0082] 4. Start fermentation. When the strain OD is 40<100, add a certain amount of carbon source and nitrogen source to the fermentation liquid, and control the carbon source to 0.05% of the total mass of the fermentation system per hour, and the carbon-nitrogen ratio is 15:1.

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

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

[0085] When the OD of the strain was ≥100, 0.1% chloroacetyl was added at the same time.

[0086] The concentration of acid-type sophorolipids in the fermentation broth was tested every hour, and the fermentation process was stopped when the concentration of acid-type sophorolipids did not increase for 2 consecutive hours.

[0087] 5. After testing and calculation, the maximum concentration of acid-type sophorolipids is 72g / L, and the palm oil conversion rate reaches 41%.

[0088] Comparative Example 2:

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

[0090] 2. Secondary seed expansion: same as step 2 in Example 1.

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

[0092] 4. Start fermentation. When the strain is 40<OD<100, add a certain amount of carbon source and nitrogen source to the fermentation liquid, and control the carbon source to 0.05% of the total mass of the fermentation system per hour, and the carbon-nitrogen ratio is 10:1.

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

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

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

[0096] The concentration of acid-type sophorolipids in the fermentation broth was tested every hour, and the fermentation process was stopped when the concentration of acid-type sophorolipids did not increase for 2 consecutive hours.

[0097] 5. After testing and calculation, the maximum concentration of acid-type sophorolipids is 68g / L, and the palm oil conversion rate reaches 44%.

[0098] It is easy to understand that the above embodiments are only examples for clear explanation and do not mean that the present invention is limited thereto. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from this are still within the protection scope of the present invention.

Claims

1. A method for producing acid-type sophorolipids, characterized in that: The method comprises the following steps: (1) Primary and secondary seed culture: The strain is activated and cultured to obtain primary seeds; the activated strain seeds are inoculated into secondary seed culture medium according to a certain inoculation amount, and expanded and cultured to obtain secondary seeds; (2) Fermentation culture: inoculating the seed solution cultured in step (1) (preferably at an inoculation rate of 1-10%) into the fermentation medium, controlling the fermentation conditions, and performing acid-type sophorolipid fermentation; Among them, the bacteria are cultured in stages. In the early stage, when 40 < bacterial OD < 100, the carbon source flow rate and the carbon-nitrogen ratio are adjusted to make the bacteria grow. In the later stage, when OD ≥ 100, the carbon source flow rate and the carbon-nitrogen ratio are adjusted again, and a promoter is added to increase the acetylation degree of sophorolipids so that the bacteria can synthesize acid-type sophorolipids.

2. The method according to claim 1, characterized in that The strain described in step (1) is selected from Pseudomonas aeruginosa or Candida tropicalis.

3. The method according to claim 1, characterized in that The step (1) of primary seed culture is as follows: taking the strain stored at low temperature into a container filled with YPD medium, and activating the culture at 180-250rpm and 27-32°C, and an OD600 between 3 and 8 indicates mature growth; wherein, preferably, the volume ratio of the strain to the YPD medium is 3%-8%.

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

5. The method according to any one of claims 1 to 4, characterized in that: The seed culture medium formula of step (1) is: 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.

6. 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 the solvent is water.

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

0.

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

9. The method according to claim 1 or 8, characterized in that: In step (2), when the bacterial strain 40<OD<100, a certain carbon source and nitrogen source are added to the fermentation liquid, and the carbon source is controlled to be 0.02-0.07% of the total mass of the fermentation system per hour, and the carbon-nitrogen ratio is 5-15:1, and the nitrogen source flow rate is adjusted according to the carbon-nitrogen ratio; When the strain OD is ≥100, the carbon source is controlled to be 0.07-0.20% of the total mass of the fermentation system per hour, the carbon-nitrogen ratio is 15-30:1, and the nitrogen source flow rate is adjusted according to the carbon-nitrogen ratio.

10. The method according to any one of claims 1 to 9, characterized in that: In step (2), the promoter is chloroacetyl, and preferably, the added amount is 0.05-0.2% by mass of the fermentation system.