Fermentation process for efficiently preparing rhamnolipid

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a fermentation process for efficiently preparing rhamnolipid. The fermentation process comprises the following steps: inoculating pseudomonas aeruginosa into an enrichment culture medium, activating and culturing for 18-24 hours to obtain a seed bacteria solution, inoculating the seed bacteria solution into a sterilized fermentation culture medium, fermenting for 90-180 hours in an aerobic environment at 25-37 DEG C, and regulating the pH in the fermentation process to obtain the pseudomonas aeruginosa strain. At the same time, adding a fermentation accelerator within 20-40th h after fermentation, and starting to supplement a carbon source and a nitrogen source at 40th h to adjust the carbon-nitrogen ratio in the fermentation medium to obtain a fermentation broth; and separating and purifying the obtained fermentation liquor to obtain the rhamnolipid. According to the method, the specific fermentation strain, the fermentation culture medium and parameter conditions in the fermentation process are selected, and the defoaming agent and the fermentation accelerant are added in the fermentation process, so that the pseudomonas aeruginosa can secrete high-yield rhamnolipid, and the production cost of the rhamnolipid is reduced to a certain extent.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and particularly relates to a fermentation process for efficiently preparing rhamnolipid. Background Art

[0002] Biosurfactant lipids are widely used in the cleaning field. They have various excellent properties, such as low toxicity, high biocompatibility, biodegradability, etc., making them ideal cleaning ingredients. Biosurfactants specifically refer to glycolipid raw materials obtained by fermentation of specific microorganisms. Currently, the two most main commercial biosurfactants are rhamnolipid and sophorolipid, with rhamnose and sophorose as the sugar structural units respectively. The application fields of biosurfactant lipids are very extensive, such as detergents and cleaning products, cosmetics, environmental remediation, petroleum industry, agriculture, food industry and pharmaceutical field.

[0003] In recent years, many related studies have been devoted to the production of biosurfactant lipids. Patent CN117946914A discloses a high-yield rhamnolipid-producing strain, a bacterial agent, a method for producing rhamnolipid and its application. The high-yield rhamnolipid-producing strain used is Pseudomonas aeruginosa, with the preservation number of CCTCC NO: M20232111. By genetic engineering, regulating the fermentation medium and fermentation conditions, the yield of rhamnolipid reaches 10.9 g / L at the 72nd hour. Patent CN 117924387 A discloses a method for separating and purifying rhamnolipid. The method comprises the following steps: acid-hydrolyzing the rhamnolipid fermentation broth to obtain an acid-hydrolyzed solution; adding an organic solvent to the acid-hydrolyzed solution to obtain an extraction solution, and separating the organic solvent phase, the emulsion phase and the water phase by phase separation; adding an alkali solution to treat the organic solvent phase, and concentrating the obtained water phase by phase separation into an aqueous rhamnolipid solution; demulsifying the emulsion phase, separating and concentrating to obtain a low-quality aqueous rhamnolipid solution. And two different-quality rhamnolipid products can be recovered simultaneously. Although the above-mentioned related studies improve the yield of rhamnolipid by optimizing fermentation conditions and using corresponding strains, further improving production efficiency and reducing production costs are still the focus of research. Moreover, compared with chemically synthesized surfactants, the production cost of rhamnolipid is relatively high, which greatly limits its wide application.

[0004] Therefore, reducing production costs is one of the key factors to promote the commercialization of rhamnolipid. Developing a method that can reduce the production cost of rhamnolipid is of great significance for the development of the industry and the application of biosurfactants. Summary of the Invention

[0005] The present invention discloses a fermentation process for efficiently preparing rhamnolipids. The fermentation process involves inoculating a seed culture solution of Pseudomonas aeruginosa into a fermentation medium, fermenting under certain fermentation conditions, and adjusting the fermentation parameters. The obtained fermentation broth is separated and purified to obtain rhamnolipids. The rhamnolipid fermentation process of the present invention has low cost and high rhamnolipid yield, and can expand the application range of rhamnolipids to a certain extent.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a fermentation process for efficiently preparing rhamnolipids, comprising the following steps:

[0008] (1) Inoculate Pseudomonas aeruginosa into an enrichment medium and activate it for 18 - 24 h to obtain a seed culture solution. Inoculate this seed culture solution into a sterilized fermentation medium and ferment it in an aerobic environment at 25 - 37 °C for 90 - 180 h. During the fermentation process, adjust the pH, and at the same time, add a fermentation promoter at the 20th h after the start of fermentation, and start supplementing carbon and nitrogen sources at the 40th h to adjust the carbon-nitrogen ratio in the fermentation medium to obtain a fermentation broth;

[0009] (2) Separate and purify the obtained fermentation broth to obtain rhamnolipids.

[0010] In some embodiments, the preservation number of the used Pseudomonas aeruginosa is CGMCC 1.12483.

[0011] In some embodiments, the OD 600 ≥ 1.

[0012] In some embodiments, the inoculation amount of the seed culture solution is 3 - 6 wt% of the fermentation medium.

[0013] Preferably, the inoculation amount of the seed culture solution is 4.5 wt% of the fermentation medium.

[0014] In some embodiments, the fermentation medium, calculated by total mass percentage as 100%, comprises the following raw material components:

[0015] Sunflower oil and / or corn oil 2 - 4%, isopropanol 1 - 2%, glucose 1 - 3%, NaNO3 1 - 2%, soybean powder 0.5 - 1%, KH2PO4 0.05 - 0.1%, Na2HPO4·12H2O 0.05 - 0.1%, MgSO4·7H2O 0.01 - 0.1%, CaCl2·2H2O 0.5 - 0.8%, FeSO4 0.01% - 0.1%, CoCl2 0.01% - 0.05%, and the balance is water; the pH of the fermentation medium is 6.5 - 7.2.

[0016] In some embodiments, the fermentation promoter is 3-hydroxytetradecanoic acid, and its addition rate is 0.5 - 1 g / h.

[0017] Preferably, the fermentation promoter is 3-hydroxytetradecanoic acid, and its addition rate is 0.75 g / h.

[0018] In some embodiments, the carbon source is selected from sunflower oil and / or corn oil, isopropanol and / or glucose; the nitrogen source is selected from NaNO3 and / or soybean powder.

[0019] Preferably, the carbon source is selected from sunflower oil and / or corn oil, isopropanol and glucose; the nitrogen source is selected from NaNO3 and soybean powder.

[0020] In some embodiments, the carbon-nitrogen ratio is adjusted to (85 - 120):1.

[0021] Preferably, the carbon-nitrogen ratio is adjusted to 105:1.

[0022] In some embodiments, 0.1 - 0.5 wt% of an antifoaming agent is further added during the fermentation process; the antifoaming agent comprises polydimethylsiloxane, block polyether, and polyethylene glycol with a mass ratio of (12 - 13):1:(1 - 1.5).

[0023] Preferably, the antifoaming agent comprises polydimethylsiloxane, block polyether, and polyethylene glycol with a mass ratio of 12.5:1:1.25.

[0024] Further preferably, the block polyether is selected from block polyether L42 and / or L43, and the number-average molecular weight of polyethylene glycol is 1000 - 5000.

[0025] In some embodiments, the preparation steps of the polydimethylsiloxane are as follows:

[0026] Mix polydimethylsiloxane, hexamethylcyclotrisiloxane, tetramethylcyclotetrasiloxane, and sulfuric acid, heat to 115 - 135 °C under stirring and keep warm for 5 - 8 h, then cool to room temperature, neutralize sulfuric acid, filter, remove water, filter again, and evaporate the low-boiling substances from the obtained filtrate to obtain polydimethylsiloxane.

[0027] In some embodiments, the mass ratio of polydimethylsiloxane, hexamethylcyclotrisiloxane, and tetramethylcyclotetrasiloxane is (0.4 - 0.7):(6 - 6.5):1.

[0028] Preferably, the mass ratio of polydimethylsiloxane, hexamethylcyclotrisiloxane, and tetramethylcyclotetrasiloxane is 0.55:6.25:1.

[0029] By selecting specific fermentation strains, fermentation media, and adjusting the parameter conditions during the fermentation process, and adding specific defoamers and fermentation promoters during the fermentation process, Pseudomonas aeruginosa CGMCC 1.12483 can secrete rhamnolipids with high yields, which is beneficial to expanding the application scope of biosurfactants.

[0030] During the microbial fermentation process, the generation of foam is a common problem, which may lead to problems such as reduced oxygen transfer efficiency, overflow of the fermentation broth, and affecting the stability of the fermentation process. Using a defoamer during the fermentation process is one of the effective methods to control foam. The defoaming effects of common defoamers on the market cannot reach the defoaming effect required by the present invention. The present invention uses polydimethylsiloxane, hexamethylcyclotrisiloxane, and tetramethylcyclotetrasiloxane as reaction raw materials, sulfuric acid as a catalyst, and prepares a polysiloxane under the conditions of stirring and heating. During the heating and insulation process, chemical reactions may occur between the three raw materials, and by changing the molecular structure and physicochemical properties of the siloxane, a polysiloxane with high defoaming performance is obtained. The defoamer obtained by compounding this polysiloxane with block polyether and polyethylene glycol is used in the process of microbial fermentation to secrete rhamnolipids. This specific defoamer has good compatibility in the fermentation system, combines the low surface tension of silicone and the foam suppression ability of polyether, can effectively control foam during the fermentation process, improve the fermentation efficiency, and thus help to increase the yield of rhamnolipids.

[0031] In addition, the applicant unexpectedly found that using 3-hydroxymyristic acid as a fermentation promoter in the present invention can improve the fermentation effect of Pseudomonas aeruginosa CGMCC 1.12483, and further increase the yield of rhamnolipids. This may be because the biosynthesis pathway of rhamnolipids involves multiple steps, and dTDP-L-rhamnose and 3-hydroxy fatty acids are the main precursor substances for the synthesis of rhamnolipids. During the fermentation process of Pseudomonas aeruginosa CGMCC 1.12483, 3-hydroxymyristic acid may be one of the key precursor substances for the synthesis of rhamnolipids. By externally providing 3-hydroxymyristic acid, the substrate availability for the synthesis of rhamnolipids can be increased, thereby increasing the yield of rhamnolipids. However, the yield of rhamnolipids is not only affected by the substrate availability, but also by fermentation conditions such as temperature, pH, oxygen supply, and the ratio of carbon source to nitrogen source. The present invention optimizes these fermentation conditions to enhance the promoting effect of 3-hydroxymyristic acid, and can significantly increase the yield of rhamnolipids secreted by Pseudomonas aeruginosa CGMCC 1.12483.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] 1. The present invention selects specific fermentation strains, fermentation media, and parameter conditions during the fermentation process, and adds specific antifoaming agents and fermentation promoters during the fermentation process, enabling Pseudomonas aeruginosa CGMCC 1.12483 to secrete rhamnolipids with high yields, reducing the production cost of rhamnolipids to a certain extent, and facilitating the expansion of the application scope of biosurfactants.

[0034] 2. The present invention prepares a specific antifoaming agent and uses it in the process of microbial fermentation to secrete rhamnolipids, which can effectively control foam during the fermentation process and improve fermentation efficiency. Detailed Embodiments

[0035] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0036] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention description, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present application are only exemplary.

[0037] It should be noted that operations such as "centrifugation", "filtration", and "stirring" described in the present invention are conventional operations for those skilled in the art and can be selected according to actual operations.

[0038] The Pseudomonas aeruginosa used in the present invention is Pseudomonas aeruginosa CGMCC 1.12483, which can be obtained through commercial channels; the number-average molecular weight of the polydimethylsiloxane used is 5000; the block polyether used is block polyether L42; the number-average molecular weight of the polyethylene glycol used is 2000.

[0039] Preparation Example 1

[0040] The preparation steps of polysiloxane are as follows:

[0041] Mix 5.5 g of polydimethylsiloxane, 62.5 g of hexamethylcyclotrisiloxane, 10 g of tetramethylcyclotetrasiloxane and 1.76 g of AR grade sulfuric acid. Heat the mixture to 125 °C under stirring at 100 rpm, keep it warm for 6 h, then cool it to room temperature. Add 2 g of calcium hydroxide to neutralize sulfuric acid, filter, add anhydrous sodium sulfate to remove water and filter again. Evaporate the obtained filtrate at 60 °C to remove low-boiling substances to obtain polysiloxane.

[0042] Preparation Example 2

[0043] The preparation steps of polysiloxane are the same as those in Preparation Example 1, except that 55 g of hexamethylcyclotrisiloxane is used.

[0044] Preparation Example 3

[0045] The preparation steps of polysiloxane are the same as those in Preparation Example 1, except that 5 g of tetramethylcyclotetrasiloxane is used.

[0046] Preparation Example 4

[0047] The preparation steps of polysiloxane are the same as those in Preparation Example 1, except that the heating temperature is 110 °C.

[0048] Preparation Example 5

[0049] The preparation steps of the defoamer are as follows:

[0050] Take the polysiloxane, block polyether and polyethylene glycol obtained in Preparation Example 1 with a mass ratio of 12.5:1:1.25, and mix them evenly to obtain the defoamer.

[0051] Preparation Example 6

[0052] The preparation steps of the defoamer are as follows:

[0053] Take the polysiloxane, block polyether and polyethylene glycol obtained in Preparation Example 2 with a mass ratio of 12.5:1:1.25, and mix them evenly to obtain the defoamer.

[0054] Preparation Example 7

[0055] The preparation steps of the defoamer are as follows:

[0056] Take the polysiloxane, block polyether and polyethylene glycol obtained in Preparation Example 3 with a mass ratio of 12.5:1:1.25, and mix them evenly to obtain the defoamer.

[0057] Preparation Example 8

[0058] The preparation steps of the defoamer are as follows:

[0059] Take the polysiloxane, block polyether, and polyethylene glycol obtained in Preparation Example 4 with a mass ratio of 12.5:1:1.25, mix them evenly to obtain the defoamer.

[0060] Example 1

[0061] An efficient fermentation process for preparing rhamnolipid includes the following steps:

[0062] (1) Inoculate Pseudomonas aeruginosa into the enrichment medium and activate it for 24 h to obtain a seed bacterial solution with an OD 600 of 1.58. Inoculate this seed bacterial solution into the sterilized fermentation medium at an inoculation amount of 4.5 wt%, ferment at 25 °C in an aerobic environment for 180 h, add 0.1 wt% of the defoamer during the fermentation process, and adjust the pH during the fermentation process within 6.5 - 7.2. At the same time, add 0.01 wt% of 3-hydroxy myristic acid at a rate of 0.5 g / h starting from the 20th h after the start of fermentation, and start supplementing the carbon source and nitrogen source from the 40th h to adjust the carbon-nitrogen ratio in the fermentation medium to 85:1 to obtain the fermentation broth;

[0063] (2) Separate and purify the obtained fermentation broth to obtain rhamnolipid.

[0064] The separation and purification steps refer to the operation in paragraph

[0059] of CN 117126904 A.

[0065] The enrichment medium used is the beef extract peptone medium with pH = 7.

[0066] The defoamer used is obtained from Preparation Example 5.

[0067] The carbon source used is sunflower oil, isopropanol, and glucose with a mass ratio of 3:1.5:2; the nitrogen source used is NaNO3 and soybean powder with a mass ratio of 2:1.

[0068] The fermentation medium used, calculated according to the total mass percentage of 100%, includes the following raw material components:

[0069] Sunflower oil 3%, isopropanol 1.5%, glucose 2%, NaNO3 1.5%, soybean powder 0.75%, KH2PO4 0.075%, Na2HPO4·12H2O 0.075%, MgSO4·7H2O 0.05%, CaCl2·2H2O 0.6%, FeSO4 0.05%, CoCl2 0.03%, and the balance is water; the pH of the fermentation medium is 6.8.

[0070] Example 2

[0071] An efficient fermentation process for preparing rhamnolipids, comprising the following steps:

[0072] (1) Inoculate Pseudomonas aeruginosa into an enrichment medium and activate it by culturing for 18 h to obtain a seed bacterial solution with an OD 600 of 1.15. Inoculate this seed bacterial solution into the sterilized fermentation medium at an inoculation amount of 4.5 wt%. Ferment at 37 °C in an aerobic environment for 90 h. During the fermentation process, add 0.5 wt% of an antifoaming agent, and adjust the pH during the fermentation process within 6.5 - 7.2. At the same time, add 0.03 wt% of 3-hydroxy myristic acid at a rate of 1 g / h starting from the 20th h after the start of fermentation, and start supplementing carbon and nitrogen sources from the 40th h to adjust the carbon-nitrogen ratio in the fermentation medium to 120:1 to obtain a fermentation broth;

[0073] (2) Separate and purify the obtained fermentation broth to obtain rhamnolipids.

[0074] The separation and purification steps refer to the operations in paragraph

[0059] of CN 117126904 A.

[0075] The enrichment medium used is a beef extract peptone medium, pH = 7.

[0076] The antifoaming agent used is obtained from Preparation Example 5.

[0077] The carbon source used is sunflower oil, isopropanol and glucose with a mass ratio of 3:1.5:2; the nitrogen source used is NaNO3 and soybean powder with a mass ratio of 2:1.

[0078] The fermentation medium used, calculated according to the total mass percentage of 100%, comprises the following raw material components:

[0079] Sunflower oil 3%, isopropanol 1.5%, glucose 2%, NaNO3 1.5%, soybean powder 0.75%, KH2PO4 0.075%, Na2HPO4·12H2O 0.075%, MgSO4·7H2O 0.05%, CaCl2·2H2O 0.6%, FeSO4 0.05%, CoCl2 0.03%, and the balance is water; the pH of the fermentation medium is 6.8.

[0080] Example 3

[0081] An efficient fermentation process for preparing rhamnolipids, comprising the following steps:

[0082] (1) Inoculate Pseudomonas aeruginosa into an enrichment medium and activate it by culturing for 20 h to obtain an OD 600A seed bacterial solution with a value of 1.33. The seed bacterial solution is inoculated into the sterilized fermentation medium at an inoculation amount of 4.5 wt%. Fermentation is carried out at 30 °C in an aerobic environment for 120 h. During the fermentation process, 0.25 wt% of an antifoaming agent is added, and the pH during the fermentation process is adjusted within 6.5 - 7.2. At the same time, 0.02 wt% of 3-hydroxy myristic acid is added at a rate of 0.75 g / h starting from the 20th h after the start of fermentation, and the carbon source and nitrogen source are supplemented starting from the 40th h to adjust the carbon-nitrogen ratio in the fermentation medium to 105:1, obtaining a fermentation broth;

[0083] (2) Separate and purify the obtained fermentation broth to obtain rhamnolipid.

[0084] The separation and purification steps refer to the operation in paragraph

[0059] of CN 117126904 A.

[0085] The enrichment medium used is beef extract peptone medium, pH = 7.

[0086] The antifoaming agent used is obtained from Preparation Example 5.

[0087] The carbon source used is sunflower seed oil, isopropanol, and glucose with a mass ratio of 3:1.5:2; the nitrogen source used is NaNO3 and soybean powder with a mass ratio of 2:1.

[0088] The fermentation medium used, calculated according to the total mass percentage of 100%, includes the following raw material components:

[0089] Sunflower seed oil 3%, isopropanol 1.5%, glucose 2%, NaNO3 1.5%, soybean powder 0.75%, KH2PO4 0.075%, Na2HPO4·12H2O 0.075%, MgSO4·7H2O 0.05%, CaCl2·2H2O 0.6%, FeSO4 0.05%, CoCl2 0.03%, and the balance is water; the pH of the fermentation medium is 6.8.

[0090] Example 4

[0091] A fermentation process for efficiently preparing rhamnolipid, the specific implementation method is the same as that of Example 3, except that the antifoaming agent is not added.

[0092] Example 5

[0093] A fermentation process for efficiently preparing rhamnolipid, the specific implementation method is the same as that of Example 3, except that in the preparation steps of the antifoaming agent used, an equal mass of polydimethylsiloxane is used to replace the polysiloxane in Preparation Example 1.

[0094] Example 6

[0095] An efficient fermentation process for preparing rhamnolipids, the specific implementation method is the same as that of Example 3, except that an equal mass of soybean oil is used to replace sunflower oil in the fermentation medium.

[0096] Example 7

[0097] An efficient fermentation process for preparing rhamnolipids, the specific implementation method is the same as that of Example 3, except that an equal mass of coconut oil is used to replace sunflower oil in the fermentation medium.

[0098] Example 8

[0099] An efficient fermentation process for preparing rhamnolipids, the specific implementation method is the same as that of Example 3, except that an equal mass of fish oil is used to replace sunflower oil in the fermentation medium.

[0100] Example 9

[0101] An efficient fermentation process for preparing rhamnolipids, the specific implementation method is the same as that of Example 3, except that the preparation steps of the defoamer are as follows: Take block polyether and polyethylene glycol with a mass ratio of 1:1.25 and mix them evenly to obtain the defoamer.

[0102] Example 10

[0103] An efficient fermentation process for preparing rhamnolipids, the specific implementation method is the same as that of Example 3, except that the carbon-nitrogen ratio in the fermentation medium is adjusted to 80:1.

[0104] Example 11

[0105] An efficient fermentation process for preparing rhamnolipids, the specific implementation method is the same as that of Example 3, except that the carbon-nitrogen ratio in the fermentation medium is adjusted to 125:1.

[0106] Comparative Example 1

[0107] An efficient fermentation process for preparing rhamnolipids, the specific implementation method is the same as that of Example 3, except that 3-hydroxy myristic acid is not added during the fermentation process.

[0108] Performance Test

[0109] 1. Defoaming property of the defoamer:

[0110] (1) Defoaming ability test: Add 100 mL of the rhamnolipid fermentation broth of Example 4 into a 500 mL graduated cylinder, and blow air into it at a speed of 3 L / min. When the bubble volume reaches the 500 mL height, stop blowing air. Then, simultaneously drop 25 mg of the defoamers from Preparation Examples 5 - 8 and 1 mL of the rhamnolipid fermentation broth after blowing air into graduated cylinders numbered 1 - 4, and measure the time taken for the foam to be completely eliminated. No. 5 is the blank group with only the rhamnolipid fermentation broth after blowing air and without adding a defoamer.

[0111] (2) Foam suppression ability test: Add 100 mL of the rhamnolipid fermentation broth of Example 4 into five 500 mL graduated cylinders numbered 1 - 5. Then, add 25 mg of the defoamers from Preparation Examples 5 - 8 into No. 1 - 4 respectively, and continuously blow air into them at a speed of 3 L / min. Record the foam height (in terms of the graduated cylinder scale) at 1 h, 12 h, 24 h, 36 h, and 48 h respectively. No. 5 is the blank group without adding a defoamer. If the foam height reaches 500 mL in the middle, record the time when the foam height reaches 500 mL.

[0112] The specific test data are shown in Table 1.

[0113] Table 1

[0114]

[0115] As can be seen from Table 1, the defoamer obtained from Preparation Example 1 has excellent defoaming and foam suppression abilities; compared with Preparation Example 1, in the defoamers of Preparation Examples 6 - 8, due to the raw materials for preparing polysiloxane or the heating temperature used, the polymerization of monomers is affected, which may lead to changes in the structure of polysiloxane, and thus affect the defoaming and foam suppression abilities of the defoamer.

[0116] Therefore, the defoamer of Preparation Example 5 is selected for use in the examples.

[0117] 2. Yield of rhamnolipid:

[0118] Detect the rhamnolipid yield of the fermentation broth in each example and comparative example (each group is set with 3 parallel samples, and the results are averaged). The detection method of rhamnolipid yield is well-known to those skilled in the art. The specific test data are shown in Table 2.

[0119] Table 2

[0120]

[0121] As can be seen from Table 2, defoamer was not added in Example 4, and the yield of rhamnolipid was 42.6 g / L. While defoamer was added in Examples 1-3, and the yields of rhamnolipid were significantly increased. Compared with Example 3, sunflower seed oil in the fermentation medium used in Examples 6-8 was replaced by other animal and vegetable oils. The utilization rate of Pseudomonas aeruginosa for soybean oil, coconut oil and fish oil was lower than that of sunflower seed oil, resulting in a decrease in the amount of rhamnolipid secreted; in Example 5, polydimethylsiloxane of equal mass was used instead of polysiloxane in the preparation steps of the defoamer used. The defoaming effect of polydimethylsiloxane was weaker than that of polysiloxane, weakening the performance of the defoamer; in Example 9, polysiloxane was not added, and the defoaming effects of block polyether and polyethylene glycol were weakened, affecting the yield of rhamnolipid; in Examples 10 and 11, the carbon-nitrogen ratio in the later stage of fermentation was changed, which was not conducive to the metabolic activities of Pseudomonas aeruginosa, resulting in a decrease in the yield of rhamnolipid; in Comparative Example 1, fermentation promoter 3-hydroxytetradecanoic acid was not added. 3-Hydroxytetradecanoic acid may be one of the key precursor substances for the synthesis of rhamnolipid, and may affect the gene expression related to rhamnolipid secretion in Pseudomonas aeruginosa, reducing the secretion amount and yield of rhamnolipid.

[0122] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on this application. Although this application is disclosed as above with preferred embodiments, it is not intended to limit this application. Any person skilled in the art, without departing from the scope of the technical solution of this application, making some changes or modifications using the technical content disclosed above is equivalent to equivalent implementation cases. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution.

Claims

1. An efficient fermentation process for preparing rhamnolipid, characterized in that, It includes the following steps: (1) Inoculate Pseudomonas aeruginosa into an enrichment medium for activation culture for 18 - 24 h to obtain a seed bacterial liquid. Inoculate the seed bacterial liquid into a sterilized fermentation medium, and ferment at 25 - 37 °C in an aerobic environment for 90 - 180 h. During the fermentation process, adjust the pH of the fermentation process. At the same time, add a fermentation promoter at the 20th h after the start of fermentation, and start supplementing carbon sources and nitrogen sources at the 40th h to adjust the carbon-nitrogen ratio in the fermentation medium to obtain a fermentation broth; (2) Separate and purify the obtained fermentation broth to obtain rhamnolipid.

2. The fermentation process for preparing rhamnolipid according to claim 1, characterized in that, The OD of the seed bacterial solution 600 ≥ 1.

3. The fermentation process for preparing rhamnolipid according to claim 2, characterized in that, The inoculation amount of the seed bacterial liquid is 3 - 6 wt% of the fermentation medium.

4. The fermentation process for preparing rhamnolipid according to claim 1, characterized in that, The fermentation medium, calculated according to the total mass percentage of 100%, includes the following raw material components: Sunflower oil and / or corn oil 2 - 4%, isopropanol 1 - 2%, glucose 1 - 3%, NaNO3 1 - 2%, soybean powder 0.5 - 1%, KH2PO4 0.05 - 0.1%, Na2HPO4·12H2O 0.05 - 0.1%, MgSO4·7H2O 0.01 - 0.1%, CaCl2·2H2O 0.5 - 0.8%, FeSO4 0.01% - 0.1%, CoCl2 0.01% - 0.05%, and the balance is water; the pH of the fermentation medium is 6.5 - 7.

2.

5. The fermentation process for preparing rhamnolipid according to claim 1, characterized in that, The fermentation promoter is 3 - hydroxy myristic acid, and its addition rate is 0.5 - 1 g / h.

6. The fermentation process for preparing rhamnolipid according to claim 1, characterized in that, The carbon source is selected from sunflower oil and / or corn oil, isopropanol and / or glucose; the nitrogen source is selected from NaNO3 and / or soybean powder.

7. The fermentation process for preparing rhamnolipid according to claim 1, characterized in that, The carbon-nitrogen ratio is adjusted to (85 - 120):

1.

8. The fermentation process for preparing rhamnolipid according to claim 1, characterized in that, During the fermentation process, 0.1 - 0.5 wt% of an antifoaming agent is also added; the antifoaming agent contains polydimethylsiloxane, block polyether, and polyethylene glycol with a mass ratio of (12 - 13):1:(1 - 1.5).

9. The fermentation process for preparing rhamnolipid according to claim 8, characterized in that, The preparation steps of the polydimethylsiloxane are as follows: Mix polydimethylsiloxane, hexamethylcyclotrisiloxane, tetramethylcyclotetrasiloxane, and sulfuric acid, heat to 115 - 135 °C under stirring conditions, keep warm for 5 - 8 h, then cool to room temperature, neutralize sulfuric acid, filter, remove water and filter again, and evaporate the obtained filtrate to remove low-boiling substances to obtain polydimethylsiloxane.

10. The fermentation process for preparing rhamnolipid according to claim 9, characterized in that, The mass ratio of polydimethylsiloxane, hexamethylcyclotrisiloxane, and tetramethylcyclotetrasiloxane is (0.4 - 0.7):(6 - 6.5):1.

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