A fermentation process for the efficient preparation of rhamnolipids

Abstract

This invention belongs to the field of microbial fermentation technology, specifically relating to a high-efficiency fermentation process for preparing rhamnolipids. The fermentation process includes the following steps: *Pseudomonas aeruginosa* is inoculated into an enrichment medium and activated for 18-24 hours to obtain a seed culture. This seed culture is then inoculated into a sterilized fermentation medium and fermented in an aerobic environment at 25-37°C for 90-180 hours. During fermentation, the pH is adjusted, and a fermentation promoter is added between the 20th and 40th hour after fermentation begins. From the 40th hour onwards, carbon and nitrogen sources are supplemented to adjust the carbon-nitrogen ratio in the fermentation medium, resulting in a fermentation broth. The fermentation broth is then separated and purified to obtain rhamnolipids. This invention, by selecting specific fermentation strains, fermentation mediums, and fermentation parameters, and by adding antifoaming agents and fermentation promoters during fermentation, enables *Pseudomonas aeruginosa* to secrete high yields of rhamnolipids, thereby reducing the production cost of rhamnolipids to a certain extent.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a fermentation process for the efficient preparation of rhamnolipids. Background Technology

[0002] Biosurfactant lipids have a wide range of applications in the cleaning industry. They possess many excellent properties, such as low toxicity, high biocompatibility, and biodegradability, making them ideal cleaning ingredients. Biosurfactants specifically refer to glycolipid raw materials obtained through fermentation by specific microorganisms. Currently, the two most important commercially available biosurfactants are rhamnose glycolipids and sophorolipids, which use rhamnose and sophorose as their structural sugar units, respectively. The applications of biosurfactant lipids are very broad, including detergents and cleaning products, cosmetics, environmental remediation, the petroleum industry, agriculture, the food industry, and the pharmaceutical field.

[0003] In recent years, many related studies have focused on the production of biosurfactant lipids. Patent CN117946914A discloses a high-yield rhamnolipin strain, inoculum, method for producing rhamnolipin, and its applications. The high-yield rhamnolipin strain used is *Pseudomonas aeruginosa*, with accession number CCTCCNO: M20232111. Through gene processes, regulation of fermentation medium and fermentation conditions, the yield of rhamnolipin reaches 10.9 g / L at 72 hours. Patent CN 117924387A discloses a method for separating and purifying rhamnolipin. The method includes the following steps: acid hydrolysis of rhamnolipin fermentation broth to obtain an acid hydrolysate; adding an organic solvent to the acid hydrolysate to obtain an extract, which is then separated into an organic solvent phase, an emulsion phase, and an aqueous phase; treating the organic solvent phase with an alkaline solution; concentrating the aqueous phase obtained after phase separation into a rhamnolipin aqueous solution; demulsifying the emulsion phase, separating and concentrating it to obtain a low-quality rhamnolipin aqueous solution. It can also simultaneously recover two different qualities of rhamnolipin products. Although the aforementioned studies have improved rhamnolipin yield by optimizing fermentation conditions and using appropriate strains, further improving production efficiency and reducing production costs remain key research areas. Furthermore, compared to chemically synthesized surfactants, the production cost of rhamnolipin is relatively high, which significantly limits its widespread application.

[0004] Therefore, reducing production costs is one of the key factors driving the commercialization of rhamnolipids. Developing a method to reduce the production cost of rhamnolipids is of great significance to industrial development and the application of biosurfactants. Summary of the Invention

[0005] This invention discloses a highly efficient fermentation process for preparing rhamnolipids. The process involves inoculating a seed culture of *Pseudomonas aeruginosa* into a fermentation medium, fermenting under specific conditions, adjusting fermentation parameters, and then separating and purifying the resulting fermentation broth to obtain rhamnolipids. This invention's rhamnolipid fermentation process is low-cost and yields high amounts of rhamnolipids, thus expanding the application range of rhamnolipids to a certain extent.

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

[0007] This invention provides a fermentation process for the efficient preparation of rhamnolipids, comprising the following steps:

[0008] (1) Pseudomonas aeruginosa was inoculated into enrichment medium and activated for 18-24 h to obtain seed culture. The seed culture was then inoculated into sterilized fermentation medium and fermented in an aerobic environment at 25-37℃ for 90-180 h. The pH was adjusted during the fermentation process. Fermentation promoter was added at 20 h after the start of fermentation, and carbon and nitrogen sources were added at 40 h to adjust the carbon-nitrogen ratio in the fermentation medium to obtain fermentation broth.

[0009] (2) The fermentation broth was separated and purified to obtain rhamnolipin.

[0010] In some embodiments, the Pseudomonas aeruginosa used has the accession number CGMCC 1.12483.

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

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

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

[0014] In some embodiments, the fermentation medium, based on a total mass percentage of 100%, comprises the following raw material components:

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

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

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

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

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

[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 added during fermentation; the antifoaming agent comprises polysiloxane, block polyether, and polyethylene glycol in a mass ratio of (12-13):1:(1-1.5).

[0023] Preferably, the defoamer comprises polysiloxane, block polyether, and polyethylene glycol in a mass ratio of 12.5:1:1.25.

[0024] More 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 polysiloxane are as follows:

[0026] Polydimethylsiloxane, hexamethylcyclotrisiloxane, tetramethylcyclotetrasiloxane and sulfuric acid are mixed, heated to 115-135℃ under stirring, kept at that temperature for 5-8 hours, cooled to room temperature, neutralized with sulfuric acid, filtered, water removed, filtered again, and the filtrate is evaporated to remove low-boiling-point substances to obtain polysiloxane.

[0027] In some embodiments, the mass ratio of the 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] This invention enables Pseudomonas aeruginosa CGMCC 1.12483 to secrete high yields of rhamnolipids by selecting specific fermentation strains and fermentation culture media, adjusting parameters during the fermentation process, and adding specific defoamers and fermentation promoters during fermentation. This is beneficial for expanding the application range of biosurfactants.

[0030] Foam formation is a common problem in microbial fermentation, potentially leading to reduced oxygen transfer efficiency, fermentation broth overflow, and instability. Using defoamers is an effective method for controlling foam during fermentation, but commercially available defoamers do not achieve the desired effect as described in this invention. This invention uses polydimethylsiloxane, hexamethylcyclotrisiloxane, and tetramethylcyclotetrasiloxane as reactants, sulfuric acid as a catalyst, and prepares a polysiloxane under stirring and heating conditions. During heating and heat preservation, a chemical reaction may occur between the three reactants, altering the molecular structure and physicochemical properties of the siloxane to obtain a polysiloxane with high defoaming performance. This polysiloxane is then compounded with block polyether and polyethylene glycol to obtain an defoamer used in the microbial fermentation process to secrete rhamnolipin. This specific defoamer exhibits good compatibility in the fermentation system and combines the low surface tension of organosilicon with the foam-suppressing ability of polyether, effectively controlling foam and improving fermentation efficiency, thereby contributing to increased rhamnolipin yield.

[0031] Furthermore, the applicant unexpectedly discovered that using 3-hydroxymyristic acid as a fermentation promoter in this invention can improve the fermentation effect of *Pseudomonas aeruginosa* CGMCC 1.12483, thereby increasing the yield of rhamnolipids. This is likely because the biosynthetic pathway of rhamnolipids involves multiple steps, among which dTDP-L-rhamnose and 3-hydroxy fatty acids are the main precursors for rhamnolipid synthesis. In the fermentation process of *Pseudomonas aeruginosa* CGMCC 1.12483, 3-hydroxymyristic acid may serve as one of the key precursors for rhamnolipid synthesis. By providing 3-hydroxymyristic acid exogenously, the substrate availability for rhamnolipid synthesis can be increased, thereby improving the yield of rhamnolipids. However, the yield of rhamnolipids is not only affected by substrate availability but also by fermentation conditions such as temperature, pH, oxygen supply, and the ratio of carbon to nitrogen sources. This invention optimizes these fermentation conditions to enhance the promoting effect of 3-hydroxymyristic acid, thereby significantly increasing the yield of rhamnolipid secreted by Pseudomonas aeruginosa CGMCC1.12483.

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

[0033] 1. This invention, by selecting specific fermentation strains, fermentation culture media, and fermentation parameters, and by adding specific defoamers and fermentation promoters during fermentation, enables Pseudomonas aeruginosa CGMCC 1.12483 to secrete high yields of rhamnolipids, thereby reducing the production cost of rhamnolipids to a certain extent and facilitating the expansion of the application range of biosurfactants.

[0034] 2. The present invention prepares a specific defoamer, which, when used in the process of microbial fermentation and secretion of rhamnolipids, can effectively control foam and improve fermentation efficiency. Detailed Implementation

[0035] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, regarding numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in the invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of the invention. Various modifications and variations to the specific embodiments described in this specification are apparent to those skilled in the art without departing from the scope or spirit of the invention. Other embodiments derived from this specification will be apparent to those skilled in the art. This application specification and embodiments are merely exemplary.

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

[0038] The Pseudomonas aeruginosa used in this invention is Pseudomonas aeruginosa CGMCC 1.12483, which can be obtained commercially; the number average molecular weight of the polydimethylsiloxane used is 5000; the block polyether used is block polyether L42; and 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] 5.5g of polydimethylsiloxane, 62.5g of hexamethylcyclotrisiloxane, 10g of tetramethylcyclotetrasiloxane, and 1.76g of AR grade sulfuric acid were mixed and heated to 125℃ under stirring at 100rpm for 6 hours. After cooling to room temperature, 2g of calcium hydroxide was added to neutralize the sulfuric acid. The mixture was filtered, and anhydrous sodium sulfate was added to remove water. After filtration, the filtrate was evaporated at 60℃ to remove low-boiling-point substances, yielding polysiloxane.

[0042] Preparation Example 2

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

[0044] Preparation Example 3

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

[0046] Preparation Example 4

[0047] The preparation steps for polysiloxane are the same as 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] The polysiloxane, block polyether, and polyethylene glycol obtained in Preparation Example 1 were mixed evenly in a mass ratio of 12.5:1:1.25 to obtain an antifoaming agent.

[0051] Preparation Example 6

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

[0053] The polysiloxane, block polyether, and polyethylene glycol obtained in Preparation Example 2 with a mass ratio of 12.5:1:1.25 were mixed evenly to obtain an antifoaming agent.

[0054] Preparation Example 7

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

[0056] The polysiloxane, block polyether, and polyethylene glycol obtained in Preparation Example 3 with a mass ratio of 12.5:1:1.25 were mixed evenly to obtain an antifoaming agent.

[0057] Preparation Example 8

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

[0059] The polysiloxane, block polyether, and polyethylene glycol obtained in Preparation Example 4 with a mass ratio of 12.5:1:1.25 were mixed evenly to obtain an antifoaming agent.

[0060] Example 1

[0061] A fermentation process for the efficient preparation of rhamnolipids includes the following steps:

[0062] (1) Pseudomonas aeruginosa was inoculated into enrichment medium and activated for 24 h to obtain OD. 600 The seed culture was 1.58g. This seed culture was inoculated into sterilized fermentation medium at an inoculum rate of 4.5wt%. Fermentation was carried out in an aerobic environment at 25℃ for 180h. During fermentation, 0.1wt% of antifoaming agent was added, and the pH was adjusted to 6.5-7.2. At the 20h mark after the start of fermentation, 0.01wt% of 3-hydroxymyristic acid was added at a rate of 0.5g / h. At the 40h mark, carbon and nitrogen sources were added to adjust the carbon-nitrogen ratio in the fermentation medium to 85:1, thus obtaining the fermentation broth.

[0063] (2) The fermentation broth was separated and purified to obtain rhamnolipin.

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

[0059] of CN 117126904 A.

[0065] The enrichment medium used was beef extract peptone medium, pH=7.

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

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

[0068] The fermentation medium used, based on a total mass percentage of 100%, includes the following raw material components:

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

[0070] Example 2

[0071] A fermentation process for the efficient preparation of rhamnolipids includes the following steps:

[0072] (1) Pseudomonas aeruginosa was inoculated into enrichment medium and activated for 18 h to obtain OD. 600 The seed culture solution was 1.15. This seed culture solution was inoculated into sterilized fermentation medium at an inoculum rate of 4.5 wt%. Fermentation was carried out in an aerobic environment at 37°C for 90 h. During the fermentation process, 0.5 wt% of antifoaming agent was added, and the pH was adjusted to 6.5-7.2. At the 20th hour after the start of fermentation, 0.03 wt% of 3-hydroxymyristic acid was added at a rate of 1 g / h. At the 40th hour, carbon and nitrogen sources were added to adjust the carbon-nitrogen ratio in the fermentation medium to 120:1, and the fermentation broth was obtained.

[0073] (2) The fermentation broth was separated and purified to obtain rhamnolipin.

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

[0059] of CN 117126904 A.

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

[0076] The defoamer used was obtained from Preparation Example 5.

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

[0078] The fermentation medium used, based on a total mass percentage of 100%, includes the following raw material components:

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

[0080] Example 3

[0081] A fermentation process for the efficient preparation of rhamnolipids includes the following steps:

[0082] (1) Pseudomonas aeruginosa was inoculated into enrichment medium and cultured for 20 h to obtain OD. 600The seed culture solution was 1.33. This seed culture solution was inoculated into sterilized fermentation medium at an inoculum rate of 4.5 wt%. Fermentation was carried out in an aerobic environment at 30°C for 120 h. During the fermentation process, 0.25 wt% of antifoaming agent was added, and the pH was adjusted to 6.5-7.2. At the 20th hour after the start of fermentation, 0.02 wt% of 3-hydroxymyristic acid was added at a rate of 0.75 g / h. Starting at the 40th hour, carbon and nitrogen sources were added to adjust the carbon-nitrogen ratio in the fermentation medium to 105:1, thus obtaining the fermentation broth.

[0083] (2) The fermentation broth was separated and purified to obtain rhamnolipin.

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

[0059] of CN 117126904 A.

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

[0086] The defoamer used was obtained from Preparation Example 5.

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

[0088] The fermentation medium used, based on a total mass percentage of 100%, includes the following raw material components:

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

[0090] Example 4

[0091] A fermentation process for the efficient preparation of rhamnolipids is described, with the specific implementation method being the same as in Example 3, except that no defoaming agent is added.

[0092] Example 5

[0093] A fermentation process for the efficient preparation of rhamnolipids is described, with the specific implementation method being the same as that in Example 3, except that an equal mass of polydimethylsiloxane is used instead of the polysiloxane in Example 1 in the preparation step of the defoamer.

[0094] Example 6

[0095] A fermentation process for efficiently preparing rhamnolipids is described, with the specific implementation method being the same as in Example 3, except that an equal mass of soybean oil is used instead of sunflower seed oil in the fermentation medium.

[0096] Example 7

[0097] A fermentation process for the efficient preparation of rhamnolipids is described, with the specific implementation method being the same as in Example 3, except that an equal mass of coconut oil is used instead of sunflower seed oil in the fermentation medium.

[0098] Example 8

[0099] A fermentation process for the efficient preparation of rhamnolipids is described, with the specific implementation method being the same as in Example 3, except that an equal mass of fish oil is used instead of sunflower seed oil in the fermentation medium.

[0100] Example 9

[0101] A fermentation process for the efficient preparation of rhamnolipids is described. The specific implementation method is the same as that in Example 3, except that the preparation step of the defoamer is as follows: block polyether and polyethylene glycol with a mass ratio of 1:1.25 are mixed evenly to obtain the defoamer.

[0102] Example 10

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

[0104] Example 11

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

[0106] Comparative Example 1

[0107] A fermentation process for the efficient preparation of rhamnolipids is described, with the specific implementation method being the same as in Example 3, except that 3-hydroxymyristic acid is not added during the fermentation process.

[0108] Performance testing

[0109] 1. Defoaming properties of defoamers:

[0110] (1) Defoaming ability test: 100 mL of the rhamnolipin fermentation broth from Example 4 was added to a 500 mL graduated cylinder. Air was bubbled in at a rate of 3 L / min. When the amount of bubbles reached a height of 500 mL, the bubbling was stopped. 25 mg of the defoaming agent from Preparation Examples 5-8 and 1 mL of the rhamnolipin fermentation broth after air bubbling were simultaneously dropped into graduated cylinders numbered 1-4. The time required for the foam to be completely eliminated was measured. Number 5 was the blank group, consisting only of the rhamnolipin fermentation broth after air bubbling, without the addition of defoaming agent.

[0111] (2) Defoaming ability test: 100 mL of the rhamnolipin fermentation broth from Example 4 was added to each of five 500 mL graduated cylinders numbered 1-5. Then, 25 mg of the defoamer from Preparation Examples 5-8 was added to each of the five cylinders. Air was continuously bubbled in at a rate of 3 L / min, and the foam height (measured on the graduated cylinder scale) was recorded at 1 h, 12 h, 24 h, 36 h, and 48 h. Cylinder number 5 was the blank group without defoamer. If the foam height reached 500 mL, the time when the foam height reached 500 mL was recorded.

[0112] Specific test data are shown in Table 1.

[0113] Table 1

[0114]

[0115] As shown in Table 1, the defoamer obtained in Preparation Example 1 has excellent defoaming and foam-suppressing abilities. Compared with Preparation Example 1, the defoamers in Preparation Examples 6-8 may have their structure changed due to the influence of the raw materials or heating temperature of the polysiloxane used in preparation, which affects the polymerization of the monomers and thus affects the defoaming and foam-suppressing abilities of the defoamer.

[0116] Therefore, the defoamer prepared in Example 5 was selected for use in the examples.

[0117] 2. Rhamnose lipid production:

[0118] The rhamnolipin yield in the fermentation broth of each embodiment and comparative example (three parallel samples were set in each group, and the average value of the results was taken) was tested. The method for detecting rhamnolipin yield is well known to those skilled in the art. Specific test data are shown in Table 2.

[0119] Table 2

[0120]

[0121] Table 2 shows that in Example 4, without the addition of defoamer, the yield of rhamnolipin was 42.6 g / L, while in Examples 1-3, with the addition of defoamer, the yield of rhamnolipin was significantly increased. Compared to Example 3, in Examples 6-8, the sunflower seed oil in the fermentation medium was replaced by other animal and vegetable oils. *Pseudomonas aeruginosa* had lower utilization rates of soybean oil, coconut oil, and fish oil than sunflower seed oil, resulting in a decrease in the amount of rhamnolipin it secreted. In Example 5, the defoamer preparation step used an equal mass of polydimethylsiloxane instead of polysiloxane. Polydimethylsiloxane has a weaker defoaming effect than polysiloxane, weakening the performance of the defoamer. In Example 9, no polysiloxane was added, and block polyethers were used instead. The defoaming effect of polyethylene glycol was weakened, affecting the yield of rhamnolipin. In Examples 10 and 11, the carbon-nitrogen ratio was changed in the later stage of fermentation, which was not conducive to the metabolic activity of Pseudomonas aeruginosa, resulting in a decrease in the yield of rhamnolipin. In Comparative Example 1, the fermentation promoter 3-hydroxymyristic acid was not added. 3-hydroxymyristic acid may be one of the key precursors for the synthesis of rhamnolipin, which may affect the gene expression related to rhamnolipin secretion in Pseudomonas aeruginosa, thereby reducing the amount of rhamnolipin secreted and the yield.

[0122] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present application in any way. Although the present application discloses the preferred embodiment as described above, it is not intended to limit the present application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of the present application using the disclosed technical content are equivalent to equivalent implementation cases. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the technical solution of the present invention are still within the scope of the technical solution.

Claims

1. A fermentation process for the efficient preparation of rhamnolipids, characterized in that, Includes the following steps: (1) Pseudomonas aeruginosa was inoculated into enrichment medium and activated for 18-24 h to obtain seed culture. The seed culture was inoculated into sterilized fermentation medium and fermented in an aerobic environment at 25-37℃ for 90-180 h. The pH was adjusted during the fermentation process. At the same time, a fermentation promoter was added at 20 h after the start of fermentation, and carbon and nitrogen sources were added at 40 h to adjust the carbon-nitrogen ratio in the fermentation medium to obtain fermentation broth. The Pseudomonas aeruginosa was Pseudomonas aeruginosa CGMCC1.12483. (2) The fermentation broth was separated and purified to obtain rhamnolipin; The fermentation medium, based on a total mass percentage of 100%, comprises the following raw material components: The fermentation medium comprises: 2-4% sunflower seed oil and / or corn oil, 1-2% isopropanol, 1-3% glucose, 1-2% NaNO3, 0.5-1% soybean flour, 0.05-0.1% KH2PO4, 0.05-0.1% Na2HPO4·12H2O, 0.01-0.1% MgSO4·7H2O, 0.5-0.8% CaCl2·2H2O, 0.01%-0.1% FeSO4, 0.01%-0.05% CoCl2, with the balance being water; the pH of the fermentation medium is 6.5-7.

2. The fermentation promoter is 3-hydroxymyristic acid; During the fermentation process, 0.1-0.5 wt% of defoamer is added; the defoamer contains polysiloxane, block polyether, and polyethylene glycol in a mass ratio of (12-13):1:(1-1.5), wherein the block polyether is block polyether L42; and the number average molecular weight of the polyethylene glycol is 2000. The preparation steps of the polysiloxane are as follows: Polydimethylsiloxane, hexamethylcyclotrisiloxane, tetramethylcyclotetrasiloxane and sulfuric acid are mixed, heated to 115-135℃ under stirring, and kept at that temperature for 5-8 hours. After cooling to room temperature, the sulfuric acid is neutralized, filtered, water is removed, and then filtered again. The filtrate is evaporated to remove low-boiling-point substances to obtain polysiloxane, wherein the number average molecular weight of polydimethylsiloxane is 5000.

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

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

4. The fermentation process for preparing rhamnolipids according to claim 1, characterized in that, The fermentation promoter is added at a rate of 0.5-1 g / h.

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

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

1.

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

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