A method for purifying rhamnolipids

Abstract

This invention provides a method for separating and purifying rhamnolipin, which can efficiently separate rhamnolipin on a large scale at low cost and obtain a light-colored, low (or no) odorless aqueous solution of rhamnolipin. The method includes the following steps: (1) Filtration: The rhamnolipin fermentation broth is pre-filtered to remove large particles and obtain filtrate a; (2) Centrifugation: Filtrate a is centrifuged and the supernatant b is collected; (3) Adsorption treatment: Activated carbon is added to the supernatant b, and the temperature, time, pH and other conditions are controlled to complete the preliminary adsorption treatment. After centrifugation, the supernatant c is obtained; (4) Decolorization and deodorization: Diatomaceous earth with different particle sizes is added to the supernatant c, stirred and mixed, and decolorized and deodorized to obtain a mixed solution d; (5) Filtration: The mixed solution d is clarified by filtration to obtain a light-colored, transparent, low-odor clear solution, which is the finished product. The separation and purification process of this invention does not use organic solvents, the product is light-colored and transparent, and has a faint caramel smell.

Description

Technical Field

[0001] This invention belongs to the field of bioextraction technology, specifically, it relates to a method for separating and purifying rhamnolipids. Background Technology

[0002] Rhamnolipids are glycolipid biosurfactants produced by the fermentation and metabolism of *Pseudomonas aeruginosa*. Compared to traditional chemically synthesized surfactants, rhamnolipid biosurfactants, in addition to having a similar amphiphilic molecular structure, also possess excellent environmental and biocompatibility, meaning they are non-toxic, harmless, and easily degradable. Therefore, they have been widely used in environmental remediation, oil extraction, biomedicine, papermaking, and cosmetics in recent years. However, the industrialization of rhamnolipids currently faces challenges such as long production cycles, low yields, small scale, and high separation and purification costs. This is particularly true in the daily chemical, food, petroleum, agricultural, and medical fields, where high product quality and stability are required. The fermentation and separation / purification processes for rhamnolipids keep costs high, and the products are often dark brown or even black in color with a humic acid-like odor, limiting their industrial application. Therefore, developing low-cost, light-colored, and low-odor rhamnolipid separation and purification processes is crucial to meeting current market demands.

[0003] Currently, the main methods for separating and purifying rhamnolipids are extraction, membrane separation, and column chromatography, but these are mostly still in the laboratory stage and rarely used for industrial-scale production. (Chinese Patent Document CN)

[0004] Patent 113150043B describes a method involving filtration to separate the supernatant, sedimentation and filtration with an acidic pH adjuster to obtain the precipitate, resuspending the supernatant in sodium bicarbonate buffer to obtain the precipitate, followed by sedimentation and filtration with an acidic pH adjuster to obtain the precipitate again, and finally adding an organic solvent and phosphate buffer for static separation to allow rhamnolipin to be extracted from the organic phase into the aqueous phase, thus obtaining a high-purity rhamnolipin solution. While this patent mentions that the organic solvent ethyl acetate has low toxicity and can be recycled, and that the equipment used is simple and readily available, it cannot control the color and odor of the rhamnolipin solution. Chinese patent document CN 112279879A describes a method using flocculation sedimentation for solid-liquid separation, activated carbon decolorization, two filtrations, and finally concentration and desalting to obtain the rhamnolipin product. Although this method uses activated carbon for decolorization, it has the advantages of being simple, low-cost, having a wide range of raw material sources, and requiring no complex equipment. However, this process can only initially decolorize rhamnolipin and remove some of its odor. It cannot guarantee the final color and odor of the product, nor can it address the issue of rhamnolipin products darkening in color and increasing in odor during storage.

[0005] Therefore, there is an urgent need to develop a method for the separation and purification of low-odor, light-colored, and transparent rhamnolipids. This method can not only produce high-purity, low-odor, light-colored, and transparent rhamnolipid solutions, but also ensure that the color and odor of the rhamnolipid solutions remain essentially unchanged during storage. Summary of the Invention

[0006] To address the problems in the prior art, this invention provides a method for separating and purifying rhamnolipin. Compared with the prior art, this method can separate rhamnolipin on a large scale and efficiently at low cost, and obtain a light-colored, low (or no) odorless aqueous solution of rhamnolipin.

[0007] This invention is achieved through the following technical solution:

[0008] A method for preparing a light-colored, transparent, low-odor rhamnolipid includes the following steps:

[0009] (1) Filtration: The rhamnolipin fermentation broth is pre-filtered to remove large particulate matter in the fermentation broth to obtain filtrate a;

[0010] (2) Centrifugation: Centrifuge the filtrate a to separate it and collect the supernatant b;

[0011] (3) Adsorption treatment: Activated carbon was added to the supernatant b, and the temperature, time, pH and other conditions were controlled to complete the preliminary adsorption treatment. After centrifugation, the supernatant c was obtained.

[0012] (4) Decolorization and deodorization: Add diatomaceous earth particles of different sizes to the supernatant c, stir and mix, and decolorize and deodorize to obtain the mixed liquid d;

[0013] (5) Filtration: The mixture d is clarified by filtration to obtain a light-colored, transparent, low-odor clear solution, which is the finished product.

[0014] In step (1), the filtration is selected from gauze filtration, mesh filtration, cotton filtration, and bulked cotton filtration, with gauze filtration being the preferred method.

[0015] The gauze used in step (1) has 10-35 layers, preferably 15-30 layers;

[0016] The pore size of the filter in step (1) is 10 mesh to 100 mesh, preferably 16 to 80 mesh;

[0017] The centrifugation speed in step (2) is 3500 rpm to 6000 rpm, preferably 4000-5500 rpm;

[0018] The centrifugation sterilization time in step (2) is 10 min to 35 min, preferably 15 min to 30 min;

[0019] In the above-described method for separating and purifying light-colored, transparent, low-odor rhamnolipids,

[0020] The weight-to-volume ratio of activated carbon to supernatant b in step (3) is 0.5-5 g:1 L, preferably 1-2 g:1 L;

[0021] The activated carbon used for adsorption treatment in step (3) is powdered activated carbon with a particle size of 100-300 mesh, preferably 180-200 mesh;

[0022] The adsorption treatment temperature in step (3) is 50℃~80℃, preferably 60℃~70℃;

[0023] The adsorption treatment time in step (3) is 1h to 3h, preferably 1.5h to 2.5h;

[0024] The pH of the adsorption treatment in step (3) is 5-8, preferably 5.5-7;

[0025] In step (4), the diatomaceous earth particles with large and small diameters in the decolorization and deodorization process are 80-400 mesh and 600 mesh, respectively.

[0026] ~2500 mesh, preferably 100-300 mesh and 800-2000 mesh;

[0027] In step (4), the mass ratio of large to small particle size diatomaceous earth in the decolorization and deodorization process is 5:1 to 10:1, preferably 6:1 to 8:1.

[0028] In step (4), the amount of diatomaceous earth with different particle sizes added during decolorization and deodorization is 50ppm to 1000ppm, preferably 80ppm to 800ppm;

[0029] In step (4), the stirring speed of the diatomaceous earth particles of different sizes during decolorization and deodorization is 100 rpm to 700 rpm, preferably 200 rpm to 500 rpm;

[0030] In step (4), the decolorization and deodorization time for diatomaceous earth particles of varying sizes is 0.5h to 5h, preferably 1h to 2h.

[0031] In step (5), the pore size of the filter membrane is 300 mesh to 900 mesh, preferably 350 mesh to 750 mesh;

[0032] The filtration pressure in step (5) is 0.1 MPa to 1 MPa, preferably 0.3 MPa to 0.5 MPa;

[0033] The filtration temperature in step (5) is 10℃~40℃, preferably 20℃~30℃.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The separation and purification method of this invention has simple process equipment, and the equipment and auxiliaries used are all conventional products, which has good universality and can realize the large-scale separation, purification and production of rhamnolipids.

[0036] This invention does not use organic solvents in the separation and purification process, which is more in line with the concept of green and natural products.

[0037] This invention uses activated carbon to initially adsorb impurities from a rhamnolipin solution and then innovatively selects diatomaceous earth particles of different sizes for decolorization and deodorization. During the decolorization and deodorization process, the smaller particle size of the diatomaceous earth effectively reduces the viscosity of the eluent and improves the elution efficiency. A filter membrane with a specific pore size is cleverly used to filter out the larger particle size of the diatomaceous earth, while a small amount of smaller particle size remains in the rhamnolipin solution. This way, the small particle size of the diatomaceous earth remaining in the rhamnolipin solution does not affect the transparent appearance of the product. Furthermore, because oxidation and Maillard reactions during storage can cause the rhamnolipin solution to darken in color and increase in odor, the small particle size of the diatomaceous earth remaining in the solution can also play a role in desorption and adsorption, thus ensuring the long-term light color and low odor of the rhamnolipin product. Detailed Implementation

[0038] The present invention will be further illustrated below with specific embodiments, but the present invention is not limited to the listed embodiments, and should also include any other known modifications within the scope of the claims of the present invention.

[0039] The equipment used in the embodiments and comparative examples:

[0040] Hitachi CP-NX Benchtop Centrifuge

[0041] Shanghai Jinghong Water Bath

[0042] Mettler FE28 Standard pH Meter

[0043] Edwards vacuum pump RV8

[0044] Raw materials used in the examples and comparative examples:

[0045] Industrial filter cloth, 16 mesh, 20 mesh, 50 mesh, 80 mesh;

[0046] Hubei Shufang 180 mesh, 190 mesh, 200 mesh activated carbon;

[0047] Haoxin wire mesh 350 mesh, 500 mesh, 750 mesh nylon mesh;

[0048] Wanhua prepared fermentation broth of Pseudomonas aeruginosa rhamnolipid.

[0049] Diatomite 100 mesh, 200 mesh, 300 mesh, 800 mesh, 1000 mesh, 1500 mesh, 2000 mesh;

[0050] Example 1:

[0051] 2000 ml of *Pseudomonas aeruginosa* rhamnolipin fermentation broth was filtered through 30 layers of 20-mesh industrial filter gauze to obtain approximately 1850 ml of filtrate A1. Filtrate A1 was centrifuged at 5500 rpm for 15 minutes, resulting in two layers: the supernatant B1, approximately 1800 ml of rhamnolipin solution, and the lower layer, containing particulate matter from the fermentation broth, including bacterial cells, proteins, and other impurities. 1.8 g of 200-mesh activated carbon was added to the supernatant B1, and the mixture was stirred at 66°C for 2 hours, while maintaining the pH at 6. The mixture was prepared by filtration under negative pressure to obtain approximately 1777 ml of preliminary eluent C1. 300 ppm of diatomaceous earth was added to the preliminary eluent C1, with a ratio of 6:1 (800 mesh to 100 mesh). The mixture was stirred at 500 rpm for 1 hour to obtain approximately 1780 ml of mixture D1. D1 was then filtered through a 350-mesh filter at 0.3 MPa and 25°C. The collected filtrate was approximately 1700 ml of the final product, a rhamnolipin solution. The initial color number was 56, and the odor was a light caramel aroma. After storage at 50°C for 3 weeks, the color number became 60, and the odor remained a light caramel aroma. In summary, this method yields high-purity rhamnolipin without the use of organic solvents.

[0052] Example 2:

[0053] 2000 ml of *Pseudomonas aeruginosa* rhamnolipin fermentation broth was filtered through 15 layers of 80-mesh industrial filter cloth to obtain approximately 1800 ml of filtrate A2. Filtrate A2 was centrifuged at 4500 rpm for 30 min, resulting in two layers: the supernatant B2, approximately 1650 ml of rhamnolipin solution, and the lower layer, containing particulate matter from the fermentation broth, including bacterial cells, proteins, and other impurities. 3.3 g of 200-mesh activated carbon was added to the supernatant B2, and the mixture was stirred at 70°C for 1.5 h, while controlling the pH period. With a pH of 5.5, a mixture was obtained, which was then centrifuged and filtered to obtain approximately 1630 ml of preliminary eluent C2. 80 ppm of diatomaceous earth was added to the preliminary eluent C2, with a ratio of 7:1 (1000 mesh to 200 mesh) to 1 h at 200 rpm, yielding approximately 1610 ml of mixture D2. D2 was then filtered through a 770-mesh filter at 0.5 MPa and 25°C, and the filtrate was collected as approximately 1590 ml of the final product, a rhamnolipin solution. The initial color number was 53, and the odor was a light caramel aroma. After storage at 50°C for 3 weeks, the color number became 62, and the odor remained a light caramel aroma. In summary, this method yields high-purity rhamnolipin without the use of organic solvents.

[0054] Example 3:

[0055] 2000 ml of *Pseudomonas aeruginosa* rhamnolipin fermentation broth was filtered through 20 layers of 50-mesh industrial filter cloth to obtain approximately 1800 ml of filtrate A3. Filtrate A3 was centrifuged at 4000 rpm for 20 minutes, resulting in two layers: the supernatant B3, approximately 1650 ml of rhamnolipin solution, and the lower layer, containing particulate matter from the fermentation broth, including bacterial cells, proteins, and other impurities. 2.5 g of 180-mesh activated carbon was added to the supernatant B3, and the mixture was stirred at 60°C for 2.5 hours, while controlling the pH period. With H set to 7, a mixture was obtained, which was then centrifuged and filtered to obtain approximately 1600 ml of preliminary eluent C3. 800 ppm of diatomaceous earth was added to the preliminary eluent C3, with a ratio of 1:8 between 2000 mesh and 300 mesh diatomaceous earth particles. The mixture was stirred at 500 rpm for 2 hours to obtain approximately 1580 ml of mixture D3. D3 was then filtered through a 500-mesh filter at 0.4 MPa and 20°C, and the collected filtrate was approximately 1550 ml of the final product, a rhamnolipin solution. The initial color number was 50, and the odor was a light caramel aroma. After storage at 50°C for 3 weeks, the color number became 59, and the odor remained a light caramel aroma. In summary, this method yields high-purity rhamnolipin without the use of organic solvents.

[0056] Example 4:

[0057] 2000 ml of *Pseudomonas aeruginosa* rhamnolipin fermentation broth was filtered through 25 layers of 16-mesh industrial filter cloth to obtain approximately 1800 ml of filtrate A4. Filtrate A4 was centrifuged at 4000 rpm for 20 minutes, resulting in two layers: the supernatant B4, approximately 1750 ml of rhamnolipin solution, and the lower layer, containing particulate matter from the fermentation broth, including bacterial cells, proteins, and other impurities. 2.6 g of 180-mesh activated carbon was added to the supernatant B4, and the mixture was stirred at 60°C for 2.5 hours, while maintaining the pH at 5. 8. The resulting mixture was then filtered under negative pressure to obtain approximately 1702 ml of preliminary eluent C4. 800 ppm of diatomaceous earth was added to the preliminary eluent C4, with a ratio of 1000 mesh to 200 mesh diatomaceous earth of 8:1. The mixture was stirred at 200 rpm for 1.5 hours to obtain approximately 1680 ml of mixture D4. D4 was then filtered through a 600-mesh filter at 0.4 MPa and 30°C. The collected filtrate was approximately 1600 ml of the final product, a rhamnolipin solution. Initial color number: 57, odor: light caramel; after 3 weeks of heat storage at 50°C, color number: 63, odor: light caramel. In summary, this method can yield high-purity rhamnolipin without the use of organic solvents.

[0058] Example 5:

[0059] 2000 ml of *Pseudomonas aeruginosa* rhamnolipin fermentation broth was filtered through 30 layers of 50-mesh industrial filter gauze to obtain approximately 1810 ml of filtrate A5. Filtrate A5 was centrifuged at 5000 rpm for 25 minutes, resulting in two layers: the supernatant B5, approximately 1740 ml of rhamnolipin solution, and the lower layer, containing particulate matter from the fermentation broth, including bacterial cells, proteins, and other impurities. 3.38 g of 190-mesh activated carbon was added to the supernatant B5, and the mixture was stirred at 68°C for 2 hours, while maintaining the pH at a controlled level. 7. Obtain the mixture, then filter under negative pressure to obtain approximately 1670 ml of preliminary eluent C5. Add 600 ppm of diatomaceous earth to the preliminary eluent C5, with a ratio of 800 mesh to 100 mesh diatomaceous earth particles of 8:1. Mix at 200 rpm for 1.5 hours to obtain approximately 1650 ml of mixture D5. Filter D5 through a 600 mesh filter at 0.5 MPa pressure and 30°C, collecting the filtrate as approximately 1600 ml of the final product, a rhamnolipin solution. Initial color number: 55, odor: light caramel; after 3 weeks of heat storage at 50°C, color number: 62, odor: light caramel. In summary, this method can obtain high-purity rhamnolipin without the use of organic solvents.

[0060] Comparative Example 1:

[0061] 2000 ml of *Pseudomonas aeruginosa* rhamnolipin fermentation broth was filtered through 30 layers of 20-mesh industrial filter gauze to obtain approximately 1840 ml of filtrate AA1. Filtrate AA1 was centrifuged at 5500 rpm for 15 minutes, resulting in two layers: the supernatant BB1, approximately 1810 ml of rhamnolipin solution, and the lower layer, containing particulate matter from the fermentation broth, including bacterial cells, proteins, and other impurities. 1.81 g of 200-mesh activated carbon was added to the supernatant BB1, and the mixture was stirred at 66°C for 2 hours while maintaining the pH at 6.0 to obtain a mixed solution. This solution was then filtered under negative pressure to obtain approximately 1771 ml of preliminary eluent CC1. CC1 was then filtered through a 350-mesh filter at 0.3 MPa and 25°C, and the collected filtrate was the final product, approximately 1750 ml of rhamnolipin solution. Initial product color: cloudy reddish-brown; odor: fermented odor. In summary, this method produces a cloudy red product with an unpleasant odor when no organic solvents are used and no diatomaceous earth is added.

[0062] Comparative Example 2:

[0063] 2000 ml of *Pseudomonas aeruginosa* rhamnolipin fermentation broth was filtered through 20 layers of 50-mesh industrial filter gauze to obtain approximately 1805 ml of filtrate AA2. Filtrate AA2 was centrifuged at 4000 rpm for 20 minutes, resulting in two layers: the supernatant BB2, approximately 1654 ml of rhamnolipin solution, and the lower layer, containing particulate matter from the fermentation broth including bacterial cells, proteins, and other impurities. 2.5 g of 180-mesh activated carbon was added to the supernatant BB2, and the mixture was stirred at 60°C for 2 minutes. The mixture was centrifuged and filtered for 5 hours, with the pH controlled at 7, to obtain a preliminary eluent, CC2, approximately 1600 ml. 800 ppm of diatomaceous earth (300 mesh) was added to CC2, and the mixture was stirred at 500 RPM for 2 hours to obtain a final eluent, DD2, approximately 1580 ml. DD2 was then filtered through a 500 mesh filter at 0.4 MPa and 20°C, and the filtrate was collected as approximately 1550 ml of the final product, a rhamnolipid solution. The initial color number was 57, and the odor was a light caramel smell. After 3 weeks of heat storage at 50°C, the color number became 205, and the odor was a mixture of caramel and fermented odor. In summary, this method, without the use of organic solvents, can further purify the color and odor of the product by adding large-particle diatomaceous earth.

[0064] Comparative Example 3:

[0065] 2000 ml of *Pseudomonas aeruginosa* rhamnolipin fermentation broth was filtered through 20 layers of 50-mesh industrial filter gauze to obtain approximately 1800 ml of filtrate AA3. Filtrate AA3 was centrifuged at 4000 rpm for 20 minutes, resulting in two layers: the supernatant BB3 (approximately 1651 ml of rhamnolipin solution) and the lower layer (particulate matter from the fermentation broth, including bacterial cells and proteins). 2.5 g of 180-mesh activated carbon was added to the supernatant BB3, and the mixture was stirred at 60°C for 2 minutes. The mixture was centrifuged and filtered for 5 hours, with the pH controlled at 7, to obtain a preliminary eluent, CC3, approximately 1600 ml. 800 ppm of diatomaceous earth (2000 mesh) was added to the CC3, and the mixture was stirred at 500 rpm for 2 hours to obtain a final eluent, DD3, approximately 1580 ml. D3 was then filtered through a 500-mesh filter at 0.4 MPa and 20°C, and the filtrate was collected as the final product, approximately 1550 ml of rhamnolipin solution. The initial color number was 100, and the odor was a light caramel smell. After 3 weeks of heat storage at 50°C, the color number became 150, and the odor changed to a mixture of caramel and fermented odor. In summary, this method, without the use of organic solvents, effectively maintains the color and odor stability of the rhamnolipin solution by adding small-particle diatomaceous earth.

Claims

1. A method for preparing a light-colored, transparent, low-odor rhamnolipid, comprising the following steps: (1) Filtration: The rhamnolipin fermentation broth is pre-filtered to remove large particulate matter in the fermentation broth to obtain filtrate a; (2) Centrifugation: Centrifuge the filtrate a to separate it and collect the supernatant b; (3) Adsorption treatment: Activated carbon was added to the supernatant b, and the temperature, time, pH and other conditions were controlled to complete the preliminary adsorption treatment. After centrifugation, the supernatant c was obtained. (4) Decolorization and deodorization: Add diatomaceous earth particles of different sizes to the supernatant c, stir and mix, and decolorize and deodorize to obtain the mixed liquid d; (5) Filtration: The mixture d is clarified by filtration to obtain a light-colored, transparent, low-odor clear solution, which is the finished product.

2. The production method according to claim 1, wherein In step (1), the filtration is selected from gauze filtration, mesh filtration, cotton filtration, and bulked cotton filtration, preferably gauze filtration; and / or, the gauze used in step (1) has 10-35 layers, preferably 15- 30 layers; and / or, the pore size of the filter in step (1) is 10 mesh to 100 mesh, preferably 16 to 80 mesh. Head.

3. The production method according to claim 1 or 2, characterized by, Step (2) The centrifugation speed is 3500 rpm to 6000 rpm, preferably 4000-5500 rpm; and / or, the centrifugation sterilization time in step (2) is 10 min to 35 min, preferably 15 min to 30 min.

4. The preparation method according to any one of claims 1-3, characterized in that, Step (3) The weight-to-volume ratio of the activated carbon used for adsorption treatment to the supernatant b is 0.5–5 g:1 L, preferably 1–2 g:1 L; and / or, the activated carbon used for adsorption treatment in step (3) is powdered activated carbon with a particle size of 100–300 mesh, preferably 180–200 mesh.

5. The preparation method according to any one of claims 1-4, characterized in that, Step (3) The adsorption treatment temperature is 50℃~80℃, preferably 60℃~70℃; and / or, step (3) The adsorption treatment time is 1h to 3h, preferably 1.5h to 2.5h; and / or, the adsorption treatment time in step (3) is... The pH for adsorption treatment is 5–8, preferably 5.5–7.

6. The preparation method according to any one of claims 1-5, characterized in that, Step (4) The diatomaceous earth particles used in the decolorization and deodorization process are 80-400 mesh and 600-2500 mesh, preferably 100-300 mesh and 800-2000 mesh; and / or, in step (4), the decolorization and deodorization process... The mass ratio of large to small particle size diatomite is 5:1 to 10:1, preferably 6:1 to 8:

1.

7. The preparation method according to any one of claims 1-6, characterized in that, Step (4) The amount of diatomaceous earth with varying particle sizes added during the decolorization and deodorization process is 50ppm to 1000ppm, preferably 80ppm to 800ppm.

8. The preparation method according to any one of claims 1-7, characterized in that, In step (4), the stirring speed of the diatomaceous earth particles of different sizes in the decolorization and deodorization process is 100 rpm to 700 rpm, preferably 200 rpm to 500 rpm; and / or, the decolorization and deodorization time of the diatomaceous earth particles of different sizes in step (4) is 0.5 h to 5 h, preferably 1 h to 2 h.

9. The preparation method according to any one of claims 1-8, characterized in that, The pore size of the filter membrane in step (5) is 300 mesh to 900 mesh, preferably 350 mesh to 750 mesh; and / or, the filtration pressure in step (5) is 0.1 MPa to 1 MPa, preferably 0.3 MPa to 0.5 MPa; and / or, the filtration temperature in step (5) is 10℃ to 40℃, preferably 20℃ to 30℃.