A method for separating and purifying di-rhamnolipids

Abstract

The present application relates to a kind of separation and purification method of di-rhamnolipid, provide di-rhamnolipid seed crystal;The pH of rhamnolipid fermentation broth removed bacterial body is adjusted to 2-4, add di-rhamnolipid seed crystal, stir uniformly after static, centrifugal and collect di-rhamnolipid crystal precipitate;Wherein, di-rhamnolipid seed crystal is 1-20% of the volume of rhamnolipid fermentation broth removed bacterial body;Di-rhamnolipid crystal is washed, dried;After washing, di-rhamnolipid crystal is added to ethyl acetate, centrifugal is obtained ethyl acetate phase, and di-rhamnolipid pure product is obtained after drying.The method of the present application can recover 50% of di-rhamnolipid from rhamnolipid fermentation broth, and the purity of di-rhamnolipid is >98%.Compared with prior art, the method of the present application can be simply separated from fermentation broth and recovered to obtain di-rhamnolipid pure product, avoid using traditional column chromatography and other operations, suitable for industrialized production of di-rhamnolipid.

Description

Technical Field

[0001] This invention relates to the field of dirhamnolipids, and more particularly to a method for the separation and purification of dirhamnolipids. Background Technology

[0002] Rhamnolipids are among the most widely studied and promising glycolipid biosurfactants. They exhibit excellent surface / interfacial activity, possessing multiple functions such as emulsification, foaming, cleaning, wetting, dispersing / dissolving hydrophobic substances, and surface modification. Furthermore, they are environmentally friendly and fully biodegradable, thus showing broad application prospects in petrochemicals, environmental remediation, and personal care. Based on the amount of rhamnose in their composition, rhamnolipids are classified into monorhamnolipids and dirhamnolipids.

[0003] Currently, the fermentation level of rhamnolipids has been significantly improved, and their industrial application prospects are promising. However, the efficient separation of rhamnolipids has not received sufficient attention, which seriously restricts the promotion and industrial application of rhamnolipids. The cost of separating rhamnolipids accounts for about 60-70% of the total production cost, and the separation of a single type of rhamnolipid (monororhamnetin or diororhamnetin) requires more complex and diversified processes, which also leads to the cost of this stage exceeding 90% of the total production cost. In order to further promote the application research of different types of rhamnolipids, it is urgent to build an efficient separation and purification process, which is also a technical problem that many researchers are currently striving to overcome.

[0004] Different types of rhamnolipides have different applications, especially dirhamnolipides, which have superior surface activity compared to monorhamnolipides and are therefore more promising. Furthermore, the purity of rhamnolipides determines their expected application scenarios, particularly in biopharmaceuticals and cosmetics, where direct contact with organisms, including humans, is required, necessitating high-purity rhamnolipides. Currently, most methods for obtaining pure mono / dirhamnolipides, both domestically and internationally, employ column chromatography. This method requires large amounts of toxic solvents, involves numerous separation steps, has extremely high separation costs, and low recovery rates, making it unsuitable for large-scale applications. Therefore, there is a need to develop efficient purification methods. Summary of the Invention

[0005] This method addresses the shortcomings of existing technologies by providing a method for the separation and purification of dirhamnolipids.

[0006] The objective of this invention can be achieved through the following technical solution: a method for separating and purifying dirhamnolipids, the method comprising the following steps:

[0007] Provides rhamnose glycolipid seed crystals;

[0008] Adjust the pH of the rhamnolipin fermentation broth (with Pseudomonas aeruginosa removed) to 2-4, add dirhamnolipin seed crystals, stir well, let stand, centrifuge, and collect the dirhamnolipin crystal precipitate; wherein, the dirhamnolipin seed crystals are 1-20% of the volume of the rhamnolipin fermentation broth (with Pseudomonas aeruginosa removed).

[0009] The dirhamnolipid crystals were washed and dried.

[0010] The washed dirhamnolipin crystals were added to ethyl acetate, centrifuged to obtain the ethyl acetate phase, and dried to obtain pure dirhamnolipin.

[0011] Furthermore, the dirhamnolipid seed crystals provided include:

[0012] Provide a pure solution of dirhamnolipin with a concentration higher than 20 g / L; adjust the pH of the pure solution of dirhamnolipin to 2-3 to obtain dirhamnolipin seed crystals.

[0013] Further, adjusting the pH of the rhamnolipin fermentation broth to 2-4 to remove Pseudomonas aeruginosa cells includes:

[0014] Sulfuric acid, hydrochloric acid, phosphoric acid, oxalic acid, citric acid, and acetic acid were added to the rhamnolipin fermentation broth to which Pseudomonas aeruginosa cells were removed, and the pH was adjusted to 2-4.

[0015] Furthermore, the dirhamnolipin seed crystals were 5% of the fermentation broth volume of rhamnolipin after removing Pseudomonas aeruginosa.

[0016] Further, add dirhamnolipin seed crystals, stir evenly, and let stand, including:

[0017] Add dirhamnolipin seed crystals, stir well, and let stand at 4°C.

[0018] Further, washing the dirhamnolipid crystals includes:

[0019] Dirhamnolipid crystals were washed with an acidic aqueous solution with a pH of 2-4.

[0020] Furthermore, the acidic aqueous solution is selected from sulfuric acid aqueous solution, hydrochloric acid aqueous solution, phosphoric acid aqueous solution, oxalic acid aqueous solution, citric acid aqueous solution or acetic acid aqueous solution.

[0021] Further drying of the dirhamnolipid crystals includes: baking or freeze-drying.

[0022] Furthermore, the dirhamnolipid crystals were freeze-dried.

[0023] Furthermore, the centrifugation conditions are 3000×g, 25℃, 1min.

[0024] Compared with existing technologies, the beneficial effects of the present invention are as follows:

[0025] This invention provides a method for the separation and purification of dirhamnolipin. The method involves adding acid to a fermentation broth (after removing bacterial cells) to a pH of 2-4, followed by the addition of dirhamnolipin crystals. This induces the dirhamnolipin in the fermentation broth to crystallize and precipitate. After washing and drying, the crystalline product of dirhamnolipin is obtained. This invention establishes a process of acidifying the fermentation broth and adding dirhamnolipin seed crystals to promote dirhamnolipin crystallization, thereby achieving efficient separation and purification of dirhamnolipin. The separation and purification method of this invention can efficiently separate and purify dirhamnolipin products from fermentation broth with excellent reproducibility. This method avoids the use of column chromatography to separate monorhamnolipin and dirhamnolipin, and has the advantages of high recovery rate and high purity. Moreover, the process is simple and easy to operate, making it suitable for large-scale industrial preparation of pure dirhamnolipin. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 A flowchart of a method for separating and purifying dirhamnolipids provided in an embodiment of the present invention. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] It should be noted that, unless otherwise specified, the features in the following embodiments and implementation methods can be combined with each other.

[0030] like Figure 1 As shown, the present invention provides a method for the isolation and purification of dirhamnolipids, the method comprising the following steps:

[0031] Step S1, providing dirhamnolipid seed crystals; including:

[0032] Provide a pure solution of dirhamnolipin with a concentration higher than 20 g / L; add acid to adjust the pH of the pure solution of dirhamnolipin to 2-3, which will result in the appearance of a large number of crystals, thus obtaining dirhamnolipin seed crystals.

[0033] Step S2: Adjust the pH of the rhamnolipin fermentation broth (with Pseudomonas aeruginosa removed) to 2-4, add dirhamnolipin seed crystals, stir well, let stand at 4°C, centrifuge, and collect the dirhamnolipin crystal precipitate; wherein, the dirhamnolipin seed crystals are 1-20% of the volume of the rhamnolipin fermentation broth (with Pseudomonas aeruginosa removed).

[0034] Furthermore, in this example, the pH value was adjusted to 2-4 by adding sulfuric acid, hydrochloric acid, phosphoric acid, oxalic acid, citric acid, and acetic acid to the rhamnolipin fermentation broth from which Pseudomonas aeruginosa cells were removed.

[0035] Preferably, the dirhamnolipin seed crystals are 5% of the volume of the rhamnolipin fermentation broth after removing Pseudomonas aeruginosa.

[0036] Furthermore, the centrifugation conditions are 3000×g, 25℃, 1min.

[0037] Step S3: Wash and dry the dirhamnolipid crystals.

[0038] Furthermore, in this example, an acidic aqueous solution with a pH of 2-4 is used to wash the dirhamnolipid crystals; wherein the acidic aqueous solution is selected from sulfuric acid aqueous solution, hydrochloric acid aqueous solution, phosphoric acid aqueous solution, oxalic acid aqueous solution, citric acid aqueous solution or acetic acid aqueous solution.

[0039] Furthermore, the drying process employs either oven drying or freeze drying; freeze drying is preferred.

[0040] Step S4: Add the washed dirhamnolipin crystals to ethyl acetate, centrifuge to obtain the ethyl acetate phase, and dry to obtain pure dirhamnolipin.

[0041] Furthermore, the centrifugation conditions are 3000×g, 25℃, 1min.

[0042] The method for separating and purifying dirhamnolipids provided by the present invention will be further described below with reference to specific examples.

[0043] Example 1: Preparation of Fermentation Broth

[0044] Pseudomonas aeruginosa PAO1 (ATCC 15692) from the American Type Culture Collection was activated for 48 h in LB medium containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl. Subsequently, it was inoculated at a 3% (v / v) in 60 mL of 250 mL flask containing 90 g / L corn oil, 8 g / L NaNO3, 0.2 g / L MgSO4, 1 g / L NaCl, 1 g / L KCl, 0.04 g / L CaCl2, 5 mL / L H3PO4 (85%), and 0.02 g / L Fe. 2+The rhamnolipin fermentation broth was prepared in a fermentation medium containing 1 mL / L trace element solution (containing 1.5 g / L ZnSO4·7H2O, 1.5 g / L MnSO4·H2O, 0.3 g / L H3BO3, 0.15 g / L CoCl2·6H2O, and 0.15 g / L CuSO4·5H2O) and cultured at 37 °C and 260 rpm for 6–8 days. After centrifugation at 10000 × g and 25 °C for 10 min, a sterile supernatant of the rhamnolipin fermentation broth was obtained, with a rhamnolipin concentration of 55 g / L.

[0045] Example 2: Isolation and purification method of dirhamnolipid

[0046] Aliquot 1 mL of sterile rhamnolipin fermentation broth into 1.5 mL conical centrifuge tubes, adjust the pH to 3 with sulfuric acid, and resuspend thoroughly. Add 10% of the volume of dirhamnolipin crystal suspension as seed crystals, after removing *Pseudomonas aeruginosa*. Resuspend thoroughly again and incubate at 4°C for 10 min. Dirhamnolipin crystals will form in the centrifuge tubes. Centrifuge to obtain the dirhamnolipin crystal precipitate. Wash the dirhamnolipin precipitate twice with sulfuric acid aqueous solution (pH=3) and centrifuge to collect the precipitate. Dissolve the dirhamnolipin in ethyl acetate, centrifuge to obtain an ethyl acetate solution of dirhamnolipin, and the precipitate consists mostly of water-soluble impurities. Dry the ethyl acetate to obtain pure dirhamnolipin. The total recovery rate of dirhamnolipin, determined by the sulfuric acid-anthrone method, was 48.2%, with a purity >98%.

[0047] Example 3: Effect of pH on the recovery rate of dirhamnolipids

[0048] Aliquot 1 mL of sterile rhamnolipin fermentation broth into 1.5 mL conical centrifuge tubes, and adjust the pH to different values ​​(2-5) with sulfuric acid. After thorough resuspending, add dirhamnolipin seed crystals, which constitute 10% of the fermentation broth volume after removing Pseudomonas aeruginosa. Resuspend again and incubate at 4°C for 10 min. Crystals form in the centrifuge tubes. Treat the precipitated dirhamnolipin crystals using the aforementioned separation method (Example 2) to obtain pure dirhamnolipin. As shown in Table 1, the results indicate that lower pH is more favorable for the recovery of dirhamnolipin. When the pH is between 2 and 4, the recovery rate of dirhamnolipin is 45%-48%, and the purity is >98%.

[0049] Table 1: pH and recovery rate of dirhamnolipid

[0050] pH Dirhamnolipid recovery rate 2 46.61％ 3 45.11％ 4 47.92％ 5 27.80％

[0051] Example 4: Effect of temperature on the recovery rate of dirhamnolipids

[0052] Aliquot 1 mL of the rhamnolipin fermentation broth (with cell removed) into 1.5 mL conical centrifuge tubes. Adjust the pH to 3 with sulfuric acid, resuspend the tubes, and add dirhamnolipin seed crystals (10% of the fermentation broth volume after removing *Pseudomonas aeruginosa* cells). After resuspending, incubate at 4°C, 15°C, and 25°C, respectively, where crystals will form in the centrifuge tubes. Treat the dirhamnolipin crystal precipitate using the aforementioned separation method (Example 2) to obtain pure dirhamnolipin. As shown in Table 2, the results indicate that lower temperatures are more conducive to the recovery of dirhamnolipin. When incubated at 4°C, crystals form within 10 minutes, with a disaccharide recovery rate of 46.34% and a purity >98%. Therefore, the preferred temperature for this process is 4°C.

[0053] Table 2: Temperature and Dirhamnolipid Recovery

[0054] temperature Dirhamnolipid recovery rate 4℃ 46.34％ 15℃ 40.77％ 25℃ 22.89％

[0055] Example 5 Effect of Seed Addition Amount

[0056] The fermentation broth of rhamnolipin, after removing the bacterial cells, was dispensed into 1 mL aliquots into 1.5 mL conical centrifuge tubes. Sulfuric acid was added to adjust the pH to 3, and the tubes were resuspended. Different amounts of dirhamnolipin crystal suspension (1%, 2%, 5%, 8%, 10%, 12%, 16%, 20%, v / v) were added as seed crystals. After resuspending and homogenizing, the mixture was incubated at 4°C. Crystals formed in the centrifuge tubes. The dirhamnolipin crystal precipitate was then treated using the aforementioned separation method (Example 2) to obtain pure dirhamnolipin. As shown in Table 3, the results indicate that the amount of seed crystals added has no significant effect on the recovery rate of dirhamnolipin, remaining between 46% and 49%, with a purity >98%. However, the higher the amount of seed crystals added, the shorter the crystallization time. Therefore, a seed crystal addition of 5% is preferred in this process.

[0057] Table 3: Seed crystal addition amount and bisrhamnolipid recovery rate

[0058] Seed crystal addition amount Crystallization time Dirhamnolipid recovery rate 1％ More than 1 hour 46.42％ 2％ No more than 1 hour 47.32％ 5％ No more than 10 minutes 46.58％ 8％ No more than 10 minutes 46.77％ 10％ No more than 10 minutes 48.15％ 12％ No more than 10 minutes 48.08％ 16％ No more than 10 minutes 47.99％ 20％ No more than 10 minutes 47.46％

[0059] Comparative Example 1: The acidified fermentation broth could not crystallize without the addition of crystals, making it impossible to directly recover dirhamnolipid.

[0060] The fermentation broth containing rhamnolipin with the bacterial cells removed was dispensed into 1 mL aliquots into 1.5 mL conical centrifuge tubes. Sulfuric acid was added to adjust the pH to 2. The centrifuge tubes were then suspended and allowed to stand at 4°C for 120 h. Only acid precipitation was produced in the centrifuge tubes, and no dirhamnolipin crystals were formed, so the separation of dirhamnolipin could not be achieved.

[0061] Comparative Example 2: Dirhamnolipids were separated and purified using a conventional column chromatography process.

[0062] The fermentation broth containing rhamnolipin (with cell removed) was taken, and sulfuric acid was added to adjust the pH to 2-3, causing acid precipitation of the rhamnolipin. The precipitate was extracted using a CHCl3:CH3OH (2:1 v / v) system. The extract was collected, concentrated by rotary evaporation, and dried under vacuum to obtain crude rhamnolipin. To further obtain dirhamnolipin, the crude product was separated by silica gel column chromatography. The column packing material was silica gel (240-425 mesh). Neutral lipids were removed by CHCl3:CH3OH (50:3 v / v), monorhamnolipin was eluted by CHCl3:CH3OH (50:5 v / v), and finally dirhamnolipin was obtained by elution with CHCl3:CH3OH (50:50 v / v). After rotary evaporation and drying, the content of dirhamnolipin was determined by the sulfuric acid-anthrone method. Finally, the recovery rate of dirhamnolipin obtained by the conventional column chromatography separation and purification process was only 30%, and the purity was 95.4%.

[0063] In summary, this invention provides a method for the separation and purification of dirhamnolipin. By adding acid to the fermentation broth (after removing bacterial cells) to a pH of 2-4, and then adding dirhamnolipin crystals, the dirhamnolipin in the fermentation broth can be induced to crystallize and precipitate. After washing and drying, the crystalline product of dirhamnolipin is obtained. This method can efficiently separate and purify dirhamnolipin from the fermentation broth, with excellent reproducibility. This method avoids the use of column chromatography to separate monorhamnolipin and dirhamnolipin, and has the advantages of high recovery rate and high purity. Moreover, the process is simple and easy to operate, making it suitable for large-scale industrial preparation of pure dirhamnolipin.

[0064] The above description is merely a preferred example of the present invention and is not intended to limit the scope of protection of the present invention. In addition to the above embodiments, the present invention may have other implementations. All technical solutions formed by equivalent substitutions or equivalent changes fall within the scope of protection claimed by the present invention.

Claims

1. A method for the isolation and purification of dirhamnolipids, characterized in that, The method includes the following steps: Provides rhamnose glycolipid seed crystals; Adjust the pH of the rhamnolipin fermentation broth (with cell removed) to 2-4, add dirhamnolipin seed crystals, stir well, let stand at 4°C, centrifuge, and collect the dirhamnolipin crystal precipitate; wherein, the dirhamnolipin seed crystals account for 5-20% of the volume of the rhamnolipin fermentation broth (with cell removed); The dirhamnolipid crystals were washed and dried. The washed dirhamnolipin crystals were added to ethyl acetate, centrifuged to obtain the ethyl acetate phase, and dried to obtain pure dirhamnolipin.

2. The method for separating and purifying dirhamnolipids according to claim 1, characterized in that, The dirhamnolipid seed crystals provided include: Provide a pure solution of bisrhamnolipin with a concentration higher than 20 g / L; The pH of the pure solution of dirhamnolipin was adjusted to 2-3 to obtain dirhamnolipin seed crystals.

3. The method for separating and purifying dirhamnolipids according to claim 1, characterized in that, Adjusting the pH of the rhamnolipin fermentation broth to 2-4 after removing the bacterial cells includes: Add sulfuric acid, hydrochloric acid, phosphoric acid, oxalic acid, citric acid, and acetic acid to the rhamnolipin fermentation broth after removing the bacterial cells to adjust the pH to 2-4.

4. The method for separating and purifying dirhamnolipids according to claim 1, characterized in that, The dirhamnolipin seed crystals were 5% of the volume of the rhamnolipin fermentation broth after removing the bacterial cells.

5. The method for separating and purifying dirhamnolipids according to claim 1, characterized in that, Washing of dirhamnolipid crystals includes: Dirhamnolipid crystals were washed with an acidic aqueous solution with a pH of 2-4.

6. The method for separating and purifying dirhamnolipids according to claim 5, characterized in that, The acidic aqueous solution is selected from sulfuric acid aqueous solution, hydrochloric acid aqueous solution, phosphoric acid aqueous solution, oxalic acid aqueous solution, citric acid aqueous solution or acetic acid aqueous solution.

7. The method for separating and purifying dirhamnolipids according to claim 1, characterized in that, Drying of dirhamnolipid crystals includes methods such as baking or freeze-drying.

8. The method for separating and purifying dirhamnolipids according to claim 7, characterized in that, The dirhamnolipid crystals were freeze-dried.

9. The method for separating and purifying dirhamnolipids according to claim 1, characterized in that, The centrifugation conditions were 3000 × g, 25°C, 1 min.

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