A method for isolating and purifying 3-hydroxydecanoic acid from pseudomonas bacteria

Abstract

The application provides a method for separating and purifying 3-hydroxydecanoic acid from Pseudomonas bacteria. First, the fermentation product of Pseudomonas bacteria is decomposed by alkali to obtain monomers of 3-hydroxydecanoic acid. Then, activated carbon is used for decolorization, and microfiltration membrane and ultrafiltration membrane are used for treatment to remove macromolecular impurities. Finally, a sodium 3-hydroxydecanoate solution is loaded onto a polystyrene-divinylbenzene macroporous resin column modified by polyethyleneimine, and eluted with an ethanol-water (volume ratio 1:1) solution with a pH of 11. The eluate is collected, ethanol is evaporated, a purified sodium 3-hydroxydecanoate solution is obtained, hydrochloric acid is added to adjust the pH to 3-4, and the precipitate is collected by centrifugation and dried to obtain a 3-hydroxydecanoic acid product. The content of 3-hydroxydecanoic acid is detected by liquid chromatography, and the total content of 3-hydroxydecanoic acid in the purified product is higher than 90%. The method uses less organic solvent, the steps are simple, the purity of the obtained product is high, and the application requirements of cosmetics and pharmaceutical intermediates are met.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for separating and purifying 3-hydroxydecanoic acid from a natural mixture. BACKGROUND

[0002] 3-hydroxy fatty acids in nature mainly come from microbial fermentation products or insect secretions. For example, many bacteria of the genus Pseudomonas can secrete polyhydroxyalkanoates (PHA), which exist in the form of intragranular inclusions in the cytoplasm (CN101270345; Zheng et al., Chinese Journal of Chemical Engineering, 2004). PHA belongs to natural polymer materials and has application prospects as biodegradable plastics. At the same time, the monomer 3-hydroxy fatty acid (including 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxylauric acid, etc.) has the functions of bacteriostasis and oil control, and can be used as an active component of cosmetics. In addition, 3-hydroxy fatty acid chains also exist in many bacterial secreted lipopolysaccharides (Kutschera et al. Science, 2019), so the source is very rich. Interestingly, most of the naturally secreted 3-hydroxy fatty acids are in the R configuration, which has the strongest biological activity (Kutschera et al. Science, 2019), and can be used as a chiral intermediate to synthesize antibiotics, vitamins, fragrances and amino acids, etc.

[0003] It is often not economical to synthesize 3-hydroxydecanoic acid by chemical method, and a mixture of R / S configurations is formed. Therefore, it is a more reasonable and green way to extract 3-hydroxydecanoic acid from natural products. Because the secreted products of bacteria are numerous, it is a complex mixture of proteins, polysaccharides, lipids and other organic matters, and there are still technical difficulties in how to effectively separate 3-hydroxydecanoic acid from impurities to obtain a product with a purity higher than 90%. For example, some patents methylate hydroxy fatty acids and use organic solvents for extraction, but they cannot obtain hydroxy fatty acid products (CN201180035259.4). There are also studies on producing 3-hydroxydecanoic acid by genetically recombining microbial strains (CN02117571.3), but it is not suitable for all strains. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings of the prior art and provide a method for separating and purifying 3-hydroxydecanoic acid from complex microbial Pseudomonas bacteria fermentation products.

[0005] The technical solution adopted by the present application is as follows:

[0006] A method for separating and purifying 3-hydroxydecanoic acid from Pseudomonas bacteria, comprising the following steps:

[0007] (1) obtaining a Pseudomonas bacterial fermentation product, wherein the Pseudomonas bacterial fermentation product mainly contains 3-hydroxy fatty acids and polymers thereof; subjecting the Pseudomonas bacterial fermentation product to alkaline treatment to decompose 3-hydroxy fatty acid polymers in the Pseudomonas bacterial fermentation product, and obtaining a first mixture;

[0008] (2) adding activated carbon to the first mixture obtained in step (1) to decolorize, and then subjecting the mixture to microfiltration and / or ultrafiltration to remove macromolecular impurities, and then adding hydrochloric acid to precipitate 3-hydroxydecanoic acid to obtain a second mixture;

[0009] (3) dissolving the second mixture obtained in step (2) with sodium hydroxide to obtain a sodium 3-hydroxydecanoate solution, and loading the sodium 3-hydroxydecanoate solution onto a resin column filled with polyethyleneimine-modified polystyrene-divinylbenzene macroporous resin to perform adsorption-elution for further purification to obtain a product, wherein the purity of 3-hydroxydecanoic acid in the product is greater than 90%;

[0010] The polyethyleneimine-modified polystyrene-divinylbenzene macroporous resin is prepared by the following method:

[0011] 1 part by weight of polystyrene-divinylbenzene macroporous resin is immersed in an aqueous solution containing 0.1-0.5 parts by weight of polyethyleneimine for 2-24 hours; then it is taken out and rinsed with pure water, and then transferred to an aqueous solution containing 0.02-0.1 parts by weight of glutaraldehyde for 24 hours, and then the resin is taken out and rinsed to obtain the polyethyleneimine-modified polystyrene-divinylbenzene macroporous resin.

[0012] Further, in the aqueous solution containing 0.1-0.5 parts by weight of polyethyleneimine, the molecular weight of polyethyleneimine is 70 KDa, and the mass fraction is 5-25 wt%; in the aqueous solution containing 0.02-0.1 parts by weight of glutaraldehyde, the mass fraction of glutaraldehyde is 0.2-1 wt%.

[0013] Further, in step (1), the Pseudomonas bacterial fermentation product is obtained by centrifuging the fermentation broth of Pseudomonas bacteria, adding n-hexane to the centrifugal precipitate at a volume ratio of 1:1, dissolving and standing, collecting the organic phase after layering, and evaporating to remove n-hexane to obtain the Pseudomonas bacterial fermentation product.

[0014] Further, in step (1), the Pseudomonas bacterial fermentation product is subjected to alkaline treatment to decompose the hydroxy fatty acid polymers therein, and a first mixture is obtained, which is specifically as follows:

[0015] In the Pseudomonas bacterial fermentation product, sodium hydroxide solution is added, wherein the mass ratio of Pseudomonas bacterial fermentation product to sodium hydroxide is 1:1-1:10, the temperature is raised to 90°C, and stirring is performed for 2-4 hours to decompose the hydroxy fatty acid polymers therein by alkaline treatment to obtain a first mixture.

[0016] Further, in the step (2), the first mixture obtained in the step (1) is added with activated carbon for decolorization, and the amount of the activated carbon added is 1-10 wt%, and after sufficient decolorization, the activated carbon is removed by filtration.

[0017] Further, in the step (2), macromolecular impurities are removed by using a microfiltration membrane and / or an ultrafiltration membrane, and then hydrochloric acid is added to precipitate 3-hydroxydecanoic acid to obtain a second mixture, specifically:

[0018] First, a microfiltration membrane with a pore size of 0.22 μm is used for filtration, and then an ultrafiltration membrane with a molecular weight cut-off of 2000 is used for filtration, and the filtrate is collected and added with hydrochloric acid to adjust the pH to 2-4 to precipitate 3-hydroxydecanoic acid, and the precipitate is collected, centrifuged and dried to obtain the second mixture.

[0019] Further, in the step (3), the concentration of the sodium 3-hydroxydecanoate solution is 50-100 g / L, and the pH is 6.5-8.0.

[0020] Further, the sodium 3-hydroxydecanoate solution is loaded onto a resin column packed with polyethyleneimine-modified polystyrene-divinylbenzene macroporous resin for adsorption-elution to further purify the product, specifically:

[0021] A resin column is packed with polyethyleneimine-modified polystyrene-divinylbenzene macroporous resin, and the sodium 3-hydroxydecanoate solution is loaded onto the resin column at a flow rate of 0.5-2 BV / h, and the content of 3-hydroxydecanoic acid at the outlet is monitored, and when it reaches 5 g / L, the feeding is stopped, and the resin column is washed with pure water until the content of 3-hydroxydecanoic acid at the outlet is less than 0.5 g / L; finally, an ethanol-water solution with a pH of 11 and a volume ratio of 1:1 is used for elution, and the elution flow rate is 0.5-2 BV / h; after the eluate is collected, the ethanol is evaporated to obtain a purified sodium 3-hydroxydecanoate solution, hydrochloric acid is added to adjust the pH to 3-4, and the precipitate is collected by centrifugation and dried to obtain the product.

[0022] Further, the Pseudomonas bacteria include one or more of P. putida, P. oleovorans and P. aeruginosa.

[0023] Compared with the existing separation and purification method of 3-hydroxydecanoic acid, the method has the following advantages and novelty: 1) the raw material is derived from bacterial fermentation broth, which is abundant and belongs to natural product; 2) the method combines microfiltration / ultrafiltration membrane process and resin adsorption-elution technology, wherein the polyethylene imine modified polystyrene-divinylbenzene macroporous resin has a large number of amine groups, which can more efficiently remove macromolecular impurities, uncharged small molecular impurities and hydrophobic impurities, so as to obtain a product with a purity of >90%. The purified product can meet the requirements of purity in the application of daily chemical and pharmaceutical intermediates. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 LC-MS spectrum of the alkali hydrolysis product of Example 3

[0025] Figure 2 HPLC spectrum of the 3-hydroxydecanoic acid product DETAILED DESCRIPTION

[0026] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0027] Example 1: Preparation of polyethylene imine modified polystyrene-divinylbenzene macroporous resin

[0028] 100 g of commercially available polystyrene-divinylbenzene (brand D101) macroporous resin was immersed in 200 ml of an aqueous solution containing 5 wt% of polyethylene imine (molecular weight 70 kDa) and gently stirred for 24 h. Subsequently, the macroporous resin was taken out, rinsed once with pure water, transferred to 100 ml of an aqueous solution containing 0.2 wt% of glutaraldehyde, and continued to be gently stirred for 24 h. The resin was taken out and soaked and rinsed thoroughly. At this time, the polyethylene imine was cross-linked on the macroporous resin, making it have a large number of amine groups.

[0029] Example 2: Preparation of polyethylene imine modified polystyrene-divinylbenzene macroporous resin

[0030] 100 g of commercially available polystyrene-divinylbenzene (brand D101) macroporous resin was immersed in 200 ml of an aqueous solution containing 5 wt% of polyethylene imine (molecular weight 70 kDa) and gently stirred for 24 h. Subsequently, the macroporous resin was taken out, rinsed once with pure water, transferred to 100 ml of an aqueous solution containing 0.2 wt% of glutaraldehyde, and continued to be gently stirred for 24 h. The resin was taken out and soaked and rinsed thoroughly. At this time, the polyethylene imine was cross-linked on the macroporous resin, making it have a large number of amine groups.

[0031] Example 3: Obtaining fermentation products of *Pseudomonas putida*

[0032] Culture of *Pseudomonas putida* ATCC49128: The culture medium consisted of 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 2 g / L glucose, and 6 vol% soybean oil. After sterilizing the culture medium at 121°C for 30 minutes (glucose was sterilized separately at 115°C for 20 minutes), the medium was cooled to room temperature and inoculated with 3 vol% *Pseudomonas putida* seeds. The medium was then cultured at 37°C on a shaker at 200 rpm for 48 hours.

[0033] 1 L of fermentation broth was collected, inactivated at 121 °C for 15 minutes, and the cells were collected by centrifugation, yielding approximately 168 g of wet cells. The cells were resuspended in a small amount of water and homogenized at high speed for 10 minutes to lyse the cells. Subsequently, 200 ml of n-hexane was added at a 1:1 volume ratio, stirred thoroughly to dissolve, and allowed to stand for separation. The organic phase was collected, and the n-hexane was evaporated to remove it, yielding approximately 82 g of solid, which was the fermentation product of *Pseudomonas putida*. The fermentation product was methylated and analyzed by gas chromatography-mass spectrometry (GC-MS), as shown in the spectrum. Figure 1 As shown in Table 1, the composition and content of the main peaks were analyzed. It is evident that the fermentation products of *Pseudomonas putida* are mainly 3-hydroxy fatty acids and their polymers, with 3-hydroxydecanoic acid being the predominant fatty acid. Other medium-chain hydroxy fatty acids and long-chain fatty acids are also present, with the long-chain fatty acids (oleic acid and linoleic acid) likely originating from incompletely metabolized soybean oil. Furthermore, the hydrolysate may contain non-fatty acid impurities, which were not detected by LC-MS.

[0034] Table 1: Analysis of fatty acid components in the product (LC-MS method)

[0035] Ingredients Residence time (min) Content, wt% 3-hydroxyoctanoic acid 13.340 2.3 3-hydroxydecanoic acid 14.627 51.1 3-hydroxydodecanoic acid 17.088～17.168 4.8 Linoleic acid 21.225 11.5 Oleic acid 21.282 11.6

[0036] Example 4: Obtaining fermentation products of Pseudomonas oleifera

[0037] Cultivation of Pseudomonas oleovorans ATCC8062: The culture medium consisted of 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium nitrate, 5 g / L ammonium sulfate, 2 wt% methyl decanoate, and 3 vol% soybean oil. After sterilizing the culture medium at 121℃ for 30 minutes, it was cooled to room temperature and inoculated with 3 vol% Pseudomonas oleovorans seeds. The culture was then incubated at 37℃ on a shaker at 200 rpm for 48-72 hours until no floating oil remained in the culture medium.

[0038] The fermentation broth 1 L was collected and inactivated at 121 °C for 15 min. The bacterial cells were collected by centrifugation, and the wet weight of the bacterial cells was about 122 g. The bacterial cells were resuspended in a small amount of water and homogenized at high speed for 10 min to break the cells. Then, 200 ml of n-hexane was added at a volume ratio of 1:1, and the mixture was stirred to dissolve the n-hexane. After the mixture was allowed to separate into layers, the organic phase was collected and the n-hexane was evaporated to obtain about 61 g of solid, which was the fermentation product of Pseudomonas oleovorans. The fermentation product was methylated and analyzed by gas chromatography-mass spectrometry. The composition and content of the main peaks were analyzed, and the results are shown in Table 2. It can be seen that the fermentation product of Pseudomonas oleovorans mainly contains 3-hydroxy fatty acids and their polymers, with 3-hydroxy decanoic acid as the main component. In addition, there are other medium-chain hydroxy fatty acids and long-chain fatty acids, which may come from the incomplete metabolism of soybean oil.

[0039] Table 2: Analysis of product composition (LC-MS method)

[0040]

[0041]

[0042] Example 5: Isolation and purification of 3-hydroxy decanoic acid from Pseudomonas bacteria

[0043] (1) Sodium hydroxide solution was added to the solid obtained in Examples 3 and 4 at a mass ratio of Pseudomonas bacteria fermentation product to sodium hydroxide of 1:1. The concentration of the sodium hydroxide solution was 10 wt%, and the mixture was heated to 90 °C and stirred for 2 h. At this time, the hydroxy fatty acid (mainly hydroxy decanoic acid) polymers were decomposed into sodium 3-hydroxy decanoate.

[0044] (2) The aqueous solutions obtained by alkali treatment in Examples 3 and 4 were labeled as 1# and 2# samples, respectively. Each sample was taken 500 ml, activated carbon was added, and the mixture was stirred to decolorize. Then, the activated carbon was removed by filtration. Subsequently, the mixture was pretreated using a microfiltration membrane with a pore size of 0.22 μm, and further treated by ultrafiltration membrane with a molecular weight cutoff of 2000 to remove large molecular impurities. Finally, hydrochloric acid was added to adjust the pH to 2-3, and 3-hydroxy decanoic acid was precipitated. After centrifugation and drying, the crude product was obtained. The content of 3-hydroxy decanoic acid was determined by HPLC refractive index method. The results are shown in Table 3. It can be seen that the addition of activated carbon has a significant effect on the decolorization rate. When 10 wt% activated carbon is added, the decolorization rate can reach more than 98.5%, and the product is basically colorless and transparent. However, the crude product after membrane treatment appears as a light yellow to dark yellow solid, and its purity is not more than 72.35%. This indicates that although a certain degree of purification has been achieved, the purity required for application has not yet been reached.

[0045] Table 3: Decolorization and membrane filtration to remove impurities

[0046]

[0047] (3) Respectively, after repeating step (2) for 2 times, the mixed obtained crude products were labeled as 1#-3 / 1#-4 and 2#-3 / 2#-4 crude products, and dissolved with sodium hydroxide, and the content of 3-hydroxydecanoic acid was adjusted to 50-100 g / L, and the pH was adjusted to 6.5-8.0, to obtain a sodium 3-hydroxydecanoate solution. A resin column was filled with 100 g of polystyrene-divinylbenzene macroporous resin modified by polyethyleneimine prepared in Examples 1 and 2, and pure water was fed into the column until the outlet pH was less than 8. The sodium 3-hydroxydecanoate solution was loaded onto the resin column at a flow rate of 0.5-2 BV / h, and the content of 3-hydroxydecanoic acid at the outlet was monitored, and when it reached 5 g / L, the feeding was stopped, and the resin column was washed with pure water until the content of 3-hydroxydecanoic acid at the outlet was less than 0.5 g / L. Finally, an ethanol-water (volume ratio 1:1) solution with a pH of 11 was used for elution, and the elution flow rate was 0.5-2 BV / h. After collecting the eluate, the ethanol was evaporated to obtain a purified sodium 3-hydroxydecanoate solution, hydrochloric acid was added to adjust the pH to 3-4, and the precipitate was collected by centrifugation and dried to obtain 3-hydroxydecanoic acid product. The unmodified polystyrene-divinylbenzene macroporous resin was used as a control group, and under the same experimental conditions as above, almost no 3-hydroxydecanoic acid product was obtained, i.e. the unmodified resin did not adsorb 3-hydroxydecanoic acid, and the 3-hydroxydecanoic acid could not be further purified.

[0048] Table 4 Resin treatment method parameters and experimental results

[0049]

[0050] The content of 3-hydroxydecanoic acid was detected by HPLC, the mobile phase was acetonitrile: water = 60:40, the pH was adjusted to 4.0 with acetic acid, the chromatographic column was a C18 column 250 mm, and the flow rate was 0.5 ml / min. 0.1 g of product was accurately weighed and diluted to 10 g / L with the mobile phase, and 20 μl of sample was injected. The content and purity were calculated according to the standard curve, and the results are shown in Table 5. Among them, a typical liquid chromatogram (1#-1) is shown in Figure 2 , in which the largest peak is 3-hydroxydecanoic acid, and the two small peaks before and after are 3-hydroxyoctanoic acid and 3-hydroxydodecanoic acid, respectively. It can also be seen from the chromatogram that the purity of the product is high.

[0051] Table 5 Purity determination results of 3-hydroxydecanoic acid product

[0052] Sample 1#-1 1#-2 1#-3 1#-4 2#-1 2#-2 2#-3 2#-4 3-hydroxydecanoic acid purity % 91.2 93.1 92.3 94.1 94.2 90.1 93.1 93.8

[0053] Obviously, the above examples are only examples for the sake of clarity, and are not limitations on the embodiments. Based on the above description, those skilled in the art can also make other different forms of changes or variations. It is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for isolating and purifying 3-hydroxydecanoic acid from Pseudomonas bacteria, characterized by, The method comprises the following steps: (1) obtaining a Pseudomonas bacterial fermentation product mainly composed of 3-hydroxy fatty acids and polymers thereof, and subjecting the Pseudomonas bacterial fermentation product to alkali treatment to decompose 3-hydroxy fatty acid polymers therein and obtain a first mixture; (2) adding activated carbon to the first mixture obtained in step (1) to decolorize, and then subjecting the mixture to microfiltration membrane and / or ultrafiltration membrane treatment to remove macromolecular impurities, and then adding hydrochloric acid to precipitate 3-hydroxydecanoic acid to obtain a second mixture; (3) dissolving the second mixture obtained in step (2) with sodium hydroxide to obtain a 3-hydroxydecanoic acid sodium solution, and loading the 3-hydroxydecanoic acid sodium solution onto a resin column filled with polyethyleneimine-modified polystyrene-divinylbenzene macroporous resin to perform adsorption-elution for further purification to obtain a product with a 3-hydroxydecanoic acid purity of greater than 90%; The polyethyleneimine-modified polystyrene-divinylbenzene macroporous resin is prepared by the following method: 1 part by weight of polystyrene-divinylbenzene macroporous resin is immersed in an aqueous solution containing 0.1-0.5 parts by weight of polyethyleneimine for 2-24 hours, then taken out and rinsed with pure water, and then transferred to an aqueous solution containing 0.02-0.1 parts by weight of glutaraldehyde for 24 hours, and then the resin is taken out and rinsed to obtain the polyethyleneimine-modified polystyrene-divinylbenzene macroporous resin.

2. The method of claim 1, wherein, In step (1), the Pseudomonas bacterial fermentation product is obtained by centrifuging a fermentation broth of Pseudomonas bacteria, adding n-hexane to the centrifugal precipitate at a volume ratio of 1:1, dissolving and standing, collecting the organic phase after layer separation, and evaporating to remove n-hexane.

3. The method of claim 1, wherein, In step (1), the Pseudomonas bacterial fermentation product is subjected to alkali treatment to decompose hydroxy fatty acid polymers therein and obtain a first mixture, specifically: Sodium hydroxide solution is added to the Pseudomonas bacterial fermentation product, the mass ratio of the Pseudomonas bacterial fermentation product to sodium hydroxide is 1:1-1:10, the temperature is raised to 90°C, and stirring is performed for 2-4 hours to decompose the hydroxy fatty acid polymers therein by alkali treatment to obtain the first mixture.

4. The method of claim 1, wherein, In step (2), activated carbon is added to the first mixture obtained in step (1) to decolorize, and the amount of activated carbon added is 1-10 wt%, and after sufficient decolorization, the activated carbon is removed by filtration.

5. The method of claim 1, wherein, In step (2), the mixture is treated with a microfiltration membrane and / or an ultrafiltration membrane to remove macromolecular impurities, and then hydrochloric acid is added to precipitate 3-hydroxydecanoic acid to obtain a second mixture, specifically: First, a microfiltration membrane with a pore size of 0.22 μm is used for filtration, and then an ultrafiltration membrane with a molecular weight cut-off of 2000 is used for filtration, the filtrate is collected, hydrochloric acid is added to adjust the pH to 2-4 to precipitate 3-hydroxydecanoic acid, the precipitate is collected, and after centrifugation and drying, the second mixture is obtained.

6. The method of claim 1, wherein, In step (3), the concentration of the 3-hydroxydecanoic acid sodium solution is 50-100 g / L, and the pH is 6.5-8.

0.

7. The method of claim 1, wherein, The 3-hydroxydecanoic acid sodium solution is loaded onto a resin column packed with polyethyleneimine modified polystyrene-divinylbenzene macroporous resin for adsorption-elution further purification to obtain the product, specifically: The polyethyleneimine modified polystyrene-divinylbenzene macroporous resin is packed into a resin column, the 3-hydroxydecanoic acid sodium solution is loaded onto the resin column at a flow rate of 0.5-2 BV / h, the content of 3-hydroxydecanoic acid at the outlet is monitored, when reaching 5 g / L, the feeding is stopped, and the resin column is washed with pure water until the content of 3-hydroxydecanoic acid at the outlet is less than 0.5 g / L; finally, an ethanol-water solution with a volume ratio of 1:1 and a pH of 11 is used for elution, the elution flow rate is 0.5-2 BV / h; after the eluate is collected, the ethanol is evaporated to dryness to obtain a purified 3-hydroxydecanoic acid sodium solution, hydrochloric acid is added to adjust the pH to 3-4, the precipitate is collected by centrifugation, and the product is obtained by drying.

8. The method of claim 1, wherein, The Pseudomonas bacteria include one or more of P. putida, P. oleovorans, and P. aeruginosa.

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