Extraction and purification method for synthesizing phytosphingosine through whole-cell fermentation

Abstract

The invention provides an extraction and purification method for synthesizing phytosphingosine through whole-cell fermentation, which comprises the following steps: carrying out enzymolysis, ceramic membrane thorough washing and concentration on fermentation liquor to obtain bacterial sludge liquid; adding strong base into the bacterial sludge liquid, and performing primary hydrolysis under a heating condition to obtain hydrolysate; adjusting the pH value of the hydrolysate to 7-8, and then extracting, centrifuging, concentrating, purifying, purifying, filtering and drying to obtain the high-purity N-acetyl phytosphingosine. Adding the high-purity N-acetyl phytosphingosine into an ethanol aqueous solution, adding a strong base into the ethanol aqueous solution, and carrying out secondary hydrolysis under a heating condition to obtain a sphingosine reaction solution; and carrying out crystallization, recrystallization, centrifugation and drying on the sphingosine reaction solution to obtain the phytosphingosine. According to the extraction and purification method disclosed by the invention, cell contents are released by utilizing lysozyme, water-soluble impurities are removed through a ceramic membrane, and tetraacetyl phytosphingosine and sphingosine are intercepted, so that the extraction and purification method is efficient and rapid.

Description

Technical Field

[0001] This invention belongs to the field of chemistry, and in particular relates to a method for the extraction and purification of plant sphingosine through whole-cell fermentation. Background Technology

[0002] Phytosphingosine is a naturally occurring sphingolipid precursor found in the epidermis. It is a crucial component of the natural lipids in the stratum corneum of human skin, playing a key role in maintaining skin barrier function, regulating cell growth and differentiation, and mediating inflammatory responses. Its main functions and applications are reflected in the following aspects: First, skin barrier repair and moisturizing. Phytosphingosine is a key component of the lipid bilayer of the stratum corneum, effectively repairing damaged skin barriers, reducing moisture loss, and enhancing the skin's moisturizing ability. Second, potent antibacterial and anti-inflammatory effects. Phytosphingosine possesses natural antibacterial properties, especially effectively inhibiting the growth of pathogenic microorganisms such as Propionibacterium acnes, which causes acne. Simultaneously, it can regulate immune responses and reduce skin inflammation, showing significant effects in improving inflammatory skin problems such as acne and dermatitis. Third, anti-aging and skin regeneration promotion effects. Phytosphingosine can stimulate keratinocyte proliferation, promote skin metabolism and self-repair, helping to delay skin aging and improve skin texture. Fourth, phytosphingosine is also widely used in cosmetics. On the one hand, phytosphingosine can serve as a key starting material for ceramides, synthesizing active ingredients such as ceramide NP and AP. On the other hand, based on its multiple biological functions, phytosphingosine has become a highly effective active ingredient in high-end skincare products, especially in anti-allergy, anti-acne, barrier repair, and anti-aging products. It is widely used in formulas for serums, creams, and lotions, achieving purifying and rejuvenating effects.

[0003] Currently, the published methods for synthesizing phytosphingosine mainly fall into two categories: chemical synthesis and bio-fermentation. Because phytosphingosine possesses three chiral centers (C2, C3, C4), precise control of the stereoconfiguration is essential to obtain the naturally active (2S, 3S, 4R) enantiomers. The diastereomeric forms have very low or no biological activity. This significantly increases the difficulty and cost of synthesis, requiring lengthy steps, expensive chiral catalysts, and large quantities of toxic, flammable, and explosive chemical reagents (such as organometallic reagents and phosphine reagents). This places extremely high demands on production operations and environmental protection. Therefore, the feasibility of large-scale production via chemical synthesis is severely limited.

[0004] Compared to chemical synthesis, bio-fermentation has unique advantages. Microbial cells can automatically complete all biochemical reactions from substrate to product. Bioenzymes can specifically catalyze the synthesis of specific stereoconfigurations, and fermentation conditions are mild, making it more environmentally friendly. The challenge in bio-fermentation synthesis of phytosphingosine lies in the economical and efficient separation and purification of phytosphingosine to produce high-quality products. Summary of the Invention

[0005] In view of this, the present invention aims to overcome the defects in the prior art and proposes a method for the extraction and purification of plant sphingosine by whole-cell fermentation.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows: This invention provides a method for extracting and purifying plant sphingosine through whole-cell fermentation, comprising the following steps: Step 1 involves enzymatically hydrolyzing, washing with a ceramic membrane, and concentrating the tetraacetyl phytosphingosine (TAPS) fermentation broth to obtain tetraacetyl phytosphingosine bacterial sludge solution. Step 2 involves adding a strong alkali to the tetraacetyl phytosphingosine bacterial sludge solution and performing a single hydrolysis under heating conditions to obtain the intermediate N-acetyl phytosphingosine hydrolysate. Step 3 involves adjusting the pH of the intermediate N-acetylphytosphoprotein (NAPS) hydrolysate to 7-8, followed by extraction, centrifugation, concentration, purification, filtration, and drying to obtain high-purity N-acetylphytosphoprotein. Step 4 involves adding the high-purity N-acetylphytase sphingosine to an aqueous ethanol solution, adding a strong alkali, and performing a secondary hydrolysis under heating conditions to obtain a sphingosine reaction solution. Step 5 involves crystallizing, recrystallizing, centrifuging, and drying the sphingosine reaction solution to obtain the plant sphingosine.

[0007] The extraction and purification method described in this invention yields N-acetyl phytosphingosine with an HPLC purity of 93% after purification, and then obtains phytosphingosine with an HPLC purity of 95% through secondary hydrolysis and recrystallization.

[0008] Furthermore, in step 1, the tetraacetyl phytosphingosine fermentation broth contains 15-30 g / kg of tetraacetyl phytosphingosine with a purity of 25-33%. In step 1, the amount of lysozyme added during the enzymatic hydrolysis step is 0.1-0.3% of the mass of the tetraacetyl phytosphingosine fermentation broth. The hydrolysis temperature is 40-45℃, the pH is 6.5-7.0, and the time is 3-5 hours. First, enzymatic hydrolysis is performed using lysozyme, followed by thorough washing and concentration using a ceramic membrane to remove water-soluble pigments and impurities, yielding a tetraacetyl phytosphingosine bacterial sludge solution.

[0009] Furthermore, the amount of strong alkali added in step 2 is 2-3% of the mass of the tetraacetyl phytosphingosine bacterial sludge liquid; the strong alkali in step 2 is sodium hydroxide or potassium hydroxide; and the temperature of the first hydrolysis step in step 2 is 45-50℃.

[0010] Furthermore, the extraction step in step 3 is performed using a solvent, wherein the solvent is at least one of ethyl acetate, butyl acetate, or dichloromethane; specifically, the extraction step in step 3 is as follows: 1-1.5 L of solvent is added to each kilogram of N-acetyl phytosphingosine hydrolysate, and the mixture is stirred and extracted at 45-55°C for 1-2 hours, then centrifuged, and the supernatant is collected. The extraction and centrifugation are repeated 3 times, and the resulting supernatants are combined.

[0011] Furthermore, the purification step in step 3 uses a macroporous resin for purification; the ethanol concentration for loading in step 3 is 50-60%, the loading temperature is 40-45℃, and the flow rate is 1.0-1.5 BV / h; the macroporous resin is at least one of D101, AB-8, ADS-17, ADS-7, D201, D301, WDT-2, MP800, D311, or WDT-3.

[0012] Furthermore, the purification step in step 3 uses acetonitrile pulping for purification; the temperature in step 3 is 10-15℃, and the time is 2-3 hours; the amount of acetonitrile added is 1-1.5 times that of the solution obtained after the purification step.

[0013] Furthermore, the mass concentration of the ethanol aqueous solution in step 4 is 50-60%; the strong base in step 4 is sodium hydroxide or potassium hydroxide; the solid-liquid ratio of high-purity N-acetylphytosphoprotein to the ethanol aqueous solution and the strong base in step 4 is 1g:12-15mL:1g; the temperature of the secondary hydrolysis step in step 4 is 70-75℃, and the time is 5-8h.

[0014] Furthermore, the crystallization step in step 5 is carried out at a temperature of 10-15°C for 3-5 hours.

[0015] Furthermore, the recrystallization step in step 5 specifically involves adding an aqueous ethanol solution to the crude crystals obtained after the crystallization step, dissolving the crystals at 65-75°C, then cooling the temperature to 10-15°C and allowing crystals to precipitate for 3-5 hours; the aqueous ethanol solution has a mass concentration of 50-60%, and 12-15 L of the aqueous ethanol solution is added per kilogram of crude crystals.

[0016] The present invention also provides a plant sphingosine prepared using the extraction and purification method described above.

[0017] Compared with the prior art, the present invention has the following advantages: The extraction and purification method for whole-cell fermentation synthesis of phytosphingosine described in this invention utilizes lysozyme to release cell contents, removes water-soluble impurities through ceramic membrane filtration, and retains tetraacetylphytosphingosine, which is highly efficient and rapid. The solvent used in the extraction and purification process can be repeatedly recycled through distillation in actual production, reducing solvent costs and consumption.

[0018] The extraction and purification method for the whole-cell fermentation synthesis of phytosphingosine described in this invention first utilizes the adsorption properties of macroporous resin to remove impurities and pigments from N-acetylphytosphingosine, and then uses acetonitrile to further purify N-acetylphytosphingosine through pulping, thereby obtaining high-purity N-acetylphytosphingosine and ensuring the purity of the final product, phytosphingosine. The yield of the extraction and purification method is greater than 90%, and the HPLC purity of phytosphingosine reaches 95%, which is superior to the purity of commercially available products, and has high commercial value and prospects. Attached Figure Description

[0019] Figure 1 This is the HPLC chromatogram of tetraacetyl phytosphingosine in the bacterial sludge solution after enzymatic hydrolysis and ceramic membrane permeation and concentration as described in Example 1 of the present invention. Figure 2 This is the HPLC chromatogram of N-acetylphytosphoprotein after purification by acetonitrile pulping following the macroporous resin purification described in this embodiment of the invention. Figure 3 This is the HPLC chromatogram of the plant sphingosine product described in the embodiments of the present invention; Figure 4 The HPLC chromatogram of the plant sphingosine product described in Comparative Example 1 of this invention is shown below. Figure 5 The HPLC chromatogram of the plant sphingosine product described in Comparative Example 2 of this invention is shown. Detailed Implementation

[0020] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0021] The preparation method of tetraacetyl phytosphingosine fermentation broth is as follows: activate the bacterial strain, use glucose as the carbon source, carry out three-stage fermentation (fermentation temperature 25-31℃, dissolved oxygen 25-35%, and fermentation with purified air for 60-96 hours) to obtain tetraacetyl phytosphingosine fermentation broth.

[0022] The present invention will be described in detail below with reference to the embodiments.

[0023] Example 1 A method for extracting and purifying plant sphingosine through whole-cell fermentation includes the following steps: Step 1: Take 10.0 kg of whole-cell fermentation synthesized tetraacetyl phytosphingosine fermentation broth (TAPS content 18.1 g / kg, HPLC purity: 30.30%), adjust pH=6.5, add 0.1% lysozyme powder (10 g), enzymatically hydrolyze at 45℃ for 4 h, and then concentrate by washing with a ceramic membrane to obtain 3.0 kg of concentrated bacterial sludge solution. Step 2: Add 2.5% NaOH (mass percentage), i.e. 75g NaOH solid, keep at 50℃ for 2h to carry out one hydrolysis to obtain N-acetyl phytosphingosine hydrolysate; Step 3: Adjust the pH of the N-acetylphytosphoprotein hydrolysate to 7-8, then add 3L of ethyl acetate, heat and stir at 50℃ for 1 hour to extract N-acetylphytosphoprotein from the hydrolysate. Centrifuge and collect the supernatant. Repeat the extraction 3 times, and combine the supernatants to obtain the N-acetylphytosphoprotein extract. Concentrate the extract to remove the solvent, obtaining the N-acetylphytosphoprotein concentrate. Add 12L of anhydrous ethanol to the above N-acetylphytosphoprotein concentrate, stir and keep warm at 40-45℃, hot filter to remove insoluble matter, collect the filtrate, then add pure water to make a 55% ethanol concentration (measured with an alcohol meter), and keep warm at 40-45℃ until ready for column loading. Pack the column with macroporous resin (model: D201). 2L of N-acetylphytosphoprotein was activated with 4% NaOH, then rinsed with water to pH 7-8, and then equilibrated with 55% ethanol aqueous solution for 1 BV. The solution was then loaded onto a column at a flow rate of 1.5 BV / h. The flow through was collected, yielding a high-purity N-acetylphytosphoprotein solution. After loading, the column was eluted with 55% ethanol aqueous solution for 2 BV to remove the remaining N-acetylphytosphoprotein. Impurities were adsorbed onto the resin and subsequently eluted with 80-95% ethanol to remove impurities and pigments. The N-acetylphytosphoprotein solution purified by the macroporous resin was concentrated to 2.5L, and 2.5L of acetonitrile was added. The mixture was stirred at 10℃ for 2 hours, centrifuged in a cloth bag, and 293g of N-acetylphytosphoprotein crystals were obtained, which weighed 143g after drying. Step 4: Add the dried 143g N-acetylphytosphoprotein crystals to 1.4L of 60% ethanol aqueous solution, and add 143g NaOH solid. Hydrolyze the mixture at 70℃ for 8h. Step 5: The above phytosphingosine solution was cooled to 10°C and crystallized for 3 hours, then centrifuged to obtain 362g of crude crystals; the 362g of crude crystals were recrystallized with 4.3L of 60% ethanol aqueous solution, and then centrifuged to obtain 328g of purified phytosphingosine wet crystals. After vacuum drying, 112g of phytosphingosine product was obtained, with a yield of 94.5%.

[0024] It should be noted that 238g of wet crystals yielded 112g after drying, equivalent to a solid content of 47% (solvent accounting for 53%). For most products, 53% solvent usually indicates a flowing solution, not a solid. However, phytosphingosine has extremely strong water- and solvent-locking capabilities, maintaining a good solid state even at a 47% solid content. The same applies to TAPS and NAPS. Precisely because of their strong water- and solvent-locking abilities, the subsequent drying time is relatively long.

[0025] Example 2 A method for extracting and purifying plant sphingosine through whole-cell fermentation includes the following steps: Step 1: Take 8.5 kg of the fermentation broth of tetraacetyl phytosphingosine synthesized by whole cell fermentation (TAPS content 16.9 g / kg, HPLC purity: 30.81%), add 0.2% lysozyme powder (17.0 g), enzymatically hydrolyze at 45℃ for 5 h, and then concentrate by washing with a ceramic membrane to obtain 2.6 kg of concentrated bacterial sludge solution. Step 2: Add 3% KOH (mass percentage), i.e. 78g KOH solid, keep at 45℃ for 2h to carry out one hydrolysis to obtain N-acetyl phytosphingosine hydrolysate; Step 3: Adjust the pH of the N-acetylphytosphoprotein hydrolysate to 7-8 with hydrochloric acid, then add 2.6 L of butyl acetate, heat and stir at 50°C for 2 h to extract N-acetylphytosphoprotein from the hydrolysate, then centrifuge and collect the supernatant; repeat the extraction 3 times, combine the supernatants to obtain the N-acetylphytosphoprotein extract; concentrate the extract to remove the solvent to obtain the N-acetylphytosphoprotein concentrate; add 11 L of hydrochloric acid to the above N-acetylphytosphoprotein concentrate. 95% ethanol, stirred and kept at 40-45℃, hot filtered to remove insoluble matter, collect the filtrate, then add pure water to dilute to a 55% ethanol concentration (measured with an alcohol meter), keep at 40-45℃ until column loading; pack 2L of macroporous resin (model: D301), activate with 4% NaOH, then rinse with water to pH=7-8, then equilibrate with 1 BV of 55% ethanol aqueous solution, then start column loading at a flow rate of 1.5 BV / h, collect the flow through, which indicates high purity. The N-acetyl phytosphingosine solution was prepared; after loading, it was washed with 55% ethanol aqueous solution for 2 BV to elute the remaining N-acetyl phytosphingosine; the impurities were adsorbed on the resin, and the impurities and pigments were then eluted with 80-95% ethanol; the N-acetyl phytosphingosine solution purified by the macroporous resin was concentrated to 1.9 L, 1.9 L of acetonitrile was added, and the mixture was stirred at 10 °C for 2 h. After centrifugation in a cloth bag, 238 g of N-acetyl phytosphingosine crystals were obtained, which weighed 121 g after drying; Step 4: Add 121g of dried N-acetylphytosphoprotein crystals to 1.6L of 60% ethanol aqueous solution, and add 121g of NaOH solid. Hydrolyze the mixture at 70℃ for 6h. Step 5: Cool the above phytosphingosine solution at 10°C for 3 hours to allow crystals to crystallize, then centrifuge to obtain 201g of crude crystals; recrystallize the 201g of crude crystals with 2.4L of 60% ethanol aqueous solution, then centrifuge to obtain 195g of purified phytosphingosine wet crystals, and vacuum dry to obtain 90g of phytosphingosine product, with a yield of 95.7%.

[0026] Example 3 A method for extracting and purifying plant sphingosine through whole-cell fermentation includes the following steps: Step 1: Take 9.1 kg of the fermentation broth of tetraacetyl phytosphingosine synthesized by whole cell fermentation (TAPS content 25.4 g / kg, HPLC purity: 32.05%), add 0.1% lysozyme powder (9.1 g), enzymatically hydrolyze at 45℃ for 4 h, and then concentrate by washing with a ceramic membrane to obtain 2.8 kg of concentrated bacterial sludge solution. Step 2: Add 2.5% NaOH (mass percentage), i.e. 70g NaOH solid, keep at 50℃ for 2h to carry out one hydrolysis to obtain N-acetyl phytosphingosine hydrolysate; Step 3: Adjust the pH of the N-acetylphytosphoprotein hydrolysate to 7-8 with hydrochloric acid, then add 2.8 L of dichloromethane and heat and stir at 50°C for 1 hour to extract N-acetylphytosphoprotein from the hydrolysate. Centrifuge and collect the supernatant. Repeat the extraction three times and combine the supernatants to obtain the N-acetylphytosphoprotein extract. Concentrate the extract to remove the solvent to obtain the N-acetylphytosphoprotein concentrate. Add 13 L of hydrochloric acid to the above N-acetylphytosphoprotein concentrate. 94% ethanol was recovered. The mixture was stirred and kept at 40-45℃, then hot-filtered to remove insoluble matter. The filtrate was collected, and then diluted with pure water to a 55% ethanol concentration (measured using an alcohol meter). The solution was kept at 40-45℃ until column loading was achieved. 2L of macroporous resin (model: WDT-2) was packed into the column. After activation with 4% NaOH, the solution was flushed with water to pH 7-8. The solution was then equilibrated to 1 BV with a 55% ethanol aqueous solution. Column loading was then initiated at a flow rate of 1.5 BV / h. Flow through was collected. The sample was prepared as a high-purity N-acetylphytosphoprotein solution. After loading, the sample was washed with 60% ethanol aqueous solution for 2 BV to remove the remaining NAPS. Impurities were adsorbed onto the resin and were subsequently washed off with 80-95% ethanol. The N-acetylphytosphoprotein solution purified by the macroporous resin was concentrated to 2.7 L, 2.7 L of acetonitrile was added, and the mixture was stirred at 10 °C for 2 h. After centrifugation in a cloth bag, 334 g of N-acetylphytosphoprotein crystals were obtained, which weighed 171 g after drying. Step 4: Add 171g of dried N-acetylphytosphoprotein crystals to 2.0L of 60% ethanol aqueous solution, and add 171g of NaOH solid. Hydrolyze the mixture at 70℃ for 7h. Step 5: The above phytosphingosine reaction solution was cooled at 10°C for 3 hours to crystallize, and then centrifuged to obtain 376g of crude crystals; the 376g of crude crystals were recrystallized with 4.5L of 60% ethanol aqueous solution, and then centrifuged to obtain 350g of purified phytosphingosine wet crystals. After vacuum drying, 141g of phytosphingosine product was obtained, with a yield of 93.2%.

[0027] Comparative Example 1 (without macroporous resin purification) Compared with Example 1, the only difference is in step 3: the pH of the N-acetylphytosphoprotein hydrolysate is adjusted to 7-8, then 3L of ethyl acetate is added, and the mixture is heated and stirred at 50°C for 1 hour to extract the N-acetylphytosphoprotein from the hydrolysate. The mixture is then centrifuged, and the supernatant is collected. The extraction is repeated 3 times, and the supernatants are combined to obtain the N-acetylphytosphoprotein extract. The extract is concentrated to remove the solvent, and the N-acetylphytosphoprotein concentrate is obtained. 2.5L of acetonitrile is added to the above N-acetylphytosphoprotein concentrate, and the mixture is stirred at 10°C for 2 hours. The mixture is then centrifuged in a cloth bag to obtain 287g of N-acetylphytosphoprotein crystals, which weigh 128g after drying.

[0028] After step 5, 114g of phytosphingosine was finally obtained, with a yield of 96.3%.

[0029] Comparative Example 2 (two hydrolysis processes combined into one) A method for extracting and purifying plant sphingosine through whole-cell fermentation includes the following steps: Step 1: Take 10.0 kg of whole-cell fermentation synthesized tetraacetyl phytosphingosine fermentation broth (TAPS content 18.1 g / kg, HPLC purity: 30.30%), adjust pH=6.5, add 0.1% lysozyme powder (10 g), enzymatically hydrolyze at 45℃ for 4 h, and then concentrate by washing with a ceramic membrane to obtain 3.0 kg of concentrated bacterial sludge solution. Step 2: Adjust the pH of the above-mentioned concentrated bacterial sludge solution to 7-8, then add 3L of ethyl acetate, heat and stir at 50℃ for 1 hour to extract tetraacetyl phytosphingosine, then centrifuge and collect the supernatant. Repeat the extraction 3 times, and combine the supernatants to obtain the tetraacetyl phytosphingosine extract. Concentrate the extract to remove the solvent, and obtain the tetraacetyl phytosphingosine extract concentrate. Add 12L of anhydrous ethanol to the above tetraacetyl phytosphingosine extract concentrate, stir and keep warm at 40-45℃, hot filter to remove insoluble matter, collect the filtrate, and then add pure water to make a 55% ethanol concentration (using an alcohol meter). (Alcohol concentration), incubate at 40-45℃ until column loading; pack 2L of macroporous resin (model: D201), activate with 4% NaOH, then rinse with water to pH=7-8, then equilibrate with 1 BV of 55% ethanol aqueous solution, and then start column loading at a flow rate of 1.5 BV / h. Collect the flow through, which is a high-purity N-acetyl phytosphingosine solution; after loading, wash with 55% ethanol aqueous solution for 2 BV to elute the remaining tetraacetyl phytosphingosine extract; impurities are adsorbed on the resin, and then elute the impurities and pigments with 80-95% ethanol; Since there is no hydrolysis, the product at this time is tetraacetyl phytosphingosine (TAPS), instead of the intermediate N-acetyl phytosphingosine (NAPS). Therefore, the specific differences between this step and Example 1 are: (1) tetraacetyl phytosphingosine is extracted; (2) tetraacetyl phytosphingosine is purified by macroporous resin (the extraction method of TAPS and NAPS is the same as the macroporous resin purification method); (3) since the purification step of intermediate N-acetyl phytosphingosine (NAPS) is not performed, the step of acetonitrile pulping and purification of intermediate is omitted.

[0030] Step 3: Add 27L of the tetraacetyl phytosphingosine solution purified by the macroporous resin to 1.08kg of 4% sodium hydroxide solid, and hydrolyze at 70℃ for 5h to directly hydrolyze tetraacetyl phytosphingosine into phytosphingosine in one step. Step 4: The above phytosphingosine solution was cooled to 10°C and crystallized for 3 hours, then centrifuged to obtain 241g of crude crystals; the 241g of crude crystals were recrystallized with 2.9L of 60% ethanol aqueous solution, and then centrifuged to obtain 218g of purified phytosphingosine wet crystals. After vacuum drying, 109g of phytosphingosine product was obtained, with a yield of 92.1%.

[0031] The purity of tetraacetyl phytosphingosine, N-acetyl phytosphingosine purified by macroporous resin and then slurryed with acetonitrile, and phytosphingosine after recrystallization and drying in the bacterial sludge solution after ceramic membrane permeation and concentration in Example 1 were analyzed by HPLC. The analytical results are as follows: Figure 1 , Figure 2 and Figure 3 As shown, the HPLC purity analysis results of phytosphingosine obtained from Comparative Example 1 and Comparative Example 2 are as follows. Figure 4 and Figure 5 As shown.

[0032] Figure 1 In the figure, the peak at 20.4 min represents tetraacetyl phytosphingosine, at which point the HPLC purity is 46.73%. Figure 2 In the figure, the peak at 12.4 min represents N-acetyl phytosphingosine, at which point the HPLC purity reaches 93.79%. At the same time, other impurities were effectively removed, indicating that the macroporous resin purification effect is very good. Figure 3 In the figure, the peak at 8.1 min represents phytosphingosine, at which point the HPLC purity reaches 95.45%.

[0033] Figure 4 In the example, the peak at 8.2 min represents phytosphingosine. In Comparative Example 1, the macroporous resin purification step was omitted, and the final purity of the phytosphingosine obtained was only 81.72%. Figure 5 In the example, the peak at 7.9 min represents phytosphingosine. In Comparative Example 2, tetraacetyl phytosphingosine was purified by macroporous resin and then hydrolyzed to phytosphingosine in one step. The final purity of the phytosphingosine obtained was 91.67%.

[0034] The method described in this invention can significantly remove impurities from phytosphingosine, meet the needs of scale-up production, is relatively simple to operate, and has a very high yield.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extraction and purification of phyto-sphingosine synthesized by whole cell fermentation, characterized by: The method comprises the following steps: Step 1 is to ferment the tetraacetyl phytosphingosine broth, to carry out enzymatic hydrolysis, ceramic membrane permeation and concentration, and to obtain tetraacetyl phytosphingosine slurry; Step 2 is to add a strong base to the tetraacetyl phytosphingosine slurry, to carry out primary hydrolysis under heating, and to obtain intermediate N-acetyl phytosphingosine hydrolysate; Step 3 is to adjust the pH value of the intermediate N-acetyl phytosphingosine hydrolysate to 7-8, and then to carry out extraction, centrifugation, concentration, purification, purification, filtration and drying, and to obtain high-purity N-acetyl phytosphingosine; Step 4 is to add the high-purity N-acetyl phytosphingosine to an aqueous ethanol solution, to add a strong base thereto, to carry out secondary hydrolysis under heating, and to obtain phytosphingosine reaction liquid; Step 5 is to carry out crystallization, recrystallization, centrifugation and drying on the phytosphingosine reaction liquid, and to obtain the phytosphingosine.

2. The method of claim 1, wherein the whole cell fermentation to synthesize phytosphingosine is extracted and purified. The content of tetraacetyl phytosphingosine in the tetraacetyl phytosphingosine broth in step 1 is 15-30 g / kg, and the purity is 25-33%; the addition amount of lysozyme in the enzymatic hydrolysis step in step 1 is 0.1-0.3% of the mass of the tetraacetyl phytosphingosine broth, the temperature of the enzymatic hydrolysis step is 40-45℃, the pH value is 6.5-7.0, and the time is 3-5 hours.

3. The method of claim 1, wherein the whole cell fermentation to synthesize phytosphingosine is purified by extraction. The addition amount of the strong base in step 2 is 2-3% of the mass of the tetraacetyl phytosphingosine slurry; the strong base in step 2 is sodium hydroxide or potassium hydroxide; and the temperature of the primary hydrolysis step in step 2 is 45-50℃.

4. The method of claim 1, wherein the whole cell fermentation to synthesize phytosphingosine is purified by extraction. The extraction step in step 3 uses a solvent for extraction, and the solvent is at least one of ethyl acetate, butyl acetate or dichloromethane; the extraction step in step 3 specifically comprises: adding 1-1.5 L of solvent to every kilogram of N-acetyl phytosphingosine hydrolysate, stirring and extracting at 45-55℃ for 1-2 h, then centrifuging, collecting the supernatant, repeating the extraction and centrifugation for 3 times, and combining the obtained centrifugal supernatant.

5. The method of claim 1, wherein the whole cell fermentation to synthesize phytosphingosine is purified by extraction. The purification step in step 3 uses a macroporous resin for purification; the loading ethanol concentration of the purification step in step 3 is 50-60%, the loading temperature is 40-45℃, and the flow rate is 1.0-1.5 BV / h; and the macroporous resin is at least one of D101, AB-8, ADS-17, ADS-7, D201, D301, WDT-2, MP800, D311 or WDT-3.

6. The method of claim 1, wherein the whole cell fermentation to synthesize phytosphingosine is purified by extraction. The purification step in step 3 uses acetonitrile beating to carry out purification; the temperature of the purification step in step 3 is 10-15℃, and the time is 2-3 h; and the addition amount of acetonitrile is 1-1.5 times of the obtained solution after the purification step.

7. The method of claim 1, wherein the whole cell fermentation to synthesize phytosphingosine is purified by extraction. The mass concentration of the ethanol aqueous solution in step 4 is 50-60%; the strong base in step 4 is sodium hydroxide or potassium hydroxide; the solid-liquid ratio of high-purity N-acetylphytosphoprotein to the ethanol aqueous solution and the strong base in step 4 is 1g:12-15mL:1g; the temperature of the secondary hydrolysis step in step 4 is 70-75℃, and the time is 5-8h.

8. The method of claim 1, wherein the whole cell fermentation to synthesize phytosphingosine is purified by extraction. The crystallization step in step 5 is carried out at a temperature of 10-15℃ for 3-5 hours.

9. The method of claim 1, wherein the whole cell fermentation to synthesize phytosphingosine is purified by extraction. The recrystallization step in step 5 is specifically as follows: an ethanol aqueous solution is added to the crude crystals obtained after the crystallization step, and the solution is dissolved at 65-75°C. Then the temperature is lowered to 10-15°C, and crystals are precipitated for 3-5 hours. The mass concentration of the ethanol aqueous solution is 50-60%, and 12-15L of the ethanol aqueous solution is added per kilogram of crude crystals.

10. A plant sphingosine prepared using the extraction and purification method according to any one of claims 1-9.

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