A method for separating and purifying triacetyl-dihydrosphingosine from a fermentation broth

Abstract

The application discloses a method for separating and purifying triacetyl dihydro sphingosine from fermentation liquor and belongs to the technical field of separation and purification. The method is combined with high-pressure homogenization crushing, low-temperature flocculation impurity removal, supercritical extraction, gradient elution silica gel column chromatography and fractional low-temperature recrystallization, and a method for efficiently separating and purifying triacetyl dihydro sphingosine is obtained. The method can fully release intracellular triacetyl dihydro sphingosine, efficiently remove protein impurities, realize directional separation of triacetyl dihydro sphingosine and tetraacetyl phytosphingosine, and obtain stable-structure and high-purity triacetyl dihydro sphingosine. The method can significantly improve the purity and yield of the product and protect the structural stability of the product. Meanwhile, the method is simple and easy to operate and is suitable for industrial production. Therefore, the method for separating and purifying triacetyl dihydro sphingosine has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of separation and purification technology, and more specifically, to a method for separating and purifying triacetyldihydrosphingosine from fermentation broth. Background Technology

[0002] Triacetyl dihydrosphingosine (TriASA) is a key precursor in the biosynthesis of ceramides. It has excellent skin barrier repair function, long-lasting moisturizing properties and anti-inflammatory activity. It can significantly improve skin dryness and sensitivity, and has broad application prospects and huge market demand in high-end skin care products, medical repair dressings and other fields.

[0003] Currently, the large-scale preparation of TriASA mainly relies on Wickham Severin yeast ( Wickerhamomyces ciferrii Fermentation production offers advantages such as low raw material costs, environmental friendliness, and high product safety. However, existing fermentation methods for preparing TriASA face several significant technical bottlenecks: the dense cell wall structure of yeast cells makes it difficult to fully release intracellularly synthesized TriASA, resulting in low extraction rates and affecting the overall yield; the complex fermentation broth system contains numerous impurities, including cell fragments, soluble proteins, nucleic acids, and polysaccharides, with separation of cell proteins from TriASA being particularly challenging and difficult to achieve efficiently using conventional impurity removal methods, increasing the burden on subsequent purification processes; the fermentation product contains tetraacetyl phytosphingosine (TAPS), which has a highly similar structure to TriASA, and their similar polarities make effective separation difficult using conventional single purification techniques such as column chromatography and recrystallization, resulting in product purity typically failing to exceed 95%, which cannot meet the purity requirements of high-end cosmetics; and the acetyl groups in the TriASA molecule are sensitive to temperature and pH conditions, easily undergoing hydrolysis during purification, damaging the structural integrity and biological activity of the product, thus limiting the selection of purification process conditions.

[0004] To address the aforementioned technical challenges, researchers have undertaken numerous attempts. For instance, supercritical CO2 extraction has been used to extract bioactive components, achieving gentle extraction, but it lacks targeted separation technology for TriASA and TAPS, and is not optimized for yeast cell disruption and protein impurity removal. Alternatively, silica gel column chromatography has been employed, but this method does not consider the stability of acetyl groups and lacks optimized impurity removal processes tailored to the characteristics of bacterial proteins, making it unsuitable for the efficient purification of TriASA.

[0005] Therefore, developing a high-purity TriASA directional purification method that can fully release intracellular TriASA, efficiently remove protein impurities, achieve directional separation of TriASA and TAPS, and protect the structural stability of the product has become an urgent technical problem to be solved in this field.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a method for separating and purifying triacetyl dihydrosphingosine from fermentation broth. Using the method of this invention, intracellular TriASA can be fully released, protein impurities can be efficiently removed, and the directional separation of TriASA and TAPS can be achieved to obtain structurally stable and highly pure TriASA.

[0008] This invention is implemented as follows: The method for separating and purifying triacetyl dihydrosphingosine from fermentation broth provided in this invention includes the following steps: S1. The fermentation broth containing bacteria that synthesize TriASA is passed into a high-pressure homogenizer to homogenize the bacterial cells.

[0009] The aforementioned fermentation broth is a bacterial culture containing triacetyldihydrophytosphoprotein obtained by fermentation of *Saccharomyces cerevisiae* or its engineered strains. However, it should be noted that the separation and purification method of this invention is not only applicable to fermentation broths but also to mixed systems containing the same or similar components. When the object of treatment is fermentation broth, it can be obtained through conventional fermentation techniques in the art. This invention does not specifically limit the fermentation method; any fermentation broth meeting the above conditions can be used as the object of treatment by the purification method of this invention. In some embodiments, the fermentation broth is obtained by fermenting *Saccharomyces cerevisiae* in a fermentation medium. The fermentation medium comprises glucose, peptone, yeast extract, ammonium sulfate, malt extract, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, chloramphenicol, and water. The fermentation broth obtained after fermentation mainly includes bacterial cells, residual culture medium, triacetyl dihydrosphingosine, taps, and other trace products.

[0010] The homogenization conditions are: a pressure of 600-900 bar, and homogenization cycles of 1-2 times. More preferably, the homogenization pressure is 650-850 bar.

[0011] This invention utilizes the shear force, impact force, and cavitation effect generated by high-pressure homogenization to fully break down the cell walls of the bacteria in the fermentation broth, thereby fully releasing the TriASA synthesized intracellularly.

[0012] S2. Adjust the pH of the crushed fermentation broth to the isoelectric point of the cell protein, add flocculant under low temperature conditions, filter after forming flocculated precipitate, and collect the filtrate.

[0013] Since the cell disruption not only releases the target substance TriASA, but also releases intracellular bacterial proteins, nucleic acids and other impurities into the liquid phase, in order to reduce the interference of impurities on the purification of the target substance, this invention designs a flocculation and impurity removal step in the extraction process.

[0014] Chitosan is used as the flocculant. Chitosan was chosen as the flocculant not only because of its high safety profile and applicability in cosmetics and food, but also because, compared to other conventional flocculants (such as gum arabic, gelatin, and ammonium alginate), chitosan can achieve flocculation, decolorization, and antibacterial effects through charge neutralization, adsorption bridging, and chelation, making it a better match for the fermentation broth conditions of this invention.

[0015] In some embodiments, the mass of the flocculant added is 0.1% to 0.3% of the fermentation liquid volume. Within this range, impurities and flocculants can be fully combined.

[0016] In some embodiments, during flocculation and sedimentation, the fermentation broth is cooled to 3-5°C, and after adding the flocculant, it is stirred at 30-50 rpm for 30-60 min, and then allowed to stand for 1-3 h. The flocculant fully binds with impurities such as bacterial proteins, bacterial fragments, and polysaccharides to form a stable flocculated precipitate, facilitating subsequent filtration and separation of the precipitate.

[0017] In some embodiments, after flocculation and sedimentation, solid-liquid separation is performed using a plate and frame filter. The plate and frame filter cloth is made of 300-500 mesh polyester, and the filtration operating pressure is 0.1-0.4 MPa. Under these circulating filtration conditions, more than 90% of solids and bacterial protein impurities can be removed.

[0018] S3. The filtrate is subjected to supercritical CO2 extraction and low-temperature concentration to obtain crude extract.

[0019] In some embodiments, the conditions for supercritical CO2 extraction are as follows: the entrainer is anhydrous ethanol, and the added volume is 5% to 10% of the filtrate volume; the extraction temperature is 33 to 37°C, the extraction pressure is 28 to 32 MPa, the CO2 flow rate is 15 to 25 L / h, and the extraction time is 2 to 4 h.

[0020] The present invention employs the above supercritical CO2 extraction conditions, which have advantages such as low temperature, high extraction efficiency, and no organic solvent residue. It can achieve efficient enrichment of TriASA under mild conditions, with an extraction rate of ≥92%.

[0021] In some embodiments, low-temperature concentration involves transferring the extract to a rotary evaporator for vacuum distillation under the following conditions: temperature 38–42°C, pressure -0.10–-0.09 MPa; simultaneously, nitrogen gas is introduced during the concentration process. Vacuum concentration removes the entrainer anhydrous ethanol, yielding a crude TriASA extract with a purity of approximately 70%. The introduction of nitrogen gas protects the product, preventing oxidative degradation of TriASA upon contact with air.

[0022] S4. Perform gradient silica gel column chromatography and fractional recrystallization on the crude extract, and collect the crystals.

[0023] In some embodiments, gradient silica column chromatography includes: loading the crude extract into a dichloromethane solution, and performing gradient elution using a dichloromethane-methanol mixture as the eluent, wherein the elution procedure is as follows: The volume ratio of dichloromethane to methanol in the eluent was 98:2 from 0 to 120 min; the volume ratio of dichloromethane to methanol in the eluent was 95:5 from 120 to 320 min, and the elution peak from 280 to 320 min was collected; the volume ratio of dichloromethane to methanol in the eluent was 90:10 from 320 to 400 min.

[0024] The reason for choosing gradient elution in this invention is that the varying proportions of eluent components in different stages allow for the elution of substances with different properties, thus improving elution efficiency. In the first stage, dichloromethane has the highest proportion, primarily eluting weakly polar impurities in the system. In the second stage, the proportion of dichloromethane decreases; this stage corresponds to the elution range of TriASA. The elution peak at 280–320 min is collected using online HPLC, and the product corresponding to this peak is the high-purity TriASA intermediate. In the third stage, the proportion of dichloromethane is even lower, primarily eluting strongly polar impurities in the system while simultaneously regenerating the chromatography column. Using the gradient silica gel column chromatography of this invention, TriASA and TAPS can be effectively separated, keeping the residual amount of TAPS below 0.5%.

[0025] The dichloromethane-methanol mixture was used as the eluent because this solution was selected through thin-layer analysis as a solvent with better selectivity and elution ability for the fermentation broth of this invention.

[0026] In some embodiments, the elution flow rate during gradient elution is 5–8 mL / min. Specifically, the flow rate is 5 mL / min in the first stage, 5 mL / min in the second stage, and 8 mL / min in the third stage. This design is based on the fact that the first and second stages are primarily separation stages, requiring slower flow rates to improve separation accuracy; the third stage is a column flushing and regeneration stage, requiring a faster flow rate to flush out any remaining compounds in the column.

[0027] In some embodiments, the concentration of the crude extract in the dichloromethane solution is 100-150 mg / mL.

[0028] In some embodiments, the sample loading volume is 5% of the column volume, and the sample loading flow rate is 3~5 mL / min.

[0029] In some embodiments, the silicone column has a size of Φ50~100 mm × 1000~1500 mm.

[0030] In some embodiments, the silica gel filling the silica gel column has a mesh size of 200-300 mesh and a moisture content of ≤0.5%. The moisture content is controlled by activating the silica gel at 110°C for 18 hours before filling.

[0031] In some embodiments, the ratio of the filling height of the silicone column to the inner diameter of the column is 20:1 to 30:1.

[0032] By selecting appropriate column specifications, silica gel mesh size, optimizing silica gel moisture content, and adjusting the packing height to inner diameter ratio of the column, the purification efficiency of the chromatography column can be improved. Furthermore, by adjusting column specifications and other conditions, the process can be scaled up for subsequent industrial production.

[0033] In some embodiments, fractional recrystallization includes: concentrating the eluent collected from gradient silica column chromatography to 200-300 mg / ml, refrigerating at -4°C for 12 h, allowing it to stand for primary crystallization to precipitate primary crystals; after solid-liquid separation, redissolving the primary crystals in anhydrous ethanol to prepare a crystallization solution of 200-300 mg / ml, refrigerating at -10°C for 8 h, and performing secondary crystallization to further remove residual trace impurities; collecting the crystals after secondary crystallization and drying them under low-temperature vacuum.

[0034] The present invention chooses fractional recrystallization because primary crystallization is for the concentrated eluent. In order to improve the yield, the concentration of the concentrated solution is 200~300 mg / ml. Although crystallization at this concentration can fully extract triacetyl dihydrosphingosine crystals, there are also relatively more impurities, and the dichloromethane residue is large, with a strong taste, which is not conducive to subsequent applications. Therefore, a secondary ethanol recrystallization was designed. This operation can reduce the residue of impurities and reduce the residue of dichloromethane to almost none, thus achieving the purpose of purification and impurity removal.

[0035] S5. The collected crystals are dried under vacuum at low temperature to obtain triacetyl dihydrosphingosine as the final product.

[0036] In some embodiments, the conditions for vacuum low-temperature drying are: temperature of 45-50℃, vacuum pressure of -0.1~-0.9MPa, and drying time of 20~28 h.

[0037] Vacuum low-temperature drying can remove moisture from crystals. Combined with the aforementioned operation method, this invention constructs a temperature control system throughout the process. The fermentation broth pretreatment, flocculation and impurity removal, plate and frame separation, extraction, concentration and crystallization steps all adopt mild temperature conditions. At the same time, nitrogen gas is introduced for protection during the concentration process, which effectively avoids the hydrolysis of acetyl groups in TriASA molecules, ensuring the structural integrity and biological activity of the product. The acetyl hydrolysis rate is ≤1%.

[0038] The present invention utilizes the above-mentioned separation and purification method to obtain a high-purity triacetyl dihydrosphingosine product with TriASA purity ≥98%, tetraacetyl phytosphingosine (TAPS) residue ≤0.5%, acetyl hydrolysis rate ≤1%, moisture content ≤0.5%, heavy metal (Pb) content ≤10 ppm, and solvent residue ≤10 ppm. Compared with existing triacetyl dihydrosphingosine products, its quality has been significantly improved, making it more suitable for use in cosmetic production.

[0039] The present invention has the following beneficial effects: This invention provides a highly efficient method for separating and purifying triacetyldihydrosphingosine (TriASA) by combining high-pressure homogenization, low-temperature flocculation for impurity removal, gradient elution silica gel column chromatography, and fractional low-temperature recrystallization, all under controlled low-temperature conditions. This method effectively releases intracellular TriASA, efficiently removes protein impurities, and achieves targeted separation of TriASA and TAPS, yielding structurally stable and highly pure TriASA. This method significantly improves product purity and yield while preserving product structural stability; furthermore, it is simple and easy to implement, making it suitable for industrial production. Therefore, the method for separating and purifying Triacetyldihydrosphingosine of this invention has promising prospects for industrial application. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0041] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0042] In a specific embodiment of the present invention, the fermentation broth used is West Wickham yeast fermentation broth; the fermentation medium includes glucose, peptone, yeast powder, ammonium sulfate, malt extract, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, chloramphenicol, and the remainder is water. The fermentation broth mainly includes bacterial cells, residual culture medium, triacetyl dihydrosphingosine, TAPS, and other trace products, wherein the content of triacetyl dihydrosphingosine is 25g / L-30g / L.

[0043] Example 1 This embodiment describes a method for separating and purifying triacetyldihydrosphingosine from fermentation broth. The specific steps are as follows: (1) High-pressure homogeneous crushing Take 10 L of fermentation broth with a TriASA content of 28 g / L, put it into a high-pressure homogenizer, adjust the homogenization pressure to 650 bar, and homogenize twice to complete cell disruption. (2) Low-temperature flocculation for impurity removal The crushed fermentation broth was introduced into a low-temperature reactor, and the pH value was adjusted to 5.5 with 1 mol / L hydrochloric acid. After cooling to 4°C, 10 g of chitosan flocculant was added, and the mixture was stirred at 50 rpm for 30 min and then allowed to stand for 1 h. Subsequently, the mixture was filtered through a plate and frame filter with the following parameters: 300 mesh polyester filter bag, 0.2 MPa. The mixture was circulated until it became clear, and 8.5 L of clear filtrate was collected. (3) Supercritical CO2 extraction The clarified filtrate was introduced into a 10 L supercritical extraction vessel, and 1000 mL of anhydrous ethanol was added as an entrainer. The extraction temperature was adjusted to 37 °C, the pressure to 28 MPa, and the CO2 flow rate to 25 L / h. Extraction was carried out for 4 h to obtain 1.2 L of extract. (4) Low-temperature concentration The extract was introduced into a rotary evaporator and distilled under reduced pressure at 42℃ and -0.09 MPa with nitrogen protection to obtain 392 g of crude TriASA extract. HPLC analysis showed that the purity of the crude extract was 68%. (5) Silica gel column chromatography A 50mm × 1000mm chromatography column was used, packed with 200-mesh silica gel (activated at 110℃ for 18 h). The crude extract was dissolved in dichloromethane to a concentration of 150 mg / mL, and the sample loading volume was 5% of the column volume. Elution was performed according to a gradient elution program (diomethyl methane to methanol volume ratio in the eluent was 98:2 from 0 to 120 min; 95:5 from 120 to 320 min; and 90:10 from 320 to 400 min). The elution peak from 280 to 320 min was collected, and after concentration, 273.91 g of TriASA intermediate was obtained. The purity was 92% as determined by HPLC. (6) Grade recrystallization The intermediate product was concentrated to a concentration of 250 mg / mL and refrigerated at -4℃ for 12 h to precipitate primary crystals. The primary crystals were then filtered under vacuum and dissolved in anhydrous ethanol to a concentration of 300 mg / mL. The primary crystals were then refrigerated at -10℃ for 8 h to carry out secondary crystallization. The crystals were collected by vacuum filtration. (7) Low-temperature vacuum drying The crystals were placed in a vacuum drying oven and dried at 50°C and -0.1 MPa for 24 h to obtain 241.37 g of off-white high-purity TriASA solid powder.

[0044] The product was found to have a TriASA purity of 98.6%, a TAPS residue of 0.3%, an acetyl hydrolysis rate of 0.8%, a moisture content of 0.3%, a heavy metal (Pb) content of 8 ppm, a solvent residue of 5 ppm, and a total yield of 85.0%.

[0045] Example 2 This embodiment describes a method for separating and purifying triacetyldihydrosphingosine from fermentation broth. The specific steps are as follows: (1) High-pressure homogeneous crushing Take 100 L of fermentation broth with a TriASA content of 25 g / L, put it into a high-pressure homogenizer, adjust the homogenization pressure to 650 bar, and homogenize twice to complete cell disruption. (2) Low-temperature flocculation for impurity removal The crushed fermentation broth was introduced into a low-temperature reactor, and the pH value was adjusted to 5.7 with 1 mol / L hydrochloric acid. After cooling to 4°C, 100 g of chitosan flocculant was added, and the mixture was stirred at 50 rpm for 30 min and then allowed to stand for 1 h. Subsequently, the mixture was filtered through a plate and frame filter with the following parameters: 300 mesh polyester filter bag, 0.2 MPa. The mixture was circulated until it became clear, and 86 L of clear filtrate was collected. (3) Supercritical CO2 extraction The clarified filtrate was introduced into a 100 L supercritical extraction vessel, and 10 L of anhydrous ethanol was added as an entrainer. The extraction temperature was adjusted to 37 °C, the pressure to 28 MPa, and the CO2 flow rate to 25 L / h. Extraction was carried out for 4 h to obtain 12.3 L of extract. (4) Low-temperature concentration The extract was introduced into a rotary evaporator and distilled under reduced pressure at 42℃ and -0.09 MPa with nitrogen protection to obtain 3393 g of crude TriASA extract. HPLC analysis showed that the purity of the crude extract was 70%. (5) Silica gel column chromatography A 50mm × 1000mm chromatography column was used, packed with 200-mesh silica gel (activated at 110℃ for 18 h). The crude extract was dissolved in dichloromethane to a concentration of 150 mg / mL, and the sample loading volume was 5% of the column volume. Elution was performed according to a gradient elution program (diocalcium chloride to methanol volume ratio of 98:2 in the eluent from 0 to 120 min; 95:5 in the eluent from 120 to 320 min; and 90:10 in the eluent from 320 to 400 min). The elution peak from 280 to 320 min was collected, concentrated, and yielded 2513.66 g of TriASA intermediate. The purity was 91.5% as determined by HPLC. (6) Grade recrystallization The intermediate product was concentrated to a concentration of 250 mg / mL and refrigerated at -4℃ for 12 h to precipitate primary crystals. The primary crystals were then filtered under vacuum and dissolved in anhydrous ethanol to a concentration of 300 mg / mL. The primary crystals were then refrigerated at -10℃ for 8 h to carry out secondary crystallization. The crystals were collected by vacuum filtration. (7) Low-temperature vacuum drying The crystals were placed in a vacuum drying oven and dried at 50°C and -0.1 MPa for 24 h to obtain 2192.27 g of off-white high-purity TriASA solid powder.

[0046] The product was tested and found to have a TriASA purity of 98.3%, a TAPS residue of 0.28%, an acetyl hydrolysis rate of 0.76%, a moisture content of 0.25%, a heavy metal (Pb) content of 7 ppm, a solvent residue of 4 ppm, and a total yield of 86.2%.

[0047] Comparative Example 1 The method for separating and purifying triacetyldihydrosphingosine from fermentation broth in this comparative example is as follows: (1) High-pressure homogeneous crushing Take 10 L of fermentation broth with a TriASA content of 24 g / L, pass it into a high-pressure homogenizer, adjust the homogenization pressure to 650 bar, and homogenize twice to complete cell disruption; Ceramic membrane-ultrafiltration was selected for protein removal at 0.3 MPa. However, due to the high bacterial concentration and viscosity of the fermentation broth, the membrane filtration efficiency was extremely low. High-pressure filtration would cause significant membrane damage and a sharp increase in the temperature of the feed solution, which would be detrimental to the stability of TriASA. Therefore, ceramic membranes were no longer considered for protein separation in this fermentation broth.

[0048] Comparative Example 2 The difference from Example 1 is that triacetyldihydrosphingosine was purified using the silica gel column chromatography-recrystallization purification method disclosed in CN200910177025.8.

[0049] The eluents used in the embodiments of this invention are dichloromethane and methanol. The petroleum ether and ethyl acetate eluents used in patent CN200910177025.8 cannot meet the separation requirements of this invention. The combination of petroleum ether and ethyl acetate has too low polarity, and triacetyl dihydrosphingosine and tetraacetyl phytosphingosine cannot be completely eluted, resulting in a huge amount of solvent used and a low product yield.

[0050] Comparative Example 3 The difference from Example 1 is that this comparative example uses a dichloromethane-methanol mixture as the eluent, and the elution program is as follows: eluent volume ratio of 98:2, 0~400min.

[0051] When the volume ratio of the eluent is 98:2, the eluent has low polarity and mainly elutes small molecule impurities in the crude extract that are less polar than triacetyl dihydrosphingosine and tetraacetyl phytosphingosine. Only a very small amount of tetraacetyl phytosphingosine is eluted, while the main component, triacetyl dihydrosphingosine, cannot be eluted.

[0052] Comparative Example 4 The difference from Example 1 is that column chromatography was not performed after the crude extract in step (4) (the purity of TriASA in the crude product in this step was 67.3%), and fractional recrystallization was performed directly.

[0053] The sample was found to have a TriASA purity of 73.4%, a TAPS residue of 18.6%, and a sticky consistency.

[0054] Comparative Example 5 The difference from Example 1 is that in step (6), the fractional recrystallization is not controlled by low temperature. The crystallization process is terminated by cooling to room temperature and the sample is directly filtered.

[0055] The sample was found to have a TriASA purity of 98.89%, a TAPS residue of 0.2%, an acetyl hydrolysis rate of 1.34%, a moisture content of 0.25%, a heavy metal (Pb) content of 5 ppm, a solvent residue of 3 ppm, and an overall yield of 44.1%.

[0056] Comparative Example 6 The difference from Example 1 is that in step (5) of the column elution procedure, the loading flow rate and the elution flow rate are both increased to 10 ml / min, while the others remain unchanged.

[0057] The results showed that a large TAPS signal peak was detected in the elution collection at 280–320 min, indicating that TAPS and TriASA were not well separated.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for isolating and purifying triacetyl-dihydrosphingosine from a fermentation broth, characterized in that, include: The pH of the fermentation broth after cell disruption is adjusted to the isoelectric point of the cell protein. Flocculant is added under low temperature conditions to form flocculated precipitate, which is then filtered and the filtrate is collected. The filtrate was then subjected to supercritical CO2 extraction and low-temperature concentration to obtain the crude extract. The crude extract was then subjected to gradient silica gel column chromatography and fractional recrystallization. The crystals were collected and dried under low temperature vacuum to obtain triacetyl dihydrosphingosine as the final product.

2. The method of claim 1, wherein, The method for breaking down the cells in the fermentation broth includes: passing the fermentation broth containing West Wickham yeast cells containing synthetic triacetyl dihydrosphingosine into a high-pressure homogenizer, adjusting the homogenization pressure to 600-900 bar, and homogenizing in cycles 1-2 times. Preferably, the homogenization pressure is 650~850 bar.

3. The method of claim 1, wherein, The flocculant includes chitosan; Preferably, the flocculant is added at a mass of 0.1% to 0.3% of the fermentation broth volume; Preferably, during flocculation and sedimentation, the fermentation broth is cooled to 3-5°C, the flocculant is added, and the mixture is stirred at 30-50 rpm for 30-60 min, and then allowed to stand for 1-3 h. Preferably, after flocculation and sedimentation, solid-liquid separation is performed using a plate and frame filter; wherein the filter cloth of the plate and frame filter is made of polyester 300~500 mesh, and the filtration operating pressure is 0.1~0.4 MPa.

4. The method according to claim 1, characterized in that, The conditions for supercritical CO2 extraction are as follows: the entrainer is anhydrous ethanol, and the added volume is 5%~10% of the filtrate volume; the extraction temperature is 33~37℃, the extraction pressure is 28~32 MPa, the CO2 flow rate is 15~25 L / h, and the extraction time is 2~4 h.

5. The method according to claim 1, characterized in that, The conditions for low-temperature concentration are: temperature 38~42℃ and pressure -0.1~-0.09MPa.

6. The method according to claim 1, characterized in that, The gradient silica column chromatography includes: loading the crude extract into a dichloromethane solution, followed by gradient elution using a dichloromethane-methanol mixture as the eluent. The elution program is as follows: The volume ratio of dichloromethane to methanol in the eluent was 98:2 from 0 to 120 min. The volume ratio of dichloromethane to methanol in the eluent was 95:5 from 120 to 320 min, and the elution peak from 280 to 320 min was collected. The volume ratio of dichloromethane to methanol in the eluent was 90:10 at 320~400 min. Preferably, the elution flow rate during gradient elution is 5~8 mL / min; Preferably, the concentration of the crude extract in the dichloromethane solution is 100-150 mg / mL; Preferably, the sample loading volume is 5% of the column volume, and the sample loading flow rate is 3~5 mL / min; Preferably, the silicone column has a size of Φ50~100 mm × 1000~1500 mm; Preferably, the silica gel filling the silica gel column has a mesh size of 200-300 and a moisture content of ≤0.5%. Preferably, the ratio of the filling height of the silicone column to the inner diameter of the column is 20:1 to 30:

1.

7. The method according to claim 1, characterized in that, The graded recrystallization includes: The eluent collected from gradient silica column chromatography was concentrated and then refrigerated at -4°C for primary crystallization to precipitate primary crystals. The primary crystals were redissolved in anhydrous ethanol to prepare a crystallization solution; the solution was then refrigerated at -10°C and allowed to stand for secondary crystallization, and the crystals after secondary crystallization were collected. Preferably, the eluent is concentrated to 200-300 mg / mL; Preferably, during the initial crystallization, the crystals are refrigerated at -4°C and allowed to stand for 12 hours. Preferably, during secondary crystallization, the concentration of the primary crystals in the crystallization solution is 200-300 mg / mL, and the time for refrigeration and standing again is 8 h.

8. The method according to claim 1, characterized in that, The conditions for vacuum low-temperature drying are: temperature 45-50℃, vacuum pressure -0.1~-0.96 MPa, and drying time 20~28 h.

9. The triacetyldihydrosphingosine product obtained by the method according to any one of claims 1 to 8, characterized in that, The finished triacetyl dihydrosphingosine product has a triacetyl dihydrosphingosine purity ≥98%, a tetraacetyl phytosphingosine residue ≤0.5%, an acetyl hydrolysis rate ≤1%, a moisture content ≤0.5%, a heavy metal content ≤10 ppm, and a solvent residue ≤10 ppm.