Method for extracting and purifying tetraacetyl phytosphingosine

Abstract

The invention provides an extraction and purification method of tetraacetyl phytosphingosine. Comprising the following steps: pretreatment and plate frame filtration of tetraacetyl phytosphingosine fermentation liquor, flash evaporation drying of a thallus wet filter cake, microwaves of bacterial powder and reflux extraction of a solvent, reverse extraction, enrichment and impurity removal of an extracting solution, preparation of a crude extract, primary crystallization of the crude extract by a polar solvent, alternate solvent crystallization of a crystallized primary product and the like. The tetra-acetyl phytosphingosine is extracted from the bacterial powder obtained by flash evaporation and drying after thallus separation by adopting a microwave and extraction solvent condensation reflux technology, and the method has the advantages of small solvent dosage, high extraction speed and high extraction yield. Besides, silica gel column chromatography separation and purification are replaced by means of reverse extraction, enrichment and impurity removal and alternate solvent crystallization, use of a mixed solvent and generation of silica gel solid waste are avoided, the purposes of saving cost and reducing environmental pollution can be achieved at the same time, and the method has wider application prospects and better industrial production potential.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic raw material preparation technology, specifically relating to a method for extracting and purifying tetraacetyl phytosphingosine. Background Technology

[0002] Tetraacetylphytosphingosine (TAPS) is a saturated long-chain amino alcohol containing eighteen carbon atoms, discovered in 1956 by American microbiologists L.J. Wickerham et al. from the culture medium of a haploid strain of *Wickerhamomyces ciferrii* NRRLY-1031F-60-10A. It belongs to the sphingolipid class of compounds and is a naturally derived active cosmetic ingredient. The phytosphingosine produced after complete deacetylation of TAPS is a lipid source that forms the skin's stratum corneum moisture barrier and is also a key ingredient in the synthesis of ceramides, a star ingredient in skincare for moisturizing and barrier repair. Therefore, TAPS is a widely used and highly promising microbial-derived natural product. The synthesis of TAPS is a very complex microbial metabolic process. The fermentation process inevitably produces some metabolic byproducts, leading to complex downstream extraction and purification processes and high difficulty in scale-up. The existing literature and patents disclose several main TAPS extraction processes:

[0003] US Patent 5627056 discloses a process for the extraction and purification of TAPS, including centrifugation to collect bacterial cells, freeze-drying the bacterial cells, extraction with a methanol / ethyl acetate mixed solvent, concentration of the extract under reduced pressure, dissolution of the concentrated extract in ethyl acetate, washing with ethyl acetate solution to remove impurities, and further concentration to obtain crude TAPS extract. The crude extract is purified by silica gel column chromatography, eluting sequentially with petroleum ether-diethyl ether at volume ratios of 1:0, 7:3, and 4:6, finally yielding TAPS with a purity of 95%. This process is limited in scale, yielding only gram-level TAPS samples. Freeze-drying is time-consuming, energy-intensive, costly, and has limited throughput. Extraction with a mixed solvent is difficult to recover and results in significant solvent loss.

[0004] The extraction and purification process of TAPS disclosed in US Patent 5618706 includes centrifugation to collect bacterial cells after fermentation, isopropanol extraction of wet bacterial cells, salt separation of the isopropanol / water co-extract, secondary ethyl acetate extraction of the bacterial residue after isopropanol extraction, and concentration of the isopropanol extract and ethyl acetate extract under reduced pressure to obtain crude TAPS extract. The crude extract is purified by silica gel column chromatography, first eluting with hexane-ethyl acetate at a volume ratio of 1:0, 9:1, 5:1, and 3:2, then eluting with ethyl acetate and isopropanol at a purity of 95%, and finally recrystallizing with isopropanol-acetic acid (99:1) or n-butanol-acetic acid (99:1). This process results in high water content in the bacterial cells after centrifugation, which inevitably leads to increased solvent consumption and difficulties in the separation of the isopropanol / water co-extract. Furthermore, the presence of isopropanol residue in the ethyl acetate secondary extract also presents challenges in recovering the mixed solvent and significant losses.

[0005] The TAPS extraction method disclosed in Chinese patent CN116803973A involves diluting, acidifying, and heating the fermentation broth to dissolve the intracellular TAPS. The TAPS is then adsorbed using an ion exchange resin, eluted using a gradient, concentrated, and dried to obtain a high-purity crude TAPS product containing water. After desalting with pure water, the purity of the TAPS can reach 90-95%. Because TAPS is easily hydrolyzed under acidic or alkaline heating conditions, the temperature of the TAPS acetic acid eluent in this process is strictly controlled at around 45℃ during concentration. Acetic acid and water have boiling points of 118℃ and 100℃, respectively. For the acetic acid-water system, the vacuum distillation temperature should generally be between 70-80℃. Lower temperatures result in slow distillation rates and low efficiency, which is detrimental to industrial production.

[0006] The above-mentioned different TAPS extraction methods can achieve the purpose of extraction and purification through different process combinations, but the purity, yield and feasibility of industrial scale-up of the product need to be further improved. Moreover, the content of the product in the crude TAPS extract is relatively low, which is not conducive to further purification of the product. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings and defects of existing technologies and provide a novel process for extracting tetraacetylphytsphingosine from the fermentation broth of tetraacetylphytsphingosine. Specifically, this process includes solid-liquid separation of the fermentation broth, flash drying of the mycelial cake, microwave and solvent reflux extraction, back-extraction enrichment and preparation of crude extract, preliminary crystallization, and alternating solvent crystallization. Using this process, the extraction yield of tetraacetylphytsphingosine is above 60%, and the purity and content of the product can reach above 97%.

[0008] The technical solution of the present invention includes the following steps:

[0009] 1) Collection of bacterial cells: Add flocculant and filter aid to the fermentation broth, stir until the flocculent is fully dispersed, and filter through a plate and frame filter press to obtain bacterial cake;

[0010] 2) Flash drying: The collected fungal cake is crushed and sent to a rotary flash dryer for flash drying to obtain fungal powder containing tetraacetyl phytosphingosine;

[0011] 3) Microwave and solvent reflux extraction: The bacterial powder was transferred into a microwave extraction tank, and a non-polar solvent was added at a material-to-liquid ratio (W / V) of 1:3 to 1:6. Microwave and solvent reflux extraction was carried out, and the extract containing tetraacetyl phytosphingosine was obtained after filtration.

[0012] 4) Back-extraction enrichment and preparation of crude extract: The extract is transferred to an extraction tank and back-extracted and enriched using a polar solvent that is immiscible with the extraction solvent. The extraction enrichment factor is more than 4.5 times. The extracts are combined and concentrated under reduced pressure to remove the organic solvent, and crude extract is obtained. The purity of the active ingredient tetraacetyl phytosphingosine is increased by more than 20%.

[0013] 5) Preliminary crystallization: Add a polar organic solvent to the crude extract at a material-to-liquid ratio (W / V) of 1:5 to 1:10 to dissolve it and prepare a concentration of 100 to 200 g / L. Crystallize by cooling while stirring. After the product precipitates, filter and separate it at low temperature. After drying with cold air, tetraacetyl phytosphoprotein is obtained as yellowish-white crystals.

[0014] 6) Secondary alternating crystallization: Add a non-polar organic solvent to the primary crystallizer at a ratio of 1:10 to 1:20 (w / v) to dissolve it, and prepare a concentration of 50 to 100 g / L. Crystallize by cooling while stirring. After the product precipitates, filter and separate it at low temperature. After drying with cold air, white crystals of tetraacetyl phytosphingosine are obtained.

[0015] Furthermore, the present invention has optimized the cell collection process in step 1). The preferred flocculants are cationic polyacrylamide and polyaluminum chloride. The preferred addition amount of cationic polyacrylamide is 0.05-0.15% (W / V) of the fermentation liquid volume, the preferred addition amount of polyaluminum chloride is 1.0-3.0% (W / V) of the fermentation liquid volume, and the preferred filter aid is diatomaceous earth or perlite, with a preferred addition amount of 4-10% (W / V) of the fermentation liquid volume.

[0016] Furthermore, the present invention has optimized the process of flash drying to prepare bacterial powder in step 2), preferably that the moisture content of the bacterial powder after flash drying is 3-6%.

[0017] Furthermore, the present invention has optimized the non-polar solvent and microwave and solvent re-extraction technology used in step 3) for bacterial powder extraction. The preferred non-polar solvent is one of hexane, heptane or petroleum ether, the preferred microwave frequency is 2450±100MHz, the preferred heating reflux temperature is 60~80℃, and the preferred microwave extraction time is 10~30min.

[0018] Furthermore, the present invention has optimized the polar solvent and extraction process used in the back-extraction enrichment in step 4). The preferred polar solvent is one of methanol or acetonitrile, the preferred number of extractions is 2 to 3, and the preferred ratio of extractant to extractant liquid is 1:10 to 1:30.

[0019] Furthermore, the present invention has optimized the polar solvent and crystallization conditions used for the preliminary crystallization in step 5). The preferred solvent is one of methanol, ethanol, n-propanol, or ethyl acetate; the preferred crystallization temperature is -20 to -10°C; and the preferred filtration temperature is -15 to -5°C.

[0020] Furthermore, the present invention has optimized the nonpolar solvent and crystallization conditions used for secondary crystallization in step 6). The preferred solvent is one of hexane, heptane or petroleum ether, the preferred crystallization temperature is -20 to 0°C, and the preferred filtration temperature is -10 to 0°C.

[0021] This invention addresses the challenges of high solvent consumption, long extraction time, and low extraction yield in the extraction of tetraacetyl phytosphingosine. It employs flash drying to dry the mycelial cake after plate and frame filtration into a mycelial powder with a moisture content reduced to 3-6%. A non-polar solvent, such as hexane, heptane, or petroleum ether, is then added for microwave and solvent reflux extraction. The microwave's cell-wall breaking and heating effects accelerate the rapid dissolution of active ingredients within the cells, significantly improving extraction efficiency and yield, while reducing production costs and saving energy.

[0022] This invention utilizes polar organic methanol or acetonitrile to back-extract tetraacetyl phytosphingosine from non-polar organic extracts, retaining some weakly polar impurities, non-polar impurities, and pigments in the upper phase, thereby simultaneously increasing the content of tetraacetyl phytosphingosine and achieving enrichment and concentration.

[0023] This invention employs an alternating solvent crystallization method to purify tetraacetyl phytosphingosine, avoiding the generation of large amounts of silica gel waste residue that occurs during silica gel column chromatography purification, thus meeting the requirements of green chemistry development.

[0024] In summary, this invention employs a combination of flash drying, microwave and solvent reflux extraction, back-extraction enrichment and impurity removal, crude extract preparation, and alternating solvent crystallization for the extraction and purification of tetraacetyl phytosphingosine. This not only reduces the use of organic solvents and lowers production costs, but also saves energy and improves production efficiency.

[0025] The tetraacetyl phytosphingosine obtained by the extraction and purification method of this invention has a purity and content of over 97.0%, which is significantly higher than that of commercially available tetraacetyl phytosphingosine. Attached Figure Description

[0026] Figure 1 The HPLC chromatogram of tetraacetyl phytosphingosine in the n-hexane extract of Example 1, step 1.3 is shown.

[0027] Figure 2 The HPLC chromatogram of tetraacetyl phytosphingosine in the crude extract in step 1.4 of Example 1 is shown.

[0028] Figure 3 The HPLC chromatogram of tetraacetyl phytosphingosine in the initial crystallized product of Example 1, step 1.5 is shown below.

[0029] Figure 4 The image shows the HPLC chromatogram of tetraacetyl phytosphingosine in the finished product from step 1.6 of Example 1. Detailed Implementation

[0030] The present invention will be further illustrated below with reference to specific embodiments, but these embodiments do not limit the present invention in any way. The scope and core content of the present invention are determined by the claims. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0031] The detection method for tetraacetyl phytosphingosine in this invention employs HPLC-UV method, specifically as follows:

[0032] Chromatographic column: HPLCONE-5C18A analytical column (250mm×4.6mm×5μm);

[0033] Ultraviolet absorption wavelength: 200nm;

[0034] Mobile phase: Acetonitrile: Water: Phosphoric acid (V / V / V) = 80:20:0.1;

[0035] Flow rate: 1.0 ml / min;

[0036] Injection volume: 20 μL;

[0037] Column temperature: 30℃.

[0038] Example 1

[0039] A method for extracting and purifying tetraacetyl phytosphingosine includes the following steps:

[0040] S1.1, Fermentation broth pretreatment and cell collection: In 1m3 0.75 ml of tetraacetyl phytosphingosine fermentation broth was added to the extraction tank. 3 (Fermentation unit 20.7 g / L) Add 15 kg of polyaluminum chloride (PAC), which is pre-prepared as a 30% aqueous solution, and slowly pour it into the fermentation broth. Stir at 70 rpm for 10 min. Then add 0.75 kg of cationic polyacrylamide (PCA), which is pre-prepared as a 0.5% aqueous solution. Adjust the stirring speed to 100 rpm and continue stirring until obvious flocculation occurs. Then add 45 kg of diatomaceous earth (6% by volume) and continue stirring for 1 h until the flocs are fully dispersed. Filter the pretreated fermentation broth using a plate and frame filter press. When no filtrate flows out, use compressed air to blow the filter cake layer, blowing the residual moisture in the filter cake into the filtrate tank. After drying, collect the wet filter cake (crystal cake), which has a wet weight of approximately 259 kg.

[0041] S1.2 Flash drying of filter cake: The filter cake after plate and frame filter press is crushed and sent to a flash dryer for drying. The inlet air temperature is controlled at 130-140℃ and the outlet air temperature is controlled at 60-70℃. 134.6 kg of dried bacterial powder containing tetraacetyl phytosphingosine is obtained, with a moisture content of 3.7%. The content of the effective component tetraacetyl phytosphingosine in the dried bacterial powder is determined to be 10.9% by the content method. The yield from the tetraacetyl phytosphingosine fermentation broth to the dried bacterial powder is 94.6%.

[0042] S1.3 Microwave and Solvent Reflux Extraction: The mycelial powder was transferred to a microwave extraction vessel, and 3.5 times its weight in hexane solution was added. Extraction was performed using microwave and solvent reflux extraction technology. The microwave frequency was 2450 Hz, the reflux temperature was 60℃, and the extraction time was 20 min. After extraction, the mixture was centrifuged and filtered to obtain 490 L of extract containing tetraacetyl phytosphingosine. An appropriate amount of the extract was placed in a glass test tube and dried under nitrogen at 50℃ using an automated nitrogen blower. The extract was dissolved in 1.0 mL of mobile phase and analyzed by HPLC. The concentration of tetraacetyl phytosphingosine in the hexane extract was found to be 28.9 g / L, and the HPLC purity was 56.15%. The yield of tetraacetyl phytosphingosine in this step was 95.9%. The HPLC chromatogram is attached. Figure 1 As shown in Table 1, the detection peak results are as follows:

[0043] Table 1: HPLC peak results of tetraacetylphytosphoethanol in n-hexane extract

[0044]

[0045] S1.4. Back-extraction enrichment and preparation of crude extract: The hexane extract was transferred to an extraction tank, and tetraacetylphytosphoside was back-extracted using acetonitrile. The extraction was performed three times at acetonitrile-to-extract ratios of 1:10, 1:20, and 1:30, with the combined acetonitrile phase volume from the three extractions being approximately 110 L, resulting in an enrichment factor of 4.5. The organic solvent was removed by vacuum concentration, yielding 18.6 kg of crude extract. A suitable amount of the extract was dissolved in the mobile phase and analyzed by HPLC. The content of the effective component tetraacetylphytosphoside in the crude extract was determined to be 69.3%, with an HPLC purity of 71.15%, representing an increase in purity of approximately 15%. The yield of tetraacetylphytosphoside in this step was 91.5%. The HPLC chromatogram is attached. Figure 2 As shown in Table 2, the detection peak results are as follows:

[0046] Table 2: HPLC Detection Results of Tetraacetylphytosphoprotein Peak in Crude Extract

[0047]

[0048]

[0049] S1.5 Preliminary Crystallization: The concentrated crude extract of acetyl phytosphingosine was transferred to a crystallization tank. Approximately 120 L of methanol was added at a material-to-liquid ratio (W / V) of 1:6.5 to prepare a crystallization solution of approximately 155 g / L. Crystals began to precipitate when the temperature was slowly lowered to -18°C. The crystals were then stirred at this temperature for 8 hours at a stirring speed of 50 rpm. The resulting crystal slurry was filtered at -10°C to obtain pale yellow wet crystals. The wet crystals were dried using a vibrating fluidized bed with cold air to obtain 12.5 kg of yellowish-white tetraacetyl phytosphingosine primary crystal. An appropriate amount of the primary crystal was dissolved in the mobile phase and analyzed by HPLC. The content of the effective component tetraacetyl phytosphingosine in the primary crystal was determined to be 84.1%, and the HPLC purity was 88.1%. The yield of tetraacetyl phytosphingosine in this step was 81.4%. The HPLC chromatogram is attached. Figure 3 As shown in Table 3, the detection peak results are as follows:

[0050] Table 3: HPLC peak results of tetraacetylphytosphoprotein in the initial crystallized product

[0051]

[0052] S1.6, Secondary Alternating Crystallization: The initial tetraacetyl phytosphingosine crystals obtained in the previous step were transferred to a crystallization tank. Approximately 160 L of n-hexane was added at a material-to-liquid ratio of 1:15 to prepare a crystallization solution of approximately 66 g / L. Crystals began to precipitate when the temperature was slowly lowered to -10°C. The crystals were then stirred at this temperature for 4 hours at a stirring speed of 50 rpm. The resulting crystal slurry was filtered at -5°C to obtain white wet crystals. The wet crystals were dried using a vibrating fluidized bed with cold air to obtain 9.9 kg of pure white tetraacetyl phytosphingosine crystals. An appropriate amount of the product was dissolved in the mobile phase and analyzed by HPLC. The content of the effective component, tetraacetyl phytosphingosine, in the product was found to be approximately 97.0%, and the HPLC purity was 97.1%. The yield of tetraacetyl phytosphingosine in this step was 94.3%. The HPLC chromatogram is attached. Figure 4 As shown in Table 4, the detection peak results are as follows:

[0053] Table 4: HPLC peak results of tetraacetylphytosphoprotein in the finished product

[0054]

[0055] In this embodiment, the overall yield from the fermentation broth to the tetraacetyl phytosphingosine product was 63.8%.

[0056] Example 2

[0057] A method for extracting and purifying tetraacetyl phytosphingosine includes the following steps:

[0058] S2.1, Fermentation broth pretreatment and cell collection: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 3 0.78 ml of tetraacetyl phytosphingosine fermentation broth was added to the extraction tank. 3 (Fermentation unit 19.8 g / L) 15.6 kg of polyaluminum chloride (2% of the original fermentation broth volume) and 0.78 kg of cationic polyacrylamide (0.1% of the original fermentation broth volume) were added, and stirred until obvious flocculation occurred. Then, 50 kg of diatomaceous earth (6.5% of the original fermentation broth volume) was added and stirred until the flocs were fully dispersed. The pretreated fermentation broth was filtered using a plate and frame filter press, and the mycelial cake was collected. The wet weight of the mycelial cake was approximately 267 kg.

[0059] S2.2 Flash drying of mycelium cake: The mycelium cake after plate and frame filter press is crushed and then sent to a flash dryer for drying to obtain about 142 kg of dried mycelium powder with a moisture content of 4.5%. The content of the effective component tetraacetyl phytosphingosine in the dried mycelium powder is 10.2%. The yield from fermentation broth to dried mycelium powder is 92.8%.

[0060] S2.3 Microwave and Solvent Reflux Extraction: The mycelial powder was transferred to a microwave extraction vessel, and four times its weight in volume of n-heptane solution was added. Extraction was performed using microwave and solvent reflux extraction technology. The microwave frequency was 2450 Hz, the reflux temperature was 65℃, and the extraction time was 15 min. After extraction, centrifugation yielded approximately 590 L of n-heptane-containing extract. HPLC analysis showed that the concentration of tetraacetylphytosphoprotein in the n-heptane extract was 23.1 g / L, the HPLC purity was 55.3%, and the yield of tetraacetylphytosphoprotein in this step was 95.1%.

[0061] S2.4. Back-extraction enrichment and preparation of crude extract: The n-heptane extract was transferred to an extraction tank, and tetraacetylphytosphoside was back-extracted with methanol. The extraction was performed three times at methanol-to-extract ratios of 1:10, 1:15, and 1:20, with the combined methanol volume from the three extractions being approximately 140 L, resulting in an enrichment factor of 4.2. The organic solvent was removed by vacuum concentration, yielding approximately 18.1 kg of crude extract. HPLC analysis showed that the content of the effective component tetraacetylphytosphoside in the extract was approximately 66.3%, with an HPLC purity of approximately 67.9%, representing an increase in purity of approximately 12%. The yield of tetraacetylphytosphoside in this step was 88.2%.

[0062] S2.5 Preliminary Crystallization: The crude extract was transferred to a crystallization tank, and approximately 145 L of n-propanol was added at a material-to-liquid ratio (W / V) of 1:8 to prepare a crystallization solution of approximately 125 g / L. Crystals began to precipitate when the temperature was slowly lowered to -10°C. The solution was then stirred at this temperature for 6 hours at a stirring speed of 50 rpm. The resulting crystal slurry was filtered at -5°C to obtain pale yellow wet crystals. The wet crystals were dried using a vibrating fluidized bed with cold air to obtain 11.6 kg of pale yellow tetraacetylphytosphoside crystalline primary product. HPLC analysis showed that the effective component tetraacetylphytosphoside in the crystalline primary product was 85.1%, and the HPLC purity was 87.3%. The yield of tetraacetylphytosphoside in this step was 82.5%.

[0063] S2.6, Secondary Alternating Crystallization: The initial tetraacetylphytosphohydrin crystals were transferred to a crystallization tank. Approximately 160 L of n-heptane was added at a feed-to-liquid ratio (W / V) of 1:14 to prepare a crystallization solution of approximately 73 g / L. The solution was slowly cooled to -5°C, at which point crystals began to precipitate. The crystals were then stirred at this temperature for 3 hours at a stirring speed of 50 rpm. The resulting crystal slurry was filtered at 0°C to obtain white wet crystals. The wet crystals were then dried using a vibrating fluidized bed with cold air to obtain 9.6 kg of yellowish-white tetraacetylphytosphohydrin crystals. The HPLC yield of the product showed a tetraacetylphytosphohydrin content of 95.8% and an HPLC purity of 97.5%, resulting in a tetraacetylphytosphohydrin yield of 92.9% in this step.

[0064] In this embodiment, the overall yield from the fermentation broth to the tetraacetyl phytosphingosine product was 62.3%.

[0065] Example 3

[0066] A method for extracting and purifying tetraacetyl phytosphingosine includes the following steps:

[0067] S3.1, Fermentation broth pretreatment and cell collection: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 3 0.72 ml of tetraacetyl phytosphingosine fermentation broth was added to the extraction tank. 3 (Fermentation unit 18.5g / L), add 14.5kg of polyaluminum chloride and 0.72kg of cationic polyacrylamide in sequence and stir evenly until obvious flocculation occurs. Then add 50kg of perlite and stir until the flocs are fully dispersed. Filter with a plate and frame filter press and collect the mycelium cake. The wet weight of the mycelium cake is about 246kg.

[0068] S3.2 Flash drying of mycelium cake: After crushing the mycelium cake, it is sent to a flash dryer for drying to obtain about 128 kg of dried mycelium powder with a moisture content of 3.8%. The content of the effective component tetraacetyl phytosphingosine in the dried mycelium powder is 9.8%. The yield from fermentation broth to dried mycelium powder is 94.0%.

[0069] S3.3 Microwave and Solvent Reflux Extraction: The mycelial powder was transferred to a microwave extraction vessel, and petroleum ether solution with a volume four times its weight in the mycelial powder was added. Extraction was performed using microwave and solvent reflux extraction technology. The microwave frequency was 2450 Hz, the reflux temperature was 68℃, and the extraction time was 18 min. After extraction, centrifugation yielded approximately 550 L of petroleum ether extract. HPLC analysis showed that the concentration of tetraacetylphytosphoprotein in the extract was 21.4 g / L, the HPLC purity was 54.1%, and the yield of tetraacetylphytosphoprotein in this step was 94.4%.

[0070] S3.4, Back-extraction and enrichment, and preparation of crude extract: The petroleum ether extract was transferred to an extraction tank, and tetraacetyl phytosphingosine was back-extracted using acetonitrile. The extraction was performed three times at acetonitrile-to-extract ratios of 1:10, 1:20, and 1:30, respectively. The combined volume of the acetonitrile phases from the three extractions was approximately 125 L, with an enrichment factor of 4.4. The organic solvent was removed by vacuum concentration, yielding approximately 15.9 kg of crude extract. HPLC analysis showed that the content of the effective component tetraacetyl phytosphingosine in the crude extract was approximately 68.5%, with an HPLC purity of approximately 68.4%, representing an increase in purity of approximately 13%. The yield of tetraacetyl phytosphingosine in this step was 92.4%.

[0071] S3.5, Preliminary Crystallization: The oil paste was transferred to a crystallization tank, and 130L of ethyl acetate was added at a material-to-liquid ratio (W / V) of approximately 1:8 to prepare a crystallization solution of approximately 1:22 g / L. The solution was slowly cooled to -8℃, at which point crystals began to precipitate. The mixture was then stirred at this temperature for 5 hours at a stirring speed of 50 rpm. The resulting crystal slurry was filtered at -5℃ to obtain pale yellow wet crystals. The wet crystals were dried using a vibrating fluidized bed with cold air to obtain 11.2 kg of pale yellow tetraacetylphytosphoside crystalline primary product. HPLC analysis showed that the effective component tetraacetylphytosphoside in the crystalline primary product was 81.5%, and the HPLC purity was 82.6%. The yield of tetraacetylphytosphoside in this step was 83.7%.

[0072] S3.6, Secondary Alternating Crystallization: The initial tetraacetyl phytosphingosine was transferred to a crystallizer. 200L of petroleum ether was added at a feed-to-liquid ratio (W / V) of 1:16.5 to prepare a crystallization solution of approximately 60g / L. The solution was slowly cooled to -5℃, at which point crystals began to precipitate. The crystals were then stirred at this temperature for 3 hours at a stirring speed of 50 rpm. The resulting crystal slurry was filtered at 0℃ to obtain white wet crystals. The wet crystals were dried using a vibrating fluidized bed with cold air to obtain 8.9kg of pure white tetraacetyl phytosphingosine crystals. The HPLC yield of the product was 95.9% tetraacetyl phytosphingosine, with an HPLC purity of 96.5%. The yield of tetraacetyl phytosphingosine in this step was 93.1%.

[0073] In this embodiment, the overall yield from the fermentation broth to the tetraacetyl phytosphingosine product was 66.9%.

[0074] The specific embodiments described in this invention are merely illustrative examples. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, but without departing from the invention or exceeding the scope defined by the appended claims.

Claims

1. A method for extracting and purifying tetraacetyl phytosphingosine, characterized in that, Includes the following steps: S1. Collect the bacterial cells in the tetraacetyl phytosphingosine fermentation broth by solid-liquid separation; Collecting the bacterial cells involves adding flocculants and filter aids to the tetraacetyl phytosphingosine fermentation broth, followed by plate and frame filtration to obtain bacterial cakes. S2. The mycelium cake obtained in step S1 is dried by flash drying: The process of drying the mushroom cake includes: breaking up the mushroom cake obtained in step S1 and flash drying it to obtain mushroom powder containing tetraacetyl phytosphingosine; S3. Preparation of extract via microwave and reflux extraction: The preparation of the extract includes: adding an extraction solvent to the bacterial powder obtained in step S2, wherein the extraction solvent is a non-polar solvent, and extracting by microwave and reflux condensation to obtain an extract containing tetraacetyl phytosphingosine; S4. Preparation of crude extract via back-extraction and enrichment: The preparation of crude extract includes: adding an extraction solvent that is immiscible with the extraction solvent to the extract obtained in step S3 for back-extraction enrichment to obtain an extract, wherein the extraction solvent is a polar solvent; The extract was concentrated under reduced pressure to obtain a crude extract containing tetraacetyl phytosphingosine; S5. Preparation of the initial crystallized product includes: adding a polar solvent to the crude extract obtained in step S4 to dissolve, crystallize, filter, and dry to obtain the initial crystalline product of tetraacetyl phytosphingosine. S6. Prepare crystalline products by alternating solvent crystallization; The preparation of the crystalline product includes: adding a non-polar solvent to the crystalline precursor obtained in step S5 to dissolve, crystallize, filter, and dry to obtain the tetraacetyl phytosphingosine crystalline product.

2. The method for extracting and purifying tetraacetyl phytosphingosine according to claim 1, characterized in that, The flocculant in step S1 includes polyaluminum chloride and cationic polyacrylamide. The amount of cationic polyacrylamide added is 0.05 to 0.15% of the volume of the fermentation liquid, and the amount of polyaluminum chloride added is 1.0 to 3.0% of the volume of the fermentation liquid.

3. The method for extracting and purifying tetraacetyl phytosphingosine according to claim 1, characterized in that, In step S1, the filter aid includes diatomaceous earth or perlite, and the amount of diatomaceous earth or perlite added is 4 to 10% of the volume of the fermentation liquid.

4. The method for extracting and purifying tetraacetyl phytosphingosine according to claim 1, characterized in that, In step S2, a bacterial powder with a moisture content of 3-6% is obtained by flash drying.

5. The method for extracting and purifying tetraacetyl phytosphingosine according to claim 1, characterized in that, The extraction solvent in step S3 is one of hexane, heptane or petroleum ether. The ratio of the bacterial powder obtained in step S2 to the extraction solvent is 1:3 to 1:

6. The microwave frequency is 2450±100MHz, the heating reflux temperature is 60 to 80℃, and the extraction time is 10 to 30 min.

6. The method for extracting and purifying tetraacetyl phytosphingosine according to claim 1, characterized in that, The extraction solvent in step S4 is either methanol or acetonitrile, and the ratio of the extraction solvent to the extract obtained in step S3 is 1:10 to 1:

30. The extraction and enrichment are performed 2 to 3 times. The extracts are combined and concentrated under reduced pressure to obtain a crude extract containing tetraacetyl phytosphingosine.

7. The method for extracting and purifying tetraacetyl phytosphingosine according to claim 1, characterized in that, In step S5: the ratio of the crude extract obtained in step S4 to the polar organic solvent is 1:5 to 1:10; the polar organic solvent is one of methanol, ethanol, n-propanol or ethyl acetate, the crystallization temperature is -20 to -10℃, and the filtration temperature is -15 to -5℃.

8. The method for extracting and purifying tetraacetyl phytosphingosine according to claim 1, characterized in that, In step S6: the ratio of the initial crystallized product obtained in step S5 to the non-polar organic solvent is 1:10 to 1:20, the non-polar organic solvent is one of hexane, heptane or petroleum ether, the crystallization temperature is -20 to 0℃, and the filtration temperature is -10 to 0℃.

9. A tetraacetyl phytosphingosine obtained by the extraction and purification method of tetraacetyl phytosphingosine according to any one of claims 1-8.

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