A high-yield wickerhamiella jordaniae producing tetraacetyl phytosphingosine and application thereof

Abstract

The application discloses a Wickerhamomyces ciferrii strain with high tetracetylated phytosphingosine production and application thereof. The strain is named Wickerhamomyces ciferrii HG025, and the preservation number is CGMCC NO.39142. The strain HG025 has extremely high utilization rate of mixed sugar from sugarcane residue and tolerance to trace inhibiting substances. The production cost is greatly reduced: the agricultural waste, i.e., sugarcane residue, is used to replace expensive glucose as a main carbon source, and the raw material cost is reduced by about 40%, and meanwhile, the resource utilization of waste is realized, which conforms to the green manufacturing concept. The product is environment-friendly, has high purity (more than 95%), maintains configuration, has high fermentation efficiency, the yield of tetracetylated phytosphingosine is more than 666.2 mg / g (DCW), the production intensity is high, and the product is a full natural biofermentation source, and has extremely high commercial value.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and resource recycling, specifically to a high-yield tetraacetyl phytosphingosine-producing Wickham Severf yeast and its applications, particularly to a high-yield tetraacetyl phytosphingosine-producing Wickham Severf yeast and its applications using refined molasses from sugarcane bagasse. Specifically, it relates to a method and application for efficiently converting agricultural waste sugarcane bagasse into specialized molasses, and then using a high-yield tetraacetyl phytosphingosine-producing Wickham Severf yeast obtained through specific screening and mutagenesis for fermentation production. Background Technology

[0002] Phytosphingosine is a long-chain base with important physiological activities and is a precursor to ceramides. It is widely used in high-end cosmetics and pharmaceuticals for skin barrier repair, anti-inflammation, and antibacterial purposes. Currently, the industrial production of phytosphingosine mainly relies on chemical synthesis or plant extraction methods. However, these two methods suffer from problems such as harsh reaction conditions, low yields, poor stereoselectivity, and limited resources.

[0003] Microbial fermentation is considered a highly promising alternative route due to its environmental friendliness and the simple stereoconfiguration of the products. Among them, *Wickerhamomyces ciferrii*, with its strong lipid metabolism network and precursor supply capacity, is considered an ideal chassis cell for synthesizing sphingolipids. However, existing technologies for the fermentation production of tetraacetylphytosphoins face two major bottlenecks: First, high raw material costs. Existing fermentation processes mostly use high-purity carbon sources such as glucose and glycerol, with carbon source costs accounting for more than 50% of the total production cost, significantly limiting the product's market competitiveness. Second, poor adaptability of strains to inexpensive carbon sources. my country's sugar industry produces tens of millions of tons of sugarcane bagasse annually. Although it can be converted into fermentable sugars through acid or enzymatic hydrolysis, the bagasse hydrolysate typically contains large amounts of fermentation inhibitors such as furfural, 5-hydroxymethylfurfural, acetic acid, and phenolic compounds. Ordinary industrial strains of *Wickerhamomyces ciferrii* experience limited growth in environments containing these inhibitors, leading to a shift in metabolic flux and resulting in extremely low yields of the target product, tetraacetylphytosphoins.

[0004] While there are reports on the resource utilization of sugarcane bagasse in existing technologies, most focus on producing low-value-added products such as ethanol or single-cell protein, and often require complex detoxification processes. Currently, there are no reports of systematically integrating the preparation process of refined molasses from sugarcane bagasse with the targeted breeding of "tolerant" high-yield tetraacetyl sphingosine-producing plant strains to achieve efficient conversion of "waste into high-value functional lipids."

[0005] Overall problems: 1) High cost of preparing tetraacetyl phytosphingosine: The fermentation substrate uses high-purity carbon sources such as glucose and glycerol, which is extremely expensive. 2) Low efficiency: Under existing fermentation conditions, the yield of tetraacetyl phytosphingosine by ordinary Wickham Severin yeast is extremely low.

[0006] Therefore, developing a high-yield, low-cost method for preparing tetraacetyl phytosphingosine is crucial for its industrial production. Summary of the Invention

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a Wickham Severus yeast that produces high levels of tetraacetyl phytosphingosine and its applications.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] In a first aspect, the present invention provides a *Wickerhamomyces ciferrii* yeast, named *Wickerhamomyces ciferrii* HG025, which was deposited on February 5, 2026, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.39142.

[0010] Secondly, the present invention provides a biological agent comprising the aforementioned Wickham Severin yeast.

[0011] Thirdly, the present invention provides an application of the aforementioned Wickham Severin yeast in high-yield tetraacetyl phytosphingosine; further, its application in the fermentation of refined molasses from sugarcane bagasse to produce tetraacetyl phytosphingosine.

[0012] Fourthly, the present invention provides an application of the aforementioned Wickham Severus yeast in constructing a recombinant engineered strain that produces high-yield tetraacetyl phytosphingosine; further, its application in constructing a recombinant engineered strain that produces tetraacetyl phytosphingosine through fermentation of refined molasses from sugarcane bagasse.

[0013] Fifthly, the present invention provides a method for producing tetraacetyl phytosphingosine by fermentation of refined molasses from sugarcane bagasse, comprising the following steps:

[0014] (1) Preparation of refined sugarcane bagasse molasses: Sugarcane bagasse is pretreated by dilute acid-steam explosion, enzymatic hydrolysis and saccharification, activated carbon adsorption and ion exchange resin purification, and concentrated to obtain refined sugarcane bagasse molasses.

[0015] (2) Fermentation production: The above-mentioned seed liquid of Wickham Severin yeast was inoculated into a fermentation medium containing refined sugarcane bagasse molasses and fermented. During the fermentation process, refined sugarcane bagasse molasses was added to maintain the carbon source supply. After the fermentation was completed, a fermentation broth containing tetraacetyl phytosphingosine was obtained.

[0016] Furthermore, in step (2), fed-batch fermentation of tetraacetyl phytosphingosine is carried out using Wickham Severin yeast, including the following process control conditions:

[0017] Fermentation temperature: 25-35℃, preferably 25-30℃, more preferably 28-30℃;

[0018] Fermentation pH: 5.0–7.5, preferably 6.0–7.5, more preferably 6.5–7.0;

[0019] Dissolved oxygen (DO) saturation: 15%–40%, preferably 20%–35%, more preferably 25%–35%;

[0020] Stirring speed: 200-800 rpm, preferably 200-600 rpm, dissolved oxygen is controlled by adjusting the stirring speed and / or the aeration ratio;

[0021] Aeration ratio: 0.5–2.0 vvm (air volume / fermentation broth volume / min), preferably 0.8–1.5 vvm, more preferably 0.8–1.2 vvm;

[0022] Can pressure: 0.02~0.08 MPa, preferably 0.03~0.06 MPa;

[0023] Fermentation cycle: 48–120 hours, preferably 56–96 hours;

[0024] Inoculation amount: 5%–20% (v / v), preferably 8%–12% (v / v).

[0025] Furthermore, in step (2), the total sugar concentration in the fermentation medium containing refined molasses from sugarcane bagasse is 30–50 g / L.

[0026] Furthermore, in step (2), the fermentation medium containing refined molasses from sugarcane bagasse also includes a precursor substance; the precursor substance is L-serine, with a concentration of 2-8 mM; preferably 5-8 mM.

[0027] Furthermore, in step (2), the fermentation medium containing refined molasses from sugarcane bagasse also includes yeast extract powder at a concentration of 1-3 g / L; and further to 2 g / L.

[0028] Furthermore, in step (2), the fermentation medium containing refined molasses from sugarcane bagasse also includes 3-8 g / L of ammonium sulfate, 2-5 g / L of potassium dihydrogen phosphate, and 0.5-1.5 g / L of magnesium sulfate heptahydrate; and further includes 5-8 g / L of ammonium sulfate, 3 g / L of potassium dihydrogen phosphate, and 0.5 g / L of magnesium sulfate heptahydrate.

[0029] Optionally, in some embodiments of the present invention, in step (2),

[0030] The conditions for adding bagasse to refine molasses are as follows: when the concentration of reducing sugar in the fermentation broth drops below 10 g / L, bagasse is added to refine molasses, and the concentration of reducing sugar in the fermentation broth is controlled to be maintained at 10-30 g / L.

[0031] Furthermore, in step (2), the method for preparing the seed culture of Wickham Severin yeast includes the following steps:

[0032] Wickham Severin yeast was inoculated into YPD liquid medium and cultured to obtain primary seed culture; it was then transferred to secondary seed culture medium at an inoculation rate of 5% to 20% (v / v) (preferably 8% to 12% (v / v)) and cultured again to obtain secondary seed culture, which is the seed culture of Wickham Severin yeast.

[0033] Preferably, the secondary seed culture medium is YPD liquid culture medium with the addition of refined sugarcane bagasse molasses at a concentration of 20–35 g / L; more preferably 30 g / L.

[0034] Preferably, the culture temperature is 25-35℃, more preferably 25-30℃, and even more preferably 28-30℃; the culture rotation speed is 150-250 rpm, more preferably 200±10 rpm; and the culture time is 20-28 h, more preferably 24 h.

[0035] Preferably, the temperature for the second culture is 25-35℃, more preferably 25-30℃, and even more preferably 28-30℃; the rotation speed for the second culture is 150-250 rpm, more preferably 200±10 rpm; and the second culture time is 20-28h, more preferably 24h.

[0036] Optionally, in some embodiments of the present invention, in step (1),

[0037] The amount of dilute acid used is as follows: dilute acid is added at a solid-liquid ratio of (0.8-2):(5-30); or more specifically, dilute acid is added at a solid-liquid ratio of 1:10.

[0038] The dilute acid includes sulfuric acid, and further includes 0.8 to 1.2 wt% sulfuric acid; and further includes 0.8 to 1.0 wt% sulfuric acid.

[0039] The conditions for steam explosion are: introducing steam to pressurize to 1.5–2.0 MPa, holding the pressure for 3–8 minutes, and then instantly depressurizing and exploding; further, introducing steam to pressurize to 1.5–1.8 MPa, holding the pressure for 5 minutes, and then instantly depressurizing and exploding.

[0040] Optionally, in some embodiments of the present invention, in step (1),

[0041] The compound enzyme preparation used for enzymatic hydrolysis includes cellulase at 10-20 FPU / g substrate and hemicellulase at 80-150 IU / g substrate.

[0042] The enzymatic hydrolysis conditions are as follows: pH adjusted to 4.8–5.2, enzymatic hydrolysis at 45–55°C and 120–180 rpm for 36–48 h; further, the conditions are as follows: pH adjusted to 5.0, enzymatic hydrolysis at 50°C and 150 rpm for 42 h.

[0043] Optionally, in some embodiments of the present invention, in step (1),

[0044] The activated carbon adsorption process involves plate and frame filtration of the enzymatic hydrolysate, heating the filtrate to 60–75°C, and passing it through an activated carbon column at a flow rate of 1–3 BV / h for decolorization; further heating the filtrate to 70°C and passing it through an activated carbon column at a flow rate of 2 BV / h for decolorization.

[0045] The ion exchange resin purification involves cooling the effluent after activated carbon adsorption and then sequentially passing it through a strong acid cation exchange resin column at a flow rate of 2–4 BV / h (preferably 3 BV / h) and a weak base anion exchange resin column at a flow rate of 2–4 BV / h (preferably 3 BV / h).

[0046] Furthermore, the strongly acidic cation exchange resin column includes the D001 strongly acidic cation exchange resin column, etc.; the weakly basic anion exchange resin column includes the D301 weakly basic anion exchange resin column, etc.

[0047] The concentration involves removing impurities from the purified solution obtained by ion exchange resin and concentrating it to a total sugar concentration of 600–700 g / L; further, it is concentrated to a total sugar concentration of 630–650 g / L.

[0048] The concentration method includes vacuum concentration.

[0049] Sixthly, the present invention provides a green process for extracting high-purity phytosphingosine from Wickham Severinia fermentation broth, comprising the following steps:

[0050] S1. Fermentation broth pretreatment and enzymatic release: The fermentation broth of Wickham Severin yeast containing tetraacetyl phytosphingosine was inactivated and the pH was adjusted. Then, enzymatic hydrolysis was performed to release intracellular products and obtain the enzymatic hydrolysate.

[0051] S2. Impurity Removal and Concentration: The enzymatic hydrolysate is separated into solid and liquid components using a ceramic membrane microfiltration system to obtain permeate and retentate. The retentate is then dialyzed and filtered (washed) with deionized water, and the dialysate is collected. The permeate and dialysate are then combined to obtain the filtrate. The filtrate is then concentrated under reduced pressure to obtain the concentrated filtrate.

[0052] S3, Alkaline hydrolysis to deacetylate: The concentrated filtrate is subjected to alkaline hydrolysis to convert tetraacetyl phytosphingosine into phytosphingosine; after the reaction is completed, the temperature is lowered and the pH is adjusted to precipitate phytosphingosine and form a suspension.

[0053] S4. Green solvent extraction and back extraction: The suspension is extracted using a bio-based solvent, cooled, and then further purified by back extraction, and the aqueous phase solution is collected.

[0054] S5. Decolorization and crystallization: Decolorize the aqueous solution, collect the filtrate, adjust the pH, and crystallize by gradient cooling to obtain crude phytosphingosine crystals.

[0055] S6. Recrystallization and drying: Dissolve crude phytosphingosine crystals in an aqueous ethanol solution by heating and recrystallize; dry the recrystallized crystals under vacuum, pulverize and sieve them, and the powder obtained after pulverization is high-purity phytosphingosine.

[0056] Optionally, in some embodiments of the present invention, in step S1,

[0057] The inactivation conditions are 80-90°C for 15-30 minutes; further, 85°C for 20 minutes.

[0058] The pH adjustment is to adjust the pH to 6.5–7.5; further, to 7.0 ± 0.2.

[0059] The enzymes used in the enzymatic hydrolysis treatment include a complex lysozyme and a glucanase; the complex lysozyme includes snailase, chitinase and polysaccharide monooxygenase; the glucanase includes β-1,3-glucanase.

[0060] The mass ratio of the snail enzyme, chitinase, and cleaving polysaccharide monooxygenase is (0.9–1.1):(1.8–2.2):(0.6–1.0); more specifically, it is 1:2:0.8.

[0061] The amount of enzyme added for the enzymatic hydrolysis treatment is 0.7-1.5%; more specifically, 1.1%.

[0062] Furthermore, the mass ratio of the compound lysozyme to glucanase is (8-6):(3-5).

[0063] The enzymatic hydrolysis conditions are as follows: enzymatic hydrolysis at 30–40°C for 6–12 hours; further, enzymatic hydrolysis at 35°C for 9 hours.

[0064] Optionally, in some embodiments of the present invention, in step S2,

[0065] The ceramic membrane has a pore size of 50–200 nm; more specifically, 150 nm.

[0066] The solid-liquid separation is carried out under the conditions of transmembrane pressure of 0.1 to 0.3 MPa and temperature of 40 to 50°C; further, it is carried out under the conditions of transmembrane pressure of 0.2 MPa and temperature of 45°C.

[0067] The volume of the concentrated filtrate is 10-30% of the filtrate volume; more specifically, 15%.

[0068] Optionally, in some embodiments of the present invention, in step S3,

[0069] The alkaline hydrolysis is performed by adjusting the alkali concentration to 1–2 mol / L using potassium hydroxide solution; further, to 1.5 mol / L.

[0070] The alkaline hydrolysis is performed at a temperature of 70–90°C for 4–8 hours; further, at a temperature of 80°C for 6 hours.

[0071] The cooling refers to reducing the temperature to 25-30°C.

[0072] The pH adjustment is to adjust the pH to 7.0–8.0; more specifically, to 7.5.

[0073] The acid used to adjust the pH is a food-grade acid; further, it is at least one of phosphoric acid and citric acid.

[0074] Optionally, in some embodiments of the present invention, in step S4,

[0075] The bio-based solvent includes at least one of 2-methyltetrahydrofuran and cyclopentyl methyl ether.

[0076] The volume ratio of the bio-based solvent to the suspension is (0.5–2) (0.6–1.8); more specifically, it is 1:1.

[0077] The extraction temperature is 40–65°C; more specifically, 50°C.

[0078] The cooling refers to reducing the temperature to 5-10°C.

[0079] The reagent used for back-extraction is acidified water; further, it is a phosphoric acid solution with a concentration of 1.5–3.5 mol / L (preferably 2 mol / L).

[0080] Optionally, in some embodiments of the present invention, in step S5,

[0081] The decolorizing agent used is activated carbon; the amount of activated carbon used is 0.8-1.5% w / w of the aqueous solution mass; more specifically, it is 1% w / w of the aqueous solution mass.

[0082] The decolorization conditions are 32–55°C for 20–50 minutes; further, decolorization is carried out at 40°C for 30 minutes.

[0083] The pH adjustment is to adjust the pH to 9.5–10.5; further, it is to adjust the pH to 10.0.

[0084] The gradient cooling conditions are as follows: first, the temperature is lowered to 10-15℃ at a rate of 0.1-0.3℃ / min, and crystals are grown for 1-3 hours; then, the temperature is lowered to 0-4℃ at a rate of 0.3-0.6℃ / min, and crystals are grown for 3-6 hours.

[0085] Furthermore, the gradient cooling conditions are as follows: first, the temperature is lowered to 15°C at a rate of 0.2–0.3°C / min, and crystallization is carried out for 2 hours; then, the temperature is lowered to 0–4°C at a rate of 0.4–0.5°C / min, and crystallization is carried out for 4 hours.

[0086] Optionally, in some embodiments of the present invention, in step S6,

[0087] The concentration of the ethanol aqueous solution is 80-90% (v / v).

[0088] The heating and melting temperature is 50–80°C; more specifically, 65°C.

[0089] The recrystallization is carried out by cooling to 2-6°C; further, it is carried out by cooling to 4°C.

[0090] The vacuum drying temperature is 30–40°C; further, it is 35°C.

[0091] The purity of the high-purity phytosphingosine is above 95%.

[0092] In a seventh aspect, the present invention provides a method for breeding Wickham Severinia HG025, comprising the following steps:

[0093] S1. Plates using sugarcane bagasse refined molasses as the sole carbon source were initially screened from oil-rich environments to obtain the starting strain;

[0094] S2. The starting strain was continuously passaged and adapted in a culture medium containing refined molasses from sugarcane bagasse to obtain an adapted strain.

[0095] S3. Adaptive evolution strains were mutagenized by ambient pressure room temperature plasma (ARTP) and then revived on plates containing refined molasses from sugarcane bagasse.

[0096] S4. Select single colonies for fermentation in 96-well plates, and use LC-MS / MS high-throughput screening to identify high-yield mutant strains of tetraacetyl phytosphingosine, which is Wickham Severin yeast HG025.

[0097] Eighthly, the present invention provides the use of the above-described Wickham Severin Yeast HG025 or the fermentation product obtained by the above-described fermentation method in the preparation of cosmetics.

[0098] The present invention has the following advantages and effects compared with the prior art:

[0099] (1) The present invention constructs a highly adapted system of “sugarcane bagasse refined molasses - special strain”: more than 95% of furfural and phenolic inhibitors in sugarcane bagasse hydrolysate are removed through a specific purification process, and the molasses is used as a stress factor for targeted domestication and breeding of strains. The obtained strain HG025 has extremely high utilization rate of mixed sugars (glucose and xylose) from sugarcane bagasse and is tolerant to residual trace inhibitors.

[0100] (2) This invention significantly reduces production costs: by using agricultural waste sugarcane bagasse to replace expensive glucose as the main carbon source, the raw material cost is reduced by about 40%, and at the same time, the resource utilization of waste is realized, which is in line with the concept of green manufacturing.

[0101] (3) Environmentally friendly: The separation and purification process mainly uses aqueous phase treatment and recyclable bio-based solvents, avoiding the use of toxic solvents such as chloroform and methanol, which meets the requirements of green chemistry.

[0102] (4) High product purity: Through the combination of ceramic membrane separation and directional crystallization process, pigments, proteins and homologues produced during fermentation are effectively removed, and the product purity can reach more than 95%.

[0103] (5) Configuration retention: The process conditions are mild, avoiding strong acid and high temperature for a long time, ensuring that the stereoconfiguration of plant sphingosine is consistent with that in human skin, thus guaranteeing its biological activity.

[0104] (6) The fermentation efficiency of the present invention is high: Under the optimized fermentation process, the yield of tetraacetyl phytosphingosine can reach more than 666.2 mg / g (DCW), which is high production intensity. Moreover, the product is a completely natural biological fermentation source and has extremely high commercial value. Attached Figure Description

[0105] Figure 1 This is a colony morphology diagram of Wickham Severin HG025. Detailed Implementation

[0106] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the materials and reagents used are commercially available.

[0107] Example 1: Breeding of Wickham Severin Yeast HG025

[0108] 1. Preparation of sugarcane bagasse molasses plates: The refined sugarcane bagasse molasses (HG001) prepared in step (1) of Example 2 was used as the sole carbon source, diluted to a total sugar concentration of 20 g / L, and ammonium sulfate 5 g / L, potassium dihydrogen phosphate 3 g / L, and agar 20 g / L were added to prepare screening plates.

[0109] 2. Screening of the starting strain: Samples were taken from the lees of a winery in Ningxia Hui Autonomous Region, enriched, and then plated onto the aforementioned plates. Single colonies with rapid growth and full growth were selected. After initial screening in shake flasks, a strain naturally producing tetraacetyl phytosphingosine was obtained, designated HG90.

[0110] 3. Adaptive evolution: The strain HG90 obtained above was inoculated into liquid medium containing 50% HG001 molasses, and subcultured every 24 hours, gradually increasing the molasses ratio to 100% (as the sole carbon source) during the subculture. After 50 consecutive generations, the evolved strain with a growth rate 30% higher than the starting strain was selected.

[0111] 4. ARTP mutagenesis: Prepare bacterial suspensions from evolved strains in the logarithmic growth phase, and use an ARTP mutagen (Wuxi Xuanshi) with a helium flow rate of 10 SLM, a power of 120 W, and a treatment time of 40 s (lethality rate of about 85%).

[0112] 5. High-throughput screening: The mutagenic bacterial culture was spread onto regeneration plates. 3000 single colonies were picked and transferred to 96-well plates (containing 40 g / L HG001 molasses) and incubated at 30℃ for 72 h. High-throughput LC-MS / MS was used for initial screening to obtain 5 high-yielding mutant strains.

[0113] 6. Genetic stability verification: The mutant strain with the highest yield was passaged 10 times consecutively, and the yield remained stable. This strain was named HG025 and identified as *Wickerhamomyces ciferrii* by ITS sequence analysis. The colony morphology is as follows: Figure 1 As shown.

[0114] The ITS sequence of Wickerhamomyces ciferrii HG025 is as follows:

[0115] .

[0116] In summary, strain HG025 is named *Wickerhamomyces ciferrii* HG025. Its preservation information is as follows: Depository institution: China General Microbiological Culture Collection Center (CGMCC); Depository address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: February 5, 2026; Deposit number: CGMCC NO.39142.

[0117] Example 2: Method for refining molasses from sugarcane bagasse and producing tetraacetyl phytosphingosine by fermentation of the molasses

[0118] (1) Preparation of refined molasses from sugarcane bagasse (HG001)

[0119] Pretreatment: Take 100 kg of air-dried sugarcane bagasse, crush it through a 40-mesh sieve, and add it to an aqueous solution containing 1.0 wt% sulfuric acid at a solid-liquid ratio of 1:10, and mix well. Place it in a steam explosion tank, introduce steam to pressurize it to 1.8 MPa, maintain the pressure for 5 min, and then release the pressure instantly for explosion.

[0120] Enzymatic hydrolysis: Collect the blasted material and adjust the pH to 5.0 with NaOH. Add a compound enzyme preparation (cellulase 15 FPU / g substrate, hemicellulase 100 IU / g substrate) and hydrolyze for 42 h at 50℃ and 150 rpm stirring.

[0121] Decolorization and desalting: The enzymatic hydrolysate was filtered using a plate and frame filter press. The filtrate was heated to 70°C and decolorized by passing it through an activated carbon column at a flow rate of 2 BV / h. After cooling, the effluent was sequentially passed through a D001 strong acid cation exchange resin column at a flow rate of 3 BV / h and then through a D301 weak base anion exchange resin column at a flow rate of 3 BV / h to obtain a purified solution. Both the D001 strong acid cation exchange resin column and the D301 weak base anion exchange resin column were purchased from Hunan Lehai Environmental Protection Technology Co., Ltd.

[0122] Concentration: The purified liquid is passed through a nanofiltration membrane to remove impurities with a molecular weight greater than 200 Da. The permeate is concentrated in a triple-effect evaporator to a total sugar concentration of 65°Bx (approximately 650 g / L), yielding refined sugarcane bagasse molasses HG001. HPLC analysis shows that it contains approximately 55% glucose, 30% xylose, 5% arabinose, and <0.05 g / L of furfural and 5-HMF.

[0123] (2) Fermentation production

[0124] Wickham Severin yeast HG025 seed culture was inoculated at a rate of 10% (v / v) into a 5 L fermenter containing refined sugarcane bagasse molasses and fermentation medium, with an initial fermentation volume of 3 L. The fermentation medium composition is as follows: refined sugarcane bagasse molasses HG001 (based on total sugar) 40 g / L, ammonium sulfate 5 g / L, yeast extract 2 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 0.5 g / L, L-serine 5 mM, and defoamer 0.2 mL / L (the defoamer is polyoxyethylene polyoxypropylene pentaerythritol ether (PPE), a common commercial product); the fermentation conditions are as follows: fermentation temperature 28℃, pH value controlled at 6.5~7.0 (adjusted by adding 12.5% ​​ammonia water and 1 mol / L dilute phosphoric acid), dissolved oxygen saturation controlled at 25%~35% (controlled by the linkage of stirring speed 200~600 rpm and aeration ratio 0.8~1.2 vvm), and tank pressure 0.04 MPa. After 18 hours of fermentation, when the residual sugar level dropped to 10 g / L, 65°Bx refined sugarcane bagasse molasses HG001 was added to maintain the residual sugar level in the fermentation broth at 10-20 g / L. Fermentation ended after 96 hours, yielding a fermentation broth containing tetraacetyl phytosphingosine, with a tetraacetyl phytosphingosine content of 666.2 mg / g (DCW).

[0125] The preparation method of Wickham Severin Yeast HG025 seed culture includes the following steps: Wickham Severin Yeast HG025 is inoculated into YPD liquid medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract) and cultured at 28℃ and 200 rpm for 24 h to obtain primary seed culture. Then, it is transferred to secondary seed medium (with an additional 30 g / L of refined sugarcane bagasse molasses HG001 added to the YPD liquid medium) at a 10% (v / v) inoculation rate and cultured at 28℃ and 200 rpm for 24 h to obtain secondary seed culture, i.e., Wickham Severin Yeast HG025 seed culture.

[0126] (3) Separation and purification

[0127] 3.1 The fermentation broth containing tetraacetyl phytosphingosine was incubated at 85°C for 20 minutes to inactivate the product and initially release some of it. The pH was adjusted to 7.0 using hydrochloric acid or potassium hydroxide solution. 0.8% of a compound lysozyme and 0.3% of a cell wall lysin β-1,3-glucanase were added. (The compound lysozyme included snail enzyme, chitinase, and polysaccharide monooxygenase in a mass ratio of 1:2:0.8, and the total amount of compound lysozyme and β-1,3-glucanase added was 1.1% (w / w)). Enzymatic hydrolysis was carried out at 35°C for 9 hours to induce cell wall rupture and efficiently release intracellular tetraacetylphagesphingosine, yielding the enzymatic hydrolysate. Snail enzyme was purchased from Shanghai Yubo Biotechnology Co., Ltd. (product number YB002K); chitinase was purchased from Shanghai Macklin Chemical Co., Ltd. (product number 9001-06-3); and polysaccharide monooxygenase was purchased from Jinpanbio (product number ACZ03002).

[0128] 3.2 The enzymatic hydrolysate was passed through a 150 nm hydrophilic ceramic membrane microfiltration system (conditions: 0.2 MPa, temperature: 45℃) for solid-liquid separation. This step effectively retained large molecular impurities such as bacterial cell fragments, proteins, and polysaccharides, obtaining a permeate and a retentate containing TAPS. The retentate was added to deionized water for dialysis (washing) to improve the product yield, obtaining a dialysate. The permeate and dialysate were then combined to obtain the filtrate.

[0129] 3.3. Concentrate the filtrate under reduced pressure (condition: -0.1 MPa) to 15% of the original filtrate volume. Add 5 mol / L potassium hydroxide solution to the concentrated filtrate to adjust the alkali concentration to 1.5 mol / L. Hydrolyze the filtrate at 80℃ for 6 hours. This step hydrolyzes tetraacetylphytosphoside to release crude phytosphoside. After the reaction is complete, cool the filtrate to 25-30℃ and adjust the pH to 7.5 with citric acid to precipitate the phytosphoside, resulting in a suspension.

[0130] 3.4. The suspension is mixed with 1 volume of 2-methyltetrahydrofuran at 50°C for extraction. Taking advantage of the high solubility of phytosphingosine in the warm solvent, it is transferred from the aqueous phase to the organic phase. After extraction, the organic phase is separated, and the temperature is lowered to 5-10°C. Acidified water (phosphoric acid, 2 mol / L) is added for back-extraction, which transfers phytosphingosine to the aqueous phase as a salt, while impurities such as fat-soluble pigments remain in the organic phase. The aqueous solution is collected. This step replaces the traditional chloroform / methanol system and significantly reduces toxicity.

[0131] 3.5 Add activated carbon to the aqueous solution at 1% w / w of the aqueous solution mass. Decolorize at 40℃ for 30 minutes, collect the filtrate, add 1 mol / L potassium hydroxide solution to the filtrate, adjust the pH to 10.0, and allow phytosphingosine to be released and slowly crystallize. Cool to 15℃ at a rate of 0.2℃ / min, and after crystallization for 2 hours, cool to 2℃ at a rate of 0.5℃ / min, and crystallize for 4 hours to obtain crude phytosphingosine crystals.

[0132] 3.6 Dissolve crude phytosphingosine crystals in an 80% (v / v) ethanol aqueous solution at 65℃ until a saturated solution is obtained. Cool the solution to 4℃ for recrystallization to remove trace amounts of homologue impurities. Dry the recrystallized crystals under vacuum at 35℃ and pulverize them to obtain high-purity phytosphingosine powder.

[0133] Examples 3-8: Preparation methods of tetraacetyl phytosphingosine under different fermentation conditions

[0134] Examples 3-8 provide preparation methods under different fermentation conditions. Except for the different parameters listed in Table 1, the other steps are the same as in Example 2.

[0135] Table 1

[0136]

[0137] Examples 9-12: Effects of molasses prepared under different pretreatment and enzymatic hydrolysis conditions on fermentation

[0138] Example 9: During the molasses preparation process, the steam explosion pressure was adjusted to 1.5 MPa, and the rest was the same as in Example 2.

[0139] Example 10: During the molasses preparation process, the sulfuric acid concentration was adjusted to 0.8%, and the rest was the same as in Example 2.

[0140] Example 11: In the molasses preparation process, the compound enzyme preparation was adjusted from cellulase 15 FPU / g substrate and hemicellulase 100 IU / g substrate to cellulase 10 FPU / g substrate and hemicellulase 150 IU / g substrate, and the rest was the same as in Example 2.

[0141] Example 12: In the molasses preparation process, the compound enzyme preparation was adjusted from cellulase 15 FPU / g substrate and hemicellulase 100 IU / g substrate to cellulase 20 FPU / g substrate and hemicellulase 80 IU / g substrate, and the rest was the same as in Example 2.

[0142] The molasses prepared in Examples 9-12 was fermented according to the fermentation process of Example 2.

[0143] Examples 13-17 Purification methods of phytosphingosine under different conditions

[0144] Examples 13-17 provide purification methods for phytosphingosine under different conditions. Except for the parameters listed in Table 2, the other steps are the same as in Example 2.

[0145] Table 2

[0146]

[0147] Comparative Example 1

[0148] In this comparative example, analytically pure glucose was used instead of sugarcane bagasse refined molasses HG001 as the carbon source. Glucose was used instead of HG001 in both the fermentation medium and the fed-batch medium, maintaining a consistent concentration (based on total sugar). The remaining strains (HG025), medium components, and fermentation conditions were identical to those in Example 2.

[0149] Comparative Example 2

[0150] This comparative example used untreated sugarcane bagasse enzymatic hydrolysate as the carbon source. In the molasses preparation step, the hydrolysate only underwent plate and frame filtration and evaporation concentration, without activated carbon adsorption or ion exchange resin treatment. The resulting crude molasses was dark brown in color and contained high concentrations of furfural (1.2 g / L) and acetic acid. This crude molasses was used to replace HG001 for fermentation, with the remaining conditions the same as in Example 2.

[0151] Comparative Example 3

[0152] This comparative example uses the unmutated starting strain HG90 for fermentation. The fermentation process and carbon source (HG001) are exactly the same as in Example 2, except that the strain is replaced with HG90.

[0153] Comparative Example 4

[0154] This comparative example uses the standard strain of Wickham's Saccharomyces cerevisiae ATCC14091 for fermentation. The fermentation process and carbon source (HG001) are exactly the same as in Example 2, except that the strain is replaced with ATCC 14091.

[0155] Comparative Example 5

[0156] This comparative example uses sucrose instead of sugarcane bagasse refined molasses HG001 as the carbon source. Although sugarcane bagasse comes from sugarcane, its composition differs from sucrose. This comparative example aims to investigate the strain's utilization of different types of sugar. Food-grade sucrose was used as the sole carbon source, and all other conditions were the same as in Example 2.

[0157] Comparative Example 6

[0158] The sugarcane bagasse pretreatment involved only 1.0% dilute sulfuric acid in a 121°C autoclave for 60 minutes, without steam explosion. Subsequent enzymatic hydrolysis and purification steps were the same as in Example 2.

[0159] Comparative Example 7

[0160] L-serine was not added to the fermentation medium in step (2). The other conditions were the same as in Example 2.

[0161] Comparative Example 8

[0162] In step (2), no yeast extract was added to the fermentation medium; only 7 g / L ammonium sulfate was used as the nitrogen source. The remaining conditions were the same as in Example 2.

[0163] Comparative Example 9

[0164] In step (2), the fermentation process is carried out without aeration or stirring, and the mixture is kept still. All other conditions are the same as in Example 2.

[0165] Comparative Example 10

[0166] In step (2), the pH is not controlled during fermentation and is allowed to decrease naturally. The remaining conditions are the same as in Example 2.

[0167] Comparative Example 11

[0168] In the separation and purification steps, the enzymatic hydrolysis treatment in step 3.1 is not performed, and the rest is the same as in Example 2.

[0169] Comparative Example 12

[0170] In the separation and purification steps, the hydrolysis reaction in step 3.3 is not performed. The concentrated filtrate is directly subjected to the next step 3.4, and the rest is the same as in Example 2.

[0171] Comparative Example 13

[0172] In the separation and purification steps, step 3.4 uses deionized water for back-extraction, and the rest is the same as in Example 2.

[0173] Comparative Example 14

[0174] In the separation and purification steps, step 3.5 involves initial and secondary cooling, where the pH-adjusted solution is placed directly in ice water for cooling and kept at that temperature for 6 hours. The rest is the same as in Example 2.

[0175] Comparative Example 15

[0176] In the separation and purification steps, the concentration of the ethanol aqueous solution in step 3.6 was changed from 80% (v / v) to 50% (v / v), and the rest was the same as in Example 2.

[0177] Experimental Example 1: Analysis of Molasses Quality and Fermentation Inhibitors

[0178] The refined molasses prepared in Examples 2 and 9-12, and the crude molasses prepared in Comparative Example 2 were subjected to component analysis, with a focus on detecting the content of fermentation inhibitors. The results are shown in Table 3.

[0179] Table 3 Comparison of the components of molasses obtained by different preparation processes

[0180]

[0181] As can be seen from the results in Table 3, the combined purification process of "activated carbon + ion exchange" used in this invention (Example 2, Examples 9-12) can effectively remove more than 95% of furfural, 5-HMF, and phenolic compounds, while significantly reducing the acetic acid content. Comparative Example 2, without purification, had extremely high concentrations of inhibitors (furfural, 5-HMF, acetic acid, total phenols, etc.), which typically severely inhibits yeast cell growth and metabolism.

[0182] Experimental Example 2: Determination of Fermentation Performance and Tetraacetyl Phytosphingosine Yield

[0183] Determination of Tetraacetylphytosphingosine (TAPS) content:

[0184] Sample preparation: Take 1 mL of fermentation broth, centrifuge at 10000 rpm for 5 min, and collect the bacterial cells. Wash once with distilled water. Add 1 mL of methanol:chloroform (2:1, v / v) mixture, sonicate to disrupt and extract for 30 min, centrifuge and collect the supernatant.

[0185] Chromatographic conditions: Agilent 1290 UPLC-6460 Triple Quad MS; Column: ZORBAX RRHDEclipse Plus C18 (2.1 × 50 mm, 1.8 μm); Mobile phase A: 0.1% formic acid aqueous solution, Mobile phase B: 0.1% formic acid acetonitrile solution; Gradient elution. Detection mode: MRM (Multiple Reaction Monitoring) mode.

[0186] Cell dry weight (DCW) determination: Take 10 mL of fermentation broth, centrifuge, wash twice with water, and dry at 105℃ to constant weight.

[0187] Test samples: Fermentation broths of Examples 2-12 and Comparative Examples 1-10.

[0188] The test results are shown in Table 4.

[0189] Table 4

[0190]

[0191] Feasibility and economics of raw material substitution (Example 2 vs. Comparative Example 1): Example 2 utilizes HG001 sugarcane bagasse refined molasses fermentation, and its TAPS yield (666.2 mg / g (DCW)) is 1.48 times that of pure glucose fermentation (Comparative Example 1, 449.1 mg / g), with very similar cell growth. This indicates that the strain HG025, selected through the "adaptive evolution" of this invention, has fully adapted to the mixed sugar components (glucose + xylose) in sugarcane bagasse refined molasses, eliminating the carbon metabolism repression effect. In terms of cost, the raw material cost of Example 2 is only 60% of that of Comparative Example 1, significantly improving economic efficiency.

[0192] The necessity of molasses purification (Example 2 vs. Comparative Example 2): When fermenting with untreated crude molasses (Comparative Example 2), cell growth was inhibited (DCW only 322.7 mg / g). This confirms that inhibitors in bagasse hydrolysate (such as high concentrations of furfural and phenols as shown in Table 3) are highly toxic to *Wickham Severus*. The purification process of this invention is a key prerequisite for waste utilization.

[0193] The decisive role of strain selection (Example 2 vs. Comparative Examples 3 and 4): In the same fermentation medium of refined sugarcane bagasse molasses HG001, the yield of the proprietary strain HG025 of this invention (666.2 mg / g (DCW)) was 2.2 times that of the starting strain HG90 (306.5 mg / g) and 2.3 times that of the commercially available strain ATCC 14091 (297.6 mg / g). This indicates that through directed evolution and mutagenesis under pressure from refined sugarcane bagasse molasses, the substrate transport and metabolic network of the strain were successfully modified, making it a high-yielding strain that specifically "eats" coarse grains. Ordinary strains cannot efficiently utilize xylose and are sensitive to trace inhibitors, resulting in extremely low yields.

[0194] Precursor and culture medium optimization (Examples 2, 5, 6 vs. Comparative Example 7): The biosynthesis of tetraacetyl phytosphingosine mainly involves the condensation of serine and palmitoyl-CoA. In Comparative Example 7, without the addition of L-serine, the yield decreased significantly to 425.1 mg / g. A comparison between Example 5 (2 mM) and Example 6 (8 mM) shows that appropriate serine addition (2-8 mM) can effectively alleviate the precursor supply bottleneck.

[0195] Robustness of process conditions: The results of Comparative Example 9 (stationary) and Comparative Example 10 (no pH control) were extremely poor, demonstrating the necessity of aerobic conditions and constant pH for the synthesis of lipid compounds by Wickham Severus yeast. Examples 3-4 and 7-8 show that this process can maintain high yields even under certain parameter fluctuations (such as sugar concentration, fermentation temperature, and nitrogen source concentration), demonstrating strong process robustness and suitability for industrial production.

[0196] Experimental Example 3: Test of Extraction Rate and Purity of Plant Sphingosine

[0197] (1) Extraction rate (%) = Total mass of high-purity phytosphingosine obtained in step 3.6 / Dry weight of cells in fermentation broth × 100%;

[0198] (2) The purity test method is as follows:

[0199] 1. Chromatographic conditions

[0200] Column: XB-C8 (4.6*150mm*3.5μm); Mobile phase A: 0.06% trifluoroacetic acid solution; Mobile phase B: acetonitrile; Column temperature: 40℃; Flow rate: 1ml / min; Injection volume: 5μL; Nebulization temperature: 50℃; Gradient program as shown in Table 5.

[0201] Table 5

[0202]

[0203] 2. Sample preparation: Accurately weigh 0.1 g of the sample and place it in a 10 mL volumetric flask. Add methanol, sonicate to dissolve and dilute to volume, then shake well. Phytosphingosine standards are prepared using the same method.

[0204] 3. Determination: Accurately measure 0.5 μL of the test solution and the standard solution, inject them into the liquid chromatograph, and record the chromatograms.

[0205] 4. Qualitative confirmation: The sample is qualitatively determined by the peak of the standard solution. The retention time of the test solution should be consistent with that of the standard solution.

[0206] 5. Blank test: Except for not weighing the sample, the sample preparation procedure shall be followed.

[0207] 6. Result calculation: Calculated by area normalization method (solvent peaks and chromatographic peaks with peak areas less than 0.05% are ignored).

[0208] Purity (%) of phytosphingosine = A 主 / A 总 ×100%

[0209] A 主 : Average peak area of ​​phytosphingosine main peak (3 parallel samples);

[0210] A 总 : The sum of the areas of all detectable peaks (excluding the blank).

[0211] The results are shown in Table 6.

[0212] Table 6

[0213]

[0214] The test results (Table 6) show that the separation and purification steps in this embodiment of the invention have a high extraction rate of phytosphingosine and a high purity of the final product. The comparative example, by changing the hydrolysis conditions, subsequent extraction steps, and extraction solvent, was affected in both the extraction rate and purity of the final product.

[0215] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A type of Wickham Severin yeast, characterized in that: The name of the *Wickerhamomyces ciferrii* is *Wickerhamomyces ciferrii* HG025, which was deposited on February 5, 2026, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.39142.

2. A biological agent, characterized in that: Includes the Wickham Severin yeast as described in claim 1.

3. The application of the Wickham Severin yeast of claim 1 or the biological agent of claim 2, characterized in that, For one of the following applications: (a) Application in high-yield tetraacetyl phytosphingosine; (b) Application in constructing recombinant engineered bacteria that produce high levels of tetraacetyl phytosphingosine; (c) Application in the preparation of cosmetics.

4. The application according to claim 3, characterized in that: (a1) Application in the fermentation of refined molasses from sugarcane bagasse to produce tetraacetyl phytosphingosine; (b1) Application in constructing recombinant engineered bacteria for the fermentation of refined molasses from sugarcane bagasse to produce tetraacetyl phytosphingosine.

5. The application according to claim 4, characterized in that: The method for preparing refined molasses from sugarcane bagasse includes the following steps: Sugarcane bagasse is pretreated by dilute acid-steam explosion, followed by enzymatic hydrolysis and saccharification, activated carbon adsorption and ion exchange resin purification, and then concentrated to obtain refined sugarcane bagasse molasses.

6. The application according to claim 5, characterized in that: The amount of dilute acid used is based on a solid-liquid ratio of (0.8-2):(5-30). Or, the dilute acid includes sulfuric acid; And / or, the conditions for the steam explosion are: introducing steam to pressurize to 1.5–2.0 MPa, holding the pressure for 3–8 minutes, and then releasing the pressure instantly to explode; And / or, the complex enzyme preparation used for the enzymatic hydrolysis includes 10-20 FPU / g substrate of cellulase and 80-150 IU / g substrate of hemicellulase; And / or, the enzymatic hydrolysis conditions are: pH adjusted to 4.8–5.2, 45–55°C, and stirring at 120–180 rpm for 36–48 h; And / or, the activated carbon adsorption involves plate and frame filtration of the enzymatic hydrolysate, heating the filtrate to 60-75°C, and passing it through an activated carbon column at a flow rate of 1-3 BV / h for decolorization; And / or, the ion exchange resin purification involves cooling the effluent after activated carbon adsorption and then sequentially passing it through a strong acid cation exchange resin column and a weak base anion exchange resin column at a flow rate of 2–4 BV / h. And / or, the concentration is to remove impurities from the purified solution obtained by ion exchange resin and concentrate it to a total sugar content of 600-700 g / L.

7. A method for producing tetraacetylphytosphoprotein by fermentation of refined molasses from sugarcane bagasse, characterized in that, Includes the following steps: The seed culture of Wickham Severin yeast as described in claim 1 was inoculated into a fermentation medium containing refined sugarcane bagasse molasses as described in any one of claims 5 to 6, and fermented. During the fermentation process, refined sugarcane bagasse molasses was added to maintain the carbon source supply. After the fermentation was completed, a Wickham Severin yeast fermentation broth containing tetraacetyl phytosphoprotein was obtained.

8. The method according to claim 7, characterized in that: The total sugar concentration in the fermentation medium containing refined molasses from sugarcane bagasse is 30–50 g / L; And / or, the fermentation medium containing refined molasses from sugarcane bagasse also includes L-serine at a concentration of 2–8 mM; And / or, the conditions for adding bagasse to refine molasses are: when the concentration of reducing sugar in the fermentation broth drops to below 10 g / L, add bagasse to refine molasses, and control the concentration of reducing sugar in the fermentation broth to be maintained at 10-30 g / L; And / or, fed-batch fermentation of tetraacetyl phytosphingosine using Wickham Severinia spp., including the following process control conditions: Fermentation temperature: 25~35℃; Fermentation pH: 5.0–7.5; Dissolved oxygen saturation: 15%–40%; Stirring speed: 200–800 rpm; Ventilation ratio: 0.5–2.0 vvm; Can pressure: 0.02~0.08 MPa; Fermentation cycle: 48–120 hours; Inoculation dosage: 5%–20% v / v.

9. A green process for extracting high-purity phytosphingosine from Wickham Severinia ferment broth, characterized in that, Includes the following steps: S1. Fermentation broth pretreatment and enzymatic hydrolysis: The fermentation broth of Wickham Severin yeast containing tetraacetyl phytosphingosine as described in claim 7 or 8 is inactivated, the pH is adjusted, and then enzymatic hydrolysis is performed to obtain the enzymatic hydrolysate. S2. Impurity Removal and Concentration: The enzymatic hydrolysate is separated into solid and liquid components using a ceramic membrane microfiltration system to obtain permeate and retentate. The retentate is then dialyzed with deionized water, and the dialysate is collected. The permeate and dialysate are then combined to obtain the filtrate. The filtrate is then concentrated under reduced pressure to obtain the concentrated filtrate. S3, Alkaline hydrolysis to deacetylate: The concentrated filtrate is subjected to alkaline hydrolysis; after the reaction is completed, the temperature is lowered and the pH is adjusted to form a suspension; S4. Green solvent extraction and back extraction: The suspension is extracted using a bio-based solvent, cooled, and then further purified by back extraction, and the aqueous phase solution is collected. S5. Decolorization and crystallization: Decolorize the aqueous solution, collect the filtrate, adjust the pH, and crystallize by gradient cooling to obtain crude phytosphingosine crystals. S6. Recrystallization and drying: Dissolve crude phytosphingosine crystals in an aqueous ethanol solution by heating and recrystallize; dry the recrystallized crystals under vacuum, pulverize and sieve them, and the powder obtained after pulverization is high-purity phytosphingosine.

10. The green process according to claim 9, characterized in that: In step S1, the inactivation conditions are: inactivation at 80-90°C for 15-30 minutes; And / or, in step S1, adjusting the pH means adjusting the pH to 6.5 to 7.5; And / or, in step S1, the enzymes used in the enzymatic hydrolysis treatment include a complex lysozyme and a glucanase; the complex lysozyme includes snailase, chitinase and polysaccharide monooxygenase; the glucanase includes β-1,3-glucanase; And / or, in step S1, the amount of enzyme added for the enzymatic hydrolysis treatment is 0.7% to 1.5%; And / or, in step S1, the enzymatic hydrolysis is performed at 30–40°C for 6–12 hours. And / or, in step S2, the pore size of the ceramic membrane is 50–200 nm; And / or, in step S2, the solid-liquid separation is carried out under the conditions of a transmembrane pressure of 0.1 to 0.3 MPa and a temperature of 40 to 50°C; And / or, in step S2, the volume of the concentrated filtrate is 10-30% of the filtrate volume; And / or, in step S3, the alkaline hydrolysis is performed by adjusting the alkali concentration to 1-2 mol / L using potassium hydroxide solution; And / or, in step S3, the alkaline hydrolysis is performed at a temperature of 70–90°C for 4–8 hours. And / or, in step S3, the cooling is to cool down to 25-30°C; And / or, in step S3, adjusting the pH means adjusting the pH to 7.0 to 8.0; And / or, in step S4, the bio-based solvent includes at least one of 2-methyltetrahydrofuran and cyclopentyl methyl ether; And / or, in step S4, the volume ratio of the bio-based solvent to the suspension is (0.5-2) (0.6-1.8). And / or, in step S4, the extraction temperature is 40–65°C; And / or, in step S4, the cooling is to cool down to 5-10°C; And / or, in step S4, the reagent used for back-extraction is acidified water; And / or, in step S5, the decolorizing agent used for decolorization is activated carbon; the amount of activated carbon used is 0.8 to 1.5% w / w of the mass of the aqueous solution; And / or, in step S5, the decolorization conditions are 32-55°C for 20-50 minutes; And / or, in step S5, adjusting the pH means adjusting the pH to 9.5 to 10.5; And / or, in step S5, the gradient cooling conditions are as follows: first, the temperature is lowered to 10-15°C at a rate of 0.1-0.3°C / min, and crystallization is carried out for 1-3 hours; then, the temperature is lowered to 0-4°C at a rate of 0.3-0.6°C / min, and crystallization is carried out for 3-6 hours. And / or, in step S6, the concentration of the ethanol aqueous solution is 80-90% v / v; And / or, in step S6, the temperature for heating and melting is 50–80°C; And / or, in step S6, recrystallization is performed by cooling the temperature to 2-6°C. And / or, in step S6, the temperature of the vacuum drying is 30-40°C; And / or, in step S6, the purity of the high-purity phytosphingosine is above 95%.

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