Integrated ceramide NP fermentation preparation process
By fermenting and synthesizing ceramide NP in situ using recombinant Saccharomyces cerevisiae engineered strains, the problems of complex purification and high cost in traditional processes have been solved, achieving efficient and low-cost production of ceramide NP, which is suitable for the cosmetics and pharmaceutical fields.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-05
- Publication Date
- 2026-03-31
AI Technical Summary
In the traditional ceramide NP production process, the intermediate purification process is complex, time-consuming and labor-intensive, the plant sphingosine loss rate is high, and the independent fermentation and synthesis systems result in high production costs and low purity.
Ceramide NP was synthesized in situ by fermentation with recombinant Saccharomyces cerevisiae engineered strains. By optimizing the compatibility between the fermentation system and the synthesis system, ceramide NP was directly synthesized from the fermentation broth. Combined with suitable culture medium and emulsifier, the process was simplified and the purity was improved.
It significantly shortens the production cycle, reduces costs, improves the utilization rate and purity of plant sphingosine, meets the requirements for high-purity applications, and is suitable for large-scale industrial production.
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Figure CN121759532A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of synthetic technology, specifically to an integrated fermentation process for preparing ceramide NP. Background Technology
[0002] Ceramide NP (N-oleoyl phytosphingosine) is a key component of the skin barrier, possessing functions such as skin barrier repair, moisturizing and water-locking, and anti-inflammatory and soothing effects. It is widely used in cosmetics, pharmaceuticals, and other fields. The traditional production process of ceramide NP consists of three independent steps: first, phytosphingosine is prepared through microbial fermentation; then, high-purity phytosphingosine raw material is obtained through multiple purification steps such as centrifugation, extraction, and column chromatography; finally, the purified phytosphingosine is reacted with ethyl oleate under the action of a catalyst to synthesize ceramide NP. After synthesis, multiple crystallization and washing processes are required to remove byproducts and impurities.
[0003] The aforementioned traditional process has significant drawbacks: 1. The intermediate purification process is complex, time-consuming, and labor-intensive, resulting in a 10%-15% loss rate of phytosphingosine and high production costs; 2. The fermentation system and the synthesis system are independent of each other, and the effective components in the fermentation broth (such as residual carbon sources and buffer substances) are not fully utilized, and the pH and solvent environment of the synthesis system need to be adjusted separately, resulting in redundant processes; 3. Impurities are easily introduced during multiple material transfers, affecting the purity of ceramide NP products.
[0004] Therefore, developing a one-step process that integrates plant sphingosine fermentation with ceramide NP synthesis to achieve in-situ conversion of fermentation products, simplify the process, and reduce losses has become an urgent technical problem to be solved in this field. Summary of the Invention
[0005] Therefore, based on the shortcomings of the prior art, the present invention provides an integrated fermentation preparation process for ceramide NP. This process optimizes the compatibility between the fermentation system and the synthesis system, eliminates the need for intermediate purification of plant sphingosine, and directly utilizes the fermentation broth to synthesize ceramide NP in situ, significantly shortening the production cycle, reducing costs, and ensuring product purity.
[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: This invention provides an integrated fermentation process for preparing ceramide NP, comprising the following steps: S1. Directed fermentation of phytosphingosine: Recombinant Saccharomyces cerevisiae engineered strains were inoculated into a suitable culture medium and fermented for 48-60 hours at 30-32℃, stirring rate of 200-300 r / min, and aeration rate of 1.0-1.5 vvm until the concentration of free phytosphingosine in the fermentation broth reached 15-20 g / L, and the proportion of free phytosphingosine was ≥90%. S2. In-situ synthesis of ceramide NP: Add emulsifier to the fermentation broth from step S1, stir and disperse, then add ethyl oleate at a molar ratio of phytosphingosine to ethyl oleate of 1:(1.2-1.3), and then add a methanol solution of 20%-30% sodium methoxide at a molar ratio of phytosphingosine to sodium methoxide of 1:(0.5-0.6), with a dropping rate of 1-2 mL / min; heat to 60-65℃, maintain reflux for 3-4 h, and control the pH of the system to 8.0-8.5 during the reaction. S3. Integrated purification: After the reaction is completed, cool to 30-35℃, add 0.1-0.2% polysaccharide flocculant, stir for 10-30 min, let stand for 1-3 h, filter to remove solid impurities; cool the filtrate to 0-5℃ to crystallize for 2-3 h, centrifuge and filter to separate the crystals, wash with methanol spray, and dry at 50-60℃ and vacuum degree -0.09MPa for 2-4 h to obtain ceramide NP product.
[0007] In step S1 of this invention, the recombinant Saccharomyces cerevisiae is a recombinant Saccharomyces cerevisiae engineered strain that is resistant to low alcohols and weak alkalinity. As a fermentation strain, it can efficiently synthesize and secrete free phytosphingosine and can maintain its activity in an environment containing 1%-2% ethanol and pH 7.0-9.0.
[0008] Further, in step S1, the adaptability culture medium is prepared by adding 1-2% (volume percentage) of pre-adaptive components, with 20-30 g / L glucose as the carbon source, 5-8 g / L yeast extract as the nitrogen source, and an initial pH of 7.0-7.5.
[0009] Furthermore, in step S1, the pre-adapted component is ethanol.
[0010] Furthermore, in step S1, the adaptable culture medium also includes 0.1-0.3 g / L magnesium sulfate and 0.05-0.1 g / L potassium dihydrogen phosphate to maintain the ion balance of the fermentation system.
[0011] In this invention, ethanol is added to the adaptability culture medium as a pre-adaptation component, which can induce the strain to adapt to the solvent environment of the subsequent synthesis system in advance; the weakly alkaline formula is close to the alkaline conditions of the synthesis reaction, reducing the need for subsequent pH adjustment steps; and an appropriate amount of mineral components maintain the ion balance of the fermentation system and ensure the metabolic activity of the strain.
[0012] Furthermore, in step S1, the concentration of phytosphingosine is monitored in real time by HPLC during the fermentation process. When the concentration reaches 15-20 g / L, the fermentation is stopped to obtain a fermentation broth containing free phytosphingosine.
[0013] The main components of the fermentation broth of this invention include: free phytosphingosine, residual glucose (≤5g / L), yeast cells (wet weight 10-15g / L), and metabolic byproducts (such as a small amount of glycerol, ≤2g / L), without any toxic impurities that would affect subsequent synthesis.
[0014] Further, in step S2, the emulsifier is Tween 80, and the amount added is 0.5%-1% of the fermentation liquid volume.
[0015] In this invention, since ethyl oleate is the oil phase, an emulsifier needs to be added first. The emulsifier is then uniformly dispersed by the high-speed stirring mechanism of the fermentation tank, achieving a two-phase mixture of oil and water (fermentation broth). The preferred emulsifier is Tween 80, which has good biocompatibility and does not affect product purity.
[0016] In this invention, the catalyst sodium methoxide is dissolved in methanol solvent and then added to the fermentation broth by slow dropwise addition. This avoids the rapid reaction between the catalyst and the small amount of water in the fermentation broth, which could lead to local overheating. At the same time, methanol can be used as a solvent for the synthesis of ceramide NP.
[0017] Furthermore, in step S2, if the pH of the system is greater than 9.0 during the reaction, 10% glacial acetic acid is added dropwise to adjust the pH to 8.0-8.5.
[0018] In step S2 of this invention, the yeast extract in the fermentation broth has a buffering effect, which can suppress the drastic pH fluctuations caused by the catalyst. The pH is monitored in real time during the reaction. If the pH is >9.0, a small amount of glacial acetic acid is added to adjust it, so as to avoid the degradation of the product due to excessive alkalinity. At the same time, glacial acetic acid can play a partial quenching role in advance, which paves the way for subsequent purification.
[0019] In step S2 of this invention, the fermenter is heated to 60-65°C and kept in a reflux state for 3-4 hours. At this temperature, the reaction requirements for ceramide NP synthesis can be met, and the yeast cells can be slightly broken to release the residual phytosphingosine in the cells and improve the raw material conversion rate.
[0020] Furthermore, in step S3, before adding the chitosan flocculant, 0.05-0.2% of the surfactant sodium dodecyl sulfate is added.
[0021] It should be noted that the chitosan flocculant and the surfactant sodium dodecyl sulfate are added based on the volume of the fermentation broth, that is, 0.1-0.2g of chitosan flocculant and 0.05-0.2g of sodium dodecyl sulfate are added per 100mL of fermentation broth.
[0022] Because fermentation cells may adsorb ceramide NP, leading to product loss, this invention adds a small amount of sodium dodecyl sulfate, a surfactant, after the reaction to disrupt the adsorption between the cells and the product, thereby improving the product recovery rate.
[0023] In step S3 of this invention, the chitosan flocculant can form a flocculent complex with the fermentation cells and insoluble reaction byproducts, facilitating subsequent separation. The solid impurities (including the fermentation cells, flocculant complex, and insoluble reaction byproducts) are then removed by filtration using a plate and frame filter press, yielding a clear filtrate containing ceramide NP. This filtrate mainly contains ceramide NP, residual solvent, and small amounts of soluble impurities such as glycerol and sodium acetate.
[0024] Furthermore, in step S3, the centrifugal filtration speed is 3000-4000 r / min, and the amount of methanol used in the spray washing is 1-1.5 times the mass of the crystals.
[0025] In step S3 of this invention, ceramide NP crystals are precipitated at low temperature, while residual metabolites (such as glycerol), soluble salts and other impurities in the fermentation broth remain in the mother liquor. After centrifugation, the crystals are further washed with methanol to remove residual impurities, thereby achieving the separation and purification of ceramide NP.
[0026] Furthermore, in step S3, before crystallization, some glycerol is removed by vacuum distillation at a temperature of 40-45°C and a vacuum degree of -0.08 MPa.
[0027] Since fermentation metabolic byproducts (such as glycerol) may affect the crystallization effect, in this invention, some glycerol is removed by vacuum distillation before crystallization, thereby avoiding glycerol from reducing the crystallization rate.
[0028] The ceramide NP product synthesized in this invention has a purity of ≥86% for component A (oleic ceramide) and a total purity of ≥96% for components A+B+C+D. Components B and D are homologous impurities / byproducts generated during fermentation or synthesis, corresponding to ceramides formed from saturated fatty acids of different chain lengths, with phase B being N-stearoyl phytosphingosine (C18:0), phase C being N-palmitoyl phytosphingosine (C16:0), and phase D being N-arachidoyl phytosphingosine (C20:0).
[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention integrates plant sphingosine fermentation and ceramide NP synthesis into a one-step process, eliminating the need for intermediate purification of plant sphingosine, avoiding raw material loss during the purification process (the utilization rate of plant sphingosine is increased from the traditional 85% to ≥92%), while shortening the production cycle by 15%-20% and significantly reducing production costs.
[0030] 2. This invention optimizes the compatibility of the culture medium and fermentation parameters, making the fermentation system highly compatible with the synthesis system. The residual carbon source and buffer substances in the fermentation broth can assist the synthesis reaction, reducing the amount of additional solvents and pH adjusters needed, and simplifying the process.
[0031] 3. In the synthesis reaction process, the present invention utilizes temperature and stirring to break the fermentation cells and release the residual phytosphingosine in the cells, thereby further improving the utilization rate of raw materials; the integrated purification process simultaneously removes fermentation impurities and synthesis byproducts, ensuring that the product purity is ≥96%, meeting the requirements for high-purity applications.
[0032] 4. The process of this invention uses conventional industrial equipment (fermentation tank, plate and frame filter, centrifugal filter, etc.) throughout the entire process, without the need for additional special devices, thus reducing equipment investment costs. Furthermore, the solvent and flocculant can be recycled, making it environmentally friendly and suitable for large-scale industrial production. Attached Figure Description
[0033] Figure 1 The structural formula of phytosphingosine; Figure 2 The structural formula of ceramide NP; Figure 3 The carbon NMR spectrum of phytosphingosine; Figure 4 The carbon NMR spectrum of ceramide NP; Figure 5 The NMR spectrum of ceramide NP is shown in the form of 1H NMR. Detailed Implementation
[0034] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.
[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.
[0036] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Example
[0037] A fermentation-integrated preparation process for ceramide NP includes the following steps: (1) Directed fermentation of phytosphingosine: Recombinant Saccharomyces cerevisiae engineered strains resistant to ethanol and weak alkali were selected. The suitable culture medium formula was: glucose 25 g / L, yeast extract 6 g / L, magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 0.08 g / L, ethanol 1.5%, and the initial pH was adjusted to 7.2. The strain was inoculated at a 6% inoculum and fermented for 54 h at 30℃, stirring rate 250 r / min, and aeration rate of 1.2 vvm. HPLC analysis showed that the concentration of free phytosphingosine in the fermentation broth was 18.2 g / L, and the free content was 93%. (2) In-situ synthesis of ceramide NP: Add 0.8% Tween 80 (based on fermentation broth volume) to the fermentation broth, stir and disperse at 380 r / min, add ethyl oleate at a molar ratio of phytosphingosine to ethyl oleate of 1:1.25, and then add 25% sodium methoxide methanol solution dropwise at a molar ratio of phytosphingosine to sodium methoxide of 1:0.55 (dropping rate 1.5 mL / min). Heat to 62℃ and reflux for 3.5 h, maintaining the pH at 8.2-8.4 during the reaction; (3) Integrated purification: After the reaction, the temperature was lowered to 32℃, 0.15% chitosan flocculant was added, stirred for 30 min, and allowed to stand for 1 h. Solid impurities were removed by plate and frame filtration. The filtrate was cooled to 3℃ and crystallized for 2.5 h. The crystals were separated by centrifugation at 3500 r / min and washed with methanol spray (the amount of methanol was 1.2 times the mass of the crystals). The crystals were dried at 55℃ and vacuum degree -0.09 MPa for 4 h to obtain the ceramide NP product.
[0038] The phytosphingosine and ceramide NP prepared in Example 1 were analyzed by NMR, and the results are as follows: Figure 3-5 As shown. Testing revealed that the ceramide NP product is a white powder, with component A having a purity of 88.3%, the total purity of components A+B+C+D being 97.2%, the utilization rate of phytosphingosine being 93.5%, and a production cycle of 72 hours. Example
[0039] A fermentation-integrated preparation process for ceramide NP includes the following steps: (1) Directed fermentation of phytosphingosine: Recombinant Saccharomyces cerevisiae engineered strains resistant to ethanol and weak alkali were selected. The suitable culture medium formula was: glucose 20 g / L, yeast extract 8 g / L, magnesium sulfate 0.15 g / L, potassium dihydrogen phosphate 0.05 g / L, ethanol 1%, and the initial pH was adjusted to 7.1. The strain was inoculated at a 5% inoculum and fermented for 48 h at 30℃, stirring rate 200 r / min, and aeration rate of 1.0 vvm. HPLC analysis showed that the concentration of free phytosphingosine in the fermentation broth was 15.6 g / L, and the free content was 91%. (2) In-situ synthesis of ceramide NP: Add 0.5% Tween 80 (based on fermentation broth volume) to the fermentation broth, stir and disperse at 350 r / min, add ethyl oleate at a molar ratio of phytosphingosine to ethyl oleate of 1:1.25, and then add 20% sodium methoxide methanol solution dropwise at a molar ratio of phytosphingosine to sodium methoxide of 1:0.5 (dropping rate 1 mL / min). Heat to 60℃ and reflux for 3 h, maintaining the pH at 8.0-8.2 during the reaction; (3) Integrated purification: After the reaction, the temperature was lowered to 30℃, 0.12% chitosan flocculant was added, stirred for 30 min, and allowed to stand for 1 h. Solid impurities were removed by plate and frame filtration. The filtrate was cooled to 0℃ and crystallized for 2 h. The crystals were separated by centrifugation at 3000 r / min and washed with methanol spray (the amount of methanol was 1 times the mass of the crystals). The crystals were dried at 52℃ and vacuum degree -0.09 MPa for 4 h to obtain the ceramide NP product.
[0040] Testing revealed that the ceramide NP product is a white powder, with a purity of 86.5% for component A, a total purity of 96.3% for components A+B+C+D, a phytosphingosine utilization rate of 92.1%, and a production cycle of 70 hours. Example
[0041] A fermentation-integrated preparation process for ceramide NP includes the following steps: (1) Directed fermentation of phytosphingosine: Recombinant Saccharomyces cerevisiae engineered strains resistant to ethanol and weak alkali were selected. The suitable culture medium formula was: glucose 28 g / L, yeast extract 6 g / L, magnesium sulfate 0.25 g / L, potassium dihydrogen phosphate 0.08 g / L, ethanol 2%, and the initial pH was adjusted to 7.5. The strain was inoculated at an inoculum of 8%, and fermented for 60 h at 30℃, stirring rate 300 r / min, and aeration rate of 1.5 vvm. HPLC analysis showed that the concentration of free phytosphingosine in the fermentation broth was 19.8 g / L, and the free content was 94%. (2) In-situ synthesis of ceramide NP: 1% Tween 80 (based on fermentation broth volume) was added to the fermentation broth and stirred at 400 r / min to disperse. Then, ethyl oleate was added at a molar ratio of phytosphingosine to ethyl oleate of 1:1.2. Next, 30% sodium methoxide methanol solution was added dropwise at a molar ratio of phytosphingosine to sodium methoxide of 1:0.6 (dropping rate 2 mL / min). The temperature was raised to 65℃ and refluxed for 4 h. The pH was maintained at 8.3-8.5 during the reaction. (3) Integrated purification: After the reaction, the temperature was lowered to 35℃, 0.2% chitosan flocculant was added, stirred for 30 min, and allowed to stand for 1 h. Solid impurities were removed by plate and frame filtration. The filtrate was cooled to 5℃ and crystallized for 3 h. The crystals were separated by centrifugation at 4000 r / min and washed with methanol spray (the amount of methanol was 1.4 times the mass of the crystals). The crystals were dried at 60℃ and vacuum degree -0.09 MPa for 4 h to obtain the ceramide NP product.
[0042] The finished product was tested and found to be a white powder with a purity of 89.1% for component A, a total purity of 97.8% for components A, B, C, and D, a phytosphingosine utilization rate of 94.3%, and a production cycle of 80 hours. Example
[0043] The only difference between this embodiment and Example 1 is that after the reaction is completed and the temperature is lowered to 32°C in step (3), 0.1% sodium dodecyl sulfate (SDS) is added before adding chitosan flocculant, and the mixture is stirred for 15 minutes before flocculation. The remaining steps and parameters are completely consistent with those in Example 1.
[0044] Testing showed that the filtrate obtained after flocculation and filtration had improved clarity, increased cell wall rupture rate to 82%, and increased phytosphingosine utilization rate to 95.1%. However, HPLC analysis of the final product revealed trace amounts of SDS residue (0.03%), which met cosmetic safety standards. The purity of component A decreased slightly to 87.5%, and the total purity of components A+B+C+D was 96.8%. Example
[0045] The only difference between this embodiment and embodiment 1 is that after filtering to remove solid impurities in step (3), a vacuum distillation step is added—the filtrate is distilled under vacuum at 40°C and a vacuum of -0.08MPa for 30 minutes to remove some low-boiling-point metabolic byproducts such as glycerol, and then cooled and crystallized. The remaining steps and parameters are completely consistent with those in embodiment 1.
[0046] Testing revealed that the glycerol removal rate in the filtrate after vacuum distillation was 75%, and the crystallization rate increased by 20%. The purity of component A in the final product was 88.5%, and the total purity of components A+B+C+D was 97.4%.
[0047] The only difference between this comparative example and Example 1 is that the adaptive culture medium is not used in step (1), but instead a conventional fermentation culture medium with the following formula: glucose 25 g / L, yeast extract 6 g / L, magnesium sulfate 0.2 g / L, potassium dihydrogen phosphate 0.08 g / L, and the initial pH is adjusted to 6.0 (a neutral to slightly acidic environment in conventional fermentation). Ethanol is not added. The remaining steps and parameters are completely consistent with those in Example 1.
[0048] Testing revealed that the concentration of free phytosphingosine in the fermentation broth was 14.8 g / L, with a free content of 78%. The pH fluctuated significantly during the synthesis reaction, requiring the addition of three times the amount of 10% glacial acetic acid to adjust the pH. The final product had a purity of 82.1% for component A, a total purity of 91.5% for components A+B+C+D, a phytosphingosine utilization rate of 85.3%, and a production cycle extended to 85 hours.
[0049] The only difference between this comparative example and Example 1 is that: in step (2), Tween 80 emulsifier is not added, and ethyl ester is added directly. The remaining steps and parameters are completely consistent with those of Example 1.
[0050] Observations showed that ethyl oleate and fermentation broth formed obvious stratification, and oil phase aggregation still occurred under high-speed stirring. After the reaction, the conversion rate of phytosphingosine was only 78.6%. The purity of component A in the final product was 80.3%, the total purity of components A+B+C+D was 90.2%, the utilization rate of phytosphingosine was 76.2%, and a small amount of unreacted ethyl oleate remained in the finished product.
[0051] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.
Claims
1. A fermentation-integrated preparation process for ceramide NP, characterized in that, Includes the following steps: S1. Directed fermentation of phytosphingosine: Recombinant Saccharomyces cerevisiae engineered strains were inoculated into a suitable culture medium and fermented for 48-60 hours at 30-32℃, stirring rate of 200-300 r / min, and aeration rate of 1.0-1.5 vvm until the concentration of free phytosphingosine in the fermentation broth reached 15-20 g / L, and the proportion of free phytosphingosine was ≥90%. S2. In-situ synthesis of ceramide NP: Add emulsifier to the fermentation broth from step S1, stir and disperse, then add ethyl oleate at a molar ratio of phytosphingosine to ethyl oleate of 1:(1.2-1.3), and then add a methanol solution of 20%-30% sodium methoxide at a molar ratio of phytosphingosine to sodium methoxide of 1:(0.5-0.6), with a dropping rate of 1-2 mL / min; heat to 60-65℃, maintain reflux for 3-4 hours, and control the pH of the system to 8.0-8.5 during the reaction. S3. Integrated purification: After the reaction is completed, cool to 30-35℃, add 0.1%-0.2% chitosan flocculant, stir for 10-30 min, let stand for 1-3 h, filter to remove solid impurities; cool the filtrate to 0-5℃ to crystallize for 2-3 h, centrifuge and filter to separate the crystals, wash with methanol spray, and dry at 50-60℃ and vacuum degree -0.09MPa for 2-4 h to obtain ceramide NP product.
2. The integrated fermentation preparation process for ceramide NP according to claim 1, characterized in that, In step S1, the adaptive culture medium is prepared by adding 1-2% pre-adaptive components with 20-30 g / L glucose as carbon source, 5-8 g / L yeast extract as nitrogen source, and the initial pH is 7.0-7.
5.
3. The integrated fermentation preparation process for ceramide NP according to claim 2, characterized in that, In step S1, the pre-adapted component is ethanol.
4. The integrated fermentation preparation process for ceramide NP according to claim 1, characterized in that, In step S1, the adaptable culture medium further includes 0.1-0.3 g / L magnesium sulfate and 0.05-0.1 g / L potassium dihydrogen phosphate.
5. The integrated fermentation preparation process for ceramide NP according to claim 1, characterized in that, In step S1, the concentration of phytosphingosine was monitored in real time by HPLC during the fermentation process, and fermentation was stopped when the concentration reached 15-20 g / L.
6. The integrated fermentation preparation process for ceramide NP according to claim 1, characterized in that, In step S2, the emulsifier is Tween 80, and the amount added is 0.5%-1% of the fermentation liquid volume.
7. The integrated fermentation preparation process for ceramide NP according to claim 1, characterized in that, In step S2, if the pH of the system is greater than 9.0 during the reaction, add 10% glacial acetic acid to adjust the pH to 8.0-8.
5.
8. The integrated fermentation preparation process for ceramide NP according to claim 1, characterized in that, In step S3, before adding the chitosan flocculant, 0.05%-0.2% of the surfactant sodium dodecyl sulfate is added.
9. The integrated fermentation preparation process for ceramide NP according to claim 1, characterized in that, In step S3, the centrifugal filtration speed is 3000-4000 r / min, and the amount of methanol used in the spray washing is 1-1.5 times the mass of the crystals.
10. The integrated fermentation preparation process for ceramide NP according to claim 1, characterized in that, In step S3, before crystallization, some glycerol is removed by vacuum distillation at a temperature of 40-45°C and a vacuum degree of -0.08 MPa.