Industrial preparation method of tetraacetyl phytosphingosine

Abstract

The invention discloses an industrial preparation method of tetraacetyl phytosphingosine, which comprises the following steps: taking thalli separated from tetraacetyl phytosphingosine fermentation liquor as a raw material, and carrying out digestion, primary extraction, alcohol precipitation, column chromatography separation, secondary extraction and multi-stage crystallization to obtain a pure tetraacetyl phytosphingosine product. The method disclosed by the invention is mild in reaction condition, low in environmental protection pressure, high in product content and yield, simple in process route, suitable for actual production and high in industrialization capability.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tetraacetyl phytosphingosine preparation, and particularly relates to an industrialized preparation method of tetraacetyl phytosphingosine. BACKGROUND

[0002] Tetraacetyl phytosphingosine (TAPS) is a natural skin care product with excellent performance, which has the effects of moisturizing, antioxidant, whitening and freckle-removing, and is remarkable in promoting the self-repair of skin barrier, and is a potential drug for treating skin diseases. Tetraacetyl phytosphingosine can generate phytosphingosine, a key precursor of a moisturizing skin care product ceramide, through deacetylation. Phytosphingosine is mainly distributed in plant seeds such as wheat, and its content is extremely low and extremely difficult to extract, and is known as "plant soft gold".

[0003] Tetraacetyl phytosphingosine is a product of complete acetylation of phytosphingosine, which has an 18-carbon carbon chain as a skeleton, and has an acetylated hydroxyl group on the 1, 3 and 4 carbons, and an acetylated amine on the 2 carbon.

[0004]

[0005] At present, the preparation methods of tetraacetyl phytosphingosine include plant extraction, chemical synthesis and microbial fermentation. Although the plant extraction is natural, the plant raw material has low content, long planting cycle and large regional influence; the chemical synthesis method has high cost and low yield, and is not suitable for food and cosmetics; the biological fermentation method has the advantages of environmental protection, low price and short cycle, and is the preferred method for realizing high yield of tetraacetyl phytosphingosine. Studies have shown that tetraacetyl phytosphingosine can be obtained through microbial fermentation, and this process route has many advantages, and has become the preferred method for large-scale production of phytosphingosine in the future.

[0006] At present, the research on tetraacetyl phytosphingosine mainly focuses on biological synthesis, such as biosynthetic pathway, mutagenesis breeding of strains, modification of high-yield engineering bacteria, and regulation of biological fermentation system, but the research on how to extract and separate the fermentation broth after biological synthesis to obtain high-quality tetraacetyl phytosphingosine is extremely insufficient. Therefore, the industrialized preparation of high-quality tetraacetyl phytosphingosine has extremely important market significance for the preparation, application and upgrading of downstream phytosphingosine and ceramide products.

[0007] There are some public data about the preparation of tetraacetyl phytosphingosine in the prior art, as follows:

[0008] CN116803973 discloses a method for extracting tetraacetylphytosphoamine. This method involves diluting fermentation broth, adjusting the pH with hydrochloric acid, adsorbing onto a cation exchange resin, washing the cation exchange resin with water, eluting with acetic acid solution, concentrating, adjusting the pH with sodium hydroxide, cooling, crystallizing, washing with pure water, and vacuum drying to obtain the tetraacetylphytosphoamine product. This method requires the use of highly corrosive chemicals hydrochloric acid and sodium hydroxide, and the activation of the cation exchange resin also requires large amounts of acid and alkali. The reaction conditions are harsh, placing high demands on workshop production conditions and operational standards, and imposing significant environmental pressure on the production company. The resulting product purity is only greater than 90%, which clearly cannot meet the current high-end market demand for high-content (95%) tetraacetylphytosphoamine. Furthermore, the low tetraacetylphytosphoamine content directly affects the content of subsequent derived phytosphoamine and ceramide products, directly leading to lower content in these derivatives.

[0009] CN115372521 discloses a method for separating and identifying phytosphingosine and / or N-acetylphytosphingosine. This method uses high-performance liquid chromatography (HPLC) with gradient elution. However, this method can only achieve the separation and identification of trace amounts of the product and cannot be used for industrial production. Furthermore, the phytosphingosine and / or N-acetylphytosphingosine separated and identified by this method are not the same product as the tetraacetylphytosphingosine of this invention.

[0010] While the above methods can prepare tetraacetylphytaseronine, they suffer from a series of problems, including harsh reaction conditions, significant environmental impact, low content, and low yield. Therefore, there is an urgent need for a large-scale production technology with milder reaction conditions, lower environmental impact, and higher content and yield. Summary of the Invention

[0011] In view of the shortcomings of the prior art, the present invention provides an industrial preparation method for tetraacetylphytosphohydrin. This method can obtain high-content tetraacetylphytosphohydrin from fermentation broth, with mild reaction conditions, low environmental pressure, high product content and yield, and a simple process route that is suitable for practical production and has strong industrialization capabilities.

[0012] The present invention adopts the following technical solution:

[0013] This invention provides an industrial method for preparing tetraacetyl phytosphingosine, comprising:

[0014] Step 1: Separate the solid and liquid components of the tetraacetyl phytosphingosine fermentation broth, collect the cell precipitate, and extract it with a 60-80% v / v low alcohol aqueous solution to obtain the extract.

[0015] According to a specific embodiment of step one of the present invention, the fermentation broth is derived from a microbial culture fermentation broth intended for the biosynthesis of tetraacetylphytosphoside. Preferably, the microorganism is one that aims to biosynthesize tetraacetylphytosphoside through microbial metabolism, including but not limited to *Pichia pastoris* (Wickham Severinia) or *Saccharomyces cerevisiae*. The acquisition of the microorganism includes, but is not limited to, one or more combinations of haploid selection, mutagenesis breeding, or metabolic engineering modification, more preferably metabolically engineered strains. Preferably, the fermentation method includes, but is not limited to, one or more combinations of batch fermentation, continuous fermentation, or semi-continuous fermentation, more preferably continuous fermentation. Preferably, the content of tetraacetylphytosphoside (TAPS) in the fermentation broth is 20–25 g / L.

[0016] According to a specific embodiment of step one of the present invention, solid-liquid separation is aimed at separating insoluble and soluble substances, including but not limited to any one or more combinations of plate and frame filter press, plate centrifugation, horizontal screw centrifugation, disc centrifugation, tubular centrifugation or hanging bag centrifugation; more preferably any one or a combination of plate and frame filter press and horizontal screw centrifugation.

[0017] According to a specific embodiment of step one of the present invention, the concentration of the lower alcohol aqueous solution is 65-75% v / v. Preferably, the lower alcohol is a C1-C2 alcohol, such as any one of ethanol and methanol, with ethanol being preferred.

[0018] According to a specific embodiment of step one of the present invention, the material-to-liquid ratio for extraction is 1g:5-10mL, more preferably 1g:7-8mL. Extraction is performed in a tank, at a temperature of 20-60℃, for 3-6 hours, with continuous stirring, and the extraction is repeated 2-4 times.

[0019] Tetraacetyl phytosphingosine is readily soluble in organic solvents such as alcohols, alkanes, and ethers, but insoluble in water. However, the inventors unexpectedly discovered that it has excellent solubility in a certain volume fraction of aqueous alcohol solution, so it can be extracted using an aqueous alcohol solution of a certain volume fraction.

[0020] This invention eliminates the need for drying the bacterial cells. After obtaining the wet bacterial cells, a certain volume fraction of a polar solvent, an alcohol-water solution, is directly added to extract the target product from the microbial cells. Firstly, extraction using only organic alcohol solutions minimizes environmental impact, with ethanol being a more environmentally friendly option. Secondly, the drying process for the bacterial cells is eliminated, saving on production costs such as time, labor, and energy associated with drying. Thirdly, compared to extraction using non-polar or weakly polar organic solvents (such as alkanes, ethers, esters, ketones, benzenes, etc.), the alcohol-water solution extraction yields a higher recovery rate.

[0021] Step 2: Remove the alcohol solvent from the extract, add C5-C10 alkanes for extraction, and separate the organic phase;

[0022] According to a specific embodiment of step two of the present invention, the method of removing alcohol solvent from the extract is to remove alcohol solvent from the extract as the main purpose, including but not limited to using any one of a single-effect evaporator, a multi-effect MED evaporator, and an MVR evaporator, with an MVR evaporator being more preferred.

[0023] According to a specific embodiment of step two of the present invention, the amount of C5-C10 alkanes added is 2 to 5 times the volume of the extract after the alcohol solvent has been removed.

[0024] According to a specific embodiment of step two of the present invention, the extraction method is to place the extract after alcohol removal in a separatory tower, let it stand at room temperature to form clear and stable upper and lower layers, and then take the upper phase.

[0025] Tetraacetylphytosphohydrin has excellent solubility in C1-C2 alcohol aqueous solutions and alkane solvents of a certain volume fraction, but its water solubility is extremely poor. Therefore, after removing alcohol from the extract and adding alkane solvents, tetraacetylphytosphohydrin will form a layer with the aqueous phase and dissolve in the alkane. It can then be separated by extraction. At the same time, water-soluble impurities are also effectively removed.

[0026] Step 3: Remove the hydrocarbon solvent from the organic phase, add 90.0-99.9% v / v ethanol, let stand to allow sufficient precipitation, then separate the solid and liquid phases and collect the filtrate.

[0027] According to a specific embodiment of step three of the present invention, the method of removing hydrocarbon solvent from the organic phase includes, but is not limited to, using any one of a single-effect evaporator, a multi-effect MED evaporator, and an MVR evaporator, with an MVR evaporator being more preferred.

[0028] According to a specific embodiment of step three of the present invention, the amount of ethanol added is such that the volume ratio of the organic phase to ethanol is 1:5 to 15. The settling temperature is -10 to 10°C, and the settling time is 6 to 12 hours. After settling, solid-liquid separation is performed to remove a small amount of insoluble matter, including but not limited to any one of ceramic membrane filtration, plate centrifuge, and filter bag filtration; more preferably, ceramic membrane filtration. The molecular weight cutoff of the ceramic membrane is 2000 to 10000 Da, more preferably 4000 to 6000 Da; the filter bag has a mesh size of 200 to 400 mesh.

[0029] Since the bacterial cells are extracted using an alcohol-water solvent, the extract contains a large amount of impurities such as proteins, tannins, and polysaccharides. Therefore, by adding a high concentration of ethanol to increase the volume fraction of ethanol in the system, alcohol precipitation is carried out, which causes impurities such as proteins, tannins, and polysaccharides in the solution to precipitate out. Then, through solid-liquid separation, the content of tetraacetyl phytosphingosine can be further increased, reducing the purification pressure for subsequent chromatography.

[0030] Step 4: Dilute the filtrate to an ethanol concentration of 45-70% v / v, load it onto a macroporous adsorption resin chromatography column for adsorption, and desorb it with an acidic alcohol solution. Combine the effluent from adsorption and the desorbed liquid from desorption to obtain a mixed solution.

[0031] According to a specific embodiment of step four of the present invention, the diluent is water; preferably, the filtrate is diluted with water to an ethanol concentration of 50-65% v / v.

[0032] According to a specific embodiment of step four of the present invention, the method further includes adding ethanol of the same concentration as the diluted solution until the tetraacetyl phytosphingosine content is 2-5% w / w, and then loading the sample onto a macroporous adsorption resin chromatography column.

[0033] According to a specific embodiment of step four of the present invention, the macroporous adsorption resin is a non-polar or weakly polar macroporous adsorption resin, preferably any one of D101, AB-8, XR320, XR601, XR920L, XR19B, and XR17SS; preferably, the column diameter-to-height ratio is 1:3 to 7, and the column loading rate is 0.5 to 2 BV / h; preferably, the loading volume is 1.3 to 1.5 times the column volume loaded when the leak point is reached. The column volume is just enough to load all the loading solution. The leak point is defined as the detection of the target substance in the effluent from the column. The leak point can be tracked by any one of TLC and HPLC, preferably TLC.

[0034] Silica gel packing material is not easily recyclable, posing a significant environmental burden, and its chromatography speed is slow. Alumina packing material undergoes a cumbersome and slow activation process, also putting considerable pressure on the environment. Ion exchange resins require alternating acid and alkali washing during activation, which is environmentally unfriendly. Dextran and agarose gel chromatography, being molecular sieves, have extremely limited throughput, making large-scale production difficult. Therefore, this invention utilizes macroporous adsorption resin packing material, which offers high throughput, a simple activation process, fast chromatography speed, and a simple activation method that allows for recycling.

[0035] As is generally known, the success of a crystallization process is directly related to the purity or content of the solute. If the purity or content of the crude solute is too low, crystallization will be impossible or the crystallization yield will be very low. This invention utilizes macroporous adsorption resin chromatography to conveniently and quickly improve the purity and content of the target substance, thereby promoting subsequent crystallization processes.

[0036] Saturated overload adsorption, also known as competitive adsorption, involves the target substance and impurities competing for adsorption sites on the resin, thereby achieving further impurity separation after adsorption saturation.

[0037] According to a specific embodiment of step four of the present invention, the acid in the acidic alcohol aqueous solution is hydrochloric acid, acetic acid, or salicylic acid, and the alcohol is a C1-C3 alcohol; preferably, the acid content in the acidic alcohol aqueous solution is 0.3-6% v / v, more preferably, hydrochloric acid 0.3-1% v / v, or acetic acid 3-6% v / v, or salicylic acid 2-5% v / v; preferably, the alcohol content in the acidic alcohol aqueous solution is 50-65% v / v; preferably, the washing column volume is 2-4 BV, and the washing column flow rate is 0.5-2 BV / h.

[0038] Hydrochloric acid, acetic acid, and salicylic acid are volatile, so any residual acid can be easily removed in subsequent processes. Acetic acid and salicylic acid are commonly used acids in cosmetics, and each has its own specific functions. For example, acetic acid has antibacterial, antioxidant, and moisturizing effects; salicylic acid has exfoliating and wrinkle-reducing effects. Therefore, they can be safely used in this invention.

[0039] After numerous experiments, the inventors unexpectedly discovered that, following competitive adsorption, acidic alcoholic aqueous solutions could effectively elute the target substance tetraacetyl phytosphingosine from the chromatography column, while impurities were not eluted.

[0040] Step 5: Remove the alcohol solvent from the mixture, add water and stir, then separate the oil phase from the water phase, separate the solid phase from the aqueous phase, and combine the oil phase and solid to obtain the mixture.

[0041] According to a specific embodiment of step five of the present invention, the method for removing alcohol solvent from the mixture includes, but is not limited to, any one of a single-effect evaporator, a multi-effect MED evaporator, or an MVR evaporator.

[0042] According to a specific embodiment of step five of the present invention, the amount of water added during the stirring process is 5 to 10 times the volume of the mixture after the alcohol solvent has been removed. The oil-water separation method involves placing the mixture in a separatory tower, allowing it to stand at room temperature to form clear and stable upper and lower layers, and then taking the upper layer, which is the oil phase. The solid-liquid separation of the lower aqueous phase aims to separate insoluble and soluble substances, and includes, but is not limited to, any one or more combinations of plate and frame filter presses, horizontal screw centrifuges, flatbed centrifuges, disc centrifuges, tubular centrifuges, or hanging bag centrifuges; more preferably, flatbed centrifuges.

[0043] Tetraacetylphytosphohydrin has excellent solubility in a certain volume fraction of alcohol-water solution. After the alcohol is removed from the mixture, tetraacetylphytosphohydrin is not easily soluble in water. Part of it will form a layer with the aqueous phase and be collected by oil-water separation. Another part of tetraacetylphytosphohydrin will be precipitated in the aqueous phase and form an insoluble substance, which can be effectively separated and collected by solid-liquid separation.

[0044] Step 6: Perform multi-stage crystallization on the mixture to obtain pure tetraacetyl phytosphingosine.

[0045] According to a specific embodiment of step six of the present invention, multi-stage crystallization includes: first adding a primary crystallization solvent to the mixture to precipitate coarse crystals, and then adding a secondary crystallization solvent to the coarse crystals to precipitate high-purity crystals;

[0046] Preferably, the primary crystallization solvent is C5-C10 alkane or petroleum ether; more preferably, the ratio of the mixture to C5-C10 alkane is 1g:5-20mL, and the ratio of the mixture to petroleum ether is 1g:3-8mL; the conditions for primary crystallization include: a temperature of -20℃ to 5℃, and continuous stirring for 3-8h.

[0047] Preferably, the secondary crystallization solvent is a mixed solvent composed of one of C5-C10 alkanes or petroleum ethers and C1-C10 alcohols. More preferably, the volume ratio of alcohol solvent to mixed solvent is 1:20 to 100, the amount ratio of crude crystals to alcohol-containing C5-C10 alkanes is 1g:5 to 15mL, and the amount ratio of crude crystals to alcohol-containing petroleum ethers is 1g:3 to 10mL. The conditions for secondary crystallization include: a temperature of -20℃ to 5℃ and continuous stirring for 4 to 12 hours.

[0048] Preferably, the C5-C10 alkane is any one of pentane, hexane, heptane, sucralane, nonane, or decane, and their isomers. The boiling range of petroleum ether is 30-60°C, 60-90°C, 90-120°C, more preferably 30-60°C. The C1-C10 alcohol solvent is any one of methanol, ethanol, propanol, butanol, pentanol, hexanol, or octanol, and also includes any one or more combinations of these alcohol isomers. The inventors unexpectedly discovered that although methanol is immiscible with alkanes such as heptane and hexane, the addition of tetraacetyl phytosphohydrin improves the miscibility between methanol and alkanes. After extensive experimentation, the inventors unexpectedly discovered that adding a certain amount of alcohol to an organic solvent can effectively further increase the crystal content.

[0049] Preferably, primary and secondary crystallization may also be accompanied by one or more combinations of ultrasound, microwave, and seed crystal addition.

[0050] Preferably, after primary crystallization, solid-liquid separation, crystal washing, and drying are required; after secondary crystallization, solid-liquid separation, crystal washing, drying, pulverization, and sieving are also required. Solid-liquid separation aims to separate the crystals from the mother liquor, specifically including but not limited to any one of a plate and frame filter press, tubular centrifuge, or hanging bag centrifuge; more preferably, any one of a plate and frame filter press or hanging bag centrifuge. The temperature for solid-liquid separation after crystallization is controlled between -20℃ and 5℃. Crystal washing is performed by directly rinsing the crystals with a low-temperature crystallization solvent at -20℃ to 5℃ near the end of solid-liquid separation. The drying temperature should not exceed 25℃, as tetraacetylphytosphohydrin is extremely sensitive to heat; the crude crystals and the crystals themselves dissolve easily when heated, thus affecting the properties of the crude crystals and the drying efficiency. Drying aims to remove residual crystallization solvent from the crude crystals or the crystal product, including but not limited to any one of natural air drying, forced-air dryer, vibrating fluidized bed dryer, vacuum dryer, freeze dryer, or flash dryer. More preferably, any one of a blower dryer, a vibrating fluidized bed dryer, or a freeze dryer is used. The material is sieved through a 50-200 mesh screen.

[0051] The principle of the industrial preparation method of this invention is as follows:

[0052] Fermentation produces a broth containing tetraacetylphytosphoamine. Microbial cells are obtained through solid-liquid separation. The wet cells can be directly extracted with an alcohol-water solution to extract the intracellular substance tetraacetylphytosphoamine. After solid-liquid separation to remove insoluble impurities, the extract is obtained. The extract is then concentrated and evaporated to remove the alcohol, and an alkane solvent is added. Due to the poor solubility of tetraacetylphytosphoamine in water, it forms a stratified layer with water and dissolves in the alkane solvent. Further purification is achieved through extraction. After extraction and concentration to remove the alkane solvent, crude tetraacetylphytosphoamine is obtained. A high volume fraction of ethanol is then added to precipitate residual protein, tannins, and other impurities in the crude product. Further purification is achieved through solid-liquid separation. The filtrate was then diluted with alcohol and water and loaded onto a macroporous adsorption resin chromatography column. Upon reaching the leak point, impurities began to compete for adsorption with tetraacetylphytosphoprotein (TAPS), substituting TAPS at the resin binding sites. The TAPS then eluted with the effluent. The TAPS on the column was further eluted with an acidic alcohol-water solution and combined with the effluent to obtain a combined solution. After concentrating the combined solution to remove the alcohol odor, TAPS and water separated again. The upper and lower insoluble phases were extracted and collected, then combined. A solvent was added for primary crystallization. The resulting crude crystals were recrystallized using an alcohol-containing solvent. After crystallization, the crystals were dried and pulverized to obtain the high-content TAPS product.

[0053] The beneficial effects of this invention are as follows:

[0054] 1. The tetraacetyl phytosphingosine obtained by the method of the present invention has a content of ≥95% and a yield of ≥80%.

[0055] 2. The tetraacetyl phytosphingosine obtained by the method of the present invention is white in color, uniform in texture, and of good quality.

[0056] 3. The method of the present invention does not require pre-drying of the bacterial cells, which greatly saves various costs associated with bacterial cell drying; it also uses relatively less organic solvent, making it more environmentally friendly.

[0057] 4. This invention uses macroporous adsorption resin as a chromatography packing material. The packing material is easy to activate, can be recycled, and is economical and environmentally friendly.

[0058] 5. The entire process route of this invention is mild and has low environmental pressure; at the same time, the process route is simple, fits actual production, and has strong industrialization capabilities. Attached Figure Description

[0059] Figure 1 This is the HPLC chromatogram of tetraacetyl phytosphingosine in Example 1. Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0061] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0062] I. Source of fermentation broth: Self-made by Dabang (Hunan) Biopharmaceutical Co., Ltd. through industrial continuous fermentation production.

[0063] II. The determination of tetraacetyl phytosphingosine was performed using HPLC-UV method, with the following specific parameters: chromatographic column: HPLCONE-5C18A analytical column (250mm×4.6mm×5μm); UV absorption wavelength: 200nm; mobile phase: acetonitrile:water:phosphoric acid (V / V / V)=80:20:0.1; flow rate: 1.0ml / min; injection volume: 20μl; column temperature: 30℃.

[0064] III. Unless otherwise specified, the raw materials or chemical reagents used in the embodiments of the present invention are obtained through conventional commercial channels.

[0065] Example 1

[0066] (1) Extraction: Take 3000L of fermentation broth with a TAPS content of 23.42g / L. After solid-liquid separation using a plate and frame filter press, wet bacterial mud is obtained. Add 65% ethanol aqueous solution to the wet bacterial mud at a material-liquid ratio of 1:7 (m / V) for tank extraction. Each extraction lasts for 5 hours and is repeated 3 times. Solid-liquid separation is performed using a plate and frame filter press, and the extracts are combined to obtain the extract.

[0067] (2) Extraction I: The alcohol in the extract of step (1) is removed and recovered using an MVR evaporator to obtain a concentrated solution. Then, 4 times the volume of n-heptane is added to the concentrated solution, and after thorough mixing, it is placed in a separatory tower for separation and the upper phase I is collected.

[0068] (3) Alcohol precipitation: After concentrating the upper phase I to remove the organic solvent, add a 95.0% alcohol-water solution at a ratio of 1:10 (V / V) of upper phase I concentrate to high-proof ethanol. After stirring thoroughly, let it stand at -5℃ for 8 hours, and then pass it through a ceramic membrane with a molecular weight cutoff of 5000 Da to collect the permeate.

[0069] (4) Column loading: Dilute the permeate with pure water to 60% ethanol by volume, and continue to add 60% ethanol aqueous solution to TAPS content to 3% to obtain the column loading solution. Then, load the solution onto an XR320 macroporous adsorption resin chromatography column with a diameter-to-height ratio of 1:5, a column loading flow rate of 1 BV / h, and a column loading volume of 1.4 times the volume loaded when the leak point is reached.

[0070] (5) Desorption: Collect the effluent from the column and desorb it with an acidic alcoholic aqueous solution of 5% acetic acid and 55% ethanol. The desorption volume is 3 BV and the flow rate is 2 BV / h to obtain the desorbed solution.

[0071] (6) Extraction II: The combined liquid was concentrated to the point of no alcohol odor using an MVR evaporator. Then, water with a volume of 7 times that of the desorbed concentrate was added and stirred evenly. Extraction was then performed, and the upper phase II was collected. The lower phase was separated into solid and liquid phases using a plate centrifuge, and the insoluble matter was collected. The upper phase II and the insoluble matter were then combined and stirred evenly to obtain a combined mixture.

[0072] (7) Crystallization: Add n-heptane to the combined mixture in step (6) at a ratio of 1:15 (m / V) of mixture: n-heptane, and crystallize at -8℃ for 5 hours with continuous stirring. Wash the crystallized filter cake with n-heptane at -8℃ and dry it to obtain coarse crystals.

[0073] (8) Recrystallization: The coarse crystals were crystallized at a ratio of coarse crystals to mixed solvent III containing alcohol petroleum ether of 1:5 (V / V) and methanol to petroleum ether of mixed solvent III of 1:60 (V / V) at -4℃ for 8 hours, accompanied by continuous stirring and sonication. The crystallized filter cake was washed with solvent at -4℃, dried by forced air at 20℃, pulverized and sieved through 200 mesh to obtain high content tetraacetyl phytosphoprotein.

[0074] After weighing and testing, tetraacetylphytosphoprotein was obtained as a pure white substance, weighing 61.03 kg, with a purity of 97.41% and a yield of 84.61%. The HPLC chromatogram is attached. Figure 1 .

[0075] Example 2

[0076] (1) Extraction: Take 3000L of fermentation broth with a TAPS content of 21.69g / L, and use a plate and frame filter press to separate the solid and liquid to obtain wet bacterial mud. Add 70% methanol aqueous solution to the wet bacterial mud at a material-liquid ratio of 1:9 (m / V) for tank extraction. Each extraction lasts for 3 hours and is repeated twice. The solid and liquid are separated by a disc centrifuge and the extract is combined to obtain the extract.

[0077] (2) Extraction I: The alcohol in the extract of step (1) is removed and recovered using an MVR evaporator to obtain a concentrated solution. Then, 3 times the volume of cyclohexane is added to the concentrated solution, and after thorough mixing, it is placed in a separatory tower for separation and the upper phase I is collected.

[0078] (3) Alcohol precipitation: After concentrating the upper phase I to remove the organic solvent, add a 92.0% alcohol-water solution at a ratio of 1:6 (V / V) of upper phase I concentrate to high-proof ethanol. After stirring thoroughly, let it stand at 0℃ for 10h, then centrifuge with a plate centrifuge and collect the permeate.

[0079] (4) Column loading: Dilute the permeate with pure water to an ethanol volume fraction of 52%, and continue to add an ethanol aqueous solution with a volume fraction of 52% until the TAPS content is 4% to obtain the column loading solution. Then, load the solution onto a D101 macroporous adsorption resin chromatography column with a diameter-to-height ratio of 1:4, a column loading flow rate of 0.5 BV / h, and a column loading volume of 1.4 times the volume loaded when the leak point is reached.

[0080] (5) Desorption: Collect the effluent from the column and desorb it with an acidic alcoholic aqueous solution of 0.5% hydrochloric acid and 60% ethanol at a desorption volume of 3 BV and a flow rate of 2 BV / h to obtain the desorbed solution;

[0081] (6) Extraction II: The combined liquid was concentrated to the point of no alcohol odor using an MVR evaporator, and then 5 times the volume of water of the desorption concentrate was added and stirred evenly before extraction. The upper phase II was collected. The lower phase was separated into solid and liquid phases using a plate centrifuge and the insoluble matter was collected. The upper phase II and the insoluble matter were then combined and stirred evenly to obtain a combined mixture.

[0082] (7) Crystallization: Add petroleum ether to the combined mixture in step (6) at a ratio of 1:6 (m / V) and crystallize at -4℃ for 5 hours with continuous stirring. Wash the crystallized filter cake with petroleum ether at -8℃ and dry it to obtain coarse crystals.

[0083] (8) Recrystallization: The coarse crystals were crystallized at a ratio of coarse crystals to mixed solvent III containing alcohol and n-heptane of 1:10 (V / V), and the ratio of methanol to petroleum ether in mixed solvent III was 1:40 (V / V). The crystals were crystallized at -5℃ for 8 hours, accompanied by continuous stirring and sonication. The crystallized filter cake was washed with solvent at -4℃, dried by forced air at 20℃, pulverized and sieved through 200 mesh to obtain high content tetraacetyl phytosphoprotein.

[0084] After weighing and testing, tetraacetylphytosphoprotein was obtained, which was pure white, weighed 56.24 kg, had a content of 96.75%, and a yield of 83.62%.

[0085] Example 3

[0086] (1) Extraction: Take 3000L of fermentation broth with a TAPS content of 24.10g / L. After solid-liquid separation using a plate and frame filter press, wet bacterial mud is obtained. Add 75% ethanol aqueous solution to the wet bacterial mud at a material-liquid ratio of 1:5 (m / V) for tank extraction. Each extraction lasts for 6 hours and is repeated 4 times. Solid-liquid separation is performed using a plate and frame filter press, and the extracts are combined to obtain the extract.

[0087] (2) Extraction I: The alcohol in the extract of step (1) is removed and recovered using a multi-effect MED evaporator to obtain a concentrated solution. Then, 5 times the volume of cyclohexane is added to the concentrated solution, and after thorough mixing, it is placed in a separatory tower for layering and the upper phase I is collected.

[0088] (3) Alcohol precipitation: After concentrating the upper phase I to remove the organic solvent, add a 98.0% alcohol-water solution at a ratio of 1:15 (V / V) of upper phase I concentrate to high-proof ethanol. After stirring thoroughly, let it stand at -10℃ for 6 hours, and then pass it through a ceramic membrane with a molecular weight cutoff of 5000 Da to collect the permeate.

[0089] (4) Column loading: Dilute the permeate with pure water to 65% ethanol by volume, and continue to add 65% ethanol aqueous solution to TAPS content to 2% to obtain the column loading solution. Then, load it onto an XR920L macroporous adsorption resin chromatography column with a diameter-to-height ratio of 1:6, a column loading flow rate of 1 BV / h, and a column loading volume of 1.3 times the volume loaded when the leak point is reached.

[0090] (5) Desorption: Collect the effluent from the column and desorb it with an acidic alcoholic aqueous solution of 4% salicylic acid and 58% ethanol. The desorption volume is 4 BV and the flow rate is 2 BV / h to obtain the desorbed solution.

[0091] (6) Extraction II: The combined liquid is concentrated until there is no alcohol odor using a multi-effect MED evaporator. Then, 8 times the volume of water is added to the desorption concentrate and stirred evenly. Extraction is then performed, and the upper phase II is collected. The lower phase is separated into solid and liquid phases using a plate centrifuge, and the insoluble matter is collected. The upper phase II and the insoluble matter are then combined and stirred evenly to obtain a combined mixture.

[0092] (7) Crystallization: Add n-hexane to the combined mixture in step (6) at a ratio of 1:10 (m / V) of mixture: n-heptane, and crystallize at -0℃ for 5h with continuous stirring. The crystallized filter cake is washed with n-hexane at 0℃ and dried to obtain coarse crystals.

[0093] (8) Recrystallization: The crude crystals were crystallized at a ratio of crude crystals to mixed solvent III containing alcohol petroleum ether of 1:8 (V / V), and the ratio of n-propanol to petroleum ether in mixed solvent III was 1:80 (V / V). The crystals were crystallized at -10℃ for 6 hours, accompanied by continuous stirring and sonication. The crystallized filter cake was washed with solvent at -10℃, dried by forced air at 20℃, pulverized and sieved through a 200-mesh sieve to obtain high content tetraacetyl phytosphingosine.

[0094] After weighing and testing, tetraacetylphytosphoprotein was obtained, which was pure white, weighed 62.45 kg, had a content of 95.93%, and a yield of 82.86%.

[0095] Comparative Example 1

[0096] Other conditions and operations were the same as in Example 1, and the TAPS content in the fermentation broth was 23.42 g / L. The difference was that in step (1), the extraction solvent was replaced with heptane instead of ethanol. After weighing and testing, tetraacetyl phytosphingosine was obtained, which was pure white, weighed 43.23 kg, had a content of 96.53%, and a yield of 59.39%.

[0097] Comparative Example 2

[0098] Other conditions and operations were the same as in Example 1, and the TAPS content in the fermentation broth was 23.42 g / L. The difference was that in step (2), after concentrating the upper phase I to remove the organic solvent, the alcohol precipitation process was omitted, and the product directly entered step (4), i.e., dilution and column loading. After weighing and testing, tetraacetyl phytosphingosine was obtained, which was pure white, with a mass of 66.91 kg, a content of 85.52%, and a yield of 81.44%.

[0099] Comparative Example 3

[0100] Other conditions and operations were the same as in Example 1, and the TAPS content in the fermentation broth was 23.42 g / L. The difference was that in step (4), the permeate was diluted with pure water to an ethanol volume fraction of 75%, and an ethanol aqueous solution with a volume fraction of 75% was added until the TAPS content was 3%. After weighing and testing, tetraacetyl phytosphingosine was obtained, which was pure white, weighed 26.04 kg, had a content of 96.83%, and a yield of 35.89%.

[0101] Comparative Example 4

[0102] Other conditions and operations were the same as in Example 1, and the TAPS content in the fermentation broth was 23.42 g / L. The difference was that in step (4), the macroporous adsorption resin was replaced with neutral alumina. After weighing and testing, the flow rate on the neutral alumina chromatography column was almost 0, and the experiment could not continue and was forced to be terminated.

[0103] Comparative Example 5

[0104] Other conditions and operations were the same as in Example 1, and the TAPS content in the fermentation broth was 23.42 g / L. The difference was that in step (4), the amount loaded onto the column was 1.0 times the volume loaded when the leakage point was reached. After weighing and testing, tetraacetyl phytosphingosine was obtained, which was pure white, weighed 64.09 kg, had a content of 78.15%, and a yield of 71.29%.

[0105] Comparative Example 6

[0106] Other conditions and operations were the same as in Example 1, and the TAPS content in the fermentation broth was 23.42 g / L. The difference was that in step (5), "washing the column with an acidic alcoholic aqueous solution of 0.5% hydrochloric acid and 55% ethanol" was changed to "washing the column with an acidic alcoholic aqueous solution of 55% ethanol". After weighing and testing, tetraacetyl phytosphingosine was obtained as a pure white substance, weighing 48.27 kg, with a content of 95.93% and a yield of 65.91%.

[0107] Comparative Example 7

[0108] Other conditions and operations were the same as in Example 1, and the TAPS content in the fermentation broth was 23.42 g / L. The difference was that in step (6), only the upper phase II was taken, and the lower insoluble matter was discarded. After weighing and testing, tetraacetyl phytosphingosine was obtained, which was pure white, with a mass of 46.91 kg, a content of 96.77%, and a yield of 64.61%.

[0109] Comparative Example 8

[0110] Other conditions and operations were the same as in Example 1, and the TAPS content in the fermentation broth was 23.42 g / L. The difference was that in step (6), the upper phase II was discarded, and only the lower insoluble matter was taken. After weighing and testing, tetraacetyl phytosphingosine was obtained, which was pure white, with a mass of 13.39 kg, a content of 97.58%, and a yield of 18.60%.

[0111] Comparative Example 9

[0112] Other conditions and operations were the same as in Example 1, and the TAPS content in the fermentation broth was 23.42 g / L. The difference was that in step (8), the crude crystals were crystallized according to a material-to-liquid ratio of crude crystals to mixed solvent III containing alcohol and petroleum ether of 1:5 (V / V), and the methanol to petroleum ether ratio of mixed solvent III was 0:60 (V / V), i.e., it did not contain alcohol. After weighing and testing, tetraacetylphytosphoprotein was obtained, which was pure white, with a mass of 64.08 kg, a content of 93.11%, and a yield of 84.92%.

[0113] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. An industrial method for preparing tetraacetyl phytosphingosine, characterized in that, The industrial preparation method includes: The solid-liquid separation of the tetraacetyl phytosphingosine fermentation broth was carried out, the cell precipitate was collected, and the precipitate was extracted with a low alcohol aqueous solution of 60-80% v / v to obtain the extract. Remove the alcohol solvent from the extract, add C5-C10 alkanes for extraction, and separate the organic phase; Remove the hydrocarbon solvent from the organic phase, add 90.0-99.9% v / v ethanol, let stand to allow sufficient precipitation, then separate the solid and liquid phases and collect the filtrate. Dilute the filtrate to an ethanol concentration of 45–70% v / v, load it onto a macroporous adsorption resin chromatography column for adsorption, and desorb it with an acidic alcohol solution. Combine the effluent from adsorption and the desorbed liquid from desorption to obtain a mixed solution. Remove the alcohol solvent from the mixture, add water and stir, then separate the oil phase from the water phase, separate the solid phase from the aqueous phase, and combine the oil phase and solid to obtain the mixture. The mixture was subjected to multi-stage crystallization to obtain pure tetraacetyl phytosphingosine.

2. The industrial preparation method according to claim 1, characterized in that, The concentration of the lower alcohol aqueous solution is 65-75% v / v.

3. The industrial preparation method according to claim 1, characterized in that, The lower alcohol is a C1-C2 alcohol, preferably ethanol.

4. The industrial preparation method according to claim 1, characterized in that, The extraction process involves a material-to-liquid ratio of 1g:5-10mL, a temperature of 20-60℃, and a time of 3-6h.

5. The industrial preparation method according to claim 1, characterized in that, The diluent is water; preferably, the filtrate is diluted with water to an ethanol concentration of 50-65% v / v.

6. The industrial preparation method according to claim 1, characterized in that, This also includes adding ethanol of the same concentration as the diluted solution until the tetraacetyl phytosphingosine content is 2-5% w / w, and then loading the sample onto a macroporous adsorption resin chromatography column.

7. The industrial preparation method according to claim 1, characterized in that, The macroporous adsorption resin is a non-polar or weakly polar macroporous adsorption resin, preferably any one of D101, AB-8, XR320, XR601, XR920L, XR19B, and XR17SS; preferably, the amount loaded onto the column is 1.3 to 1.5 times the volume of the column already loaded when the leak point is reached.

8. The industrial preparation method according to claim 1, characterized in that, The acid in the acidic alcohol aqueous solution is hydrochloric acid, acetic acid or salicylic acid, and the alcohol is a C1-C3 alcohol.

9. The industrial preparation method according to claim 1, characterized in that, The acid content in the acidic alcohol-water solution is 0.3-6% v / v, and the alcohol content is 50-65% v / v.

10. The industrial preparation method according to claim 1, characterized in that, The multi-stage crystallization includes: first adding a primary crystallization solvent to the mixture to precipitate coarse crystals, and then adding a secondary crystallization solvent to the coarse crystals to precipitate high-purity crystals; Preferably, the primary crystallization solvent is a C5-C10 alkane or petroleum ether, and the primary crystallization temperature is -20℃ to 5℃, and the time is 3 to 8 hours. Preferably, the secondary crystallization solvent is a mixed solvent composed of one of C5-C10 alkanes or petroleum ethers and C1-C10 alcohols, and the secondary crystallization temperature is -20℃ to 5℃, and the time is 4 to 12 hours.

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