A method for extracting tetraacetyl phytosphingosine

Abstract

The present invention discloses a method for extracting tetraacetylsphingosine (TAPS). By diluting the fermentation broth, the viscosity of the raw material is reduced; then, through acidification and temperature elevation, the TAPS in the cells in the fermentation broth is effectively dissolved out; under acidic conditions, the amino group in the TAPS structure is exchanged with the cation resin to achieve the preliminary extraction of TAPS; it is desorbed with an acidic solution to obtain a salt solution of TAPS; after concentration, it is adjusted to alkaline, and TAPS crystallizes in the form of an alkali; finally, the product is washed with purified water to remove the salt in the product, and the final product is obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of purification, and particularly relates to a method for extracting tetraacetyl phytosphingosine. Background Art

[0002] Sphingolipids are widely present in animals, plants, fungi, protozoa and viruses and are important components of cell membranes. There are three types of sphingolipids, including ceramides, sphingomyelins and glycosphingolipids, which are amphiphilic lipids composed of long-chain amino alcohols, fatty acids and phosphocholine. Under the action of sphingomyelinase, all sphingomyelins are ultimately degraded into ceramides. Ceramides play a crucial role in maintaining the water impermeability of the skin barrier and preventing infections and dryness of the SC. Together with cholesterol, cholesterol sulfate and free fatty acids in human skin, they can protect the skin from physical and chemical harmful substances. However, the ceramide content in the skin decreases with age. Therefore, supplementing ceramide to the skin plays an important role in protecting the skin.

[0003] By analyzing the structures of the identified ceramides, it is found that their long-chain amino alcohols include sphingosine, dihydrosphingosine, phytosphingosine, 6-hydroxy sphingosine, etc. Therefore, sphingosine, as the main sphingosine base component in human sphingolipids, is of self-evident importance for the synthesis of sphingolipids and has considerable commercial application value. Since the discovery of tetraacetyl phytosphingosine (TAPS) by Wickerham and Stodola in yeast in 1960, the prior art has been continuously researching TAPS. TAPS can stimulate the synthesis of glucosylceramide in vitro. Glucosylceramide has a protective function, helps to strengthen the skin barrier function, inhibits inflammation, inhibits angiogenesis and fades dark circles. Tetraacetyl phytosphingosine has good solubility and can be compounded with a variety of cosmetic raw materials, and is suitable for products such as anti-aging, repair and eye creams. TAPS has been commercially produced as a starting material for synthesizing phytosphingosine and ceramide, but due to its high price, large-scale production has not been achieved.

[0004] Some synthetic routes for synthesizing TAPS have been developed before, such as chemical synthesis or separation and purification from natural source substances, etc. However, the synthesis of TAPS by chemical and separation and purification methods is extremely expensive and cannot be used in food and cosmetics. Therefore, the research direction of the existing technology has turned to the biosynthesis route. Through bioengineering means, a variety of engineering strains have been successfully developed, and through fermentation engineering, the biosynthesis of TAPS has been realized, and the basic conditions for industrial production have been met. However, it has been found in actual applications that it is difficult to obtain a TAPS product with a relatively high purity from the fermentation broth containing TAPS obtained by fermentation. Usually, it is necessary to break the cell wall with a high-speed homogenizer, then extract with a large amount of organic solvents, and then obtain a high-purity product through silica gel column chromatography. In this production process, the amount of organic solvents used is large, the safety risk is high, the environmental protection pressure is large, and the production cost is high, which is not suitable for industrial production. Summary of the Invention

[0005] In order to solve the problems of the existing technology that when purifying the fermentation broth containing TAPS, the amount of organic solvents used is large, high-purity products can only be obtained through silica gel column chromatography, the safety and environmental protection risks are high, the production cost is high, and it is not conducive to industrial production, the present invention discloses a method for extracting tetraacetylsphingosine, and purifies the fermentation broth containing tetraacetylsphingosine through the following process route to obtain tetraacetylsphingosine in the fermentation broth:

[0006]

[0007] Preferably, it specifically includes the following steps:

[0008] Step 1: Obtain the fermentation broth containing tetraacetylsphingosine, dilute it 1-2 times with water, and heat it to 20-60 °C; add concentrated hydrochloric acid and stir to adjust the pH to 2-5; break the cell structure, and TAPS can be released into the aqueous phase without using cell disruption equipment and organic solvents.

[0009] Step 2: Add cationic resin to the fermentation broth treated in Step 1, stir for 2-5 hours to allow the cationic resin to fully adsorb TAPS, and recover the cationic resin through a 40-mesh sieve; since this process does not involve chemical reactions, the recovery rate of TAPS ≥ 98%.

[0010] Step 3: Elute the cationic resin with water until the pH value of the eluted water is 5-7 to remove water-soluble impurities; elute the cationic resin with acetic acid solution and collect the eluate; wherein, the volume of the acetic acid solution is 4-10 times the volume of the cationic resin, and the concentration of the acetic acid solution is 0.3-1.0 mol / L; through column elution, more than 90% of the water-soluble impurities can be removed.

[0011] Step 4: Perform reduced-pressure concentration on the eluate collected in Step 3 until it is concentrated to 1 / 8 - 1 / 3 of the original volume of the eluate to obtain a concentrated solution; then adjust the pH of the concentrated solution to 9 - 11 with a sodium hydroxide solution to remove carboxylate ions, release water-insoluble TAPS, cool it to below 10°C, crystallize for 3 - 5 h, and filter to collect the crude product. Further preferably, the mass fraction of the sodium hydroxide solution is 10%.

[0012] Step 5: Wash the crude product collected in Step 4 with 5 - 10 times of purified water at below 10°C for 2 - 5 hours to remove the residual sodium hydroxide, filter to collect the solid, and obtain the tetraacetyl phytosphingosine through vacuum drying.

[0013] Preferably, in Step 1, stirring is carried out at 20 - 60°C for 2 - 5 h, and the pH is adjusted to 2.5; the stirring temperature is further preferably 55°C.

[0014] Preferably, in Step 2, the cation resin is a strongly acidic cation resin.

[0015] Preferably, the dosage of the cation resin is 10 - 50 g of resin added per 1 g of TAPS.

[0016] Preferably, in Step 2, the stirring and adsorption are carried out at 20 - 30°C.

[0017] Preferably, in Step 3, during the elution process, the elution flow rate is controlled at 1 / 200 - 1 / 20 resin volume / min. Here, 1 / 200 - 1 / 20 resin volume / min means that the flow rate per minute is 1 / 200 - 1 / 20 of the resin volume. For example, when the resin volume is 100 ml, the flow rate is 0.5 - 5 ml / min.

[0018] Preferably, in Step 4, after reduced-pressure concentration at 30 - 60°C, the pH is adjusted to 9 - 11 with a sodium hydroxide solution.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. By diluting, heating, and adjusting the pH of the fermentation broth, the present invention dissolves the TAPS in the cells, avoids using a high-speed homogenizer for cell wall breaking, and is more conducive to industrial production; by using the ion exchange method, it avoids using a large amount of organic solvents, reduces safety and environmental protection risks, and reduces waste gas emissions; at the same time, the present invention extracts the target product by the ion exchange method, obtaining a product with high purity, without the need for silica gel column separation, which can greatly reduce costs and is more suitable for large-scale industrial production.

[0021] 2. The extraction method of the present invention is used to extract the fermentation broth containing TAPS, and the yield of TAPS is greater than 80%; the purity of the product is greater than 90%, far superior to the purity of currently commercially available products, and has higher commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the HPLC chromatogram of commercially available sample 1.

[0023] Figure 2 It is the HPLC chromatogram of commercially available sample 2.

[0024] Figure 3 It is the HPLC chromatogram of Example 1.

[0025] Figure 4 It is the HPLC chromatogram of Example 2.

[0026] Figure 5 It is the HPLC chromatogram of Example 3.

[0027] Figure 6 It is the HPLC chromatogram of Example 4.

[0028] Figure 7 It is the HPLC chromatogram of Example 5.

[0029] Figure 8 It is the HPLC chromatogram of Example 6.

[0030] Figure 9 It is the HPLC chromatogram of Example 7.

[0031] Figure 10 It is the HPLC chromatogram of Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention will be further described below in conjunction with the drawings and examples.

[0033] I. An extraction method of tetraacetyl phytosphingosine

[0034] The present invention purifies the fermentation broth containing tetraacetyl phytosphingosine through the following process route to obtain tetraacetyl phytosphingosine in the fermentation broth:

[0035]

[0036] In specific implementation, it includes the following steps:

[0037] Step 1: Obtain the fermentation broth containing tetraacetylsphingosine, dilute it 1 - 2 times with water, add concentrated hydrochloric acid and stir, adjust the pH to 2 - 5; in Step 1, stir at 20 - 60 °C for 2 - 5 h and adjust the pH to 2 - 5; further preferably, the stirring temperature is 50 °C; further preferably, the pH for TAPS dissolution is 3.0.

[0038] Step 2: Add cationic resin to the fermentation broth treated in Step 1, stir for 2 - 5 hours to allow the cationic resin to fully adsorb TAPS, and recover the cationic resin by passing through a 40 - mesh sieve; in Step 2, the cationic resin is a strongly acidic cationic resin. The dosage of the cationic resin is 10 - 50 g of resin added per 1 g of TAPS. Further preferably, the cationic resin is 001×12 strongly acidic cationic resin; further preferably, the stirring and adsorption are carried out at 20 - 30 °C; further preferably, the stirring and adsorption are carried out at 30 °C; further preferably, the optimal dosage of the cationic resin is 16.6 g of resin added per 1 g of TAPS.

[0039] Step 3: Elute the cationic resin, rinse the cationic resin with drinking water until the pH of the eluted water is 5 - 7; elute the cationic resin with acetic acid solution and collect the eluate; among them, column elution is carried out using a glass chromatography column, the volume of the acetic acid solution is 4 - 10 times the volume of the cationic resin, and the concentration of the acetic acid solution is 0.3 - 1.0 mol / L; further preferably, the pH of the eluted water of the cationic resin is 6.0; further preferably, the concentration of the acetic acid solution is 0.3 mol / L.

[0040] Step 4: Carry out vacuum concentration on the eluate collected in Step 3 to concentrate it to 1 / 8 - 1 / 3 of the original volume of the eluate to obtain a concentrated solution; then adjust the pH of the concentrated solution to 9 - 11 with sodium hydroxide solution for salting - out, cool down to below 10 °C, crystallize for 3 - 5 h, and filter to collect the crude product; among them, the mass fraction of the sodium hydroxide solution is 10%; during the elution process, control the elution flow rate to be 1 / 200 - 1 / 20 resin volume / min. Further preferably, the vacuum concentration temperature is 45 °C; further preferably, the pH for salting - out of the concentrated solution is 11.

[0041] Step 5: Pulp the crude product collected in Step 4 with purified water at below 10 °C for 2 - 5 hours, filter to collect the solid, and obtain the tetraacetylsphingosine through vacuum drying.

[0042] II. Examples

[0043] Example 1

[0044] Step 1: Take 5 L of the fermentation broth with a TAPS fermentation unit of 12 g / L, dilute it with 10 L of drinking water, adjust the pH to 3.0 with concentrated hydrochloric acid, heat to 50 °C, stir for 3 hours; cool down to 30 °C;

[0045] Step 2: Add 1000 g of 20-mesh 001×12 cationic Na-type resin, stir and adsorb for 3 hours, and detect that the unit of the waste liquid is less than 0.1 g / L; pass through a 40-mesh sieve, recover the resin, load it into an ion exchange column, and wash with drinking water until the pH is 6;

[0046] Step 3: Desorb with 0.3 mol / L acetic acid prepared with purified water, detect by TCL, and collect 5.2 L of the solution containing TAPS;

[0047] Step 4: Concentrate the desorbed solution under reduced pressure to 1000 mL; control the temperature at 45 °C, adjust the pH to 11 with 10% sodium hydroxide solution, and a large amount of crystals will precipitate during the process; slowly cool down to below 10 °C, and continue to stir and crystallize for 3 hours; filter and collect 120 g of the crude TAPS hydrate;

[0048] Step 5: Add the above-mentioned crude hydrate to 800 mL of purified water, stir and wash at below 10 °C for 3 hours; filter and collect 110 g of the wet TAPS product, and dry it under reduced pressure at 55 °C to obtain 52 g of the finished product. The yield is 86.7%.

[0049] Example 2

[0050] Step 1: Take 5 L of the fermentation broth with a TAPS fermentation unit of 12 g / L, dilute it with 10 L of drinking water, adjust the pH to 4.0 with concentrated hydrochloric acid, heat it to 50 °C, and stir for 3 hours; cool down to 30 °C;

[0051] Steps 2 to 4 are the same as those in Example 1;

[0052] Step 5: Add the above-mentioned crude hydrate to 800 mL of purified water, stir and wash at below 10 °C for 3 hours; filter and collect 102 g of the wet TAPS product, and dry it under reduced pressure at 55 °C to obtain 51.06 g of the finished product. The yield is 85.1%.

[0053] Example 3

[0054] Step 1: Take 5 L of the fermentation broth with a TAPS fermentation unit of 12 g / L, dilute it with 10 L of drinking water, adjust the pH to 5.0 with concentrated hydrochloric acid, heat it to 50 °C, and stir for 3 hours; cool down to 30 °C;

[0055] Steps 2 to 4 are the same as those in Example 1;

[0056] Step 5: Add the above-mentioned crude hydrate to 800 mL of purified water, stir and wash at below 10 °C for 3 hours; filter and collect 112 g of the wet TAPS product, and dry it under reduced pressure at 55 °C to obtain 51.54 g of the finished product. The yield is 85.9%.

[0057] According to the results of Example 1, Example 2, and Example 3, the optimal pH for TAPS dissolution is 3.0.

[0058] Example 4

[0059] Step 1: Take 5 liters of fermentation broth with a TAPS fermentation unit of 12 g / L, dilute it with 10 liters of drinking water, adjust the pH to 3.0 with concentrated hydrochloric acid, heat it to 50 °C, and stir for 3 hours; cool it down to 30 °C;

[0060] Step 2: Add 800 grams of 001×12 cationic Na-type resin with a 20-mesh specification, stir and adsorb for 3 hours, and detect that the unit of the waste liquid is less than 0.1 g / L; pass through a 40-mesh sieve, recover the resin, load it into an ion exchange column, and wash it with drinking water until the pH is 6;

[0061] Steps 3 to 4 are the same as in Example 1;

[0062] Step 5: Add 800 ml of purified water to the above-mentioned crude product containing water, stir and wash at a temperature below 10 °C for 3 hours; filter, collect 95 grams of wet TAPS product, and dry it under reduced pressure at 55 °C to obtain 48.96 grams of the finished product. The yield is 81.6%.

[0063] Example 5

[0064] Step 1: Take 5 liters of fermentation broth with a TAPS fermentation unit of 12 g / L, dilute it with 10 liters of drinking water, adjust the pH to 3.0 with concentrated hydrochloric acid, heat it to 50 °C, and stir for 3 hours; cool it down to 30 °C;

[0065] Step 2: Add 1200 grams of 001×12 cationic Na-type resin with a 20-mesh specification, stir and adsorb for 3 hours, and detect that the unit of the waste liquid is less than 0.1 g / L; pass through a 40-mesh sieve, recover the resin, load it into an ion exchange column, and wash it with drinking water until the pH is 6;

[0066] Steps 3 to 4 are the same as in Example 1;

[0067] Step 5: Add 800 ml of purified water to the above-mentioned crude product containing water, stir and wash at a temperature below 10 °C for 3 hours; filter, collect 120 grams of wet TAPS product, and dry it under reduced pressure at 55 °C to obtain 51.78 grams of the finished product. The yield is 86.3%.

[0068] According to the results of Example 1, Example 4, and Example 5, the most preferred amount of cationic resin used is 1000 grams.

[0069] Example 6

[0070] Step 1: Take 5 liters of fermentation broth with a TAPS fermentation unit of 12 g / L, dilute it with 10 liters of drinking water, adjust the pH to 3.0 with concentrated hydrochloric acid, heat it to 50 °C, and stir for 3 hours; cool it down to 30 °C;

[0071] Step 2: Put 1000 g of 001×12 cationic Na-type resin with a 20-mesh specification, stir and adsorb for 3 hours, and detect that the unit of the waste liquid is less than 0.1 g / L; pass through a 40-mesh sieve, recover the resin, load it into an ion exchange column, and wash with drinking water until the pH is 6;

[0072] Step 3: Desorb with 0.5 mol / L acetic acid prepared with purified water, detect by TCL, and collect 5.2 L of the solution containing TAPS;

[0073] Step 4 is the same as in Example 1;

[0074] Step 5: Add 800 mL of purified water to the above-mentioned crude product containing water, stir and wash at a temperature below 10°C for 3 hours; filter, collect 115 g of wet TAPS product, and dry it under reduced pressure at 55°C to obtain 50.1 g of the finished product. The yield is 83.5%.

[0075] According to the results of Example 1 and Example 6, the most preferred acetic acid concentration is 0.3 mol / L.

[0076] Example 7

[0077] Step 1: Take 5 L of the fermentation broth with a TAPS fermentation unit of 12 g / L, dilute it with 10 L of drinking water, adjust the pH to 3.0 with concentrated hydrochloric acid, heat it to 50°C, and stir for 3 hours; cool down to 30°C;

[0078] Step 2: Put 1000 g of 001×12 cationic Na-type resin with a 20-mesh specification, stir and adsorb for 3 hours, and detect that the unit of the waste liquid is less than 0.1 g / L; pass through a 40-mesh sieve, recover the resin, load it into an ion exchange column, and wash with drinking water until the pH is 6;

[0079] Step 3: Desorb with 0.3 mol / L acetic acid prepared with purified water, detect by TCL, and collect 5.2 L of the solution containing TAPS;

[0080] Step 4: Concentrate the desorbed solution under reduced pressure to 1000 mL; control the temperature at 45°C, adjust the pH to 9 with 10% sodium hydroxide solution, and a large amount of crystals will precipitate during the process; slowly cool down to below 10°C and continue to stir and crystallize for 3 hours; filter to collect 100 g of the crude TAPS product containing water;

[0081] Step 5: Add 800 mL of purified water to the above-mentioned crude product containing water, stir and wash at a temperature below 10°C for 3 hours; filter, collect 95 g of wet TAPS product, and dry it under reduced pressure at 55°C to obtain 48.6 g of the finished product. The yield is 81.0%.

[0082] Example 8

[0083] Step 1: Take 5 L of the fermentation broth with a TAPS fermentation unit of 12 g / L, dilute it with 10 L of drinking water, adjust the pH to 3.0 with concentrated hydrochloric acid, heat it to 50°C, and stir for 3 hours; cool down to 30°C;

[0084] Step 2: Add 1000 g of 20-mesh 001×12 cationic Na-type resin, stir and adsorb for 3 hours, and detect that the unit of the waste liquid is less than 0.1 g / L; sieve through a 40-mesh sieve, recover the resin, load it into an ion exchange column, and wash with drinking water until the pH is 6;

[0085] Step 3: Desorb with 0.3 mol / L acetic acid prepared with purified water, detect by TCL, and collect 5.2 L of the solution containing TAPS;

[0086] Step 4: Concentrate the desorbed solution under reduced pressure to 1000 mL; control the temperature at 45 °C, adjust the pH to 10 with 10% sodium hydroxide solution, and a large amount of crystals will precipitate during the process; slowly cool down to below 10 °C, and continue to stir and crystallize for 3 hours; filter and collect 120 g of the crude TAPS hydrate;

[0087] Step 5: Add the above-mentioned crude hydrate to 800 mL of purified water, stir and wash at below 10 °C for 3 hours; filter and collect 120 g of the wet TAPS product, and dry it under reduced pressure at 55 °C to obtain 50.4 g of the finished product. The yield is 84%.

[0088] According to the results of Example 1, Example 7, and Example 8, the most preferred salting-out pH of TAPS is 11.

[0089] Analyze the purity of the products prepared in the examples and commercially available samples, and the following results are obtained:

[0090] Table 1

[0091]

[0092]

[0093] As can be seen from Table 1, the method described in the present invention can achieve a high yield when extracting TAPS, and extract as much TAPS as possible from the fermentation broth; at the same time, the present invention further optimizes the method, improves the purity of TAPS while increasing the yield of TAPS. The present invention dilutes and heats up the fermentation broth, adjusts the pH, so that the TAPS in the cells dissolves out, avoids using a high-speed homogenizer to break the cell wall, and is more conducive to industrial production; uses the ion exchange method to avoid using a large amount of organic solvents, reduces safety and environmental protection risks, and reduces waste gas emissions; at the same time, the present invention extracts the target product by the ion exchange method, so that the obtained product has high purity, does not require silica gel column separation, can greatly reduce the cost, and is more suitable for large-scale production.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Those of ordinary skill in the art should understand that any modifications or equivalent replacements made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions should be covered by the scope of the claims of the present invention.

Claims

1. A method for extracting tetraacetylsphingosine, characterized in that, the fermentation broth containing tetraacetylsphingosine is purified through the following process route to obtain tetraacetylsphingosine in the fermentation broth: Step 1: Obtain the fermentation broth containing tetraacetylsphingosine, dilute it with water, heat it up to 20 - 60 °C, add concentrated hydrochloric acid and stir to adjust the pH to 2 - 5; Step 2: Add cationic resin to the fermentation broth treated in Step 1, stir and adsorb for 2 - 5 hours, and sieve to recover the cationic resin; the cationic resin is a strongly acidic cationic resin; Step 3: Elute the cationic resin with water until the pH value of the eluted water is 5 - 7; elute the cationic resin with acetic acid solution and collect the eluate; wherein, the volume of the acetic acid solution is 4 - 10 times the volume of the cationic resin, and the concentration of the acetic acid solution is 0.3 - 1.0 mol / L; Step 4: Carry out vacuum concentration on the eluate collected in Step 3 to concentrate it to 1 / 8 - 1 / 3 of the original volume of the eluate to obtain a concentrated solution; then adjust the pH of the concentrated solution to 9 - 11 with sodium hydroxide solution, cool it down to below 10 °C, crystallize for 3 - 5 h, and filter to collect the crude product; wherein, the mass fraction of the sodium hydroxide solution is 10 - 30%; Step 5: Wash the crude product collected in Step 4 with purified water at below 10 °C for 2 - 5 hours, filter to collect the solid, and obtain the tetraacetylsphingosine through vacuum drying.

2. The method for extracting tetraacetylsphingosine according to claim 1, characterized in that, the dosage of the cationic resin is 10 - 50 grams of resin added per 1 gram of TAPS.

3. The method for extracting tetraacetylsphingosine according to claim 1, characterized in that, in Step 2, the stirring adsorption is carried out under the condition of 20 - 30 °C.

4. The method for extracting tetraacetylsphingosine according to claim 1, characterized in that, in Step 3, during the elution process, control the elution flow rate to be 1 / 200 - 1 / 20 resin volume / min.

5. The method for extracting tetraacetylsphingosine according to claim 1, characterized in that, in Step 4, after vacuum concentration under the condition of 30 - 60 °C, adjust the pH to 9 - 11 with sodium hydroxide solution.

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