A method for preparing ceramide 3 using a complex microbial enzyme catalyst

Abstract

This invention discloses a method for preparing ceramide 3 by acylation catalyzed by a composite microbial enzyme. The method uses plant sphingosine and mixed long-chain fatty acids as raw materials, and a binary composite microbial lipase with *Candida albicans* lipase as the main enzyme and *Aspergillus oryzae* / *Rhizopus oryzae* lipase as the coenzyme as the catalyst. The enzyme-catalyzed acylation reaction is followed by filtration, vacuum distillation, recrystallization, and drying to obtain the final product. This invention's composite enzyme system achieves highly efficient synergistic catalysis of the mixed fatty acid substrate, resulting in a ceramide 3 yield ≥90%, total purity ≥93%, and oleic acid-based ceramide 3 as the main component ≥85%. The distribution of minor components closely matches that of natural ceramides. The reaction conditions are mild, with no chemical residues. The composite enzyme is recyclable and reusable. The preparation process is simple, has high tolerance for error, and is easily scaled up industrially. The product can be widely used in the pharmaceutical and cosmetic fields.

Description

Technical Field

[0001] This invention relates to the field of ceramide technology, and more particularly to the preparation technology of ceramide 3. Background Technology

[0002] Ceramide 3 (also known as ceramide NP) is a naturally occurring sphingolipid compound. As a core lipid component of the stratum corneum, it has moisturizing, skin barrier repair, and anti-aging effects. It also has anti-inflammatory and antioxidant bioactivities and is an important raw material for pharmaceutical preparations and high-end cosmetics.

[0003] Currently, the preparation methods for ceramide 3 are mainly divided into chemical synthesis and enzymatic synthesis. Chemical synthesis, for example, uses fatty acid esters and sphingosine as raw materials, involving transesterification-amidation under alkaline catalysts and high-temperature reflux conditions. This method suffers from harsh reaction conditions, chemical reagent residues, and difficulty in controlling product purity. Enzymatic synthesis often uses single fatty acids as raw materials, resulting in products with simple structures that differ significantly from the compositional characteristics of natural ceramide 3. Furthermore, some processes use chemical enzymes with low catalytic efficiency and poor substrate applicability, making industrial-scale production difficult. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing ceramide 3, so as to solve the problems of harsh conditions, reagent residues, difficulty in controlling product purity, simple product structure, and difficulty in achieving industrial production in existing preparation technologies.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for preparing ceramide 3 by catalysis using a composite microbial enzyme involves using plant sphingosine and mixed long-chain fatty acids as raw materials, and a composite microbial lipase as a catalyst, to carry out an enzymatic acylation reaction in an organic solvent system. After the reaction is completed, the ceramide 3 product is obtained through post-treatment. The mixed long-chain fatty acids consist of 80 wt% oleic acid and 20 wt% composite fatty acids, wherein the 20 wt% composite fatty acids are a mixture of linoleic acid, stearic acid, and palmitic acid in a certain proportion. The composite microbial lipase uses Candida albicans lipase as the main enzyme and one of Aspergillus oryzae lipase or Rhizopus oryzae lipase as the coenzyme, with a mass ratio of (7~8):(2~3).

[0007] Furthermore, the organic solvent is one or more of n-hexane, petroleum ether, and tert-amyl alcohol, and the amount of organic solvent added is 3 to 8 times the total mass of the raw materials.

[0008] Further, the molar ratio of the phytosphingosine to the mixed long-chain fatty acids is 1:(1.0~1.5), preferably 1:(1.2~1.3).

[0009] Furthermore, the total amount of the compound microbial lipase added is 3.5~7wt% of the total mass of the raw materials, and the enzyme activity of both the main enzyme and the coenzyme is ≥10000U / g.

[0010] Furthermore, the conditions for the enzyme-catalyzed acylation reaction are: reaction temperature 35~45℃, stirring speed 250~350r / min, and reaction time 10~16h.

[0011] Furthermore, the preferred conditions for the enzyme-catalyzed acylation reaction are: reaction temperature 40~45℃, stirring speed 350r / min, and reaction time 12~15h.

[0012] Further, the post-processing steps are as follows: after the reaction is completed, the complex microbial lipase is recovered by filtration, and the organic solvent is removed by vacuum distillation of the filtrate to obtain the crude product; 3 to 7.5 times its mass of a lower alcohol is added to the crude product, recrystallized at 0 to 5°C, and the filter cake is dried under vacuum to obtain the finished product.

[0013] Furthermore, the lower alcohol is one of methanol, ethanol, and isopropanol; the vacuum drying temperature is 40~50℃, and the drying time is 6~12h.

[0014] The advantages of this invention are:

[0015] 1. This invention relates to a complex enzyme system based on Candida albicans lipase as the main enzyme and Aspergillus oryzae / Rhizopus oryzae lipase as the auxiliary enzyme. The main enzyme and coenzyme synergistically catalyze the reaction, overcoming the substrate compatibility limitations of single enzymes. It can simultaneously and efficiently catalyze oleic acid (unsaturated 18 carbons), linoleic acid (unsaturated 18 carbons), stearic acid (saturated 18 carbons), and palmitic acid (saturated 16 carbons), achieving complete reaction of mixed fatty acid substrates with a product yield ≥90% and a total purity ≥93%.

[0016] 2. The compound enzyme system precisely regulates the distribution of product components. While ensuring that oleic acid-based ceramide 3 (main component) is ≥85%, the ratio of linoleic acid-based ceramide 3, stearic acid-based ceramide 3, and palmitic acid-based ceramide 3 meets the needs of human skin's natural ceramide 3, significantly improving the product's synergistic effects on skin barrier repair, moisturizing, and other bioactive activities.

[0017] 4. The reaction conditions are mild (near room temperature and pressure), requiring no high-temperature and high-pressure equipment, resulting in low production energy consumption; no alkaline chemical catalysts are added, avoiding chemical reagent residues, and the product meets the safety standards for pharmaceutical / cosmetic raw materials. Furthermore, the compound enzyme can be recycled and reused, significantly reducing production costs.

[0018] 5. The preparation process is simple, and the post-processing only involves filtration, vacuum distillation, and recrystallization to obtain a high-purity finished product. The process has a high tolerance for error, and the product quality is not affected by batch differences in substrates or slight fluctuations in equipment parameters. It is easy to achieve industrial-scale production.

[0019] 6. The total amount of compound enzyme added is comparable to that of single enzyme, with no additional enzyme feeding cost, and the catalytic reaction time is shorter than that of single enzyme, improving production efficiency and reducing the overall production cost per unit product. Attached Figure Description

[0020] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings:

[0021] Figure 1-2 This is the test result of Example 1. Detailed Implementation

[0022] The present invention will be described in detail below with reference to specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0023] All raw materials used in this invention are industrial-grade pure products: phytosphingosine purity ≥90%, oleic acid, linoleic acid, stearic acid, and palmitic acid purity ≥98%; organic solvents and lower alcohols are all analytical grade.

[0024] In this invention, the yield = actual mass of ceramide 3 obtained / theoretical mass of ceramide 3 produced × 100%; the purity of the product and the content of each component were determined by HPLC-UV normalization method. The detection conditions were: C18 column (250 mm × 4.6 mm, 5 μm), methanol-acetonitrile (volume ratio 9:1) as mobile phase, flow rate 1.0 mL / min, column temperature 30 ℃, and detection wavelength 202 nm.

[0025] In all embodiments, the 20wt% complex fatty acid was linoleic acid:stearic acid:palmitic acid = 1:1:1. Unless otherwise specified, methanol was used as the recrystallization solvent in the post-processing steps, the recrystallization temperature was 0℃, the vacuum drying temperature was 45℃, and the drying time was 8h.

[0026] Example 1

[0027] A method for preparing ceramide 3 by composite microbial enzyme catalysis, the specific steps of which are as follows:

[0028] 1. Prepare raw materials: Weigh 0.1 mol (30.75 g) of phytosphingosine and 0.12 mol of mixed long-chain fatty acids, wherein the mixed long-chain fatty acids consist of 80 wt% oleic acid and 20 wt% complex fatty acids;

[0029] 2. Preparation of compound microbial enzyme: Prepare compound microbial lipase at a mass ratio of 8:2.5. The main enzyme is Candida albicans lipase (CALB), and the coenzyme is Aspergillus oryzae lipase. The total enzyme activity is ≥10000U / g. Both the main enzyme and the coenzyme are solid catalysts formed by immobilizing single enzyme molecules on a solid-phase support.

[0030] 3. Reaction system construction: Add the raw materials to a three-necked reaction flask, add 6.5 times the total mass of the raw materials in n-hexane, then add 7 wt% of the total mass of the raw materials in the above-mentioned composite microbial enzyme, add a stir bar and seal the reaction flask;

[0031] 4. Catalytic acylation reaction: Place the reaction flask in a constant temperature water bath with a stirring pot, adjust the temperature to 40℃ and the stirring speed to 350r / min, and stir the reaction at the same temperature for 12h.

[0032] 5. Post-processing: The complex microbial enzymes were recovered by filtration. The filtrate was subjected to vacuum distillation at 45℃ and -0.08MPa to remove n-hexane. Methanol with a mass of 7.5 times that of the crude product was added, and the product was recrystallized at 0℃. The filter cake was then dried under vacuum at 45℃ for 7 hours to obtain ceramide 3 product.

[0033] The test results are as follows: Figure 1-2 As shown, the finished product is a white to off-white powder with a yield of 93.7% and a total purity of 96.09%. The product contains 89.519% oleic acid ceramide 3, 4.384% linoleic acid ceramide 3, 1.655% stearic acid ceramide 3, and 0.532% palmitic acid ceramide 3.

[0034] Example 2

[0035] A method for preparing ceramide 3 by composite microbial enzyme catalysis, the specific steps of which are as follows:

[0036] 1. Prepare raw materials: Weigh 0.1 mol of phytosphingosine and 0.13 mol of mixed long-chain fatty acids. The composition of the mixed long-chain fatty acids is the same as in Example 1.

[0037] 2. Preparation of compound microbial enzyme: Prepare compound microbial lipase at a mass ratio of 7:2.5. The main enzyme is Candida lipase and the coenzyme is Aspergillus oryzae lipase. The total enzyme activity is ≥10000U / g. Both the main enzyme and the coenzyme are solid catalysts formed by immobilizing single enzyme molecules on a solid support.

[0038] 3. Construction of the reaction system: Add the raw materials to a three-necked reaction flask, add n-hexane at 6.5 times the total mass of the raw materials, add the above-mentioned composite microbial enzyme at 4.5 wt% of the total mass of the raw materials, add a stir bar and seal the reaction flask;

[0039] 4. Catalytic acylation reaction: Place the reaction flask in a constant temperature water bath with a stirring pot at 45℃ and a stirring speed of 350r / min, and stir for 10h.

[0040] 5. Post-processing: Same as in Example 1.

[0041] The finished product was tested and found to be a white to off-white powder with a yield of 93.5% and a total purity of 95.3%. The product contained 85.2% oleic acid ceramide 3, 4.1% linoleic acid ceramide 3, 2.79% stearic acid ceramide 3, and 0.34% palmitic acid ceramide 3.

[0042] Example 3

[0043] A method for preparing ceramide 3 by composite microbial enzyme catalysis, the specific steps of which are as follows:

[0044] 1. Prepare raw materials: Weigh 0.1 mol of phytosphingosine and 0.15 mol of mixed long-chain fatty acids. The composition of the mixed long-chain fatty acids is the same as in Example 1.

[0045] 2. Preparation of compound microbial enzyme: Prepare compound microbial lipase at a mass ratio of 7.5:3. The main enzyme is Candida lipase and the coenzyme is Rhizopus oryzae lipase. The total enzyme activity is ≥10000U / g. Both the main enzyme and the coenzyme are solid catalysts formed by immobilizing single enzyme molecules on a solid support.

[0046] 3. Construction of the reaction system: Add the raw materials to a three-necked reaction flask, add tert-amyl alcohol at 4 times the total mass of the raw materials, add the above-mentioned compound microbial enzyme at 6 wt% of the total mass of the raw materials, add a stir bar and seal the reaction flask;

[0047] 4. Catalytic acylation reaction: The reaction flask was placed in a constant temperature water bath with a stirring speed of 350 r / min and the reaction was carried out for 16 h.

[0048] 5. Post-processing: The complex microbial enzymes were recovered by filtration, the filtrate was distilled under reduced pressure, ethanol was added to the crude product at 4 times its mass, and the product was recrystallized at 3°C. The rest of the process was the same as in Example 1.

[0049] The finished product was tested and found to be a white to off-white powder with a yield of 93.2% and a total purity of 95.0%. The product contained 86.1% oleic acid ceramide 3, 4.26% linoleic acid ceramide 3, 1.122% stearic acid ceramide 3, and 0.365% palmitic acid ceramide 3.

[0050] Example 4

[0051] A method for preparing ceramide 3 by composite microbial enzyme catalysis, the specific steps of which are as follows:

[0052] 1. Prepare raw materials: Weigh 0.1 mol of phytosphingosine and 0.10 mol of mixed long-chain fatty acids. The composition of the mixed long-chain fatty acids is the same as in Example 1.

[0053] 2. Preparation of compound microbial enzyme: Prepare compound microbial lipase at a mass ratio of 8:3. The main enzyme is Candida lipase and the coenzyme is Rhizopus oryzae lipase. The total enzyme activity is ≥10000U / g. Both the main enzyme and the coenzyme are solid catalysts formed by immobilizing single enzyme molecules on a solid support.

[0054] 3. Construction of the reaction system: Add the raw materials to a three-necked reaction flask, add petroleum ether at 7 times the total mass of the raw materials, add the above-mentioned compound microbial enzyme at 3.5 wt% of the total mass of the raw materials, add a stir bar and seal the reaction flask;

[0055] 4. Catalytic acylation reaction: Place the reaction flask in a constant temperature water bath with a stirring speed of 250 r / min at 42℃ and stir for 15 h.

[0056] 5. Post-processing: The complex microbial enzymes were recovered by filtration, the filtrate was distilled under reduced pressure, and 6.5 times its mass of isopropanol was added to the crude product. The product was recrystallized at 5°C. The rest of the process was the same as in Example 1.

[0057] The finished product was tested and found to be a white to off-white powder with a yield of 92.0% and a total purity of 94.1%. The product contained 86.3% oleic acid ceramide 3, 3.7% linoleic acid ceramide 3, 1.1% stearic acid ceramide 3, and 0.38% palmitic acid ceramide 3.

[0058] Example 5

[0059] The composite microbial lipase recovered by filtration in Example 1 was directly used to prepare ceramide 3 under the same raw material ratio and process conditions as in Example 1. The catalytic effect of the composite enzyme after the first recovery was tested: the yield of the finished product was 90.1%, the total purity of the product was 93.8%, the content of oleic acid-based ceramide 3 was 85.0%, and the catalytic activity of the composite enzyme did not decrease significantly.

[0060] Comparative Example

[0061] To verify the rationality and superiority of the compound enzyme system and process parameters of the present invention, the following comparative examples were set up. Each comparative example changed only a single variable, and the remaining steps were the same as in Example 1. The detection results were compared and analyzed with those in Example 1.

[0062] Comparative Example 1: Using a single Candida lipase

[0063] The coenzyme was removed, and only Candida lipase was used as a catalyst at a dosage of 7 wt%. The remaining steps were the same as in Example 1. Testing showed a yield of 89.2%, a total product purity of 92.0%, an oleic acid-based ceramide 3 content of 86.7%, a palmitic acid-based ceramide 3 content of only 0.28%, and other ceramide 3 contents were not significantly different from those in Example 1. The saturated fatty acid substrate reaction was incomplete.

[0064] Comparative Example 2: Imbalance in the ratio of primary and secondary enzymes in the complex enzyme

[0065] The ratio of the compound enzyme was changed to Candida lipase: Aspergillus oryzae lipase = 5:5, with a total addition of 5 wt%. The remaining steps were the same as in Example 1. Testing showed a yield of 90.2%, a total product purity of 94.5%, and an oleic acid-based ceramide 3 content of only 81.5%, with the main component content below 85%, failing to meet the product's internal control standards. Other ceramide 3 contents were not significantly different from those in Example 1.

[0066] Comparative Example 3: Insufficient amount of compound enzyme added

[0067] The compound enzyme formulation was the same as in Example 1, except the total amount added was changed to 2 wt% of the total raw material mass. The remaining steps were the same as in Example 1. Testing showed a yield of 75.8% and a total product purity of 91.2%. Insufficient catalyst led to incomplete substrate reaction, resulting in a significant decrease in yield.

[0068] Comparative Example 4: Reaction temperature too high

[0069] The reaction temperature was changed to 60℃, and the remaining steps were the same as in Example 1. Testing showed a yield of 77.9% and a total product purity of 87.8%. The high temperature caused partial inactivation of the complex enzyme and oxidative decomposition of fatty acids, resulting in a significant decrease in yield and purity.

[0070] Comparative Example 5: Using a chemical catalyst

[0071] The complex enzyme was omitted, and sodium methoxide was used as a catalyst (phytosphingosine:sodium methoxide = 1:0.5). The reaction was carried out under reflux in an oil bath at 70°C for 17 hours, with the remaining steps consistent with Example 1. Testing revealed a yield of 82.3% and a total product purity of 90.67%. Trace amounts of sodium methoxide residue were detected in the product, and the oleic acid-based ceramide 3 content was 79.8%. The contents of other ceramide 3s were not significantly different from those in Example 1, indicating the presence of residual chemical reagents.

[0072] Comparative Example 6: Using a combination of three enzymes

[0073] A compound system of three microbial lipases—Candida lipase, Aspergillus oryzae lipase, and Rhizopus oryzae lipase—was prepared at a mass ratio of 6:2.5:2, with a total addition amount of 5 wt%. The remaining steps were the same as in Example 1. Testing showed a yield of 84.3% and a total product purity of 91.3%. The multiple enzymes competed for catalytic sites, resulting in decreased catalytic efficiency and a lower yield than the binary compound enzyme system of this invention.

[0074] Experimental Data Summary and Analysis

[0075] The core experimental data of each embodiment and comparative example of the present invention are summarized in Table 1.

[0076] Table 1

[0077]

[0078] Data analysis and conclusions;

[0079] 1. The embodiments of the present invention adopt a binary complex enzyme system and optimized process, and the product yield is ≥90%, the total purity is ≥93%, the oleic acid ceramide 3 is ≥85%, there is no chemical residue, and all indicators are significantly better than all comparative examples.

[0080] 2. Although single enzyme catalysis leaves no residue, it has low catalytic efficiency for saturated fatty acid substrates (Comparative Example 1); an imbalance in the ratio of complex enzymes will lead to insufficient content of the main component (Comparative Example 2); the combination of three enzymes will cause competition for catalytic sites and reduce the yield (Comparative Example 6), proving that the binary complex enzyme ratio of the present invention is the optimal solution.

[0081] 3. The amount of enzyme added and the reaction temperature are key process parameters. Deviating from the scope of protection of this invention will lead to incomplete substrate reaction, enzyme inactivation, fatty acid oxidation, and a significant decrease in yield and purity (Comparative Examples 3 and 4).

[0082] 4. Chemical catalysis methods suffer from problems such as low yield, low purity, and chemical residues (Comparative Example 5), and cannot be compared with the composite enzyme catalysis method of this invention;

[0083] 5. The composite microbial lipase of the present invention can be recycled and reused. After the first recycling, it can still maintain high catalytic activity, which greatly reduces the cost of industrial production.

[0084] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for preparing ceramide 3 by catalysis of a composite microbial enzyme, characterized in that, Ceramide 3 was obtained by using plant sphingosine and mixed long-chain fatty acids as raw materials and a compound microbial lipase as a catalyst in an organic solvent system for enzymatic acylation. After the reaction was completed, the product was post-processed. The mixed long-chain fatty acids consisted of 80 wt% oleic acid and 20 wt% compound fatty acids, wherein the 20 wt% compound fatty acids were a mixture of linoleic acid, stearic acid and palmitic acid in a certain proportion. The compound microbial lipase used Candida albicans lipase as the main enzyme and one of Aspergillus oryzae lipase and Rhizopus oryzae lipase as the coenzyme, with a mass ratio of (7~8):(2~3).

2. The method for preparing ceramide 3 by composite microbial enzyme catalysis according to claim 1, characterized in that, The organic solvent is one or more of n-hexane, petroleum ether, and tert-amyl alcohol, and the amount of organic solvent added is 3 to 8 times the total mass of the raw materials.

3. The method for preparing ceramide 3 by composite microbial enzyme catalysis according to claim 1, characterized in that, The molar ratio of the plant sphingosine to the mixed long-chain fatty acids is 1:(1.0~1.5), preferably 1:(1.2~1.3).

4. The method for preparing ceramide 3 by composite microbial enzyme catalysis according to claim 1, characterized in that, The total amount of the compound microbial lipase added is 3.5~7wt% of the total mass of the raw materials, and the enzyme activity of both the main enzyme and the coenzyme is ≥10000U / g.

5. The method for preparing ceramide 3 by composite microbial enzyme catalysis according to claim 1, characterized in that, The conditions for the enzyme-catalyzed acylation reaction are: reaction temperature 35~45℃, stirring speed 250~350r / min, and reaction time 10~16h.

6. The method for preparing ceramide 3 by composite microbial enzyme catalysis according to claim 5, characterized in that, The preferred conditions for the enzyme-catalyzed acylation reaction are: reaction temperature 40~45℃, stirring speed 350r / min, and reaction time 12~15h.

7. The method for preparing ceramide 3 by composite microbial enzyme catalysis according to claim 1, characterized in that, The post-processing steps are as follows: after the reaction is completed, the complex microbial lipase is recovered by filtration, and the organic solvent is removed by vacuum distillation of the filtrate to obtain the crude product; 3 to 7.5 times its mass of a lower alcohol is added to the crude product, recrystallized at 0 to 5°C, and the filter cake is dried under vacuum to obtain the finished product.

8. The method for preparing ceramide 3 by composite microbial enzyme catalysis according to claim 7, characterized in that, The lower alcohol is one of methanol, ethanol, and isopropanol; the vacuum drying temperature is 40~50℃, and the drying time is 6~12h.

Images