Method for synthesizing ceramide NP through two-step enzyme catalysis

Abstract

The invention discloses a method for synthesizing ceramide NP through two-step enzyme catalysis, which comprises the following steps of: firstly, adding lipase into vegetable oil to catalyze and combine fatty acid to decompose into free fatty acid, and centrifuging to remove the lipase; a small amount of phytosphingosine is added into the hydrolyzed vegetable oil, the raw material vegetable oil is used as a solvent or a squalane system solvent for incubation, and then immobilized lipase is added for catalytic synthesis of ceramide. The invention establishes a two-enzyme method for efficiently synthesizing the ceramide NP compound, and the process is natural, environment-friendly and non-toxic. The reaction conditions are optimized through response surface analysis, meanwhile, the immobilized lipase shows excellent cycle performance, and the method is easy to operate, environmentally friendly, green and economical and has wide application prospects in the fields of cosmetics, health care products, biological medicine and the like.

Description

Technical Field

[0001] This invention relates to a method for synthesizing ceramide NP, and particularly to a two-step enzymatic synthesis method for ceramide NP. Background Technology

[0002] Ceramides are the main lipid components of the stratum corneum, accounting for 40%–50% of its total lipids. They play a crucial role in skin health, primarily in barrier function, adhesion, moisturizing, anti-aging, and anti-allergy properties. In the stratum corneum, they function by forming a lipid bilayer, helping to maintain the skin's barrier function, preventing moisture loss, and providing protection against external stimuli. As a key skincare ingredient, ceramides are increasingly used in cosmetics and medical products. To meet market demands and improve skin health, more effective ceramide acquisition pathways need to be explored.

[0003] Ceramides are a class of compounds composed of sphingosine and long-chain fatty acids linked by amide bonds. Ceramide NP, in particular, is composed of phytosphingosine and a long-chain fatty acid without hydroxyl groups. The stratum corneum of human skin mainly contains 12 different subtypes of ceramides, with ceramide NP having the highest proportion, reaching 22.1%.

[0004] Currently, ceramides are mainly obtained through three methods: extraction from plants and animals, yeast fermentation, and chemical synthesis. While extraction from plants and animals yields natural components, the low ceramide content in these organisms results in low yields and difficulty in guaranteeing purity. Yeast fermentation is a relatively efficient method, but its production involves complex control and separation steps. Chemical synthesis is a direct method, but in practical applications, it is prone to problems such as numerous byproducts and low yields. Therefore, it is necessary to explore safer and more efficient methods for obtaining ceramides. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a two-step enzymatic synthesis method for ceramide NP.

[0006] Technical solution: The present invention provides a two-step enzymatic synthesis method for ceramide NP, comprising the following steps:

[0007] (1) Hydrolysis: Add hydrolytic lipase to vegetable oil and incubate to catalyze the decomposition of bound fatty acids into free fatty acids, then remove the lipase by centrifugation;

[0008] (2) Amide synthesis: a. After centrifugation, add phytosphingosine, then add hydrolyzed peony seed oil, mix and add immobilized enzyme, and react; b. Or add hydrolyzed peony seed oil, centrifuge and then add squalane, mix and add immobilized enzyme, and react.

[0009] Furthermore, in step (1), the volume ratio of lipase to vegetable oil is 1-1.5:5-10.

[0010] Furthermore, in step (2)a, the mass ratio of phytosphingosine to immobilized lipase is 18–20:30.

[0011] Furthermore, in step (2)b, the mass ratio of phytosphingosine to immobilized lipase is 18–20:30.

[0012] Furthermore, the vegetable oil in step (1) is selected from peony seed oil.

[0013] Furthermore, the hydrolytic lipase in step (1) is selected from Aspergillus niger lipase.

[0014] Furthermore, the immobilized lipase is selected from Novozymes 435 immobilized enzyme.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] 1. This invention establishes a dual-enzyme method for synthesizing NP-type ceramide compounds, utilizing readily available natural plant-derived vegetable oils or fatty acids as raw materials. By employing hydrolyzed lipases and immobilized lipases as biocatalysts, and using the raw material vegetable oil fatty acids as the reaction medium, and by adopting appropriate reaction conditions such as substrate concentration, lipase load, and reaction temperature, the amidation reaction of plant sphingosine and fatty acid acyl donors is carried out, thereby synthesizing vegetable oil-derived NP-type ceramide compounds and improving the yield of ceramide synthesis.

[0017] 2. The synthesis method of the present invention can efficiently synthesize ceramide NP compounds, while the immobilized lipase exhibits excellent cycling performance. It is simple to operate, environmentally friendly, green and economical, and has broad application prospects. Attached Figure Description

[0018] Figure 1 The image shows the MS / MS ion diagrams of five ceramide products, where A is PA-CER, B is ALA-CER, C is LA-CER, D is OA-CER, and E is SA-CER.

[0019] Figure 2 The effect of reaction temperature on ceramide yield is shown in Figures a, b, c, d, and e, which represent average values ​​and have significant differences (p < 0.05).

[0020] Figure 3 The effect of substrate concentration on yield is shown in Figures a, b, c, d, and e, which represent the average values ​​and have significant differences (p < 0.05).

[0021] Figure 4 The effect of enzyme loading on yield is shown in Figures a, b, c, d, and e, which represent the average values ​​and have significant differences (p < 0.05).

[0022] Figure 5 The response surface and contour plots show the interaction between factors affecting enzyme-catalyzed reactions and CERs yield (a: effect of reaction temperature and enzyme dosage on yield, b: effect of reaction temperature and enzyme dosage on yield, c: effect of substrate concentration and enzyme dosage on yield).

[0023] Figure 6 The effect of lipase stability on yield is shown, where a, b, c, d, and e represent significant differences among them.

[0024] Figure 7 The effect of substrate ratio (phytosphingosine: peony seed oil fatty acids) on yield is shown in Figures a, b, c, d, and e, which represent the average values ​​and have significant differences (p < 0.05). Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be further described below.

[0026] Example 1 (Solvent-free system)

[0027] The solvent-free enzymatic synthesis method for ceramide from peony seed oil in this embodiment includes the following steps:

[0028] (1) The first step of the hydrolysis reaction was carried out in a 50mL Erlenmeyer flask. The reaction system was controlled to be 20mL. First, 20mL of peony seed oil was added, followed by 10% (wt, calculated based on the total amount of oil) of Aspergillus niger lipase and a small amount of glass beads. The mixture was thoroughly mixed and placed in a constant temperature shaker at 37℃ and 220r / min for 24h. After the reaction was completed, the lipase precipitate was removed by centrifugation at 10000rpm for 5min, and the supernatant was collected for later use.

[0029] (2) For the second step of amide synthesis, 190.2 mg of phytosphingosine was accurately weighed and placed in a 10 mL Erlenmeyer flask. Hydrolyzed peony seed oil was added and the volume was adjusted to 3 mL. After thorough mixing, 300 mg of Novozymes 435 immobilized enzyme was added, and the mixture was placed in a magnetically heated stirrer and stirred at 65 °C for 24 h. After completion, the sample was centrifuged at 10,000 rpm for 5 min, and the supernatant was collected. The product was diluted with methanol and analyzed by liquid chromatography-mass spectrometry (LC-MS).

[0030] LC-MS / MS detection was performed using a Waters Xevo TQ-S triple quadrupole mass spectrometer, and ThermoScientific was used. TM Hypersil GOLD n A 100×2.1mm, 1.9μm column was used as the analytical column. The LC-MS mobile phase detection conditions for ceramide analysis are shown in Table 1: flow rate 0.2 mL / min, injection volume 2 μL, and column temperature controlled at 40℃. Mass spectrometry detection conditions are shown in Table 2. After analysis by high-performance liquid chromatography-tandem quadrupole mass spectrometry, five expected ceramides from peony seed oil were obtained. Their mass spectrometric and chemical structural information are shown in Tables 3 and 4 below. Detailed MS / MS ion chromatograms of the five ceramides are shown below. Figure 1 As shown.

[0031] This study comprehensively investigated the effects of key parameters, such as reaction time, temperature, substrate concentration, and lipase dosage, on the overall yield of ceramide during enzymatic synthesis. The influence of temperature variation as a single factor on the yield was explored, and the results are as follows: Figure 2 As shown; the effect of substrate concentration on yield of phytosphingosine is as follows. Figure 3 As shown; the effect of enzyme loading on yield is as follows. Figure 4 As shown in the figure. After determining the optimal condition range through single-factor experiments, the Box-Behnken design method was adopted, using the total ceramide yield as the indicator, and selecting reaction temperature, substrate plant sphingosine concentration, and enzyme dosage as variables to optimize the enzymatic reaction process. The results are shown in the figure. Figure 5 As shown. To evaluate the impact of the number of recycling cycles on catalytic efficiency, seven consecutive batches of experiments were conducted, and the results are as follows. Figure 6 As shown.

[0032] Example 2 (squalane system, the difference from Example 1 is that 4 mL of hydrolyzed peony seed oil was added in the second step, followed by the addition of squalane to bring the volume to 20 mL)

[0033] The enzymatic synthesis method of ceramide from peony seed oil in the squalane system of this embodiment includes the following steps:

[0034] (1) The first step of the hydrolysis reaction was carried out in a 1L conical flask with a reaction volume of 20mL. 18mL of peony seed oil and 2mL of hydrolytic lipase were added first, followed by a small amount of glass beads. After thorough mixing, the mixture was placed in a constant temperature shaker at 37℃ and 220r / min for 24h. After the reaction was completed, the lipase precipitate was removed by centrifugation at 10000rpm for 5min, and the supernatant was collected for later use.

[0035] (2) In the second step of amide synthesis, 190.2 mg of phytosphingosine was accurately weighed and placed in a 10 mL Erlenmeyer flask. 0.6 mL of hydrolyzed peony seed oil was added, followed by squalane to a final volume of 3 mL. After thorough mixing, 300 mg of Novozymes 435 immobilized enzyme was added, and the mixture was placed in a magnetically heated stirrer and stirred at 65 °C for 24 h. After completion, the sample was centrifuged at 10,000 rpm for 5 min, and the supernatant was collected. The product was diluted with methanol and analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0036] The results are shown in Example 1. By keeping the amount of phytosphingosine constant and adjusting the amount of fatty acids added to peony seed oil, a gradient change in the molar ratio of phytosphingosine to peony seed oil fatty acids was achieved, as shown in the results. Figure 7 As shown.

[0037] Results diagram for the squalane system: Figure 7 The results showed that the highest product yield (54.50±1.44%) was achieved when the molar ratio of phytosphingosine to peony seed oil fatty acids was 1:3 (the molar ratio in this example), representing a 24.71% increase compared to a 1:1 ratio (29.79±1.11%). As the fatty acid ratio further increased to 1:8, the yield significantly decreased to 23.11±0.65%. Notably, when phytosphingosine was in excess (2:1), the yield was only 17.83±0.97%.

[0038] The following is a diagram showing the results of the peony seed oil system:

[0039] from Figure 2 It can be seen that the effect of temperature on the yield of the enzyme-catalyzed reaction product exhibits a typical "bell-shaped curve" characteristic. In the range of 55-65℃, the product yield increases significantly with increasing temperature, reaching a peak at 65℃ (34.42±1.16%) in this example. This can largely be attributed to the increased catalytic activity due to the temperature approaching the optimum of N 435. Subsequently, as the temperature gradually rises to 75℃, the yield drops sharply to 18.08±1.42%.

[0040] Figure 3 The results show that the yield is significantly positively correlated with increasing substrate concentration in the concentration range of 0.05-0.2 mol / L, reaching a maximum of 34.45 ± 2.31% at 0.2 mol / L (in this example). The yield increase slows down when the concentration exceeds 0.2 mol / L.

[0041] Figure 4 The results showed that when the enzyme dosage was increased from 50 mg to 300 mg, the yield was positively correlated with the increase of enzyme dosage (the highest yield at 300 mg was 34.35 ± 1.74%). When the enzyme dosage was > 300 mg, the yield decreased significantly to 15.40 ± 0.46% (at 400 mg).

[0042] Figure 5 Three-dimensional surface plots and contour plots are used to represent the interaction between temperature, substrate concentration, and enzyme dosage on ceramide yield. The three-dimensional response surface plot shows the relationship between the two independent variables and ceramide yield, with the surface vertices corresponding to the maximum and minimum values, providing a more comprehensive view of the relationship between the variables and the yield. The dense and elliptical contour lines in the contour plot indicate a significant interaction between the two factors. Analysis of the ceramide yield using the response surface results shows that the conditions selected in this example are the optimal conditions for response surface fitting.

[0043] Figure 6 The effect of the stability of lipase Novozym 435 on the yield was shown. As the number of cycles increased, the yield of ceramide showed a decreasing trend: the initial batch yield was 36.62±0.63%, which decreased to 27.80±2.45% by the 7th cycle. This indicates that Novozym 435 has good recyclability.

[0044] Table 1. Mobile phase composition and gradient elution for ceramide detection.

[0045]

[0046] Table 2 Ceramide Mass Spectrometry Detection Conditions

[0047]

[0048] Table 3 Mass Spectrometry Information for Five Ceramides

[0049]

[0050] Note: 1-5 are ceramides synthesized from peony seed oil by palmitic acid, α-linolenic acid, linoleic acid, oleic acid, stearic acid and phytosphingosine, respectively, after the first enzymatic reaction.

[0051] Table 4. Chemical structural information of five fatty acids and their corresponding ceramides.

[0052]

[0053] Note: 1-5 are palmitic acid, α-linolenic acid, linoleic acid, oleic acid, stearic acid and their respective ceramides synthesized from phytosphingosine.

[0054] The above are merely preferred embodiments of the present invention and do not constitute any limitation on the present invention. Any equivalent substitutions or modifications made by those skilled in the art to the technical solutions and content disclosed in the present invention without departing from the scope of the present invention shall be deemed to have remained within the protection scope of the present invention.

Claims

1. A two-step enzymatic synthesis method for ceramide NP, characterized in that, Includes the following steps: (1) Hydrolysis: Add hydrolytic lipase to vegetable oil and incubate to catalyze the decomposition of bound fatty acids into free fatty acids, then remove the lipase by centrifugation; (2) Amide synthesis: a. After centrifugation, add phytosphingosine, then add hydrolyzed peony seed oil, mix and add immobilized enzyme, and react; b. Or add hydrolyzed peony seed oil, centrifuge and then add squalane, mix and add immobilized enzyme, and react.

2. The method for two-step enzyme-catalyzed synthesis of ceramide NP according to claim 1, characterized in that, In step (1), the volume ratio of lipase to vegetable oil is 1-1.5:5-10.

3. The method for two-step enzyme-catalyzed synthesis of ceramide NP according to claim 1, characterized in that, In step (2)a, the mass ratio of phytosphingosine to immobilized lipase is 18-20:

30.

4. The method for two-step enzyme-catalyzed synthesis of ceramide NP according to claim 1, characterized in that, In step (2)b, the mass ratio of phytosphingosine to immobilized lipase is 18-20:

30.

5. The method for two-step enzyme-catalyzed synthesis of ceramide NP according to claim 1, characterized in that, The vegetable oil used in step (1) is selected from peony seed oil.

6. The two-step enzymatic synthesis method for ceramide NP according to claim 1, characterized in that, The hydrolytic lipase used in step (1) is selected from Aspergillus niger lipase.

7. The method for two-step enzyme-catalyzed synthesis of ceramide NP according to claim 1, characterized in that, The immobilized lipase was selected from Novozymes 435 immobilized enzyme.

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