Method for producing ceramide

Abstract

To provide a method for producing ceramide that can increase the yield of ceramide.SOLUTION: A method for producing ceramide includes stirring fish meat as raw material in a first solvent mixture composed of hexane and ethanol, extracting ceramide and ceramide ciliatine into the first solvent mixture, and subjecting a filtrate of the first solvent mixture to reduced-pressure distillation for drying and solidification of it, to obtain a mixture of ceramide and ceramide ciliatine. The resultant mixture of ceramide and ceramide ciliatine is treated with a solvent in a subcritical state.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing ceramide. [Background technology]

[0002] Sphingophospholipids such as ceramides are known to enhance the moisturizing effect of the skin and are used as raw materials for cosmetics, etc.

[0003] Ceramide is a compound represented by the following formula (1): On the other hand, in the case of fish meat, it exists as ceramide ciliatine represented by the following formula (2) or sphingomyelin (a compound in which the 2-aminoethylphosphonic acid group in formula (2) is replaced with a phosphorylcholine group), and it is thought that a portion of the ceramide ciliatine or sphingomyelin is converted into ceramide in its free form.

[0004] [ka]

[0005] [ka]

[0006] In the formulas (1) and (2), --- represents -(CH) n - (n is an integer).

[0007] Conventionally, a method for extracting ceramide from animals and plants using an organic solvent has been known as a method for producing ceramide. The present applicant has proposed a method in which ceramide and ceramide ciliatine are extracted from fish meat using a first mixed solvent consisting of hexane and ethanol, a second mixed solvent consisting of hexane, ethanol and water is further added to the mixture of extracted ceramide and ceramide ciliatine, the mixture is stirred, and then allowed to stand to separate the second mixed solvent into a hexane phase and an ethanol-aqueous phase, and ceramide is obtained from the hexane phase while ceramide ciliatine is obtained from the ethanol-aqueous phase (see Patent Document 1). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2017-137439 Summary of the Invention [Problem to be solved by the invention]

[0009] However, in the manufacturing method described in Patent Document 1, although ceramide can be isolated from a mixture of ceramide and ceramide ciliatine extracted from fish meat, it is difficult to convert ceramide ciliatine into ceramide, and there is a disadvantage in that the yield of ceramide cannot be increased.

[0010] An object of the present invention is to provide a method for producing ceramide that can eliminate such inconveniences and increase the yield of ceramide. [Means for solving the problem]

[0011] In order to achieve this object, the method for producing ceramide of the present invention is characterized by comprising the steps of: stirring raw fish meat in a first mixed solvent consisting of hexane and ethanol to extract ceramide and ceramide ciliatine into the first mixed solvent; filtering the first mixed solvent to obtain a filtrate; distilling the obtained filtrate to dryness under reduced pressure to obtain a mixture of ceramide and ceramide ciliatine; and treating the mixture of ceramide and ceramide ciliatine obtained by the dryness with a solvent in a subcritical state.

[0012] In the method for producing ceramide of the present invention, first, raw fish meat is stirred in a first mixed solvent consisting of hexane and ethanol, and ceramide and ceramide ciliatine are extracted into the first mixed solvent. Next, the first mixed solvent is filtered to obtain a filtrate. By the filtration, fish meat residue contained in the first mixed solvent is removed, and a filtrate containing ceramide and ceramide ciliatine can be obtained.

[0013] Next, the obtained filtrate is evaporated to dryness by vacuum distillation to obtain a mixture of ceramide and ceramide ciliatine. By vacuum distilling the filtrate, the first mixed solvent can be removed without decomposing the ceramide or ceramide ciliatine.

[0014] Next, the mixture of ceramide and ceramide ciliatine obtained by the above-mentioned drying is treated with a subcritical solvent, and the above-mentioned ceramide ciliatine is hydrolyzed by the above-mentioned subcritical solvent, and ceramide and 2-aminoethylphosphonic acid represented by the chemical formula H2NCH2CH2(PO)(OH)2 are obtained.

[0015] The term "subcritical solvent" refers to a solvent that is kept in a liquid state by applying pressure in a temperature range from the boiling point of the solvent to below its critical temperature. For example, in the case of water, the term "subcritical water" refers to water that is kept in a liquid state by applying pressure in a temperature range from 100°C, which is the boiling point of water, to 374°C, which is the critical temperature of water.

[0016] Therefore, according to the production method of the present invention, the component that has conventionally been obtained as ceramide ciliatine can also be converted into ceramide, and a sufficient amount of ceramide can be obtained.

[0017] In the production method of the present invention, the subcritical solvent is one solvent selected from the group consisting of water, ethanol, and a water-ethanol mixed solvent, and any of the solvents can be used to hydrolyze ceramide ciliatine.

[0018] The ceramide obtained by the production method of the present invention can be recovered by, for example, adding a second mixed solvent consisting of hexane, ethanol, and water to a treated product obtained by treating a mixture of the ceramide and ceramide ciliatine with the subcritical solvent, stirring the mixture, and then allowing it to stand, thereby separating the second mixed solvent into a hexane phase and an ethanol-water phase, and then drying the hexane phase to obtain ceramide. [Brief explanation of the drawings]

[0019] [Figure 1] 1 is a process diagram showing a manufacturing method of the present invention. [Figure 2] 1 is a gas chromatogram of phospholipids obtained by the production method of the present invention. [Figure 3] Mass spectrum of peak 1 in the gas chromatogram shown in Figure 2. [Figure 4] 1 shows a gas chromatogram of the components obtained by treating the phospholipids obtained by the production method of the present invention with subcritical water at 200°C for 90 minutes. [Figure 5] Figure 4 shows the mass spectrum of peak 1 in the gas chromatogram. [Figure 6] Mass spectrum of peak 3 in the gas chromatogram shown in Figure 4. [Figure 7] Mass spectrum of peak 10 in the gas chromatogram shown in Figure 4. [Figure 8] Mass spectrum of peak 11 in the gas chromatogram shown in Figure 4. [Figure 9] Mass spectrum of peak 13 in the gas chromatogram shown in Figure 4. [Figure 10] 1 shows a gas chromatogram of the components obtained by treating the phospholipids obtained by the production method of the present invention with subcritical water at 200°C for 60 minutes. [Figure 11] Mass spectrum of peak 1 in the gas chromatogram shown in Figure 10. [Figure 12] 1 shows a gas chromatogram of the components obtained by treating the phospholipids obtained by the production method of the present invention with subcritical ethanol at 200°C for 60 minutes. [Figure 13]Mass spectrum of peak 1 in the gas chromatogram shown in Figure 12. [Figure 14] 1 shows a gas chromatogram of the components obtained by treating the phospholipids obtained by the production method of the present invention with a subcritical water-ethanol mixed solvent at 200°C for 60 minutes. [Figure 15] Mass spectrum of peak 1 in the gas chromatogram shown in Figure 14. DETAILED DESCRIPTION OF THE INVENTION

[0020] Next, embodiments of the present invention will be described in more detail with reference to the accompanying drawings.

[0021] As shown in FIG. 1, in the method for producing ceramide of this embodiment, first, a first mixed solvent 2 is added to raw fish meat 1 contained in a reaction vessel, and the mixture is stirred and mixed to obtain a first mixed liquid 3.

[0022] The fish meat 1 can be, for example, a powder obtained by drying squid such as Pacific squid, spear squid, or swordtip squid in the sun for two days, then drying it in a hot air dryer at 40°C for 48 hours, cutting the dried product, and then pulverizing it. The powder contains, for example, about 10% by mass of moisture and has a particle size of 500 μm or less. The first mixed solvent 2 is made of hexane and ethanol, and contains, for example, 55 to 65 parts by mass of hexane per 100 parts by mass of ethanol.

[0023] Next, in a reaction vessel, for example, 60 to 120 ml of the first mixed solvent 2 is added to 10 g of fish meat 1, and the mixture is stirred and mixed for 1 to 4 hours. By doing so, the ceramide and ceramide ciliatin contained in the fish meat 1 are extracted into the first mixed solvent 2, and a first mixed liquid 3 is obtained.

[0024] In this embodiment, the first mixed liquid 3 is then filtered to remove fish meat residues and the like contained in the first mixed liquid 3, thereby obtaining a filtrate 4 containing ceramide and ceramide ciliatine. The filtrate 4 is then evaporated to dryness to obtain a solid 5 containing ceramide and ceramide ciliatine as phospholipids (sphingophospholipids). The evaporation to dryness can be performed, for example, by distilling the filtrate 4 under reduced pressure using a rotary evaporator or the like to remove the first mixed solvent 2.

[0025] Next, the components contained in Solid 5 were analyzed by GC / MS. GC / MS was performed using Solid 5 as a sample on a JEOL gas chromatograph time-of-flight mass spectrometer (model: JMS-T100GCV) and a non-polar column (product name: InertCap1) with an inner diameter of 0.25 mm and a length of 30 m. The column was held at 100°C for 10 minutes, then heated to 300°C at a rate of 10°C / min, and held at 300°C for 25 minutes. Helium was used as the carrier gas at 1.0 ml / min, with a split ratio of 10:1, an inlet temperature of 310°C, and chemical ionization was performed using isobutane as the reaction reagent.

[0026] The gas chromatogram of Solid 5 is shown in FIG. 2, and the mass spectrum of Peak 1 in the gas chromatogram shown in FIG. 2 is shown in FIG.

[0027] The peak with a mass-to-charge ratio (m / z) of 315.1 in Figure 3 was identified as sphingomyelin, and solid 5, whose gas chromatogram is shown in Figure 2, is presumed to be a mixture of ceramide ciliatin, a sphingophospholipid similar to sphingomyelin, and ceramide, a portion of which is released.

[0028] In this embodiment, the solid 5 is then treated with water in a subcritical state (subcritical water) (shown as subcritical water treatment 6 in FIG. 1). The subcritical water treatment 6 is performed by placing a predetermined amount of the solid 5 and a predetermined amount of water in a sealed container and heating them to a temperature equal to or higher than the boiling point of water (100°C) and lower than the critical temperature (374°C). In this manner, the water separates into a liquid phase and a gas phase in the sealed container, and the gas phase water pressurizes the container to a pressure of 0.4 to 22 MPa, converting the liquid phase water into subcritical water.

[0029] In this embodiment, the subcritical water treatment 6 is carried out at 200°C for 90 minutes, and the ceramide ciliatine is hydrolyzed by the treatment in the subcritical water, thereby obtaining ceramide and 2-aminoethylphosphonic acid represented by the chemical formula H2NCH2CH2(PO)(OH)2.

[0030] Next, the components obtained by subjecting solid 5 to subcritical water treatment 6 at 200°C for 90 minutes were analyzed by GC / MS. GC / MS was performed in exactly the same manner as when solid 5 was used as a sample, except that the components obtained by subjecting solid 5 to subcritical water treatment 6 at 200°C for 90 minutes were used as samples.

[0031] FIG. 4 shows a gas chromatogram of the components obtained by subjecting solid 5 to subcritical water treatment 6 at 200°C for 90 minutes, FIG. 5 shows a mass spectrum of peak 1 in the gas chromatogram shown in FIG. 4, FIG. 6 shows a mass spectrum of peak 3 in the gas chromatogram shown in FIG. 4, FIG. 7 shows a mass spectrum of peak 10 in the gas chromatogram shown in FIG. 4, FIG. 8 shows a mass spectrum of peak 11 in the gas chromatogram shown in FIG. 4, and FIG. 9 shows a mass spectrum of peak 13 in the gas chromatogram shown in FIG. 4.

[0032] The component corresponding to the peak with a mass-to-charge ratio (m / z) of 315.1 in Figure 5 is identical to the component corresponding to the peak with a mass-to-charge ratio (m / z) of 315.1 in Figure 3 and is identified as sphingomyelin. Furthermore, the amount of peak 1 in Figure 4 is approximately the same as that of peak 1 in Figure 2, and it is presumed that sphingomyelin is not hydrolyzed by subcritical water treatment 6 at 200°C for 90 minutes.

[0033] On the other hand, the component corresponding to the peak with a mass-to-charge ratio (m / z) of 342.1 in Figure 6 was identified as 2-aminoethylphosphonic acid, the component corresponding to the peak with a mass-to-charge ratio (m / z) of 564.5 in Figure 7 was identified as ceramide (d16:1 / 16:0), the component corresponding to the peak with a mass-to-charge ratio (m / z) of 592.5 in Figure 8 was identified as ceramide (d18:1 / 16:0), and the component corresponding to the peak with a mass-to-charge ratio (m / z) of 602.5 in Figure 9 was identified as ceramide (d19:3 / 16:0). Furthermore, peaks 3, 10, 11, and 13 in Figure 4 are also seen in Figure 2, and it is clear that Solid 5 contains ceramide and ceramide ciliatine, as described above.

[0034] It is also clear that peaks 3, 10, 11, and 13 in Fig. 4 are increased compared to the corresponding peaks in Fig. 2. Therefore, it is clear from Figs. 2 to 9 that ceramide ciliatine contained in solid 5 is hydrolyzed by subcritical water treatment 6 to produce 2-aminoethylphosphonic acid and ceramide.

[0035] Ceramide is a compound represented by the following general formula (3) (R 1 , R 2 represents an optionally unsaturated hydrocarbon group), where X is H, and where A is the number of carbon atoms in the long-chain base (LCB), B is the number of carbon-carbon double bonds, C is the number of carbon atoms in the unsaturated fatty acid (FA), and D is the number of carbon-carbon double bonds, it can be expressed as dA:B / C:D (A, B, C, and D are integers of 0 or more).

[0036] [ka]

[0037] In this embodiment, the solid 5 was then subjected to subcritical water treatment 6 at 200°C for 60 minutes, and the components obtained were analyzed by GC / MS. The GC / MS was performed in exactly the same manner as in the case of GC / MS analysis of the solid 5 as a sample shown in Fig. 2, except that the components obtained by subcritical water treatment 6 at 200°C for 60 minutes were used as samples.

[0038] FIG. 10 shows a gas chromatogram of the components obtained by subjecting solid 5 to subcritical water treatment 6 at 200° C. for 60 minutes, and FIG. 11 shows the mass spectrum of peak 1 in the gas chromatogram shown in FIG.

[0039] In this embodiment, solid 5 was then treated in subcritical 100% ethanol at 200°C for 60 minutes in the same manner as subcritical water treatment 6, except that the solvent in subcritical water treatment 6 was changed to 100% ethanol, and the components obtained by treating solid 5 in a subcritical state at 200°C for 60 minutes were analyzed by GC / MS. GC / MS was performed in exactly the same manner as in the GC / MS analysis of solid 5 shown in Figure 2, except that the components obtained by treating solid 5 with 100% ethanol in a subcritical water state at 200°C for 60 minutes were used as samples.

[0040] FIG. 12 shows a gas chromatogram of the components obtained by treating solid 5 with 100% ethanol in a subcritical water state at 200°C for 60 minutes, and FIG. 13 shows the mass spectrum of peak 1 in the gas chromatogram shown in FIG. 12.

[0041] In this embodiment, solid 5 was then treated in a subcritical ethanol-water mixed solvent at 200°C for 60 minutes in the same manner as in subcritical water treatment 6, except that the solvent in subcritical water treatment 6 was changed to an ethanol-water mixed solvent (100% ethanol:water=2:1 (volume ratio)). The components obtained by treating solid 5 in a subcritical state at 200°C for 60 minutes were then analyzed by GC / MS. GC / MS was performed in exactly the same manner as in the GC / MS analysis of solid 5 shown in FIG. 2, except that the components obtained by treating solid 5 in a subcritical water ethanol-water mixed solvent at 200°C for 60 minutes were used as samples.

[0042] FIG. 14 shows a gas chromatogram of the components obtained by treating solid 5 with a subcritical ethanol-water mixed solvent at 200°C for 60 minutes, and FIG. 15 shows the mass spectrum of peak 1 in the gas chromatogram shown in FIG. 14.

[0043] 11 and 15 clearly show that the components obtained by treating solid 5 with subcritical water or 100% ethanol at 200°C for 60 minutes contain a component corresponding to the peak with a mass-to-charge ratio (m / z) of 564. The component corresponding to the peak with a mass-to-charge ratio (m / z) of 564 in FIGS. 11 and 15 corresponds to the peak with a mass-to-charge ratio (m / z) of 564.5 in FIG. 7, i.e., is thought to correspond to ceramide (d16:1 / 16:0).

[0044] 13, it is clear that the component obtained by treating solid 5 with a subcritical ethanol-water mixed solvent at 200°C for 60 minutes contains a component corresponding to the peak with a mass-to-charge ratio (m / z) of 593. The component corresponding to the peak with a mass-to-charge ratio (m / z) of 593 in FIG. 13 is thought to correspond to the component corresponding to the peak with a mass-to-charge ratio (m / z) of 592.5 in FIG. 8, i.e., ceramide (d18:1 / 16:0).

[0045] As described above, although the components detected when water or 100% ethanol is used as the subcritical water solvent and when an ethanol-water mixed solvent is used are different, all of them are components that constitute the mass spectrum of the peaks in the gas chromatogram shown in Fig. 4. Therefore, it is clear that the production method of this embodiment can hydrolyze ceramide ciliatine regardless of whether water, 100% ethanol, or a water-ethanol mixed solvent is used as the subcritical water solvent.

[0046] In this embodiment, the treated product obtained by the subcritical water treatment 6 on the solid 5 is then placed in a separatory funnel, and a second mixed solvent is added to obtain a second mixed liquid 7. The second mixed solvent 11 is composed of hexane, ethanol, and water, and contains, for example, 100 parts by mass of ethanol, 100 to 200 parts by mass of hexane, and 3 to 50 parts by mass of water.

[0047] Next, the second mixed liquid 7 is stirred and then allowed to stand, whereby the second mixed liquid 7 separates into a hexane phase as an organic phase 8 and an ethanol-water phase 9 in the separatory funnel.

[0048] Here, organic phase 8 contains ceramide, and ethanol-aqueous phase 9 contains trace amounts of ceramide and 2-aminoethylphosphonic acid. Therefore, organic phase 8 and ethanol-aqueous phase 9 are separated, and organic phase 8 is evaporated to dryness to obtain ceramide 10. Organic phase 8 can be evaporated to dryness in the same manner as filtrate 4.

[0049] On the other hand, since ethanol-aqueous phase 9 contains trace amounts of ceramide and 2-aminoethylphosphonic acid, it is preferable to perform at least one operation of adding hexane to ethanol-aqueous phase 9, stirring, allowing to stand, separating again into a hexane phase as organic phase 8 and ethanol-aqueous phase 9, and evaporating organic phase 8 to dryness. By doing so, the ceramide contained in ethanol-aqueous phase 9 can be recovered, and the overall ceramide yield can be further improved.

[0050] In this embodiment, the organic phase 8 and the ethanol-aqueous phase 9 are separated using a separatory funnel. However, when carried out industrially, this can be done by a method such as countercurrent multistage extraction. [Explanation of symbols]

[0051] 1...fish meat, 2...first mixed solvent, 3...first mixed liquid, 4...solid (phospholipid), 6...subcritical water treatment, 7...second mixed liquid, 8...organic phase, 10...ceramide.

Claims

1. A step of stirring raw fish meat in a first mixed solvent consisting of hexane and ethanol to extract ceramide and ceramide ciliatin into the first mixed solvent; filtering the first mixed solvent to obtain a filtrate; The filtrate is evaporated to dryness under reduced pressure to obtain a mixture of ceramide and ceramide ciliatine; and treating the mixture of ceramide and ceramide ciliatine obtained by the drying process with a solvent in a subcritical state.

2. The method for producing ceramide according to claim 1, The method for producing ceramide, wherein the subcritical solvent is one solvent selected from the group consisting of water, ethanol, and a water-ethanol mixed solvent.

3. The method for producing ceramide according to claim 1 or 2, a step of adding a second mixed solvent consisting of hexane, ethanol and water to a treated product obtained by treating the mixture of ceramide and ceramide ciliatine with subcritical water, stirring the mixture, and then allowing it to stand, thereby separating the second mixed solvent into a hexane phase and an ethanol-water phase; and a step of drying the hexane phase to obtain ceramide.

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