Method for producing ceramide

Abstract

To provide a method for producing ceramide.SOLUTION: A method for producing ceramide includes the step (B) of bringing a sample containing phytoceramide 1-phosphate into contact with phytase. The pH of the reaction system in the step (B) is 2-6.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Ceramide is a type of sphingolipid, a general term for compounds in which a long-chain fatty acid is bonded to the amino group of sphingosine via an amide bond. While ceramide functions as a signaling substance for apoptosis and differentiation within cells, it also plays an important role in reducing water evaporation as a major lipid component in the stratum corneum of the skin, thereby forming a skin barrier and maintaining moisture. However, the amount of ceramide found in nature is very small, posing a challenge for industrial use.

[0003] Ceramides currently used in the cosmetics market can be broadly divided into three types: animal-derived ceramides, plant-derived ceramides, and synthetic ceramides. Animal-derived ceramides are produced from extracts of ceramides found in the brains and spinal cords of animals such as cows and horses. Because of concerns about infectious diseases such as mad cow disease, their use in food is questionable. Synthetic ceramides are chemically synthesized, making their safety difficult to ensure and unsuitable for food applications. For this reason, the most widely used ceramides are plant-derived ceramides, extracted from plants such as corn, konjac, and rice bran. However, the plant-derived ceramides currently available are ceramide precursors called glucosylceramides, in which sugars are bound to ceramides. Therefore, it is unclear whether they function as ceramides when applied to the skin. Therefore, there is a need for a method for producing ceramides that are not glycosides. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] FEBS Journal.280(2013), 3797-3809 [Non-patent document 2] J.Biochem.2017;161(2):187-195 [Non-patent document 3] J Nutr Sci Vitaminol, 2019;65:S175-S179 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention addresses the problem of providing a method for producing ceramide. [Means for solving the problem]

[0006] The present inventors have previously discovered an unknown phospholipid in cabbage lipids and determined its structure to be phytoceramide 1-phosphate (PC1P) (Non-Patent Document 1). They have also demonstrated that PC1P is produced by hydrolysis of the D-position of glycosylinositol phosphoceramide (GIPC) by the action of GIPC-specific phospholipase D (GIPC-PLD). Furthermore, the present inventors have reported that plant cells contain 50-280 nmol / g (wet wt) of GIPC and 3-60 nmol / g (wet wt) of PC1P (Non-Patent Document 2). Furthermore, the inventors have been conducting extensive research to establish a technology for extracting ceramide precursors from surplus agricultural by-products and converting them into ceramide. They discovered that treating PC1P with phytase can produce phytoceramide (PCer), a plant-derived ceramide that is not a glycoside, and have made further improvements.

[0007] The present disclosure encompasses, for example, the subject matter described in the following sections: Section 1. (B) contacting a sample containing phytoceramide 1-phosphate with phytase; A method for producing ceramide, wherein the pH of the reaction system in (B) is 2 to 6. Section 2. Before the step (B), Item 1. The method for producing a plant according to Item 1, comprising the step of: (A) contacting a water extract of the plant with glycosyl inositol phosphoceramide-specific phospholipase D to obtain a sample containing phytoceramide 1-phosphate. Section 3. Item 3. The production method according to Item 2, further comprising, before the step (A), a step of contacting the plant with an alcohol having 2 to 5 carbon atoms to obtain an insoluble matter. Section 4. Item 4. The production method according to any one of Items 1 to 3, further comprising, after step (B), a step of mixing a sample containing phytoceramide 1-phosphate contacted with phytase with a linear alkane having 5 to 8 carbon atoms and an alcohol having 1 to 5 carbon atoms, and recovering a linear alkane fraction having 5 to 8 carbon atoms. Section 5. Item 5. The production method according to any one of Items 2 to 4, wherein the glycosyl inositol phosphoceramide-specific phospholipase D is a phospholipase D derived from a plant of the Brassicaceae family. Section 6. Item 6. The method according to any one of Items 2 to 5, wherein the plant is a leafy or root vegetable, a fruit, or a mushroom. [Effects of the Invention]

[0008] A method for producing ceramides is provided. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows the results of evaluating the mixing ratio of a GIPC-PLD-containing enzyme solution and a GIPC solution. [Figure 2] 1 shows the results of evaluating the mixing ratio of a GIPC-PLD-containing enzyme solution and a GIPC solution. [Figure 3] The results of evaluating the digestibility of GIPC solutions prepared from various plants into PC1P using an enzyme solution containing GIPC-PLD are shown. [Figure 4] The results of evaluating the digestibility of cabbage-derived PC1P to PCer using phytase are shown. [Figure 5]The figure shows the amount of PCer produced when the digestibility of cabbage-derived PC1P to PCer was evaluated using phytase. [Figure 6] The results of evaluating the digestibility of cabbage-derived PC1P to PCer using acid phosphatase are shown. [Figure 7] 1 shows the results of evaluating the pH dependency of phytase on PC1P. [Figure 8] 1 shows the results of evaluating the temperature dependency of phytase on PC1P. [Figure 9] The results of extraction of PCer using hexane or heptane are shown. [Figure 10] The results of preparing PCer from cabbage using phytase are shown. [Figure 11] The figure shows the amount of PCer produced when PCer was prepared from cabbage using phytase. [Figure 12] The results of preparing PCer from cabbage using phytase are shown. [Figure 13] The results of preparing PCer from radish using phytase are shown. DETAILED DESCRIPTION OF THE INVENTION

[0010] Each embodiment included in the present disclosure will be described in further detail below.

[0011] The method for producing ceramide encompassed by the present disclosure preferably includes a step of contacting a sample containing phytoceramide 1-phosphate with phytase. In this specification, this production method may be referred to as the "production method of the present disclosure." In addition, in this specification, this step may be referred to as "step (B)" or the like.

[0012] Phytoceramide 1-phosphate (PC1P) is a plant-derived compound with a phosphate group bonded to the hydroxyl group at position 1 of ceramide. Ceramide is a type of sphingolipid, a general term for compounds in which a long-chain fatty acid is bonded to the amino group of sphingosine via an amide bond. The type of long-chain fatty acid constituting phytoceramide 1-phosphate used in the present disclosure is not particularly limited. For example, it may be a fatty acid having 12 to 28 carbon atoms. The long-chain fatty acid may or may not have a double bond. The number of double bonds in the long-chain fatty acid may be, for example, about 0 to 1.

[0013] The sample containing phytoceramide 1-phosphate may be in a liquid form (e.g., a solution, a suspension, etc.), a paste form, a solid form (e.g., a powder, a granule, etc.), etc. A liquid form is preferred.

[0014] A sample containing phytoceramide 1-phosphate can be obtained, for example, by contacting a sample containing glycosylinositol phosphoceramide (GIPC) (e.g., a water extract of a plant) with glycosylinositol phosphoceramide-specific phospholipase D, as described below. As described below, radishes contain glycosyl inositol phosphoceramide, and ground radish roots and stems (e.g., grated radish) contain glycosyl inositol phosphoceramide-specific phospholipase D. Therefore, in ground radish roots and stems (e.g., grated radish), glycosyl inositol phosphoceramide is thought to be converted to phytoceramide 1-phosphate by glycosyl inositol phosphoceramide-specific phospholipase D. Therefore, for example, ground radish roots and stems (e.g., grated radish) can also be used as a sample containing phytoceramide 1-phosphate for use in the present disclosure. For example, for example, ground radish roots and stems (e.g., grated radish) can also be used as a sample containing phytoceramide 1-phosphate for use in the present disclosure. For example, for example, samples containing glycosyl inositol phosphoceramide and glycosyl inositol phosphoceramide-specific phospholipase D (e.g., ground plant material) can also be used.

[0015] The amount of PC1P contained in the sample containing phytoceramide 1-phosphate used in the present disclosure is not particularly limited, and can be, for example, about 50 to 500 μM.

[0016] Phytase is an enzyme that hydrolyzes phosphate monoesters of phytic acid (inositol-6-phosphate) to release inorganic phosphorus. Phytases are classified into EC 3.1.3.8, EC 3.1.3.26, EC 3.1.3.62, and EC 3.1.3.72 depending on the position of the phosphate ester they hydrolyze. Any phytase can be used in the present disclosure. Among these, EC 3.1.3.8 is preferred.

[0017] The phytase used in the present disclosure is not particularly limited and may be one purified from a natural product or one that is commercially available. For example, it may be one that is available as a food additive. An example of a commercially available phytase is phytase manufactured by Shin-Nippon Chemical Industry Co., Ltd. (trade name: Sumiteam PHY).

[0018] The method for contacting a sample containing phytoceramide 1-phosphate with phytase is not particularly limited, and any method known in the art can be used, such as mixing a sample containing phytoceramide 1-phosphate with phytase in a solvent such as water. For example, when the sample containing phytoceramide 1-phosphate is in a liquid state, phytase can be added to the sample containing phytoceramide 1-phosphate.

[0019] When a sample containing phytoceramide 1-phosphate is brought into contact with phytase, the temperature of the reaction solution is not particularly limited as long as the phytase is not inactivated, but may be, for example, about 25 to 60°C, more preferably about 30 to 55°C, or may be, for example, about 35 to 50°C.

[0020] The time for which the sample containing phytoceramide 1-phosphate is brought into contact with the phytase is not particularly limited, but can be, for example, about 30 minutes to 24 hours, or about 3 to 24 hours.

[0021] When contacting a sample containing phytoceramide 1-phosphate with phytase, the pH of the reaction solution is not particularly limited as long as the phytase is not inactivated, but can be, for example, about 2 to 6, more preferably about 4 to 5.5, and even more preferably about 4 to 5.

[0022] When contacting a sample containing phytoceramide 1-phosphate with phytase, the blending ratio of the sample containing phytoceramide 1-phosphate to the phytase is not particularly limited, but can be, for example, approximately 100 to 10,000 parts by mass, or approximately 100 to 1,000 parts by mass of phytase per 1 part by mass of PC1P contained in the sample containing phytoceramide 1-phosphate.

[0023] When contacting a sample containing phytoceramide 1-phosphate with phytase, the concentration of the phytase to be added is not particularly limited, but can be, for example, about 0.05 to 8 mass%, about 0.1 to 5 mass%, about 0.5 to 5 mass%, or 1 to 5 mass% as the final concentration of phytase. For example, when a sample containing phytoceramide 1-phosphate is derived from a plant as described below, the amount of phytase to be added is not particularly limited, but can be, for example, approximately 0.001 to 0.1 parts by mass, or approximately 0.002 to 0.05 parts by mass, per 1 part by mass of wet mass of the plant.

[0024] When a sample containing phytoceramide 1-phosphate is contacted with phytase, a buffer can be further added. Examples of buffers include sodium hydrogen phosphate, sodium acetate, sodium citrate, and boric acid. These can be used alone or in combination. The concentration of the buffering agent is not particularly limited, but can be, for example, about 0.05 to 0.5M.

[0025] Ceramide (phytoceramide) can be produced from phytoceramide 1-phosphate by contacting a sample containing phytoceramide 1-phosphate with phytase.

[0026] The method for producing ceramide encompassed by the present disclosure preferably includes a step of contacting a water extract of a plant with glycosyl inositol phosphoceramide-specific phospholipase D to obtain a sample containing phytoceramide 1-phosphate. In this specification, this step may be referred to as "step (A)" or the like.

[0027] Glycosyl inositol phosphoceramide is a compound in which a uronic acid and a sugar are bound to the inositol residue of inositol phosphoceramide. Examples of the uronic acid include glucuronic acid. Examples of the sugar include hexoses such as mannose.

[0028] The plants used in the present disclosure are not particularly limited as long as they contain glycosyl inositol phosphoceramide.Furthermore, the parts of the plants used in the present disclosure (e.g., roots, leaves, seeds, rhizomes, fruits, skins (external and endodermal), flowers, etc.) are also not particularly limited as long as they contain glycosyl inositol phosphoceramide. For example, Non-Patent Document 2 reports that vegetables such as cabbage (root, inner leaves, outer leaves), radish (root, leaves), komatsuna (root, leaves), green onion (inner leaves), mung bean (root, seedling), burdock, carrot (root, leaves), and cucumber contain 50 to 280 nmol / g of GIPC per wet mass. Furthermore, Non-Patent Document 3 reports that cabbage (leaves), komatsuna (leaves), and lettuce (leaves) contain 10 to 20 mg / 100 g of GIPC; radish (root, leaves), carrot (root), burdock (root), and bean sprouts contain 7 to 9 mg / 100 g; cucumber, tomato, and potato contain 5 mg / 100 g or less; and onion and leek contain 6 to 8 mg / 100 g of GIPC. Examples of plants that can be used in the present disclosure include leafy stem vegetables such as cabbage, komatsuna, etc., Asteraceae such as lettuce, Amaryllidaceae such as leeks and onions, sprouts, etc.; root vegetables such as radish, carrot, burdock, etc.; fruit vegetables such as cucumber, tomato, etc.; tubers such as potatoes; fruits such as oranges; and mushrooms such as shiitake mushrooms. Among these, leafy stem vegetables and root vegetables are preferred. Of the leafy stem vegetables, those of the Brassicaceae family are more preferred. Particularly preferred vegetables are cabbage (e.g., inner leaves, outer leaves, etc.), radish (e.g., roots, stems, etc.), burdock (e.g., roots, etc.), and carrot (e.g., roots, stems, etc.). These can be used alone or in combination of two or more.

[0029] The water extract of a plant is not particularly limited as long as it is extracted with water from the above-mentioned plant. The water extract of a plant is not particularly limited, and may be one obtained by extracting a plant with water or a commercially available extract.

[0030] In other words, the manufacturing method of the present disclosure may further include a step of extracting the plant with water. The method of extracting the plant with water is not particularly limited, and for example, any method known in the art, such as the method of immersing the plant in water, can be used. When immersing the plant in water, heating, stirring, shaking, homogenization, etc. can be performed as necessary. These methods can be any method known in the art. The step of extracting the plant with water is not particularly limited, and can be repeated, for example, about 1 to 5 times. In addition, when the production method of the present disclosure includes the "step of contacting a plant with an alcohol having 2 to 5 carbon atoms to obtain an insoluble matter" described below, the obtained alcohol-insoluble matter can be extracted with water.

[0031] In the step of extracting the plant with water, the blending ratio of the plant to water is not particularly limited, but can be, for example, about 1 to 50 parts by mass of water per 1 part by mass of the plant (converted to dry mass).

[0032] In the step of extracting a plant with water, the temperature of the water is not particularly limited, but may be, for example, hot water (for example, about 70 to 100°C).

[0033] The time for extracting a plant with water is not particularly limited, but may be, for example, about 5 to 30 minutes.

[0034] After extracting the plant with water, the plant water extract can be obtained by removing residues, etc. The method for removing residues, etc. is not particularly limited, and any method known in the art, such as filtration, can be used.

[0035] The water extract of a plant used in the present disclosure may be in a liquid form such as a solution or suspension, or in a paste or solid form (e.g., powder, granules, etc.). A liquid form is preferred. For example, the water extract of a plant may be an extract obtained after water extraction, or may be a purified product, dried product, concentrate, etc. of the extract obtained after water extraction. These can be obtained by methods known in the art.

[0036] The concentration of GIPC contained in the water extract of the plant can be, for example, about 50 to 500 μM.

[0037] Glycosyl inositol phosphoceramide-specific phospholipase D is an enzyme that cleaves the phosphate bond in glycosyl inositol phosphoceramide. Here, "specific" means that the phospholipase D activity against glycosylinositolphosphoceramide is higher than the phospholipase D activity against lipids other than glycosylinositolphosphoceramide, such as glycerophospholipids like phosphatidylcholine and phosphatidylethanolamine; sphingomyelin, etc. For example, the phospholipase D activity against glycosylinositolphosphoceramide measured by the method described in Non-Patent Document 1 may be 2-fold, preferably 5-fold, etc. of the phospholipase D activity against lipids other than glycosylinositolphosphoceramide (more specifically, phosphatidylcholine). The method described in Non-Patent Document 1 is specifically as follows. <Method for Measuring GIPC-PLD Activity> Dissolve a certain amount of GIPC, the enzyme to be tested, and 3 mg of sodium deoxycholate in 0.2 M Tris / HCl buffer (pH 7.4), make the total volume 0.7 mL, and incubate with continuous stirring at 30°C. After incubating for 5 minutes or 30 minutes, extract the lipids in the reaction mixture by the Bligh & Dyer method, and separate the enzyme product PC1P by TLC. Quantify this PC1P and calculate the conversion rate of GIPC. <Method for Measuring Phosphatidylcholine (PC)-PLD Activity> Dissolve a certain amount of egg yolk PC, the enzyme to be tested, 0.1 mL of 0.1 M calcium chloride, and 1 mL of diethyl ether in 0.1 M acetate buffer (pH 5.6), make the total volume 1.7 mL, and incubate with continuous stirring at 30°C. After incubating for 5 minutes or 30 minutes, extract the lipids in the reaction mixture by the Bligh & Dyer method, and separate the enzyme product phosphatidic acid (PA) by TLC. Quantify this PA and calculate the conversion rate of PC.

[0038] The glycosylinositolphosphoceramide-specific phospholipase D used in the present disclosure is not particularly limited, such as those purified from natural products or commercially available ones. Non-patent document 2 reports that glycosyl inositol phosphoceramide-specific phospholipase D activity has been confirmed in cabbage (root, stem, inner leaves, outer leaves), radish (root, leaves), Japanese mustard spinach (root, leaves), broccoli (root, stem, flower), green onion (root, inner leaves, outer leaves), mung bean (radicle, hypocotyl, seedling), burdock, carrot (root), etc. The glycosyl inositol phosphoceramide-specific phospholipase D used in the present disclosure may be derived from a plant containing glycosyl inositol phosphoceramide-specific phospholipase D. Furthermore, the glycosyl inositol phosphoceramide-specific phospholipase D used in the present disclosure may be a ground product of a plant containing glycosyl inositol phosphoceramide-specific phospholipase D, or a purified product thereof, as long as it has glycosyl inositol phosphoceramide-specific phospholipase D activity. Glycosyl inositol phosphoceramide-specific phospholipase D activity is measured by the method described above. Examples of plants containing glycosyl inositol phosphoceramide-specific phospholipase D include Brassicaceae plants such as cabbage (more specifically, roots, stems, inner leaves, outer leaves, etc.), radish (more specifically, roots, stems, leaves), Japanese mustard spinach (more specifically, roots, leaves), and broccoli (more specifically, roots, stems, and flowers); Amaryllidaceae plants such as leeks (more specifically, roots, inner leaves, and outer leaves); Leguminosae plants such as mung beans (more specifically, roots, hypocotyls, and seedlings); Asteraceae plants such as burdock; and Apiaceae plants such as carrots (more specifically, roots). Among these, Brassicaceae plants are preferred, with roots or stems of Brassicaceae plants being more preferred, and radish roots or stems being particularly preferred. Examples of pulverized plants containing glycosyl inositol phosphoceramide-specific phospholipase D include the pulverized plants described above, and the purified product is preferably the liquid portion of the pulverized plant. Specifically, pulverized radish roots or stems (e.g., grated radish) are preferred, with the liquid portion of pulverized radish roots or stems being particularly preferred. These can be used alone or in combination of two or more.

[0039] The method for crushing a plant containing glycosyl inositol phosphoceramide-specific phospholipase D is not particularly limited, and any method known in the art, such as grinding the plant or crushing it in a mixer, can be used.

[0040] The method for contacting the aqueous extract of the plant with glycosyl inositol phosphoceramide-specific phospholipase D is not particularly limited, and any method known in the art can be used, such as mixing the aqueous extract of the plant with glycosyl inositol phosphoceramide-specific phospholipase D in a solvent such as water.

[0041] When contacting a water extract of a plant with glycosyl inositol phosphoceramide-specific phospholipase D, the temperature of the reaction solution is not particularly limited as long as glycosyl inositol phosphoceramide-specific phospholipase D is not inactivated, and can be, for example, about 20 to 50°C.

[0042] The time for which the water extract of the plant is brought into contact with glycosyl inositol phosphoceramide-specific phospholipase D is not particularly limited, but can be, for example, about 5 to 120 minutes.

[0043] When contacting a water extract of a plant with glycosyl inositol phosphoceramide-specific phospholipase D, the pH of the reaction solution is not particularly limited as long as glycosyl inositol phosphoceramide-specific phospholipase D is not inactivated, but can be, for example, about 5 to 8.

[0044] When contacting a water extract of a plant with glycosyl inositol phosphoceramide-specific phospholipase D, the blending ratio of the water extract of the plant to the glycosyl inositol phosphoceramide-specific phospholipase D is not particularly limited and can be appropriately set depending on, for example, the concentration of the water extract of the plant, the concentration of glycosyl inositol phosphoceramide-specific phospholipase D, etc. For example, when the liquid portion of crushed radish roots and stems is used as glycosyl inositol phosphoceramide-specific phospholipase D, the blending ratio is 1 part by mass of glycosyl inositol phosphoceramide contained in the water extract of the plant to 3 x 10 parts by mass of the liquid portion of crushed radish roots and stems. 3 ~100×10 3 It can be about parts by mass, etc. For example, when the liquid portion of ground radish roots and stems is used as glycosyl inositol phosphoceramide-specific phospholipase D, the amount of the liquid portion of ground radish roots and stems is not particularly limited, but can be, for example, about 0.01 to 2 parts by mass, or about 0.05 to 1 part by mass, per 1 part by mass of the wet mass of the plant from which the water extract of the plant is derived.

[0045] When the water extract of a plant is brought into contact with glycosyl inositol phosphoceramide-specific phospholipase D, a surfactant can be further added. Examples of surfactants include deoxycholic acid and salts thereof. Examples of salts of deoxycholic acid include sodium salt and potassium salt of deoxycholic acid. These surfactants can be used alone or in combination of two or more. The concentration of deoxycholic acid or a salt thereof is not particularly limited, but can be, for example, about 0.01 to 0.1% by mass.

[0046] When the water extract of a plant is brought into contact with glycosyl inositol phosphoceramide-specific phospholipase D, diethyl ether can be further added. The concentration of diethyl ether to be added is not particularly limited, but can be, for example, about 0.1 to 0.5% by volume as a final concentration.

[0047] When the water extract of the plant is brought into contact with glycosyl inositol phosphoceramide-specific phospholipase D, a chelating agent can be further added. Examples of the chelating agent include calcium chelating agents. Examples of calcium chelating agents include citric acid and EDTA. These can be used alone or in combination of two or more. The concentration of the chelating agent is not particularly limited, but can be, for example, about 1 mM to 200 mM. Without wishing to be bound by theory, it is expected that the incorporation of a chelating agent can inhibit phosphatidylcholine phospholipase D activity.

[0048] By contacting a water extract of a plant with glycosyl inositol phosphoceramide-specific phospholipase D, phytoceramide 1-phosphate can be produced from glycosyl inositol phosphoceramide contained in the water extract of a plant.

[0049] After contacting the water extract of the plant with glycosyl inositol phosphoceramide-specific phospholipase D, a heat treatment may be performed. The heating temperature and heating time are not particularly limited, as long as they are sufficient to inactivate glycosyl inositol phosphoceramide-specific phospholipase D. For example, the heating temperature can be about 70 to 100°C, or about 80 to 100°C. For example, the heating time can be about 3 to 15 minutes, or about 5 to 10 minutes.

[0050] Preferably, the production method of the present disclosure further comprises, after step (B), a step of mixing a sample containing phytoceramide 1-phosphate that has been contacted with phytase with a linear alkane having 5 to 8 carbon atoms and an alcohol having 1 to 5 carbon atoms, and recovering a linear alkane fraction having 5 to 8 carbon atoms. For example, after step (B), the sample containing phytoceramide 1-phosphate that has been contacted with phytase may be purified, concentrated, diluted, etc. These methods can be performed by methods known in the art.

[0051] Examples of linear alkanes having 5 to 8 carbon atoms include pentane, hexane, heptane, octane, etc. Among these, n-hexane and n-heptane are preferred.

[0052] Examples of alcohols having 1 to 5 carbon atoms include methanol, ethanol, 1-propanol, 2-propanol (isopropanol), 1-butanol, 2-butanol, 1-pentanol, and 2-pentanol. Of these, methanol and ethanol are preferred. These may be used alone or in combination of two or more.

[0053] The mixing ratio of the sample containing phytoceramide 1-phosphate contacted with phytase to the linear alkane having 5 to 8 carbon atoms and the alcohol having 1 to 5 carbon atoms can be, for example, about 1:1:1 to 1:3:1 in terms of the volume ratio of linear alkane having 5 to 8 carbon atoms: alcohol having 1 to 5 carbon atoms: sample containing phytoceramide 1-phosphate after contact with phytase and water (aqueous solution), and preferably 1:2:1.

[0054] The method for mixing a sample containing phytoceramide 1-phosphate that has been contacted with phytase with a linear alkane having 5 to 8 carbon atoms and an alcohol having 1 to 5 carbon atoms is not particularly limited, and any method known in the art can be used.

[0055] When a sample containing phytoceramide 1-phosphate that has been contacted with phytase is mixed with a linear alkane having 5 to 8 carbon atoms and an alcohol having 1 to 5 carbon atoms, the temperature of the reaction solution is not particularly limited, but can be, for example, approximately 4 to 40°C, approximately 4 to 30°C, etc.

[0056] The time for mixing a sample containing phytoceramide 1-phosphate that has been contacted with phytase with a linear alkane having 5 to 8 carbon atoms and an alcohol having 1 to 5 carbon atoms is not particularly limited, but can be, for example, about 5 to 30 minutes.

[0057] The method for collecting the C5-8 linear alkane fraction after mixing the sample containing phytoceramide 1-phosphate that has been contacted with phytase with a linear alkane having 5 to 8 carbon atoms and an alcohol having 1 to 5 carbon atoms is not particularly limited, and any method known in the art can be used. By collecting the C5-8 linear alkane fraction, ceramide (phytoceramide) can be collected.

[0058] The production method of the present disclosure may include a step of contacting a plant with hot water. This step can inactivate endogenous lipolytic enzymes contained in the plant. This step can be included, for example, before step (A).

[0059] The form of the plant is not particularly limited, and may be, for example, the plant itself or a cut plant.

[0060] The method for contacting the plant with hot water is not particularly limited, and any method known in the art, such as immersing the plant in hot water, can be used. When immersing, stirring, shaking, etc. can be performed as necessary.

[0061] When the plant is brought into contact with hot water, the temperature of the reaction solution is not particularly limited, but can be, for example, about 70 to 100°C.

[0062] The time for which the plant is in contact with hot water is not particularly limited, but can be, for example, about 1 to 30 minutes.

[0063] The production method of the present disclosure may include a step of drying the plant after the step of contacting the plant with hot water. The drying method is not particularly limited, and any method known in the art can be used.

[0064] The manufacturing method of the present disclosure may include a step of crushing the plant. The crushing method is not particularly limited, and any method known in the art can be used. For example, the crushing step may include crushing the plant after the step of contacting the plant with hot water, after the step of drying the plant, or before step (A). The form of the plant after pulverization is not particularly limited, and may be, for example, a paste, a solid (for example, powder, granules, etc.), a liquid (solution, suspension, etc.), or the like.

[0065] The production method of the present disclosure may include, prior to step (A), a step of contacting the plant with an alcohol having 2 to 5 carbon atoms to obtain an insoluble matter. This step makes it possible to remove, from among the components contained in the plant, components that are soluble in the alcohol having 2 to 5 carbon atoms.

[0066] Examples of alcohols having 2 to 5 carbon atoms include ethanol, 1-propanol, 2-propanol (isopropanol), 1-butanol, 2-butanol, 1-pentanol, and 2-pentanol. Among these, ethanol, isopropanol, and 1-butanol are preferred, and ethanol is more preferred. These may be used alone or in combination of two or more.

[0067] When a plant is brought into contact with an alcohol having 2 to 5 carbon atoms, the blending ratio of the plant to the alcohol having 2 to 5 carbon atoms is not particularly limited, but can be, for example, about 1 to 20 parts by mass of the alcohol having 2 to 5 carbon atoms per 1 part by mass of the plant (converted to dry mass).

[0068] The method for contacting the plant with the alcohol having 2 to 5 carbon atoms is not particularly limited, and any method known in the art can be used, such as immersing the plant in the alcohol having 2 to 5 carbon atoms. When immersing, stirring, shaking, etc. can be performed as necessary.

[0069] When a plant is brought into contact with an alcohol having 2 to 5 carbon atoms, the temperature of the reaction solution is not particularly limited, but can be, for example, about 20 to 70°C, or about 20 to 50°C.

[0070] The time for which the plant is brought into contact with the alcohol having 2 to 5 carbon atoms is not particularly limited, but can be, for example, about 5 to 30 minutes.

[0071] The number of times that the plant is brought into contact with the alcohol having 2 to 5 carbon atoms is not particularly limited, but can be, for example, about 1 to 5 times.

[0072] The method for recovering the insoluble matter after contacting the plant with the alcohol having 2 to 5 carbon atoms is not particularly limited, and any method known in the art can be used.

[0073] The following is an example of one embodiment. contacting the plant with hot water; drying the plant that has been contacted with hot water; grinding the dried plants; A step of contacting the crushed plant with an alcohol having 2 to 5 carbon atoms to obtain insoluble matter. A step of extracting the alcohol-insoluble matter with water; contacting the aqueous extract with glycosyl inositol phosphoceramide-specific phospholipase D to obtain a sample containing phytoceramide 1-phosphate; contacting a sample containing phytoceramide 1-phosphate with phytase; and a step of mixing a sample containing phytoceramide 1-phosphate contacted with phytase with a linear alkane having 5 to 8 carbon atoms and an alcohol having 1 to 5 carbon atoms, and recovering a linear alkane fraction having 5 to 8 carbon atoms; Including, A method for producing ceramide. Another embodiment is exemplified below. contacting the plant with hot water; drying the plant that has been contacted with hot water; grinding the dried plants; A step of extracting the dried plant with water; contacting the aqueous extract with glycosyl inositol phosphoceramide-specific phospholipase D to obtain a sample containing phytoceramide 1-phosphate; contacting a sample containing phytoceramide 1-phosphate with phytase; and a step of mixing a sample containing phytoceramide 1-phosphate contacted with phytase with a linear alkane having 5 to 8 carbon atoms and an alcohol having 1 to 5 carbon atoms, and recovering a linear alkane fraction having 5 to 8 carbon atoms; Including, A method for producing ceramide. Another embodiment is exemplified below. crushing the plant to obtain a sample containing phytoceramide 1-phosphate; contacting a sample containing phytoceramide 1-phosphate with phytase; and a step of mixing a sample containing phytoceramide 1-phosphate contacted with phytase with a linear alkane having 5 to 8 carbon atoms and an alcohol having 1 to 5 carbon atoms, and recovering a linear alkane fraction having 5 to 8 carbon atoms; Including, A method for producing ceramide.

[0074] According to the production method of the present disclosure, phytoceramide, which is a plant-derived ceramide that is not a glycoside, can be produced from agricultural products (more specifically, agricultural by-products). Phytoceramide (ceramide) obtained by the production method of the present disclosure can be used, for example, in foods and beverages, cosmetics, pharmaceuticals, etc.

[0075] It should be noted that in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Furthermore, the present disclosure encompasses all arbitrary combinations of the constituent elements described in this specification.

[0076] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0077] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these in any way. In the following, unless otherwise specified, experiments were performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "% by mass."

[0078] I.Consideration of converting GIPC to PC1P Experimental materials Commercially available cabbage (Brassica oleracea L) and radish (Raphanus sativus L) were used. Malachite green (oxalate), perchloric acid, hexaammonium heptamolybdate pentahydrate (powder), 28% aqueous ammonia, and hydrochloric acid were purchased from Kanto Chemical Co., Ltd. Tween 20 (Polyoxyethylene Sorbitan Monolaurate) and primulin were purchased from Nacalai Tesque. Thin-layer chromatography (TLC) glass plates (5721-20 x 20 cm, 60 Å) were purchased from Merck Ltd. Special-grade organic solvents were purchased from Kanto Chemical.

[0079] 1. Extraction of lipids (phospholipids) from cabbage Preparation of cabbage powder First, to inactivate the lipolytic enzymes in the cabbage, the cabbage was boiled in a water bath at 80-90°C for 5 minutes. The boiled cabbage was spread out on newspaper without overlapping and thoroughly dried in a place out of direct sunlight. It was further dried in a desiccator, crushed in a tea mill, and ground to a fine powder in a mortar to obtain cabbage powder.

[0080] Ethanol washing 0.5 g of cabbage powder was weighed into a 15 mL plastic tube. 5 mL of ethanol was added and the mixture was stirred at 60°C for 15 minutes using a concentrator. The mixture was centrifuged (1,100 x g, 5 minutes) and the supernatant was removed. This procedure was repeated three times. The mixture was then transferred to a centrifuge tube and the remaining ethanol was removed using an evaporator.

[0081] hot water extraction 0.5 g of cabbage powder or insoluble matter after ethanol washing was added with 5 mL of hot water at 80°C and homogenized. Then, hot water was added to the filtrate so that the total volume was 10 mL, and the mixture was filtered through a nonwoven fabric to obtain a GIPC solution.

[0082] Butanol Extraction After hot water extraction, 3 mL of the filtrate was placed in a test tube, 3 mL of butanol was added, and the mixture was thoroughly stirred. The mixture was then centrifuged (1,100 × g, 4 °C, 5 min) to obtain the supernatant. The supernatant was then collected, and 3 mL of butanol was added to the remaining mixture, which was thoroughly stirred and centrifuged again. The supernatant was then collected and evaporated to dryness using an evaporator.

[0083] Extraction and isolation of GIPCs from cabbage The dried butanol extract was dissolved in 500 μL of solvent A (2-propanol:hexane:HO = 110:40:50 (v / v / v)), and 100 μL of this solution was applied as a strip to the bottom 2 cm of a TLC plate preactivated at 100°C for 1 hour. A GIPC standard was also applied. The plate was placed in a developing chamber saturated with a developing solvent consisting of chloroform:methanol:28% aqueous ammonia = 45:35:10 (v / v / v) and developed. When the plate reached the top, it was removed from the developing chamber and air-dried. Primulin reagent was sprayed onto the plate, and color development was achieved by UV irradiation (365 nm). The silica gel corresponding to the GIPC band was scraped off with a scraper, and 2 mL of solvent A was added to the scraped silica gel. Centrifugation (1100×g, 4° C., 5 min) was carried out, and the solvent was removed from the resulting supernatant under a nitrogen stream, followed by quantification by the method described below.

[0084] Phospholipid quantification The organic phosphorus content was determined according to the method of Chalavardjian and Rudnicki (Chalavardjian A et al., Anal Biochem. 1970 Jul;36(1):225-6). A fixed amount of the sample solution containing phospholipids was dispensed into a test tube, and the solvent was evaporated under a stream of nitrogen. 0.1 mL of 60% perchloric acid and 0.1 mL of water were added, and the mixture was heated at 170°C for 90 minutes. After cooling to room temperature, 1 mL of water, 5 mL of 4.2% ammonium molybdate-malachite green reagent, and 0.2 mL of 1.5% Tween 20 solution were added. After thorough mixing, the absorbance at 660 nm was measured colorimetrically using a Shimadzu UV-1600 spectrophotometer.

[0085] When the amount of phospholipids extracted from dried cabbage by hot water extraction alone was compared with that extracted after washing with ethanol prior to hot water extraction, the total amount of phospholipids per gram of dried cabbage was 304 μg Pi when hot water extraction alone was performed. On the other hand, when coexisting lipids were washed away with ethanol before hot water extraction, the total amount of phospholipids per gram of dried cabbage was 77 μg Pi, indicating that the amount of total phospholipids extracted was reduced by washing with ethanol. In addition, the amount of GIPC was almost the same when only hot water extraction was performed, at 13 μg Pi per 1 g of dried cabbage, and when ethanol washing was performed, at 14 μg Pi per 1 g of dried cabbage. These results demonstrate that when hot water extraction was performed alone, the proportion of GIPCs in total phospholipids was approximately 4%, whereas when ethanol washing was performed, this increased to approximately 18%, confirming that GIPCs can be concentrated and extracted by ethanol washing.

[0086] 2. Preparation of GIPC-PLD-containing enzyme solution Since radish contains GIPC-PLD (Non-Patent Document 2), the liquid portion of grated radish was used as a GIPC-PLD-containing enzyme solution (sometimes referred to as radish enzyme solution, Japanese radish enzyme solution, etc.).

[0087] 3. Preparation of PC1P from GIPC extracted from cabbage (substrate concentration dependence) Dried cabbage was washed with ethanol and then extracted with hot water to obtain GIPC solution (GIPC 58 μg Pi / 60 mL (31 μM), equivalent to 2 g cabbage powder / 60 mL). 0 mL, 2.5 mL, 5 mL, 10 mL, and 15 mL aliquots were placed in plastic tubes. Water was added to bring the total volume of GIPC solution (cabbage solution) and water (double distilled water; DDW) to 15 mL. To each tube, 1 mL of GIPC-PLD-containing enzyme solution and 750 μL of sodium deoxycholate (NaDOC) were added. The tubes were vortexed and incubated at 30°C for 30 minutes (stirring every 5 minutes). The reaction was then terminated by heating at 80°C for 5 minutes to obtain PC1P solution. Each solution was transferred from the plastic tube to a glass centrifuge tube, and an appropriate amount of chloroform and methanol was added to form a biphasic solvent [chloroform:methanol:HO = 1:1:0.9 (v / v / v)]. The mixture was then thoroughly stirred. After centrifugation (1,100 × g, 4°C, 5 min), the lower organic layer was transferred to another centrifuge tube. The remaining upper layer was mixed with the same amount of chloroform as before, mixed thoroughly, and then centrifuged (1,100 × g, 4°C, 5 min). The lower layer was again transferred to the previous centrifuge tube. The resulting organic layer was evaporated to dryness under reduced pressure using a rotary evaporator. A 25 μL solution was made up to 250 μL with chloroform:methanol = 2:1 (v / v). A 25 μL aliquot was applied as a strip to the bottom 2 cm of a TLC plate preactivated at 100°C for 1 hour. The plate was then placed in a developing chamber saturated with a developing solvent consisting of chloroform:methanol:28% aqueous ammonia = 60:35:8 (v / v / v). Once the plate reached the top, it was removed from the developing chamber and allowed to air dry. Primulin reagent was sprayed onto the plate, and color development was performed under UV irradiation (365 nm). The color development results are shown in Figure 1. As a result, it was confirmed that PC1P was produced in a substrate concentration-dependent manner. The PC1P band was scraped off and lipids were extracted using the Bligh & Dyer method, after which quantification was performed using the phospholipid quantification method described above.The conversion rate from GIPC to PC1P was calculated based on the amount of GIPC (calculated value) contained in the GIPC solution and the quantified amount of PC1P, and the conversion rates were 65.8% (2.5 mL of GIPC solution) and 77.3% (10 mL of GIPC solution).

[0088] 4. Preparation of PC1P from GIPC extracted from cabbage (investigation of the amount of enzyme solution containing GIPC-PLD) Two heads of cabbage purchased from a supermarket were treated with hot water (80°C, 10 minutes). 1140g of the cabbage leaves were dried at 60°C for 16 hours and ground into a powder using a blender (dry mass 57g). 200mL of ethanol was added to this powder, and the mixture was heated at 60°C while stirring for 30 minutes. After centrifugation (1300g, 5 minutes), the supernatant was removed and defatted. This procedure was repeated twice more (100mL of ethanol was used from the second time onwards). 800mL of hot water (80°C, 800mL) was added to the defatted cabbage leaves, homogenized using an Ultra Disperser, and filtered through gauze while adding hot water to obtain 1140mL of GIPC solution (GIPC approximately 3.6μgPi / 3.6mL (32μM) (back-calculated from the amount of PC1P produced)). 3.6 mL of cabbage-derived GIPC solution, 0 to 0.6 mL of GIPC-PLD-containing enzyme solution, 0.4 mL of 1 M citrate buffer (pH 6.8), and 0.2 mL of 1 g / 50 mL sodium deoxycholate (NaDOC) were added and incubated at 30°C for 60 minutes, after which the reaction was stopped at 80°C for 5 minutes. An appropriate amount of chloroform and methanol was added, the pH adjusted to 2 with hydrochloric acid, and lipids were extracted by the Bligh & Dyer method. One-fifth of the extract was spotted on a TLC plate for development. The developing solution used was chloroform:methanol:28% ammonia (60:35:8, v / v). The results are shown in Figure 2. The numbers on the gel photograph in Figure 2 indicate the amount of PC1P (nmol, quantitative values ​​for non-standard samples).

[0089] As shown in Figure 2, it was confirmed that PC1P increased depending on the amount of GIPC-PLD-containing enzyme solution. It was also found that 45 nmol of PC1P could be obtained from 1 g (wet weight) of cabbage.

[0090] 5. Preparation of PC1P from GIPC Extracted from Various Plants Daikon radish, carrot, burdock, orange peel, and shiitake mushroom were purchased from a supermarket. 30 g of each was heated at 80°C for 5 minutes to inactivate the enzymes, then frozen at -80°C. The resulting material was pulverized with a hammer, added with hot water, homogenized using an Ultra Disperser, and filtered through gauze to obtain GIPC solution (50 mL). The GIPC concentrations in the GIPC solution (back-calculated from the amount of PC1P produced) were approximately 20 μM for orange, 40 μM for daikon radish, 20 μM for burdock, 20 μM for shiitake mushroom, and 60 μM for carrot. 5 mL of GIPC solution (equivalent to 3 g of starting material), 0.5 mL of 1 M citrate buffer (pH 6.8), 0.25 mL of deoxycholic acid solution (1 g / 50 mL), and 1 mL of GIPC-PLD-containing enzyme solution were added and incubated at 30°C for 1 hour. After inactivation at 90°C for 5 minutes, lipids containing PC1P were extracted using the Bligh & Dyer method, and 1 / 5 of the sample (equivalent to 0.6 g of starting material) was spotted on a TLC column and developed with chloroform:methanol:28% ammonia (60:35:8, v / v). The results are shown in Figure 3. Note that the numbers on the gel photograph in Figure 3 indicate the amount of PC1P (nmol, quantitative values ​​except for the standard value).

[0091] From this experiment, it was found that 44 nmol (73 nmol / g) of PC1P could be obtained from 0.6 g (wet mass) of radish, 27 nmol (45 nmol / g) from 0.6 g (wet mass) of orange peel, 32 nmol (53 nmol / g) from 0.6 g (wet mass) of burdock, 33 nmol (55 nmol / g) from 0.6 g (wet mass) of shiitake mushroom, and 76 nmol (127 nmol / g) from 0.6 g (wet mass) of carrot.

[0092] II. Consider converting PC1P to PCer 6. Preparation of PCer from Cabbage-derived PC1P using Acid Phosphatase or Phytase Cabbage-derived PC1P solution obtained by treatment with a GIPC-PLD-containing enzyme solution using the method described in 4 above was reacted with acid phosphatase (Sumiteam PM, EC 3.1.3.2, Shin-Nippon Chemical Industry) or phytase (Sumiteam PHY, EC 3.1.3.8, Shin-Nippon Chemical Industry), both of which are used as food additives. Acid phosphatase, a phosphate monoester hydrolase, is a dephosphorylating enzyme that hydrolyzes phosphate monoesters or polyphosphate compounds to convert them into phosphate and compounds with a hydroxyl group, and has an optimum pH on the acidic side (pH 7 or lower).

[0093] 16 nmol of cabbage-derived PC1P solution, 1.0 mL of 0.1 M citrate buffer (pH 5.0), and 0.5-50 mg of Sumiteam PHY (phytase) (final concentration: approximately 0.05-5% by mass) were added and incubated at 40°C for 60 minutes. After incubation at 80°C for 5 minutes, the reaction was stopped to obtain PCer solution. An appropriate amount of chloroform and methanol were added, and the pH was adjusted to 2 with hydrochloric acid. Lipids were extracted by the Bligh & Dyer method, and 1 / 5 of the solution was spotted and developed on a TLC column. The developing solutions are as follows: First layer: chloroform:methanol:28% ammonia = (60:35:8, v / v) Second layer: petroleum ether: diethyl ether: acetic acid = (60:40:1, v / v) The results of the TLC analysis are shown in FIG. 4, and the amount of PCer quantified from the spot intensity of the TLC analysis is shown in FIG.

[0094] The reaction was carried out in the same manner as above, except that 0.1 M citrate buffer (pH 5.0) was replaced with 0.1 M citrate buffer (pH 4, 5, 6), and 25 mg of Sumiteam PM was used as the acid phosphatase instead of 0.5 to 50 mg of Sumiteam PHY (phytase). The results are shown in Figure 6.

[0095] As shown in Figures 4 and 5, digestion of PC1P to PCer was confirmed when 0.5 to 50 mg of phytase was used. When 25 mg of phytase was used, almost no PC1P remained, indicating complete conversion to PCer. On the other hand, when acid phosphatase was used, no PCer was produced at any pH, and PC1P remained (Figure 6).

[0096] 7. Phytase Activity Measurement The pH and temperature dependence of phytase activity on the substrate PC1P was confirmed. Cabbage GIPC was converted to PC1P by the action of the radish enzyme GIPC-PLD, and PC1P was purified using TLC. This cabbage-derived purified PC1P was used in this experiment. 0.8 μg of purified PC1P (Pi) was placed in a glass test tube, and the solvent was evaporated under a stream of nitrogen. 1.0 mL of 0.1 M citrate buffer (pH 4-6), 0.05 M Tris-HCl buffer (pH 7.5), and 0.045 M ethylamino alcohol (pH 9.9) was added and vigorously stirred. 25 mg of phytase was added and vigorously stirred using a concentrator at 30°C for 60 minutes. After completion of the reaction, the reaction solution was heated at 80°C for 10 minutes to terminate the reaction, and lipids were recovered by the Bligh and Dyer method. One-fifth of the obtained lipids was dissolved in a small amount of chloroform:methanol (2:1, v / v), and one-fifth of this solution was applied to a TLC column for two-layer development (first layer: chloroform:methanol:28% ammonia = (60:35:8, v / v); second layer: petroleum ether:diethyl ether:acetic acid = (60:40:1, v / v)) to separate the lipids. The color intensity of the detected PCer band was quantified (nmol) by comparing it with the color intensity of the band of a known amount of standard PCer. This was expressed as nmol / sec / mg protein. The results are shown in Figure 7.

[0097] 7, PCer production was confirmed at pH 4 to 6, but not at pH 7.5 or 9.9. It was confirmed that a particularly large amount of PCer was produced at pH 4 to 5.

[0098] 0.5 μg of purified PC1P (Pi) was placed in a glass test tube, the solvent was evaporated under a stream of nitrogen, and 1.0 mL of 0.1 M acetate buffer (pH 5.6) was added and vigorously stirred. 25 mg of phytase was added, and the mixture was vigorously stirred using a concentrator at 40°C, 45°C, 50°C, and 55°C for 60 minutes. After completion of the reaction, the reaction was stopped by heating at 80°C for 10 minutes, and lipids were recovered by the Bligh and Dyer assay. One-fifth of the resulting lipids was dissolved in a small amount of chloroform:methanol (2:1, v / v). One-fifth of this solution was applied to a TLC column and separated by two-layer development (first layer: chloroform:methanol:28% ammonia = (60:35:8, v / v); second layer: petroleum ether:diethyl ether:acetic acid = (60:40:1, v / v)). The color intensity of the detected PCer band was quantified (nmol) by comparing it with the color intensity of a known amount of standard PCer. This was expressed as nmol / sec / mg protein. The results are shown in Figure 8.

[0099] As shown in Figure 8, the production of PCer was confirmed at temperatures between 40°C and 55°C. It was confirmed that the amount of PCer produced was particularly large at temperatures between 40 and 50°C.

[0100] 8. Extraction of PCer with hexane or heptane The PCer solution prepared by the method described in 6 above was centrifuged (2500 rpm, 10 minutes) to obtain a precipitate. 0.4 volumes of ethanol and 0.2 volumes of hexane (1:2:1 ratio) were added to 0.2 volumes of this liquid precipitate (PCer solution), and a two-layer partition was performed (centrifugation). The hexane layer was separated, and the separated amount of hexane was added again, and a two-layer partition was performed (centrifugation). The resulting hexane was added to the original hexane and concentrated using an evaporator to obtain a purified PCer. A portion of this purified PCer was separated and analyzed by TLC using the two-phase development described above. The results are shown in Figure 9. For comparison, the analysis results of lipids extracted by the Bligh & Dyer method are also shown. The rightmost lane in Figure 9 represents 10 nmol of PCer.

[0101] As shown in Figure 9, PCer was extracted more efficiently using hexane and ethanol than when fractionated with hexane alone (hexane:water = 1:1). Furthermore, a solvent ratio of hexane:ethanol:water = 1:2:1 was confirmed to extract PCer more efficiently and with fewer impurities. Similar results were obtained when methanol was used instead of ethanol. Furthermore, the color intensity of the detected PCer band was quantified (nmol) by comparing it with the color intensity of a known amount of standard PCer. The amount of PCer obtained using this method from 0.6 g of cabbage (wet mass) was calculated to be 40 nmol. It was found that 70 nmol of PCer was contained per 1 g (wet mass). Since the MW of PCer is 680, the amount of PCer obtained in this experiment was 48 μg / g (4.8 mg / 100 g). The purified PCer obtained in this experiment was thoroughly dried and its mass was measured. 20 mg of purified PCer was obtained from 100 g of cabbage (wet mass). Based on this and the previous quantitative value of PCer, the purity of PCer was calculated to be 24% (4.8 mg / 20 mg). Similar results were obtained when n-heptane (Hep) was used instead of n-hexane (Figure 9).

[0102] Furthermore, the obtained PCer was confirmed to be a ceramide containing C16:0, C22:0, C24:0, and C24:1 fatty acids by liquid chromatography-mass spectrometry.

[0103] III. Preparation of PCer from plants 9. Preparation of PCer from Cabbage 500 mL of cabbage-derived GIPC solution (approximately 100 μM (back-calculated from the amount of PCer produced)) prepared by the method described in 4 above was mixed with 300 mL of GIPC-PLD-containing enzyme solution, 50 mL of 1 M citrate buffer (pH 6.8), and 25 mL of 1 g / 50 mL sodium deoxycholate, and the mixture was incubated at 30°C for 60 minutes, followed by quenching at 80°C for 5 minutes. An appropriate amount of citric acid monohydrate was added to adjust the pH to 5, yielding 875 mL of PC1P solution (approximately 100 μM (back-calculated from the amount of PCer produced)). 25 mL of cabbage-derived PC1P solution was added to 0-0.5 g of phytase (final concentration approximately 0-2% by mass). The mixture was incubated at 40°C for 16 hours, then the reaction was stopped at 80°C for 5 minutes to obtain PCer solution. The mixture was centrifuged (1300 g, 5 minutes) and the supernatant was removed. Water was added to the liquid sediment to make a 10 mL solution. 4 mL of ethanol and 2 mL of n-hexane were added to 2 mL of this mixture, stirred, and centrifuged (1300 g, 5 minutes) to obtain the n-hexane layer. 2 mL of n-hexane was added to the remaining lower layer, stirred, and centrifuged (1300 g, 5 minutes). The n-hexane layer was then combined with the previous n-hexane layer to obtain the purified PCer. 1 / 116 of the purified PCer was spotted on a TLC column and developed into two phases. The developing solutions were as follows: First layer: chloroform:methanol:28% ammonia = (60:35:8, v / v) Second layer: petroleum ether: diethyl ether: acetic acid = (60:40:1, v / v) The results of the TLC analysis are shown in Figure 10, and the amount of PCer quantified from the spot intensity of the TLC analysis is shown in Figure 11. In Figure 10, the numbers in the center of the gel photograph indicate the amount of PCer (nmol, quantitative values ​​for values ​​other than std).

[0104] These results showed that when 10 mg of phytase was applied to 1 g of cabbage (wet mass), 160 nmol (0.1 mg) of PCer was obtained.

[0105] 10. Preparation of PCer from Cabbage One small cabbage head was heated in a hot bath (80°C, 5 minutes) and then dried (80°C, 24 hours). The dry mass was 28 g (equivalent to a wet mass of 280 g). The dried cabbage was powdered using a coffee maker blender. 200 mL of ethanol was added, the mixture was shaken well, and centrifuged (3000 g, 5 minutes). The supernatant was discarded, and the process was repeated two more times (100 mL of ethanol was used each time). The mixture was homogenized with hot water (80°C, 200 mL) and filtered. 80 mL of hot water was added to the residue, which was homogenized again and then filtered. The resulting filtrate was cooled and centrifuged (3000 g, 5 minutes). 280 mL of the supernatant was used as GIPC solution (approximately 100 μM (back-calculated from the amount of PCer produced)). 56 mL of GIPC-PLD-containing enzyme solution was added to 280 mL of GIPC solution, followed by 28 mL of 1 M citrate buffer (pH 6.8) and 14 mL of 1 g / 50 mL NaDOC. After incubation at 30°C for 1 hour, the reaction was stopped at 80°C for 5 minutes. Citric acid (crystals) was added, and the pH was adjusted to 5 using a pH meter to obtain PC1P solution (approximately 100 μM (back-calculated from the amount of PCer produced)). Three grams of phytase (approximately 0.8% by mass) was added to the PC1P solution and allowed to react at 40°C for 16 hours. The reaction was stopped by heating at 80°C for 5 minutes to obtain a PCer solution. The resulting PCer solution was cooled to 4°C and centrifuged to obtain a precipitate. Water was added to the liquid precipitate to make a 60 mL volume, followed by the addition of 120 mL of ethanol and 60 mL of hexane, mixing, and centrifugation. The hexane layer was removed, and the remaining layer was mixed with hexane, mixed, and centrifuged. The hexane layers were combined and concentrated to obtain a purified PCer. The hexane layer was concentrated to dryness, and the residue was dissolved in a 2:1 mixture of chloroform and methanol. An amount equivalent to the cabbage shown in the figure was spotted on a TLC column. The results are shown in Figure 12. Note that the numbers in the upper left corner of the gel photograph in Figure 12 indicate the amount of PCer (quantitative value).

[0106] Quantitation using ImageJ confirmed that the amount of PCer was 23 nmol when the starting cabbage weight (wet mass) was 0.14 g. The calculated MW of PCer was 15.6 μg (111 μg / g), or 31 mg / 280 g cabbage (wet mass). The total mass of the purified PCer was 180 mg, meaning that 0.6 mg of purified PCer with a purity of 17% was obtained from 1 g of cabbage.

[0107] 11. Preparation of PCer from Radish When radish is grated, endogenous GIPC-PLD may act on endogenous GIPC to produce PC1P. Therefore, we investigated whether PCer would be produced by treating grated radish juice with phytase. 100 g (wet wt) of radish was grated, and the grated juice (corresponding to PC1P solution) (approximately 130 μM (back-calculated from the amount of PCer produced)) was filtered through a nonwoven fabric. 0.1 M citrate buffer (pH 5.0) and citric acid were added to the filtrate, and the pH was adjusted to 5 using a pH meter. 1 g of phytase (final concentration approximately 1% by mass) was added, and the mixture was reacted at 40°C for 16 hours. The reaction was terminated by heating at 80°C for 5 minutes to obtain a PCer solution. The resulting PCer solution was cooled to 4°C and centrifuged (1300 g, 5 minutes) to obtain a precipitate. Water was added to the precipitate to make a 30 mL volume, followed by addition of 60 mL of ethanol and 30 mL of hexane, mixing, and centrifuging. The hexane layer was removed, and the remaining layer was again added with hexane, mixed, and centrifuged. The hexane layers were combined and concentrated to obtain a purified PCer. For comparison, extraction by Bligh & Dyer was also performed. The hexane layer was concentrated to dryness, and the residue was dissolved in a chloroform / methanol mixture (2:1), and an amount equivalent to the amount of radish (wet mass) shown in Figure 13 was spotted on a TLC. The results are shown in Figure 13. In Figure 13, "33 nmol" indicates the amount of PCer (quantitative value).

[0108] Quantitation using ImageJ confirmed that the amount of PCer extracted with a hexane:ethanol:water mixture (0.25 g) was equivalent to 33 mol. The calculated MW of PCer was 22.4 μg (90 μg / g), or 9 mg / 100 g of radish (wet wt). The total mass of the purified PCer was 30 mg, meaning that 0.3 mg of 30% pure PCer was obtained from 1 g of radish.

Claims

1. (B) A method for producing ceramide, comprising the step of contacting a sample containing phytoceramide 1-phosphate with phytase (EC 3.1.3.8), wherein the pH of the reaction system in (B) is 2 to 6.

2. Before the step (B), 2. The method according to claim 1, comprising the step of: (A) contacting a water extract of a plant with glycosyl inositol phosphoceramide-specific phospholipase D to obtain a sample containing phytoceramide 1-phosphate.

3. 3. The production method according to claim 1, further comprising, after step (B), a step of mixing a sample containing phytoceramide 1-phosphate contacted with phytase (EC 3.1.3.8) with a linear alkane having 5 to 8 carbon atoms and an alcohol having 1 to 5 carbon atoms, and recovering a linear alkane fraction having 5 to 8 carbon atoms.

4. The method according to claim 2 or 3, wherein the glycosyl inositol phosphoceramide-specific phospholipase D is a phospholipase D derived from a plant of the Brassicaceae family.

5. The method according to any one of claims 2 to 4, wherein the plant is a leafy or root vegetable, a fruit, or a mushroom.

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