JP4503263B2 - Process for producing glycosphingolipid - Google Patents

Description

  The present invention relates to an efficient method for producing a glycosphingolipid-containing product containing a high level of glycosphingolipid from plant material, and the resulting glycosphingolipid-containing product is not derived from an animal but from a plant. Moreover, because it has the feature that it does not use organic solvents that are not approved for use in foods and drinks in its manufacturing process, it is highly safe and can be used for foods and drinks and oral intake as well as cosmetics. It has special advantages.

  Traditionally, natural glycosphingolipids have been extracted from bovine brain with organic solvents and used in cosmetics, etc., but in recent years, they contain a relatively large amount of glycosphingolipids as a safe and safe material for mad cow disease. The separation from plant-derived materials has been studied. As such plant-derived materials, rice bran, soybean meal, soybean germ, wheat bran, soy sauce cake, potatoes and the like are being studied. However, these glycosphingolipid extraction and separation methods generally use organic solvents (chloroform, methanol, acetonitrile, pyridine, etc.) that should not be used in foods, such as chloroform in soybean oil (Patent Document 1). In addition, there is methanol in beer lees (Patent Document 2), and the glycosphingolipid extracted and separated in this way can be used only for limited uses such as cosmetics and cannot be used for food and drink.

In addition, even when extracted and separated from a plant-derived material with an organic solvent in this way, all the lipids contained in each plant-derived material and components soluble in the organic solvent, including glycosphingolipids, are all contained. It is difficult to increase the concentration of glycosphingolipid even in the subsequent separation and purification using an organic solvent, and the content of glycosphingolipid in the glycosphingolipid-containing product is also low. ing. Therefore, methods for purifying glycosphingolipids such as those using high-speed chromatography (Patent Document 3) and those using hydrolysis and silica gel chromatography (Patent Document 4) have been proposed after organic solvent extraction. It is inevitable that there are difficulties in industrial use due to capital investment.
JP-A-4-282317 JP 11-193283 A JP 2002-294274 A Japanese Patent Laid-Open No. 2002-30093

  The present invention has been made in view of the current state of the art, and is excellent in safety and can be used not only for cosmetics but also for foods and drinks and contains a glycosphingolipid containing a high level of glycosphingolipid. It was made for the purpose of developing a new technology for efficient industrial production of goods.

  The present invention has been made in order to achieve the above-mentioned object, and the present inventors have paid attention to plant-derived materials, not animals-derived foods and beverages, from the viewpoint of safety and utility to foods and beverages. The use of solvents that have been approved for use in Japan has been studied from various directions.

  That is, the present inventors are directed to plant-derived materials containing a relatively large amount of glycosphingolipids, and the extraction of glycosphingolipids as an organic solvent, which can be used in foods, includes ethanol, acetone, and hexane. As a result of intensive studies on a simple method for producing a high-purity glycosphingolipid-containing product using only limited species, a high-purity glycosphingolipid-containing material can be efficiently obtained by freezing the ethanol extract. Obtaining knowledge, the present invention has been completed.

  The present invention relates to a method for producing a high-sphingoglycolipid content by extracting a plant-derived material with an organic solvent, freezing the separated organic solvent solution, and obtaining a precipitate of the glycosphingolipid-containing material. The organic solvent is preferably an alcohol, particularly ethanol, and the present invention includes, for example, the following method.

  In the present invention, for example, 2 parts by weight of ethanol is added to 1 part by weight of a dried plant-derived material and extracted at 50 to 60 ° C., and then the solid content is separated, and the obtained ethanol extraction is performed. This includes a method for producing a glycosphingolipid-containing product, in which the liquid is stored at 20 ° C. or less under zero for 12 hours or more to obtain a precipitate of the glycosphingolipid-containing product. Furthermore, the resulting precipitate of glycosphingolipid-containing material is separated from the ethanol solution, and the precipitate is mixed and washed with acetone and / or hexane to remove lipids other than the glycosphingolipid, thereby achieving high purity. And a method for producing a glycosphingolipid-containing product.

Specific examples of the latter include the following. That is, the material is subjected to ethanol treatment and freezing treatment, and the obtained ethanol-containing material containing a precipitate of glycosphingolipid-containing material is treated in at least one of the following steps (1) to (5). A method for producing a glycosphingolipid.
(1) After solid-liquid separation to separate the ethanol solution part and the precipitate, the precipitate is dried to obtain a solid or powder.
(2) After solid-liquid separation to separate the ethanol solution part and the precipitate, the precipitate is washed with acetone and / or hexane and then dried to obtain a solid or powder.
(3) In step (2), after washing with acetone and / or hexane, solid-liquid separation is performed, and the resulting precipitate is dried to obtain a solid or powder.
(4) The solid or powder obtained in step (1) is washed with acetone and / or hexane and then dried to obtain a solid or powder. (5) In step (2), acetone and / or hexane After washing with, the solid-liquid separation is performed, and the resulting precipitate is dried to obtain a solid or powder.

  The raw material used in the present invention may be a plant-derived material, which contains a relatively large amount of glycosphingolipid, rice bran, soybean germ, wheat bran, sugar beet pulp, soy sauce cake, potato pulp, konnyaku tobi powder, rice cakes, It is intended for plant-derived by-products that are not generally used for food, such as defatted rice bran and soybean meal after degreasing.

  Of particular interest among the raw materials are sugar beet pulps that are produced in large quantities during the production of sugar beet sugar. Compared to other raw materials, the content of other lipids is small, which is suitable for obtaining pure glycosphingolipids, and is advantageous from the viewpoint of using by-products produced in large quantities.

  Glycosphingolipids obtained from sugar beet-derived pulp are considered to be mainly composed of ceramide, glucoceramide, sulfatide, and ganglioside, lactosylceramide, globocerceramide, etc., which have a larger sugar chain than a monosaccharide bond. For example, when beet fiber manufactured from sugar beet pulp is used, it is easy to handle because moisture, particle size, and the like are uniformly maintained. Beet fiber is a sugar beet pulp from which soluble components such as sucrose have been sufficiently extracted and removed in the process of beet sugar, that is, beet root is cut into pieces or ground, squeezed, and then immersed in hot water. The residue obtained by sufficiently extracting and removing the soluble component is further processed.

  That is, beet fiber is a fibrous material obtained by subjecting sugar beet pulp to necessary treatments such as dehydration, decolorization, deodorization, drying, pulverization, and sieving. As the beet fiber, any of the above fiber-containing materials including commercially available products can be used, but a powder body (moisture content) of 50 mesh through, preferably 30 mesh through, more preferably 10 mesh through. 25% or less, preferably 15% or less, more preferably 10% (weight) or less).

  According to the production method of the present invention, a glycosphingolipid-containing product containing glycosphingolipids with high purity can be obtained from plant-derived materials by a simple operation, and a safe glycosphingolipid for foods and cosmetics can be obtained. Obtainable.

  In addition, although a glycosphingolipid having a purity of about 4% is commercially available, it can provide a glycosphingolipid-containing product with greatly increased purity, such as sugar sugar derived from sugar beet pulp or beet fiber and polymerized. The glycosphingolipid having a basic skeleton having a sugar chain and a fatty acid and soluble in alcohol can be provided, and can be used not only for cosmetics but also for foods and drinks.

Hereinafter, the present invention will be described in more detail.
In order to carry out the present invention, it is necessary to extract a plant-derived material with an organic solvent. As the organic solvent for extracting glycosphingolipid used in the present invention, alcohol, particularly ethanol, is suitable. Ethanol is preferably a commercially available product having no moisture, but the recovered ethanol can be used if it has a moisture content of 5% or less. The amount of ethanol added is 1 to 10 parts by weight, preferably 2 to 5 parts by weight, based on 1 part by weight of the raw material. If the amount of ethanol added is small, the subsequent operation becomes easy. However, the amount added should be selected to increase the extraction rate of glycosphingolipids of each material.

  The extraction temperature is in the range of about room temperature to 70 ° C, more desirably 50-60 ° C. The extraction time is about 1 to 24 hours, preferably about 2 to 5 hours. The time can be shortened by stirring during extraction, and the extraction time varies depending on the solvent. Next, the extraction residue is separated and removed. The separation method is not particularly limited, and may be a solid-liquid separation device such as a centrifuge, a filter press, a cylinder press, or the like.

  The ethanol extract thus obtained is subjected to the freezing treatment of the present invention. In the freezing treatment, the freezing temperature is −20 ° C. or lower, preferably −50 ° C. or lower. Then, the ethanol extract is stored for 6 to 24 hours, preferably 12 to 24 hours, at the freezing temperature. By this freezing treatment, a precipitate containing glycosphingolipid is generated in the ethanol extract.

  In Table 1, 3 parts of ethanol was added to 1 part of each plant material, extracted at 60 ° C. for 5 hours, and the obtained ethanol extract was obtained by further freezing treatment (−50 ° C., 12 hours). The dried precipitate and the ethanol-concentrated concentrated dried product by an evaporator, wt% (vs. plant material) and the content of the glycosphingolipid in the dried precipitate and the concentrated evaporator-dried product were shown. The glycosphingolipid was determined by the method of Example 1 described later.

Note 1) Crude fat content% of plant material (AOAC method)
Note 2) Dry weight% of the precipitate obtained by freezing treatment (vs. plant material)
Note 3) Glycosphingolipid content% in the dried precipitate obtained by freezing treatment
Note 4)% by weight of the concentrated and dried ethanol extract (vs. plant material)
Note 5) Glycosphingolipid content in the concentrated and dried product of ethanol extract

  As is clear from the above results, comparing the precipitate from the freezing treatment of the ethanol extract with the concentrated dried product of the ethanol extract, the weight (%) of the precipitate from the freezing treatment is significantly less for each plant material. In spite of this, the content% of the glycosphingolipid (vs. plant material) is almost the same, indicating that this freezing treatment of the present invention is excellent for the purification of glycosphingolipid. In addition, the manufacturing cost can be greatly reduced compared to the conventional treatment such as evaporation in the removal of the organic solvent after the organic solvent extraction, and if the precipitate by freezing treatment is separated, the glycosphingolipid-containing product which is the object of the present invention can be obtained. Obtainable.

  In this way, a precipitate is generated by freezing the ethanol extract, and this is separated into a liquid (ethanol solution part) and a precipitate by a solid-liquid separation system. In the present specification, a product obtained by separating the produced precipitate from the liquid part is referred to as a precipitate, and a substance that is not separated from the liquid part is simply referred to as a precipitate.

  The precipitate obtained by the freezing treatment obtained as described above (that is, the separated precipitate produced by the freezing treatment) is provided as a sphingoglycolipid-containing liquid as it is or after drying (may be solid and / or powdered). be able to.

  In addition, in order to obtain a glycosphingolipid-containing product having a further increased purity, a precipitate obtained by freezing treatment and / or a dried product (solid or powdered as desired) is acetone and / or What is necessary is just to wash | clean hexane. In the washing treatment, acetone and / or hexane is added to the precipitate and / or dried product thereof, mixed, stirred, then solid-liquid separated to remove the liquid part, and the remainder (referred to as precipitate) may be obtained. . For example, in the case of a precipitate by freezing treatment, 1 to 20 parts by weight of acetone and / or hexane is mixed with 1 part by weight of the precipitate, preferably 5 to 10 parts by weight, and then the liquid part is removed. Purity can be increased. What is necessary is just to implement suitably also in the case of washing | cleaning of a dried material, following the washing | cleaning of the above-mentioned deposit.

  After washing in this way, the precipitate can be provided as it is as a glycosphingolipid-containing product, but it may be dried (solid and / or powdered) as described above. In addition, when using the liquid mixture of acetone and hexane as a washing | cleaning solvent, the liquid mixture mixed by the ratio of 1: 99-99: 1 including equal volume mixing can be used.

  Table 2 shows the results of measuring the sphingoglycolipid content of the precipitate after mixing and stirring 8 parts by weight of acetone (acetone washing) with 1 part by weight of the precipitate by ethanol solution freezing treatment and removing the acetone solution by centrifugation. It was.

Note 1) Dry weight% of precipitate obtained by freezing treatment (vs. plant material)
Note 2) Glycosphingolipid content% in the dried precipitate obtained by freezing
Note 3) Weight% of dry matter after washing the precipitate with acetone (vs. plant material)
Note 4) Content of glycosphingolipid in the dried product after washing the precipitate with acetone%

  As is clear from the above results, the weight (%) of the precipitate was greatly reduced by washing the precipitate obtained by freezing the ethanol extract with acetone in both the plant material wheat bran and defatted rice bran. However, the amount of glycosphingolipid% (vs. plant material) is almost the same, indicating that the purity of the glycosphingolipid-containing material was increased by washing with acetone.

  Further, the glycosphingolipid-containing product obtained in the present invention can be used as it is by solidifying or powdering it by a method such as spray drying or freeze drying. According to the present invention, a glycosphingolipid-containing substance having a high glycosphingolipid content can be obtained. For example, when sugar beet pulp and / or beet fiber is used as a raw material, 30% or more glycosphingolipid-containing material And a high content of 50% or more can be obtained.

  In the present invention, using only an organic solvent (ethanol, acetone, hexane) that is approved as a food additive, the ethanol extract is frozen (acquisition of precipitate) and washed with acetone and / or hexane. Thus, the present inventors have found that an edible high-purity glycosphingolipid-containing material can be easily obtained, thereby completing the present invention. In order to further increase the glycosphingolipid purity of the glycosphingolipid-containing material, a treatment such as chromatography may be performed.

  The chromatographic treatment may be performed according to a conventional method. For example, as a chromatographic separation method, the separation apparatus is a fixed bed method (one-pass method), a continuous method (pseudo moving bed method), a semi-continuous method (a fixed bed method and a continuous method). Applicable). As the packed ion exchange resin of the apparatus, chromatographic Na type, K type, Ca type and other strongly acidic ion exchange resins are used. As the resin, a styrene divinylbenzene resin having a uniform particle diameter is used. Since various chromatographic resins are sold by ion exchange resin manufacturers, they may be appropriately selected and chromatographed.

  Hereinafter, the present invention will be described with reference to examples. First, a measurement apparatus and a measurement method used in the following examples will be described.

(1) Measuring apparatus Silica gel thin layer chromatography plate: Silicagel 60 F254 type, layer thickness 0.5 mm, manufactured by Merck & Co., Densitometer: CS-9300PC, manufactured by Shimadzu Corporation

(2) Measuring method Confirmation and quantification of glycosphingolipid component: A chloroform-methanol mixture (2: 1) solution of the reagent glucotorceramide standard product and a chloroform-methanol mixture (2: 1) solution of the extract were diluted with silica gel. Spotted on a layer chromatography plate and developed with a chloroform-methanol-water mixed solvent (65: 25: 4). By using anthrone solution spray heating, a glycosphingolipid spot having the same Rf value as that of the standard product was used. In addition, quantification of glycosphingolipids, using a Sigma reagent as a standard product, creating a calibration curve with a densitometer for the color intensity of a thin layer chromatograph according to the difference in concentration, and measuring the color intensity of the sample, From the calibration curve, the amount of glycosphingolipid was determined by calculation.

  1 kg of wheat bran was put into a sealed stirring tank, 2 L of ethanol was added thereto, and the mixture was stirred at 60 ° C. for 5 hours. Thereafter, the extract and the residue were separated by suction filtration. The ethanol extract was stored in a freezer (150 ° C.) for 12 hours. The precipitate in the ethanol extract obtained by freezing was collected by centrifugation and dried by a vacuum dryer (50 ° C.) to obtain 38.2 g of a brown waxy dried product.

  For comparison, 1 kg of wheat bran was subjected to the same treatment to obtain an ethanol extract and concentrated to dryness using a rotary evaporator to obtain 73.2 g of a brown waxy dried product. Based on the above-mentioned measurement method, the presence of these sphingoglycolipids was confirmed and quantified. The results are shown in Table 3.

  As is clear from the results in Table 3, the ethanol extract obtained by extracting ethanol from wheat bran was frozen and the dried product of the precipitate had a glycosphingolipid content of 2.46%. The purity was remarkably increased as compared with 1.30% of the dried product obtained by directly concentrating. That is, it is revealed that a significant portion of the lipids other than the glycosphingolipid and the ethanol-soluble component in the ethanol extract are not transferred to the precipitate by freezing treatment.

  30 g of each of the dried product (freeze-precipitated and concentrated to dryness) obtained in Example 2 was mixed with 200 ml of acetone with stirring, and the insoluble material was centrifuged and separated by centrifugation. (50 ° C.) to obtain 6.39 g and 4.81 g of a white brown powder dried product. Based on the above measurement method, the presence and quantification of glycosphingolipids were confirmed for these dried powders. The results are shown in Table 4.

  As is clear from the results in Table 4, the precipitate obtained by the freezing treatment of the present invention was washed with acetone to obtain a glycosphingolipid-containing product having a purity of 11 and 4% higher than that of Example 1. It was. In addition, the dried product obtained by directly concentrating the ethanol extract was better in recovery and purity than the dried product obtained by washing with acetone.

  1 kg of soybean meal and 1 kg of konjac powder were put in a sealed stirring tank, 2.5 L of ethanol was added thereto, and the mixture was stirred at 60 ° C. for 6 hours. Thereafter, the extract and the residue were separated by suction filtration. The ethanol extract was stored for 12 hours in a freezer (−50 ° C.). The precipitate in the ethanol extract obtained by the freezing treatment was collected by centrifugation to obtain 14.2 g and 3.18 g of precipitate (in terms of solid content). After adding 100 ml of acetone to each of these precipitates and stirring and mixing, the insoluble matter was collected by centrifugation, dried in a vacuum dryer (50 ° C.) and dried to a white brown powder, 5.23 g, and 1 .91 g was obtained. Based on the above measurement method, the presence and quantification of the glycosphingolipids were confirmed for each dried powder. The results are shown in Table 5.

  As is clear from Table 5, the sphingo of high purity of glycosphingolipid content of 13.3% and 30.1% was obtained by subjecting soybean meal and konjac tobi powder to the freezing treatment of the present invention and washing with an organic solvent. A glycolipid-containing product was obtained.

  Each 1 kg of sugar beet pulp was put into a sealed stirring tank, and 2.5 L of ethanol was added thereto, followed by stirring at 60 ° C. for 6 hours. Thereafter, the extract and the residue were separated by suction filtration. The ethanol extract was stored for 12 hours in a freezer (−50 ° C.). The precipitate in the ethanol extract obtained by the freezing treatment was collected by centrifugation to obtain 3.7 g of precipitate (solid content converted value). After adding 100 ml of acetone to the precipitate and stirring and mixing, the insoluble matter was collected by centrifugation and dried in a vacuum dryer (50 ° C.) to obtain 2.1 g of a white brown powder dry solid. Based on the above measurement method, the presence of the glycosphingolipids was confirmed and quantified for the dried powder. The results are shown in Table 6.

  Sugar beet pulp is less concentrated by ethanol extraction than wheat bran, defatted rice bran, and soy bean meal of Example 4, but the subsequent acetone washing yields a concentrate yield similar to that of wheat bran. In addition, the glycosphingolipid content in the concentrate was remarkably high at 52.6%. As shown in Table 6, sugar beet pulp is extremely low in impurities other than glycosphingolipids in organic solvent extraction, and is commonly used in organic solvents (ethanol, acetone, hexane) as food additives commonly used in extraction. In addition, it is a plant-derived material that can obtain a glycosphingolipid-containing product having a purity of 50% or more by a simple extraction / separation method.

  In addition, sugar beet, which is a raw material for sugar beet pulp, is not a specific raw material for allergy labeling, and is a plant that is unrelated to genetic recombination. Therefore, it can be said that glycosphingolipids from sugar beet pulp are highly safe.

  Further, the obtained glycosphingolipid-containing material can be used by solidifying or powdering it by a method such as liquid (dissolved or suspended) or spray drying or freeze drying.

  Glycosphingolipids are not only effective for rough skin, prevention of skin aging, atopic dermatitis, and the like, but many techniques for use in foods have been reported. For example, use of an antihypertensive agent, a lipase inhibitor, an immunologically active agent, a cooking oil / fat composition, a cancer cell growth inhibitor, a surfactant, and the like can be considered. INDUSTRIAL APPLICABILITY The present invention can industrially produce a large amount of glycosphingolipid-containing products from sugar beet pulp, which is a by-product produced in a large amount in the beet sugar production process, with a simple operation, and can greatly contribute to society.

Claims (6)

A sugar beet pulp is extracted with ethanol, and the separated ethanol solution is stored at a temperature below 20 ° C. for 12 hours or longer to obtain a precipitate of a glycosphingolipid-containing product. A method for mass- producing a sugar beet-derived glycosphingoglycolipid-containing product having a glycosphingolipid content of 30% or more, wherein the glycosphingolipid content is 30% or more. The method for producing a large amount of a glycosphingolipid-containing material according to claim 1, wherein the freezing treatment is stored for 12 hours or more at a temperature of 50 ° C or less under zero to obtain a precipitate of the glycosphingolipid-containing material. A method for mass production of a glycosphingolipid-containing material, wherein the acetone washing treatment according to claim 1 or 2 is at least one of the following steps (1) to (3).
(1) After solid-liquid separation to separate the ethanol solution part and the precipitate, the precipitate is dried to obtain a solid or powder, which is washed with acetone and then dried to obtain a solid or powder. And
(2) After solid-liquid separation to separate the ethanol solution part and the precipitate, the precipitate is washed with acetone and then dried to obtain a solid or powder.
(3) In step (2), after washing treatment, solid-liquid separation is performed, and a precipitate obtained by separation is dried to obtain a solid or powder.   The method according to claim 3, wherein the solid-liquid separation is centrifugation.   The method according to any one of claims 1 to 4, wherein the sugar beet pulp is a beet fiber having a moisture content of 10% (weight) or less.   The method according to any one of claims 1 to 5, wherein a glycosphingolipid-containing product having a glycosphingolipid content of 50% or more is produced.