JP3992425B2 - Process for producing glycosphingolipid-containing material - Google Patents

Description

[0001]
[Technical field to which the invention belongs]
The present invention relates to a method for producing a glycosphingolipid contained in tobi powder , and more particularly to a method for efficiently producing a glycosphingolipid using tobi powder as a raw material.
[0002]
[Prior art]
Recent studies have revealed that some complex carbohydrates, especially glycolipids, have significant physiological activity. For example, ceramide composed of fatty acid and sphingosine, and cerebroside, a kind of glycosphingolipid composed of sugar, fatty acid and sphingosine, are present in the stratum corneum of human skin and function to prevent water from evaporating from the body. It has become clear. Development in use in the cosmetics field taking advantage of this high moisturizing property, as well as application in the pharmaceutical field taking advantage of the elastase inhibitory effect and free radical inhibitory effect, is progressing.
[0003]
Conventionally, these ceramide-related substances centering on glycosphingolipids have been extracted and supplied from bovine brain and the like. However, since the occurrence of mad cow disease in 1986, the supply amount has drastically decreased due to the possibility of infection to humans, and a regression phenomenon has occurred to ceramide-related substances of safe plant origin.
Plant-derived glycosphingolipids, particularly glucosylceramides, include rice (Agric. Biol. Chem., 49, 2753 (1985)) and rice bran (Japanese Patent Laid-Open No. 11-279586) and wheat (Agric. Biol. Chem., 49, 3609 (1985)), soybeans (Chem. Pharm. Bull., 38 (11), 2933 (1990), Japanese Patent Laid-Open No. 04-282317), etc. are known, Development is already underway for cosmetic ingredients and food additives.
[0004]
[Problems to be solved by the invention]
What has been utilized as plant raw materials for obtaining these plant-derived glycosphingolipids is limited to cereals and beans so far. The content of these glycosphingolipids is not so high, and it is estimated that both are about 0.01%. Moreover, all of these plant raw materials are edible for human beings, and the residue after extraction of glycosphingolipid loses its value as a food. As described above, in order to extract a very small amount of glycosphingolipid component, it is a problem of plant raw materials that a great amount of food raw materials are lost as food. On the other hand, if you look around the food processing industry, Tobi powder has a peculiar taste and pungent odor, although it produces 3000 to 4,000 tons per year as a by-product when producing konjac from konjac koji. Although it is used as a thickener for fertilizer, concrete, etc., it is a resource that is not used at all as food. Cottonseed oil cake is a by-product of squeezing cottonseed and obtaining cottonseed oil, and it was used as a vegetable protein feed until about 10 years ago. The number of farmers who feed cotton seeds directly as energy feed is increasing, and improvement in utility value is desired.
[0005]
The present invention relates to a method for producing glycosphingolipid, which has been attracting attention as a functional material for cosmetics and foods, and there has been a problem in product safety, which has been frequently pointed out by a conventionally known method based on extraction from animal tissues. It is an object of the present invention to provide a method of using a raw material that has not been valued as a food so far, although it is a plant material.
[0006]
[Means for Solving the Problems]
The present inventors have cereals conventionally used, such as legumes, outside than vegetative raw materials, high concentrations result of searching vegetable material containing glycosphingolipid, as the food as described above have been utilized The present invention has been completed by identifying that glycosphingolipids are contained in plant-derived natural resources such as powdered tobi (rice cakes) and cottonseed oil cake (oil cake) at a concentration comparable to or exceeding that of cereals and beans. I came to let you. More specifically, it is produced in large quantities during the production of konjac, and as a food, it is low in utility value tobi powder , sphingoglycolipid, which is also rich in cerebrosides, and has an appropriate polarity It has been found that the components can be efficiently extracted from these natural products by using an organic solvent, and the present invention has been completed.
[0007]
That is, the gist of the present invention is a method for producing a glycosphingolipid-containing product, characterized by adding a polar organic solvent to a powdered tobacco and extracting the glycosphingolipid.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
Tobi powder that is preferably used as an extraction raw material in the present invention is available in Japan from a national coffin raw material cooperative .
[0009]
Examples of the polar organic solvent used as the extractant in the present invention include alcohols, chloroform, acetone, acetonitrile and the like. Of these, hydrophilic alcohols are preferred. As such an alcohol, for example, any of monohydric alcohols such as methanol, ethanol, propanol, isopropanol and butanol, and dihydric or higher alcohols such as ethylene glycol and propylene glycol can be used. These solvents can be used even if they contain moisture, and two or more kinds of these solvents can be mixed and used as long as they are within the range of compositions that are mutually soluble. Furthermore, if necessary, a surfactant, an inclusion compound, or the like can be added as long as the object of the present invention is not removed and used as an extraction accelerator. In addition, when adding these extraction promoters, they can be removed by a known technique such as a column method after the completion of extraction, if necessary.
[0010]
The amount of the polar organic solvent is desirably about 1 to 30 times, more desirably about 2 to 10 times the amount of the extraction raw material to be used. If the amount of the solvent used is less than this range, the solvent does not reach the entire raw material, there is a possibility that the extraction becomes insufficient, and even if an amount of solvent exceeding this range is added, the extraction amount is no longer affected, Only the burden of solvent removal work in the subsequent concentration process is increased.
[0011]
Although the extraction temperature depends on the boiling point of the solvent to be used, when ethanol is used, the temperature is preferably from about room temperature to 70 ° C., more preferably from about room temperature to 60 ° C. If the extraction temperature is lower than this range, the extraction efficiency is lowered. Even if the temperature is higher than this range, the extraction efficiency is not greatly affected, and the amount of energy used is increased unnecessarily.
[0012]
The extraction time is 1 to 24 hours, preferably 2 to 10 hours, more preferably about 2 to 5 hours. When the extraction time is shorter than this range, the extraction is not sufficiently performed, and even if the extraction is performed for a long time exceeding this range, the extraction amount can no longer be expected to increase. Moreover, time can be shortened by stirring during extraction.
[0013]
The extraction operation is not limited to a single batch operation. A fresh solvent can be added again to the residue after extraction to perform an extraction operation, or the extraction solvent can be brought into contact with the extraction raw material a plurality of times. That is, as the extraction operation, any of batch operation, semi-continuous operation, and countercurrent multistage contact operation can be used.
[0014]
Next, the extraction residue is separated and removed. The separation method is not particularly limited, and for example, a filter press, a cylinder press, a decanter, a centrifuge, or the like can be used.
[0015]
The extract thus obtained is sent to the concentration step. In the concentration treatment, for example, concentration and drying can be performed using a vacuum concentration apparatus such as an evaporator. By this concentration treatment, a yellow to brown oily or waxy concentrate is obtained.
[0016]
When it is necessary to remove the impurities and improve the purity of the concentrate, it can be purified by a conventional method. That is, it can be performed by a method of passing through a silica gel column or the like, or simply by partitioning between a halogen-based organic solvent such as chloroform or dichloromethane and water-methanol.
[0017]
When the purity is improved by the above distribution, the water used is preferably a neutral salt aqueous solution in order to suppress the formation of a micellar structure of the amphiphile contained in the system. The salt concentration in that case depends on the type of salt, but in the case of potassium chloride, the desired concentration is in the range of 0.1 to 1.5M, more preferably 0.2 to 1.2M. This is because if the salt concentration is smaller than this range, a sufficient effect cannot be seen, and if it exceeds this range, precipitation may occur when methanol is added.
[0018]
Next, as a method for analyzing the obtained concentrate, the simplest method is a thin layer chromatographic method. Glycosphingolipid, especially glucosylceramide, is a commercially available standard product. Use this as a reference, use a commercially available silica gel thin-layer plate, develop it with a developing solvent such as chloroformic methanol, concentrate concentrated sulfuric acid, and anthrone reagent. If the color is developed, it can be easily determined that glycosphingolipids are present in a high content in the concentrate. In addition, it can be determined that glycosphingolipids are abundant by conventional methods such as high performance liquid chromatography and various chromatographic mass spectrometry methods.
[0019]
The glycosphingolipid-containing product finally obtained can be used by being dispersed in water or the like. In this case, in order to improve the dispersibility, other amphiphilic molecules, surfactants, dispersion stabilizers and the like can be blended at an appropriate blending ratio as necessary.
[0020]
Examples of such amphiphilic molecules, surfactants and dispersion stabilizers include phospholipids such as lecithin and lysolecithin, glycolipids such as sugar glycerides and sugar sterols, saponins, polysorbates, long chain fatty acids, water-soluble amphiphiles. A functional polymer.
[0021]
The obtained glycosphingolipid-containing product can be used as it is by solidifying and powdering it using a method such as freeze-drying or spray-drying.
[0022]
The glycosphingolipid component having various forms thus obtained can be used as a coating substrate for cosmetics, food additives, and the like.
[0023]
【Example】
EXAMPLES Hereinafter, although this invention is demonstrated concretely using an Example, this invention is not limited to this. First, the measuring apparatus and measuring method used in the following examples will be described.
(1) Measuring apparatus / silica gel thin layer chromatography plate: manufactured by Merck Silica gel 60 F254 type, layer thickness 0.5 mm
・ Silica gel preparative thin layer chromatography plate: manufactured by Merck Silica gel 60 F254, layer thickness 2 mm
Densitometer: CS-9300PC manufactured by Shimadzu Corporation
[0024]
(2) Measurement method (a) Confirmation of the presence of a glycosphingolipid component, quantification Along with a commercially available glucosylceramide standard ethanol solution, the ethanol solution of the extract was applied to a thin-layer chromatography plate of silica gel, and a chloroform solvent mixture ( 9: 1). A spot that gave the same Rf value (= spot moving distance / solvent tip moving distance) as that of the standard product by heating with sulfuric acid spray was defined as a glycosphingolipid spot. The cerebroside component is quantified by taking about 5 concentrations of a commercially available standard ethanol solution (for example, 0.25, 0.5, 1, 2, 5 mg / ml, or changing the amount applied). The color development intensity of these thin layer chromatographs was determined with a densitometer, a calibration curve was prepared, and the color development intensity of the sample was measured. Then, the amount of glucosylceramide (sphingoglycolipid) was calculated from the calibration curve.
[0025]
Example 1
1 kg of powdered powder was charged into a stirring tank, 2 L of ethanol was added thereto, and the mixture was stirred at room temperature for 5 hours. Thereafter, the extract and the residue were separated by filtration. The extract was concentrated by an evaporator to obtain 13.3 g of a brownish waxy concentrate. Based on the above measurement method (a), the presence and quantification of glycosphingolipids were confirmed. The results are shown in Table 1.
[0026]
[Table 1]
[0027]
Comparative Example 1
1 kg of cottonseed oil cake was charged into a stirring tank, 2 L of methanol was added thereto, and the mixture was stirred at room temperature for 5 hours. Thereafter, the extract and the residue were separated by filtration. The extract was concentrated by an evaporator to obtain 8.44 g of a brownish waxy concentrate. Based on the above measurement method (a), the presence and quantification of glycosphingolipids were confirmed. The results are shown in Table 1.
[0028]
Comparative Example 2
1 kg of wheat flour was charged into a stirring tank, 2 L of ethanol was added thereto, and the mixture was stirred at room temperature for 5 hours. Thereafter, the extract and the residue were separated by filtration. The extract was concentrated by an evaporator to obtain 9.71 g of a deep yellowish brown waxy concentrate. Based on the above measurement method (a), the presence and quantification of glycosphingolipids were confirmed. The results are shown in Table 1.
[0029]
As shown in Table 1, the extract obtained from the tobi powder using a polar organic solvent is as much as 1.33% of the total weight, and the glycosphingolipid component is 10.1% by weight based on the extract. It is high with 0.134% by weight based on the extracted raw material. In other words, it was found that the glycosphingolipid component was present in extremely high concentration and high purity in the powdered tobi powder. In addition, it can be seen that the sphingoglycolipid component is contained in the cottonseed oil cake extract in a sufficiently high concentration and purity as compared with the flour conventionally used as the extraction raw material. Thus, we succeeded in extracting glycosphingolipids from natural waste from the food processing industry at extremely high concentrations.
[0030]
Example 2
500 g of powdered tobacco was charged into a stirring tank, 1.5 L of ethanol was added thereto, stirred at room temperature for 2 hours, and the extract and the residue were separated by filtration. The extract was concentrated by an evaporator to obtain 6.4 g of a brownish waxy concentrate. This concentrate was dissolved in 1 L of a chloroform methanol mixture (1: 1), and further 400 mL of purified water was added in a decanter for partitioning. Thereafter, the chloroform layer was taken out and concentrated with an evaporator to obtain 3.9 g of a concentrate. Based on the above measurement method (a), the presence and quantification of glycosphingolipids were confirmed. The results are shown in Table 2.
[0031]
[Table 2]
[0032]
Example 3
In the same manner as in Example 2 , 500 g of tobi powder was charged into a stirring tank, 1.5 L of ethanol was added thereto, and the mixture was stirred at room temperature for 2 hours. Thereafter, the extract and the residue were separated by filtration. The extract was concentrated by an evaporator to obtain 6.3 g of a brownish waxy concentrate. This concentrate was dissolved in 1 L of a chloroform methanol mixture (1: 1), and further 400 mL of a 0.4 M aqueous potassium chloride solution was added in a decanter for partitioning. Thereafter, the chloroform layer was taken out and concentrated by an evaporator to obtain 3.3 g of a concentrate. Based on the above measurement method (a), the presence of sphingoglycolipids was confirmed and quantified. The results are shown in Table 2.
[0033]
As shown in Table 2, it was found that the purity of the glycosphingolipid in the extract can be greatly improved by a simple method only by partitioning between solvents. At this time, it was shown that the distribution efficiency was further improved by using a neutral salt aqueous solution as the water to be used.
[0034]
【The invention's effect】
According to the present invention, until now known have not been Tobi powder or al, is highly safe, it is possible to obtain a glycosphingolipid good plant origin of the image as a material.

Claims (1)

A method for producing a glycosphingolipid-containing product, comprising adding an organic solvent having polarity to a powder of tobi and extracting a glycosphingolipid.